WO2015081463A1 - Egfr inhibitor forms - Google Patents

Egfr inhibitor forms Download PDF

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Publication number
WO2015081463A1
WO2015081463A1 PCT/CN2013/088295 CN2013088295W WO2015081463A1 WO 2015081463 A1 WO2015081463 A1 WO 2015081463A1 CN 2013088295 W CN2013088295 W CN 2013088295W WO 2015081463 A1 WO2015081463 A1 WO 2015081463A1
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WO
WIPO (PCT)
Prior art keywords
mesylate
pharmaceutical composition
crystalline
compound
benzo
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PCT/CN2013/088295
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French (fr)
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WO2015081463A8 (en
Inventor
Liang MAO
Wenxian Pan
Xiaoyang Wang
Yubo ZHANG
Runyan LI
Liladhar Waykole
Vijay SETHURAMAN
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Novartis Ag
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Application filed by Novartis Ag filed Critical Novartis Ag
Priority to PCT/CN2013/088295 priority Critical patent/WO2015081463A1/en
Priority to PCT/IB2014/066475 priority patent/WO2015083059A1/en
Publication of WO2015081463A1 publication Critical patent/WO2015081463A1/en
Publication of WO2015081463A8 publication Critical patent/WO2015081463A8/en

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    • 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
    • C07D401/14—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 three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present disclosure generally relates to polymorphic form(s) of
  • the present disclosure also generally relates to a pharmaceutical composition comprising the form(s), as well of methods of using the form(s) in the treatment of cancer indications, and methods for obtaining such forms.
  • the epidermal growth factor receptor belongs to a family of proteins involved in the proliferation of normal and malignant cells. Overexpression of EGFR is found in over 70 percent of human cancers, including without limitation non-small cell lung carcinomas (NSCLC), breast cancers, gliomas, squamous cell carcinoma of the head 15 and neck, and prostate cancer.
  • NSCLC non-small cell lung carcinomas
  • breast cancers gliomas
  • squamous cell carcinoma of the head 15 and neck and prostate cancer.
  • anti-EGFR targeted molecules such as gefitinib and erlotinib.
  • the object of the present disclosure was to provide novel forms of a more potent EGFR inhibitor, the inhibitor of formula 1 :
  • Compound EGFRi (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi) that has also a better therapeutic index.
  • the forms exhibit improved hygroscopicity, stability and/or solubility. In one embodiment the form affords better handling characteristic. In another embodiment, the forms can be better used in formulating pharmaceutical compositions.
  • the novel forms of the Compound can be mixed with specific excipients, or in alternative be formulated in compositions free of other specific excipients, to further bolster their advantageous characteristics. Particularly good stability is achieved when the Compound EGFRi, or its specific forms are kept at a pH between 1 and 9, particularly between 1 and 6.8. The compound and its form exhibit the best stability at about pH 4.5.
  • Embodiments of these crystalline forms include those characterized herein as Compound EGFRi free form A, free form B, mesylate form A, mesylate form B, mesylate form C, amorphous form and HCl salt form.
  • the names used herein to characterize a specific form e.g. "free form A, free form B, mesylate form A, mesylate form B, mesylate form C, amorphous form or HCl salt form" etc., should not be considered limiting with respect to any other substance possessing similar or identical physical and chemical characteristics, but rather it should be understood that these designations are mere identifiers that should be interpreted according to the characterization information also presented herein.
  • the present disclosure provides the following aspects, advantageous features and specific embodiments, respectively alone or in combination, as listed in the following items:
  • (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises mesylate form B, the crystalline form further comprises also at least one of the forms selected from the group of free form A, free form B, mesylate form A, mesylate form C and HCl salt form.
  • x-ray powder diffraction pattern comprises 4 or more 2 ⁇ values (CuKa) selected from the group consisting 75 of:
  • 90 comprises five or more 2 ⁇ values selected from the group.
  • FIG. 1 10 essentially the same as that shown in any one of figures FIG. 2, FIG. 7, FIG. 9, FIG. 12, FIG.
  • a pharmaceutical composition comprising the crystalline form according to any one of items 1 to 9.
  • composition according to item 10 further comprising a pharmaceutically acceptable carrier or diluent.
  • composition according to item 12 wherein said form is free form A, free form B, mesylate form A, mesylate form C or HC1 salt form and wherein x-ray powder diffraction pattern comprises four 2 ⁇ values (CuKa) selected from the group consisting of:
  • composition according to item 12 wherein said form is free form A, free form B, mesylate form A, mesylate form C or HC1 salt form and wherein x-ray powder diffraction pattern comprises five 2 ⁇ values (CuKa) selected from the group consisting of:
  • composition according to item 12 wherein said form is free form A, free form B, mesylate form A, mesylate form C or HC1 salt form and wherein x-ray powder diffraction pattern comprises all 2 ⁇ values (CuKa) selected from the group consisting of: (a) 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ⁇ 0.30 for free form A,
  • a pharmaceutical composition comprising
  • a pharmaceutical composition comprising mesylate form B and a pH modifier.
  • a pharmaceutical composition comprising amorphous form and a pH modifier.
  • composition according to any one of items 10 to 20, wherein the composition is liquid.
  • a pharmaceutical formulation comprising
  • a pharmaceutical formulation comprising
  • Crystalline form according to any one of items 1 to 9 pharmaceutical composition 230 according to any one of items 10 to 27, or pharmaceutical formulation according to any one of items 28 to 33, for use in the treatment of proliferative disease.
  • proliferative disease is non-small cell lung cancer, head and neck cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, gastric cancer, glioma or prostate cancer.
  • proliferative disease is non-small cell lung cancer, head and neck cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, gastric cancer, glioma or prostate cancer.
  • Crystalline form according to any one of items 1 to 9 pharmaceutical composition 240 according to any one of items 10 to 27, or pharmaceutical formulation according to any one of items 28 to 33 for use according to item 35 or 36, wherein the proliferative disease is non-small cell lung cancer.
  • (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2 ⁇ values, 5 2 ⁇ values , 6 2 ⁇ values, 7 2 ⁇ values or all 2 ⁇ values (CuKa) selected from the group consisting of: 5.46, 14.82, 15.84, 18.67, 20.42, 255 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ⁇ 0.30 (free form A).
  • imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2 ⁇ values, 5 2 ⁇ values , 6 2 ⁇ values, 7 2 ⁇ values or all 29 values (CuKa) selected from the group consisting of: 1 1.24, 14.68, 15.66, 18.64, 19.63, 20.05, 20.31 , 22.41 , 24.91 , 25.43, 26.03, 27.18 and 28.07 ⁇ 0.30 (free form B).
  • (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2 ⁇ values, 5 2 ⁇ values , 6 2 ⁇ values, 7 2 ⁇ values or 270 all 2 ⁇ values (CuKa) selected from the group consisting of: 9.12, 12.19, 16.25, 17.38, 18.42, 19.91 , 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ⁇ 0.30 (mesylate form A).
  • (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2 ⁇ values, 5 2 ⁇ values , 6 2 ⁇ values, 7 2 ⁇ values or all 2 ⁇ values (CuKa) selected from the group consisting of: 1 1.76, 13.832, 14.41 , 15.9 17.65, 18.79, 21.46, 21.83, 22.30, 23.82, 24.51 , 24.89, 25.57, 26.66 and 27.77 ⁇ 0.30 (mesylate 280 form B).
  • (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2 ⁇ values or 5 2 ⁇ values (CuKa) selected from the 295 group consisting of: 14.89, 19.69, 25.61 , 26.13 and 27.78 ⁇ 0.30 (HC1 salt form).
  • amorphous refers to a solid form of a molecule and/or ions that is not crystalline. An amorphous solid does not display a definitive X-ray diffraction pattern.
  • substantially pure when used in reference to a form, means a compound having a purity greater than 90 weight %, including greater than 90, 91 , 92, 93, 94, 95, 96, 97, 98, and 99 weight %, and also including equal to about 100 weight % of Compound EGFRi, based on the weight of the compound.
  • the remaining material comprises other form(s) of the compound, and/or reaction impurities and/or processing impurities arising from its
  • substantially pure in that it has a purity greater than 90 weight %, as measured by means that are at this time known and generally accepted in the art, where the remaining less than 10 weight % of material comprises other form(s) of Compound EGFRi and/or reaction impurities and/or processing impurities.
  • polymorphic form 310 reference to a particular polymorphic form means that the polymorphic form includes less than 10%, preferably less than 5%, more preferably less than 3%, most preferably less than 1% by weight of any other physical forms of the Compound EGFRi.
  • free form refers to the compound per se without salt formation or association with a solvent (e.g., solvate; if solvent water - hydrate).
  • a solvent e.g., solvate; if solvent water - hydrate.
  • FIG. 1. illustrates the x-ray powder diffraction patterns of the crystalline free form A of compound
  • FIG. 2. illustrates the differential scanning calorimetry (DSC) and the thermo gravimetric analysis (TGA) of the crystalline free form A of compound
  • FIG. 3. illustrates the microscopic picture of the crystalline free form A of compound
  • FIG. 4. illustrates the x-ray powder diffraction patterns of the crystalline free form B of compound
  • FIG. 5. illustrates the differential scanning calorimetry (DSC) of the crystalline free form B of compound
  • FIG. 6. illustrates the differential scanning calorimetry (DSC) of the crystalline free form B 355 of compound
  • FIG. 7. illustrates the thermo gravimetric analysis (TGA) of the crystalline free form B of compound
  • FIG. 8. illustrates the x-ray powder diffraction patterns of the crystalline mesylate form A of compound
  • FIG. 9. illustrates the differential scanning calorimetry (DSC) and the thermo gravimetric analysis (TGA) of the crystalline mesylate form A of compound
  • FIG. 10 illustrates the microscopic picture of the crystalline mesylate form A of compound (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
  • FIG. 1 illustrates the x-ray powder diffraction patterns of the crystalline mesylate form B of compound
  • FIG. 12 illustrates the differential scanning calorimetry (DSC) and the thermogravimetric analysis (TGA) of the crystalline mesylate form B of compound
  • FIG. 13 illustrates the microscopic picture of the crystalline mesylate form B of compound (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
  • FIG. 14 illustrates the x-ray powder diffraction patterns of the crystalline mesylate form C of 395 compound
  • FIG. 15. illustrates the differential scanning calorimetry (DSC) and the thermogravimetric 400 analysis (TGA) of the crystalline mesylate form C of compound (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
  • FIG. 16 illustrates the x-ray powder diffraction patterns of the amophous form, free form A 405 and free form B of compound
  • FIG. 17 illustrates the x-ray powder diffraction patterns of the HC1 salt form of compound 410 (R,E)-N-(7-chloro- 1 -(1 -(4-(dimethylamino)but-2-enoyl)azepan-3-yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
  • FIG. 18 illustrates the differential scanning calorimetry (DSC) and the thermogravimetric analysis (TGA) of the HC1 salt form of compound
  • the disclosure relates to crystalline forms of the Compound EGFRi, which are described and characterized herein.
  • the Compound EGFRi act as inhibitor of EGFR. It can be employed in different forms for use in the treatment of cancer diseases.
  • the forms of the compound have pronounced different characteristics and can thus be appropriately selected when, for 425 example, formulating the compound in the pharmaceutical formulation.
  • the Compound EGFRi exhibits EGFR kinase activity and can be thus, particularly in specific forms as disclosed herein, used for treating proliferative disease. Inhibition of cell proliferation can be measured using methods known to those skilled in the art. For example, a
  • Viability Assay which is commercially available from Promega (Madison, Wis.). That assay determines the number of viable cells in culture based on quantitation of ATP present, which is an indication of metabolically active cells. See Crouch et al (1993) J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677.
  • the assay may be conducted in 96- or 384-well format,
  • 440 viable cells present in culture Data can be recorded by luminometer or CCD camera imaging device.
  • the luminescence output is expressed as relative light units (RLU).
  • Inhibition of cell proliferation may also be measured using colony formation assays known in the art.
  • the Compound EGFRi can be used in a method of treating a condition mediated by EGFR in a subject suffering therefrom, comprising administering to the subject a
  • the condition is a cell proliferative disease.
  • the condition is cancer, for example, non-small cell lung cancer ( SCLC), head and neck cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, gastric cancer, glioma and prostate cancer.
  • SCLC non-small cell lung cancer
  • NSCLC non-small cell lung cancer
  • Treatment of the cell proliferative disorder by administration of the Compound EGFRi results in an observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition of cancer cell infiltration into peripheral organs including the
  • Compound EGFRi may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or
  • 460 symptoms may also be felt by the patient.
  • TDP time to disease progression
  • Metastasis can be determined by staging tests and by bone scan and tests for calcium level and other enzymes to determine spread to the bone. CT scans can also be done to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastasis to the lungs and liver, respectively. Other routine methods for monitoring the
  • TRUS transrectal ultrasonography
  • TRNB transrectal needle biopsy
  • the therapeutically effective dosage of the compound, the pharmaceutical composition, or the combinations of the compound with other pharmaceutically active ingredients e.g other
  • 475 anticancer drugs thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed can also depend on the route of administration, and the seriousness of the condition
  • An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.5 mg to about 100 mg, conveniently administered, e.g. in divided 485 doses up to four times a day or in retard form.
  • administration comprise from ca. 1 to 50 mg active ingredient.
  • a therapeutic amount or dose of the compound of the present disclosure may range from about 0.1 mg/kg to about 500 mg/kg, alternatively from about 1 to 490 about 50 mg/kg.
  • treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses (such as two, three, or four times daily).
  • Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.
  • a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to 500 the desired level, treatment should cease.
  • the subject may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • crystalline free form A Tm 114.6 °C
  • crystalline free form B Tm 1 12.2 °C
  • crystalline mesylate form A Tm 168.9 °C
  • the free form dihydrate Form B is more stable in 0.1 N HC1 than the mesylate
  • Trihydrate form B Free form B can be prepared in good yield by using a simple, inexpensive and mild procedure, and in excellent purity retaining consistent water content from 30% to 90% relative humidity at 25°C. Also the dihydrate allows the use of choice of analytical methods, especially the X-ray methods, the usage of which permits clear and straightforward
  • Crystalline mesylate form A of the compound is a monohydrate. It is more stable in bulk than free forms, but more hygroscopic.
  • the best form for handling and formulating in pharmaceutical composition is mesylate form B.
  • the mesylate form B is a trihydrate. It is more stable in solid state than free forms and mesylate form A, more soluble in aqueous media, and better compatible with certain excipients.
  • Compound EGFRi HC1 salt 525 form behaves better in liquid formulations. It does not form gels with solvents.
  • 535 group consisting of : 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ⁇ 0.30, at a temperature of about 22°C.
  • a representative XRPD pattern for the free form A is presented in the following table:
  • the Compound EGFRi crystalline free form B has the following characteristic x-ray powder diffraction pattern: 1 1.24 ⁇ 0.30, 14.68 ⁇ 0.30, 15.66 ⁇ 0.30, 18.64 ⁇ 0.30, 19.63 ⁇ 0.30,
  • PXRD x-ray powder diffraction pattern
  • Compound EGFRi crystalline free form B may be characterized by a x-ray powder
  • a representative XRPD pattern for the free form B is presented in the following table:
  • a representative XRPD pattern for the mesylate form A is presented in the following table:
  • PXRD x-ray powder diffraction pattern
  • 585 selected from the group consisting of 1 1.76, 13.832, 14.41 , 15.9 17.65, 18.79, 21.46, 21.83, 22.30, 23.82, 24.51 , 24.89, 25.57, 26.66 and 27.77 ⁇ 0.30, at a temperature of about 22°C.
  • a representative XRPD pattern for the mesylate form B is presented in the following table:
  • a representative XRPD pattern for the mesylate form C is presented in the following table:
  • a representative XRPD pattern for the HCl salt form is presented in the following table:
  • the form of the Compound EGFRi is provided in substantially pure form.
  • This crystalline form of the Compound EGFRi in substantially pure 625 form may be employed in pharmaceutical compositions which may optionally include one or more other components selected, for example, from the group consisting of excipients, carriers, and one of other active pharmaceutical ingredients active chemical entities of different molecular structure.
  • the crystalline form has substantially pure phase homogeneity as indicated by less than 10%, preferably less than 5 %, and more preferably less than 2 % of the total peak area in the experimentally measured PXRD pattern arising from the extra peaks that are absent from the simulated PXRD pattern.
  • a composition consisting essentially of any one of the forms presented above, except when the form is mesylate form B, the compound contains other 640 forms as well.
  • the composition of this embodiment may comprise at least 90 weight % of the respective form, based on the weight of the Compound EGFRi in the composition.
  • reaction impurities and/or processing impurities may be determined by analytical techniques known in the art, such as, for example, chromatography, nuclear 645 magnetic resonance spectroscopy, mass spectrometry, or infrared spectroscopy.
  • the disclosed forms of the Compound EGFRi can be formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical compositions can be formulated for oral, intravenous, intradermal,
  • the pharmaceutical composition is formulated for oral administration.
  • the pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules, capsules containing liquids, powders,
  • suppositories emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • compositions can be formulated for immediate release, sustained release, or controlled release of the compounds of the invention.
  • suitable pharmaceutical excipients include, for example, 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); for example, 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); for example, a) diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or
  • 660 tablets also c) binders (e.g., magnesium aluminum silicate, starch, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone); if desired d) disintegrants, e.g., starches, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners.
  • binders e.g., magnesium aluminum silicate, starch, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone
  • disintegrants e.g., starches, alginic acid or its sodium salt, or effervescent mixtures
  • absorbents, colorants, flavors and sweeteners e.g., absorbents, colorants, flavors and sweeteners.
  • Additional suitable pharmaceutical excipients can be liquids, such as water and oils,
  • 665 including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil,
  • soybean oil, mineral oil, sesame oil and the like can be used.
  • the pharmaceutical excipients can be saline, starch, talc, keratin, colloidal silica, urea and the like.
  • auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used.
  • the pharmaceutical excipients can be saline, starch, talc, keratin, colloidal silica, urea and the like.
  • auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used.
  • the pharmaceutical excipients can be saline, starch, talc, keratin, colloidal silica, urea and the like.
  • auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used.
  • the pharmaceutical excipients can be saline, starch, talc, keratin, colloidal silica, urea and the like.
  • compositions are sterile when administered to a subject.
  • Water is a 670 useful excipient when the compound of the invention is administered intravenously.
  • the present compositions can also contain minor amounts of wetting or emulsifying agents, or pH modifier.
  • pH buffering agent can improve the stability of the Compound EGFRi, or its 675 specific forms, namely free Form A, free Form B, mesylate from A, mesylate form B, mesylate form C, amorphous form and HC1 salt form.
  • modulation of the pH through the use of pH modifiers can be very effective in improving solid dosage form stability.
  • liquid dosage forms Particularly beneficial is when pH is above pH 1 and below pH 9, particularly above pH 1 and below pH 6.8. In this range the
  • Acidic or basic ingredients can be intentionally added to a formulation to modify the pH and increase composition's stability, for example measured by amount of the Compound EGFRi degradation products or impurities.
  • pH modifier in oral solid dosage forms can be
  • the solution used for the freeze drying process can usually be buffered to the desired pH using common buffering systems and buffer concentrations used for solution formulations.
  • the pH of the reconstituted lyophile should generally correspond to solution pH prior to freeze drying.
  • Suitable pH modifiers used to enhance the stability of the forms are for example acids, bases or their salts (e.g. citric acid, fumaric acid, acetic acid, HC1, boric acid, phosphoric acid, 700 malic, formic, lactic, succinic acid, malic acid, picolinic acid or succinic acid, etc. ,or
  • pH can be measured by a pH meter using a glass electrode or micro pH electrode.
  • pH can be measured by any other suitable means, like for example neutralization
  • Compound EGFRi should be between pH 1 and pH 9, preferably between pH 2 and pH 6.8, particularly around pH 4.5; measured at 20°C.
  • a sample can be dissolved or suspended in water to prepare 1 w/w % solution or suspension of the Compound EGFRi and
  • the pH of said 1 % solution or suspension of the Compound EGFRi should be between H 1 and pH 9, preferably between pH 2 and pH 6.8, particularly around pH 4.5; measured at 25°C.
  • compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily
  • compositions can contain one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • sweetening agents such as fructose, aspartame or saccharin
  • flavoring agents such as peppermint, oil of wintergreen, or cherry
  • coloring agents such as peppermint, oil of wintergreen, or cherry
  • preserving agents to provide a pharmaceutically palatable preparation.
  • Liquid dosage forms for oral administration include pharmaceutically
  • liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular,
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • the compositions can be formulated for parenteral
  • parenteral dosage forms are administered in the form of sterile or sterilizable injectable solutions, suspensions, dry and/or lyophylized products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection (reconstitutable powders) and emulsions.
  • Vehicles used in such dosage forms include, but are not limited to, Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles.
  • compositions can be prepared according to conventional mixing, granulating or coating
  • compositions can contain, in one embodiment, from about 0.1 percent to about 99 percent; and in another embodiment from about 1 percent to about 70 percent of the compound of the invention by weight or volume.
  • the present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the compounds of the present invention as active ingredients, since water may facilitate the degradation of certain compounds.
  • Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.
  • composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits.
  • suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e. g., vials), blister packs, and strip packs.
  • compositions further comprise one or more additional therapeutic agents.
  • the compounds of the invention and the additional therapeutics agent(s) may act additively or synergistically.
  • Crystalline forms may be prepared by a variety of methods, including for example, crystallization or recrystallization from a suitable solvent, sublimation, growth from a melt, solid state transformation from another phase, crystallization from a supercritical fluid, and jet spraying. Techniques for crystallization or recrystallization of crystalline forms from a suitable solvent, sublimation, growth from a melt, solid state transformation from another phase, crystallization from a supercritical fluid, and jet spraying. Techniques for crystallization or recrystallization of crystalline forms from a
  • solvent mixture include, for example, evaporation of the solvent, decreasing the temperature of the solvent mixture, crystal seeding a supersaturated solvent mixture of the molecule and/or salt, freeze drying the solvent mixture, and addition of antisolvents (countersolvents) to the solvent mixture.
  • High throughput crystallization techniques may be employed to prepare crystalline forms including polymorphs. Crystals of drugs, including polymorphs,
  • the compound may be solubilized into a first solvent to afford a solution, followed by the addition of an antisolvent to decrease the solubility of the compound in the solution and to afford the formation of crystals.
  • An antisolvent is a solvent
  • a compound is suspended and/or stirred in a suitable solvent to afford a slurry, which may be heated to promote dissolution.
  • a suitable solvent to afford a slurry, which may be heated to promote dissolution.
  • slurry means a saturated solution of the compound, which may also contain an additional amount of the compound to afford a heterogeneous mixture of the compound and a solvent at a given temperature.
  • seed 790 may be added to any crystallization mixture to promote crystallization. Seeding may be employed to control growth of a particular polymorph or to control the particle size distribution of the crystalline product. Accordingly, calculation of the amount of seeds needed depends on the size of the seed available and the desired size of an average product particle as described, for example, in “Programmed Cooling of Batch Crystallizers," J.W.
  • seeds of small size are needed to control effectively the growth of crystals in the batch.
  • Seed of small size may be generated by sieving, milling, or micronizing of large crystals, or by micro-crystallization of solutions. Care should be taken that milling or micronizing of crystals does not result in any change in crystallinity form the desired crystal form (i.e.,
  • a cooled crystallization mixture may be
  • the isolated solids may be washed with a suitable solvent, such as cold recrystallization solvent, and dried under a nitrogen purge to afford the desired crystalline form.
  • a suitable solvent such as cold recrystallization solvent
  • the isolated solids may be analyzed by a suitable spectroscopic or analytical technique, such as solid state nuclear magnetic resonance, differential scanning
  • the resulting crystalline form is typically produced in an amount of greater than about 70 weight % isolated yield, preferably greater than 90 weight % isolated yield, based on the weight of the compound originally employed in the
  • the product may be comilled or passed through a mesh screen to
  • Crystalline forms may be prepared directly from the reaction medium of the final process for preparing EGFRi. This may be achieved, for example, by employing in the final process step a solvent or a mixture of solvents from which Compound EGFRi may be crystallized.
  • crystalline forms may be obtained by distillation or solvent addition techniques.
  • Suitable solvents for this purpose include, for example, the aforementioned nonpolar solvents and polar solvents, including protic polar solvents such as alcohols, and aprotic polar solvents such as ketones. It is also possible to obtain one form of the compound and then transform it into another form, or mixture thereof, by the methods described above.
  • the presence of more than one polymorph in a sample may be determined by techniques such as x-ray powder diffraction (PXRD) or solid state nuclear magnetic resonance spectroscopy. For example, the presence of extra peaks in the comparison of an
  • experimentally measured PXRD pattern with a simulated PXRD pattern may indicate more 825 than one polymorph in the sample.
  • the simulated PXRD may be calculated from single crystal x-ray data, see Smith, D.K., "A FORTRAN Program for Calculating X-Ray Powder Diffraction Patterns ,” Lawrence Radiation Laboratory, Livermore, California, UCRL-7196 (April 1963) or TOP AS program (Total Pattern Analysis Solution, available through Brucker AXS Inc.).
  • the pH of 1% solution or suspension of the drug substance in water was recorded at 25°C. Mettler Toledo pH-meter with Orion Micro-pH combination electrode was used.
  • Solubility was determined using UPLC. 20 mg of drug substance were equilibrated in 1 mL media at room temperature for 24 h. The solution/suspension was filtrated and the saturated solution was analyzed with UPLC.
  • an X-ray diffraction pattern may be obtained with a measurement error that is dependent upon the measurement conditions employed.
  • intensities in a X-ray diffraction pattern may fluctuate depending upon measurement conditions employed.
  • relative intensities may also vary depending upon experimental conditions and, accordingly, the exact order of intensity should not be taken into account.
  • a measurement error of diffraction angle for a conventional X-ray diffraction pattern is typically about 5% or less, and such degree of measurement error should be taken into account as pertaining to the aforementioned diffraction angles.
  • crystal forms of the instant invention are not limited to the crystal forms that provide X-ray diffraction patterns completely identical to the X-ray diffraction patterns depicted in the accompanying Figures disclosed herein. Any crystal forms that provide X- ray diffraction patterns substantially identical to those disclosed in the accompanying Figures fall within the scope of the present disclosure. The ability to ascertain substantial identities of X-ray diffraction patterns is within the purview of one of ordinary skill in the art.
  • X-ray generator power 40kV, 40 mA (1600 Watts)
  • Measuring slice 0.3 mm and 0.2 mm
  • Source slit Fixed, 0.5mm
  • Sample amount of ca 2-5 mg was placed on an objective slide and centered in the X-ray beam.
  • the DSC instrument used to test the crystalline forms was a TA Instrument® Differential Scanning Calorimetry Model 2910, TA Instruments® Modulated Differential Scanning Calorimetry Model 2920, TA Discovery series DSC or TA Instruments® Modulated
  • the DSC cell/sample chamber was purged with 100 ml/min of ultra-high purity nitrogen gas.
  • the instrument was calibrated with high purity indium. The accuracy of the measured sample temperature with this method is within about ⁇ 1°C, and the heat of fusion can be measured within a relative error of about ⁇ 5%.
  • the sample was placed into an open aluminum DSC pan and measured against an empty reference pan. About 2-6 mg of sample powder was placed into the bottom of the pan and lightly tapped down to make contact with the pan. The weight of the sample was measured accurately and recorded to a hundredth of a milligram.
  • the instrument was programmed to 885 heat at 10°C. per minute in the temperature range between 25 and 300°C.
  • the heat flow which was normalized by a sample weight, was plotted versus the measured sample temperature. The data were reported in units of watts/gram ("W/g"). The plot was made with the endothermic peaks pointing down. The endothermic melt peak was evaluated 890 for extrapolated onset temperature, peak temperature, and heat of fusion in this analysis.
  • the TGA instruments used to test the crystalline forms was TA instruments, High Resolution Thermogravimetric Analyzer Q5000. 0.5-1.0 mg of test substance was weighted and placed 895 on the open sample pan.
  • thermogram was recorded as follows: the sample was loaded into the furnace, the temperature equilibrated to 30 °C and heated to 300 °C at a heating rate of 10 °C/min, under a flow of nitrogen at 25 m L/min. The instrument was calibrated for temperature with nickel and aluminum, and calibrated for weight with a 100 mg standard.
  • Test substance was dispersed in one drop of silicon oil on a glass slide.
  • Step A A stirred solution of (S)-iert-butyl 3-aminopiperidine-l-carboxylate (0.500 g, 2.49 mmol), l-fluoro-4-methyl-2-nitrobenzene (0.387 g, 2.49 mmol) and
  • N,N-diisopropylethylamine (0.482 g, 3.74 mmol) in DMF under argon was heated to 1 10°C for 6 h (reaction completion monitored by TLC). The mixture was diluted with water and
  • Step B To a stirred solution of I-15a (0.550 g, 1.64 mmol) in MeOH (35mL) was added
  • Step A (R)-tert-butyl 3-((2-chloro-6-nitrophenyl)amino)azepane-l-carboxylate (I-26a) was prepared following procedures analogous to 1-15, Step A, using the appropriate starting materials.
  • Step B A mixture of I-26a (7.5 g, 19.5 mmol) and Zn (12.8 mg, 195 mmol) in AcOH
  • Step C The title compound (Intermediate 26) was prepared from I-26b following procedures analogous to 1-15, Step C. 3 ⁇ 4-NMR (400MHz, CDC1 3 ): d 7.34-7.26 (m, 1H), 7.04-6.97 (m, 2H), 6.05-5.85 (m, 1H), 5.84-5.72 (m, 1H), 5.50-5.37 (m, 0.5H), 5.10-4.80(m,
  • Step A A mixture of 2-methylisonicotinic acid (3.371 g, 24.6 mmol) and 2-(7-aza-lH- benzotriazole-l -yl)-l ,l ,3,3-tetramethyluronium hexafiuorophosphate (9.345 g, 24.6 mmol) in
  • Step B A solution of I-27a (8.62 g, 16.4 mmol) in MeOH (67 mL) was treated with HCI
  • the reaction mixtures contained 1 ⁇ peptide substrate, 10 ⁇ ATP, and 0.036 nM EGFR(L858R/T790M) in the reaction buffer (50 mM HEPES pH 7.1 , lOmM MgC12, 0.01 % BSA, 1 mM TCEP and 0.1
  • IC50 values were determined by 12-point (from 50 to 0.000282 ⁇ ) inhibition curves in duplicate under the assay conditions as described above.
  • no-preincubation condition the compound was added to the assay solution containing ATP and peptide, and the reaction was initiated by addition of enzyme.
  • pre-incubation conditions the compound was added to the assay solution containing enzyme and peptide, and pre-incubated
  • NIH/3T3 cell lines expressing human EGFR WT, L858R, and
  • EGFR WT-expressing cells were induced with 50 ng/ml EGF (Preprotech #AF-100-15) for 5 minutes before lysis.
  • the media was removed and cells were lysed in 25 ⁇ of Lysis buffer containing protease and phosphatase inhibitors (1% Triton X-100, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, IX complete cocktail inhibitor (Roche #11 697 498 001), IX Phosphatase Inhibitor Cocktail Set II and Set III (Sigma #P5726 and #P0044)).
  • protease and phosphatase inhibitors 1% Triton X-100, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, IX complete cocktail inhibitor (Roche #11 697 498 001), IX Phosphatase Inhibi
  • Penicillin/Streptomycin (Hyclone #SH30236.01). The cells were harvested with 0.25%
  • Phospho-EGFR (Yl 173) ELISA.
  • Solid white high-binding ELISA plates (Greiner #781074) 1085 were coated with 5 ug/ml anti-EGFR capture antibody (R&D Systems #AF231) overnight in 50 mM carbonate/bicarbonate pH 9.5 buffer. Plates were blocked with 1% BSA (Sigma #A7030) in PBS for 1 hour at room temperature. Wash steps were carried out with a Bio-Tek ELx405 SelectTM using 4 cycles of 100 ul TBS-T (20 mM Tris, 137 mM NaCl, 0.05% Tween-20) per well.
  • Control cells treated with EGF were set to 100% maximum response.
  • 10 uM afatinib (BIBW2992) was used to normalize data to 0% of maximum response.
  • the IC50's for each compound in each cell line was calculated using non-linear curve fitting analysis.
  • 1 1 15 compound was transferred to the plate containing cells by using a 50 nl Pin Head device (Perkin Elmer) and the cells were placed back in the incubator for 3 hours. Stimulation of EGFR autophosphorylation was carried out with 10 ng/ml EGF (Preprotech #AF-100-15) for 5 minutes in the tissue culture incubator.
  • Chemiluminescent detection was carried out with SuperSignal ELISA Pico substrate
  • Control cells treated with EGF were set to 100% maximum response.
  • 10 uM afatinib (BIBW2992) was used to normalize data to 0% of maximum response.
  • the IC50's for each compound in each cell line was calculated using non-linear curve fitting analysis.
  • HaCaT cells were maintained in 10% FBS RPMI supplemented with 100 ⁇ g/ml Penicillin/Streptomycin (Hyclone #SH30236.01 ). The cells were harvested with 0.25% Trypsin/EDTA (Hyclone #SH30042.1 ), re-suspended in 5% FBS/DMEM Pen/Strep and
  • Chemiluminescent detection was carried out with SuperSignal ELISA Pico substrate (ThermoScientific #37069). Signal was read on EnVision plate reader using built-in
  • Control cells treated with EGF were set to 100% maximum response.
  • 10 uM afatinib (BIBW2992) was used to normalize data to 0% of maximum response.
  • the IC50's for each compound in each cell line was calculated using non-linear curve fitting analysis.
  • the compound of the disclosure shows an inhibition IC 50 determinations obtained from EGFR target modulation in engineered NIH/3T3 cell lines for L858R/T790M and L858R, 0.01 ⁇ ⁇ and 0.015 ⁇ , respectively. For wt the value was 0.259 ⁇ .
  • L858/T790M H3255 (EGFR L858R), and HEKn (EGFR WT) cell lines were ⁇ . ⁇ , 0.030 ⁇ and 1 .180 ⁇ respectively.
  • Example 2 (i?,£')-N-(7-chloro-l -(l -(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-lH-benzo[d]imidazol-2 -yl)-2-methylisonicotinamide as obtained in Example 1 (1.0 g) was dissolved in acetone (30 mL) by heating to 55°C to form a solution. Methanesulfonic acid (325 ⁇ ) was added to acetone (50 mL), and the methanesulfonic acid/acetone (22.2 mL) was added to the solution
  • crystalline mesylate form A was prepared by dissolving 1 .0g of free form A in 1225 30mL of acetone by heating to 55°C. 325 ⁇ L ⁇ of methansulfonic acid was added to 50mL of acetone and then 22.2mL of methansulfonic acid acetone was added to free form solution at 0.05ml/min. Precipitation was formed during the addtion of methansulfonic acid, and the suspension was allowed to cool to room temperature at 0.5 °C/min. The crystal was collected by filtration and afterwards dried for 4 hours at 40°C under vacuum.
  • 850mg of amorphous form or free form of the EGFRi compound were weighed out in a 1260 20ml vial. 4.25ml of Acetonitrile were added to completely dissolve the compound. To this solution 6.86 ml of 0.6 N HCl were slowly added while stirring the solution. The solution turned yellow and solids precipitated out after 15mins. The solution was stirred for 15 mins and then let to stand without stirring overnight. The solution was filtered and dried under vacuum at 40 °C for 8hrs. A yellow solid was obtained as the final product.
  • Example 8 Evaporation of completely dissolved samples at 25 degree C
  • Example 10 Stability data for free form B and mesylate form B, both crystalline forms
  • TIs are analyzed by UPLC (method as described above). They are calculated as area-% products or against external standard 1%).
  • compositions of the excipient mixtures [mass-%]
  • Mixture 1 Lactose, spray dried (53.7%), Microcrystalline cellulose PH102 (40%),
  • Crospovidone XL (5%), Aerosil (0.3%), Mg stearate (1%)
  • Example 11 Stability data for free form A and mesylate form A, both crystalline forms
  • TIs are analyzed by UPLC (method as described above). They are calculated as area-% products or against external standard 1%).
  • Examples 10 and 1 1 show that stability of the EGFRi forms deteriorates significantly at pH below 1 and above 6.8, and even more so above pH 9. Therefore, it is best to modulate pH in
  • a pH modifier can be used for example buffers as exemplified in the examples. Excipients attaining the same effect are expected to work as well. Best stability is achieved if the micro environment of the EGFRi is between pH 2 and pH 4.7 ⁇ 0.2, particularly pH 2 and
  • PK studies for mesylate form and HCl salt form were conducted in rat. Both forms were 1350 dosed orally at 10 and 30 mg/kg in 0.5% MC/Tween 80 formulation. Both salt forms were soluble at both dose levels in the formulation. Plasma exposures of both forms and its active metabolite were determined. The mesylate form showed very similar PK profiles in the rats at both 10 and 30 mg/kg as those of HCL salt form. The exposures of both parent compound forms and active metabolite are summarized below.
  • the mesylate crystalline forms show dose proportionality from 10 to 30mg/kg, and the exposure is comparable to HCl salt.

Abstract

The present disclosure generally discloses polymorphic forms of (R,E)-N-(7-chloro-1-(1-(4-(dimethylamino)but-2-enoyl)azepan-3- yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (Compound EGFRi). A pharmaceutical composition comprising the form(s), as well of methods of using the form(s) in the treatment of cancer indication, and methods for obtaining such forms are also disclosed.

Description

EGFR INHIBITOR FORMS
FIELD OF INDUSTRIAL APPLICABILITY
The present disclosure generally relates to polymorphic form(s) of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi). The present disclosure also generally relates to a pharmaceutical composition comprising the form(s), as well of methods of using the form(s) in the treatment of cancer indications, and methods for obtaining such forms.
BACKGROUND OF THE DISCLOSURE
The epidermal growth factor receptor (EGFR, Erb-Bl) belongs to a family of proteins involved in the proliferation of normal and malignant cells. Overexpression of EGFR is found in over 70 percent of human cancers, including without limitation non-small cell lung carcinomas (NSCLC), breast cancers, gliomas, squamous cell carcinoma of the head 15 and neck, and prostate cancer. The identification of EGFR as an oncogene has led to the development of anti-EGFR targeted molecules, such as gefitinib and erlotinib.
SUMMARY OF THE DISCLOSURE
The object of the present disclosure was to provide novel forms of a more potent EGFR inhibitor, the inhibitor of formula 1 :
Figure imgf000002_0001
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi) that has also a better therapeutic index. The forms exhibit improved hygroscopicity, stability and/or solubility. In one embodiment the form affords better handling characteristic. In another embodiment, the forms can be better used in formulating pharmaceutical compositions. The novel forms of the Compound can be mixed with specific excipients, or in alternative be formulated in compositions free of other specific excipients, to further bolster their advantageous characteristics. Particularly good stability is achieved when the Compound EGFRi, or its specific forms are kept at a pH between 1 and 9, particularly between 1 and 6.8. The compound and its form exhibit the best stability at about pH 4.5.
Embodiments of these crystalline forms include those characterized herein as Compound EGFRi free form A, free form B, mesylate form A, mesylate form B, mesylate form C, amorphous form and HCl salt form. The names used herein to characterize a specific form, e.g. "free form A, free form B, mesylate form A, mesylate form B, mesylate form C, amorphous form or HCl salt form" etc., should not be considered limiting with respect to any other substance possessing similar or identical physical and chemical characteristics, but rather it should be understood that these designations are mere identifiers that should be interpreted according to the characterization information also presented herein. Specifically, the present disclosure provides the following aspects, advantageous features and specific embodiments, respectively alone or in combination, as listed in the following items:
1. A crystalline form of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprising:
(i) free form A,
(ii) free form B,
(iii) mesylate form A,
(iv) mesylate form B,
(v) mesylate form C,
(vi) HCl salt form, or
(vii) amorphous form,
wherein when the crystalline form of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises mesylate form B, the crystalline form further comprises also at least one of the forms selected from the group of free form A, free form B, mesylate form A, mesylate form C and HCl salt form.
2. The crystalline form according to item 1 consisting essentially of
(i) free form A,
(ii) free form B,
(iii) mesylate form A,
(v) mesylate form C, or
(vi) HCl salt form. 3. The crystalline form according to item 2, wherein said forms are in substantially pure form.
4. The crystalline form according to any one of items 1 to 3, wherein x-ray powder diffraction pattern comprises 4 or more 2Θ values (CuKa) selected from the group consisting 75 of:
(a) 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30 for free form A,
(b) 1 1.24, 14.68, 15.66, 18.64, 19.63, 20.05, 20.31 , 22.41 , 24.91 , 25.43, 26.03, 27.18 and 28.07± 0.30 for free form B,
80 (c) 9.12, 12.19, 16.25, 17.38, 18.42, 19.91 , 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ±
0.30 for mesylate form A,
(d) 1 1.76, 13.832, 14.41 , 15.9 17.65, 18.79, 21.46, 21.83, 22.30, 23.82, 24.51 , 24.89, 25.57, 26.66 and 27.77 ± 0.30 for mesylate form B,
(e) 1 1.51 , 13.19, 16.23, 17.50, 19.14, 23.15, 23.80, 24.65, 25.81 , 26.06, 26.70 and 28.97 ± 85 0.30 for mesylate form C, or
(f) 14.89, 19.69, 25.61 , 26.13 and 27.78 ± 0.30 for HC1 salt form,
all at a temperature of about 22°C.
5. The crystalline form according to item 4, wherein x-ray powder diffraction pattern
90 comprises five or more 2Θ values selected from the group.
6. The crystalline form according to item 4, wherein x-ray powder diffraction pattern comprises all 2Θ values selected from the group for a respective form.
95 7. A crystalline form of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide having a X-ray diffraction spectrum essentially the same as the X-ray powder diffraction spectrum shown in FIG. 1 , FIG. 4, FIG. 8, FIG. 1 1 , FIG. 14, or FIG. 17.
100
8. A crystalline form of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide having a differential scanning calorimetry (DSC) thermogram essentially the same as that shown in shown in any one of figures FIG. 2, FIG. 5, FIG. 6, FIG. 105 9, FIG. 12, FIG. 15, or FIG 18.
9. A crystalline form of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide having a thermo gravimetric analysis (TGA) diagram
1 10 essentially the same as that shown in any one of figures FIG. 2, FIG. 7, FIG. 9, FIG. 12, FIG.
15, or FIG 18.
10. A pharmaceutical composition comprising the crystalline form according to any one of items 1 to 9.
1 15 11. The pharmaceutical composition according to item 10, further comprising a pharmaceutically acceptable carrier or diluent.
12. The pharmaceutical composition according to item 10 or 1 1 , wherein the crystal form is 120 as defined in any one of items 4 to 6.
13. The pharmaceutical composition according to item 12, wherein said form is free form A, free form B, mesylate form A, mesylate form C or HC1 salt form and wherein x-ray powder diffraction pattern comprises four 2Θ values (CuKa) selected from the group consisting of:
125 (a) 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30 for free form A,
(b) 1 1.24, 14.68, 15.66, 18.64, 19.63, 20.05, 20.31 , 22.41 , 24.91 , 25.43, 26.03, 27.18 and 28.07± 0.30 for free form B,
(c) 9.12, 12.19, 16.25, 17.38, 18.42, 19.91 , 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 130 0.30 for mesylate form A,
(e) 1 1.51 , 13.19, 16.23, 17.50, 19.14, 23.15, 23.80, 24.65, 25.81 , 26.06, 26.70 and 28.97 ± 0.30 for mesylate form C, or
(f) 14.89, 19.69, 25.61 , 26.13 and 27.78 ± 0.30 for HC1 salt form,
all at a temperature of about 22°C, respectively.
135
14. The pharmaceutical composition according to item 12, wherein said form is free form A, free form B, mesylate form A, mesylate form C or HC1 salt form and wherein x-ray powder diffraction pattern comprises five 2Θ values (CuKa) selected from the group consisting of:
(a) 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30 for 140 free form A,
(b) 1 1.24, 14.68, 15.66, 18.64, 19.63, 20.05, 20.31 , 22.41 , 24.91 , 25.43, 26.03, 27.18 and 28.07± 0.30 for free form B,
(c) 9.12, 12.19, 16.25, 17.38, 18.42, 19.91 , 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 0.30 for mesylate form A,
145 (e) 1 1.51 , 13.19, 16.23, 17.50, 19.14, 23.15, 23.80, 24.65, 25.81 , 26.06, 26.70 and 28.97 ± 0.30 for mesylate form C, or
(f) 14.89, 19.69, 25.61 , 26.13 and 27.78 ± 0.30 for HC1 salt form,
all at a temperature of about 22°C, respectively.
150 15. The pharmaceutical composition according to item 12, wherein said form is free form A, free form B, mesylate form A, mesylate form C or HC1 salt form and wherein x-ray powder diffraction pattern comprises all 2Θ values (CuKa) selected from the group consisting of: (a) 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30 for free form A,
155 (b) 1 1.24, 14.68, 15.66, 18.64, 19.63, 20.05, 20.31 , 22.41 , 24.91 , 25.43, 26.03, 27.18 and 28.07± 0.30 for free form B,
(c) 9.12, 12.19, 16.25, 17.38, 18.42, 19.91 , 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 0.30 for mesylate form A,
(e) 1 1.51 , 13.19, 16.23, 17.50, 19.14, 23.15, 23.80, 24.65, 25.81 , 26.06, 26.70 and 28.97 ± 160 0.30 for mesylate form C, or
(f) 14.89, 19.69, 25.61 , 26.13 and 27.78 ± 0.30 for HC1 salt form,
all at a temperature of about 22°C, respectively.
16. A pharmaceutical composition comprising
165 (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide and a pH modifier.
17. A pharmaceutical composition comprising mesylate form B and a pH modifier.
170 18. A pharmaceutical composition comprising amorphous form and a pH modifier.
19. The pharmaceutical composition according to any one of items 10 to 16, further comprising a pH modifier.
175 20. The pharmaceutical composition according to any one of items 16 to 19, wherein the pH modifier is pharmaceutically acceptable acid, base, salt or mixtures thereof.
21. The pharmaceutical composition according to any one of items 10 to 20, wherein the composition is liquid.
180
22. The pharmaceutical composition according to any one of items 10 to 21 , wherein the (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide is in any form as defined in any one of items 1-9.
185 23. The pharmaceutical composition according to any one of items 10 to 22, wherein the pH of the formulation is between pH 1 and pH 9, measured at 25°C.
24. The pharmaceutical composition according to any one of items 10 to 22, wherein the pH of the formulation is between pH 2 and pH 9, measured at 25°C.
190
25. The pharmaceutical composition according to any one of items 10 to 22, wherein the pH of the formulation is between pH 2 and pH 6.8, measured at 25°C.
26. The pharmaceutical composition according to any one of items 10 to 22, wherein the pH 195 of the formulation is between pH 2 and pH 4.7, measured at 25°C. 27. The pharmaceutical composition according to any one of items 10 to 22, wherein the pH of the formulation is about 4.5, or pH is 4.5, measured at 25°C.
200 28. A pharmaceutical formulation comprising
(R,E)-N-(7-chloro-l-(l -(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-lH-benzo[d]imidazol-2 -yl)-2-methylisonicotinamide, wherein 1 g of the pharmaceutical formulation dissolved, suspended or slurried in 1 mL of water has pH of between pH 1 and pH 9.
205 29. A pharmaceutical formulation comprising
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide, wherein 1 w/w % solution or suspension of the pharmaceutical formulation, calculated based on the
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 210 -yl)-2-methylisonicotinamide in the pharmaceutical formulation, has pH of between pH 1 and pH 9.
30. The pharmaceutical formulation according item 28 or 29, wherein pH is between pH 1 and pH 6.8, measured at 25°C.
215
31. The pharmaceutical formulation according item 28 or 29, wherein pH is between pH 2 and pH 6.8, measured at 25°C.
32. The pharmaceutical formulation according item 28 or 29, wherein pH is between pH 2 220 and pH 4.7, measured at 25°C.
33. The pharmaceutical formulation according item 28 or 29, wherein pH is about pH 4.5, measured at 25°C.
225 34. Crystalline form according to any one of items 1 to 9, pharmaceutical composition
according to any one of items 10 to 27, or pharmaceutical formulation according to any one of items 28 to 33, for use as a medicine.
35. Crystalline form according to any one of items 1 to 9, pharmaceutical composition 230 according to any one of items 10 to 27, or pharmaceutical formulation according to any one of items 28 to 33, for use in the treatment of proliferative disease.
36. Crystalline form according to any one of items 1 to 9, pharmaceutical composition according to any one of items 10 to 27, or pharmaceutical formulation according to any one
235 of items 28 to 33 for use according to item 35, wherein the proliferative disease is non-small cell lung cancer, head and neck cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, gastric cancer, glioma or prostate cancer. 37. Crystalline form according to any one of items 1 to 9, pharmaceutical composition 240 according to any one of items 10 to 27, or pharmaceutical formulation according to any one of items 28 to 33 for use according to item 35 or 36, wherein the proliferative disease is non-small cell lung cancer.
38. Crystalline form according to any one of items 1 to 9, pharmaceutical composition 245 according to any one of items 10 to 27, or pharmaceutical formulation according to any one of items 28 to 33 for use according to any one of items 35 to 37, wherein the form is administered to a human.
39. The pharmaceutical composition according to any one of items 16 to 27, or the
250 pharmaceutical formulation according to any one of items 28 to 33, wherein x-ray powder diffraction pattern of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2Θ values, 5 2Θ values , 6 2Θ values, 7 2Θ values or all 2Θ values (CuKa) selected from the group consisting of: 5.46, 14.82, 15.84, 18.67, 20.42, 255 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30 (free form A).
40. The pharmaceutical composition according to any one of items 16 to 27, or the
pharmaceutical formulation according to any one of items 28 to 33, wherein x-ray powder diffraction pattern of
260 (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2Θ values, 5 2Θ values , 6 2Θ values, 7 2Θ values or all 29 values (CuKa) selected from the group consisting of: 1 1.24, 14.68, 15.66, 18.64, 19.63, 20.05, 20.31 , 22.41 , 24.91 , 25.43, 26.03, 27.18 and 28.07± 0.30 (free form B).
265 41. The pharmaceutical composition according to any one of items 16 to 27, or the
pharmaceutical formulation according to any one of items 28 to 33, wherein x-ray powder diffraction pattern of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2Θ values, 5 2Θ values , 6 2Θ values, 7 2Θ values or 270 all 2Θ values (CuKa) selected from the group consisting of: 9.12, 12.19, 16.25, 17.38, 18.42, 19.91 , 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 0.30 (mesylate form A).
42. The pharmaceutical composition according to any one of items 16 to 27, or the
pharmaceutical formulation according to any one of items 28 to 33, wherein x-ray powder 275 diffraction pattern of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2Θ values, 5 2Θ values , 6 2Θ values, 7 2Θ values or all 2Θ values (CuKa) selected from the group consisting of: 1 1.76, 13.832, 14.41 , 15.9 17.65, 18.79, 21.46, 21.83, 22.30, 23.82, 24.51 , 24.89, 25.57, 26.66 and 27.77 ± 0.30 (mesylate 280 form B).
43. The pharmaceutical composition according to any one of items 16 to 27, or the
pharmaceutical formulation according to any one of items 28 to 33, wherein x-ray powder diffraction pattern of
285 (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2Θ values, 5 2Θ values , 6 2Θ values, 7 2Θ values or all 2Θ values (CuKa) selected from the group consisting of: 1 1.51 , 13.19, 16.23, 17.50, 19.14, 23.15, 23.80, 24.65, 25.81 , 26.06, 26.70 and 28.97 ± 0.30 (mesylate form C).
290 44. The pharmaceutical composition according to any one of items 16 to 27, or the
pharmaceutical formulation according to any one of items 28 to 33, wherein x-ray powder diffraction pattern of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide comprises 4 2Θ values or 5 2Θ values (CuKa) selected from the 295 group consisting of: 14.89, 19.69, 25.61 , 26.13 and 27.78 ± 0.30 (HC1 salt form).
Definitions
As used herein "amorphous" refers to a solid form of a molecule and/or ions that is not crystalline. An amorphous solid does not display a definitive X-ray diffraction pattern.
300 As used herein, "substantially pure," when used in reference to a form, means a compound having a purity greater than 90 weight %, including greater than 90, 91 , 92, 93, 94, 95, 96, 97, 98, and 99 weight %, and also including equal to about 100 weight % of Compound EGFRi, based on the weight of the compound. The remaining material comprises other form(s) of the compound, and/or reaction impurities and/or processing impurities arising from its
305 preparation. For example, a crystalline form of Compound EGFRi may be deemed
substantially pure in that it has a purity greater than 90 weight %, as measured by means that are at this time known and generally accepted in the art, where the remaining less than 10 weight % of material comprises other form(s) of Compound EGFRi and/or reaction impurities and/or processing impurities. In alternative, the term "substantially pure" with
310 reference to a particular polymorphic form means that the polymorphic form includes less than 10%, preferably less than 5%, more preferably less than 3%, most preferably less than 1% by weight of any other physical forms of the Compound EGFRi.
The term "free form" refers to the compound per se without salt formation or association with a solvent (e.g., solvate; if solvent water - hydrate).
315 The term "essentially the same" with reference to X-ray diffraction peak positions, or Figures means that typical peak position and intensity variability are taken into account. For example, one skilled in the art will appreciate that the peak positions (2Θ) or other peak positions in a Figure will show some inter-apparatus variability, depending on the method used; in case of XRPD typically as much as 0.2°. Further, one skilled in the art will appreciate that relative 320 peak intensities will show inter-apparatus variability as well as variability due to degree of crystallinity, temperature, preferred orientation, prepared sample surface, and other factors known to those skilled in the art, and should be taken as qualitative measure only.
325 BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. illustrates the x-ray powder diffraction patterns of the crystalline free form A of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 330 -yl)-2-methylisonicotinamide (Compound EGFRi).
FIG. 2. illustrates the differential scanning calorimetry (DSC) and the thermo gravimetric analysis (TGA) of the crystalline free form A of compound
(R,E)-N-(7-chloro-l-(l -(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-lH-benzo[d]imidazol-2 335 -yl)-2-methylisonicotinamide (Compound EGFRi).
FIG. 3. illustrates the microscopic picture of the crystalline free form A of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
340
FIG. 4. illustrates the x-ray powder diffraction patterns of the crystalline free form B of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi) (recorded by Bruker D8 Discover X-ray 345 diffractometer).
FIG. 5. illustrates the differential scanning calorimetry (DSC) of the crystalline free form B of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 350 -yl)-2-methylisonicotinamide (Compound EGFRi) recorded by TA Discovery Series DSC in open pan DSC (a hole in DSC lid) test; the free form B lost water and changed to free form A, then the free form A melted at 1 12.2 °C
FIG. 6. illustrates the differential scanning calorimetry (DSC) of the crystalline free form B 355 of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi) by TA Discovery Series DSC in the closed pan DSC, the free form B was tested in a sealed DSC pan, which was isolated from outside. 360
FIG. 7. illustrates the thermo gravimetric analysis (TGA) of the crystalline free form B of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi) by TA Discovery Series TGA.
365
FIG. 8. illustrates the x-ray powder diffraction patterns of the crystalline mesylate form A of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
370
FIG. 9. illustrates the differential scanning calorimetry (DSC) and the thermo gravimetric analysis (TGA) of the crystalline mesylate form A of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
375
FIG. 10. illustrates the microscopic picture of the crystalline mesylate form A of compound (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
380 FIG. 1 1. illustrates the x-ray powder diffraction patterns of the crystalline mesylate form B of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
385 FIG. 12. illustrates the differential scanning calorimetry (DSC) and the thermogravimetric analysis (TGA) of the crystalline mesylate form B of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
390 FIG. 13. illustrates the microscopic picture of the crystalline mesylate form B of compound (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
FIG. 14. illustrates the x-ray powder diffraction patterns of the crystalline mesylate form C of 395 compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
FIG. 15. illustrates the differential scanning calorimetry (DSC) and the thermogravimetric 400 analysis (TGA) of the crystalline mesylate form C of compound (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
FIG. 16. illustrates the x-ray powder diffraction patterns of the amophous form, free form A 405 and free form B of compound
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi) superimposed.
FIG. 17. illustrates the x-ray powder diffraction patterns of the HC1 salt form of compound 410 (R,E)-N-(7-chloro- 1 -(1 -(4-(dimethylamino)but-2-enoyl)azepan-3-yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
FIG. 18. illustrates the differential scanning calorimetry (DSC) and the thermogravimetric analysis (TGA) of the HC1 salt form of compound
415 (R,E)-N-(7-chloro- 1 -(1 -(4-(dimethylamino)but-2-enoyl)azepan-3-yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide (Compound EGFRi).
DETAILED DESCRIPTION OF THE DISCLOSURE
420
The disclosure relates to crystalline forms of the Compound EGFRi, which are described and characterized herein. The Compound EGFRi act as inhibitor of EGFR. It can be employed in different forms for use in the treatment of cancer diseases. The forms of the compound have pronounced different characteristics and can thus be appropriately selected when, for 425 example, formulating the compound in the pharmaceutical formulation.
The Compound EGFRi exhibits EGFR kinase activity and can be thus, particularly in specific forms as disclosed herein, used for treating proliferative disease. Inhibition of cell proliferation can be measured using methods known to those skilled in the art. For example, a
430 convenient assay for measuring cell proliferation is the CellTiter-Glo™ Luminescent Cell
Viability Assay, which is commercially available from Promega (Madison, Wis.). That assay determines the number of viable cells in culture based on quantitation of ATP present, which is an indication of metabolically active cells. See Crouch et al (1993) J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may be conducted in 96- or 384-well format,
435 making it amenable to automated high-throughput screening (HTS). See Cree et al (1995) Anticancer Drugs 6:398-404. The assay procedure involves adding a single reagent
(CellTiter-Glo® Reagent) directly to cultured cells. This results in cell lysis and generation of a luminescent signal produced by a luciferase reaction. The luminescent signal is
proportional to the amount of ATP present, which is directly proportional to the number of
440 viable cells present in culture. Data can be recorded by luminometer or CCD camera imaging device. The luminescence output is expressed as relative light units (RLU). Inhibition of cell proliferation may also be measured using colony formation assays known in the art.
Furthermore, the Compound EGFRi can be used in a method of treating a condition mediated by EGFR in a subject suffering therefrom, comprising administering to the subject a
445 therapeutically effective amount of Compound EGFRi. In one embodiment, the condition is a cell proliferative disease. In a more specific embodiment, the condition is cancer, for example, non-small cell lung cancer ( SCLC), head and neck cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, gastric cancer, glioma and prostate cancer. In a specific embodiment the condition is non-small cell lung cancer (NSCLC).
450
Treatment of the cell proliferative disorder by administration of the Compound EGFRi results in an observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition of cancer cell infiltration into peripheral organs including the
455 spread of cancer into soft tissue and bone; inhibition of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. To the extent the Compound EGFRi may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or
460 symptoms may also be felt by the patient.
The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TDP) and/or
465 determining the response rate (RR). Metastasis can be determined by staging tests and by bone scan and tests for calcium level and other enzymes to determine spread to the bone. CT scans can also be done to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastasis to the lungs and liver, respectively. Other routine methods for monitoring the
470 disease include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB).
The therapeutically effective dosage of the compound, the pharmaceutical composition, or the combinations of the compound with other pharmaceutically active ingredients (e.g other
475 anticancer drugs) thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed can also depend on the route of administration, and the seriousness of the condition
480 being treated and can be decided according to the judgment of the practitioner and each
subject's circumstances in view of, e.g., published clinical studies. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.03 to 2.5 mg/kg per body weight. An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.5 mg to about 100 mg, conveniently administered, e.g. in divided 485 doses up to four times a day or in retard form. Suitable unit dosage forms for oral
administration comprise from ca. 1 to 50 mg active ingredient.
In certain embodiments, a therapeutic amount or dose of the compound of the present disclosure may range from about 0.1 mg/kg to about 500 mg/kg, alternatively from about 1 to 490 about 50 mg/kg. In general, treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses (such as two, three, or four times daily). Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.
495
Upon improvement of a subject's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to 500 the desired level, treatment should cease. The subject may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
It has been found that several forms of the
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2
505 -yl)-2-methylisonicotinamide exist. For example, amorphous form has been found to have very good solubility at lower pH. However, the compound as such is sticky and hard to formulate. Therefore, crystalline forms are deemed more suitable for formulating. Further crystalline forms were found: crystalline free form A (Tm 114.6 °C), crystalline free form B (Tm 1 12.2 °C), crystalline mesylate form A (Tm 168.9 °C) and crystalline mesylate form B
510 (Tm 170.1 °C). Mesylate Form C is an anhydrate form. It is crystalline. In the DSC curve, it shows one endotherm occurring at Tonset = 187.2 °C, but the peak is splitted into two. In the TGA curve, it shows a small weight loss, about 0.8%, up to 185 °C. Although better for formulating, crystalline free form A (anhydrous) is unstable as bulk, but is less hygroscopic than other crystalline forms. Crystalline free form B is a dihydrate and more stable than free
515 form A. The free form dihydrate Form B is more stable in 0.1 N HC1 than the mesylate
trihydrate form B. Free form B can be prepared in good yield by using a simple, inexpensive and mild procedure, and in excellent purity retaining consistent water content from 30% to 90% relative humidity at 25°C. Also the dihydrate allows the use of choice of analytical methods, especially the X-ray methods, the usage of which permits clear and straightforward
520 analysis of its release. Crystalline mesylate form A of the compound is a monohydrate. It is more stable in bulk than free forms, but more hygroscopic. The best form for handling and formulating in pharmaceutical composition is mesylate form B. The mesylate form B is a trihydrate. It is more stable in solid state than free forms and mesylate form A, more soluble in aqueous media, and better compatible with certain excipients. Compound EGFRi HC1 salt 525 form behaves better in liquid formulations. It does not form gels with solvents.
The Compound EGFRi crystalline free form A has the following characteristic x-ray powder diffraction pattern: 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30 2Θ (CuKa λ= 1.54056A). It may be characterized by a x-ray powder
530 diffraction pattern (PXRD) comprising 5 or more 2Θ values (CuKa λ= 1.54056A) selected from the group consisting of 5.46, 15.84, 20.42, 22.56, 23.77, 25.09, 25.73 ± 0.30, measured at a temperature of about 22°C and an x-ray wavelength, λ, of 1.54056A. Preferably, the Compound EGFRi crystalline free form A may be characterized by a x-ray powder diffraction pattern comprising 6 or more 2Θ values (CuKa λ=1.54056A) selected from the
535 group consisting of : 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30, at a temperature of about 22°C.
In one embodiment, a representative XRPD pattern for the free form A is presented in the following table:
540
Free form A (anhydrous form):
Angle d value Intensity %
2-Theta ° Angstrom %
5.46287 16.16386 100
9.24225 9.56081 15.1
1 1.20646 7.88906 20.7
14.82315 5.97135 38.4
15.83617 5.59158 44
17.02289 5.20435 20
18.66506 4.75 75.3
19.63889 4.51661 21.8
20.42329 4.34487 54.1
21.12807 4.20151 18.1
22.55916 3.9381 25
23.76851 3.7404 25.9
25.09203 3.54602 40.5
25.72761 3.45984 28.3
26.35253 3.3792 30.1
27.55552 3.23434 29.6
28.11551 3.17118 21.3
30.93583 2.8882 16.2
34.67523 2.58482 15.3
38.62941 2.32884 15.2
40.05239 2.24932 16.4 The Compound EGFRi crystalline free form B has the following characteristic x-ray powder diffraction pattern: 1 1.24±0.30, 14.68±0.30, 15.66±0.30, 18.64±0.30, 19.63±0.30,
545 20.05±0.30, 20.31±0.30, 22.41±0.30, 24.91±0.3025.43±0.30, 26.03±0.30, 27.18±0.30, and 28.07± 0.30 (CuKa λ= 1.54056A). It may be characterized by a x-ray powder diffraction pattern (PXRD) comprising four or more 2Θ values (CuKa λ= 1.54056A) selected from the group consisting of 18.64, 25.43, 20.31 , 26.03, 27.18, and 28.07± 0.30, measured at a temperature of about 22°C and an x-ray wavelength, λ, of 1.54056A. Preferably, the
550 Compound EGFRi crystalline free form B may be characterized by a x-ray powder
diffraction pattern comprising five or more 2Θ values (CuKa λ=1.54056 A) selected from the group consisting of 18.64, 25.43, 20.31 , 26.03, 27.18, and 28.07± 0.30, at a temperature of about 22°C.
555 In one embodiment, a representative XRPD pattern for the free form B is presented in the following table:
Free form B (dehydrate)
Angle Intensity %
-Theta 0 %
11.24 38
14.68 47
15.66 48
18.64 100
19.63 42
20.05 47
20.31 61
22.41 45
24.91 50
25.43 65
26.03 55
27.18 50
28.07 35
The Compound EGFRi crystalline mesylate form A has the following characteristic x-ray 560 powder diffraction pattern: 9.12, 12.19, 16.25, 17.38, 18.42, 19.91 , 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 0.30 2Θ (CuKa λ= 1.54056A). It may be characterized by a x-ray powder diffraction pattern (PXRD) comprising four or more 2Θ values (CuKa λ= 1.54056A) selected from the group consisting of 9.12, 12.19, 16.25, 17.38, 18.42, 19.91 , 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 0.30, measured at a temperature of about 22°C and an x-ray
565 wavelength, λ, of 1.54056A. Preferably, the Compound EGFRi crystalline mesylate form A may be characterized by a x-ray powder diffraction pattern comprising five or more 2Θ values (CuKa λ= 1.54056A) selected from the group consisting of 9.12, 12.19, 16.25, 17.38, 18.42, 19.91 , 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 0.30, at a temperature of about 22°C.
In one embodiment, a representative XRPD pattern for the mesylate form A is presented in the following table:
Mesylate form A (dihydrate):
Angle d value Intensity %
2-Theta 0 Angstrom %
9.12147 9.68713 25.9
12.19287 7.25295 100
13.43312 6.58597 8.6
14.14358 6.2567 10.9
16.24641 5.45129 14.2
17.3816 5.09775 12.5
18.41949 4.81277 74.2
19.90531 4.45675 12.4
20.85497 4.2559 13.1
21.86745 4.06109 9.5
22.43198 3.96014 14.1
22.92317 3.87639 13
23.32961 3.80976 11.7
23.79896 3.73568 9.7
24.09837 3.68994 14.2
25.43377 3.49915 10
25.85028 3.4437 10
26.63696 3.34376 14.2
27.78252 3.20843 21.8
30.98983 2.88329 8.1
34.23538 2.61701 6.7
35.41 15 2.53274 6.4
36.98372 2.4286 10.4
37.26719 2.41078 10.1
38.17953 2.35524 7.1
39.10431 2.30165 7.6
39.79202 2.26344 7.4
41.64976 2.16667 6.3
43.98388 2.05695 7.1
The Compound EGFRi crystalline mesylate form B has the following characteristic x-ray powder diffraction pattern: 1 1.76, 13.832, 14.41 , 15.9 17.65, 18.79, 21.46, 21.83, 22.30, 23.82, 24.51 , 24.89, 25.57, 26.66 and 27.77 ± 0.30 20 (CuKa λ= 1.54056A). It may be characterized by a x-ray powder diffraction pattern (PXRD) comprising five or more 2Θ 580 values (CuKa λ= 1.54056A) selected from the group consisting of 1 1.76, 13.832, 14.41 , 15.9 17.65, 18.79, 21.46, 21.83, 22.30, 23.82, 24.51 , 24.89, 25.57, 26.66 and 27.77 ± 0.30, measured at a temperature of about 22°C and an x-ray wavelength, λ, of 1.54056A.
Preferably, the Compound EGFRi crystalline mesylate form B may be characterized by a x-ray powder diffraction pattern comprising six or more 2Θ values (CuKa λ= 1.54056A)
585 selected from the group consisting of 1 1.76, 13.832, 14.41 , 15.9 17.65, 18.79, 21.46, 21.83, 22.30, 23.82, 24.51 , 24.89, 25.57, 26.66 and 27.77 ± 0.30, at a temperature of about 22°C.
In one embodiment, a representative XRPD pattern for the mesylate form B is presented in the following table:
590
Mesylate form B (trihydrate):
Angle d value Intensity %
2-Theta 0 Angstrom %
5.79532 15.23733 33.2
11.75526 7.52196 51.2
13.82942 6.39811 48.8
14.41049 6.1414 50.8
15.18888 5.82838 57.3
16.30465 5.43196 26.2
17.65193 5.02028 59
18.47867 4.79749 35.8
18.78755 4.71931 61.2
19.61655 4.5217 30.4
20.29133 4.37283 25
21.4551 4.1382 64.7
21.83466 4.06711 40.8
22.30375 3.98262 54.9
23.35057 3.80639 35.7
23.82335 3.73191 38.8
24.50774 3.62923 100
24.89097 3.57421 53
25.56678 3.48124 53.2
26.65771 3.3412 94.2
27.77467 3.20932 65.3
29.06548 3.06966 27.8
30.59466 2.91963 22.3
31.82663 2.80937 24.2
34.81147 2.57501 17.4
36.02644 2.49091 22.9
41.51816 2.17324 18.3
44.6646 2.02717 17.8 The Compound EGFRi crystalline mesylate form C has the following characteristic x-ray 595 powder diffraction pattern: 1 1.51 , 13.19, 16.23, 17.50, 19.14, 23.15, 23.80, 24.65, 25.81 , 26.06, 26.70 and 28.97 ± 0.30 2Θ (CuKa λ= 1.54056A). It may be characterized by a x-ray powder diffraction pattern (PXRD) comprising five or more 2Θ values (CuKa λ= 1.54056A) selected from the group consisting of 1 1.51 , 13.19, 16.23, 17.50, 19.14, 23.15, 23.80, 24.65, 25.81 , 26.06, 26.70 and 28.97 ± 0.30, measured at a temperature of about 22°C and an x-ray 600 wavelength, λ, of 1.54056A. Preferably, the Compound EGFRi crystalline mesylate form C may be characterized by a x-ray powder diffraction pattern comprising six or more 2Θ values (CuKa λ= 1.54056A) selected from the group consisting of 11.51 , 13.19, 16.23, 17.50, 19.14, 23.15, 23.80, 24.65, 25.81 , 26.06, 26.70 and 28.97 ± 0.30, at a temperature of about 22°C.
605 In one embodiment, a representative XRPD pattern for the mesylate form C is presented in the following table:
Mesylate form C (anhydrous form):
Angle d value Intensity %
2-Theta 0 Angstrom %
5.67913 15.54881 13.4
10.81411 8.17439 14.5
11.5087 7.68255 25.3
12.2189 7.23757 17.6
13.1893 6.70716 26.1
13.77973 6.42107 17.5
14.41634 6.13893 13.9
16.23088 5.45648 26.1
17.50003 5.06351 22.6
19.14178 4.63277 41.9
19.5925 4.5272 19.3
20.43204 4.34303 15.5
21.771 14 4.07883 22.3
22.03055 4.03139 17.1
22.53925 3.94154 19.5
23.15277 3.83846 42.6
23.80439 3.73484 26.5
24.64689 3.60905 100
25.81205 3.44872 33.8
26.06282 3.4161 23.8
26.69553 3.33655 21.9
27.27977 3.26641 14.5
27.91729 3.19325 15.3 28.97184 3.07937 22.7
29.98749 2.97735 11.7
32.8838 2.72143 15.6
35.64795 2.51648 9.5
610
The Compound EGFRi crystalline HCl salt form has the following characteristic x-ray powder diffraction pattern: 14.89, 19.69, 25.61 , 26.13 and 27.78 ± 0.30 2Θ (CuKa λ= 1.54056A), measured at a temperature of about 22°C.
615 In one embodiment, a representative XRPD pattern for the HCl salt form is presented in the following table:
HCl salt form:
Angle d value Intensity %
2-Theta 0 Angstrom %
14.88934 5.94495 90.1
15.54683 5.69499 52
18.02516 4.91716 63.2
19.69317 4.50428 90.5
20.15181 4.40279 62.6
20.87353 4.25216 55.8
22.21493 3.99834 48.5
25.61463 3.47485 100
26.12697 3.40786 72.1
26.79442 3.32446 63.3
27.78255 3.20843 78.2
32.53383 2.7499 40.2
620 All tables above contain most pronounced and characteristic peaks for each of the forms in bold.
In one embodiment of the disclosure, the form of the Compound EGFRi is provided in substantially pure form. This crystalline form of the Compound EGFRi in substantially pure 625 form may be employed in pharmaceutical compositions which may optionally include one or more other components selected, for example, from the group consisting of excipients, carriers, and one of other active pharmaceutical ingredients active chemical entities of different molecular structure.
630 Preferably, the crystalline form has substantially pure phase homogeneity as indicated by less than 10%, preferably less than 5 %, and more preferably less than 2 % of the total peak area in the experimentally measured PXRD pattern arising from the extra peaks that are absent from the simulated PXRD pattern. Most preferred is a crystalline form having substantially pure phase homogeneity with less than 1% of the total peak area in the experimentally 635 measured PXRD pattern arising from the extra peaks that are absent from the simulated
PXRD pattern.
In one embodiment, a composition is provided consisting essentially of any one of the forms presented above, except when the form is mesylate form B, the compound contains other 640 forms as well. The composition of this embodiment may comprise at least 90 weight % of the respective form, based on the weight of the Compound EGFRi in the composition.
The presence of reaction impurities and/or processing impurities may be determined by analytical techniques known in the art, such as, for example, chromatography, nuclear 645 magnetic resonance spectroscopy, mass spectrometry, or infrared spectroscopy.
The disclosed forms of the Compound EGFRi can be formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical compositions can be formulated for oral, intravenous, intradermal,
650 intramuscular, intraperitoneal, subcutaneous, intranasal, epidural, sublingual, intracerebral, intravaginal, intraventricular, intrathecal, epidural, transdermal, rectal, by inhalation, or topical administration. In one embodiment, the pharmaceutical composition is formulated for oral administration. The pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules, capsules containing liquids, powders,
655 suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
The compositions can be formulated for immediate release, sustained release, or controlled release of the compounds of the invention. Suitable pharmaceutical excipients include, for example, 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); for
660 tablets also c) binders (e.g., magnesium aluminum silicate, starch, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone); if desired d) disintegrants, e.g., starches, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners.
Additional suitable pharmaceutical excipients can be liquids, such as water and oils,
665 including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil,
soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be saline, starch, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the
pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a 670 useful excipient when the compound of the invention is administered intravenously. The present compositions can also contain minor amounts of wetting or emulsifying agents, or pH modifier.
Particularly pH buffering agent can improve the stability of the Compound EGFRi, or its 675 specific forms, namely free Form A, free Form B, mesylate from A, mesylate form B, mesylate form C, amorphous form and HC1 salt form. In solid dosage forms modulation of the pH through the use of pH modifiers can be very effective in improving solid dosage form stability. The same applies to liquid dosage forms. Particularly beneficial is when pH is above pH 1 and below pH 9, particularly above pH 1 and below pH 6.8. In this range the
680 0.1% aquous solutions or suspensions of the Compound EGFRi or its forms are most stable.
Best result in terms of improved stability is reached at pH between pH 2 and pH 4.7. Acidic or basic ingredients (pH modifiers) can be intentionally added to a formulation to modify the pH and increase composition's stability, for example measured by amount of the Compound EGFRi degradation products or impurities. pH modifier in oral solid dosage forms can be
685 selected for example on the basis of knowing pH of their saturated solution. Without wishing to be bound to any theory, it can be assumed that solid pH is reflective of the pH of the adsorbed moisture layer. Therefore, when a water soluble pH modifier dissolves in the moisture layer it achieves the pH of a saturated solution. The effect of a pH modifier on the solid pH is hence dependent on both its ionization constant (pKa) as well as its aqueous
690 solubility. Limiting the pH in a liquid formulations is much more straightforward and a
skilled person would be able to avail himself with many known buffers, acids, bases, and their salts. In case of lyophilized parenteral formulations, the solution used for the freeze drying process can usually be buffered to the desired pH using common buffering systems and buffer concentrations used for solution formulations. Again, without wishing to be bound
695 to any theory, the pH of the reconstituted lyophile should generally correspond to solution pH prior to freeze drying.
Suitable pH modifiers used to enhance the stability of the forms are for example acids, bases or their salts (e.g. citric acid, fumaric acid, acetic acid, HC1, boric acid, phosphoric acid, 700 malic, formic, lactic, succinic acid, malic acid, picolinic acid or succinic acid, etc. ,or
mixtures thereof), or mixtures thereof. pH can be measured by a pH meter using a glass electrode or micro pH electrode. In alternative, pH can be measured by any other suitable means, like for example neutralization
705 titration. In case of liquid samples, their pH can be measured directly. In order to measure pH of solid samples, they first need to be dissolved or suspended in a suitable solvent. For example, a 1 g sample of solid pharmaceutical composition comprising Compound EGFRi can be dissolved or suspended in a volume of 1 mL of water in order to prepare a solution, suspension or slurry of the pharmaceutical composition comprising Compound EGFRi and
710 optionally also other excipients of the pharmaceutical formulation (e.g. pH modifier). The pH of said solution, suspension or slurry of the pharmaceutical composition comprising
Compound EGFRi should be between pH 1 and pH 9, preferably between pH 2 and pH 6.8, particularly around pH 4.5; measured at 20°C. In alternative, a sample can be dissolved or suspended in water to prepare 1 w/w % solution or suspension of the Compound EGFRi and
715 pH measured. The pH of said 1 % solution or suspension of the Compound EGFRi should be between H 1 and pH 9, preferably between pH 2 and pH 6.8, particularly around pH 4.5; measured at 25°C.
Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily
720 suspensions, granules, powders, emulsions, capsules, syrups, or elixirs for example. Orally administered compositions can contain one or more agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Liquid dosage forms for oral administration include pharmaceutically
725 acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular,
730 cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In another embodiment, the compositions can be formulated for parenteral
735 administration by various routes, including but not limited to, intravenous (including bolus injection), subcutaneous, intramuscular, and intra-arterial administration. Such parenteral dosage forms are administered in the form of sterile or sterilizable injectable solutions, suspensions, dry and/or lyophylized products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection (reconstitutable powders) and emulsions.
740 Vehicles used in such dosage forms include, but are not limited to, Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles.
745 Compositions can be prepared according to conventional mixing, granulating or coating
methods, respectively, and the present compositions can contain, in one embodiment, from about 0.1 percent to about 99 percent; and in another embodiment from about 1 percent to about 70 percent of the compound of the invention by weight or volume.
750 The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the compounds of the present invention as active ingredients, since water may facilitate the degradation of certain compounds. Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. An anhydrous
755 pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits.
Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e. g., vials), blister packs, and strip packs.
760
In another aspect, the pharmaceutical compositions further comprise one or more additional therapeutic agents. The compounds of the invention and the additional therapeutics agent(s) may act additively or synergistically.
765 Preparation of Crystalline Materials:
Crystalline forms may be prepared by a variety of methods, including for example, crystallization or recrystallization from a suitable solvent, sublimation, growth from a melt, solid state transformation from another phase, crystallization from a supercritical fluid, and jet spraying. Techniques for crystallization or recrystallization of crystalline forms from a
770 solvent mixture include, for example, evaporation of the solvent, decreasing the temperature of the solvent mixture, crystal seeding a supersaturated solvent mixture of the molecule and/or salt, freeze drying the solvent mixture, and addition of antisolvents (countersolvents) to the solvent mixture. High throughput crystallization techniques may be employed to prepare crystalline forms including polymorphs. Crystals of drugs, including polymorphs,
775 methods of preparation, and characterization of drug crystals are discussed in Solid-State Chemistry of Drugs, S.R. Byrn, R.R. Pfeiffer, and J.G. Stowell, 2nd Edition, SSCI, West Lafayette, Indiana (1999).
For crystallization techniques that employ solvent, the choice of solvent or solvents is
780 typically dependent upon one or more factors, such as solubility of the compound,
crystallization technique, and vapor pressure of the solvent. Combinations of solvents may be employed, for example, the compound may be solubilized into a first solvent to afford a solution, followed by the addition of an antisolvent to decrease the solubility of the compound in the solution and to afford the formation of crystals. An antisolvent is a solvent
785 in which the compound has low solubility. In one method to prepare crystals, a compound is suspended and/or stirred in a suitable solvent to afford a slurry, which may be heated to promote dissolution. The term "slurry", as used herein, means a saturated solution of the compound, which may also contain an additional amount of the compound to afford a heterogeneous mixture of the compound and a solvent at a given temperature. Seed crystals
790 may be added to any crystallization mixture to promote crystallization. Seeding may be employed to control growth of a particular polymorph or to control the particle size distribution of the crystalline product. Accordingly, calculation of the amount of seeds needed depends on the size of the seed available and the desired size of an average product particle as described, for example, in "Programmed Cooling of Batch Crystallizers," J.W.
795 Mullin and J. Nyvlt, Chemical Engineering Science, 1971 ,26, 369-377. In general, seeds of small size are needed to control effectively the growth of crystals in the batch. Seed of small size may be generated by sieving, milling, or micronizing of large crystals, or by micro-crystallization of solutions. Care should be taken that milling or micronizing of crystals does not result in any change in crystallinity form the desired crystal form (i.e.,
800 change to amorphous or to another polymorph). A cooled crystallization mixture may be
filtered under vacuum, and the isolated solids may be washed with a suitable solvent, such as cold recrystallization solvent, and dried under a nitrogen purge to afford the desired crystalline form. The isolated solids may be analyzed by a suitable spectroscopic or analytical technique, such as solid state nuclear magnetic resonance, differential scanning
805 calorimetry, x-ray powder diffraction, or the like, to assure formation of the preferred
crystalline form of the product. The resulting crystalline form is typically produced in an amount of greater than about 70 weight % isolated yield, preferably greater than 90 weight % isolated yield, based on the weight of the compound originally employed in the
crystallization procedure. The product may be comilled or passed through a mesh screen to
810 delump the product, if necessary.
Crystalline forms may be prepared directly from the reaction medium of the final process for preparing EGFRi. This may be achieved, for example, by employing in the final process step a solvent or a mixture of solvents from which Compound EGFRi may be crystallized.
815 Alternatively, crystalline forms may be obtained by distillation or solvent addition techniques.
Suitable solvents for this purpose include, for example, the aforementioned nonpolar solvents and polar solvents, including protic polar solvents such as alcohols, and aprotic polar solvents such as ketones. It is also possible to obtain one form of the compound and then transform it into another form, or mixture thereof, by the methods described above.
820
The presence of more than one polymorph in a sample may be determined by techniques such as x-ray powder diffraction (PXRD) or solid state nuclear magnetic resonance spectroscopy. For example, the presence of extra peaks in the comparison of an
experimentally measured PXRD pattern with a simulated PXRD pattern may indicate more 825 than one polymorph in the sample. The simulated PXRD may be calculated from single crystal x-ray data, see Smith, D.K., "A FORTRAN Program for Calculating X-Ray Powder Diffraction Patterns ," Lawrence Radiation Laboratory, Livermore, California, UCRL-7196 (April 1963) or TOP AS program (Total Pattern Analysis Solution, available through Brucker AXS Inc.).
The following non-limiting examples are illustrative of the disclosure.
835
EXAMPLES
Details about methodology, instruments and standards used 1 pH value
The pH of 1% solution or suspension of the drug substance in water was recorded at 25°C. Mettler Toledo pH-meter with Orion Micro-pH combination electrode was used.
2 Determination of solubility
Solubility was determined using UPLC. 20 mg of drug substance were equilibrated in 1 mL media at room temperature for 24 h. The solution/suspension was filtrated and the saturated solution was analyzed with UPLC.
3 Hygroscopicity
Water sorption curves were recorded using Surface Measurement Systems dynamic vapor sorption instrument at 25 °C.
4 Polymorphism behavior
Slurries of drug substance were stirred at room temperature for 24 hours. The slurries were filtrated and the solids were analyzed with XRD instrument.
5 UPLC method
Wave length: 225 nm
Column: Waters ACQUITY HSS T3, 50 x 2.1 mm, 1.8 μιη
Column temperature: 40 °C
Flow rate: 0.60 mL/min
Mobile phase A: 0.05% TFA in 95% water/5% acetonitrile
Mobile phase B: 0.05% TFA in 95% acetonitrile 15% water
Injection volume: 1.0 \iL
Gradient:
Figure imgf000026_0001
6. X-ray Powder Diffraction Measurements
One of ordinary skill in the art will appreciate that an X-ray diffraction pattern may be obtained with a measurement error that is dependent upon the measurement conditions employed. In particular, it is generally known that intensities in a X-ray diffraction pattern may fluctuate depending upon measurement conditions employed. It should be further understood that relative intensities may also vary depending upon experimental conditions and, accordingly, the exact order of intensity should not be taken into account. Additionally, a measurement error of diffraction angle for a conventional X-ray diffraction pattern is typically about 5% or less, and such degree of measurement error should be taken into account as pertaining to the aforementioned diffraction angles. Consequently, it is to be understood that the crystal forms of the instant invention are not limited to the crystal forms that provide X-ray diffraction patterns completely identical to the X-ray diffraction patterns depicted in the accompanying Figures disclosed herein. Any crystal forms that provide X- ray diffraction patterns substantially identical to those disclosed in the accompanying Figures fall within the scope of the present disclosure. The ability to ascertain substantial identities of X-ray diffraction patterns is within the purview of one of ordinary skill in the art.
XRPD-method used to scan the samples as dislosed herein was as follows:
Instrument: Bruker D8 Discover with XYZ stage
Detector: GADDS (2D 'area' detector with 20° window)
Radiation: CuKa (0.15406 nm)
X-ray generator: power 40kV, 40 mA (1600 Watts)
Step size, resolution: 0.02 degrees
Measuring slice: 0.3 mm and 0.2 mm
Scan range: 2° - 45° (2 theta value)
Scan time: 2min
X-ray optics: Parallel (Goebel mirror)
Source slit: Fixed, 0.5mm
Detector distance: ~30cm
Sample amount of ca 2-5 mg was placed on an objective slide and centered in the X-ray beam.
7. Differential Scanning Calorimetry (DSC)
The DSC instrument used to test the crystalline forms was a TA Instrument® Differential Scanning Calorimetry Model 2910, TA Instruments® Modulated Differential Scanning Calorimetry Model 2920, TA Discovery series DSC or TA Instruments® Modulated
Differential Scanning Calorimetry Model Q1000. The DSC cell/sample chamber was purged with 100 ml/min of ultra-high purity nitrogen gas. The instrument was calibrated with high purity indium. The accuracy of the measured sample temperature with this method is within about ± 1°C, and the heat of fusion can be measured within a relative error of about ±5%. The sample was placed into an open aluminum DSC pan and measured against an empty reference pan. About 2-6 mg of sample powder was placed into the bottom of the pan and lightly tapped down to make contact with the pan. The weight of the sample was measured accurately and recorded to a hundredth of a milligram. The instrument was programmed to 885 heat at 10°C. per minute in the temperature range between 25 and 300°C.
The heat flow, which was normalized by a sample weight, was plotted versus the measured sample temperature. The data were reported in units of watts/gram ("W/g"). The plot was made with the endothermic peaks pointing down. The endothermic melt peak was evaluated 890 for extrapolated onset temperature, peak temperature, and heat of fusion in this analysis.
8. Thermogravimetric Analysis (TGA)
The TGA instruments used to test the crystalline forms was TA instruments, High Resolution Thermogravimetric Analyzer Q5000. 0.5-1.0 mg of test substance was weighted and placed 895 on the open sample pan. The TGA
thermogram was recorded as follows: the sample was loaded into the furnace, the temperature equilibrated to 30 °C and heated to 300 °C at a heating rate of 10 °C/min, under a flow of nitrogen at 25 m L/min. The instrument was calibrated for temperature with nickel and aluminum, and calibrated for weight with a 100 mg standard.
900
9. Polarized light microscope
Instrument: Olympus BX51-P polarizing microscope. 0.1-0.5 mg of test substance was dispersed in one drop of silicon oil on a glass slide.
Example 1.
(R,E)-N-(7-chloro-l-(l-(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-lH-benzo[d]imidaz ol-2-yl)-2-methylisonicotinamide
Figure imgf000028_0001
Step A 1-15a eP M 5b
Figure imgf000028_0002
910 Intermediate 15 Step A: A stirred solution of (S)-iert-butyl 3-aminopiperidine-l-carboxylate (0.500 g, 2.49 mmol), l-fluoro-4-methyl-2-nitrobenzene (0.387 g, 2.49 mmol) and
N,N-diisopropylethylamine (0.482 g, 3.74 mmol) in DMF under argon was heated to 1 10°C for 6 h (reaction completion monitored by TLC). The mixture was diluted with water and
915 extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over anhydrous a2S04 and concentrated under reduced pressure to afford (S)-iert-butyl
3-((4-methyl-2-nitrophenyl) amino) piperidine-l-carboxylate (I-15a). MS calculated for C17H24N3O4 (M-H ) 334.18, found 334.0.
Step B: To a stirred solution of I-15a (0.550 g, 1.64 mmol) in MeOH (35mL) was added
920 Pd/C (0.090 g) and the mixture was stirred at room temperature under hydrogen atmosphere (balloon) for 2h (reaction completion monitored by TLC). The mixture was filtered through Celite, washed with MeOH and concentrated under reduced pressure to afford (S)-ieri-butyl 3-((2-amino-4-methylphenyl)amino)piperidine-l-carboxylate (I-15b). MS calculated for C17H28 3O2 (M+H+) 306.22, found 306.2.
925 Step C: To a stirred solution of (»S)-tert-butyl
3-((2-amino-4-methylphenyl)amino)piperidine-l-carboxylate (I-15b) (0.500 g, 1.63 mmol) in MeOH (20 mL) was added a solution of cyanogen bromide (0.208 g, 1.96 mmol) in 1 :2 MeCN:H20 (20 mL) for a period of 5 min. The mixture was heated to 50°C for 2h (reaction completion monitored by TLC), cooled to 0°C and pH was adjusted to 10 by adding aqueous
930 Na2C03 solution. The mixture was stirred for 30 min at room temperature, the resulting solid was collected and dried under vacuum to afford the title compound (Intermediate 15).
Ή-NMR (400 MHz, CDC13): d 7.24 (s, 1H), 7.17 (d, J= 7.6 Hz, 1H), 6.85 (d, J = 8 Hz, 1H), 4.64 (br s, 2H), 4.17 (t, J= 14.8 Hz, 2H), 3.99-3.93 (m, 1H), 3.32 (d, J= 11.6 Hz, 1H), 2.79 (t, J= 12.4 Hz, 1H), 2.41 (s, 3H), 2.38-2.37 (m, 1H), 2.34 (d, J= 3.2 Hz, 1H), 1.91 (d, J =
935 13.6 Hz, 3H), 1.69-1.61 (m, 1H), 1.47 (s, 9H); MS calculated for C18H27 4O2 (M+H+) 331.21 , found 331.0.
Intermediate 26
(R)-tert-butyl 3-(2-amino-7-chloro-lH-benzo[d]imidazol-l-yl)azepane-l-carboxylate
Figure imgf000029_0001
940 Step A: (R)-tert-butyl 3-((2-chloro-6-nitrophenyl)amino)azepane-l-carboxylate (I-26a) was prepared following procedures analogous to 1-15, Step A, using the appropriate starting materials. 'H-NMR (400MHZ, CDC13): d 8.00-7.91 (m, 1H), 7.58-7.49 (m, 1H), 7.02-6.51 (m, 2H), 4.31-4.03 (m, 1H), 3.84-2.98 (m, 4H), 1.98-1.60 (m, 5H), 1.46-1.39 (m, 10H); MS calculated for Ci7H25ClN304 (M+H+) 370.15, found 370.10.
945 Step B: A mixture of I-26a (7.5 g, 19.5 mmol) and Zn (12.8 mg, 195 mmol) in AcOH
(22 mL) was stirred at room temperature for 2 h. The reaction was basified with saturated aqueous a2C03 solution, filtered, and extracted with EtOAc (3 x 80 mL). The combined organic phase was washed with brine, dried with Na2S04 and concentrated in vacuo to afford (R)-tert -butyl 3-((2-amino-6-chlorophenyl)amino)azepane-l -carboxylate (I-26b). MS
950 calculated for C17H27CIN3O2 (M+H+) 340.17, found 340.10. The crude was used in the next step without further purification.
Step C: The title compound (Intermediate 26) was prepared from I-26b following procedures analogous to 1-15, Step C. ¾-NMR (400MHz, CDC13): d 7.34-7.26 (m, 1H), 7.04-6.97 (m, 2H), 6.05-5.85 (m, 1H), 5.84-5.72 (m, 1H), 5.50-5.37 (m, 0.5H), 5.10-4.80(m,
955 0.5H), 4.41-4.23(m, 1H), 4.09-3.96(m, 0.5H), 3.94-3.81 (m, 1H), 3.76-3.57 (m, 1H),
3.22-3.14 (m, 0.5H), 2.84-2.63 (m, 1H), 2.34-2.17 (m, 1H), 2.07-1.84 (m, 1H), 1.82-1.64 (m, 2H), 1.53 (s, 9H), 1.48-1.37 (m, 1H); MS calculated for C18H26CIN4O2 (M+H+) 365.17, found 365.10.
Intermediate 27
960 (R)-N-(l-(azepan-3-yl)-7-chloro-lH-benzo[d]imidazol-2-yl)-2-methylisonicotinamide
Figure imgf000030_0001
1-26 step A '"27a Intermediate 27
Step A: A mixture of 2-methylisonicotinic acid (3.371 g, 24.6 mmol) and 2-(7-aza-lH- benzotriazole-l -yl)-l ,l ,3,3-tetramethyluronium hexafiuorophosphate (9.345 g, 24.6 mmol) in
965 CH2CI2 (120 ml) was treated at room temperature with ΝΕΪ3 (4.1 mL, 29.4 mmol). The reaction was stirred for 1 hour before it was slowly added into a CH2CI2 solution (45 ml) of 1-26 (5.98 g, 16.4 mmol). Ten minutes later, more ΝΕΪ3 (4.1 mL, 29.4 mmol) was added and the mixture stirred for 2 h. The mixture was then diluted with CH2CI2 (240 mL), washed with H2O (2 x 80 mL), saturated aqueous NaHCC^ solution (70 mL), and brine (70 mL). The
970 organic phase was dried with a2S04, and concentrated under reduced pressure. The crude material was purified by column chromatography (55% EtOAc/hexanes) to afford
(R)-tert-butyl
3-(7-chloro-2-(2-methylisonicotinamido)-lH-benzo[d]imidazol-l-yl)azepane-l -carboxylate (I-27a) as a light yellow foam. 'H-NMR (400MHZ, CDCI3): d 12.81 (br s, 1H), 8.65-8.62 (m,
975 1H), 7.95-7.85 (m, 2H), 7.27-7.11 (m, 3H), 5.64 - 5.51 (m, 1H), 4.56-4.44 (m, 1H),
4.07-3.92 (m, 1H), 3.79-3.71 (m, 0.5H), 3.41-3.35 (m, 0.5H), 3.29-3.23 (m, 1H), 2.71-2.59 (m, 1H), 2.65 (s, 3H), 2.22-2.00 (m, 3H), 1.93-1.80 (m, 1H), 1.51-1.45 (m, 1H), 1.50 (s, 3.5H), 1.41 (s, 5.5H); MS calculated for C25H3iCiN503 (M+H+) 484.20, found 484.20.
Step B: A solution of I-27a (8.62 g, 16.4 mmol) in MeOH (67 mL) was treated with HCI
980 in dioxane (4M, 67 mL) and the mixture was stirred at room temperature for 7 h. The mixture was then concentrated under reduced pressure to afford the title compound (Intermediate 27). The product was used in the next step without further purification. A sample was treated with 1 M NaOH, extracted with EtOAc, dried with Na2S04 and concentrated under reduced pressure to afford 1-27 as a free base. ¾-NMR (400MHz, CD3CN): d 8.49 (d, J=5.0 Hz, 1H), 985 7.81 (s, 1H), 7.72 (d, J=4.8 Hz, 1H), 7.50 (br d, J=7.52 Hz, 1 H), 7.16 - 7.09 (m, 2H),
5.66-5.59 (m, 1 H), 3.77 (dd, J = 6.54, 14.3 Hz, 1H), 3.18 (dd, J = 5.3, 14.3 Hz, 1H), 3.05 - 2.98 (m, 1H), 2.76-2.69 (m, 1H), 2.63-2.53 (m, 1H), 2.47 (s, 3H), 2.10-2.03 (m, 1 H),
1.96-1 .93 (m, 2H), 1.86 - 1.75 (m, 2H), 1 .61 - 1.54 (m, 2H); MS calculated for
C20H23ClN5O (M+H+) 384.15, found 384.20.
990
(i?.-E)-N-(7-chloro-l -(l -(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-lH-benzo[d]imidazol-2
-yl) -2 -methylis onico tinamide
Figure imgf000031_0001
A mixture of (E)-4-(dimethylamino)but-2-enoic acid hydrochloride (58 mg, 0.35 mmol) 995 and l -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (67 mg, 0.35 mmol) in DMF (2 mL) was treated with hydroxybenzotriazole (54 mg, 0.35 mmol) and stirred at room temperature for 1 h. The resulting mixture was added to a solution of 1-27 (100 mg, 0.22 mmol) in DMF (2 mL). Triethylamine (199 mg, 1.97 mmol) was then added and the mixture was stirred for 5 days. Water (2 mL) was added and the mixture was concentrated under
1000 reduced pressure. The residue was diluted with IN NaOH (20 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with water (50 mL) and brine (2 x 50 mL), dried over Na2S04, and concentrated under reduced pressure. The crude was purified by column chromatography (9: 1 :0.175N CH2Cl2/MeOH/NH3 in CH2C12, 0% to 100%) to afford the title compound. !H NMR (400 MHz, DMSO-de) δ 8.59 (d, J = 4.8 Hz, 1 H), 7.89 (s, 1 H),
1005 7.79 (d, J = 4.8 Hz, 1 H), 7.60 (d, J = 7.5 Hz, 1H), 7.30-7.22 (m, 2H), 6.71 -6.65 (m, 1 H),
6.57-6.54 (m, 1 H), 5.54 (br. s, 1H), 4.54 (br. s, 1H), 4.20 (br s, 1 H), 3.95 (br s, 1H), 3.48 (br s, 1H), 2.98 (br s, 2H), 2.72 (d, J = 12.0 Hz, 1 H), 2.58 (s, 3H), 2.14 (br s, 6H), 2.05 (d, J = 6.7 Hz, 3H), 1.88 (br s, 1 H), 1.46 (d, J=l 1.3 Hz, 1H); MS calculated for C26H32C1N602 (M+H+) 495.22, found 495.10. Melting point (1 14.6 °C).
1010
Example 2.
(R^)-A^-(7-chloro-l-(l-(4-(dimethylamino)but-2-enoyl)azepaii-3-yl)-lH-benzo[d]imidaz ol-2-yl)-2-methylisonicotinamide (EGFRi) Biochemical Assays
1015 IC50 determinations. All EGFR biochemical assays were carried out by HTRF method. The EGFR(L858R/T790M) enzyme were purchased from Carna (GST-a.a. 669-1210). The substrate peptide Biotin-TK-peptide was purchased from Cis-Bio. The reaction mixtures contained 1 μΜ peptide substrate, 10 μΜ ATP, and 0.036 nM EGFR(L858R/T790M) in the reaction buffer (50 mM HEPES pH 7.1 , lOmM MgC12, 0.01 % BSA, 1 mM TCEP and 0.1
1020 mM Na3 V04) at a final volume of 10 μL·. All reactions were carried out at room
temperature in white ProxiPlate™ 384-well Plus plates (PerkinElmer) and were quenched with 5 μL· of 0.2 M EDTA at 60 min. Five μL· of the detection reagents (2.5 ng PT66K and 0.05 μg SAXL per well) were added, the plates were incubated at room temperature for 1 h and then read in En Vision reader. EGFRi was diluted into assay mixture (final DMSO
1025 0.5%), and IC50 values were determined by 12-point (from 50 to 0.000282 μΜ) inhibition curves in duplicate under the assay conditions as described above. For no-preincubation condition, the compound was added to the assay solution containing ATP and peptide, and the reaction was initiated by addition of enzyme. For pre-incubation conditions, the compound was added to the assay solution containing enzyme and peptide, and pre-incubated
1030 at room temperature for desired period of time, then the reaction was initiated by addition of ATP.
EGFR Target Modulation in Engineered NIH/3T3 Cell Lines
Tissue Culture. NIH/3T3 cell lines expressing human EGFR (WT, L858R, and
1035 L858R T790M) (obtained from Matthew Meyerson's Lab at DFCI) were maintained in 10% FBS/DMEM supplemented with 100 μ^πιΐ Penicillin/Streptomycin (Hyclone #SV30010) and 2 μg/ml Puromycin. The cells were harvested with 0.05% Trypsin/EDTA (Hyclone #SH30236.01), re-suspended in 5% FBS/DMEM Pen/Strep without Puromycin and plated at 9,000 cells per well in 50 μΐ of media in a 384-well black plate with clear bottoms (Greiner
1040 #789068G). The cells were allowed to incubate overnight in a 37°C, 5% C02 humidified tissue culture incubator. A 12-point test compound curve was prepared by serial diluting a 10 μΜ stock 1 :3 in DMSO in a 384-well compound plate (Greiner #789201L). The serial diluted compound was transferred to the plate containing cells by using a 50 nl Pin Head device (Perkin Elmer) and the cells were placed back in the incubator for 3 hours. Only the
1045 EGFR WT-expressing cells were induced with 50 ng/ml EGF (Preprotech #AF-100-15) for 5 minutes before lysis. The media was removed and cells were lysed in 25 μΐ of Lysis buffer containing protease and phosphatase inhibitors (1% Triton X-100, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, IX complete cocktail inhibitor (Roche #11 697 498 001), IX Phosphatase Inhibitor Cocktail Set II and Set III (Sigma #P5726 and #P0044)).
1050 The plates were shaken at 4°C for 5 minutes with foil top at maximum speed. An aliquot of 5 μΐ from each well was transferred to ProxiPlate™ 384-well Plus plates (PE #6008289). The plates were sealed with a foil top and frozen at -80°C and thawed when needed.
AlphaLISA. The frozen aliquots were thawed and briefly centrifuged. All antibodies and beads were diluted in IX AlphaLISA HiBlock Buffer (PE #AL004C). Biotinylated
1055 anti-phospho-EGFR (Y1068) (Cell Signaling #4031) was incubated with the lysate for 1 hour at room temperature at 1 nM final concentration. Goat anti-total EGFR (R&D Systems #AF231) was added and allowed to equilibrate for 1 hour at room temperature at 1 nM final concentration. Then, 10 μΐ of mixed beads (AlphaScreen Streptavidin Donor Beads (PE #6760002S) and AlphaLISA anti-goat IgG Acceptor Beads (PE #AL107C)) was equilibrated 1060 for 1.5 hours before reading on En Vision plate reader using the built-in settings for
AlphaScreen.
Data Analysis. Cells untreated (L858R and L858R/T790M) or EGF-induced (WT) were set to 100% maximum response. For a negative control, 10 μΜ HKI-272 was used to normalize data to 0% of maximum response. With these parameters, the IC50's for each compound in 1065 each cell line was calculated using non-linear curve fitting analysis.
EGFR Target Modulation in HI 975, H3255 and HCC827 Cell Lines
Tissue Culture. Cells were maintained in 10% FBS/RPMI supplemented with 100 μg/ml
Penicillin/Streptomycin (Hyclone #SH30236.01). The cells were harvested with 0.25%
1070 Trypsin/EDTA (Hyclone #SH30042.1), re-suspended in 5% FBS/DMEM Pen/Strep and plated at 10,000 cells per well in 50 ul of media in a 384-well black plate with clear bottoms (Greiner #789068G). The cells were allowed to incubate overnight in a 37°C, 5% C02 humidified tissue culture incubator. A 12-point test compound curve was prepared by serial diluting a 10 uM stock 1 :3 in DMSO in a 384-well compound plate (Greiner #789201L). The
1075 serial diluted compound was transferred to the plate containing cells by using a 50 nl Pin Head device (Perkin Elmer) and the cells were placed back in the incubator for 3 hours. Phospho-EGFR (Yl 173) Target Modulation Assay. The media was reduced to 20 ul using a Bio-Tek ELx 405 SelectTM plate washer. Cells were lysed with 20 ul of 2X Lysis buffer containing protease and phosphatase inhibitors (2% Triton X-100, 40 mM Tris, pH 7.5, 2
1080 mM EDTA, 2 mM EGTA, 300 mM NaCl, 2X complete cocktail inhibitor (Roche #11 697
498 001), 2X Phosphatase Inhibitor Cocktail Set II and Set III (Sigma #P5726 and #P0044)). The plates were shaken at 4°C for 20 minutes. An aliquot of 25 ul from each well was transferred to pEGFR(Yl 173) ELISA plates for analysis.
Phospho-EGFR (Yl 173) ELISA. Solid white high-binding ELISA plates (Greiner #781074) 1085 were coated with 5 ug/ml anti-EGFR capture antibody (R&D Systems #AF231) overnight in 50 mM carbonate/bicarbonate pH 9.5 buffer. Plates were blocked with 1% BSA (Sigma #A7030) in PBS for 1 hour at room temperature. Wash steps were carried out with a Bio-Tek ELx405 SelectTM using 4 cycles of 100 ul TBS-T (20 mM Tris, 137 mM NaCl, 0.05% Tween-20) per well. Media was drained and cells were lysed in 40 ul Lysis buffer containing 1090 protease and phosphatase inhibitors (1 % Triton X- 100, 20 mM Tris, pH 7.5 , 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, IX complete cocktail inhibitor (Roche #1 1 697 498 001), IX Phosphatase Inhibitor Cocktail Set II and Set III (Sigma #P5726 and #P0044)). Lysis was allowed to occur for 20 minutes on ice. A 25 ul aliquot was added to each well of the blocked ELISA plate and incubated overnight at 4oC with gentle shaking. A 1 :500
1095 anti-phospho-EGFR (Cell Signaling #4407) in 0.2% BSA/TBS-T was added and incubated for 1 hour at room temperature. After washing, 1 :2,000 anti-rabbit-HRP (Cell Signaling #7074) in 0.2% BSA/TBS-T was added and incubated for 1 hour at room temperature. Chemiluminescent detection was carried out with SuperSignal ELIS A Pico substrate
(ThermoScientific #37069). Signal was read on En Vision plate reader using built-in
1 100 UltraLUM setting.
Data Analysis. Control cells treated with EGF were set to 100% maximum response. For a negative control, 10 uM afatinib (BIBW2992) was used to normalize data to 0% of maximum response. With these parameters, the IC50's for each compound in each cell line was calculated using non-linear curve fitting analysis.
1 105
EGFR Target Modulation in HEKn (EGFR-WT) Cell Line
Tissue Culture. Human neonatal epithelial keratinocytes (Invitrogen #C-001-5C) were maintained in EpiLife media (Invitrogen #M-EPI-500-CA) supplemented with growth factors (Invitrogen #S-001 -5). Cells were harvested with 0.05% Trypsin/EDTA (Hyclone
1 1 10 #SH30236.01) and quenched with 5% FBS/EpiLife media. A density of 7500 cells in 50 ul of EpiLife media (no growth factors) was plated in each well of a 384-well solid black tissue culture plate (Greiner #789168G) and incubated overnight at 37oC, 5% C02 in a humidified tissue culture incubator. A 12-point test compound curve was prepared by serial diluting a 10 uM stock 1 :3 in DMSO in a 384-well compound plate (Greiner #789201L). The serial diluted
1 1 15 compound was transferred to the plate containing cells by using a 50 nl Pin Head device (Perkin Elmer) and the cells were placed back in the incubator for 3 hours. Stimulation of EGFR autophosphorylation was carried out with 10 ng/ml EGF (Preprotech #AF-100-15) for 5 minutes in the tissue culture incubator.
Phospho-EGFR (Yl 173) ELIS A. Solid white high-binding ELISA plates (Greiner #781074)
1 120 were coated with 5 ug/ml anti-EGFR capture antibody (R&D Systems #AF231) overnight in 50 mM carbonate/bicarbonate pH 9.5 buffer. Plates were blocked with 1% BSA (Sigma #A7030) in PBS for 1 hour at room temperature. Wash steps were carried out with a Bio-Tek ELx405 SelectTM using 4 cycles of 100 ul TBS-T (20 mM Tris, 137 mM NaCl, 0.05% Tween-20) per well. Media was drained and cells were lysed in 40 ul Lysis buffer containing
1 125 protease and phosphatase inhibitors (1 % Triton X- 100, 20 mM Tris, pH 7.5 , 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, IX complete cocktail inhibitor (Roche #1 1 697 498 001), IX Phosphatase Inhibitor Cocktail Set II and Set III (Sigma #P5726 and #P0044)). Lysis was allowed to occur for 20 minutes on ice. A 25 ul aliquot was added to each well of the blocked ELISA plate and incubated overnight at 4oC with gentle shaking. A 1 :500
1 130 anti-phospho-EGFR (Cell Signaling #4407) in 0.2% BSA TBS-T was added and incubated for 1 hour at room temperature. After washing, 1 :2,000 anti-rabbit-HRP (Cell Signaling #7074) in 0.2% BSA TBS-T was added and incubated for 1 hour at room temperature.
Chemiluminescent detection was carried out with SuperSignal ELISA Pico substrate
(ThermoScientific #37069). Signal was read on En Vision plate reader using built-in
1 135 UltraLUM setting.
Data Analysis. Control cells treated with EGF were set to 100% maximum response. For a negative control, 10 uM afatinib (BIBW2992) was used to normalize data to 0% of maximum response. With these parameters, the IC50's for each compound in each cell line was calculated using non-linear curve fitting analysis.
1 140
EGFR Target Modulation in HaCaT (EGFR-WT) Cell Line
Tissue Culture. HaCaT cells were maintained in 10% FBS RPMI supplemented with 100 μg/ml Penicillin/Streptomycin (Hyclone #SH30236.01 ). The cells were harvested with 0.25% Trypsin/EDTA (Hyclone #SH30042.1 ), re-suspended in 5% FBS/DMEM Pen/Strep and
1 145 plated at 10,000 cells per well in 50 ul of media in a 384-well black plate with clear bottoms (Greiner #789068G). The cells were allowed to incubate overnight in a 37°C, 5% C02 humidified tissue culture incubator, with A 12-point test compound curve was prepared by serial diluting a 10 uM stock 1 :3 in DMSO in a 384-well compound plate (Greiner
#789201L). The serial diluted compound was transferred to the plate containing cells by
1 150 using a 50 nl Pin Head device (Perkin Elmer) and the cells were placed back in the incubator for 3 hours. Stimulation of EGFR autophosphorylation was carried out with 10 ng/ml EGF (Preprotech #AF-100-15), prepared in 2% FBS/PBS for 5 minutes in the tissue culture incubator.
Phospho-EGFR (Yl 173) ELISA. Solid white high-binding ELISA plates (Greiner #781074)
1 155 were coated with 5 ug/ml anti-EGFR capture antibody (R&D Systems #AF231) overnight in 50 mM carbonate/bicarbonate pH 9.5 buffer. Plates were blocked with 1% BSA (Sigma #A7030) in PBS for 1 hour at room temperature. Wash steps were carried out with a Bio-Tek ELx405 SelectTM using 4 cycles of 100 ul TBS-T (20 mM Tris, 137 mM NaCl, 0.05% Tween-20) per well. Media was drained and cells were lysed in 40 ul Lysis buffer containing
1 160 protease and phosphatase inhibitors (1 % Triton X- 100, 20 mM Tris, pH 7.5 , 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, IX complete cocktail inhibitor (Roche #1 1 697 498 001), IX Phosphatase Inhibitor Cocktail Set II and Set III (Sigma #P5726 and #P0044)). Lysis was allowed to occur for 20 minutes on ice. A 25 ul aliquot was added to each well of the blocked ELISA plate and incubated overnight at 4oC with gentle shaking. A 1 :500
1 165 anti-phospho-EGFR (Cell Signaling #4407) in 0.2% BSA/TBS-T was added and incubated for 1 hour at room temperature. After washing, 1 :2,000 anti-rabbit-HRP (Cell Signaling #7074) in 0.2% BSA/TBS-T was added and incubated for 1 hour at room temperature.
Chemiluminescent detection was carried out with SuperSignal ELISA Pico substrate (ThermoScientific #37069). Signal was read on EnVision plate reader using built-in
1 170 UltraLUM setting.
Data Analysis. Control cells treated with EGF were set to 100% maximum response. For a negative control, 10 uM afatinib (BIBW2992) was used to normalize data to 0% of maximum response. With these parameters, the IC50's for each compound in each cell line was calculated using non-linear curve fitting analysis.
1 175
Biological Results
- IC50 determinations for the
(i?,£')-N-(7-chloro-l -(l -(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-lH-benzo[d]imidazol-2 -yl)-2-methylisonicotinamide obtained from a EGFR biochemical assay as described above 1 180 from EGFR (L858R/T790M) without and with 90-minute pre-incubation were 0.008μΜ and Ο.ΟΟΙ μΜ, respectively.
- The compound of the disclosure shows an inhibition IC50 determinations obtained from EGFR target modulation in engineered NIH/3T3 cell lines for L858R/T790M and L858R, 0.01 Ι μΜ and 0.015μΜ, respectively. For wt the value was 0.259μΜ.
1 185 - The IC50 determinations obtained from EGFR target modulation in HI 975 (EGFR
L858/T790M), H3255 (EGFR L858R), and HEKn (EGFR WT) cell lines were Ο.ΟΒμΜ, 0.030μΜ and 1 .180μΜ respectively.
Example 3. Crystalline Mesylate form B
1 190
(i?,£')-N-(7-chloro-l -(l -(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-lH-benzo[d]imidazol-2 -yl)-2-methylisonicotinamide as obtained in Example 1 (1.0 g) was dissolved in acetone (30 mL) by heating to 55°C to form a solution. Methanesulfonic acid (325 μί) was added to acetone (50 mL), and the methanesulfonic acid/acetone (22.2 mL) was added to the solution
1 195 at 0.05ml/min. Following precipitation, the resulting suspension was cooled to room
temperature at 0.5 °C/min, and crystals were collected by filtration, and dried for 4 hours at 40°C under vacuum. The collected crystals (300 mg) were suspended in acetone/FLO (6 mL; v/v=95/5) by heating to 50°C. The suspension was kept slurrying for 16 hours, and cooled to room temperature at 0.5 °C/min. The crystal was collected by filtration and dried
1200 for 4 hours at 40°C under vacuum.
The structure of
(i?,£')-N-(7-chloro-l -(l -(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-lH-benzo[d]imidazol-2 -yl)-2-methylisonicotinamide mesylate was confirmed by Differential Scanning Calorimetry, X-Ray Powder Diffraction, and Elemental Analyses. Melting point (170.1 °C). Theoretical 1205 calculated: %C (54.8); %H (5.9); %N (14.2); %0 (13.5); %S (5.4); and %C1 (6.0); C:N ratio:
3.86. Found: %C (52.0); %H (5.8); %N (13.3); %C1 (5.9); C:N ratio : 3.91 . Stoichiometry: 1.01 .
In addition, crystalline mesylate form B was prepared by suspending 300mg of crystalline 1210 mesylate form A in 6mL of acetone/H20 (v/v=95/5) by heating to 50°C. The suspension was kept slurrying for 16 hours, and then the suspension was allowed to cool to room temperature at 0.5 °C/min. The crystal was collected by filtration and afterwards dried for 4 hours at 40°C under vacuum.
1215 Example 4. Crystalline Mesylate form A (mesylate monohydrate form)
5.0 mL of dried acetone and 800 mg of mesylate form B (mesylate trihydrate Form) as obtained in example 3 were added into a glass vial. The suspension was heated to 55 °C for 5 hours. DSC was checked to see if the transformation was complete. Another 800 mg of the 1220 mesylate form B was converted to mesylate form A with the same method, the only difference was that the suspension was allowed to equilibrate at 20 °C (the ambient temperature in the lab), overnight.
In addition, crystalline mesylate form A was prepared by dissolving 1 .0g of free form A in 1225 30mL of acetone by heating to 55°C. 325μL· of methansulfonic acid was added to 50mL of acetone and then 22.2mL of methansulfonic acid acetone was added to free form solution at 0.05ml/min. Precipitation was formed during the addtion of methansulfonic acid, and the suspension was allowed to cool to room temperature at 0.5 °C/min. The crystal was collected by filtration and afterwards dried for 4 hours at 40°C under vacuum.
1230
Example 5. Crystalline free form A
750mg of EGFRi HC1 salt form (purity: 99%) were dissolved in 15mL of mixed solvent 1235 (EtOH/H20, v/v=l/9) by heating to 60°C. 7.42mL sodium hydroxide (0.2mol/L in
EtOH/H20, v/v=l/9) was added to the HC1 salt form EtOH/ H20 solution at 0.05ml/min. Precipitation was formed during the addition of sodium hydroxide, and the suspension was allowed to cool to room temperature at 0.5 °C/min. The crystal was collected by filtration and afterwards dried for 4 hours at 40°C under vacuum.
1240
Example 6. Crystalline free form B
Crystalline free form A or the compound obtained in example 1 (2.5 g, 5.0 mmol) was dissolved into 4 mL acetone: water 5 :1 (v/v) at r.t. to result a clear solution. The solution was 1245 stirred for 24 h and white precipitate appeared after the first 1 h. The white solid was
separated with filtration then washed with 500 uL acetone:water 5 :1 (v/v) three times to remove black color. After dried under vacuum at 40 °C overnight, EGFRi free form B (2.0 g, 4.0 mmol) was obtained as white solid in 80% yield.
1250 Example 7. Crystalline HC1 salt form
1.0g of amorphous form or free form of the Compound EGFRi were dissolved in 50mL of acetone by heating to 55°C. 22.2mL of hydrochloride acid in acetone (O.l mol/L) was added to free form solution at 0.05ml/min. Precipitation was formed during the addition of
1255 hydrochloride acid, and the suspension was allowed to cool to room temperature at
0.5°C/min. The crystal was collected by filtration and afterwards dried for 4 hours at 40°C under vacuum.
Also, 850mg of amorphous form or free form of the EGFRi compound were weighed out in a 1260 20ml vial. 4.25ml of Acetonitrile were added to completely dissolve the compound. To this solution 6.86 ml of 0.6 N HCl were slowly added while stirring the solution. The solution turned yellow and solids precipitated out after 15mins. The solution was stirred for 15 mins and then let to stand without stirring overnight. The solution was filtered and dried under vacuum at 40 °C for 8hrs. A yellow solid was obtained as the final product.
Example 8. Evaporation of completely dissolved samples at 25 degree C
Mesylate form B of the EGFRi compound were completely dissolved in various solvents. The solvent was then evaporated and the obtained form measured by XRPD.
Solvents XRPD
acetonitrile/H20 = 1/1 (v/v) Mesylate form B
acetone/water = 1/1 (v/v) Mesylate form B
isopropyl alcohol/water = 1/1 (v/v) Mesylate form B
acetonitrile Mesylate form B
ethanol Mesylate form B
water Mesylate form B
propyl alcohol Mesylate form B
dichloromethane Mesylate form A
methanol Mesylate form B
Example 9. Precipitation by addition of antisolvent at 25 degree C
Mesylate form B of the EGFRi compound were completely dissolved in various solvents. The compound was then precipitated by addition of an antisolvent and the obtained form measured by XRPD. The following solvent additions were performed at 25°C
Solvent Antisolvent Ratio XRPD
Methanol tetrahydro furan 1 4 Mesylate forms A and B
1290 Ethyl acetate 1 5 Mesylate form B
tert-butylmethylether 1 10 Mesylate form A
DMF tetrahydro furan 1 5 Mesylate form B
ethyl acetate 1 1.5 Mesylate form B
tert-butylmethylether 1 10 Mesylate form B
1295 toluene 1 10 Mesylate form B
95% EtOH ethyl acetate 1 3.8 Mesylate form B
1300
Example 10. Stability data for free form B and mesylate form B, both crystalline forms
Test Conditions Salt Form Form free Form B mesylate Form B
Init .ial UPLC p furity j ( \%) n, n gng g _ w .h.it.e so .l.id.
99.5, yellow solid
color
Tl (total impurity) CL (color) Tl (total impurity) CL (color)
[%] [%]
0.1 % solutions or suspensions, 1 week, 80 °C
pH 1 (0.1 N HCI) 27.0 *c 90.4 *c pH 2 (0.01 N HCI) 7.4 *A 10.3 *A pH 4.7 acetate buffer 4.7 *c 4.9 *A pH 6.8 phosphate buffer 55.0 4A 51 .0 *A pH 9.0 borate buffer 81 .1 4A 88.9 4A pH 10.0 borate buffer 86.7 4A 93.8 4A
Water 83.3 4A 20.1 ^A
Ethanol 25.5 *c 4.7 ^A
IPA 50.7 *A 37.1 *c
Acetone 9.1 *A 5.1 *c
THF 44.1 *A 94.2 *c
DCM 81 .7 *c 7.2 *A
5% solutions, 1 day, r.t. (room temperature)
DMSO
*A 0.2 *A
Dilution, pH 6.8
Solid state, 1 week, 80 °C, tight container
Bulk (UPLC) 40.5 C 0.6 A
Bulk (XRPD) change to free Form A change to Mesylate Form A
Solid state, 1 week, 50 °C, tight container
Bulk (UPLC) 2.1 0.4
Bulk (XRPD) no change no change
Mixture, 1 week, 50 °C, tight container
1 % in mixture 1 11 .3 A 0.5 A
1 % in mixture 2 42.7 A 3.1 A
Solid state, 1 week, 80 °C, 75% RH
Bulk (UPLC) 4.0 B 1 .0 A
Bulk (XRPD) no change no change
Solid state, 1 week, 50 °C, 75% RH
Bulk (UPLC) 0.8 A 0.4 A
Bulk (XRPD) no change no change
Mixture, 1 week, 50 °C, 75% RH
1 % in mixture 1 2.2 A 0.5 A
1 % in mixture 2 1 .6 A 0.3 A
Total impurity(TI) and color (CL) ■i- Suspension * Clear solution after stress test
Test not performed A No change of color
B Slight discoloration C Medium discoloration
D Strong discoloration
TIs are analyzed by UPLC (method as described above). They are calculated as area-% products or against external standard 1%).
Purity: 100% - sum by- and total impurity
Compositions of the excipient mixtures [mass-%]
Mixture 1 : Lactose, spray dried (53.7%), Microcrystalline cellulose PH102 (40%),
Crospovidone XL (5%), Aerosil (0.3%), Mg stearate (1%)
Mixture 2: Mannitol DC (68.7%), Microcrystalline cellulose PH102 (26%), Ac-di-Sol
(4%), Aerosil (0.3%), Ca stearate (1%)
1305
Neither the free Form B nor the Mesylate Form B is stable in tested aqueous solutions at 80 °C for 1 week. Significant degradation occured in pH 1.0 (0.1 N HC1), pH 6.8, pH 9.0 and pH 10.0 buffer solutions, and in water with more than 20% TI (total impurity) after the test. Both forms are relatively more stable in pH 2.0 (0.01 N HC1) solution and pH 4.7 acetate buffer
1310 solution with 7.4% and 4.7% TI for the free Form B, and 10.3% and 4.9% TI for the Mesylate Form B, respectively. The free Form B and the Mesylate Form B are also unstable in tested organic solvents of ethanol, IP A, acetone, THF and DCM at 80 °C for 1 week. The free Form B degraded by 9.1% in acetone. The Mesylate Form B degraded by 4.7%, 5.1% and 4.2% in ethanol, acetone and DCM, respectively. The two forms degraded by more than
1315 20% in the other organic solvents. Therefore, both forms should be avoided to contact the tested solvents at high temperature for a long period.
Example 11. Stability data for free form A and mesylate form A, both crystalline forms
Test Conditions Salt Form
Form free Form A mesylate Form A
Initial UPLC purity (%),
99.9, white solid 99.5, yellow solid
color
TI (total impurity) CL (color) TI (total impurity) CL (color) [%] [%]
0.1 % solutions or suspensions, 1 week, 80 °C
pH 1 (0.1 N HCI) 97.34 *A 97.84 *A pH 2 (0.01 N HCI) 8.34 *B 1 1 .44 *B pH 4.5 acetate buffer 1 .58 *B 6.23 *B Test Conditions Salt Form
Form free Form A mesylate Form A
Initial UPLC purity (%),
99.9, white solid 99.5, yellow solid
color
Tl (total impurity) CL (color) Tl (total impurity) CL (color) [%] [%]
pH 6.8 phosphate buffer 34.95 4A 43.23 4A pH 9.0 borate buffer 85.18 4A 74.81 4A
Water 84.07 4A 14.42 4A
Acetone 0.36 *A 0.62 *A
1320
Total impurity(TI) and color (CL)
■i- Suspension * Clear solution after stress test
Test not performed A No change of color
B Slight discoloration C Medium discoloration
D Strong discoloration
TIs are analyzed by UPLC (method as described above). They are calculated as area-% products or against external standard 1%).
Purity: 100% - sum by- and total impurity
Examples 10 and 1 1 show that stability of the EGFRi forms deteriorates significantly at pH below 1 and above 6.8, and even more so above pH 9. Therefore, it is best to modulate pH in
1325 a pharmaceutical composition to keep pH within the range between pH 1 and pH 9, even better within the range between pH 1 and pH 6.8. This can be achieved by using a pH modifier. As a pH modifier can be used for example buffers as exemplified in the examples. Excipients attaining the same effect are expected to work as well. Best stability is achieved if the micro environment of the EGFRi is between pH 2 and pH 4.7±0.2, particularly pH 2 and
1330 pH 4.7.
Example 12. Solubility and pH of forms pH of 1% solution or suspension
Free form A Mesylate form A
In water 8.13 5.38 Solubility (approx. at 25°C, mg/ml) [pH final]
1340 Free form A Mesylate form A
0.1 HC1 > 11.99 [1.66] > 9.92 [1.84]
Citrate buffer, pH 3 > 11.06 [3.92] > 10.13 [3.40]
Acetate buffer, pH 4.5 > 10.20 [5.01 ] > 9.87 [4.91]
Phosphate buffer, pH 6.8 0.51 [7.37] > 9.62 [7.16]
1345 Water 0.29 [7.65] > 9.97 [5.45]
Example 13. In vivo exposure of HCl salt form and mesylate forms
PK studies for mesylate form and HCl salt form were conducted in rat. Both forms were 1350 dosed orally at 10 and 30 mg/kg in 0.5% MC/Tween 80 formulation. Both salt forms were soluble at both dose levels in the formulation. Plasma exposures of both forms and its active metabolite were determined. The mesylate form showed very similar PK profiles in the rats at both 10 and 30 mg/kg as those of HCL salt form. The exposures of both parent compound forms and active metabolite are summarized below.
1355
Salt Form Dose (mg/kg) AUC (nM*hr) Metabolite to parent ratio
HCl salt 10 9923 12%
30 23687 13%
Mesylate forms 10 8267 1 1 %
30 24092 14%
The mesylate crystalline forms show dose proportionality from 10 to 30mg/kg, and the exposure is comparable to HCl salt.

Claims

1360 CLAIMS
1. A crystalline form of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 1365 -yl)-2-methylisonicotinamide comprising:
(i) free form A,
(ii) free form B,
(iii) mesylate form A,
(iv) mesylate form B,
1370 (v) mesylate form C,
(vi) HC1 salt form, or
(vii) amorphous form,
wherein when the crystalline form of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 1375 -yl)-2-methylisonicotinamide comprises mesylate form B, the crystalline form further
comprises also at least one of the forms selected from the group of free form A, free form B, mesylate form A, mesylate form C and HC1 salt form.
2. The crystalline form according to claim 1 consisting essentially of
1380 (i) free form A,
(ii) free form B,
(iii) mesylate form A,
(v) mesylate form C, or
(vi) HC1 salt form.
1385
3. The crystalline form according to claim 2, wherein said forms are in substantially pure form.
4. The crystalline form according to any one of claims 1 to 3, wherein x-ray powder 1390 diffraction pattern comprises 4 or more 2Θ values selected from the group consisting of:
(a) 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30 for free form A,
(b) 1 1.24, 14.68, 15.66, 18.64, 19.63, 20.05, 20.31 , 22.41 , 24.91 , 25.43, 26.03, 27.18 and 28.07± 0.30 for free form B,
1395 (c) 9.12, 12.19, 16.25, 17.38, 18.42, 19.91 , 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 0.30 for mesylate form A,
(d) 1 1.76, 13.832, 14.41 , 15.9 17.65, 18.79, 21.46, 21.83, 22.30, 23.82, 24.51 , 24.89, 25.57, 26.66 and 27.77 ± 0.30 for mesylate form B,
(e) 1 1.51 , 13.19, 16.23, 17.50, 19.14, 23.15, 23.80, 24.65, 25.81 , 26.06, 26.70 and 28.97 ± 1400 0.30 for mesylate form C, or (f) 14.89, 19.69, 25.61 , 26.13 and 27.78 ± 0.30 for HC1 salt form,
all at a temperature of about 22°C.
1405 5. The crystalline form according to claim 4, wherein x-ray powder diffraction pattern
comprises five or more 2Θ values selected from the group.
6. The crystalline form according to claim 4, wherein x-ray powder diffraction pattern comprises all 2Θ values selected from the group for a respective form.
1410
7. A crystalline form of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide having a X-ray diffraction spectrum essentially the same as the X-ray powder diffraction spectrum shown in FIG. 1 , FIG. 4, FIG. 8, FIG. 1 1 , FIG. 14, or FIG. 1415 17.
8. A crystalline form of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide having a differential scanning calorimetry (DSC) thermogram 1420 essentially the same as that shown in shown in any one of figures FIG. 2, FIG. 5, FIG. 6, FIG.
9. FIG. 12, FIG. 15, or FIG 18.
9. A crystalline form of
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 1425 -yl)-2-methylisonicotinamide having a thermo gravimetric analysis (TGA) diagram
essentially the same as that shown in any one of figures FIG. 2, FIG. 7, FIG. 9, FIG. 12, FIG. 15, or FIG 18.
10. A pharmaceutical composition comprising the crystalline form according to any one of 1430 claims 1 to 9.
11. The pharmaceutical composition according to claim 10, further comprising a
pharmaceutically acceptable carrier or diluent.
1435 12. The pharmaceutical composition according to claim 10 or 11 , wherein the crystal form is as defined in any one of claims 4 to 6.
13. The pharmaceutical composition according to claim 12, wherein said form is free form A, free form B, mesylate form A, mesylate form C or HC1 salt form and wherein x-ray powder 1440 diffraction pattern comprises four 2Θ values selected from the group consisting of:
(a) 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30 for free form A,
(b) 11.24, 14.68, 15.66, 18.64, 19.63,20.05,20.31,22.41,24.91,25.43,26.03,27.18 and 28.07± 0.30 for free form B,
1445 (c)9.12, 12.19, 16.25, 17.38, 18.42, 19.91, 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 0.30 for mesylate form A,
(e) 11.51, 13.19, 16.23, 17.50, 19.14, 23.15,23.80, 24.65, 25.81, 26.06, 26.70 and 28.97 ± 0.30 for mesylate form C, or
(f) 14.89, 19.69,25.61,26.13 and 27.78 ± 0.30 for HC1 salt form,
1450 all at a temperature of about 22°C, respectively.
14. The pharmaceutical composition according to claim 12, wherein said form is free form A, free form B, mesylate form A, mesylate form C or HC1 salt form and wherein x-ray powder diffraction pattern comprises five 2Θ values selected from the group consisting of:
1455 (a) 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30 for free form A,
(b) 11.24, 14.68, 15.66, 18.64, 19.63,20.05,20.31,22.41,24.91,25.43,26.03,27.18 and 28.07± 0.30 for free form B,
(c) 9.12, 12.19, 16.25, 17.38, 18.42, 19.91, 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 1460 0.30 for mesylate form A,
(e) 11.51, 13.19, 16.23, 17.50, 19.14, 23.15,23.80, 24.65, 25.81, 26.06, 26.70 and 28.97 ± 0.30 for mesylate form C, or
(f) 14.89, 19.69,25.61,26.13 and 27.78 ± 0.30 for HC1 salt form,
all at a temperature of about 22°C, respectively.
1465
15. The pharmaceutical composition according to claim 12, wherein said form is free form A, free form B, mesylate form A, mesylate form C or HC1 salt form and wherein x-ray powder diffraction pattern comprises all 2Θ values selected from the group consisting of:
(a) 5.46, 14.82, 15.84, 18.67, 20.42, 22.56, 23.77, 25.09, 25.73, 26.35 and 27.56 ± 0.30 for 1470 free form A,
(b) 11.24, 14.68, 15.66, 18.64, 19.63,20.05,20.31,22.41,24.91,25.43,26.03,27.18 and 28.07± 0.30 for free form B,
(c) 9.12, 12.19, 16.25, 17.38, 18.42, 19.91, 20.85, 22.43, 22.92, 24.10, 26.64 and 27.78 ± 0.30 for mesylate form A,
1475 (e) 11.51, 13.19, 16.23, 17.50, 19.14, 23.15, 23.80, 24.65, 25.81, 26.06, 26.70 and 28.97 ± 0.30 for mesylate form C, or
(f) 14.89, 19.69,25.61,26.13 and 27.78 ± 0.30 for HC1 salt form,
all at a temperature of about 22°C, respectively.
1480 16. A pharmaceutical composition comprising
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide and a pH modifier.
17. A pharmaceutical composition comprising mesylate form B and a pH modifier.
1485
18. A pharmaceutical composition comprising amorphous form and a pH modifier.
19. The pharmaceutical composition according to any one of claims 10 to 16, further comprising a pH modifier.
1490
20. The pharmaceutical composition according to any one of claims 16 to 19, wherein the pH modifier is pharmaceutically acceptable acid, base, salt or mixtures thereof.
21. The pharmaceutical composition according to any one of claims 10 to 20, wherein the 1495 composition is liquid.
22. The pharmaceutical composition according to any one of claims 10 to 21 , wherein the (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide is in any form as defined in any one of claims 1 -9.
1500
23. The pharmaceutical composition according to any one of claims 10 to 22, wherein the pH of the formulation is between pH 1 and pH 9, measured at 25°C.
24. The pharmaceutical composition according to any one of claims 10 to 22, wherein the pH 1505 of the formulation is between pH 2 and pH 9, measured at 25°C.
25. The pharmaceutical composition according to any one of claims 10 to 22, wherein the pH of the formulation is between pH 2 and pH 6.8, measured at 25°C.
1510 26. The pharmaceutical composition according to any one of claims 10 to 22, wherein the pH of the formulation is between pH 2 and pH 4.7, measured at 25°C.
27. The pharmaceutical composition according to any one of claims 10 to 22, wherein the pH of the formulation is about 4.5, or pH is 4.5, measured at 25°C.
1515
28. A pharmaceutical formulation comprising
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide, wherein 1 g of the pharmaceutical formulation dissolved, suspended or slurried in 1 mL of water has pH of between pH 1 and pH 9.
1520
29. A pharmaceutical formulation comprising
(R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide, wherein 1 w/w % solution or suspension of the pharmaceutical formulation, calculated based on the
1525 (R,E)-N-(7-chloro- 1 -( 1 -(4-(dimethylamino)but-2 -enoyl)azepan-3 -yl)- 1 H-benzo [d] imidazo 1-2 -yl)-2-methylisonicotinamide in the pharmaceutical formulation, has pH of between pH 1 and pH 9.
30. The pharmaceutical formulation according claim 28 or 29, wherein pH is between pH 1 1530 and pH 6.8, measured at 25°C.
31. The pharmaceutical formulation according claim 28 or 29, wherein pH is between pH 2 and pH 6.8, measured at 25°C.
1535 32. The pharmaceutical formulation according claim 28 or 29, wherein pH is between pH 2 and pH 4.7, measured at 25°C.
33. The pharmaceutical formulation according claim 28 or 29, wherein pH is about pH 4.5, measured at 25°C.
1540
34. Crystalline form according to any one of claims 1 to 9, pharmaceutical composition according to any one of claims 10 to 27, or pharmaceutical formulation according to any one of claims 28 to 33, for use as a medicine.
1545 35. Crystalline form according to any one of claims 1 to 9, pharmaceutical composition
according to any one of claims 10 to 27, or pharmaceutical formulation according to any one of claims 28 to 33, for use in the treatment of proliferative disease.
36. Crystalline form according to any one of claims 1 to 9, pharmaceutical composition 1550 according to any one of claims 10 to 27, or pharmaceutical formulation according to any one of claims 28 to 33 for use according to claim 35, wherein the proliferative disease is non-small cell lung cancer, head and neck cancer, colorectal cancer, breast cancer, pancreatic cancer, ovarian cancer, gastric cancer, glioma or prostate cancer.
1555 37. Crystalline form according to any one of claims 1 to 9, pharmaceutical composition
according to any one of claims 10 to 27, or pharmaceutical formulation according to any one of claims 28 to 33 for use according to claim 35 or 36, wherein the proliferative disease is non-small cell lung cancer.
1560 38. Crystalline form according to any one of claims 1 to 9, pharmaceutical composition
according to any one of claims 10 to 27, or pharmaceutical formulation according to any one of claims 28 to 33 for use according to any one of claims 35 to 37, wherein the form is administered to a human.
1565
PCT/CN2013/088295 2013-12-02 2013-12-02 Egfr inhibitor forms WO2015081463A1 (en)

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Publication number Priority date Publication date Assignee Title
WO2017175144A1 (en) * 2016-04-08 2017-10-12 Novartis Ag New therapeutic uses
US11395821B2 (en) 2017-01-30 2022-07-26 G1 Therapeutics, Inc. Treatment of EGFR-driven cancer with fewer side effects

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WO2004014905A1 (en) * 2002-08-08 2004-02-19 Boehringer Ingelheim Pharmaceuticals, Inc. Substituted benzimidazole compounds
CN1612733A (en) * 2001-11-09 2005-05-04 贝林格尔·英格海姆药物公司 Benzimidazoles useful as protein kinase inhibitors
US20080021069A1 (en) * 2004-10-08 2008-01-24 Takeda Pharmaceutical Company Limited Receptor Function Regulating Agent
WO2013184757A1 (en) * 2012-06-06 2013-12-12 Irm Llc Compounds and compositions for modulating egfr activity

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CN1612733A (en) * 2001-11-09 2005-05-04 贝林格尔·英格海姆药物公司 Benzimidazoles useful as protein kinase inhibitors
WO2004014905A1 (en) * 2002-08-08 2004-02-19 Boehringer Ingelheim Pharmaceuticals, Inc. Substituted benzimidazole compounds
US20080021069A1 (en) * 2004-10-08 2008-01-24 Takeda Pharmaceutical Company Limited Receptor Function Regulating Agent
WO2013184757A1 (en) * 2012-06-06 2013-12-12 Irm Llc Compounds and compositions for modulating egfr activity

Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2017175144A1 (en) * 2016-04-08 2017-10-12 Novartis Ag New therapeutic uses
US11395821B2 (en) 2017-01-30 2022-07-26 G1 Therapeutics, Inc. Treatment of EGFR-driven cancer with fewer side effects

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