WO2017097891A1

WO2017097891A1 - Crystalline eravacycline

Info

Publication number: WO2017097891A1
Application number: PCT/EP2016/080232
Authority: WO
Inventors: Hannes Lengauer
Original assignee: Sandoz Ag
Priority date: 2015-12-10
Filing date: 2016-12-08
Publication date: 2017-06-15

Abstract

The invention relates to crystalline eravacycline and to a process for its preparation. Furthermore, the invention relates to the use of crystalline eravacycline for the preparation of a pharmaceutical composition. The pharmaceutical composition of the present invention can be used as a medicament, in particular for treatment and/or prevention of bacterial infections such as caused by Gram negative pathogens, in particular by multidrug resistant Gram negative pathogens. The pharmaceutical composition of the present invention can thus be used as a medicament for e.g. the treatment of complicated intra-abdominal and urinary tract infections.

Description

CRYSTALLINE ERAVACYCLINE

FIELD OF THE INVENTION

The invention relates to crystalline eravacycline and to a process for its preparation. Furthermore, the invention relates to the use of crystalline eravacycline for the preparation of a pharmaceutical composition. The pharmaceutical composition of the present invention can be used as a medicament, in particular for treatment and/ or prevention of bacterial infections such as caused by Gram negative pathogens, in particular by multidrug resistant Gram negative pathogens. The pharmaceutical composition of the present invention can thus be used as a medicament for e.g. the treatment of complicated intra-abdominal and urinary tract infections. BACKGROUND OF THE INVENTION

Eravacycline is a tetracycline antibiotic chemically designated (4S,4aS,5aR,l2aS)-4- (Dimethylamino)-7-fluoro-3 ,10,12,12a-tetrahydroxy- 1,11 -dioxo-9-[2(-pyrrolidin- 1 - yl)acetamido]-l,4,4a,5,5a,6,l l ,12a-octahydrotetracene-2-carboxamide and can be represented by the following chemical structure according to formula (A).

formula (A)

Eravacycline possesses antibacterial activity against Gram negative pathogens, in particular against multidrug resistant (MDR) Gram negative pathogens and is currently undergoing phase III clinical trials in patients suffering from complicated intra-abdominal infections (cIAI) and urinary tract infections (cUTI). The established mode of administration for tetracycline antibiotics is the intravenous route. Eravacycline, however, is also intended to be taken orally.

WO 2010/017470 Al discloses eravacycline as compound 34. Eravacycline is described to be prepared according to a process, which is described in more detail only for related compounds. The last step of this process involves column chromatography with diluted hydrochloric acid/ acetonitrile, followed by freeze drying. WO 2012/021829 Al discloses pharmaceutically acceptable acid and base addition salts of eravacycline in general and a general process for preparing the same involving reacting eravacycline free base with the corresponding acids and bases, respectively. On page 15, lines 3 to 6, a lyophilized powder containing an eravacycline salt and mannitol is disclosed. WO 2016/065290 Al discloses processes for the preparation of eravacycline. In example 2, the product was isolated as a dark yellow solid from a slurry in acetone: H2O (50: 1, v/v). In example 3, eravacycline was obtained as bis-hydrochloride salt after purification by preparative HPLC using acetonitrile and 0.05N HC1 as mobile phases. Xiao et. al. "Fluorocyclines. 1. 7-Fluoro-9-pyrrolidinoacetamido-6-demethyl-6- deoxytetracycline: A Potent, Broad Spectrum Antibacterial Agent" J. Med. Chem. 2012, 55, 597-605 synthesized eravacycline following the procedure for compounds 17e and 17i on page 603. After preparative reverse phase HPLC, compounds 17e and 17i were both obtained as bis- hydrochloride salts in form of yellow solids. Ronn et al. "Process R&D of Eravacycline: The First Fully Synthetic Fluorocycline in Clinical Development" Org. Process Res. Dev. 2013, 17, 838-845 describe a process yielding eravacycline bis-hydrochloride as the final product. The last step involves precipitation of eravacycline bis-hydrochloride salt by adding ethyl acetate as an antisolvent to a solution of eravacycline bis-hydrochloride in an ethanol/ methanol mixture. The authors describe in some detail the difficulties during preparation of the bis-hydrochloride salt of eravacycline. According to Ronn et al. "partial addition of ethyl acetate led to a mixture containing suspended salt and a gummy form of the salt at the bottom of the reactor. At this stage, additional ethanol was added, and the mixture was aged with vigorous stirring until the gummy material also became a suspended solid." In addition, after drying under vacuum the solid contained "higher than acceptable levels of ethanol". "The ethanol was then displaced by water by placing a tray containing the solids obtained in a vacuum oven at reduced pressure (...) in the presence of an open vessel of water." At the end eravacycline bis-hydrochloride salt containing about 4 to 6% residual moisture was obtained. The authors conclude that there is a need for additional improvements to the procedure along with an isolation step suitable for large scale manufacturing.

The cumbersome process of Ronn et al. points towards problems in obtaining eravacycline bis- hydrochloride in a suitable solid state, problems with scaleability of the available production process and problems with the isolation step of eravacycline.

It is noteworthy that eravacycline or its salts are nowhere described as being a crystalline solid and that the preparation methods used for the preparation of eravacycline are processes, like lyophilization and preparative HPLC, which typically yield amorphous material.

Amorphous solids can suffer from certain drawbacks such as low chemical purity, low chemical stability, hygroscopicity, poor isolation and powder properties, etc., which can prevent their use as active pharmaceutical ingredients. On the other hand, they often also have desirable pharmaceutical properties, such as high solubility and rapid dissolution rates. Thus, there is a need in pharmaceutical development for solid forms of an active pharmaceutical ingredient which demonstrate a favorable profile of relevant properties for formulation, such as high chemical purity, good chemical and physical stability, low hygroscopicity and high solubility in aqueous media which are indicative of a drug's in vivo behavior. SUMMARY OF THE INVENTION

The present invention solves one or more of the aforementioned problems by providing eravacycline free base in crystalline form. In particular, the invention provides crystalline eravacycline free base characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (3.2 ± 0.2)°, (5.6 ± 0.2)° and (8.5 ± 0.2)°, when measured with Cu-Kalphai,2 radiation having a wavelength of 0.15419 nm and at a temperature in the range of from 20 to 30 °C. The invention further relates to a process for the preparation of the crystalline eravacycline free base of the present invention. The invention further relates to the use of the crystalline eravacycline free base of the present invention in and/ or for the preparation of a pharmaceutical composition. The invention further relates to a pharmaceutical composition comprising an effective amount of crystalline eravacycline free base.

Definitions

In the context of the present invention the following definitions have the indicated meaning, unless explicitly stated otherwise:

As used herein the term "room temperature" refers to a temperature in the range of from 20 to 30 °C.

The term "eravacycline" as used herein refers to (45',4a5',5ai?,12a5)-4-(Dimethylamino)-7- fluoro-3 ,10,12,12a-tetrahydroxy- 1,11 -dioxo-9-[2(-pyrrolidin- 1 -yl)acetamido]- l,4,4a,5,5a,6,l l,12a-octahydrotetracene-2-carboxamide according to formula (A) disclosed herein, unless otherwise specified. The term "physical form" as used herein refers to any crystalline and/ or amorphous phase of a compound.

As used herein "solvate" refers to a crystalline form of a molecule, atom, and/ or ions that further comprises molecules of a solvent or solvents incorporated into the crystalline lattice structure. The solvent molecules in the solvate may be present in a regular arrangement and/ or a non-ordered arrangement. The solvate may comprise either a stoichiometric or nonstoichiometric amount of the solvent molecules. For example, a solvate with a nonstoichiometric amount of solvent molecules may result from partial loss of solvent from the solvate. When the solvent is water, the solvate is often referred to as a "hydrate". When the solvent is present in stoichiometric amount, the solvate may be referred to by adding greek numeral prefixes. For example, a hydrate may be referred to as monohydrate, di-hydrate, tri- hydrate etc., depending on the water/ eravacycline stoichiometry. The solvent content can be measured, for example, by gas chromatography (GC), ^-NMR or in case of water by Karl- Fischer (KF) titration.

As used herein "amorphous" refers to a solid form of a compound that is not crystalline. An amorphous compound possesses no long-range order and does not display a definitive X-ray diffraction pattern with reflections.

The term "reflection" with regards to powder X-ray diffraction as used herein, means peaks in an X-ray diffractogram, which are caused at certain diffraction angles (Bragg angles) by constructive interference from X-rays scattered by parallel planes of atoms in solid material, which are distributed in an ordered and repetitive pattern in a long-range positional order. Such a solid material is classified as crystalline material, whereas amorphous material is defined as solid material, which lacks long-range order and only displays short-range order, thus resulting in broad scattering. According to literature, long-range order e.g. extends over approximately 100 to 1000 atoms, whereas short-range order is over a few atoms only (see "Fundamentals of Powder Diffraction and Structural Characterization of Materials " by Vitalij K. Pecharsky and Peter Y. Zavalij, Kluwer Academic Publishers, 2003, page 3).

The term "essentially the same" with reference to powder X-ray diffraction means that variabilities in reflection positions and relative intensities of the reflections are to be taken into account. For example, a typical precision of the 2-theta values is in the range of ± 0.2° 2-theta, preferably in the range of ± 0.1° 2-theta. Thus, a reflection that usually appears at 3.2° 2-theta for example can appear between 3.0° and 3.4° 2-theta, preferably between 3.1 and 3.3° 2-theta on most X-ray diffractometers under standard conditions. Furthermore, one skilled in the art will appreciate that relative reflection intensities will show inter-apparatus variability as well as variability due to degree of crystallinity, preferred orientation, sample preparation and other factors known to those skilled in the art and should be taken as qualitative measure only.

The term "lath" as used herein with regards to crystal shape refers to elongated, thin and bladelike crystals.

The term "needle" as used herein with regards to crystal shape refers to acicular, thin and highly elongated crystals having similar width and breadth. The term "column" as used herein with regards to crystal shape refers to elongated, prismatic crystals with greater width and thickness than needles.

As used herein, the term "mother liquor" refers to the solution remaining after crystallization of a solid. As used herein, the term "substantially pure" with reference to a particular physical form means that the physical 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.

Crystalline eravacycline free base may be referred to herein as being characterized by graphical data "as shown in" a Figure. Such data include, for example, powder X-ray diffractograms (PXRD), Fourier transform infrared (FTIR) spectra, differential scanning calorimetry (DSC) thermograms and thermogravimetric analysis (TGA). The person skilled in the art understands that factors such as variations in instrument type, response and variations in sample directionality, sample concentration and sample purity may lead to small variations for such data when presented in graphical form, for example variations relating to the exact peak positions and intensities.

However, a comparison of the graphical data in the Figures herein with the graphical data generated for an unknown physical form and the confirmation that two sets of graphical data relate to the same crystal form is well within the knowledge of a person skilled in the art. "Reduced pressure" as used herein means a pressure in the range of from 10 mbar to 900 mbar.

"Impurities" as used herein are organic impurities as defined in the ICH tripartite guideline "Impurities in new drug substances" Q3A(R2), step4 version dated 25.10.2006. In any given sample, this means the organic components of a sample comprising eravacycline that is not eravacycline. Briefly, organic impurities can arise during the manufacturing process and/ or storage of an active pharmaceutical ingredient. They can include starting materials, byproducts, intermediates, degradation products, reagents, ligands and catalysts. In the case of eravacycline an organic impurity of particular interest is the C-4 epimer of eravacycline due to the known tendency of the tetracycline framework to epimerize at this particular position.

In the context of the present invention the content of impurities is to be determined by high performance liquid chromatography (HPLC) at 254 nm as area% of the recorded chromatogram. The method to be used is described below in the exemplifying section.

A "stabilizer" as used herein is a pharmaceutical excipient which helps to stabilize eravacycline during lyophilization. A stabilizer is an organic compound capable of reducing epimer formation in a lyophilisate wherein eravacycline and said stabilizer are the only organic compounds prepared at a pH of 4.8, when compared to a lyophilisate wherein eravacycline is the only organic compound and prepared at the same pH.

As used herein, the term "about" means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, typically within 10%, more typically within 5%, even more typically within 1% and most typically within 0.1% of the indicated value or range. Sometimes, such a range can lie within the experimental error, typical of standard methods used for the measurement and/or determination of a given value or range.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: illustrates a powder X-ray diffractogram of crystalline eravacycline free base of the present invention. The x-axis shows the diffraction position in °2-theta, the y-axis shows the number of counts in the detection unit of the diffractogram.

Figure 2: shows a photomicrographic image of eravacycline free base crystals taken with a polarized light microscope (scale bar = 100 micrometer)

Figure 3: illustrates a comparison of representative gravimetric moisture sorption isotherms of amorphous eravacycline (solid line with triangles) and crystalline eravacycline of the present invention (solid line with squares) during the sorption cycle from 0 - 65% relative humidity. The x-axis displays the relative humidity in percent (%) measured at a temperature of (25.0 ± 0.1) °C, the y-axis displays the equilibrium mass change in percent (%). The sample weight at 0% relative humidity was used as reference weight. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides eravacycline free base in crystalline form.

Crystallizing eravacycline was not straightforward. Initial attempts to crystallize eravacycline bis-hydrochloride were not successful. Also for eravacycline free base several attempts to crystallize the compound failed. Crystals were only obtained by serendipity under very specific conditions. The inventors of the present invention have surprisingly found that eravacycline free base crystallizes in the presence of a solvent comprising an aliphatic ether, such as diethyl ether, diisopropyl ether and methyl tert-butyl ether and/ or a cyclic ether like methyltetrahydrofuran. The process of the present invention for the preparation of crystalline eravacycline free base is suitable for large scale production as it does not suffer from the drawback of going through gummy material as an intermediate.

Crystalline eravacycline free base of the present invention shows excellent isolation properties such as good filtration properties, and can be easily dried after isolation. An additional advantage of the crystalline eravacycline free base of the present invention is that chemical impurities are depleted upon crystallization. Impurities, such as the C-4 epimer, are a known problem for tetracyclines, and have been described, for example, for the closely related compound tigecycline. Furthermore, crystalline eravacycline free base of the present invention shows high chemical stability and high physical stability over a broad humidity range and is less hygroscopic compared to amorphous eravacycline. A further advantage of crystalline eravacycline of the present invention is that it shows similar aqueous solubility compared to amorphous eravacycline, regardless of the pH of the solution.

Crystalline eravacycline free base thus has excellent physicochemical properties combined with high solubility in pharmaceutically relevant aqueous media. This favorable profile of relevant properties is advantageous for the use of crystalline eravacycline free base in a pharmaceutical composition, such as an oral solid dosage form, and for the preparation of a pharmaceutical composition, in particular a pharmaceutical composition comprising eravacycline, for example an eravacycline hydrochloride, intended for parenteral use. Crystalline eravacycline free base further ensures that pharmaceutical compositions comprising it have a continuous high bioavailability throughout shelf- life. Furthermore, the crystalline eravacycline free base of the present invention is a solid form of eravacycline, which is more convenient to handle during pharmaceutical processes for example which is easier to isolate in the final step of the chemical synthesis and easier to handle during formulation of a drug product. A further advantage of the crystalline eravacycline free base of the present invention is that it can be used to efficiently deplete impurities from eravacycline.

In one aspect, the present invention relates to crystalline eravacycline free base. Preferably, the present invention relates to crystalline eravacycline free base of formula (A)

formula (A)

More preferably, the present invention relates to crystalline eravacycline free base of formula

formula (A) or a solvate or a hydrate thereof.

In a preferred embodiment, crystalline eravacycline free base of the invention can be characterized by having a powder X-ray diffractogram comprising reflections at 2-Theta angles selected from:

(a) (3.2 ± 0.2)°, (5.6 ± 0.2)° and (8.5 ± 0.2)°; or

(b) (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)° and (8.5 ± 0.2)°; or

(c) (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (8.5 ± 0.2)° and (12.4 ± 0.2)°; or

(d) (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (8.5 ± 0.2)°, (12.4± 0.2)° and (12.7 ± 0.2)°; or

(e) (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (8.5± 0.2)°, (12.4 ± 0.2)°, (12.7 ± 0.2)° and (24.8 ± 0.2)°; or

(f) (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (7.4 ± 0.2)°, (8.5 ± 0.2)°, (12.4 ± 0.2)°, (12.7 ± 0.2)° and (24.8 ± 0.2)°; or

(g) (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (7.4 ± 0.2)°, (8.5 ± 0.2)°, (12.4 ± 0.2)°, (12.7 ± 0.2)°, (12.9 ± 0.2)° and (24.8 ± 0.2)°; or

(h) (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (7.4 ± 0.2)°, (8.5 ± 0.2)°, (12.4 ± 0.2)°, (12.7 ± 0.2)°, (12.9 ± 0.2)°, (22.8 ± 0.2)° and (24.8 ± 0.2)°; when measured with Cu-Kalphai,2 radiation at a wavelength of 0.15419 nm and room temperature. Alternatively, crystalline eravacycline free base of the invention can be characterized by having a powder X-ray diffractogram which is essentially the same as displayed in figure 1 of the present invention, when measured with Cu-Kalphai, 2 radiation at a wavelength of 0.15419 nm and room temperature.

In a preferred aspect, the crystalline eravacycline free base of the present invention comprises at most 20 weight%, preferably at most 10 weight%, more preferably at most 5 weight%, even more preferably at most 2 weight% and most preferably at most 1 weight%, of any other physical form of eravacycline, based on the weight of the crystalline eravacycline free base of the present invention. Most preferably, the any other physical form of eravacycline free base is amorphous eravacycline free base. In one aspect the crystalline eravacycline free base of the present invention can further be characterized by comprising crystals having needle, lath and/ or column shape.

The invention also relates to a composition comprising at least 90 weight% of crystalline eravacycline free base of the present invention, based on the total weight of the composition. Preferably, the composition comprises less than 5 weight% amorphous eravacycline, such as less than 2 weight% amorphous eravacycline. The invention also relates to a composition comprising at least 95 weight% of crystalline eravacycline free base of the present invention, based on the total weight of the composition. Preferably, the composition comprises less than 4 weight% amorphous eravacycline, such as less than 2 weight% amorphous eravacycline. In a further aspect, the invention relates to a process for the preparation of crystalline eravacycline free base of the present invention comprising slurrying amorphous eravacycline free base in a solvent comprising an aliphatic and/ or cyclic ether.

Amorphous eravacycline free base, which is used as starting material, can for example be prepared according to the procedure disclosed in Ronn et al. "Process R&D of Eravacycline: The First Fully Synthetic Fluorocycline in Clinical Development" Org. Process Res. Dev. 2013, 17, 838-845 (compound 7 corresponds to eravacycline free base).

One or more aliphatic or cyclic ether(s) can be used for the slurrying. The one or more aliphatic or cyclic ether(s) can preferably be selected from diethyl ether, diisopropyl ether, methyl tert- butyl ether and methyltetrahydrofuran. Optionally, the solvent may comprise one or more additional organic solvent(s) selected from the group of alkanes, polar aprotic solvents, polar pro tic solvents and halogenated hydrocarbons. In particular, the one or more additional organic solvent(s) are selected from the group of n-heptane, acetone, ethanol and methylene chloride. The solvent should comprise at least 50 volume%, preferably at least 80 volume% and even more preferably at least 90 volume% of an aliphatic and/ or cyclic ether, for example selected from diethyl ether, diisopropyl ether, methyl tert-butyl ether and/ or methyltetrahydrofuran. Most preferably the solvent comprises diethyl ether, diisopropyl ether, methyl tert-bu yl ether and/ or methyltetrahydrofuran at a concentration in the range of from about 98 to 100 volume%, based on the total solvent volume.

Slurrying emcompasses any kind of movement of the solid material suspended in the solvent caused by, but not limited to e.g. agitation, stirring, mixing, shaking, vibration, sonication, wet milling and the like. Eravacycline free base is slurried in a solvent comprising one or more aliphatic and/ or cyclic ether(s) preferably at a temperature of about 30 °C or less, more preferably of about 20 °C or less, even more preferably of about 10 °C or less and most preferably of about 0 °C or less. Eravacycline free base is added to the slurry until the content of amorphous eravacycline free base in the slurry is at most 20 weight%, preferably at most 10 weight%, more preferably at most 5 weight%, even more preferably at most 2 weight% and most preferably at most 1 weight%.

Usually, slurrying is conducted for a period in the range of from 1 to 168 hours, preferably from 2 to 48 hours, more preferably from 4 to 24 hours and most preferably from 6 to 18 hours. The skilled person may monitor the conversion of amorphous eravacycline free base to crystalline eravacycline free base by withdrawing samples from the slurry and analyzing the sample by powder X-ray diffraction. Optionally, seed crystals of eravacycline free base may be added to the suspension in order to facilitate crystallization.

Optionally, in a subsequent step, at least a part of the crystalline eravacycline free base is separated from its mother liquor. Preferably, the eravacycline free base crystals are separated from their mother liquor by any conventional method such as filtration, centrifugation, solvent evaporation or decantation, more preferably by filtration or centrifugation and most preferably by filtration.

Optionally, in a further step the isolated crystals are washed with a suitable solvent selected from water and/ or an organic solvent. The obtained crystalline eravacycline free base may optionally be dried. Drying may be performed at a temperature of about 40 °C or less, preferably of about 30 °C or less and most preferably drying is performed at about room temperature. Drying may be performed for a period in the range of from about 1 to 72 hours, preferably from about 2 to 48 hours, more preferably from about 4 to 24 hours and most preferably from about 6 to 18 hours. Drying may be performed at ambient pressure and/ or under reduced pressure. Preferably, drying is performed at a pressure of about 100 mbar or less, more preferably of about 50 mbar or less and most preferably of about 30 mbar or less, for example a vacuum of about 20 mbar or less.

No gummy residue is observed during the above described process. This is advantageous because gummy material tends to stick on the reactor wall and stirrer, which would be connected with yield loss and subsequent cumbersome cleaning work of the equipment. According to Ronn et. al. the occurring gummy residue observed in their procedure could only be removed by aging with vigorous stirring, which on the one hand is time-consuming and on the other hand strongly impacts particle size distribution due to the mechanical stress and therefore negatively influences the isolation properties of the material. In contrast, the crystalline eravacycline free base obtainable or obtained from the process of the present invention shows excellent isolation properties, such as good filtration properties, and can be easily dried after isolation. This is mainly due to the fact that the bulk of the material consists of well-defined lath-shaped crystals having a particle size which is suitable for filtration (see also figure 2 herein). The avoidance of gummy material throughout the process is advantageous as it saves time, effort and leads to material with improved powder properties.

Crystalline eravacycline free base exhibits physicochemical properties which enable this physical form to be used for and in the preparation of pharmaceutical compositions. One advantage of crystalline eravacycline free base of the present invention is its property that chemical impurities are not taken up into the crystal, leading to a purification effect on eravacycline. This is desirable as chemical impurities have been a concern for eravacycline so far, especially the depletion of the C4-epimer, as discussed in Ronn et al. on page 842, where 2.4% of the C-4 epimer were observed.

It was surprisingly found that the crystalline eravacycline free base of the present invention is a very efficient physical form for the removal of impurities. For example, the starting material amorphous eravacycline free base contained total impurities of about 3.2 area% (as determined by HPLC at 254 nm). This compared with only 0.8 area% total impurities in the crystalline eravacycline free base of the present invention (see also example 1 herein).

Besides good chemical stability, crystalline eravacycline free base of the present invention is also physically very stable. For example, the crystalline eravacycline free base of the present invention does not liquefy when subjected to a relative humidity in the range of from about 0 to 95% at 25.0 ± 0.1 °C, but remains a solid. In contrast, amorphous eravacycline bis- hydrochloride already showed deliquescence at 80%> relative humidity, which makes this physical form more difficult to handle and requires precautionary measures against high humidity. Also at lower relative humidities crystalline eravacycline of the present invention shows an advantageous behavior in so far as it is less hygroscopic compared to amorphous eravacycline. For example, crystalline eravacycline only takes up about 3 weight% of water between 0 and 65%>, whereas amorphous eravacycline shows a mass increase of about 6 weight% in the same range (see comparative example 1 and figure 3 herein).

Finally, the aqueous solubility of crystalline eravacycline of the present invention is surprisingly comparable to amorphous eravacycline over a broad range of pHs tested (see comparative example 2 herein). This aqueous solubility profile is highly appreciated for a drug like eravacycline, which is intended to be not only used for intravenous application, but which is the first tetracycline antibiotic also intended for oral administration, since high aqueous solubility is one of the main factors favoring good oral bioavailability of a drug. When eravacycline of the present invention is for example used for a pharmaceutical composition intended for oral use, a continuous high bioavailability can be achieved throughout shelf-life, due to the superior properties of this physical form.

Overall, crystalline eravacycline free base of the present invention shows a combination of physicochemical properties which are favorable, for example for storage, for shipping, as an intermediate in the preparation of a pharmaceutical composition, and/or also as the active pharmaceutical ingredient in a pharmaceutical composition.

In a further aspect the invention relates to the use of crystalline eravacycline free base of the present invention in the preparation of a pharmaceutical composition comprising eravacycline. Preferably, the crystalline eravacycline free base of the present invention can be used in the process for the preparation of a pharmaceutical composition comprising eravacycline, for example eravacycline hydrochloride, intended for parenteral use. Preferably, the pharmaceutical composition intended for parenteral use of the present invention is a powder for solution for infusion and most preferably a lyophilized powder for solution for infusion. The skilled person will appreciate that the lyophilized powder for solution for infusion in general will not comprise the crystalline eravacycline free base of the present invention itself, but that the crystalline eravacycline free base of the present invention is used as an intermediate before or as a starting material for the process for the preparation of a lyophilized powder for solution for infusion comprising eravacycline.

Thus, in a further aspect, the present invention relates to a process for the preparation of a pharmaceutical composition comprising eravacycline intended for parenteral use comprising the steps of: (i) providing crystalline eravacycline free base of the present invention and optionally at least one stabilizer selected from the group of sugars and/ or sugar alcohols;

(ii) dissolving or suspending crystalline eravacycline and said optionally at least one stabilizer provided in step (i) in a solvent comprising water;

(iii) adjusting the pH of the solution or suspension obtained in step (ii) by adding at least one acid or base;

(iv) optionally filtering the solution or suspension obtained in step (iii) and

(v) lyophilizing the solution or suspension obtained in any one of steps (ii) to (iv) to give a pharmaceutical composition comprising eravacycline. The order of steps (i), (ii), (iii), (iv), and (v) above can be sequential, but variations of this process are well within the scope of the skilled person. For example, in a variation of steps (i) and (ii) eravacycline may be dissolved in a solution comprising the dissolved stabilizer. Alternatively, the at least one stabilizer may be dissolved in a solution comprising eravacycline.

A suitable acid for pH adjustment in step (iii) of the above described process is, for example, hydrochloric acid, preferably in form of an aqueous solution. A suitable base for pH adjustment in step (iii) is, for example, sodium hydroxide, preferably in form of an aqueous solution.

In one aspect, the pH environment ranges from about 3.0 to about 7.0, such as pH values ranging from about 4.0 to about 5.0, or from about 4.2 to about 4.8.

The stabilizer is preferably a carbohydrate capable of reducing epimer formation in a lyophilisate comprising eravacycline prepared at a pH of 4.8 , when compared to an eravacycline lyophilisate prepared at the same pH lacking suitable carbohydrates.

An example for a stabilizer is a sugar. Suitable sugars include mono- and disaccharides, preferably monosaccharides such as mannose and glucose and disaccharides such as lactose, and sucrose. Suitable sugars may be provided as different enantiomers such as D-glucose and L-glucose and/ or solid forms. For example, lactose includes the various solid forms of lactose, e.g. anhydrous lactose, lactose monohydrate or another hydrated or solvated form of lactose.

An example for a sugar, which can be added as stabilizer in step (i) of the above described process, is lactose, preferably lactose monohydrate. An example for a sugar alcohol, which can be added as stabilizer in step (i) of the above described process, is mannitol.

A preferred molar ratio of eravacycline to stabilizer, for example lactose, in the lyophilized powder or cake is in the range of from 1.0: 0.3 to 1 :4. The molar ratio of eravacycline to stabilizer may also be in the range of from 1.0: 1.0 to :3.0.

In one example for a process of the invention, eravacycline is dissolved in water to form a solution. The pH of the solution is subsequently lowered by addition of an acid or buffer to obtain a pH from about 4.0 to about 5.0. A stabilizer, for example a carbohydrate such as lactose, is then dissolved in the solution and the solution is filtered, e.g. sterile filtered, and lyophilized to dryness to form a lyophilized powder or cake.

In another example for a process of the invention, lactose, for example 106mg lactose monohydrate, is dissolved in water and cooled to 2-8°C with nitrogen purging. Eravacycline free base according to the present invention is dissolved in the cooled lactose solution to form a solution. The pH of the solution is subsequently lowered by addition of hydrochloric acid to obtain a pH from about 4.0 to about 5.5, for example 4.9. The pH-adjusted solution is then sterile filtered. About 3.5 mL solution (comprising 53 mg eravacycline) per vial are then filled into lyophilization vials. Lyophilization is then carried out at a temperature below -40 °C until the water content is low, for example at most 1%. Vials are filled with nitrogen to about 500 mbar and then closed with a pharmaceutically acceptable stopper which contains essentially no leachable Zink and then crimped with a crimping cap.

In a further aspect the present invention relates to a pharmaceutical composition obtainable or obtained by, and preferably prepared according to, the above defined process.

The pharmaceutical compositions of the invention also include solutions prepared from the lyophilisate by e.g. reconstitution with physiological saline. The pharmaceutical composition intended for parenteral use of the present invention is preferably administered by intravenous infusion after reconstitution with sodium chloride, for example 9 mg/mL (0.9%), solution for injection, dextrose, for example 50 mg/mL (5%), solution for injection or Lactated Ringer's solution for injection.

The invention further relates to the pharmaceutical composition of the present invention for use as a medicament, in particular for use in the treatment and/ or prevention of bacterial infections, wherein the bacterial infections are caused by Gram negative bacteria, in particular by multidrug resistant Gram negative bacteria. The pharmaceutical composition of the present invention is for example suitable for the treatment and/ or prevention of complicated intraabdominal and complicated urinary tract infections.

In yet another aspect the present invention relates to a pharmaceutical composition comprising an effective amount of the crystalline eravacycline free base of the invention, and one or more pharmaceutically acceptable excipient(s). The pharmaceutical composition is preferably an oral solid dosage form, such as a tablet or a capsule.

In another aspect the present invention relates to the use of crystalline eravacycline free base for the preparation of eravacycline comprising less than 1 % impurities. In another aspect the present invention relates to the use of crystalline eravacycline free base for the preparation of eravacycline comprising less than 1% related compounds, e.g. other tetracyclines.

As mentioned above, using a process comprising the step of crystallizing eravacycline free base results in effective purification of eravacycline from related compounds, such as the C4-epimer of eravacycline and overacylated impurities resulting from side chain addition to the 9-amino group of compound 12 as described on page 839 of Ronn et al, "Process R&D of Eravacycline: The first full synthetic fluorocycline in clinical development", Org. Process Res. Dev. 2013, 17, 838-845. Therefore, the crystalline eravacycline free base of the present invention is an advantageous intermediate during the preparation of eravacycline salts, for example pharmaceutically acceptable salts of eravacycline. Pharmaceutical compositions comprising a pharmaceutically acceptable salt of eravacycline, wherein the pharmaceutically acceptable salt of eravacycline has been prepared by using the crystalline eravacycline free base of the present invention at one point during the preparation process of the pharmaceutically acceptable salt of eravacycline, will have an improved chance to meet the regulatory limits for related compounds, such as the eravacycline C-4 epimer or other related tetracyclines.

Embodiment section Aspects, advantageous features and preferred embodiments of the present invention are summarized in the following items:

1) Crystalline eravacycline free base.

2) Crystalline eravacycline free base of formula (A)

formula (A)

3) Crystalline eravacycline free base of formula (A) or a solvate or a hydrate thereof.

4) The crystalline eravacycline free base according to any one of items 1 to 3 characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (3.2 ± 0.2)°, (5.6 ± 0.2)° and (8.5 ± 0.2)°, when measured with Cu-Kalphai,2 radiation having a wavelength of 0.15419 nm and at a temperature in the range of from 20 to 30 °C.

5) The crystalline eravacycline free base according to any one of items 1 to 3 characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)° and (8.5 ± 0.2)°, when measured with Cu-Kalphai,₂ radiation having a wavelength of 0.15419 nm and at a temperature in the range of from 20 to 30 °C.

6) The crystalline eravacycline free base according to any one of items 1 to 3 characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (8.5 ± 0.2)° and (12.4 ± 0.2)°, when measured with Cu-Kalphai,₂ radiation having a wavelength of 0.15419 nm and at a temperature in the range of from 20 to 30 °C.

7) The crystalline eravacycline free base according to any one of items 1 to 3 characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (8.5 ± 0.2)°, (12.4± 0.2)° and (12.7 ± 0.2)°, when measured with Cu-Kalphai,₂ radiation having a wavelength of 0.15419 nm and at a temperature in the range of from 20 to 30 °C.

8) The crystalline eravacycline free base according to any one of items 1 to 3 characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (8.5± 0.2)°, (12.4 ± 0.2)°, (12.7 ± 0.2)° and (24.8 ± 0.2)°, when measured with Cu-Kalphai,₂ radiation having a wavelength of 0.15419 nm and at a temperature in the range of from 20 to 30 °C.

9) The crystalline eravacycline free base according to any one of items 1 to 3 characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (7.4 ± 0.2)°, (8.5 ± 0.2)°, (12.4 ± 0.2)°, (12.7 ± 0.2)° and (24.8 ± 0.2)°,when measured with Cu-Kalphai,₂ radiation having a wavelength of 0.15419 nm and at a temperature in the range of from 20 to 30 °C.

10) The crystalline eravacycline according free base to any one of items 1 to 3 characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (7.4 ± 0.2)°, (8.5 ± 0.2)°, (12.4 ± 0.2)°, (12.7 ± 0.2)°, (12.9 ± 0.2)° and (24.8 ± 0.2)° when measured with Cu-Kalphai,₂ radiation having a wavelength of 0.15419 nm and at a temperature in the range of from 20 to 30 °C.

11) The crystalline eravacycline free base according to any one of items 1 to 3 characterized by having a powder X-ray diffractogram comprising reflections at 2-theta angles of (3.2 ± 0.2)°, (5.6 ± 0.2)°, (5.8 ± 0.2)°, (7.4 ± 0.2)°, (8.5 ± 0.2)°, (12.4 ± 0.2)°, (12.7 ± 0.2)°, (12.9 ± 0.2)°, (22.8 ± 0.2)° and (24.8 ± 0.2)°, when measured with Cu-Kalphai,₂ radiation having a wavelength of 0.15419 nm and at a temperature in the range of from 20 to 30 °C.

) The crystalline eravacycline free base according to any one of items 1 to 3 characterized by a powder X-ray diffractogram essentially the same as displayed in figure 1 of the present invention, when measured with Cu-Kalphai,2 radiation at a wavelength of 0.15419 nm and at a temperature in the range of from 20 to 30 °C.

) Crystalline eravacycline free base according to any one of items 1 to 12 characterized by comprising at most 20 weight% of any other physical form of eravacycline, based on the weight of crystalline eravacycline as defined in any one of items 1 to 12.

) Crystalline eravacycline free base according to any one of items 1 to 12 comprising at most 10 weight% of any other physical form of eravacycline, based on the weight of crystalline eravacycline free base as defined in any one of items 1 to 12.

) Crystalline eravacycline free base according to any one of items 1 to 12 comprising at most 5 weight% of any other physical form of eravacycline, based on the weight of crystalline eravacycline free base as defined in any one of items 1 to 12.

) Crystalline eravacycline free base according to any one of items 1 to 12 comprising at most 2 weight% of any other physical form of eravacycline, based on the weight of crystalline eravacycline as defined in any one of items 1 to 12.

) Crystalline eravacycline free base according to any one of items 1 to 12 comprising at most 1 weight% of any other physical form of eravacycline, based on the weight of crystalline eravacycline as defined in any one of items 1 to 12.

) Crystalline eravacycline free base according to any one of items 13 to 17, wherein the other physical form of eravacycline is amorphous eravacycline.

) Crystalline eravacycline free base according to any one of items 1 to 18 characterized by comprising crystals having needle, lath and/ or column shape.

) A process for the preparation of the crystalline eravacycline free base as defined in any one of items 1 to 19 comprising slurrying eravacycline free base in a solvent comprising one or more aliphatic ether or one or more cyclic ether.

) The process according to item 20, wherein the eravacycline free base starting material is amorphous.

) The process according to item 20 or 21, wherein the one or more aliphatic ether is selected from diethyl ether, diisopropyl ether and methyl tert-butyl ether or wherein the one or more cyclic ether is methytetrahydrofuran.

) The process according to any one of items 20 to 22, wherein slurrying is performed at a temperature of 40 °C or less.

) The process according to any one of items 20 to 22, wherein slurrying is performed at a temperature of 30 °C or less.

) The process according to any one of items 20 to 22, wherein slurrying is performed at a temperature of 20 °C or less. 26) The process according to any one of items 20 to 22, wherein slurrying is performed at a temperature of 10 °C or less.

27) The process according to any one of items 20 to 22, wherein slurrying is performed at a temperature of 0 °C or less.

28) The process according to any one of items 20 to 27, wherein slurrying is performed for a period in the range of from 1 to 168 hours.

29) The process according to any one of items 20 to 27, wherein slurrying is performed for a period in the range of from 2 to 48 hours.

30) The process according to any one of items 20 to 27, wherein slurrying is performed for a period in the range of from 4 to 24 hours.

31) The process according to any one of items 20 to 27, wherein slurrying is performed for a period in the range of from 6 to 18 hours.

32) The process according to any one of items 20 to 31 further comprising the addition of eravacycline free base seed crystals.

33) The process according to any one of items 20 to 32, further comprising separating at least a part of the crystalline eravacycline free base as defined in any one of items 1 to

19 from its mother liquor.

34) The process according to item 33, wherein crystalline eravacycline free base is separated from its mother liquor by filtration, centrifugation, decantation or solvent evaporation. 35) The process according to item 33, wherein crystalline eravacycline free base is separated from its mother liquor by filtration or centrifugation.

36) The process according to any one of items 33 to 35 further comprising washing the isolated eravacycline free base crystals with a solvent selected from water and/ or an organic solvent.

37) The process according to any one of items 20 to 36 further comprising drying the eravacycline free base crystals.

38) The process according to item 37, wherein drying is performed at a temperature of 40 °C or less.

39) The process according to item 37, wherein drying is performed at a temperature of 30 °C or less.

40) The process according to item 37, wherein drying is performed at a temperature in the range of from 20 to 30 °C.

41) The process according to any one of items 37 to 40, wherein drying is performed for a period in the range of from 1 to 168 hours.

42) The process according to any one of items 37 to 40, wherein drying is performed for a period in the range of from 2 to 48 hours.

43) The process according to any one of items 37 to 40, wherein drying is performed for a period in the range of from 4 to 24 hours.

44) The process according to any one of items 37 to 40, wherein drying is performed for a period in the range of from 6 to 18 hours. 45) Use of crystalline eravacycline free base as defined in any one of items 1 to 19, or of the composition of any one of items 73 to 76, for the preparation of a pharmaceutical composition comprising eravacycline.

46) Use according to item 45, wherein the pharmaceutical composition comprising eravacycline is a pharmaceutical composition comprising eravacycline hydrochloride.

47) Use according to item 45 or 46, wherein the pharmaceutical composition is intended for parenteral use, in particular wherein the pharmaceutical composition is a powder for injection for infusion.

48) Use according to any one of items 45 to 47, wherein the pharmaceutical composition is a lyophilized powder for injection for infusion.

49) Process for the preparation of a pharmaceutical composition for parenteral use comprising:

(i) providing crystalline eravacycline free base of the present invention and optionally at least one stabilizer selected from the group of sugars and/ or sugar alcohols;

(ii) dissolving or suspending crystalline eravacycline and optionally the at least one stabilizer provided in step (i) in a solvent comprising water;

(iv) optionally filtering the solution or suspension obtained in step (iii) and

(v) lyophilizing the solution or suspension obtained in any one of steps (ii) to (iv) to give a pharmaceutical composition comprising eravacycline.

50) Process according to item 49, wherein the acid in step (iii) is hydrochloric acid.

51) Process according to item 49, wherein the base in step (iii) is sodium hydroxide.

52) Process according to any one of items 49 to 51 , wherein the sugar is lactose.

53) Process according to item 52, wherein the sugar is lactose monohydrate.

54) Process according to any one of items 49 to 53, wherein the sugar alcohol is mannitol.

55) A pharmaceutical composition obtainable or obtained by the process as defined in any one of items 49 to 54.

56) The pharmaceutical composition according to item 55 for use as a medicament.

57) The pharmaceutical composition according to item 55 for use in the treatment and/ or prevention of bacterial infections.

58) The pharmaceutical composition according to item 57, wherein the bacterial infection is caused by Gram negative bacteria.

59) The pharmaceutical composition according to item 58, wherein the bacterial infection is caused by multidrug resistant Gram negative bacteria.

60) The pharmaceutical composition according to any one of items 57 to 59, wherein the bacterial infection is selected from complicated intraabdominal and complicated urinary tract infections. 61) A pharmaceutical composition comprising an effective amount of crystalline eravacycline free base according to any one of items 1 to 19, and at least one pharmaceutically acceptable excipient.

62) The pharmaceutical composition of item 61 which is an oral solid dosage form.

63) The pharmaceutical composition of any one of items 61 to 62 which is a tablet or a capsule.

64) The pharmaceutical composition of any one of items 61 to 63, wherein eravacycline free base is present in a dose of 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 350 mg,

400 mg, 450 mg or 500 mg.

65) The pharmaceutical composition of item 63, which is a tablet and wherein eravacycline free base is present in a dose of 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 350 mg,

400 mg, 450 mg or 500 mg.

66) The pharmaceutical composition of item 63, which is a capsule and wherein eravacycline free base is present in a dose of 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 350 mg, 400 mg, 450 mg or 500 mg.

67) The pharmaceutical composition of any one of items 61 to 66, wherein the pharmaceutical composition is to be administered once daily.

68) The pharmaceutical composition of any one of items 61 to 66, wherein the pharmaceutical composition is to be administered once every second day.

69) The pharmaceutical composition of item 68, wherein eravacycline free base is present in a dose of 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 350 mg, 400 mg, 450 mg or 500 mg.

70) The pharmaceutical composition of any one of items 61 to 69, wherein the pharmaceutical composition is to be administered without food.

71) The pharmaceutical composition of any one of items 61 to 70, wherein the pharmaceutically acceptable excipient is selected from carriers, fillers, diluents, lubricants, sweeteners, stabilizing agents, solubilizing agents, antioxidants and preservatives, flavoring agents, binder, colorants, osmotic agents preservatives, buffers, surfactants, granulating and disintegrating agents, and combinations thereof.

72) Use of crystalline eravacycline free base as an intermediate in the preparation of eravacycline comprising less than 1% impurities.

73) A composition comprising at least 90 weight% of crystalline eravacycline free base according to any one of items 1 to 19, based on the total weight of the composition. 74) The composition according to item 73, comprising at least 95 weight% of crystalline eravacycline free base according to any one of items 1 to 19, based on the total weight of the composition.

75) The composition according to item 73 or item 74, comprising less than 5 weight% amorphous eravacycline.

76) The composition according to item 73 or item 74, comprising less than 2weight% amorphous eravacycline.

77) Process for the preparation of a pharmaceutical composition for parenteral use comprising:

(i) providing the composition according to any one of items 73 to 76 and optionally at least one stabilizer selected from the group of sugars and/ or sugar alcohols;

(ii) dissolving or suspending crystalline eravacycline and optionally at least one stabilizer provided in step (i) in a solvent comprising water;

(iv) optionally filtering the solution or suspension obtained in step (iii) and

78) Process according to item 77, wherein the acid in step (iii) is hydrochloric acid.

79) Process according to item 77, wherein the base in step (iii) is sodium hydroxide.

80) Process according to any one of items 77 to 79, wherein the sugar is lactose.

81) Process according to item 80, wherein the sugar is lactose monohydrate.

82) Process according to any one of items 77 to 79, wherein the sugar alcohol is mannitol.

83) A pharmaceutical composition obtainable or obtained by the process as defined in any one of items 77 to 82.

84) The use according to item 72 wherein the eravacycline comprising less than 1% impurities is an acid addition salt of eravacycline.

85) The use of item 84, wherein the acid addition salt of eravacycline is an eravacycline hydrochloride.

The following non-limiting examples are illustrative for preferred embodiments of the disclosure. They are not to be construed to be in any way limiting for the disclosure.

EXAMPLES

Powder X-ray diffraction Powder X-ray diffraction was performed with a PANalytical X'Pert PRO diffractometer equipped with a theta/theta coupled goniometer in transmission geometry, Cu-Kalphai,2 radiation (wavelength 0.15419 nm) with a focusing mirror and a solid state PIXcel detector. Diffractograms were recorded at a tube voltage of 45 kV and a tube current of 40 mA, applying a stepsize of 0.013° 2-theta with 40s per step (255 channels) in the angular range of 2° to 40° 2-theta at ambient conditions. A typical precision of the 2-theta values is in the range of ± 0.2° 2-theta, preferably of ± 0.1° 2-theta. Thus, the diffraction peak of crystalline eravacycline that appears for example at 3.2° 2-theta can appear in the range of from 3.0 to 3.4° 2-theta, preferably in the range of from 3.1 to 3.3 ° 2-theta on most X-ray diffractometers under standard conditions.

Gravimetric moisture sorption

Moisture sorption isotherms were recorded with an SPSx-Ι μ moisture sorption analyzer (proUmid, Ulm). The measurement cycle was started at ambient relative humidity (RH) of 35% and first decreased to 3% RH in one step and further to 0% RH. Then RH was increased from 0% to 95% in 5% steps. Subsequently, RH was decreased from 95% to 0% in 5% steps. Finally, RH was increased from 0% to 30% in one step. The time per step was set to a minimum of 2 hours and a maximum of 6 hours. If an equilibrium condition with a constant mass of ± 0.01% within 1 hour was reached before the maximum time for all examined samples, the sequential humidity step was applied before the maximum time of 6 hours. If no equilibrium was achieved, the consecutive humidity step was applied after the maximum time of 6 hours. The temperature was 25 ± 0.1°C.

HPLC

Instrument: Agilent HP 1 100 with Chemstation

Column: YMC-Pack Pro CI 8 RS; 150*4.6mm; 3microm

Solvent: A: 40 mmol amidosulfonic acid in water

B: 40 mmol amidosulfonic acid in water with 75% acetonitrile

Gradient:

Time [min] Amount A [%] Amount B[%>]

0 90 10

10 0 100

15 0 100

16 90 10

Injection volume: 5 microL

Flow: 0.8 mL/min

Oven temperature: 40 °C

Wavelength: 254 nm

Example 1 : Preparation of crystalline eravacycline free base A suspension of amorphous eravacycline free base, obtained according to Ronn et al. (2.00 g, 96.8 area% by HPLC) in diethyl ether (40 mL) was stirred for 17 hours at a temperature of 0 °C. Subsequently, the solid was collected by filtration, washed with cold diethyl ether (10 mL) and dried under vacuum (30 mbar) at room temperature for 3 hours to obtain 1.85 g of crystalline eravacycline free base (99.2 area% by HPLC).

Yield: 93% of theory

The powder X-ray diffractogram of crystalline eravacycline obtained according to example 1 is displayed in figure 1. A reflection list with the corresponding relative intensities is provided in table 1.

Angle Relative Intensity

[ ± 0.2 °2-Theta] [%]

3.2 100

5.6 27

5.8 13

6.4 5

7.4 7

8.5 13

9.8 4

11.4 6

12.4 12

12.7 10

12.9 7

13.9 3

14.7 3

16.7 3

22.8 7

23.6 4

24.1 3

24.8 9

Table 1 : Reflection list and relative intensities in the range of from 2.0 to 30.0°; Example 2: Preparation of crystalline eravacycline free base

A suspension of amorphous eravacycline free base (200 mg) in diisopropyl ether (4 mL) was stirred at room temperature for 24 hours. Subsequently, the solid was collected by filtration and dried under vacuum (30 mbar) at room temperature for 18 hours to obtain 153 mg of crystalline eravacycline.

Yield: 77% of theory Example 3 : Preparation of crystalline eravacycline free base

A suspension of amorphous eravacycline free base (50 mg) in methyl tert-butyl ether (1 mL) was stirred at room temperature for 24 hours. Subsequently, the solid was collected by centrifugation to obtain 45 mg of crystalline eravacycline free base. Yield: 90% of theory

Example 4: Preparation of crystalline eravacycline free base

A suspension of amorphous eravacycline free base (50 mg) in a mixture of methyltetrahydrofuran: n-heptan (1 : 1 , v/ v, 1 mL) was stirred at room temperature for 24 hours. Subsequently, the solid was collected by centrifugation to obtain crystalline eravacycline free base.

Reference example 1 : Slurry experiments in various organic solvents

Amorphous eravacycline free base (50 mg) was suspended in organic solvent (1 mL) according to table 2 and heated to reflux temperature. Subsequently, the suspension was allowed to cool to room temperature and further stirred for 18 hours. The solid was isolated by centrifugation and investigated by powder X-ray diffraction. The results are summarized in table 2.

No. Solvent PXRD

1 n-butanol/ water (1 : 1, volume: volume) amorphous

2 n-butyl acetate amorphous

3 diisopropyl ether crystalline

4 ethyl propionate amorphous

5 n-hexane amorphous

6 isopropyl acetate amorphous

7 isobutyl acetate amorphous

8 mesitylen amorphous

9 methyl acetate amorphous

10 methyl cyclohexane amorphous

11 2-propanol/ water(l : : 1, volume: volume) amorphous

12 xylene amorphous

Table 2: Summary of results from reference example 1

As can be seen from table 2 only experiment number 3, where diisopropyl ether was used as solvent, produced crystalline eravacycline, whereas all other experiments yielded amorphous material. Reference example 2: Crystallization experiments in various organic solvents

Amorphous eravacycline free base (50 mg) was dissolved at room temperature in organic solvent (1 mL) according to table 3. The solution was kept at room temperature for 1 week.

No. Solvent PXRD

1 acetone no solid obtained

2 acetonitrile/ water (1 : 1, volume: volume) no solid obtained

3 benzonitrile no solid obtained

4 benzyl alcohol no solid obtained

5 chloroform no solid obtained

6 cyclohexanone no solid obtained

7 dichloroethane no solid obtained

8 dimethoxyethane no solid obtained

9 dimethylacetamide no solid obtained

10 dimethylacetamide/ water (1 : 1, volume: volume) no solid obtained

11 dimethylformamide/ water (1 : 1, volume: volume) no solid obtained

12 dimethylformamide no solid obtained

13 dimethylsulfoxide no solid obtained

14 1 ,4-dioxane no solid obtained

15 1,4-dioxane/ water (1 : 1, volume: volume) no solid obtained

16 ethanol/ water (1 : 1, volume: volume) no solid obtained

17 methylene chloride no solid obtained

18 methyl ethyl ketone no solid obtained

19 methyl benzoate no solid obtained

20 nitromethane no solid obtained

21 piperidine no solid obtained

22 tetrahydrofurane no solid obtained

Table 3 : Summary of results from crystallization experiments As can be seen from table 3, in no case precipitation let alone crystallization were observed.

Reference example 3 : Crystallization experiments in various organic solvents

Amorphous eravacycline free base (50 mg ) was dissolved in organic solvent according to table 4 (ImL) upon heating to reflux temperature. Subsequently, the solution was allowed to cool to room temperature and stirred with a magnetic stirrer for 18 hours. In the case precipitation was observed, the solid was isolated by centrifugation. The solids were investigated by powder X- ray diffraction and the results are summarized in table 4.

Solvent PXRD

acetone/ water (1 : 1, volume : volume) no solid obtained 2 acetonitrile no solid obtained

3 anisol no solid obtained

4 1-butanol amorphous

5 2-butanol amorphous

6 chlorobenzene amorphous

7 diethylene glycol diethyl ether no solid obtained

8 dimethylsulfoxide/ water (1 : 1, volume: volume) no solid obtained

9 ethanol no solid obtained

10 ethyl acetate no solid obtained

11 isobutanol amorphous

12 isopropanol no solid obtained

13 methanol no solid obtained

14 methanol/ water (1 : 1, volume: volume) no solid obtained

15 methyl acetate/ water (1 : 1, volume: volume) no solid obtained

16 methylisobutyl ketone no solid obtained

17 methyltetrahydrofuran no solid obtained

18 2-pentanol amorphous

19 1-propanol no solid obtained

20 tert-butanol amorphous

21 toluene amorphous

22 water no solid obtained

Table 4: Summary of results from crystallization experiments

As can be seen from table 4 some of the experiments lead to precipitation of amorphous material. However, in the majority of the experiments no solid material was obtained at all.

Reference example 4:

Amorphous eravacycline free base (124 mg) was charged to a stirring solution of acetone (4.5 mL) and water (0.09 mL).The obtained solution was stirred for 2 hours at room temperature. No solid was obtained.

Reference example 5 :

Amorphous eravacycline bis-hydrochloride salt (124 mg) was charged to a stirring solution of acetone (4.5 mL) and water (0.09 mL). The obtained suspension was stirred for 2 hours at room temperature. The solid was collected by filtration, washed with acetone and dried under vacuum to yield 102 mg eravacycline bis-hydrochloride as a dark yellow solid. The obtained material was X-ray amorphous. Comparative Example 1 (gravimetric moisture sorption)

Crystalline eravacycline of the present invention and amorphous eravacycline were both subjected to a gravimetric moisture sorption experiment using an SPSx-Ιμ moisture sorption analyzer (ProUmid, Ulm). Figure 3 displays the results of the sorption cycle from 0 to 65% relative humidity, which was performed in 5% steps. The time per step was set to a minimum of 2 hours and a maximum of 6 hours. If an equilibrium condition with a constant mass of ± 0.01% within 1 hour was reached before the maximum time for all examined samples the sequential humidity step was applied before the maximum time of 6 hours. If no equilibrium was achieved the consecutive humidity step was applied after the maximum time of 6 hours. The temperature was (25 ± 0.1) °C.

As can be seen from the gravimetric moisture sorption curve displayed in Figures 3, amorphous eravacycline shows a mass increase of about 6.2 weight%, whereas the mass increase of the crystalline eravacycline of the present invention is only about 2.8 weight% in the same range from 0 to 65% relative humidity. Hence, crystalline eravacycline of the present invention is significantly less hygroscopic compared to amorphous eravacycline.

Comparative Example 2 (aqueous solubility)

The solubility of amorphous and crystalline eravacycline of the present invention was determined in water and 0. IN aqueous hydrochloric acid solution respectively using the solvent addition method. Thereby, 50 mg of each material to be tested were accurately weighed into a glass vial and solvent was added in 1 mL aliquots until the material visually dissolved.

Table 5: Results from solubility testing in H2O and 0.1N aqueous HC1 solution

The aqueous solubility of crystalline eravacycline is comparable to the solubility of amorphous eravacycline regardless of whether water or 0. IN HC1 was used.

Claims

1) Crystalline eravacycline free base.

2) The crystalline eravacycline free base of claim 1 characterized by having a powder X- ray diffractogram comprising reflections at 2-theta angles of (3.2 ± 0.2)°, (5.6 ± 0.2)° and (8.5 ± 0.2)°, when measured with Cu-Kalphai,2 radiation having a wavelength of 0.15419 nm and a temperature in the range of from 20 to 30 °C.

3) A composition comprising at least 90 weight% of crystalline eravacycline free base according to any one of the preceding claims, based on the total weight of the composition.

4) The composition according to claim 3, comprising less than 5 weight% amorphous eravacycline.

5) A process for the preparation of crystalline eravacycline free base as defined in claim 1 or 2 or the composition as defined in claim 3 or 4 comprising slurrying amorphous eravacycline free base in a solvent comprising an aliphatic and/or a cyclic ether.

6) The process according to claim 5, wherein the aliphatic ether is selected from diethyl ether, diisopropyl ether and methyl tert-butyl ether or the cyclic ether is methyltetrahydrofuran.

7) Use of crystalline eravacycline free base as defined in claim 1 or 2 or the composition as defined in claim 3 or 4 for the preparation of a pharmaceutical composition.

8) The use according to claim 7, wherein the pharmaceutical composition is intended for parenteral or oral use.

9) Process for the preparation of a pharmaceutical composition comprising eravacycline, wherein the pharmaceutical composition is intended for parenteral use, comprising the steps of:

(i) providing the crystalline eravacycline free base as defined in claim 1 or 2 or the composition as defined in claim 3 or 4 and optionally at least one stabilizer selected from the group of sugars and/ or sugar alcohols;

(ii) dissolving or suspending crystalline eravacycline free base or the composition and optionally the at least one stabilizer provided in step (i) in a solvent comprising water;

(iv) optionally filtering the solution or suspension obtained in step (iii) and

(v) lyophilizing the solution or suspension obtained in any one of steps (ii) to (iv) to give a pharmaceutical composition comprising eravacycline. 10) A pharmaceutical composition comprising an effective amount of crystalline eravacycline free base according to claim 1 or 2 or the composition as defined in claim 3 or 4, and at least one pharmaceutically acceptable excipient.

11) The pharmaceutical composition of claim 10 which is an oral solid dosage form.