CN113980014A

CN113980014A - Hydrogenated pyridopyrimidine derivative, preparation method and medical application thereof

Info

Publication number: CN113980014A
Application number: CN202110843971.2A
Authority: CN
Inventors: 陆标; 张蔡华; 贺峰; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2020-07-27
Filing date: 2021-07-26
Publication date: 2022-01-28
Anticipated expiration: 2041-07-26
Also published as: CN113980014B

Abstract

The present disclosure relates to hydrogenated pyridopyrimidine derivatives, processes for their preparation and their use in medicine. In particular, the disclosure relates to specific hydrogenated pyridopyrimidine derivatives, methods of their preparation, pharmaceutical compositions containing them and their use as therapeutic agents, particularly as KRAS inhibitors.

Description

Hydrogenated pyridopyrimidine derivative, preparation method and medical application thereof

Technical Field

The disclosure belongs to the field of medicines, and relates to a hydrogenated pyridopyrimidine derivative, a preparation method thereof, a pharmaceutical composition containing the derivative, and application of the derivative as a therapeutic agent, especially as a KRAS inhibitor.

Background

The RAS (rat Sarcoma Viral Oncogene homolog) family belongs to the small GTP enzyme superfamily and is widely expressed in various eukaryotes. There are three RAS genes (HRAS, KARS and NARS) in humans, which are expressed as four highly related RAS small gtpases (HRAS, KRAS4A, KARS4B and NRAS). It acts as a binary switch for GDP-GTP regulation. They generally exhibit two forms: a GDP (guanosine diphosphate) -bound form in the inactivated state and a GTP (guanosine triphosphate) -bound form in the activated state. RAS proteins regulate multiple downstream pathways including RAF-MEK-ERK, PI3K/Akt/mTOR by switching between two active states, thereby affecting cell growth, proliferation and differentiation (Nat Rev Cancer,2007,7, 295-. The RAS gene has high mutation rate in pancreatic cancer, colorectal cancer, non-small cell lung cancer and other tumors, and the activated mutant RAS protein can promote abnormal signal transduction, thereby causing the occurrence and development of cancer and generating drug resistance to targeted drugs. Wherein KRAS mutation is the gene with the highest mutation rate in human oncogenes and accounts for 20-30% of all tumors.

For the research of mutant forms and signal paths of KRAS protein, molecular biology has made great progress in recent years, however, the development of related targeted drugs is still challenging. In the aspect of chemical drug development, because the affinity of KRAS and GTP is very high and reaches 60pM, and the intracellular GTP concentration is at the level of mM, the affinity of the directly competing molecules to the compound is extremely high, and no successful case exists so far. In the aspect of biological drug development, the antibody drug penetrates cell membranes to target KRAS protein, and the drug delivery efficiency is low. Therefore, many researchers have tried to develop new approaches to inhibit the activity of kinases such as RAF, MEK and ERK in KRAS downstream signaling pathway to achieve the purpose of inhibiting KRAS pathway. The compounds have certain curative effect, but because the downstream inhibitor cannot completely block KRAS signals and the target point related toxic and side effects are large, the compounds have poor medicinal effect on KRAS mutant tumors. Therefore, KRAS inhibitors with new action mechanisms have great clinical application value.

KRAS mutations are predominantly point mutations, including mutations at amino acids 12, 13 and 61. Among them, the mutation of glycine at position 12 to cysteine (G12C) is most common, and the mutation is large in lung cancer, especially non-small cell lung cancer (14%); it is also expressed in some patients with colorectal (4%), pancreatic (2%) cancer. In the cancer population in the United states, the incidence of this gene mutation is even greater than the sum of ALK, RET, TRK gene mutations.

Facing the difficulty of KRAS protein becoming drug property, professor Kevan Shokat of san Francisco university, California first verifies that certain special compounds can bind KRAS G12C mutant protein through covalent bonds. Through further research, the covalent compounds can be combined with the 12 th cysteine of KRAS mutant protein and occupy a hydrophobic allosteric regulatory pocket in a II molecular switch region (switch-II regions), and the bound KRAS G12C mutant can be irreversibly locked in an inactivated state, thereby blocking a signal path dependent on the protein and the viability of cancer cells (Nature 2013, 503, 548-. The KRAS G12C small-molecule inhibitor ARS-1620 can effectively inhibit tumor growth and even completely regress tumors in various KRAS G12C mutant tumor models. Since KRAS G12C is a mutant protein in tumor cells, while wild-type KRAS does not have this mutation site, it provides a perfect tumor-selective target (Cell, 2018, 572, 578-. Companies represented by Araxas, Amgen and Mirati have issued several patents for KRAS G12C inhibitors (WO2014152588, WO2016164675, WO2017087528, WO2017201161, WO2018119183, etc.). No inhibitor drug of KRAS G12C is currently approved for marketing, and the most advanced small molecule KRAS G12C inhibitors of Amgen and Mirati enter clinical trials at 9 and 12 months in 2018, respectively, so there is a significant unmet medical need in the relevant patient population.

Disclosure of Invention

The present disclosure is directed to a plurality of compounds, or tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, which are of the following structure in table 1:

table 1 Structure and nomenclature of Compounds

Or a tautomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

The intermediate compounds of the present disclosure are shown in table 2 below:

table 2 intermediate structures and nomenclature

Another aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of table 1 of the present disclosure, or a tautomer, racemate, enantiomer, or diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

The present disclosure further relates to the use of a compound shown in table 1, or a tautomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for the preparation of a medicament for inhibiting KRAS, preferably for inhibiting KRAS G12C.

The present disclosure further relates to the use of a compound shown in table 1, or a tautomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment or prevention of cancer, inflammation, or other proliferative disease, preferably for the treatment or prevention of cancer; the cancer is selected from the group consisting of gastric cancer, esophageal cancer, melanoma, liver cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), nasopharyngeal cancer, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, endometrial cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma (e.g., multiple myeloma), astrocytoma, and glioma. Preferably, the cancer is selected from melanoma, liver cancer, kidney cancer, lung cancer (such as non-small cell lung cancer or small cell lung cancer), nasopharyngeal cancer, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, endometrial cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma (such as multiple myeloma), astrocytoma, and glioma.

The present disclosure also relates to a method of inhibiting KRAS comprising administering to a patient in need thereof a therapeutically effective amount of a compound shown in table 1 or a tautomer, racemate, enantiomer, diastereomer, mixture thereof, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.

The present disclosure also relates to a method of treating or preventing KRAS-mediated diseases comprising administering to a patient in need thereof a therapeutically effective amount of a compound shown in table 1 or its tautomers, racemates, enantiomers, diastereomers, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.

The present disclosure also relates to a method of treating or preventing cancer, inflammation, or other proliferative disease, preferably treating cancer, comprising administering to a patient in need thereof a therapeutically or prophylactically effective amount of a compound shown in table 1, or a tautomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same; wherein the cancer is selected from the group consisting of gastric cancer, esophageal cancer, melanoma, liver cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), nasopharyngeal cancer, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, endometrial cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma (e.g., multiple myeloma), astrocytoma, and glioma. Preferably, the cancer is selected from melanoma, liver cancer, kidney cancer, lung cancer (such as non-small cell lung cancer or small cell lung cancer), nasopharyngeal cancer, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, endometrial cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma (such as multiple myeloma), astrocytoma, and glioma.

The present disclosure further relates to a compound shown in table 1 or a tautomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same, for use as a medicament.

The present disclosure also relates to compounds shown in table 1 or tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the same, for use as KRAS inhibitors, preferably KRAS G12C inhibitors.

The present disclosure also relates to a compound shown in table 1 or a tautomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same, for use in treating or preventing KRAS-mediated diseases, preferably KRAS G12C-mediated diseases.

The present disclosure also relates to compounds shown in table 1, or tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the same, for use in the treatment or prevention of cancer, inflammation, or other proliferative disease, preferably cancer; wherein the cancer is selected from the group consisting of gastric cancer, esophageal cancer, melanoma, liver cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), nasopharyngeal cancer, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, endometrial cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma (e.g., multiple myeloma), astrocytoma, and glioma. Preferably, the cancer is selected from melanoma, liver cancer, kidney cancer, lung cancer (such as non-small cell lung cancer or small cell lung cancer), nasopharyngeal cancer, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, endometrial cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma (such as multiple myeloma), astrocytoma, and glioma.

The active compounds may be formulated in a form suitable for administration by any suitable route, using one or more pharmaceutically acceptable carriers to formulate compositions of the disclosure by conventional methods. Thus, the active compounds of the present disclosure may be formulated in a variety of dosage forms for oral administration, injection (e.g., intravenous, intramuscular, or subcutaneous), inhalation, or insufflation. The compounds of the present disclosure may also be formulated in sustained release dosage forms, such as tablets, hard or soft capsules, aqueous or oily suspensions, emulsions, injections, dispersible powders or granules, suppositories, lozenges, or syrups.

The dosage of the compound or composition used in the treatment methods of the present disclosure will generally vary with the severity of the disease, the weight of the patient, and the relative efficacy of the compound. However, as a general guide, the active compound is preferably in unit dosage form, or in such a way that the patient can self-administer it in a single dose. The unit dose of a compound or composition of the present disclosure may be expressed in the form of a tablet, capsule, cachet, bottled liquid, powder, granule, lozenge, suppository, reconstituted powder, or liquid. A suitable unit dose may be 0.1 to 1000 mg.

The pharmaceutical compositions of the present disclosure may contain, in addition to the active compound, one or more excipients selected from the following: fillers (diluents), binders, wetting agents, disintegrants, excipients, and the like. Depending on the method of administration, the compositions may contain from 0.1 to 99% by weight of active compound.

Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be inert excipients, granulating agents, disintegrating agents, binding agents and lubricating agents. These tablets may be uncoated or they may be coated by known techniques which mask the taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

Oral formulations may also be provided in soft gelatin capsules wherein the active ingredient is mixed with an inert solid diluent or wherein the active ingredient is mixed with a water soluble carrier or an oil vehicle.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending, dispersing or wetting agents. Aqueous suspensions may also contain one or more preservatives, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, or in a mineral oil. The oil suspension may contain a thickener. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable preparation. These compositions can be preserved by the addition of antioxidants.

The pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, or a mineral oil or a mixture thereof. Suitable emulsifiers may be naturally occurring phospholipids, and the emulsions may also contain sweetening, flavoring, preservative and antioxidant agents. Such formulations may also contain a demulcent, a preservative, a colorant and an antioxidant.

The pharmaceutical compositions of the present disclosure may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles or solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase, and the injection or microemulsion may be injected into the bloodstream of a patient by local mass injection. Alternatively, it may be desirable to administer the solution and microemulsion in a manner that maintains a constant circulating concentration of the disclosed compounds. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is an intravenous pump model Deltec CADD-PLUS. TM.5400.

The pharmaceutical compositions of the present disclosure may be in the form of sterile injectable aqueous or oleaginous suspensions for intramuscular and subcutaneous administration. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally-acceptable, non-toxic diluent or solvent. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any blend fixed oil may be used for this purpose. In addition, fatty acids can also be prepared into injections.

The compounds of the present disclosure may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and therefore will melt in the rectum to release the drug.

Dispersible powders and granules of the compounds of the present disclosure can be administered by the addition of water to prepare an aqueous suspension. These pharmaceutical compositions may be prepared by mixing the active ingredient with dispersing or wetting agents, suspending agents, or one or more preservatives.

As is well known to those skilled in the art, the dosage of a drug administered depends on a variety of factors, including, but not limited to: the activity of the particular compound employed, the age of the patient, the weight of the patient, the health condition of the patient, the behavior of the patient, the diet of the patient, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, and the like; in addition, the optimal treatment regimen, such as mode of treatment, daily amount of compound or type of pharmaceutically acceptable salt, can be verified according to conventional treatment protocols.

Detailed description of the invention

The compounds of the present disclosure may also comprise isotopic derivatives thereof. The term "isotopic derivative" refers to a compound that differs in structure only in the presence of one or more isotopically enriched atoms. For example, having the structure of the present disclosure except that "deuterium" or "tritium" is substituted for hydrogen, or¹⁸F-fluorine labeling: (¹⁸Isotope of F) instead of fluorine, or with¹¹C-，¹³C-, or¹⁴C-enriched carbon (C¹¹C-，¹³C-, or¹⁴C-carbon labeling;¹¹C-，¹³c-, or¹⁴C-isotopes) instead of carbon atoms are within the scope of the present disclosure. Such compounds are useful as analytical tools or probes in, for example, biological assays, or as tracers for in vivo diagnostic imaging of disease, or as tracers for pharmacodynamic, pharmacokinetic or receptor studies. The disclosure also includes various deuterated forms of the compounds of formula (I). Each available hydrogen atom attached to a carbon atom may be independently replaced by a deuterium atom. The person skilled in the art is able to synthesize the deuterated forms of the compounds of the formula (I) with reference to the relevant literature. Commercially available deuterated starting materials can be used in preparing the deuterated forms of the compounds of formula (I), or they can be synthesized using conventional techniques using deuterated reagents including, but not limited to, deuterated boranes, trideuteroborane tetrahydrofuran solutions, deuterated lithium aluminum hydrides, deuterated iodoethanes, deuterated iodomethanes, and the like. Deuterations can generally retain activity comparable to non-deuterated compounds and can achieve better metabolic stability when deuterated at certain specific sites, thereby achieving certain therapeutic advantages.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof in admixture with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

"pharmaceutically acceptable salts" refers to salts of the disclosed compounds which are safe and effective for use in a mammalian body and which possess the requisite biological activity. Salts may be prepared separately during the final isolation and purification of the compound, or by reacting the appropriate group with an appropriate base or acid. Bases commonly used to form pharmaceutically acceptable salts include inorganic bases such as sodium hydroxide and potassium hydroxide, and organic bases such as ammonia. Acids commonly used to form pharmaceutically acceptable salts include inorganic acids as well as organic acids.

The term "therapeutically effective amount" with respect to a drug or pharmacologically active agent refers to a sufficient amount of the drug or agent that is non-toxic but achieves the desired effect. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient and also on the particular active substance, and an appropriate effective amount in an individual case can be determined by a person skilled in the art according to routine tests.

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, and effective for the intended use.

When the term "about" is applied to a parameter such as pH, concentration, temperature, etc., it is meant that the parameter may vary by ± 10%, and sometimes more preferably within ± 5%. As will be appreciated by those skilled in the art, when the parameters are not critical, the numbers are generally given for illustrative purposes only and are not limiting.

Boc represents tert-butyloxycarbonyl;

cbz represents benzyloxycarbonyl.

Detailed Description

The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure.

Examples

The structure of the compounds was determined using Nuclear Magnetic Resonance (NMR) and/or Mass Spectrometry (MS). NMR chemical shift (. delta.) of 10^-6(ppm) is given as unit. The NMR is measured by a Bruker AVANCE II-400MHz nuclear magnetic resonance spectrometer, and the conventional deuterated solvent used for the measurement is deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDCl)₃) And deuterated Methanol (Methanol-d)₄) Tetramethylsilane (TMS) was used as an internal standard.

Determination of Mass Spectrum (MS) using a liquid chromatography-mass spectrometer (LC-MS), ionization source: electrospray ionization (ESI). The manufacturers are respectively: shimadzu, Waters and Agilent, the models are respectively: LCMS2020, UPLC-QDa and Agilent 6120, columns Sunfire C185 μm 50X 4.6mm, ACQUITY

BEH 2.1 x 50mm 1.7 μm and Xbridge C185 μm 50X 4.6 mm.

HPLC measurements were performed using an Agilent 1200DAD HPLC (column: Waters SunAire C18 (150X 4.6mm,5 μm)) and Shimadzu UFLC HPLC (column: Waters Xbridge C18 (150X 4.6mm,5 μm)).

Analytical determination of chiral HPLC using Waters-UPC 2.

The thin layer chromatography silica gel plate is produced by silica gel plate factory of Taiwan Huanghai, Xinnuo or Shandong Mount mammary gland, and the thickness of silica gel plate used by Thin Layer Chromatography (TLC) is 0.2-0.25 mm; the specification is 50X 200 mm. Thin layer preparative chromatography (prep-TLC) using silica gel plate with thickness of 0.4-0.5 mm; the specification is 200X 200 mm.

The column chromatography silica gel is generally silica gel produced by chemical industry (Shanghai) Limited, and the specification is 100-200 meshes or 200-300 meshes.

High performance liquid preparative chromatography A preparative chromatograph was used from Waters 2767 (column: Sunfire Pre C1810 μm 19X 250mm) and Waters 2767-QDa (column: Xbridge Pre C1810 μm 19X 250 mm).

Chiral preparation was performed using Waters-SFC80 (chiral column: Daciel AD/OD/OJ/IC/IA/ID 10 μm 20X 250 mm).

CombiFlash rapid preparation instrument uses MP200 medium pressure rapid purification preparation system (Agela Technologies).

The starting materials in the synthetic record, if purchased, need to be labeled as clear sources: such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shao Yuan Chemical technology (Accela ChemBio Inc), Darri Chemicals, etc.

If the reaction is carried out in argon atmosphere or nitrogen atmosphere, the specific conditions need to be marked clearly;

the pressure hydrogenation reaction uses GSH-1/12.5 type, GSH-2/12.5 type, GSH-5/12.5 type and GSH-20/12.5 type high pressure reaction kettles.

The microwave reaction uses a microwave reactor of the Monowave300 or Initiator + type.

Monitoring the reaction progress in the synthetic record by using Thin Layer Chromatography (TLC) and purifying by using thin layer preparative chromatography (prep-TLC), wherein the solvents used by the developing agent system and the exact proportion are respectively marked clearly; the addition of small amounts of basic or acidic reagents such as triethylamine, ammonia, acetic acid, etc. is also indicated clearly.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), a developing solvent used for the reaction, a system of eluents for column chromatography used for purifying compounds and a developing solvent system for thin layer chromatography including: a: dichloromethane/methanol system, B: petroleum ether/ethyl acetate system, C: the volume ratio of the n-hexane/ethyl acetate system is adjusted according to the different polarities of the compounds, and a small amount of basic or acidic reagents such as triethylamine, acetic acid and the like can be added for adjustment.

Example 1

2- ((2S,6R) -1-acryloyl-4- (7- (7-fluoro-8-methylnaphthalen-1-yl) -2- ((1- (pyrrolidinyl-1-ylmethyl) cyclopropyl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -6-methylpiperazin-2-yl) acetonitrile 1

First step of

1- (Chlorocarbonyl) cyclopropane-1-carboxylic acid methyl ester 1b

Compound 1, 1-Cyclopropyldicarboxylic acid monomethyl ester 1a (1.4g, 9.71mmol) was dissolved in 20mL of dichloromethane, 0.2mL of N, N-dimethylformamide was added, oxalyl chloride (1.26g, 9.92mmol, Tatan) was added dropwise at 0 deg.C, and the reaction was stirred at 0 deg.C for 1 hour. The resulting mixture was concentrated to give the title product, crude 1b (1.58g), which was directly used in the next reaction without purification.

Second step of

1- (pyrrolidine-1-carbonyl) cyclopropane-1-carboxylic acid methyl ester 1c

Compound 1b (1.58g, 9.71mmol) was dissolved in 500mL tetrahydrofuran and pyrrolidine (3.45g, 48.48mmol, Annaigii) was added at 0 ℃. The reaction mixture was stirred at room temperature for 16 hours, and the reaction was stopped. 50mL of water was added, extracted with ethyl acetate (50 mL. times.3), dried, filtered, and concentrated under reduced pressure to give the title product 1c (1.64g), which was directly used in the next reaction without purification.

¹H NMR(400MHz,CDCl₃)δ3.72(s,3H),3.50(t,2H),3.44(t,2H),1.94-1.87(m,4H),1.48-1.45(m,2H),1.36-1.33(m,2H)。

The third step

(1- (pyrrolidin-1-ylmethyl) cyclopropyl) methanol 1d

Compound 1c (1.0g, 5.07mmol) was dissolved in 100mL of tetrahydrofuran under argon, a solution of lithium aluminum hydride in tetrahydrofuran (1M, 10mL, 10mmol, Annage) was added at 0 deg.C, and the reaction was stirred at 0 deg.C for 4 hours. The reaction was quenched by the addition of 3.2g of sodium sulfate decahydrate solid. Drying, filtration and concentration under reduced pressure gave the title product 1d (624mg), yield: 79 percent.

¹H NMR(400MHz,CDCl₃)δ3.55(s,2H),2.61-2.57(m,6H),1.77-1.73(m,4H),0.49-0.47(m,2H),0.37-0.35(m,2H)。

The fourth step

(R) - (1- ((4-Nitrophenyl) sulfonylamino) -1-oxopropan-2-yl) carbamic acid tert-butyl ester 1f

(R) -2- ((tert-butoxycarbonyl) amino) propionic acid 1e (15.0g, 79.28mmol, Shaoyuan), 2-nitrobenzenesulfonamide (14.43g, 71.35mmol, Shaoyuan), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 22.71g, 118.90mmol, Tatan) and 4-dimethylaminopyridine (DMAP, 11.62g, 95.13mmol, Neutrol) were dissolved in N, N-dimethylformamide (500mL, Ruizie), and the reaction solution was stirred at room temperature for 16 hours. After addition of water (100mL), a small amount of the mixture was taken, citric acid was added to bring the pH of the aqueous phase to less than 7, and extraction was performed with ethyl acetate (30 mL. times.2). The combined organic phases were washed with water (30 mL. times.2), then saturated sodium bicarbonate solution was added to maintain the pH at about 7, and the mixture was washed with saturated sodium chloride solution. The organic phase was concentrated under reduced pressure to give the title compound 1f (26.4g), yield: 89 percent.

MS(ESI):372.1[M-H]^-。

The fifth step

(R) - (1- ((4-4-nitrophenyl) sulfonamido) propan-2-yl) carbamic acid tert-butyl ester 1g

Compound 1f (10.0g, 26.78mmol) was added to a 250mL three-necked flask at room temperature, borane (1M in tetrahydrofuran, 66.96mL, 66.96mmol, Fox) was added with stirring at 0 deg.C, and the reaction was allowed to slowly warm to room temperature and stirred for 16 h. The resulting mixture was quenched with methanol (30mL), then an aqueous solution of sodium hydroxide (1M, 100mL) was added, and extracted with dichloromethane (50 mL. times.3). The combined organic phases were washed with water (50 mL. times.3), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep with eluent system B to give 1g (3.35g) of the title product. Yield: 34 percent.

MS(ESI):358.0[M-H]^-。

The sixth step

(R, E) -4- (N- (2-aminopropyl) -4-nitrophenylsulfonamido) but-2-enoic acid ethyl ester

1h

Compound 1g (3.35g, 9.32mmol), ethyl 4-bromocrotonate (2.16g, 11.19mmol, Shaoyuan) and potassium carbonate (3.86g, 27.96mmol, Rujie) were dispersed in N, N-dimethylformamide (20mL, Rujie), and the reaction was stirred at room temperature under argon atmosphere for 24 hours. The resulting mixture was added with water (30mL) and extracted with ethyl acetate (50 mL. times.2). The combined organic phases were washed with water (50 mL. times.2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the crude product, and the residue was purified using CombiFlash flash prep to give the title product 1h (3.6g), yield: 81 percent.

MS(ESI):372.1[M+H]⁺。

Seventh step

Ethyl 2- ((2S,6R) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazin-2-yl) acetate 1i

Compound 1h (3.6g, 7.63mmol) was dissolved in dichloromethane (25mL) at room temperature, trifluoroacetic acid (10mL, Tatan) was added at room temperature and stirred for 2 h. And concentrating the reaction solution to obtain a crude intermediate. Toluene (50mL) was added to the residue, followed by concentration, and toluene (50mL) was added again to the residue, followed by concentration. The resulting crude intermediate was dissolved in dichloromethane (25mL), and a saturated aqueous solution of sodium bicarbonate (10mL) was added under ice-bath and stirred at room temperature for 1 hour. The reaction solution was extracted with methylene chloride (50 mL. times.2). The combined organic phases were washed with water (50 mL. times.2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give a crude product, and the residue was purified using a CombiFlash flash rapid prep. instrument with eluent system B to give the title product 1i (1.1g) in 38% yield.

MS(ESI):372.2[M+H]⁺。

Eighth step benzyl (2S,6R) -2- (2-ethoxy-2-oxoethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylate 1j

Compound 1i (1.0g, 2.69mmol), benzyl chloroformate (915mg, 5.38mmol, tetam) and sodium bicarbonate (679mg, 8.08mmol, Runjie) were dissolved in tetrahydrofuran/water (8mL/2mL, national drugs/Drech) and the reaction was stirred at room temperature under argon for 16 hours. The resulting mixture was added with water and extracted with methylene chloride (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to give a crude product and the residue was purified using CombiFlash flash prep with eluent system B to give the title product 1j (926 mg). Yield: 68 percent.

MS(ESI):506.1[M+H]⁺。

The ninth step

2- ((2S,6R) -1- ((benzyloxy) carbonyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazin-2-yl) acetic acid 1k

Compound 1g (1.5g, 2.97mmol) was dissolved in tetrahydrofuran/water (10mL/10mL, national drugs/Drech), lithium hydroxide (243mg, 5.93mmol, Rujie) was added, and the reaction was stirred at room temperature for 16 hours. The resulting mixture was extracted with ethyl acetate (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. The ethyl acetate extracted aqueous phase was adjusted to pH <7 with dilute hydrochloric acid (1M) and extracted with ethyl acetate (20mL × 2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. All filtrates were combined and concentrated to give crude 1k (1.4 g). Yield: 98 percent.

MS(ESI):478.1[M+H]⁺。

The tenth step

Benzyl (2S,6R) -2- (2-amino-2-oxoethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylate 1l

Compound 1k (1.4g, 2.93mmol), ammonium chloride (313mg, 5.86mmol, Rujie), O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 1.67g, 4.40mmol, Tatan) and N, N-diisopropylethylamine (DIPEA, 1.14g, 8.80mmol, Tatan) were dissolved in N, N-dimethylformamide (10mL, Rujie), and the reaction was stirred at room temperature for 1 hour. The resulting mixture was added with water and extracted with ethyl acetate (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep at eluent system a to give the title product 1l (1.3g), yield: 93 percent.

MS(ESI):477.1[M+H]⁺。

The eleventh step

Benzyl (2S,6R) -2- (cyanomethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylate 1m

Compound 1l (1.3g, 2.73mmol) and triethylamine (1.38g, 13.64mmol, tetam) were dissolved in dichloromethane (20mL, Rujie) at 0 deg.C, and trifluoroacetic anhydride (4.58g, 21.83mmol, tetam) was added to the reaction solution with stirring. The reaction solution was stirred at 0 ℃ for 2 hours. A saturated sodium bicarbonate solution was added to the reaction solution, followed by extraction with ethyl acetate (20 mL. times.2). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep. apparatus with eluent system B to give the title product 1m (1.0 g). Yield: 79 percent.

MS(ESI):459.1[M+H]⁺。

The twelfth step

(2S,6R) -2- (cyanomethyl) -6-methylpiperazine-1-carboxylic acid benzyl ester 1n

Compound 1m (1.0g, 2.18mmol) and cesium carbonate (1.42g, 4.36mmol, YAMCUTANGDE) were dispersed in tetrahydrofuran (10mL, national drug) and 2-mercaptoethanol (510mg, 6.54mmol, Tatan) was added with stirring. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated. To the residue were added dichloromethane (2mL) and dilute hydrochloric acid (2M, 2mL) and the mixture was stirred at room temperature for 1 hour. The reaction solution was poured into water and extracted with dichloromethane (20 mL. times.2). The organic phase was washed with water (20mL × 2) and the combined aqueous phases were adjusted to pH >7 with saturated sodium bicarbonate solution. The resulting mixture was extracted with dichloromethane (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep at eluent system B to give the title product 1n (400mg), yield: 70 percent.

MS(ESI):274.1[M+H]⁺。

Thirteenth step

4- ((3S,5R) -4- ((benzyloxy) carbonyl) -3- (cyanomethyl) -5-methylpiperazin-1-yl) -2- (methylthio) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester 1o

Compound 1N (628mg, 1.46mmol), compound 2- (methylthio) -4- (((trifluoromethyl) sulfonyl) oxy) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester (400mg, 1.46mmol, prepared using the method disclosed in example intermediate 67 ", page 76 of the description in the patent application" WO2017/201161,2017, a1 ") and N, N-diisopropylethylamine (567mg, 4.39mmol, tetam) were dissolved in N, N-dimethylformamide (10mL, wet) and the reaction was stirred under argon at 100 ℃ for 3 hours. Water was added to the cooled reaction solution, followed by extraction with ethyl acetate (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep at eluent system B to give the title product 1o (777mg), yield: 96 percent.

MS(ESI):553.1[M+H]⁺。

Fourteenth step

4- ((3S,5R) -4- ((benzyloxy) carbonyl) -3- (cyanomethyl) -5-methylpiperazin-1-yl) -2- (methylsulfonyl) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester 1p

To a solution of compound 1o (777mg, 1.41mmol) in dichloromethane (10mL, Rujie) was added m-chloroperoxybenzoic acid (710mg, 3.51mmol, 85% write, Jinan Jiabin) at 0 ℃. The reaction solution was stirred at 0 ℃ for 3 hours. The resulting mixture was concentrated at low temperature to give a crude product and the residue was purified using CombiFlash flash prep with eluent system B to give the title product 1p (750mg), yield: 91 percent.

MS(ESI):584.9[M+H]⁺。

The fifteenth step

4- ((3S,5R) -4- ((benzyloxy) carbonyl) -3- (cyanomethyl) -5-methylpiperazin-1-yl) -2- ((1- (pyrrolidin-1-ylmethyl)

Cyclopropyl) methoxy) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester 1q

Compound 1p (650mg, 1.11mmol) and compound 1d (259mg, 1.67mmol) were dissolved in tetrahydrofuran (5mL, Hengyue chemical), lithium hexamethyldisilazide (LiHMDS, 1M tetrahydrofuran solution, 2.22mL, 2.22mmol, Sichuan Weibo) was added under argon protection at 0 ℃ and stirred at room temperature for 2 hours. Water (20mL) was added to the obtained reaction solution, followed by extraction with ethyl acetate (50 mL. times.3). The combined organic phases were washed with water (50 mL. times.3), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep at eluent system a to give the title product 1q (560mg), yield: 76 percent.

MS(ESI):660.0[M+H]⁺。

Sixteenth step benzyl (2S,6R) -2- (cyanomethyl) -6-methyl-4- (2- ((1- (pyrrolidin-1-ylmethyl) cyclopropyl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) piperazine-1-carboxylate 1R

Compound 1q (560mg, 0.848mmol) was dissolved in dichloromethane (10mL, Touret) at room temperature, and a solution of hydrochloric acid/1, 4-dioxane (4M,5mL, Chemart) was added dropwise to the reaction solution with stirring. The reaction solution was stirred at room temperature for 2 hours. The resulting mixture was cryoconcentrated to give the crude product, free from saturated sodium bicarbonate solution to give the title product 1r (457mg), yield: 96 percent.

MS(ESI):560.0[M+H]⁺。

Seventeenth step

Benzyl (2S,6R) -2- (cyanomethyl) -4- (7- (7-fluoro-8-methylnaphthalen-1-yl) -2- ((1- (pyrrolidin-1-ylmethyl) cyclopropyl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -6-methylpiperazine-1-carboxylate 1S

Compound 1r (407mg, 0.728mmol), 7-fluoro-8-methylnaphthalen-1-yl-trifluoromethanesulfonate (224mg, 0.728mmol, prepared by the method disclosed in patent application "example 77 on page 76 of the specification in US2019/0270743a 1"), methanesulfonic acid (9, 9-dimethyl-4, 5-bis-diphenylphosphinoxaanthracene) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II) (Xantphos Pd G3, 136mg, 0.146mmol, nanjing yaite) and cesium carbonate (710mg, 2.184mmol, tomantard) were dispersed in toluene (10mL, national medicine) at room temperature, under argon protection, and stirred at 110 ℃ for 18 hours. Water (50mL) was added to the cooled reaction solution, followed by extraction with ethyl acetate (50 mL. times.2). The combined organic phases were washed with water, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep with eluent system a to give the title product 1s (250 mg). Yield: and 47 percent. MS (ESI) 718.1[ M + H]⁺。

Eighteenth step

2- ((2S,6R) -4- (7- (7-fluoro-8-methylnaphthalen-1-yl) -2- ((1- (pyrrolidin-1-ylmethyl) cyclopropyl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -6-methylpiperazin-2-yl) acetonitrile 1t

Compound 1s (250mg, 0.348mmol) was dissolved in methanol (10mL, Honeywell), wet palladium on carbon (50mg, weight chemical) was added, and the reaction solution was stirred at room temperature for 1 hour under hydrogen balloon protection. The resulting mixture was filtered and the filtrate was concentrated to give the title product 1t (175mg), yield: 86 percent.

MS(ESI):584.0[M+H]⁺。

Nineteenth step

Compound 1t (175mg, 0.299mmol) was dissolved in dichloromethane (5mL, Ruizi), and acryloyl chloride (27.13mg, 0.299mmol, Annage) and triethylamine (91.0mg, 0.899mmol, Tatan) were added dropwise to the reaction solution at 0 ℃. The reaction solution was stirred at 0 ℃ for 30 minutes. An aqueous sodium hydrogencarbonate solution (50mL) was added to the reaction mixture, followed by extraction with methylene chloride (50 mL. times.2). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the resulting crude residue was separated by high performance liquid preparative chromatography (acidic conditions, mobile phase containing formic acid) and the resulting fractions were freeze-dried to give the title product 1(105.01mg), yield: 54 percent.

¹H NMR(400MHz,DMSO-d₆):δ7.84-7.82(m,1H),7.73-7.71(m,1H),7.42-7.33(m,3H),6.86(brs,1H),6.22-6.18(d,1H),4.83(brs,1H),4.38(brs,1H),4.12-3.70(m,7H),3.47-3.42(m,1H),3.11-2.95(m,6H),2.79(s,3H),2.79-2.67(m,1H),2.54-2.53(m,3H),2.49-2.47(s,3H),1.68-1.67(d,4H),1.43-1.25(dd,3H),0.57-0.56(d,2H),0.43(s,2H)。

MS(ESI):638.1[M+H]⁺。

Example 2

2- ((2S,6S) -1-acryloyl-4- (7- (7-fluoro-8-methylnaphthalen-1-yl) -2- ((1- (pyrrolidinyl-1-ylmethyl) cyclopropyl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -6-methylpiperazin-2-yl) acetonitrile 2

First step of

Benzyl (2S,6S) -2- (2-ethoxy-2-oxoethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylate 2b

Ethyl 2- ((2S,6S) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazin-2-yl) acetate 2a (1.54g, 4.15mmol, prepared using the well-known method Journal of Organic Chemistry,2018,83, 6541-propan 6555), benzyl chloroformate (3.52g, 20.73mmol, Tatan) and sodium bicarbonate (1.05g, 12.44mmol, Rujie) were dissolved in tetrahydrofuran/water (10mL/10mL, national drugs/Drech), and the reaction was stirred at room temperature for 3 hours. The resulting mixture was added with water and extracted with methylene chloride (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to give the crude product and the residue was purified using CombiFlash flash prep with eluent system B to give the title product 2B (1.65g), yield: 78 percent.

MS(ESI):505.9[M+H]⁺。

Second step of

2- ((2S,6S) -1- ((benzyloxy) carbonyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazin-2-yl) acetic acid 2c

Compound 2b (1.65g, 3.26mmol) was dissolved in tetrahydrofuran/water (10mL/10mL, national drug/Drech), lithium hydroxide (215mg, 9.79mmol, Rujie) was added, and the reaction was stirred at room temperature for 16 hours. The resulting mixture was extracted with ethyl acetate (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. The aqueous phase extracted with ethyl acetate was adjusted to pH with dilute hydrochloric acid (1M)<After 7, it was extracted with ethyl acetate (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. All filtrates were combined and concentrated to give crude product 2c (1.7g) which was used in the next reaction, MS (ESI) 478.0[ M + H ] without further purification]⁺。

The third step

Benzyl (2S,6S) -2- (2-amino-2-oxoethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylate 2d

Compound 2c (1.7g, 3.56mmol), ammonium chloride (380mg, 7.12mmol, tallown), O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 1.62g, 4.27mmol, Bigelet) and N, N-diisopropylethylamine (DIPEA, 1.38g, 10.68mmol, tallown) were dissolved in N, N-dimethylformamide (10mL, wet) and the reaction was stirred at room temperature for 1 hour. The resulting mixture was added with water and extracted with ethyl acetate (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep with eluent system a to give the title product 2d (1.55g, yield: 91 percent.

MS(ESI):476.9[M+H]⁺。

The fourth step

Benzyl (2S,6S) -2- (cyanomethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylate compound 2d (1.55g, 3.25mmol) and triethylamine (1.65g, 16.26mmol, tetam) were dissolved in dichloromethane (10mL, wet) at 2e0 ℃, and trifluoroacetic anhydride (5.47g, 26.02mmol, tetam) was added to the reaction with stirring. The reaction solution was stirred at 0 ℃ for 1 hour. A saturated sodium bicarbonate solution was added to the reaction solution, followed by extraction with ethyl acetate (20 mL. times.2). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep with eluent system a to give the title product 2e (1.15g), yield: 77 percent.

MS(ESI):458.9[M+H]⁺。

The fifth step

(2S,6S) -2- (cyanomethyl) -6-methylpiperazine-1-carboxylic acid benzyl ester 2f

Compound 2e (1.15g, 2.51mmol) and cesium carbonate (1.63g, 5.02mmol, YAMINGKANDE) were dispersed in tetrahydrofuran (10mL, national drug) and 2-mercaptoethanol (586mg, 7.52mmol, Tatan) was added with stirring. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated. To the residue were added dichloromethane (2mL) and dilute hydrochloric acid (2M, 2mL) and the mixture was stirred at room temperature for 0.5 hour. The mixture was poured into water and extracted with dichloromethane (20 mL. times.2). The organic phase was washed with water (20mL × 2) and the combined aqueous phases were adjusted to pH >7 with saturated sodium bicarbonate solution. The resulting mixture was extracted with dichloromethane (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep at eluent system a to give the title product 2f (534mg), yield: 77 percent.

MS(ESI):274.0[M+H]⁺。

The sixth step

4- ((3S,5S) -4- ((benzyloxy) carbonyl) -3- (cyanomethyl) -5-methylpiperazin-1-yl) -2- (methylthio) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester 2g

The compound tert-butyl 2- (methylthio) -4- (((trifluoromethyl) sulfonyl) oxy) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylate (839mg, 1.95mmol, prepared by the method disclosed in example 67 "on page 76 of the specification, in patent application" WO2017/201161,2017, a1 "), compound 2f (534mg, 1.95mmol) and N, N-diisopropylethylamine (DIPEA, 757mg, 5.86mmol, tetam) were dissolved in N, N-dimethylformamide (10mL, rit) and the reaction solution was stirred under argon atmosphere at 100 ℃ for 2 hours. Water was added to the cooled reaction solution, followed by extraction with ethyl acetate (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2), dried over anhydrous sodium sulfate and filtered. The residue was purified using CombiFlash flash prep with eluent system B to give the title product 2g (987mg), yield: 91 percent.

MS(ESI):553.0[M+H]⁺。

Seventh step

4- ((3S,5S) -4- ((benzyloxy) carbonyl) -3- (cyanomethyl) -5-methylpiperazin-1-yl) -2- (methylsulfonyl) -5, 8-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester 2H

To a solution of compound 2g (987mg, 1.79mmol) in dichloromethane (10mL, Rujie) was added m-chloroperoxybenzoic acid (906mg, 4.46mmol, 85% write, jonan Jiabin) at 0 ℃. The reaction solution was stirred at 0 ℃ for 3 hours. The resulting mixture was concentrated at low temperature to give a crude product and the residue was purified using CombiFlash flash prep with eluent system B to give the title product 2h (980mg), yield: 93 percent.

MS(ESI):584.9[M+H]⁺。

Eighth step

4- ((3S,5S) -4- ((benzyloxy) carbonyl) -3- (cyanomethyl) -5-methylpiperazin-1-yl) -2- ((1- (pyrrolidin-1-ylmethyl) cyclopropyl) methoxy) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester 2i

Compound 2h (700mg, 1.2mmol) and compound 1d (260mg, 1.68mmol) were dissolved in tetrahydrofuran (5mL, Hengyue chemical), lithium hexamethyldisilazide (LiHMDS, 1M tetrahydrofuran solution, 2.39mL, 2.39mmol, Sichuan Weibo) was added under argon protection at 0 ℃ and stirred at room temperature for 3 hours. Water (20mL) was added to the obtained reaction solution, followed by extraction with ethyl acetate (50 mL. times.3). The combined organic phases were washed with water (50 mL. times.3), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep at eluent system a to give the title product 2i (351mg), yield: 44 percent.

MS(ESI):660.2[M+H]⁺。

The ninth step

Benzyl (2S,6S) -2- (cyanomethyl) -6-methyl-4- (2- ((1- (pyrrolidin-1-ylmethyl) cyclopropyl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) piperazine-1-carboxylate 2j

Compound 2i (351mg, 0.531mmol) was dissolved in dichloromethane (10mL, Touret) at room temperature, and a solution of hydrochloric acid/1, 4-dioxane (4M,5mL, Chemart) was added dropwise to the reaction solution with stirring. The reaction solution was stirred at room temperature for 2 hours. The resulting mixture was poured into water and extracted with dichloromethane (20 mL. times.2). The combined organic phases were washed with water (20 mL. times.2) and discarded. The combined aqueous phases were adjusted to a pH >7 with aqueous sodium bicarbonate solution and extracted with dichloromethane (20 mL. times.2). The combined organic phases were washed with water (20mL × 2), dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated to give the crude product, and the residue was purified using CombiFlash flash prep with eluent system a to give the title product 2j (160mg), yield: 53 percent.

MS(ESI):560.1[M+H]⁺。

The tenth step

Benzyl (2S,6S) -2- (cyanomethyl) -4- (7- (7-fluoro-8-methylnaphthalen-1-yl) -2- ((1- (pyrrolidin-1-ylmethyl) cyclopropyl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -6-methylpiperazine-1-carboxylate 2k

Compound 2j (159mg, 0.284mmol) and-fluoro-8-methylnaphthalen-1-yl-trifluoromethylsulfonate (131mg, 0.426mmol, prepared by the method disclosed in patent application "example 77 on page 76 of the description in US2019/0270743A 1", prepared by intermediate 77 ") were dissolved in anhydrous toluene (3.0mL, Rujie) at room temperature, followed by addition of methanesulfonic acid (9, 9-dimethyl-4, 5-bis diphenylphosphinoxaanthracene) (2 '-amino-1, 1'-biphenyl-2-yl) palladium (II) (27mg, 0.028mmol, Leyan), 4, 5-bis-diphenylphosphino-9, 9-dimethylxanthene (33mg, 0.056mmol, Bi obtained),

molecular sieves (160mg, Wako Junyaku) and cesium carbonate (231.6mg, 0.71mmol, Yamin), were substituted with argon three times, and then stirred at 110 ℃ for 18 hours. The reaction mixture was cooled to room temperature, poured into ice water, and extracted with ethyl acetate (20 mL. times.3). The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified using CombiFlash flash prep at eluent system a to give the title product 2k (106mg), yield: and 55 percent.

MS(ESI):718.8[M+H]⁺。

The eleventh step

2l of 2- ((2S,6S) -4- (7- (7-fluoro-8-methylnaphthalen-1-yl) -2- ((1- (pyrrolidin-1-ylmethyl) cyclopropyl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -6-methylpiperazin-2-yl) acetonitrile

Compound 2k (106mg, 0.148mmol) was dissolved in anhydrous tetrahydrofuran (1.5mL, Runjie) and methanol (1.5mL, Honeyville), palladium hydroxide/charcoal (50mg, 20 wt%, Annagel) was added and replaced with hydrogen three times, and the reaction was stirred under hydrogen balloon at room temperature for 2 hours. The reaction was filtered and the filtrate was concentrated to give the title product 2l (82mg), yield: 95 percent.

MS(ESI):584.3[M+H]⁺。

The twelfth step

Compound 2l (82mg, 0.141mmol) was dissolved in dichloromethane (2.0mL, national drug), and triethylamine (17mg, 0.169mmol, Rujie) and acryloyl chloride (14mg, 0.155mmol, Bi De) were added dropwise to the reaction mixture sequentially at 0 ℃ under an argon atmosphere. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was poured into ice water, and extracted with ethyl acetate (20 mL. times.2). The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated, and the residue was purified by hplc (acidic conditions, mobile phase containing formic acid) to give the title product 2(35mg) in yield: 39 percent.

¹H NMR(400MHz,DMSO-d₆)δ7.85(dd,1H),7.73(d,1H),7.52-7.34(m,3H),6.90-6.70(m,1H),6.27(d,1H),5.81(d,1H),4.68(s,1H),4.41(s,1H),4.14-4.09(m,1H),4.08(d,2H),4.05-3.98(m,1H),3.98-3.84(m,3H),3.73(d,1H),3.61(d,2H),3.50-3.40(m,3H),3.08-2.98(m,2H),2.98-2.88(m,2H),2.82(d,4H),2.42(s,2H),1.66(s,4H),1.32-1.22(m,3H),0.58-0.51(m,2H),0.43-0.36(m,2H)。

MS(ESI):638.8[M+H]⁺。

Example 3 (Final product Structure see US20190270743A1, page 211)

(S) -2- (4- (2- ((1-aminocyclobutyl) methoxy) -7- (8-chloro-7-fluoronaphthalen-1-yl) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -1- (2-fluoropropenyl) piperazin-2-yl) acetonitrile 3

First step of

2- (methylthio) -4- (((trifluoromethyl) sulfonyl) oxy) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester 3b

Tert-butyl 4-hydroxy-2- (methylthio) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylate 3a (10.3g, 34.64mmol, prepared by the method disclosed in patent application "example 32" on page 79 of the specification in WO2017/201161A 1) was dissolved in 200mL of dichloromethane, and N, N-diisopropylethylamine (6.72g, 52mmol) and trifluoromethanesulfonic anhydride (11.8g, 41.82mmol) were added under ice-cooling and stirred at room temperature for 17 hours. 50mL of saturated sodium bicarbonate solution was added, extracted with dichloromethane (300 mL. times.3), the organic phases were combined, washed with saturated sodium chloride solution (100mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography using eluent system B to give the title compound 3B (9.5g), yield: and (3.9).

Second step of

(S) -4- (4- ((benzyloxy) carbonyl) -3- (cyanomethyl) piperazin-1-yl) -2- (methylthio) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester 3c

Compound 3b (9.5g, 22.12mmol) was dissolved in 80mL acetonitrile and N, N-diisopropylethylamine (5.72g, 44.25mmol) and benzyl (2S) -2- (cyanomethyl) piperazine-1-carboxylate (6.89g, 26.57mmol, prepared using the method disclosed in patent application "example 63" on page 110 of the specification in US2018/0072723a 1) were added separately and refluxed for 17 hours. The reaction solution was concentrated under reduced pressure, 30mL of a saturated sodium bicarbonate solution was added to the residue, extracted with ethyl acetate (50mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography with eluent system B to give the title compound 3c (10.3g), yield: 86.4 percent.

The third step

(S) -4- (4- ((benzyloxy) carbonyl) -3- (cyanomethyl) piperazin-1-yl) -2- (methylsulfonyl) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester 3d

Compound 3c (9.8g, 18.19mmol) was dissolved in 200mL of dichloromethane, m-chloroperoxybenzoic acid (6.28g, 36.38mmol) was added, and the reaction was stirred at room temperature for 2 hours. 50mL of saturated sodium bicarbonate solution was added, extracted with dichloromethane (200 mL. times.3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the title product 3d (10.3g), yield: 99.2 percent.

MS m/z(ESI):571.2[M+1]⁺。

The fourth step

(1-Aminocyclobutyl) methanol 3f

The compound 1-aminocyclobutanecarboxylic acid (1.5g, 13.03mmol, Shaoyuan) was dissolved in tetrahydrofuran (30mL, hengye) at room temperature under argon protection at 0 ℃ with stirring and lithium aluminium hydride (1M tetrahydrofuran solution, 26mL, 25mmol, Annaiji) was added dropwise. After the addition was complete, the reaction was slowly warmed to room temperature and stirred under argon for 16 hours. The reaction was quenched with sodium sulfate decahydrate solid in ice bath, dried over anhydrous sodium sulfate and filtered. The filter cake was rinsed with methylene chloride/methanol (10/1,40 mL. times.8). The combined filtrates were concentrated at room temperature, and the obtained residue was purified by silica gel column chromatography with eluent system A to give the title compound 3f (600mg, yield: 45%).

¹H NMR(400MHz,Acetone-d₆):δ3.74(s,2H),2.86(brs,2H),2.20-2.12(m,2H),1.79-1.67(m,2H),1.23-1.18(m,2H)。

The fifth step

(S) -2- ((1-Aminocyclobutyl) methoxy) -4- (4- ((benzyloxy) carbonyl) -3- (cyanomethyl) piperazin-1-yl) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester 3g

Compound 3d (1g, 1.75mmol) and compound 3f (266mg, 2.63mmol) were dissolved in tetrahydrofuran (20mL, Hengyue) at room temperature, under argon protection and with stirring, lithium hexamethyldisilazide (LiHMDS, 1M in tetrahydrofuran, 3.50mL, 3.5mmol, Annagi) was added slowly dropwise. After the addition was complete, the reaction was allowed to warm to room temperature and stirred under argon for 16 h. To the reaction mixture were added a saturated ammonium chloride solution and a saturated sodium chloride solution, followed by extraction with ethyl acetate (30 mL. times.3). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography with eluent system a to give the title compound 3g (800mg), yield: 77 percent.

MS(ESI):592.8[M+H]⁺。

The sixth step

Benzyl (S) -4- (2- ((1-aminocyclobutyl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -2- (cyanomethyl) piperazine-1-carboxylate 3h

Compound 3g (800mg, 1.35mmol) was dissolved in dichloromethane (16mL, swiss) at room temperature, and hydrochloric acid/1, 4-dioxane (4M, 8mL, Chemart) was added dropwise with stirring at 0 ℃. The reaction was carried out at room temperature for 1 hour. The reaction mixture was concentrated, and the resulting residue was dissolved in water and extracted with dehydrated ether (15 mL. times.2). The resulting extract was discarded, and the aqueous phase was adjusted to pH 8 with saturated aqueous sodium bicarbonate and extracted with ethyl acetate (25 mL. times.3). The combined organic phases were dried over anhydrous sodium sulfate and filtered. Concentrating the filtrate to obtain the titled compoundCompound 3h (600mg) yield 90%. MS (ESI) 492.2[ M + H ]]⁺。

Seventh step

Benzyl (S) -4- (2- ((1-aminocyclobutyl) methoxy) -7- (8-chloro-7-fluoronaphthalen-1-yl) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -2- (cyanomethyl) piperazine-1-carboxylate 3j

The compound 3h (600mg, 1.22mmol), 8-chloro-7-fluoronaphthalen-1-yl-trifluoromethanesulfonate 3i (600mg, 1.83mmol, prepared as disclosed in patent application "US 2019/0270743A1, example intermediate 78", page 76, of the specification, 3 ", 4, 5-bis-diphenylphosphino-9, 9-dimethylxanthene (Xantphos, 141mg, 243.94. mu. mol, obtained by end), cesium carbonate (995mg, 3.05mmol, YAMINGKANGDE), methanesulfonic acid (9, 9-dimethyl-4, 5-bis-diphenylphosphinoanthracene) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II) (Xantphos Pd G3, 116mg, 122.36. mu. mol, Haohong organism) and molecular sieves were used at room temperature

(1.0g, 1.22mmol, Annage) was added to dry toluene (20mL, national drug) and stirred at 110 ℃ for 16 h under argon. The reaction solution was cooled to room temperature and then filtered. The filter cake was washed with methanol (10 mL. times.2). The combined filtrates were concentrated, and the obtained residue was purified by silica gel column chromatography with eluent system A to give the title compound 3j (200mg) in 24% yield.

MS(ESI):670.5[M+H]⁺。

Eighth step

Benzyl (S) -4- (2- ((1- ((tert-butoxycarbonyl) amino) cyclobutyl) methoxy) -7- (8-chloro-7-fluoronaphthalen-1-yl) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -2- (cyanomethyl) piperazine-1-carboxylate 3k

Compound 3j (200mg, 298.43. mu. mol) was dissolved in tetrahydrofuran (6mL, national drug) at room temperature and a solution of di-tert-butyl dicarbonate (195mg, 894.50. mu. mol, Neurospora, Shanghai) and sodium bicarbonate (326mg, 3.88mmol, Runjie) in water (3mL, Drech) was added dropwise at 0 ℃. The reaction solution was stirred for 2 hours at room temperature. Water was added to the reaction solution, followed by extraction with ethyl acetate (20 mL. times.3). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title compound 3k (110mg) in 47% yield.

MS(ESI):770.3[M+H]⁺。

The ninth step

(S) - (1- (((7- (8-chloro-7-fluoronaphthalen-1-yl) -4- (3- (cyanomethyl) piperazin-1-yl) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-2-yl) oxy) methyl) cyclobutyl) carbamic acid tert-butyl ester 3l

Compound 3k (110mg, 142.80 μmol) was dissolved in tetrahydrofuran (5mL, chinese medicine) at room temperature, palladium hydroxide/charcoal (20mg, 20 wt.%) and methanol (0.5mL, hopcalite) were added, and after four hydrogen replacements, stirring was carried out under hydrogen balloon protection at room temperature for 1 hour. The reaction was filtered and the filter cake was rinsed with methanol (5 mL. times.3). The combined filtrates were concentrated to give the title compound 3l (78mg) in 85% yield.

MS(ESI):636.2[M+H]⁺。

The tenth step

(S) - (tert-butyl 1- (((7- (8-chloro-7-fluoronaphthalen-1-yl) -4- (3- (cyanomethyl) -4- (2-fluoropropenoyl) piperazin-1-yl) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-2-yl) oxy) methyl) cyclobutyl) carbamate 3m

2-Fluoroacrylic acid (13mg, 144.36. mu. mol, Kiya) was dissolved in tetrahydrofuran (2mL, Hengyue) at room temperature and N, N-diisopropylethylamine (DIPEA, 24mg, 185.70. mu. mol, Sichuan Weibo technique) and O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 60mg, 157.80. mu. mol, Haima organism) were added sequentially. After the reaction solution was stirred at room temperature for 0.5 hour, 3l (78mg, 122.61. mu. mol) of the compound was added, and the reaction solution was stirred at room temperature for 2 hours. Water was added to the reaction solution, followed by extraction with ethyl acetate (20 mL. times.3). The combined organic phases were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography with eluent system B to give the title compound 3m (56mg) in a yield of 64%.

MS(ESI):708.5[M+H]⁺。

The eleventh step

(S) -2- (4- (2- ((1-aminocyclobutyl) methoxy) -7- (8-chloro-7-fluoronaphthalen-1-yl) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -1- (2-fluoroacryloyl) piperazin-2-yl) acetonitrile hydrochloride 3

Compound 3M (56mg, 79.07. mu. mol) was dissolved in tetrahydrofuran (3mL, pharmacia) at room temperature and hydrochloric acid/1, 4-dioxane (4M, 3mL, Chemart) was added dropwise at 0 ℃. The reaction was carried out at room temperature for 5 hours. The reaction mixture was concentrated, and the resulting residue was suspended in ethyl acetate (8mL), stirred for 2 hours and then filtered. The filter cake was washed with dry ether (3 mL. times.3). The filter cake was collected and lyophilized to give the title compound 3(30.45mg, hydrochloride salt) in 63% yield.

¹H NMR(400MHz,DMSO-d₆):δ8.34(brs,3H),8.04(dd,1H),7.81(dd,1H),7.64-7.51(m,3H),7.47-7.40(m,1H),5.41(dd,1H),5.29(d,1H),4.88(brs,1H),4.44(s,2H),4.21-3.95(m,4H),3.85-3.74(m,1H),3.35-3.28(m,2H),3.17-3.09(m,4H),2.98-2.91(m,1H),2.75-2.56(m,1H),2.33-2.22(m,3H),1.93-1.89(s,2H)。

MS(ESI):608.2[M+H]⁺。

Example 4

(S, E) -2- (4- (7- (7-fluoro-8-methylnaphthalen-1-yl) -2- ((1- (pyrrolidin-1-ylmethyl) cyclopropyl) methoxy) -5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidin-4-yl) -1- (4-fluorobut-2-enoyl) piperazin-2-yl) acetonitrile 4

The title compound 4(85mg) was prepared using the synthetic route of example 1, substituting the starting compound 1n of the thirteenth step with benzyl (2S) -2- (cyanomethyl) piperazine-1-carboxylate (prepared using the method disclosed in patent application "example intermediate 63" on page 110 of the specification, US2018/0072723a 1), substituting acryloyl chloride in the nineteenth step with the compound 4-fluorobut-2-enoic acid (prepared using the well-known method Journal of the American Chemical Society,2009, vol.131(30), 10376-10377), using the general synthetic route of example 1, yield: 36.9 percent.

MS m/z(ESI):656.0[M+1]⁺。

¹H NMR(400MHz,DMSO-d₆)δ7.88-7.83(m,1H),7.76-7.72(m,1H),7.45-7.37(m,3H),6.83-6.78(m,2H),5.20-5.19(m,1H),5.13-5.11(m,1H),5.01-4.38(m,2H),4.10-3.99(m,5H),3.76-3.55(m,2H),3.48-3.44(m,1H),3.28-3.19(m,1H),3.10-3.04(m,4H),2.81-2.79(m,1H),2.80(s,3H),2.77-2.65(m,1H),2.42-2.37(m,6H),1.66-1.63(m,4H),0.5-0.39(m,4H)。

Biological evaluation

Test example 1: biological evaluation of inhibition of H358 cell ERK phosphorylation by compounds of the present disclosure

First, test purpose

This experiment was performed by detecting the inhibitory effect of compounds on cell ERK phosphorylation according to IC₅₀Size the compounds of the disclosure were evaluated for their inhibitory effect on the KRAS target (containing the G12C mutation).

Second, Experimental methods

H358 cells (ATCC, CRL-5807) were cultured in RPMI1640(Hyclone, SH30809.01) complete medium containing 10% fetal bovine serum. The first day of the experiment, H358 cells were seeded in 96-well plates at a density of 25,000 cells/well using complete medium, 190. mu.L cell suspension per well, placed at 37 ℃, 5% CO₂The cell culture box was cultured overnight. The following day, 10. mu.L of test compound diluted in a gradient of complete medium at 9 concentration points with a 6-fold gradient starting at 10. mu.M was added to each well, a blank containing 0.5% DMSO was set, and the well plate was placed at 37 ℃ and 5% CO₂The cell culture chamber of (3) was incubated for 3 hours. After 3 hours, the 96-well cell culture plate was removed, the medium was aspirated off, and 200. mu.L of PBS (Shanghai culture Biotech Co., Ltd., B320) was added to each well and washed once. PBS was aspirated, 50. mu.L of lysis buffer (lysis buffer, Cisbio, 64KL1FDF) containing blocking solution (blocking reagent, Cisbio, 64KB1AAC) was added to each well, and the well plates were placed on a shaker and lysed for 30 minutes at room temperature with shaking. After lysis, the mixture was pipetted and mixed, 16. mu.L of lysate was transferred to two HTRF 96 well assay plates (Cisbio, 66PL96100) per well, and then 4. mu.L of premixed phospho-ERK1/2 antibody solution (Cisbio, 64AERPEG) or 4. mu.L of premixed total-ERK1/2 antibody solution (Cisbio, 64NRKPEG) was added to each plate. The plate was sealed with a sealing membrane, centrifuged for 1 min in a microplate centrifuge and incubated overnight at room temperature in the dark. On the third day, a PHERAstar multifunctional microplate reader (BMG Labtech, S)/N471-0361) read the fluorescence values emitted at wavelengths of 665nm and 620nm with excitation at a wavelength of 337 nm.

Third, data analysis

IC of inhibitory Activity of Compounds was calculated from Compound concentration and pERK/total ERK ratio using Graphpad Prism software₅₀See table 3 below for values, results.

TABLE 3 inhibitory Activity data of compounds of the present disclosure on ERK phosphorylation in H358 cells

Example numbering	IC₅₀(nM)
		1	2.2
2	0.4
		4	3.2
COMPARATIVE EXAMPLE (Compound 3)	1862.0

And (4) conclusion: the compound disclosed by the invention has a good inhibition effect on ERK phosphorylation of H358 cells.

Test example 2: biological evaluation of H358 cell proliferation inhibition by the disclosed compounds

First, test purpose

The inhibition of KRAS target (containing the G12C mutation) by the compounds of the disclosure was evaluated by testing the proliferation inhibition of H358 cells by the compounds of the disclosure.

Second, Experimental methods

H358 cells (ATCC, CRL-5807) were cultured in complete medium, RPMI1640 medium (Hyclone, SH30809.01) containing 10% fetal bovine serum (Corning, 35-076-CV). The first day of the experiment, H358 cells were seeded in 96-well plates at a density of 1500 cells/well using complete medium, 100. mu.L of cell suspension per well, placed at 37 ℃, 5% CO₂The cell culture box was cultured overnight. The following day, 10. mu.L of test compound diluted in a gradient of complete medium at 9 concentration points with 5-fold gradient starting from 10. mu.M was added to each well, a blank containing 0.5% DMSO was set, and the well plate was placed at 37 ℃ and 5% CO₂The cell culture chamber of (2) was cultured for 120 hours. On the seventh day, 96 well cell culture plates were removed and 50. mu.L of each well was added

The luminescence signal value was read by a multi-functional microplate reader (Perkinelmer, VICTOR 3) after the luminescence Cell visual Assay (Promega, G7573) was left at room temperature for 10 minutes.

Third, data analysis

IC of compound inhibitory activity was calculated using Graphpad Prism software₅₀See table 4 below for values, results.

Table 4 inhibitory Activity of compounds of the present disclosure on H358 cell proliferation

Example numbering	IC₅₀(nM)
		1	10.6
2	1.5
		4	10.9
COMPARATIVE EXAMPLE (Compound 3)	204.9

And (4) conclusion: the compound disclosed by the invention has a good inhibition effect on H358 cell proliferation.

Test example 3: experimental biological evaluation of inhibition of ERK phosphorylation in MIA PaCa-2 cells

First, test purpose

This experiment was performed by examining the inhibitory effect of compounds on the phosphorylation of MIAPaCa-2 cell ERK according to IC₅₀Size the compounds of the disclosure were evaluated for their inhibitory effect on the KRAS target (containing the G12C mutation).

Second, Experimental methods

MIA PaCa-2 cells (ATCC, CRL-1420) were cultured in DMEM/HIGH GLUCOSE (GE, SH30243.01) complete medium containing 10% fetal bovine serum and 2.5% horse serum. The first day of experiment, MIA PaCa-2 cells were seeded at a density of 25,000 cells/well in 96-well plates using complete medium, 190. mu.L of cell suspension per well, placed at 37 ℃ with 5% CO₂The cell culture box was cultured overnight. The following day, 10. mu.L of test compound diluted in a gradient of complete medium at 9 concentration points with a 6-fold gradient starting at 10. mu.M was added to each well, a blank containing 0.5% DMSO was set, and the well plate was placed at 37 ℃ and 5% CO₂The cell culture chamber of (2) was incubated for 4 hours. After 4 hours, the 96-well cell culture plate was removed, the medium was aspirated off, and 200. mu.L of PBS (Shanghai culture Biotech Co., Ltd., B320) was added to each well and washed once. PBS was aspirated, 50. mu.L of lysis buffer (lysis buffer, Cisbio, 64KL1FDF) containing blocking solution (blocking reagent, Cisbio, 64KB1AAC) was added to each well, and the well plates were placed on a shaker and lysed for 30 minutes at room temperature with shaking. After lysis, the mixture was pipetted and mixed, 16. mu.L of lysate was transferred to two HTRF 96-well assay plates (Cisbio, 66PL96100) per well, and thenBoth plates were loaded with 4. mu.L of premixed phospho-ERK1/2 antibody solution (Cisbio, 64AERPEG) or 4. mu.L of premixed total-ERK1/2 antibody solution (Cisbio, 64NRKPEG), respectively. The plate was sealed with a sealing membrane, centrifuged for 1 min in a microplate centrifuge and incubated overnight at room temperature in the dark. On the third day, the fluorescence values emitted at wavelengths of 665nm and 620nm after excitation at 337nm were read using a PHERAstar multifunctional microplate reader (BMG Labtech, S/N471-.

Third, data analysis

IC of inhibitory Activity of Compounds was calculated from Compound concentration and pERK/Total ERK ratio using Graphpad Prism software₅₀See table 5 below for values, results.

TABLE 5 IC inhibition of cellular ERK phosphorylation by compounds of this disclosure₅₀The value is obtained.

Example numbering	IC₅₀(nM)
		1	5.2
2	0.9
		4	5.2
COMPARATIVE EXAMPLE (Compound 3)	569.2

And (4) conclusion: the compound disclosed by the invention has a good inhibition effect on the ERK phosphorylation of MIA PaCa-2 cells.

Test example 4: biological evaluation of MIA PaCa-2 cell proliferation experiments

First, test purpose

The inhibitory effect of the compounds of the present disclosure on the KRAS target (containing the G12C mutation) was evaluated by testing the inhibitory effect of the compounds of the present disclosure on the proliferation of MIAPaCa-cells.

Second, Experimental methods

MIA PaCa-2 cells (ATCC, CRL-1420) were cultured in complete medium, i.e., DMEM/HIGH GLUCOSE (GE, SH30243.01) complete medium containing 10% fetal bovine serum and 2.5% horse serum. The first day of experiment, MIA PaCa-2 cells were seeded at a density of 500 cells/well in 96-well plates using complete medium, 90. mu.L of cell suspension per well, placed at 37 ℃ with 5% CO₂The cell culture box was cultured overnight. The following day, 10. mu.L of test compound diluted in a gradient of complete medium at 9 concentration points with 5-fold gradient starting from 10. mu.M was added to each well, a blank containing 0.5% DMSO was set, and the well plate was placed at 37 ℃ and 5% CO₂The cell culture chamber of (2) was cultured for 72 hours. On the fifth day, 96-well cell culture plates were removed and 50. mu.L of each well was added

Luminescence Cell Viability Assay (reagent for detecting Luminescent Cell activity) (Promega, G7573) was left at room temperature for 10 minutes, and then the luminescence signal value was read using a multi-functional microplate reader (PerkinElmer, VICTOR 3).

Third, data analysis

IC of compound inhibitory activity was calculated using Graphpad Prism software₅₀See table 6 below for values, results.

TABLE 6 IC inhibition of MIA PaCa-2 cell proliferation by compounds of the present disclosure₅₀The value is obtained.

Example numbering	IC₅₀(nM)
		1	5.1
2	0.9
		4	10.4

And (4) conclusion: the compound disclosed by the invention has a good inhibition effect on MIA PaCa-2 cell proliferation.

As mentioned belowComparative Compound AThe structure is as follows:

(Compound A see WO2019099524A1 claim 55, 7 th Compound)

(Compound B see WO2020238791A1, example 8 Compound)

Test example 5 inhibition of enzymatic Activity of human liver microsomal CYP3A4 Testosterone metabolism site by Compounds of the disclosure

The enzymatic activity of the disclosed compound on the metabolic site of human liver microsome CYP3A4 testosterone is determined by adopting the following experimental method:

first, experimental material and instrument

1. Phosphate buffer (20 XPBS, purchased from Biotech),

2.NADPH(ACROS，A2646-71-1),

3. human liver microsomes (Corning Gentest, Cat No, 452161, Lot No.905002, Donor35),

ABI QTrap 4000 liquid dual-purpose instrument (AB Sciex),

ZORBAX extended-C18, 3X 50mm,3.5 μm (Agilent, USA),

CYP probe substrate (testosterone, Vocko, CAS No. [58-22-0]/75 μ M), and positive control inhibitor (ketoconazole, SIGMA, Cat No. K1003-100 MG).

Second, the experimental procedure

100mM PBS buffer was prepared, and 7.5mM MgCl was prepared using the buffer₂And 5mM NADPH solution, followed by 7.5mM MgCl₂A0.25 mg/mL microsome solution was prepared, and a 30mM stock solution was diluted with DMSO to a 30mM, 10mM, 3mM, 1mM, 0.3mM, 0.03mM, 0.003mM, 0mM series of solutions I, which were then diluted 200-fold with Phosphate Buffered Saline (PBS) to give a series of test solutions II (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M). Testosterone working solution diluted to a concentration of 375 μ M with PBS.

Prepared in 7.5mM MgCl₂40. mu.L of the microsome solution (0.25 mg/mL) was added, and then 20. mu.L of each of a testosterone working solution and a compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, and 0. mu.M) was added thereto at 375. mu.M and mixed uniformly. The positive control group replaced the compound with ketoconazole at the same concentration. Simultaneously 5mM NADPH solution at 37 ℃ pre-incubation for 5 minutes. After 5 min 20. mu.L NADPH was added to each well, the reaction was started and incubated for 30 min. After 30 minutes 250. mu.L of acetonitrile containing the internal standard was added to all samples, mixed well, shaken at 800rpm for 10 minutes, and then centrifuged at 3700rpm for 10 minutes. mu.L of the supernatant was mixed with 80. mu.L of ultrapure water and transferred to LC-MS/MS for analysis.

The value is calculated by Graphpad Prism to obtain the IC of the drug on the metabolic site of CYP3A4 testosterone₅₀The values are shown in Table 7.

TABLE 7 IC of testosterone metabolic site of the disclosed compounds on human liver microsomal CYP3A4₅₀The value is obtained.

Example numbering	IC₅₀(μM)
		2	9.3
Comparative Compound A	2.7

And (4) conclusion: the disclosed compound has weak inhibition on testosterone metabolic sites of human liver microsome CYP3A4, and shows better safety.

Test example 6 inhibition of the enzymatic Activity of human liver microsome CYP2C9 diclofenac metabolic site by Compounds of the disclosure

The enzymatic activity of the disclosed compound on the diclofenac metabolic site of human liver microsome CYP2C9 is determined by adopting the following experimental method:

first, experimental material and instrument

1. Phosphate buffer (20 XPBS, purchased from Biotech),

2.NADPH(ACROS，A2646-71-1),

3. human liver microsomes (Corning Gentest, Cat No, 452161, Lot No.9050002, Donor,35)

ABI QTrap 4000 liquid dual-purpose instrument (AB Sciex),

ZORBAX extended-C18, 3X 50mm,3.5 μm (Agilent, USA),

CYP probe substrate (diclofenac, SIGMA, Cat No. D6899-10G/4. mu.M) and positive control inhibitor (sulfaphenazole, SIGMA, Cat No. 526-08-9).

Second, the experimental procedure

100mM PBS buffer was prepared, and 7.5mM MgCl was prepared using this buffer₂And 5mM NADPH solution, followed by 7.5mM MgCl₂A0.25 mg/mL microsome solution was prepared, and a 30mM stock solution was diluted with DMSO to a 30mM, 10mM, 3mM, 1mM, 0.3mM, 0.03mM, 0.003mM, 0mM series of solutions I, which were then diluted 200-fold with Phosphate Buffered Saline (PBS) to give a series of test solutions II (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M). Diclofenac working solution diluted to a concentration of 20. mu.M with PBS.

Respectively taking and matchingPrepared at 7.5mM MgCl₂In (4), 40. mu.L of the microsome solution (0.25 mg/mL) was added, and then 20. mu.L of each of 15. mu.M of midazolam working solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M) was mixed well. The positive control group replaced the compound with the same concentration of sulfaphenazole. Simultaneously 5mM NADPH solution at 37 ℃ pre-incubation for 5 minutes. After 5 min 20. mu.L NADPH was added to each well, the reaction was started and incubated for 30 min. All incubated samples were set up in duplicate. After 30 minutes 250. mu.L of acetonitrile containing the internal standard was added to all samples, mixed well, shaken at 800rpm for 10 minutes, and then centrifuged at 3700rpm for 10 minutes. mu.L of the supernatant was mixed with 80. mu.L of ultrapure water and transferred to LC-MS/MS for analysis.

The value is calculated by Graphpad Prism to obtain the IC of the drug on the site of metabolism of CYP2C9 diclofenac acid₅₀The values are shown in Table 8.

TABLE 8 IC of the compounds disclosed herein on the site of diclofenac metabolism by human liver microsomes CYP2C9₅₀The value is obtained.

Example numbering	IC₅₀(μM)
		2	9.0
Comparative Compound A	4.2

And (4) conclusion: the disclosed compound has weak inhibition effect on the human liver microsome CYP2C9 diclofenac metabolic site, shows better safety, and prompts that the metabolic drug interaction based on the CYP2C9 diclofenac metabolic site does not occur.

Test example 7 inhibition of enzymatic Activity of human liver microsome CYP2C19(S) -Mefenton site of metabolism by Compounds of the present disclosure

The enzymatic activity of the disclosed compound on human liver microsome CYP2C19(S) -mephenytoin metabolic site is determined by adopting the following experimental method:

first, experimental material and instrument

1. Phosphate buffer (20 XPBS, purchased from Biotech),

2.NADPH(ACROS，A2646-71-1),

ABI QTrap 4000 liquid dual-purpose instrument (AB Sciex),

ZORBAX extended-C18, 3X 50mm,3.5 μm (Agilent, USA),

CYP probe substrate ((S) -Memfungin/20. mu.M, powder from carbofuran technologies, Inc., Cat No.303768) and positive control inhibitor (ticlopidine, powder from SIGMA, Cat No. T6654-1G).

Second, the experimental procedure

100mM PBS buffer was prepared, and 7.5mM MgCl was prepared using this buffer₂And 5mM NADPH solution, followed by 7.5mM MgCl₂A0.25 mg/mL microsome solution was prepared, and a 30mM stock solution was diluted with DMSO to a 30mM, 10mM, 3mM, 1mM, 0.3mM, 0.03mM, 0.003mM, 0mM series of solutions I, which were then diluted 200-fold with Phosphate Buffered Saline (PBS) to give a series of test solutions II (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M). (S) -Mefenton-working solution diluted to a concentration of 100. mu.M with PBS.

Separately prepared in 7.5mM MgCl₂In (4), 40. mu.L of the microsome solution (0.25 mg/mL) was added, and then 20. mu.L of each of 15. mu.M of midazolam working solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. mu.M) was mixed well. The positive control group replaced the compound with ticlopidine at the same concentration. Simultaneously 5mM NADPH solution at 37 ℃ pre-incubation for 5 minutes. After 5 min 20. mu.L NADPH was added to each well, the reaction was started and incubated for 30 min. All incubated samples were set up in duplicate. After 30 minutes, 250. mu.L of a reagent containing sodium chloride was added to all the samplesAcetonitrile as an internal standard, mixed well, shaken at 800rpm for 10 minutes, and then centrifuged at 3700rpm for 10 minutes. mu.L of the supernatant was mixed with 80. mu.L of ultrapure water and transferred to LC-MS/MS for analysis.

The value is calculated by Graphpad Prism to obtain the IC of the drug on CYP2C19(S) -mephenytoin metabolic site₅₀The values are shown in Table 9.

TABLE 9 IC of the compounds of this disclosure on the human liver microsomal CYP2C19(S) -mefenton metabolic site₅₀The value is obtained.

Example numbering	IC₅₀(μM)
		2	15.8
Comparative Compound A	10.3

And (4) conclusion: the disclosed compound has weak inhibition effect on human liver microsome CYP2C19(S) -mephenytoin metabolic site, shows better safety, and suggests that metabolic drug interaction based on CYP2C19(S) -mephenytoin metabolic site does not occur.

Test example 8 in vitro plasma stability assay of Compounds of the disclosure

1 purpose

A test method for researching the stability of the in vitro plasma is established by adopting various types of plasma which is frozen or freshly prepared, and the stability of the in vitro plasma of the compound is evaluated.

2 instruments, devices and materials

2.1 instrumentation

1) Eppendorf 5804R refrigerated centrifuge, Eppendorf Co, Germany

2) Thermomixer 5355, Eppendorf, Germany

3) Deepwell plate 96/1000 μ l, Thermo Co., USA

4) API 4000Q-trap linear ion trap mass spectrometer, Applied Biosystems, USA

5) LC-30A ultra high pressure liquid chromatography System, Shimadzu corporation, Japan

2.2 materials

1) Various genera cryopreserved plasma (heparin sodium anticoagulation)

2) Freezing or fresh preparation of human plasma (heparin sodium anticoagulation)

3 procedure of operation

3.1 stock solution preparation

The compounds were weighed out separately and made up in 30mM stock in DMSO.

3.2 dilution of working solution

Stock solutions at 30mM were diluted to 1600. mu.M solution I in DMSO and 1600. mu.M solution I was diluted to 16. mu.M working solution II in 50% methanol.

3.3 sample incubation

The experiment was set up at 0, 30, 60, 90, 120, 240, 6 time points, two replicates were set up at each time point, 469. mu.L of plasma or whole blood, 31. mu.L of the above prepared working solution II at 16. mu.M concentration, giving a final concentration of 1. mu.M compound. Incubate at 37 ℃ and start timing (0 min first with 300. mu.L acetonitrile containing internal standard and then working solution II). At the other time points, the reaction was stopped with 300. mu.L of acetonitrile containing the internal standard. Then, the mixture was shaken at 850rpm on a shaker for 5 minutes, centrifuged at 3700rpm on a centrifuge for 15 minutes, 100. mu.l of the supernatant was added to 100. mu.l of deionized water, and the mixture was shaken at 450rpm on a shaker for 5 minutes before LC-MSMS analysis.

4. Data analysis

The compounds were compared for stability with respect to the percentage remaining of prototype Area ratio at 0 minutes at each time point.

％Remain of Time zero＝Area ratio _{time point}/Area ratio_{time zero％}

Taking the natural logarithm Ln of the percentage, and taking the incubation Time as the abscissa and the corresponding Ln value (Ln% remaining of Time zero) as the ordinate to perform linear regression to obtain the coefficient k value of the linear regression equation.

The metabolic half-life T in vitro plasma was calculated using the following formula_1/2

T_1/20.693/k (k is linear regression equation coefficient)

TABLE 10 in vitro plasma stability of the compounds of this disclosure

TABLE 11 in vitro plasma stability of comparative Compound B

And (4) conclusion: the compound disclosed by the invention has higher stability in vitro plasma.

Claims

1. A compound, or a tautomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, selected from any one of the following compounds:

2. a compound, or a tautomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, selected from any one of the following compounds:

3. a pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1, or a tautomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

4. Use of a compound according to claim 1 or a stereoisomer, a tautomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 3, for the manufacture of a medicament for the inhibition of KRAS, preferably for the manufacture of a medicament for the inhibition of KRAS G12C.

5. Use of a compound according to claim 1 or a stereoisomer, a tautomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 3, for the preparation of a medicament for the treatment or prevention of cancer, inflammation, or other proliferative disease, preferably for the preparation of a medicament for the treatment or prevention of cancer; the cancer is selected from gastric cancer, esophageal cancer, melanoma, liver cancer, kidney cancer, lung cancer, nasopharyngeal cancer, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, head and neck squamous cell carcinoma, endometrial cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma, and glioma.