WO2025092973A1 - Parp1-targeting compound and use thereof - Google Patents

Abstract

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

Description

PARP1 targeting compounds and uses thereof Technical Field

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

Background Art

During the growth of cells, their DNA will be constantly damaged by various internal and surrounding adverse factors. Among DNA damage, the most serious types of damage are single-strand breaks and double-strand breaks, among which single-strand breaks are more common. If these breaks are not repaired promptly and accurately, they will lead to genomic instability, causing cancer and even directly causing cell death. For single-strand breaks of DNA, its repair mainly depends on the PARP enzyme. For double-strand breaks, there are two ways to repair double-stranded DNA: non-homologous end joining repair and homologous recombination repair. Homologous recombination repair is a high-fidelity, error-free repair method, and it is also the main way to repair double-stranded DNA. There are many proteins involved in homologous recombination repair, among which the most well-known is the BRCA protein. 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 would be selectively inhibited by PARP inhibitors. Based on this research result, scholars proposed the concept of synthetic lethality: the loss of either BRCA or PARP gene itself is not lethal, but the simultaneous inactivation of both will lead to cell death. Based on the theory of synthetic lethality, PARP inhibitors (PARPi) have been developed to selectively target cancer cells with BRCA1/2 mutations.

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

Therefore, there is an unmet clinical need for effective and safe PARP inhibitors, especially for PARP1 Selective PARP inhibitor.

Summary of the invention

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

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

in,

X is selected from N or C, and when X is selected from N, represents a single bond, when X is selected from C, represents a double bond;

_X1 is selected from N or C( _R8a ), _X2 is selected from N or C( _R8b ), _X3 is selected from N or C( _R8c ), and at most one of _X1 , _X2 and _X3 is selected from N;

_R1 is selected from _C1-8 alkylthio, _C1-8 halogenated alkylthio, _C1-8 deuterated alkylthio, _C1-8 halogenated alkoxy, _C1-8 halogenated alkyl or _C2-8 alkenyl;

_R2 is selected from hydrogen, fluorine, chlorine, _C1-4 alkyl, _C1-4 fluoroalkyl or _C1-4 deuterated alkyl;

R ₃ and R ₄ are independently selected from hydrogen, deuterium, fluorine, C _1-4 alkyl, C _1-4 fluoroalkyl or C _1-4 deuterated alkyl;

Alternatively, R ₃ , R _{4 ,} together with the atoms to which they are attached, form a 3-6 membered alkyl ring;

R ₅ is selected from hydrogen, fluorine, chlorine, cyano, C _1-4 alkyl, C _1-4 fluoroalkyl or C _1-4 deuterated alkyl;

Alternatively, R ₄ , R ₅ and the atoms to which they are connected together form a 5-7 membered alkyl heterocycle; when R ₄ , R ₅ and the atoms to which they are connected are connected to form a ring, the 5-7 membered alkyl heterocycle contains 1 O heteroatom in addition to X on the main ring;

R ₃ , R ₄ and the atoms to which they are attached, and R ₄ , R ₅ and the atoms to which they are attached, will not form a ring at the same time;

R ₆ is selected from -C(O)-NH-R _6a ; R _6a is selected from C _1-4 alkyl, C _1-4 fluoroalkyl, C _1-4 deuterated alkyl or 3-6 membered cycloalkyl;

_R7 is selected from hydrogen, fluorine, chlorine, cyano, _C1-4 alkyl, _C1-4 fluoroalkyl or _C1-4 deuterated alkyl;

R _8a , R _8b , and R _8c are independently selected from hydrogen, fluorine, chlorine, C _1-4 alkyl, C _1-4 deuterated alkyl, and C _1-4 fluoroalkyl;

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

In some embodiments of the present invention, _X1 is selected from N, _X2 is selected from C( _R8b ), and _X3 is selected from C( _R8c ); or _X1 is selected from C( _R8a ), _X2 is selected from N, _{and X3} is selected from C( _R8c ); or _X1 is selected from C( _R8a ), _X2 is selected from C( _R8b ), and _X3 is selected from C( _R8c ).

In some embodiments of the present invention, _R1 is selected from _{C1-4 alkylthio, C1-4} haloalkylthio, _C1-4 deuterated alkylthio, _C1-4 haloalkoxy, _C1-4 haloalkyl, _C2-4 alkenyl; _{in R1} , the halogen is selected from F, Cl, and Br.

In some preferred embodiments of the present invention, R ₁ is selected from methylthio, halogenated methylthio, deuterated methylthio, halogenated methoxy, halogenated methyl, halogenated ethyl or vinyl; in R ₁ , the halogenated halogen is selected from F and Cl.

In some further embodiments of the present invention, _R1 is selected from -S- _CH3 , -S- _CF2H , -S- _CF3 , -S- _CF2Cl , -S-CD3, -O- _CF2H , -O- _CF3 , -O- _CF2Cl , _-CF2 - _CH3 or _-CH = _CH2 .

In some embodiments of the present invention, _R2 is selected from hydrogen, fluorine, chlorine, methyl, fluoromethyl or deuterated methyl.

In some embodiments of the present invention, when R ₃ and R ₄ do not form a ring with the atoms to which they are attached, R ₃ and R ₄ are independently selected from hydrogen, deuterium, fluorine, methyl, fluoromethyl or deuterated methyl.

In some embodiments of the present invention, when R ₄ and R ₅ do not form a ring with the atoms to which they are attached, R ₅ is selected from hydrogen, fluorine, chlorine, cyano, methyl, fluoromethyl or deuterated methyl.

In some embodiments of the present invention, R ₇ is selected from hydrogen, fluorine, chlorine, cyano, methyl, fluoromethyl or deuterated methyl.

In some embodiments of the present invention, R _8a , R _8a , R _8c are independently selected from hydrogen, fluorine, chlorine, methyl, fluoromethyl or deuterated methyl.

In some embodiments of the present invention, R _6a is selected from methyl, fluoromethyl, deuterated methyl or cyclopropyl.

In some preferred embodiments of the present invention, R _6a is selected from methyl, -CF ₃ , -CD ₃ or cyclopropyl.

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

In some embodiments of the present invention, when R ₄ and R ₅ do not form a ring with the atoms to which they are attached, the structural unit (connected to the main ring from left to right, that is, the N end is connected to the main ring methylene, and the X end is connected to the main ring pyridyl) is selected from:

In some embodiments of the present invention, when R ₄ and R ₅ do not form a ring with the atoms to which they are attached, the structural unit Selected from:

In some embodiments of the present invention, when R ₄ , R ₅ and the atoms to which they are attached form a ring, the structural unit Selected from:

In some preferred embodiments of the present invention, when R ₄ , R ₅ and the atoms to which they are attached form a ring, the structural unit Selected from:

In some embodiments of the present invention, the structural unit in formula I Replace with (Both are connected to the main ring in the same way, that is, the N end is connected to the main ring methylene, and the O end is connected to the main ring pyridyl) (In this case, R ₅ does not form a ring with the connected atoms).

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

In some embodiments of the invention, _R1 is selected from -S- _CH3 , -S- _CF2H , -S- _CF3 , -S- _CF2Cl , -S- _CD3 , -O- _CF2H , -O- _CF3 , -O- _CF2Cl , _-CF2 - _CH3 , -CH= _CH2 , -S- _CFH2 or -O- _CFH2 .

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

In some embodiments of the present invention, the structural unit in formula I Replace with (Both are connected to the main ring in the same way, that is, the N end is connected to the methylene group of the main ring, and the CH end is connected to the pyridyl group of the main ring) (In this case, R ₅ does not form a ring with the connected atoms).

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

In some embodiments of the present invention, when R ₄ and R ₅ do not form a ring with the atoms to which they are attached, the structural unit Selected from:

In some embodiments of the present invention, when R ₄ , R ₅ and the atoms to which they are attached form a ring, the structural unit Selected from:

In some preferred embodiments of the present invention, when R ₄ , R ₅ and the atoms to which they are attached form a ring, the structural unit Selected from:

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

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

In some embodiments of the present invention, the structural unit in formula I Replace with _R9 is selected from H.

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

In a second aspect, the present invention provides a pharmaceutical composition comprising the aforementioned compound or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite or prodrug as an active ingredient, supplemented with a pharmaceutically acceptable carrier.

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

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

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

In other embodiments, the administration of the compounds or pharmaceutical compositions of the present invention may be combined with another therapeutic method. The other therapeutic method may be selected from, but not limited to: radiotherapy, chemotherapy, immunotherapy, or a combination thereof.

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

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

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

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

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

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

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

In some preferred embodiments, the tumor-like disorder lacks the HR-dependent DNA DSB repair pathway.

In some preferred embodiments, the tumor-like disorder comprises one or more cancer cells having reduced or absent ability to repair DNA DSB via HR relative to normal cells.

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

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

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

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

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

Beneficial effects of the present invention:

The present invention provides a novel class of highly active and highly selective PARP1 inhibitors, which 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, and weak inhibitory effect on non-homologous recombination-deficient cells; (4) excellent pharmacokinetic properties (e.g., good bioavailability, suitable half-life and duration of action); (5) excellent safety (lower toxicity and/or fewer side effects, wider therapeutic window), etc. The PARP1 targeted compounds described in the present invention are surprisingly able to penetrate the blood-brain barrier (BBB) and can be used to treat diseases and conditions occurring in central nervous system tissues (e.g., brain and spinal cord).

Definition of terms:

Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those commonly understood by those skilled in the art. The terms "include," "comprises," "has," "contains," or "involves," and other variations thereof herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps. It should be understood by those skilled in the art that the above terms, such as "includes," encompass the meaning of "consisting of."

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

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

For example, the expression "C _1-8 " should be understood to include any sub-ranges and each point value therein, such as C _2-5 , C _3-4 , C _1-2 , C _1-3 , C _1-4 , C _1-5 , etc., as well as C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , etc. For example, the expression "C _2-8 " should also be understood in a similar manner, for example, it can include any sub-ranges and point values contained therein, such as C _6-8 , C _6-7 , C _7-8 , etc., as well as C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , etc. For another example, the expression "3-6 yuan" should be understood to include any sub-ranges and each point value therein, such as 3-4 yuan, 3-5 yuan, 3-6 yuan, 4-5 yuan, 4-6 yuan, etc., as well as 3, 4, 5, 6 yuan, etc.

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

In the present invention, unless otherwise specified, "alkyl" includes a linear or branched monovalent saturated hydrocarbon group. For example, alkyl includes methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 3-(2-methyl)butyl, 2-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-hexyl, 2-methylpentyl, etc. Similarly, _C1-4 in " _C1-4 alkyl" refers to a group containing 1, 2, 3 or 4 carbon atoms in a linear or branched form.

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

In the present invention, unless otherwise specified, "haloalkyl" refers to an alkyl group as described above, in which one or more hydrogen atoms are present. Atoms are replaced by halogen. For example, the term "C _1-6 haloalkyl" refers to a C _1-6 alkyl group optionally substituted by one or more (e.g., 1-3) halogens. It will be appreciated by those skilled in the art that when there are more than one halogen substituent, the halogens may be the same or different and may be located on the same or different C atoms. Examples of haloalkyl groups include, for example, -CH ₂ F, -CHF ₂ , -CF ₃ , -CCl ₃ , -C ₂ F ₅ , -C ₂ Cl ₅ , -CH ₂ CF ₃ , -CH ₂ Cl or -CH ₂ CH ₂ CF _3. The haloalkyl group in the present invention is optionally substituted by one or more substituents described herein.

In the present invention, unless otherwise specified, "fluoroalkyl" refers to the alkyl group described above, wherein one or more hydrogen atoms are replaced by fluorine atoms. For example, the term "C _1-4 fluoroalkyl" refers to a C _1-4 alkyl group optionally substituted by one or more (e.g., 1-3) fluorine atoms. It will be understood by those skilled in the art that when there are more than one fluorine atom substituents, the fluorine atoms may be the same or different, and may be located on the same or different C atoms. Examples of haloalkyl groups include, for example, -CH ₂ F, -CHF ₂ , -CF ₃ , -C ₂ F ₅ , -CH ₂ CF ₃ , -CH ₂ CH ₂ CF ₃ , etc. The fluoroalkyl group in the present invention is optionally substituted by one or more substituents described in the present invention.

In the present invention, unless otherwise specified, "alkenyl" refers to a straight or branched aliphatic hydrocarbon group having at least one C=C double bond. For example, " _C2-4 alkenyl" refers to an alkenyl group having 2 to 4 carbon atoms. Common alkenyl groups include (but are not limited to) vinyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, n-octenyl, n-decenyl, etc. The alkenyl group in the present invention is optionally substituted by one or more substituents described in the present invention.

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

The present invention also includes all pharmaceutically acceptable isotopically labeled compounds, which are identical to the compounds of the present invention except that one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominant in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium ( ^2H ), tritium ( ^3H )); isotopes of carbon (e.g., ^13C and _14C ); isotopes of chlorine (e.g., 37Cl); isotopes of iodine (e.g., ^125I ); isotopes of nitrogen (e.g., ^13N and ^15N ); isotopes of oxygen (e.g., ^17O and ^18O ); isotopes of phosphorus (e.g., ^32P ); and isotopes of sulfur (e.g., ^34S ).

In this application, "pharmaceutical composition" refers to a preparation of the compound of the present invention and a medium generally accepted in the art for delivering biologically active compounds to mammals (e.g., humans). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote administration of the organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

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

As used herein, the terms "drug combination", "drug combination", "combination therapy", "administering other treatments", "Administering other therapeutic agents" and the like refers to medical treatments obtained 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 of at least one compound described herein and at least one synergistic agent to a patient in the form of a single entity or a single dosage form. The term "non-fixed combination" refers to the simultaneous administration, combined administration or sequential administration of at least one compound described herein and at least one synergistic agent to a patient in the form of separate entities, at the same time, in combination or at variable intervals. These also apply to cocktail therapies, for example, the administration of three or more active ingredients.

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

In the present invention, unless otherwise indicated, "treating" means reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a curve showing the change in tumor volume in a NOD-SCID mouse subcutaneous tumor model of human breast cancer 436 cells after administration of compounds 47 and 64.

Figure 2 is a physical picture of the change in tumor volume in the human breast cancer 436 cell NOD-SCID mouse subcutaneous tumor model after administration of compounds 47 and 64.

FIG3 is a graph showing the changes in tumor volume in the breast cancer MDA-MB-436 nude mouse model after administration of compound 11.

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

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

FIG6 is a graph showing the effect of compound 11 administration on the body weight of human breast cancer MDA-MB-436 mice.

DETAILED DESCRIPTION

The scheme of the present invention will be explained below in conjunction with embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are followed.

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

Table 1 Abbreviations and their meanings in the present invention

The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements are performed using a Bruker AVANCE-400 nuclear magnetic spectrometer, with deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD) as the solvent, tetramethylsilane (TMS) as the internal standard, and chemical shifts are given in units of 10 ^-6 (ppm).

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

HPLC determination used Agilent 1200DAD high pressure liquid chromatograph (Sunfirc C18, 150X 4.6mm, 5wn, chromatographic column) and Waters 2695-2996 high pressure liquid chromatograph (Gimini C18, 150X 4.5mm, 5ym chromatographic column).

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

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

Unless otherwise specified in the following examples, the reactions were carried out under an argon atmosphere or a nitrogen atmosphere. Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a volume of about 1 L. Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a volume of about 1 L. The hydrogenation reaction is usually evacuated, filled with hydrogen, and the operation is repeated 3 times.

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

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

Step 2: Weigh compound int-1b (475 g, 1.93 mol) and add it to DMF (2.85 L), then add DMF-DMA (2.85 L) dropwise. After adding, 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 produced, then add to water and stir to precipitate. Cool to 20-30 °C, stir for 1 h, then filter, and dry the filter cake in a vacuum oven at 70 °C to constant weight to obtain a red solid int-1c (612 g, yield 95.3%).

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

Step 4: Add compounds int-1d (512 g, 1.69 mol) and int-1e (1457.0 g, 7.61 mol) to anhydrous ethanol (7.5 L), stir to dissolve, and add SnCl ₂ (1815.0 g, 9.57 mol) to the reaction system in batches at room temperature. After the addition, heat to reflux and stir for 1-2 h. Cool the reaction system to 45-50 ° C, concentrate under reduced pressure until there is no fraction, add ethyl acetate to the system and stir to dissolve, and then adjust the pH to 7-8 with saturated sodium bicarbonate. During the process, gas is released violently and a large amount of solid is precipitated. The reaction solution is centrifuged, and the filtrate is collected and allowed to stand for stratification. The organic layer is concentrated under reduced pressure at 40-45 ° C until there is no fraction, and 200-300 mesh silica gel is added to purify by column to obtain flocculent solid int-1 (230 g, yield 36.5%).

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

Step 1: Add compound int-2a (1g, 4.6mmol), int-2b (1.7g, 5.5mmol), Pd(dppf)Cl ₂ (0.3g, 0.46mmol) and potassium carbonate (1.6g, 11.5mmol) to a mixed solvent of 7ml dioxane, 3ml anhydrous ethanol and 4ml water, then replace nitrogen three times, and react at 90°C for 2h under nitrogen protection. After TLC detection, the reaction is cooled to room temperature, 30ml dichloromethane and 20ml water are added, and the layers are separated in a separatory funnel. The aqueous phase is extracted twice with dichloromethane, and the organic phases are combined and then washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried. The crude product is purified by column chromatography to obtain compound int-2c (1g, white solid).

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

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

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

Table 2 Intermediates int-3 to int-4

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

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

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

Step 3: The crude product of int-5b (171 mg, 0.5 mmol) was dissolved in methanol (5 mL), followed by addition of 4M HCl 1,4-dioxane (0.9 mL) solution, and reacted at room temperature for 12 h. The reaction was monitored by TLC. After the reaction was complete, potassium carbonate was added, stirred for 30 minutes, and potassium carbonate was removed by filtration to obtain the crude product of compound int-5 (180 mg, yellow-brown solid). MS/ESI[M+H] ⁺ =244.1.

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

Table 3 Intermediates int-6 to int-8

Intermediate int-9: N-methyl-5-(piperazine-1-yl)pyridineamide

Step 1: Add compound int-2a (5.66 g, 26.21 mmol, 1.05 eq), compound int-9a (4.65 g, 25.0 mmol, 1.00 eq), Cs ₂ CO ₃ (16.27 g, 50.0 mmol, 2.00 eq) and RuPhos-Pd-G3 (1.04 g, 1.25 mmol, 0.05 eq) to 1,4-dioxane (50 mL) and stir overnight at 120 ° C under nitrogen atmosphere. After the reaction is completed by LCMS, the mixture is allowed to cool to room temperature. The reaction solution is diluted with water (100 mL) and then extracted with ethyl acetate (2 x 100 mL). The combined organic layer is washed with brine (2 x 50 mL) and washed with anhydrous Na ₂ SO ₄ The mixture was dried, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound int-9b (white solid, 4.5 g). MS/ESI [M+H] ⁺ = 322.1.

Step 2: Compound int-9b (1.92 g, 6.0 mmol, 1.0 eq) was added to a solution of methylamine (8 mL, 25-30 wt% aqueous solution) in methanol (7 mL) and stirred at room temperature for 3 h under a nitrogen atmosphere. The reaction was monitored by LCMS. The reaction was quenched with a saturated aqueous solution of ammonium chloride (30 mL) at room temperature. The resulting mixture was extracted with dichloromethane (3 x 50 mL). The combined organic layer was washed with brine (2 x 50 mL) and dried over anhydrous Na ₂ SO _4. After filtration, the filtrate was concentrated under reduced pressure to obtain compound int-9c (1.65 g) as a light yellow oil. MS/ESI[M+H] ⁺ =321.3.

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

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

Table 4 Intermediates int-10 and int-11

Intermediate INT12: N-cyclopropyl-5-(piperazine-1-yl)pyridineamide

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

Step 2: Weigh int-12a (180 mg, 0.6 mmol), EDCI (144 mg, 0.75 mmol), HOBT (100 mg, 0.75 mmol) 2 mL DMF, N-methylmorpholine (290 mg, 3.2 mmol), cyclopropylamine (34 mg, 0.6 mmol), react at room temperature for 12 h, monitor by TLC, after the raw materials are completely consumed, dilute with water, extract with EA, wash the organic phase with water 5 times, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain the crude product int-12b (165 mg). MS/ESI [M+H] ⁺ = 347.2.

Step 3: The crude product of int-12b (150 mg, 0.4 mmol) was dissolved in 5 mL MeOH, followed by the addition of 0.9 mL 4M HCl in dioxane solution, and the reaction was carried out at room temperature for 12 h. The reaction was monitored by TLC. After the reaction was complete, potassium carbonate was added, and after stirring for 30 minutes, potassium carbonate was removed by filtration to obtain the crude product of compound int-12 (120 mg, yellow-brown solid). MS/ESI[M+H] ⁺ =247.1.

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

Table 5 Intermediates int-13 to int-15

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

Step 1: Add compound int-16a (2.0 g, 0.01305 mol) and compound int-16b (3.393 g, 0.0195 mol) to tetrahydrofuran (200 mL), cool the reaction solution to 0°C, add triphenylphosphine (20.54 g, 0.0783 mol), replace with nitrogen three times, stir for 1 hour, add DEAD (11.36 g, 0.06525 mol), stir at room temperature for 12 hours, after the reaction is completed, concentrate the reaction solution under reduced pressure, and the concentrate is subjected to column chromatography (PE:EA=10:1~1:1), and the product is collected to obtain a crude compound int-16c, LCMS=341.1.

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

Step 3: Add compound int-16d (4.5 g, 0.0146 mol) to dichloromethane (20 mL), add dioxane hydrochloride solution (20 mL), and react at room temperature for 1 h. After the reaction is completed, the reaction solution is concentrated under reduced pressure and Ethyl acetate was added to the concentrate for slurrying, and the solid was collected by filtration to obtain compound int-16 hydrochloride, LCMS=240.2.

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

Step 1: Dissolve compound int-1 (3.0 g, 9.64 mmol) in anhydrous toluene (30 mL), add compound int-17a (4.0 g, 18.34 mmol), Pd ₂ (dba) ₃ (1.0 g, 1.05 mmol), Xantphos (1.1 g, 1.9 mmol), DIPEA (4.0 mL, 24.24 mmol), replace with N ₂ three times, and stir at 110 ° C for 12 h. After cooling the reaction solution, slowly pour it into 100 mL saturated ammonium chloride solution for quenching, extract with EA 4*50 mL, dry with sodium sulfate, and separate by column chromatography (PE/EA=5/1). The obtained compound is dissolved in anhydrous EtOH (30 mL), NaOEt (1.3 g, 19.11 mmol) is added, and stirred at 25 ° C for 1 h. The reaction solution was slowly poured into HCl (1.0 M, 50 mL) for quenching, extracted with EA 5*30 mL, dried over sodium sulfate, concentrated and slurried with EA to obtain compound int-17b (2.5 g, crude) LC-MS: ESI [M+H] ⁺ = 265.2.

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

Step 3: Dissolve the compound int-17d (0.61 g, 1.84 mmol) in DCM (20 mL), slowly add DIBAL-H (1.0 M, 5.0 mL, 5.0 mmol) at 0°C, and stir at 0°C for 1 h. Slowly pour the reaction solution into 50 mL of saturated ammonium chloride solution to quench, filter with diatomaceous earth, extract with 4*30 mL of EA, and dry with sodium sulfate to obtain a yellow oily compound int-17e (0.45 g, crude). LC-MS: ESI [M+H] ⁺ = 291.1.

Step 4: Compound int-17e (0.45 g, 1.55 mmol) was dissolved in DCM (30 mL), TEA (0.7 mL, 7.36 mmol) and MsCl (0.5 mL, 3.6 mmol) were added dropwise at 0°C, and stirred at 0°C for 1 h. 30 ml of saturated sodium bicarbonate solution was slowly poured into the reaction solution for quenching, and DCM was extracted for 3*30 ml, dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain compound int-17 (0.6 g, crude). LC-MS: ESI [M+H] ⁺ = 369.2.

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

Step 1: Place compound int-1 (5.0 g, 16.07 mmol) in a sealed tube and add DMSO (50 mL) to dissolve. After solution, CuI (3.0 g, 15.7 mmol) and Zn(OAc) ₂ (6.0 g, 32.78 mmol) were added, and N ₂ was replaced three times, and then stirred at 150°C for 15 h. After cooling, the reaction solution was slowly poured into 100 mL of saturated ammonium chloride solution for quenching, filtered through diatomaceous earth, extracted with EA 5*50 mL, dried with sodium sulfate, and separated by column chromatography (PE/EA=5/1) to obtain a yellow solid compound int-18a (0.3 g, 1.08 mmol, yield 8%). LC-MS: ESI[M+H] ⁺ =279.2.

Step 2: Dissolve compound int-18a (0.3 g, 1.08 mmol) in DCM (20 mL), slowly add DIBAL-H (1.0 M, 5.0 mL, 5.0 mmol) at 0°C, and stir at 0°C for 1 h. Slowly pour the reaction solution into 50 mL saturated ammonium chloride solution to quench, filter with diatomaceous earth, extract with 4*30 mL EA, and dry with sodium sulfate to obtain yellow oily compound int-18b (0.3 g, crude). LC-MS: ESI [M+H] ⁺ = 237.2.

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

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

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

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

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

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

Step 5: The crude product of compound int-19e (140 mg, 0.4 mmol) was dissolved in 5 mL MeOH, followed by the addition of 0.9 mL 4M HCl in dioxane solution, and the reaction was carried out at room temperature for 12 h. The reaction was monitored by TLC. After the reaction was complete, potassium carbonate was added, stirred for 30 minutes, and potassium carbonate was removed by filtration to obtain the crude product of compound int-19 (120 mg). MS/ESI [M+H] ⁺ = 249.1.

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

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

Step 2: Add compound int-20b (0.25 g, 0.6 mmol), compound int-20c (0.14 g, 0.66 mmol), Pd(dppf)Cl ₂ (0.04 g, 0.06 mmol) and potassium carbonate (0.16 g, 1.2 mmol) to a mixed solvent of 20 ml dioxane and 2 ml water, then replace nitrogen, and react at 80°C for 3 hours under nitrogen protection. After TLC detection, the reaction is cooled to room temperature, concentrated by rotary evaporation, and extracted three times by adding 30 ml ethyl acetate and 20 ml water. After combining the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, filter, and spin dry. The obtained crude product is purified by column chromatography, solidified by pulping with petroleum ether, filtered, and dried to obtain compound int-20d (180 mg, light yellow liquid); LC-MS: ESI[M+H] ⁺ =434.6.

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

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

Intermediate int-21: (6-methoxy-7-((methyl-d ₃ )thio)-1,5-naphthyridin-3-yl)methyl methanesulfonate

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

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

Step 1: Sodium chlorodifluoroacetate int-22a (2.88 g, 18.9 mmol, 2 eq) and K ₂ CO ₃ (1.95 g, 14.1 mmol, 1.5 eq) were added to a 100 mL two-necked flask and dried under high vacuum for 1 hour. DMF (99%, 30 mL) was added at room temperature, and int-17b (2.5 g, 9.43 mmol) was added in batches. The reaction mixture was stirred at 95° C. for 15 minutes (exothermic reaction) and cooled to room temperature. Water (130 mL) and ethyl acetate (130 mL) were added. The layers were separated and the organic layer was washed with water (4 x 130 mL). Drying with MgSO ₄ and concentrating under vacuum, column chromatography purification (PE/EA=1/5) gave yellow oily compound int-22b (1.2 g). LC-MS: ESI[M+H] ⁺ =315.1.

Step 2: Dissolve compound int-22b (0.58 g, 1.84 mmol) in DCM (20 mL), slowly add DIBAL-H (1.0 M, 5.0 mL, 5.0 mmol) at 0°C, and stir at 0°C for 1 h. Slowly pour the reaction solution into 50 mL saturated ammonium chloride solution to quench, filter with diatomaceous earth, extract with 4*30 mL EA, and dry with sodium sulfate to obtain yellow oily compound int-22c (0.42 g, crude). LC-MS: ESI [M+H] ⁺ = 273.1.

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

Intermediate int-23: 6-Fluoro-N-methyl-5-(piperidin-4-yl)picolinamide

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

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

Step 1: Dissolve compound int-24a (0.85 g, 4.564 mmol) and compound int-9a (1.0 g, 4.149 mmol) in dioxane (40 mL), add RuPphos-Pd-G ₃ (0.35 g, 0.415 mmol), Cs ₂ CO ₃ (5.41 g, 16.595 mmol), replace with N ₂ three times, and stir at 100°C for 15 h. Pour the reaction solution into saturated sodium bicarbonate (30 mL), extract with EA (3*30 mL), dry with sodium sulfate, concentrate under reduced pressure, and purify by column chromatography (PE/EA=3/1) to obtain compound int-24b (0.7 g, 2.021 mmol) as a white solid. LC-MS: ESI[M+H]+=347.2.

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

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

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

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

Step 2: Dissolve compound int-25c (1.2 g, 2.989 mmol) in MeOH (20 mL) and add Pd/C 10% (1.2 g, 11.276 mmol), H ₂ replaced 3 times, stirred at 25°C for 1 h. Silica gel was filtered off under reduced pressure, and dried under reduced pressure to obtain compound int-25d (1.1 g, 2.726 mmol, 91.21%) as a light yellow solid. LC-MS: ESI [M+H] ⁺ = 404.2.

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

Intermediate int-26: 6-fluoro-N-(1-methyl-1H-pyrazol-4-yl)-5-(piperazin-1-yl)picolinamide

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

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

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

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

Step 1: Dissolve compound int-25a (1 g, 3.102 mmol) in MeOH (15 mL), add Pd/C 10% (1 g, 9.397 mmol), replace with H ₂ three times, and stir at 25°C for 1 h. Filter through silica gel pad, and dry the filtrate under reduced pressure to obtain compound int-27a (1 g, 3.083 mmol, 99.38%) as a light yellow solid. LC-MS: ESI [M+H] ⁺ = 325.2.

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

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

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

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

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

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

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

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

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

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

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

Step 1: Dissolve compound int-31a (2 g, 9.899 mmol) and compound int-9a (2.21 g, 11.878 mmol) in 1,4-dioxane (50 mL), add Ruphos-Pd-G3 (0.83 g, 0.990 mmol), Cs ₂ CO ₃ (9.68 g, 29.696 mmol), replace with N ₂ three times, and stir at 100°C for 12 h. Pour the reaction solution into saturated sodium bicarbonate (30 mL), extract with EA (3*30 mL), dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography (PE/EA=3/1) to obtain compound int-31b (1.7 g, 5.530 mmol, 55.87%), a light yellow liquid. LC-MS: ESI[M+H] ⁺ =308.2.

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

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

Step 1: Add (R)-3-(Hydroxymethyl)piperazine-1-carboxylic acid tert-butyl ester (5 g, 23.118 mmol) to DMF (50 mL), add p-methoxybenzyl chloride (3.62 g, 23.118 mmol) and potassium carbonate (9.58 g, 69.355 mmol), raise the temperature to 60 ° C and react for 24 hours. After the reaction is completed, water is added to quench the reaction, and ethyl acetate is added for extraction. The organic phase is washed with water 3 times, and the organic phase is concentrated under reduced pressure. The crude product is purified by column chromatography (petroleum ether: ethyl acetate = 10: 1 to 5: 1) to obtain compound int-32b (6 g, yield: 77.14%). LC-MS: ESI[M+H] ⁺ =337.4. ¹ H NMR(400MHz,Chloroform-d)δ7.14(d,J＝8.3Hz,2H),6.79(d,J＝8.3Hz,2H),3.88(d,J＝13.2Hz,1H),3.81–3.76(m,1H),3.72(s,3H), 3.60(m,1H),3.51(m,2H),3.28(d,J＝13.1Hz,2H),3.07(s,1H),2.69(d,J＝12.3Hz,1H),2.56–2.42(m,1H),2.18(m,1H),1.38(s,9H).

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

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

Step 4: Add compound int-32e (500 mg, 0.966 mmol) to acetonitrile (10 mL) and water (10 mL), cool to 0°C, add cerium ammonium nitrate (2.647 g, 4.83 mmol), stir at room temperature for 12 hours, after the reaction is completed, add the reaction solution to saturated sodium bicarbonate aqueous solution to adjust pH to 8-9, add ethyl acetate for extraction, wash the organic phase with water 3 times, concentrate the organic phase under reduced pressure, and purify the obtained crude product by column chromatography (dichloromethane: methanol = 8: 1) to obtain crude compound int-32f (100 mg, yield: 26.05%). LC-MS: ESI [M+H] ⁺ = 398.2.

Step 5: Add compound int-32f (85 mg, 0.214 mmol) and DIEA (82.93 mg, 0.642 mmol) to NMP (3 mL), heat to 180°C and stir for 6 hours. After the reaction is completed, add the reaction solution to water, extract with ethyl acetate, wash the organic phase with water three times, and concentrate the organic phase under reduced pressure to obtain compound int-32g (80 mg, yield: 99.11%). LC-MS: ESI [M+H] ⁺ = 378.2.

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

Step 7: Add compound int-32h (80 mg, 0.221 mmol) to dioxane (2 mL), add dioxane hydrochloride (1 mL), stir at room temperature for 1 hour, after the reaction is completed, concentrate the reaction solution under reduced pressure to obtain compound int-32 (hydrochloride 80 mg), LC-MS: ESI [M+H] ⁺ = 263.2.

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

Step 1: Add compound int-33a (4.0 g, 19.116 mmol) to dichloromethane (80 mL), add sodium carbonate (5.07 g, 47.790 mmol) and 4-nitrophenyl chloroacetate (4.24 g, 21.027 mmol), and react at room temperature for 12 h. After the reaction is completed, add water to quench the reaction, add dichloromethane to extract, wash the organic phase with water, dry the organic phase over anhydrous sodium sulfate, filter, collect the filtrate, and concentrate the filtrate to obtain compound int-33b (yield: 83.79%), LC-MS: ESI [M+H] ⁺ = 375.2.

Step 2: Add compound int-33b (6.0 g, 16.028 mmol) to dichloromethane (100 mL), add triethylamine (4.87 g, 48.083 mmol) and methylamine hydrochloride (1.30 g, 19.233 mmol), react at room temperature for 12 h, add water to quench the reaction, add dichloromethane to extract, wash the organic phase with water, dry the organic phase with anhydrous sodium sulfate, filter, collect the filtrate, concentrate the filtrate, and perform column chromatography (PE:EA=5:1~1:1) to collect the product to obtain compound int-33c (yield: 46.86%), LC-MS: ESI[M+H] ⁺ =267.2.

Step 3: Add compound int-33c (2.0 g, 7.51 mmol) to dichloromethane (20 mL), add trifluoroacetic acid (5 mL), react at room temperature for 1 h, and after the reaction is completed, concentrate under reduced pressure and collect the product to obtain compound int-33d trifluoroacetate, LC-MS: ESI [M+H] ⁺ = 167.20.

Step 4: Add compound int-33d trifluoroacetate (2.3 g, 8.208 mmol) to dichloromethane (20 mL), add triethylamine to adjust the pH to 6-7, add tert-butyl 3-oxyylideneazetidine-1-carboxylate (2.81 g, 16.416 mmol), add, react at room temperature for 1 h, add sodium triacetoxyborohydride (3.48 g, 16.416 mmol), after the reaction is completed, add water to quench the reaction, add dichloromethane to extract, wash the organic phase with water, dry the organic phase with anhydrous sodium sulfate, filter, collect the filtrate, concentrate the filtrate, and column chromatograph the concentrate (PE; EA = 2:1-1:1) to collect the product to obtain compound int-33e (yield: 75.82%), LC-MS: ESI [M+H] ⁺ = 322.2.

Step 5: Add compound int-33e (2.0 g, 6.223 mmol) to dichloromethane (20 mL), add trifluoroacetic acid (5 mL), react at room temperature for 1 h, and after the reaction is completed, concentrate under reduced pressure and collect the product to obtain compound int-33 trifluoroacetate. LC-MS: ESI [M+H] ⁺ = 222.1.

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

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

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

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

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

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

Step 2: Add compound int-35a (130 mg, 0.372 mmol), cyclopropylamine (34.81 mg, 0.372 mmol), DIEA (144.27 mg, 1.116 mmol) to DMF (3 mL), add HATU (212.22 mg, 0.558 mmol), and react at room temperature for 1 h. Add the reaction solution into water, extract with EA, and concentrate to remove the solvent to obtain compound int-35b (120 mg, yield: 83.02%), LC-MS: [M+H] ⁺ = 389.2.

Step 3: Add compound int-35b (120 mg, 0.309 mmol) to dichloromethane (3 mL), add hydrochloric acid dioxane solution (3 mL), and react at room temperature for 1 h. Concentrate the reaction solution under reduced pressure to dryness to obtain crude compound int-35 hydrochloride (120 mg, 0.309 mmol). mg), LC-MS: [M+H] ⁺ =289.2.

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

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

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

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

Step 1: Add compound int-37a (565 mg, 2.894 mmol), compound int-2a (625.10 mg, 2.894 mmol), bistriphenylphosphine palladium dichloride (203.10 mg, 0.289 mmol), TEA (1.207 mL, 8.681 mmol), CuI (110.22 mg, 0.579 mmol), dissolve in DMF (10 mL), replace with N2, heat to T = 80 ° C and stir for 20 h, filter the reaction solution, spin dry the filtrate, and use EA/PE = 0-40% column chromatography to obtain compound int-37b (529 mg) as a yellow oil. LC-MS: [M+H] ⁺ = 331.2.

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

Step 3: Compound int-37c (529 mg, 1.606 mmol) was dissolved in DCM (20 mL), TFA (5 mL, 65.296 mmol) was added, and the mixture was stirred at room temperature for 2 h. The reaction solution was spin-dried to obtain a crude product of compound int-37 (1.25 g) as a yellow oil (containing TFA), which was used directly in the next step without further purification. LC-MS: [M+H] ⁺ = 230.2.

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

Step 1: Add compound int-38a (400 mg, 1.852 mmol), compound int-2a (438.23 mg, 2.222 mmol), bistriphenylphosphine palladium dichloride (64.98 mg, 0.093 mmol), cuprous iodide (29.38 mg, 0.093 mmol), TEA (1.029 mL, 7.406 mmol), DMF (5 mL) into the reaction flask, replace with N2, raise the temperature to T = 50 ° C and stir overnight, add water to the reaction solution, extract with EA, dry the organic phase, spin dry, and chromatograph the residue with EA/PE = 0-90% column to obtain compound int-38b (529 mg) as a brown oil, LC-MS: [M+H] ⁺ = 333.1.

Step 2: Add compound int-38b (339 mg, 1.020 mmol) to the bottle, dissolve in methanol (10 mL), add methylamine (33%, methanol solution), and stir at room temperature overnight. The reaction solution was spin-dried, and the residue was subjected to column chromatography with MeOH/DCM = 0-9% to obtain compound int-38c (321 mg) as a light yellow oil, LC-MS: [M+H] ⁺ = 332.2.

Step 3: Add compound int-38c (321 mg, 0.969 mmol) to the bottle, dissolve in DCM (20 mL), cool to T = -78 ° C, add DAST (0.256 mL, 1.937 mmol) dropwise, and stir for 1 h. Add water to the reaction solution, separate the liquids, extract the aqueous phase with DCM, combine the organic phases, dry, and spin dry. The residue was subjected to EA/PE = 0-40% column chromatography to obtain compound int-38d (229 mg) as a colorless oil, LC-MS: [M+H] ⁺ = 334.2.

Step 4: Add compound int-38d (118 mg, 0.354 mmol) to the bottle, dissolve in DCM (10 mL), add TFA (3 mL, 39.177 mmol), stir at room temperature for 2 h, LC-MS shows: the reaction is complete, the reaction solution is concentrated under reduced pressure to obtain compound int-38 (181 mg, containing TFA) as a colorless oil, which is used directly in the next step without further purification. LC-MS: [M+H] ⁺ = 234.2.

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

Step 1: Dissolve compound int-2b (350 mg, 1.132 mmol) and compound int-39a (200 mg, 1.143 mmol) in H ₂ O (5 mL) and dioxane (20 mL), add Pd(dppf)Cl ₂ (82.82 mg, 0.113 mmol) and K ₂ CO ₃ (312.86 mg, 2.26 mmol), replace with N ₂ three times, stir at 100°C for 12 h. Concentrate under reduced pressure, and perform column chromatography (EA/PE=1/1, DCM/MeOH=20/1) to obtain compound int-39b (300 mg, 1.08 mmol, 95.55%). LC-MS: ESI [M+H] ⁺ = 278.2.

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

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

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

Step 2: Compound 1a (0.37 g, 1.34 mmol) was dissolved in DCM (10 mL), DAST (0.4 mL, 4.02 mmol) was added at 0°C, the temperature was slowly raised to 25°C, and stirred for 12 h. The reaction solution was slowly poured into 30 mL of saturated sodium bicarbonate to quench, extracted with DCM for 3*30 mL, dried with sodium sulfate, filtered, concentrated under reduced pressure, and purified by Pre-TLC (EA/PE=1/3) to obtain white solid compound 1b (0.09 g, 0.30 mmol, yield 22.7%). LC-MS: ESI[M+H] ⁺ =297.2.

Step 3: Dissolve compound 1b (0.09 g, 0.30 mmol) in dioxane (10 mL), slowly add HBr (46% in H ₂ O, 10 mL), and stir at 70°C for 1 h. After cooling, slowly pour the reaction solution into 100 mL saturated sodium bicarbonate solution to quench, extract with EA 4*40 mL, dry with sodium sulfate, and obtain white solid compound 1c (0.09 g) by Pre-TLC (DCM/EA=1/1). LC-MS: ESI[M+H] ⁺ =283.1.

Step 4: Dissolve compound 1c (0.09 g, 0.31 mmol) in DCM (10 mL), slowly add DIBAL-H (1 M, 3.0 mL, 3.0 mmol) dropwise at 0°C under _N2 atmosphere, and stir at 0°C for 1 h. Slowly pour 30 mL of The mixture was quenched with saturated sodium potassium tartrate solution, extracted with 4*40 mL of DCM, dried with sodium sulfate, and Pre-TLC (DCM/MeOH=15/1) gave white solid compound 1d (0.026 g, 0.11 mmol, yield 34.8%). LC-MS: ESI [M+H] ⁺ = 241.2.

Step 5: Compound 1d (0.026 g, 0.11 mmol) was dissolved in DCM (20 mL), and DIPEA (0.1 mL, 0.56 mmol) and MsCl (0.02 mL, 0.2 mmol) were added dropwise at 0°C, and the mixture was heated to 25°C and stirred for 1 h. 20 ml of saturated sodium bicarbonate solution was slowly poured into the reaction solution for quenching, and 3*30 ml of DCM was extracted, dried over sodium sulfate, and filtered and concentrated under reduced pressure to obtain compound 1e (0.05 g). LC-MS: ESI [M+H] ⁺ = 397.2.

Step 6: Compound 1e (0.05 g, 0.12 mmol) was dissolved in ACN (10 mL), and DIPEA (0.5 mL, 2.80 mmol), KI (0.02 g), and compound int-6 (0.02 g, 0.09 mmol) were added and stirred at 80°C for 2 h. 20 ml of saturated ammonium chloride solution was slowly poured into the reaction solution for quenching, EA was extracted with 4*30 ml, dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain compound 1f (0.05 g). LC-MS: ESI [M+H] ⁺ = 522.2.

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

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

Step 1: Dissolve compound 1b (0.68 g, 2.30 mmol) in DCM (30 mL), slowly add DIBAL-H (1 M, 11.5 mL, 11.5 mmol) at 0°C under _N2 atmosphere, and stir at 25°C for 1 h. Slowly pour 100 mL of saturated sodium potassium tartrate solution into the reaction solution to quench, extract with DCM 5*50 mL, dry with sodium sulfate, filter and concentrate under reduced pressure to obtain compound 2a (0.59 g, crude). LC-MS: ESI [M+H] ⁺ = 257.2.

Step 2: Compound 2a (0.59 g, 2.32 mmol) was dissolved in DCM (20 mL), TEA (2.0 mL, 13.0 mmol) and MsCl (0.8 mL, 7.10 mmol) were added dropwise at 0 °C, and the mixture was heated to 25 °C and stirred for 1 h. 20 ml of saturated sodium bicarbonate solution was slowly poured into the reaction solution to quench, and 4*30 ml of DCM was extracted, dried over sodium sulfate, and filtered and concentrated under reduced pressure to obtain compound Material 2b (0.60g, crude). LC-MS: ESI[M+H] ⁺ =333.2.

Step 3: Compound 2b (0.20 g, 0.60 mmol) was dissolved in acetonitrile (10 mL), and DIPEA (0.2 mL, 1.55 mmol) was added. Compound int-3 (0.2 g, 0.85 mmol) was stirred at 80°C for 2 h. 20 ml of saturated ammonium chloride solution was slowly poured into the reaction solution for quenching, 5*30 ml of EA was extracted, dried over sodium sulfate, filtered and concentrated under reduced pressure, and Pre-TLC (DCM/MeOH=20/1) obtained white solid compound 2c (0.11 g, 0.23 mmol, yield 38%). LC-MS: ESI[M+H] ⁺ =472.1.

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

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

Table 6 Compounds 3 to 9

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

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

Step 2: Compound 10a (0.09 g, 0.21 mmol) was dissolved in dioxane (10 mL), HBr (46% in H ₂ O, 5.0 mL) was slowly added dropwise, and stirred at 70°C for 1 h. The reaction solution was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution for quenching, extracted with 4*50 mL of DCM, dried with sodium sulfate, and purified by reverse preparation to obtain white solid compound 10 (30 mg, 0.07 mmol, yield 34%). LC-MS: ESI [M+H] ⁺ = 422.2. ^1H NMR(400MHz,MeOD)δ8.70(s,1H),8.51(d,J＝1.8Hz,1H),8.07–7.93(m,2H),7.78(s,1H),7.62(s,1H),6.38 (s,1H),3.82(s,2H),2.96(s,3H),2.84(t,J＝5.7Hz,2H),2.65(s,2H),2.53(s,3H),2.20(t,J＝7.6Hz,2H).

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

Table 7 Compounds 11 to 30

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

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

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

The preparation methods of compounds 32-40 of Examples 32-40 can be referred to Example 31.

Table 8 Compounds 32-40

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

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

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

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

Table 9 Compounds 42 to 49

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

Step 1: Compound int-1 (0.91 g, 2.92 mmol) was added to anhydrous THF (300 mL), the reaction solution was cooled to -20 ° C, DIBAL-H (4.9 mL, 7.3 mmol, 1.5 M toluene solution) was added under a N ₂ atmosphere at -20 ° C, and the reaction mixture was further stirred between -15-0 ° C for 3 hours. TLC showed that the reaction was complete. The reaction was slowly quenched with 3N NaOH aqueous solution between -15-0 ° C, and the internal temperature was kept at no more than 0 ° C. The volatile substances were removed under reduced pressure at 25 ° C, and the resultant was extracted with ethyl acetate (30 mL*3), and the combined organic phase was washed with water (30 mL) and brine (30 ml), dried with anhydrous Na ₂ SO ₄ 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 50a (0.54 g) was obtained as a yellow solid. LC-MS: ESI [M+H] ⁺ = 269.2.

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

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

Step 4: Compound 50c (0.3 g, 0.62 mmol) was dissolved in dioxane (10 mL), HBr (46% in H ₂ O, 5.0 mL) was slowly added dropwise, and stirred at 70°C for 1 h. The reaction solution was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution for quenching, extracted with 4*30 mL of DCM, dried with sodium sulfate, and slurried with EA/PE (10/1) to obtain white solid compound 50d (0.35 g, crude). LC-MS: ESI [M+H] ⁺ = 469.2/470.2.

Step 5: Compound 50d (0.2 g, 0.423 mmol) was dissolved in dioxane (10 mL), tri-n-butyl vinyl tin (0.27 g, 0.86 mmol), XPhos-Pd-G ₂ (0.05 g, 0.06 mmol) were added, and after N ₂ substitution three times, the mixture was heated at 90°C. The mixture was stirred for 15 h. After cooling, it was concentrated and purified by Pre-TLC (DCM/MeOH=20/1) and then by reverse phase preparative purification to obtain compound 50 (20 mg, 0.05 mmol, yield 12%). LC-MS: ESI [M+H] ⁺ = 416.2. ¹ H NMR (400 MHz, CDCl ₃ )δ10.75(s,1H),8.57(d,J＝1.7Hz,1H),8.11(s,1H),8.05(d,J＝5.0Hz,1H),7.96 (d,J＝7.9Hz,1H),7.67(s,1H),7.53(d,J＝7.9Hz,1H),7.00(dd,J＝17.7,11.4Hz,1 H),6.24(dd,J＝17.7,1.2Hz,1H),5.65(s,1H),5.54(dd,J＝11.3,1.1Hz,1H),3.8 0(s,2H),3.22(d,J＝2.9Hz,2H),3.03(d,J＝5.1Hz,5H),2.77(s,2H),2.54(s,3H).

The preparation methods of compounds 51-58 of Examples 51-58 can be referred to Example 50.

Table 10 Compounds 51 to 58

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

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

Compound 59a (0.12 g, 0.27 mmol) was dissolved in dioxane (10 mL), HBr (46% in H ₂ O, 5.0 mL) was slowly added dropwise, and stirred at 70° C. for 1 h. The reaction solution was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution for quenching, extracted with DCM 4 x 30 mL, dried with sodium sulfate, and purified by reverse preparation to obtain white solid compound 59 (35 mg, 0.082 mmol, yield 31%). LC-MS: ESI [M+H] ⁺ = 425.2. ¹ H NMR (400MHz, CDCl ₃ )δ8.52(s,1H),8.16(d,J＝2.4Hz,1H),8.05(d,J＝8.7Hz,1H),7.77(s,1H),7.64(d,J＝12.2Hz,2H), 7.22(d,J＝8.8Hz,1H),3.70(s,2H),3.36(s,4H),3.01(d,J＝5.0Hz,3H),2.67(s,4H),2.50(s,3H).

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

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

Step 2: Compound 60a (4.34 g, 16.08 mmol) was dissolved in DCM (100 mL), and DIPEA (10 mL, 80.4 mmol) was added dropwise. MOMCl (2.5 mL, 32.16 mmol) was slowly added dropwise at 0°C, and stirred at 0°C for 2 h. The reaction solution was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution for quenching, extracted with DCM 4*30 mL, dried with sodium sulfate, concentrated under reduced pressure, and column chromatography (EA/PE=1/3) was performed to obtain compound 60b (2.44 g, 7.79 mmol). LC-MS: ESI[M+H] ⁺ =313.1/315.1.

Step 3: Compound 60b (2.44 g, 7.79 mmol) was dissolved in toluene (20 mL), and DIPEA (3 mL, 18.18 mmol), Pd2(dba)3 (0.74 g, 0.8 mmol), Xantphos (0.9 g, 1.56 mmol), and compound int-17 (3.4 g, 15.63 mmol) were added. The mixture was stirred at 110°C for 12 h under N2 atmosphere. The mixture was concentrated under reduced pressure and column chromatography (EA/PE=1/3) was performed to obtain compound 60c (3.2 g, 7.34 mmol, yield 91%). LC-MS: ESI[M+H] ⁺ =437.1.

Step 4: Compound 60c (3.2 g, 7.32 mmol) was dissolved in EtOH (50 mL), EtONa (1.5 g, 22.05 mmol) was added, and the mixture was stirred at 25°C for 2 h. The reaction solution was slowly poured into an aqueous solution of HCl (0.5 M, 100 mL) for quenching, extracted with EA 5*50 mL, dried over sodium sulfate, and concentrated under reduced pressure to obtain compound 60d (3.0 g, crude). LC-MS: ESI [M+H] ⁺ = 267.2.

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

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

Step 7: Compound 60f (0.6 g, 2.35 mmol) was dissolved in DCM (10 mL), DMF (0.01 mL) was added, SOCl ₂ (0.4 mL, 4.70 mmol) was added dropwise at 25°C, and the mixture was stirred for 1 h at 25°C. The mixture was filtered and concentrated under reduced pressure to obtain compound 60g (0.6 g, crude). LC-MS: ESI [M+H] ⁺ = 273.2.

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

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

The preparation methods of compounds 61-63 of Examples 61-63 can be referred to Example 60.

Table 11 Compounds 61-63

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

Step 1: Dissolve compound int-18b (0.59 g, 2.50 mmol) in DCM (10 mL), add DMF (0.01 mL) dropwise, add SOCl ₂ (0.60 mL, 5.00 mmol) dropwise at 0°C, and stir at 25°C for 1 h. Concentrate under reduced pressure to remove the solvent to obtain compound 64a (0.64 g, crude). LC-MS: ESI [M+H] ⁺ = 255.2.

Step 2: Compound 64a (0.64 g, 2.50 mmol) was dissolved in dioxane (10 mL), HBr (38% in AcOH, 2.0 mL) was slowly added dropwise, and stirred at 40°C for 1 h. After the reaction solution was cooled, 100 mL of saturated carbonic acid was slowly poured into the mixture. The mixture was quenched in sodium hydrogen solution, filtered to obtain a filter cake, washed with DCM/MeOH (20/1), and the filtrate was concentrated under reduced pressure to obtain compound 64b (0.6 g, crude). LC-MS: ESI [M+H] ⁺ = 241.1.

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

Example 65: 5-(4-(((7-(1,1-difluoroethyl)-6-oxo-56-dihydro-1,5-naphthyridin-3-yl)methyl)piperazin-1-yl)-6-fluoro-N-methylpicolinamide

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

Step 2: Compound 65a (0.12 g, 0.43 mmol) was dissolved in dioxane (10 mL), HBr (38% in AcOH, 2.0 mL) was slowly added dropwise, and stirred at 40°C for 1 h. The reaction solution was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution for quenching, and filtered under reduced pressure to obtain white solid compound 65b (0.12 g, crude). LC-MS: ESI [M+H] ⁺ = 259.2.

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

Example 66: 5-(1-(((7-(1,1-difluoroethyl)-6-oxo-56-dihydro-1,5-naphthyridin-3-yl)methyl)piperidin-4-yl)-6-fluoro-N-methylpicolinamide

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

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

Step 1: Dissolve compound int-22c (1.0 g, 3.47 mmol) in DCM (10 mL), add DMF (0.01 mL), add SOCl ₂ (1.0 mL, 6.94 mmol) dropwise at 25°C, and stir at 25°C for 1 h. Concentrate under reduced pressure to remove the solvent to obtain compound 67a (0.6 g, crude). LC-MS: ESI [M+H] ⁺ = 291.2.

Step 2: Compound 67a (1.0, 3.44 mmol) was dissolved in dioxane (10 mL), HBr (38% in AcOH, 3.0 mL) was slowly added dropwise, and stirred at 40°C for 1 h. The reaction solution was cooled and slowly poured into 100 mL of saturated sodium bicarbonate solution for quenching, and filtered to obtain white solid compound 67b (0.90 g, crude). LC-MS: ESI [M+H] ⁺ = 277.2.

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

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

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

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

Step 1: Compound 69a (1.0 g, 5.0 mmol) was dissolved in DCM (20 mL), MnO ₂ (7.0 g, 80.0 mmol) was added, and the mixture was stirred at 25° C. for 15 h. The mixture was filtered through celite, and the filtrate was collected and concentrated under reduced pressure to obtain compound 69b (1.0 g, crude). LC-MS: ESI [M+H] ⁺ = 200.1/202.1.

Step 2: Compound 69b (1.0 g, 5.0 mmol) was dissolved in DCM (30 mL), methylthioacetic acid (0.4 mL, 5.0 mmol) was added, the temperature was lowered to 0°C, pyridine (4.0 mL, 50.0 mmol) and POCl ₃ (2.3 mL, 25 mmol) were added dropwise in sequence, and the temperature was raised to 25°C and stirred for 1 h. The reaction solution was slowly poured into a saturated sodium bicarbonate solution (100 mL) under an ice-water bath to quench the reaction, and DCM was used for extraction (5*30 mL), dried over sodium sulfate, and concentrated under reduced pressure to obtain compound 69c (1.4 g, crude). LC-MS: ESI [M+H] ⁺ = 290.2/292.2.

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

Step 4: Dissolve compound 69d (0.7 g, 2.57 mmol) in dioxane (30 mL) and add tributyltin Methanol (1.0 mL, 3.11 mmol), XPHOS-Pd-G ₂ (0.2 g, 0.26 mmol), stirred at 100° C. for 12 h under N ₂ atmosphere. After the reaction was complete, the mixture was filtered through celite pad, and the filtrate was dried by spin drying and purified by Prep-TLC to obtain compound 69e (0.17 g, 0.77 mmol, 30%). LC-MS: ESI [M+H] ⁺ = 222.2.

Step 5: Compound 69e (0.17 g, 0.77 mmol) was dissolved in toluene (10 mL), DMF (0.01 mL) was added, SOCl ₂ (0.06 mL, 0.85 mmol) was added dropwise at 25°C, and the mixture was stirred for 1 h at 25°C. The mixture was concentrated under reduced pressure to obtain compound 69f (0.2 g, crude). LC-MS: ESI [M+H] ⁺ = 240.2.

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

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

Table 12 Compounds 70-74

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

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

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

Step 1: Dissolve compound 76a (10 g, 42.731 mmol) in THF (200 mL), add BF ₃ ^. Et ₂ O (18.19 g, 128.194 mmol), add NaBH ₄ (4.85 g, 128.194 mmol) at 0°C, and stir at 0°C for 2 h. Slowly pour the reaction solution into 100 mL of saturated ammonium chloride solution to quench the reaction. Concentrate under reduced pressure, extract with EA 5*50 mL. Dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude product of compound 76b (10.0 g), LC-MS: ESI [M+H] ⁺ = 220.2/222.2.

Step 2: Compound 76b (10 g, 45.446 mmol) was dissolved in DCM (200 mL), MnO ₂ (19.76 g, 227.231 mmol) was added, and the mixture was stirred at 25° C. for 3 h. The mixture was filtered through Celite pad, and the filtrate was collected and concentrated under reduced pressure to obtain compound 76c (7.0 g, crude). LC-MS: ESI [M+H] ⁺ = 217.2/219.2.

Step 3: Compound 76c (5 g, 22.933 mmol) was dissolved in THF (150 mL), ethyl 2-methylthioacetate (3.08 g, 22.933 mmol), N ₂ was replaced, LiHMDS (68.798 mL, 68.798 mmol) was added dropwise at -78°C, and stirred at -78°C for 1 h. The reaction solution was slowly poured into a saturated ammonium chloride solution (100 mL) under an ice-water bath to quench the reaction, extracted with EA 5*30 mL, dried with sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (EA/PE=1/3) to obtain compound 76d (7.0 g, crude). LC-MS: ESI[M+H] ⁺ =288.2/290.2.

Step 4: Compound 76d (2 g, 6.941 mmol) was dissolved in dioxane (30 mL), tributyltin methanol (2.23 g, 6.941 mmol) and XPHOS-Pd-G ₂ (0.55 g, 0.694 mmol) were added, and stirred at 100° C. for 12 h under N ₂ atmosphere. Prep-TLC purification gave compound 76e (1.35 g, 5.64 mmol, 81%). LC-MS: ESI [M+H] ⁺ = 240.2.

Step 5: Compound 76e (1.35 g, 5.64 mmol) was dissolved in toluene (30 mL), DMF (0.02 mL) was added, SOCl ₂ (0.81 g, 6.771 mmol) was added dropwise at 25°C, and stirred at 25°C for 1 h. The mixture was concentrated under reduced pressure to obtain compound 76f (1.3 g, crude). LC-MS: ESI [M+H] ⁺ = 258.2.

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

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

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

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

Step 1: Compound 91a (2 g, 12.774 mmol) was dissolved in DMF (20 mL) and ACN (20 mL), selectfluor (5.43 g, 15.329 mmol) was added, _N2 was replaced 3 times, and the mixture was stirred at 80°C for 2 h. After the reaction was completed, saturated sodium bicarbonate solution (30 mL) was added to quench the reaction. EA was extracted with 5*30 mL, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by column chromatography (EA/PE=1/3) to obtain compound 91b (0.9 g, 5.15 mmol, 40.3%), LC-MS: ESI[M+H] ⁺ =175.2.

Step 2: Dissolve compound 91b (0.9 g, 5.15 mmol) in THF (30 mL), compound ethyl 2-methylthioacetate (0.83 g, 6.18 mmol), replace with N ₂ , add LiHMDS (25.7 mL, 25.7 mmol) dropwise at -78 °C, and stir at -78 °C for 1 h. Then raise the temperature to 25 °C and stir for 12 h. Slowly pour the reaction solution into saturated ammonium chloride solution (100 mL) under ice-water bath to quench the reaction, and collect the filter cake by filtration to obtain the target compound 91c (2.8 g, crude). LC-MS: ESI [M+H] ⁺ = 245.2.

Step 3: Compound 91c (2.3 g, 9.4 mmol) was dissolved in dioxane (50 mL), tributyltin methanol (3.32 g, 10.34 mmol) and XPHOS-Pd-G ₂ (0.74 g, 0.94 mmol) were added, and stirred at 100° C. for 12 h under N ₂ atmosphere. Prep-TLC purification gave compound 91d (0.8 g, 0.94 mmol, 35.2%). LC-MS: ESI [M+H] ⁺ = 241.2.

Step 4: Compound 91d (0.8 g, 3.33 mmol) was dissolved in toluene (30 mL), DMF (0.05 mL) was added, SOCl ₂ (0.40 g, 3.33 mmol) was added dropwise at 25°C, and stirred at 100°C for 4 h. The mixture was concentrated under reduced pressure and purified by Prep-TLC to obtain compound 91e (0.35 g, 1.35 mmol, 40.6%). LC-MS: ESI [M+H] ⁺ = 259.2.

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

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

Table 14 Compounds 92-95, 97 and 108

Biological activity test:

1. PARP-1 enzyme assay

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

1.1 PARP1 enzyme assay

1.1.1 Preparation of buffer: PBST: 1X PBS, 0.05% Tween-20, blocking solution: 1X PBS, 0.05% Tween-20, 5% BSA, reaction buffer: 50mM Tris-HCl (pH7.5), 0.005% Tween-20, 0.01% BSA.

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

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

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

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

1.1.5 Prepare 1000 times the compound, transfer 1uL of the compound to 199uL of reaction buffer in a 96-well plate, mix well, and transfer 5uL of the mixed compound to a 384-well reaction plate.

1.1.6 Prepare 25/10 times PARP1-DNA solution with reaction buffer, transfer 10uL PARP1-DNA solution to the 384-well reaction plate. For the negative control well, transfer 10uL DNA solution. The final concentration of PARP1 is 0.02nM and the final concentration of DNA is 0.8nM.

1.1.7 Prepare 25/10 times NAD+ solution with reaction buffer, transfer 10uL NAD+ solution to a 384-well reaction plate, the final NAD+ concentration is 3.5uM, and incubate at room temperature for 60 minutes.

1.1.8 Prepare 25/10 times NAD+ solution with reaction buffer, transfer 10uL NAD+ solution to a 384-well reaction plate, the final NAD+ concentration is 3.5uM, and incubate at room temperature for 60 minutes.

1.1.9 Washing: After the reaction is completed, discard the reaction solution and wash three times with PBST solution according to step 2, and finally pat the reaction plate dry.

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

1.1.11 Washing: Discard the primary antibody and wash three times with PBST solution according to step 2. Finally, pat dry. Should board.

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

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

1.1.14 Color development: Mix Femto-ECL Substrate A and Femto-ECL Substrate B in a 1:1 ratio and transfer 25uL to a 384 reaction plate.

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

The test results are shown in Table 15 below:

Table 15 PARP-1 enzyme test results

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

2. Cell anti-proliferation activity test:

BRCA mutant MDA-MB-436 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin, and 100μg/mL streptomycin, and cultured in a 5% saturated CO2 incubator at 37°C. When the cells grew to 80% confluence, the cells were collected, centrifuged at 300g for 10min, and plated on a 96-well plate at 1200 cells/well. After 24h, different final concentrations of PARPi (0, 0.01, 0.1, 1, 10, 100, 1000nM) were added and cultured for 72h. The cells were treated with a replacement medium (PARPi was added again at the same final concentration) and cultured for 96h. The 96-well plate was taken out, and the OD value at a wavelength of 450nM was detected by CCK8 method, and the cell inhibition rate was calculated: inhibition rate % = 1-(mean OD value of the drug group-mean OD value of the Blank group)/(mean OD value of the Control group-mean OD value of the Blank group)*100%.

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

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

The test results are shown in Table 16 below:

Table 16 Cell antiproliferative activity test results

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

3. Inhibition experiment of compounds on hERG potassium channel

Cell culture and treatment: CHO cells stably expressing hERG were cultured in a cell culture flask and placed in an incubator at 37°C and 5% CO _2. When the cell density grew to 60-80%, the cell culture medium was removed, and the cells were washed once with PBS and digested with Detachin. After complete digestion, the cells were neutralized with culture medium, centrifuged, the supernatant was removed, and the cells were resuspended with culture medium. The cell density was adjusted to 2-5×10 ⁶ /mL for later use.

Compound preparation: Dilute the compound stock solution with 100% DMSO, that is, take 10μL of the compound stock solution and add it to 20μL DMSO, and dilute it 3 times continuously to 6 concentrations. Take 4μL of the 6 concentrations of the compound respectively and add it to 396μL of extracellular fluid, that is, dilute it 100 times to get 6 intermediate concentrations. Then take 80μL of the 6 intermediate concentration compounds respectively and add them to 320μL of extracellular fluid, that is, dilute it 5 times to the final concentration to be tested. The highest test concentration is 40μM, and there are 6 concentrations of 40, 13.33, 4.44, 1.48, 0.49 and 0.16μM respectively. The DMSO content in the final test concentration does not exceed 0.2%, and this concentration of DMSO has no effect on the hERG potassium channel. The compound preparation is completed by the Bravo instrument throughout the dilution process.

Electrophysiological recording process: The single-cell high-impedance sealing and whole-cell pattern formation process are all automatically completed by the Qpatch instrument. After obtaining the whole-cell recording mode, the cell is clamped at -80 mV. Before giving a 5-second +40 mV depolarizing stimulus, a 50-millisecond -50 mV pre-voltage is given, and then repolarizes to -50 mV for 5 seconds, and then returns to -80 mV. This voltage stimulus is applied every 15 seconds. After recording for 2 minutes, the extracellular solution is given for 5 minutes, and then the drug administration process begins. The compound concentration starts from the lowest test concentration, and each test concentration is given for 2.5 minutes. After all concentrations are given continuously, the positive control compound 3μM Cisapride is given. At least 3 cells (n≥3) are tested for each concentration.

Data processing: Data analysis and processing were performed using GraphPad Prism 5.0 and Excel software. The IC50 of the compound was calculated using GraphPad Prism 5 software by fitting the following equation:

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

Among them, X is the Log value of the test sample detection concentration, Y is the inhibition percentage at the corresponding concentration, and Bottom and Top are the minimum and maximum inhibition percentages, respectively.

Table 17 Inhibition results of compounds on hERG potassium channels

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

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

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

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

Experimental steps: Weigh the compound, add a small amount of DMSO, and then add sodium chloride solution for injection to make a 1mg·mL ^-1 compound solution for administration. Mice, male mice, were orally administered at 10 mg·kg ^-1. Whole blood and whole brain were collected 1 h and 6 h after administration (n=3). Whole blood was centrifuged at 3500 rpm for 15 min, and the supernatant plasma was collected. The weight of the centrifuge tube was weighed as M1, the weight of the centrifuge tube containing the whole brain was weighed as M2, the weight of the centrifuge tube after adding water homogenate was weighed as M3, and the weight of the centrifuge tube after taking out 30μL of homogenate was weighed as M4. Take 30μL of plasma and 30μL of brain homogenate in a centrifuge tube, add 120μL of acetonitrile containing 20 ng·ml ^-1 internal standard SAHA to precipitate, vortex for 30s, centrifuge at 13000 rpm for 15 min, and take the supernatant into the injection bottle for testing.

Standard curve range: 10～10000 ng·ml ^-1 .

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

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

Table 18 Results of the brain blood distribution test in mice after compound administration

Conclusion: The plasma concentration and brain tissue concentration of compounds 10, 11, 12, 17, 22, 27, 31, 42, 47, 50 and 60 of the present invention are significantly higher than those of AZD9574 at 1h and 6h. Therefore, compounds 10, 11, 12, 17, 22, 27, 31, 42, 47, 50 and 60 have the potential for brain penetration.

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

Experimental purpose: To understand the pharmacokinetics of the compound.

Experimental basis: Technical guidelines for non-clinical pharmacokinetic studies of chemical drugs, 2014.

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

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

Sample collection: 6 male Balb/c mice (Chengdu Dashuo Experimental Animal Co., Ltd., license number: SCXK (Chuan) 2020-030), 3 mice were intravenously administered (IV) at 1 mg·kg-1, and 3 mice were gavaged (PO) at 1 mg·kg-1. About 0.05 mL of blood was collected at 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 10h, 24h and 48h after administration. The collected blood was centrifuged at 3500rpm for 15min, and the supernatant plasma was collected and frozen at -40℃ for testing. The blood drug concentration was quantitatively analyzed by LC-MS/MS analysis method, and pharmacokinetic parameters such as peak time (Cmax), area under the drug-time curve (AUC(0-t)), half-life (T _1/2 ), clearance (CL), tissue distribution (Vdss), biological Utilization (F), etc.

Table 19 Pharmacokinetic test results of compounds in Balb/c mice

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

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

(1) Main instruments and equipment

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

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

Water purifier: American Millipore brand, model F7PNO9748; vertical high pressure sterilizer: Yamato brand, model DKN812C; 1mL disposable sterile syringe: Shanghai Kangdeli Enterprise Development Group Co., Ltd.; 1mL disposable sterile insulin syringe: BD brand; weight scale: Shanghai Hengping Instrument Factory, model JY2002; analytical balance: SARTORIUS brand, model BCE95I-1CEU;

Thermometer and hygrometer: Wuqiang Thermohygrometer Manufacturing Center, measurement and manufacture number: Jizi 30260102; small centrifuge: American Thermo brand, model: SORVALL LEGEND MICRO 17Centrifuge; mini mixer: Yeasen brand, model: ES-VM25; metal bath: SCILOGEX brand, model: SCL120-S.

(2) Main software and data processing systems

Graphpad Prism, version 6.0, Graphpad Software Ltd., Graphpad Prism software is used to organize and quantify data and to create bar graphs.

(3) Experimental animals

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

(4) Modeling method

Tumor cell culture and preparation: For routine tumor cell line passage and culture, the culture medium contains 10% fetal bovine serum, 100U/mL penicillin, and 100μg/mL streptomycin. When the cells grow to 80% confluence, collect the cells and centrifuge at 300g for 10min. Wash with pre-cooled PBS three times, collect the cells by centrifugation at 300g for 10min, resuspend the cells in PBS, count the cells with a hemocytometer, adjust the cell concentration, and pre-cool the cell suspension on ice after cell collection. Take 100μL of the cell suspension and inject it subcutaneously in the dorsal armpit of the mouse. Start group treatment when the tumor volume reaches ^200-300mm3 .

(5) Observation and testing indicators

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

(6) Weight

Animals to be measured: All surviving experimental animals planned for measurement; Measurement time: Weigh all animals before modeling for experimental grouping, weigh mice once every 2 days after modeling, and record the weight changes of mice in real time. Effective judgment criteria: Tumor volume (TV), relative tumor volume (RTV) and relative tumor proliferation rate of subcutaneous transplanted tumor model. Use a vernier caliper to measure the long and short diameters of the tumor every 2 days, and dynamically observe the anti-tumor effect of the test drug. The calculation formula of tumor volume (TV) is: TV (mm3) = a×b2×0.5; where a and b represent the long diameter and short diameter, respectively.

The relative tumor volume (RTV) was calculated based on the measurement results. The calculation formula was: RTV = Vt/V0, where V0 was the TV measured for each group at the time of cage administration (i.e., d0), and Vt was the TV for each measurement of the group. The evaluation index for antitumor activity was the relative tumor growth rate T/C (%), and the calculation formula was as follows:
T/C(%)=TRTV/CRTV×100%;

TRTV: relative tumor volume of treatment group; CRTV: relative tumor volume of negative control group. Efficacy evaluation criteria: T/C (%)>60 is ineffective; T/C (%)≤60, and P<0.05 after statistical analysis is effective. Tumor weight determination and calculation of tumor inhibition rate (%) At the end of treatment, the animals were killed, the tumor was dissected and removed, the tumor weight was weighed, and photographed. The tumor inhibition rate (%) was calculated according to the following formula:

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

Effective judgment criteria: Tumor inhibition rate (i.e. tumor growth inhibition rate) < 40% is ineffective; tumor inhibition rate ≥ 40% and P < 0.05 after statistical analysis is effective. Subcutaneous tumor model comprehensive judgment criteria: It is judged to be effective when one of the above two effective judgment criteria (relative tumor proliferation rate and tumor inhibition rate) meets the effective criteria.

(7) Data collection and analysis

All raw data in the facility are collected manually or using a data acquisition system according to the experimental protocol and relevant regulations of the research institution. The manually collected data can be transcribed into software such as Excel for analysis and reporting. The data of each group are quantitative data. The experimental data of each group of animals in the table are expressed as mean ± standard deviation (Mean ± SD), and the experimental data in the picture are described as mean ± standard error (Mean ± SEM.). t-test analysis and survival analysis were performed using EXCEL and graphpad prism software.

Experimental process: 30 female NOD/SCID mice were randomly divided into blank control group, compound 47 (0.3 mg/kg) dose group, compound 64 (0.3 mg/kg) dose group, reference compound AZD5305 0.3 mg/kg and reference compound Olaparib 100 mg/kg dose group, a total of 5 groups, 6 mice in each group, all were orally administered once a day. At the same time, the body weight was weighed every 2 days and the length and width of the tumor were measured with a vernier caliper. After 30 days of administration, the tumor-bearing mice were anesthetized and killed, the tumor tissue was peeled off, weighed and photographed, and the tumor inhibition rate was calculated.

Conclusion: The experimental results are shown in Figures 1 and 2. After cell inoculation, each drug-treated group was continuously administered for 30 days. Compared with the blank control group, each drug-treated group (AZD5305 0.3 mg/kg, compound 47 0.3 mg/kg, compound 64 0.3 mg/kg, Olaparib 100 mg/kg) had a significant inhibitory effect on the growth of human breast cancer cell MM436 cell line NOD/SCID mouse subcutaneous tumor model. The anti-tumor effects of the compound 47 0.3 mg/kg dose group and the compound 64 0.3 mg/kg dose group were significantly better than the reference compound Olaparib 100 mg/kg dose group, and were equivalent to the AZD5305 0.3 mg/kg dose group; in addition, after 30 days of administration, the tumors of one mouse were completely regressed in the compound 64 0.3 mg/kg and AZD5305 0.3 mg/kg dose groups, respectively, showing excellent anti-tumor efficacy.

7. Hematological toxicity assay

CD34+ hematopoietic stem cells (hmPB34-P-SC) were cultured overnight in RPMI 1640 complete medium supplemented with 25ng/mL IL-3, 25ng/mL IL-6, 25ng/ml SCF and 10% _fetal bovine serum at 37°C and 5% CO2. The next day, the cells were resuspended and seeded in 96-well plates at a concentration of 1000 cells per well, and different concentrations of drugs were added at the same time, with 3 replicates. After 5 days of culture, the number of live cells in each well was determined using the CellTiter-Glo 2.0 kit and detected by a microplate reader.

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

Conclusion: The compounds of the present invention have weak inhibitory effects on CD34+ hematopoietic stem progenitor cells hmPB34-P-SC, especially compounds 76, 83, 89 and 95, whose IC ₅₀ values for hmPB34-P-SC inhibitory activity are greater than 1000nM, which are significantly better than the positive drug Olaparib.

8. Evaluation of the distribution of compounds in cerebrospinal fluid and plasma

Experimental steps: Weigh the compound and add a small amount of DMSO, then add sodium chloride solution for injection to make a 5mg·mL ^-1 compound solution for administration. Male rats were intravenously administered at 5mg·kg ^-1. Cerebrospinal fluid and whole blood were collected at 0.25h and 2h after administration (n=1). Whole blood was centrifuged at 3500rpm for 15min, and the supernatant plasma was collected. 10μL plasma and 10μL cerebrospinal fluid were placed in a centrifuge tube, and 40μL acetonitrile containing 20ng·ml ^-1 internal standard SAHA was added for precipitation, vortexed for 30s, centrifuged at 13000rpm for 15min, and the supernatant was placed in a sample bottle for testing. Standard curve range: 1~1000ng·ml ^-1 .

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

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

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

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

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

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

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

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

Claims (29)

The compound of formula I or a pharmaceutically acceptable form thereof, characterized in that: the structure of formula I is as follows:
in, X is selected from N or C, and when X is selected from N, represents a single bond, when X is selected from C, represents a double bond; _X1 is selected from N or C( _R8a ), _X2 is selected from N or C( _R8b ), _X3 is selected from N or C( _R8c ), and at most one of _X1 , _X2 and _X3 is selected from N; _R1 is selected from _C1-8 alkylthio, _C1-8 halogenated alkylthio, _C1-8 deuterated alkylthio, _C1-8 halogenated alkoxy, _C1-8 halogenated alkyl or _C2-8 alkenyl; _R2 is selected from hydrogen, fluorine, chlorine, _C1-4 alkyl, _C1-4 fluoroalkyl or _C1-4 deuterated alkyl; R ₃ and R ₄ are independently selected from hydrogen, deuterium, fluorine, C _1-4 alkyl, C _1-4 fluoroalkyl or C _1-4 deuterated alkyl; Alternatively, R ₃ , R _{4 ,} together with the atoms to which they are attached, form a 3-6 membered alkyl ring; R ₅ is selected from hydrogen, fluorine, chlorine, cyano, C _1-4 alkyl, C _1-4 fluoroalkyl or C _1-4 deuterated alkyl; Alternatively, R ₄ , R ₅ and the atoms to which they are connected together form a 5-7 membered alkyl heterocycle; when R ₄ , R ₅ and the atoms to which they are connected are connected to form a ring, the 5-7 membered alkyl heterocycle contains 1 O heteroatom in addition to X on the main ring; R ₃ , R ₄ and the atoms to which they are attached, and R ₄ , R ₅ and the atoms to which they are attached, will not form a ring at the same time; R ₆ is selected from -C(O)-NH-R _6a ; R _6a is selected from C _1-4 alkyl, C _1-4 fluoroalkyl, C _1-4 deuterated alkyl or 3-6 membered cycloalkyl; _R7 is selected from hydrogen, fluorine, chlorine, cyano, _C1-4 alkyl, _C1-4 fluoroalkyl or _C1-4 deuterated alkyl; R _8a , R _8b , and R _8c are independently selected from hydrogen, fluorine, chlorine, C _1-4 alkyl, C _1-4 deuterated alkyl, and C _1-4 fluoroalkyl; The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled substances, metabolites or prodrugs. The compound according to claim 1, characterized in that: _X1 is selected from N, _X2 is selected from C( _R8b ), and _X3 is selected from C( _R8c ); or _X1 is selected from C( _R8a ), _X2 is selected from N, _X3 is selected from C( _R8c ); or _X1 is selected from C( _R8a ), _X2 is selected from C( _R8b ), and _X3 is selected from C( _R8c ). The compound according to claim 1 or 2, characterized in that: R ₁ is selected from C _1-4 alkylthio, C _1-4 haloalkylthio, C _1-4 deuterated alkylthio, C _1-4 haloalkoxy, C _1-4 haloalkyl, C _2-4 alkenyl; in R ₁ , the halogen is selected from F, Cl, Br; Preferably, R ₁ is selected from methylthio, halogenated methylthio, deuterated methylthio, halogenated methoxy, halogenated methyl, halogenated ethyl or vinyl; in R ₁ , the halogenated halogen is selected from F and Cl; More preferably, R ₁ is selected from -S-CH ₃ , -S-CF ₂ H, -S-CF ₃ , -S-CF ₂ Cl, -S-CD ₃ , -O-CF ₂ H, -O-CF ₃ , -O-CF ₂ Cl, -CF ₂ -CH ₃ or -CH=CH ₂ . The compound according to any one of claims 1 to 3, characterized in that at least one of the following is satisfied: _R2 is selected from hydrogen, fluorine, chlorine, methyl, fluoromethyl or deuterated methyl; R ₃ and R ₄ are independently selected from hydrogen, deuterium, fluorine, methyl, fluoromethyl or deuterated methyl; R ₅ is selected from hydrogen, fluorine, chlorine, cyano, methyl, fluoromethyl or deuterated methyl; R ₇ is selected from hydrogen, fluorine, chlorine, cyano, methyl, fluoromethyl or deuterated methyl; R _8a , R _8a , R _8c are independently selected from hydrogen, fluorine, chlorine, methyl, fluoromethyl or deuterated methyl. The compound according to any one of claims 1 to 4, characterized in that: R _6a is selected from methyl, fluoromethyl, deuterated methyl or cyclopropyl; Preferably, R _6a is selected from methyl, -CF ₃ , -CD ₃ or cyclopropyl. The compound according to any one of claims 1 to 5, characterized in that: Selected from:

The compound according to any one of claims 1 to 5, characterized in that: when R ₄ and R ₅ do not form a ring with the atoms to which they are connected, the structural unit Selected from: The compound according to any one of claims 1 to 7, characterized in that when R ₄ and R ₅ do not form a ring with the atoms to which they are connected, the structural unit Selected from: The compound according to any one of claims 1 to 8, characterized in that: when R ₄ , R ₅ and When the atoms connected form a ring, the structural unit Selected from: Preferably, when R ₄ , R ₅ and the atoms to which they are connected form a ring, the structural unit Selected from:
The compound according to any one of claims 1 to 8, characterized in that: the structural unit in formula I Replace with The compound according to any one of claims 1 to 10, characterized in that the compound is selected from:




The compound according to claim 1 or 2, characterized in that: R ₁ is selected from -S-CH ₃ , -S-CF ₂ H, -S-CF ₃ , -S-CF ₂ Cl, -S-CD ₃ , -O-CF ₂ H, -O-CF ₃ , -O-CF ₂ Cl, -CF ₂ -CH ₃ , -CH=CH ₂ , -S-CFH ₂ or -O-CFH ₂ . The compound according to any one of claims 1 to 2, 4 to 5 or 12, characterized in that: the structural unit Selected from:

The compound according to any one of claims 1 to 2, 4 to 5, 7 to 8 or 12 to 13, characterized in that: the structural unit in formula I Replace with The compound according to any one of claims 1 to 2, 4 to 5, 7 to 10 or 12 to 14, characterized in that In that: the compound is selected from:
The compound according to any one of claims 1 to 2, 4 to 5, 7 or 12 to 13, characterized in that when R ₄ and R ₅ do not form a ring with the atoms to which they are connected, the structural unit Selected from: The compound according to any one of claims 1 to 2, 4 to 5, 7 or 12 to 13 or 16, characterized in that when R ₄ and R ₅ form a ring with the atoms to which they are connected, the structural unit Selected from: Preferably, when R ₄ , R ₅ and the atoms to which they are connected form a ring, the structural unit Selected from:
The compound according to any one of claims 1 to 2, 4 to 5, 7 or 12 to 13 or 16, characterized in that: the structural unit in formula I Replace with The compound according to any one of claims 1 to 2, 4 to 5, 7, 10, 12 to 14 or 18, characterized in that: the structural unit in formula I Replace with The compound according to any one of claims 1 to 2, 4 to 5 or 12 to 13, characterized in that: the structural unit in formula I Replace with _R9 is selected from H. The compound according to any one of claims 1 to 2, 4 to 5, 7, 10, 12 to 14 or 16 to 20, characterized in that the compound is selected from:

The pharmaceutical composition is characterized in that: it is a pharmaceutical composition according to any one of claims 11, 15 or 21 The compound or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug is an active ingredient, assisted by a pharmaceutically acceptable carrier. The compound according to any one of claims 11, 15 or 21, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug thereof, and the pharmaceutical composition according to claim 22, are used in the preparation of a medicament for preventing and/or treating PARP1 enzyme-related diseases. The use according to claim 23 is characterized in that the PARP1 enzyme-related disease is a tumor-related disease. The use according to claim 24 is characterized in that the tumor-like disease lacks the HR-dependent DNA DSB repair pathway. The use according to claim 24 or 25 is characterized in that the tumor-like disease comprises one or more cancer cells, and the cancer cells have reduced or absent ability to repair DNA DSB through HR compared to normal cells. The use according to claim 26, characterized in that the cancer cells have a BRCA1 or BRCA2 defective phenotype. The use according to any one of claims 24 to 27 is characterized in that the tumor-related disease is breast cancer, ovarian cancer, primary peritoneal cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer, glioblastoma, lung cancer, meningioma, pituitary tumor, craniopharyngioma, schwannoma, glioma, ependymoma, primitive neuroectodermal tumor, central nervous system lymphoma, germ cell tumor, metastasis, brain cancer or central nervous system cancer. Use of the compound according to any one of claims 11, 15 or 21 or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug, and the pharmaceutical composition according to claim 22 in the preparation of a PARP1 inhibitor.