Compounds useful as ATR kinase inhibitors

Abstract

The present invention relates to an ATR inhibitor as shown in Formula I, and a preparation method and application thereof. The compound shown in Formula I has high inhibitory activity against ATR, and also has excellent pharmacodynamics, in vitro / in vivo pharmacokinetic properties and safety, and has high clinical application prospects. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and in particular relates to an ATR inhibitor and a preparation method and application thereof. Background Art

[0002] ATR (Ataxia-telangiectasia and Rad3 related) is an ATM Rad3-related protein kinase, which belongs to the PI3KKs (Phosphoinositide 3kinase-related protein kinases) kinase family together with ATM (Ataxia-telangiectasia mutated) and DNA-PKcs (DNA-dependent protein kinase catalytic subunit).

[0003] Under the influence of external pressure or the internal environment of the body, cells suffer DNA damage, especially DNA single-strand or double-strand breaks (DSB) and replication stress. In normal cells, in order to maintain genome stability, DNA damage will be quickly recognized by DNA damage response (DDR) factors, thereby activating cell cycle checkpoints and directly repairing DNA. Depending on the type of damage, different DNA repair pathways are involved, which together form a highly complex and interactive defense mechanism against genotoxic damage.

[0004] Both ATM and ATR are activated by DNA damage and DNA replication stress, but their DNA damage specificity and functions are different. ATM is mainly activated by double-strand DNA breaks, while ATR responds to a wide range of DNA damage, including DSBs and various DNA damages that interfere with replication. The two work together to send signals of DNA damage and regulate downstream processes. However, many types of cancer cells have defects in some of their DNA repair processes (such as ATM signaling), thereby showing greater dependence on their remaining intact DNA repair proteins (such as ATR). In ATM-deficient cancer cells, inhibiting ATR can effectively inhibit cancer cell growth and play a role in synthetic lethality in treating cancer.

[0005] Synthetic lethality refers to the fact that in a pair of genes with similar or compensatory functions, the inactivation of a single gene is tolerable for cell survival, while the simultaneous destruction of multiple genes leads to cell death. Compared with other cancer treatments such as traditional radiotherapy and chemotherapy, synthetic lethality-based treatment strategies have three advantages: First, ATM is one of the most common abnormal genes in cancer. According to the COSMIC database, 167 different ATM somatic mutations (excluding variants of unknown origin) have been observed in tumors and hematological malignancies. Therefore, synthetic lethality-based treatment strategies can combat most cancer mutations; second, the corresponding patients can be simply identified based on the high selectivity of gene mutations; finally, combined use with chemotherapeutic drugs can improve their efficacy and reduce the dose, avoiding or reducing adverse reactions.

[0006] ATR inhibitors in the preclinical and clinical research stages include Berzosertib (VX-970), Ceralaserti (AZD6738), Elimusertib (BAY-1895344), ART0380, RP-3500, VX-803 and M1774 disclosed in WO2010071837A1. Among them, VX-970 is undergoing Phase I / II or Phase II clinical trials for indications such as prostate cancer, small cell lung cancer, ovarian cancer, urothelial carcinoma and non-small cell lung cancer. AZD6738 has entered Phase II clinical treatment for breast cancer, head and neck squamous cell carcinoma, gastric cancer and non-small cell lung cancer. ART0380 is in Phase I / II clinical trials for the treatment of ovarian cancer, RP-3500 for the treatment of solid tumors, and VX-803 for the treatment of breast cancer. BAY-1895344 is in Phase I clinical trials for the treatment of lymphoma and solid tumors, and M1774 is in Phase I clinical trials for the treatment of solid tumors.

[0007]

[0008] The above clinical trials have shown that disruption of ATR function promotes cancer cell death in the presence or absence of DNA damaging agents, suggesting that ATR inhibitors may be effective as single agents or as sensitizers for radiotherapy or genotoxic chemotherapy. Summary of the invention

[0009] Problems to be solved by the invention:

[0010] Although the compounds disclosed in the prior art have inhibitory activity against ATR, they have high cytotoxicity and low in vivo PK exposure. Therefore, there is still a need to develop more inhibitors with low cytotoxicity, high exposure and high ATR inhibitory activity.

[0011] Solutions used to solve the problem:

[0012] In order to solve the above technical problems, the present disclosure provides the following technical solutions:

[0013] A compound of formula I or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof:

[0014] in:

[0015] Ring A and Ring B are C 6-10 Aryl or 5-10 membered heteroaryl;

[0016] R 1 is selected from hydrogen, deuterium, cyano, halogen, and substituted or unsubstituted hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group, guanidinyl, urea, amide, aminosulfonamide, sulfonamide and HN-C(=NH)-C 1-6 alkyl;

[0017] R 2 and R 5 Selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, amino, C 1-6 Alkoxy;

[0018] or R 1 and R 2 Together with the atoms to which they are attached, they form a 5-6 membered heteroaryl or heterocyclic group containing 1-3 atoms selected from oxygen, nitrogen and sulfur atoms, wherein the 5-6 membered heteroaryl or heterocyclic group has 0-1 methylene groups on the ring, which can be arbitrarily replaced by -C(=O)-, or -C(=NR 7 )-substitution;

[0019] If present, each R 3 are each independently selected from hydrogen, deuterium, halogen, C 1-6 Alkyl and is selected from cyano, amino, hydroxyl, halogen, C 1-6 Alkyl and C 1-6 The C substituted by the alkoxy substituent 1-6 alkyl;

[0020] R 4 Selected from hydrogen, deuterium, C 1-6 Alkyl, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group, C 1-6 Alkylamino and (C 1-3 Alkyl)2amino;

[0021] or R 4 and R 5Together with the atoms to which they are attached, they form a 5-7 membered heterocyclic group containing 1-3 atoms selected from oxygen, nitrogen, sulfur, and the 5-7 membered heterocyclic group is optionally selected from deuterium, hydroxyl, C 1-6 Alkyl, halogen, C 1-6 Alkoxy and C 1-6 The substituents of alkylamino are substituted; 1-3 methylene groups on the ring can be arbitrarily replaced by -SO2-, -SO(=NHR 7 )-, -SO2NH-, -CO-, -C(=O)NH-,C 1-6 Alkylamino, C 3-6 Cycloalkylamino substituted, the C 1-6 Alkylamino or C 3-6 Cycloalkylamino, wherein 0-2 methylene groups may be replaced by oxygen, nitrogen, sulfur, C 1-3 Replaced by alkylamino;

[0022] If present, each R 6 are each independently selected from hydrogen, deuterium, halogen, amino, C 1-6 Alkoxy, C 1-6 Alkyl and is selected from cyano, amino, hydroxyl, halogen, C 1-6 Alkyl and C 1-6 The C substituted by the alkoxy substituent 1-6 alkyl;

[0023] Q 1 Selected from C 1-6 Alkylene, -CO-, amino, -SO-, -SO2-, -C(=NR 7 )- and 3-7 membered heterocyclic groups, wherein C 1-6 The 0-4 methylene groups in the alkylene group can be arbitrarily replaced by amino groups, -CO-, -SO-, -SO2-, -C(=NR 7 )-substitution;

[0024] Q 2 Selected from -SO2-, SO, -SO(=NR 7 )-,-SO2NR 7 - and -CO-;

[0025] Every R 7 are each independently selected from hydrogen, deuterium, C 1-6 Alkyl, cyano, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group, C 1-6 Alkoxy, C 1-6 Alkylamino and is selected from cyano, amino, hydroxy, halogen, C 1-6 Alkyl and C 1-6 The C substituted by the alkoxy substituent 1-6 Alkyl, C 3-7Cycloalkyl, 3-7 membered heterocyclic group;

[0026] n1, n2, and n3 are each any integer from 0 to 2.

[0027] Effects of the invention:

[0028] The aminopyrazine compounds provided by the present disclosure have high inhibitory activity against ATR, and also have excellent pharmacodynamics, in vitro / in vivo pharmacokinetic properties and safety, and have high prospects for clinical application. DETAILED DESCRIPTION

[0029] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following is a detailed description by listing specific embodiments. Unless otherwise defined, the technical and scientific terms used in this disclosure have the same meanings as those in the technical field to which this application belongs.

[0030] In one aspect, the present disclosure provides a compound represented by Formula I or a pharmaceutically acceptable salt, ester, isomer, solvate, prodrug or isotope-labeled substance thereof:

[0031]

[0032] in:

[0033] Ring A and Ring B are C 6-10 Aryl or 5-10 membered heteroaryl;

[0034] R 1 is selected from hydrogen, deuterium, cyano, halogen, and substituted or unsubstituted hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group, guanidinyl, urea, amide, aminosulfonamide, sulfonamide and HN-C(=NH)-C 1-6 alkyl;

[0035] R 2 and R 5 Selected from hydrogen, deuterium, halogen, C 1-6 Alkyl, amino, C 1-6 Alkoxy;

[0036] or R 1 and R 2 Together with the atoms to which they are attached, they form a 5-6 membered heteroaryl or heterocyclic group containing 1-3 atoms selected from oxygen, nitrogen and sulfur atoms, wherein the 5-6 membered heteroaryl or heterocyclic group has 0-1 methylene groups on the ring, which can be arbitrarily replaced by -C(=O)-, or -C(=NR 7 )-substitution;

[0037] If present, each R3 are each independently selected from hydrogen, deuterium, halogen, C 1-6 Alkyl and is selected from cyano, amino, hydroxyl, halogen, C 1-6 Alkyl and C 1-6 The C substituted by the alkoxy substituent 1-6 alkyl;

[0038] R 4 Selected from hydrogen, deuterium, C 1-6 Alkyl, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group, C 1-6 Alkylamino and (C 1-3 Alkyl)2amino;

[0039] or R 4 and R 5 Together with the atoms to which they are attached, they form a 5-7 membered heterocyclic group containing 1-3 atoms selected from oxygen, nitrogen, sulfur, and the 5-7 membered heterocyclic group is optionally selected from deuterium, hydroxyl, C 1-6 Alkyl, halogen, C 1-6 Alkoxy and C 1-6 The substituents of alkylamino are substituted; 1-3 methylene groups on the ring can be arbitrarily replaced by -SO2-, -SO(=NHR 7 )-, -SO2NH-, -CO-, -C(=O)NH-,C 1-6 Alkylamino, C 3-6 Cycloalkylamino substituted, the C 1-6 Alkylamino or C 3-6 Cycloalkylamino, wherein 0-2 methylene groups may be replaced by oxygen, nitrogen, sulfur, C 1-3 Replaced by alkylamino;

[0040] If present, each R 6 are each independently selected from hydrogen, deuterium, halogen, amino, C 1-6 Alkoxy, C 1-6 Alkyl and is selected from cyano, amino, hydroxyl, halogen, C 1-6 Alkyl and C 1-6 The C substituted by the alkoxy substituent 1-6 alkyl;

[0041] Q 1 Selected from C 1-6 Alkylene, -CO-, amino, -SO-, -SO2-, -C(=NR 7 )- and 3-7 membered heterocyclic groups, wherein C 1-6 The 0-4 methylene groups in the alkylene group can be arbitrarily replaced by amino groups, -CO-, -SO-, -SO2-, -C(=NR 7 )-substitution;

[0042] Q2 Selected from -SO2-, SO, -SO(=NR 7 )-,-SO2NR 7 - and -CO-;

[0043] Every R 7 are each independently selected from hydrogen, deuterium, C 1-6 Alkyl, cyano, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group, C 1-6 Alkoxy, C 1-6 Alkylamino and is selected from cyano, amino, hydroxy, halogen, C 1-6 Alkyl and C 1-6 The C substituted by the alkoxy substituent 1-6 Alkyl, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group;

[0044] n1, n2, and n3 are each any integer from 0 to 2.

[0045] In certain embodiments, n1 is 0 or 1; n2 is 1 or 2, and n3 is preferably 0 or 1.

[0046] In certain embodiments, the ring A and ring B are selected from a benzene ring and a pyridine ring, preferably a benzene ring.

[0047] In certain embodiments, the compound of formula I is Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , Q1, Q2, n1, n2, n3 are as defined above.

[0048] In certain embodiments, R 1 is selected from hydrogen, deuterium, cyano, halogen, and substituted or unsubstituted hydroxyl, amino, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-7 Cycloalkyl, 3-7 membered heterocyclic group, guanidinyl, urea, amide, aminosulfonamide, sulfonamide and HN-C(=NH)-C 1-6 Alkyl; the substitution is selected from C 1-6 Alkyl, C 3-7 The cycloalkyl group is substituted with a substituent.

[0049] In certain embodiments, R 2 and R 5 are each independently hydrogen or deuterium.

[0050] In certain embodiments, R 1 and R2 Together with the atoms to which they are attached, they form a 5-6 membered heteroaryl or heterocyclic group containing 1-3 atoms selected from oxygen, nitrogen and sulfur atoms, wherein the 5-6 membered heterocyclic group has 0-1 methylene groups on the ring which can be arbitrarily replaced by -C(=O)-, or -C(=NR 7 )-substituted; the five-membered heteroaryl or five-membered heterocyclic group is any one of the following structures:

[0051]

[0052] R 8 Selected from hydrogen, deuterium, C 1-6 Alkyl, C 3-7 Cycloalkyl.

[0053] In certain embodiments, R 3 is halogen or halogen-substituted C 1-6 Alkyl, preferably F or CF3.

[0054] In certain embodiments, R 4 Methyl or any of the following structures:

[0055]

[0056] In certain embodiments, R 4 and R 5 Together with the atoms to which they are attached, they form a 5-7 membered heterocyclic group containing 1-3 atoms selected from oxygen, nitrogen and sulfur, wherein the 5-7 membered heterocyclic group is preferably any one of the following structures:

[0057]

[0058] n4 is an integer selected from 0 to 2;

[0059] R 9 Selected from oxygen, NR 7 ;

[0060] R 10 and R 11 are each independently selected from hydrogen, deuterium, C 1-6 Alkyl, C 1-6 Alkoxy and C 1-6 Alkylamino; or R 10 and R 11 Together with the atoms to which they are attached, they form C 3-7 Cycloalkyl or a 3-7 membered heterocyclic group containing 1-2 atoms selected from oxygen, nitrogen and sulfur;

[0061] R 12 and R 13 are each independently selected from hydrogen, deuterium, halogen, hydroxyl, C 1-6 Alkyl, C 1-6Alkoxy and C 1-6 Alkylamino; or R 12 and R 13 Formation = O;

[0062] R 14 Selected from hydrogen, deuterium, substituted or unsubstituted C 1-6 Alkyl, C 1-6 Alkoxy, C 3-7 Cycloalkyl and 3-7 membered heterocyclic groups, wherein the substitution is selected from halogen, hydroxyl, amino, C 1-6 Alkylamino, (C 1-6 alkyl)2-amino substituent;

[0063] R 7 As defined above.

[0064] In certain embodiments, each R 6 are each independently selected from hydrogen, deuterium and fluorine.

[0065] In certain embodiments, Q 1 Selected from C 1-6 Alkylene, -CO-, amino, -SO-, -SO2-, -C(=NR 7 )-or any of the following structures:

[0066]

[0067] In certain embodiments, Q 2 Selected from -SO2-, -SO(=NH)-, -SO(=NMe)- and -CO-.

[0068] In certain embodiments, each R 7 are independently selected from hydrogen, deuterium, methyl and cyano.

[0069] In certain embodiments, n4 is 0 or 1.

[0070] In certain embodiments, R 9 Selected from oxygen and NH.

[0071] In certain embodiments, R 10 and R 11 Each independently selected is hydrogen and methyl.

[0072] In certain embodiments, R 10 and R 11 Together with the atoms to which they are attached, they form C 3-7 Cycloalkyl or a 3-7 membered heterocyclic group containing 1-2 atoms selected from oxygen, nitrogen and sulfur; 3-7 Cycloalkyl is cyclopropane.

[0073] In certain embodiments, R 12 and R 13 Each is independently selected from hydrogen, deuterium, methyl, fluorine, and hydroxyl.

[0074] In certain embodiments, R 14 is selected from hydrogen, methyl, ethyl, isopropyl, cyclopropyl, hydroxyethyl,

[0075] In certain embodiments, the compound is selected from:

[0076]

[0077]

[0078]

[0079] In certain embodiments, the compound is selected from:

[0080]

[0081]

[0082] The present disclosure also provides a method for preparing the compound of formula I, route 1:

[0083]

[0084] Route 2:

[0085]

[0086] The present disclosure also provides a pharmaceutical composition, comprising at least one of the aforementioned compounds or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0087] In certain embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0088] In certain embodiments, the pharmaceutical composition contains 0.01%-99.99% of the aforementioned compound based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of the aforementioned compound. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound.

[0089] In certain embodiments, the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable carrier, diluent or excipient, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable carrier, diluent or excipient. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable carrier, diluent or excipient. In certain embodiments, the pharmaceutical composition contains 1%-99% of a pharmaceutically acceptable carrier, diluent or excipient. In certain embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable carrier, diluent or excipient.

[0090] All compounds involved in the present invention and mixtures and compositions containing the compounds of the present invention can be administered to the body via any administration route. The administration route can be oral administration, intravenous injection, intramuscular injection, subcutaneous injection, rectal administration, vaginal administration, sublingual infusion, nasal inhalation, oral inhalation, eye drops, and local or systemic transdermal administration.

[0091] All compounds involved in the present invention and mixtures and compositions containing the compounds of the present invention can be formulated into a single dose, which contains the active compound of the present invention and carriers, excipients, etc., and the dosage form can be tablets, capsules, injections, granules, powders, suppositories, pills, creams, pastes, gels, powders, oral solutions, inhalants, suspensions, dry suspensions, patches, lotions, etc. These dosage forms may contain ingredients commonly used in pharmaceutical preparations, such as diluents, absorbents, wetting agents, binders, disintegrants, colorants, pH adjusters, antioxidants, antibacterial agents, isotonicity adjusters, anti-adhesive agents, etc.

[0092] Suitable formulations of the above-mentioned various dosage forms are available from public sources, such as Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins published in 2006 and Rowe, Raymond C. Handbook of Pharmaceutical Excipients, Chicago, Pharmaceutical Press published in 2005. Therefore, those skilled in the art can easily prepare them.

[0093] Different dosages can be selected according to the nature and intensity of the disease suffered by different individuals, the age, gender, weight of the patient, the route of administration and other factors. The dosage of the compound of the present invention can be 0.01 to 500 mg / kg per day, preferably 1-100 mg / kg per day, and can be administered once or multiple times.

[0094] The present disclosure also provides use of the compounds described in the present disclosure, their pharmaceutically acceptable salts or pharmaceutical compositions in the preparation of drugs for treating and / or preventing ATR kinase-mediated diseases and related diseases. The disease is selected from diseases, disorders and conditions of excessive or abnormal cell proliferation. Preferably, the diseases, disorders and conditions include cancer and myeloproliferative diseases. Preferably, the cancer is selected from skin cancer, bladder cancer, ovarian cancer, breast cancer, gastric cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, gastric cancer, kidney cancer, renal parenchymal cancer, cervical cancer, uterine body cancer, endometrial cancer, testicular cancer, urinary cancer, melanoma, astrocytic carcinoma, meningioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, adult T-cell leukemia lymphoma, hepatocellular carcinoma, bronchial cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, seminoma, rhabdomyosarcoma, chondrosarcoma, myosoma, fibrosarcoma.

[0095] The present disclosure further provides a method for treating ATR kinase-mediated diseases, wherein the mammal may be a human or a non-human mammal, for therapeutic purposes, comprising administering the compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in the present disclosure to the mammal.

[0096] Terminology explanation:

[0097] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0098] The term "C 1-6 “Alkyl”, alone or in combination, means a saturated straight or branched alkyl group containing 1 to 6, especially 1 to 4 carbon atoms, including methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, n-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3,-dimethyl-2-butyl, etc. Preferably, “C 1-6"Alkyl" is any one of methyl, ethyl, isopropyl and tert-butyl. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available attachment point. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0099] The term "C 1-6 "Alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane by removing two hydrogen atoms, which is an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and the like. Alkylene can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment.

[0100] The term "C 3-7 "Cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, which means a saturated cycloalkyl group having 3 to 7, especially 3-6 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. In particular, "C 3-7 "Cycloalkyl" refers to cyclopropyl, cyclopentyl, cyclohexyl, etc.

[0101] The term "C 1-6 "Alkylamino" alone or in combination means an amino group as defined above, wherein the hydrogen atom of the amino group is replaced by at least one C 1-6 Alkyl substituted, where "C 1-6 "C alkyl" means as defined above, and accordingly, "C 1-6“Alkylamino” includes methylamino, ethylamino, propylamino, isopropylamino, n-butylamino, isobutylamino, 2-butylamino, tert-butylamino, n-pentylamino, 2-pentylamino, 3-pentylamino, 2-methyl-2-butylamino, 3-methyl-2-butylamino, 3-methyl-1-butylamino, 2-methyl-1-butylamino, n-hexylamino, 2-hexylamino, 3-hexylamino, 2-methyl-2-pentylamino, 3-methyl-2-pentylamino, 4-methyl-2-pentylamino, 3-methyl-3-pentylamino, 2-methyl-3-pentylamino, 2,3-dimethyl-2-butylamino, 3,3-dimethyl-2-butylamino and the like. Particularly, “C 1-6 "Alkylamino" means methylamino, ethylamino, isopropylamino, tert-butylamino and the like.

[0102] The term "C 1-6 "Alkoxy" alone or in combination means a group C 1-6 Alkyl-O-, where "C 1-6 "Alkyl" means as defined above.

[0103] The term "heterocyclic group" refers to a saturated or partially unsaturated (containing 1 or 2 double bonds) non-aromatic cyclic group consisting of carbon atoms and heteroatoms such as nitrogen, oxygen or sulfur. This cyclic group can be a monocyclic or bicyclic group. In the present invention, the number of heteroatoms in the heterocyclic group is preferably 1, 2, 3 or 4, and the nitrogen, carbon or sulfur atom in the heterocyclic group can be optionally oxidized. The hydrogen atoms on the "heterocyclic group" are independently and optionally substituted by one or more substituents described in the present invention. The "heterocyclic group" can be linked to the parent molecule through any ring atom on the ring.

[0104] The term "3-7 membered heterocyclyl" refers to a monocyclic heterocyclic group containing 3-7 carbon atoms and heteroatoms; for example, aziridinyl, azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxothiomorpholinyl.

[0105] The term "C 3-7 "Cycloalkylamino" means, alone or in combination, an amino group as defined above, wherein the hydrogen atoms of the amino group are replaced by at least one C 3-7 Cycloalkyl substituted, "C 3-7 "Cycloalkyl" means as defined above.

[0106] The term "3-7 membered heterocyclylamino" means, alone or in combination, an amino group as defined above, wherein the hydrogen atoms of the amino group are replaced by at least one C 3-7 Heterocyclic group substituted, "C 3-7 "Heterocyclyl" means as defined above.

[0107] The term "aryl" refers to any stable 6-10 membered monocyclic or bicyclic aromatic group, including phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydroindanyl or biphenyl and the like.

[0108] The term "heteroaryl" refers to an aromatic ring group formed by replacing the carbon atoms on the ring with at least one heteroatom selected from sulfur, oxygen or nitrogen, and the aromatic ring group can be a 5-7-membered monocyclic or 7-12-membered bicyclic group. In the present invention, the number of heteroatoms in the heterocyclic group is preferably 1, 2, 3 or 4, such as thienyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, imidazolyl, tetrazolyl, isothiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, naphthyl, benzothienyl, indolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, quinolyl, isoquinolyl, quinazolinyl, etc.

[0109] The term "aryl C 1-6 "Alkyl" means C 1-6 The alkyl group is substituted with one or more aryl groups. 1-6 Alkyl means as defined above.

[0110] The term "heteroaryl C 1-6 "Alkyl" means C 1-6 The alkyl group is substituted with one or more heteroaryl groups, the heteroaryl and C 1-6 Alkyl means as defined above.

[0111] The term "amino" alone or in combination means a primary amino group (-NH2), a secondary amino group (-NH-) or a tertiary amino group.

[0112] The term "halogen" alone or in combination denotes fluorine, chlorine, bromine or iodine, in particular fluorine, chlorine or bromine.

[0113] The term "cyano", alone or in combination, refers to the group -CN.

[0114] The term "carboxy", alone or in combination, refers to the group -COOH.

[0115] The term "hydroxy", alone or in combination, refers to the group -OH.

[0116] The term "guanidino", alone or in combination, refers to the group -NH-C(=NH)-NH2.

[0117] The term "urea", alone or in combination, refers to the group -NH-C(=O)-NH2.

[0118] The term "amido", alone or in combination, refers to the group -C(=O)-NH2.

[0119] The term "substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and a person skilled in the art will be able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g. olefinic) bond.

[0120] In the chemical structures of the compounds disclosed herein, the bond No configuration is specified, i.e., the bond Can be or or include both and In the chemical structure of the compounds disclosed in the present invention, the bond The configuration is not specified, that is, it can be Z configuration or E configuration, or contain both configurations.

[0121] The term "isomer" includes all isomeric forms including enantiomers, diastereomers and geometric isomers including cis-trans isomers. Therefore, the single stereochemical isomers of the compounds designed in the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or cis-trans isomers) all belong to the scope of the present invention.

[0122] The term "isotope-labeled substance" refers to the introduction of an isotope into any compound of the present invention. The introduced isotope may be 2 H, 3 H, 13 C, 14 C, 15 N, 17 Oh, 18 Oh, 31 P, 32 P, 35 S, 18 F, 36 Cl, specific isotopic derivatives can be prepared by conventional techniques.

[0123] The term "pharmaceutically acceptable salt" means that the compounds of the present invention exist in the form of their pharmaceutical salts, including acid addition salts and base addition salts. Pharmaceutically acceptable salts are described in pharmaceutically salts described by SM Berge in J. Pharmaceutical Sciences (Volume 66: 1-19, 1977). In the present invention, pharmaceutically acceptable non-toxic acid addition salts refer to salts formed by the compounds of the present invention and organic or inorganic acids, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the compounds of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts, such as ammonium salts or N-containing organic bases. + (C 1-6 Alkyl)4 salt.

[0124] The terms involved in the present invention are defined above. Those skilled in the art can also understand the above terms in combination with the prior art. The following is a further description based on the content of the present invention and the definitions of the terms.

[0125] The preparation of the compounds and pharmaceutically acceptable salts described in the present disclosure is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure.

[0126] The experimental methods in the examples of this disclosure that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or product manufacturers. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0127] The abbreviations used in the present invention are as follows:

[0128] CDCl3: deuterated chloroform

[0129] Dioxane: 1,4-dioxane

[0130] CO2: Carbon dioxide

[0131] conc.H2SO4: concentrated sulfuric acid

[0132] DCM: dichloromethane

[0133] DME: Ethylene glycol dimethyl ether

[0134] DMF: N,N-dimethylformamide

[0135] DMSO: dimethyl sulfoxide

[0136] DMSO-d6: deuterated dimethyl sulfoxide

[0137] EtOH: ethanol

[0138] EtONa: Sodium ethoxide

[0139] FeCl3: Ferric chloride

[0140] g: grams

[0141] HCOOH: Formic acid

[0142] Hz:Hertz

[0143] h: hours

[0144] IC 50 : Half maximal inhibitory concentration

[0145] MeOH: Methanol

[0146] mg: milligram

[0147] mL: milliliters

[0148] mmol: millimole

[0149] MHz: Megahertz

[0150] NaBH3CN: Sodium cyanoborohydride

[0151] NaOH: Sodium hydroxide

[0152] NH4OAc: ammonium acetate

[0153] NMR: Nuclear Magnetic Resonance

[0154] M: molar concentration

[0155] Oxalyl chloride:

[0156] PBS: Phosphate buffered saline

[0157] PdCl2(dppf): [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride

[0158] TCQ: Chloranil

[0159] TLC: Thin layer chromatography

[0160] TsNHNH2: p-Toluenesulfonylhydrazine

[0161] uM: micromoles / liter

[0162] μg: microgram

[0163] μL: microliter

[0164] δ: Chemical shift

[0165] The following describes the general test conditions in the embodiments of the present invention:

[0166] First, the reactions in the examples are generally carried out under nitrogen protection.

[0167] Furthermore, the intermediates and final products were separated and purified by chromatographic columns, preparative chromatographic plates and ISCO rapid preparative chromatographic system. The chromatographic columns were filled with silica gel, the silica gel (300-400 mesh) was produced by Shanghai Titan Technology Co., Ltd., the preparative chromatographic plates were produced by Yantai Jiangyou Silica Gel Development Co., Ltd., and the chromatographic columns used by ISCO (particle size 40-63 μm, ) is produced by Changzhou Santai Technology Co., Ltd.

[0168] Furthermore, the LC-MS liquid chromatography-mass spectrometry instrument used was Waters ACQUITY Arc equipped with QDaDetector. A Waters XBridge C18 column (specifications: 2.1×50 mm, 3.5 μm) was used. The mass spectrometry (MS) used an ESI source and only indicated the molecular weight M of the parent molecule, usually reporting [M+H] + The injection volume was determined by the sample concentration; the flow rate was 1.2 mL / min; the HPLC peak was recorded and read at UV-Vis wavelengths of 220 nm and 254 nm. The mobile phases were 0.01% formic acid in ultrapure water (mobile phase A) and 0.01% formic acid in acetonitrile (mobile phase B). The gradient elution conditions are shown in Tables 1 and 2 below:

[0169] Table 1: Gradient elution conditions 1

[0170] Time (min) <![CDATA[A(H2O,0.01%HCOOH)]]> <![CDATA[B(CH3CN,0.01%HCOOH)]]> 0.0-0.3 95-85 5-15 0.3-3.2 85-20 15-80 3.2-3.8 20-5 80-95 3.8-3.81 5-95 95-5 3.81-4.0 95 5

[0171] Table 2: Gradient elution conditions 2

[0172]

[0173]

[0174] Further, NMR spectra were obtained using a Varian 400 MHz nuclear magnetic resonance spectrometer, often using CDCl3, DMSO-d6 as solvents, and chemical shifts were reported in ppm. The descriptions of the various peaks are as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets). Coupling constants are expressed in Hz.

[0175] Embodiment 1:

[0176] 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride

[0177] Step 1a: Preparation of (E)-4-((hydroxyimino)methyl)benzonitrile

[0178]

[0179] Step 1a Specific method: At room temperature, hydroxylamine hydrochloride (3.18 g, 45.75 mmol) was added to a mixed solution of 4-cyano-benzaldehyde (5 g, 38.13 mmol) in tetrahydrofuran (30 mL) and water (3 mL). The reaction mixture was stirred at room temperature (~20°C) for 16 hours. Water (40 mL) was added to the reaction system, the organic phase was separated, the aqueous phase was extracted three times with ethyl acetate (30 mL), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow solid (E)-4-((hydroxyimino)methylene)benzonitrile (4.5 g), which was a crude product and was directly used in the next step without further purification.

[0180] Step 1b: Preparation of di(tert-butyl)(5-bromo-3-(3-(4-cyanophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0181] Step 1b Specific method: To a mixed solution of di(tert-butyl)(5-bromo-3-ethynyl-2-pyrazinyl)carbamate (0.5 g, 1.26 mmol) in methanol (12 mL) and water (2.4 mL), (E)-4-((hydroxyimino)methylene)benzonitrile (0.27 g, 1.88 mmol) and bis(trifluoroacetyl)iodobenzene (0.81 g, 1.88 mmol) were added in sequence, and stirred at room temperature overnight. Ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The organic phase was then washed three times with water (20 mL). The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain di(tert-butyl)(5-bromo-3-(3-(4-cyanophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.45 g).

[0182] Step 1c: Preparation of di(tert-butyl)(3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0183]

[0184] Step 1c Specific method: Under nitrogen protection, to a mixed solution of di(tert-butyl)(5-bromo-3-(3-(4-cyanophenyl)isoxazol-5-yl)pyrazine-2-yl)carbamate (1.5 g, 3.39 mmol) in 1,4-dioxane (15 mL) and water (1.5 mL), 4-isopropylsulfonylphenylboronic acid (0.77 g, 3.39 mmol), potassium carbonate (0.94 g, 6.78 mmol) and 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (0.5 g, 0.678 mmol) were added at room temperature. After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di(tert-butyl)(3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (0.6 g).

[0185] Step 1d: Preparation of di(tert-butyl)(3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0186]

[0187] Step 1d Specific method: To a solution of di(tert-butyl)(3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfone)phenyl)pyrazin-2-yl)carbamate (540 mg, 0.99 mmol) in methanol (15 mL) at room temperature, nickel chloride hexahydrate (705 mg, 2.97 mmol) and sodium borohydride (112 mg, 2.97 mmol) were added in sequence. After the reaction solution was stirred at room temperature for 1 hour, it was quenched with ammonium chloride (2 mL). Ethyl acetate (40 mL) and saturated aqueous sodium bicarbonate solution (40 mL) were added. After the mixed solution was filtered through diatomaceous earth, the filtrate was poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40 mL), and the combined organic phases were concentrated and separated by column chromatography to give di(tert-butyl)(3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (300 mg).

[0188] Step 1e: Preparation of di(tert-butyl)(3-(3-(4-(guanidinomethylidene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0189]

[0190] Step 1e Specific method: To a solution of di(tert-butyl)(3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (36 mg, 0.053 mmol) in acetonitrile (3 mL), 1H-pyrazole-1-carboximidamide hydrochloride (7.5 mg, 0.051 mmol) and N,N-diisopropylethylamine (10 μL) were added in sequence. The mixture was stirred at room temperature for 16 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction solution, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di(tert-butyl)(3-(3-(4-(guanidinomethylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (27 mg).

[0191] Step 1f: Preparation of 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride

[0192] Step 1f Specific method: To a solution of di(tert-butyl)(3-(3-(4-(guanidinomethylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (27 mg, 0.04 mmol) in ethyl acetate (2 mL), a mixed solution of hydrochloric acid and ethyl acetate (4 M, 2 mL) was added. After the reaction solution was stirred at room temperature for 1 hour, the reaction solution was concentrated to obtain a yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine hydrochloride (22 mg). 1 H NMR (400 MHz, DMSO-d6): δ8.94 (s, 1H), 8.38–8.36 (m, 2H), 8.05–8.03 (m, 2H), 7.94–7.92 (m, 2H), 7.80 (s, 1H), 7.53–7.51 (m, 2H), 7.48 (brs, 3H), 7.35 (brs, 2H), 7.22 (brs, 3H), 4.52–4.50 (m, 2H), 3.49–3.42 (m, 1H), 1.20–1.18 (m, 6H). MS found (ESI + )[(M+H) + ]:492.

[0193] Embodiment 2:

[0194] (4-(5-amino-6-(3-(4-((methylamino)methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)phenyl)(imino)(isopropyl)-λ 6 -Sulfoxide

[0195]

[0196] Step 2a: Preparation of (4-(propyl-2-sulfonylimino)phenyl)boronic acid

[0197]

[0198] Step 2a Specific method: To a solution of 4-(isopropylthio)phenylboronic acid (2.20 g, 11.22 mmol) in ethanol (50 mL), iodobenzene diacetate (10.84 g, 33.66 mmol) and acetic acid amine (3.46 g, 44.88 mmol) were added. The reaction solution was heated to 75 ° C and stirred for 4 hours, and then ethyl acetate (50 mL) and water (50 mL) were added to the reaction solution. The aqueous phase was concentrated to a solid, and ethyl acetate (50 mL) and methanol (30 mL) were added to the solid, stirred for 0.5 hours, filtered, and the filtrate was concentrated to obtain a black oil (4-(propyl-2-sulfinyl imino) phenyl) boronic acid (1.3 g), which was a crude product and was directly used in the next step without further purification.

[0199] Step 2b: Preparation of 1-(4-(diethoxymethyl)phenyl)-N-methylmethyleneamine

[0200]

[0201] Step 2b Specific method: triethylamine (1.6 mL) and methylamine hydrochloride (0.78 g, 11.52 mmol) were added to a methanol (10 mL) solution of terephthalaldehyde diacetaldehyde (2 g, 9.60 mmol) in sequence. After the reaction solution was stirred at room temperature for 0.5 hours, sodium borohydride (0.58 g, 15.37 mmol) was added. The reaction solution was stirred at room temperature for another hour. Ice water (0.1 mL) was added to quench the reaction, and then ethyl acetate (50 mL) and water (50 mL) were added. The aqueous phase after separation was extracted three times with ethyl acetate (20 mL), and the combined organic phase was concentrated and separated by column chromatography to obtain 1-(4-(diethoxymethyl)phenyl)-N-methylmethyleneamine (2.78 g).

[0202] Step 2c: Preparation of tert-butyl (4-(diethoxymethyl)benzyl)(methylene)carbamate

[0203]

[0204] Step 2c Specific method: To a solution of 1-(4-(diethoxymethyl)phenyl)-N-methylmethyleneamine (2.78g, 9.58mmol) in dichloromethane (20mL), triethylamine (2.91g, 28.75mmol) and di-tert-butyl dicarbonate (6.27g, 28.75mmol) were added in sequence. The reaction solution was stirred at room temperature for 4 hours. Water (20mL) was added to the reaction solution. After separation, the aqueous phase was extracted three times with dichloromethane (20mL). The combined organic phase was concentrated and separated by column chromatography to obtain tert-butyl (4-(diethoxymethyl)benzyl) (methylene) carbamate (2.9g).

[0205] Step 2d: Preparation of tert-butyl (E)-(4-((hydroxyimino)methyl)benzyl)(methyl)carbamate

[0206]

[0207] Specific method of step 2d: tert-butyl (4-(diethoxymethyl)benzyl)(methyl)carbamate (2.9 g, 8.97 mmol) and hydroxylamine hydrochloride (0.75 g, 10.76 mmol) were prepared by a method similar to step 1a of example 1 to obtain tert-butyl (E)-(4-((hydroxyimino)methyl)benzyl)(methyl)carbamate (1.8 g) as a yellow oil.

[0208] Step 2e: Preparation of tert-butyl(5-bromo-3-(3-(4-(((tert-butoxycarbonyl)(methylene)amino)phenyl)isoxazol-5-yl)pyrazin-2-yl)(tert-butoxycarbonyl)carbamate

[0209]

[0210] Specific method of step 2e: tert-butyl (E)-(4-((hydroxyimino)methyl)benzyl)(methyl)carbamate (500 mg, 1.88 mmol), (E)-4-((isonitroso)methyl)benzonitrile (500 mg, 1.26 mmol) and bis(trifluoroacetyl)iodobenzene (810 mg, 1.88 mmol) were prepared by a method similar to that of step 1b of example 1 to obtain tert-butyl (5-bromo-3-(3-(4-(((tert-butoxycarbonyl)(methylene)amino)phenyl)isoxazol-5-yl)pyrazin-2-yl)(tert-butoxycarbonyl)carbamate (680 mg).

[0211] Step 2f: Preparation of tert-butyl(tert-butoxycarbonyl)(3-(3-(4-(((tert-butoxycarbonyl)methylene)amino)phenyl)isoxazol-5-yl)-5-(4-(2-propylsulfonylimino)phenyl)pyrazin-2-yl)carbamate

[0212]

[0213] Specific method of step 2f: tert-butyl (5-bromo-3-(3-(4-(((tert-butoxycarbonyl)(methylene)amino)phenyl)isoxazol-5-yl)pyrazine-2-yl)(tert-butoxycarbonyl)carbamate (100 mg, 0.151 mmol) and (4-(propyl-2-yl-sulfinylimino)phenyl)boric acid (68 mg, 0.302 mmol), potassium carbonate (42 mg, 0.302 mmol) and 1,1'-bis(diphenylphosphinoferrocenepalladium) dichloride (22 mg, 0.03 mmol) were prepared according to a method similar to step 1c of Example 1 to obtain yellow solid tert-butyl (tert-butoxycarbonyl)(3-(3-(4-(((tert-butoxycarbonyl)methylene)amino)phenyl)isoxazol-5-yl)-5-(4-(2-propylsulfinylimino)phenyl)pyrazine-2-yl)carbamate (65 mg).

[0214] Step 2g: Preparation of (4-(5-amino-6-(3-(4-((methylamino)methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)phenyl)(imino)(isopropyl)-λ 6 -Sulfoxide

[0215]

[0216] Step 2g Specific method: To a solution of tert-butyl(tert-butyloxycarbonyl)(3-(3-(4-(((tert-butyloxycarbonyl)methylene)amino)phenyl)isoxazol-5-yl)-5-(4-(2-propylsulfinylimino)phenyl)pyrazin-2-yl)carbamate (35 mg, 0.046 mmol) in ethyl acetate (2 mL), a mixed solution of hydrochloric acid and ethyl acetate (4 M, 2 mL) was added. The reaction solution was stirred at room temperature for 1 hour, and then the reaction solution was concentrated and ethyl acetate (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL) were added. After separation, the organic phase was washed once with water (20 mL), and the organic phase was concentrated and separated by column chromatography to obtain (4-(5-amino-6-(3-(4-((methylamino)methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)phenyl)(imino)(isopropyl)-λ 6 -sulfoxide (14.5 mg). 1H NMR (400MHz, DMSO-d6): δ9.29 (s, 2H), 8.93 (s, 1H), 8.33–8.31 (m, 2H), 8.11–8.09 (m, 2H), 7.95–7.92 (m, 2H), 7.84 (s, 1H), 7.74–7.72 (m, 2H), 7.23–7.17 (m, 2H), 4.20 (s, 2H), 3.30–3.24 (m, 1H), 2.58 (s, 3H), 1.17–1.15 (m, 6H). MS found (ESI + )[(M+H) + ]:463.

[0217] Example 3: 3-(3-(1-H-benzo[d][1,2,3]triazol-5-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-amine

[0218]

[0219] Step 3a: Preparation of 1-H-benzo[d][1,2,3]triazole-5-carbonitrile

[0220]

[0221] Step 3a Specific method: To a solution of 3,4-diaminobenzonitrile (1.0 g, 7.51 mmol) in acetic acid (15 mL), add an aqueous solution of sodium nitrite (0.62 g of sodium nitrite dissolved in 15 mL of water). The reaction solution was stirred at room temperature for 2 hours. Ethyl acetate (40 mL) and an aqueous solution (20 mL) were added. After separation, the organic phase was concentrated to obtain a white solid 1-H-benzo[d][1,2,3]triazole-5-carbonitrile (0.8 g), which was a crude product and was used directly in the next step without further purification.

[0222] Step 3b: Preparation of tert-butyl-5-cyano-1-H-benzo[d][1,2,3]triazole-1-carbamate

[0223]

[0224] Step 3b: 1-H-benzo[d][1,2,3]triazole-5-carbonitrile (0.8 g, 5.55 mmol), triethylamine (2.27 mL) and di-tert-butyl dicarbonate (3.63 g, 16.65 mmol) were prepared by a method similar to that of Step 2c of Example 2 to obtain tert-butyl-5-cyano-1-H-benzo[d][1,2,3]triazole-1-carbamate (1.23 g).

[0225] Step 3c: Preparation of 1-H-benzo[d][1,2,3]triazole-5-carbaldehyde

[0226]

[0227] Step 3c Specific method: DABAL-H (12 mL, 1M in PhMe) was slowly added to a toluene (10 mL) solution of tert-butyl-5-cyano-1-H-benzo[d][1,2,3]triazole-1-carbamate (1.23 g, 5.04 mmol) at -40°C. The reaction solution was stirred at -40°C for 3 hours. L-tartaric acid aqueous solution (1 mL, 1M) was added to quench. Ethyl acetate (20 mL) was added, the system was filtered, and the filtrate was washed with water (20 mL) and saturated brine (20 mL). The organic phase was concentrated to give 1-H-benzo[d][1,2,3]triazole-5-carboxaldehyde (160 mg) as a crude product and used directly in the next step without further purification.

[0228] Step 3d: Preparation of tert-butyl-5-formyl-1-H-benzo[d][1,2,3]triazole-1-carbamate

[0229]

[0230] Step 3d Specific method: 1-H-benzo[d][1,2,3]triazole-5-carboxaldehyde (0.16 g, 1.09 mmol), triethylamine (0.44 mL) and di-tert-butyl dicarbonate (0.72 g, 3.26 mmol) were prepared by a method similar to that of Step 2c of Example 2 to obtain tert-butyl-5-formyl-1-H-benzo[d][1,2,3]triazole-1-carbamate (140 mg).

[0231] Step 3e: Preparation of tert-butyl (E)-6-((hydroxyimino)methylene)-1H-benzo[d][1,2,3]triazole-1-carboxylate

[0232] Step 3e Specific method: tert-butyl-5-formyl-1-H-benzo[d][1,2,3]triazole-1-carbamate (0.14 g, 0.566 mmol) and hydroxylamine hydrochloride (0.048 g, 0.68 mmol) were prepared in a manner similar to that of Step 1a of Example 1 to obtain tert-butyl (E)-6-((hydroxyimino)methylene)-1H-benzo[d][1,2,3]triazole-1-carboxylate (75 mg) as a yellow oil.

[0233] Step 3f: Preparation of di(tert-butyl)-5-(5-(6-bromo-3-((tert-butoxycarbonyl)amino)pyrazin-2-yl)isoxazol-3-yl)-1H-benzo[d][1,2,3]triazole-1-carboxylate

[0234]

[0235] Step 3f Specific method: Tert-butyl (E)-6-((hydroxyimino)methylene)-1H-benzo[d][1,2,3]triazole-1-carboxylate (75 mg, 0.282 mmol), (E)-4-((isonitroso)methyl)benzonitrile (75 mg, 0.188 mmol) and bis(trifluoroacetyl)iodobenzene (121 mg, 0.282 mmol) were prepared in a manner similar to that of Step 1b of Example 1 to give yellow solid di(tert-butyl)-5-(5-(6-bromo-3-((tert-butoxycarbonyl)amino)pyrazin-2-yl)isoxazol-3-yl)-1H-benzo[d][1,2,3]triazole-1-carboxylate (30 mg).

[0236] Step 3g: Preparation of tert-butyl (3-(3-(1-H-benzo[d][1,2,3]triazol-5-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0237]

[0238] Specific method of step 3g: Di(tert-butyl)-5-(5-(6-bromo-3-((tert-butoxycarbonyl)amino)pyrazin-2-yl)isoxazol-3-yl)-1H-benzo[d][1,2,3]triazole-1-carboxylate (30 mg, 0.045 mmol) and 4-isopropylsulfonylphenylboronic acid (10 mg, 0.045 mmol), potassium carbonate (13 mg, 0.09 mmol) and 1,1'-bis(diphenylphosphinoferrocenepalladium) dichloride (7 mg, 0.009 mmol) were prepared according to a method similar to step 1c of Example 1 to obtain yellow solid tert-butyl (3-(3-(1-H-benzo[d][1,2,3]triazol-5-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (15 mg).

[0239] Step 3h: Preparation of 3-(3-(1-H-benzo[d][1,2,3]triazol-5-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-amine

[0240]

[0241] Step 3h Specific method: Tert-butyl (3-(3-(1-H-benzo[d][1,2,3]triazol-5-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (15 mg, 0.027 mmol) and ethyl hydrochloride (2 mL, 4 M) were prepared in a manner similar to that of Step 2g of Example 2 to give yellow solid 3-(3-(1-H-benzo[d][1,2,3]triazol-5-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-amine (6.9 mg). 1 H NMR (DMSO-d6, 400 MHz): δ8.96 (s, 1H), 8.65 (brs, 1H), 8.41–8.39 (m, 2H), 8.13–8.08 (m, 2H), 8.00 (s, 1H), 7.95–7.93 (m, 2H), 7.25–7.24 (m, 3H), 3.50–3.43 (m, 1H), 1.20–1.18 (m, 6H). MS found (ESI + )[(M+H) + ]:462.

[0242] Example 4: 1-(4-(5-(3-amino-6-(4-isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3-fluorobenzyl)guanidine dihydrochloride

[0243]

[0244] Step 4a: Preparation of (E)-3-fluoro-4-((hydroxyimino)methylene)benzonitrile

[0245]

[0246] Step 4a Specific method: Add hydroxylamine hydrochloride (1.86 g, 26.82 mmol) and potassium carbonate (1.85 g, 13.41 mmol) to a mixed solution of 4-cyano-2-fluoroformaldehyde (2.0 g, 13.41 mmol) in methanol (30 mL) and water (3 mL). Stir the reaction mixture at 75 ° C for 3 hours. Add ethyl acetate (40 mL) and water (40 mL) to the reaction system, separate the organic phase, extract the aqueous phase with ethyl acetate (30 mL) three times, dry the combined organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain a white solid (E)-3-fluoro-4-((hydroxyimino)methylene)benzonitrile (2.1 g), which is a crude product and is directly used in the next step without further purification.

[0247] Step 4b: Preparation of di(tert-butyl)(5-bromo-3-(3-(4-cyano-2-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0248]

[0249] Step 4b specific method: (E)-3-Fluoro-4-((hydroxyimino)methylene)benzonitrile (300 mg, 1.88 mmol), di(tert-butyl)(5-bromo-3-ethynyl-2-pyrazinyl)carbamate (500 mg, 1.26 mmol) and bis(trifluoroacetyl)iodobenzene (0.81 g, 1.88 mmol) were prepared in a manner similar to that of Step 1b of Example 1 to give yellow solid di(tert-butyl)(5-bromo-3-(3-(4-cyano-2-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (450 mg).

[0250] Step 4c: Preparation of di(tert-butyl)(3-(3-(4-cyano-2-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0251]

[0252] Specific method of step 4c: di(tert-butyl)(5-bromo-3-(3-(4-cyano-2-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (450 mg, 0.803 mmol), 4-isopropylsulfonylphenylboronic acid (183 mg, 0.803 mmol), potassium carbonate (222 mg, 1.61 mmol) and 1,1'-bis(diphenylphosphinoferrocenepalladium) dichloride (117 mg, 0.16 mmol) were prepared by a method similar to step 1c of Example 1 to obtain light yellow solid di(tert-butyl)(3-(3-(4-cyano-2-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (230 mg).

[0253] Step 4d: Preparation of di(tert-butyl)(3-(3-(4-(aminomethyl)-2-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0254]

[0255] Step 4d Specific method: Di(tert-butyl)(3-(3-(4-cyano-2-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (100 mg, 0.177 mmol), nickel chloride hexahydrate (127 mg, 0.532 mmol) and sodium borohydride (20 mg, 0.532 mmol) were prepared according to a method similar to Step 1d of Example 1 to obtain di(tert-butyl)(3-(3-(4-(aminomethyl)-2-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (28 mg) as a pale yellow oil.

[0256] Step 4e: Preparation of di(tert-butyl)(3-(3-(2-fluoro-4-(guanidinomethylidene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0257]

[0258] Specific method of step 4e: di(tert-butyl)(3-(3-(4-(aminomethyl)-2-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (28 mg, 0.05 mmol), 1H-pyrazole-1-carboximidamide hydrochloric acid (7 mg, 0.05 mmol) and N,N-diisopropylethylamine (10 uL) were prepared according to a method similar to step 1e of Example 1 to obtain light yellow oil di(tert-butyl)(3-(3-(2-fluoro-4-(guanidinomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (15 mg).

[0259] Step 4f: Preparation of 1-(4-(5-(3-amino-6-(4-isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3-fluorobenzyl)guanidine dihydrochloride

[0260]

[0261] Specific method of step 4f: Di(tert-butyl)(3-(3-(2-fluoro-4-(guanidinomethylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (15 mg, 0.025 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4M, 2 mL) were prepared according to a method similar to step 1f of Example 1 to obtain a yellow solid 1-(4-(5-(3-amino-6-(4-isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3-fluorobenzyl)guanidine hydrochloride (9.3 mg). 1H NMR (400 MHz, DMSO-d6): δ8.95 (s, 1H), 8.50–8.47 (m, 1H), 8.37–8.35 (m, 2H), 8.06–8.02 (m, 1H), 7.94–7.92 (m, 2H), 7.61–7.60 (m, 1H), 7.47 (brs, 3H), 7.34 (brs, 3H), 7.21 (s, 3H), 4.55–4.53 (m, 2H), 3.49–3.42 (m, 1H), 1.19–1.17 (m, 6H). MS found (ESI + )[(M+H) + ]:510.

[0262] Example 5: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-cyclopropylurea

[0263]

[0264] Step 5a: Preparation of tert-butyl(tert-butyloxycarbonyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(((phenoxycarbonyl)amino)methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0265]

[0266] Step 5a Specific method: triethylamine (0.15 mL) and phenyl chloroformate (114 mg, 0.728 mmol) were added to a solution of di(tert-butyl)(3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (200 mg, 0.364 mmol) in dichloromethane (20 mL), and the mixture was stirred at 25° C. for 2 hours. The mixture was concentrated and purified by column chromatography to give yellow oil tert-butyl(tert-butyloxycarbonyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(((phenyloxycarbonyl)amino)methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (152 mg).

[0267] Step 5b: Preparation of di(tert-butyl)(3-(3-(4-((3-cyclopropylureido)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0268]

[0269] Step 5b Specific method: Cyclopropylamine (22 mg) was added to a solution of tert-butyl (tert-butyloxycarbonyl) (5-(4-(isopropylsulfonyl) phenyl)-3-(3-(4-(((phenyloxycarbonyl)amino)methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (13 mg, 0.019 mmol) in acetonitrile (3 mL), and the mixture was stirred at 70° C. for 2 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction solution, and the aqueous phase after separation was extracted three times with ethyl acetate (20 mL). The combined organic phases were concentrated and separated by column chromatography to obtain di(tert-butyl) (3-(3-(4-((3-cyclopropylureido)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (10 mg) as a white solid.

[0270] Step 5c: Preparation of 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-cyclopropylurea

[0271]

[0272] Specific method of step 5c: Di(tert-butyl)(3-(3-(4-((3-cyclopropylureido)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (10 mg, 0.015 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4 M, 2 mL) were prepared in a manner similar to that of step 2g of Example 2 to obtain yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-cyclopropylurea (1.7 mg). 1 HNMR (DMSO-d6, 400MHz): δ8.93 (s, 1H), 8.38–8.36 (m, 2H), 7.97–7.92 (m, 4H), 7.75 (s, 1H), 7.45–7.43 (m, 2H), 7.17 (s, 2H), 6.49–6.48 (m, 1H), 6.26 (s, 1H), 4.31–4.30 (m, 2H), 3.49–3.42 (m, 1H), 2.47–2.44 (m, 1H), 1.20–1.18 (m, 6H), 0.62–0.58 (m, 2H), 0.38–0.37 (m, 2H). MS measured value (ESI + )[(M+H) + ]:533.

[0273] Example 6: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-ethylurea

[0274]

[0275] Step 6a: Preparation of di(tert-butyl)(3-(3-(4-((3-ethylureido)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0276]

[0277] Specific method of step 6a: tert-butyl(tert-butyloxycarbonyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(((phenoxycarbonyl)amino)methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (22 mg, 0.03 mmol), triethylamine (4.5 uL) and ethyl hydrochloride (27 mg, 0.3 mmol) were prepared by a method similar to that of step 5b of example 5 to obtain white solid di(tert-butyl)(3-(3-(4-((3-ethylureido)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (7 mg).

[0278] Step 6b: Preparation of 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-ethylurea

[0279]

[0280] Specific method of step 6b: Di(tert-butyl)(3-(3-(4-((3-ethylureido)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (7 mg, 0.01 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4M, 2 mL) were prepared according to a method similar to that of step 2g of Example 2 to obtain yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-ethylurea (1.6 mg). 1H NMR (DMSO-d6, 400MHz): δ8.93 (s, 1H), 8.38–8.36 (m, 2H), 7.97–7.92 (m, 4H), 7.75 (s, 1H), 7.44–7.42 (m, 2H), 7.17 (s, 2H), 6.40–6.37 (m, 1H), 5.95–5.92 (m, 1H), 4.29–4.28 (m, 2H), 3.49–3.42 (m, 1H), 3.08–3.02 (m, 2H), 1.20–1.18 (m, 6H), 1.03–1.00 (m, 3H). MS found (ESI + )[(M+H) + ]:521.

[0281] Example 7: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-methylurea

[0282]

[0283] Step 7a: Preparation of di(tert-butyl)(3-(3-(4-((3-methylureido)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0284]

[0285] Specific method of step 7a: tert-butyl(tert-butyloxycarbonyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(((phenoxycarbonyl)amino)methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (15 mg, 0.02 mmol), triethylamine (3 uL) and methyl hydrochloride (15 mg, 0.2 mmol) were prepared by a method similar to that of step 5b of Example 5 to obtain white solid di(tert-butyl)(3-(3-(4-((3-methylureido)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (11 mg).

[0286] Step 7b: Preparation of 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-methylurea

[0287]

[0288] Specific method of step 7b: Di(tert-butyl)(3-(3-(4-((3-methylureido)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (11 mg, 0.018 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4M, 2 mL) were prepared according to a method similar to that of step 2g of Example 2 to obtain yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-methylurea (6.3 mg). 1 H NMR (DMSO-d6, 400MHz): δ8.93 (s, 1H), 8.38–8.36 (m, 2H), 7.97–7.92 (m, 4H), 7.75 (s, 1H), 7.44–7.42 (m, 2H), 7.16 (s, 2H), 6.49–6.46 (m, 1H), 5.87–5.86 (m, 1H), 4.29–4.27 (m, 2H), 3.47–3.44 (m, 1H), 2.59–2.58 (m, 3H), 1.20–1.18 (m, 6H). MS found (ESI + )[(M+H) + ]:507.

[0289] Example 8: 3-(3-(4-((aminosulfonyl)aminomethylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-amine

[0290]

[0291] Step 8a: Preparation of tert-butyl(tert-butyloxycarbonyl)(3-(3-(4-(((N-(tert-butyloxycarbonyl)aminosulfonyl)amino)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0292]

[0293] Step 8a Specific method: Di(tert-butyl)(3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (30 mg, 0.05 mmol), triethylamine (20 uL) and tert-butyl chlorosulfonylaminomethyl ester (24 mg, 0.1 mmol) were prepared according to a method similar to that of Step 5a of Example 5 to obtain pale yellow solid tert-butyl(tert-butoxycarbonyl)(3-(3-(4-(((N-(tert-butoxycarbonyl)aminosulfonyl)amino)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (13 mg).

[0294] Step 8b: Preparation of 3-(3-(4-((aminosulfonyl)aminomethylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-amine

[0295]

[0296] Specific method of step 8b: tert-butyl(tert-butoxycarbonyl)(3-(3-(4-(((N-(tert-butoxycarbonyl)aminosulfonyl)amino)methylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (13 mg, 0.02 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4 M, 2 mL) were prepared in a manner similar to that of step 2g of Example 2 to give yellow solid 3-(3-(4-((aminosulfonyl)aminomethylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-amine (6.2 mg). 1 H NMR: δ (DMSO-d6, 400MHz) 8.93 (s, 1H), 8.39–8.36 (m, 2H), 7.99–7.92 (m, 4H), 7.78 (s, 1H), 7.56–7.54 (m, 2H), 7.17–7.13 (m, 3H), 6.66 (s, 2H), 4.18–4.16 (m, 2H), 3.47–3.44 (m, 1H), 1.20–1.18 (m, 6H). MS found (ESI + )[(M+H) + ]:529.

[0297] Example 9: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-urea

[0298]

[0299] Step 9a: Preparation of di(tert-butyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(ureidomethylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0300]

[0301] Step 9a Specific method: tert-butyl(tert-butyloxycarbonyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(((phenoxycarbonyl)amino)methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (22 mg, 0.03 mmol) and ammonia methanol (2 mL, 7 M) were prepared by a method similar to that of Step 5b of Example 5 to obtain white solid di(tert-butyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(ureidomethylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (16 mg).

[0302] Step 9b: Preparation of 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-urea

[0303]

[0304] Step 9b specific method: Di(tert-butyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(ureidomethylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (16 mg, 0.03 mmol) and HCl in ethyl acetate (4M, 2 mL) were prepared in a manner similar to that of Step 2g of Example 2 to give yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)-3-urea (5.7 mg). 1 H NMR (DMSO-d6, 400MHz): δ8.94 (s, 1H), 8.39–8.37 (m, 2H), 7.98–7.92 (m, 4H), 7.77 (s, 1H), 7.44–7.42 (m, 2H), 7.20 (s, 2H), 6.54–6.51 (m, 1H), 5.59 (s, 2H), 4.27–4.25 (m, 2H), 3.49–3.46 (m, 1H), 1.19–1.18 (m, 6H). MS found (ESI + )[(M+H) + ]:493.

[0305] Example 10: 4-(5-amino-6-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)-N,N-dimethylbenzenesulfonamide dihydrochloride

[0306] Step 10a: Preparation of di(tert-butyl)(3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(N,N-dimethylaminosulfonyl)phenyl)pyrazin-2-yl)carbamate

[0307]

[0308] Step 10a Specific method: Under nitrogen protection, to a mixed solution of di(tert-butyl)(5-bromo-3-(3-(4-cyanophenyl)5-isoxazolyl)2-pyrazinyl)carbamate (200 mg, 0.37 mmol) in 1,4-dioxane (3 mL) and water (0.6 mL), 4-isopropylsulfonylphenylboronic acid (127 mg, 0.41 mmol), potassium carbonate (102 mg, 0.74 mmol) and 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (27 mg, 0.037 mmol) were added in sequence at room temperature (≈20°C). After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (10 mL) and water (5 mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (10 mL), the combined organic phases were concentrated and separated by column chromatography to give di(tert-butyl)(3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(N,N-dimethylaminosulfonyl)phenyl)pyrazin-2-yl)carbamate (205 mg).

[0309] Step 10b: Preparation of di(tert-butyl)(3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(N,N-dimethylaminosulfonyl)phenyl)pyrazin-2-yl)carbamate

[0310]

[0311] Step 10b Specific method: To a solution of di(tert-butyl)(3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(N,N-dimethylaminosulfonyl)phenyl)pyrazin-2-yl)carbamate (205 mg, 0.32 mmol) in methanol (3 mL), nickel chloride hexahydrate (228 mg, 0.96 mmol) and sodium borohydride (36 mg, 0.96 mmol) were added in sequence at room temperature. After the reaction solution was stirred at room temperature for 1 hour, it was quenched with a saturated aqueous solution of ammonium chloride (2 mL). Ethyl acetate (10 mL) and a saturated aqueous solution of sodium bicarbonate (10 mL) were added. After the mixed solution was filtered through diatomaceous earth, the filtrate was poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (10 mL), and the combined organic phases were concentrated and separated by column chromatography to give di(tert-butyl)(3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(N,N-dimethylaminosulfonyl)phenyl)pyrazin-2-yl)carbamate (33 mg).

[0312] Step 10c: Preparation of di(tert-butyl)(5-(4-(N,N-dimethylaminosulfonyl)phenyl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0313]

[0314] Step 10c Specific method: To a solution of di(tert-butyl)(3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(N,N-dimethylaminosulfonyl)phenyl)pyrazin-2-yl)carbamate (33 mg, 0.050 mmol) in acetonitrile (2 mL), 1H-pyrazole-1-carboximidamide hydrochloride (7.3 mg, 0.050 mmol) and N,N-diisopropylethylamine (10 uL) were added in sequence. The mixture was stirred at room temperature (≈20°C) for 16 hours. Ethyl acetate (10 mL) and water (10 mL) were added to the reaction solution, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (10 mL), the combined organic phases were concentrated and separated by column chromatography to give di(tert-butyl)(5-(4-(N,N-dimethylaminosulfonyl)phenyl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (29 mg).

[0315] Step 10d: Preparation of 4-(5-amino-6-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)-N,N-dimethylbenzenesulfonamide dihydrochloride

[0316] Step 10d Specific method: To a solution of di(tert-butyl)(5-(4-(N,N-dimethylaminosulfonyl)phenyl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (29 mg, 0.04 mmol) in ethyl acetate (2 mL), a mixed solution of hydrochloric acid and ethyl acetate (4 M, 2 mL) was added. After the reaction solution was stirred at room temperature for 1 hour, the reaction solution was concentrated to obtain a yellow solid 4-(5-amino-6-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)-N,N-dimethylbenzenesulfonamide dihydrochloride (12.4 mg). 1H NMR (400 MHz, DMSO-d6): δ8.96 (s, 1H), 8.40–8.38 (m, 2H), 8.36-8.33 (m, 1H), 8.08–8.06 (m, 2H), 7.87–7.85 (m, 2H), 7.82 (s, 1H), 7.55–7.53 (m, 2H), 7.44 (brs, 2H), 7.31 (brs, 2H), 7.18 (brs, 3H), 4.53–4.52 (m, 2H), 2.68 (s, 6H). MS found (ESI + )[(M+H) + ]:493.

[0317] Example 11: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3,5-difluorobenzyl)guanidine dihydrochloride

[0318]

[0319] Step 11a: Preparation of tert-butyl N-[(tert-butoxy)carbonyl]-N-{3-ethynyl-5-[4-(propane-2-sulfonyl)phenyl]pyrazin-2-yl}carbamate

[0320]

[0321] Step 11a Specific method: Under nitrogen protection, to a mixed solution of 4-isopropylsulfonylphenylboronic acid (500 mg, 2.19 mmol) in 1,4-dioxane (5 mL) and water (1 mL), at room temperature (~20°C), N-(5-bromo-3-ethynylpyrazine-2-yl)-N-[(tert-butoxy)carbonyl]carbamic acid tert-butyl ester (582 mg, 1.46 mmol), potassium carbonate (606 mg, 4.38 mmol) and 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (107 mg, 0.15 mmol) were added in sequence. After replacing nitrogen three times, the reaction solution was stirred at 55°C for two hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give tert-butyl N-[(tert-butoxy)carbonyl]-N-{3-ethynyl-5-[4-(propane-2-sulfonyl)phenyl]pyrazin-2-yl}carbamate (433 mg).

[0322] Step 11b: Preparation of (E)-3,5-difluoro-4-((hydroxyimino)methyl)benzonitrile

[0323]

[0324] Step 11b Specific method: At room temperature, hydroxylamine hydrochloride (499 mg, 7.18 mmol) was added to a mixed solution of 3,5-difluoro-4-formylbenzonitrile (1 g, 5.98 mmol) in tetrahydrofuran (10 mL) and water (1 mL). The reaction mixture was stirred at room temperature (~20°C) for 16 hours. Water (60 mL) was added to the reaction system, the organic phase was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a white solid (E)-3,5-difluoro-4-((hydroxyimino)methyl)benzonitrile (433 mg), which was a crude product and was directly used in the next step without further purification.

[0325] Step 11c: Preparation of tert-butyl (3-(3-(4-cyano-2,6-difluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0326]

[0327] Step 11c Specific method: To a mixed solution of (E)-3,5-difluoro-4-((hydroxyimino)methyl)benzonitrile (80 mg, 0.44 mmol) in methanol (5 mL) and water (1 mL), tert-butyl N-[(tert-butoxy)carbonyl]-N-{3-ethynyl-5-[4-(propane-2-sulfonyl)phenyl]pyrazin-2-yl}carbamate (147 mg, 0.29 mL) and bis(trifluoroacetyl)iodobenzene (189 mg, 0.44 mmol) were added in sequence, and stirred at room temperature (~20°C) overnight. Ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The organic phase was then washed three times with water (20 mL). The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated and separated by column chromatography to give tert-butyl (3-(3-(4-cyano-2,6-difluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (177 mg).

[0328] Step 11d: Preparation of tert-butyl (3-(3-(4-(aminomethyl)-2,6-difluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0329]

[0330] Step 11d Specific method: To a solution of tert-butyl (3-(3-(4-cyano-2,6-difluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (177 mg, 0.26 mmol) in methanol (2 mL), nickel chloride hexahydrate (185 mg, 0.78 mmol) and sodium borohydride (29 mg, 0.78 mmol) were added in sequence at room temperature. After the reaction solution was stirred at room temperature for 1 hour, it was quenched with ammonium chloride (2 mL). Ethyl acetate (40 mL) and saturated aqueous sodium bicarbonate solution (40 mL) were added. After the mixed solution was filtered through diatomaceous earth, the filtrate was poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40 mL), and the combined organic phases were concentrated and separated by column chromatography to give tert-butyl (3-(3-(4-(aminomethyl)-2,6-difluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (25 mg).

[0331] Step 11e: Preparation of tert-butyl (3-(3-(2,6-difluoro-4-(guanidinomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0332]

[0333] Step 11e Specific method: To a solution of tert-butyl (3-(3-(4-(aminomethyl)-2,6-difluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (25 mg, 0.042 mmol) in acetonitrile (2 mL), 1H-pyrazole-1-carboximidamide hydrochloride (6.3 mg, 0.043 mmol) and N,N-diisopropylethylamine (6 mg, 0.045 mmol) were added in sequence. The mixture was stirred at room temperature (-20° C.) for 16 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction solution, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give tert-butyl (3-(3-(2,6-difluoro-4-(guanidinomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (20 mg).

[0334] Step 11f: Preparation of 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3,5-difluorobenzyl)guanidine dihydrochloride

[0335]

[0336] Step 11f Specific method: To a solution of tert-butyl (3-(3-(2,6-difluoro-4-(guanidinomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (20 mg, 0.032 mmol) in ethyl acetate (2 mL), a mixed solution of hydrochloric acid and ethyl acetate (4 M, 2 mL) was added. After the reaction solution was stirred at room temperature for 1 hour, the reaction solution was concentrated to obtain a yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3,5-difluorobenzyl)guanidine dihydrochloride (13 mg). 1 H NMR (400 MHz, DMSO-d6): δ8.96 (s, 1H), 8.43–8.40 (m, 1H), 8.36–8.34 (m, 2H), 7.93–7.91 (m, 2H), 7.52 (s, 1H), 7.38 (s, 2H), 7.32–7.30 (m, 2H), 7.25 (s, 2H), 7.22 (s, 1H), 7.13 (s, 2H), 4.55–4.54 (m, 2H), 3.48–3.45 (m, 1H), 1.19–1.17 (m, 6H). MS found (ESI + )[(M+H) + ]:528.

[0337] Example 12: 1-(4-(5-(3-amino-6-(4-(N,S-dimethylsulfonylimino)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride

[0338]

[0339] Step 12a: Preparation of (4-bromophenyl)(methyl)(methylimino)-λ 6 -Sulfamethoxazole

[0340]

[0341] Step 12a: Sodium hydride (60% in mineral oil, 154 mg, 3.84 mmol) was added to (4-bromophenyl)(imino)(methyl)-λ in N,N-dimethylformamide (3 mL). 6 -sulfazone (300mg, 1.28mmol) and the resulting mixture was stirred at room temperature for 5min.Iodomethane (0.4mL, 6.41mmol) was added and the reaction mixture was stirred overnight.The mixture was concentrated in a vacuum, diluted with water, and extracted with ethyl acetate.The combined extracts were washed with salt water, dried over MgSO4, and concentrated in a vacuum.The residue was purified by silica gel chromatography to obtain (4-bromophenyl) (methyl) (methylimino)-λ6 -Sulfamethoxazole (330 mg).

[0342] Step 12b: Preparation of (4-(N,S-dimethylsulfoxy)phenyl)boronic acid

[0343]

[0344] Step 12b: Under nitrogen protection, ((4-bromophenyl)(methyl)(methylimino)-λ 6 -sulfamethoxazole (230 mg, 0.93 mmol), 4,4,4,4,5,5,5,5-octamethyl-2,2-bis(1,3,2-dioxaborane) (353 mg, 1.39 mmol), potassium acetate (273 mg, 2.78 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (68 mg, 0.09 mmol) were dissolved in anhydrous 1,4-dioxane (4 mL) solution. After replacing nitrogen three times, the reaction solution was stirred at 90 ° C for two hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered with diatomaceous earth, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), and the combined organic phase was concentrated and separated by column chromatography to obtain (4-(N, S-dimethylsulfoxide)phenyl)boric acid (197 mg).

[0345] Step 12c: Preparation of tert-butyl N-[(tert-butyloxy)carbonyl]-N-{3-[3-(4-cyanophenyl)-1,2-oxazol-5-yl]-5-{4-[methyl(methylene)oxy-λ 6 -sulfonyl]phenyl}pyrazin-2-yl}carbamate

[0346]

[0347] Step 12c Specific method: Under nitrogen protection, (4-(N, S-dimethylsulfoxide)phenyl)boric acid (197 mg, 0.93 mmol), tert-butyl N-{5-bromo-3-[3-(4-cyanophenyl)-1,2-oxazol-5-yl]pyrazine-2-yl}-N-[(tert-butoxy)carbonyl]carbamate (300 mg, 0.55 mmol), potassium carbonate (299 mg, 1.66 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (41 mg, 0.06 mmol) were dissolved in a mixed solution of 1,4-dioxane (4 mL) and water (1 mL). After replacing nitrogen three times, the reaction solution was stirred at 90° C. for two hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered through diatomaceous earth, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phase was concentrated and separated by column chromatography to obtain tert-butyl N-[(tert-butoxy)carbonyl]-N-{3-[3-(4-cyanophenyl)-1,2-oxazol-5-yl]-5-{4-[methyl(methylene)oxy-λ 6 -sulfonyl]phenyl}pyrazin-2-yl}carbamate (145 mg).

[0348] Step 12d: Preparation of tert-butyl N-(3-{3-[4-(aminomethyl)phenyl)-1,2-oxazol-5-yl}-5-{4-[methyl(methylimino)oxy-λ 6 -sulfonyl]phenyl}pyrazin-2-yl)-N-[(tert-butyloxy)carbonyl]carbamate

[0349]

[0350] Step 12d: To tert-butyl N-[(tert-butyloxy)carbonyl]-N-{3-[3-(4-cyanophenyl)-1,2-oxazol-5-yl]-5-{4-[methyl(methylene)oxy-λ 6 -sulfonyl] phenyl} pyrazine-2-yl} carbamate (145mg, 0.23mmol) in methanol (2mL) solution, nickel chloride hexahydrate (164mg, 0.69mmol) and sodium borohydride (26mg, 0.69mmol) were added in sequence at room temperature. After the reaction solution was stirred at room temperature for 1 hour, it was quenched with a saturated aqueous solution of ammonium chloride (2mL). Ethyl acetate (40mL) and a saturated aqueous solution of sodium bicarbonate (40mL) were added. After the mixed solution was filtered through diatomaceous earth, the filtrate was poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40mL), and the combined organic phase was concentrated and separated by column chromatography to obtain tert-butyl N-(3-{3-[4-(aminomethyl)phenyl)-1,2-oxazol-5-yl}-5-{4-[methyl(methylimino)oxo-λ 6-sulfonyl]phenyl}pyrazin-2-yl)-N-[(tert-butyloxy)carbonyl]carbamate (15 mg).

[0351] Step 12e: Preparation of tert-butyl N-[(tert-butoxy)carbonyl]-N-(3-{3-[4-(aminocarboxamidomethyl)phenyl]-1,2-oxazol-5-yl}-5-{4-[methyl(methylimino)oxy-λ 6 -sulfonyl]phenyl}pyrazin-2-yl)carbamate

[0352]

[0353] Step 12e: tert-butyl N-(3-{3-[4-(aminomethyl)phenyl)-1,2-oxazol-5-yl}-5-{4-[methyl(methylimino)oxy-λ 6 1H-pyrazole-1-carboximidamide hydrochloric acid (7 mg, 47.26 μmol) and N,N-diisopropylethylamine (9 mg, 70.89 μmol) were added to a solution of 1H-pyrazole-1-carboximidamide hydrochloric acid (7 mg, 47.26 μmol) and N,N-diisopropylethylamine (9 mg, 70.89 μmol) in acetonitrile (1 mL). Stir for 16 hours at room temperature (~20°C). Ethyl acetate (20 mL) and water (20 mL) were added to the reaction solution, and the resulting solution was separated. The aqueous phase was extracted three times with ethyl acetate (20 mL), and the combined organic phases were concentrated and separated by column chromatography to obtain tert-butyl N-[(tert-butoxy)carbonyl]-N-(3-{3-[4-(aminocarboxamidomethyl)phenyl]-1,2-oxazol-5-yl}-5-{4-[methyl(methylimino)oxy-λ 6 -sulfonyl]phenyl}pyrazin-2-yl)carbamate (15 mg).

[0354] Step 12f: Preparation of 1-(4-(5-(3-amino-6-(4-(N,S-dimethylsulfonylimino)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride

[0355]

[0356] Step 12f Specific method: To a solution of tert-butyl (3-(3-(2,6-difluoro-4-(guanidinomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (15 mg, 22.16 μmol) in ethyl acetate (2 mL), add HCl in ethyl acetate (4 M, 2 mL). After the reaction solution was stirred at room temperature for 1 hour, the reaction solution was concentrated to obtain a yellow solid 1-(4-(5-(3-amino-6-(4-(N,S-dimethylsulfonylimino)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride (11 mg). 1 HNMR (400 MHz, DMSO-d6): δ9.01 (s, 1H), 8.50–8.48 (m, 2H), 8.30–8.27 (m, 1H), 8.13–8.11 (m, 2H), 8.05–8.03 (m, 1H), 7.81 (s, 1H), 7.52–7.51 (m, 2H), 7.37 (s, 2H), 7.24 (s, 2H), 7.12 (s, 2H), 4.50–4.48 (m, 2H), 3.93-3.92 (m, 2H), 2.66 (s, 3H), 1.23 (s, 3H). MS found (ESI + )[(M+H) + ]:477.

[0357] Example 13: 1-(4-(5-(3-amino-6-(4-(N-methylpropane-2-ylsulfonylimino)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride

[0358]

[0359] Step 13a: Preparation of (4-bromophenyl)(imino)(isopropyl)-λ 6 -Sulfamethoxazole

[0360]

[0361] Step 13a Specific method: At room temperature, add iodophenyl diacetic acid (4.18 g, 12.98 mmol) and ammonium acetate (1.34 g, 17.30 mmol) to a solution of (4-bromophenyl)(isopropyl)sulfonamide (1 g, 4.33 mmol) in ethanol (10 mL). Stir the reaction mixture at room temperature (~20°C) for 3 hours. Add water (60 mL) to the reaction system, separate the organic phase, extract the aqueous phase three times with ethyl acetate (20 mL), dry the combined organic phases with anhydrous sodium sulfate, filter, concentrate and separate by column chromatography to obtain (4-bromophenyl)(imino)(isopropyl)-λ 6 -Sulfamethoxazole (1.07 g).

[0362] Step 13b: Preparation of (4-bromophenyl)(isopropyl)(methylimino)-λ 6 -Sulfamethoxazole

[0363]

[0364] Step 13b: Sodium hydride (60%, in mineral oil, 229 mg, 5.72 mmol) was added to (4-bromophenyl)(imino)(isopropyl)-λ in N,N-dimethylformamide (5 mL). 6 -sulfazone (500mg, 1.91mmol) and the resulting mixture was stirred at room temperature for 5 minutes.Iodomethane (0.6mL, 9.54mmol) was added and the reaction mixture was stirred overnight.The mixture was concentrated in vacuo, diluted with water, and extracted with ethyl acetate.The combined extracts were washed with brine, dried over MgSO4, and concentrated in vacuo.The crude product was purified by silica gel chromatography to obtain (4-bromophenyl) (isopropyl) (methylimino)-λ 6 -Sulfamethoxazole (490 mg).

[0365] Step 13c: Preparation of (4-(N-methylpropane-2-ylsulfonylimino)phenyl)boronic acid

[0366]

[0367] Step 13c: Under nitrogen protection, (4-bromophenyl)(isopropyl)(methylimino)-λ 6 -sulfamethoxazole (250 mg, 0.91 mmol), 4,4,4,4,5,5,5,5-octamethyl-2,2-bis(1,3,2-dioxaborane) (345 mg, 1.36 mmol), potassium acetate (267 mg, 2.72 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (66 mg, 0.09 mmol) were dissolved in anhydrous 1,4-dioxane (4 mL) solution. After replacing nitrogen three times, the reaction solution was stirred at 90 ° C for two hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered with diatomaceous earth, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), and the combined organic phase was concentrated and separated by column chromatography to obtain (4-(N-methylpropane-2-ylsulfonylimino)phenyl)boric acid (218 mg).

[0368] Step 13d: Preparation of tert-butyl N-[(tert-butoxy)carbonyl]-N-{3-[3-(4-cyanophenyl)-1,2-oxazol-5-yl]-5-{4-[(methylimino)(oxy)(propan-2-yl)-λ 6-sulfonyl]phenyl}pyrazin-2-yl}carbamate

[0369]

[0370] Step 13d Specific method: Under nitrogen protection, (4-(N-methylpropane-2-ylsulfonylimino)phenyl)boric acid (218 mg, 0.91 mmol), tert-butyl N-{5-bromo-3-[3-(4-cyanophenyl)-1,2-oxazol-5-yl]pyrazine-2-yl}-N-[(tert-butoxy)carbonyl]carbamate (300 mg, 0.55 mmol), potassium carbonate (229 mg, 1.66 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (40 mg, 0.06 mmol) were dissolved in a mixed solution of 1,4-dioxane (4 mL) and water (1 mL). After replacing nitrogen three times, the reaction solution was stirred at 90° C. for two hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered through diatomaceous earth, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to obtain tert-butyl N-[(tert-butoxy)carbonyl]-N-{3-[3-(4-cyanophenyl)-1,2-oxazol-5-yl]-5-{4-[(methylimino)(oxy)(propan-2-yl)-λ 6 -sulfonyl]phenyl}pyrazin-2-yl}carbamate (180 mg).

[0371] Step 13e: Preparation of tert-butyl N-(3-{3-[4-(aminomethyl)phenyl)-1,2-oxazol-5-yl}-5-{4-[(methylimino)(oxy)(propan-2-yl)-λ 6 -sulfonyl]phenyl}pyrazin-2-yl)-N-[(tert-butyloxy)carbonyl]carbamate

[0372]

[0373] Step 13e: To tert-butyl N-[(tert-butyloxy)carbonyl]-N-{3-[3-(4-cyanophenyl)-1,2-oxazol-5-yl]-5-{4-[(methylimino)(oxy)(propan-2-yl)-λ 6-sulfonyl] phenyl} pyrazine-2-yl} carbamate (180 mg, 0.27 mmol) in methanol (2 mL) solution, nickel chloride hexahydrate (195 mg, 0.82 mmol) and sodium borohydride (31 mg, 0.82 mmol) were added in sequence at room temperature. After the reaction solution was stirred at room temperature for 1 hour, it was quenched with a saturated aqueous solution of ammonium chloride (2 mL). Ethyl acetate (40 mL) and a saturated aqueous solution of sodium bicarbonate (40 mL) were added. After the mixed solution was filtered through diatomaceous earth, the filtrate was poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40 mL), and the combined organic phase was concentrated and separated by column chromatography to obtain tert-butyl N-(3-{3-[4-(aminomethyl)phenyl)-1,2-oxazol-5-yl}-5-{4-[(methylimino)(oxy)(propane-2-yl)-λ 6 -sulfonyl]phenyl}pyrazin-2-yl)-N-[(tert-butyloxy)carbonyl]carbamate (16 mg).

[0374] Step 13f: Preparation of tert-butyl N-[(tert-butyloxy)carbonyl]-N-(3-{3-[4-(aminocarboxamidomethyl)phenyl]-1,2-oxazol-5-yl}-5-{4-[(methylimino)(oxy)(propan-2-yl)-λ 6 -sulfonyl]phenyl}pyrazin-2-yl)carbamate

[0375] Step 13f: tert-butyl N-(3-{3-[4-(aminomethyl)phenyl)-1,2-oxazol-5-yl}-5-{4-[(methylimino)(oxy)(propan-2-yl)-λ 6 1H-pyrazole-1-carboximidamide hydrochloric acid (7 mg, 48.28 μmol) and N,N-diisopropylethylamine (9 mg, 72.42 μmol) were added to a solution of 16 mg, 24.14 μmol, in acetonitrile (1 mL). The mixture was stirred at room temperature (-20 ° C) for 16 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction solution, and the resulting solution was separated. The aqueous phase was extracted three times with ethyl acetate (20 mL). The combined organic phases were concentrated and separated by column chromatography to obtain tert-butyl N-[(tert-butoxy)carbonyl]-N-(3-{3-[4-(aminocarboxamidomethyl)phenyl]-1,2-oxazol-5-yl}-5-{4-[(methylimino)(oxy)(propane-2-yl)-λ 6 -sulfonyl]phenyl}pyrazin-2-yl)carbamate (13 mg).

[0376] Step 13g: Preparation of 1-(4-(5-(3-amino-6-(4-(N-methylpropane-2-ylsulfonylimino)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride

[0377]

[0378] Step 13g: Specific method: To tert-butyl N-[(tert-butyloxy)carbonyl]-N-(3-{3-[4-(aminocarboxamidomethyl)phenyl]-1,2-oxazol-5-yl}-5-{4-[(methylimino)(oxy)(propan-2-yl)-λ 6 A solution of 1-(4-(5-(3-amino-6-(4-(N-methylpropane-2-ylsulfonylimino)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride (8 mg) was added to a solution of 1-(4-(5-(3-amino-6-(4-(N-methylpropane-2-ylsulfonylimino)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride (8 mg) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6): δ9.00 (s, 1H), 8.52–8.50 (m, 2H), 8.34–8.31 (m, 1H), 8.05–8.03 (m, 4H), 7.82 (s, 1H), 7.52–7.50 (m, 2H), 7.40 (s, 2H), 7.27 (s, 2H), 7.14 (s, 2H), 4.50–4.49 (m, 2H), 4.28 (s, 1H), 3.93 (s, 1H), 2.67 (s, 3H), 1.45–1.44 (m, 3H), 1.26–1.25 (m, 3H). MS found (ESI + )[(M+H) + ]:505.

[0379] Example 14: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)-3-methylurea

[0380] Step 14a: (E)-4-nitrobenzaldehyde oxime

[0381]

[0382] Step 14a Specific method: At room temperature, hydroxylamine hydrochloride (1.02 g, 15.89 mmol) was added to a solution of 4-nitrobenzaldehyde (2 g, 13.24 mmol) in tetrahydrofuran (30 mL). The reaction mixture was stirred at room temperature (≈20°C) for 16 hours. Water (40 mL) was added to the reaction system, the organic phase was separated, the aqueous phase was extracted three times with ethyl acetate (30 mL), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow solid (E)-4-nitrobenzaldehyde oxime (2.2 g), which was a crude product and was directly used in the next step without further purification.

[0383] Step 14b: Di-tert-butyl (5-bromo-3-(3-(4-nitrophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0384] Step 14b Specific method: To a mixed solution of di(tert-butyl)(5-bromo-3-ethynyl-2-pyrazinyl)carbamate (0.48 g, 1.20 mmol) in methanol (18 mL) and water (3.6 mL), (E)-4-nitrobenzaldehyde oxime (0.3 g, 1.81 mmol) and bis(trifluoroacetyl)iodobenzene (0.78 g, 1.81 mmol) were added in sequence, and stirred at room temperature overnight. Ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The organic phase was then washed three times with water (20 mL). The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain di-tert-butyl(5-bromo-3-(3-(4-nitrophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.41 g).

[0385] Step 14c: Preparation of di(tert-butyl)(3-(3-(4-nitrophenyl)5-isoxazolyl)-5-(4-(isopropylsulfonyl)phenyl)2-pyrazinyl)carbamate

[0386]

[0387] Step 14c Specific method: Under nitrogen protection, to a mixed solution of di(tert-butyl)(5-bromo-3-(3-(4-nitrophenyl)5-isoxazolyl)2-pyrazinyl)carbamate (0.37 g, 0.66 mmol) in 1,4-dioxane (15 mL) and water (1.5 mL), 4-isopropylsulfonylphenylboronic acid (0.17 g, 0.73 mmol), potassium carbonate (0.18 g, 1.32 mmol) and 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (0.10 g, 0.13 mmol) were added at room temperature. After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di(tert-butyl)(3-(3-(4-nitrophenyl)5-isoxazolyl)-5-(4-(isopropylsulfonyl)phenyl)2-pyrazinyl)carbamate (0.17 g).

[0388] Step 14d: Di-tert-butyl (3-(3-(4-aminophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0389]

[0390] Step 14d Specific method: To a solution of di-tert-butyl (3-(3-(4-nitrophenyl) 5-isoxazolyl)-5-(4-(isopropylsulfonyl)phenyl)2-pyrazinyl)carbamate (130 mg, 0.20 mmol) in acetic acid (8 mL), reduced iron powder (109 mg, 2.0 mmol) was added at room temperature. After the reaction solution was stirred at room temperature for 16 hours, it was diluted with ethyl acetate (40 mL), the mixed solution was filtered through diatomaceous earth, washed with sodium bicarbonate aqueous solution (40 mL) until neutral, and then poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40 mL), and the combined organic phases were concentrated and separated by column chromatography to obtain di-tert-butyl (3-(3-(4-aminophenyl)isoxazolyl-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (100 mg).

[0391] Step 14e: Preparation of di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-((phenoxycarbonyl)amino)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0392]

[0393] Step 14e Specific method: triethylamine (0.017 mL) and phenyl chloroformate (7.3 mg, 0.047 mmol) were added to a solution of di-tert-butyl (3-(3-(4-aminophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (27 mg, 0.042 mmol) in dichloromethane (2 mL), and the mixture was stirred at 25° C. for 2 hours. The mixture was concentrated and purified by thin layer chromatography to obtain a yellow oily compound di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-((phenoxycarbonyl)amino)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (20 mg).

[0394] Step 14f: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-methylureido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0395]

[0396] Step 14f Specific method: Methylamine hydrochloride (4 mg, 0.052 mmol) was added to a solution of di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-((phenoxycarbonyl)amino)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (20 mg, 0.026 mmol) in acetonitrile (3 mL), and the mixture was stirred at 50° C. for 2 hours. Ethyl acetate (10 mL) and water (10 mL) were added to the reaction solution, and the aqueous phase after separation was extracted three times with ethyl acetate (20 mL). The combined organic phases were concentrated and separated by column chromatography to obtain di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-methylureido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (12 mg) as a white solid.

[0397] Step 14g: Preparation of 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)2-pyrazinyl)3-isoxazolyl)benzyl)-3-methylurea

[0398]

[0399] Step 14g Specific method: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-methylureido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (12 mg, 0.015 mmol) and HCl in ethyl acetate (4M, 2 mL), the reaction solution was stirred at room temperature for 1 hour, concentrated, ethyl acetate (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL) were added, and the organic phase was washed with water (20 mL) after separation. The organic phase was concentrated and separated by column chromatography to obtain a yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)2-pyrazinyl)3-isoxazolyl)benzyl)-3-methylurea (6.7 mg). 1 H NMR (DMSO-d6, 400MHz): δ8.93 (s, 1H), 8.82 (s, 1H), 8.38–8.36 (m, 2H), 7.94–7.92 (m, 2H), 7.88–7.86 (m, 2H), 7.69 (s, 1H), 7.59–7.57 (m, 2H), 7.17 (s, 2H), 6.13–6.11 (m, 1H), 3.48–3.44 (m, 1H), 2.67–2.66 (m, 3H), 1.20–1.18 (m, 6H). MS found (ESI + )[(M+H) + ]:493.

[0400] Example 15: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)urea

[0401]

[0402] Step 15a: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-ureidophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0403]

[0404] Step 15a Specific method: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-((phenoxycarbonyl)amino)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (20 mg, 0.026 mmol) was dissolved in ammonia methanol solution (7M, 3 mL), and the mixture was stirred at 50° C. for 2 hours. Ethyl acetate (10 mL) and water (10 mL) were added to the reaction solution, and the aqueous phase after separation was extracted three times with ethyl acetate (20 mL), and the combined organic phase was concentrated and separated by column chromatography to obtain di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-ureidophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (12 mg) as a white solid.

[0405] Step 15b: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)urea

[0406]

[0407] Step 15b Specific method: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-methylureido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (12 mg, 0.015 mmol) and HCl in ethyl acetate (4M, 2 mL) were added, and the reaction solution was stirred at room temperature for 1 hour, concentrated, and ethyl acetate (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL) were added. After separation, the organic phase was washed once with water (20 mL), and the organic phase was concentrated and separated by column chromatography to obtain yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)urea (1.6 mg). 1 H NMR (DMSO-d6, 400MHz): δ8.92 (s, 1H), 8.86 (s, 1H), 8.38–8.36 (m, 2H), 7.94–7.92 (m, 2H), 7.88–7.86 (m, 2H), 7.68 (s, 1H), 7.59–7.57 (m, 2H), 7.15 (s, 2H), 6.97 (m, 2H), 3.48–3.44 (m, 1H), 1.20–1.18 (m, 6H). MS found (ESI + )[(M+H) + ]:479.

[0408] Example 16: 2-1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)guanidine

[0409] Step 16a: 2-1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)guanidine

[0410]

[0411] Step 16a Specific method: To a solution of di-tert-butyl (3-(3-(4-aminophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (50 mg, 0.079 mmol) in dioxane hydrochloride (4M, 3 mL) was added cyanamide (10 mg, 0.14 mmol), and the mixture was stirred at 80° C. for 2 hours. After the reaction was cooled to room temperature, ethyl acetate (20 mL) and sodium bicarbonate aqueous solution (10 mL) were added to the reaction solution, and the aqueous phase after separation was extracted three times with ethyl acetate (20 mL), and the combined organic phase was concentrated and separated by column chromatography to obtain a white solid 2-1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)guanidine (18.3 mg). 1 H NMR (DMSO-d6, 400MHz): δ8.95 (s, 1H), 8.39–8.37 (m, 2H), 8.10–8.07 (m, 2H), 7.95–7.93 (m, 2H), 7.82 (s, 1H), 7.71 (m, 3H), 7.44–7.42 (m, 2H), 7.19 (s, 2H), 5.40 (s, 2H), 3.50–3.43 (m, 1H), 1.20–1.18 (m, 6H). MS found (ESI + )[(M+H) + ]:478.

[0412] Example 17: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)-3-methylguanidine dihydrochloride

[0413]

[0414] Step 17a: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-isothiocyanatophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0415]

[0416] Step 17a Specific method: To a solution of di-tert-butyl (3-(3-(4-aminophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (110 mg, 0.21 mmol) in dichloromethane (4 mL), sodium hydroxide aqueous solution (1M, 0.6 mL) and phenyl chlorothioformate (43 mg, 0.25 mmol) were added, and the mixture was stirred at 25° C. for 2 hours. Dichloromethane (20 mL) and aqueous solution (10 mL) were added to the reaction solution, and the aqueous phase after separation was extracted three times with dichloromethane (10 mL), and the combined organic phases were concentrated and separated by column chromatography to obtain a yellow oily compound di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-isothiocyanatophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (70 mg).

[0417] Step 17b: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-methylthioureido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0418]

[0419] Step 17b Specific method: Methylamine hydrochloride (28 mg, 0.42 mmol) was added to a solution of di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-isothiocyanatophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (200 mg, 0.42 mmol) in acetonitrile (5 mL). The reaction solution was stirred at 50° C. for 1 hour, and ethyl acetate (20 mL) and water (20 mL) were added. After separation, the organic phase was washed once with water (20 mL), and the organic phase was concentrated and separated by column chromatography to obtain yellow solid di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-methylthioureido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (150 mg).

[0420] Step 17c: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-methylguanidino)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0421]

[0422] Specific method of step 17c: 2-iodoacylbenzoic acid (13 mg, 0.05 mmol) was added to the reaction solution of acetonitrile (1.5 mL) and ammonia water (1.5 mL) of di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-methylthioureido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (26 mg, 0.04 mmol), and the reaction solution was stirred at 50°C for 1 hour, and then ethyl acetate (20 mL) and water (20 mL) were added. After separation, the organic phase was washed once with water (20 mL), and the organic phase was concentrated and separated by column chromatography to obtain yellow solid di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-methylguanidino)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (5 mg).

[0423] Step 17d: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)-3-methylguanidine dihydrochloride

[0424] Specific method of step 17d: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-methylguanidino)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (5 mg, 0.007 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4M, 2 mL) were stirred at room temperature for 1 hour, and the reaction solution was concentrated to give a yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)-3-methylguanidine dihydrochloride (1.7 mg). 1 H NMR (400 MHz, DMSO-d6): δ8.98 (s, 1H), 8.42–8.40 (m, 2H), 8.12–8.10 (m, 2H), 8.02–8.00 (m, 2H), 7.86 (s, 1H), 7.46–7.44 (m, 2H), 3.51–3.48 (m, 1H), 2.89–2.88 (m, 3H), 1.22–1.21 (m, 6H). MS found (ESI + )[(M+H) + ]:492.

[0425] Example 18: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)-3-ethylguanidine dihydrochloride

[0426]

[0427] Step 18a: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-ethylthioureido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0428]

[0429] Specific method of step 18a: Ethylamine hydrochloride (25 mg, 0.13 mmol) and triethylamine (63 mg, 0.62 mmol) were added to a solution of di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-isothiocyanatophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (120 mg, 0.42 mmol) in acetonitrile (5 mL). The reaction solution was stirred at 50° C. for 1 hour, and ethyl acetate (20 mL) and water (20 mL) were added. After separation, the organic phase was washed once with water (20 mL), and the organic phase was concentrated and separated by column chromatography to obtain di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-ethylthioureido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (100 mg) as a yellow solid.

[0430] Step 18b: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-ethylguanidino)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0431]

[0432] Specific method of step 18b: 2-iodoacylbenzoic acid (50 mg, 0.18 mmol) was added to the reaction solution of acetonitrile (2 mL) and ammonia water (2 mL) of di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-ethylthioureido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (100 mg, 0.16 mmol), and the reaction solution was stirred at 50° C. for 1 hour, and then ethyl acetate (20 mL) and water (20 mL) were added. After separation, the organic phase was washed once with water (20 mL), and the organic phase was concentrated and separated by column chromatography to obtain yellow solid di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-ethylguanido)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (5 mg).

[0433] Step 18c: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)-3-ethylguanidine dihydrochloride

[0434] Specific method of step 18c: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-(3-ethylguanidino)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (45 mg, 0.063 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4M, 2 mL) were stirred at room temperature for 1 hour, and the reaction solution was concentrated to give a yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)-3-ethylguanidine dihydrochloride (34.5 mg). 1 HNMR (400MHz, DMSO-d6): δ9.94 (s, 1H), 8.96 (s, 1H), 8.39–8.37 (m, 2H), 8.17–8.15 (m, 1H), 8.09–8.07 (m, 2H), 7.95–7.93 (m, 2H) 7.83 (s, 1H), 7.42–7.40 (m, 2H), 3.50–3.45 (m, 1H), 3.33–2.29 (m, 2H), 1.20–1.15 (m, 9H). MS found (ESI + )[(M+H) + ]:506.

[0435] Example 19: 1-(6-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)pyridin-3-yl)-3-methylguanidine dihydrochloride

[0436]

[0437] Step 19a: (E)-5-Nitropyridinecarboxaldehyde oxime

[0438]

[0439] Step 19a Specific method: At room temperature, hydroxylamine hydrochloride (0.55 g, 7.89 mmol) was added to a solution of 5-nitropyridinecarboxaldehyde (1 g, 6.57 mmol) in tetrahydrofuran (15 mL). The reaction mixture was stirred at room temperature for 16 hours. Water (40 mL) was added to the reaction system, the organic phase was separated, the aqueous phase was extracted three times with ethyl acetate (30 mL), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow solid (E)-5-nitropyridinecarboxaldehyde oxime (1.1 g), which was a crude product and was directly used in the next step without further purification.

[0440] Step 19b: Di-tert-butyl (5-bromo-3-(3-(5-nitropyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0441] Step 19b Specific method: To a mixed solution of di(tert-butyl)(5-bromo-3-ethynyl-2-pyrazinyl)carbamate (0.80 g, 1.99 mmol) in methanol (18 mL) and water (3.6 mL), (E)-4-nitrobenzaldehyde oxime (0.50 g, 2.99 mmol) and bis(trifluoroacetyl)iodobenzene (1.29 g, 2.99 mmol) were added in sequence, and stirred at room temperature overnight. Ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The organic phase was then washed three times with water (20 mL). The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain di-tert-butyl(5-bromo-3-(3-(5-nitropyridine-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.20 g).

[0442] Step 19c: Preparation of di(tert-butyl)(3-(3-(5-nitropyridin-2-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)2-pyrazinyl)carbamate

[0443]

[0444] Step 19c Specific method: Under nitrogen protection, to a mixed solution of di-tert-butyl (5-bromo-3-(3-(5-nitropyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.20 g, 0.36 mmol) in 1,4-dioxane (15 mL) and water (1.5 mL), 4-isopropylsulfonylphenylboronic acid (0.082 g, 0.36 mmol), potassium carbonate (0.15 g, 1.07 mmol) and 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (0.05 g, 0.071 mmol) were added at room temperature (~20°C). After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di(tert-butyl)(3-(3-(5-nitropyridin-2-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)2-pyrazinyl)carbamate (0.12 g).

[0445] Step 19d: Di-tert-butyl (3-(3-(5-nitropyridin-2-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0446]

[0447] Step 19d Specific method: Reduced iron powder (109 mg, 2.0 mmol) was added to a solution of di(tert-butyl)(3-(3-(5-nitropyridin-2-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)2-pyrazinyl)carbamate (120 mg, 0.20 mmol) in acetic acid (8 mL) at room temperature. The reaction solution was stirred at room temperature for 16 hours, diluted with ethyl acetate (40 mL), and the mixed solution was filtered through diatomaceous earth, washed with sodium bicarbonate aqueous solution (40 mL) until neutral, and then poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40 mL), and the combined organic phases were concentrated and separated by column chromatography to obtain di-tert-butyl(3-(3-(5-nitropyridin-2-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (75 mg).

[0448] Step 19e: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(5-isothiocyanatopyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0449]

[0450] Step 19e Specific method: To a solution of di-tert-butyl (3-(3-(5-nitropyridin-2-yl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (75 mg, 0.11 mmol) in dichloromethane (4 mL) were added aqueous sodium hydroxide solution (1 M, 0.35 mL) and phenyl chlorothioate (23 mg, 0.13 mmol), and the mixture was stirred at 25 °C for 2 hours. Dichloromethane (20 mL) and aqueous solution (10 mL) were added to the reaction solution. The aqueous phase after separation was extracted three times with dichloromethane (10 mL). The combined organic phases were concentrated and separated by column chromatography to obtain a yellow oily compound, di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(5-isothiocyanatopyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (53 mg).

[0451] Step 19f: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(5-(3-methylthioureido)pyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0452]

[0453] Step 19f Specific method: To a solution of di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(5-isothiocyanatopyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (45 mg, 0.078 mmol) in acetonitrile (5 mL), methylamine hydrochloride (8 mg, 0.12 mmol) and triethylamine (24 mg, 0.23 mmol) were added, and the reaction solution was stirred at 50° C. for 1 hour, and ethyl acetate (20 mL) and water (20 mL) were added. After separation, the organic phase was washed once with water (20 mL), and the organic phase was concentrated and separated by column chromatography to obtain yellow solid di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(5-(3-methylthiourea)pyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (38 mg).

[0454] Step 19g: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(5-(3-methylguanidino)pyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0455]

[0456] Step 19g Specific method: 2-iodoacylbenzoic acid (25 mg, 0.089 mmol) was added to the reaction solution of di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(5-(3-methylthioureido)pyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (38 mg, 0.075 mmol) in acetonitrile (1.5 mL) and ammonia water (1.5 mL). The reaction solution was stirred at 50° C. for 1 hour, and ethyl acetate (20 mL) and water (20 mL) were added. After separation, the organic phase was washed once with water (20 mL), and the organic phase was concentrated and separated by column chromatography to obtain yellow solid di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(5-(3-methylguanidino)pyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (15 mg).

[0457] Step 19h: 1-(6-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)pyridin-3-yl)-3-methylguanidine dihydrochloride

[0458]

[0459] Specific method of step 19h: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(5-(3-methylguanidino)pyridin-2-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (15 mg, 0.021 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4M, 2 mL) were stirred at room temperature for 1 hour, and the reaction solution was concentrated to give a yellow solid 1-(6-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)pyridin-3-yl)-3-methylguanidine dihydrochloride (11.7 mg). 1 H NMR (400 MHz, DMSO-d6): δ10.13 (s, 1H), 8.96 (s, 1H), 8.68 (m, 1H), 8.39–8.37 (m, 2H), 8.21–8.17 (m, 2H), 7.95–7.93 (m, 2H), 7.90–7.87 (m, 2H), 7.71 (s, 1H), 3.49–3.45 (m, 1H), 2.87 (m, 3H), 1.20–1.18 (m, 6H). MS found (ESI + )[(M+H) + ]:493.

[0460] Example 20: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3-fluorophenyl)-3-methylguanidine dihydrochloride

[0461]

[0462] Step 20a: (E)-2-Fluoro-4-nitrobenzaldehyde oxime

[0463]

[0464] Step 20a Specific method: Add hydroxylamine hydrochloride (0.25 g, 3.55 mmol) to a solution of 2-fluoro-4-nitrobenzaldehyde (0.5 g, 2.96 mmol) in tetrahydrofuran (8 mL) at room temperature. Stir the reaction mixture at room temperature (~20°C) for 16 hours. Add water (40 mL) to the reaction system, separate the organic phase, extract the aqueous phase three times with ethyl acetate (30 mL), dry the combined organic phases with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain a yellow solid (E)-2-fluoro-4-nitrobenzaldehyde oxime (0.7 g), which is a crude product and is directly used in the next step without further purification.

[0465] Step 20b: Di-tert-butyl (5-bromo-3-(3-(2-fluoro-4-nitrophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0466] Step 20b Specific method: To a mixed solution of di(tert-butyl)(5-bromo-3-ethynyl-2-pyrazinyl)carbamate (0.85g, 2.14mmol) in methanol (18mL) and water (3.6mL), (E)-2-fluoro-4-nitrobenzaldehyde oxime (0.59g, 3.21mmol) and bis(trifluoroacetyl)iodobenzene (1.38g, 3.21mmol) were added in sequence, and stirred at room temperature overnight. Ethyl acetate (40mL) and water (40mL) were added to the reaction solution. The organic phase was then washed three times with water (20mL). The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain di-tert-butyl(5-bromo-3-(3-(2-fluoro-4-nitrophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.72g).

[0467] Step 20c: Preparation of di-tert-butyl (3-(3-(2-fluoro-4-nitrophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0468]

[0469] Step 20c Specific method: Under nitrogen protection, to a mixed solution of di-tert-butyl (5-bromo-3-(3-(2-fluoro-4-nitrophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.30 g, 0.52 mmol) in 1,4-dioxane (15 mL) and water (1.5 mL), 4-isopropylsulfonylphenylboronic acid (0.12 g, 0.52 mmol), potassium carbonate (0.22 g, 1.55 mmol) and 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (0.076 g, 0.01 mmol) were added at room temperature (~20°C). After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(2-fluoro-4-nitrophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (0.13 g).

[0470] Step 20d: Di-tert-butyl (3-(3-(4-amino-2-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0471]

[0472] Step 20d Specific method: To a solution of di-tert-butyl (3-(3-(2-fluoro-4-nitrophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (130 mg, 0.19 mmol) in acetic acid (8 mL), reduced iron powder (106 mg, 1.9 mmol) was added at room temperature. After the reaction solution was stirred at room temperature for 16 hours, it was diluted with ethyl acetate (40 mL), the mixed solution was filtered through diatomaceous earth, washed with sodium bicarbonate aqueous solution (40 mL) until neutral, and then poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40 mL), and the combined organic phases were concentrated and separated by column chromatography to obtain di-tert-butyl (3-(3-(4-amino-2-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (85 mg).

[0473] Step 20e: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-amino-2-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0474]

[0475] Step 20e Specific method: A sodium hydroxide aqueous solution (1M, 0.35mL) and phenyl chlorothioformate (23mg, 0.13mmol) were added to a solution of di-tert-butyl (3-(3-(4-amino-2-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (75mg, 0.11mmol) in dichloromethane (4mL), and the mixture was stirred at 25°C for 2 hours. Dichloromethane (20mL) and aqueous solution (10mL) were added to the reaction solution, and the aqueous phase after separation was extracted three times with dichloromethane (10mL). The combined organic phase was concentrated and separated by column chromatography to obtain a yellow oily compound di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-amino-2-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (56mg).

[0476] Step 20f: Di-tert-butyl (3-(3-(2-fluoro-4-(3-methylthioureido)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0477]

[0478] Step 20f Specific method: Methylamine hydrochloride (8 mg, 0.12 mmol) and triethylamine (24 mg, 0.23 mmol) were added to acetonitrile (5 mL) of di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-amino-2-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (56 mg, 0.078 mmol). The reaction solution was stirred at 50° C. for 1 hour, and ethyl acetate (20 mL) and water (20 mL) were added. After separation, the organic phase was washed once with water (20 mL), and the organic phase was concentrated and separated by column chromatography to obtain yellow solid di-tert-butyl (3-(3-(2-fluoro-4-(3-methylthioureido)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (38 mg).

[0479] Step 20g: Di-tert-butyl (3-(3-(2-fluoro-4-(3-methylguanidino)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0480]

[0481] Step 20g Specific method: 2-iodoacylbenzoic acid (25 mg, 0.089 mmol) was added to the reaction solution of acetonitrile (1.5 mL) and ammonia (1.5 mL) of di-tert-butyl (3-(3-(2-fluoro-4-(3-methylthioureido)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (38 mg, 0.075 mmol), and the reaction solution was stirred at 50°C for 1 hour, and then ethyl acetate (20 mL) and water (20 mL) were added. After separation, the organic phase was washed once with water (20 mL), and the organic phase was concentrated and separated by column chromatography to obtain yellow solid di-tert-butyl (3-(3-(2-fluoro-4-(3-methylguanidino)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (28 mg).

[0482] Step 20h: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3-fluorophenyl)-3-methylguanidine dihydrochloride

[0483]

[0484] Specific method of step 20h: Di-tert-butyl (3-(3-(2-fluoro-4-(3-methylguanidino)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (28 mg, 0.039 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4M, 2 mL) were stirred at room temperature for 1 hour, and the reaction solution was concentrated to give a yellow solid 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3-fluorophenyl)-3-methylguanidine dihydrochloride (19.1 mg). 1 H NMR (400 MHz, DMSO-d6): δ10.16 (s, 1H), 8.99 (s, 1H), 8.40–8.38 (m, 2H), 8.23–8.22 (m, 1H), 8.11–8.07 (m, 2H), 7.98–7.96 (m, 2H), 7.63–7.62 (m, 1H), 7.42–7.39 (m, 1H), 7.29–7.26 (m, 2H), 3.52–3.46 (m, 1H), 2.91–2.89 (m, 3H), 1.23–1.21 (m, 6H). MS found (ESI + )[(M+H) + ]:510.

[0485] Example 21: 5-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-1,3-dihydro-benzimidazol-2-one

[0486]

[0487] Step 21a: (E)-2-Oxo-2,3-dihydro-benzimidazole-5-carbaldehyde oxime

[0488]

[0489] Step 21a Specific method: Add hydroxylamine hydrochloride (0.36 g, 5.18 mmol) to a solution of 2-oxo-2,3-dihydro-benzimidazole-5-carboxaldehyde (0.7 g, 4.31 mmol) in tetrahydrofuran (16 mL) at room temperature. Stir the reaction mixture at room temperature (~20°C) for 16 hours. Add water (40 mL) to the reaction system, separate the organic phase, extract the aqueous phase three times with ethyl acetate (30 mL), dry the combined organic phases with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain a yellow solid (E)-2-oxo-2,3-dihydro-benzimidazole-5-carboxaldehyde oxime as a crude product and use it directly in the next step without further purification.

[0490] Step 21b: Di-tert-butyl (5-bromo-3-(3-(2-oxo-2,3-dihydro-benzoimidazol-5-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0491]

[0492] Step 21b Specific method: To a mixed solution of di(tert-butyl)(5-bromo-3-ethynyl-2-pyrazinyl)carbamate (1.05 g, 2.14 mmol) in methanol (25 mL) and water (5 mL), (E)-2-oxo-2,3-dihydro-benzimidazole-5-carbaldehyde oxime (0.7 g, 3.95 mmol) and bis(trifluoroacetyl)iodobenzene (1.7 g, 3.95 mmol) were added in sequence, and stirred at room temperature (about 20°C) overnight. Ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The organic phase was then washed three times with water (20 mL). The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated and separated by column chromatography to give di-tert-butyl (5-bromo-3-(3-(2-oxo-2,3-dihydro-benzoimidazol-5-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.33 g).

[0493] Step 21c: Di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(2-oxo-2,3-dihydro-benzoimidazol-5-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0494]

[0495] Step 21c Specific method: Under nitrogen protection, to a mixed solution of di-tert-butyl (5-bromo-3-(3-(2-oxo-2,3-dihydro-benzoimidazol-5-yl)isoxazol-5-yl)pyrazine-2-yl)carbamate (0.150, 0.26mmol) in 1,4-dioxane (7mL) and water (1.4mL), 4-isopropylsulfonylphenylboronic acid (0.066g, 0.29mmol), potassium carbonate (0.073g, 0.52mmol) and 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (0.038g, 0.05mmol) were added at room temperature. After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40mL) and water (40mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(2-oxo-2,3-dihydro-benzoimidazol-5-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.06 g).

[0496] Step 21d: 5-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-1,3-dihydro-benzimidazol-2-one

[0497]

[0498] Specific method of step 21d: The reaction solution of di-tert-butyl (5-(4-(isopropylsulfonyl)phenyl)-3-(3-(2-oxo-2,3-dihydro-benzoimidazol-5-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (60 mg, 0.13 mmol) and HCl in ethyl acetate (4M, 2 mL) was stirred at room temperature for 1 hour, then the reaction solution was diluted with ethyl acetate (400 mL), washed with saturated sodium bicarbonate solution until neutral, the organic phases were combined and dried, and concentrated to give a yellow solid 5-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-1,3-dihydro-benzoimidazol-2-one (39.4 mg). 1 H NMR (400 MHz, DMSO-d6): δ8.92 (s, 1H), 8.38–8.36 (m, 2H), 7.94–7.92 (m, 2H), 7.70 (s, 1H), 7.62–7.60 (m, 1H), 7.53 (s, 1H), 7.15 (s, 2H), 7.07–7.05 (m, 1H), 3.52–3.46 (m, 1H), 1.20–1.18 (m, 6H). MS found (ESI + )[(M+H) + ]:477.

[0499] Example 22: 5-(5-(3-amino-6-(4-(propan-2-ylsulfonylimino)phenyl)pyrazin-2-yl)isoxazol-3-yl)-1,3-dihydro-benzimidazol-2-one dihydrochloride

[0500]

[0501] Step 22a: Di-tert-butyl (3-(3-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)isoxazol-5-yl)-5-(4-(propan-2-ylsulfoxide)phenyl)pyrazin-2-yl)carbamate

[0502]

[0503] Step 22a Specific method: Under nitrogen protection, to a mixed solution of di-tert-butyl (5-bromo-3-(3-(2-oxo-2,3-dihydro-benzoimidazol-5-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.150, 0.26mmol) in 1,4-dioxane (7mL) and water (1.4mL), (4-(propan-2-ylsulfonylimide)phenyl)boric acid (0.120g, 0.52mmol), potassium carbonate (0.11g, 0.79mmol) and 1,1'-bis(diphenylphosphinoferrocenepalladium dichloride) (0.038g, 0.05mmol) were added at room temperature (~20°C). After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40mL) and water (40mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)isoxazol-5-yl)-5-(4-(propan-2-ylsulfoxide)phenyl)pyrazin-2-yl)carbamate (0.07 g).

[0504] Step 22b: 5-(5-(3-amino-6-(4-(propan-2-ylsulfonylimino)phenyl)pyrazin-2-yl)isoxazol-3-yl)-1,3-dihydro-benzimidazol-2-one dihydrochloride

[0505]

[0506] Specific method of step 22b: Di-tert-butyl (3-(3-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)isoxazol-5-yl)-5-(4-(propan-2-ylsulfoxide)phenyl)pyrazin-2-yl)carbamate (70 mg, 0.18 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4M, 2 mL) were stirred at room temperature for 1 hour, and then concentrated to give a yellow solid 5-(5-(3-amino-6-(4-(propan-2-ylsulfonylimide)phenyl)pyrazin-2-yl)isoxazol-3-yl)-1,3-dihydro-benzoimidazole-2-one dihydrochloride (48.1 mg). 1H NMR (400 MHz, DMSO-d6): δ10.97–10.95 (m, 2H), 9.04 (s, 1H), 8.55–8.53 (m, 2H), 8.15–8.13 (m, 2H), 7.78 (s, 1H), 7.67–7.65 (m, 1H), 7.57 (s, 1H), 7.13–7.11 (m, 2H), 4.25–4.18 (m, 1H), 1.44–1.42 (m, 3H), 1.33–1.31 (m, 3H). MS found (ESI + )[(M+H) + ]:476.

[0507] Example 23: 1-(4-(5-(3-amino-6-(4-(cyclopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride

[0508] Step 23a: Preparation of (4-(cyclopropylsulfonyl)phenyl)boronic acid

[0509]

[0510] Step 23a Specific method: To a mixed solution of 1-bromo-4-(cyclopropylsulfonyl)benzene (500 mg, 2.03 mmol) in N,N-dimethylformamide (12 mL), bis-pinacol borate (775 mg, 3.05 mmol), potassium acetate (497 mg, 0.51 mmol) and 1,1'-bisdiphenylphosphinoferrocene palladium dichloride (150 mg, 0.20 mmol) were added in sequence at room temperature. After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered with diatomaceous earth, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), and the combined organic phase was concentrated to obtain (4-(cyclopropylsulfonyl)phenyl)boric acid (800 mg) as a crude product and directly used in the next step without further purification.

[0511] Step 23b: Preparation of di-tert-butyl-(3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(cyclopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0512]

[0513] Step 23b Specific method: Under nitrogen protection, to a mixed solution of di(tert-butyl)(5-bromo-3-(3-(4-cyanophenyl)5-isoxazolyl)2-pyrazinyl)carbamate (0.6 g, 1.11 mmol) in 1,4-dioxane (15 mL) and water (1.5 mL), (4-(cyclopropylsulfonyl)phenyl)boric acid (0.31 g, 1.33 mmol), potassium carbonate (0.44 g, 3.33 mmol) and 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (0.16 g, 0.22 mmol) were added in sequence at room temperature. After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(cyclopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (0.13 g).

[0514] Step 23c: Preparation of di-tert-butyl (3-(3-(4-aminophenyl)isoxazol-5-yl)-5-(4-(cyclopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0515]

[0516] Step 23c Specific method: To a solution of di-tert-butyl (3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(cyclopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (125 mg, 0.20 mmol) in methanol (5 mL) at room temperature, nickel chloride hexahydrate (142 mg, 0.60 mmol) and sodium borohydride (23 mg, 0.60 mmol) were added in sequence. After the reaction solution was stirred at room temperature for 1 hour, it was quenched with ammonium chloride (2 mL). Ethyl acetate (40 mL) and saturated aqueous sodium bicarbonate solution (40 mL) were added. After the mixed solution was filtered through diatomaceous earth, the filtrate was poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40 mL), and the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(4-aminophenyl)isoxazol-5-yl)-5-(4-(cyclopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (21 mg).

[0517] Step 23d: Preparation of di-tert-butyl (5-(4-(cyclopropylsulfonyl)phenyl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0518]

[0519] Step 23d Specific method: 1H-pyrazole-1-carboximidamide hydrochloride (6 mg, 0.038 mmol) and N,N-diisopropylethylamine (8 uL) were added to a solution of di-tert-butyl (3-(3-(4-aminophenyl)isoxazol-5-yl)-5-(4-(cyclopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (21 mg, 0.034 mmol) in acetonitrile (3 mL) in sequence. Stir at room temperature for 16 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction solution, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), and the combined organic phases were concentrated and separated by column chromatography to obtain di-tert-butyl (5-(4-(cyclopropylsulfonyl)phenyl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (6 mg).

[0520] Step 23e: 1-(4-(5-(3-amino-6-(4-(cyclopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride

[0521] Step 23e Specific method: A solution of di-tert-butyl (5-(4-(cyclopropylsulfonyl)phenyl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (6 mg, 0.01 mmol) in ethyl acetate (2 mL) was added with a mixed solution of hydrochloric acid and ethyl acetate (4 M, 2 mL). After the reaction solution was stirred at room temperature for 1 hour, the reaction solution was concentrated to obtain 1-(4-(5-(3-amino-6-(4-(cyclopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride (3 mg) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6): δ8.96 (s, 1H), 8.39–8.37 (m, 2H), 8.22–8.20 (m, 1H), 8.09–8.07 (m, 2H), 8.02–7.99 (m, 2H), 7.82 (s, 1H), 7.54–7.52 (m, 2H), 7.35 (s, 2H), 7.22 (s, 3H), 7.10 (s, 2H), 4.52–4.51 (m, 2H), 2.97–2.91 (m, 1H), 1.40–1.37 (m, 2H), 1.32–1.29 (m, 2H). MS found (ESI + )[(M+H) + ]:490.

[0522] Example 24: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-2-fluorobenzyl)guanidine dihydrochloride

[0523]

[0524] Step 24a: Preparation of (E)-2-fluoro-4-((hydroxyimino)methyl)benzonitrile

[0525]

[0526] Step 24a Specific method: At room temperature, hydroxylamine hydrochloride (466 mg, 6.71 mmol) was added to a solution of 2-fluoro-4-formylbenzonitrile (500 mg, 3.36 mmol) in tetrahydrofuran (30 mL). The reaction mixture was stirred at room temperature for 16 hours. Water (40 mL) was added to the reaction system, the organic phase was separated, the aqueous phase was extracted three times with ethyl acetate (30 mL), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow solid (E)-2-fluoro-4-((hydroxyimino)methyl)benzonitrile (430 mg), which was a crude product and was directly used in the next step without further purification.

[0527] Step 24b: Preparation of di-tert-butyl (5-bromo-3-(3-(4-cyano-3-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0528] Step 24b Specific method: To a mixed solution of di(tert-butyl)(5-bromo-3-ethynyl-2-pyrazinyl)carbamate (0.4 g, 4.88 mmol) in methanol (12 mL) and water (2.4 mL), (E)-2-fluoro-4-((hydroxyimino)methyl)benzonitrile (0.65 g, 3.25 mmol) and bis(trifluoroacetyl)iodobenzene (1.05 g, 4.88 mmol) were added in sequence, and stirred at room temperature overnight. Ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The organic phase was then washed three times with water (20 mL). The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain di-tert-butyl(5-bromo-3-(3-(4-cyano-3-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.24 g).

[0529] Step 24c: Preparation of di-tert-butyl (3-(3-(4-cyano-3-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0530]

[0531] Step 24c Specific method: Under nitrogen protection, to a mixed solution of di-tert-butyl (5-bromo-3-(3-(4-cyano-3-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.2g, 0.36mmol) in 1,4-dioxane (15mL) and water (1.5mL), 4-isopropylsulfonylphenylboronic acid (0.80g, 0.36mmol), potassium carbonate (0.15g, 1.08mmol) and 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (0.052g, 0.072mmol) were added at room temperature. After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40mL) and water (40mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(4-cyano-3-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (0.11 g).

[0532] Step 24d: Preparation of di-tert-butyl (3-(3-(4-(aminomethyl)-3-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0533]

[0534] Step 24d Specific method: To a solution of di-tert-butyl (3-(3-(4-cyano-3-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (107 mg, 0.16 mmol) in methanol (5 mL) at room temperature, nickel chloride hexahydrate (115 mg, 0.48 mmol) and sodium borohydride (19 mg, 0.48 mmol) were added in sequence. After the reaction solution was stirred at room temperature for 1 hour, it was quenched with ammonium chloride (2 mL). Ethyl acetate (40 mL) and saturated aqueous sodium bicarbonate solution (40 mL) were added. After the mixed solution was filtered through diatomaceous earth, the filtrate was poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40 mL), and the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(4-(aminomethyl)-3-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (26 mg).

[0535] Step 24e: Preparation of di-tert-butyl (3-(3-(3-fluoro-4-(guanidinomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0536]

[0537] Step 24e Specific method: To a solution of di-tert-butyl (3-(3-(4-(aminomethyl)-3-fluorophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (26 mg, 0.046 mmol) in acetonitrile (3 mL), 1H-pyrazole-1-carboximidamide hydrochloride (7 mg, 0.046 mmol) and N,N-diisopropylethylamine (9 uL) were added in sequence. The mixture was stirred at room temperature (~20°C) for 16 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction solution, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(3-fluoro-4-(guanidinomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (12 mg).

[0538] Step 24f: Preparation of 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)2-pyrazinyl)3-isoxazolyl)benzyl)guanidine dihydrochloride

[0539] Step 24f Specific method: To a solution of di-tert-butyl (3-(3-(3-fluoro-4-(guanidinomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (12 mg, 0.02 mmol) in ethyl acetate (2 mL) was added HCl in ethyl acetate (4 M, 2 mL). After the reaction solution was stirred at room temperature for 1 hour, the reaction solution was concentrated to give 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-2-fluorobenzyl)guanidine dihydrochloride (8 mg) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6): δ8.99 (s, 1H), 8.42–8.40 (m, 2H), 8.06 (s, 1H), 7.98–7.94 (m, 4H), 7.93–7.90 (m, 1H), 7.60–7.56 (m, 1H), 7.25 (s, 2H), 4.57–4.56 (m, 2H), 3.54–3.48 (m, 1H), 1.23–1.21 (m, 6H). MS found (ESI + )[(M+H) + ]:510.

[0540] Example 25: 4-(5-amino-6-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)-N,N-dimethylbenzamide dihydrochloride

[0541]

[0542] Step 25a: Preparation of di-tert-butyl (3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(dimethylcarbamoyl)phenyl)pyrazin-2-yl)carbamate

[0543]

[0544] Step 25a Specific method: Under nitrogen protection, to a mixed solution of di(tert-butyl)(5-bromo-3-(3-(4-cyanophenyl)5-isoxazolyl)2-pyrazinyl)carbamate (0.4g, 0.74mmol) in 1,4-dioxane (10mL) and water (2mL), N,N-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxybenzaldehyde-2-yl)benzamide (0.24g, 0.89mmol), potassium carbonate (0.30g, 2.22mmol) and 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (0.10g, 0.15mmol) were added in sequence at room temperature. After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40mL) and water (40mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(dimethylcarbamoyl)phenyl)pyrazin-2-yl)carbamate (0.16 g).

[0545] Step 25b: Preparation of di-tert-butyl (3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(dimethylcarbamoyl)phenyl)pyrazin-2-yl)carbamate

[0546]

[0547] Step 25b Specific method: To a solution of di-tert-butyl (3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(4-(dimethylcarbamoyl)phenyl)pyrazine-2-yl)carbamate (160 mg, 0.26 mmol) in methanol (5 mL) was added nickel chloride hexahydrate (187 mg, 0.79 mmol) and sodium borohydride (30 mg, 0.79 mmol) at room temperature. After the reaction solution was stirred at room temperature for 1 hour, it was quenched with ammonium chloride (2 mL). Ethyl acetate (40 mL) and saturated sodium bicarbonate aqueous solution (40 mL) were added. After the mixed solution was filtered through diatomaceous earth, the filtrate was poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40 mL), and the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(dimethylcarbamoyl)phenyl)pyrazin-2-yl)carbamate (21 mg).

[0548] Step 25c: Preparation of di-tert-butyl (5-(4-(dimethylcarbamoyl)phenyl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0549]

[0550] Step 25c Specific method: 1H-pyrazole-1-carboximidamide hydrochloride (6 mg, 0.038 mmol) and N,N-diisopropylethylamine (8 uL) were added to a solution of di-tert-butyl (3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(dimethylcarbamoyl)phenyl)pyrazin-2-yl)carbamate (21 mg, 0.034 mmol) in acetonitrile (3 mL) in sequence. The mixture was stirred at room temperature (~20°C) for 16 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction solution, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (5-(4-(dimethylcarbamoyl)phenyl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (15 mg).

[0551] Step 25d: Preparation of 4-(5-amino-6-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)-N,N-dimethylbenzamide hydrochloride

[0552]

[0553] Step 25d Specific method: To a solution of di-tert-butyl (5-(4-(dimethylcarbamoyl)phenyl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (15 mg, 0.03 mmol) in ethyl acetate (2 mL), HCl in ethyl acetate (4 M, 2 mL) was added. After the reaction solution was stirred at room temperature for 1 hour, the reaction solution was concentrated to give a yellow solid 4-(5-amino-6-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)-N,N-dimethylbenzamide hydrochloride (5 mg). 1 H NMR (400MHz, DMSO-d6): δ8.87 (s, 1H), 8.25–8.23 (m, 1H), 8.21–8.15 (m, 2H), 8.06–8.04 (m, 2H), 7.77 (s, 1H), 7.53–7.49 (m, 4H), 7.36 (s, 3H), 7.23 (s, 2H), 7.11 (s, 2H), 4.50–4.48 (m, 2H), 3.00–2.97 (m, 6H). MS found (ESI + )[(M+H) + ]:457.

[0554] Example 26: 4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-N-carbamoylbenzamide dihydrochloride

[0555]

[0556] Step 26a: Preparation of methyl (E)-4-((hydroxyimino)methyl)benzoate

[0557]

[0558] Step 26a Specific method: At room temperature, hydroxylamine hydrochloride (0.51 g, 7.31 mmol) was added to a solution of methyl 4-formylbenzoate (1 g, 6.10 mmol) in tetrahydrofuran (30 mL). The reaction mixture was stirred at room temperature for 16 hours. Water (40 mL) was added to the reaction system, the organic phase was separated, the aqueous phase was extracted three times with ethyl acetate (30 mL), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain yellow solid methyl (E)-4-((hydroxyimino)methyl)benzoate (1.2 g) as a crude product and used directly in the next step without further purification.

[0559] Step 26b: Preparation of methyl 4-(5-(6-bromo-3-((tert-butoxycarbonyl)amino)pyrazin-2-yl)isoxazol-3-yl)benzoate

[0560] Step 26b Specific method: To a mixed solution of di(tert-butyl)(5-bromo-3-ethynyl-2-pyrazinyl)carbamate (0.89 g, 2.23 mmol) in methanol (12 mL) and water (2.4 mL), methyl (E)-4-((hydroxyimino)methyl)benzoate (0.60 g, 3.35 mmol) and bis(trifluoroacetyl)iodobenzene (1.44 g, 3.35 mmol) were added in sequence, and stirred at room temperature overnight. Ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The organic phase was then washed three times with water (20 mL). The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain methyl 4-(5-(6-bromo-3-((tert-butyloxycarbonyl)amino)pyrazin-2-yl)isoxazol-3-yl)benzoate (0.96 g).

[0561] Step 26c: Preparation of methyl 4-(5-(3-((tert-butoxycarbonyl)amino)-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzoate

[0562]

[0563] Step 26c Specific method: Under nitrogen protection, 4-(5-(6-bromo-3-((tert-butyloxycarbonyl)amino)pyrazin-2-yl)isoxazol-3-yl)benzoic acid methyl ester (0.4g, 0.70mmol) in 1,4-dioxane (15mL) and water (1.5mL) were added in turn at room temperature (~20°C) to a mixed solution of 4-isopropylsulfonylphenylboronic acid (0.16g, 0.70mmol), potassium carbonate (0.29g, 2.10mmol) and 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (0.10g, 0.14mmol). After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40mL) and water (40mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give methyl 4-(5-(3-((tert-butoxycarbonyl)amino)-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzoate (0.33 g).

[0564] Step 26d: Preparation of 4-(5-(3-((tert-butyloxycarbonyl)amino)-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzoic acid

[0565]

[0566] Step 26d Specific method: To a solution of methyl 4-(5-(3-((tert-butoxycarbonyl)amino)-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzoate (150 mg, 0.26 mmol) in methanol (5 mL) was added lithium hydroxide (33 mg, 0.78 mmol) at room temperature. After the reaction solution was stirred at room temperature for 1 hour, it was adjusted to acidity with aqueous hydrochloric acid solution (1 M), and then ethyl acetate (20 mL) was added for extraction three times. The combined organic phase was concentrated to give 4-(5-(3-((tert-butoxycarbonyl)amino)-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzoic acid (150 mg) as a crude product and used directly in the next step without further purification.

[0567] Step 26e: Preparation of tert-butyl (3-(3-(4-(carbamoylcarbamoyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0568]

[0569] Step 26e Specific method: 2-chloro-1-methylpyridinium iodide (45 mg, 0.17 mmol) was added to a solution of 4-(5-(3-((tert-butoxycarbonyl)amino)-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzoic acid (75 mg, 0.013 mmol) in N-methylpyrrolidone (3 mL) and stirred at 50°C for 1 hour, followed by the addition of 1-(tert-butoxycarbonyl)guanidine (28 mg, 0.17 mmol) and N,N-diisopropylethylamine (66 uL). The mixture was stirred at room temperature for 16 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction solution, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give tert-butyl (3-(3-(4-(carbamoylcarbamoyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (60 mg).

[0570] Step 26f: Preparation of 4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-N-carbamoylbenzamide dihydrochloride

[0571]

[0572] Step 26f Specific method: To a solution of tert-butyl (3-(3-(4-(carbamoylcarbamoyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (60 mg, 0.10 mmol) in ethyl acetate (2 mL), a mixed solution of hydrochloric acid and ethyl acetate (4 M, 2 mL) was added. After the reaction solution was stirred at room temperature for 1 hour, the reaction solution was concentrated to obtain 4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-N-carbamoylbenzamide dihydrochloride (45 mg) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6): δ12.12 (s, 1H), 8.96 (s, 1H), 8.73 (s, 2H), 8.56 (s, 2H), 8.39–8.37 (m, 2H), 8.34–8.32 (m, 2H), 8.26–8.24 (m, 2H), 7.95–7.93 (m, 3H), 7.23 (s, 1H), 3.50–3.43 (m, 1H), 1.23–1.21 (m, 6H). MS found (ESI+) [(M+H)+]: 506.

[0573] Example 27: 1-(4-(5-(3-amino-6-(1,1-dioxy-2,3-dihydrobenzothiophen-5-yl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride

[0574]

[0575] Step 27a: Preparation of 5-bromobenzothiophene 1,1-dioxide

[0576]

[0577] Step 27a Specific method: At room temperature, m-chloroperbenzoic acid (2.4 g, 14.16 mmol) was added to a solution of 5-bromobenzothiophene (1 g, 4.72 mmol) in dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 6 hours. Excess saturated aqueous sodium sulfite solution (40 mL) was added to the reaction system to quench, the organic phase was separated, the aqueous phase was extracted three times with dichloromethane (30 mL), the combined organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure and column chromatography was performed to obtain 5-bromobenzothiophene 1,1-dioxide (0.97 g) as a white solid.

[0578] Step 27b: Preparation of 5-bromo-2,3-dihydrobenzothiophene 1,1-dioxide

[0579]

[0580] Step 27b Specific method: Sodium borohydride (0.12 g, 3.07 mmol) was added to a solution of 5-bromobenzothiophene 1,1-dioxide (0.5 g, 2.05 mmol) in ethanol (8 mL) at room temperature. The reaction mixture was stirred at room temperature (~20°C) for 6 hours. Saturated aqueous ammonium chloride solution (4 mL) was added to the reaction system to quench it, followed by ethyl acetate (40 mL) and water (40 mL). The organic phase was separated, and the aqueous phase was extracted three times with ethyl acetate (30 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a white solid 5-bromo-2,3-dihydrobenzothiophene 1,1-dioxide (0.50 g) as a crude product and used directly in the next step without further purification.

[0581] Step 27c: Preparation of (1,1-dioxy-2,3-dihydrobenzothiophen-5-yl)boronic acid

[0582]

[0583] Step 27c Specific method: To a mixed solution of 5-bromo-2,3-dihydrobenzothiophene 1,1-dioxide (500 mg, 2.03 mmol) in N,N-dimethylformamide (12 mL), bis-pinacol borate (775 mg, 3.05 mmol), potassium acetate (497 mg, 0.51 mmol) and 1,1'-bisdiphenylphosphinoferrocene palladium dichloride (150 mg, 0.20 mmol) were added in sequence at room temperature. After replacing nitrogen three times, the reaction solution was stirred at 80°C for three hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered with diatomaceous earth, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), and the combined organic phase was concentrated to obtain (1,1-dioxy-2,3-dihydrobenzothiophene-5-yl) boronic acid (800 mg) as a crude product and directly used in the next step without further purification.

[0584] Step 27d: Preparation of di-tert-butyl (3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(1,1-dioxy-2,3-dihydrobenzothiophen-5-yl)pyrazin-2-yl)carbamate

[0585]

[0586] Step 27d Specific method: Under nitrogen protection, to a mixed solution of di-tert-butyl (5-bromo-3-(3-(4-cyano-3-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (0.6 g, 1.11 mmol) in 1,4-dioxane (15 mL) and water (1.5 mL), (1,1-dioxy-2,3-dihydrobenzothiophene-5-yl)boric acid (0.65 g, 2.22 mmol), potassium carbonate (0.44 g, 3.33 mmol) and 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (0.16 g, 0.22 mmol) were added at room temperature (~20°C). After replacing nitrogen three times, the reaction solution was stirred at 95°C for three hours. After cooling to room temperature, ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(1,1-dioxy-2,3-dihydrobenzothiophene-5-yl)pyrazin-2-yl)carbamate (0.25 g).

[0587] Step 27d: Preparation of di-tert-butyl (3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(1,1-dioxy-2,3-dihydrobenzothiophen-5-yl)pyrazin-2-yl)carbamate

[0588]

[0589] Step 27d Specific method: To a solution of di-tert-butyl (3-(3-(4-cyanophenyl)isoxazol-5-yl)-5-(1,1-dioxy-2,3-dihydrobenzothiophene-5-yl)pyrazine-2-yl)carbamate (250 mg, 0.40 mmol) in methanol (5 mL), nickel chloride hexahydrate (284 mg, 1.20 mmol) and sodium borohydride (23 mg, 1.2 mmol) were added in sequence at room temperature. After the reaction solution was stirred at room temperature for 1 hour, it was quenched with a saturated aqueous solution of ammonium chloride (2 mL). Ethyl acetate (40 mL) and a saturated aqueous solution of sodium bicarbonate (40 mL) were added. After the mixed solution was filtered through diatomaceous earth, the filtrate was poured into a separatory funnel. The aqueous phase was extracted three times with ethyl acetate (40 mL), and the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(1,1-dioxy-2,3-dihydrobenzothiophen-5-yl)pyrazin-2-yl)carbamate (62 mg).

[0590] Step 27e: Preparation of di-tert-butyl (5-(1,1-dioxy-2,3-dihydrobenzothiophen-5-yl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0591]

[0592] Step 27e Specific method: To a solution of di-tert-butyl (3-(3-(4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(1,1-dioxy-2,3-dihydrobenzothiophen-5-yl)pyrazin-2-yl)carbamate (62 mg, 0.10 mmol) in acetonitrile (3 mL), 1H-pyrazole-1-carboximidamide hydrochloride (17 mg, 0.11 mmol) and N,N-diisopropylethylamine (32 uL) were added in sequence. The mixture was stirred at room temperature for 16 hours. Ethyl acetate (20 mL) and water (20 mL) were added to the reaction solution, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give di-tert-butyl (5-(1,1-dioxy-2,3-dihydrobenzothiophene-5-yl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (25 mg).

[0593] Step 27f: Preparation of 1-(4-(5-(3-amino-6-(1,1-dioxy-2,3-dihydrobenzothiophen-5-yl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride

[0594]

[0595] Step 27f Specific method: To a solution of di-tert-butyl (5-(1,1-dioxy-2,3-dihydrobenzothiophen-5-yl)-3-(3-(4-(guanidinomethyl)phenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (25 mg, 0.037 mmol) in ethyl acetate (2 mL), add HCl in ethyl acetate (4 M, 2 mL). After the reaction solution was stirred at room temperature for 1 hour, the reaction solution was concentrated to give a yellow solid 1-(4-(5-(3-amino-6-(1,1-dioxy-2,3-dihydrobenzothiophen-5-yl)pyrazin-2-yl)isoxazol-3-yl)benzyl)guanidine dihydrochloride (14 mg). 1H NMR (400 MHz, DMSO-d6): δ8.92 (s, 1H), 8.27–8.25 (m, 2H), 8.19–8.16 (m, 1H), 8.06–8.04 (m, 2H), 7.84–7.82 (m, 1H), 7.79 (s, 1H), 7.51–7.49 (m, 2H), 7.32 (m, 1H), 7.19–7.17 (m, 3H), 7.06 (s, 1H), 4.50–4.48 (m, 2H), 3.67–3.63 (m, 2H), 3.46–3.43 (m, 2H). MS found (ESI + )[(M+H) + ]:476.

[0596] Example 28: N-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)acetamidine dihydrochloride

[0597] Step 28a: Preparation of (E)-4-(bromomethyl)benzaldehyde oxime

[0598]

[0599] Specific method of step 28a: 4-(bromomethyl)benzaldehyde (500 mg, 2.51 mmol) and hydroxylamine hydrochloride (346.7 mg, 5.02 mmol) were prepared in a manner similar to step 1a of Example 1 to obtain white solid (E)-4-(bromomethyl)benzaldehyde oxime (500 mg).

[0600] Step 28b: Preparation of di(tert-butyl)(5-bromo-3-(3-(4-bromomethylphenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0601]

[0602] Step 28b specific method: Di(tert-butyl)(5-bromo-3-ethynyl-pyrazin-2-yl)carbamate (0.5 g, 1.26 mmol) and (E)-4-(bromomethyl)benzaldehyde oxime (0.40 g, 1.88 mmol) were prepared by a method similar to that of Step 1b of Example 1 to obtain di(tert-butyl)(5-bromo-3-(3-(4-bromomethylphenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (350 mg).

[0603] Step 28c: Preparation of di(tert-butyl)(5-bromo-3-(3-(4-ethanaminylmethylphenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0604]

[0605] Step 28c Specific method: At room temperature, acetamidine hydrochloride (46.4 mg, 0.49 mmol), potassium carbonate (113.8 mg, 0.83 mmol) and potassium iodide (54.8 mg, 0.33 mmol) were added to a solution of di(tert-butyl)(5-bromo-3-(3-(4-bromomethylphenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (200 mg, 0.33 mmol) in acetonitrile (3 ml), and the reaction solution was stirred at room temperature for 3 hours. Ethyl acetate (40 mL) and water (40 mL) were added to the reaction solution. The organic phase was then washed three times with water (20 mL). The organic phase was separated and dried over anhydrous sodium sulfate, filtered, concentrated and separated by column chromatography to obtain di(tert-butyl)(5-bromo-3-(3-(4-acetamidine-methylphenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (60 mg).

[0606] Step 28d: Preparation of di(tert-butyl)(3-(3-(4-ethanaminylmethylphenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0607]

[0608] Step 28d Specific method: Di(tert-butyl)(5-bromo-3-(3-(4-ethoxyamidinomethylphenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (60 mg, 0.10 mmol) and 4-isopropylsulfonylphenylboronic acid (35.1 mg, 0.15 mmol) were prepared by a method similar to that in Step 1c of Example 1 to obtain di(tert-butyl)(3-(3-(4-ethoxyamidinomethylphenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (8 mg).

[0609] Step 28e: Preparation of N-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)acetamidine dihydrochloride

[0610]

[0611] Specific method of step 28e: Di(tert-butyl)(3-(3-(4-ethanaminylmethylphenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (8 mg, 0.011 mmol) was prepared by a method similar to that of step 1c of example 1 to obtain yellow solid N-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)benzyl)acetamidine dihydrochloride (4 mg).1 H NMR (400 MHz, DMSO-d6): δ10.18 (br.s, 1H), 9.45 (s, 1H), 9.01 (s, 1H), 8.98 (s, 1H), 8.42–8.40 (m, 2H), 8.09–8.07 (m, 2H), 7.97–7.95 (m, 2H), 7.85 (s, 1H), 7.62–7.60 (m, 2H), 7.48 (brs, 3H), 7.35 (brs, 3H), 7.22 (brs, 2H), 4.63–4.62 (m, 2H), 3.49–3.42 (m, 1H), 2.29 (s, 3H), 1.23–1.21 (m, 2H). MS found (ESI+) [(M+H)+]: 491.

[0612] Example 29: N-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)acetimide dihydrochloride

[0613]

[0614] Step 29a: Preparation of (E)-4-iodobenzaldehyde oxime

[0615]

[0616] Step 29a Specific method: 4-iodobenzaldehyde (500 mg, 2.16 mmol) and hydroxylamine hydrochloride (223.6 mg, 3.24 mmol) were prepared in a manner similar to that of Step 1a of Example 1 to obtain white solid (E)-4-iodobenzaldehyde oxime (400 mg).

[0617] Step 29b: Preparation of di(tert-butyl)(5-bromo-3-(3-(4-iodophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0618]

[0619] Specific method of step 29b: Di(tert-butyl)(5-bromo-3-ethynyl-pyrazin-2-yl)carbamate (500 mg, 1.26 mmol) and (E)-4-iodobenzaldehyde oxime (463.6 mg, 1.88 mmol) were prepared by a method similar to step 1b of example 1 to obtain di(tert-butyl)(5-bromo-3-(3-(4-iodophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (400 mg).

[0620] Step 29c: Preparation of di(tert-butyl)(3-(3-(4-iodophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0621]

[0622] Specific method of step 29c: Under nitrogen protection, di(tert-butyl)(5-bromo-3-(3-(4-iodophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (400.0 mg, 0.62 mmol) and 4-isopropylsulfonylphenylboronic acid (169.6 mg, 0.74 mmol) were prepared by a method similar to step 1c of example 1 to obtain di(tert-butyl)(3-(3-(4-iodophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (90 mg).

[0623] Step 29d: Preparation of N-(4-(5-(3-di(tert-butyloxycarbonyl)amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)acetimide

[0624]

[0625] Step 29d: Specific method: Under nitrogen protection, to a solution of di(tert-butyl)(3-(3-(4-iodophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (50.0 mg, 0.067 mmol) in tert-butanol (2 ml) were added acetamidine hydrochloride (12.6 mg, 0.13 mmol), sodium tert-butoxide (19.3 mmol, 0. 20mmol), 2-(dicyclohexylphosphino)-3,6-dimethoxy-2'-4'-6'-tri-1-propyl-11'-biphenyl (7.2mg, 0.013mmol) and chloro(2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethylphenyl)]palladium(II) (9.7mg, 0.013mmol). The reaction solution was stirred at 95°C for five hours. After cooling to room temperature, ethyl acetate (40mL) and water (40mL) were added to the reaction solution. The mixed solution was filtered through celite, the resulting solution was separated, the aqueous phase was extracted three times with ethyl acetate (20 mL), the combined organic phases were concentrated and separated by column chromatography to give N-(4-(5-(3-di(tert-butoxycarbonyl)amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)acetimide (8 mg).

[0626] Step 29e: Preparation of N-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)acetimidide dihydrochloride

[0627]

[0628] Step 29e specific method: N-(4-(5-(3-di(tert-butoxycarbonyl)amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)acetimide (8 mg, 0.012 mmol) was prepared by a method similar to that of Step 1f of Example 1 to obtain N-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)acetimide (4 mg). 1 H NMR (400 MHz, DMSO-d6): δ11.66 (s, 1H), 9.7 (br. s, 1H), 8.99 (s, 1H), 8.78 (s, 1H), 8.42–8.40 (m, 2H), 8.22–8.20 (m, 2H), 7.98–7.96 (m, 2H), 7.90 (s, 1H), 7.57–7.55 (m, 2H), 7.27-7.20 (m, 2H), 3.56-3.53 (m, 1H), 2.42 (s, 3H), 1.23-1.21 (m, 6H). MS found (ESI+) [(M+H)+]: 477.

[0629] Example 30: 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3-(trifluoromethyl)benzyl)guanidine dihydrochloride

[0630]

[0631] Step 30a: Preparation of 4-formyl-3-(trifluoromethyl)benzonitrile

[0632]

[0633] Step 30a Specific method: Under nitrogen protection, zinc cyanide (1.0 g, 8.8 mmol) and tetrakis(triphenylphosphine)palladium (459.7 mg, 0.40 mmol) were added to a DMF (15 ml) solution of 4-bromo-2-(trifluoromethyl)benzaldehyde (2 g, 7.96 mmol) in sequence, and the reaction solution was stirred at 90° C. for four hours. After cooling to room temperature, the reaction solution was concentrated, and then ethyl acetate (50 mL) and water (50 mL) were added to the reaction solution. The resulting solution was separated, the aqueous phase was extracted twice with ethyl acetate (30 mL), and the combined organic phase was concentrated and separated by column chromatography to obtain 4-formyl-3-(trifluoromethyl)benzonitrile (1.1 g).

[0634] Step 30b: Preparation of (E)-4-((hydroxyimino)methyl)-3-(trifluoromethyl)benzonitrile

[0635]

[0636] Step 30b: 4-Formyl-3-(trifluoromethyl)benzonitrile (500 mg, 2.5 mmol) and hydroxylamine hydrochloride (223.6 mg, 3.24 mmol) were prepared in a manner similar to that of Step 1a of Example 1 to give (E)-4-((hydroxyimino)methyl)-3-(trifluoromethyl)benzonitrile (400 mg) as a white solid.

[0637] Step 30c: Preparation of di(tert-butyl)(5-bromo-3-(3-(2-trifluoromethyl-4-cyanophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0638]

[0639] Step 30c Specific method: Di(tert-butyl)(5-bromo-3-ethynyl-pyrazin-2-yl)carbamate (500 mg, 1.26 mmol) and (E)-4-((hydroxyimino)methyl)-3-(trifluoromethyl)benzonitrile (400 mg, 1.87 mmol) were prepared by a method similar to that of Step 1b of Example 1 to obtain di(tert-butyl)(5-bromo-3-(3-(2-trifluoromethyl-4-cyanophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (300 mg).

[0640] Step 30d: Preparation of di(tert-butyl)(3-(3-(2-trifluoromethyl-4-cyanophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0641]

[0642] Specific method of step 30d: Under nitrogen protection, di(tert-butyl)(5-bromo-3-(3-(2-trifluoromethyl-4-cyanophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (300.0 mg, 0.49 mmol) and 4-isopropylsulfonylphenylboronic acid (169.6 mg, 0.74 mmol) were prepared by a method similar to step 1c of Example 1 to obtain di(tert-butyl)(3-(3-(2-trifluoromethyl-4-cyanophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (100 mg).

[0643] Step 30e: Preparation of di(tert-butyl)(3-(3-(2-trifluoromethyl-4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0644]

[0645] Step 30e Specific method: Di(tert-butyl)(3-(3-(2-trifluoromethyl-4-cyanophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (100 mg, 0.14 mmol) was prepared by a method similar to that in Step 1d of Example 1 to obtain di(tert-butyl)(3-(3-(2-trifluoromethyl-4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (10 mg).

[0646] Step 30f: Preparation of di(tert-butyl)(3-(3-(2-trifluoromethyl-4-(guanidinomethylidene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate

[0647]

[0648] Step 30f specific method: Di(tert-butyl)(3-(3-(2-trifluoromethyl-4-(aminomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (10 mg, 0.014 mmol) and 1H-pyrazole-1-carboximidamide hydrochloric acid (7.5 mg, 0.051 mmol) were prepared by a method similar to that of Step 1e of Example 1 to obtain di(tert-butyl)(3-(3-(2-trifluoromethyl-4-(guanidinomethyl)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (5 mg).

[0649] Step 30g: Preparation of 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3-(trifluoromethyl)benzyl)guanidine dihydrochloride

[0650]

[0651] Step 30g Specific method: Di(tert-butyl)(3-(3-(2-trifluoromethyl-4-(guanidinomethylene)phenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)carbamate (5 mg, 0.006 mmol) was prepared by a method similar to that of Step 1f of Example 1 to obtain 1-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-3-(trifluoromethyl)benzyl)guanidine dihydrochloride (2 mg). 1 H NMR (400 MHz, DMSO-d6): δ8.96 (s, 1H), 8.36–8.34 (m, 2H), 8.30-8.27 (m, 1H), 8.18–8.16 (m, 1H), 7.98–7.97 (m, 1H), 7.94-7.92 (m, 2H), 7.87-7.85 (m, 1H), 7.79–7.77 (m, 1H), 7.45 (s, 1H), 7.32 (s, 1H), 7.19 (s, 1H), 7.06 (s, 1H), 4.61–4.59 (m, 1H), 3.55–3.53 (m, 1H), 1.19–1.17 (m, 6H). MS found (ESI + )[(M+H) + ]:560.

[0652] Example 31: 5-(5-(3-amino-6-(4-isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-isoindol-1-one

[0653]

[0654] Step 31a: Preparation of (E)-1-oxaisoindolyl-5-carbaldehyde oxime

[0655]

[0656] Step 31a Specific method: 1-Oxaisoisoindole-5-carbaldehyde (0.1 g, 0.62 mmol) and hydroxylamine hydrochloride (0.052 g, 0.74 mmol) were prepared in a manner similar to that of Step 1a in Example 1 to obtain white solid (E)-1-oxaisoisoindole-5-carbaldehyde oxime (0.175 g), which was a crude product and was directly used in the next step without further purification.

[0657] Step 31b: Preparation of di(tert-butyl)(5-bromo-3-(3-(1-oxoisoindole-5-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0658]

[0659] Specific method of step 31b: (E)-1-oxaisoindol-5-carbaldehyde oxime (175 mg, 0.62 mmol), di(tert-butyl)(5-bromo-3-ethynyl-2-pyrazinyl)carbamate (160 mg, 0.401 mmol) and bis(trifluoroacetyl)iodobenzene (0.26 g, 0.62 mmol) were prepared by a method similar to step 1b of Example 1 to obtain yellow solid di(tert-butyl)(5-bromo-3-(3-(1-oxaisoindol-5-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (120 mg).

[0660] Step 31c: Preparation of di(tert-butyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(1-oxoisoindole-5-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate

[0661]

[0662] Specific method of step 31c: Di(tert-butyl)(5-bromo-3-(3-(1-oxoisoisoindole-5-yl)isoxazol-5-yl)pyrazine-2-yl)carbamate (120 mg, 0.209 mmol), 4-isopropylsulfonylphenylboronic acid (48 mg, 0.209 mmol), potassium carbonate (58 mg, 0.418 mmol) and 1,1'-bis(diphenylphosphinoferrocenepalladium) dichloride (31 mg, 0.042 mmol) were prepared by a method similar to step 1c of Example 1 to obtain light yellow solid di(tert-butyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(1-oxoisoisoindole-5-yl)isoxazol-5-yl)pyrazine-2-yl)carbamate (90 mg).

[0663] Step 31d: Preparation of 5-(5-(3-amino-6-(4-isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-isoindol-1-one

[0664] Specific method of step 31d: Di(tert-butyl)(5-(4-(isopropylsulfonyl)phenyl)-3-(3-(1-oxaisoindol-5-yl)isoxazol-5-yl)pyrazin-2-yl)carbamate (90 mg, 0.156 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4M, 2 mL) were prepared according to a method similar to step 1f of Example 1 to obtain a yellow solid 5-(5-(3-amino-6-(4-isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)-isoindol-1-one (24.7 mg). 1 H NMR (400 MHz, DMSO-d6): δ8.95 (s, 1H), 8.72 (s, 1H), 8.39–8.37 (m, 2H), 8.24 (s, 1H), 8.15–8.13 (m, 1H), 7.95–7.93 (m, 2H), 7.88–7.84 (m, 2H), 7.20 (s, 2H), 4.50 (s, 2H), 3.49–3.43 (m, 1H), 1.20–1.19 (m, 6H). MS found (ESI + )[(M+H) + ]:476.

[0665] Example 32: 5-(5-amino-6-(3-(4-((methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)-3-fluoro-2,2-dimethyl-2,3-dihydrobenzo[b]thiophene-1,1-dioxide

[0666]

[0667] Step 32a: Preparation of methyl-2-((4-bromophenyl)thio)acetate

[0668]

[0669] Step 32a Specific method: At room temperature, methyl bromoacetate (7.14 g, 46.65 mmol) and triethylamine (6.29 g, 62.20 mmol) were added to a solution of 4-bromothiophenol (5.88 g, 31.10 mmol) in tetrahydrofuran (50 mL). The reaction mixture was heated to 70 degrees Celsius and stirred for 3 hours. Water (100 mL) and ethyl acetate (100 mL) were added to the reaction system, the organic phase was separated, the aqueous phase was extracted three times with ethyl acetate (50 mL), the combined organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure and column chromatography was performed to obtain methyl-2-((4-bromophenyl)thio)acetate (8.6 g) as a white solid.

[0670] Step 32b: Preparation of 2-((4-bromophenyl)thio)acetic acid

[0671]

[0672] Step 32b Specific method: At room temperature, sodium hydroxide (2.49 g, 62.19 mmol) was added to a mixed solution of methyl-2-((4-bromophenyl)thio)acetate (8.12 g, 31.10 mmol) in methanol (60 mL) and water (20 mL). The reaction mixture was stirred at room temperature (~20°C) for 12 hours. After adding water (300 mL) to the reaction system, the pH was adjusted to 5-6 with 2M hydrochloric acid aqueous solution, and ethyl acetate (300 mL) was added, the organic phase was separated, the aqueous phase was extracted three times with ethyl acetate (100 mL), the combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a white solid 2-((4-bromophenyl)thio)acetic acid (6.3 g) as a crude product and directly used in the next step without further purification.

[0673] Step 32c: Preparation of 5-bromobenzo[b]thiophen-3(2H)-one

[0674]

[0675] Step 32c Specific method: 2-((4-bromophenyl)thio)acetic acid (3.0 g, 12.14 mmol) was dissolved in trifluoromethanesulfonic acid (15 mL), and the reaction solution was stirred at 80°C for 0.5 hours. After cooling to room temperature, the reaction solution was dropped into ice water, and then ethyl acetate (100 mL) was added, and the organic phase was washed three times with saturated sodium bicarbonate aqueous solution (30 mL). The obtained organic phase was concentrated and column chromatography was performed to obtain 5-bromobenzo[b]thiophene-3(2H)-one (1.5 g) as a yellow solid.

[0676] Step 32d: Preparation of 5-bromobenzo[b]thiophen-3(2H)-one-1,1-dioxide

[0677]

[0678] Specific method of step 32d: 5-bromobenzo[b]thiophene-3(2H)-one (1.5 g, 6.55 mmol) and m-chloroperbenzoic acid (3.39 g, 19.64 mmol) were prepared by a method similar to step 27a of Example 27 to obtain yellow solid 5-bromobenzo[b]thiophene-3(2H)-one-1,1-dioxide (1.4 g).

[0679] Step 32e: Preparation of 5-bromo-2,2-dimethyl-benzo[b]thiophen-3(2H)-one-1,1-dioxide

[0680]

[0681] Step 32e Specific method: To a solution of 5-bromobenzo[b]thiophene-3(2H)-one-1,1-dioxide (400 mg, 1.53 mmol) in tetrahydrofuran (12 mL) at room temperature, 1,5-diazabicyclo[4.3.0]non-5-ene (950 mg, 7.66 mmol) and iodomethane (2.17 g, 15.32 mmol) were added in sequence. The reaction solution was heated to 70 degrees Celsius and stirred for 3 hours, and then ethyl acetate (50 mL) and water (50 mL) were added to separate the organic phase, and the aqueous phase was extracted three times with ethyl acetate (40 mL). The combined organic phases were concentrated and separated by column chromatography to obtain 5-bromo-2,2-dimethyl-benzo[b]thiophene-3(2H)-one-1,1-dioxide (380 mg) as a white solid.

[0682] Step 32f: Preparation of 5-bromo-3-hydroxy-2,2-dimethyl-2,3-dihydrobenzo[b]thiophene-1,1-dioxide

[0683]

[0684] Step 32f Specific method: Sodium borohydride (137 mg, 3.63 mmol) was added to a solution of 5-bromo-2,2-dimethyl-benzo[b]thiophene-3(2H)-one-1,1-dioxide (210 mg, 0.73 mmol) in methanol (5 mL). Stir at room temperature for 1 hour. After the reaction solution was quenched with water (1 mL), ethyl acetate (20 mL) and water (20 mL) were added, and the resulting solution was separated. The aqueous phase was extracted three times with ethyl acetate (20 mL), and the combined organic phases were concentrated and separated by column chromatography to obtain 5-bromo-3-hydroxy-2,2-dimethyl-2,3-dihydrobenzo[b]thiophene-1,1-dioxide (150 mg) as a white solid.

[0685] Step 32g: Preparation of 5-bromo-3-fluoro-2,2-dimethyl-2,3-dihydrobenzo[b]thiophene-1,1-dioxide

[0686]

[0687] Step 32g Specific method: At zero degrees Celsius, diethylamino sulfur trifluoride (250 mg, 1.55 mmol) was added to a solution of 5-bromo-3-hydroxy-2,2-dimethyl-2,3-dihydrobenzo[b]thiophene-1,1-dioxide (150 mg, 0.51 mmol) in dichloromethane (5 mL). Stir at room temperature for 1 hour. After the reaction solution was quenched with water (1 mL), dichloromethane (20 mL) and water (20 mL) were added, and the resulting solution was separated. The aqueous phase was extracted three times with dichloromethane (20 mL), and the combined organic phase was concentrated and separated by column chromatography to obtain 5-bromo-3-fluoro-2,2-dimethyl-2,3-dihydrobenzo[b]thiophene-1,1-dioxide (120 mg) as a white solid.

[0688] Step 32h: Preparation of 3-fluoro-2,2-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzo[b]thiophene-1,1-dioxide

[0689]

[0690] Step 32h Specific method: 5-bromo-3-fluoro-2,2-dimethyl-2,3-dihydrobenzo[b]thiophene-1,1-dioxide (120 mg, 0.409 mmol), bipyraclostrobin (208 mg, 0.82 mmol), potassium acetate (80 mg, 0.82 mmol) and 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (60 mg, 0.082 mmol) were prepared according to a method similar to step 27c of Example 27 to obtain 3-fluoro-2,2-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzo[b]thiophene-1,1-dioxide (230 mg) as a crude product and used directly in the next step without further purification.

[0691] Step 32I: Preparation of tert-butyl(tert-butyloxycarbonyl)(3-(3-(4-(((tert-butyloxycarbonyl)(methyl)amino)methylene)phenyl)isoxazol-5-yl)-5-(3-fluoro-2,2-dimethyl-1,1-dioxido-2,3-dihydrobenzo[b]thiophen-5-yl)pyrazin-2-yl)amino acid methyl ester

[0692] Step 32I Specific method: Di-tert-butyl (5-bromo-3-(3-(4-cyano-3-fluorophenyl)isoxazol-5-yl)pyrazin-2-yl)carbamate (270 mg, 0.409 mmol), 3-fluoro-2,2-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzo[b]thiophene-1,1-dioxide (140 mg, 0.408 mmol), potassium carbonate (113 mg, 0.8 2mmol) and 1,1'-bisdiphenylphosphinoferrocenepalladium dichloride (60 mg, 0.082mmol) were prepared according to a method similar to that of Example 27, Step 27d to give tert-butyl(tert-butoxycarbonyl)(3-(3-(4-(((tert-butoxycarbonyl)(methyl)amino)methylene)phenyl)isoxazol-5-yl)-5-(3-fluoro-2,2-dimethyl-1,1-dioxido-2,3-dihydrobenzo[b]thiophen-5-yl)pyrazin-2-yl)amino acid methyl ester (130 mg).

[0693] Step 32J: Preparation of 5-(5-amino-6-(3-(4-((methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)-3-fluoro-2,2-dimethyl-2,3-dihydrobenzo[b]thiophene-1,1-dioxide

[0694]

[0695] Step 32J Specific method: Tert-butyl(tert-butyloxycarbonyl)(3-(3-(4-(((tert-butyloxycarbonyl)(methyl)amino)methylene)phenyl)isoxazol-5-yl)-5-(3-fluoro-2,2-dimethyl-1,1-dioxido-2,3-dihydrobenzo[b]thiophene-5-yl)pyrazin-2-yl)amino acid methyl ester (130 mg, 0.163 mmol) and a mixed solution of hydrochloric acid and ethyl acetate (4 M, 2 mL) were prepared according to a method similar to step 1f of Example 1 to obtain a yellow solid 5-(5-amino-6-(3-(4-((methylene)phenyl)isoxazol-5-yl)pyrazin-2-yl)-3-fluoro-2,2-dimethyl-2,3-dihydrobenzo[b]thiophene-1,1-dioxide (57.2 mg). 1 H NMR (400 MHz, DMSO-d6): δ8.99 (s, 1H), 8.53–8.51 (m, 2H), 8.02–7.98 (m, 3H), 7.79 (s, 1H), 7.56–7.54 (m, 2H), 7.22 (s, 2H), 6.06 (s, 0.5H), 5.93 (s, 0.5H), 3.82 (s, 2H), 2.36 (s, 3H), 1.50–1.49 (m, 3H), 1.37 (s, 3H). MS found (ESI +)[(M+H) + ]:494.

[0696] The following specifically describes the biological implementation data to further illustrate the technical solution of the present invention.

[0697] ATR enzyme activity inhibition assay

[0698] The HTRF (Homogeneous Time-Resolved Fluorescence) test was used to screen compounds for their ability to inhibit ATR kinase. In the reaction system, ATR phosphorylates the substrate p53, and then anti-phospho-p53-Eu is added as an energy donor to specifically bind to the phosphorylation site on p53. Anti-GST-d2 is added as an energy acceptor to specifically bind to the GST tag carried on p53. The energy donor is excited by a laser of a certain wavelength (340nm) to emit light with a wavelength of 615nm. Energy transfer occurs between the donor and the acceptor, causing the energy acceptor to emit light with a wavelength of 665nm. A plate reader is used to detect the two emitted lights and calculate the ratio of the two signals of 665nm and 615nm. The IC of the sample to be tested can be calculated by plotting and calculating. 50 .

[0699] Configure various buffer systems required for the experiment:

[0700] 1. Prepare ATR reaction buffer containing 25mM HEPES (Gibco, Cat#15630-080), 5mM DTT (Sigma, Cat#D0632-10G), 10mM MnCl2 (Sigma, Cat#7773-01-5), 5mM DTT (Sigma, Cat#D0632-10G), 1mg / ml BSA (Sigma, Cat#B2064-50G), 0.01% Brij35 (Sigma, Cat#9002-92-0), 1% Glycerol (Sigma, Cat#G5516-500ML) and H2O. Dilute the full-length ATR enzyme (eurofins, Cat#14-953M) to 60nM using the prepared ATR buffer.

[0701] 2. Prepare reaction substrate containing 80 nM p53 (eurofins, Cat#14-952M) and 300 nM ATP (Sigma, Cat#R0441).

[0702] 3. Prepare detection solution: HTRF detection buffer (Cisbio, Cat#62SDBRDF) dilute anti-phospho-p53-Eu cryptate (Cisbio, Cat#61P08KA) and anti-GST-d2 (Cisbio, Cat#61GSTDLB) to 1 unit each.

[0703] The powdered compound was dissolved in DMSO at a stock concentration of 10 mM. The test compound was diluted three-fold with DMSO starting from a final concentration of 1 μM and then added to a 384-well plate, 10.05 μl per well (containing 0.498% DMSO). 5 μl ATR was added to each well, incubated at 25°C for 10 minutes, and then 5 μl reaction substrate was added. After incubation at 25°C for 90 minutes, 10 μl detection solution was added. The reaction was allowed to react overnight, and the 665 / 615 nm data was read on the Envision 2104 Multilabel Reader. The compound inhibition curve was drawn using the four-parameter logistic regression equation of the XLfit software to calculate the compound inhibition rate.

[0704] The following table shows the relative inhibition values ​​of the compounds disclosed in this application on the ATR enzyme:

[0705]

[0706]

[0707] Analyzing the above example data, our compounds have the same or better ability to inhibit ATR than VX-970.

[0708] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A compound represented by formula I or a pharmaceutically acceptable salt thereof: in: Ring A is a benzene ring or a pyridine ring; Ring B is a benzene ring; R 1 is selected from substituted or unsubstituted guanidine, urea, aminosulfonamide and HN-C(=NH)-C 1-6 Alkyl; the substitution is selected from C 1-6 Alkyl, C 3-7 Substituted by a cycloalkyl substituent; R 2 and R 5 Selected from hydrogen, deuterium, halogen; or R 1 and R 2 Together with the atoms to which they are attached, they form a 5-membered heteroaryl or heterocyclic group containing 1-3 nitrogen atoms, wherein 0-1 methylene groups on the ring of the 5-membered heteroaryl or heterocyclic group can be arbitrarily replaced by -C(=O)-; the 5-membered heteroaryl or 5-membered heterocyclic group is any one of the following structures: R 8 Selected from hydrogen, deuterium; If present, each R 3 are each independently selected from hydrogen, deuterium, halogen, C 1-6 Alkyl and C substituted by halogen 1-6 alkyl; R 4 Selected from C 1-6 Alkyl, C 3-7 Cycloalkyl and (C 1-3 Alkyl)2amino; If present, each R 6 each independently selected from hydrogen, deuterium; Q 1 Selected from C 1-6 Alkylene, -CO-; Q 2 Selected from -SO2-, -SO(=NR 7 )- and -CO-; Every R 7 are each independently selected from hydrogen, deuterium, C 1-6 alkyl; n1 is 0 or 1; n2 is 1 or 2; n3 is 0 or 1.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The compound of formula I is Where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , Q1, Q2, n1, n2, n3 as defined in claim 1.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R 2 and R 5 are each independently hydrogen or deuterium.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R 3 is halogen or halogen-substituted C 1-6 alkyl.

5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, characterized in that: R 3 It is F or CF3.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R 4 Methyl or any of the following structures:

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: Every R 6 are each independently selected from hydrogen and deuterium.

8. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein Q 2 Selected from -SO2-, -SO(=NH)-, -SO(=NMe)- and -CO-.

9. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein Every R 7 are independently selected from hydrogen, deuterium and methyl.

10. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

11. A compound selected from:

12. A pharmaceutical composition comprising the compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof.

13. Use of the compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 12 in the preparation of a medicament for preventing and / or treating a disease involving ATR.

14. The use according to claim 13, characterized in that The ATR-implicated disease is selected from diseases, disorders and conditions of excessive or abnormal cell proliferation.

15. The use according to claim 14, characterized in that The diseases, disorders and conditions are selected from cancer and myeloproliferative disorders.

16. The use according to claim 15, characterized in that The cancer is selected from skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, kidney cancer, cervical cancer, uterine body cancer, endometrial cancer, testicular cancer, urinary cancer, melanoma, astrocytic carcinoma, meningioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, adult T-cell leukemia lymphoma, hepatocellular carcinoma, bronchial cancer, basal cell tumor, seminoma, chondrosarcoma, myosarcoma, fibrosarcoma.

17. The use according to claim 16, characterized in that The renal cancer is selected from renal parenchymal cancer; The lung cancer is selected from small cell lung cancer and non-small cell lung cancer; The bone cancer is selected from multiple myeloma; The sarcoma is selected from rhabdomyosarcoma.

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