Pyridopyrimidine derivative as small GTP enzyme KRAS mutation inhibitor

Abstract

The invention provides a pyridopyrimidine derivative as a small GTP enzyme KRAS mutation inhibitor, and particularly provides a pyridopyrimidine derivative with a structural formula as shown in a general formula (A) and pharmaceutically acceptable salts thereof, and the pyridopyrimidine derivative has inhibitory activity on mutants such as KRAS G12D and the like. The invention also provides a preparation method of the derivative, a pharmaceutical composition and a salifying compound of the derivative, and medical application of the derivative as a small GTP enzyme KRAS mutation inhibitor in treatment of different types of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry. Specifically, the present invention provides a class of pyridopyrimidine derivatives, and a preparation method thereof, as well as the medical use of the compounds, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof as small GTPase KRAS mutation inhibitors in the treatment of different tumors. Background Art

[0002] As a human oncogene, RAS is one of the most commonly mutated genes in tumors. RAS mutations are found in about 30% of tumors. If the regulatory factors of RAS and upstream and downstream mutations of the signaling pathway are combined, almost all tumors are covered. The KRAS gene (Kirsten rat sarcoma viral oncogene homolog) is an important member of the RAS gene family. The protein encoded by the KRAS gene is a GDP / GTP binding protein, a small GTPase enzyme, which belongs to the superprotein family. The KRAS protein has 188 amino acids and a molecular weight of 21.6KD. It is located on the inner side of the cell membrane and is connected to the cell membrane through the farnesyl modification gene. KRAS protein is like a switch. It can bind to GTP in an activated state (KRAS-GTP) or bind to GDP in a closed state (or inactive state) (KRAS-GDP). Subsequently, GTPase activating protein (GAP) can hydrolyze GTP bound to KRAS-GTP into GDP, prompting the formation of the KRAS-GDP closed state, thereby placing the KRAS switch in an inactive closed state. KRAS protein is a "switch" between the KRAS-GTP activated state (open state) and the KRAS-GDP inactive state (closed state). In the activated state, it can activate downstream signaling pathways including the MAPK signaling pathway, the PI3K signaling pathway, and the Ral-GDS signaling pathway. The RAS protein switch controls its downstream signaling pathways, thereby promoting cell survival, proliferation, and cytokine release, and plays an important role in life processes such as cell proliferation, differentiation, and apoptosis. KRAS can also be briefly activated by growth factors (such as EGFR). The activated KRAS can activate downstream signaling pathways such as the PI3K-AKT-mTOR signaling pathway that controls cell generation, and the RAS-RAF-MEK-ERK signaling pathway that controls cell proliferation. Mutated KRAS will continue to be activated even in the absence of kinase activation such as EGFR, leading to continued cell proliferation and ultimately cancer.

[0003] KRAS mutations are highly expressed in a variety of tumors, the most common of which include lung cancer, intestinal cancer, pancreatic cancer, colon cancer, small intestine cancer, bile duct cancer, etc. Structural studies have shown that most KRAS gene mutations interfere with KRAS's ability to hydrolyze GTP, ultimately causing KRAS to be continuously activated, making it unable to effectively regulate cell signal transduction, thereby promoting the occurrence, development, and metastasis of tumors.

[0004] For KRAS mutations, mutations at amino acid position 12 (G12) account for about 80%, while G12C mutations account for about 14% of all G12 mutations. In recent years, researchers have developed a series of covalent inhibitors for KRAS G12C mutations, but the development of inhibitors for KRASG12D mutations has encountered great challenges.

[0005] At present, no good method has been developed for covalent binding to aspartic acid. The difficulty in directly inhibiting the KRAS G12D mutant is not only that the surface of the protein encoded by KRAS is smooth and lacks binding sites, but also that KRAS binds to GTP / GDP very strongly and the concentration of intracellular GTP / GDP is also very high, which makes it impossible to develop competitive inhibitors for GTP and KRAS. It is also very difficult to develop protein-protein interactions that inhibit or disrupt KRAS mutant proteins and their effector proteins. Not only is the membrane localization of KRAS regulated by farnesyl transferases, etc., but the therapeutic window for targeting KRAS downstream signaling molecules (effector proteins) and inhibiting the wild-type signaling pathway required for growth is narrow. Moreover, due to the compensatory mechanism, it is impossible to completely and effectively inhibit the downstream signals of KRAS mutants, thereby greatly limiting the efficacy of kinase inhibitors developed for effector proteins against KRAS mutations.

[0006] In summary, there is still a great unmet clinical need for the development of small GTPase inhibitors with oral safety and efficacy, such as KRAS G12D mutation inhibitors. Summary of the invention

[0007] The purpose of the present invention is to provide a KRAS mutant inhibitor that is safe and effective for oral administration, in particular, an inhibitor for the treatment of tumors such as colorectal cancer, lung cancer, pancreatic cancer, bile duct cancer, esophageal cancer, breast cancer, and gastric cancer.

[0008] The first aspect of the present invention provides a compound as shown in the following formula A, or a pharmaceutically acceptable salt thereof:

[0009]

[0010] Wherein, Ring A is a 4-9-membered saturated or partially unsaturated carbon ring, or a 4-9-membered saturated or partially unsaturated hetero ring;

[0011] X is selected from the group consisting of N, CH or CF;

[0012] R1 is selected from the following group: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 cyanoalkyl, C1-C6 hydroxyalkyl, allyl, -C(=O)H, -CO2R 5 、-C(=O)R 6 、-SO2R 7 、-CO2N(R 5 )2、-C O 2 CH R 8 O(C=O)R 9 , aryl, heteroaryl, CONH2, CONR a R b or CONHR c ; where R a , R b , R c are respectively selected from: C1-C6 alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;

[0013] R1' is hydrogen, deuterium, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 deuterated alkyl, C2-C6 alkenyl, C2-C3 alkynyl, cyano substituted methyl;

[0014] p is 0, 1, 2, 3, 4, 5 or 6;

[0015] R2 is selected from the group consisting of hydrogen, deuterium, halogen, cyano, cyanomethyl, hydroxyl, hydroxymethyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 deuterated alkyl, C2-C6 alkenyl, C2-C3 alkynyl;

[0016] R4 is selected from the group consisting of hydrogen, deuterium, halogen, cyano, cyanomethyl, hydroxyl, hydroxymethyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 deuterated alkyl;

[0017] Each R5, R6, R7 is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl;

[0018] R8 is each independently selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C15 haloalkyl, C1-C3 alkoxy;

[0019] R9 is independently selected from the following group: hydrogen, C1-C15 saturated or unsaturated alkyl, C1-C15 saturated or unsaturated halogenated alkyl, C1-C15 saturated or unsaturated alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl; R 10Selected from the group consisting of hydrogen, deuterium, halogen, cyano, hydroxyl, nitro, amino, hydroxymethyl, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 deuterated alkyl, C1-C4 haloalkoxy, C2-C4 alkenyl, C3-C6 cycloalkyl;

[0020] R 11 is selected from the group consisting of hydrogen, deuterium, halogen, methyl, hydroxymethyl, ethyl, C3-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 cycloalkoxy, C1-C6 cycloalkanethiol, deuterated methyl, deuterated ethyl, halomethyl, halomethoxy; or two R 11 The carbon atoms connected thereto together form a 3-9-membered cycloalkyl or heterocyclic group;

[0021] m is 0, 1, 2, 3 or 4;

[0022] R 12 Selected from the group consisting of H, deuterium, halogen, cyano, methyl, hydroxymethyl, ethyl, C3-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkanethio, C1-C6 cycloalkoxy, C1-C6 cycloalkanethio, deuterated methyl, deuterated ethyl, halomethyl, halomethylenyl, halomethoxy;

[0023] n is 1, 2 or 3;

[0024] q is 0, 1, 2, 3, 4, or 5;

[0025] R 13 and R 14 Each is independently selected from the group consisting of hydrogen, deuterium, methyl, deuterated methyl, or cyclopropyl;

[0026] R 15 and R 16 Each is independently selected from the group consisting of hydrogen, deuterium, methyl, deuterated methyl, or cyclopropyl;

[0027] M is selected from the group consisting of oxygen atom, sulfur atom, NH, or NR 16 ;

[0028] R 16 is methyl, or deuterated methyl;

[0029] R 17 is H, F, chloro, bromo, methyl, trifluoromethyl, cyano, deuterated methyl, or ethynyl;

[0030] Wherein, the saturated or partially unsaturated carbocyclic ring, saturated or partially unsaturated heterocyclic ring, or heterocyclic group may be saturated or partially unsaturated, but not aromatic, and may be a monocyclic ring, a condensed ring, a bridged ring or a spirocyclic ring, and the heterocyclic ring has 1-4 heteroatoms selected from N, O or S.

[0031] In another preferred embodiment, the compound has a structure shown in the following formula:

[0032]

[0033] In another preferred embodiment, the compound has a structure shown in the following formula (I) or formula (II):

[0034]

[0035] Among them, R1, R1', R2, R 10 , R 11 , R 12 , p, m, n, q are as described in the first aspect of the present invention;

[0036] In another preferred embodiment, the compound has a structure shown in the following formula:

[0037]

[0038] In another preferred embodiment, the compound has a structure shown in the following formula:

[0039]

[0040] Among them, R1, R1', R2, R 10 , R 11 , R 12 , p, n, q are all as described in the first aspect of the present invention.

[0041] In another preferred embodiment, the compound has a structure shown in the following formula:

[0042]

[0043] Wherein, R1, R1', R2, R 10 , R 11 , p, and m are as described in the first aspect of the present invention.

[0044] In another preferred embodiment, R 1 Selected from the group consisting of hydrogen, hydroxy, C1-C6 alkyl, C1-C6 cyanoalkyl, C1-C6 hydroxyalkyl, HC(=O)-, -CO2R 5 , or -CO2N(R 5 )2.

[0045] In another preferred embodiment, the Having a structure selected from the group consisting of:

[0046]

[0047]

[0048]

[0049] In another preferred embodiment, the Having a structure selected from the group consisting of:

[0050]

[0051] In another preferred embodiment, the R 2 Selected from H, D, CD3, halogen, hydroxyl, mercapto, cyano, C1-C3 alkyl, cyanomethyl, halogenated C1-C3 alkyl, C1-C3 alkoxy, vinyl, ethynyl, allyl.

[0052] In another preferred embodiment, the R 2 Selected from H, D, CD3, halogen, C1-C3 alkyl, halogenated C1-C3 alkyl, C1-C3 alkoxy, vinyl.

[0053] In another preferred embodiment, the compound of formula (I) or formula (II) has a structure selected from the following group:

[0054]

[0055]

[0056]

[0057] The second aspect of the present invention provides the use of the compound according to the first aspect of the present invention for preparing a drug for treating a disease associated with the activity or expression of a KRAS mutant.

[0058] In another preferred embodiment, the disease associated with the activity or expression of the KRAS mutant is a tumor; preferably, it is a tumor selected from the following group: sarcoma, myxoma, rhabdomyomas, fibromas, lipomas, teratomas, bronchial carcinomas, lung cancers, bronchial adenomas, lymphomas, chondroma hamartomas, mesothelioma, esophageal cancer, gastric cancer, pancreatic cancer, small intestine cancer, colorectal cancer, cecal cancer, genitourinary tract tumors, kidney cancer, bladder cancer, urethral cancer, prostate, testicular cancer, liver cancer, bile duct cancer, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, gallbladder cancer, ampullary cancer, bone cancer, brain cancer, uterine cancer, vaginal cancer, hematological tumors, skin cancer, ovarian cancer, and breast cancer.

[0059] In another preferred embodiment, the KRAS mutant is a KRAS G12D mutant, a KRAS G12V mutant, a KRASG12S mutant or a KRAS G13D mutant.

[0060] The third aspect of the present invention provides a pharmaceutical composition, which comprises: (i) a therapeutically effective amount of the compound of formula A as described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

[0061] In another preferred embodiment, the pharmaceutical composition is used to treat diseases related to the activity or expression level of KRAS mutants.

[0062] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. DETAILED DESCRIPTION

[0063] After long-term and in-depth research, the inventors prepared a class of compounds having the structure shown in Formula A, and found that they have the activity of inhibiting KRAS-effector protein-protein interactions. Moreover, the compounds have an inhibitory effect on a series of KRAS-effector protein-protein interactions at extremely low concentrations (as low as ≤100nmol / L), and the inhibitory activity is quite excellent, so they can be used to treat diseases related to KRAS mutations such as tumors. Based on the above findings, the inventors have completed the present invention.

[0064] the term

[0065] As used herein, the term "C1-C6 alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, or the like, and "C1-C3 alkyl" and the like have similar definitions.

[0066] The term "C1-C6 alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, or the like, and "C1-C3 alkoxy" and the like have similar definitions.

[0067] In the present invention, the terms "comprising", "including" or "including" indicate that various components can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting mainly of" and "consisting of" are included in the term "comprising".

[0068] In the present invention, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reaction), that is, has a reasonable benefit / risk ratio.

[0069] In the present invention, the term "effective amount" refers to the amount of a therapeutic agent to treat, alleviate or prevent a target disease or condition, or to an amount that exhibits a detectable therapeutic or preventive effect. The exact effective amount for a subject depends on the subject's size and health, the nature and extent of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is useless to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation, which a clinician is able to judge.

[0070] In the present invention, the term "prodrug" refers to a prodrug, a drug precursor, or a prodrug, which refers to a compound that has no or low in vitro protein-interaction enzyme activity against KRAS mutants after chemical structure modification, and releases a highly active drug through enzymatic action or non-enzymatic chemical reaction in vivo to exert its efficacy.

[0071] As used herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of halogen, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C2-C6 acyl, unsubstituted or halogenated C1-C6 alkyl-hydroxy.

[0072] Unless otherwise specified, in the present invention, all compounds appearing are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all compounds of the present invention, each chiral carbon atom can be optionally in R configuration or S configuration, or a mixture of R configuration and S configuration.

[0073] The term "cycloalkyl" includes saturated and partially unsaturated cycloalkyl groups having 3 to 12 carbons, for example 3 to 8 carbons, and as a further example 3 to 6 carbons, wherein the cycloalkyl is optionally substituted with one or more. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl and cyclooctyl. The term "cycloalkyl" also includes bridged cycloalkyl groups, for example bicyclo[1.1.1]pentyl.

[0074] As used herein, the term "aryl" group is a C6-C 14 Aromatic moiety, which is optionally substituted by one or more R6 or one or more R7 as defined herein. As an example, aryl is C6-C10 aryl. Examples of aryl include, but are not limited to, phenyl, naphthyl, anthracenyl, fluorenyl and dihydrobenzofuranyl. "Aryl" also refers to a bicyclic or tricyclic ring system, wherein one or both rings of the aromatic ring system can be saturated or partially saturated, respectively, and wherein if the ring system includes two saturated rings, the saturated rings can be fused or spirocyclic, but the connection position with the rest of the compound is on the aryl moiety.

[0075] A "heterocyclyl" or "heterocyclic" group is a ring structure having 3 to 12 atoms, such as 4 to 8 atoms, wherein one or more atoms are selected from the group consisting of N, O and S, wherein the ring N atoms can be oxidized to NO, and the ring S atoms can be oxidized to SO or SO2, and the remaining ring atoms are carbon. A heterocyclyl can be a monocyclic, bicyclic, spirocyclic or bridged ring system. The heterocyclyl or heterocycle can be partially or fully unsaturated, and when the heterocyclyl is a partially unsaturated (i.e., partially non-aromatic) structure, the attachment site of the heterocyclyl or heterocycle can be located on a fully or partially saturated ring or on an aromatic ring.

[0076] The term "heteroaryl" refers to a group having 5 to 14 ring atoms, preferably 5, 6, 9 or 10 ring atoms; and in addition to carbon atoms, each ring has one to three heteroatoms selected from N, O and S. "Heteroaryl" also refers to a bicyclic ring system in which each ring has, in addition to carbon atoms, one to three heteroatoms selected from N, O and S, wherein one of the ring systems may be saturated or partially saturated.

[0077] The term "halogen" refers to F, Cl, Br and I.

[0078] As used herein, the term "compound of the present invention" refers to a compound represented by Formula A. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compound of Formula A.

[0079] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzenesulfonic acid, and acidic amino acids such as aspartic acid and glutamic acid.

[0080] Preparation of small GTPase KRAS mutant inhibitors

[0081] Once the structures of the compounds of the present invention are known, the compounds of the present invention can be prepared from commercially available reagents using the synthetic methods and reaction schemes described herein, or using other reagents and conventional methods well known to those skilled in the art.

[0082] For example, the compounds of reaction formula A, Formula I and Formula II can be prepared and synthesized according to Formula A. In step a, 7-chloro, 8-fluorobenzene ring or pyrido [4,3-d] pyrimidine dione (1) is reacted with phosphorus oxychloride to generate an intermediate 2,4,7-trichloro-8-fluorobenzene ring or pyrido pyrimidine intermediate (2). In step b, intermediate 2 is reacted with a substituted bridged ring piperazine to generate intermediate (3). In step c, intermediate 3 is reacted with a nucleophilic reagent 1,1-cyclobutane substituted hydroxymethyl alcohol to generate a nucleophilic substitution product intermediate of 4A or 4B. In step d, intermediate (4A or 4B) is reacted with a polysubstituted benzothiophene boronic acid or boric ester reagent or a corresponding trialkyl substituted tin reagent (these two types of reactions are respectively called Suzuki Cross Coupling and Stille Cross Coupling) to generate a carbon-carbon coupling intermediate (5A or 5B). In step e, the intermediate (5A or 5B) is subjected to acidic conditions (usually a TFA / dichloromethane mixed reagent) to remove the tert-butyl formate to finally generate a compound Formula I or Formula II with different structural products.

[0083] According to the preparation method similar to that of formula A, compounds of formula III and formula IV can be prepared and synthesized according to formula B.

[0084]

[0085] Similar compounds can be prepared by replacing the corresponding starting material fragments using the methods described in the examples of the present invention.

[0086] Pharmaceutical compositions and methods of administration

[0087] Since the compounds of the present invention have excellent inhibitory activity against KRAS mutants, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate diseases associated with the activity or expression of KRAS mutants.

[0088] KRAS mutants not only refer to G12D mutants, but also include G12V, G12S, G13D and G12C mutants.

[0089] In a preferred embodiment, the above-mentioned disease associated with KRAS mutant activity or expression is selected from the following group:

[0090] Heart: sarcomas (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma;

[0091] Lung: bronchial carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, enchondroma, hamartoma, mesothelioma;

[0092] Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract : Kidney (adenocarcinoma, Wilms' tumor (Nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatoid tumor, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma;

[0093] Biliary tract: gallbladder cancer, ampullary cancer, bile duct cancer;

[0094] Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulocyte sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondroma (osteocartilaginous exostosis), benign enchondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumor; Nervous system: skull (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meninges (meningiomas, meningosarcomas, gliomas), brain (astrocytomas, medulloblastomas, gliomas, ependymomas, germ cell tumors (pinealomas), glioblastoma multiforme, oligodendrogliomas, schwannomas, retinoblastomas, congenital tumors), spinal neurofibromas, meningeal Tumor, glioma, sarcoma); Gynecology: uterus (endometrial cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa cell-theca tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube (carcinoma), breast cancer, ovarian cancer; Hematology: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma);

[0095] Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi sarcoma, dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and Adrenal gland: neuroblastoma.

[0096] In a most preferred embodiment, the cancer is non-small cell lung cancer, small cell lung cancer, colorectal cancer, rectal cancer or pancreatic cancer.

[0097] The medicine or pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention / dose, more preferably, 5-600 mg of the compound of the present invention / dose. Preferably, the "one dose" is a capsule or tablet.

[0098] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0099] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0100] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0101] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0102] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0103] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0104] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0105] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0106] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required as necessary.

[0107] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0108] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage. For a person weighing 60 kg, the daily dosage is usually 1 to 1200 mg, preferably 5 to 600 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill range of a skilled physician.

[0109] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0110] Preparation Example 1

[0111]

[0112] Step a: DMAP (1.54 g) was added to a DCM (25 ml) solution of raw material 1-1 (1 g), 2 (1.03 g), EDCI.HCl (1.45 g) under nitrogen protection. The resulting reaction solution was stirred overnight at room temperature, and the reaction progress was monitored by TLC. The conversion of the raw materials was completed. The reaction solution was poured into (100 ml) water, 1N HCl was added to adjust the solution pH to 5, and extracted 3 times with 80 ml of dichloromethane, the organic phases were combined, washed twice with saturated brine (50 ml), dried over anhydrous sodium sulfate, and filtered. The filtrate was dried under reduced pressure to obtain intermediate 3-1 (1.5 g) as a light yellow oil.

[0113] Step b: LiAlH4 (896 mg) was added to a THF (50 ml) solution of intermediate 3-1 (1.4 g) at -10°C under nitrogen protection. The resulting reaction solution was stirred overnight at room temperature. The reaction progress was monitored by TLC and the reaction was completed. The reaction solution was stirred and cooled to 0°C. Water (1 ml) was slowly added, followed by 10% NaOH aqueous solution (1 ml) and water (1 ml) for quenching. The reaction solution was stirred for 30 minutes and then filtered. The filtrate was dried under reduced pressure to obtain a colorless oil Preparation Example 1 (700 mg) which was directly used in the next step.

[0114] 1 H NMR (300MHz, DMSO-d6) δ4.66(s,1H),3.56 -3.50(m,4H),3.48(s,2H),2.37-2.29(m,6H),1.87 -1.65(m,6H).

[0115] Preparation Example 2, Preparation Example 3 and Preparation Example 4

[0116]

[0117] Preparation Example 2, Preparation Example 2A, Preparation Example 3 and Preparation Example 4 can be prepared by a method similar to Preparation Example 1.

[0118] Preparation Example 5

[0119]

[0120] Step a:

[0121] 5-1 (2.0 g, 9.1 mmol) was dissolved in methanol (25), and sodium borohydride (0.67 g, 17.7 mmol) was added in batches at 0°C. The reaction was then warmed to room temperature and stirred for 1 hour. TLC showed that the reaction was complete, and the mixture was poured into a saturated ammonium chloride solution (10 mL) and extracted with DCM (10 mL*3). The organic layer was washed with water (10 ml) and a saturated sodium chloride aqueous solution (10 mL), dried over anhydrous magnesium sulfate, filtered, and the organic phase liquid was then dried under reduced pressure to obtain a white solid 5-2 (1.95 g).

[0122] 1 H NMR (400MHz, CDCl3) δ7.34 (ddd, J=8.8, 4.2, 1.9Hz, 1H), 7.05 (dd, J=17.7, 8.9Hz, 1H), 4.85 (d, J=2.2Hz, 2H).

[0123] Step b:

[0124] 5-2 (1.95 g, 8.8 mmol) was dissolved in THF (20 ml), and DIEA (1.8 ml, 10.3 mmol) was added, the mixture was cooled to 0 ° C, and then methanesulfonic anhydride (1.7 g, 9.42 mmol) was added. After stirring at ambient temperature for 16 hours, the mixture was diluted with EtOAc: methyl tert-butyl ether (1: 1) (15 mL) and washed with cold water (20 ml). The layers were separated and the aqueous solution was extracted twice with EtOAc: methyl tert-butyl ether (1: 1) (20 mL * 3). The combined organics were washed with water and saturated sodium chloride aqueous solution, dried over anhydrous magnesium sulfate, filtered, and the organic phase liquid was then spin-dried under reduced pressure to give a yellow oil 5-3 (2.60 g).

[0125] 1 H NMR (400MHz, CDCl3) δ7.41–7.35(m,1H),7.15(q,J=8.8Hz,1H),5.37(d,J=1.4Hz,2H),3.07(d,J=0.6Hz,3H).

[0126] Step c:

[0127] A mixture of compound 5-3 (2.6 g, 8.2 mmol) and potassium cyanide (0.61 g, 9.03 mmol) in ethanol (20 ml) and water (4 ml) was refluxed for 30 minutes and then cooled to ambient temperature. The solvent was removed in vacuo and the residue was suspended in DCM (20 mL). The mixture was washed with water, saturated sodium bicarbonate solution and saturated sodium chloride aqueous solution. The organic matter was dried over magnesium sulfate, filtered, and the organic phase liquid was then spin-dried under reduced pressure. The crude product was purified by silica gel flash column chromatography, eluted with 10-100% DCM / hexane to obtain 5-4 (1.8 g).

[0128] 1 H NMR (400MHz, CDCl3) δ7.40 (ddd, J=9.0, 4.4, 2.1Hz, 1H), 7.13 (dd, J=17.4, 9.1Hz, 1H), 3.89 (d, J=1.9Hz, 2H).

[0129] Step d:

[0130] A solution of 5-4 (1.8 g, 7.75 mmol) in DMF (20 ml) was cooled in an ice bath, and then potassium tert-butoxide (0.930 g, 8.12 mmol) was added to the solution in batches, the mixture was stirred for 10 minutes (the reaction turned dark red), and ethoxycarbonyl isothiocyanate (0.98 mL, 8.14 mmol) was added dropwise. The reaction mixture was stirred at ambient temperature for 1 hour and then heated at 100 ° C for 30 minutes. The mixture was then cooled in an ice bath for 10 minutes, and water (50 ml) was slowly added under stirring. The resulting precipitate was collected by filtration, rinsed with water and hexane, and then dried to obtain 5-5 (2.45 g) which was directly used in the next step.

[0131] Step e:

[0132] In a solution of 5-5 (2.45 g, 7.2 mmol) in DMSO (10 milliliters), 5N NaOH solution (0.8 milliliter, 40 mmol) was added and refluxed for 7 hours. The mixture was cooled to room temperature and treated with cold water while stirring vigorously. The resulting precipitate was collected by filtration, washed with water, and dried to give white solid compound 5-6 (1.55 g).

[0133] 1 H NMR (400MHz, DMSO-d6) δ8.22 (s, 2H), 7.51-7.48 (dd, J = 4.0, 8.0Hz, 1H), 6.96 (t, J = 8.0Hz, 1H).

[0134] Step f:

[0135] Boc2O (1.96 g, 8.71 mmol) was added to a solution of compound 5-6 (2 g, 7.9 mmol), DIEA (2.1 mL, 12 mmol) and DMAP (80 mg, 0.655 mmol) in THF (27 mL) and DMF (4 mL). The mixture was stirred under nitrogen for 4 hours at room temperature. The reaction mixture was diluted with aqueous sodium bicarbonate solution and ethyl acetate (1:1) (20 mL) and then stirred for 10 minutes. The resulting precipitate was collected by filtration, washed with cold EtOAc (20 mL), dried, and further extracted with EtOAc (20 mL*2). The organic extract was washed with water (20 mL) and saturated aqueous sodium chloride solution (20 mL), dried over magnesium sulfate, filtered, and then the organic phase liquid was spin-dried under reduced pressure and purified by silica gel flash chromatography column, eluted with 20-100% DCM / hexane to obtain 5-7 (1.2 g, 3.2 mmol).

[0136] 1H NMR (400MHz, DMSO-d6) δ11.78 (s, 1H), 7.69-7.66 (dd, J = 4.0, 8.0Hz, 1H), 7.20 (t, J = 8.0Hz, 1H), 1.54 (s, 9H).

[0137] Step g:

[0138] Intermediate 5-7 (1.60 g, 4.14 mmol) and bis-neopentyl glycol diboronate 5-8 (3.70 g, 15.7 mmol) were dissolved in 1,4-dioxane (30 ml). Potassium acetate (1.2 g, 12.4 mmol) was added and replaced with nitrogen three times. DPEpHosPdC12 (0.3 g, 0.42 mmol) was added and heated at 95 °C for 1 hour. The mixture was then cooled to room temperature and filtered. The reaction solution was concentrated to 10 mL under reduced pressure, diluted with heptane (20 mL), stirred for 10 minutes, and then filtered through celite, rinsed with heptane and heptane: methyl tert-butyl ether (1:1). The filtrate was concentrated, dissolved in minimal DCM (10 ml), and filtered through a silica gel pad, rinsed with ethyl acetate: heptane (1:1). The filtrate was rinsed with saturated ammonium chloride solution and saturated sodium chloride aqueous solution, respectively. The organics were dried over magnesium sulfate, filtered, and the organic phase liquid was then spin dried under reduced pressure and purified by flash column chromatography on silica gel eluting with 5-50% (20% acetone in DCM) / hexanes to give the boronate Preparation 5 as a white solid (1.30 g, 3.2 mmol).

[0139] 1 H NMR(DMSO-d6)δ11.6(s,1H),7.61(m,1H),7.20(m,1H),3.78(s,4H),1.54(s,9H),1.03(s,6H).

[0140] Preparation Example 6

[0141]

[0142] Similar borate esters such as Preparation Example 6 can be prepared by a similar method as Preparation Example 5.

[0143] Preparation Example 7

[0144]

[0145] Step a:

[0146] At room temperature, the raw material 7-1 (4.0 g, 18.56 mmol) was dissolved in phosphorus oxychloride (40 mL), and N, N-diisopropylethylamine (16.0 mL, 92.8 mmol) was added, and the mixture was reacted at 120°C for 3 hours. After the reaction was complete, the mixture was cooled and concentrated under reduced pressure, and the crude product was directly used in the next step to obtain a black oily 2,4,7-trichloro, 8-fluoropyridopyrimidine intermediate 7-2 (4.68 g).

[0147] Step b:

[0148] Under room temperature, the intermediate 7-2 (4.68 g, 18.4 mmol) was dissolved in dichloromethane (80 ml) and N, N-diisopropylethylamine was added to adjust (pH value was 7), and then N, N-diisopropylethylamine (8.0 mL, 45.96 mmol) and (1R, 5S)-3, 8-diazacyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (3.25 g, 15.32 mmol) were added in sequence. Stir at room temperature for 17 hours. After the reaction was complete, water (300 ml) was slowly added and extracted three times with dichloromethane (200 ml). The organic phase was washed with saturated brine (200 ml), and then dried over anhydrous sodium sulfate. The organic phase solution was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:4) to obtain yellow solid Preparation Example 7 (2.61 g,).

[0149] LCMS: m / z=428.1 (M+H) + .

[0150] 1 H NMR (400MHz, DMSO) δ9.07 (s, 1H), 4.48 (s, 2H), 4.26 (s, 2H), 3.70 (s, 2H), 1.78 (s, 2H), 1.61 (d, J = 7.4Hz, 2H), 1.46 (s, 9H).

[0151] Example 1

[0152]

[0153] Step a

[0154] Under nitrogen protection, KF (41 mg) was added to a DMSO (10 ml) solution of Preparation Example 3 (131 mg) and Preparation Example 7 (150 mg). The resulting reaction solution was stirred at 120°C overnight, and the reaction was monitored by TLC. The conversion of the raw materials was complete. The cooled reaction solution was poured into (80 ml) water, extracted 3 times with 50 ml of ethyl acetate, the organic phases were combined, washed twice with saturated brine (35 ml), dried over anhydrous sodium sulfate, and filtered. The crude product after the organic phase was dried under reduced pressure was purified by silica gel column (EA / PE, 0-20%) to obtain a light yellow solid intermediate 1-1 (95 mg)

[0155] LCMS: [M+H] + =579.0

[0156] Step b

[0157] Under nitrogen protection, intermediate 1-1 (1 g), preparation example 5 (1.4 g), DPEphosPdCl2 (502 mg), Cs2CO3 (2.25 g) and DMF (50 ml) were added to the bottle and stirred at 110°C for 7 hours under nitrogen protection. TLC monitored the reaction and the conversion of raw materials was completed. After the reaction solution was cooled and filtered, the reaction solution was poured into (150 ml) water, extracted 3 times with ethyl acetate (100 ml), combined organic phases, washed twice with saturated brine (100 ml), dried over anhydrous sodium sulfate, filtered, and the organic phase was dried under reduced pressure. The crude product after drying was purified by C18 silica gel column (methanol / water (0.05% ammonium bicarbonate) = 0-95%) to obtain white solid intermediate 1-2 (330 mg).

[0158] LCMS: [M+H] + =835.0

[0159] Step c

[0160] Trifluoroacetic acid (2 ml) was added to a solution of intermediate 1-2 (100 mg) in dichloromethane (6 ml) at room temperature. The reaction solution was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete, and the reaction solution was concentrated under reduced pressure to obtain a brown-yellow oily crude product. The crude product was purified by Prep-TLC with a developing solvent of DCM / MeOH=9 / 1 (0.5% ammonia water) to obtain Example 1 (30 mg) as a white solid.

[0161] LCMS: [M+H] + =635.0.

[0162] Example 2

[0163]

[0164] The preparation of Example 2 can refer to the similar method of Example 1 to replace the reaction intermediate of Preparation Example 1 with Preparation Example 7, and then carry out carbon-carbon coupling with Preparation Example 6 and then hydrolyze with trifluoroacetic acid to obtain white solid Example 2 (23 mg).

[0165] LCMS: m / z=651.0 (M+H) + .

[0166] Example 3

[0167]

[0168] The preparation of Example 3 can refer to the similar method of Example 1 to replace the reaction intermediate of Preparation Example 2 with Preparation Example 7, and then carry out carbon-carbon coupling with Preparation Example 6 and then hydrolyze with trifluoroacetic acid to obtain white solid Example 3 (17 mg).

[0169] LCMS: m / z=677.0 (M+H) + .

[0170] Example 4

[0171]

[0172] The preparation of Example 4 can refer to the similar method of Example 1 to replace the reaction intermediate of Preparation Example 3 and Preparation Example 7, and then carry out carbon-carbon coupling with Preparation Example 6 and then hydrolyze with trifluoroacetic acid to obtain white solid Example 4 (20 mg).

[0173] LCMS: m / z=653.0 (M+H) + .

[0174] Example 5

[0175]

[0176] The preparation of Example 5 can refer to the similar method of Example 1 to replace the reaction intermediate of Preparation Example 4 and Preparation Example 7, and then carry out carbon-carbon coupling with Preparation Example 6 and then hydrolyze with trifluoroacetic acid to obtain white solid Example 5 (26 mg).

[0177] LCMS: m / z=665.0 (M+H) + .

[0178] 1 H NMR(300MHz,DMSO-d6)δ9.07(s,1H),8.25(s,2H),7.29(t,1H),4.49(s,1H),4 .27(s,3H),3.69(d,1H),3.48(t,7H),2.47–2.32(m,6H),1.68–1.32(m,12H).

[0179] Test Example 1: KRAS G12D protein competitive inhibition activity experiment (Competition assay)

[0180] This experiment uses homogeneous time-resolved fluorescence (HTRF) to identify a class of compounds that compete for binding to the GDP form of KRAS protein and that can compete to displace the fluorescently labeled probe occupying the KRAS protein binding site. Biotinylated recombinant KRAS protein (sequence 1-188AA) was purified and expressed in Escherichia coli, and the target compound was diluted in a gradient ratio using DMSO solution to obtain a series of compound concentrations. During the experiment, 5nM KRAS G12D, 25nM fluorescently labeled probe and target compound diluted in reaction buffer (50mM HEPES pH 7.5, 5mM MgCl2, 1mM DTT) were added to a 384-well white shallow well plate (PerkinElmer), and appropriate control wells were set up. GDP-KRAS protein, fluorescently labeled probe and compound were pre-incubated in a 384-well plate for 30 minutes, and then diluted 0.5nM Tb-SA (Cisbio) was added to start the reaction. After sealing the plate and incubating at room temperature in the dark for 1 hour, the TR-FRET signal value (excitation wavelength: 320 nm, emission wavelength: 615 nm and 665 nm) was measured using EnVision microplate reader (PerkinElmer). Calculate the fluorescence signal ratio RLU = (665 nm signal / 615 nm signal) x 104; the % inhibition rate of the competitive binding between the target compound and the labeled probe at different concentrations was calculated by setting the 0% inhibition rate and 100% inhibition rate signal wells.

[0181] Compound inhibition rate IR (%) formula = (RLU 0％抑制 -RLU 化合物 ) / (RLU 0％抑制 -RLU 100％抑制 )×100%, and the 4-parameter logistic model was used to fit the compound gradient dilution concentration and the corresponding inhibition rate to calculate the IC 50 The competitive inhibitory activity (IC) of the test compound on KRAS G12D protein 50 , μM) results are shown in Table 1 below.

[0182] Test Example 2: KRAS protein interaction (PPI) experiment

[0183] In this experiment, the homogeneous time-resolved fluorescence (HTRF) method was used to detect the inhibitory activity of small molecule compounds on the interaction between KRAS protein and downstream RAF1 protein in the GTP-activated state. The purified Tag1-RAF1 protein stock solution was diluted 100 times with TR-FRET buffer. Similarly, the purified Tag2-KRAS protein and GTP mixture were diluted 100 times with buffer to ensure that the final concentration of GTP was 10μM. The working concentrations of these proteins were optimized to ensure the generation of maximum signals. The test compounds were graded and diluted with DMSO solution to obtain a series of compound concentrations, and the final concentration of DMSO was controlled to be 0.5%. 4μL GTP-KRAS protein, 4μL RAF1 protein and 2μL compound working solution were added to a 384-well white shallow well plate (PerkinElmer), and appropriate control wells were set simultaneously. GTP-KRAS protein, RAF1 protein and compound were pre-incubated in a 384-well plate for 15 minutes, and then 10μL Anti-Tag1-Eu diluted in HTRF detection buffer was added. 3+ The reaction was initiated by adding a mixture of anti-Tag2-XL665 labeled antibodies. The plates were sealed and incubated at 4°C in the dark for 2 hours. The TR-FRET signal value was measured using an EnVision microplate reader (PerkinElmer) (excitation wavelength: 320 nm, emission wavelength: 615 nm and 665 nm). The fluorescence signal ratio RLU was calculated as (665 nm signal / 615 nm signal) x 10 4 The compound % inhibition rate is calculated by setting the 0% inhibition rate well and the 100% inhibition rate well signals.

[0184] Compound inhibition rate IR (%) formula = (RLU 0％抑制 -RLU 化合物 ) / (RLU 0％抑制 -RLU 100％抑制 )×100%, and the four-parameter logistic model was used to fit the compound gradient dilution concentration and the corresponding inhibition rate to calculate the IC 50 The inhibitory activity of the test compound on KRAS G12D protein (IC 50 , μM) results are shown in Table 1 below.

[0185] Test Example 3: KRAS GDP-GTP Nucleotide Exchange Assay

[0186] In this experiment, the homogeneous time-resolved fluorescence (HTRF) method was used to detect the inhibitory activity of small molecule compounds on the conversion of KRAS protein from the GDP-inactivated state to the GTP-activated state. GDP loaded Biotin-KRAS (G12D, aa 2-186, commissioned by ChemPartner) or GDP loaded biotin-KRAS (WT, aa 2-185, commissioned by ChemPartner) and Tb cryptate-labeled Streptavidin (Cisbio) were mixed (final concentration: KRAS 10nM; Tb 0.335nM) and incubated at 25°C for 30min. Then, the target compound was diluted in a gradient ratio using DMSO solution to obtain a series of compound action concentrations, and the final concentration of DMSO was controlled to be 1%. 5μL of the mixture of Biotin-KRAS protein and Tb-SA and 5μL of compound working solution were added to a 384-well white shallow well plate (PerkinElmer). The plate was sealed and incubated at 25°C in the dark for 1 hour. Then, 5 μL of SOS1 (Cytoskeleton) / BODIPY-GTP (Sigma) mixture (final concentration: SOS1 50 nM; GTP 10 nM) was added, and appropriate control wells were set. The plate was sealed and incubated at 25°C in the dark for 20 min, and the TR-FRET signal value (excitation wavelength: 320 nm, emission wavelength: 615 nm and 665 nm) was measured using an EnVision microplate reader (PerkinElmer). Calculate the fluorescence signal ratio RLU = (665 nm signal / 615 nm signal) x104; the % inhibition rate of the target compound inhibiting the nucleotide conversion of KRAS protein at different concentrations was calculated by setting the 0% inhibition rate and 100% inhibition rate signal wells.

[0187] Compound inhibition rate IR (%) formula = (RLU 0％抑制 -RLU 化合物 ) / (RLU 0％抑制 -RLU 100％ Inhibition) × 100%, and the IC was calculated by fitting the compound gradient dilution concentration and the corresponding inhibition rate using the 4-parameter logistic model. 50 The inhibitory activity of the test compound on KRAS protein nucleotide turnover (IC 50 , μM) are shown in the following Table 1.

[0188] Example Activity Data

[0189] Table 1

[0190]

[0191] Note: NA: Not applicable. NT: Not tested.

[0192] It can be seen from the above results that when the SOS1-catalyzed KRAS-GDP G12D protein undergoes nucleotide exchange, the compounds of the present application exhibit higher selectivity relative to the wild type, and compared with the positive control compound KRpep-2d, both the activity and selectivity are greatly improved. It can also be seen from the above examples that, compared with the positive control compound, it is suggested that the compounds of the present invention not only have higher activity in inhibiting the protein-protein interaction of GTP-KRAS-RAF1, but also have higher activity and higher selectivity for the wild type in inhibiting the nucleotide exchange of the KRAS-GDP protein catalyzed by SOS1.

[0193] Test Example 4: Tumor Cell Line Proliferation Inhibition Experiment (2D)

[0194] Five tumor cell lines AGS (G12D), PANC-1 (G12D), GP2D (G12D), ASPC-1 (G12D) and BXPC-3 (WT) from commercial sources were selected for cell proliferation inhibition experiments. They were cultured in F12, DMEM, DMEM, RPMI-1640 and RPMI-1640 culture media (Gibco, ThermoFisher) containing 10% fetal bovine serum, respectively, and incubated in a 37°C, 5% CO2 incubator. The cells all grew in an adherent state, and the growth status was observed under an inverted microscope. When the number of cells was appropriate, they were subcultured.

[0195] Take five cell lines of AGS, PANC-1, GP2D, ASPC-1 and BXPC-3 in the logarithmic growth phase, inoculate them in a 96-well cell culture plate (Corning) at an appropriate cell density, and culture them at 37°C in a cell culture incubator containing 5% CO2 for 24 hours, and then add 10 μL of the test compound or positive drug to each well. At the same time, set up a positive control group (100% inhibition well) and a negative control group (0% inhibition well), repeat 2 wells for each concentration of the drug group, and repeat 6 wells for the positive control group and the negative control group. After continuing to culture in the incubator for 5 days, the subsequent AlamarBlue test operation is carried out.

[0196] AlamarBlue test operation: add 10 μL AlamarBlue reagent (ThermoFisher) to each well, incubate for 1-4 hours, shake for 1-2 minutes, measure the fluorescence value with MD microplate reader at EX: 560nm, EM: 590nm wavelength, record the results, and calculate the drug inhibition rate on cells (%) = (A 0％抑制 -A 药物 ) / (A 0％抑制 -A 100％抑制)×100%, and then the drug dose response curve was obtained by using the nonlinear regression method (usually using the four-parameter fitting curve equation, 4-parameter logistic model)) using the MATILAB software to obtain the IC 50 The proliferation inhibitory activity (IC) of the test compounds on five commercial tumor cell lines (AGS, PANC-1, GP2D, ASPC-1 and BXPC-3) was analyzed. 50 , μM) results are shown in Table 2 below.

[0197] Example Activity Data (Proliferation)

[0198] Table 2

[0199] Example AGS GP2D PANC-1 AsPc-1 BxPC-3 1 0.604 0.237 0.479 1.278 1.993 2 0.114 0.138 0.159 0.237 2.519 3 0.206 0.173 0.184 0.302 0.887 4 0.125 0.063 0.083 0.195 1.374 5 0.923 0.850 1.020 1.755 3.103

[0200] Test Example 5 Determination of phosphorylated p-ERK1 / 2 (THR202 / TYR204) in AGS and PANC-1 cells

[0201] AGS (ECACC, 89090402) and PANC-1 (ATCC, CRL-1469) both contain KRAS G12D mutation. This method uses the Advanced phospho-ERK1 / 2 (Thr202 / Tyr204) kit (Cat. No. 64AERPEH) of CISBIO to determine the inhibitory activity of the compounds of the present invention on p-ERK1 / 2 protein in AGS and PANC-1 cells.

[0202] The experimental process is briefly described as follows: AGS cells and PANC-1 cells were cultured in HAMF12 medium and DMEM medium (Gibco) containing 10% FBS (Gibco), respectively. On the first day of the experiment, AGS cells and PANC-1 cells were seeded into 96-well cell culture plates at a density of 20,000 cells / well and placed in a cell culture incubator (37°C, 5% CO2) for overnight incubation. On the second day, the culture medium in the 96-well cell culture plate was removed, and 10 μL of fresh culture medium containing the compound to be tested in a gradient dilution was added, and then the cell culture plate was placed in a cell culture incubator (37°C, 5% CO2) for 2 hours. After 2 hours, the culture plate was removed, the culture medium in the 96-well cell culture plate was removed, 50 μL of 1× cell lysis solution (Cisbio) was added to each well, and the cell culture plate was placed in a microplate constant temperature incubator and incubated at 25°C for 45 minutes. After the cells are fully lysed, mix them by pipetting, then transfer 16 μL of cell lysate to a 384-well white plate (Corning), then pipette 4 μL / well of pre-mixed phospho-ERK1 / 2 antibody and add it to the 384-well plate containing the cell lysate, seal the plate and centrifuge to mix, then place the 384-well plate in a microplate thermostat incubator and incubate at 25°C for 4 hours. Finally, use EnVision microplate reader (PerkinElmer) to measure the TR-FRET signal value (excitation wavelength: 320 nm, emission wavelength: 615 nm and 665 nm). Calculate the fluorescence signal ratio RLU = (665 nm signal / 615 nm signal) x 10 4 The % inhibition rate of the compound of the present invention at different concentrations in inhibiting the intracellular p-ERK1 / 2 protein is calculated by setting the 0% inhibition rate and 100% inhibition rate signal wells.

[0203] Compound inhibition rate IR (%) formula = (RLU 0％抑制 -RLU 化合物 ) / (RLU 0％抑制 -RLU 100％抑制 )×100%, and then the compound concentration and the corresponding inhibition rate were calculated, and the standard curve was fitted (4-parameter logistic model), and the IC 50 The inhibitory activity of the compounds of the present invention on p-ERK1 / 2 protein in AGS and PANC-1 cells (IC 50 , μM) are shown in Table 3 below.

[0204] Example p-ERK activity data

[0205] Table 3

[0206] Example AGS PANC-1 2 0.058 0.008 3 0.098 0.011 4 0.071 0.011

[0207] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound as shown in the following formula A, or a pharmaceutically acceptable salt thereof: in, Ring A is a 4-9-membered saturated or partially unsaturated carbon ring, or a 4-9-membered saturated or partially unsaturated hetero ring; X is selected from the group consisting of N, CH or CF; R1 is selected from the following group: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 cyanoalkyl, C1-C6 hydroxyalkyl, allyl, -C(=O)H, -CO2R 5 、-C(=O)R 6 、-SO2R 7 、-CO2N(R 5 )2. -CO2CHR 8 O(C=O)R 9 , aryl, heteroaryl, CONH2, CONR a R b or CONHR c ; where R a , R b , R c are respectively selected from: C1-C6 alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl; R1' is hydrogen, deuterium, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 deuterated alkyl, C2-C6 alkenyl, C2-C3 alkynyl, cyano substituted methyl; p is 0, 1, 2, 3, 4, 5 or 6; R2 is selected from the group consisting of hydrogen, deuterium, halogen, cyano, cyanomethyl, hydroxyl, hydroxymethyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 deuterated alkyl, C2-C6 alkenyl, C2-C3 alkynyl; R4 is selected from the group consisting of hydrogen, deuterium, halogen, cyano, cyanomethyl, hydroxyl, hydroxymethyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 deuterated alkyl; Each R5, R6, R7 is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl; R8 are each independently selected from the group consisting of hydrogen, C1-C3 alkyl, C1-C 15 Haloalkyl, C1-C3 alkoxy; R9 are each independently selected from the group consisting of hydrogen, C1-C 15 Saturated or unsaturated alkyl, C1-C 15 Saturated or unsaturated halogenated alkyl, C1-C 15 saturated or unsaturated alkyl-oxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl; R 10 Selected from the group consisting of hydrogen, deuterium, halogen, cyano, hydroxyl, nitro, amino, hydroxymethyl, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, C1-C4 deuterated alkyl, C1-C4 haloalkoxy, C2-C4 alkenyl, C3-C6 cycloalkyl; R 11 is selected from the group consisting of hydrogen, deuterium, halogen, methyl, hydroxymethyl, ethyl, C3-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 cycloalkoxy, C1-C6 cycloalkanethio, deuterated methyl, deuterated ethyl, halomethyl, halomethoxy; or two R 11 The carbon atoms connected thereto together form a 3-9-membered cycloalkyl or heterocyclic group; m is 0, 1, 2, 3 or 4; R 12 Selected from the group consisting of hydrogen, deuterium, halogen, cyano, methyl, hydroxymethyl, ethyl, C3-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkanethio, C1-C6 cycloalkoxy, C1-C6 cycloalkanethio, deuterated methyl, deuterated ethyl, halomethyl, halomethylenyl, halomethoxy; n is 1, 2 or 3; q is 0, 1, 2, 3, 4, or 5; R 13 and R 14 Each is independently selected from the group consisting of hydrogen, deuterium, methyl, deuterated methyl, or cyclopropyl; R 15 and R 16 Each is independently selected from the group consisting of hydrogen, deuterium, methyl, deuterated methyl, or cyclopropyl; M is selected from the group consisting of oxygen atom, sulfur atom, NH, or NR 16 ; R 16 is methyl, or deuterated methyl; R 17 is H, F, chloro, bromo, methyl, trifluoromethyl, cyano, deuterated methyl, or ethynyl; Wherein, the saturated or partially unsaturated carbocyclic ring, saturated or partially unsaturated heterocyclic ring, or heterocyclic group may be saturated or partially unsaturated, but not aromatic, and may be a monocyclic ring, a condensed ring, a bridged ring or a spirocyclic ring, and the heterocyclic ring has 1-4 heteroatoms selected from N, O or S.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that The compound has a structure shown in the following formula (I) or formula (II):

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that The compound has a structure shown in the following formula (IA1) or formula (IIB1):

4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein The compound has a structure shown in the following formula (Ia) or formula (IIb):

5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein The compounds have the structures shown in the following table:

6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein The pyridopyrimidine 2-position right-side chain oxy or amino, i.e., S-based substituent group has a structure selected from the following group:

7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that The Having a structure selected from the group consisting of:

8. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein The compound of formula A has a structure selected from the following group:

9. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein The compound has a structure selected from the group consisting of:

10. The use of the compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: Use of the method for preparing a drug for treating a disease associated with the activity or expression of a KRAS mutant; preferably, the disease is selected from the group consisting of a tumor; preferably, a tumor selected from the group consisting of a sarcoma, a myxoma, a rhabdomyoma, a fibroma, a lipoma, a teratoma, a bronchial carcinoma, a lung cancer, a bronchial adenoma, a lymphoma, a chondroma hamartoma, a mesothelioma, an esophageal cancer, a gastric cancer, a pancreatic cancer, a small intestine cancer, a large intestine cancer, a cecal cancer, a genitourinary tract tumor, a kidney cancer, a bladder cancer, a urethral cancer, a prostate cancer, a testicular cancer, a liver cancer, a bile duct cancer, a hepatoblastoma, an angiosarcoma, a hepatocellular adenoma, a hemangioma, a gallbladder cancer, an ampullary cancer, a bile duct cancer, a bone cancer, a brain cancer, a uterine cancer, a vaginal cancer, a blood tumor, a skin cancer, and a breast cancer.

11. A pharmaceutical composition, comprising: (i) a therapeutically effective amount of the compound according to claim 1, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

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