Covalent kinase inhibitor, preparation method, pharmaceutical composition and use

Abstract

Disclosed in the present invention are a covalent kinase inhibitor, a preparation method, a pharmaceutical composition and the use. The covalent kinase inhibitor has a structure as shown in formula (I), (II), (III), (IV) or (V), and further comprises a pharmaceutically acceptable salt of the covalent kinase inhibitor, which can efficiently and covalently bind to a target kinase, and simultaneously trigger the release of NO / NO2 —. Covalent binding can realize covalent inhibition of a sulfydryl-containing target kinase, and the released NO / NO2 — can further perform S-nitrosylation modification on a target kinase or a protein interacting with the target kinase, so as to inhibit the phosphorylation of the target kinase or the protein, prevent kinase signaling, and provide a synergistic effect. The covalent kinase inhibitor can act on a variety of kinases associated with malignant tumor proliferation, has good selectivity and a low off-target effect, significantly inhibits the growth of tumor cells at both cell and animal levels, and has no obvious adverse reaction. In addition, the preparation method is simple and universal, which is beneficial to the expansion of the parent nucleus structures of other multiple kinase inhibitors.

Description

Covalent kinase inhibitors, preparation methods, pharmaceutical compositions and applications Technical Field

[0001] The present invention relates to a method based on NO / NO2 — Kinase inhibitors of release-type covalent warheads, preparation methods, pharmaceutical compositions and applications, particularly a NO / NO2-based kinase inhibitor with synergistic effects — Kinase inhibitor with a releasable covalent warhead, preparation method, pharmaceutical composition and application. Background Art

[0002] NO is a water-soluble free radical gas in the body with redox properties, playing an important role in physiology and pathology. It plays an extremely important biological role in cardiovascular regulation, nerve regulation, and immune regulation. — It is one of the metabolites of NO in the body. It can be reduced to NO under ischemic and hypoxic conditions. In some cases, it can also be regarded as a type of NO donor. NO can react with protein cysteine ​​sulfhydryl groups to mediate protein cysteine ​​sulfhydryl nitrosylation (SNO), which is a very important post-translational modification. It is worth mentioning that SNO can also be converted from NO2 — Mediated occurrence.

[0003] NO / NO2 — Donor drugs generally refer to those composed of NO / NO2 — A prodrug formed by a donor and a related drug or an active compound through various connecting groups. Various structural types of NO donors have been discovered, such as nitrosothiol, nitrate, NO-metal complex (nitroprusside), furazan N-oxide, azodiol salt, etc. — Donors include 2-nitromethyl-1,3-diphenyl-2-en-1-one and the like.

[0004] Covalent kinase inhibitors irreversibly bind to target kinase residues (typically Cys) through covalent bonds, thereby exerting their target inhibitory function and achieving disease treatment. They generally contain a low-reactivity bonding functional group (warhead group) and a ligand. After the ligand locates the inhibitor at the corresponding site on the target kinase, the warhead group reacts with Cys to form a covalent bond.

[0005] Although NO / NO2 — Donor drugs and covalent inhibitors have experienced great development, but still face many shortcomings. For example, for NO / NO2 — For donor drugs, they trigger NO / NO2 — The release mechanism currently relies on chemical and biological means such as light control and sound control; NO / NO2 —The specificity and accuracy of the released NO / NO2 are low. — Due to its free diffusion nature, the target at the molecular level is uncertain. — It will act on multiple biological macromolecules and produce more side effects. — Donor drugs still need to explore NO / NO2 with higher specificity and better accuracy — Release mode.

[0006] While covalent inhibitors have a high target occupancy rate, this also results in a single mode of action and a lack of effective synergistic therapeutic mechanisms. Furthermore, existing covalent inhibitors also face drawbacks such as low bioavailability, high dosing frequency, high dosage, and a high susceptibility to target Cys mutation resistance. Therefore, exploring new synergistic mechanisms for covalent inhibitors may help enhance efficacy and, to a certain extent, overcome these drawbacks.

[0007] Summary of the Invention

[0008] Purpose of the invention: The first purpose of the present invention is to provide a NO / NO2 based — A kinase inhibitor with a releasable covalent warhead; a second purpose is to provide a method for preparing the covalent kinase inhibitor; a third purpose is to provide a pharmaceutical composition containing the covalent kinase inhibitor; and a fourth purpose is to provide an application of the covalent kinase inhibitor and its pharmaceutical composition.

[0009] Technical solution: The covalent kinase inhibitor of the present invention has a structure of Formula I, II, III, IV or V, and further includes a pharmaceutically acceptable salt thereof.

[0010] in:

[0011] R1 and R2 are selected from C1-C4 alkyl or R1, R2 and the nitrogen to which they are attached together form a 4-7 membered heterocyclic ring containing 1-2 nitrogen, oxygen, or sulfur atoms, and the ring carbon atoms are substituted with at least one hydrogen, hydroxyl, halogen, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, or hydroxyl-substituted C1-C4 alkyl;

[0012] The kinase inhibitor nucleus is selected from:

[0013] The present invention designs a novel NO / NO2 — A kinase inhibitor of a release-type covalent warhead, wherein the covalent kinase inhibitor having the structure of Formula I has 2-(α-methylene-α,β-unsaturated ketone)-azodiol salt warhead structure can covalently bind to the specific sulfhydryl group of protein kinase, and the covalent binding mediates the addition-elimination reaction to remove the O of the azodiol salt. 2 Protection, triggering NO release in situ in a very small range. The covalent kinase inhibitor with the structure of formula II has an α-nitromethyl-α, β-unsaturated ketone warhead structure and uses the same mechanism to mediate NO2 by covalent binding reaction. — Released in a very small range. Covalent kinase inhibitors with structures of formula III, IV, and V have a ring-strained nitro-substituted warhead structure, which, mediated by ring strain, undergoes a nucleophilic substitution reaction with the specific sulfhydryl group of protein kinase and releases NO2 in a very small range. — .

[0014] The present invention is to NO / NO2 — The releasable warhead groups have been newly designed, so that the kinase inhibitors covalently bound to these warhead groups can be subjected to nucleophilic attack in the presence of thiol-containing target proteins in vitro / in vivo, and then the azodiol salt fragment is removed through addition-elimination reaction. 2 Protection, release of NO, and release of NO2 by the same mechanism or ring strain mechanism — On the one hand, covalent binding can inhibit the target enzyme containing sulfhydryl groups, and on the other hand, the released NO / NO2 — It can further produce nitrosylation modification on target kinases and interacting kinases, inhibit their own phosphorylation, prevent kinase signal transduction, and exert a synergistic effect.

[0015] The compounds designed in this invention have good covalent binding triggering specificity, NO / NO2 — High release efficiency, good biological activity, and its covalent binding and NO / NO2 — The synergistic effect can effectively overcome the existing NO / NO2 — The insufficiency of donor drugs and covalent inhibitors. At the same time, due to the synergistic effect, the dosage of drugs can be reduced, thereby improving patient compliance, significantly improving safety and reducing toxic side effects.

[0016] In the design of covalent kinase inhibitors of Formula I, azodiolates have obvious advantages in terms of selective and targeted release of NO. On the one hand, azodiolates are very unstable under physiological conditions and can spontaneously release 1-2 molecules of NO with a half-life ranging from seconds to hours. On the other hand, the O 2 The O connected to the nitrogen onium ion is called O 1 , the O connected to the olefinic nitrogen atom is O 2 ) after protection, it can be converted into a stable prodrug; by removing O under specific conditions2 The protecting group is converted into an unstable azodiol anion, thereby achieving selective and targeted release of NO. At the same time, the covalent kinase inhibitor of formula II can also selectively and targetedly release NO2 through the same addition-elimination reaction mechanism. — In addition, the covalent kinase inhibitors of formula III, IV, and V are selective and targeted by ring tension. — release.

[0017] The present invention is based on covalent inhibition therapy and NO / NO2 — The application of donor drugs in the field of anti-tumor therapy utilizes the chemical group release advantage of the covalent inhibitor warhead group to obtain NO / NO2 based — A new class of azodiol salt drugs was designed. 2 Protection method: The prodrug deprotection is performed according to the mechanism of Formula 1 or Formula 2, and each HS-R3 is selected from one of the small molecule thiol compound glutathione, cysteine, and protein BTK, HER1, HER2, Fgfr4, HER3, HER4, and KRAS G12C.

[0018] Formula 1:

[0019] Formula 2:

[0020] Preferably, in the structure, R1 and R2 are selected from methyl, ethyl, or R1, R2 and the N to which they are connected together form a 5-6 membered heterocyclic ring, wherein the heterocyclic ring is a morpholine ring, a piperidine ring, or a tetrahydropyrrole ring, and the ring carbon atom is substituted by at least one hydrogen, hydroxyl, fluorine, chlorine, bromine, methyl, ethyl, trifluoromethyl, hydroxymethyl, or hydroxyethyl; NR1R2 is further preferably selected from dimethylamino, methylethylamino, diethylamino, morpholinyl, tetrahydropyrrolyl, 2-hydroxymethyltetrahydropyrrolyl, piperidinyl, and 4-hydroxypiperidinyl.

[0021] Specifically, the covalent kinase inhibitor is most preferably selected from any one of the following compounds:

[0022] Furthermore, the above-mentioned covalent kinase inhibitor forms a pharmaceutically acceptable salt with any of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, malic acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, ferulic acid or mandelic acid.

[0023] The method for preparing the covalent kinase inhibitor of the present invention comprises the following steps:

[0024] (1) When the covalent kinase inhibitor has the structural formula of Formula I or II,

[0025] (i) Compound 1 reacts in the presence of paraformaldehyde, triethylenediamine, and phenol to obtain intermediate 2;

[0026] (ii) intermediate 2 is reacted in the presence of p-toluenesulfonyl chloride and triethylamine to obtain intermediate 3;

[0027] (iii) intermediate 3 reacts with the corresponding azodicarbonium diolate or AgNO2 under the catalysis of 15-crown-5-ether to obtain the final product 4 or 5;

[0028] (2) When the covalent kinase inhibitor has the structural formula III,

[0029] (i) The kinase inhibitor core 6 reacts with 2,3-dibromopropionic acid under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to obtain compound 7;

[0030] (ii) Compound 7 reacts with nitromethane to obtain the final product 8;

[0031] (3) When the covalent kinase inhibitor has the structural formula IV,

[0032] (i) tert-Butyl 3-oxocyclobutylcarboxylate is reacted in the presence of hydroxylamine hydrochloride and sodium acetate to obtain oxime intermediate 10;

[0033] (ii) intermediate 10 reacts with urea peroxide and trifluoroacetic anhydride to obtain intermediate 11;

[0034] (iii) intermediate 11 reacts with the kinase inhibitor core 6 to give the final product 12;

[0035] (4) When the covalent kinase inhibitor has the structural formula of Formula V, the kinase inhibitor core 6 reacts with 2-bromo-1-(3,3-dinitroazetidine-1-yl)ethanone to obtain the final product 13;

[0036] Among them, R1, R2, and the kinase inhibitor core are defined as above;

[0037] The corresponding acid is reacted with the kinase inhibitor prepared by the above method to form a salt to obtain the pharmaceutically acceptable salt.

[0038] The covalent kinase inhibitors of the present invention and pharmaceutically acceptable carriers form the pharmaceutical compositions of the present invention. Common pharmaceutical preparations such as tablets, capsules, syrups, suspensions, and injections can be prepared by adding common pharmaceutical excipients such as flavorings, sweeteners, liquid / solid fillers, and diluents.

[0039] The covalent kinase inhibitors and pharmaceutical compositions of the present invention are used to prepare drugs for treating and / or preventing proliferative diseases, delaying the progression of proliferative diseases, alleviating the symptoms of proliferative diseases, and assisting in the treatment and management of proliferative diseases.

[0040] Preferably, the proliferative disease is selected from tumors, rheumatic diseases, chronic inflammation, and infectious mononucleosis.

[0041] More preferably, the proliferative disease is selected from:

[0042] Gastric cancer, colorectal cancer, lung cancer (such as lung adenocarcinoma), breast cancer, liver cancer, prostate cancer, thyroid cancer, pancreatic cancer, bladder cancer, kidney cancer, brain tumor, neck cancer, CNS (central nervous system) cancer, malignant glioma, myeloproliferative disease, atherosclerosis, leukemia, pulmonary fibrosis, lymphoma, rheumatic disease, chronic inflammatory disease, non-lymphoreticular tumors, cryoglobulinemia, papular mucinosis, familial splenic anemia, multiple myeloma, amyloidosis, solitary plasmacytoma, heavy chain disease, light chain disease, malignant lymphoma, chronic lymphocytic leukemia, monocytic leukemia, semi-molecular disease, primary macroglobulinemia, primary macroglobulinemia purpura, secondary benign monoclonal immunoglobulinemia Gammopathies, osteolytic lesions, acute lymphoblastic leukemia, lymphoblastoma, some non-Hodgkin lymphomas, Sézary syndrome, infectious mononucleosis, acute histiocytosis, hairy cell leukemia, Hodgkin lymphoma, colon cancer, rectal cancer, intestinal polyps, diverticulitis, colitis, pancreatitis, hepatitis, small cell lung cancer, neuroblastoma, neuroendocrine cell tumors, islet cell tumors, medullary thyroid carcinoma, melanoma, uterine cancer, chronic hepatitis, cirrhosis, ovarian cancer, retinoblastoma, cholecystitis, head and neck squamous cell carcinoma, digestive tract malignancies, non-small cell lung cancer, cervical cancer, testicular cancer, bladder cancer, myeloma, or bone malignancies (e.g., osteosarcoma).

[0043] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0044] 1. This type of covalent kinase inhibitor can effectively bind to the target enzyme and release NO / NO2 — On the one hand, covalent binding can inhibit the target enzyme containing sulfhydryl groups, and on the other hand, the released NO / NO2 —It can further produce nitrosylation modification on target kinases and interacting kinases, inhibit their autophosphorylation, block kinase signal transduction, and exert synergistic effects; it is also selective and reduces non-target inhibition;

[0045] 2. This type of covalent kinase inhibitor has significant inhibitory activity at the molecular, cellular, and animal levels, acting on a variety of enzymes associated with malignant proliferation and malignant proliferating cells, and inhibiting tumor growth in animals without causing significant adverse reactions;

[0046] 3. The preparation method of the compound is simple and universal, which is conducive to the expansion of various structures. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG1 is a mass spectrometry verification result of the covalent binding of compound I1 to Bruton's tyrosine kinase (BTK);

[0048] FIG2 is the mass spectrometry verification result of the covalent binding of I3 compound to Bruton's tyrosine kinase (BTK);

[0049] FIG3 shows the NO release results of compound I1 in BTK storage solution, rat liver microsomes, rat plasma, GSH solution, and the NO release results of compound I1 in B cell lymphoma Daudi cells;

[0050] FIG4 shows the stability test results of compounds I1-I6 in rat plasma and GSH solution;

[0051] FIG5 is the Kinact / Ki parameter test results of compound I1;

[0052] FIG6 is the kinase profile test results of compound I1;

[0053] FIG7 shows the results of the nude mouse experiment with compound I1;

[0054] FIG8 is the verification result of co-localization of NO release and Bruton's tyrosine kinase (BTK) in cells by compound I1;

[0055] FIG9 shows the results of NO released by compound I1 causing increased nitrosation and decreased phosphorylation of Bruton's tyrosine kinase (BTK) / inhibition of phosphorylation signal transduction. DETAILED DESCRIPTION

[0056] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0057] Example 1: Preparation of Compound I1

[0058] 1. Dissolve 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-D]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one (ibrutinib) (10 g, 0.023 mol) in 50 ml of 1,4-dioxane. Add paraformaldehyde (40.9 g, 0.454 mol), triethylenediamine (3.82 g, 0.034 mol), and phenol (1.6 g, 0.017 mol). React at 60°C for 3 days. Concentrate under reduced pressure to remove the solvent, then extract with ethyl acetate (50 mL). The organic layer is washed three times with water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1, v / v) to obtain V1 as a white solid. 1 H NMR(500MHz,Chloroform-d)δ8.25(s,1H),7.60–7.54(m,3H),7.40–7.32(m,3H),7.18(s,1 H),7.12–6.99(m,4H),6.98–6.92(m,3H),6.82(s,1H),5.76(d,J=12.5Hz,1H),5.59(d,J=12 .5Hz,1H),5.00(s,1H),4.37(t,J=5.5Hz,1H),4.28–4.14(m,3H),3.94(s,1H),3.87(s,1H) ,3.52(d,J=8.4Hz,3H),2.10(s,1H),2.01(s,1H),1.96(s,1H),1.91(s,1H).HRMS(ESI):m / z calcd.for C 26 H 26 N6O3,[M+H] + :471.2066,found:471.2061.

[0059] 2. Dissolve V1 (200 mg, 0.43 mmol) in 15 ml of dichloromethane, add p-toluenesulfonyl chloride (90 mg, 0.47 mmol) and triethylamine (18.3 mg, 0.18 mmol), and react at room temperature for 10 h under nitrogen protection. Then extract with ethyl acetate, and wash the organic layer with water and saturated brine three times each, dry over anhydrous sodium sulfate, and obtain V2 as a white solid by column chromatography (petroleum ether / ethyl acetate = 20 / 1, v / v). 1H NMR(500MHz,Chloroform-d)δ8.00(s,1H),7.81–7.75(m,3H),7.69–7.63(m,3H),7.40–7.3 0(m,5H),7.10(tt,J=7.6,2.0Hz,1H),7.04–6.94(m,7H),6.72(s,1H),5.84(d,J=12.3Hz,1H ),5.61(d,J=12.5Hz,1H),4.94(s,1H),4.49(s,2H),4.01(s,1H),3.94(s,1H),3.51(s,2H) ,2.43(t,J=1.0Hz,4H),2.08(s,1H),2.01(s,1H),1.96(s,1H),1.88(s,1H).HRMS(ESI):m / z calcd.forC 33 H 32 N6O5S,[M+H] + :625.2155,found:624.2151.

[0060] 3. The p-toluenesulfonyl chloride-substituted product V2 (300 mg, 0.48 mmol) was dissolved in 20 ml of DMSO, and dimethylaminoazolinium dialkoxide (305 mg, 2.40 mmol) was added. The reaction was carried out at room temperature under nitrogen for 10 h, and then extracted with ethyl acetate. The organic layer was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to obtain a white solid I1. 1 H NMR(500MHz,Chloroform-d)δ8.63(s,1H),7.69–7.63(m,3H),7.39–7.32(m ,3H),7.12–7.05(m,1H),7.08–6.96(m,7H),6.72(s,1H),5.84(d,J=12.4Hz, 1H),5.59(d,J=12.5Hz,1H),5.00(s,1H),4.53(d,J=0.9Hz,2H),3.94(s,1H) ,3.87(s,1H),3.52(d,J=6.2Hz,3H),2.96(s,6H),2.08(s,1H),2.01(s,1H), 1.96(s,1H),1.88(s,1H).HRMS(ESI):m / z calcd.forC 28 H 31 N9O4,[M+H] + :558.2499,found:558.2498.

[0061] Example 2: Preparation of Compound I2

[0062] Refer to the synthesis method of Example 1. 1 H NMR(500MHz,Chloroform-d)δ8.25(s,1H),7.69–7.63(m,3H),7.39–7.32(m,3H),7.12(tt,J=7.5,2.0 Hz,1H),7.04–6.75(m,7H),6.52(s,1H),5.64(d,J=12.4Hz,1H),5.59(d,J=12.5Hz,1H),4.86(s,1H),4 .53(d,J=12.3Hz,1H),4.32(d,J=12.3Hz,1H),3.78(s,1H),3.77(s,1H),3.71(s,2H),3.53(d,J=10.4 Hz,3H),2.93(s,3H),2.08(s,1H),2.01(s,1H),1.96(s,1H),1.88(s,1H),1.19(s,3H).HRMS(ESI):m / z calcd.forC 29 H 33 N9O4,[M+H] + :572.2656,found:572.2652.

[0063] Example 3: Preparation of Compound I3

[0064] Refer to the synthesis method of Example 1. 1 H NMR(500MHz,Chloroform-d)δ8.35(s,1H),7.89–7.83(m,3H),7.40–7.32(m,3H),7.12(tt,J=7.5,2.1Hz,1H ),7.13–6.99(m,7H),6.89(s,1H),5.84(d,J=12.4Hz,1H),5.65(d,J=12.5Hz,1H),5.15(s,1H),4.66(d,J=1 2.3Hz,1H),4.48(d,J=12.3Hz,1H),4.01(s,1H),3.94(s,1H),3.73(d,J=12.4Hz,2H),3.65(d,J=12.5Hz,2H ),3.53(d,J=10.4Hz,3H),2.08(s,1H),2.01(s,1H),1.96(s,1H),1.88(s,1H),1.17(s,6H).HRMS(ESI):m / z calcd.forC 30 H35 N9O4,[M+H] + :586.2812,found:586.2807.

[0065] Example 4: Preparation of Compound I4

[0066] Refer to the synthesis method of Example 1. 1 H NMR(500MHz,Chloroform-d)δ8.29(s,1H),7.69–7.63(m,3H),7.40–7.32(m,3H),7.09(tt,J=7.6 ,2.0Hz,1H),7.04–6.94(m,7H),6.72(s,1H),5.84(d,J=12.4Hz,1H),5.59(d,J=12.5Hz,1H),5.00 (s,1H),4.53(d,J=0.9Hz,2H),4.01(s,1H),3.94(s,1H),3.68(d,J=7.1Hz,5H),3.53(d,J=10.4H z,3H),3.29(d,J=8.4Hz,5H),2.08(s,1H),2.01(s,1H),1.96(s,1H),1.88(s,1H).HRMS(ESI):m / z calcd.for C 30 H 33 N9O5,[M+H] + :600.2605,found:600.2601.

[0067] Example 5: Preparation of Compound I5

[0068] Refer to the synthesis method of Example 1. 1H NMR(500MHz,Chloroform-d)δ8.25(s,1H),7.51–7.63(m,3H),7.40–7.28(m,3H),7.18(tt,J=7.5,2.0H z,1H),7.04–6.88(m,7H),6.66(s,1H),5.65(d,J=12.5Hz,1H),5.59(d,J=12.5Hz,1H),5.00(s,1H),4.5 3(d,J=12.3Hz,1H),4.48(d,J=12.3Hz,1H),4.01(s,1H),3.94(s,1H),3.53(d,J=10.4Hz,3H),3.27(d,J =2.4Hz,5H),2.08(s,1H),2.01(s,1H),1.96(s,1H),1.88(s,1H),1.83(d,J=3.7Hz,5H).HRMS(ESI):m / z calcd.for C 30 H 33 N9O4,[M+H] + :584.2656,found:584.2651.

[0069] Example 6: Preparation of Compound I6

[0070] Refer to the synthesis method of Example 1. 1 H NMR(500MHz,Chloroform-d)δ8.69(s,1H),7.69–7.63(m,3H),7.64–7.32(m,3H),7.23(tt,J=7.6,2.0Hz,1H ),7.14–6.92(m,7H),6.72(s,1H),5.84(d,J=12.4Hz,1H),5.59(d,J=12.5Hz,1H),5.25(s,1H),4.53(d,J=1 2.3Hz,1H),4.48(d,J=12.3Hz,1H),4.01(s,1H),3.94(s,1H),3.53(d,J=10.4Hz,3H),3.38(s,2H),3.31(s, 2H),2.08(s,1H),2.01(s,1H),1.96(s,1H),1.88(s,1H),1.78(d,J=2.6Hz,5H),1.60(s,2H).HRMS(ESI):m / z calcd.for C 31 H 35 N9O4,[M+H] + :598.2812,found:598.2808.

[0071] Example 7: Preparation of Compound I7

[0072] Referring to the synthesis method of Example 1, the p-toluenesulfonyl chloride substituted product V2 (300 mg, 0.48 mmol) was dissolved in 10 ml of anhydrous ether, and AgNO2 (369 mg, 2.40 mmol) was added. The reaction was carried out at room temperature in the dark under nitrogen protection for 10 h. Then the solvent was concentrated under reduced pressure and extracted with ethyl acetate. The organic layer was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to obtain I7 as a white solid. 1 H NMR(500MHz,Chloroform-d)δ8.66(s,1H),7.77–7.64(m,3H),7.47–7.39(m,3H),7.11–6.94(m,9H),6.72(s,1H),5.75(d,J=12.5Hz,1H),5 .58(d,J=12.3Hz,1H),5.15(s,1H),4.44(d,J=3.1Hz,3H),3.87(d,J=1.8Hz,3H),3.52(d,J=2.0Hz,3H),2.08(s,1H),2.01(s,1H),1.96(s, 1H),1.84(s,1H).HRMS(ESI):m / z calcd.for C 26 H 25 N7O4,[M+H]+:500.1968,found:500.1965.

[0073] Example 8: Preparation of Compound I8

[0074] 1. 3-(4-Phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine (ibrutinib intermediate) (10 g, 0.02587 mol) was dissolved in 50 ml of dichloromethane, and EDCI (9.91 g, 0.05174 mol), triethylamine (5.24 g, 0.05174 mol), and 2,3-dibromopropionic acid (5.99 g, 0.02587 mol) were added. The reaction was carried out at room temperature for 36 hours, and the solvent was removed by concentration under reduced pressure. The mixture was then extracted with ethyl acetate (100 mL). The organic layer was washed with water and saturated brine three times each, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain white solid V3. 1H NMR(500MHz,Chloroform-d)δ8.75(s,1H),7.68–7.61(m,3H),7.39–7.32(m,3 H),7.11–7.04(m,1H),7.08(s,2H),7.04–6.94(m,5H),6.72(s,1H),5.00(s,1H ),4.21(s,1H),4.01(s,1H),3.94(s,1H),3.81(d,J=1.4Hz,3H),3.47(d,J=11 .9Hz,2H),2.08(s,1H),2.01(s,1H),1.96(s,1H),1.92(s,1H).HRMS(ESI):m / z calcd.for C 25 H 24 Br2N6O2,[M+H] + :599.0327,found:599.0325.

[0075] 2. Dissolve V3 (10 g, 0.01666 mol) in 50 ml of ethanol, add nitromethane (1.5 g, 0.02499 mol), reflux for 24 hours, concentrate under reduced pressure to remove the solvent, and then extract with ethyl acetate (100 mL). The organic layer was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to obtain I8 as a white solid. 1 H NMR(500MHz,Chloroform-d)δ8.35(s,1H),7.60–7.54(m,2H),7.39–7.32(m,2H),7.11 –7.06(m,1H),7.08(s,1H),7.09–6.94(m,5H),6.81(s,1H),5.00(s,1H),4.49(s,1H), 4.12(s,1H),3.62(s,1H),3.47(d,J=1.1Hz,2H),3.40(s,1H),3.18(d,J=4.9Hz,1H),2 .68(d,J=5.1Hz,1H),2.08(s,1H),1.97(d,J=10.6Hz,2H),1.84(s,1H).HRMS(ESI):m / z calcd.for C 26 H 25 N7O4,[M+H] + :500.1968,found:500.1964.

[0076] Example 9: Preparation of Compound I9

[0077] 1. Dissolve tert-butyl 3-oxocyclobutylcarboxylate (1 g, 0.005875 mol), sodium acetate (1.60 g, 0.01175 mol), and hydroxylamine hydrochloride (0.8165 g, 0.01175 mol) in 50 ml of ethanol, reflux for 24 hours, and concentrate under reduced pressure to remove the solvent. Then, extract with ethyl acetate (100 mL). The organic layer was washed with water and saturated brine three times each, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 50 / 1, v / v) to obtain colorless oil V4. 1 H NMR(500MHz,Chloroform-d)δ2.82(s,1H),2.69(d,J=13.0Hz,2H),2.62(d,J=13.0Hz,2H),1.42(s,9H).HRMS(ESI):m / z calcd.for C9H 14 O3,[M+H] + :171.0943,found:171.0941.

[0078] 2. Dissolve V4 (500 mg, 2.94 mmol), trifluoroacetic anhydride (926.06 mg, 4.41 mmol), urea peroxide (414.85 mg, 4.41 mmol), and sodium dihydrogen phosphate (529.09 mg, 4.41 mmol) in anhydrous acetonitrile, reflux for 4 hours, filter, and concentrate under reduced pressure to remove the solvent in the filtrate. Then, extract with ethyl acetate (100 mL). The organic layer is washed three times with water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to obtain V5 as a white solid. 1 H NMR(500MHz,Chloroform-d)δ4.15(s,1H),2.78(d,J=13.0Hz,2H),2.69(d,J=13.0Hz,2H),2.50(s,1H).HRMS(ESI):m / z calcd.for C5H7NO4,[M+H] + :146.0375,found:146.0370.

[0079] 3. 3-(4-Phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine (ibrutinib intermediate) (10 g, 0.02587 mol) was dissolved in 50 ml of dichloromethane, and EDCI (9.91 g, 0.05174 mol), triethylamine (5.24 g, 0.05174 mol), and V5 (4.51 g, 0.031044) were added. The reaction was carried out at room temperature for 24 hours, and the solvent was removed by concentration under reduced pressure. The mixture was then extracted with ethyl acetate (100 mL). The organic layer was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 50 / 1, v / v) to obtain I9 as a white solid. 1 H NMR(500MHz,Chloroform-d)δ8.25(s,1H),7.60–7.54(m,3H),7.39–7.32(m,3H),7.1 2–7.05(m,1H),7.08(s,1H),7.05–6.95(m,5H),6.81(s,1H),5.00(s,1H),4.22(s,1H ),4.09(s,1H),3.59(s,1H),3.51(s,2H),2.69(d,J=13.0Hz,2H),2.64(s,1H),2.53( d,J=13.0Hz,2H),2.08(s,1H),2.01(s,1H),1.96(s,1H),1.92(s,1H).HRMS(ESI):m / z calcd.for C 27 H 27 N7O4,[M+H] + :514.2125,found:514.2119.

[0080] Example 10: Compound I 10 Preparation

[0081] 3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine (ibrutinib intermediate) (10 g, 0.02587 mol) was dissolved in 50 ml of dichloromethane and pre-cooled on an ice bath for 10 minutes. 2-Bromo-1-(3,3-dinitroazetidine-1-yl)ethanone (6.2 g, 0.02328 mol) was dissolved in 10 ml of dichloromethane and then added dropwise to the previous ibrutinib intermediate solution. After reacting in an ice bath for 30 minutes, the mixture was moved to room temperature and reacted for 2 hours. The solvent was removed by concentration under reduced pressure, and then extracted with ethyl acetate (100 mL). The organic layer was washed with water and saturated brine 3 times each, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 30 / 1, v / v) to obtain a white solid I 10 .1 H NMR(500MHz,Chloroform-d)δ8.71(s,1H),7.69–7.63(m,3H),7.40–7.32(m,3H),7.08 (tt,J=7.5,2.1Hz,1H),7.04–6.95(m,7H),6.72(s,1H),4.83(s,1H),3.56(s,1H),3.51 (s,2H),3.43(s,2H),3.18(d,J=12.4Hz,1H),3.13(d,J=12.3Hz,1H),3.06(s,1H),2.82 (s,1H),2.70(s,1H),2.00(s,1H),1.96(s,1H),1.82(d,J=12.5Hz,2H).HRMS(ESI):m / z calcd.for C 27 H 27 N9O6,[M+H] + :574.2084,found:574.2080.

[0082] Example 11: Preparation of Compound III

[0083] Refer to the synthesis method of Example 1. 1 H NMR (500MHz, Methanol-d4) δ7.90 (s, 1H), 7.63 (t, J = 8.4Hz, 1H), 7.51 (d, J = 9.1Hz, 1H), 7.29 (s, 1H), 5.57 (d, J = 105.3Hz, 1H), 4.9 7(s,2H),4.08(s,J=2.2Hz,3H),3.93(t,J=10.6Hz,2H),3.06(s,6H),2.13–2.07(m,3H),1.95(s,2H),1.45(s,2H).HRMS(ESI):m / z calcd.forC 26 H 28 Cl2FN7O5,[M+H] + :608.1513,found:608.1509.

[0084] Example 12: Preparation of Compound II2

[0085] Refer to the synthesis method of Example 1. 1H NMR(500MHz,Chloroform-d)δ8.68(s,1H),8.28(s,1H),7.45(s,1H),7.35( s,2H),5.57(s,1H),5.37(s,1H),5.00–4.89(m,2H),4.79(s,1H),4.04(s,3H ),3.93–3.87(m,2H),3.82–3.66(m,2H),3.36(q,J=7.1Hz,2H),2.96(s,3H), 2.08–2.03(m,2H),1.97–1.92(m,2H),1.13(t,J=7.1Hz,3H).HRMS(ESI):m / z calcd.for C 27 H 30 Cl2FN7O5,[M+H] + :622.1670,found:622.1670.

[0086] Example 13: Preparation of Compound II3

[0087] Refer to the synthesis method of Example 1. 1 H NMR(300MHz,Chloroform-d)δ8.62(s,1H),8.09(s,1H),8.00(s,1H),7.57–7.52(m,1H),7.39(d,J=9.5Hz,1H),5.56(s,1H),5.35(s,1H),4.96( s,2H),4.81(s,1H),4.01(s,3H),3.89(s,2H),3.67(s,2H),3.13(q,J=6.9Hz,4H),1.99(d,J=29.2Hz,4H),1.08(t,J=7.0Hz,6H).HRMS(ESI):m / z calcd.for C 28 H 32 Cl2FN7O5,[M+H] + :636.1826,found:636.1817.

[0088] Example 14: Preparation of Compound II4

[0089] Refer to the synthesis method of Example 1. 1H NMR(500MHz,Chloroform-d)δ8.68(s,1H),8.28(s,1H),7.44(s,1H),7.35(s,2H),5.57(s,1H),5.37(s,1H),4.96(s,2H),4.78(s,1H), 4.04(s,3H),3.91(t,J=9.7Hz,2H),3.85(t,J=4.8Hz,4H),3.76–3.65(m,2H),3.45(t,J=4.7Hz,4H),2.11–1.97(m,4H).HRMS(ESI):m / z calcd.for C 28 H 30 Cl2FN7O6,[M+H] + :650.1619,found:650.1619.

[0090] Example 15: Preparation of Compound II5

[0091] Refer to the synthesis method of Example 1. 1 H NMR(500MHz,Chloroform-d)δ8.41(s,1H),7.82(s,1H),7.63(t,J=8.2Hz,1H),7.46( dd,J=8.8,1.8Hz,1H),5.66(s,1H),5.45(s,1H),4.96(s,2H),4.87(dd,J=6.7,3.3Hz ,1H),4.04(s,3H),3.41–3.37(m,4H),3.37–3.30(m,4H),2.08(ddd,J=12.2,8.2,3.7 Hz,2H),1.73(p,J=5.8Hz,4H),1.58–1.50(m,2H),1.35–1.28(m,2H).HRMS(ESI):m / z calcd.for C 29 H 32 Cl2FN7O5,[M+H] + :648.1826,found:648.1826.

[0092] Example 16: Preparation of Compound II6

[0093] Refer to the synthesis method of Example 1. 1H NMR(500MHz,Chloroform-d)δ8.61(s,1H),7.95(t,J=8.5Hz,1H),7.69(s,1H),7.29(s,1H),7.24(dd,J=8.9,1.9Hz,1H),5.50(s,1H),5 .29(s,1H),4.82(s,1H),4.00(s,3H),3.73(q,J=7.0Hz,4H),3.68(s,9H),1.95(s,2H),1.93–1.87(m,2H),1.25(d,4H).HRMS(ESI):m / z calcd.for C 29 H 32 Cl2FN7O6,[M+H] + :664.1775,found:664.1764.

[0094] Example 17: Preparation of Compound II7

[0095] Refer to the synthesis method of Example 7. 1 H NMR(500MHz,Chloroform-d)δ8.76(s,1H),8.50(s,1H),7.62(d,J=7.5Hz,1H),7.50(d,J=7.5Hz,1H),7.41(s,1H),7.09(s,1H),5.76(d,J=12 .4Hz,1H),5.53(d,J=12.5Hz,1H),4.86(s,1H),4.45(s,2H),3.80(s,3H),3.49(s,2H),3.41(s,2H),2.18(s,2H),2.00(s,2H).HRMS(ESI):m / z calcd.for C 24 H 22 Cl2FN5O5,[M+H] + :550.0982,found:550.0978.

[0096] Example 18: Preparation of Compound II8

[0097] Refer to the synthesis method of Example 8. 1H NMR(500MHz,Chloroform-d)δ9.26(s,1H),8.50(s,1H),7.62(d,J=7.5Hz,1H),7.50(d,J=7.5Hz,1H),7.41(s,1H),7.09(s,1H),4.83(s,1H),4.5 3(s,1H),3.80(s,3H),3.49(s,2H),3.33(d,J=3.7Hz,4H),3.18(d,J=4.9 Hz,1H),2.68(d,J=5.1Hz,1H),2.13(s,2H),2.00(s,2H).HRMS(ESI):m / z calcd.for C 24 H 22 Cl2FN5O5,[M+H] + :550.0982,found:550.0979.

[0098] Example 19: Preparation of Compound II9

[0099] Refer to the synthesis method of Example 9. 1 H NMR(500MHz,Chloroform-d)δ9.35(s,1H),8.54(s,1H),7.62(d,J=7.5Hz,1H),7.50(d,J=7.5Hz,1H),7.37(s,1H),7.09(s,1H),4.83(s,1H ),4.22(s,1H),3.79(s,3H),3.51(s,2H),3.38(s,2H),2.69(d,J=13.0Hz,2H),2.57–2.50(m,3H),2.18(s,2H),2.03(s,2H).HRMS(ESI):m / z calcd.for C 25 H 24 Cl2FN5O5,[M+H] + :564.1139,found:564.1134.

[0100] Example 20: Compound II 10 Preparation

[0101] Refer to the synthesis method of Example 10. 1H NMR(500MHz,Chloroform-d)δ9.35(s,1H),8.54(s,1H),7.60(d,J=7.5Hz,1H),7.50(d,J=7.5Hz,1H),7.37(s,1H),7.09(s,1H),4. 83(s,1H),3.81(s,3H),3.51(s,2H),3.44(s,2H),3.17(s,2H),3.03(s,2H),2.75(s,2H),2.07(s,2H),2.03(s,2H).HRMS(ESI):m / z calcd.for C 25 H 24 Cl2FN7O7,[M+H] + :624.1098,found:624.1095.

[0102] Example 21: Preparation of Compound III1

[0103] Refer to the synthesis method of Example 1. 1 H NMR (500MHz, Chloroform-d) δ9.04 (s, 1H), 7.58 (d, J = 10.6Hz, 3H), 6.65 (s, 1H), 5. 84(s,1H),5.54(s,1H),4.86(d,J=2.5Hz,2H),4.51(d,J=7.8Hz,1H),4.28(s,1H),4 .08–3.99(m,2H),3.98(d,J=1.6Hz,6H),3.78–3.73(m,1H),3.61(td,J=12.0,2.6H z,1H),2.92(s,6H),2.10(d,J=12.9Hz,1H),1.95(d,J=12.1Hz,1H).HRMS(ESI):m / z calcd.for C 27 H 31 Cl2N7O6,[M+Na] + :642.1605,found:642.1598.

[0104] Example 22: Preparation of Compound III2

[0105] Refer to the synthesis method of Example 1. 1H NMR(500MHz,Chloroform-d)δ9.04(s,1H),7.59(d,J=7.8Hz,2H),7.55(d,J=8.8Hz,1H),6.65(s, 1H),5.79(s,1H),5.51(s,1H),4.98–4.88(m,2H),4.52–4.45(m,1H),4.26(s,1H),4.03(td,J=13. 4,12.7,7.4Hz,2H),3.98(d,J=1.8Hz,6H),3.75(d,J=11.9Hz,1H),3.65–3.57(m,1H),3.04(q,J= 7.1Hz,4H),2.12(d,J=13.7Hz,1H),1.93(d,J=12.6Hz,1H),1.01(t,J=7.1Hz,6H).HRMS(ESI):m / z calcd.for C 29 H 35 Cl2N7O6,[M+Na] + :670.1918,found:670.1915.

[0106] Example 23: Preparation of Compound III3

[0107] Refer to the synthesis method of Example 1. 1 H NMR(500MHz,Chloroform-d)δ9.05(s,1H),7.58(d,J=10.1Hz,3H),6.65(s,1H),5.82(s,1H) ,5.53(s,1H),4.87(s,2H),4.50(d,J=7.9Hz,1H),4.28(s,1H),4.04(dd,J=13.6,4.9Hz,2H), 3.97(d,J=1.5Hz,6H),3.77–3.73(m,1H),3.61(td,J=11.9,2.5Hz,1H),3.27(t,J=5.6Hz,4H) ,2.12–2.06(m,1H),1.98–1.92(m,1H),1.70–1.66(m,4H),1.47–1.43(m,2H).HRMS(ESI):m / z calcd.for C 30 H 35 Cl2N7O6,[M+Na] + :682.1918,found:670.1913.

[0108] Example 24: Preparation of Compound III4

[0109] Refer to the synthesis method of Example 1. 1 H NMR(300MHz,Chloroform-d)δ9.05(s,1H),7.65–7.52(m,3H),6.64(s,1H),5.81(s, 1H),5.53(s,1H),4.89(s,2H),4.55–4.42(m,1H),4.25(d,J=13.2Hz,1H),4.04(dd,J =10.3,3.9Hz,2H),3.98(s,6H),3.77(dd,J=5.9,3.6Hz,4H),3.61(td,J=11.9,2.5H z,1H),3.43–3.25(m,4H),2.12(d,J=12.6Hz,1H),2.06–1.82(m,2H).HRMS(ESI):m / z calcd.for C 29 H 33 Cl2N7O7,[M+Na] + :684.1711,found:684.1702.

[0110] Example 25: Preparation of Compound III5

[0111] Refer to the synthesis method of Example 1. 1 H NMR(300MHz,Chloroform-d)δ9.04(s,1H),7.60(s,3H),6.65(s,1H),5.96( s,1H),5.56(s,1H),4.85–4.67(m,2H),4.58(s,1H),4.34(s,1H),4.03(s,2H ),3.99(d,J=3.2Hz,6H),3.86(d,J=11.0Hz,1H),3.78(d,J=7.8Hz,1H),3.6 6(s,1H),3.63–3.51(m,2H),3.42(s,2H),2.10–1.75(m,7H).HRMS(ESI):m / z calcd.for C 30 H 35 Cl2N7O7,[M+Na] + :698.1867,found:698.1861.

[0112] Example 26: Preparation of Compound III6

[0113] Refer to the synthesis method of Example 1. 1H NMR(500MHz,Chloroform-d)δ9.06(s,1H),7.59(d,J=8.2Hz,3H),6.65(s,1H),5 .84(s,1H),5.54(s,1H),4.87(s,2H),4.54–4.49(m,1H),4.32–4.26(m,1H),4.07 –4.00(m,2H),3.98(s,6H),3.87–3.82(m,1H),3.75(d,J=11.8Hz,1H),3.64–3.59 (m,3H),3.23–3.18(m,2H),1.97–1.91(m,3H),1.74–1.66(m,4H).HRMS(ESI):m / z calcd.for C 30 H 35 Cl2N7O7,[M+Na] + :698.1867,found:698.1856.

[0114] Example 27: Preparation of Compound III7

[0115] Refer to the synthesis method of Example 7. 1 H NMR(500MHz,Chloroform-d)δ8.55(s,1H),7.94(s,1H),7.74(s,1H),7.65(s,1H),7.54 (s,1H),7.44(s,1H),6.79(s,1H),5.76(d,J=12.4Hz,1H),5.53(d,J=12.5Hz,1H),4.44 (d,J=12.3Hz,1H),4.35(d,J=12.5Hz,1H),4.16(s,1H),4.05(s,1H),3.99(s,1H),3.86 (s,6H),3.80(d,J=13.2Hz,2H),3.75(s,1H),2.07(s,1H),1.94(s,1H).HRMS(ESI):m / z calcd.for C 25 H 25 Cl2N5O6,[M+H] + :562.1182,found:562.1180.

[0116] Example 28: Preparation of Compound III8

[0117] Refer to the synthesis method of Example 8. 1H NMR(500MHz,Chloroform-d)δ8.55(s,1H),7.94(s,1H),7.74(s,1H),7.65(s,1H ),7.54(s,1H),7.09(s,1H),6.79(s,1H),4.58(s,1H),4.21(s,1H),3.98(s,1H), 3.92(s,1H),3.87(s,6H),3.81(d,J=3.1Hz,2H),3.75(s,1H),3.60(s,1H),2.82( d,J=4.9Hz,1H),2.75(d,J=4.9Hz,1H),2.07(s,1H),1.96(s,1H).HRMS(ESI):m / z calcd.for C 25 H 25 Cl2N5O6,[M+H] + :562.1182,found:562.1179.

[0118] Example 29: Preparation of Compound III9

[0119] Refer to the synthesis method of Example 9. 1 H NMR(500MHz,Chloroform-d)δ8.60(s,1H),7.94(s,1H),7.74(s,1H),7.63(s,1H) ,7.57(s,1H),7.12(s,1H),6.79(s,1H),4.22(s,1H),4.17(s,1H),3.99(s,1H),3 .89(s,6H),3.81(d,J=4.0Hz,2H),3.76(s,1H),3.72(s,1H),2.90(d,J=13.0Hz,2 H),2.58(s,1H),2.40(d,J=13.0Hz,2H),2.07(s,1H),1.94(s,1H).HRMS(ESI):m / z calcd.for C 26 H 27 Cl2N5O6,[M+H] + :576.1338,found:576.1334.

[0120] Example 30: Compound III 10 Preparation

[0121] Refer to the synthesis method of Example 10. 1H NMR(500MHz,Chloroform-d)δ8.60(s,1H),7.94(s,1H),7.65(s,1H),7.57(s,1H),7.27(s,1H),6.79(s,1H),4.06(s,1H),3.99(s,1H),3.91( s,6H),3.82(d,J=12.3Hz,1H),3.78–3.70(m,5H),3.62(s,1H),3.51(s,2H),3.44(s,2H),3.02(s,1H),1.86(d,J=8.1Hz,2H).HRMS(ESI):m / z calcd.for C 26 H 27 Cl2N7O8,[M+H] + :636.1298,found:636.1295.

[0122] Example 31: Preparation of Compound IV

[0123] Refer to the synthesis method of Example 10. 1 H NMR(500MHz,Chloroform-d)δ9.79(s,1H),8.27(d,J=4.9Hz,1H),7.84(d,J=0.9 Hz,2H),7.43–7.36(m,2H),7.24(d,J=6.6Hz,3H),7.09(d,J=17.7Hz,2H),6.99( s,1H),4.61(s,2H),3.82(d,J=12.4Hz,6H),3.69(d,J=12.5Hz,1H),3.57(d,J=1 2.5Hz,1H),3.43(s,4H),2.89(s,3H),2.74(s,2H),2.32(s,6H).HRMS(ESI):m / z calcd.for C 30 H 36 N 10 O6,[M+H] + :633.2814,found:633.2810.

[0124] Example 32: Preparation of Compound V

[0125] Refer to the synthesis method of Example 10. 1H NMR(500MHz,Chloroform-d)δ8.38(d,J=4.9Hz,1H),7.75(s,1H),7.31(t,J=7.5Hz,1H ),7.14(dd,J=7.5,2.0Hz,1H),7.06(d,J=4.9Hz,1H),6.88(dd,J=7.5,2.0Hz,1H),3.7 5(s,1H),3.61(s,2H),3.56(s,2H),3.24(s,1H),3.19(s,1H),3.09(d,J=0.9Hz,3H),2 .75(s,1H),2.57–2.49(m,3H),2.32(s,3H),1.33(s,3H),1.28(s,5H).HRMS(ESI):m / z calcd.for C 32 H 33 F2N9O7,[M+H] + :694.2471,found:694.2469.

[0126] Example 33: Covalent Binding Study of Compounds to Bruton's Tyrosine Kinase (BTK)

[0127] 1. Experimental methods

[0128] Covalent binding of I1 and I3 to Bruton's tyrosine kinase (BTK) was determined by mass spectrometry. The target protein was diluted to 2 μM in buffer, and then the test compound was added at varying volumes of 1 / 100–1 / 20 of the 200 μM solution to obtain a 2 μM concentration. The reaction mixture was injected into the LC / MS at room temperature for various times. For data analysis, the raw spectra were deconvoluted using a 20,000:40,000 Da window and 1 Da resolution. The percent labeling of a compound was calculated as the labeling of the specific compound divided by the total protein detected.

[0129] 2. Experimental results

[0130] The results are shown in Figures 1 and 2. It can be seen from the results that the compound can achieve complete binding with BTK after incubation at room temperature for 1 hour. The molecular weight calculation results are consistent with the addition-elimination NO release mechanism proposed in the compound design.

[0131] Example 34: NO release study of compounds

[0132] 1. Experimental methods

[0133] The Griess method was used to measure the NO release of the target compound in different pH buffer solutions, target protein storage solution, rat plasma, rat liver microsomes, etc. The NO fluorescent probe method was combined with flow cytometry to measure the NO release of the target compound in Daudi lymphoma cells.

[0134] 2. Experimental results

[0135] The results are shown in Figure 3. I1 released significantly more NO in BTK storage solution than in rat liver microsomes, rat plasma, and GSH solution, indicating that I1 has a high release selectivity. Using the DAF-FM DA probe assay, I1 produced significantly more NO release in Daudi B-cell lymphoma cells compared to ibrutinib.

[0136] Example 35: Stability study of compounds

[0137] 1. Experimental methods

[0138] The stability of compounds I1-I6 in plasma, liver microsomes and GSH was investigated by LC-MS.

[0139] 2. Experimental results

[0140] The results are shown in Figure 4, indicating that compounds I1-I6 all have high in vitro stability.

[0141] Example 36: In vitro kinase inhibitory activity study of compounds

[0142] 1. Experimental methods

[0143] The kinase activities of compounds I1-I6, III1-II6, and III1-III6 against BTK, HER2, and Fgfr4, respectively, were determined using the ADP-Glo ​​assay (IC 50 50 μL of test compound was added to a 384-well dilution plate. The test compound was serially diluted 5-fold in DMSO at a ratio of 1:3 per column. 0.025 μL of the diluted test compound solution was transferred to a 384 assay plate using an Echo. 2.5 μL of enzyme working solution was added to the 384-well assay plate. The plate was centrifuged at 1000 rpm for 1 minute. 2.5 μL of substrate (ATP and substrate) working solution was added to initiate the reaction. The plates were incubated at 25°C for 0, 2, 4, 8, 15, 30, 60, and 90 minutes, respectively. 5 μL of TK Beads solution was added to initiate the reaction. The plates were incubated at 25°C for 60 minutes. The fluorescence signals at 665 nm and 620 nm were read relative to those at BMG (ratio 665 / 620).

[0144] 2. Experimental results

[0145] The results are shown in Tables 1, 2 and 3.

[0146] Table 1. Inhibitory activity of compounds I1-I6 on BTK kinase

[0147] Table 2. Results of HER2 kinase inhibitory activity of compounds III-II6

[0148] Table 3. Inhibitory activity of compounds III1-III6 on Fgfr4 kinase

[0149] Example 37: In vitro anti-tumor cell proliferation activity study of the compound

[0150] 1. Experimental methods

[0151] The inhibitory activity of compounds I1-I6, II1-II6 and III1-III6 against tumor cell lines was tested using the CCK8 method. The cell lines were cultured in DMEM medium or PRMI 1640 medium containing 10% fetal bovine serum (FBS) (SJSA-1 and A549 cells were cultured in PRMI 1640 medium, and MDA-MB-231 and HeLa cells were cultured in DMEM medium). All cell lines were cultured in a Shellab 2323-2 CO2 constant temperature cell culture incubator under the following conditions: air containing 5% CO2 and a temperature of 37°C. Cell activity was determined using the CCK8 (Beyotime) method. Cells were seeded in 96-well plates at a density of 8,000-10,000 cells per well. After incubation in a constant temperature incubator for 24 hours, the cells were observed under a microscope to be in good condition and almost completely attached to the wall. After adding different concentrations of compounds or 0.1% DMSO, the cells were cultured for another 48 hours. After 48 h, CCK8 reagent was added and cultured for another two hours. The absorbance OD value of each well at a wavelength of 450 nM was measured using an Envision 2104 multifunctional microwell analyzer (Perkin Elmer). Finally, GraphPad 8.0 was used to fit the dose-effect curve and calculate the IC 50 Cell viability = [(experimental group - blank control group) / (control group - blank control group)] * 100%. Experimental group: cells + CCK8 solution + drug solution; control group: cells + CCK8 solution + 0.1% DMSO; blank control group: no cells + CCK8 solution. Three replicate wells were set up for each group, and each experiment was repeated three times.

[0152] 2. Experimental results

[0153] The results are shown in Tables 4, 5 and 6.

[0154] Table 4. In vitro antitumor activity test results of compounds I1-I8 (IC 50, μM)

[0155] Table 5. In vitro antitumor activity test results of compounds III-II

[0156] Table 6. In vitro antitumor activity test results of compounds III1-III6

[0157] Example 38: Binding study of compounds to kinases

[0158] 1. Experimental methods

[0159] The Kinact / ki parameter of I1 was tested using the HTRF / ADP-Glo / FI method. 50 μL of I1 was added to a 384-well dilution plate. I1 was serially diluted 5-fold in DMSO at a ratio of 1:3 per column. Using an Echo, 0.025 μL of the diluted I1 solution was transferred from each row to a 384-well assay plate. 2.5 μL of enzyme working solution was added to the 384-well assay plate. The plate was centrifuged at 1000 rpm for 1 minute. 2.5 μL of substrate (ATP and substrate) working solution was added to initiate the reaction. The reaction was incubated at 25°C for 0, 2, 4, 8, 15, 30, 60, and 90 minutes. The reaction was initiated by adding 5 μL of TK Beads solution. The reaction was incubated at 25°C for 60 minutes. The fluorescence signal at 665 nm and 620 nm was read relative to BMG (ratio: 665 / 620).

[0160] 2. Experimental results

[0161] The results are shown in Figure 5 , indicating that modification of the warhead site of I1 increased steric hindrance, making the first non-covalent binding step slower, but modification of the warhead site did not significantly affect the binding efficacy of I1 to BTK.

[0162] Example 39: Kinase profiling study of compounds

[0163] 1. Experimental methods

[0164] Compound I1 was subjected to kinase profiling using the ADP-Glo ​​Kinase Assay. 2× ATP and substrate solution and 2× kinase and metal solution were prepared in assay buffer and 20 nL of compound was transferred to a 384 assay plate via an Echo 655. 2 μL of 2× kinase and metal solution was added, mixed, and incubated in a 384 assay plate at 25°C for 10 minutes. 2 μL of 2× substrate and ATP solution was added to the wells and incubated at 25°C for 60 minutes. 4 μL of ADP-Glo ​​Reagent was added to the wells and incubated at 25°C for 40 minutes. 8 μL of Kinase Detection Reagent was added to the wells and incubated at 25°C for 40 minutes. Signals were recorded on a microtiter plate reader.

[0165] 2. Experimental results

[0166] The results, shown in Figure 6, indicate that modification of the warhead enhances the kinase selectivity of compound I1, effectively reducing off-target inhibition of JAK and ErbB kinases compared to ibrutinib. This may contribute to I1 having fewer gastrointestinal side effects compared to ibrutinib.

[0167] Example 40: In vivo activity study of compounds

[0168] 1. Experimental methods

[0169] 150 μL TMD8 cell suspension (6×10 6 cells) were subcutaneously inoculated into the right back of 8-week-old female BALB / c nude mice. When the average tumor volume reached about 60 mm 3 Mice were randomly divided into a blank control group (n=8 / group) and a compound I1 group (15 mg / kg, PO, BID). Tumor volume and body weight were recorded every other day after administration. After 20 days, all mice were sacrificed and the tumors were retained for further study. Tumor size was calculated using the formula: V (tumor volume, mm 3 ) = L (length, mm) × W2 (width, mm) × 0.5. Tumor growth inhibition (TGI) was calculated using the following formula: TGI = (1-TWt / TWc) × 100%, where TWt and TWc represent the average tumor weights on day 20 in the treatment and control groups, respectively.

[0170] 2. Experimental results

[0171] The results, shown in Figure 7, show that compared to the blank control group, the experimental group treated with A12 (15 mg / kg) significantly inhibited tumor growth. During the experiment, the mice remained in good health, with normal activity, normal water and food intake, normal skin gloss and color, no diarrhea, and no inflammation at the tumor site.

[0172] Example 41: Co-localization study of NO release from compounds and Bruton's tyrosine kinase (BTK)

[0173] 1. Experimental methods

[0174] A BTK-mCherry (excitation wavelength 587 nm, emission wavelength 610 nm) lentiviral vector was constructed using pKG-CMV-MCS-EF1-Puro as the carrier. Daudi cells were cultured and lentivirally transfected for 48 hours. DAF-FM DA (excitation wavelength 495 nm, emission wavelength 515 nm) was added and incubated for 15 minutes. The cells were washed three times to remove excess dye. Compounds were then added to achieve final concentrations of 1 μM, 5 μM, and 10 μM. Compounds were incubated for 5 minutes, 30 minutes, and 60 minutes, respectively. After incubation, the cells were washed three times with PBS. 300 μL of ready-to-use DAPI staining solution was added and incubated at room temperature in the dark for 10 minutes. The cells were then washed three times with PBS. Smears were made and the cells were observed under a focused microscope at 600x magnification.

[0175] 2. Experimental results

[0176] The results, shown in Figure 8, demonstrate clear subcellular colocalization of NO release and BTK. Subsequent addition of the NO scavenger PTIO revealed confocal imaging of NO scavenging, with no NO present at the BTK site. These results demonstrate that I1 releases NO in situ and within a very small area at the BTK site within the cell.

[0177] Example 42: Study on the Effect of NO Released by Compounds on Increased Nitrosation and Decreased Phosphorylation of Bruton's Tyrosine Kinase (BTK) / Inhibition of Phosphorylation Signal Transduction

[0178] 1. Experimental methods

[0179] Daudi cells were cultured and incubated with 1-10 μM of each compound for 1-5 hours. DMSO, ibrutinib, I1, and azotinium groups were used. After Biotin-Switch conversion, the target proteins were purified using Streptavidin Agarose (Life Technologies, Catalog No. SA100-04). Nitrosylation of the target proteins was then characterized. Western blotting was also used to evaluate the effect of nitrosylation on target protein phosphorylation in the same cell group, revealing the crosstalk between kinase nitrosylation and phosphorylation.

[0180] 2. Experimental results

[0181] The results are shown in Figure 9. After incubation with Daudi cells, I1 significantly increased BTK nitrosylation compared to ibrutinib, but did not cause global nitrosylation compared to azodicarbonium diolates (non-selective NO donors). The effect of I1 on the phosphorylation of BTK and the BTK-interacting kinase protein kinase B (AKT) was also tested compared to ibrutinib. The results showed that I1 significantly reduced the phosphorylation of BTK and AKT compared to ibrutinib, indicating that nitrosylation and covalent inhibition caused by NO play a synergistic role in suppressing phosphorylation.

Claims

1. A covalent kinase inhibitor, characterized in that Having a structure of Formula I, II, III, IV or V, and also including pharmaceutically acceptable salts thereof, in: R1 and R2 are selected from C1-C4 alkyl or R1, R2 and the nitrogen to which they are attached together form a 4-7 membered heterocyclic ring containing 1-2 nitrogen, oxygen, or sulfur atoms, and the ring carbon atom is substituted by at least one hydrogen, hydroxyl, halogen, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, or hydroxyl-substituted C1-C4 alkyl; The kinase inhibitor nucleus is selected from:

2. The covalent kinase inhibitor according to claim 1, characterized in that In the structure: R1 and R2 are selected from methyl, ethyl or R1, R2 and the N to which they are connected together form a 5-6 membered heterocyclic ring, wherein the heterocyclic ring is a morpholine ring, a piperidine ring, or a tetrahydropyrrole ring, and the ring carbon atom is substituted by at least one hydrogen, hydroxyl, fluorine, chlorine, bromine, methyl, ethyl, trifluoromethyl, hydroxymethyl, or hydroxyethyl.

3. The covalent kinase inhibitor according to claim 2, characterized in that In the structure: NR1R2 is selected from dimethylamino, methylethylamino, diethylamino, morpholinyl, tetrahydropyrrolyl, 2-hydroxymethyltetrahydropyrrolyl, piperidinyl, 4-hydroxypiperidinyl.

4. The covalent kinase inhibitor according to claim 1, characterized in that Select any of the following compounds:

5. The covalent kinase inhibitor according to claim 1, characterized in that The pharmaceutically acceptable salt is a salt of the kinase inhibitor formed with any of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, malic acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, ferulic acid or mandelic acid.

6. A method for preparing the covalent kinase inhibitor according to claim 1, characterized in that: The following steps are involved: (1) When the covalent kinase inhibitor has the structural formula of Formula I or II, (i) Compound 1 is reacted in the presence of paraformaldehyde, triethylenediamine and phenol to obtain intermediate 2; (ii) intermediate 2 is reacted in the presence of p-toluenesulfonyl chloride and triethylamine to obtain intermediate 3; (iii) intermediate 3 reacts with the corresponding azodiol salt or AgNO2 under the catalysis of 15-crown-5-ether to obtain the final product 4 or 5; (2) When the covalent kinase inhibitor has the structural formula III, (i) the kinase inhibitor core 6 is reacted with 2,3-dibromopropionic acid under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to obtain compound 7; (ii) Compound 7 reacts with nitromethane to obtain the final product 8; (3) When the covalent kinase inhibitor has the structural formula IV, (i) tert-butyl 3-oxocyclobutylcarboxylate is reacted in the presence of hydroxylamine hydrochloride and sodium acetate to obtain oxime intermediate 10; (ii) intermediate 10 reacts with urea peroxide and trifluoroacetic anhydride to obtain intermediate 11; (iii) intermediate 11 reacts with kinase inhibitor core 6 to obtain final product 12; (4) When the covalent kinase inhibitor has a structural formula of Formula V, the kinase inhibitor core 6 is reacted with 2-bromo-1-(3,3-dinitroazetidine-1-yl)ethanone to obtain the final product 13; Wherein, R1, R2, and the kinase inhibitor core are defined as in claim 1; The pharmaceutically acceptable salt is obtained by reacting the corresponding acid with the covalent kinase inhibitor prepared by the above method to form a salt.

7. A pharmaceutical composition, characterized in that The invention comprises the covalent kinase inhibitor according to claim 1 and a pharmaceutically acceptable carrier.

8. Use of the covalent kinase inhibitor according to claim 1 or the pharmaceutical composition according to claim 7 in the preparation of a drug for treating and / or preventing a proliferative disease, delaying the progression of a proliferative disease, alleviating the symptoms of a proliferative disease, assisting in the treatment of a proliferative disease, or managing a proliferative disease.

9. The use according to claim 8, characterized in that: The proliferative disease is selected from the group consisting of tumors, rheumatic diseases, chronic inflammation, and infectious mononucleosis.

10. The use according to claim 9, characterized in that: The proliferative disease is selected from the group consisting of gastric cancer, colorectal cancer, lung cancer, breast cancer, liver cancer, prostate cancer, thyroid cancer, pancreatic cancer, bladder cancer, kidney cancer, brain tumor, cervical cancer, uterine cancer, ovarian cancer, cervical cancer, testicular tumor, myeloma, osteosarcoma, malignant glioma, myeloproliferative disease, malignant lymphoma, chronic lymphocytic leukemia, monocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia, non-lymphoreticular tumor, solitary plasmacytoma, lymphoblastoma, Hodgkin's lymphoma, neuroblastoma, retinoblastoma, neuroendocrine cell tumor, pancreatic islet cell tumor, cell tumor, melanoma, atherosclerosis, pulmonary fibrosis, rheumatic diseases, chronic inflammation, papular mucinosis, familial splenic anemia, amyloidosis, heavy chain disease, light chain disease, semi-molecular disease, cryoglobulinemia, primary macroglobulinemia, primary macroglobulinemia purpura, secondary benign monoclonal gammopathy, osteolytic lesions, some non-Hodgkin's lymphomas, Sezary syndrome, infectious mononucleosis, acute histiocytosis, intestinal polyps, diverticulitis, colitis, pancreatitis, hepatitis, chronic hepatitis, cirrhosis, cholecystitis.

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