Heterocyclic compounds and uses thereof

Abstract

This invention discloses a class of heterocyclic compounds and their uses, belonging to the field of chemical and pharmaceutical technology. The heterocyclic compounds of Formula II provided by this invention can act as PARP1 inhibitors, exhibiting advantages of high activity and high selectivity, as well as excellent pharmacokinetic properties and excellent safety.

Description

Technical Field

[0001] This invention belongs to the field of chemical medicine and relates to a class of heterocyclic compounds and their uses. Background Technology

[0002] During cell growth, its DNA is constantly damaged by various internal and external adverse factors. Among DNA damage types, the most serious 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, subsequently causing carcinogenesis or even cell death. For single-strand DNA breaks, repair primarily relies on PARP enzymes. For double-strand breaks, repair mechanisms include non-homologous end linkage and homologous recombination repair. Homologous recombination repair is a high-fidelity, error-free repair method and the main pathway for double-strand DNA repair. Many proteins are involved in homologous recombination repair, the most well-known being 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 were selectively inhibited by PARP inhibitors. Based on this research, researchers proposed the concept of synthetic lethality: the deletion of either BRCA or PARP genes is not fatal in itself, but the simultaneous inactivation of both leads 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 demonstrated excellent clinical efficacy in patients with homologous recombination-deficient cancers. However, whether used as monotherapy or in combination therapy, hematological toxicities (anemia, neutropenia, and thrombocytopenia) and other toxicities limit the application of these drugs. Related research (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-benzenesulfon amide (Pazopanib), a novel and potent vascular endothelial growth factor receptor inhibitor.[J]. Journal of Medicinal Chemistry, 2008, 51(15):4632.) suggests that these adverse reactions may stem from the inhibition of PARP2 by marketed PARP inhibitors, which is not essential for therapeutic efficacy. Highly selective PARP1 inhibitors can reduce hematological toxicity, improve the therapeutic safety window, and increase the potential for combination therapy with other chemotherapy or targeted drugs.

[0004] Therefore, there is an unmet clinical need for effective and safe PARP inhibitors, particularly PARP inhibitors selective for PARP1. The novel PARP1 inhibitor described in this invention exhibits unexpectedly high selectivity for PARP1 compared to other PARP family members (such as PARP2, PARP3, PARP5a, and PARP6), and can be used to treat diseases related to PARP function. Summary of the Invention

[0005] The purpose of this invention is to provide a class of heterocyclic compounds and their uses to achieve highly selective and efficient prevention or treatment of diseases related to PARP function.

[0006] In a first aspect, the present invention provides a compound of formula II or a pharmaceutically acceptable form thereof, said formula II having the following structure:

[0007]

[0008] in:

[0009] Indicates a single bond or a double bond;

[0010] R1 is selected from halogen, C 1-6 Alkyl or 3-6 membered cycloalkyl;

[0011] X1 is selected from N or C(R) 5a X2 is selected from N or C(R). 5b X3 is selected from N or C(R) 5c X1, X2, and X3 have exactly one selected from N;

[0012] R 2a and R 2b It is independently selected from hydrogen, deuterium, methyl, or deuterated methyl;

[0013] R3 is selected from deuterium, fluorine, and C. 1-4 Alkyl or C 1-4 At least one of the deuterated alkyl groups;

[0014] R 3a Selected from hydrogen, deuterium, fluorine, hydroxyl, cyano, C 1-4 Alkyl, C 1-4 Fluorinated alkyl or C 1-4 Alkoxy;

[0015] R4 is selected from halogen or cyano groups;

[0016] R 5a R 5a and R 5c Independently selected from hydrogen, fluorine, chlorine, and C 1-4 Alkyl, C 1-4 Deuterated alkyl, C 1-4 Fluoroalkyl, C 1-4 Alkoxy or C 1-4 Fluoroalkoxy;

[0017] R6 is selected from hydrogen, fluorine, chlorine, and C. 1-4 Alkyl, C 1-4 Fluorinated alkyl or C 1-4 Deuterated alkyl groups;

[0018] X5 is selected from nitrogen or C(R) 9a X6 is selected from nitrogen or C(R) 9b X7 is selected from nitrogen or C(R) 9c X8 is selected from nitrogen or C(R) 9d );

[0019] R 9a R 9b R 9c and R 9d Independently selected from hydrogen, fluorine, chlorine, cyano, C 1-4 Alkyl or C 1-4 Fluorinated alkyl groups;

[0020] n1 is an integer between 0 and 8;

[0021] n3 is independently selected from 0 or 1;

[0022] The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs.

[0023] In some preferred embodiments of the present invention, in the compounds represented by Formula II above, or in their pharmaceutically acceptable forms, R1 is selected from fluorine, chlorine, and C. 1-4 Alkyl or 3-4 membered cycloalkyl.

[0024] In some preferred embodiments of the present invention, in the compound represented by Formula II above or in its pharmaceutically acceptable form, R1 is selected from chloro, methyl, ethyl or cyclopropyl.

[0025] In some preferred embodiments of the present invention, in the compound represented by Formula II above or in its pharmaceutically acceptable form, R3 is selected from at least one of deuterium, methyl, or deuterated methyl.

[0026] In some preferred embodiments of the present invention, in the compound represented by Formula II above or in its pharmaceutically acceptable form, R3 is selected from deuterium or methyl.

[0027] In some preferred embodiments of the invention, the compound represented by Formula II above, or its pharmaceutically acceptable form, is used. When it is a double bond, R 3a It does not exist. When it is a single bond, R 3a Selected from hydrogen and deuterium.

[0028] In some preferred embodiments of the invention, in the compound represented by Formula II above or in its pharmaceutically acceptable form, R4 is selected from chlorine or cyano.

[0029] In some preferred embodiments of the invention, in the compound represented by Formula II above or in its pharmaceutically acceptable form, R6 is selected from hydrogen, fluorine, chlorine or methyl.

[0030] In some preferred embodiments of the present invention, in the compounds represented by Formula II above or in their pharmaceutically acceptable forms, R 5a R 5b and R 5c It is independently selected from hydrogen, fluorine, chlorine, or methyl.

[0031] In some preferred embodiments of the present invention, in the compounds represented by Formula II above or in their pharmaceutically acceptable forms, R 9a R 9b R 9c and R 9d It is independently selected from hydrogen, fluorine, or methyl.

[0032] In some more preferred embodiments of the present invention, the following compounds and their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs are provided:

[0033]

[0034] In some more preferred embodiments of the present invention, the following compounds and their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs are provided:

[0035]

[0036] In some more preferred embodiments of the present invention, the following compounds and their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs are provided:

[0037]

[0038] In a second aspect, the present invention provides a pharmaceutical composition having the aforementioned compound (Formula II) or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug as the active ingredient, supplemented by a pharmaceutically acceptable carrier.

[0039] A further object of the present invention is to provide a method for preparing the pharmaceutical composition of the present invention, the method comprising combining any compound of formula II or a pharmaceutically acceptable form thereof, or a mixture thereof, with one or more pharmaceutically acceptable carriers.

[0040] The pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention are pharmaceutically acceptable carriers, and examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (2005).

[0041] Pharmaceutical compositions can be administered in any form, as long as they achieve the purpose of preventing, alleviating, preventing, or curing symptoms in human or animal patients. For example, they can be formulated into various suitable dosage forms depending on the route of administration.

[0042] In other embodiments, the administration of the compounds or pharmaceutical compositions of the present invention may be combined with other treatment methods. These other treatment methods may be selected from, but are not limited to, radiotherapy, chemotherapy, immunotherapy, or combinations thereof.

[0043] This invention also relates to a pharmaceutical formulation comprising any compound of formula II or a pharmaceutically acceptable form thereof, or a mixture thereof, as an active ingredient, or the pharmaceutical composition of this invention. In some embodiments, the formulation is in the form of a solid dosage form, a semi-solid dosage form, a liquid dosage form, or a gaseous dosage form.

[0044] A further object of the present invention is to provide an article of manufacture, for example, in the form of a kit. The article of manufacture as used herein is intended to include, but is not limited to, medicine boxes and packaging. The article of manufacture of the present invention comprises: (a) a first container; (b) a pharmaceutical composition contained in the first container, wherein the composition comprises: a first therapeutic agent, the first therapeutic agent comprising: any compound of formula II or a pharmaceutically acceptable form thereof, or a mixture thereof; (c) optionally present packaging instructions stating that the pharmaceutical composition may be used to treat oncological conditions (as defined below); and (d) a second container.

[0045] The first container is a container for containing a pharmaceutical composition. This container may be used for the preparation, storage, transportation, and / or individual / bulk sales. The first container is intended to encompass bottles, jars, vials, flasks, syringes, tubes (e.g. for cream products), or any other container for the preparation, containment, storage, or dispensing of pharmaceutical products.

[0046] The second container is a container for holding the first container and optional instruction manuals. Examples of the second container include, but are not limited to, boxes (e.g., cardboard or plastic boxes), cartons, cartons, bags (e.g., paper or plastic bags), sachets, and cloth bags. The instruction manuals may be physically attached to the outside of the first container by cable ties, glue, U-staples, or other adhesive methods, or they may be placed inside the second container without any physical means of attachment to the first container. Alternatively, the instruction manuals may be located outside the second container. When located outside the second container, it is preferable that the instruction manuals be physically attached by cable ties, glue, U-staples, or other adhesive methods. Alternatively, they may be adjacent to or in contact with the outside of the second container without physical attachment.

[0047] The package insert, such as a trademark, label, or marking, lists information relating to the pharmaceutical composition contained within the first container. The listed information is typically determined by the regulatory authority governing the region where the product is to be sold (e.g., the U.S. Food and Drug Administration). Preferably, the package insert specifically lists the approved indications for which the pharmaceutical composition is used. The package insert can be made of any material from which information contained therein or on the material can be read. Preferably, the package insert is made of a printable material (e.g., paper, plastic, cardboard, foil, adhesive paper, or plastic) on which the desired information can be formed (e.g., printed or coated).

[0048] Thirdly, the present invention provides the use of the aforementioned compounds, compounds of formula II, and related specific compounds or pharmaceutically acceptable forms thereof, or the pharmaceutical compositions of the present invention, in the preparation of medicaments for the prevention or treatment of PARP1 enzyme-related diseases.

[0049] The present invention provides a method for preventing or treating PARP1 enzyme-related diseases, the method comprising administering to an individual in need a compound of formula II or a pharmaceutically acceptable form thereof, or a pharmaceutical composition of the present invention.

[0050] The present invention provides compounds of formula II or their pharmaceutically acceptable forms, or pharmaceutical compositions thereof, for the prevention or treatment of PARP1 enzyme-related diseases.

[0051] The present invention provides a method for preventing or treating PARP1 enzyme-related diseases by combining a compound of Formula II or a pharmaceutically acceptable form thereof, or a pharmaceutical composition thereof, with other treatment methods including but not limited to: radiotherapy, chemotherapy, immunotherapy, or combinations thereof.

[0052] In some implementations, the PARP1 enzyme-related disease is a disease that is sensitive to or responsive to PARP1 enzyme inhibition.

[0053] In some implementations, the PARP1 enzyme-related disease is a tumor-related condition.

[0054] In some preferred embodiments, the tumor-related diseases lack the HR-dependent DNA DSB repair pathway.

[0055] In some preferred embodiments, the tumor-like condition comprises one or more cancer cells that have a reduced or absent ability to repair DNA DSB via HR compared to normal cells.

[0056] In some preferred embodiments, the cancer cells have a BRCA1 or BRCA2 defective phenotype.

[0057] In some embodiments, the PARP1 enzyme-related diseases are oncological conditions, including but not limited to solid and hematologic malignancies. In further embodiments, the oncological conditions include, but are 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 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 lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML)), multiple myeloma, or lymphoma.

[0058] In some preferred embodiments, the PARP1 enzyme-related diseases are breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer, or lung cancer.

[0059] In a further preferred embodiment, the compounds of the present invention can be used in combination with radiotherapy, chemotherapy, or immunotherapy to prevent or treat cancer.

[0060] The beneficial effects of this invention are:

[0061] This invention provides a novel class of highly active and selective PARP1 inhibitors that can achieve 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, with weak inhibitory effect on non-homologous recombination-deficient cells; (4) excellent pharmacokinetic properties (e.g., good bioavailability, suitable half-life, and duration of action); and (5) excellent safety (low toxicity and / or fewer side effects, and a wider therapeutic window).

[0062] Terminology definition:

[0063] 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 “comprising,” “including,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps. Those skilled in the art will understand that the foregoing term “comprising” encompasses the meaning of “consisting of.”

[0064] In this invention, the terms "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 as indicating that the composition comprises "one or more" pharmaceutically acceptable excipients.

[0065] When the lower and upper limits of a numerical range are disclosed, any numerical value falling within that range and any included range are specifically disclosed. In particular, each range of values ​​disclosed herein (in the form of “about a to b”, or equivalently, “approximately a to b”, or equivalently, “about ab”) should be understood to represent each numerical value and range encompassed within a wider range.

[0066] For example, the expression "C" 1-6 "This should be understood as encompassing any subrange and each point value, such as C." 2-5 C3-4 C1-2, C 1-3 C 1-4 C 1-5 And so on, as well as C1, C2, C3, C4, C5, C6, etc.

[0067] Unless otherwise stated, all other uses of this material are permitted. It indicates a single or double bond.

[0068] In this invention, unless otherwise stated, halogen refers to fluorine, chlorine, bromine or iodine.

[0069] In this invention, unless otherwise stated, "alkyl" includes straight-chain or branched monovalent saturated hydrocarbon groups. For example, alkyl groups include 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, "C 1-4 C in "alkyl" 1-4 It refers to a group that contains 1, 2, 3 or 4 carbon atoms arranged in a straight chain or branched chain.

[0070] In this invention, unless otherwise stated, “cycloalkyl,” “carbocyclic,” 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 spirocyclic groups, such as bicyclic [1.1.1]pentyl, bicyclic [2.2.1]heptyl, bicyclic [3.2.1]octyl, bicyclic [5.2.0]nonyl, decahydronaphthyl, etc. For example, “C 3-12 cycloalkyl” refers to a cycloalkyl group having 3-12 cyclic carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). The cycloalkyl or cycloalkylene groups in this invention may optionally be substituted by one or more substituents described in this invention.

[0071] In this invention, unless otherwise stated, "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-aryl group that is optionally substituted with one or more (e.g., 1-3) fluorine atoms. 1-4 Alkyl groups. Those skilled in the art will understand that when there is more than one fluorine atom substituent, the fluorine atoms can be the same or different, and can be located on the same or different C atoms. Examples of haloalkyl groups include, for example, -CH2F, -CHF2, -CF3, -C2F5, -CH2CF3, -CH2CH2CF3, etc. The fluoroalkyl groups in this invention are optionally substituted with one or more substituents described in this invention.

[0072] This invention also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the compounds of this invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compounds of this 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 (e.g., 37Cl); isotopes of iodine (e.g., 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes (e.g., O); 32 P); and isotopes of sulfur (e.g., ... 34 S).

[0073] In this invention, "polymorph" refers to different solid crystalline phases resulting from the presence of two or more different molecular arrangements in the solid state of certain compounds of this invention. Some compounds of this invention may exist in more than one crystal form, and this invention aims to include various crystal forms and mixtures thereof. Typically, crystallization produces solvates of the compounds of this invention. The term "solvate" as used in this invention refers to an aggregate comprising one or more molecules of the compound of this invention and one or more solvent molecules. The solvent may be water, in which case the solvate is a hydrate. Alternatively, the solvent may be an organic solvent. Therefore, the compounds of this invention can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and corresponding solvated forms. The compounds of this invention can form true solvates, but in some cases, they may also remain only as indeterminate water or a mixture of water and a portion of indeterminate solvent. The compounds of this invention can react in a solvent or precipitate or crystallize from a solvent. The solvates of the compounds of this invention are also included within the scope of this invention. This invention also covers all possible crystalline forms or polymorphs of the compounds of this invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0074] In this invention, "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of this invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, and imine-enamine tautomers. It is to be understood that the scope of this invention covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0075] In this invention, pharmaceutically acceptable salts include their acid addition salts and base addition salts. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts. A review of suitable salts can be found, for example, in “Remington’s Pharmaceutical Sciences,” Mack Publishing Company, Easton, Pa., (2005); and “Handbook of Pharmaceutical Salts: Properties, Selection, and Use,” Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of this invention are known to those skilled in the art. “Pharmaceutically acceptable acid addition salt” refers to a salt formed with an inorganic or organic acid that retains the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecanoate, glycolate, gluconate, lactate, sebate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylic acid, and naphthalenedisulfonate. These salts can be prepared by methods known in this patent. "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Preferred inorganic salts are ammonium salts, sodium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins.Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in this patent.

[0076] In this invention, unless otherwise stated, "ester" refers to an ester derived from the compounds described herein, including physiologically hydrolyzable esters (compounds of this invention that can be hydrolyzed under physiological conditions to release free acids or alcohols). The compounds of this invention may themselves be esters.

[0077] The compounds of the present invention can exist as solvates (preferably hydrates), wherein the compounds of the present invention contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, can be stoichiometric or non-stoichiometric.

[0078] Those skilled in the art will understand that not all nitrogen-containing heterocycles can form nitrogen oxides because nitrogen requires available lone pairs of electrons to be oxidized. Those skilled in the art will identify nitrogen-containing heterocycles capable of forming nitrogen oxides. They will also recognize that tertiary amines can form nitrogen oxides. Synthetic methods for preparing nitrogen oxides of heterocycles and tertiary amines are well known to those skilled in the art, including the oxidation of heterocycles and tertiary amines with peroxyacids such as peracetic acid and m-chloroperoxybenzoic acid (mCPBA), hydrogen peroxide, alkyl peroxides such as tert-butyl peroxide, sodium perborate, and dioxiranes such as dimethyldioxirane. These methods for preparing nitrogen oxides have been extensively described and reviewed in the literature, see, for example: T.L. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp. 748-750 (AR. Katritzky and A.J. Boulton, Eds., Academic Press); and G.W. H. Heeseman and E.S. G. Wierstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp. 390-392 (AR. Katritzky and A.J. Boulton, Eds., Academic Press).

[0079] In this invention, "metabolite" refers to a substance formed in the body upon administration of a compound of the present invention. Metabolites of the compound can be identified using techniques known in the art, and their activity can be characterized by experimental methods. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of the present invention, including compounds obtained by methods that expose the compounds of the present invention to mammals for a time sufficient to produce their metabolites.

[0080] In this invention, a "prodrug" refers to certain derivatives of the compounds of the invention that, when administered to or onto the body, can be converted, for example, by hydrolysis and cleavage into the compounds of the invention having the desired activity. Typically, such prodrugs are functional group derivatives of the compounds that readily convert in vivo into the desired therapeutically active compounds. Further information regarding the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems," Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella). The prodrugs of the invention can be prepared, for example, by replacing suitable functional groups present in the compounds of the invention with portions known to those skilled in the art as "pro-moiety" (e.g., as described in "Design of Prodrugs," H. Bundgaard (Elsevier, 1985)).

[0081] In this application, "pharmaceutical composition" refers to a formulation of the compounds of the present invention with a medium generally accepted in the art for delivering bioactive compounds to mammals (e.g., humans). This medium includes pharmaceutically acceptable carriers. The purpose of the pharmaceutical composition is to facilitate administration to the organism, enhance the absorption of the active ingredient, and thereby exert its bioactivity.

[0082] In this application, "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that is permitted by the relevant government regulatory authority or is acceptable for human or livestock use.

[0083] As used herein, the terms “drug combination,” “drug co-administration,” “combination therapy,” “administration of other treatments,” and “administration of other therapeutic agents” refer to pharmaceutical treatments achieved by mixing or combining more than one active ingredient, including fixed and non-fixed combinations of active ingredients. The term “fixed combination” refers to the simultaneous administration to a patient of at least one compound described herein and at least one synergistic agent in the form of a single entity or dosage form. The term “non-fixed combination” refers to the simultaneous, combined, or sequential administration to a patient of at least one compound described herein and at least one synergistic agent in the form of a single entity at variable intervals. These also apply to cocktail therapies, such as the administration of three or more active ingredients.

[0084] In this invention, unless otherwise stated, "tumor" includes, but is not limited to, diseases such as leukemia, gastrointestinal stromal tumor, histiocytic lymphoma, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, squamous cell carcinoma of the lung, adenocarcinoma of the lung, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell carcinoma, cervical cancer, ovarian cancer, intestinal cancer, nasal cancer, brain cancer, bone cancer, esophageal cancer, melanoma, kidney cancer, and oral cancer.

[0085] In this invention, unless otherwise stated, "treatment" means reversing, alleviating, or inhibiting the progression of a disease or condition or one or more symptoms of such a disease or condition, or preventing such a disease or condition or one or more symptoms of such a disease or condition.

[0086] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention. Attached Figure Description

[0087] Figure 1 The figure shows the changes in tumor volume in MDA-MB-436 nude mice after administration of compounds 2 and 5. Detailed Implementation

[0088] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product manual.

[0089] The reagents and raw materials used in the embodiments of this invention are all commercially available.

[0090] Table 1. Abbreviations and their meanings in this invention.

[0091]

[0092]

[0093] The structure of the compound described in this invention was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 NMR 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 expressed as 10⁻⁶. -6 (ppm) is given as the unit.

[0094] MS measurements were performed using an Agilent SQD (ESI) mass spectrometer (manufacturer: Agilent, signal: 6110).

[0095] HPLC determinations were performed using an Agilent 1200DAD high-performance liquid chromatograph (Sunfirc C18, 150X 4.6mm, 5µm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C18, 150X 4.5mm, 5µm column).

[0096] The silica gel plates used for thin-layer chromatography are Qingdao Ocean GF254 silica gel plates. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15mm-0.2mm, while the silica gel plates used for thin-layer chromatography separation and purification of products have a diameter of 0.4mm-0.5mm.

[0097] Column chromatography typically uses Qingdao Marine 100-200 or 200-300 mesh silica gel as a carrier.

[0098] Unless otherwise specified in the following examples, all reactions are carried out under an argon or nitrogen atmosphere. An argon or nitrogen atmosphere refers to a reaction flask connected to an approximately 1L argon or nitrogen balloon. A hydrogen atmosphere refers to a reaction flask connected to an approximately 1L hydrogen balloon. Hydrogenation reactions are typically performed under vacuum, followed by hydrogen filling, and repeated three times.

[0099] intermediate preparation

[0100] Intermediate INT1: 7-(chloromethyl)-3-ethyl-1,5-naphthidium-2(1H)-one

[0101]

[0102] Step 1: Compound INT1a (20 g, 95.1 mmol) and selenium dioxide (16 g, 144 mmol) were added to a 250 mL reaction flask, followed by 120 mL of 1,4-dioxane. The mixture was heated to 110 °C and stirred overnight. After the reaction was complete as monitored by TLC, the reaction solution was filtered, and the residue was washed with ethyl acetate. The filtrates were combined and concentrated by rotary evaporation. The crude product was purified by column chromatography to give compound INT1b (16 g, yellow solid). LC-MS: ESI [M+H] + =225.2.

[0103] Step 2: Add sodium hydride (6.86 g, 171.4 mmol) to a 250 ml reaction flask, then add 60 ml of 1,4-dioxane, and purge with nitrogen three times. Cool to 0 °C, and slowly add triethyl 2-phosphonobutyrate (43.2 g, 171.4 mmol) under nitrogen protection. Stir the reaction at 0 °C for 10 minutes, then heat to room temperature and stir for 10 minutes, then heat to 40 °C and stir for 5 minutes. Cool the reaction to -78 °C. Slowly add INT1b (16 g, 71.4 mmol) dissolved in 60 ml of 1,4-dioxane solution, and maintain the reaction at -78 °C with stirring for one small-scale test. After the reaction is complete as monitored by TLC, quench the reaction solution by slowly adding ice to a saturated ammonium chloride aqueous solution. Extract three times with 150 ml of ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, filter, and evaporate to dryness. The crude product was purified by column chromatography to give compound INT1c (13.26 g, brown liquid). LC-MS: ESI [M+H] + =323.3.

[0104] Step 3: Compound INT1c (13.26 g, 41.1 mmol) was added to 100 mL of anhydrous ethanol, followed by Pd / C (1.33 g, 10%). The mixture was stirred overnight at room temperature. After the reaction was complete as monitored by LC-MS, the reaction solution was filtered, and the residue was washed with copious amounts of ethanol. The filtrates were combined and concentrated by rotary evaporation. A 1,4-dioxane solution (50 mL) of 4 mol / L hydrochloric acid was added, and the mixture was stirred at room temperature for 30 minutes. Diethyl ether was added, precipitating a large amount of solid. The solid was filtered and dried to obtain compound INT1d (7.32 g, white solid). LC-MS: ESI [M+H] + =249.3; 1H NMR (400MHz, DMSO-d6) δ10.44(s,1H),8.62(d,J=1.9Hz,1H),7.75(d,J=1.9Hz,1H),4.34(q,J=7.1Hz,2H),3.87(s,0H),3.24(dd,J= 16.8, 6.3Hz, 1H), 2.97 (dd, J = 16.8, 10.1Hz, 1H), 1.81–1.65 (m, 1H), 1.52–1.36 (m, 1H), 1.32 (t, J = 7.1Hz, 3H), 0.93 (t, J = 7.4Hz, 3H).

[0105] Step 4: Add compound INT1d (7.32 g, 29.5 mmol) to a 250 ml reaction flask, then add 120 ml of 1,4-dioxane, followed by DDQ (7.38 g, 32.5 mmol). Reflux overnight. After the reaction is complete as monitored by LC-MS, concentrate the reaction solution by rotary evaporation, add saturated sodium bicarbonate aqueous solution, stir for 1 hour, filter, wash the residue with water, then wash with a small amount of diethyl ether, and dry to obtain compound INT1e (2.31 g, yellow solid). LC-MS: ESI [M+H] + =247.3.

[0106] Step 5: Add compound INT1e (2.0 g, 8.1 mmol) to a 150 mL reaction flask, add 60 mL of tetrahydrofuran, cool to 0 °C, then add a 2.5 mol / L lithium aluminum hydride tetrahydrofuran solution (6.48 mL, 16.2 mmol), and react at 0 °C for 2 hours. After the reaction is complete as monitored by TLC, add 5 mL of water to quench the reaction, add a large amount of anhydrous sodium sulfate to dry, filter, wash the residue with a large amount of dichloromethane, combine the filtrates, concentrate by rotary evaporation, and dry to obtain compound INT1f (1.2 g, white solid). LC-MS: ESI [M+H] + =205.3; 1 H NMR (400MHz, DMSO-d6) δ11.87(s,1H),8.03(d,J=2.0Hz,1H),7.36(d,J=1.0Hz,1H),7 .34(dd,J=2.0,0.9Hz,1H),4.51(s,2H),2.52(d,J=1.8Hz,1H),1.15(t,J=7.4Hz,3H).

[0107] Step 6: Add compound INT1f (0.82 g, 4.0 mmol) to a 50 ml reaction flask, along with 20 ml of dichloromethane and 1 ml of N,N-dimethylformamide. Cool to 0 °C, and add sulfoxide (0.87 ml, 12 mmol) dropwise. React at 0 °C for 1 hour. After the reaction is complete as monitored by TLC, concentrate the reaction solution by rotary evaporation. The crude product is purified by column chromatography to obtain compound INT1 (0.66 g, gray solid). LC-MS: ESI [M+H] + =223.7. 1 H NMR (400MHz, DMSO) δ12.16 (s, 1H), 8.55 (s, 1H), 7.97–7.73 (m, 2H), 4.95 (s, 2H), 2.56 (d, J = 7.4Hz, 2H), 1.19 (t, J = 7.4Hz, 3H).

[0108] Intermediate INT2: 7-(chloromethyl)-3-cyclopropyl-1,5-naphthyridine-2(1H)-one

[0109]

[0110] Step 1: Weigh compound INT2a (15 g, 72.4 mmol) and triethyl phosphite (24.1 g, 144.9 mmol) into a reaction flask. Under nitrogen protection, stir the mixture at 130 °C for 24 h. After the reaction, purify the solution by column chromatography to obtain a colorless liquid INT2b (10 g, 52%). LC-MS: ESI [M+H] + =265.1.

[0111] Step 2: Dissolve INT2b (10g, 37.8mmol) in 100mL THF. Under nitrogen protection, slowly add NaH (60%, 2.3g, 56.8mmol) solid at 0℃. After the addition is complete, stir for 0.5h, then stir at room temperature for 10min. Finally, slowly add INT1b (10.2g, 45.4mmol) in THF solution at -78℃. After the addition is complete, maintain -78℃ and stir for 1h. After confirming the completion of the reaction by LC-MS, quench the reaction with saturated aqueous NH4Cl solution. Extract with EA (150mL × 3). Combine the organic phases and dry with anhydrous Na2SO4. Filter and evaporate to dryness. Purify by column chromatography to obtain a yellow oily substance INT2c (10g, 79%). LC-MS: ESI [M+H] + =335.1.

[0112] Step 3: Weigh INT2c (10 g, 29.9 mmol) into a reaction flask, dissolve it in 150 mL of glacial acetic acid, then slowly add Fe powder (5.0 g, 89.7 mmol). After the addition is complete, heat to 70 °C and stir for 2 h. After cooling to room temperature, filter under vacuum and wash the filter cake with a small amount of DCM and MeOH. Concentrate the filtrate to dryness under vacuum, and then purify by column chromatography to obtain a pale yellow solid INT2d (1.85 g, 24%). LC-MS: ESI [M+H] + =259.1.

[0113] Step 4: Weigh compound INT2d (1.85 g, 7.2 mmol) into a reaction flask, add 20 mL of THF, stir at -20 °C, and slowly add DIBAL-H (1.5 M in toluene, 16 mL, 25.1 mmol) dropwise under nitrogen protection. After the addition is complete, return to room temperature and stir for 0.5 h. Quench the reaction by adding saturated potassium sodium tartrate aqueous solution dropwise at 0 °C, stir overnight at room temperature, extract three times with a mixed solution of DCM and MeOH (3:1), combine the organic phases, dry with anhydrous Na2SO4, filter and evaporate to dryness to obtain a pale yellow solid crude product INT2e (1.46 g, 94%). LC-MS: ESI [M+H] + =217.1.

[0114] Step 5: Weigh INT2e (1.46 g, 6.8 mmol) and DMF (98.7 mg, 1.4 mmol) into a reaction flask, add 50 mL of toluene to dissolve them, and slowly add SOCl2 (1.1 g, 9.5 mmol) dropwise at 0 °C. After the addition is complete, slowly raise the temperature to room temperature and stir overnight. After the reaction is complete, concentrate the solvent under vacuum, and purify by column chromatography with DCM and MeOH (0-7%) to obtain a pale yellow solid compound INT2 (1.24 g, 78%). LC-MS: ESI [M+H] + =235.1.

[0115] Example 1: 1'-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthid-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-carboxamide

[0116]

[0117] Step 1: Compounds 1a (1 g, 4.6 mmol), 1b (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 dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to give compound 1c (1 g, white solid).

[0118] Step 2: Add 1c (1g, 3mmol), methylamine aqueous solution (5g, 161.3mmol), and anhydrous methanol (20ml) to a 100ml reaction flask, and stir overnight at room temperature. After the reaction is complete as monitored by TLC, concentrate the reaction solution under reduced pressure to dryness to obtain compound 1d (0.8g, white solid).

[0119] Step 3: Add compound 1d (0.5g, 1.5mmol) to 10ml of anhydrous methanol, followed by 10ml of 4mol / L dioxane hydrochloride solution. Stir at room temperature for 0.5-1h. After the reaction is complete as monitored by TLC, concentrate the reaction solution under reduced pressure to obtain compound 1e (0.5g, white solid).

[0120] Step 4: Compound 1e (0.05 g, 0.23 mmol), INT1 (0.06 g, 0.28 mmol), N,N-diisopropylethylamine (0.15 g, 1.15 mmol), and potassium iodide (0.19 g, 1.15 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 1 (0.02 g, white solid); LC-MS: ESI [M+H] + =404.5; 1 H NMR (400MHz, DMSO): 11.85(s,1H),8.71(d,J=5.0Hz,1H),8.69(s,1H),8.41(d,J=1.3Hz,1H),8.06–7.91(m,2H),7.75(s,1H),7.6 4(s,1H),6.42(s,1H),3.72(s,2H),3.16(s,2H),2.81(d,J=4.8Hz,3H),2.70(s,2H),2.54(d,J=7.4Hz,4H),1.18(t,J=7.4Hz,3H).

[0121] Example 2: 1'-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthid-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridine]-5-nitrile

[0122]

[0123] Step 1: Compound 2a (0.5 g, 2.7 mmol), 1b (1.02 g, 3.3 mmol), Pd(dppf)Cl2 (0.2 g, 0.27 mmol), and potassium carbonate (0.94 g, 6.8 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 2b (0.7 g, white solid).

[0124] Step 2: Compound 14b (0.5 g, 1.8 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 2c (0.2 g, white solid).

[0125] Step 3: Compound 2c (0.05 g, 0.27 mmol), INT1 (0.072 g, 0.32 mmol), N,N-diisopropylethylamine (0.17 g, 1.35 mmol), and potassium iodide (0.22 g, 1.35 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 2 (0.021 g, white solid). LC-MS: ESI [M+H] + =372.1. 1H NMR (400MHz, DMSO) δ11.86(s,1H),8.94(d,J=1.9Hz,1H),8.44(t,J=19.0Hz,1H),8.34–8.13(m,1H),7.78–7.69(m,2H),7.65(s,1H),6.92(d,J=1 2.0Hz,1H),3.73(s,2H),3.55(d,J=14.7Hz,2H),3.21(d,J=2.7Hz,2H),2 .69(t,J=5.5Hz,2H), 2.54(d,J=7.4Hz,2H), 1.18(td,J=7.3,2.8Hz,3H).

[0126] Example 3: 1'-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthid-3-yl)methyl)-5-fluoro-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-nitrile

[0127]

[0128] Step 1: Compound 3a (0.5 g, 2.5 mmol), 1b (0.81 g, 2.6 mmol), Pd(dppf)Cl2 (0.18 g, 0.25 mmol), and potassium carbonate (0.86 g, 6.3 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 3b (0.52 g, white solid).

[0129] Step 2: Compound 3b (0.5 g, 1.7 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 3c (0.2 g, white solid).

[0130] Step 3: Compound 3c (0.05 g, 0.25 mmol), INT1 (0.066 g, 0.3 mmol), N,N-diisopropylethylamine (0.16 g, 1.25 mmol), and potassium iodide (0.21 g, 1.25 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 3 (0.024 g, white solid). LC-MS: ESI [M+H] + =390.1. 1 H NMR (400MHz, DMSO) δ11.85(s,1H),8.77(s,1H),8.41(d,J=1.7Hz,1H),8.12(dd,J=10.9,1.6Hz,1H),7.75(s,1H),7.64(d,J=1.1 Hz,1H),6.74–6.62(m,1H),3.73(s,2H),3.19(d,J=2.7Hz,2H),2.70(t,J=5.5Hz,2H),2.60–2.52(m,4H),1.18(t,J=7.4Hz,3H).

[0131] Example 4: 4-(1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthid-3-yl)methyl)-1,2,3,6-tetrahydropyridin-4-yl)-3-fluorobenzonitrile

[0132]

[0133] Step 1: Compound 4a (0.5 g, 2.5 mmol), 1b (0.81 g, 2.6 mmol), Pd(dppf)Cl2 (0.18 g, 0.25 mmol), and potassium carbonate (0.87 g, 6.3 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 4b (0.63 g, white solid).

[0134] Step 2: Compound 4b (0.5 g, 1.7 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 4c (0.2 g, white solid).

[0135] Step 3: Compound 4c (0.05 g, 0.25 mmol), INT1 (0.066 g, 0.3 mmol), N,N-diisopropylethylamine (0.16 g, 1.25 mmol), and potassium iodide (0.21 g, 1.25 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 4 (0.026 g, white solid). LC-MS: ESI [M+H] + =389.4. 1 H NMR (400MHz, DMSO) δ11.85(s,1H),8.41(s,1H),7.83(d,J=11.2Hz,1H),7.75(s,1H),7.67(d,J=11.7Hz,2H),7.57(t,J=7.9Hz ,1H),6.16(s,1H),3.71(s,2H),3.15(s,2H),2.67(t,J=5.1Hz,2H),2.54(d,J=7.4Hz,2H),2.49(s,2H),1.19(t,J=7.4Hz,3H).

[0136] Example 5: 3-Ethyl-7-(5-fluoro-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-methyl)-1,5-naphthidium-2(1H)-one

[0137]

[0138] Step 1: Compound 5a (0.5 g, 2.8 mmol), 1b (0.92 g, 3.4 mmol), Pd(dppf)Cl2 (0.21 g, 0.52 mmol), and potassium carbonate (0.99 g, 13 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to give compound 5b (0.7 g, white solid).

[0139] Step 2: Compound 5b (0.5 g, 1.8 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 5c (0.3 g, white solid).

[0140] Step 3: Compound 5c (0.05 g, 0.25 mmol), INT1 (0.063 g, 0.30 mmol), N,N-diisopropylethylamine (0.17 g, 1.25 mmol), and potassium iodide (0.21 g, 1.25 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 5 (0.020 g, white solid). LC-MS: ESI [M+H] + =365.2. 1 H NMR (400MHz, DMSO) δ11.85(s,1H),8.51(d,J=2.9Hz,1H),8.41(d,J=1.7Hz,1H),7.75(s,1H),7.70(dd,J=8.8,3.0Hz,1H),7.66(d,J=3.6Hz,1H) ,7.61(dd,J=8.9,4.5Hz,1H),6.63(s,1H),3.72(s,2H),3.15(d,J=2.7Hz,2H),2.68(t,J=5.6Hz,2H),2.61–2.53(m,4H),1.19(t,J=7.4Hz,3H).

[0141] Example 6: 1'-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthid-3-yl)methyl)-2-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-nitrile

[0142]

[0143] Step 1: Compound 6a (0.5 g, 2.5 mmol), 1b (0.94 g, 3.0 mmol), Pd(dppf)Cl2 (0.18 g, 0.25 mmol), and potassium carbonate (0.88 g, 6.3 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 6b (0.6 g, white solid).

[0144] Step 2: Compound 6b (0.5 g, 1.7 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 6c (0.3 g, white solid).

[0145] Step 3: Compound 6c (0.05 g, 0.25 mmol), INT1 (0.067 g, 0.3 mmol), N,N-diisopropylethylamine (0.16 g, 1.25 mmol), and potassium iodide (0.21 g, 1.25 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 6 (0.024 g, white solid). LC-MS: ESI [M+H] + =386.2. 1 H NMR (400MHz, DMSO) δ11.87(s,1H),8.42(d,J=1.4Hz,1H),7.84(d,J=7.8Hz,1H),7.74(d,J=7.6Hz,2H),7.66(s,1H),5.76(d, J=1.5Hz,1H),3.73(s,2H),3.14(t,J=9.5Hz,2H),2.75–2.64(m,2H),2.60–2.53(m,2H),2.36(s,2H),1.19(t,J=7.4Hz,3H).

[0146] Example 7: 1'-((7-ethyl-4-fluoro-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridine]-5-nitrile

[0147]

[0148] Step 1: Compound 7a (180 g, 0.763 mol) was slowly added to a methanol (2 L) solution of MeONa (618.0 g, 11.4 mol, 15.0 eq) at 0 °C. The reaction mixture was stirred at 20 °C for 1 hour, and then quenched with a saturated aqueous solution of NH4Cl (2 L) while stirring. The resulting white precipitate was filtered off, and the filter cake was washed with water (500 mL x 3) and dried under vacuum. A white solid product 7b (150 g, 84.5% yield) was given. 1 HNMR (400M Hz, DMSO-d6) δ (ppm) 9.06 (d, J = 2.4 Hz, 1H), 8.78 (d, J = 2.4 Hz, 1H), 4.07 (s, 3H).

[0149] Step 2: To a solution of compound 7b (150 g, 0.647 mol) in a mixed solvent of 1,4-dioxane (2.4 L) and water (600 mL), K₂CO₃ (178.8 g, 1.29 mol, 2 eq.), potassium vinyltrifluoroborate (104.0 g, 0.776 mmol, 1.2 eq.), and Pd(dppf)Cl₂ (14.2 g, 19.4 mmol, 0.03 eq.) were added. The mixture was degassed and backfilled three times with N₂, then stirred at 80 °C for 8 hours. Volatile substances were removed under reduced pressure, and the resulting mixture was extracted with ethyl acetate (1 L * 3), dried over anhydrous Na₂SO₄, and the combined organic layers were concentrated and purified by silica gel column chromatography (0-10% ethyl acetate / petroleum ether). This yielded a yellow solid product 7c (81.5 g, 70% yield). 1 HNMR(400M Hz,DMSO-d6)δ(ppm)8.96(d,J=2.7Hz,1H),8.57(d,J=2.8Hz,1H),6.82(dd,J=17.8,1 1.3Hz, 1H), 6.15 (dd, J=17.7, 1.0Hz, 1H), 5.56 (dd, J=11.3, 1.0Hz, 1H), 4.05 (s, 3H).

[0150] Step 3: Under N2, Pd / C (10% wt, 5.0 g) was added to a solution of 7c (50.0 g, 0.28 mol) in methanol (500 mL). The mixture was degassed and backfilled three times with hydrogen, then stirred at room temperature for 12 h under a hydrogen atmosphere. After the reaction was complete, the mixture was filtered through diatomaceous earth and washed with ethyl acetate (50 mL * 3). The combined filtrates were concentrated under vacuum to give a deep purple solid 7d (38.8 g, 92% yield). 1 H NMR (400MHz, DMSO-d6) δ7.33 (d, J=2.8Hz, 1H), 6.85 (dt, J=2.8, 0.7Hz, 1H), 4.65 (br s, 2H), 3.73 (s, 3H), 2.42 (q, J=7.5Hz, 2H), 1.08 (t, J=7.5Hz, 3H).

[0151] Step 4: Diethyl 2-(ethoxymethylene)malonate (27.3 g, 0.126 mol, 25.5 mL, 1.2 eq) was added in a single batch to a solution of 7d (16 g, 0.105 mol) in EtOH (300 mL). The reaction mixture was refluxed for 2 hours with stirring. TLC showed that the reaction was complete. After cooling to room temperature, the mixture was concentrated under vacuum to give a dark purple residue. The product was further purified by silica gel column chromatography (0-10% ethyl acetate / petroleum ether) to give compound 7e (32.0 g, 94% yield, white solid). 1 HNMR (400MHz, DMSO-d6) δ10.65(s,1H),8.28(s,1H),8.05(d,J=2.8Hz,1H),7.66(d,J=2.8Hz,1H),4.15( dd,J=32.3,6.9Hz,4H),3.86(s,3H),2.54(q,J=7.5Hz,2H),1.24(q,J=6.4Hz,6H),1.14(t,J=7.5Hz,3H).

[0152] Step 5: Compound 7e (32.0 g, 0.099 mol) was added to a 1.0 L three-necked round-bottom flask equipped with a reflux condenser and a mechanical stirrer, followed by the addition of a phenyl ether-biphenyl eutectic (CAS: 8004-13-5, 300 mL). The system was degassed and purged with N2 three times, then placed in an oil bath preheated to 240 °C. The reaction mixture was stirred at 250-260 °C for 1 hour, then cooled to room temperature. TLC showed the reaction was complete. 1.2 L of diisopropyl ether was added with stirring, and the mixture was stirred at room temperature for 1 hour. The resulting grayish-white precipitate was collected by filtration, washed with diisopropyl ethyl ether (100 mL * 3), and dried under vacuum. The product 7f (21.3 g, 77% yield) was given as a grayish-white solid. LC-MS: ESI [M+H] + =277.1.

[0153] Step 6: At 0–5 °C, DAST (37.3 g, 0.23 mol, 30.5 mL, 3.0 eq) was slowly added to a suspension of compound 7f (21.3 g, 0.077 mol) in DCM (400 mL). The ice bath was removed, and the reaction mixture was stirred at room temperature for 8 hours until a clear orange solution was formed. TLC showed that only trace amounts of the starting material remained (DCM / MeOH = 20:1, Rf = 0.3), and a major product (PE / EA = 5:1, Rf = 0.35) was produced. The reaction was quenched at 0–5 °C with a saturated aqueous solution of NaHCO3 (1.0 L) until pH = 8. The organic layer was separated, and the remaining aqueous phase was extracted with DCM (200 mL * 2). The combined DCM layers were washed with a saturated NaHCO3 solution (200 mL), then washed with water (200 mL), dried over anhydrous Na2SO4, and evaporated to dryness. The product was purified by silica gel column chromatography (PE / EA = 20:1 to 10:1) to give 7 g (18.0 g, 84% yield, white needle-like solid). 1 H NMR (400MHz, CDCl3) δ9.15(d,J=8.5Hz,1H),8.02(d,J=1.5Hz,1H),4.48(q,J=7 .1Hz, 2H), 2.79 (q, J = 7.4Hz, 2H), 1.45 (t, J = 7.1Hz, 3H), 1.33 (t, J = 7.4Hz, 3H).

[0154] Step 7: 7 g (13.0 g, 0.047 mol) of the compound was added to anhydrous THF (300 mL). The reaction mixture was cooled to -20 °C, and DIBAL-H (78.0 mL, 0.117 mol, 1.5 M toluene solution, 2.5 eq.) was added under a N2 atmosphere at -20 °C. The reaction mixture was further stirred between -15 °C and 0 °C for 3 hours. TLC showed that the reaction was complete. The reaction was slowly quenched with 3N NaOH aqueous solution between -15 °C and 0 °C, while keeping the internal temperature below 0 °C. Volatile substances were removed under reduced pressure at 25 °C. The product was extracted with ethyl acetate (300 mL * 3), and the combined organic phases were washed with water (300 mL) and brine (300 mL). The product was 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). The product was a yellow solid (8.0 g, 72% yield) for 7 hours. 1H NMR(400MHz,DMSO-d6)δ8.79(d,J=8.8Hz,1H),8.10(d,J=1.2Hz,1H),5.52(s,1 H), 4.75 (s, 2H), 4.07 (s, 3H), 2.74 (q, J = 7.9, 7.4Hz, 2H), 1.26 (t, J = 7.4Hz, 3H).

[0155] Step 8: The suspension of compound 7h (8.0 g, 0.034 mol) in acetonitrile (100 mL) was cooled to -15 to 0 °C using an ice-salt bath. TMSI (3.0 eq) was slowly added under N2 with stirring. The ice-salt bath was then removed, and the reaction mixture was slowly heated to 30 °C and stirred at the same temperature for 24 hours until the starting material was completely consumed. Volatile substances were removed under reduced pressure. Ethyl acetate (250 mL) and 1N NaOH aqueous solution (100 mL) were added to the solid residue, and the resulting mixture was stirred at room temperature for 1 hour, during which a large amount of white precipitate formed. The white solid was collected by filtration, washed with water (10 mL x 3), then washed with ethyl acetate (10 mL x 2), and dried under vacuum to give compound 7i. Yield: 5.3 g (70%). 1 H NMR (400MHz, DMSO-d6) δ12.09(s,1H),8.46(d,J=8.7Hz,1H),7.77(d,J=1.5Hz,1H),5 .50 (s, 1H), 4.66 (d, J = 3.9Hz, 2H), 2.56 (qd, J = 7.4, 1.1Hz, 2H), 1.19 (t, J = 7.5Hz, 3H).

[0156] Step 9: Under a nitrogen atmosphere at 0-5°C, slowly add SOCl2 (4.3g, 0.036mol, 2.6mL, 1.5eq) to a DMF (100mL) solution of compound 7i (5.3g, 0.024mol). Stir the mixture at 25°C for 3 hours until the starting material is completely consumed. Cool the reaction mixture to 0-5°C using an ice-water bath and quench it with 1N NaOH (70mL) to pH = 9. Then add water (180mL) with stirring. Stir the reaction mixture at room temperature for 1 hour. Collect the resulting grayish-white precipitate by filtration, wash with water (10mL*3), and dry under vacuum to obtain compound 7j (4.01g, 70% yield). 1 H NMR (400MHz, DMSO-d6) δ12.25(s,1H),8.55(d,J=8.7Hz,1H),7.79(q,J=1.4Hz,1H),4.95(s,2H),2.57(qd,J=7.4,1.3Hz,2H),1.19(t,J=7.4Hz,3H);13 C NMR(101MHz,DMSO-d6)δ161.2,152.2(d,J=266.6Hz),145.8(d,J=3.0Hz),141.67,140.1(d,J=4.0H z), 135.0 (d, J = 3.0Hz), 123.0 (d, J = 10.1Hz), 119.9 (d, J = 8.1Hz), 39.9 (d, J = 4.0Hz), 23.14, 12.25; 19 F NMR(376MHz,DMSO-d6)δ-125.12. MS(ESI):241.0(M+1) + .

[0157] Step 10: Compound 7j (40 mg, 0.17 mmol), 2c (51 mg, 0.17 mmol), N,N-diisopropylethylamine (72 mg, 0.56 mmol), and potassium iodide (3 mg, 0.02 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 85 °C for 2 hours. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 7 (42 mg, white solid); LC-MS: ESI [M+H] + =436.5; 1 H NMR (400MHz, CDCl3) δ9.58(s,1H),8.58(d,J=8.7Hz,1H),8.49(d,J=1.8Hz,1H),8.14(d,J=8.1Hz ,1H),7.96(d,J=5.0Hz,1H),7.82(d,J=1.2Hz,1H),7.74(dd,J=8.2,2.2Hz,1H),6.00(t,J=3.3Hz, 1H),3.84(s,2H),3.33(d,J=15.0Hz,1H),3.16(d,J=17.0Hz,1H),3.03(d,J=5.1Hz,3H),2.90(s,1 H), 2.83–2.67 (m, 3H), 2.58 (dd, J = 11.1, 4.2Hz, 1H), 1.31 (t, J = 7.4Hz, 3H), 1.02 (d, J = 6.9Hz, 3H).

[0158] Example 8: 4-(1-((7-ethyl-4-fluoro-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1,2,3,6-tetrahydropyridin-4-yl)-3-fluorobenzonitrile

[0159]

[0160] Compound 7j (50.0 mg, 0.21 mmol), compound 4c (62.9 mg, 0.23 mmol), DIEA (134.3 mg, 1.04 mmol), and KI (6.9 mg, 0.04 mmol) were weighed into a reaction tube, and 5 mL of acetonitrile was added. The mixture was reacted at 85 °C for 2 h. The reaction was cooled to room temperature, and a saturated aqueous solution of NaHCO3 was added and stirred for 2 h. The mixture was then extracted with DCM (30 mL × 3). The organic phases were combined and dried over anhydrous Na2SO4. The solutions were filtered and evaporated to dryness. The solutions were then column-secreted with DCM and MeOH (0-5%) to give a pale yellow solid (52 mg, 62%). Pre-HPLC was used to prepare a white solid 8 (22 mg, 26%) by freeze-drying. LC-MS: ESI [M+H] + =407.2; 1 H NMR (400MHz, DMSO) δ12.12(s,1H),8.45(d,J=8.5Hz,1H),7.82(dd,J=11.2,1.5Hz,1H),7.79(s,1H),7.67(dd,J=8.1,1.5Hz,1H),7.56(t,J=7 .9Hz,1H),6.14(s,1H),3.79(s,2H),3.17(d,J=2.8Hz,2H),2.70(t,J=5.5Hz,2H),2.57(q,J=7.4Hz,2H),2.47(s,2H),1.19(t,J=7.4Hz,3H).

[0161] Example 9: 3-Ethyl-8-fluoro-7-((5-fluoro-3',6'-dihydro-[2,4'-bipyridin]-1'(2'H)-yl)methyl)-1,5-naphthidin-2(1H)-one

[0162]

[0163] Compound 7j (50.0 mg, 0.21 mmol), compound 5c (57.4 mg, 0.23 mmol), DIEA (134.3 mg, 1.04 mmol), and KI (6.9 mg, 0.04 mmol) were weighed into a reaction tube, and 5 mL of acetonitrile was added. The mixture was reacted at 85 °C for 2 h. The reaction was cooled to room temperature, and a saturated aqueous solution of NaHCO3 was added and stirred for 2 h. The mixture was then extracted with DCM (30 mL × 3). The organic phases were combined and dried over anhydrous Na2SO4. After filtration and evaporation, the solution was lyophilized to obtain a white solid 9 (36 mg, 45%) by HPLC. LC-MS: ESI [M+H] + =383.2; 1H NMR (400MHz, DMSO) δ8.50(s,1H),8.46(d,J=8.3Hz,1H),7.79(s,1H),7.67(d,J=6.0Hz,1H),7.61(d,J =3.9Hz,1H),6.62(s,1H),3.79(s,2H),3.18(s,2H),2.70(s,2H),2.56(s,4H),1.20(t,J=7.3Hz,3H).

[0164] Example 10: 1'-((7-chloro-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridine]-5-nitrile

[0165]

[0166] Step 1: Under nitrogen protection, add 10a (500g, 1.99mol) and 2.5L of methanol, and cool to 0-5℃. Add a 1.0L methanol solution of sodium methoxide (118g) dropwise at 0-5℃. After complete addition, raise the temperature to room temperature and stir for 1 hour. Add 2.0L of water to the reaction system, stir for 30min, and then concentrate under reduced pressure at 40℃ until no liquid is dispensed. Add 4.0L of ethyl acetate, stir to separate the layers, extract the aqueous layer with ethyl acetate, and separate the layers. Combine the organic layers, wash with saturated brine, separate the layers, and concentrate the organic layer under reduced pressure at 40℃ to obtain a white solid 10b (480g).

[0167] Step 2: Weigh 475 g (1.93 mol) of compound 10b and add it to DMF (2.85 L), then add DMF-DMA (2.85 L) dropwise. After the addition is complete, heat to 100 °C and stir for 2 h. After the reaction is complete, cool to 70-80 °C, concentrate under reduced pressure until no liquid is dispensed, then add water and stir to precipitate. Cool to 20-30 °C, stir for 1 h, and filter. Dry the filter cake in a vacuum drying oven at 70 °C to constant weight to obtain red solid 10c (612 g, yield 95.3%).

[0168] Step 3: Compound 10c (500g, 1.91mol) was added to THF (2.56L) and stirred until dissolved. An aqueous solution of sodium periodate (805g, 3.72mol) (2.56L) was added dropwise to the reaction system. The mixture was stirred at room temperature for 2-4 hours. After the reaction was complete, ethyl acetate (4.0L) and water (4.0L) were added to the reaction system, and the mixture was stirred to separate the layers. The aqueous layer was extracted twice with ethyl acetate (2.0L). The organic layers were combined and washed successively with saturated sodium thiosulfate solution and saturated brine. The organic layer was concentrated under reduced pressure at 40-45℃ until no fraction remained, yielding 500g of an oily substance (10d), which was used directly in the next step.

[0169] Step 4: Compounds 10d (512 g, 1.69 mol) and 10e (1457.0 g, 7.61 mol) were added to anhydrous ethanol (7.5 L) and stirred until dissolved. SnCl2 (1815.0 g, 9.57 mol) was added to the reaction system in batches at room temperature. After the addition was complete, the mixture was heated to reflux and stirred for 1-2 h. The reaction system was cooled to 45-50 °C and concentrated under reduced pressure until no fraction remained. 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 precipitated. The reaction solution was centrifuged, and the filtrate was collected and allowed to stand to separate into layers. The organic layer was concentrated under reduced pressure at 40-45 °C until no fraction remained. The solution was purified by column chromatography using 200-300 mesh silica gel to obtain flocculent solid 10f (230 g, yield 36.5%).

[0170] Step 5: Compound 10f (1.20 g, 3.85 mmol) and CuCl (0.57 g, 5.78 mmol) were added to DMF (10 mL). The resulting mixture was stirred overnight at 120 °C. After the reaction was complete, the reaction solution was cooled to room temperature, as detected by LC-MS. The resulting mixture was diluted with ethyl acetate (20 mL). It was washed with 10% ammonia solution. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 10 g (800 mg, 77.78%) as a white solid. LC-MS: ESI [M+H] + =267.0.

[0171] Step 6: Under nitrogen protection, 10 g (800 mg, 3.00 mmol) of compound and TMSI (1.80 g, 9.00 mmol) were added to acetonitrile (8 mL). The reaction mixture was heated to 50 °C and stirred for 2 hours. After the reaction was monitored by LC-MS, the mixture was cooled to room temperature. The resulting mixture was diluted with ethyl acetate (50 mL). The aqueous layer was washed with 3 x 50 mL of water (10% triethylamine). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 10 h (740 mg, 97.64%) as a white solid. LC-MS: ESI [M+H] + =252.9.

[0172] Step 7: Compound 10i (0.74 g, 2.92 mmol) was added to anhydrous THF (300 mL). The reaction mixture was cooled to -20 °C, and DIBAL-H (4.9 mL, 7.3 mmol, 1.5 M toluene solution) was added under a N2 atmosphere at -20 °C. The reaction mixture was further stirred between -15 °C and 0 °C for 3 hours. TLC showed that the reaction was complete. The reaction was slowly quenched with 3N NaOH aqueous solution between -15 °C and 0 °C, while maintaining the internal temperature below 0 °C. Volatile substances were removed under reduced pressure at 25 °C. The product was extracted with ethyl acetate (30 mL * 3), and the combined organic phases were washed with water (30 mL) and brine (30 mL). The product was 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 10i (0.42 g) was given as a yellow solid. LC-MS: ESI [M + H] + =211.2.

[0173] Step 8: Under a nitrogen atmosphere at 0-5°C, slowly add SOCl2 (357 mg, 3.0 mmol) to a DMF (100 mL) solution of compound 10i (0.42 g, 2.0 mmol). Stir the mixture at 25°C for 3 h until the starting material is completely consumed. Cool the reaction mixture to 0-5°C using an ice-water bath and quench it with 1N NaOH to pH = 9. Then add water (10 mL) with stirring. Stir the reaction mixture at room temperature for 1 h. Collect the resulting grayish-white precipitate by filtration, wash with water (10 mL * 3), and dry under vacuum to obtain compound 10j (250 mg). LC-MS: ESI [M + H] + =229.0.

[0174] Step 9: Weigh compound 10j (40.0 mg, 0.17 mmol), compound 2c (48.2 mg, 0.19 mmol), DIEPA (112.8 mg, 0.87 mmol), and KI (6.9 mg, 0.04 mmol) into a reaction tube, add 5 mL of acetonitrile, and react at 85 °C for 2 h. Cool the reaction to room temperature, add saturated NaHCO3 aqueous solution, stir for 0.5 h, extract with DCM (50 mL × 3), combine the organic phases, dry with anhydrous Na2SO4, filter, and evaporate to dryness. Filter with MeOH (0-4%) and DCM to give a pale yellow solid, compound 10 (38.0 mg, 58%). LC-MS: ESI [M+H] + =371.1; 1H NMR (400MHz, CDCl3) δ8.81(s,1H),8.53(s,1H),8.19(s,1H),7.94(d,J=7.8Hz,1H),7.72(s,1H),7.52(d ,J=7.9Hz,1H),7.33(s,1H),6.84(s,1H),3.81(s,2H),3.37(d,J=12.5Hz,2H),2.81(s,2H),2.70(s,2H).

[0175] Example 11: 3-Chloro-7-((5-fluoro-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-yl)methyl)-1,5-naphthyridine-2(1H)-one

[0176]

[0177] Compound 10j (40.0 mg, 0.17 mmol), compound 5c (48.2 mg, 0.19 mmol), DIEPA (112.8 mg, 0.87 mmol), and KI (6.9 mg, 0.04 mmol) were weighed into a reaction tube, and 5 mL of acetonitrile was added. The mixture was reacted at 85 °C for 2 h. The reaction was cooled to room temperature, and a saturated aqueous solution of NaHCO3 was added and stirred for 0.5 h. The mixture was extracted with DCM (50 mL × 3), and the organic phases were combined and dried over anhydrous Na2SO4. The residues were filtered and evaporated to dryness. The residues were then column-sected with MeOH (0.4%) and DCM to give a pale yellow solid, compound 11 (34.0 mg, 53%). LC-MS: ESI [M+H] + =371.1; 1 H NMR (400MHz, CDCl3) δ8.45(dd,J=3.7,1.8Hz,1H),8.31(d,J=1.9Hz,1H),8.12(s,1H),7.66(d,J=1.0Hz,1H),7.35–7.31( m, 2H), 6.45 (d, J = 3.4Hz, 1H), 3.71 (d, J = 3.6Hz, 2H), 3.20 (d, J = 3.0Hz, 2H), 2.71 (t, J = 5.6Hz, 2H), 2.60 (d, J = 1.7Hz, 2H).

[0178] Example 12: 5-(1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphtholine-3-yl)methyl)-1,2,3,6-tetrahydropyridin-4-yl)pyrrolidine-2-nitrile

[0179]

[0180] Step 1: Compound 12a (0.5 g, 2.5 mmol), 1b (0.85 g, 2.6 mmol), Pd(dppf)Cl2 (0.19 g, 0.27 mmol), and potassium carbonate (0.93 g, 6.8 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After the reaction was complete as detected by TLC, the mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 12b (0.5 g, white solid).

[0181] Step 2: Compound 12b (0.5 g, 1.7 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 12c (0.4 g, white solid).

[0182] Step 3: Compound 12c (0.05 g, 0.26 mmol), INT1 (0.069 g, 0.31 mmol), N,N-diisopropylethylamine (0.16 g, 2.3 mmol), and potassium iodide (0.20 g, 1.3 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 12 (0.022 g, white solid); LC-MS: ESI [M+H] + =373.4; 1 H NMR(500MHz,DMSO)δ9.34(s,1H),9.01(s,1H),8.39(s,1H),7.79(s,1H),7.63(s,1H),6.29(s,1H),3 .66(s,2H),3.42(s,1H),2.90(s,1H),2.76(s,1H),2.58(s,1H),2.41(d,J=33.7Hz,4H),1.07(s,3H).

[0183] Example 13: 1'-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthid-3-yl)methyl)-3-methyl-1',2',3',6'-tetrahydro-[2,4'-bipyridine]-5-nitrile

[0184]

[0185] Step 1: Compound 13a (0.5 g, 2.2 mmol), 1b (0.85 g, 2.64 mmol), Pd(dppf)Cl2 (0.16 g, 0.22 mmol), and potassium carbonate (0.79 g, 5.5 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 13b (0.7 g, white solid).

[0186] Step 2: Compound 13b (0.5 g, 1.5 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 13c (0.4 g, white solid).

[0187] Step 3: Compound 13c (0.05 g, 0.22 mmol), INT1 (0.06 g, 0.27 mmol), N,N-diisopropylethylamine (0.14 g, 1.1 mmol), and potassium iodide (0.18 g, 1.11 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 13 (0.018 g, white solid); LC-MS: ESI [M+H] + =386.5; 1 H NMR (500MHz, DMSO) δ9.12(d,J=3.1Hz,1H),8.39(d,J=3.1Hz,1H),7.98(d,J=2.9Hz,1H),7.67(dd,J=45.1,2.5Hz,2H),6.7 6–6.41(m,1H),3.66(s,2H),2.84–2.72(m,2H),2.60(dd,J=12.7,10.7Hz,1H),2.49–2.40(m,4H),1.07(t,J=13.4Hz,3H).

[0188] Example 14: 4-(1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthiophene-3-yl)methyl)-1,2,3,6-tetrahydropyridin-4-yl)-2,5-difluorobenzonitrile

[0189]

[0190] Step 1: Compound 14a (0.5 g, 2.5 mmol), 1b (0.94 g, 3.75 mmol), Pd(dppf)Cl2 (0.19 g, 0.25 mmol), and potassium carbonate (0.88 g, 6.3 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 14b (0.6 g, white solid).

[0191] Step 2: Compound 14b (0.5 g, 1.7 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 14c (0.4 g, white solid).

[0192] Step 3: Compound 14c (0.05 g, 0.28 mmol), INT1 (0.065 g, 0.32 mmol), N,N-diisopropylethylamine (0.11 g, 1.4 mmol), and potassium iodide (0.14 g, 1.4 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 14 (0.025 g, white solid); LC-MS: ESI [M+H]+ = 407.2; 1 H NMR (400MHz, DMSO) δ11.84(s,1H),8.41(d,J=1.7Hz,1H),7.95(dd,J=10.5,5.5Hz,1H),7.75(s,1H),7.64(s,1H),7.58(dd,J=10.2,6.1Hz,1H), 6.26(s,1H),3.71(s,2H),3.17(d,J=8.7Hz,2H),2.67(t,J=4.7Hz,2H),2.55(dd,J=11.6,4.1Hz,2H),2.49–2.46(m,2H),1.18(t,J=7.4Hz,3H).

[0193] Example 15: 4-(1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthiophene-3-yl)methyl)-1,2,3,6-tetrahydropyridin-4-yl)-2,3-difluorobenzonitrile

[0194]

[0195] Step 1: Compound 15a (0.5 g, 2.8 mmol), 1b (0.92 g, 3.4 mmol), Pd(dppf)Cl2 (0.21 g, 0.52 mmol), and potassium carbonate (0.99 g, 13 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 15b (0.7 g, white solid).

[0196] Step 2: Compound 15b (0.5 g, 1.8 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 15c (0.3 g, white solid).

[0197] Step 3: Compound 15c (0.05 g, 0.25 mmol), INT1 (0.063 g, 0.30 mmol), N,N-diisopropylethylamine (0.17 g, 1.25 mmol), and potassium iodide (0.21 g, 1.25 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 15 (0.020 g, white solid); LC-MS: ESI [M+H] + =407.4; 1 H NMR (400MHz, DMSO) δ11.84(s,1H),8.41(s,1H),7.79–7.70(m,2H),7.64(s,1H),7.40(t,J=6.8Hz, 1H), 6.24 (s, 1H), 3.72 (s, 2H), 3.17 (s, 2H), 2.68 (s, 2H), 2.58–2.53 (m, 2H), 1.18 (t, J = 7.4Hz, 3H).

[0198] Example 16: 4-(1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthiophene-3-yl)methyl)-1,2,3,6-tetrahydropyridin-4-yl)-3,5-difluorobenzonitrile

[0199]

[0200] Step 1: Compound 16a (0.5 g, 2.2 mmol), 1b (0.73 g, 2.3 mmol), Pd(dppf)Cl2 (0.17 g, 0.22 mmol), and potassium carbonate (0.79 g, 5.5 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After the reaction was complete as detected by TLC, the mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 16b (0.6 g, white solid).

[0201] Step 2: Compound 16b (0.5 g, 1.8 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 16c (0.4 g, white solid).

[0202] Step 3: Compound 16c (0.05 g, 0.18 mmol), INT1 (0.064 g, 0.25 mmol), N,N-diisopropylethylamine (0.11 g, 0.9 mmol), and potassium iodide (0.13 g, 0.9 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 16 (0.023 g, white solid); LC-MS: ESI [M+H] + =407.4; 1 H NMR (400MHz, DMSO) δ11.84(s,1H),8.41(d,J=1.7Hz,1H),7.80(d,J=7.5Hz,2H),7.75(s,1H),7.66(d,J=1.1Hz,1H),5.94 (s,1H),3.72(s,2H),3.14(d,J=2.5Hz,2H),2.67(t,J=5.4Hz,2H),2.58–2.53(m,2H),2.36(s,2H),1.18(t,J=7.4Hz,3H).

[0203] Example 17: 4-(1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphtholin-3-yl)methyl)-1,2,3,6-tetrahydro-pyridin-4-yl)-2,6-difluorobenzonitrile

[0204]

[0205] Step 1: Compounds 17a (1 g, 4.6 mmol), 1b (1.72 g, 5.5 mmol), Pd(dppf)Cl2 (0.34 g, 0.46 mmol), and potassium carbonate (1.6 g, 11.5 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 17b (1.3 g, white solid).

[0206] Step 2: Compound 17b (1g, 3.1mmol) was added to 10ml of anhydrous methanol, followed by 20ml of aqueous methylamine solution. The mixture was stirred overnight at room temperature. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 17c (0.7g, white solid).

[0207] Step 3: Compound 17c (0.05 g, 0.25 mmol), INT1 (0.071 g, 0.231 mmol), N,N-diisopropylethylamine (0.17 g, 1.25 mmol), and potassium iodide (0.22 g, 1.25 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 17 (0.023 g, white solid); LC-MS: ESI [M+H] + =406.4; 1 H NMR (400MHz, DMSO) δ11.84(s,1H),8.41(d,J=1.6Hz,1H),7.75(s,1H),7.63(s,1H),7.52(d,J=10.2H z,2H),6.61(s,1H),3.72(s,2H),3.17(s,2H),2.68(s,2H),2.59–2.53(m,3H),1.19(t,J=7.4Hz,3H).

[0208] Example 18: 1'-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthid-3-yl)methyl)-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-nitrile

[0209]

[0210] Step 1: Compound 18a (0.5 g, 2.7 mmol), 1b (1.02 g, 3.3 mmol), Pd(dppf)Cl2 (0.2 g, 0.27 mmol), and potassium carbonate (0.94 g, 6.8 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 18b (0.5 g, white solid).

[0211] Step 2: Compound 18b (0.5 g, 1.8 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 18c (0.3 g, white solid).

[0212] Step 3: Compound 18c (0.05 g, 0.27 mmol), INT1 (0.072 g, 0.32 mmol), N,N-diisopropylethylamine (0.17 g, 1.35 mmol), and potassium iodide (0.22 g, 1.35 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 18 (0.023 g, white solid). LC-MS: ESI [M+H] + =372.1. 1 H NMR (400MHz, DMSO) δ11.86(s,1H),8.82(s,1H),8.42(d,J=1.7Hz,1H),8.01(s,2H),7.75(s,1H),7.66(s ,1H),6.48(s,1H),3.88(s,2H),3.17(s,2H),2.71(s,2H),2.55(d,J=7.4Hz,4H),1.19(t,J=7.4Hz,3H).

[0213] Example 19: 4-(1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1,2,3,6-tetrahydropyridin-4-yl)benzylnitrile

[0214]

[0215] Step 1: Compound 19a (0.5 g, 2.5 mmol), 1b (0.85 g, 2.6 mmol), Pd(dppf)Cl2 (0.19 g, 0.27 mmol), and potassium carbonate (0.93 g, 6.8 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 19b (0.5 g, white solid).

[0216] Step 2: Compound 19b (0.5 g, 1.7 mmol) was added to 10 ml of anhydrous methanol, followed by 10 ml of 4 mol / L dioxane hydrochloride solution. The mixture was stirred at room temperature for 0.5-1 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated under reduced pressure to obtain compound 19c (0.4 g, white solid).

[0217] Step 3: Compound 19c (0.05 g, 0.26 mmol), INT1 (0.069 g, 0.31 mmol), N,N-diisopropylethylamine (0.16 g, 2.3 mmol), and potassium iodide (0.20 g, 1.3 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 19 (0.022 g, white solid); LC-MS: ESI [M+H] + =371.2;1H NMR(400MHz,DMSO-d6)δ11.88(s,1H),8.40(s,1H),7.84–7.73(m,3H),7.69–7.57(m,3H) ,6.39(s,1H),3.71(s,2H),3.14(s,2H),2.68(s,2H),2.54(s,2H),1.18(t,J=6.2Hz,3H).

[0218] Example 20: 1'-((3-ethyl-2-oxo-1,2-dihydro-1,6-naphthid-7-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridine]-5-nitrile

[0219]

[0220] Step 1: Compound 20a (3.85 g, 24.6 mmol), DIEA (16.0 g, 123.3 mmol), and DMAP (0.6 g, 4.81 mmol) were added to 100 mL of dichloromethane and cooled to 0 °C. Butyryl chloride (8.10 g, 76.3 mmol) was added dropwise. After the addition was complete, the mixture was allowed to return to room temperature and reacted for 24 h. After the reaction was confirmed to be complete by LC-MS, 100 mL of ethyl acetate and 100 mL of water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with ethyl acetate, and the combined organic phases were evaporated to dryness. Then, 40 mL of dichloromethane was added to precipitate a solid. The solid was filtered, and the filter cake was evaporated to dryness to obtain compound 20b (0.8 g, pink solid).

[0221] Step 2: Compound 20b (0.2 g, 0.96 mmol), potassium vinyltrifluoroborate (141.6 mg, 1.04 mmol), Pd(dppf)Cl2 (17.4 mg, 0.024 mmol), and potassium carbonate (199 mg, 1.44 mmol) were added to 4 ml of dioxane / water = 9:1. The mixture was purged with nitrogen three times, heated to 90 °C and stirred for 4 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 20c (0.1 g, yellow solid).

[0222] Step 3: Compound 20c (0.1 g, 0.5 mmol) was added to 10 ml of dioxane and 2.5 ml of water, followed by sodium periodate (425 g, 2.0 mmol) and potassium osmium tetroxide (75 mg, 0.15 mmol). The mixture was stirred at room temperature for 2 h. After the reaction was confirmed to be complete by LC-MS, 50 ml of ethyl acetate and 50 ml of water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with ethyl acetate. The combined organic phases were washed with saturated sodium sulfite. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filter cake was evaporated to dryness to obtain compound 20d (80 mg, yellow solid).

[0223] Step 4: Add compound 2c (127 mg, 0.49 mmol) and triethylamine (198 mg, 1.96 mmol) to 10 mL of dichloromethane and stir until dissolved. Add compound 20d (100 mg, 0.49 mmol) to 10 mL of dichloromethane and stir until dissolved. Add the dichloromethane solution of compound 2c to the dichloromethane solution of compound 20d and stir at room temperature for 1 h. Add sodium triacetoxyborohydride (623 mg, 2.94 mmol) and stir at room temperature for 2 h. After the reaction is complete as detected by LC-MS, add 20 mL of saturated ammonium chloride, stir and separate the layers. Wash the organic layer successively with saturated sodium bicarbonate and saturated sodium chloride, dry with anhydrous sodium sulfate, filter, and evaporate to dryness. Pre-HPLC preparation yields compound 20 (66 mg, white solid); LC-MS: ESI [M+H] +=372.1;1H NMR (400MHz, DMSO-d6) δ11.91(s,1H),8.97(d,J=2.2Hz,1H),8.74(s,1H),8.27(dd,J=8.4,2.2Hz,1H),7.85–7.73(m,2H),7.3 4(s,1H),6.98(d,J=3.7Hz,1H),3.78(s,2H),3.28(q,J=3.1Hz,2H),2.74(t,J=5.6Hz,2H),2.63(s,2H),1.18(t,J=7.4Hz,3H).

[0224] Example 21: 3-Ethyl-7-((5-fluoro-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-yl)methyl)-1,6-naphthidium-2(1H)-one

[0225]

[0226] Compound 5c (124 mg, 0.49 mmol) and triethylamine (198 mg, 1.96 mmol) were added to 10 mL of dichloromethane and stirred until dissolved. Compound 20d (100 mg, 0.49 mmol) was added to 10 mL of dichloromethane and stirred until dissolved. The dichloromethane solution of compound 5c was added to the dichloromethane solution of compound 20d, and the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (623 mg, 2.94 mmol) was added, and the mixture was stirred at room temperature for 2 h. After the reaction was complete as detected by LC-MS, 20 mL of saturated ammonium chloride was added, and the mixture was stirred to separate the layers. The organic layer was washed successively with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness before Pre-HPLC preparation to obtain compound 21 (70 mg, white solid). LC-MS: ESI [M+H] + =365.1;1H NMR (400MHz, DMSO-d6) δ11.91(s,1H),8.74(s,1H),8.52(d,J=2.9Hz,1H),7.81(s,1H),7.74–7.61(m,2H),7.35(s,1H) ,6.66(d,J=3.7Hz,1H),3.77(s,2H),3.21(q,J=3.0Hz,2H),2.72(t,J=5.6Hz,2H),2.61(s,2H),1.18(t,J=7.4Hz,3H).

[0227] Example 22: 1'-((7-cyclopropyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridine]-5-nitrile

[0228]

[0229] Compound INT2 (50.0 mg, 0.21 mmol), compound 2c (60.5 mg, 0.23 mmol), DIEPA (137.7 mg, 1.1 mmol), and KI (7.1 mg, 0.04 mmol) were weighed into a reaction tube, and 5 mL of acetonitrile was added. The mixture was reacted at 85 °C for 3 h. The reaction was cooled to room temperature, and a saturated aqueous solution of NaHCO3 was added and stirred for 0.5 h. The mixture was then extracted with DCM (50 mL × 3). The organic phases were combined and dried over anhydrous Na2SO4. After filtration and evaporation, the mixture was purified by HPLC to obtain a white solid compound 22 (42.0 mg, 51%). LC-MS: ESI [M+H] + =384.2; 1 H NMR (400MHz, DMSO) δ11.87(s,1H),8.51(d,J=2.9Hz,1H),8.39(d,J=1.6Hz,1H),7.69(td,J=8.7,2.9Hz,1H),7.61(dd,J=8.8,4.2Hz,2H),7.42(s, 1H),6.62(s,1H),3.71(s,2H),3.14(d,J=2.6Hz,2H),2.67(t,J=5.5Hz,2 H),2.57(s,2H),2.18–2.09(m,1H),1.01–0.91(m,2H),0.86–0.77(m,2H).

[0230] Example 23: 3-Cyclopropyl-7-((5-Fluoro-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-yl)methyl)-1,5-naphthyridine-2(1H)-one

[0231]

[0232] Compound INT2 (50.0 mg, 0.21 mmol), compound 5c (58.8 mg, 0.23 mmol), DIEPA (137.7 mg, 1.1 mmol), and KI (7.1 mg, 0.04 mmol) were weighed into a reaction tube, and 5 mL of acetonitrile was added. The mixture was reacted at 85 °C for 3 h. The reaction was cooled to room temperature, and a saturated aqueous solution of NaHCO3 was added and stirred for 0.5 h. The mixture was then extracted with DCM (50 mL × 3). The organic phases were combined and dried over anhydrous Na2SO4. After filtration and evaporation, the mixture was purified by HPLC to obtain a white solid compound 23 (12.0 mg, 15%). LC-MS: ESI [M+H] + =377.2; 1H NMR (400MHz, DMSO) δ11.86(s,1H),8.95(d,J=2.0Hz,1H),8.39(d,J=1.7Hz,1H),7.74(d,J=8.4Hz,1H),7.62(d,J=1.3Hz,1H),7.42(s,1H),6 .94(s,1H),3.72(s,2H),3.21(d,J=2.8Hz,2H),2.69(t,J=5.5Hz,2H), 2.59(s,2H),2.19–2.10(m,1H),1.01–0.93(m,2H),0.85–0.79(m,2H).

[0233] Example 24: 3-Cyclopropyl-7-((5-Fluoro-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-yl)methyl)-1,5-naphthyridine-2(1H)-one

[0234]

[0235] Step 1: Weigh compound 24a (50 g, 314 mmol) and N-bromosuccinimide (67 g, 377 mmol) into a reaction flask, add 300 mL of sulfuric acid, and heat to 80 °C and react overnight. After the reaction is complete as detected by TLC, cool to room temperature, slowly add ice water to dilute, extract three times with ethyl acetate, combine the organic phases, wash with water, wash with saturated sodium bicarbonate solution, dry with anhydrous sodium sulfate, filter, and evaporate to dryness. The crude product is purified by column chromatography to give compound 24b (65 g, pale yellow solid); LC-MS: ESI [M+H] + =238.9.

[0236] Step 2: Compound 24b (43 g, 181 mmol), compound 24c (21 g, 181 mmol), N,N-diisopropylethylamine (70 g, 543 mmol), and N,N-dimethylformamide (100 ml) were added to a 500 ml reaction flask and stirred overnight at room temperature. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure, extracted three times with ethyl acetate and water, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to give compound 24d (37 g, orange-red solid); LC-MS: ESI [M+H] + =336.1.

[0237] Step 3: Compound 24d (26 g, 78 mmol) was added to 200 ml of anhydrous methanol and 5 ml of water, followed by ammonium chloride (35 g, 621 mmol). The mixture was cooled to 0 °C, and zinc powder (43 g, 621 mmol) was slowly added. The mixture was then heated to room temperature and reacted for 1 hour. After the reaction was complete as monitored by TLC, the mixture was filtered. The filtrate was concentrated under reduced pressure, and 30 ml of a 4 mol / L dioxane solution was added. The mixture was reacted at room temperature for 1 hour. After the reaction was complete as monitored by TLC, petroleum ether was added to precipitate a solid. The solid was filtered, dried, and compound 24e (16 g, grayish-white solid) was obtained. LC-MS: ESI [M+H] + =274.1.

[0238] Step 4: Compound 24e (16 g, 57 mmol) and 2,3-dichloro-5,6-dicyanobenzoquinone (16 g, 69 mmol) were added to 500 mL of dichloromethane and reacted overnight at room temperature. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure, quenched with saturated sodium bicarbonate aqueous solution, and extracted three times with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to give compound 24f (7.9 g, brown solid); LC-MS: ESI [M+H] + =272.1.

[0239] Step 5: Weigh compound 24f (0.5 g, 1.8 mmol), tributyltin methanol (0.65 g, 2.0 mmol), and XphosPd G2 (73 mg, 0.09 mmol). Add 15 mL of dioxane, replace with nitrogen, and heat to 80 °C overnight. After the reaction is complete as monitored by TLC, extract three times with water and ethyl acetate. Combine the organic phases, dry with anhydrous sodium sulfate, filter, and evaporate to dryness. The crude product is purified by column chromatography to give compound 24 g (0.4 g, pale yellow solid); LC-MS: ESI [M+H] + =223.2.

[0240] Step 6: 24 g (0.4 g, 0.8 mmol) of compound was added to 10 mL of hydrobromic acid aqueous solution, and the mixture was heated to 80 °C and reacted for 3 hours. After the reaction was complete as monitored by TLC, saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted three times with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to obtain compound 24h (0.24 g, pale yellow solid); LC-MS: ESI [M+H] + =286.1.

[0241] Step 7: Compound 24h (0.05 g, 0.23 mmol), 2c (0.065 g, 0.28 mmol), N,N-diisopropylethylamine (0.16 g, 1.15 mmol), and potassium iodide (0.2 g, 1.15 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 24 (0.026 g, white solid); LC-MS: ESI [M+H] + =390.1.

[0242] Example 25: 3-Ethyl-8-fluoro-7-((5-fluoro-3',6'-dihydro-[2,4'-bipyridin]-1'(2'H)-yl)methyl)quinoxalin-2(1H)-one

[0243]

[0244] Compound 24h (0.05 g, 0.25 mmol), 5c (0.071 g, 0.231 mmol), N,N-diisopropylethylamine (0.17 g, 1.25 mmol), and potassium iodide (0.22 g, 1.25 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 25 (0.023 g, white solid); LC-MS: ESI [M+H] + =383.1.

[0245] Example 26: 1'-((7-methyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridine]-5-nitrile

[0246]

[0247] Step 1: Compound 10f (2 g, 2.8 mmol), methylboric acid (1.4 g, 3.4 mmol), Pd(dppf)Cl2 (0.4 g, 0.52 mmol), and potassium carbonate (3.2 g, 13 mmol) were added to a mixed solvent of 7 ml dioxane, 3 ml anhydrous ethanol, and 4 ml water. The mixture was then purged with nitrogen three times and reacted at 90 °C for 2 h under nitrogen protection. After TLC detection of complete reaction, the reaction mixture was cooled to room temperature, and 30 ml dichloromethane and 20 ml water were added. The mixture was separated into layers in a separatory funnel. The aqueous phase was extracted twice with dichloromethane. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to give compound 26a (1.3 g, white solid).

[0248] Step 2: Compound 26a (1.0 g, 8.1 mmol) was added to a 150 ml reaction flask, followed by 60 ml of tetrahydrofuran. The mixture was cooled to 0 °C, and then a 2.5 mol / L lithium aluminum hydride tetrahydrofuran solution (6.48 ml, 16.2 mmol) was added. The reaction was carried out at 0 °C for 2 hours. After the reaction was monitored by TLC until complete, 5 ml of water was added to quench the reaction. The mixture was dried with a large amount of anhydrous sodium sulfate, filtered, and the residue was washed with a large amount of dichloromethane. The filtrates were combined and concentrated by rotary evaporation. After drying, compound 26b (0.8 g, white solid) was obtained.

[0249] Step 3: Compound 26b (0.7 g, 4.0 mmol) was added to a 50 ml reaction flask, along with 20 ml of dichloromethane and 1 ml of N,N-dimethylformamide. The mixture was cooled to 0 °C, and thionyl chloride (0.87 ml, 12 mmol) was added dropwise. The reaction was carried out at 0 °C for 1 hour. After the reaction was completed by TLC monitoring, the reaction solution was concentrated by rotary evaporation. The crude product was purified by column chromatography to obtain compound 26c (0.6 g, gray solid).

[0250] Step 4: Add compound 26c (0.5 g, 4.0 mmol) to a 50 ml reaction flask and add 20 ml of hydrobromic acid. React at 80 °C for 2 h. After cooling to room temperature, adjust the pH to alkaline with saturated sodium carbonate solution. A solid precipitates out. Filter and dry under reduced pressure to obtain compound 26d (0.4 g, gray solid).

[0251] Step 5: Compound 2e (0.05 g, 0.25 mmol), 26d (0.063 g, 0.30 mmol), N,N-diisopropylethylamine (0.17 g, 1.25 mmol), and potassium iodide (0.21 g, 1.25 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 26 (0.020 g, white solid); LC-MS: ESI [M+H] + =357.2; 1H NMR (400MHz, DMSO-d6) δ11.86 (s, 1H), 8.95 (dd, J = 2.3, 0.9Hz, 1H), 8.40 (d, J =1.8Hz,1H),8.25(dd,J=8.4,2.2Hz,1H),7.87–7.78(m,1H),7.74(dd,J=8.4, 1.0Hz,1H),7.64(d,J=1.8Hz,1H),6.97–6.89(m,1H),3.73(s,2H),3.21(d,J =3.3Hz,2H),2.70(t,J=5.6Hz,2H),2.63–2.55(m,2H),2.14(d,J=1.3Hz,3H).

[0252] Example 27: 7-((5-fluoro-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-yl)methyl)-3-methyl-1,5-naphthidium-2(1H)-one

[0253]

[0254] Compound 5c (0.05 g, 0.18 mmol), compound 26d (0.064 g, 0.25 mmol), N,N-diisopropylethylamine (0.11 g, 0.9 mmol), and potassium iodide (0.13 g, 0.9 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 27 (0.023 g, white solid); LC-MS: ESI [M+H] + =351.1; 1 H NMR(400MHz, DMSO-d6)δ11.86(s,1H),8.49(d,J=2.9Hz,1H),8.39(d,J=1.8Hz,1H),7.84–7.79(m,1H),7.71–7.56(m,3H) ,6.64–6.58(m,1H),3.70(s,2H),3.14(q,J=3.0Hz,2H),2.67(t,J=5.6Hz,2H),2.60–2.52(m,2H),2.13(d,J=1.3Hz,3H).

[0255] Example 28: 5-Fluoro-1'-((7-methyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-6-nitrile

[0256]

[0257] Compound 3C (0.05 g, 0.26 mmol), compound 26d (0.069 g, 0.31 mmol), N,N-diisopropylethylamine (0.16 g, 2.3 mmol), and potassium iodide (0.20 g, 1.3 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 28 (0.022 g, white solid); LC-MS: ESI [M+H] + =376.1; 1 H NMR (400MHz, DMSO-d6) δ11.86(s,1H),8.77(t,J=1.7Hz,1H),8.40(d,J=1.8Hz,1H),8.11(dd,J=10.9,1.8Hz,1H),7.86–7.81(m,1H),7.63(d,J= 1.9Hz, 1H), 6.67 (dt, J = 3.8, 2.3Hz, 1H), 3.73 (s, 2H), 3.19 (q, J = 3.0Hz, 2H), 2.70 (t, J = 5.6Hz, 2H), 2.55 (d, J = 6.4Hz, 2H), 2.14 (d, J = 1.3Hz, 3H).

[0258] Example 29: 3-Fluoro-4-(1-(((7-methyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1,2,3,6-tetrahydropyridin-4-yl)benzylnitrile

[0259]

[0260] Compound 4c (0.05 g, 0.24 mmol), compound 26d (0.066 g, 0.29 mmol), N,N-diisopropylethylamine (0.16 g, 1.2 mmol), and potassium iodide (0.2 g, 1.2 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 29 (0.023 g, white solid); LC-MS: ESI [M+H] + =375.1; 1 H NMR (400MHz, DMSO-d6) δ11.86(s,1H),8.40(s,1H),7.82(d,J=8.6Hz,2H),7.67(d,J=12.3Hz,2H), 7.59–7.47(m,1H),6.15(s,1H),3.71(s,2H),3.15(s,2H),2.67(s,2H),2.51(s,2H),2.14(s,3H).

[0261] Example 30: 4-(1-((7-methyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1,2,3,6-tetrahydropyridin-4-yl)benzylnitrile

[0262]

[0263] Compound 19c (0.05 g, 0.23 mmol), compound 26d (0.065 g, 0.28 mmol), N,N-diisopropylethylamine (0.16 g, 1.15 mmol), and potassium iodide (0.2 g, 1.15 mmol) were added to 10 mL of anhydrous acetonitrile. The mixture was stirred at 80 °C for 2 h. After the reaction was complete as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 30 (0.026 g, white body); LC-MS: ESI [M+H] + =357.1; 1 H NMR (400MHz, DMSO-d6) δ11.85(s,1H),8.40(s,1H),7.89–7.73(m,3H),7.63(d,J=7.7Hz ,3H),6.39(s,1H),3.71(s,2H),3.17–3.12(m,2H),2.69(t,J=5.6Hz,2H),2.14(s,3H).

[0264] Bioactivity testing:

[0265] 1. PARP-1 enzyme assay

[0266] 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), histones (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).

[0267] 1.1 PARP1 enzyme assay

[0268] 1.1.1 Preparation of buffer solutions: PBST: 1X PBS, 0.05% Tween-20; blocking buffer: 1X PBS, 0.05% Tween-20, 5% BSA; reaction buffer: 50mM Tris-HCl (pH 7.5), 0.005% Tween-20, 0.01% BSA.

[0269] 1.1.2 Coating: Prepare 50 ng / mL Histone coating solution with 1xPBS, transfer 25 μL of the coating solution to a 384-well reaction plate, and coat overnight at 4°C.

[0270] 1.1.3 Washing: After coating, discard the coating solution and wash with PBST solution. The method is to transfer 50 μL of PBST to a 384-well reaction plate, let it stand for 5 minutes, discard the washing solution, refill the plate, repeat the washing 3 times, and finally pat the reaction plate dry and wait for the next step of blocking.

[0271] 1.1.4 Blocking: Transfer 50 μL of blocking solution to a 384-well reaction plate and let stand for 1 hour.

[0272] Washing: After the blocking is completed, discard the blocking solution, wash the plate three times with PBST solution as described in step 2, and finally pat the reaction plate dry.

[0273] 1.1.5 Prepare a 1000-fold dilution of the compound, transfer 1 μL of the compound to a 96-well plate containing 199 μL of reaction buffer, mix well, and then transfer 5 μL of the mixed compound to a 384-well reaction plate.

[0274] 1.1.6 Prepare a 25 / 10-fold 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.

[0275] 1.1.7 Prepare a 25 / 10-fold NAD+ solution using reaction buffer, transfer 10 μL of NAD+ solution to a 384-well reaction plate, with a final NAD+ concentration of 3.5 μM, and incubate at room temperature for 60 minutes.

[0276] 1.1.8 Prepare a 25 / 10-fold NAD+ solution using reaction buffer, transfer 10 μL of NAD+ solution to a 384-well reaction plate, with a final NAD+ concentration of 3.5 μM, and incubate at room temperature for 60 minutes.

[0277] 1.1.9 Washing: After the reaction is complete, discard the reaction solution, wash the plate three times with PBST solution as described in step 2, and finally pat the plate dry.

[0278] 1.1.10 Dilute the primary antibody (anti-Poly / Mono-ADP Ribose Rabbit mAb) 2000 times with blocking buffer, add 20 μL of primary antibody, and incubate at room temperature for 1.5 hours.

[0279] 1.1.11 Washing: Discard the primary antibody, wash three times with PBST solution as described in step 2, and finally pat the reaction plate dry.

[0280] 1.1.12 Dilute the secondary antibody (anti-rabbit IgG, HRP-linked Antibody) 2000 times with blocking buffer, add 20uL of secondary antibody, and incubate at room temperature for 1 hour.

[0281] 1.1.13 Washing: Discard the secondary antibody, wash three times with PBST solution as described in step 2, and finally pat the reaction plate dry.

[0282] 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 reaction plate.

[0283] 1.1.15 Reading: Read the chemiluminescence value RLU using Envision.

[0284] The test results are shown in Table 2 below:

[0285] Table 2 PARP-1 enzyme test results

[0286]

[0287] Conclusion: The compounds of this invention have a significant inhibitory effect on PARP1.

[0288] 2. Cell anti-proliferation activity test

[0289] BRCA mutant MDA-MB-436 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and incubated at 37°C in a 5% CO2 saturated incubator. When cells reached 80% confluence, they were harvested, centrifuged at 300g for 10 min, and seeded at 1200 cells / well in 96-well plates. After 24 h, different final concentrations of PARPi (0, 0.01, 0.1, 1, 10, 100, 1000 nM) were added, and the cells were cultured for another 72 h. The medium was then changed (with the same final concentration of PARPi added), and the cells were cultured for another 96 h. The OD values ​​at 450 nM were measured using the CCK8 assay, and the cell inhibition rate was calculated as follows: Inhibition rate % = 1 - (mean OD value of the treated group - mean OD value of the Blank group) / (mean OD value of the control group - mean OD value of the Blank group) * 100%.

[0290] BRCA wild-type DLD-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and incubated at 37°C in a 5% CO2 saturated incubator. When cells reached 80% confluence, they were harvested, centrifuged at 300g for 10 min, and seeded at 1000 cells / well in 96-well plates. After 24 h, different final concentrations of PARPi (0, 1, and 10 μM) were added, and the cells were cultured for another 72 h. The medium was then changed (with the same final concentration of PARPi added), and the cells were cultured for another 96 h. The OD values ​​at 450 nM were measured using the CCK8 assay, and the cell inhibition rate was calculated.

[0291] Inhibition rate % = 1 - (mean OD value of the treated group - mean OD value of the Blank group) / (mean OD value of the control group - mean OD value of the Blank group) * 100%. Reference compound AZD9574 was purchased from MedChemExpress (MCE).

[0292] The test results are shown in Table 3 below:

[0293] Table 3 Results of cell antiproliferation activity assay

[0294]

[0295] Conclusion: Compounds 2, 3, 4, 5, etc. of the present invention have significant inhibitory effects on BRCA-mutated MDA-MB-436 cells and no obvious inhibitory effects on BRCA wild-type DLD-1 cells, indicating that the compounds of the present invention specifically inhibit homologous recombination-deficient tumor cells.

[0296] 3. Pharmacokinetic Evaluation of the Compound in Balb / c Mice

[0297] Experimental Purpose: To understand the pharmacokinetics of the compound.

[0298] Experimental Basis: Technical Guidelines for Non-Clinical Pharmacokinetic Studies of Chemical Drugs, 2014.

[0299] Experimental Scheme: The pharmacokinetics of the compound was investigated by intravenous administration (1 mg·kg -1 ) and intragastric administration (1 mg·kg -1 ) to Balb / c mice.

[0300] Sample Preparation: Weigh about 0.2 mg of the compound, dissolve it in 10 μL DMSO, and then add sodium chloride injection solution to prepare a 0.1 mg·mL-1 compound solution for use in the experiment.

[0301] Sample Collection: Six Balb / c mice (Chengdu Dashuo Experimental Animal Co., Ltd., License No.: SCXK (Chuan) 2020-030), male. Three mice were administered intravenously (IV) at 1 mg·kg-1 and three mice were administered intragastrically (PO) at 1 mg·kg-1. Approximately 0.05 mL of blood was collected at 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 to collect the supernatant plasma, which was stored at -40 °C for further analysis. The plasma drug concentration was quantitatively analyzed by LC-MS / MS, and pharmacokinetic parameters such as peak time (Cmax), area under the plasma concentration-time curve (AUC(0-t)), half-life (T 1 / 2 ), clearance (CL), volume of distribution at steady state (Vdss), bioavailability (F), etc. were calculated.

[0302] The results of the pharmacokinetic evaluation are shown in Table 4 below:

[0303] Table 4 Pharmacokinetic Test Results of the Compound in Balb / c Mice

[0304]

[0305] Conclusion: The compounds of this invention exhibit favorable pharmacokinetic properties in Balb / c mice, including good oral bioavailability, exposure, half-life, and clearance.

[0306] 5. In vivo pharmacodynamic studies of compounds 2 and 5 on the MDA-MB-436 subcutaneous xenograft model in nude mice.

[0307] Experimental Procedure: Twenty female NOD / SCID mice were randomly divided into four groups: a blank control group, a reference compound AZD-9574 (1 mg / kg), a compound 2 (1 mg / kg), and a compound 5 (1 mg / kg), with five mice in each group. All mice were administered the medication orally once daily. Body weight was measured every two days, and tumor length and width were measured using calipers.

[0308] Conclusion: The experimental results are as follows Figure 1 After cell seeding, each treatment group was administered the drug for 22 consecutive days. Compared with the blank control group, each treatment group (AZD9574 1 mg / kg dose group, compound 2 1 mg / kg dose group, and compound 5 1 mg / kg dose group) showed a significant inhibitory effect on tumor growth in the NOD / SCID mouse subcutaneous tumor model of human breast cancer cell line MM436. In particular, at the same dose, compound 2 showed better antitumor effect than the reference compound AZD9574.

[0309] 6. Evaluation of the distribution of the compound in the brain bloodstream

[0310] Experimental objective: To obtain the brain blood distribution of the compound.

[0311] Experimental protocol: The brain-blood distribution of the compound was investigated by monitoring its levels in the mouse brain and plasma.

[0312] Experimental Procedure: Weigh the compound, add a small amount of DMSO, then add sodium chloride solution for injection to prepare a 1 mg·mL⁻¹ compound solution for administration. 24 male mice were orally administered the compound at 10 mg·kg⁻¹. Whole blood and whole brain were collected at 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. Centrifuge tube M1 was weighed, the centrifuge tube containing the whole brain was weighed as M2, the centrifuge tube containing the brain after homogenization with water was weighed as M3, and 30 μL of homogenate was collected as M4. 30 μL of plasma and 30 μL of brain homogenate were homogenized into centrifuge tubes, 120 μL of acetonitrile precipitate containing 20 ng·mL⁻¹ internal standard SAHA was added, vortexed for 30 s, and centrifuged at 13000 rpm for 15 min. The supernatant was collected and placed in a sample vial for analysis.

[0313] Standard curve range: 10–10000 ng / ml -1 .

[0314] Drug content in the brain = measured value × 0.03 × (M3 - M1) / [(M2 - M1) × (M3 - M4)]

[0315] The results of the compound's distribution in cerebral blood are shown in Table 5 below:

[0316] Table 5 Results of blood distribution test in mouse brain after compound administration.

[0317]

[0318] Conclusion: The plasma and brain tissue concentrations of compounds 2, 5, and 30 of the present invention were higher than those of AZD9574 and compound 1 at 1 h and 6 h, especially at 6 h, the brain tissue concentration of compounds 2, 5, and 30 was significantly higher than that of AZD9574 and compound 1. Therefore, compounds 2, 5, and 30 have the potential for brain penetration.

Claims

1. Formula The compound or its pharmaceutically acceptable salt thereof, characterized in that: The formula The structure is as follows: ； in: Indicates a double bond; R1is selected from C 1-6 alkyl; X1 is selected from N or C(R) 5a X2 is selected from N or C(R). 5b X3 is selected from C(R) 5c X1 and X2 are selected from N, and there is exactly one of them. R 2a and R 2b Independently selected from hydrogen; R3 is selected from deuterium, fluorine, and C. 1-4 Alkyl or C 1-4 At least one of the deuterated alkyl groups; R 3a It does not exist; R4 is selected from halogen or cyano groups; R 5a R 5b and R 5c Independently selected from hydrogen; R6 is selected from hydrogen, fluorine, or chlorine; X5 is selected from nitrogen or C(R) 9a X6 is selected from nitrogen or C(R) 9b X7 is selected from nitrogen or C(R) 9c X8 is selected from nitrogen or C(R) 9d ); R 9a R 9b R 9c and R 9d Independently selected from hydrogen, fluorine, chlorine, or C 1-4 alkyl; n1 is 0; n3 is selected from 0.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R1 is selected from C 1-4 alkyl.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that: R1 is selected from methyl or ethyl.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R4 is selected from chlorine or cyano.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: R 9a R 9b R 9c and R 9d It is independently selected from hydrogen, fluorine, or methyl.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from: , , , , , , , , , , , , , , or .

7. A pharmaceutical composition, characterized in that: It uses the compound or a pharmaceutically acceptable salt of any one of claims 1 to 6 as the active ingredient, supplemented by a pharmaceutically acceptable carrier.

8. Use of the compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 7 in the preparation of a medicament for the prevention and / or treatment of PARP1 enzyme-related diseases.

9. The use according to claim 8, characterized in that: The PARP1 enzyme-related disease is tumor.

10. The use according to claim 9, characterized in that: The tumor contains one or more cancer cells that have a BRCA1 or BRCA2 defective phenotype.

11. The use according to claim 9, characterized in that: The tumors mentioned are breast cancer, ovarian cancer, primary peritoneal cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer, glioblastoma, or lung cancer.

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