CN114478548A

CN114478548A - Application of Bruton's tyrosine kinase inhibitor

Info

Publication number: CN114478548A
Application number: CN202111235137.1A
Authority: CN
Inventors: 张翱; 丁健; 耿美玉; 谢华; 刘喜宝; 杨汉煜; 高娜; 张阳; 杨凤娇
Original assignee: Shanghai Runshi Pharmaceutical Technology Co ltd; Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Runshi Pharmaceutical Technology Co ltd; Shanghai Institute of Materia Medica of CAS
Priority date: 2020-10-23
Filing date: 2021-10-22
Publication date: 2022-05-13
Also published as: WO2022083745A1

Abstract

The invention provides an application of a compound A serving as a Bruton tyrosine kinase BTK inhibitor or a pharmaceutically acceptable salt thereof in preparing a medicament for treating BTK-related tumor diseases. 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 better pharmacokinetic property and metabolic stability, can be used for developing a medicinal preparation for treating BTK-related tumor diseases, and has important clinical application value.

Description

Application of Bruton's tyrosine kinase inhibitor

Technical Field

The application belongs to the field of medicines, and particularly relates to an application of a Bruton's Tyrosine Kinase (BTK) inhibitor, in particular to an application of the compound in preparing a medicine for treating BTK-related tumor diseases.

Background

B cell antigen receptor (BCR) signaling plays a crucial role in normal B cell development and adaptive immunity, and activation of this signaling pathway contributes to the development and progression 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, is a key regulator in the B Cell Receptor (BCR) signaling pathway, is mainly expressed at various developmental stages of B lymphocytes (except plasma cells at the end of B lymphocyte development), and has important effects on proliferation, differentiation, and apoptosis of B cells.

In B cell-associated malignancies, the BCR signaling pathway is overactive, inhibiting normal differentiation and apoptosis of B cells, promoting abnormal proliferation, and aberrant regulation of the BCR pathway is often known to occur in malignancies of a variety of B cell types. Currently, 4 BTK inhibitors are marketed, Ibrutinib (Ibrutinib, trade name ibruvvica) is the first BTK Small molecule inhibitor to be marketed, belongs to a first generation of BTK inhibitors, and is approved by the FDA in the united states in 2013 for clinical treatment of Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), etc., and thereafter continuously expanding the indications on Ibrutinib, and further includes Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/Small Lymphocytic Lymphoma, SLL) carrying 17p deletion, Macroglobulinemia fahrenheit (Waldenstrom macroglobulinia, WM), Marginal Zone Lymphoma (Marginal Zone, MZL), Chronic Graft-Versus-Host Disease (Chronic gray-Host Disease, cgcd). Ibrutinib can form covalent binding with 481 th cysteine (Cys481) in the ATP binding domain of BTK kinase, irreversibly inhibit BTK activation, and block BTK signal pathway, thereby inhibiting proliferation and survival of B lymphoma cells and achieving the purpose of tumor treatment. Ibrutinib achieves substantial efficacy in clinical treatment. But because the selectivity of the ibrutinib target is poor, certain toxic and side effects exist in clinic. Acatinib (acarabutinib, ACP-196, trade name calsequence) was approved for the treatment of MCL and CLL in 2017, belonging to the second generation BTK targeting drugs. Compared with ibrutinib, the selectivity of the acatinib on BTK is higher, and off-target toxicity is lower. In addition, Zanubrutinib (BGB-3111), developed by baiji state biotechnology limited, was approved by FDA for treated adult MCL patients in 2019 at 11 months, and was the first established local anticancer drug by FDA-breakthrough therapy in china. Tirabrutinib (ONO-4059) developed by japan minifield pharmaceutical limited was approved by the japan pharmaceutical medical device integration institution (PMDA) at 3 months of 2020 for the treatment of relapsed or refractory Primary Central Nervous System Lymphoma (PCNSL) and lymphoplasmacytic lymphoma (LPL). Overall, the first generation inhibitors have high inhibitory activity against BTK, but poor target selection and bioavailability; the second generation inhibitors are selective, but have a lower rate of inhibition of BTK than the first generation inhibitors. Structurally, the core backbone of currently marketed BTK drugs is mainly a bicyclic ring system.

In order to obtain a new generation of BTK inhibitor with high activity of the first generation inhibitor and good selectivity of the second generation inhibitor, a series of compounds with pyrimido [5,4-b ] indolizine or pyrimido [5,4-b ] pyridine structure are disclosed in the patent CN108101905A of Shanghai pharmaceutical research of Chinese academy of sciences. Among them, pyrimido [5,4-b ] indolizines S1 and S10 and pyrimido [5,4-b ] pyrine S18, S19 and S20 showed higher BTK inhibitory activity, and in further work, S-configured compounds of S18, S19 and S20 (i.e., S18S, S19S and S20S) [ Yu Xue, et al discovery of4,7-Diamino-5- (4-phenoxyphenyl) -6-methyeneaziridino [5,4-b ] pyrolidone as Novel Bruton' S Tyrosine inhibitors medium. j.chem., 2018,61, 4608. sul 4627 ], but further studies found that one of these compounds S1, S34 and S19 is unstable at the end of phenyl ring (S3626), and that the end of S-phenyl ring is metabolized at the p-position of the phenyl ring 26); whereas the oral bioavailability of compound S20S is not ideal.

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

Disclosure of Invention

The invention aims to provide an application of a Bruton's tyrosine kinase BTK inhibitor compound A or a pharmaceutically acceptable salt thereof in preparing a medicament for treating BTK-related tumor diseases, wherein the compound A has the following structure:

the BTK related tumor diseases comprise hematological tumors and solid tumors.

Preferably, the hematological tumors are lymphoma and leukemia.

More preferably, the lymphoma is a B cell lymphoma.

The use, the BTK related tumor disease comprises histiocytic lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, marginal zone lymphoma, follicular lymphoma, Burkitt's lymphoma or Fahrenheit macroglobulinemia.

The use as described above, wherein the diffuse large B-cell lymphoma is selected from 1 or more of the group consisting of diffuse large B-cell lymphoma of unspecified type, other large B-cell lymphoma, high-grade B-cell lymphoma accompanied by MYC and/or Bcl gene abnormality, and high-grade B-cell lymphoma of unspecified type; preferably, the diffuse large B-cell lymphoma is selected from 1 or more of the group consisting of other large B-cell lymphomas, high grade B-cell lymphomas with MYC and/or Bcl gene abnormalities, and high grade B-cell lymphomas of unspecified type.

The above uses, the other large B-cell lymphomas include T-cell/histiocyte-rich DLBCL, primary central nervous system DLBCL, primary skin DLBCL (leg), EBV-positive DLBCL, non-finger DLBCL, large B-cell lymphoma associated with chronic inflammation, lymphomatoid granuloma, large B-cell lymphoma accompanied with IRF4 rearrangement, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablast lymphoma, HHV 8-positive DLBCL, primary effusion lymphoma, and the like;

the use, wherein the high-grade B cell lymphoma accompanied with MYC gene abnormality is selected from 1 or more of the group consisting of MYC gene amplified high-grade B cell lymphoma and MYC gene fused high-grade B cell lymphoma; the Bcl gene abnormal high-grade B cell lymphoma is selected from Bcl2 and/or Bcl6 gene abnormal high-grade B cell lymphoma, and the Bcl2 and/or Bcl6 gene abnormal high-grade B cell lymphoma is selected from 1 or more of the group consisting of Bcl2 gene amplified high-grade B cell lymphoma, Bcl2 gene fused high-grade B cell lymphoma, Bcl6 gene amplified high-grade B cell lymphoma and Bcl6 gene fused high-grade B cell lymphoma.

The diffuse large B-cell lymphoma according to the present invention may be a diffuse large B-cell lymphoma carrying an abnormal chromosome.

The above use, the mantle cell lymphoma is selected from 1 or more of the group consisting of classical mantle cell lymphoma, leukemia-like non-lymphadenitic mantle cell lymphoma and Cyclin D1 positive mantle cell lymphoma.

The above use, wherein the mantle cell lymphoma is selected from mantle cell lymphoma with Bcl gene abnormality; preferably a mantle cell lymphoma with Bcl1 and/or Bcl2 gene abnormalities; further preferred is a Bcl1 gene fused or rearranged mantle cell lymphoma or Bcl2 gene fused or rearranged mantle cell lymphoma.

The mantle cell lymphoma of the present invention may be a mantle cell lymphoma carrying an abnormal chromosome.

For the above use, the Burkitt lymphoma is a Burkitt lymphoma carrying an abnormal chromosome.

The above use, the gene abnormality refers to gene mutation, gene fusion or rearrangement and/or gene amplification.

The abnormal chromosome refers to a chromosome which is amplified, deleted, broken, rearranged and/or translocated.

In the application, the medicine can also contain one or more other targeted medicines or chemotherapeutic medicines. The other targeted drugs or chemotherapeutic drugs refer to targeted drugs or chemotherapeutic drugs clinically used for treating tumor-related diseases.

The medicine can be prepared into clinically acceptable preparations, such as oral preparations, injection preparations, external preparations and the like.

The above use, wherein the medicament contains a therapeutically effective amount of compound a or a pharmaceutically acceptable salt thereof, the therapeutically effective amount is preferably administered at a daily dose of 0.001 to 1000mg, more preferably at a daily dose of 0.01 to 500mg, even more preferably at a daily dose of 0.1 to 200mg, even more preferably at a daily dose of 0.5 to 100mg, even more preferably at a daily dose of 0.5 to 50mg, even more preferably at a daily dose of 0.5 to 30mg, even more preferably at a daily dose of 0.5 to 20 mg. Administration can be single dose or divided dose.

In another aspect, the present invention provides a method for treating a BTK-related tumor disease, which comprises administering to a subject or patient a medicament containing a therapeutically effective amount of compound a or a pharmaceutically acceptable salt thereof.

The method, wherein the BTK related tumor diseases comprise hematological tumors and solid tumors.

Preferably, the hematological tumors are lymphoma and leukemia.

More preferably, the lymphoma is a B cell lymphoma.

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's lymphoma, or fahrenheit macroglobulinemia.

The use as described above, wherein the mantle cell lymphoma is selected from 1 or more of the group consisting of classical mantle cell lymphoma, leukemia-like non-lymphadenitic mantle cell lymphoma and Cyclin D1-positive mantle cell lymphoma.

The use as described above, wherein the mantle cell lymphoma is selected from mantle cell lymphoma with Bcl gene abnormality; preferably a mantle cell lymphoma with Bcl1 and/or Bcl2 gene abnormalities; further preferred is a Bcl1 gene fused or rearranged mantle cell lymphoma or Bcl2 gene fused or rearranged mantle cell lymphoma.

The use as described above, said Burkitt lymphoma being a Burkitt lymphoma carrying an abnormal chromosome.

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 dose frequency of administration of compound a or a pharmaceutically acceptable salt thereof can be determined by conventional methods such as modeling, dose escalation studies or clinical trials and by taking into account factors such as the nature and severity of the disease to be treated, the age, general condition and body weight of the patient, as well as the particular compound administered, its pharmacokinetic properties, and the route of administration. A suitable dosage range for Compound A or a pharmaceutically acceptable salt thereof is from about 0.001mg/kg to about 1000mg/kg per day; preferably, from about 0.01mg/kg to about 100 mg/kg; further preferably, from about 0.02mg/kg to about 50 mg/kg; even more preferably, from about 0.03mg/kg to about 20 mg/kg. Preferably, the daily administration dosage of the compound A or the pharmaceutically acceptable salt thereof is 0.001mg to 1000mg, and further preferably, the daily administration dosage of the compound A or the pharmaceutically acceptable salt thereof is 0.01mg to 500 mg; still more preferably, compound a or a pharmaceutically acceptable salt thereof is administered at a dose of 0.1-200mg per day; still more preferably, compound a or a pharmaceutically acceptable salt thereof is administered at a dose of 0.5-100mg per day; still more preferably, compound a or a pharmaceutically acceptable salt thereof is administered at a dose of 0.5-50mg per day; still more preferably, the compound a or a pharmaceutically acceptable salt thereof is administered at a dose of 0.5-30mg per day; still more preferably, compound a or a pharmaceutically acceptable salt thereof is administered at a dose of 0.5-20mg per day; administered in single or divided doses.

In another aspect, the present invention also provides a method of treating a disorder in a patient by administering to the patient a medicament containing a therapeutically effective amount of compound a or a pharmaceutically acceptable salt thereof, the disorder in the patient being a BTK-related neoplastic disease.

Preferably, the hematological tumors are lymphoma and leukemia.

More preferably, the lymphoma is a B cell lymphoma.

the use, wherein the high-grade B cell lymphoma accompanied with MYC gene abnormality is selected from 1 or more of the group consisting of MYC gene amplified high-grade B cell lymphoma and MYC gene fused high-grade B cell lymphoma; the high-grade B cell lymphoma with Bcl gene abnormality is selected from 1 or more of the group consisting of Bcl2 and/or Bcl6 gene abnormality high-grade B cell lymphoma, Bcl2 and/or Bcl6 gene abnormality high-grade B cell lymphoma, Bcl2 gene amplification high-grade B cell lymphoma, Bcl2 gene fusion high-grade B cell lymphoma, Bcl6 gene amplification high-grade B cell lymphoma and Bcl6 gene fusion high-grade B cell lymphoma.

The therapeutically effective amount, administration dose or administration dose of the compound A or the pharmaceutically acceptable salt thereof is counted by the compound A.

Diffuse large B-cell lymphomas (DLBCL) were classified into four categories according to the 2017 th edition of hematopoietic and lymphoid tissue tumors (revision 4): non-specific types of DLBCL, other large B-cell lymphomas, high grade B-cell lymphomas, and B-cell lymphomas that cannot be classified between DLBCL and classical hodgkin's lymphoma. Wherein, the non-specific DLBCL comprises a central blast variant, an immunoblast variant, a anaplasia variant and other rare variants (such as spindle cell variant and signet ring cell-like variant) morphologically; other large B-cell lymphomas include T-cell/histiocyte-rich DLBCL, primary central nervous system DLBCL, primary skin DLBCL (legged), 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, plasmacytoma, HHV 8-positive DLBCL, primary effusion lymphoma, etc.; high grade B cell lymphomas include high grade B cell lymphomas with abnormalities (e.g., gene rearrangement or fusion, gene amplification, gene mutation, etc.) in MYC, BCL2, and/or BCL6, non-specific types of high grade B cell lymphomas, etc., such as U-2932 cells (with BCL2 gene amplification), WILL-2 cells (with BCL2 gene fusion), etc., as mentioned herein. (Source: ATCC official network information and DSMZ official network information)

Reference herein to "diffuse large B-cell lymphoma carrying an abnormal chromosome" refers to diffuse large B-cell lymphomas carrying abnormal chromosomes (e.g., chromosomal amplification, deletion, fragmentation, rearrangement, and/or translocation, etc.), such as the Pfeiffer cells referred to herein (presence of multiple chromosomal abnormalities, including chromosomal t (14; 18) (q 32; q21) translocations), and the like. (Source: ATCC official network information and DSMZ official network information)

Reference herein to "mantle cell lymphoma carrying an abnormal chromosome" refers to a mantle cell lymphoma carrying an abnormal chromosome (e.g., chromosomal translocation, chromosomal fusion, chromosomal deletion, etc.), such as the Z-138 cells mentioned herein (presence of a chromosomal abnormality, e.g., chromosomal t (11; 14) (q 13; q32) translocation and/or chromosomal del (5) (p15) deletion, etc.), Mino cells (presence of a chromosomal abnormality, e.g., chromosomal del (6) (q16) deletion, etc.), REC-1 cells (presence of a chromosomal abnormality, e.g., chromosomal t (11; 14) (q 13; q32) translocation, etc.). (Source: ATCC official network information and DSMZ official network information)

Reference herein to a "mantle cell lymphoma with a Bcl gene abnormality" is a mantle cell lymphoma in which a Bcl gene abnormality (e.g., gene mutation, gene amplification, gene rearrangement or/fusion, gene abnormal activation, etc.) is present, which may result in overexpression of a Bcl-associated protein, e.g., Jeko-1 cells (in which Bcl-1 gene rearrangement) as referred to herein, etc. (Source: ATCC official website information)

Reference herein to "a Burkitt lymphoma carrying an abnormal chromosome" refers to a Burkitt lymphoma carrying an abnormal chromosome (e.g., an abnormality in chromosome size and/or number, chromosome translocation, chromosome fusion, chromosome deletion, etc.), such as Raji cells (abnormal in chromosome size and/or number 1 or 4) as referred to herein. (Source: ATCC official website information)

The pharmaceutically acceptable salts of the compounds may be conventional non-toxic salts formed by reacting 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; the DIAD: diisopropyl azodicarboxylate; DIPEA: diisopropylethylamine; DMF: n, N-dimethylformamide; EA: ethyl acetate; HATU: 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate; NBS: n-bromosuccinimide; NIS: n-iodosuccinimide; PdCl₂(dppf): [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride; pd (PPh)₃)₄: tetrakis (triphenylphosphine) palladium; PdCl₂: palladium dichloride; pd (OAc)₂: palladium acetate; pd (PPh)₃)₂Cl₂: bis (triphenylphosphine) palladium dichloride; PE: petroleum ether; THF: tetrahydrofuran; DMSO, DMSO: dimethyl sulfoxide (DMSO).

The invention achieves one or more of the following beneficial technical effects:

(1) the results of in vitro and in vivo efficacy tests show that the inhibitory activity of the compound A on BTK and lymphoma cells cultured in vitro is superior to that of the early-stage compounds S1, S10, S18S, S19S and S20S, and is also superior to that of the first-generation BTK inhibitor ibrutinib and the second-generation BTK inhibitor acatinib which are on the market at present. In an in vivo transplantation tumor model, the compound A also shows a significantly better tumor growth inhibition effect than S18S and ibrutinib.

(2) The in vitro pharmacodynamic test result shows that the compound A has good inhibition effect on various tumor cells and also has good inhibition effect on different specific cell strains of the same cell type, namely the compound A can achieve good inhibition effect on the same tumor cell type and cell strains of different sources, and has good clinical application prospect.

(3) Pharmacokinetic studies show that the in vivo clearance rate of the compound A is significantly lower than that of ibrutinib and structural analogues S18S, S19S and S20S, and only is 1/10-1/20 of the ibrutinib and the structural analogues; after the compound A is orally taken, the drug exposure (AUC) in blood plasma is 70 times higher than that of ibrutinib, and the absolute bioavailability is obviously improved.

(4) S1, S10, S18S and S19S have a plurality of metabolite varieties and poor metabolic stability because the 4-position of the terminal phenyl group is easy to oxidize, and S18S can only retain 51% of the proto-drug after being incubated by rat liver microsomes. The compound A effectively overcomes the problem that the terminal benzene ring of a diphenyl ether structure in the compound is hydroxylated through structural modification, and has few metabolite types and proportions under the action of different species of liver microsomes (HLM: human liver microsomes; RLM: rat liver microsomes; MLM: mouse liver microsomes), basically takes a prototype drug as a main drug (60min: 84% -98%), and has better metabolic stability.

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

Drawings

FIG. 1: experimental results of the REC-1 xenograft tumor model are shown schematically.

FIG. 2: experimental results of the TMD8 xenograft tumor model are shown schematically.

Detailed Description

The following further provides examples which are intended to aid in the understanding of the present invention and are intended to be illustrative only and do not limit the scope of the invention.

Example 1: synthesis of Compound A

1. Synthesis of intermediate 3

4-chloro-5-iodo-7H-pyrrolo [2,3-d ] pyrimidine (starting material 2,17.28g,1eq) and anhydrous potassium carbonate (2eq) were added to a 250mL round-bottomed flask and dried under vacuum to remove water. Dried DMF was added as solvent and the crushed 2- ((tert-butoxycarbonyl) amino) -but-3-en-1-yl (starting material 1,24.6g,1.5eq) methane sulfonate was replaced with nitrogen. The mixture was stirred at 55 ℃ for 12 hours, and the time was appropriately extended to ensure completion of the reaction.

After the reaction is finished, adding water and ethyl acetate for extraction for three times, and combiningThe ester layer was combined, back extracted once with water, and washed with saturated brine. Dried over anhydrous sodium sulfate. Dry chromatography (eluent: CHCl)₃MeOH 100:1) to obtain 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 3,19.28g) in 69.5% yield.

¹H NMR(300MHz,CDCl₃)δ8.60(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

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

After the reaction was completed, it was cooled to room temperature, water and ethyl acetate were added to extract, and the ester layers were combined and washed with saturated brine. Dried over anhydrous sodium sulfate. Dry chromatography (eluent: CHCl)₃MeOH 30:1) to obtain 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 4,6.86g) in 78.0% yield.

¹H NMR(300MHz,CDCl₃)δ8.25(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

Adding (S) - (1- (4-amino-5-iodo-7H-pyrrolo [2, 3-d) into a 1L round-bottom flask]Pyrimidin-7-yl) but-3-en-2-yl) carbamic acid tert-butyl ester (mediumIntermediate 4,32.9g,1eq), N- (pyridin-2-yl) -4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzamide (starting material 5,34.8g,1.4eq) and Pd (PPh)₃)₄(17.7g,0.2 eq). 1, 4-dioxane (383mL) was added as a solvent and N was replaced₂.2M sodium carbonate solution (76.6mL) was added with stirring. The mixture was stirred at 90 ℃ under reflux for 5 hours.

Water and ethyl acetate were added for extraction, and the ester layers were combined and washed with saturated brine. Dried over anhydrous sodium sulfate. Dry-purifying with EA as eluent to remove most impurities, and then with CHCl₃A 30:1 mixture of MeOH was used as eluent. The product may contain a small amount of impurities, and the pure product can be separated out by PE recrystallization. To obtain the product (S) - (1- (4-amino-5- (4- (pyridine-2-yl carbamoyl) phenyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-7-yl) but-3-en-2-yl) carbamic acid tert-butyl ester (intermediate 6, 28.4g) in 74.3% yield. ee>99.5％。

4. Synthesis of intermediate 7

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

After the reaction, water and ethyl acetate were added for extraction, the ester layers were combined, back-extracted once with water, and washed with saturated brine. Dried over anhydrous sodium sulfate. Dry-packing with CHCl₃MeOH-50: 1 mixture as eluent, then CHCl was changed₃A 30:1 mixture of MeOH was used as eluent. To obtain the product (S) - (1- (4-amino-6-bromo-5- (4- (pyridine-2-yl carbamoyl) phenyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-7-yl) but-3-en-2-yl) carbamic acid tert-butyl ester (intermediate 7, 25.8g) in 68.2% yield. ee>99.5％。

5. Synthesis of intermediate 8

Into a 250mL 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 7,11.9g,1eq) and PdCl₂(dppf) (1.66g,0.11eq), 51mL THF as solvent was added and nitrogen was replaced several times to ensure completeness. 4M sodium hydroxide solution (8.2mL) was added with stirring. Stirring was carried out at 85 ℃ under reflux for 15 hours.

After the reaction was completed, water and ethyl acetate were added to extract, and the ester layers were combined and washed with saturated brine. Dried over anhydrous sodium sulfate. Dry chromatography (eluent: CHCl)₃MeOH ═ 30:1) to give the product (S) - (4-amino-6-methylene-5- (4- (pyridin-2-ylcarbamoyl) phenyl) -7, 8-dihydro-6H-pyrimido [5, 4-b)]Pyrin-7-yl) carbamic acid tert-butyl ester (intermediate 8, 8.79g) in 85.9% yield. ee>99.5％。

6. Synthesis of Compound A

To a 250mL round bottom flask was added tert-butyl (S) - (4-amino-6-methylene-5- (4- (pyridin-2-ylcarbamoyl) phenyl) -7, 8-dihydro-6H-pyrimido [5,4-b ] pyrazin-7-yl) carbamate (intermediate 8,2.75g,1eq) and 110mL DCM was added as a solvent. Trifluoroacetic acid (10.5mL) was added dropwise with stirring. Stirred at room temperature for 3 hours. And after the reaction is finished, directly spin-drying the reaction liquid, taking out trifluoroacetic acid by using a methanol tape for several times, and spin-drying to obtain an amino de-Boc protected crude product which is directly put into the next step.

The product from the previous step was transferred to a 250mL round bottom flask, triethylamine (1eq) was added, and after stirring for five minutes, 2-butynoic acid (0.511g,1.1eq) and HATU (2.31g,1.1eq) were added, and 100mL DCM was added as a solvent. The ice-water bath was cooled to 0 deg.C and triethylamine (1.54mL +0.77mL) 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 yellowish. AddingWater and DCM were added for extraction and the organic phases were combined and washed with brine. Dried over anhydrous sodium sulfate and subjected to column chromatography (CHCl)₃MeOH 30:1) gave the final product a (1.88g) in 73.3% yield. ee>99.5％。

1H NMR(400MHz,CDCl₃)δ8.98(s,1H),8.43(dt,J＝8.3,1.0Hz,1H),8.34(ddd,J＝5.0,1.9,0.9Hz,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 enzyme activity inhibition Activity of Bruton kinase (BTK) molecule level

An enzyme reaction substrate Poly (Glu, Tyr)_4:1After diluting the plate with PBS (10mM sodium phosphate buffer, 150mM NaCl, pH 7.2-7.4) without potassium ion to 20. mu.g/mL coated plate, reacting at 37 ℃ for 12-16 hours, washing the plate three times with 200. mu.L/well T-PBS (PBS containing 0.1% Tween-20), and drying the plate in an oven at 37 ℃ for 1-2 hours. To the above substrate-coated microplate, a reaction buffer (50mM HEPES pH 7.4, 50mM MgCl) was first added₂，0.5mM MnCl₂，0.2mM Na₃VO₄1mM DTT) was added to the sample at 49. mu.L/well (final concentration: 5. mu.M). Mu.l of the test compound (compound wells) or DMSO containing the corresponding concentration (negative control wells) was added to each well, and no enzyme control wells were required for each experiment. The reaction was initiated by adding 50. mu.L of BTK tyrosine kinase protein diluted in the reaction buffer.

The above reaction system was placed in a shaker (100rpm) at 37 ℃ for 1 hour, then the plate was washed three times with T-PBS, primary anti-PY 99100. mu.L/well (Santa Cruz) was added, and the shaker reaction at 37 ℃ was carried out for 0.5 hour. After washing the plate with T-PBS, 100. mu.L/well of horseradish peroxidase-labeled goat-anti-mouse secondary antibody diluent was added, and the mixture was subjected to shaking reaction at 37 ℃ for 0.5 hour. After washing the plate with T-PBS, 2mg/mL OPD developing solution 100. mu.L/well was added, and the reaction was carried out at 25 ℃ for 1 to 10 minutes in the dark. Then 2M H was added₂SO₄The reaction was stopped at 50. mu.L/well and read using a tunable wavelength microplate reader SPECTRA MAX Plus384 at 490 nm.

Compounds S1, S10, ibrutinib, acatinib, S18S, S19S and S20S were used as positive control compounds, wherein compounds S1, S10, S18S, S19S and S20S were prepared using methods disclosed in the prior art (e.g. CN108101905A) or similar methods, and ibrutinib and acatinib were purchased from seleck corporation.

The inhibition ratio of each compound was obtained by the following formula:

IC₅₀the values were determined by regression with a four parameter method using a microplate reader random plus software. The results are shown in table 1 below.

TABLE 1 inhibition of BTK by different compounds

Compound (I)	IC₅₀(nM)
		S1	～1
S10	<10
		Ibrutinib	～1
Acatinib	～10
		S18s	～1
S19s	～1
		S20s	～1
Compound A	0.5

The results show that the BTK inhibitory activity of the compound A is better than that of the previous compounds S1, S10, S18S, S19S and S20S, and is also better than that of the currently marketed first-generation BTK inhibitor ibrutinib and second-generation BTK inhibitor acatinib.

Experimental example 2: detection of in vitro proliferation inhibition activity of compound on human B lymphoma cells

Test cell

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

The test method comprises the following steps:

a cell suspension (Ramos: 10000 cells/well; TMD 8: 12000 cells/well) was inoculated into a 96-well plate, and after the cell state was stabilized by standing in an incubator at 37 ℃ for 2 hours, test compounds at different concentrations (3 duplicate wells per concentration) were added to each well, and a blank control (a well containing only a culture solution and no cells), a negative control (a well containing only cells and no compounds) and a positive compound control were set simultaneously. After 72h of dosing, 20. mu.L of MTT (5mg/mL) was added to each well and incubated at 37 ℃ for 4h, 100. mu.L of triple (10% SDS, 5% isobutanol, 0.01M HCl) was added and left overnight at 37 ℃. OD value was measured at a wavelength of 570nm using a wavelength-tunable microplate reader SPECTRAmax Plus 384.

The inhibition of the compound was determined by the following formula:

IC₅₀the values were determined by regression with a four parameter method using a microplate reader random plus software. The experiment was independently repeated 3 times, and the results are shown in table 2 below.

Compounds S1, S10, ibrutinib, acatinib, S18S, S19S and S20S, also described above, were used as positive control compounds.

TABLE 2 proliferation inhibitory Activity of different compounds on Ramos cells and TMD8 cells

Compound (I)	Ramos cell IC₅₀	TMD8 cell IC₅₀
			S1	94.73μM	0.006μM
S10	8.72μM	0.030μM
			Ibrutinib	12.91μM	0.005μM
AcartinNi (Ni) is	38.16μM	0.023μM
			Compound A	3.15μM	0.003μM
S18s	5.04μM	0.016μM
			S19s	—	0.017μM
S20s	14.3μM	0.004μM

The results show that the proliferation inhibition capacity of the compound A on B cell lymphoma is better than that of the previous compounds S1, S10, S18S, S19S and S20S at the cellular level, and is also better than that of the currently marketed first-generation BTK inhibitor ibrutinib and second-generation BTK inhibitor acatinib. It is further noted that compound a of the present invention has higher proliferation inhibitory activity against Ramos cells and higher proliferation inhibitory activity against TMD8 cells than other compounds.

Test cell

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

Test method

The Cell proliferation inhibitory activity of the compound was examined by the CCK8(Cell Counting Kit-8, # D3100L4057, Shanghai Liji Biotech Co., Ltd.) staining method.

(1) Tumor cells in log phase of growth were seeded in 96-well plates at an appropriate density of cell suspension at 95. mu.L per well, depending on their different growth rates. After the cell state is stabilized, 10 mu L of compound with the required concentration diluted in a gradient way is sequentially added into the incubator at 37 ℃ for standing for 2-4h, 3 multiple holes are arranged in each dose, and a solvent control hole and a blank control hole without cells are arranged at the same time. Culturing for 72h at 37 ℃ in a carbon dioxide incubator.

(2) CCK8 staining solution was added at 10. mu.L/well. The incubator is incubated for 2-4h, and read by a microplate reader SPECTRAmax PLUS384, and the measurement wavelength is 450 nm. The growth inhibition rate of the compound on the cells is calculated by the formula:

the inhibition ratio%.

Half the inhibition IC₅₀The values were calculated using a four parameter method. Each experiment was independently repeated 3 times to determine the average IC of each experiment₅₀The value serves as the final indicator of the inhibition capacity.

TABLE 3 proliferation inhibitory Activity of Compound A on B lymphoma cells (BTK inhibitor-sensitive cell line)

The results show that the compound A has stronger inhibitory activity on 2 BTK inhibitor sensitive cell strains and is superior to positive control medicaments of ibrutinib and acatinib.

TABLE 4 proliferation inhibitory Activity of Compound A on B lymphoma cells

The above results indicate that the inhibitory activity of compound a on other B lymphoma cells is either comparable to ibrutinib or intermediate to ibrutinib and acartinib, all at micromolar levels. The results in tables 3 and 4 show that, compared with the positive control drug, the compound A has a more obvious inhibition effect on BTK inhibitor sensitive cell strains, and also has a certain inhibition activity on other B lymphoma cells, namely has better BTK inhibition selectivity.

As shown by the results in tables 3 and 4 above, the inhibitory effect of compound A on U-2932 and WILL-2 suggests that the compound A of the present invention is expected to have an effect of treating high-grade B-cell lymphoma accompanied by Bcl gene abnormality in diffuse large B-cell lymphoma; the inhibition effect of the compound A on Pfeiffer indicates that the compound A is expected to have the effect of treating diffuse large B-cell lymphoma carrying abnormal chromosomes in the diffuse large B-cell lymphoma; the inhibitory effect of compound a on RL suggests that compound a of the present invention is expected to have an effect on the treatment of follicular lymphoma; the inhibitory effect of compound a on Raji, NAMALWA suggests that compound a of the present invention is expected to have an effect on treatment of Burkitt lymphoma; the inhibition effect of the compound A on Raji suggests that the compound A provided by the invention is expected to have the effect of treating Burkitt lymphoma carrying abnormal chromosomes; the inhibition effect of the compound A on Z-138, Mino and REC-1 indicates that the compound A provided by the invention is expected to have the effect of treating mantle cell lymphoma carrying abnormal chromosomes in the mantle cell lymphoma; the inhibitory effect of compound a on JeKo-1 suggests that compound a of the present invention is expected to have an effect of treating mantle cell lymphoma carrying abnormal chromosomes among mantle cell lymphomas; the inhibitory effect of compound a on KARPAS-1106P suggests that compound a of the present invention is expected to have an effect on the treatment of primary mediastinal B-cell lymphoma among diffuse large B-cell lymphomas.

In addition, considering that the inhibitory activity of compound a on OCI-LY10, REC-1(BTK inhibitor sensitive cell line) is superior to that of the positive control drugs ibrutinib and acatinib, and the inhibitory activity on other B lymphoma cells is comparable to that of ibrutinib, or the results between ibrutinib and acatinib suggest that: compound a of the invention is expected to be able to treat diseases treated by ibrutinib and acatinib, such as chronic lymphocytic leukemia, chronic lymphocytic leukemia/small lymphocytic lymphoma carrying a 17p deletion, fahrenheit 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) The species are as follows: mouse

2) Strain: CB-17SCID

3) Week age and body weight: 6-8 weeks; 18-22g

4) Sex: female

5) The supplier: beijing Wittiulihua laboratory animal technology Co Ltd

REC-1 xenograft tumor model

1) The species are as follows: mouse

2) Strain: BALB/c nude mice

3) Week age and body weight: 6-8 weeks; 17-20g

4) Sex: female

5) The supplier: shanghai Ling Chang Biotechnology Co., Ltd

Cell culture-in vitro suspension culture of human lymphoma TMD8 cells under the conditions of RPMI 1640 medium (supplier: gibco; cat # 22400; "ProductionLot # 4868546) supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin, 5% CO at 37 ℃%₂And (5) culturing. Routine treatment was performed twice a week for passages. When the saturation degree of the cells is 80% -90%, collecting the cells, counting and inoculating.

Human mantle cell lymphoma REC-1 cells were cultured in vitro in suspension in RPMI 1640 medium (supplier: gibco; cat # 22400;. production lot # 1868795) supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin at 37 ℃ in 5% CO 2. Routine treatment passages were performed twice a week. When the saturation degree of the cells is 80% -90%, collecting the cells, counting and inoculating.

Tumor cell inoculation 0.2mL 10X 10⁶The right back of each nude mouse was subcutaneously inoculated with TMD8 cells for personal lymphoma (PBS: Matrigel ═ 1: 1). The average tumor volume reaches 104mm³The grouped administration is started. Groups were made by an Excel-based random grouping software according to animal tumor volume, with 6 mice per group.

0.2mL of 5X 10⁶One REC-1 cell was inoculated subcutaneously into the right back of each nude mouse (PBS: Matrigel ═ 1: 1). The average tumor volume reaches 100mm³The grouped administration is started. Groups were made by an Excel-based random grouping software according to animal tumor volume, with 6 mice per group.

Preparation of a test substance:

see tables 5 and 6 below for the test formulation methods:

TABLE 5 TMD8 xenograft tumor model test article formulation method

Note: the sample is used as it is, the prepared sample is stored at 4 ℃, and the medicine needs to be mixed evenly and lightly before being administered to animals; the administration mode comprises the following steps: performing intragastric administration; the administration volume is 10. mu.L/g

TABLE 6 REC-1 xenograft tumor model test substance preparation method

Note: the sample is used as it is, the prepared sample is stored at 4 ℃, and the medicine needs to be mixed evenly and lightly before being administered to animals; the administration mode comprises the following steps: performing intragastric administration; the administration volume was 10. mu.L/g.

Experimental animals daily observations that the formulation and any modification of the experimental protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of Suzhou drug Mingkuda new drug development, Inc. The use and welfare of the experimental animals were performed in compliance with the provisions of the international committee for evaluation and approval of experimental animals (AAALAC). Animals are monitored daily for health and mortality, and routine examinations include observations of the effects of tumor growth and drug treatment on the animal's daily performance such as behavioral activity, food intake (visual only), weight changes (three weekly weight measurements), appearance signs, or other abnormalities. The number of deaths and side effects of animals in the groups were recorded based on the number of animals in each group.

Tumor measurements and experimental indices: the experimental index is to investigate whether the tumor growth is inhibited, delayed or cured. Tumor diameters were measured three times a week with a vernier caliper.

The formula for tumor volume is:

V＝0.5a×b²，

a and b represent the major and minor diameters of the tumor, respectively.

The tumor suppressor therapeutic effect of the compound was evaluated as TGI (%) or relative tumor proliferation rate T/C (%). TGI (%), reflecting the rate of tumor growth inhibition.

Calculation of TGI (%):

TGI (%) - [1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group)/(average tumor volume at the end of treatment of the solvent control group-average tumor volume at the start of treatment of the solvent control group) ] was 100%.

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

T/C％＝T_RTV/C_RTV×100％(T_RTV: treatment group relative tumor volume; c_RTV: negative control group relative tumor volume). Calculating Relative Tumor Volume (RTV) according to the tumor measurement result, wherein the calculation formula is that RTV is V_t/V₀In which V is₀When administered in groups (i.e. d)₀) Measurement of the mean tumor volume, V_tIs the average tumor body at a certain measurementProduct, T_RTVAnd C_RTVThe same day data was taken.

Statistical analysis: statistical analysis, including mean and Standard Error (SEM) of tumor volume for each time point for each group. Treatment groups showed the best therapeutic effect on day 15 (REC-1 xenograft tumor model) and day 17 (TMD8 xenograft tumor model) after drug administration, respectively, and therefore statistical analysis was performed based on this data to assess differences between groups. Three or more comparisons were analyzed by one-way ANOVA and if F values were significantly different, the measurements were performed using the Games-Howell method. All data analyses were performed with SPSS 17.0. Significant differences were considered with p < 0.05.

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. 15 days after the start of the administration, the tumor volume of the tumor-bearing mice in the solvent control group reaches 3501mm³The volume of the ibrutinib 25mg/kg group tumor is 1323mm³Compared with a solvent control group, the compound has obvious tumor inhibition effect (T/C is 38 percent, TGI is 64 percent, and p is 0.008). The tumor volumes of Compound A15mg/kg and 30mg/kg groups were 1034 and 680mm, respectively³Compared with a solvent control group, the compound A has obvious tumor inhibition effect (the T/C values are respectively 30% and 19%, the TGI values are respectively 73% and 83%, and the p values are both less than 0.01), and compared with a positive drug ibrutinib 25mg/kg group, the compound A15mg/kg group has better tumor inhibition effect.

TABLE 7 evaluation of antitumor drug efficacy of Compound A on REC-1 xenograft tumor model (calculated based on tumor volume on day 15 post-administration)

Note: a. mean ± SEM; b. tumor growth inhibition is mediated by T/C and TGI (%) - [1- (T)₁₅-T₀)/(V₁₅-V₀)]X 100) calculation; c. the administration mode comprises the following steps: 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 figure 2. The tumor volume of the tumor-bearing mice in the solvent control group reaches 1852mm 17 days after the start of the administration³The volume of the Ibrutinib group tumor is 661mm³Compared with a solvent control group, the compound has obvious tumor inhibition effect (T/C is 35.68%, TGI is 68.18%, p is<0.001). The tumor volumes of the compound A5 mg/kg and 10mg/kg groups were 912mm³And 553mm³Compared with a solvent control group, the compound A has obvious tumor inhibition effect (T/C values are 49.27% and 29.85% respectively, TGI values are 53.78% and 74.35% respectively, and p values are less than 0.01), and compared with a positive drug ibrutinib 25mg/kg group, the compound A10mg/kg group has better tumor inhibition effect.

TABLE 8 evaluation of antitumor drug efficacy of Compound A against the TMD8 xenograft tumor model (calculated based on tumor volume on day 17 post-administration)

Note: a. mean ± SEM; b. tumor growth inhibition is mediated by T/C and TGI (%) - [1- (T)₁₇-T₀)/(V₁₇-V₀)]X 100) calculation; c. the administration mode comprises the following steps: once a day; p<0.01。

The results show that the compound A has obvious tumor growth inhibition activity in two BTK sensitive mouse transplantation tumor models, and is obviously superior to the first generation BTK inhibitor ibrutinib which is on the market at present.

In addition, the above experiment in the human lymphoma TMD8 xenograft tumor model was repeated using compound S18S, and the T/C (%) results are shown in the following table. In the following table, the T/C (%) results for Compound A are also shown for comparison.

TABLE 9 antitumor Effect of Compounds A and S18 on TMD8 xenograft tumor model

Note: the administration mode comprises the following steps: once a day.

As can be seen from the above data, at lower doses (10mg/kg), Compound A exhibited better tumor growth inhibition than Compound S18S (15 mg/kg).

Experimental example 4: evaluation of pharmacokinetic Properties of rat

14 SD rats, male, with a weight of 200- & gt 220g, were randomly divided into 4 groups of 4/3, and the test compounds were administered by gavage and intravenous administration, respectively, according to the following specific schedule in Table 10:

TABLE 10 methods of administration of test Compounds

Group of	Number of animals	Compound (I)	Route of administration	Administration dose (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: the intragastric administration is prepared by 0.5 percent sodium carboxymethylcellulose (CMC-Na) containing 1 percent Tween 80, and the concentration of the prepared medicine is 0.3 mg/mL; the intravenous administration was made into a solution with 5% DMSO/5% Tween 80/90% physiological saline, and the administration concentration was 0.2 mg/mL.

Fasted for 12 hours before the test, water was freely available. Meals were uniformly taken 2 hours after dosing.

Blood sampling time points and sample treatment:

intragastric administration: 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 hours post-administration;

intravenous administration: 5 minutes, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24 hours post-dose;

at the above set time points, 0.3mL of venous blood was taken from the retrobulbar venous plexus of rats, placed in heparinized tubes, centrifuged at 11000rpm for 5 minutes, plasma was separated and frozen in a-20 ℃ refrigerator.

Sample testing and data analysis

The concentration of compound a in rat plasma was determined by LC/MS method.

Pharmacokinetic parameters after administration were calculated using a non-compartmental model of WinNonlin 5.3 software (Pharsight, usa).

Peak concentration of C_maxAnd time to peak T_maxIs an actual measurement value;

area under the time curve AUC_0-tThe value: calculating by adopting a trapezoidal method;

AUC_0-∞＝AUC_0-t+C_t/k_e，

C_tthe blood concentration at the last measurable time point,

k_eto eliminate the rate constant;

elimination of half-life t_1/2＝0.693/k_e；

The mean residence time MRT is AUMC/AUC.

Clearance rate CL ═ D/AUC_0-∞(ii) a Steady state volume of distribution Vss ═ CL × MRT

Absolute bioavailability F ═ AUC (AUC)_{Gavage stomach}×D_Vein)/(AUC_Vein×D_{Gavage stomach})×100％

The results of the tests are shown in Table 11 below:

TABLE 11 results of pharmacokinetic experiments for different compounds

Note: the above pharmacokinetic data of S18S, S19S and S20S were extracted from "Yu Xue, et al.discovery of4,7-Diamino-5- (4-phenoxyphenyl) -6-methyenepyrido [5,4-b ] pyridoline as Novel Bruton' S Tyrosine Kinase inhibitors J.Med.chem.,2018,61,4608-4627.

The results show that the clearance rate of the compound A in rats is obviously lower than that of ibrutinib (20 times), and the drug exposure in blood plasma is 70 times higher than that of the ibrutinib after oral administration; the clearance rate in vivo of the compound A is also obviously higher than that of S18S, S19S and S20S, and the drug exposure amount in blood plasma after oral administration is obviously higher than that of S18S, S19S and S20S. Namely, compared with ibrutinib, S18S, S19S and S20S, the compound A has better oral administration performance and good oral bioavailability under the same dosage.

Therefore, the compound A is a BTK inhibitor which has novel structure, can be orally taken, has high selectivity and high activity, has obviously better in-vivo and in-vitro activity than the BTK inhibitor which is on the market at home and abroad at present, has obviously better tumor growth inhibition activity than a positive control drug ibrutinib under the same dosage, and has great development value.

The above embodiments are merely exemplary in nature and are not intended to limit the claimed embodiments or the application or uses of such embodiments. In this document, the term "exemplary" represents "as an example, instance, or illustration. Any exemplary embodiment herein is not necessarily to be construed as preferred or advantageous over other embodiments.

Claims

1. Use of compound a or a pharmaceutically acceptable salt thereof as a bruton's tyrosine kinase BTK inhibitor for the manufacture of a medicament for the treatment of a BTK related neoplastic disease, said compound a having the 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 lymphoma is a B cell lymphoma.

3. The use according to claim 1 or 2, wherein the BTK-related tumor disease comprises histiocytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, marginal zone lymphoma, follicular lymphoma, Burkitt's lymphoma, or fahrenheit macroglobulinemia.

4. Use according to claim 1 or 2, wherein the medicament further comprises one or more other targeted, chemotherapeutic agents.

5. Use according to claim 1 or 2, wherein the medicament is formulated as a clinically acceptable formulation, preferably an oral formulation, an injectable formulation, an external formulation.

6. The use according to claim 1 or 2, wherein the medicament comprises a therapeutically effective amount of compound a or a pharmaceutically acceptable salt thereof, preferably the therapeutically effective amount is from 0.001mg to 1000mg per day, more preferably from 0.01mg to 500mg per day, even more preferably from 0.1 mg to 200mg per day, even more preferably from 0.1 mg to 100mg per day, even more preferably from 0.5 mg to 50mg per day, even more preferably from 0.5 mg to 30mg per day, even more preferably from 0.5 mg to 20mg per day; administration can be single dose or divided dose.

7. A method of treating a BTK-related neoplastic disease, comprising administering to a subject or patient a medicament containing a therapeutically effective amount of compound a, or a pharmaceutically acceptable salt thereof, having the structure:

8. the method of claim 7, wherein the BTK-associated tumor disease is B-cell lymphoma.

9. The method of 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's lymphoma, or Waldenstrom's macroglobulinemia.

10. The method according to any one of claims 7 to 9, wherein the administration is oral, injectable, topical or in vitro, preferably oral or injectable.