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 use thereof

technical field

The invention belongs to the field of medical biotechnology, and in particular relates to a compound that inhibits KRAS G12C mutation and a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing the compound and its salt in the treatment of proliferative diseases such as tumors caused by KRAS G12C mutation Applications.

Background technique

RAS proteins play a very important role in signal transduction and growth regulation. RAS proteins can be divided into three types: KRAS, HRAS and NRAS. RAS proteins enter an "activated" or "inactive" state by binding to guanine trinucleotide phosphate (GTP) or guanine dinucleotide phosphate (GDP), respectively. In the quiescent state, RAS protein binds to the cell and GDP, making RAS inactive; when the cell is activated, RAS protein binds to GTP to form GTP-RAS, and simultaneously activates RAS and its downstream signals (Nature Review cancer 3 : 11-22, 2003). When the RAS protein is mutated, the activated GTP-RAS increases, and the RAS signal is in a state of continuous activation, thereby activating downstream signals, stimulating abnormal cell proliferation, and inducing tumorigenesis.

K-RAS (Kirsten rat sarcoma virus oncogene) is a highly mutated oncogene in human tumors. The most common K-RAS mutations occur in glycine at position 12 (G12), glycine at position 13 (G13), and glutamine at position 61 (Q61); the mutation at position G12 has the highest incidence (Nat Rev. Drug Discov 2014, 13:828-851). K-RAS G12C mutation refers to the mutation of the 12th glycine of K-RAS protein to cysteine, which is the most common type of K-RAS mutation. The frequency of K-RAS G12C mutant tumors was pancreatic cancer (57%), colorectal cancer (35%), biliary tract cancer (28%), small bowel cancer (17%), lung cancer (16%), endometrial cancer ( 15%) and ovarian cancer (14%), etc. (Seminars in Cancer Biology. 2019 Jun 27). Malignant tumors with K-RAS G12C mutation are not sensitive to conventional treatment, so patients have poor prognosis and short survival time.

K-RAS G12C mutein inhibitor is a new drug target for anti-RAS targeted therapy discovered in recent years (Nature 503:548-551, 2013). K-RAS G12C covalent inhibitor is to use the nucleophilic reactivity of the mutated cysteine at position 12 to design small molecule compounds; by modification with disulfide bonds, the compound enters the K-RAS G12C allosteric pocket, and by using Disulfide bond modification blocks the activation of K-RAS G12C mutant protein and inhibits tumor growth. Since there is currently no effective drug against KRAS mutation (Cell Chem Biol. 2019, 26(10): 1338-1348), it is urgent to discover new K-RAS G12C mutation inhibitors for the treatment of K-RAS G12C mutation tumors (Cancer Treat Rev. 2020, 84:101974).

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a new compound for inhibiting KRAS G12C mutein.

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

in:

P is selected from C or N;

R ₁ is selected from H or

R ₂ , R ₃ and R ₄ are each independently selected from H, halogen, C _1-3 alkyl, 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 heteroaryl Alkylalkyl may be optionally substituted with one or more _R7 ;

R ₅ is selected from H, halogen, C _1-3 alkyl, amino, hydroxyl, cyano, haloalkyl;

e is 1, 2, 3 or 4;

R ₆ is selected from H, C _1-3 alkyl, haloalkyl, cyclopropyl, halogen, amino, oxo, cyano;

h is 0, 1, 2 or 3;

m is 0 or 1;

R ₇ is selected from H, alkyl, halogen, haloalkyl;

K is a 4-12 membered saturated or partially saturated monocyclic, bicyclic, bridged or spirocyclic ring containing 1-2 N atoms, which may optionally be replaced by one or more R ₈ is substituted, and multiple substituted R ₈ can be the same or different;

R ₈ is selected from C _1-3 alkyl, heteroalkyl, cyano, halogen, C _2-4 alkynyl, oxo, -N(R ₉ ) ₂ , -(CO)OR ₁₀ , wherein said C _{1 -3} Alkyl can be optionally substituted by cyano, halogen, heteroaryl;

R ₉ is selected from oxygen, hydroxyl, acyl, halogen, C _1-4 alkyl, oxo, haloalkyl;

R ₁₀ is selected from hydrogen, C _1-3 alkyl, hydroxyalkyl;

G is selected from

R ₁₁ is selected from hydrogen, halogen, C _1-3 alkyl, alkoxy, hydroxyalkyl, cyano, aminoalkyl;

R ₁₂ is selected from hydrogen, halogen, C _1-4 alkyl, alkoxy, cyano, amido, haloalkyl, amino, hydroxyl, hydroxyalkyl;

R ₁₃ is selected from hydrogen, alkyl, alkoxy, cyano, halogen;

R ₁₄ is selected from hydrogen, alkyl, alkoxy, aminoalkyl.

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

Wherein, the meanings of R ₁ , R ₂ , R ₃ , R ₄ , L, Q, K and G are as defined in the present invention.

In a preferred embodiment of the present invention, R ₁ is selected from hydrogen or

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

Wherein, the meaning of G is as defined in the present invention.

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

Wherein, the piperazine ring is substituted by R ₈ ; the meanings of R ₈ , R ₁₁ , R ₁₂ and R ₁₃ are as defined in the present invention.

In the third aspect, the present invention provides the compound represented by the following formula, its pharmaceutically acceptable salt, its optical isomer, its crystal form, its deuterated product, its active metabolite or its solvate:

In specific compounds 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, its pharmaceutically acceptable salt, its optical isomer, its crystal form, its deuterated product, its active metabolite or its solvent The composition also includes excipients.

In the fifth aspect, the present invention also provides the compound of the present invention, its pharmaceutically acceptable salt, its optical isomer, its crystal form, its deuterated product, its active metabolite, its solvate or the present invention Use of the pharmaceutical composition in a medicament for treating proliferative diseases caused by KRAS G12C mutation.

Preferably, the proliferative disease is a tumor.

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

In the sixth aspect, the present invention also provides the compound of the present invention, its pharmaceutically acceptable salt, its optical isomer, its crystal form, its deuterated product, its active metabolite, its solvate or the present invention Use of the pharmaceutical composition in preparing a medicament for treating proliferative diseases caused by KRAS G12C mutation.

Preferably, the proliferative disease is a tumor.

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

In the seventh aspect, the present invention also provides the compound of the present invention, its pharmaceutically acceptable salt, its optical isomer, its crystal form, its deuterated product, its active metabolite, its solvate or the present invention Use of the pharmaceutical composition in inhibiting KRASG12C mutation.

In the eighth aspect, the present invention also provides the compound of the present invention, its pharmaceutically acceptable salt, its optical isomer, its crystal form, its deuterated product, its active metabolite, its solvate or the present invention Use of the pharmaceutical composition in the preparation of a medicament for inhibiting KRAS G12C mutation.

In a ninth aspect, the present invention also provides a method for inhibiting 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, or an effective amount thereof. A crystalline form, a deuterated product thereof, an active metabolite thereof, a solvate 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 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, or a pharmaceutically acceptable salt thereof. Optical isomer, its crystal form, its deuterated product, its active metabolite, its solvate or said pharmaceutical composition of the present invention;

Preferably, the proliferative disease is a tumor;

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

Detailed ways

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

definition

Unless otherwise defined, 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 mutant form of KRAS protein resulting from the substitution of glycine at position 12 of KRAS protein with cysteine.

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

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

The term "amino" refers to _-NH2 .

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

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

The term "haloalkyl" refers to an alkyl chain in which one or more hydrogens are replaced by a halogen; exemplary examples of haloalkyl include, but are not limited to, trifluoromethyl and the like.

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

The term "alkoxy" refers to _-OC1-5 -alkyl.

The term "cycloalkyl" refers to saturated and partially saturated cyclic hydrocarbon groups of 3 to 12 carbons, 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 N atoms.

The term "heterocyclyl" or "heterocyclic group" refers to a 3- to 12-membered ring group containing one or more nitrogen, oxygen, and sulfur atoms in the ring-forming atoms in addition to carbon atoms. A "heterocyclyl" or "heterocyclic group" is a monocyclic, bicyclic, spirocyclic, or bridged ring; the "heterocyclyl" or "heterocyclic group" may optionally be a carbon or Substituted on nitrogen; exemplary "heterocyclyl" or "heterocyclic groups" include, but are not limited to, pyrrolidinyl, piperazinyl, epoxy, azetidinyl, hexahydropyridyl, tetrahydropyrrole base, tetrahydrofuranyl, morpholinyl, thiazolidinyl, pyrrolidone, thiazolyl, oxazolyl, hexahydropiperidinyl, oxiranyl, thienyl, tetrahydrothienyl, piperidinyl, imidazolyl, Indolyl, pyridyl, pyrimidinyl, etc.

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

The term "aryl" refers to a group containing an aromatic ring, which may be optionally substituted.

The term "aralkyl" refers to an aryl group covalently attached to an alkyl group, which may independently be optionally substituted; exemplary aralkyl groups are ( _C6-10 )aryl( _C1-5 )alkanes base.

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

The term "pharmaceutical composition" refers to a compound (including racemates, enantiomers, stereoisomers, deuterated compounds) of the present invention or pharmaceutically acceptable salts, hydrates, solvates, pro- The pharmaceutical composition prepared by the drug, the crystal form, the deuterated compound, the active metabolite and its pharmaceutically acceptable carrier, excipient or adjuvant to facilitate administration.

Preferably, the pharmaceutical composition of the present invention comprises the KRAS G12C-inhibiting compound of the present invention and a pharmaceutically acceptable excipient or carrier. The administration routes of the compounds and pharmaceutical compositions of the present invention can be: 1) Oral: such as tablets, capsules, etc.; 2) Injection: such as intravenous injection, subcutaneous injection, intramuscular injection, ocular injection, etc.; 3) Intrarectal: such as Suppositories, gels, etc.; (4) Nostril inhalation: such as sprays, aerosols, etc.; it can also be administered through drug release systems such as liposomes, sustained-release technology, and controlled-release technology. Among them, the preferred method is oral administration or injection administration.

The term "pharmaceutically acceptable salts" refers to salts that maintain the biological activity possessed by the compounds of the present invention without exhibiting undesired 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, sulfuric, phosphoric, nitric, hydrobromic, etc., and acetic, malic, tartaric, oxalic, Salts formed by organic acids such as succinic acid, benzoic acid, tannic acid, alginic acid, and polyglutamic acid; 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 the compounds of the present invention or pharmaceutically acceptable salts thereof, buffers, diluents, fillers, stabilizers, solubilizers and other adjuvant substances disclosed in the art.

Various dosage forms of the pharmaceutical composition composed of the compounds of the present invention are prepared by methods commonly used in the pharmaceutical industry, including but not limited to mixing, dissolving, granulating, grinding, emulsifying, sugar coating, freeze drying and the like.

The "pharmaceutically acceptable carrier" of the present invention refers to the 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, fiber plain, magnesium carbonate, acrylic polymer, methacrylic acid polymer, gelatin, ethylene glycol, castor oil, sesame oil, corn oil, peanut oil, etc.

The pharmaceutical composition and application method provided by the present invention containing a compound that inhibits KRAS G12C can be used to treat various tumors related to KRAS G12C mutation, including but not limited to the following tumor sites: lung, colorectum, pancreas, brain, head and neck, liver , stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid and soft tissues. More specifically, the compounds or pharmaceutical compositions of the present invention are useful in the treatment of: lung cancer (non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma), pancreatic cancer (ductal adenocarcinoma, insulinoma), gastric cancer, esophageal cancer (squamous cell carcinoma) squamous cell carcinoma, adenocarcinoma, leiomyosarcoma), small intestine (adenocarcinoma), kidney (adenocarcinoma, Wilms tumor), bladder and urethra (squamous cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis Carcinoma (Seminoma), Hepatocellular Carcinoma, Cholangiocarcinoma, Brain Astrocytoma, Brain Glioblastoma, Retinoblastoma, Endometrial Cancer, Cervical Cancer, Ovarian Cancer, Sarcoma, Acute Lymphoblastic Leukemia , chronic lymphocytic leukemia, osteosarcoma, fibrosarcoma, chondrosarcoma, multiple myeloma, myelodysplastic syndrome, Hodgkin's disease, non-Hodgkin's lymphoma, malignant melanoma, hemangioma.

compound

The present invention provides compounds represented by formula I, formula I-1, pharmaceutically acceptable salts thereof, optical isomers, crystal forms, deuterated products, active metabolites or solvates thereof, preferred compounds as follows:

Reaction formula and examples

The present invention will be described in detail below by way of examples, but the examples only describe the embodiments of the present invention and do not limit the scope of the present invention. The compounds of the present invention can be prepared by various synthetic methods known to those skilled in the art, including but not limited to the methods adopted in the examples of the present invention and alternative methods known to those skilled in the art. Preferred embodiments include but not limited to limited to the embodiments of the present invention. Various changes and substitutions made to the technical solutions of the present invention by those skilled in the art under the premise of the design idea of the present invention all belong to the protection scope of the present invention.

Reaction formula I

As shown in the general reaction formula I, compound (1) and urea are heated in an alkaline solvent (such as sodium ethoxide) to generate a ring-closure reaction to generate compound (2); compound (2) and POCl ₃ in an alkaline solution (such as DIEA ) heating to generate compound (3); compound (3) in a basic solvent (such as DIEA) group K (such as piperazine-1-carboxylate tert-butyl ester in some embodiments) heating reaction product is further reacted with G The reaction generates compound (4); compound (4) is hydrogenated and reduced to generate compound (5) under the catalysis of methanol and other solvents and palladium carbon; under nitrogen protection, compound (5) is heated and reacted with R ₁ to generate compound (6) ; Under nitrogen and low temperature, compound (6) is heated with LQ previously treated with NaH at low temperature to react biological end product (I).

Example 1

2-(2S)-[4-[7-(5,6,7,8-Tetrahydronaphthalene-1-yl)-2-(((S)-1-methylpyrrolidin-2-yl)methane oxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile, its synthesis Methods as below:

1. Dissolve compound 1 and Boc-protected 2(S)-(cyanomethyl)piperazine in dioxane (24ml), then add DIEA (2.64g, 20.4mmol, 3.5ml), stir at 55°C for 10 Hour. The reaction was diluted with water and extracted with EA, the combined organic layers were washed with brine, dried _over anhydrous _Na2SO4 and concentrated in vacuo, the residue was purified by trituration with MTBE (200ml) to give compound 2.

2. Dissolve (S)-(1-methylpyrrolidin-2-yl)methanol (130 mg, 1.1 mmol, 130 μl) in THF (5 ml), then cool to 0 °C, add NaH (36 mg, 0.9 mmol) and stirred for 2 h. Compound 2 was then added and stirred overnight in a closed tube at 70°C under nitrogen protection. After completion of the reaction monitored by TLC, saturated _NH4Cl (20 ml) was added to quench the reaction and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo to give compound 3.

3. Bubble ammonia gas to methanol (30 ml) for 10 min, then add compound 3 (190 mg, 338 μmol), add catalytic amount of palladium, replace with H ₂ , and stir at 40° C. under H ₂ for 12 h. After completion of the reaction, the reactant was filtered, concentrated in vacuo, and subjected to column chromatography to obtain compound 4.

4. Compound 4 (500mg, 1.06mmol) and 8-bromo-1-chloro-1,2,3,4-tetrahydronaphthalene (291mg, 1.27mmol) were dissolved in anhydrous toluene (15ml), followed by nitrogen protection Pd ₂ (dba) ₃ (121 mg, 132 μmol) and Cs ₂ CO ₃ (1.3 g, 3.98 mmol) were added and refluxed at 110° C. overnight. Extracted with EA, combined organic phases, dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo, and column chromatography gave compound 5.

5. Dissolve 500 mg of compound 5 in 8 ml of DCM, add TFA (1 ml) dropwise under an ice bath, continue the reaction, then slowly add DIPEA (200 μl, 1.21 mmol), and dropwise add 2-fluoroacryloyl chloride (50 μl, 968 μmol) under an ice bath , continue to react for 2h. The reaction was monitored by TLC. After the reaction, saturated NaHCO ₃ was added, extracted with EA, the organic phases were combined, dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo, filtered, concentrated, and purified by preparative HPLC to obtain 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-methylpyrrolidine -2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazine-2- base] acetonitrile, whose synthesis refers to the general 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-methylpyrrolidine -2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazine-2- base] acetonitrile, whose synthesis refers to the general formula 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-methylpyrrolidine -2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazine-2- base]acetonitrile, whose synthesis refers to 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile , its synthetic 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-methyl Pyrrolidin-2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazine- 2-yl]acetonitrile, whose synthesis is referenced to reaction 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile , its synthetic 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile , its synthetic 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-fluoroacryloyl) Piperazin-2-yl]acetonitrile, whose synthesis is referred to reaction formula 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-methylpyrrole Alk-2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazine-2 -yl]acetonitrile, whose synthesis refers to the general formula I, LCMS (ESI) m/z 591.3.

Example 11

2-(2S)-[4-[7-(8-Hydroxy-5,6,7,8-tetrahydronaphthalene-1-yl)-2-((S)-1-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile , its synthetic reference 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-methylpyrrole Alk-2-yl)methoxy]-5,6,7,8-tetrahydropyridine[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)-piperazine-2 -yl]acetonitrile, whose synthesis refers to the general 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyridin[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile, Its synthetic 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile , its synthetic 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile , its synthetic 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)-piperazin-2-yl] Acetonitrile, whose synthesis refers to 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-methylpyrrole Alk-2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazine-2 -yl]acetonitrile, whose synthesis refers to the general 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)-piperazin-2-yl] Acetonitrile, whose synthesis refers to 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-methyl Pyrrolidin-2-yl)methoxy)-5,6,7,8-tetrahydropyridine[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazine-2 -yl]acetonitrile, whose synthesis refers to the general formula 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-methyl Pyrrolidin-2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazine- 2-yl]acetonitrile, whose synthesis refers to the reaction formula I, LCMS (ESI) m/z 607.2.

Example 21

2-(2S)-[4-[7-(8-Chloro-5,6,7,8-tetrahydronaphthalen-1-yl)-2-(((S)-1-methylpyrrolidine-2 -yl)methoxy)-5,6,7,8-tetrahydropyridine[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile , its synthetic 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-methylpyrrole Alk-2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)-piperazine- 2-yl]acetonitrile, whose synthesis is referenced to 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-acryloyl-piperazin-2-yl]acetonitrile, the synthetic reference of which 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile , its synthetic reference reaction formula 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile , its synthetic reference reaction formula 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-methyl Pyrrolidin-2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazine- 2-yl]acetonitrile, whose synthesis refers to the 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-methylpyrrole Alk-2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)-piperazine- 2-yl]acetonitrile, whose synthesis is referenced to 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazin-2-yl]acetonitrile , its synthetic 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-methylpyrrole Alk-2-yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)piperazine-2 -yl]acetonitrile, whose synthesis refers to the general 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyridin[3,4-d]pyrimidin-4-yl]-1-(2-fluoroacryloyl)-piperazin-2-yl]acetonitrile , its synthetic reference 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-methylpyrrolidine-2- yl)methoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl]-1-(but-2-propyn-one-1-yl)piperidine Azin-2-yl]acetonitrile, whose synthesis is referenced to Reaction Formula I, LCMS (ESI) m/z 587.2.

activity detection

1. Determination of Covalent Binding of Example Compounds to KRAS G12C

Covalent binding of example compounds to cellular KRASG12C muteins was determined by gel mobility shift assay.

Three human non-small cell lung cancer cell lines: NCI-H358 cells (KRAS G12C mutation), A549 cells (KRASG12S mutation) and HCC827 cells (KRAS wild type).

Experimental reagents and instruments: RPMI1640 medium, DMEM cell medium, fetal bovine serum, 0.25% trypsin-0.53mM EDTA digestion solution, DMSO, penicillin-streptomycin, KRAS antibody (Sigma), the secondary antibody is Anti-rabbit IgG -HRP, Cell Titer-Gio detection kit. Promega microplate detector, cell culture flask, cell culture microplate (96-well or 384-well), CO ₂ incubator, FluorChemR detector (ProteinSimple).

The gel mobility shift assay measures the covalent complexes formed by the compounds with the cellular KRAS G12C mutant protein. Based on the increased molecular weight of the covalent complex formed by the combination of the compound and the K-RAS G12C mutant protein, a corresponding electrophoresis band hysteresis appeared in electrophoresis compared with the unconjugated KRAS G12C mutant protein; quantitative scanning electrophoresis with a FluorChemR detector Bands, the ratio of electrophoretic shifts of the compound-KRAS G12C mutein complex to the G12C mutein of the unbound compound was calculated for a rapid and direct determination of the covalent binding characteristics of the compound to the KRAS G12C mutein; the more the compound bound to the KRAS G12C mutein , the larger the calculated ratio.

Cell culture: After resuscitating the tumor cells cryopreserved in liquid nitrogen, culture the cells with a cell culture medium containing 10% fetal bovine serum and 10% penicillin-streptomycin. After the cells grow to an exponential growth phase, they are digested and centrifuged to collect the cells and resuspend in In the culture medium; inoculate cells according to the number of 5,000-10,000 cells per well, and place them in a carbon dioxide incubator with a constant temperature of 37° C., 5% CO ₂ and saturated humidity.

After treatment of cells with compounds at different concentrations and for different times, cells were washed with phosphate buffered saline (PBS) and then incubated in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid) Sodium, 0.1% SDS, containing protease and phosphatase inhibitors) to lyse cells, extract total cell protein, and measure protein concentration with BCA protein detection kit; take an equal amount of protein for SDS-PAGE electrophoresis. The gel was transferred to a nitrocellulose membrane; after transfer, the membrane was blocked with 5% skim milk in TBS-0.1% Tween-20, then primary antibody was added, and incubated overnight at 4°C with gentle shaking. Membranes after overnight incubation were washed and incubated with fluorophore-conjugated secondary antibodies for 1 hour at room temperature, and bands were scanned with a FluorChemR detector (ProteinSimple). The experimental results are shown in Table 1: "A"≤25% ratio; 25%<"B"≤50%; "C"≥50%, the results are shown in Table 1.

Table 1. Migration ratios of compounds forming covalent complexes with KRAS G12C muteins

The results of gel migration assay showed that KRAS G12C inhibitor can selectively form a covalent complex with KRAS G12C mutant protein in KRAS G12C-mutated human non-small cell lung cancer NCI-H358 cells, resulting in gel migration retardation.

2. Measurement of the activity of the compound to inhibit cell proliferation

The antiproliferative activity of compounds against KRAS G12C mutant tumor cells was determined by Luminometer luminescence assay.

Three human non-small cell lung cancer cell lines: NCI-H358 cells (KRAS G12C mutation), A549 cells (KRASG12S mutation) and HCC827 cells (KRAS wild type).

Experimental reagents and instruments: RPMI1640 medium, DMEM cell medium, fetal bovine serum, 0.25% trypsin-0.53mM EDTA digestion solution, DMSO, penicillin-streptomycin, Cell Titer-Gio detection kit. Promega microplate reader, cell culture flask, cell culture microplate (96-well or 384-well), CO ₂ constant temperature incubator.

Cell culture: resuscitate the tumor cells cryopreserved in liquid nitrogen, culture the cells with cell culture medium containing 10% fetal bovine serum and 10% penicillin-streptomycin, and when the cells grow to the exponential growth phase, digest and centrifuge to collect the cells and resuspend them in In the culture medium; inoculate the cells according to the number of 5000-10000 cells per well, and place them in an incubator with a constant temperature of 37° C., 5% CO ₂ , and a saturated humidity carbon dioxide incubator for overnight culture.

72h after the compound was added to the cell culture medium, the 96-well plate was taken out from the 37°C incubator and placed at room temperature for 30min for CTG detection. Do not shake the plate during the experiment. 100 μl of CTG reagent was added, mixed for 2 min, and then incubated at room temperature for 10 min. The luminescence value was detected and recorded by a GloMax 96 microplate luminometer (CellTiter-Glo Luminescent CellViability Assay, Promega), and cell viability was observed.

Each compound to be tested was diluted into 10 concentration gradients and added to the corresponding wells of the cell plate respectively, and then the cell plate was returned to the carbon dioxide incubator to continue to culture for 72 hours. After the incubation, PromegaCellTiter-Glo reagent was added to each well of the cell plate, incubated at room temperature for 10min, the luminescence signal was detected by a Promega microplate detector, and the _IC50 value was calculated. The results of the antiproliferative activity of the compounds of the present invention are expressed as A, B, and ND: 0.001≤“A”≤1 μM; “B”>1 μM; ND=not determined, the results are shown in Table 2.

Table 2 Anti-proliferative effects of example compounds on KRAS-G12C mutant tumor cells

The experimental results showed that the compounds of the present invention showed high anti-proliferation inhibitory activity on KRAS G12C-mutated NCL-H358 cells, but only weak anti-proliferative activities on A549 cells and HCC827 cells without KRAS G12S mutation (Table 2) . The results of the anti-proliferation experiments are consistent with the results obtained by the above-mentioned gel mobility assay, indicating that the compounds of the present invention have a selective inhibitory effect on KRAS G12C mutant cells.

Although the present invention has been described in detail above with general description, specific embodiments and tests, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art . Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (13)

1. The compound represented by formula I, its pharmaceutically acceptable salt, its optical isomer, its crystal form, its deuterated product, its active metabolite or its solvate:

in: P is selected from C or N; R ₁ is selected from H or

R ₂ , R ₃ and R ₄ are each independently selected from H, halogen, C _1-3 alkyl, 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 radicals and heteroarylalkyl groups may be optionally substituted with one or more R ₇ ; R ₅ is selected from H, halogen, C _1-3 alkyl, amino, hydroxyl, cyano, haloalkyl; e is 1, 2, 3 or 4; R ₆ is selected from H, C _1-3 alkyl, haloalkyl, cyclopropyl, halogen, amino, oxo, cyano; h is 0, 1, 2 or 3; m is 0 or 1; R ₇ is selected from H, alkyl, halogen, haloalkyl; K is a 4-12 membered saturated or partially saturated monocyclic, bicyclic, bridged or spirocyclic ring containing 1-2 N atoms, which may optionally be replaced by one or more R ₈ is substituted, and multiple substituted R ₈ can be the same or different; R ₈ is selected from C _1-3 alkyl, heteroalkyl, cyano, halogen, C _2-4 alkynyl, oxo, -N(R ₉ ) ₂ , -(CO)OR ₁₀ , wherein said C _{1 -3} Alkyl can be optionally substituted by cyano, halogen, heteroaryl; R ₉ is selected from oxygen, hydroxyl, acyl, halogen, C _1-4 alkyl, oxo, haloalkyl; R ₁₀ is selected from hydrogen, C _1-3 alkyl, hydroxyalkyl; G is selected from

R ₁₁ is selected from hydrogen, halogen, C _1-3 alkyl, alkoxy, hydroxyalkyl, cyano, aminoalkyl; R ₁₂ is selected from hydrogen, halogen, C _1-4 alkyl, alkoxy, cyano, amido, haloalkyl, amino, hydroxyl, hydroxyalkyl; R ₁₃ is selected from hydrogen, alkyl, alkoxy, cyano, halogen; R ₁₄ is selected from hydrogen, alkyl, alkoxy, aminoalkyl. 2. The compound represented by formula I-1, its pharmaceutically acceptable salt, its optical isomer, its crystal form, its deuterated product, its active metabolite or its solvate:

Wherein, the meanings of R ₁ , R ₂ , R ₃ , R ₄ , L, Q, K and G are as defined in claim 1 . 3. The compound of claim 1 or 2, wherein R ₁ is selected from hydrogen or

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

Wherein, the meaning of G is as 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 R ₈ ; the meanings of R ₈ , R ₁₁ , R ₁₂ and R ₁₃ are as defined in claim 1 . 6. The compound represented by the following formula, its pharmaceutically acceptable salt, its optical isomer, its crystal form, its deuterated product, its active metabolite or its solvate:

7. A pharmaceutical composition comprising the compound of any one of claims 1-6, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a crystalline form thereof, a deuterated substance thereof, an active metabolite thereof or a solvate thereof, also including excipients. 8. The compound of any one of claims 1-6, its pharmaceutically acceptable salt, its optical isomer, its crystalline form, its deuterated product, its active metabolite, its solvate or claim 7. Use of the pharmaceutical composition in a medicament for the treatment of proliferative diseases caused by KRASG12C mutation; Preferably, the proliferative disease is a tumor; More preferably, the tumor comprises the following tumorigenic sites: lung, colorectum, pancreas, brain, head and neck, liver, stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid and soft tissue. 9. The compound of any one of claims 1-6, its pharmaceutically acceptable salt, its optical isomer, its crystalline form, its deuterated product, its active metabolite, its solvate or claim 7. Use of the pharmaceutical composition in the preparation of a medicament for treating proliferative diseases caused by KRAS G12C mutation; Preferably, the proliferative disease is a tumor; More preferably, the tumor comprises the following tumorigenic sites: lung, colorectum, pancreas, brain, head and neck, liver, stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid and soft tissue. 10. The compound of any one of claims 1-6, its pharmaceutically acceptable salt, its optical isomer, its crystalline form, its deuterated product, its active metabolite, its solvate or claim 7. Use of the pharmaceutical composition for inhibiting KRASG12C mutation. 11. The compound of any one of claims 1-6, its pharmaceutically acceptable salt, its optical isomer, its crystalline form, its deuterated product, its active metabolite, its solvate or claim 7. Use of the pharmaceutical composition for preparing a medicament for inhibiting KRAS G12C mutation. 12. A method for inhibiting 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, Its crystalline form, its deuterated product, its active metabolite, its solvate or the pharmaceutical composition of claim 7. 13. A method for treating a proliferative disease caused by 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, Its optical isomer, its crystal form, its deuterated product, its active metabolite, its solvate or the pharmaceutical composition according to claim 7; Preferably, the proliferative disease is a tumor; More preferably, the tumor comprises the following tumorigenic sites: lung, colorectum, pancreas, brain, head and neck, liver, stomach, esophagus, breast, cervix, ovary, endometrium, larynx, oral cavity, prostate, thyroid and soft tissue.