Double-fused-ring compound as well as preparation method and application thereof

Abstract

The invention provides a double-fused-ring compound, a preparation method and application thereof, and particularly provides a compound as shown in a formula (I) or pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorphic substance, solvate, N-oxide, isotope labeled compound, metabolite or prodrug thereof, and a pharmaceutical composition, a preparation method and application thereof, and particularly provides a double-fused-ring compound as shown in the formula (I) or pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorphic substance, solvate, N-oxide, isotope labeled compound, metabolite or prodrug thereof. # imgabs0 #

Description

A type of bicyclic compound, preparation method and use thereof

[0001] This application is based on the application with CN application number 202211126580.X and application date September 16, 2022, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of medicinal chemistry, and specifically relates to a class of bicyclic compounds, their preparation methods and uses. Background Art

[0003] Toll-like receptors (TLRs) belong to the pattern recognition receptor (PRR) family and can specifically recognize pathogen-associated molecular patterns (PAMPs). They are a key protein involved in nonspecific immunity and serve as a bridge between nonspecific and specific immunity. To date, 13 TLRs have been discovered in mammals, 10 of which (TLR1-10) have been found in humans. TLR1 / 2 / 4 / 5 / 6 / 10 are expressed on the cell surface and can rapidly recognize products of bacterial metabolism. TLR3 / 7 / 8 / 9 are expressed in endosomes and primarily recognize and monitor exogenous nucleic acids from pathogens.

[0004] TLR7 has a restricted distribution in humans, primarily expressed by B cells and plasmacytoid dendritic cells (pDCs) in response to viral infection. It induces high levels of interferon-α, enhances pDCs' costimulatory and antigen presentation capabilities, promotes CD4+ T cell proliferation, and further activates CD8+ T cells, leading to adaptive T cell responses. TLR8 is more widely expressed in humans, primarily by monocytes, NK cells, and myeloid dendritic cells (mDCs). Stimulation of TLR8 can induce the release of various proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-6, IL-12, and interferon-γ. Furthermore, the TLR8 signaling pathway has been shown to be necessary and sufficient to reverse the suppressive function of Tregs. Therefore, TLR7 and TLR8 play important roles in both innate and adaptive immunity, making them attractive targets for anticancer and antiviral immunotherapy. There have been reports of multiple TLR7 and / or TLR8 agonists being developed by different companies, but there is still a great demand for TLR7 and / or TLR8 agonists with better activity, safety, and physicochemical properties.

[0005] A new class of immunostimulating antibody conjugates (ISACs) is emerging. These conjugates combine immunomodulators, such as TLR7 and / or TLR8 agonists, with tumor-targeting antibodies via cleavable or non-cleavable linkers. These conjugates combine the precise tumor targeting of antibodies with the long-lasting anti-tumor activity and immune memory of immunomodulators, such as TLR7 and / or TLR8 agonists, enabling systemic administration while safely eliciting anti-tumor immune responses.

[0006] This application provides a class of bicyclic compounds with TLR7 and / or TLR8 agonist activity. These compounds have improved activity, safety, and physicochemical properties. They can also be used in immunostimulatory antibody conjugates, which are of great significance for the development of tumor and virus drugs.

[0007] Summary of the Invention

[0008] On the one hand, the present application provides a class of bicyclic compounds that have a strong agonistic effect on TLR7 and / or TLR8, and thus have better uses in preventing and / or treating TLR7 and / or TLR8-mediated related diseases. The compounds are also found to have good physicochemical properties (e.g., solubility, physical and / or chemical stability) and a good safety profile.

[0009] The compound is a compound of formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0010] in,

[0011] L 1 Selected from O, S and NR 5 ;

[0012] L 2 is a covalent bond, or is selected from C 1-6 Alkylene and -C 1-6 Alkylene-O-;

[0013] R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, 5-10 membered heteroaryl and R 6 -C(O)-NH-;

[0014] R 2 Selected from hydrogen, C1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, R 7 and R 7 -L 3 -substituted by a group;

[0015] L 3 Selected from C 1-6 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-;

[0016] R 3 Selected from hydrogen, halogen, C 1-6 Alkyl and C 1-6 alkoxy;

[0017] R 4 Selected from hydrogen, halogen, C 1-6 Alkyl and C 1-6 alkoxy;

[0018] R 5 Selected from hydrogen and C 1-6 alkyl;

[0019] R 6 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with an alkoxy group;

[0020] R 7 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6substituted with alkoxy and carboxyl groups;

[0021] R 8 Selected from hydrogen and C 1-6 alkyl.

[0022] In another aspect, the present application provides a drug-linker of the following formula or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0023] Wherein, D is a fragment of the compound described above;

[0024] L' is a linker fragment.

[0025] The present application also provides an immunostimulatory antibody conjugate represented by formula (ISAC-I) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0026] in,

[0027] D is a fragment of the compound described above;

[0028] Ab is an antibody that can target the target antigen;

[0029] L is the fragment obtained by connecting L' in the drug-linker described above with Ab;

[0030] z is selected from 1-10.

[0031] On the other hand, the present application provides a composition comprising the immunostimulatory antibody conjugate or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof.

[0032] On the other hand, the present application provides the use of the compound represented by formula (I) in the preparation of immunostimulatory antibody conjugates.

[0033] On the other hand, the present application provides a pharmaceutical composition comprising a prophylactically and / or therapeutically effective amount of the compound of any one of the first aspects or its pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites or prodrugs, the immunostimulatory antibody conjugate represented by formula (ISAC-I), or the composition and one or more pharmaceutically acceptable carriers.

[0034] On the other hand, the present application provides a drug kit comprising the compound of any one of the first aspect or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug, the immunostimulatory antibody conjugate represented by formula (ISAC-I), the composition or the pharmaceutical composition.

[0035] On the other hand, the present application provides the use of the compound of any one of the first aspect or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug, the immunostimulatory antibody conjugate represented by formula (ISAC-I), the composition or pharmaceutical composition in the preparation of a medicament for preventing and / or treating TLR7 and / or TLR8-mediated related diseases.

[0036] On the other hand, the present application provides a compound according to any one of the first aspects or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, an immunostimulatory antibody conjugate represented by formula (ISAC-I), the composition or pharmaceutical composition, which is used to prevent and / or treat TLR7 and / or TLR8-mediated related diseases.

[0037] In another aspect, the present application provides a method for preparing the compound represented by formula (I).

[0038] definition

[0039] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art. References to technology used herein are intended to refer to technology commonly understood in the art, including variations of technology or substitutions of equivalent technology that would be apparent to those skilled in the art. While it is believed that the following terms are well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

[0040] As used herein, the terms "comprises," "comprising," "having," "containing," or "involving," and variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0041] As used herein, the term "alkyl" is defined as a straight-chain or branched saturated aliphatic hydrocarbon group. For example, as used herein, the term "C 1-6"Alkyl" refers to a straight or branched chain group having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl), which is optionally substituted with one or more (e.g., 1 to 3) suitable substituents such as halogen. The term "C 1-3 "Alkyl" refers to a straight or branched chain group having 1 to 3 carbon atoms (eg methyl, ethyl, n-propyl, isopropyl), which is optionally substituted by one or more (such as 1 to 3) suitable substituents such as halogen.

[0042] As used herein, the term "alkoxy" refers to "alkyl-O-," wherein "alkyl" is as defined above. For example, the term "C 1-6 "Alkoxy" refers to "C 1-6 Alkyl-O-", the "C 1-6 "C alkyl" is as defined above. 1-3 "Alkoxy" refers to "C 1-3 Alkyl-O-", the "C 1-3 "Alkyl" is as defined above. Exemplary C 1-6 Alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy or n-hexoxy.

[0043] As used herein, the term “C 1-6 "Alkylene" refers to a divalent group formed by removing two hydrogen atoms from a straight or branched alkane containing 1 to 6 carbon atoms, including "C 1-5 Alkylene", "C 1-4 Alkylene", "C 1-3 Alkylene", "C 1-2 "Alkylene", specific examples include but are not limited to: -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, etc.

[0044] As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., a monocyclic ring such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or a bicyclic ring, including spirocyclic, fused or bridged systems such as bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl or bicyclo[5.2.0]nonyl, decahydronaphthyl, etc.), which is optionally substituted with one or more (such as 1 to 3) suitable substituents. For example, the term "C 3-6"Cycloalkyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring having 3 to 6 ring carbon atoms (for example cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), which is optionally substituted by one or more (such as 1 to 3) suitable substituents, for example methyl substituted cyclopropyl.

[0045] As used herein, the term "halogen" group is defined to include fluorine, chlorine, bromine, or iodine.

[0046] As used herein, the term "halo" refers to substitution with one or more (such as 1 to 3) the same or different halogen atoms.

[0047] As used herein, the term "haloalkyl" refers to an alkyl group substituted by one or more (such as 1 to 3) the same or different halogen atoms. For example, the term "C 1-6 "Haloalkyl" refers to a haloalkyl group having 1 to 6 carbon atoms, for example, -CF3, -C2F5, -CHF2, -CH2F, -CH2CF3, -CH2Cl or -CH2CH2CF3, etc.

[0048] As used herein, the term "heterocycle" or "heterocyclyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic group, for example, having 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms and one or more (e.g., 1, 2, 3 or 4) independently selected from N, O or S(O) in the ring. t (wherein t is 0, 1 or 2) heteroatom, such as 3-12 membered heterocyclyl, 3-7 membered heterocyclyl, 3-6 membered heterocyclyl, 5-6 membered heterocyclyl, etc., such as 5-10 membered nitrogen-containing heterocyclyl, 6-10 membered oxygen-containing heterocyclyl, 6-8 membered sulfur-containing heterocyclyl, 5-8 membered oxygen-containing heterocyclyl, etc. Representative examples of heterocyclyl include, but are not limited to, oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydropyrrolidinyl, hexahydro-1H-pyrroline, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, tetrahydropyridinyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, etc.

[0049] As used herein, the term "aryl" or "aromatic ring" refers to an all-carbon monocyclic or fused-ring polycyclic aromatic group having a conjugated π electron system. For example, the term "C 6-10 Aryl" or "C 6-10 "Aromatic ring" refers to an aromatic group containing 6 to 10 carbon atoms, such as phenyl (ring) or naphthyl (ring). The aromatic group or aromatic ring is optionally substituted by one or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO2, C 1-6 alkyl, etc.) substituted.

[0050] In this article, term " heteroaryl " refers to a cyclic group with aromaticity, wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the ring structure can be oxoed. Specific examples include but are not limited to 5-10 yuan heteroaryl, 5-10 yuan nitrogen-containing heteroaryl, 6-10 yuan oxygen-containing heteroaryl, 6-8 yuan nitrogen-containing heteroaryl, 5-8 yuan oxygen-containing heteroaryl, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl oxazolyl, pyridinyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1,4-dioxadienyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, and the like.

[0051] Optionally, hydrogen in the groups referred to herein may be replaced by deuterium.

[0052] The term "substituted" means that one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on the designated atom are replaced with a group selected from the indicated group, provided that the designated atom's normal valence in the current context is not exceeded and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0053] If a substituent is described as being "optionally substituted with," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the substituents listed, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced, individually and / or collectively, with independently selected substituents or unsubstituted. If a nitrogen of a substituent is described as being optionally substituted with one or more of the substituents listed, one or more hydrogens on the nitrogen (to the extent of any hydrogens present) may each be replaced with an independently selected substituent or unsubstituted.

[0054] If a substituent is described as being "independently selected" from a group of groups, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.

[0055] As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, where reasonable.

[0056] Unless otherwise indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

[0057] When a bond to a substituent is shown to pass through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom in the substitutable ring.

[0058] The present application also provides all pharmaceutically acceptable isotopically labeled compounds, which are identical to the compounds described above, except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of suitable isotopes include, but are not limited to, isotopes of hydrogen (e.g., 2 H. 3 H, deuterium D, tritium T); carbon isotopes (such as 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 37 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); isotopes of phosphorus (such as 32 P); and sulfur isotopes (e.g. 35 S). Certain isotopically labeled compounds (e.g., those incorporating radioactive isotopes) are useful in drug and / or substrate tissue distribution studies (e.g., assays). The radioactive isotope tritium (i.e. 3 H) and carbon-14 (i.e. 14 C) are particularly useful for this purpose because they are easy to incorporate and easy to detect. 11 C. 18 F. 15 O and 13Substitution with N) can be used to examine substrate receptor occupancy in positron emission tomography (PET) studies. Isotopically labeled compounds can be prepared by methods analogous to those described in the accompanying schemes and / or examples and preparations by using appropriate isotopically labeled reagents instead of the non-labeled reagents previously employed. Pharmaceutically acceptable solvates described herein include those in which the crystallization solvent is isotopically substituted, for example, D2O, acetone-d6 or DMSO-d6.

[0059] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In a compound with one or more (e.g., 1, 2, 3, or 4) asymmetric centers, it can produce a racemic mixture, a single enantiomer, a diastereomeric mixture, and a single diastereoisomer. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compound can exist as a mixture of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0060] The present application also provides all possible crystalline forms or polymorphs of the compound, which may be a single polymorph or a mixture of more than one polymorph in any ratio.

[0061] It should also be understood that certain compounds herein may be used therapeutically in free form or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof. As used herein, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, solvates, metabolites, or prodrugs that, upon administration to a patient in need thereof, are capable of directly or indirectly providing the compound or its metabolites or residues. Therefore, references herein to "the compound" are intended to encompass all of the aforementioned derivative forms of the compound.

[0062] Pharmaceutically acceptable salts of the compounds include acid addition salts and base addition salts thereof. Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts. For a review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds are known to those skilled in the art.

[0063] The compound may exist in the form of a solvate, preferably a hydrate, wherein the compound contains a polar solvent as a structural element of the crystal lattice of the compound. The amount of the polar solvent, especially water, may be present in a stoichiometric or non-stoichiometric ratio.

[0064] Those skilled in the art will appreciate that not all nitrogen-containing heterocycles are capable of forming N-oxides, as nitrogen requires an available lone pair of electrons to oxidize to an oxide; those skilled in the art will recognize nitrogen-containing heterocycles that are capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include oxidation of heterocycles and tertiary amines with peroxyacids such as peracetic acid and meta-chloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate, and dioxirane such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see for example: TL Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp 748-750; AR Katritzky and AJ Boulton, Eds., Academic Press; and GWH Cheeseman and ESGWerstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp 390-392, AR Katritzky and AJ Boulton, Eds., Academic Press.

[0065] The present application also provides metabolites of the compounds, i.e., substances formed in vivo upon administration of the compounds. Such products can be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the administered compounds. Therefore, the present application also relates to metabolites of the compounds, including compounds produced by methods that contact the compounds with a mammal for a period of time sufficient to produce their metabolic products.

[0066] The present application further provides prodrugs of the compounds, which are certain derivatives of the compounds that may have little or no pharmacological activity themselves, and when administered to the body or thereon, can be converted into the compounds having the desired activity by, for example, hydrolytic cleavage. 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," Volume 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 (E.B. Roche, ed., American Pharmaceutical Association). The prodrugs can be prepared, for example, by replacing appropriate functional groups present in the compounds 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)).

[0067] The present application also provides compounds containing protecting groups. During any process for preparing the compounds, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved, thereby forming a chemically protected form of the compound. This can be achieved using conventional protecting groups, for example, those described in Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene & P.GM Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference. Protecting groups can be removed at an appropriate subsequent stage using methods known in the art.

[0068] The term "about" means within ±10%, preferably within ±5%, and more preferably within ±2% of the stated numerical value.

[0069] The term "conjugate" refers to a substance formed by linking a small molecule drug to a targeting moiety. In some embodiments of the present application, the small molecule drug and the targeting moiety are connected via a linker. The linker can be broken in specific environments (e.g., a low intracellular pH environment, a slightly acidic tumor microenvironment) or under specific effects (e.g., the action of lysosomal proteases), thereby separating the small molecule drug from the targeting moiety.

[0070] The term "linker" refers to a moiety that connects a small molecule drug to a targeting moiety.

[0071] The term "targeting moiety" refers to a portion of the conjugate that is capable of specifically binding to a target (or portion of a target) on the cell surface. Through the interaction of the targeting moiety with the target, the conjugate can be delivered to a specific cell population.

[0072] Compound

[0073] In a first aspect, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0074] in,

[0075] L 1 Selected from O, S and NR 5 ;

[0076] L 2 is a covalent bond, or is selected from C 1-6 Alkylene and -C 1-6 Alkylene-O-;

[0077] R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, 5-10 membered heteroaryl and R 6 -C(O)-NH-;

[0078] R 2 Selected from hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, R 7 and R7 -L 3 -substituted by a group;

[0079] L 3 Selected from C 1-6 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-;

[0080] R 3 Selected from hydrogen, halogen, C 1-6 Alkyl and C 1-6 alkoxy;

[0081] R 4 Selected from hydrogen, halogen, C 1-6 Alkyl and C 1-6 alkoxy;

[0082] R 5 Selected from hydrogen and C 1-6 alkyl;

[0083] R 6 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with an alkoxy group;

[0084] R 7 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with alkoxy and carboxyl groups;

[0085] R 8 Selected from hydrogen and C 1-6 alkyl.

[0086] In some embodiments, R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more selected from hydrogen, halogen, hydroxyl, C 1-3 Alkyl, C1-3 Alkoxy, 5-10 membered heteroaryl and R 6 -C(O)-NH-;

[0087] R 6 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with an alkoxy group.

[0088] In some embodiments, R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more selected from hydrogen, halogen, hydroxyl, C 1-3 Alkyl, C 1-3 Alkoxy, 5-10 membered heteroaryl and R 6 -C(O)-NH-;

[0089] R 6 C 1-6 alkyl.

[0090] In some embodiments, R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more groups selected from hydrogen, hydroxy, methyl, methoxy and C 1-3 substituted by an alkyl-C(O)-NH- group;

[0091] In some embodiments, R 1 C 1-6 Alkyl, the alkyl group is unsubstituted or substituted by methyl, hydroxy, methoxy or C 1-3 Alkyl-C(O)-NH-substituted.

[0092] In some embodiments, R 1 C 1-6 alkyl.

[0093] In some embodiments, R 1 It is n-butyl.

[0094] In some embodiments, R 2 Selected from hydrogen, C 1-3 Alkyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, C 6-10Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, R 7 and R 7 -L 3 -substituted by a group;

[0095] R 7 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with alkoxy and carboxyl groups;

[0096] L 3 Selected from C 1-6 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-;

[0097] R 8 Selected from hydrogen and C 1-6 alkyl.

[0098] In some embodiments, R 2 Selected from hydrogen, C 1-3 Alkyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-3 Alkyl, C 1-3 Halogenated alkyl, C 1-3 Alkoxy, R 7 and R 7 -L 3 -substituted by a group;

[0099] R 7 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C1-6 Halogenated alkyl, C 1-6 substituted with alkoxy and carboxyl groups;

[0100] L 3 Selected from C 1-6 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-;

[0101] R 8 Selected from hydrogen and C 1-6 alkyl.

[0102] In some embodiments, R 2 Selected from hydrogen, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-3 Alkyl, C 1-3 Halogenated alkyl, C 1-3 Alkoxy, R 7 and R 7 -L 3 -substituted by a group;

[0103] R 7 Selected from C 1-3 Alkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-3 Alkyl, C 1-3 Halogenated alkyl, C 1-3 substituted with alkoxy and carboxyl groups;

[0104] L 3 Selected from C 1-3 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-;

[0105] R 8 Selected from hydrogen and C 1-3 alkyl.

[0106] In some embodiments, R 2 Selected from hydrogen, C3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, C 1-3 Alkyl, C 1-3 Alkoxy, R 7 and R 7 -L 3 -substituted by a group;

[0107] R 7 Selected from C 1-3 Alkyl and 3-6 membered heterocyclic group, the alkyl and heterocyclic group are optionally substituted by one or more selected from hydrogen and C 1-3 substituted with an alkyl group;

[0108] L 3 Selected from C 1-3 Alkylene and -C 1-3 Alkylene-NR 8 -;

[0109] R 8 C 1-3 alkyl.

[0110] In some embodiments, R 2 Selected from hydrogen, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, C 1-3 Alkyl, C 1-3 The 6-membered nitrogen-containing heterocyclic group is optionally substituted by one or more hydrogen and methyl groups.

[0111] In some embodiments, L 1 is selected from O, S and NH.

[0112] In some embodiments, L 1 For NH.

[0113] In some embodiments, L 2 is a covalent bond, or is selected from C 1-3 Alkylene and -C 1-3 Alkylene-O-.

[0114] In some embodiments, L 2 For methylene.

[0115] In some embodiments, R 3Selected from hydrogen, halogen, C 1-3 Alkyl and C 1-3 Alkoxy.

[0116] In some embodiments, R 3 For hydrogen.

[0117] In some embodiments, R 4 Selected from hydrogen, halogen, C 1-3 Alkyl and C 1-3 Alkoxy.

[0118] In some embodiments, R 4 For hydrogen.

[0119] The present invention encompasses compounds of formula (I) obtained by any combination of the above-mentioned preferred groups.

[0120] In some embodiments, the compound has the structure of Formula (II-A):

[0121] Among them, each group R 1 and R 2 As defined above.

[0122] In some embodiments, R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, 5-10 membered heteroaryl and R 6 -C(O)-NH-;

[0123] R 2 Selected from hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, R 7 and R 7 -L 3 -substituted by a group;

[0124] L 3 Selected from C 1-6 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8-and-NR 8 -C(O)-;

[0125] R 6 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with an alkoxy group;

[0126] R 7 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with alkoxy and carboxyl groups;

[0127] R 8 Selected from hydrogen and C 1-6 alkyl.

[0128] In some embodiments, R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more selected from hydrogen, halogen, hydroxyl, C 1-3 Alkyl, C 1-3 Alkoxy, 5-10 membered heteroaryl and R 6 -C(O)-NH-;

[0129] R 6 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl and C 1-6 substituted with an alkoxy group;

[0130] R 2 Selected from hydrogen, C 1-3 Alkyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, C 6-10Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, R 7 and R 7 -L 3 -substituted by a group;

[0131] R 7 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with alkoxy and carboxyl groups;

[0132] L 3 Selected from C 1-6 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-;

[0133] R 8 Selected from hydrogen and C 1-6 alkyl.

[0134] In some embodiments, R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more selected from hydrogen, halogen, hydroxyl, C 1-3 Alkyl, C 1-3 Alkoxy, 5-10 membered heteroaryl and R 6 -C(O)-NH-;

[0135] R 6 C 1-6 alkyl;

[0136] R 2 Selected from hydrogen, C 1-3 Alkyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-3 Alkyl, C 1-3 Halogenated alkyl, C1-3 Alkoxy, R 7 and R 7 -L 3 -substituted by a group;

[0137] R 7 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with alkoxy and carboxyl groups;

[0138] L 3 Selected from C 1-6 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-;

[0139] R 8 Selected from hydrogen and C 1-6 alkyl.

[0140] In some embodiments, R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more groups selected from hydrogen, hydroxy, methyl, methoxy and C 1-3 substituted by an alkyl-C(O)-NH- group;

[0141] R 2 Selected from hydrogen, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-3 Alkyl, C 1-3 Halogenated alkyl, C 1-3 Alkoxy, R 7 and R 7 -L 3 -substituted by a group;

[0142] R 7 Selected from C 1-3 Alkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-3 Alkyl, C1-3 Halogenated alkyl, C 1-3 substituted with alkoxy and carboxyl groups;

[0143] L 3 Selected from C 1-3 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-;

[0144] R 8 Selected from hydrogen and C 1-3 alkyl.

[0145] In some embodiments, R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more groups selected from hydrogen, hydroxy, methyl, methoxy and C 1-3 substituted by an alkyl-C(O)-NH- group;

[0146] R 2 Selected from hydrogen, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-6 membered heteroaryl, the cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more groups selected from hydrogen, methyl, methoxy, -(CH2)N(CH3)2, -(CH2CH2)N(CH3)2, 6-membered nitrogen-containing heterocyclic and 6-membered nitrogen-containing heterocyclic-methylene-, the 6-membered nitrogen-containing heterocyclic is optionally substituted by one or more hydrogen and methyl.

[0147] In some embodiments, R 1 Selected from n-butyl, R 2 Selected from hydrogen,

[0148] In some embodiments, R 1 Selected from n-butyl, R 2 Selected from hydrogen,

[0149] In some embodiments, R 1 Selected from n-butyl, R 2 Selected from hydrogen,

[0150] In some embodiments, the compound is selected from:

[0151] In another aspect, the present application provides a drug-linker of the following formula or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof:

[0152] wherein D is a fragment of any of the above compounds;

[0153] L' is a linker fragment.

[0154] In some embodiments, L' is selected from -L 4 -L 5 -L 6 ,in,

[0155] L 4 is selected from a chemical bond, -C(=O)-, and -NH-;

[0156] L 5 is a polyethylene glycol fragment, e.g. wherein a is an integer selected from 1 to 10;

[0157] L 6 for in,

[0158] Z1 is selected from a chemical bond and -C(=O)-;

[0159] Z2 is selected from chemical bonds and C 1-20 alkylene;

[0160] Z3 is selected from chemical bonds, C 2-6 Alkenylene and C 2-6 Alkynylidene;

[0161] A is selected from

[0162] LG is a leaving group for nucleophilic substitution reaction, selected from halogen, nitro, benzenesulfonate, p-toluenesulfonate, trifluoromethanesulfonate, -S(O)2-R 9 and-S(O)-R 9 ;

[0163] R 9 Selected from C 1-6 Alkyl and C 1-6 Halogenated alkyl.

[0164] In some embodiments, L 6 for in,

[0165] Z1 is selected from a chemical bond and -C(=O)-;

[0166] Z2 is selected from chemical bonds and C 1-10 alkylene;

[0167] Z3 is selected from chemical bonds, C 2-4 Alkenylene and C 2-4 Alkynylidene;

[0168] A is selected from

[0169] LG is selected from halogen, nitro, benzenesulfonate, p-toluenesulfonate, trifluoromethanesulfonate, -S(O)2-R 9 and-S(O)-R 9 ;

[0170] R 9 Selected from C 1-6 Alkyl and C 1-6 Halogenated alkyl.

[0171] In some embodiments, L 6 for in,

[0172] Z1 is -C(=O)-;

[0173] Z2 is C 1-6 alkylene;

[0174] Z3 is a chemical bond or

[0175] A is selected from

[0176] LG is a methylsulfonyl group.

[0177] In some embodiments, L 6 for

[0178] In some embodiments, L' is selected from:

[0179] In some embodiments, the drug-linker is selected from the following structures:

[0180] The aforementioned D-L' is linked to a tumor-targeting antibody via a linker L' to obtain the immunostimulatory antibody conjugate described herein. Therefore, in another aspect, the present application also provides an immunostimulatory antibody conjugate of formula (ISAC-I) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof:

[0181] in,

[0182] D is a fragment of any of the above compounds;

[0183] Ab is an antibody that can target a target antigen; for example, an antibody that targets tumor cells, such as trastuzumab monoclonal antibody;

[0184] L is the fragment obtained by linking L' described in any of the above items with Ab;

[0185] z is selected from 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0186] In some embodiments, z is selected from 1-8, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, or 7-8.

[0187] In some embodiments, L is selected from the following structures:

[0188] In some embodiments, the immunostimulatory antibody conjugate is selected from:

[0189] z is independently selected from 1-8, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, or 7-8.

[0190] In another aspect, the present application provides a composition comprising the immunostimulatory antibody conjugate described in any one of the foregoing or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof.

[0191] The composition can comprise a plurality of immunostimulatory antibody conjugates as described herein, wherein each immunostimulatory antibody conjugate comprises a DL as described herein, wherein z is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 DLs. Thus, the composition can be characterized by a "drug-antibody" ratio (DAR) ranging from about 1 to about 10. Methods for determining DAR are well known to those skilled in the art and include methods using reverse phase chromatography or HPLC-MS. For example, in some embodiments, the DAR value of the composition is an integer or decimal selected from 1-8, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8 or 7- 8. For another example, in some embodiments, the DAR value of the pharmaceutical composition is 7.0-8.0, such as 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0192] Preparation method

[0193] In another aspect, the present application provides a method for preparing the compound of any one of the first aspects. In particular, the present application provides a method for preparing a compound of formula (II-A), comprising the following steps:

[0194] Among them, R 1 and R 2 As defined above.

[0195] (1) reacting compound II-A-1 with compound II-A-2 to obtain compound II-A-3;

[0196] The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, 1,4-dioxane, tetrahydrofuran, and any combination thereof, preferably tetrahydrofuran. The reaction is preferably carried out in the presence of a suitable organic base, which may be selected from N,N-diisopropylethylamine, triethylamine, 4-dimethylaminopyridine, and any combination thereof, preferably N,N-diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably -10-80°C. The reaction is preferably carried out for a suitable time, for example, 2-24 hours.

[0197] (2) reacting compound II-A-3 with N,N-dimethylformamide dimethyl acetal to obtain compound II-A-4;

[0198] The reaction is preferably carried out in a suitable organic solvent. The organic solvent can be selected from N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out at a suitable temperature, preferably 50-180°C. The reaction is preferably carried out for a suitable time, for example, 2-12 hours.

[0199] (3) subjecting compound II-A-4 to a ring-closure reaction with compound II-A-5 to obtain compound II-A-6;

[0200] The reaction is preferably carried out in a suitable organic solvent. The organic solvent can be selected from methanol, ethanol, isopropanol, n-butanol, tetrahydrofuran, and any combination thereof, preferably ethanol. The reaction is preferably carried out at a suitable temperature, preferably 20-100° C. The reaction is preferably carried out for a suitable time, for example, 2-24 hours.

[0201] (4) subjecting compound II-A-6 to an oxidation reaction to obtain compound II-A-7;

[0202] The reaction is preferably carried out in a suitable organic solvent. The organic solvent can be selected from dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, and any combination thereof, preferably dichloromethane. The reaction is preferably carried out in the presence of a suitable oxidant, which can be selected from meta-chloroperbenzoic acid, hydrogen peroxide, tert-butyl hydroperoxide, and any combination thereof, preferably meta-chloroperbenzoic acid. The reaction is preferably carried out at a suitable temperature, preferably -10-80°C. The reaction is preferably carried out for a suitable time, for example, 1-12 hours.

[0203] (5) reacting compound II-A-7 with an ammonia solution to obtain compound II-A;

[0204] The reaction is preferably carried out in a suitable solvent. The solvent can be selected from tetrahydrofuran, 1,4-dioxane, water, and any combination thereof, preferably 1,4-dioxane. The reaction is preferably carried out at a suitable temperature, preferably 50-150°C. The reaction is preferably carried out for a suitable time, for example, 2-24 hours.

[0205] In particular, the present application also provides another method for preparing a compound of formula (II-A), which comprises the following steps:

[0206] Among them, R 1 and R 2 As defined above;

[0207] LG represents a leaving group, which includes but is not limited to a halogen atom, a methanesulfonyloxy group, a p-toluenesulfonyloxy group, and the like.

[0208] (1) subjecting compound II-A-4 to a ring-closure reaction with an ammonia solution to obtain compound II-A-8;

[0209] The reaction is preferably carried out in a suitable organic solvent. The organic solvent can be selected from methanol, ethanol, isopropanol, n-butanol, tetrahydrofuran, and any combination thereof, preferably methanol. The reaction is preferably carried out at a suitable temperature, preferably 20-100° C. The reaction is preferably carried out for a suitable time, for example, 2-24 hours.

[0210] (2) subjecting compound II-A-8 to an oxidation reaction to obtain compound II-A-9;

[0211] The reaction is preferably carried out in a suitable organic solvent. The organic solvent can be selected from dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, and any combination thereof, preferably dichloromethane. The reaction is preferably carried out in the presence of a suitable oxidant, which can be selected from meta-chloroperbenzoic acid, hydrogen peroxide, tert-butyl hydroperoxide, and any combination thereof, preferably meta-chloroperbenzoic acid. The reaction is preferably carried out at a suitable temperature, preferably -10-80°C. The reaction is preferably carried out for a suitable time, for example, 1-12 hours.

[0212] (3) reacting compound II-A-9 with an ammonia solution to obtain compound II-A-10;

[0213] The reaction is preferably carried out in a suitable solvent. The solvent can be selected from tetrahydrofuran, 1,4-dioxane, water, and any combination thereof, preferably 1,4-dioxane. The reaction is preferably carried out at a suitable temperature, preferably 50-150°C. The reaction is preferably carried out for a suitable time, for example, 2-24 hours.

[0214] (4) reacting compound II-A-10 with compound II-A-11 to obtain compound II-A;

[0215] The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetone, and any combination thereof, preferably N,N-dimethylformamide. The reaction is preferably carried out in the presence of a suitable organic base or inorganic base, which may be selected from N,N-diisopropylethylamine, triethylamine, 4-dimethylaminopyridine, sodium carbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide, and any combination thereof, preferably cesium carbonate. The reaction is preferably carried out at a suitable temperature, preferably 20-100° C. The reaction is preferably carried out for a suitable time, for example 2-12 hours.

[0216] The specific conditions of each of the above reaction steps are well known in the art and are not specifically limited herein. Based on the teachings of this application and common knowledge in the art, those skilled in the art can select and replace the substituents in the general formula to prepare different compounds, and such selections and replacements are within the scope of protection of the present invention.

[0217] Pharmaceutical compositions and kits

[0218] In another aspect, the present application provides a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound as described above or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, the immunostimulatory antibody conjugate as described above, or the composition as described above, and one or more pharmaceutically acceptable carriers.

[0219] In another aspect, the present application provides a kit comprising a compound described in any of the foregoing, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof, an immunostimulatory antibody conjugate described in any of the foregoing, a composition described in any of the foregoing, or a pharmaceutical composition described in any of the foregoing. Optionally, the kit further comprises instructions for use.

[0220] As used herein, the term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and / or other animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0221] Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition include, but are not limited to, sterile liquids. The pharmaceutical composition can be in the form of, for example, a solid preparation, a semisolid preparation, a liquid preparation, or a gaseous preparation.

[0222] The pharmaceutical compositions can act systemically and / or locally. For this purpose, they can be administered by a suitable route, for example by injection or percutaneous administration; or by oral administration or by inhalation.

[0223] The content or dosage of the compound in the pharmaceutical composition may be about 0.001 mg to about 1000 mg, suitably 0.01-800 mg, preferably 0.05-500 mg.

[0224] In some embodiments, the present application provides a method for preparing the pharmaceutical composition, comprising combining the compound or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug with one or more pharmaceutically acceptable carriers.

[0225] Treatment methods and uses

[0226] The compound described herein or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug, or the pharmaceutical composition has a strong agonistic effect on the target TLR7 and / or TLR8.

[0227] Therefore, another object of the present application is to provide the use of the compound described herein in the preparation of an immunostimulatory antibody conjugate. In some embodiments, the immunostimulatory antibody conjugate comprises a small molecule ligand capable of interacting with TLR7 and / or TLR8.

[0228] Another object of the present application is to provide the compound or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug, the immunostimulatory antibody conjugate, the composition, or the use of the pharmaceutical composition in the preparation of a medicament, which is used to prevent and / or treat TLR7 and / or TLR8-mediated related diseases.

[0229] Another object of the present application is to provide the compound or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug, the immunostimulatory antibody conjugate, the composition, or the pharmaceutical composition, which is used to prevent and / or treat TLR7 and / or TLR8-mediated related diseases.

[0230] Another object of the present application is to provide a method for preventing and / or treating TLR7 and / or TLR8-mediated related diseases, which comprises administering to an individual in need thereof a preventive or therapeutically effective amount of the compound or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug, the immunostimulatory antibody conjugate, the composition or pharmaceutical composition.

[0231] In some embodiments, the TLR7 and / or TLR8-mediated related disease is preferably a tumor.

[0232] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired prophylactic or therapeutic effect, for example, to achieve relief of one or more symptoms associated with the disease being treated.

[0233] The dosage regimen can be adjusted to provide the optimal desired response. For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It is to be noted that dosage values ​​can vary with the type and severity of the condition to be alleviated and can include single or multiple doses. It is to be further understood that for any particular individual, the specific dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the compound.

[0234] The amount of the compound administered will depend on the individual being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound, and the judgment of the prescribing physician. In some cases, dosage levels no higher than the lower limit of the aforementioned range may be sufficient, while in other cases, larger doses may still be employed without causing any adverse side effects, provided that the larger dose is first divided into several smaller doses to be administered throughout the day.

[0235] As used herein, unless otherwise indicated, the terms "treat," ...

[0236] The term "prevention" refers to inhibiting and delaying the onset of a disease, and includes not only prevention before the development of a disease but also prevention of recurrence of a disease after treatment.

[0237] As used herein, "subject" includes humans and non-human animals. Exemplary human subjects include human subjects suffering from diseases (e.g., diseases described herein) (referred to as patients) or normal individuals. "Non-human animals" herein include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0238] The ones used in this article When it is connected to either end of a double bond, it indicates that the double bond configuration is cis or trans. When it appears in the form of The key of the position is the connection key. DETAILED DESCRIPTION

[0239] In order to make the purpose and technical scheme of the present invention clearer, the embodiments of the present invention are described in detail below in conjunction with embodiment.But those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.Unindicated specific conditions in the examples are all carried out according to the conditions of normal conditions or manufacturer's advice.Reagents used or instruments not indicated by manufacturer are all conventional products that can be obtained commercially.

[0240] The structures of the compounds were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS). 1 H NMR measurements were performed on a JEOL Eclipse 400 nuclear magnetometer. The solvents used were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6). The internal standard was tetramethylsilane (TMS). Chemical shifts (δ) were given in parts per million (ppm).

[0241] The MS measuring instrument is an Agilent (ESI) mass spectrometer, manufactured by Agilent, model: Agilent 6120B.

[0242] Preparation method for preparative high performance liquid chromatography:

[0243] Instrument model: Agilent 1260, chromatographic column: Waters SunFire Prep C18 OBD (19 mm × 150 mm × 5.0 μm); column temperature: 25°C; flow rate: 20.0 mL / min; detection wavelength: 214 nm; elution gradient: (0 min: 10% A, 90% B; 16.0 min: 90% A, 10% B); mobile phase A: acetonitrile; mobile phase B: 0.05% formic acid in water.

[0244] Aluminum plates (20×20 cm) produced by Merck were used for thin layer chromatography silica gel plates (TLC), and the specifications used for thin layer chromatography separation and purification were GF 254 (1 mm) produced in Yantai.

[0245] The reaction is monitored by thin layer chromatography (TLC) or LC-MS; the developing solvent systems used include: dichloromethane and methanol system, n-hexane and ethyl acetate system, and petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound or by adding triethylamine.

[0246] Microwave reaction was carried out using Biotage Initiator+ (400W, RT-300°C) microwave reactor.

[0247] Column chromatography generally uses 200-300 mesh silica gel as a carrier. Eluent systems include: dichloromethane and methanol systems, and petroleum ether and ethyl acetate systems. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine can also be added for adjustment.

[0248] Preparation method of reverse phase column chromatography:

[0249] Instrument model: Biotage fast medium-pressure preparative chromatography, chromatographic column: Agela C18 reversed-phase column (Spherical; 20-35 μm; 100 A); chromatographic column temperature: 25°C; flow rate: 28.0 mL / min; detection wavelength: 220 nm; mobile phase A: acetonitrile; mobile phase B: water.

[0250] Unless otherwise specified in the examples, the reaction temperature is room temperature (20°C to 35°C);

[0251] The reagents used in this article were purchased from Acros Organics, Aldrich Chemical Company, Teber Chemical and other companies.

[0252] In general synthesis methods, examples, and intermediate synthesis examples, the meanings of the abbreviations are as follows.

[0253] 1. Compound Examples

[0254] Intermediate Preparation Example 1: Preparation of 2-amino-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one

[0255] Step 1: Preparation of ethyl 4-(butylamino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate

[0256] Ethyl 6-chloro-4-methyl-2-(methylthio)pyrimidine-5-carboxylate (12 g, 48.15 mmol), N,N-diisopropylethylamine (12.6 g, 96.31 mmol), and n-butylamine (5.34 g, 72.23 mmol) were dissolved in tetrahydrofuran (200 mL) and stirred at room temperature for 12 hours. Water (1 L) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (200 mL). The organic phases were combined and washed sequentially with dilute hydrochloric acid (1N, 100 mL) and saturated brine. The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the title compound (13.3 g, yield: 97.4%).

[0257] MS m / z(ESI):284.2[M+H] + .

[0258] Step 2: Preparation of ethyl 4-(butylamino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylate

[0259] Ethyl 4-(butylamino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate (11 g, 38.8 mmol) and N,N-dimethylformamide dimethyl acetal (23 g, 194 mmol) were dissolved in N,N-dimethylformamide (100 mL), heated to 150°C, and stirred for 4 hours. After cooling the reaction system to room temperature, water (500 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (200 mL). The organic phases were combined and washed three times with saturated brine. The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the title compound (11 g, yield: 83.8%).

[0260] MS m / z(ESI):339.2[M+H] + .

[0261] Step 3: Preparation of 4-(butylamino)-2-(methylthio)pyrido[4,3-d]pyrimidin-5(6H)-one

[0262] Ethyl 4-(butylamino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylate (11 g, 32.5 mmol), methanolic ammonia solution (21 mL, 7 M), and ethanol (100 mL) were added to an autoclave and heated to 80°C with stirring for 12 hours. The reaction system was concentrated to dryness, and the resulting residue was purified by slurrying with methyl tert-butyl ether to obtain the title compound (7.8 g, yield: 90.5%).

[0263] MS m / z(ESI):265.1[M+H] + .

[0264] Step 4: Preparation of 4-(butylamino)-2-(methylsulfinyl)pyrido[4,3-d]pyrimidin-5(6H)-one

[0265] Dissolve 4-(Butylamino)-2-(methylthio)pyrido[4,3-d]pyrimidin-5(6H)-one (7.8 g, 29.3 mmol) in dichloromethane (80 mL). Cool in an ice-water bath. Add m-chloroperbenzoic acid (8.95 g, 44.2 mmol) slowly in portions at 0°C. Slowly warm to room temperature and stir for 4 hours. Pour the reaction solution into water (200 mL) and extract three times with dichloromethane (100 mL). Combine the organic phases, wash twice with saturated aqueous sodium bicarbonate (100 mL), twice with saturated aqueous sodium sulfite (100 mL), and three times with saturated brine (50 mL). Dry the organic phase over anhydrous sodium sulfate and concentrate to obtain the title compound (8.3 g, yield: 100%).

[0266] MS m / z(ESI):281.0[M+H] + .

[0267] Step 5: Preparation of 4-(butylamino)-2-((2,4-dimethoxybenzyl)amino)pyrido[4,3-d]pyrimidin-5(6H)-one

[0268] 4-(Butylamino)-2-(methylsulfinyl)pyrido[4,3-d]pyrimidin-5(6H)-one (8.3 g, 29.3 mmol) and 2,4-dimethoxybenzylamine (5.9 g, 35.2 mmol) were dissolved in N,N-dimethylformamide (100 mL), heated to 80°C, and stirred for 12 hours. After cooling the reaction system to room temperature, water (500 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (100 mL). The organic phases were combined and washed three times with saturated brine. The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the crude title compound (12 g, yield: 100%).

[0269] MS m / z(ESI):384.2[M+H] + .

[0270] Step 6: Preparation of 2-amino-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one

[0271] 4-(Butylamino)-2-((2,4-dimethoxybenzyl)amino)pyrido[4,3-d]pyrimidin-5(6H)-one (12 g, 29.3 mmol) was dissolved in trifluoroacetic acid (60 mL) and stirred at room temperature for 12 hours. The reaction system was concentrated to dryness, and saturated aqueous sodium bicarbonate (100 mL) was slowly added to the reaction system. A large amount of solid precipitated, which was filtered and the filter cake washed with water. The filter cake was dried and purified by slurrying with methyl tert-butyl ether to obtain the title compound (5.6 g, yield: 81.7%).

[0272] MS m / z(ESI):234.2[M+H] + .

[0273] Intermediate Preparation Example 2: Preparation of (6-(chloromethyl)pyridin-3-yl)methanol

[0274] Methyl 6-(chloromethyl)nicotinate (1 g, 5.39 mmol) was dissolved in dry tetrahydrofuran (3 mL), cooled to 0°C, and a solution of lithium aluminum hydride (408.93 mg, 10.78 mmol) in tetrahydrofuran (3 mL) was added dropwise. The mixture was stirred at 0°C for 0.5 hours. Ice water (2 mL) and a 15% aqueous sodium hydroxide solution (0.5 mL) were added to the reaction system, and the mixture was extracted three times with dichloromethane (30 mL). The organic phases were combined and washed twice with saturated brine (30 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the title compound (200 mg, yield: 23.6%).

[0275] MS m / z(ESI):158.0[M+H] + .

[0276] Example 1: Preparation of 2-amino-4-(butylamino)-6-methyl-5,6-dihydropyrido[4,3-d]pyrimidin-5-one (Compound 1)

[0277] Step 1: Preparation of ethyl 4-(butylamino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate

[0278] Ethyl 6-chloro-4-methyl-2-(methylthio)pyrimidine-5-carboxylate (1.0 g, 4.05 mmol), N,N-diisopropylethylamine (0.78 g, 6.02 mmol), and n-butylamine (0.44 g, 6.02 mmol) were dissolved in tetrahydrofuran (20 mL) and stirred at room temperature for 12 hours. Water (150 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (60 mL). The organic phases were combined and washed sequentially with dilute hydrochloric acid (1N, 15 mL) and saturated brine (15 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the title compound (1.1 g, yield: 95.8%).

[0279] MS m / z(ESI):284.2[M+H] + .

[0280] Step 2: Preparation of ethyl 4-(butylamino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylate

[0281] Ethyl 4-(butylamino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate (1.1 g, 3.88 mmol) and N,N-dimethylformamide dimethyl acetal (4.62 g, 38.8 mmol) were dissolved in N,N-dimethylformamide (10 mL), heated to 150°C, and stirred for 4 hours. After cooling the reaction system to room temperature, water (100 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (60 mL). The organic phases were combined and washed three times with saturated brine (45 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the title compound (1.1 g, yield: 83.8%).

[0282] MS m / z(ESI):339.2[M+H] + .

[0283] Step 3: Preparation of 4-(butylamino)-6-methyl-2-(methylthio)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one

[0284] Ethyl 4-(butylamino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylate (0.5 g, 1.48 mmol) and 30% ethanolic methylamine solution (4 mL) were dissolved in ethanol (10 mL), heated to 80°C, and stirred for 12 hours. After cooling the reaction system to room temperature, water (100 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (60 mL). The organic phases were combined and washed three times with saturated brine (45 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by slurrying with methyl tert-butyl ether (5 mL) to obtain the title compound (0.25 g, yield: 60.7%).

[0285] MS m / z(ESI):279.1[M+H] + .

[0286] Step 4: Preparation of 4-(butylamino)-6-methyl-2-(methylsulfinyl)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one

[0287] Dissolve 4-(Butylamino)-6-methyl-2-(methylthio)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one (0.1 g, 0.36 mmol) in dichloromethane (6 mL). Cool in an ice-water bath. Add m-chloroperbenzoic acid (93.0 mg, 0.54 mmol) slowly in portions at 0°C. Slowly warm to room temperature and stir for 4 hours. Pour the reaction solution into water (50 mL) and extract three times with dichloromethane (30 mL). Combine the organic phases, wash twice with saturated aqueous sodium bicarbonate (20 mL), twice with saturated aqueous sodium sulfite (20 mL), and three times with saturated brine (30 mL). Dry the organic phase over anhydrous sodium sulfate and concentrate to obtain the title compound (0.1 g, yield: 94.4%).

[0288] MS m / z(ESI):295.1[M+H] + .

[0289] Step 5: Preparation of 2-amino-4-(butylamino)-6-methyl-5,6-dihydropyrido[4,3-d]pyrimidin-5-one (Compound 1)

[0290] 4-(Butylamino)-6-methyl-2-(methylsulfinyl)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one (0.1 g, 0.34 mmol) was dissolved in a 0.5 N ammonia solution in 1,4-dioxane (5 mL), heated to 100°C, and stirred for 12 hours. After cooling the reaction system to room temperature, water (60 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (40 mL). The organic phases were combined and washed three times with saturated brine (30 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by preparative high-performance liquid chromatography to obtain the title compound (20 mg, yield: 23.8%).

[0291] MS m / z(ESI):248.1[M+H] + .

[0292] 1H NMR (400MHz, CD3OD): δ7.49(d,J=7.6Hz,1H),6.15(d,J=7.6Hz,1H),3.53-3.47( m,2H),3.46(s,3H),1.65-1.61(m,2H),1.48-1.42(m,2H),0.98(t,J=7.6Hz,3H).

[0293] Example 2: Preparation of 2-amino-4-((1,3-dimethoxypropane-2-yl)amino)-6-methyl-5,6-dihydropyrido[4,3-d]pyrimidin-5-one (Compound 2)

[0294] Step 1: Preparation of ethyl 4-((1,3-dimethoxypropane-2-yl)amino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate

[0295] The synthetic route of Example 1 was adopted, and the raw material in the first step, n-butylamine, was replaced with 2-amino-1,3-dimethoxypropane to obtain the title compound (1.4 g, yield: 89.3%).

[0296] MS m / z(ESI):330.1[M+H] + .

[0297] Step 2: Preparation of ethyl 4-((1,3-dimethoxypropane-2-yl)amino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylate

[0298] Using the synthetic route of Example 1, the second-step reaction raw material 4-(butylamino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester was replaced with 4-((1,3-dimethoxypropane-2-yl)amino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester to obtain the title compound (1.2 g, yield: 80.9%).

[0299] MS m / z(ESI):385.1[M+H] + .

[0300] Step 3: Preparation of 4-((1,3-dimethoxypropane-2-yl)amino)-6-methyl-2-(methylthio)-5,6-dihydropyridin[4,3-d]pyrimidin-5-one

[0301] Using the synthetic route of Example 1, the third step reaction raw material 4-(butylamino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester was replaced with 4-((1,3-dimethoxypropane-2-yl)amino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester to obtain the title compound (0.7 g, yield: 65.4%).

[0302] MS m / z(ESI):325.1[M+H] + .

[0303] Step 4: Preparation of 4-((1,3-dimethoxypropane-2-yl)amino)-6-methyl-2-(methylsulfinyl)-5,6-dihydropyridin[4,3-d]pyrimidin-5-one

[0304] The synthetic route of Example 1 was used to replace the raw material of the fourth step reaction, 4-(butylamino)-6-methyl-2-(methylthio)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one, with 4-((1,3-dimethoxypropan-2-yl)amino)-6-methyl-2-(methylthio)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one to give the title compound (45 mg, yield: 47.8%).

[0305] MS m / z(ESI):341.1[M+H] + .

[0306] Step 5: Preparation of 2-amino-4-((1,3-dimethoxypropane-2-yl)amino)-6-methyl-5,6-dihydropyridin[4,3-d]pyrimidin-5-one (Compound 2)

[0307] Using the synthetic route of Example 1, the raw material of the fifth step reaction, 4-(butylamino)-6-methyl-2-(methylsulfinyl)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one, was replaced with 4-((1,3-dimethoxypropan-2-yl)amino)-6-methyl-2-(methylsulfinyl)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one to give the title compound (6 mg, yield: 20.1%).

[0308] MS m / z(ESI):294.2[M+H] + .

[0309] 1H NMR (400MHz, CD3OD): δ7.54 (d, J = 7.6 Hz, 1H), 6.18 (d, J = 7.6 Hz, 1H), 3.63-3.50 (m, 5H), 3.46 (s, 3H), 3.39 (s, 6H).

[0310] Example 3: Preparation of 2-amino-4-((1,3-dihydroxypropane-2-yl)amino)-6-methyl-5,6-dihydropyrido[4,3-d]pyrimidin-5-one (Compound 3)

[0311] Step 1: Preparation of ethyl 4-((1,3-dihydroxypropane-2-yl)amino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate

[0312] The synthetic route of Example 1 was adopted, and the raw material in the first step, n-butylamine, was replaced with 2-amino-1,3-propanediol to obtain the title compound (0.54 g, yield: 83.6%).

[0313] MS m / z(ESI):302.0[M+H] + .

[0314] Step 2: Preparation of ethyl 4-((1,3-dihydroxypropane-2-yl)amino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylate

[0315] Using the synthetic route of Example 1, the second-step reaction raw material 4-(butylamino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester was replaced with 4-((1,3-dihydroxypropane-2-yl)amino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester to obtain the title compound (0.75 g, yield: 82.3%).

[0316] MS m / z(ESI):357.1[M+H] + .

[0317] Step 3: Preparation of 4-((1,3-dihydroxypropane-2-yl)amino)-6-methyl-2-(methylthio)-5,6-dihydropyridin[4,3-d]pyrimidin-5-one

[0318] Using the synthetic route of Example 1, the third step reaction raw material 4-(butylamino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester was replaced with 4-((1,3-dihydroxypropane-2-yl)amino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester to obtain the title compound (0.14 g, yield: 58.8%).

[0319] MS m / z(ESI):297.0[M+H] + .

[0320] Step 4: Preparation of 4-((1,3-dihydroxypropane-2-yl)amino)-6-methyl-2-(methylsulfinyl)-5,6-dihydropyridin[4,3-d]pyrimidin-5-one

[0321] Using the synthetic route of Example 1, the starting material of the fourth step reaction, 4-(butylamino)-6-methyl-2-(methylthio)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one, was replaced with 4-((1,3-dihydroxypropane-2-yl)amino)-6-methyl-2-(methylthio)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one to give the title compound (30 mg, yield: 44.5%).

[0322] MS m / z(ESI):313.0[M+H] + .

[0323] Step 5: Preparation of 2-amino-4-((1,3-dihydroxypropane-2-yl)amino)-6-methyl-5,6-dihydropyrido[4,3-d]pyrimidin-5-one (Compound 3)

[0324] The synthetic route of Example 1 was used to replace the raw material of the fifth step reaction, 4-(butylamino)-6-methyl-2-(methylsulfinyl)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one, with 4-((1,3-dihydroxypropane-2-yl)amino)-6-methyl-2-(methylsulfinyl)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one to give the title compound (5 mg, yield: 26.6%).

[0325] MS m / z(ESI):266.1[M+H] + .

[0326] 1 H NMR (400MHz, DMSO-d6): δ9.61-9.59(m,1H),7.57(d,J=7.6Hz,1H),6.53(s,2H),6.0 1(d,J=7.6Hz,1H),4.79(s,2H),4.18-4.16(m,1H),3.59-3.47(m,4H),3.41(s,3H).

[0327] Example 4: Preparation of 2-amino-4-butylamino)-6-(4-((dimethylamino)methyl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 4)

[0328] 2-Amino-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one (18.4 mg, 0.079 mmol), cesium carbonate (39.1 mg, 0.12 mmol), and 1-(4-(chloromethyl)phenyl)-N,N-dimethylmethanamine (16.5 mg, 0.09 mmol) were added to N,N-dimethylformamide (2 mL), and the mixture was heated to 50°C and stirred for 8 hours. After cooling the reaction system to room temperature, water (60 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (30 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by preparative high-performance liquid chromatography to obtain the title compound (12.2 mg, yield: 40.6%).

[0329] MS m / z(ESI):381.1[M+H] + .

[0330] 1 H NMR (400MHz, CD3OD): δ7.57(d,J=7.6Hz,1H),7.33-7.28(m,4H),6.18(d,J=7.6Hz, 1H),5.10(s,2H),3.52-3.49(m,4H),2.27(s,6H),1.68-1.59(m,2H),1.47-1.42(m, 2H), 0.98 (t, J = 7.6Hz, 3H).

[0331] Example 5: Preparation of 2-amino-4-(butylamino)-6-(4-((dimethylamino)methyl)-2-methoxybenzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 5)

[0332] Step 1: Preparation of methyl 4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)-3-methoxybenzoate

[0333] 2-Amino-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one (200 mg, 0.85 mmol), potassium carbonate (240 mg, 1.7 mmol), potassium iodide (13 mg, 0.08 mmol), and methyl 4-(bromomethyl)-3-methoxybenzoate (266 mg, 1.02 mmol) were added to N,N-dimethylformamide (6 mL) and stirred at 60°C for 8 hours. After cooling the reaction system to room temperature, water (50 mL) was added to the reaction system and extracted three times with dichloromethane (30 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, volume ratio) to obtain the title compound (150 mg, yield: 42.5%).

[0334] MS m / z(ESI):412.2[M+H] + .

[0335] Step 2: Preparation of 2-amino-4-(butylamino)-6-(4-(hydroxymethyl)-2-methoxybenzyl)pyrido[4,3-d]pyrimidin-5(6H)-one

[0336] Methyl 4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)-3-methoxybenzoate (115 mg, 0.28 mmol) was added to tetrahydrofuran (3 mL) and anhydrous ethanol (3 mL). Sodium borohydride (32 mg, 0.85 mmol) and anhydrous calcium chloride (31 mg, 0.28 mmol) were then added, and the mixture was heated to 60°C and stirred for 6 hours. After cooling the reaction system to room temperature, saturated aqueous ammonium chloride (50 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (30 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, volume ratio) to obtain the title compound (60 mg, yield: 55.7%).

[0337] MS m / z(ESI):384.2[M+H] + .

[0338] Step 3: Preparation of 4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)-3-methoxybenzyl methanesulfonate

[0339] 2-Amino-4-(butylamino)-6-(4-(hydroxymethyl)-2-methoxybenzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (60 mg, 0.16 mmol) and triethylamine (32 mg, 0.32 mmol) were added sequentially to dichloromethane (3 mL). Methanesulfonyl chloride (24 mg, 0.21 mmol) was added dropwise under ice-water cooling. The reaction mixture was stirred at room temperature for 12 hours. Saturated aqueous ammonium chloride (50 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (30 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the crude title compound (70 mg, yield: 94.5%).

[0340] MS m / z(ESI):462.2[M+H] + .

[0341] Step 4: Preparation of 2-amino-4-(butylamino)-6-(4-((dimethylamino)methyl)-2-methoxybenzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 5)

[0342] 4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)-3-methoxybenzyl methanesulfonate (70 mg, 0.15 mmol), cesium carbonate (100 mg, 0.30 mmol), and dimethylamine hydrochloride (18 mg, 0.23 mmol) were added to N,N-dimethylformamide (2 mL), heated to 50°C, and stirred for 8 hours. After cooling the reaction system to room temperature, water (60 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (30 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by preparative high-performance liquid chromatography to obtain the title compound (10 mg, yield: 16.1%).

[0343] MS m / z(ESI):411.2[M+H] + .

[0344] 1H NMR (400MHz, CD3OD) δ7.67(d,J=8.0Hz,1H),7.26(d,J=8.0Hz,1H),7.12(s,1H),7.00(d,J=8.0Hz,1H),6.21(d,J=8.0Hz,1H),5.0 8(s,2H),4.12(s,2H),3.92(s,3H),3.56-3.47(m,2H),2.71(s,6H),1.66-1.59(m,2H),1.46-1.38(m,2H),0.96(t,J=8.0Hz,3H).

[0345] Example 6: Preparation of 2-amino-4-(butylamino)-6-(4-(piperazin-1-ylmethyl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 6)

[0346] Step 1: Preparation of tert-butyl 4-(4-(chloromethyl)benzyl)piperazine-1-carboxylate

[0347] Dissolve 1,4-bis(chloromethyl)benzene (1 g, 5.75 mmol), N,N-diisopropylethylamine (1.1 g, 8.62 mmol), and tert-butyl piperazine-1-carboxylate (1.07 g, 5.75 mmol) in N,N-dimethylformamide (10 mL), slowly warm to 60°C, and stir for 6 hours. Pour the reaction solution into water (100 mL) and extract three times with dichloromethane (50 mL). The organic phases are combined and washed three times with saturated brine. The organic phases are dried over anhydrous sodium sulfate and concentrated. The resulting residue is purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, volume ratio) to obtain the title compound (1 g, yield: 53.8%).

[0348] MS m / z(ESI):325.2[M+H] + .

[0349] Step 2: Preparation of tert-butyl 4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazine-1-carboxylate

[0350] The synthetic method of the first step of Example 5 was used to replace the reaction raw material 4-(bromomethyl)-3-methoxybenzoic acid methyl ester with 4-(4-(chloromethyl)benzyl)piperazine-1-carboxylic acid tert-butyl ester to obtain the title compound (0.3 g, yield: 58.6%).

[0351] MS m / z(ESI):522.3[M+H] + .

[0352] Step 3: Preparation of 2-amino-4-(butylamino)-6-(4-(piperazin-1-ylmethyl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 6)

[0353] Tert-butyl 4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazine-1-carboxylate (300 mg, 0.57 mmol) was dissolved in a mixture of methanol (4 mL) and a 4N solution of hydrogen chloride in 1,4-dioxane (2 mL) and stirred at room temperature for 12 hours. After completion of the reaction, the reaction mixture was concentrated, and the resulting residue was purified by reverse-phase column chromatography (elution gradient: A% = 5% - 15%) to obtain the title compound (0.21 g, yield: 87.5%).

[0354] MS m / z(ESI):422.3[M+H] + .

[0355] 1 H NMR (400MHz, CD3OD): δ7.82-7.79(m,1H),7.35-7.31(m,4H),6.30-6.27(m,1H),5.14(s,2H),3.59-3.53( m,4H),3.19-3.17(m,4H),2.66-2.65(m,4H),1.66-1.63(m,2H),1.46-1.43(m,2H),0.96(t,J=8.0Hz,3H).

[0356] Example 7: Preparation of 2-amino-4-(butylamino)-6-(4-((4-methylpiperazin-1-yl)methyl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 7)

[0357] 2-Amino-4-(butylamino)-6-(4-(piperazin-1-ylmethyl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (50 mg, 0.12 mmol) and formaldehyde (30% aqueous solution, 15 mg, 0.15 mmol) were added sequentially to methanol (4 mL), followed by sodium cyanoborohydride (22 mg, 0.36 mmol). The reaction system was stirred at room temperature for 2 hours. The reaction solution was poured into water (50 mL) and extracted three times with dichloromethane (20 mL). The organic phases were combined and washed three times with saturated brine (15 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by preparative HPLC to obtain the title compound (10 mg, yield: 19.4%).

[0358] MS m / z(ESI):436.3[M+H] + .

[0359] 1 H NMR (400MHz, CD3OD): δ7.63(d,J=8.0Hz,1H),7.34-7.29(m,4H),6.20(d,J=8.0Hz,1H),5.11(s,2H),3.60(s,2H),3. 53-3.49(m,2H),3.12-2.64(m,4H),2.60-2.48(m,7H),1.66-1.63(m,2H),1.47-1.43(m,2H),0.96(t,J=8.0Hz,3H).

[0360] Example 8: Preparation of 2-amino-4-(butylamino)-6-(4-(piperazin-1-yl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 8)

[0361] Step 1: Preparation of 2-amino-6-(4-bromobenzyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one

[0362] The synthetic method of the first step of Example 5 was used to replace the reaction raw material 4-(bromomethyl)-3-methoxybenzoic acid methyl ester with 1-bromo-4-(bromomethyl)benzene to obtain the title compound (250 mg, yield: 58.2%).

[0363] MS m / z(ESI):402.1[M+H] + .

[0364] Step 2: Preparation of tert-butyl 4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)phenyl)piperazine-1-carboxylate

[0365] 2-Amino-6-(4-bromobenzyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one (50 mg, 0.12 mmol), tert-butyl piperazine-1-carboxylate (46 mg, 0.24 mmol), 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (11 mg, 0.02 mmol) and sodium tert-butoxide (24 mg, 0.24 mmol) were added to a mixed solvent of N,N-dimethylformamide (2 mL) and toluene (4 mL). After nitrogen substitution, tris(dibenzylideneacetone)dipalladium (22 mg, 0.02 mmol) was added, and the temperature was raised to 110°C and stirred for 12 hours. After the reaction system was cooled to room temperature, water (50 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (30 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The obtained residue was purified by preparative high performance liquid chromatography to give the title compound (25 mg, yield: 39.7%).

[0366] MS m / z(ESI):508.3[M+H] + .

[0367] Step 3: Preparation of 2-amino-4-(butylamino)-6-(4-(piperazin-1-yl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 8)

[0368] The synthesis method of the third step of Example 6 was used to replace the reaction raw material 4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazine-1-carboxylic acid tert-butyl ester with 4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)phenyl)piperazine-1-carboxylic acid tert-butyl ester to give the title compound (16 mg, yield: 80%).

[0369] MS m / z(ESI):408.2[M+H] + .

[0370] 1 H NMR (400MHz, CD3OD) δ8.02(d,J=8.0Hz,1H),7.33(d,J=8.0Hz,2H),7.01(d,J=8.0Hz,2H),6.39(d,J=8.0Hz,1H), 5.13(s,2H),3.65-3.61(m,2H),3.42-3.34(m,8H),1.71-1.63(m,2H),1.47-1.42(m,2H),0.99(t,J=8.0Hz,3H).

[0371] Example 9: Preparation of 2-amino-4-(butylamino)-6-(4-(4-methylpiperazin-1-yl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 9)

[0372] The synthetic method of the second step of Example 8 was adopted, and the reaction raw material piperazine-1-carboxylic acid tert-butyl ester was replaced with N-methylpiperazine to obtain the title compound (6 mg, yield: 10.8%).

[0373] MS m / z(ESI):422.3[M+H] + .

[0374] 1 H NMR (400MHz, CD3OD): δ7.51(d,J=8.0Hz,1H),7.20(d,J=8.0Hz,1H),6.92(d,J=8.0Hz,1H),6.14(d,J=8.0Hz,1H),4.98(s,2H),3.49 -3.46(m,2H),3.29-3.16(m,4H),2.67-2.59(m,4H),2.36-2.32(m,5H),1.65-1.61(m,2H),1.50-1.39(m,2H),0.96(d,J=8.0Hz,3H).

[0375] Example 10: Preparation of 2-amino-4-(butylamino)-6-((5-((dimethylamino)methyl)pyridin-2-yl)methyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 11)

[0376] Step 1: Preparation of 2-amino-4-(butylamino)-6-((5-(hydroxymethyl)pyridin-2-yl)methyl)pyrido[4,3-d]pyrimidin-5(6H)-one

[0377] 2-Amino-4-(butylamino)-pyrido[4,3-d]pyrimidin-5(6H)-one (200 mg, 0.86 mmol), cesium carbonate (560.73 mg, 1.7 mmol), and (6-(chloromethyl)pyridin-3-yl)methanol (148.63 mg, 0.63 mmol) were added to N,N-dimethylformamide (6 mL), heated to 80°C, and stirred for 16 hours. After cooling the reaction system to room temperature, water (60 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (30 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by preparative high-performance liquid chromatography to obtain the title compound (160 mg, yield: 53.2%).

[0378] MS m / z(ESI):355.2[M+H] + .

[0379] Step 2: Preparation of 2-amino-4-(butylamino)-6-((5-(chloromethyl)pyridin-2-yl)methyl)pyrido[4,3-d]pyrimidin-5(6H)-one

[0380] 2-Amino-4-(butylamino)-6-((5-(hydroxymethyl)pyridin-2-yl)methyl)pyrido[4,3-d]pyrimidin-5(6H)-one (160.00 mg, 0.45 mmol) was dissolved in dichloromethane (4 mL), and thionyl chloride (537.10 mg, 4.51 mmol) was added dropwise. The mixture was stirred at room temperature for 4 hours. Water (60 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (30 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, volume ratio) to obtain the title compound (12 mg, yield: 7.1%).

[0381] MS m / z(ESI):373.1[M+H] + .

[0382] Step 3: Preparation of 2-amino-4-(butylamino)-6-((5-((dimethylamino)methyl)pyridin-2-yl)methyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 11)

[0383] 2-Amino-4-(butylamino)-6-((5-(chloromethyl)pyridin-2-yl)methyl)pyrido[4,3-d]pyrimidin-5(6H)-one (12 mg, 32.18 μmol), N,N-dimethylmethanamine (3.94 mg, 48.28 μmol), sodium iodide (0.49 mg, 3.22 μmol), and cesium carbonate (21.26 mg, 64.37 μmol) were added to acetonitrile (2 mL) and reacted at room temperature for 16 hours. The reaction mixture was concentrated to obtain a residue which was purified by preparative HPLC to afford the title compound (5 mg, 40.7% yield).

[0384] MS m / z(ESI):382.2[M+H] + .

[0385] 1H NMR (400MHz, CD3OD) δ9.49(t,J=6.0Hz,1H),8.44(d,J=2.0Hz,1H),7.76(dd,J=8.0,2.0Hz,1H),7.63(d,J=8.0Hz,1H),7.29(d ,J=8.0Hz,1H),6.21(d,J=8.0Hz,1H),5.19(s,2H),3.51-3.45(m,4H),2.24(s,6H),1.66-1.27(m,5H),0.96(t,J=8.0Hz,3H).

[0386] Example 11: Preparation of 2-amino-4-(butylamino)-6-((4-((dimethylamino)methyl)cyclohexyl)methyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 12)

[0387] Step 1: Preparation of 4-(hydroxymethyl)-N,N-dimethylcyclohexane-1-carboxamide

[0388] 4-(Hydroxymethyl)cyclohexane-1-carboxylic acid (0.5 g, 3.13 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (1.56 g, 4.07 mmol), N,N-diisopropylethylamine (1.21 g, 9.4 mmol), and dimethylamine hydrochloride (0.38 g, 4.69 mmol) were added sequentially to N,N-dimethylformamide (8 mL). The reaction mixture was stirred at room temperature for 12 hours. Ethyl acetate (60 mL) was added to the reaction mixture, which was then washed sequentially with 1N sodium hydroxide solution, 1N hydrochloric acid solution, and twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to afford the crude title compound (0.52 g, yield: 89.6%).

[0389] MS m / z(ESI):186.1[M+H] + .

[0390] Step 2: Preparation of (4-((dimethylamino)methyl)cyclohexyl)methanol

[0391] 4-(Hydroxymethyl)-N,N-dimethylcyclohexane-1-carboxamide (250 mg, 1.34 mmol) was dissolved in tetrahydrofuran (10 mL). Lithium aluminum tetrahydride (256 mg, 6.68 mmol) was slowly added under ice-water cooling. The reaction mixture was warmed to 50°C and stirred for 12 hours. Solid sodium sulfate decahydrate (0.2 g) was added to the reaction system and stirred for 20 minutes. The reaction mixture was filtered through a pad of Celite, and the filter cake was washed with ethyl acetate. The organic phases were combined and concentrated to obtain the crude title compound (230 mg, yield: 100%).

[0392] MS m / z(ESI):172.2[M+H] + .

[0393] Step 3: Preparation of 1-(4-(chloromethyl)cyclohexyl)-N,N-dimethylmethanamine

[0394] Dissolve (4-((dimethylamino)methyl)cyclohexyl)methanol (230 mg, 1.34 mmol) in dichloromethane (10 mL) and add thionyl chloride (0.8 g, 6.68 mmol). Warm the reaction mixture to 45°C and stir for 12 hours. The reaction mixture was concentrated to provide the crude title compound (200 mg, 78.7% yield).

[0395] MS m / z(ESI):190.1[M+H] + .

[0396] Step 4: Preparation of 2-amino-4-(butylamino)-6-((4-((dimethylamino)methyl)cyclohexyl)methyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 12)

[0397] The synthetic method of the first step of Example 5 was used to replace the reaction raw material 4-(bromomethyl)-3-methoxybenzoic acid methyl ester with 1-(4-(chloromethyl)cyclohexyl)-N,N-dimethylmethanamine to obtain the title compound (12 mg, yield: 9.6%).

[0398] MS m / z(ESI):387.3[M+H] + .

[0399] 1 H NMR (400MHz, CD3OD): δ7.46(d,J=8.0Hz,1H),6.15(d,J=8.0Hz,1H),3.86(d,J=8.0Hz,2H),3.52- 3.47(m,2H),2.47(d,J=8.0Hz,2H),2.40-2.25(m,6H),1.70-1.31(m,14H),0.98(t,J=8.0Hz,3H).

[0400] Example 12: Preparation of 2-amino-4-(butylamino)-6-(piperidin-4-ylmethyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 13)

[0401] Step 1: Preparation of tert-butyl 4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)piperidine-1-carboxylate

[0402] Using the synthetic method of the first step of Example 10, replacing (6-(chloromethyl)pyridin-3-yl)methanol with tert-butyl 4-(chloromethyl)piperidine-1-carboxylate (551.10 mg, 2.33 mmol) to obtain the title compound (833 mg, yield: 91.2%).

[0403] MS m / z(ESI):431.3[M+H] + .

[0404] Step 2: Preparation of 2-amino-4-(butylamino)-6-(piperidin-4-ylmethyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 13)

[0405] Dissolve tert-butyl 4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)piperidine-1-carboxylate (703 mg, 1.47 mmol) in methanol (10 mL). Add a 4M solution of hydrogen chloride in 1,4-dioxane (11.02 mL). Stir at room temperature for 2 hours. Concentrate the reaction mixture to obtain a residue, which was purified by preparative high-performance liquid chromatography to obtain the title compound (400 mg, 74.2% yield).

[0406] Example 13: Preparation of 2-amino-4-(butylamino)-6-((1-methylpiperidin-4-yl)methyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 14)

[0407] Using the synthetic method of the first step of Example 10, replacing (6-(chloromethyl)pyridin-3-yl)methanol with 4-(chloromethyl)-1-methylpiperidine (69.62 mg, 0.47 mmol) to obtain the title compound (3.5 mg, yield: 2.1%).

[0408] MS m / z(ESI):345.2[M+H] + .

[0409] 1H NMR(400MHz,CD3OD)δ9.73(s,1H),8.54(s,1H),7.62-7.56(m,1H),6.24-6.22(m,1H),4.05-3.85(m,2H),3 .58-3.32(m,5H),3.08-2.97(m,1H),2.95-2.72(m,4H),2.49-2.06(m,2H),1.90-1.75(m,3H),1.67-1.60(m 2H), 1.49-1.40 (m, 2H), 0.98 (t, J = 8.0Hz, 3H).

[0410] Example 14: Preparation of 2-amino-4-(butylamino)-6-((1-(2-(dimethylamino)ethyl)piperidin-4-yl)methyl)pyrido[4,3-d]pyrimidin-5(6H)-one (Compound 15)

[0411] 2-Amino-4-(butylamino)-6-(piperidin-4-ylmethyl)pyrido[4,3-d]pyrimidin-5(6H)-one (190 mg, 466.08 μmol), 2-chloro-N,N-dimethylethanolamine (55.16 mg, 512.69 μmol), sodium iodide (6.99 mg, 46.61 μmol), and cesium carbonate (303.72 mg, 932.17 μmol) were added to N,N-dimethylformamide (4 mL) and stirred at 80°C for 16 hours. Water (30 mL) was added to the reaction system, and the mixture was extracted three times with ethyl acetate (15 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by preparative high-performance liquid chromatography to obtain the title compound (5 mg, yield: 2.7%).

[0412] MS m / z(ESI):402.3[M+H] + .

[0413] 1 H NMR (400MHz, CD3OD) δ9.61(t,J=6.0Hz,1H),7.48(d,J=8.0Hz,1H),6.15(d,J=8.0Hz,1H),3.79(d,J=8.0Hz,2H),3.53-3.46(m,2H),2.96(d,J=1 2.0Hz,2H),2.55-2.46(m,4H),2.27(s,6H),2.04-1.99(m,2H),1.89-1. 83(m,1H),1.68-1.60(m,4H),1.50-1.28(m,5H),0.98(t,J=6.0Hz,3H).

[0414] Example 15: Preparation of 2-amino-4-((1-hydroxy-2-methylhexan-2-yl)amino)-6-methylpyridone[4,3-d]pyrimidin-5(6H)-one (Compound 16)

[0415] Step 1: Preparation of 2-amino-2-methylhexanenitrile

[0416] 2-Hexanone (10 g, 0.1 mol) and ammonium chloride (15.9 g, 0.3 mol) were added to a 7N ammonia-methanol solution (100 mL). Trimethylsilyl cyanide (20 g, 0.2 mol) was slowly added dropwise to the reaction mixture. The reaction system was stirred at room temperature for 72 hours. The reaction solution was concentrated, and ethyl acetate (100 mL) was added to the system. The mixture was filtered through a pad of Celite, and the filtrate was concentrated to obtain the crude title compound (12 g, yield: 86.6%).

[0417] MS m / z(ESI):127.1[M+H] + .

[0418] Step 2: Preparation of 2-amino-2-methylhexanoic acid

[0419] 2-Amino-2-methylhexanenitrile (5 g, 39.7 mmol) was added to concentrated hydrochloric acid (30 mL). The reaction system was heated to 80°C and stirred for 12 hours. The reaction solution was concentrated to obtain the crude hydrochloride of the title compound (6.5 g, yield: 91.7%).

[0420] MS m / z(ESI):146.1[M+H] + .

[0421] Step 3: Preparation of 2-amino-2-methylhexan-1-ol

[0422] 2-Amino-2-methylhexanoic acid (2 g, 10.9 mmol) was added to tetrahydrofuran (20 mL). Under ice-water cooling, lithium aluminum tetrahydride (2.1 g, 54.5 mmol) was slowly added. The reaction system was heated to 60°C and stirred for 12 hours. Sodium sulfate decahydrate powder (5 g) was added to the reaction system and stirred for 20 minutes. The reaction mixture was filtered through a pad of Celite, and the filter cake was washed with ethyl acetate. The organic phases were combined and concentrated to obtain the crude title compound (1.1 g, yield: 76.9%).

[0423] MS m / z(ESI):132.1[M+H] + .

[0424] Step 4: Preparation of ethyl 4-((1-hydroxy-2-methylhexan-2-yl)amino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate

[0425] The synthetic method of the first step of Example 1 was adopted, and the reaction raw material n-butylamine was replaced with 2-amino-2-methylhexan-1-ol to obtain the title compound (0.8 g, yield: 45.6%).

[0426] MS m / z(ESI):342.2[M+H] + .

[0427] Step 5: Preparation of ethyl 4-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)amino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate

[0428] Ethyl 4-((1-hydroxy-2-methylhexan-2-yl)amino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylate (0.65 g, 1.71 mmol), imidazole (0.24 g, 3.43 mmol), and tert-butyldimethylsilyl chloride (0.31 g, 2.06 mmol) were added to N,N-dimethylformamide (6 mL), and the reaction was stirred at room temperature for 12 hours. Water (50 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (30 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, volume ratio) to obtain the title compound (0.8 g, yield: 100%).

[0429] MS m / z(ESI):456.3[M+H] + .

[0430] Step 6: Preparation of ethyl 4-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)amino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylate

[0431] The synthesis method of the second step of Example 1 was used to replace the reaction raw material 4-(butylamino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester with 4-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)amino)-6-methyl-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester to obtain the title compound (0.8 g, yield: 89.8%).

[0432] MS m / z(ESI):511.3[M+H] + .

[0433] Step 7: Preparation of 4-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)amino)-6-methyl-2-(methylthio)pyrido[4,3-d]pyrimidin-5(6H)-one

[0434] The synthesis method of the third step of Example 1 was used to replace the reaction raw material 4-(butylamino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester with 4-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)amino)-6-(2-(dimethylamino)vinyl)-2-(methylthio)pyrimidine-5-carboxylic acid ethyl ester to obtain the title compound (0.65 g, yield: 92.8%).

[0435] MS m / z(ESI):451.2[M+H] + .

[0436] Step 8: Preparation of 4-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)amino)-6-methyl-2-(methylsulfinyl)pyrido[4,3-d]pyrimidin-5(6H)-one

[0437] The synthesis method of the fourth step of Example 1 was used to replace the reaction raw material 4-(butylamino)-6-methyl-2-(methylthio)-5,6-dihydropyrido[4,3-d]pyrimidin-5-one with 4-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)amino)-6-methyl-2-(methylthio)pyrido[4,3-d]pyrimidin-5(6H)-one to give the title compound (0.6 g, yield: 100%).

[0438] MS m / z(ESI):467.2[M+H] + .

[0439] Step 9: Preparation of 4-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)amino)-2-((2,4-dimethoxybenzyl)amino)-6-methylpyrido[4,3-d]pyrimidin-5(6H)-one

[0440] The synthetic method of the fifth step of Intermediate Preparation Example 1 was adopted to replace the reaction raw material 4-(butylamino)-2-(methylsulfinyl)pyrido[4,3-d]pyrimidin-5(6H)-one with 4-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)amino)-6-methyl-2-(methylsulfinyl)pyrido[4,3-d]pyrimidin-5(6H)-one to obtain the title compound (0.5 g, yield: 78.3%).

[0441] MS m / z(ESI):570.3[M+H] + .

[0442] Step 10: Preparation of 2-amino-4-((1-hydroxy-2-methylhexan-2-yl)amino)-6-methylpyridone[4,3-d]pyrimidin-5-(6H)-one (Compound 16)

[0443] The synthesis method of the sixth step of Intermediate Preparation Example 1 was adopted to replace the reaction raw material 4-(butylamino)-2-((2,4-dimethoxybenzyl)amino)pyrido[4,3-d]pyrimidin-5(6H)-one with 4-((1-((tert-butyldimethylsilyl)oxy)-2-methylhexan-2-yl)amino)-2-((2,4-dimethoxybenzyl)amino)-6-methylpyrido[4,3-d]pyrimidin-5(6H)-one to obtain the title compound (25 mg, yield: 39.3%).

[0444] MS m / z(ESI):306.2[M+H] + .

[0445] 1 H NMR (400MHz, DMSO-d6): δ9.82 (s, 1H), 7.68 (d, J = 8.0Hz, 1H), 6.91 (s, 2H), 6.09 (b r,1H),5.03(s,1H),3.67(d,J=10.0Hz,1H),3.55(d,J=12.0Hz,1H),3.39(s,3H), 1.92-1.89(m,1H),1.76-1.64(m,1H),1.34(s,3H),1.25-1.18(m,4H),0.86(t,J=6.0Hz,3H).

[0446] Example 16: Preparation of N-(2-((2-amino-6-methyl-5-oxo-5,6-dihydropyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexyl)acetamide (Compound 18)

[0447] Step 1: Preparation of 2-((2-amino-6-methyl-5-oxo-5,6-dihydropyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexanal

[0448] 2-Amino-4-((1-hydroxy-2-methylhexan-2-yl)amino)-6-methylpyridone[4,3-d]pyrimidin-5(6H)-one (0.5 g, 1.64 mmol) and Dess-Martin periodinane (1.04 g, 2.46 mmol) were added sequentially to anhydrous dichloromethane (10 mL). The reaction system was stirred at room temperature for 12 hours. The reaction solution was slowly added to a mixture of saturated sodium thiosulfate solution and saturated sodium bicarbonate solution (20 mL, volume ratio 1:1). The mixture was stirred at room temperature for 0.5 hours and extracted three times with dichloromethane (15 mL). The organic phases were combined and washed three times with saturated brine (15 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the crude title compound (0.5 g, yield: 100%).

[0449] MS m / z(ESI):304.2[M+H] + .

[0450] Step 2: Preparation of 2-amino-4-((1-((2,4-dimethoxybenzyl)amino)-2-methylhexan-2-yl)amino)-6-methylpyrido[4,3-d]pyrimidin-5(6H)-one

[0451] 2-((2-amino-6-methyl-5-oxo-5,6-dihydropyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexanal (0.45 g, 1.48 mmol), 2,4-dimethoxybenzylamine (0.37 g, 2.23 mmol), and isopropyl titanate (0.64 g, 2.23 mmol) were added sequentially to anhydrous methanol (10 mL) and stirred at room temperature for 2 hours. Sodium cyanoborohydride (0.38 g, 5.92 mmol) was added, and the reaction system was stirred at room temperature for 2 hours. The reaction solution was poured into water (50 mL) and extracted three times with dichloromethane (30 mL). The organic phases were combined and washed three times with saturated brine (15 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated to obtain the crude title compound (0.7 g, yield: 100%).

[0452] MS m / z(ESI):455.3[M+H] + .

[0453] Step 3: Preparation of 2-amino-4-((1-amino-2-methylhexan-2-yl)amino)-6-methylpyrido[4,3-d]pyrimidin-5(6H)-one

[0454] The synthetic method of the sixth step of Intermediate Preparation Example 1 was adopted to replace the reaction raw material 4-(butylamino)-2-((2,4-dimethoxybenzyl)amino)pyrido[4,3-d]pyrimidin-5(6H)-one with 2-amino-4-((1-((2,4-dimethoxybenzyl)amino)-2-methylhexan-2-yl)amino)-6-methylpyrido[4,3-d]pyrimidin-5(6H)-one to obtain the title compound (0.23 g, yield: 52.3%).

[0455] MS m / z(ESI):305.2[M+H] + .

[0456] Step 4: Preparation of N-(2-((2-amino-6-methyl-5-oxo-5,6-dihydropyrido[4,3-d]pyrimidin-4-yl)amino)-2-methylhexyl)acetamide (Compound 18)

[0457] 2-Amino-4-((1-amino-2-methylhexan-2-yl)amino)-6-methylpyrido[4,3-d]pyrimidin-5(6H)-one (40 mg, 0.13 mmol) and triethylamine (66 mg, 0.65 mmol) were added to anhydrous dichloromethane (4 mL). Acetic anhydride (26.5 mmol, 0.26 mmol) was added under ice-water cooling, and the reaction system was stirred at room temperature for 4 hours. The reaction solution was poured into water (50 mL) and extracted three times with dichloromethane (30 mL). The organic phases were combined and washed three times with saturated brine (15 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by preparative high-performance liquid chromatography to obtain the title compound (7 mg, yield: 15.4%).

[0458] MS m / z(ESI):347.2[M+H] + .

[0459] 1 H NMR (400MHz, CD3OD): δ7.61(d,J=8.0Hz,1H), 6.20(d,J=8.0Hz,1H), 3.88(d,J=12.0Hz,1H), 3.73(d,J=12.0Hz, 1H), 3.49 (s, 3H), 2.23-2.10 (m, 1H), 1.94 (s, 3H), 1.61-1.51 (m, 1H), 1.44-1.26 (m, 7H), 0.91 (t, J = 6.0Hz, 3H).

[0460] Example 17: Preparation of N-(26-(4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazin-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosadecanoyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (Compound 20)

[0461] Step 1: Preparation of tert-butyl (26-oxo-3,6,9,12,15,18,21,24-octaoxahexacosadecyl)carbamate

[0462] To a solution of tert-butyl (26-hydroxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)carbamate (1 g, 19.5 mmol) in dichloromethane (20 mL) was added Dess-Martin periodinane (1.23 g, 29.3 mmol) and stirred at room temperature overnight. After the reaction was complete, the reaction was diluted with dichloromethane. Saturated sodium thiosulfate solution (10 mL) and sodium bicarbonate solution (10 mL) were added sequentially and stirred for 20 minutes. The mixture was extracted twice with dichloromethane. The combined organic phases were washed with brine and concentrated to yield a residue which was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 12 / 1, volume ratio) to afford the title compound (0.9 g, yield: 90.0%).

[0463] MS m / z(ESI):512.3[M+H] + .

[0464] Step 2: Preparation of tert-butyl (26-(4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazin-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosyl)carbamate

[0465] To a solution of 2-amino-4-(butylamino)-6-(4-(piperazin-1-ylmethyl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (90 mg, 0.21 mmol) in dichloroethane (4 mL) was added tert-butyl (26-oxo-3,6,9,12,15,18,21,24-octaoxahexacosadecanoyl)carbamate (163 mg, 0.32 mmol), glacial acetic acid (20 mg, 0.21 mmol), and sodium cyanoborohydride (69 mg, 1.06 mmol). The reaction mixture was allowed to react at room temperature for 12 hours. After completion, the reaction was diluted with water and extracted with dichloromethane. The combined organic phases were washed with brine and concentrated to give a residue which was purified by preparative HPLC to afford the title compound (30 mg, 18.6% yield).

[0466] MS m / z(ESI):917.6[M+H] + .

[0467] Step 3: Preparation of 2-amino-6-(4-((4-(26-amino-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperazin-1-yl)methyl)benzyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one

[0468] The synthesis method of the third step of Example 6 was used to replace the reaction raw material 4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazine-1-carboxylic acid tert-butyl ester with (26-(4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazin-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosadecyl)carbamic acid tert-butyl ester to give the title compound (28 mg, yield: 100%).

[0469] MS m / z(ESI):817.5[M+H] + .

[0470] Step 4: Preparation of N-(26-(4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazin-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide

[0471] 2-Amino-6-(4-((4-(2,6-amino-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperazin-1-yl)methyl)benzyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one (28 mg, 34.3 μmol), N,N-diisopropylethylamine (14 mg, 108.3 μmol), and 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (15 mg, 41.1 μmol) were added sequentially to N,N-dimethylformamide (2 mL) and stirred at room temperature for 1 hour. The reaction mixture was purified by preparative HPLC to obtain the title compound (17 mg, 46.4% yield).

[0472] MS m / z(ESI):1067.6[M+H] + .

[0473] 1 H NMR(400MHz,DMSO-d6)δ12.93(s,1H),10.08(s,1H),9.46(s,1H),9.12(s,2H),8.14(s,1H ),7.95(t,J=5.2Hz,1H),7.30(s,4H),6.41(d,J=8.0Hz,1H),5.16(s,2H),3.72(s,2H),3.6 0-3.39(m,40H),3.20(q,J=6.0Hz,3H),2.87(s,3H),2.67(s,2H),2.35(s,2H),2.26(t,J=8 .0Hz,3H),1.82-1.75(m,2H),1.63-1.51(m,2H),1.45-1.30(m,2H),0.91(t,J=6.0Hz,3H).

[0474] Example 18: Preparation of N-(24-(4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazin-1-yl)-24-oxo-3,6,9,12,15,18,21-heptaoxatetracosyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (Compound 21)

[0475] Step 1: Preparation of tert-butyl (24-(4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazin-1-yl)-24-oxo-3,6,9,12,15,18,21-heptaoxatetracosyl)carbamate

[0476] To a solution of 2-amino-4-(butylamino)-6-(4-(piperazin-1-ylmethyl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one (60 mg, 0.14 mmol) in N,N-dimethylformamide (4 mL) were added 2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26-octaoxa-5-azanonacosane-29-oic acid (76 mg, 0.15 mmol), triethylamine (70 mg, 0.7 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (80 mg, 0.21 mmol), and the reaction mixture was reacted at room temperature for 12 hours. After the reaction was completed, the mixture was diluted with water and extracted with dichloromethane. The organic phases were combined and washed with brine. The organic phases were concentrated and the residue was purified by preparative HPLC to give the title compound (80 mg, yield: 63.4%).

[0477] MS m / z(ESI):901.5[M+H] + .

[0478] Step 2: Preparation of 2-amino-6-(4-((4-(1-amino-3,6,9,12,15,18,21-heptaoxatetracosanoyl)piperazin-1-yl)methyl)benzyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one

[0479] The synthesis method of the third step of Example 6 was used to replace the reaction raw material 4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazine-1-carboxylic acid tert-butyl ester with (24-(4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazin-1-yl)-24-oxo-3,6,9,12,15,18,21-heptaoxatetracosyl)carbamic acid tert-butyl ester to give the title compound (70 mg, yield: 85.6%).

[0480] MS m / z(ESI):801.5[M+H] + .

[0481] Step 3: Preparation of N-(24-(4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazin-1-yl)-24-oxo-3,6,9,12,15,18,21-heptaoxatetracosyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (Compound 21)

[0482] The synthesis method of the fourth step of Example 17 was used to replace the reaction raw material 2-amino-6-(4-((4-(26-amino-3,6,9,12,15,18,21,24-octaoxacosacosan-24-yl)piperazin-1-yl)methyl)benzyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one with 2-amino-6-(4-((4-(1-amino-3,6,9,12,15,18,21-heptaoxatetracosan-24-yl)piperazin-1-yl)methyl)benzyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one to give the title compound (11 mg, yield: 15.9%).

[0483] MS m / z(ESI):1051.5[M+H] + .

[0484] 1 H NMR (400MHz, CD3OD) δ8.97(s,2H),7.66(d,J=8.0Hz,1H),7.31(q,J=8.0Hz,4H),6.21(d,J=8.0Hz,1H),5.11(s,2H),3.87-3.44(m,39H ),3.36(s,6H),2.65-2.55(m,4H),2.50-2.33(m,6H),2.00-1.89(m,2H),1.68-1.60(m,2H),1.49-1.38(m,2H),0.98(t,J=8.0Hz,3H).

[0485] Example 19: Preparation of N-(26-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)piperidin-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosadecanoyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (Compound 22)

[0486] Step 1: Preparation of tert-butyl (26-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)piperidin-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosyl)carbamate

[0487] The synthesis method of the second step of Example 17 was adopted to replace the reaction raw material 2-amino-4-(butylamino)-6-(4-(piperazin-1-ylmethyl)benzyl)pyrido[4,3-d]pyrimidin-5(6H)-one with 2-amino-4-(butylamino)-6-(piperidin-4-ylmethyl)pyrido[4,3-d]pyrimidin-5(6H)-one to obtain the title compound (40 mg, yield: 19.5%).

[0488] MS m / z(ESI):826.5[M+H] + .

[0489] Step 2: Preparation of 2-amino-6-((1-(26-amino-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidin-4-yl)methyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one

[0490] The synthesis method of the third step of Example 6 was used to replace the reaction raw material 4-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperazine-1-carboxylic acid tert-butyl ester with (26-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)piperidin-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosadecyl)carbamic acid tert-butyl ester to give the title compound (30 mg, yield: 81.3%).

[0491] MS m / z(ESI):726.5[M+H] + .

[0492] Step 3: Preparation of N-(26-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)piperidin-1-yl)-3,6,9,12,15,18,21,24-octaoxahexacosadecyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (Compound 22)

[0493] The synthesis method of the fourth step of Example 17 was used to replace the reaction raw material 2-amino-6-(4-((4-(26-amino-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperazin-1-yl)methyl)benzyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one with 2-amino-6-((1-(26-amino-3,6,9,12,15,18,21,24-octaoxahexacosanyl)piperidin-4-yl)methyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one to give the title compound (20 mg, yield: 37.3%).

[0494] MS m / z(ESI):976.5[M+H] + .

[0495] 1 H NMR (400MHz, CD3OD) δ9.72(t,J=4.0Hz,1H),8.98(s,2H),8.50(s,1H),7.58(d,J=8.0Hz,1H),6. 21(d,J=8.0Hz,1H),3.90(d,J=8.0Hz,2H),3.83-3.79(m,2H),3.65-3.50(m,36H),3.38-3.34(m, 5H),3.29-3.26(m,2H),2.99-2.89(t,J=12.0Hz,2H),2.59(t,J=8.0Hz,2H),2.40(t,J=8.0Hz,2 H), 2.16 (s, 1H), 1.98-1.86 (m, 4H), 1.67-1.56 (m, 4H), 1.49-1.40 (m, 2H), 0.98 (t, J = 8.0Hz, 3H).

[0496] Example 20: Preparation of 1-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)-N-(33-(2-(methylsulfonyl)pyrimidin-5-yl)-28-oxo-3,6,9,12,15,18,21,24-octaoxa-27-azatris-32-yn-1-yl)piperidine-4-carboxamide (Compound 23)

[0497] Step 1: Preparation of methyl 1-(4-(chloromethyl)benzyl)piperidine-4-carboxylate

[0498] 1,4-Bis(chloromethyl)benzene (500 mg, 2.86 mmol), methyl piperidine-4-carboxylate (449.86 mg, 3.14 mmol), and potassium carbonate (1.18 g, 8.57 mmol) were added to acetonitrile (4 mL), and the reaction mixture was heated to 75°C and stirred for 5 hours. Water (30 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (15 mL). The organic phases were combined and washed twice with saturated brine (20 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1, volume ratio) to obtain the title compound (205 mg, yield: 25.5%).

[0499] MS m / z(ESI):282.1[M+H] + .

[0500] Step 2: Preparation of methyl 1-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperidine-4-carboxylate

[0501] Using the synthesis method of the first step of Example 6, replacing (6-(chloromethyl)pyridin-3-yl)methanol with 1-(4-(chloromethyl)benzyl)piperidine-4-carboxylic acid methyl ester, the title compound (172 mg, yield: 56.5%) was obtained.

[0502] MS m / z(ESI):479.3[M+H] + .

[0503] Step 3: Preparation of 1-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperidine-4-carboxylic acid

[0504] Methyl 1-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperidine-4-carboxylate (100 mg, 188.05 μmol) was dissolved in methanol (5 mL) and water (1 mL). Sodium hydroxide (112.83 mg, 2.82 mmol) was added and stirred at room temperature for 4 hours. The reaction mixture was concentrated, and the resulting residue was purified by preparative high-performance liquid chromatography to obtain the title compound (21 mg, 24.0% yield).

[0505] MS m / z(ESI):465.3[M+H] + .

[0506] Step 4: Preparation of 1-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)-N-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidine-4-carboxamide

[0507] 1-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)piperidine-4-carboxylic acid (21 mg, 45.20 μmol), 26-azido-3,6,9,12,15,18,21,24-octaoxahexacosacan-1-amine (25.77 mg, 58.77 μmol), N,N-diisopropylethylamine (17.53 mg, 135.61 μmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (25.78 mg, 67.81 μmol) were added to N,N-dimethylformamide (2 mL) and stirred at room temperature for 2 hours. The reaction mixture was concentrated and the residue was purified by preparative HPLC to give the title compound (20 mg, yield: 50.0%).

[0508] MS m / z(ESI):885.5[M+H] + .

[0509] Step 5: Preparation of 1-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)-N-(26-amino-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidine-4-carboxamide

[0510] 1-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)-N-(26-azido-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidine-4-carboxamide (20 mg, 22.60 μmol) was dissolved in aqueous ammonia (0.5 mL) and tetrahydrofuran (3 mL). Triphenylphosphine (7.70 mg, 29.38 μmol) was added, and the reaction system was heated to 50°C and stirred for 3 hours. The reaction mixture was concentrated, and the resulting residue was purified by preparative high-performance liquid chromatography to obtain the title compound (10 mg, yield: 51.5%).

[0511] MS m / z(ESI):859.5[M+H] + .

[0512] Step 6: Preparation of 1-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)-N-(33-(2-(methylsulfonyl)pyrimidin-5-yl)-28-oxo-3,6,9,12,15,18,21,24-octaoxa-27-azatris-32-yn-1-yl)piperidine-4-carboxamide (Compound 23)

[0513] The synthesis method of the fourth step of Example 17 was used to replace the reaction raw material 2-amino-6-(4-((4-(26-amino-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperazin-1-yl)methyl)benzyl)-4-(butylamino)pyrido[4,3-d]pyrimidin-5(6H)-one with 1-(4-((2-amino-4-(butylamino)-5-oxopyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzyl)-N-(26-amino-3,6,9,12,15,18,21,24-octaoxahexacosyl)piperidine-4-carboxamide to give the title compound (4.1 mg, yield: 30.5%).

[0514] MS m / z(ESI):1109.6[M+H] + .

[0515] 1 H NMR (400MHz, CD3OD) δ9.62 (s, 1H), 8.98 (s, 2H), 8.50 (s, 1H), 7.63 (d, J = 8.0Hz, 1H) ,7.37(q,J=8.0Hz,4H),6.20(d,J=8.0Hz,1H),5.12(s,2H),3.85(s,2H),3.61-3.59 (m,26H),3.54-3.49(m,6H),3.38-3.33(m,7H),3.20-3.11(m,2H),2.61-2.28(m,9 H), 1.98-1.76 (m, 7H), 1.67-1.59 (m, 2H), 1.49-1.39 (m, 2H), 0.97 (t, J = 6.0Hz, 3H).

[0516] 2. Preparation of Immunostimulatory Antibody Conjugates (ISACs)

[0517] Example 2-1 ISAC-1

[0518] The trastuzumab monoclonal antibody stock solution was adjusted to pH 6.51 with 1M sodium hydrogen phosphate solution and then diluted to 10 mg / mL with phosphate buffered saline (PBS) at pH 6.5-6.6 to obtain a trastuzumab monoclonal antibody buffer solution. 0.1M EDTA and TCEP (6 eq, 10 mM) were added to the resulting trastuzumab monoclonal antibody buffer solution, shaken, and reacted at 25°C for 2 hours. A DMSO solution (10 mM) of compound 20 (12-fold the molar equivalent of trastuzumab) was then added and shaken. The reaction was allowed to proceed at room temperature for 2 hours and at 25°C for 2 hours. The solution was then purified using a NAP-10 gel column and washed with histidine hydrochloride buffer at pH 6.0-6.1. The filtrate was collected to obtain ISAC-1 in histidine hydrochloride buffer (1.9 mg / mL, 1.5 mL) and stored at 4°C.

[0519] Example 2-2 ISAC-2

[0520] The trastuzumab monoclonal antibody stock solution was adjusted to pH 6.51 with 1M sodium hydrogen phosphate solution and then diluted to 10 mg / mL with phosphate buffered saline (PBS) at pH 6.5-6.6 to obtain a trastuzumab monoclonal antibody buffer solution. 0.1M EDTA and TCEP (6 eq, 10 mM) were added to the resulting trastuzumab monoclonal antibody buffer solution, shaken, and reacted at 25°C for 2 hours. A DMSO solution (10 mM) of compound 21 (12 times the molar equivalent of trastuzumab) was added and shaken. The solution was reacted at room temperature for 2 hours and at 25°C for 2 hours. The solution was purified using a NAP-10 gel column and washed with histidine hydrochloride buffer at pH 6.0-6.1. The filtrate was collected to obtain ISAC-2 in histidine hydrochloride buffer (3.0 mg / mL, 1.5 mL) and stored at 4°C.

[0521] Example 2-3 ISAC-3

[0522] The trastuzumab monoclonal antibody stock solution was adjusted to pH 6.51 with 1M sodium hydrogen phosphate solution and then diluted to 10 mg / mL with phosphate buffered saline (PBS) at pH 6.5-6.6 to obtain a trastuzumab monoclonal antibody buffer solution. 0.1M EDTA and TCEP (6 eq, 10 mM) were added to the resulting trastuzumab monoclonal antibody buffer solution, shaken, and reacted at 25°C for 2 hours. A DMSO solution (10 mM) containing 11 molar equivalents of compound 22 was added, shaken, and reacted at room temperature for 2 hours and at 25°C for 2 hours. The solution was purified using a NAP-10 gel column and washed with histidine hydrochloride buffer at pH 6.0-6.1. The filtrate was collected to obtain ISAC-3 in histidine hydrochloride buffer (3.2 mg / mL, 1.5 mL) and stored at 4°C.

[0523] Example 2-4 ISAC-4

[0524] The trastuzumab stock solution was adjusted to pH 6.51 with 1M sodium hydrogen phosphate solution and then diluted to 10 mg / mL with phosphate buffered saline (PBS) at pH 6.5-6.6 to obtain a trastuzumab buffer solution. 0.1M EDTA and 6 eq of TCEP (10 mM) were added to the resulting trastuzumab buffer solution, shaken, and reacted at 25°C for 2 hours. A 10-fold molar equivalent of trastuzumab in DMSO (10 mM) was then added and the solution was shaken and reacted at room temperature for 2 hours and at 25°C for 2 hours. The solution was then purified using a NAP-10 gel column and washed with histidine hydrochloride buffer at pH 6.0-6.1. The filtrate was collected to obtain ISAC-4 in histidine hydrochloride buffer (3.2 mg / mL, 1.5 mL) and stored at 4°C.

[0525] III. Drug / Antibody Ratio of Immunostimulatory Antibody Conjugates (ISACs): Determination of DAR Value

[0526] The molecular weight of ISAC was determined by LC-MS, and the drug / antibody ratio (DAR) was calculated.

[0527] Chromatographic determination conditions:

[0528] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0529] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN;

[0530] Flow rate: 0.25 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 1 μl;

[0531] Mass spectrometry conditions:

[0532] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0533] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time 0.5s;

[0534] m / z 600-4000; Time bins to sum 40.

[0535] The DAR values ​​of the immunostimulatory antibody conjugates ISAC-1, ISAC-2, ISAC-3, ISAC-4, and ISAC-1 were calculated by CE-SDS to be 8.0, 8.0, 7.99, and 8.0, respectively.

[0536] 2. Biological Tests

[0537] Experimental Example 1: Determination of the agonist activity of compounds on HEK-Blue hTLR7 cells

[0538] Experimental steps:

[0539] 1. HEK-Blue hTLR7 cells (Invivogen) were cultured in DMEM (Hyclone) containing 10% FBS heat-inactivated fetal bovine serum (Corning). On the day of the assay, the cells were observed under a microscope, and the cells were collected, resuspended, counted, and the cell concentration was adjusted to 50 μL per well, with a total cell volume of 2 × 10 4 , seeded cells in 96-well plates.

[0540] 2. After cells have adhered overnight, add 50 μL of test compound at various concentrations to the plate, resulting in final concentrations of 100 μM, 33.3 μM, 11.11 μM, 3.70 μM, 1.23 μM, 0.41 μM, 0.14 μM, 0.05 μM, and 0 μM, respectively. The final DMSO concentration is 1%. Incubate the compounds and cells in a 37°C, 5% CO2 incubator for 20 h.

[0541] 3. After the compound incubation is completed, observe the cell status of the largest signal well or the highest and lowest compound concentrations under a microscope and whether compound crystals will precipitate at high concentrations. Transfer 10 μL of cell culture supernatant to a new 96-well transparent plate, add 90 μL of QUANTI-Blue (Invivogen) detection reagent to each well, incubate at 37°C for 3 hours, and read the OD using a multifunctional microplate reader (BMG LABTECH). 620 .

[0542] 4.EC 50 The log(agonist) vs. response--Variable slope fitting calculation was performed using Graphpad Prism software. Emax is the OD at which the activation effect of the test compound reaches its maximum. 620 .

[0543] Experimental results:

[0544] The activation activity of the compounds on HEK-Blue hTLR7 cells was determined according to the above method. The activity of the compounds was expressed as EC 50 and Emax, the results are shown in Table 1:

[0545] Table 1. Experimental results of the activation effect of compounds on HEK-Blue hTLR7 cells

[0546] The results showed that compounds 1, 4, 5, 6, 7, 8, 9, 11, 14 and 16 had a strong activation effect on human TLR7.

[0547] Experimental Example 2: Determination of the agonist activity of compounds on HEK-Blue hTLR8 cells

[0548] Experimental steps:

[0549] 1. HEK-Blue hTLR8 cells (Invivogen) were cultured in DMEM (Hyclone) containing 10% FBS heat-inactivated fetal bovine serum (Corning). On the day of the assay, the cells were observed under a microscope, and the cells were collected, resuspended, counted, and the cell concentration was adjusted to 50 μL per well, with a total cell volume of 2 × 10 4 , seeded cells in 96-well plates.

[0550] 2. After cells have adhered overnight, add 50 μL of test compound at various concentrations to the plate, resulting in final concentrations of 100 μM, 33.3 μM, 11.11 μM, 3.70 μM, 1.23 μM, 0.41 μM, 0.14 μM, 0.05 μM, and 0 μM, respectively. The final DMSO concentration is 1%. Incubate the compounds and cells in a 37°C, 5% CO2 incubator for 20 h.

[0551] 3. After the compound incubation is completed, observe the cell status of the largest signal well or the highest and lowest compound concentrations under a microscope and whether the compound crystals will precipitate at high concentrations. Transfer 10 μL of cell culture supernatant to a new 96-well transparent plate, add 90 μL of QUANTI-Blue (Invivogen-) detection reagent to each well, incubate at 37°C for 3 hours, and read the OD using a multifunctional microplate reader (BMG LABTECH). 620 .

[0552] 4.EC 50 The log(agonist) vs. response--Variable slope fitting calculation was performed using Graphpad Prism software. Emax is the OD at which the activation effect of the test compound reaches its maximum. 620 .

[0553] Experimental results:

[0554] The activation activity of the compounds on HEK-Blue hTLR8 cells was determined according to the above method. The activity of the compounds was expressed as EC 50 and Emax, the results are shown in Table 2:

[0555] Table 2. Experimental results of the activation effect of compounds on HEK-Blue hTLR8 cells

[0556] The results showed that compounds 1, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16 and 18 had a strong activation effect on human TLR8.

[0557] Experimental Example 3: Determination of the ability of immunostimulatory antibody conjugates (ISACs) to stimulate TNF-α secretion from peripheral blood mononuclear cells (PBMCs) in the presence of tumor cells

[0558] Experimental steps:

[0559] 1. Collect HER2-expressing HCC1954 tumor cells (Connotai) in the logarithmic growth phase, wash twice with PBS, and resuspend the cells in RPMI1640 + 10% heat-inactivated FBS complete medium to adjust the cell density to 2×105 HCC1954 tumor cells were seeded in 96-well plates (Corning) at 1×10 cells / mL. 4 cells.

[0560] 2. Resuscitate PBMC (Sai ​​Li Biotechnology) and resuspend the cells in RPMI1640+10% heat-inactivated FBS complete medium to adjust the density to 6×10 5 10 cells / mL were added to a 96-well plate inoculated with tumor cells, with 3×10 cells / well 4 PBMC cells.

[0561] 3. Add 100 μL of different concentrations of the test substance Trastuzumab monoclonal antibody and the immunostimulatory antibody conjugate ISAC-1 to the well plate to make the final concentrations of 500 nM, 166.7 nM, 55.6 nM, 18.52 nM, 6.17 nM, 2.06 nM, and 0.69 nM, respectively. Add 100 μL of RPMI1640 + 10% heat-inactivated FBS complete medium to the blank wells.

[0562] 4. Incubate in a 5% CO2, 37°C incubator. After 24 hours, remove 50 μL of supernatant from each well and incubate with the primary antibody and HRP-conjugated secondary antibody according to the human TNF-α ELISA kit (Invitrogen). Develop the color and read the absorbance at 450 nm. Calculate the TNF-α concentration using Graphpad Prism software based on the standard curve.

[0563] Table 3. ISAC-stimulated TNF-α secretion in the in vitro HCC1954 tumor cell and PBMC co-incubation system

[0564] The results showed that each ISAC could effectively stimulate PBMC to secrete TNF-α in the in vitro HCC1954 tumor cell and PBMC co-incubation system.

[0565] Experimental Example 4. Evaluation of the efficacy of ISAC in a SCID-beige mouse xenograft tumor model

[0566] Experimental steps:

[0567] 1. Each SCID-beige mouse (Wei Tong Li Hua) was subcutaneously inoculated with 5×10 6 HCC1954 cells (mixed with Matrigel at an equal volume of 1:1 and suspended in 0.1 mL PBS).

[0568] 2. Wait until the average tumor volume grows to about 150mm 3When the time comes, mice with irregular, too small or too large tumor volumes are excluded. The remaining mice are randomly grouped according to tumor volume and animal body weight, and are respectively intraperitoneally injected (i.p.) with normal saline (vehicle control), Trastuzumab monoclonal antibody, and immune-stimulating antibody conjugate ISAC-1 once to observe the efficacy of the test compound on this tumor-bearing mouse model and the tolerance of the animals to the test compound.

[0569] 3. During the experiment, the body weights of the mice are weighed twice a week and the tumor volumes are measured, and the data are recorded.

[0570] 4. Data statistics, the calculation formula for tumor volume (V): V = 1 / 2 × a × b 2 , where a and b represent the length and width respectively. The anti-tumor drug efficacy is evaluated by the tumor growth inhibition rate TGI (%), and the calculation formula: TGI (%) (瘤体积) = [1 - (T Vt - T V0 ) / (C Vt - C V0 )] × 100%, T V0 is the average tumor volume of the test compound group when grouped for administration, T Vt is the average tumor volume of the test compound group at t days after administration; C V0 is the average tumor volume of the vehicle group when grouped for administration; C Vt is the average tumor volume of the vehicle group at t days after administration. When the tumor regresses, TGI (%) (瘤体积) = 100% - (T Vt - T V0 ) / T V0 × 100%. If the tumor shrinks compared to the initial volume, that is, when V t < V0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR).

[0571] The above embodiments do not limit the solutions of the present invention in any way. Except those described herein, various modifications of the present invention will be obvious to those skilled in the art according to the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The protection scope of this application is given by the appended claims and any equivalents thereof.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof: in, L 1 Selected from O, S and NR 5 ; L 2 is a covalent bond, or is selected from C 1-6 Alkylene and -C 1-6 Alkylene-O-; R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more selected from hydrogen, halogen, hydroxyl, C 1-6 Alkyl, C 1-6 Alkoxy, 5-10 membered heteroaryl and R 6 -C(O)-NH-; R 2 Selected from hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, R 7 and R 7 -L 3 -substituted by a group; L 3 Selected from C 1-6 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-; R 3 Selected from hydrogen, halogen, C 1-6 Alkyl and C 1-6 alkoxy; R 4 Selected from hydrogen, halogen, C 1-6 Alkyl and C 1-6 alkoxy; R 5 Selected from hydrogen and C 1-6 alkyl; R 6 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with an alkoxy group; R 7 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl and 3-12 membered heterocyclic group, the alkyl, cycloalkyl and heterocyclic group are optionally One or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with alkoxy and carboxyl groups; R 8 Selected from hydrogen and C 1-6 alkyl.

2. The compound of claim 1 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein: R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more selected from hydrogen, halogen, hydroxyl, C 1-3 Alkyl, C 1-3 Alkoxy, 5-10 membered heteroaryl and R 6 -C(O)-NH-; R 6 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-12 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with an alkoxy group; Preferably, R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more selected from hydrogen, halogen, hydroxyl, C 1-3 Alkyl, C 1-3 Alkoxy, 5-10 membered heteroaryl and R 6 -C(O)-NH-; R 6 C 1-6 alkyl; Preferably, R 1 C 1-6 Alkyl, said alkyl being optionally substituted by one or more groups selected from hydrogen, hydroxy, methyl, methoxy and C 1-3 substituted by an alkyl-C(O)-NH- group; Preferably, R 1 C 1-6 Alkyl, the alkyl group is unsubstituted or substituted by methyl, hydroxy, methoxy or C 1-3 Alkyl-C(O)-NH-substituted.

3. The compound of any one of claims 1-2, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein: R 2 Selected from hydrogen, C 1-3 Alkyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, R 7 and R 7 -L 3 -substituted by a group; R 7 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with alkoxy and carboxyl groups; L 3 Selected from C 1-6 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-; R 8 Selected from hydrogen and C 1-6 alkyl; Preferably, R 2 Selected from hydrogen, C 1-3 Alkyl, C 3-8 Cycloalkyl, 3-8 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-3 Alkyl, C 1-3 Halogenated alkyl, C 1-3 Alkoxy, R 7 and R 7 -L 3 -substituted by a group; R 7 Selected from C 1-6 Alkyl, C 3-10 Cycloalkyl and 3-12 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 substituted with alkoxy and carboxyl groups; L 3 Selected from C 1-6 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-; R 8 Selected from hydrogen and C 1-6 alkyl; Preferably, R 2 Selected from hydrogen, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the cycloalkyl, heterocyclic, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-3 Alkyl, C 1-3 Halogenated alkyl, C 1-3 Alkoxy, R 7 and R 7 -L 3 -substituted by a group; R 7 Selected from C 1-3 Alkyl, C 3-6 Cycloalkyl and 3-6 membered heterocyclic groups, wherein the alkyl, cycloalkyl and heterocyclic groups are optionally substituted by one or more selected from hydrogen, halogen, amino, hydroxyl, cyano, C 1-3 Alkyl, C 1-3 Halogenated alkyl, C 1-3 substituted with alkoxy and carboxyl groups; L 3 Selected from C 1-3 Alkylene, O, S, NR 8 、-C 1-6 Alkylene-NR 8 -、-C(O)-、-C(O)-NR 8 -and-NR 8 -C(O)-; R 8 Selected from hydrogen and C 1-3 alkyl; Preferably, R 2 Selected from hydrogen, C 3-6 Cycloalkyl, 3-6 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, the cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted by one or more selected from hydrogen, C 1-3 Alkyl, C 1-3 Alkoxy, R 7 and R 7 -L 3 -substituted by a group; R 7 Selected from C 1-3 Alkyl and 3-6 membered heterocyclic group, the alkyl and heterocyclic group are optionally substituted by one or more selected from hydrogen and C 1-3 substituted with an alkyl group; L 3 Selected from C 1-3 Alkylene and -C 1-3 Alkylene-NR 8 -; R 8 C 1-3 alkyl.

4. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein: L 1 is selected from O, S and NH.

5. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein: L 2 is a covalent bond, or is selected from C 1-3 Alkylene and -C 1-3 Alkylene-O-.

6. The compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein: R 3 Selected from hydrogen, halogen, C 1-3 Alkyl and C 1-3 Alkoxy.

7. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein: R 4 Selected from hydrogen, halogen, C 1-3 Alkyl and C 1-3 Alkoxy.

8. The compound of any one of claims 1 to 7, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound has the structure of Formula (II-A): in, R 1 and R 2 As defined in any one of claims 1 to 7.

9. The compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound has the following structure:

10. The drug-linker of the following formula or its pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug: D-L' in, D is a fragment of the compound according to any one of claims 1 to 9; L' is the linker fragment; Preferably, L' is selected from -L 4 -L 5 -L 6 ,in, L 4 is selected from the group consisting of a covalent bond, -C(=O)-, and -NH-; L 5 is a polyethylene glycol fragment, e.g. wherein a is an integer selected from 1 to 10; L 6 for in, Z1 is selected from a chemical bond and -C(=O)-; Z2 is selected from chemical bonds and C 1-20 alkylene; Z3 is selected from chemical bonds, C 2-6 Alkenylene and C 2-6 Alkynylidene; A is selected from LG is a leaving group for nucleophilic substitution reaction, selected from halogen, nitro, benzenesulfonate, p-toluenesulfonate, trifluoromethanesulfonate, -S(O)2-R 9 and-S(O)-R 9 ; R 9 Selected from C 1-6 Alkyl and C 1-6 alkyl halide; Preferably, L' is selected from:

11. The drug-linker of claim 10 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, wherein the drug-linker is selected from the following structures:

12. An immunostimulatory antibody conjugate of formula (ISAC-I) or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug thereof: in, D is a fragment of the compound according to any one of claims 1 to 9; Ab is an antibody that can target the target antigen; L is a fragment obtained by linking L' in claim 10 or 11 with Ab; z is selected from 1-10, for example 1-8.

13. The immunostimulatory antibody conjugate of claim 12 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, wherein the immunostimulatory antibody conjugate is selected from: z is independently selected from 1-8, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8, or 7-8.

14. Use of a compound in the preparation of an immunostimulatory antibody conjugate, wherein the compound is selected from the compound according to any one of claims 1 to 9.

15. A composition comprising the immunostimulatory antibody conjugate of claim 12 or 13 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof.

16. The composition of claim 15, wherein the DAR value is an integer or decimal selected from 1-8, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 3-4, 3-5, 3-6, 3-7, 3-8, 4-5, 4-6, 4-7, 4-8, 5-6, 5-7, 5-8, 6-7, 6-8 or 7-8; Preferably, the DAR value is 7.0-8.0, such as 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.

0.

17. A pharmaceutical composition comprising a prophylactic or therapeutically effective amount of a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, or the immunostimulatory antibody conjugate according to claim 12 or 13, or the composition according to claim 15 or 16, and one or more pharmaceutically acceptable carriers.

18. Use of a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, the immunostimulatory antibody conjugate according to claim 12 or 13, or the composition according to claim 15 or 16, or the pharmaceutical composition according to claim 17, in the preparation of a medicament for preventing and / or treating TLR7 and / or TLR8-mediated diseases such as cancer and viral infection.

19. A kit comprising a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug thereof, or the immunostimulatory antibody conjugate according to claim 12 or 13, or the composition according to claim 15 or 16, or the pharmaceutical composition according to claim 17.

20. A compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, the immunostimulatory antibody conjugate according to claim 12 or 13, or the composition according to claim 15 or 16, or the pharmaceutical composition according to claim 17, for use in preventing and / or treating TLR7 and / or TLR8-mediated diseases such as cancer and viral infection.

21. A method for preventing and / or treating TLR7 and / or TLR8-mediated diseases such as cancer and viral infection, comprising administering to an individual in need thereof a preventive or therapeutically effective amount of a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite or prodrug thereof, the immunostimulatory antibody conjugate according to claim 12 or 13, or the composition according to claim 15 or 16, or the pharmaceutical composition according to claim 17.

Images