WO2012101066A1 - Pyridine biaryl amine compounds and their uses - Google Patents

Pyridine biaryl amine compounds and their uses Download PDF

Info

Publication number
WO2012101066A1
WO2012101066A1 PCT/EP2012/050914 EP2012050914W WO2012101066A1 WO 2012101066 A1 WO2012101066 A1 WO 2012101066A1 EP 2012050914 W EP2012050914 W EP 2012050914W WO 2012101066 A1 WO2012101066 A1 WO 2012101066A1
Authority
WO
WIPO (PCT)
Prior art keywords
mmol
compound
mixture
pyran
stirred
Prior art date
Application number
PCT/EP2012/050914
Other languages
French (fr)
Inventor
William R. Antonios-Mccrea
Paul A. Barsanti
Cheng Hu
Xianming Jin
Xiaodong Lin
Eric J. Martin
Yue Pan
Keith B. Pfister
Paul A. Renhowe
Martin Sendzik
James Sutton
Lifeng Wan
Original Assignee
Novartis Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis Ag filed Critical Novartis Ag
Publication of WO2012101066A1 publication Critical patent/WO2012101066A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/74—Amino or imino radicals substituted by hydrocarbon or substituted hydrocarbon radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

  • the invention provides a novel class of compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent diseases or disorders associated with aberrant cellular signaling pathways that can be modulated by inhibition of kinases, particularly diseases or disorders that involve aberrant cellular signaling pathways that can be modulated by inhibition of CDK9.
  • Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within the cell. (Hardie, G. and Hanks, S. THE PROTEIN KINASE FACTS BOOK, I and II, Academic Press, San Diego, Calif.: 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, efc.).
  • phosphorylate e.g., protein-tyrosine, protein-serine/threonine, lipids, efc.
  • CDK cyclin-dependent kinase
  • CDK1-B3 cyclin B1-B3
  • CDK2 cyclin D1-D3
  • CDK4 CDK5
  • CDK6 cyclin E
  • CDKs 7, 8, and 9 are implicated in the regulation of transcription.
  • the CDKs seem to participate in cell cycle progression and cellular transcription, and loss of growth control is linked to abnormal cell proliferation in disease (see e.g., Malumbres and Barbacid, Nat. Rev. Cancer 2001 , 1 :222). Increased activity or temporally abnormal activation of cyclin-dependent kinases has been shown to result in the development of human tumors (Sherr C. J., Science 1996, 274: 1672-1677). Indeed, human tumor development is commonly associated with alterations in either the CDK proteins themselves or their regulators (Cordon-Cardo C, Am. J. Pat1/701. 1995; 147: 545-560; Karp J. E. and Broder S., Nat. Med. 1995; 1 : 309-320; Hall M. et al., Adv.
  • CDKs 7 and 9 seem to play key roles in transcription initiation and elongation, respectively (see, e.g., Peterlin and Price, Cell 23: 297-305, 2006; Shapiro, J. Clin. Oncol. 24: 1770-83, 2006;).
  • Inhibition of CDK9 has been linked to direct induction of apoptosis in tumor cells of hematopoietic lineages through down-regulation of transcription of antiapoptotic proteins such as Mcl1 (Chao, S.-H. et al. J. Biol. Chem. 2000;275:28345- 28348; Chao, S.-H. et al. J. Biol. Chem.
  • Inhibition of transcription through CDK9 or CDK7 may have selective non-proliferative effect on the tumor cell types that are dependent on the transcription of mRNAs with short half lives, for example Cyclin D1 in Mantle Cell Lymphoma.
  • Some transcription factors such as Myc and NF-kB selectively recruit CDK9 to their promoters, and tumors dependent on activation of these signaling pathways may be sensitive to CDK9 inhibition.
  • CDK inhibitors may also be used in the treatment of
  • cardiovascular disorders such as restenosis and atherosclerosis and other vascular disorders that are due to aberrant cell proliferation.
  • Vascular smooth muscle proliferation and intimal hyperplasia following balloon angioplasty are inhibited by over-expression of the cyclin-dependent kinase inhibitor protein.
  • CDKs are important in neutrophil-mediated inflammation and CDK inhibitors promote the resolution of inflammation in animal models. (Rossi, A.G. et al, Nature Med. 2006, 12: 1056). Thus CDK inhibitors, including CDK9 inhibitors, may act as antiinflammatory agents.
  • CDK inhibitors are useful as chemoprotective agents through their ability to inhibit cell cycle progression of normal untransformed cells (Chen, et al. J. Natl.
  • CDK9 inhibitors Pyridine compounds of the formula below have been described as CDK9 inhibitors, and accordingly such compounds are useful for treating cancer and other conditions mediated by CDK9 activity:
  • Ri is Ci-8 alkyl, C 3 . 8 cycloalkyl, C 3 . 8 branched alkyl, -(CH 2 )o-3-0-Ci. 4 alkyl,
  • R 2 is hydrogen, Ci -4 alkoxy, Ci -4 haloalkyl, Ci -4 -alkyl, or halogen;
  • Ai is N or CR 3 ;
  • a 4 is N or CR 6 , with the proviso that only one of A ! and A 4 is a N;
  • R 3 is Ci -4 alkyl, H, or OCi -4 alkyl
  • R 4 is hydrogen, halogen, 5 to 7 membered heterocyclyl-aryl, or A 6 -L-R 9 ;
  • R 5 is hydrogen, Ci -4 alkyl, or halogen
  • R 6 is hydrogen, Ci -4 alkyl, or halogen
  • R 7 is hydrogen, Ci -4 alkyl, or halogen
  • a 6 is NR 8 ;
  • L is Co-3-alkylene or C 3 . 8 branched alkylene
  • R 8 is hydrogen, Ci -4 alkyl; or -C 3 . 8 branched alkyl;
  • R 9 is hydrogen, Ci. 6 alkyl, C 3 . 8 cycloalkyl, 4 to 8 member heterocycloalkyl, aryl, or heteroaryl, wherein said groups are optionally substituted with one to three substituents each independently selected from hydrogen, halogen, Ci -4 alkyl, Ci -4 haloalkyl, -OH, -O-C 1 .3 alkyl, -O-C 1 -3 haloalkyl, -0-(CH 2 ) 2 -3-0-Ci. 2 alkyl, -C(0)-Ci. 4 alkyl, and -NH-C(0)-Ci-4 alkyl.
  • CDK1 protein kinases
  • CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9 protein kinases
  • the present invention provides novel biaryl pyridine compounds having structural similarities to the compounds described above, which are also useful to treat cancer and other conditions based on their activity on CDK9.
  • Compounds of the invention are of Formulas l-VI as further described herein.
  • the invention includes pharmaceutically acceptable salts of compounds of any of Formulas l-VI. Further structural description of these compounds and of pharmaceutical compositions and methods of use of these compounds are described below.
  • the foregoing compounds are inhibitors of at least one kinase, including at least CDK9, and are therefore useful for treating conditions mediated by excessive or undesired levels of CDK9 activity, such as the conditions described herein.
  • Another embodiment provides a method of treating a disease or condition mediated by CDK9 by using a compound of Formulas l-VI, or a pharmaceutically acceptable salt thereof. Also provided in another embodiment is the manufacture of a medicament for the treatment of a disease or condition mediated by CDK9, said medicament comprising a compound of any of Formulas l-VI, or a pharmaceutically acceptable salt thereof.
  • Another aspect of the present invention provides a method of treating a disease or condition mediated by CDK9 using a compound of any of Formulas l-VI, or pharmaceutically acceptable salt thereof.
  • a preferred method comprises administering a therapeutically effective amount of a compound of any of Formulas l-VI, or a pharmaceutical composition comprising an effective amount of a compound of any of Formulas l-VI to a subject in need thereof.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of any of Formulas l-VI, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutically acceptable carrier diluent or excipient.
  • the present invention provides a method of regulating, modulating, or inhibiting protein kinase activity which comprises contacting a protein kinase with a compound of the invention.
  • Suitable protein kinases include CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9, or any combination thereof.
  • the protein kinase is selected from the group consisting of CDK1 , CDK2 and CDK9, or any combination thereof.
  • the protein kinase is in a cell culture.
  • the protein kinase is in a mammal.
  • the invention provides a method of treating a protein kinase-associated disorder comprising administering to a subject in need thereof a
  • Suitable protein kinases includeCDKI , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9 or combinations thereof (preferably, the protein kinase is selected from the group consisting of CDK1 , CDK2 and CDK9, more preferably, the protein kinase is CDK9.)
  • Suitable CDK combinations include CDK4 and CDK9; CDK1 , CDK2 and CDK9; CDK9 and CDK7; CDK9 and CDK1 ; CDK9 and CDK2; CDK4, CDK6 and CDK9; CDK1 , CDK2, CDK3, CDK4, CDK6 and CDK9.
  • the invention provides a method of treating cancer comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a compound of the invention.
  • Suitable cancers for treatment by the compounds, compositions and methods described herein include bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancer.
  • protein kinase-associated disorder includes disorders and states (e.g., a disease state) that are associated with the activity of a protein kinase, e.g., the CDKs, e.g., CDK1 , CDK2 and/or CDK9.
  • a protein kinase e.g., the CDKs, e.g., CDK1 , CDK2 and/or CDK9.
  • Non-limiting examples of protein kinase-associated disorders include abnormal cell proliferation (including protein kinase- associated cancers), viral infections, fungal infections, autoimmune diseases and neurodegenerative disorders.
  • treat includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated.
  • the treatment comprises the induction of a protein kinase-associated disorder, followed by the activation of the compound of the invention, which would in turn diminish or alleviate at least one symptom associated or caused by the protein kinase-associated disorder being treated.
  • treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.
  • use includes one or more of the following embodiments of the invention, respectively: the use in the treatment of protein kinase-associated disorders; the use for the manufacture of pharmaceutical compositions for use in the treatment of these diseases, e.g., in the manufacture of a medicament; methods of use of compounds of the invention in the treatment of these diseases; pharmaceutical preparations having compounds of the invention for the treatment of these diseases; and compounds of the invention for use in the treatment of these diseases; as appropriate and expedient, if not stated otherwise.
  • diseases to be treated and are thus preferred for use of a compound of the present invention are selected from cancer, inflammation, cardiac hypertrophy, and HIV infection, as well as those diseases that depend on the activity of protein kinases.
  • compositions herein which bind to a protein kinase sufficiently to serve as tracers or labels, so that when coupled to a fluor or tag, or made radioactive, can be used as a research reagent or as a diagnostic or an imaging agent.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a fully saturated straight-chain (linear; unbranched) or branched chain, having the number of carbon atoms specified, if designated (i.e. C Ci 0 means one to ten carbons).
  • Illustrative "alkyl” group examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. If no size is designated, the alkyl groups mentioned herein contain 1-10 carbon atoms, typically 1-8 carbon atoms, and preferably 1-6 or 1-4 carbon atoms.
  • alkoxy refers to -O-alkyl, wherein the term alkyl is as defined above.
  • cycloalkyl by itself or in combination with other terms, represents, unless otherwise stated, cyclic versions of alkyl. Additionally, cycloalkyl may contain fused rings, but is not intended to describe fused fully aromatic aryl and heteroaryl groups. Cycloalkyl groups, unless indicated otherwise, are unsubstituted, but may be substituted with those groups typically suitable for alkyl group substitutions. Illustrative examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and the like. If no ring size is specified, the cycloalkyl groups described herein generally contain 3-10 ring members, preferably 3-6 ring members.
  • heterocyclic or “heterocycloalkyl” or “heterocyclyl,” by itself or in combination with other terms, represents a cycloalkyl containing at least one annular carbon atom and at least one annular heteroatom selected from the group consisting of O, N, P, Si and S, preferably from N, O and S, wherein the ring is not aromatic but can contain unsaturations.
  • the nitrogen and sulfur atoms in a heterocyclic group may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heterocyclic groups discussed herein, if not otherwise specified, contain 3-10 ring members, and at least one ring member is a heteroatom selected from N, O, P, Si, and S.
  • heterocyclic group Preferably, not more than three of these heteroatoms are included in a heterocyclic group, and generally not more than two of these heteroatoms are present in a single ring of the heterocyclic group.
  • the heterocyclic group can be fused to an additional carbocyclic or heterocyclic ring.
  • a heterocyclic group can be attached to the remainder of the molecule at an annular carbon or annular heteroatom.
  • heterocyclic may contain fused rings, but excludes fused systems containing a heteroaryl group as part of the fused ring system.
  • heterocyclic groups include, 1- (1 ,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,
  • tetrahydrothien-3-yl 1 -piperazinyl, 2-piperazinyl, piperidin-2-one, azepane, tetrahydro- 2H-pyranyl, pyrrolidinyl, methylpyrrolidinone, alkylpiperidinyl, haloalkylpiperidinyl, 1- (alkylpiperidin-l-yl)ethanone, and the like.
  • substituents
  • 'heterocyclic' groups are piperidine, morpholine,
  • thiomorpholine piperazine, pyrrolidine, tetrahydrofuran, oxetane, oxepane, oxirane, tetrahydrothiofuran, thiepane, thiirane, and optionally substituted versions of each of these.
  • aryl represents an aromatic hydrocarbon group which can be a single ring or multiple rings (e.g., from 1 to 3 rings) which are fused together.
  • Aryl includes fused rings, wherein one or more of the fused rings is fully saturated (e.g. , cycloalkyl) or partially unsaturated (e.g. , cyclohexenyl), but not a heterocyclic or heteroaromatic ring.
  • Illustrative examples of aryl groups include, but are not limited to, phenyl, 1 -naphthyl, 2-naphthyl, and tetrahydronaphthyl.
  • heteroaryl refers to groups comprising a single ring, or a fused ring, where at least one of the rings is an aromatic ring that contain from one to four heteroatoms selected from N, O, and S as ring members (i.e. , it contains at least one heteroaromatic ring), wherein the nitrogen and sulfur atoms can be oxidized, and the nitrogen atom(s) can be quaternized.
  • a heteroaryl group can be attached to the remainder of the molecule through an annular carbon or annular heteroatom, and it can be attached through any ring of the heteroaryl moiety, if that moiety is a bicyclic, tricyclic, or a fused ring system.
  • a heteroaryl group may contain fused rings, wherein one of the fused rings is aromatic or heteroaromatic, and the other fused ring(s) are partially unsaturated (e.g., cyclohexenyl, 2,3-dihydrofuran, tetrahydropyrazine, and 3,4-dihydro- 2H-pyran), or completely saturated (e.g., cyclohexyl, cyclopentyl, tetrahydrofuran, morpholine, and piperazine).
  • the term heteroaryl is also intended to include fused rings systems that include a combination of aromatic and heteroaromatic rings systems (e.g., indoles, quinoline, quinazolines, and benzimidazoles).
  • heteroaryl groups are 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, and
  • halo or halogen
  • halo represents a fluorine, chlorine, bromine, or iodine atom. Commonly when present as a substituent, halo refers to F or CI or Br, preferably F or CI.
  • haloalkyi represents an alkyl group as defined above, wherein one or more hydrogen atoms of the alkyl group are replaced by a halogen atom which may be the same or different.
  • haloalkyi thus includes mono-haloalkyl, di-haloalkyl, tri- haloalkyi, tetra-haloalkyl, and the like as well as per-haloalkyl.
  • perhalo refers to the respective group wherein all available valences are replaced by halo groups.
  • perhaloalkyl includes -CCI 3 , -CF 3 , -CCI 2 CF 3 , and the like.
  • perfluoroalkyl and “perchloroalkyl” are a subset of perhaloalkyl wherein all available valences are replaced by fluoro and chloro groups, respectively.
  • Illustrative examples of perfluoroalkyl include -CF 3 and -CF 2 CF 3
  • perchloroalkyl include -CCI 3
  • Optionally substituted indicates that the particular group or groups being described may have no non-hydrogen substituents (i.e., it can be unsubstituted), or the group or groups may have one or more non-hydrogen substituents. If not otherwise specified, the total number of such substituents that may be present is equal to the number of H atoms present on the unsubstituted form of the group being described. Typically, an optionally substituted group will contain up to four (1-4) substituents.
  • Suitable optional substituent groups include halo, d. 4 alkyl, -NH-C(0)-CH 2 -0-Ci_4 alkyl, -NHC(0)-Ci. 4 alkyl, -C(0)-0-Ci. 4 alkyl,
  • the term "compounds of the present invention” refer to compounds of Formula I , prodrugs thereof, pharmaceutically acceptable salts of the compounds, and/or prodrugs, and hydrates or solvates of the compounds, salts, and/or prodrugs, as well as, all stereoisomers (including diastereoisomers and enantiomers), tautomers, and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties (e.g. , polymorphs, solvates and/or hydrates).
  • salts refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. It is noted that salts of the novel compounds described herein are of course useful as precursors for the neutral species or for pharmaceutically acceptable salts.
  • a therapeutically effective amount of a compound of the present invention refers to an amount of the compound of the present invention that when administered to a subject, is effective to (1) at least partially alleviating, inhibiting, preventing and/or ameliorating a condition, or a disorder or a disease (i) mediated by one or more CDK enzymes, or (ii) associated with one or more CDK enzyme activities, or (iii) characterized by activity of proteins regulated (directly or indirectly) by one or more CDK enzymes (e.g. RNA polymerase I I); or (2) reducing or inhibiting the expression of proteins whose expression is dependent (directly or indirectly) on one or more CDK enzymes (e.g. Mcl-1 , Cyclin D, Myc etc.).
  • CDK enzymes e.g. RNA polymerase I I
  • a therapeutically effective amount refers to the amount of the compound of the present invention that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reducing or inhibiting the activity of proteins regulated by one or more CDK enzymes; or at least partially reducing or inhibiting the expression of proteins whose expression is dependent (directly or indirectly) on one or more CDK enzymes.
  • the term "subject" refers to an animal. Typically the animal is a mammal. A subject also refers to for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human.
  • primates e.g., humans
  • the subject is a primate.
  • the subject is a human.
  • the invention provides compounds having a bi-aryl core structure with a pyridine ring connected to a second heterocyclic ring, wherein each ring has an additional substituent attached at a position 'meta' to the biaryl linkage.
  • R is selected from the group consisting of d_ 6 alkyl, C 3 . 6 branched alkyl,
  • each heterocycloalkyi contains one or two heteroatoms selected from N, S and O as ring members,
  • R 3 is H, CI or F
  • L is a bond or Ci_ 3 alkylene
  • L 2 is Co-3 alkylene, CD 2 , CHD, or C 3 . 8 branched alkylene;
  • a 1 is O or -NR 8 - Z 2 is N or CR 2 ;
  • Z 4 is N or CR 4 ;
  • Z 5 is N or CR 5 ;
  • Z 6 is N or CR 6 ;
  • R 2 , R 4 , R 5 , R 6 , and R 7 are each independently selected from H, C C 4 alkyl, C C 4 alkoxy, C C 4 haloalkyi, C C 4 haloalkoxy, CN, F and CI;
  • R 8 is H or methyl
  • R 9 is an optionally substituted group selected from Ci_ 3 alkyl, C 3 . 6 branched alkyl, C 3 . 7 cycloalkyl, C 3 . 7 heterocycloalkyl having up to two heteroatoms selected from N, O and S as ring members, -(CH 2 )i- 3 -0-Ci. 4 alkyl, pyridyl, and phenyl,
  • R 9 wherein the optional substituents for R 9 are up to four groups independently selected from halo, oxo, CN, C C 4 haloalkyi, C C 4 haloalkoxy, - OH, R ⁇ OR', -C(0)R ⁇ CONH2, CONHR', NH 2 , NHR', -NH-C(0)-R', where each R' is independently C C 4 straight chain or branched chain alkyl or haloalkyi.
  • R 5 or R 6 or both will represent H .
  • R 5 or R 6 or both will represent H .
  • R 7 is H and R 2 and R 4 can be H also, when they are present.
  • a 1 is NR 8 .
  • R 8 is H.
  • L is a bond; in some embodiments, L 2 is CH 2 .
  • R 9 is a heterocyclic group selected from tetrahydropyran, tetrahydrothiopyran, tetrahydrothiopyran-1 -dioxide, piperidine, pyrrolidine, dioxane, and tetrahydrofuran, oxepane, and dioxepane.
  • R 9 is substituted with at least one group selected from CN, halo, and C1-C4 alkyl. In some preferred embodiments, R 9 is a cyclopropyl or
  • the compound is selected from the group consisting of:
  • L is a bond or CH 2
  • R is cyclohexyl
  • R 3 is F or CI
  • L 2 is CH 2 , CHD, or CD 2 ;
  • R 9 is selected from pyridyl, phenyl, tetrahydropyran, cyclopropane, and tetrahydrofuran, each of which can be substituted with up to three groups independently selected from F, CI, -OH, Me, ethyl, -OMe, CN, and CONH 2 .
  • a 1 is NH and L 2 is CH 2 .
  • R 9 is optionally substituted phenyl or substituted cyclopropane or tetrahydropyran.
  • R 0 and R and R 2 each independently represent H, F,
  • R 2 is preferably CN.
  • R 0 and R are typically H, F, CI,
  • a compound of any of embodiments 1-17, for use in therapy is a compound of any of embodiments 1-17, for use in therapy.
  • a method to treat cancer comprising administering to a subject in need thereof an effective amount of a compound according to any of embodiments 1-17.
  • cancer is selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal, and pancreatic cancer.
  • the compound is administered, simultaneously or sequentially, with an antiinflammatory, antiproliferative,
  • a pharmaceutical composition comprising a compound according to any of embodiments 1-17 admixed with at least one pharmaceutically acceptable excipient.
  • composition of embodiment 25 which comprises at least one pharmaceutically acceptable carrier and at least one other pharmaceutically acceptable excipient.
  • additional therapeutic agent is an antiinflammatory, antiproliferative, chemotherapeutic agent, immunosuppressant, anti-cancer, cytotoxic agent or kinase inhibitor or a salt thereof.
  • Additional embodiments of the compounds of the invention include the following compounds and their pharmaceutically acceptable salts, as well as the novel compounds in the Tables herein. 30
  • Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wley, New York, 1999, and references cited therein.
  • the starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof.
  • many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wsconsin, USA), Bachem (Torrance,
  • the various starting materials, intermediates, and compounds of the embodiments may be isolated and purified, where appropriate, using conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation, and chromatography. Characterization of these compounds may be performed using conventional methods such as by melting point, mass spectrum, nuclear magnetic resonance, and various other spectroscopic analyses.
  • the compounds of the present invention can be isolated and used per se or as their pharmaceutical acceptable salt.
  • the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids, e.g., acetate, aspartate, benzoate, besylate,
  • bromide/hydrobromide bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate,
  • Inorganic acids from which salts can be derived include, for example,
  • hydrochloric acid hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table.
  • the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like.
  • Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.
  • the pharmaceutically acceptable salts of the present invention can be any pharmaceutically acceptable salts of the present invention.
  • salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid.
  • a stoichiometric amount of the appropriate base such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the like
  • Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two.
  • non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable, where practicable.
  • Lists of additional suitable salts can be found, e.g., in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Company, Easton, Pa., (1985); and in HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES,
  • the compounds of the present invention also include isotopically labeled forms of the compounds which may be synthesized using the processes described herein or modifications thereof known by those of skill in the art.
  • Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as 2 H, 3 H, C, 3 C, 4 C, 5 N, 8 F 3 P, 32 P, 35 S, 36 CI, 25 l respectively.
  • the invention includes various isotopically labeled compounds as defined herein, for example those into which radioactive isotopes, such as 3 H, 3 C, and 4 C, are present.
  • isotopically labeled compounds are useful in metabolic studies (with 4 C), reaction kinetic studies (with, for example 2 H or 3 H), detection or imaging techniques, such as positron emission
  • PET tomography
  • SPECT single-photon emission computed tomography
  • drug or substrate tissue distribution assays or in radioactive treatment of patients.
  • 8 F or labeled compound may be particularly desirable for PET or SPECT studies.
  • Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available
  • isotopically labeled reagent for a non-isotopically labeled reagent isotopically labeled reagent for a non-isotopically labeled reagent.
  • isotopes particularly deuterium (i.e., 2 H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index.
  • deuterium in this context is regarded as a substituent of a compound of the formula (I).
  • concentration of such a heavier isotope, specifically deuterium may be defined by the isotopic enrichment factor.
  • isotopic enrichment factor means the ratio between the isotopic abundance and the natural abundance of a specified isotope. If a substituent in a compound of this invention is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).
  • Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.
  • Compounds enriched in deuterium are referred to herein as 'deuterated versions' of the compounds of Formula I.
  • the invention includes all enantiomers of any chiral compound disclosed, in either substantially pure levorotatory or dextrorotatory form, or in a racemic mixture, or in any ratio of enantiomers.
  • the compounds disclosed herein may contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e. , as individual enantiomers or diastereomers, or as stereoisomer enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of the embodiments, unless otherwise indicated.
  • stereoisomers may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like. Unless stereochemistry is explicitly indicated in a chemical structure or chemical name, the chemical structure or chemical name is intended to embrace all possible stereoisomers, conformers, rotamers, and tautomers of the compound depicted.
  • a compound containing a chiral carbon atom is intended to embrace both the (R) enantiomer and the (S) enantiomer, as well as mixtures of enantiomers, including racemic mixtures; and a compound containing two chiral carbons is intended to embrace all enantiomers and diastereomers (including (R,R), (S, S), (R,S), and (R, S) isomers).
  • solvates refers to a molecular complex of a compound of the present invention (including pharmaceutically acceptable salts thereof) with one or more solvent molecules.
  • solvent molecules are those commonly used in the pharmaceutical art, which are known to be innocuous to the recipient, e.g. , water, ethanol, and the like.
  • hydrate refers to the complex where the solvent molecule is water.
  • solvates and hydrates of the compounds of the present invention are considered compositions, wherein the composition comprises a compound of the present invention and a solvent (including water).
  • the compounds of the present invention may exist in either amorphous or polymorphic form; therefore, all physical forms are considered to be within the scope of the present invention.
  • co-crystals i.e. compounds of the present invention that contain groups capable of acting as donors and/or acceptors for hydrogen bonds may be capable of forming co-crystals with suitable co-crystal formers.
  • co-crystals may be prepared from compounds of formula (I) by known co-crystal forming procedures. Such procedures include grinding, heating, co-subliming, co-melting, or contacting in solution compounds of formula (I) with the co-crystal former under crystallization conditions and isolating co-crystals thereby formed.
  • Suitable co-crystal formers include those described in WO 2004/078163. Hence the invention further provides co-crystals comprising a compound of formula (I).
  • pro-drugs convert in vivo to the compounds of the present invention.
  • a pro-drug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into a compound of this invention following administration of the prodrug to a subject.
  • the suitability and techniques involved in making and using prodrugs are well known by those skilled in the art.
  • Prodrugs can be conceptually divided into two non-exclusive categories, bioprecursor prodrugs and carrier prodrugs. See THE PRACTICE OF MEDICINAL CHEMISTRY, Ch. 31 -32 (Ed. Wermuth, Academic Press, San Diego, Calif., 2001 ).
  • bioprecursor prodrugs are compounds that are inactive or have low activity compared to the corresponding active drug compound, that contain one or more protective groups and are converted to an active form by metabolism or solvolysis. Both the active drug form and any released metabolic products should have acceptably low toxicity.
  • Carrier prodrugs are drug compounds that contain a transport moiety, e.g., that improve uptake and/or localized delivery to a site(s) of action.
  • a transport moiety e.g., that improve uptake and/or localized delivery to a site(s) of action.
  • the linkage between the drug moiety and the transport moiety is a covalent bond
  • the prodrug is inactive or less active than the drug compound
  • any released transport moiety is acceptably non-toxic.
  • the transport moiety is intended to enhance uptake
  • the release of the transport moiety should be rapid.
  • it is desirable to utilize a moiety that provides slow release e.g., certain polymers or other moieties, such as cyclodextrins.
  • Carrier prodrugs can, for example, be used to improve one or more of the following properties: increased lipophilicity, increased duration of pharmacological effects, increased site-specificity, decreased toxicity and adverse reactions, and/or improvement in drug formulation (e.g., stability, water solubility, suppression of an undesirable organoleptic or physiochemical property).
  • lipophilicity can be increased by esterification of (a) hydroxyl groups with lipophilic carboxylic acids (e.g., a carboxylic acid having at least one lipophilic moiety), or (b) carboxylic acid groups with lipophilic alcohols (e.g., an alcohol having at least one lipophilic moiety, for example aliphatic alcohols).
  • prodrugs are, e.g., esters of free carboxylic acids and S-acyl derivatives of thiols and O-acyl derivatives of alcohols or phenols, wherein acyl has a meaning as defined herein.
  • Suitable prodrugs are often pharmaceutically acceptable ester derivatives convertible by solvolysis under physiological conditions to the parent carboxylic acid, e.g., lower alkyl esters, cycloalkyl esters, lower alkenyl esters, benzyl esters, mono- or di-substituted lower alkyl esters, such as the -(amino, mono- or di- lower alkylamino, carboxy, lower alkoxycarbonyl)-lower alkyl esters, the -(lower alkanoyloxy, lower alkoxycarbonyl or di-lower alkylaminocarbonyl)-lower alkyl esters, such as the pivaloyloxymethyl ester and the like conventionally used in the art
  • a typical pharmaceutical composition comprises a compound of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • the term "pharmaceutically acceptable carriers, diluents or excipients” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • the pharmaceutical composition can be formulated for particular routes of administration such as oral administration, and parenteral administration, etc.
  • the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).
  • the pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifers and buffers, etc.
  • the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Tablets may be uncoated, film coated, or enteric coated according to methods known in the art.
  • compositions for oral administration include an effective amount of a compound of the invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide
  • Tablets may contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
  • Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example, peanut oil, liquid paraffin or olive oil.
  • Certain injectable compositions are aqueous isotonic solutions or suspensions, and
  • compositions are advantageously prepared from fatty emulsions or suspensions.
  • Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
  • Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 -75%, or contain about 1-50%, of the active ingredient.
  • the invention further provides pharmaceutical compositions and dosage forms that may comprise one or more agents that reduce the rate by which the compound of the present invention as an active ingredient will decompose.
  • agents which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers, etc.
  • the compounds of Formulas l-VI in free form or in pharmaceutically acceptable salt form exhibit valuable pharmacological properties, e.g. CDK inhibiting properties, e.g. as indicated in vitro and in vivo tests as provided below and are therefore suitable for use in therapy.
  • CDK inhibiting properties e.g. as indicated in vitro and in vivo tests as provided below
  • an individual “in need thereof” may be an individual who has been diagnosed with or previously treated for the condition to be treated. With respect to prevention, the individual in need thereof may also be an individual who is at risk for a condition (e.g., a family history of the condition, life-style factors indicative of risk for the condition, etc.).
  • a step of administering a compound of the invention is disclosed herein, the invention further contemplates a step of identifying an individual or subject in need of the particular treatment to be
  • Yet another aspect of the present invention provides a method of treating a disease or condition mediated by CDK9 comprising administration to a subject in need thereof a therapeutically effective amount of a compound of Formula I, or a
  • a compound of Formula I for use in a method of treating a disease or condition mediated by CDK9 is selected from cancer, cardiac hypotrophy, HIV and inflammatory diseases.
  • Another aspect of the present invention provides a method of treating a cancer selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal, and pancreatic cancer.
  • a cancer selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal, and pancreatic cancer.
  • Yet another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the invention provides a method of regulating, modulating, or inhibiting protein kinase activity which comprises contacting a protein kinase with a compound of the invention.
  • the protein kinase is selected from the group consisting of CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9, or any combination thereof.
  • the protein kinase is selected from the group consisting of CDK1 , CDK2 and CDK9, or any combination thereof.
  • the protein kinase is in a cell culture.
  • the protein kinase is in a mammal.
  • the invention provides a method of treating a protein kinase-associated disorder comprising administering to a subject in need thereof a
  • the protein kinase is selected from the group consisting of CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9.
  • the protein kinase-associated disorder is cancer.
  • the cancer is selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancer.
  • the protein kinase-associated disorder is inflammation.
  • the inflammation is related to rheumatoid arthritis, lupus, type 1 diabetes, diabetic nephropathy, multiple sclerosis, glomerulonephritis, chronic
  • the protein kinase-associated disorder is a viral infection.
  • the viral infection is associated with the HIV virus, human papilloma virus, herpes virus, poxvirus virus, Epstein-Barr virus, Sindbis virus, or adenovirus.
  • the protein kinase-associated disorder is cardiac hypertrophy.
  • the invention provides a method of treating cancer comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a compound of the invention such that the cancer is treated.
  • the cancer is selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancer.
  • the invention provides a method of treating inflammation comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a compound such that the inflammation is treated, wherein the compound is a compound of the invention.
  • the inflammation is related to rheumatoid arthritis, lupus, type 1 diabetes, diabetic nephropathy, multiple sclerosis, glomerulonephritis, chronic inflammation, and organ transplant rejections.
  • the invention provides a method of treating cardiac
  • hypertrophy comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a compound such that the cardiac hypertrophy is treated, wherein the compound is a compound of the invention.
  • the invention provides a method of treating a viral infection comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a compound such that the viral infection is treated, wherein the compound is a compound of the invention.
  • the viral infection is associated with the HIV virus, human papilloma virus, herpes virus, poxvirus virus, Epstein-Barr virus, Sindbis virus, or adenovirus.
  • the subject to be treated by the compounds of the invention is a mammal. In another embodiment, the mammal is a human.
  • the compounds of the invention is administered,
  • the compound, or salt thereof is
  • a PTK inhibitor administered, simultaneously or sequentially, with one or more of a PTK inhibitor, cyclosporin A, CTLA4-lg, antibodies selected from anti-ICAM-3, anti-IL-2 receptor, anti- CD45RB, anti-CD2, anti-CD3, anti-CD4, anti-CD80, anti-CD86, and monoclonal antibody OKT3, CVT-313, agents blocking the interaction between CD40 and gp39, fusion proteins constructed from CD40 and gp39, inhibitors of NF-kappa B function, nonsteroidal antiinflammatory drugs, steroids, gold compounds, FK506, mycophenolate mofetil, cytotoxic drugs, TNF-a inhibitors, anti-TNF antibodies or soluble TNF receptor, rapamycin, leflunimide, cyclooxygenase-2 inhibitors, paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin
  • the invention provides a packaged protein kinase-associated disorder treatment, comprising a protein kinase-modulating compound of the Formula I or Formula II, packaged with instructions for using an effective amount of the protein kinase-modulating compound to treat a protein kinase-associated disorder.
  • the compound of the present invention is further characterized as a modulator of a protein kinase, including, but not limited to, protein kinases selected from the group consisting of abl, ATK, Bcr-abl, Blk, Brk, Btk, c-fms, e- kit, c-met, c-src, CDK, cRafl, CSFIR, CSK, EGFR, ErbB2, ErbB3, ErbB4, ERK, Fak, fes, FGFRI, FGFR2, FGFR3, FGFR4, FGFR5, Fgr, FLK-4, flt-1 , Fps, Frk, Fyn, GSK, Gst-Flkl, Hck, Her-2, Her-4, IGF- IR, INS-R, Jak, JNK, KDR, Lck, Lyn, MEK, p38, panHER,
  • protein kinases selected from the group consisting of
  • PDGFR PLK, PKC, PYK2, Raf, Rho, ros, SRC, TRK, TYK2, UL97, VEGFR, Yes, Zap70, Aurora-A, GSK3-alpha, HIPK1 , HIPK2, HIP3, IRAKI , JNK1 , JNK2, JNK3, TRKB, CAMKII, CK1 , CK2, RAF, GSK3Beta, MAPK1 , MKK4, MKK7, MST2, NEK2, AAK1 , PKCalpha, PKD, RIPK2 and ROCK-II.
  • the protein kinase is selected from the group consisting of CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9 and any combination thereof, as well as any other CDK, as well as any CDK not yet identified.
  • the protein kinase is selected from the group consisting of CDK1 , CDK2 and CDK9.
  • the protein kinase is selected from the group consisting of CDK9.
  • CDK combinations of interest include CDK4 and CDK9; CDK1 , CDK2 and CDK9; CDK9 and CDK7; CDK9 and CDK1 ; CDK9 and CDK2; CDK4, CDK6 and CDK9; CDK1 , CDK2, CDK3, CDK4, CDK6 and CDK9.
  • the compounds of the present invention are used for the treatment of protein kinase-associated disorders.
  • protein kinase-associated disorder includes disorders and states (e.g., a disease state) that are associated with the activity of a protein kinase, e.g., the CDKs, e.g., CDK1 , CDK2 and/or CDK9.
  • disorders and states e.g., a disease state
  • Non-limiting examples of protein kinase-associated disorders include abnormal cell proliferation (including protein kinase-associated cancers), viral infections, fungal infections, autoimmune diseases and neurodegenerative disorders.
  • Non-limiting examples of protein-kinase associated disorders include proliferative diseases, such as viral infections, auto-immune diseases, fungal disease, cancer, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis, chronic inflammation, neurodegenerative disorders, such as Alzheimer's disease, and post-surgical stenosis and restenosis.
  • proliferative diseases such as viral infections, auto-immune diseases, fungal disease, cancer, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis, chronic inflammation
  • neurodegenerative disorders such as Alzheimer's disease, and post-surgical stenosis and restenosis.
  • Protein kinase-associated diseases also include diseases related to abnormal cell proliferation, including, but not limited to, cancers of the breast, ovary, cervix, prostate, testis, esophagus, stomach, skin, lung, bone, colon, pancreas, thyroid, biliary passages, buccal cavity and pharynx (oral), lip, tongue, mouth, pharynx, small intestine, colon-rectum, large intestine, rectum, brain and central nervous system, glioblastoma, neuroblastoma, keratoacanthoma, epidermoid carcinoma, large cell carcinoma, adenocarcinoma, adenocarcinoma, adenoma, adenocarcinoma, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma, kidney carcinoma, myeloid disorders, lymphoid disorders, Hodgkin's, hairy
  • protein kinase-associated cancers include carcinomas, hematopoietic tumors of lymphoid lineage, hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, tumors of the central and peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma
  • pigmentosum pigmentosum
  • keratoctanthoma thyroid follicular cancer
  • Kaposi's sarcoma Kaposi's sarcoma
  • Protein kinase-associated disorders include diseases associated with apoptosis, including, but not limited to, cancer, viral infections, autoimmune diseases and neurodegenerative disorders.
  • Non-limiting examples of protein-kinase associated disorders include viral infections in a patient in need thereof, wherein the viral infections include, but are not limited to, HIV, human papilloma virus, herpes virus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus.
  • Non-limiting examples of protein-kinase associated disorders include tumor angiogenesis and metastasis.
  • Non-limiting examples of protein-kinase associated disorders also include vascular smooth muscle proliferation associated with
  • Atherosclerosis postsurgical vascular stenosis and restenosis, and endometriosis.
  • protein-kinase associated disorders include those associated with infectious agents, including yeast, fungi, protozoan parasites such as Plasmodium falciparum, and DNA and RNA viruses.
  • the compound of the present invention is further characterized as a modulator of a combination of protein kinases, e.g., the CDKs, e.g., CDK1 , CDK2 and/or CDK9.
  • a compound of the present invention is used for protein kinase-associated diseases, and/or as an inhibitor of any one or more protein kinases. It is envisioned that a use can be a treatment of inhibiting one or more isoforms of protein kinases.
  • the compounds of the invention are inhibitors of cyclin-dependent kinase enzymes.
  • inhibition of the CDK4/cyclin D1 complex blocks phosphorylation of the Rb/inactive E2F complex, thereby preventing release of activated E2F and ultimately blocking E2F-dependent DNA transcription. This has the effect of inducing G1 cell cycle arrest.
  • the CDK4 pathway has been shown to have tumor-specific deregulation and cytotoxic effects. Accordingly, the ability to inhibit the activity of combinations of CDKs will be of beneficial therapeutic use.
  • CDK9 inhibition may sensitize cells to TNFalpha or TRAIL stimulation by inhibition of NF-kB, or may block growth of cells by reducing myc-dependent gene expression. CDK9 inhibition may also sensitize cells to genotoxic chemotherapies, HDAC inhibition, or other signal transduction based therapies.
  • the compounds of the invention can lead to depletion of anti- apoptotic proteins, which can directly induce apoptosis or sensitize to other apoptotic stimuli, such as cell cycle inhibition, DNA or microtubule damage or signal transduction inhibition. Depletion of anti-apoptotic proteins by the compounds of the invention may directly induce apoptosis or sensitize to other apoptotic stimuli, such as cell cycle inhibition, DNA or microtubule damage or signal transduction inhibition.
  • the compounds of the invention can be effective in combination with chemotherapy, DNA damage arresting agents, or other cell cycle arresting agents.
  • the compounds of the invention can also be effective for use in chemotherapy-resistant cells.
  • the present invention includes treatment of one or more symptoms of cancer, inflammation, cardiac hypertrophy, and HIV infection, as well as protein kinase- associated disorders as described above, but the invention is not intended to be limited to the manner by which the compound performs its intended function of treatment of a disease.
  • the present invention includes treatment of diseases described herein in any manner that allows treatment to occur, e.g., cancer, inflammation, cardiac hypertrophy, and HIV infection.
  • the invention provides a pharmaceutical composition of any of the compounds of the present invention.
  • the invention provides a pharmaceutical composition of any of the compounds of the present invention and a pharmaceutically acceptable carrier or excipient of any of these compounds.
  • the invention includes the compounds as novel chemical entities.
  • the invention includes a packaged protein kinase- associated disorder treatment.
  • the packaged treatment includes a compound of the invention packaged with instructions for using an effective amount of the compound of the invention for an intended use.
  • the compounds of the present invention are suitable as active agents in pharmaceutical compositions that are efficacious particularly for treating protein kinase- associated disorders, e.g., cancer, inflammation, cardiac hypertrophy, and HIV infection.
  • the pharmaceutical composition in various embodiments has a pharmaceutically effective amount of the present active agent along with other pharmaceutically acceptable excipients, carriers, fillers, diluents and the like.
  • pharmaceutically effective amount indicates an amount necessary to administer to a host, or to a cell, issue, or organ of a host, to achieve a therapeutic result, especially the regulating, modulating, or inhibiting protein kinase activity, e.g., inhibition of the activity of a protein kinase, or treatment of cancer, inflammation, cardiac hypertrophy, and HIV infection.
  • the present invention provides a method for inhibiting the activity of a protein kinase.
  • the method includes contacting a cell with any of the compounds of the present invention.
  • the method further provides that the compound is present in an amount effective to selectively inhibit the activity of a protein kinase.
  • the present invention provides a use of any of the compounds of the invention for manufacture of a medicament to treat cancer, inflammation, cardiac hypertrophy, and HIV infection in a subject.
  • the invention provides a method of manufacture of a medicament, including formulating any of the compounds of the present invention for treatment of a subject.
  • TLC thin layer chromatography
  • glass or plastic backed silica gel plates such as, for example, Baker-Flex Silica Gel 1 B2-F flexible sheets.
  • TLC results were readily detected visually under ultraviolet light, or by employing well known iodine vapor and other various staining techniques.
  • Mass spectrometric analysis was performed on LCMS instruments: Waters
  • GCMS analysis is performed on a Hewlett Packard instrument (HP6890 Series gas chromatograph with a Mass Selective Detector 5973; injector volume: 1 L; initial column temperature: 50 °C; final column temperature: 250 °C; ramp time: 20 minutes; gas flow rate: 1 mL/min; column: 5 % phenyl methyl siloxane, Model No. HP 190915-443, dimensions: 30.0 m x 25 m x 0.25 m).
  • NMR Nuclear magnetic resonance
  • spectral reference was either TMS or the known chemical shift of the solvent.
  • Some compound samples were run at elevated temperatures (e.g., 75 oC) to promote increased sample solubility. Melting points are determined on a Laboratory Devices Mel-Temp apparatus (Holliston, MA). Preparative separations are carried out using a Combiflash Rf system
  • Typical solvents employed for the Combiflash Rf system and flash column chromatography are dichloromethane, methanol, ethyl acetate, hexane, heptane, acetone, aqueous ammonia (or ammonium hydroxide), and triethyl amine.
  • Typical solvents employed for the reverse phase HPLC are varying concentrations of acetonitrile and water with 0.1 % trifluoroacetic acid. The following abbreviations have the following meanings. If not specifically defined, abbreviations will have their generally accepted meanings.
  • BINAP 2,2'-bis(diphenylphosphino)-1 , 1'-binapthyl
  • DIPEA N,N-diisopropylethylamine
  • HATU 2-(7-aza-1 H-benzotriazole-1-yl)-1 , 1 ,3,3-tetramethyluronium
  • NBS N-bromosuccinimide
  • protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.
  • Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W.
  • the compounds disclosed herein may contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer- enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of the embodiments, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.
  • the starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof.
  • many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), Emka- Chemce or Sigma (St. Louis, Missouri, USA).
  • synthesis can start with a functionalized pyridine I wherein LG is a leaving group such as F, CI, OTf, and the like.
  • X can be a functional group like CI, Br, I or OTf.
  • Compound I can be converted into boronic acid or boronic ester II by:
  • synthesis can start with a functionalized hyeterocyle I wherein X is Br, I, CI, OTf, and the like.
  • Compound I can be converted into boronic acid or boronic ester II by:
  • synthesis can start with a functionalized pyridine I wherein X can be a functional group like CI, Br, I, OTf, and the like.
  • Compound I can be converted into boronic acid or boronic ester II by:
  • synthesis can start with a functionalized pyridine I wherein X can be a functional group like CI, Br, I, OTf, and the like.
  • Compound I can be converted into boronic acid or boronic ester II by:
  • synthesis can start with a functionalized hyeterocyle I wherein X is Br, I, CI, OTf, and the like.
  • Compound I can be converted into boronic acid or boronic ester II by:
  • Step 3 Preparation of (R/S)-tert-butyl 6-bromopyridin-2-yl((2,2-dimethyltetrahydro-2H- pyran-4-yl)methyl)carbamate To a mixture of te/f-butyl 6-bromopyridin-2-ylcarbamate (686 mg, 2.51 mmol),
  • Step 4 Preparation of (R/S)-tert-butyl 5'-chloro-2'-fluoro-2,4'-bipyridin-6-yl((2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl)carbamate
  • 2,3,6-Trifluoropyridine (17.91 ml, 188 mmol) was dissolved in anhydrous MeOH (300 ml) and the resulting mixture was placed under argon. This mixture then was treated with a 25wt% methanolic solution of sodium methoxide (43.0 ml, 188 mmol). The resulting mixture was then heated at about 65° C for 2 hr. The reaction mixture was cooled to ambient temperature, and concentrated in vacuo to yield a residue which then was mixed with brine (200 ml_), and extracted with Et20 (3 x 200 ml).
  • Step 2 Preparation of 3,6-difluoro-2-hydroxypyridine
  • sodium iodide 66.6 g, 445 mmol
  • chlorotnmethylsilane 56.8 ml, 445 mmol
  • the resulting mixture was heated at 80-85 °C for 2.5 hr.
  • the mixture was cooled to ambient temperature and diluted with EtOAc (300 ml_) and water (300 ml_) and vigorously stirred for another hr.
  • Step 5 Preparation of 5'-chloro-N-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4- yl)methyl)-2'-fluoro-2,4'-bipyridin-6-amine
  • 6-bromo-N-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)methyl) pyridin-2-amine (1 10 mg, 0.36 mmol), 5-chloro-2-fluoro-pyridine-4-boronic acid (193 mg, 1.10 mmol), 0.55 ml 2.0M saturated sodium carbonate aqueous solution in 2 ml DME was purged with Argon for 3 min, PdCl 2 (dppf)CH 2 Cl 2 (30 mg, 0.037 mmol) was added to this purged .
  • Step 3 To a solution of (4-methoxytetrahydro-2H-pyran-4-yl) methanol (300 mg, 2.05 mmol) in pyridine (4 ml) at ambient temperature was added toluenesulfonic chloride (430 mg, 2.25 mmol) and the resulting mixture was stirred overnight at about 25 °C. The stirred mixture was concentrated and the solid residue was dissolved in DCM and purified by silica gel chromatography using a 12 g column, eluting with 0-30% ethyl acetate in heptane to yield the desired compound "O" as a light yellow solid (360 mg).1 H NMR (300 MHz, CHLOROFORM-d) .
  • reaction mixture was cooled to ambient temperature, diluted with EtOAc (20ml_), washed with saturated NaHC0 3 solution and brine, dried over sodium sulfate and concentrated in vacuo to yield 502 mg of a light brown crude liquid, which was purified by column chromatography ( 5 to 50% ethyl acetate in heptane)to yield the desired products.
  • Step 2a A mixture of 6-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-3- (trifluoromethyl)pyridin-2-amine(100 mg, 0.339 mmol), 5-chloro-2-fluoropyridin-4- ylboronic acid (89 mg, 0.509 mmol), PdCl 2 (dppf).CH 2 Cl 2 adduct (27.7 mg, 0.034 mmol), DME (1.5 mL) and 2M aqueous Na 2 C0 2 (0.5 mL, 1 mmol) was stirred in a sealed glass vessel at about 100 °C for about 3 hours.
  • Step 1 6-Bromo-2-aminopyridine (15 g, 87 mmol) and TEA (13.3 mL, 95 mmol) were dissolved in 173 mL of DCM. BOC-anhydride (20.8 g, 95 mmol) was then dissolved in 100 mL of DCM and added over 10 min using a syringe pump. The reaction mixture was stirred at ambient temperature for 72 hr. The solvents were evaporated and the resulting residue was purified by silica gel chromatography (heptane: EtOAc 1 :0 to 7:3) to give the product as a colorless solid (23.0 g, 97%).
  • Step 3 A solution of tert-Butyl 6-bromo-5-chloropyridin-2-ylcarbamate (2.32 g, 7.54 mmol) in DMF (25 mL) was mixed with sodium hydride (60% dispersion in mineral oil, 513 mg, 12.8 mmol), and the resulting mixture reaction mixture was stirred for 30 minutes at ambient temperature. (2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl 4- methylbenzenesulfonate (3.15 g, 10.56 mmol), dissolved in 5 mL DMF, was then added and the resulting mixture was stirred at about 25 °C for 3 hours. The reaction mixture was partitioned between water and EtOAc.
  • Step 4 A mixture of tert-butyl 6-bromo-5-chloropyridin-2-yl((2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl)carbamate (1.86 g, 4.29 mmol), 5-chloro-2- fluoropyridin-4-ylboronic acid (1.50 g, 8.58 mmol), PdCI2(dppf)*DCM adduct (350 mg, 0.429 mmol), DME (15.6 mL) and 2 M aqueous sodium carbonate solution (5.4 mL) were combined in a glass bomb. The bomb was sealed and heated at about 98°C for 2 hours. The reaction mixture was cooled to ambient temperature and then diluted with EtOAc.
  • Step 1 To NaH (0.366 g, 9.16 mmol) in THF (12 mL) at 0 °C was added 1 ,3- dimethoxy-2-propanol (1 g, 8.32 mmol) in THF (8 mL) solution. The mixture was warmed to ambient temperature and stirred for 0.5 hour. To this was added tosyl chloride (1.587 g, 8.32 mmol) in one portion. The resulting white cloudy mixture then was stirred at ambient temperature for 16 hours. LC/MS showed complete conversion to 1 ,3- dimethoxypropan-2-yl 4-methylbenzenesulfonate. The reaction mixture was poured into water and extracted with EtOAc.
  • the organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated in vacuo to yield 2 g of a colorless oil.
  • the crude mixture was purified by Analogix system (silica gel column 80 g, gradient: 0 min, 100%n-heptane; 5-12 min, 20% EtOAc in Heptane; 12-15 min. 30% EtOAc in Heptane and hold until 30 min).
  • the pure fractions were combined and concentrated in vacuo to yield 1.25 g of the tosylate product 1 ,3-dimethoxypropan-2-yl 4- methylbenzenesulfonate as a colorless oil, which solidified upon standing.
  • Step 2 To the tosylate obtained in Step 1 (0.8g, 2.92 mmol) in DMSO (8 ml) was added 1 ,4-trans-cyclohexane diamine (0.999 g, 8.75 mmol). The resulting brown mixture was heated in a capped vial to about 95 °C, with stirring, for 2 hours. The reaction mixture was poured into 10% HCI in water (10 ml_) at 0 °C (ice cubes in HCI) and extracted with DCM (1x20 ml_). The aqueous (light pink) was basified with 6N NaOH to a pH >12 and extracted with DCM (2x20ml_).
  • Step 1 Synthesis of 4-((3,6-difluoropyridin-2-yl-amino)methyl)tetrahydro-2H-pyran-4- carbonitrile
  • Step 2 Synthesis of 4-((6-(benzyloxy)-3-fluoropyridin-2-yl-amino)methyl)tetrahydro-2H- pyran-4-carbonitrile
  • Benzyl alcohol (352 mg, 3.26 mmol) was dissolved in anhydrous DMF (2 ml) and placed under argon. This was then treated with a 60% dispersion in oil of SODIUM HYDRIDE (78.7 mg, 3.26 mmol). This resultant suspension was then stirred at room temperature for 15 min. At this time it was treated with a solution of 4-((3,6- difluoropyridin-2-ylamino)methyl)tetrahydro-2H-pyran-4-carbonitrile (275 mg, 1.09 mmol) dissolved in anhydrous DMF (2 ml). Once the addition was complete the reaction was stirred at 90°C for 5 hours. The reaction was allowed to cool to room temperature.
  • Step 3 Synthesis of 4-((3-fluoro-6-hydroxypyridin-2-yl-amino)methyl)tetrahydro-2H- pyran-4-carbonitrile
  • Step 4 Synthesis 6-((4-cyanotetrahydro-2H-pyran-4-yl) methyl)-amino-5-fluoropyridin-2- yl trifluoromethanesulfonate
  • Step 5 Synthesis of 4-((5'-chloro-2',5-difluoro-2,4'-bipyridin-6-yl- amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (Intermediate AA)
  • Step 1 Synthesis of tert-butyl 6-bromopyridin-2-yl((4-methoxytetrahydro-2H-pyran-4- yl)methyl) carbamate
  • Step 2 Synthesis of tert-butyl 5'-chloro-2'-fluoro-2,4'-bipyridin-6-yl((4- methoxytetrahydro-2H-pyran-4-yl)methyl)carbamate
  • Step 3 Synthesis of 5'-chloro-2'-fluoro-N-((4-methoxytetrahydro-2H-pyran-4-yl)methyl)- 2,4'-bipyridin-6-amine (Intermediate AC)
  • Step 1 Preparation of (R,E)-2-methyl-N-((tetrahydro-2H-pyran-4- yl)methylene)propane-2-sulfinamide
  • Step 2 Preparation of (R)-2-methyl-N-((S)-1 -(tetrahydro-2H-pyran-4- yl)ethyl)propane-2-sulfinamide
  • Step 1 Preparation of (S,E)-2-methyl-N-((tetrahydro-2H-pyran-4- yl)methylene)propane-2-sulfinamide
  • Step 2 Preparation of (S)-2-methyl-N-((R)-1 -(tetrahydro-2H-pyran-4- yl)ethyl)propane-2-sulfinamide
  • Step 4 Preparation of (6,6-dimethyl-1 ,4-dioxan-2-yl)methanamine
  • Step 1 Preparation of methyl 4-cyanotetrahydro-2H-pyran-4-carboxylate
  • Step 1 Preparation of 1 ,6-dioxaspiro[2.5]octane
  • Step 3 Preparation of toluene-4-sulfonic acid 4-methoxy-tetrahydro-pyran-4- yl methyl ester
  • Step 1 Preparation of (2R,6S)-2,6-dimethyldihydro-2H-pyran-4(3H)-one
  • a solution of 2,6-dimethyl-4H-pyran-4-one (2 g, 16.1 mmol) in EtOH (20 mL) was stirred over Pd/C (10 wt.%, 0.2 g) under hydrogen (15 psi) for 16 hrs at ambient temperature.
  • the suspension was filtered off and the filtrate was concentrated under reduced pressure.
  • the residue was dissolved in dichloromethane (15 mL) and treated with Dess-Martin periodinane (2.3 g) at ambient temperature for 16 hrs.
  • reaction mixture was diluted with water (15 mL) and extracted with diethylether (2x 30 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered off and concentrated under reduced pressure. The residue was purified by column chromatography [silica gel,
  • Step 3 Preparation of (2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-carbaldehyde
  • the reaction mixture was cooled to 0 °C, neutralized with 1 N aqueous sodium hydroxide solution until pH ⁇ 6 and extracted with diethylether.
  • Step 1a To a solution of DIPEA (6.12 mL, 35.0 mmol) in dichloromethane (80 mL) was added trimethylsilyl trifluoromethanesulfonate (7.79 g, 35.0 mmol) and slowly a solution of tetrahydro-2H-pyran-4-carbaldehyde (2 g, 17.52 mmol) in dichloromethane (80 mL) at 0 °C. Upon completion of the addition, the reaction mixture was stirred at room temperature for 2 hrs. The mixture was concentrated under reduced pressure and the residue was treated with hexane (200 mL). The precipitate was filtered off and the solution was concentrated under reduced pressure providing crude trimethylsilyl ether, which was directly used in the next step without further purification.
  • Step 1 b To a solution of crude trimethylsilyl ether in dichloromethane (100 mL) was added dropwise a solution of N-fluorobenzenesulfonimide (5.53 g, 17.52 mmol), dissolved in dichloromethane (50 mL), at 0 °C. The mixture was stirred for 3 hrs at room temperature and the crude solution of 4-fluorotetrahydro-2H-pyran-4-carbaldehyde was directly used in the next reaction.
  • Step 2 Preparation of 6-bromo-N-((4-fluorotetrahydro-2H-pyran-4- yl)methyl)pyridin-2-amine
  • Step 1 Preparation of 1 ,3-dimethoxypropan-2-yl 4-methylbenzenesulfonate
  • Step 1 Preparation of 1 -(trideuteromethoxy)propan-2-yl 4- methylbenzenesulfonate
  • Step 1 Preparation of 2-deutero-1 -methoxypropan-2-ol To 1-methoxypropan-2-one (5.26 mL, 56.8 mmol) in MeOH-d4 (10 mL) and THF (50.00 mL) at 0 °C was added NaBD 4 (2.375 g, 56.8 mmol) portion wise. Vigorous off-gassing was seen. The reaction mixture was warmed to room temperature and stirred under argon for 5 hrs. The reaction mixture was worked up by pouring saturated aqueous NaHC0 3 solution (10 mL) and stirred for 1 hr. The product was extracted with diethyl ether (100 mL), washed with brine, dried with sodium sulfate and concentrated under reduced pressure to give 3.53 g of colorless liquid. This was used in the next step without further purification.
  • Step 3 Preparation of trans-N1-(2-deutero-1 -methoxypropan-2-yl)cyclohexane-1 ,4- diamine
  • Step 1 Preparation of tert-butyl (trans-4-((2- methoxyethyl)amino)cyclohexyl)carbamate
  • Step 2 Preparation of tert-butyl (trans-4-(cyclopropyl(2-methoxyethyl)amino)- cyclohexyl)carbamate
  • Step 3 Preparation of trans-N1-cyclopropyl-N1 -(2-methoxyethyl)cyclohexane-1,4- diamine
  • Step 1 Preparation of (R)-3-(benzyloxy)-1 ,1 ,1-trifluoropropan-2-ol
  • Step 4 Preparation of (R)-3,3,3-trifluoro-2-methoxypropyl 4- methylbenzenesulfonate Sodium hydride (412 mg, 10.31 mmol) was added to a solution of (R)-3,3,3-trifluoro-2- methoxypropan-1-ol (495 mg, 3.44 mmol) in THF (10 ml) at ambient temperature. The mixture was stirred for 30 minutes.
  • Step 5 Preparation of N1 -((R)-3,3,3-trifluoro-2-methoxypropyl)cyclohexane-trans- 1,4-diamine
  • Step 3 Preparation of trans-N1,N1 -dibenzylcyclohexane-1 ,4-diamine
  • Step 5 Preparation of 3-((trans-4-(dibenzylamino)cyclohexyl)amino)-1 ,1,1- trifluoro-2-methylpropan-2-ol
  • Step 6 Preparation of 3-((trans-4-aminocyclohexyl)amino)-1,1,1-trifluoro-2- methylpropan-2-ol (racemic m
  • Step 1 Preparation of (S)-benzyl (4-(3-methoxypyrrolidin-1- yl)cyclohexyl)carbamate
  • Step 1 Preparation of ethyl 2-(((trans)-4-aminocyclohexyl)amino)-2- methylpropanoate
  • Step 1 Preparation of rac benzyl ((1S,3S,4S)-3-hydroxy-4-((2- methoxyethyl)amino)cyclohexyl)carbamate
  • Step 2 Preparation of rac tert-butyl ((1S,2S,4S)-4-(((benzyloxy)carbonyl)amino)-2- hydroxycyclohexyl)(2-methoxyethyl)carbamate
  • Step 3 Preparation of rac tert-butyl ((1 S,2S,4S)-4-amino-2-hydroxycyclohexyl)(2- methoxyethyl)carbamate
  • Step 1 Preparation of rac (1S,3S,6R)-(7-Oxa-bicyclo[4.1.0]hept-3-yl)-carbamic acid benzyl ester
  • Step 3 Preparation of rac ((1 S,3R,4R)-3-azido-4-methoxy-cyclohexyl)-carbamic acid benzyl ester
  • Step 5 Preparation of rac ((1 S,3R,4R)-3-tert-butoxycarbonylamino-4-methoxy- cyclohexyl)-carbamic acid benzyl ester
  • Step 6 Preparation of rac ((1 R,2R,5S)-5-amino-2-methoxy-cyclohexyl)-carbamic acid tert-butyl ester
  • a mixture of rac ((1S,3R,4R)-3-tert-butoxycarbonylamino-4-methoxy-cyclohexyl)- carbamic acid benzyl ester (170 mg, 0.45 mmol), Pd/C (10%. 15 mg) in methanol (10 mL) was stirred at room temperature for 2 hr. The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure.
  • Step 1 Preparation of rac (1S,3S,6R)-(7-oxa-bicyclo[4.1.0]hept-3-yl)-carbamic acid benzyl ester
  • Step 2 Preparation of rac ((1 S,3S,4S)-4-azido-3-hydroxy-cyclohexyl)-carbamic acid benzyl ester
  • Step 3 Preparation of rac ((1 S,3S,4S)-4-azido-3-methoxy-cyclohexyl)-carbamic acid benzyl ester
  • Step 5 Preparation of rac ((1 S,2S,4S)-4-benzyloxycarbonylamino-2-methoxy- cyclohexyl)-carbamic acid tert-butyl ester
  • Step 6 Preparation of rac ((1 S,2S,4S)-4-amino-2-methoxy-cyclohexyl)-carbamic acid tert-butyl ester
  • Step 1 Preparation of rac ((1 R,3S,4S)-4-hydroxy-3-methoxy-cyclohexyl)-carbamic acid benzyl ester
  • Step 3 Preparation of rac ((1 R,3S,4R)-4-azido-3-methoxy-cyclohexyl)-carbamic acid benzyl ester
  • Step 4 Preparation of rac ((1 R,3S,4R)-4-amino-3-methoxy-cyclohexyl)-carbamic acid benzyl ester
  • Step 5 Preparation of rac ((1 R,3S,4R)-4-tert-butoxycarbonylamino-3-methoxy- cyclohexyl)-carbamic acid benzyl ester
  • Step 6 Preparation of rac ((1 R,2S,4R)-4-amino-2-methoxy-cyclohexyl)-carbamic acid tert-butyl ester
  • the TFA salt was free- based using 200 ml of ethyl acetate and washed with saturated sodium bicarbonate 35 ml (1x), water (2x), saturated brine (1x), dried over sodium sulfate, filtered and concentrated to yield a solid. The solid was dissolved in (1 :1 ACN/ water), filtered, and lyophilized to yield 80 mg of the title compound as free-base.
  • the crude reaction mixture was cooled to room temperature, diluted with 3 ml DMSO, filtered, and purified by prep HPLC. (there is a general HPLC conditions in the general experimental session).
  • the fractions were concentrated, mixed with 500 ml ethyl acetate, and basified with saturated sodium bicarbonate 120 ml.
  • the ethyl acetate layer was separated, and the basic water layer was extracted with 300 ml ethyl acetate.
  • the ethyl acetate layers were combined and washed with water (3x), saturated salt solution (1x), dried with sodium sulfate, filtered and concentrated to yield a solid.
  • Step 1 Preparation of frans-N1-(4-bromopyridin-2-yl)cyclohexane-1 ,4-diamine
  • 4-bromo-2-chloropyridine (1500 mg, 7.79 mmol)
  • DMSO 15 ml
  • frans-cyclohexane-1 ,4-diamine 4450 mg, 39.0 mmol
  • the reaction mixture was cooled to room temperature, filtered and purified by prep LC, and lyophilized to yield 393mg of the title compound as a TFA salt.
  • LCMS (m/z): 270.2/272.2 (MH+), retention time 0.31 min.
  • Step 1 Preparation of 2,5-dichloro-4-(6-fluoropyridin-2-yl)pyrimidine
  • 2,4,5-trichloropyrimidine 49.3 mg, 0.269 mmol
  • 2-fluoro-6-(4, 4,5,5- tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine 50 mg, 0.224 mmol
  • PdCl 2 dppf
  • CH 2 Cl 2 adduct (18.31 mg, 0.022 mmol
  • DME 0.7 ml
  • 2M sodium carbonate 0.247 ml, 0.493 mmol
  • cyclohexylmethanamine was removed under vacuum to yield a residue.
  • the residue was mixed with 0.5 ml DMSO, filtered, purified by prep HPLC and then lyophilized to yield 9.4 mg of the title compound as a TFA salt.
  • Example 5 (Compound 5) (5'-chloro-N6-(3-fluorobenzyl)-N2'-(piperidin-4-yl)-2,4'-bipyridine-2 ⁇ 6-diarnine
  • Step 3 Preparation of frans-4-(5'-chloro-6-(piperidin-4-yl-amino)-2,4'-bipyridin-2'-yl- amino)cyclohexanol
  • the Boc protecting group was removed from the intermediate by adding HCL 6M aq (140 ⁇ , 0.840 mmol) to the crude reaction mixture, followed by stirring the mixture at 90 °C for 45 minutes.
  • the reaction mixture was cooled, 0.5 ml of DMSO was added, filtered and purified by prep LC. Lyophilization of the material yielded 9.8 mg of the title compound, as a TFA salt.
  • Step 1 Preparation of 6-chloro-N-(3-fluorobenzyl)pyrazin-2-amine: To 2,6- dichloropyrazine (175 mg, 1.175 mmol) was added DMSO (1.5 ml), TEA (0.196 ml, 1.410 mmol) and (3-fluorophenyl)methanamine (368 mg, 2.94 mmol)l. The reaction mixture then was stirred at 90 °C until completion as indicated by LCMS, about 1 hour. To the reaction mixture was added 3 ml of DMSO, filtered and the residue was purified by prep LC. After lyophilization, 160 mg of the title compound was obtained as a TFA.
  • Step 2 Preparation of N-(3-fluorobenzyl)-6-(2-fluoropyridin-4-yl)pyrazin-2-amine: To 6-chloro-N-(3-fluorobenzyl)pyrazin-2-amine (140 mg, 0.589 mmol) was added 2- fluoropyridin-4-ylboronic acid (125 mg, 0.884 mmol), PalladiumTetrakis (82 mg, 0.071 mmol), DME (3.3 ml), and 2M sodium carbonate (1.031 ml, 2.062 mmol) . The resulting reaction mixture was stirred at 1 10 °C until completion as indicated by LCMS, about 3 hours. The reaction mixture was cooled to room temperature, diluted with 20 ml of ethyl acetate, filtered and concentrated to yield a crude solid. The solid was dissolved in
  • Step 2 Preparation of 3-fluoro-/V-(3-fluorobenzyl)-6-methoxypyridin-2-amine: 3,6-difluoro-A/-(3-fluorobenzyl)pyridine-2-amine (0.5209 g, 2.19 mmol), was dissolved in anhydrous MeOH ( 6.6 mL) and placed under argon. This mixture then was treated with sodium methoxide (0.500 mL, 0.473 g, 2.19 mmol, 25% in MeOH) by slow addition. The resulting mixture was then heated in the microwave at 150°C for four 30 min. The reaction mixture was then poured into brine (25 mL).
  • Step 3 Preparation of 5-fluoro-6-(3-fluorobenzylamino)pyridine-2-ol: 3-fluoro-/V- (3-fluorobenzyl)-6-methoxypyridin-2-amine (0.100 g, 0.400 mmol) was dissolved in anhydrous CH 3 CN (1.6 mL). This mixture was treated with sodium iodide (0.301 g, 2.01 mmol) followed by trimethylsilylchloride (0.257 mL, 0.218 g, 2.01 mmol). The resulting reaction mixture was then heated at reflux for 2 hr.

Abstract

The present invention provides a compound of formula (I): and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof. These compounds inhibit the activity of CDK9 and are thus useful as pharmaceuticals. Also provided are methods of treating a disease or condition mediated by CDK9 using the compounds of Formula I and isomers thereof, and pharmaceutical compositions comprising such compounds.

Description

PYRIDINE BIARYL AMINE COMPOUNDS AND THEIR USES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of priority to U.S. Provisional Application No.
61/437,082, filed January 28, 2011 , the contents of which are incorporated herein by reference. FIELD OF THE INVENTION
The invention provides a novel class of compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent diseases or disorders associated with aberrant cellular signaling pathways that can be modulated by inhibition of kinases, particularly diseases or disorders that involve aberrant cellular signaling pathways that can be modulated by inhibition of CDK9.
BACKGROUND
Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within the cell. (Hardie, G. and Hanks, S. THE PROTEIN KINASE FACTS BOOK, I and II, Academic Press, San Diego, Calif.: 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, efc.). Sequence motifs have been identified that generally correspond to each of these kinase families (See, for example, Hanks, S. K., Hunter, T., FASEB J. 1995, 9, 576-596; Knighton et al., Science 1991 , 253, 407-414; Hiles et al., Cell 1992, 70, 419-429; Kunz et al., Cell 1993, 73, 585-596; Garcia-Bustos et a/., EMBO J. 1994, 13, 2352-2361).
Many diseases are associated with abnormal cellular responses triggered by the protein kinase-mediated events described above. These diseases include, but are not limited to, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, Alzheimer's disease, viral diseases, and hormone- related diseases. Accordingly, there has been a substantial effort in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents. The cyclin-dependent kinase (CDK) complexes are a class of kinases that are targets of interest. These complexes comprise at least a catalytic (the CDK itself) and a regulatory (cyclin) subunit. Some of the more important complexes for cell cycle regulation include cyclin A (CDK1-also known as cdc2, and CDK2), cyclin B1-B3 (CDK1) and cyclin D1-D3 (CDK2, CDK4, CDK5, CDK6), cyclin E (CDK2). Each of these complexes is involved in a particular phase of the cell cycle. Additionally, CDKs 7, 8, and 9 are implicated in the regulation of transcription.
The CDKs seem to participate in cell cycle progression and cellular transcription, and loss of growth control is linked to abnormal cell proliferation in disease (see e.g., Malumbres and Barbacid, Nat. Rev. Cancer 2001 , 1 :222). Increased activity or temporally abnormal activation of cyclin-dependent kinases has been shown to result in the development of human tumors (Sherr C. J., Science 1996, 274: 1672-1677). Indeed, human tumor development is commonly associated with alterations in either the CDK proteins themselves or their regulators (Cordon-Cardo C, Am. J. Pat1/701. 1995; 147: 545-560; Karp J. E. and Broder S., Nat. Med. 1995; 1 : 309-320; Hall M. et al., Adv.
Cancer Res. 1996; 68: 67-108).
CDKs 7 and 9 seem to play key roles in transcription initiation and elongation, respectively (see, e.g., Peterlin and Price, Cell 23: 297-305, 2006; Shapiro, J. Clin. Oncol. 24: 1770-83, 2006;). Inhibition of CDK9 has been linked to direct induction of apoptosis in tumor cells of hematopoietic lineages through down-regulation of transcription of antiapoptotic proteins such as Mcl1 (Chao, S.-H. et al. J. Biol. Chem. 2000;275:28345- 28348; Chao, S.-H. et al. J. Biol. Chem. 2001 :276:31793-31799; Lam et. al. Genome Biology 2: 0041.1-11 , 2001 ; Chen et al. Blood 2005: 106:2513; MacCallum et al. Cancer Res. 2005;65:5399; and Alvi et al. Blood 2005; 105:4484). In solid tumor cells, transcriptional inhibition by downregulation of CDK9 activity synergizes with inhibition of cell cycle CDKs, for example CDK1 and 2, to induce apoptosis (Cai, D.-P., Cancer Res 2006, 66:9270. Inhibition of transcription through CDK9 or CDK7 may have selective non-proliferative effect on the tumor cell types that are dependent on the transcription of mRNAs with short half lives, for example Cyclin D1 in Mantle Cell Lymphoma. Some transcription factors such as Myc and NF-kB selectively recruit CDK9 to their promoters, and tumors dependent on activation of these signaling pathways may be sensitive to CDK9 inhibition.
Small molecule CDK inhibitors may also be used in the treatment of
cardiovascular disorders such as restenosis and atherosclerosis and other vascular disorders that are due to aberrant cell proliferation. Vascular smooth muscle proliferation and intimal hyperplasia following balloon angioplasty are inhibited by over-expression of the cyclin-dependent kinase inhibitor protein. Moreover, the purine CDK2 inhibitor CVT- 313 (Ki = 95 nM) resulted in greater than 80% inhibition of neointima formation in rats.
CDKs are important in neutrophil-mediated inflammation and CDK inhibitors promote the resolution of inflammation in animal models. (Rossi, A.G. et al, Nature Med. 2006, 12: 1056). Thus CDK inhibitors, including CDK9 inhibitors, may act as antiinflammatory agents.
Certain CDK inhibitors are useful as chemoprotective agents through their ability to inhibit cell cycle progression of normal untransformed cells (Chen, et al. J. Natl.
Cancer Institute. 2000; 92: 1999-2008). Pre-treatment of a cancer patient with a CDK inhibitor prior to the use of cytotoxic agents can reduce the side effects commonly associated with chemotherapy. Normal proliferating tissues are protected from the cytotoxic effects by the action of the selective CDK inhibitor.
Pyridine compounds of the formula below have been described as CDK9 inhibitors, and accordingly such compounds are useful for treating cancer and other conditions mediated by CDK9 activity:
Figure imgf000005_0001
or a pharmaceutically acceptable salt thereof, wherein:
Ri is Ci-8 alkyl, C3.8 cycloalkyl, C3.8 branched alkyl, -(CH2)o-3-0-Ci.4 alkyl,
-(CH2)o-2 heteroaryl, or a 4 to 8 membered heterocycloalkyl group, wherein said groups are each independently optionally substituted with one to three substituents selected from the group consisting of -NH2, -OH, =0, -Ci-4 alkyl, -Ci-4 haloalkyl, -C3.5 branched alkyl, -(CH2)i.3-0-Ci.2 alkyl, -NH-C(0)-CH2-0-d.4 alkyl, -NH-C(0)-d.4 alkyl, -NH-C(O)- C3.8 branched alkyl, -NH-C(0)0-d.4 alkyl, -NH-S02-d.4 alkyl, -NH-S02-C3.8 branched alkyl, -NH-S02-C3.5 cycloalkyl, -0-(CH2)2.3-0-d.2 alkyl, -0-d.4 alkyl,
-C(0)d.4 alkyl, -C(0)-0-d.4 alkyl, -C(0)-C3.8 branched alkyl, -C(0)-CH2-0-d.4 alkyl, - S02-Ci-4 alkyl, -S02-C3.8 branched alkyl, and -S02-C3.5 cycloalkyl;
R2 is hydrogen, Ci-4 alkoxy, Ci-4 haloalkyl, Ci-4-alkyl, or halogen;
Ai is N or CR3; A4 is N or CR6, with the proviso that only one of A! and A4 is a N;
R3 is Ci-4 alkyl, H, or OCi-4 alkyl;
R4 is hydrogen, halogen, 5 to 7 membered heterocyclyl-aryl, or A6-L-R9;
R5 is hydrogen, Ci-4 alkyl, or halogen;
R6 is hydrogen, Ci-4 alkyl, or halogen;
R7 is hydrogen, Ci-4 alkyl, or halogen;
A6 is NR8;
L is Co-3-alkylene or C3.8 branched alkylene;
R8 is hydrogen, Ci-4 alkyl; or -C3.8 branched alkyl; and
R9 is hydrogen, Ci.6 alkyl, C3.8 cycloalkyl, 4 to 8 member heterocycloalkyl, aryl, or heteroaryl, wherein said groups are optionally substituted with one to three substituents each independently selected from hydrogen, halogen, Ci-4 alkyl, Ci-4 haloalkyl, -OH, -O-C1.3 alkyl, -O-C1-3 haloalkyl, -0-(CH2)2-3-0-Ci.2 alkyl, -C(0)-Ci.4 alkyl, and -NH-C(0)-Ci-4 alkyl.
There remains a great need to develop novel inhibitors of protein kinases, such as CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9, as well as combinations thereof.
SUMMARY OF EMBODIMENTS OF THE INVENTION
The present invention provides novel biaryl pyridine compounds having structural similarities to the compounds described above, which are also useful to treat cancer and other conditions based on their activity on CDK9. Compounds of the invention are of Formulas l-VI as further described herein.
Figure imgf000006_0001
(I) (II)
Figure imgf000007_0001
The invention includes pharmaceutically acceptable salts of compounds of any of Formulas l-VI. Further structural description of these compounds and of pharmaceutical compositions and methods of use of these compounds are described below.
The foregoing compounds are inhibitors of at least one kinase, including at least CDK9, and are therefore useful for treating conditions mediated by excessive or undesired levels of CDK9 activity, such as the conditions described herein.
Another embodiment provides a method of treating a disease or condition mediated by CDK9 by using a compound of Formulas l-VI, or a pharmaceutically acceptable salt thereof. Also provided in another embodiment is the manufacture of a medicament for the treatment of a disease or condition mediated by CDK9, said medicament comprising a compound of any of Formulas l-VI, or a pharmaceutically acceptable salt thereof.
Another aspect of the present invention provides a method of treating a disease or condition mediated by CDK9 using a compound of any of Formulas l-VI, or pharmaceutically acceptable salt thereof. A preferred method comprises administering a therapeutically effective amount of a compound of any of Formulas l-VI, or a pharmaceutical composition comprising an effective amount of a compound of any of Formulas l-VI to a subject in need thereof.
The present invention also provides a pharmaceutical composition comprising a compound of any of Formulas l-VI, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient. Also provided in another embodiment is the use of a compound of any of Formulas l-VI, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a disease or condition mediated by CDK9. In another aspect, the present invention provides a method of regulating, modulating, or inhibiting protein kinase activity which comprises contacting a protein kinase with a compound of the invention. Suitable protein kinases include CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9, or any combination thereof.
Preferably, the protein kinase is selected from the group consisting of CDK1 , CDK2 and CDK9, or any combination thereof. In still another embodiment, the protein kinase is in a cell culture. In yet another embodiment, the protein kinase is in a mammal.
In another aspect, the invention provides a method of treating a protein kinase- associated disorder comprising administering to a subject in need thereof a
pharmaceutically acceptable amount of a compound of the invention. Suitable protein kinases includeCDKI , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9 or combinations thereof (preferably, the protein kinase is selected from the group consisting of CDK1 , CDK2 and CDK9, more preferably, the protein kinase is CDK9.) Suitable CDK combinations include CDK4 and CDK9; CDK1 , CDK2 and CDK9; CDK9 and CDK7; CDK9 and CDK1 ; CDK9 and CDK2; CDK4, CDK6 and CDK9; CDK1 , CDK2, CDK3, CDK4, CDK6 and CDK9.
In yet another aspect, the invention provides a method of treating cancer comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a compound of the invention. Suitable cancers for treatment by the compounds, compositions and methods described herein include bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancer. DETAILED DESCRIPTION
As used herein, the term "protein kinase-associated disorder" includes disorders and states (e.g., a disease state) that are associated with the activity of a protein kinase, e.g., the CDKs, e.g., CDK1 , CDK2 and/or CDK9. Non-limiting examples of protein kinase-associated disorders include abnormal cell proliferation (including protein kinase- associated cancers), viral infections, fungal infections, autoimmune diseases and neurodegenerative disorders.
The term "treat," "treated," "treating" or "treatment" includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated. In certain embodiments, the treatment comprises the induction of a protein kinase-associated disorder, followed by the activation of the compound of the invention, which would in turn diminish or alleviate at least one symptom associated or caused by the protein kinase-associated disorder being treated. For example, treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.
The term "use" includes one or more of the following embodiments of the invention, respectively: the use in the treatment of protein kinase-associated disorders; the use for the manufacture of pharmaceutical compositions for use in the treatment of these diseases, e.g., in the manufacture of a medicament; methods of use of compounds of the invention in the treatment of these diseases; pharmaceutical preparations having compounds of the invention for the treatment of these diseases; and compounds of the invention for use in the treatment of these diseases; as appropriate and expedient, if not stated otherwise. In particular, diseases to be treated and are thus preferred for use of a compound of the present invention are selected from cancer, inflammation, cardiac hypertrophy, and HIV infection, as well as those diseases that depend on the activity of protein kinases. The term "use" further includes embodiments of compositions herein which bind to a protein kinase sufficiently to serve as tracers or labels, so that when coupled to a fluor or tag, or made radioactive, can be used as a research reagent or as a diagnostic or an imaging agent.
The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a fully saturated straight-chain (linear; unbranched) or branched chain, having the number of carbon atoms specified, if designated (i.e. C Ci0 means one to ten carbons). Illustrative "alkyl" group examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. If no size is designated, the alkyl groups mentioned herein contain 1-10 carbon atoms, typically 1-8 carbon atoms, and preferably 1-6 or 1-4 carbon atoms.
The terms "alkoxy," refers to -O-alkyl, wherein the term alkyl is as defined above.
The term "cycloalkyl" by itself or in combination with other terms, represents, unless otherwise stated, cyclic versions of alkyl. Additionally, cycloalkyl may contain fused rings, but is not intended to describe fused fully aromatic aryl and heteroaryl groups. Cycloalkyl groups, unless indicated otherwise, are unsubstituted, but may be substituted with those groups typically suitable for alkyl group substitutions. Illustrative examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and the like. If no ring size is specified, the cycloalkyl groups described herein generally contain 3-10 ring members, preferably 3-6 ring members.
The term "heterocyclic" or "heterocycloalkyl" or "heterocyclyl," by itself or in combination with other terms, represents a cycloalkyl containing at least one annular carbon atom and at least one annular heteroatom selected from the group consisting of O, N, P, Si and S, preferably from N, O and S, wherein the ring is not aromatic but can contain unsaturations. The nitrogen and sulfur atoms in a heterocyclic group may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heterocyclic groups discussed herein, if not otherwise specified, contain 3-10 ring members, and at least one ring member is a heteroatom selected from N, O, P, Si, and S. Preferably, not more than three of these heteroatoms are included in a heterocyclic group, and generally not more than two of these heteroatoms are present in a single ring of the heterocyclic group. The heterocyclic group can be fused to an additional carbocyclic or heterocyclic ring. A heterocyclic group can be attached to the remainder of the molecule at an annular carbon or annular heteroatom. Additionally, heterocyclic may contain fused rings, but excludes fused systems containing a heteroaryl group as part of the fused ring system. Illustrative examples of heterocyclic groups include, 1- (1 ,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,
tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, piperidin-2-one, azepane, tetrahydro- 2H-pyranyl, pyrrolidinyl, methylpyrrolidinone, alkylpiperidinyl, haloalkylpiperidinyl, 1- (alkylpiperidin-l-yl)ethanone, and the like.
As with other moieties described herein, heterocycloalkyl moieties can be unsubstituted, or substituted with various substituents known in the art, e.g., hydroxy, halo, oxo (C=0), alkylimino (RN=, wherein R is a loweralkyl or loweralkoxy group), amino, alkylamino, dialkylamino, acylaminoalkyl, alkoxy, thioalkoxy, polyalkoxy, loweralkyl, cycloalkyl or haloalkyl. Non-limiting examples of heterocycloalkyl groups include the following, where each moiety may be attached to the parent molecule at any available valence:
Figure imgf000010_0001
Figure imgf000011_0001
Specifically included 'heterocyclic' groups are piperidine, morpholine,
thiomorpholine, piperazine, pyrrolidine, tetrahydrofuran, oxetane, oxepane, oxirane, tetrahydrothiofuran, thiepane, thiirane, and optionally substituted versions of each of these.
The term "aryl", unless otherwise stated, represents an aromatic hydrocarbon group which can be a single ring or multiple rings (e.g., from 1 to 3 rings) which are fused together. Aryl includes fused rings, wherein one or more of the fused rings is fully saturated ( e.g. , cycloalkyl) or partially unsaturated (e.g. , cyclohexenyl), but not a heterocyclic or heteroaromatic ring. Illustrative examples of aryl groups include, but are not limited to, phenyl, 1 -naphthyl, 2-naphthyl, and tetrahydronaphthyl.
The term "heteroaryl", as used herein, refers to groups comprising a single ring, or a fused ring, where at least one of the rings is an aromatic ring that contain from one to four heteroatoms selected from N, O, and S as ring members (i.e. , it contains at least one heteroaromatic ring), wherein the nitrogen and sulfur atoms can be oxidized, and the nitrogen atom(s) can be quaternized. A heteroaryl group can be attached to the remainder of the molecule through an annular carbon or annular heteroatom, and it can be attached through any ring of the heteroaryl moiety, if that moiety is a bicyclic, tricyclic, or a fused ring system. A heteroaryl group may contain fused rings, wherein one of the fused rings is aromatic or heteroaromatic, and the other fused ring(s) are partially unsaturated (e.g., cyclohexenyl, 2,3-dihydrofuran, tetrahydropyrazine, and 3,4-dihydro- 2H-pyran), or completely saturated (e.g., cyclohexyl, cyclopentyl, tetrahydrofuran, morpholine, and piperazine). The term heteroaryl is also intended to include fused rings systems that include a combination of aromatic and heteroaromatic rings systems (e.g., indoles, quinoline, quinazolines, and benzimidazoles). Illustrative examples of heteroaryl groups are 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.
The terms "halo" or "halogen," represents a fluorine, chlorine, bromine, or iodine atom. Commonly when present as a substituent, halo refers to F or CI or Br, preferably F or CI.
The term "haloalkyi," represents an alkyl group as defined above, wherein one or more hydrogen atoms of the alkyl group are replaced by a halogen atom which may be the same or different. The term haloalkyi thus includes mono-haloalkyl, di-haloalkyl, tri- haloalkyi, tetra-haloalkyl, and the like as well as per-haloalkyl. The prefix "perhalo" refers to the respective group wherein all available valences are replaced by halo groups. For example "perhaloalkyl" includes -CCI3, -CF3, -CCI2CF3, and the like. The terms
"perfluoroalkyl" and "perchloroalkyl" are a subset of perhaloalkyl wherein all available valences are replaced by fluoro and chloro groups, respectively. Illustrative examples of perfluoroalkyl include -CF3 and -CF2CF3, and of perchloroalkyl include -CCI3
and -CCI2CCI3.
Optionally substituted" as used herein indicates that the particular group or groups being described may have no non-hydrogen substituents (i.e., it can be unsubstituted), or the group or groups may have one or more non-hydrogen substituents. If not otherwise specified, the total number of such substituents that may be present is equal to the number of H atoms present on the unsubstituted form of the group being described. Typically, an optionally substituted group will contain up to four (1-4) substituents. Where an optional substituent is attached via a double bond, such as a carbonyl oxygen (=0), the group takes up two available valences on the group being substituted, so the total number of substituents that may be included is reduced according to the number of available valences. Suitable optional substituent groups include halo, d.4alkyl, -NH-C(0)-CH2-0-Ci_4 alkyl, -NHC(0)-Ci.4 alkyl, -C(0)-0-Ci.4alkyl,
-0-Ci-4alkyl, -0-Ci-4haloalkyl, -Ci-4alkylene-0-Ci-4haloalkyl, -Ci-4alkylene-0-Ci-4alkyl, - NH-Ci.4alkyl, -C(0)-CH2-0-Ci.4 alkyl, -C(0)-0-C3.6 branched alkyl, -Ci_4 haloalkyl, - (CH2)i-3-0-Ci-2 alkyl, -Ci.4-cycloalkyl, -Ci.4alkylene-0-Ci_4alkyl, -NH2, -S02-Ci.4alkyl, -NH- C(0)-Ci_4 alkyl, and -NH-S02-Ci_4 alkyl, hydroxyl, nitro, cyano, oxo, -C(0)-Ci-4alkyl, - C(O)- and the like.
Unless specified otherwise, the term "compounds of the present invention" refer to compounds of Formula I , prodrugs thereof, pharmaceutically acceptable salts of the compounds, and/or prodrugs, and hydrates or solvates of the compounds, salts, and/or prodrugs, as well as, all stereoisomers (including diastereoisomers and enantiomers), tautomers, and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties (e.g. , polymorphs, solvates and/or hydrates).
As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. It is noted that salts of the novel compounds described herein are of course useful as precursors for the neutral species or for pharmaceutically acceptable salts.
The term "a therapeutically effective amount" of a compound of the present invention refers to an amount of the compound of the present invention that when administered to a subject, is effective to (1) at least partially alleviating, inhibiting, preventing and/or ameliorating a condition, or a disorder or a disease (i) mediated by one or more CDK enzymes, or (ii) associated with one or more CDK enzyme activities, or (iii) characterized by activity of proteins regulated (directly or indirectly) by one or more CDK enzymes (e.g. RNA polymerase I I); or (2) reducing or inhibiting the expression of proteins whose expression is dependent (directly or indirectly) on one or more CDK enzymes (e.g. Mcl-1 , Cyclin D, Myc etc.). When used in conjunction with a cell, the term "a therapeutically effective amount" refers to the amount of the compound of the present invention that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reducing or inhibiting the activity of proteins regulated by one or more CDK enzymes; or at least partially reducing or inhibiting the expression of proteins whose expression is dependent (directly or indirectly) on one or more CDK enzymes.
As used herein, the term "subject" refers to an animal. Typically the animal is a mammal. A subject also refers to for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In one aspect, the invention provides compounds having a bi-aryl core structure with a pyridine ring connected to a second heterocyclic ring, wherein each ring has an additional substituent attached at a position 'meta' to the biaryl linkage. The following enumerated embodiments represent aspects and embodiments of the invention.
1. A compound of Formula I:
Figure imgf000014_0001
or a pharmaceutically acceptable salt thereof, wherein:
R is selected from the group consisting of d_6 alkyl, C3.6branched alkyl,
C3_i0cycloalkyl, C3_i 0 heterocycloalkyi, C3- 0-partially unsaturated cycloalkyl and C3_i0 partially unsaturated heterocycloalkyi;, wherein each heterocycloalkyi contains one or two heteroatoms selected from N, S and O as ring members,
wherein R is optionally substituted with up to four groups selected from halo, oxo (=0), CN, C C4 haloalkyl, C C4 haloalkoxy, -R, -OR, -NR2, -COR, COOR, -(CH2)q-R, -(CH2)q-NR2, -CONR2, -NHCOR, -NRCOOR, -SR, -S(0)qR, - S02NHR, -NHS02R, -(CH2)q-CN, -0-(CH2)q-CN, -(CH2)q-OR, and -0-(CH2)q-OR, where each R is independently H or an optionally substituted C C6 alkyl or C3-C7 cycloalkyl or C3-C7 heterocycloalkyi containing N, O or S as a ring member, wherein the optional substituents for R are up to three groups independently selected from halo, oxo, CN, C C4 haloalkyi, C C4 haloalkoxy, -OH, R\ OR', -C(0)R\ NH2, N HR\ - NH-C(0)-R', where each R' is independently C C4 straight chain or branched chain alkyl;
and where two R on the same atom or on adjacent connected atoms can optionally cyclize to form a 5-6 membered ring optionally containing 1-2 heteroatoms selected from N, O and S as ring members, and optionally substituted with up to four groups selected from halo, oxo (=0), CN, C1-C4 alkyl, C C4 alkoxy, C C4 haloalkyi, and C C4 haloalkoxy;
and each q is 1-2;
R3 is H, CI or F;
L is a bond or Ci_3 alkylene;
L2 is Co-3 alkylene, CD2, CHD, or C3.8 branched alkylene;
A1 is O or -NR8- Z2 is N or CR2;
Z4 is N or CR4;
Z5 is N or CR5;
Z6 is N or CR6;
provided that at least one but not more than two of Z2, Z4, Z5 and Z6 is N;
and if Z2 is N, either Z4 or Z6 is also N;
and if Z5 is N, either Z4 or Z6 is also N;
R2, R4, R5, R6, and R7 are each independently selected from H, C C4 alkyl, C C4 alkoxy, C C4 haloalkyi, C C4 haloalkoxy, CN, F and CI;
R8 is H or methyl;
R9 is an optionally substituted group selected from Ci_3 alkyl, C3.6 branched alkyl, C3.7 cycloalkyl, C3.7 heterocycloalkyl having up to two heteroatoms selected from N, O and S as ring members, -(CH2)i-3-0-Ci.4 alkyl, pyridyl, and phenyl,
wherein the optional substituents for R9 are up to four groups independently selected from halo, oxo, CN, C C4 haloalkyi, C C4 haloalkoxy, - OH, R\ OR', -C(0)R\ CONH2, CONHR', NH2, NHR', -NH-C(0)-R', where each R' is independently C C4 straight chain or branched chain alkyl or haloalkyi. In some embodiments, R5 or R6 or both will represent H . In some embodiments,
R7 is H and R2 and R4 can be H also, when they are present. In some embodiments, A1 is NR8. In preferred embodiments, R8 is H.
In some embodiments, L is a bond; in some embodiments, L2 is CH2.
In some embodiments, R9 is a heterocyclic group selected from tetrahydropyran, tetrahydrothiopyran, tetrahydrothiopyran-1 -dioxide, piperidine, pyrrolidine, dioxane, and tetrahydrofuran, oxepane, and dioxepane.
In particular embodiments, R9 is substituted with at least one group selected from CN, halo, and C1-C4 alkyl. In some preferred embodiments, R9 is a cyclopropyl or
tetrahydropyran, and is substituted with CN.
In certain embodiments, the compound is selected from the group consisting of:
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-isobutyl-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(pyridin-3-ylmethyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-N2'-isobutyl-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-N2'-(pyridin-3-ylmethyl)-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-N2'-((tetrahydro-2H-pyran-4-yl)methyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-aminocyclohexyl)-N2'-(3-aminopropyl)-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-((tetrahydro-2H-pyran-4-yl)methyl)-[4,4'-bipyridine]-
2,2'-diamine
N2-(trans-4-aminocyclohexyl)-N2'-(3-aminopropyl)-5-chloro-[4,4'-bipyridine]-2,2'-diamine 3-((2'-((trans-4-aminocyclohexyl)amino)-5'-chloro-[4,4'-bipyridin]-2-yl)amino)propan-1-ol
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3-methoxypropyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-isopentyl-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(cyclohexylmethyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-N2'-benzyl-5-chloro-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(2-fluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3-fluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(4-fluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(2-chlorobenzyl)-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-((R)-1-phenylethyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3-chlorobenzyl)-[4,4'-bipyridine]-2,2'-diamine N2-((1 r,4S)-4-aminocyclohexyl)-5-chloro-N2'-((S)-1-phenylethyl)-[4,4'-bipyridine]-2,2'-diarriine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(4-chlorobenzyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(2,3-difluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(2,4-difluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3,4-difluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3,5-difluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3-(trifluoromethyl)benzyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(2,5-difluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3-methylbenzyl)-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3-(difluoromethoxy)benzyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3-(trifluoromethoxy)benzyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(pyridin-2-ylmethyl)-[4,4'-bipyridine]-2,2'-diamin
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-phenethyl-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(pyridin-4-ylmethyl)-[4,4'-bipyridine]-2,2'-diamin N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3-methoxybenzyl)-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-N2'-benzyl-[4,4'-bipyridine]-2,2'-diamine N2-(trans-4-aminocyclohexyl)-N2'-(3-fluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine N2'-(trans-4-aminocyclohexyl)-5-chloro-N2-(3-fluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine trans-N1-(4-(6-((3-fluorobenzyl)amino)pyrimidin-4-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine trans-N1-(4-(5-chloro-2-((3-fluorobenzyl)amino)pyrimidin-4-yl)pyridin-2-yl)cyclohexane-1 ,4- diamine
trans-N1-(4-(2-((3-fluorobenzyl)amino)pyrimidin-4-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine trans-N1-(4-(4-((3-fluorobenzyl)amino)pyrimidin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine N2'-(trans-4-aminocyclohexyl)-N4-(3-fluorobenzyl)-[2,4'-bipyridine]-2',4-diamine N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-((5-fluoropyridin-3-yl)methyl)-[4,4'-bipyridi
diamine
3-(((2'-((trans-4-aminocyclohexyl)amino)-5'-chloro-[4,4'-bipyridin]-2-yl)amino)methyl)p
N2-(trans-4-aminocyclohexyl)-N2'-(3-bromobenzyl)-5-chloro-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-N2'-((5-bromopyridin-3-yl)methyl)-5-chloro-[4,4'-bipyridine]-2,2'^ diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(4-methylbenzyl)-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-N2'-(4-bromobenzyl)-5-chloro-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(4-(trifluoromethyl)benzyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(4-chloro-3-fluorobenzyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(4-(difluoromethoxy)benzyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(4-(trifluoromethoxy)benzyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-(aminomethyl)cyclohexyl)-5-chloro-N2'-(3-fluorobenzyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-(aminomethyl)cyclohexyl)-5-chloro-N2'-(3,5-difluorobenzyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-(aminomethyl)cyclohexyl)-5-chloro-N2'-(pyridin-4-ylmethyl)-[4,4'-bipyridine]-2,2'- diamine
N2-(trans-4-(aminomethyl)cyclohexyl)-5-chloro-N2'-(3,4-difluorobenzyl)-[4,4'-bipyridine]-2,2'- diamine
5-chloro-N2'-(3-fluorobenzyl)-N2-(piperidin-4-yl)-[4,4'-bipyridine]-2,2'-diamine 5-chloro-N2'-(3,5-difluorobenzyl)-N2-(piperidin-4-yl)-[4,4'-bipyridine]-2,2'-diamine 5-chloro-N2-(piperidin-4-yl)-N2'-(pyridin-4-ylmethyl)-[4,4'-bipyridine]-2,2'-diamine 5-chloro-N2'-(3,4-difluorobenzyl)-N2-(piperidin-4-yl)-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-5,6'-dichloro-N2'-(3-fluorobenzyl)-[4,4'-bipyridine]-2,2'-diamine
N2-(trans-4-aminocyclohexyl)-5-chloro-N2'-(3-fluorobenzyl)-6'-methyl-[4,4'-bipyridine]-2,2'- diamine
4-(((4-(5-chloro-2-((trans-4-((2-methoxyethyl)amino)cyclohexyl)amino)pyridin-4-yl)pyrimidin-2- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
and
4-(((2-(5-chloro-2-((trans-4-((2-methoxyethyl)amino)cyclohexyl)amino)pyridin-4-yl)pyrimidin-4- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile 2. The compound of embodiment 1 , wherein:
L is a bond or CH2, and R is cyclohexyl,
wherein R is substituted with up to four groups selected from halo, oxo (=0), CN, C1-C4 haloalkyl, C C4 haloalkoxy, -R, -OR, -NR2, -COR, - COOR, -CONR2, -NHCOR, -NRCOOR, -S(0)qR, -S02NHR, -NHS02R, - (CH2)q-OR, and -0-(CH2)q-OR;
R3 is F or CI;
L2 is CH2, CHD, or CD2; and
R9 is selected from pyridyl, phenyl, tetrahydropyran, cyclopropane, and tetrahydrofuran, each of which can be substituted with up to three groups independently selected from F, CI, -OH, Me, ethyl, -OMe, CN, and CONH2.
In some of these embodiments, A1 is NH and L2 is CH2. In some preferred embodiments of this type, R9 is optionally substituted phenyl or substituted cyclopropane or tetrahydropyran.
3. The com ound of embodiment 1 or 2, wherein -L-R9 is
Figure imgf000019_0001
where R 0 and R and R 2 each independently represent H, F,
CI, -OCHF2, -C(0)-Me, -OH, Me, -OMe, -CN, -Ethyl, -CONH2, or -NH-C(0)-Me. In these embodiments, R 2 is preferably CN. R 0 and R are typically H, F, CI,
Me or OMe.
4. The compound of any of the preceding embodiments, wherein R3 is CI. 5. The compound of any one of embodiments 1-4, wherein R is an optionally substituted cyclohexyl, piperidine, or pyrrolidine ring.
6. The compound of any one of embodiments 1-5, wherein R7 is H. 7. The compound of any one of embodiments 1-6, wherein A1 is NH.
8. The compound of any one of embodiments 1-7, wherein Z2 is N. The compound of any one of embodiments 1-8, wherein Z is N.
The compound of any of embodiments 1-8, wherein Z6 is CH.
The compound of any one of embodiments 1-9, wherein Z5 is CH.
The compound of any one of embodiments 1-8, wherein Z4 is N.
The compound of embodiment 1 , which is a compound of Formula II:
Figure imgf000020_0001
The compound of embodiment 1 , which is a compound of Formula III:
Figure imgf000020_0002
The compound of embodiment 1 , which is a compound of Formula (IV):
Figure imgf000020_0003
(IV). The compound of embodiment 1 , which is a compound of Formula (V)
Figure imgf000021_0001
The compound of embodiment 1 , which is a compound of Formula (VI):
Figure imgf000021_0002
A compound of any of embodiments 1-17, for use in therapy.
19. The compound of embodiment 18, which is for use to treat a condition mediated by CDK9.
The compound of embodiment 19, wherein the condition is cancer.
21. The compound of embodiment 20, wherein the cancer is selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract,
gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal, and pancreatic cancer. 22. A method to treat cancer, comprising administering to a subject in need thereof an effective amount of a compound according to any of embodiments 1-17.
23. The method of embodiment 22, wherein the cancer is selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal, and pancreatic cancer. 24. The method of embodiment 23, wherein the compound is administered, simultaneously or sequentially, with an antiinflammatory, antiproliferative,
chemotherapeutic agent, immunosuppressant, anti-cancer, cytotoxic agent or kinase inhibitor or salt thereof. 25. A pharmaceutical composition comprising a compound according to any of embodiments 1-17 admixed with at least one pharmaceutically acceptable excipient.
26. The pharmaceutical composition of embodiment 25, which comprises at least one pharmaceutically acceptable carrier and at least one other pharmaceutically acceptable excipient.
27. The pharmaceutical composition of embodiment 25 or 26, further comprising at least one additional therapeutic agent. 28. The pharmaceutical composition of embodiment 27, wherein the additional therapeutic agent is an antiinflammatory, antiproliferative, chemotherapeutic agent, immunosuppressant, anti-cancer, cytotoxic agent or kinase inhibitor or a salt thereof.
Additional embodiments of the compounds of the invention include the following compounds and their pharmaceutically acceptable salts, as well as the novel compounds in the Tables herein.
Figure imgf000023_0001
Figure imgf000023_0002
Figure imgf000023_0003
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000029_0002
Figure imgf000029_0003
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000031_0002
Figure imgf000031_0003
30
Figure imgf000032_0001
Figure imgf000032_0002
Figure imgf000032_0003
Figure imgf000033_0001
Figure imgf000033_0002
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Further details of the compounds of the invention and of methods for making and using the compounds and pharmaceutical compositions thereof are provided below. Further details of the compounds of the invention and of methods for making and using the compounds and pharmaceutical compositions thereof are provided below.
The compounds of the invention described herein can be prepared from readily available starting materials using the following general methods and procedures and adaptations thereof that will be apparent to the skilled person. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are described, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine experimentation.
Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from
undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wley, New York, 1999, and references cited therein.
The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wsconsin, USA), Bachem (Torrance,
California, USA), Emka-Chemce or Sigma (St. Louis, Missouri, USA). Others may be prepared by procedures, or obvious modifications thereof, described in standard reference texts such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1- 15 (John Wley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1- 40 (John Wley and Sons, 1991), March's Advanced Organic Chemistry, (John Wley and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH
Publishers Inc., 1989).
The various starting materials, intermediates, and compounds of the embodiments may be isolated and purified, where appropriate, using conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation, and chromatography. Characterization of these compounds may be performed using conventional methods such as by melting point, mass spectrum, nuclear magnetic resonance, and various other spectroscopic analyses.
The description of the disclosure herein should be construed in congruity with the laws and principals of chemical bonding. For example, it may be necessary to remove a hydrogen atom in order accommodate a substituent at any given location. Furthermore, it is to be understood that definitions of the variables (e.g., "R groups"), as well as the bond locations of the generic formulae of the invention (e.g., formulas I or II), will be consistent with the laws of chemical bonding known in the art. It is also to be understood that all of the compounds of the invention described above will further include bonds between adjacent atoms and/or hydrogens as required to satisfy the valence of each atom. That is, bonds and/or hydrogen atoms are added to provide the following number of total bonds to each of the following types of atoms: carbon: four bonds;
nitrogen: three bonds; oxygen: two bonds; and sulfur: two-six bonds.
Compounds of the embodiments may generally be prepared using a number of methods familiar to one skilled in the art in view of the methods described herein.
The compounds of the present invention can be isolated and used per se or as their pharmaceutical acceptable salt. In many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids, e.g., acetate, aspartate, benzoate, besylate,
bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate,
methylsulphate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate and trifluoroacetate salts.
Inorganic acids from which salts can be derived include, for example,
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.
Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.
Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.
The pharmaceutically acceptable salts of the present invention can be
synthesized from a parent compound, a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two.
Generally, use of non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable, where practicable. Lists of additional suitable salts can be found, e.g., in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Company, Easton, Pa., (1985); and in HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES,
SELECTION, AND USE, by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
The compounds of the present invention also include isotopically labeled forms of the compounds which may be synthesized using the processes described herein or modifications thereof known by those of skill in the art. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as 2H, 3H, C, 3C, 4C, 5N, 8F 3 P, 32P, 35S, 36CI, 25l respectively. The invention includes various isotopically labeled compounds as defined herein, for example those into which radioactive isotopes, such as 3H, 3C, and 4C, are present. Such isotopically labeled compounds are useful in metabolic studies (with 4C), reaction kinetic studies (with, for example 2H or 3H), detection or imaging techniques, such as positron emission
tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an 8F or labeled compound may be particularly desirable for PET or SPECT studies. Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available
isotopically labeled reagent for a non-isotopically labeled reagent.
Further, substitution with heavier isotopes, particularly deuterium (i.e., 2H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index. It is understood that deuterium in this context is regarded as a substituent of a compound of the formula (I). The concentration of such a heavier isotope, specifically deuterium, may be defined by the isotopic enrichment factor. The term
"isotopic enrichment factor" as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. If a substituent in a compound of this invention is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).
Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed. Compounds enriched in deuterium are referred to herein as 'deuterated versions' of the compounds of Formula I.
Compounds of the present invention include isomers including all stereoisomers of the compounds referred to in the formulas herein, including
enantiomers, diastereomers, as well as all conformers, rotamers, and tautomers, unless otherwise indicated. The invention includes all enantiomers of any chiral compound disclosed, in either substantially pure levorotatory or dextrorotatory form, or in a racemic mixture, or in any ratio of enantiomers. Furthermore, the compounds disclosed herein may contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e. , as individual enantiomers or diastereomers, or as stereoisomer enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of the embodiments, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like. Unless stereochemistry is explicitly indicated in a chemical structure or chemical name, the chemical structure or chemical name is intended to embrace all possible stereoisomers, conformers, rotamers, and tautomers of the compound depicted. For example, a compound containing a chiral carbon atom is intended to embrace both the (R) enantiomer and the (S) enantiomer, as well as mixtures of enantiomers, including racemic mixtures; and a compound containing two chiral carbons is intended to embrace all enantiomers and diastereomers (including (R,R), (S, S), (R,S), and (R, S) isomers).
The compounds of the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, it is intended that the invention embrace both solvated and unsolvated forms. The term "solvate" refers to a molecular complex of a compound of the present invention (including pharmaceutically acceptable salts thereof) with one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical art, which are known to be innocuous to the recipient, e.g. , water, ethanol, and the like. The term "hydrate" refers to the complex where the solvent molecule is water. As defined herein, solvates and hydrates of the compounds of the present invention are considered compositions, wherein the composition comprises a compound of the present invention and a solvent (including water).
The compounds of the present invention may exist in either amorphous or polymorphic form; therefore, all physical forms are considered to be within the scope of the present invention.
Compounds of the invention, i.e. compounds of the present invention that contain groups capable of acting as donors and/or acceptors for hydrogen bonds may be capable of forming co-crystals with suitable co-crystal formers. These co-crystals may be prepared from compounds of formula (I) by known co-crystal forming procedures. Such procedures include grinding, heating, co-subliming, co-melting, or contacting in solution compounds of formula (I) with the co-crystal former under crystallization conditions and isolating co-crystals thereby formed. Suitable co-crystal formers include those described in WO 2004/078163. Hence the invention further provides co-crystals comprising a compound of formula (I).
In certain uses of the compounds of the present invention, it may be
advantageous to use a pro-drug of the compound. In general, pro-drugs convert in vivo to the compounds of the present invention. A pro-drug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into a compound of this invention following administration of the prodrug to a subject. The suitability and techniques involved in making and using prodrugs are well known by those skilled in the art. Prodrugs can be conceptually divided into two non-exclusive categories, bioprecursor prodrugs and carrier prodrugs. See THE PRACTICE OF MEDICINAL CHEMISTRY, Ch. 31 -32 (Ed. Wermuth, Academic Press, San Diego, Calif., 2001 ). Generally, bioprecursor prodrugs are compounds that are inactive or have low activity compared to the corresponding active drug compound, that contain one or more protective groups and are converted to an active form by metabolism or solvolysis. Both the active drug form and any released metabolic products should have acceptably low toxicity.
Carrier prodrugs are drug compounds that contain a transport moiety, e.g., that improve uptake and/or localized delivery to a site(s) of action. Desirably for such a carrier prodrug, the linkage between the drug moiety and the transport moiety is a covalent bond, the prodrug is inactive or less active than the drug compound, and any released transport moiety is acceptably non-toxic. For prodrugs where the transport moiety is intended to enhance uptake, typically the release of the transport moiety should be rapid. In other cases, it is desirable to utilize a moiety that provides slow release, e.g., certain polymers or other moieties, such as cyclodextrins. Carrier prodrugs can, for example, be used to improve one or more of the following properties: increased lipophilicity, increased duration of pharmacological effects, increased site-specificity, decreased toxicity and adverse reactions, and/or improvement in drug formulation (e.g., stability, water solubility, suppression of an undesirable organoleptic or physiochemical property). For example, lipophilicity can be increased by esterification of (a) hydroxyl groups with lipophilic carboxylic acids (e.g., a carboxylic acid having at least one lipophilic moiety), or (b) carboxylic acid groups with lipophilic alcohols (e.g., an alcohol having at least one lipophilic moiety, for example aliphatic alcohols).
Exemplary prodrugs are, e.g., esters of free carboxylic acids and S-acyl derivatives of thiols and O-acyl derivatives of alcohols or phenols, wherein acyl has a meaning as defined herein. Suitable prodrugs are often pharmaceutically acceptable ester derivatives convertible by solvolysis under physiological conditions to the parent carboxylic acid, e.g., lower alkyl esters, cycloalkyl esters, lower alkenyl esters, benzyl esters, mono- or di-substituted lower alkyl esters, such as the -(amino, mono- or di- lower alkylamino, carboxy, lower alkoxycarbonyl)-lower alkyl esters, the -(lower alkanoyloxy, lower alkoxycarbonyl or di-lower alkylaminocarbonyl)-lower alkyl esters, such as the pivaloyloxymethyl ester and the like conventionally used in the art. In addition, amines have been masked as arylcarbonyloxymethyl substituted derivatives which are cleaved by esterases in vivo releasing the free drug and formaldehyde
(Bundgaard, J. Med. Chem. 2503 (1989)). Moreover, drugs containing an acidic NH group, such as imidazole, imide, indole and the like, have been masked with N- acyloxymethyl groups (Bundgaard, Design of Prodrugs, Elsevier (1985)). Hydroxy groups have been masked as esters and ethers. EP 039,051 (Sloan and Little) discloses Mannich-base hydroxamic acid prodrugs, their preparation and use. Typically, the compounds of the present invention are administered as a pharmaceutical composition. A typical pharmaceutical composition comprises a compound of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. As used herein, the term "pharmaceutically acceptable carriers, diluents or excipients" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, and parenteral administration, etc. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions). The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifers and buffers, etc.
Typically, the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Tablets may be uncoated, film coated, or enteric coated according to methods known in the art.
Suitable compositions for oral administration include an effective amount of a compound of the invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil. Certain injectable compositions are aqueous isotonic solutions or suspensions, and
suppositories are advantageously prepared from fatty emulsions or suspensions. Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 -75%, or contain about 1-50%, of the active ingredient. The invention further provides pharmaceutical compositions and dosage forms that may comprise one or more agents that reduce the rate by which the compound of the present invention as an active ingredient will decompose. Such agents, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers, etc.
The compounds of Formulas l-VI in free form or in pharmaceutically acceptable salt form, exhibit valuable pharmacological properties, e.g. CDK inhibiting properties, e.g. as indicated in vitro and in vivo tests as provided below and are therefore suitable for use in therapy.
When used with respect to methods of treatment/prevention and the use of the compounds and formulations thereof described herein, an individual "in need thereof" may be an individual who has been diagnosed with or previously treated for the condition to be treated. With respect to prevention, the individual in need thereof may also be an individual who is at risk for a condition (e.g., a family history of the condition, life-style factors indicative of risk for the condition, etc.). Typically, when a step of administering a compound of the invention is disclosed herein, the invention further contemplates a step of identifying an individual or subject in need of the particular treatment to be
administered or having the particular condition to be treated.
Another aspect of the present invention provides a compound of any of the formulae described herein, or pharmaceutically acceptable salt or solvate thereof, for use in therapy. Yet another aspect of the present invention provides a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof for use in a method of treating a disease or condition mediated by CDK9.
Yet another aspect of the present invention provides a method of treating a disease or condition mediated by CDK9 comprising administration to a subject in need thereof a therapeutically effective amount of a compound of Formula I, or a
pharmaceutically acceptable salt thereof. Provided in yet another aspect of the present invention is a compound of Formula I for use in a method of treating a disease or condition mediated by CDK9 is selected from cancer, cardiac hypotrophy, HIV and inflammatory diseases.
Another aspect of the present invention provides a method of treating a cancer selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal, and pancreatic cancer.
Yet another aspect of the present invention provides a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
In another aspect, the invention provides a method of regulating, modulating, or inhibiting protein kinase activity which comprises contacting a protein kinase with a compound of the invention. In one embodiment, the protein kinase is selected from the group consisting of CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9, or any combination thereof. In another embodiment, the protein kinase is selected from the group consisting of CDK1 , CDK2 and CDK9, or any combination thereof. In still another embodiment, the protein kinase is in a cell culture. In yet another embodiment, the protein kinase is in a mammal.
In another aspect, the invention provides a method of treating a protein kinase- associated disorder comprising administering to a subject in need thereof a
pharmaceutically acceptable amount of a compound of the invention such that the protein kinase-associated disorder is treated. In one embodiment, the protein kinase is selected from the group consisting of CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9.
In one embodiment, the protein kinase-associated disorder is cancer. In still another embodiment, the cancer is selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancer. In one embodiment, the protein kinase-associated disorder is inflammation. In another embodiment, the inflammation is related to rheumatoid arthritis, lupus, type 1 diabetes, diabetic nephropathy, multiple sclerosis, glomerulonephritis, chronic
inflammation, and organ transplant rejections.
In another embodiment, the protein kinase-associated disorder is a viral infection.
In one embodiment, the viral infection is associated with the HIV virus, human papilloma virus, herpes virus, poxvirus virus, Epstein-Barr virus, Sindbis virus, or adenovirus.
In still another embodiment, the protein kinase-associated disorder is cardiac hypertrophy.
In another aspect, the invention provides a method of treating cancer comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a compound of the invention such that the cancer is treated. In one embodiment, the cancer is selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancer.
In another aspect, the invention provides a method of treating inflammation comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a compound such that the inflammation is treated, wherein the compound is a compound of the invention. In one embodiment, the inflammation is related to rheumatoid arthritis, lupus, type 1 diabetes, diabetic nephropathy, multiple sclerosis, glomerulonephritis, chronic inflammation, and organ transplant rejections.
In another aspect, the invention provides a method of treating cardiac
hypertrophy comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a compound such that the cardiac hypertrophy is treated, wherein the compound is a compound of the invention.
In another aspect, the invention provides a method of treating a viral infection comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a compound such that the viral infection is treated, wherein the compound is a compound of the invention. In one embodiment, the viral infection is associated with the HIV virus, human papilloma virus, herpes virus, poxvirus virus, Epstein-Barr virus, Sindbis virus, or adenovirus.
In one embodiment, the subject to be treated by the compounds of the invention is a mammal. In another embodiment, the mammal is a human.
In another aspect, the compounds of the invention is administered,
simultaneously or sequentially, with an antiinflammatory, antiproliferative, chemotherapeutic agent, immunosuppressant, anti-cancer, cytotoxic agent or kinase inhibitor or salt thereof. In one embodiment, the compound, or salt thereof, is
administered, simultaneously or sequentially, with one or more of a PTK inhibitor, cyclosporin A, CTLA4-lg, antibodies selected from anti-ICAM-3, anti-IL-2 receptor, anti- CD45RB, anti-CD2, anti-CD3, anti-CD4, anti-CD80, anti-CD86, and monoclonal antibody OKT3, CVT-313, agents blocking the interaction between CD40 and gp39, fusion proteins constructed from CD40 and gp39, inhibitors of NF-kappa B function, nonsteroidal antiinflammatory drugs, steroids, gold compounds, FK506, mycophenolate mofetil, cytotoxic drugs, TNF-a inhibitors, anti-TNF antibodies or soluble TNF receptor, rapamycin, leflunimide, cyclooxygenase-2 inhibitors, paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C, ecteinascidin 743, porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin, etoposide, etoposide phosphate, teniposide, melphalan, vinblastine, vincristine, leurosidine, epothilone, vindesine, leurosine, or derivatives thereof.
In another aspect, the invention provides a packaged protein kinase-associated disorder treatment, comprising a protein kinase-modulating compound of the Formula I or Formula II, packaged with instructions for using an effective amount of the protein kinase-modulating compound to treat a protein kinase-associated disorder.
In certain embodiments, the compound of the present invention is further characterized as a modulator of a protein kinase, including, but not limited to, protein kinases selected from the group consisting of abl, ATK, Bcr-abl, Blk, Brk, Btk, c-fms, e- kit, c-met, c-src, CDK, cRafl, CSFIR, CSK, EGFR, ErbB2, ErbB3, ErbB4, ERK, Fak, fes, FGFRI, FGFR2, FGFR3, FGFR4, FGFR5, Fgr, FLK-4, flt-1 , Fps, Frk, Fyn, GSK, Gst-Flkl, Hck, Her-2, Her-4, IGF- IR, INS-R, Jak, JNK, KDR, Lck, Lyn, MEK, p38, panHER,
PDGFR, PLK, PKC, PYK2, Raf, Rho, ros, SRC, TRK, TYK2, UL97, VEGFR, Yes, Zap70, Aurora-A, GSK3-alpha, HIPK1 , HIPK2, HIP3, IRAKI , JNK1 , JNK2, JNK3, TRKB, CAMKII, CK1 , CK2, RAF, GSK3Beta, MAPK1 , MKK4, MKK7, MST2, NEK2, AAK1 , PKCalpha, PKD, RIPK2 and ROCK-II.
In a preferred embodiment, the protein kinase is selected from the group consisting of CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9 and any combination thereof, as well as any other CDK, as well as any CDK not yet identified. In a particularly preferred embodiment, the protein kinase is selected from the group consisting of CDK1 , CDK2 and CDK9. In a particularly preferred embodiment, the protein kinase is selected from the group consisting of CDK9. In a particular embodiment, CDK combinations of interest include CDK4 and CDK9; CDK1 , CDK2 and CDK9; CDK9 and CDK7; CDK9 and CDK1 ; CDK9 and CDK2; CDK4, CDK6 and CDK9; CDK1 , CDK2, CDK3, CDK4, CDK6 and CDK9.
In other embodiments, the compounds of the present invention are used for the treatment of protein kinase-associated disorders. As used herein, the term "protein kinase-associated disorder" includes disorders and states (e.g., a disease state) that are associated with the activity of a protein kinase, e.g., the CDKs, e.g., CDK1 , CDK2 and/or CDK9. Non-limiting examples of protein kinase-associated disorders include abnormal cell proliferation (including protein kinase-associated cancers), viral infections, fungal infections, autoimmune diseases and neurodegenerative disorders.
Non-limiting examples of protein-kinase associated disorders include proliferative diseases, such as viral infections, auto-immune diseases, fungal disease, cancer, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis, chronic inflammation, neurodegenerative disorders, such as Alzheimer's disease, and post-surgical stenosis and restenosis. Protein kinase- associated diseases also include diseases related to abnormal cell proliferation, including, but not limited to, cancers of the breast, ovary, cervix, prostate, testis, esophagus, stomach, skin, lung, bone, colon, pancreas, thyroid, biliary passages, buccal cavity and pharynx (oral), lip, tongue, mouth, pharynx, small intestine, colon-rectum, large intestine, rectum, brain and central nervous system, glioblastoma, neuroblastoma, keratoacanthoma, epidermoid carcinoma, large cell carcinoma, adenocarcinoma, adenocarcinoma, adenoma, adenocarcinoma, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma, kidney carcinoma, myeloid disorders, lymphoid disorders, Hodgkin's, hairy cells, and leukemia.
Additional non-limiting examples of protein kinase-associated cancers include carcinomas, hematopoietic tumors of lymphoid lineage, hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, tumors of the central and peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma
pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma.
Protein kinase-associated disorders include diseases associated with apoptosis, including, but not limited to, cancer, viral infections, autoimmune diseases and neurodegenerative disorders.
Non-limiting examples of protein-kinase associated disorders include viral infections in a patient in need thereof, wherein the viral infections include, but are not limited to, HIV, human papilloma virus, herpes virus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus.
Non-limiting examples of protein-kinase associated disorders include tumor angiogenesis and metastasis. Non-limiting examples of protein-kinase associated disorders also include vascular smooth muscle proliferation associated with
atherosclerosis, postsurgical vascular stenosis and restenosis, and endometriosis.
Further non-limiting examples of protein-kinase associated disorders include those associated with infectious agents, including yeast, fungi, protozoan parasites such as Plasmodium falciparum, and DNA and RNA viruses.
In another embodiment, the compound of the present invention is further characterized as a modulator of a combination of protein kinases, e.g., the CDKs, e.g., CDK1 , CDK2 and/or CDK9. In certain embodiments, a compound of the present invention is used for protein kinase-associated diseases, and/or as an inhibitor of any one or more protein kinases. It is envisioned that a use can be a treatment of inhibiting one or more isoforms of protein kinases.
The compounds of the invention are inhibitors of cyclin-dependent kinase enzymes. Without being bound by theory, inhibition of the CDK4/cyclin D1 complex blocks phosphorylation of the Rb/inactive E2F complex, thereby preventing release of activated E2F and ultimately blocking E2F-dependent DNA transcription. This has the effect of inducing G1 cell cycle arrest. In particular, the CDK4 pathway has been shown to have tumor-specific deregulation and cytotoxic effects. Accordingly, the ability to inhibit the activity of combinations of CDKs will be of beneficial therapeutic use.
Furthermore, the cell's ability to respond and survive chemotherapeutic assault may depend on rapid changes in transcription or on activation of pathways which are highly sensitive to CDK9/cyclinT1 (PTEF-b) activity. CDK9 inhibition may sensitize cells to TNFalpha or TRAIL stimulation by inhibition of NF-kB, or may block growth of cells by reducing myc-dependent gene expression. CDK9 inhibition may also sensitize cells to genotoxic chemotherapies, HDAC inhibition, or other signal transduction based therapies.
As such, the compounds of the invention can lead to depletion of anti- apoptotic proteins, which can directly induce apoptosis or sensitize to other apoptotic stimuli, such as cell cycle inhibition, DNA or microtubule damage or signal transduction inhibition. Depletion of anti-apoptotic proteins by the compounds of the invention may directly induce apoptosis or sensitize to other apoptotic stimuli, such as cell cycle inhibition, DNA or microtubule damage or signal transduction inhibition. The compounds of the invention can be effective in combination with chemotherapy, DNA damage arresting agents, or other cell cycle arresting agents. The compounds of the invention can also be effective for use in chemotherapy-resistant cells.
The present invention includes treatment of one or more symptoms of cancer, inflammation, cardiac hypertrophy, and HIV infection, as well as protein kinase- associated disorders as described above, but the invention is not intended to be limited to the manner by which the compound performs its intended function of treatment of a disease. The present invention includes treatment of diseases described herein in any manner that allows treatment to occur, e.g., cancer, inflammation, cardiac hypertrophy, and HIV infection.
In certain embodiments, the invention provides a pharmaceutical composition of any of the compounds of the present invention. In a related embodiment, the invention provides a pharmaceutical composition of any of the compounds of the present invention and a pharmaceutically acceptable carrier or excipient of any of these compounds. In certain embodiments, the invention includes the compounds as novel chemical entities.
In one embodiment, the invention includes a packaged protein kinase- associated disorder treatment. The packaged treatment includes a compound of the invention packaged with instructions for using an effective amount of the compound of the invention for an intended use.
The compounds of the present invention are suitable as active agents in pharmaceutical compositions that are efficacious particularly for treating protein kinase- associated disorders, e.g., cancer, inflammation, cardiac hypertrophy, and HIV infection. The pharmaceutical composition in various embodiments has a pharmaceutically effective amount of the present active agent along with other pharmaceutically acceptable excipients, carriers, fillers, diluents and the like. The phrase,
"pharmaceutically effective amount" as used herein indicates an amount necessary to administer to a host, or to a cell, issue, or organ of a host, to achieve a therapeutic result, especially the regulating, modulating, or inhibiting protein kinase activity, e.g., inhibition of the activity of a protein kinase, or treatment of cancer, inflammation, cardiac hypertrophy, and HIV infection.
In other embodiments, the present invention provides a method for inhibiting the activity of a protein kinase. The method includes contacting a cell with any of the compounds of the present invention. In a related embodiment, the method further provides that the compound is present in an amount effective to selectively inhibit the activity of a protein kinase. In other embodiments, the present invention provides a use of any of the compounds of the invention for manufacture of a medicament to treat cancer, inflammation, cardiac hypertrophy, and HIV infection in a subject.
In other embodiments, the invention provides a method of manufacture of a medicament, including formulating any of the compounds of the present invention for treatment of a subject.
EXAMPLES
Referring to the examples that follow, compounds of the invention can be synthesized using the methods described herein, along with adaptations of these and other methods known to one skilled in the art. The compounds and/or intermediates described below were characterized by high performance liquid chromatography (HPLC) using a Waters Millenium chromatography system with a 2695 Separation Module (Milford, MA). The analytical columns were reversed phase Phenomenex Luna C18 5 μ, 4.6 x 50 mm, from Alltech (Deerfield, IL). A gradient elution was used (flow 2.5 mL/min), typically starting with 5 % acetonitrile/95 % water and progressing to 100 % acetonitrile over a period of 10 minutes. All solvents contained 0.1 % trifluoroacetic acid (TFA). Compounds were detected by ultraviolet light (UV) absorption at either 220 or 254 nm. HPLC solvents were from Burdick and Jackson (Muskegan, Ml), or Fisher Scientific (Pittsburgh, PA).
In some instances, purity was assessed by thin layer chromatography (TLC) using glass or plastic backed silica gel plates, such as, for example, Baker-Flex Silica Gel 1 B2-F flexible sheets. TLC results were readily detected visually under ultraviolet light, or by employing well known iodine vapor and other various staining techniques. Mass spectrometric analysis was performed on LCMS instruments: Waters
System (Acuity UPLC and a Micromass ZQ mass spectrometer; Column: Acuity HSS C18 1.8-micron, 2.1 x 50 mm; gradient: 5-95 % acetonitrile in water with 0.05 % TFA over a 1.8 min period ; flow rate 1.2 mL/min; molecular weight range 200-1500; cone Voltage 20 V; column temperature 50 °C). All masses were reported as those of the protonated parent ions.
Specific Optical Rotation
The specific optical rotation was measured on an Autopol IV Automatic
Polarimeter (Rudolph Research Analytical) with a 100-mm path-length cylindrical glass cell at 20°C temperature. The wavelength of the light used was 589 nanometer (the sodium D line). Optical rotation of the same cell filled with solvent was subtracted as blank. The final result was the average of two measurements, each over 10 seconds. The 10 mg/mL sample solution was prepared using MeOH as solvent.
GCMS analysis is performed on a Hewlett Packard instrument (HP6890 Series gas chromatograph with a Mass Selective Detector 5973; injector volume: 1 L; initial column temperature: 50 °C; final column temperature: 250 °C; ramp time: 20 minutes; gas flow rate: 1 mL/min; column: 5 % phenyl methyl siloxane, Model No. HP 190915-443, dimensions: 30.0 m x 25 m x 0.25 m).
Nuclear magnetic resonance (NMR) analysis was performed on some of the compounds with a Varian 300 MHz NMR (Palo Alto, CA) or Varian 400 MHz MR NMR (Palo Alto, CA). The spectral reference was either TMS or the known chemical shift of the solvent. Some compound samples were run at elevated temperatures (e.g., 75 oC) to promote increased sample solubility. Melting points are determined on a Laboratory Devices Mel-Temp apparatus (Holliston, MA). Preparative separations are carried out using a Combiflash Rf system
(Teledyne Isco, Lincoln, NE) with RediSep silica gel cartridges (Teledyne Isco, Lincoln, NE) or SiliaSep silica gel cartridges (Silicycle Inc., Quebec City, Canada) or by flash column chromatography using silica gel (230-400 mesh) packing material, or by HPLC using a Waters 2767 Sample Manager, C-18 reversed phase column, 30X50 mm, flow 75 mL/min. Typical solvents employed for the Combiflash Rf system and flash column chromatography are dichloromethane, methanol, ethyl acetate, hexane, heptane, acetone, aqueous ammonia (or ammonium hydroxide), and triethyl amine. Typical solvents employed for the reverse phase HPLC are varying concentrations of acetonitrile and water with 0.1 % trifluoroacetic acid. The following abbreviations have the following meanings. If not specifically defined, abbreviations will have their generally accepted meanings.
Abbreviations
ACN: Acetonitrile
BINAP: 2,2'-bis(diphenylphosphino)-1 , 1'-binapthyl
BOC-anhydride: di-tert-butyl dicarbonate
bp: boiling point
d: days
DAST: Diethylaminosulfur trifluoride
DBU: 1 ,8-Diazabicyclo[5.4.0]undec-7-ene DCM: Dichloromethane
DIEA: diisopropylethylamine
DIPEA: N,N-diisopropylethylamine
DMAP: 4-Dimethylaminopyridine
DME: 1 ,2-dimethoxyethane
DMF: N,N-dimethylformamide
DMSO: dimethyl sulfoxide
dppf: 1 ,1 '-bis(diphenylphosphino)ferrocene
eq: equivalent
EtOAc: ethyl acetate
EtOH: ethanol
GCMS: gas chromatography-mass spectrometry
HATU: 2-(7-aza-1 H-benzotriazole-1-yl)-1 , 1 ,3,3-tetramethyluronium
hexafluorophosphate
HPLC or hplc: high performance liquid chromatography
hr: hour
hrs: hours
KO-tBu: potassium tert-butoxide
LHMDS: Lithium bis(trimethylsilyl)amide
MCPBA: mefa-chloroperoxybenzoic acid
MeOH: methanol
n.a.: not available
NaH: sodium hydride
NBS: N-bromosuccinimide
NEt3: triethylamine
NMP: N-methyl-2-pyrrolidone
Rt: retention time
THF: tetrahydrofuran
TLC: thin layer chromatography
General Synthetic Methods
Compounds of the present invention can be synthesized by procedures known to one skilled in the art, and the general schemes outlined below, adapted as needed to produce the compounds of Formulas l-VI. Some suitable methods and intermediates for making such biaryl compounds are described in WO 2008/079933. While many of the examples below have different heteroaryl rings from the compounds of the invention, the person of ordinary skill will be able to select suitable starting materials and adapt these methods to synthesize the compounds of the invention.
Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in T. W.
Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.
Furthermore, the compounds disclosed herein may contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer- enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of the embodiments, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.
The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), Emka- Chemce or Sigma (St. Louis, Missouri, USA). Others may be prepared by procedures, or obvious modifications thereof, described in standard reference texts such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wley and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).
Compounds of the present invention can be synthesized by procedures known to one skilled in the art, and the general schemes outlined below. The person of ordinary skill will be able to select suitable starting materials and adapt these methods to synthesize compounds of the invention based on knowledge in the art and the Schemes and examples provided herein.
Figure imgf000056_0001
Figure imgf000056_0002
As shown in Scheme 1 , synthesis can start with a functionalized pyridine I wherein LG is a leaving group such as F, CI, OTf, and the like. X can be a functional group like CI, Br, I or OTf. Compound I can be converted into boronic acid or boronic ester II by:
1) PdCI2(dppf) DCM adduct, potassium acetate, bis(pinacolato)diboron heating from 30 - 120 °C in solvents such as THF, DMF, DME, DMA, toluene and dioxane; and 2) In a solvent such as THF or diethylether, anion halogen exchange by addition of nBuLi or LDA followed by quenching the anion with triisopropyl borate. Upon hydrolysis a boronic acid can be obtained. Suzuki cross-coupling reaction between compound II and pyridine III then gives bi- heteroaryl intermediate IV. The SNAR reaction between IV and an amine NH2(L R1) in the presence of a base in a solvent such as DMF, THF, DMSO, NMP, dioxane with heating (30-130 °C) can give compound V. When R1' or R9'are not identical to R or R9, respectively, further functional manipulation is needed to obtain VI.
Scheme 2
Figure imgf000057_0001
IV
-B(OH)2
NhEOJ 1
SN AR
Figure imgf000057_0002
V VI
As shown in Scheme 2, synthesis can start with a functionalized hyeterocyle I wherein X is Br, I, CI, OTf, and the like. Compound I can be converted into boronic acid or boronic ester II by:
1) PdCI2(dppf) DCM adduct, potassium acetate, bis(pinacolato)diboron heating from 30 - 120 °C in solvents such as THF, DMF, DME, DMA, toluene and dioxane; and 2) In a solvent such as THF or diethylether, anion halogen exchange by addition of nBuLi or LDA followed by quenching the anion with triisopropyl borate. Upon hydrolysis a boronic acid can be obtained.
Suzuki cross-coupling reaction between compound II and pyridine III where X is Br, I, CI or OTF and LG is a CI, F, and the like then gives bi-heteroaryl intermediate IV. The SNAR reaction between IV and an amine NH2(L R1) in the presence of a base in a solvent such as DMF, THF, DMSO, NMP, dioxane with heating (30-130 °C) can give compound V. When R1' or R9'are not identical to R or R9, respectively, further functional manipulation is needed to obtain VI. Scheme 3
Figure imgf000058_0001
As shown in Scheme 3, synthesis can start with a functionalized pyridine I wherein X can be a functional group like CI, Br, I, OTf, and the like. Compound I can be converted into boronic acid or boronic ester II by:
1) PdCI2(dppf) DCM adduct, potassium acetate, bis(pinacolato)diboron heating from 30 - 120 °C in solvents such as THF, DMF, DME, DMA, toluene and dioxane; and 2) In a solvent such as THF or diethylether, anion halogen exchange by addition of nBuLi or LDA followed by quenching the anion with triisopropyl borate. Upon hydrolysis a boronic acid can be obtained. Suzuki cross-coupling reaction between compound II and pyridine III then gives bi- heteroaryl compound V. When R1' or R9'are not identical to R or R9, respectively, further functional manipulation is needed to obtain VI. Scheme 4
Figure imgf000059_0001
BR'2= — B(OH)2 V
Deprotections
Further Functionalization
Figure imgf000059_0002
VI
As shown in Scheme 4, synthesis can start with a functionalized pyridine I wherein X can be a functional group like CI, Br, I, OTf, and the like. Compound I can be converted into boronic acid or boronic ester II by:
1) PdCI2(dppf) DCM adduct, potassium acetate, bis(pinacolato)diboron heating from 30 - 120 °C in solvents such as THF, DMF, DME, DMA, toluene and dioxane; and 2) In a solvent such as THF or diethylether, anion halogen exchange by addition of nBuLi or LDA followed by quenching the anion with triisopropyl borate. Upon hydrolysis a boronic acid can be obtained. Suzuki cross-coupling reaction between compound II and pyridine III then gives bi- heteroaryl compound V. When R1' or R9'are not identical to R or R9, respectively, further functional manipulation is needed to obtain VI. Scheme 5
Figure imgf000060_0001
As shown in Scheme 5, synthesis can start with a functionalized hyeterocyle I wherein X is Br, I, CI, OTf, and the like. Compound I can be converted into boronic acid or boronic ester II by:
1) PdCI2(dppf) DCM adduct, potassium acetate, bis(pinacolato)diboron heating from 30 - 120 °C in solvents such as THF, DMF, DME, DMA, toluene and dioxane; and 2) In a solvent such as THF or diethylether, anion halogen exchange by addition of nBuLi or LDA followed by quenching the anion with triisopropyl borate. Upon hydrolysis a boronic acid can be obtained.
Suzuki cross-coupling reaction between compound II and pyridine III where X is Br, I, CI or OTF and LG is a CI, F, and the like then gives bi-heteroaryl intermediate IV. The SNAR reaction between IV and an alcohol or amine of formula A L2R9 in the presence of a base in a solvent such as DMF, THF, DMSO, NMP, dioxane with heating (30-130 °C) can give compound V. When R1' or R9'are not identical to R or R9, respectively, further functional manipulation is needed to obtain VI.
Synthesis of intermediates
Synthesis of 6-bromo-N-(3-fluorobenzyl) pyridin-2-amine (Intermediate A)
Figure imgf000061_0001
A solution of 2,6-dibromopyridine (7.1 g, 30.0 mmol) in NMP (16 mL) was mixed with a mixture of (3-fluorophenyl)methanamine (4.13 g, 33.0 mmol) and Huenig's Base (5.76 mL, 33.0 mmol). The resulting mixture was stirred under argon at 115-120 °C for about 168 hr. The mixture was then cooled to ambient temperature and diluted with EtOAc (250 mL). The organic layer was separated, washed with saturated aqueous sodium bicarbonate (2x), water (2x), brine (1x), dried over sodium sulfate, filtered, and concentrated in vacuo to yield a crude material. The crude material was purified by column chromatography [Si02, 120 g, EtOAc/hexane = 0/100 to 20/80] providing 6- bromo-N-(3-fluorobenzyl) pyridin-2-amine (7.11 g) as an off-white solid. LCMS (m/z): 281.1/283.1 [M+H]+; Retention time = 1.03 min.
Synthesis of 5'-chloro-2'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-6-amine (Intermediate B)
Figure imgf000061_0002
A mixture of 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine (A, 2.0 g, 7.11 mmol), 5- chloro-2-fluoropyridin-4-ylboronic acid (2.0 g, 1 1.4 mmol), PdCl2(dppf).CH2Cl2 adduct (0.465 g, 0.569 mmol), DME (27 mL) and 2M aqueous Na2C02 (9.25 mL, 18.50 mmol) was stirred at about 100 °C for 3 hr. After cooling to ambient temperature, the mixture was diluted with EtOAc (25 mL) and MeOH (20 mL), filtered, and concentrated in vacuo to yield a crude material. The crude material was purified by column chromatography [silica gel, 120g, EtOAc/hexane = 0/100 to 20/80] providing 5'-chloro-2'-fluoro-N-(3- fluorobenzyl)-2,4'-bipyridin-6-amine (1.26 g) as an off-white solid. LCMS (m/z): 332.2 [M+H]+; Retention time = 0.92 min.
Synthesis of 6-bromo-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine (Intermediate C)
Figure imgf000062_0001
A mixture of 2-bromo-6-fluoropyridine (750 mg, 4.26 mmol) in DMSO (3 mL) was mixed with (tetrahydro-2H-pyran-4-yl)methanamine hydrochloride (775 mg, 5.1 1 mmol) and NEt3 (1.426 mL, 10.23 mmol). The resulting mixture was heated at about 110 °C for 18 hr. The mixture was cooled to ambient temperature and diluted with EtOAc. The organic layer was separated, washed with saturated aqueous sodium bicarbonate solution, water, and brine, dried over sodium sulfate, filtered and concentrated in vacuo to yield a resulting residue. The resulting residue was purified by column
chromatography [Si02, 40 g, EtOAc/heptane = 0/100 to 30/70]. Pure fractions were combined and concentrated in vacuo providing 6-bromo-N-((tetrahydro-2H-pyran-4- yl)methyl)pyridin-2-amine (B1 , 940 mg) as a white solid. LCMS (m/z): 271.0/272.9
[M+H]+; Retention time = 0.81 min.
Synthesis of 5'-chloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6- amine (Intermediate D)
Figure imgf000063_0001
A mixture of 6-bromo-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine (C, 271 mg, 1 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (351 mg, 2.000 mmol), PdCl2(dppf).CH2Cl2 adduct (82 mg, 0.100 mmol) in DME (4.5 ml_) and 2M Na2C03 (318 mg, 3.00 mmol) was heated in a sealed tube at about 103 °C for about 2 hr. The mixture then was cooled to ambient temperature, diluted with EtOAc (-25 ml_) and MeOH (~5 ml_), filtered, and concentrated in vacuo to yield a resulting residue. The resulting residue was purified by column chromatography [Si02, 12 g, EtOAc/heptane = 10/90 to 50/50]. Fractions were combined and concentrated in vacuo providing 5'-chloro-2'- fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-amine (260 mg) as a yellow thick oil. LCMS (m/z): 322.1/323.9 [M+H]+; Retention time = 0.60 min.
Synthesis of 6-bromo-5-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine (E) and 6-bromo-3-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine (Intermediate F)
Figure imgf000063_0002
A solution of 6-bromo-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine (C,
1000 mg, 3.69 mmol) in chloroform (15 ml_) was diluted with 1-chloropyrrolidine-2,5- dione (NCS, 492 mg, 3.69 mmol). The mixture then was heated in a sealed tube at about 33 °C for about 16 hr, followed by heating the reaction mixture for about 24 hr at about 37 °C, and then for an additional 5 days at about 43 °C. The reaction mixture then was cooled to ambient temperature, diluted with 1 N aqueous sodium hydroxide solution and DCM. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [ISCO, Si02, 80g, EtOAc/heptane = 5/95 2 min, 5/95 to 30/70 2-15 min, to 35/65 15-18 min, then 35%]. Fractions were combined and concentrated in vacuo yielding 6-bromo-3-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine (F, 453 mg), and 6-bromo-5-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine (E, -500 mg). (F): LCMS (m/z): 305.0 [M+H]+; Retention time = 1.01 min. (E): LCMS (m/z): 305.0 [M+H]+; Retention time = 0.96 min.
Synthesis of 3,5'-dichloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6- amine (Intermediate G)
Figure imgf000064_0001
A mixture of 6-bromo-5-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine (E, 300 mg, 0.982 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (344 mg, 1.963 mmol), PdCI2(dppf).CH2CI2 adduct (80 mg, 0.098 mmol) in DME (4.5 mL) and 2M aqueous sodium carbonate (4.5 mL, 4.50 mmol) was heated in a sealed tube at about 103 °C for about 16 hr. The reaction mixture was cooled to ambient temperature, diluted with EtOAc (-100 mL) and saturated aqueous sodium carbonate solution. The organic layer was separated, washed with saturated aqueous sodium carbonate solution (2x), dried over sodium sulfate, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [ISCO, Si02, 25g, EtOAc/heptane = 0/100 to 25/75]. Fractions were combined and concentrated in vacuo providing 3,5'-dichloro-2'- fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-amine (140 mg) as a light brown liquid. LCMS (m/z): 356.1 [M+H]+; Retention time = 0.96 min. Synthesis of 5,5'-dichloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6- amine (Intermediate H)
Figure imgf000065_0001
A mixture of 6-bromo-3-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine (F, 200 mg, 0.654 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (230 mg, 1.309 mmol), PdCI2(dppf).CH2CI2 adduct (53.4 mg, 0.065 mmol) in DME (3 mL) and 2M aqueous sodium carbonate (3 mL, 6.00 mmol) was heated in a sealed tube at about 103 °C for 16 hr. The reaction mixture was cooled to ambient temperature, diluted with EtOAc (-100 mL) and saturated aqueous sodium bicarbonate solution. The organic layer was separated, washed with saturated aqueous sodium bicarbonate solution (2x), dried over sodium sulfate, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [ISCO, Si02, 25 g, EtOAc/heptane = 0/100 to 30/70].
Fractions were combined and concentrated in vacuo providing 5,5'-dichloro-2'-fluoro-N- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-amine (130 mg) as a nearly colorless liquid. LCMS (m/z): 356.1 [M+H]+; Retention time = 1.10 min.
Synthesis of 5'-chloro-2', 5-difluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6- amine Intermediate I)
Figure imgf000065_0002
Step 1. Preparation of 3,6-difluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine
A mixture of 2,3,6-trifluoropyridine (3 g, 22.54 mmol), (tetrahydro-2H-pyran-4- yl)methanamine (3.89 g, 33.8 mmol) and triethylamine (7.86 mL, 56.4 mmol) in NMP (60 mL) was heated at about 70 °C for about 1 hr. The reaction mixture was cooled to ambient temperature, diluted with EtOAc (-100 mL), brine (-50 mL) and water (-50 mL). The separated organic layer was washed with brine (1x), 0.3N aqueous HCI (2x), saturated aqueous NaHC03 solution (1x), brine (1x), dried over Na2S04, filtered off and concentrated in vacuo providing crude 3,6-difluoro-N-((tetrahydro-2H-pyran-4- yl)methyl)pyridin-2-amine, which was directly used in the next reaction without further purification. Yield: 3.5 g. LCMS (m/z): 229.1 [M+H]+; Retention time = 0.79 min. Step 2. Preparation of 3-fluoro-6-methoxy-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine
To a solution of 3,6-difluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine (5 g, 21.91 mmol) in MeOH (35 mL) was added sodium methoxide (25 wt.% in MeOH, 15.03 mL, 65.7 mmol). The resulting mixture was heated in a steel bomb at about 135 °C for -18 hr. The mixture then was cooled to ambient temperature and concentrated in vacuo. The resulting residue was taken up in water (-250 mL) yielding a precipitate, which was collected by filtration, and then washed with water. The solid then was dissolved in toluene (10 mL)/DCM (10 mL), decanted from the dark brownish film and concentrated in vacuo. The resulting residue was dried in high vacuo providing crude 3- fluoro-6-methoxy-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine as a nearly colorless oil, which was directly used in the next reaction without further purification. Yield: 4.96 g. LCMS (m/z): 241.1 [M+H]+; Retention time = 0.87 min.
Step 3. Preparation of 5-fluoro-6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyridin-2-ol
To a solution of 3-fluoro-6-methoxy-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine
(4.6 g, 19.14 mmol) in acetonitrile (50 mL) was added sodium iodide (20.09 g, 134 mmol) and TMS-chloride (17.13 mL, 134 mmol). The resulting mixture was stirred at about 95 °C for 20 hr. The reaction mixture was cooled to ambient temperature and then diluted with EtOAc (80 mL) and water (40 mL). The diluted mixture was stirred vigorously for about 30 min. The organic layer was separated and washed with 0.1 N aqueous HCI solution. The combined aqueous layers were carefully neutralized (pH -7) with solid NaHCOs solution and extracted with EtOAc (1x 100 mL) and DCM (2x 50mL). The combined organic layers were washed with saturated aqueous NaHC03 solution and brine, dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 80 g, EtOAc/heptane = 10/90 for 2 min, EtOAc/heptane = 10/90 to 100/0 over 23 min, then EtOAc/heptane = 100/0] providing 5- fluoro-6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyridin-2-ol as a highly viscous oil which turned to purple upon standing at room temperature. Yield: 780 mg. LCMS (m/z): 227.1 [M+H]+; Retention time = 0.42 min. Step 4. Preparation of 5-fluoro-6-((tetrahydro-2H-pyran-4-yl)rnethyl)arninopyridin-2-yl trifluoromethanesulfonate
A solution of 5-fluoro-6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyridin-2-ol (500 mg, 2.210 mmol) and triethylamine (0.462 ml_, 3.31 mmol) in DCM (20 ml_) was gradually diluted at about 0°C with trifluoromethanesulfonic anhydride (1.120 ml_, 6.63 mmol). The resulting mixture was stirred for about 2 hr at 0 °C and carefully mixed with ice-cooled saturated aqueous NaHC03 solution. The aqueous layer was separated, and extracted with DCM (2x). The combined organic layers were dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column
chromatography [Si02, 40 g, 30 min, EtOAc/heptane = 5/95 to 40/60] providing 5-fluoro- 6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyridin-2-yl trifluoromethanesulfonate as a colorless oil. Yield: 743 mg. LCMS (m/z): 359.0 [M+H]+; Retention time = 1.02 min. Step 5. Preparation of 5'-chloro-2',5-difluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine
A mixture of 5-fluoro-6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyridin-2-yl trifluoromethanesulfonate (712 mg, 1.987 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (697 mg, 3.97 mmol), PdCl2(dppf).CH2Cl2 adduct (162 mg, 0.199 mmol) in DME (8 ml_) and 2 M aqueous Na2C03 solution (2.6 ml_, 1.987 mmol) in a sealed tube was heated at 95 °C for 3 hr. The mixture was allowed to cool to ambient temperature and was diluted with EtOAc (-100 ml_) and saturated aqueous NaHC03 solution. The separated organic layer was washed with saturated aqueous NaHC03 (2x), dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 40 g, EtOAc/heptane = 0/100 to 25/75 over 20 min] providing 5'-chloro-2',5-difluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6- amine as a white solid. Yield: 570 mg. LCMS (m/z): 340.1 [M+H]+; Retention time = 0.99 min. Synthesis of (R/S)-5'-chloro-N-((2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)-2'-fluoro- '-bipyridin-6-amine (Intermediate J)
J
Figure imgf000067_0001
Step 1. Preparation of te/f-butyl 6-bromopyridin-2-ylcarbamate
To a solution of 6-bromopyridin-2-amine (3 g, 17.34 mmol), triethylamine (3.14 mL, 22.54 mmol) and DMAP (0.424 g, 3.47 mmol) in DCM (24 mL) was added slowly a solution of BOC-anhydride (4.83 mL, 20.81 mmol) in DCM (6 mL). The reaction mixture was stirred at ambient temperature for -24 hr. The mixture was diluted with water, brine and EtOAc. The separated aqueous layer was extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated in vacuo. The resulting residue was purified by column chromatography providing te/f-butyl 6-bromopyridin-2- ylcarbamate as a white solid. Yield: 1.67 g. LCMS (m/z): 274.9 [M+H]+; Retention time = 0.95 min.
Step 2: Preparation of (R/S)-(2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl 4- methylbenzenesulfonate
To a solution of (2,2-dimethyltetrahydro-2H-pyran-4-yl)methanol (1 g, 6.93 mmol) in DCM (5 mL) and pyridine (5 mL, 61.8 mmol) was added para-toluenesulfonyl chloride (1.586 g, 8.32 mmol) and DMAP (0.042 g, 0.347 mmol). The mixture was stirred for 18 hr at ambient temperature. The reaction mixture was concentrated in vacuo and the resulting residue was diluted with water and DCM. The separated organic layer was washed with 0.2N aqueous HCI (1x), 1 N aqueous HCI (2x), brine, dried over sodium sulfate, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 40 g, EtOAc/hexane = 0/100 to 50/50; 25 min] providing (R/S)-(2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl 4-methylbenzenesulfonate as a colorless oil. Yield: 2.05 g. LCMS (m/z): 299.1 [M+H]+; Retention time = 0.96 min.
Step 3: Preparation of (R/S)-tert-butyl 6-bromopyridin-2-yl((2,2-dimethyltetrahydro-2H- pyran-4-yl)methyl)carbamate To a mixture of te/f-butyl 6-bromopyridin-2-ylcarbamate (686 mg, 2.51 mmol),
K2C03 (347 mg, 2.51 mmol), (2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl 4- methylbenzenesulfonate (750 mg, 2.51 mmol) in DMF (10 mL) was added carefully NaH (60 wt.%, 141 mg, 3.52 mmol) in portions [Caution: gas development!]. The resulting mixture was stirred at about 45 °C for 4 hr. The mixture was warmed to ambient temperature and was diluted with EtOAc (-50 mL) and saturated aqueous NaHC03. The organic layer was separated, washed with saturated aqueous NaHC03 solution (1x), dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 40 g, 25 min, EtOAc/heptane = 0/100 to 25/75 over 25 min] providing (R/S)-tert-butyl 6-bromopyridin-2-yl((2,2-dimethyltetrahydro-2H- pyran-4-yl)methyl)carbamate as highly viscous, colorless oil. Yield: 723 mg. LCMS (m/z): 344.9 {loss of tert Bu-group}/(399.0).[M+H]+; Retention time = 1.22 min.
Step 4: Preparation of (R/S)-tert-butyl 5'-chloro-2'-fluoro-2,4'-bipyridin-6-yl((2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl)carbamate A mixture of tert-butyl 6-bromopyridin-2-yl((2,2-dimethyltetrahydro-2H-pyran-4- yl)methyl)carbamate (710 mg, 1.778 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid, PdCl2(dppf).CH2Cl2 adduct (145 mg, 0.178 mmol) in DME (7 mL) and 2M aqueous Na2C03 solution (2.3 mL, 1.778 mmol) was heated in a sealed tube at about 98 °C for 2 hr. The mixture was cooled to ambient temperature and diluted with EtOAc (-100 mL) and saturated aqueous NaHC03 solution. The separated organic layer was washed with saturated aqueous NaHC03 (2x), dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 40 g, 25 min, EtOAc/heptane = 0/100 to 25/75 over 25 min] providing (R/S)-tert-butyl 5'-chloro-2'- fluoro-2,4'-bipyridin-6-yl((2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)carbamate as a highly viscous, colorless oil. Yield: 605 mg. LCMS (m/z): 394.1 {loss of tert Bu- group}/450.2 [M+H]+; Retention time = 1.24 min.
Step 5. Preparation of (R/S)-5'-chloro-N-((2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)- 2'-fluoro-2,4'-bipyridin-6-amine
To a solution of tert-butyl 5'-chloro-2'-fluoro-2,4'-bipyridin-6-yl((2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl)carbamate (950 mg, 2.11 1 mmol) in methanol (5 mL) was added 4M HCI/dioxane (15 mL, 494 mmol). The resulting mixture was stirred for -45 min at ambient temperature. The mixture then was concentrated in vacuo and the resulting residue was dissolved in EtOAc (-50 mL) and saturated aqueous NaHC03 solution (-50 mL). The separated organic layer was washed with saturated aqueous NaHC03 solution (1x), brine (1x), dried over Na2S04, filtered off and concentrated in vacuo providing crude (R/S)-5'-chloro-N-((2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)- 2'-fluoro-2,4'-bipyridin-6-amine as a colorless oil, which was directly used in the next reaction without further purification. Yield: 740 mg. LCMS (m/z): 350.1 [M+H]+;
Retention time = 0.69 min. Synthesis of 5'-chloro-2',3,6-trifluoro-2,4'-bipyridine (Intermediate K)
Figure imgf000070_0001
Step 1. Preparation of 3, 6-difluoro-2-methoxypyridine
2,3,6-Trifluoropyridine (17.91 ml, 188 mmol) was dissolved in anhydrous MeOH (300 ml) and the resulting mixture was placed under argon. This mixture then was treated with a 25wt% methanolic solution of sodium methoxide (43.0 ml, 188 mmol). The resulting mixture was then heated at about 65° C for 2 hr. The reaction mixture was cooled to ambient temperature, and concentrated in vacuo to yield a residue which then was mixed with brine (200 ml_), and extracted with Et20 (3 x 200 ml). The combined extracts were dried (Na2S04), filtered, and concentrated in vacuo to give 21.5 g (79% yield) of crude 3,6-difluoro-2-methoxypyridine as a white solid which was carried on to the next step without purification.
Step 2. Preparation of 3,6-difluoro-2-hydroxypyridine To 3,6-difluoro-2-methoxypyridine (21.5 g, 148 mmol) in acetonitrile (250 ml) was added sodium iodide (66.6 g, 445 mmol) and chlorotnmethylsilane (56.8 ml, 445 mmol). The resulting mixture was heated at 80-85 °C for 2.5 hr. The mixture was cooled to ambient temperature and diluted with EtOAc (300 ml_) and water (300 ml_) and vigorously stirred for another hr. The layers were separated, and the aqueous phase was extracted with additional ethyl acetate (200 ml_). The combined organic layers were washed sequentially with 0.6 N aqueous HCI (250 ml_) and brine (250 ml_) and concentrated in vacuo to yield a slurry. The slurry was filtered and rinsed three times with cold acetonitrile to yield 10.8 g of desired product as a white solid. The filtrate was concentrated and purified by flash chromatography over silica gel (heptanes:ethyl acetate gradient) to give an additional 4.2 g (77% yield combined) of 3,6-difluoro-2- hydroxypyridine as a white solid, LCMS (m/z): 132.0 [M+H]+; retention time = 0.47 min. Step 3. Preparation of 3,6-difluoropyridin-2-yl trifluoromethanesulfonate
An ice water bath-cooled solution of 3,6-difluoro-2-hydroxypyridine (10.75 g, 82 mmol) and triethylamine (22.86 ml, 164 mmol) in DCM (550 ml) was mixed with a solution of trifluoromethanesulfonic anhydride (16.63 ml, 98 mmol) in DCM (100 ml) over 20 min. The resulting mixture then was stirred for 2 hr at 0 °C, with the progress of the reaction followed by TLC (2: 1 heptanes:ethyl acetate). The reaction mixture was quenched with saturated aqueous NaHC03 solution (200 ml_). The separated aqueous layer was extracted with DCM (2x). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography over silica gel (EtOAc/heptane gradient) to give 16.3 g (76% yield) of 3,6-difluoropyridin-2-yl trifluoromethanesulfonate as a yellow oil.
Step 4. Preparation of 5'-chloro-2',3,6-trifluoro-2,4'-bipyridine
A mixture of 3,6-difluoropyridin-2-yl trifluoromethanesulfonate (3.50 g, 13.30 mmol) and 5-chloro-2-fluoropyridine-4-boronic acid (3.27 g, 18.62 mmol) in THF (27 ml) was degassed by bubbling Argon gas for 10 min. Aqueous sodium carbonate (13.30 ml, 26.6 mmol) and PdCl2(dppf).CH2Cl2 adduct (0.652 g, 0.798 mmol)were added, and the mixture was degassed for an additional 5 min. The resulting reaction mixture was stirred at about 100 °C for 2 hr in a sealed vessel. The reaction mixture was cooled to ambient temperature, diluted with EtOAc and water. The separated organic layer was dried over Na2S04, filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography over silica gel (heptanes/ethyl acetate gradient) to yield 2.78 g (85% yield) of 5'-chloro-2',3,6-trifluoro-2,4'-bipyridine as a crystalline solid. LCMS (m/z): 244.9 [M+H]+; retention time = 0.86 min.
Synthesis of 5'-chloro-N-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)methyl)-2'- fluoro-2,4'-bipyridin-6-amine (Intermediate L)
Figure imgf000071_0001
Step 1. Preparation of (2R,6S)-2,6-dimethyldihydro-2H-pyran-4(3H)-one
A solution of 2,6-dimethyl-4H-pyran-4-one (2g, 16.1 mmol) in 20ml ethanol was stirred over 10% Pd/C (0.2g) under hydrogen (15 psi) for 16 hours at ambient temperature. TLC showed two spots; one was desired product and second one was side product in a 1 : 1 ratio. GCMS M+ 128 for product, and M+ 130 for side product.
Suspension was filtered off, and the filtrate was concentrated to remove solvent to give 2.3g crude product which contained -30% of the side product. The resulting oily residue was treated with 2.3g Dess-Martin periodinane in 15ml DCM at ambient temperature for 16 hours. GCMS showed oxidation was complete, desired product formation was confirmed by GCMS at M+ 128. ~3ml NaS2C03 was added to the suspension and the resulting mixture was stirred for 1 hour at ambient temperature, then 20ml saturated sodium bicarbonate solution was added to, and new mixture was stirred for another hour. The organic phase was separated, washed with water, brine, dried and filtered through celite. The filtrate was concentrated and resulting residue was purified by ISCO eluting with 10% ethyl acetate in heptane to yield 600 mg of the desired product. GCMS: M=128. HNMR: 1.5ppm (6H), 2.3ppm (4H), 3.75ppm (2H).
Step 2. Preparation of (2R,6S,E)-4-(methoxymethylene)-2,6-dimethyltetrahydro-2H-pyran
To a suspension of (methoxymethyl)triphenyl phosphine chloride (1.5g, 4.45 mmol) in 8ml THF at -10°C, was added dropwise 4.45ml 1.0M/THF solution of sodium bis(trimethylsilyl) amide. The resulting reaction mixture was stirred for 1 hour, followed by addition of a solution of (2R,6S)-2,6-dimethyldihydro-2H-pyran-4(3H)-one (380mg, 2.96 mmol) in 2ml THF. The resulting mixture was warmed to ambient temperature and stirred for an additional 3 hours. GCMS showed formation of desired product at M+156, as major component. The reaction mixture was quenched with 15ml water, and was extracted with diethyl ether (2x30ml). The combined organic phase was washed with brine, dried and concentrated. The resulting residue was purified by ISCO eluting with 10% ethyl acetate in heptane to yield 240 mg of the desired product as a colorless oil, GCMS showed M=156. HNMR: 5.9ppm (1 H), 3.45ppm (3H), 3.25ppm (2H), 2.45ppm (1 H), 1.85ppm (1 H), 1.6ppm (1 H), 1.38ppm (1 H), 1.1 ppm (6H).
Step 3. Preparation of (2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-carbaldehyde A mixture of (2R,6S,E)-4-(methoxymethylene)-2,6-dimethyltetrahydro-2H-pyran (240mg, 1.53 mmol) and 88% formic acid (1.5ml, 34.4 mmol) in water was heated in an oil bath under Argon to about 90°C for 1 hour. GCMS indicated that reaction was complete under the condition. The reaction mixture was cooled in an ice bath, neutralized with 6N NaOH to a pH=6, and extracted with diethyl ether. The organic phase were dried and concentrated to dryness to yield 120 mg of the desired product as yellow colored oil. GCMS M=142. HNMR showed 9.51 ppm (s, 1 H, CHO).
Step 4. Preparation of 6-bromo-N-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl) methyl) pyridin-2-amine
The mixture of (2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-carbaldehyde (120mg, 0.84 mmol) and 6-bromo-2-aminopyridine (219mg, 1.26 mmol) in 5ml DCM was stirred at ambient temperature for about 40 min. To this solution was added sodium triacetoxy borohydride (268mg, 1.26 mmol), followed by the addition of 0.01 ml acetic acid. The resulting solution was stirred at ambient temperature for about 40 hours. The reaction mixture was concentrated in vacuo to yield a residue was diluted with ethyl acetate, washed with sodium bicarbonate, brine, dried, concentrated. The resulting residue was purified by ISCO eluting with 10% to 20% ethyl acetate in heptane to yield 1 10 mg of the desired product as colorless oil. LCMS (m/z): 299/301 (MH+), retention time = 1.01 min.
Step 5. Preparation of 5'-chloro-N-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4- yl)methyl)-2'-fluoro-2,4'-bipyridin-6-amine A mixture of 6-bromo-N-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)methyl) pyridin-2-amine (1 10 mg, 0.36 mmol), 5-chloro-2-fluoro-pyridine-4-boronic acid (193 mg, 1.10 mmol), 0.55 ml 2.0M saturated sodium carbonate aqueous solution in 2 ml DME was purged with Argon for 3 min, PdCl2(dppf)CH2Cl2 (30 mg, 0.037 mmol) was added to this purged . The resulting mixture was heated at about 95°C in an oil bath for 3.5 hours. Formation of the desired product was confirmed by LCMS: MH+ 350, 0.70 min. The preceding reaction mixture was diluted with ethyl acetate, washed with water, brine, dried over sodium sulfate and concentrated. The resulting residue was purified by ISCO eluting with 10% ethyl acetate in heptane to give 90mg desired product as colorless oil. LCMS (m/z): 350 (MH+), retention time = 0.70 min. Synthesis of 5'-chloro-N6-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2',6-diamine (Intermediate M)
Figure imgf000074_0001
A mixture of 5'-chloro-N-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)methyl)- 2'-fluoro-2,4'-bipyridin-6-amine (60mg, 0.17 mmol), and 3.0ml 28% ammonium hydroxide aqueous solution was heated at about 130°C in an oil bath for 17 hours. Formation of compound M was Reaction confirmed by LCMS/LC data. The reaction mixture was diluted with ethyl acetate, washed with water, saturated sodium bicarbonate, and brine, dried over sodium sulfate and concentrated to yield 50 mg of the desired product. LCMS (m/z): 347 (MH+), retention time = 0.53 min.
Synthesis of 3-bromo-5'-chloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine (Intermediate N)
Figure imgf000074_0002
A mixture of 5'-chloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine (516 mg, 1.60 mmol) and N-bromosuccinimide (286 mg, 1.60 mmol) in acetonitrile (12 ml_) was stirred at 90° C for 3 hr in a sealed vessel. Volatiles were removed under reduced pressure. The resulting residue was dissolved in ethyl acetate and washed sequentially with saturated aqueous sodium bicarbonate and brine. The organic phase was dried (Na2S04), filtered, and concentrated. The crude material was purified by column chromatography over silica gel (heptanes/ethyl acetate gradient) to yield 608 mg of the desired product. LCMS (m/z): 402.0 [M+H]+; Retention time = 1.03 min.
Synthesis of intermediate (4-methoxytetrahydro-2H-pyran-4-yl) methyl 4- methylbenzen (Intermediate O)
Figure imgf000075_0001
Step 1. Synthesis of 1 ,6-dioxaspiro[2.5]octane
To a clear solution of trimethylsulfonium iodide (3.27 g, 16 mmol) in 20 ml of DMSO was added dihydro-2H-pyran-4(3H)-one (1.0g, 10 mmol) with stirring. To this mixture, under nitrogen, was then slowly added KOtBu (1.68g, 15 mmol) in 15 ml of DMSO. The resulting solution was then stirred overnight at ambient temperature.
Water (50ml) was slowly added to the mixture, and the resulting mixture was extracted with diethyl ether (3x20ml). The ether layers were combined, dried and concentrated in vacuo to yield 650 mg of the crude product. 1 H NMR (300 MHz, CHLOROFORM-d) . ppm 1.44 - 1.62 (m, 2 H) 1.76 - 1.98 (m, 2 H) 2.70 (s, 2 H) 3.70 -3.98 (m, 4 H).
Step 2. Synthesis of (4-methoxytetrahydro-2H-pyran-4-yl) methanol
To a solution of 1 ,6-dioxaspiro[2.5]octane (600 mg, 5.26mmol) in methanol (10 ml) at 0 °C (ice-water) under nitrogen was added camphorsulfonic acid (50 mg, 0.21 mmol) and the resulting mixture was stirred at about 0 °C for 2 hours. The mixture was concentrated in vacuo and the crude residue was used in the next step without purification. The desired product was obtained as a light yellow oil (707 mg).
Step 3. To a solution of (4-methoxytetrahydro-2H-pyran-4-yl) methanol (300 mg, 2.05 mmol) in pyridine (4 ml) at ambient temperature was added toluenesulfonic chloride (430 mg, 2.25 mmol) and the resulting mixture was stirred overnight at about 25 °C. The stirred mixture was concentrated and the solid residue was dissolved in DCM and purified by silica gel chromatography using a 12 g column, eluting with 0-30% ethyl acetate in heptane to yield the desired compound "O" as a light yellow solid (360 mg).1 H NMR (300 MHz, CHLOROFORM-d) . ppm 1.45 - 1.63 (m, 2 H) 1.61 - 1.79 (m, 2 H) 2.46 (s, 3 H), 3.16 (s, 3 H) 3.53 - 3.75 (m, 4 H) 3.93 (s, 2 H), 7.36 (d, J = 8.20 Hz, 2 H) 7.81 (d, J = 8.20 Hz, 2 H).
Synthesis of tert-butyl 6-bromo-5-chloropyridin-2-yl((4-methoxytetrahydro-2H-pyran-4- yl)methyl)carbamate (Intermediate P)
Figure imgf000076_0001
To a stirred solution of tert-butyl 6-bromo-5-chloropyridin-2-ylcarbamate (140 mg, 0.455 mmol) in DMF (2 ml) under nitrogen was added NaH (60%, 30 mg, 0.774 mmol). The resulting mixture was stirred at ambient temperature for one hour. A solution of (4- methoxytetrahydro-2H-pyran-4-yl)methyl 4-methylbenzenesulfonate (intermediate O, 164 mg, 0.546 mmol) in DMF ( 1.5ml) was then added to the preceding mixture. The resulting mixture was then stirred overnight at about 85 °C. The stirred mixture was diluted with 30 ml of ethyl acetate, washed with water (20 ml x3) and dried. After concentration the resulting residue was purified by silica gel chromatography using a 12g column, eluting with 5-20% ethyl acetate in hexane to yield the desired compound "P" as a viscous oil (92 mg), which solidified upon standing overnight. LCMS (m/z): 437.0
[M+H]+; Retention time = 1.158 min. Synthesis of (l cyclohexyl)methyl 4-methylbenzenesulfonate (Intermediate Q)
Figure imgf000076_0002
This compound was synthesized from cyclohexanone following the procedure described for (4-methoxytetrahydro-2H-pyran-4-yl)methyl 4-methylbenzenesulfonate (Intermediate O).
LCMS (m/z): 299.2 [M+H]+; Retention time = 1.055 min.
Synthesis of 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile (Intermediate R)
Figure imgf000077_0001
Step 1. Synthesis of dihydro-2H-pyran-4,4(3H)-dicarbonitrile
A mixture of malononitrile (0.991 g, 15 mmol), 1-bromo-2-(2-bromoethoxy)ethane (3.83 g, 16.50 mmol) and DBU (4.97 ml, 33.0 mmol) in DMF (6 ml) was heated at about 85 °C for 3 hours, and then cooled to ambient temperature. The mixture was concentrated in vacuo, the resulting residue was diluted with ethyl acetate, washed three times with water and dried overnight under high vacuum to yield the desired product as a light brown solid (1.65 g). GC-MS: 136 [M]; Retention time = 5.76 min. H NMR (300 MHz, CHLOROFORM-d) . ppm 2.14-2.32 (m, 4 H) 3.77-3.96 (m, 4 H).
Step 2. A mixture of dihydro-2H-pyran-4,4(3H)-dicarbonitrile (450 mg, 3.31 mmol) and Sodium borohydride (375 mg, 9.92 mmol) in EtOH (15 ml) was stirred at ambient temperature for about 4 hours. The mixture was concentrated and the resulting residue was diluted with ethyl acetate, washed with water and dried. Concentration in vacuo afforded 388 mg of the crude product which was used directly in the next step. LCMS (m/z): 141.0 [M+H]+; Retention time = 0.18 min.
Synthesis of 4-((6-bromopyridin-2-yl-amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (Intermediate S)
Figure imgf000077_0002
To 2-bromo-6-fluoropyridine (400 mg, 2.273 mmol) in DMSO (4 ml) at ambient temperature was sequentially added 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile (Intermediate R, 382 mg, 2.73 mmol) and triethylamine (0.792 ml, 5.68 mmol). The resulting light brown mixture was heated at 110 °C in a sealed glass bomb for 18 hours. The reaction mixture then was cooled to ambient temperature, reaction mixture diluted with EtOAc, washed with saturated NaHC03 solution and brine, dried over sodium sulfate and concentrated in vacuo to yield 890 mg of a light brown liquid. The crude material was purified by silica gel chromatography using a 12g column, eluting with 5%- 20% ethyl acetate in hexane to afford 410 mg (60.9 %) of the desired product "S". LCMS (m/z): 297.9 [M+H]+; Retention time = 0.823 min. H NMR (400 MHz, CHLOROFORM- cO . ppm: 1.67-1.96 (m, 4H), 3.59-3.78 (m, 4H), 3.98 (m, 2H),4.82 (t, J=6.65Hz,1 H), 6.39 (d, J=8.22,1 H), 6.72-6.84 ( m, 1 H), 7.16-7.33 (m, 1 H). Synthesis of 5'-chloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-5-(trifluoromethyl)- 2,4'-bipyridin-6-amine (Intermediate T) and 5'-chloro-2'-fluoro-N-((tetrahydro-2H-pyran-4- yl)methyl)-3-(trifluoromethyl)-2,4'-bipyridin-6-amine (Intermediate U).
Figure imgf000078_0001
Step 1. Synthesis of 6-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-5- (trifluoromethyl)pyridin-2-amine and 6-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-3- (trifluoromethyl)pyrid -2-amine
Figure imgf000078_0002
To a solution of 2,6-dichloro-3-(trifluoromethyl)pyridine (320 mg, 1.482 mmol) in DMSO (1.5ml) at ambient temperature was added (tetrahydro-2H-pyran-4- yl)methanamine (188 mg, 1.630 mmol) and triethylamine (0.207 ml, 1.482 mmol). The resulting light brown mixture was heated at about 120 °C in a sealed glass bomb for about 18 hours. The reaction mixture was cooled to ambient temperature, diluted with EtOAc (20ml_), washed with saturated NaHC03 solution and brine, dried over sodium sulfate and concentrated in vacuo to yield 502 mg of a light brown crude liquid, which was purified by column chromatography ( 5 to 50% ethyl acetate in heptane)to yield the desired products.
6-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-5-(trifluoromethyl)pyridin-2-amine: 340 mg, 78 %: LCMS (m/z): 295.2 [M+H]+; Retention time = 0.971 min; and 6-chloro-N- ((tetrahydro-2H-pyran-4-yl)methyl)-3-(trifluoromethyl)pyridin-2-amine: 80 mg, 18 %. LCMS (m/z): 295.1 [M+H]+; Retention time = 1.033 min.
Step 2a. A mixture of 6-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-3- (trifluoromethyl)pyridin-2-amine(100 mg, 0.339 mmol), 5-chloro-2-fluoropyridin-4- ylboronic acid (89 mg, 0.509 mmol), PdCl2(dppf).CH2Cl2 adduct (27.7 mg, 0.034 mmol), DME (1.5 mL) and 2M aqueous Na2C02 (0.5 mL, 1 mmol) was stirred in a sealed glass vessel at about 100 °C for about 3 hours. After cooling to ambient temperature the mixture was diluted with EtOAc (25 mL) and MeOH (20 mL), filtered and concentrated in vacuo. The resulting crude material was purified by column chromatography [silica gel, 12g, EtOAc/hexane = 5/100 to 50/50] to yield 5'-chloro-2'-fluoro-N-((tetrahydro-2H- pyran-4-yl)methyl)-5-(trifluoromethyl)-2,4'-bipyridin-6-amine (Intermediate T, 102 mg, , 77 % ). LCMS (m/z): 390.2 [M+H]+; Retention time = 1.12 min. Step 2b. Intermediate U was synthesized following the procedure described for5'- chloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-5-(trifluoromethyl)-2,4'-bipyridin-6- amine
LCMS (m/z): 390.2 [M+H]+; Retention time = 1.01 min. Synthesis of 3,5'-dichloro-N-((2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)-2'-fluoro-2,4'- bipyridin-6-amine (Intermediate V)
Figure imgf000079_0001
Step 1. 6-Bromo-2-aminopyridine (15 g, 87 mmol) and TEA (13.3 mL, 95 mmol) were dissolved in 173 mL of DCM. BOC-anhydride (20.8 g, 95 mmol) was then dissolved in 100 mL of DCM and added over 10 min using a syringe pump. The reaction mixture was stirred at ambient temperature for 72 hr. The solvents were evaporated and the resulting residue was purified by silica gel chromatography (heptane: EtOAc 1 :0 to 7:3) to give the product as a colorless solid (23.0 g, 97%). LCMS (m/z): 272.8/274.8 (M+H), retention time = 0.97 min. Step 2. tert-Butyl 6-bromopyridin-2-ylcarbamate (23.0 g, 84 mmol) was mixed with acetonitrile, (CH3CN, 281 mL), and NCS (1 1.24 g, 84 mmol). The reaction mixture was heated at about 85°C for 3 hours, and an additional 5.5 g of NCS was then added. Heating was continued at about 85oC for an additional 3 hours, followed by addition of 5.5 g of NCS. All starting materials were consumed after about 1 hour. Brine (50 mL) was added and acetonitrile was evaporated under vacuum. The residual aqueous solution was extracted three times with EtOAc. All EtOAc layers were combined, dried over Na2S04, filtered through a fritted filter and concentrated under vacuum. The resulting residue was purified on silica gel, eluting with 3% EtOAc in heptane to afford the product as a colorless solid (14.6 g, 56.3%). LCMS (m/z): 306.9/308.9/310.9 (M+H), retention time = 1.14 min.
Step 3. A solution of tert-Butyl 6-bromo-5-chloropyridin-2-ylcarbamate (2.32 g, 7.54 mmol) in DMF (25 mL) was mixed with sodium hydride (60% dispersion in mineral oil, 513 mg, 12.8 mmol), and the resulting mixture reaction mixture was stirred for 30 minutes at ambient temperature. (2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl 4- methylbenzenesulfonate (3.15 g, 10.56 mmol), dissolved in 5 mL DMF, was then added and the resulting mixture was stirred at about 25 °C for 3 hours. The reaction mixture was partitioned between water and EtOAc. The layers were separated and the EtOAc layer was washed twice with water. The EtOAc layer was then dried over sodium sulfate, filtered through a fritted filter and concentrated under vacuum. The resulting residue was purified using silica gel chromatography (0 to 30% EtOAc in heptane) to yield the product as a colorless solid (2.16 g, 66%). LCMS (m/z): 432.9/434.9/436.9 (M+H), retention time = 1.28 min.
Step 4. A mixture of tert-butyl 6-bromo-5-chloropyridin-2-yl((2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl)carbamate (1.86 g, 4.29 mmol), 5-chloro-2- fluoropyridin-4-ylboronic acid (1.50 g, 8.58 mmol), PdCI2(dppf)*DCM adduct (350 mg, 0.429 mmol), DME (15.6 mL) and 2 M aqueous sodium carbonate solution (5.4 mL) were combined in a glass bomb. The bomb was sealed and heated at about 98°C for 2 hours. The reaction mixture was cooled to ambient temperature and then diluted with EtOAc. The diluted mixture was washed three times with saturated aqueous NaHC03 solution, dried over sodium sulfate, filtered through a fritted filter and concentrated under vacuum. Purification was done using silica gel chromatography (15% EtOAc in heptane) to yield the product as a colorless solid (1.5 g, 72%). LCMS (m/z): 484.2/486.1 (M+H), retention time = 1.33 min. Step 5. tert-Butyl 3,5'-dichloro-2'-fluoro-2,4'-bipyridin-6-yl((2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl)carbamate (8 mg, 0.017 mmol), DCM (1 mL) and TFA (0.1 mL, 1.3 mmol) were combined in a 4 mL screw cap vial. The vial was capped and the reaction mixture was stirred at ambient temperature for 1 hour. The solvent was evaporated under vacuum and the residual material was converted to the free base using sodium bicarbonate. (5.8 mg, 91 %). LCMS (m/z): 3484.2/386.1/388.2 (M+H), retention time = 1.07 min. Synthesis of 2,3-difluoropyridin-4-ylboronic acid (Intermediate W)
Figure imgf000081_0001
A mixture of THF and hexanes (6mL, 1 : 1 v:v), and diisopropyl amine (0.681 mL, 4.78 mmol) was cooled to -78 °C. BuLi (2.5 M in hexanes, 2.00 mL, 5.00 mmol) was added to the cooled mixture, followed by addition of 2,3-difluoropyridine after about 15 minutes. The mixture was stirred for 1 hour at -78 °C before being transferred to a 3 mL THF solution of triisopropyl borate (1 .1 1 mL, 4.78 mmol) at -78 °C via a cannula. The resulting solution was stirred at -78 °C for 1 hour, slowly warmed up to ambient temperature and then quenched with 2 M NaOH solution (20 mL). The two layers were separated and the aqueous phase was washed once with ether. The aqueous phase was then acidified with HCI to pH 5 and extracted three times with EtOAc. The organic layers were combined, dried over sodium sulfate and concentrated to yield the product as a light yellow solid, which was used in the next step without purification. LCMS (m/z): 159.9 (M+H), retention time = 0.35 min.
Synthesis of trans-N 1 -(1 ,3-dimethoxypropan-2-yl)cyclohexane-1 ,4-diamine (Intermediate
Figure imgf000081_0002
Step 1 . To NaH (0.366 g, 9.16 mmol) in THF (12 mL) at 0 °C was added 1 ,3- dimethoxy-2-propanol (1 g, 8.32 mmol) in THF (8 mL) solution. The mixture was warmed to ambient temperature and stirred for 0.5 hour. To this was added tosyl chloride (1.587 g, 8.32 mmol) in one portion. The resulting white cloudy mixture then was stirred at ambient temperature for 16 hours. LC/MS showed complete conversion to 1 ,3- dimethoxypropan-2-yl 4-methylbenzenesulfonate. The reaction mixture was poured into water and extracted with EtOAc. The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated in vacuo to yield 2 g of a colorless oil. The crude mixture was purified by Analogix system (silica gel column 80 g, gradient: 0 min, 100%n-heptane; 5-12 min, 20% EtOAc in Heptane; 12-15 min. 30% EtOAc in Heptane and hold until 30 min). The pure fractions were combined and concentrated in vacuo to yield 1.25 g of the tosylate product 1 ,3-dimethoxypropan-2-yl 4- methylbenzenesulfonate as a colorless oil, which solidified upon standing.
Step 2. To the tosylate obtained in Step 1 (0.8g, 2.92 mmol) in DMSO (8 ml) was added 1 ,4-trans-cyclohexane diamine (0.999 g, 8.75 mmol). The resulting brown mixture was heated in a capped vial to about 95 °C, with stirring, for 2 hours. The reaction mixture was poured into 10% HCI in water (10 ml_) at 0 °C (ice cubes in HCI) and extracted with DCM (1x20 ml_). The aqueous (light pink) was basified with 6N NaOH to a pH >12 and extracted with DCM (2x20ml_). The organic extracts were combined, dried with sodium sulfate and concentrated in vacuo to yield compound "X" as a purple liquid. LC/MS showed containing desired product (M+1 =217, Rt=0.32min, no UV absorption at 214nm wavelength). This was used in the next step without further purification.
Synthesis of 4-((5'-chloro-2',5-difluoro-2,4'-bipyridin-6-yl-amino)methyl)tetrahydro-2H- rmediate AA)
Figure imgf000082_0001
Step 1 : Synthesis of 4-((3,6-difluoropyridin-2-yl-amino)methyl)tetrahydro-2H-pyran-4- carbonitrile
Figure imgf000082_0002
To 2,3,6-Trifluoropyridine (0.6g, 4.5 mmol) in DMSO (5 ml) at room temperature was added 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile (Intermediate R, 1.01 g, 7.23 mmol) and triethylamine (1.57 ml, 11.24 mmol) sequentially. The light brown mixture was heated at 105 °C in a sealed glass bomb for 18 hours. After cooled to room temperature the reaction mixture was extracted with EtOAc (40 ml), washed with saturated NaHC03 solution and brine, dried over sodium sulfate and concentrated in vacuo to give a light brown liquid. This crude material was purified by silica gel chromatography using a 12 g column, eluting with 5%-20% ethyl acetate in hexane to afford 550 mg (48.2 % yield) of the desired product. LCMS (m/z): 254.1 [M+H]+;
retention time = 0.743 min. 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.69 - 1.95 (m, 4 H) 3.60 - 3.82 (m, 4 H) 4.00 (ddd, J=12.13, 4.30, 1.96 Hz, 2 H) 5.02 (br. S., 1 H) 6.12 (td, J=5.58, 2.54 Hz, 1 H) 7.19 - 7.33 (m, 1 H).
Step 2: Synthesis of 4-((6-(benzyloxy)-3-fluoropyridin-2-yl-amino)methyl)tetrahydro-2H- pyran-4-carbonitrile
Figure imgf000083_0001
Benzyl alcohol (352 mg, 3.26 mmol) was dissolved in anhydrous DMF (2 ml) and placed under argon. This was then treated with a 60% dispersion in oil of SODIUM HYDRIDE (78.7 mg, 3.26 mmol). This resultant suspension was then stirred at room temperature for 15 min. At this time it was treated with a solution of 4-((3,6- difluoropyridin-2-ylamino)methyl)tetrahydro-2H-pyran-4-carbonitrile (275 mg, 1.09 mmol) dissolved in anhydrous DMF (2 ml). Once the addition was complete the reaction was stirred at 90°C for 5 hours. The reaction was allowed to cool to room temperature. It was then poured into brine (20 ml). This was extracted with EtOAc (3 x 15 ml). The combined extracts were washed with H20 (3 x 10 ml) followed by brine (1 x 10 ml). The organic layer was dried (Na2S04), filtered, and the solvent removed in vacuo to give the crude material which was purified using the ISCO and a 12 g Si02 column. Eluted using 100 hexanes to 30 EtOAc / 70 hexanes over 20 min. 245 mg (66% yield) of the desired product was obtained as a viscous liquid. LCMS (m/z): 342.1 [M+H]+; retention time = 1.017 min.
Step 3: Synthesis of 4-((3-fluoro-6-hydroxypyridin-2-yl-amino)methyl)tetrahydro-2H- pyran-4-carbonitrile
Figure imgf000084_0001
A mixture of 4-((6-(benzyloxy)-3-fluoropyridin-2-ylamino)methyl)tetrahydro-2H- pyran-4-carbonitrile (200 mg, 0.586 mmol), AMMONIUM FORMATE (1 1 1.3 mg, 1.758 mmol) and Pd-C (10%, wet, 25mg) in methanol (4 ml) was stirred at 70 °C for 45 min and cooled. The mixture was then filtered to remove Pd-C and inorganics, the filtrate was then concentrated and dried further via high vacuum to afford 141 mg ( 96% yield) of the crude product as a light pink solid. LCMS (m/z): 252.1 [M+H]+; retention time = 0.540 min.
Step 4: Synthesis 6-((4-cyanotetrahydro-2H-pyran-4-yl) methyl)-amino-5-fluoropyridin-2- yl trifluoromethanesulfonate
Figure imgf000084_0002
To a solution of 5-fluoro-6-((4-cyano-tetrahydro-2H-pyran-4- yl)methyl)aminopyridin-2-ol (141 mg, 0.562 mmol) and TEA (0.782 ml, 5.60 mmol) in DCM (6 ml) was added trifluoromethanesulfonic anhydride (0.142 ml, 0.842 mmol) slowly at 0 °C. The mixture was stirred for 2 hours at 0 °C and one hour at room temperature and poured carefully into ice-cooled saturated aqueous NaHC03 solution. The separated aqueous layer was extracted with DCM (2x10ml). The combined organic layers were dried over Na2S04, filtered off and concentrated in vacuo. The residue was purified by column chromatography [ISCO, Si02, 12g, 15 min, EtOAc/heptane = 5/95 for 2 min, then EtOAc/heptane = 5/95 to 40/60 for 2min-17min]. Pure fractions were combined and concentrated in vacuo to give a colorless oil (200 mg, 0.522 mmol, 93 % yield) as the desired product. LCMS (m/z): 384.0 [M+H]+; Rt = 0.946 min.
Step 5: Synthesis of 4-((5'-chloro-2',5-difluoro-2,4'-bipyridin-6-yl- amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (Intermediate AA)
A mixture of 5-fluoro-6-((4-cyano-tetrahydro-2H-pyran-4-yl)methylamino)pyridin- 2-yl trifluoromethanesulfonate (200 mg, 0.522 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (183.2 mg, 1.044 mmol), PdCl2(dppf)-CH2Cl2 adduct (85.1 mg, 0.104 mmol), and SODIUM CARBONATE (221.6 mg, 2.08 mmol, in 1 ml of water) in DME (3 ml) was de- gassed and heated at 1 10 °C for 20 min in a sealed microwave vial, cooled. The upper layer of mixture was separated, the bottom one was extracted with ethyl acetates, the organic layers were combined and concentrated to afford the crude product, which was purified by ISCO ( 10 to 50% ethyl acetate in heptane, 20 min) to afford 150 mg ( 79% yield) of the desired product was an off-white solid. LCMS (m/z): 365.1 [M+H]+; retention time = 0.929 min.
Synthesis of 4-((5'-chloro-2'-fluoro-2,4'-bipyridin-6-yl-amino)methyl)tetrahydro-2H-pyran- 4-carbonitrile Intermediate AB)
Figure imgf000085_0001
A mixture of 4-((6-bromopyridin-2-yl-amino)methyl)tetrahydro-2H-pyran-4- carbonitrile ( Intermediate S, 410 mg, 1.384 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (362.2mg, 2.07 mmol), PdCI2(dppf).CH2Cl2 adduct (113 mg, 0.14 mmol), DME (5 Ml) and 2 M aqueous Na2C02 (1.75 Ml, 3.5 mmol) was sealed and stirred at 110 °C for 20 min using microwave reactor. After cooling to room temperature the mixture was extracted with EtOAc (35 Ml), filtered and concentrated in vacuo. The crude material was purified by column chromatography [silica gel, 24g, EtOAc/hexane = 5/100 to 50/50] to provide 4-((5'-chloro-2'-fluoro-2,4'-bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4- carbonitrile (360 mg, 75 % yield). LCMS (m/z): 347 [M+H]+; retention time = 0.814 min.
Synthesis of 5'-chloro-2'-fluoro-N-((4-methoxytetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine (Intermediate AC)
Figure imgf000086_0001
Step 1 : Synthesis of tert-butyl 6-bromopyridin-2-yl((4-methoxytetrahydro-2H-pyran-4- yl)methyl) carbamate
Figure imgf000086_0002
To a solution of tert-butyl 6-bromo-pyridin-2-ylcarbamate (136mg, 0.50mmol) in DMF (2ml) under nitrogen was added NaH (60%, 40mg, 1.0 mmol) under stirring. The resultant mixture was stirred at room temperature for one hour. A solution of (4- methoxytetrahydro-2H-pyran-4-yl)methyl 4-methylbenzenesulfonate (Intermediate O, 152 mg, 0.506mmol) in DMF (1.5ml) was then added. The resulting mixture was then stirred at 85 °C for about 18 hours. The mixture was diluted with 30 ml of ethyl acetate, washed with water (20 ml x3) and dried with sodium sulfate. After concentration the residue was purified by silica gel chromatography using a 12g column, eluting with 5-20% ethyl acetate in hexane to give the desired title compound as a viscous oil (92 mg, 46% yield), which solidified upon standing overnight. LCMS (m/z): 403.1 [M+H]+; Rt = 1.026 min.
Step 2: Synthesis of tert-butyl 5'-chloro-2'-fluoro-2,4'-bipyridin-6-yl((4- methoxytetrahydro-2H-pyran-4-yl)methyl)carbamate
Figure imgf000087_0001
A mixture of tert-butyl 6-bromo-pyridin-2-yl((4-methoxytetrahydro-2H-pyran-4- yl)methyl)carbamate (50 mg, 0.125 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (43.7mg, 0.249 mmol), PdCl2(dppf).CH2Cl2 adduct (15.2 mg, 0.019 mmol), DME (1.5 mL) and 2M aqueous Na2C02 (0.25 mL, 0.5 mmol) was sealed and stirred at 100 °C for 3 hours. After cooling to room temperature the mixture was diluted with EtOAc (15 mL), filtered and concentrated in vacuo. The crude material was purified by column chromatography [silica gel, 12g, EtOAc/hexane = 5/100 to 50/50] to provide tert-butyl 5'- chloro-2'-fluoro-2,4'-bipyridin-6-yl((4-methoxytetrahydro-2H-pyran-4-yl)methyl)carbamate (32 mg, 57 % yield). LCMS (m/z): 452.2 [M+H]+; retention time = 1.068 min.
Step 3: Synthesis of 5'-chloro-2'-fluoro-N-((4-methoxytetrahydro-2H-pyran-4-yl)methyl)- 2,4'-bipyridin-6-amine (Intermediate AC)
A solution of tert-butyl 5'-chloro-2'-fluoro-2,4'-bipyridin-6-yl((4-methoxytetrahydro- 2H-pyran-4-yl)methyl)carbamate (32 mg, 0.071 mmol) and TRIFLUOROACETIC ACID (0.982 ml, 12.75 mmol) in DCM (2ml) was stirred at room temperature for 40 min. The mixture was then concentrated to afford 22 mg of the crude material which was used in the next step without purification. LCMS (m/z): 352.2 [M+H]+; Rt = 0.634 min.
Synthesis of (R)-2-methyl-2-(trifluoromethyl)oxirane
(Reference: A. Harada, Y. Fujiwara, T. Katagiri, Tetrahedron: Asymmetry (2008) 1210- 1214.) F3C
To a solution of (R)-2-(trifluoromethyl)oxirane (0.5 g, 4.46 mmol) under argon at -100 °C was added n-BuLi (1.89 mL, 4.91 mmol) and the mixture was stirred at this temperature for 10 min. To the solution was added iodomethane (0.558 mL, 8.92 mmol) and the mixture was stirred at -80 °C for 3 hours. The mixture was allowed to warm to 0 °C and directly usded in the next reaction. Total volumen: -24.8 mL; 0.18 M solution. To 1 mL of this solution was added triethylamine (139 μί, 0.997 mmol). The mixture was stirred for -30 min and the formed precipitate was removed over a syringe filter. The clear solution was directly used.
Synthesis of 2,5-difluoropyridin-4-ylboronic acid
Figure imgf000088_0001
To a solution of diisopropylamine (1.74 mL, 12.20 mmol) in anhydrous tetrahydrofuran (22 mL) under argon at -20 °C was added n-butyllithium (7.66 mL, 1.6M in hexanes) slowly over 10 min. The newly formed LDA was then cooled to -78 °C. A solution of 2,5- difluoropyridine (1.05 mL, 1 1.5 mmol) in anhydrous tetrahydrofuran (3 mL) was added slowly over 30 min and the mixture was stirred at -78 °C for 4 hrs. A solution of triisopropyl borate (5.90 mL, 25.4 mmol) in anhydrous tetrahydrofuran (8.6 mL) was added dropwise. Once the addition was complete the reaction mixturre was warmed to room temperature and stirring was continued for an additional hour. The reaction mixture was diluted with aqueous sodium hydroxide solution (4 wt.%, 34 mL). The separated aqueous layer was cooled to 0 °C and then slowly acidified to pH = 4 with 6N aqueous hydrochloride solution (-10 mL). The mixture was extracted with EtOAc (3x 50 mL). The combined organic layers washed with brine (50 mL), dried over sodium sulfate, filtered off and concentrated under reduced pressure. The residue was triturated with diethylether to give 2,5-difluoropyridin-4-ylboronic acid (808 mg).
Synthesis of (l-cyanocvclopropyl)methyl methanesulfonate
Figure imgf000089_0001
Stepl : Preparation of methyl 1-cyanocyclopropanecarboxylate
Figure imgf000089_0002
In a 100 mL flask at 0 °C, 1-cyanocyclopropanecarboxylic acid (3 g, 27.0 mmol) was dissolved in toluene (45 mL) and MeOH (5 mL). Reaction was treated dropwise with TMS-Diazomethane (27.0 mL, 27.0 mmol) and reaction stirred at 0 °C for 2 hr. Reaction was concentrated under reduced pressure providing a yellow oil, which was used without further purification (3.21 g, 25.7 mmol) GC/MS Rt = 5.0 min, m/z = 125.
Step2: Preparation of 1-(hydroxymethyl)cyclopropanecarbonitrile
Figure imgf000089_0003
In a 100 mL flask at 0 °C, methyl 1-cyanocyclopropanecarboxylate (1 g, 7.99 mmol) was dissolved in 1 ,2-Dimethoxyethane (20 ml) and MeOH (2 mL). Reaction was treated portion wise with NaBH4 (0.605 g, 15.98 mmol) and reaction stirred at 0 °C for 2 hr and then 20 hrs overnight. Reaction was quenched with 20 mL of saturated NH4CI solution. Reaction was diluted with Et20 and stirred vigorously for 2 hrs. Organics were isolated, dried (MgS04), filtered and concentrated under reduced pressure to provide the title compound as a yellow oil which was used without further purification (755 mg) GC/MS Rt = 4.8 min, m/z = 98. Step 3: Preparation of (l-cyanocyclopropyl)methyl methanesulfonate
Figure imgf000090_0001
In a 250 mL RBR at 0 °C, 1-(hydroxymethyl)cyclopropanecarbonitrile (400 mg, 4.12 mmol) was dissolved in methylene chloride (15 mL) and triethylamine (1.148 mL, 8.24 mmol). Reaction was treated drop wise with methanesulfonyl chloride (0.353 mL, 4.53 mmol) and reaction stirred at 0 °C for 2 hr. Reaction was quenched with 20 mL of saturated aqueous Na2C03 solution. Reaction mixture was diluted with Et20 and stirred vigorously for 30 minutes. Organics were isolated, dried (MgS04), filtered and concentrated under reduced pressure providing the title compound as a yellow oil which was used without further purification (622 mg).
Synthesis of (S)-1-(tetrahvdro-2H-pyran-4-yl)ethanamine
Figure imgf000090_0002
Step 1 : Preparation of (R,E)-2-methyl-N-((tetrahydro-2H-pyran-4- yl)methylene)propane-2-sulfinamide
A mixture of tetrahydro-2H-pyran-4-carbaldehyde (2.0 g, 17.52 mmol), (R)-2- methylpropane-2-sulfinamide (1.062 g, 8.76 mmol), pyridine 4-methylbenzenesulfonate (0.110 g, 0.438 mmol) and magnesium sulfate (5.27 g, 43.8 mmol) in dichloroethane (13 mL) was stirred at room temperature for 18 hrs. The solids were filtered off and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography [silica gel] providing (R,E)-2-methyl-N-((tetrahydro-2H-pyran- 4-yl)methylene)propane-2-sulfinamide (1.9 g). LCMS (m/z): 218.1 [M+H]+; Retention time = 0.58 min.
Step 2: Preparation of (R)-2-methyl-N-((S)-1 -(tetrahydro-2H-pyran-4- yl)ethyl)propane-2-sulfinamide
To a solution of (R,E)-2-methyl-N-((tetrahydro-2H-pyran-4-yl)methylene)propane-2- sulfinamide (0.93 g, 4.28 mmol) in dichloromethane (21.4 mL) at 0 °C was added slowly methylmagnesium bromide (2.0 M in tetrahydrofuran, 4.28 mL, 8.56 mmol). The reaction mixture was warmed to room temperature and stirred for 3 hrs. The mixture was diluted with saturated aqueous ammonium chloride solution (5 ml_). The separated organic layer was washed with water and brine, dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by column chromatography providing (R)-2-methyl-N-((S)-1-(tetrahydro-2H-pyran-4-yl)ethyl)propane-2-sulfinamide (910 mg). LCMS (m/z): 234.0 [M+H]+; Retention time = 0.58 min.
Step 3: Preparation of (S)-1 -(tetrahydro-2H-pyran-4-yl)ethanamine
To a solution of (R)-2-methyl-N-((S)-1-(tetrahydro-2H-pyran-4-yl)ethyl)propane-2- sulfinamide (400 mg, 1.714 mmol) in MeOH (5 ml_) was added 4M hydrochloride in dioxane (5 ml_). The reaction mixture was stirred at room temperature for 30 min. The mixture was concentrated under reduced pressure and the residue was diluted with diethylether (10 ml_). The precipitate was collected by filtration and washed with diethylether providing crude (S)-1-(tetrahydro-2H-pyran-4-yl)ethanamine hydrochloride salt. The hydrochloride salt was dissolved in water (10 ml_) and neutralized with saturated aqueous sodium bicarbonate solution. The mixture was extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered off and concentrated under reduced pressure providing crude (S)-1-(tetrahydro-2H-pyran-4- yl)ethanamine (212 mg), which was directly used in the next reaction without further purification. LCMS (m/z): 130.1 [M+H]+; Retention time = 0.34 min.
Synthesis of (R)-1-(tetrahvdro-2H-pyran-4-yl) ethanamine
Figure imgf000091_0001
Step 1 : Preparation of (S,E)-2-methyl-N-((tetrahydro-2H-pyran-4- yl)methylene)propane-2-sulfinamide
A mixture of tetrahydro-2H-pyran-4-carbaldehyde (2.0 g, 17.52 mmol), (S)-2- methylpropane-2-sulfinamide (1.062 g, 8.76 mmol), pyridine 4-methylbenzenesulfonate (0.110 g, 0.438 mmol) and magnesium sulfate (5.27 g, 43.8 mmol) in dichloroethane (13 ml_) was stirred at room temperature for 18 hrs. The solids were filtered off and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography [silica gel] providing (S,E)-2-methyl-N-((tetrahydro-2H-pyran- 4-yl)methylene)propane-2-sulfinamide (1.50 g). LCMS (m/z): 218.1 [M+H]+; Retention time = 0.58 min.
Step 2: Preparation of (S)-2-methyl-N-((R)-1 -(tetrahydro-2H-pyran-4- yl)ethyl)propane-2-sulfinamide
To a solution of (S,E)-2-methyl-N-((tetrahydro-2H-pyran-4-yl)methylene)propane- 2-sulfinamide (1.5 g, 6.90 mmol) in dichloromethane (34.5 mL) at 0 °C was slowly added methylmagnesium bromide (1.646 g, 13.80 mmol). The reaction mixture was warmed to room temperature and stirred for 3 hrs. The mixture was diluted with saturated aqueous ammonium chloride solution (5 mL). The separated organic layer was washed with water and brine, dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by column chromatograph providing (S)-2-methyl-N- ((R)-1-(tetrahydro-2H-pyran-4-yl)ethyl)propane-2-sulfinamide (1.40 g). LCMS (m/z): 234.3 [M+H]+; Retention time = 0.57 min.
Step 3: Preparation of (R)-1-(tetrahydro-2H-pyran-4-yl) ethanamine
To a solution of (S)-2-methyl-N-((R)-1-(tetrahydro-2H-pyran-4-yl)ethyl)propane-2- sulfinamide (400 mg, 1.714 mmol) in MeOH (5 mL) was added 4M hydrochloride in dioxane (5 mL). The reaction mixture was stirred at room temperature for 30 min. The mixture was concentrated under reduced pressure and the residue was diluted with diethylether (10 mL). The precipitate was collected by filtration and washed with diethylether providing crude (R)-1-(tetrahydro-2H-pyran-4-yl)ethanamine hydrochloride salt. The hydrochloride salt was dissolved in water (10 mL) and neutralized with saturated aqueous sodium bicarbonate solution. The mixture was extracted with dichloromethane (2x). The combined organic layers were dried over sodium sulfate, filtered off and concentrated under reduced pressure providing crude (R)-l-(tetrahydro- 2H-pyran-4-yl)ethanamine (200 mg), which was directly used in the next reaction without further purification. LCMS (m/z): 130.1 [M+H]+; Retention time = 0.34 min.
Synthesis of (2,2-dimethyltetrahydro-2H-pyran-4-yl)methanamine
Figure imgf000092_0001
Step 1 : Preparation of (2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl 4- methylbenzenesulfonate
To a solution of (2,2-dimethyltetrahydro-2H-pyran-4-yl)methanol (1 g, 6.93 mmol) in dichloromethane (5 ml_) and pyridine (5 ml_, 61.8 mmol) was added para-toluenesulfonyl chloride (1.586 g, 8.32 mmol) and DMAP (0.042 g, 0.347 mmol). The resulting mixture was stirred for 18 hrs at room temperature. The reaction mixture was concentrated under reduced pressure and the residue was diluted with water and dichloromethane. The separated organic phase was washed with 0.2N aqueous hydrochloride solution (1x), 1 N aqueous hydrochloride solution (2x), brine, dried over sodium sulfate, filtered off and concentrated under reduced pressure. The residue was purified by column
chromatography [silica gel, 40 g, EtOAc/hexane = 0/100 to 50/50] providing (2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl 4-methylbenzenesulfonate (2.05 g) as a colorless oil. LCMS (m/z): 299.1 [M+H]+; Retention time = 0.96 min. Step 2: Preparation of (2,2-dimethyltetrahydro-2H-pyran-4-yl)methanamine
Into a solution of (2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl 4- methylbenzenesulfonate (3 g, 10.05 mmol) in tetrahydrofuran (25 ml_) in a steel bomb was condensed ammonia (-5.00 ml_) at -78 °C. The mixture was heated in the steel bomb at 125 °C for -18 hrs. The mixture was cooled to -78 °C, the steel bomb was opened, and the mixture was allowed to warm up to room temperature under a stream of nitrogen. The mixture was concentrated under reduced pressure and the residue was partitioned between a aqueous sodium hydroxide solution (5 wt.%) and dichloromethane. The separated aqueous layer was extracted with dichloromethane (1x). The combined organic layers were washed with aqueous sodium hydroxide solution (5 wt.%), dried over sodium sulfate, filtered off and concentrated under reduced pressure providing crude (2,2-dimethyltetrahydro-2H-pyran-4-yl)methanamine (-2.36 g) as yellow liquid, which was directly used in the next reaction without further purification. LCMS (m/z): 144.1 [M+H]+; Retention time = 0.26 min. Synthesis of (6,6-dimethyl-1 ,4-dioxan-2-yl)methanamine
Figure imgf000093_0001
Step 1 : Preparation of 1-(allyloxy)-2-methylpropan-2-ol
To allylic alcohol (57.4 ml_, 844 mmol) was added sodium hydride (60 wt.% in mineral oil, 2.43 g, 101 mmol) at O °C. After stirring for 20 min 2,2-dimethyloxirane (15 ml_, 169 mmol) was added and the solution was refluxed overnight. The mixture was allowed to cool to room temperature, diluted with saturated aqueous ammonium chloride solution and extracted with diethylether (3x). The combined organic layers were dried over sodium sulfate, filtered off and concentrated under reduced pressure to remove diethylether. The residue was distilled providing 1-(allyloxy)-2-methylpropan-2-ol (12.3 g, 42 torr, bp 58-60 °C) as a colorless oil. H NMR (400 MHz, chloroform-d) δ [ppm]: 5.87 - 5.96 (m, 1 H) 5.26 - 5.31 (m, 1 H) 5.18 - 5.21 (m, 1 H) 4.03 - 4.05 (m, 2 H) 3.28 (s, 2 H) 2.31 (br. s, H) 1.23 (s, 3 H) 1.22 (s, 3 H). Step 2: Preparation of 6-(iodomethyl)-2,2-dimethyl-1,4-dioxane
To a solution of 1-(allyloxy)-2-methylpropan-2-ol (5.0 g, 38 mmol) in acetonitrile (400 mL) was added sodium bicarbonate (19.5 g, 77 mmol) and the mixture was cooled to 0 °C. Iodine (1 1.7 g, 46.1 mmol) was added and the reaction mixture was allowed to warm up to room temperature and stirred overnight. To the mixture was added triethylamine (6.42 mL, 46.1 mmol) and additional iodine (7.8 g, 30.7 mmol) and stirring was continued for additional 5 hrs at 0 °C. To the mixture was added potassium carbonate (6.37 g, 46.1 mmol) and the suspension was stirred at room temperature for ~3 days. The reaction mixture was diluted with saturated aqueous sodium thiosulfate solution (200 mL) and EtOAc (300 mL). The separated aqueous layer was extracted with EtOAc (2x) and the combined organic layers were dried over sodium sulfate, filtered off and concentrated under reduced pressure. The residue was purified by column chromatography [silica gel, EtOAc/hexane = 10/100 to 10/40] providing 6-(iodomethyl)-2,2-dimethyl-1 ,4-dioxane as a yellow oil (2.07 g). H NMR (400 MHz, chloroform-d) δ [ppm]: 4.01 (dd, J = 1 1.2, 2.8 Hz, 1 H) 3.81 - 3.88 (m, 1 H) 3.44 (d, J = 1 1.2 Hz, 1 H) 3.22 (dd, J = 11.6, 0.8 Hz, 1 H) 2.97- 3.13 (m, 3 H) 1.33 (s, 3 H) 1.14 (s, 3 H). 1-(Allyloxy)-2-methylpropan-2-ol (1.63 g) was recovered.
Step3: Preparation of 6-(azidomethyl)-2,2-dimethyl-1,4-dioxane
To a solution of 6-(iodomethyl)-2,2-dimethyl-1 ,4-dioxane (1.80 g, 7.03 mmol) in anhydrous DMF (9 mL) was added sodium azide (0.685 g, 10.5 mmol) and the suspension was heated at 80 °C for 2.5 hrs. The mixture was diluted with water (30 mL) and EtOAc (30 mL). The separated organic layer was washed with water (3x). The aqueous layers were combined and extracted with EtOAc (1x). The combined organic layers, dried over sodium sulfate, filtered off and concentrated under reduced pressure. The residue was purified by column chromatography [silica gel, EtOAc/hexane = 10/40 to 20/40] providing 6-(azidomethyl)-2,2-dimethyl-1 ,4-dioxane (0.93 g) as a colorless oil. H NMR (400 MHz, chloroform-d) δ [ppm]: 4.00-4.06 (m, 1 H) 3.75 (ddd, J = 1 1.2, 2.4, 0.4 Hz, 1 H) 3.49 (d, J = 11.2 Hz, 1 H) 3.14-3.29 (m, 4 H) 1.35 (s, 3 H), 1.14 (s, 3 H).
Step 4: Preparation of (6,6-dimethyl-1 ,4-dioxan-2-yl)methanamine
To a solution of 6-(azidomethyl)-2,2-dimethyl-1 ,4-dioxane (502 mg, 2.93 mmol) in anhydrous tetrahydrofuran (15 ml_) was added slowly a solution of lithium
aluminumhydride (1 M in tetrahydrofuran, 3.81 ml_) 0 °C and the mixture was stirred at 0 °C for 1 hr and at room temperature for 0.5 hr. The reaction mixture was cooled to 0 °C and sodium sulfate decahydrate (excess) was slowly added and the suspension was vigorously stirred overnight. The suspension was filtered through cotton and the filtrate was concentrated under reduced pressure providing crude (6,6-dimethyl-1 ,4-dioxan-2- yl)methanamine (410 mg) as a colorless oil, which was directly used in the next step without purification. LCMS (m/z): 146.1 [M+H]+; Retention time = 0.42 min. Synthesis of (5,5-dimethyl-1 ,4-dioxan-2-yl)methanamine
Figure imgf000095_0001
Step 1 : Preparation of 2-(allyloxy)-2-methylpropan-1-ol
To a solution of 2,2-dimethyloxirane (15.0 ml_, 169 mmol) in allylic alcohol (57.4 ml_) was added perchloric acid (70 wt.%, 7.26 ml_, 84 mmol) slowly at 0°C. The solution was warmed to room temperature and stirred for 1.5 hrs. The reaction mixture was diluted with saturated aqueous sodium bicarbonate solution and extracted with diethylether (3x). The combined organic layers were dried over sodium sulfate, filtered off and
concentrated under reduced pressure to remove diethylether. The residue was distilled providing 2-(allyloxy)-2-methylpropan-1-ol (9.70 g, 38 torr, bp 74-76 °C) as a colorless oil. H NMR (400 MHz, chloroform-d) δ [ppm]: 5.87 - 5.97 (m, 1 H) 5.25 - 5.31 (m, 1 H) 5.12 - 5.16 (m, 1 H) 3.92 - 3.94 (m, 2 H) 3.45 (m, 2 H) 1.19 (s, 6 H). Step 2: Preparation of 5-(iodomethyl)-2,2-dimethyl-1,4-dioxane
To a solution of 2-(allyloxy)-2-methylpropan-1-ol (5.0 g, 38.4 mmol) in acetonitrile (350 ml_) was added sodium bicarbonate (9.68 g, 1 15 mmol) and the mixture was cooled to 0 °C. Iodine (29.2 g, 1 15 mmol) was added and the reaction mixture was allowed to warm up to room temperature and stirred for 6 hrs. The reaction mixture was diluted with saturated aqueous sodium thiosulfate solution and concentrated under reduced pressure removing most of the organic solvent. The residue was extracted with EtOAc (2x) and the combined organic layers were dried over sodium sulfate, filtered off and concentrated under reduced pressure. The residue was purified by column chromatography [silica gel, EtOAc/hexane = 10/100 to 10/40] providing 6-(iodomethyl)-2,2-dimethyl-1 ,4-dioxane as a colorless oil (7.04 g). H NMR (400 MHz, chloroform-d) δ [ppm]: 3.70-3.73 (m, 1 H) 3.57 - 3.64 (m, 2 H) 3.43 - 3.50 (m, 2 H) 3.13 - 3.15 (m, 2 H) 1.32 (s, 3 H) 1.13 (s, 3 H).
Step 3: Preparation of 5-(azidomethyl)-2,2-dimethyl-1 ,4-dioxane
To a solution of 5-(iodomethyl)-2,2-dimethyl-1 ,4-dioxane (2.58 g, 10.1 mmol) in anhydrous DMF (13 mL) was added sodium azide (0.982 g, 15.1 mmol) and the suspension was heated at 80 °C for 2.5 hrs. The mixture was diluted with water (40 mL) and EtOAc (40 mL). The separated organic layer was washed with water (3x). The aqueous layers were combined and extracted with EtOAc (1x). The combined organic layers, dried over sodium sulfate, filtered off and concentrated under reduced pressure. The residue was purified by column chromatography [silica gel, EtOAc/hexane = 10/40 to 50/50] providing 6-(azidomethyl)-2,2-dimethyl-1 ,4-dioxane (1.61 g) as a colorless oil. NMR (400 MHz, chloroform-d) δ [ppm]: 3.63 - 3.72 (m, 2 H) 3.52 - 3.59 (m, 2 H) 3.42 (d, J = 11.6 Hz, 1 H) 3.29 (d, J = 4.4 Hz, 2 H) 1.33 (s, 3 H) 1.13 (s, 3 H). Step 4: Preparation of (5,5-dimethyl-1 ,4-dioxan-2-yl)methanamine
To a solution of 5-(azidomethyl)-2,2-dimethyl-1 ,4-dioxane (810 mg, 4.73 mmol) in anhydrous tetrahydrofuran (20 mL) was added slowly a solution of lithium
aluminumhydride (1.0 M tetrahydrofuran, 6.2 mL) 0 °C and the mixture was stirred at 0 °C for 1 hr and at room temperature for 0.5 hr. The reaction mixture was cooled to 0 °C and sodium sulfate decahydrate (excess) was slowly added and the suspension was vigorously stirred overnight. The suspension was filtered through cotton and the filtrate was concentrated under reduced pressure providing crude (5,5-dimethyl-1 ,4-dioxan-2- yl)methanamine (673 mg) as a colorless oil, which was directly used in the next step without purification. LCMS (m/z): 146.1 [M+H]+; Retention time = 0.42 min.
Synthesis of (4-methyltetrahvdro-2H-pyran-4-yl)methanamine
Figure imgf000096_0001
Preparation of 4-methyltetrahydro-2H-pyran-4-carbonitrile To a solution of tetrahydro-2H-pyran-4-carbonitrile (2 g, 18.00 mmol) in tetrahydrofuran (10 mL) at 0 - 5 °C was added slowly LHMDS (21.59 mL, 21.59 mmol). The mixture was stirred for 1.5 hrs at 0 °C. lodomethane (3.37 mL, 54.0 mmol) was added slowly and stirring was continued for 30 min at ~0 °C and then for ~2 hrs at room temperature. The mixture was cooled to 0 °C and carefully diluted with 1 N aqueous hydrochloride solution (30 mL) and EtOAc (5 mL) and concentrated under reduced pressure. The residue was taken up in diethylether and the separated organic layer was washed with brine, dried over sodium sulfate, filtered off and concentrated under reduced pressure providing crude 4-methyltetrahydro-2H-pyran-4-carbonitrile (1.8 g) as an orange oil, which was directly used in the next reaction without further purification. LCMS (m/z): 126.1 [M+H]+; Retention time = 0.44 min.
Step 2: Preparation of (4-methyltetrahydro-2H-pyran-4-yl)methanamine
To a solution of 4-methyltetrahydro-2H-pyran-4-carbonitrile (1.8 g, 14.38 mmol) in tetrahydrofuran (30 mL) was carefully added lithium aluminum hydride (1 M solution in tetrahydrofuran, 21.57 mL, 21.57 mmol) at 0 °C. The reaction mixture was stirred for 15 min at 0 °C, allowed to warm to room temperature and stirred for additional 3 hrs at room temperature. To the reaction mixture was carefully added water (0.9 mL) [Caution: gas development!], 1 N aqueous sodium hydroxide solution (2.7 mL) and water (0.9 mL). The mixture was vigorously stirred for 30 min. The precipitate was filtered off and rinsed with tetrahydrofuran. The solution was concentrated under reduced pressure providing crude (4-methyltetrahydro-2H-pyran-4-yl)methanamine (1.54 g) as a yellowish solid, which was directly used in the next step without further purification. LCMS (m/z): 130.1 [M+H]+; Retention time = 0.21 min.
Synthesis of 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000097_0001
Step 1 : Preparation of dihydro-2H-pyran-4,4(3H)-dicarbonitrile
A mixture of malononitrile (0.991 g, 15 mmol), 1-bromo-2-(2-bromoethoxy)ethane (3.83 g, 16.50 mmol) and DBU (4.97 mL, 33.0 mmol) in DMF (6 mL) was heated at 85 °C for 3 hrs. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was diluted with EtOAc (25 mL), washed with water (2x 10 mL), dried over sodium sulfat, filtered off and concentrated under reduced pressure and further dried in high vacuo providing crude dihydro-2H-pyran-4,4(3H)-dicarbonitrile (1.65 g) as a light brown solid, which was directly used in the next step without further purification. GCMS: 136 [M]; Retention time = 5.76 min. H NMR (300 MHz, chloroform- d) δ [ppm]: 2.14-2.32 (m, 4 H) 3.77-3.96 (m, 4 H).
Step 2: Preparation of 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile
To a solution of dihydro-2H-pyran-4,4(3H)-dicarbonitrile (450 mg, 3.31 mmol in EtOH (15 ml_) was added sodium borohydride (375 mg, 9.92 mmol) in portions and the mixture was stirred at room temperature for 4 hrs. The mixture was concentrated under reduced pressure and the residue was diluted with EtOAc (30 ml_), washed with water (10 ml_), dried over sodium sulfate, filtered off and concentrated under reduced pressure providing crude 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile (388 mg), which was directly used in the next step without further purification. LCMS (m/z): 141.0 [M+H]+; Retention time = 0.18 min.
Synthesis of 4-(hvdroxymethyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000098_0001
Step 1 : Preparation of methyl 4-cyanotetrahydro-2H-pyran-4-carboxylate
To methylcyanoacetate (7.87 ml, 101 mmol) in DM F (60 ml_) at room temperature was added a solution of 1-bromo-2-(2-bromoethoxy)ethane (25.7 g, 1 1 1 mmol) in 20 ml_ DMF. To this mixture was added a solution of DBU (33.2 ml_, 222 mmol) in 20 ml_ DMF dropwise via an addition funnel. The brown mixture was heated to 85 °C under argon for 3 hours. The reaction mixture was allowed to cool to room temperature, poured into water and extracted with EtOAc. The organic extracts were combined, washed with water and brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 120 g, EtOAc/heptane]. Fractions were combined and concentrated under reduced pressure providing methyl 4- cyanotetrahydro-2H-pyran-4-carboxylate (1 1.2 g) as a nearly colorless oil.
Step 2: Preparation of 4-(hydroxymethyl)tetrahydro-2H-pyran-4-carbonitrile
To a solution of methyl 4-cyanotetrahydro-2H-pyran-4-carboxylate (1 1.2 g, 66.2 mmol) in DME (60 ml_) and MeOH (6 ml_) at 0 °C was added sodium borohydride (1 .454 g, 38.4 mmol) in one portion. The reaction mixture was stirred under argon at room temperature for 16 hrs. The resulting mixture was was poured into saturated aqueous ammonium chloride solution (30ml_) and extracted with EtOAc (2x 20 mL). The organic extracts were combined, washed with brine, dried over sodium sulfate and concentrated under reduced pressure providing crude 4-(hydroxymethyl)tetrahydro-2H-pyran-4-carbonitrile (7.8 g) as a nearly colorless oil, which was diectly used without further purification. H NMR (400 MHz, chloroform-d3) δ ppm 1.58 - 1.70 (m, 2 H) 1.91 (dd, J=13.69, 1.96 Hz, 2 H) 2.31 (br. s., 1 H) 3.64 - 3.76 (m, 4 H) 3.94 - 4.06 (m, 2 H). Synthesis of toluene-4-sulfonic acid 4-methoxy-tetrahvdro-pyran-4-ylmethyl ester
Figure imgf000099_0001
Step 1 : Preparation of 1 ,6-dioxaspiro[2.5]octane
To a solution of trimethylsulfonium iodide (3.27 g, 16 mmol) in DMSO (20 mL) under nitrogen atmosphere was added dihydro-2H-pyran-4(3H)-one (1.0 g, 10 mmol). To the mixture was added slowly a solution of tert-butoxide (1.68 g, 15 mmol) in DMSO (15 mL) and the solution was stirred at room temperature overnight. The reaction mixture was diluted slowly with water (50 mL) and extracted with diethylether (3x 20 mL). The combined organic layers were dried over sodium sulfate, filtered off and concentrated under reduced pressure providing crude 1 ,6-dioxaspiro[2.5]octane (650 mg), which was directly used without further purification. H NMR (300 MHz, chloroform-d) 5 [ppm]: 1.44 - 1.62 (m, 2 H) 1.76 - 1.98 (m, 2 H) 2.70 (s, 2 H) 3.70 -3.98 (m, 4 H).
Step 2: Preparation of (4-methoxytetrahydro-2H-pyran-4-yl) MeOH
To a solution of 1 ,6-dioxaspiro[2.5]octane (600 mg, 5.26 mmol) in MeOH (10 mL) under nitrogen was added camphorsulfonic acid (50 mg, 0.21 mmol) at 0 °C and the mixture was stirred at 0 °C for 2 hrs. The mixture was concentrated under reduced pressure providing crude (4-methoxytetrahydro-2H-pyran-4-yl)methanol (707 mg) as a light yellow oil, which was directly used in the next step without further purification. H NMR (300 MHz, chloroform-d) δ [ppm]: 1.89 - 2.08 (m, 4 H), 3.18 - 3.30 (m, 3 H), 3.47 - 3.59 (m, 2 H), 3.64 - 3.78 (m, 4 H).
Step 3: Preparation of toluene-4-sulfonic acid 4-methoxy-tetrahydro-pyran-4- yl methyl ester
To a solution of (4-methoxytetrahydro-2H-pyran-4-yl) MeOH (300 mg, 2.05 mmol) in pyridine (4 mL) was added toluenesulfonic chloride (430 mg, 2.25 mmol) at room temperature and the mixture was stirred at 25 °C overnight. The mixture was
concentrated under reduced pressure and the residue was dissolved in dichloromethane (2 mL). Purification by column chromatography [silica gel, 12 g, EtOAc/hexane = 0/100 to 30/70] provided toluene-4-sulfonic acid 4-methoxy-tetrahydro-pyran-4-ylmethyl ester (360 mg) as a light yellow solid. H NMR (300 MHz, chloroform-d) 5. [ppm]: 1.45 - 1.63 (m, 2 H) 1.61 - 1.79 (m, 2 H) 2.46 (s, 3 H), 3.16 (s, 3 H) 3.53 - 3.75 (m, 4 H) 3.93 (s, 2 H), 7.36 (d, J = 8.20 Hz, 2 H) 7.81 (d, J = 8.20 Hz, 2 H).
Synthesis of (4-methoxytetrahvdro-2H-pyran-4-yl)methanamine
Figure imgf000100_0001
Step 1 : Preparation of 4,4-dimethoxytetrahydro-2H-pyran
A mixture of dihydro-2H-pyran-4(3H)-one (501 mg, 5 mmol), trimethyl orthoformate
(0.608 mL, 5.50 mmol) and toluenesulfonic acid monohydrate (2.85 mg, 0.015 mmol) in MeOH (1 mL) was stirred in a sealed tube at 80 °C for 30 min. The reaction mixture was allowed to cool to room temperature and was concentrated under reduced pressure providing crude 4,4-dimethoxytetrahydro-2H-pyran (703 mg), which was used in the next step without further purification. H NMR (400 MHz, chloroform-d) 5. [ppm]: 1.61 - 1.90 (m, 4 H) 3.20 (s, 6 H) 3.60 - 3.78 (m, 4 H).
Step 2: Preparation of 4-methoxytetrahydro-2H-pyran-4-carbonitrile
To a solution of 4,4-dimethoxytetrahydro-2H-pyran (0.703 g, 4.81 mmol) and
tin(IV)chloride (0.564 mL, 4.81 mmol) in dichloromethane (15 mL) was added slowly 2- isocyano-2-methylpropane (0.400 g, 4.81 mmol) at -70 °C and the mixture was allowed to warm to room temperature over 2-3 hrs. The mixture was diluted with aqueous sodium bicarbonate solution (10 mL) and dichloromethane (20 mL). The separated organic layer was washed with water (3x 10 mL) and dried over sodium sulfate, filtered off and concentrated under reduced pressure providing crude 4-methoxytetrahydro-2H- pyran-4-carbonitrile (51 1 mg), which was used in the next step without further purification. GCMS: 109 [M-MeOH]; Retention time = 5.44 min.
Step 3: Preparation of (4-methoxytetrahydro-2H-pyran-4-yl)methanamine
To a mixture of LiAIH4 (275 mg, 7.24 mmol) in tetrahydrofuran (10 ml_) at room
temperature was slowly added a solution of 4-methoxytetrahydro-2H-pyran-4-carbonitrile (51 1 mg, 3.62 mmol) in tetrahydrofuran (10 ml_). The mixture was stirred at room temperature for 1 hr and heated to reflux for 3 hrs. The reaction mixture was cooled to 0 °C and water (3 ml_) was carefully added dropwise. The resulting mixture was stirred for additional 30 min and filtered to remove all solids. The filtrate was dried over sodium sulfate for 2 hrs, filtered off and concentrated under reduced pressure providing crude (4- methoxytetrahydro-2H-pyran-4-yl)methanamine (370 mg), which was used in the next step without further purification. LCMS (m/z): 146.1 [M+H]+, 114.0 [M-MeOH]; Retention time = 0.19 min.
Synthesis of toluene-4-sulfonic acid 1',1 '-dioxo-hexahvdro-1-thiopyran-4-yl-methyl ester
Figure imgf000101_0001
A mixture of (1 ', 1'-dioxo-hexahydro-1-thiopyran-4-yl)-methanol (2.5 g, 15.22 mmol)
[Organic Process Research & Development 2008, 72, 892-895.], pyridine (25 ml_) and tosyl-CI (2.90 g, 15.22 mmol) was stirred for 18 hrs at 50 °C. The reaction mixture was concentrated under reduced pressure. The residue was purified by column
chromatography [silica gel, EtOAc/hexane = 0/100 to 70/30]. Fractions were combined and concentrated under reduced pressure providing toluene-4-sulfonic acid 1', 1 '-dioxo- hexahydro-1-thiopyran-4-yl-methyl ester (3.78 g). LCMS (m/z): 319.0 [M+H]+; Retention time = 0.71 min.
Synthesis of (2R,6S)-2,6-dimethyltetrahvdro-2H-pyran-4-carbaldehyde
Figure imgf000101_0002
Step 1 : Preparation of (2R,6S)-2,6-dimethyldihydro-2H-pyran-4(3H)-one A solution of 2,6-dimethyl-4H-pyran-4-one (2 g, 16.1 mmol) in EtOH (20 mL) was stirred over Pd/C (10 wt.%, 0.2 g) under hydrogen (15 psi) for 16 hrs at ambient temperature. The suspension was filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved in dichloromethane (15 mL) and treated with Dess-Martin periodinane (2.3 g) at ambient temperature for 16 hrs. To the suspension was added saturated aqueous sodium thiosulfate solution (~3 mL) and the mixture was stirred for 1 hr. The mixture was diluted with saturated aqueous sodium bicarbonate solution (20 mL) and stirred for an additional 1 hr. The separated organic phase was washed with water and brine, dried over sodium sulfate, filtered through celite and concentrated under reduced pressure. The residue was purified by column chromatography [silica gel, EtOAc/heptane = 10/90]. Fractions were combined and concentrated under reduced pressure providing (2R,6S)-2,6-dimethyldihydro-2H-pyran-4(3H)-one (600 mg). GCMS: 128 [M]; Retention time = 4.25 min. H NMR (400 MHz, DMSO-d6) δ [ppm]: 1.18 (d, J=6.26 Hz, 6 H) 2.11 - 2.25 (m, 4 H) 3.58 - 3.77 (m, 2 H).
Step 2: Preparation of (2R,6S)-4-(methoxymethylene)-2,6-dimethyltetrahydro-2H- pyran
To a suspension of (methoxymethyl)triphenyl phosphine chloride (1.5 g, 4.45 mmol) in tetrahydrofuran (8 mL) was added slowly sodium bis(trimethylsilyl) amide (1 M solution in tetrahydrofuran, 4.45 mL) at -10 °C. The reaction mixture was stirred for 1 hr and a solution of (2R,6S)-2,6-dimethyldihydro-2H-pyran-4(3H)-one (380 mg, 2.96 mmol) in tetrahydrofuran (2 mL) was added slowly. The resulting mixture was allowed to warm to ambient temperature and stirred for 3 hrs. The reaction mixture was diluted with water (15 mL) and extracted with diethylether (2x 30 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered off and concentrated under reduced pressure. The residue was purified by column chromatography [silica gel,
EtOAc/heptane = 10/90] providing (2R,6S)-4-(methoxymethylene)-2,6- dimethyltetrahydro-2H-pyran (240 mg) as a colorless oil. GCMS: 156 [M]; Retention time = 5.40 min. H NMR (400 MHz, DMSO-d6) δ [ppm]: 1.07 (t, J=6.06 Hz, 6 H) 1.18 - 1.29 (m, 1 H) 1.31 - 1.46 (m, 1 H) 1.61 (t, J=12.13 Hz, 1 H) 1.93 (d, J=13.30 Hz, 1 H) 3.17 - 3.28 (m, 2 H) 3.46 (s, 3 H) 5.89 (s, 1 H).
Step 3: Preparation of (2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-carbaldehyde A mixture of (2R,6S)-4-(methoxymethylene)-2,6-dimethyltetrahydro-2H-pyran (240 mg, 1.53 mmol) and formic acid (-88 wt.% in water, 1.5 mL, 34.4 mmol) under argon was heated at 90 °C for 1 hr. The reaction mixture was cooled to 0 °C, neutralized with 1 N aqueous sodium hydroxide solution until pH~6 and extracted with diethylether. The organic layer were dried over sodium sulfate, filtered off and concentrated under reduced pressure providing crude (2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-carbaldehyde (120 mg) as a yellow oil, which was directly used in the next reaction without further purification. GCMS: 142 [M]; Retention time = 5.0 min. H NMR (400 MHz, DMSO-d6) δ [ppm]: 0.89 - 1.00 (m, 2 H) 1.09 (d, J=6.26 Hz, 6 H) 1.77 (ddd, J=12.33, 1.96, 1.76 Hz, 2 H) 3.35 (t, J=7.04 Hz, 1 H) 3.38 - 3.48 (m, 2 H) 9.51 (s, 1 H). Synthesis of 6-bromo-N-((4-fluorotetrahvdro-2H-pyran-4-yl)methyl)pyridin-2-amine
Figure imgf000103_0001
Step 1 : Preparation of 4-fluorotetrahydro-2H-pyran-4-carbaldehyde
Step 1a: To a solution of DIPEA (6.12 mL, 35.0 mmol) in dichloromethane (80 mL) was added trimethylsilyl trifluoromethanesulfonate (7.79 g, 35.0 mmol) and slowly a solution of tetrahydro-2H-pyran-4-carbaldehyde (2 g, 17.52 mmol) in dichloromethane (80 mL) at 0 °C. Upon completion of the addition, the reaction mixture was stirred at room temperature for 2 hrs. The mixture was concentrated under reduced pressure and the residue was treated with hexane (200 mL). The precipitate was filtered off and the solution was concentrated under reduced pressure providing crude trimethylsilyl ether, which was directly used in the next step without further purification.
Step 1 b: To a solution of crude trimethylsilyl ether in dichloromethane (100 mL) was added dropwise a solution of N-fluorobenzenesulfonimide (5.53 g, 17.52 mmol), dissolved in dichloromethane (50 mL), at 0 °C. The mixture was stirred for 3 hrs at room temperature and the crude solution of 4-fluorotetrahydro-2H-pyran-4-carbaldehyde was directly used in the next reaction.
Step 2: Preparation of 6-bromo-N-((4-fluorotetrahydro-2H-pyran-4- yl)methyl)pyridin-2-amine
To 6-bromopyridin-2-amine (3.03 g, 17.50 mmol) was added the crude solution of 4- fluorotetrahydro-2H-pyran-4-carbaldehyde in dichloromethane. To the resulting mixture was added acetic acid (1.002 mL, 17.50 mmol) and sodium triacetoxyborohydride (5.56 g, 26.3 mmol) in portions. The mixture was stirred for 2 hrs at room temperature. The mixture was diluted carefully with saturated aqueous sodium bicarbonate solution. The separated aqueous layer was extracted with dichloromethane (1x). The combined organic layers were washed with water (1x), saturated aqueous sodium bicarbonate solution (1x) and concentrated under reduced pressure. The solid residue was dissolved in dichloromethane (100 ml_) and 3M aqueous hydrochloride solution (60 ml_). The separated organic layer was extracted with 3M aqueous hydrochloride solution (3x 20 ml_). The combined acidic layers were washed with dichloromethane (1x). Solid sodium bicarbonate was added carefully to the acidic solution [Caution: gas development!] until pH>~8. The aqueous mixture was extraction with dichloromethane (2x) and EtOAc (2x). The combined organic layers were concentrated under reduced pressure. The residue was dissolved in EtOAc. The solution was washed with 0.3M aqueous hydrochloride solution and brine, dried over sodium sulfate, filtered off and concentrated under reduced pressure. The residue was purified by column chromatography [silica gel, 40 g, EtOAc/heptane = 5/95 to 30/70] providing 6-bromo-N-((4-fluorotetrahydro-2H-pyran-4- yl)methyl)pyridin-2-amine (1.82 g) as a white solid. LCMS (m/z): 288.9/291.0 [M+H]+; Retention time = 0.84 min.
Synthesis of N-(1 ,3-dimethoxypropan-2-yl)cvclohexane-trans-1 ,4-diamine
Figure imgf000104_0001
Step 1 : Preparation of 1 ,3-dimethoxypropan-2-yl 4-methylbenzenesulfonate
To NaH (0.366 g, 9.16 mmol) in THF (12 ml_) at 0 °C was added 1 ,3-dimethoxy-2- propanol (1 g, 8.32 mmol) in THF (8 ml_) solution. The mixture was warmed to room temperature and stirred for 0.5 hour. To this was added tosyl chloride (1.587 g, 8.32 mmol) in one portion. The white cloudy mixture was stirred at room temperature for 16 hours. LC/MS showed complete conversion. The reaction mixture was poured into water and extracted with EtOAc. The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated in vacuo to give 2 g of colorless oil. The crude mixture was purified by column chromatography (silica gel column 80 g, gradient: 0 min, 100%n-heptane; 5-12 min, 20% EtOAc in Heptane; 12-15 min. 30% EtOAc in Heptane and hold until 30 min). The pure fractions were combined and concentrated under reduced pressure to give 1.25 g of product as colorless oil which solidified upon standing. Step 2: Preparation of N-(1,3-dimethoxypropan-2-yl)cyclohexane-trans-1 ,4- diamine
To the tosylate obtained in Step 1 (0.8g, 2.92 mmol) in DMSO (8 ml) was added 1 ,4- trans-cyclohexane diamine (0.999 g, 8.75 mmol). The brown mixture in a capped vial was heated to 95 °C with stirring for 2 hours. The reaction mixture was poured into 10% HCI in water (10 mL) at 0 °C (ice cubes in HCI) and extracted with DCM (1x 20 mL). The aqueous (light pink) was basified with 6N aqueous NaOH to pH >12 and extracted with DCM (2x 20mL). The organic extracts were combined, dried with sodium sulfate and concentrated under reduced pressure to give a purple liquid. LCMS (m/z): 217 [M+H]+; Retention time = 0.32 min; no UV absorption at 214 nm wavelength. This was used in the next step without further purification.
Synthesis of cis- and frans-4-(2,2-dimethylmorpholino)cyclohexanamine
Figure imgf000105_0001
Stepl : Preparation of tert-butyl cis/trans-4-(2,2-dimethylmorpholino)
cyclohexylcarbamate
Figure imgf000105_0002
To a solution of tert-butyl 4-oxocyclohexylcarbamate (350 mg, 1.641 mmol) in methylene chloride (8 mL) was added 2,2-dimethylmorpholine (189 mg, 1.641 mmol) followed by sodium triacetoxyborohydride (1.739 g, 8.21 mmol). Reaction mixture was stirred at 25 °C for 6 hr. Reaction mixture was diluted with EtOAc and washed with water.
Organics were isolated, dried (MgS04), filtered and concentrated under reduced pressure. The residue was purified by column chromatography [Si02; 12 g] to provide the title compound as a yellow oil. LCMS (m/z): 313.1 [M+H]+; Retention time = 0.60 min. Step2: Preparation of cis- and irans-4-(2,2-dimethylmorpholino)cyclohexanamine
Figure imgf000106_0001
To a solution of tert-butyl cis/trans-4-(2,2-dimethylmorpholino)cyclohexylcarbamate (419 mg, 1.341 mmol) in methylene chloride (10 mL) was added trifluoroacetic acid (0.103 mL, 1.341 mmol). Reaction was stirred at 25 °C for 2 hr. Reaction was concentrated to provide the title compounds as trifluoroacetic acid salts as a white solid which was used without further purification. (400 mg, 1.884 mmol). LCMS (m/z): 213.1 [M+H]+; Retention time = 0.19 min LC/MS Rt = 0.19 min, m/z (H+)= 213.1
Synthesis of trans-N1-((R)-1-methoxypropan-2-yl)cvclohexane-1 ,4-diamine
Figure imgf000106_0002
Preparation of (S)-1-methoxypropan-2-yl 4-methylbenzenesulfonate
Figure imgf000106_0003
To sodium hydride (5.99 g, 150 mmol) in THF (200mL) at 0 °C was added (S)-1- methoxypropan-2-ol (13.5 g, 150 mmol) dropwise. The mixture was warmed to room temperature and stirred under argon for 1 hr. The resulting white cloudy mixture was cooled to 0 °C. To this was added 4-methylbenzene-1-sulfonyl chloride (28.6 g, 150 mmol) in THF (200 mL). The reaction mixture was stirred at room temperature for 18 hr. The reaction mixture was poured into water and extracted with EtOAc (3x 150 mL). The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give 45 g of oil. The crude mixture was purified by column chromatography [Si02, 330 g, EtOAc /heptane = 0/100 for 10 min, 10/90 for 20 min, then 30/70], providing 27.33 g of (S)-1-methoxypropan-2-yl 4- methylbenzenesulfonate as colorless oil. H NMR (400 MHz, chloroform-d) δ ppm 1.28 (d, 3 H) 2.45 (s, 3 H) 3.25 (s, 3 H) 3.33 - 3.47 (m, 2 H) 4.72 (td, 1 H) 7.34 (d, 2 H) 7.82 (d, 2 H).
Step 2: Preparation of trans-N1-((R)-1-methoxypropan-2-yl)cyclohexane-1,4- diamine
Figure imgf000107_0001
To (S)-1-methoxypropan-2-yl 4-methylbenzenesulfonate (15 g, 61.4 mmol) in acetonitrile (100 mL) at room temperature was added 1 ,4-trans-cyclohexane-diamine (17.53 g, 153 mmol). The light brown mixture was heated to 90 °C in a sealed steel bomb for 18 hr. The resulting mixture was cloudy light brown. LC/MS showed formation of desired product and side bis-alkylated product. A second batch of the same reaction mixture was set up in a similar fashion (12.33 g of (S)-1-methoxypropan-2-yl 4- methylbenzenesulfonate, 14.41 g of 1 ,4-trans-cyclohexane-diamine) and the two reactions were cooled to room temperature, combined and worked up as below. To the cooled reaction mixture, ether (-200 mL) was added. The solid was removed by filtration. The filtrate was concentrated then heptane (80 mL) and EtOAc (15 mL) were added. The precipitates were removed by filtration. The filtrate was concentrated under reduced pressure to give brown oil and some solid. The residue was dissolved with 100 mL of water and extracted with ether (1x 100 mL) and DCM (4x 45 mL). Ether extract was discarded. The DCM extracts were combined, dried with sodium sulfate and
concentrated under reduced pressure to give 10.4 g (50% yield) of brown oil. LC/MS showed this contained trans-N1-((R)-1-methoxypropan-2-yl)cyclohexane-1 ,4-diamine (major) along with bis-alkylated side product (-5%). This was used in the next step without further purification. LCMS (m/z): 187.1 [M+H]+; Retention time = 0.15 min. H NMR (400 MHz, chloroform-d) δ ppm 1.02 (d, 3 H) 1.05 - 1.23 (m, 4 H) 1.77 - 2.03 (m, 4 H) 2.49 (br. s., 1 H) 2.65 (d, 1 H) 2.95 - 3.06 (m, 1 H) 3.18 - 3.31 (m, 2 H) 3.34 (s, 3 H).
Synthesis of trans-N1-(1-(trideuteromethoxy)propan-2-yl)cyclohexane-1 ,4-diamine
Figure imgf000107_0002
Step 1 : Preparation of 1 -(trideuteromethoxy)propan-2-yl 4- methylbenzenesulfonate
Figure imgf000108_0001
To 2-methyloxirane (0.603 mL, 8.61 mmol) in DMF (10 ml_) at room temperature was added methanol-d4 (0.310 g, 8.61 mmol) dropwise. The resulting grey cloudy mixture was stirred at room temperature under argon for 30 min followed by addition of 2- methyloxirane (0.603 mL, 8.61 mmol). The mixture was heated to 50 °C in a sealed scintillation vial for 18 hr. The resulting mixture was dark brown and cloudy. To this was added tosyl-CI (1.641 g, 8.61 mmol) in one portion and the mixture was stirred at room temperature for 3 hr. The reaction mixture was poured into aqueous saturated NaHC03 solution (50 mL) and extracted with EtOAc (2 x 50 mL). The organic extracts were combined, washed with brine, dried with sodium sulfate, filtered and concentrated under reduced pressure to give a brown oil. The crude mixture was purified by column chromatography [Si02, 40 g, EtOAc/heptane = 0/100 for 4 min, 30/70 for 4-8 min, then 50/50 for 20 min] providing 0.77g of 1-(trideuteromethoxy)propan-2-yl 4- methylbenzenesulfonate as a light yellow oil.
Step 2: Preparation of trans-N1-(1-(trideuteromethoxy)propan-2-yl)cyclohexane- 1,4-diamine
Figure imgf000108_0002
To 1-(trideuteromethoxy)propan-2-yl 4-methylbenzenesulfonate (0.77 g, 3.1 1 mmol) in acetonitrile (10 mL) at room temperature was added 1 ,4-trans-cyclohexane-diamine (0.71 1 g, 6.23 mmol). The light brown mixture was heated to 90 °C in a sealed steel bomb for 18 hr. The resulting mixture was cloudy light brown. LC/MS showed formation of desired product and side bis-alkylated product in a ratio about 2: 1. The reaction mixture was cooled to room temperature and ether was added. The solid was removed by filtration. The filtrate was concentrated under reduced pressure to give a brown oil. The residue was dissolved with saturated aqueous sodium bicarbonate solution (5 mL) and extracted with ether (1x 10 mL) and DCM (4x 5 mL). LC/MS showed ether extract mainly contained bis-alkylated side product and little product, this was discarded. The DCM extracts were combined, dried with sodium sulfate, filtered and concentrated under reduced pressure to give 0.19 g of trans-N1-(1-(trideuteromethoxy)propan-2- yl)cyclohexane-1 ,4-diamine as a brown oil. LC/MS showed this contained desired product (major) along with bis-alkylated side product and other impurity (with UV absorption). This was used in the next step without further purification. LCMS (m/z): 188.1 [M+H]+; Retention time = 0.17 min. Synthesis of trans-N1-(2-deutero-1-methoxypropan-2-yl)cvclohexane-1 ,4-diamine
Figure imgf000109_0001
H
Step 1 : Preparation of 2-deutero-1 -methoxypropan-2-ol
Figure imgf000109_0002
To 1-methoxypropan-2-one (5.26 mL, 56.8 mmol) in MeOH-d4 (10 mL) and THF (50.00 mL) at 0 °C was added NaBD4 (2.375 g, 56.8 mmol) portion wise. Vigorous off-gassing was seen. The reaction mixture was warmed to room temperature and stirred under argon for 5 hrs. The reaction mixture was worked up by pouring saturated aqueous NaHC03 solution (10 mL) and stirred for 1 hr. The product was extracted with diethyl ether (100 mL), washed with brine, dried with sodium sulfate and concentrated under reduced pressure to give 3.53 g of colorless liquid. This was used in the next step without further purification.
Step 2: Preparation of 2-deutero-1-methoxypropan-2-yl 4-methylbenzenesulfonate
Ov D
Figure imgf000109_0003
To NaH (1.549 g, 38.7 mmol) in THF (10 ml_) was added 2-deutero-1-methoxypropan-2- ol (3.53 g, 38.7 mmol) in THF (10 ml_) dropwise. The mixture was stirred at room temperature for 10 min to give a grey cloudy mixture. To this was added tosyl-CI (7.39 g, 38.7 mmol) in one portion. The reaction mixture was stirred under argon at room temperature for 2 days. The reaction mixture was poured into water and extracted with ethyl acetate. The organic extracts were combined, washed with brine, dried with sodium sulfate, filtered, and concentrated under reduced pressure to give 7.2 g of colorless oil. The crude mixture was purified by column chromatography [Si02, 120 g, EtOAc/heptane = 0/100 for 4 min, 30/70 until 12 min, then 50/50 until 20 min] providing 4.3 g of 2-deutero-1-methoxypropan-2-yl 4-methylbenzenesulfonate as a colorless oil. H NMR (400 MHz, chloroform-d) δ ppm 1.27 (s, 3 H) 2.45 (s, 3 H) 3.25 (s, 3 H) 3.33 - 3.46 (m, 2 H) 7.34 (d, 2 H) 7.81 (d, 2 H).
Step 3: Preparation of trans-N1-(2-deutero-1 -methoxypropan-2-yl)cyclohexane-1 ,4- diamine
Figure imgf000110_0001
To 2-deutero-1-methoxypropan-2-yl 4-methylbenzenesulfonate (4.3g, 17.53 mmol) in acetonitrile (80 ml_) at room temperature was added 1 ,4-trans-cyclohexane-diamine (4.00 g, 35.1 mmol). The light brown mixture was heated to 90 °C in a sealed steel bomb for 18 hr. The resulting mixture was cloudy light brown. LC/MS showed formation of desired product and side bis-alkylated product in a ratio of 2: 1. The reaction mixture was cooled to room temperature and ether was added. The solid was removed by filtration. The filtrate was concentrated under reduced pressure to give a brown oil. To this was added ether (80 ml_) and heptane (80 ml_). A lot of precipitates formed which were removed by filtration. The filtrate was concentrated under reduced pressure to give 2.85 g of brown oil. The residue was dissolved with 20 ml_ of saturated aqueous sodium bicarbonate solution and extracted with ether (1x 40 ml_) and DCM (4x 20 ml_). LC/MS showed ether extract only contained bis-alkylated side product and little product. The DCM extracts were combined, dried with sodium sulfate and concentrated under reduced pressure to give 1.19 g of brown oil. LC/MS showed this contained desired product (major) along with bis-alkylated side product. This was used in the next step without further purification. LCMS (m/z): 188.1 [M+H]+; Retention time = 0.17 min. H NMR (400 MHz, chloroform-d) δ ppm 0.97 - 1.27 (m, 7 H) 1.81 - 2.03 (m, 4 H) 2.42 - 2.55 (m, 1 H) 2.59 - 2.71 (m, 1 H) 3.19 - 3.31 (m, 2H) 3.34 (s, 3 H).
Synthesis of trans-N 1 -cvclopropyl-N 1 -(2-methoxyethyl)cvclohexane- 1 ,4-diamine
Figure imgf000111_0001
Step 1 : Preparation of tert-butyl (trans-4-((2- methoxyethyl)amino)cyclohexyl)carbamate
BocHN
Figure imgf000111_0002
To 2-methoxyethyl 4-methylbenzenesulfonate (2.68 g, 11.64 mmol) in acetonitrile (50 mL) at room temperature was added N-Boc-trans-cyclohexane-1 ,4-diamine (4.99 g, 23.28 mmol). The off-white suspension was heated to 95 °C in a sealed glass bomb for 18 hr. The resulting mixture was light brown with white precipitate. LC/MS showed no starting materials with desired product and side product in a ratio of -1 : 1. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to give 3 g of brown oil. The crude product was purified by column chromatography [silica gel, 40 g, MeOH/DCM = 0/100 for 5 min, 5/95 for 5 min, then 1/9 for 30 min]. The pure fractions were combined and concentrated under reduced pressure to give 2.08 g of product as white foam. LC/MS showed the material was not very clean, but contains desired product as main component, showed no UV absorption. LCMS (m/z): 273.1 [M+H]+; Retention time = 0.45 min. H NMR showed as mono- tosylate salt. H NMR (400 MHz, methanol-d4) δ ppm 1.17 - 1.51 (m, 13 H) 1.93 - 2.19 (m, 4 H) 2.37 (s, 3 H) 2.88 - 3.03 (m, 1 H) 3.10 - 3.17 (m, 2 H) 3.40(s, 3 H) 3.55 - 3.64 (m, 2 H) 7.16 - 7.27 (m, 2 H) 7.67 - 7.75 (m, 2 H).
Step 2: Preparation of tert-butyl (trans-4-(cyclopropyl(2-methoxyethyl)amino)- cyclohexyl)carbamate
BocHN
Figure imgf000111_0003
Similar to procedure as described in Gillaspy et ai, Tetrahedron Lett. 1995, 36, 7399-
7402:
To tert-butyl (trans-4-((2-methoxyethyl)amino)cyclohexyl)carbamate (0.5 g, 1.836 mmol) in MeOH (10 mL) at room temperature was added acetic acid (1.051 mL, 18.36 mmol), 3 A molecular sieves (0.7 g, 1.836 mmol) (powder, dried at 150 °C in oven overnight) and (l-ethoxycyclopropoxy)trimethylsilane (1.600 mL, 9.18 mmol) sequentially. To the mixture was added sodium cyanoborohydride (0.461 g, 7.34 mmol). The reaction mixture was heated to 70 °C under argon for 16 hr. The reaction mixture was cooled and filtered through filter paper. The collected solids were washed with MeOH (30 mL). The filtrate was concentrated under reduced pressure to dryness. The residue was re- dissolved in 30 mL of 2N aqueous NaOH solution and extracted with EtOAc (3x 30 mL). The organic extracts were combined, washed with brine, dried with sodium sulfate, filtered and concentrated under reduced pressure to give 0.34 g of while solid. This was used in the next step without further purification. LCMS (m/z): 313.1 [M+H]+; Retention time = 0.54 min.
Step 3: Preparation of trans-N1-cyclopropyl-N1 -(2-methoxyethyl)cyclohexane-1,4- diamine
Figure imgf000112_0001
To tert-butyl (trans-4-(cyclopropyl(2-methoxyethyl)amino)cyclohexyl)carbamate (0.33g, 1.056 mmol) in DCM (1 mL) was added trifluoroacetic acid (1 mL, 12.98 mmol). The homogeneous reaction mixture was stirred at room temperature for 2 hr. LC/MS showed complete conversion. Methanol was added to the reaction and the mixture was concentrated under reduced pressure to give a light brown oil. This was diluted with methanol (30 mL). To this was added PL-HC03 MR-Resin (1.87 mmol/g, 6 g) until the pH 8. The resin was filtered and washed with MeOH. The filtrate was concentrated under reduced pressure to give 0.25 g of colorless oil. LCMS (m/z): 213.1 [M+H]+; Retention time = 0.19 min. This was used in the next step without further purification. Synthesis of trans-4-(morpholinomethyl)cvclohexanamine
Figure imgf000112_0002
Step 1 : Preparation of benzyl (trans-4-(morpholinomethyl)cyclohexyl)carbamate
Figure imgf000113_0001
To the solution of benzyl (trans-4-formylcyclohexyl)carbamate (525 mg, 2.0 mmol) and morpholine (0.175 mL, 2.0 mmol) in DCE (13 mL), was added sodium
triacetoxhydroborate (596 mg, 2.81 mmol) and acetic acid (0.1 15 mL, 2.0 mmol). The reaction mixture was stirred at room temperature for 18 hr. The reaction solution was diluted with ethyl acetate and aqueous sodium bicarbonate solution. The separated organic layer was washed with saturated aqueous sodium bicarbonate solution, water and brine. The organic phase was dried over sodium sulfate, filtered and concentrated under reduced pressure to give a white residue as benzyl (trans-4- (morpholinomethyl)cyclohexyl)carbamate (652 mg) without further purification. LCMS (m/z): 333.1 [M+H]+; Retention time = 0.55 min. Step 2: Preparation of trans-4-(morpholinomethyl)cyclohexanamine
A mixture of benzyl (trans-4-(morpholinomethyl)cyclohexyl)carbamate (652 mg, 1.96 mmol) and 10% palladium on carbon (208 mg, 0.2 mmol) in a solution of EtOH (20 mL) and THF (5 mL) was stirred in a round bottom flask under hydrogen atmosphere at 25 °C for 16 hr. The reaction mixture was filtered through a pad of celite and washed with methanol (80 mL). All organic filtrate was concentrated under reduced pressure to give trans-4-(morpholinomethyl)cyclohexanamine (395 mg) as an oil, which was used without further purification. LCMS (m/z): 199.1 [M+H]+; Retention time = 0.13 min.
Synthesis of N1-((R)-3,3,3-trifluoro-2-methoxypropyl)cvclohexane-trans-1 ,4-diamine
Figure imgf000113_0003
Step 1 : Preparation of (R)-3-(benzyloxy)-1 ,1 ,1-trifluoropropan-2-ol
(R)-(+)-3,3,3-Trifluoro-1 ,2-epoxypropane (700 μΙ_, 8.08 mmol) and benzyl alcohol (1.68 ml_, 16.17 mmol) were dissolved in DCM (20 ml). Boron trifluoride diethyl etherate (102 μΙ_, 0.808 mmol) was added. The reaction mixture was stirred for about 16 hours at 60 °C in a sealed vessel. The reaction was judged to be complete by TLC (2: 1 heptanes:ethyl acetate). The reaction mixture was cooled to ambient temperature, diluted with DCM, and washed sequentially with saturated sodium bicarbonate and brine. The organic phase was dried over sodium sulfate, filtered, and concentrated. The crude material was purified by flash chromatography (heptanes/ethyl acetate gradient) to give 998 mg of (R)-3-(benzyloxy)-1 , 1 , 1-trifluoropropan-2-ol as a colorless oil.
Step 2: Preparation of (R)-((3,3,3-trifluoro-2-methoxypropoxy)methyl)benzene
(R)-3-(benzyloxy)-1 , 1 , 1-trifluoropropan-2-ol (998 mg, 4.53 mmol) was dissolved in THF (20 ml) at ambient temperature. Sodium hydride (190 mg, 4.76 mmol) was added. The mixture was stirred for 10 minutes at ambient temperature and 20 minutes at 50 °C. lodomethane (0.312 ml, 4.99 mmol) was added. The reaction vessel was sealed and stirred at 50 °C for about 16 hours. TLC (2: 1 heptanes:ethyl acetate) showed clean conversion to product. The cooled reaction was quenched by the addition of saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to give 1.05 g of crude (R)-((3,3,3-trifluoro-2- methoxypropoxy)methyl)benzene which was used without further purification.
Step 3: Preparation of (R)-3,3,3-trifluoro-2-methoxypropan-1 -ol
(R)-((3,3,3-trifluoro-2-methoxypropoxy)methyl)benzene (1.05 g, 4.48 mmol) was dissolved in methanol (90 ml). Argon was bubbled through the solution for 5 minutes, and 20% palladium hydroxide on carbon (0.079 g, 0.1 12 mmol) was added. The flask was purged and flushed twice with hydrogen. The mixture was stirred for about 16 hours at ambient temperature under a hydrogen balloon. The mixture was filtered through a pad of celite. The filter cake was rinsed with additional methanol. The filtrate was concentrated at ambient temperature to give 495 mg of (R)-3,3,3-trifluoro-2- methoxypropan-1-ol as a colorless oil. This was used in the next step without further purification. Step 4: Preparation of (R)-3,3,3-trifluoro-2-methoxypropyl 4- methylbenzenesulfonate Sodium hydride (412 mg, 10.31 mmol) was added to a solution of (R)-3,3,3-trifluoro-2- methoxypropan-1-ol (495 mg, 3.44 mmol) in THF (10 ml) at ambient temperature. The mixture was stirred for 30 minutes. P-Toluenesulfonyl chloride (1965 mg, 10.31 mmol) was added. The white cloudy solution was stirred at ambient temperature for 18 hours. The reaction mixture was diluted with saturated aqueous sodium bicarbonate and extracted with EtOAc. The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated in vacuo. The crude mixture was purified by flash chromatography (heptanes : EtOAc gradient) to give 0.51 g of (R)-3,3,3-trifluoro-2- methoxypropyl 4-methylbenzenesulfonate as a colorless crystalline solid. LCMS (m/z): 298.9 [M+H]+; Retention time = 1.01 min.
Step 5: Preparation of N1 -((R)-3,3,3-trifluoro-2-methoxypropyl)cyclohexane-trans- 1,4-diamine
(R)-3,3,3-trifluoro-2-methoxypropyl 4-methylbenzenesulfonate (510 mg, 1.71 mmol) and trans- 1 ,4-diaminocyclohexane (586 mg, 5.13 mmol) were suspended in DMSO (4 ml). The reaction mixture was stirred at 100 °C for 3 hours. The cooled reaction mixture was diluted with water (40 ml_) and extracted with DCM. The combined extracts were washed sequentially with water and brine, dried over sodium sulfate, filtered, and concentrated to give 400 mg of crude N1-((R)-3,3,3-trifluoro-2- methoxypropyl)cyclohexane-trans-1 ,4-diamine which was used without further purification. LCMS (m/z): 241.1 [M+H]+; Retention time = 0.33 min. H NMR (400 MHz, chloroform-d) δ ppm 0.93 - 1.20 (m, 4 H) 1.83 (br. s., 4 H) 2.25 - 2.41 (m, 2 H) 2.65 - 2.85 (m, 4 H) 3.52 (s, 3 H) 3.54 - 3.66 (m, 2 H). Synthesis of 3-((trans-4-aminocvclohexyl)amino)-1 ,1 ,1-trifluoro-2-methylpropan-2-ol (racemic mixture)
Figure imgf000115_0001
Preparation of trans-tert-butyl-4-aminocyclohexylcarbamate
Figure imgf000115_0002
To a stirred solution of trans-cyclohexane-1 ,4-diamine (40.0 g, 350 mmol) in CHCI3 (400 ml_) at 0 °C was added di-tert-butyl dicarbonate (40.6 ml_, 175 mmol), in one portion. The reaction mixture was allowed to warm to room temperature and stirred for -72 hrs. The solvent was removed under reduced pressure and water (150 mL) was added. The product was filtered off. Toluene was added and the water was evaporated off until material precipitated, which was filtered off. Further evaporation yielded more precipitate which was collected by filtration. The combined precipitates were stirred in ether (250 mL) and filtered, providing trans-tert-butyl-4-aminocyclohexylcarbamate (34.2 g, 160 mmol). H NMR (400 MHz, chloroform-d) δ ppm 1.10 - 1.34 (m, 4 H) 1.43 (s, 9 H) 1.79 - 2.03 (m, 4 H) 3.17 - 3.30 (m, 2 H). Step 2: Preparation of trans-tert-butyl-4-(dibenzylamino)cyclohexylcarbamate
Figure imgf000116_0001
To trans-tert-butyl-4-aminocyclohexylcarbamate (6 g, 28.0 mmol) in acetonitrile (40 mL) was added benzyl bromide (7.33 mL, 61.6 mmol) and potassium carbonate (15.48 g, 1 12 mmol). The mixture was heated at 80 °C for -20 hrs. The mixture was allowed to cool to room temperature and water (-100 mL) was added. The precipitate was filtered off and washed with water, dried under educed pressure providing crude trans-tert-butyl-4- (dibenzylamino)cyclohexylcarbamate as a white solid, which was directly used in the next step without further purification. LCMS (m/z): 395.0 [M+H]+; Retention time = 0.81 min.
Step 3: Preparation of trans-N1,N1 -dibenzylcyclohexane-1 ,4-diamine
Figure imgf000116_0002
Crude trans-tert-butyl-4-(dibenzylamino)cyclohexylcarbamate (-28 mmol) was suspended in MeOH (10 mL). 4M HCI (60 mL; solution in dioxane) was added and the mixture was stirred ~1 hr. Additional 4M HCI (10 mL) were added and stirring was continued for 30 min. The mixture was concentrated under reduced pressure. The residue was suspended in diethylether, filtered off and washed with diethylether to give trans-N1 ,N1-dibenzylcyclohexane-1 ,4-diamine as its HCI-salt. LCMS (m/z): 295.1
[M+H]+; Retention time = 0.48 min. The HCI-salt was suspended in DCM and basified with potassium carbonate. The aqueous mixture was extracted with DCM (3x) and ethyl acetate (2x). The organic mixtures were (seperately) washed with saturated NaHC03 solution and brine, filtered through celite. The organic solutions were combined and concentrated under reduced pressure providing trans-N1 ,N1-dibenzylcyclohexane-1 ,4- diamine (2.46 g), which was directly used in the next step without further purification. Step 4: Preparation of N-(trans-4-(dibenzylamino)cyclohexyl)-3,3,3-trifluoro-2- hydroxy-2-methylpropanamide
Figure imgf000117_0001
To a solution of 2-hydroxy-2-(trifloromethyl)propionic acid (0.846 g, 5.35 mmol), HOBT (0.819 g, 5.35 mmol), and diisopropylethylamine (1.1 12 mL, 6.37 mmol) in DCM (45 mL) was added trans-N1 ,N1-dibenzylcyclohexane-1 ,4-diamine (1.5 g, 5.09 mmol) and EDC (1.025 g, 5.35 mmol). The reaction solution was stirred at 25 °C for 5 hr. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution. It was diluted with dichloromethane (100 mL) and stirred vigorously for 15 min. The separated organic layer was washed with saturated aqueous sodium bicarbonate solution and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography [silica gel, 40 g, EtOAc/heptane = 0/100 to 60/40] providing N-(trans-4-(dibenzylamino)cyclohexyl)-3,3,3- trifluoro-2-hydroxy-2-methylpropanamide (497 mg). LCMS (m/z): 435.2 [M+H]+;
Retention time = 0.66 min.
Step 5: Preparation of 3-((trans-4-(dibenzylamino)cyclohexyl)amino)-1 ,1,1- trifluoro-2-methylpropan-2-ol
Figure imgf000118_0001
To a solution of N-(trans-4-(dibenzylamino)cyclohexyl)-3,3,3-trifluoro-2-hydroxy-2- methylpropanamide (322 mg, 0.741 mmol) in THF (6 ml_) was added 1 M borane tetrahydrofuran complex (7 ml_, 7 mmol). The reaction mixture was stirred at 55 °C for 3 hr, but was not complete. The solution was quenched with saturated aqueous sodium bicarbonate solution and stirred vigorously overnight. It was diluted with ethyl acetate (60 ml_). The organic layer was washed with saturated aqueous sodium bicarbonate solution (2x) and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was filtered by column chromatography [silica gel, 24 g, ethyl acetate/dichloromethane = 0/100 to 35/65] providing product 3-((trans-4- (dibenzylamino)cyclohexyl)amino)-1 , 1 ,1-trifluoro-2-methylpropan-2-ol (84 mg, 80% pure) with impurity N-(trans-4-(dibenzylamino)cyclohexyl)-3,3,3-trifluoro-2-hydroxy-2- methylpropanamide (200 mg). LCMS (m/z): 421.1 [M+H]+; Retention time = 0.53 min for the product. LCMS (m/z): 421.1 [M+H]+; Retention time = 0.69 min for the impurity.
Step 6: Preparation of 3-((trans-4-aminocyclohexyl)amino)-1,1,1-trifluoro-2- methylpropan-2-ol (racemic m
Figure imgf000118_0002
A mixture of 50/50 3-((trans-4-(dibenzylamino)cyclohexyl)amino)-1 , 1 , 1-trifluoro-2- methylpropan-2-ol and N-(trans-4-(dibenzylamino)cyclohexyl)-3,3,3-trifluoro-2-hydroxy-2- methylpropanamide (314 mg), and 20% by weight palladium hydroxide on carbon (1 15 mg, 0.164 mmol) in ethanol (7 ml_) was stirred in a steel bomb under hydrogen atmosphere (60 psi) at 25 °C for 18 hr. The reaction mixture was filtered through a pad of celites and washed with ethyl acetate (100 ml_). The filtrate was concentrated under reduced pressure providing crude product mixture of 3-((trans-4- aminocyclohexyl)amino)-1 , 1 , 1-trifluoro-2-methylpropan-2-ol as a solid, which was directly used in the next step without further purification. LCMS (m/z): 241.1 [M+H]+; Retention time = 0.16 min. Purity -50%. Synthesis of (S)-4-(3-methoxypyrrolidin-1-yl)cvclohexanamine
Figure imgf000119_0001
Step 1 : Preparation of (S)-benzyl (4-(3-methoxypyrrolidin-1- yl)cyclohexyl)carbamate
Figure imgf000119_0002
To the solution of benzyl (4-oxocyclohexyl)carbamate (1.5 g, 6.07 mmol) and (S)-3- methoxypyrrolidine (0.876 g, 6.37 mmol) in DCE (30 mL) was added sodium
triacetoxhydroborate (1.8 g, 8.5 mmol). The reaction mixture was stirred at room temperature for 18 hr and became a brown solution. The reaction solution was diluted with ethyl acetate and sodium bicarbonate solution. The separated organic layer was washed with saturated aqueous sodium bicarbonate solution, water, and brine. The organic phase was dried over sodium sulfate, filtered and concentrated under reduced pressure to give a beige color residue as (S)-benzyl (4-(3-methoxypyrrolidin-1- yl)cyclohexyl)carbamate (1.99 g) without further purification. LCMS (m/z): 333.2 [M+H]+; Retention time = 0.55 min.
Step 2: Preparation of (S)-4-(3-methoxypyrrolidin-1-yl)cyclohexanamine
Figure imgf000119_0003
A mixture of (S)-benzyl (4-(3-methoxypyrrolidin-1-yl)cyclohexyl)carbamate (1.99 g, 5.69 mmol) and 10% palladium on carbon (1.21 g, 1.14 mmol) in EtOH (40 mL) was stirred in a round bottom flask under hydrogen atmosphere at 25 °C for 16 hr. The reaction mixture was filtered through a pad of celite and washed with methanol (300 mL). All organic filtrate was concentrated under reduced pressure to give (S)-4-(3- methoxypyrrolidin-1-yl)cyclohexanamine (1.12 g) as a brown oil without further purification. LCMS (m/z): 199.1 [M+H]+; Retention time = 0.18 min. Synthesis of2-(((trans)-4-aminocvclohexyl)amino)-2-methylpropan-1-ol
Figure imgf000120_0001
Step 1 : Preparation of ethyl 2-(((trans)-4-aminocyclohexyl)amino)-2- methylpropanoate
Figure imgf000120_0002
A mixture of ethyl 2-bromo-2-methylpropanoate (4.51 ml, 30.8 mmol) and cyclohexane- 1 ,4-diamine (7.03 g, 61.5 mmol) in DME (60 ml) was heated to 100 °C in a sealed glass bomb for 16 hours. The reaction became cloudy. LCMS indicated mono- and bisalkylation. The reaction was allowed to cool to room temperature and the solid was filtered off (filter paper). The filtrate was concentrated under reduced pressure to give an off white solid. The solid was dissolved in DCM (50 ml_), neutralized with saturated sodium bicarbonate aqueous solution (-20 ml_), and diluted with brine (20 ml_). The organic layer was dried with sodium sulfate and concentrated under reduced pressure to give 4 g of crude ethyl 2-(((trans)-4-aminocyclohexyl)amino)-2-methylpropanoate as a light brown oil. LCMS (m/z): 229.2 [M+H]+; Retention time = 0.27 min. This crude material was used in the next step without further purification. Step2: Preparation of 2-(((trans)-4-aminocyclohexyl)amino)-2-methylpropan-1 -ol
To crude ethyl 2-(((trans)-4-aminocyclohexyl)amino)-2-methylpropanoate (3.5 g) inTHF (25 ml) at 0 °C was added lithium aluminium hydride (THF solution) (7.66 mmol) via syringe over 15 minutes. The brown mixture was warmed to room temperature and stirred for 16 hours. The reaction mixture was diluted with THF(30 ml_) and cooled to 0 °C. To the mixture was added sodium sulfate decahydrate in one portion. The mixture was stirred under argon for 2 hours. The light brown cloudy solution was diluted with DCM (30 ml_) and filtered through a filter paper. The filtrate was concentrated under reduced pressure to give 1.51 g of crude 2-(((trans)-4-aminocyclohexyl)amino)-2- methylpropan-1-ol as a brown sticky oil. LCMS (m/z): 187.1 [M+H]+; Retention time = 0.14 min (desired product) and LCMS (m/z): 259.2 [M+H]+; Retention time = 0.17 min (side product) in a ratio about 2: 1. This mixture was used in the next step without further purification.
Synthesis of rac tert-butyl ((1S,2S,4S)-4-amino-2-hvdroxycvclohexyl)(2- methoxyethvDcarbamate
Figure imgf000121_0001
Step 1 : Preparation of rac benzyl ((1S,3S,4S)-3-hydroxy-4-((2- methoxyethyl)amino)cyclohexyl)carbamate
Figure imgf000121_0002
To a solution of rac benzyl (1 S,3S,6R)-7-oxabicyclo[4.1.0]heptan-3-ylcarbamate (450 mg, 1.820 mmol) in acetonitrile (8 mL) was added lithium perchlorate (968 mg, 9.10 mmol) and reaction was stirred at room temperature for 15 minutes until a homogeneous solution resulted. To this was added 2-methoxyethanamine (0.784 mL, 9.10 mmol) and reaction was heated at 50 °C for 12 hr. Reaction was diluted with EtOAc and washed with water. Organics were isolated, dried (MgS04), filtered and concentrated to 889 mg of yellow oil which was purified by column chromatography [Si02, 12 g, EtOAc/heptane] to provide 505 mg of rac benzyl ((1 S,3S,4S)-3-hydroxy-4-((2- methoxyethyl)amino)cyclohexyl)carbamate as a colorless oil. LCMS (m/z): 322.9 [M+H]+; Retention time = 0.49 min.
Step 2: Preparation of rac tert-butyl ((1S,2S,4S)-4-(((benzyloxy)carbonyl)amino)-2- hydroxycyclohexyl)(2-methoxyethyl)carbamate
Figure imgf000122_0001
To a solution of rac benzyl ((1S,3S,4S)-3-hydroxy-4-((2- methoxyethyl)amino)cyclohexyl)carbamate (1.33 g, 4.13 mmol) in THF (15 ml_) was added 1 N aqueous sodium hydroxide solution (15 ml_, 15.00 mmol) followed by di-tert- butyl dicarbonate (1.92 ml_, 8.25 mmol). Reaction was stirred at 25 °C for 12 hr.
Reaction was diluted with EtOAc and washed with water. Organics were isolated, dried (MgS04), filtered and concentrated under reduced pressure to 1.7 g of yellow oil which was purified by column chromatography [Si02, 80 g, EtOAc/Heptane) to provide 1.2 g of rac tert-butyl ((1 S,2S,4S)-4-(((benzyloxy)carbonyl)amino)-2-hydroxycyclohexyl)(2- methoxyethyl)carbamate as a colorless oil. LCMS (m/z): 423.1 [M+H]+; Retention time = 0.58 min.
Step 3: Preparation of rac tert-butyl ((1 S,2S,4S)-4-amino-2-hydroxycyclohexyl)(2- methoxyethyl)carbamate
To a solution of rac tert-butyl ((1 S,2S,4S)-4-(((benzyloxy)carbonyl)amino)-2- hydroxycyclohexyl)(2-methoxyethyl)carbamate (550 mg, 1.302 mmol) in EtOH (10 ml) was passed through hydrogen-Cube at 1 mL/min flow rate under full hydrogen conditions (20 bar-reading). Reaction was concentrated to 360 mg of rac tert-butyl ((1 S,2S,4S)-4- amino-2-hydroxycyclohexyl)(2-methoxyethyl)carbamate as a colorless oil which was used without further purification. LCMS (m/z): 289.2 [M+H]+; Retention time = 0.52 min.
Synthesis of rac ((1 R,2R,5S)-5-amino-2-methoxy-cvclohexyl)-carbamic acid tert-butyl ester
Figure imgf000122_0002
Step 1 : Preparation of rac (1S,3S,6R)-(7-Oxa-bicyclo[4.1.0]hept-3-yl)-carbamic acid benzyl ester
Figure imgf000123_0001
Titled compound was prepared following the procedure described in Tetrahydron 61 (2005) 1207-1219. LCMS (m/z): 248.1 [M+H]+, Retention time = 0.77 min. Step 2: Preparation of rac ((1 S,3R,4R)-3-azido-4-hydroxy-cyclohexyl)-carbamic acid benzyl ester
Figure imgf000123_0002
To a solution of benzyl rac (1S,3S,6R)-(7-oxa-bicyclo[4.1.0]hept-3-yl)-carbamic acid benzyl ester in methanol (25 mL) was added lithium perchlorate (6.56 g, 61.7 mmol) and sodium azide (1.82 g, 28.0 mmol). The mixture was stirred at 23 °C for 18 hr. To the reaction mixture was added 50 mL of water. Saturated aqueous sodium
bicarbonatesolution was added to adjust pH=8. The mixture was taken up by 200 mL of ethyl acetate. The organic layer was collected then washed with water, brine and dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography [Si02, 40 g, EtOAc/heptane = 0/100 to 30/70]. Pure fractions were combined and concentrated under reduced pressure giving 1.21 g of titled compound. LCMS (m/z): 291.1 [M+H]+; Retention time = 0.75 min.
Step 3: Preparation of rac ((1 S,3R,4R)-3-azido-4-methoxy-cyclohexyl)-carbamic acid benzyl ester
Figure imgf000123_0003
To a solution of rac ((1S,3R,4R)-3-azido-4-hydroxy-cyclohexyl)-carbamic acid benzyl ester (1.21 g, 4.17 mmol) in acetonitrile (20 mL) was added silver(l) oxide (2.90 g, 12.50 mmol) and methyl iodide (5.92 g, 41.7 mmol). The reaction mixture was stirred at 80 °C for 18 h. The mixture was diluted with EtOAc (-25 mL) and MeOH (~5 mL), filtered off and concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 40 g, EtOAc/heptane = 10/90 to 50/50]. Fractions were combined and concentrated under reduced pressure giving 320 mg of titled compound. LCMS (m/z): 305.0 [M+H]+; Retention time = 0.93min. Step 4: Preparation of rac ((1 S,3R,4R)-3-amino-4-methoxy-cyclohexyl)-carbamic acid benzyl ester
Figure imgf000124_0001
A mixture of rac ((1S,3R,4R)-3-azido-4-methoxy-cyclohexyl)-carbamic acid benzyl ester (230 mg, 0.76 mmol), zinc powder (148 mg g, 2.67 mmol) in acetic acid (3 mL) was stirred at room temperature for 1 hr. The mixture was diluted with 50 mL of methanol and filtered to remove the solid. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 12 g,
methanol/dichloromethane = 1/99 to 10/90]. Fractions were combined and concentrated under reduced pressure giving 140 mg of titled compound. LCMS (m/z): 279.1 [M+H]+; Retention time = 0.54 min.
Step 5: Preparation of rac ((1 S,3R,4R)-3-tert-butoxycarbonylamino-4-methoxy- cyclohexyl)-carbamic acid benzyl ester
Figure imgf000124_0002
A mixture of rac ((1S,3R,4R)-3-amino-4-methoxy-cyclohexyl)-carbamic acid benzyl ester (140 mg, 0.50 mmol), boc anhydride (110 mg, 0.50 mmol) in DCM (5 mL) was stirred at for 2 hr. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 12 g, ethyl acetate/heptane = 0/100 to 60/40]. Fractions were combined and concentrated under reduced pressure giving 145 mg of titled compound. LCMS (m/z): 379.1 [M+H]+; Retention time = 1.01 min.
Step 6: Preparation of rac ((1 R,2R,5S)-5-amino-2-methoxy-cyclohexyl)-carbamic acid tert-butyl ester A mixture of rac ((1S,3R,4R)-3-tert-butoxycarbonylamino-4-methoxy-cyclohexyl)- carbamic acid benzyl ester (170 mg, 0.45 mmol), Pd/C (10%. 15 mg) in methanol (10 mL) was stirred at room temperature for 2 hr. The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 12 g, ethyl methanol/dichloromethane = 0/100 to 10/90]. Fractions were combined and concentrated under reduced pressure giving 89 mg of titled compound. LCMS (m/z): 245.1 [M+H]+; Retention time = 0.46 min.
Synthesis of rac ((1S,2S,4S)-4-amino-2-methoxy-cvclohexyl)-carbamic acid tert-butyl ester
Figure imgf000125_0001
Step 1 : Preparation of rac (1S,3S,6R)-(7-oxa-bicyclo[4.1.0]hept-3-yl)-carbamic acid benzyl ester
Figure imgf000125_0002
Titled compound was prepared following the procedure described in Tetrahydron 61 (2005) 1207-1219. LCMS (m/z): 248.1 [M+H]+; Retention time = 0.77 min.
Step 2: Preparation of rac ((1 S,3S,4S)-4-azido-3-hydroxy-cyclohexyl)-carbamic acid benzyl ester
Figure imgf000125_0003
To a solution of rac benzyl (1S,3S,6R)-(7-oxa-bicyclo[4.1.0]hept-3-yl)-carbamic acid benzyl ester (1.24 g, 5.0 mmol) in methanol (25 mL) was added lithium perchlorate (6.56 g, 61.7 mmol) and sodium azide (1.82 g, 28.0 mmol). The mixture was stirred at 23 °C for 18 hr. To the reaction mixture was added 50 mL of water. Saturated aqueous sodium bicarbonate solution was added to adjust pH=8. The mixture was taken up by 200 mL of ethyl acetate. The organic layer was collected then washed with water, brine and dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography [Si02, 40 g, EtOAc/heptane = 0/100 to 30/70]. Pure fractions were combined and concentrated under reduced pressure giving 1.21 g of titled compound. LCMS (m/z): 291.1 [M+H]+; Retention time = 0.75 min.
Step 3: Preparation of rac ((1 S,3S,4S)-4-azido-3-methoxy-cyclohexyl)-carbamic acid benzyl ester
Figure imgf000126_0001
To a solution of benzyl rac ((1 S,3S,4S)-4-azido-3-hydroxy-cyclohexyl)-carbamic acid benzyl ester (1.21 g, 4.17 mmol) in acetonitrile (20 mL) was added silver(l) oxide (2.90 g, 12.50 mmol) and methyl iodide (5.92 g, 41.7 mmol). The reaction mixture was stirred at 80 °C for 18 hr. The mixture was diluted with EtOAc (-25 mL) and MeOH (~5 mL), filtered off and concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 40 g, EtOAc/heptane = 10/90 to 50/50]. Fractions were combined and concentrated under reduced pressure giving 320 mg of titled compound. LCMS (m/z): 305.0 [M+H]+; Retention time = 0.93min. Step 4: Preparation of rac ((1 S,3S,4S)-4-amino-3-methoxy-cyclohexyl)-carbamic acid benzyl ester
Figure imgf000126_0002
A mixture of rac ((1S,3S,4S)-4-azido-3-methoxy-cyclohexyl)-carbamic acid benzyl ester (230 mg, 0.76 mmol), zinc powder (148 mg g, 2.67 mmol) in acetic acid (3 mL) was stirred at room temperature for 1 hr. The mixture was diluted with 50 mL of methanol and filtered to remove the solid. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 12 g,
methanol/dichloromethane = 1/99 to 10/90]. Fractions were combined and concentrated under reduced pressure giving 140 mg of titled compound. LCMS (m/z): 279.1 [M+H]+; Retention time = 0.54 min.
Step 5: Preparation of rac ((1 S,2S,4S)-4-benzyloxycarbonylamino-2-methoxy- cyclohexyl)-carbamic acid tert-butyl ester
Figure imgf000127_0001
A mixture of rac ((1S,3S,4S)-4-amino-3-methoxy-cyclohexyl)-carbamic acid benzyl ester (140 mg, 0.50 mmol), boc anhydride (110 mg, 0.50 mmol) in DCM (5 mL) was stirred at room temperature for 2 hr. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 12 g, ethyl acetate/heptane = 0/100 to 60/40]. Fractions were combined and concentrated under reduced pressure giving 145 mg of titled compound. LCMS (m/z): 379.1 [M+H]+; Retention time = 1.01 min.
Step 6: Preparation of rac ((1 S,2S,4S)-4-amino-2-methoxy-cyclohexyl)-carbamic acid tert-butyl ester
A mixture of rac ((1S,2S,4S)-4-benzyloxycarbonylamino-2-methoxy-cyclohexyl)-carbamic acid tert-butyl ester (170 mg, 0.45 mmol), Pd/C (10%. 15 mg) in methanol (10 mL) was stirred at room temperature for 2 hr. The catalyst was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 12 g, ethyl methanol/dichloromethane = 0/100 to 10/90].
Fractions were combined and concentrated under reduced pressure giving 89 mg of titled compound. LCMS (m/z): 245.1 [M+H]+; Retention time = 0.46 min.
Synthesis of rac ((1 R,2S,4R)-4-amino-2-methoxy-cvclohexyl)-carbamic acid tert-butyl ester
Figure imgf000127_0002
Step 1 : Preparation of rac ((1 R,3S,4S)-4-hydroxy-3-methoxy-cyclohexyl)-carbamic acid benzyl ester
Figure imgf000128_0001
To a solution of rac (1 R,3R,6S)-(7-oxa-bicyclo[4.1.0]hept-3-yl)-carbamic acid benzyl ester (350 mg, 1.415 mmol) in methanol (0.057 mL, 1.415 mmol) was added sulfuric acid (0.015 mL, 0.281 mmol). The mixture was stirred at 23 °C for 18 hr. To the reaction mixture was added saturated aqueous sodium bicarbonate solution to adjust pH=8. The mixture was concentrated to dryness then re-dissolved in ethyl acetate (25 mL). The organic solution washed with water, brine and dried over sodium sulfate, filtered off and concentrated under reduced pressure. The crude product was purified column chromatography [Si02, 24 g, EtOAc/heptane = 0/100 to 30/70]. Pure fractions were combined and concentrated under reduced pressure giving 280 mg of titled compound. LCMS (m/z): 280.1 [M+H]+; Retention time = 0.66 min. Step 2: Preparation of rac methanesulfonic acid (1 S,2S,4R)-4- benzyloxycarbonylamino-2-methoxy-cyclohexyl ester
Figure imgf000128_0002
s
To a solution of benzyl rac ((1 R,3S,4S)-4-hydroxy-3-methoxy-cyclohexyl)-carbamic acid benzyl ester (280 mg, 1.00 mmol) in pyridine (10 mL) was added methanesulfonyl chloride (0.117 mL, 1.504 mmol. The reaction mixture was stirred at room temperature for 18 hr. The mixture was diluted with EtOAc (-25 mL) and MeOH (~5 mL), filtered off and concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 12 g, EtOAc/heptane = 10/90 to 50/50]. Fractions were combined and concentrated under reduced pressure giving 1 10 mg of titled compound. LCMS (m/z): 358.1 [M+H]+; Retention time = 0.83min.
Step 3: Preparation of rac ((1 R,3S,4R)-4-azido-3-methoxy-cyclohexyl)-carbamic acid benzyl ester
Figure imgf000129_0001
A mixture of rac methanesulfonic acid (1S,2S,4R)-4-benzyloxycarbonylamino-2-methoxy- cyclohexyl ester (1.7 g, 4.76 mmol), sodium azide (0.928 g, 14.3 mmol) in DMF (15 mL) in a sealed tube was heated at 110 °C for 18 hr. The mixture was allowed to cool to room temperature. The mixture was diluted with 150 mL of ethyl acetate, washed with water, brine and dried over sodium sulfate, concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 12 g, EtOAc/heptane = 10/90 to 50/50]. Fractions were combined and concentrated under reduced pressure giving 1.10 g of titled compound. LCMS (m/z): 305.1 [M+H]+; Retention time = 0.99 min.
Step 4: Preparation of rac ((1 R,3S,4R)-4-amino-3-methoxy-cyclohexyl)-carbamic acid benzyl ester
Figure imgf000129_0002
A mixture of rac ((1 R,3S,4R)-4-azido-3-methoxy-cyclohexyl)-carbamic acid benzyl ester (750 mg, 2.45 mmol), zinc powder (0.484 g, 7.39 mmol) in acetic acid (15 mL) was stirred at room temperature for 1 hr. The mixture was diluted with 50 mL of methanol and filtered to remove the solid. The filtrate was concentrated under reduced pressure.
The residue was purified by column chromatography [Si02, 12 g,
methanol/dichloromethane = 1/99 to 10/90]. Fractions were combined and concentrated under reduced pressure giving 460 mg of titled compound.
LCMS (m/z): 279.1 [M+H]+; Retention time = 0.54 min.
Step 5: Preparation of rac ((1 R,3S,4R)-4-tert-butoxycarbonylamino-3-methoxy- cyclohexyl)-carbamic acid benzyl ester
Figure imgf000129_0003
A mixture of rac ((1 R,3S,4R)-4-amino-3-methoxy-cyclohexyl)-carbamic acid benzyl ester (688 mg, 2.47 mmol), boc anhydride (539 mg, 2.47 mmol) in THF (10 mL) was stirred at room temperature for 2 hr. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 12 g, ethyl acetate/heptane = 0/100 to 60/40]. Fractions were combined and concentrated under reduced pressure giving 810 mg of titled compound. LCMS (m/z): 379.1 [M+H]+; Retention time = 0.99 min.
Step 6: Preparation of rac ((1 R,2S,4R)-4-amino-2-methoxy-cyclohexyl)-carbamic acid tert-butyl ester
A mixture of rac ((1 R,3S,4R)-4-tert-butoxycarbonylamino-3-methoxy-cyclohexyl)- carbamic acid benzyl ester (1.66 g, 4.39 mmol), 10% Pd/C (140 mg, 0.13 mmol) in methanol (40 mL) was stirred under hydrogen at room temperature for 2 hr. The mixture was filtered to remove the solid. Filtrate was concentrated under reduced pressure. The residue was purified by column chromatography [Si02, 40 g, methanol/dichloromehtane = 0/100 to 10/90]. Fractions were combined and concentrated under reduced pressure giving 510 mg of titled compound. LCMS (m/z): 245.1 [M+H]+; Retention time = 0.59 min.
Example 1a (Compound 1)
N2'-(frans-4-aminocyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000130_0001
Step 1. Preparation of frans-N1-(5-chloro-4-iodopyridin-2-yl)cyclohexane-1 ,4-diamine
A mixture of 5-chloro-2-fluoro-4-iodopyridine (1000 mg, 3.88 mmol), DMSO (7 ml), and frans-cyclohexane-1 ,4-diamine (2661 mg, 23.31 mmol) reaction mixture was stirred at about 85 °C for 2 hours, followed by LCMS. The crude reaction mixture then was mixed with 5 ml DMSO, filtered and purified by prep LC. After lyophilization, 1.17 grams of the title compound was obtained as a TFA salt. LCMS (m/z): 352.1 (MH+), retention time = 0.50 min. Step 2. Preparation of frans-N1-(5'-chloro-6-fluoro-2,4'-bipyridin-2'-yl)cyclohexane-1 ,4- diamine
A mixture of frans-N1-(5-chloro-4-iodopyridin-2-yl)cyclohexane-1 ,4-diamine (from step 1 above, 300 mg, 0.853 mmol), 2-fluoro-6-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan- 2-yl)pyridine (285 mg, 1.280 mmol), PdCI2(dppf).CH2CI2 adduct (84 mg, 0.102 mmol), DME (4 ml), Ethanol (1 ml), and 2M sodium carbonate (1.706 ml, 3.41 mmol) reaction mixture was stirred at about 90 °C until done by LCMS. The reaction mixture was cooled, then diluted with 25 ml of ethyl acetate and 10 ml of methanol, filtered, and concentrated to yield a crude solid. The crude solid was dissolved in DMSO, filtered and purified by prep LC. After lyophilization, 200 mg of the title compound was obtained as a TFA salt. LCMS (m/z): 321.0 (MH+), retention time = 0.48 min.
Step 3. Preparation of N2'-(frans-4-aminocyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine
To frans-N1-(5'-chloro-6-fluoro-2,4'-bipyridin-2'-yl)cyclohexane-1 ,4-diamine (from Step 2 above, 200 mg, 0.623 mmol) was added DMSO (2 ml) and (3- fluorophenyl)methanamine (351 mg, 2.81 mmol). The crude reaction mixture was stirred at 115 °C until done, as indicated by LCMS. The excess amine was removed under reduced pressure. The resulting crude residue was dissolved in 2 ml of DMSO, filtered, purified by prep LC and lyophilized to yield a TFA salt. The TFA salt was free- based using 200 ml of ethyl acetate and washed with saturated sodium bicarbonate 35 ml (1x), water (2x), saturated brine (1x), dried over sodium sulfate, filtered and concentrated to yield a solid. The solid was dissolved in (1 :1 ACN/ water), filtered, and lyophilized to yield 80 mg of the title compound as free-base. LCMS (m/z): 426.1 (MH+), retention time = 0.61 min.; 1 H NMR (300 MHz, METHANOL-d4, 25 °C) 1.21 - 1.40 (m, 4 H) 1.89 - 2.00 (m, 2 H) 2.07 (d, J=10.56 Hz, 2 H) 2.69 - 2.79 (m, 1 H) 3.55 - 3.64 (m, 1 H) 4.57 (s, 2 H) 6.53 (d, J=8.61 Hz, 1 H) 6.59 (s, 1 H) 6.80 (d, J=7.04 Hz, 1 H) 6.90 - 6.97 (m, 1 H) 7.09 (d, J=10.17 Hz, 1 H) 7.14 - 7.20 (m, 1 H) 7.25 - 7.34 (m, 1 H) 7.48 (t, J=7.83 Hz, 1 H) 7.93 (s, 1 H) Example 1 b (Compound 1)
N2'-(franss-4-aminocyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000132_0001
Step! Preparation of 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine
A mixture of 2,6-dibromopyridine (7.1 g, 30.0 mmol), NMP (16 ml), (3- fluorophenyl)methanamine (4.13 g, 33.0 mmol) and Hunig's Base (5.76 ml, 33.0 mmol) was flushed with argon. The crude reaction mixture was stirred at 1 15-120 °C for about 168 hours. LC/MS was used to monitor the reaction. The crude mixture was then cooled to room temperature, and then diluted with 250 ml of ethyl acetate, washed with saturated sodium bicarbonate (2x), water (2x), saturated, salt solution (1x), dried over sodium sulfate, filtered, and concentrated under reduced pressure to yield a residue. The residue was purified by silica gel chromatography using a 120 g column, eluting from 0%-20% ethyl acetate with hexane. The desired fractions were concentrated to yield, 7.11 grams of the titled compound as a free base, which was used in the next step without further purification. LCMS (m/z): 281.1/283.1 (MH+), retention time = 1.03 min. Step 2. Preparation of 5'-chloro-2'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-6-amine
A mixture of 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine (2.0 g, 7.11 mmol), 5- chloro-2-fluoropyridin-4-ylboronic acid (1.996 g, 1 1.38 mmol), PdCl2(dppf).CH2Cl2 adduct (0.465 g, 0.569 mmol), DME (27 ml), and 2M sodium carbonate (9.25 ml, 18.50 mmol) reaction mixture was stirred at about100 °C for 3 hours. The crude mixture was cooled to room temperature, diluted with 25 ml ethyl acetate and 20 ml methanol, filtered and concentrated to yield crude residue. The crude residue was purified by silica gel chromatography using a 120 g column, eluting from 0%-20% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, to yield 1.259 grams of titled compound as free base, which was used in the next step without further purification. LCMS (m/z): 332.2 (MH+), retention time = 0.92 min. Step 3. Preparation of N2'-(frans-4-aminocyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine A mixture of 5'-chloro-2'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-6-amine (725 mg, 2.185 mmol) was added DMSO (7 ml), frans-cyclohexane-1 ,4-diamine (1996 mg, 17.48 mmol) and TEA (0.609 ml, 4.37 mmol) was stirred at about 100 °C for 20 hours. The reaction was monitored by LC/MS. The crude reaction mixture was cooled to room temperature, diluted with 3 ml DMSO, filtered, and purified by prep HPLC. (there is a general HPLC conditions in the general experimental session). The fractions were concentrated, mixed with 500 ml ethyl acetate, and basified with saturated sodium bicarbonate 120 ml. The ethyl acetate layer was separated, and the basic water layer was extracted with 300 ml ethyl acetate. The ethyl acetate layers were combined and washed with water (3x), saturated salt solution (1x), dried with sodium sulfate, filtered and concentrated to yield a solid. The solid was dissolved in (1 : 1 ACN/ water) filtered and lyophilized to yield 755 mg of the title compound as free-base. LCMS (m/z): 426.3 (MH+), retention time = 0.59 min.; 1 H NMR (300 MHz, METHANOL-d4, 25 °C) δ ppm 1.10 - 1.43 (m, 4 H) 1.90 (d, J=12.01 Hz, 2 H) 2.01 (d, J=12.01 Hz, 2 H) 2.70 - 2.84 (m, 1 H) 3.47 - 3.60 (m, 1 H) 4.48 (s, 2 H) 6.44 (d, J=8.50 Hz, 1 H) 6.51 (s, 1 H) 6.71 (d, J=7.33 Hz, 1 H) 6.79 - 6.91 (m, 1 H) 7.00 (d, J=9.96 Hz, 1 H) 7.05 - 7.13 (m, 1 H) 7.15 - 7.27 (m, 1 H) 7.40 (t, J=7.77 Hz, 1 H) 7.85 (s, 1 H)
Example 2 (Compound 2)
N2'-(franss-4-aminocyclohexyl)-N6-(cyclohexylmethyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000133_0001
Step 1. Preparation of frans-N1-(4-bromopyridin-2-yl)cyclohexane-1 ,4-diamine A mixture of 4-bromo-2-chloropyridine (1500 mg, 7.79 mmol), DMSO (15 ml), and frans-cyclohexane-1 ,4-diamine (4450 mg, 39.0 mmol)was stirred at 100 °C until the formation of the product, as indicated by LCMS. The reaction mixture was cooled to room temperature, filtered and purified by prep LC, and lyophilized to yield 393mg of the title compound as a TFA salt. LCMS (m/z): 270.2/272.2 (MH+), retention time = 0.31 min.
Step 2. Preparation of frans-N1-(6-fluoro-2,4'-bipyridin-2'-yl)cyclohexane-1 ,4-diamine
A mixture of frans-N1-(4-bromopyridin-2-yl)cyclohexane-1 ,4-diamine (102 mg, 0.377 mmol), 2-fluoro-6-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine (80 mg, 0.359 mmol), PdCI2(dppf).CH2CI2 adduct (29.3 mg, 0.036 mmol), DME (2 ml), Ethanol (0.2 ml), and 2M sodium carbonate (0.717 ml, 1.435 mmol) reaction mixture was stirred at about 85 °C until completion, as indicated by LCMS. The crude mixture was cooled to room temperature, diluted with 5 ml of ethyl acetate and 2 ml of methanol, filtered and concentrated to yield a crude solid. The solid was dissolved in DMSO, refiltered, purified by prep LC, and lyophilized to yield 64 mg of the title compound as its TFA salt. LCMS (m/z): 287.2 (MH+), retention time = 0.43 min.
Step 3. Preparation of N2'-(franss-4-aminocyclohexyl)-N6-(cyclohexylmethyl)-2,4'- bipyridine-2',6-diamine
A mixture of transs-N 1-(6-fluoro-2,4'-bipyridin-2'-yl)cyclohexane-1 ,4-diamine (15 mg, 0.052 mmol), DMSO (0.4 ml), and cyclohexylmethanamine (59.3 mg, 0.524 mmol) was heated at about 105 °C for about 24 hours, or until the product formation was completed, as indicated by LCMS. The excess amine was removed under reduced pressure to yield a residue. The residue was mixed with 0.5 ml of DMSO, filtered and purified by prep LC. After lyophilization, 1 1.3 mg of the title compound was obtained as a TFA salt. LCMS (m/z): 380.3 (MH+), retention time = 0.61 min. H NMR (400 MHz, METHANOL-d4, 45 °C) δ ppm 0.97 - 1.1 1 (m, 2 H) 1.17 - 1.36 (m, 3 H) 1.49 - 1.72 (m, 6 H) 1.71 - 1.80 (m, 2 H) 1.84 (d, J=12.91 Hz, 2 H) 2.11 - 2.28 (m, 4 H) 3.13 - 3.25 (m, 1 H) 3.28 (d, 2 H, App.) 3.65 - 3.75 (m, 1 H) 6.65 (d, J=8.61 Hz, 1 H) 7.16 (d, J=7.43 Hz, 1 H) 7.43 - 7.48 (m, 1 H) 7.52 (t, J=7.83 Hz, 1 H) 7.64 (s, 1 H) 7.85 (d, J=7.04 Hz, 1 H)
Example 3 (Compound 3)
frans-N1-(5-chloro-4-(6-(cyclohexylmethylamino)pyridin-2-yl)pyrimidin-2-yl)cyclohexane- 1 ,4-diamine
Figure imgf000135_0001
Step 1. Preparation of 2,5-dichloro-4-(6-fluoropyridin-2-yl)pyrimidine A mixture of 2,4,5-trichloropyrimidine (49.3 mg, 0.269 mmol), 2-fluoro-6-(4, 4,5,5- tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine (50 mg, 0.224 mmol), PdCl2(dppf).CH2Cl2 adduct (18.31 mg, 0.022 mmol), DME (0.7 ml), and 2M sodium carbonate (0.247 ml, 0.493 mmol) reaction mixture was stirred at about 80 °C until the reaction mixture was complete, as indicated by LCMS. The reaction mixture was cooled, diluted with 5 ml of ethyl acetate and 1 ml of methanol, filtered and concentrated to yield a crude solid. The crude material was purified by silica gel chromatography using a 12g column, eluting from 0%-40% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, to yield 39.5 mg of titled compound as a free base. LCMS (m/z): 244.0 (MH+), retention time = 0.89 min.
Step 2. Preparation of frans-N1-(5-chloro-4-(6-fluoropyridin-2-yl)pyrimidin-2- yl)cyclohexane-1 ,4-diamine
A mixture of 2,5-dichloro-4-(6-fluoropyridin-2-yl)pyrimidine (37 mg, 0.152 mmol), DMSO (1.5 ml) and frans-cyclohexane-1 ,4-diamine (87 mg, 0.758 mmol)reaction mixture was stirred at about 75 °C for about 2 hours. The reaction mixture was cooled, filtered and purified by prep LC, and then lyophilized to yield 39.5 mg of the title compound as a TFA salt. LCMS (m/z): 322.2(MH+), retention time = 0.59 min. Step 3. Preparation of frans-N1-(5-chloro-4-(6-(cyclohexylmethylamino)pyridin-2- yl)pyrimidin-2-yl)cyclohexane-1 ,4-diamine
A mixture of frans-N1-(5-chloro-4-(6-fluoropyridin-2-yl)pyrimidin-2-yl)cyclohexane- 1 ,4-diamine (12 mg, 0.037 mmol), cyclohexylmethanamine (42.2 mg, 0.373 mmol), and DMSO (0.35 ml) was stirred at about 105 °C for about 24 hours. The excess
cyclohexylmethanamine was removed under vacuum to yield a residue. The residue was mixed with 0.5 ml DMSO, filtered, purified by prep HPLC and then lyophilized to yield 9.4 mg of the title compound as a TFA salt. LCMS (m/z): 415.3 (MH+), retention time = 0.67 min.; 1 H NMR (400 MHz, METHANOL-d4, 45 °C) δ ppm 0.89 - 1.07 (m, 2 H) 1.10 - 1.30 (m, 3 H) 1.30 - 1.54 (m, 4 H) 1.55 - 1.65 (m, 2 H) 1.69 (d, J=12.91 Hz, 2 H) 1.76 (d, J=12.91 Hz, 2 H) 1.96 - 2.14 (m, 4 H) 2.98 - 3.10 (m, 1 H) 3.18 (d, J=6.65 Hz, 2 H) 3.71 - 3.82 (m, 1 H) 7.03 (d, J=9.00 Hz, 1 H) 7.49 (br. s., 1 H) 7.83 (t, J=8.22 Hz, 1 H) 8.35 (s, 1 H)
Example 4 (Compound 4)
(N2'-(frans-4-(aminomethyl)cyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6- diamine
Figure imgf000136_0001
Step! Preparation of tert-butyl (frans-4-(5-chloro-4-iodopyridin-2-yl- amino)cyclohexyl)methylcarbamate
A mixture of 5-chloro-2-fluoro-4-iodopyridine (517 mg, 2.008 mmol), tert-butyl (frans-4-aminocyclohexyl)methylcarbamate (550 mg, 2.410 mmol), DMSO (2 ml) and TEA (0.336 ml, 2.410 mmol) reaction mixture was stirred at about 95 °C for about 26 hours. The crude reaction mixture was cooled to room temperature, mixed with 125 ml ethyl acetate, washed with saturated sodium bicarbonate (2x), water (3x), saturated salt solution (1x), dried sodium sulfate, filtered and concentrated under reduced pressure to yield a residue. The residue was purified by silica gel chromatography using a 40g column, eluting from 0%-35% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, yielding 656 mg of titled compound as free base. LCMS (m/z): 466.1 (MH+), retention time = 0.93 min.
Step 2. Preparation of tert-butyl (frans-4-(5'-chloro-6-fluoro-2,4'-bipyridin-2'-yl- amino)cyclohexyl)methylcarbamate
A mixture of tert-butyl (frans-4-(5-chloro-4-iodopyridin-2-yl- amino)cyclohexyl)methylcarbamate (510 mg, 1.095 mmol), 2-fluoro-6-(4, 4,5,5- tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine (440 mg, 1.971 mmol), PdCl2(dppf).CH2Cl2 adduct (89 mg, 0.109 mmol), DME (7.5 ml), and 2M sodium carbonate (2.464 ml, 4.93 mmol) reaction mixture was stirred at about 100 °C for about 2 hours. The reaction mixture was cooled to room temperature, mixed with 20 ml ethyl acetate, filtered and concentrated to yield a crude solid. The crude solid was purified by silica gel chromatography using 40g column, eluting from 0%-45% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, yielding 396 mg of titled compound as a free base. LCMS (m/z): 435.2(MH+), retention time = 0.85 min..
Step 3. Preparation of N-(frans-4-(aminomethyl)cyclohexyl)-5'-chloro-6-fluoro-2,4'- bipyridin-2'-amine
A mixture of tert-butyl (frans-4-(5'-chloro-6-fluoro-2,4'-bipyridin-2'-yl- amino)cyclohexyl)methylcarbamate (390 mg, 0.897 mmol), 4M HCI in Dioxane (5604 μΙ, 22.42 mmol) reaction mixture was stirred at ambient temperature for 1 hr. The crude reaction mixture was concentrated, and then dried under high vacuum to a constant mass giving 335 mg of the title compound as a HCL salt. LCMS (m/z): 335.1 (MH+), retention time = 0.51 min.
Step 4. Preparation of N2'-(frans-4-(aminomethyl)cyclohexyl)-5'-chloro-N6-(3- fluorobenzyl)-2,4'-bipyridine-2',6-diamine
A mixture of N-(frans-4-(aminomethyl)cyclohexyl)-5'-chloro-6-fluoro-2,4'-bipyridin- 2'-amine (15 mg, 0.045 mmol), DMSO (0.35 ml), TEA (0.012 ml, 0.090 mmol) and (3- fluorophenyl)methanamine (50.5 mg, 0.403 mmol) reaction mixture was flushed with argon and then stirred at about 105 °C for about 40 hours. The excess (3- fluorophenyl)methanamine was removed under reduced pressure to yield a crude material, which was mixed with 0.5 ml DMSO, filtered, purified by prep LC, and then lyophilized to yield 11.2 mg of the title compound, as a TFA salt. LCMS (m/z):
440.2(MH+), retention time = 0.62 min. H NMR (300 MHz, METHANOL-d4, 25 °C) δ ppm 1.11 - 1.28 (m, 2 H) 1.28 - 1.47 (m, 2 H) 1.67 (ddd, J=10.92, 7.40, 3.66 Hz, 1 H)
1.92 (d, J=1 1.72 Hz, 2 H) 2.14 (d, J=10.55 Hz, 2 H) 2.83 (d, J=6.74 Hz, 2 H) 3.57 - 3.69 (m, 1 H) 4.63 (s, 2 H) 6.84 (d, J=8.79 Hz, 1 H) 6.90 (s, 1 H) 6.94 (d, J=7.03 Hz, 1 H) 6.96 - 7.03 (m, 1 H) 7.10 (d, J=9.96 Hz, 1 H) 7.18 (d, J=7.62 Hz, 1 H) 7.29 - 7.39 (m, 1 H) 7.69 - 7.77 (m, 1 H) 8.01 (s, 1 H)
Example 5 (Compound 5) (5'-chloro-N6-(3-fluorobenzyl)-N2'-(piperidin-4-yl)-2,4'-bipyridine-2\6-diarnine
Figure imgf000138_0001
Step 1. Preparation of tert-butyl 4-(5-chloro-4-iodopyridin-2-yl-amino)piperidine-1- carboxylate
A mixture of 5-chloro-2-fluoro-4-iodopyridine (517 mg, 2.008 mmol), tert-butyl 4- aminopiperidine-1 -carboxylate (603 mg, 3.01 mmol), DMSO (2 ml) and TEA (0.420 ml, 3.01 mmol) reaction mixture was stirred at 90 °C for 18 hours. The reaction mixture was cooled to room temperature, mixed with 150 ml of ethyl acetate, washed with saturated sodium bicarbonate (2x), water (3x), saturated salt solution (1x), dried sodium sulfate, filtered and concentrated to yield a crude material, which was purified by silica gel chromatography using a 40g column, eluting from 0%-40% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, giving 585 mg of the title compound as free base. LCMS (m/z): 438.1 (MH+), retention time = 1.00 min.
Step 2. Preparation of tert-butyl 4-(5'-chloro-6-fluoro-2,4'-bipyridin-2'-yl-amino)piperidine- 1 -carboxylate A mixture of tert-butyl 4-(5-chloro-4-iodopyridin-2-yl-amino)piperidine-1- carboxylate (468 mg, 1.069 mmol), 2-fluoro-6-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)pyridine (429 mg, 1.925 mmol), PdCI2(dppf).CH2CI2 adduct (87 mg, 0.107 mmol), DME (7.5 ml), and 2M sodium carbonate (2.406 ml, 4.81 mmol) reaction mixture was stirred at 100 °C for 2 hr. The reaction mixture was cooled to room temperature, mixed with 20 ml of ethyl acetate, filtered and concentrated to yield a crude material. The crude material was purified by silica gel chromatography using a 40g column, eluting from 0%-40% ethyl acetate with hexane. The desired fractions were combined and concentrated to constant mass, giving 360 mg of the title compound as free base. LCMS (m/z): 407.2 (MH+), retention time = 0.85 min. Step 3. Preparation of tert-butyl 4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridi amino)piperidine-1-carboxylate
A mixture of tert-butyl 4-(5'-chloro-6-fluoro-2,4'-bipyridin-2'-yl-amino)piperidine-1- carboxylate (200 mg, 0.492 mmol), DMSO (2 ml), TEA (0.137 ml, 0.983 mmol) and (3- fluorophenyl)methanamine (554 mg, 4.42 mmol) reaction mixture was flushed with argon and stirred at 100 °C for 40 hr, as the reaction mixture progress was followed by LCMS. The reaction mixture was cooled to room temperature, mixed with 150 ml of ethyl acetate, washed with saturated sodium bicarbonate (2x), water (3), saturated salt solution (1x), dried over sodium sulfate, filtered and concentrated to yield a crude material, which was purified by silica gel chromatography using a 12g column, eluting from 0%-35% ethyl acetate with hexane. The desired fractions were collected and concentrated to constant mass, giving 225 mg of the title compound as a free base. LCMS (m/z): 512.3 (MH+), retention time = 0.91 min.
Step 4. Preparation of 5'-chloro-N6-(3-fluorobenzyl)-N2'-(piperidin-4-yl)-2,4'-bipyridine- 2',6-diamine
A mixture of tert-butyl 4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl- amino)piperidine-1-carboxylate (220 mg, 0.430 mmol), HCI 4M in Dioxane (7 mL, 28.0 mmol) was stirred at ambient temperature for 1 hr. The solvent was evaporated under reduced pressure to yield a solid which was further dried under high vacuum to yield 250mg of the title compound as a HCI salt. A portion of the title compound was purified by prep LC, and then lyophilized to yield 19.0 mg of the title compound as a TFA salt. . LCMS (m/z): 412.2 (MH+), retention time = 0.60 min.; 1 H NMR (300 MHz, METHANOL- d4, 25 °C ) δ ppm 1.66 - 1.83 (m, 2 H) 2.25 (dd, J=14.21 , 3.08 Hz, 2 H) 3.08 - 3.21 (m, 2 H) 3.36 - 3.51 (m, 2 H) 3.96 - 4.12 (m, 1 H) 4.65 (s, 2 H) 6.74 (s, 1 H) 6.91 (s, 1 H) 6.94 (s, 1 H) 6.98 - 7.06 (m, 1 H) 7.12 (d, J=9.96 Hz, 1 H) 7.19 (d, J=7.62 Hz, 1 H) 7.31 - 7.43 (m, 1 H) 7.77 - 7.85 (m, 1 H) 8.09 (s, 1 H)
Example 6 (Compound 6)
5'-chloro-N2'-(1-(ethylsulfonyl)piperidin-4-yl)-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6- diamine
Figure imgf000140_0001
Preparation of 5'-chloro-N2'-(1-(ethylsulfonyl)piperidin-4-yl)-N6-(3-fluoro benzyl)-2,4'- bipyridine-2',6-diamine
A mixture of 5'-chloro-N6-(3-fluorobenzyl)-N2'-(piperidin-4-yl)-2,4'-bipyridine-2',6- diamine (Example 6, 16 mg, 0.039 mmol), dichloromethane (0.5 ml), and TEA (0.022 ml, 0.155 mmol) was cooled to 0 °C. This cooled mixture was then diluted with a solution of 0.03 ml of dichloromethane with ethanesulfonyl chloride (6.99 mg, 0.054 mmol). The reaction mixture then was warmed to ambient temperature and stirred for 1 hour, followed by LCMS. The reaction mixture solvent was removed under reduced pressure, to yield a residue which was dissolved in 0.75 ml DMSO, filtered, purified by prep LC and then lyophilized to yield 9.9 mg of the title compound, as a TFA salt. LCMS (m/z): 504.2 (MH+), retention time = 0.77 min.; 1 H NMR (300 MHz, METHANOL-d4, 25 °C) δ ppm 1.32 (t, J=7.33 Hz, 3 H) 1.47 - 1.67 (m, 2 H) 2.08 (d, J=10.84 Hz, 2 H) 2.96 - 3.12 (m, 4 H) 3.75 (d, J=12.89 Hz, 2 H) 3.80 - 3.92 (m, 1 H) 4.65 (s, 2 H) 6.83 (s, 1 H) 6.92 (d, J=9.08 Hz, 1 H) 6.95 (d, J=7.62 Hz, 1 H) 7.01 (t, J=8.64 Hz, 1 H) 7.1 1 (d, J=9.96 Hz, 1 H) 7.19 (d, J=7.62 Hz, 1 H) 7.30 - 7.41 (m, 1 H) 7.75 - 7.85 (m, 1 H) 8.06 (s, 1 H) Example 7 (Compound 7)
N-(frans-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)cyclohexyl)-2- (dimethylamino)acetamide
Figure imgf000140_0002
Preparation of N-(frans-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl- amino)cyclohexyl)-2-(dimethylamino)acetamide
A mixture of 2-(dimethylamino)acetic acid (6.05 mg, 0.059 mmol), NMP (0.5 ml), Huenig's Base (0.023 ml, 0.132 mmol), and HATU (24.55 mg, 0.065 mmol) was stirred at ambient temperature for 5 minutes, folowed by addition of N2'-(trans-4- aminocyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6-diamine (Example 1) (12.5 mg, 0.029 mmol). The resulting mixture was stirred at ambient temperature for 4 hours. The crude reaction mixture was diluted with 0.25 ml of DMSO, filtered, purified by prep LC and then lyophilized to yield 6.8 mg of the title compound, as a TFA salt.
LCMS (m/z): 511.3 (MH+), retention time = 0.62 min.; 1 H NMR (300 MHz, METHANOL- d4, 25 °C) δ ppm 1.32 - 1.53 (m, 4 H) 1.98 - 2.07 (m, 2 H) 2.07 - 2.18 (m, 2 H) 2.92 (s, 6 H) 3.60 - 3.68 (m, 1 H) 3.70 - 3.82 (m, 1 H) 3.90 (s, 2 H) 4.63 (s, 2 H) 6.83 (d, J=8.79 Hz, 1 H) 6.86 (s, 1 H) 6.93 (d, J=7.03 Hz, 1 H) 6.99 (s, 1 H) 7.10 (d, J=9.67 Hz, 1 H) 7.18 (d, J=7.62 Hz, 1 H) 7.28 - 7.40 (m, 1 H) 7.68 - 7.77 (m, 1 H) 8.01 (s, 1 H)
Example 8 (Compound 8)
frans-4-(5'-chloro-6-(piperidin-4-yl-amino)-2,4'-bipyridin-2'-yl-amino)cyclohexanol
Figure imgf000141_0001
Step 1. Preparation of frans-4-(5-chloro-4-iodopyridin-2-yl-amino)cyclohexanol
To 5-chloro-2-fluoro-4-iodopyridine (600 mg, 2.331 mmol) was added DMSO (2.2 ml), frans-4-aminocyclohexanol (1074 mg, 9.32 mmol) and TEA (0.390 ml, 2.80 mmol). The resulting reaction mixture was stirred at 75 °C for 24 hr, followed by LCMS. The reaction mixture was cooled to room temperature, mixed with 150 ml of ethyl acetate, washed with saturated sodium bicarbonate (1x), water (1x), saturated salt solution (1x), dried over sodium sulfate, filtered and concentrated to yield a crude material. The crude material was purified by silica gel chromatography using a 40g column eluting from15%- 75% ethyl acetate with hexane. The desired fractions were combined and concentrated to constant mass, giving 750 mg of the title compound as free base, which was used in the next step without further purification. LCMS (m/z): 353.0 (MH+), retention time = 0.56 min.
Step 2. Preparation of frans-4-(5'-chloro-6-fluoro-2,4'-bipyridin-2'-yl-amino)cyclohexanol
A mixture of frans-4-(5-chloro-4-iodopyridin-2-yl-amino)cyclohexanol (575 mg, 1.631 mmol), 2-fluoro-6-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine (655 mg, 2.94 mmol), PdCI2(dppf).CH2Cl2 adduct (133 mg, 0.163 mmol), DME (15 ml), and 1 2M sodium carbonate (4.48 ml, 8.97 mmol) reaction mixture was stirred at 95 °C for 2 hr, followed by LCMS. The reaction mixture was cooled to room temperature, mixed with 20 ml of ethyl acetate, 15 ml of methanol, , filtered and concentrated to yield a crude product. The crude was purified by silica gel chromatography using a 40g column, eluting from 35%-85% ethyl acetate with hexane. The desired fractions were combined and concentrated to constant mass, giving 440 mg of titled compound as free base. LCMS (m/z): 322.2(MH+), retention time = 0.53 min.
Step 3. Preparation of frans-4-(5'-chloro-6-(piperidin-4-yl-amino)-2,4'-bipyridin-2'-yl- amino)cyclohexanol A mixture of frans-4-(5'-chloro-6-fluoro-2,4'-bipyridin-2'-yl-amino)cyclohexanol
(15.5 mg, 0.048 mmol), DMSO (0.4 ml), and tert-butyl 4-aminopiperidine-1-carboxylate (48.2 mg, 0.241 mmol) reaction mixture was stirred at 105 °C for 40 hr. LCMS indicated formation of the intermediate tert-butyl 4-(5'-chloro-2'-(trans-4-hydroxycyclohexylamino)- 2,4'-bipyridin-6-yl-amino)piperidine-1-carboxylate (LCMS (m/z): 502.4(MH+), retention time = 0.70 min.). The Boc protecting group was removed from the intermediate by adding HCL 6M aq (140 μΙ, 0.840 mmol) to the crude reaction mixture, followed by stirring the mixture at 90 °C for 45 minutes. The reaction mixture was cooled, 0.5 ml of DMSO was added, filtered and purified by prep LC. Lyophilization of the material yielded 9.8 mg of the title compound, as a TFA salt. LCMS (m/z): 402.3 (MH+), retention time = 0.41 min.; 1 H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.32 - 1.52 (m, 4 H) 1.71 - 1.87 (m, 2 H) 1.96 - 2.12 (m, 4 H) 2.27 (dd, J=14.21 , 3.37 Hz, 2 H) 3.06 - 3.18 (m, 2 H) 3.39 - 3.50 (m, 2 H) 3.54 - 3.68 (m, 2 H) 4.05 - 4.17 (m, 1 H) 6.72 (d, J=8.50 Hz, 1 H) 6.90 (d, J=7.33 Hz, 1 H) 7.00 (s, 1 H) 7.56 - 7.64 (m, 1 H) 8.01 (s, 1 H) Example 9 (Compound 9) N-(frans-4-(aminomethyl)cyclohexyl)-5'-chloro-6-(3-fluorobenzyloxy)-2,4'-bipyridin-2L amine
Figure imgf000143_0001
Step 1. Preparation of 2-bromo-6-(3-fluorobenzyloxy)pyridine
To 2-bromo-6-fluoropyridine (176 mg, 1.000 mmol) was added DMF (1.5 ml) and (3-fluorophenyl)methanol (139 mg, 1.100 mmol) and cesium carbonate (391 mg, 1.200 mmol), and the resulting mixture reaction mixture was stirred at 95 °C for 6 hr, as the progress of the reaction mixture was followed by LCMS. The reaction mixture was cooled to room temperature, diluted with 120 ml of ethyl acetate, washed with saturated sodium bicarbonate (1x), water (1x), saturated salt solution (1x), dried over sodium sulfate, filtered and concentrated to yield a crude product which was purified by silica gel chromatography using a12g column eluting from 0%-20% ethyl acetate with hexane. The desired fractions were combined and concentrated to constant mass, giving 156 mg of the title compound as a free base. LCMS (m/z): 282.0/284.0 (MH+), retention time = 1.19 min. Step 2. Preparation of 5'-chloro-2'-fluoro-6-(3-fluorobenzyloxy)-2,4'-bipyridine
A mixture of 2-bromo-6-(3-fluorobenzyloxy)pyridine (145 mg, 0.514 mmol), 5- chloro-2-fluoropyridin-4-ylboronic acid (144 mg, 0.822 mmol), Palladium Tetrakis (71.3 mg, 0.062 mmol), DME (3 ml), and 1 2M sodium carbonate (1.028 ml, 2.056 mmol) was reaction mixture was stirred at 100 °C for 3 hr, followed by LCMS. The reaction mixture was cooled, diluted with 10 ml of ethyl acetate, filtered and concentrated to yield a crude product, which was purified by silica gel chromatography using a 12g column eluting from 0%-20% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, giving 100 mg of titled compound as a free base. LCMS (m/z): 333.1 (MH+), retention time = 1.26 min. Step 3. Preparation of N-(frans-4-(aminomethyl)cyclohexyl)-5'-chloro-6-(3- fluorobenzyloxy)-2,4'-bipyridin-2'-amine
To 5'-chloro-2'-fluoro-6-(3-fluorobenzyloxy)-2,4'-bipyridine (30 mg, 0.090 mmol) was added DMSO (0.8 ml), TEA (0.025 ml, 0.180 mmol), and tert-butyl {trans-4- aminocyclohexyl)methylcarbamate (41.2 mg, 0.180 mmol). The reaction mixture was flushed with argon and stirred at 100-105 °C for 40 hr. Formation of the intermediate product tert-butyl (trans-4-(5'-chloro-6-(3-fluorobenzyloxy)-2,4'-bipyridin-2'-yl- amino)cyclohexyl)methylcarbamatewas indicated by LCMS. (LCMS (m/z): 541.4 (MH+), retention time = 1.05 min.). The solvent DMSO was removed under reduce pressure. The Boc group was removed from the intermediate by adding 4M HCI in Dioxane (1.5 ml, 6.00 mmol), followed with stirring at ambient temperature for 90 minutes. The solvent was removed under reduced pressure. The crude product was dissolved in 1.0 ml of DMSO with 0.075 ml of water, filtered and purified by prep LC. After lyophilization, 28.3 mg of the title compound was obtained as a TFA salt. LCMS (m/z): 441.3(MH+), retention time = 0.76 min.; H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.12 - 1.29 (m, 2 H) 1.29 - 1.47 (m, 2 H) 1.60 - 1.76 (m, J=14.76, 7.51 , 3.66, 3.66 Hz, 1 H) 1.92 (d, J=12.60 Hz, 2 H) 2.16 (d, J=10.55 Hz, 2 H) 2.84 (d, J=6.74 Hz, 2H) 3.58 - 3.71 (m, 1 H) 5.43 (s, 2 H) 6.92 - 6.99 (m, 2 H) 6.99 - 7.08 (m, 1 H) 7.18 (d, J=9.67 Hz, 1 H) 7.25 (d, J=7.62 Hz, 1 H) 7.30 - 7.42 (m, 2 H) 7.83 (t, J=7.77 Hz, 1 H) 8.01 (s, 1 H)
Example 10 (Compound 10)
frans-N1-benzyl-N4-(4-(6-(3-fluorobenzylamino)pyrazin-2-yl)pyridin-2-yl)cyclohexane- 1 ,4-diamine
Figure imgf000144_0001
Step 1. Preparation of 6-chloro-N-(3-fluorobenzyl)pyrazin-2-amine: To 2,6- dichloropyrazine (175 mg, 1.175 mmol) was added DMSO (1.5 ml), TEA (0.196 ml, 1.410 mmol) and (3-fluorophenyl)methanamine (368 mg, 2.94 mmol)l. The reaction mixture then was stirred at 90 °C until completion as indicated by LCMS, about 1 hour. To the reaction mixture was added 3 ml of DMSO, filtered and the residue was purified by prep LC. After lyophilization, 160 mg of the title compound was obtained as a TFA.
(m/z): 238.1 (MH+), retention time = 0.96 min.
Step 2. Preparation of N-(3-fluorobenzyl)-6-(2-fluoropyridin-4-yl)pyrazin-2-amine: To 6-chloro-N-(3-fluorobenzyl)pyrazin-2-amine (140 mg, 0.589 mmol) was added 2- fluoropyridin-4-ylboronic acid (125 mg, 0.884 mmol), PalladiumTetrakis (82 mg, 0.071 mmol), DME (3.3 ml), and 2M sodium carbonate (1.031 ml, 2.062 mmol) . The resulting reaction mixture was stirred at 1 10 °C until completion as indicated by LCMS, about 3 hours. The reaction mixture was cooled to room temperature, diluted with 20 ml of ethyl acetate, filtered and concentrated to yield a crude solid. The solid was dissolved in
DMSO, filtered and purified by prep LC. After lyophilization, 72 mg of the title compound was obtained as a TFA salt,. LCMS (m/z): 299.1 (MH+), retention time = 0.89 min.
Step 3. Preparation of frans-N1-(4-(6-(3-fluorobenzylamino)pyrazin-2-yl)pyridin-2- yl)cyclohexane-1 ,4-diamine:
To N-(3-fluorobenzyl)-6-(2-fluoropyridin-4-yl)pyrazin-2-amine (30 mg, 0.101 mmol) was added DMSO (0.6 ml) and frans-cyclohexane-1 ,4-diamine (115 mg, 1.006 mmol). The reaction mixture then was stirred at 105 °C until completion as indicated by LCMS, about 40 hours. To the crude reaction mixture, after cooling to room
temperature, the mixture was added 0.75 ml of DMSO, the resulting mixture filtered and purified by prep LC. After lyophilization, 34 mg of the title compound was obtained as a TFA salt. LCMS (m/z): 393.2 (MH+), retention time = 0.54 min.
Step 4. Preparation of frans-N1-benzyl-N4-(4-(6-(3-fluorobenzylamino) pyrazin-2- yl)pyridin-2-yl)cyclohexane-1 ,4-diamine:
To frans-N1-(4-(6-(3-fluorobenzyl amino)pyrazin-2-yl)pyridin-2-yl)cyclohexane- 1 ,4-diamine (19 mg, 0.048 mmol) was added NMP (0.6 ml), acetic acid (0.042 ml, 0.726 mmol) and benzaldehyde (10.27 mg, 0.097 mmol). The resulting reaction mixture was stirred overnight at ambient temperature. To the stirred reaction mixture was added sodium triacetoxyborohydride (41.0 mg, 0.194 mmol) and the resulting mixture was stirred overnight (24 hours) at ambient temperature. To the reaction mixture then was added additional sodium triacetoxyborohydride (21.0 mg, 0.099 mmol) and the resulting mixture was stirred for an additional 2 more hours. To the crude mixture then was added 0.8 ml of DMSO, filtered and purified by prep LC. After lyophilization, 7.0 mg of the title compound was obtained as a TFA salt. LCMS (m/z): 483.2 (MH+), retention time = 0.65 min.; 1 H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.35 - 1.51 (m, 2 H) 1.51 - 1.69 (m, 2 H) 2.04 - 2.36 (m, 4 H) 3.08 - 3.18 (m, 1 H) 3.56 - 3.70 (m, 1 H) 4.16 (s, 2 H) 4.60 (s, 2 H) 6.82 - 6.93 (m, 1 H) 7.03 (d, J=9.67 Hz, 1 H) 7.11 (d, J=7.62 Hz, 1 H) 7.19 - 7.29 (m, 1 H) 7.32 (d, J=6.74 Hz, 1 H) 7.35 - 7.46 (m, 5 H) 7.54 (s, 1 H) 7.80 (d, J=6.74 Hz, 1 H) 7.97 (s, 1 H) 8.25 (s, 1 H)
Example 11 (Compound 1 1)
N2'-(frans-4-aminocyclohexyl)-N6-(3-fluorobenzyl)-4-(trifluoromethyl)-2,4'-bipyridine-2',6- diamine
Figure imgf000146_0001
Step 1. Preparation of 6-chloro-N-(3-fluorobenzyl)-4-(trifluoromethyl)pyridin-2-amine:
To 2,6-dichloro-4-(trifluoromethyl)pyridine (250 mg, 1.157 mmol) was added DMSO (2 ml), TEA (0.194 ml, 1.389 mmol), and (3-fluorophenyl)methanamine (290 mg, 2.315 mmol). The reaction mixture was stirred at 90 °C until completion as indicated by LCMS, about 1 hour. To the crude reaction mixture was added 1.5 ml of DMSO, filtered and purified by prep LC. After lyophilization, 158 mg of the title compound was obtained as a TFA salt. LCMS (m/z): 305.1 (MH+), rt= 1.21 min. Step 2. Preparation of 2'-fluoro-N-(3-fluorobenzyl)-4-(trifluoromethyl)-2,4'-bipyridin-6- amine:
To 6-chloro-N-(3-fluorobenzyl)-4-(trifluoromethyl)pyridin-2-amine (70 mg, 0.230 mmol) was added 2-fluoropyridin-4-ylboronic acid (58.3 mg, 0.414 mmol),
PdCl2(dppf).CH2Cl2 adduct (22.52 mg, 0.028 mmol), DME (1.2 ml), and 2M sodium carbonate (0.460 ml, 0.919 mmol). The resulting reaction mixture was stirred at 105 °C until completion as indicated by LCMS, about 6 hours. The reaction mixture was cooled, 15 ml of ethyl acetate and 5 ml of methanol was added, filtered and concentrated to yield a crude solid. The solid was purified by prep LC. The product was free-based using 200 ml of ethyl acetate and washed with saturated sodium bicarbonate (1x), water (2x), saturated salt solution (1x), dried sodium sulfate, filtered and concentrated to a constant mass, yielding 35 mg of titled compound as free base. LCMS (m/z): 366.2 (MH+), retention time = 1.20 min.
Step 3. preparation of N2'-(frans-4-aminocyclohexyl)-N6-(3-fluorobenzyl)-4- (trifluoromethyl)-2,4'-bipyridine-2',6-diamine:
To 2'-fluoro-N-(3-fluorobenzyl)-4-(trifluoromethyl)-2,4'-bipyridin-6-amine (34 mg, 0.093 mmol) was added DMSO (1.7ml) and frans-cyclohexane-1 ,4-diamine (159 mg, 1.396 mmol). The resulting reaction mixture was stirred at 105 °C until completion as indicated by LCMS, about 40 hours. To the crude reaction mixture was added 0.75 ml of DMSO, filtered and purified by prep LC. After lyophilization, 28.1 mg of the title compound was obtained as a TFA salt. LCMS (m/z): 460.3 (MH+), retention time = 0.72 min.; 1 H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.40 - 1.72 (m, 4 H) 2.18 (t, J=13.77 Hz, 4 H) 3.1 1 - 3.24 (m, 1 H) 3.62 - 3.76 (m, 1 H) 4.72 (s, 2 H) 6.95 (s, 1 H) 7.12 (d, J=9.96 Hz, 1 H) 7.17 - 7.24 (m, 1 H) 7.27 - 7.37 (m, 1 H) 7.40 (s, 1 H) 7.42 - 7.48 (m, 1 H) 7.69 (s, 1 H) 7.87 (d, J=6.74 Hz, 1 H)
Example 12 (Compound 12)
^'-(frans^-aminocyclohexy -S'-chloro-S-fluoro-A^-iS-fluorobenzy ^^'-bipyridine^'.e- diamine
Figure imgf000147_0001
Step 1. Preparation of 3,6-difluoro-/V-(3-fluorobenzyl)pyridine-2-amine:
2,3,6-trifluoropyridine (1.07 mL, 1.5 g, 1 1.27 mmol), 3-fluorobenzylamine (3.18 mL, 3.53 g, 28.2 mmol), and triethylamine (4.71 mL, 3.42 g, 33.8 mmol) were dissolved in NMP (39 mL) to form a mixture This mixture reaction mixture was stirred at 100°C for 1 hr. The reaction mixture was then extracted with EtOAc (3 x 75 mL). The combined extracts were washed with H20 (4 x 75 mL) followed by brine (1 x 75 mL). The organic layer was dried (Na2S04), filtered, and the solvent removed in vacuo. The resulting residue was subjected to silica gel column chromatography. Elution using 100 hexanes to 30 EtOAc / 70 hexanes yielded 2.63 g (98%) of 3,6-difluoro-/V-(3-fluorobenzyl)pyridine- 2-amine. LCMS (m/z): 239.1 (MH+), retention time = 1.01 min.
Step 2. Preparation of 3-fluoro-/V-(3-fluorobenzyl)-6-methoxypyridin-2-amine: 3,6-difluoro-A/-(3-fluorobenzyl)pyridine-2-amine (0.5209 g, 2.19 mmol), was dissolved in anhydrous MeOH ( 6.6 mL) and placed under argon. This mixture then was treated with sodium methoxide (0.500 mL, 0.473 g, 2.19 mmol, 25% in MeOH) by slow addition. The resulting mixture was then heated in the microwave at 150°C for four 30 min. The reaction mixture was then poured into brine (25 mL). This mixture was extracted with EtOAc (3 x 25 mL), the combined extracts were washed with brine (1 x 25 mL) and dried (Na2S04). After filtration the solvent removed in vacuo. The resulting residue was subjected to silica gel column chromatography. Elution using 100 hexanes to 25 EtOAc / 75 hexanes afforded 0.3408 g (62%) of 3-fluoro-/V-(3-fluorobenzyl)-6-methoxypyridin-2- amine. LCMS (m/z): 251.1 (MH+), retention time = 1.07 min.
Step 3. Preparation of 5-fluoro-6-(3-fluorobenzylamino)pyridine-2-ol: 3-fluoro-/V- (3-fluorobenzyl)-6-methoxypyridin-2-amine (0.100 g, 0.400 mmol) was dissolved in anhydrous CH3CN (1.6 mL). This mixture was treated with sodium iodide (0.301 g, 2.01 mmol) followed by trimethylsilylchloride (0.257 mL, 0.218 g, 2.01 mmol). The resulting reaction mixture was then heated at reflux for 2 hr. The reaction mixture was then treated with MeOH (1 ml), and the resulting mixture was stirred at ambient temperature for 2 hr, and then concentrated in vacuo. The resulting residue was dissolved in EtOAc (25 ml) and partitioned with H20 (25 ml). The H20 layer was extracted with EtOAc (2 x 25 ml). The organic layers were combined and washed with brine (1 x 25 ml). The organic layer was dried (Na2S04), filtered, and the solvent removed in vacuo. The resulting residue was subjected to silica gel column chromatography. Elution using 10 EtOAc / 90 hexanes to 60 EtOAc / 40 hexanes gave 0.060 g (64%) of 5-fluoro-6-(3- fluorobenzylamino)pyridine-2-ol. LCMS (m/z): 237.2 (MH+), retention time = 0.74 min. Step 4. Preparation of 5-fluoro-6-(3-fluorobenzylamino)pyridine-2-yl
trifluoromethanesulfonate
5-fluoro-6-(3-fluorobenzylamino)pyridine-2-ol (0.060 g, 0.254 mmol) was dissolved in anhydrous CH2CI2 (2.0 mL) and placed under argon. The solution was cooled to 0°C in an ice bath. It was then treated with triethylamine (0.096 mL, 0.070 g, 0.691 mmol) followed by dropwise addition of trifluoromethanesulfonic anhydride (0.058 ml_, 0.096 g, 0.340 mmol). Once the addition was complete, the reaction mixture was stirred at 0°C for 2 hr. The reaction mixture was then poured into saturated NaHC03 (25 ml_). This mixture was extracted with EtOAc (2 x 25 ml_). The combined extracts were washed with brine (1 x 25 ml_), dried (Na2S04), filtered, and the solvent removed in vacuo. The resulting residue was subjected to silica gel column chromatography.
Elution using 5 EtOAc / 95 hexanes to 60 EtOAc / 40 hexanes yielded 0.081 g (87%) of 5-fluoro-6-(3-fluorobenzylamino)pyridine-2-yl trifluoromethanesulfonate. LCMS (m/z): 369.1 (MH+), retention time = 1.15 min. Step 5. Preparation of 5'-chloro-2',5-difluoro-/V-(3-fluorobenzyl)-2,4'-bipyridin-6-amine
5-fluoro-6-(3-fluorobenzylamino)pyridine-2-yl trifluoromethanesulfonate (0.081 1 g, 0.220 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (0.1 16 g, 0.661 mmol), and sodium carbonate (0.286 ml_, 0.573 mmol, 2M in H20) were dissolved in DME (2 ml_). The solution was then degassed by sparging with argon for 5 min. It was then treated with PdCI2(dppf) CH2CI2 adduct (0.036 g, 0.044 mmol). The reaction mixture was then heated in the microwave at 120°C for 10 min. The reaction mixture was then filtered through a pad of Celite. The filtrate was concentrated in vacuo. The resulting residue was subjected to silica gel column chromatography. Elution using 5 EtOAc / 95 hexanes to 60 EtOAc / 40 hexanes yielded 0.044 g (57%) of 5'-chloro-2',5-difluoro-/V-(3- fluorobenzyl)-2,4'-bipyridin-6-amine. LCMS (m/z): 350.0 (MH+), retention time = 1 .16 min.
Step 6. Preparation of A/2'-(frans-4-aminocyclohexyl)-5'-chloro-5-fluoro-/\/6-(3- fluorobenzyl)-2,4'-bipyridine-2',6-diamine
5'-chloro-2',5-difluoro-/\/-(3-fluorobenzyl)-2,4'-bipyridin-6-amine (0.022 g, 0.063 mmol) was dissolved in anhydrous DMSO (0.93 ml_) and charged to a microwave vial, and then treated with frans-cyclohexane-1 ,4-diamine (0.072 g, 0.629 mmol). The reaction mixture then was heated at 100°C for 18 hr. The material was purified by preparative reverse-phase HPLC to yield 0.0151 g (44%) of N2' -(trans-A- aminocyclohexyl)-5'-chloro-5-fluoro-/V6-(3-fluorobenzyl)-2,4'-bipyridine-2',6-diamine as the TFA salt. LCMS (m/z): 444.2 (M H+), retention time = 0.7 min.
Example 13 (Compound 13)
2'-((1 r, 4r)-4-amonocyclohexylamino)-5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridine-5- carbonitrile
Figure imgf000150_0001
Step 1. Preparation of 6-chloro-2-(3-fluorobenzylamino)nicotinonitrile
2,6-dichloronicotinonitrile (0.500 g, 2.89 mmol), (4-fluorophenyl)methanamine (0.816 mL, 0.904 g, 7.23 mmol), and triethylamine (1 .21 mL, 0.877 g, 8.67 mmol) were all mixed in NMP (10 mL). The resulting solution then was heated at 50°C for 18 hr. The reaction mixture was then poured in H20 (25 mL), and extracted with EtOAc (3 x 25 mL). The combined extracts were washed with H20 (4 x 25 mL), and brine (1 x 25 mL). The organic layer was separated and dried (Na2S04), filtered, and the solvent removed in vacuo. The resulting residue was purified using silica gel column chromatography.
Elution using 1 EtOAc / 3 hexanes to 3 EtOAc / 1 hexanes afforded 0.6024 g (80%) of 6- chloro-2-(3-fluorobenzylamino)nicotinonitrile. LCMS (m/z): 350.0 (MH+), retention time = 0.96 min. H NMR (300 MHz, CDCI3) δ 4.58(d, J=5.86 Hz, 2 H) 5.48 (br. s. , 1 H) 6.30 (d, J=8.50 Hz, 1 H) 6.96 - 7.06 (m, 2 H) 7.10 (d, J=7.62 Hz, 1 H) 7.28 - 7.38 (m, 1 H) 7.58 (d, J=8.79 Hz, 1 H).
Step 2. Preparation of 5'-chloro-2'-fluoro-6-(3-fluorobenzylamino)-2,4'-bipyridine-5- carbonitrile
6-chloro-2-(3-fluorobenzylamino)nicotinonitrile (0.602 g, 2.30 mmol), 5-chloro-2- fluoropyridin-4-ylboronic acid (1 .21 g, 6.91 mmol), and sodium carbonate (2.99 mL, 5.99 mmol, 2M in H20) were dissolved in DME (10.5 mL). The resulting solution was then degassed by sparging with argon for 5 min. It was then treated with PdCI2(dppf) CH2CI2 adduct (0.376 g, 0.460 mmol). The resulting reaction mixture was heated in the microwave at 120°C for 10 min. It was then filtered through a pad of Celite. The filtrate was concentrated in vacuo. The resulting residue was subjected to silica gel column chromatography. Elution using 5 EtOAc / 95 hexanes to 50 EtOAc / 50 hexanes yielded 0.2689 g (33%) of 5'-chloro-2'-fluoro-6-(3-fluorobenzylamino)-2,4'-bipyridine-5- carbonitrile. LCMS (m/z): 357.2 (MH+), retention time = 1 .02 min. Step 3. Preparation of 2'-((1 r, 4r)-4-amonocyclohexylamino)-5'-chloro-6-(3- fluorobenzylamino)-2,4'-bipyridine-5-carbonitrile 5'-chloro-2'-fluoro-6-(3-fluorobenzylamino)-2,4'-bipyridine-5-carbonitrile (0.2689 g,
0.754 mmol) was dissolved in anhydrous DMSO (1 1.0 mL) and charged to a microwave vial. This mixture was treated with frans-cyclohexane-1 ,4-diamine (0.861 g, 7.54 mmol), and the reaction mixture was then heated at 100°C for 5 hr. The reaction mixture was cooled to ambient temperature, and the material was purified by preparative reverse- phase HPLC and freebased to yield 0.2539 g (75%) of 2'-((1 r, 4r)-4- amonocyclohexylamino)-5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridine-5-carbonitrile. LCMS (m/z): 451 .2 (MH+), retention time = 0.67 min.
Example 14 (Compound 14)
2'-((1 r, 4r)-4-aminocyclohexylamino)-5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridine-5- carboxamide
Figure imgf000151_0001
Step 1. Preparation of 2'-((1 r, 4r)-4-aminocyclohexylamino)-5'-chloro-6-(3- fluorobenzylamino)-2,4'-bipyridine-5-carboxamide
2'-((1 r, 4r)-4-amonocyclohexyl amino)-5'-chloro-6-(3-fluorobenzylamino)-2,4'- bipyridine-5-carbonitrile (0.028 g, 0.055 mmol) was dissolved in DMSO (0.5 mL), and the solution was cooled to 0°C in an ice bath. The cooled solution was treated with potassium carbonate (0.001 1 g, 0.0078 mmol) followed by hydrogen peroxide (0.007 mL, 0.0069 mmol). The ice bath was removed and the reaction mixture was stirred at ambient temperature for 2 hr. More of the reagents in the same amounts were added and the reaction mixture was heated to 50°C for 16 hr. This procedure was repeated and the reaction mixture was heated at 65°C for an additional 4 hr. The reaction mixture was diluted with brine (10 mL), extracted with EtOAc (3 x 10 mL), the combined extracts were washed with brine (1 x 10 ml_) and dried (Na2S04), filtered and concentrated in vacuo. The material was purified by preparative reverse-phase HPLC to afford 0.0042 g (13%) of 2'-((1 r, 4r)-4-aminocyclohexylamino)-5'-chloro-6-(3-fluorobenzylamino)-2,4'- bipyridine-5-carboxamide as the TFA salt. LCMS (m/z): 469.1 (MH+), retention time = 0.56 min.
Example 15 (Compound 15)
2'-(frans-4-aminocyclohexylamino)-5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridine-4- carbonitrile
Figure imgf000152_0001
Step 1. Preparation of 2-chloro-6-(3-fluorobenzylamino)isonicotinonitrile
To a scintillation vial containing 2,6-dichloroisonicotinonitrile (500 mg, 2.89 mmol) was added NMP (6 ml) and (3-fluorophenyl)methanamine (868 mg, 6.94 mmol). The homogenous reaction mixture was capped and heated to 1 10 °C in a oil bath for 1 hr. The reaction mixture was diluted with EtOAc and washed with sat NaHC03, H20 and sat NaCI. The organic layer was dried Na2S04, filtered and concentrated. The crude residue was purified by column chromatography on silica gel (0-20%EtOAc/Hexane) to give 2- chloro-6-(3-fluorobenzylamino)isonicotinonitrile (750 mg, 95%). LCMS (m/z): 262.0 (MH+), retention time = 1.03 min.
Step 2. Preparation of 5'-chloro-2'-fluoro-6-(3-fluorobenzylamino)-2,4'-bipyridine-4- carbonitrile
To a degassed suspension of 2-chloro-6-(3-fluorobenzylamino) isonicotinonitrile (150 mg, 0.573 mmol) and 5-chloro-2-fluoropyridin-4-ylboronic acid (151 mg, 0.860 mmol) in DME (5 ml) was added Na2C03 (1.433 ml, 2M, 2.87 mmol) and Pd(Ph3P)4 (66.2 mg, 0.057 mmol) . The reaction mixture was capped and heated to 1 10 °C in an oil bath for 2 hr. The reaction mixture was diluted with EtOAc and washed with sat NaHC03, and then sat NaCI. The organic layer was dried over Na2S04, filtered and concentrated. The resulting residue was purified by column chromatography on silica gel (0- 20%EtOAc/Hexane) to give 5'-chloro-2'-fluoro-6-(3-fluorobenzylamino)-2,4'-bipyridine-4- carbonitrile (95 mg, 47%). LCMS (m/z): 357.0 (MH+), retention time = 1.09 min Step 3. Preparation of 2'-(frans-4-aminocyclohexylamino)-5'-chloro-6-(3- fluorobenzylamino)-2,4'-bipyridine-4-carbonitrile
To a scintillation vial containing 5'-chloro-2'-fluoro-6-(3-fluorobenzylamino)-2,4'- bipyridine-4-carbonitrile (72 mg, 0.202 mmol) was added DMSO (3 ml) and trans- cyclohexane-1 ,4-diamine (230 mg, 2.018 mmol) . The homogenous yellow reaction mixture was capped and heated to 105 °C in a oil bath for 3 hr. The reaction mixture was diluted with EtOAc and washed with sat NaHC03, sat NaCI. The organic layer was dried Na2S04, filtered and concentrated. The crude solid was purified by Prep HPLC and the collected fractions were combined and diluted with EtOAc and neutralized with sat NaHC03 and then sat NaCI. The organic layer was dried over Na2S04, filtered and concentrated to afford 2'-(frans-4-aminocyclohexylamino)-5'-chloro-6-(3- fluorobenzylamino)-2,4'-bipyridine-4-carbonitrile (73 mg, 80%). LCMS (m/z): 451.2 (MH+), retention time = 0.70 min. 1 H NMR (400 MHz, METHANOL-^) d ppm 1.34 - 1.48 (m, 2 H) 1.50 - 1.64 (m, 2 H) 2.06 - 2.22 (m, 4 H) 3.08 - 3.20 (m, 1 H) 3.63 - 3.74 (m, 1 H) 4.61 (s, 2 H) 6.81 (s, 1 H) 6.87 (s, 1 H) 6.91 - 6.99 (m, 1 H) 7.02 (s, 1 H) 7.04 - 7.10 (m, 1 H) 7.12 - 7.18 (m, 1 H) 7.25 - 7.36 (m, 1 H) 8.00 (s, 1 H).
Examples 16 and 17 (Compounds 16 and 17)
2'-(frans-4-aminocyclohexylamino)-5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridine-4- carboxamide & 2'-(frans-4-aminocyclohexylamino)-5'-chloro-6-(3-fluorobenzylamino)- 2,4'-bipyridine-4-carboxylic acid
Figure imgf000153_0001
Step 4. Preparation of 2'-(frans-4-aminocyclohexylamino)-5'-chloro-6-(3- fluorobenzylamino)-2,4'-bipyridine-4-carboxamide: To a scintillation vial containing 2'- (frans-4-aminocyclohexylamino)-5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridine-4- carbonitrile (1 1 mg, 0.024 mmol) and K2C03 (33.7 mg, 0.244 mmol) at 0 °C was added DMSO (1 ml) and H202 (10.68 μΙ, 0.122 mmol) . The reaction mixture was capped and stirred at 0 °C for 10 min and rt for 10 min. The reaction mixture was diluted with EtOAc and washed with H20, sat NaCI. The organic layer was dried over Na2S04, filtered and concentrated. The crude oil/solid was purified by reverse phase preparative HPLC to yield a TFA salt of 2'-(frans-4-aminocyclohexylamino)-5'-chloro-6-(3-fluorobenzylamino)- 2,4'-bipyridine-4-carboxamide (3.5 mg, 25% ), LCMS (m/z): 469.2 (MH+), retention time = 0.56 min and 2'-(frans-4-aminocyclohexylamino)-5'-chloro-6-(3-fluorobenzylamino)-2,4'- bipyridine-4-carboxylic acid (3.2 mg, 22%), LCMS (m/z): 470.2 (MH+), retention time = 0.61 min.
Example 18 (Compound 18)
5'-chloro-N2'-(frans-4-(dimethylamino)cyclohexyl)-N6-(3-fluorobenzyl)-2,4'-bipyridine- 2',6-diamine
Figure imgf000154_0001
Step 1. Preparation of 5'-chloro-N2'-(frans-4-(dimethylamino)cyclohexyl)-N6-(3- fluorobenzyl)-2,4'-bipyridine-2',6-diamine: To a scintillation vial containing N2'-(trans-4- aminocyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6-diamine (7 mg, 0.016 mmol) and formaldehyde (6.12 μΙ, 0.082 mmol) was added MeOH (0.3 ml) and Pd/C (5.25 mg, 4.93 μηιοΙ) . The reaction mixture was stirred under hydrogen at room temperature for 16 hours. The reaction mixture was filtered over celite and concentrated. The crude solid was purified by reverse phase preparative HPLC to yield 5'-chloro-N2'- (frans-4-(dimethylamino)cyclohexyl)-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6-diamine (2.0 mg, 24%). LCMS (m/z): 454.2 (MH+), retention time = 0.61 min. as a TFA salt after lyophilizing.
Example 19 (Compound 19)
2-(frans-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)
cyclohexylamino)ethanol
Figure imgf000155_0001
Step 1 . Preparation of N2'-(trans-4-(2-(tert-butyldimethylsilyloxy) ethylamino) cyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6-diamine: To a scintillation vial containing N2'-(frans-4-aminocyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine (17 mg, 0.040 mmol) and K2C03 (22.06 mg, 0.160 mmol) was added DMF (0.3 ml) and (2-bromoethoxy)(tert-butyl)dimethylsilane (9.55 mg, 0.040 mmol) . The reaction mixture was capped and heated to 75 °C for 7hr. The reaction mixture was diluted with DCM and washed with H20, sat NaCI. The organic layer was dried over Na2S04, filtered and concentrated. The crude solid was purified by reverse phase preparative HPLC. Collected fractions were combined, neutralized with Saturated NaHC03 and extracted with EtOAc. The organic layer was dried over Na2S04, filtered, concentrated and used directly in next step. LCMS (m/z): 584.3 (MH+), retention time = 0.87 min.
Step 2. Preparation of 2-(frans-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-
2'-yl-amino) cyclohexylamino)ethanol: To a scintillation vial containing N2'-(frans-4-(2- (tert-butyldimethylsilyloxy) ethylamino) cyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine (from step 1) was added THF (0.300 ml) and TBAF (0.160 ml, 0.319 mmol) . The homogenous reaction mixture was capped, and stirred at ambient temperature for 3 hours. The reaction mixture was concentrated and purified by reverse phase preparative HPLC to yield 2-(frans-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'- bipyridin-2'-yl-amino) cyclohexylamino)ethanol (2.2 mg, 9%). LCMS (m/z): 470.3 (MH+), retention time = 0.58 min as a TFA salt after lyophilizing. 1 H NMR (400 M Hz,
METHANOL-cH) δ ppm 1.31 - 1 .46 (m, 2 H) 1.51 - 1.67 (m, 1 H) 2.21 (d, J=10.56 Hz, 2H) 3.1 1 - 3.20 (m, 2 H) 3.66 - 3.77 (m, 1 H) 3.77 - 3.83 (m, 1 H) 4.62 (s, 1 H) 6.74 (s, 1 H) 6.78 - 6.84 (m, 1 H) 6.87 - 6.92 (m, 1 H) 6.96 - 7.03 (m, 1 H) 7.08 - 7.14 (m, 1 H) 7.15 - 7.21 (m, 1 H) 7.31 - 7.38 (m, 1 H) 7.68- 7.76 (m, 1 H) 8.02 (s, 1 H).
Example 20 (Compound 20) 5'-chloro-N6-(3-fluorobenzyl)-N2'-(frans-4-(2-(methylsulfonyl) ethylamino) cyclohexyl)- 2,4'-bipyridine-2',6-diamine
Figure imgf000156_0001
Step 1 . Preparation of 2-(methylsulfonyl)ethyl methanesulfonate: To a round- bottom flask containing 2-(methylsulfonyl)ethanol (400 mg, 3.22 mmol) at 0 °C was added DCM (10 ml) and triethylamine (4.91 μΙ, 0.035 mmol), followed by dropwise addition of mesyl chloride (2.96 mg, 0.026 mmol) . The ice bath was removed and the reaction mixture was stirred at ambient temperature for 2 hr. The reaction mixture was diluted with DCM and washed with sat NaHC03 and then sat NaCI. The organic layer was dried over Na2S04, filtered and concentrated. The resulting residue was purified via ISCO(0-60% EtOAc/Hexane) to yield 2-(methylsulfonyl)ethyl methanesulfonate (400 mg, 61 %). LCMS (m/z): 203.0 (MH+), retention time = 0.37 min.
Step 2. Preparation of 5'-chloro-N6-(3-fluorobenzyl)-N2'-(frans-4-(2- (methylsulfonyl)ethylamino) cyclohexyl)-2,4'-bipyridine-2',6-diamine: To a scintillation vial containing N2'-(frans-4-aminocyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine (10 mg, 0.023 mmol) and K2C03 (8 mg, 0.058 mmol) was added DMSO (0.5 ml) and 2-(methylsulfonyl)ethyl methanesulfonate (30 mg). The reaction mixture was capped and heated to 120 °C in an oil bath for 4 hr. The resulting solution was purified by reverse phase preparative HPLC to yield 5'-chloro-N6-(3-fluorobenzyl)- N2'-(frans-4-(2-(methylsulfonyl)ethylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine (3.7 mg, 24%). LCMS (m/z): 532.2 (MH+), retention time = 0.62 min as a TFA salt after lyophilizing.
Example 21 (Compound 21)
5'-chloro-N6-(3-fluorobenzyl)-N2'-(frans-4-(methylamino)cyclohexyl)-2,4'-bipyridine-2',6- diamine
Figure imgf000157_0001
Step 1 . Preparation of (1 s,4s)-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin- 2'-yl-amino)cyclohexyl methanesulfonate: To a round-bottom flask containing (1 s,4s)-4- (5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)cyclohexanol (obtained following example 2) (85 mg, 0.199 mmol) at 0 °C was added DCM (2 ml) and triethylamine (0.042 ml, 0.299 mmol), followed by dropwise addition of Mesyl Chloride (0.020 ml, 0.259 mmol) . The ice bath was removed and the reaction mixture was stirred at ambient temperature for 2 hr. The reaction mixture was diluted with DCM and washed with sat NaHC03, and then sat NaCI. The organic layer was dried over Na2S04, filtered and concentrated to yield (1 s,4s)-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl- amino) cyclohexyl methanesulfonate (90 mg, 90 % yield), LCMS (m/z): 505.3 (MH+), retention time = 0.77 min. The resulting residue was used in next step without further purification.
Step 2. Preparation of 5'-chloro-N6-(3-fluorobenzyl)-N2'-(frans-4-(methylamino) cyclohexyl)-2,4'-bipyridine-2',6-diamine:
To a scintillation vial containing (1 s,4s)-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'- bipyridin-2'-yl-amino)cyclohexyl methanesulfonate (20 mg, 0.040 mmol) was added
MeOH (1 ml) and methyl amine (0.594 ml, 2M, 1 .188 mmol) . The reaction mixture was capped and heated to 70 °C in a oil bath for 16 hr. Solvent was evaporated and recharge the vial with 0.6 ml 30% methyl amine in ethanol. After heating at 70 °C for another 6 hr, the reaction mixture was concentrated and purified by reverse phase preparative HPLC to yield 5'-chloro-N6-(3-fluorobenzyl)-N2'-(frans-4-(methylamino)cyclohexyl)-2,4'- bipyridine-2',6-diamine (6.5 mg, 0.015 mmol, 37.3 %), LCMS (m/z): 440.3 (MH+), retention time = 0.61 min and 5'-chloro-N2'-(cyclohex-3-enyl)-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine (3.5 mg, 22%) LCMS (m/z): 409.2 (MH+), retention time = 0.82 min. 1 H NMR (400 MHz, M ETHANOL-^) δ ppm 1 .32 - 1 .46 (m, 2 H) 1 .47 - 1 .62 (m, 2 H) 2.20 (d, J=1 1.35 Hz, 4H) 3.01 - 3.1 1 (m, 1 H) 3.67 - 3.78 (m, 1 H) 4.64 (s, 2 H) 6.81 (s, 1 H) 6.88 - 6.97 (m, 3 H) 6.97 - 7.05 (m, 1 H) 7.08 - 7.14 (m, 1 H) 7.16 - 7.22 (m, 1 H) 7.31 - 7.41 (m, 1 H) 7.75 - 7.83 (m, 1 H) 8.05 (s, 1 H).
Example 22 (Compound 22)
5'-chloro-N6-(3-fluorobenzyl)-N2'-(frans-4-((methylamino)methyl)cyclohexyl)-2,4'- bipyridine-2',6-diamine
Figure imgf000158_0001
Step 1. Preparation of (frans-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl- amino)cyclohexyl)methyl methanesulfonate:
To a round-bottom flask containing (frans-4-(5'-chloro-6-(3-fluorobenzylamino)- 2,4'-bipyridin-2'-yl-amino)cyclohexyl) methanol (obtained following example 2) (102 mg, 0.231 mmol) at 0 °C was added DCM (2 ml) and triethylamine (0.048 ml, 0.347 mmol), followed by dropwise addition of Mesyl Chloride (0.023 ml, 0.301 mmol). The ice bath was removed and the reaction mixture was stirred at rt for 2 hr. The reaction mixture was diluted with DCM and washed with sat NaHC03 and then sat NaCI. The organic layer was dried over Na2S04, filtered and concentrated to yield (trans-4-(5'-chloro-6-(3- fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)cyclohexyl)methyl methanesulfonate
(1 10mg, 92 % yield), LCMS (m/z): 519.2 (MH+), retention time = 0.80 min. The resulting residue was used in next step without further purification.
Step 2. Preparation of 5'-chloro-N6-(3-fluorobenzyl)-N2'-(frans-4-((methylamino) methyl)cyclohexyl)-2,4'-bipyridine-2',6-diamine: To a scintillation vial containing (trans-4- (5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)cyclohexyl) methyl methanesulfonate (15 mg, 0.029 mmol) was added MeOH (1 ml) and a solution of methyl amine (0.144 ml, 0.289 mmol) in MeOH . The reaction mixture was capped and heated to 70 °C in a oil bath for 16 hr. The resulting solution was concentrated and purified by reverse phase preparative HPLC to yield 5'-chloro-N6-(3-fluorobenzyl)-N2'-(frans-4- ((methylamino) methyl)cyclohexyl)-2,4'-bipyridine-2',6-diamine (8.6 mg, 52%), LCMS (m/z): 454.2 (MH+), retention time = 0.64 min as a TFA salt after lyophilizing. 1 H NMR (400 MHz, METHANOL-cH) δ ppm 1.15 - 1.29 (m, 2 H) 1.29 - 1.42 (m, 2 H) 1.67 - 1.80 (m, 1 H) 1.86 - 1.96 (m, 2 H) 2.09 - 2.21 (m, 2 H) 2.71 (s, 3 H) 2.90 (d, J=7.04 Hz, 2 H) 3.58 - 3.70 (m, 1 H) 4.63 (s, 2H) 6.88 (s, 2 H) 6.94 (d, J=7.43 Hz, 1 H) 6.96 - 7.03 (m, 1 H) 7.07 - 7.13 (m, 1 H) 7.15 - 7.21 (m, 1 H) 7.29 -7.39 (m, 1 H) 7.69 - 7.78 (m, 1 H) 8.01 (s, 1 H).
Example 23 (Compound 23)
5'-chloro-N6-(3,5-difluorobenzyl)-N2'-(frans-4-(pyrrolidin-1-yl)cyclohexyl)-2,4'-bipyridine- 2',6-diamine
Figure imgf000159_0001
Step 1. Preparation of 5'-chloro-N6-(3,5-difluorobenzyl)-N2'-(frans-4-(pyrrolidin-1- yl)cyclohexyl)-2,4'-bipyridine-2',6-diamine : To a scintillation vial containing N2'-(trans-4- aminocyclohexyl)-5'-chloro-N6-(3,5-difluorobenzyl)-2,4'-bipyridine-2',6-diamine (12.3 mg, 0.028 mmol) (obtained following example 2) and K2C03 (15.32 mg, 0.11 1 mmol) was added DMSO (0.5 ml) and 1 ,4-dibromobutane (5.98 mg, 0.028 mmol). The reaction mixture was capped and heated at 60 °C for 7 hr. The reaction mixture was diluted with DCM and washed with H20, sat NaCI. The organic layer was dried over Na2S04, filtered and concentrated. The crude solid was purified by reverse phase preparative HPLC to yield 5'-chloro-N6-(3,5-difluorobenzyl)-N2'-(frans-4-(pyrrolidin-1-yl)cyclohexyl)-2,4'- bipyridine-2',6-diamine (7.8 mg, 46.0 %), LCMS (m/z): 498.3 (MH+), retention time = 0.65 min as a TFA salt after lyophilizing. 1 H NMR (400 MHz, METHANOL-d4) δ ppm 1.26 - 1.40 (m, 2 H) 1.48 - 1.62 (m, 2 H) 1.85 - 1.98 (m, 2 H)1.99 - 2.24 (m, 7 H) 2.99 - 3.14 (m, 4 H) 3.51 - 3.68 (m, 3 H) 4.54 (s, 2 H) 6.69 - 6.80 (m, 3 H) 6.81 - 6.90 (m, 3 H) 7.60 - 7.69 (m, 1 H) 7.94 (s, 1 H).
Example 24 (Compounds 256 + 257) N2'-trans-4-aminocyclohexyl)-5'-chloro-N6-(((R)-2,2-dimethyltetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2',6-diamine and N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6- (((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diarnine
Figure imgf000160_0001
Step 1 : Preparation of N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-(((R/S)-2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diarnine A mixture of (R/S)-5'-chloro-N-((2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)-2'- fluoro-2,4'-bipyridin-6-amine (35 mg, 0.100 mmol), trans-cyclohexane-1 ,4-diamine (91 mg, 0.800 mmol), DIPEA (20.25 mg, 0.200 mmol) in DMSO (0.35 mL) was heated at 109 °C for 16 hr. The mixture was diluted with DMSO, filtered through a syringe filter and purified by HPLC to give N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-(((R/S)-2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine as its trifluoroacetic acid salt. Yield: 29 mg. LCMS (m/z): 444.2 [M+H]+; Retention time = 0.51 min.
N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-(((R)-2,2-dimethyltetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine and N2'-(trans-4-aminocyclohexyl)-5'- chloro-N6-(((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-(((R/S)-2,2-dimethyltetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine trifluoroacetic acid salt was dissolved in MeOH (2 mL) and filtered through VariPure(TM)IPE [500 mg per 6 mL tube; 0.9 mmol (nominal); part no.: PL3540-C603VP], eluted with MeOH (6 mL) and concentrated in vacuo providing N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-(((R/S)-2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine as a colorless oil. Yield: 20 mg. The enantiomers were resolved by chiral HPLC [Chiralpak AD column 21 x 50 mm, 20mic; 20 mg/2 mL EtOH; heptane/IPA; 85: 15 (v:v); 20 mL/min, 330 psi].
Fraction 1 : White solid. Yield: 7.2 mg. Retention time: 10.4 min. [Chiralpak AD-H, column 4.6 x 100 mm, 5 mic; 20 mg/2 mL EtOH; heptane/IPA; 85: 15 (v:v); 1 mL/min]. H NMR (400 MHz, METHANOL-d4) δ [ppm]1.07 - 1.18 (m, 2 H) 1.20 (s, 3 H) 1.21 (s, 3 H) 1.23 - 1.41 (m, 4 H) 1.65 - 1.74 (m, 2 H) 1.90 - 1.99 (m, 2 H) 2.09 (m, 3 H) 2.71 (br. s., 1 H) 3.19 (d, J=6.65 Hz, 2 H) 3.57 - 3.67 (m, 1 H) 3.67 - 3.74 (m, 2 H) 6.52 (d, 1 H) 6.61 (s, 1 H) 6.71 (d, 1 H) 7.42 - 7.50 (m, 1 H) 7.94 (s, 1 H).
Fraction 2: White solid. Yield: 6.6 mg. Retention time: 17.4 min. [Chiralpak AD-
H, column 4.6 x 100 mm, 5 mic; 20 mg/2 mL EtOH; heptane/IPA; 85: 15 (v:v); 1 mL/min]. H NMR (400 MHz, METHANOL-d4) δ [ppm]1.06 - 1.18 (m, 2 H) 1.20 (s, 3 H) 1.21 (s, 3 H) 1.24 - 1.42 (m, 4 H) 1.63 - 1.74 (m, 2 H) 1.91 - 2.01 (m, 2 H) 2.04 - 2.19 (m, 3 H) 2.75 (br. s., 1 H) 3.19 (d, J=7.04 Hz, 2 H) 3.57 - 3.66 (m, 1 H) 3.66 - 3.74 (m, 2 H) 6.52 (d, 1 H) 6.61 (s, 1 H) 6.72 (d, 1 H) 7.43 - 7.50 (m, 1 H) 7.94 (s, 1 H). Absolute
stereochemistry of compounds in Fraction 1 and Fraction 2 is not determined.
Example 25 (Compound 269)
N2'-(trans-4-aminocyclohexyl)-5'-chloro-5-fluoro-N6-((tetrahydro-2H-pyran-4-yl)methyl)- 2,4'-bipyridine-2',6-diamine
Figure imgf000161_0001
A mixture of 5'-chloro-2',5-difluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine (30 mg, 0.088 mmol) and trans-cyclohexane-1 ,4-diamine (81 mg, 0.706 mmol) in DMSO (0.3 mL) under argon in a sealed tube was heated at 103 °C for 18 hr. The mixture was allowed to cool to ambient temperature. The mixture was diluted with DMSO and filtered through a syringe filter. Purification by HPLC provided N2'- (trans-4-aminocyclohexyl)-5'-chloro-5-fluoro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2',6-diamine as its trifluoroacetic acid salt. Yield: 22.3 mg. LCMS (m/z): 434.1 [M+H]+; Retention time = 0.57 min.
H NMR (400 MHz, METHANOL-d4) δ [ppm] 1.22 (dd, J=12.91 , 4.30 Hz, 2 H) 1.31 - 1.65 (m, 6 H) 1.87 (ddd, J=11.05, 7.34, 3.91 Hz, 1 H) 2.07 (dd, 4 H) 3.00 - 3.13 (m, 1 H) 3.24 - 3.34 (m, 4 H) 3.50 - 3.64 (m, 1 H) 3.84 (dd, J=1 1.15, 2.93 Hz, 2 H) 6.79 (dd, 1 H) 6.93 (s, 1 H) 7.20 (dd, 1 H) 7.93 (s, 1 H).
Example 26 (Compound 155) Ethyl 2-(trans-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl- amino)cyclohexylamino)oxazole-4-carboxylate
Figure imgf000162_0001
A mixture of N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine (25 mg, 0.059 mmol), ethyl 2-chlorooxazole-4-carboxylate (12.88 mg, 0.073 mmol), triethylamine (0.041 mL, 0.293 mmol) in dioxane (1 mL) was heated at 80 °C for -20 hr. The mixture was concentrated in vacuo. The resulting residue was dissolved in DMSO and purified by HPLC providing ethyl 2-(trans-4-(5'-chloro-6-(3- fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)cyclohexylamino)oxazole-4-carboxylate as its trifluoroacetic acid salt. Yield: 7.1 mg. LCMS (m/z): 565.2 [M+H]+; Retention time = 0.85 min. Example 27 (Compound 156)
5'-chloro-N2'-(trans-4-(6-chloropyrimidin-4-yl-amino)cyclohexyl)-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine
Figure imgf000162_0002
A mixture of N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine (25 mg, 0.059 mmol), 4,6-dichloropyrimidine (10.93 mg, 0.073 mmol), triethylamine (0.020 mL, 0.147 mmol) in dioxane (1 mL) was heated at 80 °C for -16 hr. The mixture was concentrated in vacuo. The resulting residue was dissolved in DMSO and purified by HPLC providing 5'-chloro-N2'-(trans-4-(6-chloropyrimidin-4-yl- amino)cyclohexyl)-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6-diamine as its trifluoroacetic acid salt. Yield: 18 mg. LCMS (m/z): 538.1 [M+H]+; Retention time = 0.82 min.
Example 28 (Compound 266)
N2'-(trans-4-aminocyclohexyl)-3,5,5'-trichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2',6-diamine
Figure imgf000163_0001
Step 1 : Preparation of 6-bromo-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine
To a solution of 2-bromo-6-fluoropyridine (3 g, 17.05 mmol) in DMSO (8 ml_) was added (tetrahydro-2H-pyran-4-yl)methanamine (3.10 g, 20.46 mmol) and triethylamine (5.68 ml_, 40.9 mmol). The mixture was heated at 1 10 °C for 18 hr. The mixture was allowed to cool to ambient temperature and diluted with EtOAc. The organic layer was washed with saturated aqueous NaHC03 solution (1x), water (1x), brine (1x), dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography over silica gel providing 6-bromo-N-((tetrahydro-2H-pyran-4- yl)methyl)pyridin-2-amine as a white solid. Yield: 4.24 g. LCMS (m/z): 270.9/273.0
[M+H]+; Retention time = 0.78 min.
Step 2: Preparation of 6-bromo-3,5-dichloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin- 2-amine
Step 2a: To a solution of 6-bromo-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine (20 g, 74 mmol) in acetonitrile (240 ml_) was added NCS (9.85 g, 74 mmol). The mixture was heated to 80 °C for 3 hr. The reaction mixture was allowed to cool to ambient temperature and concentrated in vacuo. The resulting residue was diluted with brine (200 ml_) and extracted with EtOAc (3x 200 ml_). The combined organic layers were concentrated in vacuo. The resulting residue was purified by column
chromatography [Si02, EtOAc/heptane = 0/100 to 50/50] providing 6-bromo-5-chloro-N- ((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine (12 g) and a mixture of 6-bromo-3,5- dichloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine/6-bromo-3-ch
((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine (5 g, ratio -2:3).
Step 2b: To a solution of a mixture of 6-bromo-3,5-dichloro-N-((tetrahydro-2H- pyran-4-yl)methyl)pyridin-2-amine/6-bromo-3-chloro-N-((tetrahydro-2H-pyran-4- yl)methyl)pyridin-2-amine (4.5g, ratio -2:3) in acetonitrile (40 mL) was added NCS (1.250 g, 9.36 mmol). The mixture was heated to 80 °C for 50 min. The mixture was allowed to cool to ambient temperature and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 120 g, EtOAc/heptane] providing 6-bromo-3,5- dichloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2-amine as white solid. Yield: 2.25 g. LCMS (m/z): 340.9 [M+H]+; Retention time = 1.1 1 min.
Step 3: Preparation of 3,5,5'-trichloro-2'-fluoro-N-((tetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridin-6-amine
A mixture of 6-bromo-3,5-dichloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine (1 g, 2.94 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (0.774 g, 4.41 mmol), PdCl2(dppf).CH2Cl2 adduct (0.240 g, 0.294 mmol) in DME (12 mL) and 2M aqueous Na2C03 solution (4 mL, 2.94 mmol) in a sealed tube was heated at 90 °C for 2 hr. The mixture was allowed to cool to ambient temperature and was diluted with EtOAc (-100 mL) and saturated aqueous NaHC03. The separated organic layer was washed with saturated aqueous NaHC03 (2x), brine, dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 80 g, EtOAc/heptane = 0/100 to 30/70 over 25 min] providing 3,5,5'-trichloro-2'-fluoro-N- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-amine as a colorless liquid. Yield: 510 mg. LCMS (m/z): 391.9 [M+H]+; Retention time = 1.14 min.
Step 4: Preparation of N2'-(trans-4-aminocyclohexyl)-3,5,5'-trichloro-N6-((tetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
A mixture of 3,5,5'-trichloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine (35 mg, 0.090 mmol) and trans-cyclohexane-1 ,4-diamine (10.23 mg, 0.090 mmol) in DMSO (0.3 mL) under argon in a sealed tube was heated at 100 °C for 18 hr. The mixture was allowed to cool to ambient temperature. The mixture was diluted with DMSO, filtered through a syringe filter. Purification by HPLC provided N2'-(trans-4- aminocyclohexyl)-3,5,5'-trichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine- 2',6-diamine as its trifluoroacetic acid salt. Yield: 38 mg. LCMS (m/z): 486.0 [M+H]+; Retention time = 0.70 min. H NMR (400 MHz, METHANOL-d4) δ [ppm]1.28 (dd, J=13.11 , 4.11 Hz, 2 H) 1.34 - 1.48 (m, 2 H) 1.49 - 1.69 (m, 4 H) 1.85 - 2.01 (m, 1 H) 2.10 (d, J=12.13 Hz, 2 H) 2.15 - 2.26 (m, 2 H) 3.07 - 3.20 (m, 1 H) 3.31 - 3.40 (m, 4 H) 3.65 - 3.75 (m, 1 H) 3.91 (dd, J=11.35, 2.74 Hz, 2 H) 6.59 (s, 1 H) 7.69 (s, 1 H) 8.02 (s, 1 H).
Example 29 (Compound 311)
N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((4-methyltetrahydro-2H-pyran-4-yl)methyl)- 2,4'-bipyridine-2',6-diamine
Figure imgf000165_0001
Step 1 : Preparation of 4-methyltetrahydro-2H-pyran-4-carbonitrile (following reference: WO2005/058860)
To a solution of tetrahydro-2H-pyran-4-carbonitrile (2 g, 18.00 mmol) in THF (10 mL) at 0 - 5 °C was slowly added LHMDS (21.59 mL, 21.59 mmol). The mixture was stirred for 1.5 hr 0 °C. lodomethane (3.37 mL, 54.0 mmol) was added slowly and stirring was continued for 30 min at ~0 °C and ~2 hr at ambient temperature. The mixture was cooled to 0 °C and carefully diluted with 1 N aqueous HCI (30 mL) and EtOAc (5 mL) and concentrated. The resulting residue was taken up in diethylether and the separated organic layer was washed with brine, dried over Na2S04, filtered off and concentrated in vacuo providing crude 4-methyltetrahydro-2H-pyran-4-carbonitrile as an orange oil, which was directly used in the next reaction without further purification. Yield: 1.8 g. LCMS (m/z): 126.1 [M+H]+; Retention time = 0.44 min.
Step 2: Preparation of (4-methyltetrahydro-2H-pyran-4-yl)methanamine
To a solution of 4-methyltetrahydro-2H-pyran-4-carbonitrile (1.8 g, 14.38 mmol) in THF (30 mL) was added carefully 1 M LAH/THF (21.57 mL, 21.57 mmol) at 0 °C. The reaction mixture was stirred for 15 min at 0 °C, allowed to warm to ambient temperature and stirred for ~3 hours at ambient temperature. To the reaction mixture was carefully added water (0.9 mL), 1 N aqueous NaOH (2.7 mL) and water (0.9 mL) [Caution: gas development!]. The mixture was vigorously stirred for 30 min. The precipitate was filtered off and rinsed with THF. The solution was concentrated in vacuo providing crude (4-methyltetrahydro-2H-pyran-4-yl)methanamine as a yellowish solid, which was directly used in the next step without further purification. Yield: 1.54 g. LCMS (m/z): 130.1
[M+H]+; Retention time = 0.21 min.
Step 3: Preparation of 6-bromo-N-((4-methyltetrahydro-2H-pyran-4- yl)methyl)pyridin-2-amine
To a solution of 2-bromo-6-fluoropyridine (619 mg, 3.52 mmol) in DMSO (3 mL) was added (4-methyltetrahydro-2H-pyran-4-yl)methanamine (500 mg, 3.87 mmol) and triethylamine (498 mg, 4.93 mmol). The mixture was heated at 1 10 °C for 18 hr. The mixture was allowed to cool to ambient temperature and diluted with EtOAc. The organic layer was washed with saturated aqueous NaHC03 solution (1x), water (1x), brine (1x), dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatographyJSi02, 24 g, EtOAc/heptane = 0/100 2 min, 0/100 to 40/60 2-25 min] providing 6-bromo-N-((4-methyltetrahydro-2H-pyran-4-yl)methyl)pyridin- 2-amine as a white solid. Yield: 750 mg. LCMS (m/z): 285.0/287.0 [M+H]+; Retention time = 0.88 min.
Step 4: Preparation of 5'-chloro-2'-fluoro-N-((4-methyltetrahydro-2H-pyran-4-yl)methyl)- 2,4'-bipyridin-6-amine
A mixture of 6-bromo-N-((4-methyltetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine (750 mg, 2.63 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (830 mg, 4.73 mmol), PdCI2(dppf)-CH2Cl2Adduct (215 mg, 0.263 mmol) in DME (12 mL) and 2M aqueous Na2C03 (4 mL, 8.00 mmol) in a sealed tube was heated at 103 °C for 4 hr. The mixture was allowed to cool to ambient temperature and was diluted with EtOAc (-50 mL) and saturated aqueous NaHC03 solution. The separated organic layer was washed with saturated aqueous NaHC03 solution (2x), dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 40 g, 20 min, EtOAc/heptane = 0/100 for 2 min, then EtOAc/heptane = 5/95 to 50/50 over 18 min, then EtOAc/heptane = 50/50] providing 5'-chloro-2'-fluoro-N-((4- methyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-amine as a colorless oil. Yield: 691 mg. LCMS (m/z): 336.2 [M+H]+; Retention time = 0.66 min. Step 5: Preparation of N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((4-methyltetrahydro- 2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
A mixture of 5'-chloro-2'-fluoro-N-((4-methyltetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine (50 mg, 0.149 mmol), trans-cyclohexane-1 ,4-diamine (136 mg, 1.191 mmol), DIPEA (30.1 mg, 0.298 mmol) in DMSO (0.5 mL) was heated at 107 °C for 16 hr. The mixture was diluted with EtOAc and saturated aqueous NaHC03 solution. The separated aqueous layer was extracted with EtOAc (2x). The combined organic layers were dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was dissolved in DMSO/water (1/1), filtered through a syringe filter and purified by HPLC providing N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((4-methyltetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2',6-diamine as its trifluoroacetic acid salt. Yield: 59.5 mg. LCMS (m/z): 430.3 [M+H]+; Retention time = 0.48 min.
H NMR (400 MHz, METHANOL-d4) δ [ppm]1.13 (s, 3 H) 1.33 - 1.49 (m, 4 H) 1.49 - 1.68 (m, 4 H) 2.06 - 2.23 (m, 4 H) 3.07 - 3.22 (m, 1 H) 3.37 (s, 2 H) 3.60 - 3.69 (m, 2 H) 3.70 - 3.80 (m, 3 H) 6.77 (s, 1 H) 6.90 (d, J=7.04 Hz, 1 H) 7.12 (d, J=9.00 Hz, 1 H) 7.81 - 7.91 (m, 1 H) 8.09 (s, 1 H).
Example 30 (Compound 312)
N2'-(trans-4-aminocyclohexyl)-5'-chloro-5-fluoro-N6-((4-methyltetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000167_0001
Step 1 : Preparation of 3, 6-difluoro-N-((4-methyltetrahydro-2H-pyran-4-yl)methyl)pyridin- 2-amine
A mixture of 2,3,6-trifluoropyridine (858 mg, 6.45 mmol), (4-methyltetrahydro-2H- pyran-4-yl)methanamine (1000 mg, 7.74 mmol) and triethylamine (2.158 mL, 15.48 mmol) in NMP (16 mL) was heated at 70 °C for 1 hr. The reaction mixture was allowed to ambient temperature and was diluted with EtOAc (-100 mL), brine (-50 mL) and water (-50 mL). The separated organic layer was washed with brine (1x), 0.3N aqueous HCI (2x), saturated aqueous NaHC03 solution (1x), brine (1x), dried over Na2S04, filtered off and concentrated in vacuo to provide crude 3,6-difluoro-N-((4-methyltetrahydro-2H- pyran-4-yl)methyl)pyridin-2-amine as a colorless oil, which was directly used in the next reaction without further purification. Yield: 1.4 g. LCMS (m/z): 243.1 [M+H]+; Retention time = 0.86 min.
Step 2: Preparation of 3-fluoro-6-methoxy-N-((4-methyltetrahydro-2H-pyran-4- yl)methyl)pyridin-2-amine To a solution of 3,6-difluoro-N-((4-methyltetrahydro-2H-pyran-4-yl)methyl)pyridin-
2-amine (1.4 g, 5.78 mmol) in MeOH (14 mL) was added sodium methoxide (25 wt.% in MeOH, 7 mL, 30.8 mmol). The mixture was heated in a steel bomb at 135 °C for 3days. The mixture was cooled to ambient temperature and concentrated in vacuo. The resulting residue was taken up in water (200 mL), and the resulting precipitate was filtered off and rinsed with water. The solid was dissolved in DCM. The organic solution was washed with brine, dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 80 g, 20 min,
EtOAc/heptane = 0/100 for 2 min, then EtOAc/heptane = 5/95 to 25/75 over 23 min, EtOAc/heptane = 25/75] providing 3-fluoro-6-methoxy-N-((4-methyltetrahydro-2H-pyran- 4-yl)methyl)pyridin-2-amine as an off-white solid. Yield: 1.22 g. LCMS (m/z): 255.1
[M+H]+; Retention time = 0.89 min.
Step 3: Preparation of 5-fluoro-6-((4-methyltetrahydro-2H-pyran-4- yl)methyl)aminopyridin-2-ol
To 3-fluoro-6-methoxy-N-((4-methyltetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine in acetonitrile (12 mL) was added sodium iodide (4.24 g, 28.3 mmol) and slowly TMS-CI (3.62 mL, 28.3 mmol). The mixture was heated to reflux (oil bath: 83 °C) for 4 hr. The mixture was allowed to cool to ambient temperature and was diluted with EtOAc and saturated aqueous NaHC03 solution. The mixture was vigorously stirred for 15 min and acidified with 0.5N aqueous NaHS04 solution and stirring was continued for 5 min. The mixture was neutralized with saturated aqueous NaHC03 solution. The separated aqueous phase was extracted with EtOAc (3x). The combined organic layers were dried over sodium sulfate, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 40 g, 25 min, EtOAc/heptane = 5/95 for 2 min, 5/95 to 50/50 over 18 min, then 50/50] providing 5-fluoro-6-((4-methyltetrahydro-2H- pyran-4-yl)methyl)aminopyridin-2-ol as colorless highly viscous oil. Yield: 420 mg.
LCMS (m/z): 241.1 [M+H]+; Retention time = 0.55 min.
Step 4: Preparation of 5-fluoro-6-((4-methyltetrahydro-2H-pyran-4- yl)methyl)aminopyridin-2-yl tnfluoromethanesulfonate
To a solution of 5-fluoro-6-((4-methyltetrahydro-2H-pyran-4- yl)methyl)aminopyridin-2-ol (420 mg, 1.748 mmol) and triethylamine (0.731 mL, 5.24 mmol) in DCM (16 mL) was added trifluoromethanesulfonic anhydride (0.443 mL, 2.62 mmol) slowly at 0 °C. The mixture was stirred for 2 hr at 0 °C and poured carefully into ice-cooled saturated aqueous NaHC03 solution. The separated aqueous layer was extracted with DCM (2x). The combined organic layers were dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column
chromatography [Si02, 24 g, EtOAc/heptane = 5/95 for 2 min, then EtOAc/heptane = 5/95 to 40/60 over 13 min, then EtOAc/heptane = 40/60] providing 5-fluoro-6-((4- methyltetrahydro-2H-pyran-4-yl)methyl)aminopyridin-2-yl tnfluoromethanesulfonate as colorless oil. Yield: 600 mg.
Step 5: Preparation of 5'-chloro-2',5-difluoro-N-((4-methyltetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridin-6-amine
A mixture of 5-fluoro-6-((4-methyltetrahydro-2H-pyran-4-yl)methyl)aminopyridin-2- yl tnfluoromethanesulfonate (600 mg, 1.61 1 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (565 mg, 3.22 mmol), PdCI2(dppf)-CH2CI2 adduct (132 mg, 0.161 mmol) in DME (8 mL) and 2M aqueous Na2C03 (3 mL, 6.00 mmol) in a sealed tube was heated at 102 °C for 10 hr. The mixture was cooled to ambient temperature and was diluted with EtOAc (-100 mL) and saturated aqueous NaHC03 solution. The separated organic layer was washed with saturated aqueous NaHC03 solution (2x), dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column
chromatography [Si02, 40 g, EtOAc/heptane = 0/100 for 3 min, EtOAc/heptane = 0/100 to 30/70 over 17 min, then EtOAc/heptane = 30/70] providing 5'-chloro-2',5-difluoro-N- ((4-methyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-amine as a colorless oil. Yield: 490 mg. LCMS (m/z): 354.2 [M+H]+; Retention time = 1.05 min.
Step 6: Preparation of N2'-(trans-4-aminocyclohexyl)-5'-chloro-5-fluoro-N6-((4- methyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine A mixture of 5'-chloro-2',5-difluoro-N-((4-methyltetrahydro-2H-pyran-4-yl)methyl)- 2,4'-bipyridin-6-amine (50 mg, 0.141 mmol), trans-cyclohexane-1 ,4-diamine (129 mg, 1.131 mmol), DIPEA (28.6 mg, 0.283 mmol) in DMSO (0.5 mL) was heated at 107 °C for 16 hr. The mixture was diluted with EtOAc and saturated aqueous NaHC03 solution. The separated aqueous layer was extracted with EtOAc (2x). The combined organic layers were dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was dissolved in DMSO/water (1/1), filtered through a syringe filter and purified by HPLC providing N2'-(trans-4-aminocyclohexyl)-5'-chloro-5-fluoro-N6-((4- methyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine as its trifluoroacetic acid salt. Yield: 61.3 mg. LCMS (m/z): 448.2 [M+H]+; Retention time = 0.62 min.
H NMR (400 MHz, METHANOL-d4) δ [ppm]1.06 (s, 3 H) 1.28 - 1.54 (m, 4 H) 1.54 - 1.65 (m, 4 H) 2.06 - 2.25 (m, 4 H) 3.09 - 3.22 (m, 1 H) 3.49 (s, 2 H) 3.57 - 3.72 (m, 3 H) 3.72 - 3.81 (m, 2 H) 6.86 (dd, 1 H) 6.92 (s, 1 H) 7.31 (dd, 1 H) 7.99 (s, 1 H) Example 31 (Compound 313)
N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((4-fluorotetrahydro-2H-pyran-4-yl)methyl)- 2,4'-bipyridine-2',6-diamine
Figure imgf000170_0001
Step 1 : Preparation of 4-fluorotetrahydro-2H-pyran-4-carbaldehyde (as described in WO2009/01 1836)
Step 1 a: To a solution of DIPEA (6.12 mL, 35.0 mmol) in DCM (80 mL) was added trimethylsilyl tnfluoromethanesulfonate (7.79 g, 35.0 mmol) followed by a solution of tetrahydro-2H-pyran-4-carbaldehyde (2 g, 17.52 mmol) in DCM (80 mL) at 0 °C. Upon completion of the addition, the reaction mixture was allowed to stir at ambient temperature for 2 hr. The mixture was concentrated in vacuo and the resulting residue was treated with hexane (200 mL). The precipitate was filtered off and the solution was concentrated in vacuo providing crude trimethylsilyl ether, which was directly used in the next step without further purification. Step 1 b: To a solution of crude trimethylsilyl ether in DCM (100 mL) was added dropwise a solution of N-fluorobenzenesulfonimide (5.53 g, 17.52 mmol), dissolved in DCM (50 mL), at 0 °C. The mixture was stirred for 3 hr at ambient temperature and the crude solution of 4-fluorotetrahydro-2H-pyran-4-carbaldehyde was directly used in the next reaction.
Step 2: Preparation of 6-bromo-N-((4-fluorotetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine To 6-bromopyridin-2-amine (3.03 g, 17.50 mmol) was added the crude solution of
4-fluorotetrahydro-2H-pyran-4-carbaldehyde in DCM. To the mixture was added acetic acid (1.002 mL, 17.50 mmol) and sodium triacetoxyborohydride (5.56 g, 26.3 mmol) in portions. The mixture was stirred for 2 hr at ambient temperature. The mixture was diluted carefully with saturated aqueous NaHC03 solution. The separated aqueous layer was extracted with DCM (1x). The combined organic layers were washed with water (1x), saturated aqueous NaHC03 solution (1x) and concentrated in vacuo. The solid resulting residue was dissolved in DCM (100 mL) and 3M aqueous HCI (60 mL). The separated organic layer was extracted with 3M aqueous HCI (3x 20 mL). The combined acidic layers were washed with DCM (1x). Solid NaHC03 was added carefully to the acidic solution [Caution: gas development!] until pH>~8. The aqueous mixture was extracted with DCM (2x) and EtOAc (2x). The combined organic layers were
concentrated in vacuo. The resulting residue was dissolved in EtOAc. The solution was washed with 0.3M aqueous HCI, and brine, dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 40 g, EtOAc/heptane = 5/95 for 3 min, then EtOAc/heptane = 5/95 to 30/70 over 15 min, then EtOAc/heptane = 30/70] providing 6-bromo-N-((4-fluorotetrahydro-2H- pyran-4-yl)methyl)pyridin-2-amine as a white solid. Yield: 1.82 g. LCMS (m/z):
288.9/291.0 [M+H]+; Retention time = 0.84 min. Step 3: Preparation of 5'-chloro-2'-fluoro-N-((4-fluorotetrahydro-2H-pyran-4-yl)methyl)- 2,4'-bipyridin-6-amine
A mixture of 6-bromo-N-((4-fluorotetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine (1 g, 3.46 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (1.092 g, 6.23 mmol), PdCl2(dppf)-CH2Cl2 adduct (0.282 g, 0.346 mmol) in DME (13 mL) and 2M aqueous Na2C03 (5.19 mL, 10.38 mmol) in a sealed tube was heated at 100 °C for 2 hr. The mixture was cooled to ambient temperature and was diluted with EtOAc (-50 mL) and saturated aqueous NaHC03. The separated organic layer was washed with saturated aqueous NaHC03 (2x), dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 80 g, EtOAc/heptane = 5/95 for 4 min, then EtOAc/heptane = 5/95 to 50/50 over 18 min, then EtOAc/heptane = 50/50] providing 5'-chloro-2'-fluoro-N-((4-fluorotetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine as a colorless oil. Yield: 1.00 g. LCMS (m/z): 340.1 [M+H]+; Retention time = 0.67 min. Step 4: Preparation of N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((4-fluorotetrahydro- 2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
A mixture of 5'-chloro-2'-fluoro-N-((4-fluorotetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine (75 mg, 0.221 mmol) and trans-cyclohexane-1 ,4-diamine (202 mg, 1.766 mmol) in DMSO (1 mL) under argon in a sealed tube was heated at 103 °C for 18 hr. The mixture was cooled to ambient temperature and diluted with EtOAc and water. The separated organic layer was washed with saturated aqueous NaHC03 solution and concentrated in vacuo. The resulting residue was dissolved in DMSO/water (-2/1), filtered through a syringe filter. Purification by HPLC provided N2'-(trans-4- aminocyclohexyl)-5'-chloro-N6-((4-fluorotetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine- 2',6-diamine as its trifluoroacetic acid salt. The material was dissolved in MeOH (-3 mL), filtered through VariPure(TM)IPE [500 mg per 6 mL tube; 0.9 mmol (nominal); part no.: PL3540-C603VP], eluted with MeOH (15 mL) and concentrated in vacuo. The resulting residue was dissolved in acetonitrile/water (-3/1) and lyophilized providing N2'-(trans-4- aminocyclohexyl)-5'-chloro-N6-((4-fluorotetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine- 2',6-diamine. Yield: 58 mg. LCMS (m/z): 434.2 [M+H]+; Retention time = 0.50 min. H NMR (400 MHz, METHANOL-d4) δ [ppm]1.32 (d, J=9.78 Hz, 4 H) 1.73 - 1.88 (m, 4 H) 1.91 - 1.99 (m, 2 H) 2.08 (d, J=9.78 Hz, 2 H) 2.67 - 2.78 (m, 1 H) 3.57 - 3.73 (m, 5 H) 3.75 - 3.84 (m, 2 H) 6.60 (d, J=8.61 Hz, 1 H) 6.63 (s, 1 H) 6.78 (d, J=7.43 Hz, 1 H) 7.34 - 7.55 (m, 1 H) 7.94 (s, 1 H).
Example 32 (Compound 152)
N2'-((1S,3S,4S)-4-amino-3-methylcyclohexyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine/N2'-((1 R,3R,4R)-4-amino-3-methylcyclohexyl)-5'-chloro-N6-(3- fluorobenzyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000173_0001
Step 1 : Preparation of 4-(dibenzylamino)cyclohexanol
To a mixture of 4-aminocyclohexanol (3.51 g, 23.15 mmol) and K2C03 (12.80 g,
93 mmol) in acetonitrile (100 mL) was added benzylbromide (5.64 mL, 47.5 mmol) and the mixture was stirred at reflux for 17 hr. The crude mixture was concentrated in vacuo and the resulting residue was dissolved in water and EtOAc. The separated aqueous layer was extracted with EtOAc (2x -100 mL). The combined organic layers were washed with brine, dried over Na2S04, filtered off and concentrated in vacuo providing crude 4-(dibenzylamino)cyclohexanol as a viscous oil, which was directly used in the next step without further purification. Yield: 6.12 g. LCMS (m/z): 296.1 [M+H]+;
Retention time = 0.59 min. Step 2: Preparation of 4-(dibenzylamino)cyclohexanone (following reference
WO96/07657)
To a solution of oxalic acid (2.03 mL, 20.31 mmol) in DCM (80 mL) at -60 °C was added dropwise DMSO (3.46 mL, 48.8 mmol). After stirring for 5 min, a solution of 4- (dibenzylamino)cyclohexanol (6 g, 20.31 mmol) in DCM (40 mL) was added slowly. The mixture was stirred for 15 min and NEt3 (14.3 mL, 103 mmol) was added slowly. After stirring for 15 min the ice bath was removed and the mixture was stirred for additional 16 hr. The mixture was diluted with water (100 mL). The separated organic layer was washed with brine (1x -75 mL), dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 120 g, EtOAc/hexane = 10/90 to 50/50] providing 4-(dibenzylamino)cyclohexanone as a white solid. Yield: 5.5 g. LCMS (m/z): 294.1 [M+H]+; Retention time = 0.58 min.
Step 3: Preparation of (2S,4S)-4-(dibenzylamino)-2-methylcyclohexanone/(2R,4R)-4- (dibenzylamino)-2-methylcyclohexanone
A solution of 4-(dibenzylamino)cyclohexanone (4 g, 13.63 mmol) in THF (27 mL) was added to KHMDS/toluene (32.7 mL, 16.36 mmol) at ambient temperature. The mixture was stirred for 15 min at ambient temperature. Triethylborane (1 M in THF, 17.72 mL, 17.72 mmol) was added dropwise and the mixture was allowed to stir an additional 30 min. lodomethane (1.6 mL, 25.7 mmol) was added and the mixture was stirred for 20 hr at ambient temperature. Aqueous 1 M NaOH solution was added (-25 mL) and the mixture was vigorously stirred for 3 hr. The mixture was extracted with EtOAc (4x -100 mL) and the combined organic layers were washed with brine, dried over Na2S04, filtered off, and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 125 g, EtOAc/hexane = 0/100 to 20/80]. Fractions were combined and concentrated in vacuo providing (2S,4S)-4-(dibenzylamino)-2- methylcyclohexanone/(2R,4R)-4-(dibenzylamino)-2-methylcyclohexanone as a highly viscous oil, which became partially a white solid. Yield: 3.1 g. LCMS (m/z): 308.2[M+H]+; Retention time = 0.65 min (major isomer). Ratio major/ minor isomer: -9: 1.
Step 4: Preparation of (1 R,2S,4S)-4-(dibenzylamino)-2-methylcyclohexanol/(1S,2R,4R)- 4-(dibenzylamino)-2-methylcyclohexanol.
To a solution of (2S,4S)-4-(dibenzylamino)-2-methylcyclohexanone/(2R,4R)-4- (dibenzylamino)-2-methylcyclohexanone (3.1 g, 10.08 mmol) in THF (55 mL) at -78 °C was added L-selectride (15.13 mL, 15.13 mmol) dropwise. After stirring for 5 min at -78 °C the mixture was allowed to warm up to 0 °C. Stirring was continued for 18 hr as the reaction mixture was warmed from 0 °C to ambient temperature. The mixture was diluted carefully with 1 N aq NaOH (15 mL) and stirred vigorously for 3 hr. The mixture was extracted with EtOAc (3x -100 mL). The combined organic layers were washed with brine (-100 mL), dried over Na2S04, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 120 g, EtOAc/hexane= 0/100 to 20/80 over-25 min; EtOAc/hexane = 20/80 to 40/60 over 5 min] providing (1 R,2S,4S)-4-(dibenzylamino)-2-methylcyclohexanol/(1S,2R,4R)-4-(dibenzylamino)-2- methylcyclohexanol as a colorless liquid. Yield: 2.83 g. LCMS (m/z): 310.3 [M+H]+; Retention time = 0.66 min.
Step 5: Preparation of (1S,3S,4S)-4-azido-N,N-dibenzyl-3- methylcyclohexanamine/(1 R,3R,4R)-4-azido-N,N-dibenzyl-3-methylcyclohexanamine A mixture of DIAD (5.03 mL, 25.9 mmol) and triphenylphosphine (6.78 g, 25.9 mmol) in THF (35 mL) was allowed to form a salt. After 30 min a solution of (1 R,2S,4S)- 4-(dibenzylamino)-2-methylcyclohexanol/(1S,2R,4R)-4-(dibenzylamino)-2- methylcyclohexanol (2 g, 6.46 mmol) and diphenyl phosphorazidate (2.507 mL, 1 1.63 mmol) in THF (25 mL) was added and the mixture was stirred for 20 hr at 55 °C. The mixture was cooled to ambient temperature and diluted with EtOAc and brine. The separated organic layer was dried over Na2S04, filtered off and concentrated in vacuo providing crude (1S,3S,4S)-4-azido-N,N-dibenzyl-3-methylcyclohexanamine/(1 R,3R,4R)- 4-azido-N,N-dibenzyl-3-methylcyclohexanamine as orange oil, which was directly used in the next step without further purification. LCMS (m/z): 335.1 [M+H]+; Retention time = 0.81 min.
Step 6: Preparation of (1S,3S,4S)-N1 ,N1-dibenzyl-3-methylcyclohexane-1 ,4- diamine/(1 R,3R,4R)-N1 ,N1-dibenzyl-3-methylcyclohexane-1 ,4-diamine
To a solution of (1S,3S,4S)-4-azido-N,N-dibenzyl-3- methylcyclohexanamine/(1 R,3R,4R)-4-azido-N,N-dibenzyl-3-methylcyclohexanamine (2.174 g, 6.5 mmol) in acetic acid (50 mL) was added slowly Zn-dust (0.638 g, 9.75 mmol). The mixture was stirred for 30 min at ambient temperature. Additional Zn-dust was added (150 mg) and stirring was continued for ~15min. The mixture was diluted carefully with 1 N aqueous HCI and diethylether. The separated aqueous layer was extracted with diethylether (5x -100 mL). The aqueous layer was partially lyophilized and concentrated to dryness in vacuo. The resulting residue was diluted with 1 N aqueous HCI and concentration to dryness was repeated. Dilution with 1 N HCI and concentration was repeated. The resulting residue was dissolved in water/acetonitrile and lyophilized to provide crude (1S,3S,4S)-N1 ,N1-dibenzyl-3-methylcyclohexane-1 ,4- diamine/(1 R,3R,4R)-N 1 ,N1-dibenzyl-3-methylcyclohexane-1 ,4-diamine as fluffy white solid. The crude material was directly used in the next step without further purification. Yield: 2.292 g. LCMS (m/z): 309.3 [M+H]+; Retention time = 0.50 min.
Step 7: Preparation of tert-butyl (1S,2S,4S)-4-(dibenzylamino)-2- methylcyclohexylcarbamate/tert-butyl (1 R,2R,4R)-4-(dibenzylamino)-2- methylcyclohexylcarbamate
To (1S,3S,4S)-N1 ,N1-dibenzyl-3-methylcyclohexane-1 ,4-diamine/(1 R,3R,4R)- N1 ,N1-dibenzyl-3-methylcyclohexane-1 ,4-diamine (1.851 g, 6 mmol) in dioxane (200 mL) and saturated aqueous NaHC03 solution (100 mL) was added BOC-anhydride (2.438 mL, 10.50 mmol), dissolved in dioxane (~5 mL). The resulting white suspension was stirred vigorously for 18 hr. The mixture was extracted with DCM (4x 300 mL) and EtOAc (1x 100 mL). The combined organic layers were concentrated in vacuo. The resulting residue was dissolved in EtOAc, washed with brine, dried over Na2S04, filtered of and concentrated in vacuo. The resulting residue was purified by column
chromatography [Si02, 120 g, DCM/MeOH = 100/0 to 95/5]. Fractions containing product were combined, concentrated in vacuo providing tert-butyl (1S,2S,4S)-4- (dibenzylamino)-2-methylcyclohexylcarbamate/tert-butyl (1 R,2R,4R)-4-(dibenzylamino)- 2-methylcyclohexylcarbamate. Yield: 778 mg. LCMS (m/z): 409.2 [M+H]+; Retention time = 0.84 min.
Step 8: Preparation of tert-butyl (1S,2S,4S)-4-amino-2-methylcyclohexylcarbamate/tert- butyl (1 R,2R,4R)-4-amino-2-methylcyclohexylcarbamate
A mixture of tert-butyl (1 S,2S,4S)-4-(dibenzylamino)-2- methylcyclohexylcarbamate/tert-butyl (1 R,2R,4R)-4-(dibenzylamino)-2- methylcyclohexylcarbamate (750 mg, 1.836 mmol) and Pearlman's catalyst (290 mg, 2.73 mmol) in EtOH (35 ml_) was hydrogenated in a steel bomb under H2-atmosphere (pressure -75 psi) for 16 hr. The steel bomb was flushed with Argon, Celite and methanol were added. The mixture was filtered and concentrated in vacuo. The white resulting residue was dissolved in acetonitrile/water (1 : 1) and lyophilized giving crude tert-butyl (1S,2S,4S)-4-amino-2-methylcyclohexylcarbamate/tert-butyl (1 R,2R,4R)-4- amino-2-methylcyclohexylcarbamate, which was directly used in the next step without further purification. Yield: 412 mg. LCMS (m/z): 173.2/229.3 [M+H]+; Retention time = 0.54 min.
Step 9: Preparation of N2'-((1S,3S,4S)-4-amino-3-methylcyclohexyl)-5'-chloro-N6-(3- fluorobenzyl)-2,4'-bipyridine-2',6-diamine/N2'-((1 R,3R,4R)-4-amino-3-methylcyclohexyl)- 5'-chloro-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6-diamine
Step 9a: A mixture of Intermediate B (preparation of intermediate B is described in the intermediate session which is in front of the Examples) (25 mg, 0.075 mmol), tert-butyl (1S,2S,4S)-4-amino-2-methylcyclohexylcarbamate/tert-butyl
(1 R,2R,4R)-4-amino-2-methylcyclohexylcarbamate (25.8 mg, 0.113 mmol), triethylamine (28 μΙ, 0.201 mmol) in DMSO (0.25 ml_) was heated at 100 °C for 3days. The mixture was allowed to cool to ambient temperature and diluted with EtOAc (20 ml_) and saturated aqueous NaHC03 solution (10 ml_). The separated aqueous layer was extracted with EtOAc (3x). The combined organic layers were dried over Na2S04, filtered off and concentrated in vacuo providing crude tert-butyl (1 S,2S,4S)-4-(5'-chloro- 6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)-2-methylcyclohexylcarbamate/ tert- butyl (1 R,2R,4R)-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)-2- methylcyclohexylcarbamate, which was directly used in the next step without further purification.
Step 9b: To a solution of crude tert-butyl (1 S,2S,4S)-4-(5'-chloro-6-(3- fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)-2-methylcyclohexylcarbarnate/tert-butyl (1 R,2R,4R)-4-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)-2- methylcyclohexylcarbamate was dissolved in MeOH (3 mL) was added 4M HCI/dioxane (9 mL, 36.0 mmol). The mixture was stirred for 1 hr and concentrated in vacuo. The resulting residue was dissolved in DMSO, filtered over a syringe filter and purified by HPLC providing N2'-((1 S,3S,4S)-4-amino-3-methylcyclohexyl)-5'-chloro-N6-(3- fluorobenzyl)-2,4'-bipyridine-2',6-diamine/N2'-((1 R,3R,4R)-4-amino-3-methylcyclohexyl)- 5'-chloro-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6-diamine as the trifluoroacetic acid salt. Yield: 28.1 mg. LCMS (m/z): 440.1 [M+H]+; Retention time = 0.62 min.
Example 33 (Compound 224)
5-(2-(trans-4-aminocyclohexylamino)-5-chloropyridin-4-yl)-3-((tetrahydro-2/-/-pyran-4- yl)methyl)aminopyrazine-2-carboxamide
Figure imgf000177_0001
Step 1. Preparation of 5-(5-chloro-2-fluoropyridin-4-yl)-3-((tetrahydro-2/-/-pyran-4- yl)methyl)aminopyrazine-2-carboxamide:
3-chloro-6-(5-chloro-2-fluoropyridin-4-yl)-/V-((tetrahydro-2/-/-pyran-4- yl)methyl)pyrazin-2-amine (0.0342 g, 0.096 mmol), CuCN (0.034 g, 0.383 mmol), and dppf (0.085 g, 0.153 mmol) were dissolved in dioxane (1.5 ml). The solution was then degassed by sparging with argon for 5 min. It was then treated with Pd2(dba)3 (0.035 g, 0.038 mmol). The reaction mixture was then heated at 100°C for 5 hr. The reaction mixture was filtered through a pad of Celite then it was concentrated in vacuo to give 0.1 10 g of 5-(5-chloro-2-fluoropyridin-4-yl)-3-((tetrahydro-2/-/-pyran-4- yl)methyl)aminopyrazine-2-carboxamide. LCMS (m/z): 366 (MH+), retention time = 0.89 min. Step 2. Preparation of 5-(2-(trans-4-aminocyclohexylamino)-5-chloropyridin-4-yl)-3- ((tetrahydro-2/-/-pyran-4-yl)methyl)aminopyrazine-2-carboxamide:
5-(5-chloro-2-fluoropyridin-4-yl)-3-((tetrahydro-2/-/-pyran-4- yl)methyl)aminopyrazine-2-carboxamide (0.035 g, 0.096 mmol) was dissolved in DMSO (2 ml). This was treated with 1 ,4-diaminocyclohexane (0.109 g, 0.957 mmol). The reaction mixture was then heated at 100°C for 4 hr. The material was purified by preparative reverse-phase HPLC to give 0.0053 g of 5-(2-(trans-4- aminocyclohexylamino)-5-chloropyridin-4-yl)-3-((tetrahydro-2/-/-pyran-4- yl)methyl)aminopyrazine-2-carboxamide as the TFA salt. LCMS (m/z): 460.1 (MH+), retention time = 0.54 min.
Example 34 (Compound 231 )
trans-N 1-(5-chloro-4-(5-methyl-6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2- yl)pyridin-2-yl)cyclohexane-1 ,4-diamine
Figure imgf000178_0001
Step 1 . Preparation of 6-(5-chloro-2-fluoropyridin-4-yl)-3-methyl-N-(tetrahydro-2H-pyran- 4-yl-methyl)pyrazine-2-amine:
3-chloro-6-(5-chloro-2-fluoropyridin-4-yl)-Ay-((tetrahydro-2/-/-pyran-4- yl)methyl)pyrazin-2-amine (0.0275 g, 0.077 mmol), methylboronic acid (0.014 g, 0.231 mmol), and sodium carbonate (0.100 ml, 0.200 mmol, 2M aq solution) were dissolved in DME (1 .0 ml). The solution was then degassed by sparging with argon for 5 min. It was then treated with PdCl2(dppf).CH2Cl2 adduct (0.013 g, 0.015 mmol). The reaction mixture was then heated in the microwave at 105°C for 20 min. More of the above reagents in the same amounts were added to the reaction mixture and heating in the microwave was continued at 1 15°C for 20 min. The reaction mixture was allowed to cool to ambient temperature. It was then filtered through a pad of Celite. The filtrate was concentrated in vacuo to yield 0.0497 g of a mixture of 6-(5-chloro-2-fluoropyridin-4-yl)- 3-methyl-/V-((tetrahydro-2/-/-pyran-4-yl)pyrazine-2-amine and 6-(2-fluoro-5-methylpyridin- 4-yl)-3-methyl-/V-((tetrahydro-2/-/-pyran-4-yl_methyl)pyrazine-2-amine.
Step 2. Preparation of trans-N 1-(5-chloro-4-(5-methyl-6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine:
The mixture of 6-(5-chloro-2-fluoropyridin-4-yl)-3-methyl-/V-((tetrahydro-2/-/-pyran- 4-yl)pyrazine-2-amine and 6-(2-fluoro-5-methylpyridin-4-yl)-3-methyl-/\/-((tetrahydro-2/-/- pyran-4-yl_methyl)pyrazine-2-amine (0.025 g, 0.074 mmol) and (0.023 g, 0.074 mmol) respectively was dissolved in DMSO (1 ml). This was treated with 1 ,4- diaminocyclohexane (0.085 g, 0.742 mmol). The reaction mixture was then heated at 100°C for 18 hr. The material was purified by preparative reverse-phase HPLC to give 0.0047 g of trans-N 1 -(5-chloro-4-(5-methyl-6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine as the TFA salt.
LCMS (m/z): 431 .2 (MH+), retention time = 0.49 min.
Example 35 (Compound 240)
trans- A/ -(5-chloro-4-(5-cyclopropyl-6-((tetrahydro-2/-/-pyran-4-yl)methyl)aminopyrazin-2- yl)pyridin-2-yl)cyclohexane-1 ,4-diamine
Figure imgf000179_0001
Step 1. Preparation of 6-(5-chloro-2-fluoropyridin-4-tl)-3-cyclopropyl-/V-((tetrahydro-2/-/- pyran-4-yl)methyl)pyrazin-2-amine: 3-chloro-6-(5-chloro-2-fluoropyridin-4-yl)-/\/-((tetrahydro-2/-/-pyran-4- yl)methyl)pyrazin-2-amine (0.0316 g, 0.088 mmol), potassium cyclopropyltrifluoroborate (0.026 g, 0.177 mmol), and potassium phosphate (0.1 13 g, 0.531 mmol) were dissolved in a mixture of toluene (1 ml) and H20 (0.170 ml). The solution was then degassed by sparging with argon for 5 min. At this time it was treated with PdCl2(dppf).CH2Cl2 adduct (0.014 g, 0.018 mmol). The reaction mixture was then heated in the microwave at 1 15°C for 25 min. The reaction mixture was filtered through a plug of Celite and the filtrate was concentrated in vacuo to give 0.0445 g of the crude product. The resulting residue was subjected to silica gel column chromatography. Elution using 20 EtOAc / 80 heptane to 70 EtOAc / 30 heptane gave 0.0271 g (84%) of 6-(5-chloro-2-fluoropyridin-4-tl)-3- cyclopropyl-/V-((tetrahydro-2/-/-pyran-4-yl)methyl)pyrazin-2-amine. LCMS (m/z): 363.1 (MH+), retention time = 1.06 min.
Step 2. Preparation of trans-/V -(5-chloro-4-(5-cyclopropyl-6-((tetrahydro-2/-/-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine: 6-(5-chloro-2-fluoropyridin-4-yl)-3-cyclopropyl-/V-((tetrahydro-2/-/-pyran-4- yl)methyl)pyrazin-2-amine (0.0267 g, 0.074 mmol) was dissolved in DMSO (1 ml). This was treated with 1 ,4-diaminocyclohexane (0.084 g, 0.736 mmol). The reaction mixture was then heated at 100°C for 4 hr. Additional 1 ,4-diaminocyclohexane (0.084 g, 0.736 mmol) and triethylamine (0.0204 ml, 0.028 g, 0.294 mmol) were added. Heating at 100°C was continued for 17 hr. The reaction mixture was purified using prep HPLC. The material was purified by preparative reverse-phase HPLC to yield 0.0240 g of trans- A/ -(5-chloro-4-(5-cyclopropyl-6-((tetrahydro-2/-/-pyran-4-yl)methyl)aminopyrazin-2- yl)pyridin-2-yl)cyclohexane-1 ,4-diamine as the TFA salt. LCMS (m/z): 457.2 (MH+), retention time = 0.60 min.
Example 36 (Compound 241 )
trans- A/ -(5-chloro-4-(5-ethyl-6-((tetrahydro-2/-/-pyran-4-yl)methyl)aminopyrazin-2- yl)pyridin-2-yl)cyclohexane-1 ,4-diamine
Figure imgf000180_0001
Step 1. Preparation of 6-(5-chloro-2-fluoropyridin-4-yl)-3-ethyl-/\/-((tetrahydro-2/-/-pyran- 4-yl)methyl)pyrazine-2-amine:
3-chloro-6-(5-chloro-2-fluoropyridin-4-yl)-/V-((tetrahydro-2/-/-pyran-4- yl)methyl)pyrazin-2-amine (0.0347 g, 0.097 mmol), ethylboronic acid (0.014 g, 0.194 mmol), and sodium carbonate (0.126 ml g, 0.253 mmol, 2 M aq solution) were dissolved in DME (1 ml). The solution was then degassed by sparging with argon for 5 min. At this time it was treated with PdCI2(dppf).CH2Cl2 adduct (0.016 g, 0.019 mmol). The reaction mixture was then heated in the microwave at 1 15°C for 25 min. More ethylboronic acid (0.014 g, 0.194 mmol) and PdCI2(dppf).CH2CI2 adduct (0.016 g, 0.019 mmol) were added. The reaction mixture was then heated in the microwave at 1 15°C for 25 min. The reaction mixture was filtered through a plug of Celite and the filtrate was concentrated in vacuo to afford 0.0709 g of crude product. The material was purified using the Isco with a 4 g Si02 column. The resulting residue was subjected to silica gel column chromatography. Elution using 20 EtOAc / 80 heptane to 70 EtOAc / 30 heptane gave 0.0049 g (14%) of 6-(5-chloro-2-fluoropyridin-4-yl)-3-ethyl-/V-((tetrahydro-2/-/-pyran- 4-yl)methyl)pyrazine-2-amine. LCMS (m/z): 351.1 (M H+), retention time = 0.97 min.
Step 2. Preparation of trans-/V -(5-chloro-4-(5-ethyl-6-((tetrahydro-2/-/-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine:
6-(5-chloro-2-fluoropyridin-4-yl)-3-ethyl-/V-((tetrahydro-2/-/-pyran-4- yl)methyl)pyrazine-2-amine (0.0053 g, 0.015 mmol) was dissolved in DMSO (1 ml). This was treated with 1 ,4-diaminocyclohexane (0.017 g, 0.151 mmol). The reaction mixture was then heated at 100°C for 4 hr. Additional 1 ,4-diaminocyclohexane (0.017 g, 0.151 mmol) and triethylamine (0.0084 ml, 0.012 g, 0.060 mmol) were added. Heating at 100°C was continued for 17 hr. The reaction mixture was purified using prep HPLC. The material was purified by preparative reverse-phase HPLC to give 0.0040 g of trans- A/ -(5-chloro-4-(5-ethyl-6-((tetrahydro-2/-/-pyran-4-yl)methyl)aminopyrazin-2-yl)pyridin-2- yl)cyclohexane-1 ,4-diamine as the TFA salt. LCMS (m/z): 445.2 (MH+), retention time = 0.54 min.
Example 37 (Compound 255)
trans- A/1-(5-chloro-4-(6-((tetrahydro-2/-/-pyran-4-yl)methyl)amino-3- (trifluoromethyl)pyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine
Figure imgf000182_0001
Step 1. Preparation of 6-chloro-5-iodo-/V-((tetrahydro-2/-/-pyran-4-yl)methyl)pyrazin-2- amine:
6-chloro-/V-((tetrahydro-2/-/-pyran-4-yl)methyl)pyrazin-2-amine (0.250 g, 1.098 mmol) was dissolved in a mixture of DMSO (4.30 ml) and H20 (0.105 ml). It was cooled to 0°C in an ice bath and was then treated with /V-iodosuccinimide (0.247 g, 1.098 mmol) by portion-wise addition. Once the addition was complete the reaction mixture was stirred at ambient temperature for 24 hr. Additional NIS (0.025 g, 0.1 1 1 mmol) was added. Stirring at ambient temperature was continued for 24 hr. The reaction mixture was diluted with H20 (50 ml). This was extracted with EtOAc (3 x 50 ml). The organic layers were combined and washed with brine (1 x 50 ml). The organic layer was dried (Na2S04), filtered, and the solvent removed in vacuo to give 0.410 g of crude product. The resulting residue was subjected to silica gel column chromatography. Elution using 30 EtOAc / 70 heptane to 100 EtOAc gave 0.2144 g (55%) of 6-chloro-5-iodo-/V- ((tetrahydro-2/-/-pyran-4-yl)methyl)pyrazin-2-amine. LCMS (m/z): 353.9 (MH+), retention time = 0.92 min. H NMR (400 MHz, CH LOROFORM-d) d ppm 1.37 (qd, 2 H) 1.59 (s, 2 H) 1.67 (d, J=12.91 Hz, 2 H) 1.77 -1.94 (m, J=14.87, 7.63, 7.63, 3.52 Hz, 1 H) 3.25 (t, J=6.46 Hz, 2 H) 3.39 (td, J=1 1.74, 1.96 Hz, 2 H) 4.00 (dd, J=1 1.15, 3.72 Hz, 2 H) 4.80 (br. s. , 1 H) 7.62 (s, 1 H).
Step 2. Preparation of f-butyl-6-chloro-5-iodopyrazin-2-yl((tetrahydro-2H-pyran-4- yl)methyl)carbamate: 6-chloro-5-iodo-A/-((tetrahydro-2/-/-pyran-4-yl)methyl)pyrazin-2- amine (0.0801 g, 0.227 mmol) was dissolved in anhydrous DM F and placed under nitrogen. It was then treated with sodium hydride (0.0109 g, 0.272 mmol, 60% dispersion in mineral oil) followed by di-f-butyldicarbonate (0.099 g, 0.453 mmol). The reaction mixture was then stirred at 50°C for 24 hr. More NaH (0.0109 g, 0.072 mmol) and Boc20 (0.099 g, 0.453 mmol) were added. The reaction mixture was then heated at 70°C for 18 hr. The reaction mixture was cooled to ambient temperature, and then it was poured into brine (25 ml). This was extracted with EtOAc (3 x 25 ml). The combined extracts were washed with H20 (3 x 25 ml) followed by brine (1 x 25 ml). The organic layer was dried (Na2S04), filtered, and the solvent removed in vacuo to yield 0.0846 g of crude product. The resulting residue was subjected to silica gel column chromatography. Elution using 25 EtOAc / 75 heptane to 75 EtOAc / 25 heptane gave 0.0569 g (55%) of f-butyl-6-chloro-5-iodopyrazin-2-yl((tetrahydro-2/-/-pyran-4- yl)methyl)carbamate. LCMS (m/z): 454.0 (MH+), retention time = 1 .20 min. H NM R (400 MHz, CHLOROFORM-d) d ppm 1 .28 - 1 .46 (m, 4 H) 1.46 - 1 .64 (m, 26 H) 1.81 - 2.02 (m, 2H) 3.26 - 3.42 (m, 3 H) 3.86 (d, J=7.04 Hz, 3 H) 3.96 (dd, J=1 1 .54, 2.93 Hz, 3 H) 8.86 (s, 1 H).
Step 3. Preparation of 6-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-5- (trifluoromethyl)pyrazin-2-amine f-butyl-6-chloro-5-iodopyrazin-2-yl((tetrahydro-2H-pyran-4-yl)methyl)carbamate (0.0569 g, 0.125 mmol), methyl 2-chloro-2,2-difluoroacetate (0.047 ml, 0.063 g, 0.439 mmol), potassium fluoride (0.015 g, 0.251 mmol), and copper (I) iodide (0.100 g, 0.527 mmol) were dissolved in anhydrous DM F (0.80 ml) and placed under argon. The reaction mixture was then heated at 1 15°C for 17 hr. It was allowed to cool to ambient temperature. The reaction mixture was filtered through a pad of Celite. The filtrate was poured into brine (25 ml). This was extracted with EtOAc (3 x 25 ml). The combined extracts were washed with H20 (1 x 25 ml) followed by brine (1 x 25 ml). The organic layer was dried (Na2S04), filtered, and the solvent removed in vacuo to give 0.0401 g of crude product. The resulting residue was subjected to silica gel column chromatography. Elution using 25 EtOAc / 75 heptane to 100 EtOAc gave 0.0569 g (55%) of 6-chloro-/V- ((tetrahydro-2/-/-pyran-4-yl)methyl)-5-(trifluoromethyl)pyrazin-2-amine. LCMS (m/z): 296.0 (MH+), retention time = 0.93 min. 1 H NMR (400 MHz, CHLOROFORM-d) d ppm 1.39 (qd, J=12.33, 4.50 Hz, 2 H) 1 .68 (d, J=1 1.35 Hz, 3 H) 1.80 - 2.00 (m, J=14.87, 7.63, 7.63, 3.52 Hz, 1 H) 3.32 - 3.47 (m, 4 H) 4.01 (dd, J=1 1.35, 3.52 Hz, 2 H) 5.26 (br. s., 1 H) 7.76 (s, 1 H).
Step 4. Preparation of 6-(5-chloro-2-fluoropyridin-4-yl)-N-((tetrahydro-2H-pyran-4- yl)methyl)-5-(trifluoromethyl)pyrazin-2-amine 6-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-5-(trifluoromethyl)pyrazin-2-amine (0.020 g, 0.068 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (0.036 g, 0.203 mmol), and sodium carbonate (0.088 ml, 0.176 mmol, 2 M in H20) were dissolved in DME (0.70 ml). The solution was then degassed by sparging with argon for 5 min. It was then treated with PdCI2(dppf) CH2CI2 adduct (0.01 1 g, 0.014 mmol). The reaction mixture was then heated in a microwave at 1 10°C for 25 min. Boronic acid (-0.036 g, 0.203 mmol) and PdCI2(dppf) CH2CI2 adduct (-0.01 1 g, 0.014 mmol) were added. Heating in the microwave was continued at 1 10°C for 25 min. The reaction mixture was then filtered through a pad of Celite. The filtrate was then concentrated in vacuo to give 0.0759 g of crude product. The resulting residue was subjected to silica gel column chromatography. Elution using 25 EtOAc / 75 heptane to 100 EtOAc gave 0.0178 g (67%) of 6-(5-chloro- 2-fluoropyridin-4-yl)-/V-((tetrahydro-2/-/-pyran-4-yl)methyl)-5-(trifluoromethyl)pyrazin-2- amine. LCMS (m/z): 391 .1 (MH+), retention time = 0.96 min.
Step 5. Preparation of trans-/V -(5-chloro-4-(6-((tetrahydro-2/-/-pyran-4-yl)methyl)amino- 3-(trifluoromethyl)pyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine
6-(5-chloro-2-fluoropyridin-4-yl)-N-((tetrahydro-2/-/-pyran-4-yl)methyl)-5- (trifluoromethyl)pyrazin-2-amine (0.0178 g, 0.046 mmol) was dissolved in anhydrous DMSO (1.0 ml) and charged to a microwave vial. This was treated with trans- cyclohexane-1 ,4-diamine (0.052 g, 0.456 mmol). The reaction mixture was then heated at 100°C for 18 hr. The reaction mixture was allowed to cool to ambient temperature. The material was purified by preparative reverse-phase HPLC to give 0.0086 g (32%) of trans- A/ -(5-chloro-4-(6-((tetrahydro-2/-/-pyran-4-yl)methyl)amino-3- (trifluoromethyl)pyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine as the TFA salt. LCMS (m/z): 485.3 (MH+), retention time = 0.63 min.
Example 38 (Compound 260)
N2'-(trans-4-aminocyclohexyl)-3-chloro-5'-fluoro-N6-((tetrahydro-2H-pyran-4-yl)methyl)- 2,4'-bipyridine-2',6-diamine
Figure imgf000184_0001
Step 1. Preparation of 2,5-difluoropyridin-4-ylboronic acid
Diisopropylamine (1.74 ml, 1.24 g, 12.20 mmol) was dissolved in anhydrous THF (22 ml) and placed under argon. The solution was cooled to -20°C and then treated with n-butyllithium (7.66 ml, 12.25 mmol, 1 .6 M in hexanes) by slow addition over 10 min. The newly formed LDA (LDA = lithium diisopropylamide) was then cooled to -78°C and treated with a solution of 2,5-difluoropyridine (1 .05 ml, 1 .33 g, 1 1.56 mmol) dissolved in anhydrous THF (3 ml) by slow addition over 30 min. Once the addition was complete the reaction mixture was allowed to stir at -78°C for 4 hr. At this time the reaction mixture was treated with a solution of triisopropyl borate (5.90 ml, 4.78 g, 25.4 mmol) dissolved in anhydrous THF (8.6 ml) by dropwise addition. Once the addition was complete the reaction mixture was allowed to warm to ambient temperature then stirred at ambient temperature for an additional hour. The reaction mixture was then quenched by adding 4% aq NaOH (34 ml). The layers were separated and the aqueous layer was cooled in an ice bath. It was then acidified to pH = 4 with 6N HCI (- 10 ml) not letting the temperature go above 10°C. This was then extracted with EtOAc (3 x 50 ml). The extracts were then washed with brine (1 x 50 ml), dried (Na2S04), filtered, and the solvent removed in vacuo. The resulting residue was triturated with Et20 to give 0.8084 g (44%) of 2,5-difluoropyridin-4-ylboronic acid. Step 2. Preparation of 3-chloro-2',5'-difluoro-/\/-((tetrahydro-2/-/-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine
6-bromo-5-chloro-/V-((tetrahydro-2/-/-pyran-4-yl)methyl)pyridin-2-amine (0.500 g, 1.64 mmol), 2,5-difluoropyridin-4-ylboronic acid (0.260 g, 1.64 mmol), and sodium carbonate (2.45 ml, 4.91 mmol, 2 M in H20) were dissolved in DME (7.36 ml). The solution was then degassed by sparging with argon for 5 min. It was then treated with PdCI2(dppf) CH2CI2 adduct (0.267 g, 0.327 mmol). The reaction mixture was then heated in the microwave at 105°C for 25 min. More boronic acid (0.260 g, 1 .64 mmol) and PdCI2(dppf) CH2CI2 adduct (0.267 g, 0.327 mmol), and H20 (~2 ml) were added. Heating in the microwave was continued at 1 10°C for 30 min. The reaction mixture was then filtered through a pad of Celite. The filtrate was then concentrated in vacuo to give 1.2090 g of crude product. The resulting residue was subjected to silica gel column chromatography. Elution using 10 EtOAc / 90 heptane to 80 EtOAc / 20 heptane gave 0.3584 g (65%) of 3-chloro-2',5'-difluoro-/V-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine. LCMS (m/z): 340.0 (MH+), retention time = 0.90 min. 1 H NMR (400 MHz, CHLOROFORM-d) d ppm 1 .37 (qd, 3 H) 1.60 (br. s. , 2 H) 1.68 (d, J=12.91 Hz, 3 1-1)1.84 (ddd, J=11.15, 7.24, 4.30 Hz, 1 H) 3.21 (t, J=6.26 Hz, 2 H) 3.32 - 3.45 (m, 3 H) 4.00 (dd, J=11.15, 3.72 Hz, 2 H) 4.74 (br. s., 1 H) 6.45 (d, J=9.00 Hz, 1 H) 6.99 - 7.07 (m, 1 H) 7.51 (d, J=8.61 Hz, 1 H) 8.12 (s, 1 H). Step 3. Preparation of A/2'-(trans-4-aminocyclohexyl)-3-chloro-5'-fluoro-/\/6-((tetrahydro- 2/-/-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
3-chloro-2\5'-difluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-amine (0.0509 g, 0.150 mmol) was dissolved in anhydrous DMSO (3.0 ml) and charged to a microwave vial. This was treated with trans-cyclohexane-1 ,4-diamine (0.171 g, 1.498 mmol). The reaction mixture was then heated at 100°C for 18 hr. More trans- cyclohexane-1 ,4-diamine (0.171 g, 1.498 mmol) was added and the reaction mixture was stirred at 120°C for 18 hr. The reaction mixture was allowed to cool to ambient temperature. The material was purified by preparative reverse-phase HPLC to give 0.2410 g (30%) of A/2'-(trans-4-aminocyclohexyl)-3-chloro-5'-fluoro-/\/6-((tetrahydro-2/-/- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine as the TFA salt. LCMS (m/z): 434.2 (MH+), retention time = 0.55 min.
Example 39 (Compound 282)
N2'-(trans-4-aminocyclohexyl)-5'-chloro-3-fluoro-N6-((tetrahydro-2H-pyran-4-yl)methyl)- 2,4'-bipyridine-2',6-diamine
Figure imgf000186_0001
Step 1. Preparation of trans-N1-(5'-chloro-3,6-difluoro-2,4'-bipyridin-2'- yl)cyclohexane-1 ,4-diamine: To a solution of 5'-chloro-2',3,6-trifluoro-2,4'-bipyridine (95 mg, 0.388 mmol) in DMSO (2.5 ml_) was added trans- 1 ,4-diaminocyclohexane (177 mg, 1.55 mmol). The mixture was stirred at 90 °C for 2 hr. The cooled reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was dried
(Na2S04), filtered, and concentrated to give 137 mg of crude trans-N1-(5'-chloro-3,6- difluoro-2,4'-bipyridin-2'-yl)cyclohexane-1 ,4-diamine which was used without further purification. LCMS (m/z): 339.0 (MH+), retention time = 0.54 min
Step 2. Preparation of N2'-(trans-4-aminocyclohexyl)-5'-chloro-3-fluoro-N6- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine: To a solution of trans- N1-(5'-chloro-3,6-difluoro-2,4'-bipyridin-2'-yl)cyclohexane-1 ,4-diamine (79 mg, 0.388 mmol) in DMSO (1.5 ml) was added 4-aminomethyltetrahydropyran (161 mg, 1.40 mmol). The mixture was irradiated by microwave at 180 °C for 1 hr in a sealed microwave vial. The crude reaction mixture was purified by reverse phase HPLC and lyophilized to give N2'-(trans-4-aminocyclohexyl)-5'-chloro-3-fluoro-N6-((tetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine as its TFA salt. LCMS (m/z): 434.2 (MH+), retention time = 0.57 min.; 1 H NMR (400 MHz, DMSO-d6) ppm 1.07 - 1.32 (m, 2 H) 1.32 - 1.49 (m, 1 H) 1.59 (d, J=12.91 Hz, 1 H) 1.68 - 1.83 (m, 1 H) 1.96 (dd, 2 H) 2.93 - 3.04 (m, 1 H) 3.06 (d, J=6.65 Hz, 1 H) 3.24 (t, J=10.76 Hz, 1 H) 3.54 - 3.70 (m, 1 H) 3.82 (dd, J=10.96, 2.74 Hz, 1 H) 6.53 (s, 1 H) 6.57 (dd, J=9.19, 2.93 Hz, 1 H) 7.41 (t, 1 H) 7.79 (d, J=3.91 Hz, 2 H) 8.04 (s, 1 H)
Example 40 (Compound 283)
5'-chloro-3-flu oro-N2'-(trans-4-(2-methoxyethylamino)cyclohexyl)-N6-((tetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000188_0001
Preparation of 5'-chloro-3-fluoro-N2'-(trans-4-(2-methoxyethylamino)cyclohexyl)- N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine: To a mixture of N2'- (trans-4-aminocyclohexyl)-5'-chloro-3-fluoro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2', 6-diamine trifluoroacetate (30 mg, 0.055 mmol) and sodium carbonate (23 mg, 0.22 mmol) in DMSO (0.75 ml) was added p-toluenesulfonic acid 2-methoxyethyl ester (15 mg, 0.066 mmol). The mixture was stirred at 85 °C for 20 hr in a sealed microwave vial. The cooled reaction mixture was filtered. The filtrate was purified by reverse phase HPLC and lyophilized to give 5.0 mg of 5'-chloro-3-fluoro-N2'-(trans-4-(2- methoxyethylamino)cyclohexyl)-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine- 2',6-diamine as its TFA salt. LCMS (m/z): 492.2 (MH+), retention time = 0.57 min.; 1 H NMR (400 MHz, CHLOROFORM-d) . ppm 1.10 - 1.46 (m, 6 H) 1.64 - 1.74 (m, 2 H) 1.86 (br. s, 2 H) 1.95 - 2.09 (m, 2 H) 2.09 - 2.26 (m, 2 H) 2.58 (br. s, 1 H) 2.88 (t, J=5.09 Hz, 2 H) 3.17 (t, J=6.26 Hz, 2 H) 3.29 - 3.45 (m, 5 H) 3.53 (t, J=5.09 Hz, 3 H) 4.00 (dd,
J=1 1.35, 3.52 Hz, 2 H) 4.34 - 4.47 (m, 1 H) 4.54 - 4.68 (m, 1 H) 6.35 - 6.48 (m, 2 H) 7.31 (t, J=8.80 Hz, 1 H) 8.11 (s, 1 H).
Example 41 (Compound 286)
N2'-(trans-4-aminocyclohexyl)-3-bromo-5'-chloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)- 2, 4'-bipyridine-2', 6-diamine
Figure imgf000189_0001
Preparation of N2'-(trans-4-aminocyclohexyl)-3-bromo-5'-chloro-N6-((tetrahydro- 2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine: To a solution of 3-bromo-5'-chloro-2'- fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-arnine (100 mg, 0.250 mmol) in DMSO (1 ml_) was added trans-1 ,4-diaminocyclohexane (114 mg, 0.998 mmol). The mixture was stirred at 1 10 °C for 19 hr. The crude reaction mixture was purified by reverse phase HPLC and lyophilized to give 51 mg of N2'-(trans-4-aminocyclohexyl)-3- bromo-5'-chloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine as its TFA salt. LCMS (m/z): 494.2/496.1 (MH+), retention time = 0.61 min; 1 H NMR (400 MHz, DMSO-d6) d ppm 1.06 - 1.31 (m, 4 H) 1.31 - 1.49 (m, 2 H) 1.49 - 1.64 (m, 2 H) 1.64 - 1.82 (m, 1 H) 1.85 - 2.11 (m, 4 H) 2.93 - 3.12 (m, 3 H) 3.22 (t, J=10.96 Hz, 2 H) 3.61 (t, J=10.76 Hz, 1 H) 3.81 (dd, J=11.35, 2.74 Hz, 2 H) 6.39 (s, 1 H) 6.48 (d, 1 H) 6.82 (br. s., 1 H) 6.94 (br. s., 1 H) 7.59 (d, J=9.00 Hz, 1 H) 7.78 (d, J=3.91 Hz, 2 H) 8.02 (s, 1 H)
Example 42 (Compound 288)
(R)-3-(trans-4-(5'-chloro-6-((tetrahydro-2H-pyran-4-yl)methyl)amino-2,4'-bipyridin-2'-yl- amino)cyclohexylamino)-1 ,1 , 1-trifluoropropan-2-ol
Figure imgf000189_0002
Step 1. Preparation of N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((tetrahydro- 2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine: To a solution of 5'-chloro-2'-fluoro-N- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-amine (500 mg, 1.55 mmol) in DMSO (7 ml_) was added trans-1 ,4-diaminocyclohexane (710 mg, 6.22 mmol). The mixture was stirred at 110 °C for 19 hr. The cooled reaction mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed sequentially with water and brine, dried over sodium sulfate, filtered, and concentrated to give 600 mg of N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2',6-diamine. LCMS (m/z): 416.1 (MH+), retention time = 0.48 min.
Step 2. Preparation of (R)-3-(trans-4-(5'-chloro-6-((tetrahydro-2H-pyran-4- yl)methyl)amino-2,4'-bipyridin-2'-yl-amino)cyclohexylamino)-1 , 1 , 1-trifluoropropan-2-ol: To a solution of N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((tetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2',6-diamine (50 mg, 0.120 mmol) in 2-propanol (0.8 ml_) was added (R)-(+)-3,3,3-trifluoro-1 ,2-epoxypropane (10.4 uL, 0.120 mmol). The mixture was stirred at 60 °C for 17 hr. The reaction mixture was concentrated. The resulting residue was purified by reverse phase HPLC and lyophilized to give 63 mg of (R)-3-(trans-4-(5'- chloro-6-((tetrahydro-2H-pyran-4-yl)methyl)amino-2,4'-bipyridin-2'-yl- amino)cyclohexylamino)-1 ,1 , 1-trifluoropropan-2-ol as its TFA salt. LCMS (m/z): 528.3 (MH+), retention time = 0.53 min; 1 H NMR (400 MHz, DMSO-d6) ppm 1.08 - 1.34 (m, 4 H) 1.36 - 1.56 (m, 2 H) 1.61 (d, J=12.52 Hz, 2 H) 1.70 - 1.90 (m, 1 H) 2.04 (d, J=9.39 Hz, 3 H) 2.13 (d, J=1 1.74 Hz, 1 H) 2.97 - 3.19 (m, 4 H) 3.24 (t, J=10.76 Hz, 3 H) 3.64 (d, J=10.96 Hz, 1 H) 3.83 (dd, J=10.96, 2.74 Hz, 2 H) 4.36 - 4.50 (m, 2 H) 6.54 - 6.68 (m, 2 H) 6.70 (d, J=7.04 Hz, 0 H) 6.94 (br. s., 0 H) 7.23 (br. s., 0 H) 7.53 (br. s., 0 H) 8.04 (s, 0 H) 8.76 (br. s., 2 H)
Example 43 (Compound 289)
(S)-3-(trans-4-(3,5'-dichloro-6-((tetrahydro-2H-pyran-4-yl)methyl)amino-2,4'-bipyridin-2'- yl-amino)cyclohexylamino)-1 , 1 , 1 -trifluoropropan-2-ol
Figure imgf000190_0001
Step 1. Preparation of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine: To a solution of 3,5'- dichloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-amine (500 mg, 1.40 mmol) in DMSO (8 ml_) was added trans-1 ,4-diaminocyclohexane (641 mg, 5.61 mmol). The mixture was stirred at 95 °C for 38 hr. The cooled reaction mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed sequentially with water and brine, dried over sodium sulfate, filtered, and concentrated. The crude material was purified by flash chromatography over silica gel
(dichloromethane/methanol gradient) to give 480 mg of N2'-(trans-4-aminocyclohexyl)- 3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine. LCMS (m/z): 450.2 (MH+), retention time = 0.55 min.
Step 2. Preparation of (S)-3-(trans-4-(3,5'-dichloro-6-((tetrahydro-2H-pyran-4- yl)methyl)amino-2,4'-bipyridin-2'-yl-amino)cyclohexylamino)-1 , 1 , 1-trifluoropropan-2-ol: To a solution of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6-((tetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2',6-diamine (54 mg, 0.120 mmol) in 2-propanol (0.4 ml_) was added (S)-(-)-3,3,3-trifluoro-1 ,2-epoxypropane (10.4 uL, 0.120 mmol). The mixture was stirred at 70 °C for 2 hr. The reaction mixture was concentrated. The resulting residue was purified by reverse phase HPLC and lyophilized to give 32 mg of (S)-3-(trans-4-(3,5'- dichloro-6-((tetrahydro-2H-pyran-4-yl)methyl)amino-2,4'-bipyridin-2'-yl- amino)cyclohexylamino)-1 ,1 , 1-trifluoropropan-2-ol as its TFA salt. LCMS (m/z): 562.3 (MH+), retention time = 0.70 min; 1 H NMR (400 MHz, DMSO-d6) ppm 1.01 - 1.33 (m, 4 H) 1.35 - 1.65 (m, 4 H) 1.64 - 1.84 (m, 1 H) 1.93 -2.23 (m, 4 H) 2.94 - 3.18 (m, 4 H) 3.17 - 3.35 (m, 3 H) 3.53 - 3.69 (m, 1 H) 3.81 (dd, =1 1.35, 2.74 Hz, 2 H)4.33 - 4.48 (m, 1 H) 6.38 (s, 1 H) 6.55 (d, 1 H) 6.82 (br. s., 1 H) 6.93 (br. s., 1 H) 7.23 (br. s., 1 H) 7.48 (d, 1 H) 8.02 (s, 1 H) 8.72 (br. s., 2 H)
Example 44 (Compound 292)
3-bromo-5'-chloro-N2'-(trans-4-(2-methoxyethylamino)cyclohexyl)-N6-((tetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000191_0001
Preparation of 3-bromo-5'-chloro-N2'-(trans-4-(2-methoxyethylamino)cyclohexyl)- N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine: To a mixture of N2'- (trans-4-aminocyclohexyl)-3-bromo-5'-chloro-N6-((tetrahydro
bipyridine-2', 6-diamine (30 mg, 0.061 mmol) and sodium carbonate (19 mg, 0.18 mmol) in DMSO (0.6 ml) was added p-toluenesulfonic acid 2-methoxyethyl ester (21 mg, 0.091 mmol). The mixture was stirred at 85 °C for 20 hr in a sealed microwave vial. The cooled reaction mixture was filtered. The filtrate was concentrated and the resulting residue was purified by reverse phase HPLC and lyophilized to give 3.8 mg of 3-bromo- 5'-chloro-N2'-(trans-4-(2-methoxyethylamino)cyclohexyl)-N6-((tetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2', 6-diamine as its TFA salt. LCMS (m/z): 554.1 (MH+), retention time = 0.61 min.
Example 45 (Compound 295)
3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-((R)-3,3,3-trifluoro-2- methoxypropylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000193_0001
Preparation of 3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4- ((R)-3,3,3-trifluoro-2-methoxypropylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine: To a solution of 3,5'-dichloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6- amine (36 mg, 0.10 mmol) in DMSO (0.4 mL) was added trans-N1-((R)-3,3,3-trifluoro-2- methoxypropyl)cyclohexane-1 ,4-diamine (48 mg, 0.20 mmol) and 2,6-lutidine (0.023 mL, 0.20 mmol). The mixture was stirred at 120 °C for 20 hr. The cooled reaction mixture was purified by reverse phase HPLC and lyophilized to give 1 1.4 mg of 3,5'-dichloro-N6- ((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-((R)-3,3,3-trifluoro-2- methoxypropylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine as its TFA salt. LCMS (m/z): 576.2 (MH+), retention time = 0.68 min.
Example 46 (Compound 297)
trans-4-(3,5'-dichloro-6-((2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)amino-2,4'- bipyridin-2'-yl-amino)cyclohexanol
Figure imgf000193_0002
Preparation of trans-4-(3,5'-dichloro-6-((2,2-dimethyltetrahydro-2H-pyran-4- yl)methyl)amino-2,4'-bipyridin-2'-yl-amino)cyclohexanol: To a solution of tert-butyl 3,5'- dichloro-2'-fluoro-2,4'-bipyridin-6-yl((2,2-dimethyltetrahydro-2H-pyran-4- yl)methyl)carbamate (30 mg, 0.062 mmol) in DMSO (0.4 mL) was added trans-4- aminocyclohexanol (36 mg, 0.31 mmol) and DIEA (0.022 mL, 0.12 mmol). The mixture was stirred at 135 °C for 3 hr. The cooled reaction mixture was diluted with water and extracted with ethyl acetate. The combined extracts were dried (Na2S04), filtered, and concentrated. The resulting residue was re-dissolved in trifluoroacetic acid (1 mL), stirred for 15 min at ambient temperature, and then concentrated under reduced pressure. The crude resulting residue was purified by reverse phase HPLC and lyophilized to give 23 mg of trans-4-(3,5'-dichloro-6-((2,2-dimethyltetrahydro-2H-pyran-4- yl)methyl)amino-2,4'-bipyridin-2'-yl-amino)cyclohexanol as its TFA salt. LCMS (m/z): 479.3 (MH+), retention time = 0.72 min; H NMR (400 MHz, DMSO-d6) ppm 0.96 (d, J=12.91 Hz, 2 H) 1.08 (s, 6 H) 1.15 - 1.35 (m, 4 H) 1.54 (d, J=12.91 Hz, 2 H) 1.71 - 2.10 (m, 5 H) 3.00 (d, J=6.65 Hz, 2 H) 3.31 - 3.63 (m, 5 H) 6.47 (s, 1 H) 6.58 (d, 1 H) 7.50 (d, J=9.00 Hz, 1 H) 8.05 (s, 1 H)
Example 47 (Compound 298)
(2S)-3-(trans-4-(3,5'-dichloro-6-((2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)amino- 2,4'-bipyridin-2'-yl-amino)cyclohexylamino)-1 , 1 ,1-trifluoropropan-2-ol
Figure imgf000194_0001
Step 1. Preparation of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6-((2,2- dimethyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine: To a solution of tert-butyl 3,5'-dichloro-2'-fluoro-2,4'-bipyridin-6-yl((2,2-dimethyltetrahydro-2H-pyran-4- yl)methyl)carbamate (40 mg, 0.083 mmol) in DMSO (0.4 mL) was added trans-1 ,4- diaminocyclohexane (47 mg, 0.41 mmol) and DIEA (0.029 mL, 0.17 mmol). The mixture was stirred at 120 °C for 2 hr. The cooled reaction mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed sequentially with water and brine, dried over sodium sulfate, filtered, and concentrated. The resulting residue was re-dissolved in trifluoroacetic acid (1 mL), stirred for 15 min at ambient temperature, and then concentrated under reduced pressure. The resulting residue was taken up in DCM, washed with saturated aqueous sodium bicarbonate, dried (Na2S04), filtered, and concentrated to give 39 mg of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro- N6-((2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine. LCMS (m/z): 478.4 (MH+), retention time = 0.64 min.
Step 2. Preparation of (S)-3-(trans-4-(3,5'-dichloro-6-((tetrahydro-2H-pyran-4- yl)methyl)amino-2,4'-bipyridin-2'-yl-amino)cyclohexylamino)-1 , 1 , 1-trifluoropropan-2-ol: To a solution of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6-((2,2-dimethyltetrahydro- 2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine (19 mg, 0.040 mmol) in 2-propanol (0.3 mL) was added (S)-(-)-3,3,3-trifluoro-1 ,2-epoxypropane (3.4 uL, 0.040 mmol). The mixture was stirred at 70 °C for 2 hr. The reaction mixture was concentrated. The resulting residue was purified by reverse phase HPLC and lyophilized to give 9.1 mg of (2S)-3-(trans-4-(3,5'-dichloro-6-((2,2-dimethyltetrahydro-2H-pyran-4-yl)methyl)amino- 2,4'-bipyridin-2'-yl-amino)cyclohexylamino)-1 ,1 , 1-trifluoropropan-2-ol as its TFA salt. LCMS (m/z): 590.5 (MH+), retention time = 0.71 min; H NMR (400 MHz, DMSO-d6) ppm 0.81 - 1.32 (m, 12 H) 1.33 - 1.66 (m, 4 H) 1.82 - 1.99 (m, 1 H) 1.99 -2.21 (m, 4 H) 2.89 - 3.04 (m, 2 H) 3.04 - 3.19 (m, 2 H) 3.27 (d, J=2.35 Hz, 2 H) 4.40 (br. s., 1 H) 6.38 (s, 1 H)6.55 (d, J=9.00 Hz, 1 H) 6.77 (br. s., 1 H) 6.91 (br. s., 1 H) 7.21 (br. s., 1 H) 7.48 (d, J=9.00 Hz, 1 H) 8.03 (s, 1 H)
Example 48 (Compound 301)
5'-chloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-(2- (trifluoromethoxy)ethylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000195_0001
Preparation of 5'-chloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-(2-
(trifluoromethoxy)ethylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine: To a mixture of N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2',6-diamine (42 mg, 0.10 mmol) and triethylamine (0.028 mL, 0.20 mmol) in chloroform (0.4 ml) was added 2-(trifluoromethoxy)ethyl trifluoromethanesulfonate (39 mg, 0.15 mmol). The mixture was stirred at ambient temperature for 1 hr. The reaction mixture was concentrated under reduced pressure, purified by reverse phase HPLC, and lyophilized to give 32 mg of 5'-chloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4- (2-(trifluoromethoxy)ethylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine as its TFA salt. LCMS (m/z): 528.4 (MH+), retention time = 0.53 min.; H NMR (400 MHz, DMSO-d6) d ppm 1.09 - 1.34 (m, 4 H) 1.35 - 1.54 (m, 2 H) 1.55 - 1.69 (m, 2 H) 1.73 - 1.89 (m, 1 H) 1.94 - 2.17 (m, 4 H) 3.04 - 3.15 (m, 1 H) 3.14 - 3.20 (m, 2 H) 3.20 - 3.30 (m, 2 H) 3.30 - 3.47 (m, 2 H) 3.55 - 3.72 (m, 1 H) 3.84 (dd, J=11.15, 2.54 Hz, 2 H) 4.35 (t, J=4.70 Hz, 2 H) 6.65 (s, 1 H) 6.67 - 6.83 (m, 2 H) 7.05 (br. s., 0 H) 7.46 - 7.68 (m, 0 H) 8.06 (s, 0 H) 8.82 (d, J=3.52 Hz, 2 H)
Example 49 (Compound 302)
3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-(2- (trifluoromethoxy)ethylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000196_0001
Preparation of 3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4- (2-(trifluoromethoxy)ethylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine: To a mixture of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2',6-diamine (45 mg, 0.10 mmol) and triethylamine (0.028 mL, 0.20 mmol) in chloroform (0.4 ml) was added 2-(trifluoromethoxy)ethyl trifluoromethanesulfonate (39 mg, 0.15 mmol). The mixture was stirred at ambient temperature for 1 hr. The reaction mixture was concentrated under reduced pressure, purified by reverse phase HPLC, and lyophilized to give 29 mg of 3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'- (trans-4-(2-(trifluoromethoxy)ethylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine as its TFA salt. LCMS (m/z): 562.4 (MH+), retention time = 0.67 min.; H NMR (400 MHz, DMSO-d6) ppm 1.07 - 1.32 (m, 4 H) 1.36 - 1.52 (m, 2 H) 1.58 (d, J=12.91 Hz, 2 H) 1.65 - 1.84 (m, 1 H) 2.07 (d, J=10.56 Hz, 4 H) 2.99 - 3.17 (m, 3 H) 3.23 (t, J=10.76 Hz, 2 H) 3.35 (br. s., 2 H) 3.64 (br. s., 1 H) 3.72 - 3.89 (m, 2 H) 4.34 (t, J=4.89 Hz, 2 H) 6.32 - 6.47 (m, 1 H) 6.49 - 6.65 (m, 1 H) 6.67 - 7.10 (m, 2 H) 7.49 (d, J=9.00 Hz, 1 H) 8.03 (s, 1 H) 8.75 (d, J=3.91 Hz, 1 H)
Example 50 (Compound 284)
N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000197_0001
The mixture of 5'-chloro-N-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4- yl)methyl)-2'-fluoro-2,4'-bipyridin-6-amine L (30mg, 0.08 mmol), trans-1 ,4- cyclohexanediamine (49mg, 0.43 mmol) and triethylamine (26mg, 0.25 mmol) in 1.5ml DMSO was heated in a reaction vessel at 110°C in an oil bath for 16h. Formation of desired product was confirmed by LC/MS. The reaction mixture solution was diluted with ethyl acetate, washed with water, dried over sodium sulfate and concentrated. Crude compound was purified by HPLC to give desired product as TFA salt. LCMS (m/z):
444.2/446.2 (MH+), retention time = 0.54 min.
Example 51 (Compound 285)
5'-chloro-N6-(((2R,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-(2- methoxyethylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000197_0002
The mixture of N2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-(((2R,6S)-2,6-dimethyl tetrahydro-2H-pyran-4-yl) methyl)-2,4'-bipyridine-2',6-diamine Compound 284
(20mg, 0.045 mmol), p-toluenesulfonic acid 2-methoxyethyl ester (14mg, 0.06 mmol) and sodium carbonate (9.6mg, 0.09 mmol) in 1 ml DMSO was heated in a reaction vessel at 105°C in an oil bath for 3h. Formation of desired product was confirmed by LCMS, MH+ 502/504, 0.58min, with -50% conversion. Mixture was diluted with ethyl acetate, washed with water, dried over sodium sulfate, and concentrated. Crude product was purified by HPLC to give desired product as TFA salt. LCMS (m/z): 502.2/504.2, retention time = 0.56 min.
Example 52 (Compound 191)
N2'-((1 R,3R)-3-aminocyclopentyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6- diamine
Figure imgf000198_0001
Step ! Preparation of 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine: To 2,6- dibromopyridine (7.1 g, 30.0 mmol) was added NMP (16 ml), (3- fluorophenyl)methanamine (4.13 g, 33.0 mmol) and Huenig's Base (5.76 ml, 33.0 mmol) flushed with argon. The crude reaction mixture was stirred at 115-120 °C for 168 hr, followed by LCMS. The crude mixture was cooled, 250 ml of ethyl acetate was added, washed with saturated sodium bicarbonate (2x), water (2x), saturated salt solution (1x), dried sodium sulfate, filtered, concentrate. The crude was purified by silica gel chromatography using 120g column, eluting from 0%-20% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, giving 7.11 grams of the title compound as a free base used without further purification. LCMS (m/z): 281.1/283.1 (MH+), retention time = 1.03 min. Step 2. Preparation of 5'-chloro-2'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-6-amine: To 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine (2.0 g, 7.1 1 mmol) was added 5- chloro-2-fluoropyridin-4-ylboronic acid (1.996 g, 1 1.38 mmol), PdCI2(dppf).CH2CI2 adduct (0.465 g, 0.569 mmol), DME (27 ml) and last 2M sodium carbonate (9.25 ml, 18.50 mmol). The crude reaction was stirred at 100 °C for 3 hr, followed by LCMS. The crude mixture was cooled, 25 ml of ethyl acetate and 20 ml of methanol was added, filtered and concentrated to provide a crude product. The crude was purified by silica gel chromatography using a 120g column, eluting from 0%-20% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, giving 1.259 grams of titled compound as free base use without further purification. LCMS (m/z): 332.2 (MH+), retention time = 0.92 min.
Step3. Preparation of (1 S,3R)-3-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin- 2'-yl-amino)cyclopentanol: To 5'-chloro-2'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-6-amine (75 mg, 0.226 mmol), was added (1S,3R)-3-aminocyclopentanol (68.6 mg, 0.678 mmol), NMP (0.75 ml) and triethylamine (0.158 ml, 1.130 mmol) . The crude reaction mixture was stirred at 100 °C for 18 hr, and the reaction progress followed by LCMS. The crude reaction mixture was cooled, filtered, and purified by prep LC. The product fractions were collected, 50 mL of 1 M NaOH and 50 mL of EtOAc were added. The aqueous layer was removed, the organic layer was washed with 50 mL of saturated salt solution, dried over sodium sulfate, and reduced to constant mass. 28 mg of the desired compound was obtained. LCMS (m/z): 413.1 (MH+), retention time = 0.67 min.; H NMR (400 MHz, CHLOROFORM-d, 25°C) δ ppm 1.71 (d, J = 14.09 Hz, 1 H) 1.75 - 1.91 (m, 2 H) 1.97 - 2.05 (m, 1 H) 2.10 - 2.16 (m, 1 H) 2.61 (br. s., 1 H) 4.03 - 4.18 (m, 1 H) 4.39 (tt, J = 4.84, 2.59 Hz, 1 H) 4.55 (d, J = 5.09 Hz, 2 H) 5.19 (br. s., 2 H) 6.41 (d, J = 8.22 Hz, 1 H) 6.55 (s, 1 H) 6.90 - 7.02 (m, 2 H) 7.05 - 7.18 (m, 2 H) 7.24 - 7.34 (m, 1 H) 7.43 - 7.55 (m, 1 H) 8.07 (s, 1 H).
Step 4. Preparation of N2'-((1 R,3R)-3-aminocyclopentyl)-5'-chloro-N6-(3- fluorobenzyl)-2,4'-bipyridine-2',6-diamine: To (1 S,3R)-3-(5'-chloro-6-(3- fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)cyclopentanol (28 mg, 0.068 mmol) was added DCM (1 ml), diisopropyl ethylamine (0.030 ml, 0.170 mmol) then mesyl chloride (5.81 μΙ, 0.075 mmol), stirred at ambient temperature for 1 hr, and followed by LCMS. Another 3 uL of mesyl chloride was added and the reaction mixture was stirred an additional 30 minutes at ambient temperature. DCM was removed by rotary evaporation, and crude (1 S,3R)-3-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl- amino)cyclopentyl LCMS (m/z): 491.2 (MH+), retention time = 0.76 min. was redissolved in 2 ml_ DMF. Sodium azide (8.82 mg, 0.136 mmol) and diisopropyl ethylamine (0.030 ml, 0.170 mmol) were added, and the reaction mixture was heated at 50 °C for 18 hours, at which point only N2'-((1 R,3R)-3-azidocyclopentyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'- bipyridine-2',6-diamine was observed by LCMS (m/z): 438.2 (MH+), retention time = 0.83 min. The resulting reaction mixture was partitioned between ethyl acetate and water. The aqueous layer was removed, and the organic layer was washed with water (1x) then saturated salt solution (1x), dried over sodium sulfate, and reduced to constant mass. Crude N2'-((1 R,3R)-3-azidocyclopentyl)-5'-chloro-N6-(3-fluorobenzyl)-2,4'-bipyridine-2',6- diamine (20 mg, 0.046 mmol, LCMS (m/z): 438.2 (MH+), retention time = 0.83 min.) was dissolved in 1 ml_ of methanol, and 10% palladium on charcoal (4.86 mg, 0.046 mmol) was added under argon. H2 was bubbled through the solution while stirring for 1 hr at ambient temperature, and the reaction was followed by LCMS. The crude reaction mixture was filtered over celite washed with methanol, reduced, redissolved in DMSO, filtered and purified through prep LC. The resulting product fractions were combined, then 50 mL of 1 M NaOH and 50 mL of EtOAC were added. The aqueous layer was removed, the organic layer was washed with saturated salt solution, dried over sodium sulfate, and reduced to constant mass. 8 mg of the desired compound was obtained. LCMS (m/z): 412.1 (MH+), retention time = 0.58 min.; H NMR (300 MHz,
CHLOROFORM-d, 25 °C) δ ppm 1.31 - 1.54 (m, 2 H) 1.71 - 1.86 (m, 4 H) 1.98 - 2.13 (m, 1 H) 2.20 - 2.35 (m, 1 H) 3.54 (qd, J=6.35, 6.15 Hz, 1 H) 4.14 (sxt, J=6.56 Hz, 1 H) 4.55 - 4.67 (m, 3 H) 5.11 (t, J=5.86 Hz, 1 H) 6.40 (d, J=8.50 Hz, 1 H) 6.56 (s, 1 H) 6.88 - 7.02 (m, 1 H) 7.12 - 7.16 (m, 1 H) 7.29 - 7.34 (m, 1 H) 7.47 - 7.52 (m, 1 H) 8.09 (s, 1 H).
Example 53 (Compound 205)
(1S,3R)-3-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)-N,N- dimethylcyclopentanecarboxamide
Figure imgf000200_0001
Step ! Preparation of 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine: To 2,6- dibromopyridine (7.1 g, 30.0 mmol) was added NMP (16 ml), (3- fluorophenyl)methanamine (4.13 g, 33.0 mmol) and Huenig's Base (5.76 ml, 33.0 mmol) flushed with argon. The crude reaction mixture was stirred at 115-120 °C for 168 hr , followed by LCMS. The crude mixture was cooled, 250 ml of ethyl acetate was added, washed with saturated sodium bicarbonate (2x), water (2x), saturated salt solution (1x), dried sodium sulfate, filtered, concentrate. The crude was purified by silica gel chromatography using 120g column, eluting from 0%-20% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, giving 7.11 grams of the titled compound as a free base used without further purification. LCMS (m/z): 281.1/283.1 (MH+), retention time = 1.03 min.
Step 2. Preparation of 5'-chloro-2'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-6- amine: To 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine (2.0 g, 7.11 mmol) was added 5- chloro-2-fluoropyridin-4-ylboronic acid (1.996 g, 1 1.38 mmol), PdCI2(dppf).CH2CI2 adduct (0.465 g, 0.569 mmol), DME (27 ml) and last 2M sodium carbonate (9.25 ml, 18.50 mmol). The crude reaction mixture was stirred at 100 °C for 3 hr, followed by
LCMS. The crude mixture was cooled, 25 ml of ethyl acetate and 20 ml of methanol was added, filtered and concentrated to crude product. The crude was purified by silica gel chromatography using a 120g column, eluting from 0%-20% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, giving 1.259 grams of title compound as free base use without further purification. LCMS (m/z): 332.2 (MH+), retention time = 0.92 min.
Step 3: Preparation of (1S,3R)-3-(5'-chloro-6-(3-fluorobenzylamino)-2,4'- bipyridin-2'-yl-amino)cyclopentanecarboxylic acid: To 5'-chloro-2'-fluoro-N-(3- fluorobenzyl)-2,4'-bipyridin-6-amine (100 mg, 0.301 mmol), was added (1S,3R)-3- aminocyclopentanecarboxylic acid (117 mg, 0.904 mmol), powdered potassium hydroxide (85 mg, 1.507 mmol) and dioxane (1 ml). The reaction mixture was stirred at 100 °C for 18 hr in a sealed vessel and followed by LCMS. The crude reaction mixture was partitioned between 30 mL saturated ammonium chloride and 30 mL ethyl acetate. The organic layer was removed, dried over sodium sulfate, and reduced. This was redissolved in 1.5 mL DMSO, filtered, and purified through prep LC. The product fractions were combined and extracted with 50 mL ethyl acetate, which was dried over sodium sulfate, and concentrated to constant mass. 10 mg of the desired compound was obtained. LCMS (m/z): 441.2 (MH+), retention time = 0.68 min. H NMR (400 MHz, CHLOROFORM-d, 25 °C) δ ppm 1.59 (m, 2 H) 1.83 (m, 2 H) 1.99 (m, 1 H) 2.72 (m, 1 H) 3.40 (br. s., 1 H) 3.78 (br. s., 1 H) 4.42 (br. s., 1 H) 5.48 (br. s., 1 H) 6.29 (d, J=8.22 Hz, 1 H) 6.50 (s, 1 H) 6.80 - 6.92 (m, 2 H) 6.95 - 7.10 (m, 2 H) 7.16 - 7.25 (m, 1 H) 7.38 (t, J=8.02 Hz, 1 H) 7.89 (s, 1 H).
Step 4. Preparation of (1 S,3R)-3-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin- 2'-yl-amino)-N,N-dimethylcyclopentanecarboxamide: To (1 S,3R)-3-(5'-chloro-6-(3- fluorobenzylamino)-2,4'-bipyridin-2'-yl-amino)cyclopentanecarboxylic acid U-31332- EXP080 (10 mg, 0.023 mmol), 2M dimethyl amine in THF (0.011 ml, 0.023 mmol), N1- ((ethylimino)methylene)-N3,N3-dimethylpropane-1 ,3-diamine hydrochloride (8.70 mg, 0.045 mmol), 3H-[1 ,2,3]triazolo[4,5-b]pyridin-3-ol (4.32 mg, 0.032 mmol) were added then dimethylformamide (1 ml) and diisopropyl ethylamine (0.016 ml, 0.091 mmol) were added, and the reaction mixture was stirred at ambient temperature for 18 hr and the progress followed by LCMS. The crude reaction mixture was filtered and purified by preparative LC. The product fractions were combined, 50 mL of 1 M NaOH and 50 mL of ethyl acetate were added. The organic layer was removed, washed with 50 mL 1 M NaOH, 50 mL saturated salt solution, dried over sodium sulfate, and reduced to constant mass. 3 mg of the desired compound was obtained. LCMS (m/z): 468.1 (MH+), retention time = 0.72 min., H NMR (300 MHz, CHLOROFORM-d) ppm 1.77 - 2.16 (m, 6 H) 2.96 (s, 3 H) 3.07 (s, 3 H) 3.10 - 3.25 (m, 1 H) 4.29 (m, 1 H) 4.56 (d, J=5.27 Hz, 2 H) 5.12 (br. s., 1 H) 5.87 (br. s., 1 H) 6.38 (d, J=8.50 Hz, 1 H) 6.58 (s, 1 H) 6.91 - 7.01 (m, 1 H) 7.06 - 7.20 (m, 1 H) 7.26 - 7.37 (m, 2 H) 7.44 - 7.53 (m, 1 H) 8.09 (s, 1 H).
Example 54 (Compound 235)
5'-chloro-N6-(3-fluorobenzyl)-N2'-((1 R,3S)-3-((methylamino)methyl)cyclopentyl)-2,4'- bipyridine-2',6-diamine
Figure imgf000202_0001
Step ! Preparation of 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine: To 2,6- dibromopyridine (7.1 g, 30.0 mmol) was added NMP (16 ml), (3- fluorophenyl)methanamine (4.13 g, 33.0 mmol) and Huenig's Base (5.76 ml, 33.0 mmol) flushed with argon. The crude reaction mixture was stirred at 115-120 °C for 168 hr, followed by LCMS. The crude mixture was cooled, 250 ml of ethyl acetate was added, washed with saturated sodium bicarbonate (2x), water (2x), saturated salt solution (1x), dried sodium sulfate, filtered, concentrate. The crude was purified by silica gel chromatography using 120g column, eluting from 0%-20% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, giving 7.11 grams of the titled compound as a free base used without further purification. LCMS (m/z): 281.1/283.1 (MH+), retention time = 1.03 min.
Step 2. Preparation of 5'-chloro-2'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-6- amine: To 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine (2.0 g, 7.11 mmol) was added 5- chloro-2-fluoropyridin-4-ylboronic acid (1.996 g, 1 1.38 mmol), PdCI2(dppf).CH2CI2 adduct (0.465 g, 0.569 mmol), DME (27 ml), and 2M sodium carbonate (9.25 ml, 18.50 mmol). The crude reaction mixture was stirred at 100 °C for 3 hr, and the reaction progress followed by LCMS. The crude mixture was cooled, 25 ml of ethyl acetate and 20 ml of methanol were added, filtered and concentrated to yield a crude product. The crude was purified by silica gel chromatography using a 120g ISCO column, eluting from 0%- 20% ethyl acetate with hexane. The desired fractions were concentrated to constant mass, giving 1.259 grams of title compound as free base use without further purification. LCMS (m/z): 332.2 (MH+), retention time = 0.92 min. Step 3. Preparation of (1 R,4S)-tert-butyl 3-oxo-2-azabicyclo[2.2.1]heptane-2- carboxylate: A mixture of trans-2-azabicyclo[2.2.1]hept-5-en-3-one (2 g, 18.33 mmol) and 10% Pd/C (0.780 g, 0.733 mmol) in MeOH (100 ml) was stirred under atmospheric pressure of H2 at ambient temperature for 2 hr, and the reaction progress was followed by LCMS. Pd/C was filtered off over Celite and the filter cake was washed with MeOH. The combined organics were concentrated to afford crude (1 R,4S)-2- azabicyclo[2.2.1]heptan-3-one. LCMS (m/z): 1 12.1 (MH+), retention time = 0.30 min. The resulting residue was redissolved in DCM (100 ml), to which di-tert-butyl dicarbonate (8.51 ml, 36.7 mmol) and DMAP (1.231 g, 10.08 mmol) were added and stirred at ambient temperature for 18 hr and the reaction progress was followed by LCMS. Solvent was removed, and the crude reaction mixture was purified through column
chromatography, 10-40% EtOAc: Heptane. The desired fractions were concentrated to constant mass, yielding (1 R,4S)-tert-butyl 3-oxo-2-azabicyclo[2.2.1]heptane-2- carboxylate (2.99 g, 14.15 mmol) of a white solid. LCMS (m/z): 156.2 (M-tBu), retention time = 0.75 min.
Step 4. Preparation of tert-butyl (1 R,3S)-3-(hydroxymethyl)cyclopentylcarbamate (1 R,4S)-tert-butyl 3-oxo-2-azabicyclo[2.2.1]heptane-2-carboxylate (2.99 g, 14.15 mmol) was dissolved in MeOH (40 ml) and cooled to 0 °C. Sodium Borohydride (1.071 g, 28.3 mmol) was added and the reaction was stirred at 0 °C for 1 hr, and the reaction progress was followed by LCMS. MeOH was removed and the resulting residue was partitioned between EtOAc (250 mL) and H20 (250 mL). The organic layer was washed with brine (250 mL), dried over Na2S04, and concentrated under reduced pressure. The crude material was purified by column chromatography, 50-100% EtOAc in heptane to yield tert-butyl (1 R,3S)-3-(hydroxymethyl)cyclopentylcarbamate (2.92 g, 13.56 mmol) as a white solid. LCMS (m/z): 160.2 (M - tBu), retention time = 0.65 min.
Step 5. Preparation of ((1S,3R)-3-aminocyclopentyl)methanol: Tert-butyl (1 R,3S)-3-(hydroxymethyl)cyclopentylcarbamate (2.92 g, 13.56 mmol) was dispersed in H20 (50 ml) and refluxed at 100 °C for 18 hr, followed by LCMS. Water was removed by azeotroping with toluene (50 mL x 3). Collected ((1S,3R)-3-aminocyclopentyl)methanol (1.92 g, 12.50 mmol) as a clear, viscous oil which was used without further purification. LCMS (m/z): 1 16.1 (MH+), retention time = 0.67 min.
Step 6. Preparation of ((1S,3R)-3-(5'-chloro-6-(3-fluorobenzylamino)-2,4'- bipyridin-2'-yl-amino)cyclopentyl)methanol: To 5'-chloro-2'-fluoro-N-(3-fluorobenzyl)-2,4'- bipyridin-6-amine (100 mg, 0.301 mmol) was added DMSO (1 ml), ((1S,3R)-3- aminocyclopentyl)methanol (104 mg, 0.903 mmol) and TEA (0.21 ml, 1.51 mmol). The crude mixture was stirred at 100 °C for 20 hours, followed by LCMS. The crude reaction mixture was cooled, was diluted with EtOAc (60 mL), washed H20 (60 mL x 2), brine (60 mL), dried over Na2S04, and reduced. The crude was adsorbed onto silica gel, and purified by silica gel chromatography, 40-80% EtOAc/Heptane, 12g ISCO silica column, resulting in ((1 S,3R)-3-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl- amino)cyclopentyl)methanol (101 mg, 0.237 mmol). LCMS (m/z): 427.1 (MH+). retention time = 0.69 min.
Step 7. Preparation of (1S,3R)-3-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl- amino)cyclopentanecarbaldehyde
In a flame-dried argon purged 20 mL conical flask, oxalyl chloride (0.025 ml, 0.281 mmol) was dissolved in DCM (0.5 ml) and cooled to -78 °C under argon. DMSO (0.030 ml, 0.422 mmol) was dissolved in DCM (0.5 ml) and added dropwise to the previous solution ( I don't see issues here). This was stirred for 30 min at -78 °C.
((1S,3R)-3-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl- amino)cyclopentyl)methanol (60 mg, 0.141 mmol) was dissolved in DCM (0.5 ml) and added dropwise to the reaction mixture. The resulting mixture was stirred for 60 min at - 78 °C. TEA (0.078 ml, 0.562 mmol) was dissolved in DCM (0.5 ml) and added dropwise to the reaction mixture, after which the reaction mixture was allowed to stir and warm to ambient temp over 2 hr. The reaction mixture was diluted with EtOAc, washed with saturated NH4CI (30 ml_ x 3), H20 (30 ml_), brine (30 ml_), dried over Na2S04 and reduced. The resulting residue was used without further purification. LCMS (m/z): 425.2 (MH+), retention time = 0.72.
Step 8. Preparation of 5'-chloro-N6-(3-fluorobenzyl)-N2'-((1 R,3S)-3- ((methylamino)methyl)cyclopentyl)-2,4'-bipyridine-2',6-diamine To (1S,3R)-3-(5'-chloro-6-(3-fluorobenzylamino)-2,4'-bipyridin-2'-yl- amino)cyclopentanecarbaldehyde (20 mg, 0.047 mmol) was added methyl amine in THF (0.5 ml, 1.0 mmol) and DCM (0.5 ml_). Acetic acid (2.69 μΙ, 0.047 mmol), and sodium triacetoxyborohydride (14.96 mg, 0.071 mmol) were added and stirred for 2 hr at ambient temperature, and the reaction progress was followed by LCMS. Solvents were removed, and the crude reaction mixture redissolved in 1.5 mL of DMSO, followed by purification using preparative HPLC. Product fractions were combined and lyophilized to afford 5'- chloro-N6-(3-fluorobenzyl)-N2'-((1 R,3S)-3-((methylamino)methyl)cyclopentyl)-2,4'- bipyridine-2',6-diamine (2.5 mg, 0.006 mmol) as a TFA salt. LCMS (m/z): 440.2 (MH+), retention time = 0.62 min.
Example 56 (Compound 212)
N-2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((R)-6-oxaspiro[2.5]octan-1-yl)-2,4'- bipyridine-2',6-diamine
212
Figure imgf000205_0001
Step 1 : Preparation of (R)-6-bromo-N-(6-oxaspiro[2.5]octan-1-yl)pyridin-2-amine: To a solution of 2, 6-dibromopyridine (200 mg, 0.84 mmol) in NMP (0.42 ml_) was added (R)-6-oxaspiro[2.5]octan-1-amine hydrochloride (138 mg, 0.84 mmol) and potassium carbonate (350 mg, 2.53 mmol). The mixture was heated at 110 °C for 18 hr. The mixture was allowed to cool to ambient temperature and diluted with EtOAc. The organic layer was washed with saturated aqueous sodium bicarbonate solution, water, and brine and dried over sodium sulfate, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 40 g, EtOAc/heptane = 0/100 to 30/70]. Pure fractions were combined and concentrated in vacuo giving 210 mg of titled compound. LCMS (m/z): 282.9/284.9 [M+H]+, retention time = 0.85 min.
Step 2. Preparation of (R)-5'-chloro-2'-fluoro-N-(6-oxaspiro[2.5]octan-1-yl)-2,4'-bipyridin- 6-amine: A mixture of (R)-6-bromo-N-(6-oxaspiro[2.5]octan-1-yl)pyridin-2-amine (C, 100 mg, 0.35 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (136 mg, 0.77 mmol),
PdCl2(dppf).CH2Cl2 adduct (23 mg, 0.028 mmol) in DME (1 ml_) and 2M Na2C03 (97 mg, 0.92 mmol) in a sealed tube was heated at 103 °C for 2 hr. The mixture was allowed to cool to ambient temperature and was diluted with EtOAc (-25 ml_) and MeOH (~5 ml_), filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 12 g, EtOAc/heptane = 10/90 to 50/50]. Fractions were combined and concentrated in vacuo giving 105 mg of titled compound. LCMS (m/z): 334.0/336.0 [M+H]+, retention time = 0.64 min. Step3. Preparation of N-2'-(trans-4-aminocyclohexyl)-5'-chloro-N6-((R)-6- oxaspiro[2.5]octan-1-yl)-2,4'-bipyridine-2',6-diamine: A mixture of (R)-5'-chloro-2'-fluoro- N-(6-oxaspiro[2.5]octan-1-yl)-2,4'-bipyridin-6-amine (15 mg, 0.045 mmol), trans- cyclohexane-1 ,4-diamine (10.3 mg, 0.090 mmol), in DMSO (0.2 mmol) in a sealed tube was heated at 110 °C for 18 hr. The mixture was allowed to cool to ambient
temperature. To the reaction mixture was added 0.5 ml of DMSO, filtered and purified by prep LC. After lyophilization, 5.0 mg of the titled compound as a TFA salt was obtained. LCMS (m/z): 428.3/430.3 (MH+), retention time = 0.46 min.
Example 57 (Compound 230)
N-(4-Amino-cyclohexyl)-5'-chloro-N-(1 ,1-dioxo-hexahydro-1-thiopyran-4-yl-methyl)- [2,4']bipyridinyl-6,2'-diamine
Figure imgf000207_0001
Step 1. Preparation of toluene-4-sulfonic acid 1 , 1-dioxo-hexahydro-1-thiopyran-4- yl-methyl ester: A solution of (1 , 1-Dioxo-hexahydro-1-thiopyran-4-yl)-methanol (500 mg, 3.04 mmol) in pyridine (10 mL) was added 4-methylbenzene-1-sulfonyl chloride (871 mg, 4.57 mmol). The mixture was stirred at ambient temperature for 18 hr. The mixture was diluted with EtOAc. The organic layer was washed with saturated aqueous sodium bicarbonate solution, water, and brine and dried over sodium sulfate, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 12 g, EtOAc/heptane = 0/100 to 30/70]. Pure fractions were combined and concentrated in vacuo giving 736 mg of title compound. LCMS (m/z): 319.1 (MH+), retention time = 0.69 min. Step 2. Preparation of (6-Bromo-pyridin-2-yl)-(1 , 1-dioxo-hexahydro-1-thiopyran-4- yl-methyl)-amine: A mixture of toluene-4-sulfonic acid 1 , 1-dioxo-hexahydro-1-thiopyran- 4-yl-methyl ester (736 mg, 2.31 mmol), 6-bromopyridin-2-amine (400 mg, 2.312 mmol), potassium carbonate (639 mg, 4.62 mmol), sodium hydride (1 11 mg, 4.62 mmol) in a sealed tube was heated at 68 °C for 18 hr. The mixture was allowed to cool to ambient. The mixture was diluted with EtOAc. The organic layer was washed with saturated aqueous sodium bicarbonate solution, water, and brine and dried over sodium sulfate, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 12 g, EtOAc/heptane = 0/100 to 30/70]. Pure fractions were combined and concentrated in vacuo giving 240 mg of titled compound. LCMS (m/z): 318.8/320.9 (MH+), retention time = 0.71 min.
Step 3. Preparation of (5'-Chloro-2'-fluoro-[2,4']bipyridinyl-6-yl)-(1 , 1-dioxo-hexahydro-1- thiopyran-4-yl-methyl)-amine A mixture of (6-bromo-pyridin-2-yl)-(1 , 1-dioxo-hexahydro-1-thiopyran-4-yl- methyl)-amine (238 mg, 0.746 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (261 mg, 1.491 mmol), adduct (48.7 mg, 0.060 mmol) in DME (2 ml_) and 2M Na2C03 (205 mg, 1.938 mmol) in a sealed tube was heated at 103 °C for 2 hr. The mixture was allowed to cool to ambient temperature and was diluted with EtOAc (-25 ml_) and MeOH (~5 ml_), filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 12 g, EtOAc/heptane = 10/90 to 50/50]. Fractions were combined and concentrated in vacuo giving 150 mg of the title compound. LCMS (m/z): 370.0/372.0 (MH+); Retention time = 0.56 min.
Step 4. Preparation of N-(4-amino-cyclohexyl)-5'-chloro-N-(1 , 1-dioxo-hexahydro- 1-thiopyran-4-yl-methyl)-[2,4']bipyridinyl-6,2'-diamine: A mixture of (R)-5'-chloro-2'-fluoro- N-(6-oxaspiro[2.5]octan-1-yl)-2,4'-bipyridin-6-amine (40 mg, 0.108 mmol), and trans- cyclohexane-1 ,4-diamine (124 mg, 1.082 mmol) in DMSO (0.4 mmol) was heated in a sealed tube at 100 °C for 4 hr. The mixture was allowed to cool to ambient temperature. To the cooled reaction mixture was added 0.5 ml of DMSO, filtered and purified by prep LC. After lyophilization, 10.0 mg of the titled compound as a TFA salt was obtained. LCMS (m/z): 464.1/466.1 (MH+), retention time = 0.44 min. Example 58 (Compound 317)
5'-chloro-N6-(dideutero-(tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-(((S)- tetrahydrofuran-2-yl)methyl)aminocyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000208_0001
Step! Preparation of dideutero-(tetrahydro-2H-pyran-4-yl)methanamine: To a solution of tetrahydro-2H-pyran-4-carbonitrile (800 mg, 7.20 mmol) in THF (20 ml_) was added aluminum(lll) lithium deuteride at 0 °C. The mixture was stirred at 0 °C for 2 hr. To the stirred reaction mixture was sequentially added 300 uL of water, 900 μΙ_ of 1 N NaOH and 300 μΙ_ of water. The mixture was filtered through a thin layer of celite to remove the solid. The filtrate was dried over sodium sulfate, filtered off and concentrated in vacuo giving 700 mg of titled compound. LCMS (m/z): 1 18.2 [M+H]+, retention time = 0.25 min. The crude product was used directly for next step.
Step2. Preparation of 6-bromo-N-(dideutero(tetrahydro-2H-pyran-4- yl)methyl)pyridin-2-amine: To a solution of 2, 6-dibromopyridine (1051 mg, 5.97 mmol) in DMSO (5 mL) was added dideutero(tetrahydro-2H-pyran-4-yl)methanamine (700 mg, 5.97 mmol) and diisopropylethylamine (926 mg, 7.17 mmol). The mixture was heated at 80 °C for 2 hr. The mixture was allowed to cool to ambient temperature and diluted with EtOAc. The organic layer was washed with saturated aqueous sodium bicarbonate solution, water, and brine and dried over sodium sulfate, filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 40 g, EtOAc/heptane = 0/100 to 30/70]. Pure fractions were combined and concentrated in vacuo giving 780 mg of titled compound. LCMS (m/z): 272.9/274.9 [M+H]+, retention time = 0.77 min.
Step3. Preparation of 5'-chloro-N-(dideutero(tetrahydro-2H-pyran-4-yl)methyl)-2'-fluoro- 2,4'-bipyridin-6-amine:
A mixture of 6-bromo-N-(dideutero(tetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine (500 mg, 1.83 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (642 mg, 3.66 mmol), PdCI2(dppf).CH2CI2 adduct (120 mg, 0.146 mmol) in DME (1 mL) and 2M Na2C03 (2.38 ml, 4.76 mmol) was heated in a sealed tube at 80 °C for 48 hr. The mixture was allowed to cool to ambient temperature and was diluted with EtOAc (-25 mL) and MeOH (-5 mL), filtered off and concentrated in vacuo. The resulting residue was purified by column chromatography [Si02, 12 g, EtOAc/heptane = 10/90 to 50/50]. Fractions were combined and concentrated in vacuo giving 180 mg of titled compound. LCMS (m/z): 324.0/325.8 [M+H]+, retention time = 0.58 min.
Step4. Preparation of 5'-chloro-N6-(dideutero(tetrahydro-2H-pyran-4-yl)methyl)- N2'-(trans-4-(((S)-tetrahydrofuran-2-yl)methyl)aminocyclohexyl)-2,4'-bipyridine-2',6- diamine: A mixture of 5'-chloro-N-(dideutero(tetrahydro-2H-pyran-4-yl)methyl)-2'-fluoro- 2,4'-bipyridin-6-amine (30 mg, 0.093 mmol), trans-N1-(((S)-tetrahydrofuran-2- yl)methyl)cyclohexane-1 ,4-diamine (60 mg, 0.30 mmol), in DMSO (0.4 mmol) was heated in a sealed tube at 110 °C for 68 hr. The mixture was allowed to cool to ambient temperature. To the reaction mixture was added 0.5 ml of DMSO, filtered and purified by prep LC. After lyophilization, 10.0 mg of the titled compound as a TFA salt was obtained. LCMS (m/z): 502.3/504.3 (MH+), retention time = 0.49 min.
Example 59 (Compound 324)
5'-chloro-5-fluoro-N2'-(trans-4-(oxetan-2-yl-methylamino)cyclohexyl)-N6-((tetrahydro-2H- pyran-4-yl)methyl)-2,4'
Figure imgf000210_0001
To a stirred solution of N2'-(trans-4-aminocyclohexyl)-5'-chloro-5-fluoro-N6- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine (90 mg, 0.207 mmol)) in DMSO (1.0 ml) was add potassium carbonate (71 .7 mg, 0.518 mmol), followed by
oxetan-2-yl-methyl 4-methylbenzenesulfonate (151 mg, 0.622 mmol). The mixture was heated at 83 °C for 2h. The mixture was allowed to cool to ambient temperature, then diluted with water and then extracted with EtOAc (x3). The organics were combined then washed with water (x2), saturated brine (x2), then dried (Na2S04), filtered and evaporated under reduced pressure. The resulting residue was purified by reverse phase prep HPLC and lyophilized to yield titled compound. LCMS (m/z): 504.4/506.5 (MH+) retention time = 0.60 min as a TFA salt. Example 60 (Compound 222)
trans-4-(5-chloro-4-(5-chloro-6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2- yl)pyridin-2-yl-amino)cyclohexanol
Figure imgf000210_0002
Step 1. Preparation of 6-bromo-3-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2- amine.
To a scintillation vial containing 3,5-dibromo-2-chloropyrazine (1 g, 3.67 mmol) and TEA (1 .024 ml, 7.34 mmol) was added MeCN (5 ml) and (tetrahydro-2H-pyran-4- yl)methanamine (0.557 g, 3.67 mmol). The homogenous reaction mixture was capped, and heated to 80 °C in a oil bath for 4 hr. The reaction mixture was concentrated to dryness, diluted with EtOAc and sequentially washed with sat NaHC03, and sat NaCI. The organic layer was dried Na2S04, filtered and concentrated. The crude was purified by column chromatography on silica gel ( 20%EtOAc/Hexane) to yield 6-bromo-3-chloro- N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (688 mg, 2.244 mmol, 61 .1 % yield), yield), LCMS (m/z): 308.0 (MH+), retention time = 0.94 min, and 6-bromo-5- chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (55 mg, 0.179 mmol, 4.89 % yield), LCMS (m/z): 308.0 (MH+), retention time = 0.91 min. Step 2. Preparation of 3-chloro-6-(5-chloro-2-fluoropyridin-4-yl)-N-((tetrahydro-2H-pyran- 4-yl)methyl)pyrazin-2-amine
To a degassed suspension of 6-bromo-3-chloro-N-((tetrahydro-2H-pyran-4- yl)methyl)pyrazin-2-amine (358 mg, 1.168 mmol), Na2C03 (1 .518 ml, 3.04 mmol) and 5- chloro-2-fluoropyridin-4-ylboronic acid (307 mg, 1 .752 mmol) in DME (5 ml) was added PdCl2(dppf).CH2Cl2 adduct (76 mg, 0.093 mmol) . The reaction mixture was capped in a flask and heated to 100 °C for 4 hr an oil bath. The reaction mixture was diluted with EtOAc and washed with H20 saturated NaCI. The organic layer was dried Na2S04, filtered and concentrated. The crude oil/solid was purified column chromatography on silica gel (30%EtOAc/Hexane) to yield 3-chloro-6-(5-chloro-2-fluoropyridin-4-yl)-N-
((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (160 mg, 0.448 mmol, 38.4 % yield), LCMS (m/z): 357.0 (MH+), retention time = 1.02 min.
Step 3. Preparation of trans-4-(5-chloro-4-(5-chloro-6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl-amino)cyclohexanol :To a scintillation vial containing 3-chloro-6-(5-chloro-2-fluoropyridin-4-yl)-N-((tetrahydro-2H-pyran-4- yl)methyl)pyrazin-2-amine (20 mg, 0.056 mmol) was added DMSO (1 ml) andtrans-4- aminocyclohexanol (32.2 mg, 0.280 mmol). The reaction mixture was capped and heated to 120 °C in an oil bath for 3 hr. The reaction product was purified by reverse phase preparative HPLC to yield trans-4-(5-chloro-4-(5-chloro-6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl-amino)cyclohexanol (2.2 mg, 4.86 μηιοΙ, 8.69 % yield), LCMS (m/z): 452.1 (MH+), retention time = 0.76 min as a TFA salt after lyophilizing. H NMR (400 MHz, M ETHANOL-^) . ppm 1.17 - 1.26 (m, 4 H) 1.27 - 1.39 (m, 2 H) 1.58 (dd, J=13.1 1 , 1 .76 Hz, 2 H) 1.84 - 2.02 (m, 5 H) 3.30 (d, J=7.04 Hz, 4 H) 3.43 - 3.61 (m, 2 H) 3.84 (dd, J=1 1.35, 3.13 Hz, 2H) 6.58 (s, 1 H) 7.66 (s, 1 H) 7.90 (s, 1 H).
Example 61 (Compound 223)
trans-N 1-(5-chloro-4-(3-chloro-6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2- yl)pyridin-2-yl)cyclohexane-1 ,4-diamine
Figure imgf000212_0001
Step 1 . Preparation of 5-chloro-6-(5-chloro-2-fluoropyridin-4-yl)-N-((tetrahydro- 2H-pyran-4-yl)methyl)pyrazin-2-amine. To a suspension of 6-bromo-5-chloro-N- ((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (20 mg, 0.065 mmol), Na2C03 (17.98 mg, 0.170 mmoi and 5-chloro-2-fluoropyridin-4-ylboronic acid (17.16 mg, 0.098 mmol) in DME (1 ml) was added PdCl2(dppf).CH2Cl2 adduct (4.26 mg, 5.22 μηιοΙ) . The reaction mixture was capped in a flask and heated to 100 °C for 4 hr an oil bath. The reaction mixture was diluted with EtOAc and washed with H20 sat NaCI. The organic layer was dried Na2S04, filtered and concentrated. The crude was purified by column
chromatography on silica gel (50%EtOAc/Hexane) to yield 5-chloro-6-(5-chloro-2- fluoropyridin-4-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (10 mg, 0.028 mmol, 42.9 % yield). LCMS (m/z): 357.0 (MH+), retention time = 0.95 min. Step 2. Preparation of trans-N 1-(5-chloro-4-(3-chloro-6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine. To a scintillation vial containing 5-chloro-6-(5-chloro-2-fluoropyridin-4-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl) pyrazin-2-amine (10 mg, 0.028 mmol) and TEA (7.80 μΙ, 0.056 mmol) was added DMSO (1 ml) and trans-cyclohexane-1 ,4-diamine (32.0 mg, 0.280 mmol) . The resulting homogenous reaction mixture was capped and heated to 100 °C in an oil bath for 3 hr. The reaction product was purified by reverse phase preparative HPLC to yield trans-N 1 - (5-chloro-4-(3-chloro-6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2-yl)pyridi yl)cyclohexane-1 ,4-diamine (7.7 mg, 0.014 mmol, 48.6 % yield), LCMS (m/z): 451 .1 (MH+), retention time = 0.63 min and a TFA salt after lyophilization.
H NMR (400 MHz, METHANOL-cH) ppm 1.23 - 1.36 (m, 3 H) 1.36 - 1.49 (m, 2 H) 1.51 - 1 .71 (m, 4 H), 1 .80 - 1 .94 (m, 1 H) 2.06 - 2.25 (m, 4 H) 3.08 - 3.19 (m, 1 H) 3.23 (d, J=6.65 Hz, 2 H) 3.33 - 3.43 (m, 2 H) 3.66 - 3.77 (m, 1 H) 3.92 (dd, J=1 1 .35, 3.13 Hz, 2 H) 6.69 (s, 1 H) 7.76 (s, 1 H) 8.05 (s, 1 H).
Example 62 (Compound 225)
3-chloro-6-(5-chloro-2-(trans-4-(pyrrolidin-1 -yl)cyclohexylamino)pyridin-4-yl)-N- ((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine
Figure imgf000213_0001
Step 1. Preparation of 3-chloro-6-(5-chloro-2-(trans-4-(pyrrolidin-1 - yl)cyclohexylamino) pyridin-4-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine: To a scintillation vial containing trans-N 1-(5-chloro-4-(5-chloro-6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine (12 mg, 0.027 mmol) and K2C03 (3.67 mg, 0.027 mmol) was added DMF (1 ml) and 1 ,4-dibromobutane (3.15 μΙ, 0.027 mmol) . The reaction mixture was capped and heated to 60 °C for 3 hr. The crude solution was concentrated and purified by reverse phase preparative H PLC to yield 3-chloro-6-(5-chloro-2-(trans-4-(pyrrolidin-1 -yl)cyclohexylamino)pyridin-4-yl)-N- ((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (3.8 mg, 6.13 μηιοΙ, 23.07 % yield), LCMS (m/z): 505.2 (MH+), retention time = 0.64 min, and a TFA salt after lyophilization.
H NMR (400 MHz, METHANOL-d4) δ ppm 1.26 - 1.47 (m, 4 H) 1.56 - 1.73 (m, 4 H) 2.01 (m, 3 H) 2.10 -2.32 (m, 6 H) 3.09 - 3.23 (m, 3 H) 3.36 - 3.44 (m, 4 H) 3.60 - 3.78 (m, 3 H) 3.89 - 3.98 (m, 2 H) 6.76 (s, 1 H) 7.76 (s, 1 H) 8.03 (s, 1 H). Example 63 (Compound 226)
6-(2-(trans-4-aminocyclohexylamino)-5-chloropyridin-4-yl)-N2-((tetrahydro-2H-pyran-4- yl)methyl)pyrazine-2,3-diamine
Figure imgf000214_0001
Step 1. Preparation of 6-bromo-N2-((tetrahydro-2H-pyran-4-yl)methyl)pyrazine- 2,3-diamine : To a scintillation vial containing 3,5-dibromopyrazin-2-amine (500 mg, 1.977 mmol) and TEA (0.551 ml, 3.95 mmol) was added MeCN (6 ml) and (tetrahydro- 2H-pyran-4-yl)methanamine (300 mg, 1 .977 mmol). The homogenous reaction mixture was capped and heated to 80 °C in a oil bath for 36 hr. The reaction mixture was concentrated to dryness, diluted with EtOAc and washed with sat NaHC03, sat NaCI. The organic layer was dried Na2S04, filtered and concentrated. The crude was purified by column chromatography on silica gel (30%EtOAc/Hexane) to yield 6-bromo-N2- ((tetrahydro-2H-pyran-4-yl)methyl)pyrazine-2,3-diamine (351 mg, 1 .222 mmol, 61 .8 % yield).
Step 2. Preparation of 6-(5-chloro-2-fluoropyridin-4-yl)-N2-((tetrahydro-2H-pyran-4- yl)methyl)pyrazine-2,3-diamine
To a degassed suspension of 6-bromo-N2-((tetrahydro-2H-pyran-4- yl)methyl)pyrazine-2,3-diamine (100 mg, 0.348 mmol), Na2C03 (96 mg, 0.905 mmoiy and 5-chloro-2-fluoropyridin-4-ylboronic acid (92 mg, 0.522 mmol) in DME (3 ml) was added PdCl2(dppf).CH2Cl2 adduct (22.75 mg, 0.028 mmol). The reaction mixture was capped in a flask and heated to 100 °C for 4 hr an oil bath. The reaction mixture was diluted with EtOAc and washed with H20, sat NaCI. The organic layer was dried Na2S04, filtered and concentrated. The crude was purified by column chromatography on silica gel (100%EtOAc/Hexane) to yield 6-(5-chloro-2-fluoropyridin-4-yl)-N2- ((tetrahydro-2H-pyran-4-yl)methyl)pyrazine-2,3-diamine (34 mg, 0.101 mmol, 28.9 % yield). ). LCMS (m/z): 338.2 (MH+), retention time = 0.65 min. Step 3. Preparation of 6-(2-(trans-4-aminocyclohexylamino)-5-chloropyridin-4- yl)-N2-((tetrahydro-2H-pyran-4-yl)methyl)pyrazine-2,3-diamine : To a scintillation vial containing 6-(5-chloro-2-fluoropyridin-4-yl)-N2-((tetrahydro-2H-pyran-4- yl)methyl)pyrazine-2,3-diamine (17 mg, 0.050 mmol) was added DMSO (1.3 ml) and trans-cyclohexane-1 ,4-diamine R2 (57.5 mg, 0.503 mmol). The homogenous reaction mixture was capped and heated to 100 °C in a oil bath for 16 hr. The reaction mixture was purified by reverse phase preparative HPLC to yield 6-(2-(trans-4- aminocyclohexylamino)-5-chloropyridin-4-yl)-N2-((tetrahydro-2H-pyran-4- yl)methyl)pyrazine-2,3-diamine (13.7 mg, 0.025 mmol, 49.9 % yield), LCMS (m/z): 432.1 (MH+), retention time = 0.41 min as a TFA salt after lyophilizing. H NM R (400 MHz, METHANOL-cH) d ppm 1.30 - 1 .50 (m, 4 H) 1.51 - 1.65 (m, 2 H) 1.69 - 1.78 (m, 2 H) 1.93 - 2.06 (m, 1 H) 2.07 - 2.24 (m, 4 H) 3.10 - 3.19 (m, 1 H) 3.36 - 3.45 (m, 2 H) 3.48 (d, J=6.65 Hz, 2 H) 3.64 - 3.75 (m, 1 H) 3.96 (dd, J=1 1.35, 3.13 Hz, 2 H) 7.04 - 7.10 (m, 1 H) 7.64 (s, 1 H) 8.01 (s, 1 H).
Example 64 (Compound 233)
trans-N 1-(5-chloro-4-(3-methyl-6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2- yl)pyridin-2-yl)cyclohexane-1 ,4-diamine
Figure imgf000215_0001
Step 1 . Preparation of 6-(5-chloro-2-fluoropyridin-4-yl)-5-methyl-N-((tetrahydro- 2H-pyran-4-yl)methyl)pyrazin-2-amine : To a degassed suspension of 5-chloro-6-(5- chloro-2-fluoropyridin-4-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (10 mg, 0.028 mmol), Na2C03 (0.036 ml, 2 M, 0.072 mmoiy and methylboronic acid (5 mg, 0.084 mmol) in DME (1 ml) was added PdCI2(dppf).CH2CI2 adduct (6 mg, 7.35 μηιοΙ) . The reaction was capped and heated to 105 °C for 4 hr an oil bath. The reaction was diluted with EtOAc and washed with H20, sat NaCI. The organic layer was dried
Na2S04, filtered and concentrated. The crude was purified by column chromatography on silica gel (50%EtOAc/Hexane) to yield 6-(5-chloro-2-fluoropyridin-4-yl)-5-methyl-N- ((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (7 mg, 0.021 mmol, 74.2 % yield). LCMS (m/z): 337.2 (MH+), retention time = 0.81 min.
Step 2. Preparation of trans-N 1-(5-chloro-4-(3-methyl-6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine
To a scintillation vial containing 6-(5-chloro-2-fluoropyridin-4-yl)-5-methyl-N- ((tetrahydro-2H-pyran-4-yl)methyl) pyrazin-2-amine (7 mg, 0.021 mmol) was added DMSO and trans-cyclohexane-1 ,4-diamine (23.73 mg, 0.208 mmol). The homogenous reaction mixture was capped and heated to 100 °C in an oil bath for 4 hr. The crude solution was purified by reverse phase preparative HPLC to yield trans-N1 -(5-chloro-4- (3-methyl-6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2-yl)pyridin-2- yl)cyclohexane-1 ,4-diamine (1 .1 mg, 2.018 μηιοΙ, 9.71 % yield), LCMS (m/z): 431.2 (MH+), retention time = 0.47 min as a TFA salt after lyophilizing.
Example 65 (Compound 316)
5'-chloro-N6-((6,6-dimethyl-1 ,4-dioxan-2-yl)methyl)-N2'-(trans-4-((R)-1 -methoxypropan-2- yl-amino)cyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000216_0001
Step 1 . Preparation of 1-(allyloxy)-2-methylpropan-2-ol.
To allylic alcohol (57.4 ml_, 844 mmol) at 0 °C was added NaH (60% in mineral oil, 2.43 g, 101 mmol). After 20 min 2,2-dimethyloxirane (15 ml_, 169 mmol) was added and the solution was refluxed overnight. Saturated NH4CI solution was added and extracted three times with ether. The organic layers were combined, dried over Na2S04 and concentrated to remove ether. The resulting residue was distilled (allylic alcohol was distilled first then the product was collected at 42 torr, bp 58-60 °C) to give the product as a colorless oil (12.3 g, 56%). H NMR (400 MHz, CDCI3) δ ppm 5.87-5.96 (1 H, m), 5.26- 5.31 (1 H, m), 5.18-5.21 (1 H, m), 4.03-4.05 (2H, m), 3.28 (2H, s), 2.31 (1 H, br s), 1.23, (3H, s), 1.22 (3H, s). Step 2. Preparation of 2-methyl-1-(oxiran-2-ylmethoxy)propan-2-ol.
1-(Allyloxy)-2-methylpropan-2-ol (1.50 g, 1 1.5 mmol) was dissolved in DCM (50 ml_) and cooled to 0 °C. mCPBA (77% max, 9.94 g) was added. The suspension was stirred at 0 °C for 6.5 hr. and then saturated NaHC03 solution (~20ml) and Na2S203 solution (~20ml) were added. The resulting mixture was stirred at 0 °C for 15 min and the two layers were separated. The aqueous layer was extracted twice with DCM. The organic layers were combined, dried over Na2S04 and concentrated. The resulting residue was purified on a silica gel column (heptane: EtOAc 1 :0 to 1 :2) to give the product as a colorless oil (620 mg, 37%). H NMR (400 MHz, CDCI3) δ ppm 3.64 (1 H, ddd, J = 12.0, 5.2, 2.8 Hz), 3.24-3.29 (1 H, m), 3.17-3.21 (1 H, m), 3.11-3.14 (1 H, m), 2.97-3.00 (1 H, m), 2.88 (1 H, br s), 2.60-2.64 (1 H, m), 2.44-2.47 (1 H, m), 1.02 (6H, s).
Step 3. Preparation of (6,6-dimethyl-1 ,4-dioxan-2-yl)methanol.
2-methyl-1-(oxiran-2-ylmethoxy)propan-2-ol (620 mg, 4.24 mmol) and 10-CSA
(300 mg, 1.29 mmol) were dissolved in DCM (30 ml_) and stirred at ambient temperature for 24 hr. Saturated NaHC03 solution was added and the two layers were separated. The aqueous phase was extracted four times with DCM. The organic layers were combined, dried over Na2S04 and concentrated. The resulting residue was purified on a silica gel column (heptane: EtOAc 1 :0 to 1 :2) to give the desired product as a colorless oil (400 mg, 64%). Some starting material was recovered. H NMR (400 MHz, CDCI3) δ ppm 3.90-3.96 (1 H, m), 3.76 (1 H, dd, J = 11.2, 2.8 Hz), 3.56 (1 H, dd, J = 11.6, 4.0 Hz), 3.46-3.50 (2H, m), 3.29 (1 H, t, J = 11.2 Hz), 3.24 (1 H, dd, J = 11.6, 1.2 Hz), 2.69 (1 H, br s), 1.35 (3H, s), 1.13 (3H, s).
Step 4. Preparation of (6,6-dimethyl-1 ,4-dioxan-2-yl)methyl methanesulfonate
TEA (0.52 ml_, 3.74 mmol) and (6,6-dimethyl-1 ,4-dioxan-2-yl)methanol (390 mg, 2.67 mmol) were dissolved in DCM (10 ml_). Methanesulfonyl chloride (0.249 ml_, 3.20 mmol) was slowly added at 0 °C. After the addition was completed the solution was warmed to ambient temperature and stirred for 1 hr. Saturated NaHC03 solution was added and the two layers were separated. The aqueous layer was extracted three times with DCM. The organic layers were combined, dried over Na2S04 and concentrated. The resulting residue was purified on a silica gel column (heptane: EtOAc 4: 1 to 1 :1) to give the product as a colorless oil (584 mg, 98%). H NMR (400 MHz, CDCI3) δ ppm 4.00-4.09 (3H, m), 3.74 (1 H, dd, J = 1 1.2, 2.8 Hz), 3.42 (1 H, d, J = 11.6 Hz), 3.16-3.23 (2H, m), 2.99 (3H, s), 1.27 (3H, s), 1.05 (3H, s). Step 5. 6-bromo-N-((6,6-dimethyl-1 ,4-dioxan-2-yl)methyl)pyridin-2-amine
6-Bromopyridin-2-amine (722 mg, 4.17 mmol) was dissolved in 8 ml_ of anhydrous DMF and cooled to 0 °C. NaH (60% in mineral oil, 195 mg, 4.87 mmol) was added. After 10 min the solution was warmed to ambient temperature and stirred for 45 min until bubbling ceased. The solution was cooled to 0 °C again and (6,6-dimethyl-1 ,4- dioxan-2-yl)methyl methanesulfonate (520 mg, 2.32 mmol) in 2 ml_ of DMF was added. After the addition was completed the solution was warmed to ambient temperature and stirred overnight. It was diluted with EtOAc and washed four times with water. The aqueous layers were combined and extracted once with EtOAc. The organic layers were combined, dried over Na2S04 and concentrated. The resulting residue was purified on prep HPLC and the collected fractions were combined, concentrated, basified with Na2C03 and extracted with EtOAc three times. The organic layers were combined, dried over Na2S04 and concentrated to give the product as a light yellow oil (270 mg, 39%). LC-MS (m/z): 301.0/303.0 (M+H), retention time = 0.86 min.
Example 66 (Compound 307)
5'-chloro-N6-((5,5-dimethyl-1 ,4-dioxan-2-yl)methyl)-N2'-(trans-4-((R)-1-methoxypropan-2- yl-amino)cyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000219_0001
Step 1. Preparation of 2-(allyloxy)-2-methylpropan-1-ol.
2,2-Dimethyloxirane (15.0 mL, 169 mmol) was dissolved in allylic alcohol (57.4 mL) and cooled to 0°C. Perchloric acid (70%, 7.26 mL, 84 mmol) was slowly added. The solution was then warmed to ambient temperature and stirred for 1.5 hr. Saturated NaHC03 solution was added and extracted three times with ether. The organic layers were combined, dried over Na2S04 and concentrated to remove ether. The resulting residue was distilled (allylic alcohol was distilled first then the product was collected at 38 torr, bp 74-76 °C) to give the product as a colorless oil (9.70 g, 44%). H NMR (400
MHz, CDCI3) δ ppm 5.87-5.97 (1 H, m), 5.25-5.31 (1 H, m), 5.12-5.16 (1 H, m), 3.92-3.94 (2H, m), 3.45 (2H, m), 1.19 (6H, s).
Step 2. Preparation of 2-methyl-2-(oxiran-2-ylmethoxy)propan-1-ol.
2-(allyloxy)-2-methylpropan-1-ol (2.37 g, 18.2 mmol) was dissolved in DCM (70 mL) and cooled to 0 °C. mCPBA (77% max, 15.71 g) was added. The suspension was stirred at 0 °C for 6.5 hr before saturated NaHC03 solution and Na2S203 solution were added. It was stirred at 0 °C for 15 min and the two layers were separated. The aqueous layer was extracted twice with DCM. The organic layers were combined, dried over Na2S04 and concentrated. The resulting residue was purified on a silica gel column (heptane: EtOAc 1 :0 to 1 :2) to give the product as a colorless oil (910 mg, 34%). H NMR (400 MHz, CDCI3) δ ppm 3.65 (1 H, dd, J = 11.2, 2.8 Hz), 3.47 (1 H, br s), 3.31-3.41 (3H, m), 3.07-3.09 (1 H, m), 2.74 (1 H, t, J = 4.8 Hz), 2.63-2.65 (1 H, m), 1.12 (6H, s). Step 3. Preparation of (5, 5-dimethyl-1 ,4-dioxan-2-yl)methanol. 2-Methyl-2-(oxiran-2-ylmethoxy)propan-1-ol (870 mg, 5.95 mmol) and 10-CSA (207 mg, 15%) were dissolved in DCM (70 ml_) and stirred at ambient temperature for 24 hr. More 10-CSA (100 mg) was added and the solution was stirred overnight. Saturated NaHC03 solution was added. The two layers were separated and the aqueous layer was extracted twice with DCM. The organic layers were combined, dried over Na2S04 and concentrated to give the product as a colorless oil (750 mg, 86%). H NMR (400 MHz, CDCI3) δ ppm 3.69-3.74 (1 H, m), 3.52-3.64 (5H, m), 3.43 (1 H, dd, J = 1 1.6, 0.8 Hz), 2.57 (1 H, br s), 1.32 (3H, s), 1.13 (3H, s). Step 4. Preparation of (5, 5-dimethyl-1 ,4-dioxan-2-yl)methyl methanesulfonate.
(5,5-Dimethyl-1 ,4-dioxan-2-yl)methanol (740 mg, 5.06 mmol) and TEA (0.988 ml_, 7.09 mmol) were dissolved in DCM (20 ml_). At 0 °C MsCI (0.473 ml_, 6.07 mmol) was added dropwise. After the addition the solution was warmed to ambient temperature and stirred for 1 hr. Saturated NaHC03 solution was added and the two layers were separated. The aqueous layer was extracted three times with DCM. The organic layers were combined, dried over Na2S04 and concentrated. The resulting residue was purified on a silica gel column (heptane: EtOAc 4:1 to 1 :1) to give the product as a colorless oil (805 mg, 71 %). H NMR (400 MHz, CDCI3) δ ppm 4.18-4.19 (2H, m), 3.71-3.76 (1 H, m), 3.66 (1 H, t, J = 10.8 Hz), 3.52-3.57 (2H, m), 3.37 (1 H, d, J = 11.6 Hz), 3.03 (3H, s), 1.28 (3H, s), 1.09 (3H, s).
Step 5. Preparation of 6-bromo-N-((5,5-dimethyl-1 ,4-dioxan-2-yl)methyl)pyridin-2-amine.
6-Bromopyridin-2-amine (771 mg, 4.46 mmol) was dissolved in 10 mL of anhydrous DMF and cooled to 0 °C. NaH (60% in mineral oil, 214 mg, 5.35 mmol) was added. After 10 min the solution was warmed to ambient temperature and stirred for 15 min until bubbling ceased, to give a dark green solution. (5,5-Dimethyl-1 ,4-dioxan-2- yl)methyl methanesulfonate (500 mg, 2.23 mmol) in 2 mL of DMF was added. After the addition was completed the solution was stirred at ambient temperature for 20 min, then heated at 60 °C for 1.5 hr. It was diluted with EtOAc and washed four times with water. The aqueous layers were combined and extracted once with EtOAc. The organic layers were combined, dried over Na2S04 and concentrated. The resulting residue was purified on a silica gel column (heptane: EtOAc 1 :0 to 1 :1) to give the product contaminated with the starting aminopyridine. Another purification on silica gel column (DCM:ether 20: 1) gave the pure product (306 mg, 46%). LC-MS (m/z): 301.0/303.0 (M+H), retention time = 0.89 min. Example 67 (Compound 291 )
Synthesis of 5'-chloro-N2'-(trans-4-((R)-1-methoxypropan-2-yl-amino)cyclohexyl)-N6- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000221_0001
Step 1. To sodium hydride (0.488 g, 12.21 mmol) in 5 mL of THF was added via syringe(S)-(+)-3-methoxy-2-propanol (1 .000 ml, 1 1 .10 mmol) in 25 mL of THF at ambient temperature. The mixture was stirred for 20 min. and followed by addition of p- toluenesulfonyl chloride (2.327 g, 12.21 mmol). The white cloudy solution was stirred at ambient temperature for 18 hrs. The reaction mixture was diluted with saturated aq. NaHC03 and extracted with EtOAc. The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated in vacuo to give 2 g of colorless liquid. The crude mixture was purified by Analogix system (silica gel column 40 g, gradient: 100%n-heptane to 30% EtOAc in Heptane; 30 min.). The pure fractions were concentrated in vacuo to give 1 .22 g of colorless oil. LC-MS (m/z): 245 (M+H), retention time = 0.83 min.
Step 2. To the tosylate obtained from step 1 (0.6 g, 2.45 mmol) in DMSO (6 ml) at ambient temperature was added trans-cyclohexane-1 ,4-diamine (0.84 g, 7.37 mmol). The light brown mixture was heated to 99 °C in a capped glass vial for 1 hr. LC/MS showed nearly complete consumption of the starting material. The mixture was diluted with water and extracted with DCM. The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated in vacuo to give 0.39 g of light brown liquid. This was used in the next step without further purification. LC-MS (m/z): 187 (M+H), Retention time = 0.14 min.
Step 3. A mixture of Intermediate G (60 mg, 0.168 mmol), the above cyclohexadiamine (100 mg, 0.537 mmol) and 2,6-LUTI DINE (0.039 ml, 0.0.337 mmol) in DMSO (1 ml) was heated in a capped vial on a heating block for 18 hrs. LC/MS showed containing about 50% product. The reaction mixture was purified by HPLC (ACN in water with gradient 10% - 50% in 16 minutes) and lyophilized to give 25 mg of light yellow powder. LC-MS (m/z): 522/524 (M+H), retention time = 0.62 min. H NMR (400 MHz, CDCI3) δ ppm 1.24 - 1.47 (m, 5 H) 1.50 - 1.79 (m, 2 H) 1 .79 - 2.01 (m, 4 H) 2.1 1 - 2.31 (m, 4 H) 3.16 - 3.26 (m, 2H) 3.28 - 3.45 (m, 5 H) 3.45 - 3.66 (m, 4 H) 6.82 (d, J=9.39 Hz, 1 H) 7.05 (br. s., 1 H) 7.59 (s, 1 H) 7.78 (d, J=9.39 Hz, 1 H) 7.95 (s, 1 H) 8.76 (br. s. , 1 H)
Example 68 (Compound 197)
5'-chloro-N6-(3-fluorobenzyl)-N2'-(frans-4-((methylamino)methyl)cyclohexyl)-2,4'- bipyridine-2',6-diamine
Figure imgf000222_0001
Step 1. Preparation of 2'-chloro-5'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-6- amine: To a solution of 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine (636 mg, 2.262 mmol) and 2-chloro-5-fluoropyridin-4-yl-boronic acid (555 mg, 3.17 mmol) in DME (4 ml) and 2M Na2C03 aq (2 ml) was added PdCI2(dppf). CH2CI2 adduct (92 mg, 0.1 13 mmol). This was then heated at 1 10 °C for 16 hours. The reaction mixture was allowed to cool and then the DME was evaporated under reduced pressure. The resulting residue was partitioned between EtOAc and water. The organics were combined, then washed with H20 (x3), saturated aq. brine (x3), then dried (Na2S04), filtered and evaporated under reduced pressure. The resulting residue was purified by flash column chromatography (silica gel; 20% EtOAc/hexane) to give 2'-chloro-5'-fluoro-N-(3-fluorobenzyl)-2,4'- bipyridin-6-amine (84mg).
Step 2. Preparation of 5'-chloro-N6-(3-fluorobenzyl)-N2'-(frans-4- ((methylamino)methyl)cyclohexyl)-2,4'-bipyridine-2',6-diamine: To a scintillation vial were added 2'-chloro-5'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-6-amine (34 mg, 0.102 mmol), trans-cyclohexane-1 ,4-diamine (52.7 mg, 0.461 mmol), 1 ,3-bis(2,6-di- isopropylphenyl)imidazol-2-ylidene(1 ,4-naphthoquinone)palladium(0) (13.39 mg, 10.25 μηιοΙ), KOH (51 .8 mg, 0.922 mmol) and Dioxane (0.6 ml_). The resulting mixture was stirred with heating at 70 °C for 16h and then concentrated in vacuo. The resulting residue was dissolved in EtOAc and washed with H20 (x2) followed by saturated brine (x2), then dried (Na2S04), filtered and evaporated under reduced pressure. The resulting residue was purified by reverse phase preparative HPLC and then lyophilized to yield 5'- chloro-N6-(3-fluorobenzyl)-N2'-(frans-4-((methylamino)rnethyl)cyclohexyl)-2,4'-bipyridine- 2',6-diamine (7.9mg), LCMS (m/z): 410.3 (MH+), retention time = 0.60 min as a TFA salt. H-NMR (400 MHz, METHANOL-d4, 25 °C) 1.40 - 1.70 (m, 4 H) 2.05 - 2.25 (m, 4 H) 3.10 - 3.25 (m, 1 H) 3.55 - 3.64 (m, 1 H) 4.57 (s, 2 H) 6.76 (d, J=8.4 Hz, 1 H) 6.93 -7.00 (m, 1 H) 7.11 (d, J=10.4 Hz, 1 H) 7.20 (m, 2 H) 7.28 - 7.36 (m, 1 H) 7.52 (d, J=6.4 Hz, 1 H) 7.61 (t, J=8.0 Hz, 1 H) 7.96 (d, J=4.8 Hz, 1 H).
Example 69 (Compound 180)
N2'-(trans-4-aminocyclohexyl)-N6-(3-fluorobenzyl)-5'-methoxy-2,4'-bipyridine-2',6- diamine
Step 1. Preparation of 2'-chloro-N-(3-fluorobenzyl)-5'-methoxy-2,4'-bipyridin-6- amine: To a solution of 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine (555 mg, 1.974 mmol) and 2-chloro-5-methoxypyridin-4-ylboronic acid (518 mg, 2.76 mmol) in DME (4 ml) and 2M Na2C03 aq (2 ml) was added PdCI2(dppf).CH2Cl2 adduct (81 mg, 0.099 mmol). This was then heated at 1 10 °C for 5h. The reaction mixture was allowed to cool and then the DME was evaporated under reduced pressure. The resulting residue was partitioned between EtOAc and water. The organics were combined, then washed with H20 (x3), saturated aq. brine (x3), then dried (Na2S04), filtered and evaporated under reduced pressure. The resulting residue was purified by flash column chromatography (silica gel; 15% to 25% EtOAc/hexane) to give 2'-chloro-N-(3-fluorobenzyl)-5'-methoxy-2,4'- bipyridin-6-amine (53mg).
Step 2. Preparation of N2'-(trans-4-aminocyclohexyl)-N6-(3-fluorobenzyl)-5'- methoxy-2,4'-bipyridine-2',6-diamine: To a scintillation vial was added 2'-chloro-N-(3- fluorobenzyl)-5'-methoxy-2,4'-bipyridin-6-amine (30 mg, 0.087 mmol), trans-cyclohexane- 1 ,4-diamine (45 mg, 0.394 mmol), 1 ,3-bis(2,6-di-isopropylphenyl)imidazol-2-ylidene(1 ,4- naphthoquinone)palladium(O) (11.4 mg, 8.73 μηιοΙ), KOH (45 mg, 0.802 mmol) and Dioxane (0.3 ml_). The resulting mixture was stirred at 100 °C for 18h. The mixture was concentrated in vacuo and then diluted with water. The resultant solid was filtered and washed with water (x3). The solid was then purified by reverse phase preparative HPLC and then lyophilized to yield N2'-(trans-4-aminocyclohexyl)-N6-(3-fluorobenzyl)-5'- methoxy-2,4'-bipyridine-2',6-diamine (6.5 mg), LCMS (m/z): 422.3 (MH+), retention time = 0.54 min as a TFA salt. H-NMR (400 MHz, METHANOL-d4, 25 °C) 1.40 - 1.66 (m, 4 H) 2.05 - 2.25 (m, 4 H) 3.10 - 3.25 (m, 1 H) 3.55 - 3.64 (m, 1 H) 3.86 (s, 3 H) 4.57 (s, 2 H) 6.69 (d, J=8.4 Hz, 1 H) 6.92 -7.00 (m, 1 H) 7.10 (d, J=10.0 Hz, 1 H) 7.17 (d, J=7.6 Hz, 1 H) 7.28-7.33 (m, 2 H) 7.48 - 7.52 (m, 2 H) 7.53 - 7.58 (m, 1 H).
Example 70 (Compound 211)
N2'-(trans-4-aminocyclohexyl)-N6-(3-fluorobenzyl)-5'-methyl-2,4'-bipyridine-2',6-diamine
Figure imgf000224_0001
Step 1. Preparation of 2'-fluoro-N-(3-fluorobenzyl)-5'-methyl-2,4'-bipyridin-6- amine: To a solution of 6-bromo-N-(3-fluorobenzyl)pyridin-2-amine (85 mg, 0.302 mmol) and 2-fluoro-5-methyl-4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)pyridine (102 mg, 0.430 mmol) in DME (2 ml_) and 2M Na2C03 aq (1 ml_) was added PdCI2(dppf).CH2CI2 adduct (21 mg, 0.026 mmol). This was then heated at 1 10 °C for 16h. The reaction mixture was allowed to cool and then the DME was evaporated under reduced pressure. The resulting residue was partitioned between EtOAc and water. The organics were combined, then washed with H20 (x3), saturated aq. brine (x3), then dried (Na2S04), filtered and evaporated under reduced pressure. The resulting residue was purified by flash column chromatography (silica gel; 15% to 25% EtOAc/ hexane) to give 2'-fluoro-N- (3-fluorobenzyl)-5'-methyl-2,4'-bipyridin-6-amine (43 mg).
Step 2. Preparation of N2'-(trans-4-aminocyclohexyl)-N6-(3-fluorobenzyl)-5'- methyl-2,4'-bipyridine-2',6-diamine: To a solution of 2'-fluoro-N-(3-fluorobenzyl)-5'- methyl-2,4'-bipyridin-6-amine (18 mg, 0.058 mmol) and trans-cyclohexane-1 ,4-diamine (39.6 mg, 0.347 mmol), in NMP (0.3ml_) was added DIPEA (20 μΙ_, 0.115 mmol). The mixture was heated at 130 °C for 48h. The mixture was allowed to cool then diluted with water and then extracted with EtOAc (x3). The combined organics were washed with saturated brine (x2), then dried (Na2S04), filtered and evaporated under reduced pressure. The resulting residue was purified by reverse phase preparative HPLC and then lyophilized to yield N2'-(trans-4-aminocyclohexyl)-N6-(3-fluorobenzyl)-5'-methyl-2,4'- bipyridine-2',6-diamine (4.2 mg), LCMS (m/z): 406.3 (MH+), retention time
a TFA salt.
Example 71 (Compound 280)
Racemic 3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4- (tetrahydrofuran-3-yl-amino)cyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000225_0001
Step 1. Preparation of racemic benzyl trans-4-(tetrahydrofuran-3-yl- amino)cyclohexylcarbamate:
To a stirred solution of benzyl trans-4-aminocyclohexylcarbamate (396 mg, 1.595 mmol) in CH2CI2 (9 ml) was added dihydrofuran-3(2H)-one (151 mg, 1.754 mmol) followed by acetic acid (150 μΙ_, 2.62 mmol) and sodium triacetoxyborohydride (439 mg, 2.073 mmol) under Argon. Stirred at 25 °C for 16h, then concentrated in vacuo. The resulting residue was partitioned between EtOAc and 1 M NaOH. The organics were combined, then washed with 1 M NaOH (x2), water (x2), saturated brine (x2), then dried (Na2S04), filtered and evaporated under reduced pressure to give racemic benzyl trans- 4-(tetrahydrofuran-3-yl-amino)cyclohexylcarbamate (495 mg). The resulting residue was used in next step without further purification.
Step 2. Preparation of racemic tert-butyl trans-4-aminocyclohexyl(tetrahydrofuran-3- yl)carbamate:
To a stirred solution of racemic benzyl trans-4-(tetrahydrofuran-3-yl- amino)cyclohexylcarbamate (495 mg, 1.555 mmol) in CH2CI2 (5 ml) was added BOC- Anhydride (0.397 ml, 1.710 mmol)and the resulting mixture was stirred at 25°C under Argon for 21 hours. The mixture was evaporated under reduced pressure and purified by flash column chromatography (silica gel; 15% to 25% EtOAc/ hexane). A solution of the resultant Boc protected intermediate (135 mg, 0.323 mmol) in MeOH (5 ml_) was hydrogenated under an atmosphere of hydrogen in the presence of 10% Pd/C (24 mg, 0.226 mmol) for 18h. The mixture was then filtered through Celite and the filtrate evaporated under reduced pressure to give racemic tert-butyl trans-4- aminocyclohexyl(tetrahydrofuran-3-yl)carbamate (100mg). The resulting residue was used in next step without further purification Step 3. Preparation of racemic 3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'- (trans-4-(tetrahydrofuran-3-yl-amino)cyclohexyl)-2,4'-bipyridine-2',6-diamine: To a scintillation vial was added 3,5'-dichloro-2'-fluoro-N-((tetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridin-6-amine (25 mg, 0.070 mmol), racemic tert-butyl trans-4- aminocyclohexyl(tetrahydrofuran-3-yl)carbamate (21.95 mg, 0.077 mmol), DIPEA (24.51 μΙ, 0.140 mmol) and NMP (0.2 ml). This was heated at 110 °C for 48h. The mixture was diluted with EtOAc and washed with water (x2), saturated brine (x2), then dried
(Na2S04), filtered and evaporated under reduced pressure. The resulting residue was dissolved in CH2CI2 (0.4 ml_) and treated with TFA (100 μΙ, 1.298 mmol). After 30 minutes, the mixture was concentrated in vacuo and the resulting residue was purified by reverse phase preparative HPLC and then lyophilized to yield racemic 3,5'-dichloro-N6- ((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-(tetrahydrofuran-3-yl-amino)cyclohexyl)- 2,4'-bipyridine-2',6-diamine (10.8 mg), LCMS (m/z): 520.1/522.0 (bis-chloro isotopic signature for MH+), retention time = 0.59 min as a TFA salt.
Example 72 (Compound 320)
3,5'-dichloro-N2'-(trans-4-(((R)-2,2-dimethyl-1 ,3-dioxolan-4-yl)methyl)aminocyclohexyl)- N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000226_0001
To a stirred solution of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine (68 mg, 0.151 mmol) in DMF (0.2 ml) was added DIPEA (80 μΙ_, 0.458 mmol) followed by (S)-(2,2-dimethyl-1 ,3- dioxolan-4-yl)methyl 4-methylbenzenesulfonate (42 mg, 0.147 mmol). The mixture was heated at 75 °C for 19 hours. The mixture was allowed to cool, then diluted with water and then extracted with EtOAc (x3). The organics were combined then washed with water (x2), saturated brine (x2), then dried (Na2S04), filtered and evaporated under reduced pressure. The resulting residue was purified by reverse phase prep HPLC and lyophilized to yield 3,5'-dichloro-N2'-(trans-4-(((R)-2,2-dimethyl-1 ,3-dioxolan-4- yl)methyl)aminocyclohexyl)-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyri diamine (4.4 mg), LCMS (m/z): 564.4/566.3 (bis-chloro isotopic signature for MH+) retention time = 0.65 min as a TFA salt. Example 73 (Compounds 323 and 327)
3,5'-dichloro-N2'-(trans-4-((R)-1-methoxypropan-2-yl-amino)cyclohexyl)-N6-(((S)- tetrahydro-2H-pyran-3-yl)methyl)-2,4'-bipyridine-2',6-diamine and 3,5'-dichloro-N2'- (trans-4-((R)-1-methoxypropan-2-yl-amino)cyclohexyl)-N6-(((R)-tetrahydro-2H-pyran-3- yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000227_0001
Step 1. Preparation of racemic (tetrahydro-2H-pyran-3-yl)methyl 4- methylbenzenesulfonate: To a stirred solution of (tetrahydro-2H-pyran-3-yl)methanol (1.0 g, 8.61 mmol) and DMAP (0.053 g, 0.430 mmol) in CH2CI2 (5.0 ml_) and pyridine (6.96 ml_, 86 mmol) was added Tosyl-CI (1.805 g, 9.47 mmol). (11 :23am). After 16h the mixture was evaporated under reduced pressure and the resulting residue partitioned between EtOAc and water. The organics were separated, then washed with 0.1 M HCI (x3), H20 (x1), saturated aq. NaHC03 (x2), H20 (x1), saturated brine (x1), then dried (Na2S04), filtered and evaporated under reduced pressure to give racemic (tetrahydro- 2H-pyran-3-yl)methyl 4-methylbenzenesulfonate (2.034g). The resulting residue was used in next step without further purification.
Step 2. Preparation of racemic tert-butyl 6-bromo-5-chloropyridin-2- yl((tetrahydro-2H-pyran-3-yl)methyl)carbamate: To a cooled (0 °C), stirred solution of tert-butyl 6-bromo-5-chloropyridin-2-ylcarbamate (1.00 g, 3.25 mmol) in DMF (13.0 ml_) was added 60% dispersion NaH (0.156 g, 3.90 mmol) under Argon. Stirred at 0 °C for 30 mins then added racemic (tetrahydro-2H-pyran-3-yl)methyl 4-methylbenzenesulfonate (1.143 g, 4.23 mmol). The mixture was then allowed to warm to 25 °C and stirring continued for 19h. The reaction mixture then was diluted with saturated NH4CI and then extracted with EtOAc (x3). Organics washed with water (x2), saturated brine (x2), then dried (Na2S04), filtered and evaporated under reduced pressure. The resulting residue was purified by flash column chromatography (silica gel; 5% to 15% EtOAc/ heptanes) to give racemic tert-butyl 6-bromo-5-chloropyridin-2-yl((tetrahydro-2H-pyran-3- yl)methyl)carbamate (938 mg).
Step 3. Preparation of tert-butyl 3,5'-dichloro-2'-fluoro-2,4'-bipyridin-6- yl((tetrahydro-2H-pyran-3-yl)methyl)carbamate: To a scintillation vial was added racemic tert-butyl 6-bromo-5-chloropyridin-2-yl((tetrahydro-2H-pyran-3-yl)methyl)carbamate (832 mg, 2.051 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (719 mg, 4.10 mmol) and PdCl2(dppf).CH2Cl2 adduct (167 mg, 0.205 mmol) followed by DME (3 ml_) and 2M Na2C03 aq (2 ml_). The mixture was heated at 90 °C for 20h, then allowed to cool and added water and then extracted with EtOAc (x3). The organics were washed with water (x2), saturated brine (x2), then dried (Na2S04), filtered and evaporated under reduced pressure. The resulting residue was purified by flash column chromatography (silica gel; 5% to 15% EtOAc/ heptanes) to give racemic tert-butyl 3,5'-dichloro-2'-fluoro-2,4'- bipyridin-6-yl((tetrahydro-2H-pyran-3-yl)methyl)carbamate (374 mg)
Step 4. Preparation of 3,5'-dichloro-N2'-(trans-4-((R)-1-methoxypropan-2-yl- amino)cyclohexyl)-N6-(((S)-tetrahydro-2H-pyran-3-yl)methyl)-2,4'-bipyridine-2',6-diamine and 3,5'-dichloro-N2'-(trans-4-((R)-1-methoxypropan-2-yl-amino)cyclohexyl)-N6-(((R)- tetrahydro-2H-pyran-3-yl)methyl)-2,4'-bipyridine-2',6-diamine:
To a scintillation vial was added racemic tert-butyl 3,5'-dichloro-2'-fluoro-2,4'- bipyridin-6-yl((tetrahydro-2H-pyran-3-yl)methyl)carbamate (114 mg, 0.250 mmol), trans- N1-((R)-1-methoxypropan-2-yl)cyclohexane-1 ,4-diamine (70 mg, 0.376 mmol) and DIPEA (0.088 ml, 0.501 mmol) followed by NMP (0.1 ml). The mixture was heated at 1 10 °C for 60 hr then concentrated in vacuo. The resulting residue was purified by reverse phase prep HPLC and lyophilized. The resulting white solid was free based by dissolving in EtOAc and then washing with 1 M NaOH (x3), water (x2), saturated brine (x2), then dried (Na2S04), filtered and evaporated under reduced pressure. The resulting residue was then purified by chiral separation chromatography to yield 3,5'-dichloro-N2'-(trans-4- ((R)-1-methoxypropan-2-yl-amino)cyclohexyl)-N6-(((S)-tetrahydro-2H-pyran-3-yl)methyl)- 2,4'-bipyridine-2',6-diamine (mg), LCMS (m/z): 522.1/523.9 (MH+), tR = 0.675 min. and 3,5'-dichloro-N2'-(trans-4-((R)-1-methoxypropan-2-yl-amino)cyclohexyl)-N6-(((R)- tetrahydro-2H-pyran-3-yl)methyl)-2,4'-bipyridine-2',6-diamine (mg) LCMS 522.1/523.9 (m/z): (MH+), retention time = 0.675 min. Example 74 (Compounds 321 and 325)
3,5'-dichloro-N2'-(trans-4-((R)-1 -methoxypropan-2-yl-amino)cyclohexyl)-N6-(((S)- tetrahydro-2H-pyran-2-yl)methyl)-2,4'-bipyridine-2',6-diamine and 3,5'-dichloro-N2'- (trans-4-((R)-1-methoxypropan-2-yl-amino)cyclohexyl)-N6-(((R)-tetrahydro-2H-pyran-2- yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000229_0001
The compounds were prepared according to Example 73, except using tetrahydro-2H-pyran-2-yl)methanol to give 3,5'-dichloro-N2'-(trans-4-((R)-1- methoxypropan-2-yl-amino)cyclohexyl)-N6-(((S)-tetrahydro-2H-pyran-2-yl)methyl)-2,4'- bipyridine-2',6-diamine LCMS (m/z): 522.1/524.1 (MH+), retention time = 0.708 min and 3,5'-dichloro-N2'-(trans-4-((R)-1 -methoxypropan-2-yl-amino)cyclohexyl)-N6-(((R)- tetrahydro-2H-pyran-2-yl)methyl)-2,4'-bipyridine-2',6-diamine LCMS (m/z): 522.1/524.1 (MH+), retention time = 0.708 min.
Example 75 (Compound 208)
trans-4-(5-chloro-4-(6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2-yl)pyridin-2-yl- amino)cyclohexanol
Figure imgf000229_0002
Step 1 : Preparation of 6-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine:
A mixture of 2,6-dichloropyrazine (950 mg, 6.38 mmol), DMSO (14 ml), TEA (1.067 ml, 7.65 mmol) and (tetrahydro-2H-pyran-4-yl)methanamine (771 mg, 6.70 mmol) was stirred at 75 °C for 6 hours, and the reaction progress was followed by LCMS. The crude reaction mixture was cooled to ambient temperature, diluted with 300 ml of ethyl acetate, washed with 1 M NaOH soln.(1x), water (1x), saturated salt soln. (1x), dried with sodium sulfate, filtered, and concentrated to constant mass, giving 1185 mg of titled compound as free base, used without further purification. LCMS (m/z): 228.0 (MH+), retention time = 0.73 min.
Step 2. Preparation of 6-(5-chloro-2-fluoropyridin-4-yl)-N-((tetrahydro-2H-pyran-4- yl)methyl)pyrazin-2-amine:
A mixture of 6-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (1390 mg, 6.10 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (2141 mg, 12.21 mmol), PdCl2(dppf).CH2Cl2 adduct (399 mg, 0.488 mmol), DME (24 ml) and 2M sodium carbonate (9.16 ml, 18.31 mmol) was stirred at1 10-1 15 °C for 90 min and the reaction progress was followed by LCMS. The reaction mixture was cooled, 30 ml of ethyl acetate and 20 ml of methanol were added, filtered and concentrated to crude product. The crude was purified by silica gel chromatography using 80g column eluting with 20- 75% ethyl acetate in heptane. The desired fractions were concentrated to constant mass, giving 980 mg of titled compound as free base. LCMS (m/z): 323.0 (MH+), retention time = 0.81 min.
Step 3. Preparation of trans-4-(5-chloro-4-(6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl-amino)cyclohexanol:
A mixture of 6-(5-chloro-2-fluoropyridin-4-yl)-N-((tetrahydro-2H-pyran-4- yl)methyl)pyrazin-2-amine (375 mg, 1.162 mmol), DMSO (3.5 ml) and trans-4- aminocyclohexanol (1204 mg, 10.46 mmol) was stirred at 100 °C for 18 hours and the progress was followed by LCMS. The reaction mixture was let cool, added 300 ml of ethyl acetate, washed with saturated sodium bicarbonate solution (3x), water (2x), saturated salt solution (1x), dried with sodium sulfate, filtered and concentrated to crude solid. The crude material was purified by silica gel chromatography using 40g column, eluting slowly from (80% ethyl acetate 20% heptane with 2% MeOH) to 100% ethyl acetate with 2% MeOH. The desired fractions are concentrated to a constant mass, lyophilized from 1 :1 ACN/water (does not fully dissolve), re-lyophilized from 80 ml of (60/40) ACN/water solution with sonicating to dissolve solid, giving 270 mg of title compound as free base. LCMS (m/z): 418.3 (MH+), retention time = 0.52 min.; H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.19 - 1.55 (m, 6 H) 1.71 (d, J=12.89 Hz, 2 H) 1.85 - 2.15 (m, 5 H) 3.28 - 3.32 (dMeOH, 2H App.) 3.40 (td, J=1 1.72, 1 .76 Hz, 2 H) 3.50 - 3.73 (m, 2 H) 3.94 (dd, J=1 1 .28, 3.08 Hz, 2 H) 6.66 (s, 1 H) 7.86 (s, 2 H) 7.99 (s, 1 H)
Example 76 (Compound 215 and 216)
1-((R)-3-((2'-(trans-4-aminocyclohexylamino)-5'-chloro-2,4'-bipyridin-6-yl- amino)methyl)piperidin-1 -yl)ethanone and 1 -((R)-3-((2'-(trans-4-aminocyclohexylamino)- 2,4'-bipyridin-6-yl-amino)methyl)piperidin-1 -yl)ethanone
Figure imgf000231_0001
Step 1 . Preparation of (R)-tert-butyl 3-((2'-(trans-4-aminocyclohexylamino)-5'-chloro-2,4'- bipyridin-6-yl-amino)methyl)piperidine-1 -carboxylate:
A mixture of trans-N 1-(5'-chloro-6-fluoro-2,4'-bipyridin-2'-yl)cyclohexane-1 ,4- diamine (Example 1 a, step 2) (50 mg, 0.156 mmol), DMSO (0.75 ml), (R)-tert-butyl 3- (aminomethyl)piperidine-l -carboxylate (167 mg, 0.779 mmol) and TEA (0.033 ml, 0.234 mmol) was stirred at 100-105 °C for 40 hours and the reaction progress was followed by LCMS. The reaction mixture was let cool, added 0.75 ml of DMSO, filtered and purified by prep LC, and lyophilized to yield 36 mg of titled compound as a TFA salt. LCMS (m/z): 515.4 (MH+), retention time = 0.64 min. ;
Step 2. Preparation of benzyl trans-4-(5'-chloro-6-((S)-piperidin-3-yl-methylamino)-2,4'- bipyridin-2'-yl-amino)cyclohexyl carbamate:
A mixture of (R)-tert-butyl 3-((2'-(trans-4-aminocyclohexylamino)-5'-chloro-2,4'- bipyridin-6-yl-amino)methyl)piperidine-1-carboxylate (36 mg, 0.070 mmol), DCM (1.2 ml), TEA (0.019 ml, 0.140 mmol) and benzyl 2,5-dioxopyrrolidin-1-yl carbonate (26.1 mg, 0.105 mmol) was stirred at ambient temperature for 2 hours and the reaction progress was followed by LCMS. To this crude reaction mixture was added 25 ml of ethyl acetate, washed with 2M sodium carbonate, water (2x) and saturated salt solution (1x), dried with sodium sulfate, filtered, concentrated to crude intermediate. To the crude intermediate was added 4M HCI in Dioxane (2 ml, 8.00 mmol) and stirred at ambient temperature for 1 hour. The crude reaction mixture was concentrated to constant mass, dissolved in 1 ml of DMSO and purified by prep LC. After lyophilization, 15 mg of the title compound was obtained as a TFA salt. LCMS (m/z): 549.4 (MH+), retention time = 0.67 min. Step 3. Preparation of 1-((R)-3-((2'-(trans-4-aminocyclohexylamino)-5'-chloro-2,4'- bipyridin-6-yl-amino)methyl)piperidin-1-yl)ethanone and 1-((R)-3-((2'-(trans-4- aminocyclohexylamino)-2,4'-bipyridin-6-yl-amino)methyl)piperidin-1-yl)ethanone:
A mixture of benzyl trans-4-(5'-chloro-6-((S)-piperidin-3-yl-methylamino)-2,4'- bipyridin-2'-yl-amino)cyclohexylcarbamate (15 mg, 0.027 mmol), DCM (2 mL), TEA (0.01 1 mL, 0.082 mmol) and acetic anhydride (3.09 μί, 0.033 mmol) was stirred at ambient temperature for 2 hours and the reaction progress was followed by LCMS. The solvent was concentrated off. The reaction mixture flask was flushed with argon, 10% palladium on activated carbon (5 mg, 4.70 μηιοΙ) was added and followed by careful addition of MeOH (0.8 mL). The resulting mixture was stirred under hydrogen for 45 minutes at ambient temperature and monitored by LCMS. To the crude reaction mixture was added 2 ml of DCM, filtered and the solvent was concentrated off. The resulting residue was dissolved in 1.0 ml of DMSO, filtered and purified by prep HPLC to give two fractions corresponding to the two title compounds respectively. After lyophilization, 4.0 mg of 1-((R)-3-((2'-(trans-4-aminocyclohexylamino)-5'-chloro-2,4'-bipyridin-6-yl- amino)methyl)piperidin-1-yl)ethanone, was obtained as a TFA salt. LCMS (m/z): 457.2 (MH+), retention time = 0.46 min. In addition, 1.0 mg of 1-((R)-3-((2'-(trans-4- aminocyclohexylamino)-2,4'-bipyridin-6-yl-amino)methyl)piperidin-1-yl)ethanone, as TFA salt was also obtained. LCMS (m/z): 423.2(MH+), retention time =0.45 min. This reaction yielded two products which are separated and purified by HPLC.
Example 77 (Compound 249)
6-(2-(trans-4-(aminomethyl)cyclohexylamino)-5-chloropyridin-4-yl)-N-methyl-N- ((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine
H
Figure imgf000232_0001
Step 1. Preparation of 6-chloro-N-methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin- 2-amine:
A mixture of 2,6-dichloropyrazine (298 mg, 2.000 mmol), DMSO (6 ml), TEA (0.418 ml, 3.00 mmol) and N-methyl-1-(tetrahydro-2H-pyran-4-yl)methanamine (264 mg, 2.040 mmol) was stirred at 70 °C for 16 hours, and the reaction progress was followed by LCMS. The crude reaction mixture was let cool to room temperature, diluted with 150 ml of ethyl acetate, washed with 1 M NaOH soln.(1x), water (2x), saturated salt soln. (1x), dried with sodium sulfate, filtered, and concentrated to constant mass, giving 475 mg of the title compound as free base, which was used without further purification. LCMS (m/z): 242.0 (MH+), retention time = 0.85 min.
Step 2. Preparation of 6-(5-chloro-2-fluoropyridin-4-yl)-N-methyl-N-((tetrahydro-2H- pyran-4-yl)methyl)pyrazin-2-amine:
To 6-chloro-N-methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine (450 mg, 1.862 mmol) was added 5-chloro-2-fluoropyridin-4-ylboronic acid (588 mg, 3.35 mmol), PdCI2(dppf).CH2CI2 adduct (182 mg, 0.223 mmol), DME (8 ml) and 2M sodium carbonate (2.79 ml, 5.59 mmol). The resulting reaction mixture was stirred at 110-1 15°C for 90 minutes, and the reaction progress was followed by LCMS. The reaction mixture was cooled, 20 ml of ethyl acetate and 10 ml of methanol were added, filtered and concentrated to crude product. The crude was purified by silica gel chromatography using 24g column eluting with 20-75% ethyl acetate in heptane. The desired fractions were concentrated to constant mass, giving 499 mg of titled compound as free base. LCMS (m/z): 337.1 (MH+), retention time = 0.90 min. Step 3. Preparation of 6-(2-(trans-4-(aminomethyl)cyclohexylamino)-5-chloropyridin-4- yl)-N-methyl-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-2-amine:
A mixture of 6-(5-chloro-2-fluoropyridin-4-yl)-N-methyl-N-((tetrahydro-2H-pyran-4- yl)methyl)pyrazin-2-amine (15 mg, 0.045 mmol), DMSO (0.4 ml), and tert-butyl (trans- 4-aminocyclohexyl)methylcarbamate (92 mg, 0.401 mmol) was stirred at 100-105 °C for 18 hours, and the reaction progress was followed by LCMS. To the crude intermediate was added 6 M aq. HCI (120 μΙ, 0.720 mmol) and heated at 80°C for 40 minutes, and the reaction progress was followed by LCMS. The reaction mixture was let cool, added 0.5 ml of DMSO, filtered and purified by prep LC. After lyophilization, 15.6 mg of the title compound, as a TFA salt was obtained. LCMS (m/z): 445.2 (MH+), retention time = 0.59 min.; H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.12 - 1.47 (m, 6 H) 1.59 (d,
J=12.60 Hz, 2 H) 1.67 (ddd, J=7.18, 3.81 , 3.66 Hz, 1 H) 1.92 (d, J=12.31 Hz, 2 H) 2.01 - 2.11 (m, 1 H) 2.16 (d, J=1 1.43 Hz, 2 H) 2.83 (d, =7.03 Hz, 2 H) 3.17 (s, 3 H) 3.33 - 3.45 (m, 2 H) 3.56 (d, J=7.33 Hz, 2 H) 3.60 - 3.72 (m, 1 H) 3.93 (dd, J=11.14, 2.93 Hz, 2 H) 6.92 (s, 1 H) 8.02 (d, J=2.64 Hz, 2 H) 8.1 1 (s, 1 H). Example 78 (Compound 244)
N-(trans-4-(5-chloro-4-(6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2-yl)pyridin-2- yl-amino)cyclohexyl)acetamide
Figure imgf000234_0001
Step 1 : Preparation of N-(trans-4-(5-chloro-4-(6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl-amino)cyclohexyl)acetamide :
A mixture of trans-N1-(5-chloro-4-(6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine (example 85) (14 mg, 0.034 mmol), DCM (0.5 ml), THF (0.500 ml), TEA (0.014 ml, 0.101 mmol) and acetic anhydride (3.48 μΙ, 0.037 mmol) was stirred at ambient temperature for 1 hour, and the reaction progress was followed by LCMS. The solvent was concentrated off, added 1.0 ml of DMSO, filtered and purified by prep LC. After lyophilization 6.3 mg of title compound was obtained as a TFA salt. LCMS (m/z): 445.2 (MH+), retention time = 0.59 min.; 1 H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.21 - 1.55 (m, 6 H) 1.70 (d,
J=12.89 Hz, 2 H) 1.92 (s, 3 H) 1.93 - 2.06 (m, 3 H) 2.10 (br. s., 2 H) 3.28 - 3.32 (dMeOH, 2H App.) 3.34 - 3.47 (m, 2 H) 3.55 - 3.73 (m, 2 H) 3.94 (dd, J=1 1.28, 3.08 Hz, 2 H) 7.00 (s, 1 H) 7.94 (s, 2 H) 8.01 (s, 1 H). Example 79 (Compound 254)
3,5'-dichloro-N2'-(trans-4-(2-(methylsulfonyl)ethylamino)cyclohexyl)-N6-((tetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000235_0001
Step ! Preparation of 3,5'-dichloro-N2'-(trans-4-(2-
(methylsulfonyl)ethylamino)cyclohexyl)-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2',6-diamine:
A mixture of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6-((tetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine (Example 87) (40 mg, 0.089 mmol), potassium carbonate (30.7 mg, 0.222 mmol), DMSO (0.4 ml) and 2-(methylsulfonyl)ethyl methanesulfonate (Example 20, stepl) (26.9 mg, 0.133 mmol) was stirred at 100 °C and the reaction progress was followed by LCMS. After 4 hours, to the crude reaction mixture was added 2-(methylsulfonyl)ethyl methanesulfonate (26.9 mg, 0.133 mmol) and stirred at 100 °C for an additional 4 hours. The reaction mixture was cooled to room temperature, 0.5 ml_ of DMSO added, filtered and purified by prep. LC. After
lyophilization to TFA salt, 16.9 mg of title compound was obtained. LCMS (m/z): 556.2 (MH+), retention time = 0.61 min.; 1 H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.18 - 1.53 (m, 4 H) 1.53 - 1.72 (m, 4 H) 1.86 (dddd, J=14.83, 7.58, 3.96, 3.81 Hz, 1 H) 2.24 (d, J=10.55 Hz, 4 H) 3.11 (s, 3 H) 3.19 (d, J=6.74 Hz, 2 H) 3.25 (br. s., 1 H) 3.38 (td, J=1 1.72, 1.76 Hz, 2 H) 3.56 (s, 4 H) 3.72 (t, J=11.28 Hz, 1 H) 3.92 (dd, J=11.28, 2.78 Hz, 2 H) 6.61 (d, J=9.08 Hz, 1 H) 6.67 - 6.77 (m, 1 H) 7.50 (d, J=9.08 Hz, 1 H) 8.05 (s, 1 H).
Example 80 (Compound 258)
3,5'-dichloro-N2'-(trans-4-(2-methoxyethylamino)cyclohexyl)-N6-((tetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2',6-diamine
H
Figure imgf000235_0002
Step 1 : Preparation of 3,5'-dichloro-N2'-(trans-4-(2-methoxyethylamino)cyclohexyl)-N6- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine:
A mixture of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6-((tetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine (example 87)(40 mg, 0.089 mmol), potassium carbonate (30.7 mg, 0.222 mmol), DMSO (0.4 ml) and 1-bromo-2- methoxyethane (18.52 mg, 0.133 mmol) was stirred at 80 °C for 2 hours and the reaction progress was followed by LCMS. To the crude reaction mixture was added BOC- Anhydride (0.041 mL, 0.178 mmol) and stirred at ambient temperature for 2 hr. The BOC intermediate was purified by prep. LC and lyophilized to TFA salt, which was then mixed with 4M HCL (1 mL, 4.00 mmol) and stirred at ambient temperature for 1 hour. The solvent was concentrated off, the resulting residue dissolved in 1 ml DMSO, filtered and purified by prep. LC. After lyophilization to TFA salt, 5.3 mg of the title compound was obtained. LCMS (m/z): 508.2 (MH+), retention time = 0.63 min, Example 81 (Compound 259)
2-(trans-4-(3,5'-dichloro-6-((tetrahydro-2H-pyran-4-yl)methyl)amino-2,4'-bipyridin-2'-yl- amino)cyclohexylamino)ethanol
Figure imgf000236_0001
Step 1. Preparation of 2-(trans-4-(3,5'-dichloro-6-((tetrahydro-2H-pyran-4- yl)methyl)amino-2,4'-bipyridin-2'-yl-amino)cyclohexylamino)ethanol:
A mixture of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6-((tetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine (example 87)(40 mg, 0.089 mmol), potassium carbonate (30.7 mg, 0.222 mmol), DMSO (0.4 ml) and 2-bromoethanol (16.65 mg, 0.133 mmol) was stirred at 80 °C for 2 hours and the reaction progress was followed by LCMS. To this crude reaction mixture was added BOC-Anhydride (0.041 mL, 0.178 mmol) and stirred at ambient temperature for 2 hr. The BOC intermediate was purified by prep LC, and lyophilized to a TFA salt. Then was added 4M HCI in Dioxane (1 mL, 4.00 mmol) and stirred at ambient temperature for 1 hour. The solvent was concentrated off, the resulting residue dissolved in DMSO, purified by prep. LC. After lyophilization to TFA salt, 6.1 mg of the title compound was obtained. LCMS (m/z): 494.2 (MH+), retention time = 0.60 min.; H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.18 - 1.51 (m, 4 H) 1.50 - 1.73 (m, 4 H) 1.78 - 1.95 (m, J=14.80, 7.62, 7.47, 3.66, 3.66 Hz, 1 H) 2.23 (d, J=1 1.43 Hz, 4 H) 3.09 - 3.24 (m, 5 H) 3.38 (td, J=1 1.79, 1.61 Hz, 2 H) 3.64 - 3.77 (m, 1 H) 3.77 - 3.84 (m, 2 H) 3.92 (dd, J=1 1.28, 3.08 Hz, 2 H) 6.59 (d, J=9.08 Hz, 1 H) 6.66 (s, 1 H) 7.49 (d, J=8.79 Hz, 1 H) 8.03 (s, 1 H)
Example 82 (Compound 265)
N2'-(trans-4-aminocyclohexyl)-3-chloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2',6-diamine
Figure imgf000237_0001
Step ! Preparation of 3-chloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridin-6-amine
A mixture of 6-bromo-5-chloro-N-((tetrahydro-2H-pyran-4-yl)methyl)pyridin-2- amine (intermediate E) (630 mg, 2.062 mmol), 2-fluoropyridin-4-ylboronic acid (639 mg, 4.54 mmol), PdCI2(dppf).CH2CI2 adduct (168 mg, 0.206 mmol), DME (9 ml) and 2M sodium carbonate (3.09 ml, 6.18 mmol) was stirred at 105 °C for 2 hours, and the reaction progress was followed by LCMS. The reaction mixture was let cool to room temperature, diluted with 30ml of ethyl acetate, 10 ml of methanol, filtered and concentrated. The crude material was purified by silica gel chromatography using 40g column and eluting with 5-45% ethyl acetate in heptane. The desired fractions were concentrated to constant mass giving, 516 mg of the title compound as free base. LCMS (m/z): 332.0 (MH+), retention time = 0.88 min
Step 2. Preparation of N2'-(trans-4-aminocyclohexyl)-3-chloro-N6-((tetrahydro- 2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine: A mixture of 3-chloro-2'-fluoro-N- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6-amine (250 mg, 0.777 mmol), DMSO (2 ml), and trans-cyclohexane-1 ,4-diamine (798 mg, 6.99 mmol) was stirred at 105 °C for 20 hours and the reaction progress was followed by LCMS. The crude reaction mixture was cooled to room temperature, diluted with 250 ml of ethyl acetate, washed with saturated sodium bicarbonate (1x), water (2x), filtered and the solvent was concentrated off. The crude was dissolved in 5 ml DMSO, filtered and purified by prep. LC. After lyophilization to TFA salt, 180 mg of the title compound was obtained. LCMS (m/z): 416.2 (MH+), retention time = 0.52 min.; H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.20 - 1.41 (m, 2 H) 1.46 - 1.74 (m, 6 H) 1.85 (ddd, J=10.99, 7.33, 4.25 Hz, 1 H) 2.06 - 2.30 (m, 4 H) 3.19 (br. s., 1 H) 3.26 (d, J=7.03 Hz, 2 H) 3.33 - 3.46 (m, 2 H) 3.59 - 3.76 (m, 1 H) 3.93 (dd, J=1 1.14, 3.22 Hz, 2 H) 6.60 (d, J=8.79 Hz, 1 H) 7.23 (d, J=6.74 Hz, 1 H) 7.39 (s, 1 H) 7.49 (d, J=8.79 Hz, 1 H) 7.88 (d, J=6.74 Hz, 1 H)
Example 83 (Compound 268)
3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-(((R)-tetrahydrofuran-2- yl)methyl)aminocyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000238_0001
Step 1. Preparation of (R)-(tetrahydrofuran-2-yl)methyl methanesulfonate:
A mixture of (R)-(tetrahydrofuran-2-yl)methanol (600 mg, 5.87 mmol), DCM (35 ml), TEA (0.983 ml, 7.05 mmol) was diluted with methanesulfonyl chloride (0.467 ml, 5.99 mmol), via a dropwise addition. The reaction mixture was stirred at ambient temperature for 5 hours and the reaction progress was followed by LCMS. The crude reaction mixture was washed with saturated sodium bicarbonate (1x), water (2x), filtered and concentrated to a constant mass, giving 980 mg of the title compound, which was used without further purification. LCMS (m/z): 181.0 (MH+), retention time = 0.40 min
Step 2. Preparation of 3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4- (((R)-tetrahydrofuran-2-yl)methyl)aminocyclohexyl)-2,4'-bipyridine-2',6-diamine:
To N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6-((tetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2',6-diamine (example 87) (40 mg, 0.089 mmol) was added potassium carbonate (30.7 mg, 0.222 mmol), DMSO (0.4 ml) and (R)-(tetrahydrofuran-2- yl)methyl methanesulfonate (24.01 mg, 0.133 mmol), and the resulting reaction mixture was stirred at 100 °C for 4 hours and the reaction progress was followed by LCMS. After about 4 hours (R)-(tetrahydrofuran-2-yl)methyl methanesulfonate (24.01 mg, 0.133 mmol) was added and the resulting mixture was stirred at 100 °C for 4 hours more. The reaction mixture was cooled to room temperature, 0.5 ml_ of DMSO added, filtered and purified by prep. LC. After lyophilization to a TFA salt, 9.1 mg of the title compound was obtained. LCMS (m/z): 534.3 (MH+), retention time = 0.62 min.
Example 84 (Compound 272)
3,5'-dichloro-N2'-(trans-4-((2-methoxyethyl)(methyl)amino)cyclohexyl)-N6-((tetrahydro- 2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine:
Figure imgf000239_0001
Step 1. Preparation of (1s, 4s)-4-(3,5'-dichloro-6-((tetrahydro-2H-pyran-4- yl)methyl)amino-2,4'-bipyridin-2'-yl-amino)cyclohexanol:
To 3,5'-dichloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6- amine (intermediate G) (712 mg, 1.999 mmol) was added DMSO (4.5 ml), TEA (1.1 14 ml, 8.00 mmol) and (1 s,4s)-4-aminocyclohexanol (607 mg, 4 mmol), and the reaction mixture was stirred at 95-100 °C for 96 hours and the reaction progress was followed by LCMS. The reaction mixture was cooled, 250 ml of ethyl acetate was added, washed with saturated sodium bicarbonate (1x) water (2x) and concentrated to constant mass. The crude was purified by silica gel chromatography using 40g column eluting with 25-95 % ethyl acetate in heptane. The desired fractions were concentrated to constant mass, giving 380 mg of title compound as free base. LCMS (m/z): 451.1 (MH+), retention time = 0.65 min
Step 2. Preparation of (1s,4s)-4-(3,5'-dichloro-6-((tetrahydro-2H-pyran-4- yl)methyl)amino-2,4'-bipyridin-2'-yl-amino)cyclohexyl methanesulfonate:
To (1s,4s)-4-(3,5'-dichloro-6-((tetrahydro-2H-pyran-4-yl)methyl)amino-2,4'- bipyridin-2'-yl-amino)cyclohexanol (375 mg, 0.831 mmol) was added DCM (8 ml), and TEA (0.174 ml, 1.246 mmol) and the resulting mixture was cooled in an ice bath to 0 °C. Then with stirring was added methanesulfonyl chloride (0.071 ml, 0.914 mmol). The reaction mixture was allowed to warm to ambient temperature and stirred for 2 hours, and the reaction progress was followed by LCMS. To the crude reaction mixture was added 250 ml of ethyl acetate, washed with saturated sodium bicarbonate (1x) water (2x) and concentrated to constant mass giving, 441 mg of title compound as free base, used without further purification. LCMS (m/z): 529.3 (MH+), retention time = 0.75 min.
Step 3. Preparation of 3,5'-dichloro-N2'-(trans-4-((2- methoxyethyl)(methyl)amino)cyclohexyl)-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2',6-diamine:
To (1s,4s)-4-(3,5'-dichloro-6-((tetrahydro-2H-pyran-4-yl)methyl)amino-2,4'- bipyridin-2'-yl-amino)cyclohexyl methanesulfonate (48 mg, 0.091 mmol) was added t- Butanol (0.22 ml) and 2-methoxy-N-methylethanamine (202 mg, 2.266 mmol). The reaction mixture was stirred at 95-100 °C for 5 hours and the reaction progress was followed by LCMS. The reaction mixture was cooled to room temperature, 12 ml of ethyl acetate was added then washed with saturated sodium bicarbonate (1x) water (2x) and the solvent concentrated off. The resulting residue was dissolved in 1 ml of DMSO, filtered and purified by prep LC. After lyophilization to TFA salt, 8.61 mg of the title compound was obtained. LCMS (m/z): 522.2 (MH+), retention time = 0.63 min.; H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.18 - 1.56 (m, 4 H) 1.59 - 1.79 (m, 4 H) 1.79 - 1.95 (m, 1 H) 2.02 - 2.35 (m, 4 H) 2.87 (s, 3 H) 3.19 (d, J=6.74 Hz, 2 H) 3.24 (d, J=3.52 Hz, 1 H) 3.32 - 3.41 (m, 3 H) 3.42 (s, 3 H) 3.46 - 3.58 (m, 1 H) 3.63 - 3.78 (m, 3 H) 3.92 (dd, J=11.14, 2.93 Hz, 2 H) 6.60 (d, J=9.08 Hz, 1 H) 6.70 (s, 1 H) 7.49 (d, J=9.08 Hz, 1 H) 8.04 (s, 1 H)
Example 85 (Compound 203)
trans-N1-(5-chloro-4-(6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2-yl)pyridin-2- yl)cyclohexane-1 ,4-diamine
Figure imgf000241_0001
Step 1. Preparation of trans-N1-(5-chloro-4-(6-((tetrahydro-2H-pyran-4- yl)methyl)arninopyrazin-2-yl)pyridin-2-yl)cyclohexane-1 ,4-diamine
To 6-(5-chloro-2-fluoropyridin-4-yl)-N-((tetrahydro-2H-pyran-4-yl)methyl)pyrazin-
2-amine (example 75 step 2) (20 mg, 0.062 mmol) was added DMSO (0.6 ml) and trans- cyclohexane-1 ,4-diamine (63.7 mg, 0.558 mmol). The reaction mixture then was stirred at 100-105 °C for 18 hours and the reaction progress was followed by LCMS. The reaction mixture was let cool, diluted with 0.5 ml of DMSO, filtered and purified by prep LC. After lyophilization to TFA salt, 13.7 mg of the title compound was obtained. LCMS (m/z): 417.3 (MH+), retention time = 0.46 min.; 1 H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.22 - 1.78 (m, 8 H) 1.81 - 2.01 (m, 1 H) 2.03 - 2.28 (m, 4 H) 3.05 - 3.21 (m, 1 H) 3.28 - 3.32 (dMeOH, 2H App.) 3.39 (td, J=1 1.72, 1.76 Hz, 2 H) 3.62 - 3.79 (m, 1 H) 3.94 (dd, J=11.14, 3.22 Hz, 2 H) 6.95 (s, 1 H) 7.92 (d, J=2.93 Hz, 2 H) 8.05 (s, 1 H).
Example 86 (Compound 243)
trans-N1-(5-chloro-4-(6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2-yl)pyridin-2- yl)-N4-(2-methoxyethyl)cyclohexane-1 ,4-diamine
Figure imgf000241_0002
Step 1. Preparation of trans-N1-(5-chloro-4-(6-((tetrahydro-2H-pyran-4- yl)methyl)aminopyrazin-2-yl)pyridin-2-yl)-N4-(2-methoxyethyl)cyclohexane-1 ,4-diamine To trans-N1-(5-chloro-4-(6-((tetrahydro-2H-pyran-4-yl)methyl)aminopyrazin-2- yl)pyridin-2-yl)cyclohexane-1 ,4-diamine (Example 85) (16 mg, 0.038 mmol) was added DMSO (0.4 ml), potassium carbonate (15.91 mg, 0.115 mmol) and 1-bromo-2- methoxyethane (7.47 mg, 0.054 mmol). The reaction mixture then was stirred at 70 °C for 6 hours and the reaction progress was followed by LCMS. The reaction mixture was cooled to room temperature, 0.5 ml of DMSO was added, filtered and purified by prep LC. After lyophilization to TFA salt, 2.7 mg of the title compound was obtained. LCMS (m/z): 475.2 (MH+), retention time = 0.51 min.
Example 87 (Compound 253)
N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'- bipyridine-2',6-diamine
Figure imgf000242_0001
Step 1. Preparation of N2'-(trans-4-aminocyclohexyl)-3,5'-dichloro-N6-((tetrahydro-2H- pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine:
To 3,5'-dichloro-2'-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridin-6- amine (Intermediate G)(250 mg, 0.702 mmol) was added DMSO (3 ml) and trans- cyclohexane-1 ,4-diamine (952 mg, 6.32 mmol). The reaction mixture was stirred at 100 °C for 20 hours and the reaction progress was followed by LCMS. The reaction mixture was cooled, diluted with 250 ml ethyl acetate, washed with saturated sodium bicarbonate (1x), water (3x) and concentrated to constant mass, giving 320 mg of product as a free base, which was used without further purification. A portion of the title compound, 25 mg was purified by prep LC and lyophilized to give 17.6 mg of the title compound as a TFA salt. LCMS (m/z): 450.2 (MH+), retention time = 0.58 min.; 1 H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 1.16 - 1.76 (m, 8 H) 1.76 - 1.98 (m, 1 H) 2.04 - 2.27 (m, 4 H) 3.06 - 3.16 (m, 1 H) 3.19 (d, J=6.74 Hz, 2 H) 3.37 (t, J=11.87 Hz, 2 H) 3.62 - 3.77 (m, 1 H) 3.92 (dd, J=11.28, 3.08 Hz, 2 H) 6.61 (d, J=8.79 Hz, 1 H) 6.73 (s, 1 H) 7.50 (d, J=9.08 Hz, 1 H) 8.04 (s, 1 H).
Example 88 (Compound 178)
5'-chloro-N6-(3-fluorobenzyl)-N2'-methyl-2,4'-bipyridine-2',6-diamine
Figure imgf000243_0001
Step 1. Preparation of 5'-chloro-N6-(3-fluorobenzyl)-N2'-methyl-2,4'-bipyridine-2',6- diamine:
A mixture of 5'-chloro-2'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-6-amine
(Intermediate B ) (15 mg, 0.045 mmol) was added DMSO (0.4 ml) and methyl amine 40% in water (200 μΙ, 2.293 mmol) in a microwave tube was microwaved at 145 °C for 900 seconds and the reaction progress was followed by LCMS. Most of the amine was removed under vacuum, 0.5 ml of DMSO was added, filtered and purified by prep LC. After lyophilization 13.9 mg of the title compound was obtained as a TFA salt. LCMS (m/z): 343.0 (MH+), retention time = 0.67 min.; 1 H NMR (300 MHz, METHANOL-d4, 25°C) δ ppm 2.97 (s, 3 H) 4.62 (s, 2 H) 6.81 (d, J=8.50 Hz, 1 H) 6.91 - 7.02 (m, 3 H) 7.09 (d, J=9.96 Hz, 1 H) 7.17 (d, J=7.62 Hz, 1 H) 7.27 - 7.39 (m, 1 H) 7.69 (dd, J=8.50, 7.33 Hz, 1 H) 8.03 (s, 1 H).
Example 89 (Compound 332)
5'-chloro-5-fluoro-N6-((4-methyltetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-((1 , 1- dioxo-hexahydro-1-thiopyran-4-yl)-3-ylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000243_0002
To a solution of N2'-(trans-4-aminocyclohexyl)-5'-chloro-5-fluoro-N6-((4- methyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine (40 mg, 0.089 mmol) in DMF (0.5 ml) was added potassium carbonate (49.4 mg, 0.357 mmol), 3-chloro-1 , 1- dioxo-tetrahydro-1-thiophene (83 mg, 0.536 mmol) and sodium iodide (40.2 mg, 0.268 mmol). The reaction mixture was stirred at 100 °C for 42 hours. The cooled reaction mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed sequentially with water and brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by reverse phase HPLC and lyophilized to give 3.8 mg off-white powder of the title compound as its TFA salt. LCMS (m/z): 566.2 (MH+), retention time = 0.64 min. Example 90 (Compound 333)
5'-chloro-5-fluoro-N2'-(trans-4-((2-methyl-1 ,3-dioxolan-2-yl)methyl)aminocyclohexyl)-N6- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000244_0001
To a solution of N2'-(trans-4-aminocyclohexyl)-5'-chloro-5-fluoro-N6-((4- methyltetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine (21 mg, 0.048 mmol) in DCM (1.0 ml) was added 2-methyl-1 ,3-dioxolane-2-carbaldehyde (synthesized following the procedure reported in Org. Lett., 2009, 11, 3542-3545), sodium
triacetoxyborohydride (20.51 mg, 0.097 mmol). The reaction mixture was stirred at ambient temperature for 2 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed sequentially with water and brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by reverse phase HPLC and lyophilized to give 12 mg off-white powder of the title compound as its TFA salt. LCMS (m/z): 534.1 (MH+), retention time = 0.62 min.
Example 91 (Compound 349)
(4-((5'-chloro-2'-(trans-4-((R)-1-methoxypropan-2-ylamino)cyclohexylamino)-2,4'- bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4-yl)methanol
Figure imgf000245_0001
Step 1. Synthesis of methyl 4-cyanotetrahydro-2H-pyran-4-carboxylate
To 1-bromo-2-(2-bromoethoxy)ethane (2.57 g, 11.10 mmol) in DMSO (6 ml_, by mistake, should use DMF) at room temperature was added methylcyanoacetate (1 g, 10.09 mmol) and DBU (3.35 ml, 22.20 mmol) sequentially. The brown mixture was heated to 85 °C in a capped glass vial for 3 hours. The resulting solution was dark brown.
The reaction mixture was poured into water and extracted with EtOAc. The organic extracts were combined, washed with water, brine, dried with sodium sulfate and concentrated in vacuo to give 0.944 g of brown oil. This crude material was used in the next step without further purification.
Step 2. Synthesis of (4-(aminomethyl)tetrahydro-2H-pyran-4-yl)methanol
To the crude product from step 1 (0.944 g, 5.58 mmol) in THF (5 ml) (a dark brown solution) at 0 °C was added LAH (5.58 ml, 5.58 mmol) dropwise via a syringe.
The brown mixture was warmed to room temperature and stirred for 18 hours. The resulting mixture was yellow cloudy. LC/MS showed containing desired product. To the reaction was added sodium sulfate decahydrate solid at 0 °C. The mixture was stirred at room temperature for 20 min., then filtered and washed with DCM. The yellow filtrate was concentrated in vacuo to give 0.74 g of orange oil. This crude material was used in the next step without further purification.
Step 3. Synthesis of (4-((6-bromopyridin-2-ylamino)methyl)tetrahydro-2H-pyran-4- yl)methanol
To 2-bromo-6-fluoropyridine (0.448 g, 2.55 mmol) in NMP (4 ml) at room temperature was added TRIETHYLAMINE (0.852 ml, 6.12 mmol) and the crude product obtained in step 2 (370 mg, 2.55 mmol) sequentially. The yellow mixture was heated to 75 °C in a capped glass vial for 3 hours. LC/MS showed about 20% conversion to the product. Continued heating at 1 10 °C for 16 hrs. The reaction mixture was cooled to room temperature, poured into water and extracted with EtOAc. The organic extracts were combined, washed with water, brine, dried with sodium sulfate and concentrated in vacuo to give 0.5 g of brown oil. The crude mixture was purified by Analogix system (silica gel column 24 g, gradient: 0 min, 100%n-hexane; 2-127 min, 10% EtOAc in Hex; 7-13 min. 20% EtOAc in Hex; 13-16 min. 30% EtOAc in Hex; 16-30 min. 50% EtOAc in Hex; 30-35 min. 100% EtOAc). The pure fractions were combined and concentrated in vacuo to give 0.13 g of desired product as a white crystal. LCMS (m/z): 301/303 (MH+), retention time = 0.67 min.
Step 4. Synthesis of (4-((5'-chloro-2'-fluoro-2,4'-bipyridin-6-ylamino)methyl)tetrahydro- 2H-pyran-4-yl)methanol
Following the same procedure as in Example 1 b using (4-((6-bromopyridin-2- ylamino)methyl)tetrahydro-2H-pyran-4-yl)methanol (from step 3) and 5-chloro-2- fluoropyridin-4-ylboronic acid, the desired product was obtained. LCMS (m/z): 352 (MH+), retention time = 0.54 min.
Step 5. Synthesis of (4-((5'-chloro-2'-(trans-4-((R)-1-methoxypropan-2- ylamino)cyclohexylamino)-2,4'-bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4- yl)methanol
Following the same procedure as in Example 1 b using (4-((5'-chloro-2'-fluoro- 2,4'-bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4-yl)methanol (from step 4) and trans-N1-((R)-1-methoxypropan-2-yl)cyclohexane-1 ,4-diamine, the desired product was obtained. LCMS (m/z): 518.2 (MH+), retention time = 0.47 min.
Example 92 (Compound 348)
3,5'-dichloro-N6-((tetrahydro-2H-pyran-4-yl)methyl)-N2'-(trans-4-(1 ,1-dioxo-tetrahydro- 2H-thiopyran-4-ylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000246_0001
Compound N2'-(4-aminocyclohexyl)-3,5'-dichloro-N6-((tetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2',6-diamine (0.100 g, 0.222 mmol) (synthesized in the same manner as in Example 1 b), 2,3,5,6-tetrahydro-4H-thiopyran-4-one 1 ,1-dioxide (0.036 g, 0.244 mmol), and triethylamine (0.251 ml, 0.182 g, 1.798 mmol) were dissolved in anhydrous CH2CI2 (1.0 ml) and placed under argon. This solution was then treated with sodium triacetoxyborohydride (0.094 g, 0.444 mmol). The reaction was then stirred at room temperature for 18 hours. At this time a LC-MS was run. The reaction was about 25% complete. Additional 2,3,5,6-tetrahydro-4H-thiopyran-4-one 1 , 1-dioxide (~ 4 equivalents) and sodium triacetoxy borohydride (~8 equivalents) were added and the reaction continued for additional 27 hours. The reaction was about 60% complete as indicated by LC/MS. The reaction was quenched with sat NaHC03 (15 ml). This was extracted with EtOAc (3 x 15 ml). The combined extracts were washed with brine (1 x 15 ml), dried (Na2S04), filtered and the solvent removed in vacuo. The material was purified using the HPLC and lyophilized to give 19.7 mg off-white powder of the title compound as its TFA salt. LCMS (m/z): 582/584 (MH+), retention time = 0.58 min.
Example 93 (Compound 310)
4-((5'-chloro-2'-(trans-4-((R)-1-methoxypropan-2-yl-amino)cyclohexylamino)-2,4'- bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000247_0001
This compound was synthesized following the procedure of Example 1 b using Intermediates AB (40 mg, 0.1 15mmol) and N1-((R)-1-methoxypropan-2-yl)cyclohexane- trans-1 ,4-diamine (synthesized in step 2 of Example 67, 107mg, 0.577mmol). The product was obtained as an off white powder (30.2 mg, 35.5% yield). LCMS (m/z): 513.2 [M+H]+; retention time = 0.531 min. 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.04 (d, J=6.26 Hz, 2 H) 1.12 - 1.37 (m, 3 H) 1.84 - 2.06 (m,3 H) 2.10 - 2.25 (m, 2 H) 2.44 - 2.69 (m, 1 H) 2.91 - 3.1 1 (m, 1 H) 3.20 - 3.39 (m, 3 H) 3.43 - 3.60 (m, 1 H) 3.61 - 3.83 (m, 3 H) 3.90 - 4.08 (m, 2 H) 4.41 (d, J=8.22 Hz, 1 H) 4.67 - 4.93 (m, 1 H) 6.37 - 6.62 (m, 2 H) 6.97 (d, J=7.43 Hz, 1 H) 7.26 (s, 1 H) 7.39 - 7.58 (m, 1 H). Example 94 (Synthesis of Compound 340)
Synthesis of 4-((5'-chloro-5-fluoro-2'-(trans-4-((R)-1-methoxypropan-2-yl- amino)cyclohexylamino)-2,4'-bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4- carbonitrile
Figure imgf000248_0001
This compound was synthesized following the procedure of Example 1 b using intermediates AA( 50 mg, 0.137mmol) and N1-((R)-1-methoxypropan-2-yl)cyclohexane- trans-1 ,4-diamine (synthesized in step 2 of Example 67, 128mg, 0.685mmol). The product was obtained as an off white powder 35mg (33.6% yield). LCMS (m/z): 531.2 [M+H]+; retention time = 0.595 min.
Examples in Table 1 were prepared using methods analogous to those described above. The method column in Table 1 indicates the synthetic procedure, from a specific example, used to synthesize a given compound. Thus for example, Compound 7 is synthesized by the procedure outlined in Example 7, while compound 25 is synthesized by the procedure outlined in Example 1 a, and the like.
Compound/Ex. 357: 4-((5'-chloro-5-fluoro-2'-(frans-4-hydroxycyclohexylamino)-2,4'- bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000248_0002
M+1 (LC/MS): 460.1 ; Retention Time (min. LC/MS): 0.62.
H NMR (400 MHz, METHANOL-cH) ppm 1.29 - 1.42 (m, 3 H) 1.59 - 1.71 (m, 2 H) 1.75 - 1.80 (m, 1 H) 1.80 - 1.83 (m, 1 H) 1.88 - 1.96 (m, 2 H) 1.96 - 2.02 (m, 2 H) 2.02 - 2.13 (m, 1 H) 3.46 - 3.60 (m, 4 H) 3.72 (s, 2 H) 3.86 (m, J=12.13, 2.35 Hz, 2 H) 6.95 (dd, J=8.02, 2.93 Hz, 1 H) 7.10 (s, 1 H) 7.32 (dd, J=10.96, 8.22 Hz, 1 H) 7.92 (s, 1 H).
Compound/Ex. 358: 4-((5'-chloro-2'-(frans-4-((R)-1-methoxypropan-2- ylamino)cyclohexylamino)-2,4'-bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4- carbonitrile
Figure imgf000249_0001
Compound/Ex. 359: 4-((5'-chloro-2'-(frans-4-hydroxycyclohexylamino)-2,4'-bipyridin-6- ylamino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000249_0002
M+1 (LC/MS): 442.1 ; Retention Time (min. LC/MS): 0.55.
1 H NMR (400 MHz, METHANOL-d4) ppm 1.29 - 1.42 (m, 4 H) 1.58 - 1.70 (m, 2 H) 1.75 - 1.84 (m, 2 H) 1.87 - 2.04 (m, 4 H) 3.45 - 3.60 (m, 4 H) 3.66 (s, 2 H) 3.86 (m, J=12.13, 2.74 Hz, 2 H) 6.66 (d, J=8.22 Hz, 1 H) 6.88 (d, J=7.43 Hz, 1 H) 7.07 (s, 1 H) 7.46 - 7.53 (m, 1 H) 7.92 (s, 1 H).
Compound/Ex. 360: 4-((5'-chloro-2'-(frans-4-(ethylamino)cyclohexylamino)-2,4'- bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000250_0001
M+1 (LC/MS): 469.2; Retention Time (min. LC/MS): 0.55.
H NMR (400 MHz, METHANOL-d4) ppm 1.32 (t, J=7.24 Hz, 3 H) 1.49 (br. s., 4 H) 1.66 - 1.82 (m, 2 H) 1.84 - 1.99 (m, 2 H) 2.22 (d, J=12.52 Hz, 4 H) 3.11 (t, J=7.24 Hz, 3 3.56 - 3.72 (m, 3 H) 3.76 (s, 2 H) 3.87 -4.06 (m, 2 H) 6.81 (d, J=8.61 Hz, 1 H) 6.96 (d, J=6.65 Hz, 1 H) 7.06 (s, 1 H) 7.54 - 7.69 (m, 1 H) 8.06 (s, 1 H).
Compound/Ex. 361 : 4-((5'-chloro-2'-(frans-4-(dimethylamino)cyclohexylamino)-2,4'- bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000250_0002
M+1 (LC/MS): 469.2; Retention Time (min. LC/MS): 0.52
H NMR (400 MHz, METHANOL-^) ppm 1.39 - 1.58 (m, 2 H) 1.64 - 1.83 (m, 4 H) 1.90 (dd, J=13.50, 1.76 Hz, 2 H) 2.10 - 2.35 (m, 4 H) 2.87 (s, 6 H) 3.57 - 3.72 (m, 3 H) 3.76 (s, 2 H) 3.96 (ddd,J=9.98, 2.35, 2.15 Hz, 2 H) 6.82 (d, J=7.83 Hz, 1 H) 6.97 (d, J=6.65 Hz, 1 H) 7.06 (s, 1 H) 7.55 - 7.77 (m, 1 H) 8.07 (s, 1 H). Compound/Ex. 362: 4-((5'-chloro-2'-(frans-4-(2-
(trifluoromethoxy)ethylamino)cyclohexylamino)-2,4'-bipyridin-6- ylamino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000251_0001
M+1 (LC/MS): 553.3; Retention Time (min. LC/MS): 0.58.
Compound/Ex. 363: 4-((5'-chloro-2'-(frans-4-(tetrahydro-2H-pyran-4- ylamino)cyclohexylamino)-2,4'-bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4- carbonitrile
Figure imgf000251_0002
M+1 (LC/MS): 525.1 ; Retention Time (min. LC/MS): 0.54.
H NMR (400 MHz, METHANOL-cH) ppm 1.38 - 1.82 (m, 8 H) 1.85 - 1.95 (m, 2 H) 1.96 2.06 (m, 2 H)2.15 - 2.26 (m, 4 H) 3.40 - 3.56 (m, 3 H) 3.58 - 3.73 (m, 3 H) 3.75 (s, 2 H) 3.90 - 4.10 (m, 4 H) 6.71 - 6.80 (m, 1 H) 6.94 (s, 2 H) 7.54 - 7.65 (m, 1 H) 8.04 (s, 1 H).
Compound/Ex. 364: 5'-chloro-N6-((4-fluorotetrahydro-2H-pyran-4-yl)methyl)-N2'-( frans-4-(2-methoxyethylamino)cyclohexyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000251_0003
M+1 (LC/MS): 492.2; Retention Time (min. LC/MS): 0.34.
H NMR (400 MHz, METHANOL-cH) ppm 1.32 - 1.48 (m, 2 H) 1.49 - 1.65 (m, 2 H) 1.72 - 1.88 (m, 4 H) 2.16 - 2.26 (m, 4 H) 3.20 - 3.27 (m, 2 H) 3.42 (s, 2 H) 3.60 - 3.76 (m, 6 H) 3.77 - 3.86 (m, 2 H) 6.78 (s, 1 H) 6.91 (d, J=7.04 Hz, 1 H) 6.96 (d, J=8.61 Hz, 1 H) 7.76 (t, J=8.02 Hz, 1 H) 8.06 (s, 1 H). Compound/Ex. 365:
Figure imgf000252_0001
Compound/Ex. 366: 4-((5'-chloro-2'-(frans-4-(diethylamino)cyclohexylamino)-2,4'- bipyridin-6-ylamino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000252_0002
M+1 (LC/MS): 497.2; Retention Time (min. LC/MS): 0.58.
Compound/Ex. 367: 2-((5'-chloro-5-fluoro-2'-(frans-4-((R)-1-methoxypropan-2- ylamino)cyclohexylamino)-2,4'-bipyridin-6-ylarriino)methyl)propane-1 ,3-diol
Figure imgf000252_0003
M+1 (LC/MS): 496.2; Retention Time (min. LC/MS): 0.49. Example 368
Figure imgf000253_0001
Step 1 : Preparation of (2,5-difluoropyridin-4-yl)boronic acid
To the solution of diisopropylamine (5.25 ml, 36.8 mmol) in THF (44 ml) in a 3- neck round bottom flask at -40°C, 2.5 molar of n-butyl lithium in hexane (11.97 ml, 29.9 mmol) was added slowly. This solution was stirred at -20°C for 0.5 hour. To the reaction solution at -78°V, 2,5-difluoropyridine (2.1 ml, 23.03 mmol) in THF (4 ml) was added dropwise and the mixture was stirred at -78°C for 3 hours under argon. To the reaction mixture at -78°c, triisopropyl borate (11.76 ml, 23.03 mmol) in THF (12 ml) was added slowly and then stirred at room temperature for an hour. The mixture was quenched slowly with 4% sodium hydroxide solution (69.1 ml, 69.1 mmol) and stirred at room temperature for 45 min. The separated aqueous layer was acidified with 6N hydrochloric acid to pH = 3 and then was extracted with ethyl acetate. The separated organic layers were combined and concentrated in vacuo to give white solid. The solid product was triturated with diethyl ether and then dried under vacuo to provide white solid as (2,5- difluoropyridin-4-yl)boronic acid (3.47 g).
LCMS (m/z): 160.0 [M+H]+; Rt = 0.29 min.
Figure imgf000253_0002
Step 2A: 4-(((2',5'-difluoro-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile
To the mixture of 4-(((6-bromopyridin-2-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile (500 mg, 1.69 mmol) and (2,5-difluoropyridin-4-yl)boronic acid (295 mg, 1.86 mmol) in DME (9 ml) were added PdCl2(dppf).CH2Cl2 adduct (221 mg, 0.27 mmol) and 2 molar aqueous sodium carbonate solution (3 ml, 6 mmol). The reaction mixture was stirred at 92°C for 22 hour under argon. The mixture was diluted with ethyl acetate and stirred for additional 30 min. The separated organic layer was washed with saturated aqueous sodium bicarbonate solution, water and brine. The organic phase was dried over sodium sulfate, filtered off and concentrated in vacuo. The residue was purified by column chromatography [Si02, 24 g, EtOAc/heptane = 0/100 to 75/25] providing 4- (((2',5'-difluoro-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (204 mg).
LCMS (m/z): 331.1 [M+H]+; Rt = 0.80 min.
Figure imgf000254_0001
Step 2B: 4-(((2',5'-difluoro-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile
To the mixture of 4-(((6-bromopyridin-2-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile (3.0 g, 10.13 mmol) and (2,5-difluoropyridin-4-yl)boronic acid (3.54 g, 22.29 mmol) in THF (60 ml) were added PdCI(X-PHOS). (239 mg, 0.304 mmol) and 0.5 molar aqueous potassium phosphate tribasic solution (50.6 ml, 25.3 mmol). The reaction mixture was stirred at 60°C for 1 hour under argon. The mixture was diluted with ethyl acetate and stirred for additional 45 min. The separated organic layer was washed with saturated aqueous sodium bicarbonate solution, water and brine. The organic phase was dried over sodium sulfate, filtered off and concentrated in vacuo. The residue was purified by column chromatography [Si02, 220 g, EtOAc/heptane = 10/90 to 45/55] providing 4-(((2',5'-difluoro-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile (2.6 g).
LCMS (m/z): 331.0 [M+H]+; Rt = 0.79 min.
Figure imgf000254_0002
Step 3: Preparation of 4-(((2'-((trans-4-aminocyclohexyl)amino)-5'-fluoro-[2,4'-bipyridin]- 6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile To the mixture of 4-(((2',5'-difluoro-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro- 2H-pyran-4-carbonitrile (1 g, 3.03 mmol) and potassium carbonate (0.42 g, 3.03 mmol) in DMA (15 ml) was added trans-1 ,4-diaminocyclohexane (2.77 g, 24.22 mmol). The brown reaction solution was stirred at 1 10°C for 5 days. The reaction solution was diluted with ethyl acetate and sodium bicarbonate solution. The separated organic layer was washed with saturated aqueous sodium bicarbonate solution, water and brine. The organic phase was dried over sodium sulfate, filtered off and concentrated in vacuo. The residue was purified by column chromatography [Si02, 4 g, DCM/MeOH/NH4OH = 90/10/0% to 90/10/1.5%] providing 4-(((2'-((trans-4-aminocyclohexyl)amino)-5'-fluoro-[2,4'-bipyridin]- 6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile as solid (430 mg).
LCMS (m/z): 425.2 [M+H]+; Rt = 0.51 min.
Figure imgf000255_0001
Step 4A: Preparation of 4-(((5'-fluoro-2'-((trans-4-((1-methoxypropan-2- yl)amino)cyclohexyl)amino)-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile
To the solution of 4-(((2'-((trans-4-aminocyclohexyl)amino)-5'-fluoro-[2,4'- bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (129 mg, 0.304 mmol), methoxyacetone (0.031 ml, 0.334 mmol) and acetic acid (0.052 ml, 0.912 mmol) in DCE (2 ml) was added sodium triacetoxhydroborate (90 mg, 0.425 mmol). The reaction mixture was stirred at room temperature for 20 hours. The reaction solution was diluted with ethyl acetate and sodium bicarbonate solution. The separated organic layer was washed with saturated aqueous sodium bicarbonate solution, water and brine. The organic phase was dried over sodium sulfate, filtered off and concentrated in vacuo. The residue was purified by column chromatography [Si02, 4 g, DCM/MeOH/NH4OH = 90/10/0% to 90/10/1.5%] providing 4-(((5'-fluoro-2'-((trans-4-((1-methoxypropan-2- yl)amino)cyclohexyl)amino)-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile as solid (94 mg).
LCMS (m/z): 497.2 [M+H]+; Rt = 0.55 min.
Figure imgf000256_0001
Step 4B: Preparation of 4-(((5'-fluoro-2'-((trans-4-(((R)-1-methoxypropan-2- yl)amino)cyclohexyl)amino)-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile and 4-(((5'-fluoro-2'-((trans-4-(((S)-1-methoxypropan-2- yl)amino)cyclohexyl)amino)-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile
The residue 4-(((5'-fluoro-2'-((trans-4-((1-methoxypropan-2-yl)amino) cyclohexyl) amino)-[2,4'-bipyridin]-6-yl)amino)rnethyl)tetrahydro-2H-pyran-4-carbonitrile was purified by chiral column chromatography [the chiral chromatography condition was listed as below] to provide 4-(((5'-fluoro-2'-((trans-4-(((R)-1-methoxypropan-2- yl)amino)cyclohexyl)amino)-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile (37 mg).
LCMS (m/z): 497.1 [M+H]+; Rt = 0.56 min.
and 4-(((5'-fluoro-2'-((trans-4-(((S)-1-methoxypropan-2-yl)amino)cyclohexyl)amino)-[2,4'- bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (37 mg).
LCMS (m/z): 497.1 [M+H]+; Rt = 0.55 min. Chiral separation of 92 mg, 19 ma/mL in EtOH (LJX283)
Analytical separation:
Column: CHIRALPAK AD-H (5 um) 100 x 4.6 mm (Daicel Chemical Industries, LTD.). Solvent: C02 : ethyl alcohol+0.1 %DEA = 80 : 20
Flow rate: 5.0 mL/min; detection: UV = 220 nm.
Fraction 1 : Retention time: 2.72 min.
Fraction 2: Retention time: 3.36 min.
Preparative separation:
Column: CHIRALPAK AD-prep (10 um) 1 x 25 cm.
Solvent: C02 : ethyl alcohol+0.1 %DEA = 80 : 20
Flow rate: 15 mL/min injection: 92 mg / 5 mL detection: UV = 220 nm.
Fraction 1 : white powder. Yield: 37 mg; ee = 99% (UV, 220 nm); Fraction 2: white powder. Yield: 37 mg; ee = 99% (UV, 220 nm);
Figure imgf000257_0001
Step 5: Preparation of 4-(((5'-fluoro-2'-((trans-4-(((R)-1-methoxypropan-2- yl)amino)cyclohexyl)amino)-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile
To the solution of 4-(((2',5'-difluoro-[2,4'-bipyridin]-6-yl)amino)methyl)tetrahydro- 2H-pyran-4-carbonitrile (143 mg, 0.433 mmol) and triethylamine (0.151 ml, 1.082 mmol) in DMSO (1.8 ml) was added trans-N1-((R)-1-methoxypropan-2-yl)cyclohexane-1 ,4- diamine (386 mg, 1.558 mmol). The brown reaction solution was stirred at 1 10°C for 7 days. The reaction solution was diluted with ethyl acetate and sodium bicarbonate solution. The separated organic layer was washed with saturated aqueous sodium bicarbonate solution, water and brine. The organic phase was dried over sodium sulfate, filtered off and concentrated in vacuo. The residue was purified by using reversed phase liquid chromatography and then lyophilized to dryness as 4-(((5'-fluoro-2'-((trans-4-(((R)- 1-methoxypropan-2-yl)amino)cyclohexyl)amino)-[2,4'-bipyridin]-6- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (41 mg).
LCMS (m/z): 497.3 [M+H]+; Rt = 0.52 min. H NMR (CD3OD) ppm 7.86 (d, J = 3.5 Hz, 1 H), 7.49 (s, 1 H), 7.06 - 7.18 (m, 2H), 6.63 (d, J = 8.2 Hz, 1 H), 3.98 (dd, J = 1 1.9, 2.9 Hz, 2H), 3.81 (s, 2H), 3.66 (td, J = 11.9, 2.0 Hz, 2H), 3.53 - 3.60 (m, 1 H), 3.35 (s, 4H), 3.21 - 3.28 (m, 1 H), 3.02 - 3.1 1 (m, 1 H), 2.58 - 2.68 (m, 1 H), 1.96 - 2.17 (m, 4H), 1.87 - 1.95 (m, 2H), 1.75 - 1.85 (m, 2H), 1.17 - 1.37 (m, 4H), 1.04 (d, J = 6.7 Hz, 3H)
Example 369
1-((5'-chloro-2'-(trans-4-((R)-1-methoxypropan-2-ylamino)cyclohexylamino)-2,4'-bipyridin- 6-ylamino)methyl)cyclopropanecarbonitrile
Figure imgf000258_0001
Stepl : Preparation of methyl 1-cyanocyclopropanecarboxylate
Figure imgf000258_0002
In a 100 ml flask at 0 °C, 1-cyanocyclopropanecarboxylic acid (3 g, 27.0 mmol) was dissolved in toluene (45 ml) and MeOH (5 ml). Reaction was treated drop wise with TMS-Diazomethane (27.0 ml, 27.0 mmol) and reaction stirred at 0 °C for 2 hr. Reaction was concentrated to 3.21 g of yellow oil and utilized without further purification. (3.21 g, 25.7 mmol) GC/MS Rt = 5.0 min, m/z = 125 Step2: Preparation of 1-(hydroxymethyl)cyclopropanecarbonitrile
Figure imgf000258_0003
In a 100 ml flask at 0 °C, methyl 1-cyanocyclopropanecarboxylate (1 g, 7.99 mmol) was dissolved in 1 ,2-Dimethoxyethane (20 ml) and MeOH (2 ml). Reaction was treated portion wise with NaBH4 (0.605 g, 15.98 mmol) and reaction stirred at 0 °C for 2 hr and then 20 hrs overnight. Reaction was quenched with 20 mis of sat NH4CI.
Reaction was diluted with Et20 and stirred vigorously for 2 hrs. Organics were isolated, dried (MgS04), filtered and concentrated to provide 755 mg of the title compound as a yellow oil which was used without further purification. (755 mg, 7.77 mmol, 97 % yield) GC/MS Rt = 4.8 min, m/z = 98
Ste 3: Preparation of (l-cyanocyclopropyl)methyl methanesulfonate
Figure imgf000258_0004
In a 250 ml RBR at 0 °C, 1-(hydroxymethyl)cyclopropanecarbonitrile (400 mg, 4.12 mmol) was dissolved in methylene chloride (15 ml) and triethylamine (1.148 ml, 8.24 mmol). Reaction was treated drop wise with methanesulfonyl chloride (0.353 ml, 4.53 mmol) and reaction stirred at 0 °C for 2 hr. Reaction was quenched with 20 mis of saturated aqueous Na2C03. Reaction was diluted with Et20 and stirred vigorously for 30 minutes. Organics were isolated, dried (MgS04), filtered and concentrated to 622 mg of the title compound as a yellow oil which was used without further purification. (622 mg, 3.55 mmol, 86 % yield)
Step 4: Preparation of 1-((6-bromopyridin-2-ylamino)methyl)cyclopropanecarbonitrile
Figure imgf000259_0001
To a mixture of 6-bromopyridin-2-amine (1303 mg, 7.53 mmol) and K2C03 (260 mg, 1.884 mmol) in DMF (20 ml_) was added (1-cyanocyclopropyl) methyl
methanesulfonate (660 mg, 3.77 mmol) followed by NaH (75 mg, 1.884 mmol). The mixture was stirred in a sealed tube at 40 °C for 18 hr. The reaction mixture was diluted with EtOAc, washed with water, saturated aqueous sodium bicarbonate solution and brine. The organic layer was dried over sodium sulphate, filtered and concentrated in vacuo. The crude solid was purified by column chromatography [Si02, EtOAc/hexane = 0/100 to 50/50]. Fractions were combined and concentrated in vacuo to provide the title compound (300 mg, 1.190 mmol, 31.6 % yield) LC/MS Rt = 0.83 min, m/z (H+)=
251.9/253.9
Step 5: Preparation of 1-((5'-chloro-2'-fluoro-2,4'-bipyridin-6-ylamino)methyl)
c clopropane carbonitrile
Figure imgf000259_0002
1-((6-bromopyridin-2-ylamino)methyl)cyclopropanecarbonitrile (56 mg, 0.222 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (58.4 mg, 0.333 mmol), PdCI2(dppf).CH2Cl2 adduct (18.14 mg, 0.022 mmol) and Na2C03 (0.555 ml, 1.1 11 mmol) were combined in 1 ,2-Dimethoxyethane (1 ml). Reaction was capped and heated in the microwave at 125 °C for 10 min. Reaction was diluted with EtOAc and filtered through a pad of basic alumina with EtOAc elution. Filtrate was concentrated to 33 mg of yellow oil which was purified on ISCO (Si02) (4g default 0-100% EA/Heptanes) to provide 22 mg of the title compound as a yellow solid. (22 mg, 0.073 mmol, 32.7 % yield) LC/MS Rt = 0.80 min, m/z (H+)= 303.0
Step 6: Preparation of 1-((5'-chloro-2'-(trans-4-((R)-1-methoxypropan-2- ylamino)cyclohexylamino)-2,4'-bipyridin-6-ylamino)methyl)cyclopropanecarbonitrile
Figure imgf000260_0001
To a solution of 1-((5'-chloro-2'-fluoro-2,4'-bipyridin-6-ylamino)methyl)
cyclopropanecarbonitrile (200 mg, 0.661 mmol) in DMSO (5 ml) was added trans-N1- ((R)-1-methoxypropan-2-yl)cyclohexane-1 ,4-diamine (308 mg, 1.652 mmol). Reaction was stirred at 120 °C for 12 hr. Reaction was purified by reverse phase HPLC to provide 58.1 mg of the title compound as a white solid. (58.1 mg, 0.124 mmol, 18.75 % yield) LC/MS Rt = 0.53 min, m/z (H+)= z469.1 , 1 H N MR (400 MHz, <CD3OD>) ppm 1.07 (d, J=6.26 Hz, 3 H) 1.11 - 1.18 (m, 3 H) 1.18 - 1.25 (m, 3 H) 1.26 - 1.40 (m, 4 H) 1.96 - 2.19 (m, 4 H) 2.66 (br. s., 1 H) 3.10 (br. s., 1 H) 3.37 (s, 7 H) 3.56 - 3.72 (m, 3 H) 6.60 (d, J=8.22 Hz, 1 H) 6.72 (s, 1 H) 6.86 (d, J=7.04 Hz, 1 H) 7.51 (dd, J=8.22, 7.43 Hz, 1 H) 7.97 (s, 1 H)
Example 370
5'-chloro-5-fluoro-N2'-((trans)-4-((S)-1-(methylsulfonyl)propan-2- ylamino)cyclohexyl)-N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine and 5'-chloro-5-fluoro-N2'-((trans)-4-((R)-1-(methylsulfonyl)propan-2-ylamino)cyclohexyl)- N6-((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine
Figure imgf000261_0001
COMPOUND 269 (30 mg, 0.069 mmol), 1-(methylsulfonyl)propan-2-one (11.30 mg, 0.083 mmol) and Sodium triacetoxyborohydride (44.0 mg, 0.207 mmol) were combined in methylene chloride (1 ml). Reaction was heated at 50 °C for 6 hr. LC/MS indicated reaction complete. Reaction was concentrated and purified by reverse phase HPLC and purified fractions were combined and lyophilized to give 17 mg of a light tan solid. (17 mg, 0.031 mmol, 44.4 % yield) LC/MS Rt = 0.59 min, m/z (H+)= 554.1
The mixture of enantiomers of (20 mg, 0.036 mmol) was subjected to chiral phase separation utilizing a 20x250 mm CHIRALPAK IA column and an 80%/20% Heptanes/Ethanol isocratic gradient at 12ml/min flow rate. Loading was 20 mg in 2.5 mis of Ethanol per injection. This provided the title compounds 5'-chloro-5-fluoro-N2'-((trans)-
4- ((R)-1-(methylsulfonyl)propan-2-ylamino)cyclohexyl)-N6-((tetrahydro-2H-pyran-4- yl)methyl)-2,4'-bipyridine-2',6-diamine (7.3 mg, 0.013 mmol, 36.5 % yield) and 5'-chloro-
5- fluoro-N2'-((trans)-4-((S)-1-(methylsulfonyl)propan-2-ylamino)cyclohexyl)-N6- ((tetrahydro-2H-pyran-4-yl)methyl)-2,4'-bipyridine-2',6-diamine (7.8 mg, 0.014 mmol, 39.0 % yield). LC/MS Rt = 0.59 min, m/z (H+)= 554.1
Example 371
4-((5'-chloro-2'-((trans)-4-(2,2-dimethylmorpholino)cyclohexylamino)-2,4'-bipyridin-6- ylamino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000261_0002
Stepl : Preparation of tert-butyl cis/trans-4-(2,2-dimethylmorpholino)
cyclohexylcarbamate
Figure imgf000262_0001
To a solution of tert-butyl 4-oxocyclohexylcarbamate (350 mg, 1.641 mmol) in methylene chloride (8 ml) was added 2,2-dimethylmorpholine (189 mg, 1.641 mmol) followed by sodium triacetoxyborohydride (1.739 g, 8.21 mmol). Reaction was stirred at 25 °C for 6 hr. LC/MS indicated reaction complete. Reaction was diluted with EtOAc and washed with water. Organics were isolated, dried (MgS04), filtered and
concentrated to 489 mg of yellow oil which was purified by ISCO (Si02) (12g default 0- 100% EA/Heptanes) to provide 419 mg of the title compound as a yellow oil. LC/MS Rt = 0.60 min, m/z (H+)= 313.1
Step2: Preparation of cis- and frans-4-(2,2-dimethylmorpholino)cyclohexanamine
Figure imgf000262_0002
U-32185-EXP045 U-32185-EXP048
To a solution of tert-butyl cis/trans-4-(2,2- dimethylmorpholino)cyclohexylcarbamate (419 mg, 1.341 mmol) in methylene chloride (10 ml) was added trifluoroacetic acid (0.103 ml, 1.341 mmol). Reaction was stirred at 25 °C for 2 hr. LC/MS indicated reaction complete. Reaction was concentrated to provide 400 mg of the title compounds as a white solid which was used without further purification. (400 mg, 1.884 mmol) LC/MS Rt = 0.19 min, m/z (H+)= 213.1 Step3: Preparation of 4-((5'-chloro-2'-((trans)-4-(2,2- dimethylmorpholino)cyclohexylamino)-2,4'-bipyridin-6-ylamino)methyl)tetrahydro-2H- pyran-4-carbonitrile
Figure imgf000263_0001
To a solution of 4-((5'-chloro-2'-fluoro-2,4'-bipyridin-6-ylamino)methyl)tetrahydro- 2H-pyran-4-carbonitrile (20 mg, 0.058 mmol) was added DMSO (300 μΙ), triethylamine (40.2 μΙ, 0.288 mmol), and cis- and frans-4-(2,2-dimethylmorpholino)cyclohexanamine (43.0 mg, 0.173 mmol). The reaction was stirred at 125 °C for 12 hr. LC/MS indicated product present. Reaction was purified directly on reverse phase HPLC to provide 7.3 mg of the title compound. (7.3 mg, 0.014 mmol) LC/MS Rt = 0.62 min, m/z (H+)= 539.2 Example 372 (Cmpd No. 564 in Table 1)
Synthesis of 4-(((2-(5-chloro-2-((trans-4-((2- methoxyethyl)amino)cyclohexyl)amino)pyridin-4-yl)pyrimidin-4- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000263_0002
Step 1. Synthesis of tert-butyl (trans-4-((2-methoxyethyl)amino)cyclohexyl)carbamate A mixture of p-toluenesulfonic acid 2-methoxyethyl ester (2.7 g, 1 1.72 mmol), DIEA (6.14 mL, 35.2 mmol) and 1-N-Boc-trans-1 ,4-cyclohexyldiamine (5.03 g, 23.45 mmol) in DMSO (50 mL) was heated in 100°C oil bath for 29 hours. The formation of desired product was confirmed by LC/MS. The reaction was cooled to room temperature (orange solution with some solids), diluted in 300 mL water, 20 mL of 2 M Na2C03 and extracted with EtOAc (2 x 150 mL). Some solid NaCI was added to the aqueous and extracted with EtOAc (1 x 150 mL). The combined organic extract was washed with brine with 10 mL of 2M Na2C03 solution added, dried over Na2S04, and concentrated to yield a dark orange semi-solid (4.1 g). This was diluted in 30 mL DCM, filtered off solids (impurity), rinsed with 20 mL DCM, then concentrated to a dark orange oil.
The resulting crude material was purified by ISCO system [220 g silica gel column.
Eluted with 100% DCM for 2.5 min, then 50% DCM to 100% solution A
(90%DCM/10%MeOH/0.5% NH4OH) over 30 min, held for 10 min]. The desired fractions (identified by LC/MS and TLC (90%MeCI2/10%MeOH/0.5% NH4OH with Iodine stain) were combined and concentrated in vacuo to give the title compound as a dark orange oil that solidifies to a yellow solid upon standing (2.44 g, 8.87 mmol, 76 % yield). LCMS (m/z): 273.1 (M+H), retention time = 0.53 min.
Step 2. Synthesis of trans-N1-(2-methoxyethyl)cyclohexane-1 ,4-diamine
To a solution of the carbamate obtained in step 1 (2.44 g, 8.96 mmol) in 9 mL DCM was added TFA (3 mL, 38.9 mmol) and the reaction mixture was stirred at room temperature with oil bubbler for off-gasing for about 18 hours until LC/MS showed complete
conversion to desired product. The reaction mixture was concentrated to give an orange oil (6.4 g). This TFA salt was diluted in MeOH (50 mL) and to it was added in portions PL-HCOs MP-resin (9.0 g, 1.87 mmol/g, 100A 150-200 μΓΠ, Varian Cat# PL3540-4603). The mixture was stirred until no off-gas remains. Reaction is pH 1. Additional resin was added. Reaction became basic when a total of 37 g of resin was added and another 50 mL MeOH was added to help stirring. Reaction was stirred for 15 min after additon until no off-gas was observed. Resin was filtered off through glass-fritted funnel and rinsed several times with MeOH. The filtrate was concentrated in vacuo to dryness to yield desired product as free base as a thick brown oil (1.79 g, 8.94 mmol, 100 % yield).
LCMS (m/z): 173.1 (M+H), retention time = 0.13 min; 1 H NMR (400 MHz,
CHLOROFORM-d) . ppm 1.18 (t, J=9.00 Hz, 4 H) 1.91 (d, J=16.43 Hz, 4 H) 2.08 (br.s., 6 H) 2.44 (d, J=3.52 Hz, 1 H) 2.71 (br. s., 1 H) 2.81 (t, J=5.09 Hz, 2 H) 3.35 (s, 3 H) 3.48 (s, 1 H) 3.50 (t, J=5.09 Hz, 2 H).
Step 3. Synthesis of dihydro-2H-pyran-4,4(3H)-dicarbonitrile
A solution of malononitrile (16 g, 242 mmol) in DMF (175 mL) was cooled to 0°C (ice bath) in a 500 mL 3-neck round-bottom flask with addition funnel, thermometer, stir bar, and argon sweep. To this was added bis-(2-bromoethyl)ether (31.6 mL, 242 mmol) all at once. Then DBU (77 mL, 509 mmol) was slowly added dropwise over 30 minutes. The reaction was warmed by removing the ice bath, then heated at 60°C for 2 hours and GC/MS showed only product (retention time =5.83 min). The reaction was cooled to room temperature, poured into water (750 mL), added 250 mL saturated brine, and extracted with EtOAc (3 x 750 ml_). The organic extracts were combined, washed with 1 : 1 water/saturated brine (2 x 1 L), dried with sodium sulfate, and concentrated at 30°C to a tan solid (26.7 g). Further dried under high vacuum to give the desired product as tan solids (24.1 g, 177 mmol, 73.1 % yield), GC/MS shows 93% product. 1 H NMR shows product and few impuritites. 1 H NMR (400 MHz, CHLOROFORM-d) . ppm 3.85 - 3.90 (m, 4H), 2.23 - 2.27 (m, 4H).
Step 4. Synthesis of 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile
To a solution of the bis-nitrile obtained from step 3 (8.5 g, 62.4 mmol) in EtOH (1 10 ml_) at 0 °C (ice bath) in a 3-neck 500 ml_ round-bottom flask under argon was added NaBH4 (5.25 g, 125 mmol) in one portion. The cloudy yellow mixture was allowed to warm to room temperature (slowly exothermed to 29°C) and stirred under argon for 5 hours. The slightly cloudy mixture was concentrated to remove EtOH. The residue was suspended in EtOAc (1 15 ml_) and water (-18 ml_) was added. The mixture was stirred for 30 minutes. The organic layer was separated, washed with brine (20 ml_), dried over sodium sulfate and concentrated in vacuo at room temperature and under high vacuum for 15 minutes to give the title compound as cloudy brown oil (8.9 g, 31.7 mmol, 50.8 % yield). Assume 50% purity and take on as is without further purification. Step 5. Synthesis of 4-(((2-chloropyrimidin-4-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile
To 2,4-dichloropyrimidine (0.4 g, 2.68 mmol) in acetonitrile (6 ml) at room temperature was added the amine from step 4 (0.414 g, 2.95 mmol) and TEA (0.412 ml, 2.95 mmol) sequentially. The light brown mixture was heated at 65 °C for 18 hours. LC/MS showed nearly no starting materials and it contained desired product (LCMS (m/z): 253.1 (M+H), retention time = 0.46 min). The reaction mixture was poured into water and extracted with DCM (3x20ml_). The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated in vacuo to give 0.68 g of brown oil.
The crude mixture was purified by Analogix system (silica gel column 40 g, elution - isocratic: 0-4 min, 100%n-heptane; 4-10 min, 10% EtOAc in Heptane; 10-16 min. 20% EtOAc in Heptane; 16-24 min. 30% EtOAc in Heptane, 24-40 min. 1 % TEA in methanol (by mistake). The pure fractions were combined and concentrated in vacuo to give 0.39 g of product as brown oil. LC/MS showed the material was not very clean, but contains desired product. This was used in the next step. Step 6. Synthesis of 4-(((2-(5-chloro-2-fluoropyridin-4-yl)pyrimidin-4- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
A mixture of the chloropyrimidine obtained in step 5 (0.39 g, 1.543 mmol), 5-chloro-2- fluoropyridin-4-ylboronic acid (0.271 g, 1.543 mmol), PdCl2(dppf).CH2Cl2 adduct (0.126 g, 0.154 mmol) and sodium carbonate (2M aqueous, 1.543 ml, 3.09 mmol) in DME (4 ml) was sealed and heated in microwave to 100 °C for 15 minutes. LC/MS showed formation of desired product (LCMS (m/z): 347.9(M+H), retention time = 0.74 min) and other impurities along with -50% of the chloropyrimidine. More boronic acid (0.27 g) and PdCI2(dppf) (0.1 g) was added and the mixture was heated in microwave for 15 minutes at 1 10 °C. The resulting mixture was poured into water and extracted with DCM
(3x20ml_). The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated in vacuo to give 0.85 g of black residue. The crude mixture was purified by Analogix system (silica gel column 40 g, elution - isocratic: 0-6 min, 100%n- DCM; 6-20 min, 0-5% MeOH in DCM; 10-16 min. 20% EtOAc in Heptane; 16-24 min. 90% EtOAc in Heptane). The pure fractions were combined and concentrated in vacuo to give 90 mg of brown oil (16% yield) which was used in the next step.
Step 7. Synthesis of 4-(((2-(5-chloro-2-((trans-4-((2- methoxyethyl)amino)cyclohexyl)amino)pyridin-4-yl)pyrimidin-4- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
To a solution of fluoropyridine obtained in step 6 (90 mg, 0.207 mmol) in DMSO (1 ml) at room temperature was added trans-N1-(2-methoxyethyl)cyclohexane-1 ,4-diamine obtained in step 2 (71.3 mg, 0.414 mmol) and 2,6-lutidine (0.048 ml, 0.414 mmol) sequestially. The light brown mixture was heated to 110 °C in a sealed scintilation vial for 18 hours. LC/MS showed containing desired product (M+1 =500.0, Rt=0.52 min) and starting material along with other impurities. The reaction mixture was cooled to room temperature, filtered and purified by HPLC (ACN in water with gradient 10% - 50% in 16 minutes) and lyophilized to give desired product as a beige powder. LCMS (m/z): 500.2 (M+H), retention time = 0.52 min.
Example 373 (Cmpd No. 563 in Table 1)
Synthesis of 4-(((4-(5-chloro-2-((trans-4-((2- methoxyethyl)amino)cyclohexyl)amino)pyridin-4-yl)pyrimidin-2- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000267_0001
Step 1. Synthesis of tert-butyl (trans-4-((2-methoxyethyl)amino)cyclohexyl)carbamate A mixture of p-toluenesulfonic acid 2-methoxyethyl ester (2.7 g, 1 1.72 mmol), DIEA (6.14 mL, 35.2 mmol) and 1-N-Boc-trans-1 ,4-cyclohexyldiamine (5.03 g, 23.45 mmol) in DMSO (50 mL) was heated in 100°C oil bath for 29 hours. The formation of desired product was confirmed by LC/MS. The reaction was cooled to room temperature (orange solution with some solids), diluted in 300 mL water, 20 mL of 2 M Na2C03 and extracted with EtOAc (2 x 150 mL). Some solid NaCI was added to the aqueous and extracted with EtOAc (1 x 150 mL). The combined organic extract was washed with brine with 10 mL of 2M Na2C03 solution added, dried over Na2S04, and concentrated to yield a dark orange semi-solid (4.1 g). This was diluted in 30 mL DCM, filtered off solids (impurity), rinsed with 20 mL DCM, then concentrated to a dark orange oil.
The resulting crude material was purified by ISCO system [220 g silica gel column.
Eluted with 100% DCM for 2.5 min, then 50% DCM to 100% solution A
(90%DCM/10%MeOH/0.5% NH4OH) over 30 min, held for 10 min]. The desired fractions (identified by LC/MS and TLC (90%MeCI2/10%MeOH/0.5% NH4OH with Iodine stain) were combined and concentrated in vacuo to give the title compound as a dark orange oil that solidifies to a yellow solid upon standing (2.44 g, 8.87 mmol, 76 % yield). LC/MS (mass only): LCMS (m/z) : 273.1 (M+H), retention time = 0.53 min.
Step 2. Synthesis of trans-N1-(2-methoxyethyl)cyclohexane-1 ,4-diamine
To a solution of the carbamate obtained in step 1 (2.44 g, 8.96 mmol) in 9 mL DCM was added TFA (3 mL, 38.9 mmol) and the reaction mixture was stirred at room temperature with oil bubbler for off-gasing for about 18 hours until LC/MS showed complete conversion to desired product. The reaction mixture was concentratee to an orange oil (6.4 g). This TFA salt was diluted in MeOH (50 mL) and to it was added in portions PL- HCOs MP-resin (9.0 g, 1.87 mmol/g, 100A 150-200 μΓΠ, Varian Cat# PL3540-4603). The mixture was stirred until no off-gas remains. Reaction is pH 1. Additional resin was added. Reaction became basic when a total of 37 g of resin was added and another 50 mL MeOH to help stirring. Reaction was stirred for 15 min after addition until no off-gas was observed. Resin was filtered off through glass-fritted funnel and rinsed several times with MeOH. The filtrate was concentrated in vacuo to dryness to yield desired product as free base as a thick brown oil (1.79 g, 8.94 mmol, 100 % yield). LCMS (m/z): 173.1 (M+H), retention time = 0.13 min; 1 H NMR (400 MHz, CHLOROFORM-d) . ppm 1.18 (t, J=9.00 Hz, 4 H) 1.91 (d, J=16.43 Hz, 4 H) 2.08 (br.s., 6 H) 2.44 (d, J=3.52 Hz, 1 H) 2.71 (br. s., 1 H) 2.81 (t, J=5.09 Hz, 2 H) 3.35 (s, 3 H) 3.48 (s, 1 H) 3.50 (t, J=5.09 Hz, 2 H).
Step 3. Synthesis of dihydro-2H-pyran-4,4(3H)-dicarbonitrile
A solution of malononitrile (16 g, 242 mmol) in DMF (175 ml_) was cooled to 0°C (ice bath) in a 500 ml_ 3-neck round-bottom flask with addition funnel, thermometer, stir bar, and argon sweep. To this was added bis-(2-bromoethyl)ether (31.6 ml_, 242 mmol) all at once. Then DBU (77 ml_, 509 mmol) was slowly added dropwise over 30 min. The reaction was warmed by removing the ice bath, then heated at 60°C for 2 hours and GC/MS showed only product (retention time =5.83 min). The reaction was cooled to room temperature, poured into water (750 ml_), added 250 ml_ saturated brine, and extracted with EtOAc (3 x 750 ml_). The organic extracts were combined, washed with 1 : 1 water/saturated brine (2 x 1 L), dried with sodium sulfate, and concentrated at 30°C to a tan solid (26.7 g). Further dried under high vacuum to give the desired product as tan solids (24.1 g, 177 mmol, 73.1 % yield), GC/MS shows 93% product. 1 H NMR shows product and few impuritites. 1 H NMR (400 MHz, CHLOROFORM-d) . ppm 3.85 - 3.90 (m, 4H), 2.23 - 2.27 (m, 4H).
Step 4. Synthesis of 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile
To a solution of the bis-nitrile obtained from step 3 (8.5 g, 62.4 mmol) in EtOH (1 10 mL) at 0 °C (ice bath) in a 3-neck 500 mL round-bottom flask under argon was added NaBH4 (5.25 g, 125 mmol) in one portion. The cloudy yellow mixture was allowed to warm to room temperature (slowly exothermed to 29°C) and stirred under argon for 5 hours. The slightly cloudy mixture was concentrated to remove EtOH. The residue was suspended in EtOAc (1 15 mL) and water (-18 mL) was added. The mixture was stirred for 30 minutes. The organic layer was separated, washed with brine (20 mL), dried over sodium sulfate and concentrated in vacuo at room temperature and under high vacuum for 15 minutes to give the title compound as cloudy brown oil (8.9 g, 31.7 mmol, 50.8 % yield). Assume 50% purity and take on as is without further purification. Step 5. Synthesis of 2-chloro-4-(5-chloro-2-fluoropyridin-4-yl)pyrimidine A mixture of 2,4-dichloropyrimidine (0.4 g, 2.68 mmol), 5-chloro-2-fluoropyridin-4- ylboronic acid (0.471 g, 2.68 mmol), PdCl2(dppf).CH2Cl2 adduct (0.219 g, 0.268 mmol) and sodium carbonate (2.68 ml, 5.37 mmol) in DME (5 ml) was sealed and heated in microwave to 100 °C for 15 minutes. LC/MS showed formation of desired product (LCMS (m/z) 243.9/245.8 (M+H), retention time = 0.76 min) and other impurities. The reaction mixture was poured into water and extracted with DCM. The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated in vacuo to give 0.76 g of black residue. The crude mixture was purified by Analogix system (silica gel column 80 g, elution - isocratic: 0-4 min, 100%n-heptane; 4-10 min, 10% EtOAc in Heptane; 10-14 min. 20% EtOAc in Heptane; 14-20 min. 30% EtOAc in Heptane). The pure fractions were combined and concentrated in vacuo to give two fractions based upon TLC retention time. Between them, F2 yielded 0.21 g (32%) of desired product as a white powder. LCMS (m/z): 243.9/245.9 (M+H), 1 :1 , retention time = 0.77 min.). 1 H NMR (400 MHz, CDCI3) . ppm 7.35 - 7.44 (m, 1 H) 7.81 (d, J=5.09 Hz, 1 H) 8.30 - 8.46 (m, 1 H) 8.82 (d, J=5.09 Hz, 1 H).
Step 6. Synthesis of 4-(((4-(5-chloro-2-fluoropyridin-4-yl)pyrimidin-2- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
To the above obtained 2-chloro-4-(5-chloro-2-fluoropyridin-4-yl)pyrimidine (0.21 g, 0.860 mmol) in DMSO (3 ml) at room temperature was added the amine from step 4 (0.151 g, 0.860 mmol) and 2,6-lutidine (0.200 ml, 1.721 mmol) sequentially. The light brown mixture was heated to 45 °C in a sealed scintillation vial for 1 hour. LC/MS showed as a mixture of desired product (-50%) and starting materials. Additional amine from step 4 (0.3 g) was added. The reaction mixture was heated at 65 °C for another 18 hours. LC/MS showed nearly no starting materials and it contained desired product (LCMS (m/z): 348(M+H), retention time = 0.74 min) as major component. The reaction mixture was poured into water and extracted with DCM (3x20mL). The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated in vacuo to give 0.98 g of brown oil. This was used in the next step without purification.
Step 7. Synthesis of 4-(((4-(5-chloro-2-((trans-4-((2- methoxyethyl)amino)cyclohexyl)amino)pyridin-4-yl)pyrimidin-2- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
To the above obtained material (210 mg, 0.181 mmol) in DMSO (1 ml) at room temperature was added trans-N1-(2-methoxyethyl)cyclohexane-1 ,4-diamine obtained in step 2 (78 mg, 0.362 mmol) and 2,6-lutidine (0.042 ml, 0.362 mmol) sequestially. The light brown mixture was heated to 120 °C in a sealed scintilation vial for 16 hours. LC/MS showed containing desired product (LCMS (m/z): 500.2(M+H), retention time = 0.51 min) and other impurities. The reaction was cooled to room temperature, filtered and purified by HPLC (ACN in water with gradient 10% - 50% in 16 minutes) and lyophilized to give desired product as yellow powder (15 mg, 15% yield). LCMS (m/z): 500.2 (M+H), retention time = 0.51 min. 1 H NMR (400 MHz, CHLOROFORM-d) . ppm 1.50 - 1.85 (m, 6 H) 1.90 - 2.01 (m, 2 H) 2.21 (t, J=12.33 Hz, 4 H) 3.07 - 3.27 (m, 3 H) 3.38 (s, 3H) 3.49 - 3.77 (m, 6 H) 3.83 (d, J=6.65 Hz, 2 H) 4.01 (dd, J=12.13, 2.74 Hz, 2 H) 7.00 - 7.08 (m, 1 H) 7.15 (br. s., 1 H) 7.42 - 7.51 (m, 1 H) 7.57 -7.64 (m, 1 H) 8.08 (d, J=7.04 Hz, 1 H) 8.41 (br. s., 1 H).
Example 374 (Cmpd No. 504 in Table 1)
N2-(trans-4-aminocyclohexyl)-N2'-((tetrahydro-2H-pyran-4-yl)methyl)-4,4'- bipyridine-2,2'-diamine
Figure imgf000270_0001
Step 1 : Preparation of trans-N1-(4-bromopyridin-2-yl)cyclohexane-1 ,4-diamine:
To 4-bromo-2-chloropyridine (900 mg, 4.68 mmol) was added NMP (9 ml), trans- cyclohexane-1 ,4-diamine (2670 mg, 23.38 mmol)and triethylamine (0.717 ml, 5.14 mmol). The reaction was stirred at 95 °C for 62 hours or until done by LCMS. The reaction was let cool and the solids filtered off. The crude material was purified by prep LC and lyophilized to give 272 mg of title compound as TFA salt. LCMS (m/z): 270.1/272.1 (MH+), rt = 0.32 min. Step 2: Preparation of trans-N1-(2'-fluoro-4,4'-bipyridin-2-yl)cyclohexane-1 ,4-diamine:
To trans-N1-(4-bromopyridin-2-yl)cyclohexane-1 ,4-diamine (160 mg, 0.592 mmol) was added 2-fluoropyridin-4-ylboronic acid (150 mg, 1.066 mmol), PdCI2(dppf).CH2CI2 adduct (48.4 mg, 0.059 mmol), DME (4.5 ml) and then last 2M sodium carbonate (1.481 ml, 2.96 mmol). The reaction was stirred at 85 °C for 6 hours or until done by LCMS. The reaction was cooled, 20 ml of ethyl acetate was added filtered and the solvent concentrated off to crude residue. The crude material was purified by prep LC and lyophilized to give 44 mg of title compound as TFA salt. LCMS (m/z): 287.2 (MH+), rt = 0.37 min.
Step 3: Preparation of N2-(trans-4-aminocyclohexyl)-N2'-((tetrahydro-2H-pyran-4- yl)methyl)-4,4'-bipyridine-2,2'-diamine:
To trans-N1-(2'-fluoro-4,4'-bipyridin-2-yl)cyclohexane-1 ,4-diamine (10 mg, 0.035 mmol) was added DMSO (0.4 ml) and then (tetrahydro-2H-pyran-4-yl)methanamine (48.3 mg, 0.419 mmol). The reaction was capped and heated at 100 °C for 20 hours. The excess amine was remove under vacuum, 0.5 ml of DMSO was added, filtered, purify by prep LC and lyophilized to give 9.4 mg of title compound as TFA salt. LCMS (m/z): 382.3 (MH+), rt = 0.32 min.
Example 375 (Cmpd No. 537 in Table 1)
trans-N1-(4-(6-(3-fluorobenzylamino)pyrimidin-4-yl)pyridin-2-yl)cyclohexane-1,4- diamine
Figure imgf000271_0001
Step 1 : Preparation of 6-chloro-N-(3-fluorobenzyl)pyrimidin-4-amine:
To 4,6-dichloropyrimidine (300 mg, 2.014 mmol) was added DMSO (3.5 ml), triethylamine (0.337 ml, 2.416 mmol) and (3-fluorophenyl)methanamine (630 mg, 5.03 mmol). The reaction was stirred at 55 °C for 3 hours or until done by LCMS. The excess amine was remove under vacuum, 4 ml of DMSO was added, filtered, purify by prep LC and lyophilized to give 225 mg of title compound as TFA salt. LCMS (m/z): 238.1 (MH+), rt = 0.79 min.
Step 2: Preparation of N-(3-fluorobenzyl)-6-(2-fluoropyridin-4-yl)pyrimidin-4-amine:
To 6-chloro-N-(3-fluorobenzyl)pyrimidin-4-amine (32 mg, 0.135 mmol) was added 2-fluoropyridin-4-ylboronic acid (34.2 mg, 0.242 mmol), PdCI2(dppf).CH2CI2 adduct (16.49 mg, 0.020 mmol), DME (0.9 ml), Ethanol (0.2 ml) and then 2M sodium carbonate (0.269 ml, 0.539 mmol). The reaction was heated at 85 °C for 3 hours or until done by LCMS. The reaction was let cool, 3 ml of ethyl acetate and 1 ml of methanol was added. The mixture was filtered and concentrated to crude residue. The residue was dissolved in 1.2ml of DMSO, filtered, purify by prep LC and lyophilized to give 25 mg of title compound as TFA salt. LCMS (m/z): 299.1 (MH+), rt = 0.63 min.
Step 3: Preparation of trans-N1-(4-(6-(3-fluorobenzylamino)pyrimidin-4-yl)pyridin-2- yl)cyclohexane-1 ,4-diamine:
To N-(3-fluorobenzyl)-6-(2-fluoropyridin-4-yl)pyrimidin-4-amine (21 mg, 0.070 mmol) was added DMSO (0.6 ml) and trans-cyclohexane-1 ,4-diamine (64.3 mg, 0.563 mmol). The reaction was capped and heated at 105 °C for 22 hours, followed by LCMS. The reaction was let cool, 0.5 ml of DMSO was added, filtered, purify by prep LC and lyophilized to give 14.5 mg of title compound as TFA salt. LCMS (m/z): 393.3 (MH+), rt = 0.50 min. Example 376 (Cmpd No. 541 in Table 1)
N2,-(trans-4-aminocyclohexyl)-N4-(3-fluorobenzyl)-2,4,-bipyridine-2',4-diamine
Figure imgf000272_0001
Step 1 : Preparation of 2-chloro-N-(3-fluorobenzyl)pyridin-4-amine:
To 2-chloro-4-fluoropyridine (150 mg, 1.140 mmol) was added NMP (1.5 ml), triethylamine (0.191 ml, 1.368 mmol) and (3-fluorophenyl)methanamine (228 mg, 1.825 mmol). The reaction was stirred at 55-60 °C for 3 hours or until done by LCMS. The reaction was let cool, 2 ml of DMSO was added, filtered, purified by prep LC and lyophilized to give 204 mg of title compound as TFA salt. LCMS (m/z): 237.1 (MH+), rt = 0.53 min.
Step 2: Preparation of 2'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-4-amine:
To 2-chloro-N-(3-fluorobenzyl)pyridin-4-amine (150 mg, 0.634 mmol) was added 2-fluoropyridin-4-ylboronic acid (134 mg, 0.951 mmol), Palladium Tetrakis (88 mg, 0.076 mmol), DME (3 ml) and then 2M sodium carbonate (0.951 ml, 1.901 mmol). The reaction was heated at 110 °C for 4 hours or until done by LCMS. The reaction was let cool, 20 ml of ethyl acetate was added, filtered and concentrated to crude residue. The residue was dissolved in 2 ml of DMSO, filtered, purified by prep LC and lyophilized to give 29.5 mg of title compound as TFA salt. LCMS (m/z): 298.1 (MH+), rt = 0.59 min. Step 3: Preparation of N2'-(trans-4-aminocyclohexyl)-N4-(3-fluorobenzyl)-2,4'-bipyridine- 2',4-diamine:
To 2'-fluoro-N-(3-fluorobenzyl)-2,4'-bipyridin-4-amine (21 mg, 0.071 mmol) was added DMSO (0.6 ml) and trans-cyclohexane-1 ,4-diamine (81 mg, 0.706 mmol). The reaction was capped and heated at 110 °C for 24 hours, followed by LCMS. The reaction was let cool, 0.75 ml of DMSO was added, filtered, purify by prep LC and lyophilized to give 13.5 mg of title compound as TFA salt. LCMS (m/z): 392.1 (MH+), rt = 0.43 min.
Synthesis of 4-(((4-(5-chloro-2-(((trans)-4-((2- methoxyethyl)amino)cvclohexyl)amino)pyridin-4-yl)pyrimidin-2- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000273_0001
Step 1 : Preparation of 2-chloro-4-(5-chloro-2-fluoropyridin-4-yl)pyrimidine
Figure imgf000273_0002
Reactants 2,4-dichloropyrimidine (0.4 g, 2.68 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (0.471 g, 2.68 mmol), PdCI2(dppf) CH2CI2 adduct (0.219 g, 0.268 mmol) and sodium carbonate (2 M, 2.68 mL, 5.37 mmol) are mixed in DME (5 mL). The microwave tube was sealed and heated to 100 °C for 15 min. LC/MS showed formation of desired product (M+1 = 243.9/245.8, Rt=0.76 min) and other impurities. The reaction mixture was poured into water and extracted with DCM. The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated under reduced pressure to give 0.76 g of black residue. The crude mixture was purified by column
chromatography [Si02, 80 g, heptane/EtOAc]. The fractions were combined and concentrated under reduced pressure to give 0.21 g of 2-chloro-4-(5-chloro-2- fluoropyridin-4-yl)pyrimidine as a white powder. LCMS (m/z): 243.9/245.9 [M+H]+; Retention time = 0.77 min. H NMR (400 MHz, chloroform-d) δ pm 7.35 - 7.44 (m, 1 H) 7.81 (d, J=5.09 Hz, 1 H) 8.30 - 8.46 (m, 1 H) 8.82 (d, J=5.09 Hz, 1 H).
Step 2: Preparation of 4-(((4-(5-chloro-2-fluoropyridin-4-yl)pyrimidin-2
yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000274_0001
To 2-chloro-4-(5-chloro-2-fluoropyridin-4-yl)pyrimidine (0.21 g, 0.860 mmol) in DMSO (3 mL) at room temperature was added 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile (0.151 g, 0.860 mmol) and 2,6-lutidine (0.200 mL, 1.721 mmol) sequentially. The light brown mixture was heated to 45 °C in a sealed vial for 1 hour. Additional 4- (aminomethyl)tetrahydro-2H-pyran-4-carbonitrile (0.3 g) was added. The reaction mixture was heated at 65 °C for additional 18 hours. The reaction mixture was poured into water and extracted with DCM (3x 20 mL). The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated under reduced pressure to give 0.98 g of crude 4-(((4-(5-chloro-2-fluoropyridin-4-yl)pyrimidin-2
yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile as a brown oil. This was used in the next step without purification. LCMS (m/z): 348 [M+H]+; Retention time = 0.74 min. Step 3: Preparation of 4-(((4-(5-chloro-2-(((trans)-4-((2- methoxyethyl)amino)cyclohexyl)amino)pyridin-4-yl)pyrimidin-2- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile H
Figure imgf000275_0001
To 4-(((4-(5-chloro-2-fluoropyridin-4-yl)pyrimidin-2 yl)amino)methyl)tetrahydro-2H-pyran- 4-carbonitrile (210 mg, 0.181 mmol) inDMSO (1 mL) at room temperature was added N1-(2-methoxyethyl)cyclohexane-1 ,4-trans-diamine (78 mg, 0.362 mmol) and 2,6-lutidine (0.042 mL, 0.362 mmol) sequentially. The light brown mixture was heated to 120 °C in a sealed vial for 16 hours. The reaction was cooled to room temperature, filtered and purified by HPLC (ACN in water with gradient 10% - 50% in 16 minutes). Fractions were lyophilized to give 4-(((4-(5-chloro-2-(((trans)-4-((2- methoxyethyl)amino)cyclohexyl)amino)pyridin-4-yl)pyrimidin-2- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (15 mg) as its trifluoroacetic acid salt as a yellow powder. LCMS (m/z): 500.2 [M+H]+; Retention time = 0.51 min.
Synthesis of 4-(((2-(5-chloro-2-(((1 r,4r)-4-((2- methoxyethyl)amino)cvclohexyl)amino)pyridin-4-yl)pyrimidin-4- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000275_0002
Step 1 : Preparation of 4-(((2-chloropyrimidin-4-yl)amino)methyl)tetrahydro-2H-pyran-4- carbonitrile
Figure imgf000276_0001
To 2,4-dichloropyrimidine (0.4 g, 2.68 mmol) in acetonitrile (6 mL) at room temperature was added 4-(aminomethyl)tetrahydro-2H-pyran-4-carbonitrile (0.414 g, 2.95 mmol) and triethylamine (0.412 mL, 2.95 mmol) sequentially. The light brown mixture was heated to 45 °C in a sealed vial for 1 hour. The reaction mixture was heated at 65 °C for another 18 hours. The reaction mixture was poured into water and extracted with DCM (3x 20 mL). The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated under reduced pressure to give 0.68 g of brown oil. The crude mixture was purified by column chromatography [Si02, 40 g, heptane/EtOAc]. The fractions were combined and concentrated under reduced pressure to give 4-(((2-chloropyrimidin-4- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (0.39 g) as a brown oil, which was used in the next step without further purification. LCMS (m/z): 253.1 [M+H]+; Retention time = 0.46 min. Step 2: Preparation of 4-(((2-(5-chloro-2-fluoropyridin-4-yl)pyrimidin-4- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000276_0002
The 4-(((2-chloropyrimidin-4-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (0.39 g, 1.543 mmol), 5-chloro-2-fluoropyridin-4-ylboronic acid (0.271 g, 1.543 mmol), PdCI2(dppf) CH2CI2 adduct (0.126 g, 0.154 mmol) and sodium carbonate (1.543 mL, 3.09 mmol) were mixed in DME (4 mL). The microwave tube was sealed and radiated to 100 °C for 15 min. Additional boronic acid (0.27 g) and PdCI2(dppf) CH2CI2 adduct (0.1 g) were added and the mixture was heated in microwave for additional 15 minutes at 110 °C. The reaction mixture was poured into water and extracted with DCM (3x 20mL). The organic extracts were combined, washed with brine, dried with sodium sulfate and concentrated under reduced pressure to give 0.85 g of black residue. The crude mixture was purified by column chromatography [Si02, 40 g]. The fractions were combined and concentrated under reduced pressure to give 4-(((2-(5-chloro-2-fluoropyridin-4-yl)pyrimidin-4- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (90 mg) of a brown oil, which was used in the next step. LCMS (m/z): 347.9 [M+H]+; Retention time = 0.74 min.
Step 3: Preparation of 4-(((2-(5-chloro-2-(((trans)-4-((2- methoxyethyl)amino)cyclohexyl)amino)pyridin-4-yl)pyrimidin-4- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile
Figure imgf000277_0001
To 4-(((2-(5-chloro-2-fluoropyridin-4-yl)pyrimidin-4-yl)amino)methyl)tetrahydro-2H-pyran- 4-carbonitrile (90 mg, 0.207 mmol) in DMSO (1 mL) at room temperature was added N1- (2-methoxyethyl)cyclohexane-1 ,4-trans-diamine (71.3 mg, 0.414 mmol) and 2,6-lutidine (0.048 mL, 0.414 mmol) sequentially. The light brown mixture was heated to 1 10 °C in a sealed vial for 18 hours. The reaction mixture was cooled to room temperature, filtered through a syringe filter and purified by HPLC (ACN in water with gradient 10% - 50% in 16 minutes). Fractions were lyophilized to give 4-(((2-(5-chloro-2-(((trans)-4-((2- methoxyethyl)amino)cyclohexyl)amino)-pyridin-4-yl)pyrimidin-4- yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile (3.5 mg) as its trifluoroacetic acid salt as a beige powder. LCMS (m/z): 500.2 [M+H]+; Retention time = 0.52 min.
Using the methods described above with selection of appropriate starting materials, the compounds in Table 1 were prepared. The compound names were generated from the structures and thus refer to the associated structure regardless of whether they conform to nomenclature conventions, and the names refer to 'trans' or 'cis' relative
stereochemistry on 1 ,4-disubstituted cyclohexane compounds in some cases. As shown in the Table, these compounds are potent inhibitors of CDK9 as indicated by the data generated using the Cdk9/cyclinT1 IMAP Protocol described below; IC50s are reported in micromolar units unless otherwise specified. Table 1
Figure imgf000278_0001
Figure imgf000279_0001
Figure imgf000280_0001
Figure imgf000281_0001
Figure imgf000282_0001
Figure imgf000283_0001
Figure imgf000284_0001
Figure imgf000285_0001
Figure imgf000286_0001
Figure imgf000287_0001
Figure imgf000288_0001
Biological Methods Cdk9/cvclinT1 I MAP Protocol
The biological activity of the compounds of the invention can be determined using the assay described below.
Cdk9/cyclinT1 is purchased from Millipore, cat #14-685. The final total protein concentration in the assay is 4 nM. The 5TAMRA-cdk7tide peptide substrate, 5TAMRA- YSPTSPSYSPTSPSYSTPSPS-COOH, is purchased from Molecular Devices, cat#R7352. The final concentration of peptide substrate is 100 nM. The ATP substrate (Adenosine-5'-triphosphate) is purchased from Roche Diagnostics, cat#1140965. The final concentration of ATP substrate is 6 uM. IMAP (Immobilized Metal Assay for Phosphochemicals) Progressive Binding reagent is purchased from Molecular Devices, cat#R8139. Fluorescence polarization (FP) is used for detection. The 5TAMRA- cdk7tide peptide is phosphorylated by Cdk9/cyclinT1 kinase using the ATP substrate. The Phospho-5TAMRA-cdk7tide peptide substrate is bound to the IMAP Progressive Binding Reagent. The binding of the IMAP Progressive Binding Reagent changes the fluorescence polarization of the 5TAMRA-cdk7tide peptide which is measured at an excitation of 531 nm and FP emission of 595 nm. Assays are carried out in 100 mM Tris, pH=7.2, 10 mM MgCI2, 0.05% NaN3, 0.01 % Tween-20, 1 mM dithiothreitol and 2.5 % dimethyl sulfoxide. IMAP Progressive Binding Reagent is diluted 1 :800 in 100 % 1X Solution A from Molecular Devices, cat#R7285.
General protocol is as follows: To 10 uL of cdk9/cyclinT1 , 0.5 uL of test compound in dimethyl sulfoxide is added. 5TAMRA-cdk7tide and ATP are mixed. 10 uL of the 5TAMRA-cdk7tide /ATP mix is added to start the reaction. The reaction is allowed to proceed for 4.5 hrs. 60 uL of IMAP Progressive Binding Reagent is added. After >1 hr of incubation, plates are read on the Envision 2101 from Perkin-Elmer. The assay is run in a 384-well format using black Corning plates, cat#3573.
Cdk9/cvclinT1 Alpha Screen Protocol
Full length wild type Cdk9/cyclin T1 is purchased from Invitrogen, cat#PV4131. The final total protein concentration in the assay is 1 nM. The cdk7tide peptide substrate, biotin-GGGGYSPTSPSYSPTSPSYSPTSPS-OH, is a custom synthesis purchased from the Tufts University Core Facility. The final concentration of cdk7tide peptide substrate is 200nM. The ATP substrate (Adenosine-5'-triphosphate) is purchased from Roche Diagnostics. The final concentration of ATP substrate is 6 uM. Phospho-Rpb1 CTD (ser2/5) substrate antibody is purchased from Cell Signaling Technology. The final concentration of antibody is 0.67 ug/mL. The Alpha Screen Protein A detection kit containing donor and acceptor beads is purchased from
PerkinElmer Life Sciences. The final concentration of both donor and acceptor beads is 15 ug/mL. Alpha Screen is used for detection. The biotinylated-cdk7tide peptide is phosphorylated by cdk9/cyclinT1 using the ATP substrate. The biotinylated-cdk7tide peptide substrate is bound to the streptavidin coated donor bead. The antibody is bound to the protein A coated acceptor bead. The antibody will bind to the phosphorylated form of the biotinylated-cdk7tide peptide substrate, bringing the donor and acceptor beads into close proximity. Laser irradiation of the donor bead at 680 nm generates a flow of shortlived singlet oxygen molecules. When the donor and acceptor beads are in close proximity, the reactive oxygen generated by the irradiation of the donor beads initiates a luminescence/fluorescence cascade in the acceptor beads. This process leads to a highly amplified signal with output in the 530-620nm range. Assays are carried out in 50 mM Hepes, pH=7.5, 10 mM MgCI2, 0.1 % Bovine Serum Albumin, 0.01 % Tween-20, 1 mM Dithiolthreitol, 2.5 % Dimethyl Sulfoxide. Stop and detection steps are combined using 50 mM Hepes, pH=7.5, 18 mM EDTA, 0.1 % Bovine Serum Albumin, 0.01 % Tween-20.
General protocol is as follows: To 5 uL of cdk9/cyclinT1 , 0.25 uL of test compound in dimethyl sulfoxide is added. Cdk7tide peptide and ATP are mixed. 5 uL of the cdk7tide peptide/ATP mix is added to start the reaction. The reaction is allowed to proceed for 5hrs. 10 uL of Ab/ Alpha Screen beads/Stop-detection buffer is added. Care is taken to keep Alpha Screen beads in the dark at all times. Plates are incubated at room temperature overnight, in the dark, to allow for detection development before being read. The assay is run in a 384-well format using white polypropylene Greiner plates.

Claims

1. A compound of Formula I:
Figure imgf000291_0001
or a pharmaceutically acceptable salt thereof, wherein:
R is selected from the group consisting of Ci-6 alkyl, C3.6branched alkyl,
C3-i0cycloalkyl, C3.i0 heterocycloalkyi, C3-i0-partially unsaturated cycloalkyi and C3.i0 partially unsaturated heterocycloalkyi;, wherein each heterocycloalkyi contains one or two heteroatoms selected from N, S and O as ring members,
wherein R is optionally substituted with up to four groups selected from halo, oxo (=0), CN, C C4 haloalkyl, C C4 haloalkoxy, -R, -OR, -NR2, -COR, COOR, -(CH2)q-R, -(CH2)q-NR2, -CONR2, -NHCOR, -NRCOOR, -SR, -S(0)qR, - S02NHR, -NHS02R, -(CH2)q-CN, -0-(CH2)q-CN, -(CH2)q-OR, and -0-(CH2)q-OR, where each R is independently H or an optionally substituted C C6 alkyl or C3-C7 cycloalkyi or C3-C7 heterocycloalkyi containing N, O or S as a ring member,
wherein the optional substituents for R are up to three groups independently selected from halo, oxo, CN, C C4 haloalkyl, C C4 haloalkoxy, -OH, R', OR', -C(0)R', NH2, NHR', -
NH-C(0)-R', where each R' is independently C C4 straight chain or branched chain alkyl;
and where two R on the same atom or on adjacent connected atoms can optionally cyclize to form a 5-6 membered ring optionally containing 1-2 heteroatoms selected from N, O and S as ring members, and optionally substituted with up to four groups selected from halo, oxo (=0), CN, Ci-C4 alkyl, C C4 alkoxy, C C4 haloalkyl, and C C4 haloalkoxy; and each q is 1-2;
R3 is H, CI or F;
L is a bond or C1.3 alkylene;
L2 is Co-3 alkylene, CD2, CHD, or C3.8 branched alkylene;
A is O or -NR8- Z2 is N or CR2;
Z4 is N or CR4;
Z5 is N or CR5;
Z6 is N or CR6;
provided that at least one but not more than two of Z2, Z4, Z5 and Z6 is N;
and if Z2 is N, either Z4 or Z6 is also N;
and if Z5 is N, either Z4 or Z6 is also N;
R2, R4, R5, R6, and R7 are each independently selected from H, C C4 alkyl, C C4 alkoxy, C C4 haloalkyi, C C4 haloalkoxy, CN, F and CI;
R8 is H or methyl;
R9 is an optionally substituted group selected from Ci_3 alkyl, C3.6 branched alkyl, C3.7 cycloalkyl, C3.7 heterocycloalkyl having up to two heteroatoms selected from N, O and S as ring members, -(CH2)i-3-0-Ci_4 alkyl, pyridyl, and phenyl,
wherein the optional substituents for R9 are up to four groups independently selected from halo, oxo, CN, C C4 haloalkyi, C C4 haloalkoxy, -
OH, R', OR', -C(0)R', CONH2, CONHR', NH2, NHR', -NH-C(0)-R', where each R' is independently C C4 straight chain or branched chain alkyl or haloalkyi.
2. The compound of claim 1 , wherein:
L is a bond or CH2, and R is cyclohexyl,
wherein R is substituted with up to four groups selected from halo, oxo (=0), CN, Ci-C4 haloalkyi, C C4 haloalkoxy, -R, -OR, -NR2, -COR, - COOR, -CONR2, -NHCOR, -NRCOOR, -S(0)qR, -S02NHR, -NHS02R, - (CH2)q-OR, and -0-(CH2)q-OR;
R3 is F or CI;
L2 is CH2, CHD, or CD2; and
R9 is selected from pyridyl, phenyl, tetrahydropyran, cyclopropane, and tetrahydrofuran, each of which can be substituted with up to three groups independently selected from F, CI, -OH, Me, ethyl, -OMe, CN, and CONH2.
3. The compound of claim 1 or 2, wherein -L-R is
Figure imgf000293_0001
where R 0 and R and R 2 each independently represent H, F,
CI, -OCHF2, -C(0)-Me, -OH, Me, -OMe, -CN, -Ethyl, -CONH2, or -NH-C(0)-Me.
4. The compound of any of the preceding claims, wherein R3 is CI.
5. The compound of any one of claims 1-4, wherein R is an optionally substituted cyclohexyl, piperidine, or pyrrolidine ring.
6. The compound of any one of claims 1-5, wherein R7 is H.
7. The compound of any one of claims 1-6, wherein A1 is NH.
8. The compound of any one of claims 1-7, wherein Z2 is N.
9. The compound of any one of claims 1-8, wherein Z6 is N.
10. The compound of any of claims 1-8, wherein Z6 is CH.
11. The compound of any one of claims 1-9, wherein Z5 is CH.
12. The compound of any one of claims 1-8, wherein Z4 is N.
13. The compound of claim 1 , which is a compound of Formula II:
Figure imgf000293_0002
14. The compound of claim 1 , which is a compound of Formula III:
Figure imgf000294_0001
The compound of claim 1 , which is a compound of Formula (IV):
Figure imgf000294_0002
The compound of claim 1 , which is a compound of Formula (V):
Figure imgf000294_0003
17. The compound of claim 1 , which is a compound of Formula (VI):
Figure imgf000295_0001
18. A compound of any of claims 1-17, for use in therapy.
19. The compound of claim 18, which is for use to treat a condition mediated by CDK9.
20. The compound of claim 19, wherein the condition is cancer.
21. The compound of claim 20, wherein the cancer is selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal, and pancreatic cancer.
22. A method to treat cancer, comprising administering to a subject in need thereof an effective amount of a compound according to any of claims 1-17.
23. The method of claim 22, wherein the cancer is selected from the group consisting of bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal, and pancreatic cancer.
24. The method of claim 23, wherein the compound is administered, simultaneously or sequentially, with an antiinflammatory, antiproliferative,
chemotherapeutic agent, immunosuppressant, anti-cancer, cytotoxic agent or kinase inhibitor or salt thereof.
25. A pharmaceutical composition comprising a compound according to any of claims 1-17 admixed with at least one pharmaceutically acceptable excipient.
26. The pharmaceutical composition of claim 25, which comprises at least one pharmaceutically acceptable carrier and at least one other pharmaceutically acceptable excipient.
27. The pharmaceutical composition of claim 25 or 26, further comprising at least one additional therapeutic agent.
28. The pharmaceutical composition of claim 27, wherein the additional therapeutic agent is an antiinflammatory, antiproliferative, chemotherapeutic agent, immunosuppressant, anti-cancer, cytotoxic agent or kinase inhibitor or a salt thereof.
PCT/EP2012/050914 2011-01-28 2012-01-20 Pyridine biaryl amine compounds and their uses WO2012101066A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161437082P 2011-01-28 2011-01-28
US61/437,082 2011-01-28

Publications (1)

Publication Number Publication Date
WO2012101066A1 true WO2012101066A1 (en) 2012-08-02

Family

ID=45498007

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2012/050914 WO2012101066A1 (en) 2011-01-28 2012-01-20 Pyridine biaryl amine compounds and their uses

Country Status (1)

Country Link
WO (1) WO2012101066A1 (en)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013037896A1 (en) 2011-09-16 2013-03-21 Bayer Intellectual Property Gmbh Disubstituted 5-fluoro-pyrimidines
WO2014060376A1 (en) 2012-10-18 2014-04-24 Bayer Pharma Aktiengesellschaft 4-(ortho)-fluorophenyl-5-fluoropyrimidin-2-yl amines containing a sulfone group
WO2014076111A1 (en) 2012-11-15 2014-05-22 Bayer Pharma Aktiengesellschaft N-(pyridin-2-yl)pyrimidin-4-amine derivatives containing a sulfoximine group
WO2016059011A1 (en) 2014-10-16 2016-04-21 Bayer Pharma Aktiengesellschaft Fluorinated benzofuranyl-pyrimidine derivatives containing a sulfone group
WO2016061144A1 (en) 2014-10-14 2016-04-21 The Regents Of The University Of California Use of cdk9 and brd4 inhibitors to inhibit inflammation
US9498471B2 (en) 2011-10-20 2016-11-22 The Regents Of The University Of California Use of CDK9 inhibitors to reduce cartilage degradation
WO2017055196A1 (en) 2015-09-29 2017-04-06 Bayer Pharma Aktiengesellschaft Novel macrocyclic sulfondiimine compounds
WO2017060167A1 (en) 2015-10-08 2017-04-13 Bayer Pharma Aktiengesellschaft Novel modified macrocyclic compounds
WO2017060322A2 (en) 2015-10-10 2017-04-13 Bayer Pharma Aktiengesellschaft Ptefb-inhibitor-adc
US9670161B2 (en) 2012-10-18 2017-06-06 Bayer Pharma Aktiengesellschaft 5-fluoro-N-(pyridin-2-yl)pyridin-2-amine derivatives containing a sulfone group
US9751854B2 (en) 2014-01-14 2017-09-05 Millennium Pharmaceuticals, Inc. Heteroaryls and uses thereof
WO2018177889A1 (en) 2017-03-28 2018-10-04 Bayer Aktiengesellschaft Novel ptefb inhibiting macrocyclic compounds
WO2018177899A1 (en) 2017-03-28 2018-10-04 Bayer Aktiengesellschaft Novel ptefb inhibiting macrocyclic compounds
US10202373B2 (en) 2014-01-14 2019-02-12 Millennium Pharmaceuticals, Inc. Heteroaryls and uses thereof
US11591322B2 (en) 2020-05-15 2023-02-28 Algen Biotechnologies, Inc. Certain chemical compositions and methods of use thereof
US11701347B2 (en) 2018-02-13 2023-07-18 Bayer Aktiengesellschaft Use of 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-{4-[(S-methylsulfonimidoyl)methyl]pyridin-2-yl}pyridin-2-amine for treating diffuse large B-cell lymphoma

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0039051A2 (en) 1980-04-24 1981-11-04 Merck & Co. Inc. Mannich-base hydroxamic acid prodrugs for the improved bioavailability of non-steroidal anti-inflammatory agents, a process for preparing and a pharmaceutical composition containing them
WO2001093682A1 (en) * 2000-06-08 2001-12-13 Syngenta Participations Ag N-phenyl-4-(4-pyridyl)-2-pyrimidineamine derivatives
WO2004078163A2 (en) 2003-02-28 2004-09-16 Transform Pharmaceuticals, Inc. Pharmaceutical co-crystal compositions of drugs such as carbamazepine, celecoxib, olanzapine, itraconazole, topiramate, modafinil, 5-fluorouracil, hydrochlorothiazide, acetaminophen, aspirin, flurbiprofen, phenytoin and ibuprofen
WO2008079933A2 (en) 2006-12-22 2008-07-03 Novartis Ag Heteroaryl-heteroaryl compounds as cdk inhibitors for the treatment of cancer, inflammation and viral infections

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0039051A2 (en) 1980-04-24 1981-11-04 Merck & Co. Inc. Mannich-base hydroxamic acid prodrugs for the improved bioavailability of non-steroidal anti-inflammatory agents, a process for preparing and a pharmaceutical composition containing them
WO2001093682A1 (en) * 2000-06-08 2001-12-13 Syngenta Participations Ag N-phenyl-4-(4-pyridyl)-2-pyrimidineamine derivatives
WO2004078163A2 (en) 2003-02-28 2004-09-16 Transform Pharmaceuticals, Inc. Pharmaceutical co-crystal compositions of drugs such as carbamazepine, celecoxib, olanzapine, itraconazole, topiramate, modafinil, 5-fluorouracil, hydrochlorothiazide, acetaminophen, aspirin, flurbiprofen, phenytoin and ibuprofen
WO2008079933A2 (en) 2006-12-22 2008-07-03 Novartis Ag Heteroaryl-heteroaryl compounds as cdk inhibitors for the treatment of cancer, inflammation and viral infections

Non-Patent Citations (38)

* Cited by examiner, † Cited by third party
Title
"Fieser and Fieser's Reagents for Organic Synthesis", vol. 1, 1991, JOHN WILEY AND SONS
"Fieser's Reagents for Organic Synthesis", vol. 1-15, 1991, JOHN WILEY AND SONS
"HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION, AND USE", 2002, WILEY-VCH
"Larock's Comprehensive Organic Transformations", 1989, VCH PUBLISHERS INC.
"March's Advanced Organic Chemistry", JOHN WILEY AND SONS
"Organic Reactions", vol. 1-40, 1991, JOHN WILEY AND SONS
"REMINGTON'S PHARMACEUTICAL SCIENCES", 1985, MACK PUBLISHING COMPANY
"REMINGTON'S PHARMACEUTICAL SCIENCES", 1990, MACK PRINTING COMPANY, pages: 1289 - 1329
"Rodd's Chemistry of Carbon Compounds", vol. 1-5, 1989, ELSEVIER SCIENCE PUBLISHERS
"THE PRACTICE OF MEDICINAL CHEMISTRY", 2001, ACADEMIC PRESS
ALVI ET AL., BLOOD, vol. 105, 2005, pages 4484
BUNDGAARD, J. MED. CHEM., 1989, pages 2503
BUNDGAARD: "Design of Prodrugs", 1985, ELSEVIER
CAI, D.-P., CANCER RES, vol. 66, 2006, pages 9270
CHAO, S.-H. ET AL., J. BIOL. CHEM., vol. 275, 2000, pages 28345 - 28348
CHAO, S.-H. ET AL., J. BIOL. CHEM., vol. 276, 2001, pages 31793 - 31799
CHEN ET AL., BLOOD, vol. 106, 2005, pages 2513
CHEN ET AL., J. NATL. CANCER INSTITUTE, vol. 92, 2000, pages 1999 - 2008
CORDON-CARDO C., AM. J. PAT1/701, vol. 147, 1995, pages 545 - 560
GARCIA-BUSTOS ET AL., EMBO J., vol. 13, 1994, pages 2352 - 2361
GILLASPY, TETRAHEDRON LETT., vol. 36, 1995, pages 7399 - 7402
HALL M. ET AL., ADV. CANCER RES., vol. 68, 1996, pages 67 - 108
HANKS, S. K.; HUNTER, T., FASEB J., vol. 9, 1995, pages 576 - 596
HARDIE, G.; HANKS, S.: "THE PROTEIN KINASE FACTS BOOK", 1995, ACADEMIC PRESS
HILES ET AL., CELL, vol. 70, 1992, pages 419 - 429
KARP J. E.; BRODER S., NAT. MED., vol. 1, 1995, pages 309 - 320
KNIGHTON ET AL., SCIENCE, vol. 253, 1991, pages 407 - 414
KUNZ ET AL., CELL, vol. 73, 1993, pages 585 - 596
LAM, GENOME BIOLOGY, vol. 2, 2001
MACCALLUM ET AL., CANCER RES., vol. 65, 2005, pages 5399
MALUMBRES; BARBACID, NAT. REV. CANCER, vol. 1, 2001, pages 222
ORG. LETT., vol. 11, 2009, pages 3542 - 3545
PETERLIN; PRICE, CELL, vol. 23, 2006, pages 297 - 305
ROSSI, A.G. ET AL., NATURE MED., vol. 12, 2006, pages 1056
SHAPIRO, J. CLIN. ONCOL., vol. 24, 2006, pages 1770 - 83
SHERR C. J., SCIENCE, vol. 274, 1996, pages 1672 - 1677
T. W. GREENE; G. M. WUTS: "Protecting Groups in Organic Synthesis", 1999, WILEY
TETRAHYDRON, vol. 61, 2005, pages 1207 - 1219

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013037896A1 (en) 2011-09-16 2013-03-21 Bayer Intellectual Property Gmbh Disubstituted 5-fluoro-pyrimidines
US11351161B2 (en) 2011-10-20 2022-06-07 The Regents Of The University Of California Use of CDK9 inhibitors to reduce cartilage degradation
US9498471B2 (en) 2011-10-20 2016-11-22 The Regents Of The University Of California Use of CDK9 inhibitors to reduce cartilage degradation
US10172844B2 (en) 2011-10-20 2019-01-08 The Regents Of The University Of California Use of CDK9 inhibitors to reduce cartilage degradation
WO2014060376A1 (en) 2012-10-18 2014-04-24 Bayer Pharma Aktiengesellschaft 4-(ortho)-fluorophenyl-5-fluoropyrimidin-2-yl amines containing a sulfone group
US9670161B2 (en) 2012-10-18 2017-06-06 Bayer Pharma Aktiengesellschaft 5-fluoro-N-(pyridin-2-yl)pyridin-2-amine derivatives containing a sulfone group
US9650361B2 (en) 2012-11-15 2017-05-16 Bayer Pharam Aktiengesellschaft N-(pyridin-2-yl)pyrimidin-4-amine derivatives containing a sulfoximine group
WO2014076111A1 (en) 2012-11-15 2014-05-22 Bayer Pharma Aktiengesellschaft N-(pyridin-2-yl)pyrimidin-4-amine derivatives containing a sulfoximine group
US10538533B2 (en) 2014-01-14 2020-01-21 Millennium Pharmaceuticals, Inc. Heteroaryls and uses thereof
US9751854B2 (en) 2014-01-14 2017-09-05 Millennium Pharmaceuticals, Inc. Heteroaryls and uses thereof
US9802960B2 (en) 2014-01-14 2017-10-31 Millennium Pharmaceuticals, Inc. Heteroaryls and uses thereof
US10202373B2 (en) 2014-01-14 2019-02-12 Millennium Pharmaceuticals, Inc. Heteroaryls and uses thereof
US11020404B2 (en) 2014-10-14 2021-06-01 The Regents of the University of California, Davis Use of CDK9 and BRD4 inhibitors to inhibit inflammation
US10300073B2 (en) 2014-10-14 2019-05-28 The Regents Of The University Of California Use of CDK9 and BRD4 inhibitors to inhibit inflammation
WO2016061144A1 (en) 2014-10-14 2016-04-21 The Regents Of The University Of California Use of cdk9 and brd4 inhibitors to inhibit inflammation
WO2016059011A1 (en) 2014-10-16 2016-04-21 Bayer Pharma Aktiengesellschaft Fluorinated benzofuranyl-pyrimidine derivatives containing a sulfone group
WO2017055196A1 (en) 2015-09-29 2017-04-06 Bayer Pharma Aktiengesellschaft Novel macrocyclic sulfondiimine compounds
WO2017060167A1 (en) 2015-10-08 2017-04-13 Bayer Pharma Aktiengesellschaft Novel modified macrocyclic compounds
WO2017060322A2 (en) 2015-10-10 2017-04-13 Bayer Pharma Aktiengesellschaft Ptefb-inhibitor-adc
WO2018177889A1 (en) 2017-03-28 2018-10-04 Bayer Aktiengesellschaft Novel ptefb inhibiting macrocyclic compounds
US11242356B2 (en) 2017-03-28 2022-02-08 Bayer Aktiengesellschaft PTEFb inhibiting macrocyclic compounds
US11254690B2 (en) 2017-03-28 2022-02-22 Bayer Pharma Aktiengesellschaft PTEFb inhibiting macrocyclic compounds
WO2018177899A1 (en) 2017-03-28 2018-10-04 Bayer Aktiengesellschaft Novel ptefb inhibiting macrocyclic compounds
US11691986B2 (en) 2017-03-28 2023-07-04 Bayer Aktiengesellschaft PTEFB inhibiting macrocyclic compounds
US11701347B2 (en) 2018-02-13 2023-07-18 Bayer Aktiengesellschaft Use of 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-{4-[(S-methylsulfonimidoyl)methyl]pyridin-2-yl}pyridin-2-amine for treating diffuse large B-cell lymphoma
US11591322B2 (en) 2020-05-15 2023-02-28 Algen Biotechnologies, Inc. Certain chemical compositions and methods of use thereof

Similar Documents

Publication Publication Date Title
EP2459535B1 (en) Pyridine and pyrazine derivatives as protein kinase modulators
WO2012101066A1 (en) Pyridine biaryl amine compounds and their uses
WO2012101065A2 (en) Pyrimidine biaryl amine compounds and their uses
EP2668162A1 (en) Substituted bi-heteroaryl compounds as cdk9 inhibitors and their uses
EP2640702A1 (en) 3-(aminoaryl)-pyridine compounds
WO2012066065A1 (en) Phenyl-heteroaryl amine compounds and their uses
WO2012101064A1 (en) N-acyl pyrimidine biaryl compounds as protein kinase inhibitors
US20120157433A1 (en) Heteroaryl Compounds as Kinase Inhibitors
WO2012101063A1 (en) N-acyl pyridine biaryl compounds and their uses
US20120165306A1 (en) Pyrazinylpyridines useful for the treatment of proliferative diseases
US20110130380A1 (en) Heteroaryl Kinase Inhibitors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12700503

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12700503

Country of ref document: EP

Kind code of ref document: A1