Use of a bruton's tyrosine kinase inhibitor

Abstract

The application provides use of a compound A or a pharmaceutically acceptable salt thereof as a Bruton's tyrosine kinase (BTK) inhibitor in preparation of a drug for treating a BTK-related tumor disease. In vitro and in vivo test results show that the compound A has good inhibitory activity on BTK kinase, good inhibitory activity on human B-cell lymphoma cells with high BTK expression, and good in vivo anti-tumor activity. In addition, the compound A has good pharmacokinetic properties and metabolic stability, and can be used for developing a drug preparation for treating a BTK-related tumor disease, and has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and in particular relates to the use of a Bruton's tyrosine kinase (BTK) inhibitor, especially the use of the compound in the preparation of a medicament for treating a BTK-related tumor disease. BACKGROUND

[0002] B cell receptor (BCR) signaling plays a crucial role in normal B cell development and adaptive immunity, and the activation of this signaling pathway contributes to the occurrence and development of B cell malignancies and autoimmune diseases. Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase belonging to the TEC tyrosine kinase family, and is a key regulator in the B cell receptor (BCR) signaling pathway, mainly expressed at various stages of B lymphocyte development (except for plasma cells at the end of B lymphocyte development), and has an important influence on the proliferation, differentiation and apoptosis of B cells.

[0003] In B-cell related malignancies, the BCR signaling pathway is overactive, which inhibits the normal differentiation and apoptosis of B cells and promotes abnormal proliferation. Abnormal regulation of the BCR pathway is often found in various B cell type malignancies. Currently, there are four BTK inhibitors approved for marketing. Ibrutinib (Imbruvica) is the first small molecule BTK inhibitor approved for marketing, which belongs to the first generation of BTK inhibitors. It was approved by the US FDA in 2013 for the clinical treatment of mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Since then, Ibrutinib has expanded its indications, and currently approved indications also include chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL) with 17p deletion, Waldenstrom macroglobulinemia (WM), marginal zone lymphoma (MZL), and chronic graft-versus-host disease (cGVHD). Ibrutinib can form a covalent bond with the 481st cysteine (Cys481) in the ATP binding domain of BTK, irreversibly inhibit BTK activation, block the BTK signaling pathway, and thus inhibit the proliferation and survival of B lymphoma cells, achieving the purpose of tumor treatment. Ibrutinib has achieved substantial efficacy in clinical treatment. However, due to the poor selectivity of Ibrutinib target, there are certain toxic side effects in clinical treatment. Acalabrutinib (ACP-196, Calquence) was approved for the treatment of MCL and CLL in 2017, which belongs to the second generation of BTK targeted drugs. Compared with Ibrutinib, Acalabrutinib has higher selectivity for BTK and lower off-target toxicity. In addition, Zanubrutinib (BGB-3111) developed by Bejing Junshi Bio-technology Co., Ltd. was approved by the FDA in November 2019 for the treatment of adult patients with relapsed MCL, becoming the first Chinese anti-cancer drug approved by the FDA as a breakthrough therapy. Tirabrutinib (ONO-4059) developed by Japan's Ono Pharmaceutical Co., Ltd. was approved for marketing by the Japan Pharmaceutical and Medical Device Agency (PMDA) in March 2020, and was used for the treatment of relapsed or refractory primary central nervous system lymphoma (PCNSL) and lymphoplasmacytic lymphoma (LPL). In general, the first generation of inhibitors has high inhibitory activity on BTK, but poor target selectivity and bioavailability; the second generation of inhibitors has good selectivity, but lower inhibition rate on BTK than the first generation of inhibitors.Structurally, the core skeleton of the currently marketed BTK drugs is mainly a bicyclic system.

[0004] In order to obtain a new generation of BTK inhibitors with high activity of the first generation of inhibitors and good selectivity of the second generation of inhibitors, the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences discloses a series of compounds with pyrimido[5,4-b]indolizine or pyrimido[5,4-b]pyrimidine structure in CN108101905A patent. Among them, pyrimido[5,4-b]indolizine compounds S1 and S10 and pyrimido[5,4-b]pyrimidine compounds S18, S19 and S20 show high BTK inhibitory activity. In further work, compounds of S configuration of S18, S19 and S20 (i.e. S18s, S19s and S20s) are also synthesized [Yu Xue, et al. Discovery of 4,7-Diamino-5-(4-phenoxyphenyl)-6-methylenepyrimido[5,4-b]pyrrolizines as Novel Bruton’s Tyrosine Kinase Inhibitors. J. Med. Chem., 2018, 61, 4608-4627.]. However, further research found that these compounds S1, S10, S18s and S19s are not stable during metabolism (the active metabolic site is one of the para positions of the terminal benzene ring), and the 4-position of the terminal phenyl group is prone to oxidation; and the oral bioavailability of compound S20s is not ideal.

[0005]

[0006] Based on the above problems, it is very important to develop a BTK kinase inhibitor with excellent BTK inhibitory activity and selectivity, high in vivo anti-tumor activity, and excellent oral administration performance and metabolic stability for the treatment of BTK related tumor diseases. SUMMARY

[0007] The purpose of the present application is to provide a use of a Bruton's tyrosine kinase (BTK) inhibitor compound A or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a BTK related tumor disease, wherein the compound A has the following structure:

[0008]

[0009] The above use, the BTK related tumor disease includes hematological tumors and solid tumors.

[0010] Preferably, the hematological tumor is lymphoma and leukemia.

[0011] Further preferably, the lymphoma is B-cell lymphoma.

[0012] The use described above, the BTK-related tumor disease includes histiocytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, marginal zone lymphoma, follicular lymphoma, Burkitt lymphoma or Waldenstrom macroglobulinemia.

[0013] The use described above, the diffuse large B-cell lymphoma is selected from one or more of the group consisting of non-special type diffuse large B-cell lymphoma, other large B-cell lymphoma, high-grade B-cell lymphoma with MYC and / or Bcl gene abnormalities and non-special type high-grade B-cell lymphoma; preferably, the diffuse large B-cell lymphoma is selected from one or more of the group consisting of other large B-cell lymphoma, high-grade B-cell lymphoma with MYC and / or Bcl gene abnormalities and non-special type high-grade B-cell lymphoma.

[0014] The use described above, the other large B-cell lymphoma includes T cell / histiocyte-rich DLBCL, primary central nervous system DLBCL, primary cutaneous DLBCL (leg type), EBV-positive DLBCL, non-specified DLBCL, chronic inflammation-associated large B-cell lymphoma, lymphomatoid granulomatosis, large B-cell lymphoma with IRF4 rearrangement, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, etc.

[0015] The use described above, the high-grade B-cell lymphoma with MYC gene abnormalities is selected from one or more of the group consisting of high-grade B-cell lymphoma with MYC gene amplification, high-grade B-cell lymphoma with MYC gene fusion; the high-grade B-cell lymphoma with Bcl gene abnormalities is selected from high-grade B-cell lymphoma with Bcl2 and / or Bcl6 gene abnormalities, which is selected from one or more of the group consisting of high-grade B-cell lymphoma with Bcl2 gene amplification, high-grade B-cell lymphoma with Bcl2 gene fusion, high-grade B-cell lymphoma with Bcl6 gene amplification and high-grade B-cell lymphoma with Bcl6 gene fusion.

[0016] The use described above, the diffuse large B-cell lymphoma involved in the present application can also be a diffuse large B-cell lymphoma carrying abnormal chromosomes.

[0017] The use described above, the mantle cell lymphoma is selected from one or more of the group consisting of classical mantle cell lymphoma, leukemia-like non-nodular mantle cell lymphoma and Cyclin D1-positive mantle cell lymphoma.

[0018] The use as described above, the mantle cell lymphoma is selected from mantle cell lymphoma with abnormal Bcl gene; preferably is mantle cell lymphoma with abnormal Bcl1 and / or Bcl2 gene; further preferably is mantle cell lymphoma with Bcl1 gene fusion or rearrangement or mantle cell lymphoma with Bcl2 gene fusion or rearrangement.

[0019] The use as described above, the mantle cell lymphoma involved in the application can also be mantle cell lymphoma carrying abnormal chromosome.

[0020] The use as described above, the Burkitt lymphoma is Burkitt lymphoma carrying abnormal chromosome.

[0021] The use as described above, the gene abnormality refers to gene mutation, gene fusion or rearrangement and / or gene amplification.

[0022] The use as described above, the abnormal chromosome refers to chromosome with amplification, deletion, breakage, rearrangement and / or translocation.

[0023] The use as described above, the medicine can also contain one or more other targeted drugs or chemotherapy drugs. The other targeted drugs or chemotherapy drugs refer to targeted drugs or chemotherapy drugs clinically used for treating tumor-related diseases.

[0024] The use as described above, the medicine is prepared into clinically acceptable preparation, such as oral preparation, injection preparation, external preparation, etc.

[0025] The use as described above, the medicine contains therapeutically effective amount of Compound A or pharmaceutically acceptable salt thereof, and the therapeutically effective amount is preferably 0.001-1000 mg per day, further preferably 0.01-500 mg per day, more further preferably 0.1-200 mg per day, more further preferably 0.5-100 mg per day, more further preferably 0.5-50 mg per day, more further preferably 0.5-30 mg per day, more further preferably 0.5-20 mg per day. It can be administered in single dose or in divided doses.

[0026] In another aspect, the application also provides a method for treating BTK-related tumor diseases, characterized in that a medicine containing therapeutically effective amount of Compound A or pharmaceutically acceptable salt thereof is administered to a subject or patient.

[0027] The method as described above, the BTK-related tumor diseases include hematological tumors and solid tumors.

[0028] Preferably, the hematological tumors are lymphoma and leukemia.

[0029] Further preferably, the lymphoma is B-cell lymphoma.

[0030] The method, wherein the BTK-related neoplastic disease comprises histiocytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, marginal zone lymphoma, follicular lymphoma, Burkitt lymphoma, or Waldenstrom macroglobulinemia.

[0031] The use, wherein the diffuse large B-cell lymphoma is selected from one or more of the group consisting of non-specialized diffuse large B-cell lymphoma, other large B-cell lymphoma, high-grade B-cell lymphoma with MYC and / or Bcl gene abnormalities, and non-specialized high-grade B-cell lymphoma; preferably, the diffuse large B-cell lymphoma is selected from one or more of the group consisting of other large B-cell lymphoma, high-grade B-cell lymphoma with MYC and / or Bcl gene abnormalities, and non-specialized high-grade B-cell lymphoma.

[0032] The use, wherein the other large B-cell lymphoma comprises T-cell / histiocyte-rich DLBCL, primary central nervous system DLBCL, primary cutaneous DLBCL (leg type), EBV-positive DLBCL, non-specific DLBCL, chronic inflammation-associated large B-cell lymphoma, lymphomatoid granulomatosis, large B-cell lymphoma with IRF4 rearrangement, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, and the like.

[0033] The use, wherein the high-grade B-cell lymphoma with MYC gene abnormalities is selected from one or more of the group consisting of high-grade B-cell lymphoma with MYC gene amplification, high-grade B-cell lymphoma with MYC gene fusion; and the high-grade B-cell lymphoma with Bcl gene abnormalities is selected from high-grade B-cell lymphoma with Bcl2 and / or Bcl6 gene abnormalities, which is selected from one or more of the group consisting of high-grade B-cell lymphoma with Bcl2 gene amplification, high-grade B-cell lymphoma with Bcl2 gene fusion, high-grade B-cell lymphoma with Bcl6 gene amplification, and high-grade B-cell lymphoma with Bcl6 gene fusion.

[0034] The use, wherein the diffuse large B-cell lymphoma involved in the present application can also be a diffuse large B-cell lymphoma carrying abnormal chromosomes.

[0035] The use, wherein the mantle cell lymphoma is selected from one or more of the group consisting of classical mantle cell lymphoma, leukemia-like non-nodular mantle cell lymphoma, and Cyclin D1-positive mantle cell lymphoma.

[0036] The use as described above, the mantle cell lymphoma is selected from the group consisting of Bcl gene abnormal mantle cell lymphoma; preferably Bcl1 and / or Bcl2 gene abnormal mantle cell lymphoma; further preferably Bcl1 gene fusion or rearrangement of mantle cell lymphoma or Bcl2 gene fusion or rearrangement of mantle cell lymphoma.

[0037] The use as described above, the mantle cell lymphoma involved in the present application can also be a mantle cell lymphoma carrying abnormal chromosomes.

[0038] The use as described above, the Burkitt lymphoma is a Burkitt lymphoma carrying abnormal chromosomes.

[0039] The use as described above, the gene abnormality refers to gene mutation, gene fusion or rearrangement and / or gene amplification.

[0040] The use as described above, the abnormal chromosome refers to the chromosome that has undergone amplification, deletion, fragmentation, rearrangement and / or translocation.

[0041] The method as described above, the administration can be oral administration, injection administration, local administration or in vitro administration, preferably oral administration or injection administration.

[0042] The method as described above, the administration dose and dose frequency of Compound A or its pharmaceutically acceptable salt can be determined by conventional methods such as modeling, dose escalation study or conventional methods of clinical trials and by considering factors such as the characteristics and severity of the disease to be treated, the age, general condition and body weight of the patient, and the specific compound to be administered, its pharmacokinetic properties, and the route of administration. The suitable dose range of Compound A or its pharmaceutically acceptable salt is from about 0.001 mg / kg to about 1000 mg / kg per day; preferably, from about 0.01 mg / kg to about 100 mg / kg; further preferably, from about 0.02 mg / kg to about 50 mg / kg; more further preferably, from about 0.03 mg / kg to about 20 mg / kg. Preferably, the daily administration dose of Compound A or its pharmaceutically acceptable salt is 0.001 mg-1000 mg, further preferably, the daily administration dose of Compound A or its pharmaceutically acceptable salt is 0.01-500 mg; more further preferably, the daily administration dose of Compound A or its pharmaceutically acceptable salt is 0.1-200 mg; more further preferably, the daily administration dose of Compound A or its pharmaceutically acceptable salt is 0.5-100 mg; more further preferably, the daily administration dose of Compound A or its pharmaceutically acceptable salt is 0.5-50 mg; more further preferably, the daily administration dose of Compound A or its pharmaceutically acceptable salt is 0.5-30 mg; more further preferably, the daily administration dose of Compound A or its pharmaceutically acceptable salt is 0.5-20 mg; administered in single dose or divided doses.

[0043] In another aspect, the present application also provides a method for treating a patient's condition, which is a BTK-related neoplastic disease, by administering to the patient a medicament containing a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof.

[0044] The above method, wherein the BTK-related neoplastic disease comprises hematological neoplasms and solid tumors.

[0045] Preferably, the above hematological neoplasms are lymphomas and leukemias.

[0046] Further preferably, the above lymphomas are B-cell lymphomas.

[0047] The above method, wherein the BTK-related neoplastic disease comprises histiocytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, marginal zone lymphoma, follicular lymphoma, Burkitt lymphoma, or Waldenstrom macroglobulinemia.

[0048] The above use, wherein the diffuse large B-cell lymphoma is selected from one or more of the group consisting of non-specialized type diffuse large B-cell lymphoma, other large B-cell lymphoma, high-grade B-cell lymphoma with MYC and / or Bcl gene abnormalities, and non-specialized type high-grade B-cell lymphoma; preferably, the diffuse large B-cell lymphoma is selected from one or more of the group consisting of other large B-cell lymphoma, high-grade B-cell lymphoma with MYC and / or Bcl gene abnormalities, and non-specialized type high-grade B-cell lymphoma.

[0049] The above use, wherein the other large B-cell lymphoma comprises T-cell / histiocyte-rich DLBCL, primary central nervous system DLBCL, primary cutaneous DLBCL (leg type), EBV-positive DLBCL, DLBCL not otherwise specified, chronic inflammation-associated large B-cell lymphoma, lymphomatoid granulomatosis, large B-cell lymphoma with IRF4 rearrangement, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, etc.

[0050] The use as described above, the high-grade B-cell lymphoma with MYC gene abnormality is selected from one or more of the group consisting of high-grade B-cell lymphoma with MYC gene amplification, high-grade B-cell lymphoma with MYC gene fusion; the high-grade B-cell lymphoma with Bcl gene abnormality is selected from high-grade B-cell lymphoma with Bcl2 and / or Bcl6 gene abnormality, which is selected from one or more of the group consisting of high-grade B-cell lymphoma with Bcl2 gene amplification, high-grade B-cell lymphoma with Bcl2 gene fusion, high-grade B-cell lymphoma with Bcl6 gene amplification, and high-grade B-cell lymphoma with Bcl6 gene fusion.

[0051] The use as described above, the diffuse large B-cell lymphoma involved in the present application can also be a diffuse large B-cell lymphoma carrying abnormal chromosomes.

[0052] The use as described above, the mantle cell lymphoma is selected from one or more of the group consisting of classical mantle cell lymphoma, leukemia-like non-nodular mantle cell lymphoma, and Cyclin D1-positive mantle cell lymphoma.

[0053] The use as described above, the mantle cell lymphoma is selected from a mantle cell lymphoma with Bcl gene abnormality; preferably a mantle cell lymphoma with Bcl1 and / or Bcl2 gene abnormality; further preferably a mantle cell lymphoma with Bcl1 gene fusion or rearrangement or a mantle cell lymphoma with Bcl2 gene fusion or rearrangement.

[0054] The use as described above, the mantle cell lymphoma involved in the present application can also be a mantle cell lymphoma carrying abnormal chromosomes.

[0055] The use as described above, the Burkitt lymphoma is a Burkitt lymphoma carrying abnormal chromosomes.

[0056] The use as described above, the gene abnormality refers to gene mutation, gene fusion or rearrangement, and / or gene amplification.

[0057] The use as described above, the abnormal chromosome refers to a chromosome that has undergone amplification, deletion, fragmentation, rearrangement, and / or translocation.

[0058] The therapeutically effective amount, administration dose or dosage of the compound A or its pharmaceutically acceptable salt according to the present application is calculated based on the compound A.

[0059] According to the 2017 edition of hematopoietic and lymphoid tumors (4th revised edition), diffuse large B-cell lymphoma (DLBCL) is classified into four categories: DLBCL of non-specific type, other large B-cell lymphoma, high-grade B-cell lymphoma, and B-cell lymphoma that cannot be classified between DLBCL and classical Hodgkin's lymphoma. Among them, the DLBCL of non-specific type includes morphologically including centroblastic variant, immunoblastic variant, anaplastic variant, and other rare variants (such as spindle cell variant, signet ring cell-like variant), etc.; other large B-cell lymphoma includes T cell / histiocyte-rich DLBCL, primary central nervous system DLBCL, primary cutaneous DLBCL (leg type), EBV-positive DLBCL, DLBCL of non-specific type, chronic inflammation-associated large B-cell lymphoma, lymphomatoid granulomatosis, large B-cell lymphoma with IRF4 rearrangement, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, etc.; high-grade B-cell lymphoma includes high-grade B-cell lymphoma with MYC, BCL2 and / or BCL6 abnormalities (for example, gene rearrangement or fusion, gene amplification, gene mutation, etc.), high-grade B-cell lymphoma of non-specific type, etc., such as U-2932 cells mentioned in the present application (with Bcl2 gene amplification), WILL-2 cells (with Bcl2 gene fusion), etc. (Source: ATCC website information and DSMZ website information)

[0060] The "diffuse large B-cell lymphoma carrying abnormal chromosomes" mentioned in the present application refers to a diffuse large B-cell lymphoma carrying abnormal chromosomes (for example, chromosome amplification, deletion, breakage, rearrangement and / or translocation, etc.), such as Pfeiffer cells mentioned in the present application (with multiple chromosomal abnormalities, including chromosome t(14; 18)(q32; q21) translocation), etc. (Source: ATCC website information and DSMZ website information)

[0061] The "mantle cell lymphoma carrying abnormal chromosomes" mentioned in the present application refers to a mantle cell lymphoma carrying abnormal chromosomes (for example, chromosome translocation, chromosome fusion, chromosome deletion, etc.), such as Z-138 cells mentioned in the present application (with chromosomal abnormalities, such as chromosome t(11; 14)(q13; q32) translocation and / or chromosome del(5)(p15) deletion, etc.), Mino cells (with chromosomal abnormalities, such as chromosome del(6)(q16) deletion, etc.), REC-1 cells (with chromosomal abnormalities, such as chromosome t(11; 14)(q13; q32) translocation, etc.). (Source: ATCC website information and DSMZ website information)

[0062] The "Bcl gene abnormal mantle cell lymphoma" mentioned in the present application refers to the mantle cell lymphoma with Bcl gene abnormality (e.g., gene mutation, gene amplification, gene rearrangement or / fusion, gene abnormal activation, etc.), which can cause overexpression of Bcl-related proteins, such as Jeko-1 cells mentioned in the present application (with Bcl-1 gene rearrangement), etc. (Source: ATCC official website information)

[0063] The "Burkitt lymphoma carrying abnormal chromosome" mentioned in the present application refers to the Burkitt lymphoma carrying abnormal chromosome (e.g., abnormality of chromosome size and / or quantity, chromosome translocation, chromosome fusion, chromosome deletion, etc.), such as Raji cells mentioned in the present application (abnormality of chromosome size and / or quantity of chromosome 1 or 4).(Source: ATCC official website information)

[0064] The pharmaceutically acceptable salt of the compound can be a conventional non-toxic salt formed by the reaction of the compound with inorganic acids, organic acids, inorganic bases or organic bases.

[0065] In the preparation method and the present application, the terms used are as follows:

[0066] DCM: dichloromethane; DIAD: diisopropyl azodicarboxylate; DIPEA: diisopropylethylamine; DMF: N,N-dimethylformamide; EA: ethyl acetate; HATU: 2-(7-oxabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; NBS: N-bromosuccinimide; NIS: N-iodosuccinimide; PdCl2(dppf): [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium; Pd(PPh3)4: tetrakis(triphenylphosphine)palladium; PdCl2: palladium dichloride; Pd(OAc)2: palladium acetate; Pd(PPh3)2Cl2: bis(triphenylphosphine)palladium dichloride; PE: petroleum ether; THF: tetrahydrofuran; DMSO: dimethyl sulfoxide.

[0067] The present application achieves one or more of the following beneficial technical effects:

[0068] (1) The in vitro and in vivo pharmacodynamic test results show that the inhibitory activity of compound A on BTK and in vitro cultured lymphoma cells is not only superior to the previous compounds S1, S10, S18s, S19s and S20s, but also superior to the first-generation BTK inhibitor ibrutinib and the second-generation BTK inhibitor acalabrutinib. In the in vivo tumor transplantation model, compound A also showed a significantly better effect on inhibiting tumor growth than S18s and ibrutinib.

[0069] (2) In vitro pharmacodynamic test results show that compound A has good inhibitory effect on various types of tumor cells, and also has good inhibitory effect on different specific cell strains of the same cell type, that is, compound A can achieve good inhibitory effect on the same tumor cell type and different source cell strains, and has good clinical application prospect.

[0070] (3) Pharmacokinetic studies show that the in vivo clearance rate of compound A is significantly lower than that of ibrutinib and its structural analogs S18s, S19s and S20s, only 1 / 10-1 / 20 of the latter; the drug exposure amount (AUC) in the plasma after oral administration of compound A is 70 times higher than that of ibrutinib, and the absolute bioavailability is significantly increased.

[0071] (4) S1, S10, S18s and S19s are easily oxidized at the 4-position of the terminal phenyl group, have many types of metabolites, and have poor metabolic stability, and only 51% of the original drug can be retained after S18s is incubated with rat liver microsomes. Compound A effectively overcomes the hydroxylation of the terminal benzene ring of the diphenyl ether structure in the above-mentioned compounds by structural modification. Under the action of different species of liver microsomes (HLM: human liver microsomes; RLM: rat liver microsomes; MLM: mouse liver microsomes), the types and proportions of metabolites are small, and the metabolic stability is better, mainly in the form of the original drug (60min: 84%-98%).

[0072] In summary, compound A has good BTK inhibitory activity and tumor inhibition effect, low in vivo clearance rate, good oral bioavailability, metabolic stability, and important clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 : Schematic diagram of experimental results of REC-1 xenograft tumor model.

[0074] Figure 2 : Schematic diagram of experimental results of TMD8 xenograft tumor model. DETAILED DESCRIPTION

[0075] The following further provides implementation examples which are helpful for understanding the present application and are only used as illustration and do not limit the application range of the present application.

[0076] Example 1: Synthesis of compound A

[0077] 1. Synthesis of intermediate 3

[0078]

[0079] Into a 250 mL round bottom flask was added 4-chloro-5-iodo-7H-pyrrolo[2,3- d]pyrimidine (starting material 2, 17.28 g, 1 eq) and anhydrous potassium carbonate (2 eq) and dried under vacuum to remove water. Dry DMF was added as solvent and powdered (S)- methylsulfonic acid 2-((tert-butoxycarbonyl)amino)-but-3-en-1-yl ester (starting material 1, 24.6 g, 1.5 eq) was added and the nitrogen was replaced. The reaction was heated to 55 °C and stirred for 12 hours, the time can be extended to ensure completion of the reaction.

[0080] After the reaction was complete, water was added and the reaction was extracted with ethyl acetate three times and the organic layers were combined and washed with water once and saturated brine. The organic layer was dried over anhydrous sodium sulfate. The product, (S)-(1-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)but-3-en-2-yl)carbamic acid tert-butyl ester (intermediate 3, 19.28 g) was obtained in 69.5% yield after drying and column chromatography (eluent: CHCl3:MeOH = 100:1).

[0081] 1 H NMR (300 MHz, CDC13) δ 8.60 (s, 1H), 7.39 (s, 1H), 5.82 (ddd, J = 17.1, 10.5, 5.5 Hz, 1H), 5.33-5.14 (m, 2H), 4.80 (s, 1H), 4.63-4.51 (m, 1H), 4.51-4.42 (m, 1H), 4.35 (s, 1H), 1.33 (s, 9H). ee > 99.5%.

[0082] 2. Synthesis of Intermediate 4

[0083]

[0084] Into a 350 mL pressure tube was added (S)-(1-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7- yl)but-3-en-2-yl)carbamic acid tert-butyl ester (intermediate 3, 9.2 g) and 1,4-dioxane (40 mL) was added as solvent and aqueous ammonia (40 mL) was added. The reaction was heated to 120 °C for 2.5 hours.

[0085] After the reaction was complete, the reaction was cooled to room temperature and water was added and the reaction was extracted with ethyl acetate and the organic layers were combined and washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate. The product, (S)-(1-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)but-3-en-2-yl)carbamic acid tert-butyl ester (intermediate 4, 6.86 g) was obtained in 78.0% yield after drying and column chromatography (eluent: CHCl3:MeOH = 30:1).

[0086] 1H NMR (300 MHz, CDC13) δ 8.25 (s, 1H), 7.05 (s, 1H), 5.87-5.74 (m, 1H), 5.72 (s, 2H), 5.34-5.13 (m, 3H), 4.56-4.43 (m, 1H), 4.34 (dd, J = 14.8, 4.9 Hz, 1H), 4.30-4.15 (m, 1H), 1.35 (s, 9H). ee > 99.5%.

[0087] 3. Synthesis of Intermediate 6

[0088]

[0089] In a 1 L round bottom flask, add (S)-(1-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7- yl)but-3-en-2-yl)carbamic acid tert-butyl ester (Intermediate 4, 32.9 g, 1 eq), N-(pyridin-2- yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (raw material 5, 34.8 g, 1.4 eq) and Pd(PPh3)4(17.7 g, 0.2 eq). Add 1,4-dioxane (383 mL) as solvent and replace N2. Add 2M sodium carbonate solution (76.6 mL) under stirring. Stir at 90 °C for 5 hours under reflux.

[0090] Add water and extract with ethyl acetate, combine the ester layers and wash with saturated brine. Dry over anhydrous sodium sulfate. Dry column, first use EA as eluent to remove most of the impurities, then use CHCl3:MeOH = 30:1 mixture as eluent. The product might contain a small amount of impurities, which can be recrystallized with PE to obtain pure product. Obtain product (S)-(1-(4-amino-5-(4-(pyridin-2-ylcarbamoyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-7- yl)but-3-en-2-yl)carbamic acid tert-butyl ester (Intermediate 6, 28.4 g) with a yield of 74.3%. ee > 99.5%.

[0091] 4. Synthesis of Intermediate 7

[0092]

[0093] In a 1 L round bottom flask, add (S)-(1-(4-amino-5-(4-(pyridin-2-ylcarbamoyl)phenyl)-7H- pyrrolo[2,3-d]pyrimidin-7-yl)but-3-en-2-yl)carbamic acid tert-butyl ester (Intermediate 6, 32.7 g, 1 eq), add 600 mL DMF as solvent. Slowly add NBS (12.8 g, 1.1 eq) under stirring and stir overnight at room temperature.

[0094] After the reaction was completed, water was added and extracted with ethyl acetate, the ester layers were combined, back-extracted once with water, and washed with saturated brine. Dried over anhydrous sodium sulfate. Dry column (eluent: CHCl3:MeOH = 50:1, then CHCl3:MeOH = 30:1). Obtained the product (S)-(1-(4-amino-6-bromo-5-(4-(pyridin-2-ylcarbamoyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)but-3-en-2-yl)carbamic acid tert-butyl ester (Intermediate 7, 25.8 g) with a yield of 68.2%. ee > 99.5%.

[0095] 5. Synthesis of Intermediate 8

[0096]

[0097] In a 250 mL round bottom flask was added (S)-(1-(4-amino-6-bromo-5-(4-(pyridin-2-ylcarbamoyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)but-3-en-2-yl)carbamic acid tert-butyl ester (Intermediate 7, 11.9 g, 1 eq) and PdCl2(dppf) (1.66 g, 0.11 eq), added 51 mL THF as solvent and replaced nitrogen several times to ensure complete. Added 4M sodium hydroxide solution (8.2 mL) under stirring. Stirred at 85°C under reflux for 15 hours.

[0098] After the reaction was completed, water was added and extracted with ethyl acetate, the ester layers were combined, back-extracted once with water, and washed with saturated brine. Dried over anhydrous sodium sulfate. Dry column (eluent: CHCl3:MeOH = 50:1, then CHCl3:MeOH = 30:1). Obtained the product (S)-(1-(4-amino-6-bromo-5-(4-(pyridin-2-ylcarbamoyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)but-3-en-2-yl)carbamic acid tert-butyl ester (Intermediate 7, 25.8 g) with a yield of 68.2%. ee > 99.5%.

[0099] 6. Synthesis of Compound A

[0100]

[0101] Into a 250 mL round bottom flask, (S)-(4-amino-6-methylene-5-(4-(pyridin-2-ylcarbamoyl)phenyl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrimidin-7-yl)carbamic acid tert-butyl ester (Intermediate 8, 2.75 g, 1 eq) was added as solvent 110 mL DCM. Added trifluoroacetic acid (10.5 mL) dropwise with stirring. Stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was directly spin-dried, and the trifluoroacetic acid was carried out with methanol several times, and after spin-drying, the Boc-protected amino crude product was obtained, which was directly used in the next step.

[0102] The product of the previous step was moved to a 250 mL round bottom flask, and triethylamine (1 eq) was added, stirred for five minutes, then 2-butynoic acid (0.511 g, 1.1 eq) and HATU (2.31 g, 1.1 eq) were added, and 100 mL DCM was added as solvent. The ice water bath was cooled to 0°C, and triethylamine (1.54 mL + 0.77 mL) was added dropwise. Gradually warmed to room temperature, and stirred at room temperature for 1.5 hours. The reaction solution was slightly yellow. Added water and DCM for extraction, combined the organic phase, and washed with saturated brine. Dried over anhydrous sodium sulfate, and column chromatography (CHCl3:MeOH = 30:1) to obtain the final product A (1.88 g) with a yield of 73.3%. ee > 99.5%.

[0103] 1H NMR (400 MHz, CDCl3) δ 8.98 (s, 1H), 8.43 (dt, J = 8.3, 1.0 Hz, 1H), 8.34 (ddd, J = 5.0, 1.9, 0.9 Hz, 1H), 8.22 (s, 1H), 8.09-8.03 (m, 2H), 7.81 (ddd, J = 8.4, 7.4, 1.9 Hz, 1H), 7.69-7.63 (m, 2H), 7.13 (ddd, J = 7.4, 4.9, 1.0 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 5.67 (m, J = 8.1, 5.7, 2.6 Hz, 1H), 5.56 (d, J = 2.3 Hz, 1H), 5.40 (s, 2H), 5.27 (d, J = 2.3 Hz, 1H), 4.70 (dd, J = 11.7, 8.1 Hz, 1H), 4.09-3.99 (m, 1H), 1.99 (s, 3H).

[0104] Experimental Example 1: Evaluation of the activity of inhibiting the enzyme activity at the molecular level of Bruton's kinase (BTK)

[0105] The enzyme reaction substrate Poly(Glu, Tyr) 4:1Dilute the coated enzyme plate with PBS (10 mM sodium phosphate buffer, 150 mM NaCl, pH 7.2-7.4) without potassium ions to 20 μg / mL, after 12-16 hours of reaction at 37 ℃, wash the plate with 200 μL / well of T-PBS (PBS containing 0.1% Tween-20) three times, and dry the enzyme plate in an oven at 37 ℃ for 1-2 hours. In the above coated substrate enzyme plate, first add 49 μL / well of ATP solution diluted with reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) (final concentration 5 μM). Add 1 μL of the compound to be tested (compound well) or DMSO containing the corresponding concentration (negative control well) to each well, and set up the enzyme-free control well for each experiment. Add 50 μL of BTK tyrosine kinase protein diluted with reaction buffer to start the reaction.

[0106] Place the above reaction system in a 37 ℃ shaker (100 rpm) for 1 hour, then wash the plate with T-PBS three times, add 100 μL / well of primary antibody PY9910 (Santa Cruz) and react for 0.5 hours at 37 ℃ on a shaker. After washing the plate with T-PBS, add 100 μL / well of horseradish peroxidase-labeled goat anti-mouse secondary antibody dilution, and react for 0.5 hours at 37 ℃ on a shaker. After washing the plate with T-PBS, add 100 μL / well of 2 mg / mL OPD developing solution, and react for 1-10 minutes at 25 ℃ in the dark. Then add 50 μL / well of 2 M H2SO4 to stop the reaction, and read the results with an adjustable wavelength microplate reader SPECTRA MAX Plus384 at a wavelength of 490 nm.

[0107] Compound S1, S10, ibrutinib, acalabrutinib, S18s, S19s and S20s were used as positive control compounds, wherein compounds S1, S10, S18s, S19s and S20s were prepared by the method disclosed in the prior art (for example CN108101905A) or a similar method, and ibrutinib and acalabrutinib were purchased from Selleck Company.

[0108] The inhibition rate of each compound was calculated by the following formula:

[0109]

[0110] IC 50 The IC50values were obtained by four-parameter regression with the random software attached to the enzyme reader. The results are shown in Table 1 below.

[0111] Table 1 Inhibition of BTK by different compounds

[0112] Compound IC 50 (nM) S1 ～1 S10 <10 Ibrutinib ～1 Acalabrutinib ～10 S18s ～1 S19s ～1 S20s ～1 Compound A 0.5

[0113] The above results show that the inhibitory activity of compound A on BTK is better than that of previous compounds S1, S10, S18s, S19s and S20s, and also better than that of the first-generation BTK inhibitor ibrutinib and the second-generation BTK inhibitor acalabrutinib.

[0114] Experimental Example 2: Detection of in vitro proliferation inhibition activity of compounds on human B lymphoma cells

[0115] Experimental cells

[0116]

[0117]

[0118] Note: DLBCL: diffuse large B-cell lymphoma; FBS: fetal bovine serum; 2-mercaptoethanol: 2-mercaptoethanol

[0119] Test method:

[0120] The cell suspension (Ramos: 10,000 cells / well; TMD8: 12,000 cells / well) was inoculated in a 96-well plate and incubated in a 37°C incubator for 2 hours to stabilize the cell state. Different concentrations of the test compound were added to each well (3 replicate wells for each concentration), and at the same time, a blank control (a well containing only the culture solution without cells), a negative control (a well containing only cells without the compound), and a positive compound control were set. After 72 hours of drug treatment, 20 μL of MTT (5 mg / mL) was added to each well and incubated at 37°C for 4 hours. 100 μL of triplex solution (10% SDS, 5% isobutanol, 0.01M HCl) was added and incubated at 37°C overnight. The OD value was determined at 570 nm wavelength using an adjustable wavelength microplate reader SPECTRAmax Plus384.

[0121] The inhibition rate of the compound was calculated by the following formula:

[0122]

[0123] IC 50 The IC50value was obtained by four-parameter regression using the random software attached to the enzyme marker. The experiment was independently repeated 3 times, and the results are shown in Table 2 below.

[0124] Similarly, the above-mentioned compounds S1, S10, ibrutinib, acalabrutinib, S18s, S19s and S20s were used as positive control compounds.

[0125] Table 2 Proliferation inhibition activity of different compounds on Ramos cells and TMD8 cells

[0126] Compound Ramos cell IC 50 ]] TMD8 cells IC 50 ]] S1 94.73 μΜ 0.006 μΜ S10 8.72 μΜ 0.030 μΜ Ibrutinib 12.91 μΜ 0.005 μΜ Acalabrutinib 38.16 μΜ 0.023 μΜ Compound A 3.15 μΜ 0.003 μΜ S18s 5.04 μΜ 0.016 μΜ S19s — 0.017 μΜ S20s 14.3 μΜ 0.004 μΜ

[0127] The above results show that, at the cellular level, the proliferation inhibition ability of compound A on B-cell lymphoma is better than that of previous compounds S1, S10, S18s, S19s and S20s, and also better than that of the first-generation BTK inhibitor Ibrutinib and the second-generation BTK inhibitor Acalabrutinib. Further, it needs to be explained that, compared with other compounds, compound A of the present application has higher proliferation inhibition activity on Ramos cells and higher proliferation inhibition activity on TMD8 cells.

[0128] Experimental cells

[0129]

[0130]

[0131] Note: DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; MCL: mantle cell lymphoma; PMBCL: primary mediastinal B-cell lymphoma

[0132] Test method

[0133] The CCK8 (Cell Counting Kit-8, #D3100L4057, Shanghai Li Ji Biological Technology Co., Ltd.) staining method was used to detect the proliferation inhibition activity of the compound on cells.

[0134] (1) The tumor cells in the logarithmic growth phase were inoculated with cell suspensions of appropriate density in 96-well plates, 95 μL per well. After the cells were incubated in a 37°C incubator for 2-4 h to stabilize the cell state, 10 μL of the desired concentration of the compound was added in gradient, 3 replicates were set for each dose, and solvent control and cell-free blank control wells were also set. Incubate in a 37°C carbon dioxide incubator for 72 h.

[0135] (2) Add CCK8 staining solution, 10 μL / well. Incubate in an incubator for 2-4 h, and use a microplate reader SPECTRAmax PLUS 384 to read at a wavelength of 450 nm. The formula for calculating the growth inhibition rate of the compound on cells is:

[0136] Inhibition rate % = (control group OD value - drug group OD value) / control group OD value x 100%.

[0137] Half-inhibitory amount IC 50 The four-parameter method was used to calculate the value. Each experiment was independently repeated 3 times, and the average IC 50 value of each experiment was taken as the final indicator of inhibition ability.

[0138] Table 3 Proliferation inhibition activity of Compound A on B lymphoma cells (BTK inhibitor-sensitive cell lines)

[0139]

[0140] The above results show that Compound A has strong inhibitory activity on 2 BTK inhibitor-sensitive cell lines, and is better than the positive control drugs Ibrutinib and Acalabrutinib.

[0141] Table 4 Proliferation inhibition activity of Compound A on B lymphoma cells

[0142]

[0143]

[0144] The above results show that the inhibitory activity of Compound A on other B lymphoma cells is either comparable to Ibrutinib or between Ibrutinib and Acalabrutinib, all at micromolar level. The results of Table 3 and Table 4 collectively show that, compared with the positive control drugs, Compound A has more obvious inhibitory effect on BTK inhibitor-sensitive cell lines, and also has certain inhibitory activity on other B lymphoma cells, i.e. has better BTK inhibition selectivity.

[0145] The above results of Table 3 and Table 4 show that the inhibitory effect of Compound A on U-2932 and WILL-2 indicates that the Compound A of the present application is expected to have the effect of treating high-grade B-cell lymphoma with Bcl gene abnormalities in diffuse large B-cell lymphoma; the inhibitory effect of Compound A on Pfeiffer indicates that the Compound A of the present application is expected to have the effect of treating diffuse large B-cell lymphoma carrying abnormal chromosomes in diffuse large B-cell lymphoma; the inhibitory effect of Compound A on RL indicates that the Compound A of the present application is expected to have the effect of treating follicular lymphoma; the inhibitory effect of Compound A on Raji and NAMALWA indicates that the Compound A of the present application is expected to have the effect of treating Burkitt lymphoma; the inhibitory effect of Compound A on Raji indicates that the Compound A of the present application is expected to have the effect of treating Burkitt lymphoma carrying abnormal chromosomes; the inhibitory effect of Compound A on Z-138, Mino and REC-1 indicates that the Compound A of the present application is expected to have the effect of treating mantle cell lymphoma carrying abnormal chromosomes in mantle cell lymphoma; the inhibitory effect of Compound A on JeKo-1 indicates that the Compound A of the present application is expected to have the effect of treating mantle cell lymphoma carrying abnormal chromosomes in mantle cell lymphoma; the inhibitory effect of Compound A on KARPAS-1106P indicates that the Compound A of the present application is expected to have the effect of treating primary mediastinal B-cell lymphoma in diffuse large B-cell lymphoma.

[0146] In addition, since the inhibitory activity of Compound A on OCI-LY10, REC-1 (a BTK inhibitor-sensitive cell line) is superior to that of the positive control drugs Ibrutinib and Acalabrutinib, and the inhibitory activity on other B lymphoma cells is equivalent to Ibrutinib or between Ibrutinib and Acalabrutinib, it is suggested that Compound A of the present application is expected to be able to treat diseases treated by Ibrutinib and Acalabrutinib, such as chronic lymphocytic leukemia, chronic lymphocytic leukemia / small lymphocytic lymphoma carrying 17p deletion, Waldenstrom's macroglobulinemia, marginal zone lymphoma, and chronic graft-versus-host disease.

[0147] Experimental Example 3: Evaluation of in vivo anti-tumor activity

[0148] Experimental animals:

[0149] TMD8 xenograft tumor model

[0150] 1) Species: mouse

[0151] 2) Strain: CB-17 SCID

[0152] 3) Week age and weight: 6-8 weeks; 18-22 g

[0153] 4) Gender: female

[0154] 5) Supplier: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0155] REC-1 xenograft tumor model

[0156] 1) Species: mouse

[0157] 2) Strain: BALB / c nude mice

[0158] 3) Week age and weight: 6-8 weeks; 17-20 g

[0159] 4) Gender: female

[0160] 5) Supplier: Shanghai Lingchang Biotechnology Co., Ltd.

[0161] Cell culture: human lymphoma TMD8 cells were cultured in vitro in suspension, and the culture conditions were 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin in RPMI 1640 medium (supplier: gibco; item number: 22400-089; production batch number: 4868546), 37°C, 5% CO2. Routine handling was performed twice a week for passage. When the cell saturation was 80%-90%, the cells were collected, counted, and inoculated.

[0162] Human cell lymphoma REC-1 cells were cultured in suspension in vitro in RPMI 1640 medium (Supplier: gibco; Item No.: 22400-089; Production Batch No.: 1868795) with 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured at 37°C in 5% CO2. Routine treatment was performed twice a week for subculture. When the cell saturation was 80%-90%, the cells were collected, counted, and inoculated.

[0163] Tumor cell inoculation: 0.2 mL of 10 x 10 6 Personal lymphoma TMD8 cells were inoculated subcutaneously on the right back of each nude mouse (PBS: Matrigel = 1:1). When the average tumor volume reached 104 mm 3 The grouping and administration were started when the average tumor volume reached 104 mm

[0164] 0.2 mL of 5 x 10 6 Personal lymphoma TMD8 cells were inoculated subcutaneously on the right back of each nude mouse (PBS: Matrigel = 1:1). When the average tumor volume reached 104 mm 3 The grouping and administration were started when the average tumor volume reached 104 mm

[0165] Preparation of the test substance:

[0166] The preparation method of the test substance is shown in Tables 5 and 6 below:

[0167] Table 5 Preparation method of test substance for TMD8 xenograft tumor model

[0168]

[0169] Note: The sample was prepared immediately before use, and the prepared sample was stored at 4°C, and needed to be mixed thoroughly before administration to the animal; the administration method was gavage; the administration volume was 10 μL / g.

[0170] Table 6 Preparation method of test substance for REC-1 xenograft tumor model

[0171]

[0172]

[0173] Note: The sample was prepared immediately before use, and the prepared sample was stored at 4°C, and needed to be mixed thoroughly before administration to the animal; the administration method was gavage; the administration volume was 10 μL / g.

[0174] Daily observation of experimental animals: The experimental protocol and any modification were approved by the Institutional Animal Care and Use Committee (IACUC) of Suzhou Drugphy New Drug Development Co., Ltd. The use and welfare of experimental animals were performed in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health status and death of animals were monitored every day, and routine examinations included observation of tumor growth and the effects of drug treatment on the daily behavior of animals, such as behavioral activity, food and water intake (only visually observed), body weight changes (body weight was measured three times per week), appearance, signs or other abnormalities. The number of animal deaths and side effects within each group were recorded based on the number of animals in each group.

[0175] Tumor measurement and experimental indexes: The experimental index was to investigate whether the tumor growth was inhibited, delayed or cured. The tumor diameter was measured three times a week using a vernier caliper.

[0176] The formula for calculating the tumor volume was:

[0177] V = 0.5a x b 2 ,

[0178] a and b represent the long diameter and short diameter of the tumor, respectively.

[0179] The antitumor efficacy of the compound was evaluated by TGI (%) or relative tumor proliferation rate T / C (%). TGI (%) reflects the tumor growth inhibition rate.

[0180] Calculation of TGI (%):

[0181] TGI (%) = 【1- (the average tumor volume at the end of administration in a certain treatment group - the average tumor volume at the start of administration in the treatment group) / (the average tumor volume at the end of treatment in the solvent control group - the average tumor volume at the start of treatment in the solvent control group) 】 x 100%.

[0182] Relative tumor proliferation rate T / C (%): the calculation formula is as follows:

[0183] T / C% = T RTV / C RTV x 100% (T RTV : relative tumor volume of the treatment group; C RTV : relative tumor volume of the negative control group). According to the results of tumor measurement, the relative tumor volume (RTV) was calculated, and the calculation formula was RTV = V t / V0, wherein V0 is the average tumor volume measured at the time of grouping administration (i.e. d0), V t is the average tumor volume at a certain time of measurement, T RTV and C RTV take data on the same day.

[0184] Statistical analysis: Statistical analysis, including the mean and standard error (SEM) of tumor volume at each time point for each group. The treatment groups showed the best therapeutic effect at day 15 (REC-1 xenograft tumor model) and day 17 (TMD8 xenograft tumor model) after administration, respectively, so the statistical analysis was based on this data to evaluate the differences between groups. Comparison between three or more groups was analyzed by one-way ANOVA, and if the F value was significantly different, Games-Howell method was used for testing. All data analysis was performed using SPSS 17.0. p<0.05 was considered to be significantly different.

[0185] The in vivo efficacy of Compound A in the human mantle cell lymphoma REC-1 xenograft tumor model is shown in Table 7 and Figure 1 The tumor volume of the solvent control group reached 3501 mm 3 , and the tumor volume of the Compound A 15 mg / kg group was 1034 mm 3 , which had a significant tumor inhibition effect compared with the solvent control group (T / C = 30%, TGI = 73%, p<0.01). 3 The tumor volume of the Compound A 15 mg / kg group was better than that of the Compound A 30 mg / kg group and the positive drug 25 mg / kg group.

[0186] Table 7 Evaluation of the tumor inhibition efficacy of Compound A on the REC-1 xenograft tumor model (calculated based on the tumor volume at day 15 after administration)

[0187]

[0188] Note: a. Mean ± SEM; b. Tumor growth inhibition was calculated by T / C and TGI (TGI (%) = [1- (T 15 -T0) / (V 15 -V0)] x 100); c. Administration method: once a day; **: p<0.01.

[0189] The in vivo efficacy of Compound A in the human mantle cell lymphoma REC-1 xenograft tumor model is shown in Table 7 and Figure 2 The tumor volume of the solvent control group reached 3501 mm 3 , and the tumor volume of the Compound A 15 mg / kg group was 1034 mm 3Compound A 10 mg / kg group had significant tumor inhibition effect (T / C = 35.68%, TGI = 68.18%, p < 0.001) compared with the solvent control group. The tumor volumes of Compound A 5 mg / kg and 10 mg / kg groups were 912 mm 3 and 553 mm 3 respectively, which had significant tumor inhibition effect (T / C values were 49.27% and 29.85% respectively, TGI values were 53.78% and 74.35% respectively, p values were less than 0.01) compared with the solvent control group, and the tumor inhibition effect of Compound A 10 mg / kg group was better than that of the positive drug Ibrutinib 25 mg / kg group.

[0190] Table 8 Evaluation of the tumor inhibition effect of Compound A on TMD8 xenograft tumor model (calculated based on the tumor volume on day 17 after administration)

[0191]

[0192] Note: a. Mean ± SEM; b. Tumor growth inhibition was calculated by T / C and TGI (TGI (%) = [1- (T 17 -T0) / (V 17 -V0)] x 100); c. Administration method: once a day; **: p < 0.01.

[0193] The results showed that Compound A had significant tumor growth inhibition activity in two BTK-sensitive mouse xenograft tumor models, and was significantly better than the first generation BTK inhibitor Ibrutinib which was currently on the market.

[0194] In addition, using Compound S18s, the above experiment in the human lymphoma TMD8 xenograft tumor model was repeated, and the T / C (%) results are listed in the following table. In the following table, the T / C (%) results of Compound A are also listed for comparison.

[0195] Table 9 Tumor inhibition effect of Compound A and S18 on TMD8 xenograft tumor model

[0196]

[0197]

[0198] Note: Administration method: once a day.

[0199] From the above data, it can be seen that at a lower dose (10 mg / kg), Compound A showed better tumor growth inhibition effect than Compound S18s (15 mg / kg).

[0200] Experimental Example 4: Evaluation of pharmacokinetic properties of rats

[0201] SD rats, 14, male, weight 200-220 g, were randomly divided into 4 groups, 4 / 3 rats in each group, and were given the test compound by gavage and intravenous administration respectively, and the specific arrangement is shown in Table 10 below:

[0202] Table 10 Administration method of test compound

[0203] Group Number of animals Compound Route of administration Dose (mg / kg) 1 4 Compound A Po 3 2 3 Compound A Iv 1 3 4 Ibrutinib Po 3 4 3 Ibrutinib Iv 1

[0204] Note: The gavage administration was prepared with 1% Tween 80 in 0.5% sodium carboxymethyl cellulose (CMC-Na), and the drug concentration was 0.3 mg / mL; the intravenous administration was prepared into a solution with 5% DMSO / 5% Tween 80 / 90% normal saline, and the administration concentration was 0.2 mg / mL.

[0205] The rats were fasted for 12 hours before the test and allowed to drink water freely. Two hours after administration, the rats were fed uniformly.

[0206] Blood sampling time points and sample processing:

[0207] Gavage administration: 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 hours after administration;

[0208] Intravenous administration: 5 minutes, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 hours after administration;

[0209] At the above set time points, 0.3 mL of venous blood was taken from the rat retrobulbar venous plexus into a heparinized test tube, centrifuged at 11000 rpm for 5 minutes, and the plasma was separated and frozen in a-20℃ refrigerator.

[0210] Sample testing and data analysis

[0211] LC / MS / MS method was used to determine the concentration of compound A in rat plasma.

[0212] The WinNonlin 5.3 software (Pharsight Corporation, USA) was used to calculate the pharmacokinetic parameters after administration by non-compartment model.

[0213] Peak concentration C max and peak time T max are measured values;

[0214] Area under the curve AUC 0-t value: calculated by trapezoidal method;

[0215] AUC 0-∞ = AUC 0-t + C t / k e ,

[0216] Ct Blood concentration at the last measurable time point,

[0217] k e To eliminate the rate constant;

[0218] Elimination half-life t 1 / 2 = 0.693 / k e ;

[0219] Mean residence time MRT = AUMC / AUC.

[0220] Clearance CL = D / AUC 0-∞ ; Steady-state volume of distribution Vss = CL x MRT

[0221] Absolute bioavailability F = (AUC 灌胃 x D 静脉 ) / (AUC 静脉 x D 灌胃 ) x 100%

[0222] The test results are shown in Table 11 below:

[0223] Table 11 Pharmacokinetic test results of different compounds

[0224]

[0225] Note: The pharmacokinetic data of S18s, S19s and S20s above are extracted from “Yu Xue, et al. Discovery of 4,7-Diamino-5-(4-phenoxyphenyl)-6-methylenepyrimido[5,4-b]pyrrolizines as Novel Bruton’s Tyrosine Kinase Inhibitors. J. Med. Chem., 2018, 61, 4608-4627.”

[0226] The above results show that the clearance of compound A in rats is significantly lower than that of ibrutinib (20 times), and the drug exposure in plasma after oral administration is also as high as 70 times that of ibrutinib; the in vivo clearance of compound A is also significantly higher than that of S18s, S19s and S20s, and the drug exposure in plasma after oral administration is significantly higher than that of S18s, S19s and S20s. That is, under the same dosage, compared with ibrutinib, S18s, S19s and S20s, compound A has better oral administration performance and good oral bioavailability.

[0227] Therefore, compound A is a novel, orally available, highly selective and highly active BTK inhibitor, which has significantly better in vivo and in vitro activities than the currently marketed BTK inhibitor abroad, and has significantly better tumor growth inhibition activity than the positive control drug ibrutinib at the same dose, and has great development value.

[0228] The above implementations are merely auxiliary descriptions in nature and are not intended to limit the embodiments of the application or the application or use of the embodiments. In this text, the term "exemplary" represents "as an example, an example or an illustration". Any one of the exemplary embodiments herein does not necessarily mean that it is preferred or more advantageous than other embodiments.

Claims

1. Use of Compound A or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a BTK-related neoplastic disease, said Compound A having the following structure: , the BTK-related neoplastic disease is small lymphocytic lymphoma, marginal zone lymphoma, follicular lymphoma, Waldenstrom's macroglobulinemia, high-grade B-cell lymphoma with Bcl2 gene abnormalities, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma with anaplastic lymphoma kinase rearrangements, or Burkitt lymphoma with anaplastic lymphoma kinase rearrangements.

2. Use according to claim 1, characterized in that, The genetic abnormality refers to a gene mutation, a gene fusion or rearrangement and / or a gene amplification.

3. Use according to claim 1, characterized in that, The abnormal chromosome refers to a chromosome that has undergone amplification, deletion, fragmentation, rearrangement and / or translocation.

4. Use according to any one of claims 1 to 3, characterized in that, The medicament is prepared into a clinically acceptable formulation, which is an oral formulation, an injection formulation or a topical formulation.

5. Use according to any one of claims 1 to 3, characterized in that, The medicament contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, which is administered at a dosage of 0.001 mg-1000 mg per day in a single dose or in divided doses.

6. Use according to any one of claims 1 to 3, characterized in that, The medicament contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, which is administered at a dosage of 0.01 mg-200 mg per day in a single dose or in divided doses.

7. Use according to any one of claims 1 to 3, characterized in that, The medicament contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, which is administered at a dosage of 0.1 mg-100 mg per day in a single dose or in divided doses.

8. Use according to any one of claims 1 to 3, characterized in that, The medicament contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, which is administered at a dosage of 0.5 mg-50 mg per day in a single dose or in divided doses.

9. Use according to any one of claims 1 to 3, characterized in that, The medicament contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, which is administered at a dosage of 0.5 mg-20 mg per day in a single dose or in divided doses.

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