Crystalline form of (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[1,5-A]pyrimidine-3-carboxamide, its preparation, and its use.

Abstract

To develop a new form of (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 1), which has properties suitable for formulation, such as a high melting point and high stability. [Solution] The present invention relates to a crystalline form of compound 1 that inhibits Btk, a method for preparing the same, a pharmaceutical composition thereof, and the use of the above crystalline form in the treatment of a disease or in the manufacture of a pharmaceutical for the treatment of a disease.

Description

[Technical Field]

[0001] This invention relates to the crystalline form of (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide. The invention also includes a method for preparing a crystalline form, and a method for utilizing the crystalline form as a Btk inhibitor. Regarding. [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 primarily affects most hematopoiesis, including B cells, mast cells, and macrophages. It is expressed in lineage cells (Smith et al., J. Immunol. 152: 557-565, 1994) and is also localized in bone marrow, spleen, and lymph node tissue. Btk is involved in B cell receptor (BCR) and FcR signaling. Although they play an important role in the pathway, they are involved in B cell development and differentiation (Khan, Immunol.Res. 23: 147, 2001). Btk is activated by upstream Src family kinases. It is converted. Once activated, Btk then phosphorylates PLC gamma, which affects the function and life of B cells. It has an effect on survival (Humphries et al., J. Biol.Chem. 279: 37651, 2004).

[0003] These signaling pathways must be precisely regulated. Mutations in the gene that controls B cells cause hereditary 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 affect B cell activity. This can lead to impaired regulation of metabolism, potentially resulting in many autoimmune and inflammatory diseases. Preclinical studies have shown that Btk-deficient mice are resistant to the development of collagen-induced arthritis. This indicates that... Furthermore, clinical research on Rituxan, a CD20 antibody that can deplete mature B cells, has shown that B cells are involved in rheumatoid arthritis and systemic... It has been shown to play an important role in numerous inflammatory diseases, including lupus erythematosus and multiple sclerosis (Gurcan et al., Int. Immunopharmacol. 9: 10-25, 2009). Thus, Btk inhibitors are used to treat autoimmune diseases and / or inflammatory diseases. It is possible.

[0004] Furthermore, abnormal activation of Btk plays a crucial role in the development of B-cell lymphoma, suggesting that inhibiting Btk may be useful in treating hematological malignancies (Davis et al., Nature). 463:88-92, 2010). Preliminary clinical trial results showed that the Btk inhibitor PCI-32765 is effective in treating several types of B-cell lymphoma (for example, 54th American Society of Hematology (ASH) annual meeting abstract, Dec. 2012: 686 The Bruton's Tyrosine Kinas e(Btk)Inhibitor, Ibrutinib (PCI- 32765), Has Preferential Activity in the ABC SubtypeofRelapsed / Refractory De Novo Diffuse Large B-Cell Lymphoma (DLBCL): In terim Resultsofa Multicenter, Open-Label, Phase I Study). Btk plays a central role as a mediator in multiple signal transduction pathways. Therefore, Btk inhibitors have been strongly interested as anti-inflammatory agents and / or anti-cancer agents (Mohamed et al., Immunol. Rev.228: 58-73, 2009; Pan, Drug Newsperspect 21: 357-362, 2008; Rokosz et al.,ExpertOpin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer AgentsMed.Chem. 7: 624-632, 2007; Lou et al, J. Med. Chem. 55(10): 4539-4550, 2012).

[0005] International Application WO2014173289A discloses a series of condensed 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). [Chemical Structure] Compound 1

[0006] Compound 1 is a potent, specific and irreversible BTK kinase inhibitor. Biochemical studies ​Data obtained from preclinical studies using cell-based and animal models suggested that compound 1 has a significant effect in suppressing tumor growth in B-cell malignancies. EGFR, FGR, FRK, HER2, HER4, ITK, JAK3, LCK, and TEC were used as comparison targets. In this case, it was shown to be more selective than BTK inhibition by ibrutinib, so the compound Compound 1 is expected to have fewer side effects than ibrutinib in clinical practice. Furthermore, because compound 1 has weak inhibitory activity against ITK, it is less likely to cause rituximab-induced, antigen-dependent, cell-mediated cell damage. Because the degree of inhibition against ADCC is significantly less than that of ibrutinib, it may therefore be highly effective in treating B-cell malignancies when combined with rituximab or other ADCC-dependent antibodies.

[0007] In preclinical safety evaluations, single-dose and repeated-dose (up to 28 days) toxicity studies in rats and dogs demonstrated that compound 1 was safer than ibrutinib in terms of overall tolerance and severe toxicity. Furthermore, compound 1 exhibited superior bioavailability, as it did not have the accumulation problems observed with ibrutinib. These unique characteristics of compound 1 will lead to further clinical trials for evaluation. [Overview of the project] [Problems that the invention aims to solve]

[0008] However, in the preparation method for compound 27 in International Publication No. 2014173289, compound 1 was amorphous, which was further confirmed by the X-ray powder diffraction pattern in Figure 7A. As shown in Figure 7B, this amorphous compound 1 was shown to have a low glass transition temperature, highlighting the formulation problems of amorphous compounds, such as low stability and difficulty in purification. Therefore, it is necessary to develop a new form of compound 1, and it must have properties suitable for formulation, such as a high melting point and high stability. [Means for solving the problem]

[0009] The crystalline form of compound 1 discovered by the present inventors has a high melting point and is extremely stable even when stored at 25°C / 60%RH for up to 24 months or at 40°C / 75%RH for up to 6 months. Although the situation was as described, this discovery was unexpected.

[0010] The first aspect disclosed herein is the crystalline form of compound 1. [ka] compound 1

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

[0012] Disclosed herein as a second aspect is the crystalline form of compound BG-13 having substantially the X-ray powder diffraction pattern shown in Figure 11. [ka] Compound BG-13

[0013] A third aspect disclosed herein is a method for preparing compound 1.

[0014] Furthermore, disclosed herein are intermediate compounds of formula Ie or salts thereof, or formula If or salts thereof, which are used in the preparation of compound 1. [ka] Formula Ie Formula If

[0015] A fourth aspect disclosed herein is a method for preparing crystal form A.

[0016] Disclosed herein as a fifth aspect is a pharmaceutical composition containing a therapeutically effective amount of the crystalline form A disclosed herein.

[0017] A sixth aspect disclosed herein relates to the treatment of diseases associated with undesirable Btk activity in subjects by administering the crystalline form A disclosed herein. This is a method of treatment.

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

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

[0020] Disclosed herein as a ninth aspect is a disease associated with abnormal Btk activity in the subject. The invention relates to the use of crystalline form A disclosed herein for the manufacture of a pharmaceutical product for at least one therapeutic purpose.

[0021] Disclosed in a tenth aspect is the use of the crystalline form A disclosed herein for the manufacture of a pharmaceutical product for the treatment of an allergic disease, autoimmune disease, 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 disclosed herein in the manufacture of a pharmaceutical product for the treatment of B-cell proliferative disorders selected from B-cell malignancies or relapsed / refractory B-cell malignancies in a subject. Disclosed herein as several aspects of this aspect is the use of crystalline form A disclosed herein in the manufacture of a pharmaceutical product for the treatment of chronic lymphocytic, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenstrom macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or two or more combinations thereof in a subject.

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

[0024] In one embodiment, the ee value of amorphous compound 1 exceeds 90%. In another embodiment, the ee value of amorphous compound 1 is 97%. [Brief explanation of the drawing]

[0025] [Figure 1] XRPD pattern of crystal form A. [Figure 2] DSC curve of crystal form A. [Figure 3] TGA curve for crystal form A. [Figure 4] 1H-NMR spectrum of crystal form A. [Figure 5] 13C-NMR spectrum of crystal form A. [Figure 6] DVS plot of crystal form A. [Figure 7A] XRPD pattern of amorphous compound 1. [Figure 7B] The mDSC curve for amorphous 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] Diagram illustrating hydrogen bonding in BG-13 single crystal. [Figure 10] Crystal packing for single crystals of BG-13. [Figure 11] XRPD pattern of BG-13 single crystal. [Modes for carrying out the invention]

[0026] The crystalline compound 1 discovered by the inventors was unexpected and was named crystalline form A, but it could only be obtained under specific conditions depending on the ee value of the starting materials and the ratio of the cosolvent. Polymorphism studies were also conducted using methods such as slow evaporation, addition of poor solvents, slow cooling, vapor diffusion, and polymer-induced crystallization. Most experiments failed to yield the crystalline form, indicating that obtaining crystalline form A is not easy.

[0027] Further investigation revealed that crystal form A is an anhydrous form with a melting point of 139.4±2°C (starting temperature). To evaluate stability, samples of crystal form A were stored at 80°C for 2 days, at 25°C / 60%RH for 24 months, or at 40°C / 75%RH for 6 months, and their characteristics were identified by XRPD before, during, and after the stability tests. The results indicate that no change in crystal form was observed for all of the above periods, demonstrating good physical stability of crystal form A at 80°C, or crystal form A stored at 25°C / 60%RH for 24 months and 40°C / 75%RH for 6 months. did.

[0028] In one embodiment of crystal form A, its X-ray powder diffraction pattern includes diffraction peaks with 2θ angular values ​​individually selected from approximately 14.8±0.2°, 16.4±0.2°, and 21.4±0.2°. ru.

[0029] In one embodiment of crystal form A, its X-ray powder diffraction pattern includes diffraction peaks with 2θ angle values ​​individually selected from approximately 14.8±0.2°, 15.6±0.2°, 16.4±0.2°, and 21.4±0.2°.

[0030] In one embodiment of crystal form A, its X-ray powder diffraction pattern includes diffraction peaks with 2θ angle values ​​individually selected from approximately 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 one embodiment of crystal form A, its X-ray powder diffraction pattern includes diffraction peaks with 2θ angle values ​​individually selected from approximately 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] In some embodiments of crystal form A, an X-ray powder diffraction pattern substantially similar to that shown in Figure 1 is observed.

[0033] In some embodiments of crystal form A, the X-ray powder diffraction patterns summarized in Table 1 are observed.

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

[0035] In some preferred embodiments, the melting point of crystal form A is 139±2℃ (starting temperature).

[0036] In some preferred embodiments, crystal form A exhibits a DSC substantially as shown in Figure 2.

[0037] In some preferred embodiments, crystal form A exhibits a TGA substantially as shown in Figure 3.

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

[0039] In some embodiments, after storing crystal form A at 40°C and 75%RH for 6 months, They also exhibit substantially the same X-ray powder diffraction (XRPD) patterns. In some embodiments, crystal form A exhibits substantially the same X-ray powder diffraction (XRPD) patterns even after being stored at 25°C and 60% RH for 24 months.

[0040] Furthermore, disclosed herein is the crystalline form of compound BG-13 having substantially the X-ray powder diffraction pattern shown in Figure 11. [ka] Compound BG-13

[0041] In some embodiments, the crystal form of BG-13 is a single crystal, with 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 It has a unit cell dimension of °.

[0042] The inventors determined from single-crystal X-ray structural analysis of intermediate BG-13 that the absolute configuration of compound 1 is S. It was estimated that...

[0043] Also disclosed herein is a method for preparing Compound 1 and deuterium-labeled Compound 1, following the procedure shown in Scheme 1. The novel synthesis method and crystallization / recrystallization procedure for Compound 1 via crystal form A disclosed herein overcomes many problems of previously reported methods, such as the preparation of important chiral intermediates with optical purity exceeding 98%, improves the purity of Compound 1 to meet the acceptable levels described herein, controls impurities in Compound 1, and offers numerous advantages compared to existing methods. In particular, the method disclosed herein is highly reproducible and particularly suitable for the commercial production of Compound 1, while also providing improved quality and good yield. Other methods have allowed for the asymmetric reduction of BG-9 or its analogues in Scheme 1 from low to high enantioselectivity (5%ee to 95%ee). The other steps are the same as 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 method for preparing the compound of formula Ia, which involves asymmetric reduction of the compound of formula I in the presence of a catalyst and / or reducing agent for producing the compound of formula Ia. [ka] Equation I Equation Ia In the formula, R 1 is a hydrogen or amino protecting group.

[0046] In some embodiments, the amino protecting group is acetyl, propionyl, butyryl, phenylacetyl, benzoyl, toluyl, phenoxyacetyl (POA), methoxycarbonyl Lu, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, tert-butyloxycarbonyl Carbonyl (BOC), 2-iodoethoxycarbonyl, carbobenzoxy (CBZ), 4-methoxybenzyloxycarbonyl, (fluoren-9-ylmethoxy)carbonyl (Fmoc), 4-Me This includes, but is not limited to, toxic-2,3,6-trimethylbenzenesulfonyl (Mtr), benzyl, methyl, or 4-methoxybenzyl.

[0047] In some embodiments, the catalyst is a neutral catalytic system or a cationic catalytic 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 / (S,SorR,R)-Diop, [Ir(COD)Cl]2 / (R or S)-P-Phos, [Ir(COD)Cl]2 / (R or S)-Tol-PP hos, [Ir(COD)Cl]2 / (RorS)-Xyl-P-Phos, [Ir(COD)Cl]2 / (R,R or S,S)-Me-DuPhos, [Ir( COD)Cl]2 / (RorS)-SegPhos, [Ir(μ-Cl)(cod)]2 / (R or S)-Ship, [Ir(μ-Cl)(cod)]2 / (R orS)-Siphos, [Ir(μ-Cl)(cod)]2 / (Ror S)-Siphos-PE, [Ir(μ-Cl)(cod)]2 / (Ror S)- MonoPhos, [Ir(μ-Cl)(cod)]2 / (RorS)-tol-SDP, [Ir(μ-Cl)(cod)]2 / (S,S or R,R)-Dio p, [Ir(μ-Cl)(cod)]2 / (S,RorR,S)-Josiphos, [Ir(μ-Cl)(cod)]2 / (R or S)-Binap, [I r(μ-Cl)(cod)]2 / (RorS)-MeO-Biphep, [Ir(μ-Cl)(cod)]2 / (R or S)-Synphos, or [Ir (μ-Cl)(cod)]2 / (RorS)-Difluorphosor [Ir(cod)2] + X - (X: eg BF4,NO3,OTf, PF6, Iridium catalyst systems containing the aforementioned ligands attached to SbF6 and BarF (Wen-Boetal., 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.) However, it is not limited to these; rhodium catalyst systems including those in which the above ligands are attached to [Rh(COD)2]BF4 (Xiang-Ping et al., Top Organomet Chem 36, 313-354, 2011), but it is not limited to these; or RuCl2(RorS)-BINAP / (R or S)-DAIPEN, RuCl2( RorS)-BINAP / (R,R or S,S)-DPEN, RuCl2(S or R)-BINAP (S,S or R,R)-DACH, RuCl2[(R orS)-Tol-BINAP][(S,Sor R,R)-DPEN], RuCl2(R,R or S,S)-Me-DuPHOS / (R,R orS,S)-D PEN, RuCl2(R,RorS,S)-Et-DuPHOS / (R,R or S,S)-DPEN, RuCl2(R,R or S,S)-Et-DuPHOS / (R,RorS,S)-DACH, RuCl2(S,S or R,R)-i-Pr-DuPHOS / (R,R or S,S)-DPEN, RuCl2(RorS Includes )-HexaPHEMP / (R,RorS,S)-DPEN and RuCl2(R or S)-MeO-BIPHEP / (R,R or S,S)-DPEN Ruthenium catalyst system (Christopheretal., Adv. Synth. Catal. 345, 195-201, 2003. Jul) (Ian et al., Adv. Synth. Catal. 345, 300-307, 2003.) However, it is not limited to these.

[0048] The above method achieves excellent enantiochemical results, reaching 95% ee by using the catalyst described above. It was found that selectivity could be obtained, particularly with neutral or cationic iridium catalyst systems.

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

[0050] In some embodiments, the chiral acid may be, but is not limited to, L-malic acid, D-malic acid, L-mandelic acid, D-mandelic acid, L-camphor sulfonic acid, D-camphor sulfonic acid, L-tartaric acid, D-tartaric acid, L-DBTA, D-DBTA, L-DTTA, or D-DTTA.

[0051] Also disclosed herein is a method for dissolving a compound of formula Ic in order to produce a compound of formula Id or to increase the chiral purity of a compound of formula Id, comprising the step of 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 conventionally known amino protecting groups as described above.

[0052] In some embodiments, the chiral acid may be, but is not limited to, L-malic acid, D-malic acid, L-mandelic acid, D-mandelic acid, L-camphor sulfonic acid, D-camphor sulfonic acid, L-tartaric acid, D-tartaric acid, L-DBTA, D-DBTA, L-DTTA, or D-DTTA.

[0053] Furthermore, disclosed herein are compounds of formula Ie or salts thereof, or compounds of formula If or salts thereof, used in the preparation of compound 1. [ka] Formula Ie [ka] If expression

[0054] Furthermore, a method for preparing crystalline form A is also provided. The crystalline form disclosed herein can be prepared from a suitable solvent system containing at least one solvent by crystallizing the compound disclosed herein, and is obtained by spontaneous precipitation (evaporation) to supersaturate the solvent system, cooling, and / or by adding a poor solvent (anti-solvent) (in which the compound disclosed herein has relatively low solubility). Crystallization may also be carried out using a crystal species suitable for crystallizing the crystalline form disclosed herein, or without such a species.

[0055] In some embodiments, the method for preparing crystalline form A involves (S)-7-(1-acryloyl pipette Lysine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyri Midine-3-carboxamide (compound 1) is dissolved in DCM, the solvent is changed to EA, and the EA / MTBE is used to... This includes obtaining the desired crystal form through a recrystallization step.

[0056] In some embodiments, a method for preparing crystalline form A includes obtaining the desired crystalline form by dissolving compound 1 in EA and adding hexane.

[0057] In some embodiments, the method for preparing crystalline form A involves adding a poor solvent (anti-solvent) to a solution in which solid compound 1 or crude purified form A is present in a solvent for dissolving these solids. Examples of poor solvents include H2O and n-heptane. This does not limit the solvents used to dissolve solids, and examples of solvents for dissolving solids include, but are not limited to, acetone, DMAc, siRNA, DCM, toluene, and 2-MeTHF.

[0058] In some embodiments, a method for preparing crystalline form A involves adding a solution containing solid compound 1 or crude purified form A to a poor solvent (anti-solvent), and then allowing sufficient time for the organic vapor to interact with the solution in a sealed reactor. Examples of solvents include, but are not limited to, acetone and ¼, and examples of poor solvents include n-heptane. These are some examples, but they are not limited to these.

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

[0060] The present invention also provides a method for treating or preventing diseases related to undesirable Btk activity in a subject by administering crystalline form A to the subject. ru.

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

[0062] The present invention also provides a method for treating or preventing B-cell proliferative disorders in a subject by administering crystalline form A to that subject.

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

[0064] In some embodiments, B-cell proliferative disorders include R / R MCL, R / R CLL, and R / R SLL. This includes, but is not limited to, relapsed / refractory (R / R) B-cell malignancies, including R / R WM. I can't.

[0065] Crystal form A disclosed herein is an undesirable Btk activity in the subject. It can be used to manufacture medicines for the treatment of at least one disease related to ).

[0066] Crystal form A disclosed herein can be used to manufacture pharmaceuticals for the treatment of allergic diseases, autoimmune diseases, inflammatory diseases, cancer, or a combination of two or more thereof in a subject.

[0067] Crystal form A disclosed herein can be used in the manufacture of pharmaceuticals for the treatment of B-cell malignancies or B-cell proliferative disorders selected from relapsed / refractory B-cell malignancies in the subject.

[0068] In the latest clinical trials, compound 1 was found to be effective in untreated (TN) and relapsed / refractory (R / R) B-cell malignancies. The drug continues to demonstrate good resistance in tumors. For example, in WM, the rate of very good partial response (VGPR) exceeded 40% in a group of 42 evaluable patients, and the overall response rate (ORR) was 90% in a group of 42 evaluable patients with a median follow-up period of 12.3 months. Furthermore, in CLL / SLL, the overall response rate was high (94%) and the treatment discontinuation rate was very low (3) with a median follow-up period of 10.5 months for efficacy evaluation. %).

[0069] definition Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein shall have the meanings generally understood by those skilled in the art to which this invention pertains.

[0070] In the attached claims, singular words such as "a," "an," and "the" shall include their corresponding multiple references unless the context clearly indicates otherwise. Thus, for example, "crystal form" shall include one or more different crystal forms, etc., and "method" shall include equivalent steps and methods known to those skilled in the art, which may modify or substitute for the method described herein.

[0071] As disclosed herein, the crystalline form is a nearly pure crystal. The term “nearly pure” as used herein means that the crystalline form A disclosed herein constitutes at least 85% by weight, preferably at least 95% by weight, and more preferably at least 99% by weight.

[0072] For the crystal forms disclosed herein, only the major peaks (i.e., the most characteristic, significant, unique, and / or reproducible peaks) are summarized; additional peaks are obtained from the diffraction spectrum using conventional methods. The aforementioned major peaks are reproducible within the margin of error (±2 in the last decimal place, or ±0.2 relative to the indicated value).

[0073] As disclosed herein, "X-ray powder diffraction pattern substantially consistent with Figure 1" refers to an X-ray powder diffraction pattern showing the same major peaks as in Figure 1, where the major peaks refer to peaks showing a relative intensity exceeding 10%, preferably exceeding 20%, of the highest peak in Figure 1 (where the relative intensity is 100%).

[0074] Throughout this specification and the subsequent claims, unless the context requires otherwise, the word "comprise," as well as its conjugations such as "comprises" and "comprising," are not described. It should be understood that this means encompassing an integer or step, or a group of integers or steps. In this context, the term "comprising" is replaceable with the term "containing," and sometimes with the term "having."

[0075] The term "therapeutically effective amount" as used herein refers to the amount of a compound sufficient to treat a disease or to affect the treatment of at least one of the clinical symptoms of a disease or disorder when administered to a subject. The "therapeutic effective amount" may vary depending on the compound, the disease, disorder, and / or the symptoms of the disease or disorder, the severity of the disease, disorder, and / or the symptoms of the disease or disorder, the age of the subject receiving treatment, and / or the weight of the subject receiving treatment. In a given example, the appropriate amount may be obvious to those skilled in the art or may be determined by conventional experimentation. In the case of combination therapy, the "therapeutic effective amount" refers to the total amount of substances used in combination to provide an effective treatment for the disease, disorder, or condition.

[0076] Pharmaceutical compositions containing the compounds disclosed herein can be administered orally, by inhalation, rectally, parenterally, or topically to subjects requiring them. For oral administration, the pharmaceutical composition may be a conventional solid formulation such as a tablet, powder, granules, or capsule, or a liquid formulation such as a suspension of water or oil, or other liquid formulations such as a syrup, solution, or suspension; for parenteral administration, the pharmaceutical composition may be a solution, aqueous solution, concentrate of an oily suspension, or lyophilized powder. The formulation of the pharmaceutical composition is preferably selected from tablets, coated tablets, capsules, suppositories, nasal sprays, or injections, and more preferably selected from tablets or capsules. The pharmaceutical composition may also be a single unit administered in a precise dose. The pharmaceutical composition may further contain additional active ingredients.

[0077] All formulations of the pharmaceutical compositions disclosed herein can be manufactured by conventional methods in the pharmaceutical field. For example, a desired formulation can be manufactured by mixing an active ingredient with one or more excipients. "Pharmacologically acceptable excipients" refers to conventional pharmaceutical carriers 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); and humectants such as glycerin. It is a disintegrant such as agar, calcium carbonate, and sodium bicarbonate; an absorption enhancer such as quaternary ammonium compounds; a surfactant such as hexadecanol; an absorbent carrier such as kaolin and soap clay; and talc and calcium stearate. These include lubricants such as magnesium stearate and polyethylene glycol. The pharmaceutical composition further contains other pharmaceutically acceptable excipients such as dispersants, stabilizers, thickeners, complexing agents, buffers, penetration enhancers, polymers, fragrances, sweeteners, and dyes.

[0078] The term "disease" refers to any condition such as illness, discomfort, disease, symptom, or sign, and is replaceable with terms such as "disorder" or "condition."

[0079] Abbreviation: Acetic acid (ACOH) Adverse Events (AEs) BID_1 day twice CLL (Chronic Lymphocytic Leukemia) Con._Concentrated D-DBTA_(2S,3S)-dibenzoyl tartaric 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 dimethylacetal DMSO (Dimethyl Sulfoxide) DSC (Differential Scanning Calorimetry) DVS_Dynamic Vapor Solvation EA_ethyl acetate, hydroxy 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 Isopropyl Acetate (IPAc) IPC_In-Process Control KF_Carl Fischer L-DBTA_(2R,3R)-dibenzoyl tartaric acid Limitations of Quantification of LOQ MCL (Mantle Cell Lymphoma) MeCN or ACN_acetonitrile MeMgBr (Methyl Magnesium Bromide) MeOH_ Methanol 2-MeTHF_2-methyltetrahydrofuran MIBE_4-methyl-2-pentanone MsOH (Methanesulfonic Acid) MTBE (Methyl Tertiary Butyl Ether) NHL (Non-Hodgkin Lymphoma) NLT_ or greater (notless than) NMP1-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 Lymphocyte Lymphoma) RT_room temperature TEA (Triethylamine) TGA_Thermogravimetric analysis THF (Tetrahydrofuran) TN_No prior treatment experience VGPR - Very good partial response XRPD_X-ray Powder Diffraction WM_Waldenström macroglobulinemia [Examples]

[0080] The following embodiments illustrate the present invention, but do not limit it. Example 1: Preparation of (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 1) and Crystal form A Step 1: Synthesis of BG-2 [ka]

[0081] Under a nitrogen atmosphere, TEA (2.4 equivalents) is mixed with EA (5V), HOBT (1.2 equivalents), EDCI (1.2 equivalents), and 4 - Phenoxybenzoic acid (BG-1.80 kg, 1.0 equivalent) and malononitrile (1.2 equivalents) at 10°C It was added to the solution. The mixture was then stirred at room temperature until the reaction was complete. The mixture was then centrifuged and the cake (precipitate) was washed with EA. The filtrate was washed twice with aqueous NaHCO3 solution. The mixture was then washed with an NH4Cl aqueous solution. The organic phase was washed twice with 1.5N H2SO4 and stirred. The mixture was concentrated and then mixed. The material was precipitated from tanol and purified water. The solid was collected by centrifugation and then vacuum-dried. This yielded 79.9 kg of BG-2. 1 H NMR (DMSO-d6) δ 7.62 (d, J= 8.6 Hz, 2 H), 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 = 8.6Hz, 2H).

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

[0083] Under a nitrogen atmosphere, a solution of BG-2 (79.9 kg, 1.0 equivalent) in MeCN (5.0 v) was trimethylated at 85°C. It was added to simethan (12.0V). The resulting mixture was stirred until the reaction was complete. HPLC analysis was performed. Samples were taken for analysis. The mixture was concentrated under vacuum. The residue was precipitated with i-PrOH and hexane. The mixture was centrifuged, the cake was washed with hexane, and vacuum dried. This yielded 71.7 kg of The product was obtained. 1 H NMR (400 MHz, DMSO-d6) δ7.70(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] Under a nitrogen atmosphere, a solution of BG-3 (71.6 kg, 1.0 equivalent) in ethanol (2.5 v) was prepared. Hydrazinium hydroxide (1.0 equivalent) dissolved in 0.6 V iodine was added dropwise to the reactor at a temperature below 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. It was added. Next, the solution was cooled to 5°C, centrifuged, and the cake was washed with water (1.0 V). The product was vacuum-dried. This yielded 66.9 kg of product. 1 H NMR (DMSO-d6) δ 12.11 (brs, 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] DCM (8.0v), BG-5 (80.0kg, 1.0 equivalent), N,O-dimethylhydroxylamine hydrochloride ( A mixture of 1.2 equivalents of HOBt (1.2 equivalents) and EDCI (1.2 equivalents) is mixed with TEA (2.6 equivalents) at 15°C. It was dropped below. 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 solution (3 × 4.0 v). The filtrate was concentrated under vacuum to obtain 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, and MeMgBr (1.4 equivalents ) was added dropwise at 10 °C, and then the mixture was stirred at room temperature until the reaction was complete. The solution was cooled to 10 °C or below. Saturated aqueous NH4Cl solution was added dropwise at 10 °C or below. The mixture was centrifuged, separated, and filtered , and then the organic phase was washed twice with aqueous NaCl solution. The organic phase was concentrated to obtain 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 solution. The organic layer was concentrated, and precipitation was achieved by adding hexane . The mixture was centrifuged, and the cake was collected. The cake was dried under vacuum. As a result, 82.2 Kg of the desired product was obtained. 1 1H 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), 2.8 9 - 2.61 (m, 6H), 2.49 - 2.37 (m, 1H), 1.66 - 1.56 (m, 2H), 1.39 (s, 9H), 1.3९ - 1.20 (m , 2H).

[0088] Step 7: Synthesis of BG-9

Chemical Structure

[0089] Under a nitrogen atmosphere, a mixture of toluene (8.0 v), AcOH (0.5 v), BG-8 (1.2 equivalents), and BG-4 (66.9 kg, 1.0 equivalent) was heated to 95°C and stirred until the reaction was complete. The mixture was then cooled. The mixture was concentrated and then precipitated with methanol. The mixture was centrifuged, and the cake was washed with methanol. The cake was vacuum-dried. This yielded 107.8 kg of product. 1 1H NMR (DMSO-d6)δ8.78 (d, J = 4.6 Hz, 1H), 8.15-8.07 (m, 2H), 7.51-7.41 (m, 2H), 7. 34 (d, J =4.6Hz, 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), 1.4 3 (s, 9H).

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

[0091] Mixture of THF (10.0v), BG-9 (13.0kg, 1.0 equivalent), and D-DBTA (1.0 equivalent) under N2, with water. The primary gas was introduced into the reactor at a Pd / C ratio (10% w / w), and the hydrogen pressure was maintained at 1.8 MPa. The reactor was then operated at time The mixture 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 obtain the desired compound (yield: 94.8% and purity: 98.5%). 1 H 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] A solution of BG-10 (100.0 kg, 1.0 equivalent) in DCM (6.0 v) is prepared under a nitrogen atmosphere by adding HCl-containing EtOH. (20.9% w / w, 2.0 v) was added dropwise. The mixture was stirred until the reaction was complete. MTBE (4.0 v) The solution was cooled after adding the ) to it. The cake was collected by centrifugation and washed with hexane (2.0 V). Next, the cake was slurryed in hexane (5 V) and then centrifuged again. The sample was washed with hexane (2.0V) and vacuum-dried. This yielded 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 =7.4Hz, 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 water (6.0V) and NaOH (3.0E equivalent) containing BG-11 (85.0Kg, 1.0 Equivalent) at room temperature. The mixture was stirred until the reaction was complete. The cake was collected and then made into a slurry in MTBE (6.0 V). Next, the mixture was centrifuged to collect the cake. The cake was then vacuum-dried. This yielded 71.3 kg of product. 1 HNMR (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) in a reactor and BG-11A (30 kg, 1.0 equivalent) is heated to 70 ± 5°C under a nitrogen atmosphere, and then D-DBTA (1.20 equivalents) is added to the ethanol / water / A solution of acetic acid (7:3:1,4v) was added dropwise at a temperature of 65°C or higher. The resulting solution was heated at 60-65°C for 16 minutes. After stirring for a certain time, the mixture was cooled to room temperature. The solids were collected by centrifugation and then washed with ethanol (2.0 V). The cake was then immersed in a mixed solvent of ethanol / water / AcOH (7:3:1,20 V) at 55°C. The mixture was allowed to form a slurry for 16 hours and then cooled to room temperature. The solid was collected by centrifugation and then ethanol (2.0 The cake was washed (v). The cake was vacuum-dried (yield: 37.9%). 1 HNMR (DMSO-d6) δ 8.76 (b rs,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), 4.0 8-4.00 (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] A mixture of dichloromethane (15.0V) and a 20.0% KOH aqueous solution (3.0V) was subjected to a nitrogen atmosphere in a room. At warm temperature, BG-11B (48.0 kg, 1.0 equivalent) was added in batches. After the reaction was complete, the organic layer was added. The layers were collected, and the aqueous layer was extracted with dichloromethane (5.0 V). The organic layers were combined. Con. HCl was added at room temperature. (0.36 V) was added to the above organic layer. The resulting mixture was stirred until the reaction was complete. The solid was collected by centrifugation and then washed with dichloromethane (1.0 V). Collected solid The mixture was slurryed using MTBE (6.0V). The solid material was collected by centrifugation, and then MTBE (1.0V) was used. The product was washed and then vacuum-dried. This yielded 31.5 kg of product (yield: 100%).

[0100] Step 12: Synthesis of BG-11D (alternative intermediate) Add ACN (5.0V), soft water (10.0V), and KOH (5.0 equivalents) to the reactor and stir for at least 15 minutes. The mixture was stirred. BG-11B (1.0 equivalent) was gradually added to the reactor. The mixture was stirred until the reaction was complete. The cake was collected by centrifugation and the slurry was prepared in ACN (1.0 v) and soft water (5.0 v). The product was obtained by fermentation and then drying under vacuum.

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

[0102] A solution of BG-11C (15.0 kg, 1.0 equivalent) in MsOH (2.5 V) is incubated under a nitrogen atmosphere until the reaction is complete. The mixture was stirred at 85°C. After cooling to 5°C, purified water (4.0V) was added dropwise to the system to lower the temperature to below 35°C. The temperature was maintained (clearly rising). The resulting solution was stirred at 30°C for 16 hours and then washed with DCM (2 × 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 was adjusted to 11-12 with a 20% NaOH aqueous solution (the temperature clearly rose) while stirring at a temperature below 30°C. The organic phase was separated and collected. The aqueous phase was extracted with DCM (3.0v). Organic layer The mixture was concentrated. MTBE (4.0 v) was added to the residue. The mixture was then concentrated, and n-hept was added. The mixture was precipitated in a tung tank. The solid was collected by centrifugation and then dried in a vacuum oven. This yielded 12.55 kg of product (yield: 94.9%). 11H NMR (DMSO-d6) δ7.52- 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), 1.65-1.1 5 (m, 3H).

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

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

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

[0106] Under an N2 atmosphere, ACN (12.0V), water (12.5V), BG-13 (8.0 kg, 1.0 equivalent), and NaHCO3 are mixed. 3 (2.5 equivalents) was added to the reactor. The mixture was then cooled to -5 to 0°C. A solution of acryloyl (1.1 equivalents) in MeCN (0.5V) was added dropwise, and the mixture was stirred until the reaction was complete. Next, EA (6.0V) was added to the reactor and then stirred. The organic phase was collected. The aqueous layer was further mixed with EA. Extraction was performed at (3.0 V). The organic phases were combined and washed with brine. The organic layer was collected and concentrated.

[0107] The residue was purified using a silica gel (2 wt) column and dissolved in 3% w / w methanol (21.0 v) in DCM. 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-acryloylpiperidine-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 (10.0v), purified water (5.0v), NaOH (1.5 equivalents), and BG-13 (1.0 equivalent). The mixture was stirred to obtain a clear solution. Then toluene (6.0 v) was added to the reactants and separated. The organic phase was collected and washed twice with 15% brine (3.0 v). The organic phase prepared above was concentrated and the solvent was replaced with CH3CN (residual volume: NMT 5.0 v). CH3CN (7.5 v) and purified water (12.5 v) were added. The mixture was then cooled to 15-20°C. L-(+)-tartaric acid (0.5 equivalents) and NaHCO3 (2.5 equivalents) were added to the reaction mixture. The CH3CN(0.5v) solution of acryloyl chloride (1.1 equivalents) was mixed with the reaction mixture. The solution was added dropwise to the material. After the reaction was complete, HCl (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 and washed with 15% brine (5.0 V). The solution was then concentrated. The solvent was replaced with DCM (residue volume: 1.5-2.0 V), and the solution was collected in a silica gel column. (Silica gel: 100-200 mush, 2.0 w / w; Elutate: DCM containing 3% w / w MeOH (approx. 50 v)) The solution was purified. The collected solution was concentrated and then replaced with ÃO(4.0 v); MTBE(6.4 v) was added dropwise to the residue at 50°C, the mixture was then cooled to 5°C, and the cake was collected by centrifugation.

[0110] Step 16: Preparation of crystalline form A of compound 1 The above compound 1 cake was dissolved in 7.0 volume of DCM, and then the solvent was replaced with EA. EA / MTBE After recrystallization, the cake was collected by centrifugation and then dried under vacuum. This yielded 4.44 kg of product (yield: 70.2%).

[0111] Next, the product was identified by X-ray powder diffraction (XRPD) patterning, using the following XRPD parameters on a PANalyticalEmpyrean X-ray powder diffractometer: X-Raywavelen gth (Cu, kα, Kα1(Å):1.540598, Kα2(Å): 1.544426; Kα2 / Kα1 intensity ratio : 0.50);X-Raytube setting (45 Kv, 40mA); divergence slit (automatic); scan mod e(Continuous);scan range (°2TH) (3°-40); step size (°2TH) (0.0131); scan sp eed (° / min) (about 10). XRPD results showed that the obtained product was the crystal shown in Figure 1. I understand.

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

[0113] The thermogravimetric analysis (TGA) curve shown in Figure 3 was generated using TA Instruments' TA Q5000 TGA. The TGA parameters used are as follows: temperature(RT-desiredtemperature); heati ngrate(10oC / min); method (ramp); sample pan (platinum, open); purge gas (N2). The TGA results showed no weight loss up to 110°C, indicating that the product remained anhydrous.

[0114] Proton nuclear magnetic resonance shown in Figure 4 ( 1 The 1H NMR spectrum was measured in DMSO-d6 using a Bruker 400M NMR spectrometer. 1 HNMR (DMSO-d6) δ 7.50 (d,J = 8.6 Hz, 2H), 7.46-7.38 (m, 2H), 7 .17 (t, J =7.6Hz, 1H), 7.08 (d, J = 7.6 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 6.8 5-6.72 (m,1H),6.67 (s, 1H), 6.07 (dd, J = 16.8, 2.2 Hz, 1H), 5.64 (dd, J = 10. 4 Hz, 2.2Hz,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), 1.79-1.6 5 (m, 1H), 1.63-1.49 (m, 1H), 1.38-1.08 (m, 2H).

[0115] As shown in Figure 5, carbon nuclear magnetic resonance ( 13 The 1C-NMR spectrum was measured in DMSO-d6 using a Bruker 400M NMR spectrometer. The crystalline form A of compound 1 was measured. 13 C-NMR spectrum.

[0116] Example 2: Preparation of crystalline form A of compound 1 (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-teto Lahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 1) is WO2014173289A Compound 1 was prepared by the method disclosed herein and then freeze-dried to obtain amorphous compound 1. The above solution was obtained by heating a solution of compound 1 (200 mg, ee value > 97%) in EA (8 mL) to 50°C. Hexane (8 mL) was added dropwise at 50°C. The mixture was cooled to room temperature, stirred for 16 hours, and then filtered. After processing, 110 mg was obtained as a white solid. The obtained solid was identified as type A by XRPD.

[0117] Example 3: Preparation of crystalline form A of compound 1 (addition of poor solvent) Approximately 15 mg of the sample (crystal form A) was weighed into a 20 mL glass vial, followed by 0.4-1.2 Add the corresponding solvent (see Table 2) in mL to dissolve all the solids. Then add the mixture to 800 mL. The mixture was magnetically stirred at a speed of rpm to obtain a clear solution at room temperature. Subsequently, a relatively poor solvent (see Table 2) was added to the solution to induce precipitation, or until the total amount of poor solvent reached 15.0 mL. The solution was added up to this point. If precipitation did not occur, the solution was then evaporated at room temperature over a period of time. The resulting solid was identified as type A by XRPD.

[0118] Table 2 Poor Solvent Addition Test [Table 2]

[0119] Example 4: Preparation of crystalline form A of compound 1 (solution vapor diffusion) Dissolve approximately 15 mg of the sample (crystalline form A) in 0.5-1.5 mL of the corresponding solvent (acetone or methoxy). A clear solution was obtained in a 3 mL vial. Next, the solution was placed in a 20 mL vial containing 3 mL of a relatively poor solvent (n-heptane). The 20 mL vial was sealed with a cap and kept at room temperature for sufficient time for the organic vapors to interact with the solution. At the end of day 11, the clear solution was converted to evaporation at room temperature. The resulting solid was identified as type A by XRPD.

[0120] Example 5 Stability test of crystalline 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 two days. No change in crystalline form was observed in the XRPD pattern before and after the test.

[0121] Long-term stability tests of crystalline form A of compound 1 showed that no significant change 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, at 25°C No changes in crystal form or optical purity were observed when stored at 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 DVS Intrinsic, a surface measurement system (SMS). The DVS parameters used are as follows: temperature (2 5 °C); dm / dt (0.002% / min); Min. dm / dt stability duration (10 min); Max. equilibri um 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 slight mass increase at 80%RH, which was approximately 0.8% for crystalline form A of compound 1. There was a very slight mass increase at 80%RH, which was approximately 0.8% for crystalline form A of compound 1.

[0123] (3) Crystallization / recrystallization of compound 1 by form A to improve its purity. Crystallization / recrystallization in form A improves the purity of compound 1 and is an effective method for suppressing impurities in compound 1 to further meet the acceptance criteria of this specification. See the examples shown in Table 3.

[0124] Table 3 Changes in purity after crystallization / recrystallization in form A

Table 3

[0125] Example 6 Deuterium-labeled (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxy Preparation of phenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (Deuterium-labeled compound 1)

Chem.

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

[0127] (S)-2-(4-phenoxyphenyl)-7-(piperidine-4-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (dissociated from BG-13, step 15, compound) Substance 1, see alternative method; add 278 mg, 0.67 mmol) of DCM (20 mL) and an aqueous solution of NaHCO3 (10 mL) dropwise to the above acryloyl-d3 chloride solution (5 mL) at 0-5°C and stir at room temperature for 2 hours. The mixture was mixed. The combined organic layer was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by preparative TLC to obtain 55 mg (17.5%) of (S)-7-(1-(acryloyl-d3)piperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide as a grayish-white solid. 1 HNMR (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: Study of polymorphism 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-tet Hydro-1H-pyrazolo[1,5-a]pyrimidine-3-carboxamide was prepared according to the method disclosed in WO2014173289A, and further lyophilized to obtain amorphous Compound 1.

[0129] For each experiment in Tables 4a - 4k, Tables 5a - 5e, and Table 6, approximately 20 mg of Compound 1 as an amorphous form was weighed into a glass vial, and then the corresponding solvent was added. If necessary, the mixture was heated to obtain a clear solution. Subsequently, the mixture was kept at room temperature without stirring for 1 - 2 days until solids formed from the clear solution became visible. The solids were observed using a polarized light microscope.

[0130] Table 4 Compound 1 as the 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

[0141] Table 5 Extracted substances: Compound 1 (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 5a-5e were conducted on the same scale (i.e., the amount of starting material-amorphous compound 1 was approximately 20 mg). However, the ee values ​​of the starting materials differed in each experiment. This appeared to have a significant effect on the amount of solid material that should be formed. In the experiments in Tables 4a-4k, amorphous compound 1 with 90% ee was used as the starting material, but only a small amount of solid material was formed. In the experiments in Tables 5a-5e, amorphous compound 1 with 97% ee was used as the starting material, but a very large amount of solid material was obtained. Also, when crystallized from the 90% ee starting material, the solid material obtained in the experiments in Tables 4a-4k had a low ee value. In one of the solid material samples from Tables 4a-4k, EA / hexane was used as the crystallization system, but the ee value was only 45%.

[0147] Further verification of the results in Table 5a through a scale-up experiment similar to that in Example 2 confirmed that the obtained solid material was in the desired crystalline form (morphology A).

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

[0149] The results in Table 6 further support the idea that the formation of crystalline solids depends on specific solvent ratios. [Table 6]

[0150] (2) Study of polymorphism from crystalline form - Preparation of morphology A from crystalline form Evaporation that takes time Approximately 15 mg of the sample (crystal form A) is weighed into a 3 mL glass vial, followed by the corresponding solvent or A solvent mixture (see Table 7) was added to obtain a clear solution. The vial was then covered with Parafilm having 3-4 pinholes, and the solution was allowed to evaporate over time at room temperature. The solid was isolated for XRPD analysis. However, as summarized in Table 7, no crystalline form was produced.

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

[0152] Poor solvent addition Approximately 15 mg of the sample (crystal form A) was weighed into a 20 mL glass vial, followed by 0.4-1.2 mL of the pair The corresponding solvent was added (see Table 8). The mixture was then stirred magnetically at a speed of 800 rpm. A clear solution was obtained at room temperature. Subsequently, a relatively poor solvent (see Table 8) was added to the solution to induce precipitation, or until the total volume of the poor solvent reached 15.0 mL. If precipitation did not occur... If the solution was not suitable, it was then evaporated at room temperature over time. The results are summarized in Table 8.

[0153] Table 8 Poor Solvent Addition Experiment [Table 8] N / A: No solid material was obtained.

[0154] Cooling over time Approximately 20 mg of the sample (crystal form A) is placed in a 3 mL glass vial and then 1.0 mL of the corresponding solution is added at room temperature. The suspension was suspended in a solvent (see Table 9). The suspension was heated at 50°C while magnetically stirring at a speed of 800 rpm. The sample was transferred to a rally. The sample was equilibrated at 50°C for 2 hours and filtered using a 0.45 μm nylon membrane. The filtrate was then cooled slowly 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 form was obtained.

[0155] Table 9 Cooling experiments performed over time [Table 9] N / A: No solid material was obtained. *: The clear solution was transferred and evaporated at room temperature.

[0156] Solution vapor diffusion Dissolve approximately 15 mg of the sample (crystal form A) in 0.5-1.5 mL of the corresponding solvent (see Table 10), and 3 A clear solution was obtained in a mL vial. Subsequently, the solution was added to a 20 mL vial containing 3 mL of the relative poor solvent. It was placed in a vial. The 20 mL vial was sealed with a cap and kept at room temperature, allowing the organic vapors to dissolve. Sufficient time was allowed for interaction with the liquid. At the end of day 11, 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 Experiments on solution vapor diffusion [Table 10] N / A: No solid material was obtained. *: Solid matter was formed by evaporation over time.

[0158] Experiments in polymer-induced crystallization Dissolve approximately 15 mg of the sample (crystal form A) in 1.0 mL of the corresponding solvent (see Table 11), and prepare 3 m A clear solution was obtained in an L 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. As summarized in Table 11, no crystalline form was obtained.

[0159] Table 11 Experiments on polymer-induced crystallization [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 hydroxyethylcellulose (HEC) (mass ratio 1:1:1:1:1).

[0160] Example 8: Determination of the absolute configuration of compound 1 Fabrication of BG-13 single crystals Single crystal growth experiments (see Table 12) were performed six times using a method of slow cooling. By cooling slowly in MeOH / H2O (1:1, v / v), BG-13 was grown in the desired form. Crystals were obtained. The crystal data and precise structure are listed in Table 13.

[0161] Table 12 Single Crystal Growth Experiment [Table 12]

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

[0163] BG-13 was confirmed to be (2R,3R)-dibenzoyl tartrate (L-DBTA) salt, and was also released. The molar ratio of base pairs to L-DBTA was 2:1. Both carbon atoms (C32 and C32') in L-DBTA The stereochemistry was confirmed to be R. As shown in Figures 8 to 10, the C6 in the free base The stereochemistry was determined to be S. Furthermore, as shown in Figure 11, the structure of the single crystal was identified using powder X-ray diffraction patterning.

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

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

[0166] Common procedure: A chiral acid was added at high temperature to a solution containing compound BG-11A in the prepared solvent system. After stirring at this temperature, it was cooled to room temperature and then stirred overnight at room temperature. The solid was filtered and then washed with the prepared solvent system. The ee value was determined for the relevant salt or its Boc derivative. The compounds were then directly tested by chiral HPLC (see Table 14). With other chiral acids or solvent systems, chiral compounds with no ee value, chiral compounds with low ee values, or undesirable chiral compounds were observed.

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

[0168] Example 10: Chiral separation and improvement of chiral purity of BG-12A [ka]

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

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

[0171] The obtained L-DBTA salt (31 g, 85.6% ee) was added to THF / H2O (1 / 1, 1034 mL), and the suspension was prepared in 70 ml. The mixture was heated to ℃ and stirred until all solids dissolved. Then 517 mL of water was added. Next, the solution was slowly cooled to 40℃ and seed crystals (10 mg) were added. After stirring for about 2 hours, the solution was stirred. The mixture was slowly cooled to ambient temperature and then stirred for two days. After filtration, the solid was washed with THF / H2O = 1 / 1 (20 mL), dried under reduced pressure, and the product was obtained as a white solid (22.5 g, 72% yield). (, >98.5 ee value).

[0172] The obtained free base (6.02 g, 79.1% ee) was dissolved in EtOH / H2O (6 / 1, 90 mL) at a ratio of 1 g / 15 mL, and stirred at 78 °C to dissolve all the starting materials. Then, L-DBTA (2.84 g, 7.9 mmol) was added. A solution of EtOH / H2O (6 / 1, 7 mL) (0.55 equivalents) was added. A solid rapidly formed, and the mixture was stirred at this temperature for 1 hour, after which the heating system was removed. The mixture was cooled to room temperature. Filtered, the solid was washed with EtOH / H2O (6 / 1, 10 mL). The collected solid was treated with an aqueous NaOH solution and The product was converted to a free base using DCM, yielding a white, foamy substance (4.7 g, yield: 32.6%, 93% ee).

[0173] The obtained free base (70.0 g, 90.5% ee) was suspended in CH3CN / H2O (1 / 1, 700 mL) and heated to 60°C to obtain a clear solution. L-DBTA (33 g, 0.55 equivalents) was then added to the solution. After stirring at 60°C for approximately 2 hours, the mixture was slowly cooled to room temperature and stirred overnight. The mixture was filtered, washed with CH3CN / H2O (1 / 1, 50 mL), and dried under reduced pressure to obtain a grayish-white solid. We obtained a product (80g, yield: 80%, ee value > 98%).

[0174] Example 11: Efficacy Test (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-teto Lahydropyrazolo[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 test The crystal form A of compound 1 is BTK quinate The inhibition of the enzyme (aa2-659, Carna Biosciences) was tested using time-resolved fluorescence resonance energy transfer (TR-FRET) methodology. The experiment was performed in a small volume black plate with 384 wells, and involved BTK kinase, 5μMATP, The reaction was carried out in a reaction mixture containing 2 μM peptide substrate and 0-10 μM of compound, which consisted of 50 mM Tris pH 7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DTT, 0.005% Tween-20, and 20 nM The kinase was found in a buffer solution containing SEB and 0.01% BSA. Incubate at room temperature for 60 minutes, then open the reaction by adding ATP and peptide substrate. Started the reaction. After allowing it to react at room temperature for 60 minutes, followed the manufacturer's instructions (CisBioBioassays), etc. A certain amount of stop / detection solution was added. The stop / detection solution is Eu 3+ Cryptate-conjugated mouse monoclonal antibody (PT66) anti-phosphotyrosine and XL665-conjugated streptavidin It contains a buffer solution with 50 mM MHE PES pH 7.0, 800 mM KF, 20 mM EDTA, and 0.1% BSA. It was included in the following. The plate was sealed and incubated at room temperature for 1 hour, then the TR-FRET signal (fluorescent at 665 nm for emission at 620 nm with excitation at wavelength 337 nm) was observed. The ratio of photoluminescence was recorded using a PHERStarFS plate reader (BMGLabtech). Peptide substrate Phosphorylation of the anti-phosphotyrosine antibody binds to the biotinylated peptide substrate, but here the fluorescent donor (Eu 3+ Because the cryptotate is positioned in close proximity to the acceptor (streptavidin-XL665), the donor fluorophore (620nm) is able to reach the acceptor. High fluorescence resonance energy transfer occurs towards the ruolophore (665 nm). Inhibition of BTK kinase activity reduced the TR-FRET signal. (GraphpadPrism software) By fitting the data to the four-parameter logistic equation, the IC of compound 1 can be determined. 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. IC of crystal form A 50 (See Table 13) The tests were conducted at BeiGene's facility, and included ITK, TEC, JAK3, and EGFR tests. The procedure was performed using the TR-FRET test and the corresponding peptide as substrates.

[0177] ITK IC 50 Determination: The procedure for the ITK test is the same as that for the BTK test, with the following change: 3 μM ATP and 2 μM of TK substrate were used in the kinase reaction.

[0178] TEC IC 50 Determination: The procedure for the TEC test is the same as the BTK test, with the following changes: 1) 280 μMATP and 2 nMPoly-GT substrates were used in the kinase reaction. 2) SEB was added to the reaction buffer. It is not included.

[0179] JAK3 IC 50 Decision: The procedure for the JAK3 test is the same as the BTK test, with the following changes: 1 ) 3.4 μMATP and 3 μM peptide substrate (B-EE-15, biotin-EQEDEPEGDYFEWLE) are used in Kiner Used in the 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 Decision: The protocol for the EGFR trial is the same as that for the BTK trial, with the following modifications. 1) 20 μM MATP, 1.44 μM MTK substrate - biotin (one of the substrates common to tyrosine kinases) ) and 1% DMSO of the final concentration was used for compound kinase reactions of 0-1000 nM. 2) Reaction slow The casing solution is 50 mM MHE PES pH 7.5, containing 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2.5 mM DTT, and Contains 0.1% BSA. 3) Stop / detection solution buffer is 25 mM HEPES pH 7.5, 400 mM KF, 50 mM Contains EDTA, 0.01% Triton-X100, and 0.1% BSA.

[0181] result: IC for BTK kinase of crystalline form A 50 It was 0.27 nM. Crystal form A is potent and specific. Furthermore, it was found to be an irreversible BTK kinase inhibitor. Regarding its selectivity, when characterizing a panel of 342 human kinases at 1 μM, crystal form A inhibited more than 70% of only 13 other kinases.

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

[0183] Test 2: BTKpY223 cell test using crystalline form A method: The BTKpY223 cell assay quantitatively determines the endogenous phosphorylation level in BTKTyr223. This is an HTRF-based test aimed at [the following]. Phosphorylated Tyr223 is required for the complete activation of BTK. Yes. The test used the BTKpY223 test kit (63IDC000, Cisbio) to analyze Ramos cells (CRL-1596). It was held at ATCC.

[0184] In short, Ramos cells were subjected to serum starvation for 2 hours in RPMI1640 containing 0.5% FBS. After starving the cells, incubate them with crystalline form A and incubate them in CO2 for 1 hour. The antibodies were detected at various concentrations within the test tube. After incubation, cells were stimulated with 1 mM pervanadate (PV) or Na3VO4 (OV) for 20 minutes. The cells were then spun down and lysed in 1× lysis buffer (4× lysis buffer provided in the kit) at room temperature for 10 minutes. During incubation, a 1× antibody mixture was prepared by diluting anti-BTK-d2 and anti-pBTK-K with detection buffer (supplied in the kit). The 1× antibody mixture was distributed in 2 μl / well portions onto an OptiPlate-384 test plate (6005620, PerkinElmer). Then, 18 μL of cell lysate was transferred to a test plate pre-added with antibody solution. After gentle mixing and a short spin, the plate was 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 (PHERAstarFS, BMG). The potency of compound 1 was calculated based on the inhibition ratio between the signal intensities at 665 nm and 620 nm. IC was performed using GraphPad Prism software with the sigmoid dose-response function. 50 The value was calculated.

[0185] result: Crystal form A was found at a low concentration of 1.8 ± 0.2 nM (n=3) in the B-cell lymphoma cell line, Ramos. It inhibited the phosphorylation of BTK.

[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 ABC-type diffuse large B-cell lymphocytes The tumor cell line (TMD8) was used in this study. The cell line was prepared using 10% fetal bovine serum / FBS (ThermoSc). The samples were maintained in RPMI-1640 with added ientific (Penicillin). They were then maintained at 37°C in a humidified atmosphere of 100 units / ml penicillin (Gibco), 0.1 mg / ml streptomycin (Gibco), and 5% CO2 in the air. The cell line was seeded (laid down) within 30 passages from the original cell line obtained. He recovered from his illness.

[0187] The growth inhibitory activity of the compound in Rec-1, Mino, JEKO-1, and TMD-8 cells was observed in CellTiter-Glo Determined using the Promega bioluminescent cell viability test. Per well of a 96-well plate. The number of cells seeded was optimized for each cell line to ensure logarithmic growth over a 6-day treatment period. Cells were treated three times with a 10-point dilution series. After 6 days of exposure to the compound, an amount of CellTiter-Glo reagent equal to the volume of cell culture medium present in each well was added. The mixture was mixed on an orbital shaker for 2 minutes to lyse the cells, followed by incubation at room temperature for 10 minutes to induce and stabilize the luminescence signal. The fermentation signal represents the amount of ATP, i.e., the amount of metabolically active cells. The luminescence signal was measured using a PHERAstar FS reader (BMGLabtech). IC for cell viability... 50 The value The results were determined using GraphPadPrism software and were averaged from three independent trials.

[0188] result: Crystalline form A of compound 1 showed a specific and potent inhibitory effect 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 hematomatous tumor cell proliferation by crystalline form A [Table 17]

[0189] Study 4: Pharmacokinetic studies of crystalline form A in mice method: For a longitudinal study, mice were randomly divided into seven groups, with four mice per group. Mice were treated with a single dose of crystalline form A of compound 1 and euthanized with carbon dioxide at different time points after administration (30 minutes, 1, 2, 4, 12, and 24 hours). For dose-dependent studies, mice were randomly divided into 9 groups of 4 mice each. Mice were treated with crystalline form A of compound 1 at different dose levels and euthanized with carbon dioxide 4 hours after administration. Treatment was administered orally via gastric tube feeding at a volume of 10 ml / kg body weight. Body weight was assessed immediately before administration, and the dose was adjusted accordingly.

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

[0191] PK analysis: The maximum plasma concentration (Cmax) and the time to reach Cmax (Tmax) are determined by the plasma concentration. This was obtained directly from the time-versus-time profile.

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

[0193] Study 5: Efficacy study of crystal form A in a TMD-8 xenograft model Tumor transplantation methods: Animals were treated with cyclophosphamide (150 mg / kg in physiological saline, prepared for intraperitoneal use) and disulfide. Rufiram (prepared in physiological saline containing 0.8% Tween80, administered orally at a dose of 125 mg / kg, cyclo Pre-treatment was performed on each of the two days, one hour after each administration of phosphamide. Then, 24 hours after the second administration of cyclophosphamide, TMD-8 cells were inoculated into the animals. On the day of transplantation, the cells were... The culture medium was replaced with fresh medium. After 4 hours, the medium was removed and the cells were collected as described above. The cells were resuspended in cold PBS (4°C) and the same amount of Matrigel (BD, catalog number 356237) was added. ) is added to bring the final concentration to 2.5 × 10 7 The cells were adjusted to a concentration of cells / ml. The resuspended cells were placed on ice before inoculation. The right axillary region of each mouse was washed with 75% ethanol before cell inoculation. Each animal was inoculated with 5 × 10⁶ cells into the right anterior flank via a 26-gauge needle. 6 A 200 μl cell suspension containing cells was administered by subcutaneous injection.

[0194] The in vivo efficacy test will begin on the third day after cell inoculation, and for this purpose, first, The mice were randomly assigned to a desired number of groups, each containing 10 mice. The mice were fed twice daily (BID) on a vehicle (0.5% carboxymethylcellulose (CMC) + 0.2% Tween 80). The patients were treated with crystalline form A of compound 1 at different dose levels for 39 days. Treatment was administered orally via gastric tube feeding at a volume of 10 ml / kg body weight. Body weight was measured immediately before administration. The dosage was adjusted accordingly. Tumor volume was measured two-dimensionally twice a week using calipers (in this study, measurement was possible from day 11 after vaccination). Formula: V = 0.5 × (a × b 2 Tumor volume was calculated using the formula ), 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 period. Body weight was also recorded twice a week. Mice were also monitored daily for clinical signs of toxicity during the study period.

[0195] result: The in vivo efficacy of crystalline form A was demonstrated by TMD-8DLBCL heterologous cells grown subcutaneously in NOD / SCID mice. The study was conducted using grafts. Oral administration was performed twice daily (BID) at different dose levels. Compound 1 was tolerable, and crystalline form A showed dose-dependent antitumor effects. Crystalline form A of compound 1 already exhibited strong antitumor activity in the lowest dose test. No significant effects on animal body weight were observed throughout the study in any of the treatment groups.

[0196] Study 6: Efficacy study of crystal form A in a whole-body REC-1 xenograft model Tumor transplantation methods: The animals were treated with cyclophosphamide (prepared in saline at 150 mpk ip) and disulfiram (0.8 Prepared with saline containing % TW-80, 125 mpkp.o., 1 hour after each administration of cyclophosphamide. The cells were pre-treated for two days each day. Subsequently, REC-1 cells were inoculated into the animals 24 hours after the second dose of cyclophosphamide. On the day of transplantation, the cell culture medium was replaced with fresh medium. After 4 hours, the culture medium was removed and the cells were collected as described above. The cells were then re-inserted into cold (4°C) PBS. Suspend, 1 × 10 8 The final concentration was determined to be cells / ml. The resuspended cells were placed on ice before transplantation. For animals, 1 x 10 7A 100 μl cell suspension containing cells was administered intravenously via the tail vein.

[0197] The in vivo efficacy test will begin on day 8 after cell inoculation, and for this purpose, one group of animals will be used. The mice were randomly assigned to any desired number of groups, each containing 10 mice. The mice were fed twice daily (BID) with a vehicle (0.5% carboxymethylcellulose (CMC) + 0.2% Tween80) and different diets. Treatment was administered with crystalline form A of compound 1 at a dose level for 71 days. All administration was discontinued on day 78 after inoculation. Treatment was performed by oral gastric tube feeding (oral) at a volume of 10 ml / kg body weight. The patient's weight was assessed immediately before administration, and the dosage was adjusted accordingly. The patient's weight was recorded twice a week. (From day 33 onwards, this was changed to three times a week). Clinical signs of disease in the mice were also observed daily during the study period. This study was terminated after the overall survival period had elapsed. In cases where severe toxic effects such as loss of movement were observed, the mice were euthanized and recorded as deceased.

[0198] For data analysis: Survival analysis was performed using the Kaplan-Meier method. Survival time 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, median survival time (MST), survival range with a 95% confidence interval (RST), and life extension (ILS) were calculated. Median survival time was defined as the point at which 50% of the mice died. ILS was calculated using the following formula: %ILS=(MST-MST(vehicle)) / MST(vehicle)×100 Statistical analysis was performed between each group using the Gehan-Breslow-Wilcoxon test. A p-value of < 0.05 was considered statistically significant.

[0199] result: Crystalline form A of compound 1 is used in NOD / SCID mice to treat systemic REC-1 MCL grafts. It showed dependent antitumor activity. The efficacy of crystalline form A of compound 1 was significant in this xenograft model.

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

[0201] Test 8: Pharmacokinetics of crystalline form A The well-validated LC-MS / MS method was frequently used in pharmacokinetic (PK) studies of crystalline form A of compound 1 after single and multiple administrations in Sprague-Dawley rats and beagle dogs.

[0202] Crystalline form A of compound 1 exhibits good oral bioavailability in rats. It was rapidly absorbed and had high plasma clearance (CL) in rats. The pharmacokinetics were linear over the dose range in female rats. Linearity was less favorable in male rats. Accumulation of compound 1 after multiple oral administrations was not statistically significant in both male and female rats. Crystalline form A of compound 1 showed moderate clearance (CL), moderately good bioavailability (F%), linear PK over the dose range, and no accumulation of compound 1 after multiple oral administrations in dogs.

[0203] Test 9: ADME of crystal form A Compound 1 was widely distributed in various tissues, but less so in brain tissue, indicating that this drug does not easily cross the blood-brain barrier.

[0204] The seven major drug-metabolizing CYP isozymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, IC of crystalline form A of compound 1 (and CYP3A) 50 The value is found in human liver microsomes. We determined the compound and further evaluated its time-dependent inhibitory ability against major CYP isozymes. . CYP2C8(IC 50 =4.03μM), CYP2C9(IC 50 =5.69μM) and CYP2C19(IC) 50 = 7.58 μ Weak inhibition was observed against M), but inhibition against other CYP isozymes was even lower. Compound 1 does not appear to be a time-dependent CYP inhibitor against these seven major human CYPs. CYP3A is a major CYP isoform involved in metabolism in human liver microsomes.

[0205] Example 12: Clinical trial study (1) Results of compound 1 in ongoing Phase I clinical trial in patients with advanced B-cell malignancy The initial multicenter, open-label Phase I trial of Compound 1 is being conducted in Australia and New Zealand and consists of two phases—a dose escalation phase and a dose expansion phase involving 25 patients each, with a total of 100 patients to be enrolled. Of the 39 patients enrolled, the initial This includes all 25 patients from the dose escalation phase and 14 patients from the ongoing dose expansion phase. Based on the pharmacokinetics, pharmacodynamics, safety, and efficacy of compound 1 in the dose escalation phase, 320 mg once daily (QD) and 160 mg twice daily (BID) were selected in the ongoing dose expansion study. Further investigation is underway.

[0206] The deadline for data analysis was October 19, 2015. 29 objective responses were observed, including 3 complete responses (CR), 1 very good partial response (VGPR), and 25 partial responses (PR). Histological responses are summarized in Table 18. Of the 39 patients, 31 are continuing the study treatment, and this includes all patients who have responded to date.

[0207] Table 18 Histological response in patients [Table 18] 1 This includes five patients recently diagnosed with lymphocytosis; 2 One patient who had VGPR Including persons Note: CR = Complete response; PR = Partial response; SD = Stable disease; PD = Progressive disease; ORR = Response rate

[0208] Eight patients discontinued compound 1; six due to disease progression and two due to adverse events related to the original malignant tumor. Three patients died during the study as a result of disease progression or complications of disease progression. There were no serious drug-related adverse events (SAEs). Regardless of their relationship to treatment, the majority of adverse events were grade 1 or 2 in severity and did not limit treatment. Of the 19 grade 3 or higher AEs, four were assessed by researchers as potentially drug-related—all were spontaneously resolved neutropenia and did not require discontinuation of treatment. There was one case of major bleeding, a grade 3 or higher bleeding event or an intracranial hemorrhage event of any grade: identified as GI bleeding in a patient with mantle cell lymphoma with lymphomatous infiltration of the GI duct; however, this bleeding event occurred during drughold and resolved rapidly with resumption of treatment with compound 1, and therefore was not drug-related. It was not determined that they were experiencing any adverse events. Six patients had a baseline history of atrial fibrillation / flutter (AF), but no worsening of AF or new events were reported.

[0209] (2) Results of ongoing Phase I clinical trials of compound 1 in patients with Waldenström's macroglobulinemia (WM) A multicenter, open-label Phase I trial of compound 1 in B-cell malignancies was conducted in Australia, New Zealand, South Korea, and the United States, with dose escalation phases set within disease-specific cohorts, including treatment-naïve and relapsed / refractory Waldenström macroglobulinemia (R / R WM). The dose escalation phase in the trail was also included. In the initial phase, the total daily dose was tested in the range of 40 mg to 320 mg, while in the ongoing dose expansion phase... The study doses being tested are 160 mg twice daily (BID) or 320 mg once daily (QD). (2017) As of March 31st of that year, 48 WM patients were enrolled in this study. The response was based on the revised WM criteria. The decision was made in accordance with the criteria of the 6th International Workshop on Modern Warfare (IWWM).

[0210] Compound 1 has not been discontinued due to toxicity associated with Compound 1, and has been shown to date to be well-tolerated. Adverse events (AEs) were generally not severe and resolved spontaneously. The most frequently observed AE among the various attributes of the 48 patients whose safety was evaluated. (>10%) included 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 thought to be associated with compound 1; these included cases of hemothorax, atrial fibrillation, colitis, febrile neutropenia, and headache. Among the AEs of particular interest were a total of three cases of atrial fibrillation (all grade 1). There were cases of grade 1 or 2, and one case of severe bleeding (hemothorax) defined as grade 3 or higher bleeding or central nervous system bleeding of any grade. Treatment was discontinued in three cases: bronchiectasis, prostate adenocarcinoma, and pyloric adenocarcinoma, one case each.

[0211] At the data cutoff, response was evaluable for 42 patients. Among patients for whom the effect could not be evaluated were two patients with a follow-up period of less than 12 weeks, three patients with baseline IgM less than 500 mg / dl, and one patient whose baseline IgM was inaccurate due to being a frozen protein. With a median follow-up period of 12.3 months (4.4–30.5 months), the ORR was 90% of patients (38 / 42 patients), the primary response rate was 76% (32 / 42 patients), but the VGPR was 43% (18 / 42 patients) and the partial response rate was 33% (14 / 42).

[0212] (3) Compound 1 in patients with chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL / SLL) Results of the ongoing Phase I clinical trial A multicenter, open-label Phase I trial of compound 1 in patients with B-cell malignancies was 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. In the trail dose escalation portion, the total daily dose was tested in the range of 40 mg to 320 mg. During the ongoing dose escalation phase, the test dose is 160 mg twice daily (BID) or 320 mg once daily (QD). As of March 31, 2017, 69 CLL or SLL patients (18TN, 51R / R) have participated in the trial. It is recorded.

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

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

Claims

1. Compound 1 【Chemistry 1】 A solid, wherein the solid has an enantiomer excess value of at least 97%.

2. The solid according to claim 1, wherein the solid has a value of 97% enantiomer excess.

3. The solid according to claim 1, wherein the solid has an enantiomer excess value greater than 97%.

4. The solid according to claim 1, wherein the solid is an anhydrous substance.

5. A composition comprising the solid substance described in Claim 1 for treating a B-cell proliferative disorder in a subject requiring such treatment, wherein the B-cell proliferative disorder is selected from the group consisting of chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, Waldenstrom macroglobulinemia, marginal zone lymphoma, and follicular lymphoma, and compound 1 is administered in a dose of 160 mg twice daily (BID) or 320 mg once daily (QD).

6. The composition according to claim 5, characterized in that compound 1 is administered in a dose of 160 mg twice daily (BID).

7. The composition according to claim 5, characterized in that compound 1 is administered in a dose of 320 mg once daily (QD).

8. The composition according to claim 5, wherein the B-cell proliferative disorder is chronic lymphocytic leukemia or small lymphocytic lymphoma.

9. The composition according to claim 5, wherein the B-cell proliferative disorder is mantle cell lymphoma.

10. The composition according to claim 9, wherein the subject has received at least one prior therapy.

11. The composition according to claim 5, wherein the B-cell proliferative disorder is Waldenstrom macroglobulinemia.

12. The composition according to claim 5, wherein the B-cell proliferative disorder is marginal zone lymphoma.

13. The composition according to claim 12, wherein the marginal zone lymphoma is recurrent or refractory.

14. The composition according to claim 5, wherein the B-cell proliferative disorder is follicular lymphoma.

15. The composition according to claim 14, wherein the follicular lymphoma is recurrent or refractory.

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