Crystalline forms of (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[1,5-A]pyrimidine-3-carboxamide, their preparation, and their uses

Abstract

To develop a new form of (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 1) which possesses characteristics such as a high melting point and better stability, suitable for drug formulation.SOLUTION: The present invention relates to a crystalline form of Compound 1 to inhibit Btk, methods of preparation thereof, pharmaceutical compositions thereof, and use of the crystalline form in treatment of a disease or in manufacture of a medicament for treatment of a disease.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to crystalline forms of (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide. The present invention also relates to methods for preparing the crystalline forms and methods for utilizing the crystalline forms as Btk inhibitors. [Background technology]

[0002] Bruton's tyrosine kinase (Btk) belongs to the Tec tyrosine kinase family (Vetrie et al., Nature 361: 226-233, 1993; Bradshaw, Cell Signal. 22: 1175-84, 2010). Btk is mainly expressed in most hematopoietic cells, such as B cells, mast cells, and macrophages (Smith et al., J. Immunol. 152: 557-565, 1994), and is also localized in bone marrow, spleen, and lymph node tissues. Btk plays an important role in the B cell receptor (BCR) and FcR signaling pathways, which are involved in the development and differentiation of B cells (Khan, Immunol. Res. 23: 147, 2001). Btk is activated by upstream Src family kinases. Once activated, Btk then phosphorylates PLC gamma, affecting B cell function and survival (Humphries et al., J. Biol. Chem. 279: 37651, 2004).

[0003] These signaling pathways must be precisely regulated. Mutations in the gene encoding Btk cause an inherited B cell-specific immunodeficiency in humans, known as X-linked agammaglobulinemia (XLA) (Conley et al., Annu. Rev. Immunol. 27: 199-227, 2009). Abnormalities in BCR-mediated signaling can lead to dysregulated B cell activation, resulting in many autoimmune and inflammatory diseases. Preclinical studies have shown that Btk-deficient mice are resistant to the development of collagen-induced arthritis. Furthermore, clinical studies with Rituxan, a CD20 antibody that can deplete mature B cells, have revealed that B cells play a key role in many inflammatory diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis (Gurcan et al., Int. Immunopharmacol. 9: 10-25, 2009). Thus, Btk inhibitors can be used to treat autoimmune and / or inflammatory diseases.

[0004] Furthermore, aberrant activation of Btk plays an important role in the pathogenesis of B-cell lymphomas, which indicates that inhibition of Btk is useful for the treatment of hematological malignancies (Davis et al., Nature 463:88-92, 2010). Preliminary clinical trial results have shown that the Btk inhibitor PCI-32765 is effective in the treatment of several types of B-cell lymphoma (see, e.g., 54thAmerican Society of Hematology (ASH) annual meeting abstract, Dec. 2012: 686 The Bruton's Tyrosine Kinase (Btk) Inhibitor, Ibrutinib (PCI- 32765), Has Preferential Activity in the ABC Subtype of Relapsed / Refractory De Novo Diffuse Large B-Cell Lymphoma (DLBCL): Interim Results of a Multicenter, Open-Label, Phase I Study). Due to the central role of Btk as a mediator in multiple signaling pathways, inhibitors of Btk are of great interest as anti-inflammatory and / or anti-cancer agents (Mohamed et al., Immunol. Rev. 228: 58-73, 2009; Pan, Drug News perspect 21: 357-362, 2008; Rokosz et al., Expert Opin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer Agents Med. Chem. 7: 624-632, 2007; Lou et al, J. Med. Chem. 55(10): 4539-4550, 2012).

[0005] International Application WO2014173289A discloses a series of fused heterocyclic compounds as Btk inhibitors. In particular, WO2014173289A discloses (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (hereinafter referred to as compound 1). [ka] compound 1

[0006] Compound 1 is a potent, specific, and irreversible BTK kinase inhibitor. Preclinical data from biochemical, cell-based, and animal studies suggest that compound 1 exerts significant effects in suppressing tumor growth in B-cell malignancies. Compound 1 is expected to have fewer side effects than ibrutinib in clinical practice, as it has been shown to be more selective in BTK inhibition compared to ibrutinib when comparing EGFR, FGR, FRK, HER2, HER4, ITK, JAK3, LCK, and TEC. Furthermore, compound 1 is expected to have fewer side effects than ibrutinib in clinical practice, as its weaker inhibition of ITK results in significantly less inhibition of rituximab-induced antigen-dependent cell-mediated cytotoxicity (ADCC) than ibrutinib, and therefore may be of good efficacy in the treatment of B-cell malignancies when combined with rituximab or other ADCC-dependent antibodies.

[0007] In preclinical safety evaluation, compound 1 was demonstrated to be safer than ibrutinib in terms of overall tolerability and severe toxicity in single-dose and repeated-dose toxicity studies up to 28 days in rats and dogs. In addition, compound 1 had better bioavailability, without the accumulation issues observed with ibrutinib. These unique features of compound 1 lead to clinical trials for further evaluation. Summary of the Invention [Problem to be solved by the invention]

[0008] However, in the preparation method of compound 27 in WO 2014173289, compound 1 was amorphous, which was further confirmed by the X-ray powder diffraction pattern in FIG. 7A. This amorphous form of compound 1 was shown to have a low glass transition temperature as shown in FIG. 7B, revealing problems in the formulation of amorphous compounds, such as low stability and difficulty in purification. For this reason, it is necessary to develop a new form of compound 1, which must have characteristics such as a high melting point and high stability, making it suitable for formulation. [Means for solving the problem]

[0009] The inventors have unexpectedly discovered that the crystalline form of Compound 1 has a high melting point and is extremely stable when stored at 25° C. / 60% RH for up to 24 months or at 40° C. / 75% RH for up to 6 months.

[0010] Disclosed herein as a first aspect is a crystalline form of Compound 1. [ka] compound 1

[0011] In some embodiments, the crystalline form of Compound 1 is anhydrous crystalline (referred to herein as "crystalline form A").

[0012] Disclosed herein as a second embodiment is a crystalline form of compound BG-13 having an X-ray powder diffraction pattern substantially as shown in FIG. [ka] Compound BG-13

[0013] Disclosed in a third aspect is a method for preparing Compound 1.

[0014] Further disclosed herein are intermediate compounds of formula Ie or a salt thereof, or formula If or a salt thereof, which are used to prepare compound 1. [ka] Formula Ie Formula If

[0015] In a fourth aspect, disclosed herein is a method for preparing crystalline form A.

[0016] In a fifth aspect, disclosed herein is a pharmaceutical composition comprising a therapeutically effective amount of crystalline Form A as disclosed herein.

[0017] In a sixth aspect, disclosed herein is a method of treating a disease associated with undesirable Btk activity in a subject by administering crystalline form A disclosed herein to the subject.

[0018] Disclosed herein in a seventh aspect is a method of treating a disease in a subject by administering crystalline form A to the subject, wherein the disease is selected from an allergic disease, an autoimmune disease, an inflammatory disease, cancer, or a combination of two or more thereof.

[0019] In an eighth aspect, disclosed herein is a method of treating a B cell proliferative disorder selected from a B cell malignancy or a relapsed / refractory B cell malignancy in a subject by administering to the subject crystalline form A as disclosed herein. In some aspects of this aspect, disclosed herein is a method of treating a B cell proliferative disorder selected from chronic lymphocytic, non-Hodgkin's lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof, by administering to the subject crystalline form A as disclosed herein.

[0020] Disclosed herein as a ninth aspect is the use of crystalline form A as disclosed herein for the manufacture of a medicament for the treatment of at least one disease associated with abnormal Btk activity in a subject.

[0021] Disclosed herein as a tenth aspect is the use of crystalline form A as disclosed herein for the manufacture of a medicament for the treatment of a disease selected from an allergic disease, an autoimmune disease, an inflammatory disease, cancer, or a combination of two or more thereof in a subject.

[0022] Disclosed herein as an eleventh aspect is the use of crystalline form A as disclosed herein in the manufacture of a medicament for the treatment of a B cell proliferative disorder selected from a B cell malignancy or a relapsed / refractory B cell malignancy in a subject. Disclosed herein as some aspects of this aspect is the use of crystalline form A as disclosed herein in the manufacture of a medicament for the treatment of chronic lymphocytic, non-Hodgkin's lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof, in a subject.

[0023] Disclosed herein as a twelfth aspect is a method for preparing crystalline form A of compound 1, comprising mixing amorphous compound 1 with the following solvent system to form a clear solution; and precipitating crystalline form A by keeping the solution at room temperature or heating for a period of time with or without stirring, wherein the solution system is: Ethyl acetate:hexane = 1:0.6-0.7 (volume ratio); Ethyl acetate:heptane=1:0.6-0.7 (volume ratio); Ethyl acetate:cyclohexane = 1:0.6-1.2 (volume ratio); Methyl acetate:hexane = 1:0.6-1.2 (volume ratio); Toluene:hexane = 1.0:0.2-0.4 (volume ratio); Toluene:cyclohexane = 1.0:0.1-0.2 (volume ratio); Methyl acetate:cyclohexane = 0.6-0.8:1.0 (volume ratio); IPAC:cyclohexane = 1.0:0.2-1.0 (volume ratio); or Isobutyl acetate:cyclohexane = 1.0:0.2-1.0 (volume ratio).

[0024] In one embodiment, the ee value of the amorphous form of Compound 1 is greater than 90%. In another embodiment, the ee value of the amorphous form of Compound 1 is 97%. [Brief description of the drawings]

[0025] [Figure 1] XRPD pattern of crystalline form A. [Diagram 2] DSC curve of crystalline form A. [Diagram 3] TGA curve of crystalline form A. [Figure 4] 1H-NMR of crystalline form A. [Diagram 5] 13C-NMR of crystalline form A. [Figure 6] DVS plot of crystalline form A. [Figure 7A] XRPD pattern of compound 1 in amorphous form. [Figure 7B] The mDSC curve of the amorphous form of Compound 1 shows that the glass transition temperature of the amorphous form is 79.7° C. (midpoint temperature). [Figure 8] Absolute structure of BG-13 single crystal. [Figure 9] An illustration of hydrogen bonds in a BG-13 single crystal. [Figure 10] Crystal packing of single crystals of BG-13. [Figure 11] XRPD pattern of BG-13 single crystal. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The crystalline form of compound 1 discovered by the inventors was unexpected and was named crystalline form A, but it could only be obtained under certain conditions depending on the ee value of the starting material and the ratio of co-solvents. Polymorphism was also studied by methods such as evaporation over time, anti-solvent addition, cooling over time, vapor diffusion, and polymer-induced crystallization. Most of the experiments failed to obtain a crystalline form, which indicates that obtaining crystalline form A is not easy.

[0027] Further characterization revealed that Form A was anhydrous with a melting point of 139.4±2°C (onset temperature). To assess stability, samples of Form A were stored at 80°C for 2 days, 25°C / 60%RH for 24 months, or 40°C / 75%RH for 6 months and characterized by XRPD before, during and after the stability studies. The results showed that no change in crystal form was observed for all the above time periods, indicating good physical stability of Form A at 80°C, or stored at 25°C / 60%RH for 24 months and 40°C / 75%RH for 6 months.

[0028] In certain embodiments of Form A, the X-ray powder diffraction pattern comprises diffraction peaks at 2θ angle values ​​independently selected from about 14.8±0.2°, 16.4±0.2°, and 21.4±0.2°.

[0029] In one embodiment of crystalline form A, the X-ray powder diffraction pattern comprises diffraction peaks at 2θ angle values ​​independently selected from about 14.8±0.2°, 15.6±0.2°, 16.4±0.2°, and 21.4±0.2°.

[0030] In certain embodiments of crystalline form A, the X-ray powder diffraction pattern comprises diffraction peaks at 2θ angle values ​​independently selected from about 12.2±0.2°, 12.9±0.2°, 14.8±0.2°, 15.6±0.2°, 16.4±0.2°, and 21.4±0.2°.

[0031] In certain embodiments of crystalline form A, the X-ray powder diffraction pattern comprises diffraction peaks at 2θ angle values ​​individually selected from about 12.2±0.2°, 12.9±0.2°, 14.8±0.2°, 15.6±0.2°, 16.4±0.2°, 17.7±0.2°, 18.5±0.2°, 20.7±0.2°, and 21.4±0.2°.

[0032] Some embodiments of crystalline Form A have an X-ray powder diffraction pattern substantially like that of FIG.

[0033] Some embodiments of crystalline Form A have the X-ray powder diffraction patterns summarized in Table 1.

[0034] Table 1 X-ray diffraction pattern of crystalline form A [Table 1]

[0035] In some preferred embodiments, the melting point of crystalline form A is 139±2° C. (onset temperature).

[0036] In some preferred embodiments, crystalline form A exhibits a DSC substantially as shown in FIG.

[0037] In some preferred embodiments, crystalline form A exhibits a TGA substantially as shown in FIG.

[0038] In some embodiments, crystalline form A is slightly hygroscopic. In some embodiments, crystalline form A is not solvated.

[0039] In some embodiments, crystalline form A exhibits substantially the same X-ray powder diffraction (XRPD) pattern after storage at 40° C. and 75% RH for 6 months. In some embodiments, crystalline form A exhibits substantially the same X-ray powder diffraction (XRPD) pattern after storage at 25° C. and 60% RH for 24 months.

[0040] Also disclosed herein is a crystalline form of compound BG-13 having an X-ray powder diffraction pattern substantially in accordance with FIG. [ka] Compound BG-13

[0041] In some embodiments, the crystalline form of BG-13 is single crystalline and has unit cell dimensions of a=16.7939(4) Å, b=7.9871(2) Å, c = 23.5438(5) Å, alpha = 90.00°, beta = 108.0460(10)°, gamma = 90.00°.

[0042] The present inventors estimated that the absolute configuration of compound 1 is S based on single crystal X-ray structure analysis of intermediate BG-13.

[0043] Also disclosed herein are methods for preparing compound 1 and deuterium-labeled compound 1, as shown in the procedure in Scheme 1. The novel synthesis method and crystallization / recrystallization procedure of compound 1 via crystalline form A disclosed herein overcomes many of the problems associated with previously reported methods, such as preparation of key chiral intermediates with optical purity of over 98%, improves the purity of compound 1 to meet the tolerance levels described herein, controls impurities in compound 1, and has many advantages over existing methods. In particular, the method disclosed herein is particularly suitable for producing compound 1 reproducibly and on a commercial scale, while providing improved quality and good yields. In another method, BG-9 or its analogs in Scheme 1 could be asymmetrically reduced with low to high enantioselectivities (5% ee to 95% ee). Other steps are similar to those listed in Scheme 1.

[0044] Scheme 1: Preparation of Compound 1 and Deuterium-Labeled Compound 1 [ka] [ka]

[0045] Also disclosed herein is a process for preparing a compound of formula Ia, which comprises asymmetric reduction of a compound of formula I in the presence of a catalyst and / or reducing agent to produce a compound of formula Ia. [ka] Formula I Formula Ia In the formula, R 1 is hydrogen or an amino protecting group.

[0046] In some embodiments, amino protecting groups include, but are not limited to, acetyl, propionyl, butyryl, phenylacetyl, benzoyl, toluyl, phenoxyacetyl (POA), methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, tert-butyloxycarbonyl (BOC), 2-iodoethoxycarbonyl, carbobenzoxy (CBZ), 4-methoxybenzyloxycarbonyl, (fluoren-9-ylmethoxy)carbonyl (Fmoc), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), benzyl, methyl, or 4-methoxybenzyl.

[0047] In some embodiments, the catalyst is a neutral catalyst system or a cationic catalyst system. In some embodiments, the catalyst is [Ir(COD)Cl]2 / (R or S)-MeO-Biphep, [Ir(COD)Cl]2 / (R or S)-Binap, [Ir(COD)Cl]2 / (R or S)-Tol-Binap, [Ir(COD)Cl]2 / (R or S)-xyl-Binap, [Ir(COD)Cl]2 / (S,S or R,R)-Diop, [Ir(COD)Cl]2 / (R or S)-P-Phos, [Ir(COD)Cl]2 / (R or S)-Tol-P-Phos, [Ir(COD)Cl]2 / (R or S)-Xyl-P-Phos, [Ir(COD)Cl]2 / (R,R or S,S)-Me-DuPhos, [Ir(COD)Cl]2 / (R or S)-SegPhos, [Ir(μ-Cl)(cod)]2 / (R or S)-Ship, [Ir(μ-Cl)(cod)]2 / (R or S)-Siphos, [Ir(μ-Cl)(cod)]2 / (R or S)-Siphos-PE, [Ir(μ-Cl)(cod)]2 / (R or S)-MonoPhos, [Ir(μ-Cl)(cod)]2 / (R or S)-tol-SDP, [Ir(μ-Cl)(cod)]2 / (S,S or R,R)-Diop, [Ir(μ-Cl)(cod)]2 / (S,R or R,S)-Josiphos, [Ir(μ-Cl)(cod)]2 / (R or S)-Binap, [Ir(μ-Cl)(cod)]2 / (R or S)-MeO-Biphep, [Ir(μ-Cl)(cod)]2 / (R or S)-Synphos, or [Ir(μ-Cl)(cod)]2 / (R or S)-Difluorphosor [Ir(cod)2] + X -iridium catalyst systems including, but not limited to, the above-mentioned ligands attached to (X: e.g. BF4, NO3, OTf, PF6, SbF6 and BarF) (Wen-Bo et al., J. AM. CHEM. SOC. 125, 10536-10537 2003. Damien et al., J. Org. Chem. 77, 4544-4556, 2012. Milos et al., Org. Process Res. Dev. 16, 1293-1300, 2012.); rhodium catalyst systems including, but not limited to, the above-mentioned ligands attached to [Rh(COD)2]BF4 (Xiang-Ping et al., Top Organomet Chem 36, 313-354,2011); or RuCl2(R or S)-BINAP / (R or S)-DAIPEN, RuCl2(R or S)-BINAP / (R,R or S,S)-DPEN, RuCl2(S or R)-BINAP (S,S or R,R)-DACH, RuCl2[(R or S)-Tol-BINAP][(S,S or R,R)-DPEN], RuCl2(R,R or S,S)-Me-DuPHOS / (R,R or S,S)-DPEN, RuCl2(R,R or S,S)-Et-DuPHOS / (R,R or S,S)-DPEN, RuCl2(R,R or S,S)-Et-DuPHOS / (R,R or S,S)-DACH, RuCl2(S,S or R,R)-i-Pr-DuPHOS / (R,R or S,S)-DPEN, RuCl2(R or S)-HexaPHEMP / (R,R or Ruthenium catalyst systems including RuCl(R or S)-MeO-BIPHEP / (R,R or S,S)-DPEN (Christopher et al., Adv. Synth. Catal. 345, 195-201, 2003. Julian et al., Adv. Synth. Catal. 345, 300-307, 2003.) are also known.

[0048] It has been found that in the above process, excellent enantioselectivities of up to 95% ee can be obtained by using the above catalysts, especially with neutral or cationic iridium catalyst systems.

[0049] Disclosed herein is a method for dissolving a compound of formula IIa to produce a compound of formula IIb or to enhance the chiral purity of a compound of formula IIb, comprising treating a racemic compound of formula IIa with a chiral acid. [ka] Formula IIa Formula IIb Here, R 1 is hydrogen, methyl, benzyl, 4-methoxybenzyl or other conventional amino protecting groups as described above.

[0050] In some embodiments, chiral acids include, but are not limited to, L-malic acid, D-malic acid, L-mandelic acid, D-mandelic acid, L-camphorsulfonic acid, D-camphorsulfonic acid, L-tartaric acid, D-tartaric acid, L-DBTA, D-DBTA, L-DTTA, or D-DTTA.

[0051] Also disclosed herein is a method for resolving a compound of formula Ic to produce a compound of formula Id or to increase the chiral purity of a compound of formula Id, comprising treating a racemic compound of formula Ic with a chiral acid. [ka] Formula Ic Formula Id Here, R 1 is hydrogen, methyl, benzyl, 4-methoxybenzyl or other conventional amino protecting groups as described above.

[0052] In some embodiments, chiral acids include, but are not limited to, L-malic acid, D-malic acid, L-mandelic acid, D-mandelic acid, L-camphorsulfonic acid, D-camphorsulfonic acid, L-tartaric acid, D-tartaric acid, L-DBTA, D-DBTA, L-DTTA, or D-DTTA.

[0053] Further disclosed herein is a compound of formula Ie, or a salt thereof, or a compound of formula If, or a salt thereof, for use in preparing compound 1. [ka] Formula Ie [ka] Expression If

[0054] Additionally, methods of preparing crystalline form A are provided. The crystalline forms disclosed herein can be prepared by crystallizing the compounds disclosed herein from a suitable solvent system comprising at least one solvent, followed by spontaneous precipitation (evaporation), cooling, and / or addition of an anti-solvent (in which the compounds disclosed herein have relatively low solubility) to achieve supersaturation with the solvent system. Crystallization can also be performed with or without the use of a suitable crystal seed to crystallize the crystalline forms disclosed herein.

[0055] In some embodiments, the method for preparing crystalline form A includes dissolving (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 1) in DCM, exchanging into solvent EA, and recrystallizing from EA / MTBE to obtain the desired crystalline form.

[0056] In some embodiments, the method of preparing crystalline form A comprises dissolving compound 1 in EA and adding hexane to obtain the desired crystalline form.

[0057] In some embodiments, the method of preparing crystalline form A is carried out by adding an anti-solvent to a solution of solid compound 1 or crude form A in a solvent for dissolving these solids, where anti-solvents include, but are not limited to, HO and n-heptane, and solvents for dissolving solids include, but are not limited to, acetone, DMAc, EtOAc, DCM, toluene, and 2-MeTHF.

[0058] In some embodiments, the method of preparing crystalline form A involves adding a solution of solid compound 1 or crude form A in a solvent to an anti-solvent, including but not limited to, acetone and EtOAc, and an anti-solvent, including but not limited to, n-heptane, in a sealed reactor, allowing sufficient time for the organic vapor to interact with the solution.

[0059] Also disclosed herein is a pharmaceutical composition comprising a therapeutically effective amount of crystalline form A and a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition is administered orally. In some preferred embodiments, the pharmaceutical composition comprises 1% to 99% by weight of crystalline form A. In some more preferred embodiments, the pharmaceutical composition comprises 1% to 70% by weight of crystalline form A. In some most preferred embodiments, the pharmaceutical composition comprises 10% to 30% by weight of crystalline form A.

[0060] The present invention also provides a method of treating or preventing a disease associated with undesirable Btk activity in a subject by administering crystalline form A to the subject.

[0061] The present invention also provides a method of treating or preventing a disease selected from an allergic disease, an autoimmune disease, an inflammatory disease, cancer, or a combination of two or more thereof in a subject by administering crystalline form A to the subject.

[0062] The present invention also provides a method of treating or preventing a B cell proliferative disorder in a subject by administering crystalline form A to the subject.

[0063] In some embodiments, B cell proliferative disorders include B cell malignancies, including, but not limited to, lymphoma, non-Hodgkin's lymphoma (NHL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Waldenstrom's macroglobulinemia (WM), marginal zone lymphoma (MZL), hairy cell leukemia (HCL), Burkitt's leukemia (BL).

[0064] In some embodiments, B cell proliferative disorders include, but are not limited to, relapsed / refractory (R / R) B cell malignancies, including R / R MCL, R / R CLL, R / R SLL, R / R WM.

[0065] Crystalline Form A disclosed herein can be used to manufacture a medicament for treating at least one disease associated with undesirable Btk activity in a subject.

[0066] The presently disclosed crystalline form A can be used to manufacture a medicament for treating a disease selected from an allergic disease, an autoimmune disease, an inflammatory disease, cancer, or a combination of two or more thereof in a subject.

[0067] The presently disclosed crystalline form A can be used for the manufacture of a medicament for the treatment of a B cell proliferative disorder selected from a B cell malignancy, or a relapsed / refractory B cell malignancy, in a subject.

[0068] Recent clinical trials continue to demonstrate that compound 1 is well tolerated in previously untreated (TN) and relapsed / refractory (R / R) B-cell malignancies, including WM with a very good partial response (VGPR) rate of over 40% in 42 evaluable patients and an overall response rate (ORR) of 90% in 42 evaluable patients with a median follow-up of 12.3 months, and CLL / SLL with a high overall response rate (94%) and very low treatment discontinuation rates (3%) with a median efficacy follow-up of 10.5 months.

[0069] definition Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0070] In the appended claims, including those contained herein, singular words such as "a," "an," and "the" are intended to include their corresponding plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "crystalline form" is intended to include one or more different crystalline forms, and reference to a "method" is intended to include equivalent steps and methods known to those of skill in the art that may modify or substitute for the method described herein.

[0071] As disclosed herein, the crystalline form is a substantially pure crystal. As used herein, the term "substantially pure" refers to at least 85% by weight, preferably at least 95% by weight, and more preferably at least 99% by weight, of the crystalline form A disclosed herein.

[0072] For the crystalline forms disclosed herein, only the major peaks (i.e., the most characteristic, significant, unique and / or reproducible peaks) are summarized; additional peaks are routinely obtained from the diffraction spectra. The major peaks listed above are reproducible within the margin of error (±2 in the last decimal place, or ±0.2 relative to the stated value).

[0073] As disclosed herein, an "X-ray powder diffraction pattern substantially in accordance with FIG. 1" refers to an X-ray powder diffraction pattern that exhibits the same major peaks as in FIG. 1, where a major peak is a peak that exhibits a relative intensity of more than 10%, preferably more than 20%, relative to the highest peak in FIG. 1 (which has a relative intensity of 100%).

[0074] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise," and conjugations such as "comprises" and "comprising," will be understood to mean the inclusion of any stated integers or steps or group of integers or steps. As used herein, the term "comprising" is interchangeable with the term "containing," and, as used herein, is sometimes interchangeable with the term "having."

[0075] The term "therapeutically effective amount" as used herein refers to an amount of a compound that, when administered to a subject to treat a disease, is sufficient to affect the treatment of the disease, disorder, or condition, or at least one of the clinical symptoms of the disease or disorder. A "therapeutically effective amount" may vary depending on the compound, the illness, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in a given instance may be apparent to one of skill in the art or may be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the combined amounts used to provide effective treatment for the disease, disorder, or condition.

[0076] The pharmaceutical composition containing the compound disclosed herein can be administered orally, inhaled, rectally, parenterally or topically to a subject in need thereof. For oral administration, the pharmaceutical composition may be a conventional solid formulation such as tablet, powder, granule, capsule, etc., or a liquid formulation such as water or oil suspension or other liquid formulation such as syrup, solution, suspension, etc.; for parenteral administration, the pharmaceutical composition may be a solution, aqueous solution, oil suspension concentrate, lyophilized powder, etc. The pharmaceutical composition formulation is preferably selected from tablets, coated tablets, capsules, suppositories, nasal sprays or injections, more preferably selected from tablets or capsules. The pharmaceutical composition may be a single unit administered in a precise dosage. The pharmaceutical composition may further comprise an active ingredient.

[0077] All formulations of the pharmaceutical compositions disclosed herein can be prepared by conventional methods in the pharmaceutical field. For example, the active ingredient can be mixed with one or more excipients to prepare the desired formulation. The term "pharmaceutical acceptable excipient" refers to a conventional pharmaceutical carrier suitable for the desired pharmaceutical formulation, such as: diluents, vehicles such as water, various organic solvents, fillers such as starch and sucrose, binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone (PVP); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as hexadecanol; absorbent carriers such as kaolin and soap clay; and lubricants such as talc, calcium stearate, magnesium stearate and polyethylene glycol. The pharmaceutical composition may further include other pharmaceutical acceptable excipients, such as dispersants, stabilizers, thickeners, complexing agents, buffers, penetration enhancers, polymers, flavors, sweeteners and dyes.

[0078] The term "disease" refers to any illness, ailment, disease, symptom, or symptom, and is interchangeable with the terms "disorder" or "condition."

[0079] Abbreviation: AcOH_Acetic acid AEs - Adverse events BID_2 times a day CLL_Chronic Lymphocytic Leukemia Con._Concentrated D-DBTA_(2S,3S)-Dibenzoyltartaric acid DDQ_2,3-Dichloro-5,6-dicyano-1,4-benzoquinone DCM_Dichloromethane DIEA_N,N-Diisopropylethylamine DLBCL_Diffuse large B-cell lymphoma DMAc_N,N-Dimethylacetamide DMF_N,N-Dimethylformamide DMF-DMA_N,N-Dimethylformamide Dimethyl Acetal DMSO - Dimethyl sulfoxide DSC - Differential Scanning Calorimetry DVS_Dynamic Vapor Sorption EA_Ethyl acetate, EtOAc EDCI_1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EtOH_ethanol FL_Follicular lymphoma GC_Gas chromatograph GCMS_Gas Chromatography-Mass Spectrometry HOAc_Acetic acid HOBt_Hydroxybenzotriazole HPLC - High performance liquid chromatography IPA_Isopropyl alcohol IPAc_Isopropyl acetate IPC_In-Process Control KF_Karl Fischer L-DBTA_(2R,3R)-Dibenzoyltartaric acid LOQ - Limit of quantification MCL_Mantle cell lymphoma MeCN or ACN_Acetonitrile MeMgBr - Methylmagnesium bromide MeOH_methanol 2-MeTHF_2-Methyltetrahydrofuran MIBE_4-Methyl-2-pentanone MsOH_methanesulfonic acid MTBE - Methyl tertiary butyl ether NHL_Non-Hodgkin's Lymphoma NLT_ or more (not less than) NMP_1-Methyl-2-pyrrolidone NMR_Nuclear magnetic resonance Not more than NMT_ ORR_Overall response rate Pd_Palladium pH_hydrogen ion concentration POA_Phenoxyacetyl QD_Once a day RH_Relative humidity SLL_Small lymphocytic lymphoma RT_room temperature TEA_Triethylamine TGA_Thermogravimetric analysis THF - Tetrahydrofuran TN_No experience with treatment VGPR - very good partial response XRPD - X-ray powder diffraction WM Waldenstrom macroglobulinemia EXAMPLES

[0080] The present invention is further illustrated by the following examples which are illustrative of the invention but are not intended to be limiting. Example 1 Preparation of (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 1) and its Crystalline Form A Step 1: Synthesis of BG-2 [ka]

[0081] Under nitrogen atmosphere, TEA (2.4 equiv.) was added to a solution of EA (5 v), HOBT (1.2 equiv.), EDCI (1.2 equiv.), 4-phenoxybenzoic acid (BG-1, 80 Kg, 1.0 equiv.) and malononitrile (1.2 equiv.) at 10°C. The mixture was then stirred at room temperature until the reaction was complete. The mixture was then centrifuged and the cake was washed with EA. The filtrate was washed twice with aqueous NaHCO3 and then with aqueous NH4Cl. The organic phase was washed twice with 1.5N H2SO4 and stirred. It was concentrated and precipitated from methanol and purified water. The solid was collected by centrifugation and then dried under vacuum. This gave 79.9 Kg of BG-2. 1 H NMR (DMSO-d6) δ 7.62 (d, J = 8.6 Hz, 2H), 7.46-7.38 (m, 2H), 7.18 (t, J = 7.4 Hz, 1H), 7.06 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H).

[0082] Step 2: Synthesis of BG-3 [ka]

[0083] Under nitrogen atmosphere, a solution of BG-2 (79.9 kg, 1.0 equiv.) in MeCN (5.0 v) was added to trimethoxymethane (12.0 v) at 85° C. The resulting mixture was stirred until the reaction was complete. It was sampled for HPLC analysis and concentrated under vacuum. The residue was precipitated with i-PrOH and hexane. The mixture was centrifuged and the cake was washed with hexane and dried under vacuum. This gave 71.7 Kg of product. 1 H NMR (400 MHz, DMSO-d6) δ 7.70 (d, J = 8.4 Hz, 2H), 7.52-7.45 (m, 2H), 7.28 (t, J = 7.6 Hz, 1H), 7.22-7.06 (m, 4H), 3.93 (s, 3H).

[0084] Step 3: Synthesis of BG-4 [ka]

[0085] To a solution of BG-3 (71.6 kg, 1.0 equiv.) in ethanol (2.5 v) under nitrogen atmosphere, hydrazinium hydroxide (1.0 equiv.) dissolved in ethanol (0.6 v) was added dropwise to the reactor at less than 15° C. The solution was heated to room temperature and stirred until the reaction was complete. Water (4.0 v) was added to the reactor. The solution was then cooled to 5° C., centrifuged, and the cake was washed with water (1.0 v). The cake was dried under vacuum. This gave 66.9 Kg of product. 1 H NMR (DMSO-d6) δ 12.11 (br s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.46-7.39 (m, 2H), 7.18 (t, J = 7.6 Hz, 1H), 7.12-7.04 (m, 4H), 6.43 (br s, 2H).

[0086] Steps 4 to 6: Synthesis of BG-8 [ka]

[0087] To a mixture of DCM (8.0 v), BG-5 (80.0 Kg, 1.0 equiv), N,O-dimethylhydroxylamine hydrochloride (1.2 equiv), HOBt (1.2 equiv) and EDCI (1.2 equiv) was added TEA (2.6 equiv) dropwise below 15°C. The mixture was stirred at room temperature until the reaction was complete, centrifuged and the cake was washed twice with DCM (1.0 v). The filtrate was washed with 20% aqueous NH4Cl (3 x 4.0 v). The filtrate was concentrated under vacuum to give the crude product BG-6, which was used in the next step without further purification. The residue was dissolved in toluene (5.0 v) and THF (1.0 v), cooled to 10°C, MeMgBr (1.4 equiv) was added dropwise at 10°C, and then stirred at room temperature until the reaction was complete. The solution was cooled below 10°C. Saturated aqueous NH4Cl was added dropwise below 10°C. The mixture was centrifuged, separated, filtered and then the organic phase was washed twice with aqueous NaCl. The organic phase was concentrated to give the crude product which was used in the next step without further purification. The residue in DMF (2.5 v) and DMF-DMA (2.5 v) was stirred at 110° C. until the reaction was complete. The reaction mixture was cooled, concentrated and then DCM was added. The final mixture was washed with saturated aqueous NH4Cl. The organic layer was concentrated and precipitated by pouring in hexane. The mixture was centrifuged and the cake was collected. The cake was dried under vacuum. This gave 82.2 Kg of the desired product. 1 H NMR (DMSO-d6) δ 7.49 (d, J = 12.6 Hz, 1H), 5.01 (d, J = 12.6 Hz, 1H), 3.99-3.82 (m, 2H), 3.14-2.94 (m, 2H), 2.89-2.61 (m, 6H), 2.49-2.37 (m, 1H), 1.66-1.56 (m, 2H), 1.39 (s, 9H), 1.39-1.20 (m, 2H).

[0088] Step 7: Synthesis of BG-9 [ka]

[0089] Under a nitrogen atmosphere, a mixture of toluene (8.0 v), AcOH (0.5 v), BG-8 (1.2 equiv.) and BG-4 (66.9 Kg, 1.0 equiv.) was heated to 95° C. and stirred until the reaction was complete. The mixture was cooled, concentrated, and then precipitated with methanol. The mixture was centrifuged and the cake was washed with methanol. The cake was dried under vacuum. This gave 107.8 Kg of product. 1 H NMR (DMSO-d6) δ 8.78 (d, J = 4.6 Hz, 1H), 8.15-8.07 (m, 2H), 7.51-7.41 (m, 2H), 7.34 (d, J = 4.6 Hz, 1H), 7.27-7.19 (m, 3H), 7.17-7.10 (m, 2H), 4.24-4.02 (m, 2H), 3.81-3.69 (m, 1H), 3.12-3.82 (m, 2H), 2.15-2.04 (m, 2H), 1.76-1.60 (m, 2H), 1.43 (s, 9H).

[0090] Step 8: Synthesis of BG-10 [ka]

[0091] Hydrogen gas was introduced into the reactor with Pd / C (10% w / w) to a mixture of THF (10.0 v), BG-9 (13.0 Kg, 1.0 equiv.) and D-DBTA (1.0 equiv.) under N2 and hydrogen pressure was maintained at 1.8 MPa. The reactor was heated to 40° C. over time and stirred until the reaction was complete. The mixture was then cooled, filtered and the cake was washed with THF. The filtrate was collected and then concentrated under vacuum. DCM was added. The residue was washed with NaHCO3 water, concentrated, precipitated with MTBE and hexane, and then centrifuged. The cake was collected and dried under vacuum to give the desired compound (yield: 94.8% and purity: 98.5%). 1H NMR (DMSO-d6) δ 7.82-7.76 (m, 2H), 7.56-7.51 (m, 1H), 7.45-7.37 (m, 2H), 7.21-7.14 (m, 1H), 7.12-7.03 (m, 4H), 4.09-3.91 (m, 3H), 3.30-3.22 (m, 2H), 2.82-2.55 (m, 2H), 2.18-1.99 (m, 2H), 1.98-1.86 (m, 1H), 1.69-1.58 (m, 1H), 1.56-1.45 (m, 1H), 1.38 (s, 9H), 1.32-1.13 (m, 2H).

[0092] Step 9: Synthesis of BG-11 [ka]

[0093] To a solution of BG-10 (100.0 Kg, 1.0 equiv.) in DCM (6.0 v) under nitrogen atmosphere was added HCl in EtOH (20.9% w / w, 2.0 v) dropwise. The mixture was stirred until the reaction was complete. MTBE (4.0 v) was added to the solution and cooled. The cake was collected by centrifugation and washed with hexane (2.0 v), then the cake was slurried in hexane (5 v) and centrifuged again. The cake was washed with hexane (2.0 v) and dried under vacuum. This gave 85.2 Kg of product. 1 H NMR (DMSO-d6) δ 9.25-8.85 (m, 2H), 7.84-7.70 (m, 2H), 7.47-7.37 (m, 2H), 7.18 (t, J = 7.4 Hz, 1H), 7.12-7.03 (m, 4H), 5.73 (br s, 2H), 4.12-4.03 (m, 1H), 3.25-3.19 (m, 4H), 2.90-2.73 (m, 2H), 2.28-2.12 (m, 1H), 2.10-2.00 (m, 1H), 1.99-1.86 (m, 1H), 1.84-1.52 (m, 4H).

[0094] Step 10: Synthesis of BG-11A [ka]

[0095] A mixture of BG-11 (85.0 Kg, 1.0 equiv.) in water (6.0 v) and NaOH (3.0 equiv.) was stirred at room temperature until the reaction was complete. The cake was collected and then slurried in MTBE (6.0 v). The mixture was then centrifuged to collect the cake. The cake was dried under vacuum. This gave 71.3 Kg of product. 1 H NMR (DMSO-d6) δ 7.82-7.74 (m, 2H), 7.54-7.49 (m, 1H), 7.45-7.38 (m, 2H), 7.21-7.14 (m, 1H), 7.12-7.04 (m, 4H), 4.03-3.95 (m, 1H), 3.29-3.21 (m, 2H), 3.00-2.87 (m, 2H), 2.46-2.31 (m, 2H), 2.11-1.83 (m, 3H), 1.58-1.12 (m, 4H).

[0096] Step 11: Synthesis of BG-11B [ka]

[0097] A mixture of ethanol / water / acetic acid (7:3:1, 46 v) and BG-11A (30 kg, 1.0 equiv.) in a reactor was heated to 70±5°C under nitrogen atmosphere, and then a solution of D-DBTA (1.20 equiv.) in ethanol / water / acetic acid (7:3:1, 4 v) was added dropwise at a temperature above 65°C. The resulting solution was stirred at 60-65°C for 16 h and then cooled to room temperature. The solid was collected by centrifugation and then washed with ethanol (2.0 v). The cake was slurried in a mixed solvent of ethanol / water / AcOH (7:3:1, 20 v) at 55°C for 16 h and cooled to room temperature. The solid was collected by centrifugation and washed with ethanol (2.0 v). The cake was dried under vacuum (yield: 37.9%). 1H NMR (DMSO-d6) δ 8.76 (br s, 2H), 7.99-7.89 (m, 4H), 7.83-7.75 (m, 2H), 7.66-7.57 (m, 3H), 7.52-7.45 (m, 4H), 7.45-7.39 (m, 2H), 7.21-7.14 (m, 1H), 7.13-7.03 (m, 4H), 5.64 (s, 2H), 4.08-4.00 (m, 1H), 3.29-3.19 (m, 4H), 2.85-2.72 (m, 2H), 2.21-1.40 (m, 7H).

[0098] Step 12: Synthesis of BG-11C [ka]

[0099] BG-11B (48.0 kg, 1.0 equiv.) was added batchwise to a mixture of dichloromethane (15.0 v) and 20.0% aqueous KOH (3.0 v) under nitrogen atmosphere at room temperature. After the reaction was complete, the organic layer was collected and the aqueous layer was extracted with dichloromethane (5.0 v). The organic layers were combined. Con. HCl (0.36 v) was added to the above organic layer at room temperature. The resulting mixture was stirred until the reaction was complete. The solid was collected by centrifugation and then washed with dichloromethane (1.0 v). The collected solid was slurried with MTBE (6.0 v). The solid was collected by centrifugation and then washed with MTBE (1.0 v) and then dried under vacuum. This gave 31.5 Kg of product (yield: 100%).

[0100] Step 12: Synthesis of BG-11D (alternative intermediate) ACN (5.0 v), soft water (10.0 v), KOH (5.0 equiv.) were charged to the reactor and stirred for at least 15 min. BG-11B (1.0 equiv.) was charged to the reactor in small portions. The mixture was stirred until the reaction was complete. The cake was collected by centrifugation, slurried in ACN (1.0 v) and soft water (5.0 v), and then dried under vacuum to obtain the product.

[0101] Step 13: Synthesis of BG-12 [ka]

[0102] A solution of BG-11C (15.0 Kg 1.0 equiv.) in MsOH (2.5 v) was stirred at 85°C under nitrogen atmosphere until the reaction was complete. After cooling to 5°C. Purified water (4.0 v) was added dropwise to the system to keep the temperature below 35°C (the temperature increased obviously). The resulting solution was stirred at 30°C for 16 h and then washed with DCM (2 x 3.0 v). The aqueous phase was collected. DCM (6.0 v) was added to the aqueous phase and the mixture was cooled to 5°C. The pH value was adjusted to 11-12 with 20% NaOH aqueous solution (the temperature increased obviously) while stirring at a temperature below 30°C. The organic phase was separated and collected. The aqueous phase was extracted with DCM (3.0 v). The organic layers were combined and concentrated. MTBE (4.0 v) was added to the residue. The mixture was then concentrated and precipitated with n-heptane. The solid was collected by centrifugation and then dried in a vacuum oven, which gave 12.55 Kg of product (yield: 94.9%). 1 H NMR (DMSO-d6) δ 7.52-7.46 (m, 2H), 7.45-7.38 (m, 2H), 7.21-7.13 (m, 1H), 7.12-7.03 (m, 4H), 6.64 (s, 1H), 3.99-3.90 (m, 1H), 3.29-3.22 (m, 2H), 3.03-2.90 (m, 2H), 2.48-2.36 (m, 2H), 2.03 (dd, J = 13.9, 5.6 Hz, 2H), 2.14-1.99 (m, 1H), 1.97-1.85 (m, 1H), 1.65-1.15 (m, 3H).

[0103] Step 14: Synthesis of BG-13 [ka]

[0104] A mixture of MeOH (13.5 v), purified water (4.5 v) and BG-12 (8.5 Kg, 1.0 equiv.) in a reactor was heated to 50° C. under N2 atmosphere. A solution of L-DBTA (0.7 equiv.) in MeOH / purified water (1.5 v / 0.5 v) was added dropwise to the mixture while maintaining the temperature at 50° C. After addition, the mixture was stirred at 50° C. for at least 2 h, then cooled to room temperature and stirred at room temperature for at least 16 h. The cake was collected by centrifugation and then washed with MeOH (2.0 v). The cake was dried in a vacuum oven. This gave 9.08 Kg of product (yield: 74.8%, ee value > 98%).

[0105] Step 15: Synthesis of (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 1) [ka]

[0106] Under N2 atmosphere, ACN (12.0 v), water (12.5 v), BG-13 (8.0 Kg, 1.0 equiv.), and NaHCO3 (2.5 equiv.) were added to the reactor. The mixture was then cooled to -5 to 0°C. To the mixture, a solution of acryloyl chloride (1.1 equiv.) in MeCN (0.5 v) was added dropwise and stirred until the reaction was complete. EA (6.0 v) was then added to the reactor followed by stirring. The organic phase was collected. The aqueous layer was further extracted with EA (3.0 v). The combined organic phase was washed with brine. The organic layer was collected and concentrated.

[0107] The residue was purified on a silica gel (2 wt) column and eluted with 3% w / w methanol in DCM (21.0 v). The solution of compound 1 was collected and then concentrated under vacuum. The residue was precipitated with EA / MTBE (2.0 v). The cake was collected as the product by centrifugation.

[0108] Step 15: Synthesis of (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 1, alternative method) [ka]

[0109] A mixture of CH3CN (10.0 v), purified water (5.0 v), NaOH (1.5 eq.) and BG-13 (1.0 eq.) was stirred to obtain a clear solution. Then EtOAc (6.0 v) was added to the reaction, which was then separated. The organic phase was collected and then washed twice with 15% brine (3.0 v). The organic phase prepared above was concentrated and solvent exchanged into CH3CN (residual amount: NMT 5.0 v). CH3CN (7.5 v) and purified water (12.5 v) were added, which was then cooled to 15-20 °C. L-(+)-tartaric acid (0.5 eq.) and NaHCO3 (2.5 eq.) were added to the reaction mixture. A solution of acryloyl chloride (1.1 eq.) in CH3CN (0.5 v) was added dropwise to the reaction mixture. After the reaction was completed, EtOAc (6.0 v) was added to the reaction mixture, and the organic layer was collected. The aqueous phase was further extracted with EA (3.0 v). The organic layers were combined, washed with 15% brine (5.0 v) and then concentrated. The solvent was exchanged into DCM (volume of residue: 1.5-2.0 v) and purified on a silica gel column (silica gel: 100-200 mush, 2.0 w / w; eluent: DCM containing 3% w / w MeOH (ca. 50 v)). The collected solution was concentrated and then exchanged into EtOAc (4.0 v); MTBE (6.4 v) was added dropwise to the residue at 50° C., then the mixture was cooled to 5° C., and the cake was collected by centrifugation.

[0110] Step 16: Preparation of Crystalline Form A of Compound 1 The above cake of compound 1 was dissolved in 7.0 volumes of DCM and then solvent exchanged into EA. After recrystallization from EA / MTBE, the cake was collected by centrifugation and then dried under vacuum. This gave 4.44 Kg of product (yield: 70.2%).

[0111] The product was then characterized by X-ray powder diffraction (XRPD) pattern analysis performed on a PANalytical Empyrean X-ray powder diffractometer with the following XRPD parameters: X-Ray wavelength (Cu, kα, Kα1 (Å): 1.540598, Kα2(Å): 1.544426; Kα2 / Kα1 intensity ratio: 0.50); ​​X-Ray tube setting (45 Kv, 40mA); divergence slit (automatic); scan mode (Continuous); scan range (°2TH) (3°-40); step size (°2TH) (0.0131); scan speed (° / min) (about 10). The XRPD results showed that the product obtained was a crystal as shown in Figure 1.

[0112] The differential scanning calorimetry (DSC) curve shown in Figure 2 was generated on a TA Instruments TA Q2000 DSC. The DSC parameters used were: temperature (25°C-desired temperature); heating rate (10°C / min); method (ramp); sample pan (aluminum, crimped); purge gas (N2). The DSC results showed a sharp melting point at 139.4°C (onset temperature).

[0113] The thermogravimetric analysis (TGA) curve shown in Figure 3 was generated on a TA Instruments TA Q5000 TGA. The TGA parameters used were: temperature (RT-desired temperature); heating rate (10oC / min); method (ramp); sample pan (platinum, open); purge gas (N2). The TGA results showed no weight loss up to 110°C, indicating anhydrous nature.

[0114] The proton nuclear magnetic resonance (1 1 H NMR was measured in DMSO-d6 on a Bruker 400M NMR spectrometer. 1 H NMR (DMSO-d6) δ 7.50 (d, J = 8.6 Hz, 2H), 7.46-7.38 (m, 2H), 7.17 (t, J = 7.6 Hz, 1H), 7.08 (d, J = 7.6 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 6.85-6.72 (m, 1H), 6.67 (s, 1H), 6.07 (dd, J = 16.8, 2.2 Hz, 1H), 5.64 (dd, J = 10.4 Hz, 2.2 Hz, 1H), 4.55-4.38 (m, 1H), 4.17-3.94 (m, 2H), 3.33-3.22 (m, 2H), 3.08-2.88 (m, 1H), 2.67-2.51 (m, 1H), 2.36-2.15 (m, 1H), 2.12-1.82 (m, 2H), 1.79-1.65 (m, 1H), 1.63-1.49 (m, 1H), 1.38-1.08 (m, 2H).

[0115] As shown in FIG. 5, the carbon nuclear magnetic resonance ( 13 C-NMR) was measured in DMSO-d6 using a Bruker 400M NMR spectrometer. 13 C-NMR spectrum.

[0116] Example 2 Preparation of Crystal Form A of Compound 1 (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 1) was prepared by the method disclosed in WO2014173289A and further lyophilized to obtain compound 1 in amorphous form. A solution of compound 1 (200 mg, ee value >97%) in EA (8 mL) was heated to 50° C. to obtain the above solution. Hexane (8 mL) was added dropwise at 50° C. The mixture was cooled to room temperature and stirred for 16 h, then filtered to obtain 110 mg as a white solid. The obtained solid was identified as Form A by XRPD.

[0117] Example 3 Preparation of Crystal Form A of Compound 1 (Anti-solvent Addition) Approximately 15 mg of sample (crystalline form A) was weighed into a 20 mL glass vial, followed by the addition of 0.4-1.2 mL of the corresponding solvent (see Table 2) to dissolve all solids. The mixture was then magnetically stirred at a speed of 800 rpm to obtain a clear solution at room temperature. The relative anti-solvent (see Table 2) was then added to the solution to induce precipitation or until a total amount of anti-solvent reached 15.0 mL. If no precipitation occurred, the solution was then transferred to slow evaporation at room temperature. The resulting solid was identified as Form A by XRPD.

[0118] Table 2. Antisolvent addition test [Table 2]

[0119] Example 4 Preparation of Crystal Form A of Compound 1 (Solution Vapor Diffusion) Approximately 15 mg of sample (crystalline form A) was dissolved in 0.5-1.5 mL of the corresponding solvent (acetone or EtOAc) to obtain a clear solution in a 3 mL vial. The solution was subsequently placed in a 20 mL vial containing 3 mL of a relative poor solvent (n-heptane). The 20 mL vial was sealed with a cap and kept at room temperature, allowing sufficient time for the organic vapors to interact with the solution. At the end of the 11th day, the clear solution was converted to evaporation at room temperature. The resulting solid was identified as Form A by XRPD.

[0120] Example 5 Stability Test of Crystal Form A of Compound 1 and Purity of Compound 1 (1) Physical stability test As a thermal stability test, crystalline form A of compound 1 was stored at 80° C. for 2 days, and the XRPD pattern showed no change in the crystalline form before and after the test.

[0121] Long-term stability studies of crystalline form A of compound 1 showed that no significant changes in chemical purity occurred when stored at 25° C. / 60% RH for 24 months (% area: T0=99.2% and T12=99.2%) or at 40° C. / 75% RH for 6 months (% area: T0=99.1% and T6=99.4%). Furthermore, no changes in crystal form and optical purity were observed when stored at 25° C. / 60% RH for 24 months or at 40° C. / 75% RH for 6 months.

[0122] (2) Moisture absorption test The Dynamic Vapor Sorption (DVS) plot shown in Figure 6 was collected by a SMS (surface measurement system), DVS Intrinsic. The DVS parameters used were: temperature (25oC); dm / dt (0.002% / min); Min. dm / dt stability duration (10 min); Max. equilibrium time (180 min); RH range (0%RH to 95%RH); RH step size (10%RH from 0%RH to 90%RH, 5%RH from 90%RH to 95%RH). As shown in Figure 6, there was a very small mass gain at 80%RH, which was about 0.8% for the crystalline form A of compound 1.

[0123] (3) Crystallization / recrystallization from Form A to improve the purity of Compound 1. Crystallization / recrystallization from Form A is an effective method to improve the purity of Compound 1 and further suppress impurities in Compound 1 to meet the acceptance criteria herein. See examples shown in Table 3.

[0124] Table 3 Purity change after crystallization / recrystallization with Form A [Table 3]

[0125] Example 6 Preparation of deuterium-labeled (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (deuterium-labeled compound 1) [ka]

[0126] To a solution of acryloyl-2,3,3-d3 acid (50 mg, 0.67 mmol) and DMF (1 drop) in DCM (20 mL) was added oxalyl chloride (1.6 N, 40.9 mL, 65.5 mmol) dropwise at 0-5° C. and stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure to give crude acryloyl-d3 chloride.

[0127] A solution of (S)-2-(4-phenoxyphenyl)-7-(piperidin-4-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (dissociated from BG-13, step 15, compound 1, see alternative method; 278 mg, 0.67 mmol) in DCM (20 mL) and an aqueous solution of NaHCO3 (10 mL) was added dropwise to a solution of the above acryloyl-d3 chloride in DCM (5 mL) at 0-5 °C and stirred at room temperature for 2 h. The combined organic layers were dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by preparative TLC to give 55 mg (17.5%) of (S)-7-(1-(acryloyl-d3)piperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide as an off-white solid. 1 H NMR (400 MHz, DMSO) δ 7.50-7.44 (m, 2H), 7.42-7.35 (m, 2H), 7.17-7.10 (m, 1H), 7.09-6.99 (m, 4H), 6.64 (s, 1H), 4.52-4.40 (m, 1H), 4.10-3.95 (m, 2H), 2.29-3.25 (m, 2H), 3.04-2.86 (m, 1H), 2.63-2.50 (m, 1H), 2.32-2.13 (m, 1H), 2.06-1.81 (m, 2H), 1.75-1.45 (m, 2H), 1.35-1.08 (m, 2H). MS (ESI, m / e) [M+1] + 475.2.

[0128] Example 7 Polymorphic Study of Compound 1 (1) Study of polymorphism from amorphous form - Preparation of Form A from amorphous compound 1 (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide was prepared according to the method disclosed in WO2014173289A and further lyophilized to obtain compound 1 in amorphous form.

[0129] For each experiment in Tables 4a-4k, 5a-5e and 6, about 20 mg of compound 1 in amorphous form was weighed into a glass vial, followed by the addition of the corresponding solvent. If necessary, the mixture was heated to obtain a clear solution. The mixture was then kept at room temperature without stirring for 1-2 days, at which point the solid matter formed from the clear solution became visible. The solid matter was observed under a polarizing microscope.

[0130] Table 4 Compound 1 as starting material (ee value = 90%) [Table 4a]

[0131] [Table 4b]

[0132] [Table 4c]

[0133] [Table 4d]

[0134] [Table 4e]

[0135] Table 4f

[0136] [Table 4g]

[0137] [Table 4h]

[0138] [Table 4i]

[0139] [Table 4j]

[0140] [Table 4k] Y=Yes, and N=No.

[0141] Table 5. Compound 1 as starting material (ee value = 97%) [Table 5a]

[0142] [Table 5b]

[0143] [Table 5c]

[0144] [Table 5d]

[0145] [Table 5e] Y=Yes, and N=No.

[0146] The experiments in Tables 4a-4k and Tables 5a-5e were carried out on the same scale (i.e., the amount of starting material-amorphous Compound 1 was about 20 mg). However, the ee value of the starting material appeared to have a significant effect on the amount of solid to be formed in each experiment. The experiments in Tables 4a-4k started with 90% ee of amorphous Compound 1, thus forming small amounts of solid. The experiments in Tables 5a-5e started with 97% ee of amorphous Compound 1, yet yielded a very large amount of solid. Also, the solids obtained in the experiments in Tables 4a-4k had low ee values ​​when crystallized from the 90% ee starting material. One of the solid samples from Tables 4a-4k used EA / Hexane as the crystallization system, but had an ee value of only 45%.

[0147] The results in Table 5a were further confirmed by a scale-up experiment similar to Example 2, which confirmed that the resulting solid was the desired crystalline form (Form A).

[0148] As shown above in Tables 4a-4k and Tables 5a-5e, the formation of crystalline solids can vary depending on the particular solvent, ratio of solvents, etc.

[0149] The results in Table 6 further support the dependence of crystalline solid formation on specific solvent ratios. [Table 6]

[0150] (2) Study of polymorphism from crystalline form - Preparation of Form A from crystalline form Slow evaporation Approximately 15 mg of sample (form A) was weighed into a 3 mL glass vial, followed by addition of the corresponding solvent or solvent mixture (see Table 7) to obtain a clear solution. The vial was then covered with parafilm with 3-4 pinholes and kept at room temperature to allow the solution to evaporate slowly. The solid was isolated for XRPD analysis. However, no crystalline form was produced, as summarized in Table 7.

[0151] Table 7. Evaporation experiment over time [Table 7]

[0152] Antisolvent addition Approximately 15 mg of sample (crystalline form A) was weighed into a 20 mL glass vial, followed by the addition of 0.4-1.2 mL of the corresponding solvent (see Table 8). The mixture was then magnetically stirred at a speed of 800 rpm to obtain a clear solution at room temperature. The relative anti-solvent (see Table 8) was then added to the solution to induce precipitation or until the total amount of anti-solvent reached 15.0 mL. If no precipitation occurred, the solution was then allowed to evaporate slowly at room temperature. The results are summarized in Table 8.

[0153] Table 8 Antisolvent addition experiment [Table 8] N / A: No solids were obtained.

[0154] Slow cooling Approximately 20 mg of sample (crystalline form A) was suspended in 1.0 mL of the corresponding solvent (see Table 9) in a 3 mL glass vial at room temperature. The suspension was transferred to a slurry at 50° C. with magnetic stirring at a speed of 800 rpm. The sample was equilibrated at 50° C. for 2 h and filtered using a 0.45 μm nylon membrane. The filtrate was then slowly cooled from 50° C. to 5° C. at a rate of 0.1° C. / min. The resulting solid was kept isothermal at 5° C. until isolation for XRPD analysis. As summarized in Table 9, no crystalline forms were obtained.

[0155] Table 9. Long-term cooling experiment [Table 9] N / A: No solids were obtained. *: The clear solution was decanted and evaporated at room temperature.

[0156] Solution Vapor Diffusion Approximately 15 mg of sample (crystalline form A) was dissolved in 0.5–1.5 mL of the corresponding solvent (see Table 10) to obtain a clear solution in a 3 mL vial. The solution was subsequently placed in a 20 mL vial containing 3 mL of the relative antisolvent. The 20 mL vial was sealed with a cap and kept at room temperature to allow sufficient time for the organic vapors to interact with the solution. At the end of the 11th day, the clear solution was transferred to evaporation at room temperature. The resulting solid was identified by XRPD. The results are summarized in Table 10.

[0157] Table 10. Solution vapor diffusion experiment [Table 10] N / A: No solids were obtained. *: A solid was produced by evaporation over a long period of time.

[0158] Polymer-induced crystallization experiments Approximately 15 mg of sample (crystalline form A) was dissolved in 1.0 mL of the corresponding solvent (see Table 11) to obtain a clear solution in a 3 mL vial. The solution was then filtered using a 0.45 μm nylon membrane. Approximately 2 mg of the polymer mixture was added to the filtrate. The mixture was stirred at room temperature to induce precipitation. The solid was isolated for XRPD analysis. No crystalline form was obtained, as summarized in Table 11.

[0159] Table 11 Polymer-induced crystallization experiments [Table 11] Polymer mixture A: polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyvinyl chloride (PVC), polyvinyl acetate (PVAC), hypromellose (HPMC), methylcellulose (MC) (mass ratio 1:1:1:1:1:1) Polymer mixture B: polycaprolactone (PCL), polyethylene glycol (PEG), poly(methyl methacrylate) (PMMA), sodium alginate (SA), and hydroxyethyl cellulose (HEC) (mass ratio 1:1:1:1:1).

[0160] Example 8 Determination of the absolute configuration of compound 1 Preparation of single crystals of BG-13 Single crystal growth experiments (see Table 12) were carried out six times by slow cooling method. Single crystals of BG-13 were obtained in the desired form by slow cooling in MeOH / H2O (1:1, v / v). The crystal data and refined structure are listed in Table 13.

[0161] Table 12 Single crystal growth experiment [Table 12]

[0162] Table 13. Single crystal data and refined structure of BG-13 Single crystal data were generated on a Bruker APEX DUO single crystal diffractometer equipped with a CCD detector (Cu Kα, λ=1.54178 Å, 173.15 K). [Table 13]

[0163] BG-13 was confirmed to be the (2R,3R)-dibenzoyltartaric acid (L-DBTA) salt, and the molar ratio of the free base to L-DBTA was 2:1. The configuration of both carbons (C32 and C32') in L-DBTA was confirmed to be R. The configuration of C6 in the free base was determined to be S, as shown in Figures 8 to 10. The structure of the single crystal was also identified using powder X-ray diffraction patterning, as shown in Figure 11.

[0164] Absolute configuration of compound 1 The absolute configuration of compound 1 was estimated to be S based on the single crystal X-ray structure analysis of intermediate BG-13.

[0165] Example 9 Chiral Resolution of BG-11A [ka]

[0166] Common procedure: Chiral acid was added at elevated temperature to a solution of compound BG-11A in the prepared solvent system. After stirring at this temperature, it was cooled to room temperature and then stirred at room temperature overnight. The solid was filtered and then washed with the prepared solvent system. The ee values ​​were tested by chiral HPLC directly on the relevant salts or their Boc derivatives (see Table 14). With other chiral acids or solvent systems, chiral compounds with no ee value, low ee value, or undesired chiral compounds were observed.

[0167] Table 14 Chiral resolution of BG-11A [Table 14]

[0168] Example 10 Chiral Resolution of BG-12A and Improvement of Chiral Purity [ka]

[0169] Common procedure: Chiral acid was added to a solution containing compound BG-12A in the prepared solvent system at elevated temperature. After stirring at this temperature, it was cooled to room temperature and then stirred at room temperature overnight. The solid was filtered and then washed with the prepared solvent system. Chiral purity was tested by chiral HPLC directly on the relevant salt or free base (see Table 15). With other chiral acids or solvent systems, chiral compounds with no ee value, low ee value, or undesired chiral compounds were observed.

[0170] Table 15 Chiral resolution of BG-12A [Table 15]

[0171] The resulting L-DBTA salt (31 g, 85.6% ee) was added to THF / HO (1 / 1, 1034 mL) and the suspension was warmed to 70° C. and stirred until all solids were dissolved. 517 mL of water was then added. The solution was then slowly cooled to 40° C. and seed crystals (10 mg) were added. After stirring for about 2 h, the solution was slowly cooled to ambient temperature and then stirred for 2 days. After filtration, the solid was washed with THF / HO=1 / 1 (20 mL) and dried under vacuum to give the product as a white solid (22.5 g, 72% yield, >98.5 ee value).

[0172] The resulting free base (6.02 g, 79.1% ee) was dissolved (1 g / 15 mL) in EtOH / H2O (6 / 1, 90 mL) and stirred at 78 °C to dissolve all starting material. Then, a solution of L-DBTA (2.84 g, 7.9 mmol, 0.55 equiv) in EtOH / H2O (6 / 1, 7 mL) was added. A solid formed rapidly and the mixture was stirred at this temperature for 1 h before removing the heating system. The mixture was cooled to room temperature. It was filtered and the solid was washed with EtOH / H2O (6 / 1, 10 mL). The collected solid was converted to the free base using aqueous NaOH and DCM to give the product as a white foam (4.7 g, yield: 32.6%, 93% ee).

[0173] A suspension of the resulting free base (70.0 g, 90.5% ee) in CH3CN / H2O (1 / 1, 700 mL) was heated to 60 °C to obtain a clear solution. To the above solution was then added L-DBTA (33 g, 0.55 equiv.). After stirring at 60 °C for about 2 h, the mixture was slowly cooled to room temperature and stirred overnight. After filtration, the solid was washed with CH3CN / H2O (1 / 1, 50 mL) and dried under reduced pressure to give the product as an off-white solid (80 g, yield: 80%, ee value >98%).

[0174] Example 11 Efficacy Testing (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide was subsequently tested using its crystalline form A.

[0175] Test 1: Kinase inhibition and selectivity method: (1) BTK kinase enzyme assay Crystal form A of compound 1 was synthesized by BTK quinone The kinase was tested for inhibition of the BTK kinase (aa2-659, Carna Biosciences) by a test based on time-resolved fluorescence resonance energy transfer (TR-FRET) methodology. The test was performed in a 384-well small volume black plate in a reaction mixture containing BTK kinase, 5 μM ATP, 2 μM peptide substrate and 0-10 μM compound in a buffer containing 50 mM Tris pH 7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DTT, 0.005% Tween-20, 20 nM SEB and 0.01% BSA. The kinase was incubated with the compound for 60 min at room temperature and the reaction was started by adding ATP and peptide substrate. After 60 min at room temperature, an equal volume of stop / detection solution was added according to the manufacturer's instructions (CisBio Bioassays). The stop / detection solution contained Eu 3+ The plates contained a cryptate-conjugated mouse monoclonal antibody (PT66) anti-phosphotyrosine and XL665-conjugated streptavidin in a buffer containing 50 mM HEPES pH 7.0, 800 mM KF, 20 mM EDTA and 0.1% BSA. The plates were sealed and then incubated at room temperature for 1 h, after which the TR-FRET signal (ratio of fluorescence emission at 665 nm to emission at 620 nm with excitation at a wavelength of 337 nm) was recorded on a PHERStar FS plate reader (BMG Labtech). Phosphorylation of the peptide substrate results in the binding of the anti-phosphotyrosine antibody to the biotinylated peptide substrate, where a fluorescent donor (Eu 3+The close proximity of the donor fluorophore (cryptate) to the acceptor (streptavidin-XL665) results in a high degree of fluorescence resonance energy transfer from the donor fluorophore (620 nm) to the acceptor fluorophore (665 nm). Inhibition of BTK kinase activity reduced the TR-FRET signal. The IC of compound 1 was determined by fitting the data to a four-parameter logistic equation using Graphpad Prism software. 50 This led to the conclusion.

[0176] (2) Biochemical kinase selectivity To characterize the selectivity of crystal form A, a panel of 342 kinases at 1 μM was used at Reaction Biology Corp. Crystal form A showed less than 70% inhibition against 329 kinases and more than 70% inhibition against 13 kinases, including BTK. The IC of crystal form A 50 (see Table 13) were performed in-house at BeiGene, but further included testing of ITK, TEC, JAK3 and EGFR, using TR-FRET assays and corresponding peptides as substrates.

[0177] ITK's IC 50 Determination of: The procedure of the ITK test was similar to that of the BTK test, except for the following modifications: 3 μM ATP and 2 μM TK substrate were used in the kinase reaction.

[0178] TEC IC 50 Determination of TEC: The procedure of the TEC test was similar to that of the BTK test, except for the following modifications: 1) 280 μM ATP and 2 nM Poly-GT substrate were used in the kinase reaction; 2) No SEB was included in the reaction buffer.

[0179] JAK3 IC 50Determination of: The procedure of JAK3 test is similar to that of BTK test, except for the following changes: 1) 3.4 μM ATP and 3 μM peptide substrate (B-EE-15, Biotin-EQEDEPEGDYFEWLE) were used for the kinase reaction. 2) The reaction buffer contains 50 mM Tris pH 7.8, 10 mM MgCl2, 5 mM DTT, 0.01% TritonX-100 and 0.01% BSA.

[0180] EGFR IC 50 Determination of: The protocol for EGFR testing is similar to that for BTK testing, except for the following modifications: 1) 20 μM ATP, 1.44 μM TK substrate-biotin (one of the common substrates for tyrosine kinases) and 0-1000 nM compound. 1% DMSO was used as the final concentration for the kinase reaction. 2) The reaction buffer contains 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2.5 mM DTT and 0.1% BSA. 3) The stop / detection solution buffer contains 25 mM HEPES pH 7.5, 400 mM KF, 50 mM EDTA, 0.01% Triton-X100 and 0.1% BSA.

[0181] result: IC for BTK kinase of crystal form A 50 The cytotoxicity of crystalline form A was 0.27 nM. Crystal form A was found to be a potent, specific, and irreversible BTK kinase inhibitor. With regard to its selectivity, when characterized at 1 μM on a panel of 342 human kinases, crystal form A inhibited only 13 other kinases by 70% or more.

[0182] Table 16. Enzyme inhibitory activity of crystalline form A [Table 16] Note: BTK, EGFR, ITK, TEC and JAK3 studies were performed by using TR-FRET assay and corresponding peptides as substrates. IC of crystal form A was measured after 1 hour of pre-incubation. 50The K of ATP for five kinases M The HER4, BMX, TXK, BLKFGR, LCK, and FRK / PTK5 studies were 33 Using P-ATP and filter binding assays, the IC of crystal form A was measured using a 1-hour pre-incubation. 50 was measured with 1 μM ATP.

[0183] Study 2: BTKpY223 cell study with crystal form A method: The BTKpY223 cell assay is an HTRF-based assay aimed at quantitatively determining the endogenous phosphorylation level at BTK Tyr223. Phosphorylated Tyr223 is required for full activation of BTK. The assay was performed in Ramos cells (CRL-1596, ATCC) using the BTKpY223 assay kit (63IDC000, Cisbio).

[0184] Briefly, Ramos cells were serum-starved for 2 hours in RPMI1640 containing 0.5% FBS. After starvation, cells were incubated with crystalline form A and detected at various concentrations in a CO2 incubator for 1 hour. After incubation, cells were stimulated with 1 mM pervanadate (PV) or Na3VO4 (OV) for 20 minutes. Then, cells were spun down and lysed with 1x lysis buffer (4x lysis buffer provided in the kit) for 10 minutes at room temperature. During incubation, 1x antibody mixture was prepared by diluting anti-BTK-d2 and anti-pBTK-K in detection buffer (supplied in the kit). 2 μl / well of the 1x antibody mixture was dispensed into an OptiPlate-384 test plate (6005620, Perkin Elmer). Then, 18 μL of cell lysate was transferred to the test plate where the antibody solution was previously added. After gentle mixing and a brief spin, the plates were sealed and stored in the dark at room temperature for 18 hours. Fluorescence emission was measured at two different wavelengths (665 nm and 620 nm) using a compatible HTRF reader (PHERAstar FS, BMG). The potency of compound 1 was calculated based on the inhibition of the ratio between the signal intensities at 665 nm and 620 nm. IC was calculated with GraphPad Prism software using a sigmoidal dose-response function. 50 values ​​were calculated.

[0185] result: Crystalline form A inhibited phosphorylation of BTK in the B-cell lymphoma cell line, Ramos, at concentrations as low as 1.8±0.2 nM (n=3).

[0186] Study 3: Effect of crystalline form A on tumor cell proliferation in hematological cancer cell lines (Rec-1, Mino, JEKO-1 and TMD-8) method: Three MCL cell lines (Rec-1, Mino, and JEKO-1) and an ABC type diffuse large B cell lymphoma cell line (TMD8) were used in this study. Cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum / FBS (Thermo Scientific), 100 units / ml penicillin (Gibco) and 0.1 mg / ml streptomycin (Gibco) and maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Cell lines were recovered from frozen stocks that were laid down within 30 passages from the original cells obtained.

[0187] Growth inhibitory activity of compounds in Rec-1, Mino, JEKO-1 and TMD-8 cells was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). The number of cells seeded per well of a 96-well plate was optimized for each cell line to ensure logarithmic growth over the 6-day treatment period. Cells were treated in triplicate with a 10-point dilution series. After 6 days of exposure to compounds, a volume of CellTiter-Glo reagent equal to the volume of cell culture medium present in each well was added. The mixture was mixed for 2 minutes on an orbital shaker to lyse the cells, followed by incubation for 10 minutes at room temperature to allow development and stabilization of a luminescent signal, which represents the amount of ATP, i.e., the amount of metabolically active cells. Luminescent signals were measured using a PHERAstar FS reader (BMG Labtech). IC for cell viability 50 Values ​​were determined using GraphPad Prism software and were the average of three independent experiments.

[0188] result: Crystalline form A of compound 1 exhibited specific and potent inhibitory effects on cell proliferation in three MCL cell lines and an ABC type diffuse large B-cell lymphoma cell line (TMD8) (Table 17). Table 17. Inhibition of hematoma tumor cell proliferation by crystalline form A [Table 17]

[0189] Study 4: Pharmacokinetic study of crystalline form A in mice method: For time course studies, mice were randomly divided into 7 groups, 4 mice per group. Mice were treated with a single dose of crystalline form A of compound 1 and euthanized using carbon dioxide at different time points after dosing (30 min, 1, 2, 4, 12, 24 h). For dose-response studies, mice were randomly divided into 9 groups, 4 mice per group. Mice were treated with different dose levels of crystalline form A of compound 1 and euthanized using carbon dioxide 4 h after dosing. Treatments were administered by oral gavage (orally) in a volume of 10 ml / kg body weight. Body weight was assessed immediately before dosing and the dose was adjusted accordingly.

[0190] PK sample preparation: For time course studies, blood samples (50 μL per mouse) were collected from the retro-orbital sinus under isoflurane / oxygen anesthesia 15 min after dosing (mice from this group were also used at 24 h) or under cardiac puncture after euthanasia at other time points. For dose-response studies, blood samples were collected from the retro-orbital sinus under isoflurane / oxygen anesthesia 30 min after dosing. Plasma was collected by centrifugation at 3,000 g for 10 min and stored frozen at -80°C until analysis.

[0191] PK Analysis: Maximum plasma concentrations (C max ) and time to reach C max (T max ) were obtained directly from the plasma concentration versus time profiles.

[0192] result: Crystalline form A was rapidly absorbed and eliminated in ICR mice.

[0193] Study 5: Efficacy study of crystalline form A in TMD-8 xenograft model Tumor implantation method: Animals were pretreated with cyclophosphamide (150 mg / kg in saline, prepared intraperitoneally) and disulfiram (125 mg / kg in saline with 0.8% Tween 80, given orally, 1 hour after each dose of cyclophosphamide) on each day for 2 days. Animals were then inoculated with TMD-8 cells 24 hours after the second dose of cyclophosphamide. On the day of implantation, the cell culture medium was replaced with fresh medium. After 4 hours, the medium was removed and cells were collected as above. Cells were resuspended in cold PBS (4°C) and an equal volume of Matrigel (BD, Cat. No. 356237) was added to a final concentration of 2.5 × 10 7 The resuspended cells were placed on ice prior to inoculation. The right axillary region of each mouse was washed with 75% ethanol prior to cell inoculation. Each animal was inoculated with 5 × 10 6 200 μl of the cell suspension containing the cells was injected subcutaneously.

[0194] In vivo efficacy testing was started 3 days after cell inoculation, and for this purpose, animals were randomly assigned to the desired number of groups with 10 mice per group. Mice were treated twice daily (BID) with vehicle (0.5% carboxymethylcellulose (CMC) + 0.2% Tween 80) and crystalline form A of compound 1 at different dose levels for 39 days. Treatment was administered by oral gavage (orally) in a volume of 10 ml / kg body weight. Body weight was measured immediately before administration and the dose was adjusted accordingly. Tumor volumes were measured in two dimensions twice weekly using calipers (measurements were possible from day 11 after inoculation in this study). Formula: V = 0.5 × (a × b 2 ) was used to calculate tumor volume, where a and b are the long and short diameters of the tumor, respectively. Statistical analysis was performed using the Student's T-test. P<0.05 was considered statistically significant. One individual was responsible for tumor measurements throughout the entire study. Body weights were also recorded twice weekly. Mice were also monitored daily for clinical signs of toxicity during the study.

[0195] result: The in vivo efficacy of crystalline form A was tested in TMD-8 DLBCL xenografts grown subcutaneously in NOD / SCID mice. Daily oral administration twice daily (BID) was well tolerated at different dose levels, and crystalline form A of compound 1 showed a dose-dependent antitumor effect. Crystalline form A of compound 1 showed strong antitumor activity already at the lowest dose tested. No significant effect on animal body weight was observed throughout the study in all treatment groups.

[0196] Study 6: Efficacy study of crystalline form A in a systemic REC-1 xenograft model Tumor implantation method: Animals were pretreated with cyclophosphamide (150 mpk ip in saline) and disulfiram (125 mpk po, 1 hour after each dose of cyclophosphamide) for 2 days each day. Animals were then inoculated with REC-1 cells 24 hours after the second dose of cyclophosphamide. On the day of implantation, the cell culture medium was replaced with fresh medium. After 4 hours, the medium was removed and cells were collected as above. Cells were resuspended in cold (4°C) PBS and diluted to 1 × 10 8 The final concentration of cells / ml was 1 × 10 7 The cells were injected intravenously via the tail vein in 100 μl of the cell suspension.

[0197] In vivo efficacy testing was to begin 8 days after cell inoculation, for which animals were randomly assigned to the desired number of groups with 10 mice per group. Mice were treated twice daily (BID) with vehicle (0.5% carboxymethylcellulose (CMC) + 0.2% Tween 80) and crystalline form A of compound 1 at different dose levels for 71 days. All treatments were discontinued 78 days after inoculation. Treatments were administered by oral gavage (orally) in a volume of 10 ml / kg body weight. Body weights were assessed immediately before dosing and doses were adjusted accordingly. Body weights were recorded twice weekly (changed to 3 times weekly from day 33). Mice were also observed daily for clinical signs of illness during the study. The study was terminated after the overall survival period. In case of severe toxic effects such as loss of movement, mice were euthanized and recorded as dead.

[0198] For data analysis: Survival analysis was performed by the Kaplan-Meier method. Survival was defined as the time from the date of tumor cell inoculation to the date of death or euthanasia of the animals. For each group, the median survival time (MST), the range of survival times (RST) with 95% confidence intervals, and the extension of life span (ILS) were calculated. Median survival time was defined as the time when 50% of the mice died. ILS was calculated using the following formula: %ILS=(MST-MST(vehicle)) / MST(vehicle)×100 Statistical analysis between groups was performed using the Gehan-Breslow-Wilcoxon test, and P<0.05 was considered statistically significant.

[0199] result: Compound 1, Form A, exhibited dose-dependent antitumor activity against systemic REC-1 MCL xenografts in NOD / SCID mice. The efficacy of Compound 1, Form A, was significant in this xenograft model.

[0200] Test 7: Toxicity of Form A A comprehensive non-clinical toxicology program was conducted, including several 28-day GLP and research studies in rats and dogs, to evaluate the preclinical safety of crystalline form A of compound 1 at different dose levels. These studies followed the available regulatory guidelines for the preclinical development of anticancer drugs. These studies showed a favorable toxicological and safety pharmacological profile of compound 1. No study-related deaths occurred at any dose level throughout the study. No toxicologically significant changes in clinical chemistry or coagulation were observed throughout the study. None of these changes were observed after the recovery phase.

[0201] Study 8: Pharmacokinetics of Crystalline Form A A well-validated LC-MS / MS method was successfully used for single- and multiple-dose pharmacokinetic (PK) studies of crystalline form A of compound 1 in Sprague-Dawley rats and beagle dogs.

[0202] Compound 1 crystalline form A has good oral bioavailability in rats. It was rapidly absorbed in rats and had high plasma clearance (CL). Kinetics were linear across the dose range in female rats. Linearity was less good in male rats. Compound 1 accumulation was not statistically significant after multiple oral doses in both male and female rats. Compound 1 crystalline form A after multiple oral doses in dogs showed moderate clearance (CL), moderately good bioavailability (F%), linear PK across the dose range, and no accumulation of Compound 1.

[0203] Study 9: ADME of Form A Compound 1 was widely distributed in various tissues but was less abundant in brain tissue, indicating that the drug does not readily cross the blood-brain barrier.

[0204] IC of crystalline form A of compound 1 for the seven major drug-metabolizing CYP isoenzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A) 50The IC values ​​were determined in human liver microsomes, and the time-dependent inhibitory potency of compound 1 against the major CYP isoenzymes was also evaluated. 50 = 4.03 μM), CYP2C9 (IC 50 =5.69 μM) and CYP2C19 (IC 50 = 7.58 μM), but the inhibition against other CYP isoenzymes was even lower. Compound 1 seems unlikely to be a time-dependent CYP inhibitor against these seven major human CYPs. CYP3A is the major CYP isoform involved in metabolism in human liver microsomes.

[0205] Example 12: Clinical Trial Study (1) Ongoing Phase I Clinical Trial Results of Compound 1 in Patients with Advanced B-Cell Malignancies The first multicenter, open-label Phase I study of Compound 1 is being conducted in Australia and New Zealand and consists of two parts - a 25-patient dose escalation phase and a dose expansion phase, with a total planned enrollment of 100 patients. The total of 39 patients enrolled includes all 25 patients from the initial dose escalation portion and 14 patients from the ongoing dose expansion portion. Based on the pharmacokinetics, pharmacodynamics, safety, and efficacy of Compound 1 in the dose escalation phase, doses of 320 mg once daily (QD) and 160 mg twice daily (BID) are being further explored in the ongoing dose expansion study.

[0206] By the data analysis cutoff date of October 19, 2015, 29 objective responses had been observed, including 3 complete responses (CR), 1 very good partial response (VGPR), and 25 partial responses (PR). Histologic responses are summarized in Table 18. Thirty-one of the 39 patients remain on study treatment as ongoing, including all patients who responded to date.

[0207] Table 18. Histological response to patients [Table 18] 1including five patients with lymphocytosis as their most recent diagnosis; 2 Includes one patient with VGPR Note: CR = complete response; PR = partial response; SD = stable disease; PD = progressive disease; ORR = overall response rate.

[0208] Eight patients discontinued Compound 1, six due to disease progression and two due to adverse events related to the original malignancy. Three patients died during the study as a result of disease progression or complications of disease progression. There were no drug-related serious adverse events (SAEs). Regardless of relationship to treatment, the majority of adverse events were grade 1 or 2 in severity and were not treatment-limiting. Of the 19 grade ≥ 3 AEs, four were assessed by the investigator as possibly drug-related—all were self-limited neutropenias and did not require treatment discontinuation. There was one case of major bleeding, identified as a grade ≥ 3 hemorrhagic event or any grade intracranial hemorrhagic event: GI hemorrhage in a patient with mantle cell lymphoma with lymphomatous infiltration of the GI tract; however, this bleeding event was not determined to be drug-related because it occurred during drug hold and resolved rapidly with resumption of Compound 1 treatment. Six patients had a baseline history of atrial fibrillation / flutter (AF), but no worsening or new events of AF were reported.

[0209] (2) Results from an ongoing Phase I clinical trial of Compound 1 in patients with Waldenstrom's Macroglobulinemia (WM) A multicenter, open-label, phase I study of compound 1 in B-cell malignancies is being conducted in Australia, New Zealand, South Korea, and the United States with dose-escalation and dose-escalation phases within disease-specific cohorts, including untreated and relapsed / refractory Waldenström's macroglobulinemia (R / R WM). The dose-escalation portion of Trail tested total daily doses ranging from 40 mg to 320 mg, while the ongoing dose-expansion phase is testing test doses of 160 mg twice daily (BID) or 320 mg once daily (QD). As of March 31, 2017, 48 patients with WM had been enrolled in the study. Response was determined according to the revised 6th International Workshop on WM (IWWM) criteria.

[0210] Compound 1 has been shown to be well tolerated to date, with no discontinuations due to compound 1-related toxicity. Adverse events (AEs) were generally non-serious and self-limited. The most common AEs (>10%) among any attribution in the 48 patients evaluated for safety were petechiae / purpura / bruising (35%), upper respiratory tract infection (31%), constipation (25%), diarrhea (19%), epistaxis (19%), nausea (17%), cough (15%), anemia (15%), headache (15%), neutropenia (13%), and rash (13%), all of which were grade 1 or 2 in severity, with the exception of grade 3 or 4 anemia and neutropenia (8% each) and grade 3 or 4 diarrhea and headache (2% each). Five serious adverse events were considered related to Compound 1; these included cases of hemothorax, atrial fibrillation, colitis, febrile neutropenia, and headache. Among AEs of particular interest were a total of three cases of atrial fibrillation (all grade 1 or 2), and one case of severe hemorrhage (hemothorax), defined as grade 3 or greater hemorrhage or central nervous system hemorrhage of any grade. Three events led to treatment discontinuation: one case each of bronchiectasis, prostatic adenocarcinoma, and pyloric adenocarcinoma.

[0211] At data cutoff, 42 patients were evaluable for response. Among patients not evaluable for response were 2 patients with <12 weeks of follow-up, 3 patients with baseline IgM <500 mg / dl, and 1 patient with inaccurate baseline IgM due to frozen protein. With a median follow-up of 12.3 months (range 4.4-30.5 months), the ORR was 90% (38 / 42 patients) of patients, the major response rate was 76% (32 / 42 patients), whereas the VGPR was 43% (18 / 42 patients), and the partial response was 33% (14 / 42).

[0212] (3) Results of an ongoing Phase I clinical trial of Compound 1 in patients with chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL / SLL) A multicenter, open-label, phase I study of compound 1 in patients with B-cell malignancies is being conducted in Australia, New Zealand, South Korea, and the United States with dose escalation and dose expansion phases in disease-specific cohorts, including treatment-naïve (TN) and relapsed / refractory (R / R) CLL / SLL. The dose escalation portion of the trail tested total daily doses ranging from 40 mg to 320 mg, while the ongoing dose escalation portion has tested doses of 160 mg twice daily (BID) or 320 mg once daily (QD). As of March 31, 2017, 69 CLL or SLL patients (18 TN, 51 R / R) have been enrolled in the study.

[0213] Compound 1 was well tolerated in CLL / SLL. The most common adverse events (AEs) (≥10%) by any attribution were petechiae / purpura / bruising (46%), fatigue (29%), upper respiratory tract infection (28%), cough (23%), diarrhea (22%), headache (19%), hematuria (15%), nausea (13%), rash (13%), arthralgia (12%), muscle spasms (12%), and urinary tract infection (12%); however, all of these events were grade 1 or 2, with the exception of one case of grade 3 purpura (subcutaneous bleeding), which was the only significant bleeding event. Other adverse events of note included one case each of grade 2 diarrhea and grade 2 atrial fibrillation. A total of 18 serious AEs (SAEs) occurred in 13 patients, with no SAE occurring in more than one patient. Only one patient discontinued treatment due to an AE, a grade 2 pleural effusion.

[0214] At data cutoff, 66 patients (16 TN and 50 R / R) had ≥12 weeks of follow-up and were evaluable for efficacy, while the other 3 patients had <12 weeks of follow-up. After a median follow-up of 10.5 months (range 2.2-26.8 months), among patients, the overall response rate (ORR) was 94% (62 / 66), with 3% (2 / 66) complete responses (CR), 82% (54 / 66) partial responses (PR), and 9% (6 / 66) PRs with lymphocytosis (PR-Ls). Stable disease (SD) was observed in 5% (3 / 66) of patients. Patients with pleural effusion discontinued treatment before 12 weeks and therefore could not be evaluated for response. One case of Hodgkin's transformation was observed. In TN CLL / SLL, the median follow-up was 7.6 months (range, 3.7-11.6 months), and among patients, the ORR was 100% (16 / 16), CR was 6% (1 / 16), PR was 81% (13 / 16), and PR-L was 13% (2 / 16). In R / R CLL / SLL, the median follow-up was 14.0 months (range, 2.2-26.8 months), and among patients, the ORR was 92% (46 / 50), CR was 2% (1 / 50), PR was 82% (41 / 50), and PR-L was 8% (4 / 50). Stable disease was observed in 6% (3 / 50) of patients.

Claims

1. 1. A pharmaceutical composition for treating a B cell proliferative disorder in a subject, comprising compound 1, 【Chemistry 1】 Compound 1 Compound 1 has a purity of at least 99.3%; A pharmaceutical composition in which Compound 1 is administered at a dose of 160 mg twice daily (BID) or 320 mg once daily (QD).

2. 2. The pharmaceutical composition of claim 1, wherein the B-cell proliferative disorder is selected from the group consisting of chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, and follicular lymphoma.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein compound 1 has a purity of at least 99.5%.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the B cell proliferative disease is mantle cell lymphoma.

5. The method of claim 4, wherein the subject has undergone at least one prior treatment.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the B cell proliferative disorder is Waldenstrom's macroglobulinemia.

7. The pharmaceutical composition according to any one of claims 1 to 3, wherein the B cell proliferative disease is marginal zone lymphoma.

8. The method of claim 7, wherein the subject has undergone at least one prior treatment.

9. The pharmaceutical composition according to claim 7 or 8, wherein the marginal zone lymphoma is relapsed / refractory marginal zone lymphoma.

10. The pharmaceutical composition according to any one of claims 1 to 3, wherein the B cell proliferative disorder is chronic lymphocytic leukemia.

11. The pharmaceutical composition according to any one of claims 1 to 3, wherein the B cell proliferative disorder is small lymphocytic lymphoma.

12. The pharmaceutical composition according to any one of claims 1 to 3, wherein the B cell proliferative disorder is follicular lymphoma.

13. The pharmaceutical composition according to any one of claims 1 to 3, wherein amorphous Compound 1 or a crystal of Compound 1 is administered.

14. The pharmaceutical composition according to any one of claims 1 to 3, wherein a crystal of Compound 1 is administered.

15. The pharmaceutical composition of claim 14, wherein the crystal of Compound 1 exhibits an X-ray powder diffraction pattern including diffraction peaks having 2θ angle values ​​of 14.8±0.2°, 16.4±0.2°, and 21.4±0.2°.

16. The pharmaceutical composition of claim 14, wherein the crystal of Compound 1 exhibits an X-ray powder diffraction pattern including diffraction peaks having 2θ angle values ​​of 14.8±0.2°, 15.6±0.2°, 16.4±0.2°, and 21.4±0.2°.

17. The pharmaceutical composition according to any one of claims 14 to 16, wherein the B cell proliferative disorder is mantle cell lymphoma and the subject has undergone at least one prior treatment.

18. The pharmaceutical composition according to any one of claims 14 to 16, wherein the B cell proliferative disorder is Waldenstrom's macroglobulinemia.

19. The pharmaceutical composition according to any one of claims 14 to 16, wherein the B cell proliferative disorder is relapsed / refractory marginal zone lymphoma and the subject has undergone at least one prior treatment.

20. The pharmaceutical composition according to any one of claims 14 to 16, wherein the B cell proliferative disorder is chronic lymphocytic leukemia or small lymphocytic lymphoma.

21. The pharmaceutical composition according to any one of claims 1 to 3, wherein amorphous Compound 1 is administered.

22. 22. The pharmaceutical composition of claim 21, wherein amorphous Compound 1 has a glass transition midpoint temperature of 79.7°C.

23. 23. The pharmaceutical composition of claim 21 or 22, wherein the amorphous Compound 1 has an ee value of at least 97%.

24. The pharmaceutical composition according to any one of claims 21 to 23, wherein the B cell proliferative disorder is mantle cell lymphoma and the subject has undergone at least one prior treatment.

25. The pharmaceutical composition according to any one of claims 21 to 23, wherein the B cell proliferative disorder is Waldenstrom's macroglobulinemia.

26. The pharmaceutical composition according to any one of claims 21 to 23, wherein the B cell proliferative disorder is relapsed / refractory marginal zone lymphoma and the subject has undergone at least one prior treatment.

27. The pharmaceutical composition according to any one of claims 21 to 23, wherein the B cell proliferative disorder is chronic lymphocytic leukemia or small lymphocytic lymphoma.

28. The pharmaceutical composition according to any one of claims 1 to 27, wherein compound 1 is administered orally.

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