CN113980014B - Hydrogenated pyridopyrimidine derivative, preparation method and medical application thereof - Google Patents

Abstract

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

Description

Hydrogenated pyridopyrimidine derivative, preparation method and medical application thereof

Technical Field

The present disclosure relates to a hydrogenated pyridopyrimidine derivative, a method for preparing the same, a pharmaceutical composition containing the same, and a use thereof as a therapeutic agent, particularly as a KRAS inhibitor, and a pharmaceutical composition containing the same.

Background

The RAS (Rat Sarcoma Viral Oncogene Homolog) family belongs to the superfamily of small gtpases and is widely expressed in various eukaryotes. There are three RAS genes (HRAS, KARS and NARS) in humans that can be expressed as four highly related RAS small gtpases (HRAS, KRAS4A, KARS B and NRAS). It acts as a binary switch for GDP-GTP regulation. They generally take two forms: a GDP (guanosine diphosphate) bound form in the inactive state and a GTP (guanosine triphosphate) bound form in the active 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,2007,7,295-308). The RAS gene has higher mutation rate in various tumors such as pancreatic cancer, colorectal cancer, non-small cell lung cancer and the like, and activated mutant RAS protein can promote abnormal signal transduction, so that the occurrence and development of cancer and drug resistance to targeted drugs are caused. Wherein the KRAS mutation is the highest mutation rate gene in human oncogenes, accounting for 20-30% of all tumors.

For mutant forms of KRAS proteins and signal pathway studies, significant advances in molecular biology have been made in recent years, however, the development of related targeted drugs remains a challenge. In chemical development, since the affinity of KRAS and GTP is very high, reaching 60pM, and the intracellular GTP concentration is at the level of mM, such directly competing molecules have extremely high affinity requirements for compounds, and so far there has been no successful case. In terms of development of biological drugs, antibody drugs penetrate cell membrane targeting KRAS proteins, and drug delivery efficiency is low. Therefore, many researchers have tried to develop a new way to inhibit the activities of RAF, MEK, ERK and other kinases in the KRAS downstream signal channel, so as to achieve the purpose of inhibiting the KRAS channel. The compounds have certain curative effects, but the downstream inhibitors can not completely block KRAS signals, and the target related toxic and side effects are large, so that the compounds have poor medicinal effects on KRAS mutant tumors. Therefore, the KRAS inhibitor for developing a new action mechanism has great clinical application value.

KRAS mutations are predominantly point mutations, including mutations at amino acids 12, 13 and 61. Of these, glycine at position 12 is most commonly mutated to cysteine (G12C), which is a relatively large proportion (14%) in lung cancer, especially non-small cell lung cancer; in addition, expression was also in some colorectal (4%) and pancreatic (2%) patients. In the U.S. cancer population, the incidence of this gene mutation is even greater than the sum of ALK, RET, TRK gene mutations.

Faced with the difficulty of KRAS protein drug formulation, the professor Kevan Shokat, san Francisco, california university, first verified that certain specific compounds bind KRAS G12C muteins via covalent bonds. Through further studies, these covalent compounds were found to bind to cysteine at position 12 of KRAS muteins and occupy a hydrophobic allosteric regulatory pocket in the molecular switch-II region (switch-II regions), and the bound KRAS G12C mutations could be irreversibly locked in an inactivated state, blocking signaling pathways and cancer cell viability dependent on the protein (Nature 2013, 503, 548-551). The KRAS G12C small molecule inhibitor ARS-1620 can effectively inhibit tumor growth and even completely regress tumors on various KRAS G12C mutant tumor models. Since KRAS G12C is a mutein in tumor cells, whereas wild-type KRAS does not have this mutation site, a perfect tumor-selective target is provided (Cell, 2018, 572, 578-589). Several KRAS G12C inhibitors have been issued by companies typified by Araxas, amgen and Mirati (WO 2014152588, WO2016164675, WO2017087528, WO2017201161, WO2018119183, etc.). At present, no inhibitor drugs of KRAS G12C are approved for marketing, and the fastest-growing small molecule KRAS G12C inhibitors of Amgen and Mirati enter clinical primary trials in 9 and 12 months of 2018, respectively, thus there is a significant unmet medical need in the relevant patient population.

Disclosure of Invention

It is an object of the present disclosure to provide a plurality of compounds, or tautomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, which are in the structure of table 1 below:

table 1 Structure and nomenclature of Compounds

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Or a tautomer, racemate, enantiomer, diastereomer, or 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

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

Another aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound shown in table 1 of the present disclosure, or a tautomer, racemate, enantiomer, diastereomer, 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, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, in the manufacture of a medicament for inhibiting KRAS, preferably KRAS G12C.

The present disclosure further relates to the use 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, in the manufacture of a medicament for treating or preventing cancer, inflammation, or other proliferative disease, preferably for treating or preventing cancer; the cancer is selected from stomach cancer, esophageal cancer, 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. Preferably, the cancer is selected from melanoma, liver cancer, kidney 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.

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 a 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 a tautomer, racemate, enantiomer, diastereomer, 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 a method of 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, kidney 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 (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.

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 a tautomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use as KRAS inhibitors, preferably KRAS G12C inhibitors.

The 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 the treatment or prevention of KRAS-mediated diseases, preferably KRAS G12C-mediated diseases.

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 the treatment or prevention of cancer, inflammation, or other proliferative diseases, preferably cancer; wherein the cancer is selected from the group consisting of gastric cancer, esophageal cancer, melanoma, liver cancer, kidney 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 (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.

The active compounds can be formulated in a form suitable for administration by any suitable route, using one or more pharmaceutically acceptable carriers by conventional methods to formulate the compositions of the present disclosure. Accordingly, 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) administration, 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, troches or syrups.

The dosage of the compound or composition used in the disclosed methods of treatment 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 guideline, the active compounds are preferably administered in unit doses, or in a manner whereby the patient can self-administer a single dose. The unit dosage of a compound or composition of the present disclosure may be expressed in the form of a tablet, capsule, cachet, bottled lotion, powder, granule, lozenge, suppository, reconstituted powder or liquid formulation. Suitable unit doses may be in the range 0.1 to 1000mg.

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

The 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. The above-described sweeteners 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 an oil-in-water emulsion. The oil phase may be a vegetable oil, or a mineral oil or a mixture thereof. Suitable emulsifiers may be naturally occurring phospholipids, and 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 sterile injectable aqueous solutions. Acceptable vehicles or solvents that may be used 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, which is prepared by injecting a liquid or microemulsion into the blood stream of a patient by topical mass injection. Alternatively, it may be desirable to administer the solutions and microemulsions in a manner that maintains a constant circulating concentration of the compounds of the present disclosure. To maintain this constant concentration, a continuous intravenous delivery device may be used. An example of such a device is a Deltec CADD-PLUS. TM.5400 model intravenous pump.

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 suspensions may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents as described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally-acceptable, nontoxic diluent or solvent. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any blend fixed oil may be used. In addition, fatty acids can also be used to prepare 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 will therefore melt in the rectum to release the drug.

The compounds of the present disclosure may be administered by adding water to prepare water-suspended dispersible powders and granules. These pharmaceutical compositions may be prepared by mixing the active ingredient with a dispersing or wetting agent, suspending agent or one or more preservatives.

As is well known to those skilled in the art, the amount of drug administered depends on a variety of factors, including, but not limited to, the following: the activity of the specific compound used, the age of the patient, the weight of the patient, the health 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, etc.; in addition, the optimal mode of treatment, such as the mode of treatment, the daily amount of the compound, or the 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 include isotopic derivatives thereof. The term "isotopically-enriched 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 used in place of hydrogen, or ¹⁸ F-fluorine labeling [ ] ¹⁸ F isotope) instead of fluorine, or with ¹¹ C-， ¹³ C-, or ¹⁴ C-enriched carbon ¹¹ C-， ¹³ C-, or ¹⁴ C-carbon labeling; ¹¹ C-， ¹³ c-, or ¹⁴ C-isotopes) are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays, or as diagnostic imaging tracers in vivo for diseases, or as tracers for pharmacodynamic, pharmacokinetic or receptor studies. The present 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 with a deuterium atom. Those skilled in the art are able to refer to the relevant literature for the synthesis of deuterated forms of the compounds of formula (I). Commercially available deuterated starting materials may be used in preparing the deuterated form of the compound of formula (I) or they may be synthesized using conventional techniques with deuterated reagents including, but not limited to, deuterated boranes, trideuteroborane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like. Deuterated compounds generally retain activity comparable to non-deuterated compounds and may achieve better metabolic stability when deuterated at certain specific sites, thus achieving certain therapeutic advantages.

"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

By "pharmaceutically acceptable salts" is meant salts of the compounds of the present disclosure which are safe and effective when used in a mammal, and which possess the desired biological activity. Salts may be prepared separately during the final isolation and purification of the compounds, or by reacting the appropriate groups 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 and 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 intended effect. 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, a suitable effective amount in an individual case can be determined by one skilled in the art according to routine experimentation.

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 are effective for the intended use.

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

Boc represents t-butoxycarbonyl;

cbz represents a benzyloxycarbonyl group.

Detailed Description

The present disclosure is further described below in connection with the examples, which are not intended to limit the scope of the present disclosure.

Examples

The structural determination of the compounds employs Nuclear Magnetic Resonance (NMR) and/or Mass Spectrometry (MS). NMR chemical shift (delta) of 10 ^-6 (ppm) is given in units. Determination by NMR Using Bruker AVANCE II-400MHz Nuclear magnetic resonance spectrometer, the conventional deuterated solvent used was deuterated dimethyl sulfoxide (DMSO-d ₆ ) Deuterated chloroform (CDCl) ₃ ) And deuterated Methanol (Methanol-d) ₄ ) Tetramethylsilane (TMS) was used as an internal standard.

Mass Spectrometry (MS) was performed using a liquid chromatograph-mass spectrometer (LC-MS), ionization source: electrospray ionization (ESI). The manufacturers are respectively: shimadzu, waters and Agilent, model is respectively: LCMS2020, UPLC-QDa and Agilent 6120, chromatographic columns are Sunfire C18 μm 50×4.6mm, ACQUITY, respectively

BEH 2.1×50mm 1.7 μm and Xbridge C18 μm 50×4.6mm.

HPLC was performed using an Agilent 1200DAD high performance liquid chromatograph (column: waters SunFire C18 (150. Times.4.6 mm,5 μm)) and a Shimadzu UFLC high performance liquid chromatograph (column: waters XB ridge C18 (150. Times.4.6 mm,5 μm)).

Analysis and determination by chiral HPLC used Waters-UPC2.

The thin layer chromatography silica gel plate is produced by Sonchus, xinno or Shandong lactan silica gel plate works, and the thickness of silica gel plate used by Thin Layer Chromatography (TLC) is 0.2-0.25mm; specification 50X 200mm. The thickness of silica gel plate used for thin layer preparation chromatography (prep-TLC) is 0.4-0.5mm; specification 200X 200mm.

Column chromatography silica gel is generally used as silica gel produced by chemical industry (Shanghai) limited, and has a specification of 100 to 200 mesh or 200 to 300 mesh.

High performance liquid chromatography was performed using a Waters 2767 (column: sunfire Pre C18. Mu.m 19X 250 mm) and a Waters 2767-QDa (column: xbridge Pre C18. Mu.m 19X 250 mm) preparative chromatograph.

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

CombiFlash rapid prep machine using MP200 medium pressure rapid purification prep system (Agela Technologies).

If the starting materials in the synthesis record are commercially available compounds, it is necessary to identify the source of clarity: such as the companies ABCR GmbH & Co.KG, acros Organics, aldrich Chemical Company, shaoshao far chemical technology (Accela ChemBio Inc), darui chemical, etc.

If the reaction is required to be carried out under argon atmosphere or nitrogen atmosphere, specific conditions are required to be marked clearly;

the pressurized hydrogenation reaction uses GSH-1/12.5 type, GSH-2/12.5 type, GSH-5/12.5 type and GSH-20/12.5 type autoclaves.

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

Monitoring the reaction progress in the synthesis record, if Thin Layer Chromatography (TLC) is adopted, and if thin layer preparative chromatography (prep-TLC) is adopted for purification, the solvent and the exact proportion used by the developing agent system are respectively marked clearly; the addition of small amounts of alkaline or acidic reagents such as triethylamine, ammonia, acetic acid, etc. is also clearly indicated.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), the developing reagent used for the reaction, the system of eluent for column chromatography employed for purifying the compound and the developing reagent system of thin layer chromatography included: a: dichloromethane/methanol system, B: petroleum ether/ethyl acetate system, C: in the n-hexane/ethyl acetate system, the volume ratio of the solvent is regulated according to the polarity of the compound, and small amounts of alkaline or acidic reagents such as triethylamine, acetic acid and the like can be added for regulation.

Example 1

2- ((2 s,6 r) -1-propenoyl-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

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

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

Second step

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

Compound 1b (1.58 g,9.71 mmol) was dissolved in 500mL tetrahydrofuran and pyrrolidine (3.45 g,48.48mmol, an Naiji) was added at 0deg.C. The reaction solution 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 (1.64 g), which was then used in the next reaction without purification.

¹ H NMR(400MHz,CDCl ₃ )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)。

Third step

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

Compound 1c (1.0 g,5.07 mmol) was dissolved in 100mL of tetrahydrofuran under argon, lithium aluminum tetrahydrofuran solution (1M, 10mL,10mmol, an Naiji) was added at 0deg.C, and the reaction solution was stirred at 0deg.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 (624 mg), yield: 79%.

¹ H NMR(400MHz,CDCl ₃ )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)。

Fourth step

(R) - (1- ((4-nitrophenyl) sulfonamide) -1-oxopropan-2-yl) carbamic acid tert-butyl ester 1f

(R) -2- ((tert-Butoxycarbonyl) amino) propionic acid 1e (15.0 g,79.28mmol, shao) 2-nitrobenzenesulfonamide (14.43 g,71.35mmol, shao), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 22.71g,118.90mmol, tetany) and 4-dimethylaminopyridine (DMAP, 11.62g,95.13mmol, medium) were dissolved in N, N-dimethylformamide (500 mL, medium), and the reaction was stirred at room temperature for 16 hours. After addition of water (100 mL), 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 (26.4 g), yield: 89%.

MS(ESI):372.1[M-H] ^- 。

Fifth step

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

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

MS(ESI):358.0[M-H] ^- 。

Sixth step

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

1h

1g (3.35 g,9.32 mmol) of the compound, ethyl 4-bromocrotonate (2.16 g,11.19mmol, shao) and potassium carbonate (3.86 g,27.96mmol, run) were dispersed in N, N-dimethylformamide (20 mL, run), and the reaction mixture was stirred at room temperature under argon for 24 hours. The resulting mixture was added with water (30 mL) and extracted with ethyl acetate (50 mL. Times.2). The combined organic phases were washed with water (50 ml×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 rapid prep. with eluent system B to give the title product 1h (3.6 g), yield: 81%.

MS(ESI):372.1[M+H] ⁺ 。

Seventh step

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

Compound 1h (3.6 g,7.63 mmol) was dissolved in dichloromethane (25 mL) at room temperature, trifluoroacetic acid (10 mL, tatam) was added at room temperature and stirred for 2 h. The reaction solution was concentrated to obtain a crude intermediate. Toluene (50 mL) was added to the residue, which was then concentrated, and toluene (50 mL) was added again to the residue, which was then concentrated. The crude intermediate was dissolved in dichloromethane (25 mL), and saturated aqueous sodium bicarbonate (10 mL) was added in an ice bath and stirred at room temperature for 1 hour. The reaction mixture was extracted with methylene chloride (50 mL. Times.2). The combined organic phases were washed with water (50 ml×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 a CombiFlash flash rapid prep. with eluent system B to give the title product 1i (1.1 g) in 38% yield.

MS(ESI):372.2[M+H] ⁺ 。

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

Compound 1i (1.0 g,2.69 mmol), benzyl chloroformate (915.38 mmol, taitan) and sodium bicarbonate (679 mg,8.08mmol, run) were dissolved in tetrahydrofuran/water (8 mL/2mL, chinese medicine/dropen), and the reaction was stirred at room temperature under argon for 16 hours. The resulting mixture was extracted with dichloromethane (20 mL. Times.2) after addition of water. 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 a CombiFlash flash rapid prep machine with eluent system B to give the title product 1j (926 mg). Yield: 68%.

MS(ESI):506.1[M+H] ⁺ 。

Ninth step

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

1g (1.5 g,2.97 mmol) of the compound was dissolved in tetrahydrofuran/water (10 mL/10mL, guozhen/Chen), lithium hydroxide (243 mg,5.93mmol, run) was added, and the reaction solution 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 <7 with dilute hydrochloric acid (1M) and extracted with ethyl acetate (20 mL x 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 1k (1.4 g). Yield: 98%.

MS(ESI):478.1[M+H] ⁺ 。

Tenth step

(2S, 6R) -2- (2-amino-2-oxoethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylic acid benzyl ester 1l

Compound 1k (1.4 g,2.93 mmol), ammonium chloride (313 mg,5.86mmol, run), O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 1.67g,4.40mmol, tatanium) and N, N-diisopropylethylamine (DIPEA, 1.14g,8.80mmol, tatanium) were dissolved in N, N-dimethylformamide (10 mL, run) and the reaction stirred at room temperature for 1 hour. The resulting mixture was extracted with ethyl acetate (20 mL. Times.2) after adding water. 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 with a CombiFlash flash rapid prep machine using eluent system a to give the title product 1l (1.3 g), yield: 93%.

MS(ESI):477.1[M+H] ⁺ 。

Eleventh step

(2S, 6R) -2- (cyanomethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylic acid benzyl ester 1m

1l (1.3 g,2.73 mmol) of the compound and triethylamine (1.38 g,13.64mmol, tatam) were dissolved in methylene chloride (20 mL, run) at 0℃and trifluoroacetic anhydride (4.58 g,21.83mmol, tatam) was added to the reaction solution with stirring. The reaction solution was stirred at 0℃for 2 hours. To the reaction mixture was added a saturated sodium hydrogencarbonate 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 a CombiFlash flash rapid prep machine with eluent system B to give the title product 1m (1.0 g). Yield: 79%.

MS(ESI):459.1[M+H] ⁺ 。

Twelfth step

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

Compound 1m (1.0 g,2.18 mmol) and cesium carbonate (1.42 g,4.36mmol, MINGKANG) were dispersed in tetrahydrofuran (10 mL, country) and 2-mercaptoethanol (510 mg,6.54mmol, tatanum) was added with stirring. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated. Dichloromethane (2 mL) and diluted hydrochloric acid (2 m,2 mL) were added to the residue, followed by stirring at room temperature for 1 hour. The reaction mixture was poured into water, and extracted with methylene chloride (20 mL. Times.2). The organic phase was washed with water (20 ml×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 with CombiFlash flash rapid prep. using eluent system B to give the title product 1n (400 mg), yield: 70%.

MS(ESI):274.1[M+H] ⁺ 。

Thirteenth step

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

Compound 1N (6278 mg,1.46 mmol), compound 2- (methylthio) -4- (((trifluoromethyl) sulfonyl) oxy) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester (400 mg,1.46mmol, prepared using the method disclosed as example intermediate 67 "on page 76 of the specification in patent application" WO 2017/20161, 2017, A1 "), and N, N-diisopropylethylamine (567 mg,4.39mmol, taitant) were dissolved in N, N-dimethylformamide (10 mL, run) and the reaction was stirred at 100℃for 3 hours under argon. The cooled reaction 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 with CombiFlash flash rapid prep. using eluent system B to give the title product 1o (777 mg), yield: 96%.

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

MS(ESI):584.9[M+H] ⁺ 。

Fifteenth step

4- ((3 s,5 r) -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 (650 mg,1.11 mmol) and compound 1d (319 mg,1.67 mmol) were dissolved in tetrahydrofuran (5 mL, hengyue chemical) at room temperature, and hexamethyldisilazide lithium (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. To the resulting reaction mixture was added water (20 mL), followed by extraction with ethyl acetate (50 mL. Times.3). The combined organic phases were washed with water (50 ml×3), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and the residue was purified with CombiFlash flash rapid prep. using eluent system a to give the title product 1q (560 mg), yield: 76%.

MS(ESI):660.0[M+H] ⁺ 。

Sixteenth step (2 s,6 r) -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-carboxylic acid benzyl ester 1r

Compound 1q (560 mg,0.848 mmol) was dissolved in dichloromethane (10 mL, run) at room temperature, and hydrochloric acid/1, 4-dioxane solution (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 concentrated at low temperature to give the crude product, which was free from saturated sodium bicarbonate solution to give the title product 1r (457 mg), yield: 96%.

MS(ESI):560.0[M+H] ⁺ 。

Seventeenth step

(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-carboxylic acid benzyl ester 1s

Compound 1r (407 mg, 0.258 mmol), 7-fluoro-8-methylnaphthalen-1-yl triflate (224 mg, 0.258 mmol, prepared as disclosed in patent application "example intermediate 77 on page 76 of the specification in US2019/0270743A 1"), methanesulfonic acid (9, 9-dimethyl-4, 5-bis-methylsulfonate)Diphenylphosphinoxanes) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II) (Xantphos Pd G3, 136mg,0.146mmol, nanjing's medical stone) and cesium carbonate (710 mg,2.184mmol, ming's Kangde) were dispersed in toluene (10 mL, national medicine), under argon, and stirred at 110℃for 18 hours. To the cooled reaction solution was added water (50 mL) and extracted 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 a CombiFlash flash rapid prep machine with eluent system a to give the title product 1s (250 mg). Yield: 47%. MS (ESI): 718.1[ M+H ] ] ⁺ 。

Eighteenth step

2- ((2 s,6 r) -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 (250 mg,0.348 mmol) was dissolved in methanol (10 mL, honival), wet palladium on carbon (50 mg, chemical engineering) was added, and the reaction mixture was stirred at room temperature under hydrogen balloon for 1 hour. The resulting mixture was filtered, and the filtrate was concentrated to give the title product 1t (175 mg), yield: 86%.

MS(ESI):584.0[M+H] ⁺ 。

Nineteenth step

2- ((2 s,6 r) -1-propenoyl-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

Compound 1t (175 mg,0.299 mmol) was dissolved in dichloromethane (5 mL, run) and acryloyl chloride (27.13 mg,0.299mmol, an Naiji) and triethylamine (91.0 mg,0.899mmol, tatam) were added dropwise to the reaction solution in this order at 0 ℃. The reaction solution was stirred at 0℃for 30 minutes. To the reaction mixture was added an aqueous sodium hydrogencarbonate solution (50 mL) and then extracted 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 chromatography (acidic condition, mobile phase contained formic acid) and the resulting fractions were freeze-dried to give the title product 1 (105.01 mg), yield: 54%.

¹ H NMR(400MHz,DMSO-d ₆ ):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- ((2 s,6 s) -1-propenoyl-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

(2S, 6S) -2- (2-ethoxy-2-oxoethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylic acid benzyl ester 2b

Ethyl 2- ((2 s,6 s) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazin-2-yl) acetate 2a (1.54 g,4.15mmol, prepared by well-known methods Journal of Organic Chemistry,2018,83,6541-6555), benzyl chloroformate (3.52 g,20.73mmol, tatanum) and sodium bicarbonate (1.05 g,12.44mmol, swiftly) were dissolved in tetrahydrofuran/water (10 mL/10mL, chinese medicine/dropen) and the reaction stirred at room temperature for 3 hours. The resulting mixture was extracted with dichloromethane (20 mL. Times.2) after addition of water. 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 rapid prep. with eluent system B to give the title product 2B (1.65 g), yield: 78%.

MS(ESI):505.9[M+H] ⁺ 。

Second step

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

Compound 2b (1.65 g,3.26 mmol) was dissolved in tetrahydrofuran/water (10 mL/10mL, guozheng/Chen),lithium hydroxide (215 mg,9.79mmol, run) was added and the reaction 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, extraction was performed 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 2c (1.7 g) which was used directly in the next reaction without purification MS (ESI): 478.0[ M+H)] ⁺ 。

Third step

(2S, 6S) -2- (2-amino-2-oxoethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylic acid benzyl ester 2d

Compound 2c (1.7 g,3.56 mmol), ammonium chloride (380 mg,7.12mmol, tetany), O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 1.62g,4.27mmol, pickle) and N, N-diisopropylethylamine (DIPEA, 1.38g,10.68mmol, tetany) were dissolved in N, N-dimethylformamide (10 mL, run) and the reaction stirred at room temperature for 1 hour. The resulting mixture was extracted with ethyl acetate (20 mL. Times.2) after adding water. 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 with CombiFlash flash rapid prep. using eluent system a to give the title product 2d (1.55 g), yield: 91%.

MS(ESI):476.9[M+H] ⁺ 。

Fourth step

(2S, 6S) -2- (cyanomethyl) -6-methyl-4- ((4-nitrophenyl) sulfonyl) piperazine-1-carboxylic acid benzyl ester 2e Compound 2d (1.55 g,3.25 mmol) and triethylamine (1.65 g,16.26mmol, tatam) were dissolved in dichloromethane (10 mL, run) at 0℃and trifluoroacetic anhydride (5.47 g,26.02mmol, tatam) was added to the reaction solution with stirring. The reaction solution was stirred at 0℃for 1 hour. To the reaction mixture was added a saturated sodium hydrogencarbonate 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 with CombiFlash flash rapid prep. using eluent system a to give the title product 2e (1.15 g), yield: 77%.

MS(ESI):458.9[M+H] ⁺ 。

Fifth step

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

Compound 2e (1.15 g,2.51 mmol) and cesium carbonate (1.63 g,5.02mmol, MINGKANG) were dispersed in tetrahydrofuran (10 mL, country) and 2-mercaptoethanol (586 mg,7.52mmol, tatanum) was added with stirring. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was concentrated. Dichloromethane (2 mL) and diluted hydrochloric acid (2 m,2 mL) were added to the residue, followed by stirring at room temperature for 0.5 hours. The mixture was poured into water and extracted with dichloromethane (20 ml×2). The organic phase was washed with water (20 ml×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 with CombiFlash flash rapid prep. using eluent system a to give the title product 2f (534 mg), yield: 77%.

MS(ESI):274.0[M+H] ⁺ 。

Sixth step

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

The compound 2- (methylthio) -4- (((trifluoromethyl) sulfonyl) oxy) -5, 6-dihydropyrido [3,4-d ] pyrimidine-7 (8H) -carboxylic acid tert-butyl ester (839 mg,1.95mmol, prepared as disclosed in patent application "WO2017/201161,2017, A1", page 76, example intermediate 67 "), compound 2f (284 mg,1.95 mmol) and N, N-diisopropylethylamine (DIPEA, 757mg,5.86mmol, tatam) were dissolved in N, N-dimethylformamide (10 mL, run) and the reaction stirred at 100℃for 2 hours under argon. The cooled reaction 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. Purification of the residue with a CombiFlash flash rapid prep machine using eluent system B gave the title product 2g (987 mg), yield: 91%.

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 (987 mg,1.79 mmol) in dichloromethane (10 mL, run) was added m-chloroperoxybenzoic acid (906 mg,4.46mmol,85% purity, jienen fir) at 0deg.C. The reaction solution was stirred at 0℃for 3 hours. The resulting mixture was concentrated at low temperature to give the crude product, and the residue was purified using CombiFlash flash rapid prep. with eluent system B to give the title product 2h (980 mg), yield: 93%.

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

MS(ESI):660.2[M+H] ⁺ 。

Ninth step

(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-carboxylic acid benzyl ester 2j

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

MS(ESI):560.1[M+H] ⁺ 。

Tenth step

(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-carboxylic acid benzyl ester 2k

Compound 2j (1599 mg,0.284 mmol) and-fluoro-8-methylnaphthalen-1-yl triflate (131 mg,0.426mmol, prepared as disclosed in example intermediate 77 "page 76 of patent application" U.S. Pat. No. 5,2019/0270743A 1) were dissolved in anhydrous toluene (3.0 mL, moist) at room temperature, followed by the addition of methanesulfonic acid (9, 9-dimethyl-4, 5-bis-diphenylphosphinoxa-ne) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II) (27 mg,0.028mmol, le-m), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (33 mg,0.056mmol, bi),

molecular sieves (160 mg, national medicine) and cesium carbonate (231.6 mg,0.71mmol, medicinal min), argon was substituted three times and 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 with CombiFlash flash rapid prep. using eluent system a to give the title product 2k (106 mg), yield: 55%.

MS(ESI):718.8[M+H] ⁺ 。

Eleventh step

2- ((2 s,6 s) -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 2l

Compound 2k (106 mg,0.148 mmol) was dissolved in anhydrous tetrahydrofuran (1.5 mL, run) and methanol (1.5 mL, horniwell), palladium hydroxide on charcoal (50 mg,20wt%, an Naiji) was added, and then hydrogen was replaced three times, and the reaction mixture was stirred at room temperature under hydrogen balloon protection for 2 hours. The reaction solution was filtered, and the filtrate was concentrated to give the title product 2l (82 mg), yield: 95%.

MS(ESI):584.3[M+H] ⁺ 。

Twelfth step

2- ((2 s,6 s) -1-propenoyl-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

2l (82 mg,0.141 mmol) of the compound was dissolved in methylene chloride (2.0 mL, national drug), and triethylamine (17 mg,0.169mmol, moist) and acryloyl chloride (14 mg,0.155mmol, bi) were sequentially added dropwise to the reaction solution under argon protection at 0 ℃. The reaction solution was stirred at room temperature for 2 hours. The reaction mixture 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 high performance liquid chromatography (acidic condition, mobile phase contained formic acid) to give the title product 2 (35 mg), yield: 39%.

¹ H NMR(400MHz,DMSO-d ₆ )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 U.S. Pat. No. 3,262A 1, 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-fluoroacryloyl) piperazin-2-yl) acetonitrile 3

First step

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 (10.3 g,34.64mmol, prepared as disclosed in patent application "example intermediate 32 on page 79 of the specification in WO2017/201161A 1") was dissolved in 200mL of dichloromethane, N-diisopropylethylamine (6.72 g,52 mmol) and trifluoromethanesulfonic anhydride (11.8 g,41.82 mmol) were added separately under ice-bath and stirred at room temperature for 17 hours. 50mL of saturated sodium bicarbonate solution, dichloromethane extraction (300 mL. Times.3), combined organic phases, washing with saturated sodium chloride solution (100 mL), drying over anhydrous sodium sulfate, filtration, concentration of the filtrate under reduced pressure, purification with silica gel column chromatography using eluent system B afforded the title compound 3B (9.5 g), yield: 63.9%.

Second step

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

Compound 3b (9.5 g,22.12 mmol) was dissolved in 80mL of acetonitrile and N, N-diisopropylethylamine (5.72 g,44.25 mmol) and benzyl (2S) -2- (cyanomethyl) piperazine-1-carboxylate (6.89 g,26.57mmol, prepared as disclosed in patent application "U.S. Pat. No. 5,108/0074123A 1 at page 110, example intermediate 63"), were added, respectively, and refluxed for 17 hours. The reaction solution was concentrated under reduced pressure, 30mL of saturated sodium hydrogencarbonate solution, ethyl acetate extraction (50 ml×3) was added to the residue, 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 (10.3 g), yield: 86.4%.

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

MS m/z(ESI):571.2[M+1] ⁺ 。

Fourth step

(1-aminocyclobutyl) methanol 3f

Compound 1-aminocyclo Ding Jiasuan (1.5 g,13.03mmol, shaoyuan) was dissolved in tetrahydrofuran (30 mL, hengye) at room temperature, and lithium aluminum hydride (1M tetrahydrofuran solution, 26mL,25mmol, an Naiji) was added dropwise under argon atmosphere and stirring. After the completion of the dropwise addition, the reaction solution was slowly warmed to room temperature and stirred under argon atmosphere 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 dichloromethane/methanol (10/1, 40 mL. Times.8). The combined filtrates were concentrated at room temperature, and the resulting residue was purified by silica gel column chromatography with eluent system A to give the title compound 3f (600 mg, yield: 45%).

¹ H NMR(400MHz,Acetone-d ₆ ):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)。

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 (1 g,1.75 mmol) and compound 3f (266 mg,2.63 mmol) were dissolved in tetrahydrofuran (20 mL, hengyue) at room temperature and lithium hexamethyldisilazide (LiHMDS, 1M tetrahydrofuran solution, 3.50mL,3.5mmol, an Naiji) was slowly added dropwise under argon at 0℃and stirring. After the completion of the dropwise addition, the reaction solution was warmed to room temperature and stirred under argon atmosphere for 16 hours. A saturated ammonium chloride solution and a saturated sodium chloride solution were added to the reaction 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 (800 mg), yield: 77%.

MS(ESI):592.8[M+H] ⁺ 。

Sixth step

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

3g (800 mg,1.35 mmol) of the compound was dissolved in dichloromethane (16 mL, run) 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 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. The filtrate was concentrated to give the title compound in a yield of 3h (600 mg) of 90%. MS (ESI): 492.2[ M+H ]] ⁺ 。

Seventh step

(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-carboxylic acid benzyl ester 3j

Compound 3h (600 mg,1.22 mmol), 8-chloro-7-fluoronaphthalen-1-yl triflate 3i (600 mg,1.83mmol, prepared as disclosed in patent application "example intermediate 78 on page 76 of U.S. Pat. No. 5,2019/0270743A 1"), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (XantPhos, 141mg, 243.94. Mu. Mol, obtained), cesium carbonate (995 mg,3.05mmol, ming Kangde), methanesulfonic acid (9, 9-dimethyl-4, 5-bis-diphenylphosphinoxanthracene) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II) (XantPhos Pd G3, 116mg, 122.36. Mu. Mol, hahong biological) and molecular sieves were taken at room temperature

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

MS(ESI):670.5[M+H] ⁺ 。

Eighth step

(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-carboxylic acid benzyl ester 3k

Compound 3j (200 mg, 298.43. Mu. Mol) was dissolved in tetrahydrofuran (6 mL, country drug) at room temperature, and a solution of di-tert-butyl dicarbonate (195 mg, 894.50. Mu. Mol, shanghai, miao) and sodium bicarbonate (326 mg,3.88mmol, runner) in water (3 mL, chengsu) was added dropwise at 0 ℃. The reaction solution was stirred at room temperature for 2 hours. Water was added to the reaction mixture, 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 (110 mg), yield: 47%.

MS(ESI):770.3[M+H] ⁺ 。

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 (110 mg, 142.80. Mu. Mol) was dissolved in tetrahydrofuran (5 mL, country drug) at room temperature, palladium hydroxide on charcoal (20 mg,20 wt.%) and methanol (0.5 mL, huo Niwei mol) were added, and after four hydrogen substitutions, the mixture was stirred at room temperature under hydrogen balloon protection for 1 hour. The reaction solution was filtered, and the cake was rinsed with methanol (5 mL. Times.3). The combined filtrates were concentrated to give the title compound 3l (78 mg) in 85% yield.

MS(ESI):636.2[M+H] ⁺ 。

Tenth step

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

2-fluoroacrylic acid (13 mg, 144.36. Mu. Mol, style.) was dissolved in tetrahydrofuran (2 mL, heng Yue) at room temperature, and N, N-diisopropylethylamine (DIPEA, 24mg, 185.70. Mu. Mol, sichuan Weibo technology) and O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 60mg, 157.80. Mu. Mol, hairda. Organism) were added sequentially. After the reaction mixture was stirred at room temperature for 0.5 hour, 3l (78 mg, 122.61. Mu. Mol) of the compound was added thereto, and the reaction mixture was stirred at room temperature for 2 hours. Water was added to the reaction mixture, 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 (56 mg) in 64% yield.

MS(ESI):708.5[M+H] ⁺ 。

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 (56 mg, 79.07. Mu. Mol) was dissolved in tetrahydrofuran (3 mL, national drug) 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 solution was concentrated, and the resulting residue was suspended in ethyl acetate (8 mL), stirred for 2 hours, and filtered. The filter cake was washed with anhydrous diethyl ether (3 mL. Times.3). The filter cake was collected and lyophilized to give the title compound 3 (30.45 mg, hydrochloride) in 63% yield.

¹ H NMR(400MHz,DMSO-d ₆ ):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

Using the synthetic route of example 1, substituting the thirteenth starting compound 1n 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 in US 2018/0074123 A1), substituting the nineteenth step of acryloyl chloride with compound 4-fluorobut-2-enoic acid (prepared using the well-known methods Journal of the American Chemical Society,2009, vol.131 (30), 10376-10377) gave the title compound 4 (85 mg), yield: 36.9%.

MS m/z(ESI):656.0[M+1] ⁺ 。

¹ H NMR(400MHz,DMSO-d ₆ )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 ERK phosphorylation of H358 cells by compounds of the present disclosure

1. Purpose of testing

The test is based on IC by detecting the inhibition of ERK phosphorylation in cells by compounds ₅₀ Size evaluation the inhibition of KRAS targets (containing G12C mutations) by the compounds of the present disclosure.

2. Experimental method

H358 cells (ATCC, CRL-5807) were cultured in complete medium of RPMI1640 (Hyclone, SH 30809.01) containing 10% fetal bovine serum. On the first day of the experiment, H358 cells were seeded at a density of 25,000 cells/well in 96-well plates with 190. Mu.L of cell suspension per well using complete medium and placed37℃，5％CO ₂ The cell culture incubator was incubated overnight. The next day, 10. Mu.L of a test compound in a gradient dilution with complete medium was added to each well, the final concentration of the compound was 9 concentration points at 6-fold gradient dilutions from 10. Mu.M, a blank containing 0.5% DMSO was set, the well plate was placed at 37℃and 5% CO ₂ Is incubated for 3 hours. After 3 hours, the 96-well cell culture plate was removed, the medium was aspirated, and 200. Mu.L of PBS (Shanghai Source culture Biotech Co., ltd., B320) was added to each well and washed once. The PBS was pipetted off, 50. Mu.L of lysis buffer (lysbuffer, cisbio,64KL1 FDF) containing blocking reagent (Cisbio, 64KB1 AAC) was added to each well, and the well plate was placed on a shaker and lysed for 30 minutes with shaking at room temperature. After lysis, 16. Mu.L of lysate was transferred to each well separately to two HTRF 96-well assay plates (Cisbio, 66PL 96100) after which 4. Mu.L of either pre-mixed phospho-ERK1/2 antibody solution (Cisbio, 64 AERPEG) or 4. Mu.L of pre-mixed total-ERK1/2 antibody solution (Cisbio, 64 NRKPEG) was added to each plate. The microplate was sealed with a sealing plate membrane, centrifuged in a microplate centrifuge for 1 min, and incubated overnight at room temperature in the absence of light. On the third day, the fluorescence values of the 337nm wavelength excitation, 665nm and 620nm wavelength emissions were read using a PHERAstar multifunctional microplate reader (BMG Labtech, S/N471-0361).

3. Data analysis

IC for compound inhibitory activity was calculated using Graphpad Prism software based on the ratio of compound concentration and pERK/total ERK ₅₀ Values, results are presented in table 3 below.

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

Examples numbering	IC 50 (nM)
		1	2.2
2	0.4
		4	3.2
Comparative example (Compound 3)	1862.0

Conclusion: the compound disclosed by the disclosure has a good inhibition effect on the ERK phosphorylation of H358 cells.

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

1. Purpose of testing

Inhibition of KRAS targets (containing G12C mutations) by compounds of the present disclosure was evaluated by testing the proliferation inhibition of H358 cells by compounds of the present disclosure.

2. Experimental method

H358 cells (ATCC, CRL-5807) were cultured in complete medium, namely RPMI1640 medium (Hyclone, SH 30809.01) containing 10% fetal bovine serum (Corning, 35-076-CV). On the first day of the experiment, H358 cells were seeded at a density of 1500 cells/well in 96-well plates using complete medium, 100. Mu.L of cell suspension per well, placed at 37℃and 5% CO ₂ The cell culture incubator was incubated overnight. The next day, 10. Mu.L of a test compound in a gradient dilution with complete medium was added to each well, the final concentration of the compound was 9 concentration points at 5-fold gradient dilution starting from 10. Mu.M, a blank containing 0.5% DMSO was set, the well plate was placed at 37℃and 5% CO ₂ Is cultured in a cell culture incubator for 120 hours. On day seven, 96-well cell culture plates were removed and 50. Mu.L of each well was added

Luminescent Cell Viability Assay (Promega, G7573), after 10 minutes at room temperature, multiple uses were madeThe function microplate reader (PerkinElmer, VICTOR 3) reads the luminescence signal value.

3. Data analysis

IC for calculating Compound inhibitory Activity Using Graphpad Prism software ₅₀ Values, results are presented in table 4 below.

TABLE 4 inhibitory Activity data of the presently disclosed compounds against H358 cell proliferation

Examples numbering	IC 50 (nM)
		1	10.6
2	1.5
		4	10.9
Comparative example (Compound 3)	204.9

Conclusion: the compound disclosed by the disclosure has a good inhibition effect on proliferation of H358 cells.

Test example 3: biological evaluation of MIA PaCa-2 cell ERK phosphorylation inhibition experiments

1. Purpose of testing

The test is based on IC by detecting the inhibition of MIAPaCa-2 cell ERK phosphorylation by compounds ₅₀ Size evaluation the inhibition of KRAS targets (containing G12C mutations) by the compounds of the present disclosure.

2. Experimental method

MIA PaCa-2 cells (ATCC, CRL-1420) were cultured in complete medium of DMEM/HIGH GLUCOSE (GE, SH 30243.01) containing 10% fetal bovine serum and 2.5% horse serum. On the first day of the experiment, MIA PaCa-2 cells were seeded in 96-well plates at a density of 25,000 cells/well using complete medium, 190. Mu.L of cell suspension per well, placed at 37℃and 5% CO ₂ The cell culture incubator was incubated overnight. The next day, 10. Mu.L of a test compound in a gradient dilution with complete medium was added to each well, the final concentration of the compound was 9 concentration points at 6-fold gradient dilutions from 10. Mu.M, a blank containing 0.5% DMSO was set, the well plate was placed at 37℃and 5% CO ₂ For 4 hours. After 4 hours, the 96-well cell culture plate was removed, the medium was aspirated, and 200. Mu.L of PBS (Shanghai Source culture Biotech Co., ltd., B320) was added to each well and washed once. The PBS was pipetted off, 50. Mu.L of lysis buffer (lysbuffer, cisbio,64KL1 FDF) containing blocking reagent (Cisbio, 64KB1 AAC) was added to each well, and the well plate was placed on a shaker and lysed for 30 minutes with shaking at room temperature. After lysis, 16. Mu.L of lysate was transferred to each well separately to two HTRF 96-well assay plates (Cisbio, 66PL 96100) after which 4. Mu.L of either pre-mixed phospho-ERK1/2 antibody solution (Cisbio, 64 AERPEG) or 4. Mu.L of pre-mixed total-ERK1/2 antibody solution (Cisbio, 64 NRKPEG) was added to each plate. The microplate was sealed with a sealing plate membrane, centrifuged in a microplate centrifuge for 1 min, and incubated overnight at room temperature in the absence of light. On the third day, the fluorescence values of the 337nm wavelength excitation, 665nm and 620nm wavelength emissions were read using a PHERAstar multifunctional microplate reader (BMG Labtech, S/N471-0361).

3. Data analysis

IC for compound inhibitory activity was calculated using Graphpad Prism software based on the compound concentration and pERK/total ERK ratio ₅₀ Values, results are shown in table 5 below.

TABLE 5 IC of the inhibition of cellular ERK phosphorylation by compounds of the present disclosure ₅₀ Values.

Examples numbering	IC 50 (nM)
		1	5.2
2	0.9
		4	5.2
Comparative example (Compound 3)	569.2

Conclusion: the compound disclosed by the disclosure has a good inhibition effect on MIA PaCa-2 cell ERK phosphorylation.

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

1. Purpose of testing

Inhibition of KRAS targets (containing G12C mutations) by compounds of the present disclosure was evaluated by testing the compounds of the present disclosure for their proliferation inhibition on miappa-cells.

2. Experimental method

MIA PaCa-2 cells (ATCC, CRL-1420) were cultured in complete medium, i.e., DMEM/HIGH GLUCOSE (GE, SH 30243.01) complete medium containing 10% fetal bovine serum and 2.5% horse serum. On the first day of the experiment, MIA PaCa-2 cells were seeded in 96-well plates at a density of 500 cells/well using complete medium, 90. Mu.L of cell suspension per well, placed at 37℃and 5% CO ₂ The cell culture incubator was incubated overnight. The next day, 10. Mu.L of a test compound in a gradient dilution with complete medium was added to each well, the final concentration of the compound was 9 concentration points at 5-fold gradient dilution starting from 10. Mu.M, a blank containing 0.5% DMSO was set, the well plate was placed at 37℃and 5% CO ₂ Is cultured in a cell culture incubator for 72 hours. On the fifth day, 96-well cell culture plates were removed and 50. Mu.L of each well was added

Luminescent Cell Viability Assay (luminescence cell activity assay reagent) (Promega, G7573), after 10 minutes at room temperature, luminescence signal values were read using a multifunctional microplate reader (PerkinElmer, VICTOR 3).

3. Data analysis

IC for calculating Compound inhibitory Activity Using Graphpad Prism software ₅₀ Values, results are presented in table 6 below.

TABLE 6 IC of the compounds of the present disclosure for MIA PaCa-2 cell proliferation inhibition ₅₀ Values.

Examples numbering	IC 50 (nM)
		1	5.1
2	0.9
		4	10.4

Conclusion: the compound disclosed by the disclosure has a good inhibition effect on MIA PaCa-2 cell proliferation.

The following are mentionedComparative 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 enzyme Activity of the human liver microsomal CYP3A4 testosterone metabolism site by Compounds of the disclosure

The enzymatic activity of the disclosed compounds on human liver microsomal CYP3A4 testosterone metabolic sites was determined using the following experimental method:

1. experimental material and instrument

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

2.NADPH(ACROS，A2646-71-1),

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

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

ZORBAX extension-C18, 3X 50mm,3.5 μm (Agilent Co., USA),

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

2. Experimental procedure

Preparing 100mM PBS buffer, and preparing 7.5mM MgCl with the buffer ₂ And 5mM NADPH solution, followed by the use of the 7.5mM MgCl ₂ A microsome solution (0.25 mg/mL) was prepared, and a stock solution (30 mM) was diluted with DMSO to give a series of solutions (I) having concentrations of 30mM, 10mM, 3mM, 1mM, 0.3mM, 0.03mM, 0.003mM and 0mM, and the series of solutions (I) was further diluted 200-fold with Phosphate Buffer (PBS) to give a series of solutions (II) (150, 50, 15, 5, 1.5, 0.15, 0.015 and 0. Mu.M). Testosterone working solution diluted to 375 μm concentration with PBS.

The preparation of MgCl was carried out at 7.5mM ₂ 40. Mu.L of the microsome solution of 0.25mg/mL, and 375. Mu.M testosterone working solution and 20. Mu.L of compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. Mu.M) respectively were mixed uniformly. The positive control group replaced the compound with the same concentration of ketoconazole. At the same time5mM NADPH solution together were preincubated for 5 min at 37 ℃. After 5 minutes 20. Mu.L of NADPH was added to each well, the reaction was started and incubated for 30 minutes. After 30 minutes 250. Mu.L of acetonitrile containing an internal standard was added to all samples, mixed well, shaken at 800rpm for 10 minutes, and centrifuged at 3700rpm for 10 minutes. 100. Mu.L of the supernatant was mixed with 80. Mu.L of ultrapure water and transferred to LC-MS/MS analysis.

The IC of the medicine to CYP3A4 testosterone metabolic site is obtained by calculating the numerical value through Graphpad Prism ₅₀ The values are shown in Table 7.

TABLE 7 IC of compounds of the present disclosure for testosterone metabolic sites of human liver microsomal CYP3A4 ₅₀ Values.

Examples numbering	IC 50 (μM)
		2	9.3
Comparative Compound A	2.7

Conclusion: the compounds of the present disclosure have weaker inhibition of testosterone metabolic sites of human liver microsome CYP3A4, and exhibit better safety.

Test example 6 inhibition of enzyme Activity of the presently disclosed Compounds on human liver microsomal CYP2C9 diclofenac Metabolic site

The enzymatic activity of the disclosed compounds on human liver microsomal CYP2C9 diclofenac metabolic sites was determined using the following experimental method:

1. experimental material and instrument

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

2.NADPH(ACROS，A2646-71-1),

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

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

ZORBAX extension-C18, 3X 50mm,3.5 μm (Agilent Co., USA),

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

2. Experimental procedure

100mM PBS buffer was used to prepare 7.5mM MgCl ₂ And 5mM NADPH solution, followed by the use of the 7.5mM MgCl ₂ A microsome solution (0.25 mg/mL) was prepared, and a stock solution (30 mM) was diluted with DMSO to give a series of solutions (I) having concentrations of 30mM, 10mM, 3mM, 1mM, 0.3mM, 0.03mM, 0.003mM and 0mM, and the series of solutions (I) was further diluted 200-fold with Phosphate Buffer (PBS) to give a series of solutions (II) (150, 50, 15, 5, 1.5, 0.15, 0.015 and 0. Mu.M). Diclofenac working solution diluted to 20 μm concentration with PBS.

The MgCl solution prepared at 7.5mM is taken out ₂ In the above solution, 40. Mu.L of 0.25mg/mL microsome solution was mixed with 20. Mu.L of each of 15. Mu.M midazolam working solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. Mu.M). The positive control group replaced the compound with the same concentration of sulfanilic acid. While 5mM NADPH solution was preincubated together at 37℃for 5 minutes. After 5 minutes 20. Mu.L of NADPH was added to each well, the reaction was started and incubated for 30 minutes. All incubation samples were double-sampled. After 30 minutes 250. Mu.L of acetonitrile containing an internal standard was added to all samples, mixed well, shaken at 800rpm for 10 minutes, and centrifuged at 3700rpm for 10 minutes. 100. Mu.L of the supernatant was mixed with 80. Mu.L of ultrapure water and transferred to LC-MS/MS analysis.

The numerical value is calculated by Graphpad Prism to obtain the IC of the drug to CYP2C9 diclofenac metabolic site ₅₀ The values are shown in Table 8.

Table 8 IC of the presently disclosed compounds for human liver microsomal CYP2C9 diclofenac metabolic sites ₅₀ Values.

Examples numbering	IC 50 (μM)
		2	9.0
Comparative Compound A	4.2

Conclusion: the compound disclosed by the disclosure has weak inhibition effect on the CYP2C9 diclofenac metabolic site of human liver microsomes, shows better safety, and suggests that metabolic drug interaction based on the CYP2C9 diclofenac metabolic site cannot occur.

Test example 7 inhibition of enzyme Activity of human liver microsomal CYP2C19 (S) -Mephenytoin Metabolic site by Compounds of the disclosure

The enzymatic activity of the disclosed compounds on human liver microsomal CYP2C19 (S) -mephenytoin metabolic site was determined using the following experimental method:

1. experimental material and instrument

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

2.NADPH(ACROS，A2646-71-1),

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

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

ZORBAX extension-C18, 3X 50mm,3.5 μm (Agilent Co., USA),

CYP probe substrate ((S) -mephenytoin/20. Mu.M, powder from carbofuran technologies Co., ltd., cat No. 303768) and positive control inhibitor (ticlopidine, powder from SIGMA, cat No. T6654-1G).

2. Experimental procedure

100mM PBS buffer was used to prepare 7.5mM MgCl ₂ And 5mM NADPH solution, followed by the use of the 7.5mM MgCl ₂ A microsome solution (0.25 mg/mL) was prepared, and a stock solution (30 mM) was diluted with DMSO to give a series of solutions (I) having concentrations of 30mM, 10mM, 3mM, 1mM, 0.3mM, 0.03mM, 0.003mM and 0mM, and the series of solutions (I) was further diluted 200-fold with Phosphate Buffer (PBS) to give a series of solutions (II) (150, 50, 15, 5, 1.5, 0.15, 0.015 and 0. Mu.M). (S) -Mephenytoin working solution diluted to 100. Mu.M concentration with PBS.

The MgCl solution prepared at 7.5mM is taken out ₂ In the above solution, 40. Mu.L of 0.25mg/mL microsome solution was mixed with 20. Mu.L of each of 15. Mu.M midazolam working solution and compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0. Mu.M). The positive control group replaced the compound with ticlopidine at the same concentration. While 5mM NADPH solution was preincubated together at 37℃for 5 minutes. After 5 minutes 20. Mu.L of NADPH was added to each well, the reaction was started and incubated for 30 minutes. All incubation samples were double-sampled. After 30 minutes 250. Mu.L of acetonitrile containing an internal standard was added to all samples, mixed well, shaken at 800rpm for 10 minutes, and centrifuged at 3700rpm for 10 minutes. 100. Mu.L of the supernatant was mixed with 80. Mu.L of ultrapure water and transferred to LC-MS/MS analysis.

The numerical value is calculated by Graphpad Prism to obtain the IC of the drug to CYP2C19 (S) -mephenytoin metabolic site ₅₀ The values are shown in Table 9.

Table 9 IC of compounds of the present disclosure for human liver microsomal CYP2C19 (S) -mephenytoin metabolic site ₅₀ Values.

Examples numbering	IC 50 (μM)
		2	15.8
Comparative Compound A	10.3

Conclusion: the compounds of the present disclosure have weak inhibitory effect on human liver microsomal CYP2C19 (S) -mephenytoin metabolic site, exhibit better safety, suggesting that no metabolic drug interactions based on CYP2C19 (S) -mephenytoin metabolic site occur.

Test example 8 in vitro plasma stability test of the presently disclosed compounds

1 purpose

The in vitro plasma stability test method for the compound is established and studied by adopting frozen or freshly prepared plasma of various species, and the in vitro plasma stability of the compound is evaluated.

2 instrument and materials

2.1 instrumentation

1) Eppendorf 5804R refrigerated centrifuge, eppendorf, germany

2) Thermomixer 5355, eppendorf, germany

3) Deep plate 96/1000. Mu.l, thermo company, USA

4) API 4000Q-trap linear ion trap mass spectrometer, company Applied Biosystems U.S.A

5) LC-30A ultra-high pressure liquid chromatography system, shimadzu corporation, japan

2.2 materials

1) Frozen blood plasma of various kinds (heparin sodium anticoagulation)

2) Frozen or freshly prepared human plasma (heparin sodium anticoagulation)

3 procedure

3.1 preparation of stock solution

The compounds were weighed separately and prepared in 30mM stock with DMSO.

3.2 diluting working solution

Stock solution at a concentration of 30mM was diluted with DMSO solution to a solution I at a concentration of 1600. Mu.M, and then 50% methanol was used to dilute solution I at a concentration of 1600. Mu.M to working solution II at a concentration of 16. Mu.M.

3.3 sample incubation

Experiments were performed at 0, 30, 60, 90, 120, 240,6 time points, each time point being provided with two replicates, 469 μl of plasma or whole blood, 31 μl of the above-prepared working solution II at a concentration of 16 μm was added to give a final concentration of 1 μm. Incubation at 37℃was started and timing was started (0 min after 300. Mu.L acetonitrile containing internal standard was added to working solution II). At the end of the other time points, the reaction was stopped with 300. Mu.L of acetonitrile containing internal standard. Then shaking table 850rpm for 5 minutes, centrifuge 3700rpm for 15 minutes, adding 100. Mu.l of deionized water to 100. Mu.l of the supernatant, shaking table 450rpm for 5 minutes, and LC-MSMS analysis.

4. Data analysis

Stability of the compounds was compared at each time point as a percentage of the remaining Area ratio relative to the 0 minute prototype Area ratio.

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

Taking natural logarithm Ln of the percentages, and carrying out linear regression by taking incubation time as an abscissa and a corresponding Ln value (Ln% Remain of Time zero) as an ordinate to obtain a coefficient k value of a linear regression equation.

Calculation of metabolic half-life T in vitro plasma using the following formula _1/2

T _1/2 =0.693/k (k is a linear regression equation coefficient)

TABLE 10 in vitro plasma stability of the disclosed compounds

Table 11 comparison of in vitro plasma stability of compound B

Conclusion: the in vitro plasma stability of the public compound is higher.

Claims (7)

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

2. a compound or tautomer, racemate, enantiomer, diastereomer, or 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, diastereomer, or 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, tautomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 3, in the manufacture of a medicament for inhibiting KRAS.

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

6. Use of a compound according to claim 1 or a stereoisomer, tautomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 3, in the manufacture of a medicament for the treatment or prevention of cancer, inflammation, or other proliferative disease.

7. Use of a compound according to claim 1, or a stereoisomer, tautomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 3, in the manufacture of a medicament for the treatment or prophylaxis of cancer, wherein said 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.