CN114716436A - KRAS G12C mutation inhibitor and application thereof - Google Patents

Abstract

The invention discloses a compound for inhibiting KRAS G12C mutation, a pharmaceutically acceptable salt, an optical isomer, an active metabolite and a prodrug of the compound, and application of a pharmaceutical composition containing the compound or the salt thereof in treating proliferative diseases such as tumors and the like caused by KRAS G12C mutation.

Description

KRAS G12C mutation inhibitor and application thereof

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to a compound for inhibiting KRAS G12C mutation, pharmaceutically acceptable salts thereof, and application of a pharmaceutical composition containing the compound and the salts thereof in treating proliferative diseases such as tumors caused by KRAS G12C mutation.

Background

RAS proteins play a very important role in signal transduction and growth regulation. RAS proteins can be classified into KRAS, HRAS and NRAS. RAS proteins enter an "activated" or "inactivated" state by binding to Guanine Trinucleotide Phosphate (GTP) or Guanine Dinucleotide Phosphate (GDP), respectively. In a resting state, RAS proteins bind to cells and GDP, leaving RAS in an inactive state; when cells are activated, RAS proteins bind to GTP, forming GTP-RAS, and activate RAS and its downstream signals simultaneously (Nature Review cancer 3:11-22, 2003). When RAS protein is mutated, activated GTP-RAS is increased and RAS signal is in a sustained activation state, thereby activating downstream signals, stimulating abnormal proliferation of cells, and inducing tumorigenesis.

K-RAS (Kirsten rat sarcoma virus oncogene) is an oncogene with high mutation in human tumors. The most common K-RAS mutations occur in the glycine at position 12 (G12), glycine at position 13 (G13) and glutamine at position 61 (Q61) for disabilities; among them, the mutation at position G12 has the highest incidence (Nat Rev Drug Discov 2014,13: 828-851). The K-RAS G12C mutation refers to the mutation of glycine at position 12 of K-RAS protein to cysteine, and is the most common type of K-RAS mutation. The K-RAS G12C mutant tumors were developed in the order of pancreatic Cancer (57%), large intestinal Cancer (35%), biliary Cancer (28%), small intestinal Cancer (17%), lung Cancer (16%), endometrial Cancer (15%), and ovarian Cancer (14%) (Seminars in Cancer biology.2019Jun 27). K-RAS G12C mutated malignancies are not susceptible to conventional treatment and thus patients have poor recovery and short survival times.

K-RAS G12C mutein inhibitors are novel drug targets for anti-RAS targeted therapy in recent years (Nature 503: 548-containing 551, 2013). The K-RAS G12C covalent inhibitor is a small molecule compound designed by utilizing nucleophilic reactivity of the 12 th cysteine after mutation; the compounds enter the K-RAS G12C allosteric pocket by modification with disulfide bonds, and inhibit tumor growth by blocking activation of the K-RAS G12C mutein by modification with disulfide bonds. Since there is no drug effective for KRAS mutation (Cell Chem biol.2019, 26(10):1338-1348), there is a strong need to find new K-RAS G12C mutation inhibitors for the treatment of K-RAS G12C mutant tumors (Cancer Treat Rev.2020, 84: 101974).

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a novel compound for inhibiting KRAS G12C mutant protein.

In a first aspect, the present invention provides a compound represented by formula I, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystal form thereof, or a solvate thereof:

wherein:

p is selected from C or N;

R₁is selected from H or

R₂、R₃And R₄Each independently selected from H, halogen, C_1-3Alkyl, oxoalkyl or haloalkyl;

l is a bond, O, S or NR₇；

Q is selected from H, alkyl, hydroxyalkyl, heterocyclyl, heterocyclylalkyl, aryl, heteroaryl, heteroarylalkyl, wherein heterocyclyl, aryl, heteroaryl, and heteroarylalkyl may optionally be substituted with one or more R₇Substitution;

R₅selected from H, halogen, C_1-3Alkyl, amino, hydroxy, cyano, haloalkyl;

e is 1,2,3 or 4;

R₆selected from H, C_1-3Alkyl, haloalkyl, cyclopropyl, halogen, amino, oxo, cyano;

h is 0, 1,2 or 3;

m is 0 or 1;

R₇selected from H, alkyl, halogen, haloalkyl;

k is a 4-12 membered saturated or partially saturated monocyclic, bicyclic, bridged or spirocyclic ring containing 1-2N atoms, which may optionally be substituted with one or more R₈Substituted, multiply substituted R₈May be the same or different;

R₈is selected from C_1-3Alkyl, heteroalkyl, cyano, halogen, C_2-4Alkynyl, oxo, -N (R)₉)₂，-(CO)OR₁₀Wherein said C_1-3Alkyl may be optionally substituted with cyano, halogen, heteroaryl;

R₉selected from oxygen, hydroxy, acyl, halogen, C_1-4Alkyl, oxo, haloalkyl;

R₁₀selected from hydrogen, C_1-3Alkyl, hydroxyalkyl;

g is selected from

R₁₁Selected from hydrogen, halogen, C_1-3Alkyl, alkoxy, hydroxyalkyl, cyano, aminoalkyl;

R₁₂selected from hydrogen, halogen, C_1-4Alkyl, alkoxy, cyano, amido, haloalkyl, amino, hydroxy, hydroxyalkyl;

R₁₃selected from hydrogen, alkyl, alkoxy, cyano, halogen;

R₁₄selected from hydrogen, alkyl, alkoxy, aminoalkyl.

In a second aspect, the present invention provides a compound represented by formula I-1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, or a solvate thereof:

wherein R is₁、R₂、R₃、R₄The meanings of L, Q, K and G are as defined in the present invention.

In a preferred embodiment of the invention, R₁Selected from hydrogen or

In a preferred embodiment of the invention, K-G is selected from

Wherein G is as defined herein.

In a preferred embodiment of the invention, K-G is selected from:

wherein the piperazine ring is substituted by R₈Substitution; r₈、R₁₁、R₁₂And R₁₃The meaning of (a) is as defined in the present invention.

In a third aspect, the present invention provides a compound represented by the following formula, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystal form thereof, a deutero-compound thereof, an active metabolite thereof, or a solvate thereof:

in a specific compound of the present invention, R and S each represent a stereoconfiguration.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising the compound of the present invention, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuterogen thereof, an active metabolite thereof, or a solvate thereof, and further comprising an excipient.

In a fifth aspect, the present invention also provides the use of the compound of the present invention, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuteride thereof, an active metabolite thereof, a solvate thereof, or the pharmaceutical composition of the present invention, in a medicament for treating a proliferative disease caused by a mutation of KRAS G12C.

Preferably, the proliferative disease is a tumor.

More preferably, the tumor comprises the following tumorigenesis sites: lung, colorectal, pancreatic, brain, head and neck, liver, stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid, and soft tissue.

In a sixth aspect, the present invention also provides a use of the compound of the present invention, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deutero-compound thereof, an active metabolite thereof, a solvate thereof, or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a proliferative disease caused by the mutation KRAS G12C.

Preferably, the proliferative disease is a tumor.

More preferably, the tumor comprises the following tumorigenic sites: lung, colorectal, pancreatic, brain, head and neck, liver, stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid, and soft tissue.

In a seventh aspect, the present invention also provides a use of the compound of the present invention, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuterio thereof, an active metabolite thereof, a solvate thereof, or the pharmaceutical composition of the present invention for inhibiting KRAS G12C mutation.

In an eighth aspect, the present invention also provides a use of the compound of the present invention, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuterogen thereof, an active metabolite thereof, a solvate thereof, or the pharmaceutical composition of the present invention in the preparation of a medicament for inhibiting KRAS G12C mutation.

In a ninth aspect, the present invention also provides a method of inhibiting a mutation of KRAS G12C comprising administering to a mammal in need thereof an effective amount of the compound of the present invention, pharmaceutically acceptable salts thereof, optical isomers thereof, crystalline forms thereof, deuterides thereof, active metabolites thereof, solvates thereof or the pharmaceutical composition of the present invention.

In a tenth aspect, the present invention also provides a method of treating a proliferative disease caused by a KRAS G12C mutation, comprising administering to a mammal in need thereof an effective amount of the compound of the present invention, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuteride thereof, an active metabolite thereof, a solvate thereof, or the pharmaceutical composition of the present invention;

preferably, the proliferative disease is a tumor;

more preferably, the tumor comprises the following tumorigenesis sites: lung, colorectal, pancreatic, brain, head and neck, liver, stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid, and soft tissue.

Detailed Description

The following embodiments are provided to illustrate the present invention, but do not limit the scope of the present invention.

Definition of

Unless defined otherwise, the terms and techniques used herein have the same meaning as understood by one of ordinary skill in the art to which this invention belongs.

"KRAS G12C" as used herein refers to a mutated form of KRAS protein resulting from substitution of the 12 th glycine of KRAS protein with a cysteine.

"KRAS G12C inhibitor" as used herein refers to a compound of the present invention represented by formula I and formula I-1; these compounds irreversibly bind KRAS G12C by forming a covalent bond with the cysteine residue at position 12 of the KRAS mutein, resulting in KRAS G12C inhibition.

As used herein, "KRAS G12C-associated cancer or disease" or "proliferative disease caused by KRAS G12C mutation" refers to a cancer or disease associated with KRAS G12C mutation or caused by KRAS G12C mutation.

The term "amino" refers to-NH₂。

The term "alkyl" refers to straight and branched chain aliphatic groups of 1 to 10 carbon atoms, optionally substituted with one or more substituents; exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and the like.

The term "alkylene" refers to a group as defined above in which an alkyl group is located between and connects two chemical groups; exemplary alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, and the like.

The term "haloalkyl" refers to an alkyl chain wherein one or more hydrogens are replaced with a halogen; illustrative examples of haloalkyl groups include, but are not limited to, trifluoromethyl and the like.

The term "hydroxyalkyl" refers to-alkyl-OH.

The term "alkoxy" means-OC_1-5-an alkyl group.

The term "cycloalkyl" refers to saturated and partially saturated cyclic hydrocarbon groups having a carbon composition of 3-12, which may be optionally substituted. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

The term "heteroalkyl" refers to an alkyl group as defined above wherein one or more carbon atoms are replaced with O, S or a N atom.

The term "heterocyclyl" or "heterocyclic group" refers to a group of 3 to 12 membered rings containing one or more nitrogen atoms, oxygen atoms and sulfur atoms in addition to carbon atoms in the ring-forming atoms. "heterocyclyl" or "heterocyclic group" is a monocyclic, bicyclic, spiro or bridged ring; the "heterocyclyl" or "heterocyclic group" may be optionally substituted at one or more positions on carbon or nitrogen; exemplary "heterocyclyl" or "heterocyclic group" include, but are not limited to, pyrrolidinyl, piperazinyl, epoxy, azetidinyl, hexahydropyridyl, tetrahydropyrrolyl, tetrahydrofuranyl, morpholinyl, thiazolidinyl, pyrrolidinonyl, thiazolyl, oxazolyl, hexahydropiperidinyl, oxacyclopropaneyl, thienyl, tetrahydrothienyl, piperidinyl, imidazolyl, indolyl, pyridinyl, pyrimidinyl and the like.

The term "heterocyclylalkyl" refers to a group attached to the rest of the molecule through an alkyl group attached to a heterocyclyl.

The term "aryl" refers to a group consisting of aromatic rings, which may be optionally substituted.

The term "aralkyl" refers to an aryl group covalently linked to an alkyl group, which may independently be optionally substituted; an exemplary aralkyl group is (C)_6-10) Aryl radical (C)_1-5) An alkyl group.

The term "heteroaryl" refers to a monocyclic, bicyclic or tricyclic group consisting of 5 to 14 ring atoms.

The term "pharmaceutical composition" refers to a pharmaceutical composition prepared from a compound of the present invention (including racemates, enantiomers, stereoisomers, deuterons) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, crystal form, deuterons, active metabolites and pharmaceutically acceptable carriers, excipients or adjuvants thereof, which is useful for drug administration.

Preferably, the pharmaceutical composition of the present invention comprises a compound of the present invention that inhibits KRAS G12C and a pharmaceutically acceptable excipient or carrier. The routes of administration of the compounds and pharmaceutical compositions of the invention may be: 1) oral administration: such as tablets, capsules, and the like; 2) and (3) injection: such as intravenous injection, subcutaneous injection, intramuscular injection, ophthalmic injection, etc.; 3) in the rectum: such as suppositories, gels, and the like; (4) nasal inhalation: such as sprays, aerosols, and the like; can also be administered by liposome, sustained release technique, controlled release technique, etc. The preferred method is oral administration or injection administration.

The term "pharmaceutically acceptable salt" refers to salts that maintain the biological activity possessed by the compounds of the present invention without exhibiting undesirable toxicological effects. Examples of pharmaceutically acceptable salts of the compounds of the present invention include, but are not limited to, acid addition salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrochloric acid, and the like, and salts with organic acids such as acetic acid, malic acid, tartaric acid, oxalic acid, succinic acid, benzoic acid, tannic acid, alginic acid, polyglutamic acid, and the like; the compounds of the present invention can also be administered as pharmaceutically acceptable quaternary ammonium salts.

The pharmaceutical compositions herein may contain, in addition to a compound of the present invention or a pharmaceutically acceptable salt thereof, buffers, diluents, fillers, stabilizers, solubilizers and other adjuvant materials as disclosed in the art.

Various dosage forms of pharmaceutical compositions comprising the compounds of the present invention are prepared by methods commonly employed in the pharmaceutical industry, including, but not limited to, mixing, dissolving, granulating, levigating, emulsifying, sugar-coating, lyophilizing, and the like.

The "pharmaceutically acceptable carrier" of the present invention refers to inactive ingredients in the pharmaceutical composition, including but not limited to: various sugars such as calcium phosphate, calcium carbonate, lactose, or mannitol, starch, magnesium stearate, cellulose, magnesium carbonate, acrylic acid polymer, methacrylic acid polymer, gelatin, ethylene glycol, castor oil, sesame oil, corn oil, peanut oil, and the like.

The pharmaceutical composition containing the compound inhibiting KRAS G12C and the application method provided by the invention can be used for treating various tumors related to KRAS G12C mutation, including but not limited to the following tumor occurrence parts: lung, colorectal, pancreatic, brain, head and neck, liver, stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid, and soft tissue. More specifically, the compounds or pharmaceutical compositions of the invention are useful in the treatment of: lung cancer (non-small cell lung cancer, squamous cell carcinoma), pancreatic cancer (ductal adenocarcinoma, insulinoma), gastric cancer, esophageal cancer (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma), small intestine (adenocarcinoma), kidney (adenocarcinoma, wilms ' tumor), bladder and urethra (squamous cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testicular cancer (seminoma), hepatocellular carcinoma, cholangiocarcinoma, brain astrocytoma, glioblastoma, retinoblastoma, endometrial carcinoma, cervical carcinoma, ovarian carcinoma, sarcoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, osteosarcoma, fibrosarcoma, chondrosarcoma, multiple myeloma, myelodysplastic syndrome, hodgkin's disease, non-hodgkin's lymphoma, malignant melanoma, hemangioma.

Compound (I)

The invention provides a compound shown in formula I and formula I-1, a pharmaceutically acceptable salt, an optical isomer, a crystal form, a deuteron, an active metabolite or a solvate thereof, wherein the preferred compound is as follows:

general reaction scheme and examples

The present invention is described in detail below by way of examples, which are intended to describe embodiments of the present invention only and are not intended to limit the scope of the present invention. The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including but not limited to those employed in the examples of the present invention and alternatives well known to those skilled in the art, with preferred embodiments including but not limited to the examples of the present invention. Various changes and substitutions of the technical scheme of the invention by those skilled in the art on the premise of the design idea of the invention belong to the protection scope of the invention.

Reaction general formula I

As shown in a reaction general formula I, heating a compound (1) and urea in an alkaline solvent (such as sodium ethoxide) to perform a ring-closing reaction to generate a compound (2); compound (2) with POCl₃Heating in alkaline solution (such as DIEA) to generate compound (3); the product of the heating reaction of compound (3) in a basic solvent (e.g., DIEA) with a group K (e.g., in some embodiments, piperazine-1-carboxylic acid tert-butyl ester) is further reacted with G to produce compound (4); hydrogenating and reducing the compound (4) in a solvent such as methanol and the like under the catalysis of palladium carbon to generate a compound (5); under the protection of nitrogen, the compound (5) is reacted withR₁Heating to react to generate a combined compound (6); compound (6) is reacted with L-Q previously treated with NaH at low temperature under nitrogen and at low temperature by heating the biological end product (I).

Example 1

2- (2S) - [4- [7- (5,6,7, 8-tetrahydronaphthalen-1-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, which is synthesized as follows:

1. compound 1 and Boc-protected 2(S) - (cyanomethyl) piperazine were dissolved in dioxane (24ml), DIEA (2.64g, 20.4mmol, 3.5ml) was added and stirred at 55 ℃ for 10 hours. The reaction was diluted with water and extracted with EA, the combined organic layers were washed with brine, over anhydrous Na₂SO₄Dried and concentrated in vacuo and the residue purified by trituration with MTBE (200ml) to give compound 2.

2. (S) - (1-Methylpyrrolidin-2-yl) methanol (130mg, 1.1mmol, 130. mu.l) was dissolved in THF (5ml) and then cooled to 0 ℃ and NaH (36mg, 0.9mmol) was added to the solution and stirred for 2 h. Then compound 2 is added and stirred overnight in a closed tube at 70 ℃ under the protection of nitrogen. After TLC monitoring of the reaction completion, saturated NH was added₄The reaction was quenched with Cl (20ml) and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, over anhydrous Na₂SO₄Drying and concentration in vacuo afforded compound 3.

3. Ammonia gas was bubbled through methanol (30ml) for 10min, then Compound 3(190mg, 338. mu. mol) was added, and catalytic amounts of palladium, H, were added₂By replacement and in H₂Stirred at 40 ℃ for 12 h. After the reaction is finished, the reactant is filtered, concentrated in vacuum and subjected to column chromatography to obtain a compound 4.

4. Compound 4(500mg,1.06mmol) and 8-bromo-1-chloro-1, 2,3, 4-tetrahydronaphthalene (29)1mg, 1.27mmol) in dry toluene (15ml) and Pd added successively under nitrogen protection₂(dba)₃(121mg, 132. mu. mol) and Cs₂CO₃(1.3g, 3.98mmol) and refluxed at 110 ℃ overnight. Extracting with EA, mixing the organic phases, and passing through anhydrous Na₂SO₄Drying, vacuum concentrating, and performing column chromatography to obtain compound 5.

5. 500mg of Compound 5 was dissolved in 8ml of DCM, and TFA (1ml) was added dropwise in ice bath to continue the reaction, and DIPEA (200. mu.l, 1.21mmol) was added slowly and 2-fluoroacryloyl chloride (50. mu.l, 968. mu. mol) was added dropwise in ice bath to continue the reaction for 2 hours. The reaction was monitored by TLC. After the reaction is finished, saturated NaHCO is added₃Extracting with EA, mixing the organic phases, and passing through anhydrous Na₂SO₄Dried and concentrated in vacuo, filtered, concentrated and purified by preparative HPLC to give Exp1, the compound of example 1, LCMS (ESI) m/z 573.3.

Example 2

2- (2S) - [4- [7- (S) -3-fluoro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropy loyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 593.2.

Example 3

2- (2S) - [4- [7- (R) -3-fluoro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, which synthesis is referenced to general reaction I, lcms (esi) m/z 593.2.

Example 4

2- (2S) - [4- [7- (R) -3-fluoro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropy loyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 577.3.

Example 5

2- (2S) - [4- [7- (3-fluoro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 593.2.

Example 6

2- (2S) - [4- [7- (R) -8-fluoro- (5,6,7, 8-tetrahydronaphthalen-1-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, which synthesis is referenced to reaction general formula I, lcms (esi) m/z 591.3.

Example 7

2- (2S) - [4- [7- (5-fluoro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 577.3.

Example 8

2- (2S) - [4- [7- (6-fluoro-2, 3-dihydro-1H-inden-4-yl) -2- (((R) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropoyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 577.3.

Example 9

2- (2S) - [4- [7- (2',3' -dihydrospiro [ cyclopropane-1, 1 '-indene ] -7' -yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, the synthesis of which is referenced to reaction scheme I, lcms (esi) m/z 585.3.

Example 10

2- (2S) - [4- [7- (S) -8-fluoro-5, 6,7, 8-tetrahydronaphthalen-1-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropy-loyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 591.3.

Example 11

2- (2S) - [4- [7- (8-hydroxy-5, 6,7, 8-tetrahydronaphthalen-1-yl) -2- ((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropoyl) piperazin-2-yl ] acetonitrile, the synthesis of which is referenced to reaction formula I, lcms (esi) m/z 589.3.

Example 12

2- (2S) - [4- [7- (R) -8-hydroxy-5, 6,7, 8-tetrahydronaphthalen-1-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy ] -5,6,7, 8-tetrahydropyridine [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) -piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 589.3.

Example 13

2- (2S) - [4- [7- (5-chloro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyridine [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 593.2.

Example 14

2- (2S) - [4- [7- (6-chloro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 593.2.

Example 15

2- (2S) - [4- [7- (6-fluoro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 577.3.

Example 16

2- (2S) - [4- [7- (3-hydroxy-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoroacryloyl) -piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 575.3.

Example 17

2- (2S) - [4- [7- ((R) -3-hydroxy-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropy l) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 575.3.

Example 18

2- (2S) - [4- [7- (6-hydroxy-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoroacryloyl) -piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 575.3.

Example 19

2- (2S) - [4- [7- ((R) -8-chloro-5, 6,7, 8-tetrahydronaphthalen-1-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyridine [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, the synthesis of which is referenced to general reaction I, lcms (esi) m/z 607.2.

Example 20

2- (2S) - [4- [7- ((S) -8-chloro-5, 6,7, 8-tetrahydronaphthalen-1-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropy loyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/607 z.2.

Example 21

2- (2S) - [4- [7- (8-chloro-5, 6,7, 8-tetrahydronaphthalen-1-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyridine [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropoyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 607.2.

Example 22

2- (2S) - [4- [7- ((S) -3-hydroxy-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoroacryloyl) -piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 575.3.

Example 23

2- (2S) - [4- [7- (5-chloro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1-acryloyl-piperazin-2-yl ] acetonitrile, the synthesis of which is referenced to reaction formula I, lcms (esi) m/z 575.2.

Example 24

2- (2S) - [4- [7- (5-chloro-2, 3-dihydro-1H-inden-4-yl) -2- (((R) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, reference for its synthesis to general reaction I, lcms (esi) m/z 575.2.

Example 25

2- (2S) - [4- [7- (6-fluoro-2, 3-dihydro-1H-inden-4-yl) -2- (((R) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, which synthesis is referenced to general reaction I, lcms (esi) m/z 577.3.

Example 26

2- (2S) - [4- [7- ((R) -8-fluoro-5, 6,7, 8-tetrahydronaphthalen-1-yl) -2- (((R) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropy loyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 591.1.

Example 27

2- (2S) - [4- [7- (R) -3-hydroxy-2, 3-dihydro-1H-inden-4-yl) -2- ((((R) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoroacryloyl) -piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 575.3.

Example 28

2- (2R) - [4- [7- (5-chloro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropenyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 593.2.

Example 29

2- (2S) - [4- [7- ((S) -3-fluoro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoropropy loyl) piperazin-2-yl ] acetonitrile, for synthesis reference reaction formula I, lcms (esi) m/z 577.3.

Example 30

2- (2S) - [4- [ (7- (3-hydroxy-2, 3-dihydro-1H-inden-4-yl) -2- ((R) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyridine [3,4-d ] pyrimidin-4-yl ] -1- (2-fluoroacryloyl) -piperazin-2-yl ] acetonitrile, the synthesis of which is referenced to reaction formula I, lcms (esi) m/z 577.3.

Example 31

2- (2R) - [4- [7- (5-chloro-2, 3-dihydro-1H-inden-4-yl) -2- (((S) -1-methylpyrrolidin-2-yl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl ] -1- (but-2-propyn-1-yl) piperazin-2-yl ] acetonitrile, which synthesis is referenced to reaction formula I, lcms (esi) m/z 587.2.

Activity detection

1. Examples assay of covalent binding of Compounds to KRAS G12C

Gel mobility shift assay (gel mobility shift assay) the covalent binding of the compounds of the examples to the KRAS G12C mutein of the cell was determined.

Three human non-small cell lung cancer cell lines: NCI-H358 cells (KRAS G12C mutant), a549 cells (KRAS G12S mutant) and HCC827 cells (KRAS wild-type).

Experimental reagents and instrumentation: RPMI1640 Medium, DMEM Cell culture medium, fetal bovine serum, 0.25% trypsin-0.53 mM EDTA digestive solution, DMSO, penicillin-streptomycin, KRAS antibody (Sigma), and Anti-rabbitIgG-HRP and Cell Titer-Gio detection kit as secondary antibody. Promega microplate detector, cell culture flask, cell culture microplate (96-well or 384-well), CO₂Incubator, FluorChemR detector (ProteinSimple).

Gel mobility shift assay (gel mobility shift assay) determined the formation of covalent complexes of compounds with the cellular KRAS G12C mutein. Based on the compound and K-RAS G12C mutant protein combination to form covalent complexes with increased molecular weight, and the unbound KRAS G12C mutant protein when electrophoresis, corresponding electrophoretic band hysteresis appears; quantitatively scanning an electrophoresis band by using a FluorChemR detector, calculating the electrophoretic migration ratio of a compound-KRAS G12C mutant protein compound to a G12C mutant protein of an unbound compound, and quickly and directly determining the covalent binding characteristics of the compound and the KRAS G12C mutant protein; the more compound binds to KRAS G12C mutant protein, the greater its calculated ratio.

Cell culture: after the tumor cells frozen by liquid nitrogen are recovered, culturing the cells by using a cell culture solution containing 10 percent of fetal calf serum and 10 percent of penicillin-streptomycin, and after the cells grow to an exponential growth period, digesting, centrifuging and collecting the cells and then suspending the cells in the culture solution; inoculating cells according to 10000 cells per well, placing at constant temperature of 37 ℃ and 5% CO₂And culturing in a carbon dioxide incubator with saturated humidity.

After treating cells with the compound at different concentrations and at different times, the cells were washed with Phosphate Buffered Saline (PBS), then lysed in RIPA buffer (50mm Tris, pH7.5, 150mm NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, containing protease and phosphatase inhibitors), total cell protein was extracted, and protein concentration was determined using BCA protein assay kit; carrying out SDS-PAGE electrophoresis on equivalent protein, and transferring electrophoresis gel to a nitrocellulose membrane after the electrophoresis is finished; after membrane transfer, the membrane was blocked with 5% skim milk in TBS-0.1% Tween-20, followed by addition of primary antibody and incubation overnight with gentle shaking at 4 ℃. After overnight incubation, the membrane was washed, incubated with fluorescently conjugated secondary antibody for 1 hour at room temperature, and the band was scanned with a fluorochemr detector (ProteinSimple). The results are shown in table 1: the ratio of A to A is less than or equal to 25 percent; 25 percent of less than or equal to 50 percent of less than B; the "C" was not less than 50%, and the results are shown in Table 1.

TABLE 1 migration ratio of Compounds to KRAS G12C mutant proteins to form covalent complexes

The results of gel migration experiments show that the KRAS G12C inhibitor can selectively form a covalent complex with KRAS G12C mutant protein of KRAS G12C mutant human non-small cell lung cancer NCI-H358 cells, and the phenomenon of gel migration retardation occurs.

2. Assays for cell proliferation-inhibiting Activity of Compounds

The anti-proliferation activity of the compound on KRAS G12C mutant tumor cells is determined by a Luminometer luminescence method.

Three human non-small cell lung cancer cell lines: NCI-H358 cells (KRAS G12C mutant), a549 cells (KRAS G12S mutant) and HCC827 cells (KRAS wild-type).

Experimental reagents and instrumentation: RPMI1640 medium, DMEM Cell culture medium, fetal calf serum, 0.25% trypsin-0.53 mM EDTA digestive juice, DMSO, penicillin-streptomycin, Cell Titer-Gio detection kit. Promega plate tester, cell culture bottle, cell culture plate (96-well or 384-well), CO₂A constant temperature incubator.

Cell culture: resuscitating tumor cells cryopreserved by liquid nitrogen, culturing the cells by using a cell culture solution containing 10% fetal calf serum and 10% penicillin-streptomycin, and after the cells grow to an exponential growth period, digesting, centrifuging and collecting the cells and resuspending the cells in the culture solution; inoculating cells according to 10000 cells per well, placing at constant temperature of 37 ℃ and 5% CO₂And culturing overnight in a carbon dioxide incubator with saturated humidity.

After the compound is added into the cell culture solution for 72h, the 96-well plate is taken out from the incubator at 37 ℃ and placed at room temperature for 30min for CTG detection, and a plate is not required to be shaken in the experimental process. Adding 100. mu.l of CTG reagent, mixing uniformly for 2min, then incubating for 10min at room temperature, detecting and recording the luminescence value by a GloMax 96 microplate luminometer (CellTiter-Glo luminescence Cell vitality Assay, Promega), and observing the Cell vitality.

Each test compound was diluted to 10 concentration gradients, added to the corresponding wells of the cell plate, and the cell plate was returned to the carbon dioxide incubator for further incubation for 72 hours. After the culture is finished, adding a Promega CellTiter-Glo reagent into each hole of the cell plate, incubating for 10min at room temperature, detecting a luminescent signal by adopting a Promega micropore plate detector, and calculating IC₅₀The value is obtained. The result of the antiproliferative activity of the compounds of the invention is represented by A, B, ND: a is more than or equal to 0.001 and less than or equal to 1 mu M; "B">1 mu M; ND was not measured, and the results are shown in table 2.

TABLE 2 antiproliferative effect of the compounds of the examples on KRAS-G12C mutant tumor cells

The experimental results show that the compound of the invention shows higher antiproliferative inhibition activity on KRAS G12C mutant NCL-H358 cells, but only weak antiproliferative activity on KRAS G12S non-mutant A549 cells and HCC827 cells (Table 2). The results of the antiproliferation experiments are consistent with the results obtained by the gel mobility analysis method, and show that the compound has selective inhibition effect on KRAS G12C mutant cells.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (13)

1. A compound represented by formula I, pharmaceutically acceptable salts thereof, optical isomers thereof, crystal forms thereof, deuterides thereof, active metabolites thereof, or solvates thereof:

wherein:

p is selected from C or N;

R₁is selected from H or

R₂、R₃And R₄Each independently selected from H, halogen, C_1-3Alkyl, oxoalkyl or haloalkyl;

l is a bond, O, S or NR₇；

Q is selected from H, alkyl, hydroxyalkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aryl, heteroaryl, heteroarylalkyl, wherein heterocyclyl, aryl, heteroaryl, and heteroarylalkyl may optionally be substituted with one or more R₇Substitution;

R₅selected from H, halogen, C_1-3Alkyl, amino, hydroxy, cyano, haloalkyl;

e is 1,2,3 or 4;

R₆selected from H, C_1-3Alkyl, haloalkyl, cyclopropyl, halogen, amino, oxo, cyano;

h is 0, 1,2 or 3;

m is 0 or 1;

R₇selected from H, alkyl, halogen, haloalkyl;

k is a 4-12 membered saturated or partially saturated monocyclic, bicyclic, bridged or spirocyclic ring containing 1-2N atoms, which may optionally be substituted with one or more R₈Substituted, multiply substituted R₈May be the same or different;

R₈is selected from C_1-3Alkyl, heteroalkyl, cyano, halogen, C_2-4Alkynyl, oxo, -N (R)₉)₂，-(CO)OR₁₀Therein is describedC is_1-3Alkyl may be optionally substituted with cyano, halogen, heteroaryl;

R₉selected from oxygen, hydroxy, acyl, halogen, C_1-4Alkyl, oxo, haloalkyl;

R₁₀selected from hydrogen, C_1-3Alkyl, hydroxyalkyl;

g is selected from

R₁₁Selected from hydrogen, halogen, C_1-3Alkyl, alkoxy, hydroxyalkyl, cyano, aminoalkyl;

R₁₂selected from hydrogen, halogen, C_1-4Alkyl, alkoxy, cyano, amido, haloalkyl, amino, hydroxy, hydroxyalkyl;

R₁₃selected from hydrogen, alkyl, alkoxy, cyano, halogen;

R₁₄selected from hydrogen, alkyl, alkoxy, aminoalkyl.

2. A compound represented by formula I-1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystal form thereof, a deuteride thereof, an active metabolite thereof, or a solvate thereof:

wherein R is₁、R₂、R₃、R₄The meanings of L, Q, K and G are as defined in claim 1.

3. A compound according to claim 1 or 2, wherein R₁Selected from hydrogen or

4. A compound according to any one of claims 1-3, wherein K-G is selected from:

wherein G has the meaning defined in claim 1.

5. The compound of any one of claims 1-4, wherein K-G is selected from:

wherein the piperazine ring is substituted by R₈Substitution; r₈、R₁₁、R₁₂And R₁₃Has the meaning as defined in claim 1.

6. A compound represented by the following formula, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystal form thereof, a deuteride thereof, an active metabolite thereof, or a solvate thereof:

7. a pharmaceutical composition comprising a compound of any one of claims 1-6, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuteride thereof, an active metabolite thereof, or a solvate thereof, and an excipient.

8. Use of a compound of any one of claims 1-6, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuteride thereof, an active metabolite thereof, a solvate thereof, or a pharmaceutical composition of claim 7, in a medicament for treating a proliferative disease caused by a KRAS G12C mutation;

preferably, the proliferative disease is a tumor;

more preferably, the tumor comprises the following tumorigenic sites: lung, colorectal, pancreatic, brain, head and neck, liver, stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid, and soft tissue.

9. Use of a compound of any one of claims 1-6, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuteride thereof, an active metabolite thereof, a solvate thereof, or a pharmaceutical composition of claim 7, in the manufacture of a medicament for treating a proliferative disease caused by a KRAS G12C mutation;

preferably, the proliferative disease is a tumor;

more preferably, the tumor comprises the following tumorigenic sites: lung, colorectal, pancreatic, brain, head and neck, liver, stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid, and soft tissue.

10. Use of the compound of any one of claims 1-6, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuteride thereof, an active metabolite thereof, a solvate thereof, or the pharmaceutical composition of claim 7, for inhibiting KRAS G12C mutation.

11. Use of the compound of any one of claims 1-6, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuteride thereof, an active metabolite thereof, a solvate thereof, or the pharmaceutical composition of claim 7, in the manufacture of a medicament for inhibiting the KRAS G12C mutation.

12. A method of inhibiting a KRAS G12C mutation comprising administering to a mammal in need thereof an effective amount of a compound of any one of claims 1-6, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuteride thereof, an active metabolite thereof, a solvate thereof, or a pharmaceutical composition of claim 7.

13. A method of treating a proliferative disease caused by a KRAS G12C mutation, comprising administering to a mammal in need thereof an effective amount of the compound of any one of claims 1-6, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuteride thereof, an active metabolite thereof, a solvate thereof, or the pharmaceutical composition of claim 7;

preferably, the proliferative disease is a tumor;

more preferably, the tumor comprises the following tumorigenic sites: lung, colorectal, pancreatic, brain, head and neck, liver, stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid, and soft tissue.