CN108610295B

CN108610295B - Pyrimidines, compositions and their use in the treatment of lymphoma leukemia

Info

Publication number: CN108610295B
Application number: CN201810299255.0A
Authority: CN
Inventors: 袁宏; 李思; 赵婧媛; 吴向东; 王晓曦; 王楠; 姚冰洁; 赵丹; 姚瑶; 孙秀丽; 马晓东
Original assignee: Dalian Medical University; First Affiliated Hospital of Dalian Medical University
Current assignee: Dalian Medical University; First Affiliated Hospital of Dalian Medical University
Priority date: 2018-04-04
Filing date: 2018-04-04
Publication date: 2023-01-10
Anticipated expiration: 2038-04-04
Also published as: CN108610295A

Abstract

The invention relates to a pyrimidine compound, a composition and application thereof in treating lymphoma leukemia, wherein the pyrimidine compound is specifically a compound shown in a general formula (I), and each substituent group of the general formula (I) is defined in the specification. The invention also relates to the use of the compound shown in the general formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition containing the compound for treating tumor diseases by inhibiting Bruton's tyrosine kinase, in particular for treating Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma or chronic lymphocytic leukemia;

Description

Pyrimidines, compositions and their use in the treatment of lymphoma leukemia

Technical Field

The invention relates to a pyrimidine compound, a composition and application thereof in treating lymphoma leukemia, belonging to the technical field of medicines.

Background

Protein Tyrosine Kinases (PTKs) regulate a series of physiological and biochemical processes such as growth, differentiation and apoptosis of cells by controlling signal transduction pathways of the cells. Receptor-type tyrosine kinases are a class of relatively large kinases that span the cell membrane, having a ligand-binding extracellular domain, a transmembrane domain, and an intracellular domain that functions as a kinase-phosphorylating specific tyrosine residues and thereby affecting cell proliferation. Abnormal expression of the kinase has been found in common human cancers (e.g., lung, breast, stomach, ovarian, lymphoma). Protein tyrosine kinase has become one of the important targets for research and development of antitumor drugs.

Bruton's Tyrosine Kinase (BTK) is a cytoplasmic protein, a non-receptor protein tyrosine kinase Tec family, that is expressed in most hematopoietic cells, such as B cells, mast cells, megakaryocytes, etc., but is not expressed in T cells, NK cells, and plasma cells. In a BCR signal pathway, with the activation of BCR, BTK is activated depending on Syk and Lyn, and the activated BTK can further phosphorylate PLC gamma 2, thereby causing the activation of downstream signals including MAPK, NF kappa B and the like. BTK plays an irreplaceable role in the production of B lymphocytes. BTK can control the development and differentiation of B cells by activating cell cycle forward regulatory factors and differentiation factors, and can also control the survival and proliferation of B cells by regulating the expression of pro-apoptotic and anti-apoptotic proteins. Sustained activation of BTK is a prerequisite for the development of Chronic Lymphocytic Leukemia (CLL), and aberrant BCR-BTK signaling promotes survival of activated B-cell subsets in diffuse large B-cell lymphoma (DLBCL). BTK small molecule inhibitors have good prospects for treating hematological malignancies and autoimmune disorders. In view of the urgent need for cancer treatment, there is a need in the art to develop new drugs with better efficacy.

Disclosure of Invention

The invention aims to provide a pyrimidine compound or pharmaceutically acceptable salt thereof, wherein the pyrimidine compound has good antitumor activity.

The invention also aims to provide a pharmaceutical composition containing the pyrimidine compound or the pharmaceutically acceptable salt thereof.

Still another object of the present invention is to provide the use of the pyrimidine compound or the pharmaceutically acceptable salt thereof, or the composition.

To this end, in one aspect, the present invention provides a compound of formula (i) or a pharmaceutically acceptable salt thereof, wherein the compound of formula (i) has the following structure:

wherein the content of the first and second substances,

r is selected from hydrogen, chlorine, fluorine or trifluoromethyl;

R ₁ selected from hydrogen, methyl, fluorine or chlorine;

m is selected from

As some embodiments of the present invention, the compounds represented by the general formula (I) of the present invention have the structures represented by I-1 to I-20:

the structural compound is a pyrimidine compound, and the screening of the antitumor activity of the invention shows that the compound has stronger ability of inhibiting the proliferation of lymphocyte leukemia cells (Ramos, raji and NAMALWA), and part of the compound shows more excellent BTK resistance activity than Ibrutinib (Ibrutinib). As a molecule with novel structure, the compound has the potential of being developed into a novel high-efficiency BTK inhibitor, and has great application value for treating related tumor diseases, particularly Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma or chronic lymphocytic leukemia.

In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a compound represented by general formula (i) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition contains an effective dose of the structures shown in I-1 to I-20 or pharmaceutically acceptable salts thereof and a pharmaceutical carrier.

The compounds of the invention are preferably pharmaceutically acceptable salts of the compounds of formula (i) because of their potential use in medicine. The compounds of the present invention are bases, wherein the desired salt form can be prepared by suitable methods known in the art, including treatment of the free base with a mineral acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or treating the free base with an organic acid, such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid (pyranosidyl 1 acid), such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, and the like. Examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, chloride, bromide, iodide, acetate, propionate, caprate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propionate (propiolates), oxalate, malonate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, phenylacetate, phenylpropionate, phenylbutyrate (phenylbuterates), citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, mandelate and sulfonate, such as xylenesulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate and naphthalene-2-sulfonate.

The pharmaceutical compositions of the invention generally contain one compound of the invention. However, in some embodiments, a pharmaceutical composition of the invention may contain more than one compound of the invention. In addition, the pharmaceutical compositions of the present invention may optionally further comprise one or more other pharmaceutically active compounds.

The invention also provides the pyrimidine compound or the pharmaceutically acceptable salt thereof, and the application of the pharmaceutical composition in inhibiting tumor proliferation by inhibiting Bruton's tyrosine kinase. Specifically, the application is mainly the application in preparing the medicine for treating burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma or chronic lymphocytic leukemia.

The invention provides an application of a compound shown in the specification or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition in preparation of a Bruton's tyrosine kinase inhibitor.

The invention provides a compound shown in a general formula (I) or a pharmaceutically acceptable salt thereof, or application of a pharmaceutical composition in preparing a medicament for treating tumors. Preferably, the tumor is selected from burkitt's lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma or chronic lymphocytic leukemia, more preferably chronic lymphocytic leukemia. More preferably, the use is primarily through inhibition of bruton's tyrosine kinase.

Drawings

FIG. 1 is a graph showing the result of AO-EB staining experiments according to the embodiment of the present invention.

FIG. 2 is a graph showing the results of toxicity data of the compounds of the present invention on PBMCs.

FIG. 3 is an image of a gel obtained in an example of the present invention.

FIG. 4 is a graph showing the results of apoptosis in the examples of the present invention.

Detailed Description

The present invention is further described and explained below in conjunction with specific examples, which are not intended to limit the scope of the present invention.

The experimental method of the present invention, in which the specific conditions are not specified, is generally carried out under the conventional conditions or the conditions recommended by the manufacturers of the raw materials or the commercial products. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

EXAMPLE 1 preparation of target molecules

The thin layer chromatography silica gel plate is HSGF254 of tobacco yellow sea or GF254 of Qingdao, the silica-amine plate used in Thin Layer Chromatography (TLC) is 0.15-0.2 mm, and the thin layer chromatography separation and purification product is 0.4-0.5 mm.

The raw materials used in the present invention are mainly purchased from chemical reagents of national medicine group, beijing coupled technology, inc., aladdin chemical reagents, inc., darril Chemicals, etc.

In the examples, the solution means an aqueous solution unless otherwise specified.

In the examples, the reaction temperature is, unless otherwise specified, from 20 ℃ to 30 ℃ at room temperature.

The technical scheme adopted by the invention is as follows:

the synthetic route, reagent and condition of compound (I) are a) acryloyl chloride and NaHCO ₃ ，CH ₃ CN, rt,0.5 hour, 95%; b) Fe-NH ₄ Cl，MeOH-H ₂ O,2 hours, 70 ℃,72%; c) 2,4-dichloro-5-R-pyrimidine, DIPEA,1,4-dioxane, 60 ℃,2 hours, 91%; d) Trifluoroacetic acid, intermediate 5,2-BuOH,100 ℃,12 hours, 21% to 33%.

3 Synthesis of

1 (23.44 mmol) and NaHCO were taken ₃ (4.5g, 35.16mmol) is added into 50mL acetonitrile, acryloyl chloride (3.8g, 23.44mmol) is slowly added, the reaction is carried out at room temperature for 0.5 hour, 400mL water is added after the reaction is finished, white solid is precipitated, suction filtration and drying are carried out, so as to obtain white solid 2, the white solid 2 (19g, 68mmol) and ammonium chloride (7.3g, 136mmol) are taken into a reaction bottle, meOH (25 mL) and water (25 mL) are added, iron powder (15g, 272mmol) are added under stirring, the temperature is increased to 70 ℃ for reaction for 2 hours, suction filtration is carried out while the water phase is hot, ethyl acetate layer is combined, saturated common salt is washed once, anhydrous sodium sulfate is dried, and evaporation under reduced pressure is carried out, so as to obtain yellow white solid 3.

4 Synthesis of

3 (23.44 mmol) and DIPEA (4.5g, 35.16mmol) are taken to be put into 50mL1, 4-dioxane, 2,4-dichloro-5-R-pyrimidine (3.8g, 23.44mmol) is slowly added, the temperature is raised to 60 ℃, after 2 hours of reaction, the reaction is finished, the mixture is cooled, 400mL of water is added, yellow white solid is separated out, and the mixture is filtered, dried to obtain white-like solid which is directly reacted in the next step without purification.

Synthesis of object (I)

4 (23.44 mmol) and trifluoroacetic acid (4.5g, 35.16mmol) are taken to be put into 2-BuOH (50 ml), 5 (3.44 mmol) is slowly added, the temperature is raised to 100 ℃, after 12 hours of reaction, the reaction is finished, cooling is carried out, the mixture is poured into saturated sodium bicarbonate solution, solid is separated out, and the target molecule (I) is obtained after suction filtration, water washing, and silica gel column chromatography separation by drying.

The target molecule was synthesized according to the above method, and the physicochemical data of the synthesized target molecule were as follows:

(I-1) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.14(s,1H),9.39(s,1H),9.28(s,1H),8.26(d,J＝8.0Hz,1H),8.34(s,1H),7.84(s,1H),7.45–7.40(m,3H),7.31(dd,J＝16.0,8.0Hz,2H),6.81–6.76(m,2H),6.35(dd,J＝16.0,12.0Hz,1H),6.24(dd,J＝16.0,4.0Hz,1H),5.86(dd,J＝8.0,4.0Hz,1H),4.67(s,2H),4.60–4.53(m,1H),3.69(s,3H),1.48(d,J＝4.0Hz,3H)；HRMS(ESI)for C ₂₅ H ₂₅ ClN ₆ O ₅ ,[M+H]+ theoretical calculation 524.3576 and found 524.3506.

(I-2) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.20(s,1H),9.47(s,1H),9.29(s,1H),8.16(s,1H),7.87(s,1H),7.47–7.39(m,3H),7.34(dd,J＝16.0,8.0Hz,2H),6.78–6.66(m,2H),6.45(dd,J＝16.0,12.0Hz,1H),6.26(dd,J＝16.0,4.0Hz,1H),5.76(dd,J＝8.0,4.0Hz,1H),4.67(s,2H),4.36–4.31(m,1H),3.70–3.61(m,2H),3.59(s,3H),2.27–2.06(m,2H),1.98–1.90(m,1H),1.88–1.80(m,1H)；HRMS(ESI)for C ₂₇ H ₂₇ ClN ₆ O ₅ ,[M+H] ⁺ The theoretical calculation is 550.1081, and the actual calculation is 550.1371.

(I-3) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.18(s,1H),9.43(s,1H),9.11(s,1H),8.16(d,J＝18Hz,1H),8.0(s,1H),7.76(s,1H),7.49–7.36(m,3H),7.24(dd,J＝16.0,8.0Hz,2H),6.78–6.52(m,2H),6.35(dd,J＝16.0,12.0Hz,1H),6.25(dd,J＝16.0,4.0Hz,1H),5.84(dd,J＝8.0,4.0Hz,1H),4.84(s,2H),4.60–4.16(m,2H),3.67(s,3H)；HRMS(ESI)for C ₂₃ H ₂₂ ClN ₆ O ₅ ,[M+H] ⁺ The theoretical calculation is 497.546, and the actual measurement is 497.646.

(I-4) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.22(s,1H),9.47(s,1H),9.31(s,1H),8.38(s,1H),8.16(s,1H),8.11(s,1H),7.83(s,1H),7.54–7.49(m,3H),7.45(dd,J＝16.0,8.0Hz,2H),6.68–6.57(m,2H),6.43(dd,J＝16.0,12.0Hz,1H),6.23(dd,J＝16.0,4.0Hz,1H),5.65(dd,J＝8.0,4.0Hz,1H),4.89(s,2H),4.83–4.71(m,1H),4.60–4.50(m,1H),3.67(s,3H),1.58(d,J＝8.0Hz,3H),1.48(d,J＝7.0Hz,3H)；HRMS(ESI)for C ₂₈ H ₃₀ ClN ₇ O ₆ ,[M+H] ⁺ The theoretical calculation is 595.1681, and the actual calculation is 595.1381.

(I-5) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.27(s,1H),9.43(s,1H),9.18(s,1H),8.87(s,1H),8.37(s,1H),8.01(s,1H),7.78(s,1H),7.49–7.36(m,3H),7.23(dd,J＝16.0,8.0Hz,1H),7.18–7.08(m,4H),6.80(d,J＝12.0Hz,1H),6.78–6.66(m,2H),6.45(dd,J＝16.0,12.0Hz,1H),6.26(dd,J＝16.0,4.0Hz,1H),5.76(dd,J＝8.0,4.0Hz,1H),4.18(m,1H),4.89(s,2H),3.68(s,3H),3.18–2.81(m,2H)；HRMS(ESI)for C ₃₃ H ₂₉ Cl ₂ N ₇ O ₅ ,[M+H] ⁺ The theoretical calculation is 673.1607, and the actual measurement is 673.1397.

(I-6) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.36(s,1H),9.34(s,1H),9.23(s,1H),8.57(s,1H),7.65(s,1H),7.47–7.35(m,3H),7.29(dd,J＝16.0,8.0Hz,1H),6.78–6.66(m,2H),6.45(dd,J＝16.0,12.0Hz,1H),6.24(dd,J＝16.0,4.0Hz,1H),5.63(dd,J＝8.0,4.0Hz,1H),4.85(s,2H),4.39(m,1H),4.22(m,2H),3.73–3.58(m,2H),3.40(m,1H),2.70–2.49(m,2H),2.20(m,1H),1.50(m,3H)；HRMS(ESI)for C ₂₈ H ₂₈ Cl ₂ N ₆ O ₆ ,[M+H] ⁺ The theoretical calculation is 614.1447, and the actual measurement is 614.2387.

(I-7) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.20(s,1H),9.36(s,1H),9.27(s,1H),8.56(s,1H),8.36(s,1H),7.67(s,1H),7.57–7.43(m,3H),7.30(dd,J＝16.0,8.0Hz,1H),6.68–6.54(m,2H),6.40(dd,J＝16.0,12.0Hz,1H),6.20(dd,J＝16.0,4.0Hz,1H),5.73(dd,J＝8.0,4.0Hz,1H),4.93(s,2H),4.56(s,1H),4.28–4.08(m,2H),4.16(m,2H),2.50–2.40(m,1H),1.39(m,3H)；HRMS(ESI)for C ₂₆ H ₂₆ Cl ₂ N ₆ O ₆ [M+H] ⁺ The theoretical calculation is 588.1291, and the actual measurement is 588.1346.

(I-8) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.39(s,1H),9.20(s,1H),9.17(s,1H),8.80(s,1H),8.36(s,1H),8.16(s,1H),7.53(s,1H),7.43–7.29(m,3H),7.16(dd,J＝16.0,8.0Hz,1H),6.84–6.69(m,2H),6.40(dd,J＝16.0,12.0Hz,1H),6.15(dd,J＝16.0,4.0Hz,1H),5.60(dd,J＝8.0,4.0Hz,1H),4.36(d,J＝10.0Hz,2H),4.16–4.10(m,2H),3.30(s,2H),3.19–3.10(m,1H),2.31–2.21(m,2H),1.98–1.70(m,2H),1.64–1.53(m,2H),1.50–1.31(m,3H)；HRMS(ESI)for C ₃₀ H ₃₂ Cl ₂ N ₈ O ₅ ,[M+H] ⁺ The theoretical calculation is 654.1873, and the actual measurement is 654.1854.

(I-9) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.41(s,1H),9.59(s,1H),9.24(s,1H),8.78(s,1H),8.36(s,1H),8.15(s,1H),7.80(s,1H),7.46–7.30(m,3H),7.29(dd,J＝16.0,8.0Hz,1H),6.68–6.56(m,2H),6.40(dd,J＝16.0,12.0Hz,1H),6.23(dd,J＝16.0,4.0Hz,1H),5.56(dd,J＝8.0,4.0Hz,1H),4.60–4.51(m,1H),4.22–4.10(m,2H),3.39(s,2H),3.19–3.10(m,1H),2.31–2.10(m,2H),1.98–1.70(m,2H),1.65–1.43(m,2H),1.58(d,J＝5.0Hz,3H),1.32–1.20(m,3H)；HRMS(ESI)for C ₃₁ H ₃₄ ClN ₈ O ₅ ,[M+H] ⁺ The theoretical calculation is 652.2325, and the actual measurement is 652.1325.

(I-10) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.48(s,1H),9.46(s,1H),9.21(s,1H),8.70(s,1H),8.21(s,1H),8.10(s,1H),7.79(s,1H),7.43–7.30(m,3H),7.26(dd,J＝16.0,8.0Hz,1H),6.59–6.45(m,2H),6.30(dd,J＝16.0,12.0Hz,1H),6.21(dd,J＝16.0,4.0Hz,1H),5.50(dd,J＝8.0,4.0Hz,1H),4.18(d,J＝8.0Hz,2H),4.10–4.01(m,2H),3.50(s,2H),3.36–3.30(m,1H),3.19–3.10(m,1H),2.63–2.38(m,2H),2.50–2.36(m,1H),2.17–1.86(m,2H),1.60(s,3H)；HRMS(ESI)for C ₃₀ H ₃₂ ClN ₈ O ₆ ,[M+H] ⁺ The theoretical calculation is 654.2117, and the actual measurement is 654.2632.

(I-11) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.38(s,1H),9.45(s,1H),9.26(s,1H),8.57(s,1H),8.20(s,1H),8.08(s,1H),7.69(s,1H),7.53–7.36(m,3H),7.25(dd,J＝16.0,8.0Hz,1H),6.50–6.43(m,2H),6.30(dd,J＝16.0,12.0Hz,1H),6.11(dd,J＝16.0,4.0Hz,1H),5.65(dd,J＝8.0,4.0Hz,1H),4.61–4,51(m,1H),4.26–4.05(m,2H),4.12–4.02(m,2H),3.65–3.50(m,1H),3.39(s,2H),3.29–3.10(m,1H),2.33–2.28(m,2H),1.98–1.71(m,2H),1.69–1.59(m,2H),1.60(s,3H)；HRMS(ESI)for C ₃₁ H ₃₄ ClFN ₈ O ₆ ,[M+H] ⁺ The theoretical calculation is 668.2274, and the actual measurement is 668.2168.

(I-12) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.49(s,1H),9.49(s,1H),9.37(s,1H),8.55(s,1H),8.24(s,1H),7.97(s,1H),7.53–7.36(m,3H),7.25(dd,J＝16.0,8.0Hz,1H),6.50–6.43(m,2H),6.30(dd,J＝16.0,12.0Hz,1H),6.11(dd,J＝16.0,4.0Hz,1H),5.65(dd,J＝8.0,4.0Hz,1H),4.83(s,2H),4.69–4.60(m,1H),3.67(s,3H),1.58(d,J＝10.0Hz,3H)；HRMS(ESI)for C ₂₅ H ₂₄ ClFN ₆ O ₅ ,[M+H] ⁺ The theoretical calculation is 542.1481, and the actual measurement is 542.1283.

(I-13) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.58(s,1H),9.31(s,1H),9.27(s,1H),8.50(s,1H),8.32(s,1H),7.86(s,1H),7.58–7.44(m,3H),7.29(dd,J＝16.0,8.0Hz,1H),6.56–6.33(m,2H),6.28(dd,J＝16.0,12.0Hz,1H),6.18(dd,J＝16.0,4.0Hz,1H),5.85(dd,J＝8.0,4.0Hz,1H),4.89(s,2H),4.16–4.06(d,J＝10.0Hz,2H),3.69(s,3H),2.65(s,3H)；HRMS(ESI)for C ₂₆ H ₂₅ F ₃ N ₆ O ₅ ,[M+H] ⁺ The theoretical calculation is 558.1839, and the actual measurement is 558.1743.

(I-14) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.38(s,1H),9.45(s,1H),8.57(s,1H),8.20(s,1H),7.69(s,1H),7.53–7.36(m,3H),7.25(dd,J＝16.0,8.0Hz,1H),6.50–6.43(m,2H),6.30(dd,J＝16.0,12.0Hz,1H),6.11(dd,J＝16.0,4.0Hz,1H),5.65(dd,J＝8.0,4.0Hz,1H),4.89(s,2H),4.36–4.29(m,1H),3.60–3.51(m,2H),2.47–2.16(m,2H),2.18–1.98(m,2H),3.69(s,3H)；HRMS(ESI)for C ₂₈ H ₂₆ F ₄ N ₆ O ₅ ,[M+H] ⁺ The theoretical calculation is 602.1905, and the actual calculation is 602.1875.

(I-15) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.38(s,1H),9.76(s,1H),9.45(s,1H),9.08(s,1H),8.57(s,1H),8.20(s,1H),7.69(s,1H),7.53–7.36(m,3H),7.28–7.09(m,4H),7.25(dd,J＝16.0,8.0Hz,1H),6.80(m,1H),6.50–6.43(m,2H),6.30(dd,J＝16.0,12.0Hz,1H),6.11(dd,J＝16.0,4.0Hz,1H),5.65(dd,J＝8.0,4.0Hz,1H),4.91(m,1H),4.86(s,2H),3.76(s,3H),3.16–2.87(d,J＝2.4Hz,2H),2.45(s,3H)；HRMS(ESI)forC ₃₄ H ₃₂ FN ₇ O ₅ ,[M+H] ⁺ The theoretical calculation is 637.2449, and the actual measurement is 637.2345.

(I-16) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.28(s,1H),9.34(s,1H),9.20(s,1H),8.68(s,1H),7.59(s,1H),7.46–7.32(m,3H),7.27(dd,J＝16.0,8.0Hz,1H),6.85–6.76(m,2H),6.40(dd,J＝16.0,12.0Hz,1H),6.36(dd,J＝16.0,4.0Hz,1H),5.61(dd,J＝8.0,4.0Hz,1H),4.80(s,2H),4.54(m,1H),4.21(m,2H),3.78–3.50(m,2H),3.41(m,1H),2.78–2.45(m,2H),2.29(m,1H),1.59(m,3H)；HRMS(ESI)for C ₂₈ H ₂₈ F ₂ N ₆ O ₆ ,[M+H] ⁺ The theoretical calculation is 582.2038, and the actual measurement is 582.2435.

(I-17) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.28(s,1H),9.34(s,1H),9.27(s,1H),8.78(s,1H),8.49(s,1H),8.16(d,1H),8.0(s,1H),7.94(d,1H),7.49–7.36(m,3H),7.24(dd,J＝16.0,8.0Hz,2H),6.78–6.52(m,2H),6.35(dd,J＝16.0,12.0Hz,1H),6.25(dd,J＝16.0,4.0Hz,1H),5.84(dd,J＝8.0,4.0Hz,1H),4.16(d,2H),4.12–4.02(m,2H),3.30(s,2H),3.19–3.09(m,1H),2.30–2.20(m,2H),1.95–1.72(m,2H),1.65–1.55(m,2H),1.43–1.30(m,3H)；HRMS(ESI)for C ₃₁ H ₃₃ F ₃ N ₈ O ₅ ,[M+H] ⁺ The theoretical calculation is 654.2526, and the actual measurement is 654.2532.

(I-18) ¹ H NMR(400MHz,DMSO-d ₆ ):10.46(s,1H),9.56(s,1H),9.18(s,1H),8.70(s,1H),8.53(s,1H),8.10(s,1H),7.80(s,1H),7.48–7.35(m,3H),7.19(dd,J＝16.0,8.0Hz,1H),6.78–6.50(m,2H),6.43(dd,J＝16.0,12.0Hz,1H),6.21(dd,J＝16.0,4.0Hz,1H),5.43(dd,J＝8.0,4.0Hz,1H),4.60–4.53(m,1H),4.22–4.11(m,2H),3.39(s,2H),3.29–3.08(m,1H),2.51–2.39(m,2H),1.97–1.77(m,2H),1.65–1.43(m,2H),1.68(d,J＝5.0Hz,3H),1.42–1.30(m,3H)；HRMS(ESI)for C ₃₂ H ₃₄ ClF ₃ N ₉ O ₅ ,[M+H] ⁺ The theoretical calculation is 702.2293, and the actual measurement is 702.1412.

(I-19) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.22(s,1H),9.24(s,1H),8.76(s,1H),8.39(s,1H),8.24(d,1H),8.0(s,1H),7.89(d,1H),7.47–7.32(m,3H),7.20(dd,J＝16.0,8.0Hz,2H),6.76–6.52(m,2H),6.30(dd,J＝16.0,12.0Hz,1H),6.24(dd,J＝16.0,4.0Hz,1H),5.75(dd,J＝8.0,4.0Hz,1H),4.36(d,J＝10.0Hz,1H),4.16(d,J＝10.0Hz,2H),4.12–4.02(m,2H),3.32(m,1H),3.30(s,2H),3.28–3.18(m,1H),2.53–2.28(m,2H),2.11–1.88(m,2H),1.50(m,3H)；HRMS(ESI)for C ₃₀ H ₃₃ FN ₈ O ₆ ,[M+H] ⁺ The theoretical calculation is 620.2507, and the actual measurement is 620.2408.

(I-20) ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.28(s,1H),9.30(s,1H),9.11(s,1H),8.76(s,1H),8.35(s,1H),8.11(s,1H),7.89(s,1H),7.36–7.20(m,3H),7.16(dd,J＝16.0,8.0Hz,1H),6.69–6.40(m,2H),6.35(dd,J＝16.0,12.0Hz,1H),6.28(dd,J＝16.0,4.0Hz,1H),5.56(dd,J＝8.0,4.0Hz,1H),4.38(d,J＝8.0Hz,2H),4.18–4.08(m,2H),3.52(s,2H),3.56–3.41(m,1H),3.19–3.10(m,1H),2.63–2.38(m,2H),2.50–2.36(m,1H),2.17–1.86(m,2H),1.60(s,3H)；HRMS(ESI)for C ₃₁ H ₃₄ ClFN ₈ O ₆ ,[M+H] ⁺ The theoretical calculation is 668.2274, and the actual measurement is 668.2365.

Method for salifying target molecule

The preparation method of the inorganic acid salt comprises the following steps: dissolving a target molecule (1 mmol) in 10mL of anhydrous methanol, slowly dropwise adding a 5mL of anhydrous methanol solution of inorganic acid (1 mmol) in ice bath, stirring for 30 minutes at the temperature after dropwise adding, and then evaporating the methanol at normal temperature to obtain the inorganic acid salt of the target molecule.

The preparation method of the organic acid salt comprises the following steps: dissolving a target molecule (1 mmol) in 10mL of anhydrous methanol, slowly dropwise adding 5mL of dry ether of organic acid (1 mmol) in ice bath, stirring for 30 minutes at the temperature after dropwise adding, and then evaporating the solvent at normal temperature to obtain the organic acid salt of the target molecule.

Preparation of a mixture of two target molecules

And (3) putting the two target molecules with equal molar weight (1 mmol) into anhydrous methanol (5 mL), stirring for 10 minutes at room temperature, and evaporating the solvent at room temperature to obtain a mixture of the target molecules.

Example 2 evaluation of biological Activity of target molecule

1. Method for testing in vitro inhibitory activity of receptor tyrosine kinase

(1) Preparation of kinase assay buffer

(1) The Kinase Detection Buffer (Kinase Detection Buffer) was dissolved at room temperature and the presence or absence of the precipitate was observed.

(2) If precipitation occurs, the precipitate is dissolved by incubation (Kinase Detection Buffer) for 15 minutes at 37 ℃ and shaking frequently. Alternatively, the supernatant was carefully aspirated to remove the precipitate.

(2) Preparation of kinase assay reagent

(1) Kinase Detection buffers (Kinase Detection buffer) and (Kinase Detection Substrate) were equilibrated at room temperature prior to use.

(2) And (3) pouring the Kinase Detection Buffer solution (Kinase Detection Buffer) into a brown bottle filled with a Kinase Detection Substrate (Kinase Detection Substrate) to dissolve the freeze-dried powder Substrate, thus preparing the Kinase Detection reagent.

(3) Mix by gentle shaking, vortexing or inversion to form a homogeneous solution, and the substrate should be dissolved within 1 minute.

(4) The kinase detection reagent is used immediately after being prepared or is subpackaged at-20 ℃, and the activity of the circulating signal is not lost after the prepared reagent is frozen and thawed for several times.

(3) Standard Curve for conversion of ATP to ADP was generated

(1) The Ultra Pure ATP and ADP provided by the kit were diluted with 1 Xkinase reaction buffer (kinase reaction buffer) to make 900. Mu.L of 50. Mu.M ATP and 500. Mu.L of 50. Mu.M ADP.

(2) The 50 μ M ATP and 50 μ M ADP solutions prepared in the previous step were mixed in 384 well plates A1-A12 as shown in Table 1, simulating the concentration of ATP and ADP for each percent conversion, and mixed well.

TABLE 1 preparation of 50. Mu.M series of ATP + ADP standards

(3) Add 5. Mu.L of ADP-Glo per well ^TM Reagents to terminate the kinase reaction. Incubate at room temperature for 40 minutes.

(4) Add 10. Mu.L of Kinase Detection Reagent (Kinase Detection Reagent) per well to convert ADP to ATP and introduce luciferase and luciferin to detect ATP.

(5) Incubate at room temperature for 30-60 minutes, measure fluorescence with a multifunctional microplate reader and record the fluorescence value.

(6) Standard curves for conversion of ATP to ADP were plotted.

(4) IC to determine kinase inhibitors ₅₀ Value of

(1) 1 Xkinase reaction buffer (kinase reaction buffer), 2.5X 50 ng/. Mu.L kinase and 2.5X 0.5. Mu.g/. Mu.L substrate and 125. Mu.M ATP were prepared according to the Promega kit instructions.

(2) mu.L of 1 Xkinase reaction buffer (kinase reaction buffer), 2. Mu.L of 2.5X 0.5. Mu.g/. Mu.L substrate and 125. Mu.M ATP were added to the enzyme-free control wells. mu.L of 1 Xkinase reaction buffer (kinase reaction buffer), 2. Mu.L of 2.5X 50 ng/. Mu.L kinase, 2. Mu.L of 2.5X 0.5. Mu.g/. Mu.L substrate and 125. Mu.M ATP were added to the negative control wells. mu.L of 5 Xtest drug, 2. Mu.L of 2.5X 50 ng/. Mu.L kinase, 2. Mu.L of 2.5X 0.5. Mu.g/. Mu.L substrate and 125. Mu.M ATP are added to the test wells.

(3) The plates were mixed and incubated for 60 minutes.

(4) Add 5. Mu.L of ADP-Glo per well ^TM Reagents to terminate the kinase reaction. Incubate at room temperature for 40 minutes.

(5) Add 10. Mu.L of Kinase Detection Reagent (Kinase Detection Reagent) per well to convert ADP to ATP and introduce luciferase and luciferin to detect ATP. Incubate at room temperature for 30-60 minutes, measure fluorescence with a multifunctional microplate reader and record the fluorescence value.

(6) Results analysis, the results are shown in table 2.

2. Experiment for inhibiting BTK high expression cell growth (CCK-8 detection method)

(1) Cell type and selection: ramos cells (human Burkitt's lymphoma cells, high expression of BTK kinase), raji cells (human Burkitt's lymphoma cells, high expression of BTK kinase), NAMALWA cells (human Burkitt's lymphoma cells, high expression of BTK kinase).

(2) Cell inoculation: collecting cells in logarithmic growth phase, and adjusting cell suspension concentrationAt 5x10 per well ³ Each cell, 100 μ L per well volume, was seeded into 96-well plates, each set of 3 replicates (marginal wells filled with sterile PBS);

(3) Cell culture: after cell inoculation, the control group was incubated with 10% FBS RPMI-1640, the experimental groups were each intervened with 10. Mu.L of Ibrutinib (Ibrutinib) in different concentration gradients (1.25-40. Mu. Mol/L), with different drugs (1.25-40. Mu. Mol/L), 37 ℃,5% CO ₂ Continuously culturing in an incubator (culturing for different times according to experimental requirements);

(4) Color generation: adding 10 μ L CCK-8 solution (5 mg/mL) after culturing for 72h, terminating the culture after 4h, and oscillating on a shaker at low speed for 10min to fully dissolve crystals;

(5) Color comparison: the absorbance (OD) of each well was measured on an ELISA detector, the wavelength of 570nm was selected, and the absorbance of each well was measured using cell-free RPMl-1640 culture blank Kong Diaoling. The experiment was repeated three times;

(6) The results are recorded: cell growth inhibition rate = (control absorbance value-experimental absorbance value)/control absorbance value x 100%, cell proliferation rate = (experimental absorbance value/control absorbance value) × 100;

(7) Drawing a cell growth curve: the cell growth curve was plotted with time as abscissa and inhibition/proliferation rate as ordinate.

(8) Inhibitor concentrations were plotted in GraphPad Prism mapping software in GraphPad software to determine log [ inhibitor [ ]]Variable slope model estimation of IC versus response ₅₀ 。

The test results are shown in table 2, and table 2 shows the activity effect of the obtained compounds in inhibiting BTK tyrosine kinase and inhibiting proliferation of tumor cells.

TABLE 2

a:IC ₅₀ : ramos, raji, NAMALWA are typical B-lymphocyte leukemia cells, highly expressed by BTK kinase.

3. Acridine orange/ethidium bromide double-fluorescence staining (AO/EB)

(1) Cell culture: culturing NAMALWA cells in conventional scale-up manner, taking cells in logarithmic growth phase at 2x10 ⁵ cells/well density were seeded in 6-well plates;

(2) And (3) drug treatment: well-adherent cells were treated with different concentrations of drug (inhibitor) for 48 hours the following day; the specific drugs and the use concentrations are shown in the following table 3:

TABLE 3

(3) AO-EB staining: the culture medium was discarded, washed twice with PBS, each well was stained with 20. Mu.l of AO-EB solution (1. Mu.g/mL AO + 1. Mu.g/mL EB in PBS), and the change of apoptosis, death, etc. at each stage of the cells was observed under an inverted fluorescence microscope and photographed, and the results are shown in FIG. 1.

4. Cytotoxicity assays (PBMC assays)

Peripheral Blood Mononuclear Cells (PBMC) comprise lymphocytes, monocytes, dendritic cells and other small numbers of cells (hematopoietic stem cells, etc.). Toxicity tests can prove whether the medicine has lethality to normal immune cells. At present, the common method for separating PBMC at home and abroad is a dextran-diatrizoate density gradient centrifugation method, and the experimental steps are as follows:

(1) Blood collection and dilution: taking 2ml of blood from vein, adding into test tube containing ACD anticoagulation solution, and mixing to anticoagulate blood. Diluting the anticoagulated blood by one time with PBS;

(2) Sample adding: sucking 2ml of lymphocyte layering liquid (Tianjin TBD) and placing the lymphocyte layering liquid into a graduated centrifuge tube, inclining the centrifuge tube by an angle of 45 degrees, slowly adding diluted whole blood onto the separating liquid along the tube wall by using a capillary dropper, and keeping the interface between the lymphocyte layering liquid and the separating liquid clear;

(3) Centrifuging: centrifuging at 2000r/min for 20min at 18-20 deg.c in a horizontal centrifuge, and separating into four layers including red blood cell and granulocyte layer, layered liquid layer, mononuclear cell layer and plasma layer;

(4) And (3) recovering: the turbid strip was gently inserted with a capillary pipette, and the cell layer was gently aspirated along the tube wall and transferred to another centrifuge tube. Not only sucking all mononuclear cells, but also avoiding sucking excessive layering liquid and blood plasma so as to avoid mixing other cell components;

(5) Washing: cells were washed three times with PBS. The first time is 2000r/min and 10min; 1500r/min for 2-3 times, 10min, most mixed platelets can be removed;

(6) Suspending the pelleted cells in culture medium for use;

(7) Counting and paving: the concentration of the cell suspension was adjusted to 2.5X 10 per well ⁵ Each cell, 400 mu L of each pore volume is inoculated in a 24-pore plate, and each group is provided with two multiple pores;

(8) Cell culture: after cell inoculation, the control group was incubated with 10% FBS RPMI-1640, the experimental groups were respectively intervened with 50. Mu.L of Ibrutinib (Ibrutinib) at different concentration gradients (5-20. Mu. Mol/L), active drug (5-20. Mu. Mol/L), 37 ℃,5% CO ₂ Continuously culturing in an incubator (culturing for different times according to experimental requirements);

(9) Dyeing: after 24h incubation, 20. Mu.L of 1. Mu.g/. Mu.L AO (acridine orange), 20. Mu.L of 1. Mu.g/. Mu.L PI (propidium iodide) were added for staining for 5min, observed under an inverted fluorescence microscope and photographed;

the results of the test are shown in FIG. 2, and FIG. 2 is toxicity data of the compound to PBMC.

5. Western Blotting (Western Blotting)

5.1 extraction of Total cellular protein

1. After the NA cells were expanded, the cells were treated with the following conventional drugs as shown in Table 4, for 48 hours:

TABLE 4

2. After 48 hours, washing the cells twice with PBS, adding 150ul of protein lysate, vortexing on a vortexing instrument for 8-10 times, and lysing on ice for 35-40min;

3. after completion of lysis, centrifugation was carried out at 12000rpm at 4 ℃ for 15min;

4. determination of protein concentration: a Biyuntian Bradford protein concentration determination kit is adopted;

5. adding the centrifuged supernatant into the sample buffer, keeping at 100 deg.C for 5min, and storing at-80 deg.C.

5.2 quantification of Total cellular protein (Bradford method)

1. Making a standard curve

(1) 1mg/ml BSA was taken out from-20 ℃ and thawed at room temperature for use.

(2) 6 1.5ml centrifuge tubes were labeled 0. Mu.g, 2.5. Mu.g, 5.0. Mu.g, 10.0. Mu.g, 20.0. Mu.g, and 40.0. Mu.g, respectively.

(3) The reagents were added to each tube as in Table 5 below.

TABLE 5

Pipe pin

0μg

2.5μg

5.0μg

10.0μg

20.0μg

40.0μg

1mg/ml BSA

－

2.5μl

5.0μl

10.0μl

20.0μl

40.0μl

0.15mol/L NaCl

100μl

97.5μl

95.0μl

90.0μl

80.0μl

60.0μl

Coomassie Brilliant blue solution G250

1ml

(4) After mixing, the mixture was left at room temperature for 2min. Adding each group of samples into a 96-well plate, wherein each hole is 200ul, each concentration is provided with three multiple holes, and determining OD value at 595nm of an enzyme labeling instrument to prepare a standard curve.

2. Detecting the protein content of a sample

(1) A sufficient amount of 1.5ml centrifuge tubes were taken and 1ml of Coomassie Brilliant blue solution stored at 4 ℃ was added to each tube. Standing at room temperature for 30min to detect protein.

(2) One tube of Coomassie brilliant blue is taken, 100 mu L of 0.15mol/L NaCl solution is added, the mixture is uniformly mixed and placed for 2 minutes to be used as a blank sample, the OD value is determined by the method, the experiment is repeated for three times, and the average value is taken.

(3) A tube of Coomassie brilliant blue is taken, 95 mu L of 0.15mol/L NaCl solution and 5 mu L of protein sample to be detected are added, mixed uniformly and then are kept stand for 2min, the OD value is determined by the method, the experiment is repeated three times, and the average value is taken.

(4) And (5) obtaining the protein concentration of the sample to be detected according to the standard curve and the sample absorbance conversion.

5.3 sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)

After the protein content is measured, the volume of the solution containing 100 mug of protein is calculated to be the sample loading amount. The sample was removed to a 0.5ml centrifuge tube and 5 XSDS loading buffer was added to a final concentration of 1X. (the total volume of the sample is generally not more than 15 μ l, and 20 μ l of sample can be added to the sample well at the maximum.) before the sample is loaded, the sample is boiled in boiling water for 5min to denature the protein, and then the sample is placed on ice for standby.

1. Glue pouring: the glass plate was first cleaned before potting: one hand clasps the glass plate and the other hand dips in the detergent and gently scrubs it. Washing the two sides with tap water repeatedly until no water is hung, washing with distilled water, standing, and air drying.

(1) And (4) after aligning, putting the glass plate into a glue making frame for clamping. The card is then vertically mounted on a rack ready for potting. (the two glasses are aligned to avoid glue leakage in operation.)

(2) Preparing 10% separation gel, adding TEMED, immediately shaking up, and filling gel. When pouring the glue, a 10ml liquid-transfering gun can be used to suck 5ml of glue and discharge it along the glass at a certain speed, and when the glue surface rises to the height of the middle line of the green band, the glue can be poured out. Then a layer of water is added on the glue surface, and the gelation speed after liquid sealing is greatly improved. ( The glue filling can be started faster, and the speed is slowed down when the glue surface is as high as required. The glue must flow down the glass plate during operation to avoid bubbles in the glue. The sealing speed is slow when water is added, otherwise the separation gel is scattered and deformed. )

(3) When a line of refraction occurs between the water and the gel, it is suggested that the separation gel has solidified. Wait for another 3min to fully solidify the glue, pour off the upper liquid seal water, and carefully suck the water dry with filter paper, taking care not to touch the glue surface.

(4) 4 percent of concentrated glue is prepared, and the glue can be filled after being immediately shaken up after TEMED is added. The remaining space was filled with the gel concentrate and a comb was then inserted into the gel concentrate. The glue is also poured down the glass plate to avoid air bubbles. When the comb is inserted, the comb is kept horizontal as much as possible. Since the volume of the gel is reduced by shrinkage during solidification, the sample loading volume of the sample loading hole is reduced, and therefore, the gel is frequently supplemented on two sides in the solidification process of the concentrated gel. After the concentrated gel is solidified, the two hands respectively hold the two sides of the comb to vertically and upwards slightly pull out the comb.

(5) The concentrated gel was washed with water and placed in an electrophoresis tank. (Small glass facing inwards, big glass facing outwards.)

2. Sample application

(1) Sufficient running buffer is added to the running bath to prepare for loading. (the electrophoretic fluid should submerge at least the upper edge of the inner small glass.)

(2) The pretreated protein sample is taken out, and the sample is sucked by a microsyringe in an adherent way, taking care not to suck air bubbles. Insert the applicator needle into the well and add the sample slowly. ( Too fast loading may cause the sample to rush out of the loading well, and may cause the sample to overflow if there are air bubbles. The sample injector was washed 3 times in the external bath running buffer for the next sample addition to avoid cross contamination. )

3. Electrophoresis

(1) The power supply is plugged in, and the direction of the joint electrode is noted.

(2) Setting a constant voltage of 60V (6V/cm), increasing the voltage to 100-120V (15V/cm) for about 30min after the leading edge of the bromophenol blue dye enters the separation gel, continuing electrophoresis until the bromophenol blue reaches about 1cm above the bottom of the separation gel, turning off the power supply, and terminating electrophoresis.

5.4 transfer film

1. Preparation work

(1) For one membrane, 6 pieces of 7.0-8.3cm filter paper and 1 piece of 7.3-8.6cm PVDF membrane were prepared. Gloves are worn when the filter paper and the membrane are cut, so that the membrane is prevented from being polluted by hand protein. The cut PVDF membrane is marked and is soaked in water for 2 hours. (holding one side of the membrane gently in a dish with ultrapure water with tweezers, so that the membrane floats on water, only the lower layer comes into contact with water.

(2) The clip for transferring the membrane, two sponge pads, a glass rod, filter paper and the soaked membrane were placed in an enamel dish with transfer buffer.

(3) The clamp is opened to keep the black side horizontal. A spongy cushion is arranged on the upper surface of the mattress, and a glass rod is used for rolling back and forth for several times to roll away air bubbles inside the mattress. (one hand is rolled and the other hand is pressed to press the pad so that the pad cannot move freely.) three layers of filter paper are laid on the pad (three pieces of paper can be stacked on the pad first), the filter paper is fixed by one hand, and air bubbles are rolled by a glass rod.

2. Glue taking device

(1) The glass plate was carefully removed by gently prying it apart on both sides. The concentrated gel is slightly cut off (the concentrated gel influences the operation) to avoid scraping the separation gel, and an oblique angle is cut at the upper left corner of the separation gel to be used as a mark.

(2) The gel was equilibrated by immersion in transfer buffer.

3. Rotary film

(1) And covering the balanced separation gel on filter paper, aligning the separation gel with the filter paper by hand adjustment, and slightly rolling bubbles by using a glass rod. The film was covered with glue, the whole glue was covered (the film was not allowed to move after covering) and the air bubbles were removed. The membrane was covered with 3 sheets of filter paper and the air bubbles were removed. Finally, another spongy cushion is covered, and the clamp can be folded after rolling for a few times. The whole operation is carried out in the transfer liquid, and air bubbles are continuously rolled out. The filter papers on both sides of the membrane do not touch each other and short circuits occur after contact. (the transfer liquid contains methanol, gloves are worn during operation, and the air circulation of the experimental space is kept.)

(2) The clip is placed in the transfer tank so that the black side of the clip faces the black side of the tank and the white side of the clip faces the red side of the tank. When the electricity is transferred, heat is generated, and ice blocks can be placed at one side of the groove to reduce the temperature (100V/100 mA).

(3) After the transfer of the membrane, the membrane was stained with 1 XLichun red stain for 5min (shaking on a decolorizing shaker). The membrane proteins were then observed by rinsing the unstained dye solution with water. And drying the membrane for later use.

5.5 immune response

1. Sealing of

(1) A confining liquid (5% skim milk in TBST) was prepared.

(2) The membrane was soaked with TBST from bottom to top, transferred to a dish containing blocking solution, shaken slowly on a decolorization shaker at room temperature, and blocked at room temperature for 2 hours.

2. Anti-bonding

(1) Preparing a primary anti-dilution solution: BSAin TBST 5%, pH 7.4 was adjusted to ensure that the antigen-antibody reaction was carried out under appropriate pH conditions).

(2) The membranes were removed from the blocking solution, washed once with TBST, immersed in hybridization cassettes containing primary antibody (see Table 6 below), placed on a shaker at 4 ℃ and incubated overnight. The phosphorylated antibody is incubated for at least 12 hours. Other antibodies may also be incubated for 2-3h at room temperature.

TABLE 6

First antibody name	Source	Dilution factor	Second antibody
				BTK primary antibody	Bioworld Technology	1:300	1:6000
p-BTK primary antibody	Bioworld Technology	1:300	1:6000

3. Binding of secondary antibody

(1) After the primary antibody incubation was completed, the primary antibody was recovered and stored at-20 ℃. PVDF membrane at TBST room temperature decolorizing shaking table washing three times, each time 10min.

(2) The corresponding secondary antibody is selected according to the source of the primary antibody, and the secondary antibody is diluted with blocking solution in a certain proportion (1.

4. Chemiluminescence, development, and fixation

(1) After the secondary antibody incubation is finished, the secondary antibody can be recycled and discarded after being used twice. The membrane was washed three times with TBST on a decolorizing shaker at room temperature for 10min each time.

(2) Preparing an exposure box, a color developing solution, a preservative film and the like.

(3) Mixing ECL-A and ECL-B reagents in equal volume on a preservative film; after 1min, the PVDF membrane protein face is downwards contacted with the mixed solution fully; after 1min, the film was transferred to another preservative film, the residual liquid was removed, wrapped and placed in an X-ray film holder.

(4) In a dark room, 1 × developing solution and fixing solution are poured into a plastic tray respectively; taking out the X-ray film under a red light, and cutting the film into a proper size (the size is 1cm larger than the length and the width of the film) by using a paper cutter; opening the X-ray film holder, placing the X-ray film on the film, once the X-ray film is placed on the film, the X-ray film holder can not move any more, closing the X-ray film holder, and starting timing; properly adjusting the exposure time according to the intensity of the signal, generally 1min or 5min, and optionally pressing for multiple times at different times to obtain the best effect; after exposure, opening the X-ray film clamp, taking out the X-ray film, quickly immersing the X-ray film in a developing solution for development, and stopping development immediately after an obvious strip appears. The developing time is generally 1-2min (20-25 ℃), and when the temperature is too low (lower than 16 ℃), the developing time needs to be properly prolonged; after the development is finished, immediately immersing the X-ray film into the fixing solution, wherein the fixing time is generally 5-10min, and the film is transparent; after washing off the residual fixer with tap water, the plate was dried at room temperature. ( Note that: when the film needs to be moved for development and fixation, one corner of the film is taken as much as possible, the fingernail does not scratch the film, and otherwise the result is influenced. )

5. Gel image analysis: the film was scanned or photographed, and the molecular weight and optical density values of the target band were analyzed by gel image analysis software (Quantity One), and the results are shown in fig. 3.

6. Apoptosis assay

(1) And (3) cell culture: culturing NAMALWA cells in conventional scale-up manner, taking cells in logarithmic growth phase at 2x10 ⁵ cells/well density were seeded in 6-well plates;

(2) And (3) drug treatment: the next day, well-adherent cells were treated with different concentrations of the drug (inhibitor) for 48 hours, with the specific drug and use concentrations as in table 7;

TABLE 7

(3) Cell collection: the cells after drug treatment are digested and collected by pancreatin without EDTA, the cells are washed twice by PBS, and the total number of the collected cells in each treatment group is not less than 1 × 10 ⁵ A plurality of;

(4) And (3) detecting apoptosis: adding 5uL annexin V-FITC into the fixed cells, uniformly mixing, and then adding 5uL PI; reacting for 10min at room temperature in a dark place; apoptosis was detected by flow cytometry (Ex = 488nm.

The biological activity results show that the molecule has stronger inhibition effect on BTK kinase, most compounds reach the activity level of nanomolar level, and nearly half of the compounds have effective inhibition concentration IC50 value less than 10nmol, which is equivalent to Ibrutinib (Ibrutinib). The results of anti-cell proliferation activity revealed that most of the compounds had very potent inhibitory effects on lymphocytic leukemia cells (Ramos, raji, NAMALWA), wherein the compounds I-2, I-5, I-7, I-8, I-10, I-12, I-14, I-15, I-16 also showed superior activity to Ibrutinib (Ibrutinib), and especially the effects of I-5, I-7, I-8, I-14, I-16 were unexpected by those skilled in the art. The AO/EB fluorescent double-staining method result shows that the I-2 acts on the lymphocyte leukemia cell NAMALWA cell and can cause the morphological change of cell apoptosis. Flow analysis results show that the I-2 can obviously increase the apoptosis rate of the lymphoblastic leukemia NAMALWA cells, is dose-dependent, and has better effect than Ibrutinib (Ibrutinib). Meanwhile, as shown in figure 2, the cytotoxicity of the compound I-2 is also obviously reduced compared with that of Ibrutinib (Ibrutinib), the toxicity of the medicine is reduced while the activity of the medicine is ensured, and the compound has potential medicinal value. Western blot experiments show that I-2 can remarkably inhibit expression of related proteins in a BTK signaling pathway, and the expression level of p-BTK is down-regulated along with the sequential increase of administration concentration, so that the dosage dependence is presented. The molecules are predicted to have the potential of being developed into novel high-efficiency BTK inhibitors, and have great application value in treating related tumor diseases, particularly Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma or chronic lymphocytic leukemia.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A compound represented by the formula (I-2) or a pharmaceutically acceptable salt thereof:

2. a pharmaceutical composition comprising an effective amount of a compound represented by the formula (i-2) or a pharmaceutically acceptable salt thereof as claimed in claim 1, and a pharmaceutically acceptable carrier.

3. Use of a compound represented by formula (i-2) or a pharmaceutically acceptable salt thereof as claimed in claim 1, or a pharmaceutical composition as claimed in claim 2, for the preparation of a bruton's tyrosine kinase inhibitor.

4. Use of a compound represented by formula (i-2) or a pharmaceutically acceptable salt thereof as defined in claim 1, or a pharmaceutical composition as defined in claim 2 for the preparation of a medicament for treating tumors.

5. The use of claim 4, wherein the tumor is selected from one or more of Burkitt's lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, or chronic lymphocytic leukemia.

6. The use of claim 5, wherein the tumor is chronic lymphocytic leukemia.

7. Use according to any one of claims 4 to 6, wherein said use is effected primarily by inhibiting Bruton's tyrosine kinase.