A triazine compound, and a preparation method and use thereof

Abstract

The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a triazine compound shown in formula (I) and a preparation method and use thereof.

Description

Technical Field

[0001] This application belongs to the field of medicinal chemistry, specifically relating to a triazine compound, its preparation method, and its uses. Background Technology

[0002] KRAS mutations are among the most common oncogenic drivers in solid tumors, with G12D mutations being prevalent in pancreatic cancer (39%), colorectal cancer (44%), and lung adenocarcinoma. Developing covalent inhibitors is extremely challenging due to the lack of traditional small molecule binding sites on the KRAS protein surface and the absence of active residues (such as cysteine ​​in G12C) in G12D mutants. Current technologies utilize KRAS G12C inhibitors (such as AMG510 and MRTX849) for targeting via covalent binding to cysteine, but they are ineffective against G12D. Non-covalent inhibitors (such as MRTX1133), while designed for G12D, suffer from insufficient affinity (high IC50 value), poor selectivity (significant inhibition of wild-type KRAS), and drug resistance. Combination therapy strategies, such as combining KRAS inhibitors with MEK inhibitors or EGFR inhibitors, only partially alleviate drug resistance and are often highly toxic.

[0003] In summary, the existing technologies for KRAS G12D inhibitors have the following drawbacks:

[0004] Insufficient selectivity: Existing G12D inhibitors show significant cross-inhibition of wild-type KRAS or other mutant subtypes (such as G12V, G12S, etc.), leading to off-target toxicity. They are mainly effective against cancers with high KRAS G12D incidence, such as pancreatic cancer and colorectal cancer, but have little effect on other mutation types or tumors with low expression, limiting their market application potential. In addition, their efficacy in treating non-KRAS-dependent tumors is limited, which may restrict their clinical application scope.

[0005] Limited efficacy: Monotherapy is insufficient to overcome compensatory activation of KRAS signaling pathways (such as the MEK / ERK pathway).

[0006] Drug resistance: Mutant cells escape inhibition through bypass signaling or microenvironmental remodeling. Preclinical studies have shown that bypass activation of the KRAS signaling pathway (such as the EGFR or PI3K pathway) may lead to resistance to monotherapy, requiring combination therapy to enhance efficacy.

[0007] Complex Synthesis: The preparation of some compounds (such as MRTX1133) involves cumbersome steps and results in low yields. For example, the pyrido[4,3-d]pyrimidine skeleton of MRTX1133 requires multiple substituent optimization steps (such as R2, R4, and R7 sites), making the synthetic route complex and potentially increasing production costs and the difficulty of impurity control.

[0008] Pharmacokinetic challenges exist: non-covalent binding mechanisms may lead to lower drug stability in vivo, shorter half-life, and the need for frequent dosing to maintain effective concentrations.

[0009] Limitations of clinical data: Currently, only preclinical research data (such as mouse xenograft models) are publicly available, and results from human clinical trials are lacking. Long-term toxicity, dose tolerability, and duration of efficacy are still unclear.

[0010] Therefore, there is a demand for KRAS G12D inhibitors. Summary of the Invention

[0011] The first aspect of this application provides a compound of formula (I), or a stereoisomer thereof, or a tautomer thereof, or a solvate thereof, or an isotopically labeled compound thereof, or a pharmaceutically acceptable salt thereof.

[0012]

[0013] in,

[0014] R1 and R2 are each independently selected from: H, C1-C6 alkyl groups, or

[0015] R1 and R2, together with the N atom they contain, form a 4-7 member monocyclic heterocyclic group, a 5-10 member nitrogen-containing bridged ring heterocyclic group, a 5-10 member nitrogen-containing fused ring heterocyclic group, or a 5-10 member nitrogen-containing spirocyclic heterocyclic group, comprising 1-2 heteroatoms selected from N, O, and S. The 4-7 member monocyclic heterocyclic group, the 5-10 member nitrogen-containing bridged ring heterocyclic group, the 5-10 member nitrogen-containing fused ring heterocyclic group, and the 5-10 member nitrogen-containing spirocyclic heterocyclic group may optionally be substituted with the following groups: C1-C6 alkyl, halogen, C1-C6 haloalkyl, hydroxyl, amino, oxo, aminoC1-C6 alkyl, hydroxyC1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, or C2-C6 alkenyl carbonyl.

[0016] R3 and R4 are each selected independently from:

[0017] H, C1-C6 alkyl, C3-C7 cycloalkyl optionally substituted with hydroxyl or C1-C6 alkyl carbonyl, 4-7 membered monocyclic heterocyclic group comprising 1-2 heteroatoms selected from N, O, S optionally substituted with hydroxyl or C1-C6 alkyl carbonyl;

[0018] C6-C10 aryl groups optionally substituted with the following groups: C1-C6 alkyl, hydroxyl, cyano, amino C1-C6 alkyl, amino C1-C6 alkyl (wherein the amino group is optionally substituted with one or two C1-C6 alkyl groups), halogen, C1-C6 haloalkyl, C1-C6 alkoxy.

[0019] Among them, Rc R d Each is independently selected from: C1-C6 alkyl, hydroxyl, cyano, aminoC1-C6 alkyl, aminoC1-C6 alkyl (wherein the amino group is optionally substituted by one or two C1-C6 alkyl groups), halogen, C1-C6 haloalkyl, C1-C6 alkoxy.

[0020] Among them, R e R f R g Each is independently selected from: H, C1-C6 alkyl, hydroxy C1-C6 alkyl, carbamoyl, C1-C6 alkylaminocarbonyl, C1-C6 alkyloxycarbonylC3-C7 cycloalkylene, amino C1-C6 alkyl (wherein the amino group is optionally substituted by one or two C1-C6 alkyl groups), amino C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkyloxycarbonyl, C3-C7 cycloalkyloxyC1-C6 alkyl, any The C1-C6 alkyl group is selected from those substituted with a 4-7 membered monocyclic heterocyclic group containing 1-2 heteroatoms selected from N, O, and S; the C1-C6 alkyl group is selected from those substituted with a C1-C6 haloalkyl group and a hydroxyl group; the C3-C7 cycloalkyl group is selected from those substituted with a C1-C6 alkyloxycarbonylamino group; and the 4-7 membered monocyclic heterocyclic group containing 1-2 heteroatoms selected from N, O, and S is selected from those substituted with a C1-C6 alkyloxycarbonylamino group.

[0021] R5 is selected from: Where R a Selected from: 4-7 membered monocyclic heterocyclic groups comprising 1-2 heteroatoms selected from N, O, and S, wherein the 4-7 membered monocyclic heterocyclic group is optionally substituted with a C1-C6 alkyl group; R b Selected from: H, C1-C6 alkyl groups.

[0022] In some implementations, R1 and R2 are each independently selected from: H, methyl, or

[0023] R1 and R2, together with the N atom they contain, form a 5-10 member nitrogen-bridged heterocyclic group or a 5-6 member monocyclic heterocyclic group containing 1-2 heteroatoms selected from N and O. The 5-10 member nitrogen-bridged heterocyclic group or the 5-6 member monocyclic heterocyclic group containing 1-2 heteroatoms selected from N and O may optionally be substituted with C1-C6 alkyl or C2-C6 alkenyl carbonyl groups.

[0024] In some implementations, R1 and R2 are each independently selected from: H, methyl, or

[0025] R1, R2, together with the N atom they contain, form rings selected from the following:

[0026] In some embodiments, R3 and R4 are each independently selected from: H, C1-C6 alkyl, C3-C7 cycloalkyl optionally substituted with a hydroxyl group, or a six-membered monocyclic heterocyclic group containing one or two nitrogen atoms optionally substituted with a C1-C6 alkyl carbonyl group.

[0027] Among them, R c R d Each group is independently selected from: C1-C6 alkyl, hydroxyl, cyano, aminoC1-C6 alkyl, and aminoC1-C6 alkyl (wherein the amino group is optionally substituted by one or two C1-C6 alkyl groups).

[0028] Among them, R e R f R g Each is independently selected from: H, C1-C6 alkyl, hydroxy C1-C6 alkyl, carbamoyl, C1-C6 alkylaminocarbonyl, C1-C6 alkyloxycarbonylC3-C7 cycloalkylene, amino C1-C6 alkyl (wherein the amino group is optionally substituted with one or two C1-C6 alkyl groups), amino C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkyloxycarbonyl, C3-C7 cycloalkyloxyC1-C6 alkyl, C1-C6 alkyl optionally substituted with a six-membered monocyclic heterocyclic group containing one oxygen atom, C1-C6 alkyl optionally substituted with both C1-C6 haloalkyl and hydroxyl groups, C3-C7 cycloalkyl optionally substituted with C1-C6 alkyloxycarbonylaminoC1-C6 alkyl groups, and six-membered monocyclic heterocyclic groups containing one or two nitrogen atoms optionally substituted with C1-C6 alkyloxycarbonylamino groups.

[0029] In some embodiments, R3 and R4 are each independently selected from: H, C1-C6 alkyl (e.g., ethyl),

[0030] In some embodiments, R3 is selected from: H, C1-C6 alkyl (e.g., ethyl), and R4 is selected from:

[0031] In some implementations, R5 is selected from: Where R a1 R b1 Each is independently selected from: H, C1-C6 alkyl groups.

[0032] In some implementations, R5 is selected from:

[0033] In some embodiments, the compound is selected from:

[0034]

[0035]

[0036]

[0037] The second aspect of this application provides a method for preparing the compound described in the first aspect:

[0038]

[0039] Reagents and conditions: (a) N-R1R2, DIPEA, DCM, -40℃, 1h; (b) OH-R5, DIPEA, DCM, -20℃, overnight; (c) N-R3R4, DIPEA, DMSO, room temperature, overnight (for XS-1 to 19); or, N-R3R4, DIPEA, acetonitrile, 40℃, overnight (for XS-20 to 22, XS-25 to 34); or N-R3R4, Na2CO3, acetonitrile, 50℃, overnight (for XS-23 to 24).

[0040] The variables R1, R2, R3, R4, and R5 in the above synthesis route are defined as in the first aspect.

[0041] The third aspect of this application provides a pharmaceutical composition comprising at least one compound described in the first aspect of this application, or a stereoisomer thereof, a tautomer thereof, a solvate thereof, an isotopically labeled compound thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutical carriers and / or excipients.

[0042] The fourth aspect of this application provides the use of the compounds described in the first aspect of this application, or their stereoisomers, tautomers, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions described in the third aspect of this application, in the preparation of a medicament for treating and / or preventing diseases or conditions associated with KRAS G12D (e.g., cancer, such as KRAS G12D-related cancers).

[0043] In some embodiments, the cancer is selected from solid tumors (e.g., melanoma, carcinoma, or blastoma), primary tumors, secondary tumors (e.g., metastatic tumors), colorectal cancer (CRC) (e.g., rectal cancer), small bowel cancer, lung cancer (e.g., non-small cell lung cancer NSCLC, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), pancreatic cancer (e.g., adenocarcinoma), breast cancer (e.g., ductal breast cancer or breast adenocarcinoma), prostate cancer, ovarian cancer, brain cancer (e.g., glioblastoma), cervical cancer, gastric cancer, skin cancer, bile duct cancer, nervous system cancer (e.g., neuroblastoma), and melanoma.

[0044] The fifth aspect of this application provides the compounds described in the first aspect of this application, or their stereoisomers, tautomers, solvates, isotopically labeled compounds, or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions described in the third aspect of this application, for the treatment and / or prevention of diseases or conditions associated with KRAS G12D (e.g., cancer, such as KRAS G12D-related cancers).

[0045] In some embodiments, the cancer is selected from solid tumors (e.g., melanoma, carcinoma, or blastoma), primary tumors, secondary tumors (e.g., metastatic tumors), colorectal cancer (CRC) (e.g., rectal cancer), small bowel cancer, lung cancer (e.g., non-small cell lung cancer NSCLC, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), pancreatic cancer (e.g., adenocarcinoma), breast cancer (e.g., ductal breast cancer or breast adenocarcinoma), prostate cancer, ovarian cancer, brain cancer (e.g., glioblastoma), cervical cancer, gastric cancer, skin cancer, bile duct cancer, nervous system cancer (e.g., neuroblastoma), and melanoma.

[0046] The sixth aspect of this application provides a method for treating and / or preventing diseases or conditions associated with KRAS G12D (e.g., cancer, such as KRAS G12D-associated cancer), comprising administering to an individual in need an effective amount of the compound described in the first aspect of this application, or a stereoisomer thereof, or a tautomer thereof, or a solvate thereof, or an isotopically labeled compound thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in the third aspect of this application.

[0047] In some embodiments, the cancer is selected from solid tumors (e.g., melanoma, carcinoma, or blastoma), primary tumors, secondary tumors (e.g., metastatic tumors), colorectal cancer (CRC) (e.g., rectal cancer), small bowel cancer, lung cancer (e.g., non-small cell lung cancer NSCLC, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), pancreatic cancer (e.g., adenocarcinoma), breast cancer (e.g., ductal breast cancer or breast adenocarcinoma), prostate cancer, ovarian cancer, brain cancer (e.g., glioblastoma), cervical cancer, gastric cancer, skin cancer, bile duct cancer, nervous system cancer (e.g., neuroblastoma), and melanoma.

[0048] The seventh aspect of this application provides a method for screening KRAS G12D inhibitors. The corresponding KRAS-G12D protein sequence is shown in the sequence portion.

[0049] Terminology Definition

[0050] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this application, definitions and explanations of relevant terms are provided below.

[0051] In the event that the compound name used in this application is inconsistent with the chemical structural formula, the chemical structural formula shall prevail.

[0052] As used in this application, the term "stereoisomer" refers to an isomer formed by at least one asymmetric center. In compounds having one or more (e.g., 1, 2, 3, or 4) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules may also exist as geometric isomers (cis / trans).

[0053] Similarly, the compounds of this application may exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0054] Unless otherwise stated, the compounds of this application may exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof). The compounds of this application may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0055] The compounds of this application may exist in the form of solvates (such as hydrates), wherein the compounds of this application contain a solvent, such as water, methanol, or ethanol, as a structural element of the compound's crystal lattice. The amount of the solvent may be stoichiometric or non-stoichiometric.

[0056] As used herein, the term "isotope-labeled compound" refers to a compound in which one or more atoms are replaced by atoms of the same number but with a different atomic mass or mass number than the dominant atomic mass or mass number found in nature. Examples of isotopes suitable for inclusion in compounds of this application include, but are not limited to, hydrogen isotopes such as... 2 H, 3 H; carbon isotopes, for example 11 C, 13 C and 14C; Chlorine isotopes, for example 36 Cl; fluorine isotopes, for example 18 F; Iodine isotopes, for example 123 I and 125 I; nitrogen isotopes, for example 13 N and 15 N; oxygen isotopes, for example 15 O, 17 O and 18 O; and sulfur isotopes such as 35 S.

[0057] As used in this application, the term "pharmaceutically acceptable salt" refers to a salt of the compound of this application that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Such salts include: salts formed by addition to inorganic acids or acids formed with organic acids, or salts containing acidic protons on the parent compound but surrounded by metal ions, or coordination compounds formed with organic bases.

[0058] Pharmaceutically acceptable salts of the compounds described in this application include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by amino groups with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by other methods used in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioate, glucono-heptahydrate, glyceryl phosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, p-pentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and similar salts.

[0059] Salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium, and nitrogen. +(C1-4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and similar salts. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using the relative ions of halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0060] Pharmaceutically acceptable salts are also intended to include hemisalts, wherein the ratio of compound to acid is 2:1. Exemplary hemisalts are those derived from acids containing two carboxylic acid groups, such as malic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, glutaric acid, oxalic acid, adipic acid, and citric acid. Other exemplary hemisalts are those derived from diprotic mineral acids (e.g., sulfuric acid). Preferred exemplary hemisalts include (but are not limited to) hemi-maleic acid salts, hemi-fumaric acid salts, and hemi-succinic acid salts.

[0061] As used in this application, the term "optionally substituted" means that the group may be unsubstituted or substituted. For example, "C1-C6 alkyl optionally substituted with a halogen" means that the C1-C6 alkyl group may be unsubstituted or substituted with a halogen to obtain a haloalkyl group. It should be understood that when stating "R is selected from C1-C6 alkyl, -C1-C6 alkoxy, and -NH-C1-C6 alkyl, wherein the C1-C6 alkyl group is optionally substituted with a halogen," it means that the C1-C6 alkyl group in C1-C6 alkyl, -C1-C6 alkoxy, and -NH-C1-C6 alkyl is optionally substituted with a halogen. It should be understood that -N-(C1-C6 alkyl)2 indicates that two C1-C6 alkyl groups are attached to the nitrogen atom, and these two alkyl groups may be the same or different.

[0062] As used herein, unless otherwise expressly indicated, the descriptive phrase “...each independently selected” used throughout may mean either that the specific options expressed by the same or different symbols in different groups do not affect each other, or that the specific options expressed by the same or different symbols in the same group do not affect each other.

[0063] As used in this application, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0064] As used in this application, the term "alkyl" refers to a straight-chain or branched monovalent saturated hydrocarbon group, such as C1-C6 alkyl, which refers to a group having 1 to 6 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms. Examples of C1-C6 alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, etc., wherein the propyl group includes n-propyl and isopropyl, and the butyl group includes n-butyl, isobutyl, and neobutyl.

[0065] As used in this application, the term "halogenated" means that the modified group is substituted with one or more halogens, for example, substituted with 1, 2, 3, 4, 5 or 6 halogens. For example, "C1-C6 haloalkyl" means that a C1-C6 alkyl group as defined above is substituted with one or more halogens, examples of which include, but are not limited to, CF3, CHF2 or CF2CF3.

[0066] As used herein, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon group consisting of carbon atoms. For example, C3-C7 cycloalkyl groups have 3 to 6 carbon atoms, such as 3, 4, 5, 6, or 7 carbon atoms. The cycloalkyl group includes monocyclic, bicyclic, or polycyclic rings, including spirocyclic, fused, or bridged rings. Examples include, but are not limited to, cyclohexyl, cycloheptyl, and adamantyl.

[0067] As used in this application, the term "cycloalkylene" refers to a divalent cycloalkyl group formed by substituting one hydrogen atom of a cycloalkyl group. For example, C3-C6 cycloalkylene is a divalent cycloalkyl group formed by substituting one hydrogen atom of a C3-C6 cycloalkyl group, such as cyclopropylene, cyclobutylene, etc.

[0068] As used in this application, the term "alkoxy" refers to any of the above-described alkyl groups (e.g., C1-C6 alkyl groups) that are attached to the remainder of the molecule by an oxygen atom (-O-). For example, C1-C6 alkoxy groups.

[0069] As used in this application, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group with 2 to 6 carbon atoms, examples of which include vinyl, allyl, etc. A C2-6 alkenyl group must have at least one double bond.

[0070] As used in this application, the term "C2-C6 ynyl" refers to a straight or branched chain of 2-6 carbon atoms, such as ethynyl or propynyl. An ynyl group must have one triple bond and may optionally have one or more double bonds.

[0071] As used herein, the term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic group composed of ring atoms, wherein one, two, three, or four ring atoms are heteroatoms, and the remainder are carbon atoms; preferably, the heteroatoms are selected from N, O, or S, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may optionally be oxidized. For example, a 5-10 membered heterocyclic group refers to a group composed of 5-10 ring atoms, including 5-7 membered heterocyclic groups, 4-7 membered heterocyclic groups, etc. The heterocyclic group includes monocyclic, bicyclic, or polycyclic rings, including spirocyclic, fused, or bridged rings. Examples include, but are not limited to, oxocyclic butyl, azircyclic butyl, azircyclic pentyl, piperidinyl, piperazinyl, or morpholinyl. The term "nitrogen-containing monocyclic heterocyclic group" means that at least one heteroatom in the heterocyclic group is a nitrogen atom, and the heterocyclic group is monocyclic; examples include, but are not limited to, piperidinyl and piperazinyl. The term "nitrogen-bridged heterocyclic group" refers to a heterocyclic group in which at least one heteroatom is a nitrogen atom, and the heterocyclic group is a bridged ring. Examples include, but are not limited to, those containing nitrogen atoms. The term "nitrogen-containing fused heterocyclic group" refers to a heterocyclic group in which at least one heteroatom is a nitrogen atom, and the heterocyclic group is fused ring, examples of which include, but are not limited to, nitrogen-containing fused rings. The term "nitrogen-containing spirocyclic heterocyclic group" refers to a heterocyclic group in which at least one heteroatom is a nitrogen atom, and the heterocyclic group is a fused ring, examples of which include, but are not limited to, nitrogen-containing spirocyclic heterocyclic groups. wait.

[0072] As used in this application, the term "partially unsaturated" refers to a ring system that is neither saturated (i.e., does not contain double bonds) nor completely unsaturated (i.e., contains the maximum possible number of double bonds). In other words, a partially unsaturated ring system contains at least one double bond, but not the maximum possible number of double bonds.

[0073] From all the above descriptions, it will be apparent to those skilled in the art that any group of a compound name, such as "fluoroalkoxy," should refer to a composition conventionally derived from it, for example, from a fluorinated alkoxy group, wherein the alkyl group is as defined above. Similarly, "C1-C6 alkylaminocarbonyl" can be understood as: based on an aminocarbonyl group, wherein the amino group is replaced by a C1-C6 alkyl group. Similarly, the meanings of "C1-C6 alkyloxycarbonyl C3-C7 cycloalkylene", "C3-C7 cycloalkyloxy C1-C6 alkyl", etc., can be understood.

[0074] Similarly, any term such as alkylamino, dialkylamino, alkoxycarbonyl, alkoxycarbonylamino, heterocyclic carbonyl, heterocyclic carbonylamino, cycloalkyloxycarbonyl, alkoxyformyl, etc. includes groups, wherein the alkyl, alkoxy, aryl, C3-C7 cycloalkyl, and heterocyclic moieties are as defined above.

[0075] As used herein, the term "pharmaceutical carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, as is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutical carriers and / or excipients include, but are not limited to: pH adjusters, surfactants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, and stabilizers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and their analogues. Agents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in a pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0076] As used in this application, the term "effective amount" refers to the amount of a compound that, when administered, will alleviate one or more symptoms of the treated condition to a certain extent.

[0077] As used herein, the term "treatment" aims to alleviate, reduce, improve, or eliminate a targeted disease state or symptom. A subject is successfully "treated" if, in accordance with the methods described herein, a therapeutic amount of the antibody-drug conjugate or its racemic, enantiomer, diastereomer, pharmaceutically acceptable salt, or mixture of the foregoing forms is received, and one or more indications and symptoms exhibit an observable and / or detectable reduction or improvement. It should also be understood that treatment of the disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

[0078] As used in this application, the term "prevention" aims to avoid, reduce, prevent, or delay the onset of a disease or disease-related symptoms before the onset of the relevant drug. "Prevention" does not necessarily require the complete prevention of the onset of a disease or disease-related symptoms. For example, reducing the risk of a subject developing a specific disease or disease-related symptoms after the administration of the relevant drug, or lessening the severity of subsequently occurring related symptoms, can be considered as "prevention" of the onset or development of the disease. Detailed Implementation

[0079] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available products or can be prepared by methods known to those skilled in the art.

[0080] The English words or abbreviations used below have the following meanings:

[0081]

[0082]

[0083] Synthetic route

[0084]

[0085] Reagents and conditions: (a) N-R1R2, DIPEA, DCM, -40℃, 1h; (b) OH-R5, DIPEA, DCM, -20℃, overnight; (c) N-R3R4, DIPEA, DMSO, room temperature, overnight (for XS-1 to 19); or, N-R3R4, DIPEA, acetonitrile, 40℃, overnight (for XS-20 to 22, XS-25 to 34); or N-R3R4, Na2CO3, acetonitrile, 50℃, overnight (for XS-23 to 24).

[0086] The variables R1, R2, R3, R4, and R5 in the above synthetic route are defined as in the first aspect of the invention.

[0087] Preparation Examples 1-19: Compounds XS-1 to 19

[0088] Operating procedures (taking XS-1 as an example)

[0089] (2S)-2-(1-hydroxyethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (1)

[0090]

[0091] (S)-2-formylpyrrolidine-1-carboxylic acid tert-butyl ester (6.0 g, 30.1 mmol, 1.00 eq.) (Biode Pharmaceuticals, CAS No.: 69610-41-9) was added to a 500 mL round-bottom flask, dissolved in anhydrous tetrahydrofuran (100 mL), and stirred until homogeneous. Under a nitrogen atmosphere, the mixture was cooled to -80 °C, and methyl magnesium chloride (60 mL, 3 M in THF, 60.6 mmol, 6.00 eq.) was slowly added. The reaction was carried out at -80 °C for 4 h. The reaction was monitored by TLC (DCM:MeOH = 10:1, 1 / 100NH3OH). After the reaction of starting material a was complete, saturated ammonium chloride solution was slowly added dropwise to quench the reaction. The mixture was then transferred to room temperature, and the reaction solution was transferred to a separatory funnel. Extraction was performed by adding EA and H2O. The organic phase was washed three times with water, and the combined organic phases were dried over Na2SO4. The mixture was then concentrated under vacuum to obtain a pale yellow oily compound 1 (6.2 g, 95.7%).

[0092] (S)-1-((S)-1-methylpyrrolidone-2-yl)-ethanol(2)

[0093]

[0094] Compound 1 (6.2 g, 28.8 mmol, 1.00 eq.) was added to a 250 mL three-necked round-bottom flask, dissolved in anhydrous tetrahydrofuran (100 mL), and stirred until homogeneous. Under a nitrogen atmosphere, the mixture was cooled to 0 °C, and LiAlH4 (23 mL, 2.5 min THF, 57.6 mmol, 2.00 eq.) was slowly added. The mixture was reacted at 0 °C for 1 h, then refluxed at 70 °C for 3 h. The reaction was monitored by TLC (DCM:MeOH = 5:1, 1 / 100 NH3OH). After the reaction of starting material 1 was complete, the mixture was transferred to 0 °C, and water was slowly added dropwise to quench the reaction. The mixture was then transferred to room temperature, filtered, and concentrated under vacuum to obtain a colorless, transparent oily compound 2 (3 g, 80.6%).

[0095] 4,6-Dichloro-2-dimethylamino-1,3,5-triazine (3)

[0096]

[0097] Cyanuron chloride (5 g, 27.3 mmol, 1.00 eq.) was added to a 100 mL round-bottom flask, dissolved in dichloromethane (20 mL), and stirred until homogeneous. The mixture was cooled to -40 °C, and dimethylamine hydrochloride (1.84 g, 27.3 mmol, 1.00 eq.) and DIPEA (8 mL, 46.4 mmol, 2.00 eq.) were added. The mixture was reacted at -40 °C for 1 h. After the reaction of starting material b was complete, the mixture was transferred to room temperature, and the reaction solution was transferred to a separatory funnel. DCM and H2O were added for extraction. The organic phase was washed three times with water, and the combined organic phases were dried over Na2SO4. The crude product was concentrated under vacuum and purified by rapid silica gel chromatography (PE:EA = 5:1). The product was then concentrated under vacuum to obtain a pale yellow solid compound 3 (5 g, 95.4%). MS (ESI) 192.00 [M+H] + .

[0098] 4-Chloro-N,N-dimethyl-6-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-1,3,5-triazine-2-amine (4)

[0099]

[0100] Compound 3 (4 g, 23.2 mmol, 1.00 eq.) was added to a 100 mL round-bottom flask, dissolved in 20 mL of dichloromethane, and stirred until homogeneous. The mixture was cooled to -20 °C, and (S)-1-((S)-1-methylpyrrolidin-2-yl)-ethanol (3 g, 23.2 mmol, 1.00 eq.) and DIPEA (8 mL, 46.4 mmol, 2.00 eq.) were added. The mixture was reacted overnight at -20 °C, and the reaction was performed by TLC (Phase I). After the reaction of starting material 3 was complete, monitored by LC-MS (PE:EA = 1:1, 1 / 100NH3OH), the mixture was transferred to room temperature and then to a separatory funnel. DCM and H2O were added for extraction. The organic phase was washed three times with water, and the combined organic phases were dried over Na2SO4. The crude product was obtained by vacuum concentration and purified by rapid silica gel chromatography (PE:EA = 1:1, 1 / 100NH3OH). The product was then concentrated under vacuum to obtain compound 4 (4.3 g, 71.7%), a white solid. MS (ESI) 285.13 [M+H] + .

[0101] 2-((4-(dimethylamino)-6-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-1,3,5-triazin-2-yl)amino)-5-ethylphenol (XS-1)

[0102]

[0103] Compound 4 (0.2 g, 0.74 mmol, 1.00 eq.) and 6-amino-3-ethylphenol hydrochloride (0.2 g, 1.48 mmol, 2 eq.) were added to a 50 mL reaction flask. Dimethyl sulfoxide (10 mL) was added to dissolve the compound, and the mixture was stirred until homogeneous. Dipyraclostrobin (0.26 mL, 1.5 mmol, 2.00 eq.) was added, and the mixture was reacted overnight at room temperature. After the reaction of starting material 4 was monitored by TLC and LC-MS, the reaction solution was transferred to a separatory funnel, and EA and H2O were added for extraction. The organic phase was washed three times with water, and the combined organic phases were dried over Na2SO4. The crude product was obtained by vacuum concentration and purified by rapid silica gel chromatography (PE:EA = 2:1, 1 / 100 NH3OH). The product was then purified by vacuum concentration to obtain compound XS-1, or dissolved in DMF and purified by semi-preparative chromatography to obtain a brown solid (83 mg, 33%) with a purity of 100.00%. Molecular formula: C 20 H 30 N6O2; MS (ESI) 387.04 [M+H] + .

[0104] Preparation Examples 20-22: Compounds XS-20~22

[0105] Operating procedures (taking compound XS-20 as an example)

[0106] tert-Butyl(1R,5S)-3-(4,6-dichloro-1,3,5-triazin-2-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (5)

[0107]

[0108] Cyanuric chloride (5 g, 27.3 mmol, 1.00 eq.) was added to a 100 mL round-bottom flask, and dichloromethane (20 mL) was added to dissolve it. The mixture was stirred until homogeneous and cooled to -40 °C. 3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (5.79 g, 27.3 mmol, 1.00 eq.) and DIPEA (8 mL, 46.4 mmol, 2.00 eq.) were added. The mixture was reacted at -40 °C for 1 h. After the reaction of starting material b was monitored by TLC (PE:EA = 10:1) and LC-MS, the mixture was transferred to room temperature. The reaction solution was transferred to a separatory funnel and extracted with DCM and H2O. The organic phase was washed three times with water, and the organic phases were combined and dried with Na2SO4. The crude product was obtained by vacuum concentration. The crude product was purified and separated by rapid silica gel chromatography (PE:EA = 10:1). The crude product was then concentrated under vacuum to obtain a white solid compound 5 (9.6 g, 98.3%). MS(ESI) 360.09 [M+H] + .

[0109] tert-Butyl(1R,5S)-3-(4-chloro-6-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-1,3,5-triazin-2-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid ester (6)

[0110]

[0111] Compound 5 (9.6 g, 26.7 mmol, 1.00 eq.) was added to a 100 mL round-bottom flask, dissolved in 20 mL of dichloromethane, and stirred until homogeneous. The mixture was cooled to -20 °C, and (S)-1-((S)-1-methylpyrrolidin-2-yl)-ethanol (3.4 g, 26.7 mmol, 1.00 eq.) and DIPEA (8 mL, 46.4 mmol, 2.00 eq.) were added. The mixture was reacted overnight at 0 °C, and the reaction was performed by TLC (DCM:M). After the reaction of starting material 5 was complete, monitored by LC-MS (eOH = 20:1, NH3OH), the mixture was transferred to room temperature and then to a separatory funnel. DCM and H2O were added for extraction. The organic phase was washed three times with water, and the combined organic phases were dried over Na2SO4. The crude product was obtained by vacuum concentration and purified by rapid silica gel chromatography (DCM:MeOH = 20:1, 1 / 100NH3OH). The product was then concentrated under vacuum to obtain a white solid compound 6 (5.2 g, 49.5%). MS (ESI) 453.23 [M+H] + .

[0112] tert-Butyl(1R,5S)-3-(4-((4-ethyl-2-hydroxyphenyl)amino)-6-((S)-1-(S)-1-methylpyrrolidone-2-yl)ethoxy)-1,3,5-triazin-2-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid ester (7)

[0113]

[0114] Compound 6 (300 mg, 0.74 mmol, 1.00 eq.) and 6-amino-3-ethylphenol hydrochloride (300 mg, 1.48 mmol, 2 eq.) were added to a 50 mL reaction flask, dissolved in 10 mL of acetonitrile, and stirred until homogeneous. DIPEA (0.26 mL, 1.5 mmol, 2.00 eq.) was added, and the mixture was reacted overnight at room temperature. After the reaction of starting material 6 was monitored by TLC and LC-MS, the reaction solution was transferred to a separatory funnel, extracted with EA and H2O, and the organic phase was washed three times with water. The combined organic phases were dried over Na2SO4 and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid silica gel chromatography (PE:EA = 2:1, 1 / 100 NH3OH) to obtain compound 7, a brown solid (167 mg, 38%); MS (ESI) 554.34 [M+H]. + .

[0115] 2-((4-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-6-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-1,3,5-triazin-2-yl)amino)-5-ethylphenol (XS-20)

[0116]

[0117] Compound 7 (167 mg, 0.31 mmol, 1.00 eq.) was added to a 10 mL reaction flask, dissolved in 2 mL of DCM, and stirred until homogeneous. TFA (0.47 mL, 6.2 mmol, 20 eq.) was then added, and the mixture was reacted overnight at room temperature. After the reaction of starting material 7 was monitored by TLC and LC-MS, the mixture was concentrated under vacuum to obtain a crude product. This crude product was dissolved in DMF, purified by semi-preparative chromatography, and lyophilized to obtain compound XS-20, a brown solid with a purity of 100.00%. Molecular formula: C 24 H 35 N7O2(48mg,63.5%);MS(ESI)454.29[M+H] + .

[0118] Preparation Examples 23-24: Compounds XS-23-24

[0119] Operating procedures (taking XS-23 as an example)

[0120] Benzyl 8-(4,6-dichloro-1,3,5-triazin-2-yl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylic acid ester (8)

[0121]

[0122] Cyanurium chloride (5 g, 27.3 mmol, 1.00 eq.) was added to a 100 mL round-bottom flask, and dichloromethane (20 mL) was added to dissolve it. The mixture was stirred until homogeneous and cooled to -40 °C. Benzyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate (6.72 g, 27.3 mmol, 1.00 eq.) and DIPEA (8 mL, 46.4 mmol, 2.00 eq.) were added. The mixture was reacted at -40 °C for 1 h. After the reaction of starting material b was monitored by TLC (PE:EA = 10:1) and LC-MS, the mixture was transferred to room temperature. The reaction solution was transferred to a separatory funnel, and DCM and H2O were added for extraction. The organic phase was washed three times with water, and the combined organic phases were dried with Na2SO4. The crude product was obtained by vacuum concentration. The crude product was purified and separated by rapid silica gel chromatography (PE:EA = 10:1). The crude product was then concentrated under vacuum to obtain a white solid compound 8 (8.6 g, 80.0%). MS(ESI) 394.08 [M+H] + .

[0123] Benzyl(1R,5S)-3-(4,6-dichloro-1,3,5-triazin-2-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid ester (9)

[0124]

[0125] Compound 8 (8.6 g, 21.9 mmol, 1.00 eq.) was added to a 100 mL round-bottom flask, dissolved in 20 mL of dichloromethane, and stirred until homogeneous. The mixture was cooled to -20 °C, and (S)-1-((S)-1-methylpyrrolidin-2-yl)-ethanol (2.8 g, 21.9 mmol, 1.00 eq.) and DIPEA (8 mL, 46.4 mmol, 2.00 eq.) were added. The mixture was reacted overnight at 0 °C, and the reaction was analyzed by TLC (DCM:M). After the reaction of starting material 8 was complete, monitored by LC-MS (eOH = 20:1, NH3OH), the mixture was transferred to room temperature and then to a separatory funnel. DCM and H2O were added for extraction. The organic phase was washed three times with water, and the combined organic phases were dried over Na2SO4. The crude product was obtained by vacuum concentration and purified by rapid silica gel chromatography (DCM:MeOH = 20:1, 1 / 100NH3OH). The product was then concentrated under vacuum to obtain a white solid compound 9 (4.8 g, 45.1%). MS (ESI) 487.21 [M+H] + .

[0126] Benzyl(1R,5S)-3-(4-((1R,4S)-4-((tert-butoxycarbonyl)amino)methyl)cyclohexyl)methyl)-6-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-1,3,5-triazin-2-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid ester)(10)

[0127]

[0128] Compound 9 (300 mg, 0.62 mmol, 1.00 eq.) and N-tert-butoxycarbonyl-1,4-TRANS-diaminomethylcyclohexane (300 mg, 1.24 mmol, 2 eq.) (Biode Pharmaceuticals, CAS No.: 166168-16-7) were added to a 50 mL reaction flask, dissolved in acetonitrile (10 mL), and stirred until homogeneous. Na₂CO₃ (0.26 mL, 1.5 mmol, 2.00 eq.) was then added, and the reaction was allowed to proceed overnight at room temperature. T After the reaction of raw material 9 was complete, the reaction solution was transferred to a separatory funnel and extracted with EA and H2O. The organic phase was washed three times with water, and the combined organic phases were dried with Na2SO4. The crude product was concentrated under vacuum and purified by rapid silica gel chromatography (PE:EA = 2:1, 1 / 100NH3OH). Compound 10 was then concentrated under vacuum to obtain a white solid (221 mg, 51.5%). MS (ESI) 693.44 [M+H] + .

[0129] tert-Butyl((1S,4R)-4-(((4-(((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-6-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-1,3,5-triazin-2-yl)amino)methyl)cyclohexyl)methyl)carbamate (XS-23)

[0130]

[0131] Compound 10 (221 mg, 0.32 mmol, 1.00 eq.) was added to a 10 mL reaction flask, dissolved in 2 mL of MeOH, and stirred until homogeneous. Pd (6.81 mg, 0.06 mmol, 0.2 eq.) was then added. The reaction was carried out overnight at room temperature under a hydrogen atmosphere. After the reaction of starting material 10 was monitored by TLC and LC-MS, the mixture was filtered, concentrated under vacuum to obtain the crude product, dissolved in DMF, and purified by semi-preparative chromatography. After lyophilization, compound XS-23 was obtained as a white solid with a purity of 98.00%. Molecular formula: C 29 H 50N8O3(64mg,35.9%);MS(ESI)559.4[M+H] + .

[0132] Preparation Examples 25-29: Compounds XS-25~29

[0133] Operating procedures (taking XS-25 as an example)

[0134] tert-Butyl 4-(4,6-dichloro-1,3,5-triazin-2-yl)piperazine-1-carboxylate (11)

[0135]

[0136] Cyanurium chloride (5 g, 27.3 mmol, 1.00 eq.) was added to a 100 mL round-bottom flask, and dichloromethane (20 mL) was added to dissolve it. The mixture was stirred until homogeneous and cooled to -40 °C. Tert-butylpiperazine-1-carboxylic acid ester (5.08 g, 27.3 mmol, 1.00 eq.) and DIPEA (8 mL, 46.4 mmol, 2.00 eq.) were added. The mixture was reacted at -40 °C for 1 h. After the reaction of starting material b was completed, the mixture was transferred to room temperature and the reaction solution was transferred to a separatory funnel. DCM and H2O were added for extraction. The organic phase was washed three times with water, and the combined organic phases were dried with Na2SO4. The crude product was obtained by vacuum concentration. The crude product was purified and separated by rapid silica gel chromatography (PE:EA = 10:1). The crude product was then concentrated under vacuum to obtain a white solid compound 11 (7.8 g, 86.7%). MS(ESI) 334.08 [M+H] + .

[0137] tert-Butyl 4-(4-chloro-6-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-1,3,5-triazin-2-yl)piperazine-1-carboxylic acid ester (12)

[0138]

[0139] Compound 11 (7.8 g, 23.4 mmol, 1.00 eq.) was added to a 100 mL round-bottom flask, dissolved in 20 mL of dichloromethane, and stirred until homogeneous. The mixture was cooled to -20 °C, and (S)-1-((S)-1-methylpyrrolidin-2-yl)-ethanol (3.0 g, 23.4 mmol, 1.00 eq.) and DIPEA (8 mL, 46.4 mmol, 2.00 eq.) were added. The mixture was reacted overnight at 0 °C, and the reaction was analyzed by TLC (DCM:Me). After the reaction of starting material 11 was complete, monitored by LC-MS (OH = 20:1, NH3OH), the mixture was transferred to room temperature and then to a separatory funnel. DCM and H2O were added for extraction. The organic phase was washed three times with water, and the combined organic phases were dried over Na2SO4. The crude product was obtained by vacuum concentration and purified by rapid silica gel chromatography (DCM:MeOH = 20:1, 1 / 100NH3OH). The product was then concentrated under vacuum to obtain a white solid compound 12 (5.7 g, 57.2%). MS (ESI) 427.21 [M+H] + .

[0140] 2-Chloro-4-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-6-(piperazin-1-yl)-1,3,5-triazine (13)

[0141]

[0142] Compound 12 (5.7 g, 13.4 mmol, 1.00 eq.) was added to a 100 mL reaction flask, dissolved in 10 mL of DCM, and stirred until homogeneous. TFA (20.52 mL, 268 mmol, 20 eq.) was then added, and the mixture was reacted overnight at room temperature. After the reaction of starting material 12 was complete, it was monitored by TLC and LC-MS. The mixture was then concentrated under vacuum to obtain the crude product, compound 13 (4.5 g, 97.3%), a white solid. MS (ESI) 327.21 [M+H] + .

[0143] 1-(4-(4-chloro-6-)((S)-1-methylpyrrolidone-2-yl)ethoxy)-1,3,5-triazin-2-yl)piperazin-1-yl)prop-2-en-1-one (14)

[0144]

[0145] Compound 13 (4.5 g, 13.8 mmol, 1.00 eq.) was added to a 100 mL reaction flask, dissolved in 10 mL of DCM, and stirred until homogeneous. The mixture was cooled to 0 °C, and then TEA (3.84 mL, 27.6 mmol, 2 eq.) and acryloyl chloride (1.68 mL, 20.7 mmol, 1.5 eq.) were added. The mixture was reacted at room temperature for 1 h. After the reaction of starting material 13 was complete, it was extracted three times with DCM and saturated sodium bicarbonate solution. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the crude product, a white solid compound 14 (4.2 g, 80.8%). MS (ESI) 381.17 [M+H] + .

[0146] 1-(4-(4-((4-ethyl-2-hydroxyphenyl)amino)-6-((S)-1-((S)-1-methylpyrrolidone-2-yl)ethoxy)-1,3,5-triazin-2-yl)piperazin-1-yl)prop-2-en-1-one (XS-25)

[0147]

[0148] Compound 14 (200 mg, 0.53 mmol, 1.00 eq.) and 6-amino-3-ethylphenol hydrochloride (150 mg, 1.06 mmol, 2 eq.) were added to a 50 mL reaction flask, dissolved in acetonitrile (10 mL), and stirred until homogeneous. DIPEA (0.18 mL, 1.06 mmol, 2.00 eq.) was added, and the reaction was allowed to proceed overnight at room temperature. After the reaction of starting material 14 was complete, the reaction mixture was transferred to a separatory funnel, extracted with EA and H2O, and the organic phase was washed three times with water. The combined organic phases were dried over Na2SO4 and concentrated under vacuum to obtain a crude product. The crude product was purified by rapid silica gel chromatography (PE:EA = 2:1, 1 / 100 NH3OH). The crude product was then dissolved in DMF and purified by semi-preparative chromatography to obtain a brown solid compound XS-25 (63 mg, 24.7%) with a purity of 97.00%. Molecular formula: C 25 H 35 N7O3; MS(ESI) 482.28 [M+H]+.

[0149] Preparation Examples 30-34: Compounds XS-30~34

[0150] Operating steps

[0151] 3-Hydroxy-4-nitrobenzenenitrile (15)

[0152]

[0153] Compound c (5 g, 30.12 mmol, 1.00 eq.) was dissolved in dimethyl sulfoxide (50 mL), and then an aqueous solution of potassium hydroxide (9.6 g, 171.68 mmol, 5.70 eq.) (10 mL) was slowly added. The reaction mixture was kept at room temperature overnight. After the reaction of starting material c was completed by TLC, the pH of the solution was adjusted to 2 with dilute hydrochloric acid, and then extracted three times with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum to obtain the crude product. The crude product, compound 15 (4.3 g, 86.0%), was purified by rapid silica gel chromatography (PE:EA = 20:1).

[0154] 4-Amino-3-hydroxybenzonitrile (16)

[0155]

[0156] Compound 15 (4.3 g, 26.21 mmol, 1.00 eq.) and palladium on carbon (2 mg, 10%) were added to tetrahydrofuran (20 ml). The mixture was heated to 40 °C and stirred overnight under a hydrogen atmosphere. After the reaction of starting material 15 was completed, the reaction solution was cooled to room temperature, palladium on carbon was filtered off, and the filtrate was concentrated under reduced pressure to obtain crude product compound 16 (3.2 g, 88.6%).

[0157] tert-Butyl(1R,5S)-3-(4-chloro-6-(((S)-1-methylpyrrolidone-2-yl)methoxy)-1,3,5-triazin-2-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid ester (17)

[0158]

[0159] Compound 5 (5 g, 13.9 mmol, 1.00 eq.) was added to a 100 mL round-bottom flask, dissolved in 20 mL of dichloromethane, and stirred until homogeneous. The mixture was cooled to -20 °C, and (S)-(-)-1-methyl-2-pyrrolidinemethanol (1.6 g, 13.9 mmol, 1.00 eq.) and DIPEA (4.8 mL, 27.8 mmol, 2.00 eq.) were added. The mixture was reacted overnight at 0 °C. The reaction was then analyzed by TLC (DCM: MeOH = ... After the reaction of starting material 5 was complete, monitored by LC-MS (20:1, NH3OH), the mixture was transferred to room temperature and then to a separatory funnel. DCM and H2O were added for extraction. The organic phase was washed three times with water, and the combined organic phases were dried over Na2SO4. The crude product was obtained by vacuum concentration and purified by rapid silica gel chromatography (DCM:MeOH = 20:1, 1 / 100NH3OH). The product was then concentrated under vacuum to obtain a white solid compound 17 (5.3 g, 86.9%). MS (ESI) 439.21 [M+H] + .

[0160] tert-Butyl(1R,5S)-3-(4-((4-cyano-2-hydroxyphenyl)amino)-6-(((S)-1-methylpyrrolidone-2-yl)methoxy)-1,3,5-triazin-2-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid ester (18)

[0161]

[0162] Compound 17 (5.3 g, 12.1 mmol, 1.00 eq.) and 4-amino-3-hydroxybenzonitrile (3.2 g, 24.2 mmol, 2 eq.) were added to a 50 mL reaction flask, dissolved in 10 mL of acetonitrile, and stirred until homogeneous. DIPEA (4.2 mL, 24.2 mmol, 2.00 eq.) was added, and the mixture was reacted overnight at room temperature. After the reaction of starting material 17 was monitored by TLC and LC-MS, the reaction solution was transferred to a separatory funnel, extracted with EA and H2O, and the organic phase was washed three times with water. The combined organic phases were dried over Na2SO4 and concentrated under vacuum to obtain the crude product. The crude product was purified by rapid silica gel chromatography (PE:EA = 2:1, 1 / 100 NH3OH) to obtain compound 18, a brown solid (6.1 g, 93.8%); MS (ESI) 537.29 [M+H]. + .

[0163] 4-((4-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-6-(((S)-1-methylpyrrolidone-2-yl)methoxy)-1,3,5-triazin-2-yl)amino)-3-hydroxybenzonitrile (XS-30)

[0164]

[0165] Compound 18 (6.1 g, 11.4 mmol, 1.00 eq.) was added to a 100 mL reaction flask, dissolved in 2 mL of DCM, and stirred until homogeneous. TFA (17.3 mL, 228 mmol, 20 eq.) was then added, and the mixture was reacted overnight at room temperature. After the reaction of starting material 18 was monitored by TLC and LC-MS, the mixture was concentrated under vacuum to obtain a crude product. This crude product was dissolved in DMF, purified by semi-preparative chromatography, and lyophilized to obtain compound XS-30, a brown solid with a purity of 98.00%. Molecular formula: C 22 H 28 N8O2 (4.1g, 83.7%); MS (ESI) 437.29 [M+H] + .

[0166] 2-((4-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-6-(((S)-1-methylpyrrolidone-2-yl)methoxy)-1,3,5-triazin-2-yl)amino)-5-(aminomethyl)phenol (XS-31)

[0167]

[0168] Compound XS-30 (2 g, 4.6 mmol, 1.00 eq.) was added to a 100 mL reaction flask, dissolved in tetrahydrofuran, and cooled to 0°C. Lithium aluminum hydride (5.5 mL, 2.5 M in THF, 13.8 mmol, 3.00 eq.) was slowly added dropwise under a nitrogen atmosphere. The mixture was refluxed at 70°C for 3 h. After the reaction of the starting material XS-30 was complete, the mixture was cooled to room temperature, and potassium hydroxide solution was slowly added dropwise with vigorous stirring overnight. The mixture was filtered, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in DMF, purified by semi-preparative chromatography, and lyophilized to obtain compound XS-31 with a purity of 96.00%. Molecular formula: C 22 H 32 N8O2 (0.9g, 45.0%); MS (ESI) 441.26 [M+H] + .

[0169] 2-((4-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-6-(((S)-1-methylpyrrolidone-2-yl)methoxy)-1,3,5-triazin-2-yl)amino)-5-((methylamino)methyl)phenol (XS-32)

[0170] 2-((4-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-6-(((S)-1-methylpyrrolidone-2-yl)methoxy)-1,3,5-triazin-2-yl)amino)-5-(dimethylamino)methyl)phenol (XS-33)

[0171]

[0172] Compound XS-31 (0.8 g, 0.8 mmol, 1.00 eq.) was added to a 50 mL reaction flask, dissolved in hexafluoroisopropanol, and then methyl trifluoromethanesulfonate (0.2 g, 1.2 mmol, 1.5 eq.) was added. The mixture was stirred at room temperature for 1 hour. After the reaction of the starting material XS-31 was complete, it was quenched with hydrochloric acid solution, evaporated under reduced pressure, and purified by rapid silica gel chromatography (PE:EA = 2:1, 1 / 100NH3OH) to obtain the product compound XS-32 with a purity of 98.14%. Molecular formula: C23 H 34 N8O2 (0.25g, 68.1%); MS (ESI) 455.28 [M+H] + Compound XS-33, purity: 95.27%, molecular formula: C 24 H 36 N8O2 (0.08g, 20.3%); MS (ESI) 469.32 [M+H] + .

[0173] 2-((4-((1R,5S)-3,8-diazabicyclo[3.2.1]octane-3-yl)-6-(((S)-1-methylpyrrolidone-2-yl)methoxy)-1,3,5-triazin-2-yl)amino)-5-(2-aminopropyl-2-yl)phenol (XS-34)

[0174]

[0175] Compound XS-30 (2 g, 4.6 mmol, 1.00 eq.) was added to a 250 mL reaction flask, dissolved in tetrahydrofuran, and then tert-butylmagnesium bromide (3 M in THF, 3 mL, 9.2 mmol, 2 eq.) was added under a nitrogen atmosphere. The reaction was carried out at room temperature for 3 hours, followed by the addition of methanol. Excess Grignard reagent was quenched at 0°C, and sodium borohydride (0.26 g, 6.9 mmol, 1.5 eq.) was added. The reaction was carried out overnight at room temperature. After the reaction was completed, the reaction was monitored by TLC and LC-MS. The reaction was then quenched with saturated ammonium chloride solution, evaporated under reduced pressure, and purified by rapid silica gel chromatography (PE:EA = 10:1, 1 / 100 NH3OH) to obtain the product compound XS-34 with a purity of 96.32%. Molecular formula: C 24 H 36 N8O2(1.1g,52.4%);MS(ESI)469.32[M+H] + .

[0176] The specific compound structures and characterization data are as follows:

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] Activity test example:

[0186] Test Example 1: Cell viability assay to assess the selectivity of compounds:

[0187] The Ba / F3 modified KRAS-WT / G12C / G12D / G12V cell line was purchased from Yuanjing Biotechnology. The cells were seeded in white 384-well plates with 1000 cells per well. The plates were incubated at 37°C and 5% CO2 for 4 hours. The test compound was then added in serially diluted form, ensuring that the final concentration of DMSO was less than 0.5%. Incubation continued for 3 days. After that, 30 μl of Cell titer glo reagent (Beyotime, C0069XL) was added. The plates were read using a Biotech microplate reader, and the total fluorescence signal was used to reflect the degree of inhibition of cell viability by the test compound.

[0188] Test results show that compound 21 prepared in this application has an effect on the IC50 of Ba / F3-KRAS-G12D cells. 50 The IC50 concentration was 5.1 μM, and it had an effect on Ba / F3-KRAS-wild type cells. 50 The IC50 concentration was 4.1 μM, and its effect on Ba / F3-KRAS-G12C cells was [not specified]. 50 Greater than 20 μM, IC50 in Ba / F3-KRAS-G12V cells 50 It has a concentration greater than 20 μM, thus exhibiting strong cell selectivity.

[0189] Compound 22 prepared in this application has an effect on the IC50 of Ba / F3-KRAS-G12D cells. 50 The IC50 concentration for Ba / F3-KRAS-wild type cells was 6.5 μM. 50 The concentration was 3.7 μM, and the IC50 concentration against Ba / F3-KRAS-G12C cells was [missing value]. 50 Greater than 20 μM, IC50 in Ba / F3-KRAS-G12V cells 50 It has a concentration greater than 20 μM, thus exhibiting strong cell selectivity.

[0190] Test Example 2: KRAS-RAF1 PPI assay for compound activity:

[0191] The procedure was performed using a kit (63ADK000CB47PEH) purchased from CISBIO. First, the 500X tag1-KRAS-G12D protein was slowly dissolved on ice and diluted to 5X with diluent buffer (50mM HEPES, pH 7.5, 5mM MgCl2, 0.005% Tween 20, 1mM DTT). The 500X tag2-RAF1 protein was also slowly dissolved on ice and diluted to 5X with diluent buffer. The 100X anti-tag1-Tb3+ protein was slowly dissolved on ice and diluted to 1X with detection buffer (50mM HEPES, pH 7.5, 5mM MgCl2, 0.005% Tween 20, 1mM DTT). The 100X anti-tag1-d2 protein was also slowly dissolved on ice and diluted to 1X with detection buffer. The 34 test compounds prepared in this application were then analyzed using diluent buffer. The buffer was diluted to different concentrations to ensure that the DMSO content was consistent and less than 0.5%. 4 μl of tag1-KRAS-G12D, 4 μl of tag2-RAF1, and 2 μl of the test compound were added to a black 384-well plate and incubated at room temperature for 15 min. 5 μl of anti-tag1-Tb3+ and 5 μl of anti-tag1-d2 were added to the black 384-well plate and incubated at 4 °C for 2 h. The plate was read using a Biotech microplate reader, and the activity of the test compound was reflected by detecting the Em620 / Ex665 signal.

[0192] The results showed that all compounds were effective against KRAS-G12D IC50. 50 All are greater than 20 μM.

[0193] Test Example 3: CY5-tracer assay to evaluate compound activity:

[0194] First, a Cy5-tagged MRTX1133 analogue was synthesized as a probe tracer, and a high-concentration stock solution was prepared using DMSO and stored at -20°C. Next, the test compound was diluted: the 10 mM stock solution was diluted to 0.4 mM with DMSO, followed by a 1:3 serial dilution for 8 spots. 0.5 μl of the test compound was added to 49.5 μl of assay buffer (25 mM Hepes pH 7.4, 200 mM NaCl, 1 mM DTT, 0.005% Tween-20, 5 mM MgCl2), and the highest concentration was 4 μM (4x). KRAS-G12D protein was diluted with assay buffer to a concentration of 240 nM (4x). 5 μl of protein and 5 μl of the test compound were added to a black 384-well plate and incubated at room temperature for 10 min. Anit-his-Tb was diluted with assay buffer to a concentration of 4 nM (4x). Dilute Cy5-tracer with assay buffer to a concentration of 200 nM (4x). Mix equal volumes of Anti-his-Tb and Cy5-tracer. Add 10 μl of the mixture to a black 384-well plate and incubate at room temperature for 10 min.

[0195] The activity of the test compound is reflected by detecting the Em620 / Ex665 signal in the plate reading of the Biotech microplate reader.

[0196]

[0197] + indicates IC 50 >20μM

[0198] ++ indicates 10μM <IC 50 <20μM

[0199] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit them; although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of this application or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of this application, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in this application.

Claims

1. The compound represented by formula (I), or its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, Formula (I) in, R1 and R2 are each independently selected from: H, C1-C6 alkyl groups, or R1 and R2, together with the N atoms they contain, form Piperazine group, the The piperazine group may optionally be substituted with a C1-C6 alkyl group or a C2-C6 alkenyl carbonyl group; R3 and R4 are each selected independently from: H, C1-C6 alkyl, C3-C7 cycloalkyl optionally substituted with hydroxyl, or a six-membered monocyclic heterocyclic group containing one nitrogen atom optionally substituted with a C1-C6 alkyl carbonyl group. Among them, R c R d Each group is independently selected from: C1-C6 alkyl, hydroxy, cyano, aminoC1-C6 alkyl, and aminoC1-C6 alkyl, wherein the amino group in the aminoC1-C6 alkyl group is optionally substituted by one or two C1-C6 alkyl groups. Among them, R e R f R g Each is independently selected from: H, C1-C6 alkyl, hydroxy C1-C6 alkyl, carbamoyl, C1-C6 alkylaminocarbonyl, C1-C6 alkyloxycarbonylC3-C7 cycloalkylene, amino C1-C6 alkyl, wherein the amino group in the amino C1-C6 alkyl is optionally substituted with one or two C1-C6 alkyl groups, amino C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkyloxycarbonyl, C3-C7 cycloalkyloxyC1-C6 alkyl, C1-C6 alkyl optionally substituted with a six-membered monocyclic heterocyclic group containing one oxygen atom, C1-C6 alkyl optionally substituted with both C1-C6 haloalkyl and hydroxyl groups, C3-C7 cycloalkyl optionally substituted with C1-C6 alkyloxycarbonylaminoC1-C6 alkyl, and a six-membered monocyclic heterocyclic group containing one nitrogen atom optionally substituted with C1-C6 alkyloxycarbonylamino. R5 is selected from: , where R a1 R b1 Each is independently selected from: H, C1-C6 alkyl groups.

2. The compound of claim 1, or its stereoisomer, its tautomer, or its pharmaceutically acceptable salt, wherein, R1 and R2 are each independently selected from: H, methyl, or R1, R2, together with the N atom they contain, form rings selected from the following: , .

3. The compound of claim 1 or 2, or its stereoisomer, its tautomer, or its pharmaceutically acceptable salt, wherein, R3 and R4 are each independently selected from: H, C1-C6 alkyl groups. , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

4. The compound of claim 1 or 2, or its stereoisomer, its tautomer, or its pharmaceutically acceptable salt, wherein, R3 is selected from: H, C1-C6 alkyl, R4 is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

5. The compound of claim 3, or its stereoisomer, its tautomer, or its pharmaceutically acceptable salt, wherein, C1-C6 alkyl groups are ethyl groups.

6. The compound of claim 4, or its stereoisomer, its tautomer, or its pharmaceutically acceptable salt, wherein, C1-C6 alkyl groups are ethyl groups.

7. The compound of claim 1, or its stereoisomer, its tautomer, or its pharmaceutically acceptable salt, wherein, R5 is selected from: , , , , .

8. The compound of claim 1, or its stereoisomer, its tautomer, or its pharmaceutically acceptable salt, wherein, The compound is selected from:

9. A method for preparing the compound according to any one of claims 1 to 8: #imgpt109# Reagents and conditions: (a) N-R1R2, DIPEA, DCM, -40 o C, 1h; (b) OH-R5, DIPEA, DCM, -20 o C, overnight; (c) For XS-1~19: N-R3R4, DIPEA, DMSO, room temperature, overnight; or, For XS-20~22, XS-25~34: N-R3R4, DIPEA, acetonitrile, 40 o C, overnight; or For XS-23~24: N-R3R4, Na2CO3, acetonitrile, 50 o C, overnight; The variables R1, R2, R3, R4, and R5 in the above synthesis route are defined as described in any one of claims 1 to 8.

10. A pharmaceutical composition comprising at least one compound of any one of claims 1 to 8, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutical carriers and / or excipients.

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