FIELD OF THE INVENTION

The present invention is generally directed to crystalline forms of (1 S,3S,4f?)-4-

((3aS,4f?,5S,7aS)-4-(aminomethyl)-7a-methyl-1-methyleneoc tahydro-1 /-/-inden-5-yl)-3- (hydroxymethyl)-4-methylcyclohexanol, processes for their preparation, and

pharmaceutical compositions comprising the crystalline forms.

BACKGROUND OF THE INVENTION

Dysregulated activation of the PI3K pathway contributes to

inflammatory/immune disorders and cancer. Efforts have been made to develop modulators of PI3K as well as downstream kinases (Workman et al., Nat. Biotechnol. 24, 794-796, 2006; Simon, Cell 125, 647-649, 2006; Hennessy et al., Nat. Rev. Drug Discov. 4, 988-1004, 2005; Knight et al., Cell 125, 733-747, 2006; Ong et al., Blood (2007), Vol. 110, No. 6, pp 1942-1949). A number of promising new PI3K isoform specific inhibitors with minimal toxicities have recently been developed and used in mouse models of inflammatory disease (Camps et al., Nat. Med. 11 , 936-943, 2005; Barber et ai, Nat. Med. 11 , 933-935, 2005) and glioma (Fan et al., Cancer Cell §, 341- 349, 2006). However, because of the dynamic interplay between phosphatases and kinases in regulating biological processes, inositol phosphatase activators represent a complementary, alternative approach to reduce PIP ₃ levels. Of the phosphoinositol phosphatases that degrade PIP _3i SHIP1 is a particularly ideal target for development of therapeutics for treating immune and hemopoietic disorders because of its

hematopietic-restricted expression (Hazen et ai, Blood 113, 2924-2933, 2009;

Rohrschneider et ai, Genes Dev. 14, 505-520, 2000).

Small molecule SHIP1 modulators have been disclosed, including

sesquiterpene derivatives such as pelorol. Pelorol is a natural product isolated from the tropical marine sponge Dactylospongia elegans (Kwak et ai, J. Nat. Prod. 63, 1153-1 156, 2000; Goclik ef a/., J. Nat. Prod. 63, 1150-1 152, 2000). Other small molecule SHIP1 modulators have been described in Ong et ai, Blood (2007), 110(6) 1942-1949, Yang et al., Org. Lett. (2005) 7(6) 1073-1076 and Meimetis et al., Eur. J. Org. Chem. (2012), 27, 5195-5207 and PCT Published Patent Applications Nos.

WO 2003/033517, WO 2004/035601 , WO 2004/092100, WO 2007/147251 , WO 2007/147252, WO 201 1/0691 18, WO 2014/110036, WO 2014/143561 and WO 2014/158654 and in U.S. Patent Nos. 7,601 ,874, 7,999,010, 8,084,503,

8, 101 ,605, 8,673,975, 8,765,994 and 9,000,050.

While significant strides have been made in this field, there remains a need for effective small molecule SHIP1 modulators.

One such small molecule SHIP1 modulator is (1 S,3S,4 )-4-((3aS,4 ,5S,7aS)- 4-(aminomethyl)-7a-methyl-1-methyleneoctahydro-1 /-/-inden-5-yl)-3-(hydroxymethyl)-4- methylcyclohexanol (referred to herein as Compound A). Compound A has antiinflammatory activity as a SHIP1 modulator and is described in U.S. Patent Nos.

7,601 ,874 and 7,999,010, the relevant disclosures of which are incorporated in full by reference in their entirety, particularly with respect to the preparation of Compound A, pharmaceutical compositions comprising Compound A and methods of using

Compound A.

Compound A has the molecular formula of C2 ₀ H ₃ 5NO2, a molecular weight of 321.5 g/mole and has the follow

Compound A

Compound A is useful in treating disorders and conditions that benefit from SHIP1 modulation, such as cancers, inflammatory disorders and conditions and immune disorders and conditions. Compound A is also useful in the preparation of a medicament for the treatment of such disorders and conditions.

SUMMARY OF THE INVENTION

The present invention is generally directed to crystalline forms of Compound A, processes for their preparation, pharmaceutical compositions containing them and methods of using the crystalline forms and their compositions.

Accordingly, in one aspect, this invention is directed to a first crystalline form of

Compound A, referred to herein as Compound A Form A.

In another aspect, this invention is directed to a second crystalline form of Compound A, referred to herein as Compound A Form B. In another aspect, this invention is directed to compositions comprising a pharmaceutically acceptable excipient, carrier and/or diluent and Compound A Form A.

In another aspect, this invention is directed to compositions comprising a pharmaceutically acceptable excipient, carrier and/or diluent and Compound A Form B.

In another aspect, this invention is directed to a method for modulating SHIP1 activity in a mammal comprising administering an effective amount of Compound A Form A or an effective amount of a composition comprising Compound A Form A to the mammal in need thereof.

In another aspect, this invention is directed to a method for modulating SHIP1 activity in a mammal comprising administering an effective amount of Compound A Form B or an effective amount of a composition comprising Compound A Form B to the mammal in need thereof.

In another aspect, this invention is directed to a method for treating a disease, disorder or condition associated with SHIP1 activity in a mammal comprising administering an effective amount of Compound A Form A or an effective amount of a composition comprising Compound A Form A to the mammal in need thereof.

In another aspect, this invention is directed to a method for treating a disease, disorder or condition associated with SHIP1 activity in a mammal comprising administering an effective amount of Compound A Form B or an effective amount of a composition comprising Compound A Form B to the mammal in need thereof.

In another aspect, this invention is directed to methods for the preparation of Compound A Form A.

In another aspect, this invention is directed to methods for the preparation of Compound A Form B.

These aspects, and embodiments thereof, are described in more detail below.

To this end, various references are set forth herein which describe in more detail certain background information, procedures, compounds and/or compositions, and are each hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 illustrates the X-Ray Powder Diffraction pattern of amorphous

Compound A.

Figure 2 illustrates the X-Ray Powder Diffraction pattern of

Compound A Form A.

Figure 3 illustrates the DSC thermogram of Compound A Form A. Figure 4 illustrates the TGA thermogram of Compound A Form A.

Figure 5 illustrates the TGA/MS analysis of Compound A Form A.

Figure 6 illustrates the molecular structure of Compound A Form A, as determined from XRPD analysis.

Figure 7 illustrates the crystal packing (along the [100] direction) and the hydrogen bond scheme for Compound A Form A.

Figure 8 illustrates the Rietveld (Quantitative Phase) Analysis of Compound A Form A. Figure 8(A) is the experimental HR-XRPD pattern superimposed with calculated HR-XRPD pattern; Figure 8(B) is the difference between the experimental HR-XRPD pattern and the calculated HR-XRPD pattern; Figure 8(C) is the simulated XRPD pattern (based on the crystal model); and Figure 8(D) is the expected Bragg reflections (triangles on baseline).

Figure 9 illustrates the X-Ray Powder Diffraction pattern of Compound A

Form B.

Figure 10 illustrates the DSC thermogram of Compound A Form B.

Figure 11 illustrates the TGA thermogram of Compound A Form B.

Figure 12 illustrates the TGA/MS analysis of Compound A Form B.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention is generally directed to crystalline forms of Compound A, processes for their preparation, pharmaceutical compositions containing them and methods of using the crystalline forms.

In general, most pharmaceutical compounds, i.e., those compounds which are useful as pharmaceutical agents, are initially produced in amorphous forms, which can be characterized by only short range ordering. These compounds may be challenging to develop, as the amorphous form is often unstable relative to a crystalline form and may convert under certain conditions to any crystalline form, not necessarily the most stable one. In an embodiment of the invention, molecules of Compound A in the crystalline form have both short and long range ordering and have different physical properties as compared to the amorphous form.

Solid state physical properties of a material affect the ease with which the material is handled during processing into a pharmaceutical product, such as a tablet or capsule formulation. The physical properties affect the types of excipients, for example, to be added to a formulation for a pharmaceutical compound. Furthermore, the solid state physical property of a pharmaceutical compound is important to its dissolution in aqueous and liquid milieus, including gastric juices, thereby having therapeutic consequences. The solid state form of a pharmaceutical compound may also affect its storage requirements. From a physicochemical perspective, the crystalline form of a pharmaceutical compound is the preferred form. Organization of the molecules in an ordered fashion to form a crystal lattice provides improved chemical stability, flowability, and other powder properties including reduced moisture sorption. All of these properties are of importance to the manufacturing, formulation, storage and overall manageability of a pharmaceutical drug product.

Thus, practical physical characteristics are influenced by the particular solid form of a substance. One solid form may give rise to different thermal behavior from that of the amorphous material or other solid forms. Thermal behavior is measured in the laboratory by such techniques as capillary melting point, thermo-gravimetric analysis (TGA) and differential scanning calorimetry (DSC) and can be used to distinguish some polymorphic solid forms from others. A particular polymorphic solid form may also give rise to distinct physical properties that may be detectable by X-ray powder diffraction (XRPD), solid state ³ C-Nuclear Magnetic Resonance spectroscopy, and infrared or Raman spectrometry.

Compound A exists in an amorphous form, referred to herein as amorphous Compound A. This invention is therefore directed to stable crystalline forms of Compound A, i.e., Compound A Form A and Compound A Form B, whose properties can be influenced by controlling the conditions under which Compound A is obtained in solid form. The characteristics and properties of Compound A Form A and Compound A Form B are each described detail below.

Abbreviations

The following abbreviations may be used herein as needed:

DAD for diode array detector;

DSC for differential scanning calorimetry;

DHEA for 5-dehydroepiandrosterone;

FT-IR for fourier transform infrared spectroscopy;

FWHM for full width at half maximum;

HPLC for high performance liquid chromatography;

HR-XRPD for high resolution X-ray powder diffraction;

HT-XRPD for high throughput X-ray powder diffraction;

m.p. for melting point; PTFE for polytetrafluoroethylene;

rpm for revolutions per minute;

SDTA for simultaneous differential thermal analysis;

TFA for trifluoroacetic acid;

TGA for thermogravimetric analysis;

TGA/MS for thermogravimetric analysis coupled with mass spectroscopy;

THF for tetrahydrofuran; and

XRPD for X-ray powder diffraction.

Compound A Form A

In one embodiment of the invention, Compound A Form A is provided, characterized by the selection of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or twenty-one X-ray powder diffraction peaks selected from the group consisting of 8.943°, 9.536°, 10.497°, 11.951 °, 13.097°, 13.335°, 14.047°, 16.091 °, 16.654°, 17.940°, 18.135°, 18.496°, 18.960°, 19.724°, 20.143°, 20.461 °, 21.1 17°, 24.320°, 24.695°, 25.572°, and 25.959° 2Θ ± 0.3° 2Θ, more preferably ± 0.2° 2Θ, even more preferably ± 0.1 ° 2Θ, most preferably ± 0.05° 2Θ.

In another embodiment, Compound A Form A is characterized by the following set of XRPD peaks and, optionally, by the associated intensities listed in Table 1 :

Table 1. HR-XRPD peak table for Compound A Form A

Peak ID Angle (2Θ) d-Value Intensity

10 17.940 4.94031 L

11 18.135 4.88767 H

12 18.496 4.79309 L

13 18.960 4.67668 H

14 19.724 4.49723 M

15 20.143 4.40464 M

16 20.461 4.33705 M

17 21.117 4.20374 M

18 24.320 3.65679 L

19 24.695 3.60214 L

20 25.572 3.48058 L

21 25.959 3.42959 M

For intensity values: L = 3-25, M = 26-50, H = 51-100 (See Example 4).

In another embodiment, Compound A Form A is characterized by an XRPD substantially according to Figure 2.

In a preferred embodiment, Compound A Form A is characterized by an XRPD containing at least one of the following peaks: 8.943°, 9.536°, 10.497°, and 11.951 ° 2Θ ± 0.3° 2Θ, more preferably ± 0.2° 2Θ, even more preferably ± 0.1 ° 2Θ, most preferably ± 0.05° 2Θ. In a more preferred embodiment, Compound A Form A is characterized by an XRPD containing at least two of the following peaks: 8.943°, 9.536°, 10.497°, and 11.951 ° 2Θ ± 0.3° 2Θ, more preferably ± 0.2° 2Θ, even more preferably ± 0.1 ° 2Θ, most preferably ± 0.05° 2Θ.

In another embodiment, Compound A Form A is characterized by a DSC thermogram substantially according to Figure 3.

In another embodiment, Compound A Form A is characterized by a TGA thermogram substantially according to Figure 4.

In another embodiment, Compound A Form A is characterized by a DSC thermogram with an endothermic event with an onset at 181.3 °C ± 0.3 °C, more preferably ± 0.2 °C, most preferably ± 0.1 °C, and a characterizing endothermic peak at 182.7 °C ± 0.3 °C, more preferably ± 0.2 °C, most preferably ± 0.1 °C. From the analysis of the DSC thermogram, it was concluded that Compound A Form A is anhydrous. Residual chloroform was present (0.9% w/w) in the crystal as shown in the TGA/MS analysis (Figure 5).

In another embodiment, Compound A Form A is anhydrous and is stable as indicated by the DSC thermogram in Figure 3, which shows a sharp peak near the melting point. Compound A Form A was stable for 2 days under accelerated aging conditions (Method C). Based on XRPD data, Compound A Form A crystallizes in an orthorhombic crystal system P 2i2i2i space group with four monomers, hydrogen bonded, in the asymmetric unit, as seen in Figure 7. The crystal is held together by a network of intermolecular hydrogen bonds which likely provides the unexpected high stability/melting point of Compound A. Rietveld analysis (Figure 8) indicates the presence of only one polymorph in the Compund A Form A crystalline material.

In another embodiment, Compound A Form A is in a substantially pure form, and preferably substantially free from other amorphous, crystalline and/or polymorphic forms. In this respect, "substantially pure" means the form contains at least about 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % of the Compound A Form A. In this respect, "substantially free from other amorphous, crystalline and/or polymorphic forms" means that no more than about 20 %, 15 %, 10 %, 5 %, 4 %, 3 %, 2 %, 1 % of these other amorphous, crystalline and/or polymorphic forms are present.

In embodiments of the invention, a method for the preparation of Compound A Form A is provided, including the steps of preparing a suspension of amorphous Compound A in a solvent selected from the group consisting of water, methanol, ethanol, isopropanol, 1 ,4-dioxane, terf-butyl methyl ether, tetrahydrofuran, acetonitrile, chloroform, cyclohexane, heptane, toluene, p-xylene, cumene, isopropyl acetate, anisole, 1 ,2-dimethoxyethane, and dichloromethane, or mixtures thereof, and crystallizing Compound A Form A by methods known to those skilled in the art, such as, but not limited to, cooling crystallization, crystallization by slurry conversion, evaporative crystallization by anti-solvent addition, vapor diffusion into liquid crystallization, vapor diffusion onto a solid crystallization, and crystallization by wet milling. In a preferred embodiment, the solvent is selected from the group consisting of water, acetonitrile, chloroform, ethanol, isopropanol, te/f-butyl methyl ether, heptane, isopropyl acetate, and anisole, and mixtures thereof. In a more preferred embodiment, the solvent is chloroform. In certain embodiments, a second solvent (co-solvent or anti-solvent), selected from the group consisting of water, acetonitrile and tetrahydrofuran is used in an amount between 5 % and 95 % (v/v) with an amount of first solvent between 95 % and 5 % (v/v), preferably between 10 % and 35 % (v/v) with an amount of first solvent between 90 % and 65 % (v/v), more preferably between 15 % and 30 % (v/v) with an amount of first solvent between 85 % and 70 % (v/v), and most preferably between 20 % and 25 % (v/v), with an amount of first solvent between 80 % and 75 % (v/v). In a preferred embodiment, water, acetonitrile, or tetrahydrofuran is used as a second solvent.

In a more preferred embodiment, the first solvent/second solvent pairs are selected from the group consisting of water/tetrahydrofuran (20:80, v/v), and water/acetonitrile (10:90, v/v) were used.

The crystal of Compound A Form A of the invention has also been

characterized based on XRPD analysis of Compound A Form A as depicted in Figure 6 and/or Figure 7 and/or in Table 2:

Table 2. Crystal data and structure refinement based on XRPD analysis for Compound A Form A.

* The absolute configuration of Compound A could not be determined based on the calculated crystal data. However, the absolute stereochemistry of Compound A is set by the raw starting material DHEA; consequently, it is expected that the configuration can be assigned as S(C2)S(C4)R(C7)S(C1 1)S(C12)S(C15)S(C20) Compound A Form B

In another embodiment of the invention, there is disclosed crystalline

Compound A Form B , characterized by the selection of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty X-ray powder diffraction peaks selected from the group consisting of 6.321 °, 9.110°, 12.290°, 12.756°, 13.779°, 14.656°, 17.224°, 18.1 11 °, 19.051 °, 20.321 °, 22.059°, 23.869°, 24.644°, 25.844°, 26.870°, 28.641 °, 31.196°, 33.572°, 35.320°, and 36.896° 2Θ ± 0.3° 2Θ, more preferably ± 0.2° 2Θ, even more preferably ± 0.1 ° 2Θ, most preferably ± 0.05° 2Θ.

In another embodiment, Compound A Form B can be characterized by the following set of XRPD peaks and, optionally, by the associated intensities listed in Table 3:

Table 3. HT-XRPD peak table for Compound A Form B

Peak ID Angle (2Θ) d-Value Intensity

7 17.224 5.1441 H

8 18.11 1 4.8940 H

9 19.051 4.6547 H

10 20.321 4.3664 M

11 22.059 4.0262 M

12 23.869 3.7249 M

13 24.644 3.6095 M

14 25.844 3.4445 M

15 26.870 3.3153 M

16 28.641 3.1 142 M

17 31.196 2.8647 L

18 33.572 2.6672 L

19 35.320 2.5391 L

20 36.896 2.4342 L

For intensity values: L = 3-25, M = 26-50, H = 51-100 (See Example 4).

In another embodiment, Compound A Form B is characterized by an XRPD substantially according to Figure 9.

In a preferred embodiment, Compound A Form B is characterized by an XRPD pattern containing at least one of the following peaks: 6.321 °, 9.110°, 12.290°, and 12.756° 2Θ ± 0.3° 2Θ, more preferably ± 0.2° 2Θ, even more preferably ± 0.1 ° 2Θ, most preferably ± 0.05° 2Θ. In a more preferred embodiment, Compound A Form B is characterized by an XRPD containing at least two of the following peaks: 6.321 °, 9.1 10°, 12.290°, and 12.756° 2Θ ± 0.3° 2Θ, more preferably ± 0.2° 2Θ, even more preferably ± 0.1 ° 2Θ, most preferably ± 0.05° 2Θ.

In another embodiment, Compound A Form B is characterized by a DSC substantially according to Figure 10.

In another embodiment, Compound A Form B is characterized by a TGA substantially according to Figure 1 1. In another embodiment, Compound A Form B of the present invention is characterized by DSC with an endothermic event with an onset at 172.3 °C ± 0.3 °C, more preferably ± 0.2 °C, most preferably ± 0.1 °C and a characterizing endothermic peak at 176.5 °C ± 0.3 °C, more preferably ± 0.2 °C, most preferably ± 0.1 °C. From thermal analysis, it is concluded that solid Compound A Form B is a methanol solvate.

In another embodiment, Compound A Form B is a methanol solvate as in the TGA/MS Analysis of Figure 12

In another embodiment, Compound A Form B is in a substantially pure form, preferably substantially free from other amorphous, crystalline and/or polymorphic forms. In this respect, "substantially pure" relates to a form which contains at least about 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % of Compound A Form B. In this respect, "substantially free from other amorphous, crystalline and/or polymorphic forms" means that no more than about 20 % 15 %, 10 %, 5 %, 4 %, 3 %, 2 %, 1 % of these other amorphous, crystalline and/or polymorphic forms are present in the form according to the invention.

In embodiments of the invention, a method for the preparation of Compound A Form B is provided comprising the steps of preparing a suspension of Compound A in a mixture of solvents selected from the group consisting of methanol and water and crystallizing Compound A Form B by cooling crystallization, crystallization by slurry conversion, evaporative crystallization by anti-solvent addition, vapor diffusion into liquid crystallization, vapor diffusion onto a solid crystallization, or crystallization by wet milling.

In a preferred embodiment, the solvent is a mixture of water and methanol. In a more preferred embodiment, the solvent is a 9: 1 (v/v) mixture of methanol and water. Pharmaceutical Compositions

Compound A Form A and Compound A Form B may be formulated as a pharmaceutical composition in a manner similar to the pharmaceutical compositions disclosed in U.S. Patent Nos. 7,601 ,874 and 7,999,010. Such pharmaceutical compositions comprise Compound A Form A or Compound A Form B and one or more pharmaceutically acceptable carriers, wherein the Compound A Form A or Compound A Form B is present in the composition in an amount that is effective to treat the condition of interest. Typically, the pharmaceutical compositions of the present invention include Compound A Form A or Compound A Form B in an amount ranging from 0.1 mg to 250 mg per dosage depending upon the route of administration, and more typically from 1 mg to 60 mg. Appropriate concentrations and dosages can be readily determined by one skilled in the art.

Pharmaceutically acceptable carriers are familiar to those skilled in the art. For compositions formulated as liquid solutions, acceptable carriers include saline and sterile water, and may optionally include antioxidants, buffers, bacteriostats and other common additives. The compositions can also be formulated as pills, capsules, granules, or tablets which contain, in addition to an effective amount of Compound A Form A or Compound A Form B, diluents, dispersing and surface-active agents, binders, lubricants, and/or delayed releases agents. One skilled in this art may further formulate Compound A Form A or Compound A Form B in an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton, PA (current edition, the relevant sections of which are incorporated herein by reference in their entirety). Utility and Methods of Administration

Compound A and its crystalline forms, i.e., Compound A Form A and

Compound A Form B, have activity as SHIP1 modulators and therefore may be used to treat any of a variety of diseases, disorders or conditions in a mammal, preferably a human, that would benefit from SHIP1 modulation. Such diseases, disorders or conditions are disclosed in PCT Published Patent Application Nos. WO 2014/143561 and WO 2014/158654.

Accordingly, an embodiment of the invention is a method modulating SHIP1 activity in a mammal comprising administering an effective amount of Compound A Form A or Compound A Form B or an effective amount of a composition comprising Compound A Form A or Compound A Form B to the mammal in need thereof.

Another embodiment is a method for treating a disease, disorder or condition associated with SHIP1 activity in a mammal comprising administering an effective amount of Compound A Form A or Compound A Form B or an effective amount of a composition comprising Compound A Form A or Compound A Form B to the mammal in need thereof.

Such methods include administering to a mammal, preferably a human, Compound A Form A or Compound A Form B in an amount sufficient to treat the disease, disorder or condition. In this context, "treat" includes prophylactic

administration. Such methods include systemic administration of Compound A Form A or Compound A Form B, preferably in the form of a pharmaceutical composition as discussed above. As used herein, systemic administration includes oral and parenteral methods of administration. For oral administration, suitable pharmaceutical compositions include powders, granules, pills, tablets, and capsules as well as liquids, syrups, suspensions, and emulsions. These compositions may also include flavorants, preservatives, suspending, thickening and emulsifying agents, and other

pharmaceutically acceptable additives. For parenteral administration, the crystalline forms of Compound A of the present invention can be prepared in aqueous injection solutions which may contain buffers, antioxidants, bacteriostats, and other additives commonly employed in such solutions.

Methods of Preparation

Representative crystalline forms of Compound A of the invention were prepared according to Methods A to C, as described below and subsequently analyzed. Representative crystalline forms of Compound A were aged by Method C and subsequently analyzed. It will be appreciated that in the following general methods, solvents used, relative amounts of solvents, and other parameters such as cooling rates, temperatures, times, etc. can be altered to suit needs, up or down by up to 50 % without significant change in expected results.

Method A: Crystallization by slurry conversion and evaporative crystallization

An approximately 20 mg aliquot of amorphous solid was solid dosed in a 1.8 ml_ glass vial. Ethanol was added in defined aliquots (ex. 100 μΙ_) until about 50 % of the solid had dissolved (ex. 100 μΙ_ final volume). The vial was stirred at ambient or elevated temperature for a period of time followed by cooling to ambient temperature. After prolonged incubation, the solid was separated from the liquid by centrifugation. The solid was dried under ambient conditions and analyzed by XRPD. The mother liquors and solvent were further evaporated under ambient conditions and the remaining solids analyzed by XRPD.

By similar techniques, a person skilled in the art would be able to obtain similar results utilizing solvents selected from water, methanol, acetone, isopropanol, ethyl acetate, 2-butanone, te/f-butyl methyl ether, 1 ,4-dioxane, acetonitrile, tetrahydrofuran, cyclohexane, chloroform, dichloromethane, toluene, heptane, cumene, p-xylene, anisole, isopropyl acetate, and 1 ,2-dimethoxyethane, or mixtures thereof. Method B: Quantitative solubility assessment and crystallization

The solubility of Compound A in a solvent or solvent mixture was determined at room temperature. A 20 mg aliquot of Compound A (amorphous) was weighed in a standard 1.8 mL HPLC vial. Subsequently, chloroform (300 μΙ_) was added and the vial was left to equilibrate at room temperature with continuous stirring. After 24 hours, the solid was separated from the liquid by centrifugation and analyzed by XRPD.

Subsequently the remaining liquid phase was further filtered through a 0.45 μηι PTFE filter to remove any particulate matter. The concentration of the Compound A in solution (67 mg/mL) was determined by HPLC-DAD analysis using a calibration curve made from two independent stock solutions of the Compound A prepared in 0.1 % TFA in water/acetonitrile (50:50).

By similar techniques, a person skilled in the art would be able to obtain similar results utilizing a variety of solvents.

Method C: Accelerated Aging Analysis by XRPD

Samples collected from the crystallization conditions were subjected, as is, to accelerated aging conditions of 40 °C and 75 % relative humidity for 48 hours, via standard methods known to one skilled in the art and analyzed by XRPD.

The following Examples are provided for purposes of illustration, not limitation. In summary, the following Examples disclose the preparation, analysis and

characterization of Compound A Form A and Compound A Form B of the invention. One of ordinary skill in the art understands that experimental differences may arise due to differences in instrumentation, sample preparation, or other factors.

EXAMPLE 1

Preparation of amorphous Compound A

Amorphous Compound A was prepared by freeze-drying a THF/water (80:20, v/v) solution of Compound A. Amorphous Compound A material was further used, as is, in crystallization experiments.

EXAMPLE 2

Preparation of Compound A Form A for XRPD, DSC and TGA Analysis Compound A Form A was generated through a modified Method A procedure.

Amorphous Compound A (20 mg) was suspended in a chloroform (100 μί) in a 1.8 mL HPLC vial. The mixture was kept at room temperature, with stirring (magnetic stirring bar), for about two weeks under ambient conditions. Solids were then separated from the liquid by centrifugation and some of the solids were harvested from the vials and placed in a 96-well plate for wet analysis. The remaining solid was dried in the vial under vacuum (at room temperature and 200 mbar) and analyzed by XRPD, DSC and TGA as well as subjected to aging studies (Method C).

EXAMPLE 3

Preparation of Compound A Form B for XRPD, DSC, and TGA Analysis

Compound A Form B was generated through a modified Method A procedure. Amorphous Compound A (37.5 mg) was suspended in methanol/water (200 μΙ_,

(90: 10, v/v)) in a 1.8 ml_ HPLC vial. The mixture was kept at room temperature, with stirring (magnetiuc stirring bar), for about two weeks under ambient conditions. Solids were then separated from the liquid by centrifugation and some of the solids were harvested from the vials and placed in a 96-well plate for wet analysis. The remaining solid was dried in the vial under vacuum (at room temperature and 200 mbar) and analyzed by XRPD, DSC and TGA.

EXAMPLE 4

High Resolution X-Ray Diffraction Spectrometry Experimental Conditions

Dry solid sample from Example 2, was transferred into a boron-glass capillary with 0.3 mm outer diameter, 8 mm length. The capillary was mounted on the goniometer head and placed in the D8 Advance Bruker-AXS Diffractometer equipped with solid state LynxEye detector. The capillary was spinning during data recording, at 15 rpm. The XRPD platform was calibrated using Silver Behenate for the long d-spacings and Corundum for the short d-spacings.

Data collection for Compound A Form A was carried out, in transmission mode, at ambient conditions (-23 °C and -100 kPa) using CuK^ radiation (1.54056 A), monochromatized by germanium crystal, in the 2Θ region between 4° and 45° 2Θ, with an exposure time of 90 s for each frame and 0.016° 2Θ increments. No additional corrections were made during data collection.

Peak selection was performed using DIFFRAC ^p,us EVA software package

(Bruker-AXS, 2007), using a second derivative method working on data prepared by Savitzky-Golay (Savitzky, A. & Golay, M.J.E. (1964) Anal. Chem. 36, 1627) smoothing filter with the following criteria:

1. Peak width: The algorithm uses 5 to 57 data points centered on the point of interest, and a peak is selected from the second derivate if the peak width lays within the range FWHM < Peak width < 4 x FWHM. Peak widths for 1 Form A and 1 Form B were 0.2 and 0.3°, respectively.

2. Threshold: Based on the comparison of the computed maximum of the peak with the middle of the chord joining 2 inflection points of both sides of maximum. According to Equation 1 , a peak was accepted if l _P was greater than the intensity at chord center (l _M ) plus a factor comprising a threshold value of

1.0 multiplied by the square root of l _M , as described in the software manual (DIFFRAC ^PLUS £W» Manual (2007) Bruker-AXS, Karlsruhe):

I _P > I _M + T x Jl^ (Equation 1)

Where: l _P = peak intensity; l _M = intensity at chord center; T = threshold. The XRPD diffractogram from Example 4 is shown in Figure 2 for Compound A

Form A, and is indicative of diffractograms generated from material for Compound A Form A performed by alternative crystallization methods.

The diffractogram presented in Figure 1 was recorded on D8 Discover Bruker- AXS diffractometer using Cu K _a radiation (1.54178 A) equipped with 2D GADDS detector in transmission mode. The sample of amorphous Compound A was placed in the flat transmission sandwich-like 4.5 mm diameter sample holder protected by X-Ray transparent mylar foil. During the measurement sample was oscillated in x,y direction (perpendicular to the primary beam) with 1.75 mm radius. Data collection was carried out in two frames 1.5 < 2Θ < 21.5° and 19.5 < 2Θ < 41.5° and separately integrated with step size 0.04°. The final powder pattern was obtained by merging both frames using area between 19.5 < 2Θ < 21.5° as common share. Peaks were analyzed as described above.

EXAMPLE 5

High Throughput X-Ray Diffraction Spectrometry Experimental Conditions A sample from Example 3, was transferred to a T2 high-throughput XRPD setup. Plates were mounted on a Bruker GADDS diffractometer equipped with a Hi- Star area detector. The XRPD platform was calibrated using Silver Behenate for the long d-spacings and Corundum for the short d spacings. Data collection for Compound A Form B was carried out, in transmission mode, at ambient conditions (-23 °C and -100 kPa) using monochromatic CuK _a i radiation (1.54056 A) in two 2Θ ranges (1.5 < 2Θ < 21.5° and 19.5 < 2Θ < 41.5°) with an exposure time of 90 s for each frame. No additional corrections were made during data collection.

The XRPD diffractogram from Example 5 is shown in Figure 9 for Compound A Form B, and is indicative of diffractograms generated from material for Compound A Form B performed by alternative crystallization methods.

EXAMPLE 6

Quantitative Phase Analysis Experimental Details

Quantitative Phase Analysis (sometimes called also Standardless Phase Analysis, Multiphase Rietveld Phase Quantification, Rietveld Quantitative Analysis or Rietveld XRD Quantification) is a powerful method for determining the quantities of different polymorphs in multiphase mixtures. The method relies on the simple relationship: where W is the relative weight fraction of phase p in a mixture of n phases, and S, Z, M, and V are, respectively, the Rietveld scale factor, the number of formula units per cell, the mass of the formula unit (in atomic mass units) and the unit cell volume (in A ³ ).

Advantages of Quantitative Phase Analysis in analyzing polymorphs include a) calibration constants are computed from reliable structural data, b) all reflections in the pattern are explicitly included for calculation, and c) the effects of preferred orientation and extinction are reduced, since all reflection types are considered.

Crystal structural and peak profile parameters, particle statistics, micro- absorption etc are refined as part of the same analysis. Also, the phase analysis allows for the determination of the weight (or volume) and quantity of crystalline and amorphous phases which are present in the sample.

The most intensive diffraction peaks combined with chemical and

crystallographical knowledge should lead to indexing powder pattern and use it as reference. Using this attempt, the ratio between forms directly depends on the area of the indexed to unindexed parts of the pattern. The method used in this project is the Pawley fit based on the High Resolution powder pattern. The main purpose of the Pawley is to refine cell parameters from the whole pattern. In the Pawley method, profiles are analytical, their width is constrained to follow a Caglioti law with the three refinable parameters U, V, W as defined in most of the Rietveld-derived software.

The software used for these calculations in this project was Topas™ (Coelho, RW (2005), TOPAS- R software, Bruker AXS, Karlsruhe, Germany). The following criteria of fit were used in this particular project:

• Ycm and Y _c , _m are the observed and calculated data, respectively at data point m,

• M the number of data points,

• P the number of parameters,

• w _m the weighting given to data point m which for counting statistics is given by w _m =1/a(Y ₀ , J ² where a(Y _0im ) is the error in Y ₀ , _m ,

A representation of the crystal structure from this Example is shown in Figures 6 and 7 and details of the crystal are shown in Table 2. Calculated data and comparison to experimental (Rietveld Analysis as per Example 6) is shown in Figure 8

EXAMPLE 7

Thermal Analysis Experimental Conditions

Compound A Form A and Compound A Form B material from Examples 2 and 3, respectively, were used for thermal analysis.

DSC Analysis: Melting properties were obtained from DSC thermograms, recorded with a heat flux DSC822e instrument (Mettler-Toledo GmbH, Switzerland). The DSC822e was calibrated for temperature and enthalpy with a small piece of indium (m.p. = 156.6 °C; AH _f = 28.45 J/g). Samples were sealed in standard 40 microliter aluminum pans and heated in the DSC from 25 °C to 300 °C, at a heating rate of 10 °C/min. Dry N ₂ gas, at a flow rate of 50 mL/min, was used to purge the DSC equipment during measurement. Representative DSC data from Example 7 can be found in Figure 3 for Compound A Form A and Figure 10 for Compound A Form B and are indicative of DSC data generated from material for Compound A Form A and Compound A Form B performed by alternative crystallization methods.

TGA/MS Analysis: Mass loss due to solvent or water loss from the crystals, from Examples 2 and 3, was determined by TGA/SDTA analysis. Monitoring of the sample weight, during heating in a TGA/SDTA851 e instrument (Mettler-Toledo GmbH, Switzerland), resulted in a weight vs. temperature curve for each sample. The TGA/SDTA curves for Compound A Form A and Compound A Form B are in Figures 4 and 1 1 , respectively and are indicative of TGA/SDTA data generated from material for Compound A Form A and Compound A Form B performed by alternative crystallization methods. The TGA/SDTA851e was calibrated for temperature with indium and aluminum. Samples, from Examples 2 and 3, were weighed into 100 μΙ_ aluminum crucibles and sealed. The seals were pin-holed and the crucibles heated in the TGA from 25 °C to 300 °C at a heating rate of 10 °C/min. Dry N ₂ gas is used for purging. The gases evolved from the TGA samples were analysed by an Omnistar GSD 301 T2 (Pfeiffer Vacuum GmbH, Germany) quadrapole mass spectrometer, which analyzes masses in the range of 0 to 200 amu. TGA/MS data for Compound A Form A is in Figure 5 and Compound A Form B is in Figure 12 and are indicative of TGA/MS data generated from material for Compound A Form A and Compound A form B, respectively, performed by alternative crystallization methods.

* * * * *

All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference in their entireties.

Although the foregoing invention has been described in some detail to facilitate understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. Accordingly, the described embodiments are to be considered as illustrative and not restrictive, and the invention is not to be limited to the details given herein, but may be modified within the scope and equivalents of the appended claims.