Crystalline form of (s)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[1,5-a]pyrimidine-3-carboxamide, preparation thereof, and uses thereof

Abstract

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

Description

Technical Field

[0001] The present invention relates to a crystalline form of (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5 ,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide. The present invention also relates to a method for preparing the crystalline form and a method for using the crystalline form as a Btk inhibitor .

Background Art

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

[0003] These signaling pathways must be precisely regulated. Btk is encoded Mutations in the gene that do this cause hereditary B cell-specific immunodeficiency in humans which is known as X-linked agammaglobulinemia (XLA) (Conley et al., Annu. Rev. Immunol. 27: 199-227, 2009). Abnormalities in BCR-mediated signal transduction cause disorders in the regulation of B cell activation, resulting in the possibility of many autoimmune and inflammatory diseases. Preclinical studies have shown that Btk-deficient mice are resistant to the development of collagen-induced arthritis. Furthermore, clinical studies on the CD20 antibody rituximab, which can deplete mature B cells, have revealed that B cells play important roles in many inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis (Gurcan et al., Int. Immunopharmacol. 9: 10-25, 2009). Thus, Btk inhibitors can be used to treat autoimmune and / or inflammatory diseases.

[0004] Furthermore, abnormal activation of Btk plays an important role in the development of B cell lymphoma from which it can be seen that inhibition of Btk is useful for the treatment of hematological malignancies (Davis et al., Nature 463:88-92, 2010). From the results of preliminary clinical trials, it was found that the Btk inhibitor PCI-32765 is effective in the treatment of several types of B cell lymphomas (for example, 54th American Society of Hema tology (ASH) annual meeting abstract, Dec. 2012: 686 The Bruton's Tyrosine Kinas The Bruton tyrosine kinase (Btk) inhibitor, ibrutinib (PCI-32765), has preferential activity in the ABC subtype of relapsed / refractory de novo diffuse large B-cell lymphoma (DLBCL): Interim results of a multicenter, open-label, phase I study). Since Btk plays a central role as a mediator in multiple signaling pathways, Btk inhibitors have been of great interest as anti-inflammatory and / or anti-cancer agents (Mohamed et al., Immunol. Rev. 228: 58-73, 2009; Pan, Drug News Perspect 21: 357-362, 2008; Rokosz et al., Expert Opin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer Agents Med. Chem. 7: 624-632, 2007; Lou et al., J. Med. Chem. 55(10): 4539-4550, 2012). Subtype of Relapsed / Refractory De Novo Diffuse Large B-Cell Lymphoma (DLBCL): In terim Results of a Multicenter, Open-Label, Phase I Study). Btk plays a central role as a mediator in multiple signaling pathways, so Btk inhibitors have been of great interest as anti-inflammatory and / or anti-cancer agents (Mohamed et al., Immunol. Rev. 228: 58-73, 2009; Pan, Drug News Perspect 21: 357-362, 2008; Rokosz et al., Expert Opin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer Agents Med. Chem. 7: 624-632, 2007; Lou et al., J. Med. Chem. 55(10): 4539-4550, 2012). Since Btk plays a central role as a mediator in multiple signaling pathways, Btk inhibitors have been of great interest as anti-inflammatory and / or anti-cancer agents (Mohamed et al., Immunol. Rev. 228: 58-73, 2009; Pan, Drug News Perspect 21: 357-362, 2008; Rokosz et al., Expert Opin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer Agents Med. Chem. 7: 624-632, 2007; Lou et al., J. Med. Chem. 55(10): 4539-4550, 2012). Since Btk plays a central role as a mediator in multiple signaling pathways, Btk inhibitors have been of great interest as anti-inflammatory and / or anti-cancer agents (Mohamed et al., Immunol. Rev. 228: 58-73, 2009; Pan, Drug News Perspect 21: 357-362, 2008; Rokosz et al., Expert Opin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer Agents Med. Chem. 7: 624-632, 2007; Lou et al., J. Med. Chem. 55(10): 4539-4550, 2012). Since Btk plays a central role as a mediator in multiple signaling pathways, Btk inhibitors have been of great interest as anti-inflammatory and / or anti-cancer agents (Mohamed et al., Immunol. Rev. 228: 58-73, 2009; Pan, Drug News Perspect 21: 357-362, 2008; Rokosz et al., Expert Opin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer Agents Med. Chem. 7: 624-632, 2007; Lou et al., J. Med. Chem. 55(10): 4539-4550, 2012). Since Btk plays a central role as a mediator in multiple signaling pathways, Btk inhibitors have been of great interest as anti-inflammatory and / or anti-cancer agents (Mohamed et al., Immunol. Rev. 228: 58-73, 2009; Pan, Drug News Perspect 21: 357-362, 2008; Rokosz et al., Expert Opin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer Agents Med. Chem. 7: 624-632, 2007; Lou et al., J. Med. Chem. 55(10): 4539-4550, 2012). Since Btk plays a central role as a mediator in multiple signaling pathways, Btk inhibitors have been of great interest as anti-inflammatory and / or anti-cancer agents (Mohamed et al., Immunol. Rev. 228: 58-73, 2009; Pan, Drug News Perspect 21: 357-362, 2008; Rokosz et al., Expert Opin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer Agents Med. Chem. 7: 624-632, 2007; Lou et al., J. Med. Chem. 55(10): 4539-4550, 2012). Since Btk plays a central role as a mediator in multiple signaling pathways, Btk inhibitors have been of great interest as anti-inflammatory and / or anti-cancer agents (Mohamed et al., Immunol. Rev. 228: 58-73, 2009; Pan, Drug News Perspect 21: 357-362, 2008; Rokosz et al., Expert Opin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer Agents Med. Chem. 7: 624-632, 2007; Lou et al., J. Med. Chem. 55(10): 4539-4550, 2012).

[0005] International Application WO2014173289A discloses a series of 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). 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). 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). 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

[0006] Compound 1 is a potent, specific, and irreversible BTK kinase inhibitor. From the data obtained in biochemical research, cell-based research, and preclinical trials with animal studies, it is suggested that the effect brought about by Compound 1 regarding the suppression of tumor growth in B-cell malignancies is significant. When compared with EGFR, FGR, FRK, HER2, HER4, ITK, JAK3, LCK, and TEC, Compound 1 was shown to be more selective than the inhibition of BTK by ibrutinib. Therefore, Compound 1 is expected to have fewer side effects than ibrutinib clinically. Furthermore, since Compound 1 has a weak inhibitory ability against ITK, the degree of inhibition of rituximab-induced, antigen-dependent, cell-mediated cytotoxicity (ADCC) is significantly less than that of ibrutinib. Therefore, when combined with rituximab or other ADCC-dependent antibodies, it may be well effective in the treatment of B-cell malignancies. In the single-dose and up to 28-day repeated-dose toxicity tests on rats and dogs in preclinical safety evaluations, Compound 1 was demonstrated to be safer than ibrutinib regarding overall tolerance and severe toxicity. Furthermore, since Compound 1 has no accumulation problems as observed for ibrutinib, its bioavailability was even better. Based on these unique characteristics of Compound 1, clinical trials are conducted for further evaluation. However, in the preparation method of Compound 27 of International Publication No. WO2014173289, Compound 1 is ... ...

[0007] In the toxicity tests of single-dose and repeated-dose up to 28 days on rats and dogs in preclinical safety evaluations, Compound 1 was demonstrated to be safer than ibrutinib in terms of overall tolerance and severe toxicity. Furthermore, since Compound 1 has no accumulation problems as observed for ibrutinib, its bioavailability was even better. Based on these unique characteristics of Compound 1, clinical trials are conducted for further evaluation. ... ...

Summary of the Invention

Problems to be Solved by the Invention

[0008] However, in the preparation method of Compound 27 of International Publication No. WO2014173289, Compound 1 It is amorphous, which was further confirmed by the X-ray powder diffraction pattern of Fig. 7A. This amorphous Compound 1 was shown to have a low glass transition temperature as shown in Fig. 7B, from which problems in the formulation of amorphous compounds, such as low stability and difficulty in purification, became apparent. . Therefore, it is necessary to develop a new form of Compound 1, and its properties, such as a high melting point and high stability, are required to be suitable for formulation.

Means for Solving the Problems

[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 24 months or at 40 °C / 75% RH for 6 months. However, this discovery was unexpected.

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

Chemical Formula

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

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

Chemical Formula

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

[0014] Also disclosed herein is an intermediate compound of formula Ie or a salt thereof, or a compound of formula If or a salt thereof, which are used in the preparation of Compound 1.

Chemical formula

[0015] Disclosed herein as a fourth aspect is a method for preparing crystalline form A.

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

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

[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 disease selected from B cell malignancies or relapsed / refractory B cell malignancies in a subject by administering crystalline form A disclosed herein to the subject. Some aspects in this regard disclosed herein are that, by administering crystalline form A disclosed herein to the subject, those of chronic lymphocytic nature, non- ​​​​​​A method for treating B-cell proliferative diseases selected from Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof. chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof. A method for treating B-cell proliferative diseases selected from Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof. A method for treating B-cell proliferative diseases selected from Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof.

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

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

[0022] As an eleventh aspect, disclosed herein is the use of crystalline form A disclosed herein in the manufacture of a medicament for the treatment of B-cell proliferative diseases selected from B-cell malignancies or recurrent / refractory B-cell malignancies in a subject. Some aspects of this perspective disclosed herein are for the manufacture of a medicament for the treatment of, in a subject, chronic lymphocytic ones, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof. As an eleventh aspect, disclosed herein is the use of crystalline form A disclosed herein in the manufacture of a medicament for the treatment of B-cell proliferative diseases selected from B-cell malignancies or recurrent / refractory B-cell malignancies in a subject. Some aspects of this perspective disclosed herein are for the manufacture of a medicament for the treatment of, in a subject, chronic lymphocytic ones, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof. As an eleventh aspect, disclosed herein is the use of crystalline form A disclosed herein in the manufacture of a medicament for the treatment of B-cell proliferative diseases selected from B-cell malignancies or recurrent / refractory B-cell malignancies in a subject. Some aspects of this perspective disclosed herein are for the manufacture of a medicament for the treatment of, in a subject, chronic lymphocytic ones, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof. As an eleventh aspect, disclosed herein is the use of crystalline form A disclosed herein in the manufacture of a medicament for the treatment of B-cell proliferative diseases selected from B-cell malignancies or recurrent / refractory B-cell malignancies in a subject. Some aspects of this perspective disclosed herein are for the manufacture of a medicament for the treatment of, in a subject, chronic lymphocytic ones, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof. As an eleventh aspect, disclosed herein is the use of crystalline form A disclosed herein in the manufacture of a medicament for the treatment of B-cell proliferative diseases selected from B-cell malignancies or recurrent / refractory B-cell malignancies in a subject. Some aspects of this perspective disclosed herein are for the manufacture of a medicament for the treatment of, in a subject, chronic lymphocytic ones, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof. As an eleventh aspect, disclosed herein is the use of crystalline form A disclosed herein in the manufacture of a medicament for the treatment of B-cell proliferative diseases selected from B-cell malignancies or recurrent / refractory B-cell malignancies in a subject. Some aspects of this perspective disclosed herein are for the manufacture of a medicament for the treatment of, in a subject, chronic lymphocytic ones, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof. As an eleventh aspect, disclosed herein is the use of crystalline form A disclosed herein in the manufacture of a medicament for the treatment of B-cell proliferative diseases selected from B-cell malignancies or recurrent / refractory B-cell malignancies in a subject. Some aspects of this perspective disclosed herein are for the manufacture of a medicament for the treatment of, in a subject, chronic lymphocytic ones, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof. As an eleventh aspect, disclosed herein is the use of crystalline form A disclosed herein in the manufacture of a medicament for the treatment of B-cell proliferative diseases selected from B-cell malignancies or recurrent / refractory B-cell malignancies in a subject. Some aspects of this perspective disclosed herein are for the manufacture of a medicament for the treatment of, in a subject, chronic lymphocytic ones, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, hairy cell leukemia, Burkitt-like leukemia, or a combination of two or more thereof.

[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 systems to form a transparent solution; while stirring the solution or without stirring, keep it at room temperature for a certain period of time or precipitate crystalline form A while heating and the solution system is as follows: Ethyl acetate: hexane = 1: 0.6 - 0.7 (volume ratio); Ethyl acetate: heptane = 1: 0.6 - 0.7 (volume ratio); Ethyl acetate: cyclohexane = 1: 0.6 - 1.2 (volume ratio); Methyl acetate: hexane = 1: 0.6 - 1.2 (volume ratio); Toluene: hexane = 1.0: 0.2 - 0.4 (volume ratio); Toluene: cyclohexane = 1.0: 0.1 - 0.2 (volume ratio); Methyl acetate: cyclohexane = 0.6 - 0.8: 1.0 (volume ratio); IPAC: cyclohexane = 1.0: 0.2 - 1.0 (volume ratio); or Isobutyl acetate: cyclohexane = 1.0: 0.2 - 1.0 (volume ratio).

[0024] In one embodiment, the ee value of amorphous compound 1 exceeds 90%. In other embodiments the ee value of amorphous compound 1 is 97%.

Brief Description of the Drawings

[0025]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7A

Figure 7B

Figure 8

Figure 9

Figure 10

Figure 11

Mode for Carrying Out the Invention

[0026] The crystalline Compound 1 discovered by the inventors was unexpected and was named Crystal Form A, but it could only be obtained under specific conditions depending on the ee value of the starting material and the ratio of the co-solvent, etc. It could only be obtained under specific conditions depending on the ee value of the starting material and the ratio of the co-solvent, etc. Evaporation was carried out over time, a poor solvent was added, cooling was carried out over time, vapor diffusion was carried out, and polymorph studies were also carried out by methods such as polymer-induced crystallization. Most of the experiments could not obtain crystalline forms, indicating that it is not easy to obtain Crystal Form A. Evaporation was carried out over time, a poor solvent was added, cooling was carried out over time, vapor diffusion was carried out, and polymorph studies were also carried out by methods such as polymer-induced crystallization. Most of the experiments could not obtain crystalline forms, indicating that it is not easy to obtain Crystal Form A. Evaporation was carried out over time, a poor solvent was added, cooling was carried out over time, vapor diffusion was carried out, and polymorph studies were also carried out by methods such as polymer-induced crystallization. Most of the experiments could not obtain crystalline forms, indicating that it is not easy to obtain Crystal Form A. indicating that it is not easy to obtain Crystal Form A.

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

[0028] In one embodiment of crystalline form A, its X-ray powder diffraction pattern includes diffraction peaks at 2θ angular values individually selected from about 14.8 ± 0.2°, 16.4 ± 0 .2°, and 21.4 ± 0.2°. are included.

[0029] In one embodiment of crystalline form A, its X-ray powder diffraction pattern includes diffraction peaks at 2θ angular values individually selected from about 14.8 ± 0.2°, 15.6 ± 0 .2°, 16.4 ± 0.2°, and 21.4 ± 0.2°. are included.

[0030] In one embodiment of crystalline form A, its X-ray powder diffraction pattern includes diffraction peaks at 2θ angular values individually selected from about 12.2 ± 0.2°, 12.9 ± 0 .2°, 14.8 ± 0.2°, 15.6 ± 0.2°, 16.4 ± 0.2°, and 21.4 ± 0.2°. are included.

[0031] In one embodiment of crystalline form A, its X-ray powder diffraction pattern includes diffraction peaks at 2θ angular values individually selected from about 12.2 ± 0.2°, 12.9 ± 0 .2°, 14.8 ± 0.2°, 15.6 ± 0.2°, 16.4 ± 0.2°, 17.7 ± 0.2°, 18.5 ± 0.2°, 20.7 ± 0.2 °, and 21.4 ± 0.2°. .

[0032] In some embodiments of crystalline form A, an X-ray powder diffraction pattern substantially as shown in FIG. 1 is observed. is seen.

[0033] In some embodiments of crystalline form A, an X-ray powder diffraction pattern summarized in Table 1 is observed. is seen.

[0034] Table 1 X-ray Diffraction Pattern of Crystalline Form A

Table 1

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

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

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

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

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

[0040] Also disclosed herein is a crystalline form of compound BG-13 having an X-ray powder diffraction pattern substantially as shown in Figure 11.

Chemical formula

[0041] In some embodiments, the crystalline 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] From the single crystal X-ray structure analysis of intermediate BG-13, the present inventors presumed that the absolute configuration of compound 1 is S.

[0043] Also disclosed herein is a method for preparing compound 1 and deuterium-labeled compound 1, which is as per the procedure shown in Scheme 1. The novel synthesis method and crystallization / recrystallization procedure of compound 1 via crystalline form A disclosed herein overcome many problems associated with previously reported methods, such as the preparation of important chiral intermediates having an optical purity exceeding 98%. It meets the acceptable levels described herein by improving the purity of compound 1, controls the impurities in compound 1, and has numerous advantages compared to existing methods. In particular, the method disclosed herein is highly reproducible and particularly suitable for producing compound 1 on a commercial scale, resulting in improved quality and good yields. In other methods, BG-9 or its analog in Scheme 1 could be asymmetrically reduced with low to high enantioselectivity (from 5% ee to 95% ee). The other steps are the same as those listed in Scheme 1. The other steps are the same as those listed in Scheme 1.

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

Chemical formula

Chemical formula

[0045] ​​​​​​​​​​Also disclosed herein is a process for preparing a compound of formula Ia, which comprises performing an asymmetric reduction on a compound of formula I in the presence of a catalyst and / or a reducing agent for producing the compound of formula Ia wherein the asymmetric reduction is carried out in the presence of a catalyst and / or a reducing agent for producing the compound of formula Ia is as follows. [Chemical formula] Formula I Formula Ia wherein R 1 is hydrogen or an amino protecting group.

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

[0047] In some embodiments, the catalyst is a neutral catalyst system or a cationic catalyst system. In some embodiments, the catalyst is [Ir(COD)Cl]2 / (R or S)-MeO-Biphep, [Ir(COD)Cl]2 / (R or S)-Binap, [Ir(COD)Cl]2 / (R or S)-Tol-Binap, [Ir(COD)Cl]2 / (R or S)-xyl-Binap, [Ir(COD)Cl]2 / (S,S or R,R)-Diop, [Ir(COD)Cl]2 / (R or S)-P-Phos, [Ir(COD)Cl]2 / (R or S)-Tol-P-P ​​​​​​​​​hos, [Ir(COD)Cl]2 / (R or S)-Xyl-P-Phos, [Ir(COD)Cl] 2 / (R,R or S,S)-Me-DuPhos, [Ir( COD)Cl] 2 / (R or S)-SegPhos, [Ir(μ-Cl)(cod)] 2 / (R or S)-Ship, [Ir(μ-Cl)(cod)] 2 / (R or S)-Siphos, [Ir(μ-Cl)(cod)] 2 / (R or S)-Siphos-PE, [Ir(μ-Cl)(cod)] 2 / (R or S)- MonoPhos, [Ir(μ-Cl)(cod)] 2 / (R or S)-tol-SDP, [Ir(μ-Cl)(cod)] 2 / (S,S or R,R)-Dio p, [Ir(μ-Cl)(cod)] 2 / (S,R or R,S)-Josiphos, [Ir(μ-Cl)(cod)] 2 / (R or S)-Binap, [I r(μ-Cl)(cod)] 2 / (R or S)-MeO-Biphep, [Ir(μ-Cl)(cod)] 2 / (R or S)-Synphos, or [Ir (μ-Cl)(cod)] 2 / (R or S)-Difluorphos or [Ir(cod) 2 + X - (X: e.g. BF 4 , NO 3 , OTf, PF 6 , SbF 6 and the iridium catalyst systems containing the above-mentioned ligands added thereto for BarF) (Wen-Bo et al., J. AM. CHEM. SOC. 125, 10536-10537 2003. Damien et al., J. Org. ​Chem. 77,4544-4556, 2012. Milos et al., Org. Process Res. Dev. 16, 1293-1300, 2012.) but not limited thereto; [Rh(COD) 2 BF 4 to which the above-mentioned ligand is added including rhodium catalyst systems (Xiang-Ping et al., Top Organomet Chem 36, 313-354, 201 1) but not limited thereto; or RuCl 2 (R or S)-BINAP / (R or S)-DAIPEN, RuCl 2 ( (R or S)-BINAP / (R,R or S,S)-DPEN, RuCl 2 (S or R)-BINAP (S,S or R,R)-DACH, RuCl 2 [(R (R or S)-Tol-BINAP][(S,S or R,R)-DPEN], RuCl 2 (R,R or S,S)-Me-DuPHOS / (R,R or S,S)-D PEN, RuCl 2 (R,R or S,S)-Et-DuPHOS / (R,R or S,S)-DPEN, RuCl 2 (R,R or S,S)-Et-DuPHOS / (R,R or S,S)-DACH, RuCl 2 (S,S or R,R)-i-Pr-DuPHOS / (R,R or S,S)-DPEN, RuCl 2 (R or S )-HexaPHEMP / (R,R or S,S)-DPEN, RuCl 2 (R or S)-MeO-BIPHEP / (R,R or S,S)-DPEN-containing ruthenium catalyst systems (Christopher et al., Adv. Synth. Catal. 345, 195-201, 2003. Jul ian et al., Adv. Synth. Catal. 345, 300-307, 2003.), but is not limited thereto No.

[0048] In the above method, it has been found that excellent enantioselectivity reaching 95% ee can be obtained by using the above catalyst, especially with a neutral or cationic iridium catalyst system. It has been found. No.

[0049] Disclosed herein is a method for dissolving a compound of formula IIa to produce a compound of formula IIb or to increase the chiral purity of the compound of formula IIb, which comprises treating a racemic compound of formula IIa with a chiral acid. Including the step of treating a racemic compound of formula IIa with a chiral acid. No. [Chemical formula] Formula IIa Formula IIb Where R 1 Is hydrogen, methyl, benzyl, 4-methoxybenzyl or other Conventionally known amino protecting groups as described above.

[0050] In some embodiments, the chiral acid includes, but is not limited to, L-malic acid, D-malic acid, L-mandelic acid, D-mandelic acid, L-camphorsulfonic acid, D-camphorsulfonic acid, L-tartaric acid, D-tartaric acid, L-DBTA, D-DBTA, L-DTTA, or D-DTTA. No.

[0051] Also disclosed herein is a method for dissolving a compound of formula Ic to produce a compound of formula Id or to increase the chiral purity of the compound of formula Id, which comprises treating a racemic compound of formula Ic with a chiral acid. Including the step of treating a racemic compound of formula Ic with a chiral acid. No. [Chemical formula] Formula Ic and Formula Id Here, R 1 is hydrogen, methyl, benzyl, 4-methoxybenzyl or other commonly known amino protecting groups as described above.

[0052] In some embodiments, the chiral acid is L-malic acid, D-malic acid, L-mandelic acid , D-mandelic acid, L-camphorsulfonic acid, D-camphorsulfonic acid, L-tartaric acid, D-tartaric acid, L-DBTA, D-DBTA, L-DTTA, or D-DTTA, but is not limited thereto.

[0053] Further disclosed herein are compounds of Formula Ie or salts thereof, or compounds of Formula If or salts thereof that are used in the preparation of Compound 1.

Chemical formula

Chemical formula

[0054] Furthermore, a method for preparing crystalline form A is also provided. The crystalline forms disclosed herein can be prepared from a suitable solvent system containing at least one solvent by crystallizing the compounds disclosed herein, and by spontaneous precipitation (evaporation), cooling, and / or addition of an anti-solvent (where the compounds disclosed herein have relatively low solubility) to supersaturate the solvent system. The crystallization can be obtained by the method. Crystallization can also be carried out by using a crystal seed suitable for crystallizing the crystalline forms disclosed herein, and can also be crystallized without using it.

[0055] ​​​​In some embodiments, in the method for preparing crystalline form A, (S)-7-(1-acryloylpiper lysin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyr imidine-3-carboxamide (Compound 1) is dissolved in DCM, exchanged with the solvent EA, and recrystallized from EA / MTBE to obtain the desired crystalline form. This includes obtaining the desired crystalline form through the step of

[0056] In some embodiments, the method for preparing crystalline form A includes dissolving Compound 1 in EA and obtaining the desired crystalline form through the step of adding hexane.

[0057] In some embodiments, the method for preparing crystalline form A is performed by adding an anti-solvent to a solution in which a solid Compound 1 or a crude purified form A is in a solvent that dissolves these solids. Examples of anti-solvents include H 2O and n-heptane, but are not limited thereto. Examples of solvents for dissolving the solids include acetone, DMAc, EtOAc, D CM, toluene, and 2-MeTHF, but are not limited thereto. 2 In some embodiments, the method for preparing crystalline form A is to add a solution in which a solid Compound 1 or a crude purified form A is in a solvent to an anti-solvent, and then take a time sufficient for the organic vapor to interact with the solution in a sealed reactor. Examples of solvents include acetone and

[0058] EtOAc, but are not limited thereto. Examples of anti-solvents include n-heptane, but are not limited thereto.

[0059] Also disclosed herein are a therapeutically effective amount of crystalline form A and a pharmaceutically acceptable excipient It is a pharmaceutical composition. In some embodiments, the pharmaceutical composition is used by oral administration . 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 3 0% by weight of crystalline form A.

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

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

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

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

[0064] In some embodiments, B cell proliferative diseases include relapsed / refractory (R / R) B cell malignancies such as R / R MCL, R / R CLL, R / R SLL , R / R WM, but are not limited thereto .

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

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

[0067] The crystalline form A disclosed herein can be used to manufacture a medicament for treating a B cell proliferative disease selected from B cell malignancies or relapsed / refractory B cell malignancies in a subject .

[0068] In recent clinical trials, compound 1 has continued to be demonstrated to be well tolerated in untreated (TN) and relapsed / refractory (R / R) B cell malignancies. For example, in WM, in a cohort of 42 evaluable patients, the rate of very good partial response (VGPR) exceeded 40%, and in a cohort of 42 evaluable patients with a median follow-up of 12.3 months, the overall response rate (ORR) was 90%. Furthermore, in CLL / SLL, at the median follow-up of 10.5 months for efficacy evaluation, the overall response rate was high (94%) and the treatment discontinuation rate was very low (3 %). %.​​​​​​

[0069] Definition Unless otherwise specifically defined elsewhere in this document, all other technical terms and scientific terms used herein shall have the meanings generally understood by those skilled in the technical field to which the present invention

[0070] belongs. Words in the singular such as "a", "an", and "the", including the appended claims, shall include their corresponding plurals unless the context clearly indicates otherwise. Thus, for example, reference to "crystal form" includes one or more different crystal forms, etc., and reference to "method" includes equivalent

[0071] steps and methods known to those skilled in the art and which may modify or replace the methods described herein. As disclosed herein, the crystal form is a substantially pure crystal. The term

[0072] "substantially pure" as used herein refers to the crystal form A disclosed herein being at least 85% by weight, preferably at least 95% by weight, more preferably at least 99% by weight. For the crystal forms disclosed herein, only the major peaks (i.e., the most

[0073] characteristic, significant, unique and / or reproducible peaks) are summarized; refers to an X-ray powder diffraction pattern showing the same major peaks as 1, where the major peaks are those having a relative intensity exceeding 10%, preferably exceeding 20%, relative to the highest peak (with a relative intensity of 100%) in FIG. 1.

[0074] Throughout this specification and the appended claims, unless the context requires otherwise , the words "comprise", and the conjugations "comprises" and "comprising", are to be understood to mean including the stated integer or step or group of integers or steps. As used herein, the term "comprising" may be replaced by the term "containing" and may sometimes be replaced by the term "having" as used herein.

[0075] As used herein, the term "therapeutically effective amount" is the amount of a compound which, when administered to a subject for treating a disease, affects treatment of the disease, disorder, or condition, or at least one clinical manifestation of the disease, disorder, or condition, in a manner sufficient to effect treatment. A "therapeutically effective amount" may vary depending on the compound, the disease, the disorder, and / or the symptoms of the disease or disorder, the severity of the disease, disorder, and / or the disease or disorder symptoms, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in a given instance may be apparent to those skilled in the art or may be determined by routine experimentation. In the case of combination therapy,

[0076] The pharmaceutical composition containing the compounds disclosed herein can be administered to a subject in need thereof by oral administration, inhalation administration, rectal administration, parenteral administration or topical administration. In the case of oral administration, the pharmaceutical composition may be in the form of ordinary solid preparations such as tablets, powders, granules, capsules, etc., or liquid preparations such as suspensions in water or oil, or other liquid preparations such as syrups, solutions, suspensions, etc.; in the case of parenteral administration, the pharmaceutical composition may be a solution, aqueous solution, concentrate of oily suspension, lyophilized powder, etc. The preparation 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 be a single unit administered in an exact dosage. The pharmaceutical composition may further additionally contain an active ingredient. All formulations of the pharmaceutical compositions disclosed herein can be manufactured by ordinary methods in the pharmaceutical field. For example, the desired formulation can be manufactured by mixing the active ingredient with one or more excipients. "Pharmaceutically acceptable excipients" refers to ordinary pharmaceutical carriers suitable for the desired pharmaceutical formulation, such as, for example: diluents, vehicles such as water, various organic solvents, fillers such as starch, sucrose, cellulose derivatives, alginates, gelatin and binders such as polyvinylpyrrolidone (PVP); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate, sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as hexadecanol; absorbent carriers such as kaolin, soapclay; talc, calcium stearate In the case of oral administration, the pharmaceutical composition may be in the form of ordinary solid preparations such as tablets, powders, granules, capsules, etc., or liquid preparations such as suspensions in water or oil, or other liquid preparations such as syrups, solutions, suspensions, etc. In the case of oral administration, the pharmaceutical composition may be in the form of ordinary solid preparations such as tablets, powders, granules, capsules, etc., or liquid preparations such as suspensions in water or oil, or other liquid preparations such as syrups, solutions, suspensions, etc. In the case of parenteral administration, the pharmaceutical composition may be a solution, aqueous solution, concentrate of oily suspension, lyophilized powder, etc. In the case of parenteral administration, the pharmaceutical composition may be a solution, aqueous solution, concentrate of oily suspension, lyophilized powder, etc. The preparation 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 preparation 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 be a single unit administered in an exact dosage.

[0077] All formulations of the pharmaceutical compositions disclosed herein can be manufactured by ordinary methods in the pharmaceutical field. For example, the desired formulation can be manufactured by mixing the active ingredient with one or more excipients. For example, the desired formulation can be manufactured by mixing the active ingredient with one or more excipients. "Pharmaceutically acceptable excipients" refers to ordinary pharmaceutical carriers suitable for the desired pharmaceutical formulation, such as, for example: diluents, vehicles such as water, various organic solvents, fillers such as starch, sucrose, cellulose derivatives, alginates, gelatin and binders such as polyvinylpyrrolidone (PVP); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate, sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as hexadecanol; absorbent carriers such as kaolin, soapclay; talc, calcium stearate "Pharmaceutically acceptable excipients" refers to ordinary pharmaceutical carriers suitable for the desired pharmaceutical formulation, such as, for example: diluents, vehicles such as water, various organic solvents, fillers such as starch, sucrose, cellulose derivatives, alginates, gelatin and binders such as polyvinylpyrrolidone (PVP); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate, sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as hexadecanol; absorbent carriers such as kaolin, soapclay; talc, calcium stearate "Pharmaceutically acceptable excipients" refers to ordinary pharmaceutical carriers suitable for the desired pharmaceutical formulation, such as, for example: diluents, vehicles such as water, various organic solvents, fillers such as starch, sucrose, cellulose derivatives, alginates, gelatin and binders such as polyvinylpyrrolidone (PVP); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate, sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as hexadecanol; absorbent carriers such as kaolin, soapclay; talc, calcium stearate "Pharmaceutically acceptable excipients" refers to ordinary pharmaceutical carriers suitable for the desired pharmaceutical formulation, such as, for example: diluents, vehicles such as water, various organic solvents, fillers such as starch, sucrose, cellulose derivatives, alginates, gelatin and binders such as polyvinylpyrrolidone (PVP); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate, sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as hexadecanol; absorbent carriers such as kaolin, soapclay; talc, calcium stearate "Pharmaceutically acceptable excipients" refers to ordinary pharmaceutical carriers suitable for the desired pharmaceutical formulation, such as, for example: diluents, vehicles such as water, various organic solvents, fillers such as starch, sucrose, cellulose derivatives, alginates, gelatin and binders such as polyvinylpyrrolidone (PVP); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate, sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as hexadecanol; absorbent carriers such as kaolin, soapclay; talc, calcium stearate "Pharmaceutically acceptable excipients" refers to ordinary pharmaceutical carriers suitable for the desired pharmaceutical formulation, such as, for example: diluents, vehicles such as water, various organic solvents, fillers such as starch, sucrose, cellulose derivatives, alginates, gelatin and binders such as polyvinylpyrrolidone (PVP); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate, sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as hexadecanol; absorbent carriers such as kaolin, soapclay; talc, calcium stearate , such as lubricants like magnesium stearate and polyethylene glycol. Also, 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 anything such as a disease, discomfort, illness, symptom or sign, and is replaceable with terms such as "disorder" or "condition".

[0079] Abbreviations: AcOH_Acetic acid AEs_Adverse events BID_Twice a day 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 sorption EA_Ethyl acetate, EtOAc EDCI_1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EtOH_Ethanol FL_Follicular lymphoma GC_Gas chromatograph GCMS_Gas chromatography - mass spectrometry HOAc_Acetic acid HOBt - Hydroxybenzotriazole HPLC - High Performance Liquid Chromatography IPA - Isopropyl Alcohol IPAc - Isopropyl Acetate IPC - In - process Control KF - Karl Fischer L - DBTA - (2R,3R) - Dibenzoyl tartaric acid LOQ - Limit of Quantification MCL - Mantle Cell Lymphoma MeCN or ACN - Acetonitrile MeMgBr - Methylmagnesium Bromide MeOH - Methanol 2 - MeTHF - 2 - Methyltetrahydrofuran MIBE - 4 - Methyl - 2 - pentanone MsOH - Methanesulfonic Acid MTBE - Methyl tert - butyl Ether NHL - Non - Hodgkin Lymphoma NLT - Not less than NMP - 1 - Methyl - 2 - pyrrolidone NMR - Nuclear Magnetic Resonance NMT - Not more than ORR - Overall Response Rate Pd - Palladium pH - Hydrogen Ion Concentration POA - Phenoxyacetyl QD - Once a day RH - Relative Humidity SLL - Small Lymphocytic Lymphoma RT - Room Temperature TEA - Triethylamine TGA - Thermogravimetric Analysis THF - Tetrahydrofuran TN - Treatment - naive VGPR - Very Good Partial Response XRPD - X - ray Powder Diffraction WM - Waldenstrom Macroglobulinemia

Examples

[0080] The present invention will be further illustrated by the following examples for explaining the present invention, but it does not limit the present invention. to be. Example 1 Preparation of (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7 -tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 1) and its Crystal Form A Step 1: Synthesis of BG-2 [Chemical formula]

[0081] Under a nitrogen atmosphere, TEA (2.4 equivalents) was added to a solution of EA (5 v), HOBT (1.2 equivalents), EDCI (1.2 equivalents), 4- phenoxybenzoic acid (BG-1, 80 Kg, 1.0 equivalent) and malononitrile (1.2 equivalents) at 10 °C. Then the mixture was stirred at room temperature until the reaction was complete. Then the mixture was centrifuged separated, and the cake (precipitate) was washed with EA. The filtrate was washed twice with an aqueous solution of NaHCO 3 and then further washed with an aqueous solution of NH 4 Cl. The organic phase was washed twice with 1.5 N H 2 SO 4 and stirred. It was concentrated and precipitated from methanol and purified water. The solid was collected by centrifugation and then dried under vacuum . As a result, 79.9 Kg of BG-2 was obtained. . 1 H NMR (DMSO-d 6 ) 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 = 8.6 Hz, 2H).

[0082] Step 2: Synthesis of BG-3 [Chemical formula]

[0083] Under a nitrogen atmosphere, a solution of BG-2 (79.9 kg, 1.0 equiv.) in MeCN (5.0 v) was heated at 85 °C to trimethoxy Cimethane (12.0v) was added. The resulting mixture was stirred until the reaction was complete. HPLC analysis showed that the reaction was The mixture was sampled for precipitation and concentrated under vacuum, and the residue was precipitated with i-PrOH and hexane. The mixture was centrifuged, the cake was washed with hexane and dried under vacuum, yielding 71.7 kg. The product was obtained. 1 H NMR (400 MHz, DMSO-d 6 ) 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 equiv.) in ethanol (2.5 v) was added to the Hydrazinium hydroxide (1.0 equiv.) dissolved in alcohol (0.6 v) was added dropwise to the reactor at less than 15 °C. The solution was heated to room temperature and stirred until the reaction was complete. Water (4.0 v) was added to the reactor. The solution was then cooled to 5°C, centrifuged, and the cake was washed with water (1.0 v). The cake was dried under vacuum, which gave 66.9 Kg of product. 1 H NMR (DMSO-d 6 ) δ 12. 11 (broad s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.46 - 7.39 (m, 2H), 7.18 (t, J = 7.6 Hz, 1H), 7.12 - 7.04 (m, 4H), 6.43 (broad s, 2H).

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

Chem.

[0087] A mixture of DCM (8.0 v), BG - 5 (80.0 Kg, 1.0 equivalent), N,O - dimethylhydroxylamine hydrochloride ( 1.2 equivalents), HOBt (1.2 equivalents) and EDCI (1.2 equivalents) was added dropwise with TEA (2.6 equivalents) at 15 °C or 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 NH Cl 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 4 . 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 NH Cl solution was added dropwise at 10 °C or below. The mixture was centrifuged, separated, 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 4 added. The final mixture was saturated with NH , 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 saturated with NH Cl solution at 10 °C or below. The mixture was centrifuged, separated, filtered 4It was washed with an aqueous solution of Cl. The organic layer was concentrated, and hexane was added to precipitate. 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-d 6 ) 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), 1.39 - 1.20 (m , 2H).

[0088] Step 7: Synthesis of BG-9 [Chemical formula]

[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 cooled and concentrated, and then precipitated with methanol. The mixture was centrifuged, and the cake was washed with methanol . The cake was dried under vacuum. As a result, 107.8 Kg of the product was obtained. 1 1H NMR (DMSO-d 6 ) δ 8.78 (d, J = 4.6 Hz, 1H), 8.15 - 8.07 (m, 2H), 7.51 - 7.41 (m, 2H), 7. 34 (d, J = 4.6 Hz, 1H), 7.27 - 7.19 (m, 3H), 7.17 - 7.10 (m, 2H), 4.24 - 4.02 (m, 2H), 3.81 - 3.69 (m, 1H), 3.12 - 3.82 (m, 2H), 2.15 - 2.04 (m, 2H), 1.76 - 1.60 (m, 2H), 1.4 3 (s, 9H).

[0090] Step 8: Synthesis of BG-10

Chem.

[0091] N 2 To a mixture of the following THF (10.0 v), BG - 9 (13.0 Kg, 1.0 eq), and D - DBTA (1.0 eq) was introduced hydrogen gas into the reactor with Pd / C (10% w / w), and the hydrogen pressure was maintained at 1.8 MPa. The reactor was heated to 40 °C over time and stirred until the reaction was complete. Then the mixture was 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 NaHCO 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%). NaHCO 3 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 - d 6 ) 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 [Chemical formula]

[0093] To a solution of BG-10 (100.0 Kg, 1.0 equivalent) in DCM (6.0 v) was added EtOH containing HCl (20.9% w / w, 2.0 v) dropwise under a nitrogen atmosphere. The mixture was stirred until the reaction was complete. MTBE (4.0 v ) was added to the solution and it was cooled. The cake was collected by centrifugation and washed with hexane (2.0 v) and then the cake was slurried in hexane (5 v) and centrifuged again. The cake was washed with hexane (2.0 v) and dried under vacuum. Thus, 85.2 Kg of the product was obtained . 1 1H NMR (DMSO-d 6 ) δ 9.25 - 8.85 (m, 2H), 7.84 - 7.70 (m, 2H), 7.47 - 7.37 (m, 2H), 7 .18 (t, J = 7.4 Hz, 1H), 7.12 - 7.03 (m, 4H), 5.73 (br s, 2H), 4.12 - 4.03 (m, 1H), 3.25 - 3.19 (m, 4H), 2.90 - 2.73 (m, 2H), 2.28 - 2.12 (m, 1H), 2.10 - 2.00 (m, 1H), 1.99 - 1.86 (m, 1H), 1.84 - 1.52 (m, 4H).

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

[0095] A mixture of water (6.0 v) and NaOH (3.0 equivalents) containing BG-11 (85.0 Kg, 1.0 equivalent) was at room temperature The reaction was stirred until completion. The cake was collected and then slurried in MTBE (6.0 v). The mixture was then centrifuged to collect the cake. The cake was then dried under vacuum. This resulted in a 71.3 K g of product was obtained. 1 HNMR (DMSO-d 6 ) 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] Mixture of ethanol / water / acetic acid (7:3:1, 46 v) with BG-11A (30 kg, 1.0 equiv.) in a reactor. The mixture was heated to 70±5° C. under nitrogen atmosphere and then dissolved in D-DBTA (1.20 equiv.) in 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 h. After stirring for 2 hours, the mixture was cooled to room temperature. The solids were collected by centrifugation and then diluted with ethanol (2. The cake was washed with ethanol / water / AcOH (7:3:1, 20v) at 55°C. The slurry was allowed to stand for 16 hours and then cooled to room temperature. The solids were collected by centrifugation and diluted with ethanol (2.0 v) and the cake was dried in vacuum (yield: 37.9%). 1 HNMR (DMSO-d 6 ) δ 8.76 (b r s, 2H), 7.99 - 7.89 (m, 4H), 7.83 - 7.75 (m, 2H), 7.66 - 7.57 (m, 3H), 7.52 - 7.45 (m, 4H), 7.45 - 7.39 (m, 2H), 7.21 - 7.14 (m, 1H), 7.13 - 7.03 (m, 4H), 5.64 (s, 2H), 4.0 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

Chemical formula

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

[0100] Step 12: Synthesis of BG-11D (Alternative Intermediate) ACN (5.0 v), soft water (10.0 v), and KOH (5.0 equivalents) were placed in a reactor and stirred for at least 15 minutes It was stirred. BG-11B (1.0 equivalent) was charged into the reactor little by little. The mixture was stirred until the reaction was completed. The cake was collected by centrifugation and slurried in ACN (1.0 v) and soft water (5.0 v), and then dried under vacuum to obtain the product.

[0101] Step 13: Synthesis of BG-12

Chemical formula

[0102] A solution of BG-11C (15.0 Kg, 1.0 equivalent) in MsOH (2.5 v) was stirred at 85 °C under a nitrogen atmosphere until the reaction was completed. After cooling to 5 °C, purified water (4.0 v) was added dropwise to the system while maintaining the temperature below 35 °C (obviously the temperature increased). 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. While stirring at a temperature below 30 °C, the pH value was adjusted to 11 - 12 with 20% aqueous NaOH solution (obviously the temperature increased). The organic phase was separated and collected. The aqueous phase was extracted with DCM (3.0 v). The organic layers were combined and concentrated. MTBE (4.0 v) was added to the residue. Then the mixture was concentrated and precipitated with n-heptane. The solid was collected by centrifugation and then dried in a vacuum oven. After cooling to 5 °C, purified water (4.0 v) was added dropwise to the system while maintaining the temperature below 35 °C (obviously the temperature increased). 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. While stirring at a temperature below 30 °C, the pH value was adjusted to 11 - 12 with 20% aqueous NaOH solution (obviously the temperature increased). The organic phase was separated and collected. The aqueous phase was extracted with DCM (3.0 v). The organic layers were combined and concentrated. MTBE (4.0 v) was added to the residue. Then the mixture was concentrated and precipitated with n-heptane. The solid was collected by centrifugation and then dried in a vacuum oven. As a result, 12.55 Kg of the product was obtained (yield: 94.9%). 1 1H NMR (DMSO-d 6 ) δ 7.52 - 7.46 (m, 2H), 7.45 - 7.38 (m, 2H), 7.21 - 7.13 (m, 1H), 7.12 - 7.03 (m, 4H), 6.64 (s, 1H), 3.99 - 3.90 (m, 1H), 3.29 - 3.22 (m, 2H), 3.03 - 2.90 (m, 2H), 2.48 - 2.36 (m, 2H), 2.03 (dd, J = 13.9, 5.6 Hz, 2H), 2.14 - 1.99 (m, 1H), 1.97 - 1.85 (m, 1H), 1.65 - 1.1 5 (m, 3H).

[0103] Step 14: Synthesis of BG-13

Chem.

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

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

Chem.

[0106] Under a N2 atmosphere, ACN (12.0 v), water (12.5 v), BG - 13 (8.0 Kg, 1.0 equivalent), and NaHCO 3 (2.5 equivalents) were added to the reactor. Then the mixture was cooled to - 5 - 0 °C. To the mixture, A solution of acryloyl (1.1 eq) in MeCN (0.5 v) was added dropwise and stirred until the reaction was complete. . Next, EA (6.0 v) was added to the reactor and then stirred. The organic phase was collected. The aqueous layer was further extracted with EA (3.0 v). The combined organic phases were washed with brine. The organic layer was collected and concentrated .

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

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

Chemical formula

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

[0110] Step 16: Preparation of Crystal Form A of Compound 1 The cake of the above compound 1 was dissolved in 7.0 volumes of DCM and then exchanged with the solvent EA. After recrystallization from EA / MTBE, the cake was collected by centrifugation and then dried under vacuum. As a result, 4. 44 Kg of the product (yield: 70.2%) was obtained.

[0111] Next, the product was identified by X-ray powder diffraction (XRPD) pattern method, which was performed on a PANalytical Empyrean X-ray powder diffractometer using the following XRPD parameters: X-Ray wavelength (Cu, kα, Kα1(Å): 1.540598, Kα2(Å): 1.544426; Kα2 / Kα1 intensity ratio : 0.50); X-Ray tube setting (45 Kv, 40mA); divergence slit (automatic); scan mode (Continuous); scan range (°2TH) (3° - 40); step size (°2TH) (0.0131); scan speed (Continuous); scan range (°2TH) (3° - 40); step size (°2TH) (0.0131); scan speed eed (° / min)(about 10). As a result of XRPD, it was found that the obtained product was the crystal shown in Fig. 1. It was found.

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

[0113] The thermogravimetric analysis (TGA) curve shown in Fig. 3 was generated by TA Instruments' TA Q5000 TGA. The TGA parameters used were as follows: temperature (RT - desired temperature); heating rate (10°C / min); method (ramp); sample pan (platinum, open); purge gas (N2). ng rate(10oC / min); method (ramp); sample pan (platinum, open); purge gas (N2). ng rate(10°C / min); method (ramp); sample pan (platinum, open); purge gas (N2). In the TGA results, there was no weight loss up to 110°C, indicating that it was anhydrous.

[0114] The proton nuclear magnetic resonance ( 1 1H NMR) shown in Fig. 4 was measured with a Bruker 400M NMR spectrometer in DMSO-d 6 6. It was measured in. 1 1H NMR (DMSO-d 6 6) 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, 2.2 Hz,1H), 4.55-4.38 (m, 1H), 4.17-3.94 (m, 2H), 3.33-3.22 (m, 2H), 3.08 -2.88 (m, 1H),2.67-2.51 (m, 1H), 2.36-2.15 (m, 1H), 2.12-1.82 (m, 2H), 1.79-1.6 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 C-NMR) was measured in DMSO-d using a Bruker 400M NMR spectrometer. 6 C-NMR spectrum of crystalline form A of compound 1. 13

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

[0117] Example 3 Preparation of Crystal Form A of Compound 1 (Addition of Poor Solvent) Weighed out a sample of about 15 mg (crystalline form A) into a 20 mL glass vial, and then added 0.4 - 1.2 mL of the corresponding solvent (see Table 2) to dissolve all the solids. Then the mixture was magnetically stirred at a speed of 800 rpm to obtain a clear solution at room temperature. Subsequently, the relative poor solvent (see Table 2) was added to the solution to induce precipitation, or added until the total amount of the poor solvent reached 15.0 mL . If precipitation did not occur, the solution was then transferred to evaporation carried out at room temperature over time . The obtained solid was identified as form A by XRPD.

[0118] Table 2 Poor Solvent Addition Test

Table 2

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

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

[0121] In the long-term stability test of crystalline form A of Compound 1, 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 = 9 9.4%), it was shown that no significant change in chemical purity occurred. Furthermore, when stored at 25 °C / 60% RH for 24 months or at 40 °C / 75% RH for 6 months, no changes in crystal form and optical purity were observed.

[0122] (2) Moisture Absorption Test The dynamic vapor sorption (DVS) plot shown in Figure 6 was collected by DVS Intrinsic, an SMS (Surface Measurement System). 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.

[0123] (3) Crystallization / Recrystallization with Form A to Improve the Purity of Compound 1. Crystallization / recrystallization with Form A improves the purity of Compound 1 and is an effective method for suppressing impurities in Compound 1 to meet the acceptance criteria of this specification. See the examples shown in Table 3.

[0124] Table 3 Purity Changes after Crystallization / Recrystallization with Form A

Table 3

[0125] Example 6 Preparation of Deuterium-Labeled (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxy 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) was added oxalyl chloride (1.6 N, 40.9 mL, 65.5 mmol) dropwise at 0 - 5 °C, and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure to obtain crude acryloyl-d 3 chloride.

[0127] (S)-2-(4-Phenoxyphenyl)-7-(piperidin-4-yl)-4,5,6,7-tetrahydropyrazolo [1,5-a]pyrimidine-3-carboxamide (dissociated from BG-13, step 15, compound 1, see alternative method; 278 mg, 0.67 mmol) in DCM (20 mL) and an aqueous solution of NaHCO (10 mL) 3 was added dropwise at 0 - 5 °C to a solution of the above acryloyl-d chloride in DCM (5 mL), and the mixture was stirred at room temperature for 2 hours. 3 The combined organic layers were 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)piperidin-4-yl)-2 (4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carbox amide as an off-white solid. amide. amide as an off-white solid. 1H NMR (400 MHz, DMSO) δ 7.50-7.44 (m,2H), 7 .42-7.35 (m,2H), 7.17-7.10 (m, 1H), 7.09-6.99 (m, 4H), 6.64 (s, 1H), 4.52-4.40 (m, 1H),4.10-3.95 (m, 2H), 2.29-3.25 (m, 2H), 3.04-2.86 (m, 1H), 2.63-2.50 (m, 1H), 2.32-2.13(m, 1H), 2.06-1.81 (m, 2H), 1.75-1.45 (m, 2H), 1.35-1.08 (m, 2H) MS (ESI, m / e) [M+1] + 475.2.

[0128] Example 7 Study on Polymorphs of Compound 1 (1) Study on Polymorphs from Amorphous - Preparation of Form A from Amorphous Compound 1 (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydrofuran The present invention relates to a method for producing a hydropyrazolo[1,5-a]pyrimidine-3-carboxamide, which is disclosed in WO2014173289A. Compound 1 was prepared according to the method described above and then freeze-dried to obtain amorphous compound 1.

[0129] For each experiment in Tables 4a to 4k, 5a to 5e, and 6, Compound 1 was dissolved in an amorphous form at approximately 100 mL. 20 mg was weighed into a glass vial and the corresponding solvent was then added. Mix if necessary. The mixture was then heated to obtain a clear solution. The mixture was then kept at room temperature without stirring for 1-2 days. The solid matter formed became visible from the transparent solution. I understand.

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

[0131]

Table 4b

[0132]

Table 4c

[0133]

Table 4d

[0134]

Table 4e

[0135]

Table 4f

[0136]

Table 4g

[0137]

Table 4h

[0138]

Table 4i

[0139]

Table 4j

[0140]

Table 4k

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

Table 5a

[0142]

Table 5b

[0143]

Table 5c

[0144]

Table 5d

[0145]

Table 5e

[0146] The experiments in Tables 4a - 4k and Tables 5a - 5e were conducted on the same scale (i.e., the amount of the starting material - the amount of the amorphous compound 1 was about 20 mg). However, the ee value of the starting material seemed to have a significant effect on the amount of solid to be formed in each experiment. In the experiments in Tables 4a - 4k, starting from the amorphous compound 1 with 90% ee, the amount of solid thus formed was small. In the experiments in Tables 5a - 5e, starting from 97% ee of the amorphous compound 1, a very large amount of solid was obtained. Also, when crystallized from the starting material with 90% ee, the solids obtained in the experiments in Tables 4a - 4k had a low ee value. One of the solid samples from Tables 4a - 4k used EA / hexane as the crystallization system, but the ee value was only 45%. In one of the solid samples from Tables 4a - 4k EA / hexane was used as the crystallization system, but the ee value was only 45%.

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

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

[0149] The results in Table 6 further support that the formation of the crystalline solid depends on a specific solvent ratio. ing. [Table 6]

[0150] (2) Study on Polymorphs from Crystal Form - Preparation of Form A from Crystal Form Evaporation over Time Approximately 15 mg of the sample (crystalline form A) was weighed into a 3 mL glass vial, and then the corresponding solvent or solvent mixture (see Table 7) was added to obtain a clear solution. Subsequently, the vial was covered with parafilm having three or four pinholes and kept at room temperature to allow the solution to evaporate over time. The solid was isolated for XRPD analysis. However, as summarized in Table 7, no crystalline form was produced.

[0151] Table 7 Evaporation experiment carried out over time [Table 7]

[0152] Addition of Poor Solvent Approximately 15 mg of the sample (crystalline form A) was weighed into a 20 mL glass vial, and then 0.4 to 1.2 mL of the corresponding ​​The corresponding solvent was added (see Table 8). The mixture was then stirred magnetically at 800 rpm to obtain a clear solution at room temperature. Subsequently, the relative poor solvent (see Table 8) was added to the solution to induce precipitation or added until the total amount of the poor solvent reached 15.0 mL. If no precipitation occurred, the solution was then evaporated at room temperature over time. The results are summarized in Table 8.

[0153] Table 8 Poor Solvent Addition Experiment

Table 8

[0154] Cooling over Time Approximately 20 mg of the sample (crystalline form A) was suspended in 1.0 mL of the corresponding solvent (see Table 9) in a 3 mL glass vial at room temperature. The suspension was transferred to a screw-cap vial and stirred magnetically at 800 rpm while heating to 50 °C. The sample was equilibrated at 50 °C for 2 hours and filtered using a 0.45 μm nylon membrane. The filtrate was then cooled from 50 °C to 5 °C at a rate of 0.1 °C / min over time. The resulting solid was kept isothermal at 5 °C until isolated for XRPD analysis. As summarized in Table 9, no crystalline form was obtained.

[0155] Table 9 Cooling Experiment over Time

Table 9

[0156] Solution Vapor Diffusion ​Dissolve approximately 15 mg of the sample (crystalline form A) in 0.5 - 1.5 mL of the corresponding solvent (see Table 10), and obtain a clear solution in a 3 mL vial. Subsequently, transfer the solution into a 20 mL vial containing 3 mL of the relative poor solvent. Seal the 20 mL vial with a cap, keep it at room temperature, and allow sufficient time for the organic vapor to interact with the solution. At the end of the 11th day, transfer the clear solution to evaporation at room temperature. Identify the obtained solid by XRPD. Summarize the results in Table 10.

[0157] Table 10 Experiment on solution vapor diffusion

Table 10

[0158] Experiment on Polymer-Induced Crystallization Dissolve approximately 15 mg of the sample (crystalline form A) in 1.0 mL of the corresponding solvent (see Table 11), and obtain a clear solution in a 3 m L vial. Then filter the solution using a 0.45 μm nylon membrane . Add approximately 2 mg of the polymer mixture to the filtrate. Stir the mixture at room temperature to induce precipitation. Isolate the solid for XRPD analysis. As summarized in Table 11, no crystalline form was obtained .

[0159] Table 11 Experiment on polymer - induced crystallization

Table 11

[0160] Example 8 Determination of the Absolute Configuration of Compound 1 Preparation of Single Crystal of BG-13 The single crystal growth experiment (see Table 12) was carried out 6 times by the method of cooling over time. Me OH / H 2 O (1:1, v / v) by cooling over time, single crystals of BG-13 in the desired form were obtained. The crystal data and precise structure are shown as a list in Table 13.

[0161] Table 12 Single crystal growth experiment

Table 12

[0162] Table 13 Data and precise structure of single crystals of BG-13 The single crystal data were generated with a Bruker APEX DUO single crystal diffractometer equipped with a CCD detector (Cu K α, λ = 1.54178 Å, 173.15 K).

Table 13

[0163] BG-13 was confirmed to be the (2R,3R)-dibenzoyl tartaric acid (L-DBTA) salt, and the molar ratio of the free base to L-DBTA was 2:1. The configuration of both carbons (C32 and C32’) in L-DBTA was confirmed to be R. As shown in Figures 8 to 10, C6 in the free base ​​​The three-dimensional configuration was determined to be S. Also, as shown in Fig. 11, the structure of the single crystal was identified using the powder X-ray diffraction pattern method. The structure of the single crystal was identified using the powder X-ray diffraction pattern method.

[0164] Absolute Configuration of Compound 1 The absolute configuration of Compound 1 was estimated to be S from the single crystal X-ray structural analysis of Intermediate BG-13.

[0165] Example 9: Chiral Resolution of BG-11A [Chemical formula]

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

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

[0168] Example 10: Chiral Resolution of BG-12A and Improvement of Chiral Purity [Chemical formula]

[0169] Common procedure: To a solution containing Compound BG-12A in the prepared solvent system, a chiral acid was added at a high temperature. ​​​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 chiral purity was tested directly by chiral HPLC with the relevant salt or free base (see Table 15). With other chiral acids or solvent systems, no chiral compounds with no ee value, chiral compounds with low ee values, or undesirable chiral compounds were found .

[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 / H 2 O (1 / 1, 1034 mL), and the suspension was warmed to 70 °C and stirred until all solids were dissolved. Then 517 mL of water was added. Next, the solution was slowly cooled to 40 °C, and a seed crystal (10 mg) was added. After stirring for about 2 hours, the solution was slowly cooled to ambient temperature and then stirred for 2 days. It was filtered, and the solid was washed with THF / H O = 1 / 1 (20 m 2 L) and dried under reduced pressure to obtain the product as a white solid (22.5 g, yield 72% , >98.5 ee value).

[0172] The obtained free base (6.02 g, 79.1% ee) was dissolved in EtOH / H 2 O (6 / 1, 90 mL) at (1 g / 15 mL), and stirred at 78 °C to dissolve all starting materials. Then, a solution of L-DBTA (2.84 g, 7.9 mmol , 0.55 equivalent) in EtOH / H 2 O (6 / 1, 7 mL) was added. Solids formed rapidly, and the mixture ​​After stirring at this temperature for 1 hour, the heating system was removed. The mixture was cooled to room temperature. It was filtered, and the solid was washed with EtOH / H 2 O (6 / 1, 10 mL). The collected solid was converted to the free base using an aqueous NaOH solution and DCM to obtain the product as a white foamy substance (4.7 g, yield: 32. 6%, 93% ee).

[0173] A suspension of the obtained free base (70.0 g, 90.5% ee) in CH 3 CN / H 2 O (1 / 1, 700 mL) was heated to 60 °C to obtain a clear solution. Then, L-DBTA (33 g, 0.55 equivalents) was added to the above solution . After stirring at 60 °C for about 2 hours, the mixture was slowly cooled to room temperature and stirred overnight. It was filtered, and the solid was washed with CH 3 CN / H 2 O (1 / 1, 50 mL) and dried under reduced pressure to obtain the product as an off-white solid (80 g, yield: 80%, ee value > 98%).

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

[0175] Test 1: Inhibition and Selectivity of Kinase Method: (1) BTK Kinase Enzyme Test Crystal Form A of Compound 1 was used for the BTK kin -ase (aa2-659, Carna Biosciences) for inhibition, but by a test based on the time-resolved fluorescence resonance energy transfer (TR-FRET) methodology . It was carried out. The test was performed in a 384-well small-volume black plate, and was carried out in a reaction mixture containing BTK kinase, 5 μM ATP, 2 μM peptide substrate and 0 - 10 μM compound, and these were in a buffer containing 50 mM Tris pH 7.4, 10 mM MgCl 2 2, 2 mM MnCl 2 2, 0.1 mM EDTA, 1 mM DTT, 0.005% Tween-20, 20 nM SEB and 0.01% BSA. The kinase was incubated with the compound at room temperature for 60 minutes, and the reaction was initiated by adding ATP and the peptide substrate. After reacting at room temperature for 60 minutes, an equal volume of stop / detection solution was added according to the manufacturer's instructions (CisBio Bioassays). The stop / detection solution contained an anti-phosphotyrosine Eu cryptate-conjugated mouse monoclonal 3+ antibody (PT66) and XL665-conjugated streptavidin, and was in a buffer containing 50 mM HEPES pH 7.0, 800 mM KF, 20 mM EDTA and 0.1% BSA. The plate was sealed and then incubated at room temperature for 1 hour, and then the TR-FRET signal (the ratio of luminescence at 665 nm to luminescence at 620 nm with excitation at 337 nm) was recorded with a PHERStar FS plate reader (BMG Labtech). Phosphorylation of the peptide substrate causes the anti-phosphotyrosine antibody to bind to the biotinylated peptide substrate, where the fluorescence donor (Eu cryptate) is placed very close to the acceptor (streptavidin-XL665), so that a high degree of fluorescence resonance energy transfer occurs from the donor fluorophore (620 nm) to the acceptor fluorophore (665 nm). BTK kinase Phosphorylation of the peptide substrate causes the anti-phosphotyrosine antibody to bind to the biotinylated peptide substrate, where the fluorescence donor (Eu 3+ cryptate) is placed very close to the acceptor (streptavidin-XL665), so that a high degree of fluorescence resonance energy transfer occurs from the donor fluorophore (620 nm) to the acceptor fluorophore (665 nm). Since the donor fluorophore (620 nm) is placed very close to the acceptor fluorophore (665 nm), a high degree of fluorescence resonance energy transfer occurs from the donor fluorophore (620 nm) to the acceptor fluorophore (665 nm). A high degree of fluorescence resonance energy transfer occurs from the donor fluorophore (620 nm) to the acceptor fluorophore (665 nm). BTK kinase The TR-FRET signal decreased due to the inhibition of enzyme activity. The data was fitted to a four-parameter logistic equation using Graphpad Prism software to derive the IC of compound 1. 50

[0176] (2) Biochemical Kinase Selectivity For the selectivity characterization of crystalline form A, a panel of 342 kinases at 1 μM was used at Reaction Biology Corp. Crystalline form A showed less than 70% inhibition against 329 kinases and more than 70% inhibition against 13 kinases including BTK. The IC of crystalline form A (Table 1 50 see reference 3) was performed in BeiGene's facility and further included tests for ITK, TEC, JAK3, and EGFR, and was conducted using a TR-FRET assay and the corresponding peptides as substrates.

[0177] Determination of the IC 50 of ITK: The procedure for the ITK assay was the same as the BTK assay except for the following changes: 3 μM ATP and 2 μM TK substrate were used in the kinase reaction.

[0178] Determination of the IC 50 of TEC: The procedure for the TEC assay was the same as the BTK assay except for the following changes: 1) 2 80 μM ATP and 2 nM Poly-GT substrate were used in the kinase reaction. 2) SEB was not included in the reaction buffer.

[0179] Determination of the IC 50 of JAK3: The procedure for the JAK3 assay was the same as the BTK assay except for the following changes: 1 ) 3.4 μM ATP and 3 μM peptide substrate (B-EE-15, biotin-EQEDEPEGDYFEWLE) were used in the kinase ​​​​​​Used in the ζ reaction. 2) The reaction buffer was 50 mM Tris pH 7.8, 10 mM MgCl 2 , 5 mM DTT, 0.01% T riton X-100 and 0.01% BSA.

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

[0181] Results: The IC 50 of crystalline form A against BTK kinase was 0.27 nM. Crystalline form A was found to be a potent, specific , and irreversible inhibitor of BTK kinase. Regarding its selectivity, when characterized with a panel of 342 human kinases at 1 μM, crystalline form A inhibited more than 70% of only 13 other kinases.

[0182] Table 16 Enzyme inhibitory activity of crystalline form A

Table 16

[0183] Test 2: BTKpY223 Cell Test with Crystal Form A Method: The BTKpY223 cell test is an HTRF-based test aimed at quantitatively determining the endogenous phosphorylation level at BTKTyr223. Phosphorylated Tyr223 is required for the complete activation of BTK . The test was performed in Ramos cells (CRL-1596 , ATCC) using the BTKpY223 test kit (63IDC000, Cisbio).

[0184] Briefly, Ramos cells were serum-starved for 2 hours in RPMI1640 containing 0.5% FBS . After serum starvation, the cells were incubated with crystal form A and detected at various concentrations in a 1-hour CO 2 incubation chamber. After incubation, the cells were stimulated with 1 mM sodium orthovanadate( PV) or Na 3 VO 4 (OV) for 20 minutes. Then, the cells were spun down and lysed with 1× lysis buffer (4× lysis buffer attached to the kit) at room temperature for 10 minutes . During incubation ​In the detection buffer (supplied in the kit), dilute anti-BTK-d2 and anti-pBTK-K to prepare a 1× antibody mixture at 2 μl / well and dispense it into an OptiPlate-384 test plate (6005620, PerkinElmer). Then, transfer 18 μL of cell lysate to the test plate pre-added with the antibody solution, mix gently, spin briefly, seal the plate, and store it in the dark at room temperature for 18 hours. Measure the fluorescence emission at two different wavelengths (665 nm and 620 nm) using a compatible HTRF reader (PHERAstar FS, BMG). Calculate the potency of Compound 1 based on the inhibition of the ratio between the signal intensities at 665 nm and 620 nm. Calculate the IC value using GraphPad Prism software with a sigmoid dose-response function. Thus, a 1× antibody mixture was prepared. The 1× antibody mixture was dispensed into an OptiPlate-384 test plate (6005620, PerkinElmer) at 2 μl / well. Subsequently, 18 μL of cell lysate was transferred to the test plate pre-added with the antibody solution. Mix gently and spin briefly, then seal the plate and store it in the dark at room temperature for 18 hours. Measure the fluorescence emission at two different wavelengths (665 nm and 620 nm) using a compatible HTRF reader (PHERAstar FS, BMG). The potency of Compound 1 was calculated based on the inhibition of the ratio between the signal intensities at 665 nm and 620 nm. Calculate the IC value using GraphPad Prism software with a sigmoid dose-response function. 50

[0185] Results: Crystalline Form A inhibited the phosphorylation of BTK in the B-cell lymphoma cell line, Ramos, at a low concentration of 1.8 ± 0.2 nM (n = 3).

[0186] Test 3: Effect of Crystal Form A on Tumor Cell Proliferation in Hematological Cancer Cell Lines (Rec-1, Mino, JEK O-1 and TMD-8) Method: Three MCL cell lines (Rec-1, Mino, and JEKO-1) and the ABC-type diffuse large B-cell lymphoma cell line (TMD8) were used in this study. The cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum / FBS (Thermo Scientific), 100 units / ml penicillin (Gibco), 0.1 mg / ml streptomycin (Gibco), and 5% CO in the air in a humidified atmosphere at 37°C. 2 It was done. The cell line was recovered from the obtained original cells from the frozen stock seeded within 30 passages.

[0187] The growth inhibitory activity of the compounds in Rec-1, Mino, JEKO-1 and TMD-8 cells was determined using the CellTiter-Glo luminescent cell viability assay (Promega). The number of cells seeded per well in a 96-well plate was optimized for each cell line to ensure logarithmic growth over a 6-day treatment period. The cells were treated 3 times in a 10-fold dilution series. After 6 days of exposure to the compound, an equal volume of CellTiter-Glo reagent 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 generate and stabilize the luminescence signal, which represents the amount of ATP, i.e., the amount of metabolically active cells. The luminescence signal was measured using a PHERAstar FS reader (BMGLabtech). The IC values for cell viability were determined using GraphPad Prism software and averaged over 3 independent tests. 50

[0188] Results: Crystal form A of compound 1 showed a specific and potent inhibitory effect on cell growth in three MCL cell lines and an ABC-type diffuse large B-cell lymphoma cell line (TMD8) (Table 17). Table 17 Inhibition of hematological tumor cell growth by crystal form A

Table 17

[0189] Test 4: Pharmacokinetic Study of Crystal Form A in Mice Method: For the time-course study, mice were randomly divided into 7 groups of 4 mice each. The mice were treated with a single dose of crystalline form A of Compound 1 and euthanized using carbon dioxide at different time points (30 minutes, 1, 2 , 4, 12, 24 hours) after dosing. For the dose-dependency study, , mice were randomly divided into 9 groups of 4 mice each. The mice were treated with different dose levels of crystalline form A of Compound 1 and euthanized using carbon dioxide 4 hours after dosing. The treatment was administered by oral gavage (oral) at a volume of 10 ml / kg body weight. Body weight was evaluated immediately before dosing and the dose was adjusted accordingly.

[0190] PK sample preparation: For the time-course study, blood samples (50 μL per mouse) were collected from the retro-orbital sinus under isoflurane / oxygen anesthesia 15 minutes after dosing (mice in this group were also used at the 24-hour time point), or under cardiac puncture after euthanasia at other time points. For the dose-dependency study, blood samples were collected from the retro-orbital sinus under isoflurane / oxygen anesthesia 30 minutes after dosing. 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) were obtained directly from the plasma concentration versus time profile.

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

[0193] Test 5: Efficacy Test of Crystal Form A in the TMD-8 Xenograft Model Tumor Implantation Method: The animals were treated with cyclophosphamide (150 mg / kg in saline, prepared intraperitoneally) and disodium. Rufilam (prepared in saline containing 0.8% Tween 80 and administered orally at 125 mg / kg) and cyclosporine (prepared in saline containing 0.8% Tween 80 and administered orally at 125 mg / kg) were administered. The mice were pretreated with cyclophosphamide (1 hour after each dose) on each day for 2 days. 24 hours after the second dose of Famid, animals were inoculated with TMD-8 cells. On the day of implantation, cells The culture medium was replaced with fresh medium. After 4 hours, the medium was removed and the cells were incubated as described above. The cells were resuspended in cold PBS (4°C) and added to an equal volume of Matrigel (BD, Cat. No. 356237 ) to a final concentration of 2.5 × 10 7 cells / ml. Resuspended cells were kept on ice prior to inoculation. The right axillary region of each mouse was washed with 75% ethanol prior to cell inoculation. 5 x 10 in the right anterior flank via needle 6 200 μl of the cell suspension containing the cells was injected subcutaneously.

[0194] In vivo efficacy testing will begin on the third day after cell inoculation. The animals were randomly assigned to the desired number of groups with 10 mice per group. twice (BID), vehicle (0.5% carboxymethylcellulose (CMC) + 0.2% Tween 80) and different dose levels of Compound 1, crystalline form A, for 39 days. Treatment was by oral gavage. The animals were orally administered with a volume of 10 ml / kg body weight. Tumor volumes were measured bidimensionally twice weekly using calipers. (In this test, measurements were possible from the 11th day after inoculation.) Formula: V = 0.5 × (a × b 2) was used to calculate the tumor volume. In the formula, a and b are the major and minor diameters of the tumor, respectively. Statistics cal 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. During the test period, the mice were also monitored daily for clinical signs of toxicity. The in vivo efficacy of crystalline form A was tested in subcutaneous TMD-8 DLBCL xenografts grown in NOD / SCID mice. It was administered orally twice a day (BID) daily, but was well tolerated at different dose levels. Crystalline form A of compound 1 showed a dose-dependent antitumor effect. Crystalline form A of compound 1 already showed strong antitumor activity in the lowest-dose test. In all treatment groups, no significant effect on the body weight of the animals was observed throughout the test.

[0195] Results: The animals were pretreated with cyclophosphamide (prepared in saline at 150 mpk i.p.) and disulfiram (prepared in saline containing 0.8% TW-80, 125 mpk p.o.) for 2 days, once a day, 1 hour after each administration of cyclophosphamide. Then, 24 hours after the second administration of cyclophosphamide, the animals were inoculated with REC-1 cells. On the day of transplantation, the cell 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 (4°C) PBS to a final concentration of 1 × 10 cells / ml. The resuspended cells were placed on ice before transplantation. Each animal received 1 × 10 cells / ml

[0196] Test 6: Efficacy Test of Crystal Form A in the Systemic REC-1 Xenograft Model Tumor Implantation Method: The animals were pretreated with cyclophosphamide (prepared in saline at 150 mpk i.p.) and disulfiram (prepared in saline containing 0.8% TW-80, 125 mpk p.o.) for 2 days, once a day, 1 hour after each administration of cyclophosphamide. Then, 24 hours after the second administration of cyclophosphamide, the animals were inoculated with REC-1 cells. On the day of transplantation, the cell 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 (4°C) PBS to a final concentration of 1 × 10 cells / ml. The resuspended cells were placed on ice before transplantation. Each animal received 1 × 10 cells / ml 8 cells / ml of the final concentration. The resuspended cells were placed on ice before transplantation. Each animal received 1 × 10 7A 100 μl cell suspension containing cells was intravenously injected via the tail vein.

[0197] The in vivo efficacy test was started on the 8th day after cell inoculation. For this purpose, animals were randomly assigned to a desired number of groups, each group having 1 or 10 mice. The mice were treated with crystalline form A of Compound 1 at different dose levels twice a day (BID) with vehicle (0.5% carboxymethylcellulose (CMC) + 0.2% Tween 80) for 71 days. All administrations were terminated on the 78th day after inoculation. The treatment was performed by oral gavage at a volume of 10 ml / kg body weight. The body weight was evaluated immediately before administration, and the dose was adjusted accordingly. The body weight was recorded twice a week (changed to three times a week from the 33rd day). During the test period, the clinical signs of the mice's illness were also observed daily. This test was terminated after the entire survival period. In case of severe toxic effects such as loss of movement, the mice were euthanized and recorded as dead.

[0198] For data analysis: Survival analysis was performed by the Kaplan - Meier method. The survival period was defined as the time from the day of tumor cell inoculation to the day the animal died or was euthanized. For each group, the median survival time (MST), the range of the survival period with 95% confidence interval (RST), and the increase in lifespan (ILS) were calculated. The median survival time was defined as the time point when 50% of the mice died. ILS was calculated using the following formula: %ILS = (MST - MST (vehicle)) / MST (vehicle) × 100 Statistical analysis was performed between groups using the Gehan - Breslow - Wilcoxon test. If P < 0.05, it was considered statistically significant.

[0199] Results: Crystal form A of Compound 1 showed dose dependent antitumor activity against systemic REC-1 MCL xenografts in NOD / SCID mice. The efficacy of crystal form A of Compound 1 was significant in this xenograft model.

[0200] Test 7: Toxicity of Crystal Form A An extensive nonclinical toxicity testing program was conducted, including some of the 28-day GLP tests and studies in rats and dogs, to evaluate the preclinical safety of crystal form A of Compound 1 at different doses. These studies followed the regulatory guidelines available for the preclinical development of anticancer drugs. In these studies, the toxicological and safety pharmacological profiles of Compound 1 were shown to be favorable. No deaths occurred in relation to those tested at any dose level throughout the tests. No toxicologically significant changes or coagulations in clinical chemistry were observed throughout the tests. None of these changes were observed even after the recovery phase.

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

[0202] Crystal form A of Compound 1 has good oral bioavailability in rats. It was rapidly absorbed in rats and had a high plasma clearance (CL). The kinetics were linear over the dose range in female rats. The linearity in male rats was not as good. In both male and female rats, the accumulation of Compound 1 after multiple oral administrations was not statistically significant. ​​​​​​​​​​​There was no accumulation of Compound 1 after multiple oral administrations to dogs in crystalline form A. Moderate clearance ( CL), moderately good bioavailability (F%), and linear PK across the dose range were observed , and no accumulation of Compound 1 was seen.

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

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

[0205] Example 12: Clinical Trial Research (1) Results of Compound 1 in Phase I Clinical Trials in Patients with Advanced B-Cell Malignancies The first multicenter, open-label, Phase I trial of Compound 1 was conducted in Australia and New Zealand and consisted of two parts - a dose escalation phase and a dose expansion phase involving 25 patients and plans to enroll a total of 100 patients. Among the total of 39 enrolled patients, all 25 patients from the initial dose escalation part and 14 patients from the ongoing dose expansion part are included. Based on the pharmacokinetics, pharmacodynamics, safety and efficacy of Compound 1 in the dose escalation phase, once daily (QD) at 320 mg and twice daily (BID) at 160 mg are being further explored in the ongoing dose expansion study.

[0206] The data analysis deadline was October 19, 2015. Although 29 objective responses were observed, among them, 3 complete responses (CR), 1 very good partial response (VGPR), and 25 partial responses (PR) were seen. The responses on tissue diagnosis were summarized in Table 18. Among the 39 patients, 31 are continuing the study treatment while still in progress, and this includes all patients who have had a response so far.

[0207] Table 18 Responses on tissue diagnosis for patients

Table 18

[0208] Eight patients discontinued Compound 1, of which 6 were due to disease progression and 2 were due to adverse events related to the original malignancy. Three patients died during the study as a result of disease progression or complications of disease progression. There were no drug-related serious adverse events (SAEs). ​​​​​Regardless of the relationship with 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, 4 were evaluated by the researchers as possibly drug-related―all were neutropenia that resolved spontaneously and did not require treatment discontinuation. There was 1 case of major hemorrhage, Grade 3 or higher bleeding event or any grade of intracranial bleeding event: identified as GI bleeding in a patient with mantle cell lymphoma with lymphomatous infiltration of the GI tract; this bleeding event occurred during drug hold and resolved rapidly with resumption of treatment with Compound 1, so it was not judged to be drug-related. Six patients had a baseline history of atrial fibrillation / flutter (AF), but no worsening or new events of AF were reported.

[0209] (2) Clinical Trials of Compound 1 in Patients with Waldenström Macroglobulinemia (WM) Results of the Ongoing Phase I Trials The 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 and dose expansion phases set within disease-specific cohorts, which included untreated and relapsed / refractory Waldenström macroglobulinemia (R / R WM). In the dose escalation part of Trail, the total daily dose was tested in the range of 40 mg to 320 mg, while in the ongoing dose expansion phase, the test doses of 160 mg twice daily (BID) or 320 mg once daily (QD) are being tested. As of March 31, 2017, 48 WM patients were registered in this study. Efficacy was determined according to the criteria of the 6th International Workshop on WM (IWWM) revised for WM.

[0210] Compound 1 has shown good tolerance and has not been interrupted due to the toxicity associated with Compound 1 to date. Adverse events (AEs) generally have low severity and are self-limiting and resolve spontaneously. The most common AEs (> 10%) among any of the 48 patients evaluated for safety (> 10%) were petechiae / purpura / contusions (35%), upper respiratory tract infections (31%), constipation (25%), diarrhea (19%), epistaxis (19%), nausea (17%), cough (15%), anemia (15%), headache (15 %), neutropenia (13%), and rash (13%). Except for grade 3 or 4 anemia and neutropenia (8% each), and grade 3 or 4 diarrhea and headache (2% each), all were grade 1 or 2 in severity. Five serious adverse events were considered to be related to Compound 1 ; these included one case each of hemothorax, atrial fibrillation, colitis, febrile neutropenia, and headache. Among the AEs of particular interest, there were a total of three cases of atrial fibrillation (all grade 1 or 2), and one case of severe bleeding (hemothorax), which was defined as bleeding of grade 3 or higher or any grade of central nervous system bleeding. Three events: one case each of bronchiectasis , prostate adenocarcinoma, and pyloric adenocarcinoma led to treatment discontinuation. At the data cutoff, 42 patients were evaluable for response. Among the patients for whom efficacy could not be evaluated, there were two patients with a follow-up period of less than 12 weeks, three patients with a baseline IgM of less than 500 mg / dl, and one patient with an inaccurate baseline IgM due to frozen protein. The median follow-up period was 12.3 months (4.4 - 30.5 months), and the ORR was 90% among patients

[0211] ​​​​% (38 / 42 patients), the major response rate was 76% (32 / 42 patients), but the VGPR was 43% (18 / 42 patients), with a partial response rate of 33% (14 / 42).

[0212] (3) Ongoing Phase I Clinical Trial Results of Compound 1 in Patients with Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma (CLL / SLL) Results of the Ongoing Phase I Clinical Trials A multi-centre, open-label Phase I study of Compound 1 in patients with B-cell malignancies is being conducted in Australia, It is being conducted in New Zealand, South Korea, and the United States, with dose escalation in disease-specific cohorts. The study included a dose-expansion phase and a phase with no prior treatment, including previously untreated (TN) and relapsed / refractory (R / R) CLL / SLL. The dose escalation portion of the trail tested total daily doses ranging from 40 mg to 320 mg. , and in the ongoing dose escalation portion, the study is using 160 mg twice daily (BID) or 320 mg once daily (QD). As of March 31, 2017, 69 patients with CLL or SLL (18 TN, 51 R / R) were enrolled in the study. It has been recorded.

[0213] Compound 1 was well tolerated in CLL / SLL. The most common adverse events (AEs) (≥10%) were petechiae / purpura / bruising (46%) and fatigue (2 9%), upper respiratory tract infection (28%), cough (23%), diarrhea (22%), headache (19%), and hematuria (15%) ), nausea (13%), rash (13%), joint pain (12%), muscle spasms (12%), urinary tract infections The only serious bleeding event was grade 3 purpura (subcutaneous bleeding). All of these events were grade 1 or 2, with the exception of one patient. Other adverse events included one case each of grade 2 diarrhea and grade 2 atrial fibrillation. It occurred. A total of 18 severe AEs (SAEs) occurred in 13 patients, but no SAE occurred in more than two patients. Only one patient with grade 2 pleural effusion discontinued treatment due to AE.

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

Claims

1. Compound 1 【Chemistry 1】 wherein said amorphous form has an enantiomeric excess value of at least 90%.

2. The amorphous form of claim 1, wherein the amorphous form has a glass transition midpoint temperature of about 79.7°C.

3. The amorphous form of claim 2, wherein the amorphous form has an enantiomeric excess value of at least 97%.

4. The amorphous form of claim 3, wherein the amorphous form has a purity of at least 99.3%.

5. The amorphous form of claim 4, wherein the amorphous form has a purity of at least 99.5%.

6. A pharmaceutical composition comprising the amorphous form of claim 5 and a pharma- ceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, wherein the composition is in a single unit form.

8. The pharmaceutical composition of claim 7, wherein the single unit form contains approximately 40 mg of the amorphous form of compound 1.

9. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition comprises approximately 160 mg of the amorphous form of compound 1.

10. Compound 1 【Chemistry 2】 Lyophilized amorphous form of.

11. The freeze-dried amorphous form of claim 10, wherein the freeze-dried amorphous form has an enantiomeric excess value of at least 97%.

12. The freeze-dried amorphous form of claim 11, wherein the freeze-dried amorphous form has a purity of at least 99.3%.

13. The freeze-dried amorphous form of claim 12, wherein the freeze-dried amorphous form has a purity of at least 99.5%.

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