Crystal form of N-heteroaryl sulfonamide, composition and preparation method thereof

Abstract

The invention relates to a crystal form of N-heteroaryl sulfonamide, namely, 2, 6-dichloro-N-(3-isobutyl-1, 5-dimethyl-1H-pyrazol-4-yl)-4-(2-(piperazin-1-yl) pyridine-4-yl) benzenesulfonamide, and a pharmaceutical composition of the N-heteroaryl sulfonamide. The present invention relates to crystalline forms, methods of making the crystalline forms, and methods of using the crystalline forms in the treatment of disease.

Description

Technical Field

[0001] The present disclosure generally relates to crystalline forms of N-heteroarylsulfonamides, namely 2,6-dichloro-N-(3-isobutyl-1,5-dimethyl-1H-pyrazol-4-yl)-4-(2-(piperazin-1-yl)pyridin-4-yl)benzenesulfonamide, pharmaceutical compositions thereof, methods of preparing the crystalline forms, and methods of using the crystalline forms in the treatment of diseases. Background Art

[0002] The discovery and characterization of new crystalline forms of pharmaceutical compounds is important for the research and development of new drugs. This is because different crystalline forms of the same pharmaceutical compound can have different physicochemical properties, such as solubility, physical / chemical stability, stability during transportation and storage, ease of formulation, bioavailability, and / or pharmacokinetic properties.

[0003] A new crystal form of a pharmaceutical compound arises when the compound crystallizes into a different internal structure, such as a different lattice arrangement. This different internal structure (i.e., new crystal form) arises because the system tends to a thermodynamically stable (i.e., low-energy) state.

[0004] Because of the ongoing need for pharmaceutical compounds that exhibit improved pharmacokinetic properties, bioavailability, and / or improved stability, etc., there remains a need for new crystalline forms of pharmaceutical compounds. Summary of the Invention

[0005] In one aspect of the present disclosure, there is provided a compound (I)

[0006] The crystal form,

[0007] The crystalline form is characterized by Figure 37 The X-ray powder diffraction pattern is shown.

[0008] In another aspect of the present disclosure, there is provided a compound (I)

[0009] The crystal form,

[0010] The crystalline form is characterized by an X-ray powder diffraction pattern having peaks at 10.2 degrees, 11.4 degrees, and 20.5 degrees 2Θ ± 0.2 degrees 2Θ.

[0011] In one embodiment of the present disclosure, a crystalline form is provided, wherein the crystalline form is further characterized by an X-ray powder diffraction pattern having peaks at 14.6, 22.9, 26.1, and 31.0 degrees 2θ ± 0.2 degrees 2θ. In another embodiment, the crystalline form is further characterized by an X-ray powder diffraction pattern having peaks at 18.6, 21.4, 23.4, and 33.5 degrees 2θ ± 0.2 degrees 2θ.

[0012] In another aspect of the present disclosure, there is provided a compound (I)

[0013] The crystal form,

[0014] The crystalline form is characterized by having an X-ray powder diffraction pattern with peaks at ±0.2 degrees 2Θ substantially as provided in Table 1.0' below.

[0015] Table 1.0'

[0016]

[0017]

[0018] In another embodiment of the present disclosure, a crystalline form is provided, wherein the crystalline form is characterized by a DSC thermogram obtained using a heating rate of 10°C / min, the DSC thermogram comprising an endothermic event with an onset temperature of 220.2°C ± 0.2°C. In another embodiment, the crystalline form is characterized by being substantially as Figure 41 DSC thermogram shown.

[0019] In one embodiment of the present disclosure, a crystalline form is provided, wherein the crystalline form is characterized by comprising 103.2 cm -1 、993.0cm -1 and 1602.8cm -1 ±0.2cm -1 Wavelength value (cm -1 In another embodiment, the crystalline form is further characterized by comprising a Raman spectrum at 126.5 cm -1 、144.9cm -1 、227.3cm -1 、456.1cm -1 、1043.5cm -1 、1164.7cm -1 and 1582.5cm -1 ±0.2cm -1 Wavelength value (cm -1 In another embodiment, the crystalline form is characterized by comprising wave numbers (cm) substantially as provided in Table 2.0' below. -1 )±0.2cm -1 Raman spectrum.

[0020] Table 2.0'

[0021]

[0022]

[0023] In another aspect of the present disclosure, a pharmaceutical composition is provided, comprising a crystalline form of Compound (I) described herein; and a pharmaceutically acceptable adjuvant, diluent, carrier or vehicle.

[0024] In another aspect of the present disclosure, there is provided a method for preparing a crystalline form of Compound (I) as described herein, comprising the following steps:

[0025] adding the HCl form of Compound (I) to a first organic solvent to form a first mixture;

[0026] adding a base to the first mixture to form a first solution; and

[0027] isolating the free form of compound (I) from the first solution;

[0028] adding the free form of compound (I) to a second organic solvent to form a second mixture;

[0029] aging the second mixture to form a third mixture; and

[0030] The crystalline form of Compound (I) is isolated from the third mixture.

[0031] In another embodiment of the present disclosure, a method is provided, wherein adding the HCl form of Compound (I) to the first organic solvent further comprises heating the first mixture to a temperature of about 30° C. In another embodiment of the present disclosure, a method is provided, 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.

[0032] In another embodiment of the present disclosure, a method is provided, wherein adding the free form of compound (I) to a second organic solvent to form a second mixture further comprises:

[0033] stirring the second mixture at ambient temperature;

[0034] Optionally ramping up from ambient temperature to about 50°C and then ramping down from about 50°C to ambient temperature, and

[0035] optionally adding additional organic solvent to the second mixture until the free form of Compound (I) is dissolved in the second organic solvent; and

[0036] The temperature was lowered from about 50°C to about 5°C at a rate of about 0.1°C / min.

[0037] In another embodiment, a method is provided wherein aging the second mixture to form a third mixture further comprises aging at 25 / 50°C with a 4 hour cycle.

[0038] In another embodiment, a method is provided 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, a method is described wherein the first organic solvent is selected from the group consisting of ethanol, acetone, water, or ethyl acetate.

[0039] In another embodiment, a method is provided wherein the base is NaOH.

[0040] In another embodiment, a method is provided, wherein the second organic solvent is selected from the group consisting of 2-propanol; 2-methylTHF; 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, a method is described, wherein the second organic solvent is selected from the group consisting of 2-propanol; 2-methylTHF; 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.

[0041] In another embodiment of the present disclosure, there is provided a method further comprising filtering insoluble particles from the first solution before isolating the free form of Compound (I) from the first solution.

[0042] In another aspect of the present disclosure, there is provided a method for recrystallizing a crystalline form of Compound (I) as described herein, or a crystalline form of Compound (I) prepared by a method described herein, comprising the steps of:

[0043] dissolving the crystalline form of compound (I) in an organic solvent at a first temperature to form a first solution;

[0044] cooling from a first temperature to a second temperature;

[0045] adding seed crystals of the crystalline form of compound (I) to the first solution to form a first mixture;

[0046] cooling from the second temperature to a third temperature;

[0047] adding an antisolvent to the first mixture to form a second mixture;

[0048] aging the second mixture for about 24 hours to form a third mixture; and

[0049] The recrystallized crystalline form of Compound (I) is isolated from the third mixture.

[0050] In another embodiment of the present disclosure, a method is provided 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, a method is described wherein the organic solvent is selected from the group consisting of DMSO; DMA; and NMP. In another example, the organic solvent is DMSO.

[0051] In one embodiment, a method is provided wherein the anti-solvent comprises water, a C1-C3 alcohol, or a combination thereof. In one embodiment, the anti-solvent comprises water, a C1-C3 alcohol, or a combination thereof in an amount ranging from about 100% water to about 100% C1-C3 alcohol, or any combination of water and C1-C3 alcohol from 100% water to 100% C1-C3 alcohol.

[0052] In one embodiment, the C1-C3 alcohol comprises a C1-C3 straight chain or branched chain or cyclic alcohol. In one embodiment, the C1-C3 alcohol comprises methanol, ethanol, n-propanol, branched chain propanol or a combination thereof.

[0053] In another embodiment, a method is provided wherein the antisolvent is selected from the group consisting of ethanol and water. In one embodiment, the antisolvent is selected from the group consisting of ethanol and water, and the amount ranges from about 100% water to about 100% ethanol, or any combination of water and ethanol from 100% water to 100% ethanol. In another embodiment, a method is provided wherein the antisolvent is selected from the group consisting of EtOH:HO (1:1); EtOH:HO (1:2); and EtOH:HO (2:1).

[0054] In another embodiment, a method is provided, wherein the organic solvent is DMSO and the antisolvent is EtOH:H2O (1:1), EtOH:H2O (1:2), or EtOH:H2O (2:1). In another embodiment, the organic solvent is DMSO and the antisolvent is EtOH:H2O (1:2).

[0055] In another embodiment, a method is provided wherein the antisolvent is added at a volume of about 10% to about 20% of the volume of the organic solvent. In another embodiment, the antisolvent is added at a volume of about 10% of the volume of the organic solvent.

[0056] In another embodiment, a method is provided wherein the first temperature is about 65°C.

[0057] In another embodiment, a method is provided wherein the second temperature is about 59°C.

[0058] In another embodiment, a method is provided wherein the third temperature is about 25°C.

[0059] In another embodiment, a method is provided wherein decreasing the temperature from the second temperature to the third temperature comprises decreasing the temperature at a rate of 0.5°C / min.

[0060] In another embodiment, a method is provided, further comprising filtering insoluble particles from the first solution before cooling from the first temperature to the second temperature.

[0061] In another aspect of the present disclosure, provided is a method of treating a subject having cancer, comprising administering to the subject the crystalline form of Compound (I) described herein or the pharmaceutical composition described herein.

[0062] In another aspect of the present disclosure, provided is a method of treating a subject having an NMT2-deficient cancer, comprising administering to the subject a crystalline form of Compound (I) described herein or a pharmaceutical composition described herein.

[0063] In another embodiment of the present disclosure, a method is provided, wherein the cancer is a lymphoma. In another embodiment, the lymphoma is a B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom's, MALT-type / monocytic B-cell lymphoma, or Burkitt's lymphoma.

[0064] In another embodiment, a method is provided, wherein the cancer is anaplastic large cell lymphoma, acute myeloid leukemia, blast phase chronic myeloid leukemia, Burkitt lymphoma, plasma cell myeloma, intestinal adenocarcinoma, mixed adenosquamous lung carcinoma, small cell lung carcinoma, lung carcinoma, esophageal squamous cell carcinoma, bone tumor, breast ductal carcinoma, diffuse gastric adenocarcinoma, medullary thyroid carcinoma, urinary tract transitional cell carcinoma, myeloma, ovarian clear cell carcinoma, transitional cell carcinoma (ureter and bladder), chronic myeloid leukemia (CML), lymphoma-CLL, breast cancer, colorectal adenocarcinoma, pancreatic cancer, ovarian cancer, non-small cell lung cancer, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, hepatobiliary carcinoma (bile duct cancer), gallbladder cancer, liver cancer, or esophageal squamous cell carcinoma.

[0065] In another embodiment, a method is provided wherein the subject is a child, teenager, adult, or elderly. In another embodiment, the subject is male or female. In another embodiment, the subject is human. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Embodiments of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings.

[0067] Figure 1 Depicted is the XRPD diffractogram of PCLX-001 (J09899, HCl pattern 1).

[0068] Figure 2 Depicts PCLX-001 (J09899, HCl Model 1) 1 H-NMR spectrum.

[0069] Figure 3 Depicted is the HPLC chromatogram of PCLX-001 (J09899, HCl mode 1).

[0070] Figure 4 Thermal analysis of PCLX-001 (J09899, HCl Mode 1) is depicted.

[0071] Figure 5 Depicted is the GVS isotherm of PCLX-001 (J09899, HCl mode 1).

[0072] Figure 6 Depicted are the GVS kinetics of PCLX-001 (J09899, HCl model 1).

[0073] Figure 7 Depicted are XRPD overlays of PCLX-001 (J09899, HCl pattern 1) before and after storage at 25°C / 97% RH and 40°C / 75% RH.

[0074] Figure 8 Depicted is the HPLC chromatogram of PCLX-001 (J09899, HCl mode 1) after storage at 25°C / 97% RH.

[0075] Figure 9 Depicted is the HPLC chromatogram of PCLX-001 (J09899, HCl mode 1) after storage at 40°C / 75% RH.

[0076] Figure 10 Depicted are overlays of XRPD patterns of PCLX-001 (J09899, HCl pattern 1) before and after GVS analysis.

[0077] Figure 11The XRPD diffractograms of PCLX-001 (J09898, J09899, J09951, J09952, and J09953, HCl pattern 1) are depicted.

[0078] Figure 12A Depicted are the XRPD diffractograms of the salt breaking experiments (EG-1826-10-04; free form pattern 1 and EG-1826-10-05; mixed salt) and PCLX-001 (J09899 and J09898).

[0079] Figure 12B Describes the salt breaking experiments (EG-1826-10-04 and EG-1826-10-05) and PCLX-001 (J09899) 1 H-NMR overlay.

[0080] Figure 13 Depicted is the HPLC chromatogram of EG-1826-10-04.

[0081] Figure 14 Depicted is the HPLC chromatogram of EG-1826-10-05.

[0082] Figure 15 Depicting EG-1826-10-04 and EG-1826-12-02 1 H-NMR overlay.

[0083] Figure 16 Thermal analysis of EG-1826-12-02 is depicted.

[0084] Figure 17 Depicted is the HPLC chromatogram of EG-1826-12-02.

[0085] Figure 18 The XRPD diffraction patterns of the scaled-up salt breaking experiments (EG-1826-10-04, EG-1826-12-02, and EG-1826-22-01→05) and PCLX-001 (J09899) are depicted.

[0086] Figure 19 XRPD diffractograms of the free form Pattern 1 (EG-1826-12-02 and EG-1826-25-01) are depicted.

[0087] Figure 20 Depicted are the 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).

[0088] Figure 21 Depicted are the 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).

[0089] Figure 22 Thermal analysis of EG-1826-28-04 is depicted.

[0090] Figure 23 Thermal analysis of EG-1826-28-14 is depicted.

[0091] Figure 24 Depicting EG-1826-28-04 1 H-NMR.

[0092] Figure 25 Depicting EG-1826-28-14 1 H-NMR.

[0093] Figure 26 Depicted is a SEM image of EG-1826-28-09.

[0094] Figure 27 Depicted is a SEM image of EG-1826-28-14.

[0095] Figure 28 Depicted is a PLM image of EG-1826-28-09.

[0096] Figure 29 Depicted are XRPD overlays of EG-1826-28-21 and EG-1826-28-19 before storage and after storage of EG-1826-28-21 at 40°C / 75% RH and EG-1826-28-19 at 25°C / 97% RH.

[0097] Figure 30 Depicted are XRPD diffractograms of the parent form (EG-1826-25-01), the free form, Pattern 2, before heating to 220°C (EG-1826-28-10), and after heating to 220°C (EG-1826-39-01).

[0098] Figure 31 Solubility curves of PCLX-001 free form Model 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) are depicted.

[0099] FIG32 depicts (A) solubility predictions as a function of the time of seed addition and (B) solubility predictions as a function of increasing antisolvent volume.

[0100] Figure 33 Depicted is the DynoChem prediction of PCLX-001 free form Model 1 in DMSO.

[0101] Figure 34 Depicted are DynoChem predictions of PCLX-001 free form pattern 1 in DMSO and antisolvent EtOH:H2O (1:2) (5%).

[0102] Figure 35 Depicted are DynoChem predictions of PCLX-001 free form pattern 1 in DMSO and antisolvent EtOH:H2O (1:2) (10%).

[0103] Figure 36 Depicted are DynoChem predictions of PCLX-001 free form pattern 1 in DMSO and antisolvent EtOH:H2O (1:2) (20%).

[0104] Figure 37 The XRPD of J10206 (PCLX-001 free form pattern 2) is depicted.

[0105] Figure 38 Depicted are overlays of XRPD patterns of J10206 and PCLX-001 free form Pattern 2 reference (EG-1826-28-11).

[0106] Figure 39 Describes J10206 (PCLX-001 free form model 2) 1 H-NMR spectrum.

[0107] Figure 40 Depicts the free form of J10206 and PCLX-001 Model 2 reference (EG-1826-28-11) 1 H-NMR overlay.

[0108] Figure 41 Thermal analysis of J10206 (PCLX-001 free form mode 2) is depicted.

[0109] Figure 42 Depicted is the HPLC chromatogram of J10206 (PCLX-001 free form pattern 2).

[0110] Figure 43 Depicted is the Raman spectrum of J10206 (PCLX-001 free form mode 2).

[0111] Figure 44 Depicted is a close-up Raman spectrum of J10206 (PCLX-001 free form mode 2).

[0112] Figure 45 Depicted are overlays of the XRPD patterns of J10206 and EG-1826-48-07.

[0113] Figure 46A Depicted are overlays of XRPD patterns of J10206, EG-1826-49-XX (XX=01→15) determined for solubility at 60°C.

[0114] Figure 46B Depicted are overlays of XRPD patterns of J10206, EG-1826-50-XX (XX=01→15) determined for solubility at 25°C.

[0115] Figure 47 Depicted are overlays of XRPD patterns of J10206, EG-1826-53-XX (XX=01→12) from replicate solubility measurements.

[0116] Figure 48 Depicted are overlays of XRPD patterns of EG-1826-62-02 and EG-1826-63-01.

[0117] Figure 49 Depicted is the HPLC chromatogram of EG-1826-63-01.

[0118] Figure 50 Depicted is a PLM image of EG-1826-63-01.

[0119] Figure 51 A solubility versus pH graph depicting the solubility pH curve of J10206 in a 7 pH buffer medium.

[0120] Figure 52 Depicted are summary plasma concentrations (± SD) of PCLX-001 salt form (Test Item 1) and free base (Test Item 2) in male Sprague-Dawley rats on day 1 after oral dosing.

[0121] Figure 53 Depicted are summary (±SD) plasma concentrations of PCLX-001 salt form (Test Item 1) and free base (Test Item 2) in female beagle dogs on day 1 following oral dosing.

[0122] Figure 54 Depicted are summary (±SD) of plasma free base PCLX-001 concentrations in female mice on day 1 following oral administration of PCLX-001.

[0123] Figure 55 Depicted is the HPLC chromatogram of EG-1826-62-02.

[0124] Figure 56 Depicted is the HPLC chromatogram of EG-1826-63-01.

[0125] Figure 57 Depicted is the HPLC chromatogram of EG-1826-63-02.

[0126] Figure 58 Depicted is the HPLC chromatogram of J10206 (PCLX-001 free form pattern 2). DETAILED DESCRIPTION

[0127] definition

[0128] 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.

[0129] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0130] As used herein, the term "comprising" will be understood to mean that the following list is non-exhaustive and may or may not include any other suitable items, such as one or more additional features, components and / or ingredients, as appropriate.

[0131] As used herein, the term "cancer" refers to various conditions caused by the abnormal, uncontrolled growth of cells. Cells that can cause cancer, called "cancer cells," have characteristic properties, such as uncontrolled proliferation, immortalization, metastatic potential, rapid growth and proliferation rate, and / or certain typical morphological features. Cancer cells can be in the form of tumors, but such cells can also be present only in the subject's body, or can be non-tumorigenic cancer cells. Cancer can be detected by any of many methods, including but not limited to detecting the presence of one or more tumors (e.g., by clinical or radiological methods), examining cells in the tumor or from another biological sample (e.g., from a tissue biopsy), measuring blood markers indicating cancer, and detecting the genotype of the cancer. However, a negative result in one or more of the above-mentioned detection methods does not necessarily indicate the absence of cancer. For example, a patient who demonstrates complete response to cancer treatment may still have cancer, as evidenced by subsequent recurrence.

[0132] As used herein, the term "subject" refers to an animal and can include, for example, domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animals. In a specific example, the subject is a human.

[0133] The terms "treatment" or "treat" as used herein refer to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, alleviation of the extent of the disease, stabilization of the disease state (i.e., no worsening), prevention of the spread of the disease, delaying or slowing the progression of the disease, improvement or alleviation of the disease state, reduction and alleviation of disease recurrence (whether partial or complete), whether detectable or undetectable. "Treating" and "treatment" may also refer to prolonged survival compared to the expected survival if not treated. "Treating" and "treatment" as used herein also include prophylactic treatments. For example, a subject with early-stage cancer (e.g., early-stage lymphoma) can be treated to prevent its progression, or a subject in remission can be treated with a compound or composition as described herein to prevent its recurrence.

[0134] 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 being sought by a researcher or clinician. This amount can be a "therapeutically effective amount". These terms refer to the amount of the compound and / or composition described herein that treats a subject with a disease or condition after single or multiple dose administration. The attending diagnostician, as a person skilled in the art, can easily determine the effective amount by using known techniques and by observing the results obtained under similar circumstances. When determining the effective amount or dosage, the attending diagnostician considers many factors, including but not limited to: the species of the subject; its size, age and general health; the specific condition, disorder or disease involved; the extent or involvement or severity of the condition, disorder or disease, the response of the individual subject; the specific compound administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; the use of concomitant drugs; and other relevant circumstances.

[0135] The term "pharmaceutically acceptable" as used herein includes compounds, materials, compositions and / or dosage forms (such as unit doses) that are suitable for use in contact with the tissues of subjects, with a reasonable benefit / risk ratio without excessive toxicity, irritation, allergic response or other problems or complications. Each carrier, excipient, etc. is also "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0136] The term "excipient" refers to a pharmacologically inactive ingredient, such as a diluent, lubricant, surfactant, carrier, or the like. Excipients used to prepare pharmaceutical compositions are generally safe, non-toxic, and acceptable for human pharmaceutical use. Reference to an excipient includes both one and more than one such excipient.

[0137] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier including, but not limited to, phosphate-buffered saline, water, emulsions (e.g., 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, preservatives, and the like.

[0138] As used herein, "treatment or dosing regimen" refers to the dosage, frequency of administration, or duration of treatment, with or without the addition of a second drug in combination.

[0139] As used herein, the term "diagnosis" refers to the identification of a molecular and / or pathological state, disease or condition, such as the identification of lymphoma or other types of cancer.

[0140] As used herein, the term "alleviate" refers to the lessening, reduction, or elimination of a condition, disease, disorder, or phenotype, including abnormalities or symptoms.

[0141] Compound (I) (PCLX-001) is an N-heteroarylsulfonamide named 2,6-dichloro-N-(3-isobutyl-1,5-dimethyl-1H-pyrazol-4-yl)-4-(2-(piperazin-1-yl)pyridin-4-yl)benzenesulfonamide.

[0142] As used herein, the term "crystalline form of Compound (I)" refers to the form of Compound (I) (PCLX-001) identified and characterized herein as the free form Form 2 (see Examples 1 and 2).

[0143] As used herein, the term "HCl form of Compound (I)" refers to the form of Compound (I) (PCLX-001) identified and characterized herein as HCl Pattern 1 (see Example 1).

[0144] As used herein, the term "the 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).

[0145] Crystal form

[0146] In general, the present disclosure provides a compound (I)

[0147] The new crystal form,

[0148] Methods of preparing this novel crystalline form, compositions containing it, and its use in treating diseases such as cancer, microbial infections, neurological diseases / disorders, diabetes, ischemia, osteoporosis, and related conditions.

[0149] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized as follows Figure 37 The X-ray powder diffraction pattern is shown.

[0150] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by having peaks at 10.167 degrees, 11.385 degrees, and 20.505 degrees 2θ ± 0.2 degrees 2θ; or an X-ray powder diffraction pattern having peaks at 10.2 degrees, 11.4 degrees, and 20.5 degrees 2θ ± 0.2 degrees 2θ.

[0151] 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, the DSC thermogram comprising an endothermic event with an onset temperature of 220.2°C ± 0.2°C.

[0152] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by being substantially as Figure 41 DSC thermogram shown.

[0153] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by comprising 103.24 cm -1 、993.01cm -1 and 1602.81cm -1 Wavelength value (cm -1 ); or 103.2cm -1 、993.0cm -1 、1602.8cm -1 ±0.2cm -1 Wavelength value (cm -1 )’s Raman spectrum.

[0154] In one embodiment of the present disclosure, a compound (I) is described

[0155] The crystal form,

[0156] The crystalline form is characterized by Figure 37 The X-ray powder diffraction pattern is shown.

[0157] In another embodiment, a compound (I) is described

[0158] The crystal form,

[0159] The crystalline form is characterized by an X-ray powder diffraction pattern having peaks at 10.2 degrees, 11.4 degrees, and 20.5 degrees 2Θ ± 0.2 degrees 2Θ.

[0160] In another example of the present disclosure, a crystalline form is described, wherein the crystalline form is further characterized by an X-ray powder diffraction pattern having peaks at 14.6 degrees, 22.9 degrees, 26.1 degrees, and 31.0 degrees 2θ ± 0.2 degrees 2θ. In another example, the crystalline form is further characterized by an X-ray powder diffraction pattern having peaks at 18.6 degrees, 21.4 degrees, 23.4 degrees, and 33.5 degrees 2θ ± 0.2 degrees 2θ.

[0161] In another embodiment of the present disclosure, a compound (I) is described

[0162] The crystal form,

[0163] The crystalline form is characterized by having an X-ray powder diffraction pattern with peaks at ±0.2 degrees 2Θ substantially as provided in Table 1.0' below.

[0164] Table 1.0'

[0165]

[0166]

[0167] In another example of the present disclosure, a crystalline form is described, wherein the crystalline form is characterized by a DSC thermogram obtained using a heating rate of 10°C / min, the DSC thermogram comprising an endothermic event with an onset temperature of 220.2°C ± 0.2°C. In another example, the crystalline form is characterized by being substantially as Figure 41 DSC thermogram shown.

[0168] In another embodiment of the present disclosure, a crystalline form is described wherein the crystalline form is characterized by comprising 103.2 cm -1 、993.0cm -1 and 1602.8cm-1 ±0.2cm -1 Wavelength value (cm -1 In another embodiment, the crystalline form is further characterized by comprising a Raman spectrum of 126.5 cm -1 、144.9cm -1 、227.3cm -1 、456.1cm -1 、1043.5cm -1 、1164.7cm -1 and 1582.5cm -1 ±0.2cm -1 Wavelength value (cm -1 In another embodiment, the crystalline form is characterized by comprising wave numbers (cm) substantially as provided in Table 2.0' below. -1 )±0.2cm -1 Raman spectrum.

[0169] Table 2.0'

[0170]

[0171]

[0172]

[0173] Methods of preparation and recrystallization

[0174] In one or more embodiments of the present disclosure, there is provided a method for preparing the compound (I) characterized as described herein.

[0175] The method of crystal form.

[0176] Typically, a crystalline form of Compound (I) is prepared by providing an HCl form of Compound (I) and breaking the HCl form (e.g., by 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., comprising cooling, adding an antisolvent, and maturation) to form the crystalline form of Compound (I).

[0177] In one or more embodiments, the method for preparing a crystalline form of compound (I) as characterized herein comprises adding the HCl 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 the free form of compound (I) from the first solution; adding the free form of compound (I) to a second organic solvent to form a second mixture; aging the second mixture to form a third mixture; and isolating the crystalline form of compound (I) from the third mixture.

[0178] In one or more embodiments, the method further comprises filtering insoluble particles from the first solution prior to isolating the free form of Compound (I) from the first solution,

[0179] In one or more embodiments of the present disclosure, the HCl form of Compound (I) is characterized as follows Figure 1 The X-ray powder diffraction pattern is shown.

[0180] In one or more embodiments, the HCl form of Compound (I) is characterized by having peaks at 7.220, 11.00, and 13.457 degrees 2θ ± 0.2 degrees 2θ; or an X-ray powder diffraction pattern having peaks at 7.2, 11.0, and 13.5 degrees 2θ ± 0.2 degrees 2θ. In one or more embodiments, the HCl form of Compound (I) is characterized by having an X-ray powder diffraction pattern having peaks at ± 0.2 degrees 2θ substantially as provided in Table 3.0' below.

[0181] Table 3.0'

[0182] Angle -2θ° strength% 7.2 76.4 10.6 29.3 11.0 53.4 12.2 18.8 12.5 13.5 13.5 100.0 14.4 21.2 14.7 15.4 16.7 28.4 16.9 25.0 18.2 15.9 18.9 35.6 19.1 37.5 19.8 26.0 20.2 13.5 20.9 26.0 21.5 23.6 21.7 19.7 22.1 14.9 22.3 15.4 22.5 15.9 23.3 53.8 23.9 28.8 24.4 62.5 25.1 19.7 25.5 29.3 26.1 56.7 26.3 24.5 27.2 19.7 27.4 16.3 28.1 16.3 28.5 13.5 28.9 22.6 29.4 20.2 30.0 16.8 31.7 38.9

[0183] In one or more embodiments, the HCl form of Compound (I) is characterized by a DSC thermogram obtained using a heating rate of 10°C / min, the DSC thermogram comprising an endothermic event with an onset temperature of 64.7°C ± 0.2°C.

[0184] In one or more embodiments, the HCl form of Compound (I) is characterized by being substantially as Figure 4 DSC thermograms as shown or listed in Table 15 (J09899, DSC).

[0185] In one or more embodiments of the present disclosure, the free form of Compound (I) is characterized as follows Figure 19 (represented by EG-1826-12-02) shows the X-ray powder diffraction pattern.

[0186] 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 degrees, 8.095 degrees, and 19.010 degrees 2θ ± 0.2 degrees 2θ; or peaks at 6.3 degrees, 8.1 degrees, and 19.0 degrees 2θ ± 0.2 degrees 2θ. In one or more embodiments, the free form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks at ± 0.2 degrees 2θ substantially as provided in Table 4.0' below.

[0187] Table 4.0'

[0188] Angle -2θ° strength% 6.3 100 8.1 13.8 11.8 16.8 12.2 11 15.6 10.7 19.0 20.1 23.5 12.8

[0189] 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.

[0190] In one or more embodiments, the free form of Compound (I) is characterized by being substantially as follows Figure 16 DSC thermograms as shown or listed in Table 19 (DSC).

[0191] In one or more embodiments of the present disclosure, there is provided a method of recrystallizing the compound (I) characterized herein.

[0192] The method of crystal form.

[0193] Typically, recrystallizing a crystalline form of Compound (I) involves dissolving the crystalline form in an organic solvent at elevated temperature to form a solution. The hot solution may be filtered to remove any insoluble particles. The hot solution is then seeded with a small amount of the crystalline form, followed by cooling the seeded solution. An antisolvent is added to the seeded cooled solution, followed by aging the solution. The crystalline form of Compound (I) is recrystallized from the aging solution and can be isolated.

[0194] 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; cooling from the first temperature to a second temperature; adding seed crystals of the crystalline form of compound (I) to the first solution to form a first mixture; cooling from the second temperature to a third temperature; adding an anti-solvent to the first mixture to form a second mixture; aging the second mixture for about 24 hours to form a third mixture; and separating the recrystallized crystalline form of compound (I) from the third mixture.

[0195] In one or more embodiments, the method further comprises filtering insoluble particles from the first solution before cooling from the first temperature to the second temperature.

[0196] In one or more embodiments of the present application, the compound (I) characterized as described herein

[0197] The crystal form,

[0198] Prepared and / or recrystallized by the procedures detailed in Example 1 and / or Example 2.

[0199] In one embodiment of the present disclosure, a method for preparing a crystalline form of Compound (I) as described herein is described, comprising the following steps:

[0200] adding the HCl form of Compound (I) to a first organic solvent to form a first mixture;

[0201] adding a base to the first mixture to form a first solution; and

[0202] isolating the free form of compound (I) from the first solution;

[0203] adding the free form of compound (I) to a second organic solvent to form a second mixture;

[0204] aging the second mixture to form a third mixture; and

[0205] The crystalline form of Compound (I) is isolated from the third mixture.

[0206] In another example of the present disclosure, a method is described, wherein adding the HCl 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, a method is described, 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.

[0207] In another example of the present disclosure, a method is described, wherein adding the free form of compound (I) to a second organic solvent to form a second mixture further comprises:

[0208] stirring the second mixture at ambient temperature;

[0209] Optionally, heating from ambient temperature to about 50°C and then cooling from about 50°C to ambient temperature, and

[0210] optionally adding additional organic solvent to the second mixture until the free form of Compound (I) is dissolved in the second organic solvent; and

[0211] The temperature was lowered from about 50°C to about 5°C at a rate of about 0.1°C / min.

[0212] In another example, a method is described wherein aging the second mixture to form a third mixture further comprises aging at 25 / 50° C. for a 4 hour cycle.

[0213] In another example, a method is described 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, a method is described wherein the first organic solvent is selected from the group consisting of ethanol, acetone, water, or ethyl acetate.

[0214] In another example, a method is described wherein the base is NaOH.

[0215] In another example, a method is described in which the second organic solvent is selected from the group consisting of 2-propanol; 2-methylTHF; 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, a method is described in which the second organic solvent is selected from the group consisting of 2-propanol; 2-methylTHF; 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.

[0216] In another example of the present disclosure, a method is described, further comprising filtering insoluble particles from the first solution before isolating the free form of Compound (I) from the first solution.

[0217] In another embodiment of the present disclosure, a method of recrystallizing a crystalline form of Compound (I) as described herein, or a crystalline form of Compound (I) prepared by a method described herein, is described, comprising the steps of:

[0218] dissolving the crystalline form of compound (I) in an organic solvent at a first temperature to form a first solution;

[0219] cooling from a first temperature to a second temperature;

[0220] adding seed crystals of the crystalline form of compound (I) to the first solution to form a first mixture;

[0221] cooling from the second temperature to a third temperature;

[0222] adding an antisolvent to the first mixture to form a second mixture;

[0223] aging the second mixture for about 24 hours to form a third mixture; and

[0224] The recrystallized crystalline form of Compound (I) is isolated from the third mixture.

[0225] In another embodiment of the present disclosure, a method is described 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, a method is described wherein the organic solvent is selected from the group consisting of DMSO; DMA; and NMP. In another embodiment, the organic solvent is DMSO.

[0226] In one embodiment, a method is provided wherein the anti-solvent comprises water, a C1-C3 alcohol, or a combination thereof. In one embodiment, the anti-solvent comprises water, a C1-C3 alcohol, or a combination thereof in an amount ranging from about 100% water to about 100% C1-C3 alcohol, or any combination of water and C1-C3 alcohol from 100% water to 100% C1-C3 alcohol.

[0227] In one embodiment, the C1-C3 alcohol comprises a C1-C3 straight chain or branched chain or cyclic alcohol. In one embodiment, the C1-C3 alcohol comprises methanol, ethanol, n-propanol, branched chain propanol or a combination thereof.

[0228] In another embodiment, a method is provided wherein the antisolvent is selected from the group consisting of ethanol and water. In one embodiment, the antisolvent is selected from the group consisting of ethanol and water, and the amount ranges from about 100% water to about 100% ethanol, or any combination of water and ethanol from 100% water to 100% ethanol. In another example, a method is described wherein the antisolvent is selected from the group consisting of EtOH:HO (1:1); EtOH:HO (1:2); and EtOH:HO (2:1).

[0229] In another example, a method is described wherein the organic solvent is DMSO and the antisolvent is EtOH:H2O (1:1), EtOH:H2O (1:2), or EtOH:H2O (2:1). In another example, the organic solvent is DMSO and the antisolvent is EtOH:H2O (1:2).

[0230] In another example, a method is described wherein the antisolvent is added at a volume of about 10% to about 20% of the volume of the organic solvent. In another example, the antisolvent is added at a volume of about 10% of the volume of the organic solvent.

[0231] In another example, a method is described wherein the first temperature is about 65°C.

[0232] In another example, a method is described wherein the second temperature is about 59°C.

[0233] In another example, a method is described wherein the third temperature is about 25°C.

[0234] In another example, a method is described wherein decreasing the temperature from the second temperature to the third temperature comprises decreasing the temperature at a rate of 0.5° C. / min.

[0235] In another example, a method is described that also includes filtering insoluble particles from the first solution before cooling the solution from the first temperature to the second temperature.

[0236] Pharmaceutical composition and use thereof

[0237] In one or more aspects of the present disclosure, the compound (I) characterized as described herein

[0238] The crystalline form of Compound (I) is pharmacologically active. In one or more embodiments of the present disclosure, the crystalline form of Compound (I) inhibits N-myristoyltransferase (NMT) activity. In one or more embodiments, the crystalline form of Compound (I) is used to treat a subject suffering from cancer. In one or more embodiments, the crystalline form of Compound (I) is used to treat a subject suffering from NMT2-deficient cancer.

[0239] N-myristoylation of proteins is a modification in which myristic acid, a 14-carbon saturated fatty acid, is covalently linked to the NH2-terminal glycine of a variety of cellular, viral, and tumor proteins (e.g., oncogenic Src-related tyrosine kinase, heterotrimeric Gα subunit, etc.).

[0240] Myristoylated proteins in cells have various biological functions in signal transduction and tumorigenesis. Modification of proteins by myristoylation is essential 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.

[0241] Myristoylation of proteins is catalyzed by N-myristoyltransferase (NMT). NMT is responsible for this activity in eukaryotic cells and acts by modifying its polypeptide substrate after removing the starting methionine residue with methionyl aminopeptidase. This modification occurs primarily as a co-translational process, although post-translational myristoylation can also occur after proteolytic cleavage of proteins, usually during apoptosis. Two isoenzymes of the mammalian NMT enzyme have been cloned and named NMT1 and NMT2.

[0242] NMT plays a pro-survival role in cells. Both NMTs are present in all normal cells. Increased NMT activity and expression have also been shown in many tumor types, suggesting that NMT inhibitors may be potential anti-cancer agents.

[0243] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) characterized as described herein can be used to treat or prevent diseases or conditions that can be prevented, alleviated or treated by regulating / inhibiting N-myristoyltransferase (NMT) activity (referred to herein as NMT-related diseases or conditions). Such NMT-related diseases or conditions include, but are not limited to, hyperproliferative disorders, such as cancer, microbial infections, neurological diseases / disorders, inflammatory diseases, immune diseases, autoimmune diseases, diabetes, ischemia, osteoporosis, and related conditions.

[0244] In one or more embodiments, the cancer to be prevented, ameliorated or treated includes all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasiveness.

[0245] In one or more embodiments, the cancer to be prevented, alleviated or treated includes malignant tumors of various organ systems, such as those affecting, for example, the lung, breast, thyroid, lymphoid, gastrointestinal and genitourinary tracts, and adenocarcinomas, including malignant tumors such as most colon cancers, renal cell carcinomas, prostate cancer and / or testicular tumors, non-small cell lung cancer, small intestine cancer and esophageal cancer. The term "cancer" is known in the art and refers to malignant tumors of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, genitourinary cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer and melanoma. Exemplary cancers include cancers formed by tissues of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term "cancer" also includes carcinosarcoma, for example, which includes malignant tumors composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or a cancer in which tumor cells form a recognizable glandular structure. The term "sarcoma" is known in the art and refers to a mesenchymal derivation malignant tumor. Other types of cancer include leukemia, skin cancer, intracranial cancer, and brain cancer.

[0246] 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 various types of malignant growth, 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 Hodgkin's lymphoma (characterized by, for example, the presence of Reed-Sternberg cells in the cancerous area). Non-Hodgkin's lymphoma includes more than 29 types of lymphoma, the distinction between which is based on the type of cancer cell.

[0247] In one embodiment, cancer is B lymphoma. Therefore, in one embodiment of the present disclosure, the crystal form of compound (I) and its pharmaceutical composition are suitable for treating subjects suffering from B cell lymphoma. The example of B cell lymphoma includes but is not limited to, such as follicular lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom's macroglobulinemia, MALT type / monocyte-like B cell lymphoma. It is also contemplated to treat 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.

[0248] In one or more embodiments, 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 macroglobulinemia, MALT-type / monocytic B cell lymphoma, Burkitt's lymphoma, pediatric lymphoma, anaplastic large cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia blast crisis, Burkitt's lymphoma, plasma cell myeloma, intestinal adenocarcinoma, mixed adenosquamous lung carcinoma , small cell lung cancer, lung cancer, squamous cell carcinoma of the esophagus, bone tumor, ductal carcinoma of the breast, diffuse gastric adenocarcinoma, medullary thyroid cancer, transitional cell carcinoma of the urinary tract, myeloma, clear cell ovarian carcinoma, transitional cell carcinoma (ureter and bladder cancer), chronic myeloid leukemia (CML), lymphoma-CLL, breast cancer, colorectal adenocarcinoma, pancreatic cancer, ovarian cancer, non-small cell lung cancer, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, hepatobiliary carcinoma (bile duct cancer), gallbladder cancer, liver cancer, or squamous cell carcinoma of the esophagus.

[0249] In one or more embodiments, the microbial infection to be prevented, alleviated or treated is an infection caused by bacteria, parasites, protozoa, viruses or fungi (including yeast).

[0250] A "pathogen" is generally defined as any disease-causing organism. The parasitic pathogen may be 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), Cryptosporidium spp., Isospora spp., Balantidium spp., Loa Loa, Ascaris lumbricoides, Dirofilaria immitis, Toxoplasma ssp. (e.g., Toxoplasma gondii), Onchocerca spp. (e.g., O. volualno).

[0251] The viral pathogen may be from a virus selected from, but not limited to, the group consisting of: human immunodeficiency virus (HIV1 and 2); human T-cell leukemia virus (HTLV 1 and 2); Ebola virus; human papillomavirus (e.g., HPV-2, HPV-5, HPV-8, HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, HPV-54, and HPV-56); papillomasalis virus; rhinovirus; poliovirus; herpes virus; adenovirus; Epstein-Barr virus; influenza virus, hepatitis B and C virus, smallpox virus, rotavirus, or SARS coronavirus.

[0252] The fungal pathogen may be from a fungus (including yeast) selected from, but not limited to, the group consisting of: 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).

[0253] In one or more embodiments, the neurological diseases / disorders to be prevented, alleviated or treated may include: neuropsychiatric disorders, including Parkinson's disease, attention deficit hyperactivity disorder (ADHD), depression (bipolar disorder), schizophrenia and addiction; neurodegenerative disorders (e.g., Alzheimer's disease, Tourette syndrome, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, senile chorea, Sydenham chorea, autism, head and spinal cord trauma, acute and chronic pain, epilepsy and seizures, dementia, distonia, tremor, autism, cerebral ischemia and neuronal cell death) and apoptosis-related disorders (particularly neuronal apoptosis).

[0254] When used to treat a subject, the crystalline form of Compound (I) characterized as described herein can be administered as a free compound or as part of a pharmaceutical composition. It can be administered orally, intravenously, subcutaneously, buccal, rectal, cutaneous, nasal, tracheal, bronchial, by any other parenteral route, as an oral or nasal spray, or by inhalation.

[0255] The crystalline form of Compound (I) can be administered in a pharmaceutically acceptable dosage form as a free compound or as part of a pharmaceutical composition. Depending on the disease and subject to be treated and the route of administration, the compound or pharmaceutical composition can be administered in different doses.

[0256] The crystalline form of Compound (I) can be administered orally or parenterally ("parenteral" as used herein refers to administration methods including 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) can be administered alone or in combination with a pharmaceutically acceptable diluent, excipient, or carrier as a pharmaceutical composition.

[0257] The actual dosage level of the crystalline form of compound (I) in the pharmaceutical composition can be changed to obtain the amount of the crystalline form of active compound (I) that effectively achieves the desired therapeutic response of a particular subject, composition and / or mode of administration. The selected dosage level will depend on the activity of the crystalline form of compound (I), the route of administration, the severity of the condition being treated, and the condition and previous medical history of the subject being treated. However, those skilled in the art can use a starting dose of the crystalline form of compound (I) at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0258] In one or more embodiments of the present disclosure, where inhibition of kinase activity is required for treating, preventing, controlling, ameliorating, or reducing the risk of a condition, an appropriate dosage level may be about 0.01 to 500 mg per kg of patient body weight per day, which may be administered in a single dose or multiple doses, or as 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. Suitable dosage levels 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.

[0259] For oral administration, the pharmaceutical composition comprising the crystalline form of compound (I) can be provided in the form of tablets containing 1.0 to 1000 mg of 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 mg of the crystalline form of compound (I), the dosage of which is adjusted according to the symptoms of the subject to be treated. The crystalline form of compound (I) can be administered as a free compound or as part of a pharmaceutical composition, 1 to 4 times a day; or once or twice a day. The dosage regimen can be adjusted to provide the best therapeutic response.

[0260] In another aspect of the present disclosure, a pharmaceutical composition is provided, comprising the crystalline form of Compound (I) mixed with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0261] The disclosure is suitable for the pharmaceutical composition of parenteral injection and comprises pharmaceutically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion and for being reformulated as the sterile powder of sterile injection solution or dispersion before use.The example of suitable aqueous and non-aqueous carrier, diluent, solvent or vehicle comprises water, ethanol, polyol (such as glycerol, propylene glycol, Polyethylene Glycol etc.) and suitable mixture thereof, vegetable oil (such as olive oil) and injectable organic ester such as ethyl oleate.Suitable mobility can be kept, for example, by using coating material such as lecithin, by keeping required granularity in the case of dispersion, and by using surfactant.

[0262] Pharmaceutical compositions of the present disclosure may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. By adding various antibacterial and antifungal agents, such as parabens, chlorobutanol, or phenol sorbic acid, it is possible to ensure that the activity of microorganisms is prevented. It may also be desirable to include, for example, isotonic agents, such as sugars or sodium chloride.

[0263] Prolonged absorption of the injectable pharmaceutical form can be brought about by the addition of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0264] In some cases, in order to prolong the effect of the pharmaceutical composition of the present disclosure, it is necessary to slow down the absorption of the composition or the crystalline form of Compound (I) from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension, wherein the crystalline form of Compound (I) is suspended in a liquid in which the crystalline form has poor solubility. The absorption rate of the crystalline form of Compound (I) depends on its dissolution rate.

[0265] Alternatively, the delayed absorption of the pharmaceutical composition for parenteral administration of the present disclosure is achieved by dissolving or suspending the crystal form of the compound (I) in an oil vehicle. By forming a microencapsulated matrix of the crystal form of the compound (I) in a biodegradable polymer (e.g., polylactide-polyglycolide), an injectable depot form is suitably prepared. According to the ratio of the crystal form of the compound (I) to the polymer and the properties of the specific polymer used, the release rate can be controlled. Examples of other biodegradable polymers include polyorthoesters and polyanhydrides.

[0266] Depot injectable formulations can also be prepared by encapsulating a crystalline form of Compound (I) in liposomes or microemulsions that are compatible with body tissues. The injectable formulation can be sterilized, for example, by filtration through a bacteria-retaining filter or by adding a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0267] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound (e.g., a crystalline form of Compound (I)) is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier (e.g., sodium citrate or dicalcium phosphate) and / or one or more of the following: 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) disintegrants, 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 glyceryl monostearate; h) absorbents, such as kaolin and bentonite clays, 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 contain buffering agents.Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using, for example, such excipients as lactose or milk sugar and high molecular weight polyethylene glycols.

[0268] Suitably, the oral formulations include a dissolution aid. The characteristics of the dissolution aid are not limited as long as it is pharmaceutically acceptable. Examples include nonionic surfactants 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 sulfides, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monooleate, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monooleate, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl sulfides, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monooleate, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene alkyl ... Fatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorbitol fatty acid esters, fatty acid alkanolamides and alkylamine oxides; bile acids and their salts (e.g., chenodeoxycholic acid, cholic acid, deoxycholic acid, dehydrocholic acid and their salts, and glycine or taurine conjugates thereof); ionic surfactants such as sodium lauryl sulfate, fatty acid soaps, alkyl sulfonates, alkyl phosphates, ether phosphates, basic amino acid fatty acid salts; triethanolamine soaps and alkyl quaternary ammonium salts; and amphoteric surfactants such as betaines and aminocarboxylates.

[0269] 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 formulation art. They may optionally contain an opacifying agent and may be a composition that releases the active ingredient only, or preferably in a specific part of the intestinal tract, and / or in a delayed manner. Examples of embedding compositions include polymeric substances and waxes.

[0270] If appropriate, the crystalline form of Compound (I) may also be in microencapsulated form, 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 micronized.

[0271] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the crystalline form of compound (I), liquid dosage forms can contain inert diluents commonly used in the art such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and aromatics. In addition to the active compound, the suspension can also contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth gum and mixtures thereof.

[0272] The pharmaceutical composition for rectal or vaginal administration of the present invention is preferably a suppository, which can be prepared by mixing the crystalline form of Compound (I) with a suitable non-irritating excipient or carrier (such as cocoa butter, polyethylene glycol or suppository wax), which is solid at room temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the crystalline form of Compound (I).

[0273] The crystal form of compound (I) can 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 monolayer or multilayer hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. In addition to the crystal form of compound (I), the pharmaceutical composition of the present disclosure in liposome form may contain stabilizers, preservatives, excipients, etc. Preferred lipids are natural and synthetic phospholipids and phosphatidylcholines (lecithins). Methods for forming liposomes are known in the art.

[0274] Dosage forms for topical administration of the crystalline forms of Compound (I) include powders, sprays, ointments and inhalants. The crystalline forms of Compound (I) are mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives, buffers or propellants. The crystalline forms of Compound (I) can also be administered, for example, as part of a pharmaceutical composition in the form of an ophthalmic preparation or ophthalmic ointment, powder and solution.

[0275] In another embodiment of the present disclosure, a pharmaceutical composition is described, comprising a crystalline form of Compound (I) as described herein; and a pharmaceutically acceptable adjuvant, diluent, carrier or vehicle.

[0276] In another example of the present disclosure, a method of treating a subject having cancer is described, comprising administering to the subject a crystalline form of Compound (I) described herein or a pharmaceutical composition described herein.

[0277] In another example of the present disclosure, a method of treating a subject having an NMT2-deficient cancer is described, comprising administering to the subject a crystalline form of Compound (I) described herein or a pharmaceutical composition described herein.

[0278] In another example of the present disclosure, a method is described wherein the cancer is a lymphoma. In another example, the lymphoma is a B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom's macroglobulinemia, MALT-type / monocytic B-cell lymphoma, or Burkitt's lymphoma.

[0279] In another example, a method is described wherein the cancer is anaplastic large cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia blast crisis, Burkitt lymphoma, plasma cell myeloma, intestinal adenocarcinoma, mixed adenosquamous lung carcinoma, small cell lung carcinoma, lung carcinoma, esophageal squamous cell carcinoma, bone tumor, breast ductal carcinoma, diffuse gastric adenocarcinoma, medullary thyroid carcinoma, urinary tract transitional cell carcinoma, myeloma, ovarian clear cell carcinoma, transitional cell carcinoma (ureter and bladder), chronic myeloid leukemia (CML), lymphoma-CLL, breast cancer, colorectal adenocarcinoma, pancreatic cancer, ovarian cancer, non-small cell lung cancer, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, hepatobiliary carcinoma (bile duct cancer), gallbladder cancer, liver cancer, or esophageal squamous cell carcinoma.

[0280] In another example, a method is described 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 human.

[0281] In order to better understand 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 the present invention in any way.

[0282] Example

[0283] Example 1 - Screening studies of compound (I) (PCLX-001)

[0284] summary

[0285] Solid-state characterization was performed on the supplied batches of PCLX-001. Three batches were labeled HCl Mode 1, and one batch was labeled HCl Mode 2. All batches exhibited a peak at ~32.1° 2θ, representing sodium chloride. Ion chromatography confirmed the presence of excess chloride and sodium. One batch underwent a salt breaking step.

[0286] The use of sodium hydroxide by ion exchange desalting was successful and was scaled up several times to a high 1g scale. The recovered material did not contain chloride ions and sodium (free form) and was in a crystalline form (expressed as free form pattern 1). The purity of this material was 96.3%, comparable to that of the starting material. The melt (start) of this form was recorded at 161.5°C.

[0287] Polymorphic landscape screening was performed using free form Pattern 1. Twenty-two solvents / solvent systems were selected for this stage, starting with free form Pattern 1. The following methods were used: cooling, maturation, and antisolvent addition. Free form Pattern 2 was obtained in almost all cases, indicating that it may be the most stable form under the conditions tested.

[0288] The free form, Pattern 2, is a crystalline, non-solvated form, and its XRPD pattern remained virtually unchanged after storage for one week at elevated temperatures and high humidity levels (40°C / 75% RH and 25°C / 97% RH). Melting / degradation temperatures were observed between 209°C and 217°C. Only a gradual weight loss due to loss of unbound water was observed on the TGA thermogram, followed by degradation of the sample above 210°C.

[0289] Of all the polymorphs found during testing, free form Pattern 2 was found to exhibit the most desirable solid state properties and was further investigated to produce an optimized crystallization method for forming free form Pattern 2 (see Example 2).

[0290] Abbreviations

[0291]

[0292]

[0293] Instrument and method details

[0294] X-ray powder diffraction (XRPD)

[0295] Bruker AXSD8 Advance

[0296] XRPD diffractograms were collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA) and a θ-2θ goniometer equipped with a Ge monochromator. The incident beam passed through a 2.0 mm divergence slit, followed by a 0.2 mm anti-scatter slit and a knife-edge. The diffracted beam passed through an 8.0 mm receiving slit and a 2.5° Soller slit before detection by a Lynxeye detector. The software used for data acquisition and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA, respectively.

[0297] The samples were tested as flat samples using powder under ambient conditions. The samples were prepared on polished zero-background (510) silicon wafers by gently pressing them onto a flat surface or by inserting them into a cutting chamber. The samples were rotated in their own plane.

[0298] Details of the standard data collection method are:

[0299] Angle range: 2 to 42°2θ

[0300] Step size: 0.05°2θ

[0301] Acquisition time: 0.5s / step (total acquisition time: 6.40min)

[0302] PANalytical Empyrean

[0303] XRPD diffractograms were collected on a PANalytical Empyrean diffractometer using Cu Kα radiation (45 kV, 40 mA) in transmission geometry. A 0.5° slit, a 4 mm mask, and a 0.04 rad Soller slit with a focusing lens were used for the incident beam. A PIXcel3D detector placed on the diffracted beam was equipped with a receiving slit and a 0.04 rad Soller slit. The software used for data acquisition was X'Pert Data Collector using the X'Pert user interface. Data were analyzed and presented using Diffrac Plus EVA or HighScorePlus.

[0304] Samples were prepared and analyzed in transmission mode in metal or Millipore 96-well plates. Powders (approximately 1-2 mg) were used as received, using an X-ray transparent film between the metal sheets of the metal well plates. Millipore plates were used to separate and analyze solids in suspensions by adding small amounts of the suspension directly to the plate and filtering under light vacuum.

[0305] The scanning mode for the metal plates used a side angle scan axis, while the Millipore plates were scanned in 2θ.

[0306] Details of the standard screening data collection method are:

[0307] Angular range: 2.5 to 32.0° 2θ

[0308] Step size: 0.0130°2θ

[0309] Acquisition time: 12.75s / step (total acquisition time is 2.07min)

[0310] Nuclear magnetic resonance (NMR)

[0311] Solution-state NMR

[0312] 1 H NMR and / or 13 C NMR spectra were collected on a Bruker 400 MHz instrument equipped with an autosampler and controlled by a DRX400 console. Unless otherwise stated, samples were prepared in DMSO-d6 solvent. Automated experiments were performed using the ICON-NMR configuration within Topspin software, using standard Bruker sample loading experiments (1H, 13C{1H}, DEPT135). Offline analysis was performed using an ACD Spectrus Processor. For unconventional spectroscopy (2D NMR and variable temperature NMR), data were collected using Topspin only.

[0313] Differential Scanning Calorimetry (DSC)

[0314] TA Instruments Q2000

[0315] DSC data were collected on a TA Instruments Q2000 equipped with a 50-position autosampler. Typically, 0.5-3 mg of each sample was placed in a perforated aluminum dish and heated from 25° C. to 300° C. at 10° C. / min. A dry nitrogen purge at 50 ml / min was maintained.

[0316] Modulated temperature DSC was performed at a base heating rate of 2°C / min with a temperature modulation parameter of ±0.636°C (amplitude) per 60 seconds (cycle).

[0317] The instrument control software was Advantage for Q Series and Thermal Advantage, and the data were analyzed using Universal Analysis or TRIOS.

[0318] TA Instruments Discovery DSC

[0319] DSC data were acquired on a TA Instruments Discovery DSC equipped with a 50-position autosampler. Typically, 0.5-3 mg of each sample was placed in a perforated aluminum dish and heated from 25°C to 300°C at 10°C / min. A dry nitrogen purge was maintained at 50 ml / min. The instrument was controlled by TRIOS software, and data were analyzed using TRIOS or Universal Analysis.

[0320] Thermogravimetric analysis (TGA)

[0321] TA Instruments Q500

[0322] TGA data were acquired on a TA Instruments Q500 TGA equipped with a 16-position autosampler. Typically, 5-10 mg of each sample was loaded onto a pre-tared aluminum DSC dish and heated from ambient temperature to 350°C at 10°C / min. A nitrogen purge was maintained at 60 ml / min. The instrument was controlled by Advantage for Q Series and ThermalAdvantage software, and data were analyzed using Universal Analysis or TRIOS.

[0323] TA Instruments Discovery TGA

[0324] TGA data were acquired on a TA Instruments Discovery TGA equipped with a 25-position autosampler. Typically, 5-10 mg of each sample was loaded onto a pre-tared aluminum DSC dish and heated from ambient temperature to 350°C at 10°C / min. A nitrogen purge was maintained at 25 ml / min. The instrument was controlled by TRIOS software, and data were analyzed using TRIOS or UniversalAnalysis.

[0325] Polarized Light Microscopy (PLM)

[0326] Leica LM / DM polarized light microscope

[0327] Samples were analyzed on a Leica LM / DM polarized light microscope equipped with a digital 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 coverslip. Samples were observed at appropriate magnification using partially polarized light and a lambda pseudocolor filter. Images were acquired using StudioCapture or Image ProPlus software.

[0328] Scanning electron microscopy (SEM)

[0329] Data were collected on a Phenom Pro scanning electron microscope. A small sample was mounted on an aluminum stub using conductive double-sided tape. A thin layer of gold was applied using a sputter coater (20 mA, 120 s).

[0330] Gravimetric Vapor Sorption (GVS)

[0331] Adsorption isotherms were obtained using a SMSDVS Intrinsic moisture sorption analyzer controlled by DVS Intrinsic Control software. The sample temperature was maintained at 25°C by instrument control. Humidity was controlled by a mixed dry and wet nitrogen flow with a total flow rate of 200 ml / min. Relative humidity was measured near the sample by a calibrated Rotronic probe (dynamic range 1.0-100% RH). The weight change (mass relaxation) of the sample as a function of % RH was continuously monitored by a microbalance (accuracy ±0.005 mg).

[0332] Typically, 5-30 mg of sample is placed in a tared stainless steel basket under ambient conditions. Samples are loaded and unloaded at 40% RH and 25° C. (typical room temperature conditions). Water adsorption isotherms are performed as follows (2 scans per complete cycle). Standard isotherms are performed at 25° C. in a 0-90% RH range at 10% RH intervals. Typically, a double cycle (4 scans) is performed. Data analysis is performed in Microsoft Excel using DVS Analysis Suite.

[0333] Table 1 Methods of SMSDVS intrinsic experiment

[0334]

[0335]

[0336] After completion of the isotherm a sample was recovered and reanalyzed by XRPD (see above).

[0337] Chemical purity determined by HPLC

[0338] Purity analysis was performed on an Agilent HP1100 / Infinity II 1260 Series system equipped with a diode array detector using OpenLAB software. Full method details are provided below:

[0339] Table 2 HPLC method for chemical purity determination

[0340]

[0341] Karl Fischer titration (KF) water determination

[0342] The water content of each sample was measured using an 851 Titrano coulometer on a Metrohm 874 oven sample processor at 150°C with a Hydranal Coulomat AG oven reagent and nitrogen purge. Weighed solid samples were placed in sealed sample vials. Each titration used approximately 10 mg of sample, and replicates were performed. Unless otherwise stated, results are presented as averages. Data acquisition and analysis were performed using Tiamo software.

[0343] Thermodynamic water solubility

[0344] Water solubility is determined by suspending enough compound in the relevant medium to a maximum final concentration of ≥10 mg / ml of the free form of the compound parent. The suspension is balanced for 24 hours at 25 ° C on a Heidolph plate shaker set at 750 rpm. The saturated solution pH is subsequently measured, and the suspension is filtered through a glass fiber C filter (particle retention 1.2 μm) and appropriately diluted. Quantification is performed by HPLC with reference to a standard solution of approximately 0.15 mg / ml in DMSO. Different volumes of standard solution, dilution, and undiluted sample solutions are injected. Solubility is calculated using the peak area determined by the integration of the peak found at the same retention time as the main peak in the standard injection.

[0345] Table 3 HPLC method used for solubility measurement

[0346]

[0347] Analyses were performed on an Agilent HP1100 / Infinity II 1260 Series system equipped with a diode array detector and using OpenLAB software.

[0348] Thermodynamic solubility of J09899 in FaSSGF, FeSSIF, FaSSIF and deionized water

[0349]

[0350]

[0351] Methods and Instructions

[0352] Sufficient sample was suspended in 0.5 ml of medium to achieve a maximum expected concentration of 100 mg / ml of the compound's hydrochloride salt. The resulting suspension was then shaken at 25°C / 750 rpm for 24 hours. After equilibrium, the appearance was recorded and the pH of the saturated solution was measured. The sample was then filtered through a glass "C" fiber filter (particle cutoff 1.2 μm). The sample was diluted ×10 and ×100 in the appropriate medium.

[0353] Quantification was performed by HPLC with reference to a standard solution of approximately 0.15 mg / ml. Different volumes of standard, diluted, and undiluted sample solutions were injected. Solubility was calculated using the peak area determined by integrating the peak found at the same retention time as the main peak in the standard injection.

[0354] Observation results

[0355] Co-elution of the SIF peak with the parent peak was observed based on the universal solubility method for this sample. The sample was analyzed using a universal purity method (higher resolution column) to separate these peaks.

[0356]

[0357]

[0358]

[0359]

[0360] Summary and Conclusion

[0361] In FaSSGF medium, FaSSIF medium, and deionized water, the samples were classified as "slightly soluble" according to USP classification. In FeSSIF, the samples were classified as "very slightly soluble" according to USP classification. These values were calculated based on IC and KF analysis based on samples with 1.4 equivalents of HCl.

[0362] Due to issues with co-elution of SIF peaks when using the universal solubility method, all subsequent analyses will be performed using the universal purity method as this allows for separation of the peaks.

[0363] Ion chromatography (IC)

[0364] Data were acquired on a Metrohm 930 Compact IC Flex equipped with an 858 Professional autosampler and an 800 Dosino dosage unit monitor using IC MagicNet software. Accurately weighed samples were prepared as stock solutions in appropriate solvents. Quantification was achieved by comparison with standard solutions of known concentrations of the analyte. Analyses were performed in duplicate, and the average values are presented unless otherwise stated.

[0365] Table 11 IC method of cation chromatography

[0366]

[0367] Table 12 IC method of anion chromatography

[0368]

[0369] Determination and prediction of pKa and LogP

[0370] result

[0371]

[0372] Structure and prediction

[0373]

[0374] By Yasuda-Shedlovsky extrapolation, a high pKa of 8.95 was confirmed as acidic, and a pKa of 8.19 was confirmed as basic (which was inconsistent with the prediction software).

[0375] Methods and Instructions

[0376] Data were collected on a Sirius T3 instrument equipped with a Ag / AgCl double-junction pH electrode and a D-PAS accessory. The electrode was calibrated using four positive parameters obtained from a blank titration. Alkaline titrants were standardized by titration with KHP. 0.5M aqueous HCl and KOH were used as the acid and base titrants, respectively. Titrations were performed against a background of ISA 0.15M KCl(aq). Data were optimized using a Sirius T3 Refine. pKa and LogP values were predicted using ACD / Labs Percepta.

[0377] UV quantitative pKa (aqueous phase)

[0378] Samples were prepared as 11.07 mM stock solutions in DMSO (5 μL stock solution for analysis). Data were acquired by UV quantitative single titration from pH 1.5-4.0 (low to high) under aqueous conditions at 25°C.

[0379] Fast UV pKa (aqueous phase)

[0380] Samples were prepared as 10 mM stock solutions in DMSO (5 μL stock solution for analysis). Data were acquired by rapid UV single titration at 25°C under aqueous conditions, pH 2.0-12.0 (low to high).

[0381] pH quantification LogP

[0382] 0.96 mg of sample was weighed directly into a T3 vial. Data was collected using potentiometric titration using three ratios of octanol (0.020 / 0.050 / 1.0 mL) and ionic strength adjusted (ISA) water with pH values ranging from low to high (pH 2.0-12.0). The collected potentiometric data was used to calculate LogP, LogP ion, and LogD values.

[0383] LogD shake flask method

[0384] The stock solution of 1mg / ml in the preparation octanol, and at room temperature vibrated 1 hour to guarantee to dissolve completely. An equal-volume octanol solution and USP pH 7.4 phosphate buffer (50mM) were vibrated 1 hour at room temperature in duplicate. The sample was centrifuged for 10min at 13400rpm and separated into each phase with a glass pipette. The layer of suitable dilution separation (if necessary, with acetonitrile dilution octanol sample, with buffer dilution water sample) was analyzed using the method described in detail below by HPLC.

[0385] Table 13 HPLC method for determining LogD

[0386]

[0387]

[0388] Experimental crystallization method

[0389] The choice of crystallization method has a great influence on which crystalline form is produced, so it is important to use a variety of methods and conditions for crystallization when searching for polymorphs.

[0390] Table 14 lists the classical crystallization methods used in this paper and the degrees of freedom available for each method.

[0391] Table 14 Classical crystallization methods used in this project

[0392]

[0393] Solvent-mediated technology

[0394] These are classic techniques for producing crystalline materials. In theory, crystallization occurs when the concentration of a compound in a solvent is higher than its solubility product. Generally, crystallization is kinetically hindered, and crystals can only grow from supersaturated solutions.

[0395] For crystallization screening, solvents should be selected with a high diversity of properties (hydrogen bond donor / acceptor propensity, dipole moment, dielectric constant, viscosity, etc.). Often, solvent mixtures can be used to obtain systems with appropriate solubility, polarity, etc. The substance also needs to be chemically stable in the given solvent or solvent mixture. Several methods can be used to achieve a supersaturated metastable state.

[0396] Maturation / Slurry Maturation

[0397] To investigate crystal forms, aging experiments (or slurry aging) are typically performed in different solvents or solvent mixtures, followed by heating-cooling cycles. Repeated heating and cooling cycles can increase crystallinity or convert a metastable state (or, in the case of amorphous materials, an unbalanced state) into a more thermodynamically stable crystal form. The rate and extent of this conversion depends on the solubility of the input material.

[0398] Due to 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 wider variety of forms can be obtained.

[0399] Curing room procedures

[0400] The suspension for maturation was placed in a platform shaking incubator (Heidolph Titramax / Incubator 1000) and subjected to a series of heating-cooling cycles from room temperature to approximately 50° C. This was achieved by turning the heating on and off every 4 hours. The shaking was continuous.

[0401] Polar Bear Program

[0402] The suspension was stirred (500 rpm) for 1 hour at 50° C. in a Polar Bear (Cambridge Reactor Design). The sample was then cooled to 5° C. at 0.1° C. / min and stirred for a further 4 hours.

[0403] Cooling crystallization

[0404] Crystallization can be achieved by lowering the temperature of a clear solution. The solubility of most substances decreases with decreasing temperature, so cooling can be used to produce supersaturation. However, in many cases, the material retains a high solubility even at low temperatures, or the solubility changes little over the target temperature range. In these cases, other methods of producing supersaturation must be considered (such as controlled evaporation below).

[0405] program

[0406] The solution was 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 minutes and preliminarily analyzed by XRPD.

[0407] Controlled evaporation

[0408] Crystallization can be produced by controlled evaporation of a clear, particle-free solution. This is particularly true when the solvent has a relatively high vapor pressure. At a nearly constant temperature, the solvent is removed from the system, thereby increasing the solute concentration. When maximum supersaturation is reached, crystals nucleate and grow. This technique also has the advantage that, due to the slow evaporation of the sample, large single crystals suitable for SCXRD can usually be produced.

[0409] program

[0410] Evaporate the solution under ambient conditions by removing the cap of the vial and replacing it with a perforated seal or by inserting a 25 gauge syringe needle through the cap. Allow the sample to evaporate slowly to dryness or until a solid appears under ambient conditions.

[0411] Characterization of compound (I) (PCLX-001)

[0412] Compound (I) (PCLX-001) (Batch J09898 & Batch J09899) were characterized using a variety of techniques to investigate the solid form and chemical properties of PCLX-001. The results are summarized in Table 15.

[0413] Table 15 Characteristic data of PCLX-001 (J09898&J09899)

[0414]

[0415] By XRPD analysis ( Figure 1 ) Characterization of PCLX-001 (J09899, HCl Model 1) confirmed it to be crystalline and designated as HCl Model 1 with a purity of 97.2% ( Figure 3 ). 1 H-NMR spectrum ( Figure 2) is consistent with the proposed molecular structure, with the presence of trace amounts of acetone. 1.3 molar equivalents of chloride ion were observed in the anionic IC, which is lower than the expected 3 equivalents of chloride ion (since the compound is considered to be in the trichloride form). In addition, 0.2 molar equivalents of sodium and 0.1 molar equivalents of calcium were observed in the cationic IC, which may have been generated during the compound's manufacturing process.

[0416] Thermal analysis of PCLX-001 (J 09899, HCl mode 1) Figure 4 ) showed that the material had a significant 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% was observed from -50° C. to 225° C., followed by a small weight loss of 1%, with the material degrading at 250° C. (onset). The weight loss may be related to the loss of water in the sample, as 9.7% w / w water was observed in the sample by KF analysis.

[0417] GVS analysis of PCLX-001 (J09899, HCl model 1) Figure 5 and Figure 6 ) showed that the sample was very hygroscopic, absorbing 39.76% w / w water at 90% RH. However, no significant changes in the appearance of the solid or the XRPD pattern were observed after GVS analysis.

[0418] Static stability experiments were also conducted at two sets of increased storage conditions, 40°C / 75% RH and 25°C / 97% RH, which showed no significant changes in the solid appearance or XRPD pattern ( Figure 7 and 10 ), and in both cases high purity is maintained ( Figure 8 and Figure 9 ), leading to the conclusion that it is stable at elevated humidity and temperature.

[0419] Thermodynamic solubility revealed high solubility in FaSSGF (9.9 mg / ml) and in water (6.0 mg / ml), with relatively low solubility in FeSSIF (1.9 mg / ml) and FaSSIF (0.64 mg / ml). The difference between the predicted and measured pKa values was minimal. Analysis revealed a Log P of 2.2 at pH 7.4. Furthermore, the difference in the measured pKa values was sufficient to exclude zwitterions.

[0420] The received replacement batch of PCLX-001 (J09898) was confirmed to have a different crystallization pattern by XRPD analysis and was designated as HCl Pattern 2. This material showed a lower purity of 96.7%. However, 1The H-NMR spectrum matches the proposed molecular structure, with trace amounts of acetone and TBME present. Thermal analysis was also performed on PCLX-001 (J09898), where a significant endotherm of 196 J / g was observed at 40.2°C (onset), followed by an exotherm of 8 J / g at 138.7°C (onset), and an endotherm of 10 J / g at 165.8°C (onset). A weight loss of 11.4% w / w was observed from ~50°C to 225°C, followed by material degradation at 250°C (onset). In addition, 2 molar equivalents of chloride ion were observed in the anionic IC, still less than the expected 3 molar equivalents of chloride ion for the trisalt, and no cations were observed in the cationic IC.

[0421] The characteristics of other batches of PCLX-001 (J09951, J09952, and J09953) were confirmed to be crystalline HCl Pattern 1, all maintaining high purity of 98.5%, 98.3%, and 98.5%, respectively. 1 The H-NMR spectra match the proposed molecular structure. In the XRPD diffractograms of J09951, J09952, and J09953, a sharp peak was observed at 32° 2θ, indicating the presence of a higher proportion of NaCl in the samples compared to batch J09899. Table 16 and Figure 11 Summarizes all features.

[0422] Table 16 Characteristic data of PCLX-001 (J09951, J09952 & J09953)

[0423] technology PCLX-001(J09951) PCLX-001(J09952) PCLX-001(J09953) XRPD Crystalline, HCl pattern 1 Crystalline, HCl pattern 1 Crystalline, HCl pattern 1 <![CDATA[ 1 H-NMR]]> Consistent with the structure Consistent with the structure Consistent with the structure HPLC 98.45％ 98.28％ 98.48％

[0424] Salt breaking experiment of PCLX-001

[0425] program

[0426] PCLX-001 (J09899, HCl mode 1, 50 mg ± 1 mg) was weighed into 5 × 4 ml vials and treated with increasing volumes (20 vol, 30 vol, 40 vol, 50 vol) of the relevant solvent (ethanol, acetone, water, DCM and ethyl acetate) until the material was completely dissolved or until a maximum of 50 vol (2.5 ml) was added. After each addition of solvent, the vial was stirred at room temperature (RT) for 5 minutes. If no dissolution occurred, it was heated to 50 ° C and stirred for an additional 5 minutes. Each vial was allowed to stand at RT for 5 minutes before adding a new aliquot of solvent.

[0427] After the evaluation was complete, 1 equivalent (119.2 μl, relative to the Cl ions calculated by IC, added as a 1 M stock solution in water) of NaOH was added at 50°C and the sample was slowly cooled to 5°C at 0.1°C / min. All solids were separated by filtration and dried under suction. The DCM system produced a yellow solution. The system was washed with 1 part of water, the organic layer was separated, and then dried in a vacuum oven at RT. All separated solids were analyzed by XRPD.

[0428] Results and discussion

[0429] In the salt breaking experiments of PCLX-001 (J09899), no dissolution was observed in four of the five solvents tested, and a yellow suspension was formed in all cases. Ethanol was the only exception, where dissolution was observed at 50 vol at 50°C, forming a colorless solution. Upon addition of 1 equivalent of NaOH relative to the chloride ion calculated from the IC analysis of the J09899 material, the sample in ethanol formed a yellow solution, while all the remaining samples remained yellow suspensions.

[0430] After cooling to 5°C, the sample in DCM formed a clear yellow solution, while all the remaining samples formed a light yellow suspension. The sample in DCM was selected for further processing and separated by a separatory funnel. The organic layer was dried in a vacuum oven at RT overnight to form a new solid, which was designated as free form pattern 1 in the subsequent characterization stage. The sample in ethyl acetate was also further processed, in which the sample was filtered and dried under suction, resulting in a new pattern, designated as "mixed salts", because IC analysis showed evidence of the presence of chloride ions. See Table 18 and Figures 12A to 14 .

[0431] Table 18 Characterization of salt breaking experiment of J09899

[0432]

[0433] The new samples obtained from the salt breaking test of J09899 were characterized. XRPD analysis of the samples obtained from DCM (see Figure 12A ) showed the formation of a new pattern, which was later designated as free form pattern 1. This was also due to the 1 The forward shift observed in the H-NMR spectrum indicates the formation of the parent form (see Figure 12B ). In addition, IC analysis showed that no anions or cations were observed, confirming the production of free form model 1. A high purity of 96.26% was also maintained in the free form model 1 (see Figure 13 ).

[0434] XRPD analysis (see Figure 12A) also confirmed the formation of a new solid termed a "mixed salt" from ethyl acetate as 1.3 molar equivalents of chloride ion and 1.4 molar equivalents of sodium were observed by IC analysis. 1 H-NMR analysis (see Figure 12B ) also showed a forward shift and maintained a high purity of 97.8% (see Figure 14 ). This indicates that the NMR shift eliminates the presence of the zwitterion.

[0435] Scaled-up formation of the parent / free form from J09899

[0436] program

[0437] Try 1-1g scale up

[0438] PCLX-001 (J09899, HCl pattern 1, 1 g ± 10 mg) was weighed into a 20 ml vial and transferred to a 100 ml round-bottom flask and treated with 50 volumes (50 ml) of DCM at 50°C to form a yellow suspension. Subsequently, 1 equivalent of NaOH (added as a 1 M aqueous solution relative to the Cl ions calculated by IC) was added at 50°C and the sample was slowly cooled to 5°C at 0.1°C / min to form a light yellow solid. All isolated solids were analyzed by XRPD. Sample ID: EG-1826-12-01. Results: XRPD analysis showed that the sample conformed to the "mixed salt" pattern observed in the salt breaking experiment using ethyl acetate as solvent.

[0439] Try 2-1g scale up

[0440] PCLX-001 (J09899, HCl mode 1, 1 g ± 10 mg) was weighed into a 20 ml vial and transferred to a 100 ml round-bottom flask and treated with 50 volumes (50 ml) of DCM at 30° C. to form a yellow suspension. Subsequently, 1 equivalent of NaOH (2.4 mL, added as a 1 M aqueous solution relative to Cl ions calculated by IC) was added at 30° C. and stirred for 1 hour to form a yellow solution.

[0441] Water (50 ml) was added to produce separated aqueous and organic layers. The organic layer was removed using a separatory 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 the magnesium sulfate and the remaining solution was dried under vacuum at RT overnight. The isolated solid was analyzed by XRPD. Sample ID: EG-1826-12-02

[0442] Try 3-200mg to scale up

[0443] PCLX-001 (J09899, HCl mode 1, 200 mg ± 5 mg) was weighed into a 20 ml vial and treated with 50 volumes (10 ml) of DCM at 30° C. to form a yellow suspension. 1 equivalent of NaOH (447 μl, added as a 1 M aqueous solution relative to Cl ions calculated by IC) was then added at 30° C. and stirred for 1 hour to form a yellow solution.

[0444] Water (10 ml) was added to produce a separated aqueous and organic layer. The organic layer was removed using a separatory funnel and magnesium sulfate was added as a drying agent. The aqueous layer was washed with DCM to remove any remaining organic layer. After the magnesium sulfate agglomerated to indicate the drying endpoint, the organic layer was filtered from the magnesium sulfate and the remaining solution was dried under vacuum at RT overnight. All isolated solids were analyzed by XRPD. Sample ID: EG-1826-22-01→05.

[0445] Attempt 3 - Repeat 1g scale up

[0446] PCLX-001 (J09899, HCl mode 1, 1 g ± 10 mg) was weighed into a 20 ml vial and transferred to a 100 ml round-bottom flask and treated with 50 volumes (50 ml) of DCM at 30° C. to form a yellow suspension. Subsequently, 1 equivalent of NaOH (2.4 mL, added as a 1 M aqueous solution relative to Cl ions calculated by IC) was added at 30° C. and stirred for 1 hour to form a yellow solution.

[0447] Water (50 ml) was added to produce separated aqueous and organic layers. The organic layer was removed using a separatory funnel and magnesium sulfate was added as a drying agent. The aqueous layer was washed with DCM to remove any remaining organic layer. After the magnesium sulfate agglomerated to indicate the drying endpoint, the organic layer was filtered from the magnesium sulfate and the remaining solution was dried under vacuum at RT overnight. All isolated solids were analyzed by XRPD. Sample ID: EG-1826-25-01.

[0448] Results and discussion

[0449] Scale-up of free form Pattern 1 was successful at both the 200 mg and 1 g scales and was confirmed by XRPD.Fully characterised the parent / free form Pattern 1 (EG-1826-12-02) at the 1 g scale-up and are summarized in Table 19.

[0450] Table 19 Characteristics of the parent form scaled up

[0451]

[0452]

[0453] 1 H-NMR analysis ( Figure 15 ) further confirmed the formation of free form model 1 and maintained a high purity of 96.1% ( Figure 17 Thermal analysis ( Figure 16 ) showed a weight loss of 0.5% w / w from 50°C to 100°C, degradation at 250°C (onset), and a significant, broad endotherm between 50°C and 170°C (T peak = 169.6°C, 74 J / g). Static stability experiments showed no significant change in solid appearance or XRPD pattern, and high purity was retained under elevated storage conditions, confirming stability under high temperature and humidity. IC analysis indicated the presence of 0.1 molar equivalents of sodium in the sample, which could be eliminated in an additional washing step.

[0454] Additional scale-up procedures were confirmed using XRPD analysis of the free form Pattern 1 using EG-1865-12-02 as the primary reference. Figure 18 and Figure 19 An XRPD overlay of the free form of Pattern 1 is shown successfully scaled up.

[0455] Solubility test of free form mode 1

[0456] Solvent List

[0457] Table 20 summarizes the list of solvents used in the initial solubility testing of free form Model 1.

[0458] Table 20 List of solvents for initial solubility evaluation

[0459] serial number solvent ICH classification 1 2-Propanol 3 2 acetone 3 3 Acetonitrile 2 4 Ethanol:water (90:10 v / v) 3 5 Ethyl acetate 3 6 heptane 3 7 Methyl ethyl ketone 3 8 tert-Butyl methyl ether 3 9 THF 2 10 Toluene 2 11 Methanol 2 12 ethanol 3 14 DMSO 2 15 Isobutanol 3

[0460] Program 1

[0461] EG-1826-12-02 (free form model 1, 20 mg ± 1 mg) was weighed into 10 x 4 ml vials. The selected solvent system (Table 20) was added to each vial in equal portions (10 vol, 20 vol, 40 vol, 60 vol, 80 vol) until the sample dissolved or a maximum of 80 volumes (1.6 ml) was reached.

[0462] After adding each aliquot, the sample was first stirred at RT for 5 minutes, and then stirred for another 5 minutes at 50°C, 500 rpm on a PolarBear if the sample did not dissolve. If no dissolution occurred, the sample was cooled to RT before the next portion of solvent was added. All suspensions remaining after the solubility assessment had 2 equivalents of HCl (74.5 μl, added as a 1 M stock solution in THF, relative to 20 mg of parent / free form) added to each vial at 50°C. All solids obtained were initially analyzed by XRPD.

[0463] Results and discussion

[0464] The results of initial solubility testing of the parent form are summarized below.Solubility testing was performed to select appropriate solvents for salt screening rather than to perform full solubility assessments (which are listed in Table 23).

[0465] No solubility was observed in any solvent except DMSO. DMSO showed very high solubility, forming a clear yellow solution upon addition of 10 volumes of solvent at RT.

[0466] After the solubility evaluation was completed, two molar equivalents of HCl were added to each sample to evaluate whether dissolution occurred after the addition of the counterion. Dissolution was not achieved in toluene, TBME, and heptane, but was successful with all remaining solvents.

[0467] Extended solubility evaluation of free form model 1

[0468] program

[0469] EG-1826-25-01 (parent / free form, 10 mg ± 1 mg from 1 g scale-up attempt 3) was weighed into 22×HPLC vials. The selected solvent system (see Table 22) was added to each vial in equal portions (10 vol, 20 vol, 40 vol, 60 vol, 80 vol) until the sample dissolved or a maximum of 80 volumes was reached.

[0470] After adding each aliquot, the sample was first stirred at RT for 5 minutes. If the sample did not dissolve, it was then stirred for another 5 minutes at 50°C and 500 rpm on a PolarBear. If dissolution did not occur, the sample was cooled to RT before the next portion of solvent was added. All solutions were slowly cooled to 5°C at 0.1°C / min. All suspensions were matured in a 25 / 50°C maturation chamber in 4 hour cycles for a maximum of 2 days. All solids obtained were initially analyzed by XRPD.

[0471] Table 22 List of solvents used for extended solubility evaluation

[0472] serial number solvent ICH classification 1 1,4-Dioxane 2 2 2-Propanol 3 3 2-MethylTHF Uncategorized 4 acetone 3 5 Acetonitrile 2 6 dichloromethane 2 7 dimethyl sulfoxide 2 9 N,N'-dimethylacetamide 2 10 ethanol 3 11 MeOH:water (90:10 v / v) N / A 12 Ethyl acetate 3 13 Ethanol:water 50:50 N / A 14 Isopropyl acetate 3 15 Methanol 2 16 Isobutanol 3 17 NMP 2 18 tert-Butyl methyl ether 3 19 THF 2 20 Anisole 3 21 water N / A 22 2-Methoxyethanol 2

[0473] Results and discussion

[0474] The results of the extended solubility evaluation are summarized in Tables 23-26 and Figures 20 to 29 Only free form pattern 2 was generated from the extended solubility evaluation.

[0475] Table 23 Observations from the Extended Solubility Evaluation of EG-1826-25-01 (Parent Form) (1 of 2)

[0476]

[0477] ×=not dissolved, √=dissolved

[0478] Table 24 Observations from the Extended Solubility Evaluation of EG-1826-25-01 (Parent Form) (2 of 2)

[0479]

[0480] ×=not dissolved, √=dissolved

[0481] Table 25 Results of extended solubility evaluation of EG-1826-25-01 (parent form)

[0482]

[0483] Table 26 Characterization of free form model 2

[0484]

[0485] Two samples resulting from the solubility evaluation were further characterized. 1 H-NMR showed that the spectrum was consistent with the structure of 0.14 equivalents of acetone and 0.2 equivalents of isopropyl acetate present in EG-1826-28-04 ( Figure 24 and Figure 25 ).

[0486] Thermal analysis of EG-1826-28-04 ( Figure 22 ) showed a weight loss of 0.2% at 50-60°C, followed by a weight loss of 0.8% at 200-220°C, with the degradation onset at 250°C. A significant and sharp endotherm of 223 J / g was observed at 209.1°C (onset), followed by a small and sharp recrystallization peak of 15 J / g at 219°C (onset). Figure 23 ), where a 0.2% weight loss was observed at 70-80° C., followed by a greater weight loss of 4.1% at 190-240° C., and a degradation onset of 250° C. A significant and sharp endotherm of 93 J / g was observed at 214.6° C. (onset), followed by a sharp recrystallization peak of 63 J / g at 218.6° C. (onset).

[0487] Static stability testing showed no significant changes in appearance or XRPD pattern when stored for 7 days at elevated storage conditions of 25°C / 97% relative humidity and 40°C / 75% relative humidity. The amount of solvent noted in the NMR spectrum of the free form, Mode 2, represents residual / unbound solvent. This amount can be removed by heating and drying under vacuum. Controlled crystallization / optimization of the crystallization process can avoid issues with residual solvent content.

[0488] Heat the free form Mode 2 to 220°C

[0489] Since a significant recrystallization peak was observed in TGA analysis during characterization of samples from solubility evaluations, heating of free form Pattern 2 was performed. This experiment was performed to assess whether the recrystallization peak was associated with the formation of a new form or the same free form Pattern 2.

[0490] EG-1826-28-10 (free form pattern 2, 5 mg) was heated to 220° C. at 10° C. / min by TGA. The solid obtained was reanalyzed by XRPD. Sample ID: EG-1826-39-01.

[0491] Results and discussion

[0492] XRPD analysis of the sample heated to 220°C ( Figure 30 ) confirmed that the recrystallization peaks were consistent with the free form mode 2, and it could be inferred that the sample did not convert to another form at higher temperatures.

[0493] in conclusion

[0494] Characterization of compound (I) designated as HCl Pattern 1 (PCLX-001, batch J09899) confirmed that only 1.3 equivalents of chloride ion were observed from the anionic IC, which was lower than the expected 3 equivalents of chloride ion. In addition, 0.2 equivalents of sodium and 0.1 equivalents of calcium were observed in the cationic IC, which may have been generated during the manufacturing process of the compound. Characterization of other batches of PCLX-001 (J09951, J09952 and J09953, designated as HCl Pattern 1) observed sharp peaks in the XRPD diffraction pattern, which is consistent with the presence of a higher proportion of NaCl in the sample than in PCLX-001 (J09899).

[0495] Salt breaking tests were performed on five process solvents using sodium hydroxide. XRPD analysis of samples obtained from DCM showed the formation of a new pattern which was later assigned as free form pattern 1. This was due to the 1The forward shift observed in the H-NMR spectrum indicated the formation of the free form. This was confirmed by IC analysis, which showed no anions or cations, confirming the production of free form Model 1. A high purity of 96.3% was also maintained in free form Model 1. Free form Model 1 was also successfully scaled up to 200 mg and 1 g.

[0496] The polymorphic screening performed on free form pattern 1 resulted in only one new pattern, designated free form pattern 2. Since a significant recrystallization peak was observed in DSC analysis during characterization of free form pattern 2, free form pattern 2 was also heated.

[0497] In conclusion, among all the polymorphs found, the free form Pattern 2 showed the most desirable solid-state properties.

[0498] Example 2 - Solubility Measurement and Crystallization Evaluation of PCLX-001 Free Form Model 2

[0499] summary

[0500] This article summarizes the solubility measurements and crystal development performed on the free form of PCLX-001, Model 2.

[0501] Characterization of PCLX-001 free form Pattern 2 (J10206) by XRPD analysis was confirmed to be crystalline with a purity of 98.85%. 1 H-NMR and Raman spectra matched the proposed molecular structure, with no anions or cations observed.

[0502] Solubility experiments were performed using gravimetric analysis. Based on the solubility evaluation results described in Example 1, solubility was measured in DMSO, DMA, and NMP. The maximum solubility range between 60°C and 25°C was observed in DMSO, with a calculated solubility of 32 mg / ml at 60°C and 12.3 mg / ml at 25°C. DMSO was selected as the preferred pure solvent for process development because it had already been used in the crystallization process.

[0503] In addition, to investigate solvent ratios to determine the ratio with the best results, the solvent systems were ranked using gravimetric analysis to determine solubility at both 25°C and 60°C. The solvent systems selected for solubility curve generation 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).

[0504] Solubility curves were successfully generated in all selected solvent systems. As can be seen from the solubility curves, the solubility curves in DMSO EtOH:H2O (1:2) (70:30) and DMSO EtOH:H2O (2:1) (50:50) were significantly lower than those in pure DMSO and DMSO EtOH:H2O (1:2) (95:5). Since the addition of antisolvents showed a favorable effect on increasing the yield of PCLX-001 free form Mode 2, the solubility data from DMSO EtOH:H2O (1:2) (95:5) were input into DynoChem software for process development.

[0505] Trial seeding crystallisations were performed using two different procedures. The first trial seeding crystallisation involved the addition of 5% anti-solvent followed by the addition of seeds at elevated temperature. During this trial seeding crystallisation it was observed that the sample fragmented upon the addition of 5% anti-solvent prior to the addition of seeds. This was expected from the predictions generated on DynoChem. Another trial seeding crystallisation was performed where the seeds were followed by the addition of 10% anti-solvent. This successfully crystallised PCLX-001 free form Pattern 2 on a 50mg scale with an 84% yield. This was slightly lower than the 95% yield predicted by DynoChem; however, as the crystallisation process had not yet been fully optimised, a slightly lower yield was expected.

[0506] Successful scale-up of the selected solvent system DMSO EtOH:H2O (1:2) (10%) on a 700 mg scale was performed. Further characterization confirmed that the material was consistent with PCLX-001 free form Model 2.

[0507] Abbreviations

[0508]

[0509]

[0510] Instrument and method details

[0511] X-ray powder diffraction (XRPD)

[0512] Bruker AXSD8 Advance

[0513] XRPD diffractograms were collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA) and a θ-2θ goniometer equipped with a Ge monochromator. The incident beam passed through a 2.0 mm divergence slit, followed by a 0.2 mm anti-scatter slit and a knife-edge. The diffracted beam passed through an 8.0 mm receiving slit and a 2.5° Soller slit before detection by a Lynxeye detector. The software used for data acquisition and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA, respectively.

[0514] The samples were tested as flat samples using powder under ambient conditions. The samples were prepared on polished zero-background (510) silicon wafers by gently pressing them onto a flat surface or by inserting them into a cutting chamber. The samples were rotated in their own plane.

[0515] Details of the standard data collection method are:

[0516] Angle range: 2 to 42°2θ

[0517] Step size: 0.05°2θ

[0518] Acquisition time: 0.5s / step (total acquisition time: 6.40min)

[0519] PANalytical Empyrean

[0520] XRPD diffractograms were collected on a PANalytical Empyrean diffractometer in transmission geometry using Cu Kα radiation (45 kV, 40 mA). A 0.5° slit, a 4 mm mask, and a 0.04 rad Soller slit with a focusing lens were used for the incident beam. A PIXcel placed on the diffracted beam 3D The detector was equipped with a receiving slit and a 0.04 rad Soller slit. Data were collected using the X'Pert Data Collector software with the X'Pert user interface. Data were analyzed and presented using Diffrac Plus EVA or HighScorePlus.

[0521] Samples were prepared and analyzed in transmission mode in metal or Millipore 96-well plates. X-ray transmission film was used between the metal sheets of the metal well plates and powder (approximately 1-2 mg) was used. Millipore plates were used to separate and analyze solids in suspensions by adding small amounts of suspensions directly to the plates and filtering under light vacuum.

[0522] The scanning mode for the metal plates used a side angle scan axis, while the Millipore plates were scanned in 2θ.

[0523] Details of the standard screening data collection method are:

[0524] Angular range: 2.5 to 32.0° 2θ

[0525] Step size: 0.0130°2θ

[0526] Acquisition time: 12.75s / step (total acquisition time is 2.07min)

[0527] Nuclear magnetic resonance (NMR)

[0528] Solution-state NMR

[0529] 1 H NMR and / or 13 C NMR spectra were collected on a Bruker 400 MHz instrument equipped with an autosampler and controlled by a DRX400 console. Unless otherwise stated, samples were prepared in DMSO-d6 solvent. Automated experiments were performed using the ICON NMR configuration within Topspin software, using standard Bruker loading protocols ( 1 H, 13 C{ 1 H}, DEPT135). Offline analysis was performed using an ACD Spectrus Processor.

[0530] Differential Scanning Calorimetry (DSC)

[0531] TA Instruments Q2000

[0532] DSC data were collected on a TA Instruments Q2000 equipped with a 50-position autosampler. Typically, 0.5-3 mg of each sample was placed in a perforated aluminum dish and heated from 25° C. to 300° C. at 10° C. / min. A dry nitrogen purge at 50 ml / min was maintained.

[0533] The instrument control software was Advantage for Q Series and Thermal Advantage, and the data were analyzed using Universal Analysis or TRIOS.

[0534] Thermogravimetric analysis (TGA)

[0535] TAInstruments Q500

[0536] TGA data were collected on a TA Instruments Q500 TGA equipped with a 16-position autosampler. Typically, 5-10 mg of each sample was loaded onto a pre-tared aluminum DSC dish and heated from ambient temperature to 350° C. at 10° C. / min. A nitrogen purge of 60 ml / min was maintained over the sample.

[0537] The instrument control software was Advantage for Q Series and Thermal Advantage, and the data were analyzed using Universal Analysis or TRIOS.

[0538] Polarized Light Microscopy (PLM)

[0539] Leica LM / DM polarized light microscope

[0540] Samples were analyzed on a Leica LM / DM polarized light microscope equipped with a digital 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 coverslip. Samples were observed at appropriate magnification using partially polarized light and a lambda pseudocolor filter. Images were acquired using StudioCapture or Image ProPlus software.

[0541] Scanning electron microscopy (SEM)

[0542] Data were collected on a Phenom Pro scanning electron microscope. A small sample was mounted on an aluminum stub using conductive double-sided tape. A thin layer of gold was applied using a sputter coater (20 mA, 120 s).

[0543] Chemical purity determination by HPLC

[0544] Purity analysis was performed on an Agilent HP1100 / Infinity II 1260 Series system equipped with a diode array detector using OpenLAB software. Full method details are provided below:

[0545] Table 27 HPLC method for chemical purity determination

[0546]

[0547] Karl Fischer titration (KF) water determination

[0548] The water content of each sample was measured using an 851 Titrano coulometer on a Metrohm 874 oven sample processor at 150°C with a Hydranal Coulomat AG oven reagent and nitrogen purge. Weighed solid samples were placed in sealed sample vials. Each titration used approximately 10 mg of sample, and replicates were performed. Unless otherwise stated, results are presented as averages. Data acquisition and analysis were performed using Tiamo software.

[0549] Ion chromatography (IC)

[0550] Data were acquired on a Metrohm 930 Compact IC Flex equipped with an 858 Professional autosampler and an 800 Dosino dosage unit monitor using IC MagicNet software. Accurately weighed samples were prepared as stock solutions in appropriate solvents. Quantification was achieved by comparison with standard solutions of known concentrations of the analyte. Analyses were performed in duplicate, and the average values are presented unless otherwise stated.

[0551] Table 28 IC method for cation chromatography

[0552]

[0553] Table 29 IC method of anion chromatography

[0554]

[0555] Raman spectroscopy

[0556] Data were acquired on a Renishaw inVia Qontor. Instrument control, data analysis, and display software were WiRE.

[0557] Method: Excitation source, λ ex =785nm laser, properly attenuated to avoid sample degradation. Raman shift range: 100-5000cm -1 ; Exposure time: 0.02-10s; Cumulative times: 1-3.

[0558] Crystal16

[0559] A Crystal 16 crystallization system (Technobis, NL) was used to determine the material solubility and metastable zone as a function of temperature. API slurries of varying total concentrations were prepared by adding a known amount of solid to a known amount of cooled solvent (0.5 to 1.5 ml) and stirring at 500 rpm using a magnetic bar. Saturation temperature was measured by heating and cooling cycles from 65°C to 23°C at a rate of 0.5°C / min.

[0560] When the temperature is raised, the solid is completely dissolved and the suspension becomes a clear solution, so that the transmittance reaches a maximum. This temperature is designated as the clearing point, which is considered to be consistent with the saturation temperature. The solution is then cooled at a rate of 0.5°C / min and the temperature at which particles first form is detected by a decrease in light transmittance. This is designated as the cloud point. These points are fitted by the Van'tHoff equation, and the difference between the cloud point and the clearing point defines the metastable zone width (MSZW) of the system. The instrument control software is Crystallisation Systems, and the data are analyzed using Crystal Clear and Microsoft Excel.

[0561] Characterization of Compound (I) (PCLX-001, free form model 2, batch J10206)

[0562] The material (Batch J10206) was characterized using a variety of techniques to investigate the solid form and chemical properties of PCLX-001 free form Model 2. The results are summarized in Table 30.

[0563] Table 30 Characterization data of PCLX-001 free form model 2 (J10206)

[0564]

[0565] By XRPD analysis ( Figure 37 and Figure 38 ) Characterization of PCLX-001 free form model 2 (J10206) confirmed that it was crystalline with a purity of 98.85% ( Figure 42 ). 1 H-NMR ( Figure 39 and Figure 40 ) and Raman ( Figure 43 and Figure 44 ) spectrum matches the proposed molecular structure, with no anions or cations observed. Thermal analysis of PCLX-001 free form mode 2 (J10206) ( Figure 41 ) showed that the material had a significant endotherm of 92 J / g at 220°C (onset), followed by a significant exotherm of 65 J / g at 225°C (peak). A weight loss of 0.6% w / w was observed from 200°C to 240°C, with the material degrading at 260°C (onset). The weight loss was associated with the loss of water from the sample, as 0.3% water was observed in the sample by KF analysis. Solubility (pH profile) analysis showed that the compound became increasingly soluble in acidic media, with samples suspended at pH <4.5 becoming clear.

[0566] Solubility determination and solubility curve construction

[0567] Solubility of J10206 (pH distribution in 7 pH buffer media)

[0568]

[0569] Methods and Instructions

[0570] pH distribution

[0571] Sufficient sample is suspended in 1.0 ml of medium to achieve a maximum expected concentration of 10 mg / ml of the free form of the compound. The resulting suspension is then shaken at 25°C / 750 rpm for 24 hours. The pH of the sample solution is checked regularly and adjusted with 0.2 M HCl / NaOH as necessary to ensure that the desired pH value (± 0.2) is always maintained. After equilibrium, the appearance is recorded and the final pH of the saturated solution is measured. The sample is then diluted with the appropriate relevant buffer solution after filtering through a glass "C" fiber filter (particle cutoff size 1.2 μm).

[0572] Quantification was performed by HPLC with reference to a standard solution of approximately 0.15 mg / ml. Different volumes of standard solution, diluted and undiluted sample solutions were injected. Solubility was calculated using the peak area determined by integrating the peak found at the same retention time as the main peak in the standard injection.

[0573]

[0574] *Adjusted with 60 and 70 μL of 0.2 and 0.5 M HCl, respectively.

[0575] **pH adjusted with 0.5 M HCl.

[0576] ***pH was adjusted with 5 and 2 μL of 0.2 M HCl and NaOH, respectively.

[0577] observe

[0578] The sample suspended in pH 1.2 buffer became clear 1 hour before pH adjustment, while all other samples were either turbid or contained residual solids. After 1 hour of pH adjustment, the sample suspended in pH 2.0 buffer became clear. During the 24-hour equilibration process, all remaining samples were either turbid or contained residual solids.

[0579]

[0580]

[0581]

[0582] Results, Summary, and Conclusions

[0583] Compounds become increasingly soluble in acidic media, where samples suspended at pH < 4.5 become clear. Samples suspended at pH 1.2 were clear 1 hour before pH adjustment, so the concentrations before pH adjustment are quoted. Samples suspended at pH 2.0 became clear 1 hour after pH adjustment, so the concentrations are quoted for the total volume of medium after adjustment. See Figure 51 , where the two points between pH 0 and 2 are greater than the solubility value.

[0584] Solubility determination in DMSO, DMA, and NMP

[0585] The solubility in DMSO, DMA and NMP was measured based on the solubility evaluation results of Example 1. This was done at two different temperatures: 60°C and RT.

[0586] program

[0587] Solubility determination at 60°C

[0588] J10206 (30 mg ± 1 mg) was weighed into 4 x HPLC vials and a stir bar was added. The sample was dissolved in the selected solvents DMSO, DMA, and NMP (1 ml) and heated to 60°C on a Polar Bear with stirring for 2-3 hours to allow the sample to equilibrate. The sample was then filtered and the mother liquor was placed in a pre-weighed vial and evaporated in a vacuum oven at room temperature overnight. The solid isolated after filtration was analyzed by XRPD.

[0589] Solubility determination at 25°C

[0590] J10206 (30 mg ± 1 mg) was weighed into 4 x HPLC vials and a stir bar was added. The sample was dissolved in the selected solvents DMSO, DMA, and NMP (1 ml) and stirred in a stirrer at 25°C for 2-3 hours to allow the sample to equilibrate. The sample was then filtered, and the mother liquor was placed in a pre-weighed vial and evaporated in a vacuum oven at room temperature overnight. The solid isolated after filtration was analyzed by XRPD.

[0591] Results and discussion

[0592] The maximum solubility range between 60°C and 25°C was observed in DMSO, with a calculated solubility of 32 mg / ml at 60°C and 12.3 mg / ml at 25°C in DMSO. No large solubility range between 60°C and 25°C was observed using DMA and NMP as solvents. DMSO was selected as the preferred pure solvent for process development because it had already been used in the crystallization process. See Tables 31 and 32. Figure 45 .

[0593] Table 31 Observation results of solubility determination of PCLX-001 free form model 2 (J10206) in DMSO, DMA and NMP

[0594]

[0595] Table 32 Gravimetric solubility results of PCLX-001 free form model 2 (J10206) solubility determination in DMSO, DMA and NMP

[0596]

[0597] Solubility determination using EtOH:H2O mixture as antisolvent

[0598] In parallel, to investigate solvent ratios to determine the ratio that yielded the best results, a series of solvent systems were run to determine solubility using gravimetric analysis at both 25° C. and 60° C. Observations were recorded during the solubility array experiment to monitor whether oiling out occurred during the process.

[0599] program

[0600] Solubility determination at 60°C

[0601] J10206 (30 mg ± 1 mg) was weighed into a 15× HPLC vial and a stir bar was added. The sample was dissolved in a selected ratio of DMSO and EtOH:H2O (1:1), EtOH:H2O (1:2), or EtOH:H2O (2:1) (1 ml) solvent and heated to 60°C on a PolarBear and stirred for 2-3 hours to allow the sample to equilibrate. The sample was then filtered and the mother liquor was placed in a pre-weighed vial and evaporated in a vacuum oven at RT overnight. The solid isolated after filtration was analyzed by XRPD.

[0602] Solubility determination at 25°C

[0603] J10206 (30 mg ± 1 mg) was weighed into a 15× HPLC vial and a stir bar was added. The sample was dissolved in a selected ratio of DMSO and EtOH:H2O (1:1), EtOH:H2O (1:2), or EtOH:H2O (2:1) (1 ml) solvent and heated to 25°C on a PolarBear and stirred for 2-3 hours to allow the sample to equilibrate. The sample was then filtered and the mother liquor was placed in a pre-weighed vial and evaporated in a vacuum oven at RT overnight. The solid isolated after filtration was analyzed by XRPD.

[0604] Results and discussion

[0605] Due to the controlled range of solubility observed from 60°C to 25°C, the DMSO to antisolvent ratios selected for process development were DMSO:EtOH:H2O (1:2) (70:30) and DMSO EtOH:H2O (1:1) (50:50). See Tables 33 to 36, Figure 46A and Figure 46B .

[0606] Table 33 Observations of solubility measurements of PCLX-001 free form Model 2 (J10206) at 60°C using EtOH:H2O mixture as antisolvent

[0607]

[0608] Table 34 Gravimetric solubility results for PCLX-001 free form Model 2 (J10206) at 60°C using EtOH:H2O mixture as antisolvent

[0609]

[0610] *Since the suspension is thick, filtration takes a long time, so cooling the solvent may result in lower solubility

[0611] Table 35 Observations of solubility measurements of PCLX-001 free form Model 2 (J10206) at 25°C using EtOH:H2O mixture as antisolvent

[0612]

[0613] Table 36 Gravimetric solubility results for PCLX-001 free form Model 2 (J10206) at 25°C using EtOH:H2O mixture as antisolvent

[0614]

[0615] Repeated solubility measurements using EtOH:H2O mixtures as antisolvents

[0616] Previous solubility determination experiments were held for 2-3 hours to allow the samples to equilibrate (see above and Tables 33 to 36). The solubility determination experiments were repeated with an extended equilibrium time of 24 hours to obtain more accurate solubility values. These experiments were repeated for extreme ratios of DMSO to antisolvent.

[0617] program

[0618] Solubility determination at 60°C

[0619] J10206 (30 mg ± 1 mg) was weighed into a 15× HPLC vial and a stir bar was added. The sample was dissolved in a selected ratio of DMSO and EtOH:H2O (1:1) or EtOH:H2O (2:1) (1 ml) solvent and heated to 60°C on a Polar Bear with stirring for 24 hours to allow the sample to equilibrate. The sample was then filtered and the mother liquor was placed in a pre-weighed vial and evaporated in a vacuum oven at RT for 72 hours. The solid isolated after filtration was analyzed by XRPD.

[0620] Solubility determination at 25°C

[0621] J10206 (30 mg ± 1 mg) was weighed into a 15× HPLC vial and a stir bar was added. The sample was dissolved in a selected ratio of DMSO and EtOH:H2O (1:1), EtOH:H2O (1:2), or EtOH:H2O (2:1) (1 ml) solvent and heated to 25°C on a PolarBear and stirred for 24 hours to allow the sample to equilibrate. The sample was then filtered and the mother liquor was placed in a pre-weighed vial and evaporated in a vacuum oven at RT for 72 hours. The solid isolated after filtration was analyzed by XRPD.

[0622] Results and discussion

[0623] Compared to previous solubility determination experiments where samples were allowed to equilibrate for 2-3 hours, the extended equilibration time of 24 hours showed no significant difference in solubility values. Therefore, it was determined that the selected solvent systems DMSO:EtOH:H2O (1:2) (70:30) and DMSO EtOH:H2O (1:1) (50:50) would generate solubility curves for process development. See Tables 37 to 38, Figure 47 .

[0624] Table 37 Observations from repeated solubility measurements of PCLX-001 free form Model 2 (J10206) at 60°C and 25°C using EtOH:H2O mixture as antisolvent

[0625]

[0626] Table 38 Gravimetric solubility results of replicate solubility measurements of PCLX-001 free form Model 2 (J10206) at 60°C and 25°C using EtOH:H2O mixture as antisolvent

[0627]

[0628] *Abnormal, repeat the experiment to evaluate whether the initial value is accurate

[0629] Extended solubility assay using EtOH:H2O mixture as antisolvent

[0630] program

[0631] Solubility determination in DMSO EtOH:H2O (1:2) (80:20)

[0632] J10206 was weighed into 2 x HPLC vials and suspended in increasing volumes of DMSO, EtOH:H2O (1:2) (80:20) up to 100 vol (1 ml) at 60° C. Observations were made at each solvent addition.

[0633] Solubility determination in DMSO EtOH:H2O (1:2) (95:5)

[0634] J10206 was weighed into an HPLC vial and suspended in increasing volumes of DMSO, EtOH:H2O (1:2) (95:5) up to 100 vol (1 ml) at 60° C. Observations were made at each addition of solvent.

[0635] Results and discussion

[0636] No dissolution was observed in either 10 mg or 3 mg samples using up to 100 volumes of DMSO EtOH:HO (1:2) (80:20) solvent. The sample was observed to be soluble in 80 volumes of DMSO EtOH:HO (1:2) (95:5) at 60°C, which yielded a good solubility range at a lower temperature of 25°C. Therefore, a solubility curve was generated using DMSO EtOH:HO (1:2) (95:5) for process development.

[0637] Table 39 Observations from extended solubility assay of PCLX-001 free form Model 2 (J10206) using EtOH:H2O mixture as antisolvent

[0638]

[0639] Construction of solubility curves

[0640] Solubility curves for J10206 (PCLX-001 free form, Model 2) were generated in DMSO, DMSO EtOH:H2O (2:1) (50:50), DMSO EtOH:H2O (1:2) (70:30), and DMSO EtOH:H2O (1:2) (95:5). Solubility curves were generated using Crystal 16 to select solvents for process development ( Figure 31 ).

[0641] Solubility curves were successfully generated in all selected solvent systems. As can be seen from the solubility curves, the solubility curves in DMSO EtOH: HO (1:2) (70:30) and DMSO EtOH: HO (2:1) (50:50) were lower than those in pure DMSO and DMSO EtOH: HO (1:2) (95:5). Since the addition of antisolvent is believed to have a beneficial effect in increasing the yield of PCLX-001 free form Mode 2 (see DynoChem below), the solubility data from DMSO EtOH: HO (1:2) (95:5) was entered into the DynoChem software for process development.

[0642] Differences in the solubility of Crystal 16

[0643] During the Crystal 16 experiment, a peak was observed at the beginning of the Crystal 16 experiment, which may indicate conversion or oiling out of the sample. Solubility of the sample was also observed at 55°C, which was unexpected as solubility of the sample was expected to be observed at 25°C. Due to the differences in solubility and peaks observed in Crystal 16, one of the Crystal 16 experiments was repeated in Polar Bear and observed at 10°C intervals.

[0644] Repeating the Crystal16 experiment on PolarBear

[0645] program

[0646] J10206 (3.83 mg) was weighed into an HPLC vial and suspended in DMSO, EtOH:H2O (2:1) (50:50). The sample was gradually heated from 23°C to 65°C at 0.5°C / min on a Polar Bear to simulate the conditions observed in Crystal 16. Photos of the sample were taken at 35, 55, and 65°C, and a micrograph of the sample was taken at 45°C to assess the solubility of the sample upon heating and whether the solubility values observed in Crystal 16 were accurate.

[0647] Results and discussion

[0648] Photos taken at 23, 35, and 55°C showed a yellowish, hazy suspension, indicating that the sample was not completely dissolved. Microscopic examination at 45°C showed crystals up to 100 μm in size, which confirmed that the sample was not completely dissolved, but also ruled out oiling. Complete dissolution of the sample was observed at 65°C, confirming that the results of the Crystal 16 experiment were accurate.

[0649] DynoChem

[0650] On DynoChem, graphs were generated from the solubility data for DMSO EtOH:H2O (1:2) (95:5) to predict the optimal crystallization process based on the time point of seed addition and the amount of antisolvent required. From these graphs, it was recommended to add seeds after cooling the crystals, e.g. Figure 32A This is shown by the middle blue dashed line. This is due to a gradual, controlled decrease in solubility upon cooling from 60°C to 25°C, followed by a small drop in solubility upon seeding at 25°C. Compared to the red dashed line, a large drop in solubility is observed, as expected, due to sample precipitation caused by the presence of the antisolvent. This is considered undesirable due to the lack of control over crystallization.

[0651] Based on the prediction of the amount of antisolvent required for crystallization, e.g. Figure 32B As shown, increasing the antisolvent beyond 11% begins to reduce solubility returns, which means lower yields. Therefore, antisolvents up to 10% antisolvent were used.

[0652] Crystallization in DMSO

[0653] Solubility data from DMSO and DMSO, EtOH:H2O (1:2) were input into DynoChem software to gain insights into the predicted crystallization of PCLX-001 free form Model 2 in pure DMSO. Figure 33 As shown, to produce 200 mg of PCLX-001 free Form 2 in 20 vol of DMSO and without antisolvent, the expected yield is 84%.

[0654] Crystallization in DMSO, EtOH:H2O (1:2) (5% antisolvent)

[0655] The solubility data from DMSO and DMSO, EtOH:H2O (1:2) were input into DynoChem software to gain insights into the crystallization predictions for PCLX-001 free form Model 2 in DMSO and 5% antisolvent EtOH:H2O (1:2). Figure 34 As shown, a 92% yield is expected to produce 200 mg of PCLX-001 free form 2 in 20 vol of DMSO and 5% antisolvent EtOH:H2O (1:2). This 7% increase in yield compared to pure DMSO highlights the beneficial effect of the antisolvent and that only a small amount of antisolvent is needed to have a large impact on the yield increase.

[0656] Crystallization in DMSO, EtOH:H2O (1:2) (10% antisolvent)

[0657] Solubility data from DMSO and DMSO, EtOH:H2O (1:2) were input into DynoChem software to gain insights into the crystallization predictions for PCLX-001 free form Model 2 in DMSO and 10% antisolvent EtOH:H2O (1:2). Figure 35 As shown, to produce 200 mg of PCLX-001 free form 2 in 20 vol of DMSO and 10% antisolvent EtOH:HO (1:2), a 95% yield is expected. For a 5% increase in antisolvent, the yield increased by 10% compared to pure DMSO and by 4% compared to DMSO EtOH:HO (1:2) (5%).

[0658] Crystallization in DMSO, EtOH:H2O (1:2) (20% antisolvent)

[0659] Solubility data from DMSO and DMSO, EtOH:H2O (1:2) were input into DynoChem software to gain insights into the crystallization predictions for PCLX-001 free form Model 2 in DMSO and 20% antisolvent EtOH:H2O (1:2). Figure 36 As shown, to produce 200 mg of PCLX-001 free form 2 in 20 vol of DMSO and 20% antisolvent EtOH:HO (1:2), a 98% yield is expected. Compared to DMSO EtOH:HO (1:2) (10%), a 10% increase in total antisolvent resulted in a 2.5% increase in yield, indicating that there is little yield gain for the additional volume in the solvent.

[0660] Trial crystallization of J10206

[0661] program

[0662] J10206 (50 mg ± 1 mg) was weighed into 2 x 4 ml vials and dissolved in DMSO (20 vol, 1 ml) on a Polar Bear at 65° C. The sample was then cooled to 59° C. to ensure a supersaturation ratio of 1.3.

[0663] Add seed crystals before adding 10% antisolvent

[0664] ~10 mg of seed crystals were added to another vial at 59 ° C and stirred for 5 mins to evaluate whether the seed crystals were retained. Observation showed that the seed crystals were able to be retained, so the sample was cooled to 25 ° C at 0.5 ° C / min and 10% anti-solvent EtOH:H2O (1:2) (2 vol, 100 μl) was added to the vial. The sample was stirred at 25 ° C for 24 hours to form a light yellow suspension. The sample was filtered by positive pressure under nitrogen and dried in a vacuum oven to calculate the wet yield and dry yield. Sample ID: EG-1826-62-02. The sample was analyzed by XRPD, NMR, PLM, SEM and HPLC ( Figure 55 , sample ID EG-1826-62-02).

[0665] DMSO-ethanol-water purification:

[0666] The crude material (PCLX-001; 7.0 g) was diluted with DMSO (20.0 Vol.) and gradually heated to 55 ± 5 ° C until the solution was clear for 10 ± 5 min. A mixture of ethanol (7.0 Vol.) and pure water (7.0 Vol.) was added to the reaction mass at 55 ± 5 ° C, and then stirred at 55 ± 5 ° C for 10 ± 5 min. The reaction mass was gradually cooled to 0 ± 5 ° C, stirred at 0 ± 5 ° C for 16 h, and then filtered. The wet material was dried at 50-55 ° C for 2 h. 7.0 g was taken for DMSO-ethanol-water purification, and 5.6 g of the final compound was isolated. HPLC purity after DMSO-ethanol-water purification: 99.58%. No seed crystals were present.

[0667] Results and discussion

[0668] Table 40 Characterization data of EG-1826-62-02

[0669]

[0670]

[0671] Characterization of EG-1826-62-02 confirmed the successful generation of crystalline PCLX-001 free form Pattern 2 ( Figure 48 ; Table 5.0A'). By 1 Further characterization by H-NMR analysis confirmed that EG-1826-62-02 was consistent with the proposed structure of PCLX-001 free form Mode 2, and SEM images showed that the sample consisted of irregular plate-like particles of ∼250 μm covered with smaller particles. Smaller particles in the 20-50 μm range and primary particles in the 2-5 μm range were also observed.

[0672] The yield of the crystallization was calculated to be 84%, slightly lower than the expected 95%. However, the lower yield was expected because the crystallization process was not fully optimized.

[0673] Table 5. Peaks at ±0.2 degrees 2θ of the X-ray powder diffraction pattern of EG-1826-62-02

[0674]

[0675]

[0676] Scale-up of selected systems (700 mg scale)

[0677] Procedure: Add 10% anti-solvent after adding seed crystals

[0678] J10206 (700 mg ± 10 mg) was weighed into a 50 ml container 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 that a supersaturation ratio of 1.3 was obtained. ~ 40 mg of seed crystals were added to the solution at 59 ° C and stirred for 5 minutes to maintain the seed crystals. 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 stirred at 25 ° C for 24 hours to form a light yellow suspension. The sample was filtered using a Buchner funnel and dried under vacuum for 20 min. The XRPD ( Figure 48 , Table 5.0B'), NMR, HPLC ( Figure 49 ) and PLM( Figure 50 ) Analyzed sample. Sample ID: EG-1826-63-01.

[0679] The same experiment was repeated to check that the purity profile remained unchanged. HPLC analysis was performed on a 200 mg scale. Sample ID: EG-1826-63-02. The data obtained from the experiments were consistent, with similar improvements in impurities observed in all cases, with a reduction in impurities at RRT 0.83.

[0680] See also Figures 56 to 58 .

[0681] Results and discussion

[0682] Table 41 Characterization data of J10206, EG-1826-63-01 and EG-1826-63-02

[0683]

[0684]

[0685] Characterization of EG-1826-63-01 confirmed the successful production of crystalline PCLX-001 free form Model 2. 1 Further characterization by H-NMR analysis confirmed that EG-1826-63-01 was consistent with the proposed structure of PCLX-001 free form Mode 2, and PLM images showed that the sample consisted of irregular plate-like particles of ∼100 μm covered with smaller particles. Smaller particles in the 20-50 μm range and primary particles in the 2-5 μm range were also observed.

[0686] The yield of the scaled-up crystallization was calculated to be 87%, slightly lower than the expected value of 95%. However, since the crystallization process was not fully optimized, the lower yield was expected.

[0687] Table 5. Peaks at ±0.2 degrees 2θ of X-ray powder diffraction pattern of 0B'EG-1826-63-01

[0688]

[0689]

[0690] in conclusion

[0691] Characterization of PCLX-001 free form Pattern 2 (J10206) by XRPD analysis confirmed it to be crystalline with a purity of 98.85%. 1 H-NMR and Raman spectra were consistent with the proposed molecular structure, with no anions or cations observed. Thermal analysis of PCLX-001 free form mode 2 (J10206) showed that the material had a significant endotherm of 92 J / g at 220°C (onset), followed by a significant exotherm of 65 J / g at 225°C (peak). A weight loss of 0.6% w / w was observed from 200°C to 240°C, with the material degrading at 260°C (onset). The weight loss was associated with the loss of water from the sample, as 0.3% water was observed in the sample by KF analysis. Solubility (pH profile) analysis showed that the compound became increasingly soluble in acidic media, with samples suspended at pH < 4.5 becoming clear.

[0692] Solubility experiments were performed using gravimetric analysis. Based on the solubility evaluation results from Example 1, solubility was measured in DMSO, DMA, and NMP. The maximum solubility range between 60°C and 25°C was observed in DMSO, with a calculated solubility of 32 mg / ml at 60°C and 12.3 mg / ml at 25°C. DMSO was selected as the preferred pure solvent for process development because it had already been used in the crystallization process.

[0693] In parallel, in order to investigate the solvent ratios to determine the ratio with the best results, a series of solvent systems were run to determine the solubility at two temperatures, 25°C and 60°C, using gravimetric analysis. The solvent systems selected for generating 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. As can be seen from the solubility curves, the solubility curves in DMSO EtOH:H2O (1:2) (70:30) and DMSO EtOH:H2O (2:1) (50:50) were lower than those in pure DMSO and DMSO EtOH:H2O (1:2) (95:5). Since the addition of antisolvent was shown to have a beneficial effect on increasing the yield of PCLX-001 free form Pattern 2, solubility data from DMSO EtOH: H2O (1:2) (95:5) was input into DynoChem software to develop the process. Based on the prediction of the amount of antisolvent required for crystallization, it was noted that increasing the antisolvent beyond 11% began to reduce the solubility return, which meant a lower yield return. Therefore, it was recommended to use up to 10% antisolvent.

[0694] Two different seed crystallization procedures were used. The first seed crystallization involved adding 5% antisolvent followed by seeding at elevated temperature. During this seed crystallization, it was observed that the sample fragmented upon adding 5% antisolvent, followed by seeding. This was expected from predictions generated on DynoChem. Another seed crystallization was performed, following seeding with 10% antisolvent. This successfully crystallized PCLX-001 free form Pattern 2 on a 50 mg scale with an 84% yield. This was slightly lower than the 95% yield predicted by DynoChem; however, a slightly lower yield was expected as the crystallization process was not fully optimized.

[0695] Successful scale-up of the selected solvent system DMSO EtOH:H2O (1:2) (10%) on a 700 mg scale was performed. 1 Further characterization by H-NMR analysis confirmed that EG-1826-63-01 was consistent with the proposed structure of PCLX-001 free form Model 2.

[0696] Example 3 - Bioequivalence PK Study of Salt and Free Base Forms of PCLX-001

[0697] Bioequivalence studies in rats and dogs

[0698] The pharmacokinetic characteristics of the free base and salt forms of PCLX-001 were compared in Sprague Dawley rats (Study 6902337) and dogs (Study 6902338). PCLX-001 (salt form; PCLX-001 HCl Model 1, Lot No. PYA / 19 / 001#I-036) and PCLX-001, PYAStage-H (free base form; PCLX-001 free form Model 2, Lot No. PYA / 19 / 001#H-124), adjusted to pH 2.5±0.5 in ultrapure water, were administered once by oral gavage at 125 mg free base / kg to male Sprague Dawley rats (n=6 / group) and 4 mg / kg to female beagle dogs (n=5 / group). These dose levels are the highest non-serious toxic doses (HNSTD) achieved in each species in a GLP-compliant 4-week study with a 2-week recovery period (rat study number 8002835, dog study number 8002836). Blood samples for pharmacokinetic evaluation were collected from all animals before and 30 minutes, 1, 2, 4, 7, and 24 hours after dosing. PK blood samples were processed into plasma and analyzed for PCLX-001 concentration using a validated LC-MS / MS method. The lower limit of quantification (LLOQ) was 5 ng / mL.

[0699] Test details:

[0700]

[0701]

[0702] Comparative PK study in rats

[0703] Following administration of both forms to rats, plasma concentrations of PCLX-001 were quantifiable throughout the 24-hour sampling period. Mean T values of both forms of PCLX-001 at 7 hours post-dose were observed. 最大 The average C of the salt form and the free base 最大 The AUC values of the salt form and free base were 44900 and 40000 ng / mL, respectively. 最后 In rats, the exposure of the two forms was similar, and the salt form had a C 最大 The ratio is 1.12, and the AUC 最后 The ratio is 1.13. 最大 The time points after administration were limited, and the elimination period was not characterized in any rats. Figure 52 and Table 42.

[0704] Table 42: Summary of Mean (±SE) Pharmacokinetic Parameters of Salt and Free Base PCLX-001 in Sprague-Dawley Rat Plasma Following Oral Administration of 125 mg / kg PCLX-001 on Day 1*

[0705]

[0706] * Due to C 最大 The time after that is limited, T 1 / 2 and AUC(0-inf) cannot be calculated R c最大 =C 最大 Salt form / C 最大 Free base; R AUC =AUC 最后 Salt form / AUC 最后 Free base; NA = not applicable

[0707] Comparative PK study in dogs

[0708] Following administration of the salt form to dogs, plasma concentrations of PCLX-001 were quantifiable throughout the 24-hour sampling period, with the exception of one animal where concentrations were quantifiable up to 7 hours post-dose. Following administration of the free base to dogs, plasma concentrations of PCLX-001 were quantifiable up to 24 hours post-dose in two animals and up to 7 hours post-dose in three animals. The mean C for PCLX-001 was 1.38 mmol / l. 最大 It was observed 1 hour after administration of the salt form and 0.5 or 1 hour after administration of the free base. 最大 After that, the concentration decreased. 1 / 2 The average C of the salt form and the free base after administration of PCLX-001 was 3.88 to 5.35 hours and 2.39 to 3.94 hours, respectively. 最大 The AUCs for the salt form and free base were 582 and 523 ng / mL, respectively. 最后 The salt and free base exposures were similar in dogs, with the salt form having a C 最大 The ratio is 1.1, AUC 最后 The ratio is 1.32. Figure 53 and Table 43.

[0709] Table 43: Summary of Mean (±SD) Pharmacokinetic Parameters of Salt and Free Base PCLX-001 in Beagle Dogs Following Oral Administration of 4 mg / kg PCLX-001 on Day 1

[0710]

[0711] In conclusion, the free base and salt forms of PCLX-001 exhibited similar pharmacokinetic profiles when administered orally as a single dose in HNSTD to rats and dogs and were therefore considered bioequivalent.

[0712] PK study of free base PCLX-001 in mice

[0713] In addition to the bioequivalence studies conducted above, a third PK study was conducted in Crl:CD1 (ICR) mice to obtain exposure data for the free base form of PCLX-001 at 35 mg / kg / day, a dose that was effective in previous mouse xenograft studies. PCLX-001 was orally administered to female mice at a dose of 35 mg / kg / day for 7 consecutive days. Blood samples for pharmacokinetic evaluation were collected from all animals before dosing and at 30 minutes, 1, 2, 4, 7, and 24 hours after dosing on days 1 and 7. Three mice were bled at each time point, and the data are expressed as the mean for each time point. C values of PCLX-001 were observed 4 hours after dosing on day 1 and 1 hour after dosing on day 7. 最大 Since C 最大 Due to limited time points after d1, the terminal elimination phase was not characterized on day 1. However, the maximum plasma concentration subsequently declined and the terminal half-life (T 1 / 2 ) was estimated to be 1.68 hours. After repeated daily dosing, the exposure on day 7 was similar to that on day 1, with AUC 最后 The ratio was 0.914 (91.4%). 最大 and AUC 最后 46900 ng / mL and 470000 hr*ng / mL respectively. Figure 54 and Table 44.

[0714] Table 44: Summary of pharmacokinetic parameters of free base PCLX-001 in plasma of female mice after oral administration of 35 mg / kg PCLX-001 on days 1 and 7 (± SE)

[0715]

[0716] NA = Not Applicable; NC = Not Calculated

[0717] R AUC =AUC on day 7 最后 / Day 1 AUC 最后

[0718] The embodiments described herein are merely exemplary. Those skilled in the art may make substitutions, modifications, and variations to the specific embodiments. The scope of the claims should not be limited by the specific embodiments set forth herein, but should be interpreted in a manner consistent with the specification as a whole.

[0719] All publications, patents, and patent applications mentioned in this specification are indicative of the levels of skill of 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.

[0720] The invention being thus described, it will be obvious that the invention 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 modifications obvious to one skilled in the art are intended to be included within the scope of the appended claims.

Claims

1. A compound (I) The crystal form, The crystalline form is characterized by the X-ray powder diffraction pattern shown in Figure 37.

2. A compound (I) The crystal form, The crystalline form is characterized by an X-ray powder diffraction pattern having peaks at 10.2 degrees, 11.4 degrees, and 20.5 degrees 2Θ ± 0.2 degrees 2Θ.

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

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

5. A compound (I) The crystal form, The crystalline form is characterized by having an X-ray powder diffraction pattern with peaks at ±0.2 degrees 2Θ substantially as provided in Table 1.0'.

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

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

8. The crystalline form according to any one of claims 1 to 7, wherein the crystalline form is characterized by comprising 103.2 cm -1 、993.0cm -1 and 1602.8cm -1 ±0.2cm -1 Wavelength value (cm -1 )’s Raman spectrum.

9. The crystalline form according to claim 8, wherein the crystalline form is further characterized by comprising 126.5 cm -1 、144.9cm -1 、227.3cm -1 、456.1cm -1 、1043.5cm -1 、1164.7cm -1 and 1582.5cm -1 ±0.2cm -1 Wavelength value (cm -1 )’s Raman spectrum.

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

11. A method for preparing a crystalline form of compound (I) according to any one of claims 1 to 9, comprising the following steps: adding the HCl 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 the free form of compound (I) from the first solution; adding the free form of the compound (I) to a second organic solvent to form a second mixture; aging the second mixture to form a third mixture; and The crystalline form of Compound (I) is isolated from the third mixture.

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

13. The method according to 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 The first solution was stirred for about 1 hour.

14. The method according to 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, heating from ambient temperature to about 50°C and then cooling from about 50°C to ambient temperature, and optionally adding additional organic solvent to the second mixture until the free form of the compound (I) is dissolved in the second organic solvent; and The temperature was lowered from about 50°C to about 5°C at a rate of about 0.1°C / min.

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

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

18. The process according to 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-methylTHF; 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 according to any one of claims 11 to 19, further comprising filtering insoluble particles from the first solution before isolating the free form of Compound (I) from the first solution.

21. A method for recrystallizing the crystalline form of compound (I) according to any one of claims 1 to 9, or the crystalline form of compound (I) prepared by the method according to any one of claims 11 to 20, comprising the following steps: dissolving the crystalline form of compound (I) in an organic solvent at a first temperature to form a first solution; cooling from the first temperature to a second temperature; adding seed crystals of the crystalline form of compound (I) to the first solution to form a first mixture; cooling from the second temperature to a third temperature; adding an antisolvent to the first mixture to form a second mixture; aging the second mixture for about 24 hours to form a third mixture; and The recrystallized crystalline form of Compound (I) is isolated 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 according to claim 21 or 22, wherein the organic solvent is DMSO.

24. The method of any one of claims 21 to 23, wherein the antisolvent 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 antisolvent 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 antisolvent is added in a volume of about 10% to about 20% of the volume of the organic solvent.

28. The method of claim 27, wherein the antisolvent is added in a volume of about 10% of the volume of the 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 decreasing the temperature from the second temperature to the third temperature comprises decreasing the temperature at 0.5°C / min.

33. The method of any one of claims 21 to 32, further comprising filtering insoluble particles from the first solution before cooling from the first temperature to the second temperature.

34. A method of treating a subject suffering from cancer, comprising: The subject is administered the crystalline form of Compound (I) according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 10.

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

36. The method of claim 34 or 35, wherein the cancer is 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 macroglobulinemia, MALT-type / monocytic 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, chronic myeloid leukemia blast crisis, Burkitt lymphoma, plasma cell myeloma, intestinal adenocarcinoma, mixed adenosquamous lung carcinoma, small cell lung carcinoma, lung carcinoma, esophageal squamous cell carcinoma, bone tumor, breast ductal carcinoma, diffuse gastric adenocarcinoma, medullary thyroid carcinoma, urinary tract transitional cell carcinoma, myeloma, ovarian clear cell carcinoma, transitional cell carcinoma (ureter and bladder), chronic myeloid leukemia (CML), lymphoma-CLL, breast cancer, colorectal adenocarcinoma, pancreatic cancer, ovarian cancer, non-small cell lung cancer, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, hepatobiliary carcinoma (bile duct cancer), gallbladder cancer, liver cancer, or esophageal squamous cell carcinoma.

39. The method of any one of claims 34 to 38, wherein the subject is a child, teenager, 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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