Thia-fused cyclic compound, and preparation method therefor and use thereof

Abstract

Disclosed are a thia-fused cyclic compound, and a preparation method therefor and the use thereof. The compound has a structure represented by formula (I), and exhibits a potent inhibitory activity against various KRAS protein subtypes, such as KRAS G12D, KRAS G12C, KRAS G12V, KRAS WT, KRAS G12R, KRAS G12S and KRAS G12A. The compound can be used for preparing a drug for treating and / or preventing diseases related to multi-subtype KRAS mutations, including pancreatic cancer, lung cancer, colorectal cancer, etc., and thus has broad clinical application prospects.

Description

Thionid ring compounds, their preparation methods and applications Technical Field

[0001] This invention relates to a thiohexacyclic compound, its preparation method and application, and more particularly to a thiohexacyclic compound that can be prepared as a drug for treating and / or preventing cancer, its preparation method and application. Background Technology

[0002] KRAS (a homolog of the Kirsten rat sarcoma virus oncogene) is a G protein that acts as an intracellular "on / off" switch, linking extracellular mitotic signaling with intracellular proliferative responses. Mitogen stimulation induces GTP binding to KRAS, causing a conformational change that allows KRAS to interact with downstream effector proteins, leading to cell proliferation. Normally, proliferative signaling is regulated by GTPase activator proteins (GAPs) to restore KRAS to its GDP-bound, non-proliferative state. However, mutations in KRAS weaken the regulatory cycle between these GDP and GTP-bound states, leading to the accumulation of GTP-bound active states and dysregulated cell proliferation. For example, genetic alterations at codon 12 of KRAS replace the naturally occurring glycine residue at this position with different amino acids, particularly aspartic acid (G12D mutation or KRAS G12D), cysteine ​​(G12C mutation or KRASG12C), and valine (G12V mutation or KRAS G12V). Similarly, mutations in codons 13, 61, and 146 of KRAS are commonly found in the KRAS gene. Mutations in the KRAS protein are an important factor in inducing tumors, with different mutant KRAS proteins being overexpressed in different tumors, such as pancreatic cancer, non-small cell lung cancer, and colorectal cancer. In addition, overactivated endothelial cell KRAS G12V can also cause arteriovenous malformations in the brain.

[0003] Existing KRAS protein inhibitors mainly target mutant proteins of KRAS G12C and KRAS G12D subtypes, such as AMG510, MRTX849, and JDQ443. These inhibitors primarily target specific subtypes of KRAS protein and are difficult to achieve broad-spectrum inhibitory effects. Summary of the Invention

[0004] Purpose of the invention: The first purpose of this invention is to provide a thiohexacyclic compound with antitumor activity; the second purpose is to provide a method for preparing the compound; and the third purpose is to provide a pharmaceutical application of the compound.

[0005] Technical solution: The compound having the structure of formula (I) or its stereoisomer, or a pharmaceutically acceptable salt, as described in this invention.

[0006] In the structure of formula (I):

[0007] R 1a R 1b R 2a R 2b R 3a R 3b Selected from hydrogen, deuterium, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, halogen, -NH2, -NH(C1-C4 alkyl), -N(C1-C4 alkyl)2, C3-C5 cycloalkyl or 3-5 member heterocyclic groups containing 1-2 N, O, S;

[0008] Or R 1a R 1b R 2a R 2b R 3a R 3b Together with the carbon atom it is attached to, they form a cyclopropane or cyclobutane ring in the form of (1) or (2):

[0009] (1)R 1a R 1b One of them and R 2a R 2b one of;

[0010] (2)R 2a R 2b One of them and R 3a R 3b one of;

[0011] X selects N or CR a ;

[0012] R1, R a Selected from hydrogen, halogen, -OH, -NH2, cyano, or C1-C4 alkyl;

[0013] Ring A is selected from pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, oxadiazole, isoxazole, thiazole, thiadiazole, isothiazole, or triazole;

[0014] Z, V, and Y are selected from N or C;

[0015] R2 is selected from a 3-10 heterocyclic group containing 1-2 N, O, or S members that is substituted with hydrogen, one or more identical or different C1-C6 alkyl groups, C1-C6 alkoxy groups, or 5-6 heterocyclic groups containing 1-2 N, O, or S members, or -O (C1-C6 alkyl) that is substituted with hydrogen or 3-10 heterocyclic groups containing 1-2 N, O, or S members, wherein the C1-C6 alkyl group is substituted with hydrogen or 3-8 heterocyclic groups containing 1-2 N, O, or S members, and the 3-10 heterocyclic group is substituted with hydrogen or one or more identical or different R5 groups.

[0016] R5 is selected from C1-C6 alkyl, C1-C6 alkoxy, halogen or 3-10 heterocyclic groups containing 1-2 N, O, S groups;

[0017] W is selected from N, O, or C;

[0018] R3 and R4 are selected from hydrogen, -NH2, C1-C6 alkyl, C1-C6 haloalkyl, C3-C 10 Cycloalkyl, 3-10 member heterocyclic groups containing 1-2 N, O, S atoms, C6-C 10 Aryl, 5-10 membered heteroaryl containing 1-4 N, O, S or absent, wherein C1-C6 alkyl, C1-C6 haloalkyl, C3-C 10 Cycloalkyl, 3-10 member heterocyclic groups containing 1-2 N, O, S atoms, C6-C 10 The aryl or 5-10-membered heteroaryl group containing 1-4 N, O, S is substituted with hydrogen, or one or more identical or different R6 or R7.

[0019] Alternatively, R3, R4, and the W connected to them can ring together to form a C3-C structure replaced by one or more R6s. 10 Cycloalkanes or C3-C 10 Heterocyclic alkanes containing 1-2 N, O, or S atoms;

[0020] R6 is selected from hydrogen, C1-C6 alkyl, -OR7, -N(R7)2, halogen, -CN, -NH2, -C(=O)R7, -C(=O)OR7, -C(=O)N(R7)2, -NHC(=O)OR7 or (=O);

[0021] R7 is selected from hydrogen, C1-C6 alkyl, C3-C6 alkyl, and C7-C6 alkyl groups. 10 Cycloalkyl, 3-10 member heterocyclic groups containing 1-2 N, O, S atoms, C6-C 10 Aryl or 5-10 membered heteroaryl groups containing 1-4 N, O, S groups, of which C1-C6 alkyl, C3-C6 alkyl, C4-C6 alkyl, C5-C6 alkyl, C6 ... 10 Cycloalkyl, 3-10 member heterocyclic groups containing 1-2 N, O, S atoms, C6-C 10The aryl or 5-10-membered heteroaryl group containing 1-4 N, O, S is substituted by one or more identical or different R8, R9;

[0022] R8 is selected from hydrogen, -OR9, -N(R9)2 or -C(O)N(R9)2;

[0023] R9 is selected from hydrogen, C1-C6 alkyl, C3-C6 alkyl, and C4-C6 alkyl groups. 10 Cycloalkyl, 3-10 membered heterocyclic group containing 1-2 N, O, S or 5-10 membered heteroaryl group containing 1-4 N, O, S, wherein the C1-C6 alkyl group is substituted by the following substituents: hydrogen, C1-C6 alkoxy, C3-C 10 Cycloalkyl or heterocyclic compounds consisting of 3-10 members containing 1-2 N, O, or S groups substituted with hydrogen or C1-C6 alkyl groups.

[0024] This invention designs a broad-spectrum inhibitor of KRAS protein. Broad-spectrum inhibition of various KRAS protein subtypes effectively prevents KRAS protein mutations and enhances the inhibitory effect on KRAS protein-related pathways. When KRAS protein is inhibited, NRAS and HRAS can be compensatorily upregulated, compensating for the normal physiological function of KRAS protein and avoiding the toxicity of broad-spectrum inhibition.

[0025] The compounds disclosed herein can exist in specific stereoisomer forms. The term "isomer" refers to isomers with the same structure but different spatial arrangements of atoms. These include cis and trans (or Z and E) isomers, (-)- and (+)- isomers, (R)- and (S)- enantiomers, diastereomers, (D)- and (L)- isomers, tautomers, transisomers, conformational isomers, and mixtures thereof (such as racemic mixtures and mixtures of diastereomers). Substituents in the compounds disclosed herein may contain additional asymmetric atoms. All such stereoisomers and mixtures thereof are included within the scope of this disclosure. Optically active (-)- and (+)- isomers, (R)- and (S)- enantiomers, and (D)- and (L)- isomers can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. This disclosure discloses an isomer of a compound, which can be prepared by asymmetric synthesis or with chiral auxiliaries, or, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), by forming a salt of the diastereomer with a suitable optically active acid or base, followed by diastereomer resolution using conventional methods known in the art to obtain the pure isomer. Furthermore, the separation of enantiomers and diastereomers is typically performed by chromatography.

[0026] In the chemical structure of the compounds described in this disclosure, the bonds... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be or Or simultaneously include or Two configurations.

[0027] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0028] The term "C1-C6 alkyl" refers to saturated straight-chain and branched hydrocarbon groups having 1-6 carbon atoms, including but not limited to other examples of alkyl groups such as methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 3-methyl-1-butyl, 2-methyl-2-butyl, 2,2-dimethyl-1-propyl, 1-hexyl, 3-hexyl, 2-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 3,3-dimethyl-2-butyl, 2,3-dimethyl-1-butyl, 2,2-dimethyl-1-butyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, etc.

[0029] The term "C1-C6 alkoxy" refers to a group consisting of a saturated cycloalkyl group having 1-6 carbon atoms combined with an oxygen group, including but not limited to methoxy, ethoxy, 1-propoxy, 2-propoxy, 1-butoxy, 2-methyl-1-propoxy, 2-butoxy, 2-methyl-2-propoxy, 1-pentoxy, 2-pentoxy, 3-pentoxy, 3-methyl-1-butoxy, 2-methyl-2-butoxy, 2,2-dimethyl-1-propoxy, 1-hexoxy, 3-hexoxy, 2-methyl-2-pentoxy, 4-methyl-2-pentoxy, 3-methyl-3-pentoxy, 2-methyl-3-pentoxy, 3,3-dimethyl-2-butoxy, 2,3-dimethyl-1-butoxy, 2,2-dimethyl-1-butoxy, 2-methyl-1-pentoxy, 3-methyl-1-pentoxy, etc.

[0030] Alkyl halides are derived from previously defined alkyl groups by the independent substitution of one or more hydrogen atoms in the chain with the same or different halogen atoms. If the alkyl halide is further substituted, the substitution can occur in mono- or poly-substituted forms, independently of each other on all hydrogen-carrying carbon atoms. Examples of alkyl halide are -CF-CHF-CHF, -CFCF, -CHFCF, -CHCFCFCH2-CHFCH2-CF, CFCF2-CHFCHCH, -CHFCHF-, etc.

[0031] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic heterocycle (i.e., monocyclic heterocyclic group) or polycyclic heterocyclic system (i.e., polycyclic heterocyclic group) containing at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may optionally be oxidized, i.e., to form nitrogen oxides; the sulfur may optionally be oxidized, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS- or -SS-), and has 4 to 20 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., 4 to 20 membered heterocyclic groups), including 4-10 membered monocyclic heterocyclic groups, 6-20 membered spirocyclic heterocyclic groups, 6-18 membered fused heterocyclic groups, and 7-10 membered bridged heteroalkyl groups. The heterocyclic group is preferably a heterocyclic group having 4 to 16 ring atoms (i.e., a 4 to 16-membered heterocyclic group), wherein one or two atoms are nitrogen atoms; more preferably a heterocyclic group having 4 to 11 ring atoms (i.e., a 4 to 11-membered heterocyclic group), wherein one or two atoms are nitrogen atoms. "Spiroheterocyclic group" refers to a polycyclic heterocyclic system in which the rings share a single atom (called a spiro atom), and the rings may contain one or more double bonds, and the rings contain at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may be optionally oxidized, i.e., to form nitrogen oxides; the sulfur may be optionally oxidized, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS- or -SS-), provided that at least one monocyclic heterocyclic group is present and the bonding point is on the monocyclic heterocyclic group, which has 6 to 20 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., 6 to 20 membered spiroheterocyclic groups). The spiroheterocyclic group is preferably a spiroheterocyclic group having 6 to 14 ring atoms (i.e., a 6 to 14-membered spiroheterocyclic group), more preferably a spiroheterocyclic group having 7 to 14 ring atoms (i.e., a 7 to 14-membered spiroheterocyclic group) or a spiroheterocyclic group having 7 to 11 ring atoms (i.e., a 7 to 11-membered spiroheterocyclic group). The spiroheterocyclic group includes monospirocyclic groups and multispirocyclic groups (such as bispirocyclic groups, etc.), preferably monospirocyclic groups or bispirocyclic groups, more preferably 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered or 7-membered / 6-membered monospirocyclic groups."Fused heterocyclic group" refers to a polycyclic heterocyclic system in which two adjacent atoms are shared between the rings. The ring may contain one or more double bonds and at least one (e.g., 1, 2, 3 or 4) heteroatom selected from nitrogen, oxygen and sulfur (the nitrogen may optionally be oxidized to form nitrogen oxides; the sulfur may optionally be oxidized to form sulfoxides or sulfones, but excluding -OO-, -OS- or -SS-). It is a monocyclic heterocyclic group fused with one or more monocyclic heterocyclic groups, or a monocyclic heterocyclic group fused with one or more cycloalkyl, aryl or heteroaryl groups, wherein the bonding point is on the monocyclic heterocyclic group and has 6 to 18 (e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) ring atoms (i.e., 6 to 20 membered fused heterocyclic group). The fused heterocyclic group is preferably a fused heterocyclic group having 6 to 14 ring atoms (i.e., a 6 to 14-membered fused heterocyclic group), more preferably a fused heterocyclic group having 7 to 10 ring atoms (i.e., a 7 to 10-membered fused heterocyclic group). The fused heterocyclic group includes bicyclic and polycyclic fused heterocyclic groups (such as tricyclic fused heterocyclic groups, tetracyclic fused heterocyclic groups, etc.), preferably bicyclic or tricyclic fused heterocyclic groups, more preferably 3 / 4, 3 / 5, 3 / 6, 4 / 4, 4 / 5, 4 / 6, 5 / 3, 5 / 4, 5 / 5, 5 / 6, 5 / 7, 6 / 3, 6 / 4, 6 / 5, 6 / 6, 6 / 7, 7 / 5, or 7 / 6 bicyclic fused heterocyclic groups. Examples of heterocyclic groups include, but are not limited to:

[0032] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic carbocyclic ring having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 3 to 20 membered cycloalkyl). The cycloalkyl is preferably a cycloalkyl having 3 to 12 ring atoms (i.e., 3 to 12 membered cycloalkyl) or a cycloalkyl having 3 to 10 ring atoms (i.e., 3 to 10 membered cycloalkyl), more preferably a cycloalkyl having 3 to 8 ring atoms (i.e., 3 to 8 membered cycloalkyl), most preferably a cycloalkyl having 3 to 6 ring atoms (i.e., 3 to 6 membered cycloalkyl), and even more preferably a cycloalkyl having 5 or 6 ring atoms (i.e., 5 or 6 membered cycloalkyl). Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0033] Preferably, the compound has the structure of formula (Ia):

[0034] The definitions of rings A, X, W, Z, V, Y, R1, R2, R3, and R4 are as described above.

[0035] Preferably, the compound has the structure of formula (Ib):

[0036] The definitions of rings A, X, W, Z, V, Y, R1, R2, R3, and R4 are as described above.

[0037] Preferably, in the structure, ring A is selected from:

[0038] In a further preferred embodiment, in the aforementioned structure, ring A is selected from:

[0039] Those skilled in the art will understand that, according to the conventions used in the art, the structural formulas of the descriptive groups described in this application... and This refers to the connection of the corresponding group to other segments or groups in the compound shown in formula (I) through this site.

[0040] Preferably, in the structure described above, R1 is a methyl group.

[0041] Further preferably, the compound has the structure of formula (I*):

[0042] The definitions of rings A, X, W, Z, V, Y, R2, R3, and R4 are as described above.

[0043] More preferably, the compound has the structure of formula (I*a):

[0044] The definitions of X, W, Z, V, Y, R2, R3, and R4 are as described above.

[0045] Furthermore, the compound has the structure of formula (I*b):

[0046] The definitions of X, W, Z, V, Y, R2, R3, and R4 are as described above.

[0047] Preferably, in the structure described above:

[0048] R3 and R4 are selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C 10 Cycloalkyl, 3-10 member heterocyclic groups containing 1-2 N, O, S atoms, C6-C 10 Aryl or 5-10-membered heteroaryl groups containing 1-4 N, O, S groups, with R3 and R4 substituted by hydrogen or one or more identical or different R6 and R7 groups;

[0049] Alternatively, R3, R4, and the W connected to them can ring together to form a C3-C structure replaced by one or more R6s. 10 Cycloalkanes or C3-C 10Heterocyclic alkanes containing 1-2 N, O, or S atoms;

[0050] The definitions of W, R6, and R7 are as described above.

[0051] Preferably, in the structure, R2 is selected from:

[0052] In a further preferred embodiment, in the aforementioned structure, R2 is selected from:

[0053] Furthermore, in the aforementioned structure, R2 is selected from:

[0054] Preferably, in the aforementioned structure, Selected from the following groups substituted with hydrogen, or one or more identical or different R6 or R7 groups:

[0055] The definitions of R6 and R7 are as described above.

[0056] Further preferred, in the aforementioned structure, Selected from:

[0057] Preferably, the compound is selected from any one of the following compounds:

[0058] Preferably, the pharmaceutically acceptable salt is a salt formed by the compound with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, or ferulic acid.

[0059] The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention, prepared by reacting a compound having specific substituents discovered in this invention with a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, etc.); and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Acids such as citric acid, tartaric acid, and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (e.g., arginine), glucuronic acid, etc. Certain specific compounds of the present invention contain basic and acidic functional groups, thus allowing them to be converted into any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and then the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubilities in polar solvents.

[0060] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0061] The preparation methods of the compounds or their stereoisomers and pharmaceutically acceptable salts described in this invention are selected from any of the following methods:

[0062] Method 1: When ring A is Compound I is obtained from compound I-1 through substitution, coupling, aminolysis, condensation and cyclization.

[0063] Method 2: When ring A is Compound I is obtained from compound I-1 through substitution, coupling, reduction, condensation and cyclization.

[0064] Method 3: When ring A is Compound I is obtained from compound I-1 through substitution, coupling, thiolation, condensation and cyclization.

[0065] Among them, X, W, Z, V, Y, R 1a R 1b R 2a R 2b R 3a R 3b The definitions of R1, R2, R3, and R4 are as described above;

[0066] The corresponding acid is salted with compound I prepared by the above method to obtain the pharmaceutically acceptable salt.

[0067] The pharmaceutical compositions of the present invention comprise the compound described herein or a stereoisomer thereof, a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

[0068] The pharmaceutically acceptable carrier can be an excipient widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to allow the active ingredient to dissolve at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipient can be an inert filler, or it may provide a function such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipient may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0069] The pharmaceutical compositions of the present invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0070] The pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0071] The compounds described in this invention, or their stereoisomers, pharmaceutically acceptable salts, or pharmaceutical compositions thereof, are used in the preparation of medicaments for the treatment and / or prevention of cancer.

[0072] Preferably, the drug is a drug for treating and / or preventing tumors with KRAS mutations, KRAS wild-type amplification, or KRAS overexpression.

[0073] More preferably, the drug is a drug for treating and / or preventing tumors with KRAS G12C, KRAS G12D, KRAS G12V, KRAS WT, KRAS G12S, KRAS G12A or KRAS G12R mutations.

[0074] Preferably, the drug is a drug for treating and / or preventing pancreatic cancer, lung cancer, colorectal cancer, bile duct cancer, urothelial carcinoma, multiple myeloma, melanoma, uterine cancer, endometrial cancer, thyroid cancer, acute myeloid leukemia, bladder cancer, gastric cancer, cervical cancer, head and neck squamous cell carcinoma, diffuse large B-cell lymphoma, esophageal cancer, chronic lymphocytic leukemia, hepatocellular carcinoma, breast cancer, ovarian cancer, prostate cancer, glioblastoma, renal cancer, or sarcoma.

[0075] Further preferably, the drug is a drug for treating and / or preventing adenocarcinoma, lung cancer, ovarian cancer, colorectal cancer, gastric cancer, gastroesophageal junction cancer, or esophageal cancer.

[0076] Further preferred cancers include adenocarcinoma, lung cancer, ovarian cancer, colorectal cancer, gastric cancer, gastroesophageal junction cancer, or esophageal cancer that are KRAS G12D or KRAS G12V mutated.

[0077] Further optimization is made, pancreatic cancer is pancreatic duct adenocarcinoma, lung cancer is small cell lung cancer (NSCLC), and colorectal cancer is colorectal adenocarcinoma.

[0078] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0079] The compounds designed in this invention can effectively inhibit multiple KRAS protein isoforms, such as KRAS G12D, KRAS G12C, KRAS G12V, KRAS WT, KRAS G12R, KRAS G12S, and KRAS G12A, and can also inhibit the proliferation of various tumor cells, with protein and cell inhibitory activity IC50. 50 The optimal concentration values ​​are all below 1 μM, reaching nanomolar concentration levels, which can be prepared as drugs for the treatment and / or prevention of various diseases, including pancreatic cancer, lung cancer, and colorectal cancer, with broad prospects for clinical application. Detailed Implementation

[0080] The technical solution of the present invention will be further described below with reference to the embodiments.

[0081] 1 H-NMR was performed using a BRUKER AVANCE-300 NMR spectrometer (Brucker, Switzerland), with TMS as the internal standard. Shift values ​​(δ) were expressed in ppm. Low-resolution mass spectrometry was performed using an Expression compact Fourier transform mass spectrometer.

[0082] Example 1: Synthesis of 4-methyl-4-[4-((S)-1-methylpyrrolidin-2-yl)methoxy)-6-(4,7-diazaspiro[2.5]octyl-7-yl)pyrimidin-2-yl]-1,2,4-oxadiazol-5-yl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine (compound 1)

[0083] (1) Synthesis of 2-amino-4-methyl-4,5,6,7-tetrahydrobenzo[d]thiazole-4-carboxylic acid (intermediate I-1)

[0084] Sodium hydride (800 mg, 20 mmol) was added to 30 mL of anhydrous tetrahydrofuran under ice bath conditions. After stirring for 15 minutes in an ice bath, methylcyclohexane-1-methyllactone (3.14 g, 20 mmol) was added, and the reaction was stirred for 0.5 hours. Then, iodomethane (3.10 g, 22 mmol) was added, and the reaction was allowed to proceed overnight at room temperature in an ice bath until the starting material was completely reacted. After the reaction was completed, 50 mL of saturated ammonium chloride aqueous solution was added to quench the reaction. The mixture was extracted with 50 mL of ethyl acetate, and the organic phase was separated. After concentrating to remove the organic solvent, 2.90 g of crude methyl-1-methyl-2-oxocyclohexane-1-carboxylate was obtained. The crude product was dissolved in 30 mL of dichloromethane, and liquid bromine (2.70 g, 17 mmol) was added dropwise under ice bath conditions. After reacting for 1 hour in an ice bath, the starting material was completely reacted. After the reaction was complete, 20 mL of saturated sodium sulfite aqueous solution was added, and the mixture was extracted with 50 mL of ethyl acetate. The organic phase was separated, and the organic solvent was removed by concentration to obtain 2.60 g of crude methyl-3-bromo-1-methyl-2-oxocyclohexane-1-carboxylate. The crude product was dissolved in 30 mL of methanol, and thiourea (1.55 g, 20 mmol) was added. The mixture was heated to 60 °C and reacted for 3 hours until the reaction was complete. After removing the organic solvent by concentration, 50 mL of water and 50 mL of dichloromethane were added, and the organic phase was extracted and separated. After concentration, the mixture was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain 820 mg of grayish-white solid methyl-2-amino-4-methyl-4,5,6,7-tetrahydrobenzo[d]thiazole-4-carboxylate. The product was dissolved in 30 mL of methanol, and 6M potassium hydroxide (1.19 g, 21 mmol) aqueous solution was added dropwise. The mixture was heated to 70 °C and refluxed for 12 hours until the reaction was complete. After concentration to remove the organic solvent, 50 mL of water and 50 mL of ethyl acetate were added. After separating the aqueous phase, the pH was adjusted to 5-6 with 1 M HCl aqueous solution, and 50 mL of ethyl acetate was added to extract and separate the organic phase. After concentration, 470 mg of grayish-white solid product of 2-amino-4-methyl-4,5,6,7-tetrahydrobenzo[d]thiazol-4-carboxylic acid was obtained, with an overall yield of 11.1%. 1 H NMR(400MHz,DMSO-d)δ6.72(d,J=6.8Hz,1H),6.63(d,J=7.0Hz,1H),2.81–2.71(m,1H),2.7 1–2.65(m,1H),2.34(ddd,J=28.8,11.3,8.6Hz,2H),2.16–2.03(m,2H),1.66(s,3H).; EI-MS m / z:213[M+H] + .

[0085] (2) Synthesis of 4-methyl-4-[4-((S)-1-methylpyrrolidin-2-yl)methoxy)-6-(4,7-diazaspiro[2.5]octyl-7-yl)pyrimidin-2-yl]-1,2,4-oxadiazol-5-yl]-4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine (compound 1)

[0086] 4,6-Chloro-2-cyanopyrimidine (300 mg, 2.15 mmol) was dissolved in 10 mL of dichloromethane, and (S)-(1-methylpyrrolidone-2-yl)methanol (272 mg, 2.37 mmol) and N,N-diisopropylethylamine (555 mg, 4.3 mmol) were added. The reaction was carried out overnight at room temperature until complete. After the reaction was completed, the organic solvent was removed by vacuum distillation, and the crude product was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to give 352 mg of a white oily product. The white oily product was dissolved in 10 mL of 1,4-dioxane, and 4,7-diazaspiro[2.5]octane-4-carboxylic acid tert-butyl ester (326 mg, 1.54 mmol) and triethylamine (282 mg, 2.8 mmol) were added. The mixture was heated to 90 °C and reacted for 5 hours until complete. After the reaction was complete, 30 mL of water and 30 mL of ethyl acetate were added, and the organic phase was extracted and separated. After removing the organic solvent by vacuum distillation, 400 mg of a white solid product was obtained. The white solid product was dissolved in 10 mL of anhydrous ethanol, and 123 mg (3.72 mmol) of 50% hydroxylamine aqueous solution was added. The mixture was heated to 70 °C and reacted for 0.5 h until the reaction was complete. After the reaction was complete, the organic solvent and excess hydroxylamine were removed by vacuum distillation to obtain 410 mg of a white solid intermediate, with a total yield of 40.7%. Intermediate I-1 (50 mg, 0.21 mmol) was dissolved in 10 mL of dichloromethane, and triethylamine (42 mg, 0.42 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (80 mg, 0.21 mmol) were added. After stirring at room temperature for 0.5 hours, the white solid intermediate from the previous step (96 mg, 0.21 mmol) was added, and the mixture was reacted at room temperature for 2 hours until the reaction was complete. After the reaction was complete, 20 mL of water and dichloromethane were added, and the organic phase was extracted and separated. The organic solvent was removed by vacuum distillation, yielding 64 mg of a brownish-yellow oily crude product. The brownish-yellow oily crude product was dissolved in 10 mL of tetrahydrofuran, and 1,8-diazabispyrospirocyclic [5.4.0]undecyl-7-ene (115 mg, 0.76 mmol) was added. The mixture was heated to 90 °C and refluxed for 12 hours until the reaction was complete. After the reaction was complete, the organic solvent was removed by vacuum distillation. The crude product was separated by silica gel column chromatography (dichloromethane:methanol = 100:1 to 30:1) to obtain 37 mg of a white solid product. The white solid product was dissolved in 5 mL of dioxane, and 5 mL of 5M hydrochloric acid-dioxane solution was added. The mixture was stirred at room temperature for 3 hours until the reaction was complete. After the reaction was complete, the organic solvent was removed by vacuum distillation. After adding saturated sodium bicarbonate aqueous solution, the mixture was extracted with dichloromethane to separate the organic phase. After removing the organic solvent by vacuum distillation, 22 mg of a white solid product was obtained, with a total yield of 19.4%.1H NMR(400MHz,DMSO-d)δ6.91(d,J=6.8Hz,1H),6.74(d,J=7.0Hz,1H),6.18(s,1H),4.49(t,J=3.7 Hz,1H),4.18(dd,J=7.7,1.7Hz,2H),3.69–3.64(m,1H),3.63(d,J=4.5Hz,3H),3.27–3.17(m,1H ),3.08(ddt,J=9.3,6.8,3.9Hz,2H),2.88–2.65(m,4H),2.58–2.50(m,1H),2.50–2.44(m,1H),2 .28(d,J=1.4Hz,3H),2.13–1.96(m,2H),1.92(dd,J=8.1,4.6Hz,7H),1.79–1.65(m,4H).; EI-MS m / z:537[M]. + .

[0087] The following compounds were prepared using a procedure similar to that in Example 1:

[0088] Example 2: Synthesis of 2-amino-4-methyl-4-(3-(4-((S)-1-methylpyrrolidine-2-ylmethoxy)-6-(4,7-diazaspiro[2.5]octane-7-yl)pyrimidin-2-yl)-1,2,4-oxadiazol-5-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-cyanide (compound 8)

[0089] (1) Synthesis of 2-amino-3-cyano-4-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-4-carboxylic acid (intermediate I-2)

[0090] Sodium hydride (800 mg, 20 mmol) was added to 30 mL of anhydrous tetrahydrofuran under ice bath conditions. After stirring for 15 minutes in an ice bath, methylcyclohexane-1-methyllactone (3.14 g, 20 mmol) was added, and the mixture was stirred for 0.5 hours. Then, iodomethane (3.10 g, 22 mmol) was added, and the mixture was allowed to react overnight at room temperature in an ice bath until the starting material was completely reacted. After the reaction was complete, 50 mL of saturated ammonium chloride aqueous solution was added to quench the reaction. The mixture was extracted with 50 mL of ethyl acetate, and the organic phase was separated. After concentrating to remove the organic solvent, 2.90 g of crude methyl-1-methyl-2-oxocyclohexane-1-carboxylate was obtained. The crude product was dissolved in 30 mL of toluene, and ammonium acetate (261 mg, 3.39 mmol) and acetic acid (224 mg, 3.39 mmol) were added. The mixture was reacted at 100 °C for 2 hours until the starting material was completely reacted. After the reaction was completed, the organic solvent was removed by concentration. 50 mL of water and 50 mL of ethyl acetate were added, and the organic phase was extracted and separated. The product was then concentrated to obtain 2.5 g of crude methyl dicyanomethane-1-methylcyclohexane-1-carboxylic acid ester. The crude product was dissolved in 10 mL of N,N-dimethylformamide, and precipitated sulfur (814 mg, 3.18 mmol) and L-proline (390 mg, 3.39 mmol) were added. After reacting at 80 °C for 1 hour, the reaction was complete. 50 mL of water and 50 mL of ethyl acetate were added, and the organic phase was extracted and separated. After concentration with the organic solvent, the crude product was separated by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1) to obtain 750 mg of a bright yellow solid product of methyl-2-amino-3-cyano-4-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-4-carboxylic acid ester. The product was dissolved in 30 mL of methanol, and 6 M potassium hydroxide (1008 mg, 18 mmol) aqueous solution was added dropwise. The mixture was heated to 70 °C and refluxed for 12 hours until the reaction was complete. After concentrating to remove the organic solvent, 50 mL of water and 50 mL of ethyl acetate were added. After separating the aqueous phase, the pH was adjusted to 5-6 with 1 M HCl aqueous solution, and 50 mL of ethyl acetate was added to extract and separate the organic phase. The product was concentrated to give 650 mg of 2-amino-3-cyano-4-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-4-carboxylic acid brownish-yellow solid product, with an overall yield of 13.7%. 1 H NMR(400MHz,DMSO-d)δ6.03(d,J=6.6Hz,1H),5.89(d,J=6.6Hz,1H),2.90–2.75(m,2H),2.36(dd,J=10 .7,8.0Hz,1H),2.31(dd,J=10.7,8.0Hz,1H),1.99(ddtd,J=11.2,10.0,8.1,3.2Hz,2H),1.56(s,3H).

[0091] (2) Synthesis of 2-amino-4-methyl-4-(3-(4-((S)-1-methylpyrrolidine-2-ylmethoxy)-6-(4,7-diazaspiro[2.5]octane-7-yl)pyrimidin-2-yl)-1,2,4-oxadiazol-5-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-cyanide (compound 8)

[0092] 4,6-Chloro-2-cyanopyrimidine (300 mg, 2.15 mmol) was dissolved in 10 mL of dichloromethane, and (S)-(1-methylpyrrolidone-2-yl)methanol (272 mg, 2.37 mmol) and N,N-diisopropylethylamine (555 mg, 4.3 mmol) were added. The reaction was carried out overnight at room temperature until complete. After the reaction, the organic solvent was removed by vacuum distillation, and the crude product was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to give 310 mg of a white oily product. The white oily product was dissolved in 10 mL of 1,4-dioxane, and 4,7-diazaspiro[2.5]octane-4-carboxylic acid tert-butyl ester (260 mg, 1.23 mmol) and triethylamine (248 mg, 2.46 mmol) were added. The mixture was heated to 90 °C and reacted for 5 hours until complete. After the reaction was complete, 30 mL of water and 30 mL of ethyl acetate were added to extract and separate the organic phase. The organic solvent was removed by vacuum distillation, and the crude product was separated by column chromatography (petroleum ether:ethyl acetate = 3:1 to 1:2) to give 262 mg of a white solid product. The white solid product was dissolved in 10 mL of anhydrous ethanol, and 75 mg (1.13 mmol) of 50% hydroxylamine aqueous solution was added. The mixture was heated to 70 °C and reacted for 0.5 hours until the reaction was complete. After the reaction was complete, the organic solvent and excess hydroxylamine were removed by vacuum distillation to give 276 mg of a white solid intermediate, with an overall yield of 25.8%. 2-Amino-3-cyano-4-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-4-carboxylic acid (50 mg, 0.21 mmol) was dissolved in 10 mL of dichloromethane. Triethylamine (42 mg, 0.42 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (80 mg, 0.21 mmol) were added. After stirring at room temperature for 0.5 hours, the white solid intermediate from the previous step (97 mg, 0.21 mmol) was added, and the reaction was allowed to proceed for 2 hours at room temperature until complete. After the reaction was complete, 20 mL of water and dichloromethane were added, and the organic phase was extracted and separated. The organic solvent was removed by vacuum distillation to obtain 65 mg of a brownish-yellow oily crude product. The crude, brownish-yellow oily product was dissolved in 10 mL of tetrahydrofuran, and 1,8-diazabispyrocyclo[5.4.0]undecyl-7-ene (115 mg, 0.76 mmol) was added. The mixture was heated to 90°C and refluxed for 12 hours until the reaction was complete. After the reaction, the organic solvent was removed by vacuum distillation. The crude product was separated by silica gel column chromatography (dichloromethane:methanol = 100:1 to 30:1) to obtain 32 mg of white solid product. The white solid product was dissolved in 5 mL of dioxane, and 5 mL of 5M hydrochloric acid-dioxane solution was added. The mixture was stirred at room temperature for 3 hours until the reaction was complete. After the reaction, the organic solvent was removed by vacuum distillation. After adding saturated sodium bicarbonate aqueous solution, the mixture was extracted with dichloromethane to separate the organic phase. After removing the organic solvent by vacuum distillation, 13 mg of white solid product was obtained, with a total yield of 11.0%. 1H NMR (400MHz, DMSO-d) δ6.19 (s, 2H), 5.87 (s, 4H), 4.49 (t, J = 3.7Hz, 2H), 4.18 (dd, J = 7.8 ,1.7Hz,4H),3.69–3.64(m,2H),3.64–3.60(m,5H),3.24–3.14(m,2H),3.08(ddt,J=9.3 ,6.8,3.9Hz,4H),2.88–2.75(m,7H),2.37(ddd,J=11.8,9.9,9.0Hz,4H),2.28(d,J=1.4 Hz,5H),2.01–1.88(m,11H),1.86(s,5H),1.78–1.70(m,6H),1.70–1.65(m,1H).; EI-MS m / z:561[M] + .

[0093] The following compounds were prepared using a similar procedure to that in Example 2:

[0094] Example 3: Synthesis of (S)-2-amino-4-[3-[2-[(S)-4-isopropyl-2-methyl-1,4-diazacycloheptane-1-yl]-6-(methylamino)pyrimidin-4-yl]-1,2,4-oxadiazol-5-yl]-4-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-cyanide (compound 24)

[0095] 2,6-Chloro-4-cyanopyrimidine (300 mg, 2.15 mmol) was dissolved in 10 mL of dichloromethane, and methylamine hydrochloride (160 mg, 2.37 mmol) and N,N-diisopropylethylamine (555 mg, 4.3 mmol) were added. The reaction was carried out at room temperature for 3 hours until completion. After the reaction, the organic solvent was removed by vacuum distillation, and the crude product was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to give 290 mg of a white powder. The white powder was dissolved in 10 mL of 1,4-dioxane, and tert-butyl-(S)-3-methyl-1,4-diazazolium chloride was added. 1-Carboxylic acid ester (442 mg, 2.07 mmol) and triethylamine (248 mg, 2.46 mmol) were reacted at 120 °C for 8 hours until complete. After the reaction, 30 mL of water and 30 mL of ethyl acetate were added, and the organic phase was extracted and separated. After removing the organic solvent by vacuum distillation, the crude product was separated by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain 310 mg of a transparent oil. The transparent oil was dissolved in 10 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added. The reaction was carried out at room temperature for 2 hours until complete. 30 mL of dichloromethane and 30 mL of saturated sodium bicarbonate aqueous solution were added, and the mixture was extracted and separated to wash away excess trifluoroacetic acid. The organic solvent was removed by vacuum distillation of the organic phase to obtain 230 mg of a pale yellow oil. The pale yellow oil was dissolved in 10 mL of anhydrous ethanol, and 1 mL of acetone was added. The mixture was stirred at room temperature for 0.5 hours, followed by the addition of sodium cyanoborohydride (315 mg, 5 mmol). The reaction was carried out at 60 °C for 1 hour until complete. After removing the organic solvent by vacuum distillation, 30 mL of water and 30 mL of dichloromethane were added, and the organic phase was extracted and separated. After removing the organic solvent by vacuum distillation, the crude product was separated by column chromatography (dichloromethane:methanol = 50:1) to obtain 215 mg of a colorless, transparent oil. The above product was dissolved in 10 mL of anhydrous ethanol, and 50% aqueous hydroxylamine (98 mg, 1.49 mmol) was added. The mixture was heated to 60 °C and reacted for 0.5 hours. After the reaction was complete, the organic solvent and excess hydroxylamine were removed by vacuum distillation to obtain 210 mg of a white solid product, with an overall yield of 30.2%.

[0096] Dissolve (S)-2-amino-3-cyano-4-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-4-carboxylic acid (50 mg, 0.21 mmol) in 10 mL of dichloromethane, add triethylamine (42 mg, 0.42 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (80 mg, 0.21 mmol), stir at room temperature for 0.5 hours, then add the white solid intermediate from the previous step (67 mg, 0.21 mmol), and react at room temperature for 2 hours until the reaction is complete. After the reaction is complete, add 20 mL of water and dichloromethane, extract and separate the organic phase, remove the organic solvent by vacuum distillation, and obtain 50 mg of brownish-yellow oily crude product. The brownish-yellow oily crude product was dissolved in 10 mL of tetrahydrofuran, and 1,8-diazabispyrocyclo[5.4.0]undecyl-7-ene (115 mg, 0.76 mmol) was added. The mixture was heated to 90 °C and refluxed for 12 hours until the reaction was complete. After the reaction was completed, the organic solvent was removed by vacuum distillation, and the crude product was separated by silica gel column chromatography (dichloromethane:methanol = 100:1 to 30:1) to give 12 mg of white solid product, with an overall yield of 11.0%. 1H NMR(400MHz,DMSO-d)δ6.94(s,2H),6.46(q,J=4.7Hz,2H),4.94(d,J=6.6Hz,2H),4.77(d,J=6.6Hz,2H),4.06– 3.94(m,2H),3.57(dt,J=12.5,8.5Hz,2H),3.38(dt,J=12.3,8.6Hz,2H),2.96(d,J=4.8Hz,6H),2.91(dd,J=12. 4,6.5Hz,2H),2.87–2.77(m,2H),2.77–2.63(m,8H),2.59–2.50(m,2H),2.37(ddd,J=12.5,8.8,7.8Hz,2H),2.2 5–2.16(m,2H),1.94–1.77(m,14H),1.21(d,J=8.8Hz,6H),1.08(d,J=6.4Hz,6H),1.03(d,J=6.4Hz,6H).; EI-MS m / z:521[M] + .

[0097] The following compounds were prepared using a similar procedure to that in Example 3:

[0098] Example 4: Inhibitory effect of compounds on various KRAS protein subtypes

[0099] 1. Experimental Principle

[0100] This invention involves co-incubating KRAS protein with FITC-labeled fluorescent molecules (disclosed in CN118791557A) and polypeptide fluorescent probes, using compound solutions of varying concentrations, to investigate the competitive binding ability of the compounds to the protein. The compounds can occupy the protein binding pocket, preventing the fluorescent molecules from binding. Unbound KRAS fluorescent molecules exhibit rapid rotation and low fluorescence polarization values ​​in the solution. Based on this characteristic, this invention indirectly reflects the inhibitory activity of the compounds against KRAS protein by measuring their fluorescence polarization values.

[0101] 2. Experimental Methods

[0102] The compound was three-fold diluted to create 12 concentration gradients. 20 μL of each gradient was added to a 384-well plate (Corning #3575), followed by equal volumes of KRAS protein and a fluorescent molecule, bringing the final concentrations to 200 nM and 10 nM, respectively. The plates were incubated on a shaker for 1 hour. Detection was performed using a TecanSPARK multi-mode microplate reader with excitation at 485 nm and emission at 535 nm. Results were analyzed using a Graphpad Prism 9. The blank control consisted of 20 μL of the fluorescent molecule + 40 μL of buffer; the negative control consisted of 20 μL of the fluorescent molecule + 20 μL of KRAS protein + 20 μL of buffer; and the positive control was the pan-KRAS inhibitor BI-2865. The buffer formulation was 50 mM Hepes, 5 mM MgCl2, pH 6.80.

[0103] 3. Experimental Results

[0104] The calculation formula is: Inhibition rate % = 100 × (1 - (Measured value - Blank) / (Negative value - Blank)), which gives the inhibition rate corresponding to a specific concentration. The obtained inhibition rate and logarithmic concentration value are then imported into GraphPadprism 9.0 for analysis and fitting to obtain the IC50. 50 The values ​​and analysis results are shown in Table 1.

[0105] Table 1. Inhibitory effects of compounds on various KRAS protein subtypes (IC50, 100 mg / L). 50 (μM) Note: A represents IC 50 Values ​​less than 1 μM, B indicates IC 50 Values ​​range from 1 μM to 10 μM, where C represents IC. 50 The value is greater than 10 μM.

[0106] As shown in Table 1, all tested compounds exhibited inhibitory effects against multiple KRAS protein isoforms. Among them, compound 7 showed the highest IC50 value against all KRAS protein isoforms. 50 All values ​​reached concentration levels below 1 μM; the IC50 values ​​of the remaining compounds for some KRAS protein subtypes were... 50 The values ​​can reach concentration levels below 1 μM or between 1 μM and 10 μM.

[0107] Example 5: Inhibitory effect of compounds on the proliferation of various tumor cells

[0108] Cells selected: MKN1 (KRAS) WT ), AGS (KRAS) G12D ), SK-CO1 (KRAS G12V ), Mia paca2(KRAS G12C ).

[0109] Drug setup: Compounds 24, 25, 26, 28, 32 and BI-2865 were serially diluted 3-fold starting from 100 μM to 10 concentrations, and blank wells and negative control wells were set up.

[0110] Various tumor cells were seeded at a density of 1000 cells / well in 96-well white blood cell culture plates, with 100 μL of cell suspension in each well. Cells were allowed to adhere for 12–14 h. The culture medium in the 96-well plates was then removed, and culture medium containing different concentrations of the drug was added, with three accessory wells for each concentration. The plates were incubated for 72 h. The cell culture plates were then removed and allowed to equilibrate at room temperature for 10 min. 100 μL of CellTiter-Lumi luminescent reagent (CellTiter-Lumi) was added to each well. TM The Luminescent Cell Viability Assay Kit was used. The cells were shaken at room temperature for 2 min, incubated at room temperature for 10 min, and the relative fluorescence units (RLU) of the chemiluminescence were measured. The inhibition rate was calculated based on the RLU value of the blank wells. The IC50 was fitted using the log(inhibitor) vs. normalized response-variable slope method in Graphpad 8.0.1 software. 50 The values ​​and analysis results are shown in Table 2.

[0111] Table 2. Inhibitory effects of compounds on the proliferation of various tumor cells (IC50) 50 (μM) Note: A represents IC 50 Values ​​less than 1 μM, B indicates IC 50 Values ​​range from 1 μM to 5 μM, where C represents IC. 50 The value is greater than 5 μM.

[0112] As shown in Table 2, all tested compounds exhibited inhibitory effects on tumor cells carrying different mutant KRAS proteins. Among them, compound 24 showed the highest IC50 activity against various tumor cells. 50 The concentrations of all compounds reached below 1 μM, comparable to the positive control compound BI-2865; the IC50 values ​​of the remaining compounds were lower than those of some tumor cells. 50 The value can reach below 1 μM.

Claims

1. A compound having the structure of formula (I) or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that, In the structure of formula (I): R 1a R 1b R 2a R 2b R 3a R 3b Selected from hydrogen, deuterium, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, halogen, -NH2, -NH(C1-C4 alkyl), -N(C1-C4 alkyl)2, C3-C5 cycloalkyl or 3-5 member heterocyclic groups containing 1-2 N, O, S; Or R 1a R 1b R 2a R 2b R 3a R 3b Together with the carbon atom it is attached to, they form a cyclopropane or cyclobutane ring in the form of (1) or (2): (1)R 1a R 1b One of them and R 2a R 2b one of; (2)R 2a R 2b One of them and R 3a R 3b one of; X selects N or CR a ; R1, R a Selected from hydrogen, halogen, -OH, -NH2, cyano, or C1-C4 alkyl; Ring A is selected from pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, oxadiazole, isoxazole, thiazole, thiadiazole, isothiazole, or triazole; Z, V, and Y are selected from N or C; R2 is selected from a 3-10 heterocyclic group containing 1-2 N, O, or S members that is substituted with hydrogen, one or more identical or different C1-C6 alkyl groups, C1-C6 alkoxy groups, or 5-6 heterocyclic groups containing 1-2 N, O, or S members, or -O (C1-C6 alkyl) that is substituted with hydrogen or 3-10 heterocyclic groups containing 1-2 N, O, or S members, wherein the C1-C6 alkyl group is substituted with hydrogen or 3-8 heterocyclic groups containing 1-2 N, O, or S members, and the 3-10 heterocyclic group is substituted with hydrogen or one or more identical or different R5 groups. R5 is selected from C1-C6 alkyl, C1-C6 alkoxy, halogen or 3-10 heterocyclic groups containing 1-2 N, O, S groups; W is selected from N, O, or C; R3 and R4 are selected from hydrogen, -NH2, C1-C6 alkyl, C1-C6 haloalkyl, C3-C 10 Cycloalkyl, 3-10 member heterocyclic groups containing 1-2 N, O, S atoms, C6-C 10 Aryl, 5-10 membered heteroaryl containing 1-4 N, O, S or absent, wherein C1-C6 alkyl, C1-C6 haloalkyl, C3-C 10 Cycloalkyl, 3-10 member heterocyclic groups containing 1-2 N, O, S atoms, C6-C 10 The aryl or 5-10-membered heteroaryl group containing 1-4 N, O, S is substituted with hydrogen, or one or more identical or different R6 or R7. Alternatively, R3, R4, and the W connected to them can ring together to form a C3-C structure replaced by one or more R6s. 10 Cycloalkanes or C3-C 10 Heterocyclic alkanes containing 1-2 N, O, or S atoms; R6 is selected from hydrogen, C1-C6 alkyl, -OR7, -N(R7)2, halogen, -CN, -NH2, -C(=O)R7, -C(=O)OR7, -C(=O)N(R7)2 or -NHC(=O)OR7 or (=O); R7 is selected from hydrogen, C1-C6 alkyl, C3-C6 alkyl, and C7-C6 alkyl groups. 10 Cycloalkyl, 3-10 member heterocyclic groups containing 1-2 N, O, S atoms, C6-C 10 Aryl or 5-10-membered heteroaryl groups containing 1-4 N, O, S groups, of which C1-C6 alkyl, C3-C6 alkyl, and C4-C6 alkyl groups are present. 10 Cycloalkyl, 3-10 member heterocyclic groups containing 1-2 N, O, S atoms, C6-C 10 The aryl or 5-10 member heteroaryl group containing 1-4 N, O, S is substituted by one or more identical or different R8, R9; R8 is selected from hydrogen, -OR9, -N(R9)2 or -C(O)N(R9)2; R9 is selected from hydrogen, C1-C6 alkyl, C3-C6 alkyl, and C4-C6 alkyl groups. 10 Cycloalkyl, 3-10 membered heterocyclic group containing 1-2 N, O, S or 5-10 membered heteroaryl group containing 1-4 N, O, S, wherein the C1-C6 alkyl group is substituted by the following substituents: hydrogen, C1-C6 alkoxy, C3-C 10 Cycloalkyl or heterocyclic compounds consisting of 3-10 members containing 1-2 N, O, or S groups substituted with hydrogen or C1-C6 alkyl groups.

2. The compound or its stereoisomer, or a pharmaceutically acceptable salt, according to claim 1, characterized in that, The compound has the structure of formula (Ia): The definitions of rings A, X, W, Z, V, Y, R1, R2, R3, and R4 are as described in claim 1.

3. The compound or its stereoisomer, or a pharmaceutically acceptable salt, according to claim 1, characterized in that, The compound has the structure of formula (Ib): The definitions of rings A, X, W, Z, V, Y, R1, R2, R3, and R4 are as described in claim 1.

4. The compound or its stereoisomer, or a pharmaceutically acceptable salt, according to claim 1, characterized in that, In the aforementioned structure, ring A is selected from:

5. The compound or its stereoisomer, or a pharmaceutically acceptable salt, according to claim 1, characterized in that... In the aforementioned structure: R3 and R4 are selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C 10 Cycloalkyl, 3-10 member heterocyclic groups containing 1-2 N, O, S atoms, C6-C 10 Aryl or 5-10-membered heteroaryl groups containing 1-4 N, O, S groups, with R3 and R4 substituted by hydrogen or one or more identical or different R6 and R7 groups; Alternatively, R3, R4, and the W connected to them can ring together to form a C3-C structure replaced by one or more R6s. 10 Cycloalkanes or C3-C 10 Heterocyclic alkanes containing 1-2 N, O, or S atoms; The definitions of W, R6, and R7 are as described in claim 1.

6. The compound or its stereoisomer, or a pharmaceutically acceptable salt, according to claim 1, characterized in that, In the aforementioned structure, R2 is selected from:

7. The compound or its stereoisomer, or a pharmaceutically acceptable salt, according to claim 1, characterized in that, In the aforementioned structure, Selected from the following groups substituted with hydrogen, or one or more identical or different R6 or R7 groups: The definitions of R6 and R7 are as described in claim 1.

8. The compound or its stereoisomer, or a pharmaceutically acceptable salt, according to claim 1, characterized in that, The compound is selected from any one of the following compounds:

9. The compound or its stereoisomer, or a pharmaceutically acceptable salt, according to claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by the compound with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, or ferulic acid.

10. A method for preparing the compound of claim 1 or its stereoisomers, or a pharmaceutically acceptable salt thereof, characterized in that, Choose from any of the following methods: Method 1: When ring A is Compound I is obtained from compound I-1 through substitution, coupling, aminolysis, condensation and cyclization. Method 2: When ring A is Compound I is obtained from compound I-1 through substitution, coupling, reduction, condensation and cyclization. Method 3: When ring A is Compound I is obtained from compound I-1 through substitution, coupling, aminolysis, condensation and cyclization. Among them, X, W, Z, V, Y, R 1a R 1b R 2a R 2b R 3a R 3b The definitions of R1, R2, R3, and R4 are as described in claim 1; The corresponding acid is salted with compound I prepared by the above method to obtain the pharmaceutically acceptable salt.

11. The use of a compound or its stereoisomer, or a pharmaceutically acceptable salt thereof, according to claim 1, in the preparation of a medicament for treating and / or preventing cancer.

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