WO2024083211A1

WO2024083211A1 - Heterocyclic deuterated compound and use thereof

Info

Publication number: WO2024083211A1
Application number: PCT/CN2023/125568
Authority: WO
Inventors: 贾涛; 王太津; 白忠友; 李钢
Original assignee: 成都赜灵生物医药科技有限公司
Priority date: 2022-10-20
Filing date: 2023-10-20
Publication date: 2024-04-25

Abstract

Disclosed is a heterocyclic deuterated compound and a use thereof, relating to the technical field of chemical medicine. The heterocyclic deuterated compound represented by formula (I) provided by the present invention can be used as a PARP1 inhibitor, has the advantages of high activity and selectivity, and further has excellent pharmacokinetic properties and excellent safety.

Description

Heterocyclic deuterated compounds and uses thereof

Technical Field

The invention belongs to the field of chemical medicine and relates to a class of heterocyclic deuterated compounds and uses thereof.

Background technique

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 alone 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 inhibitors. The novel PARP1 inhibitors of the present invention have unexpectedly higher selectivity for PARP1 than other PARP family members (such as PARP2, PARP3, PARP5a and PARP6), and can be used to treat diseases related to PARP function.

Summary of the invention

The purpose of the present invention is to provide a class of heterocyclic deuterated compounds and uses thereof, so as to achieve highly selective and efficient prevention or treatment of diseases related to PARP function.

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

in:

_R1 is selected from halogen, _C1-4 alkyl, _C1-4 fluoroalkyl, _C1-4 alkoxy, _C1-4 fluoroalkoxy, 3-6 membered cycloalkyl or 3-6 membered fluorocycloalkyl;

_X1 is selected from N or _CR5a , _X2 is selected from N or _CR5b , and _X3 is selected from N or _CR5c ;

_R6 is selected from hydrogen or halogen;

R ₄ is -CONHR ₇ , R ₇ is selected from C _1-4 alkyl, C _1-4 fluoroalkyl, C _1-4 deuterated alkyl, 3-6 membered cycloalkyl or 3-6 membered fluorocycloalkyl;

R _9a is selected from hydrogen, halogen, C _1-4 alkyl, C _1-4 fluoroalkyl, C _1-4 alkoxy, C _1-4 fluoroalkoxy, 3-6 membered cycloalkyl, 3-6 membered fluorocycloalkyl or cyano;

R _9c is selected from hydrogen or halogen;

_R2 is selected from hydrogen or _C1-4 alkyl;

R _5a is selected from hydrogen, halogen or C _1-4 alkyl, R _5b is selected from hydrogen or halogen, and R _5c is selected from hydrogen or halogen;

Ring A is selected from (Ring A right end is connected to the main ring pyridine)

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

In some preferred embodiments of the present invention, in the compound represented by the above formula I or a pharmaceutically acceptable form thereof, R ₁ is selected from chloro, methyl, ethyl, fluoromethyl, fluoroethyl, cyclopropyl or fluorocyclopropyl.

In some preferred embodiments of the present invention, in the compound represented by the above formula I or a pharmaceutically acceptable form thereof, R ₆ is selected from hydrogen or fluorine.

In some preferred embodiments of the present invention, in the compound represented by the above formula I or a pharmaceutically acceptable form thereof, _R7 is selected from methyl, ethyl, deuterated methyl, deuterated ethyl, fluoromethyl, fluoroethyl, cyclopropyl or fluorocyclopropyl.

In some preferred embodiments of the present invention, in the compound represented by the above formula I or a pharmaceutically acceptable form thereof, R _9a is selected from hydrogen, fluorine, chlorine, methyl, ethyl, fluoromethyl, fluoroethyl, cyclopropyl or fluorocyclopropyl.

In some preferred embodiments of the present invention, in the compound represented by the above formula I or a pharmaceutically acceptable form thereof, R _9c is selected from hydrogen or fluorine.

In some preferred embodiments of the present invention, in the compound represented by the above formula I or a pharmaceutically acceptable form thereof, R ₂ is selected from hydrogen or methyl.

In some preferred embodiments of the present invention, in the compound represented by the above formula I or a pharmaceutically acceptable form thereof, R _5a is selected from hydrogen, fluorine or methyl, R _5b is selected from hydrogen or fluorine, and R _5c is selected from hydrogen or fluorine.

In some preferred embodiments of the present invention, in the compound represented by the above formula I or a pharmaceutically acceptable form thereof, the structural unit Selected from the following structures:

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

In a second aspect, the present invention provides a pharmaceutical composition, which comprises the aforementioned compound of formula I 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.

A further object of the present invention is to provide an article of manufacture, for example, in the form of a kit. Articles of manufacture as used herein are intended to include, but are not limited to, kits and packages. The article of manufacture of the present invention comprises: (a) a first container; (b) a pharmaceutical composition located in the first container, wherein the composition comprises: a first therapeutic agent, the first therapeutic agent comprising: any compound of Formula I or a pharmaceutically acceptable form thereof, or a mixture thereof; (c) an optional package insert indicating that the pharmaceutical composition can be used to treat a neoplastic condition (as defined below); and (d) a second container.

The first container is a container for holding a pharmaceutical composition. This container can be used for preparation, storage, transportation and/or individual/batch sales. The first container is intended to cover bottles, jars, vials, flasks, syringes, tubes (e.g., for cream products), or any other container for preparing, holding, storing or dispensing pharmaceutical products.

The second container is a container for containing the first container and optional package insert. Examples include, but are not limited to, boxes (e.g., paper or plastic boxes), boxes, cartons, bags (e.g., paper or plastic bags), pouches, and sacks. The package instructions may be physically adhered to the exterior of the first container via a cable tie, glue, staples, or other means of adhesion, or they may be placed inside the second container without any physical means of adhesion to the first container. Alternatively, the package instructions are located outside the second container. When located outside the second container, it is preferred that the package instructions are physically adhered via a cable tie, glue, staples, or other means of adhesion. Alternatively, they may abut or contact the exterior of the second container without physical adhesion.

The package insert is a trademark, label, label, or the like, which lists information related to the pharmaceutical composition located in the first container. The listed information is usually determined by the regulatory agency (e.g., the U.S. Food and Drug Administration) that governs the area where the product is to be sold. Preferably, the package insert specifically lists the indications for which the pharmaceutical composition is approved. The package insert can be made of any material from which the information contained therein or thereon can be read. Preferably, the package insert is a printable material (e.g., paper, plastic, cardboard, foil, adhesive paper or plastic, etc.) on which the desired information can be formed (e.g., printed or applied).

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

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-related disorder, including but not limited to solid and hematological malignancies. In further embodiments, the tumor-related disorder is but not limited to breast cancer, colorectal cancer, Cancer of the bowel, colon, lung (including small cell lung cancer, non-small cell lung cancer and bronchioloalveolar carcinoma) and prostate, as well as cancer of the bile duct, bone, bladder, head and neck, kidney, liver, gastrointestinal tissue, oesophagus, ovary, pancreas, skin, testis, thyroid, uterus, cervix and vulva, and leukaemia (including chronic lymphocytic leukaemia (CLL), acute lymphocytic leukaemia (ALL) and chronic myeloid leukaemia (CML)), multiple myeloma or lymphoma.

In some preferred embodiments, the tumor-like disorder is breast cancer, ovarian cancer, primary peritoneal cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer, glioblastoma or lung cancer.

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.

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.

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. Therefore, 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 _1-4 " should be understood to cover any sub-ranges and each point value therein, such as C _2-4 , C _3-4 , C _1-2 , C _1-3 , C _{1-4 ,} etc., as well as C ₁ , C ₂ , C ₃ , C ₄ , 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, "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.

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 the present invention, "polymorph" refers to different solid crystalline phases produced by the presence of two or more different molecular arrangements in the solid state of certain compounds of the present invention. Some compounds of the present invention may exist in more than one crystal form, and the present invention is intended to include various crystal forms and mixtures thereof. Generally, crystallization will produce solvates of the compounds of the present invention. The term "solvate" used in the present invention refers to an aggregate comprising one or more molecules of the compound of the present invention and one or more solvent molecules. The solvent may be water, in which case the solvate is a hydrate. Alternatively, the solvent may be an organic solvent. Therefore, the compounds of the present invention may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and corresponding solvated forms. The compounds of the present invention may form true solvates, but in some cases, may also only retain adventitious water or water. The compounds of the present invention may be reacted in a solvent or precipitated or crystallized from a solvent. Solvates of the compounds of the present invention are also included within the scope of the present invention. The present invention also encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

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

In the present invention, pharmaceutically acceptable salts include acid addition salts and base addition salts thereof. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts. For a review of suitable salts, see, for example, "Remington's Pharmaceutical Sciences", Mack Publishing Company, Easton, Pa., (2005); and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use", Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art. "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that can retain the biological effectiveness of free bases without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, caprylate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalene disulfonate, etc. These salts can be prepared by methods known in this patent. "Pharmaceutically acceptable base addition salt" refers to salts formed with inorganic or organic bases that can maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following salts: primary, secondary, and tertiary amines, substituted amines, including natural Substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline hexyl caffeine. These salts can be prepared by methods known to this patent.

In the present invention, unless otherwise indicated, "ester" refers to esters derived from the compounds described herein, including physiologically hydrolyzable esters (which can be hydrolyzed under physiological conditions to release the compounds of the present invention in free acid or alcohol form). The compounds of the present invention themselves may also be esters.

The compounds of the present invention may exist in the form of solvates (preferably hydrates), wherein the compounds of the present invention contain polar solvents as structural elements of the crystal lattice of the compounds, in particular water, methanol or ethanol. The amount of polar solvents, in particular water, may exist in a stoichiometric or non-stoichiometric ratio.

Those skilled in the art will appreciate that, since nitrogen requires available lone pairs of electrons to be oxidized to oxides, not all nitrogen-containing heterocycles are capable of forming nitrogen oxides. Those skilled in the art will recognize nitrogen-containing heterocycles that are capable of forming nitrogen oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming nitrogen oxides. The synthetic methods for preparing nitrogen oxides of heterocycles and tertiary amines are well known to those skilled in the art, including oxidation of heterocycles and tertiary amines with peroxyacids such as peracetic acid and metachloroperbenzoic acid (mCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate and dioxirane such as dimethyl dioxirane. These methods for preparing nitrogen oxides have been widely described and reviewed in the literature, see, for example: T.L.Gilchrist, Comprehensive Organic Synthesis, vol.7, pp748-750 (A.R.Katritzky and A.J.Boulton, Eds., Academic Press); and G.W.H.Cheeseman and E.S.G.Werstiuk, Advances in Heterocyclic Chemistry, vol.22, pp390-392 (A.R.Katritzky and A.J.Boulton, Eds., Academic Press).

In the present invention, "metabolite" refers to a substance formed in vivo when a compound of the present invention is administered. The metabolites of a compound can be identified by techniques known in the art, and their activity can be characterized by experimental methods. Such products can be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compound. Therefore, the present invention includes metabolites of the compounds of the present invention, including compounds prepared by contacting the compounds of the present invention with a mammal for a period of time sufficient to produce their metabolites.

In the present invention, "prodrug" refers to certain derivatives of the compounds of the present invention that can be converted into compounds of the present invention having the desired activity by, for example, hydrolytic cleavage when administered to the body or thereon. Typically, such prodrugs will be functional group derivatives of the compounds that are easily converted into the desired therapeutically active compounds in vivo. Additional information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella). Prodrugs of the present invention can be prepared, for example, by replacing appropriate functional groups present in the compounds of the present invention with certain moieties known to those skilled in the art as "pro-moieties" (e.g., as described in "Design of Prodrugs", H. Bundgaard (Elsevier, 1985)).

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", "administration of other treatments", "administration of other therapeutic agents" and the like refer to drug treatments obtained by mixing or combining more than one active ingredient, including fixed and non-fixed combinations of the active ingredients. The term "fixed combination" refers to the simultaneous administration of at least one compound described herein and at least one synergistic agent to a patient 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 at variable intervals of at least one compound described herein and at least one synergistic agent to a patient in the form of separate entities. 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 and other diseases.

As used herein, 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.

On the basis of not 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.

Detailed ways

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 structure of the compound of the present invention is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR is determined by a Bruker AVANCE-400 nuclear magnetic spectrometer, and the determination solvent is deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated Deuterated chloroform (CDCl ₃ ) and deuterated methanol (CD ₃ OD) were used as the internal standard. Tetramethylsilane (TMS) is used as the internal standard. 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 determinations were performed using an Agilent 1200DAD high pressure liquid chromatograph (Sunfirc C18, 150X 4.6mm, 5wn, chromatographic column) and a 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 preparation

Intermediate INT1: 7-(Chloromethyl)-3-ethyl-1,5-naphthyridin-2(1H)-one

Step 1: Add compound INT1a (20 g, 95.1 mmol) and selenium dioxide (16 g, 144 mmol) to a 250 ml reaction bottle, add 120 ml 1,4-dioxane, heat to 110 ° C and stir to react overnight. After TLC monitoring, the reaction solution was filtered, the filter residue was rinsed with ethyl acetate, and the filtrate was combined and concentrated by rotary evaporation. The crude product was purified by column chromatography to obtain compound INT1b (16 g, yellow solid). LC-MS: ESI [M + H] ⁺ = 225.2.

Step 2: Add sodium hydride (6.86g, 171.4mmol) and 60ml 1,4-dioxane to a 250ml reaction bottle, and then replace nitrogen three times. Cool to 0℃, slowly drop the compound 2-phosphonobutyric acid triethyl ester (43.2g, 171.4mmol) under nitrogen protection, stir and react at 0℃ for 10 minutes, warm to room temperature and stir and react for 10 minutes, then warm to 40℃ and stir and react for 5 minutes, and cool the reaction to -78℃. Slowly drop the compound INT1b (16g, 71.4mmol) dissolved in 60ml 1,4-dioxane solution, keep stirring and react at -78℃ for 1 test. After the reaction is complete as monitored by TLC, slowly add the reaction solution into an ice-cold saturated aqueous ammonium chloride solution to quench, extract three times with 150ml ethyl acetate, and combine. The organic phase was dried over anhydrous sodium sulfate, filtered and spin-dried. The crude product was purified by column chromatography to obtain compound INT1c (13.26 g, brown liquid). LC-MS: ESI [M+H] ⁺ = 323.3.

Step 3: Add compound INT1c (13.26 g, 41.1 mmol) to 100 ml of anhydrous ethanol, then add Pd/C (1.33 g, 10%), and stir at room temperature to react overnight. After the reaction is complete as monitored by LC-MS, filter the reaction solution, rinse the filter residue with a large amount of ethanol, combine the filtrates and concentrate by rotary evaporation. Add 4 mol/L hydrochloric acid in 1,4-dioxane solution (50 ml), stir at room temperature for 30 minutes, add ether to precipitate a large amount of solid, filter and dry to obtain compound INT1d (7.32 g, white solid). LC-MS: ESI [M+H] ⁺ ＝249.3; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.44 (s, 1H), 8.62 (d, J＝1.9 Hz, 1H), 7.75 (d, J＝1.9 Hz, 1H), 4.34 (q, J＝7.1 Hz, 2H), 3.87 (s, 0H), 3.24 (dd, J＝16.8, 6.3 Hz, 1H), 2.97 (dd, J＝16.8, 10.1 Hz, 1H), 1.81–1.65 (m, 1H), 1.52–1.36 (m, 1H), 1.32 (t, J＝7.1 Hz, 3H), 0.93 (t, J＝7.4 Hz, 3H).

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

Step 5: Add compound INT1e (2.0 g, 8.1 mmol) and 60 ml of tetrahydrofuran to a 150 ml reaction bottle, cool to 0°C, then add 2.5 mol/L lithium aluminum hydride tetrahydrofuran solution (6.48 ml, 16.2 mmol), and react at 0°C for 2 hours. After TLC monitoring, add 5 ml of water to quench the reaction, add a large amount of anhydrous sodium sulfate to dry, filter, rinse the filter residue with a large amount of dichloromethane, combine the filtrates, and concentrate by rotary evaporation. After drying, compound INT1f (1.2 g, white solid) is obtained. LC-MS: ESI [M+H] ⁺ ＝205.3; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.87 (s, 1H), 8.03 (d, J＝2.0 Hz, 1H), 7.36 (d, J＝1.0 Hz, 1H), 7.34 (dd, J＝2.0, 0.9 Hz, 1H), 4.51 (s, 2H), 2.52 (d, J＝1.8 Hz, 1H), 1.15 (t, J＝7.4 Hz, 3H).

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

Intermediate INT2: 7-(Chloromethyl)-3-cyclopropyl-1,5-naphthyridin-2(1H)-one

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

Step 2: INT2b (10 g, 37.8 mmol) was dissolved in 100 mL THF. Solid NaH (60%, 2.3 g, 56.8 mmol) was slowly added at 0°C under nitrogen protection. The mixture was stirred for 0.5 h after addition and then stirred at room temperature for 10 min. Finally, a THF solution of INT1b (10.2 g, 45.4 mmol) was slowly added at -78°C. The mixture was stirred at -78°C for 1 h after addition. After LC-MS confirmed the completion of the reaction, a saturated aqueous solution of NH ₄ Cl was added to quench the reaction. The mixture was extracted with EA (150 mL×3). The organic phases were combined and dried over anhydrous Na ₂ SO _4. The mixture was filtered and dried, and purified by column chromatography to obtain a yellow oil INT2c (10 g, 79%). LC-MS: ESI[M+H] ⁺ ＝335.1.

Step 3: Weigh compound INT2c (10 g, 29.9 mmol) into a reaction bottle, add 150 mL of glacial acetic acid to dissolve, then slowly add Fe (5.0 g, 89.7 mmol) powder, raise the temperature to 70°C and stir for 2 h, cool to room temperature and filter, wash the filter cake with a small amount of DCM and MeOH, concentrate the filtrate in vacuo, and then purify by column chromatography to obtain a light yellow solid INT2d (1.85 g, 24%), LC-MS: ESI [M+H] ⁺ ＝259.1.

Step 4: Weigh compound INT2d (1.85 g, 7.2 mmol) into a reaction bottle, add 20 mL THF and stir at -20°C, slowly add DIBAL-H (1.5 M in toluene, 16 mL, 25.1 mmol) dropwise under nitrogen protection, return to room temperature and stir for 0.5 h after completion of the addition, add saturated sodium potassium tartrate aqueous solution dropwise at 0°C to quench the reaction, stir at room temperature overnight, extract three times with a mixed solution of DCM and MeOH (3:1), combine the organic phases and dry with anhydrous Na ₂ SO ₄ , filter and spin-dry to obtain a pale yellow solid crude product INT2e (1.46 g, 94%), LC-MS: ESI [M+H] ⁺ ＝217.1.

Step 5: Weigh compound INT2e (1.46 g, 6.8 mmol) and DMF (98.7 mg, 1.4 mmol) into a reaction bottle, add 50 mL of toluene to dissolve, slowly drop SOCl ₂ (1.1 g, 9.5 mmol) at 0°C, slowly warm to room temperature after the addition is complete, and stir overnight to react. After the reaction is complete, vacuum concentrate the solvent, and purify with DCM and MeOH (0-7%) through a column to obtain a light yellow solid compound INT2 (1.24 g, 78%), LC-MS: ESI [M+H] ⁺ = 235.1.

Example 1: 1'-(7-ethyl-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: Add compound 1a (1g, 4.6mmol), 1b (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 spin-dried. The crude product is purified by column chromatography to obtain compound 1c (1g, white solid).

Step 2: Add 1c (1 g, 3 mmol), methylamine aqueous solution (5 g, 161.3 mmol) and anhydrous methanol (20 ml) into 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 1d (0.8 g, white solid).

Step 3: Add compound 1d (0.5 g, 1.5 mmol) to 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, and after the reaction is completed as monitored by TLC, concentrate the reaction solution under reduced pressure to dryness to obtain compound 1e (0.5 g, white solid).

Step 4: Compound 1e (0.05 g, 0.23 mmol), INT1 (0.06 g, 0.28 mmol), N,N-diisopropylethylamine (0.15 g, 1.15 mmol) and potassium iodide (0.19 g, 1.15 mmol) were added to 10 ml of anhydrous acetonitrile and stirred at 80° C. for 2 h. After the reaction was completed as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 1 (0.02 g, white solid); LC-MS: ESI[M+H] ⁺ =404.5; ¹ H NMR (400 MHz, DMSO): 11.85 (s, 1H), 8.71 (d, J = 5.0 Hz, 1H), 8.69 (s, 1H), 8.41 (d, J = 1.3 Hz, 1H), 8.06–7.91 (m, 2H), 7.75 (s, 1H), 7.64 (s, 1H), 6.42 (s, 1H), 3.72 (s, 2H), 3.16 (s, 2H), 2.81 (d, J = 4.8 Hz, 3H), 2.70 (s, 2H), 2.54 (d, J = 7.4 Hz, 4H), 1.18 (t, J = 7.4 Hz, 3H).

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

Step 1: Add compound 2a (6.0 g, 49.2 mmol) to 50 ml of tetrahydrofuran, cool to -78°C, control the temperature at -60 to -78°C, add LDA (2M, 29.5 ml, 59.0 mmol) dropwise, stir for 15 min after addition, add 40 ml of tetrahydrofuran solution of 2b (19.3 g, 54.1 mmol) dropwise, return to room temperature naturally after addition, stir for 2 h and take a sample. After TLC detection, add 60 ml of saturated aqueous ammonium chloride solution and stir, separate the layers, add 100 ml of ethyl acetate to the aqueous layer for extraction, separate the layers, wash the combined organic layers with 100 ml of saturated brine, separate the layers, and concentrate the organic phase under reduced pressure to remove the solvent to obtain compound 2c (20.0 g, oil).

Step 2: 2c (19.3 g, 28.7 mmol), potassium carbonate (7.93 g, 57.4 mmol), and 2d (8.32 g, 31.6 mmol) (100 ml) were added to 1,4-dioxane/water = 10:1 solution 100 ml, and the atmosphere was replaced with nitrogen three times. The temperature was raised to 80 ° C. and stirred for 2 h. After the reaction was completed by LC-MS detection, the temperature was lowered to room temperature, 100 ml of water and 100 ml of ethyl acetate were added, and the layers were separated. The aqueous layer was extracted three times with 100 ml of ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and spin-dried. The crude product was purified by column chromatography to obtain compound 2e (4.2 g, yellow solid).

Step 3: Add 2e (4.2 g, 13.0 mmol) and 40% methylamine aqueous solution (5.1 g, 65.2 mmol) to 21 ml of anhydrous methanol, stir at room temperature for 1 h, and after LC-MS detection, the reaction solution is concentrated to dryness, 40 ml of 1,4-dioxane is added, and 4M/hydrochloric acid dioxane solution (16.3 ml, 65.2 mmol) is added dropwise, and stirred at room temperature for 2 h. After LC-MS detection, the reaction is complete, and the mixture is filtered and dried to obtain compound 2f (3.4 g, off-white solid).

Step 4: 2f (3.4 g, 11.5 mmol), DIEA (5.6 g, 46.0 mmol), INT1 (2.4 g, 10.5 mmol), KI (340 mg, 2.1 mmol) were added to 48 ml of acetonitrile, and the mixture was heated to 80°C and stirred for 2 h. After the reaction was complete by LC-MS, 200 ml of saturated sodium bicarbonate solution was added, the mixture was stirred and filtered, and the mixture was purified by spin column chromatography to obtain compound 2 (2.4 g, white solid); LC-MS: ESI [M+H] ⁺ = 408.2; ¹ H NMR (400 MHz, DMSO-d ₆ )δ11.90(s,1H),8.82-8.72(m,2H),8.48(d,J＝1.9Hz,1H),8.10-7.99(m,2H),7.82(q,J＝1.0Hz,1H),7.75-7.66(m,1H),6.48(d,J＝1.5Hz,1H),3.78(s,2H),2.88(d,J＝4.9Hz,3H),2.64-2.58(m,4H),1.25(t,J＝7.4Hz,3H).

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

Step 1: Compound 2c (20 g, 60 mmol), bipyralidone (15.2 g, 72 mmol), potassium acetate (11.8 g, 120 mmol), and PdCl ₂ (dppf) (2.1 g, 3 mmol) were added to a reaction flask in sequence, protected by N ₂ , and reacted at 80°C overnight. After the reaction was completed, the mixture was filtered, washed with EA, and the filtrate was concentrated. The residue was subjected to column chromatography to obtain compound 3a (15 g, oil).

Step 2: 3a (15 g, 22.3 mmol), potassium carbonate (7.93 g, 57.4 mmol), and 3b (8.32 g, 31.6 mmol) (100 ml) were added to 1,4-dioxane/water = 10:1 solution 100 ml, and the mixture was replaced with nitrogen three times. The temperature was raised to 80 ° C. and stirred for 2 h. After the reaction was completed by LC-MS detection, the temperature was lowered to room temperature, 100 ml of water and 100 ml of ethyl acetate were added, and the layers were separated. The aqueous layer was extracted three times with 100 ml of ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and spin-dried. The crude product was purified by column chromatography to obtain compound 3c (12 g, yellow solid).

Step 3: 3c (4.2 g, 13.0 mmol) and 40% methylamine aqueous solution (5.1 g, 65.2 mmol) were added to 21 ml of anhydrous methanol, and the mixture was stirred at room temperature for 1 h. After the reaction was completed as detected by LC-MS, the reaction solution was concentrated to dryness, 40 ml of 1,4-dioxane was added, and 4M/dioxane hydrochloride solution (16.3 ml, 65.2 mmol) was added dropwise. The mixture was stirred at room temperature for 2 h. After the reaction was completed as detected by LC-MS, the mixture was filtered and the filtrate was dried to obtain compound 3d (3.4 g, off-white solid).

Step 4: 3d (3.4 g, 11.5 mmol), DIEA (5.6 g, 46.0 mmol), INT1 (2.4 g, 10.5 mmol), KI (340 mg, 2.1 mmol) were added to 48 ml of acetonitrile, and the mixture was heated to 80°C and stirred for 2 h. After the reaction was complete as detected by LC-MS, 200 ml of saturated sodium bicarbonate solution was added, the mixture was stirred and filtered, and the mixture was purified by spin column chromatography to obtain compound 3 (2.4 g, white solid); LC-MS: ESI [M+H] ⁺ = 426.5; ¹ H NMR (400 MHz, DMSO) δ 11.84 (s, 1H), 8.63 (d, J = 4.8 Hz, 1H), 8.42 (t, J = 4.4 Hz, 1H), 8.16–8.03 (m, 1H), 7.92 (dd, J = 7.7, 1.5 Hz, 1H), 7.75 (s, 1H), 7.65 (s, 1H), 6.24 (s, 1H), 3.72 (s, 2H), 2.88 (t, J = 8.1 Hz, 1H), 2.80 (d, J = 4.8 Hz, 3H), 2.60–2.52 (m, 3H), 1.18 (t, J = 7.4 Hz, 3H).

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

Step 1: In 100 ml of 1,4-dioxane/water = 10:1 solution, add 3a (13 g, 21.2 mmol), potassium carbonate (7.8 g, 55 mmol), 4a (8.1 g, 28.6 mmol) 100 ml, replace with nitrogen three times, heat to 80 ° C and stir for 2 h. After the reaction is complete by LC-MS detection, cool to room temperature, add 100 ml of water and 100 ml of ethyl acetate, separate the layers, extract the aqueous layer with 100 ml of ethyl acetate three times, combine the organic layers, add anhydrous sodium sulfate, dry, filter, and spin dry. The crude product is purified by column chromatography to obtain compound 4b (10.6 g, yellow solid).

Step 2: Add 4b (3.8 g, 11.3 mmol) and 40% methylamine aqueous solution (4.5 g, 60.5mmol), stirred at room temperature for 1h. After the reaction was completed by LC-MS, the reaction solution was concentrated to dryness, 40ml of 1,4-dioxane was added, and 4M/hydrochloric acid dioxane solution (15ml, 64mmol) was added dropwise. The mixture was stirred at room temperature for 2h. After the reaction was completed by LC-MS, the mixture was filtered and the filtrate was dried to obtain compound 4c (3.2g, off-white solid).

Step 3: 4c (3.2 g, 11.2 mmol), DIEA (5.4 g, 45.2 mmol), INT1 (2.1 g, 10.5 mmol), KI (320 mg, 1.9 mmol) were added to 48 ml of acetonitrile, and the mixture was heated to 80°C and stirred for 2 h. After the reaction was complete as detected by LC-MS, 200 ml of saturated sodium bicarbonate solution was added, and the mixture was stirred and filtered. The mixture was purified by spin column chromatography to obtain compound 4 (1.8 g, white solid); LC-MS: ESI [M+H] ⁺ = 426.5; ¹ H NMR (400 MHz, DMSO) δ 11.84 (s, 1H), 8.63 (d, J = 4.8 Hz, 1H), 8.42 (t, J = 4.4 Hz, 1H), 8.16–8.03 (m, 1H), 7.92 (dd, J = 7.7, 1.5 Hz, 1H), 7.75 (s, 1H), 7.65 (s, 1H), 6.24 (s, 1H), 3.72 (s, 2H), 2.88 (t, J = 8.1 Hz, 1H), 2.80 (d, J = 4.8 Hz, 3H), 2.60–2.52 (m, 3H), 1.18 (t, J = 7.4 Hz, 3H).

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

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

Step 2: Weigh compound 5b (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 5c (612 g, yield 95.3%).

Step 3: Add compound 5c (500 g, 1.91 mol) to THF (2.56 L) and stir to dissolve. Add an aqueous solution (2.56 L) of sodium periodate (805 g, 3.72 mol) to the reaction system. Stir at room temperature for 2-4 hours. After the reaction is completed, add ethyl acetate (4.0 L) and water (4.0 L) to the reaction system, stir to separate the layers, and extract the aqueous layer with ethyl acetate (2.0 L) twice. After the organic layers are combined, saturated sodium thiosulfate solution and saturated brine are added in turn for washing. The organic layer is concentrated under reduced pressure at 40-45 ° C until there is no fraction, and 500 g of oily substance 5d is obtained, which is directly used in the next step.

Step 4: Compound 5d (512 g, 1.69 mol) and 5e (1457.0 g, 7.61 mol) were added to anhydrous ethanol (7.5 L), stirred to dissolve, and SnCl ₂ (1815.0 g, 9.57 mol) was added to the reaction system in batches at room temperature. After the addition, the temperature was raised to reflux and stirred for 1-2 h. The reaction system was cooled to 45-50 ° C, concentrated under reduced pressure until no fraction was left, ethyl acetate was added to the system and stirred to dissolve, and then saturated sodium bicarbonate was used to adjust the pH to 7-8. During the process, gas was released violently and a large amount of solid was precipitated. The reaction solution was centrifuged, and the filtrate was collected and allowed to stand for stratification. The organic layer was concentrated under reduced pressure at 40-45 ° C until no fraction was left, and purified by column chromatography to obtain flocculent solid 5f (230 g, yield 36.5%).

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

Step 6: Under nitrogen protection, compound 5g (800 mg, 3.00 mmol) and TMSI (1.80 g, 9.00 mmol) were added to acetonitrile (8 mL). The reaction solution was heated to 50 ° C and stirred for 2 hours. After the reaction was detected by LCMS, the reaction solution was cooled to room temperature. The resulting mixture was diluted with ethyl acetate (50 mL). The aqueous layer was washed with 3x50 mL of water (10% triethylamine). The combined organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 5h (740 mg, 97.64%) as a white solid. LC-MS: ESI [M + H] ⁺ = 252.9.

Step 7: Compound 5h (0.74 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 5i (0.42 g) was obtained as a yellow solid. LC-MS: ESI [M+H] ⁺ = 211.2.

Step 8: SOCl ₂ (357 mg, 3.0 mmol) was slowly added to a DMF (100 mL) solution of compound 5i (0.42 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 5j (250 mg). LC-MS: ESI [M+H] ⁺ = 229.0.

Step 9: Compound 5j (229 mg, 1.0 mmmol), 2f (331.5 mg, 1.5 mmol), N,N-diisopropyl Ethylamine (0.55 g, 5 mmol) and potassium iodide (332 mg, 2 mmol) were added to 10 ml of anhydrous acetonitrile and stirred at 85°C for 2 hours. After the reaction was completed as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 5 (142 mg, white solid); LC-MS: ESI [M+H] ⁺ = 414.2.

Example 6: 1'-((7-chloro-8-methyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-2',2',6',6'-d ₄ -6-carboxamide

Step 1: Compound 6a (50 g, 0.23 mol), isopropenylboronic acid pinacol ester (77.6 g, 0.46 mol), potassium carbonate (150.4 g, 0.46 mol), Pd(dppf)Cl ₂ (8.4 g, 0.012 mmol) were weighed into a reaction bottle, nitrogen was replaced 3 times, 550 mL dioxane/H ₂ O (10:1) was added, nitrogen was replaced 3 times, and then the reaction was carried out at 110°C for 5 h under nitrogen protection. After the reaction was completed, the reaction was cooled to room temperature, and then filtered through diatomaceous earth. The filter cake was washed with EA (100 mL×3), and the mother liquor was concentrated. Then EA (200 mL×5) and H ₂ O were added for extraction, the organic phases were combined and dried with anhydrous Na ₂ SO ₄ , filtered and dried, and purified by column chromatography to obtain a yellow oil 6b (44.5 g, 87%), LC-MS: ESI [M+H] ⁺ = 223.1.

Step 2: Weigh 6b (44.5 g, 0.20 mol) into a reaction bottle, add 400 mL of glacial acetic acid, slowly add iron powder (35.4 g, 0.60 mol), and react at 70°C for 4 h. After the reaction is completed, cool the reaction to room temperature, remove excess iron powder by suction filtration, spin dry the mother liquor, then add EA (150 mL × 4) and saturated NaHCO ₃ aqueous solution for extraction, combine the organic phases and dry them with anhydrous Na ₂ SO ₄ , filter and spin dry, and purify by column chromatography to obtain a light yellow solid 6c (32.3 g, 84%), LC-MS: ESI [M+H] ⁺ = 193.1.

Step 3: Weigh 6c (10 g, 52.0 mmol) and TEA (15.8 g, 156.1 mmol) into a reaction bottle, add 70 mL of toluene to dissolve, add triphosgene (7.7 g, 26.0 mmol) in toluene solution (30 mL) dropwise at 0°C, and stir at 60°C for 5 h. After the reaction is complete, add a small amount of methanol to quench the reaction, then vacuum concentrate to remove the solvent, slurry with ether to remove some impurities, and purify the solid residue by column chromatography to obtain a white solid 6d (3.2 g, 28%), LC-MS: ESI [M+H] ⁺ = 219.1.

Step 4: Weigh 6d (3.2 g, 14.7 mmol) and NCS (3.1 g, 23.5 mmol) into a reaction bottle, add 35 mL of glacial acetic acid to dissolve, add dichloroacetic acid (0.38 g, 2.9 mmol) under nitrogen protection, and stir and react at 90°C overnight. After the reaction is completed, vacuum concentrate to remove most of the solvent, wash with saturated NaHCO ₃ aqueous solution, extract with DCM three times, combine the organic phases and dry with anhydrous Na ₂ SO ₄ , filter and spin dry, and purify by column chromatography to obtain a light yellow solid 6e (3.1 g, 84%), LC-MS: ESI [M+H] ⁺ = 253.0.

Step 5: Weigh 6e (3.1 g, 12.3 mmol) into a reaction bottle, add 30 mL of THF and stir at -20°C, slowly add DIBAL-H (1.5 M in toluene, 65.4 mL) dropwise under nitrogen protection, return to room temperature and stir for 0.5 h after completion of the addition, add saturated sodium potassium tartrate aqueous solution dropwise at 0°C to quench the reaction, stir at room temperature overnight, extract 5 times with a mixed solution of DCM and MeOH (3:1), combine the organic phases and dry over anhydrous Na ₂ SO ₄ , filter and spin dry to obtain a light yellow solid 6f (810 mg, 29%), LC-MS: ESI [M+H] ⁺ ＝225.0.

Step 6: Weigh 6f (810 mg, 3.6 mmol) and DMF (26.4 mg, 0.36 mmol) into a reaction bottle, add DCM to dissolve, slowly add SOCl ₂ (2.1 g, 21.6 mmol) at 0°C, slowly warm to room temperature after the addition, and stir overnight to react. After the reaction, the solvent was concentrated in vacuo, and purified by column chromatography to obtain 6 g (172 mg, 20%) of a light yellow solid, LC-MS: ESI [M+H] ⁺ = 243.0.

Step 7: Compound 6g (40.0 mg, 0.16 mmol), 2f (53.3 mg, 0.18 mmol), DIEPA (106.3 mg, 0.82 mmol), and KI (2.7 mg, 0.016 mmol) were weighed into a reaction tube, and 5 mL of acetonitrile was added to react at 80°C for 3 h. The reaction was cooled to room temperature, the solvent was concentrated, and extracted with DCM (20 mL×3), the organic phases were combined and dried over anhydrous Na ₂ SO ₄ , filtered and dried, and filtered with MeOH (0-10%) and DCM to obtain a light yellow solid 6 (45 mg, yield 64%). LC-MS: ESI [M+H] ⁺ = 428.2; ¹ H NMR (400 MHz, DMSO) δ 12.28 (s, 1H), 8.71 (d, J = 5.2 Hz, 2H), 8.53 (d, J = 1.8 Hz, 1H), 8.02–7.95 (m, 2H), 7.71 (d, J = 1.8 Hz, 1H), 6.42 (s, 1H), 3.76 (s, 2H), 2.82 (d, J = 4.9 Hz, 3H), 2.65 (s, 3H), 2.54 (s, 2H).

Example 7: 1-(3-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)methyl)-N-methyl-1,2,3,6-tetrahydrobipyridine-2,2,6,6-d ₄ -6-carboxamide

Step 1: Add compound 7a (3.85 g, 24.6 mmol), DIEA (16.0 g, 123.3 mmol), DMAP (0.6 g, 4.81 mmol) to 100 ml of dichloromethane, cool to 0°C, add n-butyryl chloride (8.10 g, 76.3 mmol) dropwise, return to room temperature and react for 24 h. After the reaction is complete as detected by LC-MS, add 100 ml of ethyl acetate and 100 ml of water, separate the layers in a separatory funnel, extract the aqueous phase twice with ethyl acetate, combine the organic phases and spin dry, then add 40 ml of dichloromethane to precipitate the solid, filter, and spin dry the filter cake to obtain compound 7b (0.8 g, pink solid).

Step 2: Compound 7b (0.2 g, 0.96 mmol), potassium vinyl trifluoroborate (141.6 mg, 1.04 mmol), Pd(dppf)Cl ₂ (17.4 mg, 0.024 mmol) and potassium carbonate (199 mg, 1.44 mmol) were added to 4 ml of dioxane/water = 9:1, and the atmosphere was replaced with nitrogen three times. The temperature was raised to 90°C and stirred for 4 h. After the reaction was completed as monitored by TLC, the reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography to obtain compound 7c (0.1 g, yellow solid).

Step 3: Add compound 7c (0.1 g, 0.5 mmol) to 10 ml of dioxane and 2.5 ml of water, then add sodium periodate (425 g, 2.0 mmol) and potassium osmate (75 mg, 0.15 mmol), stir at room temperature for 2 h, and after LC-MS detection, add 50 ml of ethyl acetate and 50 ml of water, separate the layers in a separatory funnel, extract the aqueous phase twice with ethyl acetate, combine the organic phases, wash with saturated sodium sulfite, dry the organic layer over anhydrous sodium sulfate, filter, and spin-dry the filter cake to obtain compound 7d (80 mg, yellow solid).

Step 4: 7d (167 mg, 0.54 mmol) and triethylamine (198 mg, 1.96 mmol) were added to 10 ml of dichloromethane and stirred to dissolve; a DCM solution (10 ml) of 2f (100 mg, 0.49 mmol) was added to the reaction solution and stirred at room temperature for 1 h, sodium triacetoxyborohydride (623 mg, 2.94 mmol) was added and stirred at room temperature for 2 h. After the reaction was complete as detected by LC-MS, 20 ml of saturated ammonium chloride was added, stirred and separated, and the organic layer was washed with saturated sodium bicarbonate and saturated sodium chloride in turn, dried over anhydrous sodium sulfate, filtered, and purified by spin column chromatography to obtain compound 7 (60 mg, white solid); LC-MS: ESI [M + H] ⁺ = 408.5; 1H NMR (400 MHz, DMSO-d6) δ 11.90 (s, 1H), 8.79-8.62 (m, 3H), 8.06-7.93 (m, 2H), 7.81 (d, J = 1.8 Hz, 1H), 7.35 (s, 1H), 6.45 (d, J = 1.6 Hz, 1H), 3.77 (s, 2H), 2.82 (d, J = 4.9 Hz, 3H), 2.57 (d, J = 1.6 Hz, 2H), 1.17 (t, J = 7.4 Hz, 3H).

Example 8: 1'-((7-ethyl-4-fluoro-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-2',2',6',6'-d ₄ -6-carboxamide

Step 1: At 0°C, slowly add compound 8a (180 g, 0.763 mol) to a solution of MeONa (618.0 g, 11.4 mol, 15.0 eq) in methanol (2 L). The reaction mixture was stirred at 20°C for 1 hour, and then quenched with saturated NH ₄ Cl aqueous solution (2 L) under stirring. The formed white precipitate was filtered, and the filter cake was washed with water (500 mL*3) and dried under vacuum. A white solid product 8b (150 g, 84.5% yield) was obtained. ¹ HNMR (400 MHz, DMSO-d ₆ ) δ (ppm) 9.06 (d, J=2.4 Hz, 1H), 8.78 (d, J=2.4 Hz, 1H), 4.07 (s, 3H).

Step 2: To a solution of compound 8b (150 g, 0.647 mol) in a mixed solvent of 1,4-dioxane (2.4 L) and water (600 mL) were added K ₂ CO ₃ (178.8 g, 1.29 mol, 2 eq.), potassium vinyl trifluoroborate (104.0 g, 0.776 mmol, 1.2 eq.) and Pd(dppf)Cl ₂ (14.2 g, 19.4 mmol, 0.03 eq.). The mixture was degassed and backfilled with N ₂ three times, and then stirred at 80° C. for 8 hours. The volatiles were removed under reduced pressure, and the resulting mixture was extracted with ethyl acetate (1 L*3), dried over anhydrous Na ₂ SO ₄ , and the combined organic layers were concentrated and purified by silica gel column chromatography (0-10% ethyl acetate/petroleum ether). The yellow solid product 8c (81.5 g, 70% yield) was obtained. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm) 8.96 (d, J=2.7 Hz, 1H), 8.57 (d, J=2.8 Hz, 1H), 6.82 (dd, J=17.8, 11.3 Hz, 1H), 6.15 (dd, J=17.7, 1.0 Hz, 1H), 5.56 (dd, J=11.3, 1.0 Hz, 1H), 4.05 (s, 3H).

Step 3: Pd/C (10% wt, 5.0 g) was added to a solution of 8c (50.0 g, 0.28 mol) in methanol (500 mL) under _N2 , the mixture was degassed and backfilled with hydrogen three times, and then stirred at room temperature for 12 h under hydrogen atmosphere. After the reaction was completed, the mixture was filtered through celite and washed with ethyl acetate (50 mL*3). The combined filtrate was concentrated under vacuum to give a dark purple solid 8d. (38.8 g, 92% yield). ¹ H NMR (400 MHz, DMSO- _d6 ) δ7.33 (d, J = 2.8 Hz, 1H), 6.85 (dt, J = 2.8, 0.7 Hz, 1H), 4.65 (br s, 2H), 3.73 (s, 3H), 2.42 (q, J = 7.5 Hz, 2H), 1.08 (t, J = 7.5 Hz, 3H).

The fourth step: 8d (16g, 0.105mol) was added to a solution of EtOH (300mL) at once with diethyl 2-(ethoxymethylene) malonate (27.3g, 0.126mol, 25.5mL, 1.2eq), and the reaction mixture was refluxed for 2 hours under agitation. TLC showed that the reaction was complete. After being cooled to room temperature, the mixture was concentrated under vacuum to obtain a dark purple residue. The product was further purified to give compound 8e (32.0g, 94% yield, white solid) by silica gel column chromatography (0-10% ethyl acetate/petroleum ether). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.65 (s, 1H), 8.28 (s, 1H), 8.05 (d, J=2.8 Hz, 1H), 7.66 (d, J=2.8 Hz, 1H), 4.15 (dd, J=32.3, 6.9 Hz, 4H), 3.86 (s, 3H), 2.54 (q, J=7.5 Hz, 2H), 1.24 (q, J=6.4 Hz, 6H), 1.14 (t, J=7.5 Hz, 3H).

Step 5: Compound 8e (32.0 g, 0.099 mol) was added to a three-necked round bottom flask (1.0 L) equipped with a reflux condenser and a mechanical stirrer, followed by the addition of phenyl ether-biphenyl eutectic (CAS: 8004-13-5, 300 mL). The system was degassed and filled with _N2 three times, then placed in an oil bath preheated to 240 ° C. The reaction mixture was stirred at 250-260 ° C for 1 hour, then cooled to room temperature. TLC showed that the reaction was complete. 1.2 L of diisopropyl ether was added under stirring, and the mixture was stirred at room temperature for 1 hour. The off-white precipitate formed was collected by filtration, washed with diisopropyl ether (100 mL * 3), and dried in vacuo. The product 8f (21.3 g, 77% yield) was obtained as an off-white solid. LC-MS: ESI [M + H] ⁺ = 277.1.

Step 6: DAST (37.3 g, 0.23 mol, 30.5 mL, 3.0 eq) was slowly added to a suspension of compound 8f (21.3 g, 0.077 mol) in DCM (400 mL) at 0-5°C. The ice-water bath was removed and the reaction mixture was stirred at room temperature for 8 hours until a clear orange solution was formed. TLC showed that only a trace amount of starting material remained. (DCM/MeOH=20:1, Rf=0.3), and produce a major product (PE/EA=5:1, Rf=0.35). The reaction was quenched with saturated NaHCO ₃ aqueous solution (1.0L) at 0-5°C until pH=8, the organic layer was separated, and the remaining aqueous phase was extracted with DCM (200mL*2). The combined DCM layer was washed with saturated NaHCO ₃ solution (200mL), then washed with water (200ml), dried over anhydrous Na ₂ SO ₄ , and spin-dried. Purification by silica gel column chromatography (PE/EA=20:1 to 10:1) gave the product 8g (18.0g, 84% yield, white needle-shaped solid). ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.15 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 1.5 Hz, 1H), 4.48 (q, J = 7.1 Hz, 2H), 2.79 (q, J = 7.4 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H), 1.33 (t, J = 7.4 Hz, 3H).

Step 7: Compound 8g (13.0 g, 0.047 mol) was added to anhydrous THF (300 mL), the reaction solution was cooled to -20 ° C, DIBAL-H (78.0 mL, 0.117 mol, 1.5 M toluene solution, 2.5 eq.) 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 (300 mL*3), and the combined organic phase was washed with water (300 mL) and brine (300 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 8h (8.0 g, 72% yield) was obtained as a yellow solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.79 (d, J=8.8 Hz, 1H), 8.10 (d, J=1.2 Hz, 1H), 5.52 (s, 1H), 4.75 (s, 2H), 4.07 (s, 3H), 2.74 (q, J=7.9, 7.4 Hz, 2H), 1.26 (t, J=7.4 Hz, 3H).

Step 8: Cool a suspension of compound 8h (8.0 g, 0.034 mol) in acetonitrile (100 mL) to -15-0 ° C with an ice-salt bath, and slowly add TMSI (3.0 eq) under stirring under N _2. Afterwards, remove the ice-salt bath, slowly warm the reaction mixture to 30 ° C, and stir at the same temperature for 24 hours until the raw material is completely consumed. The volatile substances are removed under reduced pressure, ethyl acetate (250 mL) and 1N NaOH aqueous solution (100 mL) are added to the solid residue, and the resulting mixture is stirred at room temperature for 1 hour, during which a large amount of white precipitate is formed. The white solid is collected by filtration, washed with water (10 mL * 3), then washed with ethyl acetate (10 mL * 2) and dried in vacuo to obtain compound 8i. Yield: 5.3 g (70%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.09 (s, 1H), 8.46 (d, J=8.7 Hz, 1H), 7.77 (d, J=1.5 Hz, 1H), 5.50 (s, 1H), 4.66 (d, J=3.9 Hz, 2H), 2.56 (qd, J=7.4, 1.1 Hz, 2H), 1.19 (t, J=7.5 Hz, 3H).

Step 9: SOCl ₂ (4.3 g, 0.036 mol, 2.6 mL, 1.5 eq) was slowly added to a DMF (100 mL) solution of compound 8i (5.3 g, 0.024 mol) 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, and quenched with 1N NaOH (70 mL) to pH = 9, and then water (180 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 8j (4.01 g, 70% yield). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ12.25 (s, 1H), 8.55 (d, J = 8.7 Hz, 1H), 7.79 (q, 1H), 4.95 (s, 2H), 2.57 (qd, J=7.4, 1.3 Hz, 2H), 1.19 (t, J=7.4 Hz, 3H); ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 161.2, 152.2 (d, J=266.6 Hz), 145.8 (d, J=3.0 Hz), 141.67, 140.1 (d, J=4.0 Hz), 135.0 (d, J=3.0 Hz), 123.0 (d, J=10.1 Hz), 119.9 (d, J=8.1 Hz), 39.9 (d, J=4.0 Hz), 23.14, 12.25; ¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -125.12. MS (ESI): 241.0 (M+1) ⁺ .

Step 10: Compound 8j (40 mg, 0.17 mmol), 2f (51 mg, 0.17 mmol), N,N-diisopropylethylamine (72 mg, 0.56 mmol) and potassium iodide (3 mg, 0.02 mmol) were added to 10 ml of anhydrous acetonitrile, stirred at 85°C for 2 hours, and after the reaction was completed as monitored by TLC, the reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography to obtain compound 8 (42 mg, white solid). LC-MS: ESI [M+H] ⁺ = 426.2. ¹ H NMR (400 MHz, Chloroform-d) δ 9.64 (s, 1H), 8.48 (dd, J = 5.5, 3.2 Hz, 2H), 8.07 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 5.3 Hz, 1H), 7.83-7.64 (m, 2H), 6.14 (d, J = 1.6 Hz, 1H), 3.79 (s, 2H), 2.96 (d, J = 5.1 Hz, 3H), 2.66 (qd, J = 7.4, 1.4 Hz, 2H), 2.51 (d, J = 1.6 Hz, 2H), 1.24 (t, J = 7.4 Hz, 3H).

Example 9: 1'-((7-cyclopropyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-2',2',6',6'-d ₄ -6-carboxamide

Preparation of 1'-((7-cyclopropyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-N-methyl-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-2',2',6',6'-d ₄ -6-carboxamide Reference Example 2. LC-MS: ESI [M+H] ⁺ =420.2. ¹ H NMR (400 MHz, Chloroform-d) δ 11.85 (s, 1H), 8.47 (dd, J = 11.1, 2.0 Hz, 2H), 8.07 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 5.3 Hz, 1H), 7.72 (dd, J = 8.2, 2.3 Hz, 1H), 7.66 (d, J = 1.9 Hz, 1H), 7.44 (s, 1H), 6.15 (d, J = 1.6 Hz, 1H), 3.71 (s, 2H), 2.97 (d, J = 5.1 Hz, 3H), 2.51 (d, J = 1.6 Hz, 2H), 2.36–2.18 (m, 1H), 1.06–0.96 (m, 2H), 0.84–0.70 (m, 2H).

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

Step 1: Weigh 2e (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 10a (730 mg, light yellow solid) was obtained.

Step 2: Weigh 10a (230 mg, 0.7 mmol), EDCI (191 mg, 1 mmol), HOBT (100 mg, 1 mmol), 2 mL DMF, N-methylmorpholine (250 mg, 2.8 mmol), d ₃ -methylamine hydrochloride (49 mg, 0.7 mmol), react at warm 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 spin dry to obtain a crude product 10b (220 mg, light yellow oily liquid).

Step 3: Dissolve the crude product of 10b (220 mg, 0.7 mmol) in 4 mL MeOH, then add 1.1 mL 4 M HCl in dioxane solution, react at room temperature for 12 h, monitor the reaction by TLC, add potassium carbonate after the reaction is complete, stir for 30 minutes, filter and remove potassium carbonate to obtain the crude product of compound 10c (270 mg).

Step 4: Compound 10c (40 mg, 0.17 mmol), INT2 (51 mg, 0.17 mmol), N,N-diisopropylethylamine (72 mg, 0.56 mmol) and potassium iodide (3 mg, 0.02 mmol) were added to 10 ml of anhydrous acetonitrile, stirred at 85°C for 2 hours, and after the reaction was completed as monitored by TLC, the reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography to obtain compound 10 (42 mg, white solid). LC-MS: ESI [M+H] ⁺ = 423.2. ¹ H NMR (400 MHz, Chloroform-d) δ 11.85 (s, 1H), 8.47 (dd, J = 11.1, 2.0 Hz, 2H), 8.07 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 5.3 Hz, 1H), 7.72 (dd, J = 8.2, 2.3 Hz, 1H), 7.66 (d, J = 1.9 Hz, 1H), 7.44 (s, 1H), 6.15 (d, J = 1.6 Hz, 1H), 3.71 (s, 2H), 2.51 (d, J = 1.6 Hz, 2H), 2.36–2.18 (m, 1H), 1.06–0.96 (m, 2H), 0.84–0.70 (m, 2H).

Example 11: N-cyclopropyl-1'-((7-cyclopropyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-2',2',6',6'-d ₄ -6-carboxamide

Preparation of N-cyclopropyl-1'-((7-cyclopropyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[3,4'-bipyridine]-2',2',6',6'-d ₄ -6-carboxamide Reference Example 10. LC-MS: ESI [M+H] ⁺ =446.2.

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), and reference compound AZD5305 were purchased from MedChemExpress (MCE).

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 the plate three times with PBST solution according to step 2. Finally, pat the reaction plate dry.

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

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

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 2:

Table 2 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 3:

Table 3 Cell antiproliferative activity test results

Conclusion: The compounds of the present invention have a 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.

3. 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 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), bioavailability (F), etc. were calculated.

The results of pharmacokinetic evaluation are shown in Table 4 below:

Table 4 Pharmacokinetic test results of the compounds in Balb/c mice

Conclusion: The compounds of the present invention have good pharmacokinetic properties in Balb/c mice, including good oral bioavailability, exposure, half-life and clearance, etc. After oral administration of 1 mg/kg, the C _max of compounds 2 and 9 is better than that of the reference compound AZD5305.

4. Pharmacokinetic evaluation of the compound in SD rats

Experimental purpose: To understand the pharmacokinetics of the compound.

Experimental plan: The pharmacokinetics of the compound were investigated by oral and intravenous administration in SD rats.

Experimental steps: weigh the compound, add a small amount of DMSO, and then add sodium chloride solution for injection to make a solution for administration. Six SD rats, male, were administered intravenously and orally. About 0.1 mL of blood was collected at 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 10h and 24h after administration, centrifuged at 3500rpm for 15min, and the supernatant plasma was collected. Take 5μL of plasma in an EP tube, add 100μL of acetonitrile containing 20ng·ml-1 internal standard SAHA to precipitate protein, vortex for 30s, centrifuge at 13000rpm for 15min, and take the supernatant into the injection bottle for testing. Standard curve range: 10~10000ng·ml-1.

The results of pharmacokinetic evaluation are shown in Table 5 below:

Table 5 Pharmacokinetic test results of the compounds in SD rats

Conclusion: The compounds of the present invention have good pharmacokinetic properties in SD rats, including good oral Bioavailability, exposure, half-life and clearance, etc. The C _max and AUC (0-t) of compound 2 were better than those of the reference compound AZD5305.

5. Evaluation of PARP enzyme selectivity

Table 6 Reagents

experiment procedure:

(1) Coating histone substrate: 5x histone was diluted to 1x with PARP buffer solution, 25uL per well was coated at 4℃ overnight

(2) Add 100uL of PBST to the 384 reaction plate and wash the plate three times, 5 minutes each time.

(3) Add 25uL of blocking buffer solution to the 384 reaction plate and leave at room temperature for 90 minutes. Add 100uL of PBST to the 384 reaction plate and wash the plate 3 times, 5 minutes each time.

(4) Compound preparation: Add 100 nL of compound to the 384 reaction plate and centrifuge for 1 minute.

(5) Add 5uL PARP protein to the 384 reaction plate and centrifuge at 1000rpm for 1 minute.

(6) Add 5uL PARP substrate mixture to the 384 reaction plate and centrifuge at 1000rpm for 1 minute. Incubate at room temperature for 1 hour.

(7) Detection: Add 100uL of PBST to the 384 reaction plate and wash the plate 3 times for 5 minutes each time. Dilute Stre-HRP 2000 times in blocking solution and add 25uL to each well, react at room temperature for 30 minutes.

(8) Add 100uL of PBST to the 384 reaction plate and wash the plate 3 times, 5 minutes each time. Mix ELISA ECL substrate A and substrate B 1:1 and add to the 384 reaction plate, 25uL per well.

(9) Read the luminescent signal of the compound using a BMG microplate reader.

The results of the enzyme selectivity experiment are shown in Table 7 below:

Table 7 Results of the test on the selectivity of compounds for PARP family enzymes

Conclusion: The compounds of the present invention have high inhibitory activity against PARP-1 enzyme, and weaker inhibitory effects on PARP-2, PARP-5A and PARP-11 of the same family. Compound 2 has better selectivity for PARP-2 and PARP-5A than the reference compound AZD5305, and compound 9 has better selectivity for PARP-2, PARP-5A and PARP1 than the reference compound AZD5305.

Claims

The compound of formula I or a pharmaceutically acceptable form thereof, characterized in that: the structure of formula I is as follows:

in:

R1 is selected from halogen, C1-4 alkyl, C1-4 fluoroalkyl, C1-4 alkoxy, C1-4 fluoroalkoxy, 3-6 membered cycloalkyl or 3-6 membered fluorocycloalkyl;

X1 is selected from N or CR5a , X2 is selected from N or CR5b , and X3 is selected from N or CR5c ;

R6 is selected from hydrogen or halogen;

R 4 is -CONHR 7 , R 7 is selected from C 1-4 alkyl, C 1-4 fluoroalkyl, C 1-4 deuterated alkyl, 3-6 membered cycloalkyl or 3-6 membered fluorocycloalkyl;

R 9a is selected from hydrogen, halogen, C 1-4 alkyl, C 1-4 fluoroalkyl, C 1-4 alkoxy, C 1-4 fluoroalkoxy, 3-6 membered cycloalkyl, 3-6 membered fluorocycloalkyl or cyano;

R 9c is selected from hydrogen or halogen;

R2 is selected from hydrogen or C1-4 alkyl;

R 5a is selected from hydrogen, halogen or C 1-4 alkyl, R 5b is selected from hydrogen or halogen, and R 5c is selected from hydrogen or halogen;

Ring A is selected from

The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotopically labeled substances, metabolites or prodrugs.
The compound according to claim 1, characterized in that: R 1 is selected from chlorine, methyl, ethyl, fluoromethyl, fluoroethyl, cyclopropyl or fluorocyclopropyl.
The compound according to claim 1, characterized in that: R 6 is selected from hydrogen or fluorine.
The compound according to claim 1, characterized in that: R7 is selected from methyl, ethyl, deuterated methyl, deuterated ethyl, fluoromethyl, fluoroethyl, cyclopropyl or fluorocyclopropyl.
The compound according to claim 1, characterized in that: R 9a is selected from hydrogen, fluorine, chlorine, methyl, ethyl, fluoromethyl, fluoroethyl or cyclopropyl.
The compound according to claim 1, characterized in that: R 9c is selected from hydrogen or fluorine.
The compound according to claim 1, characterized in that: R2 is selected from hydrogen or methyl.
The compound according to claim 1, characterized in that: R 5a is selected from hydrogen, fluorine or methyl, R 5b is selected from hydrogen or fluorine, and R 5c is selected from hydrogen or fluorine.
The compound according to any one of claims 1 to 8, characterized in that: Selected from the following structures:
The compound according to any one of claims 1 to 9, characterized in that: Selected from the following structures:
The compound according to any one of claims 1 to 10, characterized in that the compound is selected from:
The compound according to any one of claims 1 to 10, characterized in that the compound is selected from:
A pharmaceutical composition, characterized in that it uses the compound according to any one of claims 1 to 12 or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug as an active ingredient, supplemented with a pharmaceutically acceptable carrier.
The compound according to any one of claims 1 to 12 or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite or prodrug, and the pharmaceutical composition according to claim 13 are used in the preparation of drugs for preventing and/or treating PARP1 enzyme-related diseases.
The use according to claim 14 is characterized in that the PARP1 enzyme-related disease is a tumor-related disease.
The use according to claim 15 is characterized in that the tumor-like disease lacks the HR-dependent DNA DSB repair pathway.
The use according to claim 15 or 16 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 17, characterized in that the cancer cells have a BRCA1 or BRCA2 defective phenotype.
The use according to any one of claims 15 to 18, characterized in that the tumor disease is breast cancer, ovarian cancer, primary peritoneal cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer, glioblastoma or lung cancer.