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WO2024244029A1 - 共价激酶抑制剂、制备方法、药物组合物和应用 - Google Patents

共价激酶抑制剂、制备方法、药物组合物和应用 Download PDF

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WO2024244029A1
WO2024244029A1 PCT/CN2023/098265 CN2023098265W WO2024244029A1 WO 2024244029 A1 WO2024244029 A1 WO 2024244029A1 CN 2023098265 W CN2023098265 W CN 2023098265W WO 2024244029 A1 WO2024244029 A1 WO 2024244029A1
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kinase inhibitor
acid
covalent
cancer
kinase
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French (fr)
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黄张建
吴建兵
张奕华
张宸
叶辉
纪多睿
熊涛
李存睿
梁壮壮
袁逊
孙玉泽
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中国药科大学
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    • C07D487/04—Ortho-condensed systems
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
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    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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    • C07B2200/07—Optical isomers

Definitions

  • the present invention relates to a kinase inhibitor based on NO/ NO2 - releasing covalent warhead, a preparation method, a pharmaceutical composition and an application, and in particular to a kinase inhibitor based on NO/ NO2 - releasing covalent warhead with synergistic effect, a preparation method, a pharmaceutical composition and an application.
  • NO is a water-soluble free radical gas in organisms with redox properties, playing an important role in physiology and pathology. It plays an extremely important biological role in cardiovascular and cerebrovascular regulation, nerve and immune regulation.
  • NO 2 — is one of the metabolites of NO in the body, which 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 mediated by NO 2 — .
  • NO/NO 2 -donor drugs generally refer to prodrugs formed by NO/NO 2 -donors and related drugs or certain active compounds through various connecting groups.
  • NO donors include nitrosothiol, nitrate ester, NO-metal complex (nitroprusside), furazan N-oxide, azodiol salt, etc.;
  • NO 2 -donors include 2-nitromethyl-1,3-diphenyl-2-ene-1-one, etc.
  • Covalent kinase inhibitors irreversibly bind to target kinase residues (generally Cys) through covalent bonds, thereby exerting target inhibition function and achieving the purpose of disease treatment. They generally contain a less reactive bonding functional group (warhead group) and a ligand part. After the ligand locates the inhibitor at the corresponding site of the target kinase, the warhead group reacts with Cys to form a covalent bond.
  • warhead group a less reactive bonding functional group
  • NO/NO 2 donor drugs and covalent inhibitors have experienced great development, they still face many shortcomings.
  • NO/NO 2 donor drugs
  • the mechanism of triggering NO/NO 2 release currently relies on chemical and biological means such as light control and sound control; NO/NO 2 —release has low specificity and poor precision.
  • the released NO/NO 2 is uncertain at the molecular level due to its free diffusion nature, and NO/NO 2 —will act on multiple biomacromolecules and produce more side effects. Therefore, for NO/NO 2 —donor drugs, it is still necessary to explore NO/NO 2 —release modes with higher specificity and better precision.
  • covalent inhibitors Although they have a high target occupancy rate, this also leads to a single mode of action and a lack of effective synergistic treatment mechanism.
  • existing covalent inhibitors also face the disadvantages of low bioavailability, high frequency of administration, large dosage, and easy to produce target Cys mutation resistance. Therefore, for covalent inhibitors, exploring new synergistic mechanisms may help enhance the efficacy and overcome the above shortcomings to a certain extent.
  • the first objective of the present invention is to provide a kinase inhibitor based on NO/ NO2 - releasing covalent warheads; the second objective is to provide a method for preparing the covalent kinase inhibitor; the third objective is to provide a pharmaceutical composition containing the covalent kinase inhibitor; and the fourth objective is to provide an application of the covalent kinase inhibitor and its pharmaceutical composition.
  • the covalent kinase inhibitor of the present invention has a structure of Formula I, II, III, IV or V, and also includes a pharmaceutically acceptable salt thereof,
  • R 1 and R 2 are selected from C1-C4 alkyl or R 1 and R 2 and the nitrogen to which they are connected together form a 4-7 membered heterocyclic ring containing 1-2 nitrogen, oxygen, and 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:
  • the present invention designs a kinase inhibitor based on NO/NO 2 — releasing covalent warhead, wherein the covalent kinase inhibitor with the structure of formula I has an O 2 -( ⁇ -methylene- ⁇ , ⁇ -unsaturated ketone)-azodiol salt warhead structure, which can covalently bind to the specific thiol group of protein kinase, and remove the O 2 protection of the azodiol salt by covalent binding-mediated addition-elimination reaction, triggering NO to be released in situ in a very small range.
  • the covalent kinase inhibitor with the structure of formula II has an ⁇ -nitromethyl- ⁇ , ⁇ -unsaturated ketone warhead structure, and uses the same mechanism to mediate the release of NO 2 — in a very small range by covalent binding reaction.
  • the covalent kinase inhibitor with the structure of formula III, IV, and V has a ring strain nitro substituted warhead structure, which is mediated by ring strain and releases NO 2 — in a very small range after a nucleophilic substitution reaction with the specific thiol group of protein kinase.
  • the present invention has made a new design for NO/NO 2 — releasing warhead groups, so that the kinase inhibitors covalently bound to these warhead groups can release NO by removing the azodiol salt fragment O 2 protection through addition-elimination reaction after nucleophilic attack in the presence of thiol-containing target proteins in vitro/in vivo, and release NO 2 — through the same mechanism or ring tension mechanism.
  • covalent binding can play a covalent inhibitory role on thiol-containing target enzymes, and on the other hand, the released NO/NO 2 — can further produce nitrosylation modification on target kinases and interacting kinases, inhibit their autophosphorylation, prevent kinase signal transduction, and play a synergistic role.
  • the compounds designed by the present invention have good covalent binding triggering specificity, high NO/NO 2 -releasing efficiency, good biological activity, and the covalent binding and NO/NO 2 -synergistic effect thereof can effectively overcome the deficiencies of existing NO/NO 2 -donor drugs and covalent inhibitors. At the same time, due to the synergistic effect, the dosage of the drug can be reduced, thereby improving the compliance of the patient, and significantly improving the safety and reducing the toxic side effects.
  • diazenium diolates have obvious advantages in selective and targeted release of NO.
  • diazenium diolates are very unstable under physiological conditions. It can automatically release 1-2 molecules of NO, and its half-life ranges from a few seconds to a few hours.
  • the covalent kinase inhibitor of formula II can also selectively and targetedly release NO2- through the same addition- elimination reaction mechanism.
  • the covalent kinase inhibitors of formula III, IV, and V are selectively and targetedly released by ring tension.
  • the present invention is based on the application of covalent inhibition therapy and NO/ NO2 - donor drugs in the field of anti-tumor therapy, and utilizes the chemical group release advantage of the covalent inhibitor warhead group to obtain a kinase inhibitor based on NO/ NO2 - releasing covalent warhead, and at the same time designs a new O2 protection method for a class of azodiol salt drugs.
  • the prodrug deprotection is carried out 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, KRAS G12C.
  • R 1 and R 2 are selected from methyl, ethyl, or R 1 , R 2 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;
  • NR 1 R 2 is further preferably selected from dimethylamino, methylethylamino, diethylamino, morpholinyl, tetrahydropyrrolyl, 2-hydroxymethyltetrahydropyrrolyl, piperidinyl, and 4-hydroxypiperidinyl.
  • the covalent kinase inhibitor is most preferably selected from any of the following compounds:
  • the above 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.
  • the method for preparing the covalent kinase inhibitor of the present invention comprises the following steps:
  • intermediate 2 is reacted in the presence of p-toluenesulfonyl chloride and triethylamine to obtain intermediate 3;
  • intermediate 10 reacts with urea peroxide and trifluoroacetic anhydride to obtain intermediate 11;
  • R 1 , R 2 , and the kinase inhibitor nucleus are as defined above;
  • the corresponding acid is reacted with the kinase inhibitor prepared by the above method to form a salt, thereby obtaining the pharmaceutically acceptable salt.
  • the covalent kinase inhibitor of the present invention and a pharmaceutically acceptable carrier form the pharmaceutical composition of the present invention, and can be specifically added with common pharmaceutical excipients such as flavorings, sweeteners, liquid/solid fillers, diluents, etc. to prepare common pharmaceutical preparations, such as tablets, capsules, syrups, suspensions, injections, etc.
  • common pharmaceutical excipients such as flavorings, sweeteners, liquid/solid fillers, diluents, etc.
  • the covalent kinase inhibitor and the pharmaceutical composition thereof described in 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 of proliferative diseases and managing proliferative diseases.
  • the proliferative disease is selected from tumors, rheumatic diseases, chronic inflammation, infectious mononucleosis.
  • the proliferative disease is selected from:
  • 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 inflammation, 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 gamma Gammopathies, osteolytic lesions, acute lymphocytic leukemia,
  • This type of covalent kinase inhibitor can effectively covalently bind to the target enzyme and can effectively release NO/NO 2 — ; on the one hand, the covalent binding can play a covalent inhibitory role on the sulfhydryl-containing target enzyme, and on the other hand, the released NO/NO 2 — can further produce nitrosylation modification on the target kinase and the interacting kinase, inhibit its autophosphorylation, prevent kinase signal transduction, and exert a synergistic effect; and it is selective and reduces non-target inhibition;
  • This type of covalent kinase inhibitor has significant inhibitory activity at the molecular level, cellular level and animal level, acting on a variety of enzymes related to malignant proliferation and malignant proliferating cells, and inhibiting tumor growth in animals without producing obvious adverse reactions;
  • the preparation method of the compound is simple and universal, which is conducive to the expansion of various structures.
  • FIG1 is a mass spectrometry verification result of the covalent binding of compound I1 to Bruton's tyrosine kinase (BTK);
  • FIG2 is a mass spectrometry verification result of the covalent binding of I3 compound to Bruton's tyrosine kinase (BTK);
  • 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;
  • FIG4 is the stability test results of compounds I 1 -I 6 in rat plasma and GSH solution
  • FIG5 is a test result of the Kinact/Ki parameter of compound I1 ;
  • FIG6 is the kinase spectrum test result of compound I1 ;
  • FIG7 is the result of the nude mouse experiment of compound I1 ;
  • FIG8 is the verification result of the co-localization of NO release and Bruton's tyrosine kinase (BTK) of compound I1 in cells;
  • FIG. 9 shows the results that NO released by compound I 1 causes increased nitrosylation and decreased phosphorylation/blocked phosphorylation signal transduction of Bruton's tyrosine kinase (BTK).
  • BTK Bruton's tyrosine kinase
  • the covalent binding of I 1 and I 3 to Bruton's tyrosine kinase (BTK) was determined by mass spectrometry.
  • the target protein was diluted to 2 ⁇ M in buffer, and then 1/100-1/20 of the test compound was added from the 200 ⁇ M solution to obtain a 2 ⁇ M concentration of the test compound.
  • the reaction mixture was injected into the LC/MS at different times at room temperature. For data analysis, the raw spectra were deconvoluted using a 20000:40000Da window and 1Da resolution.
  • the labeling percentage of the compound is the labeling of the specific compound divided by the total protein detected.
  • the NO release of the target compound was determined by Griess method 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 determine the NO release of the target compound in lymphoma Daudi cells.
  • Example 36 In vitro kinase inhibitory activity study of compounds
  • kinase activities (IC 50 ) of compounds I 1 -I 6 , II 1 -II 6 and III 1 -III 6 against BTK, HER2 and Fgfr4 were determined using the ADP-Glo method. 50 ⁇ L of the test compound was added to a 384-well dilution plate, and each column was serially diluted 5 times in DMSO at a ratio of 1:3.
  • 0.025 ⁇ L of the diluted test compound solution in each row was transferred to the 384
  • 2.5 ⁇ L of enzyme working solution was added to a 384-well assay plate, centrifuged at 1000 prm for 1 min, 2.5 ⁇ L of substrate (ATP and substrate) working solution was added to start the reaction, incubated at 25°C for 0, 2, 4, 8, 15, 30, 60, and 90 min, 5 ⁇ L of TK Beads solution was added to initiate the reaction, incubated at 25°C for 60 min, and the fluorescence signals at 665 nm and 620 nm were read with BMG (ratio 665/620).
  • the CCK8 method was used to test the inhibitory activity of compounds I 1 -I 6 , II 1 -II 6 and III 1 -III 6 on tumor cell lines.
  • 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, MDA-MB-231 and HeLa cells were cultured in DMEM medium). All cell lines were cultured in a Shellab 2323-2 CO 2 constant temperature cell culture incubator under the following conditions: air containing 5% CO 2 at a temperature of 37°C. Cell activity was determined using the CCK8 (Beyotime) method.
  • the cells were seeded in a 96-well plate at a cell density of 8000-10000/well. After incubation in a constant temperature incubator for 24 hours, it was observed under a microscope that the cells were in good condition and almost completely adherent to the wall. Different concentrations of compounds or 0.1% DMSO were added and cultured for 48 hours. After 48 hours CCK8 reagent was added, and the culture was continued for two hours. The absorbance OD value of each well at a wavelength of 450 nM was measured using Envision 2104 multifunctional microwell analyzer (Perkin Elmer), and the IC 50 value was calculated by fitting the dose-effect curve using GraphPad 8.0.
  • cell survival rate [(experimental group-blank control group)/(control group-blank control group)]*100%.
  • 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.
  • the Kinact/ki parameter of I 1 was tested using the HTRF/ADP-Glo/FI method. 50 ⁇ L of I 1 was added to a 384-well dilution plate, and each column was serially diluted 1:3 in DMSO by 5 times. 0.025 ⁇ L of diluted I 1 solution was transferred to a 384-well assay plate using Echo. 2.5 ⁇ L of enzyme working solution was added to the 384-well assay plate, and centrifuged at 1000 prm for 1 minute.
  • Compound I 1 was subjected to kinase profiling using the ADP-Glo Kinase Assay.
  • TMD8 cell suspension 150 ⁇ L of TMD8 cell suspension (6 ⁇ 10 6 cells in PBS) was subcutaneously inoculated into the right back of 8-week-old female BALB/c nude mice.
  • n 8/group
  • a compound I 1 group 15 mg/kg, PO, BID
  • Tumor volume and body weight were recorded every other day after administration. After 20 days, all mice were killed and the tumors were retained for further study.
  • the size of the tumor was calculated by the formula.
  • V (tumor volume, mm 3 ) L (length, mm) ⁇ W2 (width, mm) ⁇ 0.5.
  • the experimental group A12 (15 mg/kg) can significantly inhibit the growth of tumors.
  • the mice were in good health, with normal activities, normal water and food intake, normal skin gloss and color, no diarrhea, and no inflammation at the tumor site.
  • Example 41 Co-localization study of NO release of compounds and Bruton's tyrosine kinase (BTK)
  • BTK-mCherry (excitation wavelength 587nm, emission wavelength 610nm) lentiviral vector with pKG-CMV-MCS-EF1-Puro as the carrier.
  • Culture Daudi cells transfect them with lentivirus for 48h, add DAF-FM DA (excitation wavelength 495nm, emission wavelength 515nm) and incubate for 15min, discard and wash 3 times, wash off the excess dye, add compounds and incubate, so that the final concentration of the compounds is 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, etc.; incubate the compounds for 5min/30min/60min, etc.; after incubation, add PBS and wash 3 times; add 300 ⁇ L of ready-to-use DAPI dye solution, incubate at room temperature in the dark for 10min; wash 3 times with PBS. Smear, observe the cells under a focusing microscope, and observe the images at 600 times.
  • Example 42 NO release from compounds causes increased nitrosylation and phosphorylation of Bruton's tyrosine kinase (BTK) Study on decreased phosphorylation/blocked phosphorylation signal transduction
  • BTK Bruton's tyrosine kinase
  • Daudi cells were cultured, and the cell suspension was incubated with 1-10 ⁇ M of each group of compounds for 1-5 hours.
  • DMSO group, ibrutinib group, I1 group, and azo group were set up.
  • Streptavidin Agarose lifetechnologies, catalog number: SA100-04
  • the same group of cells was used to simultaneously evaluate the effect of nitrosylation on the phosphorylation degree of the target protein by WB, revealing the crosstalk mechanism between kinase nitrosylation and phosphorylation.

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Abstract

本发明公开了一种共价激酶抑制剂、制备方法、药物组合物和应用，该共价激酶抑制剂的结构如式(I)、(II)、(III)、(IV)或(V)，还包含其药学上可接受的盐，其可与靶激酶高效共价结合，同时触发NO/NO2 —的释放；一方面共价结合可对含巯基靶激酶的共价抑制，另一方面所释放的NO/NO2 —可进一步对靶激酶或与其相互作用的蛋白产生巯基亚硝基化修饰，进而抑制其自身磷酸化，阻止激酶信号转导，发挥协同作用；可作用于多种与恶性肿瘤增殖相关的激酶，具有较好的选择性和较低的脱靶效应，在细胞和动物水平显著地抑制肿瘤细胞地生长，并且无明显不良反应。此外，制备方法简便、通用，有利于其他多种激酶抑制剂母核结构的扩展。

Description

共价激酶抑制剂、制备方法、药物组合物和应用 技术领域
本发明涉及一种基于NO/NO2 —释放型共价弹头的激酶抑制剂、制备方法、药物组合物和应用，尤其涉及一种具有协同增效作用的基于NO/NO2 —释放型共价弹头的激酶抑制剂、制备方法、药物组合物和应用。
背景技术
NO是生物体内一种水溶性的自由基气体，具有氧化还原特性，在生理、病理方面扮演重要角色。在心脑血管调节、神经、免疫调节等方面扮演着极其重要的生物学作用。NO2 —是NO在体内的代谢产物之一，其在缺血缺氧条件下可被还原为NO，在某些情况下，其也可被视为一类NO供体。NO可与蛋白半胱氨酸巯基发生反应，介导蛋白半胱氨酸巯基亚硝基化(SNO)，这是一种非常重要的翻译后修饰。值得一提的是，SNO也可由NO2 —介导发生。
NO/NO2 —供体型药物一般是指由NO/NO2 —供体及相关药物或某种活性化合物通过各种连接基团而形成的前药。现已发现多种结构类型的NO供体，如亚硝基硫醇、硝酸酯、NO-金属复合物(硝普盐)、呋咱N-氧化物、偶氮鎓二醇盐等；NO2 —供体包括2-硝甲基-1,3-二苯基-2-烯-1-酮等。
共价激酶抑制剂通过共价键与靶激酶残基(一般为Cys)发生不可逆结合，从而发挥靶标抑制功能，实现疾病治疗目的。其一般含有一个反应性较低的成键官能团(弹头基团)和一个配体部分，配体将抑制剂定位于靶激酶相应位点后，弹头基团与Cys反应，形成共价键。
虽然NO/NO2 —供体药物及共价抑制剂经历了长足的发展，但仍面临诸多不足。例如，对于NO/NO2 —供体药物而言，其触发NO/NO2 —释放的机制目前多依赖于光控、声控等化学生物学手段；NO/NO2 —释放的特异性低、精准度差，所释放的NO/NO2 —由于其自由扩散性质导致分子层面靶标的不确定，NO/NO2 —将会作用于多个生物大分子产生较多的副作用。因此，对于NO/NO2 —供体药物，仍需探索特异性更高、精准度更佳的NO/NO2 —释放模式。
对于共价抑制剂而言，其虽有较高的靶标占有率，但同时这也导致了其作用模式单一，缺少有效的协同治疗机制；此外，现有共价抑制剂也面临着生物利用度较低、给药频次高、给药剂量大和较易产生目标Cys突变耐药等缺点。因此，对于共价抑制剂，探索新的协同作用机制，可能有助于增强药效，在一定程度上克服上述缺点。
发明内容
发明目的：本发明的第一目的是提供一种基于NO/NO2 —释放型共价弹头的激酶抑制剂；第二目的是提供一种所述共价激酶抑制剂的制备方法；第三目的是提供一种含有所述共价激酶抑制剂的药物组合物，第四目的是提供一种所述共价激酶抑制剂及其药物组合物的应用。
技术方案：本发明所述的共价激酶抑制剂具有式I、II、III、IV或V的结构，还包含其药学上可接受的盐，

其中：
R1、R2选自C1-C4烷基或者R1、R2与其连接的氮共同形成4-7元杂环，所述杂环含有1-2个氮、氧、硫杂原子，并且环系碳原子被至少一个氢、羟基、卤素、C1-C4烷基、卤素取代的C1-C4烷基、羟基取代的C1-C4烷基取代；
激酶抑制剂母核选自：
本发明设计了一种基于NO/NO2 —释放型共价弹头的激酶抑制剂，其中具有式I结构的共价激酶抑制剂具有O2-(α-亚甲基-α,β-不饱和酮)-偶氮鎓二醇盐弹头结构，可与蛋白激酶特定巯基发生共价结合，并且由共价结合介导加成-消除反应脱去偶氮鎓二醇盐的O2保护，触发NO在极小范围内原位释放。具有式II结构的共价激酶抑制剂具有α-硝甲基-α,β-不饱和酮弹头结构，使用同样机理，由共价结合反应介导NO2 —在极小范围内释放。具有式III、IV、V结构的共价激酶抑制剂具有环张力硝基取代弹头结构，由环张力介导，与蛋白激酶特定巯基发生亲核取代反应后，在极小范围内释放NO2 —。
本发明对NO/NO2 —释放型弹头基团进行了全新的设计，使共价结合这些弹头基团的激酶抑制剂在体外/体内含巯基的靶标蛋白质存在下，经亲核进攻后通过加成-消除反应脱去偶氮鎓二醇盐片段O2保护，释放NO，以及通过相同机理或环张力机理释放NO2 —。一方面共价结合可起到对含巯基靶标酶的共价抑制作用，另一方面所释放的NO/NO2 —可进一步对靶标激酶及相互作用激酶产生亚硝基化修饰，抑制其自身磷酸化，阻止激酶信号转导，发挥协同作用。
本发明设计的化合物共价结合触发特异性较好，NO/NO2 —释放效率高，生物活性好，其具有的共价结合及NO/NO2 —的协同作用，可有效克服现有NO/NO2 —供体药物和共价抑制剂的不足。同时由于协同效应可以减少服用药物的剂量，从而提高患者服药依从性，并能显著提高安全性并降低毒副作用。
在式I的共价激酶抑制剂的设计中，偶氮鎓二醇盐(diazeniumdiolates)在选择性和靶向性释放NO方面具有明显优势。一方面是偶氮鎓二醇盐在生理条件下极易不稳定， 能自动释放1～2分子NO，其半衰期从几秒钟到几小时不等。另一方面，将偶氮鎓二醇盐的O2位(与氮鎓离子相连的O称之为O1，与烯键氮原子相连的O为O2)保护后，可转化为稳定的前药；通过特定条件去除O2保护基团，转化为不稳定的偶氮鎓二醇盐阴离子，从而达到选择性和靶向性释放NO。同时，式II的共价激酶抑制剂也可通过相同的加成-消除反应机理进行选择性和靶向性NO2 —释放。此外，式III、IV、V的共价激酶抑制剂则由环张力介导选择性和靶向性NO2 —释放。
本发明基于共价抑制疗法以及NO/NO2 —供体药物在抗肿瘤治疗领域的应用，利用共价抑制剂弹头基团的化学基团释放优势，得到基于NO/NO2 —释放型共价弹头的激酶抑制剂，并同时设计出一类偶氮鎓二醇盐药物的新O2保护方法。其按照如式1或式2机理进行前药脱保护，各HS-R3选自小分子巯基化合物谷胱甘肽、半胱氨酸及蛋白质BTK、HER1、HER2、Fgfr4、HER3、HER4、KRAS G12C中的一种。
式1：
式2：
优选，所述结构中，R1、R2选自甲基、乙基或者R1、R2与其连接的N共同形成5-6元杂环，所述杂环为吗啉环、哌啶环、四氢吡咯环，并且环系碳原子被至少一个氢、羟基、氟、氯、溴、甲基、乙基、三氟甲基、羟甲基、羟乙基取代；NR1R2进一步优选自二甲氨基、甲基乙基氨基、二乙氨基、吗啡啉基、四氢吡咯基、2-羟甲基四氢吡咯基、哌啶基、4-羟基哌啶基。
具体地，所述共价激酶抑制剂最优选自以下任一化合物：


进一步地，上述共价激酶抑制剂与以下任一酸形成其药效上可接受的盐：盐酸、氢溴酸、硫酸、磷酸、碳酸、甲磺酸、苯磺酸、对甲苯磺酸、萘磺酸、樟脑磺酸、柠檬酸、酒石酸、苹果酸、乳酸、丙酮酸、乙酸、马来酸、琥珀酸、富马酸、水杨酸、苯基乙酸、阿魏酸或杏仁酸。
本发明所述的共价激酶抑制剂的制备方法，包含以下步骤：
(1)当所述共价激酶抑制剂具有式I或II的结构式时，
(i)化合物1在多聚甲醛、三乙烯二胺、苯酚的存在下反应得到中间体2；
(ii)中间体2在对甲苯磺酰氯、三乙胺的存在下反应得到中间体3；
(iii)中间体3在15-冠-5-醚的催化下与相应偶氮鎓二醇盐或AgNO2反应得到终产物4或5；
(2)当所述共价激酶抑制剂具有式III的结构式时，
(i)激酶抑制剂母核6在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的催化下与2,3二溴丙酸反应得到化合物7；
(ii)化合物7与硝基甲烷反应得到终产物8；
(3)当所述共价激酶抑制剂具有式IV的结构式时，
(i)3-氧代环丁基羧酸叔丁酯在盐酸羟胺、醋酸钠的存在下反应得到肟中间体10；
(ii)中间体10与过氧化脲、三氟醋酸酐反应得到中间体11；
(iii)中间体11与激酶抑制剂母核6反应得到终产物12；
(4)当所述共价激酶抑制剂具有式V的结构式时，激酶抑制剂母核6与2-溴-1-(3,3- 二硝基吖丁啶-1-基)乙酮反应得到终产物13；
其中，R1、R2、激酶抑制剂母核的定义如前；
将相应的酸与以上方法制备的激酶抑制剂成盐，即得所述药学上可接受的盐。
本发明所述的共价激酶抑制剂以及药学上可接受的载体形成本发明所述的药物组合物，具体可以添加香料、甜味剂、液体/固体填料、稀释剂等常用药用辅料制成常见的药用制剂，如片剂、胶囊、糖浆、悬浮剂、注射剂等。
本发明所述的共价激酶抑制剂及其药物组合物应用于制备为治疗和/或预防增殖性疾病、延缓增殖性疾病的进程、减轻增殖性疾病的症状和、辅助治疗增殖性疾病、处理增殖性疾病的药物。
优选，所述增殖性疾病选自肿瘤、风湿性疾病、慢性炎症、传染性单核细胞增多症。
进一步优选，所述增殖性疾病选自：
胃癌，结直肠癌，肺癌(如肺腺癌)，乳腺癌，肝癌，前列腺癌，甲状腺癌，胰腺癌，膀胱癌，肾癌，脑瘤，颈癌，CNS(中枢神经系统)的癌症，恶性胶质瘤，骨髓增生病，动脉粥样硬化，白血病，肺纤维化，淋巴癌，风湿性疾病，慢性炎症，非淋巴网状系统肿瘤，冷球蛋白血症，丘疹性黏蛋白沉积症，家族性脾性贫血，多发性骨髓瘤，淀粉样变，孤立性浆细胞瘤，重链病，轻链病，恶性淋巴瘤，慢性淋巴细胞白血病，单核细胞白血病，半分子病，原发性巨球蛋白血症，原发性巨球蛋白血症紫癜，继发性良性单克隆丙种球蛋白病，溶骨性病变，急性淋巴细胞白血病，淋巴母细胞瘤，部分非霍奇金淋巴瘤，Sezary综合征，传染性单核细胞增多症，急性组织细胞增多症，毛细胞白血病，霍奇金淋巴瘤，结肠癌，直肠癌，肠道息肉，憩室炎，结肠炎，胰腺炎，肝炎，小细胞肺癌，神经母细胞瘤，神经内分泌细胞肿瘤，胰岛细胞瘤，甲状腺髓样癌，黑色素瘤，子宫癌，慢性肝炎，肝硬化，卵巢癌，视网膜母细胞瘤，胆囊炎，头颈部鳞癌，消化道恶性肿瘤，非小细胞肺癌，宫颈癌，睾丸肿瘤，膀胱癌、骨髓瘤或骨组织恶性肿瘤(如骨肉瘤)。
有益效果：与现有技术相比，本发明具有如下显著优点：
1、该类共价激酶抑制剂可有效与靶标酶共价结合，同时能够有效释放NO/NO2 —；一方面共价结合可起到对含巯基靶标酶的共价抑制作用，另一方面所释放的NO/NO2 —可进一步对靶标激酶及相互作用激酶产生亚硝基化修饰，抑制其自身磷酸化，阻止激酶信号转导，发挥协同作用；并且具有选择性，降低非靶标抑制；
2、该类共价激酶抑制剂在分子水平、细胞水平和动物水平具有显著的抑制活性，作用于多种与恶性增殖相关的酶以及恶性增殖细胞，并且抑制动物体内肿瘤增长的同时未产生明显不良反应；
3、化合物制备方法简便、通用，利于多种结构的拓展。
附图说明
图1是I1化合物与布鲁顿氏酪氨酸激酶(BTK)共价结合的质谱验证结果；
图2是I3化合物与布鲁顿氏酪氨酸激酶(BTK)共价结合的质谱验证结果；
图3是I1化合物分别在BTK储存液、大鼠肝微粒体、大鼠血浆、GSH溶液中的NO释放结果以及I1化合物在B细胞淋巴瘤Daudi细胞中的NO释放结果；
图4是I1-I6化合物在大鼠血浆和GSH溶液中的稳定性测试结果；
图5是I1化合物的Kinact/Ki参数测试结果；
图6是I1化合物的激酶谱测试结果；
图7是I1化合物的裸鼠实验结果；
图8是I1化合物在细胞中的NO释放与布鲁顿氏酪氨酸激酶(BTK)共定位验证结果；
图9是I1化合物释放的NO造成布鲁顿氏酪氨酸激酶(BTK)亚硝化升高及磷酸化下降/磷酸化信号转导受阻结果。
具体实施方式
下面结合实施例对本发明的技术方案作进一步说明。
实施例1：化合物I1的制备
1、将1-[(3R)-3-[4-氨基-3-(4-苯氧基苯基)-1H-吡唑并[3,4-D]嘧啶-1-基]-1-哌啶基]-2-丙烯-1-酮(伊布替尼)(10g,0.023mol)，溶于50ml 1，4二氧六环中，加入多聚甲醛(40.9g,0.454mol)、三乙烯二胺(3.82g,0.034mol)、苯酚(1.6g,0.017mol),60℃反应3天。减压浓缩除去溶剂，然后用乙酸乙酯(50mL)萃取，有机层用水、饱和食盐水各洗涤3次，无水硫酸钠干燥，浓缩，柱层析(石油醚/乙酸乙酯＝5/1,v/v)得白色固体V1。1H NMR(500MHz,Chloroform-d)δ8.25(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＝12.5Hz,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 C26H26N6O3,[M+H]+:471.2066,found:471.2061.
2、将V1(200mg,0.43mmol)，溶于15ml二氯甲烷中，加入对甲苯磺酰氯(90mg，0.47mmol)、三乙胺(18.3mg，0.18mmol)，氮气保护下室温反应10h，然后用乙酸乙酯萃取，有机层用水、饱和食盐水各洗涤3次，无水硫酸钠干燥，柱层析(石油醚/乙酸乙酯＝20/1,v/v)得白色固体V2。1H NMR(500MHz,Chloroform-d)δ8.00(s,1H),7.81–7.75(m,3H),7.69–7.63(m,3H),7.40–7.30(m,5H),7.10(tt,J＝7.6,2.0Hz,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.forC33H32N6O5S,[M+H]+:625.2155,found:624.2151.
3、将对甲苯磺酰氯取代产物V2(300mg,0.48mmol)溶于20ml DMSO中，加入二甲氨基偶氮鎓二醇盐(305mg,2.40mmol)，氮气保护下室温反应10h，然后用乙酸乙酯萃取，有机层用水、饱和食盐水各洗涤3次，无水硫酸钠干燥，柱层析(石油醚/乙酸乙酯＝10/1,v/v)即得白色固体I1。1H 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.forC28H31N9O4,[M+H]+:558.2499,found:558.2498.
实施例2：化合物I2的制备
参照实施例1的合成方法。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＝7.5,2.0Hz,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＝10.4Hz,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.forC29H33N9O4,[M+H]+:572.2656,found:572.2652.
实施例3：化合物I3的制备
参照实施例1的合成方法。1H 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＝12.3Hz,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.forC30H35N9O4,[M+H]+:586.2812,found:586.2807.
实施例4：化合物I4的制备
参照实施例1的合成方法。1H 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＝10.4Hz,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 C30H33N9O5,[M+H]+:600.2605,found:600.2601.
实施例5：化合物I5的制备
参照实施例1的合成方法。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＝7.5,2.0Hz,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),4.53(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 C30H33N9O4,[M+H]+:584.2656,found:584.2651.
实施例6：化合物I6的制备
参照实施例1的合成方法。1H 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＝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),3.38(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 C31H35N9O4,[M+H]+:598.2812,found:598.2808.
实施例7：化合物I7的制备
参照实施例1的合成方法，将对甲苯磺酰氯取代产物V2(300mg,0.48mmol)溶于10ml无水乙醚中，加入AgNO2(369mg,2.40mmol)，氮气保护下室温避光反应10h，然后减压浓缩溶剂，用乙酸乙酯萃取，有机层用水、饱和食盐水各洗涤3次，无水硫酸钠干燥，柱层析(石油醚/乙酸乙酯＝10/1,v/v)即得白色固体I7。1H 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 C26H25N7O4,[M+H]+:500.1968,found:500.1965.
实施例8：化合物I8的制备
1、将3-(4-苯氧基苯基)-1-(哌啶-3-基)-1H-吡唑并[3,4-D]嘧啶-4-胺(伊布替尼中间体)(10g，0.02587mol)溶于50ml二氯甲烷中，加入EDCI(9.91g，0.05174mol)、三乙胺(5.24g，0.05174mol)、2,3-二溴丙酸(5.99g，0.02587mol)，室温反应36小时，减压浓缩除去溶剂，然后用乙酸乙酯(100mL)萃取，有机层用水、饱和食盐水各洗涤3次，无水硫酸钠干燥，浓缩，柱层析(石油醚/乙酸乙酯＝1/1,v/v)得白色固体V3。1H NMR(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,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 C25H24Br2N6O2,[M+H]+:599.0327,found:599.0325.
2、将V3(10g，0.01666mol)溶解于50ml乙醇中，加入硝基甲烷(1.5g，0.02499mol)，回流反应24小时，减压浓缩除去溶剂，然后用乙酸乙酯(100mL)萃取，有机层用水、饱和食盐水各洗涤3次，无水硫酸钠干燥，浓缩，柱层析(石油醚/乙酸乙酯＝10/1,v/v)得白色固体I8。1H 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 C26H25N7O4,[M+H]+:500.1968,found:500.1964.
实施例9：化合物I9的制备
1、将3-氧代环丁基羧酸叔丁酯(1g，0.005875mol)、乙酸钠(1.60g，0.01175mol)、盐酸羟胺(0.8165g，0.01175mol)溶于50ml乙醇中，回流反应24小时，减压浓缩除去溶剂，然后用乙酸乙酯(100mL)萃取，有机层用水、饱和食盐水各洗涤3次，无水硫酸钠干燥，浓缩，柱层析(石油醚/乙酸乙酯＝50/1,v/v)得无色油状物V4。1H 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 C9H14O3,[M+H]+:171.0943,found:171.0941.
2、将V4(500mg，2.94mmol)、三氟乙酸酐(926.06mg，4.41mmol)、过氧化脲(414.85mg，4.41mmol)、磷酸二氢钠(529.09mg，4.41mmol)溶于无水乙腈中，回流反应4小时，过滤，减压浓缩除去滤液中的溶剂，然后用乙酸乙酯(100mL)萃取，有机层用水、饱和食盐水各洗涤3次，无水硫酸钠干燥，浓缩，柱层析(石油醚/乙酸乙酯＝10/1,v/v)得白色固体V5。1H 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.
3、将3-(4-苯氧基苯基)-1-(哌啶-3-基)-1H-吡唑并[3,4-D]嘧啶-4-胺(伊布替尼中间体)(10g，0.02587mol)溶于50ml二氯甲烷中，加入EDCI(9.91g，0.05174mol)、三乙胺(5.24g，0.05174mol)、V5(4.51g，0.031044)，室温反应24小时，减压浓缩除去溶剂，然后用乙酸乙酯(100mL)萃取，有机层用水、饱和食盐水各洗涤3次，无水硫酸钠干燥，浓缩，柱层析(石油醚/乙酸乙酯＝50/1,v/v)得白色固体I9。1H NMR(500MHz,Chloroform-d)δ8.25(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＝13.0Hz,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 C27H27N7O4,[M+H]+:514.2125,found:514.2119.
实施例10：化合物I10的制备
将3-(4-苯氧基苯基)-1-(哌啶-3-基)-1H-吡唑并[3,4-D]嘧啶-4-胺(伊布替尼中间体)(10g，0.02587mol)溶于50ml二氯甲烷中，置于冰浴上预冷10分钟。将2-溴-1-(3,3-二硝基吖丁啶-1-基)乙酮(6.2g，0.02328mol)溶于10ml二氯甲烷中，随后滴入先前伊布替尼中间体溶液中，冰浴反应30分钟后移至室温反应2小时。减压浓缩除去溶剂，然后用乙酸乙酯(100mL)萃取，有机层用水、饱和食盐水各洗涤3次，无水硫酸钠干燥，浓缩，柱层析(石油醚/乙酸乙酯＝30/1,v/v)得白色固体I10。1H 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 C27H27N9O6,[M+H]+:574.2084,found:574.2080.
实施例11：化合物II1的制备
参照实施例1的合成方法。1H 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),4.97(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.forC26H28Cl2FN7O5,[M+H]+:608.1513,found:608.1509.
实施例12：化合物II2的制备
参照实施例1的合成方法。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 C27H30Cl2FN7O5,[M+H]+:622.1670,found:622.1670.
实施例13：化合物II3的制备
参照实施例1的合成方法。1H 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 C28H32Cl2FN7O5,[M+H]+:636.1826,found:636.1817.
实施例14：化合物II4的制备
参照实施例1的合成方法。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 C28H30Cl2FN7O6,[M+H]+:650.1619,found:650.1619.
实施例15：化合物II5的制备
参照实施例1的合成方法。1H 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＝12.2,8.2,3.7Hz,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 C29H32Cl2FN7O5,[M+H]+:648.1826,found:648.1826.
实施例16：化合物II6的制备
参照实施例1的合成方法。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 C29H32Cl2FN7O6,[M+H]+:664.1775,found:664.1764.
实施例17：化合物II7的制备
参照实施例7的合成方法。1H 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 C24H22Cl2FN5O5,[M+H]+:550.0982,found:550.0978.
实施例18：化合物II8的制备
参照实施例8的合成方法。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),4.53(s,1H),3.80(s,3H),3.49(s,2H),3.33(d,J＝3.7Hz,4H),3.18(d,J＝4.9Hz,1H),2.68(d,J＝5.1Hz,1H),2.13(s,2H),2.00(s,2H).HRMS(ESI):m/z calcd.for C24H22Cl2FN5O5,[M+H]+:550.0982,found:550.0979.
实施例19：化合物II9的制备
参照实施例9的合成方法。1H 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 C25H24Cl2FN5O5,[M+H]+:564.1139,found:564.1134.
实施例20：化合物II10的制备
参照实施例10的合成方法。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 C25H24Cl2FN7O7,[M+H]+:624.1098,found:624.1095.
实施例21：化合物III1的制备
参照实施例1的合成方法。1H 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＝12.0,2.6Hz,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 C27H31Cl2N7O6,[M+Na]+:642.1605,found:642.1598.
实施例22：化合物III2的制备
参照实施例1的合成方法。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 C29H35Cl2N7O6,[M+Na]+:670.1918,found:670.1915.
实施例23：化合物III3的制备
参照实施例1的合成方法。1H 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 C30H35Cl2N7O6,[M+Na]+:682.1918,found:670.1913.
实施例24：化合物III4的制备
参照实施例1的合成方法。1H 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＝11.9,2.5Hz,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 C29H33Cl2N7O7,[M+Na]+:684.1711,found:684.1702.
实施例25：化合物III5的制备
参照实施例1的合成方法。1H 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),3.66(s,1H),3.63–3.51(m,2H),3.42(s,2H),2.10–1.75(m,7H).HRMS(ESI):m/z calcd.for C30H35Cl2N7O7,[M+Na]+:698.1867,found:698.1861.
实施例26：化合物III6的制备
参照实施例1的合成方法。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 C30H35Cl2N7O7,[M+Na]+:698.1867,found:698.1856.
实施例27：化合物III7的制备
参照实施例7的合成方法。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),7.44(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 C25H25Cl2N5O6,[M+H]+:562.1182,found:562.1180.
实施例28：化合物III8的制备
参照实施例8的合成方法。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 C25H25Cl2N5O6,[M+H]+:562.1182,found:562.1179.
实施例29：化合物III9的制备
参照实施例9的合成方法。1H 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,2H),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 C26H27Cl2N5O6,[M+H]+:576.1338,found:576.1334.
实施例30：化合物III10的制备
参照实施例10的合成方法。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 C26H27Cl2N7O8,[M+H]+:636.1298,found:636.1295.
实施例31：化合物IV的制备
参照实施例10的合成方法。1H NMR(500MHz,Chloroform-d)δ9.79(s,1H),8.27(d,J＝4.9Hz,1H),7.84(d,J＝0.9Hz,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＝12.5Hz,1H),3.43(s,4H),2.89(s,3H),2.74(s,2H),2.32(s,6H).HRMS(ESI):m/z calcd.for C30H36N10O6,[M+H]+:633.2814,found:633.2810.
实施例32：化合物V的制备
参照实施例10的合成方法。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),3.75(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 C32H33F2N9O7,[M+H]+:694.2471,found:694.2469.
实施例33：化合物与布鲁顿氏酪氨酸激酶(BTK)的共价结合研究
1、实验方法
结合质谱手段测定I1、I3与布鲁顿氏酪氨酸激酶(BTK)的共价结合。目标蛋白在缓冲液中稀释至2μM，然后从200μM溶液中加入1/100-1/20不等体积的待测化合物，得到2μM浓度的测试化合物。将反应混合物在室温下不同时间注入LC/MS。进行数据分析，使用20000:40000Da窗口和1Da分辨率对原始谱图进行反卷积处理。化合物的标记百分比为特定化合物的标记除以检测到的总体蛋白质。
2、实验结果
结果如图1和图2所示，由结果可知化合物与BTK在室温孵育1h条件下即可达到完全的结合，分子量计算结果符合化合物设计提出的加成-消除释放NO机理。
实施例34：化合物的NO释放研究
1、实验方法
利用Griess法在不同pH缓冲盐溶液、靶蛋白储存液、大鼠血浆、大鼠肝微粒体等体系中测定目标化合物的NO释放结果。采用NO荧光探针法，结合流式细胞技术，测定目标化合物在淋巴瘤细胞Daudi中NO的释放。
2、实验结果
结果如图3，I1在BTK储存液中的NO释放显著高于大鼠肝微粒体、大鼠血浆及GSH溶液，表明I1具有较高的释放选择性。采用DAF-FM DA探针法测定I1在细胞中的NO释放，相较于伊布替尼，在B细胞淋巴瘤Daudi细胞中，其具有显著的NO释放。
实施例35：化合物的稳定性研究
1、实验方法
采用LC-MS法考察化合物I1-I6在血浆、肝微粒体和GSH等介质中的稳定性。
2、实验结果
结果如图4，表明化合物I1-I6均具有较高的体外稳定性。
实施例36：化合物的体外激酶抑制活性研究
1、实验方法
使用ADP-Glo法测定化合物I1-I6，II1-II6和III1-III6分别对BTK、HER2和Fgfr4的激酶活性(IC50)。向384孔稀释板中加入50μL待测化合物，每列在DMSO中按1:3连续稀释待测化合物5倍，使用Echo将每行0.025μL稀释的待测化合物溶液转移至384 检测板，向384孔测定板中加入2.5μL酶工作溶液，1000prm离心1分钟，加入2.5μL底物(ATP和底物)工作溶液以启动反应，在25℃分别孵育0、2、4、8、15、30、60、90分钟，加入5μL TK Beads溶液引发反应，25℃孵育60分钟，读数665nm和620nm荧光信号与BMG(比率665/620)。
2、实验结果
结果如表1、表2和表3
表1.化合物I1-I6对BTK激酶抑制活性结果
表2.化合物II1-II6对HER2激酶抑制活性结果
表3.化合物III1-III6对Fgfr4激酶抑制活性结果
实施例37：化合物的体外抗肿瘤细胞增殖活性研究
1、实验方法
使用CCK8法测试化合物I1-I6，II1-II6和III1-III6对肿瘤细胞株的抑制活性。细胞株在含10％胎牛血清(FBS)的DMEM培养基或PRMI 1640培养基中培养(SJSA-1和A549细胞使用PRMI 1640培养基培养，MDA-MB-231和HeLa细胞使用DMEM培养基培养)，所有细胞株均置于Shellab 2323-2 CO2恒温细胞培养箱中培养，条件：含5％CO2的空气，温度为37℃。细胞活性采用CCK8(Beyotime)方法测定。以8000-10000/孔的细胞密度接种于96孔板，恒温培养箱孵育24h后，显微镜下观察细胞状态良好且几乎完全贴壁生长，加入不同浓度的化合物或0.1％的DMSO后继续培养48h。48h后 加入CCK8试剂，继续培养两小时后用Envision 2104多功能微孔分析仪(Perkin Elmer)测定450nM波长下各孔吸光度OD值，最后用GraphPad 8.0处理拟合量效曲线计算IC50值。其中，细胞存活率＝[(实验组-空白对照组)/(对照组-空白对照组)]*100％。其中，实验组：细胞+CCK8溶液+药物溶液；对照组：细胞+CCK8溶液+0.1％DMSO；空白对照组：无细胞+CCK8溶液。每组设置3个平行复孔，每个实验重复三次。
2、实验结果
结果如表4、表5和表6。
表4.化合物I1-I8的体外抗肿瘤活性测试结果(IC50，μM)
表5.化合物II1-II6的体外抗肿瘤活性测试结果
表6.化合物III1-III6的体外抗肿瘤活性测试结果
实施例38：化合物与激酶的结合研究
1、实验方法
使用HTRF/ADP-Glo/FI法测试I1的Kinact/ki参数。向384孔稀释板中加入50μL I1，每列在DMSO中按1:3连续稀释I1 5倍，使用Echo将每行0.025μL稀释的I1溶液转移至384检测板，向384孔测定板中加入2.5μL酶工作溶液，1000prm离心1分钟，加入2.5μL底物(ATP和底物)工作溶液以启动反应，在25℃分别孵育0、2、4、8、15、 30、60、90分钟，加入5μL TK Beads溶液引发反应，25℃孵育60分钟，读数665nm和620nm荧光信号与BMG(比率：665/620)。
2、实验结果
结果如图5，表明I1弹头部位改造增加空间位阻，使得第一步非共价结合步骤较慢，但对弹头部位进行改造并未显著影响I1与BTK的结合效力。
实施例39：化合物的激酶谱研究
1、实验方法
使用ADP-Glo Kinase Assay对化合物I1进行激酶谱检测。使用测定缓冲液制备2×ATP和底物溶液以及2×激酶和金属溶液，通过Echo 655将20nL化合物转移至384测定板。添加2μL的2×激酶和金属溶液，混合并于25℃在384测定板中孵育10分钟。向孔中加入2μL 2×底物和ATP溶液，并于25℃孵育60分钟。向孔中加入4μL ADP-Glo Reagent，并于25℃孵育40分钟。向孔中加入8μL Kinase Detection Reagent，25℃孵育40分钟。在微量滴定板读数器上记录信号。
2、实验结果
结果如图6，表明对弹头部位的改造使得化合物I1的激酶选择性得到提升，相比于伊布替尼，有效减少了对JAK家族激酶、ErbB家族激酶等的脱靶抑制。这可能使得I1相比于伊布替尼具有更小的胃肠道副作用等。
实施例40：化合物的体内活性研究
1、实验方法
将150μL TMD8细胞悬液(PBS中6×106个细胞)皮下接种到8周龄雌性BALB/c裸鼠的右背部。当肿瘤的平均体积达到约60mm3时，将小鼠随机分为空白对照组(n＝8/组)、化合物I1组(15mg/kg，PO，BID)。给药后每隔一天记录肿瘤体积和体重。20天后，所有的小鼠被处死，保留肿瘤用于进一步研究。肿瘤的大小是通过公式计算。V(肿瘤体积，mm3)＝L(长度，mm)×W2(宽度，mm)×0.5。肿瘤生长抑制率(TGI)用以下公式计算。TGI＝(1-TWt/TWc)×100％，其中TWt和TWc分别代表给药组和对照组第20天的平均肿瘤重量。
2、实验结果
结果如图7，与空白对照组相比，实验组A12(15mg/kg)可显著抑制肿瘤的增长。且实验期间小鼠健康状况良好，活动情况正常、进水进食情况正常、小鼠皮肤光泽度及颜色正常、无腹泻现象发生，肿瘤部位无炎症出现。
实施例41：化合物的NO释放与布鲁顿氏酪氨酸激酶(BTK)共定位研究
1、实验方法
构建以pKG-CMV-MCS-EF1-Puro为载体的BTK-mCherry(激发波长587nm，发射波长610nm)慢病毒载体。培养Daudi细胞，对其进行慢病毒转染48h后，加入DAF-FM DA(激发波长495nm，发射波长515nm)共孵育15min，弃上清洗3次，洗除多余染料后，加入化合物孵育，使化合物终浓度分别为1μM，5μM，10μM等；化合物孵育5min/30min/60min等；孵育完成后加PBS浸洗3次；加入即用型DAPI染液300μL，室温避光孵育10min；PBS浸洗3次。涂片，聚焦显微镜下观察细胞，600倍进行成像观察。
2、实验结果
结果如图8，结果显示NO释放与BTK有较清晰的亚细胞共定位。随后加入NO清除剂PTIO，共聚焦成像则显示NO被清除，在BTK处无NO。以上结果表明，I1在细胞内BTK处原位、极小范围内释放NO。
实施例42：化合物的NO释放造成布鲁顿氏酪氨酸激酶(BTK)亚硝化升高及磷酸 化下降/磷酸化信号转导受阻研究
1、实验方法
培养Daudi细胞，取细胞悬液与1-10μM各组化合物孵育1-5h，设置DMSO组，伊布替尼组，I1组，偶氮鎓组。进行Biotin-Switch转化后使用Streptavidin Agarose(lifetechnologies，货号：SA100-04)纯化后，进行目的蛋白的亚硝化表征。同时，同一组细胞利用WB同时评价亚硝基化对靶蛋白磷酸化程度的影响，揭示激酶亚硝基化与磷酸化的串扰机制。
2、实验结果
结果如图9，化合物与Daudi细胞孵育后，I1相较于伊布替尼引起了BTK亚硝基化的显著性升高，但相较于偶氮鎓二醇盐(无选择性的NO供体)并未引起全局的亚硝基化升高。同时测试了I1相较于伊布替尼对BTK及与BTK有相互作用的激酶蛋白激酶B(AKT)的磷酸化影响程度，结果显示I1相较于伊布替尼使得BTK、AKT的磷酸化程度显著降低，表明NO带来的亚硝基化和共价抑制起到了抑制磷酸化的协同作用。

Claims (10)

  1. 一种共价激酶抑制剂，其特征在于，具有式I、II、III、IV或V的结构，还包含其药学上可接受的盐，
    其中：
    R1、R2选自C1-C4烷基或者R1、R2与其连接的氮共同形成4-7元杂环，所述杂环含有1-2个氮、氧、硫杂原子，并且环系碳原子被至少一个氢、羟基、卤素、C1-C4烷基、卤素取代的C1-C4烷基、羟基取代的C1-C4烷基取代；
    激酶抑制剂母核选自：
  2. 根据权利要求1所述的共价激酶抑制剂，其特征在于，所述结构中：
    R1、R2选自甲基、乙基或者R1、R2与其连接的N共同形成5-6元杂环，所述杂环为吗啉环、哌啶环、四氢吡咯环，并且环系碳原子被至少一个氢、羟基、氟、氯、溴、甲基、乙基、三氟甲基、羟甲基、羟乙基取代。
  3. 根据权利要求2所述的共价激酶抑制剂，其特征在于，所述结构中：
    NR1R2选自二甲氨基、甲基乙基氨基、二乙氨基、吗啡啉基、四氢吡咯基、2-羟甲基四氢吡咯基、哌啶基、4-羟基哌啶基。
  4. 根据权利要求1所述的共价激酶抑制剂，其特征在于，选自以下任一化合物：


  5. 根据权利要求1所述的共价激酶抑制剂，其特征在于，所述药学上可接 受的盐为所述激酶抑制剂与以下任一酸形成的盐：盐酸、氢溴酸、硫酸、磷酸、碳酸、甲磺酸、苯磺酸、对甲苯磺酸、萘磺酸、樟脑磺酸、柠檬酸、酒石酸、苹果酸、乳酸、丙酮酸、乙酸、马来酸、琥珀酸、富马酸、水杨酸、苯基乙酸、阿魏酸或杏仁酸。
  6. 一种权利要求1所述的共价激酶抑制剂的制备方法，其特征在于，包含以下步骤：
    (1)当所述共价激酶抑制剂具有式I或II的结构式时，
    (i)化合物1在多聚甲醛、三乙烯二胺、苯酚的存在下反应得到中间体2；
    (ii)中间体2在对甲苯磺酰氯、三乙胺的存在下反应得到中间体3；
    (iii)中间体3在15-冠-5-醚的催化下与相应偶氮鎓二醇盐或AgNO2反应得到终产物4或5；
    (2)当所述共价激酶抑制剂具有式III的结构式时，
    (i)激酶抑制剂母核6在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的催化下与2,3二溴丙酸反应得到化合物7；
    (ii)化合物7与硝基甲烷反应得到终产物8；
    (3)当所述共价激酶抑制剂具有式IV的结构式时，
    (i)3-氧代环丁基羧酸叔丁酯在盐酸羟胺、醋酸钠的存在下反应得到肟中间体10；
    (ii)中间体10与过氧化脲、三氟醋酸酐反应得到中间体11；
    (iii)中间体11与激酶抑制剂母核6反应得到终产物12；
    (4)当所述共价激酶抑制剂具有式V的结构式时，激酶抑制剂母核6与2-溴-1-(3,3-二硝基吖丁啶-1-基)乙酮反应得到终产物13；
    其中，R1、R2、激酶抑制剂母核的定义如权利要求1所述；
    将相应的酸与以上方法制得的共价激酶抑制剂成盐，即得所述药学上可接受的盐。
  7. 一种药物组合物，其特征在于，包含权利要求1所述的共价激酶抑制剂以及药学上可接受的载体。
  8. 一种权利要求1所述的共价激酶抑制剂或者权利要求7所述的药物组合物在制备治疗和/或预防增殖性疾病、延缓增殖性疾病的进程、减轻增殖性疾病的症状、辅助治疗增殖性疾病、处理增殖性疾病的药物中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述增殖性疾病选自肿瘤、风湿性疾病、慢性炎症、传染性单核细胞增多症。
  10. 根据权利要求9所述的应用，其特征在于，所述增殖性疾病选自胃癌、结直肠癌、肺癌、乳腺癌，肝癌、前列腺癌、甲状腺癌、胰腺癌、膀胱癌、肾癌、脑瘤、颈癌、子宫癌、卵巢癌、宫颈癌、睾丸肿瘤、骨髓瘤、骨肉瘤恶性胶质瘤、骨髓增生病、恶性淋巴瘤、慢性淋巴细胞白血病、单核细胞白血病、毛细胞白血病、急性淋巴细胞白血病、非淋巴网状系统肿瘤、孤立性浆细胞瘤、淋巴母细胞瘤、霍奇金淋巴瘤、神经母细胞瘤、视网膜母细胞瘤、神经内分泌细胞肿瘤、胰岛细胞瘤、黑色素瘤、动脉粥样硬化、肺纤维化、风湿性疾病、慢性炎症、丘疹性黏蛋白沉积症、家族性脾性贫血、淀粉样变、重链病、轻链病、半分子病、冷球蛋白血症、原发性巨球蛋白血症、原发性巨球蛋白血症紫癜、继发性良性单克隆丙种球蛋白病、溶骨性病变、部分非霍奇金淋巴瘤、Sezary综合征、传染性单核细胞增多症、急性组织细胞增多症、肠道息肉、憩室炎、结肠炎、胰腺炎、肝炎、慢性肝炎、肝硬化、胆囊炎。
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