Crystalline form of an n-heteroarylsulphonamide, compositions and methods thereof

Abstract

A crystalline form of N-heteroarylsulphonamide 2,6-dichloro-N-(3-isobutyl-1,5- dimethyl-1H-pyrazol-4-yl)-4-(2-(piperazin-1-yl)pyridin-4-yl)benzenesulfonamide and pharmaceutical compositions thereof; and methods of preparing said crystalline form, and methods of using said crystalline form in the treatment of disease.

Description

CRYSTALLINE FORM OF AN N-HETEROARYLSULPHONAMIDE.COMPOSITIONS AND METHODS THEREOFFIELD

[0001] The present disclosure relates generally to a crystalline form of / V- heteroarylsulphonamide, 2,6-dichloro- / V-(3-isobutyl-1 ,5-dimethyl-1 H-pyrazol-4-yl)-4-(2- (piperazin-1-yl)pyridin-4-yl)benzenesulfonamide, to pharmaceutical compositions thereof, to methods of preparing said crystalline form, and to methods of using said crystalline form in the treatment of disease.BACKGROUND

[0002] Discovery and characterization of new crystalline forms of pharmaceutical compounds are important for researching and developing new medicines. This is because different crystalline forms of the same pharmaceutical compound can have different physicochemical properties, such as solubility, physical / chemical stability, stability during distribution and storage, ease of formulation, bioavailability, and / or pharmacokinetic properties.

[0003] New crystalline forms of pharmaceutical compounds result from a compound crystallizing into different internal structures, such as different crystal lattice arrangements. Such different internal structures (i.e., new crystalline forms) occur because systems tend towards thermodynamically stable (i.e., low energy) states.

[0004] As there is a continuing need for pharmaceutical compounds that exhibit better pharmacokinetic properties, bioavailability, and / or better stability, and the like, there remains a need for new crystalline forms of pharmaceutical compounds.SUMMARY

[0005] In an aspect of the present disclosure, there is provided a crystalline form of Compound (I)(said crystalline form being characterized by an X-ray powder diffraction pattern as shown in FIG. 37.

[0006] In another aspect of the present disclosure, there is provided a crystalline form of Compound (I)said crystalline form being characterized by an X-ray powder diffraction pattern having peaks at 10.2, 11 .4, and 20.5 degrees two theta ± 0.2 degrees two theta.

[0007] In an embodiment of the present disclosure, there is provided a crystalline form wherein said crystalline form is further characterized by the X-ray powder diffraction pattern having peaks at 14.6, 22.9, 26.1 , and 31.0 degrees two theta ± 0.2 degrees two theta. In another embodiment, said crystalline form is further characterized by the X-ray powder diffraction pattern having peaks at 18.6, 21.4, 23.4, and 33.5 degrees two theta ± 0.2 degrees two theta.

[0008] In another aspect of the present disclosure, there is provided a crystalline form of Compound (I)said crystalline form being characterized by an X-ray powder diffraction pattern having peaks substantially as provided below in Table 1 .0’ ± 0.2 degrees two theta.

[0009] Table 1.0’

[0010] In another embodiment of the present disclosure, there is provided a crystalline form wherein said crystalline form is characterized by a DSC thermogram obtained using a heating rate of 10 °C / min comprising an endothermic event with an onset temperature of 220.2 °C ± 0.2 °C. In another embodiment, said crystalline form is characterized by a DSC thermogram substantially as shown in FIG. 41.

[0011] In an embodiment of the present disclosure, there is provided a crystalline form wherein said crystalline form is characterized by a Raman spectrum comprising wavenumber values (cm-1) 103.2 cm-1, and 993.0 cm1, and 1602.8 cm1±0.2 cm1. In another embodiment, said crystalline form is further characterized by the Raman spectrum comprising wavenumber values (cm1) of 126.5 cm1, 144.9 cm1, 227.3 cm1, 456.1 cm1,1043.5 cm1, 1164.7 cm1, and 1582.5 cm'1± 0.2 cm'1. In another embodiment, said crystalline form is characterized by a Raman spectrum comprising wavenumber values (cm1) substantially as provided below in Table 2.0’± 0.2 cm1.

[0012] Table 2.0’

[0013] In another aspect of the present disclosure, there is provided a pharmaceutical composition, comprising a crystalline form of Compound (I) as described herein; and a pharmaceutically acceptable adjuvant, diluent, carrier, or vehicle.

[0014] In another aspect of the present disclosure, there is provided a method of preparing a crystalline form of Compound (I) as described herein, comprising the steps of: adding a HCI form of Compound (I) to a first organic solvent to form a first mixture; adding a base to the first mixture to form a first solution; and isolating from the first solution a free form of Compound (I); adding the free form of Compound (I) to a second organic solvent to form a second mixture; maturing the second mixture to form a third mixture; andisolating from the third mixture the crystalline form of Compound (I).

[0015] In another embodiment of the present disclosure, there is provided a method wherein adding a HCI form of Compound (I) to a first organic solvent further comprises heating the first mixture to a temperature of about 30 °C. In another embodiment of the present disclosure, there is provided a method wherein adding a base to the first mixture to form a first solution further comprises: maintaining the first solution at a temperature of about 30 °C; and stirring the first solution for about 1 hour.

[0016] In another embodiment of the present disclosure, there is provided a method wherein adding the free form of Compound (I) to a second organic solvent to form a second mixture further comprises: stirring the second mixture at ambient temperature; optionally ramping up the temperature from ambient temperature to about 50 °C and then ramping down the temperature from about 50 °C to ambient temperature, and optionally adding additional organic solvent to the second mixture until the free form of Compound (I) dissolves in the second organic solvent; and ramping down the temperature from about 50 °C to about 5 °C at a rate of about 0.1 °C / min.

[0017] In another embodiment, there is provided a method wherein maturing the second mixture to form a third mixture further comprises maturing at 25 / 50 °C in 4-hour cycles.

[0018] In another embodiment, there is provided a method wherein the first organic solvent is selected from the group consisting of ethanol, acetone, water, DCM, or ethyl acetate. In another embodiment, the first organic solvent is DCM. In another example, there is described a method wherein the first organic solvent is selected from the group consisting of ethanol, acetone, water, or ethyl acetate.

[0019] In another embodiment, there is provided a method wherein the base is NaOH.

[0020] In another embodiment, there is provided a method wherein the second organic solvent is selected from the group consisting of 2-propanol; 2-methyl THF; acetone; acetonitrile; DMSO; N,N'-dimethylacetamide; MeOH:water (90:10 v / v); ethyl acetate; ethanol:water (50:50 v / v); isopropyl acetate; isobutanol; tert-butyl methyl ether; THF; and water. In another example, there is described a method wherein the second organic solvent is selected from the group consisting of 2-propanol; 2-methyl THF; acetone; acetonitrile; DMSO; N,N'-dimethylacetamide; MeOH:water (90:10 v / v); ethyl acetate; ethanol:water (50:50 v / v); isopropyl acetate; isobutanol; tert-butyl methyl ether; THF; and water.

[0021] In another embodiment of the present disclosure, there is provided a method further comprising filtering insoluble particulates from the first solution before isolating from the first solution a free form of Compound (I).

[0022] In another aspect of the present disclosure, there is provided a method of recrystallizing the crystalline form of Compound (I) as described herein, or the crystalline form of Compound (I) prepared by the method described herein, comprising the steps of: dissolving the crystalline form of Compound (I) in an organic solvent at a first temperature to form a first solution; ramping down the temperature from the first temperature to a second temperature; adding a seed of the crystalline form of Compound (I) to the first solution to form a first mixture; ramping down the temperature from the second temperature to a third temperature; adding an anti-solvent to the first mixture to form a second mixture; maturing the second mixture for about 24 hours to form a third mixture; and isolating the recrystallized, crystalline form of Compound (I) from the third mixture.

[0023] In another embodiment of the present disclosure, there is provided a method wherein the organic solvent is selected from the group consisting of DMSO; DMA; and NMP. In another embodiment the organic solvent is DMSO. In another embodiment of the present disclosure, there is described a method wherein the organic solvent is selected from the group consisting of DMSO; DMA; and NMP. In another example, the organic solvent is DMSO.

[0024] In an embodiment, there is provided a method wherein the anti-solvent comprises water, a C-1-C3 alcohol, or a combination thereof. In an embodiment, the antisolvent comprises water, C1-C3 alcohol, or combination thereof in amounts ranging between about 100% water and about 100% C1-C3 alcohol, or any combination of water and CrC3alcohol between 100% water and 100% C1-C3 alcohol.

[0025] In an embodiment, the C1-C3 alcohol comprises a C1-C3 linear or branched or cyclic alcohol. In an embodiment, the C1-C3 alcohol comprises methanol, ethanol, n- propanol, branched propanol, or a combination thereof.

[0026] In another embodiment, there is provided a method wherein the anti-solvent is selected from the group consisting of ethanol and water. In an embodiment, the antisolvent is selected from the group consisting of ethanol and water in amounts ranging between about 100% water and about 100% ethanol, or any combination of water andethanol between 100% water and 100% ethanol. In another embodiment, there is provided a method wherein the anti-solvent is selected from the group consisting of EtOH:H2O (1 :1); EtOH:H2O (1 :2); and EtOH:H2O (2:1).

[0027] In another embodiment, there is provided a method wherein the organic solvent is DMSO and the anti-solvent is EtOH:H2O (1 :1), EtOH:H2O (1 :2), or EtOH:H2O (2:1). In another embodiment, the organic solvent is DMSO and the anti-solvent is EtOH:H2O (1 :2).

[0028] In another embodiment, there is provided a method wherein the anti-solvent is added at a volume that is about 10% to about 20% the volume of organic solvent. In another embodiment, the anti-solvent is added at a volume that is about 10% the volume of organic solvent.

[0029] In another embodiment, there is provided a method wherein the first temperature is about 65 °C.

[0030] In another embodiment, there is provided a method wherein the second temperature is about 59 °C.

[0031] In another embodiment, there is provided a method wherein the third temperature is about 25°C.

[0032] In another embodiment, there is provided a method wherein ramping down the temperature from the second temperature to the third temperature comprises a ramp down of 0.5 °C / min.

[0033] In another embodiment, there is provided a method further comprising filtering insoluble particulates from the first solution before ramping down the temperature from the first temperature to a second temperature.

[0034] In another aspect of the present disclosure, there is provided a method of treating a subject having a cancer comprising: administering to said subject a crystalline form of Compound (I) as described herein, or the pharmaceutical composition as described herein.

[0035] In another aspect of the present disclosure, there is provided a method of treating a subject having a cancer deficient in NMT2 comprising administering to said subject a crystalline form of Compound (I) as described herein, or the pharmaceutical composition as described herein.

[0036] In another embodiment of the present disclosure, there is provided a method wherein the cancer is a lymphoma. In another embodiment, the lymphoma is B cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B- CLL / SLL, immunocytoma / Waldenstrom's, MALT-type / monocytoid B cell lymphoma, or Burkitt’s lymphoma.

[0037] In another embodiment, there is provided a method wherein the cancer is anaplastic large cell lymphoma, acute myeloid leukemia, Blast Phase Chronic Myeloid Leukemia, Burkitt’s Lymphoma, Plasma Cell Myeloma, Intestinal Adenocarcinoma, Lung mixed Adenosquamous Carcinoma, Lung Small Cell Carcinoma, Lung, Oesophagus Squamous Cell Carcinoma, Bone, Breast Ductal Carcinoma, Stomach Diffuse Adenocarcinoma, Thyroid Medullary Carcinoma, urinary T ract T ransitional Cell Carcinoma, myeloma, ovarian clear cell carcinoma, transition cell carcinoma (ureter and bladder cancer), chronic myelogenous leukemia (CML), lymphoma-CLL, breast carcinoma, colorectal adenocarcinoma, pancreas adenocarcinoma, ovarian carcinoma, non-small cell lunch carcinoma, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, cholangiocarcinoma (bile duct cancer), gallbladder cancer, liver cancer(s), or esophageal squamous carcinoma.

[0038] In another embodiment, there is provided a method wherein wherein the subject is a child, adolescent, adult, or elderly. In another embodiment, the subject is male or female. In another embodiment, the subject is a human.BRIEF DESCRIPTION OF THE FIGURES

[0039] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0040] FIG. 1 depicts XRPD diffractogram of PCLX-001 (J09899, HCI Pattern 1).

[0041] FIG. 2 depicts 1H-NMR spectra of PCLX-001 (J09899, HCI Pattern 1).

[0042] FIG. 3 depicts HPLC chromatogram of PCLX-001 (J09899, HCI Pattern 1).

[0043] FIG. 4 depicts thermal analysis of PCLX-001 (J09899, HCI Pattern 1).

[0044] FIG. 5 depicts GVS isothermal plot of PCLX-001 (J09899, HCI Pattern 1).

[0045] FIG. 6 depicts GVS kinetic plot of PCLX-001 (J09899, HCI Pattern 1).

[0046] FIG. 7 depicts XRPD Overlay of PCLX-001 (J09899, HCI Pattern 1) Pre- and Post-storage at 25°C / 97% RH and 40°C / 75% RH.

[0047] FIG. 8 depicts HPLC chromatogram of PCLX-001 (J09899, HCI Pattern 1) Post-storage at 25 °C I 97% RH.

[0048] FIG. 9 depicts HPLC chromatogram of PCLX-001 (J09899, HCI Pattern 1) Post-storage at 40 °C I 75% RH.

[0049] FIG. 10 depicts XRPD overlay of PCLX-001 (J09899, HCI Pattern 1) Pre- and Post-GVS analysis.

[0050] FIG. 11 depictsXRPD diffractogram of PCLX-001 (J09898, J09899, J09951 , J09952 & J09953, HCI Pattern 1).

[0051] FIG. 12A depicts XRPD diffractogram of Salt Breaking Experiments (EG- 1826-10-04; Free Form Pattern 1 & EG-1826-10-05; Mixed Salt) and PCLX-001 (J09899 & J09898).

[0052] FIG. 12B depicts1H-NMR Spectra Overlay of Salt Breaking Experiments (EG-1826-10-04 & EG-1826-10-05) and PCLX-001 (J09899).

[0053] FIG. 13 depicts HPLC chromatogram of EG-1826-10-04.

[0054] FIG. 14 depicts HPLC chromatogram of EG-1826-10-05.

[0055] FIG. 15 depicts1H-NMR Overlay of EG-1826-10-04 and EG-1826-12-02.

[0056] FIG. 16 depicts thermal analysis of EG-1826-12-02.

[0057] FIG. 17 depicts HPLC chromatogram of EG-1826-12-02.

[0058] FIG. 18 depicts XRPD diffractogram of Scale-up Salt Breaking Experiments(EG-1826-10-04, EG-1826-12-02 & EG-1826-22-01 ^05) and PCLX-001 (J09899).

[0059] FIG. 19 depicts XRPD diffractogram of Free Form Pattern 1 (EG-1826-12-02 and EG-1826-25-01).

[0060] FIG. 20 depicts XRPD diffractograms of J09899, Free Form Pattern 1 (EG- 1826-25-01) and Free Form Pattern 2 (EG-1826-28-07 and EG-1826-28-09).

[0061] FIG. 21 depicts XRPD diffractograms of J09899, Free Form Pattern 1 (EG- 1826-25-01) and Free Form Pattern 2 (EG-1826-28-02, 03, 04, 05, 10, 11 , 12, 13, 14, 16,18, 19, 21).

[0062] FIG. 22 depicts Thermal Analysis of EG-1826-28-04.

[0063] FIG. 23 depicts Thermal Analysis of EG-1826-28-14.

[0064] FIG. 24 depicts1H-NMR of EG-1826-28-04.

[0065] FIG. 25 depicts1H-NMR of EG-1826-28-14.

[0066] FIG. 26 depicts SEM images of EG-1826-28-09.

[0067] FIG. 27 depicts SEM images of EG-1826-28-14.

[0068] FIG. 28 depicts PLM images of EG-1826-28-09.

[0069] FIG. 29 depicts XRPD Overlay of EG-1826-28-21 and EG-1826-28-19 Prestorage and EG- 1826-28-21 Post- storage at 40°C / 75% RH and EG-1826-28-19 Poststorage at 25°C / 97% RH.10070] FIG. 30 depicts XRPD diffractogram of Parent Form (EG-1826-25-01), Free Form Pattern 2 Pre-heating to 220°C (EG-1826-28-10) and Post-heating to 220°C (EG- 1826-39-01).

[0071] FIG. 31 depicts Solubility curves for PCLX-001 Free Form Pattern 2 (J10206) in DMSO, DMSO EtOH:H2O (2:1) (50:50), DMSO EtOH:H2O (1 :2) (70:30) and DMSO EtOH:H2O (1 :2) (95:5).

[0072] FIG. 32 depicts (A) Graph of solubility predictions depending timepoint of addition of seeds, (B) Graph of solubility predictions on increasing volumes of anti-solvent.

[0073] FIG. 33 depicts DynoChem predictions of PCLX-001 Free Form Pattern 1 in DMSO.

[0074] FIG. 34 depicts DynoChem predictions of PCLX-001 Free Form Pattern 1 in DMSO and anti-solvent EtOH:H2O (1 :2) (5%).

[0075] FIG. 35 depicts DynoChem predictions of PCLX-001 Free Form Pattern 1 in DMSO and anti-solvent EtOH:H2O (1 :2) (10%).

[0076] FIG. 36 depicts DynoChem predictions of PCLX-001 Free Form Pattern 1 in DMSO and anti-solvent EtOH:H2O (1 :2) (20%).

[0077] FIG. 37 depicts XRPD of J10206 (PCLX-001 Free Form Pattern 2).

[0078] FIG. 38 depicts XRPD Overlay of J10206 and PCLX-001 Free Form Pattern2 Reference (EG-1826-28-11).

[0079] FIG. 39 depicts1H-NMR spectra of J10206 (PCLX-001 Free Form Pattern 2).

[0080] FIG. 40 depicts1H-NMR Overlay of J10206 and PCLX-001 Free Form Pattern 2 Reference (EG- 1826-28-11).

[0081] FIG. 41 depicts Thermal Analysis of J10206 (PCLX-001 Free Form Pattern 2).

[0082] FIG. 42 depicts HPLC chromatogram of J10206 (PCLX-001 Free Form Pattern 2).

[0083] FIG. 43 depicts Raman spectra of J10206 (PCLX-001 Free Form Pattern 2).

[0084] FIG. 44 depicts Close-up Raman spectra of J10206 (PCLX-001 Free FormPattern 2).

[0085] FIG. 45 depicts XRPD Overlay of J10206 and EG-1826-48-07.

[0086] FIG. 46A depicts XRPD Overlay of J 10206, EG-1826-49-XX (XX = 01 -> 15) from solubility determination at 60 °C.

[0087] FIG. 46B depicts XRPD Overlay of J 10206, EG-1826-50-XX (XX = 01 -> 15) from solubility determination at 25 °C.

[0088] FIG. 47 depicts XRPD Overlay of J 10206, EG-1826-53-XX (XX = 01 -> 12) from repeat solubility determination.

[0089] FIG. 48 depicts XRPD Overlay of EG-1826-62-02 and EG- 1826-63-01 .

[0090] FIG. 49 depicts HPLC chromatogram of EG-1826-63-01 .

[0091] FIG. 50 depicts PLM images of EG-1826-63-01 .

[0092] FIG. 51 depicts graph of solubility vs pH for solubility pH profiling in 7 pH buffered media on J 10206.

[0093] FIG. 52 depicts Summary (± SD) Male Sprague-Dawley Rat Plasma Concentrations of PCLX-001 Salt Form (Test Item 1) and Free Base (Test Item 2) on Day 1 Following Oral Administration.

[0094] FIG. 53 depicts Summary (± SD) Female Beagle Dog Plasma of PCLX-001 Salt Form (Test Item 1) and Free Base (Test Item 2) on Day 1 Following Oral Administration.

[0095] FIG. 54 depicts Summary (± SD) Female Mouse Plasma Free Base PCLX- 001 Concentrations Following Oral Administration of PCLX-001 on Day 1.

[0096] FIG. 55 depicts HPLC chromatogram of EG-1826-62-02.

[0097] FIG. 56 depicts HPLC chromatogram of EG-1826-63-01 .

[0098] FIG. 57 depicts HPLC chromatogram of EG-1826-63-02.

[0099] FIG. 58 depicts HPLC chromatogram of J10206 (PCLX-001 Free Form Pattern 2).DETAILED DESCRIPTION

[0100] Definitions

[0101] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0102] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0103] The term "comprising" as used herein will be understood to mean that the list following is non-exhaustive and may or may not include any other additional suitable items, for example one or more further feature(s), component(s) and / or ingredient(s) as appropriate.

[0104] The term “cancer”, as used herein, refers to a variety of conditions caused by the abnormal, uncontrolled growth of cells. Cells capable of causing cancer, referred to as “cancer cells”, possess characteristic properties such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or certain typical morphological features. Cancer cells may be in the form of a tumor, but such cells may also exist alone within a subject, or may be a non-tumorigenic cancer cell. A cancer can be detected in any of a number of ways, including, but not limited to, detecting the presence of a tumor or tumors (e.g., by clinical or radiological means), examining cells within a tumor or from another biological sample (e.g., from a tissue biopsy), measuring blood markers indicative of cancer, and detecting a genotype indicative of a cancer. However, a negative result in one or more of the above detection methods does not necessarily indicate theabsence of cancer, e.g., a patient who has exhibited a complete response to a cancer treatment may still have a cancer, as evidenced by a subsequent relapse.

[0105] The term “subject”, as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. In a specific example, the subject is a human.

[0106] The term “treatment” or “treat” as used herein, refers to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also include prophylactic treatment. For example, a subject with early cancer, for example an early stage lymphoma, can be treated to prevent progression or alternatively a subject in remission can be treated with a compound or composition described herein to prevent recurrence.

[0107] The term "pharmaceutically effective amount" or “effective amount” as used herein refers to the amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician. This amount can be a “therapeutically effective amount”. These terms refer to the amount of a compound and / or compositions described herein which treats, upon single or multiple dose administration, a subject with a disease or condition. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount, the dose, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of the subject; its size, age, and general health; the specific condition, disorder, or disease involved; the degree of or involvement or the severity of the condition, disorder, or disease, the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0108] The term "pharmaceutically acceptable" as used herein includes compounds, materials, compositions, and / or dosage forms (such as unit dosages) which are suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. is also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0109] The term "excipient" means a pharmacologically inactive component such as a diluent, lubricant, surfactant, carrier, or the like. Excipients that are useful in preparing a pharmaceutical composition are generally safe, non-toxic and are acceptable for human pharmaceutical use. Reference to an excipient includes both one and more than one such excipient.

[0110] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), stabilizers and preservatives, and the like.

[0111] A "treatment or dosage regimen" as used herein refers to a combination of dosage, frequency of administration, or duration of treatment, with or without addition of a second medication.

[0112] The term “diagnosis” as used herein, refers to the identification of a molecular and / or pathological state, disease or condition, such as the identification of lymphoma, or other type of cancer.

[0113] The term "alleviates" as used herein refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.

[0114] Compound (I) (PCLX-001) is a / V-heteroarylsulphonamide named 2,6- dichloro- / V-(3-isobutyl-1 ,5-dimethyl-1 H-pyrazol-4-yl)-4-(2-(piperazin-1-yl)pyridin-4- yl)benzene sulfonamide.

[0115] As used herein, the term “crystalline form of Compound (I)” refers to the form of Compound (I) (PCLX-001) identified and characterized herein as Free Form Pattern 2 (see Example 1 and Example 2).

[0116] As used herein, the term “HCI form of Compound (I)” refers to the form of Compound (I) (PCLX-001) identified and characterized herein as HCI Pattern 1 (see Example 1).

[0117] As used herein, the term “free form of Compound (I)” refers to the form of Compound (I) (PCLX-001) identified and characterized herein as Free Form Pattern 1 (see Example 1).

[0118] Crystalline Form

[0119] Generally, the present disclosure provides a new crystalline form of Compound (I)methods of preparing this new crystalline form, compositions containing it, and its use in the treatment of diseases such as cancer, microbial infections, neurological diseases / disorders, diabetes, ischemia, osteoporosis, and associated conditions.

[0120] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by an X-ray powder diffraction pattern as shown in FIG. 37.

[0121] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks at 10.167, 11.385, and 20.505 degrees two theta ± 0.2 degrees two theta; or at 10.2, 11.4, and 20.5 degrees two theta ± 0.2 degrees two theta.

[0122] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by a DSC thermogram obtained using a heating rate of 10 °C / min comprising an endothermic event with an onset temperature of 220.2 °C± 0.2 °C.

[0123] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by a DSC thermogram substantially as shown in FIG. 41 .

[0124] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by a Raman spectrum comprising wavenumber values (cm1) of 103.24 cm1, and 993.01 cm1, and 1602.81 cm1; or 103.2 cm1, and 993.0 cm1, and 1602.8 cm1± 0.2 cm1.

[0125] In an example of the present disclosure, there is described a crystalline form of Compound (I)said crystalline form being characterized by an X-ray powder diffraction pattern as shown in FIG. 37.

[0126] In another example, there is described a crystalline form of Compound (I)said crystalline form being characterized by an X-ray powder diffraction pattern having peaks at 10.2, 11 .4, and 20.5 degrees two theta ± 0.2 degrees two theta.

[0127] In another example of the present disclosure, there is described a crystalline form wherein said crystalline form is further characterized by the X-ray powder diffraction pattern having peaks at 14.6, 22.9, 26.1 , and 31.0 degrees two theta ± 0.2 degrees two theta. In another example, said crystalline form is further characterized by the X-ray powder diffraction pattern having peaks at 18.6, 21.4, 23.4, and 33.5 degrees two theta ± 0.2 degrees two theta.

[0128] In another example of the present disclosure, there is described a crystalline form of Compound (I)said crystalline form being characterized by an X-ray powder diffraction pattern having peaks substantially as provided below in Table 1 .0’ ± 0.2 degrees two theta.

[0129] Table 1.0’

[0130] In another example of the present disclosure, there is described a crystalline form wherein said crystalline form is characterized by a DSC thermogram obtained using a heating rate of 10 °C / min comprising an endothermic event with an onset temperature of 220.2 °C ± 0.2 °C. In another example, said crystalline form is characterized by a DSC thermogram substantially as shown in FIG. 41 .

[0131] In another example of the present disclosure, there is described a crystalline form wherein said crystalline form is characterized by a Raman spectrum comprisingwavenumber values (cm1) 103.2 cm1, and 993.0 cm1, and 1602.8 cm1±0.2 cm1. In another example, said crystalline form is further characterized by the Raman spectrum comprising wavenumber values (cm1) of 126.5 cm1, 144.9 cm1, 227.3 cm1, 456.1 cm1, 1043.5 cm1, 1164.7 cm1, and 1582.5 cm-1± 0.2 cm'1. In another embodiment, said crystalline form is characterized by a Raman spectrum comprising wavenumber values (cm1) substantially as provided below in Table 2.0’± 0.2 cm1.

[0132] Table 2.0’

[0133] Methods of Preparing and Recrystallizing

[0134] In one or more embodiments of the present disclosure, there is provided a method of preparing a crystalline form of Compound (I), as characterized herein.

[0135] Generally, the crystalline form of Compound (I) is prepared by providing a HCI form of Compound (I) and salt-breaking the HCI form (e.g., via ion-exchange using a base) to form a free form of Compound (I). The free form of Compound (I) is then added to a solvent and the mixture is matured (e.g., including cooling, anti-solvent addition, maturation) to form the crystalline form of Compound (I).

[0136] In one or more embodiments, the method of preparing a crystalline form of Compound (I) as characterized herein comprises adding a HCI form of Compound (I) to a first organic solvent to form a first mixture; adding a base to the first mixture to form a first solution; and isolating from the first solution a free form of Compound (I); adding the free form of Compound (I) to a second organic solvent to form a second mixture; maturing the second mixture to form a third mixture; and isolating from the third mixture the crystalline form of Compound (I).

[0137] In one or more embodiments, the method further comprises filtering insoluble particulates from the first solution before isolating from the first solution a free form of Compound (I),

[0138] In one or more embodiments of the present disclosure, the HCI form of Compound (I) is characterized by an X-ray powder diffraction pattern as shown in FIG. 1 .

[0139] In one or more embodiments, the HCI form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks at 7.220, 11.00, and 13.457 degrees two theta ± 0.2 degrees two theta; or at 7.2, 11 .0, and 13.5 degrees two theta ± 0.2 degrees two theta. In one or more embodiments, the HCI form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks substantially as provided below in Table 3.0’ ± 0.2 degrees two theta.

[0140] Table 3.0’

[0141] In one or more embodiments, the HCI form of Compound (I) is characterized by a DSC thermogram obtained using a heating rate of 10°C / min comprising an endothermic event with an onset temperature of 64.7 °C ± 0.2 °C.

[0142] In one or more embodiments, the HCI form of Compound (I) is characterized by a DSC thermogram substantially as shown in FIG. 4 or as listed in Table 15 (J09899, DSC).

[0143] In one or more embodiments of the present disclosure, the free form of Compound (I) is characterized by an X-ray powder diffraction pattern as shown in FIG. 19 (indicated by EG- 1826- 12-02).

[0144] In one or more embodiments, the free form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks at 6.301 , 8.095, and 19.010 degreestwo theta ± 0.2 degrees two theta; or 6.3, 8.1 , and 19.0 degrees two theta ± 0.2 degrees two theta. In one or more embodiments, the free form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks substantially as provided below in Table 4.0’ ± 0.2 degrees two theta.

[0145] Table 4.0’

[0146] In one or more embodiments, the free form of Compound (I) is characterized by a DSC thermogram obtained using a heating rate of 10 °C / min comprising an endothermic event with an onset temperature of 161 .5 °C ± 0.2 °C.

[0147] In one or more embodiments, the free form of Compound (I) is characterized by a DSC thermogram substantially as shown in FIG. 16 or as listed in Table 19 (DSC).

[0148] In one or more embodiments of the present disclosure, there is provided a method of recrystallizing a crystalline form of Compound (I), as characterized herein.

[0149] Generally, recrystallizing the crystalline form of Compound (I) involves dissolving the crystalline form in an organic solvent at elevated temperatures to form a solution. Said hot solution may be filtered to remove any insoluble particulates. This heated solution is then seeded with a small amount of the crystalline form, following which the seeded solution is cooled. An anti-solvent is added to the seeded, cooled solution, following which the solution is left to mature. From this matured solution, the crystalline form of Compound (I) recrystallizes and can be isolated.

[0150] In one or more embodiments, the method of recrystallizing the crystalline form of Compound (I) comprises dissolving the crystalline form of Compound (I) in an organic solvent at a first temperature to form a first solution; ramping down the temperaturefrom the first temperature to a second temperature; adding a seed of the crystalline form of Compound (I) to the first solution to form a first mixture; ramping down the temperature from the second temperature to a third temperature; adding an anti-solvent to the first mixture to form a second mixture; maturing the second mixture for about 24 hours to form a third mixture; and isolating the recrystallized, crystalline form of Compound (I) from the third mixture.

[0151] In one or more embodiments, the method further comprises filtering insoluble particulates from the first solution before ramping down the temperature from the first temperature to a second temperature.

[0152] In one or more embodiments of the present application, the crystalline form of Compound (I)Cl, as characterized herein, is prepared and / or recrystallized via the procedures detailed in Example 1 and / or Example 2.

[0153] In an example of the present disclosure, there is described a method of preparing a crystalline form of Compound (I) as described herein, comprising the steps of: adding a HCI form of Compound (I) to a first organic solvent to form a first mixture; adding a base to the first mixture to form a first solution; and isolating from the first solution a free form of Compound (I); adding the free form of Compound (I) to a second organic solvent to form a second mixture; maturing the second mixture to form a third mixture; and isolating from the third mixture the crystalline form of Compound (I).

[0154] In another example of the present disclosure, there is described a method wherein adding a HCI form of Compound (I) to a first organic solvent further comprises heating the first mixture to a temperature of about 30 °C. In another example of the present disclosure, there is described a method wherein adding a base to the first mixture to form a first solution further comprises: maintaining the first solution at a temperature of about 30 °C; and stirring the first solution for about 1 hour.

[0155] In another example of the present disclosure, there is described a method wherein adding the free form of Compound (I) to a second organic solvent to form a second mixture further comprises: stirring the second mixture at ambient temperature; optionally ramping up the temperature from ambient temperature to about 50 °C and then ramping down the temperature from about 50 °C to ambient temperature, and optionally adding additional organic solvent to the second mixture until the free form of Compound (I) dissolves in the second organic solvent; and ramping down the temperature from about 50 °C to about 5 °C at a rate of about 0.1 °C / min.

[0156] In another example, there is described a method wherein maturing the second mixture to form a third mixture further comprises maturing at 25 / 50°C in 4-hour cycles.

[0157] In another example, there is described a method wherein the first organic solvent is selected from the group consisting of ethanol, acetone, water, DCM, or ethyl acetate. In another embodiment, the first organic solvent is DCM. In another example, there is described a method wherein the first organic solvent is selected from the group consisting of ethanol, acetone, water, or ethyl acetate.

[0158] In another example, there is described a method wherein the base is NaOH.

[0159] In another example, there is described a method wherein the second organic solvent is selected from the group consisting of 2-propanol; 2-methyl THF; acetone; acetonitrile; DMSO; N,N'-dimethylacetamide; MeOH:water (90:10 v / v); ethyl acetate; ethanol:water (50:50 v / v); isopropyl acetate; isobutanol; tert-butyl methyl ether; THF; and water. In another example, there is described a method wherein the second organic solvent is selected from the group consisting of 2-propanol; 2-methyl THF; acetone; acetonitrile; DMSO; N,N'-dimethylacetamide; MeOH:water (90:10 v / v); ethyl acetate; ethanol:water (50:50 v / v); isopropyl acetate; isobutanol; tert-butyl methyl ether; THF; and water.

[0160] In another example of the present disclosure, there is described a method further comprising filtering insoluble particulates from the first solution before isolating from the first solution a free form of Compound (I).

[0161] In another example of the present disclosure, there is described a method of recrystallizing the crystalline form of Compound (I) as described herein, or the crystalline form of Compound (I) prepared by the method described herein, comprising the steps of: dissolving the crystalline form of Compound (I) in an organic solvent at a first temperature to form a first solution; ramping down the temperature from the first temperature to a second temperature;adding a seed of the crystalline form of Compound (I) to the first solution to form a first mixture; ramping down the temperature from the second temperature to a third temperature; adding an anti-solvent to the first mixture to form a second mixture; maturing the second mixture for about 24 hours to form a third mixture; and isolating the recrystallized, crystalline form of Compound (I) from the third mixture.

[0162] In another example of the present disclosure, there is described a method wherein the organic solvent is selected from the group consisting of DMSO; DMA; and NMP. In another example, the organic solvent is DMSO. In another example of the present disclosure, there is described a method wherein the organic solvent is selected from the group consisting of DMSO; DMA; and NMP. In another example, the organic solvent is DMSO.

[0163] In an embodiment, there is provided a method wherein the anti-solvent comprises water, a C1-C3 alcohol, or a combination thereof. In an embodiment, the antisolvent comprises water, C1-C3 alcohol, or combination thereof in amounts ranging between about 100% water and about 100% C-i-C3alcohol, or any combination of water and Ci-C3alcohol between 100% water and 100% Ci-C3alcohol.

[0164] In an embodiment, the CrC3alcohol comprises a Ci-C3linear or branched or cyclic alcohol. In an embodiment, the Ci-C3alcohol comprises methanol, ethanol, n- propanol, branched propanol, or a combination thereof.

[0165] In another embodiment, there is provided a method wherein the anti-solvent is selected from the group consisting of ethanol and water. In an embodiment, the antisolvent is selected from the group consisting of ethanol and water in amounts ranging between about 100% water and about 100% ethanol, or any combination of water and ethanol between 100% water and 100% ethanol. In another example, there is described a method wherein the anti-solvent is selected from the group consisting of EtOH:H2O (1 :1); EtOH:H2O (1 :2); and EtOH:H2O (2:1).

[0166] In another example, there is described a method wherein the organic solvent is DMSO and the anti-solvent is EtOH:H2O (1 :1), EtOH:H2O (1 :2), or EtOH:H2O (2:1). In another example, the organic solvent is DMSO and the anti-solvent is EtOH:H2O (1 :2).

[0167] In another example, there is described a method wherein the anti-solvent is added at a volume that is about 10% to about 20% the volume of organic solvent. In another example, the anti-solvent is added at a volume that is about 10% the volume of organic solvent.

[0168] In another example, there is described a method wherein the first temperature is about 65 °C.

[0169] In another example, there is described a method wherein the second temperature is about 59 °C.

[0170] In another example, there is described a method wherein the third temperature is about 25°C.

[0171] In another example, there is described a method wherein ramping down the temperature from the second temperature to the third temperature comprises a ramp down of 0.5 °C / min.

[0172] In another example, there is described a method further comprising filtering insoluble particulates from the first solution before ramping down the temperature from the first temperature to a second temperature.

[0173] Pharmaceutical Compositions and Uses Thereof

[0174] In one or more aspects of the present disclosure, the crystalline form of Compound (I), as characterized herein, possesses pharmacological activity. In one or more embodiments of the present disclosure, the crystalline form of Compound (I) inhibits / V-myristoyl transferase (NMT) activity. In one or more embodiments, the crystalline form of Compound (I) is used for treating a subject having a cancer. In one or more embodiments, the crystalline form of Compound (I) is used for treating a subject having a cancer the deficient in NMT2.

[0175] / V-myristoylation of proteins is a modification in which myristate (a 14-carbon saturated fatty acid) is covalently attached to the NH2terminal glycine of a variety of cellular, viral, and onco-proteins (e.g., oncogenic Src-related tyrosine kinases, heterotrimeric G alpha subunits, etc.).

[0176] Cellular myristoylated proteins have diverse biological functions in signal transduction and oncogenesis. Modification of proteins by myristoylation is required for the subcellular targeting, protein conformation and biological activity of many important proteins in eukaryotic cells, including those required for signal transduction and regulatory functions important in cell growth. Tyrosine kinases of the Src family (proto-oncogenes) are among the most extensively studied myristoylated proteins.

[0177] Myristoylation of proteins is catalyzed by / V-myristoyltransferase (NMT). NMT is responsible for this activity in eukaryotic cells and works by modifying its polypeptide substrate after the removal of the initiator methionine residue by methionyl aminopeptidase. This modification occurs primarily as a cotranslational process, although myristoylation can also occur post-translationally after proteolytic cleavage of proteins, typically during apoptosis. Two isozymes of the mammalian NMT enzymes have been cloned and are designated NMT1 and NMT2.

[0178] NMTs play a pro-survival role in cells. The two NMTs are present in all normal cells. An increase in NMT activity and expression has also been shown in a number of tumour types, suggesting inhibitors of NMT could be potential anti-cancer agents.

[0179] In one of more embodiments of the present disclosure, the crystalline form of Compound (I), as characterized herein, may be useful in the treatment or prevention of diseases or disorders which can be prevented, alleviated ortreated by modulation / inhibition of / V-myristoyl transferase (NMT) activity (referred to herein as NMT related diseases or disorders). Such NMT related diseases or disorders include but are not limited to hyperproliferative disorders, for example cancer, microbial infections, neurological diseases / disorders, inflammatory diseases, immune diseases, autoimmune diseases, diabetes, ischemia, osteoporosis, and associated conditions.

[0180] In one or more embodiments, the cancer to be prevented, alleviated or treated includes all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.

[0181] In one or more embodiments, the cancer to be prevented, alleviated or treated includes malignancies of the various organ systems, such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term "carcinoma" also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizableglandular structures. The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation. Further types of cancer include leukaemia, skin, intracranial and brain cancer.

[0182] In one or more embodiments, the cancer to be prevented, alleviated or treated includes lymphoma. The term “lymphoma” as used herein refers to a malignant growth of B or T cells in the lymphatic system. “Lymphoma” includes numerous types of malignant growths, including Hodgkin's Lymphoma and non-Hodgkin's lymphoma. The term “non-Hodgkin's Lymphoma” as used herein, refers to a malignant growth of B or T cells in the lymphatic system that is not a Hodgkin's Lymphoma (which is characterized, e.g., by the presence of Reed-Sternberg cells in the cancerous area). Non-Hodgkin's lymphomas encompass over 29 types of lymphoma, the distinctions between which are based on the type of cancer cells.

[0183] In an embodiment, the cancer is a B-lymphoma. Thus, in an embodiment of the present disclosure, the crystalline form of Compound (I) and pharmaceutical compositions thereof are suitable for the treatment of a subject with B cell lymphoma. Examples of B-cell lymphomas include, but are not limited to, for example, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom's, and MALT-type / monocytoid B cell lymphoma. Also contemplated are the treatment of pediatric lymphomas such as Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, precursor B-LBL, precursor T-LBL, and anaplastic large cell lymphoma.

[0184] In one or more embodiments embodiment, the cancer to be prevented, alleviated or treated is lymphoma, B cell lymphoma, follicular lymphoma, diffuse large B- cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom's, MALT- type / monocytoid B cell lymphoma, Burkitt’s lymphoma, a pediatric lymphoma, anaplastic large cell lymphoma, acute myeloid leukemia, Blast Phase Chronic Myeloid Leukaemia, Burkitt’s Lymphoma, Plasma Cell Myeloma, Intestinal Adenocarcinoma, Lung mixed Adenosquamous Carcinoma, Lung Small Cell Carcinoma, Lung, Oesophagus Squamous Cell Carcinoma, Bone, Breast Ductal Carcinoma, Stomach Diffuse Adenocarcinoma, Thyroid Medullary Carcinoma, urinary T ract T ransitional Cell Carcinoma, myeloma, ovarian clear cell carcinoma, transition cell carcinoma (ureter and bladder cancer), chronic myelogenous leukemia (CML), lymphoma-CLL, breast carcinoma, colorectal adenocarcinoma, pancreas adenocarcinoma, ovarian carcinoma, non-small cell lunch carcinoma, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, cholangiocarcinoma (bile duct cancer), gallbladder cancer, liver cancer(s), or esophageal squamous carcinoma.

[0185] In one or more embodiments, the microbial infection to be prevented, alleviated or treated is an infection caused by a bacterium, parasite, protozoa, virus or fungus including yeast.

[0186] A "pathogen" is generally defined as any disease-causing organism. A parasitic pathogen may be derived from a parasite selected from, but not limited to, the group consisting of Trypanosoma spp. ( e.g. T. cruzi, T. brucei, T. congolense), Leishmania spp. (e.g. L. major, L. donovani, L. braziliensis), Giardia spp., Trichomonas spp. (e.g. Tr. vaginalis), Entamoeba spp. (e.g. E. histolytica), Naegleria spp., Acanthamoeba spp. (e.g. A. castelleni), Schistosoma spp. (e.g. S. mansoni, S. japonicam), Plasmodium spp. (e.g. P. falciparum), Crytosporidium spp., Isospora spp., Balantidium spp., Loa Loa, Ascaris lumbricoides, Dirofilaria immitis, Toxoplasma ssp. (e.g To. gondii), Onchocerca spp. (e.g. O. volualno).

[0187] A viral pathogen may be derived from a virus selected from, but not limited to, the group consisting of: Human Immunodeficiency Virus (HIV1 & 2); Human T Cell Leukaemia Virus (HTLV 1 & 2); Ebola virus; human papilloma virus (e.g. HPV-2, HPV-5, HPV-8 HPV-16, HPV-18, HPV-31 , HPV-33, HPV-52, HPV-54 and HPV- 56); papovavirus; rhinovirus; poliovirus; herpesvirus; adenovirus; Epstein Barr virus; influenza virus, hepatitis B and C viruses, Variola virus, rotavirus or SARS coronavirus.

[0188] A fungal pathogen may be derived from a fungus (including yeast) selected from, but not limited to, the genera Candida spp., (e.g. C. albicans, C. tropicalis), Aspergillus spp. (e.g. A. fumigatus), Cryptococcus spp. (e.g. Cryptococcus neoformans), and Saccharomyces spp. (e.g. Saccharomyces cerevisiae), Pneumocystis spp. (e.g. Pneumocystis carinii).

[0189] In one or more embodiments, the neurological diseases / disorder to be prevented, alleviated or treated may include neuropsychiatric disorders, including Parkinson's Disease, Attention Deficit Hyperactivity Disorder (ADHD), depression (bipolar disorder) and schizophrenia and addiction; neurodegenerative disorders (e.g. Alzheimer's disease, Tourette Syndrome, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, senile chorea, Sydenham's chorea, autism, head and spinal cord trauma, acute and chromic pain, epilepsy and seizures, dementia, distonia, tremor, autism, cerebral ischemia and neuronal cell death) and disorders linked to apoptosis (particularly neuronal apoptosis).

[0190] When used for treatment of a subject, the crystalline form of Compound (I) as characterized herein may be administered either as a free compound or as part of a pharmaceutical composition. It may be administered orally, intravenously, subcutaneously,buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray or via inhalation.

[0191] The crystalline form of Compound (I) may be administered, either as a free compound or as part of a pharmaceutical composition, in a pharmaceutically acceptable dosage form. Depending upon the disorder and subject to be treated and the route of administration, the compound or pharmaceutical composition may be administered at varying doses.

[0192] The crystalline form of Compound (I) may be administered orally or parenterally ("parenterally" as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion) to a subject to obtain an inhibitory effect. In the case of larger animals, such as humans, the crystalline form of Compound (I) may be administered alone or as a pharmaceutical composition in combination with pharmaceutically acceptable diluents, excipients or carriers.

[0193] Actual dosage levels of the crystalline form of Compound (I) in pharmaceutical compositions may be varied so as to obtain an amount of the active crystalline form of Compound (I) that is effective to achieve the desired therapeutic response for a particular subject, compositions, and / or mode of administration. The selected dosage level will depend upon the activity of the crystalline form of Compound (I), the route of administration, the severity of the condition being treated, and the condition and prior medical history of the subject being treated. However, it is within the skill of the art to start doses of the crystalline form of Compound (I) at levels lower than required for achieving the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0194] In one or more embodiments of the present disclosure where treatment, prevention, control, amelioration, or reduction of risk of conditions require inhibition of kinase activity, an appropriate dosage level may be about 0.01 to 500 mg per kg patient body weight per day, which can be administered in single or multiple doses, or in a continuous infusion. In one or more embodiments, the dosage level will be about 0.1 to about 250 mg / kg per day; or about 0.5 to about 100 mg / kg per day. A suitable dosage level may be about 0.01 to 250 mg / kg per day, about 0.05 to 100 mg / kg per day, or about 0.1 to 50 mg / kg per day. Within this range, the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg / kg per day.

[0195] For oral administration, pharmaceutical compositions comprising the crystalline form of Compound (I) may be provided in the form of tablets containing 1 .0 to 1000 milligrams of the the crystalline form of Compound (I), particularly 1.0, 5.0, 10.0, 15.0,20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0 and 1000.0 milligrams of the crystalline form of Compound (I) for the symptomatic adjustment of the dosage to the subject to be treated. The crystalline form of Compound (I) may be administered, either as a free compound or as part of a pharmaceutical composition, on a regimen of 1 to 4 times per day; or once or twice per day. The dosage regimen may be adjusted to provide the optimal therapeutic response.

[0196] In another aspect of the present disclosure there is provided a pharmaceutical composition comprising the crystalline form of Compound (I) in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0197] Pharmaceutical compositions of the present disclosure for parenteral injection suitably comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0198] Pharmaceutical compositions of the present disclosure may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol or phenol sorbic acid. It may also be desirable to include isotonic agents such as sugars or sodium chloride, for example.

[0199] Prolonged absorption of an injectable pharmaceutical form may be brought about by the inclusion of agents (for example aluminum monostearate and gelatin) which delay absorption.

[0200] In some cases, in order to prolong the effect of the pharmaceutical compositions of the present disclosure, it is desirable to slow the absorption of the composition, or the crystalline form of Compound (I) from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension, where the crystalline form of Compound (I) is suspended in a liquid in which the crystalline form has poor solubility. The rate of absorption of the crystalline form of Compound (I) then depends upon its rate of dissolution.

[0201] Alternatively, delayed absorption of a parenterally administered pharmaceutical composition of the present disclosure is accomplished by dissolving or suspending the crystalline form of Compound (I) in an oil vehicle. Injectable depot forms are suitably made by forming microencapsulated matrices of the crystalline form of Compound (I) in biodegradable polymers, for example polylactide-polyglycolide. Depending upon the ratio of the crystalline form of Compound (I) to polymer and the nature of the particular polymer employed, the rate of release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).

[0202] Depot injectable formulations may also be prepared by entrapping the crystalline form of Compound (I) in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.

[0203] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, an active compound (e.g., the crystalline form of Compound (I)) is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or one or more: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example.

[0204] Suitably, oral formulations contain a dissolution aid. The dissolution aid is not limited as to its identity so long as it is pharmaceutically acceptable. Examples include nonionic surface active agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g. sorbitan trioleate), polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers,polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorbitol fatty acid esters, fatty acid alkylolamides, and alkylamine oxides; bile acid and salts thereof (e.g. chenodeoxycholic acid, cholic acid, deoxycholic acid, dehydrocholic acid and salts thereof, and glycine or taurine conjugate thereof); ionic surface active agents, such as sodium laurylsulfate, fatty acid soaps, alkylsulfonates, alkylphosphates, ether phosphates, fatty acid salts of basic amino acids; triethanolamine soap, and alkyl quaternary ammonium salts; and amphoteric surface active agents, such as betaines and aminocarboxylic acid salts.

[0205] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, and / or in delayed fashion. Examples of embedding compositions include polymeric substances and waxes.

[0206] The crystalline form of Compound (I) may also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients. The crystalline form of Compound (I) may also be in finely divided form, for example it may be micronised.

[0207] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the crystalline form of Compound (I), the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents. Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof.

[0208] Pharmaceutical compositions of the present disclosure for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the crystallineform of Compound (I) with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the crystalline form of Compound (I).

[0209] The crystalline form of Compound (I) may also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. Pharmaceutical compositions of the present disclosure in liposome form can contain, in addition to the crystalline form of Compound (I), stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art.

[0210] Dosage forms for topical administration of the crystalline form of Compound(I) include powders, sprays, ointments and inhalants. The crystalline form of Compound (I) is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers or propellants which may be required. The crystalline form of Compound (I) may also be administered, for example as part of a pharmaceutical composition, in ophthalmic formulations, or eye ointments, powders and solutions.

[0211] In another example of the present disclosure, there is described a pharmaceutical composition, comprising a crystalline form of Compound (I) as described herein; and a pharmaceutically acceptable adjuvant, diluent, carrier, or vehicle.

[0212] In another example of the present disclosure, there is described a method of treating a subject having a cancer comprising: administering to said subject a crystalline form of Compound (I) as described herein, or the pharmaceutical composition as described herein.

[0213] In another example of the present disclosure, there is described a method of treating a subject having a cancer deficient in NMT2 comprising administering to said subject a crystalline form of Compound (I) as described herein, or the pharmaceutical composition as described herein.

[0214] In another example of the present disclosure, there is described a method wherein the cancer is a lymphoma. In another example, the lymphoma is B cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom's, MALT-type / monocytoid B cell lymphoma, or Burkitt’s lymphoma.

[0215] In another example, there is described a method wherein the cancer is anaplastic large cell lymphoma, acute myeloid leukemia, Blast Phase Chronic Myeloid Leukaemia, Burkitt’s Lymphoma, Plasma Cell Myeloma, Intestinal Adenocarcinoma, Lung mixed Adenosquamous Carcinoma, Lung Small Cell Carcinoma, Lung, Oesophagus Squamous Cell Carcinoma, Bone, Breast Ductal Carcinoma, Stomach Diffuse Adenocarcinoma, Thyroid Medullary Carcinoma, urinary Tract Transitional Cell Carcinoma, myeloma, ovarian clear cell carcinoma, transition cell carcinoma (ureter and bladder cancer), chronic myelogenous leukemia (CML), lymphoma-CLL, breast carcinoma, colorectal adenocarcinoma, pancreas adenocarcinoma, ovarian carcinoma, non-small cell lunch carcinoma, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, cholangiocarcinoma (bile duct cancer), gallbladder cancer, liver cancer(s), or esophageal squamous carcinoma.

[0216] In another example, there is described a method wherein wherein the subject is a child, adolescent, adult, or elderly. In another example, the subject is male or female. In another example, the subject is a human.

[0217] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.

[0218] EXAMPLES

[0219] EXAMPLE 1 - Screening Studies on Compound (I) (PCLX-001)

[0220] SUMMARY

[0221] Solid state characterisation was performed on supplied batches of PCLX-001. Three batches were denoted as HCI Pattern 1 , and one batch was denoted HCI Pattern 2. All the batches contained a peak at ~ 32.1 °2-theta, which is representative of sodium chloride. Ion chromatography confirmed the presence of excess chloride as well as sodium content. One batch was taken forward for the salt breaking step.

[0222] Salt breaking via ion exchange using sodium hydroxide was successful and was scaled up a few times, up to 1 g scale. The recovered material was chloride and sodium free (free form) and it was crystalline form (denoted, free form Pattern 1). The purity of this material was 96.3 %, comparable to that of the starting material. The melt of this form was recorded at 161.5 °C (onset).

[0223] The free form Pattern 1 was taken forward to explore its polymorphic landscape. Twenty-two solvents / solvent systems were selected for this phase, with the starting material being free form Pattern 1. The following approaches where employed;cooling, maturation and anti-solvent addition. Free form Pattern 2 was obtained in almost all cases, which suggested that it is possibly the most stable form under the tested conditions.

[0224] Free form Pattern 2 was a crystalline, non solvated form that remained unchanged by XRPD after one week storage at elevated temperature and humidity levels almost (40 °C / 75%RH and 25 °C / 97%RH). The melt I degradation temperature was observed between 209-217 °C. Only gradual weight loss, due to loss of unbound water, was observed on the TGA thermogram followed by sample degradation above 210 °C.

[0225] Free form Pattern 2 was found to display the most desirable solid-state properties out of all polymorphs discovered during testing, and was further studied to generate an optimised crystallisation method for the formation of Free Form Pattern 2 (see Example 2).

[0226] ABBREVIATIONS

[0227] INSTRUMENT AND METHODOLOGY DETAILS

[0228] X-ray Powder Diffraction (XRPD)

[0229] Bruker AXS D8 Advance

[0230] XRPD diffractograms were collected on a Bruker D8 diffractometer using CuKa radiation (40 kV, 40 mA) and a 9-29 goniometer fitted with a Ge monochromator. The incident beam passes through a 2.0 mm divergence slit followed by a 0.2 mm antiscatter slit and knife edge. The diffracted beam passes through an 8.0 mm receiving slit with 2.5° Soller slits followed by the Lynxeye Detector. The software used for data collection and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA respectively.

[0231] Samples were run under ambient conditions as flat plate specimens using powder. The sample was prepared on a polished, zero-background (510) silicon wafer by gently pressing onto the flat surface or packed into a cut cavity. The sample was rotated in its own plane.

[0232] The details of the standard data collection method are:• Angular range: 2 to 42° 29• Step size: 0.05° 29• Collection time: 0.5 s / step (total collection time: 6.40 min)

[0233] PANalytical Empyrean

[0234] XRPD diffractograms were collected on a PANalytical Empyrean diffractometer using Cu Ka radiation (45 kV, 40 mA) in transmission geometry. A 0.5° slit, 4 mm mask and 0.04 rad Soller slits with a focusing mirror were used on the incident beam. A PIXcel3D detector, placed on the diffracted beam, was fitted with a receiving slit and 0.04 rad Soller slits. The software used for data collection was X’Pert Data Collector using X’Pert Operator Interface. The data were analysed and presented using Diffrac Plus EVA or HighScore Plus.

[0235] Samples were prepared and analysed in either a metal or Millipore 96 wellplate in transmission mode. X-ray transparent film was used between the metal sheets on the metal well-plate and powders (approximately 1 - 2 mg) were used as received. The Millipore plate was used to isolate and analyse solids from suspensions by adding a small amount of suspension directly to the plate before filtration under a light vacuum.

[0236] The scan mode for the metal plate used the gonio scan axis, whereas a 29 scan was utilised for the Millipore plate.

[0237] The details of the standard screening data collection method are:• Angular range: 2.5 to 32.0° 29• Step size: 0.0130° 29• Collection time: 12.75 s / step (total collection time of 2.07 min)

[0238] Nuclear Magnetic Resonance (NMR)

[0239] Solution State NMR

[0240] 1 H NMR and / or 13C NMR spectra were collected on a Bruker 400 MHz instrument equipped with an auto-sampler and controlled by a DRX400 console. Samples were prepared in DMSO-c / 6 solvent, unless otherwise stated. Automated experiments were acquired using ICON-NMR configuration within Topspin software, using standard Bruker- loaded experiments (1 H, 13C {1 H}, DEPT135). Off-line analysis was performed using ACD Spectrus Processor. For non-routine spectroscopy (2D NMR and variable temperature NMR), data were acquired through the use of Topspin alone.

[0241] Differential Scanning Calorimetry (DSC)

[0242] TA Instruments Q2000

[0243] DSC data were collected on a TA Instruments Q2000 equipped with a 50 position auto-sampler. Typically, 0.5 - 3 mg of each sample, in a pin-holed aluminium pan, was heated at 10 °C / min from 25 °C to 300 °C. A purge of dry nitrogen at 50 ml / min was maintained over the sample.

[0244] Modulated temperature DSC was carried out using an underlying heating rate of 2 °C / min and temperature modulation parameters of ±0.636 °C (amplitude) every 60 seconds (period).

[0245] The instrument control software was Advantage for Q Series and Thermal Advantage and the data were analysed using Universal Analysis or TRIOS.

[0246] TA Instruments Discovery DSC

[0247] DSC data were collected on a TA Instruments Discovery DSC equipped with a 50 position auto-sampler. Typically, 0.5 - 3 mg of each sample, in a pin-holed aluminium pan, was heated at 10 °C / min from 25 °C to 300 °C. A purge of dry nitrogen at 50 ml / minwas maintained over the sample. The instrument control software was TRIOS and the data were analysed using TRIOS or Universal Analysis.

[0248] Thermo-Gravimetric Analysis (TGA)

[0249] TA Instruments Q500

[0250] TGA data were collected on a TA Instruments Q500 TGA, equipped with a 16 position auto-sampler. Typically, 5-10 mg of each sample was loaded onto a pre-tared aluminium DSC pan and heated at 10 °C / min from ambient temperature to 350 °C. A nitrogen purge at 60 ml / min was maintained over the sample. The instrument control software was Advantage for Q Series and Thermal Advantage and the data were analysed using Universal Analysis or TRIOS.

[0251] TA Instruments Discovery TGA

[0252] TGA data were collected on a TA Instruments Discovery TGA, equipped with a 25 position auto-sampler. Typically, 5 - 10 mg of each sample was loaded onto a pre-tared aluminium DSC pan and heated at 10 °C / min from ambient temperature to 350 °C. A nitrogen purge at 25 ml / min was maintained over the sample. The instrument control software was TRIOS and the data were analysed using TRIOS or Universal Analysis.

[0253] Polarised Light Microscopy (PLM)

[0254] Leica LM / DM Polarised Light Microscope

[0255] Samples were analysed on a Leica LM / DM polarised light microscope with a digital video camera for image capture. A small amount of each sample was placed on a glass slide, with or without immersion oil, and covered with a glass slip. The sample was viewed with appropriate magnification and partially polarised light, coupled to a A false- colour filter. Images were captured using StudioCapture or Image ProPlus software.

[0256] Scanning Electron Microscopy (SEM)

[0257] Data were collected on a Phenom Pro Scanning Electron Microscope. A small quantity of sample was mounted onto an aluminium stub using conducting doublesided adhesive tape. A thin layer of gold was applied using a sputter coater (20 mA, 120 s).

[0258] Gravimetric Vapour Sorption (GVS)

[0259] Sorption isotherms were obtained using a SMS DVS Intrinsic moisture sorption analyser, controlled by DVS Intrinsic Control software. The sample temperature was maintained at 25 °C by the instrument controls. The humidity was controlled by mixing streams of dry and wet nitrogen, with a total flow rate of 200 ml / min. The relative humidity was measured by a calibrated Rotronic probe (dynamic range of 1.0 - 100 %RH), located near the sample. The weight change (mass relaxation) of the sample as a function of %RH was constantly monitored by a microbalance (accuracy ±0.005 mg).

[0260] Typically, 5 - 30 mg of sample was placed in a fared mesh stainless steel basket under ambient conditions. 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 per complete cycle). The standard isotherm was performed at 25 °C at 10 %RH intervals over a 0 - 90 %RH range. Typically, a double cycle (4 scans) was carried out. Data analysis was carried out within Microsoft Excel using the DVS Analysis Suite.

[0261] Table 1 Method for SMS DVS Intrinsic experiments

[0262] The sample was recovered after completion of the isotherm and re-analysed by XRPD (see above).

[0263] Chemical Purity Determination by HPLC

[0264] Purity analysis was performed on an Agilent HP1100 / 1 nfinity II 1260 series system equipped with a diode array detector and using OpenLAB software. The full method details are provided below:

[0265] Table 2 HPLC method for chemical purity determinations

[0266] Water Determination by Karl Fischer Titration (KF)

[0267] The water content of each sample was measured on a Metrohm 874 Oven Sample Processor at 150 °C with 851 Titrano Coulometer using Hydranal Coulomat AG oven reagent and nitrogen purge. Weighed solid samples were introduced into a sealed sample vial. Approximately 10 mg of sample was used per titration and duplicate determinations were made. An average of these results is presented unless otherwise stated. Data collection and analysis were performed using Tiamo software.

[0268] Thermodynamic Aqueous Solubility

[0269] Aqueous solubility was determined by suspending sufficient compound in relevant media to give a maximum final concentration of >10 mg / ml of the parent free-form of the compound. The suspension was equilibrated at 25 °C, on a Heidolph plate shaker set to 750 rpm for 24 hours. The pH of the saturated solution was then measured, and the suspension filtered through a glass fiber C filter (particle retention 1.2 pm) and diluted appropriately. Quantitation was by HPLC with reference to a standard solution of approximately 0.15 mg / ml in DMSO. Different volumes of the standard, diluted and undiluted sample solutions were injected. The solubility was calculated using the peak areas determined by integration of the peak found at the same retention time as the principal peak in the standard injection.

[0270] Table 3 HPLC method for solubility measurements

[0271] Analysis was performed on an Agilent HP1100 / Infinity I1 1260 series system equipped with a diode array detector and using OpenLAB software.

[0272] Thermodynamic Solubility of J09899 in FaSSGF, FeSSIF, FaSSIF and Deionised Water

[0273] Methods and Comments

[0274] Sufficient sample was suspended in 0.5 ml media for a maximum anticipated concentration of 100 mg / ml of the Hydrochloric Acid salt of the compound. The resulting suspensions were then shaken at 25 °CZ 750 rpm for 24 hours. After equilibration, the appearance was noted and the pH of the saturated solution was measured. Samples were then filtered through a glass ‘C’ fiber filter (Particle retention 1 .2 pm). Samples were diluted x10 and x100 in appropriate media.

[0275] Quantitation was by HPLC with reference to a standard solution of approximately 0.15 mg / ml. Different volumes of the standard, diluted and undiluted sample solutions were injected. The solubility was calculated using the peak areas determined by integration of the peak found at the same retention time as the principal peak in the standard injection.

[0276] Observations

[0277] Co-elution of the SIF peak with the parent peak observed on the generic solubility method for this sample. Sample was run using the Generic Purity method (higher resolution column) to resolve these peaks.Table 5 JM Generic Solubility HPLC Method

[0278] Summary and Conclusions

[0279] In FaSSGF media, FaSSIF media and deionised water the sample was categorized as ‘slightly soluble’ under USP classification. In FeSSIF, the sample was classified as ‘very slightly soluble’ under USP classification. These values were calculated based on the sample having 1 .4 equivalents of HCI, based on IC and KF analysis.

[0280] As there was an issue with co-elution of the SIF peak in using the Generic solubility method, all subsequent analysis is to be run using the Generic purity method as this can resolve the peaks.

[0281] Ion Chromatography (IC)

[0282] Data were collected on a Metrohm 930 Compact IC Flex with 858 Professional autosampler and 800 Dosino dosage unit monitor, using IC MagicNet software. Accurately weighed samples were prepared as stock solutions in a suitable solvent. Quantification was achieved by comparison with standard solutions of known concentration of the ion being analysed. Analyses were performed in duplicate and an average of the values is given unless otherwise stated.

[0283] Table 11 IC method for cation chromatography

[0284] Table 12 IC method for anion chromatography

[0285] pKa and LoqP Determination and Prediction

[0286] Results

[0287] Structure and Predictions

[0288] High pKa at 8.95 confirmed as acidic and pKa at 8.19 confirmed as basic via Yasuda-Shedlovsky extrapolation (disagrees with prediction software).

[0289] Method and Comments

[0290] Data were collected on a Sirius T3 instrument with a D-PAS attachment fitted with a Ag / AgCI double junction pH electrode. The electrode was calibrated using the four plus parameters derived from a blank titration. The base titrant was standardised by titration with KHP. 0.5 M HCI and KOH aqueous solutions were used as the acid and base titrants respectively for the testing. Titration was performed in a background of ISA 0.15 M KCI (aq). The data were refined using Sirius T3 Refine. Prediction of pKa and LogP values was made using ACD / Labs Percepta.

[0291] UV-Metric pKa (Aqueous)

[0292] The sample was prepared as a 11 .07 mM stock solution in DMSO (5pL stock used for analysis). Data were obtained by a UV-metric single titration from pH 1 .5-4.0 (low to high) under aqueous conditions at 25 °C.

[0293] Fast-UV pKa (Aqueous)

[0294] The sample was prepared as a 10 mM stock solution in DMSO (5pL stock used for analysis). Data were obtained by a Fast-UV single titration from pH 2.0-12.0 (low to high) under aqueous conditions at 25 °C.

[0295] pH-Metric LogP

[0296] 0.96 mg of the sample was weighed directly into a T3 vial. Data were collected using the potentiometric titration method using the high LogP assay with three ratios of octanol (0.020 / 0.050 / 1.0 mL), ionic-strength adjusted (ISA) water from pH 2.0-12.0 (low to high). The potentiometric data collected were used to calculate the LogP, LogPiOn, and LogD values.

[0297] LogP Shake Flask

[0298] A 1 mg / ml stock solution in octanol was prepared and shaken at room temperature for 1 hour to ensure complete dissolution. Equal volumes of the octanol solution and USP pH 7.4 phosphate buffer (50 mM) were shaken, in duplicate, for 1 hour at room temperature. The sample was centrifuged for 10 min at 13400 rpm and the phases separated using a glass pipette. The separated layers were diluted appropriately (octanol samples with acetonitrile and aqueous samples with buffer if required) and analysed by HPLC using the method detailed below.

[0299] Table 13 HPLC method for LogD determinations

[0300] EXPERIMENTAL CRYSTALLISATION METHODOLOGIES

[0301] The choice of crystallisation method has a major influence on which form is produced, and it is therefore important to perform crystallisations using various methods and conditions when looking for polymorphs.

[0302] Classical crystallisation methods used herein are listed in Table 14 together with the degrees of freedom available for each process.

[0303] Table 14 Classical crystallization methods used in this project

[0304] Solvent Mediated Techniques

[0305] These are classical techniques used for generating crystalline material. Theoretically, crystallisation occurs when the concentration of a compound in a solvent is higher than its solubility product. Generally, crystallisation is kinetically hindered, and crystals grow only from supersaturated solutions.

[0306] For a crystallisation screen, solvents with highly diverse properties should be chosen (hydrogen bond donor / acceptor propensity, dipole moment, dielectric constant, viscosity, etc.). Often solvent mixtures are useful in obtaining systems with suitable solubilities, polarities etc. The substance also needs to be chemically stable in the givensolvents or solvent mixtures. There are several ways to achieve the metastable state of supersaturation.

[0307] Maturation / Slurry Ripening

[0308] For the investigation of crystalline forms, often maturation experiments (or slurry ripening) are performed in various solvents or solvent mixtures and subjected to heatcool cycles. Repeated heating and cooling cycles may increase the degree of crystallinity or convert a meta-stable state (or out-of-equilibrium state in the case of amorphous material) into a more thermodynamically stable crystalline form. The rate and extent of conversion is dependent upon solubility of the input material.

[0309] For thermodynamic reasons, the system can only evolve towards more stable forms. Therefore, if the starting material is crystalline, it is impossible to obtain a less stable crystalline phase. If the starting material is amorphous, a much greater variety of forms may be obtained.

[0310] Maturation Chamber Procedure

[0311] Suspensions for maturation were placed in a platform shaker incubator (Heidolph Titramax I Incubator 1000) and subjected to a series of heat-cool cycles from ambient to approximately 50 °C. This was achieved by switching the heating on or off every 4 hours. Shaking was maintained throughout.

[0312] Polar Bear Procedure

[0313] Suspensions were stirred (500 rpm) in a Polar Bear (Cambridge Reactor Design) for 1 hr at 50 °C. The samples were then cooled to 5 °C at 0.1 °C / min and stirred for a further 4 hours.

[0314] Cooling Crystallisation

[0315] Crystallisation can be obtained by lowering the temperature of a clear solution. The solubility of most materials decreases with decreasing temperature, so cooling can be used to generate supersaturation. In many cases however, the solubility of a material remains high even at low temperatures or the solubility changes very little over the temperature range of interest. In these cases, other methods for creation of supersaturation must be considered (such as controlled evaporation below).

[0316] Procedure

[0317] Solutions were cooled to 5 °C at 0.1 °C / min in a Polar Bear and stirred at this temperature for 24 hours. All solids were filtered and dried under suction for 20 min and initially analysed by XRPD.

[0318] Controlled Evaporation

[0319] Crystallisation can be generated by controlled evaporation of a clear, particulate free, solution. This is especially true when the solvent has a relatively highvapour pressure. At approximately constant temperature, the solvent is being removed from the system, thereby increasing the solute concentration. The crystal nucleation and growth are obtained when some maximum supersaturation is reached. This technique also has the advantage that since the samples are slowly evaporated it is often possible to generate large single crystals suitable for SCXRD.

[0320] Procedure

[0321] Solutions were evaporated at ambient conditions by removing the lids of the vials and replacing them with pin-holed sealing film; or by inserting a 25-gauge syringe needle through the lids. The samples were allowed to slowly evaporate to dryness I until a solid appeared at ambient conditions.

[0322] CHARACTERISATION OF COMPOUND (I) (PCLX-001)

[0323] Compound (I) (PCLX-001)(J09898 & batch J09899) was characterised using a wide range of techniques to investigate the solid form and chemical properties of PCLX-001. A summary of the results is shown in Table 15.

[0324] Table 15 Characterization data for PCLX-001 (J09898 & J09899)

[0325] The characterisation of PCLX-001 (J09899, HCI Pattern 1) was confirmed to be crystalline, assigned HCI Pattern 1 by XRPD analysis (Fig. 1), with a purity of 97.2 % (Fig. 3). 1 H-NMR spectra (Fig. 2) matched the proposed structure of the molecule with trace amount of acetone present. 1.3 molar equivalents of chloride were observed from anion IC, which was lower than an anticipated 3 equivalents of chloride as the compound was believed to be a tri-chloride form. In addition, 0.2 molar equivalents of sodium and 0.1 molar equivalents of calcium were also observed in cation IC which may have arisen in the manufacturing process of the compound.

[0326] Thermal analysis of PCLX-001 (J09899, HCI Pattern 1) (Fig. 4) showed the material to have a large endotherm at 64.7 °C (onset, 174 J / g) followed by an endotherm at 154.5 °C (onset, 23 J / g). A weight loss of 9.7 % followed by a small weight loss of 1 % from ~50°C and 225 °C was observed with degradation of the material at 250 °C (onset). The weight loss may have been related to the loss of water from the sample as 9.7 % w / w water was observed in the sample by KF analysis.

[0327] GVS analysis on PCLX-001 (J09899, HCI Pattern 1) (Fig. 5 and Fig. 6) showed the sample to be very hygroscopic with an uptake of 39.76 % w / w water at 90% RH. However, no visible change in appearance of the solid or XRPD pattern post-GVS analysis was observed.

[0328] Static stability experiments were also conducted at two sets of elevated storage conditions at 40°C / 75% RH and 25°C / 97% RH which showed no visible change in the appearance of the solid or XRPD pattern (Fig. 7 and 10) and retained high purity in both cases (Fig. 8 and Fig. 9), concluding stability at both elevated humidity and temperature.

[0329] Thermodynamic solubility showed high solubility in FaSSGF at 9.9 mg / ml and water at 6.0 mg / ml and relatively lower solubility in FeSSIF at 1.9 mg / ml and FaSSIF - 0.64 mg / ml. Slight differences between the predicted pKa values and the measured pKa values. Log P analysis was found to be 2.2 at pH 7.4. Further, measured difference in pKas were enough to rule out zwitterion.

[0330] The characterization of the alternate batch of the received PCLX-001 (J09898) was confirmed to have a different crystalline pattern and was assigned HCI Pattern 2, by XRPD analysis. The material displayed lower purity of 96.7 %. However, the1H-NMR spectra matched the proposed structure of the molecule with trace amounts of acetone and TBME present. Thermal analysis was also carried out on PCLX-001 (J09898) where a large endotherm was observed at 40.2 °C (onset) 196 J / g followed by an exotherm at 138.7 °C (onset) 8 J / g and endotherm at 165.8 °C (onset) 10 J / g. A weight loss of 11.4 % w / w from ~50 °C to 225 °C followed by degradation of the material at 250 °C (onset). In addition, 2 molar equivalents of chloride were observed in anion IC, still lower than the expected 3 molar equivalents of chloride for a tris-salt, and no cations were observed in cation IC.

[0331] Characterization of additional batches of PCLX-001 (J09951 , J09952 and J09953) were confirmed to be crystalline HCI Pattern 1 , all retaining high purity of 98.5%, 98.3% and 98.5%, respectively. The 1 H-NMR spectra matched the proposed structure of the molecule. A sharp peak was observed in the XRPD diffractograms of J09951 , J09952 and J09953 at 32° 29 which represents the presence of NaCI within the sample at a higher ratio compared to batch J09899. Full characterization is summarized in Table 16 and Fig. 11.

[0332] Table 16 Characterisation data for PCLX-001 (J09951 , J09952 & J09953)

[0333] Trial Salt Breaking Experiments of PCLX-001

[0334] Procedure

[0335] PCLX-001 (J09899,HCI Pattern 1 , 50 mg ± 1 mg) was weighed into 5 x 4 ml vials and was treated with increasing volumes (20 vol, 30 vol, 40 vol, 50 vol) of relevant solvent (ethanol, acetone, water, DCM, and ethyl acetate) either until the material fullydissolved or until a maximum of 50 vol (2.5 ml) had been added. After each addition of solvent, the vials were stirred at room temperature (RT) for 5 mins and if dissolution did not occur, was heated to 50 °C and stirred for a further 5 mins. Each vial was allowed to stand at RT for 5 mins before the addition of a new aliquot of solvent.

[0336] After the assessment was complete, 1 eq (119.2 pl, relative to Cl ions calculated from IC, added as a 1 M stock solution in water) of NaOH was added at 50 °C and the samples were slowly cooled to 5 °C at 0.1 °C / min. All solids were isolated by filtration and dried under suction. The DCM system produced a yellow solution. This system was washed with 1 portion of water and the organic layer was separated, and then dried in the vacuum oven at RT. All isolated solids were analysed by XRPD.

[0337] Results and Discussion

[0338] No dissolution was observed in four out of five solvents trialled for the salt breaking experiments of PCLX-001 (J09899) and yellow suspensions were formed in all cases. Ethanol was the only exception were dissolution was observed at 50°C in 50 volumes and a colourless solution was formed. Upon addition of 1 equivalent of NaOH relative to chloride ions calculated from IC analysis on the J09899 material, the sample in ethanol formed a yellow solution and all remaining samples retained a yellow suspension.

[0339] After cooling to 5°C, the sample in DCM formed a clear yellow solution whereas all remaining samples formed a light-yellow suspension. The sample in DCM was selected to undergo further treatment and was separated via a separating funnel. The organic layer was dried in a vacuum oven at RT overnight to form a new solid, assigned Free Form Pattern 1 in later characterisation stage. Further treatment was also performed on the sample in ethyl acetate where the sample was filtered and dried under suction and yielded a new pattern, denoted as “Mixed Salt” as the IC analysis revealed evidence of chloride ion to be present. See Table 18 and Fig. 12A to Fig. 14.

[0340] Table 18 Characterisation of salt breaking experiments of J09899

[0341] Characterisation was carried out on the new samples obtained from the salt breaking trials on J09899. XRPD analysis (see Fig. 12A) on the sample obtained from DCM showed the formation of a new pattern, later assigned Free Form Pattern 1 . This was also due to a shift upfield being observed in the1H-NMR spectra of the sample, suggesting the formation of a parent form (see Fig. 12B). In addition, IC analysis showed no anions or cations being observed, confirming the generation of Free Form Pattern 1. High purity was also retained in Free Form Pattern 1 at 96.26% (see Fig. 13).

[0342] XRPD analysis (see Fig. 12A) also confirmed the formation of a new solid from ethyl acetate denoted as a “Mixed Salt” due to IC analysis observing 1.3 molar equivalents of chloride and 1 .4 molar equivalents of sodium to be present.1H-NMR analysis (see Fig. 12B) also showed the shift upfield and a high purity being retained at 97.8% (see Fig. 14). The suggestion was that the NMR shifts eliminated zwitterion presence.

[0343] Scale-up Formation of Parent / Free Form from J09899

[0344] Procedures

[0345] Attempt 1 - 1g Scale-up

[0346] PCLX-001 (J09899, HCI Pattern 1 , 1g ± 10mg) was weighed into a 20 ml vial and transferred to a 100 ml round bottom flask which was treated with 50 volumes (50 ml) of DCM at 50 °C to form a yellow suspension. This was followed by the addition of 1 eq. of NaOH (relative to Cl ions calculated from IC, added as a 1M solution in water) at 50 °C and the sample was slowly cooled to 5 °C at 0.1 °C / min to form pale yellow solid. All isolated solids were analysed by XRPD. Sample ID: EG- 1826- 12-01. Results: XRPD analysis revealed the sample was consistent with the “mixed salt” pattern as observed in the trial experiments for salt breaking using ethyl acetate as a solvent.

[0347] Attempt 2 - 1g Scale-up

[0348] PCLX-001 (J09899, HCI Pattern 1 , 1g ± 10mg) was weighed into a 20 ml vial and transferred to a 100 ml round bottom flask which was treated with 50 volumes (50 ml) of DCM at 30 °C to form a yellow suspension. This was followed by the addition of 1 eq. of NaOH (2.4 ml_, relative to Cl ions calculated from IC, added as a 1M solution in water) at 30 °C and left to stir for 1 hour to a form yellow solution.

[0349] Water (50ml) was added to produce separate aqueous and organic layers. The organic layer was removed using a separating funnel and magnesium sulfate was added as a drying agent. The aqueous layer was washed with DCM to remove any remaining organic layer. The organic layer was filtered from magnesium sulfate and the remaining solution was dried in a vacuum over at RT overnight. The isolated solid was analysed by XRPD. Sample ID: EG-1826-12-02

[0350] Attempt 3 - 200 mg Scale-up

[0351] PCLX-001 (J09899, HCI Pattern 1 , 200 mg ± 5 mg) was weighed into a 20 ml vial and was treated with 50 volumes (10 ml) of DCM at 30 °C to form a yellow suspension. This was followed by the addition of 1 eq. of NaOH (447 pl, relative to Cl ions calculated from IC, added as a 1 M solution in water) at 30 °C and left to stir for 1 hour to a form yellow solution.

[0352] Water (10 ml) was added to produce separate aqueous and organic layers. The organic layer was removed using a separating funnel and magnesium sulfate was added as a drying agent. The aqueous layer was washed with DCM to remove any remaining organic layer. The organic layer was filtered from magnesium sulfate after the magnesium sulfate had clumped to indicate the drying end point and the remaining solution was dried in a vacuum over at RT overnight. All isolated solids were analysed by XRPD. Sample ID: EG- 1826-22-01 ^05.

[0353] Attempt 3 - Repeat of 1g Scale-up

[0354] PCLX-001 (J09899, HCI Pattern 1 , 1g ± 10mg) was weighed into a 20 ml vial and transferred to a 100 ml round bottom flask which was treated with 50 volumes (50 ml) of DCM at 30 °C to form a yellow suspension. This was followed by the addition of 1 eq. of NaOH (2.4 mL, relative to Cl ions calculated from IC, added as a 1M solution in water) at 30 °C and left to stir for 1 hour to a form yellow solution.

[0355] Water (50ml) was added to produce separate aqueous and organic layers. The organic layer was removed using a separating funnel and magnesium sulfate was added as a drying agent. The aqueous layer was washed with DCM to remove any remaining organic layer. The organic layer was filtered from magnesium sulfate after the magnesium sulfate had clumped to indicate the drying end point and the remaining solutionwas dried in a vacuum over at RT overnight. All isolated solids were analysed by XRPD. Sample ID: EG-1826-25-01 .

[0356] Results and Discussion

[0357] Scale-up of Free Form Pattern 1 was successful on both 200 mg scale and 1 g scale and confirmed by XRPD. Full characterisation was performed on 1 g scale-up of Parent / Free Form Pattern 1 (EG-1826-12-02) and is summarised in Table 19.

[0358] Table 19 Characterisation of scale-up formation of parent form

[0359] 1H-NMR analysis (Fig. 15) further confirmed the formation of Free Form Pattern 1 and retained a high purity at 96.1 % (Fig. 17). Thermal analysis (Fig. 16) showed a weight loss of 0.5 % w / w from 50 °C to 100 °C with degradation at 250 °C (onset) and a large, broad endotherm at between 50 °C and 170 °C (Tpeak = 169.6 °C, 74 J / g). Static stability experiments showed no visible change in the appearance of the solid or XRPD pattern and retained high purity at elevated storage conditions confirming stability at elevated temperature and humidity. IC analysis showed the presence of 0.1 molar equivalents of sodium in the sample which could be eliminated in an extra washing step.

[0360] XRPD analysis was used to confirm other scale-up processes on Free Form Pattern 1 using EG-1865-12-02 as the main reference. XRPD overlays of successful scale- up of Free Form Pattern 1 are shown in Fig. 18 and Fig. 19.

[0361] Solubility Test on Free Form Pattern 1

[0362] List of Solvents

[0363] The list of solvents used in the initial solubility test on Free Form Pattern 1 are summarised in Table 20.

[0364] Table 20 List of solvents for initial solubility assessment

[0365] Procedure 1

[0366] EG-1826-12-02 (Free Form Pattern 1 , 20 mg ± 1 mg) was weighed into 10 x 4ml vials. To each vial, the selected solvent system (Table 20) was added in aliquots (10 vol, 20 vol, 40 vol, 60 vol, 80 vol) until either sample dissolution occurred or a maximum of 80 volumes (1.6ml) had been reached.

[0367] After the addition of each aliquot, the samples were stirred firstly at RT for 5 minutes and if the sample did not dissolve, it was then stirred at 50 °C, 500 rpm on a Polar Bear for a further 5 minutes. If no dissolution occurred, the samples were cooled back to RT before the addition of the next aliquot of solvent. All suspensions remaining after the solubility assessment had 2 eq. of HCI (74.5 pl, added as 1 M stock solution in THF, relative to 20 mg parent / free form) added to each vial at 50 °C. All solids obtained were initially analysed by XRPD.

[0368] Results and Discussion

[0369] The results from the initial solubility test on the parent form are summarised below. This solubility was performed in order to select a suitable solvent for the salt screen and was not intended to constitute a full solubility assessment (a full solubility assessment tabulated in Table 23).

[0370] No dissolution was observed in all solvents except DMSO. DMSO showed extremely high solubility, forming a clear yellow solution upon the addition of 10 volumes of solvent at RT.

[0371] Two molar equivalents of HCI was added to each of the samples following completion of the solubility assessment to assess whether dissolution occurs upon addition of a counter-ion. Dissolution was not achieved in toluene, TBME and heptane, but otherwise was successful for all remaining solvents.

[0372] Extended Solubility Assessment on Free Form Pattern 1

[0373] Procedure

[0374] EG-1826-25-01 (Parent / Free Form from 1g Scale-Up Attempt 3, 10 mg ± 1 mg) was weighed into 22 x HPLC vials. To each vial, the selected solvent system (see Table 22) was added in aliquots (10 vol, 20 vol, 40 vol, 60 vol, 80 vol) until either sample dissolution occurred or a maximum of 80 volumes had been reached.

[0375] After the addition of each aliquot, the samples were stirred firstly at RT for 5 minutes and if the sample did not dissolve, it was then stirred at 50 °C, 500 rpm on a Polar Bear for a further 5 minutes. If no dissolution occurred, the samples were cooled back to RT before the addition of the next aliquot of solvent. All solutions were cooled slowly to 5°C at 0.1 °C / min. All suspensions were matured in a maturation chamber at 25 / 50°C in 4-hour cycles for a maximum of 2 days. All solids obtained were initially analysed by XRPD.

[0376] Table 22 List of solvents for extended solubility assessment

[0377] Results and Discussion

[0378] The results from the extended solubility assessment are summarised in Table 23 - 26 and Fig. 20 to Fig. 29. Only Free Form Pattern 2 was yielded from the extended solubility assessment.5

[0379] Table 23 Observations from extended solubility assessment on EG-1826-25-01(Parent Form) (1 of 2)* = did not dissolve, = did dissolve

[0380] Table 24 Observations from extended solubility assessment on EG-1826-25-01 0 (Parent Form) (2 of 2)* = did not dissolve, = did dissolve

[0381] Table 25 Results from extended solubility assessment on EG-1826-25-01 (Parent Form)

[0382] Table 26 Characterisation of Free Form Pattern 2

[0383] Further characterisation was carried out on two of the samples yielded from the solubility assessment. 1 H-NMR on the samples showed the spectra was consistent with the structure with 0.14 equivalents of acetone present in EG- 1826-28-04 and 0.2 equivalents of isopropyl acetate in EG-1826-28-14 (Fig. 24 and Fig. 25).

[0384] Thermal analysis of EG-1826-28-04 (Fig. 22) showed a weight loss of 0.2% between 50-60°C followed by 0.8% weight loss between 200-220°C and degradation onset at 250°C. A large, sharp endotherm at 209.1 °C (onset) of 223 J / g was observed followed by a smaller, sharp recrystallisation peak at 219°C (onset) of 15 J / g. Similar thermal analysis was observed in EG-1826-28-14 (Fig. 23) where a weight loss of 0.2% between 70-80°C was seen followed by a larger weight loss of 4.1% between 190-240°C and degradation onset at 250°C. A large sharp endotherm at 214.6°C (onset) of 93 J / g was observed followed by a sharp recrystallisation peak at 218.6°C (onset) of 63 J / g.

[0385] Static stability experiments showed no visible change in appearance or XRPD pattern when held at elevated storage conditions of 25°C / 97% relative humidity and 40°C / 75% relative humidity for 7 days. The amounts of solvents noted on the NMR spectra of the free form Pattern 2 were residual amount / unbound solvent. This amount could be removed by heating and drying under the vacuum. A control crystallization / optimization of the crystallization method can circumvent the issue of residual solvent content.

[0386] Heating of Free Form Pattern 2 to 220 °C

[0387] The heating of Free Form Pattern 2 was carried out due to a large recrystallisation peak being observed in the TGA analysis during the characterisation of a sample from the solubility assessment. This experiment was performed to assess whether the recrystallisation peak was related to the formation of a new form or the same Free Form Pattern 2.

[0388] EG-1826-28-10 (Free Form Pattern 2, ~5 mg) was heated 220 °C @ 10°C / min via TGA. The solid obtained was reanalysed by XRPD. Sample ID: EG-1826-39- 01.

[0389] Results and Discussion

[0390] XRPD analysis (Fig. 30) on the sample post-heating to 220 °C confirmed that the recrystallisation peak was consistent with Free Form Pattern 2 and it could be concluded that the sample did not convert to another form at higher temperatures.

[0391] CONCLUSIONS

[0392] Characterisation of Compound (I) (PCLX-001 , batch J09899), assigned HCIPattern 1 , confirmed only 1 .3 equivalents of chloride were observed from anion IC, lower than the anticipated 3 equivalents of chloride. In addition, 0.2 equivalents of sodium and 0.1 equivalents of calcium were also observed in cation IC which may have arisen in the manufacturing process of the compound. The characterisation of the additional batches of PCLX-001 (J09951 , J09952 and J09953, assigned HCI Pattern 1) observed a sharp peak was in XRPD diffractogram consistent with the presence of NaCI within the sample at a higher ratio compared to PCLX-001 (J09899).

[0393] Salt breaking trials using sodium hydroxide were conducted on five process solvents. XRPD analysis on the sample obtained from DCM showed the formation of a new pattern which was later assigned Free Form Pattern 1 . This was due to a shift upfield being observed in the 1 H-NMR spectra suggesting the formation of a free form. IC analysis confirmed this, showing no anions or cations being observed, confirming the generation of Free Form Pattern 1. High purity was also retained in Free Form Pattern 1 at 96.3%. Free Form Pattern 1 was also successfully scaled up on 200 mg and 1 g scales.

[0394] The polymorphism screen carried out on Free Form Pattern 1 only yielded one new pattern, denoted Free Form Pattern 2. The heating of Free Form Pattern 2 was also carried out due to a large recrystallisation peak being observed in the DSC analysis during the characterisation of Free Form Pattern 2.

[0395] In conclusion, Free Form Pattern 2 showed the most desirable solid-state properties out of all polymorphs discovered.

[0396] EXAMPLE 2 - Solubility Measurements and Crystallization Assessment on PCLX-001 Free Form Pattern 2

[0397] SUMMARY

[0398] Herein is summarised the solubility measurements and crystallisation development conducted on PCLX-001 Free Form Pattern 2.

[0399] The characterisation of the PCLX-001 Free Form Pattern 2 (J10206) was confirmed to be crystalline by XRPD analysis, with a purity of 98.85%. The1H-NMR and Raman spectra matched the proposed structure of the molecule and no anions or cations were observed.

[0400] Solubility determination experiments were conducted using a gravimetric method. Based on the solubility assessment outcome as described in Example 1 , solubility was measured in DMSO, DMA and NMP. The largest range in solubility between 60 °C and 25 °C was observed in DMSO, with a solubility of 32 mg / ml at 60 °C and solubility of 12.3 mg / ml at 25 °C calculated in DMSO. DMSO was selected as the preferred neat solvent to progress into process development as it was already being used in the crystallisation process.

[0401] In parallel, and to investigate solvent ratios to determine the ratio for the best outcome, arrays of solvent systems were conducted to determine the solubility at two temperatures, 25 °C and 60 °C using gravimetric method. The solvent systems selected to be taken forward for generation of solubility curves and process development were DMSO EtOH:H2O (1 :2) (70:30), DMSO EtOH:H2O (1 :2) (95:5) and DMSO EtOH:H2O (2:1) (50:50).

[0402] Solubility curves were successfully generated in all selected solvent systems. From the solubility curves, it could be seen that the solubility curve in DMSO EtOH:H2O (1 :2) (70:30) and DMSO EtOH:H2O (2:1) (50:50) are significantly lower in comparison to neat DMSO and DMSO EtOH:H2O (1 :2) (95:5). As the addition of antisolvent was shown to have an advantageous effect in increasing the yield of PCLX-001 Free Form Pattern 2, solubility data from DMSO EtOH:H2O (1 :2) (95:5) was inputted into DynoChem software to develop a process.

[0403] Trial seeding crystallisation were conducted using two different procedures. The first trial seeding crystallisation consisted of the addition of 5% anti-solvent prior to the addition of seed at high temperature. Observations during this trial seeding crystallisation saw, upon the addition of 5% anti-solvent, sample crashed out prior to the addition of seed. This was expected from the predictions generated on DynoChem. The other trial seeding crystallisation was conducted with the addition of seed prior to the addition of 10% antisolvent. This successfully crystallised PCLX-001 Free Form Pattern 2 on 50 mg scale with a yield of 84%. This was slightly lower than the predicted 95% yield from DynoChem;however, as that crystallisation process had not been fully optimised, a slightly lower yield was expected.

[0404] A successful scale-up of the selected solvent system DMSO EtOH:H2O (1 :2) (10%) was conducted on a 700 mg scale. Further characterisation confirmed that the material was consistent with PCLX-001 Free Form Pattern 2.

[0405] ABBREVIATIONS

[0406] INSTRUMENT AND METHODOLOGY DETAILS

[0407] X-ray Powder Diffraction (XRPD)

[0408] Bruker AXS D8 Advance

[0409] XRPD diffractograms were collected on a Bruker D8 diffractometer using Cu Ka radiation (40 kV, 40 mA) and a 9-29 goniometer fitted with a Ge monochromator. The incident beam passes through a 2.0 mm divergence slit followed by a 0.2 mm anti-scatter slit and knife edge. The diffracted beam passes through an 8.0 mm receivingslit with 2.5° Soller slits followed by the Lynxeye Detector. The software used for data collection and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA respectively.

[0410] Samples were run under ambient conditions as flat plate specimens using powder. The sample was prepared on a polished, zero-background (510) silicon wafer by gently pressing onto the flat surface or packed into a cut cavity. The sample was rotated in its own plane.

[0411] The details of the standarddata collection method are:• Angular range: 2 to 42° 29• Step size: 0.05° 29• Collection time: 0.5 s / step (total collection time: 6.40 min)

[0412] PANalytical Empyrean

[0413] XRPD diffractograms were collected on a PANalytical Empyrean diffractometer using Cu Ka radiation (45 kV, 40 mA) in transmission geometry. A 0.5° slit, 4 mm mask and 0.04 rad Soller slits with a focusing mirror were used on the incident beam. A PIXcel3Ddetector, placed on the diffracted beam, was fitted with a receiving slit and 0.04 rad Soller slits. The software used for data collection was X’Pert Data Collector using X’Pert Operator Interface. The data were analysed and presented using Diffrac Plus EVA or HighScore Plus.

[0414] Samples were prepared and analysed in either a metal or Millipore 96 wellplate in transmission mode. X-ray transparent film was used between the metal sheets on the metal well-plate and powders (approximately 1 - 2 mg) were used. The Millipore plate was used to isolate and analyse solids from suspensions by adding a small amount of suspension directly to the plate before filtration under a light vacuum.

[0415] The scan mode for the metal plate used the gonio scan axis, whereas a 29 scan was utilised for the Millipore plate.

[0416] The details of the standard screening data collection method are:• Angular range: 2.5 to 32.0° 29• Step size: 0.0130° 29• Collection time: 12.75 s / step (total collection time of 2.07 min)

[0417] Nuclear Magnetic Resonance (NMR)

[0418] Solution State NMR

[0419] 1H NMR and / or13C NMR spectra were collected on a Bruker 400 MHz instrument equipped with an auto-sampler and controlled by a DRX400 console. Samples were prepared in DMSO-c / 6solvent, unless otherwise stated. Automated experiments were acquired using ICON-NMR configuration within Topspin software, using standard Bruker-loaded experiments (1H,13C {1H}, DEPT135). Off-line analysis was performed using ACD Spectrus Processor.

[0420] Differential Scanning Calorimetry (DSC)

[0421] TA Instruments Q2000

[0422] DSC data were collected on a TA Instruments Q2000 equipped with a 50 position auto-sampler. Typically, 0.5 - 3 mg of each sample, in a pin-holed aluminium pan, was heated at 10 °C / min from 25 °C to 300 °C. A purge of dry nitrogen at 50 ml / min was maintained over the sample.

[0423] The instrument control software was Advantage for Q Series and Thermal Advantage and the data were analysed using Universal Analysis or TRIOS.

[0424] Thermo-Gravimetric Analysis (TGA)

[0425] TA Instruments Q500

[0426] TGA data were collected on a TA Instruments Q500 TGA, equipped with a 16 position auto-sampler. Typically, 5 - 10 mg of each sample was loaded onto a pre-tared aluminium DSC pan and heated at 10 °C / min from ambient temperature to 350 °C. A nitrogen purge at 60 ml / min was maintained over the sample.

[0427] The instrument control software was Advantage for Q Series and Thermal Advantage and the data were analysed using Universal Analysis or TRIOS.

[0428] Polarised Light Microscopy (PLM)

[0429] Leica LM / DM Polarised Light Microscope

[0430] Samples were analysed on a Leica LM / DM polarised light microscope with a digital video camera for image capture. A small amount of each sample was placed on a glass slide, with or without immersion oil, and covered with a glass slip. The sample was viewed with appropriate magnification and partially polarised light, coupled to a A false- colour filter. Images were captured using StudioCapture or Image ProPlus software.

[0431] Scanning Electron Microscopy (SEM)

[0432] Data were collected on a Phenom Pro Scanning Electron Microscope. A small quantity of sample was mounted onto an aluminium stub using conducting doublesided adhesive tape. A thin layer of gold was applied using a sputter coater (20 mA, 120 s).

[0433] Chemical Purity Determination by HPLC

[0434] Purity analysis was performed on an Agilent HP1100 / 1 nfinity II 1260 series system equipped with a diode array detector and using OpenLAB software. The full method details are provided below:

[0435] Table 27 HPLC method for chemical purity determinations

[0436] Water Determination by Karl Fischer Titration (KF)

[0437] Water content of each sample was measured on a Metrohm 874 Oven Sample Processor at 150 °C with 851 Titrano Coulometer using Hydranal Coulomat AG oven reagent and nitrogen purge. Weighed solid samples were introduced into a sealed sample vial. Approximately 10 mg of sample was used per titration and duplicate determinations were made. An average of these results is presented unless otherwise stated. Data collection and analysis were performed using Tiamo software.

[0438] Ion Chromatography (IC)

[0439] Data were collected on a Metrohm 930 Compact IC Flex with 858Professional autosampler and 800 Dosino dosage unit monitor, using IC MagicNet software. Accurately weighed samples were prepared as stock solutions in a suitable solvent. Quantification was achieved by comparison with standard solutions of known concentration of the ion being analysed. Analyses were performed in duplicate and an average of the values is given unless otherwise stated.

[0440] Table 28 - IC method for cation chromatography

[0441] Table 29 IC method for anion chromatography

[0442] Raman Spectroscopy

[0443] Data were collected on a Renishaw inVia Qontor. Instrument control, data analysis and presentation software was WiRE.

[0444] Method: excitation source, Aex= 785 nm laser, attenuated appropriately to avoid sample degradation. Raman shift range: 100 - 5000 cm1; Exposure time: 0.02 -10 s; Accumulations: 1 - 3.

[0445] Crystal 16

[0446] A Crystal 16 crystallisation system (Technobis, NL) was used to determine the solubility and metastable zone of the material as a function of temperature. Slurries of the API, in different overall concentrations, were prepared by adding a known amount of solid to a known amount of chilled solvent (between 0.5 and 1 .5 ml) and stirred at 500 rpm using a magnetic bar. The saturation temperature was measured through cycles of heating and cooling from 65 to 23 °C at 0.5 °C / min.

[0447] Upon increasing the temperature, the solid completely dissolved and the suspension became a clear solution such that the light transmission reached its maximum value. This temperature was assigned as the clear point, which was assumed to coincide with the saturation temperature. Then, by cooling the solution at a rate of 0.5 °C / min, the temperature at which particles first formed was detected by a decrease in the light transmission. This was assigned as the cloud point. The points were fitted by a Van’t Hoff equation and the difference between the cloud and the clear points defined the metastable zone width (MSZW) of the system. The instrument control software was Crystallisation Systems and the data were analysed using Crystal Clear and Microsoft Excel.

[0448] CHARACTERISATION OF COMPOUND (I) (PCLX-001, FREE FORM PATTERN 2, BATCH J10206)

[0449] The material (batch J 10206) was characterised using a wide range of techniques to investigate the solid form and chemical properties of PCLX-001 Free Form Pattern 2. A summary of the results is shown in Table 30.

[0450] Table 30 Characterisation data for PCLX-001 Free Form Pattern 2 (J10206)

[0451] The characterisation of the PCLX-001 Free Form Pattern 2 (J10206) was confirmed to be crystalline by XRPD analysis (Fig. 37 and Fig. 38), with a purity of 98.85% (Fig. 42). The1H-NMR (Fig. 39 and Fig. 40) and Raman (Fig. 43 and Fig. 44) spectrums matched the proposed structure of the molecule and no anions or cations were observed. Thermal analysis (Fig. 41) of PCLX-001 Free Form Pattern 2 (J10206) showed the material to have a large endotherm at 220 °C (onset) of 92 J / g followed by a large exotherm at 225 °C (peak) of 65 J / g. A weight loss of 0.6 % w / w between 200 and 240 °C was observed with a degradation of the material at 260 °C (onset). The weight loss relates to the loss of water from the sample as 0.3 % water was observed in the sample by KF analysis. The solubility (pH profiling) analysis showed the compound becomes increasingly soluble in acidic media, where samples suspended in pH < 4.5 became clear.

[0452] SOLUBILITY DETERMINATION AND CONSTRUCTION ON SOLUBILITY CURVE

[0453] Solubility (pH profiling in 7 pH buffered media) on J10206

[0454] Method and Comments

[0455] pH Profiling

[0456] Sufficient sample was suspended in 1 .0 ml media for a maximum anticipated concentration of 10 mg / ml of the free form of the compound. The resulting suspensions were then shaken at 25 °C / 750 rpm for 24 hours. The pH of the sample solutions was periodically checked and adjusted as necessary with 0.2M HCI / NaOH to ensure that the desired pH was maintained (± 0.2) throughout. After equilibration, the appearance was noted and the final pH of the saturated solution was measured. Samples were then filtered through a glass ‘C’ Fiber filter (Particle retention size 1.2 pm), before dilution with the relevant buffer solution as appropriate.

[0457] Quantitation was by HPLC with reference to a standard solution of approximately 0.15 mg / ml. Different volumes of the standard, diluted and undiluted sample solutions were injected. The solubility was calculated using the peak areas determined by integration of the peak found at the same retention time as the principal peak in the standard injection.* 60 and 70 pL of 0.2 and 0.5 M HCI used for adjustment respectively. ** pH adjusted with 0.5 M HCI.*** pH adjusted with 5 and 2 pL of 0.2 M HCI and NaOH respectively.

[0458] Observations

[0459] Samples suspended in pH 1 .2 buffer became clear prior to pH adjustment at 1 hour, while all other samples were either turbid or contained residual solid. After 1 hour pH adjustment, samples suspended in pH 2.0 buffer became clear. All remaining samples were either turbid or contained residual solid up to 24 hours of equilibration.PCLX-001 PYA Stage-H (Free Form) Solubility Results

[0460] Results, Summary and Conclusions

[0461] The compound became increasingly soluble in acidic media, where samples suspended in pH < 4.5 became clear. The samples suspended in pH 1.2 were clear priorto pH adjustment at 1 hour, so concentration quoted from before pH adjustment. Whereas samples suspended in pH 2.0 became clear after pH adjustment at 1 hour so concentration quoted using total volume of media after adjustment. See Fig. 51 , where the two points between pH 0 and 2 are greater than values for solubility.

[0462] Solubility Determination in DMSO, DMA and NMP

[0463] Based on the solubility assessment outcome of Example 1 , solubility was measured in DMSO, DMA and NMP. This was performed at two different temperatures at 60 °C and RT.

[0464] Procedure

[0465] Solubility Determination at 60 °C

[0466] J 10206 (30 mg ± 1 mg) was weighed into 4 x HPLC vials and stirrer bars were added. The sample was dissolved in selected solvent DMSO, DMA, and NMP (1 ml) and was heated to 60 °C on a Polar Bear and stirred for 2-3 hours to allow sample to equilibrate. The sample was then filtered and mother liquor was placed into a pre-weighed vial and evaporated in a vacuum oven overnight at RT. The isolated solid following filtration was analysed by XRPD.

[0467] Solubility Determination at 25 °C

[0468] J 10206 (30 mg ± 1 mg) was weighed into 4 x HPLC vials and stirrer bars were added. The sample was dissolved in selected solvent DMSO, DMA, and NMP (1 ml) and was stirrer at 25 °C on a stirrer bar and stirred for 2-3 hours to allow sample to equilibrate. The sample was then filtered and mother liquor was placed into a pre-weighed vial and evaporated in a vacuum oven overnight at RT. The isolated solid following filtration was analysed by XRPD.

[0469] Results and Discussion

[0470] The largest range in solubility between 60 °C and 25 °C was observed in DMSO - with a solubility of 32 mg / ml at 60 °C and solubility of 12.3 mg / ml at 25 °C calculated in DMSO. A large range in solubility between 60 °C and 25 °C was not observed using DMA and NMP as solvents. DMSO was selected as the preferred neat solvent to progress into process development as it was already being used in the crystallisation process. See Table 31 and Table 32, Fig. 45.

[0471] Table 31 Observations from solubility determination in DMSO, DMA and NMP on PCLX-001 Free Form Pattern 2 (J10206)

[0472] Table 32 Gravimetric solubility results from solubility determination in DMSO, DMA and NMP on PCLX-001 Free Form Pattern 2 (J10206)

[0473] Solubility Determination using EtOH:H2O mixtures as anti-solvent

[0474] In parallel, to investigate solvent ratios to determine the ratio for the best outcome, array of solvent systems were conducted to determine the solubility at two temperatures, 25 °C and 60 °C using gravimetric method. Observations were noted during the solubility array experiments to monitor whether oiling out phenomenon was occurring during the process.

[0475] Procedure

[0476] Solubility Determination at 60 °C

[0477] J 10206 (30 mg ± 1 mg) was weighed into 15 x HPLC vials and stirrer bars were added. The sample was dissolved in selected solvent ratios of DMSO and EtOH:H2O (1 :1), EtOH:H2O (1 :2) or EtOH:H2O (2:1) (1 ml) and was heated to 60 °C on a Polar Bear and stirred for 2-3 hours to allow sample to equilibrate. The sample was then filtered and mother liquor was placed into a pre-weighed vial and evaporated in a vacuum oven overnight at RT. The isolated solid following filtration was analysed by XRPD.

[0478] Solubility Determination at 25 °C

[0479] J10206 (30 mg ± 1 mg) was weighed into 15 x HPLC vials and stirrer bars were added. The sample was dissolved in selected solvent ratios of DMSO and EtOH:H2O (1 :1), EtOH:H2O (1 :2) or EtOH:H2O (2:1) (1 ml) and was heated to 25 °C on a Polar Bear and stirred for 2-3 hours to allow sample to equilibrate. The sample was then filtered and mother liquor was placed into a pre-weighed vial and evaporated in a vacuum oven overnight at RT. The isolated solid following filtration was analysed by XRPD.

[0480] Results and Discussion

[0481] The selected ratios of DMSO to antisolvent to progress forward into process development were DMSO:EtOH:H2O (1 :2) (70:30) and DMSO EtOH:H2O (1 :1) (50:50) due a controlled range in solubility being observed between 60 °C and 25 °C. See Table 33 to Table 36, Fig. 46A and Fig. 46B.

[0482] Table 33 Observations from solubility determination using EtOH:H2O mixtures as anti-solvent on PCLX-001 Free Form Pattern 2 (J10206) at 60 °C

[0483] Table 34 Gravimetric solubility results from solubility determination using EtOH:H2O mixtures as anti-solvent on PCLX-001 Free Form Pattern 2 (J10206) at 60 °C*Filtration required longer time due to thick suspension so solvent cooling may have resulted in lower solubility

[0484] Table 35 Observations from solubility determination using EtOH:H2O mixtures as anti-solvent on PCLX-001 Free Form Pattern 2 (J10206) at 25 °C

[0485] Table 36 Gravimetric solubility results from solubility determination using EtOH:H2O mixtures as anti-solvent on PCLX-001 Free Form Pattern 2 (J10206) at 25 °C

[0486] Repeated Solubility Determination using EtOH:H2O mixtures as anti- solvent

[0487] Previous solubility determination experiments were held for 2-3 hours to allow for sample equilibration (see above, and Tables 33 to 36). Solubility determination experiments were repeated with an extended period of equilibration for 24 hours to obtain more accurate solubility values. These experiments were repeated for extreme ratios of DMSO to anti-solvent.

[0488] Procedure

[0489] Solubility Determination at 60 °C

[0490] J10206 (30 mg ± 1 mg) was weighed into 15 x HPLC vials and stirrer bars were added. The sample was dissolved in selected solvent ratios of DMSO and EtOH:H2O (1 :1), EtOH:H2O (1 :2) or EtOH:H2O (2:1) (1 ml) and was heated to 60 °C on a Polar Bear and stirred for 24 hours to allow sample to equilibrate. The sample was then filtered and mother liquor was placed into a pre-weighed vial and evaporated in a vacuum oven for 72 hours at RT. The isolated solid following filtration was analysed by XRPD.

[0491] Solubility Determination at 25 °C

[0492] J10206 (30 mg ± 1 mg) was weighed into 15 x HPLC vials and stirrer bars were added. The sample was dissolved in selected solvent ratios of DMSO and EtOH:H2O (1 :1), EtOH:H2O (1 :2) or EtOH:H2O (2:1) (1 ml) and was heated to 25 °C on a Polar Bear and stirred for 24 hours to allow sample to equilibrate. The sample was then filtered andmother liquor was placed into a pre-weighed vial and evaporated in a vacuum oven for 72 hours at RT. The isolated solid following filtration was analysed by XRPD.

[0493] Results and Discussion

[0494] Extended period of equilibration for 24 hours showed no significant differences in the solubility values compared to previous solubility determination experiments where sample was left to equilibrate for 2-3 hours. Therefore, it was confirmed that the selected solvent systems DMSO:EtOH:H2O (1 :2) (70:30) and DMSO EtOH:H2O (1 :1) (50:50) were to be taken forward to generate solubility curves for process development. See Table 37 to Table 38, Fig. 47.

[0495] Table 37 Observations from repeat solubility determination using EtOH:H2O mixtures as anti-solvent on PCLX-001 Free Form Pattern 2 (J10206) at 60 and 25 °C

[0496] Table 38 Gravimetric solubility results from repeat solubility determination using EtOH:H2O mixtures as anti-solvent on PCLX-001 Free Form Pattern 2 (J10206) at 60 and 25 °C*Anomaly, experiment was repeated to assess whether initial value was accurate.

[0497] Extended Solubility Determination using EtOH:H2O mixtures as antisolvent

[0498] Procedure

[0499] Solubility Determination in DMSO EtOH:H2O (1 :2) (80:20)

[0500] J10206 was weighed into 2 x HPLC vials and suspended in increasing volumes of DMSO, EtOH:H2O (1 :2) (80:20) up to a maximum of 100 vol (1 ml) at 60 °C. Observations were taken upon each addition of solvent.

[0501] Solubility Determination in DMSO EtOH:H2O (1 :2) (95:5)

[0502] J10206 was weighed into a HPLC vial and suspended in increasing volumes of DMSO, EtOH:H2O (1 :2) (95:5) up to a maximum of 100 vol (1 ml) at 60 °C. Observations were taken upon each addition of solvent.

[0503] Results and Discussion

[0504] No dissolution was observed using DMSO EtOH:H2O (1 :2) (80:20) at up to 100 volumes of solvent with either 10 mg or 3 mg of sample. The sample was observed to be soluble in DMSO EtOH:H2O (1 :2) (95:5) at 80 volumes at 60 °C which would produce a good solubility range at a lower temperature of 25 °C. Therefore, DMSO EtOH:H2O (1 :2) (95:5) was taken forward to generate solubility curves for process development.

[0505] Table 39 Observations from extended solubility determination using EtOH:H2O mixtures as anti-solvent on PCLX-001 Free Form Pattern 2 (J10206)

[0506] Construction of Solubility Curves

[0507] Solubility curves were generated for J10206 (PCLX-001 Free Form Pattern 2) in DMSO, DMSO EtOH:H2O (2:1) (50:50), DMSO EtOH:H2O (1 :2) (70:30), DMSO EtOH:H2O (1 :2) (95:5). Solubility curves were generated using the Crystal 16 to select a solvent for process development (Fig. 31).

[0508] Solubility curves were successfully generated in all selected solvent systems. From the solubility curves, it can be seen that the solubility curve in DMSO EtOH:H2O (1 :2) (70:30) and DMSO EtOH:H2O (2:1) (50:50) are lower in comparison to neat DMSO and DMSO EtOH:H2O (1 :2) (95:5). As the addition of anti-solvent was seen to have an advantageous effect in increasing the yield of PCLX-001 Free Form Pattern 2 (see DynoChem below), solubility data from DMSO EtOH:H2O (1 :2) (95:5) was inputted into DynoChem software to develop a process.

[0509] Differences in solubility on Crystal 16

[0510] During Crystal 16 experiments, a peak was observed at the start of the Crystal 16 experiments which may have indicated conversion or oiling out of the sample. Solubility of the sample was also observed at 55 °C which was unexpected due to solubility of the sample predicted to be observed at 25 °C. Due to the differences in solubility and a peak in the Crystal 16 being observed, one of the Crystal 16 experiments was replicated in a Polar Bear and observations were taken at 10 °C intervals.

[0511] Repeat Crystal 16 Experiments on Polar Bear

[0512] Procedure

[0513] J10206 (3.83 mg) was weighed into a HPLC vial and was suspended inDMSO, EtOH:H2O (2:1) (50:50). The sample was heated gradually from 23 °C to 65 °C at 0.5 °C / min on a Polar Bear to imitate conditions observed in Crystal 16. Photos were taken of the sample at 35, 55 and 65 °C and microscopy was taken of the sample at 45 °C to assess the solubility of the sample upon heating and whether solubility value observed in Crystal 16 is accurate.

[0514] Results and Discussion

[0515] Photos taken at 23, 35, and 55 °C show a light yellow, hazy suspension which suggest the sample has not fully dissolved. Microscopy at 45 °C show crystals up to ~100 pm which confirm the sample has not fully dissolved but in addition, negated the sample oiling out. The sample was observed to have fully dissolved at 65 °C which confirmed the results from Crystal 16 experiments were accurate.

[0516] DynoChem

[0517] On DynoChem, graphs were generated from the solubility data from DMSO EtOH:H2O (1 :2) (95:5) to predict optimal crystallisation process depending on the timepoint of the addition of seed and amount of anti-solvent required. From these graphs, it was recommended to add seed after cooling the crystallisation as indicated by the dotted blue line in graph Fig. 32A. This was due to a gradual, controlled decrease in solubility from 60 °C to 25 °C upon cooling followed by a small drop in solubility once seeds were added at 25 °C. In comparison to the red dotted line, a large, sharp drop in solubility was observed as expected due to presence of anti-solvent causing sample to precipitate out. This was considered not ideal as it lacked control of the crystallisation.

[0518] From the predictions on amount of anti-solvent required for the crystallisation, as depicted in graph Fig. 32B, increasing the anti-solvent past 11% began to reduce return from solubility which meant lower return on yield. Therefore, use antisolvent, up to a maximum of 10% anti-solvent.

[0519] Crystallisation in DMSO

[0520] The solubility data from DMSO and DMSO, EtOH:H2O (1 :2) was inputted into the DynoChem software to gain an understanding on the predictions of the crystallisation of PCLX-001 Free Form Pattern 2 in neat DMSO. As seen in Fig. 33, to generate 200 mg of PCLX-001 Free Form Pattern 2 in 20 vol of DMSO and no anti-solvent, a yield of 84% was expected.

[0521] Crystallisation in DMSO, EtOH:H2O (1 :2) (5% anti-solvent)

[0522] The solubility data from DMSO and DMSO, EtOH:H2O (1 :2) was inputted into the DynoChem software to gain an understanding on the predictions of the crystallisation of PCLX-001 Free Form Pattern 2 in DMSO and 5% anti-solvent EtOH:H2O (1 :2). As seen in Fig. 34, to generate 200 mg of PCLX-001 Free Form Pattern 2 in 20 vol of DMSO and 5% anti-solvent EtOH:H2O (1 :2), a yield of 92 % was expected. This was an increase of 7% in yield compared to neat DMSO which highlighted the advantageous effect of the presence of anti-solvent and that little addition of anti-solvent was required to have a large effect on the increase of yield.

[0523] Crystallisation in DMSO, EtOH:H2O (1 :2) (10% anti-solvent)

[0524] The solubility data from DMSO and DMSO, EtOH:H2O (1 :2) was inputted into the DynoChem software to gain an understanding on the predictions of the crystallisation of PCLX-001 Free Form Pattern 2 in DMSO and 10% anti-solvent EtOH:H2O (1 :2). As seen in Fig. 35, to generate 200 mg of PCLX-001 Free Form Pattern 2 in 20 vol of DMSO and 10% anti-solvent EtOH:H2O (1 :2), a yield of 95 % was expected. This was an increase of 10% in yield compared to neat DMSO and 4% in yield compared to DMSO EtOH:H2O (1 :2) (5%) for an increase in 5% anti-solvent.

[0525] Crystallisation in DMSO, EtOH:H2O (1 :2) (20% anti-solvent)

[0526] The solubility data from DMSO and DMSO, EtOH:H2O (1 :2) was inputted into the DynoChem software to gain an understanding on the predictions of the crystallisation of PCLX-001 Free Form Pattern 2 in DMSO and 20% anti-solvent EtOH:H2O (1 :2). As seen in Fig. 36, to generate 200 mg of PCLX-001 Free Form Pattern 2 in 20 vol of DMSO and 20% anti-solvent EtOH:H2O (1 :2), a yield of 98 % was expected. This was an increase of 2.5% in yield compared to DMSO EtOH:H2O (1 :2) (10%) for an increase in 10% total anti-solvent, suggesting that for the additional volume in solvent, there was little return.

[0527] Trial Seeding Crystallisation on J10206

[0528] Procedure

[0529] J10206 (50 mg ± 1 mg) was weighed into 2 x 4 ml vials and dissolved inDMSO (20 vol, 1 ml) at 65 °C on a Polar Bear. The sample was then cooled to 59 °C to ensure a supersaturation ratio of 1.3 could be obtained.

[0530] Addition of seed prior to addition of 10% anti-solvent

[0531] To the other vial, ~10 mg seed was added at 59 °C and was allowed to stir for 5 mins to assess whether seed sustained. Observations showed seed to sustain and therefore, sample was cooled to 25 °C at 0.5 °C / min and 10% anti-solvent EtOH:H2O (1 :2) (2 vol, 100 pl) was added to the vial. The sample was left to stir at 25 °C for 24 hours to form a light yellow suspension. The sample was filtered via positive pressure under nitrogen and dried in a vacuum oven to calculate both wet and dry yield. Sample ID: EG-1826-62- 02. Sample was analysed by XRPD, NMR, PLM, SEM, and HPLC (Fig. 55, Sample ID EG- 1826-62-02).

[0532] DMSO-Ethanol-Water Purification:

[0533] Crude material (PCLX-001 ; 7.0g) was diluted with DMSO (20.0 Vol.) and gradually heated to 55 ± 5 °C until clean solution for 10 ± 5 min. Added mixture of ethanol (7.0 Vol.) and purified water (7.0 Vol.) to reaction mass at 55±5 °C, and then stirred at 55±5 °C for 10± 5 min . Gradually cooled the reaction mass to 0±5 °C. Stirred the reaction mass for 16 h at 0±5 °C and filtered. The wet material was dried at 50-55°C for 2 h. 7.0 g wastaken for DMSO-Ethanol-Water purification, and isolated 5.6 g of final compound). Purity by HPLC after DMSO-Ethanol-Water purification: 99.58%. No seeding occurred.

[0534] Results and Discussion

[0535] Table 40 Characterisation data for EG-1826-62-02

[0536] The characterisation of EG- 1826-62-02 confirmed the successful production of crystalline PCLX-001 Free Form Pattern 2 (Fig. 48; Table 5.0A’) using the suggested method of the addition of seeds prior to the addition of 10% anti-solvent generated on DynoChem. Further characterisation by1H-NMR analysis confirmed EG-1826-62-02 was consistent with the proposed structure of PCLX-001 Free Form Pattern 2 and SEM images showed the sample to consist of irregular plate particles ~ 250 pm coated with smaller particles. Smaller particles were also observed in the range of 20 - 50 pm with primary particles in the range of 2 - 5 pm.

[0537] The yield of the crystallisation was calculated to be 84% which was slightly lower than the predicted 95%. However, as the crystallisation process was not fully optimised, a lower yield was expected.

[0538] Table 5.0A’ Peaks ± 0.2 degrees two theta of EG-1826-62-02 X-ray powder diffraction pattern

[0539] Scale-up of Selected System (700 mg scale)

[0540] Procedure: Addition of seed prior to addition of 10% anti-solvent

[0541] J10206 (700 mg ± 10 mg) was weighed into 50 ml vessel on EasyMax and dissolved in DMSO (20 vol, 14 ml) at 65 °C to produce a clear yellow solution. The sample was then cooled to 59 °C to ensure a supersaturation ratio of 1.3 was obtained. To the solution, ~40 mg seed was added at 59 °C and was allowed to stir for 5 mins for seed to sustain. The sample was cooled to 25 °C at 0.5 °C / min and 10% anti-solvent EtOH:H2O (1 :2, 2 vol, 1.4 ml) was added to the vial. The sample was left to stir at 25 °C for 24 hours to form a light yellow suspension. The sample was filtered with a Buchner funnel and allowed to dry under vacuum for 20 mins. Sample was analysed by XRPD (Fig. 48, Table 5. OB’), NMR, HPLC (Fig. 49) and PLM (Fig. 50). Sample ID: EG-1826-63-01 .

[0542] The same experiment was repeated to check that the purity profile remained the same. HPLC analysis was applied in this case, at a scale of 200 mg. Sample ID: EG- 1826-63-02. The data obtained for the experiments was consistent, with a similar improvement in impurity was seen in all cases, with a decrease in the impurity at RRT 0.83.

[0543] See FIG. 56 to 58.

[0544] Results and Discussion

[0545] Table 41 Characterisation data for J10206, EG- 1826-63-01 , and EG-1826- 63-02

[0546] The characterisation of EG- 1826-63-01 confirmed the successful production of crystalline PCLX-001 Free Form Pattern 2. Further characterisation by 1 H-NMR analysis confirmed EG-1826-63-01 was consistent with the proposed structure of PCLX-001 Free Form Pattern 2 and PLM images showed the sample to consist of irregular plate particles~ 100 pm coated with smaller particles. Smaller particles were also observed in the range of 20 - 50 pm with primary particles in the range of 2 - 5 pm.

[0547] The yield of the scale-up crystallisation was calculated at 87% which was slightly lower than the predicted value of 95%. However as the crystallisation process was not fully optimised, a lower yield was expected.

[0548] Table 5. OB’ Peaks ± 0.2 degrees two theta of EG-1826-63-01 X-ray powder diffraction pattern

[0549] CONCLUSIONS

[0550] Characterisation of PCLX-001 Free Form Pattern 2 (J10206) was confirmed to be crystalline by XRPD analysis, with a purity of 98.85%. The1H-NMR and Raman spectrums matched the proposed structure of the molecule and no anions or cations were observed. Thermal analysis of PCLX-001 Free Form Pattern 2 (J10206) showed the material to have a large endotherm at 220 °C (onset) of 92 J / g followed by a large exotherm at 225 °C (peak) of 65 J / g. A weight loss of 0.6 % w / w between 200 and 240 °C was observed with a degradation of the material at 260 °C (onset). The weight loss related to the loss of water from the sample as 0.3 % water was observed in the sample by KF analysis. The solubility (pH profiling) analysis showed the compound becomes increasingly soluble in acidic media, where samples suspended in pH < 4.5 became clear.

[0551] Solubility determination experiments were conducted using a gravimetric method. Based on the solubility assessment outcome from Example 1 , solubility was measured in DMSO, DMA and NMP. The largest range in solubility between 60 °C and 25 °C was observed in DMSO, with a solubility of 32 mg / ml at 60 °C and solubility of 12.3 mg / ml at 25 °C calculated in DMSO. DMSO was selected as the preferred neat solvent to progress into process development as it was already being used in the crystallisation process.

[0552] In parallel, and to investigate solvent ratios to determine the ratio for the best outcome, array of solvent systems were conducted to determine the solubility at two temperatures, 25 °C and 60 °C using gravimetric method. The solvent systems selected for generation of solubility curves and process development were DMSO EtOH:H2O (1 :2) (70:30), DMSO EtOH:H2O (1 :2) (95:5) and DMSO EtOH:H2O (2:1) (50:50). Solubility curves were successfully generated in all selected solvent systems. From the solubility curves, it could be seen that the solubility curve in DMSO EtOH:H2O (1 :2) (70:30) and DMSO EtOH:H2O (2:1) (50:50) are lower in comparison to neat DMSO and DMSO EtOH:H2O (1 :2) (95:5). As the addition of anti-solvent was shown to have an advantageous effect in increasing the yield of PCLX-001 Free Form Pattern 2, solubility data from DMSO EtOH:H2O (1 :2) (95:5) was inputted into DynoChem software to develop a process. From the predictions on amount of anti-solvent required for the crystallization, it was noted that increasing the anti-solvent past 11% begins to reduce return from solubility which means lower return on yield. Therefore, it was recommended to use anti-solvent up to a maximum of 10% anti-solvent.

[0553] Trial seeding crystallisation were conducted using two different procedures. The first trial seeding crystallisation consisted of the addition of 5% anti-solvent prior to the addition of seed at high temperature. Observations during this trial seeding crystallisation saw upon the addition of 5% anti-solvent, sample crashed out prior to the addition of seed. This was expected from the predictions generated on DynoChem. The other trial seeding crystallisation was conducted with the addition of seed prior to the addition of 10% antisolvent. This successfully crystallised PCLX-001 Free Form Pattern 2 on 50 mg scale with a yield of 84%. This was slightly lower than the predicted 95% yield from DynoChem however, as this crystallisation process was not fully optimised, a slightly lower yield was expected.

[0554] A successful scale-up of the selected solvent system DMSO EtOH:H2O (1 :2) (10%) was conducted on a 700 mg scale. Further characterisation by1H-NMR analysis confirmed EG-1826-63-01 was consistent with the proposed structure of PCLX-001 Free Form Pattern 2.

[0555] EXAMPLE 3 - Bioequivalence PK Studies of Salt vs. Free Base Form of PCLX-001

[0556] Bioequivalence Studies in Rats and Dogs

[0557] Studies were performed to compare pharmacokinetic characteristics of PCLX-001 in the free base form and in the salt form in Sprague Dawley rats (Study 6902337) and in dogs (Study 6902338). PCLX-001 (salt form; PCLX-001 HCI Pattern 1 , Batch No. PYA / 19 / 001#l-036) and PCLX-001 , PYA Stage-H (free base form; PCLX-001 Free Form Pattern 2, Batch No. PYA / 19 / 001#H- 124) were dosed by oral gavage on a single occasion at 125 mg free base / kg in Ultra Pure Water adjusted to pH 2.5 ± 0.5 to male Sprague Dawley rats (n= 6 / group) and at 4 mg / kg in female beagle dogs (n = 5 / group).These dose levels were the highest non-severely toxic dose (HNSTD) for each species in GLP 4-Week Studies with a 2-Week Recovery period (Rat Study 8002835, Dog Study 8002836). Blood samples for pharmacokinetcs evaluation were collected from all animals at pre-dose and at 30 minutes, 1 , 2, 4, 7, and 24 hours post-dose. PK blood samples were processed to plasma and analyzed for PCLX-001 concentrations using a validated LC- MS / MS method. The lower limit of quantitation (LLOQ) was 5 ng / mL.

[0558] Test Details:

[0559] Comparison PK Study in Rats

[0560] Following administration of the two forms in rats, plasma concentrations of PCLX-001 were quantifiable throughout the 24-hour sampling period. Mean Tmax of PCLX- 001 was observed at 7 hours post-dose for both forms. Mean Cmax was 44900 and 40000 ng / mL for the salt form and the free base respectively. The AUCiastwas 627000 and 557000 ng / mL, for the salt form and the free base respectively. In rats, exposure was similar between the two forms with salt form-to-free base ratio of 1 .12 for Cmax and 1 .13 for AUCiast. Due to limited timepoints following Cmax, the elimination phase was not characterized for any rats. See Fig. 52 and Table 42.

[0561] Table 42: Summary Mean (± SE) Salt vs. Free Base PCLX-001 Pharmacokinetic Parameters in Sprague-Dawley Rat Plasma Following 125 mg / kg Oral Administration of PCLX-001 on Day 1**T 1 / 2 and AUC(o-int) were not calculable due to limited time points following CmaxRcmax = Cmax Salt form / Cmax Free base; RAUC = AUCtiast Salt form / AUCtiast Free base; NA = Not applicable

[0562] Comparison PK Study in Dogs

[0563] Following administration of the salt form in dogs, plasma concentrations of PCLX-001 were quantifiable throughout the 24-hour sampling period except for one animal where concentrations were quantifiable up to 7 hours post-dose. Following administration of free base in dogs, plasma concentrations of PCLX-001 were quantifiable up to 24-hours post-dose in two animals and were quantifiable up to 7 hours post-dose in three animals. Mean Cmax of PCLX-001 were observed at 1 hour postdose for salt form and at 0.5 or 1 hour post-dose for free base. Following Cmax there was a decline and TI / 2, when estimable, ranged from 3.88 to 5.35 hours for salt form, and from 2.39 to 3.94 hours for free base. Following administration t of PCLX-001 , mean Cmax was 582 and 523 ng / mL for salt form and free base respectively, AUCiastwas 3440 and 2610 hr*ng / mL salt form and free base respectively. Exposure in dogs was similar between salt and free base, with salt form-to- free base ratios of 1.1 for Cmax and 1.32 for AUCiast. See Fig. 53 and Table 43.

[0564] Table 43: Summary Mean (± SD) Salt vs. Free Base PCLX-001 Pharmacokinetic Parameters in Beagle Dogs Following 4 mg / kg Oral Administration of PCLX-001 on Day 1

[0565] In conclusion, when administered to rats and to dogs in a single oral dose at the HNSTD for each species, the free base form and the salt form of PCLX-001 exhibited similar pharmacokinetic profiles and so are considered to be bioequivalent.

[0566] PK Study of Free Base PCLX-001 in Mice

[0567] In addition to the bioequivalence studies performed above, a third PK study was performed in Crl:CD1 (ICR) mice in order to generate exposure data for the free base form of PCLX-001 at 35 mg / kg / day, a dose that was efficacious in a previous mouse xenograft studies. PCLX-001 was administered orally to female mice at 35 mg / kg / day up to seven consecutive days. Blood samples for pharmacokinetcs evaluation were collectedfrom all animals at pre-dose and at 30 minutes, 1 , 2, 4, 7, and 24 hours post-dose on Day 1 and on Day 7. Three mice were bled per time point and data is presented as the mean per time point. Cmax of PCLX-001 was observed at 4 hours post-dose on Day 1 and at 1 hour post-dose on Day 7. Due to limited time points following Cmax, the terminal elimination phase was not characterized on Day 1. On Day 7, however, maximum plasma concentrations were followed by a decline and the terminal half-life (T1 / 2) was estimated at 1.68 hours. Following repeated daily administration, exposure on Day 7 was similar compared to Day 1 with an AUCtiast ratio of 0.914 (91.4%). Mean Cmax and AUCtiast in mice were 46900 ng / mL and 470000 hr*ng / ml_, respectively. See Fig. 54 and Table 44.

[0568] Table 44: Summary (± SE) Free Base PCLX-001 Pharmacokinetic Parameters in Female Mouse Plasma Following 35 mg / kg Oral Administration of PCLX- 001 on Days 1 and 7NA = Not applicable; NC = Not calculatedRAUC=Day 7 Day

[0569] The embodiments described herein are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[0570] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0571] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:1 . A crystalline form of Compound (I)said crystalline form being characterized by an X-ray powder diffraction pattern as shown in FIG. 37.

2. A crystalline form of Compound (I)said crystalline form being characterized by an X-ray powder diffraction pattern having peaks at 10.2, 11 .4, and 20.5 degrees two theta ± 0.2 degrees two theta.

3. The crystalline form of claim 2, wherein said crystalline form is further characterized by the X-ray powder diffraction pattern having peaks at 14.6, 22.9, 26.1 , and 31.0 degrees two theta ± 0.2 degrees two theta.

4. The crystalline form of claim 2 or 3, wherein said crystalline form is further characterized by the X-ray powder diffraction pattern having peaks at 18.6, 21.4, 23.4, and 33.5 degrees two theta ± 0.2 degrees two theta.

5. A crystalline form of Compound (I)said crystalline form being characterized by an X-ray powder diffraction pattern having peaks substantially as provided in Table 1 .0’ ± 0.2 degrees two theta.

6. The crystalline form of any one of claims 1 to 5, wherein said crystalline form is characterized by a DSC thermogram obtained using a heating rate of 10 °C / min comprising an endothermic event with an onset temperature of 220.2 °C ± 0.2 °C.

7. The crystalline form of any one of claims 1 to 5, wherein said crystalline form is characterized by a DSC thermogram substantially as shown in FIG. 41.

8. The crystalline form of any one of claims 1 to 7, wherein said crystalline form is characterized by a Raman spectrum comprising wavenumber values (cm1) 103.2 cm1, and 993.0 cm1, and 1602.8 cm1± 0.2 cm1.

9. The crystalline form of claim 8, wherein said crystalline form is further characterized by the Raman spectrum comprising wavenumber values (cm1) of 126.5 cm1, 144.9 cm1, 227.3 cm1, 456.1 cm1, 1043.5 cm1, 1164.7 cm1, and 1582.5 cm1± 0.2 cm1.

10. A pharmaceutical composition, comprising: a crystalline form of Compound (I) of any one of claims 1 to 9; and a pharmaceutically acceptable adjuvant, diluent, carrier, or vehicle.

11. A method of preparing a crystalline form of Compound (I) of any one of claims 1 to 9, comprising the steps of: adding a HCI form of Compound (I) to a first organic solvent to form a first mixture; adding a base to the first mixture to form a first solution; and isolating from the first solution a free form of Compound (I); adding the free form of Compound (I) to a second organic solvent to form a second mixture;maturing the second mixture to form a third mixture; and isolating from the third mixture the crystalline form of Compound (I).

12. The method of claim 11 , wherein adding a HCI form of Compound (I) to a first organic solvent further comprises heating the first mixture to a temperature of about 30 °C.

13. The method of claim 11 or 12, wherein adding a base to the first mixture to form a first solution further comprises: maintaining the first solution at a temperature of about 30 °C; and stirring the first solution for about 1 hour.

14. The method of any one of claims 11 to 13, wherein adding the free form of Compound (I) to a second organic solvent to form a second mixture further comprises: stirring the second mixture at ambient temperature; optionally ramping up the temperature from ambient temperature to about 50 °C and then ramping down the temperature from about 50 °C to ambient temperature, and optionally adding additional organic solvent to the second mixture until the free form of Compound (I) dissolves in the second organic solvent; and ramping down the temperature from about 50 °C to about 5 °C at a rate of about 0.1 °C / min.

15. The method of any one of claims 11 to 14, wherein maturing the second mixture to form a third mixture further comprises maturing at 25 / 50°C in 4-hour cycles.

16. The method of any one of claims 11 to 13, wherein the first organic solvent is selected from the group consisting of ethanol, acetone, water, DCM, or ethyl acetate.

17. The method of claim 16, wherein the first organic solvent is DCM.

18. The method of any one of claims 11 to 17, wherein the base is NaOH.

19. The method of any one of claims 11 to 18, wherein the second organic solvent is selected from the group consisting of 2-propanol; 2-methyl THF; acetone; acetonitrile; DMSO; N,N'-dimethylacetamide; MeOH:water (90:10 v / v); ethyl acetate; ethanol:water (50:50 v / v); isopropyl acetate; isobutanol; tert-butyl methyl ether; THF; and water.

20. The method of any one of claims 11 to 19, further comprising filtering insoluble particulates from the first solution before isolating from the first solution a free form of Compound (I).21 . A method of recrystallizing the crystalline form of Compound (I) of any one of claims 1 to 9, or the crystalline form of Compound (I) prepared by the method of any one of claims 11 to 20, comprising the steps of: dissolving the crystalline form of Compound (I) in an organic solvent at a first temperature to form a first solution; ramping down the temperature from the first temperature to a second temperature; adding a seed of the crystalline form of Compound (I) to the first solution to form a first mixture; ramping down the temperature from the second temperature to a third temperature; adding an anti-solvent to the first mixture to form a second mixture; maturing the second mixture for about 24 hours to form a third mixture; and isolating the recrystallized, crystalline form of Compound (I) from the third mixture.

22. The method of claim 21 , wherein the organic solvent is selected from the group consisting of DMSO; DMA; and NMP.

23. The method of claim 21 or 22, wherein the organic solvent is DMSO.

24. The method of any one of claims 21 to 23, wherein the anti-solvent is selected from the group consisting of EtOH:H2O (1 :1); EtOH:H2O (1 :2); and EtOH:H2O (2:1).

25. The method of any one of claims 21 to 24, wherein the organic solvent is DMSO and the anti-solvent is EtOH:H2O (1 :1), EtOH:H2O (1 :2), or EtOH:H2O (2:1).

26. The method of claim 25, wherein the organic solvent is DMSO and the anti-solvent is EtOH:H2O (1 :2).

27. The method of claim 26, wherein the anti-solvent is added at a volume that is about 10% to about 20% the volume of organic solvent.

28. The method of claim 27, wherein the anti-solvent is added at a volume that is about 10% the volume of organic solvent.

29. The method of any one of claims 21 to 28, wherein the first temperature is about 65 °C.

30. The method of any one of claims 21 to 29, wherein the second temperature is about 59 °C.31 . The method of any one of claims 21 to 30, wherein the third temperature is about 25°C.

32. The method of any one of claims 21 to 31 , wherein ramping down the temperature from the second temperature to the third temperature comprises a ramp down of 0.5 °C / min.

33. The method of any one of claims 21 to 32, further comprising filtering insoluble particulates from the first solution before ramping down the temperature from the first temperature to a second temperature.

34. A method of treating a subject having a cancer comprising: administering to said subject a crystalline form of Compound (I) of any one of claims 1 to 9, or the pharmaceutical composition of claim 10.

35. A method of treating a subject having a cancer deficient in NMT2 comprising administering to said subject a crystalline form of Compound (I) of any one of claims 1 to 9, or the pharmaceutical composition of claim 10.

36. The method of claim 34 or 35, wherein the cancer is a lymphoma.

37. The method of claim 36, wherein the lymphoma is B cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom's, MALT-type / monocytoid B cell lymphoma, or Burkitt’s lymphoma.

38. The method of claim 34 or 35, wherein the cancer is anaplastic large cell lymphoma, acute myeloid leukemia, Blast Phase Chronic Myeloid Leukaemia, Burkitt’s Lymphoma, Plasma Cell Myeloma, Intestinal Adenocarcinoma, Lung mixed Adenosquamous Carcinoma, Lung Small Cell Carcinoma, Lung, Oesophagus Squamous Cell Carcinoma, Bone, Breast Ductal Carcinoma, Stomach Diffuse Adenocarcinoma, Thyroid Medullary Carcinoma, urinary Tract Transitional Cell Carcinoma, myeloma, ovarian clear cell carcinoma, transition cellcarcinoma (ureter and bladder cancer), chronic myelogenous leukemia (CML), lymphoma- CLL, breast carcinoma, colorectal adenocarcinoma, pancreas adenocarcinoma, ovarian carcinoma, non-small cell lunch carcinoma, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, cholangiocarcinoma (bile duct cancer), gallbladder cancer, liver cancer(s), or esophageal squamous carcinoma.

39. The method of any one of claims 34 to 38, wherein the subject is a child, adolescent, adult, or elderly.

40. The method of any one of claims 34 to 39, wherein the subject is male, or female.41 . The method of any one of claims 34 to 40, wherein the subject is a human.

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