WO2016086080A1

WO2016086080A1 - Crystalline forms of parp inhibitors

Info

Publication number: WO2016086080A1
Application number: PCT/US2015/062572
Authority: WO
Inventors: Stephen J. BIERLMAIER; Ralph C. HALTIWANGER; Martin J. Jacobs
Original assignee: Cephalon, Inc.
Priority date: 2014-11-26
Filing date: 2015-11-25
Publication date: 2016-06-02
Also published as: CN107207511A; EP3224259A1; RU2017120655A3; CA2967828A1; BR112017010588A2; US10150772B2; US20170267683A1; MX2017006679A; JP2017535564A; HK1244006A1; IL252162A0; RU2017120655A; KR20170088894A; AU2015353549A1

Abstract

The present disclosure relates to crystalline forms of 4,5,6,7-tetrahydro-l l-methoxy-2- [(4-methyl-1-piperazinyl)methyl]- 1H-cyclopenta[a]pyrrolo [3,4-c]carbazole- 1,3 (2H)-dione, including salts forms and free base forms.

Description

CRYSTALLINE FORMS OF PARP INHIBITORS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No.

62/084,652, filed November 26, 2014, the entirety of which is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure relates to crystalline forms of 4,5,6,7-tetrahydro- 11- methoxy-2-[(4-methyl-l-piperazinyl)methyl]-lH-cyclopenta[a]pyrrolo[3,4-c]carbazole-l,3(2H)- dione and salts thereof.

BACKGROUND

[0003] Compound A (4,5,6,7-Tetrahydro- 11 -methoxy-2-[(4-methyl- 1 - piperazinyl)methyl]-lH-cyclopenta[a]pyrrolo[3,4-c]carbazole-l,3(2H)-dione) is a PARP (poly ADP-ribose polymerase) inhibitor for use in the treatment of breast, ovarian, and other cancers, either alone or in conjunction with chemotherapy or radiotherapy. See, e.g., U.S. Patent Nos. 7,122,679; 8,716,493; and 8 633,314.

Compound A

[0004] Compound A is a prodrug of Compound B:

Compound B [0005] The free base form of Compound A forms hydrates, which are undesirable. In addition, the free base form of Compound A has a low bulk density, impeding manufacturing. Alternative forms of Compound A are needed.

SUMMARY

[0006] The disclosure is directed to Compound A, acetate salt Form Ai_.5; Compound A, glycolate salt hydrate Form Ai; Compound A, L-malate salt Form Ai; Compound A, L-malate salt Form Ai_.5; Compound A, L-pyroglutamate salt Form Ai; Compound A, free base Form Co; Compound A, hydrochloride salt Form A; Compound A, fumarate salt Form A; and Compound A, p-toluenesulfonate salt Form A. Pharmaceutical compositions comprising one or more of these forms are also described. Methods of using these forms is described, as well.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 shows an XRPD Pattern for Compound A Free Base, Form Ao.

[0008] Figure 2 shows a DSC/TGA Overlay for Compound A Free Base, Form A₀.

[0009] Figure 3 shows an XRPD Pattern of Compound A Acetate Salt, Form A1.5.

[0010] Figure 4 shows VT-XRPD Patterns of Compound A Acetate Salt, Form A1.5 - Requested Mode.

[0011] Figure 5 shows VT-XRPD Patterns of Compound A Acetate Salt, Form A1.5 - Continuous Mode.

[0012] Figure 6 shows a DSC and TGA Overlay of Compound A Acetate Salt, Form

[0013] Figure 7 shows a DVS Overlay of Compound A Acetate Salt, Form A1.5.

[0014] Figure 8 shows a photomicrograph of Compound A Acetate Salt, Form A1.5.

[0015] Figure 9 shows an XRPD Pattern of Compound A Glycolate Salt Hydrate, Form AL

[0016] Figure 10 shows thermal XRPD Patterns of Compound A Glycolate Salt

Hydrate, Form Ai_.

[0017] Figure 1 1 shows a DSC and TGA Overlay of Compound A Glycolate Salt

Hydrate, Form Ai.

[0018] Figure 12 shows a DVS Overlay of Compound A Glycolate Salt Hydrate, Form Ai.

[0019] Figure 13 shows a photomicrograph of Compound A Glycolate Salt Hydrate, Form Ai_. [0020] Figure 14 shows an XRPD Pattern of Compound A L-Malate Salt, Form A_L

[0021] Figure 15 shows VT-XRPD Patterns of Compound A Malate Salt, Form A_x.

[0022] Figure 16 shows a DSC and TGA Overlay of Compound A L-Malate Salt, Form Ai.

[0023] Figure 17 shows a DVS of Compound A L-Malate Salt, Form A

[0024] Figure 18 shows a photomicrograph of Compound A L-Malate Salt, Form Ai.

[0025] Figure 19 shows an XRPD Pattern of Compound A L-Malate Salt, Form Ai_.5

[0026] Figure 20 shows a DSC and TGA Overlay of Compound A L-Malate Salt, Form

[0027] Figure 21 shows an XRPD Pattern of Compound A L-Pyroglutamate Salt, Form Ai.

[0028] Figure 22 shows VT-XRPD Patterns of Compound A L-Pyroglutamate Salt, Form Ai.

[0029] Figure 23 shows a DSC and TGA Overlay of Compound A L-Pyroglutamate Salt, Form Ai.

[0030] Figure 24 shows a DVS of Compound A L-Pyroglutamate Salt, Form Ai.

[0031] Figure 25 shows a photomicrograph of Compound A L-Pyroglutamate Salt, Form Ai.

[0032] Figure 26 shows an XRPD Pattern of Compound A Free Base, Form Co.

[0033] Figure 27 shows thermal XRPD Patterns of Compound A Free Base, Form Co.

[0034] Figure 28 shows a DSC and TGA Overlay of Compound A Free Base, Form Co.

[0035] Figure 29 shows a photomicrograph of Compound A A Free Base, Form Co_.

[0036] Figure 30 shows an XRPD Pattern of Compound A Hydrochloride Salt, Form A.

[0037] Figure 31 shows a DSC and TGA Overlay of Compound A Hydrochloride Salt, Form A.

[0038] Figure 32 shows a DVS of Compound A Hydrochloride Salt, Form A.

[0039] Figure 33 shows an XRPD Pattern of Compound A Fumarate Salt, Form A.

[0040] Figure 34 shows a DSC and TGA Overlay of Compound A Fumarate Salt, Form A.

[0041] Figure 35 shows a XRPD Pattern of Compound A p-Toluenesulfonate Salt, Form A. [0042] Figure 36 shows a DSC and TGA Overlay of Compound A p-Toluenesulfonate Salt, Form A.

[0043] Figure 37 shows plasma levels of Compound B, 1 mg/kg intravenous,

Compound A, ascorbic acid salt, 30 mg/kg oral, and Compound A, glycolate hydrate salt, 30 mg/kg oral in rat.

[0044] Figure 38 shows the single crystal structure of Compound A, glycolate hydrate salt.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0045] The present disclosure addresses a need in the art by providing new forms of Compound A, including new crystalline free base forms of Compound A and new crystalline salt forms of Compound A.

[0046] The disclosure is directed to, among other things, Compound A, acetate salt Form A_{1 5}; Compound A, glycolate salt hydrate Form Ai; Compound A, L-malate salt Form Ai; Compound A, L-malate salt Form Ai.₅; Compound A, L-pyroglutamate salt Form Ai; Compound A, free base Form Co; Compound A, hydrochloride salt Form A; Compound A, fumarate salt Form A; and Compound A, p-toluenesulfonate salt Form A. Pharmaceutical compositions comprising one or more of these forms are also described.

[0047] In one embodiment, the present disclosure pertains to Compound A, acetate salt Form A1.5. In one aspect, this crystalline form is characterized by an X-ray diffraction pattern comprising one or more of the following peaks: 6.4, 9.2, 12.7, 13.0, 15.2, 17.4, 18.4, 19.0, 19.3, 21.3, 21.5, 23.1, 24.1, 24.2, and/or 28.2 ± 0.2 degrees 2-theta. In another aspect, this crystalline form comprises at least 3 of the foregoing peaks. In yet another aspect, this crystalline for comprises at least 4, 5, 6, 7, 8, 9, or 10 of the foregoing peaks. In another aspect, this crystalline form has an X-ray powder diffraction pattern substantially as depicted in Figure 3.

[0048] The disclosure is also directed to Compound A, glycolate hydrate salts. These salts can have varying amounts of water within the crystal structure. For example, the ratio of Compound A to water can be from about 1 :0.1 to about 1 : 1. In other embodiments, the ratio of Compound A to water is 1 :0.1; 1 :0.2; 1 :0.3; 1 :0.4; 1 :0.5; 1 :0.6; 1 :0.7; 1 :0.8; 1 :0.9 or 1 : 1.

[0049] Another embodiment of the present disclosure pertains to Compound A, glycolate hydrate salt Form Ai. In one aspect, this crystalline form is characterized by an X-ray diffraction pattern comprising one or more of the following peaks: 8.1, 8.2, 8.7, 13.9, 14.7, 14.9, 16.3, 17.4, 17.6, 18.2, 18.5, 19.0, 20.2, 20.6, 21.2, 21.4, 23.0, 24.5, 24.7, 26.1, 26.3, 28.0, 30.0, 30.1, 30.2, and/or 32.8±0.2 degrees 2-theta. In another aspect, this crystalline form comprises at least 3 of the foregoing peaks. In yet another aspect, this crystalline for comprises at least 4, 5, 6, 7, 8, 9, or 10 of the foregoing peaks. In another aspect, this crystalline form has an X-ray powder diffraction pattern substantially as depicted in Figure 9.

[0050] Yet another embodiment of the disclosure pertains to Compound A, L-malate salt Form Αχ. In one aspect, this crystalline form is characterized by an X-ray diffraction pattern comprising one or more of the following peaks: 8.6, 9.2, 10.1, 10.4, 11.7, 1 1.9, 14.7, 15.3, 15.6,

17.2, 17.8, 18.5, 20.3, 20.7, 21.2, 22.4, 23.5, 24.3, and/or 27.0 ± 0.2 degrees 2-theta. In another aspect, this crystalline form comprises at least 3 of the foregoing peaks. In yet another aspect, this crystalline for comprises at least 4, 5, 6, 7, 8, 9, or 10 of the foregoing peaks. In another aspect, this crystalline form has an X-ray powder diffraction pattern substantially as depicted in Figure 14.

[0051] In another embodiment, the disclosure pertains to Compound A, L-malate salt Form A1.5. In one aspect, this crystalline form is characterized by an X-ray diffraction pattern comprising one or more of the following peaks: 5.5, 6.8, 8.0, 8.4, 8.8, 9.2, 1 1.8, 12.8, 13.1, 13.6, 14.4, 16.0, 16.7, 18.1, 18.5, 19.4, 20.2, 20.5, 21.1, 21.9, 23.4, and/or 24.6 ± 0.2 degrees 2-theta. In another aspect, this crystalline form comprises at least 3 of the foregoing peaks. In yet another aspect, this crystalline for comprises at least 4, 5, 6, 7, 8, 9, or 10 of the foregoing peaks. In another aspect, this crystalline form has an X-ray powder diffraction pattern substantially as depicted in Figure 19.

[0052] Also described herein is Compound A, L-pyroglutamate salt Form Ai. In one aspect, this crystalline form is characterized by an X-ray diffraction pattern comprising one or more of the following peaks: 6.0, 9.6, 10.3, 10.5, 1 1.0, 12.0, 13.2, 15.0, 16.7, 17.5, 17.8, 18.0, 19.0, 20.8, 21.0, 21.1, 22.0, 22.1, 23.1, 23.4, 23.5, 24.8, and/or 26.6 ± 0.2 degrees 2-theta. In another aspect, this crystalline form comprises at least 3 of the foregoing peaks. In yet another aspect, this crystalline for comprises at least 4, 5, 6, 7, 8, 9, or 10 of the foregoing peaks. In another aspect, this crystalline form has an X-ray powder diffraction pattern substantially as depicted in Figure 21.

[0053] The present disclosure also pertains to Compound A, free base Form Co. In one aspect, this crystalline form is characterized by an X-ray diffraction pattern comprising one or more of the following peaks: 8.5, 8.8, 13.9, 14.4, 15.4, 17.6, 18.1, 18.5, 19.2, 19.7, 20.4, 21.1, 21.4, 21.9, 23.6, 24.6, 29.4 and/or 30.1 ± 0.2 degrees 2-theta. In another aspect, this crystalline form comprises at least 3 of the foregoing peaks. In yet another aspect, this crystalline for comprises at least 4, 5, 6, 7, 8, 9, or 10 of the foregoing peaks. In another aspect, this crystalline form has an X-ray powder diffraction pattern substantially as depicted in Figure 27.

[0054] Another embodiment of the present disclosure pertains to Compound A, hydrochloride salt Form A. In one aspect, this crystalline form is characterized by an X-ray diffraction pattern comprising one or more of the following peaks: 7.5, 8.6, 12.2, 17.1, 18.8, 18.9, 22.3, 24.5, 25.6, 26.1, 33.5, and/or 34.1 ± 0.2 degrees 2-theta. In another aspect, this crystalline form comprises at least 3 of the foregoing peaks. In yet another aspect, this crystalline for comprises at least 4, 5, 6, 7, 8, 9, or 10 of the foregoing peaks. In another aspect, this crystalline form has an X-ray powder diffraction pattern substantially as depicted in Figure 30.

[0055] Yet another embodiment of the present disclosure pertains to Compound A, fumarate salt Form A. In one aspect, this crystalline form is characterized by an X-ray diffraction pattern comprising one or more of the following peaks: 9.0, 10.5, 1 1.1, 14.9, 17.1, 17.7, 19.3, 21.1, 22.3, 22.9, 23.5, 24.0, 24.2, 25.7, 25.9, 27.3, 29.0, and/or 31.1 ± 0.2 degrees 2- theta. In another aspect, this crystalline form comprises at least 3 of the foregoing peaks. In yet another aspect, this crystalline for comprises at least 4, 5, 6, 7, 8, 9, or 10 of the foregoing peaks. In another aspect, this crystalline form has an X-ray powder diffraction pattern substantially as depicted in Figure 33.

[0056] And yet another embodiment of the present disclosure pertains to Compound A, p-toluenesulfonate salt Form A. In one aspect, this crystalline form is characterized by an X-ray diffraction pattern comprising one or more of the following peaks: 6.0, 9.6, 10.3, 10.5, 1 1.0, 12.0, 12.9, 13.2, 15.0, 16.7, 17.0, 17.5, 17.8, 18.0, 19.0, 20.8, 21.0, 21.1, 22.1, 22.7, 23.1, 23.4, 23.5, 24.8, , and/or 26.6 ± 0.2 degrees 2-theta. In another aspect, this crystalline form comprises at least 3 of the foregoing peaks. In yet another aspect, this crystalline for comprises at least 4, 5, 6, 7, 8, 9, or 10 of the foregoing peaks. In another aspect, this crystalline form has an X-ray powder diffraction pattern substantially as depicted in Figure 35.

[0057] In some embodiments, the polymorphic forms of the disclosure are substantially free of any other polymorphic forms, or of specified polymorphic forms. In any embodiment of the present invention, by "substantially free" is meant that the forms of the present invention contain 20% (w/w) or less, 10% (w/w) or less, 5% (w/w) or less, 2% (w/w) or less, particularly 1% (w/w) or less, more particularly 0.5% (w/w) or less, and most particularly 0.2% (w/w) or less of either any other polymorphs, or of a specified polymorph or polymorphs. In other

embodiments, the polymorphs of the disclosure contain from 1% to 20% (w/w), from 5% to 20% (w/w), or from 5% to 10% (w/w) of any other polymorphs or of a specified polymorph or polymorphs.

[0058] The salts and solid state forms of the present invention have advantageous properties including at least one of: high crystallinity, solubility, dissolution rate, morphology, thermal and mechanical stability to polymorphic conversion and/or to dehydration, storage stability, low content of residual solvent, a lower degree of hygroscopicity, flowability, and advantageous processing and handling characteristics such as compressibility, and bulk density.

[0059] A crystal form may be referred to herein as being characterized by graphical data "as substantially depicted in" a Figure. Such data include, for example, powder X-ray diffractograms. The skilled person will understand that such graphical representations of data may be subject to small variations, e.g., in peak relative intensities and peak positions due to factors such as variations in instrument response and variations in sample concentration and purity, which are well known to the skilled person. Nonetheless, the skilled person would readily be capable of comparing the graphical data in the Figures herein with graphical data generated for an unknown crystal form and confirm whether the two sets of graphical data are

characterizing the same crystal form or two different crystal forms.

[0060] The term "amorphous," as used herein, means lacking a characteristic crystal shape or crystalline structure.

[0061] The term "crystalline," as used herein, means having a regularly repeating arrangement of molecules or external face planes.

[0062] The term "crystalline form," as used in herein, refers to a solid chemical compound or mixture of compounds that provides a characteristic pattern of peaks when analyzed by x-ray powder diffraction; this includes, but is not limited to, polymorphs, solvates, hydrates, co-crystals, and de-solvated solvates.

[0063] The term "polymorphic" or "polymorphism" is defined as the possibility of at least two different crystalline arrangements for the same chemical molecule.

[0064] The term "solution," as used herein, refers to a mixture containing at least one solvent and at least one compound at least partially dissolved in the solvent.

[0065] The term "pharmaceutically acceptable excipients," as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art, such as in Remington: The Science and Practice of Pharmacy, 20th ed.; Gennaro, A. R., Ed.; Lippincott Williams & Wilkins: Philadelphia, Pa., 2000. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0066] The pharmaceutical compositions of the present invention may be used in a variety of ways, including but not limited to the enhancement of the anti-tumor activity of radiation or DNA-damaging chemotherapeutic agents (Griffin, R. J.; Curtin, N. J.; Newell, D. R.; Golding, B. T.; Durkacz. B. W.; Calvert, A. H. The role of inhibitors of poly(ADP-ribose) polymerase as resistance-modifying agents in cancer therapy. Biochemie 1995, 77, 408).

[0067] For therapeutic purposes, the crystalline forms of the present invention can be administered by any means that results in the contact of the active agent with the agent's site of action in the body of the subject. The crystalline forms may be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in combination with other therapeutic agents, such as, for example, analgesics. The crystalline forms of the present invention are preferably administered in therapeutically effective amounts for the treatment of the diseases and disorders described herein to a subject in need thereof.

[0068] In therapeutic or prophylactic use, the crystalline forms of the present invention may be administered by any route that drugs are conventionally administered. Such routes of administration include intraperitoneal, intravenous, intramuscular, subcutaneous, intrathecal, intracheal, intraventricular, oral, buccal, rectal, parenteral, intranasal, transdermal or intradermal. Administration may be systemic or localized.

[0069] The crystalline forms described herein may be administered in pure form, combined with other active ingredients, or combined with pharmaceutically acceptable nontoxic excipients or carriers. Oral compositions will generally include an inert diluent carrier or an edible carrier. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a dispersing agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents. Further, a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes, colorings, and flavorings.

[0070] Alternative preparations for administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are dimethylsulfoxide, alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include mixtures of alcohols and water, buffered media, and saline. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.

[0071] Preferred methods of administration of the crystalline forms to mammals include intraperitoneal injection, intramuscular injection, and intravenous infusion. Various liquid formulations are possible for these delivery methods, including saline, alcohol, DMSO, and water based solutions. The concentration may vary according to dose and volume to be delivered and can range from about 1 to about 1000 mg/mL. Other constituents of the liquid formulations can include preservatives, inorganic salts, acids, bases, buffers, nutrients, vitamins, or other pharmaceuticals such as analgesics or additional PARP and kinase inhibitors.

[0072] Having thus described the invention with reference to particular preferred embodiments and illustrative examples, those in the art can appreciate modifications to the invention as described and illustrated that do not depart from the spirit and scope of the invention as disclosed in the specification. The Examples are set forth to aid in understanding the invention but are not intended to, and should not be construed to limit its scope in any way.

EXAMPLES

[0073] Solvents used in the following examples were of reagent-grade quality and were used without further purification. Known forms of Compound A are indicated by Ao and Bo for anhydrous material and ¾ for hydrate.

X-Ray Powder Diffraction (XRPD)

[0074] Standard Reflection Mode Measurements: Powder X-ray diffraction patterns were recorded on a PANalytical X Pert Pro diffractometer equipped with an X'celerator detector using CuK_a radiation at 45 kV and 40 mA. K_al radiation was obtained with a highly oriented crystal (Gel 1 1) incident beam monochromator. A 10mm beam mask, and fixed (1/4°) divergence and anti-scatter (1/8°) slits were inserted on the incident beam side. A fixed 5mm receiving slit and a 0.04 radian Soller block were inserted on the diffracted beam side. The X- ray powder pattern scan was collected from ca. 2 to 40° 2Θ with a 0.0080° step size and 96.06 sec counting time which resulted in a scan rate of approximately 0.5°/min. The sample was spread on silicon zero background (ZBG) plate for the measurement. The sample was rotated using a PANalytical PW3064 Spinner (15 revolutions / min.).

[0075] Measurement of the Si reference standard before the data collection resulted in values for 2Θ and intensity that were well within the tolerances of 28.44 < 2Θ < 28.50 and significantly greater than the minimum peak height of 150cps.

[0076] SCXRD - Single Crystal X-ray Diffraction: For data collection, a piece (0.12 x 0.04 x 0.03 mm3) was broken from a clump of about three or four separate pieces to give an apparently single crystal. The crystal was mounted on a fine glass fiber with the aid of polyisobutene oil (also known as PARATONE) onto a Bruker-Nonius X8 Proteum

diffractometer attached to a Nonius FR-591 rotating anode (CuKa) with 'Helios' focusing optics. The crystal was maintained at 90K throughout with a CryoCool LT2 from Cryolndustries of America. Diffraction images for indexing clearly showed split reflections, consistent with either cracking or twinning, but with spot components that were close enough to be integrated together. The relative intensities of pairs of split reflections suggested that cracking was more likely than twinning.

[0077] The crystal was indexed from the reflections found in 72 diffraction images (six sets of twelve 0.5 ° frames). Data collection consisted of 1485 2 ° frames in 15 scans at three detector swing angles (two 360° φ-scans at -40° in 2Θ, three 90° ω-scans at -45° in 2Θ, four 360° φ-scans at -96° in 2Θ and six 90° co-scans at -96° in 2Θ) sufficient to cover reciprocal space for an arbitrarily oriented triclinic crystal to a resolution of 0.83 A with four-fold redundancy. Data were integrated, scaled, averaged and merged using the programs in the APEX2 package from Bruker-AXS. Final cell parameters were derived from the output diagnostics of the integration process. The structure was solved by standard direct methods using SHELXS and refined using SHELXL, both from the SHELX97 package. Diagrams were drawn using XP from the

SHELXTL suite and with Mercury from the CCDC. Additional molecular graphics and void calculation were done with Platon.

[0078] Positional and anisotropic displacement parameters of all non-hydrogen atoms were refined. The H atoms were located in a difference Fourier's map, but those attached to carbon atoms were repositioned geometrically. The H atoms were initially refined with soft restraints on the bond lengths and angles to regularize their geometry (C-H in the range 0.93- 0.98 and N-H to 0.86 A) and Uiso(H) (in the range 1.2-1.5 times Ueq of the parent atom), after which the positions were refined with riding constraints.

[0079] Default Reitveld refinement of the single crystal unit cell parameters against the measured XRPD pattern gave a good fit with no unexplained peaks.

[0080] Variable Temperature X-Ray Powder Diffraction (VT-XRPD): Variable temperature studies were performed with an Anton Paar CHC temperature/humidity chamber under computer control through an Anton Paar TCUl 10 temperature control unit.

[0081] Typically the measurements were done with a nitrogen flow through the camera. Two measurement schemes were used, restricted and continuous. In the restricted mode, measurements were made, only after the CHC chamber reached the requested temperature. In the continuous mode, the sample was heated at 10°C/minute and fast scans were measured as the temperature changed. In both cases, after the requested temperature was reached, the sample was cooled at 35°C/minute and a slow scan was measured at 25°C. The slow 2Θ scans were collected from ca. 3 to 30° or 40° with a 0.0080° step size and 100.97 sec counting time which resulted in a scan rate of approximately 0.5°/min. The fast scans were collected from ca. 3 to 30° 2Θ with a 0.0167° step size and 1.905 sec counting time which resulted in a scan rate of approximately 44°/min.

[0082] The temperatures chosen were based on DSC results.

[0083] For the diffractometer set-up a 10mm beam mask, 0.04 radian Soller slits, and fixed (1/4°) divergence and anti-scatter (1/8°) slits were inserted on the incident beam side. A fixed 5 mm receiving slit, 0.04 radian Soller slits and a 0.02 mm Nickel filter were inserted on the diffracted beam side.

[0084] Differential Scanning Calorimetry (DSC): Thermal curves were acquired using a Perkin-Elmer Sapphire DSC unit equipped with an autosampler running Pyris software version 6.0 calibrated with Indium prior to analysis. Solid samples of 1-10 mg were weighed into 20 μϊ_^ aluminum pin hole sample pans. The DSC cell was then purged with nitrogen and the temperature heated from 0 to 270°C at 10°C / min. Indium (T_m = 156.6°C; AH_FUS = 28.45 J g^_1) was used for calibration.

[0085] Modulated Differential Scanning Calorimetry (MDSC): Thermal curves were acquired using a TA Q200 Modulated DSC unit. Solid samples of 5-20 mg were weighed into 50 μϊ_^ aluminum pinhole hermetically sealed pans. The MDSC cell was then purged with nitrogen and the temperature heated at 2°C/min from 0°C to 350°C at 2°C/min with a modulation amplitude of +/- 1°C over a 60 second period.

[0086] Thermogravimetric Mass Spectrometry (TGA/MS): Thermal curves were acquired using a Perkin-Elmer Pyris 1 TGA unit running Pyris software version 6.0 calibrated with alumel (95% nickel, 2% manganese, 2% aluminum and 1% silicon), nickel and calcium oxalate monohydrate. TGA samples between 1-5 mg were monitored for percent weight loss as heated from 25 to 250°C at 10°C/min in a furnace purged with Helium at ca. 50 mL/min. To simultaneously follow the evolution of the gaseous decomposition products over the temperature range investigated, the thermobalance was connected to a ThermoStar Quadrupole Mass Spectrometer (Asslar, Germany). The transfer line to introduce gaseous decomposition products into the mass spectrometer was a deactivated fused silica capillary (SGE Analytical science, Fused Silica (100% Methyl Deactivated), 220 mm OD, 150 mm ID, Australia) temperature controlled to 200°C to avoid possible condensation of the evolved gases. In this way the TGA weight loss and the mass spectrometric ion intensity curves of the selected ionic species could be recorded simultaneously.

[0087] Dynamic Vapor Sorption (DVS): DVS experiments have been carried out using the DVS-HT instrument (Surface Measurement Systems, London, UK). This instrument measures the uptake and loss of vapor gravimetrically using a recording ultra- microbalance with a mass resolution of ± 0.1 μg. The vapor partial pressure (±1.0%) around the sample is controlled by mixing saturated and dry carrier gas streams using electronic mass flow controllers. The desired temperature is maintained at ± 0.1 °C. The samples (1 - 10 mg) were placed into the DVS-HT and DVS-1 instruments at the desired temperature.

[0088] The sample was loaded and unloaded at 40% RH and 25°C (typical room conditions). A moisture sorption isotherm was performed as outlined below (2 scans giving 1 complete cycle). The software uses a least squares minimization procedure together with a model of the mass relaxation, to predict an asymptotic value. The measured mass equilibration value must be within 2% of that predicted by the software before proceeding to the next % RH value. The minimum equilibration time was set to 1 hour and the maximum to 4 hours.

[0089] Optical Microscopy: Microscopic observation of the sample morphology was performed using an Olympus B60 polarized light microscope. Samples were suspended in mineral oil and compressed on a glass slide with a cover slip prior to observation. Images were taken with a FW-24 (PAX CAM) camera. A lOx objective coupled with an additional lOx magnification from the microscope optics gave a total magnification of lOOx. PAX-it software (Version 6.2) was used to capture and analyze the images.

[0090] Nuclear Magnetic Resonance Spectroscopy ( H-NMR): The stoichiometry of the salts were determined by ^XH -NMR spectroscopy using a Bruker DPX400 instrument running under conditions optimized to give the best available spectrum for each sample. Each sample (2-4 mg) was dissolved in 0.75 mL DMSO-d6 and spectrum obtained in thin walled glass tubes (4 xl4 mm).

Identity, Assay, and Purity by HPLC

[0091] Equipment: Testing was performed on a calibrated and validated Agilent 1200 Rapid Resolution High Performance Liquid Chromatography (HPLC) system designated LC-0430-AD or LC-418-1D. The system comprises a binary SL pump, degasser, high performance autosampler SL with a fraction collector, thermostated column compartment with a 2 valve column switcher, and a DAD SL detector. All standard solutions and samples were prepared in Class A glass volumetric flasks and were placed in autosampler vials. Standard weighings were done using a calibrated Mettler analytical balance. The sample preparations were centrifuged using an Eppendorf microcentrifuge. The primary chromatography data was acquired and integrated using Empower 2 software. Microsoft Office Excel 2003 was used for the calculation of results.

[0092] Reagents: Acetonitrile was obtained from CCI. Trifluoroacetic acid was obtained from EMD. HPLC grade water (18 ΜΩ-cm) was obtained from the laboratory

Barnstead Nanopure system UPW-0403-AD located in laboratory A21 1. Compounds A and B were prepared as previously described.

Instrument Parameters:

Column: Zorbax Eclipse XDB-C18, 100 x 3.0 mm ID, 1.8 μ packing

Detector: UV/vis @ 290 nm

Column Temperature: 25 °C

Flow Rate: 0.64 mL/min

Mobile Phase A: 0.1% TFA in water

Mobile Phase B: 0.1% TFA in ACN

Gradient:

Time (min) Mobile Phase A (%) Mobile Phase B (%)

0 75 25

10 55 45

12 5 95

13 5 95

13.1 75 25

16.7 75 25 [0094] Solid State Stability of Salts at 40°C and 75% Humidity: Samples of the form to be studied (15-20 mg) were weighed into standard 1.5 mL HPLC vials (32 x 11.6 mm) and stored uncapped for 0, 7, 14 and 28 days in a 40°C and 75% RH stability chamber. Samples were removed on the indicated day and capped. Measurements of XRPD, DSC, TGA and HPLC Identity by Purity and Assay measurements were completed on each time point sample.

[0095] Estimation of Water Solubility: Ten mg portions of the salt forms to be studied were weighed into a standard 1.5 mL HPLC vial (32 x 1 1.6 mm). A stir bar and 100 μϊ_^ of water were added to each vial. The samples were capped and stirred for 5-10 minutes. If a clear solution was not obtained by visual inspection, an additional 100-300 portion of water was added and stirred. This process was repeated until the sample dissolved or until 1000 of water was added. An estimation of solubility was based on the volume of water necessary to dissolve the known weight of sample. The results from these measurements are presented in Table 1 1.

Table 1. Estimated Water Solubility and HPLC analyses of Salts with One Equivalent of

Example 1. Salts with Two Equivalents of Acid in Acetone by Maturation

[0096] 200mg of Compound A (0.478 mmoles) was dissolved with warming and stirring in each of five- 20 mL scintillation vials in 15 mL of acetone. 1.95 equivalents of acetic, glycolic, L-malic, or L-malic (1 Eq., 0.48 mmoles) acids were added to the clear Compound A solutions. As soon as these acids were added, the clear solutions became cloudy and began crystallizing. The vials were subject to two cycles of maturation on the HEL unit. Each cycle of maturation consisted of heating to 50° C over a period of one hour, holding at 50°C for four hours, cooling over a period of one hour to 5°C, and holding at 5°C for four hours. The solid was isolated by suction filtration and solid dried overnight at 50°C and house vacuum (-200 mm) to give yellow solids. The results are presented in Table 2.

Table 2

Example 2. Acid Screening (Two Equivalents) in Acetone using Quick Cooling

[0097] To seven HPLC vials containing a stirring bar and 1.5 mL of Compound A solution (13.3 mg/mL), the quantities of acids to give two equivalents (0.096 mmoles) were weighed or added by pipette. The samples were capped and heated to the boiling point and then chilled overnight in the refrigerator at 2-8 °C. The solid was isolated by suction filtration and solid dried overnight at 50°C and house vacuum (~200mm) to give yellow solids. The results are presented in Table 3.

Table 3

Example 3. Salts with Two Equivalents of Acid in Acetone by Slurry Conversion

[0098] 400mg of Compound A (0.956 mmoles) was slurried with warming and stirring in each of five 20 mL glass scintillation vials with 18 mL of acetone. Two equivalents of acetic, glycolic, L-malic, L-pyroglutamic or L-malic (1 Eq. (0.956 mmoles) acids were added to the COMPOUND A suspension in each vial. These mixtures were capped and warmed to near the boiling point. In all cases a heavy yellow solid was noted. The samples were allowed to cool to ambient temperature on the laboratory bench and chilled overnight in the refrigerator at 2-8 °C. The solid was isolated by suction filtration and the product dried overnight at 50 °C and house vacuum (-200 mm) to give yellow solids. The results are presented in Table 4. Table 4

Example 4. Acid Screening (Two Equivalents) in Acetone - Maturation

[0099] 240 mg of Compound A (0.574 mmoles) in 18 mL of acetone and warmed with stirring by a magnetic stirring bar to dissolve. This solution was dispensed equally to 12 1.5 mL HPLC vials.

[00100] To each of 5 vials containing an aliquot of the Compound A solution and a stirring bar, the quantities of acid to give two equivalents (0.096 mmoles) were weighed or added by pipette. The samples were capped and subject to two cycles of maturation on the HEL unit. Each cycle of maturation consisted of heating to 50° C over a period of one hour, holding at 50° C for four hours, cooling over a period of one hour to 5°C, and holding at 5°C for four hours. The solid was isolated by suction filtration and solid dried overnight at 50°C and house vacuum (-200 mm) to give yellow solids. The results are presented in Table 5.

Table 5

Example 5. One Equivalent in Acetone - Slow Cooling

[00101] A solution of 240 mg of Compound A (0.57 mmoles) was prepared in 12 mL of acetone and warmed with stirring to dissolve. Twelve equal aliquots of this solution will give 20 mg (0.0478mmoles) of Compound A in 1 mL of acetone in each vial. The weight of acid corresponding to 1.05 equivalents (0.06 mmoles) of acid was weighed or added by pipette if liquid to 12 1.5 mL HPLC vials. To each vial one of the aliquots of Compound A was added. The vials were capped and warmed with stirring to mix and subject to 2 cycles of slow cooling on the HEL unit. Each cycle of slow cooling on the HEL unit consisted of heating over a period of 1 hour to 80°C holding for 1 hour at 80°C and then cooling over a period of 5 hours to 5°C and holding at 5°C for 16-18 hours. Solid was isolated by suction filtration and samples were dried at 50°C overnight at house vacuum (-200 mm). The results are presented in Table 6.

Table 6

EXO = exotherm

Example 6. Preparation of Ascorbate Salt

[00102] 200 mg of Compound A (0.478 mmoles) was weighed into a 20 mL glass scintillation vial with a stirring bar followed by 88.4mg (0.503 mmoles, 1.05 equivalents) of ascorbic acid (J.T. Baker Anhydrous Lot B36597). 2.5 ml of 2,2,2-trifluoroethanol was added by pipette and the sample was warmed. The slurry that formed was subject to 2 cycles of slow cooling on the HEL unit. Each cycle of slow cooling on the HEL unit consisted of heating over a period of 1 hour to 80°C, holding for 1 hour at 80°C, and then cooling over a period of 5 hours to 5°C and holding at 5°C for 16-18 hours. Solid was isolated by suction filtration and samples were dried at 50°C overnight at house vacuum (-200 mm) to give 142mg of yellow solid (49% yield). The crystalline product was analyzed by HPLC and gave 96.2% of Compound B and 0.8% of Compound A. The structure of the Compound B salt was confirmed by ^- MR.

Compound A, Free Base, Form An

XRPD

[00103] The XRPD is depicted in Figure 1.

Thermal Analysis

[00104] Thermal data is depicted in Figure 2.

Preparation

[00105] The salt was prepared according to the procedure in Example 1. XRPD

[00106] The X-ray diffraction data for the acetate salt, Form A₁.5, is given in Figure 3 and Table 7. Variable temperature XRPD measurements in requested mode (165°C and 200°C) showed two changes in Form - from the acetate to Form Bo and then conversion to Form Ao. In continuous mode, using one minute scans from 5.5° to 11.5° and a l°C/minute temperature ramp, three changes in form were noted, acetate to Freebase B₀, B₀ to A₀ and A₀ to amorphous (Figure 4). The acetate slowly converts to freebase Form Bo over the temperature range 91°C to 130°C. The form changes from B₀ to A₀ between 197°C and 200°C (Figure 5).

Table 7. XRPD Peaks for the Acetate Salt, Form A₁.5

*The use of ZBG or glass plates typically introduces a positive sample height displacement and results in small (0.05° to 0.2°) offset in 2Θ values. The highest peak (intensity 100%) is set in bold letters.

Thermal Analysis

[00107] The DSC curve of the acetate salt, Form A₁.5, shows the presence of one endothermic/degradation peak; at 185.4°C having a AHF_us of 172.0 J/g (Figure 6). The acetate salt had a weight loss of 29.5% between 25 and 150°C.

Water Sorption

[00108] The DVS plot in Figure 7 indicates that the sample appears to be saturated from the onset. There is a steady weight loss during the drying curves with no equilibration reached. The sample was dried at 0%RH for 4 hours for each cycle. There were 4 cycles run, showing a continuing weight loss. The experiment was repeated on another DVS unit and showed similar results. H-NMR Spectoscopy

[00109] The ^- MR spectrum showed all of the peaks expected for Compound A. The peak at about 7.5 ppm was normalized to the one aromatic proton expected to absorb in this region. The remainder of the peaks associated with Compound A then followed in the proper ratio. For the acetate salt, only one peak is expected at 1.9-2.0 ppm. This peak should integrate for 3 protons. Instead, it showed about 4.5 protons, about 1.5 acetic acid molecules per

Compound A molecule.

Stability

[00110] The data is given in Table 8 for the aging of the acetate salt, Form A1.5, at 40° C and 75% RH. The XRPD, changes throughout the 28 day test period. The TGA and

Compound A Assay values are probably reflecting loss of acetic acid as seen in the thermal and XRPD work cited above. DSC, HPLC Purity and Compound B assay are relatively constant during the study. A monoacetate salt should assay as 87.5% Compound A. A diacetate salt should Assay as 77.7% Compound A. The values in Table 8, suggest that the salt is changing composition as it aged. The ^- MR measured 1.5 molecules of acetic acid per molecule of Compound A. The XRPD pattern showed peaks for a hydrate Compound A Free Base, Form Ha. Possibly as the sample aged the excess acetic acid volatilized. The volatility of acetic acid and the changing XRPD pattern suggest that another candidate be chosen.

Table 8. Stability at 40°C and 75% RH of the Acetate Salt, Form A1.5

Optical Microscopy

[00111] The sample as shown in Figure 8 presented agglomerates of irregular shaped crystals. The sampled showed birefringence under plane-polarized light. Compound A, Glycolate Salt Hydrate, Form

Preparation

[00112] The salt was prepared according to Example 1.

XRPD

[00113] The X-ray diffraction data for the glycolate hydrate salt, Form Ai, is given in Figure 9 and Table 9. Overlaid scans for variable temperature XRPD measurements are shown in Figure 10. The initial XRPD pattern compared to glycolate hydrate Form Ai. There was no change on exposure to a dry 2 atmosphere. During the one hour slow scan measurement at 175°C, the pattern changed. There is an increase in peak intensities on heating from 175°C to 225°C. It did not compare to known Compound A freebase patterns. The sample on the plate at the end of the measurements was a dark brown powder which did not have the appearance of passing through a melt. The patterns observed after heating to 175°C and 225°C partially compares to Compound B. This is consistent with the DSC which shows changes after 130°C and a melt at 205°C. Both the VT-XRPD and the DSC were consistent with the loss of glycolic acid and conversion to Compound B.

Table 9. XRPD Peaks for the Glycolate Hydrate Salt, Form Ai

Pos. [°2Θ] Position calc. h k 1 d-spacing [A] Height [cts] Rel. Int. [%]

8.12 8.13 0 0 1 10.8850 781 8.7

8.24 8.25 0 1 0 10.7261 5010 55.9

8.68 8.69 0 1 1 10.1821 6898 77.0

11.96 11.98 1 1 1 7.3925 501 5.6

13.62 13.63 1 1 0 6.4987 275 3.1

13.90 13.91 0 1 -1 6.3683 4729 52.8

14.62 14.63 1 0 -1 6.0549 581 6.5

14.68 14.70 0 1 2 6.0279 692 7.7

14.89 14.90 0 2 1 5.9468 456 5.1

16.29 16.30 0 0 2 5.4374 321 3.6

17.42 17.44 0 2 2 5.0866 3502 39.1

17.59 17.61 1 2 1 5.0367 994 1 1.1

18.20 18.22 1 -2 -1 4.8706 557 6.2

18.48 18.50 1 2 2 4.7970 927 10.3

18.98 18.99 2 0 1 4.6728 252 2.8

19.84 19.85 2 0 0 4.4719 328 3.7

20.23 20.24 2 1 1 4.3864 1426 15.9 Pos. [°2Θ] Position calc. h k 1 d-spacing [A] Height [cts] Rel. Int. [%]

20.58 20.59 2 -1 0 4.3131 1969 22.0

21.21 21.22 2 -1 1 4.1864 3681 41.1

21.30 21.32 0 1 -2 4.1681 1097 12.2

21.44 21.46 1 1 3 4.1409 926 10.3

21.48 21.49 2 0 2 4.1337 2196 24.5

21.54 21.56 1 -2 -2 4.1216 273 3.0

21.66 21.68 1 -2 1 4.0988 240 2.7

22.82 22.84 0 2 3 3.8938 297 3.3

23.04 23.06 0 3 2 3.8571 2250 25.1

23.07 23.08 2 -1 -1 3.8523 1182 13.2

23.71 23.73 2 0 -1 3.7491 239 2.7

24.45 24.47 2 2 1 3.6373 464 5.2

24.73 24.75 2 -1 2 3.5969 8960 100.0

25.95 25.96 1 -3 -2 3.4310 312 3.5

26.07 26.09 2 -2 1 3.4148 209 2.3

26.27 26.28 0 3 3 3.3900 267 3.0

26.41 26.43 1 3 3 3.3716 308 3.4

27.08 27.09 2 1 -1 3.2907 249 2.8

27.90 27.92 2 -1 -2 3.1952 271 3.0

27.96 27.98 1 3 0 3.1881 219 2.4

28.53 28.55 1 2 4 3.1260 206 2.3

29.96 29.97 3 0 0 2.9805 486 5.4

30.05 30.06 0 4 2 2.9718 224 2.5

30.08 30.10 2 -2 2 2.9682 1322 14.7

30.13 30.14 3 -1 0 2.9639 546 6.1

30.21 30.23 2 -1 3 2.9557 1534 17.1

31.57 31.58 3 -1 2 2.8318 240 2.7

32.01 32.03 3 2 2 2.7934 298 3.3

32.76 32.77 1 4 1 2.7319 202 2.3

33.11 33.12 3 2 1 2.7038 276 3.1

33.51 33.53 3 0 -1 2.6721 371 4.1

34.01 34.02 2 -2 -3 2.6343 249 2.8

37.51 37.52 0 1 -4 2.3960 240 2.7 *The use of ZBG or glass plates typically introduces a positive sample height displacement and results in small (0.05° to 0.2°) offset in 2Θ values. The highest peak (intensity 100%) is set in bold letters.

Single Crystal Structure

[00114] The single crystal X-ray structure confirmed the presence of the glycolate anion and showed that the piperazine nitrogen atom carries the hydrogen atom. The molecule is shown in Figure 38. The structure also shows a water molecule which is present at 60% occupancy, that is, the ratio of Compound A to water is 1 :0.6. Structural details are given in the below table.

[00115] Fractional coordinates and isotropic displacement parameters for nonhydi atoms of Compound A glycolate hydrate are below.

Atom x/a y/b z/c Ueq or Uiso

N(l) -257(2) -899(1) 12193(1) 20(1)

N(2) 5139(2) 694(1) 7829(1) 20(1)

N(3) 6756(2) 2109(1) 5718(1) 19(1)

N(4) 6028(2) 3909(1) 3603(1) 20(1)

0(1) 3205(2) 2569(1) 10538(1) 28(1)

0(2) 4938(2) 2063(1) 8709(1) 29(1)

0(3) 4772(1) -997(1) 7440(1) 24(1)

C(l) 125(2) 99(2) 12375(2) 19(1)

C(2) -591(2) 359(2) 13379(2) 24(1)

C(3) 17(2) 1385(2) 13408(2) 26(1)

C(4) 1276(2) 2140(2) 12470(2) 25(1)

C(5) 1979(2) 1871(2) 11474(2) 21(1)

C(6) 1426(2) 814(2) 11409(2) 18(1)

C(7) 1877(2) 171(2) 10607(2) 18(1)

C(8) 3033(2) 251(2) 9554(2) 18(1)

C(9) 3028(2) -663(2) 9123(2) 18(1)

C(10) 1928(2) -1682(2) 9680(2) 18(1)

C(l l) 1733(2) -2727(2) 9343(2) 21(1)

C(12) 438(2) -3632(2) 10444(2) 32(1)

C(13) -315(2) -2918(2) 11209(2) 22(1)

C(14) 786(2) -1790(2) 10718(2) 18(1)

C(15) 769(2) -890(2) 11162(2) 18(1)

C(16) 3936(2) 3508(2) 10681(2) 29(1)

C(17) 4427(2) 1141(2) 8708(2) 20(1)

C(18) 4362(2) -404(2) 8046(2) 19(1)

C(19) 6654(2) 1170(2) 6943(2) 20(1)

C(20) 6273(2) 3305(2) 5683(2) 19(1)

C(21) 6719(2) 4290(2) 4353(2) 20(1)

C(22) 6426(2) 2644(2) 3709(2) 24(1)

C(23) 6001(2) 1698(2) 5052(2) 21(1)

C(24) 6476(2) 4852(2) 2287(2) 25(1)

C(1G) 539(2) 3469(2) 4989(2) 28(1)

0(1G) 335(2) 4218(1) 3828(1) 36(1)

C(2G) 2165(2) 3395(2) 4961(2) 22(1) Atom x/a y/b z/c Ueq or Uiso

0(2G) 3132(1) 4059(1) 3938(1) 28(1)

0(3 G) 2455(1) 2720(1) 5939(1) 26(1)

0(1W) 2887(3) 5938(2) 1816(2) 33(1)

[00116] Fractional coordinates and isotropic displacement parameters for hydi atoms of Compound A glycolate hydrate are below.

Atom x/a y/b z/c Ueq or Uiso

H(1N) -1000(20) -1530(20) 12750(20) 24

H(4N) 4940(30) 3842(19) 3953(19) 23

H(2) -1457 -148 14013 29

H(3) -433 1583 14085 31

H(4) 1659 2849 12512 30

H(1 1A) 2661 -3146 9246 26

H(1 1B) 1469 -2413 8561 26

H(12A) 830 -4388 10960 38

H(12B) -296 -3897 10139 38

H(13A) -1292 -2670 1 1078 26

H(13B) -472 -3436 12105 26

H(16A) 3290 4181 10650 44

H(16B) 4885 3850 10010 44

H(16C) 4137 3140 1 1482 44

H(19A) 7229 1522 7305 24

H(19B) 7157 450 6862 24

H(20A) 6749 3560 6170 23

H(20B) 5171 3219 6054 23

H(21A) 6384 5100 4325 24

H(21B) 7825 4402 3996 24

H(22A) 7518 2687 3319 28

H(22B) 5895 2376 3262 28

H(23A) 4900 1613 5432 25

H(23B) 6297 871 5108 25

H(24A) 6170 5666 2242 38

H(24B) 5986 4590 1822 38

H(24C) 7567 4923 1929 38 Atom x/a y/b z/c Ueq or Uiso

H(1G1) 58 261 1 5299 33

H(1G2) 21 3808 5583 33

H(1G) 11 12 4714 3360 53

H(1W) 3200(50) 5350(40) 2430(40) 42(1 1)

H(2W) 3340(60) 6690(40) 1640(40) 70(16)

Thermal Analysis

[00117] The DSC curve of the glycolate hydrate salt, Form Ai, shows the presence of two different endothermic peaks; one at 77.4°C having a AHF_us of 63.4 J/g and a second peak at 209.0°C and a AH_Fus of 170.9 J/g (Figure 11). The glycolate hydrate salt had a weight loss of 1.9% between 25 and 150°C.

Water Sorption

[00118] The DVS plot in Figure 12 indicated that there was surface adsorption with limited bulk absorption throughout the entire RH range. The total uptake in moisture at 90% RH is -3.5%.

H-NMR Spectroscopy

[00119] The spectrum gives all of the peaks necessary for Compound A. After normalization of the integration to one proton in the aromatic region at about 7.5 ppm for Compound A, there is a two proton singlet at about 3.9 ppm for the two protons associated with the methylene group of glycolic acid. This indicated a 1 : 1 mole ratio of Compound A to glycolic acid in the salt.

Stability

[00120] The data given in Table 10 indicate that this salt is fairly stable to the test conditions. A modest increase in Compound B is noted after 28 days. A monoglycolate salt, as the ^XH- NMR indicated, should have a Compound A Assay of 84.5% Compound A. Increasing loss in TGA suggests increasing water content, for example, 3.5% loss would be expected for a water to Compound A ratio of 1 : 1. Table 10. Stability at 40 °C and 75% RH of Glycolate Salt Hydrate, Form Ai

Day XRPD DSC,°C TGA, COMPOUND COMPOUND HPLC

% A Assay, % B Assay, % Purity, %

0 Al 69.7, 207.9 2.1 69.9 0.1 99.8

7 No change 208.3 2.3 68.4 0.1 99.6

14 No change 68.8, 207.3 2.6 73.2 0.2 99.7

28 No change 207.4 3.5 66.8 0.6 99.5

Optical Microscopy

[00121] In Figure 13, the sample presented individual and agglomerates of crystals. The sample showed birefringence under plane polarized light.

Compound A, L-Malate Salt, Form Ai

Preparation

[00122] The salt was prepared according to Example 1.

XRPD

[00123] The X-ray diffraction data for the malate salt, Form Ai, is given in Figure 14 and Table 11. Overlaid slow scans for a VT-XRPD study are shown in Figure 15.

[00124] The initial XRPD pattern is as expected. There is no change in form on exposure to a dry N2 atmosphere (Figure 15). There is a change when the sample is held at 175°C for an hour. The fast scan measured when 175°C was first reached compares to the starting pattern. The crystalinity is almost completely gone in the fast scan measured after 175°C. The slow scan pattern observed for this sample after heating to 175°C and cooling to 25°C partially compares to the pattern for Compound B. This observation is consistent with thermal decomposition to Compound B.

Table 1 1. XRPD Peaks for Malate Salt, Form Ai

Thermal Analysis

[00125] The DSC curve of the malate salt, Form Ai shows the presence of one endothermic peak; at 186.4°C having a AHF_us of 75.7 J/g (Figure 16). The malate salt had a weight loss of 1.0% between 25 and 150°C.

Water Sorption

[00126] The DVS plot in (Figure 17) indicated there was very little water absorption during the first cycle from 40%RH to 70%RH. Only surface adsorption is occurring. At 80%RH is an increase in water uptake. The large hysteresis gap is due to bulk absorption. The total uptake is ~2%. The isotherm is irreversible.

H-NMR Spectroscopy

[00127] All of the peaks expected for Compound A are present. After normalization of the one aromatic proton at 7.5 ppm, there is a one proton triplet at about 4.05ppm that is consistent with L- malic acid. This established the 1 : 1 stoichiometry for the Compound A L- malic acid salt in Form Ai.

Stability

[00128] The data in Table 12 show that the L-malate salt is stable to the test conditions with a constant XRPD, DSC, TGA and HPLC Purity values (MJJ3331-49). An increase in Compound B is observed after 28 days. As with the glycolate hydrate salt, the L-malate Assay value for Compound A is lower than the 75.8% value expected.

Table 12. Stability at 40°C and 75% RH of the L-Malate Salt, Form Ai Day XRPD DSC TGA COMPOUND COMPOUND HPLC

A Assay B Assay Purity

0 i 193.0°C 0.1% 69.9% 0.2% 99.5%

7 No change 192.0°C 0.2% 71.8% 0.4% 99.3%

14 No change 191.4°C 0.8% 72.0% 0.5% 98.8%

28 No change 191.1°C 0.3% 71.7% 0.8% 98.4%

Optical Microscopy

[00129] In Figure 18, the sample showed individual crystals and agglomerates of irregular shaped crystals. The sample showed birefringence under plane polarized light.

Compound A, L-Malate Salt, Form A^s

Preparation

[00130] The salt was prepared according to Example 2.

XRPD

[00131] The X-ray diffraction data for the malate salt, Form A1.5, is given in Figure 19 and Table 13.

Table 13. XRPD Peaks for Malate Salt, Form A1.5

Thermal Analysis

[00132] The DSC curve of the L-malate salt, Form A_L5, shows the presence of one endothermic peak; at 160.4°C having a AHF_us of 39.2 J/g (Figure 20). The L-malate salt had a weight loss of 3.6% between 25 and 150°C. This Form melts at a much lower temperature and has a larger weight loss than the malate salt, Form Ai_.

H-NMR Spectroscopy

[00133] The ^- MR spectrum of the L-malate salt, Form A₁.5 showed all of the peaks were present for Compound A and the normalized integration showed about 3 moles of L-malic acid for two moles of Compound A. This preparation represented a new form for Compound A L-malate salt.

Compound A, L-Pyroglutamate Salt, Form

Preparation

[00134] The sale was prepared according to Example 3.

XRPD

[00135] The X-ray diffraction data for the L-pyroglutamate salt, Form Ai is given in Table 14 and Figure 21. The XRPD pattern showed a highly crystalline solid.

[00136] Variable temperature XRPD measurements are shown in Figure 22. The initial XRPD pattern is as expected. There is no change in form on heating to 175°C. At the end of the experiment a black glass was left on the ZBG plate. Comparison of the expected pattern for Compound B and the sample after heating to 210°C shows small differences. This suggests conversion of Compound A to Compound B and a possible second component.

Table 14. XRPD Peaks for L-Pyroglutamate Salt, Form Ai

No. Pos. d-spacing Rel. No. Pos. d-spacing Rel.

[20°]* [A] Int.[%] [20°]* [A] Int.[%]

9 14.32 6.180 12 29 23.39 3.800 84

10 15.00 5.900 24 30 23.51 3.781 56

11 16.71 5.301 36 31 24.11 3.689 14

12 17.02 5.206 22 32 24.53 3.626 8

13 17.51 5.061 59 33 24.84 3.582 54

14 17.79 4.983 68 34 25.08 3.547 9

15 18.02 4.919 78 35 26.56 3.353 33

16 18.68 4.747 19 36 27.57 3.232 8

17 18.98 4.672 29 37 28.15 3.168 13

18 19.37 4.578 7 38 28.78 3.099 9

19 20.22 4.388 7 39 30.22 2.955 11

20 20.76 4.276 35 40 30.43 2.935 9

Thermal Analysis

[00137] The DSC curve of the L-pyroglutamate salt, Form Ai, shows the presence of two endothermic peaks; at 50.4°C having a AHF_us of 35.6 J/g and 198.2°C having a AHF_u of 76.8 J/g (Figure 23). The pyroglutamate salt had a weight loss of 3.5% between 25 and 150°C.

Water Sorption

[00138] In the DVS Plot (Figure 24) indicated that during the first cycle there is very little water absorption over the RH range of 40-75% (~2%). Only surface adsorption is occurring. At 80% RH there is a massive uptake in moisture. The large hysteresis gap at 50-90% RH is due to bulk absorption with a possible hydrate formation. The total uptake is -27%.

H-NMR Spectroscopy

[00139] All of the peaks are present for Compound A. After normalization of the integration for one proton for the aromatic peak in Compound A at about 7.5 ppm, there is an additional one proton singlet at about 7.85ppm for the hydrogen atom on the amide nitrogen in pyroglutamic acid. In addition, there is an additional one proton multiplet at about 4.05 ppm from the one hydrogen atom attached to the carbon atom adjacent to the carboxylic acid group. This establishes this salt as a mono L-pyroglutamate salt of Compound A.

Stability

[00140] This salt was stable over a 28 day test period, except for a slow increase in Compound B content (Table 15). Table 15. Stability at 40°C and 75% RH of the L-Pyroglutamate Salt, Form Ai (Prepared with

Optical Microscopy

[00141] The sample presented agglomerates of irregular shaped crystals as shown in Figure 25. The sample showed birefringence under plane polarized light.

Comparison of Salts

[00142] In Table 16, glycolate hydrate Form Ai, L-malate Form Ai and the one and two equivalent preparations of L-pyroglutamate Form Ai are compared. The glycolate hydrate salt, Form Ai, generated the least amount of Compound B during 40° C and 75% RH stability testing. The glycolate hydrate exhibited a preference for water absorption since the TGA value increased to 3.5 % during stability testing (Table 10).

Table 16. Comparison of Compound A Salts

Compound A, Free Base, Form Cn

Preparation [00143] The free base was prepared according to Example 4.

XRPD

[00144] The X-ray diffraction data for free base, Form Co, is given in Figure 26 and Table 17. The XRPD pattern showed a crystalline solid.

[00145] Variable temperature XRPD measurements are shown in Figure 27. The initial XRPD pattern compares to the expected pattern for Form C₀. There is no change in form on exposure to a dry N2 atmosphere. There is no change in form after heating to 175°C. After heating to 235°C the XRPD pattern is changed and is similar to, but not the same as, the pattern observed for Compound B. Similar patterns have been seen for other VT samples. There seem to be two components present in this decomposition product.

Table 17. XRPD Peaks for Free Base, Form Co

Thermal Analysis [00146] The DSC curve of the free base, Form Co, shows the presence of one endothermic peak; at 207.3°C having a AH_Fus of 71.4 J/g (Figure 28). Form Co had a weight loss of 2.3% between 25 and 150°C.

Optical Microscopy

[00147] In Figure 29, the sample presented agglomerates and individual irregular shaped crystals. The sample showed birefringence under plane polarized light.

Compound A, Hydrochloride Salt, Form A

Preparation

[00148] The salt was prepared according to Example 5.

XRPD

[00149] The X-ray diffraction data for the chloride salt, Form A, is given in Figure 30 and Table 18.

Table 18. XRPD Peaks for the Hydrochloride Salt, Form A

*The use of ZBG or glass plates typically introduces a positive sample height displacement and results in small (0.05° to 0.2°) offset in 2Θ values. The highest peak (intensity 100%) is set in bold letters. Thermal Analysis

[00150] The DSC curve of the hydrochloride salt, Form A, shows one endothermic peak at 247.3°C having a AH_Fus of 41.6 J/g (Figure 31). The hydrochloride salt, Form A, had a weight loss of 0.2% between 25 and 150°C.

Water Sorption

[00151] The DVS Plot (Figure 32) indicated there is surface adsorption with limited bulk absorption throughout the entire RH range. The total uptake in moisture is -2.25%.

Stability

[00152] The data in Table 19 show a relatively constant XRPD pattern and DSC value with modest changes in TGA value. The HPLC values are quite different with Assay value decreasing to nearly half after 28 days of testing. Also noted was a steady decline in HPLC purity and an increase in Compound B content to 1.5%. The theoretical value for Compound A content in a Compound A monohydrochloride salt is 92.0%.

Table 19. Stability at 40°C and 75% RH of the Hydrochloride Form A

Compound A, Fumarate Salt, Form A

Preparation

[0100] The salt was prepared according to Example 5.

XRPD

[00153] The X-ray diffraction data for Compound A Fumarate Salt, Form A, is given in Figure 33 and Table 20.

Table 20. XRPD Peaks for the Fumarate Salt, Form A

Thermal Analysis

[00154] The DSC curve of the fumarate salt, Form A, showed the presence of one endothermic peak; at 231.3°C having a AH_Fus of 106.9 J/g (Figure 34). Form A had a weight loss of 0.2% between 25 and 150°C.

Compound A, p-Toluenesulfonate Salt, Form A

Preparation

[00155] The salt was prepared according to Example 5.

XRPD

[00156] Characterization of the p-Toluenesulfonate Salt, Form A is depicted in Figure 35 and Table 21. Table 21. XRPD Peaks for the p-Toluenesulfonate Salt, Form A

Thermal Analysis

[00157] The DSC curve of the p-toluenesulfonate salt, Form A, shows the presence of one endothermic peak; at 239.6°C having a AH_Fus of 38.5 J/g (Figure 36). Form A had a weight loss of 0.04% between 25 and 150°C.

Claims

What is Claimed:

1. A crystalline form of 4,5,6,7-tetrahydro-l l-methoxy-2-[(4-methyl-l-piperazinyl)methyl]- lH-cyclopenta[a]pyrrolo 3,4-c]carbazole-l,3(2H)-dione (Compound A)

Compound A

that is

Compound A, acetate salt Form Ai.₅;

Compound A, glycolate salt hydrate Form Ai;

Compound A, L-malate salt Form Ai;

Compound A, L-malate salt Form A1.5 ;

Compound A, L-pyroglutamate salt Form Ai;

Compound A, free base Form Co;

Compound A, hydrochloride salt Form A;

Compound A, fumarate salt Form A; or

Compound A, p-toluenesulfonate salt Form A.

2. The crystalline form of claim 1 that is Compound A, glycolate salt hydrate Form Ai.

3. The crystalline form of claim 2, characterized by an X-ray powder diffraction pattern having at least three peaks selected from the group consisting of 8.2, 8.7, 13.8, 14.9, 16.4, 17.5, 18.2, 18.5, 20.2, 20.6, 21.2, 21.4, 23.0, 24.6, 27.8, 29.9, 30.1, and 30.5 degrees two theta ± 0.2 degrees two theta.

4. The crystalline form of claim 2 or claim 3, further characterized by an X-ray powder diffraction pattern substantially as depicted in Figure 9 or Figure 10.

5. The crystalline form of any one of claims 2 to 4, further characterized by an X-ray powder diffraction pattern substantially as depicted in Figure 9.

6. The crystalline form of any one of claims 2 to 5, further characterized by a DSC

substantially as depicted in Figure 11.

7. The crystalline form of any one of claims 2 to 6, further characterized by a DVS

substantially as depicted in Figure 12.

8. The crystalline form of claim 1 that is Compound A, acetate salt Form A1.5.

9. The crystalline form of claim 8, characterized by an X-ray powder diffraction pattern having at least three peaks selected from the group consisting of 6.4, 9.2, 12.7, 13.0, 15.2, 17.4, 18.4, 19.0, 19.3, 21.3, 21.5, 23.1, 24.1, 24.2, and 28.2 ± 0.2 degrees 2-theta.

10. The crystalline form of claim 8 or claim 9, further characterized by an X-ray powder diffraction pattern substantially as depicted in Figure 3, Figure 4, or Figure 5.

11. The crystalline form of any one of claims 8 to 10, further characterized by a DSC

substantially as depicted in Figure 6.

12. The crystalline form of any one of claims 8 to 1 1, further characterized by a DVS

substantially as depicted in Figure 7.

13. The crystalline form of claim 1 that is Compound A, L-malate salt Form Αχ.

14. The crystalline form of claim 13, characterized by an X-ray powder diffraction pattern having at least three peaks selected from the group consisting of 8.6, 9.2, 10.1, 10.4, 1 1.7, 1 1.9, 14.7, 15.3, 15.6, 17.2, 17.8, 18.5, 20.3, 20.7, 21.2, 22.4, 23.5, 24.3, and 27.0 ± 0.2 degrees 2-theta.

15. The crystalline form of claim 13 or claim 14, further characterized by an X-ray powder diffraction pattern substantially as depicted in Figure 14 or Figure 15.

16. The crystalline form of any one of claims 13 to 15, further characterized by a DSC substantially as depicted in Figure 16.

17. The crystalline form of any one of claims 13 to 16, further characterized by a DVS

substantially as depicted in Figure 17.

18. The crystalline form of claim 1 that is Compound A, L-malate salt Form A1.5.

19. The crystalline form of claim 18, characterized by an X-ray powder diffraction pattern having at least three peaks selected from the group consisting of 5.5, 6.8, 8.0, 8.4, 8.8, 9.2, 1 1.8, 12.8, 13.1, 13.6, 14.4, 16.0, 16.7, 18.1, 18.5, 19.4, 20.2, 20.5, 21.1, 21.9, 23.4, and 24.6 ± 0.2 degrees 2-theta.

20. The crystalline form of claim 18 or claim 19, further characterized by an X-ray powder diffraction pattern substantially as depicted in Figure 19.

21. The crystalline form of any one of claims 18 to 20, further characterized by a DSC

substantially as depicted in Figure 20.

22. The crystalline form of claim 1 that is Compound A, L-pyroglutamate salt Form Ai.

23. The crystalline form of claim 22, characterized by an X-ray powder diffraction pattern having at least three peaks selected from the group consisting of 6.0, 9.6, 10.3, 10.5, 1 1.0, 12.0, 13.2, 15.0, 16.7, 17.5, 17.8, 18.0, 19.0, 20.8, 21.0, 21.1, 22.0, 22.1, 23.1, 23.4, 23.5, 24.8, and 26.6 ± 0.2 degrees 2-theta.

24. The crystalline form of claim 22 or claim 23, further characterized by an X-ray powder diffraction pattern substantially as depicted in Figure 21 or Figure 22.

25. The crystalline form of any one of claims 22 to 24, further characterized by a DSC

substantially as depicted in Figure 23.

26. The crystalline form of any one of claims 22 to 25, further characterized by a DVS

substantially as depicted in Figure 24.

27. The crystalline form of claim 1 that is Compound A, free base Form Co.

28. The crystalline form of claim 27, characterized by an X-ray powder diffraction pattern having at least three peaks selected from the group consisting of 8.5, 8.8, 13.9, 14.4, 15.4, 17.6, 18.1, 18.5, 19.2, 19.7, 20.4, 21.1, 21.4, 21.9, 23.6, 24.6, 29.4 and 30.1 ± 0.2 degrees 2-theta.

29. The crystalline form of claim 27 or claim 28, further characterized by an X-ray powder diffraction pattern substantially as depicted in Figure 26 or Figure 27.

30. The crystalline form of any one of claims 27 to 29, further characterized by a DSC

substantially as depicted in Figure 28.

31. The crystalline form of claim 1 that is Compound A, hydrochloride salt Form A.

32. The crystalline form of claim 31, characterized by an X-ray powder diffraction pattern having at least three peaks selected from the group consisting of 7.5, 8.6, 12.2, 17.1, 18.8, 18.9, 22.3, 24.5, 25.6, 26.1, 33.5, and 34.1 ± 0.2 degrees 2-theta.

33. The crystalline form of claim 31 or claim 32, further characterized by an X-ray powder diffraction pattern substantially as depicted in Figure 30.

34. The crystalline form of any one of claims 31 to 33, further characterized by a DSC

substantially as depicted in Figure 31.

35. The crystalline form of any one of claims 31 to 34, further characterized by a DVS

substantially as depicted in Figure 32.

36. The crystalline form of claim 1 that is Compound A, fumarate salt Form A.

37. The crystalline form of claim 36, characterized by an X-ray powder diffraction pattern having at least three peaks selected from the group consisting of 9.0, 10.5, 1 1.1, 14.9, 17.1, 17.7, 19.3, 21.1, 22.3, 22.9, 23.5, 24.0, 24.2, 25.7, 25.9, 27.3, 29.0, and 31.1 ± 0.2 degrees 2-theta.

38. The crystalline form of claim 36 or claim 37, further characterized by an X-ray powder diffraction pattern substantially as depicted in Figure 33.

39. The crystalline form of any one of claims 36 to 38, further characterized by a DSC substantially as depicted in Figure 34.

40. The crystalline form of claim 1 that is Compound A, p-toluenesulfonate salt Form A.

41. The crystalline form of claim 40, characterized by an X-ray powder diffraction pattern having at least three peaks selected from the group consisting of 6.0, 9.6, 10.3, 10.5, 1 1.0, 12.0, 12.9, 13.2, 15.0, 16.7, 17.0, 17.5, 17.8, 18.0, 19.0, 20.8, 21.0, 21.1, 22.1, 22.7, 23.1, 23.4, 23.5, 24.8, , and 26.6 ± 0.2 degrees 2-theta.

42. The crystalline form of claim 40 or claim 41, further characterized by an X-ray powder diffraction pattern substantially as depicted in Figure 35.

43. The crystalline form of any one of claims 40 to 42, further characterized by a DSC

substantially as depicted in Figure 36.

44. A pharmaceutical composition comprising the crystalline form of any one of the

preceding claims and at least one pharmaceutically acceptable excipient.

45. A method of treating cancer in a patient comprising administering to the patient a

crystalline form of 4,5,6,7-tetrahydro-l l-methoxy-2-[(4-methyl-l-piperazinyl)methyl]- lH-cyclopenta[a]pyrrolo[3,4-c]carbazole-l,3(2H)-dione (Compound A) according to any one of claims 1 to 43.

46. The method of claim 45, wherein the cancer is breast cancer or ovarian cancer.