PARP1-targeting compound and use thereof

Abstract

A PARP1-targeting compound represented by formula (I) and a use thereof, relating to the technical field of chemical medicines. The PARP1-targeting compound can be used as a PARP1 inhibitor, has the advantages of high activity and high selectivity, and also has excellent pharmacokinetic properties, excellent safety, and brain penetration potential.

Description

PARP1 targeting compounds and uses thereof Technical Field

[0001] The present invention belongs to the field of chemical medicine and relates to a class of PARP1 targeting compounds and uses thereof. Background Art

[0002] As cells grow, their DNA is constantly damaged by various internal and external factors. The most severe types of DNA damage are single-strand breaks and double-strand breaks, with single-strand breaks being more common. If these breaks are not repaired promptly and accurately, they can lead to genomic instability, cancer, and even direct cell death. The repair of single-strand breaks in DNA primarily relies on the enzyme PARP. Double-strand breaks can be repaired through two methods: non-homologous end joining repair and homologous recombination repair. Homologous recombination repair is a high-fidelity, error-free repair method and the primary pathway for double-stranded DNA repair. Numerous proteins are involved in homologous recombination repair, the most well-known of which are the BRCA proteins. Two studies in 2005 (Farmer H, McCabe N, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy [J]. Nature, 2005, 434(7035): 917-921. Bryant, H., Schultz, N., Thomas, H. et al. Specific killing of BRCA2-deficient tumors with inhibitors of poly(ADP-ribose)polymerase. Nature 434, 913–917 (2005)) showed that tumor cells lacking BRCA1 or BRCA2 are selectively inhibited by PARP inhibitors. Based on this research result, scholars proposed the concept of synthetic lethality: the loss of either BRCA or PARP gene is not lethal in itself, but the simultaneous inactivation of both will lead to cell death. Based on the theory of synthetic lethality, PARP inhibitors (PARPi) have been developed to selectively target cancer cells with BRCA1 / 2 mutations.

[0003] PARP inhibitors have shown excellent clinical efficacy in patients with homologous recombination-deficient cancers. However, whether used as a single agent or in combination therapy, hematological toxicity (anemia, neutropenia, and thrombocytopenia) and other toxicities limit the application of this type of drug. Related studies have shown (Harris PA, Boloor A, Cheung M, et al. Discovery of 5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methyl-benzenesulfonamide (Pazopanib), a novel and potent vascular endothelial growth factor receptor inhibitor. [J]. Journal of Medicinal Chemistry, 2008, 51(15): 4632.) These adverse reactions may be due to the inhibition of PARP2 by marketed PARP inhibitors, which is not essential for efficacy. Highly selective PARP1 inhibitors can reduce hematological toxicity, increase the therapeutic safety window, and increase the potential for combination with other chemotherapy or targeted drugs.

[0004] Therefore, there is an unmet clinical need for effective and safe PARP inhibitors, particularly PARP inhibitors that are selective for PARP1.

[0005] Summary of the Invention

[0006] The object of the present invention is to provide a class of PARP1-targeted compounds and uses thereof, which have unexpectedly higher selectivity for PARP1 than other PARP family members (such as PARP2, PARP3, PARP5a and PARP6), and can achieve highly selective and highly effective prevention or treatment of diseases related to PARP function; in particular, the PARP1-targeted compounds of the present invention are surprisingly able to penetrate the blood-brain barrier (BBB); therefore, the PARP1-targeted compounds of the present invention can be used to treat diseases and conditions occurring in central nervous system tissues (such as the brain and spinal cord).

[0007] In a first aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable form thereof, the structure of which is as follows:

[0008] in,

[0009] X is selected from N or C, and when X is selected from N, represents a single bond. When X is selected from C, represents a double bond;

[0010] X1 is selected from N or C(R 8a ), X2 is selected from N or C(R 8b ), X3 is selected from N or C(R 8c ), and at most one of X1, X2 and X3 is selected from N;

[0011] R1 is selected from C 1～8 Alkylthio, C 1～8 Halogenated alkylthio, C 1～8 Deuterated alkylthio, C 1～8 Halogenated alkoxy, C 1～8 Haloalkyl or C 2～8 alkenyl;

[0012] R2 is selected from hydrogen, fluorine, chlorine, C 1-4 Alkyl, C 1-4 Fluorinated alkyl or C 1-4 deuterated alkyl;

[0013] R3, R4 are independently selected from hydrogen, deuterium, fluorine, C 1-4 Alkyl, C 1-4 Fluorinated alkyl or C 1-4 deuterated alkyl;

[0014] Alternatively, R3, R4, and the atoms to which they are attached, together form a 3-6 membered alkyl ring;

[0015] R5 is selected from hydrogen, fluorine, chlorine, cyano, C 1-4 Alkyl, C 1-4 Fluorinated alkyl or C 1-4 deuterated alkyl;

[0016] Alternatively, R4, R5 and the atoms to which they are attached together form a 5-7 membered alkyl heterocyclic ring; when R4, R5 and the atoms to which they are attached are connected to form a ring, the 5-7 membered alkyl heterocyclic ring contains 1 O heteroatom in addition to X on the main ring;

[0017] R3, R4 and the atoms to which they are attached, and R4, R5 and the atoms to which they are attached, do not simultaneously form a ring;

[0018] R6 is selected from -C(O)-NH-R 6a ; R 6a Selected from C 1-4 Alkyl, C 1-4 Fluorinated alkyl, C 1-4 Deuterated alkyl or 3-6 membered cycloalkyl;

[0019] R7 is selected from hydrogen, fluorine, chlorine, cyano, C 1-4 Alkyl, C 1-4 Fluorinated alkyl or C1-4 deuterated alkyl;

[0020] R 8a 、R 8b 、R 8c independently selected from hydrogen, fluorine, chlorine, C 1-4 Alkyl, C 1-4 Deuterated alkyl, C 1-4 Fluorinated alkyl;

[0021] The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled substances, metabolites, or prodrugs.

[0022] In some embodiments of the present invention, X1 is selected from N, X2 is selected from C(R 8b ), X3 is selected from C(R 8c ); or X1 is selected from C(R 8a ), X2 is selected from N, X3 is selected from C(R 8c ); or X1 is selected from C(R 8a ), X2 is selected from C(R 8b ), X3 is selected from C(R 8c ).

[0023] In some embodiments of the present invention, R1 is selected from C 1～4 Alkylthio, C 1～4 Halogenated alkylthio, C 1～4 Deuterated alkylthio, C 1～4 Halogenated alkoxy, C 1～4 Halogenated alkyl, C 2～4 alkenyl; in R1, the halogen of the halogenated group is selected from F, Cl, and Br.

[0024] In some preferred embodiments of the present invention, R1 is selected from methylthio, halogenated methylthio, deuterated methylthio, halogenated methoxy, halogenated methyl, halogenated ethyl or vinyl; in R1, the halogenated halogen is selected from F and Cl.

[0025] In some further embodiments of the present invention, R1 is selected from -S-CH3, -S-CF2H, -S-CF3, -S-CF2Cl, -S-CD3, -O-CF2H, -O-CF3, -O-CF2Cl, -CF2-CH3 or -CH=CH2.

[0026] In some embodiments of the present invention, R2 is selected from hydrogen, fluorine, chlorine, methyl, fluoromethyl or deuterated methyl.

[0027] In some embodiments of the present invention, when R3 and R4 do not form a ring with the atoms to which they are attached, R3 and R4 are independently selected from hydrogen, deuterium, fluorine, methyl, fluoromethyl or deuterated methyl.

[0028] In some embodiments of the present invention, when R4 and R5 do not form a ring with the atoms to which they are attached, R5 is selected from hydrogen, fluorine, chlorine, cyano, methyl, fluoromethyl or deuterated methyl.

[0029] In some embodiments of the present invention, R7 is selected from hydrogen, fluorine, chlorine, cyano, methyl, fluoromethyl or deuterated methyl.

[0030] In some embodiments of the present invention, R 8a 、R 8a 、R 8c are independently selected from hydrogen, fluorine, chlorine, methyl, fluoromethyl or deuterated methyl.

[0031] In some embodiments of the present invention, R 6a is selected from methyl, fluoromethyl, deuterated methyl or cyclopropyl.

[0032] In some preferred embodiments of the present invention, R 6a Selected from methyl, -CF3, -CD3 or cyclopropyl.

[0033] In some embodiments of the present invention, the structural unit Selected from:

[0034] In some embodiments of the present invention, when R4 and R5 do not form a ring with the atoms to which they are attached, the structural unit (connected to the main ring from left to right, that is, the N end is connected to the main ring methylene, and the X end is connected to the main ring pyridyl) is selected from:

[0035] In some embodiments of the present invention, when R4 and R5 do not form a ring with the atoms to which they are attached, the structural unit Selected from:

[0036] In some embodiments of the present invention, when R4, R5 and the atoms to which they are attached form a ring, the structural unit Selected from:

[0037] In some preferred embodiments of the present invention, when R4, R5 and the atoms to which they are connected form a ring, the structural unit Selected from:

[0038] In some embodiments of the present invention, the structural unit in formula I Replace with (The connection mode of the two is consistent with the main ring, that is, the N end is connected to the main ring methylene, and the O end is connected to the main ring pyridyl) (In this case, R5 does not form a ring with the connected atoms).

[0039] The present invention also provides some specific compounds, which are selected from:

[0040] In some embodiments of the present invention, R1 is selected from -S-CH3, -S-CF2H, -S-CF3, -S-CF2Cl, -S-CD3, -O-CF2H, -O-CF3, -O-CF2Cl, -CF2-CH3, -CH=CH2, -S-CFH2 or -O-CFH2.

[0041] In some embodiments of the present invention, the structural unit Selected from:

[0042] In some embodiments of the present invention, the structural unit in formula I Replace with (The connection mode of the two is consistent with the main ring, that is, the N end is connected to the main ring methylene, and the CH end is connected to the main ring pyridyl) (In this case, R5 does not form a ring with the connected atoms).

[0043] The present invention also provides some specific compounds, which are selected from:

[0044] In some embodiments of the present invention, when R4 and R5 do not form a ring with the atoms to which they are attached, the structural unit Selected from:

[0045] In some embodiments of the present invention, when R4, R5 and the atoms to which they are attached form a ring, the structural unit Selected from:

[0046] In some preferred embodiments of the present invention, when R4, R5 and the atoms to which they are connected form a ring, the structural unit Selected from:

[0047] In some embodiments of the present invention, the structural unit in formula I Replace with (Connected to the main ring from left to right, that is, the N end is connected to the main ring methylene, and the alkynyl end is connected to the main ring pyridyl).

[0048] In some embodiments of the present invention, the structural unit in formula I Replace with

[0049] In some embodiments of the present invention, the structural unit in formula I Replace with R9 is selected from H.

[0050] The present invention also provides some specific compounds, which are selected from:

[0051] In a second aspect, the present invention provides a pharmaceutical composition comprising the aforementioned compound or its pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites or prodrugs as active ingredients, supplemented with a pharmaceutically acceptable carrier.

[0052] A further object of the present invention is to provide a method for preparing the pharmaceutical composition of the present invention, which comprises combining any compound of Formula I or a pharmaceutically acceptable form thereof, or a mixture thereof, with one or more pharmaceutically acceptable carriers.

[0053] The pharmaceutically acceptable carrier that can be used in the pharmaceutical composition of the present invention is a pharmaceutically acceptable carrier. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (2005).

[0054] The pharmaceutical composition can be administered in any form, as long as it prevents, alleviates, prevents or cures the symptoms of a human or animal patient. For example, it can be prepared into various suitable dosage forms according to the route of administration.

[0055] In other embodiments, the administration of the compound or pharmaceutical composition of the present invention can be combined with another treatment method. The other treatment method can be selected from, but not limited to: radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

[0056] The present invention also relates to a pharmaceutical preparation comprising any compound of Formula I or a pharmaceutically acceptable form thereof, or a mixture thereof as an active ingredient, or a pharmaceutical composition of the present invention. In some embodiments, the preparation is in the form of a solid preparation, a semi-solid preparation, a liquid preparation, or a gaseous preparation.

[0057] In a third aspect, the present invention provides the use of the aforementioned compound, the compound of formula I, and related specific compounds or pharmaceutically acceptable forms thereof, or the pharmaceutical composition of the present invention in the preparation of a medicament for preventing or treating PARP1 enzyme-related diseases.

[0058] The present invention provides a method for preventing or treating PARP1 enzyme-related diseases, comprising administering a compound of formula I or a pharmaceutically acceptable form thereof, or a pharmaceutical composition of the present invention to a subject in need thereof.

[0059] The present invention provides a compound of formula I or a pharmaceutically acceptable form thereof, or a pharmaceutical composition of the present invention, for use in preventing or treating PARP1 enzyme-related diseases.

[0060] The present invention provides a method for preventing or treating PARP1 enzyme-related diseases in combination with a compound of Formula I or a pharmaceutically acceptable form thereof, or a pharmaceutical composition of the present invention, wherein the additional treatment method includes but is not limited to: radiotherapy, chemotherapy, immunotherapy, or a combination thereof.

[0061] In some embodiments, the PARP1 enzyme-related disease is a disease that is sensitive or responsive to PARP1 enzyme inhibition.

[0062] In some embodiments, the PARP1 enzyme-related disease is a tumor-related disorder.

[0063] In some preferred embodiments, the oncological disorder is deficient in a HR-dependent DNA DSB repair pathway.

[0064] In some preferred embodiments, the neoplastic disorder comprises one or more cancer cells that have a reduced or absent ability to repair DNA DSBs by HR relative to normal cells.

[0065] In some preferred embodiments, the cancer cells have a BRCA1 or BRCA2 deficient phenotype.

[0066] In some embodiments, the PARP1 enzyme-related disease is a tumor-related disorder, including but not limited to solid and hematological malignancies. In further embodiments, the tumor-related disorder includes but is not limited to breast cancer, colorectal cancer, colon cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer and bronchioloalveolar carcinoma) and prostate cancer, as well as bile duct cancer, bone cancer, bladder cancer, head and neck cancer, kidney cancer, liver cancer, gastrointestinal tissue cancer, esophageal cancer, ovarian cancer, pancreatic cancer, skin cancer, testicular cancer, thyroid cancer, uterine cancer, cervical cancer and vulvar cancer, as well as leukemia (including chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML)), multiple myeloma, lymphoma, meningioma, pituitary tumor, craniopharyngioma, schwannoma, glioma, ependymoma, primitive neuroectodermal tumor, central nervous system lymphoma, germ cell tumor, metastasis, brain cancer or central nervous system cancer.

[0067] In some preferred embodiments, the PARP1 enzyme-related disease is breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer, lung cancer or glioma.

[0068] In a further preferred embodiment, the compounds of the present invention can be used in combination with chemoradiotherapy or immunotherapy to prevent or treat cancer.

[0069] In a fourth aspect, the present invention provides the use of the aforementioned compound, the compound of formula I, and related specific compounds or pharmaceutically acceptable forms thereof, or the pharmaceutical composition of the present invention in the preparation of PARP1 inhibitors.

[0070] Beneficial effects of the present invention:

[0071] The present invention provides a novel class of highly active and highly selective PARP1 inhibitors capable of achieving at least one of the following technical effects: (1) high inhibitory activity against PARP1 enzyme; (2) selective inhibition of PARP1 enzyme, with high selectivity for other PARP family enzymes such as PARP2, PARP5a, and PARP5b; (3) strong inhibitory activity against homologous recombination-deficient tumor cells and weak inhibitory effect on non-homologous recombination-deficient cells; (4) excellent pharmacokinetic properties (e.g., good bioavailability, appropriate half-life and duration of action); (5) excellent safety (lower toxicity and / or fewer side effects, wider therapeutic window), etc.; the PARP1 targeting compounds described in the present invention are surprisingly capable of penetrating the blood-brain barrier (BBB) ​​and can be used to treat diseases and conditions occurring in central nervous system tissues (e.g., brain and spinal cord).

[0072] Definition of terms:

[0073] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. The terms "include," "comprising," "having," "containing," or "involving," and their variations herein, are inclusive or open-ended and do not exclude other unrecited elements or method steps. It should be understood by those skilled in the art that the above terms, such as "comprising," encompass the meaning of "consisting of."

[0074] In the present invention, "a", "an", "the", "at least one" and "one or more" are used interchangeably. Thus, for example, a composition comprising "a" pharmaceutically acceptable excipient can be interpreted to mean that the composition includes "one or more" pharmaceutically acceptable excipients.

[0075] When the lower and upper limits of a numerical range are disclosed, any value and any included range falling within the range are specifically disclosed. In particular, each range of values ​​disclosed herein (in the form "about a to b," or equivalently, "approximately a to b," or equivalently, "about a b") should be understood to represent each value and range encompassed within the broader range.

[0076] For example, the statement "C 1-8 " should be understood to include any sub-ranges therein and each point value, such as C 2-5 、C 3-4 、C 1-2 、C 1-3 、C 1-4 、C 1-5 etc., as well as C1, C2, C3, C4, C5, C6, etc. For example, the expression “C 2-8 ” should also be understood in a similar manner, for example, any sub-ranges and point values ​​contained therein may be included, for example, C 6-8 、C 6-7 、C 7-8 etc. and C3, C4, C5, C6, C7, C8, etc. For another example, the expression "3-6 yuan" should be understood to cover any sub-range therein and every point value, such as 3-4 yuan, 3-5 yuan, 3-6 yuan, 4-5 yuan, 4-6 yuan, etc. and 3, 4, 5, 6 yuan, etc.

[0077] In the present invention, unless otherwise specified, halogen means fluorine, chlorine, bromine or iodine.

[0078] In the present invention, unless otherwise specified, "alkyl" includes a linear or branched monovalent saturated hydrocarbon group. For example, alkyl includes methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 3-(2-methyl)butyl, 2-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-hexyl, 2-methylpentyl, etc. Similarly, "C1-4 C in "alkyl" 1-4 It refers to a group containing 1, 2, 3 or 4 carbon atoms in a straight or branched chain.

[0079] In the present invention, unless otherwise specified, "cycloalkyl", "carbocycle" or "cycloalkylene" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic hydrocarbon group. Common cycloalkyl groups include (but are not limited to) monocyclic cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclobutene, cyclopentene, cyclohexene, etc.; or bicyclic cycloalkyl groups, including fused rings, bridged rings or spiro rings, such as bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[5.2.0]nonyl, decalinyl, etc. For example, "C 3-12 cycloalkyl" refers to a cycloalkyl group having 3-12 ring carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12). The cycloalkyl or cycloalkylene group in the present invention is optionally substituted with one or more substituents described herein.

[0080] In the present invention, unless otherwise specified, "haloalkyl" refers to an alkyl group as described above, wherein one or more hydrogen atoms are replaced by halogen. For example, the term "C 1-6 "Haloalkyl" refers to a C 1-6 Alkyl. It will be understood by those skilled in the art that when there are more than one halogen substituent, the halogens may be the same or different and may be located on the same or different C atoms. Examples of haloalkyl include, for example, -CH2F, -CHF2, -CF3, -CCl3, -C2F5, -C2Cl5, -CH2CF3, -CH2Cl or -CH2CH2CF3. The haloalkyl groups of the present invention are optionally substituted with one or more substituents described herein.

[0081] In the present invention, unless otherwise specified, "fluoroalkyl" refers to the alkyl group described above, wherein one or more hydrogen atoms are replaced by fluorine atoms. For example, the term "C 1-4 "Fluoroalkyl" refers to a C group optionally substituted by one or more (e.g. 1-3) fluorine atoms. 1-4 Alkyl. It will be understood by those skilled in the art that when there are more than one fluorine atom substituent, the fluorine atoms may be the same or different and may be located on the same or different C atoms. Examples of haloalkyl groups include -CH2F, -CHF2, -CF3, -C2F5, -CH2CF3, -CH2CH2CF3, etc. The fluoroalkyl groups of the present invention are optionally substituted with one or more substituents described herein.

[0082] In the present invention, unless otherwise specified, "alkenyl" refers to a linear or branched aliphatic hydrocarbon group having at least one C=C double bond. For example, "C 2-4 "Alkenyl" refers to an alkenyl group having 2 to 4 carbon atoms. Common alkenyl groups include (but are not limited to) ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, n-octenyl, n-decenyl, etc. The alkenyl group in the present invention is optionally substituted with one or more substituents described herein.

[0083] In the present invention, unless otherwise specified, "substituted" means that one or more hydrogen atoms in a group are replaced by the same or different substituents.

[0084] The present invention also includes all pharmaceutically acceptable isotopically labeled compounds, which are identical to the compounds of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium ( 2 H), tritium ( 3 H)); carbon isotopes (e.g. 13 C and 14 C); isotopes of chlorine (such as 37Cl); isotopes of iodine (such as 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 17 O and 18 O); isotopes of phosphorus (such as 32 P); and sulfur isotopes (e.g. 34 S).

[0085] As used herein, a "pharmaceutical composition" refers to a formulation of a compound of the present invention and a medium generally accepted in the art for delivering a biologically active compound to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient, and thereby exert its biological activity.

[0086] In this application, "pharmaceutically acceptable carrier" includes but is not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved or accepted by relevant governmental regulatory authorities for use in humans or livestock.

[0087] As used herein, the terms "drug combination," "drug combination," "combination therapy," "administration of another therapy," "administration of another therapeutic agent," and the like refer to a drug therapy obtained by mixing or combining more than one active ingredient, and include both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" refers to the simultaneous administration of at least one compound described herein and at least one synergistic agent to a patient as a single entity or single dosage form. The term "non-fixed combination" refers to the simultaneous administration of at least one compound described herein and at least one synergistic agent to a patient as separate entities, either in combination or sequentially at variable intervals. This also applies to cocktail therapies, e.g., administration of three or more active ingredients.

[0088] In the present invention, unless otherwise specified, "tumor" includes but is not limited to leukemia, gastrointestinal stromal tumor, histiocytic lymphoma, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, squamous cell lung cancer, lung adenocarcinoma, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, cervical cancer, ovarian cancer, intestinal cancer, rhinitis cancer, brain cancer, bone cancer, esophageal cancer, melanoma, kidney cancer, oral cancer, brain cancer and central nervous system cancer.

[0089] As used herein, unless otherwise indicated, "treating" or "treating" means reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

[0090] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 shows the curve of tumor volume change in the human breast cancer 436 cell NOD-SCID mouse subcutaneous tumor model after administration of compounds 47 and 64.

[0092] Figure 2 is a photo of the changes in tumor volume in a NOD-SCID mouse subcutaneous tumor model of human breast cancer 436 cells after administration of compounds 47 and 64.

[0093] Figure 3 shows the changes in tumor volume in the breast cancer MDA-MB-436 nude mouse model after administration of compound 11.

[0094] FIG4 is a graph showing the effect of compound 11 administration on the intracranial fluorescence intensity of the human breast cancer MDA-MB-436 mouse model.

[0095] FIG5 is a graph showing the effect of compound 11 on the survival of mice bearing human breast cancer MDA-MB-436.

[0096] FIG6 is a graph showing the effect of compound 11 on the body weight of human breast cancer MDA-MB-436 mice after administration. DETAILED DESCRIPTION

[0097] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area or the product specifications shall be followed.

[0098] The reagents and raw materials used in the examples of the present invention are all commercially available.

[0099] Table 1 Abbreviations and their meanings in the present invention

[0100] The structures of the compounds of the present invention were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 nuclear magnetic spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). Chemical shifts were measured at 10 -6 The units are given in ppm.

[0101] MS was measured using an Agilent SQD (ESI) mass spectrometer (manufacturer: Agilent, signal: 6110).

[0102] HPLC analysis was performed using an Agilent 1200DAD high pressure liquid chromatograph (Sunfirc C18, 150×4.6 mm, 5 μm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18, 150×4.5 mm, 5 μm column).

[0103] The thin layer chromatography silica gel plate used was Qingdao Ocean GF254 silica gel plate. The specification of the silica gel plate used in thin layer chromatography (TLC) was 0.15mm-0.2mm. The specification of the thin layer chromatography separation and purification product was 0.4mm-0.5mm silica gel plate.

[0104] Column chromatography generally uses Qingdao Ocean 100-200, 200-300 mesh silica gel as the carrier.

[0105] Unless otherwise specified, all reactions in the following examples were conducted under an argon or nitrogen atmosphere. Argon or nitrogen atmosphere refers to the reaction flask being connected to an approximately 1 L argon or nitrogen balloon. Hydrogen atmosphere refers to the reaction flask being connected to an approximately 1 L hydrogen balloon. Hydrogenation reactions were typically performed by evacuating the flask and then filling it with hydrogen, repeating this process three times.

[0106] Intermediate int-1: 7-bromo-6-methoxy-1,5-naphthyridine-3-carboxylic acid ethyl ester

[0107] Step 1: Add int-1a (500g, 1.99mol) and 2.5L of methanol under nitrogen protection and cool to 0-5°C. Add a solution of sodium methoxide (118g) in methanol (1.0L) dropwise at 0-5°C. After complete addition, warm to room temperature and stir for 1 hour. Add water (2.0L) to the reaction system, stir for 30min, and then concentrate under reduced pressure at 40°C. Concentrate until no liquid is discharged, then add ethyl acetate (4.0L), stir and separate, add ethyl acetate to the aqueous layer, extract, and separate the liquids. Combine the organic layers, add saturated brine to wash, separate the liquids, and concentrate the organic layer under reduced pressure at 40°C to obtain int-1b (480g) as a white solid.

[0108] Step 2: Weigh compound int-1b (475 g, 1.93 mol) into DMF (2.85 L), then add DMF-DMA (2.85 L) dropwise. After addition, heat to 100°C and stir for 2 h. After the reaction is complete, cool to 70-80°C and concentrate under reduced pressure until no liquid is released. Add to water and stir to precipitate. Cool to 20-30°C, stir for 1 h, and filter. Dry the filter cake in a vacuum oven at 70°C to constant weight to obtain a red solid, int-1c (612 g, 95.3% yield).

[0109] Step 3: Add compound int-1c (500g, 1.91mol) to THF (2.56L) and stir to dissolve. Add sodium periodate (805g, 3.72mol) in water (2.56L) dropwise to the reaction system. Stir at room temperature for 2-4h. After the reaction is completed, add ethyl acetate (4.0L) and water (4.0L) to the reaction system, stir and separate, and extract the aqueous layer twice with ethyl acetate (2.0L). After the organic layers are combined, saturated sodium thiosulfate solution and saturated brine are added in turn for washing. The organic layer is concentrated under reduced pressure at 40-45°C until there is no fraction to obtain 500g of oily product int-1d, which is used directly in the next step.

[0110] Step 4: Compounds int-1d (512 g, 1.69 mol) and int-1e (1457.0 g, 7.61 mol) were added to anhydrous ethanol (7.5 L) and stirred to dissolve. SnCl2 (1815.0 g, 9.57 mol) was added to the reaction system in batches at room temperature. After the addition, the temperature was raised to reflux and stirred for 1-2 hours. The reaction system was cooled to 45-50°C and concentrated under reduced pressure until no fractions were present. Ethyl acetate was added to the system and stirred to dissolve. The pH was then adjusted to 7-8 with saturated sodium bicarbonate. During this process, gas was released violently, and a large amount of solid was precipitated. The reaction solution was centrifuged, and the filtrate was collected and allowed to stand for stratification. The organic layer was concentrated under reduced pressure at 40-45°C until no fractions were present. 200-300 mesh silica gel was added and purified by column chromatography to obtain int-1 (230 g, 36.5% yield) as a flocculent solid.

[0111] Intermediate int-2: N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0112] Step 1: Compound int-2a (1 g, 4.6 mmol), int-2b (1.7 g, 5.5 mmol), Pd(dppf)Cl2 (0.3 g, 0.46 mmol), and potassium carbonate (1.6 g, 11.5 mmol) were added to a mixed solvent of 7 ml of dioxane, 3 ml of anhydrous ethanol, and 4 ml of water. The nitrogen atmosphere was then replaced three times and the mixture was reacted at 90°C under nitrogen for 2 h. After TLC analysis, the reaction mixture was cooled to room temperature, 30 ml of dichloromethane and 20 ml of water were added, and the layers were separated in a separatory funnel. The aqueous phase was extracted twice with dichloromethane, and the combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. The resulting crude product was purified by column chromatography to yield compound int-2c (1 g, white solid).

[0113] Step 2: Int-2c (1 g, 3 mmol), aqueous methylamine (5 g, 161.3 mmol), and anhydrous methanol (20 ml) were added to a 100 ml reaction flask and stirred overnight at room temperature. After completion of the reaction as monitored by TLC, the reaction solution was concentrated under reduced pressure to dryness to afford int-2d (0.8 g, white solid).

[0114] Step 3: Add compound int-2d (0.5 g, 1.5 mmol) to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L hydrochloric acid and dioxane solution. Stir at room temperature for 0.5-1 h. After the reaction is complete as monitored by TLC, the reaction solution is concentrated under reduced pressure to dryness to obtain compound int-2 (0.5 g, white solid).

[0115] The preparation of intermediates int-3 and int-4 refers to intermediate int-2.

[0116] Table 2 Intermediates int-3 to int-4

[0117] Intermediate int-5: N-cyclopropyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0118] Step 1: Weigh int-2c (1.4 g, 4.5 mmol), add 20 mL of MeOH to dissolve, add 5 mL of water, add lithium hydroxide (570 mg, 13.5 mmol), react at room temperature for 12 h, monitor the reaction by TLC, and after the reaction is complete, add 2 M HCl to adjust the pH to 6. Add EA (3 x 25 mL) for extraction, combine the organic phases, dry over anhydrous sodium sulfate, and vacuum dry to obtain the product int-5a (460 mg, light yellow solid).

[0119] Step 2: Weigh int-5a (180 mg, 0.6 mmol), EDCI (144 mg, 0.75 mmol), HOBT (100 mg, 0.75 mmol) 2 mL DMF, N-methylmorpholine (290 mg, 3.2 mmol), cyclopropylamine (34 mg, 0.6 mmol), react at room temperature for 12 h, monitor by TLC, dilute with water after the raw materials are completely consumed, extract with EA, wash the organic phase with water 5 times, dry over anhydrous sodium sulfate, and spin dry to give the crude product int-5b (180 mg, yellow oily liquid).

[0120] Step 3: Dissolve the crude product of int-5b (171 mg, 0.5 mmol) in methanol (5 mL), then add 4M HCl 1,4-dioxane (0.9 mL) and react at room temperature for 12 h. Monitor the reaction by TLC. After the reaction is complete, add potassium carbonate and stir for 30 minutes. Remove the potassium carbonate by filtration to obtain the crude product of compound int-5 (180 mg, yellow-brown solid). MS / ESI [M+H] + =244.1.

[0121] The preparation of intermediates int-6 to int-8 refers to intermediate int-5.

[0122] Table 3 Intermediates int-6 to int-8

[0123] Intermediate int-9: N-methyl-5-(piperazin-1-yl)pyridineamide

[0124] Step 1: Compound int-2a (5.66 g, 26.21 mmol, 1.05 eq), compound int-9a (4.65 g, 25.0 mmol, 1.00 eq), Cs2CO3 (16.27 g, 50.0 mmol, 2.00 eq), and RuPhos-Pd-G3 (1.04 g, 1.25 mmol, 0.05 eq) were added to 1,4-dioxane (50 mL) and stirred at 120°C overnight under a nitrogen atmosphere. After the reaction was complete, the mixture was cooled to room temperature by LCMS. The reaction solution was diluted with water (100 mL) and then extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with brine (2 x 50 mL), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound int-9b (white solid, 4.5 g). MS / ESI [M+H] + =322.1.

[0125] Step 2: Compound int-9b (1.92 g, 6.0 mmol, 1.0 eq) was added to a solution of methylamine (8 mL, 25-30 wt% aqueous solution) in methanol (7 mL) and stirred at room temperature for 3 h under a nitrogen atmosphere. The reaction was monitored by LCMS. The reaction was quenched with saturated aqueous ammonium chloride solution (30 mL) at room temperature. The resulting mixture was extracted with dichloromethane (3 x 50 mL). The combined organic layer was washed with brine (2 x 50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to obtain compound int-9c (1.65 g) as a light yellow oil. MS / ESI[M+H] + =321.3.

[0126] Step 3: Compound int-9c (482 mg, 1.50 mmol, 1.00 eq) and HCl in 1,4-dioxane (3.7 mL, 15.0 mmol, 10.0 eq, 4.0 M) were stirred at room temperature for 2 hours. After the reaction was completed, the resulting mixture was concentrated under reduced pressure by LCMS monitoring to obtain compound int-9 (392 mg, crude product) as a yellow solid. The crude product was used directly in the next step without further purification. MS / ESI[M+H] + =221.3.

[0127] The preparation of intermediates int-10 and int-11 refers to intermediate int-9.

[0128] Table 4 Intermediates int-10 and int-11

[0129] Intermediate INT12: N-cyclopropyl-5-(piperazin-1-yl)pyridineamide

[0130] Step 1: Weigh int-9b (1.4 g, 4.5 mmol), add 20 mL of MeOH to dissolve, add 5 mL of water, add lithium hydroxide (570 mg, 13.5 mmol), react at room temperature for 12 h, monitor the reaction by TLC, add 2 M HCl after the reaction is complete to adjust the pH to 6, add EA (3 x 25 mL) to extract, combine the organic phases, dry over anhydrous sodium sulfate, and vacuum dry to obtain the product int-12a (845 mg).

[0131] Step 2: Weigh int-12a (180 mg, 0.6 mmol), EDCI (144 mg, 0.75 mmol), HOBT (100 mg, 0.75 mmol), 2 mL DMF, N-methylmorpholine (290 mg, 3.2 mmol), and cyclopropylamine (34 mg, 0.6 mmol). React at room temperature for 12 h. Monitor by TLC. After the starting material is completely consumed, dilute with water and extract with EA. The organic phase is washed five times with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to obtain the crude product int-12b (165 mg). MS / ESI [M+H] + =347.2.

[0132] Step 3: Dissolve the crude product of int-12b (150 mg, 0.4 mmol) in 5 mL of MeOH, then add 0.9 mL of 4 M HCl in dioxane solution. React at room temperature for 12 h. Monitor the reaction by TLC. After completion, add potassium carbonate. Stir for 30 minutes, then filter and remove the potassium carbonate to obtain the crude product of compound int-12 (120 mg, yellow-brown solid). MS / ESI [M+H] + =247.1.

[0133] The preparation method of intermediates int-13 to int-15 refers to intermediate int-12.

[0134] Table 5 Intermediates int-13 to int-15

[0135] Intermediate int-16: N-methyl-5-(((2R,3S)-2-methylazetidin-3-yl)oxy)picolinamide

[0136] Step 1: Compound int-16a (2.0 g, 0.01305 mol) and compound int-16b (3.393 g, 0.0195 mol) were added to tetrahydrofuran (200 mL), the reaction solution was cooled to 0°C, triphenylphosphine (20.54 g, 0.0783 mol) was added, and nitrogen was replaced three times. The mixture was stirred for 1 hour, and DEAD (11.36 g, 0.06525 mol) was added. After the addition was completed, the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the concentrate was subjected to column chromatography (PE:EA=10:1~1:1). The product was collected to obtain a crude compound int-16c, LCMS=341.1.

[0137] Step 2: Add compound int-16c (4.5 g, 0.0146 mol) to methanol (20 mL), add methylamine alcohol solution (20 mL), and react at room temperature for 1 hour. After the reaction, the reaction solution is concentrated under reduced pressure to obtain the crude compound int-16d, LCMS = 340.2.

[0138] Step 3: Add compound int-16d (4.5 g, 0.0146 mol) to dichloromethane (20 mL), add dioxane hydrochloride solution (20 mL), and react at room temperature for 1 hour. After the reaction, the reaction solution is concentrated under reduced pressure, ethyl acetate is added to the concentrate to make a pulp, and the filtered solid is collected to obtain compound int-16 hydrochloride, LCMS = 240.2.

[0139] Intermediate int-17: (6-methoxy-7-((trifluoromethyl)thio)-1,5-naphthyridin-3-yl)methyl methanesulfonate

[0140] Step 1: Dissolve compound int-1 (3.0 g, 9.64 mmol) in anhydrous toluene (30 mL). Add compound int-17a (4.0 g, 18.34 mmol), Pd2(dba)3 (1.0 g, 1.05 mmol), Xantphos (1.1 g, 1.9 mmol), and DIPEA (4.0 mL, 24.24 mmol). After nitrogen displacement three times, stir at 110°C for 12 h. Cool the reaction solution and slowly pour it into 100 mL of saturated ammonium chloride solution for quenching. Extract with 4 x 50 mL of EA, dry with sodium sulfate, and separate by column chromatography (PE / EA = 5 / 1). The resulting compound is dissolved in anhydrous EtOH (30 mL), and NaOEt (1.3 g, 19.11 mmol) is added. Stir at 25°C for 1 h. The reaction solution was slowly poured into HCl (1.0 M, 50 mL) for quenching, extracted with EA (5*30 mL), dried over sodium sulfate, concentrated, and slurried with EA to obtain compound int-17b (2.5 g, crude). LC-MS: ESI [M+H] + =265.2.

[0141] Step 2: Dissolve int-17b (2.5 g, 9.43 mmol) in DCM (30 mL). Add int-17c (2.7 g, 8.2 mmol) at 25°C and stir at 20°C for 1 h. Concentrate and filter through a column (PE / EA = 1 / 5) to afford int-17d (0.61 g, 1.84 mmol, 20%) as a yellow oil. LC-MS: ESI [M+H] + =333.2.

[0142] Step 3: Dissolve compound int-17d (0.61 g, 1.84 mmol) in DCM (20 mL) and slowly add DIBAL-H (1.0 M, 5.0 mL, 5.0 mmol) dropwise at 0°C. Stir at 0°C for 1 h. The reaction mixture is slowly poured into 50 mL of saturated ammonium chloride solution to quench the mixture. Filter the mixture through Celite and extract with 4 x 30 mL of EA. Dry the mixture through sodium sulfate to obtain int-17e (0.45 g, crude) as a yellow oil. LC-MS: ESI [M+H] + =291.1.

[0143] Step 4: Dissolve compound int-17e (0.45 g, 1.55 mmol) in DCM (30 mL). Add TEA (0.7 mL, 7.36 mmol) and MsCl (0.5 mL, 3.6 mmol) dropwise at 0°C and stir at 0°C for 1 h. Slowly pour 30 mL of saturated sodium bicarbonate solution into the reaction mixture to quench. Extract with 30 mL of DCM, dry with sodium sulfate, and filter and concentrate under reduced pressure to obtain compound int-17 (0.6 g, crude). LC-MS: ESI [M+H] + =369.2.

[0144] Intermediate int-18: (6-methoxy-7-(methylthio)-1,5-naphthyridin-3-yl)methyl methanesulfonate

[0145] Step 1: Compound int-1 (5.0 g, 16.07 mmol) was placed in a sealed tube and dissolved in DMSO (50 mL). CuI (3.0 g, 15.7 mmol) and Zn(OAc)2 (6.0 g, 32.78 mmol) were added. After nitrogen displacement three times, the reaction mixture was stirred at 150°C for 15 h. The reaction mixture was cooled and slowly poured into 100 mL of saturated ammonium chloride solution to quench the mixture. The mixture was filtered through celite and extracted with 5 x 50 mL of EA. The mixture was dried over sodium sulfate and separated by column chromatography (PE / EA = 5 / 1) to afford int-18a (0.3 g, 1.08 mmol, 8% yield) as a yellow solid. LC-MS: ESI [M+H] + =279.2.

[0146] Step 2: Dissolve compound int-18a (0.3 g, 1.08 mmol) in DCM (20 mL) and slowly add DIBAL-H (1.0 M, 5.0 mL, 5.0 mmol) dropwise at 0°C. Stir at 0°C for 1 h. The reaction mixture is slowly poured into 50 mL of saturated ammonium chloride solution to quench the mixture. Filter the mixture through Celite and extract with 4 x 30 mL of EA. Dry the mixture with sodium sulfate to obtain compound int-18b (0.3 g, crude) as a yellow oil. LC-MS: ESI [M+H] + =237.2.

[0147] Step 3: Dissolve compound int-18b (0.3 g, 1.26 mmol) in DCM (10 mL). Add TEA (0.5 mL, 4.32 mmol) and MsCl (0.3 mL, 2.62 mmol) dropwise at 0°C. Stir at 0°C for 1 h. Slowly pour 20 mL of saturated sodium bicarbonate solution into the reaction mixture to quench. Extract with 30 mL of DCM, dry with sodium sulfate, and filter and concentrate under reduced pressure to obtain compound int-18 (0.3 g, crude). LC-MS: ESI [M+H]+ =315.2.

[0148] Intermediate int-19: (R)-N-methyl-1,2,3,4,4a,5-hexahydropyrazino[1,2-d]pyrido[2,3-b][1,4]oxazine-8-carboxamide

[0149] Step 1: Dissolve compound int-19a (4.2 g, 19.4 mmol) in dichloromethane (100 mL), add triethylamine (117 mg, 38.9 mmol), tert-butyldimethylsilyl chloride (2.17 g, 14.39 mmol), and 4-dimethylaminopyridine (3.94 g, 0.94 mmol), and stir for 14 hours. The reaction solution is concentrated under reduced pressure, and the residue is purified by silica gel column chromatography to obtain compound int-19b (5.2 g). LC-MS: ESI [M+H] + =331.2.

[0150] Step 2: Dissolve compound int-19b (1.3 g, 5.55 mmol) and compound int-3a (2.02 g, 6.1 mmol) in 1,4-dioxane (15 mL). Add (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II) methanesulfonate (464 mg, 554.7 μmol) and cesium carbonate (3.6 g, 11.1 mmol). Under a nitrogen atmosphere, react at 110°C for 14 hours. The reaction solution is concentrated under reduced pressure, and the residue is purified by silica gel column chromatography to obtain compound int-19c (1 g, yield: 37.2%). LC-MS: ESI [M+H] + =484.2.

[0151] Step 3: Dissolve compound int-19c (1 g, 2.06 mmol) in tetrahydrofuran (6 mL), add tetrabutylammonium fluoride in tetrahydrofuran (1 M / L, 6 mL), and stir for 2 hours. The reaction solution is concentrated under reduced pressure to obtain the crude title compound int-19d (722 mg, yield: 99%). The product is used directly in the next step without purification. LC-MS: ESI [M+H] + =350.2.

[0152] Step 4: Dissolve the crude product, int-19d (722 mg, 2.06 mmol), in methylamine (8 mL, 25-30 wt% aqueous solution) in methanol (7 mL) and stir for 2 hours. The reaction solution was concentrated under reduced pressure to afford the crude title compound, int-19e (700 mg, 97% yield), which was used directly in the next step without purification. LC-MS: ESI [M+H]+ =349.2.

[0153] Step 5: Dissolve the crude product of compound int-19e (140 mg, 0.4 mmol) in 5 mL of MeOH, then add 0.9 mL of 4 M HCl in dioxane solution. React at room temperature for 12 h. Monitor the reaction by TLC. After completion, add potassium carbonate. Stir for 30 minutes, then filter and remove the potassium carbonate to obtain the crude product of compound int-19 (120 mg). MS / ESI [M+H] + =249.1.

[0154] Intermediate int-20: (R)-N-methyl-3,4,4a,5-tetrahydro-2H-pyrano[2,3-b:5,4-c']bipyridine-8-carboxamide

[0155] The first step: Weigh the compound int-20a (0.25 g, 0.83 mmol), add 20 ml of tetrahydrofuran, replace nitrogen, cool to -78 ° C, add lithium diisopropylamide (0.46 mL, 0.91 mmol) dropwise, stir for 30 minutes, add N-phenylbis(trifluoromethanesulfonyl)imide (0.36 g, 1.0 mmol) dissolved in 10 mL of tetrahydrofuran dropwise, warm to room temperature and react for 2 hours. After TLC detection, the reaction is complete, saturated ammonium chloride aqueous solution is added to quench, and the mixture is extracted three times with ethyl acetate. After the organic phases are combined, they are dried over anhydrous sodium sulfate, filtered, and spin-dried. Purification by column chromatography gives compound int-20b (0.25 g, colorless liquid).

[0156] Step 2: Compound int-20b (0.25 g, 0.6 mmol), compound int-20c (0.14 g, 0.66 mmol), Pd(dppf)Cl2 (0.04 g, 0.06 mmol) and potassium carbonate (0.16 g, 1.2 mmol) were added to a mixed solvent of 20 ml of dioxane and 2 ml of water, and then the nitrogen was replaced and the reaction was carried out at 80 ° C for 3 hours under nitrogen protection. After TLC detection, the reaction was cooled to room temperature, concentrated by rotary evaporation, and extracted three times with 30 ml of ethyl acetate and 20 ml of water. The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried. The crude product was purified by column chromatography, solidified by beating with petroleum ether, filtered, and dried to obtain compound int-20d (180 mg, light yellow liquid); LC-MS: ESI [M+H] + =434.6.

[0157] Step 3: Int-20d (180 mg, 0.4 mmol), methylamine aqueous solution (0.12 mL, 1.6 mmol), and anhydrous methanol (15 mL) were added to a 25 mL reaction flask and stirred at room temperature for 3 hours. After the reaction was complete as monitored by LC-MS, the reaction solution was concentrated under reduced pressure, slurried with petroleum ether, and filtered and dried to obtain compound int-20e (160 mg, light yellow solid); LC-MS: ESI [M+H] + =439.6.

[0158] Step 4: Compound int-20e (160 mg, 0.38 mmol) was added to 10 ml of anhydrous methanol, followed by the addition of 0.4 ml of a 4 mol / L hydrochloric acid solution in dioxane. The mixture was stirred at room temperature for 2 hours. After the reaction was complete as monitored by TLC, dioxane was added to precipitate a solid, which was filtered and dried to obtain compound int-20 (80 mg, yellow solid); LC-MS: ESI [M+H] + =246.3.

[0159] Intermediate int-21: (6-methoxy-7-((methyl-d3)thio)-1,5-naphthyridin-3-yl)methyl methanesulfonate

[0160] Preparation of (6-methoxy-7-((methyl-d3)thio)-1,5-naphthyridin-3-yl)methyl methanesulfonate reference intermediate int-18. LC-MS: ESI [M+H] + =318.3.

[0161] Intermediate int-22: (7-((difluoromethyl)thio)-6-methoxy-1,5-naphthyridin-3-yl)methyl methanesulfonate

[0162] Step 1: Sodium chlorodifluoroacetate int-22a (2.88 g, 18.9 mmol, 2 eq) and K2CO3 (1.95 g, 14.1 mmol, 1.5 eq) were added to a 100 mL two-necked flask and dried under high vacuum for 1 hour. DMF (99%, 30 mL) was added at room temperature, and int-17b (2.5 g, 9.43 mmol) was added portionwise. The reaction mixture was stirred at 95°C for 15 minutes (exothermic reaction) and cooled to room temperature. Water (130 mL) and ethyl acetate (130 mL) were added. The layers were separated and the organic layer was washed with water (4 x 130 mL). Drying was performed over MgSO4 and concentration was performed under vacuum. Purification by column chromatography (PE / EA = 1 / 5) gave int-22b (1.2 g) as a yellow oil. LC-MS: ESI [M+H] + =315.1.

[0163] Step 2: Dissolve compound int-22b (0.58 g, 1.84 mmol) in DCM (20 mL) and slowly add DIBAL-H (1.0 M, 5.0 mL, 5.0 mmol) dropwise at 0°C. Stir at 0°C for 1 h. The reaction mixture is slowly poured into 50 mL of saturated ammonium chloride solution to quench the mixture. Filter the mixture through Celite and extract with 4 x 30 mL of EA. Dry the mixture with sodium sulfate to obtain compound int-22c (0.42 g, crude) as a yellow oil. LC-MS: ESI [M+H] + =273.1.

[0164] Step 3: Dissolve compound int-12c (0.42 g, 1.55 mmol) in DCM (30 mL). Add TEA (0.7 mL, 7.36 mmol) and MsCl (0.5 mL, 3.6 mmol) dropwise at 0°C and stir at 0°C for 1 h. Slowly pour 30 mL of saturated sodium bicarbonate solution into the reaction mixture to quench. Extract with 30 mL of DCM, dry with sodium sulfate, and filter and concentrate under reduced pressure to obtain compound int-22 (0.5 g, crude). LC-MS: ESI [M+H] + =351.2.

[0165] Intermediate int-23: 6-fluoro-N-methyl-5-(piperidin-4-yl)picolinamide

[0166] Compound int-3 (0.2 g, 0.84 mmol) was dissolved in DCM (20 mL) and Pd / C (10%, 0.1 g) was added. After hydrogen replacement three times, the mixture was stirred at 25°C under a hydrogen atmosphere for 12 h. The reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to remove the solvent to obtain compound int-23 (0.2 g, crude). LC-MS: ESI [M+H] + =238.2.

[0167] Intermediate int-24: 6-cyano-N-methyl-5-(piperazin-1-yl)picolinamide

[0168] Step 1: Dissolve int-24a (0.85 g, 4.564 mmol) and int-9a (1.0 g, 4.149 mmol) in dioxane (40 mL). Add RuPphos-Pd-G3 (0.35 g, 0.415 mmol) and Cs2CO3 (5.41 g, 16.595 mmol). N2 was purged three times and stirred at 100°C for 15 h. The reaction mixture was poured into saturated sodium bicarbonate (30 mL) and extracted with EA (3 x 30 mL). Dry with sodium sulfate, concentrate under reduced pressure, and purify by column chromatography (PE / EA = 3 / 1) to obtain int-24b (0.7 g, 2.021 mmol) as a white solid. LC-MS: ESI [M+H]+ = 347.2.

[0169] Step 2: Dissolve compound int-24b (0.7 g, 2.021 mmol) in MeOH (10 mL) and add methylamine methanol solution (10 mL). Stir at 25°C for 1 h. Concentrate the reaction mixture under reduced pressure to obtain compound int-24c (0.7 g, 2.03 mmol, crude). LC-MS: ESI [M+H]+ = 346.2.

[0170] Step 3: Dissolve compound int-24c (0.7 g, 2.027 mmol) in HCl / dioxane (10 mL, 4.0 M) and stir at 25°C for 1 h. The reaction solution was concentrated under reduced pressure to afford compound int-24 (0.6 g, 2.130 mmol, crude). LC-MS: ESI [M+H]+ = 246.2.

[0171] Intermediate int-25: 6-fluoro-N-(1-methyl-1H-pyrazol-4-yl)-5-(piperidin-4-yl)picolinamide

[0172] Step 1: Dissolve compound int-25a (1 g, 3.102 mmol) in DCM (30 mL), add compound int-25b (0.36 g, 3.723 mmol), HATU (1.42 g, 3.723 mmol), and DIPEA (1.20 g, 9.307 mmol), and stir at 25°C for 1 h. The reaction solution was poured into saturated sodium bicarbonate (30 mL), extracted with DCM (3*30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE / EA=1 / 1) to obtain compound int-25c (1.2 g, 2.989 mmol, 96.36%) as a light yellow oily liquid. LC-MS: ESI [M+H] + =402.2.

[0173] Step 2: Dissolve compound int-25c (1.2 g, 2.989 mmol) in MeOH (20 mL), add 10% Pd / C (1.2 g, 11.276 mmol), replace with H2 three times, and stir at 25°C for 1 h. Filter through silica gel under reduced pressure and dry under reduced pressure to obtain compound int-25d (1.1 g, 2.726 mmol, 91.21%) as a light yellow solid. LC-MS: ESI [M+H] + =404.2.

[0174] Step 3: Dissolve compound int-25d (1.1 g, 2.726 mmol) in HCl / MeOH (10 mL, 4.0 M), stir at 25°C for 0.5 h, and concentrate under reduced pressure to obtain compound int-25 (1 g, 2.943 mmol, 98.95%) as a white solid. LC-MS: ESI [M+H] + =304.2.

[0175] Intermediate int-26: 6-Fluoro-N-(1-methyl-1H-pyrazol-4-yl)-5-(piperazin-1-yl)picolinamide

[0176] Step 1: Dissolve compound int-26a (1 g, 2.9 mmol) in THF (10 mL) and H₂O (10 mL). Add LiOH (0.62 g, 14.7 mmol) and stir at 0°C for 2 h. Pour the reaction mixture into saturated aqueous ammonium chloride (30 mL). Extract with DCM (3 x 30 mL), dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain compound int-26b (1 g, 3.1 mmol, crude) as a white solid. LC-MS: ESI [M+H] + =326.2.

[0177] Step 2: Dissolve compound int-26b (1 g, 3.1 mmol) in DCM (30 mL), add compound int-25b (0.36 g, 3.723 mmol), HATU (1.42 g, 3.7 mmol), and DIPEA (1.20 g, 9.3 mmol), and stir at 25°C for 1 h. The reaction solution was poured into saturated sodium bicarbonate (30 mL) and extracted with DCM (3*30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE / EA=1 / 1) to obtain compound int-26c (1.1 g, 2.7 mmol, 88.48%) as a light yellow oily liquid. LC-MS: ESI[M+H] + =405.2.

[0178] Step 3: Dissolve compound int-26c (1.1 g, 2.7 mmol) in HCl / MeOH (10 mL, 4.0 M) and stir at 25°C for 0.5 h. Concentrate under reduced pressure to obtain compound int-26 (0.8 g, 2.6 mmol, 96.65%) as a white solid. LC-MS: ESI [M+H] + =305.2.

[0179] Intermediate int-27: N-cyclopropyl-6-fluoro-5-(piperidin-4-yl)picolinamide

[0180] Step 1: Dissolve compound int-25a (1 g, 3.102 mmol) in MeOH (15 mL), add 10% Pd / C (1 g, 9.397 mmol), replace with H2 three times, and stir at 25°C for 1 h. Filter through a pad of silica gel, and dry the filtrate under reduced pressure to obtain compound int-27a (1 g, 3.083 mmol, 99.38%) as a pale yellow solid. LC-MS: ESI [M+H] + =325.2.

[0181] Step 2: Dissolve compound int-27a (1 g, 3.083 mmol) in DCM (30 mL), add cyclopropylamine hydrochloride (0.58 g, 6.166 mmol), DIPEA (1.20 g, 9.249 mmol), and HATU (1.76 g, 4.625 mmol), and stir at 25°C for 1 h. The reaction solution was poured into saturated sodium bicarbonate (30 mL), extracted with DCM (3*30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE / EA=1 / 1) to obtain compound int-27b (1 g, 2.752 mmol, 89.25%) as a light yellow solid. LC-MS: ESI [M+H] + =364.2.

[0182] Step 3: Dissolve compound int-27b (1.0 g, 2.752 mmol) in dioxane hydrochloride solution (10 mL, 4.0 M) and stir at 25°C for 1 h. Concentrate under reduced pressure to obtain compound int-27 (0.7 g, 2.658 mmol, 96.61%) as a white solid. LC-MS: ESI [M+H] + =264.3.

[0183] Intermediate int-28: N,6-dimethyl-5-(piperidin-4-yl)picolinamide

[0184] The preparation method of N,6-dimethyl-5-(piperidin-4-yl)picolinamide refers to the intermediate int-23. LC-MS:ESI[M+H] + =234.2.

[0185] Intermediate int-29: 2-chloro-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0186] The preparation method of 2-chloro-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide refers to intermediate int-2. LC-MS:ESI[M+H] + =252.7.

[0187] Intermediate int-30: 6-chloro-N-methyl-5-(piperidin-4-yl)picolinamide

[0188] The preparation method of 6-chloro-N-methyl-5-(piperidin-4-yl)picolinamide refers to intermediate int-28. LC-MS:ESI[M+H] + =254.2.

[0189] Intermediate int-31: 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide

[0190] The preparation method of 6-chloro-N-methyl-5-(piperazin-1-yl)picolinamide refers to the intermediate int-9. LC-MS:ESI[M+H] + =255.2.

[0191] Intermediate int-32: 1-(5-methoxy-3-methylpyridin-2-yl)piperazine

[0192] Step 1: Dissolve compound int-31a (2 g, 9.899 mmol) and compound int-9a (2.21 g, 11.878 mmol) in 1,4-dioxane (50 mL). Add Ruphos-Pd-G3 (0.83 g, 0.990 mmol) and Cs2CO3 (9.68 g, 29.696 mmol). N2 was replaced three times and stirred at 100°C for 12 h. The reaction mixture was poured into saturated sodium bicarbonate (30 mL) and extracted with EA (3 x 30 mL). Dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography (PE / EA = 3 / 1) to obtain compound int-31b (1.7 g, 5.530 mmol, 55.87%) as a light yellow liquid. LC-MS: ESI [M+H]+ =308.2.

[0193] Step 2: Dissolve compound int-31b (1.7 g, 5.530 mmol) in methanolic hydrochloric acid (20 mL) and stir at 25°C for 1 h. Concentrate under reduced pressure to obtain compound int-31 (1.1 g, 5.307 mmol, 95.96%) as a white solid. LC-MS: ESI [M+H] + =208.2.

[0194] Intermediate int-32: Preparation of (R)-N-methyl-1,2,3,4,4a,5-hexahydro-7H-pyrazino[2,1-c]pyrido[3,2-e][1,4]oxazolidine-9-carboxamide hydrochloride

[0195] Step 1: (R)-tert-Butyl 3-(hydroxymethyl)piperazine-1-carboxylate (5 g, 23.118 mmol) was added to DMF (50 mL), followed by p-methoxybenzyl chloride (3.62 g, 23.118 mmol) and potassium carbonate (9.58 g, 69.355 mmol). The mixture was heated to 60°C for 24 h. Upon completion of the reaction, water was added to quench the reaction, followed by extraction with ethyl acetate. The organic phase was washed three times with water and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 10:1 to 5:1) to afford compound int-32b (6 g, yield: 77.14%). LC-MS: ESI [M+H] + =337.4. 1 H NMR(400MHz,Chloroform-d)δ7.14(d,J=8.3Hz,2H),6.79(d,J=8.3Hz,2H),3.88(d,J=13.2Hz,1H),3.81–3.76(m,1H),3.72(s,3H), 3.60(m,1H),3.51(m,2H),3.28(d,J=13.1Hz,2H),3.07(s,1H),2.69(d,J=12.3Hz,1H),2.56–2.42(m,1H),2.18(m,1H),1.38(s,9H).

[0196] Step 2: Compound int-32b (6 g, 17.834 mmol) and 6-bromo-2-(bromomethyl)-3-fluoropyridine (4.80 g, 17.834 mmol) were added to DMF (100 mL), cooled to 0°C, and sodium hydroxide (1.07 g, 26.751 mmol) was added. The reaction was allowed to react at room temperature for 12 h. After the reaction was complete, water was added to quench the reaction, and ethyl acetate was added for extraction. The organic phase was washed with water three times and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 5:1 to 1:1) to obtain compound int-32d (4.2 g). LC-MS: ESI [M+H] + =525.4. 1 H NMR(400MHz,Chloroform-d)δ7.37-7.34(m,1H),7.23-7.19(m,1H),7.16–7.10(m,2H),6.79–6.73(m,2H),4.66–4.51(m,2H),3.84(s,1H), 3.72(s,3H),3.59-3.55(m,2H),3.43(d,J=13.1Hz,1H),3.29(d,J=13.3Hz,1H),3.11(s,2H),2.61-2.53(m,2H),2.09(s,1H),1.37(s,9H).

[0197] Step 3: Compound int-32d (4.2 g, 8.009 mmol), PdCl2(PPh3)2 (1.12 g, 1.602 mmol), and DIEA (3.11 g, 24.026 mmol) were added to DMF (30 mL) and ethanol (30 mL). Under carbon monoxide protection, the temperature was raised to 90°C for 12 h. The reaction solution was added to water and extracted with ethyl acetate. The organic phase was washed with water three times and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 5:1 to 2:1) to obtain compound int-32e (2 g, yield: 48.25%). LC-MS: ESI [M+H] + =518.2.

[0198] Step 4: Compound int-32e (500 mg, 0.966 mmol) was added to acetonitrile (10 mL) and water (10 mL), cooled to 0°C, and cerium ammonium nitrate (2.647 g, 4.83 mmol) was added. The mixture was stirred at room temperature for 12 hours. After the reaction, the reaction solution was added to a saturated aqueous sodium bicarbonate solution to adjust the pH to 8-9. Ethyl acetate was added for extraction, and the organic phase was washed with water three times. The organic phase was concentrated under reduced pressure. The crude product was purified by column chromatography (dichloromethane:methanol=8:1) to obtain crude compound int-32f (100 mg, yield: 26.05%). LC-MS: ESI[M+H] + =398.2.

[0199] Step 5: Compound int-32f (85 mg, 0.214 mmol) and DIEA (82.93 mg, 0.642 mmol) were added to NMP (3 mL), heated to 180°C, and stirred for 6 hours. After the reaction, the reaction solution was added to water and extracted with ethyl acetate. The organic phase was washed with water three times and concentrated under reduced pressure to obtain compound int-32g (80 mg, yield: 99.11%). LC-MS: ESI [M+H] + =378.2.

[0200] Step 6: Add compound int-32g (80 mg, 0.212 mmol) and methylamine alcohol solution (2 mL) to methanol (2 mL) and stir at room temperature for 1 hour. After the reaction is complete, the reaction solution is concentrated under reduced pressure to obtain crude compound int-32h (80 mg, yield: 100%). LC-MS: ESI [M+H] + =363.4.

[0201] Step 7: Compound int-32h (80 mg, 0.221 mmol) was added to dioxane (2 mL), and dioxane hydrochloride (1 mL) was added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain compound int-32 (hydrochloride 80 mg). LC-MS: ESI [M+H] + =263.2.

[0202] Intermediate int-33: 5-(azetidin-3-yl)-N-methyl-5,6-dihydropyrrolo[3,4-c]pyrazole-2(4H)-carboxamide

[0203] Step 1: Compound int-33a (4.0 g, 19.116 mmol) was added to dichloromethane (80 mL), and sodium carbonate (5.07 g, 47.790 mmol) and 4-nitrophenyl chloroacetate (4.24 g, 21.027 mmol) were added. After the addition was complete, the reaction was allowed to react at room temperature for 12 h. After the reaction was completed, water was added to quench the reaction, and dichloromethane was added to extract. The organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated to obtain compound int-33b (yield: 83.79%). LC-MS: ESI [M+H] + =375.2.

[0204] Step 2: Compound int-33b (6.0 g, 16.028 mmol) was added to dichloromethane (100 mL), and triethylamine (4.87 g, 48.083 mmol) and methylamine hydrochloride (1.30 g, 19.233 mmol) were added. After the addition was complete, the reaction was allowed to react at room temperature for 12 h. After the reaction was completed, water was added to quench the reaction, and dichloromethane was added to extract. The organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated to dryness and the concentrate was subjected to column chromatography (PE:EA=5:1 to 1:1) to collect the product to obtain compound int-33c (yield: 46.86%). LC-MS: ESI[M+H] + =267.2.

[0205] Step 3: Compound int-33c (2.0 g, 7.51 mmol) was added to dichloromethane (20 mL), and trifluoroacetic acid (5 mL) was added. The mixture was reacted at room temperature for 1 h. After the reaction was completed, the mixture was concentrated under reduced pressure and the product was collected to obtain compound int-33d trifluoroacetate. LC-MS: ESI [M+H] + =167.20.

[0206] Step 4: Compound int-33d trifluoroacetate (2.3 g, 8.208 mmol) was added to dichloromethane (20 mL), triethylamine was added to adjust the pH to 6-7, tert-butyl 3-oxyazetidine-1-carboxylate (2.81 g, 16.416 mmol) was added, and the reaction was allowed to proceed at room temperature for 1 h. Sodium triacetoxyborohydride (3.48 g, 16.416 mmol) was added. After the reaction was completed, water was added to quench the reaction, and dichloromethane was added for extraction. The organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected. The filtrate was concentrated to dryness, and the concentrate was subjected to column chromatography (PE; EA = 2:1 to 1:1) to collect the product to obtain compound int-33e (yield: 75.82%), LC-MS: ESI [M+H] + =322.2.

[0207] Step 5: Compound int-33e (2.0 g, 6.223 mmol) was added to dichloromethane (20 mL), and trifluoroacetic acid (5 mL) was added. The mixture was reacted at room temperature for 1 h. After the reaction, the mixture was concentrated under reduced pressure and the product was collected to obtain compound int-33 trifluoroacetate. LC-MS: ESI [M+H] + =222.1.

[0208] Intermediate int-34: 5-(azetidin-3-ylethynyl)-6-fluoro-N-methylpicolinamide

[0209] Step 1: Add compound int-3a (537.47 mg, 2.306 mmol), compound int-34a (418 mg, 2.306 mmol), CuI (87.85 mg, 0.461 mmol), bistriphenylphosphine palladium dichloride (161.88 mg, 0.231 mmol), TEA (0.962 mL, 6.919 mmol), DMF (10 mL) to the bottle, replace with N2, warm to 80 ° C and stir overnight, pour the reaction solution into ice water to quench, extract with EA, dry the organic phase, and spin dry. The residue was purified by column chromatography with EA / PE = 0-25% to obtain the product int-34b (357 mg, color solid), LC-MS: [M+H] + = 335.2.

[0210] Step 2: Add compound int-34b (314 mg, 0.939 mmol) to the bottle, dissolve it in DCM (10 mL), cool to 0-10°C, add methylamine methanol solution (0.8 mL, 0.120 mmol) dropwise, stir for 1.5 h, slowly return to room temperature, continue stirring for 3 h, control the temperature at 10-20°C, and concentrate under reduced pressure. The residue is purified by column chromatography with EA / PE = 0-26% to obtain the product int-34c (110 mg, oily liquid), LC-MS: [M+H] + = 334.2.

[0211] Step 3: Add compound int-34c (86 mg, 0.258 mmol) to the bottle, dissolve in DCM (10 mL), add trifluoroacetic acid (2 mL, 26.118 mmol), stir at room temperature for 5 h, and spin dry to obtain the crude trifluoroacetate of compound int-34 (130 mg). It was used directly in the next step without further purification. LC-MS: [M+H]+=234.1.

[0212] Intermediate int-35: (R)-N-cyclopropyl-1,2,3,4,4a,5-hexahydro-7H-pyrazino[2,1-c]pyrido[3,2-e][1,4]oxazolidine-9-carboxamide

[0213] Step 1: Compound int-32g (210 mg, 0.578 mmol) was added to methanol (2.00 mL), tetrahydrofuran (2 mL), and water (2 mL). Lithium hydroxide monohydrate (121.35 mg, 2.889 mmol) was added and allowed to react at room temperature for 2 h. After completion of the reaction, the reaction solution was adjusted to pH 3-4 with 1N hydrochloric acid, extracted with ethyl acetate, and concentrated to dryness to obtain compound int-35a (160 mg, yield: 79.25%). LC-MS: [M+H] + =350.1.

[0214] Step 2: Compound int-35a (130 mg, 0.372 mmol), cyclopropylamine (34.81 mg, 0.372 mmol), and DIEA (144.27 mg, 1.116 mmol) were added to DMF (3 mL), and HATU (212.22 mg, 0.558 mmol) was added. The mixture was reacted at room temperature for 1 h. The reaction solution was added to water, extracted with EA, and concentrated to remove the solvent to obtain compound int-35b (120 mg, yield: 83.02%). LC-MS: [M+H] + =389.2.

[0215] Step 3: Compound int-35b (120 mg, 0.309 mmol) was added to dichloromethane (3 mL), followed by a dioxane hydrochloride solution (3 mL), and the mixture was allowed to react at room temperature for 1 h. The reaction mixture was concentrated to dryness under reduced pressure to obtain the crude compound int-35 hydrochloride (120 mg). LC-MS: [M+H] + =289.2.

[0216] Intermediate int-36: N-methyl-5-((2S,3R)-2-methylazetidin-3-yl)-5,6-dihydropyrrolo[3,4-c]pyrazole-2(4H)-carboxamide

[0217] Step 1: Add compound int-33d (1.1 g, 2.790 mmol) to the reaction flask, dissolve in DCM (25 mL), add triethylamine (1.801 mL, 12.958 mmol), stir for 10 min, add compound int-36a (0.4 g, 2.160 mmol), stir for 10 min, add sodium triacetoxyborohydride (1.6 g, 7.559 mmol), stir at room temperature for 5 h, pour the reaction solution into ice water, separate the organic phase, extract the aqueous phase with DCM, combine the organic phases, dry, spin dry, and concentrate under reduced pressure. The residue is purified by EA / PE = 0-95% column chromatography to obtain an oily compound int-36b (315 mg, white solid).

[0218] Step 2: Add compound int-36b (315 mg, 0.94 mmol) to the reaction flask, dissolve in DCM (10 mL), cool to T = 0-10 ° C, add TFA (2 mL, 26.118 mmol), and stir at room temperature for 3 h. LC-MS shows that the reaction is complete. The crude product of compound int-36 (766 mg, containing TFA) is obtained by spin drying. LC-MS: [M+H] + =236.2, used directly in the next step without further purification.

[0219] Intermediate int-37: N-methyl-5-(pyrrolidin-3-ylethynyl)picolinamide

[0220] Step 1: Add compound int-37a (565 mg, 2.894 mmol), compound int-2a (625.10 mg, 2.894 mmol), bistriphenylphosphine palladium dichloride (203.10 mg, 0.289 mmol), TEA (1.207 mL, 8.681 mmol), and CuI (110.22 mg, 0.579 mmol) to a reaction flask. Dissolve in DMF (10 mL). Replace with nitrogen. Heat to 80°C and stir for 20 h. Filter the reaction mixture, spin dry, and purify the filtrate. Purify the residue by column chromatography using 0-40% EA / PE to obtain compound int-37b (529 mg) as a yellow oil. LC-MS: [M+H] + =331.2.

[0221] Step 2: Add compound int-37b (529 mg, 1.601 mmol) to the reaction flask, dissolve in methanol (15 mL), add methylamine (15 mL), stir at room temperature for 5 h, and spin dry the reaction mixture to obtain crude compound int-37c (529 mg) as a light yellow oil. LC-MS: [M+H] + =330.2, used directly in the next step without further purification.

[0222] Step 3: Dissolve compound int-37c (529 mg, 1.606 mmol) in DCM (20 mL) and add TFA (5 mL, 65.296 mmol). Stir at room temperature for 2 h. The reaction solution is then dried to give crude compound int-37 (1.25 g) as a yellow oil (containing TFA). This crude product is used directly in the next step without further purification. LC-MS: [M+H] + =230.2.

[0223] Intermediate int-38: 5-((3-fluoroazetidin-3-yl)ethynyl)-N-methylpicolinamide

[0224] Step 1: Compound int-38a (400 mg, 1.852 mmol), compound int-2a (438.23 mg, 2.222 mmol), bistriphenylphosphine palladium dichloride (64.98 mg, 0.093 mmol), cuprous iodide (29.38 mg, 0.093 mmol), TEA (1.029 mL, 7.406 mmol), DMF (5 mL) were added to the reaction flask, the temperature was raised to T = 50 ° C and stirred overnight, water was added to the reaction solution, EA was extracted, the organic phase was dried, and the residue was purified by column chromatography with EA / PE = 0-90% to obtain compound int-38b (529 mg) as a brown oil, LC-MS: [M+H] + =333.1.

[0225] Step 2: Compound int-38b (339 mg, 1.020 mmol) was added to the vial and dissolved in methanol (10 mL). Methylamine (33% methanol solution) was added and stirred at room temperature overnight. The reaction solution was dried and the residue was purified by column chromatography using MeOH / DCM = 0-9% to obtain compound int-38c (321 mg) as a light yellow oil. LC-MS: [M+H] + =332.2.

[0226] Step 3: Compound int-38c (321 mg, 0.969 mmol) was added to the vial and dissolved in DCM (20 mL). The temperature was lowered to -78°C, and DAST (0.256 mL, 1.937 mmol) was added dropwise. The mixture was stirred for 1 h. Water was added to the reaction mixture, and the layers were separated. The aqueous phase was extracted with DCM, and the organic phases were combined, dried, and spin-dried. The residue was purified by column chromatography using 0-40% EA / PE to obtain compound int-38d (229 mg) as a colorless oil. LC-MS: [M+H] + =334.2.

[0227] Step 4: Compound int-38d (118 mg, 0.354 mmol) was added to the vial and dissolved in DCM (10 mL). TFA (3 mL, 39.177 mmol) was added and stirred at room temperature for 2 h. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain compound int-38 (181 mg, containing TFA) as a colorless oil. It was used directly in the next step without further purification. LC-MS: [M+H] + =234.2.

[0228] Intermediate int-39: 4-(1,5-dimethyl-1H-imidazol-4-yl)-1,2,3,6-tetrahydropyridine

[0229] Step 1: Dissolve compound int-2b (350 mg, 1.132 mmol) and compound int-39a (200 mg, 1.143 mmol) in H2O (5 mL) and dioxane (20 mL). Add Pd(dppf)Cl2 (82.82 mg, 0.113 mmol) and K2CO3 (312.86 mg, 2.26 mmol). Replace the atmosphere with N2 three times and stir at 100°C for 12 h. Concentrate under reduced pressure and column chromatography (EA / PE = 1 / 1, DCM / MeOH = 20 / 1) yields compound int-39b (300 mg, 1.08 mmol, 95.55%). LC-MS: ESI [M+H] + =278.2.

[0230] Step 2: Dissolve compound int-39b (300 mg, 1.08 mmol) in a standard hydrochloric acid-methanol solution (4.0 M, 10 mL) and stir at 25°C for 1 h. Concentrate under reduced pressure to obtain compound int-39 (180 mg, 1.016 mmol, 93.89%). LC-MS: ESI [M+H] + =178.2.

[0231] Example 1: 1'-((7-(1,1-difluoroethyl)-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0232] Step 1: Dissolve compound int-1 (1.80 g, 1.61 mmol) in dioxane (30 mL), add XPhos-Pd-G2 (0.40 g, 0.51 mmol) and tributyl(1-ethoxyethylene)tin (2.0 g, 11.6 mmol), and heat at 80°C under N2 with stirring for 2 h. Pour the reaction solution into 30 mL of saturated sodium bicarbonate solution, extract with ethyl acetate (5 x 30 mL), dry over sodium sulfate, filter, and concentrate under reduced pressure. Then, add THF (10 mL) and HCl (3.0 M, 20 mL), and stir for 12 h. Pour the reaction solution into 30 mL of saturated sodium bicarbonate solution, extract with ethyl acetate (3 x 40 mL), dry over sodium sulfate, and filter. Column chromatography (EA / PE = 1 / 5) afforded compound 1a (2.30 g) as a yellow oil. LC-MS: ESI [M+H] + =275.2.

[0233] Step 2: Dissolve compound 1a (0.37 g, 1.34 mmol) in DCM (10 mL). Add DAST (0.4 mL, 4.02 mmol) at 0°C, slowly warm to 25°C, and stir for 12 h. The reaction mixture was slowly poured into 30 mL of saturated sodium bicarbonate to quench. Extract with 30 mL of DCM, dry with sodium sulfate, filter, and concentrate under reduced pressure. Purify by Pre-TLC (EA / PE = 1 / 3) to obtain compound 1b (0.09 g, 0.30 mmol, 22.7% yield) as a white solid. LC-MS: ESI [M+H] + =297.2.

[0234] Step 3: Dissolve compound 1b (0.09 g, 0.30 mmol) in dioxane (10 mL) and slowly add HBr (46% in H2O, 10 mL) dropwise. Stir at 70°C for 1 h. Cool the reaction mixture and slowly pour it into 100 mL of saturated sodium bicarbonate solution to quench the mixture. Extract with 4 x 40 mL of EA, dry with sodium sulfate, and analyze by Pre-TLC (DCM / EA = 1 / 1) to obtain compound 1c (0.09 g) as a white solid. LC-MS: ESI [M+H] + =283.1.

[0235] Step 4: Dissolve compound 1c (0.09 g, 0.31 mmol) in DCM (10 mL). Under a nitrogen atmosphere at 0°C, slowly add DIBAL-H (1 M, 3.0 mL, 3.0 mmol) dropwise. Stir at 0°C for 1 h. Slowly pour 30 mL of saturated sodium potassium tartrate solution into the reaction mixture to quench. Extract with 4 x 40 mL of DCM, dry with sodium sulfate, and analyze by pre-TLC (DCM / MeOH = 15 / 1) to obtain compound 1d (0.026 g, 0.11 mmol, 34.8% yield) as a white solid. LC-MS: ESI [M+H] + =241.2.

[0236] Step 5: Dissolve compound 1d (0.026 g, 0.11 mmol) in DCM (20 mL). Add DIPEA (0.1 mL, 0.56 mmol) and MsCl (0.02 mL, 0.2 mmol) dropwise at 0°C. Heat the temperature to 25°C and stir for 1 h. Slowly pour 20 mL of saturated sodium bicarbonate solution into the reaction mixture to quench. Extract with 30 mL of DCM, dry with sodium sulfate, and filter and concentrate under reduced pressure to obtain compound 1e (0.05 g). LC-MS: ESI [M+H] + =397.2.

[0237] Step 6: Dissolve compound 1e (0.05 g, 0.12 mmol) in ACN (10 mL), add DIPEA (0.5 mL, 2.80 mmol), KI (0.02 g), and compound int-6 (0.02 g, 0.09 mmol), and stir at 80°C for 2 h. Slowly pour 20 mL of saturated ammonium chloride solution into the reaction mixture to quench. Extract with 4 x 30 mL of EA, dry over sodium sulfate, and filter and concentrate under reduced pressure to obtain compound 1f (0.05 g). LC-MS: ESI [M+H] + =522.2.

[0238] Step 7: Compound 1f (0.05 g, 0.09 mmol) was dissolved in MeOH (5 mL), and KOH (5 mL, 3.0 M) was added and stirred at 25°C for 1 h. The reaction solution was slowly poured into 30 mL of saturated ammonium chloride solution to quench the reaction. The mixture was extracted with 4 x 30 mL of EA, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The white solid compound 1 (6.0 mg, 0.013 mmol, yield 13.5%) was purified by reverse phase chromatography. LC-MS: ESI [M+H] + =443.2. 1 H NMR (400MHz, MeOD) δ8.68(s,1H),8.59(s,1H),8.22(s,1H),8.03(d,J=8.4Hz,1H),7.95(dd,J=8.4,2.0Hz,1H),7.80(s,1H) ,6.36(s,1H),3.85(s,2H),3.01(s,1H),2.84(t,J=5.6Hz,2H),2.65(s,2H),2.19(t,J=7.6Hz,1H),2.07(t,J=14.4Hz,3H).

[0239] Example 2: 1'-((7-(1,1-difluoroethyl)-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-2-fluoro-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0240] Step 1: Dissolve compound 1b (0.68 g, 2.30 mmol) in DCM (30 mL). Under a nitrogen atmosphere at 0°C, slowly add DIBAL-H (1 M, 11.5 mL, 11.5 mmol) dropwise. Stir at 25°C for 1 h. Slowly pour 100 mL of saturated sodium potassium tartrate solution into the reaction mixture to quench. Extract with 5 x 50 mL of DCM, dry over sodium sulfate, and filter and concentrate under reduced pressure to obtain compound 2a (0.59 g, crude). LC-MS: ESI [M+H] + =257.2.

[0241] Step 2: Dissolve compound 2a (0.59 g, 2.32 mmol) in DCM (20 mL). Add TEA (2.0 mL, 13.0 mmol) and MsCl (0.8 mL, 7.10 mmol) dropwise at 0°C. Heat the temperature to 25°C and stir for 1 h. Slowly pour 20 mL of saturated sodium bicarbonate solution into the reaction mixture to quench. Extract with 4 x 30 mL of DCM, dry with sodium sulfate, and filter and concentrate under reduced pressure to obtain compound 2b (0.60 g, crude). LC-MS: ESI [M+H] + =333.2.

[0242] Step 3: Dissolve compound 2b (0.20 g, 0.60 mmol) in acetonitrile (10 mL), add DIPEA (0.2 mL, 1.55 mmol), and stir compound int-3 (0.2 g, 0.85 mmol) at 80°C for 2 h. Slowly pour 20 mL of saturated ammonium chloride solution into the reaction mixture to quench the mixture. Extract with 5 x 30 mL of EA, dry with sodium sulfate, filter, and concentrate under reduced pressure. Pre-TLC (DCM / MeOH = 20 / 1) yields compound 2c (0.11 g, 0.23 mmol, 38% yield) as a white solid. LC-MS: ESI [M+H] + =472.1.

[0243] Step 4: Compound 2c (0.11 g, 0.23 mmol) was dissolved in dioxane (10 mL), and HBr (46% in H2O, 5.0 mL) was slowly added dropwise. The mixture was stirred at 70°C for 1 h. The reaction mixture was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution for quenching. The mixture was extracted with DCM / MeOH (20 / 1) (5 x 30 mL), dried over sodium sulfate, and purified by reverse preparative method to obtain compound 2 (20 mg, 0.043 mmol, 19% yield) as a white solid. LC-MS: ESI [M+H] + =458.2. 1 H NMR (400MHz, CDCl3) δ8.52(d,J=1.6Hz,1H),8.24(s,1H),7.99(dd,J=7.6,1.6Hz,1H),7.86–7.71(m,2H),7.65(s, 1H), 6.16 (s, 1H), 3.75 (s, 2H), 3.23 (d, J = 3.2Hz, 2H), 2.97 (d, J = 5.2Hz, 3H), 2.73 (m, 4H), 2.06 (t, J = 18.8Hz, 3H).

[0244] The preparation methods of compounds 3 to 9 of Examples 3 to 9 can be referred to Example 2.

[0245] Table 6 Compounds 3 to 9

[0246] Example 10: N-methyl-1'-((7-(methylthio)-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1'-, 2', 3', 6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0247] Step 1: Dissolve compound int-18 (0.1 g, 0.3 mmol) in acetonitrile (5 mL), add DIPEA (0.3 mL, 1.80 mmol), KI (0.02 g), and compound int-2 (0.1 g, 0.46 mmol), and stir at 80°C for 2 h. Slowly pour 20 mL of saturated ammonium chloride solution into the reaction mixture for quenching. Extract with 4 x 30 mL of EA, dry with sodium sulfate, and purify by Prep-TLC (DCM / MeOH = 20 / 1) to afford compound 10a (0.09 g, 0.21 mmol, 68% yield) as a yellow oil. LC-MS: ESI [M+H] + =436.2.

[0248] Step 2: Dissolve compound 10a (0.09 g, 0.21 mmol) in dioxane (10 mL) and slowly add HBr (46% in H2O, 5.0 mL) dropwise. Stir at 70°C for 1 h. Cool the reaction mixture and slowly pour it into 100 mL of saturated sodium bicarbonate solution to quench the mixture. Extract with 4 x 50 mL of DCM, dry with sodium sulfate, and purify by reverse preparative method to obtain compound 10 (30 mg, 0.07 mmol, 34% yield) as a white solid. LC-MS: ESI [M+H] + =422.2. 1 H NMR(400MHz,MeOD)δ8.70(s,1H),8.51(d,J=1.8Hz,1H),8.07–7.93(m,2H),7.78(s,1H),7.62(s,1H),6.38 (s,1H),3.82(s,2H),2.96(s,3H),2.84(t,J=5.7Hz,2H),2.65(s,2H),2.53(s,3H),2.20(t,J=7.6Hz,2H).

[0249] The preparation methods of compounds 11 to 30 of Examples 11 to 30 can be referred to Example 10.

[0250] Table 7 Compounds 11-30

[0251] Example 31: 2-Fluoro-N-methyl-1'-((6-oxo-7-((trifluoromethyl)thio)-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1'-, 2', 3', 6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0252] Step 1: Dissolve compound int-17 (0.2 g, 0.54 mmol) in acetonitrile (5 mL), add DIPEA (0.45 mL, 2.70 mmol), KI (0.02 g), and compound int-3 (0.2 g, 0.85 mmol), and stir at 80°C for 2 h. Slowly pour 20 mL of saturated ammonium chloride solution into the reaction mixture for quenching. Extract with 4 x 30 mL of EA, dry with sodium sulfate, and purify by Pre-TLC (DCM / MeOH = 20 / 1) to afford compound 31a (0.09 g, 0.18 mmol, 32% yield) as a yellow oil. LC-MS: ESI [M+H] + =436.2.

[0253] Step 2: Compound 31a (0.09 g, 0.18 mmol) was dissolved in dioxane (10 mL), and HBr (46% in H2O, 5.0 mL) was slowly added dropwise. The mixture was stirred at 70°C for 1 h. The reaction mixture was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution for quenching. The mixture was extracted with 4 x 50 mL of DCM, dried over sodium sulfate, and purified by reverse preparative method to obtain compound 31 (20 mg, 0.04 mmol, 22% yield) as a white solid. LC-MS: ESI [M+H] + =494.2. 1 H NMR (400MHz, DMSO) δ8.64(d,J=4.9Hz,1H),8.54(d,J=1.5Hz,1H),8.33(s,1H),8.09(dd,J=9.8,7.8Hz,1H),7.96–7.83(m,1 H), 7.75 (s, 1H), 6.26 (s, 1H), 3.78 (s, 2H), 3.19 (d, J = 2.5Hz, 2H), 2.79 (d, J = 4.8Hz, 3H), 2.70 (t, J = 5.5Hz, 2H), 2.52 (s, 2H).

[0254] The preparation methods of compounds 32 to 40 in Examples 32 to 40 can be referred to Example 31.

[0255] Table 8 Compounds 32-40

[0256] Example 41: 1'-((7-(difluoromethyl)thio)-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0257] Step 1: Dissolve compound int-22 (0.2 g, 0.5 mmol) in acetonitrile (5 mL), add DIPEA (0.45 mL, 2.70 mmol), KI (0.02 g), and compound int-2 (0.2 g, 0.85 mmol), and stir at 80°C for 2 h. Slowly pour 20 mL of saturated ammonium chloride solution into the reaction mixture for quenching. Extract with 4 x 30 mL of EA, dry with sodium sulfate, and purify by Pre-TLC (DCM / MeOH = 20 / 1) to afford compound 41a (0.1 g, 0.22 mmol, 32% yield) as a yellow oil. LC-MS: ESI [M+H] + =472.2.

[0258] Step 2: Compound 41a (0.084 g, 0.18 mmol) was dissolved in dioxane (10 mL). HBr (46% in H2O, 5.0 mL) was slowly added dropwise and stirred at 70°C for 1 h. The reaction mixture was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution to quench the mixture. The mixture was extracted with 4 x 50 mL of DCM and dried over sodium sulfate. The mixture was purified by reverse preparative method to obtain compound 41 (18 mg, 0.04 mmol, 22% yield) as a white solid. LC-MS: ESI [M+H] + =458.2. 1 H NMR (400MHz, DMSO) δ8.69(s,2H),8.48(s,1H),8.08(s,1H),7.99(dd,J=6.0,4.0Hz,2H),7.85(t,J=46.9Hz,1H),7.69 (s,1H),6.43(s,1H),3.75(s,2H),3.17(s,3H),2.81(d,J=4.8Hz,3H),2.72(t,J=5.5Hz,2H),2.54(d,J=13.7Hz,2H).

[0259] The preparation methods of compounds 42 to 49 of Examples 42 to 49 can be referred to Example 41.

[0260] Table 9 Compounds 42-49

[0261] Example 50: 2-Fluoro-N-methyl-1'-((6-oxo-7-vinyl-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0262] Step 1: Compound int-1 (0.91 g, 2.92 mmol) was added to anhydrous THF (300 mL), the reaction solution was cooled to -20°C, DIBAL-H (4.9 mL, 7.3 mmol, 1.5 M toluene solution) was added under a -20°C N2 atmosphere, and the reaction mixture was further stirred between -15-0°C for 3 hours. TLC showed that the reaction was complete. The reaction was slowly quenched with 3N NaOH aqueous solution between -15-0°C, and the internal temperature was kept above 0°C. The volatile substances were removed under reduced pressure at 25°C, and the resultant was extracted with ethyl acetate (30 mL*3). The combined organic phase was washed with water (30 mL) and brine (30 ml), dried over anhydrous Na2SO4 and evaporated to dryness. The crude product was purified by silica gel column chromatography (pure DCM, then DCM / acetone = 30:1 to 10:1). Product 50a (0.54 g) was obtained as a yellow solid. LC-MS: ESI [M+H] + =269.2.

[0263] Step 2: Under a nitrogen atmosphere at 0-5°C, SOCl2 (357 mg, 3.0 mmol) was slowly added to a solution of compound 50a (0.54 g, 2.0 mmol) in DMF (100 mL). The mixture was stirred at 25°C for 3 h until the starting material was completely consumed. The reaction mixture was cooled to 0-5°C with an ice-water bath and quenched with 1N NaOH to pH = 9, followed by the addition of water (10 mL) with stirring. The reaction mixture was stirred at room temperature for 1 hour, and the resulting off-white precipitate was collected by filtration, washed with water (10 mL*3), and dried under vacuum to obtain compound 50b (0.32 g). LC-MS: ESI [M+H] + =287.0.

[0264] Step 3: Compound 50b (0.42 g, 1.45 mmol) was dissolved in 10 mL of ethanol, and compound int-3 (0.35 g, 1.45 mmol) and KI (0.10 g, 0.6 mmol) were added dropwise. DIPEA (3.0 mL, 23 mmol) was added dropwise, and the mixture was stirred at 80°C for 2 h. After cooling, the reaction solution was slowly poured into 30 mL of saturated ammonium chloride solution for quenching. The mixture was extracted with 4 x 30 mL of EA, dried over sodium sulfate, and separated by column chromatography (DCM / MeOH = 20 / 1, supplemented with 5% triethylamine) to afford compound 50c (0.47 g, 0.98 mmol, 67% yield) as a white solid. LC-MS: ESI [M+H] + =482.2 / 484.1.

[0265] Step 4: Compound 50c (0.3 g, 0.62 mmol) was dissolved in dioxane (10 mL), and HBr (46% in H2O, 5.0 mL) was slowly added dropwise. The mixture was stirred at 70°C for 1 h. The reaction mixture was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution to quench the mixture. The mixture was extracted with 4 x 30 mL of DCM, dried over sodium sulfate, and slurried with EA / PE (10 / 1) to afford compound 50d (0.35 g, crude) as a white solid. LC-MS: ESI [M+H] + =469.2 / 470.2.

[0266] Step 5: Compound 50d (0.2 g, 0.423 mmol) was dissolved in dioxane (10 mL), and tri-n-butylvinyltin (0.27 g, 0.86 mmol) and XPhos-Pd-G2 (0.05 g, 0.06 mmol) were added. After nitrogen displacement three times, the mixture was stirred at 90°C for 15 h. After cooling, the mixture was concentrated and purified by pre-TLC (DCM / MeOH = 20 / 1) followed by reverse phase preparative purification to afford compound 50 (20 mg, 0.05 mmol, 12% yield). LC-MS: ESI [M+H] + =416.2. 1H NMR (400MHz, CDCl3) δ10.75 (s, 1H), 8.57 (d, J = 1.7Hz, 1H), 8.11 (s, 1H), 8.05 (d, J = 5. 0Hz,1H),7.96(d,J=7.9Hz,1H),7.67(s,1H),7.53(d,J=7.9Hz,1H),7.00(dd,J=17.7, 11.4Hz,1H),6.24(dd,J=17.7,1.2Hz,1H),5.65(s,1H),5.54(dd,J=11.3,1.1Hz,1H) ,3.80(s,2H),3.22(d,J=2.9Hz,2H),3.03(d,J=5.1Hz,5H),2.77(s,2H),2.54(s,3H).

[0267] The preparation methods of compounds 51 to 58 of Examples 51 to 58 can be referred to Example 50.

[0268] Table 10 Compounds 51-58

[0269] Example 59: N-methyl-5-(4-(((7-(methylthio)-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)piperazin-1-yl)picolinamide

[0270] Step 1: Dissolve compound int-18 (0.1 g, 0.3 mmol) in ACN (10 mL), add DIPEA (0.3 mL, 1.8 mmol), KI (0.02 g), and compound int-9 (0.1 g, 0.45 mmol), and stir at 80°C for 2 h. Slowly pour 20 mL of saturated ammonium chloride solution into the reaction mixture to quench. Extract with 4 x 30 mL of EA, dry over sodium sulfate, and purify by Prep-TLC (DCM / MeOH = 10 / 1) to afford compound 59a (0.12 g, 0.27 mmol, 91% yield) as a white solid. LC-MS: ESI [M+H] + =439.

[0271] Compound 59a (0.12 g, 0.27 mmol) was dissolved in dioxane (10 mL), and HBr (46% in H2O, 5.0 mL) was slowly added dropwise. The mixture was stirred at 70°C for 1 h. The reaction mixture was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution for quenching. The mixture was extracted with DCM (4 x 30 mL), dried over sodium sulfate, and purified by reverse preparative method to obtain Compound 59 (35 mg, 0.082 mmol, 31% yield) as a white solid. LC-MS: ESI [M+H] +=425.2. 1 H NMR (400MHz, CDCl3) δ8.52 (s, 1H), 8.16 (d, J = 2.4Hz, 1H), 8.05 (d, J = 8.7Hz, 1H), 7.77 (s, 1H), 7.64 (d, J = 12. 2Hz, 2H), 7.22 (d, J = 8.8Hz, 1H), 3.70 (s, 2H), 3.36 (s, 4H), 3.01 (d, J = 5.0Hz, 3H), 2.67 (s, 4H), 2.50 (s, 3H).

[0272] Example 60: 2-Fluoro-1-((7-((fluoromethyl)thio)-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0273] Step 1: Dissolve compound int-1 (5.0 g, 16.08 mmol) in DCM (100 mL). Add DIBAL-H (1.0 M, 50 mL, 50.0 mmol) dropwise at 0°C and stir at 0°C for 1 h. Slowly add 100 mL of saturated sodium potassium tartrate solution dropwise. Extract with 4 x 30 mL of DCM, dry over sodium sulfate, and concentrate under reduced pressure to obtain compound 60a (4.34 g, crude). LC-MS: ESI [M+H] + =270.1.

[0274] Step 2: Dissolve compound 60a (4.34 g, 16.08 mmol) in DCM (100 mL), add DIPEA (10 mL, 80.4 mmol) dropwise, and slowly add MOMCl (2.5 mL, 32.16 mmol) dropwise at 0°C. Stir at 0°C for 2 h. Cool the reaction solution and slowly pour it into 100 mL of saturated sodium bicarbonate solution to quench. Extract with 4 x 30 mL of DCM, dry with sodium sulfate, and concentrate under reduced pressure. Column chromatography (EA / PE = 1 / 3) yields compound 60b (2.44 g, 7.79 mmol). LC-MS: ESI [M+H] + =313.1 / 315.1.

[0275] Step 3: Dissolve compound 60b (2.44 g, 7.79 mmol) in toluene (20 mL), add DIPEA (3 mL, 18.18 mmol), Pd2(dba)3 (0.74 g, 0.8 mmol), Xantphos (0.9 g, 1.56 mmol), and int-17 (3.4 g, 15.63 mmol), and stir at 110°C under N2 atmosphere for 12 h. Concentrate under reduced pressure and column chromatography (EA / PE = 1 / 3) afford compound 60c (3.2 g, 7.34 mmol, 91% yield). LC-MS: ESI [M+H] + =437.1.

[0276] Step 4: Dissolve compound 60c (3.2 g, 7.32 mmol) in EtOH (50 mL), add EtONa (1.5 g, 22.05 mmol), and stir at 25°C for 2 h. The reaction solution was slowly poured into a 0.5 M aqueous solution of HCl (100 mL) to quench, extract with 5 x 50 mL of EA, dry over sodium sulfate, and concentrate under reduced pressure to obtain compound 60d (3.0 g, crude). LC-MS: ESI [M+H] + =267.2.

[0277] Step 5: Compound 60d (1.0 g, 3.76 mmol) was dissolved in MeCN (10 mL), and Cs2CO3 (21.2 g, 3.76 mmol) was added. Iodofluoromethane was added dropwise to the reaction mixture, and stirred at 25°C for 3 hours. The reaction mixture was poured into saturated ammonium chloride (50 mL) for quenching, extracted with 5 x 30 mL of EA, dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (EA / PE = 1 / 2) to afford compound 60e (0.65 g, 2.17 mmol, 59%). LC-MS: ESI [M+H] + =299.2.

[0278] Step 6: Compound 60e (0.65 g, 2.17 mmol) was dissolved in HCl (4.0 M in MeOH, 10 mL), stirred at 25°C for 2 h, concentrated under reduced pressure, and quenched with 100 mL of saturated sodium bicarbonate solution. The mixture was extracted with 4 x 30 mL of DCM, dried over sodium sulfate, and concentrated under reduced pressure to afford compound 60f (0.6 g, crude). LC-MS: ESI [M+H] + =255.2.

[0279] Step 7: Dissolve compound 60f (0.6 g, 2.35 mmol) in DCM (10 mL), add DMF (0.01 mL), and add SOCl2 (0.4 mL, 4.70 mmol) dropwise at 25°C. Stir at 25°C for 1 h. Filter and concentrate under reduced pressure to obtain compound 60g (0.6 g, crude). LC-MS: ESI [M+H] + =273.2.

[0280] Step 8: Dissolve compound 60g (0.60g, 2.20mmol) in dioxane (10mL) and slowly add HBr (48% in AcOH, 3.0mL) dropwise. Stir at 40°C for 1h. Cool the reaction mixture and slowly pour it into 100mL of saturated sodium bicarbonate solution to quench the mixture. Filter and obtain compound 60h (0.70g, crude) as a white solid. LC-MS: ESI [M+H] + =259.1.

[0281] Step 9: Dissolve compound 60h (0.1 g, 0.39 mmol) in ACN (10 mL), add DIPEA (0.2 mL, 1.21 mmol), KI (0.02 g), and compound 7 (0.1 g, 0.42 mmol), and stir at 80°C for 2 h. Reverse phase chromatography was performed under reduced pressure to obtain compound 60 (0.08 g, 0.17 mmol, 45% yield) as a white solid. LC-MS: ESI [M+H] + =458.1. 1 H NMR (400MHz, DMSO) δ12.66 (s, 1H), 8.76-8.54 (m, 2H), 8.14 (dd, J = 9.8, 7.8Hz, 1H), 8.04-7.92 (m, 2H), 7 .87(d,J=1.3Hz,1H),6.32-6.05(m,3H),4.59(s,2H),3.90(s,3H),3.36(s,1H),2.80(d,J=4.8Hz,5H).

[0282] The preparation methods of compounds 61 to 63 of Examples 61 to 63 can be referred to Example 60.

[0283] Table 11 Compounds 61-63

[0284] Example 64: 6-Fluoro-N-methyl-5-(1-((7-(methylthio)-6-oxo-5-(6-dihydro-1,5-naphthyridin-3-yl)methyl)piperidin-4-yl)picolinamide

[0285] Step 1: Dissolve compound int-18b (0.59 g, 2.50 mmol) in DCM (10 mL), add DMF (0.01 mL) dropwise, then add SOCl2 (0.60 mL, 5.00 mmol) dropwise at 0°C. Stir at 25°C for 1 h. Concentrate under reduced pressure to remove the solvent to yield compound 64a (0.64 g, crude). LC-MS: ESI [M+H] + =255.2.

[0286] Step 2: Dissolve compound 64a (0.64 g, 2.50 mmol) in dioxane (10 mL) and slowly add HBr (38% in AcOH, 2.0 mL) dropwise. Stir at 40°C for 1 h. Cool the reaction mixture and slowly pour it into 100 mL of saturated sodium bicarbonate solution to quench the mixture. Filter the resulting filter cake, wash it with DCM / MeOH (20 / 1), and concentrate the filtrate under reduced pressure to yield compound 64b (0.6 g, crude). LC-MS: ESI [M+H] + =241.1.

[0287] Step 3: Dissolve compound 64b (0.1 g, 0.42 mmol) in acetonitrile (10 mL), add DIPEA (0.3 mL, 2.1 mmol), KI (0.02 g), and int-23 (0.1 g, 0.42 mmol), and stir at 80°C for 2 h. Concentrate under reduced pressure and perform reverse phase reaction to obtain compound 64 (0.03 g, 0.068 mmol, 16% yield). LC-MS: ESI [M+H] + =442.2. 1 H NMR (400MHz, DMSO) δ12.11(s,1H),8.61(q,J=4.4Hz,1H),8.40(d,J=1.6Hz,1H),8.06(dd,J=9.5,7.9Hz,1H),7.96-7.82(m,1H),7.62(d,J=1.1 Hz,1H),7.54(s,1H),3.62(s,2H),2.94(d,J=11.4Hz,2H),2.79(t,J=8.8Hz,4H),2.45(s,3H)2.13(dd,J=11.4,9.0Hz,2H),1.81-1.62(m,4H).

[0288] Example 65: 5-(4-(((7-(1,1-difluoroethyl)-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)piperazin-1-yl)-6-fluoro-N-methylpicolinamide

[0289] Step 1: Dissolve compound 2a (0.12 g, 0.47 mmol) in DCM (10 mL), add DMF (0.01 mL), add SOCl2 (0.1 mL, 0.87 mmol) dropwise at 25°C, and stir at 25°C for 1 h. Concentrate under reduced pressure to remove the solvent to give compound 65a (0.12 g, crude). LC-MS: ESI [M+H] + =273.2.

[0290] Step 2: Dissolve compound 65a (0.12 g, 0.43 mmol) in dioxane (10 mL) and slowly add HBr (38% in AcOH, 2.0 mL) dropwise. Stir at 40°C for 1 h. Cool the reaction mixture and slowly pour it into 100 mL of saturated sodium bicarbonate solution to quench the mixture. Filter under reduced pressure to obtain compound 65b (0.12 g, crude) as a white solid. LC-MS: ESI [M+H] + =259.2.

[0291] Step 3: Dissolve compound 65b (0.06 g, 0.23 mmol) in acetonitrile (10 mL), add DIPEA (0.15 mL, 1.0 mmol), KI (0.01 g), and compound int-10 (0.05 g, 0.21 mmol), and stir at 80°C for 2 h. Concentrate under reduced pressure to remove the solvent, and the residue is purified by Pre-HPLC to afford compound 65 (0.02 g, 0.043 mmol, 19% yield) as a white solid. LC-MS: ESI [M+H] + =461.2. 1 H NMR (400MHz, DMSO) δ12.24(s,1H),8.53(d,J=1.7Hz,1H),8.41(q,J=4.6Hz,1H),8.09(s,1H),7.85(dd,J=8.0,1.2Hz,1H),7.71(d,J=1.1Hz, 1H), 7.58 (dd, J=10.6, 8.2Hz, 1H), 3.72 (s, 2H), 3.19 (d, J=4.7Hz, 4H), 2.77 (d, J=4.8Hz, 3H), 2.60 (d, J=4.3Hz, 4H), 2.06 (t, J=19.4Hz, 3H).

[0292] Example 66: 5-(1-(((7-(1,1-difluoroethyl)-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)piperidin-4-yl)-6-fluoro-N-methylpicolinamide

[0293] Compound 65b (0.06 g, 0.23 mmol) was dissolved in acetonitrile (10 mL), and DIPEA (0.15 mL, 1.0 mmol), KI (0.01 g), and compound int-23 (0.06 g, 0.21 mmol) were added. The mixture was stirred at 80°C for 2 h. The solvent was removed by concentration under reduced pressure, and the residue was purified by Pre-HPLC to afford compound 66 (0.02 g, 0.043 mmol, 19% yield). LC-MS: ESI [M+H] + =460.2. 1 H NMR (400MHz, DMSO) δ8.61(d,J=4.8Hz,1H),8.51(d,J=1.7Hz,1H),8.12-7.99(m,2H),7.90(dd,J=7.6,1.3Hz,1H),7.69(d,J=1.0Hz,1H),3.67(s ,2H),2.94(d,J=11.4Hz,2H),2.78(d,J=4.8Hz,4H),2.16(dd,J=11.3,8.7Hz,2H),2.05(dd,J=23.9,14.9Hz,3H),1.75(dt,J=12.0,6.9Hz,4H).

[0294] Example 67: 5-(4-((7-(difluoromethyl)thio)-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)piperazin-1-yl)-N-methylpicolinamide

[0295] Step 1: Dissolve compound int-22c (1.0 g, 3.47 mmol) in DCM (10 mL), add DMF (0.01 mL), and add SOCl2 (1.0 mL, 6.94 mmol) dropwise at 25°C. Stir at 25°C for 1 h. Concentrate under reduced pressure to remove the solvent to afford compound 67a (0.6 g, crude). LC-MS: ESI [M+H] + =291.2.

[0296] Step 2: Dissolve compound 67a (1.0, 3.44 mmol) in dioxane (10 mL) and slowly add HBr (38% in AcOH, 3.0 mL) dropwise. Stir at 40°C for 1 h. Cool the reaction mixture and slowly pour it into 100 mL of saturated sodium bicarbonate solution to quench the mixture. Filter the mixture to obtain compound 67b (0.90 g, crude) as a white solid. LC-MS: ESI [M+H] + =277.2.

[0297] Step 3: Dissolve compound 67b (0.1 g, 0.36 mmol) in acetonitrile (10 mL), add DIPEA (0.2 mL, 1.21 mmol), KI (0.02 g), and compound int-2 (0.1 g, 0.45 mmol), and stir at 80°C for 2 h. Concentrate under reduced pressure to remove the solvent, and the residue is purified by Pre-HPLC to obtain compound 67 (0.05 g, 0.11 mmol, 30% yield). LC-MS: ESI [M+H] + =461.2. 1 H NMR (400MHz, DMSO) δ8.48(d,J=1.3Hz,1H),8.39(d,J=4.9Hz,1H),8.27(d,J=2.8Hz,1H),8.08(s,1H),8.03-7 .62(m,3H),7.39(dd,J=8.8,2.8Hz,1H),3.68(s,2H),3.38(s,4H),2.78(d,J=4.8Hz,3H),2.60-2.51(m,4H).

[0298] Example 68: 5-(1-((7-(difluoromethyl)thio)-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)piperidin-4-yl)-6-fluoro-N-methylpicolinamide

[0299] Compound 67b (0.1 g, 0.36 mmol) was dissolved in acetonitrile (10 mL), and DIPEA (0.2 mL, 1.21 mmol), KI (0.02 g), and int-23 (0.1 g, 0.41 mmol) were added. The mixture was stirred at 80°C for 2 h. The solvent was removed by concentration under reduced pressure, and the residue was purified by Pre-HPLC to afford compound 68 (0.04 g, 0.08 mmol, 23% yield). LC-MS: ESI [M+H] + =478.2. 1 H NMR (400MHz, DMSO) δ12.79 (s, 1H), 8.74-8.46 (m, 2H), 8.14 (s, 1H), 7.95 (ddd, J=66.2, 41.2, 26.0Hz, 4 H), 4.54 (s, 2H), 3.56 (d, J = 11.6Hz, 2H), 3.28-3.04 (m, 3H), 2.80 (d, J = 4.7Hz, 3H), 2.11-1.84 (m, 4H).

[0300] Example 69: 2-Fluoro-N-methyl-1'-(3-(methylthio)-2-oxo-1,2-dihydroquinolin-7-yl)methyl)-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0301] Step 1: Dissolve compound 69a (1.0 g, 5.0 mmol) in DCM (20 mL), add MnO2 (7.0 g, 80.0 mmol), and stir at 25°C for 15 h. Filter through a pad of Celite, collect the filtrate, and concentrate under reduced pressure to obtain compound 69b (1.0 g, crude). LC-MS: ESI [M+H] + =200.1 / 202.1.

[0302] Step 2: Dissolve compound 69b (1.0 g, 5.0 mmol) in DCM (30 mL), add methylthioacetic acid (0.4 mL, 5.0 mmol), cool to 0°C, and sequentially add pyridine (4.0 mL, 50.0 mmol) and POCl3 (2.3 mL, 25 mmol) dropwise. After complete addition, warm to 25°C and stir for 1 h. Under an ice-water bath, slowly pour the reaction solution into saturated sodium bicarbonate solution (100 mL) to quench the reaction. Extract with 5 x 30 mL of DCM, dry with sodium sulfate, and concentrate under reduced pressure to obtain compound 69c (1.4 g, crude). LC-MS: ESI [M+H] + =290.2 / 292.2.

[0303] Step 3: Compound 69c (1.4 g, 5.0 mmol) was dissolved in MeOH (20 mL), and MeONa (0.8 g, 15.0 mmol) was added. The mixture was stirred at 25°C for 21 h. The reaction solution was concentrated and quenched with saturated ammonium chloride solution (20 mL). The mixture was extracted with 4 x 30 mL of DCM, dried over sodium sulfate, and concentrated under reduced pressure. The mixture was then purified by column chromatography (EA / PE = 1 / 1) to afford compound 69d (0.7 g, 2.57 mmol, 51%). LC-MS: ESI [M+H] + =270.2 / 272.1.

[0304] Step 4: Compound 69d (0.7 g, 2.57 mmol) was dissolved in dioxane (30 mL), and tributyltin methanol (1.0 mL, 3.11 mmol) and XPHOS-Pd-G2 (0.2 g, 0.26 mmol) were added. The mixture was stirred at 100°C under a nitrogen atmosphere for 12 h. After the reaction was complete, the mixture was filtered through celite and the filtrate was dried and purified by Prep-TLC to afford compound 69e (0.17 g, 0.77 mmol, 30%). LC-MS: ESI [M+H] + =222.2.

[0305] Step 5: Dissolve compound 69e (0.17 g, 0.77 mmol) in toluene (10 mL), add DMF (0.01 mL), add SOCl2 (0.06 mL, 0.85 mmol) dropwise at 25°C, and stir at 25°C for 1 h. Concentrate under reduced pressure to obtain compound 69f (0.2 g, crude). LC-MS: ESI [M+H] + =240.2.

[0306] Step 7: Compound 69f (0.1 g, 0.42 mmol) was dissolved in acetonitrile (10 mL), and DIPEA (0.3 mL, 1.68 mmol), KI (0.02 g), and int-3 (0.1 g, 0.42 mmol) were added. The mixture was stirred at 80°C for 2 h. The solvent was removed by concentration under reduced pressure, and the residue was purified by Pre-HPLC to afford compound 69 (0.03 g, 0.07 mmol, 16% yield) as a white solid. LC-MS: ESI [M+H] + =439.2. 1 H NMR (400MHz, DMSO) δ11.96 (s, 1H), 8.67-8.55 (m, 1H), 8.08 (dd, J = 9.9, 7.8Hz, 1H),7.92(dd,J=7.7,1.7Hz,1H),7.65-7.49(m,2H),7.30(d,J=16.6Hz,1H),7. 17(dd,J=8.1,1.2Hz,1H),6.26(s,1H),3.66(s,2H),3.14(d,J=2.8Hz,2H),2. 79 (d, J = 4.8 Hz, 3H), 2.67 (t, J = 5.5 Hz, 2H), 2.51 (d, J = 1.7 Hz, 2H), 2.41 (s, 3H).

[0307] The preparation methods of compounds 70-74 of Examples 70-74 can be referred to Example 69.

[0308] Table 12 Compounds 70-74

[0309] Example 75: N-methyl-5-(4-((7-(methyl-d3)thio)-6-oxo-5,6-dihydro-1,5-naphthopyridin-3-yl)methyl)piperazin-1-yl)picolinamide

[0310] Preparation of N-methyl-5-(4-((7-(methyl-d3)thio)-6-oxo-5,6-dihydro-1,5-naphthopyridin-3-yl)methyl)piperazin-1-yl)picolinamide Reference Example 59 to obtain Compound 75 (0.04 g, 0.09 mmol, 23% yield). LC-MS: ESI [M+H] + =428.5. 1 H NMR (400MHz, CDCl3) δ11.49(s,1H),8.53(s,1H),8.16(d,J=2.4Hz,1H),8.05(d,J=8.7Hz,1H),7.78(d,J=4.5Hz,1H),7.72(s,1 H), 7.64 (s, 1H), 7.22 (dd, J = 8.8, 2.7Hz, 1H), 3.71 (s, 2H), 3.36 (d, J = 4.6Hz, 4H), 3.01 (d, J = 5.0Hz, 3H), 2.68 (d, J = 4.5Hz, 4H).

[0311] Example 76: 2-Chloro-1'-((8-fluoro-3-(methylthio)-2-oxo-1,2-dihydroquinolin-7-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4-bipyridine]-6-carboxamide

[0312] Step 1: Dissolve compound 76a (10 g, 42.731 mmol) in THF (200 mL) and add BF3 . Et2O (18.19 g, 128.194 mmol) was added to NaBH4 (4.85 g, 128.194 mmol) at 0°C and stirred at 0°C for 2 h. The reaction solution was slowly poured into 100 mL of saturated ammonium chloride solution to quench the reaction. The solution was concentrated under reduced pressure and extracted with 50 mL of EA. The product was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product of compound 76b (10.0 g). LC-MS: ESI [M+H] + =220.2 / 222.2.

[0313] Step 2: Dissolve compound 76b (10 g, 45.446 mmol) in DCM (200 mL), add MnO2 (19.76 g, 227.231 mmol), and stir at 25°C for 3 h. Filter through a pad of Celite, collect the filtrate, and concentrate under reduced pressure to obtain compound 76c (7.0 g, crude). LC-MS: ESI [M+H] + =217.2 / 219.2.

[0314] Step 3: Dissolve compound 76c (5 g, 22.933 mmol) in THF (150 mL) and ethyl 2-methylthioacetate (3.08 g, 22.933 mmol). Displace the atmosphere with nitrogen and add LiHMDS (68.798 mL, 68.798 mmol) dropwise at -78°C. Stir at -78°C for 1 h. Quench the reaction by slowly pouring the reaction solution into saturated ammonium chloride solution (100 mL) under an ice-water bath. Extract with 5 x 30 mL of EA, dry with sodium sulfate, and concentrate under reduced pressure. Separate by column chromatography (EA / PE = 1 / 3) to afford compound 76d (7.0 g, crude). LC-MS: ESI [M+H] + =288.2 / 290.2.

[0315] Step 4: Dissolve compound 76d (2 g, 6.941 mmol) in dioxane (30 mL), add tributyltin methanol (2.23 g, 6.941 mmol) and XPHOS-Pd-G2 (0.55 g, 0.694 mmol), and stir at 100°C under N2 atmosphere for 12 h. Prep-TLC purification afforded compound 76e (1.35 g, 5.64 mmol, 81%). LC-MS: ESI [M+H] + =240.2.

[0316] Step 5: Dissolve compound 76e (1.35 g, 5.64 mmol) in toluene (30 mL), add DMF (0.02 mL), add SOCl2 (0.81 g, 6.771 mmol) dropwise at 25°C, and stir at 25°C for 1 h. Concentrate under reduced pressure to obtain compound 76f (1.3 g, crude). LC-MS: ESI [M+H] + =258.2.

[0317] Step 6: Dissolve compound 76f (0.045 g, 0.179 mmol) and int-29 (0.05 g, 0.179 mmol) in acetonitrile (20 mL). Add DIEA (0.12 g, 0.894 mmol) and potassium iodide (0.03 g, 0.179 mmol). Stir at 80°C for 2 h. Concentrate under reduced pressure and perform reverse phase reaction to obtain compound 76 (45 mg, 0.095 mmol, 53.22%). LC-MS: ESI [M+H] + =473.2. 1H NMR (400MHz, DMSO) δ12.08(s,1H),8.60(q,J=4.5Hz,1H),7.95(d,J=7.8Hz,1H),7.88(d,J=7.7Hz,1H),7.61(s,1H),7.46(d,J=8 .1Hz,1H),7.28–7.20(m,1H),5.84(s,1H),3.70(d,J=43.0Hz,2H),3.13(s,3H),2.80(d,J=4.8Hz,3H),2.70(s,2H),2.42(s,3H).

[0318] The preparation methods of compounds 77-90, 96, 98-99 and 100-107 of Examples 77-90, 96, 98-99 and 100-107 of Examples 90, 96, 98-99 and 100-107 can be referred to Example 76.

[0319] Table 13 Compounds 77-90, 96, 98-99 and 100-107

[0320] Example 91: 5-(4-((8-fluoro-3-(methylthio)-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)methyl)piperazin-1-yl)-N,6-dimethylpicolinamide

[0321] Step 1: Compound 91a (2 g, 12.774 mmol) was dissolved in DMF (20 mL) and ACN (20 mL). Selectfluor (5.43 g, 15.329 mmol) was added, and the atmosphere was replaced with N2 three times. The mixture was stirred at 80°C for 2 h. After completion of the reaction, saturated sodium bicarbonate solution (30 mL) was added to quench the reaction. The mixture was extracted with 5 x 30 mL of EA, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The mixture was isolated and purified by column chromatography (EA / PE = 1 / 3) to afford compound 91b (0.9 g, 5.15 mmol, 40.3%). LC-MS: ESI [M+H] + =175.2.

[0322] Step 2: Dissolve compound 91b (0.9 g, 5.15 mmol) in THF (30 mL) and ethyl 2-methylthioacetate (0.83 g, 6.18 mmol). Displace the atmosphere with nitrogen and add LiHMDS (25.7 mL, 25.7 mmol) dropwise at -78°C. Stir at -78°C for 1 hour. Then, warm the temperature to 25°C and stir for 12 hours. Under an ice-water bath, slowly pour the reaction mixture into saturated ammonium chloride solution (100 mL) to quench the reaction. The filter cake is collected by filtration to yield the target compound 91c (2.8 g, crude). LC-MS: ESI [M+H] + =245.2.

[0323] Step 3: Dissolve compound 91c (2.3 g, 9.4 mmol) in dioxane (50 mL), add tributyltin methanol (3.32 g, 10.34 mmol) and XPHOS-Pd-G2 (0.74 g, 0.94 mmol), and stir at 100°C under N2 atmosphere for 12 h. Prep-TLC purification afforded compound 91d (0.8 g, 0.94 mmol, 35.2%). LC-MS: ESI [M+H] + =241.2.

[0324] Step 4: Dissolve compound 91d (0.8 g, 3.33 mmol) in toluene (30 mL), add DMF (0.05 mL), add SOCl2 (0.40 g, 3.33 mmol) dropwise at 25°C, and stir at 100°C for 4 h. Concentrate under reduced pressure and purify by Prep-TLC to afford compound 91e (0.35 g, 1.35 mmol, 40.6%). LC-MS: ESI [M+H] + =259.2.

[0325] Step 5: Compound 91e (30 mg, 0.116 mmol) and int-11 (27.63 mg, 0.116 mmol) were dissolved in acetonitrile (20 mL). DIEA (44.97 mg, 0.348 mmol) and potassium iodide (38.50 mg, 0.232 mmol) were added and stirred at 80°C for 2 h. The mixture was concentrated under reduced pressure and then subjected to reverse phase reaction to afford compound 91 (45 mg, 0.095 mmol, 53.22%). LC-MS: ESI [M+H] + =457.2. 1H NMR (400MHz, DMSO) δ12.57(s,1H),8.65(s,1H),8.41(q,J=4.7Hz,1H),7.83–7.66(m,2H),7.45(d,J=8. 3Hz,1H),3.79(s,2H),2.91(s,4H),2.79(d,J=4.9Hz,3H),2.70–2.60(m,4H),2.47(s,3H),2.45(s,3H).

[0326] The preparation methods of compounds 92-95 of Examples 92-95, compound 97 of Example 97 and compound 108 of Example 108 were prepared with reference to Example 91.

[0327] Table 14 Compounds 92-95, 97 and 108

[0328] Biological activity test:

[0329] 1. PARP-1 enzyme assay

[0330] Experimental materials: PARP1 protein (BPS, Cat. No. 80501), PARP2 protein (BPS, Cat. No. 80502), PARP5A protein (BPS, Cat. No. 80504), Biotin-NAD+ (R&D, Cat. No. 6573), Strep-HRP (Thermo Pierce, Cat. No. 21127), NAD+ (TCI, Cat. No. D0919-5G), Quantitative enhanced chemiluminescence HRP substrate kit (Thermo Pierce, Cat. No. 15159), Histone (Active Motif, Cat. No. 81167), Activated DNA (Genscript, Cat. No. L05182-01&02&03), anti-rabbit IgG, HRP-linked Antibody (CST, Cat. No. 7074P2), anti-Poly / Mono-ADP Ribose (E6F6A) Rabbit mAb (CST, Cat. No. 83732S), SuperSignal ELISA Femto Substrate (THERMO PIERCE, Cat. No. 37074).

[0331] 1.1 PARP1 enzyme assay

[0332] 1.1.1 Buffer preparation: PBST: 1X PBS, 0.05% Tween-20, blocking solution: 1X PBS, 0.05% Tween-20, 5% BSA, reaction buffer: 50 mM Tris-HCl (pH 7.5), 0.005% Tween-20, 0.01% BSA.

[0333] 1.1.2 Coating: Prepare 50 ng / mL Histone coating solution in 1xPBS, transfer 25 μL of the coating solution to a 384-well reaction plate, and coat overnight at 4°C.

[0334] 1.1.3 Washing: After coating, discard the coating solution and wash with PBST solution. Transfer 50uL PBST to a 384-well reaction plate, let it stand for 5 minutes, discard the wash solution, refill the plate, and repeat the washing process three times. Finally, pat the reaction plate dry and wait for the next step of blocking.

[0335] 1.1.4 Blocking: Transfer 50 μL of blocking solution to a 384-well reaction plate and let it stand for 1 hour.

[0336] Washing: After blocking, discard the blocking solution and wash the plate three times with PBST solution according to step 2. Finally, pat the reaction plate dry.

[0337] 1.1.5 Prepare a 1000-fold dilution of the compound, transfer 1 μL of the compound to 199 μL of reaction buffer in a 96-well plate, mix thoroughly, and transfer 5 μL of the mixed compound to a 384-well reaction plate.

[0338] 1.1.6 Prepare a 25 / 10x PARP1-DNA solution using reaction buffer. Transfer 10 μL of PARP1-DNA solution to a 384-well reaction plate. For the negative control wells, transfer 10 μL of DNA solution. The final concentration of PARP1 is 0.02 nM, and the final concentration of DNA is 0.8 nM.

[0339] 1.1.7 Prepare a 25 / 10x NAD+ solution in reaction buffer. Transfer 10 μL of NAD+ solution to a 384-well reaction plate. The final NAD+ concentration is 3.5 μM. Incubate at room temperature for 60 minutes.

[0340] 1.1.8 Prepare a 25 / 10x NAD+ solution in reaction buffer. Transfer 10 μL of NAD+ solution to a 384-well reaction plate. The final NAD+ concentration is 3.5 μM. Incubate at room temperature for 60 minutes.

[0341] 1.1.9 Washing: After the reaction is completed, discard the reaction solution and wash the plate three times with PBST solution according to the method in step 2. Finally, pat the reaction plate dry.

[0342] 1.1.10 Dilute the primary antibody (anti-Poly / Mono-ADP Ribose Rabbit mAb) 2000-fold with blocking buffer, add 20 μL of primary antibody, and incubate at room temperature for 1.5 hours.

[0343] 1.1.11 Washing: Discard the primary antibody and wash the plate three times with PBST solution according to the method in step 2. Finally, pat the reaction plate dry.

[0344] 1.1.12 Dilute the secondary antibody (anti-rabbit IgG, HRP-linked Antibody) 2000-fold with blocking solution, add 20 μL of secondary antibody, and incubate at room temperature for 1 hour.

[0345] 1.1.13 Washing: Discard the secondary antibody and wash the plate three times with PBST solution according to step 2. Finally, pat the reaction plate dry.

[0346] 1.1.14 Color development: Mix Femto-ECL Substrate A and Femto-ECL Substrate B in a 1:1 ratio and transfer 25 μL to a 384-well reaction plate.

[0347] 1.1.15 Reading: Use Envision to read the chemiluminescence value RLU.

[0348] The test results are shown in Table 15 below:

[0349] Table 15 PARP-1 enzyme test results

[0350] Conclusion: The compounds of the present invention have a significant inhibitory effect on PARP1.

[0351] 2. Cell anti-proliferation activity test:

[0352] BRCA-mutant MDA-MB-436 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% saturated CO2 incubator. When cells reached 80% confluency, they were harvested, centrifuged at 300 g for 10 minutes, and plated at 1200 cells / well in a 96-well plate. After 24 hours, PARPi was added at various final concentrations (0, 0.01, 0.1, 1, 10, 100, and 1000 nM) and cultured for an additional 72 hours. The medium was then replaced (with the same final concentration of PARPi added) and cultured for an additional 96 hours. The 96-well plate was removed and the OD values ​​at a wavelength of 450 nM were measured using the CCK8 assay. The cell inhibition rate was calculated as follows: % inhibition = 1 - (mean OD value of the treatment group - mean OD value of the blank group) / (mean OD value of the control group - mean OD value of the blank group) * 100%.

[0353] BRCA wild-type cells DLD-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100μg / mL streptomycin, and cultured in a 5% saturated CO2 incubator at 37°C. When the cells grew to 80% confluence, the cells were collected, centrifuged at 300g for 10 minutes, and plated on a 96-well plate at 1000 cells / well. After 24 hours, different final concentrations of PARPi (0, 1, 10μM) were added and cultured for 72 hours. The cells were treated with a new medium (PARPi was re-added with the same final concentration) and cultured for 96 hours. The 96-well plate was removed, and the OD value at a wavelength of 450nM was detected by CCK8, and the cell inhibition rate was calculated:

[0354] Inhibition rate %=1-(average OD value of the drug administration group-average OD value of the Blank group) / (average OD value of the Control group-average OD value of the Blank group)*100%.

[0355] The test results are shown in Table 16 below:

[0356] Table 16 Cell antiproliferative activity test results

[0357] Conclusion: The compounds of the present invention have significant inhibitory effect on BRCA mutant MDA-MB-436 cells, but have no significant inhibitory effect on BRCA wild-type DLD-1 cells, indicating that the compounds of the present invention specifically inhibit homologous recombination-deficient tumor cells. In addition, a large number of preferred compounds of the present invention, such as 2, 11, 17, 21, 28, and 59, have significantly better activity than the reference compounds AZD5305 and AZD9574.

[0358] 3. Inhibition experiment of compounds on hERG potassium channels

[0359] Cell culture and treatment: CHO cells stably expressing hERG were cultured in a cell culture flask at 37°C in a 5% CO2 incubator. When the cell density reached 60-80%, the cell culture medium was removed, the cells were washed once with PBS, and then digested with Detachin. After digestion was complete, the cells were neutralized with culture medium, centrifuged, and the supernatant was removed. The cells were then resuspended in culture medium to adjust the cell density to 2-5×10 6 / mL for future use.

[0360] Compound Preparation: Dilute the compound stock solution with 100% DMSO by adding 10 μL of compound stock solution to 20 μL of DMSO and serially diluting the solution 3-fold to six concentrations. Add 4 μL of each of the six concentrations to 396 μL of extracellular fluid, achieving a 100-fold dilution to obtain six intermediate concentrations. Then, add 80 μL of each of the six intermediate concentrations to 320 μL of extracellular fluid, achieving a 5-fold dilution to the desired final concentration. The highest concentration tested was 40 μM, followed by six concentrations of 40, 13.33, 4.44, 1.48, 0.49, and 0.16 μM. The DMSO content in the final test concentration did not exceed 0.2%, as this concentration has no effect on hERG potassium channels. Compound preparation was performed using the Bravo instrument throughout the entire dilution process.

[0361] Electrophysiological recordings: Single-cell high-impedance sealing and whole-cell pattern formation were automated by the Qpatch instrument. After acquiring whole-cell recording mode, cells were clamped at -80 mV. A 50-millisecond pre-depolarization of -50 mV was applied before a 5-second depolarization of +40 mV. The cells then repolarized to -50 mV for 5 seconds before returning to -80 mV. This voltage was applied every 15 seconds. After recording for 2 minutes, extracellular solution was added for 5 minutes. Drug administration then began. Compound concentrations were administered for 2.5 minutes at each test concentration, starting with the lowest tested concentration. After all concentrations were administered, the positive control compound, 3 μM Cisapride, was administered. At least three cells were tested for each concentration (n ≥ 3).

[0362] Data processing: Data analysis and processing were performed using GraphPad Prism 5.0 and Excel software. Compound IC50 was calculated using GraphPad Prism 5 software by fitting the following equation:

[0363] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))

[0364] Where X is the Log value of the test sample concentration, Y is the inhibition percentage at the corresponding concentration, and Bottom and Top are the minimum and maximum inhibition percentages, respectively.

[0365] Table 17 Inhibition results of compounds on hERG potassium channels

[0366] Conclusion: Compounds 11, 12, 17, 31, 47, 50, 64, 73, 89 and 95 of the present invention have weak inhibitory effects on hERG potassium ion channels.

[0367] 4. Evaluation of the brain and blood distribution of compounds in vivo

[0368] Experimental purpose: To obtain the brain-blood distribution of the compound.

[0369] Experimental plan: The brain-blood distribution of the compound was investigated by monitoring the content of the compound in the mouse brain and plasma.

[0370] Experimental steps: Weigh the compound, add a small amount of DMSO, and then add sodium chloride solution for injection to make 1 mg mL -1 The compound solution is ready for administration. Mice, male, 10 mg·kg -1 Oral administration was performed, and whole blood and whole brain were collected 1 h and 6 h after administration (n = 3). Whole blood was centrifuged at 3500 rpm for 15 min, and the supernatant plasma was collected. The weight of the centrifuge tube was weighed as M1, the weight of the centrifuge tube containing the whole brain was weighed as M2, the weight of the centrifuge tube after adding water to homogenize was weighed as M3, and the weight of the centrifuge tube after taking out 30 μL of homogenate was weighed as M4. 30 μL of plasma and 30 μL of brain homogenate were taken into the centrifuge tube, and 120 μL of 20 ng·ml -1 The internal standard SAHA was precipitated with acetonitrile, vortexed for 30 seconds, centrifuged at 13,000 rpm for 15 minutes, and the supernatant was collected and placed in a sample vial for testing.

[0371] Standard curve range: 10~10000 ng·ml -1 .

[0372] Drug content in brain = measured value × 0.03 × (M3-M1) / [(M2-M1) × (M3-M4)].

[0373] The results of the brain blood distribution of the compound are shown in Table 18 below:

[0374] Table 18 Results of brain blood distribution test in mice after compound administration

[0375] Conclusion: The plasma and brain tissue concentrations of compounds 10, 11, 12, 17, 22, 27, 31, 42, 47, 50, and 60 of the present invention were significantly higher than those of AZD9574 at 1 and 6 hours. Therefore, compounds 10, 11, 12, 17, 22, 27, 31, 42, 47, 50, and 60 have the potential to penetrate the brain.

[0376] 5. Pharmacokinetic evaluation of the compound in Balb / c mice

[0377] Experimental purpose: To understand the pharmacokinetics of the compound.

[0378] Experimental basis: Technical Guidelines for Nonclinical Pharmacokinetic Studies of Chemical Drugs, 2014.

[0379] Experimental plan: The pharmacokinetics of the compound were investigated by intravenous administration (1 mg·kg-1) and oral administration (1 mg·kg-1) to Balb / c mice.

[0380] Sample preparation: weigh about 0.2 mg of the compound, add 10 μL of DMSO to dissolve it, and then add sodium chloride solution for injection to make a 0.1 mg·mL-1 compound solution for administration.

[0381] Sample collection: 6 male Balb / c mice (Chengdu Dashuo Experimental Animal Co., Ltd., license number: SCXK (Chuan) 2020-030), 3 were intravenously administered (IV) at 1 mg·kg-1, and 3 were gavage administered (PO) at 1 mg·kg-1. Approximately 0.05 mL of blood was collected 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, and 48 h after administration. The collected blood was centrifuged at 3500 rpm for 15 min, the supernatant plasma was collected, and frozen at -40 ° C for testing. The plasma concentration was quantitatively analyzed by LC-MS / MS analysis, and pharmacokinetic parameters such as peak time (Cmax), area under the concentration-time curve (AUC(0-t)), and half-life (T) were calculated. 1 / 2 ), clearance (CL), tissue distribution (Vdss), bioavailability (F), etc.

[0382] Table 19 Pharmacokinetic test results of the compound in Balb / c mice

[0383] Conclusion: The compounds of the present invention have good pharmacokinetic properties in Balb / c mice.

[0384] 6. In vivo pharmacodynamic study of compounds 47 and 64 on the subcutaneous transplanted tumor model of breast cancer MDA-MB-436 in nude mice

[0385] (1) Main instruments and equipment

[0386] CO2 cell culture incubator: Yamato brand, model IP610; biological safety cabinet: Suzhou Antai brand, model BSC-1304ⅡA2; normal temperature centrifuge: Thermo Scientific brand, model SORVALLST 16;

[0387] Digital inverted microscope: Olympus brand, model CKX3-SLP; constant temperature water bath: Shanghai Yuejin Medical Instrument Co., Ltd., model HSW-420; cell counting chamber: Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd., model XB.K.25; liquid nitrogen tank: Thermo brand, model CY50935-70; refrigerator: Qingdao Haier brand, model BCD-601WDGX; medical low-temperature refrigerator: American Thermo brand, model ULTS1651;

[0388] Water purifier: Millipore, USA, model F7PNO9748; vertical autoclave: Yamato, model DKN812C; 1 mL disposable sterile syringe: Shanghai Kindly Enterprise Development Group Co., Ltd.; 1 mL disposable sterile insulin syringe: BD; weight scale: Shanghai Hengping Instrument Factory, model JY2002; analytical balance: SARTORIUS, model BCE95I-1CEU.

[0389] Thermohygrometer: Wuqiang Thermohygrometer Manufacturing Center, measurement number Ji 30260102; small centrifuge: American Thermo brand, model SORVALL LEGEND MICRO 17 Centrifuge; mini mixer: Yeasen brand, model ES-VM25; metal bath: SCILOGEX brand, model SCL120-S.

[0390] (2) Main software and data processing systems

[0391] Graphpad Prism, version 6.0, Graphpad Software Ltd., was used to organize and quantify data and to create bar graphs.

[0392] (3) Experimental animals

[0393] Strain: NOD / SCID, Grade: SPF, Source: Beijing Weitonglihua Biotechnology Co., Ltd.

[0394] (4) Modeling method

[0395] Tumor cell culture and preparation: For routine tumor cell line passage and culture, the culture medium contains 10% fetal bovine serum, 100U / mL penicillin, and 100μg / mL streptomycin. When the cells grow to 80% confluence, collect the cells and centrifuge at 300g for 10 minutes. Wash 3 times with pre-cooled PBS, collect the cells by centrifugation at 300g for 10 minutes, resuspend the cells with PBS, count the cells with a hemocytometer, adjust the cell concentration, and pre-cool the cell suspension on ice after the cells are collected. Take 100μL of cell suspension and inject it subcutaneously in the dorsal armpit of the mouse. When the tumor volume reaches 200-300mm 3 Group treatment started at that time.

[0396] (5) Observation and testing indicators

[0397] General status observation: Observed animals: All surviving experimental animals planned for observation; Observation time: Twice a day; Observation content: including but not limited to general manifestations, behavioral status, eyes, mouth, nose and mouth, ears, hair, feces, urine, genitals and other toxic symptoms. If any abnormalities are found, a detailed description is required.

[0398] (6)Weight

[0399] All surviving experimental animals planned for measurement were weighed before modeling for experimental grouping. After modeling, mice were weighed every two days, and weight changes were recorded in real time. Effectiveness criteria included tumor volume (TV), relative tumor volume (RTV), and relative tumor growth rate in the subcutaneous transplanted tumor model. The long and short diameters of the tumors were measured with a vernier caliper every two days to dynamically observe the antitumor effects of the test drug. The formula for calculating tumor volume (TV) is: TV (mm3) = a × b2 × 0.5; where a and b represent the long and short diameters, respectively.

[0400] Relative tumor volume (RTV) was calculated based on the measurement results using the formula: RTV = Vt / V0, where V0 is the TV measured at the time of dosing (d0) for each group, and Vt is the TV at each measurement for that group. The antitumor activity was evaluated using the relative tumor growth rate (T / C) (%), calculated as follows: T / C (%) = TRTV / CRTV × 100%.

[0401] TRTV: relative tumor volume of the treatment group; CRTV: relative tumor volume of the negative control group. Efficacy evaluation criteria: T / C (%) > 60 indicates ineffectiveness; T / C (%) ≤ 60, with a P < 0.05 as determined by statistical analysis, indicates efficacy. Tumor Weight Determination and Calculation of Tumor Inhibition Rate (%): At the end of treatment, animals were sacrificed, tumors were dissected, weighed, and photographed. The tumor inhibition rate (%) was calculated using the following formula:

[0402] Tumor inhibition rate (tumor growth inhibition rate, %) = (average tumor weight of negative control group (g) - average tumor weight of drug-treated group (g)) / average tumor weight of negative control group (g) × 100%;

[0403] Effectiveness criteria: Tumor inhibition rate (i.e., tumor growth inhibition rate) < 40% is considered ineffective; tumor inhibition rate ≥ 40% and P < 0.05 after statistical analysis is considered effective. Comprehensive criteria for subcutaneous tumor model effectiveness: Effectiveness is determined when either of the two effectiveness criteria (relative tumor growth rate and tumor inhibition rate) is met.

[0404] (7) Data collection and analysis

[0405] All raw data within the facility were collected manually or using a data acquisition system according to the experimental protocol and the relevant regulations of the research institution. Manually collected data can be transcribed into software such as Excel for analysis and reporting. All data in each group are quantitative data. Tables show the mean ± standard deviation (SD) for each animal experimental data group, and figures show the mean ± standard error (SEM). T-tests and survival analyses were performed using Excel and GraphPad Prism software.

[0406] Experimental Procedure: Thirty female NOD / SCID mice were randomly divided into five groups, each consisting of six mice: a blank control group, a compound 47 (0.3 mg / kg) group, a compound 64 (0.3 mg / kg) group, a reference compound AZD5305 (0.3 mg / kg), and a reference compound olaparib (100 mg / kg) group. All mice were orally administered once daily. Body weights were measured every two days, and tumor length and width were measured with a vernier caliper. After 30 days of administration, tumor-bearing mice were anesthetized and sacrificed. Tumor tissue was excised, weighed, and photographed, and tumor inhibition rates were calculated.

[0407] Conclusion: The experimental results are shown in Figures 1 and 2. Following cell inoculation, each treatment group was administered for 30 consecutive days. Compared to the blank control group, each treatment group (AZD5305 0.3 mg / kg, compound 47 0.3 mg / kg, compound 64 0.3 mg / kg, and olaparib 100 mg / kg) demonstrated significant inhibitory effects on tumor growth in a NOD / SCID mouse subcutaneous tumor model of human breast cancer MM436 cells. The antitumor effects of the compound 47 0.3 mg / kg and compound 64 0.3 mg / kg groups were significantly superior to those of the reference compound olaparib 100 mg / kg group and comparable to those of the AZD5305 0.3 mg / kg group. Furthermore, after 30 days of treatment, complete tumor regression was observed in one mouse each receiving compound 64 0.3 mg / kg and AZD5305 0.3 mg / kg, demonstrating excellent antitumor efficacy.

[0408] 7. Hematological toxicity assay

[0409] CD34+ hematopoietic stem and progenitor cells (hmPB34-P-SC) were cultured overnight at 37°C in 5% CO2 in RPMI 1640 complete medium supplemented with 25 ng / mL IL-3, 25 ng / mL IL-6, 25 ng / mL SCF, and 10% fetal bovine serum. The next day, cells were resuspended and seeded at 1000 cells per well in 96-well plates. Drug treatments were added at various concentrations in triplicate. After 5 days of culture, the number of viable cells in each well was determined using the CellTiter-Glo 2.0 kit and analyzed by microplate reader.

[0410] Table 20 Compounds' inhibitory activity test results on CD34+ hematopoietic stem cell hmPB34-P-SC

[0411] Conclusion: The compounds of the present invention have weak inhibitory effects on CD34+ hematopoietic stem and progenitor cells hmPB34-P-SC, especially compounds 76, 83, 89 and 95, which have a weak inhibitory effect on hmPB34-P-SC. 50 The value was greater than 1000nM, which was significantly better than the positive drug Olaparib.

[0412] 8. Evaluation of compound distribution in cerebrospinal fluid and plasma

[0413] Experimental steps: Weigh the compound and add a small amount of DMSO, then add sodium chloride solution for injection to make 5 mg mL -1 The compound solution is ready for administration. Rats, male rats, 5 mg kg -1After intravenous administration, cerebrospinal fluid and whole blood were collected 0.25 h and 2 h after administration (n=1). The whole blood was centrifuged at 3500 rpm for 15 min, and the supernatant plasma was collected. 10 μL of plasma and 10 μL of cerebrospinal fluid were placed in a centrifuge tube and 40 μL of 20 ng·ml -1 The internal standard SAHA was precipitated with acetonitrile, vortexed for 30 seconds, centrifuged at 13000 rpm for 15 minutes, and the supernatant was transferred to a sample vial for testing. Standard curve range: 1-1000 ng ml -1 .

[0414] Table 21 Results of the test on the distribution of the compound in rat cerebrospinal fluid and plasma after administration

[0415] Conclusion: Some compounds of the present invention have good brain penetration potential, especially compounds 81, 83, 88, 89, 90, 95, 96 and 101, which have higher distribution concentrations in rat cerebrospinal fluid.

[0416] 9. In vivo pharmacodynamic study of compound 11 on the subcutaneous transplanted tumor model of breast cancer MDA-MB-436 in nude mice

[0417] Experimental procedure: 18 female NOD / SCID mice were randomly divided into a blank control group, a compound AZD9574 (3 mg / kg) group, and a compound 11 (1 mg / kg) group, with a total of 3 groups, 6 mice in each group, all of which were orally administered once a day. At the same time, the body weight was weighed every 2 days and the length and width of the tumor were measured with a vernier caliper. After 30 days of administration, the tumor-bearing mice were anesthetized and killed, the tumor tissue was removed, weighed, and photographed, and the tumor inhibition rate was calculated. Compound AZD9574 was synthesized according to the synthesis method of Example 20 of patent WO2021260092.

[0418] Conclusion: The experimental results are shown in Figure 3. After cell inoculation, each treatment group was administered for 30 consecutive days. Compared with the blank control group, the compound AZD9574 (3 mg / kg) group and the compound 11 (1 mg / kg) group showed a significant inhibitory effect on tumor growth in the NOD / SCID mouse subcutaneous tumor model of human breast cancer MDA-MB-436 cell line. The anti-tumor effect of the compound 11 1 mg / kg group was superior to that of the AZD9574 3 mg / kg group.

[0419] 10. Pharmacodynamic study of compound 11 on the intracranial model of breast cancer MDA-MB-436 nude mice

[0420] Experimental Procedure: MDA-MB-436-Luc cells (purchased from American Type Culture Collection) were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. When grown to 80% confluency, cells were harvested and centrifuged at 300 g for 10 minutes. Cells were washed three times with pre-chilled PBS and centrifuged at 300 g for 10 minutes. Cells were resuspended in PBS and counted using a hemocytometer. The cell concentration was adjusted to 2.5 × 10 7 / mL, after the cells were collected, the cell suspension was pre-cooled on ice. Take 5μL of cell suspension and inject it into the mouse skull (2mm outside, 1mm in front, 3mm deep). Ten days after inoculation, group treatment was started based on the results of in vivo imaging. The experiment used 18 female NOD / SCID mice and randomly divided them into a blank control group, a compound 11 3mg / kg dose group, and a reference compound AZD9574 3mg / kg group, for a total of 3 groups, with 6 mice in each group. Each group was orally administered once a day, and the body weight was weighed every 2 days. The fluorescence intensity of the mouse brain was measured every 7 days. The drug was stopped after 42 days of administration, and the changes in the fluorescence intensity of the mouse brain and the survival rate of the mouse were counted.

[0421] Conclusion: The experimental results are shown in Figures 4 to 6. The experimental animals were divided into experimental groups 10 days after inoculation, and each treatment group was treated continuously for 42 days. Compared with the blank control group, each treatment group (AZD9574 3 mg / kg, compound 11 3 mg / kg) had a significant inhibitory effect on the growth of intracranial tumors in the NOD / SCID mouse model of human breast cancer MDA-MB-436 cell line. The fluorescence intensity of each treatment group was significantly lower than that of the blank control group. The fluorescence intensity of the compound 11 3 mg / kg group was weaker than that of the AZD9574 3 mg / kg group, indicating a stronger intracranial anti-tumor effect. At the observation endpoint, the median survival time of the blank control group, AZD9574 3 mg / kg group, and compound 11 3 mg / kg group was NA, 37.5 days, and 61.5 days, respectively. At the observation endpoint, the survival rate of compound 11 was significantly better than that of the reference compound AZD9574, and the weight loss of compound 11 was weaker than that of the reference compound AZD9574, indicating that compound 11 has a better therapeutic effect on intracranial tumors.

Claims

1. A compound of formula I or a pharmaceutically acceptable form thereof, characterized in that: The structure of Formula I is as follows: in, X is selected from N or C, and when X is selected from N, represents a single bond, when X is selected from C, represents a double bond; X1 is selected from N or C (R 8a ), X2 is selected from N or C(R 8b ), X3 is selected from N or C(R 8c ), and at most one of X1, X2 and X3 is selected from N; R1 is selected from C 1～8 Alkylthio, C 1～8 Halogenated alkylthio, C 1～8 Deuterated alkylthio, C 1～8 Halogenated alkoxy, C 1～8 Haloalkyl or C 2～8 alkenyl; R2 is selected from hydrogen, fluorine, chlorine, C 1-4 Alkyl, C 1-4 Fluorinated alkyl or C 1-4 Deuterated alkyl; R3 and R4 are independently selected from hydrogen, deuterium, fluorine, C 1-4 Alkyl, C 1-4 Fluorinated alkyl or C 1-4 Deuterated alkyl; Alternatively, R3, R4 and the atoms to which they are attached together form a 3-6 membered alkyl ring; R5 is selected from hydrogen, fluorine, chlorine, cyano, C 1-4 Alkyl, C 1-4 Fluorinated alkyl or C 1-4 Deuterated alkyl; Alternatively, R4, R5 and the atoms to which they are attached together form a 5-7 membered alkyl heterocycle; when R4, R5 and the atoms to which they are attached are connected to form a ring, the 5-7 membered alkyl heterocycle contains 1 O heteroatom in addition to X on the main ring; R3, R4 and the atoms to which they are attached, and R4, R5 and the atoms to which they are attached, will not form a ring at the same time; R6 is selected from -C(O)-NH-R 6a ; R 6a Selected from C 1-4 Alkyl, C 1-4 Fluorinated alkyl, C 1-4 Deuterated alkyl or 3-6 membered cycloalkyl; R7 is selected from hydrogen, fluorine, chlorine, cyano, C 1-4 Alkyl, C 1-4 Fluorinated alkyl or C 1-4 Deuterated alkyl; R 8a , R 8b , R 8c independently selected from hydrogen, fluorine, chlorine, C 1-4 Alkyl, C 1-4 Deuterated alkyl, C 1-4 Fluorinated alkyl; The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled substances, metabolites or prodrugs.

2. The compound according to claim 1, characterized in that: X1 is selected from N, X2 is selected from C(R 8b ), X3 is selected from C(R 8c ); or X1 is selected from C(R 8a ), X2 is selected from N, X3 is selected from C(R 8c ); or X1 is selected from C(R 8a ), X2 is selected from C(R 8b ), X3 is selected from C(R 8c ).

3. The compound according to claim 1 or 2, characterized in that: R1 is selected from C 1～4 Alkylthio, C 1～4 Halogenated alkylthio, C 1～4 Deuterated alkylthio, C 1～4 Halogenated alkoxy, C 1～4 Haloalkyl, C 2～4 alkenyl; in R1, the halogenated halogen is selected from F, Cl, Br; Preferably, R1 is selected from methylthio, halogenated methylthio, deuterated methylthio, halogenated methoxy, halogenated methyl, halogenated ethyl or vinyl; in R1, the halogenated halogen is selected from F and Cl; More preferably, R1 is selected from -S-CH3, -S-CF2H, -S-CF3, -S-CF2Cl, -S-CD3, -O-CF2H, -O-CF3, -O-CF2Cl, -CF2-CH3 or -CH=CH2.

4. The compound according to any one of claims 1 to 3, characterized in that: At least one of the following is met: R2 is selected from hydrogen, fluorine, chlorine, methyl, fluoromethyl or deuterated methyl; R3 and R4 are independently selected from hydrogen, deuterium, fluorine, methyl, fluoromethyl or deuterated methyl; R5 is selected from hydrogen, fluorine, chlorine, cyano, methyl, fluoromethyl or deuterated methyl; R7 is selected from hydrogen, fluorine, chlorine, cyano, methyl, fluoromethyl or deuterated methyl; R 8a , R 8a , R 8c are independently selected from hydrogen, fluorine, chlorine, methyl, fluoromethyl or deuterated methyl.

5. The compound according to any one of claims 1 to 4, characterized in that: R 6a is selected from methyl, fluoromethyl, deuterated methyl or cyclopropyl; Preferably, R 6a Selected from methyl, -CF3, -CD3 or cyclopropyl.

6. The compound according to any one of claims 1 to 5, characterized in that: Structural unit Selected from:

7. The compound according to any one of claims 1 to 5, characterized in that: When R4 and R5 do not form a ring with the atoms to which they are connected, the structural unit Selected from:

8. The compound according to any one of claims 1 to 7, characterized in that: When R4 and R5 do not form a ring with the atoms to which they are connected, the structural unit Selected from:

9. The compound according to any one of claims 1 to 8, characterized in that: When R4, R5 and the atoms to which they are connected form a ring, the structural unit Selected from: Preferably, when R4, R5 and the atoms to which they are connected form a ring, the structural unit Selected from:

10. The compound according to any one of claims 1 to 8, characterized in that: The structural unit in formula I Replace with 11. The compound according to any one of claims 1 to 10, characterized in that: The compound is selected from:

12. The compound according to claim 1 or 2, characterized in that: R1 is selected from -S-CH3, -S-CF2H, -S-CF3, -S-CF2Cl, -S-CD3, -O-CF2H, -O-CF3, -O-CF2Cl, -CF2-CH3, -CH=CH2, -S-CFH2 or -O-CFH2.

13. The compound according to any one of claims 1 to 2, 4 to 5 or 12, characterized in that: Structural unit Selected from:

14. The compound according to any one of claims 1 to 2, 4 to 5, 7 to 8 or 12 to 13, characterized in that: The structural unit in formula I Replace with 15. The compound according to any one of claims 1 to 2, 4 to 5, 7 to 10 or 12 to 14, characterized in that: The compound is selected from:

16. The compound according to any one of claims 1 to 2, 4 to 5, 7 or 12 to 13, characterized in that: When R4 and R5 do not form a ring with the atoms to which they are connected, the structural unit Selected from:

17. The compound according to any one of claims 1 to 2, 4 to 5, 7 or 12 to 13 or 16, characterized in that: When R4, R5 and the atoms to which they are connected form a ring, the structural unit Selected from: Preferably, when R4, R5 and the atoms to which they are connected form a ring, the structural unit Selected from:

18. The compound according to any one of claims 1 to 2, 4 to 5, 7 or 12 to 13 or 16, characterized in that: The structural unit in formula I Replace with 19. The compound according to any one of claims 1 to 2, 4 to 5, 7, 10, 12 to 14 or 18, characterized in that: The structural unit in formula I Replace with 20. The compound according to any one of claims 1 to 2, 4 to 5 or 12 to 13, characterized in that: The structural unit in formula I Replace with R9 is selected from H.

21. The compound according to any one of claims 1 to 2, 4 to 5, 7, 10, 12 to 14 or 16 to 20, characterized in that: The compound is selected from:

22. A pharmaceutical composition, characterized in that: The active ingredient is a compound according to any one of claims 11, 15 or 21 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof, assisted by a pharmaceutically acceptable carrier.

23. The compound of any one of claims 11, 15 or 21 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite or prodrug thereof, and the pharmaceutical composition of claim 22, are used in the preparation of a medicament for preventing and / or treating PARP1 enzyme-related diseases.

24. The use according to claim 23, characterized in that: The PARP1 enzyme-related disease is a tumor-related disease.

25. The use according to claim 24, characterized in that: The tumor-like disorders are deficient in the HR-dependent DNA DSB repair pathway.

26. The use according to claim 24 or 25, characterized in that: The neoplastic disorder comprises one or more cancer cells having a reduced or absent ability to repair DNA DSBs by HR relative to normal cells.

27. The use according to claim 26, characterized in that: The cancer cells have a BRCA1 or BRCA2 deficient phenotype.

28. The use according to any one of claims 24 to 27, characterized in that: The tumor-like diseases are breast cancer, ovarian cancer, primary peritoneal cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer, glioblastoma, lung cancer, meningioma, pituitary tumor, craniopharyngioma, schwannoma, glioma, ependymoma, primitive neuroectodermal tumor, central nervous system lymphoma, germ cell tumor, metastasis, brain cancer or central nervous system cancer.

29. Use of the compound of any one of claims 11, 15 or 21 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite or prodrug thereof, and the pharmaceutical composition of claim 22 in the preparation of a PARP1 inhibitor.

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