Pan-kras inhibitor compounds and methods of making and using the same

Abstract

This invention relates to the pan-KRAS inhibitor compound shown in Formula 43, its synthesis method, and its application.

Description

Technical Field

[0001] This invention relates to a compound, and more particularly to a highly active pan-KRAS inhibitor, its formulation, and its use. Background Technology

[0002] RAS is one of the most frequently mutated genes in human tumors, occurring in approximately 30% of cancer patients, with KRAS accounting for about 85% of RAS mutations. KRAS mutations are found in 88% of pancreatic cancers, 50% of colorectal adenocarcinomas, and 32% of lung adenocarcinomas, making the development of KRAS-targeting inhibitors of great clinical significance and value.

[0003] KRAS is a membrane-bound protein with GTPase activity. It acts as a "molecular switch" by cycling between the GDP-binding inactive conformation and the GTP-binding active conformation through nucleotide exchange. In its GTP-bound state, KRAS can activate multiple downstream signaling pathways, including RAF-MEK-ERK and PI3K-AKT, to regulate life processes such as cell growth, proliferation, differentiation, and apoptosis.

[0004] KRAS mutations (such as G12C, G12D, G12V, and G13D) affect GTP hydrolysis mediated by GTPase-activating proteins (GAPs), increasing the number of KRAS in a GTP-bound activated state. This overactivation of downstream signaling pathways ultimately leads to tumor development and progression. However, due to the lack of a suitable hydrophobic pocket for drug binding in the KRAS protein, and its affinity for GTP and GDP being in the picomolar range (~20 pM), the development of inhibitors that competitively bind to KRAS is extremely difficult. For decades, KRAS has been considered an untreatable target.

[0005] In May 2021, AMG510 was approved by the FDA for the treatment of KRAS carriers. G12C The emergence of mutations in locally advanced or metastatic non-small cell lung cancer has broken the historical barrier of KRAS being "untreatable." However, G12C mutations account for only a small fraction of KRAS mutations. For mutations at other KRAS sites, there is currently a lack of satisfactory and effective inhibitory compounds, leaving a large unmet clinical need. Therefore, the development of effective pan-KRAS inhibitory compounds is a necessity given the current technology. Summary of the Invention

[0006] This invention provides a pan-KRAS inhibitor. This structure differs from existing KRAS inhibitors that function through covalent binding. G12CInstead of acting as an inhibitor, KRAS exerts its effects by mediating the formation of a ternary complex between KRAS and ubiquitous intracellular chaperone proteins (such as Cyclophilin A). The formation of this ternary complex can sterically block the binding of KRAS to its downstream effector molecules (such as RAF), inhibiting the activation of the MAPK and PI3K-AKT signaling pathways, thereby suppressing tumor development and progression, and playing a therapeutic role in diseases such as cancer.

[0007] In one aspect, the present invention provides a KRas inhibitor compound having the structure of Formula 43:

[0008]

[0009] In some embodiments of the present invention, the compound is prepared via the intermediate compound INT-3:

[0010]

[0011] In a summary of some embodiments of the present invention, the preparation of the intermediate compound INT-3 includes the following steps:

[0012]

[0013] Step 1: Compound INT-2e was dissolved in dichloromethane, and trifluoroacetic acid was added. The reaction was carried out at room temperature for 2 hours. The reaction mixture was monitored by LCMS until the reactants were fully reacted. The reaction solution was directly concentrated under reduced pressure. The residue was dissolved in DCM, washed twice with saturated NaHCO3 aqueous solution, washed with water with the organic phase, dried over sodium sulfate, filtered, and concentrated to obtain a yellow solid compound INT-3a. Step 2: Compounds INT-3a and INT-3b were dissolved in acetonitrile. N,N,N',N'-tetramethylchloromethanemida hexafluorophosphate and 1-methylimidazole were added at 0°C. The reaction was carried out at 0°C for 1 hour. After 1 hour, the reaction of the starting materials was monitored by LCMS until complete. The reaction solution was poured into water, extracted with dichloromethane, washed with water, and purified by column chromatography to obtain a white solid compound INT-3c. In the third step, compound INT-3c, 2-dicyclohexylphosphine-2′,6′-dimethylbiphenyl, tris(dibenzylacetone)dipalladium, and potassium acetate were dissolved in toluene. Pinacol borane was added under nitrogen protection. After the addition was complete, the reaction was carried out at 50°C for 3 hours under nitrogen protection. The reaction of the starting materials was monitored by LCMS until complete. The reaction solution was filtered and purified by silica gel column chromatography to obtain a yellow solid compound INT-3.

[0014] In some embodiments of the present invention, the preparation of compound 43 includes the following steps:

[0015]

[0016] Step 1: Intermediates INT-3 and INT-38 were dissolved in a mixed solvent of 1,4-dioxane and water. 1,1-bis(diphenylphosphine)diferroferric palladium dichloride and potassium phosphate were added. After purging the reaction system with nitrogen, it was heated to 70°C and stirred for 12 hours. The reaction solution was cooled to room temperature and filtered through diatomaceous earth. The filtrate was concentrated. The residue was purified by preparative thin-layer chromatography (dichloromethane / methanol = 30 / 1) to obtain compound 43a. Step 2: Compound 43a was dissolved in DMF, and cesium carbonate was added. Iodoethane was then added dropwise to the reaction solution. The reaction solution was stirred at room temperature for 6 hours. LCMS analysis confirmed the reaction was complete. Saturated brine was added to the reaction system, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 43.

[0017] In another aspect, the present invention also provides pharmaceutical compositions comprising any of the foregoing compounds or pharmaceutically acceptable salts, isotope derivatives, or stereoisomers thereof.

[0018] In one aspect, the present invention also provides the use of the aforementioned compounds or pharmaceutically acceptable salts, isotope derivatives, stereoisomers, and pharmaceutical compositions thereof in the preparation of medicaments for the prevention and / or treatment of cancer, tumors, inflammatory diseases, autoimmune diseases, or immune-mediated diseases.

[0019] It is particularly noteworthy that, in this article, when referring to “compounds” with structures of formula (I) and (II), the term generally also includes their stereoisomers, diastereomers, enantiomers, racemic mixtures, and isotopic derivatives.

[0020] As is known to those skilled in the art, the salts, solvates, and hydrates of a compound are alternative forms of the compound, and they can all be converted into the compound under certain conditions. Therefore, it is particularly noteworthy that when referring to compounds with the structure of formula (I) and formula (II) herein, pharmaceutically acceptable salts are generally also included, and further include their solvates and hydrates.

[0021] Similarly, when referring to a compound in this article, its prodrug, metabolites, and nitrogen oxides are generally also included.

[0022] The pharmaceutically acceptable salts described in this invention can be formed using, for example, inorganic or organic acids: "Pharmaceutically acceptable salt" means a salt that, within a reasonable medical judgment, is suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., and has a reasonable benefit / risk ratio. The salts can be prepared in situ during the final separation and purification of the compounds of this invention, or solely by reacting a free base or free acid with a suitable reagent, as outlined below. For example, the free base function can react with a suitable acid.

[0023] The pharmaceutically acceptable salts of the present invention can be prepared by conventional methods, for example by dissolving the compounds of the present invention in a water-miscible organic solvent (e.g., acetone, methanol, ethanol, and acetonitrile), adding an excess of an aqueous solution of an organic or inorganic acid to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid, and then separating the precipitated salt.

[0024] The precursors or metabolites described in this invention can be precursors or metabolites known in the art, as long as they are metabolized and transformed in vivo to form compounds. For example, "prodrug" refers to those prodrugs of the compounds of this invention that, within a reasonable medical judgment, are suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and have a reasonable benefit / risk ratio and are effective for their intended use. The term "prodrug" refers to a compound that is rapidly transformed in vivo to produce the parent compound of the above formula, for example, through in vivo metabolism, or through N-demethylation of the compounds of this invention.

[0025] The term "solvate" as used in this invention refers to the physical association of the compound of this invention with one or more solvent molecules (organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. The solvent molecules in the solvate may be present in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric solvent molecules. "Solvate" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0026] The "stereoisomerism" described in this invention is divided into conformational isomerism and configurational isomerism. Configurational isomerism can be further divided into cis-trans isomerism and optical isomerism. Conformational isomerism refers to the phenomenon where organic molecules with a certain configuration undergo different spatial arrangements of atoms or groups of atoms due to the rotation or twisting of carbon or carbon single bonds. Common examples include the structures of alkanes and cycloalkanes, such as the chair conformation and boat conformation in the cyclohexane structure. "Stereoisomers" refer to compounds of this invention containing one or more asymmetric centers, thus allowing them to exist as racemic mixtures and racemic mixtures, single enantiomers, diastereomer mixtures, and single diastereomers. The compounds of this invention have asymmetric centers, each of which produces two optical isomers. The scope of this invention includes all possible optical isomers and diastereomer mixtures, as well as pure or partially pure compounds. The compounds of this invention can exist as tautomers, which have different hydrogen bonding sites through one or more double bond shifts. For example, ketones and their enol forms are ketone-enol tautomers. All tautomers and mixtures thereof are included in the compounds of this invention. All enantiomers, diastereomers, racemates, mesomates, cis-trans isomers, tautomers, geometric isomers, epimers, and mixtures thereof are included within the scope of this invention.

[0027] The term "isotope derivative" in this invention refers to molecules in which the compounds described herein are isotopically labeled. Commonly used isotopes for isotopic labeling are hydrogen isotopes. 2 H and 3 H; Carbon isotopes: 11 C, 13 C and 14 C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O, 17 O and 18 O and sulfur isotopes 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Especially deuterium. 3 H and carbon 13 C, because they are easy to label and convenient to detect, are more widely used. Some heavy isotopes, such as deuterium (… 2Substitution with H can enhance metabolic stability and prolong the half-life, thereby reducing the dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques, just like non-isotope-labeled compounds.

[0028] The present invention also provides the use of the compounds of the present invention in the preparation of medicaments for the prevention and / or treatment of cancer, tumors, inflammatory diseases, autoimmune diseases or immune-mediated diseases.

[0029] Furthermore, the present invention provides pharmaceutical compositions for the prevention and / or treatment of cancer, tumors, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, attention-related diseases, or immune-mediated diseases, comprising compounds of the present invention as active ingredients. The pharmaceutical compositions may optionally comprise a pharmaceutically acceptable carrier.

[0030] Furthermore, the present invention provides a method for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, attention-related diseases, or immune-mediated diseases, comprising administering the compound of the present invention to a mammal in need of such treatment.

[0031] Representative examples of cancers or tumors may include, but are not limited to, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neurocytoma, rectal cancer, colon cancer, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, testicular cancer, urinary tract cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma, and peripheral thyroid cancer. Neuroectodermal tumors, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, diffuse large B-cell lymphoma (DLBCL), hepatocellular carcinoma, gallbladder cancer, bronchial carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myoma, liposarcoma, fibrosarcoma, Ewing's sarcoma, or plasmacytoma.

[0032] When the compounds of the present invention or their pharmaceutically acceptable salts are administered in combination with other anticancer agents or immune checkpoint inhibitors used to treat cancer or tumors, the compounds of the present invention or their pharmaceutically acceptable salts may provide enhanced anticancer effects.

[0033] When the compounds of the present invention or their pharmaceutically acceptable salts are administered in combination with other therapeutic agents for treating inflammatory diseases, autoimmune diseases and immune-mediated diseases, the compounds of the present invention or their pharmaceutically acceptable salts may provide enhanced therapeutic effects.

[0034] In addition, the present invention provides a method for preventing and / or treating tumors, cancer, viral infections, organ transplant rejection, neurodegenerative diseases, attention-related diseases, or autoimmune diseases, comprising administering the compounds of the present invention or the pharmaceutical compositions of the present invention to mammals in need of such treatment.

[0035] The pharmaceutical compositions of the present invention can be formulated into dosage forms for oral or parenteral administration (including intramuscular, intravenous, and subcutaneous routes, and intratumoral injection) according to any of the conventional methods, such as tablets, granules, powders, capsules, syrups, emulsions, microemulsions, solutions, or suspensions.

[0036] Other features of the invention will become apparent in the process of describing exemplary embodiments. The embodiments described are given to illustrate the invention and are not intended to be limiting. The following examples use the methods disclosed in the invention to prepare, separate and characterize.

[0037] The compounds of the present invention can be prepared in a variety of ways known to those skilled in the art of organic synthesis. They can be synthesized using the methods described below, as well as synthetic methods known in the field of organic synthetic chemistry, or by variations thereof understood by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reaction is carried out in a solvent or solvent mixture suitable for the kit materials used and suitable for the transformation achieved. Those skilled in the art of organic synthesis will understand that the functionalities present on the molecule are consistent with the proposed transformation. This sometimes necessitates determining whether to change the order of synthetic steps or the starting materials to obtain the desired compound of the present invention. Detailed Implementation

[0038] the term

[0039] Unless otherwise specified, the terms used in this application, including those in the specification and claims, are defined as follows. Unless otherwise specified, conventional methods such as mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are used. In this application, unless otherwise specified, "or" or "and" refers to "and / or".

[0040] All features described in this specification (including any claims or abstracts) and / or all steps involved in any method or process may exist in any combination unless certain features or steps are mutually exclusive in the same combination.

[0041] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0042] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0043] The units used in weight-volume percentages in this invention are well known to those skilled in the art, for example, referring to the weight (g) of the solute in 100 ml of solution. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as known to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0044] Example

[0045] General process

[0046] When the preparation method is not specified, all raw materials and reagents used in this invention are known products that can be synthesized according to methods known in the art, or can be obtained by purchasing commercially available products. None of the commercially available reagents used require further purification.

[0047] Room temperature refers to 20-30℃.

[0048] Unless otherwise specified in the reaction examples, all reactions were carried out under a nitrogen atmosphere. A nitrogen atmosphere refers to a reaction flask connected to a nitrogen balloon of approximately 1L.

[0049] Hydrogenation reactions are typically carried out under vacuum, filled with hydrogen gas, and repeated three times. A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon of approximately 1L.

[0050] Microwave reaction use Initiator + Microwave Reactor.

[0051] The structure of the compounds of this invention was determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). NMR shifts (δ) were expressed in terms of 10⁻¹⁰. -6 The measurements are given in units of (ppm). NMR determinations are performed using (Bruker Ascend) TMA Model 500 NMR spectrometer was used. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). The following abbreviations are used for NMR signal multiplicity: s = singlet, brs = broad peak, d = doublet, t = triplet, m = multiplet. Coupling constants are listed in J values ​​and measured in Hz.

[0052] Reversed-phase preparative chromatography was performed using a Thermo (UltiMate 3000) reversed-phase preparative chromatograph. Rapid column chromatography was performed using an Agilent (FS-9200T) automated column press, and pre-packed silica gel columns were obtained from Sante. Pre-packed column. Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The thickness used for thin-layer chromatography separation and purification of products is 0.4mm to 0.5mm.

[0053] The LC-MS analysis method is as follows:

[0054] 1) Mass spectrometry method: Thermo Fisher MSQ PLUS mass spectrometer, ESI source, positive ion mode. Ion source parameter settings: drying gas temperature 350℃; drying gas flow rate 10L / min; MS range: 120-1000.

[0055] 2) Liquid chromatography conditions: Column: Waters XBridge (3.5 μm, 50 mm × 4.6 mm); Mobile phase A is an aqueous solution containing 0.1% ammonium bicarbonate, and mobile phase B is an acetonitrile solution, with linear gradient elution according to Table 1; Flow rate: 2 mL / min; Column temperature: 30 ℃; UV detection wavelength: 214 nm, 254 nm, 280 nm; Injection volume: 2 μL.

[0056] Table 1. Gradient elution conditions

[0057]

[0058] The HPLC analysis method is as follows:

[0059] Chromatographic column: Waters XBridge phenyl (3.5 μm, 150 mm × 4.6 mm); mobile phase A was an aqueous solution containing 0.1% ammonium bicarbonate, and mobile phase B was an acetonitrile solution, with linear gradient elution performed according to Table 2; flow rate: 1 mL / min; column temperature: 30 ℃; UV detection wavelengths: 214 nm, 254 nm, 280 nm; injection volume: 2 μL.

[0060] Table 2. Gradient elution conditions

[0061]

[0062] The synthesis methods of some intermediates in the invention are as follows:

[0063] Intermediate 1

[0064]

[0065] Intermediate 1 is prepared by the following steps:

[0066]

[0067] Step 1: Dissolve methyl 2,2-dimethyl-3-hydroxypropionate INT-1a (100 g, 757 mmol) in 1 L of N,N-dimethylformamide, add imidazole (129 g, 1.89 mol), stir to dissolve, and add tert-butyldiphenylchlorosilane (229 g, 832 mmol) dropwise at room temperature. After the addition is complete, continue stirring for 4 hours. After the reaction is complete, pour the reaction solution into 3 L of ice water. Extract the suspension with ethyl acetate (1 L * 2). Wash the organic phase three times with water, concentrate under reduced pressure to obtain a colorless oily substance INT-1b, which can be used directly in the next step without purification. ESI-MS (m / z): 371.2 [M+H] + .

[0068] Step 2: Add the residual INT-1b obtained in the previous step to 2L of methanol, then add 360g of a prepared 33% sodium hydroxide aqueous solution, and stir at room temperature for 17 hours. After the reaction is complete, add 1L of water, remove methanol under reduced pressure, and extract the residual liquid with petroleum ether (1L*5). After extraction, adjust the pH of the aqueous phase to 4-5 with hydrochloric acid, continue stirring for 30 minutes, filter, and dry to obtain a white solid INT-1c (269g, yield 90%). ESI-MS (m / z): 357.8 [M+H] + .

[0069] Step 3: Dissolve INT-1c (130g, 365mmol) in 500mL of dichloromethane, add thionyl chloride (130g, 1.09mol, 79.4mL) at room temperature, stir at 60℃ for 3 hours. After the reaction is complete, remove dichloromethane and the remaining thionyl chloride under reduced pressure to obtain a pale yellow oily substance INT-1d. Without purification, add 200mL of dichloromethane for later use.

[0070] Step 4: Dissolve INT-1e (64.8 g, 331 mmol) in 400 mL of dichloromethane. Add 198 mL of diethylaluminum chloride solution (2 M in hexanes) dropwise at 0 °C, controlling the temperature to not exceed 5 °C during the addition. Stir for 30 minutes after the addition is complete. Add the resulting dichloromethane solution of INT-1d dropwise to the reaction flask. Control the temperature to not exceed 10 °C during the addition. Continue stirring for 2 hours after the addition is complete. After the reaction is complete, pour the reaction solution into 1 L of ice water, stir for 30 minutes, concentrate under reduced pressure to remove dichloromethane, and extract the residue with ethyl acetate (1 L * 2). Wash with water, and rotary evaporate the organic phase to obtain a brown oily substance. Add the oily substance to 2 L of a 10 / 1 mixture of petroleum ether / ethyl acetate, stir to precipitate a solid, filter, and obtain a yellow solid INT-1f (139 g, yield 78%). ESI-MS (m / z): 534.8 [M + H] + .

[0071] Step 5: Dissolve INT-1f (100g, 187mmol) in 500mL of tetrahydrofuran, add lithium borohydride (12.2g, 561mmol), stir overnight at 60°C. After the starting material disappears, quench the reaction solution in 200mL of ice water, extract with ethyl acetate (500mL*3), wash the organic phase with water, dry it, concentrate under reduced pressure, dissolve the residue in 500mL of dichloromethane, add diethyl 2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate (28.4g, 112mmol) and p-toluenesulfonic acid (21.4g, 112mmol), stir at room temperature for 3 hours, concentrate under reduced pressure after the reaction is complete, remove dichloromethane, dissolve the residue in 500mL of methanol, add 100mL of pre-prepared 14% lithium hydroxide aqueous solution, stir at room temperature for 3 hours, and filter to obtain yellow solid INT-1g (84g, yield 86.3%). ESI-MS (m / z): 520.2 [M+H] + .

[0072] Step 6: Dissolve INT-1 g (50 g, 96 mmol) in 250 mL of tetrahydrofuran, add tetrabutylammonium fluoride (1 M inTHF, 197 mL), stir overnight at 60 °C. After the reaction is complete, add the reaction solution to 300 mL of water, extract with ethyl acetate (200 mL * 3), wash with water, concentrate under reduced pressure to obtain a brown oil. Dissolve the residue in 40 mL of methanol, add 20 mL of water, wash the mixture with petroleum ether (40 mL * 5), concentrate under reduced pressure to remove methanol, extract the residue with ethyl acetate (50 mL * 2), wash the organic phase with water, dry to obtain a pale yellow oil INT-1h (25 g, yield 90.4%). ESI-MS (m / z): 282.8 [M + H] + .

[0073] Step 7: Dissolve compound INT-1h (22 g, 77 mmol) in 100 mL of dichloromethane. Add 4-dimethylaminopyridine (467 mg, 3.82 mmol) and triethylamine (23.2 g, 230 mmol). Add acetic anhydride (7.9 g, 77 mmol) dropwise at 0 °C. After the addition is complete, allow the mixture to heat naturally and stir overnight. Once the reaction is complete, wash the reaction solution with water, dry it, and concentrate it to obtain a brown oil. Purify the oil by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to obtain a pale yellow oil INT-1i (22.5 g, yield 90.7%). ESI-MS (m / z): 324.2 [M+H] + .

[0074] Step 8: Compound INT-1i (40 g, 123 mmol) was dissolved in dioxane (400 mL), and potassium acetate (30.3 g, 308.4 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (10 g, 12.3 mmol), and pinacol diboronate (78.3 g, 308 mmol) were added. The reaction was carried out at 90 °C for 3 hours under nitrogen protection. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure. The residue was dissolved in ethyl acetate (300 mL), washed with water and brine, and the organic phase was purified by silica gel column chromatography to obtain a white solid compound INT-1j (35 g, yield 76.4%). ESI-MS (m / z): 372.5 [M+H] + .

[0075] Step 9: Compound INT-1j (35 g, 94.3 mmol) and compound INT-1k (37.9 g, 104 mmol) were dissolved in dioxane (300 mL) and water (30 mL). Potassium phosphate (50 g, 236 mmol) and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (6.89 g, 9.43 mmol) were added. The reaction was carried out overnight at 90 °C under nitrogen protection. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure. The residue was dissolved in ethyl acetate (300 mL), washed with water and brine, and the organic phase was purified by silica gel column chromatography to obtain a yellow oily compound INT-1l (28 g, yield 56.1%). ESI-MS (m / z): 530.7 [M+H] + .

[0076] Step 10: Compound INT-1l (28 g, 52.9 mmol) was dissolved in N,N-dimethylformamide (280 mL), and N-iodosuccinimide (11.9 g, 52.9 mmol) was added. The reaction was carried out at 50 °C for 2 hours. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was poured into water (800 mL), extracted with ethyl acetate (200 mL * 2), washed with saturated brine, dried, filtered, and purified by silica gel column chromatography to obtain a yellow solid compound INT-1m (22 g, yield 63.5%). ESI-MS (m / z): 656.6 [M + H] + .

[0077] Step 11: Compound INT-1m (5.0 g, 7.63 mmol), 2-dicyclohexylphosphine-2′,6′-dimethoxy-biphenyl (939 mg, 2.29 mmol), tris(dibenzylacetone)dipalladium (838 mg, 0.915 mmol), and potassium acetate (2.6 g, 26.7 mmol) were dissolved in toluene (100 mL). Pinara-borane (4.9 g, 38.1 mmol) was added dropwise under nitrogen protection. After the addition was complete, the reaction was carried out at 50 °C for 5 hours under nitrogen protection. The reaction mixture was monitored by LC-MS to ensure complete reaction of the starting material. The reaction solution was filtered and purified by silica gel column chromatography to obtain a yellow oily compound INT-1 (4.5 g, 90% yield). ESI-MS (m / z): 656.5 [M+H] + .

[0078] Intermediate 2

[0079]

[0080] Intermediate 2 is prepared by the following steps:

[0081]

[0082] Step 1: Compound INT-1m (12 g, 18.3 mmol) was dissolved in tetrahydrofuran (120 mL) and water (20 mL). Lithium hydroxide monohydrate (3.84 g, 91.5 mmol) was added, and the mixture was reacted overnight at room temperature. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure. The residue was dissolved in water (100 mL), and the pH was adjusted to 4–5 with 4 M hydrochloric acid. The mixture was extracted with dichloromethane (100 mL * 3), the organic phase was washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid compound INT-2a (10.6 g, yield 96.6%). ESI-MS (m / z): 600.7 [M + H] + .

[0083] Step 2: Compounds INT-2a (9.5 g, 15.9 mmol) and INT-2b (11.7 g, 31.7 mmol) were dissolved in acetonitrile (190 mL). N,N,N',N'-tetramethylchloromethanesulfonyl hexafluorophosphate (6.67 g, 23.8 mmol) and 1-methylimidazole (6.51 g, 79.2 mmol) were added at 0 °C. The reaction was carried out at 0 °C for 1 hour. LC-MS was used to monitor the reaction until complete. The reaction solution was poured into water (200 mL), extracted with dichloromethane (100 mL x 3), and the organic phase was washed with water. The solution was purified by silica gel column chromatography to obtain a yellow solid compound INT-2c (9.6 g, yield 83.5%). ESI-MS (m / z): 726.3 [M+H] + .

[0084] Step 3: Compound INT-2c (9.6 g, 13.2 mmol) was dissolved in tetrahydrofuran (100 mL) and water (10 mL). Lithium hydroxide monohydrate (1.39 g, 33.1 mmol) was added, and the reaction was carried out at room temperature for 4 hours. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure, and the residue was dissolved in water (100 mL). The pH was adjusted to 4-5 with 4 M hydrochloric acid, and a white solid precipitated. The solid was filtered, washed with water, and dried to obtain a white solid compound INT-2d (8.3 g, yield 88.2%). ESI-MS (m / z): 712.6 [M+H] + .

[0085] Step 4: Compound INT-2d (3.5 g, 4.9 mmol), 1-hydroxybenzotriazole (1.99 g, 14.8 mmol), and 4-dimethylaminopyridine (1.8 g, 14.8 mmol) were dissolved in dichloromethane (170 mL). N,N-diisopropylethylamine (6 mL, 34.4 mmol) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.71 g, 24.6 mmol). The reaction was carried out overnight at room temperature. The reaction mixture was monitored by LCMS to ensure complete reaction. The reaction solution was washed with saturated ammonium chloride aqueous solution, dried over sodium sulfate, and purified by silica gel column chromatography to obtain a yellow solid compound INT-2e (2 g, yield 58.6%). ESI-MS (m / z): 694.6 [M+H] + .

[0086] Step 5: Compound INT-2e (500 mg, 0.721 mmol), 2-dicyclohexylphosphine-2′,6′-dimethylbiphenyl (88.8 mg, 0.216 mmol), tris(dibenzylacetone)dipalladium (79 mg, 0.086 mmol), and potassium acetate (247 mg, 2.52 mmol) were dissolved in tetrahydrofuran (20 mL). Pinara-borane (461 mg, 3.6 mmol) was added dropwise under nitrogen protection. After the addition was complete, the reaction was carried out at 50 °C for 3 hours under nitrogen protection. LC-MS was used to monitor the reaction until complete. The reaction solution was filtered and purified by silica gel column chromatography to obtain a yellow solid compound INT-2 (400 mg, 80% yield). ESI-MS (m / z): 694.6 [M+H] + .

[0087] Intermediate 3

[0088]

[0089] Intermediate 3 is prepared by the following steps:

[0090]

[0091] Step 1: Compound INT-2e (1.7 g, 2.45 mmol) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (5 mL) was added. The reaction was carried out at room temperature for 2 hours. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure. The residue was dissolved in DCM (50 mL), washed twice with saturated NaHCO3 aqueous solution, washed with water with the organic phase, dried over sodium sulfate, filtered, and concentrated to give a yellow solid compound INT-3a (1.3 g, yield 89.4%). ESI-MS (m / z): 594.7 [M+H] + .

[0092] Step 2: Compounds INT-3a (1.3 g, 2.19 mmol) and INT-3b (0.24 g, 2.41 mmol) were dissolved in acetonitrile (30 mL). N,N,N',N'-tetramethylchloromethanesulfonyl hexafluorophosphate (922 mg, 3.29 mmol) and 1-methylimidazole (414 mg, 5.04 mmol) were added at 0 °C. The reaction was carried out at 0 °C for 1 hour. LC-MS was used to monitor the reaction until complete. The reaction solution was poured into water (50 mL), extracted with dichloromethane (50 mL x 3), and the organic phase was washed with water. The mixture was then purified by column chromatography to obtain a white solid compound INT-3c (1.3 g, yield 87.9%). ESI-MS (m / z): 675.7 [M+H] + .

[0093] Step 3: Compound INT-3c (1.1 g, 1.63 mmol), 2-dicyclohexylphosphine-2′,6′-dimethylbiphenyl (200 mg, 0.188 mmol), tris(dibenzylacetone)dipalladium (179 mg, 0.195 mmol), and potassium acetate (559 mg, 5.7 mmol) were dissolved in toluene (30 mL). Pinara-borane (1.04 g, 8.14 mmol) was added dropwise under nitrogen protection. After the addition was complete, the reaction was carried out at 50 °C for 3 hours under nitrogen protection. LC-MS was used to monitor the reaction until complete. The reaction solution was filtered and purified by silica gel column chromatography to obtain a yellow solid compound INT-3 (990 mg, 90% yield). ESI-MS (m / z): 676.9 [M+H] + .

[0094] Intermediate 4

[0095]

[0096] Intermediate 4 is prepared by the following steps:

[0097]

[0098] Step 1: Compounds INT-3a (2.2 g, 3.71 mmol) and INT-4a (0.47 g, 4.08 mmol) were dissolved in dichloromethane (50 mL). N,N,N',N'-tetramethylchloromethanesulfonyl hexafluorophosphate (1.56 g, 5.56 mmol) and 1-methylimidazole (0.70 g, 8.53 mmol) were added at 0 °C. The reaction was carried out at 0 °C for 1 hour. LC-MS was used to monitor the reaction until complete. The reaction solution was poured into water (50 mL), extracted with dichloromethane (50 mL x 3), and the organic phase was washed with water. The mixture was then purified by column chromatography to obtain a white solid compound INT-4b (2.3 g, yield 90.0%). ESI-MS (m / z): 690.2 [M+H] + .

[0099] Step 2: Compound INT-4b (2.1 g, 3.05 mmol), 2-dicyclohexylphosphine-2′,6′-dimethylbiphenyl (375 mg, 0.91 mmol), tris(dibenzylacetone)dipalladium (335 mg, 0.365 mmol), and potassium acetate (1.05 g, 10.7 mmol) were dissolved in toluene (30 mL). Pinara-borane (1.95 g, 15.2 mmol) was added dropwise under nitrogen protection. After the addition was complete, the reaction was carried out at 50 °C for 3 hours under nitrogen protection. LC-MS was used to monitor the reaction until complete. The reaction solution was filtered and purified by silica gel column chromatography to obtain a yellow solid compound INT-4 (1.8 g, yield 85.7%). ESI-MS (m / z): 690.3 [M+H] + .

[0100] Intermediate 5

[0101]

[0102] Intermediate 5 is prepared by the following steps:

[0103]

[0104] Step 1: (S)-3-bromo-5-iodo-2-(1-methoxyethyl)pyridine INT-5a (2.0 g, 5.85 mmol) was dissolved in tetrahydrofuran (20 mL), followed by the addition of cuprous iodide (111 mg, 0.585 mmol), bis(triphenylphosphine)palladium dichloride (410 mg, 0.585 mmol), triethylamine (1.18 g, 11.7 mmol), and 4-propynyl-1-morpholine INT-5b (878 mg, 7.02 mmol). The reaction mixture was stirred at room temperature for 3 hours under nitrogen protection. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / ethyl acetate = 1 / 1) to give a pale yellow oily compound INT-5 (1.8 g, yield 90.7%). ESI-MS (m / z): 339.4 [M+H] + .

[0105] Intermediate 15

[0106]

[0107] Intermediate 15 is prepared by the following steps:

[0108]

[0109] Step 1: Compound INT-15a (600 mg, 3.0 mmol) was dissolved in methanol (5 mL), and potassium carbonate (1.25 g, 9.0 mmol) and dimethyl (1.16 g, 6 mmol) phosphonate were added at room temperature. The reaction mixture was stirred at room temperature for 12 h. The reaction was stopped by TLC. Saturated brine was added to the reaction system, and the mixture was extracted with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound INT-15b (587 mg, 99% yield).

[0110] Step 2: Compound INT-15b (570 mg, 2.92 mmol) and compound INT-5a (1 g, 2.92 mmol) were dissolved in tetrahydrofuran (8 mL), and bis(triphenylphosphine)palladium dichloride (204 mg, 0.29 mmol), cuprous iodide (56 mg, 0.29 mmol), and triethylamine (591 mg, 5.85 mmol) were added. The reaction system was purged with nitrogen and stirred at room temperature for 8 h. The reaction was confirmed by LCMS. Saturated brine was added to the reaction system, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1) to give a yellow oily liquid INT-15 (1.08 g, 90% yield). ESI-MS (m / z): 409.6 [M+H] + .

[0111] Intermediate 38

[0112]

[0113] By replacing INT-5b in intermediate INT-5 with 4-propargylthiomorpholine-1,1-dioxide, compound INT-38 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 387.5 [M+H] + .

[0114] The synthesis method of the compounds in the embodiments of this invention is as follows:

[0115] Example 10

[0116] (1S,2S)-N-((6 3 S,4S,Z)-1 1 -ethyl-1 2 -(2-((S)-1-methoxyethyl)-5-(((R)-1-

[0117] methylpyrrolidin-2-yl)ethynyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-

[0118] 6 1 6 2 6 3 6 4 6 5 6 6 -hexahydro-1 1 H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-

[0119] pyridazinacyclodecaphane-4-yl)-2-methylcyclopropane-1-carboxamide

[0120]

[0121] Example 10 was prepared by the following steps:

[0122]

[0123] Step 1: Intermediate INT-3 (55 mg, 0.08 mmol) and intermediate INT-15 (30 mg, 0.08 mmol) were dissolved in a mixed solvent of 1,4-dioxane (3 mL) and water (0.2 mL). 1,1-bis(diphenylphosphine)dimerferropalladium dichloride (6 mg, 0.01 mmol) and potassium phosphate (47 mg, 0.02 mmol) were added. The reaction system was purged with nitrogen and heated to 70 °C with stirring for 12 hours. After cooling to room temperature, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated. The residue was purified by preparative thin-layer chromatography (dichloromethane / methanol = 30 / 1) to give compound 10a (62 mg, 96% yield). ESI-MS (m / z): 879.2 [M+H] + .

[0124] Step 2: Compound 10a (62 mg, 0.07 mmol) was dissolved in DMF (2 mL), and cesium carbonate (47 mg, 0.14 mmol) was added. Then, iodoethane (23 mg, 0.14 mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred at room temperature for 6 hours. The reaction was confirmed by LCMS. Saturated brine was added to the reaction system, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound 10b (64 mg, 99% yield). ESI-MS (m / z): 908.3 [M+H] + .

[0125] Step 3: Compound 10b (64 mg, 0.07 mmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (41 mg, 0.36 mmol) was added dropwise to the reaction solution at 0 °C. The reaction solution was stirred at 0 °C for 1 hour. The reaction was confirmed by LCMS. The reaction was quenched by adding saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound 10c (57 mg, 99% yield). ESI-MS (m / z): 807.6 [M+H] + .

[0126] Step 4: The crude compound 10c (57 mg, 0.07 mmol) was dissolved in methanol (2 mL). Formaldehyde aqueous solution (0.05 mL, 37% w / w) was added dropwise to the reaction solution at room temperature, and the mixture was stirred for 10 min at room temperature. Subsequently, sodium cyanoborohydride (14 mg, 0.21 mmol) was slowly added to the reaction solution, and the mixture was stirred for another 3 hours at room temperature. The reaction was confirmed to be complete by LC-MS. The reaction was quenched by adding saturated ammonium chloride aqueous solution, extracted with dichloromethane, and the organic phases were combined and concentrated. The residue was purified by preparative liquid chromatography to give a white solid compound 10 (3.0 mg, 5.2% yield) and its epimer 10' (4.0 mg, 6.9% yield). The absolute configurations of the two compounds are assumed based on experience. In existing analytical methods, 10 is a compound with relatively low polarity and relatively long LC-MS and HPLC retention times, while 10' is a compound with relatively high polarity and relatively short LC-MS and HPLC retention times.

[0127] Compound 10:

[0128] ESI-MS (m / z): 820.0 [M+H] + LC-MS retention time RT = 1.97 min. HPLC retention time RT = 14.55 min.

[0129] 1 H NMR (500MHz, DMSO) δ8.79(d,J=2.0Hz,1H),8.55–8.47(m,2H),7.82(d,J=2.0Hz,1H),7.81(s,1H),7.75(dd,J=8.5,2.0Hz,1H),7.58(d,J =8.5Hz,1H),5.56(t,J=9.0Hz,1H),5.10–5.05(m,1H),4.36–4.15(m,4H),4.12–4.03(m,1H),3.57(s,2H),3.41–3.35(m,1H),3.25(s,3H) ,3.17–3.11(m,1H),3.00–2.94(m,1H),2.83–2.72(m,2H),2.41–2.31(m,5H),2.19–2.11(m,1H),2.13–2.05(m,1H),1.94–1.68(m,6H),1 .56–1.46(m,2H),1.35(d,J=6.0Hz,3H),1.26–1.20(m,1H),1.06(s,3H),0.91(s,3H),0.93–0.88(m,4H),0.57–0.52(m,1H),0.34(s,3H).

[0130] Compound 10':

[0131] ESI-MS (m / z): 820.0 [M+H] + LC-MS retention time RT = 1.90 min. HPLC retention time RT = 13.87 min.

[0132] 1 H NMR (500MHz, DMSO) δ8.80(d,J=2.0Hz,1H),8.53(dd,J=9.0,2.0Hz,2H),7.96(d,J=2.0Hz,1H),7.81(s,1H),7.74(dd,J=8.5,1.5Hz,1H),7.54 (d,J=8.5Hz,1H),5.54(t,J=9.0Hz,1H),5.07–5.02(m,1H),4.27–4.18 (m,2H),3.99–3.90(m,2H),3.85–3.80(m,1H),3.70–3.66(m,1H),3.57– 3.52(m,1H),3.42–3.37(m,1H),3.31(s,2H),3.19–3.01(m,5H),2.80– 2.75(m,2H),2.40–2.28(m,5H),2.18–2.12(m,2H),1.92–1.69(m,5H),1 .57–1.47(m,2H),1.21(d,J=6.0Hz,3H),1.10(t,J=7.0Hz,3H),1.07(s ,3H),0.93(s,3H),0.90–0.85(m,1H),0.59–1.47(m,2H),,0.50(s,3H).

[0133] Example 43

[0134] (1S,2S)-N-((6 3 S,4S,Z)-1 2 -(5-(3-(1,1-dioxidothiomorpholino)prop-1-yn-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1 1 -ethyl-10,10-dimethyl-5,7-dioxo-

[0135] 6 1 6 2 6 3 6 4 6 5 6 6 -hexahydro-11 H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-

[0136] pyridazinacyclodecaphane-4-yl)-2-methylcyclopropane-1-carboxamide

[0137]

[0138] By replacing INT-15 in Example 10 with INT-38 and using similar methods and reaction steps, compound 43 can be obtained.

[0139]

[0140] Specifically, in the first step: intermediates INT-3 and INT-38 were dissolved in a mixed solvent of 1,4-dioxane and water. 1,1-bis(diphenylphosphine)diferroferric palladium dichloride and potassium phosphate were added. After purging the reaction system with nitrogen, it was heated to 70°C and stirred for 12 hours. The reaction solution was cooled to room temperature and filtered through diatomaceous earth. The filtrate was concentrated. The residue was purified by preparative thin-layer chromatography (dichloromethane / methanol = 30 / 1) to obtain compound 43a.

[0141] Step 2: Compound 43a was dissolved in DMF, cesium carbonate was added, and then iodoethane was added dropwise to the reaction solution. The reaction solution was stirred at room temperature for 6 hours. The reaction was confirmed to be complete by LCMS. Saturated brine was added to the reaction system, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 43. ESI-MS (m / z): 884.8 [M+H] + LC-MS retention time RT = 1.70 min. HPLC retention time RT = 12.44 min.

[0142] 1H NMR(500MHz, DMSO-d6)δ8.81(d,J=2.0Hz,1H),8.53–8.48(m,2H),7.86(d,J=2.0Hz,1H),7.80(s,1H),7.75(d,J=8.5Hz,1H),7.58(d,J=8.5Hz,1H),5 .56(t,J=9.0Hz,1H),5.09–5.05(m,1H),4.34–4.32(m,1H),4.29–4.25(m, 2H),4.21–4.15(m,2H),4.09–4.06(m,1H),3.77–3.75(m,2H),3.58–3.57(m ,2H),3.31–3.30(m,2H),3.25(s,3H),3.18–3.14(m,5H),3.06–3.03(m,3H ),2.98–2.94(m,1H),2.79–2.74(m,1H),2.41–2.36(m,1H),2.10–2.06(m, 1H),1.82–1.77(m,2H),1.52–1.47(m,2H),1.35(d,J=6.0Hz,3H),1.08–1. 05(m,4H),0.91(s,3H),0.90–0.86(m,4H),0.57–0.53(m,1H),0.34(s,3H).

[0143] Biological screening and results of RAS inhibitors

[0144] Experimental Example 1: In vitro cell proliferation inhibition assay

[0145] Due to the diversity of RAS mutations, and in order to evaluate the activity of compounds in different RAS mutant cell lines, we selected KRAS. WT KRAS G12C KRAS G12D KRAS G12V In vitro activity assessment and screening of compounds were performed using BRAF-mutant cell lines (see table below).

[0146] Cell Line Histotype Mutant NCI-H358 Lung;Bronchiole KRAS(p.G12C) MIA PaCa-2 Pancreas KRAS(p.G12C) LS513 Large intestine;Cecum KRAS(p.G12D) AsPC-1 Pancreas KRAS(p.G12D) HCC1588 Lung KRAS(p.G12D);BRAF(p.E204L) SW480 Large intestine;Colon KRAS(p.G12V) NCI-H727 Lung;Bronchus KRAS(p.G12V) NCI-H520 Lung <![CDATA[KRAS WT ;]]> HT-29 Colon <![CDATA[KRAS WT ;BRAF(p.V600E)]]>

[0147] Experimental plan: Cell Luminescent Viability Assay(Promega)

[0148] Depending on the doubling time of different cell lines, varying numbers of cells (1000-5000 cells / well) were seeded into 96-well plates containing 180 μl of the corresponding culture medium and cultured overnight in a 37°C cell culture incubator containing 5% CO2. The next day, the test compound was pre-diluted 3-fold serially with the culture medium, with a maximum concentration of 100 μM, for a total of 10 concentration gradients. Then, 20 μl of culture medium containing different concentrations of the compound was added to the cells in the 96-well plates, ensuring the final concentration of the compound was at a maximum of 10 μM, representing 10 concentration gradients of 3-fold dilutions. After co-incubating the cells and compound for 72 h, the 96-well plates were removed from the incubator and equilibrated at room temperature for 30 min. Then, 25 μl of the culture medium was added to each well. Mix the Reagent thoroughly and incubate at room temperature for 10 min. Then transfer 100 μl of sample to a white 96-well plate (OptiPlate). TM -96, PerkinElmer), using a multi-functional microplate reader ( The i3x (Molecular Devices) were used to read the fluorescence signal values. The signal values ​​were then standardized, and a four-parameter regression equation was used to fit a curve to calculate the half maximal inhibitory concentration (IC50) of the compound on the cell line.

[0149] Table 3: Antiproliferative activity of the compounds of this invention against KRAS cell mutants

[0150]

Claims

1. A KRas inhibitor compound having the structure of Formula 43: 。 2. A pharmaceutical composition comprising compound 43 as described in claim 1.

3. Use of compound 43 of claim 1 and the pharmaceutical composition of claim 2 in the preparation of a medicament for the prevention and / or treatment of tumors, inflammatory diseases or immune-mediated diseases.

4. Use of compound 43 of claim 1 and the pharmaceutical composition of claim 2 in the preparation of a medicament for the prevention and / or treatment of cancer, inflammatory diseases or autoimmune diseases.

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