WO2022083745A1 - Use of bruton's tyrosine kinase inhibitor - Google Patents

Abstract

A use of compound A or a pharmaceutically acceptable salt thereof as a Bruton's tyrosine kinase (BTK) inhibitor in the preparation of drugs for treating BTK-related tumor diseases. Compound A has good inhibitory activity on BTK, has good inhibitory activity on BTK highly expressed human B-cell lymphoma cells, and also has good in-vivo anti-tumor activity. Compound A has good pharmacokinetic properties and metabolic stability, and can be used for developing pharmaceutical preparations for treating BTK-related tumor diseases.

Description

Use of a Bruton's tyrosine kinase inhibitor technical field

The 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 medicine for the treatment of BTK-related tumor diseases.

Background technique

B cell receptor (BCR) signaling plays a crucial role in normal B cell development and adaptive immunity, and activation of this signaling pathway contributes to the occurrence and development of B cell malignancies and autoimmune diseases. develop. 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. It is mainly expressed in various developmental stages of B lymphocytes (except plasma cells at the end of B lymphocyte development), and has an important effect on the proliferation, differentiation and apoptosis of B cells.

In B cell-related malignancies, the BCR signaling pathway is overactive, thereby inhibiting the normal differentiation and apoptosis of B cells and promoting abnormal proliferation. It is known that abnormal regulation of the BCR pathway often exists in malignant tumors of various B cell types. At present, 4 BTK inhibitors have been approved for marketing. Ibrutinib (trade name Imbruvica) is the first BTK small molecule inhibitor to be marketed and belongs to the first generation of BTK inhibitors. It was approved by the US FDA in 2013 for Clinical treatment of Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), etc. Since then, ibrutinib has continued to expand its indications. The currently approved indications also include 17p deletion chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/Small Lymphocytic Lymphoma, SLL), Waldenstrom Macroglobulinemia (WM), marginal zone lymphoma (Marginal Zone Lymphoma, MZL), chronic graft-resistant Host disease (chronic Graft-Versus-Host Disease, cGVHD). Ibrutinib can form covalent binding with cysteine 481 (Cys481) in the ATP-binding domain of BTK kinase, irreversibly inhibit BTK activation, block BTK signaling pathway, thereby inhibiting the proliferation and survival of B lymphoma cells. achieve the purpose of tumor treatment. Ibrutinib has achieved substantial efficacy in clinical treatment. However, due to the poor target selectivity of ibrutinib, there are certain toxic and side effects in clinical practice. Acalabrutinib (ACP-196, trade name Calquence) was approved for the treatment of MCL and CLL in 2017 and belongs to the second generation of BTK targeted drugs. Compared with ibrutinib, acaltinib is more selective for BTK and has lower off-target toxicity. In addition, Zanubrutinib (Zabrutinib, BGB-3111), developed by BeiGene Biotechnology Co., Ltd., was approved by the FDA in November 2019 for the treatment of adult MCL patients, becoming the first Chinese local antibody to receive FDA breakthrough therapy designation. cancer drugs. Tirabrutinib (ONO-4059) developed by Japan's Ono Pharmaceutical Co., Ltd. was approved by the Japan Pharmaceuticals and Medical Devices Agency (PMDA) in March 2020 for relapsed or refractory primary central nervous system lymphoma (PCNSL) and treatment of lymphoplasmacytic lymphoma (LPL). In general, the first-generation inhibitors have high inhibitory activity against BTK, but the target selection and bioavailability are poor; the second-generation inhibitors have good selectivity, but the inhibition rate of BTK is lower than that of the first-generation inhibitors. Structurally, the core skeleton of currently marketed BTK drugs is mainly a bicyclic system.

In order to obtain a new generation of BTK inhibitors that have both the high activity of the first-generation inhibitors and the good selectivity of the second-generation inhibitors, the Shanghai Institute of Materia Medica, Chinese Academy of Sciences disclosed a series of pyrimido[5,4 -b] Compounds of indolizine or pyrimido[5,4-b]pyrine structure. Among them, pyrimido[5,4-b]indolizine compounds S1 and S10 and pyrimido[5,4-b]pyrine compounds S18, S19 and S20 showed higher BTK inhibitory activity, and further work In , the S-configuration compounds of S18, S19 and S20 (ie S18s, S19s and S20s) were 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.], but further research found that these compounds S1, S10, S18s and S19s are unstable during metabolism (The para-position of the terminal phenyl ring is one of the active metabolic sites), and the 4-position of the terminal phenyl ring is easily oxidized; however, the oral bioavailability of compound S20s is not ideal.

Based on the above problems, further development of BTK kinase inhibitors with excellent BTK inhibitory activity and selectivity, high in vivo antitumor activity, and excellent oral administration performance and metabolic stability is very important for the treatment of BTK-related tumor diseases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the use of a Bruton's tyrosine kinase BTK inhibitor compound A or a pharmaceutically acceptable salt thereof in the preparation of a medicine for the treatment of BTK-related tumor diseases, the compound A has the following structure:

For the above purposes, the BTK-related tumor diseases include hematological tumors and solid tumors.

Preferably, the above-mentioned hematological tumors are lymphomas and leukemias.

Further preferably, the above-mentioned lymphoma is a B-cell lymphoma.

For the above purposes, the BTK-related tumor diseases include histiocytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, marginal zone lymphoma, and follicular lymphoma , Burkitt lymphoma, or Waldenstrom's macroglobulinemia.

For the above purposes, the diffuse large B-cell lymphoma is selected from the group consisting of non-special types of diffuse large B-cell lymphoma, other large B-cell lymphomas, high-grade B-cell lymphomas with MYC and/or Bcl gene abnormalities, and non-specific types of diffuse large B-cell lymphomas. One or more of the group consisting of a special type of high-grade B-cell lymphoma; preferably, the diffuse large B-cell lymphoma is selected from other large B-cell lymphomas, those with MYC and/or Bcl gene abnormalities One or more of the group consisting of high-grade B-cell lymphoma and non-specialized type of high-grade B-cell lymphoma.

For the above purposes, the other large B-cell lymphomas include T-cell/histiocyte-rich DLBCL, primary central nervous system DLBCL, primary cutaneous DLBCL (leg type), EBV-positive DLBCL, unspecified DLBCL, chronic inflammation Associated large B-cell lymphoma, lymphomatoid granuloma, 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 exudative lymphoma, etc.;

For the above purposes, the high-grade B-cell lymphoma with MYC gene abnormality is one or more selected from the group consisting of high-grade B-cell lymphoma with MYC gene amplification and high-grade B-cell lymphoma with MYC gene fusion The high-grade B-cell lymphoma with abnormal Bcl gene is selected from high-grade B-cell lymphoma with abnormal Bcl2 and/or Bcl6 gene, and the high-grade B-cell lymphoma with abnormal Bcl2 and/or Bcl6 gene is selected from Bcl2 gene 1 in the group consisting of amplified high-grade B-cell lymphoma, Bcl2-fused high-grade B-cell lymphoma, Bcl6-amplified high-grade B-cell lymphoma, and Bcl6-fused high-grade B-cell lymphoma or more.

For the above purposes, the diffuse large B-cell lymphoma involved in the present invention can also be a diffuse large B-cell lymphoma carrying abnormal chromosomes.

For the above purposes, the mantle cell lymphoma is one or more selected from the group consisting of classic mantle cell lymphoma, leukemia-like non-scarring mantle cell lymphoma and Cyclin D1 positive mantle cell lymphoma.

For the above purposes, the mantle cell lymphoma is selected from Bcl gene abnormal mantle cell lymphoma; preferably Bcl1 and/or Bcl2 gene abnormal mantle cell lymphoma; more preferably Bcl1 gene fusion or rearrangement mantle cell lymphoma or Mantle cell lymphoma with Bcl2 gene fusion or rearrangement.

For the above purposes, the mantle cell lymphoma involved in the present invention may also be a mantle cell lymphoma carrying abnormal chromosomes.

For the above purposes, the Burkitt lymphoma is a Burkitt lymphoma carrying abnormal chromosomes.

For the above purposes, the gene abnormality refers to gene mutation, gene fusion or rearrangement and/or gene amplification.

For the above purposes, the abnormal chromosome refers to a chromosome that has undergone amplification, deletion, fragmentation, rearrangement and/or translocation.

For the above purposes, the medicament may also contain one or more other targeted drugs or chemotherapeutic drugs. The other targeted drugs or chemotherapeutic drugs refer to targeted drugs or chemotherapeutic drugs clinically used for the treatment of tumor-related diseases.

For the above purposes, the medicine is prepared into clinically acceptable preparations, such as oral preparations, injection preparations, external preparations and the like.

For the above purposes, the medicine contains a therapeutically effective dose of Compound A or a pharmaceutically acceptable salt thereof, and the therapeutically effective dose is preferably 0.001-1000 mg per day, more preferably 0.01-500 mg per day, and more More preferably the daily dose is 0.1-200mg, still more preferably the daily dose is 0.5-100mg, still more preferably the daily dose is 0.5-50mg, still more preferably the daily dose is 0.5-30mg, even more preferably The daily dose is 0.5-20 mg. Administration can be single dose or divided doses.

In another aspect, the present invention also provides a method for treating BTK-related tumor diseases, characterized by administering to a subject or patient a drug containing a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof.

In the above method, the BTK-related tumor diseases include hematological tumors and solid tumors.

Preferably, the above-mentioned hematological tumors are lymphomas and leukemias.

Further preferably, the above-mentioned lymphoma is a B-cell lymphoma.

The above method, the BTK-related tumor diseases include histiocytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, marginal zone lymphoma, and follicular lymphoma , Burkitt lymphoma, or Waldenstrom's macroglobulinemia.

For the above purposes, the diffuse large B-cell lymphoma is selected from the group consisting of non-special types of diffuse large B-cell lymphoma, other large B-cell lymphomas, high-grade B-cell lymphomas with MYC and/or Bcl gene abnormalities, and non-specific types of diffuse large B-cell lymphomas. One or more of the group consisting of a special type of high-grade B-cell lymphoma; preferably, the diffuse large B-cell lymphoma is selected from other large B-cell lymphomas, those with MYC and/or Bcl gene abnormalities One or more of the group consisting of high-grade B-cell lymphoma and non-specialized type of high-grade B-cell lymphoma.

For the above purposes, the other large B-cell lymphomas include T-cell/histiocyte-rich DLBCL, primary central nervous system DLBCL, primary cutaneous DLBCL (leg type), EBV-positive DLBCL, unspecified DLBCL, chronic inflammation Associated large B-cell lymphoma, lymphomatoid granuloma, 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 exudative lymphoma, etc.;

For the above purposes, the high-grade B-cell lymphoma with MYC gene abnormality is one or more selected from the group consisting of high-grade B-cell lymphoma with MYC gene amplification and high-grade B-cell lymphoma with MYC gene fusion The high-grade B-cell lymphoma with abnormal Bcl gene is selected from high-grade B-cell lymphoma with abnormal Bcl2 and/or Bcl6 gene, and the high-grade B-cell lymphoma with abnormal Bcl2 and/or Bcl6 gene is selected from Bcl2 gene One of the group consisting of amplified high-grade B-cell lymphoma, Bcl2-fused high-grade B-cell lymphoma, Bcl6-amplified high-grade B-cell lymphoma, and Bcl6-fused high-grade B-cell lymphoma or more.

For the above purposes, the diffuse large B-cell lymphoma involved in the present invention can also be a diffuse large B-cell lymphoma carrying abnormal chromosomes.

For the above purposes, the mantle cell lymphoma is selected from one or more of the group consisting of classic mantle cell lymphoma, leukemia-like non-scarring mantle cell lymphoma and Cyclin D1 positive mantle cell lymphoma.

For the above purposes, the mantle cell lymphoma is selected from Bcl gene abnormal mantle cell lymphoma; preferably Bcl1 and/or Bcl2 gene abnormal mantle cell lymphoma; more preferably Bcl1 gene fusion or rearrangement mantle cell lymphoma or Bcl2 gene fusion or rearrangement of mantle cell lymphoma.

For the above purposes, the mantle cell lymphoma involved in the present invention may also be a mantle cell lymphoma carrying abnormal chromosomes.

For the above purposes, the Burkitt lymphoma is a Burkitt lymphoma carrying abnormal chromosomes.

For the above purposes, the gene abnormality refers to gene mutation, gene fusion or rearrangement and/or gene amplification.

For the above purposes, the abnormal chromosome refers to a chromosome that has undergone amplification, deletion, fragmentation, rearrangement and/or translocation.

In the above method, the administration may be oral administration, injection administration, topical administration or in vitro administration, preferably oral administration or injection administration.

The above methods, the dose and frequency of administration of Compound A or a pharmaceutically acceptable salt thereof can be achieved by conventional methods such as modeling, dose escalation studies or conventional methods of clinical trials and by taking into account factors such as the nature and severity of the disease to be treated, the patient age, general condition and body weight, as well as the specific compound administered, its pharmacokinetic properties, and the route of administration. A suitable dosage range of Compound A or a pharmaceutically acceptable salt thereof 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; even more preferably, from about 0.03 mg/kg to about 20 mg/kg. Preferably, the daily dose of Compound A or a pharmaceutically acceptable salt thereof is 0.001 mg-1000 mg, further preferably, the daily dose of Compound A or its pharmaceutically acceptable salt is 0.01-500 mg; even more preferably , the daily dose of Compound A or its pharmaceutically acceptable salt is 0.1-200 mg; more preferably, the daily dose of Compound A or its pharmaceutically acceptable salt is 0.5-100 mg; more preferably, the compound The daily dose of A or its pharmaceutically acceptable salt is 0.5-50 mg; more preferably, the daily dose of Compound A or its pharmaceutically acceptable salt is 0.5-30 mg; even more preferably, Compound A or A pharmaceutically acceptable salt thereof is administered in a daily dose of 0.5-20 mg; administered in single or divided doses.

In another aspect, the present invention also provides a method of treating a disorder in a patient, whose disorder is a BTK-related tumor disease, by administering to the patient a drug containing a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof.

In the above method, the BTK-related tumor diseases include hematological tumors and solid tumors.

Preferably, the above-mentioned hematological tumors are lymphomas and leukemias.

Further preferably, the above-mentioned lymphoma is a B-cell lymphoma.

The above method, the BTK-related tumor diseases include histiocytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, marginal zone lymphoma, and follicular lymphoma , Burkitt lymphoma, or Waldenstrom's macroglobulinemia.

For the above purposes, the diffuse large B-cell lymphoma is selected from the group consisting of non-special types of diffuse large B-cell lymphoma, other large B-cell lymphomas, high-grade B-cell lymphomas with MYC and/or Bcl gene abnormalities, and non-specific types of diffuse large B-cell lymphomas. One or more of the group consisting of a special type of high-grade B-cell lymphoma; preferably, the diffuse large B-cell lymphoma is selected from other large B-cell lymphomas, those with MYC and/or Bcl gene abnormalities One or more of the group consisting of high-grade B-cell lymphoma and non-specialized type of high-grade B-cell lymphoma.

For the above purposes, the other large B-cell lymphomas include T-cell/histiocyte-rich DLBCL, primary central nervous system DLBCL, primary cutaneous DLBCL (leg type), EBV-positive DLBCL, unspecified DLBCL, chronic inflammation Associated large B-cell lymphoma, lymphomatoid granuloma, 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 exudative lymphoma, etc.;

For the above purposes, the high-grade B-cell lymphoma with MYC gene abnormality is one or more selected from the group consisting of high-grade B-cell lymphoma with MYC gene amplification and high-grade B-cell lymphoma with MYC gene fusion The high-grade B-cell lymphoma with abnormal Bcl gene is selected from high-grade B-cell lymphoma with abnormal Bcl2 and/or Bcl6 gene, and the high-grade B-cell lymphoma with abnormal Bcl2 and/or Bcl6 gene is selected from Bcl2 gene One of the group consisting of amplified high-grade B-cell lymphoma, Bcl2-fused high-grade B-cell lymphoma, Bcl6-amplified high-grade B-cell lymphoma, and Bcl6-fused high-grade B-cell lymphoma or more.

For the above purposes, the diffuse large B-cell lymphoma involved in the present invention can also be a diffuse large B-cell lymphoma carrying abnormal chromosomes.

For the above purposes, the mantle cell lymphoma is selected from one or more of the group consisting of classic mantle cell lymphoma, leukemia-like non-scarring mantle cell lymphoma and Cyclin D1 positive mantle cell lymphoma.

For the above purposes, the mantle cell lymphoma is selected from Bcl gene abnormal mantle cell lymphoma; preferably Bcl1 and/or Bcl2 gene abnormal mantle cell lymphoma; more preferably Bcl1 gene fusion or rearrangement mantle cell lymphoma or Bcl2 gene fusion or rearrangement of mantle cell lymphoma.

For the above purposes, the mantle cell lymphoma involved in the present invention may also be a mantle cell lymphoma carrying abnormal chromosomes.

For the above purposes, the Burkitt lymphoma is a Burkitt lymphoma carrying abnormal chromosomes.

For the above purposes, the gene abnormality refers to gene mutation, gene fusion or rearrangement and/or gene amplification.

For the above purposes, the abnormal chromosome refers to a chromosome that has undergone amplification, deletion, fragmentation, rearrangement and/or translocation.

The therapeutically effective amount, administration dose or administration dose of Compound A or a pharmaceutically acceptable salt thereof of the present invention are all calculated as Compound A.

According to the 2017 edition of Hematopoietic and Lymphoid Tissue Neoplasms (4th revised edition), diffuse large B-cell lymphoma (DLBCL) is divided into four categories: non-specialized DLBCL, other large B-cell lymphoma, high-grade B-cell lymphoma and unclassifiable B-cell lymphomas intermediate between DLBCL and classic Hodgkin lymphoma. Among them, non-special types of DLBCL include morphologically including centroblastoid variant, immunoblastoid variant, anaplastic variant and other rare variants (such as spindle cell variant, signet ring cell variant), etc.; other large B-cell lymphocytes Tumors include T-cell/histiocyte-rich DLBCL, primary central nervous system DLBCL, primary cutaneous DLBCL (leg type), EBV-positive DLBCL, unspecified DLBCL, chronic inflammation-related large B-cell lymphoma, lymphoma Granuloma-like, 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 (eg, gene rearrangement or fusion, gene amplification, gene mutation, etc.) Grade B-cell lymphoma, non-special type of high-grade B-cell lymphoma, etc., such as U-2932 cells (with Bcl2 gene amplification), WILL-2 cells (with Bcl2 gene fusion), etc. mentioned in this application. (Source: ATCC official website information and DSMZ official website information)

"Diffuse large B-cell lymphoma carrying abnormal chromosomes" referred to in this application refers to diffuse large B-cell lymphoma carrying abnormal chromosomes (eg, chromosomal amplification, deletion, breakage, rearrangement and/or translocation, etc.). Cellular lymphomas, such as Pfeiffer cells mentioned in this application (there are multiple chromosomal abnormalities, including translocation of chromosome t(14;18)(q32;q21)) and the like. (Source: ATCC official website information and DSMZ official website information)

The "mantle cell lymphoma carrying abnormal chromosomes" mentioned in this application refers to mantle cell lymphomas carrying abnormal chromosomes (eg, chromosomal translocations, chromosomal fusions, chromosomal deletions, etc.), such as Z mentioned in this application. -138 cells (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 (chromosomal abnormalities such as chromosome t(11;14)(q13;q32) translocation, etc.). (Source: ATCC official website information and DSMZ official website information)

The "Bcl gene abnormal mantle cell lymphoma" mentioned in the present application refers to a mantle cell lymphoma with Bcl gene abnormality (eg, gene mutation, gene amplification, gene rearrangement or/fusion, abnormal gene activation, etc.), It can lead to overexpression of Bcl-related proteins, such as the Jeko-1 cells mentioned in this application (with Bcl-1 gene rearrangement) and the like. (Source: ATCC official website information)

"Burkitt lymphoma carrying abnormal chromosomes" referred to in this application refers to Burkitt lymphomas carrying abnormal chromosomes (eg, abnormalities in chromosomal size and/or number, chromosomal translocations, chromosomal fusions, chromosomal deletions, etc.), such as Raji cells referred to in this application (abnormal size and/or number of chromosomes 1 or 4). (Source: ATCC official website information)

The pharmaceutically acceptable salts of the compounds can be conventional non-toxic salts formed by the reaction of the compounds with inorganic acids, organic acids, inorganic bases or organic bases.

In this preparation method and in the present invention, the terms used are as follows:

DCM: dichloromethane; DIAD: diisopropyl azodicarboxylate; DIPEA: diisopropylethylamine; DMF: N,N-dimethylformamide; EA: ethyl acetate; HATU: 2-(7 -benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate; NBS: N-bromosuccinimide; NIS: N-iodosuccinimide Amine; PdCl ₂ (dppf): [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride; Pd(PPh ₃ ) ₄ : tetrakis(triphenylphosphine)palladium; PdCl ₂ : Palladium dichloride; Pd(OAc) ₂ : palladium acetate; Pd(PPh ₃ ) ₂ Cl ₂ : bistriphenylphosphonium palladium dichloride; PE: petroleum ether; THF: tetrahydrofuran; DMSO: dimethyl sulfoxide.

The present invention has achieved one or more of the following beneficial technical effects:

(1) The results of in vitro and in vivo efficacy tests showed that the inhibitory activity of compound A on BTK and lymphoma cells cultured in vitro was not only better than that of the previous compounds S1, S10, S18s, S19s and S20s, but also better than that of the first-generation BTK currently on the market The inhibitor ibrutinib and the second-generation BTK inhibitor acalatinib. In the in vivo xenograft model, compound A also showed significantly better inhibitory effect on tumor growth than S18s and ibrutinib.

(2) The results of the in vitro pharmacodynamic test showed that Compound A has a good inhibitory effect on various types of tumor cells, and also has a good inhibitory effect on different specific cell lines of the same cell type, that is, Compound A has a good inhibitory effect on the same cell type. Tumor cell types and cell lines from different sources can achieve better inhibitory effects and have better clinical application prospects.

(3) Pharmacokinetic studies showed that the in vivo clearance rate of Compound A was significantly lower than that of ibrutinib and its structural analogs S18s, S19s, and S20s, and was only 1/10 to 1/20 of the latter; Compound A was administered orally The post-plasma drug exposure (AUC) was 70-fold higher than that of ibrutinib, and the absolute bioavailability was significantly higher.

(4) S1, S10, S18s and S19s are easy to be oxidized at the 4-position of the terminal phenyl group, have many kinds of metabolites, and have poor metabolic stability. S18s can only retain 51% of the prototype drug after incubation with rat liver microsomes. Compound A effectively overcomes the hydroxylation of the terminal benzene ring of the diphenyl ether structure in the above compounds through structural modification, and can be detected in different species of liver microsomes (HLM: human liver microsomes; RLM: rat liver microsomes; MLM: mouse liver microsomes). Under the action of microsomes), the types and proportions of metabolites are small, and the prototype drugs are basically the main ones (60min: 84%-98%), and the metabolic stability is better.

In conclusion, compound A has good BTK inhibitory activity and tumor inhibitory effect, and has low in vivo clearance rate, good oral bioavailability, and stable metabolism, which has important clinical application value.

Description of drawings

Figure 1: Schematic diagram of the experimental results of the REC-1 xenograft tumor model.

Figure 2: Schematic diagram of experimental results of the TMD8 xenograft tumor model.

Detailed ways

Embodiments are further provided below, which are helpful for understanding the present invention, are only used for illustration and do not limit the scope of application of the present invention.

Example 1: Synthesis of Compound A

1. Synthesis of Intermediate 3

4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (raw material 2, 17.28g, 1eq) and anhydrous potassium carbonate (2eq) were added to a 250mL round-bottom flask, and the water was removed by vacuum drying. Add dry DMF as solvent, pulverized (S)-methanesulfonic acid 2-((tert-butoxycarbonyl)amino)-but-3-en-1-yl ester (raw material 1, 24.6g, 1.5eq), Replace nitrogen. Heating and stirring at 55°C for 12 hours, the time can be appropriately extended to ensure the completion of the reaction.

After the reaction was completed, water and ethyl acetate were added for extraction three times, the ester layers were combined, back-extracted once with water, and washed with saturated brine. Dry over anhydrous sodium sulfate. Dry column (eluent: CHCl ₃ :MeOH=100:1) to obtain the product (S)-(1-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine-7) -yl)but-3-en-2-yl)carbamate tert-butyl ester (Intermediate 3, 19.28 g) in 69.5% yield.

¹ H NMR (300MHz, CDCl ₃ ) δ 8.60 (s, 1H), 7.39 (s, 1H), 5.82 (ddd, J=17.1, 10.5, 5.5Hz, 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%.

2. Synthesis of Intermediate 4

Add (S)-(1-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)but-3-en-2-yl) to a 350mL pressure tube tert-Butyl carbamate (Intermediate 3, 9.2 g), 1,4-dioxane (40 mL) was added as a solvent, and aqueous ammonia (40 mL) was added. The reaction was sealed at 120°C for 2.5 hours.

After completion of the reaction, the mixture was cooled to room temperature, and extracted with water and ethyl acetate. The ester layers were combined and washed with saturated brine. Dry over anhydrous sodium sulfate. Dry column (eluent: CHCl ₃ :MeOH=30:1) to obtain the product (S)-(1-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidine-7) -yl)but-3-en-2-yl)carbamate tert-butyl ester (Intermediate 4, 6.86 g) in 78.0% yield.

¹ H NMR (300MHz, CDCl ₃ ) 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, 4.9Hz, 1H), 4.30-4.15 (m, 1H), 1.35 (s, 9H). ee>99.5%.

3. Synthesis of Intermediate 6

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) tert-Butyl carbamate (Intermediate 4, 32.9g, 1eq), N-(pyridin-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxa Borol-2-yl)benzamide (starting material 5, 34.8g, 1.4eq) and Pd( _PPh3 ) ₄ (17.7g, 0.2eq). 1,4-Dioxane (383 mL) was added as solvent and _N2 was displaced. 2M sodium carbonate solution (76.6 mL) was added with stirring. It was stirred under reflux at 90°C for 5 hours.

Water and ethyl acetate were added for extraction, and the ester layers were combined and washed with saturated brine. Dry over anhydrous sodium sulfate. The column was dry-passed, and EA was used as the eluent to remove most of the impurities, and then the mixture of CHCl ₃ :MeOH=30:1 was used as the eluent. The product may contain a small amount of impurities, which can be recrystallized from PE to separate out the pure product. The product (S)-(1-(4-amino-5-(4-(pyridin-2-ylcarbamoyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl) was obtained But-3-en-2-yl)carbamate tert-butyl ester (Intermediate 6, 28.4 g) in 74.3% yield. ee>99.5%.

4. Synthesis of Intermediate 7

Add (S)-(1-(4-amino-5-(4-(pyridin-2-ylcarbamoyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine to a 1 L round bottom flask -7-yl)but-3-en-2-yl)carbamate tert-butyl ester (Intermediate 6, 32.7 g, 1 eq), 600 mL of DMF was added as solvent. NBS (12.8 g, 1.1 eq) was added slowly with stirring and stirred at room temperature overnight.

After the reaction was completed, water and ethyl acetate were added for extraction, and the ester layers were combined, back-extracted once with water, and washed with saturated brine. Dry over anhydrous sodium sulfate. The column was dry-passed, firstly using the mixture of CHCl ₃ :MeOH=50:1 as the eluent, and then changing the mixture of CHCl ₃ :MeOH=30:1 as the eluent. The product (S)-(1-(4-amino-6-bromo-5-(4-(pyridin-2-ylcarbamoyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine- 7-yl)but-3-en-2-yl)carbamate tert-butyl ester (Intermediate 7, 25.8 g) in 68.2% yield. ee>99.5%.

5. Synthesis of Intermediate 8

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)carbamate tert-butyl ester (Intermediate 7, 11.9g, 1eq) and _PdCl2 (dppf) (1.66g, 0.11eq), 51mL was added THF was used as solvent and nitrogen was replaced several times to ensure completeness. 4M sodium hydroxide solution (8.2 mL) was added with stirring. Stir at reflux for 15 hours at 85°C.

After completion of the reaction, water and ethyl acetate were added for extraction, and the ester layers were combined and washed with saturated brine. Dry over anhydrous sodium sulfate. Dry column (eluent: CHCl ₃ :MeOH=30:1) to obtain the product (S)-(4-amino-6-methylene-5-(4-(pyridin-2-ylcarbamoyl)) Phenyl)-7,8-dihydro-6H-pyrimido[5,4-b]pyrin-7-yl)carbamate tert-butyl ester (Intermediate 8, 8.79 g) in 85.9% yield. ee>99.5%.

6. Synthesis of Compound A

In a 250 mL round bottom flask, add (S)-(4-amino-6-methylene-5-(4-(pyridin-2-ylcarbamoyl)phenyl)-7,8-dihydro-6H- Pyrimido[5,4-b]pyrin-7-yl)carbamate tert-butyl ester (Intermediate 8, 2.75 g, 1 eq), 110 mL of DCM was added as solvent. Trifluoroacetic acid (10.5 mL) was added dropwise with stirring. Stir at room temperature for 3 hours. After the reaction is completed, the reaction solution is directly spin-dried, and the trifluoroacetic acid is taken out with methanol several times.

Transfer the product of the previous step to a 250mL round-bottom flask, add triethylamine (1eq), stir for five minutes, then add 2-butynoic acid (0.511g, 1.1eq) and HATU (2.31g, 1.1eq), add 100 mL of DCM was used as solvent. The ice-water bath was cooled to 0°C, and triethylamine (1.54 mL+0.77 mL) was added dropwise. The temperature was gradually raised to room temperature, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was slightly yellow. Water and DCM were added for extraction, and the organic phases were combined and washed with saturated brine. After drying over anhydrous sodium sulfate, the final product A (1.88 g) was obtained after column chromatography (CHCl ₃ :MeOH=30:1), and the yield was 73.3%. ee>99.5%.

1H NMR (400MHz, CDCl ₃ ) 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=8.4,7.4,1.9Hz,1H),7.69-7.63(m,2H),7.13(ddd,J=7.4,4.9,1.0Hz ,1H),6.55(d,J＝8.2Hz,1H),5.67(m,J＝8.1,5.7,2.6Hz,1H),5.56(d,J＝2.3Hz,1H),5.40(s,2H) , 5.27 (d, J=2.3Hz, 1H), 4.70 (dd, J=11.7, 8.1Hz, 1H), 4.09-3.99 (m, 1H), 1.99 (s, 3H).

Experimental Example 1: Evaluation of Bruton Kinase (BTK) Molecular Level Enzyme Activity Inhibitory Activity

Dilute the enzyme reaction substrate Poly(Glu,Tyr) _4:1 with potassium-free PBS (10mM sodium phosphate buffer, 150mM NaCl, pH 7.2-7.4) to 20μg/mL coated microtiter plate at 37°C After 12-16 hours of reaction, the plate was washed three times with 200 μL/well of T-PBS (PBS containing 0.1% Tween-20), and the ELISA plate was dried in a 37° C. oven for 1-2 hours. In the above substrate-coated ELISA plate, first add 49 μL/well of ATP solution diluted with reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl ₂ , 0.5 mM MnCl ₂ , 0.2 mM Na ₃ VO ₄ , 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 a no-enzyme control well is required for each experiment. An additional 50 μL of BTK tyrosine kinase protein diluted in reaction buffer was added to initiate the reaction.

The above reaction system was placed in a shaker at 37°C (100 rpm) for 1 hour, then the plate was washed three times with T-PBS, and the primary antibody PY99 100 μL/well (Santa Cruz) was added, and the reaction was shaken at 37°C for 0.5 hour. After washing the plate with T-PBS, 100 μL/well of horseradish peroxidase-labeled goat anti-mouse secondary antibody diluent was added, and the reaction was shaken at 37°C for 0.5 hours. After washing the plate with T-PBS, add 100 μL/well of 2 mg/mL OPD chromogenic solution, and react at 25°C for 1-10 minutes in the dark. Then, 50 μL/well of 2M H ₂ SO ₄ was added to stop the reaction, and the reaction was read with a tunable wavelength microplate reader SPECTRA MAX Plus384 with a wavelength of 490 nm.

Compounds S1, S10, ibrutinib, acalatinib, S18s, S19s and S20s are used as positive control compounds, wherein compounds S1, S10, S18s, S19s and S20s adopt the methods disclosed in the prior art (eg CN108101905A) or similar methods, ibrutinib and acalatinib were purchased from Selleck Company.

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

The IC ₅₀ value was obtained by four-parameter regression using the software attached to the microplate reader. The results are listed in Table 1 below.

Table 1 Inhibitory effects of different compounds on BTK

compound	IC50 (nM)
S1	~1
S10	<10
ibrutinib	~1
acalatinib	~10
S18s	~1
S19s	~1
S20s	~1
Compound A	0.5

The above results show that the inhibitory activity of compound A on BTK is better than that of the 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 that are currently on the market. Catinib.

Experimental Example 2: Detection of the In vitro Proliferation Inhibitory Activity of Compounds on Human B Lymphoma Cells

experimental cells

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

experiment method:

The cell suspension (Ramos: 10,000 cells/well; TMD8: 12,000 cells/well) was inoculated into a 96-well plate, and left for 2 hours in a 37°C incubator until the cells were in a stable state, and then different concentrations of test compounds were added to each well. (3 duplicate wells for each concentration), and a blank control (well containing only culture medium, no cells), negative control (well with only cells, no compound) and positive compound control were set at the same time. After dosing for 72 h, 20 μL of MTT (5 mg/mL) was added to each well and incubated at 37°C for 4 h, followed by 100 μL of triple solution (10% SDS, 5% isobutanol, 0.01M HCl), and placed at 37°C overnight. The OD value was measured with a wavelength-tunable microplate reader SPECTRAmax Plus384 at a wavelength of 570 nm.

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

The IC ₅₀ value was obtained by four-parameter regression using the software attached to the microplate reader. The experiment was repeated 3 times independently and the results are listed in Table 2 below.

The compounds S1, S10, ibrutinib, acalatinib, S18s, S19s and S20s described above were also used as positive control compounds.

Table 2 Proliferation inhibitory activities of different compounds on Ramos cells and TMD8 cells

compound	Ramos cell IC 50	IC50 in TMD8 cells
S1	94.73μM	0.006μM
S10	8.72μM	0.030μM
ibrutinib	12.91μM	0.005μM
acalatinib	38.16μM	0.023μM
Compound A	3.15μM	0.003μM
S18s	5.04μM	0.016μM
S19s	—	0.017μM
S20s	14.3μM	0.004μM

The above results show that, at the cellular level, compound A has better inhibitory effect on the proliferation of B-cell lymphoma than the previous compounds S1, S10, S18s, S19s and S20s, and is also better than the currently marketed first-generation BTK inhibitor ibrutinib and acaltinib, a second-generation BTK inhibitor. It should be further noted that, compared with other compounds, Compound A of the present invention has higher proliferation inhibitory activity on Ramos cells, and has higher proliferation inhibitory activity on TMD8 cells.

experimental cells

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

experiment method

CCK8 (Cell Counting Kit-8, #D3100L4057, Shanghai Liji Biotechnology Co., Ltd.) staining method was used to detect the proliferation inhibitory activity of the compounds on cells.

(1) For tumor cells in the logarithmic growth phase, according to their different growth rates, a cell suspension of an appropriate density was inoculated into a 96-well plate, 95 μL per well. Incubate at 37°C for 2-4h until the cells are in a stable state, then add 10 μL of compound with the desired concentration of gradient dilution in sequence, set 3 duplicate wells for each dose, and set solvent control and cell-free blank control wells at the same time. Incubate in a carbon dioxide incubator at 37°C for 72h.

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

Inhibition rate %=(OD value of control group-OD value of administration group)/OD value of control group×100%.

The IC ₅₀ value of the median inhibitory dose was calculated by the four-parameter method. Each experiment was independently repeated 3 times, and the average IC ₅₀ value of each experiment was obtained as the final indicator of inhibitory ability.

Table 3 The proliferation inhibitory activity of compound A on B lymphoma cells (BTK inhibitor-sensitive cell line)

The above results showed that compound A had strong inhibitory activity on the two BTK inhibitor-sensitive cell lines, and was superior to the positive control drugs ibrutinib and acaltinib.

Table 4 The proliferation inhibitory activity of compound A on B lymphoma cells

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 acaltinib, all at the micromolar level. The results in Table 3 and Table 4 together show that, compared with the positive control drug, Compound A has a more obvious inhibitory effect on BTK inhibitor-sensitive cell lines, and can also have a certain inhibitory activity on other B lymphoma cells, that is, it has better inhibitory activity. Selectivity of BTK inhibition.

The results in Table 3 and Table 4 above show that the inhibitory effect of compound A on U-2932 and WILL-2 suggests that compound A of the present invention is expected to have the effect of treating high-grade B cells with abnormal Bcl gene in diffuse large B-cell lymphoma The effect of lymphoma; the inhibitory effect of compound A on Pfeiffer suggests that the compound A of the present invention 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 It is suggested that the compound A of the present invention is expected to have the effect of treating follicular lymphoma; the inhibitory effect of compound A on Raji and NAMALWA suggests that the compound A of the present invention is expected to have the effect of treating Burkitt lymphoma; the inhibitory effect of compound A on Raji suggests the present invention The compound A of the present invention 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 suggests that the compound A of the present invention is expected to have the effect of treating the mantle cell lymphoma carrying abnormal chromosomes The effect of compound A on JeKo-1 suggests that the compound A of the present invention 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 suggests Compound A of the present invention is expected to have an effect on the treatment of primary mediastinal B-cell lymphoma in diffuse large B-cell lymphoma.

In addition, given that the inhibitory activity of compound A on OCI-LY10 and REC-1 (BTK inhibitor-sensitive cell lines) is better than that of the positive control drugs ibrutinib and acalatinib, and the inhibitory activity on other B lymphoma cells The inhibitory activity is comparable to that of ibrutinib, or is between ibrutinib and acalatinib, suggesting that Compound A of the present invention is expected to be able to treat diseases treated by ibrutinib and acalatinib, such as Chronic lymphocytic leukemia, chronic lymphocytic leukemia with 17p deletion/small lymphocytic lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, chronic graft-versus-host disease.

Experimental example 3: Evaluation of antitumor activity in vivo

Experimental animals:

TMD8 xenograft tumor model

1) Species: mouse

2) Strain: CB-17SCID

3) Age and weight: 6-8 weeks; 18-22g

4) Gender: Female

5) Supplier: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

REC-1 xenograft tumor model

1) Species: mouse

2) Strain: BALB/c nude mice

3) Age and weight: 6-8 weeks; 17-20g

4) Gender: Female

5) Supplier: Shanghai Lingchang Biotechnology Co., Ltd.

Cell culture: Human lymphoma TMD8 cells were cultured in vitro in suspension, and the culture conditions were RPMI 1640 medium (supplier: gibco; product number: 22400-089; production batch number: 4868546) with 10% fetal bovine serum, 100U/mL penicillin and 100μg /mL streptomycin, incubate at 37°C with 5% CO ₂ . Routine treatment passaging was performed twice a week. When the cell saturation was 80%-90%, cells were harvested, counted, and seeded.

Human mantle cell lymphoma REC-1 cells were cultured in vitro in suspension in RPMI 1640 medium (supplier: gibco; product number: 22400-089; production batch number: 1868795) with 10% fetal bovine serum, 100U/mL penicillin and 100 μg/mL streptomycin, 37 ℃ 5% CO2 culture. Routine treatment passaging was performed twice a week. When the cell saturation was 80%-90%, cells were harvested, counted, and seeded.

Tumor cell inoculation: 0.2 mL of 10×10 ⁶ human lymphoma TMD8 cells were subcutaneously inoculated into the right back of each nude mouse (PBS:Matrigel=1:1). Group administration was started when the average tumor volume reached 104 mm ³ . Animals were grouped according to tumor volume by an Excel-based randomization software, with 6 mice per group.

0.2 mL of 5×10 ⁶ REC-1 cells were subcutaneously inoculated into the right back of each nude mouse (PBS:Matrigel=1:1). Group administration was started when the average tumor volume reached 100 mm ³ . Animals were grouped according to tumor volume by an Excel-based randomization software, with 6 mice per group.

Preparation of test substance:

The preparation method of the test substance is shown in Table 5 and Table 6 below:

Table 5 Preparation method of test substance in TMD8 xenograft tumor model

Note: The samples are prepared and used now, and the prepared samples are stored at 4°C, and the drug needs to be gently mixed thoroughly before administration to animals; administration method: gavage; administration volume 10 μL/g

Table 6 Preparation method of test substance in REC-1 xenograft tumor model

Note: The samples are prepared and used now, and the prepared samples are stored at 4°C, and the drug needs to be gently mixed thoroughly before administration to animals; administration method: gavage; administration volume 10 μL/g.

Daily observation of experimental animals: The formulation and any modification of this experimental protocol have been evaluated and approved by the Laboratory Animal Care and Use Committee (IACUC) of Suzhou WuXi AppTec New Drug Development Co., Ltd. The use and welfare of experimental animals were carried out in accordance with the regulations of the International Association for the Assessment and Accreditation of Laboratory Animals (AAALAC). The health status and death of the animals were monitored daily. Routine examinations included observing the effects of tumor growth and drug treatment on the animals' daily behaviors such as behavioral activities, food and water intake (visual observation only), body weight changes (weight measurement three times a week), appearance signs or other abnormalities. Animal deaths and side effects within groups were recorded based on the number of animals in each group.

Tumor Measurements and Experimental Indicators: Experimental indicators examine whether tumor growth is inhibited, delayed or cured. Tumor diameters were measured with vernier calipers three times a week.

The formula for calculating tumor volume is:

V=0.5a×b ² ,

a and b represent the long and short diameters of the tumor, respectively.

The antitumor efficacy of the compounds was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%), reflecting tumor growth inhibition rate.

Calculation of TGI(%):

TGI(%)=[1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group)/(average tumor volume at the end of treatment in the solvent control group-average at the beginning of treatment in the solvent control group Tumor volume)] × 100%.

Relative tumor proliferation rate T/C (%): The calculation formula is as follows:

T/C%=T _RTV /C _RTV ×100% (T _RTV : relative tumor volume of the treatment group; C _RTV : relative tumor volume of the negative control group). The relative tumor volume (RTV) is calculated according to the results of tumor measurement, and the formula is RTV=V _t /V ₀ , where V ₀ is the average tumor volume measured during group administration (ie d ₀ ), and V _t is a measurement The mean tumor volume at the time of T _RTV and C _RTV were taken on the same day.

Statistical analysis: Statistical analysis including mean and standard error (SEM) of tumor volume for each time point for each group. The treatment group showed the best therapeutic effect on the 15th day (REC-1 xenograft tumor model) and 17th day (TMD8 xenograft tumor model) after administration, so a statistical analysis was performed based on this data to evaluate between groups difference. One-way ANOVA was used for comparison among three or more groups, and if there was a significant difference in F value, the Games-Howell method was used to test. All data analyses were performed with SPSS 17.0. p<0.05 was considered a significant difference.

The in vivo efficacy of compound A in the human mantle cell lymphoma REC-1 xenograft tumor model is shown in Table 7 and FIG. 1 . 15 days after the start of administration, the tumor volume of the tumor-bearing mice in the solvent control group reached 3501 mm ³ , and the tumor volume in the ibrutinib 25 mg/kg group was 1323 mm ³ , which had a significant tumor-inhibiting effect compared with the solvent control group (T/C = 38%, TGI = 64%, p = 0.008). The tumor volume of Compound A 15mg/kg and 30mg/kg groups were 1034 and 680mm ³ , respectively, which had a significant tumor inhibitory effect compared with the solvent control group (T/C values were 30% and 19%, respectively, TGI values were 73 % and 83%, both p values were less than 0.01), compared with the positive drug ibrutinib 25 mg/kg group, the compound A 15 mg/kg group had better tumor-inhibiting effect.

Table 7 Evaluation of antitumor efficacy of compound A on REC-1 xenograft tumor model (calculated based on tumor volume on the 15th day after administration)

Note: a. Mean±SEM; b. Tumor growth inhibition was calculated by T/C and TGI (TGI(%)=[1-(T ₁₅ -T ₀ )/(V ₁₅ -V ₀ )]×100); c. Mode of administration: once a day; **: p<0.01.

The in vivo efficacy of Compound A in the human lymphoma TMD8 xenograft tumor model is shown in Table 8 and FIG. 2 . 17 days after the start of administration, the tumor volume of the tumor-bearing mice in the solvent control group reached 1852 mm ³ , and the tumor volume of the ibrutinib 25 mg/kg group was 661 mm ³ . Compared with the solvent control group, the tumor volume was significantly inhibited (T/C = 35.68%, TGI = 68.18%, p < 0.001). The tumor volume of compound A 5mg/kg and 10mg/kg groups were 912mm ³ and 553mm ³ , respectively, which had a significant tumor inhibitory effect compared with the solvent control group (T/C values were 49.27% and 29.85%, TGI values were 53.78% and 74.35%, both p values were less than 0.01), compared with the positive drug ibrutinib 25 mg/kg group, the compound A 10 mg/kg group had better tumor-inhibiting effect.

Table 8 Evaluation of antitumor efficacy of compound A on TMD8 xenograft tumor model (calculated based on tumor volume on the 17th day after administration)

Note: a. Mean±SEM; b. Tumor growth inhibition was calculated by T/C and TGI (TGI(%)=[1-(T ₁₇ -T ₀ )/(V ₁₇ -V ₀ )]×100); c. Mode of administration: once a day; **: p<0.01.

The results show that in two BTK-sensitive mouse xenograft models, compound A has significant tumor growth inhibitory activity, which is significantly better than that of ibrutinib, the first-generation BTK inhibitor currently on the market.

In addition, using compound S18s, the above experiments in the human lymphoma cancer TMD8 xenograft tumor model were repeated, and the T/C (%) results are listed in the table below. In the table below, the T/C (%) results for Compound A are also listed for comparison.

Table 9 Inhibitory effect of compound A and S18 on TMD8 xenograft tumor model

Note: Mode of administration: once a day.

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

Experimental Example 4: Evaluation of Pharmacokinetic Properties in Rats

14 SD rats, male, weighing 200-220 g, were randomly divided into 4 groups, with 4/3 rats in each group, and the test compounds were administered by gavage and intravenous respectively. The specific arrangement is shown in Table 10 below:

Table 10 Test compound administration methods

group	number of animals	compound	Route of administration	Dosage (mg/kg)
1	4	Compound A	gavage (po)	3
2	3	Compound A	vein (iv)	1
3	4	ibrutinib	gavage (po)	3
4	3	ibrutinib	vein (iv)	1

Note: For intragastric administration, it is formulated with 0.5% carboxymethylcellulose sodium (CMC-Na) containing 1% Tween 80, and the drug concentration is 0.3 mg/mL; for intravenous administration, it is formulated with 5% DMSO/5% Tween 80/90% physiological saline was formulated into a solution, and the dose concentration was 0.2 mg/mL.

Fasting for 12 hours before the test, free access to water. 2 hours after the administration of a unified meal.

Blood collection time point and sample processing:

Oral administration: 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 hours after administration;

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

At the above set time points, 0.3 mL of venous blood was collected from the retroocular venous plexus of rats, placed in a heparinized test tube, centrifuged at 11,000 rpm for 5 minutes, and the plasma was separated and frozen in a -20°C refrigerator.

Sample testing and data analysis

The concentration of Compound A in rat plasma was determined by LC/MS/MS method.

The non-compartmental model of WinNonlin 5.3 software (Pharsight, USA) was used to calculate the pharmacokinetic parameters after administration.

The peak concentration _Cmax and the peak time _Tmax are measured values;

AUC _0-t value of the area under the drug-time curve: calculated by trapezoidal method;

AUC _0-∞ =AUC _0-t +C _t / _ke ,

C _t is the blood drug concentration at the last measurable time point,

_ke is the elimination rate constant;

elimination half-life t _1/2 =0.693/ _ke ;

Mean residence time MRT=AUMC/AUC.

Clearance rate CL=D/AUC _0-∞ ; steady-state volume of distribution Vss=CL×MRT

Absolute bioavailability F = (AUC _gavage × D _vein )/(AUC _vein × D _gavage ) × 100%

The test results are shown in Table 11 below:

Table 11 Pharmacokinetic experimental results of different compounds

Note: The pharmacokinetic data of S18s, S19s and S20s above are taken 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.”

The above results show that the clearance rate 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 70 times higher than that of ibrutinib; the clearance rate of compound A in vivo is also significantly higher In S18s, S19s and S20s, the drug exposure in plasma after oral administration was significantly higher than that in S18s, S19s and S20s. That is, at the same dose, compared with ibrutinib, S18s, S19s and S20s, compound A has better oral administration performance and good oral bioavailability.

Therefore, Compound A is a novel, oral, highly selective and highly active BTK inhibitor with significantly better in vitro and in vivo activity than the currently marketed BTK inhibitors. At the same dose, it inhibits tumor growth. The activity is significantly better than that of the positive control drug ibrutinib, which is of great development value.

The above embodiments are merely auxiliary descriptions in nature, and are not intended to limit the embodiments of the application target or the applications or uses of these embodiments. As used herein, the term "exemplary" means "serving as an instance, instance, or illustration." Any exemplary embodiment herein is not necessarily to be construed as preferred or advantageous over other embodiments.

Claims (10)

1. A use of a compound A as a Bruton's tyrosine kinase BTK inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of BTK-related tumor diseases, the compound A has the following structure:

   
2. The use according to claim 1, wherein the BTK-related tumor diseases include hematological tumors and solid tumors; preferably, the hematological tumors are lymphomas and leukemias; further preferably, the lymphomas are B cells lymphoma.
3. The use according to claim 1 or 2, wherein the BTK-related tumor diseases include histiocytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, and small lymphocytic lymphoma tumor, marginal zone lymphoma, follicular lymphoma, Burkitt lymphoma, or Waldenstrom's macroglobulinemia.
4. The use according to claim 1 or 2, wherein the medicament further contains one or more other targeted drugs and chemotherapeutic drugs.
5. The use according to claim 1 or 2, wherein the medicine is prepared into a clinically acceptable preparation, and the preparation is preferably an oral preparation, an injection preparation, and an external preparation.
6. The use according to claim 1 or 2, wherein the medicament contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, and the therapeutically effective dose is preferably 0.001 mg-1000 mg per day, More preferably the daily dosage is 0.01-500mg, still more preferably the daily dosage is 0.1-200mg, still more preferably the daily dosage is 0.1-100mg, still more preferably the daily dosage is 0.5-50mg, even more preferably The daily dose is 0.5-30 mg, more preferably 0.5-20 mg per day; it can be administered in a single dose or in divided doses.
7. A method for treating BTK-related tumor diseases, characterized in that, administering to a subject or patient a drug containing a therapeutically effective dose of Compound A or a pharmaceutically acceptable salt thereof, wherein Compound A has the following structure:

   
8. The method according to claim 7, wherein the BTK-related tumor disease is B-cell lymphoma.
9. The method according to claim 8, wherein the B-cell lymphoma 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's macroglobulinemia.
10. The method according to any one of claims 7-9, wherein the administration can be oral administration, injection administration, topical administration or in vitro administration, preferably oral administration or injection administration.

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