(S)-1-(1-acryloylpiperidin-3-yl)-2-fluoro-5,6,7,8,9,10-hexahydrocyclohepta[b]indole-4-carboxamide and related crystalline forms, compositions, and methods thereof

Abstract

Crystalline forms of (S)-1-(1-acryloylpiperidin-3-yl)-2-fluoro-5,6,7,8,9,10-hexahydrocyclohepta[b]indole-4-carboxamide are provided. Pharmaceutical compositions containing (S)-1-(1-acryloylpiperidin-3-yl)-2-fluoro-5,6,7,8,9,10-hexahydrocyclohepta[b]indole-4-carboxamide are also provided, as well as related methods for their preparation and use in modulating kinases in general, and for the treatment of cancer in particular.

Description

[Technical field]

[0001] The present invention relates to crystalline forms of (S)-1-(1-acryloylpiperidin-3-yl)-2-fluoro-5,6,7,8,9,10-hexahydrocyclohepta[b]indole-4-carboxamide, as well as products comprising (S)-1-(1-acryloylpiperidin-3-yl)-2-fluoro-5,6,7,8,9,10-hexahydrocyclohepta[b]indole-4-carboxamide, and related methods of their use and preparation. [Background technology]

[0002] Protein kinases are a large group of intracellular and transmembrane signaling proteins in eukaryotic cells. These enzymes are responsible for the transfer of the terminal (gamma) phosphate from ATP to specific amino acid residues in target proteins. Phosphorylation of specific amino acid residues in target proteins can modulate their activity, leading to profound changes in cell signaling and metabolism. Protein kinases can be found in cell membranes, cytosol, and organelles, such as the nucleus, and are involved in mediating multiple cellular functions, including metabolism, cell growth and differentiation, cell signaling, modulation of immune responses, and cell death. Serine kinases specifically phosphorylate serine or threonine residues in target proteins. Similarly, tyrosine kinases, including tyrosine receptor kinases, phosphorylate tyrosine residues in target proteins. Tyrosine kinase families include: TEC, SRC, ABL, JAK, CSK, FAK, SYK, FER, ACK, and receptor tyrosine kinase subfamilies, including ERBB, FGFR, VEGFR, RET, and EPH. Subclass I of the receptor tyrosine kinase superfamily includes the ERBB receptors, which include four members: ErbB1 (also called epidermal growth factor receptor (EGFR)), ErbB2, ErbB3 and ErbB4.

[0003] Kinases exert control over key biological processes related to health and disease. Furthermore, aberrant activation or overexpression of various protein kinases is implicated in multiple diseases and disorders characterized by benign and malignant proliferation, as well as disease mechanisms resulting from inappropriate activation of the immune system. Thus, inhibitors of select kinases or kinase families are believed to be useful in the treatment of cancer, vascular diseases, autoimmune diseases, and inflammatory conditions, including, but not limited to, solid tumors, hematological malignancies, thrombosis, arthritis, graft versus host disease, lupus erythematosus, psoriasis, colitis, ileitis, multiple sclerosis, uveitis, coronary vasculopathy, systemic sclerosis, atherosclerosis, asthma, transplant rejection, allergy, ischemia, dermatomyositis, pemphigus, and the like.

[0004] The Tec kinases are a family of non-receptor tyrosine kinases that are predominantly, but not exclusively, expressed in cells of hematopoietic origin. The Tec family includes TEC, Bruton's tyrosine kinase (BTK), inducible T-cell kinase (ITK), resting lymphocyte kinase (RLK / TXK for tyrosine protein kinase), and myeloid expressed kinase (BMX / ETK).

[0005] BTK is important in regulating B cell receptor signaling and B cell development and activation. Mutations in the gene encoding BTK in humans lead to X-linked agammaglobulinemia, which is characterized by reduced immune function, including defective B cell maturation, reduced levels of immunoglobulins and peripheral B cells, and impaired T cell-independent immune responses. BTK is activated by Src-family kinases, phosphorylating PLC gamma, leading to effects on B cell function and survival. Additionally, BTK is important for the cellular functions of mast cells, macrophages, and neutrophils, indicating that BTK inhibition is effective in treating diseases mediated by these and related cells, including inflammation, bone disorders, and allergic diseases. BTK inhibition is also important in lymphoma cell survival, indicating that inhibition of BTK is useful in the treatment of lymphoma and other cancers. Thus, inhibitors of BTK and related kinases are of great interest as anti-inflammatory and anti-cancer agents. BTK is also important for platelet function and thrombus formation, and selective BTK inhibitors have been shown to be useful as antithrombotic agents. Furthermore, BTK is required for inflammasome activation, and inhibition of BTK can be used in the treatment of inflammasome-related disorders, including stroke, gout, type 2 diabetes, obesity-induced insulin resistance, atherosclerosis, and Muckle-Wells syndrome. In addition, BTK is expressed in HIV-infected T cells, and treatment with a BTK inhibitor sensitizes infected cells to apoptotic death, resulting in reduced viral production. Thus, BTK inhibitors may be useful in the treatment of HIV-AIDS and other viral infections.

[0006] (S)-1-(1-acryloylpiperidin-3-yl)-2-fluoro-5,6,7,8,9,10-hexahydrocyclohepta[b]indole-4-carboxamide, hereafter referred to as "Compound 1", is an orally available, selective, potent inhibitor of Bruton's tyrosine kinase (BTK), thereby offering a potential treatment option in BTK-driven diseases. Compound 1's potency for BTK inhibition is in the nM range in both cell-free enzyme assays and whole blood functional assays. It is central nervous system (CNS) penetrant and demonstrates rapid BTK inactivation kinetics in both peripheral and CNS tissues. In a kinome scan, Compound 1 exhibits high kinase selectivity against 349 kinases, with only two kinases (TEC and TXK) demonstrating >50% inhibition at 1 μM.

[0007] Amorphous forms of Compound 1 (i.e., Compounds 5-6) are described in U.S. Patent Application 17 / 225,984, published as U.S. Patent Application Publication No. US2022 / 0009920 (herein incorporated by reference in its entirety). Compound 1 has the chemical formula C 22 H 26 Molecular weight of FN3O2, 383.47, and below: [ka] It has the structure: In view of the clinical promise of Compound 1, there is a need for new, improved and / or enhanced forms of Compound 1 in the context of pharmaceutical drug products suitable for oral administration, as well as compositions comprising Compound 1 and related methods of making and using same. The present invention meets these and related needs, as evidenced by the following detailed description and the attached drawings.

[0008] A solid drug form can exist in either an amorphous or crystalline state. In the case of crystalline forms, the molecules are positioned in three-dimensional lattice sites. When a compound recrystallizes from a solution or slurry, it crystallizes in different spatial lattice arrangements, a property called "polymorphism," and the different crystalline forms are referred to as "polymorphs" or individually as "polymorph." Different polymorphs of a given substance may differ from each other with respect to one or more physical properties, such as solubility and dissolution, true density, crystal shape, compaction behavior, flow properties, and / or solid-state stability. In the case of a chemical substance that exists in two (or more) polymorphic forms, the unstable form(s) will generally convert to the more thermodynamically stable form(s) at a given temperature after a sufficient period of time. If this conversion is not rapid, the thermodynamically unstable form is referred to as a "metastable" form. In general, the stable form exhibits the lowest solubility and the greatest chemical stability. However, a metastable form may exhibit sufficient chemical and physical stability under normal storage conditions to permit its use in a commercial form, in which case the less stable metastable form may exhibit more desirable properties than the stable form, such as enhanced solubility or better oral bioavailability. Summary of the Invention [Means for solving the problem]

[0009] Thus, in one embodiment, there is provided a novel solid crystalline form of Compound 1. In a more particular embodiment, the novel solid crystalline forms are six distinct polymorphs of Compound 1, which are referred to herein as "Form I," "Form II," "Form III," "Form IV," "Form V," "Form VI," and "Form VII."

[0010] In certain embodiments, a crystalline form of Compound 1 is provided, which is Form I, and in further embodiments, is substantially pure Form I. Form I can be characterized by various analytical techniques disclosed herein, including (for example) X-ray powder diffraction (XRPD) and the characteristic diffractograms generated thereby.

[0011] In certain embodiments, a crystalline form of Compound 1 is provided, the crystalline form being Form II, and in further embodiments being substantially pure Form II. Form II can be characterized by various analytical techniques disclosed herein, including (for example) X-ray powder diffraction (XRPD) and the characteristic diffractograms generated thereby.

[0012] In certain embodiments, a crystalline form of Compound 1 is provided, the crystalline form being Form III, and in further embodiments being substantially pure Form III. Form III can be characterized by various analytical techniques disclosed herein, including (for example) X-ray powder diffraction (XRPD) and the characteristic diffractograms generated thereby.

[0013] In certain embodiments, a crystalline form of Compound 1 is provided, the crystalline form being Form IV, and in further embodiments being substantially pure Form IV. Form IV can be characterized by various analytical techniques disclosed herein, including (for example) X-ray powder diffraction (XRPD) and the characteristic diffractograms generated thereby.

[0014] In certain embodiments, a crystalline form of Compound 1 is provided, the crystalline form being Form V, and in further embodiments, substantially pure Form V. Form V can be characterized by various analytical techniques disclosed herein, including (for example) X-ray powder diffraction (XRPD) and the characteristic diffractograms generated thereby.

[0015] In certain embodiments, a crystalline form of Compound 1 is provided, the crystalline form being Form VI, and in further embodiments being substantially pure Form VI. Form VI can be characterized by various analytical techniques disclosed herein, including (for example) X-ray powder diffraction (XRPD) and the characteristic diffractograms generated thereby.

[0016] In certain embodiments, a crystalline form of Compound 1 is provided, the crystalline form being Form VII, and in further embodiments being substantially pure Form VII. Form VII can be characterized by various analytical techniques disclosed herein, including (for example) X-ray powder diffraction (XRPD) and the characteristic diffractograms generated thereby.

[0017] In other embodiments, crystalline forms of Compound 1 are provided, which are mixtures of two or more forms. As defined below, mixtures are provided where one crystalline form is present in a ratio ranging from 5 to 95% by weight of the other crystalline form(s) or forms (ratios above or below this range are characteristic of substantially pure crystalline forms).

[0018] In other embodiments, a process for preparing a solid crystalline form of Compound 1 is provided.

[0019] In another embodiment, a pharmaceutical composition is provided that comprises Compound 1 in combination with one or more pharma- ceutically acceptable carriers. Such compositions can be formulated in a variety of different forms. For example, the compositions can be formulated for oral administration.

[0020] In certain embodiments, the pharmaceutical composition may include an additional therapeutically active agent (i.e., in addition to Compound 1), or such additional therapeutically active agent may be present as a separate pharmaceutical composition and co-administered with Compound 1 (e.g., at the same time).

[0021] In a further embodiment, the additional therapeutically active agent is a corticosteroid, a corticosteroid agent, an immunosuppressant, and / or an anti-inflammatory agent. In more specific embodiments, the immunosuppressant is selected from the group consisting of interferon alpha, interferon gamma, cyclophosphamide, tacrolimus, mycophenolate mofetil, methotrexate, dapsone, sulfasalazine, azathioprine, anti-CD20 agents (such as rituximab, ofatumumab, obinutuzumab, or veltuzumab, or biosimilar versions thereof), anti-TNF alpha agents (such as etanercept, infliximab, golimumab, adalimumab, or certolizumab pegol, or biosimilar versions thereof), anti-IL6 agents directed against a ligand or its receptor (such as tocilizumab, sarilumab, olokizumab, elcirilumab, or siltuximab), anti-IL17 agents directed against a ligand or its receptor (such as secukinumab, ustekinumab, brodalumab ... anti-IL1 agents against a ligand or its receptor (such as rilonacept, canakinumab, or anakinra), anti-IL2 agents against a ligand or its receptor (such as basiliximab or daclizumab), anti-CD2 agents such as alefacept, anti-CD3 agents such as muromonab-cd3, anti-CD80 / 86 agents such as abatacept or belatacept, anti-sphingosine-1-phosphate receptor agents such as fingolimod, anti-C5 agents such as eculizumab, anti-integrin alpha4 agents such as natalizumab, anti-α4β7 agents such as vedolizumab, anti-mTOR agents such as sirolimus or everolimus, anti-calcineurin agents such as tacrolimus, anti-BAFF / BlyS agents (such as belimumab, VAY736, or blisibimod), leflunomide and teriflunomide.

[0022] In a further embodiment, the additional therapeutically active agent is an immunosuppressant such as rituximab, ofatumumab, obinutuzumab, veltuzumab, or a biosimilar version thereof.

[0023] In further embodiments, the additional therapeutically active agent is an immunomodulatory imide (IMiD) such as thalidomide and its analogues (lenalidomide, pomalidomide and iverdimide), checkpoint blockade agents such as anti-PD1, anti-CTLA4, anti-Tim3 and anti-Lag3 monoclonal antibodies, anti-CD19 monoclonal antibodies such as inebilizumab and tafasitamab, IRAK inhibitors, chemotherapeutic agents such as methotrexate and temozolomide, or anti-CD19 CAR T cell therapy.

[0024] In another embodiment, the pharmaceutical composition comprises polyethylene glycol. In another embodiment, the pharmaceutical composition comprises polyethylene glycol and / or propylene glycol monolaurate. In another embodiment, the pharmaceutical composition comprises vitamin E. In another embodiment, the pharmaceutical composition comprises butylated hydroxytoluene (BHT). In some embodiments, the pharmaceutical composition comprises 1-25 mg of Compound 1.

[0025] In another embodiment, a method is provided for treating a disease or condition modulated by kinase inhibition, comprising administering to a subject in need thereof an effective amount of, or a pharmaceutical composition comprising, Compound 1. In a more particular embodiment, the kinase is a tyrosine kinase, such as (but not limited to) BTK.

[0026] In certain embodiments, the disease or condition is cancer, an autoimmune disease, an inflammatory disease, or a thromboembolic disease. In one embodiment, there is provided the use of Compound 1, or a crystalline form of Compound 1, or a pharmaceutical composition thereof in the manufacture of a medicament. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 shows a Form I XRPD pattern. [Diagram 2] FIG. 1 shows Form I TGA and DSC thermograms. [Diagram 3] FIG. 1 shows a Form II XRPD pattern. [Figure 4] FIG. 1 shows Form II TGA and DSC thermograms. [Diagram 5] FIG. 1 shows a Form III XRPD pattern. [Figure 6] FIG. 1 shows Form III TGA and DSC thermograms. [Figure 7] FIG. 13 shows Form III DVS plot. [Figure 8] FIG. 1 shows Form III XRPD patterns before and after DVS testing. [Figure 9] FIG. 1 shows a Form IV XRPD pattern. [Figure 10] FIG. 1 shows Form IV TGA and DSC thermograms. [Figure 11] FIG. 1 shows XRPD patterns of Form V heated to 85° C. and 155° C. [Figure 12] FIG. 1 shows Form V TGA and DSC thermograms. [Figure 13] FIG. 1 shows the XRPD pattern of Form VI. [Figure 14] FIG. 13 shows Form VI TGA and DSC thermograms. [Figure 15] FIG. 1 shows a Form VII XRPD pattern. [Figure 16] FIG. 13 shows a Form VII DSC thermogram. [Figure 17] FIG. 1 shows Form VII TGA thermogram. [Figure 18] FIG. 1 shows an overlay of XRPD patterns of Forms I-VII. [Figure 19] FIG. 1 shows the proposed crystal morphology transformation. [Figure 20] FIG. 1 shows a Form III DSC thermogram. [Figure 21] FIG. 1 shows Form III TGA and DSC thermograms. [Figure 22] FIG. 1 shows Form III XRPD pattern stability analysis. [Figure 23]FIG. 1 shows the Form III single crystal X-ray structure. [Figure 24] FIG. 1 is a diagram showing an outline of a manufacturing process. [Diagram 25] FIG. 1 shows plasma profile mean±SD. [Figure 26] FIG. 1 shows CSF to unbound plasma ratios by dose. [Figure 27] FIG. 1 shows single and multiple dose plasma profiles (mean±SD). [Figure 28] FIG. 1 shows plasma profiles (mean±SD) of 15 mg fasted, medium fat meal, and high fat meal. [Figure 29] FIG. 1 shows the plasma profile (mean±SD) of Compound 1 alone and with itraconazole. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] According to the present disclosure, novel solid crystalline forms of Compound 1 are provided. In more particular embodiments, the novel solid crystalline forms are seven different polymorphs of Compound 1; namely, Form I, Form II, Form III, Form IV, Form V, Form VI, or Form VII. These forms differ from the amorphous form of Compound 1 in the structure of the crystal lattice, and each form gives a unique X-ray powder diffraction (XRPD) pattern and differential scanning calorimetry (DSC) thermogram.

[0029] As used herein, "amorphous" refers to the lack of well-ordered diffraction lines due to the absence of a repeating crystal lattice.

[0030] In one embodiment, the present disclosure provides Form I characterized by an XRPD pattern having peaks at 9.2011, 13.9620, and 16.1506±0.2 degrees two-theta. In another embodiment, Form I is provided, further characterized by an XRPD pattern substantially as shown in FIG.

[0031] In one embodiment, the present disclosure provides Form II characterized by an XRPD pattern having peaks at 4.2759, 8.5794, and 24.2411±0.2 degrees two-theta. In another embodiment, Form II is provided further characterized by an XRPD pattern substantially as shown in FIG.

[0032] In one embodiment, the present disclosure provides Form III characterized by an XRPD pattern having peaks at 10.2543, 13.5006, and 13.9691±0.2 degrees two-theta. In another embodiment, Form III is provided further characterized by an XRPD pattern substantially as shown in FIG.

[0033] In one embodiment, the present disclosure provides Form IV characterized by an XRPD pattern having peaks at 8.6027, 11.9598, 13.9360, 21.5845, and 25.4090±0.2 degrees two-theta. In another embodiment, Form IV is provided further characterized by an XRPD pattern substantially as shown in FIG.

[0034] In one embodiment, the present disclosure provides Form V characterized by an XRPD pattern having peaks at 6.4014, 9.1908, 14.8143, 17.5539, 21.5891, 23.9883, and 25.5807±0.2 degrees two-theta. In another embodiment, Form V is provided, further characterized by an XRPD pattern substantially as shown in FIG.

[0035] In one embodiment, the present disclosure provides Form VI characterized by an XRPD pattern having peaks at 6.8339, 10.1404, 15.6784, 16.1217, 17.5940, 20.6765, 25.5122, and 26.7363±0.2 degrees two-theta. In another embodiment, Form VI is provided further characterized by an XRPD pattern substantially as shown in FIG.

[0036] In one embodiment, the present disclosure provides Form VII characterized by an XRPD pattern having peaks at 6.727, 8.4799, 9.4854, 12.0161, 17.1901, 18.8407, 19.0691, 19.7285, and 20.2268±0.2 degrees two-theta. In another embodiment, Form VII is provided further characterized by an XRPD pattern substantially as shown in FIG.

[0037] In the practice of this invention, a single polymorph (i.e., Form I, Form II, Form III, Form IV, Form V, Form VI, or Form VII) may be utilized in substantially pure form or as a mixture of one or more polymorphs.

[0038] In a further embodiment, a method is provided for treating a disease or condition modulated by kinase inhibition comprising administering to a subject in need thereof an effective amount of a solid crystalline form of Compound 1; i.e., Form I, Form II, Form III, Form IV, Form V, Form VI, or Form VII.

[0039] In one embodiment, the kinase is a tyrosine kinase, and in a more particular embodiment, Bruton's tyrosine kinase (BTK).

[0040] In one embodiment, the disease or condition modulated by kinase inhibition is cancer.

[0041] This invention is further illustrated by the following examples, which should not be construed as limiting in any way. EXAMPLES

[0042] Polymorphism Screening Polymorph screening of compound 1 was performed using a variety of crystallization methods, including slurry, cooling and evaporative crystallization, antisolvent precipitation, thermal and mechanical treatments.

[0043] (Example 1) Analysis method (Example 1A) X-ray powder diffraction (XRPD) XRPD patterns were identified using an X-ray diffractometer (PANalytical Empyrean). The system was PIXcel 1D The detector was equipped with a 1000 nm NMR spectroscopy (NMR spectroscopy) microscope. Samples were scanned from 3 to 40 degrees 2θ with a step size of 0.013 degrees 2θ. The tube voltage and current were 45 KV and 40 mA, respectively. (Form VII was scanned from 4 to 40 degrees 2θ with a step size of 0.011 degrees 2θ, the tube voltage and current were 40 KV and 15 mA, respectively.)

[0044] (Example 1B) Differential Scanning Calorimetry (DSC) DSC was performed using a Discovery DSC 250 (TA Instruments, US) (for Form VII, a Discovery DSC Q2000 was used). The samples were placed in aluminum pinhole airtight pans and the weight was accurately recorded. The samples were then heated from 25°C to the final temperature at a rate of 10°C / min.

[0045] (Example 1C) Thermogravimetric analysis (TGA) TGA was performed on a Discovery TGA 55 (TA Instruments, US) (for Form VII, a Discovery TGA Q500 was used). Samples were placed in open tared aluminum pans, auto-weighed, and inserted into the TGA furnace. Samples were heated from room temperature (RT) to the final temperature at a rate of 10° C. / min.

[0046] (Example 1D) Dynamic Vapor Sorption (DVS) Moisture sorption / desorption data were collected on a Vsorp Dynamic Moisture Sorption Analyzer (ProUmid GmbH&Co.KG, Germany). Samples were placed into a tared sample chamber and automatically weighed. [Table A]

[0047] (Example 1E) Proton nuclear magnetic resonance ( 1 H-NMR) d as a solvent 6 - Using a Bruker AVANCE III HD 300 or 400 equipped with an autosampler (SampleXpress 60) using DMSO, 1 H-NMR was performed.

[0048] (Example 1F) High-performance liquid chromatography (HPLC) HPLC analyses were performed using an Agilent HPLC 1260 series instrument. [Table B]

[0049] (Example 2) Characterization of starting materials A single batch of compound 1 as a light yellow solid (2.66 g, 99.78% purity) was used as the starting material for the polymorph screen. The material was mostly rod-like crystals with low crystallinity and particle size of 10-20 μm. XRPD characterization revealed that the material was a mixture of Form I and Form II, which converted to Form I after heating to 170 °C.

[0050] (Example 3) Multi-shape screen Various techniques for producing crystalline materials were evaluated. (Example 3A) Evaporative crystallization Evaporative crystallization studies were performed in THF, methanol, acetone, isopropanol, and dichloromethane (i.e., solvents providing a solubility >3 mg / mL) under fast and slow evaporation rates: Fast: Solution dried by nitrogen purging at room temperature (approximately 25°C) Slow: Solution evaporated to dryness at room temperature (approximately 25°C) in a draft chamber Solubility was measured by HPLC and the residual solids were analyzed by XRPD after evaporation. In all cases only amorphous material was obtained.

[0051] (Example 3B) slurry The starting materials were added to the 13 single solvents and the resulting suspensions were stirred for 3 days at room temperature (approximately 24° C.) or at 50° C. Any solids obtained were characterized and the results are summarized in Table 1 (loading concentrations are in mg / mL). Forms II, III, IV, and V were isolated as indicated. [Table 1]

[0052] (Example 3C) Cooling crystallization Starting material (approximately 20 mg) was weighed into a vial and solvent was added to give a near clear saturated solution or suspension, which was stirred at 50° C. These were then cooled to room temperature (approximately 24° C., slow cool) or the filtrate was placed directly in the refrigerator (2-8° C., fast cool). Solids were obtained from methanol and ethanol; cooling of these solids gave Form II. All other conditions gave solutions. Results are presented in the solvents (Table 2). [Table 2]

[0053] (Example 3D) Anti-solvent precipitation Solvent / anti-solvent experiments were performed on 12 systems. Solvents providing high solubility included DMSO, THA, acetone, ethyl acetate, 2-butanone, and IPA. Solvents providing low solubility included IPAc, ACN, MTBE, and heptane was selected as the anti-solvent. A saturated solution was prepared by dissolving starting material (approximately 20 mg) in the solvent. After filtration, anti-solvent was gradually added to the filtrate in aliquots of 20-100 μL until turbidity was observed or 10 V was reached at room temperature (approximately 25 °C). If precipitation occurred, the product was properly characterized and the results are shown in Table 3.

[0054] Form I was obtained from DMSO / water (1:1), EtOc / heptane (1 / 3) and IPA / water (1 / 3).

[0055] Form II was obtained from acetone / water (1 / 2).

[0056] Form VI was obtained from 2-butanone / water (1 / 2). The remaining conditions either remained as solution or provided insufficient solid for analysis. [Table 3]

[0057] (Example 4) Characterization of identified polymorphic forms Seven crystalline forms were identified and assigned as Forms I, II, III, IV, V, VI and VII. Form I Form I, irregularly shaped crystals with high crystallinity and fine particle size, was obtained from a heptane slurry or by heating the starting material to about 170° C. The Form I XRPD is shown in FIG. 1 and significant peaks from the XRPD trace are listed below in Table 4: [Table 4-1] [Table 4-2] [Table 4-3]

[0058] There was a weight loss of 0.6% before 165° C. in the TGA profile (FIG. 2).

[0059] Form II Form II is an irregular crystal with high crystallinity obtained by slurrying and cooling from methanol or ethanol. The XRPD of Form II is shown in Figure 3, and significant peaks from the XRPD trace are listed below in Table 5: [Table 5-1] [Table 5-2]

[0060] Multiple thermal events were observed in the DSC curve (Figure 4). The first endothermic peak was the dehydration / desolvation of Form II, and the second endothermic peak was the melting of Form I. Approximately 4.0% weight loss was observed in the TGA profile before 140°C, and 1.7% ethanol was detected by NMR.

[0061] Form III Form III is an irregular crystal with high crystallinity obtained from a slurry in a single solvent. The XRPD of Form III is shown in Figure 5, and significant peaks from the XRPD trace are listed below in Table 6: [Table 6-1] [Table 6-2]

[0062] One melting peak was observed in the DSC curve and there was no obvious weight loss in the TGA profile between RT and 165° C. (FIG. 6), indicating that Form III is an anhydrous form. The competitive slurry results of Form I and Form III indicate that Form III is the most stable form between RT and 80° C. DVS data showed that Form III is non-hygroscopic (<0.5% weight gain up to 90% RH, see FIG. 7) and the crystalline morphology remains unchanged after the DVS study (FIG. 8).

[0063] Form IV Form IV is an irregular crystal with high crystallinity obtained only from MTBE slurry. The XRPD of Form IV is shown in Figure 9, and significant peaks from the XRPD trace are listed below in Table 7: [Table 7-1] [Table 7-2]

[0064] The DSC of Form IV (FIG. 10) showed two endothermic peaks before 200° C., corresponding to the desolvation of Form IV and the melting of Form I, respectively. Approximately 9.5% weight loss was observed in the TGA profile between 105 and 170° C. 9.8% MTBE was detected by NMR.

[0065] Form V Form V is an irregular crystal with high crystallinity obtained only from a toluene slurry. The XRPD of Form V is shown in Figure 11, and significant peaks from the XRPD trace are listed below in Table 8: [Table 8-1] [Table 8-2] [Table 8-3]

[0066] Form V was heated at 85° C. and 155° C. and the resulting solid forms were analyzed by PXRD (FIG. 11, top trace). Form V heated to 85° C. remained unchanged; heating to 155° C. resulted in the formation of an amorphous form. DSC showed two endothermic peaks before 150° C. (FIG. 12). The TGA profile showed approximately 1.0% weight loss between 90 and 170° C. 1.0% toluene was detected by NMR.

[0067] Form VI Form VI is an irregular crystal with high crystallinity obtained from MEK / HO anti-solvent crystallization. The XRPD of Form VI is shown in Figure 13, and significant peaks from the XRPD trace are listed below in Table 9: [Table 9-1] [Table 9-2] [Table 9-3]

[0068] DSC showed a broad endothermic peak with a weight loss of 13% before 120° C. (FIG. 14). 12% MEK was detected by NMR.

[0069] Form VII Form VII was observed during isolation of compound 1 via heptane / 2-methyltetrahydrofuran (2-MeTHF) recrystallization and was determined to be a 2-MeTHF solvate. More specifically, a solution of compound 1 was dissolved in 2-MeTHF (3 volumes) at 60° C. Heptane anti-solvent (4.1 volumes) was then added over 1 hour and the temperature was maintained at 60° C. The mixture was then cooled to 20° C. and stirred at 20° C. for 4 hours. The resulting solid was isolated by filtration and the wet cake was washed with heptane (1.4 volumes) and dried for 30 minutes and then dried in a vacuum oven at 55° C. for at least 12 hours.

[0070] The XRPD of Form VII is shown in FIG. 15, and significant peaks from the XRPD trace are listed below in Table 10: [Table 10-1] [Table 10-2] [Table 10-3]

[0071] DSC and TGA of Form VII (FIGS. 16 and 17, respectively) show one endothermic peak at 122-124° C., corresponding to desolvation of Form VII. Approximately 6.6% weight loss was observed in the TGA profile between 102-105° C. 9.9% 2-methyl-THG was detected by NMR.

[0072] Summary of identified solid forms Seven new crystalline forms were identified and assigned as Forms I-VII. Figure 18 shows the XRPD overlay of all seven forms. Forms I and III are anhydrates; Forms II, IV, V, VI, and VII are solvates or hydrates. Forms II and IV convert to Form I upon heating, see Table 11. Table 12 shows the characterization data for the various forms. [Table 11] [Table 12]

[0073] Mutual conversion research Equal amounts of Form I and Form III were mixed in water and heptane to form a slurry and stirred at room temperature (about 25° C.) or at 80° C. The residual solids were isolated, characterized, and all showed conversion to Form III. Thus, Form III is considered more stable than Form I. A proposed conversion map of the seven crystalline forms is shown in FIG.

[0074] (Example 5) Form III Preparation & Characterization (Example 5A) 120 mg preparation of Form III A suspension of the starting material (150 mg) in isopropyl acetate (2.5 mL) was stirred at room temperature for 3 days. The resulting solid was isolated by filtration to provide irregularly shaped crystals (120 mg; 80% yield). XRPD analysis of the resulting material compared to reference Form III material confirms that the material is Form III. This Form III material was used for the studies described below.

[0075] (Example 5B) DSC&TGA analysis Heat treatment of Compound 1, Form III was carried out using DSC as follows: Equilibrate at 25 °C; Ramp 10°C / min to 190°C; Equilibrate at -40°C; and Ramp 5°C / min to 300°C.

[0076] Figure 20 shows the resulting DSC curves, the lower curve is ramped 10°C / min to 190°C; the middle curve is equilibrated at -40°C, and the upper curve is ramped 5°C / min to 300°C. The glass transition temperature is 110°C. An endothermic peak with an onset temperature of 175.8°C was detected.

[0077] Figure 21 shows an overlay of the TGA and DSC thermograms of Form III. No obvious weight loss prior to melting was observed in the TGA profile.

[0078] (Example 5C) Solid State Stability The solid state stability of Compound 1, Form III, was examined in duplicate (Sample 1 and Sample 2) for 7 days under the following conditions and the resulting solids were analyzed by XRPD: 40°C / 75% relative humidity (open); and 60°C (covered).

[0079] FIG. 22 shows the XRPD patterns of the starting material and the material isolated after exposure to the above conditions, indicating that Form III was both chemically and physically stable under the conditions determined.

[0080] The solid state stability of Compound 1, Form III was also examined for three months under the following conditions: 40°C / 75% relative humidity; and 25℃ / 60% relative humidity Form III was stable under the conditions determined.

[0081] (Example 5D) Solubility Test The solvent addition method via visual inspection of the samples was used to determine the approximate solubility of the initial solids, and the results are summarized in Table 13. The starting material is freely soluble in DMSO (>250 mg / mL), sparingly soluble in THF and dioxane (>20 mg / mL), and has very low solubility (<0.6 mg / mL) in MTBE, water, and n-heptane. [Table 13] Values ​​are rounded to the nearest integer and are reported as "<" if no dissolution was observed or ">" if dissolution occurred after addition of the first aliquot.

[0082] Form III is non-hygroscopic and was chemically and physically stable at 40°C / 75% humidity and 60°C for 1 week.

[0083] Form III remained unchanged after milling, although the crystallinity was slightly reduced.

[0084] Form III can be prepared by slurry from a single solvent, such as IPAC, IPA and CAN.

[0085] Characterization of Compound 1, Form III In each of the following studies (Examples 6-10), the starting material was Compound 1 (free form, no salt) Form III. The material was a light yellow solid containing non-hygroscopic irregularly shaped crystals with aggregates. The same analytical methods as described in Example 1 were used.

[0086] (Example 6) pKa determination Using the Sirius T3 titrator, pK a The acidic and basic sites were very weak and undetectable by the Sirius T3. The average pKa individual results (for the increments performed at 25°C) were as follows: Titration Ionic Strength Chi-square Point 3-5 10.168 0.6254 Points 52-102 0.1830.8024 Points 103-153 0.1960.6051

[0087] (Example 7) Solubility test in vehicle The vehicle solution was prepared as follows: [Table C]

[0088] (Example 8A) Solubility Test Solubility was determined at room temperature for 24 and 72 hours, and at 45°C for 4 and 24 hours. Approximately 30 / 100 mg of Compound 1 was added to the vehicle (2 mL) and the mixture was stirred at room temperature for 24 or 72 hours. The suspension was filtered or centrifuged, and the filtrate was analyzed by HPLC. Solubility and pH results are presented in Tables 14 and 15. After 24 hours, high solubility (>50 mg / mL) was observed in Cremophor HS 15, Gelucire 44 / 14, Gelucire 48 / 16, and PEG400 / TPGS (3 / 1). After 72 hours, high solubility (>50 mg / mL) was observed in PG, PEG400, Capryol 90, and Labrasol. [Table 14] [Table 15]

[0089] (Example 8B) Further solubility testing Compound 1 (approximately 0.5 g) was added to each excipient until visually saturated. The mixtures were incubated at room temperature (liquid excipients) or 40° C. (semi-solid excipients) for at least 48 hours on a temperature-controlled mixer using glass beads to facilitate mixing. The samples were then centrifuged using a 0.45 μm PVDF filter to separate the liquid and solid portions. The amount of dissolved compound in the filtrate was quantified by HPLC. XRPD analysis of any solid powders confirmed there was no change in morphology. The solubility results are presented in Table 16 and show that Compound 1 had high solubility (>30 mg / g) in most of the excipients screened. [Table 16]

[0090] (Example 9) Solubility tests in biological media Preparation of FaSSIF FaSSIF buffer solution: 6 mL of 0.2 M NaOH, 388.7 mg of NaH2PO4, and 608.2 mg of NaCl were dissolved and diluted to 100 mL with water. The pH was 6.51. FaSSIF medium: 34.56 mg of SIF powder was dissolved in 15 mL of FaSSIF buffer solution. The solution was stirred and equilibrated for 2 hours at ambient temperature with light protection. SIF powder was purchased from Biorelevant.com.

[0091] Preparation of FeSSIF FeSSIF buffer solution: 408.4 mg NaOH, 868.5 mg acetic acid, and 1.1802 g NaCl were dissolved and diluted to 100 mL with water. The pH of the solution was 4.98. FeSSIF medium: 168.42 mg SIF powder was dissolved in 15 mL FeSSIF buffer solution. The solution was protected from light.

[0092] Preparation of FaSSGF FaSSGF buffer solution: 222 mg of NaCl was dissolved in water, then the pH was adjusted to 1.6 by adding 1N HCl. FaSSGF medium: 1.2 mg of SIF powder was dissolved in 10 mL of FaSSIF buffer solution. The solution was stirred and equilibrated at ambient temperature with light protection for 2 hours.

[0093] Preparation of FaSSIF-V2 FaSSIF-V2 buffer solution: 154.4 mg NaOH, 246.7 mg maleic acid and 445.6 mg NaCl were dissolved and diluted to 100 mL with water. The pH was 6.51. FaSSIF-V2 medium: 28.64 mg FaSSIF-V2 powder (purchased from Biorelevant.com) was dissolved in 8 mL FaSSIF-V2 buffer solution. The solution was stirred and equilibrated for 2 hours at ambient temperature with light protection.

[0094] Preparation of FeSSIF-V2 FeSSIF-V2 buffer solution: 363.3.4 mg NaOH, 710 mg maleic acid, and 814.4 mg NaCl were dissolved and diluted to 100 mL with water. The pH of the solution was 5.8. FeSSIF-V2 medium: 156.16 mg FeSSIF-V2 powder was dissolved in 8 mL FeSSIF-V2 buffer solution. The solution was protected from light.

[0095] Solubility tests in biological media Solubility was measured in simulated gastrointestinal fluids (FaSSGF, FaSSIF, FeSSIF, FaSSIF-V2, and FeSSIF-V2) and pH 6.5 buffer, pH 5.8 buffer media at 37° C. for 0.5, 2, and 24 hours. Approximately 15 mg of sample was weighed into a sample vial and then 3.0 mL of FaSSGF, FaSSIF, FeSSIF, FaSSIF-V2, FeSSIF-V2, pH 6.5 buffer, and pH 5.8 buffer media (solubility in buffer solution without SIF powder was performed for comparison) was added, respectively. Samples were prepared in duplicate for each media. The suspensions were shaken at 37° C. for up to 24 hours. At 1, 4, and 24 hours, the suspensions were filtered and the filtrates were analyzed by HPLC, with the results presented in Table 17. The remaining solid was collected for XRPD analysis and showed no morphological changes. [Table 17] (Example 10) Solubility test in aqueous media at various pH levels Preparation of pH 1.0 solution (0.1N HCl) 0.833 mL of concentrated hydrochloric acid was diluted to 100 mL with water (pH=1.0). Preparation of pH 1.2 buffer solution Adjust the pH to 1.2 by adding an appropriate amount of water to 0.1 N HCl. Preparation of pH 7.4 buffer solution KH2PO4 (1360.0 mg) and NaOH (310.0 mg) are mixed in a 200 mL volumetric flask. Water is added to dissolve the solids and dilute the volume. Good mixing is achieved by shaking the flask. (pH=7.42).

[0096] Solubility tests in media of different pH and in water Solubility was measured in pH 1.0 water, pH 1.2 buffer, and pH 7.4 buffer at room temperature and 37°C. Approximately 10-15 mg of sample was weighed into a sample vial, then 2.0 / 3.0 mL of 0.1 N HCl solution, pH 1.2 buffer, and pH 7.4 buffer media, and water were added, respectively. Samples were run in duplicate for each media. The suspensions were shaken at room temperature or 37°C. At 4, 24, and 48 hours, the suspensions were centrifuged and the filtrates were analyzed by HPLC; the results are presented in Table 18). The residual solids were collected for XRPD analysis, but no morphological changes occurred during the study. In all cases, very low solubility was observed. [Table 18]

[0097] (Example 11) Single crystal X-ray structure of compound 1, form III Cu K α Single crystal X-ray diffraction studies were carried out on a Bruker Smart APEX II CCD diffractometer equipped with a 1000 nm radiation (λ=1.54178 Å).

[0098] Crystals of compound 1, Form III, were grown from EtOAc / pentane.

[0099] A 0.23 x 0.2 x 0.17 mm piece of colorless crystal was mounted on a Cryoloop with paratone oil. Data were collected in a stream of nitrogen gas at 100(2) K using Φ and ω scans. The crystal-to-detector distance was 40 mm and exposure times were 1, 2, 3, or 5 seconds depending on the 2θ range per frame using a scan width of 1.25°. Data collection was 97.7% complete for 67.679° in θ. A total of 27594 reflections were collected covering indices -11<=h<=11, -11<=k<=11, -13<=l<=13. 6852 reflections were measured with an R of 0.0232. intand was found to be symmetry independent. Indexing and refinement of the unit cell indicated a triclinic lattice. The space group was found to be P1. The data were integrated using the Bruker SAINT software program and scaled using the SADABS software program. Solution by direct methods (SHELXT) produced a complete phasing model that was consistent with the proposed structure.

[0100] All non-hydrogen atoms were anisotropically refined by full matrix least squares (SHELXL-2014). A riding model was used to place all carbon-bonded hydrogen atoms. Their positions were constrained relative to their parent atoms using the appropriate HFIX command in SHELXL-2014. Absolute stereochemistry was finally assigned (Frac=0.04(3)). The structure is shown in FIG. 23. There are two copies of the compound in the asymmetric unit. The crystallographic data was as follows: Crystal system: triclinic space group P1 Unit cell dimensions a=9.61280(10)Å α=110.7050(10)° b=9.78860(10)Å β=90.9170(10)° c=10.85140(10)Å γ=96.4060(10)° Volume 947.470(17)Å 3 Z2 Density (calculated) 1.344Mg / m 3 Absorption coefficient 0.764mm -1 F(000) 408 Crystal size: 0.23 x 0.2 x 0.17 mm 3 Theta range for data collection: 4.363 to 70.453° Exponent range -11<=h<=11, -11<=k<=11, -13<=l<=13 Reflections collected: 27594 Independent reflection 6852[R(int)=0.0232] Perfect for Theta = 67.679° 97.7% Absorption correction: semi-empirical from equivalents Maximum and minimum transmittance 0.5220 and 0.4322 Refinement method F 2 Full matrix least squares for Data / Suppression / Parameter 6852 / 3 / 505 F 2 Goodness of fit for 1.026 Final R index [I>2 sigma (I)] R1 = 0.0262, wR2 = 0.0715 R-index (all data) R1=0.0266, wR2=0.0720 Absolute structural parameter 0.04(3) The largest diffraction peak and hole are 0.181 and -0.168e. -3

[0101] Formulation Development Formulation development and batch manufacturing of Compound 1 softgel capsules was investigated. Compound 1, Form III was used as the starting material. Seven formulations were prepared and their solubility, kinetic solubility, permeability, physical stability and chemical stability were evaluated. Formulation A7 contains Compound 1, PEG400, Lauroglycol 90, Vitamin ETPGS and BHT. Formulation A7 softgel capsules were manufactured in three strengths (2mg, 5mg & 25mg). The softgel shell contains gelatin (Type 195), Sorbitol Special-Glycerin Blend, Titanium Dioxide, FD&C Blue #1, Red Iron Oxide and Purified Water.

[0102] (Example 12) Preparation of Formulations A1-A6 Six formulations (A1, A2, A3, A4, A5 and A6) were prepared with a target compound 1 concentration of 22.7 mg / g. These formulations were designed to create different formulation types based on the ratio of glyceride, surfactant, and hydrophilic co-solvent. Butylated hydroxytoluene (BHT) was included in each of the formulations as an antioxidant to minimize any potential oxidative degradation. Table 19 shows the formulation composition. [Table 19]

[0103] (Example 13) Solubility of Formulations A1-A6 The solubility of formulations A1-A6 was evaluated via fiber optic dissolution using a Pion Rainbow Dynamic Dissolution Monitor with Fiber Optic and a Distek Dissolution System 2500 (Dissolution Apparatus Type II). Three biorelevant media were evaluated: FaSSGF (pH=1.3), FaSSIF (pH=6.5), and FeSSIF (pH=5.5), as detailed in Table 20. [Table 20]

[0104] The solubility of each formulation was continuously monitored by a fiber optic probe over a 6 hour period in each dissolution bath (N=1, 500 mL volume, 37° C., paddle speed of 75 rpm). All formulations showed increased solubility in each medium over crystalline Compound 1 alone (used as control), with A1, A3 and A4 exhibiting the highest solubilities.

[0105] (Example 14) Permeability of Formulations A1, A3 and A4 The permeability of formulations A1, A3 and A4 was evaluated using a Pion μFlux instrument (two compartments) and a Pion Rainbow Dynamic Dissolution Monitor with a fiber optic probe. The test formulation was added to the compartment containing FaSSIF medium, and acceptor sink buffer was placed in the second compartment. The two compartments were separated by a membrane filter coated with GIT-0 lipid solution. (This setup mimics passive diffusion in the intestinal membrane). The fiber optic probe in each compartment continuously monitored the compound 1 concentration on each side of the membrane over a period of 6 hours. Table 21 shows the percentage compound 1 permeability for each formulation after 6 hours. [Table 21]

[0106] (Example 15) Physical stability evaluation (temperature maintained at room temperature and 40℃) The physical stability of Formulations A1, A3 and A4 was evaluated at room temperature (15-25°C) and at 40°C. Two sets of samples (approximately 3 g each) were aliquoted into single glass vials. One set was kept at room temperature for 7 days and the other at 40°C for 7 days. Changes in appearance such as color change, phase separation or precipitation were monitored via visual observation. No signs of physical instability were observed at room temperature or at 40°C.

[0107] (Example 16) Physical stability evaluation (temperature cycle approx. 40℃ / -20℃) The physical stability of formulations A1, A3 and A4 was evaluated under temperature cycling conditions. Samples (about 3 g each) of each test formulation (A1, A3 and A4) were aliquoted into glass vials and exposed to three cycles of high (40° C.) and low (−20° C.) temperatures for about 24 hours at each temperature condition. As shown in Table 22, no color change, phase separation or precipitation was observed. [Table 22]

[0108] (Example 17) Plasticizer Loading Plasticizer loading evaluates the possible effect of plasticizer migration into the fill solution after encapsulation in softgel capsules. Two plasticizers were evaluated: Sorbitol Sorbitan Solution and Sorbitol Special-Glycerin Blend A810. Loading was performed by spiking 5% of each plasticizer into each formulation. Spiked samples were stored at room temperature (15-25°C) and 40°C and observations were noted at T=0 and then daily for up to 7 days. No precipitation was observed. Phase separation was observed as clear droplets at the bottom of the vial in all of the formulations in the presence of both plasticizers, suggesting that the plasticizer and fill migration were not miscible and that the plasticizer was not likely to migrate out of the capsule shell. Tables 23 and 24 summarize the results. [Table 23] [Table 24]

[0109] (Example 18) Formulation A7 Formulation A7 was prepared according to the same conditions as formulations A1-A6. Formulation A7 contains the same components and amounts as formulation A4, except that Lauroglycol 90 (propylene glycol monolaurate (type II)) was used instead of Lauroglycol FCC (propylene glycol monolaurate (type I)). These excipients contain different ratios of mono- and diesters. [Table D]

[0110] (Example 19) Maximum drug solubility The maximum solubility of Compound 1 in Formulations A1, A3, A4 and A7 was assessed to determine the maximum possible drug loading per formulation. Placebo formulations of each prototype were prepared and supersaturated with Compound 1. The mixtures were incubated at 40° C. for at least 48 hours on a temperature-controlled shaker with glass mixing beads to ensure maximum solubility was achieved. The samples were then centrifuged using 0.45 μm PVDF filters to separate the liquid and solid portions. The amount of dissolved Compound 1 in the filtrate was quantified by HPLC analysis and is shown in Table 25. [Table 25]

[0111] (Example 20) Stability testing Formulations A1, A3 and A7 were prepared and divided into four sets of samples to which the following were added: Nothing (control) 5% water Gel 1: Gel 004007-L3DXHBHM contains Sorbitol Special-Glycerin Blend as a plasticizer. Gel 2: Gel 004013-LSMHRS1HM contains sorbitol sorbitan solution as a plasticizer

[0112] Gels were prepared using titanium dioxide, iron oxide, red and FD&C Blue #1 as colorants. One dried swatch of Gel 004007 was added to Gel 1 sample and one dried swatch of 004013 was added to Gel 2.

[0113] Samples were stored in 6 mL amber glass vials at room temperature, 40° C., 50° C., and 5° C. for up to 8 weeks. Control samples (compound 1 alone) were analyzed in parallel with the study samples, and the control peak was subtracted from the total impurities. Compound 1 related impurities after storage at 40° C. are listed in Table 26. [Table 26]

[0114] The stability results are presented in Tables 27-34. Most of the impurities observed at T=0 were present in the Compound 1 starting material alone and therefore were not considered formulation-related impurities. [Table 27] [Table 28] [Table 29] [Table 30] [Table 31] [Table 32] [Table 33] [Table 34]

[0115] All three formulations (A1, A3, and A7) showed similar stability up to 2 weeks at 40° C., 50° C., and 5° C. After 8 weeks, the stability of A7 at 40° C. was analyzed.

[0116] (Example 21) Batch Production - 2mg, 5mg & 25mg Capsules based on A7 Figure 24 depicts a flow diagram of the batch manufacturing process. Three strengths of softgel capsules containing 2 mg, 5 mg, and 25 mg of Compound 1 were manufactured. The composition of the fill formulation was equivalent and the dose was adjusted by varying the capsule size. The fill mix was split for the 5 mg and 2 mg batches. The composition of the fill formulation is depicted in Table 35. The composition of the softgel shell is shown in Table 36. [Table 35] [Table 36]

[0117] The fill mixture was prepared by mixing the tank using a Becomix 2.5L. The tank was preheated to 40°C. Propylene glycol monolaurate, vitamin ETPGS, and BHT were added to the Becomix tank and mixed under vacuum maintained at -0.9 bar for 15 minutes or more with an agitator speed of 1.0 m / s (range 0.5-1.5 m / s in right mode) and a homogenizer speed of 5.0 m / s (range 5-10 m / s). The product temperature was maintained between 38°C and 42°C throughout the mixing process by adjusting the agitator and homogenizer speeds as required within their respective validated ranges. Compound 1 was then added with a yellow light turned on to protect from light. The Compound 1 container was rinsed with polyethylene glycol 400 to ensure complete transfer. The mixture was mixed for an additional 60 minutes at 40°C. The solution was then degassed for 30 minutes at 40°C and filtered through a 325-mesh in-line filter upon discharge from the Becomix tank to remove any undissolved material. In-process assays were performed on the fill material and the target fill weight was adjusted accordingly (Table 37). The fill material was stored in a stainless steel hopper at 40°C until ready for encapsulation. [Table 37]

[0118] A heated closed head hopper was used during encapsulation at 40° C. Three dies were evaluated for capsule strength; all three dies produced adequate seals. G2VD was used for 2mg. G4VH was used for 5mg. G20BA was used for 25mg. The gel mass was prepared using gelatin, sorbitol special-glycerin blend A810 as a plasticizer, and purified water, and color converted to opaque blue by adding titanium dioxide, red iron oxide, FD&C Blue #1, and purified water. In-process fill weight, shell weight, and seal thickness were monitored every 10 minutes throughout the run. A summary of the in-process checks is presented in Table 38. [Table 38-1] [Table 38-2]

[0119] (Example 22) Drying and moisture content The drying of the filled capsules was monitored by measuring the fill moisture and hardness over several days. The capsules were dried to hardness within the following ranges: 25mg capsule: 7.0-10.0 Newtons 2mg & 5mg capsules: 7.0-11.0 Newtons At each time point, five capsules were tested for hardness using a Bareiss Hardness Durometer. The capsules were placed on a test plate with the seam parallel to the plate. The hardness tester plunger was adjusted until it just made contact with the capsule. The hardness test measures the applied force required to displace the softgel 2 mm. The moisture content (FM) of the filled capsules was also tested using a Karl Fischer Titrator. The fill material from five capsules was removed and tested in duplicate. The hardness and moisture results are presented in Table 39. [Table 39]

[0120] (Example 23) Moisture ingress Moisture ingress studies were performed on each of the batches. The fill material was tested at the beginning, middle, and end of the encapsulation process to test for moisture ingress from the capsule shell into the fill material. Fill material was removed from selected capsules immediately and after 5, 10, and 15 minute hold times and tested for water content, indicating that there was not any significant water ingress occurring during encapsulation. The results are summarized in Table 40. [Table 40A]

[0121] (Example 24) 2mg & placebo; 5mg & placebo; 10mg, 20mg, 25mg & placebo capsules "Immediate release", soft gelatin capsules (softgels) for oral administration were prepared in 2 mg, 5 mg, 10 mg, and 25 mg strengths. The 2 mg, 5 mg, and 10 mg soft gelatin capsules were opaque, oval, blue colored without print, and contained a pale yellow to green opaque semi-solid measuring <22 mm in size in its largest dimension. The 25 mg soft gelatin capsules were opaque, oval, blue colored without print, and contained a pale yellow to green opaque semi-solid. [Table E]

[0122] Capsule content uniformity, residual solvents and elemental impurities meet pharmaceutical standards (USP <905> ;USP <467> Option 1; and USP <232> / <233> Any unspecified degradation products were present at <1.0% and total degradation products were <3.0%.

[0123] All capsules containing Compound 1 have a common fill and the same gelatin composition and thickness. Table 40 lists the capsule fill components, and Table 41 lists the capsule shell components. Table 42 lists the source and function of the capsule components. [Table 40B] [Table 41]

[0124] The following processing aids (typically used in soft gelatin encapsulation) were also used: fractionated coconut oil (triglyceride, medium chain) lubricates the gelatin ribbons to prevent them from sticking to the tooling; unbleached soy lecithin minimizes the capsules from sticking to each other during processing. [Table 42]

[0125] Encapsulation of 2 mg capsules resulted in a suboptimal seal. Leakage was observed at both 30° C. / 65% RH and 40° C. / 75% RH conditions at the 3 month stable and accelerated conditions and was determined to be caused by the die used for encapsulation.

[0126] Tooling studies were performed to establish the appropriate die set. All pockets were tested for each die and it was determined that the G2VD (fill die, 8 pockets across and 200 pockets total) produced the optimal seal. The G3VAL die produced the best seal of those tested.

[0127] A study was conducted to determine the appropriate tooling for the 10 mg capsules, and die set G7.5VK produced optimal results. In-process checks (IPC) for fill weight, shell weight, and seal thickness were performed at the beginning and end of encapsulation (Table 43). [Table 43]

[0128] Bricking occurs when softgels packaged in a bottle usually stick together and form a "brick". The severity of bricking can range from mild, where the brick can be removed by tapping the bottle, to severe, where the softgels do not separate. Bricking is typically observed at accelerated conditions during stability determination, most commonly in hydrophilic fill formulations. Bricking has been observed for 2 mg, 5 mg, and 25 mg capsules under accelerated conditions. Drying studies were conducted to determine the optimal drying time to achieve equilibrium water content of the fill material. Capsules were tested for hardness, fill moisture, and water activity until equilibrium was reached before completing drying to ensure that as much water as possible was removed.

[0129] (Example 25) Finished Product Testing The finished capsules were analyzed and the results are shown in Table 44. [Table 44]

[0130] (Example 26) Phase I clinical trial A Phase 1 dose escalation study in healthy subjects to evaluate: Safety, tolerability, pharmacokinetics, and pharmacodynamics of Compound 1; Effect of food on Compound 1 pharmacokinetics, The effect of formulation on Compound 1 pharmacokinetics, and CYP3A-Mediated Drug-Drug Interactions with Compound 1.

[0131] The study will investigate administration of Compound 1 to healthy human subjects to evaluate the safety, tolerability and PK of single and multiple ascending doses. Additional objectives include determining the effect of formulation on PK, the effect of food on PK, the effect of CYP3A inhibitors on PK, and the effect of Compound 1 on CYP3A activity. AUC, AUC, C max , and T max PK parameters, including pharmacokinetics, pharmacokinetics, and pharmacokinetics, will be determined. Additional exploratory objectives will evaluate the pharmacodynamics of BTK inhibition over time. The study will include patients aged 18-55 years, weighing more than 48 kg, and with a BMI of 18.5-30.0 kg / m 2 The study will be conducted in up to 144 healthy male and female subjects with a history of pulmonary circulation and no clinically significant abnormalities.

[0132] The study consisted of three parts (Parts A–C) as described below.

[0133] Part A (Dose Escalation, Single and Multiple Ascending Dose) Part A is a randomized, double-blind, placebo-controlled, single and multiple dose study with reciprocal dose escalation administered in the fed state (standard moderate fat meal). Part A consists of nine cohorts (up to seven single ascending dose [SAD] cohorts [Cohorts 1-6 & 14], and up to three multiple ascending dose [MAD] cohorts [Cohorts 7-9]). Subjects are randomized in a 3:1 ratio per cohort to receive either: Compound 1 Formulation A (N=6 for Cohorts 1-4 and N=9 for Cohorts 5-9 & 14); or placebo (PBO; N=2 for Cohorts 1-4 and N=3 for Cohorts 5-9 & 14). Formulation A used in this example clinical trial protocol is equivalent to Formulation A7 in Example 18. Single Ascending Dose Cohorts: Cohorts 1-6 Cohorts 1-4: On Day 1, all subjects will receive a single dose of Formulation A Compound 1 or placebo. Cohorts 5, 6 & 14: On Day -1, all subjects receive placebo (baseline ECG measurements established). On day 1, subjects receive a single oral dose of Compound 1 or placebo. [Table F] Multiple Ascending Dose Cohorts: (Cohorts 7-9) Cohorts 7 and 8: On Day 1, all subjects begin multiple doses of Formulation A Compound 1 or placebo once daily for 10 days. Cohort 9: On Day -1, all subjects receive placebo. On day 1, all subjects begin multiple doses of Compound 1 or placebo once daily for 10 days. [Table G] Initiation of a single dose cohort at a higher dose will be determined by review of safety data through 7 days after the last dose from all subjects enrolled in the previous dosing cohort. In the absence of dose-limiting toxicity, the next higher dose cohort will begin.

[0134] Part B Based on safety data from Part A, Part B will begin at a dose up to the highest dose evaluated in Part A.

[0135] Formulation Effects Cohort 10 is a randomized, two-treatment, two-period, two-sequence, single dose level formulation efficacy evaluation. In treatment sequence AB, subjects (N=5) receive Compound 1 Formulation A in the fed state on day 1, followed by Formulation B on day 8 in the fed state. In treatment sequence BA, subjects (N=5) receive Compound 1 Formulation B in the fed state on day 1, followed by Formulation A in the fed state on day 8. Days 2 to 7 are drug-free days. [Table H]

[0136] Food Effects Cohort 11 is a randomized, two-treatment, two-period, two-sequence, single dose level food effect evaluation. The formulation used in cohort 11 is Formulation A. In treatment sequence FH, subjects receive compound 1 in the fasted state on day 1 and a second dose in the fed state (high fat, high calorie meal) on day 8. In treatment sequence HF, subjects receive compound 1 in the fed state (high fat, high calorie meal) on day 1 and a second dose in the fasted state on day 8. Days 2-7 are drug holidays. Summary of Treatment Duration for Part B (Cohorts 10 and 11) [Table I]

[0137] Part C (CYP3A drug-drug interactions) In Part C, fed dosing will consist of a standard moderate fat meal. The formulation utilized is Formulation A. Dosing for Part C will be based on safety data from Part A.

[0138] Cohort 12 is an open-label, fixed-sequence, multiple-dose DDI study with itraconazole (ITZ). Subjects receive a single oral dose of Compound 1 administered in the fasted state on day 1. Beginning on day 3 and continuing through day 6, subjects receive itraconazole 200 mg once daily (QD) in the fasted state. On day 5, subjects receive Compound 1 administered in the fasted state 1 hour after administration of itraconazole. [Table J]

[0139] Cohort 13 is an open-label, fixed-sequence, multiple-dose DDI study with midazolam (MDZ). Subjects receive an oral dose of midazolam (2 mg) in the fed state on day 1. Beginning on day 3 and continuing through day 12, subjects receive Compound 1 twice daily (BID) in the fed state. On days 3 and 11, subjects receive a single oral dose of midazolam (2 mg) administered simultaneously with the morning dose of Compound 1 in the fed state. [Table K] Number of targets Part A (N=104) Cohorts 1-4: 8 subjects per cohort, 6 compound 1 + 2 placebo (32 total) Cohorts 5-9 & 14: 12 subjects per cohort, 9 compound 1 + 3 placebo (60 total) Part B (N=22) Cohort 10: 10 subjects, all Compound 1 Cohort 11: 12 subjects, all Compound 1 Part C (N=18) Cohorts 12-13: 9 subjects per cohort, all Compound 1 Study duration Screening: 28 days Constraints: Cohorts 1-4: 4 days, 3 nights (days -1 to 3) Cohort 5, 6% 14: 5 days, 4 nights (days -2 to 3) Cohorts 7 & 8: 13th, 12th night (Days -1 to 12) Cohort 9: Day 14, Night 13 (Days -2 to 12) Cohorts 10 & 11: 11 days, 10 nights (days -1 to 10) Cohort 12: 8 days, 7 nights (days -1 to 7) Cohort 13: 14 days, 13 nights (days -1 to 13) Follow-up visit: Approximately 7 days after the last dose Total study duration (screening, detention, and follow-up): Approximately 39 to 49 days

[0140] Formulation A and Formulation B Compound 1 is provided as a blue soft gelatin capsule for oral administration. Each capsule contains a lipid-based fill solution composed of Compound 1 dissolved in PEG 400, propylene glycol monolaurate, vitamin E polyethylene glycol succinate (TPGS), and butylated hydroxytoluene (BHT). Capsules are filled with sufficient fill solution to contain 2 mg, 5 mg, or 25 mg of Compound 1. The soft gelatin capsule shell material contains gelatin (Type 195), sorbitol-glycerin blend (A810), titanium dioxide, FD&C blue #1 color, and red iron oxide. Formulation B is evaluated based on the safety and PK data from Part A.

[0141] (Example 27) Clinical trial results Safety, tolerability, and pharmacokinetic profile of single and multiple ascending doses of Compound 1 in healthy subjects This first-in-human study was designed to evaluate single and multiple dose safety, tolerability, pharmacokinetics (PK; including CNS penetration) and pharmacodynamics (PD) of Compound 1, food effects, drug interactions, and cardiac safety to inform dosing and concomitant drug therapy in clinical studies, as described in Example 25, Part A.

[0142] Methods: This is an ongoing Phase 1, double-blind, randomized, placebo-controlled, single ascending and multiple ascending dose (SAD, MAD) study in healthy subjects with reciprocal dose escalation and adaptive dose selection.

[0143] Safety will be determined throughout the study, and PK and PD will be characterized using serial blood samples collected up to 48 hours post-dose for Compound 1 plasma concentrations, BTK target occupancy (TO) and target engagement (TE). Cerebrospinal fluid (CSF) samples will be collected 2 hours post-dose to characterize the relationship between Compound 1 plasma concentrations and CNS penetration (CSF / unbound plasma concentrations). Compound 1 PK parameters (e.g., AUC last , AUC inf , C max , T max, t 1 / 2 ) is estimated by standard non-compartmental methods.

[0144] Results: Preliminary pharmacokinetic results from the three completed SAD cohorts are presented including 41 subjects (7 receiving 5 mg, 8 receiving 15 mg, 8 receiving 45 mg, 8 receiving 5 mg + 10 mg placebo, 12 receiving 100 mg, and 12 receiving 200 mg; randomized in a 3:1 ratio to receive Compound 1 or placebo) in the fed state (moderate fat meal). Review of the blinded safety data thus demonstrated that the study drug was generally well tolerated with no serious adverse events or premature study discontinuations reported. Preliminary PK parameters are presented in Table 45 (all pharmacokinetic parameters in Table 45 are reported as median (Min, Max) T max and t 1 / 2 The mean (reported as % coefficient of variation) except for , and the mean (SD) plasma concentration-time profiles of Compound 1 are presented in FIG. [Table 45]

[0145] Compound 1 increased mean plasma T max It is rapidly absorbed following oral administration and exhibits a terminal half-life of approximately 3-4 hours. Single-dose Compound 1 exposure (AUC inf ) increased in a dose-proportional manner from 5 mg to 15 mg and more than nearly dose-proportional from 15 mg to 200 mg. Mean Compound 1 CSF:unbound plasma concentration ratios approached 1, indicating unimpeded access of plasma unbound drug into the CNS (see, e.g., Table 46, reported as Mean (% Coefficient of Variation), and Figure 26, showing CSF to unbound plasma ratios by dose). The observed Compound 1 plasma exposure, CNS penetration, and preliminary TO and TEPD data were consistent with translational PK / PD models that showed low doses of Compound 1 resulted in high levels of BTK inhibition in the blood and CNS. [Table 46]

[0146] Results: Preliminary pharmacokinetic results from one completed MAD cohort are presented, including 12 subjects randomized in a 3:1 ratio to receive Compound 1 or placebo in the fed state (medium fat meal), receiving 15 mg once daily for 10 days. Review of blinded safety data thus demonstrated that the study drug was generally well tolerated with no serious adverse events or premature study discontinuations reported. Preliminary PK parameters are shown in Table 47 (all pharmacokinetic parameters in Table 46 are reported as median (Min, Max) T max and t 1 / 2 The data are presented in Figure 27, which shows the mean (reported as % coefficient of variation) except for . [Table 47]

[0147] Compound 1 increased the mean plasma T max and was rapidly absorbed following oral administration, exhibiting a terminal half-life of approximately 3 hours. Minimal to none accumulation of Compound 1 was observed with once daily dosing.

[0148] Results: Preliminary pharmacokinetic results evaluating the effect of food on Compound 1 PK are presented for subjects in Cohorts 11-12 and Cohorts 2 and 15-15 receiving a single dose of 15 mg under fasting conditions or in the fed state (with a moderate fat meal or with a high fat high calorie meal). Preliminary PK parameters are presented in Table 48 (all pharmacokinetic parameters in Table 47 are reported as median (Min, Max)). max and t 1 / 2 are reported as mean (% coefficient of variation) except for , and are presented in FIG. [Table 48]

[0149] Compound 1 increased the mean plasma T max Compound 1 was rapidly absorbed following oral administration, with a terminal half-life of approximately 3 hours. Food (moderate-fat or high-fat high-calorie meals) had no clinically significant effect on the PK of Compound 1.

[0150] Results: Preliminary pharmacokinetic results evaluating the drug-drug interaction effect of a strong CYP3A inhibitor (itraconazole) on Compound 1 PK are presented, including 9 subjects from Cohort 12 receiving a single dose of 2 mg Compound 1 alone or in combination with 200 mg itraconazole once daily under fasting conditions. Preliminary PK parameters are shown in Table 49 (all pharmacokinetic parameters in Table 49 are reported as median (Min, Max)). max and t 1 / 2 The mean (reported as % coefficient of variation) except for , and the mean (SD) plasma concentration-time profiles of Compound 1 are presented in FIG. [Table 49]

[0151] Compound 1 increased the mean plasma T max Compound 1 AUC inf (15%) and C max (23%). Coadministration of strong CYP3A inhibitors with Compound 1 had no clinically significant effect on the PK of Compound 1.

[0152] Conclusions: Data from the first six SAD cohorts and the first MAD cohort in healthy subjects indicate that low doses of Compound 1 provided adequate plasma exposure and CSF penetration to drive the desired BTK inhibition in the CNS. Neither food nor coadministration of a strong CYP3A inhibitor had clinically significant effects on the PK of Compound 1. Blinded safety data indicate that single-dose study drug administration was well tolerated across the dose range.

[0153] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, using concepts from various patents, applications, and publications to provide still further embodiments.

[0154] These and other changes can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Thus, the claims are not limited by this disclosure.

[0155] All publications, patents, and patent applications mentioned in this specification 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.

[0156] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 257,509, filed October 19, 2021, and U.S. Provisional Application No. 63 / 393,163, filed July 28, 2022, which applications are hereby incorporated by reference in their entireties.

Claims

1. A solid crystalline form of (S)-1-(1-acryloylpiperidin-3-yl)-2-fluoro-5,6,7,8,9,10-hexahydrocyclohepta[b]indole-4-carboxamide, Form III, characterized by an XRPD pattern having peaks at 10.2543±0.2, 13.5006±0.2, and 13.9691±0.2 degrees two-theta; Solid crystalline form.

2. 10. The solid crystalline form of claim 1, further characterized by an XRPD pattern having peaks at 22.22±0.2, 19.27±0.2, 20.81±0.2, and 8.70±0.2 degrees two-theta.

3. 10. The solid crystalline form of claim 1, further characterized by an XRPD pattern substantially as shown in Figure 5.

4. 2. The solid crystalline form of claim 1, wherein greater than 95% by weight of the crystalline form is Form III.

5. A pharmaceutical composition comprising the solid crystalline form of any one of claims 1 to 4.

6. 6. The pharmaceutical composition of claim 5, comprising an additional therapeutically active compound.

7. 6. The pharmaceutical composition of claim 5, formulated for oral administration.

8. 6. The pharmaceutical composition of claim 5, in the form of a gel capsule.

9. 6. The pharmaceutical composition of claim 5, further comprising polyethylene glycol.

10. 6. The pharmaceutical composition of claim 5, further comprising polyethylene glycol monolaurate.

11. 6. The pharmaceutical composition of claim 5, further comprising vitamin E.

12. 6. The pharmaceutical composition of claim 5, further comprising butylated hydroxytoluene.

13. Use of a solid crystalline form described in any one of claims 1 to 4 for the manufacture of a medicament for treating a disease or condition modulated by kinase inhibition in a subject in need thereof.

14. 14. The use according to claim 13, wherein the tyrosine kinase is Bruton's tyrosine kinase (BTK).

15. 14. The use according to claim 13, wherein the disease or condition is cancer.

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