Crystalline forms of N-heteroaryl sulfonamides, compositions and methods thereof

Abstract

Crystalline forms of the N-heteroarylsulfonamide 2,6-dichloro-N-(3-isobutyl-1,5-dimethyl-1H-pyrazol-4-yl)-4-(2-(piperazin-1-yl)pyridin-4-yl)benzenesulfonamide and pharmaceutical compositions thereof; and methods of preparing the crystalline forms and methods of using the crystalline forms in the treatment of diseases.

Description

[Technical Field]

[0001] Field The present disclosure relates generally to crystalline forms of the N-heteroaryl sulfonamide 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 for preparing the crystalline forms, and methods of using the crystalline forms in the treatment of disease. [Background technology]

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

[0003] New crystalline forms of pharmaceutical compounds arise from the crystallization of the compound into different internal structures, e.g., different crystal lattice configurations. Such different internal structures (i.e., new crystalline forms) arise because the system tends toward a thermodynamically stable (i.e., lower energy) state. Summary of the Invention [Problem to be solved by the invention]

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

[0005] overview In certain embodiments of the present disclosure, a crystalline form of Compound (I) characterized by the X-ray powder diffraction pattern shown in Figure 37 is provided.

[0006] [ka]

[0007] is provided.

[0008] In another aspect of the disclosure, a crystalline form of Compound (I) characterized by an X-ray powder diffraction pattern having peaks at 10.2, 11.4, and 20.5 degrees two-theta ± 0.2 degrees two-theta.

[0009] [ka]

[0010] is provided.

[0011] In one embodiment of the present disclosure, a crystalline form is provided further characterized by an X-ray powder diffraction pattern having peaks at 14.6, 22.9, 26.1, and 31.0 degrees two-theta ± 0.2 degrees two-theta, hi 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 two-theta ± 0.2 degrees two-theta.

[0012] In another embodiment of the present disclosure, a crystalline form of Compound (I) characterized by an X-ray powder diffraction pattern having peaks ±0.2 degrees two-theta substantially as provided below in Table 1.0' (Table 1).

[0013] [ka]

[0014] is provided.

[0015] [Table 1]

[0016] In another embodiment of the present disclosure, there is provided a crystalline form characterized by a DSC thermogram obtained using a heating rate of 10° C. / min, comprising an endothermic event with an onset temperature of 220.2° C.±0.2° C. In another embodiment, the crystalline form is characterized by a DSC thermogram substantially as shown in FIG.

[0017] In one embodiment of the present disclosure, 103.2 cm -1 , and 993.0 cm -1 , and 1602.8 cm -1 ±0.2cm -1 Wave value (cm -1 In another embodiment, the crystalline form is characterized by a Raman spectrum comprising a peak at 126.5 cm -1 , 144.9cm -1 , 227.3cm -1 , 456.1cm -1 , 1043.5cm -1 , 1164.7cm -1 , and 1582.5 cm -1 ±0.2cm -1 Wave value (cm -1 In another embodiment, the crystalline form is further characterized by a Raman spectrum comprising wavevalues ​​(cm) substantially as provided below in Table 2.0'. -1 )±0.2cm -1 The Raman spectrum includes:

[0018] [Table 2A]

[0019] [Table 2B]

[0020] In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising a crystalline form of Compound (I) described herein and a pharmaceutically acceptable adjuvant, diluent, carrier, or vehicle.

[0021] In another aspect of the present disclosure, there is provided a method for preparing a crystalline form of Compound (I) described herein, comprising: 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; maturing the second mixture to form a third mixture; and isolating the crystalline form of Compound (I) from the third mixture. A method is provided that includes:

[0022] In another embodiment of the present disclosure, a method is provided, wherein the step of 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 the step of 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.

[0023] In another embodiment of the present disclosure, the step of adding the free form of Compound (I) to a second organic solvent to form a second mixture comprises: stirring the second mixture at ambient temperature; Optionally, increasing the temperature from ambient to about 50°C, and then decreasing the temperature from about 50°C to ambient; and Optionally, adding additional organic solvent to the second mixture until the free form of Compound (I) is dissolved in the second organic solvent; and Decrease the temperature from about 50°C to about 5°C at a rate of about 0.1°C / min. A method is provided, further comprising:

[0024] In another embodiment, a method is provided wherein maturing the second mixture to form a third mixture further comprises maturing at 25 / 50°C in a 4 hour cycle.

[0025] 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.

[0026] In another embodiment, a method is provided wherein the base is NaOH.

[0027] In another embodiment, a method is provided in which the second organic solvent is selected from the group consisting of 2-propanol; 2-methyl THF; acetone; acetonitrile; DMSO; N,N'-dimethylacetamide; MeOH:water (90:10 v / v); ethyl acetate; ethanol:water (50:50 v / v); isopropyl acetate; isobutanol; tert-butyl methyl ether; THF; and water. In another example, a method is described in which the second organic solvent is selected from the group consisting of 2-propanol; 2-methyl THF; acetone; acetonitrile; DMSO; N,N'-dimethylacetamide; MeOH:water (90:10 v / v); ethyl acetate; ethanol:water (50:50 v / v); isopropyl acetate; isobutanol; tert-butyl methyl ether; THF; and water.

[0028] In another embodiment of the present disclosure, a method is provided further comprising the step of filtering insoluble particulates from the first solution prior to the step of isolating the free form of Compound (I) from the first solution.

[0029] In another aspect of the disclosure, there is provided a method of recrystallizing a crystalline form of Compound (I) described herein or prepared by a method described herein, comprising: dissolving a crystalline form of Compound (I) in an organic solvent at a first temperature to form a first solution; decreasing the temperature from the first temperature to a second temperature; adding seed crystals of a crystalline form of Compound (I) to a first solution to form a first mixture; decreasing the temperature from the second temperature to a third temperature; adding an anti-solvent to the first mixture to form a second mixture; maturing the second mixture for about 24 hours to form a third mixture; isolating the recrystallized crystalline form of Compound (I) from the third mixture. A method is provided that includes:

[0030] 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.

[0031] In some embodiments, methods are provided in which the anti-solvent comprises water, a C1-C3 alcohol, or a combination thereof, hi some embodiments, the anti-solvent comprises water, a C1-C3 alcohol, or a combination thereof in an amount ranging between about 100% water and about 100% C1-C3 alcohol, or any combination of water and a C1-C3 alcohol between 100% water and 100% C1-C3 alcohol.

[0032] In some embodiments, the C1-C3 alcohol comprises a C1-C3 linear, branched, or cyclic alcohol. In some embodiments, the C1-C3 alcohol comprises methanol, ethanol, n-propanol, branched propanol, or a combination thereof.

[0033] In another embodiment, a method is provided wherein the anti-solvent is selected from the group consisting of ethanol and water. In some embodiments, the anti-solvent is selected from the group consisting of ethanol and water in amounts ranging between about 100% water and about 100% ethanol, or any combination of water and ethanol between 100% water and 100% ethanol. In another embodiment, a method is provided wherein the anti-solvent is selected from the group consisting of EtOH:HO (1:1); EtOH:HO (1:2); and EtOH:HO (2:1).

[0034] In another embodiment, a method is provided in which the organic solvent is DMSO and the anti-solvent is EtOH:H2O (1:1), EtOH:H2O (1:2), or EtOH:H2O (2:1). In another embodiment, the organic solvent is DMSO and the anti-solvent is EtOH:H2O (1:2).

[0035] In another embodiment, a method is provided in which the anti-solvent is added in a volume that is about 10% to about 20% of the volume of the organic solvent. In another embodiment, the anti-solvent is added in a volume that is about 10% of the volume of the organic solvent.

[0036] In another embodiment, a method is provided wherein the first temperature is about 65°C.

[0037] In another embodiment, a method is provided wherein the second temperature is about 59°C.

[0038] In another embodiment, a method is provided wherein the third temperature is about 25°C.

[0039] In another embodiment, a method is provided wherein decreasing the temperature from the second temperature to the third temperature comprises a decrease of 0.5° C. / min.

[0040] In another embodiment, a method is provided further comprising filtering insoluble particulates from the first solution prior to the step of lowering the temperature from the first temperature to the second temperature.

[0041] In another aspect of the present disclosure, there is provided a method of treating a subject having cancer, comprising administering to the subject a crystalline form of Compound (I) described herein, or a pharmaceutical composition described herein.

[0042] In another aspect of the present disclosure, there is provided a method of treating a subject having a cancer deficient in NMT2, comprising administering to the subject a crystalline form of Compound (I) described herein, or a pharmaceutical composition described herein.

[0043] In another embodiment of the present disclosure, a method is provided wherein the cancer is lymphoma, hi another embodiment, the lymphoma is B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom, MALT / monocytoid B-cell lymphoma, or Burkitt lymphoma.

[0044] In another embodiment, a method is provided wherein the cancer is anaplastic large cell lymphoma, acute myeloid leukemia, blastic phase chronic myeloid leukemia, Burkitt's lymphoma, plasma cell myeloma, intestinal adenocarcinoma, mixed adenosquamous carcinoma of the lung, small cell lung carcinoma, lung, esophageal squamous cell carcinoma, bone, ductal carcinoma of the breast, diffuse gastric adenocarcinoma, medullary thyroid carcinoma, transitional cell carcinoma of the urinary tract, myeloma, ovarian clear cell carcinoma, transitional cell carcinoma (ureter and bladder cancer), chronic myeloid leukemia (CML), lymphoma-CLL, breast cancer, colorectal adenocarcinoma, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung carcinoma, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, bile duct carcinoma (cholangiocarcinoma), gallbladder carcinoma, liver cancer, or esophageal squamous cell carcinoma.

[0045] In another embodiment, a method is provided wherein the subject is a child, adolescent, adult, or elderly. In another embodiment, the subject is male or female. In another embodiment, the subject is human.

[0046] Embodiments of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0047] [Figure 1] FIG. 1 shows the XRPD diffractogram of PCLX-001 (J09899, ​​HCl pattern 1). [Figure 2] FIG. 1 shows the H-NMR spectrum of PCLX-001 (J09899, ​​HCl pattern 1). [Figure 3] FIG. 1 shows the HPLC chromatogram of PCLX-001 (J09899, ​​HCl pattern 1). [Figure 4] FIG. 1 shows thermal analysis of PCLX-001 (J09899, ​​HCl pattern 1). [Figure 5] FIG. 1 shows a GVS isotherm plot of PCLX-001 (J09899, ​​HCl pattern 1). [Figure 6] FIG. 1 shows a GVS kinetic plot of PCLX-001 (J09899, ​​HCl pattern 1). [Figure 7] FIG. 1 shows an XRPD overlay of PCLX-001 (J09899, ​​HCl pattern 1) before storage and after storage at 25° C. / 97% RH and 40° C. / 75% RH. [Figure 8] FIG. 1 shows the HPLC chromatogram of PCLX-001 (J09899, ​​HCl pattern 1) after storage at 25° C. / 97% RH. [Figure 9] FIG. 1 shows the HPLC chromatogram of PCLX-001 (J09899, ​​HCl pattern 1) after storage at 40° C. / 75% RH. [Figure 10] FIG. 1 shows an XRPD overlay of PCLX-001 (J09899, ​​HCl pattern 1) before and after GVS analysis. [Figure 11] FIG. 1 shows the XRPD diffractogram of PCLX-001 (J09898, J09899, ​​J09951, J09952, and J09953, HCl pattern 1). [Figure 12A]FIG. 1 shows XRPD diffractograms of salt breaking experiments (EG-1826-10-04; free form pattern 1 and EG-1826-10-05; mixed salt) and PCLX-001 (J09899 and J09898). [Figure 12B] FIG. 1 shows a H-NMR spectral overlay of salt-disruption experiments (EG-1826-10-04 and EG-1826-10-05) and PCLX-001 (J09899). [Figure 13] FIG. 1 shows an HPLC chromatogram of EG-1826-10-04. [Figure 14] FIG. 1 shows an HPLC chromatogram of EG-1826-10-05. [Figure 15] FIG. 1 shows a H-NMR overlay of EG-1826-10-04 and EG-1826-12-02. [Figure 16] FIG. 1 shows the thermal analysis of EG-1826-12-02. [Figure 17] FIG. 1 shows an HPLC chromatogram of EG-1826-12-02. [Figure 18] FIG. 1 shows XRPD diffractograms of scale-up salt-breaking experiments (EG-1826-10-04, EG-1826-12-02 and EG-1826-22-01→05) and PCLX-001 (J09899). [Figure 19] FIG. 1 shows the XRPD diffractograms of free form Pattern 1 (EG-1826-12-02 and EG-1826-25-01). [Figure 20] FIG. 1 shows 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). [Figure 21] FIG. 1 shows 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). [Figure 22] FIG. 1 shows the thermal analysis of EG-1826-28-04. [Figure 23] FIG. 1 shows the thermal analysis of EG-1826-28-14. [Figure 24] FIG. 1 shows the H-NMR of EG-1826-28-04. [Figure 25] FIG. 1 shows the H-NMR of EG-1826-28-14. [Figure 26] FIG. 1 shows an SEM image of EG-1826-28-09. [Figure 27] FIG. 1 shows an SEM image of EG-1826-28-14. [Figure 28] FIG. 1 shows a PLM image of EG-1826-28-09. [Figure 29] FIG. 1 shows XRPD overlays of EG-1826-28-21 and EG-1826-28-19 before storage and EG-1826-28-21 after storage at 40° C. / 75% RH and EG-1826-28-19 after storage at 25° C. / 97% RH. [Figure 30] FIG. 1 shows XRPD diffractograms of the parent form (EG-1826-25-01), free form Pattern 2 before heating to 220° C. (EG-1826-28-10) and after heating to 220° C. (EG-1826-39-01). [Figure 31] FIG. 1 shows the solubility curves of PCLX-001 free form pattern 2 (J10206) in DMSO, DMSO EtOH:HO (2:1) (50:50), DMSO EtOH:HO (1:2) (70:30), and DMSO EtOH:HO (1:2) (95:5). [Figure 32] FIG. 1 shows (A) a graph of predicted solubility as a function of the time point of seed addition, and (B) a graph of predicted solubility as the volume of antisolvent is increased. [Figure 33] FIG. 1 shows DynoChem prediction of PCLX-001 free form pattern 1 in DMSO. [Figure 34]FIG. 1 shows DynoChem prediction of PCLX-001 free form pattern 1 in DMSO and antisolvent EtOH:H2O (1:2) (5%). [Figure 35] FIG. 1 shows DynoChem prediction of PCLX-001 free form pattern 1 in DMSO and antisolvent EtOH:H2O (1:2) (10%). [Figure 36] FIG. 1 shows DynoChem prediction of PCLX-001 free form pattern 1 in DMSO and antisolvent EtOH:H2O (1:2) (20%). [Figure 37] FIG. 1 shows the XRPD of J10206 (PCLX-001 free form pattern 2). [Figure 38] FIG. 1 shows an XRPD overlay of J10206 and PCLX-001 free form Pattern 2 reference (EG-1826-28-11). [Figure 39] FIG. 1 shows the H-NMR spectrum of J10206 (PCLX-001 free form pattern 2). [Figure 40] FIG. 1 shows a H-NMR overlay of J10206 and PCLX-001 free form pattern 2 reference (EG-1826-28-11). [Figure 41] FIG. 1 shows thermal analysis of J10206 (PCLX-001 free form pattern 2). [Figure 42] FIG. 1 shows an HPLC chromatogram of J10206 (PCLX-001 free form pattern 2). [Figure 43] FIG. 1 shows the Raman spectrum of J10206 (PCLX-001 free form pattern 2). [Figure 44] FIG. 1 shows a close-up Raman spectrum of J10206 (PCLX-001 free form pattern 2). [Figure 45] FIG. 1 shows an XRPD overlay of J10206 and EG-1826-48-07. [Figure 46A] FIG. 1 shows an XRPD overlay of J10206, EG-1826-49-XX (XX=01→15) from a solubility determination at 60° C. [Figure 46B] FIG. 1 shows an XRPD overlay of J10206, EG-1826-50-XX (XX=01→15) from a solubility determination at 25° C. [Figure 47] FIG. 1 shows an XRPD overlay of J10206, EG-1826-53-XX (XX=01→12) from repeated solubility determinations. [Figure 48] FIG. 1 shows an XRPD overlay of EG-1826-62-02 and EG-1826-63-01. [Figure 49] FIG. 1 shows an HPLC chromatogram of EG-1826-63-01. [Figure 50] FIG. 1 shows a PLM image of EG-1826-63-01. [Figure 51] 1 is a graph of solubility versus pH for solubility pH profiling of J10206 in seven pH buffer media. [Figure 52] FIG. 1 shows a summary of (±SD) male Sprague-Dawley rat plasma concentrations of PCLX-001 salt form (Test Article 1) and free base (Test Article 2) 1 day after oral administration. [Figure 53] FIG. 1 shows a summary of (±SD) female beagle dog plasma concentrations of PCLX-001 salt form (Test Article 1) and free base (Test Article 2) 1 day after oral administration. [Figure 54] FIG. 1 shows a summary of (±SD) female mouse plasma free base PCLX-001 concentrations after oral administration of PCLX-001 on day 1. [Figure 55] FIG. 1 shows an HPLC chromatogram of EG-1826-62-02. [Figure 56] FIG. 1 shows an HPLC chromatogram of EG-1826-63-01. [Figure 57] FIG. 1 shows an HPLC chromatogram of EG-1826-63-02. [Figure 58] FIG. 1 shows an HPLC chromatogram of J10206 (PCLX-001 free form pattern 2). DETAILED DESCRIPTION OF THE INVENTION

[0048] Detailed Description definition Unless otherwise defined, 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.

[0049] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0050] The term "comprising," as used herein, will be understood to mean that the following list is not inclusive and may or may not include any other additional suitable items as appropriate, such as one or more further features, components and / or ingredients.

[0051] The term "cancer," as used herein, refers to various conditions caused by abnormal, uncontrolled proliferation of cells. Cells capable of causing cancer are called "cancer cells" and have characteristic properties, such as uncontrolled proliferation, immortality, metastatic potential, rapid proliferation and growth rate, and / or certain typical morphological features. Cancer cells can be in the form of a tumor, but such cells can also exist alone in a subject or be non-tumorigenic cancer cells. Cancer can be detected by several methods, including, but not limited to, detecting the presence of one or more tumors (e.g., by clinical or radiological means), examining cells within a tumor or from another biological sample (e.g., from a tissue biopsy), measuring blood markers indicative of cancer, and detecting a genotype indicative of cancer. However, negative results from one or more of the above detection methods do not necessarily indicate the absence of cancer; for example, a patient who has shown a complete response to cancer treatment may still have cancer, as evidenced by a subsequent recurrence.

[0052] The term "subject," as used herein, refers to an animal and can include, for example, domestic animals, such as cats, dogs, etc., livestock (e.g., cows, 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 animal. In a specific embodiment, the subject is a human.

[0053] The terms "treatment" or "treating," as used herein, refer to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of disease, stabilized (i.e., not worsening) disease, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the disease state, reduction in disease recurrence, and remission (partial or complete), whether detectable or undetectable. "Treat" and "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treat" and "treatment," as used herein, also include prophylactic treatment. For example, a subject with early-stage cancer, such as early-stage lymphoma, can be treated with a compound or composition described herein to prevent progression, or alternatively, a subject in remission can be treated with a compound or composition described herein to prevent recurrence.

[0054] The term "pharmaceutically effective amount" or "effective amount," as used herein, refers to an amount of a drug or pharmaceutical agent that elicits the biological or medical response in a tissue, system, animal, or human that is being sought by a researcher or clinician. This amount may be a "therapeutically effective amount." These terms refer to an amount of a compound and / or composition described herein that, in single or multiple administrations, will treat a subject with a disease or condition. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by using known techniques and observing results obtained under analogous circumstances. In determining an effective amount, dosage, several factors will be considered by the attending diagnostician, including, but not limited to, the subject's species; its size, age, and general health; the specific condition, disorder, or disease involved; the extent or coexistence or severity of the condition, disorder, or disease; the individual subject's response; the particular compound administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosing regimen; the use of concomitant medications; and other relevant circumstances.

[0055] The term "pharmaceutically acceptable," as used herein, includes compounds, materials, compositions, and / or dosage forms (e.g., unit dosages) that are suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc., is also "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0056] The term "excipient" refers to a pharmacologically inactive ingredient, such as a diluent, lubricant, surfactant, carrier, etc. Excipients useful in preparing pharmaceutical compositions are generally safe, non-toxic, and acceptable for human pharmaceutical use. Reference to an excipient includes both one such excipient and two or more such excipients.

[0057] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as, but not limited to, phosphate buffered saline solution, 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, disintegrating agents (e.g., potato starch or sodium starch glycolate), stabilizers and preservatives, and the like.

[0058] "Treatment or dosing regimen," as used herein, refers to a combination of dosage, frequency of administration, or duration of treatment, with or without the addition of a second medication.

[0059] The term "diagnosis" as used herein refers to the identification of a molecular and / or pathological state, disease or condition, for example, the identification of lymphoma or other cancer types.

[0060] The term "alleviate," as used herein, refers to the lessening, diminution, or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.

[0061] 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.

[0062] As used herein, the term "crystalline form of Compound (I)" refers to the form of Compound (I) (PCLX-001) identified and characterized herein as free form Pattern 2 (see Examples 1 and 2).

[0063] 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).

[0064] As used herein, the term "free form of Compound (I)" refers to the form of Compound (I) (PCLX-001) identified and characterized herein as Free Form Pattern 1 (see Example 1).

[0065] Crystalline morphology Generally, the present disclosure relates to a novel crystalline form of Compound (I).

[0066] [ka]

[0067] Provided are methods for preparing this new crystalline form, compositions containing it, and its use in the treatment of diseases such as cancer, microbial infections, neurological diseases / disorders, diabetes, ischemia, osteoporosis, and related conditions.

[0068] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by the X-ray powder diffraction pattern shown in FIG.

[0069] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks at 10.167, 11.385, and 20.505 degrees two-theta ± 0.2 degrees two-theta; or 10.2, 11.4, and 20.5 degrees two-theta ± 0.2 degrees two-theta.

[0070] 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, which includes an endothermic event with an onset temperature of 220.2°C ± 0.2°C.

[0071] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) is characterized by a DSC thermogram substantially as shown in FIG.

[0072] In one or more embodiments of the present disclosure, the crystalline form of Compound (I) has a molecular weight of 103.24 cm -1, and 993.01 cm -1 , and 1602.81 cm -1 ; or 103.2 cm -1 , and 993.0 cm -1 , and 1602.8 cm -1 ±0.2cm -1 Wave value (cm -1 ) is characterized by a Raman spectrum containing

[0073] In certain examples of the present disclosure, a crystalline form of Compound (I) characterized by the X-ray powder diffraction pattern shown in Figure 37.

[0074] [ka]

[0075] is described.

[0076] In another example, a crystalline form of Compound (I) characterized by an X-ray powder diffraction pattern having peaks at 10.2, 11.4, and 20.5 degrees two-theta ± 0.2 degrees two-theta.

[0077] [ka]

[0078] is described.

[0079] In another example of the disclosure, a crystalline form is described that is further characterized by an X-ray powder diffraction pattern with peaks at 14.6, 22.9, 26.1, and 31.0 degrees two-theta ± 0.2 degrees two-theta. In another example, the crystalline form is further characterized by an X-ray powder diffraction pattern with peaks at 18.6, 21.4, 23.4, and 33.5 degrees two-theta ± 0.2 degrees two-theta.

[0080] In another example of the present disclosure, a crystalline form of Compound (I) characterized by an X-ray powder diffraction pattern having peaks ±0.2 degrees two-theta substantially as provided below in Table 1.0' (Table 3).

[0081] [ka]

[0082] is described.

[0083] [Table 3]

[0084] Another example of the disclosure describes a crystalline form characterized by a DSC thermogram obtained using a heating rate of 10° C. / min, which includes an endothermic event with an onset temperature of 220.2° C.±0.2° C. In another example, the crystalline form is characterized by a DSC thermogram substantially as shown in FIG.

[0085] In another example of the present disclosure, 103.2 cm -1 , and 993.0 cm -1 , and 1602.8 cm -1 ±0.2cm -1 Wave value (cm -1 In another example, the crystalline form is described as having a Raman spectrum comprising a band at 126.5 cm -1 , 144.9cm -1 , 227.3cm -1 , 456.1cm -1 , 1043.5cm -1 , 1164.7cm -1 , and 1582.5 cm -1 ±0.2cm -1 Wave value (cm -1 In another embodiment, the crystalline form is further characterized by a Raman spectrum comprising wavevalues ​​(cm) substantially as provided below in Table 2.0'. -1 )±0.2cm -1 The Raman spectrum includes:

[0086] [Table 4A]

[0087] [Table 4B]

[0088] Preparation and recrystallization methods In one or more embodiments of the present disclosure, the crystalline forms of Compound (I) characterized herein

[0089] [ka]

[0090] A method for preparing the compound is provided.

[0091] Generally, the crystalline form of Compound (I) is prepared by providing the HCl form of Compound (I) and salt-destroying the HCl form (e.g., by ion exchange using a base) to form the free form of Compound (I). The free form of Compound (I) is then added to a solvent and the mixture is aged (e.g., including cooling, antisolvent addition, and aging) to form the crystalline form of Compound (I).

[0092] In one or more embodiments, a method for preparing the crystalline form of Compound (I) 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; maturing the second mixture to form a third mixture; and isolating the crystalline form of Compound (I) from the third mixture.

[0093] In one or more embodiments, the method further comprises filtering insoluble particulates from the first solution prior to isolating the free form of Compound (I) from the first solution.

[0094] In one or more embodiments of the present disclosure, the HCl form of Compound (I) is characterized by the X-ray powder diffraction pattern shown in FIG.

[0095] In one or more embodiments, the HCl form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks at 7.220, 11.00, and 13.457 degrees two-theta ± 0.2 degrees two-theta; or 7.2, 11.0, and 13.5 degrees two-theta ± 0.2 degrees two-theta. In one or more embodiments, the HCl form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks substantially as provided below in Table 3.0' (Table 5) ± 0.2 degrees two-theta.

[0096] [Table 5]

[0097] 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, which includes an endothermic event with an onset temperature of 64.7°C ± 0.2°C.

[0098] In one or more embodiments, the HCl form of Compound (I) is characterized by a DSC thermogram substantially as shown in Figure 4 or listed in Table 15 (J09899, ​​DSC).

[0099] In one or more embodiments of the present disclosure, the free form of Compound (I) is characterized by the X-ray powder diffraction pattern shown in Figure 19 (referred to by EG-1826-12-02).

[0100] In one or more embodiments, the free form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks at 6.301, 8.095, and 19.010 degrees two-theta ± 0.2 degrees two-theta; or 6.3, 8.1, and 19.0 degrees two-theta ± 0.2 degrees two-theta. In one or more embodiments, the free form of Compound (I) is characterized by an X-ray powder diffraction pattern having peaks substantially as shown below in Table 4.0' (Table 6) ± 0.2 degrees two-theta.

[0101] [Table 6]

[0102] 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, which contains an endothermic event with an onset temperature of 161.5°C ± 0.2°C.

[0103] In one or more embodiments, the free form of Compound (I) is characterized by a DSC thermogram substantially as shown in Figure 16 or listed in Table 19 (DSC).

[0104] In one or more embodiments of the present disclosure, the crystalline forms of Compound (I) characterized herein

[0105] [ka]

[0106] A method for recrystallizing

[0107] Generally, 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 can be filtered to remove insoluble particulates. The heated solution is then seeded with a small amount of the crystalline form, after which the seeded solution is cooled. An antisolvent is added to the cooled, seeded solution, after which the solution is allowed to mature. From this mature solution, the crystalline form of Compound (I) recrystallizes and can be isolated.

[0108] In one or more embodiments, a method of recrystallizing a 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; reducing the temperature 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; reducing the temperature 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 isolating the recrystallized crystalline form of Compound (I) from the third mixture.

[0109] In one or more embodiments, the method further comprises filtering insoluble particulates from the first solution prior to lowering the temperature from the first temperature to the second temperature.

[0110] In one or more embodiments of the present application, the crystalline forms of Compound (I) characterized herein

[0111] [ka]

[0112] is prepared and / or recrystallized by the procedures detailed in Example 1 and / or Example 2.

[0113] In one example of the present disclosure, there is provided a method for preparing a crystalline form of Compound (I) described herein, comprising: 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; maturing the second mixture to form a third mixture; and isolating the crystalline form of Compound (I) from the third mixture. A method is described that includes:

[0114] In another example of the disclosure, a method is described wherein the step of 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 disclosure, a method is described wherein the step of 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.

[0115] In another example of the present disclosure, the step of adding the free form of Compound (I) to a second organic solvent to form a second mixture comprises: stirring the second mixture at ambient temperature; Optionally, increasing the temperature from ambient to about 50°C, and then decreasing the temperature from about 50°C to ambient; and Optionally, adding additional organic solvent to the second mixture until the free form of Compound (I) is dissolved in the second organic solvent; and Decrease the temperature from about 50°C to about 5°C at a rate of about 0.1°C / min. A method is described further comprising:

[0116] In another example, a method is described wherein the step of maturing the second mixture to form a third mixture further comprises maturing at 25 / 50° C. in a 4 hour cycle.

[0117] 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.

[0118] In another example, a method is described in which the base is NaOH.

[0119] In another example, a method is described in which the second organic solvent is selected from the group consisting of 2-propanol; 2-methyl THF; acetone; acetonitrile; DMSO; N,N'-dimethylacetamide; MeOH:water (90:10 v / v); ethyl acetate; ethanol:water (50:50 v / v); isopropyl acetate; isobutanol; tert-butyl methyl ether; THF; and water. In another example, a method is described in which the second organic solvent is selected from the group consisting of 2-propanol; 2-methyl THF; acetone; acetonitrile; DMSO; N,N'-dimethylacetamide; MeOH:water (90:10 v / v); ethyl acetate; ethanol:water (50:50 v / v); isopropyl acetate; isobutanol; tert-butyl methyl ether; THF; and water.

[0120] In another example of the present disclosure, a method is described further comprising filtering insoluble particulates from the first solution prior to isolating the free form of Compound (I) from the first solution.

[0121] In another example of the present disclosure, there is provided a method of recrystallizing a crystalline form of Compound (I) described herein or prepared by a method described herein, comprising: dissolving a crystalline form of Compound (I) in an organic solvent at a first temperature to form a first solution; decreasing the temperature from the first temperature to a second temperature; adding seed crystals of a crystalline form of Compound (I) to a first solution to form a first mixture; decreasing the temperature from the second temperature to a third temperature; adding an anti-solvent to the first mixture to form a second mixture; maturing the second mixture for about 24 hours to form a third mixture; isolating the recrystallized crystalline form of Compound (I) from the third mixture. A method is described that includes:

[0122] In another example of the present disclosure, a method is described in which the organic solvent is selected from the group consisting of DMSO; DMA; and NMP. In another example, the organic solvent is DMSO. In another example of the present disclosure, a method is described in which the organic solvent is selected from the group consisting of DMSO; DMA; and NMP. In another example, the organic solvent is DMSO.

[0123] In some embodiments, methods are provided in which the anti-solvent comprises water, a C1-C3 alcohol, or a combination thereof, hi some embodiments, the anti-solvent comprises water, a C1-C3 alcohol, or a combination thereof in an amount ranging between about 100% water and about 100% C1-C3 alcohol, or any combination of water and a C1-C3 alcohol between 100% water and 100% C1-C3 alcohol.

[0124] In some embodiments, the C1-C3 alcohol comprises a C1-C3 linear, branched, or cyclic alcohol. In some embodiments, the C1-C3 alcohol comprises methanol, ethanol, n-propanol, branched propanol, or a combination thereof.

[0125] In another embodiment, a method is provided wherein the anti-solvent is selected from the group consisting of ethanol and water. In some embodiments, the anti-solvent is selected from the group consisting of ethanol and water in amounts ranging between about 100% water and about 100% ethanol, or any combination of water and ethanol between 100% water and 100% ethanol. In another example, a method is described wherein the anti-solvent is selected from the group consisting of EtOH:HO (1:1); EtOH:HO (1:2); and EtOH:HO (2:1).

[0126] In another example, a method is described in which the organic solvent is DMSO and the anti-solvent is EtOH:H2O (1:1), EtOH:H2O (1:2), or EtOH:H2O (2:1). In another example, the organic solvent is DMSO and the anti-solvent is EtOH:H2O (1:2).

[0127] In another example, a method is described in which the anti-solvent is added in a volume that is about 10% to about 20% of the volume of the organic solvent. In another example, the anti-solvent is added in a volume that is about 10% of the volume of the organic solvent.

[0128] In another example, a method is described wherein the first temperature is about 65°C.

[0129] In another example, a method is described wherein the second temperature is about 59°C.

[0130] In another example, a method is described wherein the third temperature is about 25°C.

[0131] In another example, a method is described wherein the step of decreasing the temperature from the second temperature to the third temperature comprises a decrease of 0.5° C. / minute.

[0132] In another example, a method is described that further comprises filtering insoluble particulates from the first solution before lowering the temperature from the first temperature to the second temperature.

[0133] Pharmaceutical compositions and uses thereof In one or more embodiments of the present disclosure, the crystalline forms of Compound (I) characterized herein

[0134] [ka]

[0135] has pharmacological activity. In one or more embodiments of the present disclosure, a crystalline form of Compound (I) inhibits N-myristoyltransferase (NMT) activity. In one or more embodiments, a crystalline form of Compound (I) is used to treat a subject with cancer. In one or more embodiments, a crystalline form of Compound (I) is used to treat a subject with cancer deficient in NMT2.

[0136] Protein N-myristoylation is a modification in which myristic acid (a 14-carbon saturated fatty acid) is covalently attached to the NH2-terminal glycine of a variety of cellular, viral, and oncoproteins (e.g., oncogenic Src-related tyrosine kinase, heterotrimeric G alpha subunit, etc.).

[0137] Myristoylated proteins in cells have diverse biological functions in signal transduction and carcinogenesis. Protein modification by myristoylation is required for subcellular targeting, protein conformation, and the biological activity of many important proteins in eukaryotic cells, including those required for signal transduction and regulatory functions important in cellular proliferation. The Src family (proto-oncogene) tyrosine kinases are among the most extensively studied myristoylated proteins.

[0138] Protein myristoylation is catalyzed by N-myristoyltransferase (NMT). NMT performs this activity in eukaryotic cells by modifying its polypeptide substrate after removal of the initiator methionine residue by methionyl aminopeptidase. This modification occurs primarily as a cotranslational process, but myristoylation can also occur posttranslationally, typically after proteolytic cleavage of the protein during apoptosis. Two isozymes of mammalian NMT enzymes have been cloned and designated NMT1 and NMT2.

[0139] NMTs play a pro-survival role in cells. Two NMTs are present in all normal cells. Increased NMT activity and expression have also been shown in several tumor types, suggesting that NMT inhibitors may be potential anticancer drugs.

[0140] In one or more embodiments of the present disclosure, the crystalline forms of Compound (I) characterized herein may be useful in the treatment or prevention of diseases or disorders that can be prevented, alleviated, or treated by modulation / inhibition of N-myristoyltransferase (NMT) activity (herein referred to as NMT-associated diseases or disorders). Such NMT-associated diseases or disorders 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.

[0141] In one or more embodiments, the cancer to be prevented, alleviated, or treated includes all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness.

[0142] In one or more embodiments, the cancers to be prevented, ameliorated, or treated include malignant tumors of various organ systems, such as those affecting the lung, breast, thyroid, lymphatic system, gastrointestinal, and genitourinary tract, as well as malignant tumors such as adenocarcinomas, including most colon cancers, renal cell carcinoma, prostate cancer, and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus. The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissue, such as respiratory system cancer, gastrointestinal system cancer, genitourinary system cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term "carcinoma" also includes carcinosarcomas, including, for example, malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to malignant tumors of mesenchymal origin. Additional cancer types include leukemia, skin cancer, intracranial cancer, and brain cancer.

[0143] 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 proliferation of B cells or T cells in the lymphatic system. "Lymphoma" includes many types of malignant proliferation, including, for example, Hodgkin's lymphoma and non-Hodgkin's lymphoma. The term "non-Hodgkin's lymphoma," as used herein, refers to a malignant proliferation of B cells or T cells in the lymphatic system that is not Hodgkin's lymphoma (e.g., characterized by the presence of Reed-Sternberg cells in the cancerous area). Non-Hodgkin's lymphoma encompasses more than 29 types of lymphoma, the distinction between which is based on the type of cancer cells.

[0144] In some embodiments, the cancer is B-lymphoma. Thus, in some embodiments of the present disclosure, the crystalline forms of Compound (I) and pharmaceutical compositions thereof are suitable for treating subjects with B-cell lymphoma. Examples of B-cell lymphoma include, but are not limited to, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom's lymphoma, and MALT / monocytic B-cell lymphoma. Treatment of childhood lymphomas, such as Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, precursor B-LBL, precursor T-LBL, and anaplastic large cell lymphoma, is also contemplated.

[0145] In one or more embodiments, the cancer to be prevented, ameliorated, or treated is lymphoma, B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom, MALT / monocytoid B-cell lymphoma, Burkitt lymphoma, childhood lymphoma, anaplastic large cell lymphoma, acute myeloid leukemia, blastic phase chronic myeloid leukemia, Burkitt lymphoma, plasma cell myeloid leukemia, tumors, intestinal adenocarcinoma, mixed adenosquamous carcinoma of the lung, small cell lung carcinoma, lung, esophageal squamous cell carcinoma, bone, ductal carcinoma, diffuse gastric adenocarcinoma, medullary thyroid carcinoma, transitional cell carcinoma of the urinary tract, myeloma, ovarian clear cell carcinoma, transitional cell carcinoma (ureter and bladder cancer), chronic myeloid leukemia (CML), lymphoma-CLL, breast cancer, colorectal adenocarcinoma, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung cancer, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, bile duct carcinoma (cholangiocarcinoma), gallbladder carcinoma, liver cancer, or esophageal squamous cell carcinoma.

[0146] In one or more embodiments, the microbial infection to be prevented, alleviated, or treated is an infection caused by a bacterium, a parasite, a protozoan, a virus, or a fungus, including a yeast.

[0147] A "pathogen" is generally defined as any disease-causing organism. Parasitic pathogens include Trypanosoma species (e.g., T. cruzi, T. brucei, T. congolense), Leishmania species (e.g., L. major, L. donovani, L. braziliensis), Giardia species, Trichomonas species (e.g., Trichomonas vaginalis), Entamoeba species (e.g., E. histolytica), and the like. histolytica), Naegleria species, Acanthamoeba species (e.g., A. castelleni), Schistosoma species (e.g., S. mansoni, S. japonicum), Plasmodium species (e.g., P. falciparum), Crytosporidium species, Isospora species, Balantidium species, Loa loa, Ascaris lumbricoides, Dirofilaria immitis, Toxoplasma species (e.g., Toxoplasma The parasite may be derived from a parasite selected from the group consisting of, but not limited to, Onchocerca species (e.g., O. gondii), Onchocerca species (e.g., O. volualno).

[0148] The viral pathogen may be derived from a virus selected from, but not limited to, the group consisting of human immunodeficiency viruses (HIV1 and 2); human T-cell leukemia viruses (HTLV1 and 2); Ebola virus; human papillomaviruses (e.g., HPV-2, HPV-5, HPV-8, HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, HPV-54, and HPV-56); papovavirus; rhinovirus; poliovirus; herpesvirus; adenovirus; Epstein-Barr virus; influenza virus, hepatitis B and C virus, smallpox virus, rotavirus, or SARS coronavirus.

[0149] The fungal pathogen may be derived from a fungus (including yeast) selected from, but not limited to, Candida species (e.g., C. albicans, C. tropicalis), Aspergillus species (e.g., A. fumigatus), Cryptococcus species (e.g., Cryptococcus neoformans), and Saccharomyces species (e.g., Saccharomyces cerevisiae), Pneumocystis species (e.g., Pneumocystis carinii).

[0150] In one or more embodiments, the neurological disease / disorder to be prevented, alleviated or treated may include neuropsychiatric disorders such as Parkinson's disease, attention deficit hyperactivity disorder (ADHD), depression (bipolar disorder) and schizophrenia and addiction; neurodegenerative disorders (e.g., Alzheimer's disease, Tourette's syndrome, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, senile chorea, Sydenham's chorea, autism, head and spinal trauma, acute and chronic pain, epilepsy and seizures, dementia, dystonia, tremor, autism, cerebral ischemia and neuronal cell death) and disorders associated with apoptosis (particularly neuronal apoptosis).

[0151] When used to treat a subject, the crystalline forms of Compound (I) characterized herein can be administered as the free compound or as part of a pharmaceutical composition, which can be administered orally, intravenously, subcutaneously, buccal, rectally, transdermally, intranasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray, or by inhalation.

[0152] The crystalline forms of Compound (I) can be administered in a pharmaceutically acceptable dosage form, either as a free compound or as part of a pharmaceutical composition. Depending on the disorder and subject being treated and the route of administration, the compound or pharmaceutical composition can be administered in various doses.

[0153] The crystalline forms of Compound (I) can be administered orally or parenterally ("parenteral," as used herein, refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injection and infusion) to a subject to achieve an inhibitory effect. In the case of larger animals, such as humans, the crystalline forms of Compound (I) can be administered alone or as a pharmaceutical composition in combination with a pharmaceutically acceptable diluent, excipient, or carrier.

[0154] The actual dosage level of the crystalline form of Compound (I) in the pharmaceutical composition can be varied to obtain an amount of the active crystalline form of Compound (I) effective to achieve the desired therapeutic response for 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 medical history of the subject being treated. However, it is within the skill of one of ordinary skill in the art to start the dosage of the crystalline form of Compound (I) at a level lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0155] In one or more embodiments of the present disclosure, when the treatment, prevention, control, amelioration, or reduction of risk of a condition requires inhibition of kinase activity, a suitable dosage level may be about 0.01 to 500 mg / kg of patient body weight per day, which may be administered in single or multiple doses or by continuous infusion. In one or more embodiments, the dosage level may 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.

[0156] For oral administration, pharmaceutical compositions containing the crystalline form of Compound (I) can be provided in the form of tablets containing 1.0 to 1000 milligrams 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 milligrams of the crystalline form of Compound (I), for symptomatic adjustment of the dosage to the subject being treated. The crystalline form of Compound (I) can be administered as a free compound or as part of a pharmaceutical composition on a regimen of 1 to 4 times per day; or once or twice daily. The administration regimen can be adjusted to provide the optimal therapeutic response.

[0157] In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising a crystalline form of Compound (I) in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0158] Pharmaceutical compositions of the present disclosure for parenteral injection preferably include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0159] The pharmaceutical compositions of the present disclosure may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, or phenol sorbic acid. It may also be desirable to include isotonic agents, such as sugars or sodium chloride.

[0160] Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0161] In some cases, to prolong the effect of the pharmaceutical composition of the present disclosure, it is desirable to slow the absorption of the composition or crystalline form of Compound (I) from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension in which the crystalline form of Compound (I) is suspended in a liquid in which the crystalline form has low solubility. In this case, the rate of absorption of the crystalline form of Compound (I) depends on its dissolution rate.

[0162] Alternatively, delayed absorption of the parenterally administered pharmaceutical composition of the present disclosure is accomplished by dissolving or suspending a crystalline form of Compound (I) in an oil vehicle. Injectable depot forms are preferably made by forming microencapsulated matrices of the crystalline form of Compound (I) in biodegradable polymers such as polylactide-polyglycolide. The release rate can be controlled depending on the ratio of the crystalline form of Compound (I) to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).

[0163] Depot injectable formulations can also be prepared by entrapping the crystalline form of Compound (I) in liposomes or microemulsions compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

[0164] 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 incorporated into at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or one or more of: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) humectants, such as glycerol; d) disintegrating agents. Disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarders such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, 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 can also be used as fillers for soft and hard-filled gelatin capsules, for example, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0165] Preferably, the oral formulation contains a solubilizing agent. There are no limitations on the type of solubilizing agent, 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 thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol mono-fatty acid esters, polyoxyethylene propylene glycol mono-fatty acid esters, polyoxyethylene sorbitol fatty acid esters, fatty acid alkylolamides, and the like. alkylolamides, and alkylamine oxides; bile acids and their salts (e.g., chenodeoxycholic acid, cholic acid, deoxycholic acid, dehydrocholic acid and their salts, and glycine or their taurine conjugates); ionic surfactants such as sodium lauryl sulfate, fatty acid soaps, alkyl sulfonates, alkyl phosphates, ether phosphates, fatty acid salts of basic amino acids; triethanolamine soaps, and alkyl quaternary ammonium salts; and amphoteric surfactants such as betaine and aminocarboxylate salts.

[0166] Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can be of a composition so that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, and / or in a delayed manner. Examples of embedding compositions include polymeric substances and waxes.

[0167] The crystalline forms of Compound (I) may also be in microencapsulated form, if appropriate, containing one or more of the above-mentioned excipients. The crystalline forms of Compound (I) may also be in micronized form, for example, micronized.

[0168] 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 ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially 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 contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and fragrances. Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0169] Pharmaceutical compositions of the present disclosure for rectal or vaginal administration are preferably suppositories that 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 a suppository wax that is solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the crystalline form of Compound (I).

[0170] The crystalline form of Compound (I) can also be administered in the form of liposomes. As known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes can be used. The pharmaceutical composition of the present disclosure in the form of liposomes can contain stabilizers, preservatives, excipients, etc. in addition to the crystalline form of Compound (I). Preferred lipids are phospholipids and phosphatidylcholines (lecithins), both natural and synthetic. Methods for forming liposomes are known in the art.

[0171] Dosage forms for topical administration of the crystalline form of Compound (I) include powders, sprays, ointments, and inhalants. The crystalline form of Compound (I) is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives, buffers, or propellants that may be required. The crystalline form of Compound (I) can also be administered as part of a pharmaceutical composition, such as in eye preparations, or eye ointments, powders, and solutions.

[0172] Another example of the present disclosure describes a pharmaceutical composition comprising a crystalline form of Compound (I) described herein and a pharmaceutically acceptable adjuvant, diluent, carrier, or vehicle.

[0173] Another example of the present disclosure describes a method of treating a subject having cancer, comprising administering to the subject a crystalline form of Compound (I) described herein, or a pharmaceutical composition described herein.

[0174] In another example of the present disclosure, there is described a method of treating a subject having a cancer deficient in NMT2, comprising administering to the subject a crystalline form of Compound (I) described herein, or a pharmaceutical composition described herein.

[0175] In another example of the disclosure, a method is described wherein the cancer is lymphoma. In another example, the lymphoma is B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-CLL / SLL, immunocytoma / Waldenstrom, MALT / monocytoid B-cell lymphoma, or Burkitt lymphoma.

[0176] In another example, methods are described wherein the cancer is anaplastic large cell lymphoma, acute myeloid leukemia, blastic phase chronic myeloid leukemia, Burkitt's lymphoma, plasma cell myeloma, intestinal adenocarcinoma, mixed adenosquamous carcinoma of the lung, small cell lung carcinoma, lung, esophageal squamous cell carcinoma, bone, ductal carcinoma of the breast, diffuse gastric adenocarcinoma, medullary thyroid carcinoma, transitional cell carcinoma of the urinary tract, myeloma, ovarian clear cell carcinoma, transitional cell carcinoma (ureter and bladder cancer), chronic myeloid leukemia (CML), lymphoma-CLL, breast cancer, colorectal adenocarcinoma, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung cancer, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, bile duct carcinoma (cholangiocarcinoma), gallbladder carcinoma, liver cancer, or esophageal squamous cell carcinoma.

[0177] In another example, methods are 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.

[0178] In order to further understand the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only, and therefore, they should not in any way limit the scope of the invention. [Example]

[0179] Example 1 Screening test for compound (I) (PCLX-001) overview Solid-state characterization was performed on the supplied batches of PCLX-001. Three batches were shown as HCl Pattern 1 and one batch was shown as HCl Pattern 2. All batches contained a peak at approximately 32.1° 2-theta, representing sodium chloride. Ion chromatography confirmed the presence of excess chloride and sodium content. One batch was carried forward to the salt destruction step.

[0180] Salt destruction by ion exchange using sodium hydroxide was successful and was scaled up several times to the 1 g scale. The recovered material was chloride- and sodium-free (free form) and in crystalline form (designated free form pattern 1). The purity of this material was 96.3%, comparable to that of the starting material. The melting of this form was recorded at 161.5 °C (onset).

[0181] Free form Pattern 1 was carried forward to investigate its polymorphic properties. 22 solvents / solvent systems were selected for this step, with free form Pattern 1 as the starting material. The following procedure was used: cooling, maturation, and antisolvent addition. Free form Pattern 2 was obtained in almost all cases, suggesting that this was likely the most stable form under the conditions tested.

[0182] Free form Pattern 2 was a crystalline, nonsolvated form that remained unchanged by XRPD after 1 week of storage at elevated temperatures and humidity levels (40°C / 75% RH and 25°C / 97% RH). Melting / decomposition temperatures were observed between 209 and 217°C. Only gradual mass loss due to loss of unbound water was observed in the TGA thermogram, followed by sample decomposition above 210°C.

[0183] Free form Pattern 2 was found to exhibit the most desirable solid-state properties of all polymorphs discovered during testing, and was therefore further tested to create an optimized crystallization method for forming free form Pattern 2 (see Example 2).

[0184] [Table 7]

[0185] Equipment and methodology details X-ray powder diffraction (XRPD) Bruker AXS D8 Advance 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, followed by a Lynxeye detector. The software used for data collection and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA, respectively.

[0186] Samples were run under ambient conditions as flat specimens using powders. Samples were prepared on polished, zero-background (510) silicon wafers by gently pressing them onto a flat surface or filling a cut cavity. The samples were rotated within their own plane.

[0187] Details of standard data collection methods are as follows: Angle range: 2~42°2θ Step size: 0.05° 2θ Acquisition time: 0.5 seconds / step (total acquisition time: 6.40 minutes)

[0188] PANalytical's Empyrean 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 and focusing mirror were used in the incident beam. A PIXcel3D detector, positioned in the diffracted beam, was fitted with a receiving slit and a 0.04 rad Soller slit. The software used for data collection was X'Pert Data Collector with the X'Pert Operator Interface. Data were analyzed and presented using Diffrac Plus EVA or HighScore Plus.

[0189] Samples were prepared and analyzed in transmission mode in metal or Millipore 96-well plates. X-ray transparent film was used between metal sheets on metal well plates, and the powder (approximately 1-2 mg) was used as is. Millipore plates were used to isolate and analyze solids from suspension by adding a small amount of suspension directly to the plate before filtration under light vacuum.

[0190] The scan mode for the metal plate used the gonioscan axis, while the Millipore plate utilized a 2θ scan.

[0191] Details of standard screening data collection methods are as follows: Angle range: 2.5 to 32.0° 2θ Step size: 0.0130°2θ Acquisition time: 12.75 seconds per step (total acquisition time 2.07 minutes)

[0192] nuclear magnetic resonance (NMR) Solution-state NMR 1H NMR and / or 13C NMR spectra were collected on a Bruker 400 MHz instrument equipped with an autosampler and controlled by a DRX400 console. Samples were prepared in DMSO-d6 solvent unless otherwise noted. Automated experiments were acquired using the ICON-NMR setting in Topspin software using standard Bruker on-board experiments (1H, 13C{1H}, DEPT135). Offline analysis was performed using an ACD Spectrus processor. For atypical spectroscopy (2D NMR and variable temperature NMR), data were acquired using Topspin alone.

[0193] Differential scanning calorimetry (DSC) TA Instruments Q2000 DSC data were collected on a TA Instruments Q2000 equipped with a 50-position autosampler. Typically, 0.5–3 mg of each sample was heated in a pinhole aluminum pan from 25°C to 300°C at 10°C / min. A 50 ml / min purge of dry nitrogen was maintained over the sample.

[0194] Temperature modulated DSC was performed using a base heating rate of 2°C / min and temperature modulation parameters of ±0.636°C (amplitude) every 60 seconds (duration).

[0195] The instrument control software was Advantage for Q Series and Thermal Advantage, and data were analyzed using Universal Analysis or TRIOS.

[0196] TA Instruments Discovery DSC DSC data were collected on a TA Instruments Discovery DSC equipped with a 50-position autosampler. Typically, 0.5–3 mg of each sample was heated in a pinhole aluminum pan from 25°C to 300°C at 10°C / min. A 50 ml / min purge of dry nitrogen was maintained over the sample. The instrument control software was TRIOS, and data were analyzed using TRIOS or Universal Analysis.

[0197] Thermogravimetric analysis (TGA) TA Instruments Q500 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 into a pre-tared aluminum DSC pan and heated from ambient temperature to 350 °C at 10 °C / min. A 60 ml / min nitrogen purge was maintained throughout the sample. The instrument control software was Advantage for Q Series and Thermal Advantage, and data were analyzed using Universal Analysis or TRIOS.

[0198] TA Instruments Discovery TGA TGA data were collected on a TA Instruments Discovery TGA equipped with a 25-position autosampler. Typically, 5-10 mg of each sample was loaded into a pre-tared aluminum DSC pan and heated from ambient temperature to 350 °C at 10 °C / min. A 25 ml / min nitrogen purge was maintained throughout the sample. The instrument control software was TRIOS, and data were analyzed using TRIOS or Universal Analysis.

[0199] Polarized Light Microscopy (PLM) Leica LM / DM polarizing microscope Samples were analyzed with a Leica LM / DM polarized light microscope using a digital video camera for image capture. A small amount of each sample was mounted on a glass slide with or without immersion oil and covered with a coverslip. Samples were viewed under appropriate magnification and partially polarized light coupled with a λ false color filter. Images were acquired using StudioCapture or Image ProPlus software.

[0200] Scanning Electron Microscopy (SEM) Data were collected on a Phenom Pro scanning electron microscope. A small sample was mounted on an aluminum stub using conductive double-sided adhesive tape. A thin layer of gold was applied using a sputter coater (20 mA, 120 s).

[0201] Gravimetric Vapor Sorption (GVS) Adsorption isotherms were obtained using an SMS DVS Intrinsic moisture sorption analyzer controlled by DVS Intrinsic Control software. Sample temperature was maintained at 25 °C by the instrument control. Humidity was controlled by mixing dry and humid nitrogen streams at a total flow rate of 200 ml / min. Relative humidity was measured by a calibrated Rotronic probe (dynamic range 1.0–100% RH) located near the sample. Sample mass change (mass relaxation) as a function of % RH was continuously monitored by a microbalance (accuracy ±0.005 mg).

[0202] Typically, 5-30 mg of sample was placed in a tared mesh stainless steel basket under ambient conditions. Samples were loaded and unloaded at 40% RH and 25°C (typical room conditions). Moisture sorption isotherms were performed as outlined below (two scans per complete cycle). Standard isotherms were performed at 25°C over a 0-90% RH range at 10% RH intervals. Duplicate cycles (four scans) were typically performed. Data analysis was performed in Microsoft Excel using the DVS Analysis Suite.

[0203] [Table 8]

[0204] After completion of the isotherm, samples were withdrawn and re-analyzed by XRPD (see above).

[0205] Chemical Purity Measurement by HPLC 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:

[0206] [Table 9]

[0207] Determining Water by Karl Fischer Titration (KF) The water content of each sample was measured in a Metrohm 874 Oven Sample Processor at 150°C with an 851 Titrano coulometer using Hydranal Coulomat AG oven reagent and a nitrogen purge. A weighed solid sample was introduced into a sealed sample vial. Duplicate determinations were performed using approximately 10 mg of sample per titration. The average of these results is presented unless otherwise stated. Data collection and analysis were performed using Tiamo software.

[0208] thermodynamic water solubility Aqueous solubility was determined by suspending sufficient compound in the relevant vehicle to achieve a maximum final concentration of 10 mg / ml or greater of the parent-free form of compound. The suspension was equilibrated for 24 hours at 25°C on a Heidolph plate shaker set at 750 rpm. The pH of the saturated solution was then measured, and the suspension was filtered through a glass fiber C filter (particle retention 1.2 μm) and diluted appropriately. Quantification was by HPLC referenced to a standard solution of approximately 0.15 mg / ml in DMSO. Various volumes of standard, diluted, and undiluted sample solutions were injected. Solubility was calculated using peak areas, determined by integration of the peaks found at the same retention time as the main peak in the standard injection.

[0209] [Table 10]

[0210] Analyses were performed on an Agilent HP1100 / Infinity II 1260 series system equipped with a diode array detector using OpenLAB software.

[0211] [Table 11]

[0212] Methods and Comments Sufficient sample for the highest anticipated concentration of 100 mg / ml of the compound's hydrochloride salt was suspended in 0.5 ml of vehicle. The resulting suspension was then shaken at 25°C / 750 rpm for 24 hours. After equilibration, the appearance was noted and the pH of the saturated solution was measured. The sample was then filtered through a glass "C" fiber filter (particle retention 1.2 μm). Samples were diluted 10-fold and 100-fold into the appropriate vehicle.

[0213] Quantification was by HPLC with reference to a standard solution of approximately 0.15 mg / ml. Various volumes of standard, diluted, and undiluted sample solutions were injected. Solubility was calculated using the peak area determined by integration of the peak found at the same retention time as the main peak in the standard injection.

[0214] observation For this sample, co-elution of the SIF peak and the parent peak was observed using the generic solubility method. The sample was run using the generic purity method (higher resolution column) to resolve these peaks.

[0215] [Table 12]

[0216] [Table 13]

[0217] [Table 14]

[0218] Summary and Conclusion In FaSSGF medium, FaSSIF medium, and deionized water, the sample was classified as "slightly soluble" by the USP classification. In FeSSIF, the sample was classified as "very slightly soluble" by the USP classification. These values ​​were calculated based on a sample containing 1.4 equivalents of HCl, based on IC and KF analysis.

[0219] Since there were issues with co-elution of the SIF peaks when using the generic solubility method, all subsequent analyses will be performed using the generic purity method as this method is able to resolve the peaks.

[0220] Ion Chromatography (IC) Data were collected using IC MagicNet software on a Metrohm 930 Compact IC Flex equipped with an 858 Professional autosampler and an 800 Dosino dosage unit monitor. Accurately weighed samples were prepared as stock solutions in appropriate solvents. Quantitation was achieved by comparison with standard solutions of known concentrations of the analyte ions. Analyses were performed in duplicate, and values ​​are averages unless otherwise stated.

[0221] [Table 15]

[0222] [Table 16]

[0223] Determination and prediction of pKa and LoqP result

[0224] [Table 17]

[0225] Structure and Prediction

[0226] [ka]

[0227] By Yasuda-Shedlovsky extrapolation, the high pKa of 8.95 was identified as acidic, and the pKa of 8.19 was identified as basic (not in agreement with the prediction software).

[0228] Methods and Comments Data were collected on a Sirius T3 instrument equipped with a D-PAS attachment fitted with an Ag / AgCl dual injection pH electrode. The electrode was calibrated using four positive parameters derived from a blank titration. Base titrants were standardized by titration with KHP. 0.5 M HCl and KOH aqueous solutions were used as acid and base titrants, respectively, for the tests. Titrations were performed in a background of ISA 0.15 M KCl (aq). Data were refined using Sirius T3 Refine. Predictions of pKa and LogP values ​​were performed using ACD / Labs Percepta.

[0229] UV metric pKa (aqueous) Samples were prepared as 11.07 mM stock solutions in DMSO (5 μL of stock was used for analysis). Data were obtained by UV-metric single titration under aqueous conditions at 25 °C from pH 1.5 to 4.0 (low to high).

[0230] Fast UV pKa (aqueous) Samples were prepared as 10 mM stock solutions in DMSO (5 μL of stock was used for analysis). Data were obtained by fast UV single titration from pH 2.0 to 12.0 (low to high) under aqueous conditions at 25 °C.

[0231] pH metric LogP 0.96 mg of sample was weighed directly into a T3 vial. Data were collected using a potentiometric titration method using a high LogP assay with three ratios of octanol (0.020 / 0.050 / 1.0 mL) and ionic strength adjusted (ISA) water at pH 2.0 to 12.0 (low to high). The collected potentiometric data were used to calculate LogP, LogP ion , and LogD values ​​were calculated.

[0232] LogD flask shake A 1 mg / ml stock solution in octanol was prepared and shaken for 1 hour at room temperature to ensure complete dissolution. Equal volumes of the octanol solution and USP pH 7.4 phosphate buffer (50 mM) were shaken in duplicate for 1 hour at room temperature. Samples were centrifuged at 13,400 rpm for 10 minutes, and the phases were separated using a glass pipette. The separated layers were diluted appropriately (with acetonitrile for octanol samples and buffer for aqueous samples, as necessary) and analyzed by HPLC using the method detailed below.

[0233] [Table 18]

[0234] Experimental crystallization methodology The choice of crystallization method has a large effect on which form is produced, so when searching for polymorphs it is important to perform crystallization using a variety of methods and conditions.

[0235] The classical crystallization methods used herein are listed in Table 14 along with the degrees of freedom available for each process.

[0236] [Table 19]

[0237] Solvent-Mediated Technology These are classic techniques used to produce 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 grow only from supersaturated solutions.

[0238] For crystallization screening, solvents should be selected with very different properties (hydrogen bond donor / acceptor tendencies, dipole moments, dielectric constants, viscosities, etc.). Usually, solvent mixtures are useful to obtain systems with appropriate solubility, polarity, etc. The substance also needs to be chemically stable in a given solvent or solvent mixture. There are several ways to achieve a metastable state of supersaturation.

[0239] Maturation / Slurry Maturation To investigate crystalline morphology, maturation experiments (or slurry maturation) are typically performed in various solvents or solvent mixtures and subjected to heating-cooling cycles. Repeated heating and cooling cycles can increase the degree of crystallinity, or can convert a metastable state (or a non-equilibrium state in the case of amorphous materials) to a more thermodynamically stable crystalline form. The rate and extent of conversion depend on the solubility of the input materials.

[0240] For thermodynamic reasons, a system can only evolve to a more stable form. Therefore, if the starting material is crystalline, it is impossible to obtain a less stable crystalline phase. If the starting material is amorphous, a much more diverse range of morphologies can be obtained.

[0241] Maturation chamber procedure The maturation suspension was placed in a platform shaker incubator (Heidolph Titramax / Incubator 1000) and subjected to a series of heating-cooling cycles from ambient temperature to approximately 50°C. This was achieved by turning the heat on and off every 4 hours. Shaking was maintained throughout.

[0242] Polar Bear Instructions The suspension was stirred (500 rpm) in a Polar Bear (Cambridge Reactor Design) for 1 hour at 50° C. The sample was then cooled to 5° C. at 0.1° C. / min and stirred for a further 4 hours.

[0243] Cooling crystallization Crystallization can be achieved by lowering the temperature of a clear solution. Because the solubility of most materials decreases with decreasing temperature, cooling can be used to create supersaturation. However, in many cases, the solubility of a material remains high even at low temperatures, or the solubility changes only slightly over the temperature range of interest. In these cases, other methods of creating supersaturation must be considered (e.g., controlled evaporation as described below).

[0244] procedure The solution was cooled to 5° C. at 0.1° C. / min in a Polar Bear and stirred at this temperature for 24 h. All solids were filtered, dried under vacuum for 20 min and initially analyzed by XRPD.

[0245] Controlled Evaporation Crystallization can be achieved by controlled evaporation of a clear, particulate-free solution. This is especially 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. Once a certain maximum supersaturation is reached, crystal nucleation and growth occur. This technique also has the advantage that, because the sample is evaporated slowly, it is usually possible to produce large single crystals suitable for SCXRD.

[0246] procedure The solution was allowed to evaporate at ambient conditions by removing the vial lid and replacing it with a pinhole sealing film or by inserting a 25 gauge syringe needle into the lid. The sample was allowed to evaporate slowly until dry / solid appeared at ambient conditions.

[0247] Characterization of Compound (I) (PCLX-001) Compound (I) (PCLX-001) (J09898 and batch J09899) was characterized by investigating the solid-state morphology and chemical properties of PCLX-001 using a range of techniques. A summary of the results is shown in Table 15.

[0248] [Table 20]

[0249] Characterization of PCLX-001 (J09899, ​​HCl Pattern 1) confirmed its crystalline nature, assigned HCl Pattern 1 by XRPD analysis (Figure 1), with a purity of 97.2% (Figure 3). The 1H-NMR spectrum (Figure 2) was consistent with the proposed molecular structure, with traces of acetone present. 1.3 molar equivalents of chloride were observed in the anion IC, which was lower than the expected 3 equivalents of chloride since the compound was believed to be in the trichloride form. Additionally, 0.2 molar equivalents of sodium and 0.1 molar equivalents of calcium were also observed in the cation IC, which may have arisen during the compound's manufacturing process.

[0250] Thermal analysis (Figure 4) of PCLX-001 (J09899, ​​HCl Pattern 1) showed that the material had a large endotherm at 64.7 °C (onset, 174 J / g) followed by an endotherm at 154.5 °C (onset, 23 J / g). A mass loss of 9.7% was observed from approximately 50 °C and 225 °C, followed by a small mass loss of 1%, and decomposition of the material was observed at 250 °C (onset). Since 9.7% w / w water was observed in the sample by KF analysis, the mass loss may have been related to the loss of water from the sample.

[0251] GVS analysis of PCLX-001 (J09899, ​​HCl Pattern 1) (Figures 5 and 6) showed that the sample was highly hygroscopic, with a water uptake of 39.76% w / w at 90% RH. However, no visible changes were observed in the appearance or XRPD pattern of the solid after GVS analysis.

[0252] Static stability experiments were also performed at two sets of high storage conditions, 40°C / 75% RH and 25°C / 97% RH, which showed no visible changes in the appearance or XRPD patterns of the solids (Figures 7 and 10), and the purity remained high in both cases (Figures 8 and 9), concluding stability at both high humidity and high temperature.

[0253] Thermodynamic solubility showed high solubility in FaSSGF at 9.9 mg / ml and in water at 6.0 mg / ml, and relatively low solubility in FeSSIF at 1.9 mg / ml and FaSSIF at 0.64 mg / ml. There was little difference between the predicted and measured pKa values. Log P analysis was found to be 2.2 at pH 7.4. Furthermore, the difference in measured pKa was sufficient to rule out a zwitterion.

[0254] Characterization of a replacement batch of received PCLX-001 (J09898) was confirmed to have a different crystalline pattern and was assigned HCl pattern 2 by XRPD analysis. The material exhibited a lower purity of 96.7%. However, 1 The H-NMR spectrum was consistent with the proposed molecular structure, with traces of acetone and TBME present. Thermal analysis was also performed on PCLX-001 (J09898), revealing a large endotherm of 196 J / g at 40.2 °C (onset), followed by an 8 J / g exotherm at 138.7 °C (onset) and a 10 J / g endotherm at 165.8 °C (onset). An 11.4% w / w mass loss occurred from approximately 50 °C to 225 °C, followed by decomposition of the material at 250 °C (onset). Additionally, 2 molar equivalents of chloride were observed in the anionic IC, still lower than the 3 molar equivalents expected for the Tris salt, and no cation was observed in the cationic IC.

[0255] Characterization of additional batches of PCLX-001 (J09951, J09952, and J09953) confirmed crystalline HCl Pattern 1, and all retained high purity of 98.5%, 98.3%, and 98.5%, respectively. 1H-NMR spectra were consistent with the proposed molecular structure. A sharp peak at 32°2θ was observed in the XRPD diffractograms of J09951, J09952, and J09953, representing the presence of a higher ratio of NaCl in the samples compared to batch J09899. Full characterization is summarized in Table 16 and Figure 11.

[0256] [Table 21]

[0257] Trial salt destruction experiment of PCLX-001 procedure PCLX-001 (J09899, ​​HCl Pattern 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 up to 50 volumes (2.5 ml) were added. After each solvent addition, the vial was stirred at room temperature (RT) for 5 minutes; if dissolution did not occur, 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.

[0258] After the assay was complete, 1 equivalent (119.2 μl, added as a 1 M stock solution in water relative to the Cl ions calculated from the IC) of NaOH was added at 50° C., and the sample was slowly cooled to 5° C. at 0.1° C. / min. All solids were isolated by filtration and dried under vacuum. The DCM system produced a yellow solution. The system was washed once with water, and the organic layer was separated and then dried in a vacuum oven at RT. All isolated solids were analyzed by XRPD.

[0259] Results and Discussion In salt-breaking experiments of PCLX-001 (J09899), no dissolution was observed in four of the five solvents tested; in all cases, a yellow suspension formed. Ethanol was the only exception, where dissolution was observed at 50°C and 50 times the volume, forming a colorless solution. Upon addition of 1 equivalent of NaOH relative to chloride ions, calculated from IC analysis of the J09899 material, the sample in ethanol formed a yellow solution, while all remaining samples remained yellow suspensions.

[0260] After cooling to 5°C, the sample in DCM formed a clear yellow solution, while all remaining material formed a pale yellow suspension. The sample in DCM was selected for further processing and separated by separatory funnel. The organic layer was dried overnight at RT in a vacuum oven, resulting in the formation of a new solid that was assigned free form Pattern 1 for later characterization. Further processing was also performed on the sample in ethyl acetate; here, the sample was filtered and dried under vacuum, and IC analysis revealed evidence of the presence of chloride ions, resulting in a new pattern designated as "mixed salt." See Table 18 and Figures 12A-14.

[0261] [Table 22]

[0262] Characterization was performed on a new sample obtained from the salt-break test of J09899. XRPD analysis of the sample obtained from DCM (see Figure 12A) showed the formation of a new pattern, which was later assigned as free form pattern 1. This also indicates that the upfield shift of the sample 1 This was because the anion and cation were observed in the H-NMR spectrum, suggesting the formation of the parent form (see Figure 12B). Furthermore, IC analysis showed that neither anions nor cations were observed, confirming the formation of free form Pattern 1. Free form Pattern 1 also retained a high purity of 96.26% (see Figure 13).

[0263] XRPD analysis (see FIG. 12A) also confirmed the formation of a new solid from ethyl acetate, designated as "mixed salt," since IC analysis showed that 1.3 molar equivalents of chloride and 1.4 molar equivalents of sodium were present. 1 H-NMR analysis (see Figure 12B) also showed an upfield shift, suggesting that the high purity was maintained at 97.8% (see Figure 14), excluding the presence of zwitterions.

[0264] Scale-up formation of parent / free forms from J09899 procedure Attempt 1 - 1g scale up 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, which was treated with 50 volumes (50 ml) of DCM at 50° C. to form a yellow suspension. Following this, 1 equivalent of NaOH (added as a 1 M solution in water relative to the Cl ions calculated from the IC) was added at 50° C., and the sample was slowly cooled to 5° C. at 0.1° C. / min to form a pale yellow solid. All isolated solids were analyzed by XRPD. Sample ID: EG-1826-12-01. Results: XRPD analysis revealed the sample to be consistent with the "mixed salt" pattern observed in trial salt destruction experiments using ethyl acetate as the solvent.

[0265] Attempt 2 - 1g scale up 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, which was treated with 50 volumes (50 ml) of DCM at 30° C. to form a yellow suspension. Following this, 1 equivalent of NaOH (2.4 ml, added as a 1 M solution in water relative to Cl ions calculated from the IC) was added at 30° C. and left to stir for 1 hour to form a yellow solution.

[0266] Water (50 ml) was added to produce separate 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 the 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.

[0267] Attempt 3 - 200mg scale up PCLX-001 (J09899, ​​HCl Pattern 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. This was followed by the addition of 1 equivalent of NaOH (447 μl, added as a 1 M solution in water relative to Cl ions calculated from the IC) at 30° C. and left stirring for 1 hour to form a yellow solution.

[0268] Water (10 ml) was added to produce separate 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 the remaining organic layer. After the magnesium sulfate condensed to indicate a drying endpoint, the organic layer was filtered from the magnesium sulfate and the remaining solution was dried overnight at RT under vacuum. All isolated solids were analyzed by XRPD. Sample ID: EG-1826-22-01→05.

[0269] Attempt 3 - 1g scale-up repeat 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, which was treated with 50 volumes (50 ml) of DCM at 30° C. to form a yellow suspension. Following this, 1 equivalent of NaOH (2.4 ml, added as a 1 M solution in water relative to Cl ions calculated from the IC) was added at 30° C. and left to stir for 1 hour to form a yellow solution.

[0270] Water (50 ml) was added to produce separate 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 the remaining organic layer. After the magnesium sulfate condensed to indicate a drying endpoint, the organic layer was filtered from the magnesium sulfate and the remaining solution was dried overnight at RT under vacuum. All isolated solids were analyzed by XRPD. Sample ID: EG-1826-25-01.

[0271] Results and Discussion Scale-up of free form Pattern 1 was successful at both the 200 mg and 1 g scales and was confirmed by XRPD. Full characterization was performed on the 1 g scale-up of parent / free form Pattern 1 (EG-1826-12-02) and is summarized in Table 19.

[0272] [Table 23]

[0273] 1 H-NMR analysis (Figure 15) further confirmed the formation of free form Pattern 1, retaining a high purity of 96.1% (Figure 17). Thermal analysis (Figure 16) showed a mass loss of 0.5% w / w from 50 to 100 °C, with decomposition at 250 °C (onset) and a large, broad endotherm between 50 and 170 °C (Tpeak = 169.6 °C, 74 J / g). Static stability experiments showed no visible changes in the solid's appearance or XRPD pattern, retaining high purity under elevated storage conditions and confirming stability at elevated temperature and humidity. IC analysis indicated the presence of 0.1 molar equivalents of sodium in the sample, which could be removed with an additional washing step.

[0274] XRPD analysis was used to confirm other scale-up processes of Free Form Pattern 1 using EG-1865-12-02 as the primary reference. XRPD overlays of successful scale-up of Free Form Pattern 1 are shown in Figures 18 and 19.

[0275] Solubility test of free form pattern 1 Solvent List The list of solvents used in the initial solubility studies of free form Pattern 1 is summarized in Table 20.

[0276] [Table 24]

[0277] Step 1 EG-1826-12-02 (free form pattern 1, 20 mg ± 1 mg) was weighed into 10 x 4 ml vials. To each vial, the selected solvent system (Table 20) was added in aliquots (10x, 20x, 40x, 60x, 80x) until dissolution of the sample occurred or a maximum of 80x volume (1.6 ml) was reached.

[0278] After each aliquot was added, the sample was first stirred at RT for 5 min; if the sample did not dissolve, it was then stirred in the Polar Bear at 500 rpm at 50° C. for an additional 5 min. If dissolution did not occur, the sample was cooled back to RT before adding the next aliquot of solvent. All suspensions remaining after the solubility assessment had 2 equivalents of HCl (74.5 μl, added as a 1 M stock solution in THF, per 20 mg of parent / free form) added to each vial at 50° C. All resulting solids were initially analyzed by XRPD.

[0279] Results and Discussion The results of the initial solubility testing of the parent form are summarized below. This solubility was performed to select suitable solvents for salt screening and was not intended to constitute a total solubility assessment (tabulated in Table 23).

[0280] No dissolution was observed in any solvent except DMSO, which showed extremely high solubility, forming a clear yellow solution upon addition of 10 volumes of solvent at RT.

[0281] After the solubility evaluation was completed, 2 molar equivalents of HCl were added to each of the samples to assess whether dissolution would occur with the addition of the counterion. Dissolution was not achieved in toluene, TBME, and heptane, but was otherwise successful in all remaining solvents.

[0282] Extended solubility assessment of free form Pattern 1 procedure EG-1826-25-01 (parent / free form from 1 g scale-up attempt 3, 10 mg ± 1 mg) was weighed into 22 HPLC vials. To each vial, the selected solvent system (see Table 22) was added in aliquots (10x volumes, 20x volumes, 40x volumes, 60x volumes, 80x volumes) until dissolution of the sample occurred or a maximum of 80x volume was reached.

[0283] After each aliquot was added, the sample was first stirred at RT for 5 min; if the sample did not dissolve, it was then stirred in the Polar Bear at 50°C and 500 rpm for an additional 5 min. If dissolution did not occur, the sample was cooled back to RT before adding the next aliquot of solvent. All solutions were slowly cooled to 5°C at 0.1°C / min. All suspensions were matured in a maturation chamber at 25 / 50°C in a 4-hour cycle for up to 2 days. All resulting solids were initially analyzed by XRPD.

[0284] [Table 25]

[0285] Results and Discussion The results of the extended solubility evaluation are summarized in Tables 23-26 and Figures 20-29. Only free form Pattern 2 was obtained from the extended solubility evaluation.

[0286] [Table 26]

[0287] [Table 27]

[0288] [Table 28]

[0289] [Table 29]

[0290] Further characterization was performed on two of the samples obtained from the solubility study. H-NMR of the samples showed that the spectra were consistent with the structures, with 0.14 equivalents of acetone present in EG-1826-28-04 and 0.2 equivalents of isopropyl acetate present in EG-1826-28-14 (Figures 24 and 25).

[0291] Thermal analysis of EG-1826-28-04 (Figure 22) showed a 0.2% mass loss between 50 and 60°C, followed by a 0.8% mass loss between 200 and 220°C and an onset of decomposition at 250°C. A large, sharp endotherm at 209.1°C (onset) of 223 J / g was observed, followed by a smaller, sharp recrystallization peak at 219°C (onset) of 15 J / g. A similar thermal analysis was observed for EG-1826-28-14 (Figure 23), which showed a 0.2% mass loss between 70 and 80°C, followed by a larger mass loss of 4.1% between 190 and 240°C and an onset of decomposition at 250°C. A large, sharp endotherm at 214.6°C (onset) of 93 J / g was observed, followed by a sharp recrystallization peak at 218.6°C (onset) of 63 J / g.

[0292] Static stability experiments showed no visible changes in appearance or XRPD pattern when kept at elevated storage conditions of 25°C / 97% relative humidity and 40°C / 75% relative humidity for 7 days. The amount of solvent observed in the NMR spectrum of free form Pattern 2 was residual / unbound solvent. This amount could be removed by heating and drying under vacuum. Controlled crystallization / optimization of the crystallization method can avoid the issue of residual solvent content.

[0293] Heating of free form pattern 2 to 220°C During the characterization of the sample from the solubility evaluation, a large recrystallization peak was observed in the TGA analysis, leading to heating of the free form Pattern 2. This experiment was performed to evaluate whether the recrystallization peak was related to the formation of a new form or to the same free form Pattern 2.

[0294] EG-1826-28-10 (free form pattern 2, approximately 5 mg) was heated to 220° C. at 10° C. / min by TGA. The resulting solid was reanalyzed by XRPD. Sample ID: EG-1826-39-01.

[0295] Results and Discussion XRPD analysis of the sample after heating to 220°C (Figure 30) confirmed that the recrystallization peaks were consistent with free form pattern 2, allowing us to conclude that the sample does not convert to another form at higher temperatures.

[0296] conclusion Characterization of compound (I) (PCLX-001, batch J09899), assigned HCl Pattern 1, confirmed that only 1.3 equivalents of chloride were observed in the anion IC, less than the expected 3 equivalents of chloride. Additionally, 0.2 equivalents of sodium and 0.1 equivalents of calcium were observed in the cation IC, which may have arisen during the compound's manufacturing process. Characterization of additional batches of PCLX-001 (J09951, J09952, and J09953, also assigned HCl Pattern 1), revealed sharp peaks in the XRPD diffractogram consistent with the presence of higher NaCl ratios in the sample compared to PCLX-001 (J09899).

[0297] A salt break test using sodium hydroxide was performed with five process solvents. XRPD analysis of a sample obtained from DCM showed the formation of a new pattern that was later assigned Free Form Pattern 1. This was because an upfield shift was observed in the H-NMR spectrum, suggesting the formation of the free form. IC analysis confirmed this, showing that no anions or cations were observed, confirming the production of Free Form Pattern 1. A high purity of 96.3% was also retained for Free Form Pattern 1. Free Form Pattern 1 was also successfully scaled up at the 200 mg and 1 g scales.

[0298] Polymorphism screening performed on free form Pattern 1 yielded only one new pattern, designated Free Form Pattern 2. Heating of Free Form Pattern 2 was also performed because a large recrystallization peak was observed in DSC analysis during characterization of Free Form Pattern 2.

[0299] In conclusion, free form pattern 2 exhibited the most desirable solid-state properties of all the polymorphs discovered.

[0300] Example 2 Solubility measurement and crystallization evaluation of PCLX-001 free form pattern 2 overview Herein, the solubility measurements and crystallization occurrences performed on PCLX-001 free form Pattern 2 are summarized.

[0301] Characterization of PCLX-001 free form pattern 2 (J10206) was confirmed to be crystalline by XRPD analysis, 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.

[0302] Solubility experiments were performed using gravimetric methods. Based on the solubility evaluation results described in Example 1, solubility was measured in DMSO, DMA, and NMP. The largest range of solubility was observed between 60°C and 25°C in DMSO, with a calculated solubility of 32 mg / ml at 60°C and 12.3 mg / ml at 25°C in DMSO. DMSO was selected as the preferred neat solvent to proceed with process development because it had already been used in the crystallization process.

[0303] In parallel, and using gravimetric methods to explore solvent ratios to determine the ratio for best results, an array of solvent systems was performed to determine solubility at two temperatures: 25° C. and 60° C. The solvent systems selected to generate solubility curves and proceed to process development were DMSO EtOH:HO (1:2) (70:30), DMSO EtOH:HO (1:2) (95:5), and DMSO EtOH:HO (2:1) (50:50).

[0304] Solubility curves were successfully generated for all selected solvent systems. From the solubility curves, it can be seen that the solubility curves for DMSO EtOH:HO (1:2) (70:30) and DMSO EtOH:HO (2:1) (50:50) were significantly lower than those for neat DMSO and DMSO EtOH:HO (1:2) (95:5). Since the addition of an antisolvent was shown to have a beneficial effect on increasing the yield of PCLX-001 free form Pattern 2, the solubility data from DMSO EtOH:HO (1:2) (95:5) was entered into DynoChem software for process development.

[0305] Two different procedures were used to conduct trial seeded crystallizations. The first trial seeded crystallization consisted of adding 5% antisolvent before adding seeds at elevated temperatures. Observations during this trial seeded crystallization indicated that adding 5% antisolvent caused the sample to crash out before adding seeds, as predicted by the predictions generated in DynoChem. Another trial seeded crystallization was conducted by adding seeds before adding 10% antisolvent. This successfully crystallized PCLX-001 free form pattern 2 at a 50 mg scale in 84% yield. This was slightly lower than the 95% yield predicted by DynoChem; however, a slightly lower yield was expected because the crystallization process was not fully optimized.

[0306] Successful scale-up of the selected solvent system DMSO EtOH:HO (1:2) (10%) was performed at the 700 mg scale. Further characterization confirmed that the material was consistent with PCLX-001 free form pattern 2.

[0307] [Table 30]

[0308] Equipment and methodology details X-ray powder diffraction (XRPD) Bruker AXS D8 Advance 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, followed by a Lynxeye detector. The software used for data collection and analysis was Diffrac Plus XRD Commander and Diffrac Plus EVA, respectively.

[0309] Samples were run under ambient conditions as flat plate specimens using powder. Samples were prepared on polished, zero-background (510) silicon wafers by gently pressing them onto a flat surface or filling a cut cavity. The samples were rotated within their own plane.

[0310] Details of standard data collection methods are as follows: Angle range: 2~42°2θ Step size: 0.05° 2θ Acquisition time: 0.5 seconds / step (total acquisition time: 6.40 minutes)

[0311] PANalytical's Empyrean XRPD diffractograms were collected on a PANalytical Empyrean diffractometer using Cu Ka radiation (45 kV, 40 mA) in transmission geometry. A 0.5° slit, a 4 mm mask, and a 0.04 rad Soller slit and focusing mirror were used in the incident beam. A PIXcel 3D The detector was fitted with a receiving slit and a 0.04 rad Soller slit. The software used for data collection was X'Pert Data Collector using the X'Pert Operator Interface. Data were analysed and presented using Diffrac Plus EVA or HighScore Plus.

[0312] Samples were prepared and analyzed in transmission mode in metal or Millipore 96-well plates. X-ray transparent film was used between metal sheets on metal well plates, and powder (approximately 1-2 mg) was used. Millipore plates were used to isolate and analyze solids from suspension by adding a small amount of suspension directly to the plate before filtration under light vacuum.

[0313] The scan mode for the metal plate used the gonioscan axis, while the Millipore plate utilized a 2θ scan.

[0314] Details of standard screening data collection methods are as follows: Angle range: 2.5 to 32.0° 2θ Step size: 0.0130°2θ Acquisition time: 12.75 seconds per step (total acquisition time 2.07 minutes)

[0315] nuclear magnetic resonance (NMR) Solution-state NMR 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. Samples were prepared in DMSO-d6 solvent unless otherwise stated. Automated experiments were performed using standard Bruker on-board experiments ( 1 H, 13 C{ 1 H}, DEPT135) were acquired using the ICON-NMR settings in Topspin software. Offline analysis was performed using an ACD Spectrus processor.

[0316] Differential scanning calorimetry (DSC) TA Instruments Q2000 DSC data were collected on a TA Instruments Q2000 equipped with a 50-position autosampler. Typically, 0.5–3 mg of each sample was heated in a pinhole aluminum pan from 25°C to 300°C at 10°C / min. A 50 ml / min purge of dry nitrogen was maintained over the sample.

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

[0318] Thermogravimetric analysis (TGA) TA Instruments Q500 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 into a pre-tared aluminum DSC pan and heated from ambient temperature to 350°C at 10°C / min. A 60 ml / min nitrogen purge was maintained over the entire sample.

[0319] The instrument control software was Advantage for Q Series and Thermal Advantage, and data were analyzed using Universal Analysis or TRIOS.

[0320] Polarized Light Microscopy (PLM) Leica LM / DM polarizing microscope Samples were analyzed with a Leica LM / DM polarized light microscope using a digital video camera for image capture. A small amount of each sample was mounted on a glass slide with or without immersion oil and covered with a coverslip. Samples were viewed under appropriate magnification and partially polarized light coupled with a λ false color filter. Images were acquired using StudioCapture or Image ProPlus software.

[0321] Scanning Electron Microscopy (SEM) Data were collected on a Phenom Pro scanning electron microscope. A small sample was mounted on an aluminum stub using conductive double-sided adhesive tape. A thin layer of gold was applied using a sputter coater (20 mA, 120 s).

[0322] Chemical purity determination by HPLC 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:

[0323] [Table 31]

[0324] Determining Water by Karl Fischer Titration (KF) The water content of each sample was measured in a Metrohm 874 Oven Sample Processor at 150°C with an 851 Titrano coulometer using Hydranal Coulomat AG oven reagent and a nitrogen purge. A weighed solid sample was introduced into a sealed sample vial. Duplicate determinations were performed using approximately 10 mg of sample per titration. The average of these results is presented unless otherwise stated. Data collection and analysis were performed using Tiamo software.

[0325] Ion Chromatography (IC) Data were collected using IC MagicNet software on a Metrohm 930 Compact IC Flex equipped with an 858 Professional autosampler and an 800 Dosino dosage unit monitor. Accurately weighed samples were prepared as stock solutions in appropriate solvents. Quantitation was achieved by comparison with standard solutions of known concentrations of the analyte ions. Analyses were performed in duplicate, and values ​​are averages unless otherwise stated.

[0326] [Table 32]

[0327] [Table 33]

[0328] Raman spectroscopy Data were collected using a Renishaw inVia QWertor. The instrument control, data analysis and presentation software was WiRE.

[0329] Method: Excitation source, λ ex = 785 nm laser, appropriately attenuated to avoid sample degradation. Raman shift range: 100–5000 cm-1 ;Exposure time: 0.02~10s;Accumulation: 1~3.

[0330] Crystal 16 The solubility and metastable zone of the materials were determined as a function of temperature using a Crystal 16 crystallization system (Technobis, NL). Slurries of different total concentrations of API were prepared by adding a known amount of solid to a known amount of cooled solvent (between 0.5 and 1.5 ml) and stirred at 500 rpm using a magnetic bar. The saturation temperature was measured through heating and cooling cycles from 65 to 23 °C at 0.5 °C / min.

[0331] As the temperature was increased, the solids dissolved completely, the suspension became a clear solution, and the light transmittance reached a maximum. This temperature was assigned as the clearing point, which was assumed to correspond to the saturation temperature. The solution was then cooled at a rate of 0.5°C / min, and the temperature at which particles first formed was detected by the decrease in light transmittance. This was assigned as the cloud point. These points were fitted by the van't Hoff equation, and the difference between the cloud point and the clearing point defined the metastable zone width (MSZW) of the system. The instrument control software was Crystallisation Systems, and data were analyzed using Crystal Clear and Microsoft Excel.

[0332] Characterization of Compound (I) (PCLX-001, Free Form Pattern 2, Batch J10206) The material (Batch J10206) was characterized using a range of techniques to investigate the solid state morphology and chemical properties of PCLX-001 free form Pattern 2. A summary of the results is shown in Table 30.

[0333] [Table 34]

[0334] Characterization of PCLX-001 free form Pattern 2 (J10206) was confirmed to be crystalline by XRPD analysis (Figures 37 and 38) and was 98.85% pure (Figure 42). 1 H-NMR (Figures 39 and 40) and Raman (Figures 43 and 44) ​​spectra were consistent with the proposed molecular structure, with neither anions nor cations observed. Thermal analysis (Figure 41) of PCLX-001 free form Pattern 2 (J10206) showed that the material had a large endotherm at 220 °C (onset) of 92 J / g, followed by a large exotherm at 225 °C (peak) of 65 J / g. A mass loss of 0.6% w / w was observed between 200 and 240 °C, with decomposition of the material observed at 260 °C (onset). Since 0.3% water was observed in the sample by KF analysis, the mass loss is related to the loss of water from the sample. Solubility (pH profiling) analysis showed that the compound became increasingly soluble in acidic media; samples suspended below pH 4.5 became clear.

[0335] Solubility determination and construction of solubility curves

[0336] [Table 35]

[0337] Methods and Comments pH profiling Sufficient sample for a maximum expected concentration of 10 mg / ml of the free form of the compound was suspended in 1.0 ml of medium. The resulting suspension was then shaken at 25°C / 750 rpm for 24 hours. The pH of the sample solution was checked periodically and adjusted with 0.2 M HCl / NaOH as needed to ensure the desired pH was maintained throughout (±0.2). After equilibration, the appearance was noted and the final pH of the saturated solution was measured. The sample was then filtered through a glass "C" fiber filter (particle retention size 1.2 μm) before being diluted with the relevant buffer as needed.

[0338] Quantification was by HPLC with reference to a standard solution of approximately 0.15 mg / ml. Various volumes of standard, diluted, and undiluted sample solutions were injected. Solubility was calculated using the peak area determined by integration of the peak found at the same retention time as the main peak in the standard injection.

[0339] [Table 36]

[0340] observation The sample suspended in pH 1.2 buffer became clear at 1 hour before pH adjustment, while all other samples remained cloudy or contained residual solids. After 1 hour of pH adjustment, the sample suspended in pH 2.0 buffer became clear. All remaining samples remained cloudy or contained residual solids until 24 hours of equilibration.

[0341] [Table 37]

[0342] [Table 38]

[0343] Results, Summary and Conclusions The compound became increasingly soluble in acidic media, with samples suspended below pH 4.5 becoming clear. The sample suspended at pH 1.2 was clear before the 1-hour pH adjustment, so the concentration was quoted from before the pH adjustment. On the other hand, the sample suspended at pH 2.0 became clear after the 1-hour pH adjustment, so the concentration was quoted using the total volume of the medium after the adjustment. Two points between pH 0 and 2 are greater than the solubility value, see Figure 51.

[0344] Solubility determination in DMSO, DMA and NMP Based on the solubility evaluation results of Example 1, the solubility was measured in DMSO, DMA, and NMP at two different temperatures: 60° C. and RT.

[0345] procedure Solubility measurement at 60℃ J10206 (30 mg ± 1 mg) was weighed into four HPLC vials and a stir bar was added. The samples were dissolved in the selected solvents DMSO, DMA, and NMP (1 ml), heated to 60 °C in a Polar Bear, and stirred for 2-3 hours to allow the samples to equilibrate. The samples were then filtered, and the mother liquor was placed in a pre-weighed vial and evaporated overnight at RT in a vacuum oven. The isolated solids after filtration were analyzed by XRPD.

[0346] Solubility determination at 25°C J10206 (30 mg ± 1 mg) was weighed into four HPLC vials and a stir bar was added. The samples were dissolved in the selected solvents DMSO, DMA, and NMP (1 ml) and stirred with a stir bar at 25 °C for 2-3 hours to allow the samples to equilibrate. The samples were then filtered, and the mother liquor was placed in a pre-weighed vial and evaporated overnight at RT in a vacuum oven. The isolated solids after filtration were analyzed by XRPD.

[0347] Results and Discussion The largest range of solubility was observed in DMSO between 60°C and 25°C, with a calculated solubility of 32 mg / ml at 60°C and 12.3 mg / ml at 25°C in DMSO. This large range of solubility between 60°C and 25°C was not observed when DMA and NMP were used as solvents. DMSO was selected as the preferred neat solvent to proceed into process development because it had already been used in the crystallization process. See Table 31 and Table 32, Figure 45.

[0348] [Table 39]

[0349] [Table 40]

[0350] Solubility determination using EtOH:H2O mixture as antisolvent In parallel, solvent ratios were investigated to determine the ratio for best results, and a solvent system sequencing was performed using gravimetric methods to determine solubility at two temperatures: 25° C. and 60° C. Observations during the solubility sequencing experiments were recorded to monitor whether oiling out phenomenon was occurring during the process.

[0351] procedure Solubility determination at 60°C J10206 (30 mg ± 1 mg) was weighed into 15 HPLC vials and a stir bar was added. Samples were dissolved in the selected solvent ratios of DMSO and EtOH:HO (1:1), EtOH:HO (1:2), or EtOH:HO (2:1) (1 ml), heated to 60 °C in a Polar Bear, and stirred for 2–3 hours to equilibrate the samples. The samples were then filtered, and the mother liquor was placed in a pre-weighed vial and evaporated overnight at RT in a vacuum oven. The isolated solids after filtration were analyzed by XRPD.

[0352] Solubility determination at 25°C J10206 (30 mg ± 1 mg) was weighed into 15 HPLC vials and a stir bar was added. Samples were dissolved in the selected solvent ratios of DMSO and EtOH:HO (1:1), EtOH:HO (1:2), or EtOH:HO (2:1) (1 ml), heated to 25 °C in a Polar Bear, and stirred for 2–3 hours to equilibrate the samples. The samples were then filtered, and the mother liquor was placed in a pre-weighed vial and evaporated overnight at RT in a vacuum oven. The isolated solids after filtration were analyzed by XRPD.

[0353] Results and Discussion The DMSO to antisolvent ratios selected for process development were DMSO:EtOH:HO (1:2) (70:30) and DMSO:EtOH:HO (1:1) (50:50) because a controlled range of solubility was observed between 60° C. and 25° C. See Tables 33-36, Figures 46A and 46B.

[0354] [Table 41]

[0355] [Table 42]

[0356] [Table 43]

[0357] [Table 44]

[0358] Repeated solubility determinations using EtOH:H2O mixtures as antisolvents Previous solubility determination experiments were held for 2-3 hours to allow the samples to equilibrate (see above and Tables 33-36). The solubility determination experiments were repeated with extended equilibration over 24 hours to obtain more accurate solubility values. These experiments were repeated at extreme DMSO to antisolvent ratios.

[0359] procedure Solubility determination at 60°C J10206 (30 mg ± 1 mg) was weighed into 15 HPLC vials and a stir bar was added. Samples were dissolved in the selected solvent ratio of DMSO and EtOH:HO (1:1), EtOH:HO (1:2), or EtOH:HO (2:1) (1 ml), heated to 60 °C in a Polar Bear, and stirred for 24 hours to allow the samples to equilibrate. The samples were 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 isolated solids after filtration were analyzed by XRPD.

[0360] Solubility determination at 25°C J10206 (30 mg ± 1 mg) was weighed into 15 HPLC vials and a stir bar was added. Samples were dissolved in the selected solvent ratio of DMSO and EtOH:HO (1:1), EtOH:HO (1:2), or EtOH:HO (2:1) (1 ml), heated to 25 °C in a Polar Bear, and stirred for 24 hours to allow the samples to equilibrate. The samples were 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 isolated solids after filtration were analyzed by XRPD.

[0361] Results and Discussion The extended equilibration period of 24 hours did not show a significant difference in solubility values ​​compared to the previous solubility determination experiment, in which samples were left to equilibrate for 2-3 hours. Therefore, the selected solvent systems DMSO:EtOH:HO (1:2) (70:30) and DMSO EtOH:HO (1:1) (50:50) were confirmed to proceed with generating solubility curves for process development. See Table 37-Table 38, Figure 47.

[0362] [Table 45]

[0363] [Table 46]

[0364] Extended solubility measurements using EtOH:H2O mixtures as antisolvents procedure Solubility determination in DMSO EtOH:H2O (1:2) (80:20) J10206 was weighed into two HPLC vials and suspended in increasing volumes of DMSO, EtOH:HO (1:2) (80:20) up to 100-fold volume (1 ml) at 60° C. Observations were made upon each addition of solvent.

[0365] Solubility determination in DMSO EtOH:H2O (1:2) (95:5) J10206 was weighed into an HPLC vial and suspended in increasing volumes of DMSO, EtOH:HO (1:2) (95:5) up to 100-fold volume (1 ml) at 60° C. Observations were made with each addition of solvent.

[0366] Results and Discussion No dissolution was observed using up to 100 volumes of the solvent DMSO EtOH:HO (1:2) (80:20) with 10 mg or 3 mg of sample. The sample was observed to be soluble in 80 volumes of DMSO EtOH:HO (1:2) (95:5) at 60 °C, which would provide a good solubility range at the lower temperature of 25 °C. Therefore, DMSO EtOH:HO (1:2) (95:5) was carried forward for process development to generate the solubility curve.

[0367] [Table 47]

[0368] Construction of solubility curves Solubility curves were generated for J10206 (PCLX-001 free form pattern 2) in DMSO, DMSO EtOH:HO (2:1) (50:50), DMSO EtOH:HO (1:2) (70:30), and DMSO EtOH:HO (1:2) (95:5). Solubility curves were generated using Crystal 16 to select solvents for process development (Figure 31).

[0369] Solubility curves were successfully generated for all selected solvent systems. The solubility curves show that the solubility curves for DMSO EtOH:HO (1:2) (70:30) and DMSO EtOH:HO (2:1) (50:50) are lower than those for neat DMSO and DMSO EtOH:HO (1:2) (95:5). Since the addition of an antisolvent was found to have a beneficial effect on increasing the yield of PCLX-001 free form Pattern 2 (see DynoChem below), the solubility data from DMSO EtOH:HO (1:2) (95:5) was entered into DynoChem software for process development.

[0370] Difference in solubility in Crystal 16 During the Crystal 16 experiments, a peak was observed at the beginning of the Crystal 16 experiment, which may have indicated sample conversion or oiling out. Sample solubility was also observed at 55°C, which was unexpected as sample solubility was expected to be observed at 25°C. Due to the difference in solubility and the observed Crystal 16 peak, one of the Crystal 16 experiments was reproduced on Polar Bear and observations were made at 10°C intervals.

[0371] Repeated Crystal 16 experiments on Polar Bear procedure J10206 (3.83 mg) was weighed into an HPLC vial and suspended in DMSO, EtOH:HO (2:1) (50:50). The sample was heated stepwise in a Polar Bear from 23 to 65 °C at 0.5 °C / min to mimic the conditions observed with Crystal 16. Photographs of the sample were taken at 35, 55, and 65 °C, and a microscopic examination of the sample was performed at 45 °C to assess the solubility of the sample upon heating and whether the solubility values ​​observed with Crystal 16 were accurate.

[0372] Results and Discussion Photographs taken at 23, 35, and 55°C showed a pale yellow, cloudy suspension, suggesting that the sample had not completely dissolved. Microscopic observation at 45°C showed crystals up to approximately 100 μm in size, confirming that the sample had not completely dissolved, further ruling out oiling out of the sample. The sample was observed to have completely dissolved at 65°C, confirming that the results of the Crystal 16 experiment were accurate.

[0373] DynoChem Graphs were generated in DynoChem from the solubility data from DMSO EtOH:HO (1:2) (95:5) to predict the optimal crystallization process depending on the time of seed addition and the amount of antisolvent required. From these graphs, it was recommended to add seeds after cooling the crystallization, as indicated by the blue dotted line in the graph in Figure 32A. This was because cooling resulted in a stepwise, controlled decrease in solubility from 60 °C to 25 °C, followed by a smaller decrease once seeding at 25 °C. Compared to the red dotted line, a large, sharp decrease in solubility was observed, as expected, due to the presence of the antisolvent, which precipitated the sample. This was considered less than ideal due to the lack of control over the crystallization.

[0374] From the prediction of the amount of antisolvent required for crystallization shown in the graph in Figure 32B, increasing the antisolvent beyond 11% began to reduce the benefit from solubility, which meant a lower yield benefit. Therefore, antisolvent up to a maximum of 10% antisolvent was used.

[0375] Crystallization in DMSO The solubility data from DMSO and DMSO, EtOH:HO (1:2) was entered into DynoChem software to understand the prediction of crystallization of PCLX-001 free form Pattern 2 in neat DMSO. As seen in Figure 33, a yield of 84% was predicted to produce 200 mg of PCLX-001 free form Pattern 2 in 20 volumes of DMSO and no antisolvent.

[0376] Crystallization in DMSO, EtOH:H2O (1:2) (5% antisolvent) The solubility data from DMSO and DMSO, EtOH:HO (1:2) was entered into DynoChem software to understand the prediction of crystallization of PCLX-001 free form Pattern 2 in DMSO and 5% anti-solvent EtOH:HO (1:2). As seen in Figure 34, a 92% yield was predicted to produce 200 mg of PCLX-001 free form Pattern 2 in 20 volumes of DMSO and 5% anti-solvent EtOH:HO (1:2). This is a 7% yield increase compared to neat DMSO, highlighting the beneficial effect of the presence of the anti-solvent and the fact that only a small addition of anti-solvent is required to have a significant effect on the yield increase.

[0377] Crystallization in DMSO, EtOH:H2O (1:2) (10% antisolvent) The solubility data from DMSO and DMSO, EtOH:HO (1:2) was entered into DynoChem software to understand the prediction of crystallization of PCLX-001 free form Pattern 2 in DMSO and 10% anti-solvent EtOH:HO (1:2). As seen in Figure 35, a 95% yield was predicted to produce 200 mg of PCLX-001 free form Pattern 2 in 20-fold volume of DMSO and 10% anti-solvent EtOH:HO (1:2). This was a 10% yield increase compared to neat DMSO and a 4% yield increase compared to the 5% anti-solvent DMSO EtOH:HO (1:2) (5%).

[0378] Crystallization in DMSO, EtOH:H2O (1:2) (20% antisolvent) The solubility data from DMSO and DMSO, EtOH:HO (1:2) was entered into DynoChem software to understand the prediction of crystallization of PCLX-001 free form Pattern 2 in DMSO and 20% anti-solvent EtOH:HO (1:2). As seen in Figure 36, a 98% yield was predicted to produce 200 mg of PCLX-001 free form Pattern 2 in 20-fold volume of DMSO and 20% anti-solvent EtOH:HO (1:2). This is a 2.5% yield increase compared to DMSO EtOH:HO (1:2) (10%) for a 10% total anti-solvent increase, suggesting there was little benefit from the additional volume of solvent.

[0379] Trial seeded crystallization of J10206 procedure J10206 (50 mg±1 mg) was weighed into two 4 ml vials and dissolved in DMSO (20 volumes, 1 ml) in a Polar Bear at 65° C. The samples were then cooled to 59° C. to ensure a supersaturation ratio of 1.3.

[0380] Seeding before adding 10% antisolvent To another vial, approximately 10 mg of seeds were added at 59°C and allowed to stir for 5 minutes to assess whether the seeds would persist. Observation indicated that the seeds would persist, so the sample was cooled to 25°C at 0.5°C / min, and 10% anti-solvent EtOH:HO (1:2) (2 volumes, 100 μl) was added to the vial. The sample was left stirring at 25°C for 24 hours, forming a pale yellow suspension. The sample was filtered under nitrogen with positive pressure and dried in a vacuum oven, and both wet and dry yields were calculated. 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).

[0381] DMSO-ethanol-water purification: The crude material (PCLX-001; 7.0 g) was diluted with DMSO (20.0 vol) and gradually heated to 55 ± 5 °C for 10 ± 5 minutes until a clear solution was obtained. A mixture of ethanol (7.0 vol) and purified water (7.0 vol) was added to the reaction mass at 55 ± 5 °C and then stirred at 55 ± 5 °C for 10 ± 5 minutes. The reaction mass was gradually cooled to 0 ± 5 °C. The reaction mass was stirred at 0 ± 5 °C for 16 hours and filtered. The wet material was dried at 50-55 °C for 2 hours. 7.0 g was taken up for DMSO-ethanol-water purification, and 5.6 g of the final compound was isolated. Purity by HPLC after DMSO-ethanol-water purification: 99.58%. No seeding was performed.

[0382] Results and Discussion

[0383] [Table 48]

[0384] Characterization of EG-1826-62-02 confirmed the successful generation of crystalline PCLX-001 free form Pattern 2 (Figure 48; Table 5.0A') using the proposed seeding method prior to the addition of 10% antisolvent, produced by DynoChem. 1 Further characterization by H-NMR analysis confirmed that EG-1826-62-02 was consistent with the proposed structure of PCLX-001 free form pattern 2, and SEM images showed that the sample consisted of irregular plate-like particles of approximately 250 μm coated with smaller particles. Smaller particles were also observed in the 20–50 μm range, with the main particles in the 2–5 μm range.

[0385] The crystallization yield was calculated to be 84%, slightly lower than the predicted 95%, however, the lower yield was expected as the crystallization process was not fully optimized.

[0386] [Table 49]

[0387] Scale-up of selected systems (700 mg scale) Procedure: Seed crystals added before adding 10% antisolvent J10206 (700 mg ± 10 mg) was weighed into a 50 ml EasyMax vial and dissolved in DMSO (20 volumes, 14 ml) at 65 °C to produce a clear yellow solution. The sample was then cooled to 59 °C to ensure a supersaturation ratio of 1.3. Approximately 40 mg of seed crystals were added to this solution at 59 °C and allowed to stir for 5 minutes to allow seeding to continue. The sample was cooled to 25 °C at 0.5 °C / min, and 10% anti-solvent EtOH:HO (1:2, 2 volumes, 1.4 ml) was added to the vial. The sample was left stirring at 25 °C for 24 hours to form a pale yellow suspension. The sample was filtered through a Buchner funnel and dried under vacuum for 20 minutes. The sample was analyzed by XRPD (Figure 48, Table 5.OB' (Table 51)), NMR, HPLC (Figure 49), and PLM (Figure 50). Sample ID: EG-1826-63-01.

[0388] The same experiment was repeated to ensure that the purity profile remained the same. HPLC analysis was applied in this case on a 200 mg scale. Sample ID: EG-1826-63-02. The data obtained for the experiment was consistent, with a similar improvement in impurities seen in all cases, with a reduction in the impurity with an RRT of 0.83.

[0389] Please refer to Figures 56 to 58.

[0390] Results and Discussion

[0391] [Table 50]

[0392] Characterization of EG-1826-63-01 confirmed the successful production of crystalline PCLX-001 free form pattern 2. Further characterization by 1H-NMR analysis confirmed that EG-1826-63-01 was consistent with the proposed structure of PCLX-001 free form pattern 2, and PLM images showed the sample consisted of irregular plate-like particles of approximately 100 μm coated with smaller particles. Small particles were also observed in the 20-50 μm range, with the majority of particles in the 2-5 μm range.

[0393] The yield of the scale-up crystallization was calculated to be 87%, slightly lower than the predicted value of 95%, however, a lower yield was expected as the crystallization process was not fully optimized.

[0394] [Table 51]

[0395] conclusion Characterization of PCLX-001 free form pattern 2 (J10206) was confirmed to be crystalline by XRPD analysis with a purity of 98.85%. 1 H-NMR and Raman spectra were consistent with the proposed molecular structure, with neither anions nor cations observed. Thermal analysis of PCLX-001 free form Pattern 2 (J10206) showed the material to have a large endotherm at 220 °C (onset) of 92 J / g, followed by a large exotherm at 225 °C (peak) of 65 J / g. A mass loss of 0.6% w / w was observed between 200 and 240 °C, with decomposition of the material observed at 260 °C (onset). Since 0.3% water was observed in the sample by KF analysis, the mass loss was related to the loss of water from the sample. Solubility (pH profiling) analysis showed that the compound became increasingly soluble in acidic media; samples suspended below pH 4.5 became clear.

[0396] Solubility measurements were performed using gravimetric methods. Based on the solubility evaluation results from Example 1, solubility was measured in DMSO, DMA, and NMP. The maximum range of solubility was observed between 60°C and 25°C in DMSO, with a calculated solubility of 32 mg / ml at 60°C and 12.3 mg / ml at 25°C in DMSO. DMSO was selected as the preferred neat solvent to proceed with process development because it had already been used in the crystallization process.

[0397] Solubility was determined at two temperatures, 25°C and 60°C, using a gravimetric method to investigate parallel and solvent ratios to determine the ratio for best results. The solvent systems selected for solubility curve generation and process development were DMSO EtOH:HO (1:2) (70:30), DMSO EtOH:HO (1:2) (95:5), and DMSO EtOH:HO (2:1) (50:50). Solubility curves were successfully generated for all selected solvent systems. The solubility curves showed lower solubility values ​​for DMSO EtOH:HO (1:2) (70:30) and DMSO EtOH:HO (2:1) (50:50) compared to neat DMSO and DMSO EtOH:HO (1:2) (95:5). Because 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:HO (1:2) (95:5) was entered into DynoChem software to develop the process. From the prediction of the amount of antisolvent required for crystallization, it was noted that by increasing the antisolvent beyond 11%, the benefit from solubility began to decrease, which meant a lower yield benefit. Therefore, it was recommended to use up to 10% antisolvent.

[0398] Two different procedures were used to conduct trial seeded crystallizations. The first trial seeded crystallization consisted of adding 5% antisolvent before adding seeds at elevated temperatures. Observations during this trial seeded crystallization indicated that the addition of 5% antisolvent caused the sample to precipitate before the addition of seeds. This was expected from the predictions generated in DynoChem. Another trial seeded crystallization was conducted by adding seeds before adding 10% antisolvent. This successfully crystallized PCLX-001 free form pattern 2 at a 50 mg scale in 84% yield, slightly lower than the 95% yield predicted by DynoChem. However, the slightly lower yield was expected because this crystallization process was not fully optimized.

[0399] Successful scale-up of the selected solvent system DMSO EtOH:H2O (1:2) (10%) was performed at the 700 mg scale. 1 Further characterization by H-NMR analysis confirmed that EG-1826-63-01 conforms to the proposed structure of PCLX-001 free form pattern 2.

[0400] Example 3 Bioequivalence PK Study of Salt Versus Free Base Form of PCLX-001 Bioequivalence studies in rats and dogs A study was conducted to compare the pharmacokinetic characteristics of the free base and salt forms of PCLX-001 in Sprague-Dawley rats (Study 6902337) and dogs (Study 6902338). PCLX-001 (salt form; PCLX-001 HCl Pattern 1, Batch No. PYA / 19 / 001#1-036) and PCLX-001, PYA Stage-H (free base form; PCLX-001 Free Form Pattern 2, Batch No. PYA / 19 / 001#H-124) were administered by oral gavage to male Sprague-Dawley rats (n=6 / group) at 125 mg free base / kg and to female beagle dogs (n=5 / group) at 4 mg / kg in ultrapure water adjusted to pH 2.5±0.5. These dose levels were the highest non-serious toxicity doses (HNSTD) for each species in GLP 4-week studies (rat study 8002835, dog study 8002836) with a 2-week recovery period. Blood samples for pharmacokinetic evaluation were collected from all animals pre-dose and 30 minutes, 1 hour, 2 hours, 4 hours, 7 hours, and 24 hours post-dose. PK blood samples were processed to plasma and analyzed for PCLX-001 concentrations using a validated LC-MS / MS method. The lower limit of quantitation (LLOQ) was 5 ng / mL.

[0401] Exam Details:

[0402] [Table 52]

[0403] Comparative PK study in rats After administration of both forms in rats, plasma concentrations of PCLX-001 were quantifiable throughout the 24-hour sampling period. max was observed at 7 hours post-dose for both formulations. Mean C max were 44,900 ng / mL and 40,000 ng / mL for the salt form and free base, respectively. AUC last were 627,000 ng / mL and 557,000 ng / mL for the salt form and free base, respectively. maxFor , the salt form to free base ratio was 1.12, and the AUC last For C, the mean value was 1.13, and exposure was similar between the two forms. max Due to limited later time points, the elimination phase was not characterized for any of the rats. See Figure 52 and Table 42.

[0404] [Table 53]

[0405] Comparative PK study in dogs After administration of the salt form in dogs, plasma concentrations of PCLX-001 were quantifiable throughout the 24-hour sampling period, except in one dog where concentrations were quantifiable up to 7 hours post-dose. After administration of the free base in dogs, plasma concentrations of PCLX-001 were quantifiable up to 24 hours post-dose in two dogs and 7 hours post-dose in three dogs. The mean C of PCLX-001 max was observed at 1 hour after administration for the salt form and at 0.5 or 1 hour after administration for the free base. max After that, there is a decrease in T 1 / 2 Where estimable, the mean C ranged from 3.88 to 5.35 hours for salt forms and 2.39 to 3.94 hours for the free base. max were 582 ng / mL and 523 ng / mL for the salt form and free base, respectively, and AUC last The C values ​​were 3440 hr*ng / mL and 2610 hr*ng / mL for the salt form and free base, respectively. max For , the salt form to free base ratio is 1.1, and the AUC last was 1.32 and was similar between the salt and free base. See Figure 53 and Table 43.

[0406] [Table 54]

[0407] In conclusion, when administered as a single oral dose to rats and dogs at various HNSTDs, the free base and salt forms of PCLX-001 exhibited similar pharmacokinetic profiles and are therefore considered bioequivalent.

[0408] PK study of free base PCLX-001 in mice In addition to the bioequivalence studies performed above, a third PK study was conducted in Crl:CD1 (ICR) mice to generate 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 35 mg / kg / day for up to 7 consecutive days. Blood samples for pharmacokinetic evaluation were collected from all animals pre-dose and 30 minutes, 1 hour, 2 hours, 4 hours, 7 hours, and 24 hours post-dose on days 1 and 7. Three mice were bled per time point, and data are presented as the average per time point. C of PCLX-001 max was observed 4 hours after administration on Day 1 and 1 hour after administration on Day 7. max Due to limited later time points, the terminal elimination phase was not characterized on day 1. However, on day 7, the maximum plasma concentration was followed by a decline, and the terminal half-life (T 1 / 2 ) was estimated to be 1.68 hours. After repeated daily dosing, exposure on day 7 was tlast The ratio was 0.914 (91.4%), which was similar to that on day 1. The mean C max and AUC tlast were 46,900 ng / mL and 470,000 hr*ng / ml, respectively. See Figure 54 and Table 44.

[0409] [Table 55]

[0410] The embodiments described herein are intended to be examples only. Changes, modifications, and variations to the specific embodiments may occur to those skilled in the art. The claims should not be limited by the specific embodiments shown herein, but should be construed in a manner consistent with the specification as a whole.

[0411] All publications, patents, and patent applications mentioned in this specification are indicative of the level 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.

[0412] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and it is intended to include within the scope of the following claims all such modifications which would be obvious to those skilled in the art.

Claims

1. Compound (I) 【Chemistry 1】 37. A crystalline form of claim 36, wherein said crystalline form is characterized by the X-ray powder diffraction pattern shown in FIG.

2. Compound (I) 【Chemistry 2】 1. A crystalline form of claim 1, wherein said crystalline form is characterized by an X-ray powder diffraction pattern having peaks at 10.2, 11.4, and 20.5 degrees two-theta ± 0.2 degrees two-theta.

3. 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, 22.9, 26.1, and 31.0 degrees two-theta ± 0.2 degrees two-theta.

4. 4. The crystalline form of claim 2 or 3, wherein 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 two-theta ± 0.2 degrees two-theta.

5. Compound (I) 【Transformation 3】 1. A crystalline form of claim 1, wherein said crystalline form is characterized by an X-ray powder diffraction pattern having peaks substantially as set forth in Table 1.0' ±0.2 degrees two-theta.

6. 6. The crystalline form of 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, comprising an endothermic event with an onset temperature of 220.2°C ± 0.2°C.

7. 6. The crystalline form of 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 crystal form has a diameter of 103.2 cm -1 , and 993.0 cm -1 , and 1602.8 cm -1 ±0.2cm -1 Wave value (cm -1 8. The crystalline form of any one of claims 1 to 7, characterized by a Raman spectrum comprising:

9. The crystalline form has a molecular weight of 126.5 cm -1 , 144.9cm -1 , 227.3cm -1 , 456.1cm -1 , 1043.5cm -1 , 1164.7cm -1 , and 1582.5 cm -1 ±0.2cm -1 Wave value (cm -1 9. The crystalline form of claim 8, further characterized by a Raman spectrum comprising:

10. A crystalline form of compound (I) according to any one of claims 1 to 9, and Pharmaceutically acceptable adjuvants, diluents, carriers, or vehicles A pharmaceutical composition comprising:

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

12. 12. The method of claim 11, wherein the step of 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. adding a base to the first mixture to form a first solution; maintaining the first solution at a temperature of about 30°C; and Stirring the first solution for about 1 hour.

13. The method of claim 11 or 12, further comprising:

14. adding the free form of Compound (I) to a second organic solvent to form a second mixture; stirring the second mixture at ambient temperature; Optionally, increasing the temperature from ambient to about 50°C, and then decreasing the temperature from about 50°C to ambient; and Optionally, adding additional organic solvent to the second mixture until the free form of Compound (I) is dissolved in the second organic solvent; and Decrease the temperature from about 50°C to about 5°C at a rate of about 0.1°C / min.

14. The method of claim 11, further comprising:

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

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

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

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

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

20. 20. The method of claim 11, further comprising filtering insoluble particulates from the first solution prior to isolating the free form of Compound (I) from the first solution.

21. 21. A method for recrystallizing a crystalline form of compound (I) according to any one of claims 1 to 9 or a crystalline form of compound (I) prepared by a method according to any one of claims 11 to 20, comprising: dissolving said crystalline form of Compound (I) in an organic solvent at a first temperature to form a first solution; decreasing the temperature from the first temperature to a second temperature; adding seed crystals of said crystalline form of Compound (I) to said first solution to form a first mixture; decreasing the temperature from the second temperature to a third temperature; adding an anti-solvent to the first mixture to form a second mixture; maturing the second mixture for about 24 hours to form a third mixture; isolating the recrystallized crystalline form of Compound (I) from said third mixture. A method comprising:

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

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

24. The anti-solvent is EtOH:H 2 HO(1:1);EtOH:H 2 0 (1:2); and EtOH:H 2 24. The method of any one of claims 21 to 23, wherein the nucleotide sequence is selected from the group consisting of: O(2:1).

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

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

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

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

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

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

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

32. 32. The method of any one of claims 21 to 31, wherein decreasing the temperature from the second temperature to the third temperature comprises a decrease of 0.5°C / minute.

33. 33. The method of any one of claims 21 to 32, further comprising filtering insoluble particulates from the first solution prior to lowering the temperature from the first temperature to the second temperature.

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

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

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

37. 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, MALT / monocytoid B-cell lymphoma, or Burkitt lymphoma.

38. 36. The method of claim 34 or 35, wherein the cancer is anaplastic large cell lymphoma, acute myeloid leukemia, blastic phase chronic myeloid leukemia, Burkitt's lymphoma, plasma cell myeloma, intestinal adenocarcinoma, mixed adenosquamous carcinoma of the lung, small cell lung carcinoma, lung, esophageal squamous cell carcinoma, bone, ductal carcinoma, diffuse gastric adenocarcinoma, medullary thyroid carcinoma, transitional cell carcinoma of the urinary tract, myeloma, ovarian clear cell carcinoma, transitional cell carcinoma (ureter and bladder cancer), chronic myeloid leukemia (CML), lymphoma-CLL, breast cancer, colorectal adenocarcinoma, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung carcinoma, osteosarcoma, melanoma, gastric adenocarcinoma, endometrial adenocarcinoma, bile duct carcinoma (cholangiocarcinoma), gallbladder carcinoma, liver cancer, or esophageal squamous cell carcinoma.

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

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

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

Images