Covalent kinase inhibitors, methods of preparation, pharmaceutical compositions, and uses

Abstract

The application discloses a covalent kinase inhibitor, a preparation method, a pharmaceutical composition and application, the structure of the covalent kinase inhibitor is as shown in formula I, II, III, IV or V, and the covalent kinase inhibitor further comprises a pharmaceutically acceptable salt thereof, the covalent kinase inhibitor can be efficiently covalently combined with a target kinase, and simultaneously triggers the release of NO / NO2 — ; on one hand, the covalent combination can covalently inhibit the target kinase containing a sulfydryl group, and on the other hand, the released NO / NO2 — can further produce sulfydryl nitroso modification on the target kinase or a protein interacting with the target kinase, thereby inhibiting self-phosphorylation, preventing kinase signal transduction and playing a synergistic effect; the covalent kinase inhibitor can act on various kinases related to malignant tumor proliferation, has good selectivity and a low off-target effect, can significantly inhibit the growth of tumor cells at a cell and animal level, and has no obvious adverse reactions. In addition, the preparation method is simple and universal, and is conducive to the expansion of a mother nucleus structure of various other kinase inhibitors.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of NO / NO2 — Releasing covalent warhead-based kinase inhibitors, preparation method, pharmaceutical composition and application, especially to a kind of NO / NO2 — Releasing covalent warhead-based kinase inhibitors, preparation method, pharmaceutical composition and application. BACKGROUND

[0002] NO is a water-soluble free radical gas in vivo, with redox properties, plays an important role in physiology and pathology. In cardiovascular regulation, nerve, immune regulation and other aspects, it plays an extremely important biological role. NO2 — Is one of the metabolites of NO in vivo, which can be reduced to NO under ischemia and hypoxia, and in some cases, it can also be regarded as a kind of NO donor. NO can react with protein cysteine thiol to mediate protein cysteine thiol nitrosylation (SNO), which is a very important post-translational modification. It is worth mentioning that SNO can also be mediated by NO2 — .

[0003] NO / NO2 — Donor type drugs generally refer to prodrugs formed by NO / NO2 — Donor and related drugs or some active compounds through various linking groups. It has been found that there are many structural types of NO donors, such as nitrosothiol, nitrate, NO-metal complex (nitroprusside), furazan N-oxide, azo imine glycolate, etc.; NO2 — Donor includes 2-nitromethyl-1,3-diphenyl-2-en-1-ketone, etc.

[0004] Covalent kinase inhibitors bind irreversibly to target kinase residues (usually Cys) through covalent bonds, thereby exerting target inhibition function and achieving disease treatment purposes. It generally contains a less reactive bonding functional group (warhead group) and a ligand part. After the ligand locates the corresponding site of the target kinase, the warhead group reacts with Cys to form a covalent bond.

[0005] Although NO / NO2 — Donor drugs and covalent inhibitors have undergone great development, but still face many deficiencies. For example, for NO / NO2 — Donor drugs, the mechanism of triggering NO / NO2 — Release currently depends on photocontrol, sound control and other chemical biological means; NO / NO2 — Release has low specificity and poor precision, and the released NO / NO2 —Due to its free diffusion property, NO / NO2 — will act on multiple biological macromolecules to produce more side effects. Therefore, for NO / NO2 — donor drugs, it is still necessary to explore NO / NO2 — release mode with higher specificity and better accuracy.

[0006] For covalent inhibitors, although they have higher target occupancy, they also lead to single mode of action, lack of effective synergistic treatment mechanism; in addition, existing covalent inhibitors also have the disadvantages of low bioavailability, high frequency of drug administration, large dose of drug administration and easy to produce target Cys mutation drug resistance. Therefore, for covalent inhibitors, exploring new synergistic mechanism may help to enhance efficacy and overcome the above shortcomings to some extent. SUMMARY

[0007] The purpose of the present application is to provide a covalent kinase inhibitor based on NO / NO2 — release type covalent warhead; the second purpose is to provide a preparation method of the covalent kinase inhibitor; the third purpose is to provide a pharmaceutical composition containing the covalent kinase inhibitor, and the fourth purpose is to provide the covalent kinase inhibitor and the application of the pharmaceutical composition thereof.

[0008] Technical scheme: the covalent kinase inhibitor provided by the present application has the structure of formula I, II, III, IV or V, and also comprises a pharmaceutically acceptable salt thereof,

[0009]

[0010]

[0011] Wherein:

[0012] R1, R2 are selected from C1-C4 alkyl or R1, R2 and the nitrogen connected therewith jointly form a 4-7 membered heterocyclic ring, the heterocyclic ring contains 1-2 nitrogen, oxygen, sulfur heteroatoms, and the ring system carbon atoms are substituted by at least one hydrogen, hydroxyl, halogen, C1-C4 alkyl, halogen-substituted C1-C4 alkyl, hydroxyl-substituted C1-C4 alkyl;

[0013] The kinase inhibitor mother nucleus is selected from:

[0014]

[0015] The present application designs a covalent kinase inhibitor based on NO / NO2 — release type covalent warhead, wherein the covalent kinase inhibitor with formula I structure has O 2-(α-methylene-α,β-unsaturated ketone)-diazenium diolates warhead structure, which can covalently bind to specific thiol of protein kinase and release NO2 via addition-elimination reaction mediated by covalent binding 2 protection, triggering in situ release of NO in a very small range. Covalent kinase inhibitors with structure of formula II have α-nitromethyl-α,β-unsaturated ketone warhead structure, using the same mechanism, release NO2 via covalent binding reaction — in a very small range. Covalent kinase inhibitors with structure of formula III, IV, V have ring strain nitro-substituted warhead structure, which releases NO2 in a very small range via ring strain after nucleophilic substitution reaction with specific thiol of protein kinase — .

[0016] The present application designs NO / NO2 — releasing warhead groups for the first time, so that covalent binding of these warhead groups to kinase inhibitors can release diazenium diolates fragment O 2 protection, release NO, and release NO2 via the same mechanism or ring strain mechanism — . On the one hand, covalent binding can play a covalent inhibitory role on thiol-containing target enzymes, and on the other hand, released NO / NO2 — can further produce nitrosylation modification on target kinases and interacting kinases, inhibit their own phosphorylation, prevent kinase signal transduction, and play a synergistic role.

[0017] The designed compounds of the present application have good specificity in covalent binding, high efficiency in NO / NO2 — release, and good biological activity. The synergistic effect of covalent binding and NO / NO2 — can effectively overcome the shortcomings of existing NO / NO2 — donor drugs and covalent inhibitors. At the same time, due to the synergistic effect, the dose of the drug can be reduced, thereby improving patient compliance, significantly improving safety, and reducing side effects.

[0018] In the design of covalent kinase inhibitors of formula I, diazenium diolates have obvious advantages in selective and targeted release of NO. On the one hand, diazenium diolates are extremely unstable under physiological conditions and can automatically release 1-2 molecules of NO, with a half-life of several seconds to several hours. On the other hand, the O 2 position (O 1 connected to the nitrogenium ion is called O 2 connected to the olefinic nitrogen atom is called O ) of diazenium diolates can be protected to convert it into a stable prodrug; and the O2 protecting group, which is converted to an unstable diazohydrate anion, thus achieving selective and targeted release of NO. At the same time, the covalent kinase inhibitors of formula II can also release NO2 — selectively and targetedly via the same addition-elimination reaction mechanism. —

[0019] The present application is based on covalent inhibition therapy and NO / NO2 — application in the field of anti-tumor therapy, taking advantage of the chemical group release of the covalent inhibitor warhead group, to obtain a new class of NO / NO2 — release type covalent warhead kinase inhibitors, and at the same time, a new O 2 protecting method. It deprotects the prodrug according to the mechanism of formula 1 or formula 2, and each HS-R3 is selected from one of the small molecule sulfhydryl compounds glutathione, cysteine and proteins BTK, HER1, HER2, Fgfr4, HER3, HER4, KRAS G12C.

[0020] Formula 1:

[0021]

[0022] Formula 2:

[0023]

[0024] Preferably, in the structure, R1, R2 are selected from methyl, ethyl, or R1, R2 together with the N to which they are connected form a 5-6 membered heterocyclic ring, and the heterocyclic ring is a morpholine ring, a piperidine ring, a tetrahydropyrrole ring, and the ring system carbon atom is substituted with at least one hydrogen, hydroxyl, fluorine, chlorine, bromine, methyl, ethyl, trifluoromethyl, hydroxymethyl, hydroxyethyl; NR1R2 is further preferably selected from dimethylamino, methylethylamino, diethylamino, morpholinyl, tetrahydropyrrolyl, 2-hydroxymethyl tetrahydropyrrolyl, piperidyl, 4-hydroxypiperidyl.

[0025] Specifically, the covalent kinase inhibitor is most preferably selected from any one of the following compounds:

[0026]

[0027]

[0028]

[0029] ​Further, the covalent kinase inhibitors described above form pharmaceutically acceptable salts 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.

[0030] The method for preparing the covalent kinase inhibitors described in the present application comprises the following steps:

[0031] (1) when the covalent kinase inhibitors have the structural formula of formula I or II,

[0032] (i) compound 1 is reacted in the presence of paraformaldehyde, triethylenediamine, phenol to obtain intermediate 2;

[0033] (ii) intermediate 2 is reacted in the presence of p-toluenesulfonyl chloride, triethylamine to obtain intermediate 3;

[0034] (iii) intermediate 3 is reacted with the corresponding azonia salt or AgNO2 under the catalysis of 15-crown-5-ether to obtain the final product 4 or 5;

[0035]

[0036] (2) when the covalent kinase inhibitors have the structural formula of formula III,

[0037] (i) the kinase inhibitor mother nucleus 6 is reacted with 2,3 dibromopropionic acid under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to obtain compound 7;

[0038] (ii) compound 7 is reacted with nitromethane to obtain the final product 8;

[0039]

[0040] (3) when the covalent kinase inhibitors have the structural formula of formula IV,

[0041] (i) tert-butyl 3-oxocyclobutylcarboxylate is reacted in the presence of hydroxylamine hydrochloride, sodium acetate to obtain oxime intermediate 10;

[0042] (ii) intermediate 10 is reacted with urea peroxide, trifluoroacetic anhydride to obtain intermediate 11;

[0043] (iii) intermediate 11 is reacted with the kinase inhibitor mother nucleus 6 to obtain the final product 12;

[0044]

[0045] (4) when the covalent kinase inhibitor has the structural formula of Formula V, the kinase inhibitor mother nucleus 6 is reacted with 2-bromo-1-(3,3-dinitroazetidin-1-yl) ethanone to obtain the final product 13;

[0046]

[0047] wherein, R1, R2, the definition of the kinase inhibitor mother nucleus is as described above;

[0048] The corresponding acid is reacted with the kinase inhibitor prepared by the above method to form a salt, thereby obtaining the pharmaceutically acceptable salt.

[0049] The covalent kinase inhibitor and the pharmaceutically acceptable carrier form the pharmaceutical composition of the present application, and common pharmaceutical excipients such as flavors, sweeteners, liquid / solid fillers, diluents, etc. can be added to prepare common pharmaceutical preparations such as tablets, capsules, syrups, suspensions, injections, etc.

[0050] The covalent kinase inhibitor and the pharmaceutical composition thereof of the present application are applied to prepare a drug for treating and / or preventing a proliferative disease, delaying the progress of a proliferative disease, alleviating the symptoms of a proliferative disease, assisting in the treatment of a proliferative disease, and treating a proliferative disease.

[0051] Preferably, the proliferative disease is selected from tumors, rheumatic diseases, chronic inflammation, infectious mononucleosis.

[0052] Further preferably, the proliferative disease is selected from:

[0053] gastric cancer, colorectal cancer, lung cancer (e.g. 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, glioblastoma, myeloproliferative disease, atherosclerosis, leukemia, pulmonary fibrosis, lymphoma, rheumatic disease, chronic inflammation, non-lymphoid reticuloendothelial system tumor, cryoglobulinemia, papulosis, familial splenic anemia, multiple myeloma, amyloidosis, solitary plasmacytoma, heavy chain disease, light chain disease, malignant lymphoma, chronic lymphocytic leukemia, monocytic leukemia, half-molecule disease, primary macroglobulinemia, primary macroglobulinemia purpura, secondary benign monoclonal gammopathy, osteolytic lesion, acute lymphocytic leukemia, lymphoblastoma, partial non-Hodgkin lymphoma, Sezary syndrome, infectious mononucleosis, acute histiocytosis, hairy cell leukemia, Hodgkin lymphoma, colon cancer, rectal cancer, intestinal polyp, diverticulitis, colitis, pancreatitis, hepatitis, small cell lung cancer, neuroblastoma, neuroendocrine cell tumor, islet cell tumor, medullary thyroid cancer, melanoma, uterine cancer, chronic hepatitis, cirrhosis, ovarian cancer, retinoblastoma, cholecystitis, head and neck squamous cell carcinoma, gastrointestinal malignancy, non-small cell lung cancer, cervical cancer, testicular tumor, bladder cancer, myeloma or bone tissue malignancy (e.g. osteosarcoma).

[0054] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages:

[0055] 1. The covalent kinase inhibitors can effectively covalently bind to target enzymes and effectively release NO / NO2 — ; on the one hand, covalent binding can covalently inhibit target enzymes containing sulfydryl groups, and on the other hand, the released NO / NO2 — can further produce nitrosylation modification on target kinases and interacting kinases, inhibit their own phosphorylation, prevent kinase signal transduction, and play a synergistic role; and has selectivity, reduces non-target inhibition;

[0056] 2. The covalent kinase inhibitors have remarkable inhibitory activity at the molecular level, cell level and animal level, act on a variety of enzymes related to malignant proliferation and malignant proliferative cells, and inhibit tumor growth in animals without obvious adverse reactions;

[0057] 3. The compound preparation method is simple and universal, and is beneficial to the expansion of various structures. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 is the mass spectrometry verification result of covalent binding of the I1 compound to Bruton's tyrosine kinase (BTK);

[0059] Figure 2is the mass spectrometry verification result of the covalent binding of I3 compound to Bruton's tyrosine kinase (BTK);

[0060] Figure 3 is the NO release result of I1 compound in BTK stock solution, rat liver microsomes, rat plasma, GSH solution respectively and the NO release result of I1 compound in Daudi cells of B-cell lymphoma;

[0061] Figure 4 is the stability test result of I1-I6 compounds in rat plasma and GSH solution;

[0062] Figure 5 is the Kinact / Ki parameter test result of I1 compound;

[0063] Figure 6 is the kinase profile test result of I1 compound;

[0064] Figure 7 is the nude mouse experiment result of I1 compound;

[0065] Figure 8 is the co-localization verification result of the NO release of I1 compound in cells and Bruton's tyrosine kinase (BTK);

[0066] Figure 9 is the result of the increase of Bruton's tyrosine kinase (BTK) nitrosylation and the decrease of phosphorylation / phosphorylation signal transduction obstruction caused by the NO released by I1 compound. DETAILED DESCRIPTION

[0067] The technical solutions of the present application are further described below in combination with examples.

[0068] Example 1: Preparation of compound I1

[0069]

[0070] 1. 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) was dissolved in 50 ml of 1,4-dioxane, and paraformaldehyde (40.9 g, 0.454 mol), triethylenediamine (3.82 g, 0.034 mol), and phenol (1.6 g, 0.017 mol) were added, and the mixture was reacted at 60°C for 3 days. The solvent was removed by concentration under reduced pressure, and then extracted with ethyl acetate (50 mL). The organic layer was washed with water and saturated brine three times each, dried over anhydrous sodium sulfate, concentrated, and column chromatographed (petroleum ether / ethyl acetate = 5 / 1, v / v) to obtain white solid V1. 1H NMR (500 MHz, Chloroform-d) δ 8.25 (s, 1H), 7.60 - 7.54 (m, 3H), 7.40 - 7.32 (m, 3H), 7.18 (s, 1H), 7.12 - 6.99 (m, 4H), 6.98 - 6.92 (m, 3H), 6.82 (s, 1H), 5.76 (d, J = 12.5 Hz, 1H), 5.59 (d, J = 12.5 Hz, 1H), 5.00 (s, 1H), 4.37 (t, J = 5.5 Hz, 1H), 4.28 - 4.14 (m, 3H), 3.94 (s, 1H), 3.87 (s, 1H), 3.52 (d, J = 8.4 Hz, 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.

[0071] 2, V1 (200 mg, 0.43 mmol) was dissolved in 15 ml of dichloromethane, p-toluenesulfonyl chloride (90 mg, 0.47 mmol), triethylamine (18.3 mg, 0.18 mmol) were added, and the reaction was carried out at room temperature for 10 h under nitrogen protection, and then extracted with ethyl acetate, the organic layer was washed with water, saturated brine 3 times, dried over anhydrous sodium sulfate, and column chromatography (petroleum ether / ethyl acetate = 20 / 1, v / v) to obtain white solid V2. 1 H NMR (500 MHz, Chloroform-d) δ 8.00 (s, 1H), 7.81 - 7.75 (m, 3H), 7.69 - 7.63 (m, 3H), 7.40 - 7.30 (m, 5H), 7.10 (tt, J = 7.6, 2.0 Hz, 1H), 7.04 - 6.94 (m, 7H), 6.72 (s, 1H), 5.84 (d, J = 12.3 Hz, 1H), 5.61 (d, J = 12.5 Hz, 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 = 1.0 Hz, 4H), 2.08 (s, 1H), 2.01 (s, 1H), 1.96 (s, 1H), 1.88 (s, 1H). HRMS (ESI): m / z calcd for C 33 H 32 N6O5S, [M+H] + : 625.2155, found: 624.2151.

[0072] 3. Dissolve the p-toluenesulfonyl chloride substitution product V2 (300 mg, 0.48 mmol) in 20 ml of DMSO, add dimethylaminoazepine glycolate (305 mg, 2.40 mmol), and react at room temperature for 10 h under nitrogen protection, then extract with ethyl acetate, wash the organic layer with water and saturated brine 3 times each, dry over anhydrous sodium sulfate, and column chromatograph (petroleum ether / ethyl acetate = 10 / 1, v / v) to obtain white solid I1. 1 H NMR (500 MHz, Chloroform-d) δ 8.63 (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 = 12.4 Hz, 1H), 5.59 (d, J = 12.5 Hz, 1H), 5.00 (s, 1H), 4.53 (d, J = 0.9 Hz, 2H), 3.94 (s, 1H), 3.87 (s, 1H), 3.52 (d, J = 6.2 Hz, 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. for C 28 H 31 N9O4, [M+H] + :558.2499, found:558.2498.

[0073] Example 2: Preparation of compound I2

[0074]

[0075] Refer to the synthetic method of Example 1. 1H 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.

[0076] Example 3: Preparation of compound I3

[0077]

[0078] The synthesis method is as described in 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 H 35 N9O4, [M+H] +: 586.2812, found: 586.2807.

[0079] Example 4: Preparation of compound I4

[0080]

[0081] The synthesis method of Reference Example 1 was referred to. 1 H NMR (500 MHz, Chloroform-d) δ 8.29 (s, 1H), 7.69 - 7.63 (m, 3H), 7.40 - 7.32 (m, 3H), 7.09 (tt, J = 7.6, 2.0 Hz, 1H), 7.04 - 6.94 (m, 7H), 6.72 (s, 1H), 5.84 (d, J = 12.4 Hz, 1H), 5.59 (d, J = 12.5 Hz, 1H), 5.00 (s, 1H), 4.53 (d, J = 0.9 Hz, 2H), 4.01 (s, 1H), 3.94 (s, 1H), 3.68 (d, J = 7.1 Hz, 5H), 3.53 (d, J = 10.4 Hz, 3H), 3.29 (d, J = 8.4 Hz, 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.

[0082] Example 5: Preparation of compound I5

[0083]

[0084] The synthesis method of Reference Example 1 was referred to. 1H NMR (500 MHz, Chloroform-d) δ 8.25 (s, 1H), 7.51-7.63 (m, 3H), 7.40-7.28 (m, 3H), 7.18 (tt, J = 7.5, 2.0 Hz, 1H), 7.04-6.88 (m, 7H), 6.66 (s, 1H), 5.65 (d, J = 12.5 Hz, 1H), 5.59 (d, J = 12.5 Hz, 1H), 5.00 (s, 1H), 4.53 (d, J = 12.3 Hz, 1H), 4.48 (d, J = 12.3 Hz, 1H), 4.01 (s, 1H), 3.94 (s, 1H), 3.53 (d, J = 10.4 Hz, 3H), 3.27 (d, J = 2.4 Hz, 5H), 2.08 (s, 1H), 2.01 (s, 1H), 1.96 (s, 1H), 1.88 (s, 1H), 1.83 (d, J = 3.7 Hz, 5H). HRMS (ESI): m / z calcd for C 30 H 33 N9O4,[M+H] + : 584.2656, found: 584.2651.

[0085] Example 6: Preparation of compound I6

[0086]

[0087] Reference to the synthesis method of Example 1. 1 H NMR (500 MHz, Chloroform-d) δ 8.25 (s, 1H), 7.51-7.63 (m, 3H), 7.40-7.28 (m, 3H), 7.18 (tt, J = 7.5, 2.0 Hz, 1H), 7.04-6.88 (m, 7H), 6.66 (s, 1H), 5.65 (d, J = 12.5 Hz, 1H), 5.59 (d, J = 12.5 Hz, 1H), 5.00 (s, 1H), 4.53 (d, J = 12.3 Hz, 1H), 4.48 (d, J = 12.3 Hz, 1H), 4.01 (s, 1H), 3.94 (s, 1H), 3.53 (d, J = 10.4 Hz, 3H), 3.27 (d, J = 2.4 Hz, 5H), 2.08 (s, 1H), 2.01 (s, 1H), 1.96 (s, 1H), 1.88 (s, 1H), 1.83 (d, J = 3.7 Hz, 5H). HRMS (ESI): m / z calcd for C 31 H 35 N9O4,[M+H] +:598.2812,found:598.2808.

[0088] Example 7: Preparation of compound I7

[0089]

[0090] Following 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 diethyl ether, and AgNO2 (369 mg, 2.40 mmol) was added. The reaction was carried out at room temperature in the dark for 10 h under nitrogen protection. The solvent was then concentrated under reduced pressure, extracted with ethyl acetate, and the organic layer was washed three times each with water and saturated brine. The mixture was dried over anhydrous sodium sulfate and subjected to column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to obtain a white solid I7. 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 / zcalcd.for C 26 H 25 N7O4,[M+H]+:500.1968,found:500.1965.

[0091] Example 8: Preparation of compound I8

[0092]

[0093] 1. Dissolve 10 g (0.02587 mol) of 3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidine-4-amine (ibrutinib intermediate) in 50 mL of dichloromethane, add 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), react at room temperature for 36 hours, concentrate under reduced pressure to remove solvent, and then extract with ethyl acetate (100 mL). Wash the organic layer three times with water and three times with saturated brine, dry with anhydrous sodium sulfate, concentrate, and column chromatography (petroleum ether / ethyl acetate = 1 / 1, v / v) to give white solid V3.1 HNMR(500MHz,Chloroform-d)δ8.75(s,1H),7.68–7.61(m,3H),7.39–7.32(m, 3H),7.11–7.04(m,1H),7.08(s,2H),7.04–6.94(m,5H),6.72(s,1H),5.00(s,1 H),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.

[0094] 2. Dissolve V3 (10g, 0.01666mol) in 50ml of ethanol, add nitromethane (1.5g, 0.02499mol), reflux for 24 hours, concentrate under reduced pressure to remove solvent, then extract with ethyl acetate (100mL), wash the organic layer three times with water and three times with saturated brine, dry with anhydrous sodium sulfate, concentrate, and column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to give white solid I8. 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 / zcalcd.for C 26 H 25 N7O4,[M+H] + :500.1968,found:500.1964.

[0095] Example 9: Preparation of compound I9

[0096]

[0097] 1. Dissolve 3-oxocyclobutylcarboxylic acid tert-butyl ester (1 g, 0.005875 mol), sodium acetate (1.60 g, 0.01175 mol), hydroxylamine hydrochloride (0.8165 g, 0.01175 mol) in 50 mL of ethanol, reflux for 24 hours, remove the solvent under reduced pressure, then extract with ethyl acetate (100 mL), wash the organic layer with water, saturated brine for 3 times, dry over anhydrous sodium sulfate, concentrate, column chromatography (petroleum ether / ethyl acetate = 50 / 1, v / v) to give colorless oil V4. 1 H NMR (500 MHz, Chloroform-d) δ 2.82 (s, 1H), 2.69 (d, J = 13.0 Hz, 2H), 2.62 (d, J = 13.0 Hz, 2H), 1.42 (s, 9H). HRMS (ESI): m / z calcd for C9H 14 O3, [M+H] + : 171.0943, found: 171.0941.

[0098] 2. Dissolve V4 (500 mg, 2.94 mmol), trifluoroacetic anhydride (926.06 mg, 4.41 mmol), urea peroxide (414.85 mg, 4.41 mmol), sodium dihydrogen phosphate (529.09 mg, 4.41 mmol) in anhydrous acetonitrile, reflux for 4 hours, filter, remove the solvent of the filtrate under reduced pressure, then extract with ethyl acetate (100 mL), wash the organic layer with water, saturated brine for 3 times, dry over anhydrous sodium sulfate, concentrate, column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to give white solid V5. 1 H NMR (500 MHz, Chloroform-d) δ 4.15 (s, 1H), 2.78 (d, J = 13.0 Hz, 2H), 2.69 (d, J = 13.0 Hz, 2H), 2.50 (s, 1H). HRMS (ESI): m / z calcd for C5H7NO4, [M+H] + : 146.0375, found: 146.0370.

[0099] 3. 3-(4-Phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine (intermediate of Ibrutinib) (10 g, 0.02587 mol) was dissolved in 50 ml of dichloromethane, EDCI (9.91 g, 0.05174 mol), triethylamine (5.24 g, 0.05174 mol), V5 (4.51 g, 0.031044) were added, and the reaction was allowed to proceed at room temperature for 24 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, saturated brine three times each, dried over anhydrous sodium sulfate, concentrated, and column chromatographed (petroleum ether / ethyl acetate = 50 / 1, v / v) to obtain I9 as a white solid. 1 H NMR (500 MHz, Chloroform-d) δ 8.25 (s, 1H), 7.60 - 7.54 (m, 3H), 7.39 - 7.32 (m, 3H), 7.12 - 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 = 13.0 Hz, 2H), 2.64 (s, 1H), 2.53 (d, J = 13.0 Hz, 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.

[0100] Example 10: Preparation of compound I 10

[0101]

[0102] ​Dissolve 3-(4-phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine (intermediate of Ibrutinib) (10 g, 0.02587 mol) in 50 ml dichloromethane, and pre-cool in ice bath for 10 minutes. Dissolve 2-bromo-1-(3,3-dinitroazetidin-1-yl)ethanone (6.2 g, 0.02328 mol) in 10 ml dichloromethane, then drop into the previous Ibrutinib intermediate solution, and after 30 minutes of reaction in ice bath, move to room temperature for 2 hours. Concentrate to remove the solvent under reduced pressure, then extract with ethyl acetate (100 mL), wash the organic layer with water, saturated brine for 3 times each, dry over anhydrous sodium sulfate, concentrate, and column chromatography (petroleum ether / ethyl acetate = 30 / 1, v / v) to obtain white solid I 10 . 1 H NMR (500 MHz, Chloroform-d) δ 8.71 (s, 1H), 7.69-7.63 (m, 3H), 7.40-7.32 (m, 3H), 7.08 (tt, J = 7.5, 2.1 Hz, 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 = 12.4 Hz, 1H), 3.13 (d, J = 12.3 Hz, 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 = 12.5 Hz, 2H). HRMS (ESI): m / z calcd. for C 27 H 27 N9O6, [M+H] + : 574.2084, found: 574.2080.

[0103] Example 11: Preparation of compound II1

[0104]

[0105] Refer to the synthetic method of Example 1. 1H NMR (500 MHz, Methanol-d4) δ 7.90 (s, 1H), 7.63 (t, J = 8.4 Hz, 1H), 7.51 (d, J = 9.1 Hz, 1H), 7.29 (s, 1H), 5.57 (d, J = 105.3 Hz, 1H), 4.97 (s, 2H), 4.08 (s, J = 2.2 Hz, 3H), 3.93 (t, J = 10.6 Hz, 2H), 3.06 (s, 6H), 2.13 - 2.07 (m, 3H), 1.95 (s, 2H), 1.45 (s, 2H). HRMS (ESI): m / z calcd for C 26 H 28 Cl2FN7O5, [M+H] + : 608.1513, found: 608.1509.

[0106] Example 12: Preparation of compound II2

[0107]

[0108] Reference to the synthesis method of Example 1. 1 H NMR (500 MHz, Chloroform-d) δ 8.68 (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 = 7.1 Hz, 2H), 2.96 (s, 3H), 2.08 - 2.03 (m, 2H), 1.97 - 1.92 (m, 2H), 1.13 (t, J = 7.1 Hz, 3H). HRMS (ESI): m / z calcd for C 27 H 30 Cl2FN7O5, [M+H] + : 622.1670, found: 622.1670.

[0109] Example 13: Preparation of compound II3

[0110]

[0111] Reference to the synthesis method of Example 1. 1H NMR (300 MHz, Chloroform-d) δ 8.62 (s, 1H), 8.09 (s, 1H), 8.00 (s, 1H), 7.57-7.52 (m, 1H), 7.39 (d, J = 9.5 Hz, 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 = 6.9 Hz, 4H), 1.99 (d, J = 29.2 Hz, 4H), 1.08 (t, J = 7.0 Hz, 6H). HRMS (ESI): m / z calcd for C 28 H 32 Cl2FN7O5, [M+H] + : 636.1826, found: 636.1817.

[0112] Example 14: Preparation of compound II4

[0113]

[0114] The synthetic method of Reference Example 1 was referred to. 1 H NMR (500 MHz, Chloroform-d) δ 8.68 (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 = 9.7 Hz, 2H), 3.85 (t, J = 4.8 Hz, 4H), 3.76-3.65 (m, 2H), 3.45 (t, J = 4.7 Hz, 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.

[0115] Example 15: Preparation of compound II5

[0116]

[0117] The synthetic method of Reference Example 1 was referred to. 1H NMR (500 MHz, Chloroform-d) δ 8.41 (s, 1H), 7.82 (s, 1H), 7.63 (t, J = 8.2 Hz, 1H), 7.46 (dd, J = 8.8, 1.8 Hz, 1H), 5.66 (s, 1H), 5.45 (s, 1H), 4.96 (s, 2H), 4.87 (dd, J = 6.7, 3.3 Hz, 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 = 5.8 Hz, 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.

[0118] Example 16: Preparation of compound II6

[0119]

[0120] Reference to the synthesis method of Example 1. 1 H NMR (500 MHz, Chloroform-d) δ 8.61 (s, 1H), 7.95 (t, J = 8.5 Hz, 1H), 7.69 (s, 1H), 7.29 (s, 1H), 7.24 (dd, J = 8.9, 1.9 Hz, 1H), 5.50 (s, 1H), 5.29 (s, 1H), 4.82 (s, 1H), 4.00 (s, 3H), 3.73 (q, J = 7.0 Hz, 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.

[0121] Example 17: Preparation of compound II7

[0122]

[0123] Reference to the synthesis method of Example 7. 1H NMR (500 MHz, Chloroform-d) δ 8.76 (s, 1H), 8.50 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 7.41 (s, 1H), 7.09 (s, 1H), 5.76 (d, J = 12.4 Hz, 1H), 5.53 (d, J = 12.5 Hz, 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.

[0124] Example 18: Preparation of compound II8

[0125]

[0126] Reference to the synthesis method of Example 8. 1 H NMR (500 MHz, Chloroform-d) δ 9.26 (s, 1H), 8.50 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 7.41 (s, 1H), 7.09 (s, 1H), 4.83 (s, 1H), 4.53 (s, 1H), 3.80 (s, 3H), 3.49 (s, 2H), 3.33 (d, J = 3.7 Hz, 4H), 3.18 (d, J = 4.9 Hz, 1H), 2.68 (d, J = 5.1 Hz, 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.

[0127] Example 19: Preparation of compound II9

[0128]

[0129] Reference to the synthesis method of Example 9. 1H NMR (500 MHz, Chloroform-d) δ 9.35 (s, 1H), 8.54 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.5 Hz, 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 = 13.0 Hz, 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.

[0130] Example 20: Preparation of compound II 10

[0131]

[0132] Refer to the synthetic method of Example 10. 1 H NMR (500 MHz, Chloroform-d) δ 9.35 (s, 1H), 8.54 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.5 Hz, 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 = 13.0 Hz, 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 Cl2FN7O7, [M+H] + : 624.1098, found: 624.1095.

[0133] Example 21: Preparation of compound III1

[0134]

[0135] Refer to the synthetic method of Example 1. 1 ​H NMR (500 MHz, Chloroform-d) δ 9.04 (s, 1H), 7.58 (d, J = 10.6 Hz, 3H), 6.65 (s, 1H), 5.84 (s, 1H), 5.54 (s, 1H), 4.86 (d, J = 2.5 Hz, 2H), 4.51 (d, J = 7.8 Hz, 1H), 4.28 (s, 1H), 4.08 - 3.99 (m, 2H), 3.98 (d, J = 1.6 Hz, 6H), 3.78 - 3.73 (m, 1H), 3.61 (td, J = 12.0, 2.6 Hz, 1H), 2.92 (s, 6H), 2.10 (d, J = 12.9 Hz, 1H), 1.95 (d, J = 12.1 Hz, 1H). HRMS (ESI): m / z calcd for C 27 H 31 Cl2N7O6, [M+Na] + : 642.1605, found: 642.1598.

[0136] Example 22: Preparation of compound III2

[0137]

[0138] Reference to the synthesis method of Example 1. 1 H NMR (500 MHz, Chloroform-d) δ 9.04 (s, 1H), 7.59 (d, J = 7.8 Hz, 2H), 7.55 (d, J = 8.8 Hz, 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, 7.4 Hz, 2H), 3.98 (d, J = 1.8 Hz, 6H), 3.75 (d, J = 11.9 Hz, 1H), 3.65 - 3.57 (m, 1H), 3.04 (q, J = 7.1 Hz, 4H), 2.12 (d, J = 13.7 Hz, 1H), 1.93 (d, J = 12.6 Hz, 1H), 1.01 (t, J = 7.1 Hz, 6H). HRMS (ESI): m / z calcd for C 29 H 35 Cl2N7O6, [M+Na] + : 670.1918, found: 670.1915.

[0139] Example 23: Preparation of compound III3

[0140]

[0141] Reference is made to the synthesis method of Example 1. 1 H NMR (500 MHz, Chloroform-d) δ 9.05 (s, 1H), 7.58 (d, J = 10.1 Hz, 3H), 6.65 (s, 1H), 5.82 (s, 1H), 5.53 (s, 1H), 4.87 (s, 2H), 4.50 (d, J = 7.9 Hz, 1H), 4.28 (s, 1H), 4.04 (dd, J = 13.6, 4.9 Hz, 2H), 3.97 (d, J = 1.5 Hz, 6H), 3.77 - 3.73 (m, 1H), 3.61 (td, J = 11.9, 2.5 Hz, 1H), 3.27 (t, J = 5.6 Hz, 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.

[0142] Example 24: Preparation of compound III4

[0143]

[0144] Reference is made to the synthesis method of Example 1. 1 H NMR (300 MHz, Chloroform-d) δ 9.05 (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 = 13.2 Hz, 1H), 4.04 (dd, J = 10.3, 3.9 Hz, 2H), 3.98 (s, 6H), 3.77 (dd, J = 5.9, 3.6 Hz, 4H), 3.61 (td, J = 11.9, 2.5 Hz, 1H), 3.43 - 3.25 (m, 4H), 2.12 (d, J = 12.6 Hz, 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.

[0145] Example 25: Preparation of compound III5

[0146]

[0147] The synthetic method of Example 1 was referred to. 1 H NMR (300 MHz, Chloroform-d) δ 9.04 (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 = 3.2 Hz, 6H), 3.86 (d, J = 11.0 Hz, 1H), 3.78 (d, J = 7.8 Hz, 1H), 3.66 (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.

[0148] Example 26: Preparation of compound III6

[0149]

[0150] The synthetic method of Example 1 was referred to. 1 H NMR (500 MHz, Chloroform-d) δ 9.06 (s, 1H), 7.59 (d, J = 8.2 Hz, 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 = 11.8 Hz, 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.

[0151] Example 27: Preparation of compound III7

[0152]

[0153] Referring to the synthesis method of Example 7. 1 H NMR (500 MHz, Chloroform-d) δ 8.55 (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 = 12.4 Hz, 1H), 5.53 (d, J = 12.5 Hz, 1H), 4.44 (d, J = 12.3 Hz, 1H), 4.35 (d, J = 12.5 Hz, 1H), 4.16 (s, 1H), 4.05 (s, 1H), 3.99 (s, 1H), 3.86 (s, 6H), 3.80 (d, J = 13.2 Hz, 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.

[0154] Example 28: Preparation of compound III8

[0155]

[0156] Referring to the synthesis method of Example 8. 1 H NMR (500 MHz, Chloroform-d) δ 8.55 (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 = 3.1 Hz, 2H), 3.75 (s, 1H), 3.60 (s, 1H), 2.82 (d, J = 4.9 Hz, 1H), 2.75 (d, J = 4.9 Hz, 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.

[0157] Example 29: Preparation of compound III9

[0158]

[0159] The synthesis method is described in 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.

[0160] Example 30: Compound III 10 Preparation

[0161]

[0162] The synthesis method is described in Example 10. 1 H 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.

[0163] Example 31: Preparation of Compound IV

[0164]

[0165] Reference is made to the synthesis method of Example 10. 1 H NMR (500 MHz, Chloroform-d) δ 9.79 (s, 1H), 8.27 (d, J = 4.9 Hz, 1H), 7.84 (d, J = 0.9 Hz, 2H), 7.43 - 7.36 (m, 2H), 7.24 (d, J = 6.6 Hz, 3H), 7.09 (d, J = 17.7 Hz, 2H), 6.99 (s, 1H), 4.61 (s, 2H), 3.82 (d, J = 12.4 Hz, 6H), 3.69 (d, J = 12.5 Hz, 1H), 3.57 (d, J = 12.5 Hz, 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.

[0166] Example 32: Preparation of compound V

[0167]

[0168] Reference is made to the synthesis method of Example 10. 1 H NMR (500 MHz, Chloroform-d) δ 8.38 (d, J = 4.9 Hz, 1H), 7.75 (s, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.14 (dd, J = 7.5, 2.0 Hz, 1H), 7.06 (d, J = 4.9 Hz, 1H), 6.88 (dd, J = 7.5, 2.0 Hz, 1H), 3.75 (s, 1H), 3.61 (s, 2H), 3.56 (s, 2H), 3.24 (s, 1H), 3.19 (s, 1H), 3.09 (d, J = 0.9 Hz, 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.

[0169] Example 33: Covalent binding study of compound with Bruton's tyrosine kinase (BTK)

[0170] 1. Experimental method

[0171] 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 1 / 100-1 / 20 of the volume of the test compound was added from a 200 μM solution to obtain a 2 μM concentration of the test compound. The reaction mixture was injected into LC / MS at room temperature for different times. Data analysis was performed, and the original spectrum was deconvoluted using a 20000:40000 Da window and a 1 Da resolution. The labeling percentage of the compound was the labeling of the specific compound divided by the total protein detected.

[0172] 2. Experimental results

[0173] The results are shown in Figure 1 and Figure 2 From the results, it can be seen that the compound can reach complete binding with BTK at room temperature for 1 h, and the molecular weight calculation result is consistent with the addition-elimination release of NO mechanism proposed by the compound design.

[0174] Example 34: NO release study of the compound

[0175] 1. Experimental method

[0176] The Griess method was used to determine the NO release results of the target compound in different pH buffer salt solutions, target protein storage solutions, rat plasma, rat liver microsomes, etc. The NO fluorescent probe method was used in combination with flow cytometry to determine the NO release of the target compound in lymphoma cells Daudi.

[0177] 2. Experimental results

[0178] The results are shown in Figure 3 The NO release of I1 in the BTK storage solution was significantly higher than that in the rat liver microsomes, rat plasma and GSH solution, indicating that I1 has high release selectivity. The DAF-FM DA probe method was used to determine the NO release of I1 in cells, and compared with ibutinib, it has significant NO release in B-cell lymphoma Daudi cells.

[0179] Example 35: Stability study of the compound

[0180] 1. Experimental method

[0181] LC-MS method was used to investigate the stability of compounds I1-I6 in plasma, liver microsomes and GSH, etc.

[0182] 2. Experimental results

[0183] The results are shown inFigure 4 , indicating that compounds I1-I6 all have high in vitro stability.

[0184] Example 36: Study on in vitro kinase inhibition activity of compounds

[0185] 1. Experimental method

[0186] The ADP-Glo method was used to determine the kinase activity (IC 50 ) of compounds I1-I6, II1-II6 and III1-III6 on BTK, HER2 and Fgfr4, respectively. 50 μL of the test compound was added to a 384-well dilution plate, and the test compound was diluted 5 times in DMSO at a ratio of 1:3 in each column. 0.025 μL of the diluted test compound solution was transferred to a 384-well detection plate using an Echo, 2.5 μL of enzyme working solution was added to the 384-well detection plate, 1000 rpm was centrifuged for 1 minute, 2.5 μL of substrate (ATP and substrate) working solution was added to start the reaction, and the reaction was incubated at 25°C for 0, 2, 4, 8, 15, 30, 60, 90 minutes, respectively. 5 μL of TK Beads solution was added to initiate the reaction, and the reaction was incubated at 25°C for 60 minutes. The fluorescence signals at 665 nm and 620 nm were read and the BMG (ratio 665 / 620) was determined.

[0187] 2. Experimental results

[0188] The results are shown in Tables 1, 2 and 3

[0189] Table 1. Results of BTK kinase inhibition activity of compounds I1-I6

[0190]

[0191] Table 2. Results of HER2 kinase inhibition activity of compounds II1-II6

[0192]

[0193] Table 3. Results of Fgfr4 kinase inhibition activity of compounds III1-III6

[0194]

[0195] Example 37: Study on in vitro anti-tumor cell proliferation activity of compounds

[0196] 1. Experimental method

[0197] The inhibitory activities of compounds I1-I6, II1-II6 and III1-III6 on tumor cell lines were tested by 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), and all cell lines were placed in a Shellab 2323-2 CO2 incubator for culture, with conditions of 5% CO2-containing air and a temperature of 37°C. Cell activity was determined by CCK8 (Beyotime) method. Cells were seeded in a 96-well plate at a density of 8000-10000 / well, and after incubation in a constant temperature incubator for 24 h, the cells were observed under a microscope to be in good condition and almost completely adherent growth. After adding different concentrations of compounds or 0.1% DMSO, the culture was continued for 48 h. After 48 h, CCK8 reagent was added, and after two hours of further incubation, the absorbance OD value of each well at 450 nM wavelength was determined by Envision 2104 multifunctional microplate analyzer (Perkin Elmer), and finally the IC 50 values were calculated by processing the dose-effect curve with GraphPad 8.0. Among them, the cell survival rate = [(experimental group-blank control group) / (control group-blank control group)]*100%. Among them, the experimental group: cells+CCK8 solution+drug solution; the control group: cells+CCK8 solution+0.1% DMSO; the blank control group: no cells+CCK8 solution. Three parallel duplicate wells were set in each group, and each experiment was repeated three times.

[0198] 2、Experimental results

[0199] The results are shown in Tables 4, 5 and 6.

[0200] Table 4. Test results of in vitro anti-tumor activity of compounds I1-I8 (IC 50 , μM)

[0201]

[0202] Table 5. Test results of in vitro anti-tumor activity of compounds II1-II6

[0203]

[0204] Table 6. Test results of in vitro anti-tumor activity of compounds III1-III6

[0205]

[0206] Example 38: Study on the binding of compounds to kinases

[0207] 1、Experimental method

[0208] Kinact / ki parameters of I1 were tested using HTRF / ADP-Glo / FI method. 50 μL I1 was added to 384-well dilution plate, 5-fold serial dilution of I1 in DMSO per column, 0.025 μL diluted I1 solution per row was transferred to 384 assay plate using Echo, 2.5 μL enzyme working solution was added to 384-well assay plate, 1000 rpm centrifugation for 1 min, 2.5 μL substrate (ATP and substrate) working solution was added to initiate the reaction, 0, 2, 4, 8, 15, 30, 60, 90 min incubation at 25 °C, 5 μL TK Beads solution was added to initiate the reaction, 25 °C incubation for 60 min, read 665 nm and 620 nm fluorescence signal with BMG (ratio: 665 / 620).

[0209] 2. Experimental results

[0210] The results are shown in Table 1, which indicate that the modification of the warhead part of I1 increases the steric hindrance, making the first step of non-covalent binding slower, but the modification of the warhead part does not significantly affect the binding potency of I1 to BTK. Figure 5

[0211] Example 39: Kinase profiling of compounds

[0212] 1. Experimental method

[0213] Kinase profiling of compound I1 was tested using ADP-Glo Kinase Assay. 2x ATP and substrate solution and 2x kinase and metal solution were prepared using assay buffer, 20 nL compound was transferred to 384 assay plate by Echo 655. 2 μL of 2x kinase and metal solution was added, mixed and incubated at 25 °C for 10 min in 384 assay plate. 2 μL of 2x substrate and ATP solution was added to the well and incubated at 25 °C for 60 min. 4 μL of ADP-Glo Reagent was added to the well and incubated at 25 °C for 40 min. 8 μL of Kinase Detection Reagent was added to the well and incubated at 25 °C for 40 min. The signal was recorded on a microtiter plate reader.

[0214] 2. Experimental results

[0215] The results are shown in Table 1, which indicate that the modification of the warhead part of I1 increases the steric hindrance, making the first step of non-covalent binding slower, but the modification of the warhead part does not significantly affect the binding potency of I1 to BTK. Figure 6

[0216] Example 40: In vivo activity study of compounds​​

[0217] 1. Experimental Methods

[0218] 150 μL of TMD8 cell suspension (6 × 10⁻⁶ in PBS) was added. 6 (1 cell) was subcutaneously injected into the right back of 8-week-old female BALB / c nude mice. When the average tumor volume reached approximately 60 mm², the tumor was... 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 preserved for further research. Tumor size was calculated using the formula: V(tumor volume, mm). 3 = L (length, mm) × W2 (width, mm) × 0.5. The tumor growth inhibition rate (TGI) is calculated using the following formula: TGI = (1 - TWt / TWc) × 100%, where TWt and TWc represent the mean tumor weight on day 20 in the treatment group and the control group, respectively.

[0219] 2. Experimental Results

[0220] The results are as follows Figure 7 Compared with the blank control group, the experimental group A12 (15mg / kg) significantly inhibited tumor growth. Furthermore, the mice were in good health during the experiment, with normal activity levels, normal food and water intake, normal skin luster and color, no diarrhea, and no inflammation at the tumor site.

[0221] Example 41: Co-localization study of NO release from the compound and Bruton's tyrosine kinase (BTK)

[0222] 1. Experimental Methods

[0223] A BTK-mCherry lentiviral vector (excitation wavelength 587 nm, emission wavelength 610 nm) using pKG-CMV-MCS-EF1-Puro as the vector was constructed. Daudi cells were cultured and transfected with lentiviral agent for 48 h. Then, DAF-FM DA (excitation wavelength 495 nm, emission wavelength 515 nm) was added and incubated for 15 min. The cells were washed three times to remove excess dye, and then the compound was added for incubation at final concentrations of 1 μM, 5 μM, and 10 μM. Incubation was performed for 5 min, 30 min, and 60 min, 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 min. The cells were then washed three times with PBS. Smears were prepared and observed under a focusing microscope at 600x magnification.

[0224] 2. Experimental Results

[0225] The results are as followsFigure 8 The results show that NO release has a clear subcellular co-localization with BTK. Subsequent addition of the NO scavenger PTIO shows that NO is scavenged and there is no NO at BTK by confocal imaging. The above results show that I1 releases NO in situ and in a very small range at BTK in the cell.

[0226] Example 42: Study on the increase of nitrosylation and the decrease of phosphorylation / phosphorylation signal transduction of Bruton's tyrosine kinase (BTK) caused by NO release of the compound

[0227] 1. Experimental method

[0228] Daudi cells were cultured, and cell suspensions were incubated with 1-10 μM of each group of compounds for 1-5 h, and a DMSO group, an Ibrutinib group, an I1 group, and an azoium group were set. After Biotin-Switch conversion, Streptavidin Agarose (lifetechnologies, item number: SA100-04) was used for purification, and then the nitrosylation of the target protein was characterized. At the same time, the same group of cells was used to evaluate the influence of nitrosylation on the phosphorylation degree of the target protein by WB, and the crosstalk mechanism of kinase nitrosylation and phosphorylation was revealed.

[0229] 2. Experimental results

[0230] The results are as follows Figure 9 After incubation of the compound with Daudi cells, I1 caused a significant increase in BTK nitrosylation compared with Ibrutinib, but did not cause a global increase in nitrosylation compared with azoium glycolate (non-selective NO donor). The influence of I1 on the phosphorylation of BTK and kinase B (AKT) interacting with BTK was tested compared with Ibrutinib, and the results showed that I1 caused a significant decrease in the phosphorylation of BTK and AKT compared with Ibrutinib, indicating that the nitrosylation and covalent inhibition brought by NO played a synergistic role in inhibiting phosphorylation.

Claims

1. A covalent kinase inhibitor, characterized in that, having the structure of Formula I or a pharmaceutically acceptable salt thereof, ， wherein: R1, R2 are selected from C1-C4 alkyl or R1, R2 together with the nitrogen to which they are attached form a 4-7 membered heterocyclic ring, said 4-7 membered heterocyclic ring being a morpholine ring, a piperidine ring, a tetrahydropyrrole ring, and the ring system carbon atoms are substituted with at least one hydrogen, hydroxyl, fluorine, chlorine, bromine, methyl, ethyl, trifluoromethyl, hydroxymethyl, hydroxyethyl; the kinase inhibitor parent nucleus is selected from: 。 2. The covalent kinase inhibitor of claim 1, wherein, in the structure: R1, R2 are selected from methyl, ethyl.

3. The covalent kinase inhibitor of claim 1, wherein, in the structure: NR1R2 is selected from dimethylamino, methylethylamino, diethylamino, morpholino, tetrahydropyrrolo, 2-hydroxymethyltetrahydropyrrolo, piperidino, 4-hydroxypiperidino.

4. The covalent kinase inhibitor of claim 1, wherein, is selected from any one of the following compounds: 。 5. The covalent kinase inhibitor of claim 1, wherein, the pharmaceutically acceptable salt is a salt of the compound of Formula I with any one 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, comprising the following steps: (i) reacting compound 1 in the presence of paraformaldehyde, triethylenediamine, phenol to obtain intermediate 2; (ii) reacting intermediate 2 in the presence of p-toluenesulfonyl chloride, triethylamine to obtain intermediate 3; (iii) reacting intermediate 3 with the corresponding azonia salt of glycol in the presence of 15-crown-5-ether to obtain the final product 4; ； wherein R1, R2, the definition of the kinase inhibitor parent nucleus is as described in claim 1; salting the corresponding acid with the final product 4 prepared by the above method to obtain the pharmaceutically acceptable salt.

7. A pharmaceutical composition, characterized by, comprising the covalent kinase inhibitor of claim 1 and a pharmaceutically acceptable carrier.

8. Use of the covalent kinase inhibitor of claim 1 or the pharmaceutical composition of claim 7 in the manufacture of a medicament for treating and / or preventing a proliferative disease selected from the group consisting of malignant lymphoma, monocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia.

9. Use of the covalent kinase inhibitor of claim 1 or the pharmaceutical composition of claim 7 in the manufacture of a medicament for delaying the progression of a proliferative disease, or alleviating the symptoms of a proliferative disease selected from the group consisting of malignant lymphoma, monocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia.

10. Use according to claim 8 or 9, characterized in that, the medicament has BTK kinase inhibitory activity.

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