RAS inhibitors for the treatment of cancer

Abstract

The application discloses a RAS inhibitor for treating cancer, and belongs to the field of chemical medicines. The application provides a compound as shown in formula (I) or a pharmaceutically acceptable salt, a prodrug, a hydrate or a solvent compound, a crystal form, a stereoisomer or an isotopic variant thereof, which can be used as a RAS inhibitor for treating or alleviating a RAS protein related disease, such as a RAS mediated related disease, for example, pancreatic cancer, lung cancer or colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of chemical medicine, specifically relating to RAS inhibitors for the treatment of cancer. Background Technology

[0002] RAS oncogenes are frequently mutated in human cancers. Their expression products, Ras proteins (K-Ras, H-Ras, and N-Ras), are widely distributed in eukaryotes. Studies have confirmed that RAS gene expression products are key components of signaling pathways controlling eukaryotic cell proliferation, survival, and differentiation. Ras are classified into HRAs, NRAs, and KRas, with KRAS mutations accounting for 86% of all RAS mutations. Due to the high prevalence of KRAS mutations in cancer patients, it plays a crucial role in various human cancers and is therefore a suitable target for anticancer therapies. In fact, approximately 30% of all human cancers are caused by Ras protein mutations, many of which are fatal. Dysregulation of Ras proteins caused by activating mutations, overexpression, or upstream activation is common in human tumors, and activating mutations of Ras are frequently found in human cancers. For example, the activation mutation at codon 12 in the Ras protein significantly biases the Ras mutant protein population towards the "on" (GTP-binding) state (Ras(ON)) by inhibiting GTPase-activated protein (GAP) dependence and intrinsic GTP hydrolysis rate, thereby exerting its effect through oncogenic MAPK signaling. Notably, Ras exhibits a picomolar affinity for GTP, allowing it to be activated even in the presence of low concentrations of this nucleotide.

[0003] Currently, only KRAS-targeting G12C Inhibitors targeting KRAS mutations have been launched. G12D Inhibitors of the mutation are also in clinical trials; small molecules effectively target KRAS. G12D Mutation remains a challenge, and safe and effective KRAS G12D Inhibitors have been a huge unmet clinical need. Summary of the Invention

[0004] [Technical Issues]

[0005] This invention provides a novel class of indole-substituted macrocyclic derivatives with superior inhibitory activity and a longer metabolic half-life.

[0006] [Technical Solution]

[0007] The purpose of this invention is to provide a class of novel compounds that can be used as RAS protein inhibitors, including novel compounds and their pharmaceutically acceptable salts, with novel RAS protein inhibitory activity and / or good pharmacodynamic / pharmacokinetic properties, and their use in treating or alleviating RAS-mediated diseases such as pancreatic cancer, lung cancer, or colorectal cancer.

[0008] One object of the present invention is to provide compounds of the structure shown in general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope-labeled compounds, or prodrugs thereof:

[0009]

[0010] In the formula:

[0011] X is CR3, Y is N; or X is N, Y is CR3;

[0012] R1, R2, and R3 are independently selected from H and C, respectively. 1-3 Alkyl, Halogenated C 1-3 Alkyl, or C 3-6 cycloalkyl;

[0013] L is selected from -CH2- or empty bond.

[0014] In one embodiment of the present invention, R1 is cyclopropyl and R2 is trifluoromethyl (CF3).

[0015] In one embodiment of the present invention, the present invention specifically provides the following compound:

[0016]

[0017] In one embodiment of the present invention, the pharmaceutically acceptable salt is an inorganic salt or an organic salt. The inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, nitrate, phosphate, and acid phosphate. The organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, pyruvic acid, acetic acid, succinic acid, phenylacetic acid, mandelic acid, and ferulic acid.

[0018] The present invention also provides a method for preparing compounds of general formula (I).

[0019] The present invention also provides pharmaceutical compositions comprising the above-described compound of general formula (I) of the present invention or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label or prodrug, and pharmaceutical excipients.

[0020] In one embodiment of the present invention, the pharmaceutical excipient includes a pharmaceutically acceptable carrier, excipient, or diluent.

[0021] Pharmaceutically acceptable carriers include microspheres, nanoparticles, and liposomes.

[0022] In one embodiment of the present invention, the dosage form of the pharmaceutical composition includes injection, lyophilized powder for injection, suspension, implant, embolization, capsule, tablet, pill and oral liquid.

[0023] The present invention also provides the use of compounds of the above general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope labels or prodrugs thereof in the preparation of drugs for inhibiting RAS proteins.

[0024] The present invention also provides the use of compounds of the above general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope labels or prodrugs in medicaments for the prevention or treatment of diseases such as human malignant melanoma, pancreatic cancer, lung cancer or colorectal cancer.

[0025] The present invention also provides the use of compounds of the above general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope labels or prodrugs thereof in medicaments for the prevention or treatment of RAS-mutant and RAS-dependent tumors.

[0026] Treatment of RAS-mutated and RAS-dependent tumors, such as solid tumors; further, said solid tumors are malignant melanoma, pancreatic cancer, lung cancer, or colorectal cancer. More specifically, cancers that can be treated by the compounds of the present invention or their salts, pharmaceutical compositions comprising such compounds or salts, and methods include, but are not limited to, the following tumor types: astrocytic, breast, cervical, colorectal, endometrial, esophageal, gastric, head and neck, hepatocellular, laryngeal, lung, oral cavity, ovarian, prostate, and thyroid cancers and sarcomas. Other cancers include, for example: the heart, such as sarcomas (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyosarcoma, fibroma, lipoma, and teratoma; the lungs, such as bronchogenic carcinomas (squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) carcinomas, bronchial adenomas, sarcomas, lymphomas, chondromatous hamartomas, and mesotheliomas; the gastrointestinal tract, such as the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), the stomach (carcinoma, lymphoma, leiomyosarcoma), the pancreas (ductal adenocarcinoma, islet tumor, glucagonoma, gastrinoma, carcinoid tumor, vasoactive intestinal peptide tumor), and the small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma). Sarcoma, leiomyomas, hemangiomas, lipomas, neurofibromas, fibromas; colon (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyomas); genitourinary tract, such as: kidney (adenocarcinoma, Wilms' tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminomatous sarcoma, teratoma, embryonal carcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoma-like tumor, lipoma); liver, such as: hepatocellular carcinoma, bile duct carcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bile duct, such as: gallbladder carcinoma, ampullary carcinoma, bile duct carcinoma; bone, such as: osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma. Sarcoma), malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondrogenic osteoma), benign chondroma, chondroblastoma, chondromycinoid fibroma, osteoid osteoma and giant cell tumor; nervous system, such as: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningeal sarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor), spinal neurofibroma, neurofibromatosis type 1, meningioma, glioma, sarcoma;Gynecological diseases, such as: uterus (endometrial cancer, uterine cancer, endometrial cancer), cervix (cervical cancer, cervical dysplasia), ovary (ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-theca cell tumor, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma), fallopian tubes (cancer); hematopoietic system diseases, such as: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders (e.g., myelofibrosis and myeloproliferative neoplasms, multiple myeloma, myelodyplasia syndrome), Hodgkin's disease). Diseases such as non-Hodgkin's lymphoma (malignant lymphoma); skin conditions such as malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, nevus dysplasia, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal gland conditions such as neuroblastoma.

[0027] Beneficial effects:

[0028] The compounds of this invention can be used as RAS inhibitors, exhibiting significantly improved activity or selectivity compared to the positive control (RMC-9805), and can be used for the treatment and / or prevention of diseases regulated by RAS proteins, such as pancreatic cancer, lung cancer, or colorectal cancer. Furthermore, the compounds of this invention, or their salts, have virtually no side effects and exhibit favorable drug metabolism and pharmacokinetics. Therefore, the compounds designed in this invention have excellent prospects for development and application. Detailed Implementation

[0029] The technical solution of the present invention will be described in detail below with reference to the embodiments.

[0030] In this invention, "C" 1-3 "Alkyl" refers to a saturated, straight-chain or branched monovalent hydrocarbon group having one to three carbon atoms. Examples include, but are not limited to, methyl, ethyl, 1-propyl, and 2-propyl.

[0031] In this invention, "C" 3-6 "Cycloalkyl" refers to a cycloalkyl group having 3 to 6 carbon atoms;

[0032] In this invention, "substituted" means that one or more hydrogen atoms in a group are replaced by the same or different substituents.

[0033] In this invention, “administering” or “giving” an individual compound means providing the compound of this invention to an individual in need of treatment.

[0034] The term "pharmaceutically acceptable salt" refers to a salt that, within reasonable medical judgment, is suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and has a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art.

[0035] The following examples and definitions used elsewhere in this document are as follows:

[0036]

[0037] <Pharmaceutical Composition>

[0038] The present invention also provides pharmaceutical compositions comprising the above-described compound of general formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0039] The compounds of the present invention or pharmaceutically acceptable salts thereof can be formulated into solid dosage forms for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, etc. In these solid dosage forms, the compound of general formula (I) of the present invention is mixed as the active ingredient with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate. Or it may be mixed with the following components: (1) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol and silica; (2) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, gum arabic; (3) humectants, such as glycerin; (4) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginic acid, certain silicates and sodium carbonate; (5) slowing agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glyceryl monostearate; (8) adsorbents, such as kaolin; (9) lubricants, such as talc, calcium stearate, solid polyethylene glycol, sodium dodecyl sulfate, etc., or mixtures thereof. Buffers may also be included in capsules, tablets and pills.

[0040] The solid dosage forms, such as tablets, sugar pills, capsules, pellets, and granules, can be coated or microencapsulated with coating and shell materials such as enteric coatings and other materials known in the art. They may contain opaque agents, and the release of the active ingredient from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active ingredient may also be formed into microcapsules with one or more of the excipients described above.

[0041] The compounds of the present invention or pharmaceutically acceptable salts thereof can be formulated into liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, tinctures, etc. In addition to the compound of general formula (I) or its pharmaceutically acceptable salt as the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water and other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil, etc., or mixtures thereof. In addition to these inert diluents, the liquid dosage forms of the present invention may also include conventional adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0042] The suspending agent includes, for example, ethoxylated octadecyl alcohol, polyoxyethylene sorbitol, and dehydrated sorbitol, microcrystalline cellulose, agar, or mixtures thereof.

[0043] The compounds of this invention and their pharmaceutically acceptable salts can be formulated into dosage forms for parenteral injection, including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, and excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0044] The compounds of this invention or pharmaceutically acceptable salts thereof can be formulated into dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays, and inhalers. The compounds of general formula (I) of this invention or pharmaceutically acceptable salts thereof, as active ingredients, are mixed under sterile conditions with a physiologically acceptable carrier and optionally with preservatives, buffers, and propellants, if necessary.

[0045] The pharmaceutical compositions of the present invention comprise a compound of general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier, excipient, and diluent. In preparing the pharmaceutical compositions, the compound of general formula (I) or a pharmaceutically acceptable salt thereof is typically mixed with a pharmaceutically acceptable carrier, excipient, or diluent. The content of the compound of general formula (I) or a pharmaceutically acceptable salt thereof can be 0.01-1000 mg, for example 0.05-800 mg, 0.1-500 mg, 0.01-300 mg, 0.01-200 mg, 0.05-150 mg, 0.05-50 mg, etc.

[0046] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are contemplated. Compounds of the present invention containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic form. Methods for preparing optically active forms from optically inactive raw materials are known in the art, for example by resolving racemic mixtures or by stereoselective synthesis. Geometric isomers of alkenes, C=N double bonds, etc., may also be present in the compounds described herein, and all such stable isomers are covered in this invention. Cis and trans geometric isomers of the compounds of the present invention can be isolated in the form of mixtures of isomers or as separate isomers.

[0047] The compounds described herein can exist as trans-block isomers, which are conformational isomers that occur when rotation around a single bond in the molecule is prevented or significantly slowed due to steric interactions with other parts of the molecule. The compounds disclosed in this invention include all trans-block isomers, which can be pure, single trans-block isomers, or mixtures enriched with one of the trans-block isomers, or nonspecific mixtures of each. Separation of isomers is permitted if the rotational potential around the single bond is sufficiently high and the interconversion between conformations is sufficiently slow.

[0048] Unless otherwise specified, the term “compound” as used herein is intended to include all stereoisomers, geometric isomers, tautomers and isotopes of the structure described.

[0049] All compounds and their pharmaceutically acceptable salts can be found together with other substances, such as water and solvents (e.g., in hydrate and solvate form) or can be separated.

[0050] In some embodiments, the compounds of the present invention or their salts are substantially isolated. "Substantially isolated" means that the compound is at least partially or substantially separated from its formation or detection environment. Partial separation may include, for example, the enrichment of a composition containing a compound of the present invention. Substantialtive separation may include a composition containing at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 80 wt%, at least about 90 wt%, at least about 95 wt%, at least about 97 wt%, or at least about 99 wt% of a compound of the present invention or its salt. Methods for separating the compounds and their salts are conventional methods in the art.

[0051] 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. The technical solutions of the present invention will be described in detail below with reference to the embodiments.

[0052] The following examples are illustrative and not limiting of the synthesis of compounds of general formula (I). All temperatures are in degrees Celsius. Unless otherwise stated, all evaporations were performed under reduced pressure. Unless otherwise stated, reagents were purchased from commercial suppliers and used without further purification. The structures of the final products, intermediates, and starting materials were confirmed by standard analytical methods, such as elemental analysis and spectroscopic characterization, such as MS and NMR. Abbreviations used are conventional abbreviations in the art.

[0053] Preparation of intermediate I-5: 5-bromo-3-(3-((tert-butyldiphenylsilyl)oxo)-2,2-dimethylpropyl)-2-iodo-1H-indole (I-5)

[0054]

[0055] Preparation of the first-step intermediate: 1-(5-bromo-1H-indol-3-yl)-3-((tert-butyldiphenylsilyl)-2,2-dimethylpropane-1-one (I-3)

[0056] A solution of 1 M SnCl4 in DCM (2 L) of 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropyl acyl chloride (starting material I-1, 100.0 g, 267 mmol) was added dropwise under N2 protection and stirred at 0 °C for 30 min. Then, a solution of 5-bromo-1H-indole (I-2) in DCM (500 mL) (52.3 g, 267 mmol) was added dropwise to the mixture at the same temperature and stirred at 0 °C for 1 h. LC / MS indicated the reaction was complete. The reactants were quenched by adding 1.2 L of water. The resulting solutions were extracted with 3 × 1.5 L of DCM and the organic layers were combined. The organic layers were washed with 3 × 5 L of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography to give intermediate I-3 (116.2 g, 81% yield). MS-ESI (m / z): 534.2 [M+l] + .

[0057] Preparation of the second-step intermediate: 5-bromo-3-(3-((tert-butyldiphenylsilyl)oxo)-2,2-dimethylpropyl)-1H-indole (I-4)

[0058] LiBH4 (14.2 g, 652.1 mmol) was added fractionally to a THF (1.5 L) solution of I-3 (116.2 g, 216 mmol) under N2 protection at 0 °C. The mixture was then slowly heated to 60 °C and stirred for 20 h. LC / MS indicated that the reaction was complete. The reaction mixture was quenched by adding 1.2 L of water. The resulting solution was extracted with 3 × 1.5 L of EtOAc and the organic layers were combined. The organic layers were washed with 3 × 5 L of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was diluted with DCM (1 L) and then mixed with dihydropyridine (7.1 g, 87.0 mmol) and p-toluenesulfonic acid monohydrate (2.1 g, 10.9 mmol) and stirred at 10 °C for 2 h. The mixture was then filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography to give intermediate I-4 (102.1 g, 90% yield). MS-ESI (m / z): 520.2 [M+l] + .

[0059] Preparation of the third intermediate: 5-bromo-3-(3-((tert-butyldiphenylsilyl)oxo)-2,2-dimethylpropyl)-2-iodo-1H-indole (I-5)

[0060] AgOTf (60.5 g, 235.3 mmol) was added fractionally to a THF (1.5 L) solution of I-4 (102.1 g, 196.1 mmol) and I2 (49.8 g, 196.1 mmol) under N2 protection at 25 °C. The mixture was then stirred at 25 °C for 2 h. LC / MS indicated that the reaction was complete. The reactants were diluted with 1.2 L of water. The resulting solutions were extracted with 3 × 1.5 L EtOAc and the organic layers were combined. The organic layers were washed with 3 × 5 L brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography to give intermediate I-5 (103.5 g, 82% yield). MS-ESI (m / z): 646.1 [M+l] + .

[0061] Preparation of intermediate I-10: (S)-3-bromo-5-iodo-2-(1-methoxyethyl)pyridine (I-10)

[0062]

[0063] Preparation of the first-step intermediate: (S)-1-(3-bromopyridin-2-yl)ethyl-1-ol (I-7)

[0064] At 0 °C, a mixture of TEA (834 mL, 6.0 mol) and FA (45.3 g, 1.20 mol) was added fractionally under N2 protection with (4S,5S)-2-chloro-2-methyl-1-(4-methylbenzenesulfonyl)-4,5-diphenyl-1,3-diaza-2-rutheniumcyclopentaneisopropyltoluene (3.25 g, 5.0 mmol) and stirred at 40 °C for 15 min. Then, 1-(3-bromopyridin-2-yl)ethyl-1-one (starting material I-6, 100.0 g, 500 mmol) was added fractionally to the mixture at room temperature. The reaction mixture was stirred at 40 °C for 2 h. LC / MS indicated that the reaction was complete. The reaction mixture was diluted with 1.2 L of saturated NaCl aqueous solution. The resulting solution was extracted with 3 × 1.5 L EtOAc and the organic layers were combined. The organic layer was washed with 3 × 5 L of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography to give intermediate I-7 (87.6 g, 86% yield). MS-ESI (m / z): 202.0 [M+l] + .

[0065] Preparation of the second intermediate: (S)-3-bromo-2-(1-methoxyethyl)pyridine (I-8)

[0066] NaH (20.8 g, 520.3 mmol) was added fractionally to a DMF (1 L) solution of I-7 (87.6 g, 433.6 mmol) under N2 protection at 0 °C, and the mixture was stirred at 0 °C for 1 h. Then, MeI (123.1 g, 867.1 mmol) was added fractionally to the mixture at 0 °C. The reaction mixture was heated to 25 °C and stirred for 2 h. LC / MS indicated that the reaction was complete. The reaction mixture was cooled to 0 °C, and a saturated NH4Cl aqueous solution (1 L) was slowly added dropwise at 0 °C, while stirring at this temperature for 1 h. The resulting solution was extracted by adding 3 × 1.5 L of EtOAc, and the organic layers were combined. The organic layers were washed with 3 × 5 L of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography to give intermediate I-8 (79.5 g, 85% yield). MS-ESI (m / z): 216.1 [M+l] + .

[0067] Preparation of the third intermediate: (S)-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)boronic acid (I-9)

[0068] A solution of I-8 (79.5 g, 367.9 mmol) and bis(pinacol)diboron (140.1 g, 551.8 mmol) in THF (2 L) was added in portions under N2 protection to dtbpy (14.8 g, 55.19 mmol) and chloro(1,5-cyclooctadiene)iridium(I) dimer (7.4 g, 11.04 mmol), and the mixture was stirred at 75 °C for 16 h. LC / MS indicated that the reaction was complete. The reaction solution was concentrated under reduced pressure, diluted with EtOAc (1 L), and a mixed aqueous solution of Na2CO3 (40 g) and NaOH (10 g) (600 mL) was added to adjust the pH to 10. The aqueous phase was then back-extracted with EtOAc (1 L), and the pH was adjusted to 6 with 6 M HCl aqueous solution until a large amount of solid precipitated. The mixture was filtered under reduced pressure to obtain residue I-9 (49 g, 51% yield). MS-ESI (m / z): 260.1 [M+l] + .

[0069] Preparation of the fourth intermediate: (S)-3-bromo-5-iodo-2-(1-methoxyethyl)pyridine (I-10)

[0070] A solution of I-9 (49 g, 188.5 mmol) in 2 L of ACN was added fractionally under N2 protection with NIS (106.1 g, 471.3 mmol) and stirred at 80 °C for 16 h. LC / MS indicated that the reaction was complete. The reaction solution was concentrated under reduced pressure, diluted with DCM (1 L), and extracted with 10% Na2S2O3 aqueous solution (600 mL x 3) to obtain the organic layer. The organic layer was washed with 3 × 2 L of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography to give intermediate I-10 (45 g, 69% yield).

[0071] MS-ESI (m / z): 341.9 [M+l] + .

[0072] Preparation of intermediate I-17: (S)-1-cyclopropyl-4-(6-(1-methoxyethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)pyridin-3-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (I-17)

[0073]

[0074] Preparation of the first-step intermediate: (S)-2-((5-bromo-6-(1-methoxyethyl)pyridin-3-yl)amino)ethyl-1-ol (I-12)

[0075] DIEA (12 mL, 72.4 mmol) was added to a solution of I-10 (22.5 g, 65.8 mmol) in 300 mL of DMF, followed by the addition of 2-aminoethyl-1-ol (16.1 g, 263.2 mmol). The reaction mixture was shaken at room temperature for 2 h. LC / MS indicated that the reaction was complete. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give intermediate I-12 (15.1 g, 83% yield). MS-ESI (m / z): 275.1 [M+l] + .

[0076] Preparation of the second-step intermediate: (S)-2-((5-bromo-6-(1-methoxyethyl)pyridin-3-yl)amino)ethyl methanesulfonate (I-13)

[0077] Intermediate I-12 (15.1 g, 54.9 mmol) was dissolved in DCM (300 mL). TEA (12.2 mL, 87.8 mmol) and methanesulfonyl chloride (7.54 g, 65.8 mmol) were added sequentially under ice bath conditions. After the addition was complete, the reaction was allowed to proceed at room temperature for 3 hours. LC / MS analysis showed that the reaction was complete. The reaction was then quenched with water (500 mL), extracted with DCM (400 mL x 3), and the organic phases were combined. After washing with water and saturated brine, the mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude intermediate I-13 (18.1 g, yield: 93%), which was used directly in the next step without purification. MS-ESI (m / z): 353.1 [M+l] +

[0078] Preparation of the third intermediate: (S)-5-bromo-N-(2-hydrazinoethyl)-6-(1-methoxyethyl)pyridine-3-amine (I-14)

[0079] Intermediate I-13 (18.1 g, 51.2 mmol) was dissolved in ethanol (300 mL), and 80% hydrazine hydrate (4.5 g, 71.7 mmol) was added. The mixture was heated to 60 °C and reacted for 5 hours. LC / MS analysis showed that the reaction was complete. The mixture was then concentrated under reduced pressure to give intermediate I-14 (13.1 g, yield: 88%), which was used directly in the next step without purification. MS-ESI (m / z): 289.1 [M+l] +

[0080] Preparation of the fourth intermediate: (S)-4-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (I-15)

[0081] A methanol (200 mL) solution of intermediate I-14 (13.1 g, 45.3 mmol) was added to the reaction mixture, which was then heated to 65 °C for 1 hour. LC / MS analysis showed that the reaction was complete. The reaction mixture was cooled at room temperature and concentrated under reduced pressure to obtain the residue. Further purification by column chromatography yielded intermediate I-15 (12.2 g, yield: 90%).

[0082] MS-ESI (m / z): 299.1 [M+l] +

[0083] Preparation of the fifth intermediate: (S)-4-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)-1-cyclopropyl-1,4,5,6-tetrahydro-1,2,4-triazine (I-16)

[0084] A solution of intermediate I-15 (6.1 g, 20.4 mmol) in THF (200 mL) was added with bromocyclopropane (3.6 g, 30 mmol), followed by KOtBu (6.6 g, 60 mmol), and the mixture was heated to boiling for 16 hours. LC / MS indicated that the reaction was complete. After the reaction, the residue was concentrated under reduced pressure and then dissolved in ethyl acetate (200 mL) and washed with water. The aqueous layer was extracted with ethyl acetate (200 mL x 2). The organic phases were combined, washed with brine, dried over Na2SO4, concentrated, and further purified by column chromatography to give intermediate I-16 (2.83 g, 41% yield). MS-ESI (m / z): 339.1 [M+1] + .

[0085] Preparation of intermediate in step 6: (S)-1-cyclopropyl-4-(6-(1-methoxyethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)pyridin-3-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (I-17)

[0086] A solution of intermediate I-16 (10.5 g, 31.0 mmol) and bis(pinacol)diboron (9.2 g, 36.2 mmol) in toluene (200 mL) was added fractionally under N2 protection with KOAc (8.1 g, 82.3 mmol) and Pd(dppf)Cl2 (2.4 g, 3.3 mmol) and stirred at 90 °C for 48 hours. LC / MS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure, extracted with DCM (400 mL x 3), and the organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified by column chromatography to give intermediate I-17 (9.2 g, 77% yield). MS-ESI (m / z): 361.2 [M+l] + .

[0087] Preparation of intermediate I-23: (S)-4-cyclopropyl-1-(6-(1-methoxyethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)pyridin-3-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (I-23)

[0088]

[0089] Preparation of intermediate: tert-butyl-(S)-(2-((5-bromo-6-(1-methoxyethyl)pyridin-3-yl)amino)ethyl)carboxylic acid ester (I-18)

[0090] DIEA (27 mL, 161 mmol) was added to a 1.5 L solution of I-10 (50 g, 146 mmol), followed by tert-butyl (2-aminoethyl)carbamate (72.6 g, 453 mmol). The reaction mixture was stirred at room temperature for 2 h. LC / MS indicated that the reaction was complete. The solvent was removed under reduced pressure. The residue was further purified to give intermediate I-18 (45 g, 82% yield). MS-ESI (m / z): 374.1 [M+l] + .

[0091] Preparation of intermediate: tert-butyl(S)-(2-(1-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)hydrazyl)ethyl)carboxylic acid ester (I-19)

[0092] I-18 (45 g, 121 mmol) was suspended in 6N HCl aqueous solution (1 L), cooled to 0 °C, and a solution of NaNO2 (12.44 g, 181 mmol) in water (120 mL) was slowly added. The mixture was stirred at 0 °C for 30 minutes, then at room temperature for 30 minutes. Stannous chloride dihydrate (54.3 g, 241 mmol) was then added at 0 °C, and the mixture was stirred at room temperature for 2 hours. LC / MS indicated that the reaction was complete. After the reaction, the solution was alkalized to pH 9 with 1 M NaOH aqueous solution, extracted with EtOAc (1 L x 3), and the organic phases were combined. The mixture was washed with water, then with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified by column chromatography to obtain intermediate I-19 (37 g, 79% yield). MS-ESI (m / z): 389.1 [M+l] + .

[0093] Preparation of intermediate: (S)-2-(1-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)hydrazino)ethyl-1-amine (I-20)

[0094] TFA (200 mL) was added sequentially to a DCM (1 L) solution of I-19 (37 g, 95 mmol) at 0 °C, and the reaction was carried out at 25 °C for 12 hours. LC / MS indicated that the reaction was complete. After the reaction was complete, the residue was concentrated under reduced pressure, and then 1 L of methyl tert-butyl ether was added. After stirring at room temperature for 1 hour, the mixture was filtered, and the filter cake was dried to give intermediate I-20 (30 g, crude product), which was used directly in the next step without purification. MS-ESI (m / z): 289.1 [M+l] + .

[0095] Preparation of intermediate: (S)-1-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (I-21)

[0096] A methanol (500 mL) solution of intermediate I-20 (30 g, 103.7 mmol) was added to a reaction mixture of TMO (9.86 g, 518.7 mmol), and the mixture was heated to 65 °C for 1 hour. LC / MS analysis showed that the reaction was complete. The reaction mixture was cooled at room temperature and concentrated under reduced pressure to obtain the residue. Further purification by column chromatography gave intermediate I-21 (27 g, yield: 87%). MS-ESI (m / z): 299.1 [M+l] +

[0097] Preparation of intermediate: (S)-1-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)-4-cyclopropyl-1,4,5,6-tetrahydro-1,2,4-triazine (I-22)

[0098] Following the synthetic method for intermediate (I-16), compound I-21 was reacted to give intermediate (I-22) (5.9 g, 63% yield). MS-ESI (m / z): 339.1 [M+1] + .

[0099] Preparation of intermediate: (S)-4-cyclopropyl-1-(6-(1-methoxyethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)pyridin-3-yl)-1,4,5,6-tetrahydro-1,2,4-triazine (I-23)

[0100] Following the synthetic method for intermediate (I-17), compound I-22 was reacted to give intermediate (I-23) (8.4 g, 75% yield). MS-ESI (m / z): 387.2 [M+l] + .

[0101] Preparation of intermediate I-27: (S)-3-(5-bromo-2-(5-(1-cyclopropyl-5,6-dihydro-1,2,4-triazine-4(1H)-yl)-2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (I-27)

[0102]

[0103] Preparation of intermediate: (S)-5-bromo-3-(3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropyl)-2-(5-(1-cyclopropyl-5,6-dihydro-1,2,4-triazin-4(1H)yl)-2-(1-methoxyethyl)pyridin-3-yl)-1H-indole (I-24)

[0104] A mixture of dioxane (400 mL) and water (40 mL) of intermediates I-5 (26.4 g, 41 mmol) and I-17 (14.8 g, 41 mmol) was added fractionally under N2 protection with K3PO4 (21.8 g, 100 mmol) and Pd(dppf)Cl2 (2.92 g, 4.0 mmol) and stirred at 70 °C for 16 hours. LC / MS indicated that the reaction was complete. The reaction solution was concentrated under reduced pressure, extracted with EtOAc (500 mL x 3), and the organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified by column chromatography to give intermediate I-24 (19.6 g, 763% yield). MS-ESI (m / z): 778.4 [M+l] + .

[0105] Preparation of intermediate: (S)-5-bromo-3-(3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropyl)-2-(5-(1-cyclopropyl-5,6-dihydro-1,2,4-triazin-4(1H)yl)-2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indole (I-25)

[0106] Cs₂CO₃ (28.5 g, 86 mmol) was added to a DMF (400 mL) solution of intermediate I-24 (23.2 g, 30 mmol), followed by 1,1,1-trifluoro-2-iodoethane (12.6 g, 60 mmol), and the reaction was carried out at 25 °C for 12 h. LC / MS indicated that the reaction was complete. After the reaction, the residue was concentrated under reduced pressure and then dissolved in ethyl acetate (200 mL) and washed with water. The aqueous layer was extracted with ethyl acetate (200 mL x 2). The organic phases were combined, washed with brine, dried over Na₂SO₄, and concentrated to obtain crude intermediate I-25 (20.3 g, crude product), which was used directly in the next step without purification. MS-ESI (m / z): 860.4 [M+1] + .

[0107] Preparation of intermediate: (S)-3-(5-bromo-2-(5-(1-cyclopropyl-5,6-dihydro-1,2,4-triazine-4(1H)-yl)-2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (I-26)

[0108] I-25 (21.6 g, 25.1 mmol) was added fractionally to a solution of TBAF (53.4 g, 170 mmol) in THF (500 mL), and the reaction was carried out at 50 °C for 16 h. LC / MS indicated that the reaction was complete. After the reaction, the residue was concentrated under reduced pressure, extracted with EtOAc (500 mL x 3), and the organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified by column chromatography to give intermediate I-26 (8.59 g, 55% yield). MS-ESI (m / z): 622.3 [M+l] + .

[0109] Preparation of intermediate: (S)-3-(5-bromo-2-(5-(1-cyclopropyl-5,6-dihydro-1,2,4-triazine-4(1H)-yl)-2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (I-27)

[0110] I-26 (37.0 mg) was purified by HPLC chirality under the following conditions (column: CHIRAL ART Cellulose-SB, 2*25 cm, 5 μm; mobile phase A: MtBE (10 mM NH3-MeOH), mobile phase B: EtOH; flow rate: 20 mL / min; gradient: 15% B to 50% B over 7 min; 275 / 210 nm), yielding (S)-3-(5-bromo-2-(5-(1-cyclopropyl-5,6-dihydro-1,2,4-triazine-4(1H)-yl)-2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (I-27) (13.0 mg, yield: 35%, ee% = 98.7%).

[0111] MS-ESI (m / z): 622.2 [M+l] + .

[0112] And the corresponding transisomer (S)-3-(5-bromo-2-(5-(1-cyclopropyl-5,6-dihydro-1,2,4-triazin-4(1H)-yl)-2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (I-28) (11.0 mg, yield: 30%, ee% = 99.1%)

[0113] MS-ESI (m / z): 622.2 [M+l] + .

[0114] Preparation of intermediate I-32: (S)-3-(5-bromo-2-(5-(4-cyclopropyl-5,6-dihydro-1,2,4-triazine-1(4H)-yl)-2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (I-32)

[0115]

[0116] Following the synthetic method of intermediate (I-27), intermediate (I-32) was obtained.

[0117] MS-ESI (m / z): 622.2 [M+l] + .

[0118] MS-ESI (m / z) of the resisted transisomer (I-33): 622.2 [M+l] + .

[0119] Preparation of intermediate II-9: methyl-(S)-2,3-diazabicyclo[3.1.1]heptane-4-carboxylate (II-9)

[0120]

[0121] Preparation of intermediate: (S)-4-benzyl-3-(2-(3-oxocyclobutyl)acetyl)tetrahydrooxazol-2-one (II-3)

[0122] To a solution of II-1 (50 g, 390.2 mmol), II-2 (76.1 g, 429.3 mmol), 4-dimethylaminopyridine (4.8 g, 39.1 mmol), and TEA (118.5 g, 1175 mmol) in DCM (1.5 L), 2-chloro-1-methylpyridine (salt) iodide (169.5 g, 663.4 mmol) was added in portions and the mixture was reacted at 25 °C for 1 h. LC / MS indicated that the reaction was complete. After the reaction was complete, the mixture was diluted with an appropriate amount of water and extracted with EtOAc (1 L x 3). The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude intermediate II-3 (93 g, 83% yield). The residue was not purified and proceeded directly to the next step. MS-ESI (m / z): 288.2 [M+l] + .

[0123] Preparation of intermediate: (S)-4-benzyl-3-(2-(3-hydroxycyclobutyl)acetyl)tetrahydrooxazol-2-one (II-4)

[0124] NaBH4 (9.8 g, 258 mmol) was added fractionally to a THF (1.5 L) solution of II-3 (93 g, 324 mmol) and AcOH (37.1 mL, 648 mmol) under N2 protection at 0 °C. The mixture was then stirred at this temperature for 2 hours. LC / MS indicated that the reaction was complete. The reactants were quenched by adding 1.2 L of saturated NH4Cl aqueous solution. The mixture was concentrated under reduced pressure, and the resulting solution was extracted with 3 × 1.5 L of EtOAc and the organic layers were combined. The organic layers were washed with 3 × 5 L of saturated NaHCO3 aqueous solution to pH ~8, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude intermediate II-4 (97 g, crude). MS-ESI (m / z): 290.2 [M+l] + .

[0125] Preparation of intermediate: (S)-3-(2-(4-benzyl-2-oxotetrahydrooxazol-3-yl)-2-carbonylethyl)cyclobutyl-4-methylbenzenesulfonate (II-5)

[0126] 4-Toluenesulfonyl chloride (89.5 g, 470 mmol) was added fractionally to a DCM (1.5 L) solution of II-4 (97 g, 335 mmol), 4-dimethylaminopyridine (53.3 g, 436 mmol), and DIEA (86.7 g, 671 mmol) under N2 protection at 0 °C. The mixture was then stirred at 25 °C for 3 h. LC / MS indicated that the reaction was complete. The reaction solution was washed with 1 L of water. The aqueous phase was concentrated under reduced pressure, and the resulting solution was extracted with 2 × 1.5 L of DCM, and the organic layers were combined. The organic layers were washed with saturated saline solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified to give intermediate II-5 (132 g, 88% yield). MS-ESI (m / z): 444.2 [M+l] + .

[0127] Preparation of intermediate: (S)-4-benzyl-3-(2-(3-bromocyclobutyl)acetyl)tetrahydrooxazol-2-one (II-6)

[0128] BrLi (51.7 g, 596 mmol) was added fractionally to a 2 L NMP solution of II-5 (132 g, 298 mmol), and the mixture was heated to 90 °C and reacted for 13 h. LC / MS indicated that the reaction was complete. The mixture was diluted with 3 L of water. The resulting solution was extracted with 2 × 3 L EtOAc, and the organic layers were combined. The organic layers were washed with saturated saline solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified to give intermediate II-6 (94 g, 89% yield). MS-ESI (m / z): 352.1 [M+l]+.

[0129] Preparation of intermediate: (S)-2,3-bis(tert-butoxycarbonyl)-2,3-diazabicyclo[3.1.1]heptane-4-carboxylic acid (II-7)

[0130] A mixture of LDA (2M) and THF (n-heptane) in 174mL (347mmol) was slowly added dropwise to a solution of II-4 (94g, 267mmol) in 2L of THF at -78°C, and the mixture was stirred at this temperature for 30 minutes. Then, a solution of DBAD (73.7g, 321mmol) in 1L of DCM was slowly added to the mixture, and the mixture was stirred at this temperature for another 30 minutes. Subsequently, DMPU (1.03kg, 8mol) was slowly added to the mixture, and the temperature was slowly raised to 25°C for 13 hours. LC / MS indicated that the reaction was complete. After quenching with water (1L), LiOH·H₂O (33.6g, 801mmol) was added, and the mixture was stirred at 25°C for 1 hour. LC / MS indicated that the reaction was complete. The mixture was extracted with 2×3L of EtOAc, and the organic layers were combined. The organic layers were washed with saturated saline solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified to give intermediate II-7 (76 g, 83% yield). MS-ESI (m / z): 343.3 [M+l] + .

[0131] Preparation of intermediate: 2,3-di-tert-butyl-4-methyl-(S)-2,3-diazabicyclo[3.1.1]heptane-2,3,4-tricarboxylic acid ester (II-8)

[0132] A solution of 2 M TMSCHN2 in n-hexane (555 mL, 1.1 mol) was slowly added dropwise to a solution of II-7 (76 g, 222 mmol) in MeOH (2 L), and the reaction was carried out at 25 °C for 1 h. LC / MS indicated that the reaction was complete. The mixture was diluted with 5 mL of AcOH. The mixture was concentrated under reduced pressure to give crude intermediate II-6 (63 g, 79% yield). MS-ESI (m / z): 357.2 [M+l] + SFC analysis (column: Cellulose-4 (100mm*4.6mm, 3μm); mobile phase: phase A: supercritical carbon dioxide, phase B: [0.05% ammonia in ethanol]; B%: from 5% to 40% in 4 minutes, then held at 40% for 0.5 minutes, then held at 5% for 1.5 minutes) showed a retention time of 1.345 min and a chiral purity of 97.31% for its enantiomers. MS-ESI (m / z): 357.2 [M+l] + .

[0133] Preparation of intermediate: methyl-(S)-2,3-diazabicyclo[3.1.1]heptane-4-carboxylate (II-9)

[0134] TFA (200 mL) was added sequentially to a DCM (1 L) solution of II-8 (63 g, 177 mmol) at 0 °C, and the reaction was carried out at 25 °C for 12 h. LC / MS indicated that the reaction was complete. After the reaction was completed, the residue was concentrated under reduced pressure, and then 1 L of methyl tert-butyl ether was added. After stirring at room temperature for 1 hour, the mixture was filtered, and the filter cake was dried to give intermediate II-9 (19 g, 69% yield), which was used directly in the next step without purification.

[0135] MS-ESI (m / z): 157.1 [M+l] + .

[0136] Preparation of intermediate a-7: (S)-1-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-morpholin-2-yl)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester (a-7)

[0137]

[0138] Step 1: Synthesis of (2R)-2-formylmorpholino-4-ylcarboxylate tert-butyl ester

[0139] At room temperature, TEMPO (310 mg, 2.3 mmol) and NaHCO3 (30 g, 348 mmol) were added to a solution of (2R)-2-(hydroxymethyl)morpholino-4-ylcarboxylate (a-1, 25.1 g, 115 mmol) in EtOAc (400 mL). The mixture was cooled to -50 °C, and then TCCA (27.5 g, 120 mmol) was added dropwise to EtOAc (100 mL) over 30 minutes. The reaction mixture was heated to 5 °C and maintained for 2 hours, then quenched with 10% Na2S2O3 (100 mL) and stirred for 20 minutes. The resulting mixture was filtered and the organic phase was separated. The aqueous phase was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with H2O (100 mL) and brine (100 mL), and then dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure to obtain an oily product (24.8 g, crude product). This product was used directly in the next reaction.

[0140] Step 2: Synthesis of tert-butyl (S,Z)-2-(2-(((benzoxy)carbonyl)amino)-3-methoxy-3-oxoprop-1-en-1-yl)morpholine-4-carboxylic acid

[0141] Tetramethylguanidine (35 g, 306 mmol) was added to a solution of (2R)-2-formylmorpholino-4-ylcarboxylate (48.0 g, 150 mmol) and methyl 2-{[(benzyloxy)carbonyl]amino}-2-(dimethoxyphosphoryl)acetate (60.4 g, 184 mmol) in MeCN (300 mL) at 0–10 °C. The reaction mixture was stirred at 10 °C for 30 min, then heated to room temperature and maintained for 2 h. The reaction mixture was diluted with DCM (200 mL) and washed with 10% citric acid (200 mL) and 10% NaHCO3 aqueous solution (200 mL). The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography to give a solid product (34.7 g, 87% yield). MS-ESI (m / z): 421.2 [M+l] + .

[0142] Step 3: Synthesis of tert-butyl (S)-2-((S)-2-(((benzoxy)carbonyl)amino)-3-methoxy-3-oxopropyl)morpholine-4-carboxylic acid

[0143] (S,S)-Et-DUPHOS-Rh (250 mg, 0.35 mmol) was added to a solution of (S,Z)-2-(2-(((benzyloxy)carbonyl)amino)-3-methoxy-3-oxoprop-1-en-1-yl)morpholino4-carboxylate (25.0 g, 60 mol) in MeOH (300 mL). The mixture was stirred at room temperature for 48 hours under a H2 (60 psi) atmosphere. The reaction mixture was concentrated and purified by silica gel column chromatography to give the product (20.3 g, 82% yield). MS-ESI (m / z): 423.2 [M+l] + .

[0144] Step 4: Synthesis of (S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-4-(tert-butoxycarbonyl)morpholin-2-yl)propionic acid

[0145] LiOH·H2O (3.5 g, 82.7 mmol) was added to a mixed solution of (S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-methoxy-3-oxopropyl)morpholine-4-carboxylic acid tert-butyl ester (11.5 g, 27.3 mmol) in THF (150 mL) and H2O (60 mL), and the mixture was reacted at 25 °C for 1 h. LC / MS indicated that the reaction was complete. After the reaction, the residue was concentrated under reduced pressure, and the mixture was acidified to pH 6 with 1 M HCl aqueous solution after adding an appropriate amount of water. Then, it was extracted with DCM (200 mL x 3), the organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude intermediate a-5 (9.2 g, 96% yield), which was used directly for the next step without purification. MS-ESI (m / z): 409.1 [M+l] + .

[0146] Step 5: Synthesis of tert-butyl (S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-3-(methoxycarbonyl)tetrahydropyridazine-1(2H)-yl)-3-oxopropyl)morpholine-4-carboxylic acid

[0147] To a DCM (50 mL) solution of intermediate a-5 (5.36 g, 13.1 mmol), methyl methyl (S)-hexahydropyridazine-3-carboxylate hydrochloride (3.6 g, 20 mmol), HOBt (350 mg, 2.6 mmol), EDCI (5.0 g, 26.0 mmol), and NMM (13.3 g, 131.0 mmol) were added at 0 °C, and the mixture was heated to 25 °C for 1 h. LC / MS indicated that the reaction was complete. After the reaction was complete, the mixture was quenched with an appropriate amount of water, then extracted with EtOAc (100 mL x 3). The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified by column chromatography to give intermediate a-6 (5.68 g, 81% yield). MS-ESI (m / z): 535.3 [M+l] + .

[0148] Step 6: Synthesis of methyl (S)-1-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-morpholin-2-yl)propionyl)hexahydropyridazine-3-carboxylate

[0149] At 15°C, HCl / EtOAc (15 mL) was added to a stirred solution of (S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-3-(methoxycarbonyl)tetrahydropyridazine-1(2H)-yl)-3-oxopropyl)morpholin-4-carboxylic acid tert-butyl ester (1.39 g, 2.6 mmol) in EtOAc (2 mL). The reaction was stirred at 15°C for 2 hours, followed by concentration under reduced pressure to give an oily intermediate a-7 (0.89 g, 79% yield).

[0150] MS-ESI (m / z): 435.3 [M+l] + .

[0151] Preparation of intermediate: (S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-morpholin-2-yl)propionyl)-2,3-diazabicyclo[3.1.1]heptane-4-carboxylic acid methyl ester (a-9)

[0152]

[0153] Following the method used for intermediate a-7, intermediate (a-9) (0.98 g, yield: 83%) was synthesized.

[0154] MS-ESI (m / z): 447.2 [M+l] + .

[0155] Intermediate b-6: (2 2 S, 6 3 S,4S)-4-amino-1 2 -(5-(1-Cyclopropyl-5,6-dihydro-1,2,4-triazin-4(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-1 1 -(2,2-trifluoroethyl)-6 1 6 2 6 3 6 4 6 5 6 6 -hexahydro-1 1 Preparation of H-8-oxa-2(4,2)-morpholin-1(5,3)-indole-6(1,3)-pyridazinylcycloundecane-5,7-dione (b-6)

[0156]

[0157] Step 1: Synthesis of (S)-5-bromo-3-(3-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpropyl)-2-(5-(1-cyclopropyl-5,6-dihydro-1,2,4-triazin-4(1H)-yl)-2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indole (b-1)

[0158] At 0 °C, TBSCl (51.0 g, 0.35 mol) in DCM (200 mL) was added to a solution of intermediate I-27 (136.8 g, 0.22 mol) and imidazole (31.0 g, 0.46 mol) in DCM (500 mL). The reaction was stirred at room temperature for 2 hours. The resulting solution was washed with H2O (3 × 300 mL) and brine (2 × 200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the product as a solid (137.6 g, 85% yield).

[0159] MS-ESI (m / z): 736.3 [M+l] + .

[0160] Step 2: Synthesis of (S)-1-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-4-(3-((tert-butyldimethylsiloxy)-2,2-dimethylpropyl)-2-(5-(1-cyclopropyl-5,6-dihydro-1,2,4-triazine-4(1H)-yl)-2-((S)-1-methoxyethylpyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)morpholin-2-yl)propionyl)hexahydropyridazine-3-carboxylate (b-2)

[0161] Intermediate a-7 (32.0 g, 99.0 mmol), RuPhos (16.7 g, 35.7 mmol), di-μ-chlorobis(2-amino-1,1-biphenyl-2-yl-C,N)dipalladium(II) (2.8 g, 4.4 mmol), and cesium carbonate (99 g, 305 mmol) were added to a stirred solution of intermediate b-1 (66.2 g, 90.0 mmol) in dioxane (500 mL). Then, RuPhos Pd-G2 (3.5 g, 4.4 mmol) was added at 105 °C under a N2 atmosphere. The reaction mixture was stirred at 105 °C for 6 hours under a N2 atmosphere. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC to give a solid product (65.7 g, 67% yield). MS-ESI (m / z): 1102.6 [M+l] + .

[0162] Step 3: Synthesis of (S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-4-(3-((tert-butyldimethylsiloxy)-2,2-dimethylpropyl)-2-(5-(1-cyclopropyl-5,6-dihydro-1,((S)-1-methoxyethylpyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)morpholin-2-yl)propionyl)-2,3-diazabicyclo[3.1.1]heptane-4-carboxylic acid (b-3)

[0163] At room temperature, LiOH (1.1 g, 30 mmol) in H₂O (50 mL) was added to a solution of intermediate b-2 (10.9 g, 10 mmol) in THF (270 mL). The reaction was stirred at room temperature for 2 hours, followed by treatment with 1 N HCl at 0–5 °C to adjust the pH to 4–5. The resulting mixture was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The organic phase was then concentrated under reduced pressure to give intermediate b-3 (10.0 g, 93% yield) as a solid, which was used directly in the next reaction.

[0164] MS-ESI (m / z): 1076.6 [M+l] + .

[0165] Step 4: Synthesis of (S)-1-((S)-2-((benzyloxy)carbonyl)amino)-3-(S)-4-(2-(5-(1-cyclopropyl-5,6-dihydro-1,2,4-triazine-4(1H)-yl)-2-(S)-1-methoxyethyl)pyridin-3-yl)-3-(3-hydroxy-2,2-dimethylpropyl)-1-(2,2-trifluoroethyl)-1H-indole)morpholin-2-yl)propionyl)hexahydropyridazine-3-carboxylic acid (b-4)

[0166] At room temperature, a mixture of tetrabutylammonium fluoride (1M in THF, 200mL, 200mmol) and AcOH (11g, 200mmol) was added to a solution of intermediate b-3 (10.0g, 10mmol) in THF (10mL). The reaction mixture was stirred at 75°C for 3 hours. The resulting mixture was diluted with EtOAc (150mL) and washed with HO (3 x 20mL). The organic phase was concentrated under reduced pressure to give intermediate b-4 as a solid product (8.66g, 90% yield), which was used directly for the next reaction. MS-ESI (m / z): 962.5 [M+l] + .

[0167] Step 5: Benzyl((2) 2 S, 6 3S, 4S)-1 2 -(5-(1-Cyclopropyl-5,6-dihydro-1,2,4-triazin-4(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1 1 -(2,2-trifluoroethyl)-6 1 6 2 6 3 6 4 6 5 6 6 -hexahydro-1 1 Synthesis of H-8-oxa-2-(4,2)-morpholin-1(5,3)-indole-6-(1,3-pyridazinylundecane-4-yl)carbamate (b-5)

[0168] Under an argon atmosphere, at room temperature, EDCI (88 g, 458 mmol) and HOBt (27.6 g, 204 mmol) were added to a stirred solution of intermediate b-4 (8 g, 10.2 mmol) and DIPEA (59 g, 459 mmol) in DCM (800 mL). The reaction mixture was stirred for 16 hours at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give intermediate b-5 (5 g, 66% yield) as a solid. MS-ESI (m / z): 944.5 [M+l] + .

[0169] Step 6: (2) 2 S, 6 3 S,4S)-4-amino-1 2 -(5-(1-Cyclopropyl-5,6-dihydro-1,2,4-triazin-4(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-1 1 -(2,2-trifluoroethyl)-6 1 6 2 6 3 6 4 6 5 6 6 -hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-morpholin-1(5,3)-indole-6(1,3)-pyridazinylcycloundecane-5,7-dione (b-6)

[0170] Under H2 conditions, at room temperature, Pd / C (400 mg) and ammonium acetate (1.67 g, 32 mmol) were added to a solution of intermediate b-5 (944 mg, 1.0 mmol) in MeOH (40 mL), and the mixture was stirred for 2 hours. The resulting mixture was filtered and concentrated under reduced pressure. The residue was redissolved in DCM (50 mL) and washed with H2O (10 mL × 2), followed by concentration under reduced pressure to give a solid product (320 mg, 97% yield), which was used directly for the next reaction. MS-ESI (m / z): 810.4 [M+l] + .

[0171] Following the method used for intermediate b-6, intermediates b-7, b-8, and b-9 were synthesized, and their corresponding structural information is shown below:

[0172]

[0173]

[0174] Preparation of intermediate c5: (S)-2-cyclopentyl-2-((S)-2,7-diazaspiro[4.4]nonane-2-yl)benzyl acetate ((c5))

[0175]

[0176] Step 1: Synthesis of (R)-2-cyclopentyl-2-(((trifluoromethyl)sulfonyl)oxy)benzyl acetate (c2)

[0177] Under N2 conditions, Tf2O (3.8 g, 13.5 mmol) and 2,6-dimethylpyridine (1.5 g, 14.0 mmol) were added to a solution of (R)-2-cyclopentyl-2-hydroxyacetic acid benzyl ester (c1, 2.3 g, 10 mmol) in DCM (50 mL) at 0 °C, and the mixture was stirred at 0 °C for 2 hours. The residue was diluted with H2O (30 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (2 × 50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give product c2, which was used directly without further purification.

[0178] MS-ESI (m / z): 367.1 [M+l] + .

[0179] Step 2: Synthesis of tert-butyl 7-((S)-2-(benzoxy)-1-cyclopentyl-2-oxoethyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate (c3)

[0180] To a solution of intermediate c2 (4.8 g, 13.0 mmol) in THF (20 mL), tert-butyl 2,7-diazaspiro[4.4]nonane-2-carboxylate (1.96 g, 8.8 mmol) and Cs₂CO₃ (10.6 g, 30.0 mmol) were added. The mixture was stirred for 30 min at room temperature. The residue was diluted with H₂O (30 mL) and extracted with EtOAc (3 × 30 mL). The combined organic layers were washed with brine (2 × 40 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give intermediate c3 (2.2 g, 56% yield) as a solid.

[0181] MS-ESI (m / z): 443.3 [M+l] + .

[0182] Step 3: Synthesize (S)-2-((R)-7-(tert-butoxycarbonyl)-2,7-diazaspiro[4.4]non-2-yl)-2-cyclopentylacetic acid and (S)-2-((S)-7-(tert-butoxycarbonyl)-2,7-diazaspiro[4.4]non-2-yl)-2-cyclopentylacetic acid c4.

[0183] At 15°C, HCl / EtOAc (15 mL) was added to a stirred solution of intermediate c3 (1.15 g, 2.6 mmol) in EtOAc (2 mL). The mixture was stirred at 15°C for 2 hours, followed by concentration under reduced pressure to obtain an oily intermediate (c4).

[0184] The residual intermediate C4 was purified by silica gel column chromatography (20→50% EtOAc / petroleum ether) to obtain intermediate C5: (S)-2-cyclopentyl-2-((S)-2,7-diazaspiro[4.4]nonane-2-yl)benzyl acetate (394 mg, 20% yield).

[0185] MS-ESI (m / z): 343.2 [M+l] + .

[0186] Intermediate C6: (S)-2-cyclopentyl-2-((R)-2,7-diazaspiro[4.4]nonane-2-yl)benzyl acetate (355 mg, 18% yield). MS-ESI (m / z): 343.2 [M+l] + .

[0187] Preparation of intermediate C12: (2R,3R)-3-cyclopropyl-1-methylazacyclopropane-2-carboxylic acid (C12)

[0188]

[0189] Step 1: Synthesis of (R,E)-N-(cyclopropylmethylene)-4-methylbenzenesulfinamide (C8)

[0190] Under N2, at room temperature, (R)-4-methylbenzenesulfinamide (15.53 g, 85.60 mmol) and Ti(OEt)4 (40.0 g, 172.1 mmol) were added to a solution of cyclopropaneformaldehyde (7.0 g, 100.0 mmol) in THF (150 mL). The mixture was stirred at 75 °C for 2 hours. The reaction mixture was poured into brine / H2O (1:1, 600 mL) at 0–15 °C. The mixture was filtered through a diatomaceous earth mat, and the mat was washed with EtOAc (6 × 200 mL). The combined filtrates were extracted with EtOAc (2 × 200 mL). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The crude product was purified by silica gel column chromatography to give product c8 (14.3 g, 76% yield) as a solid.

[0191] MS-ESI (m / z): 188.2 [M+l] + .

[0192] Step 2: Synthesis of (2R,3R)-3-cyclopropyl-1-((R)-p-tolylsulfinyl)azacyclopropane-2-carboxylic acid ethyl ester (c9)

[0193] Under N2, LiHMDS (1M, 142.0mL) was added to a solution of ethyl 2-bromoacetate (24.2g, 141.6mmol) in THF (500mL) for 10 minutes at -70°C. The mixture was stirred at -70°C for 20 minutes. An intermediate c8 solution (14.3g, 70.0mmol) in THF (150mL) was added to the reaction solution at -70°C and maintained for 10 minutes. The mixture was then stirred at -70°C for 1 hour under N2. The reaction mixture was poured into cold H2O (1.0L) and stirred at room temperature for 5 minutes. The aqueous layer was extracted with EtOAc (3 × 300mL). The combined organic layers were washed with brine (300mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The crude product was purified by silica gel column chromatography to give an oily product c9 (11g, 53% yield).

[0194] MS-ESI (m / z): 294.2 [M+l] + .

[0195] Step 3: Synthesis of (2R,3R)-3-cyclopropylazacyclopropane-2-carboxylic acid ethyl ester (C10)

[0196] Intermediate C9 (6.1 g, 20.8 mmol) was dissolved in anhydrous THF (300 mL). MeMgBr (3 M, 14.0 mL) was added dropwise over 40 minutes at -65 °C under N2. The reaction mixture was stirred for 10 minutes. A saturated aqueous solution of NH4Cl (90 mL) was added dropwise at -65 °C. The cooling bath was removed, and the reaction mixture was warmed to room temperature. EtOAc (300 mL) was added, and the organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the oily product C10 (2.21 g, 68% yield). MS-ESI (m / z): 156.1 [M+l] + .

[0197] Step 4: Synthesis of (2R,3R)-3-cyclopropyl-1-methylazacyclopropane-2-carboxylic acid ethyl ester (C11)

[0198] Methylboric acid (1.85 g, 30.9 mmol), 2,2'-bipyridine (1.61 g, 10.3 mmol), Cu(OAc)₂ (1.87 g, 10.3 mmol), and Na₂CO₃ (32.8 g, 30.9 mmol) were added to a solution of intermediate c10 (2.0 g, 10.3 mmol) in DCE (30 mL). The reaction mixture was stirred at 45 °C for 40 h. The mixture was poured into an aqueous solution of NH₄Cl (50 mL) and extracted with DCM (3 × 50 mL). The combined organic phases were washed with brine (80 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the oily product c11 (823.0 mg, 47% yield). MS-ESI (m / z): 170.1 [M+l] + .

[0199] Step 5: (2R,3R)-3-cyclopropyl-1-methylazacyclopropane-2-carboxylic acid (C12)

[0200] A solution of LiOH•H₂O (168.0 mg, 4.0 mmol) in H₂O (2 mL) was added to a solution of intermediate c11 (340 mg, 2.0 mmol) in THF (2 mL). The reaction mixture was stirred for 1 hour at room temperature. The pH was adjusted to approximately 3 with 0.5 N HCl at 0 °C, and the solution was directly lyophilized to obtain product c12 (244 mg, 86%) as a solid, which was used directly in the next reaction.

[0201] MS-ESI (m / z): 142.1 [M+l] + .

[0202] Preparation of intermediate: (2S)-2-cyclopentyl-2-((5S)-7-((3R)-3-cyclopropyl-1-methylaziridin-2-carbonyl)-2,7-diazaspiro[4.4]non-2-yl)acetic acid (c14)

[0203]

[0204] Step 1: Synthesis of (2S)-2-cyclopentyl-2-((5S)-7-((3R)-3-cyclopropyl-1-methylaziridin-2-carbonyl)-2,7-diazaspiro[4.4]non-2-yl)benzyl acetate (c13)

[0205] Intermediate c-12 (141.0 mg, 1.0 mmol), HOBt (350 mg, 2.6 mmol), EDCI (384.6 mg, 2.0 mmol), and NMM (1.3 g, 13.0 mmol) were added to a DCM (20 mL) solution of intermediate c-5 (342.0 mg, 1.0 mmol) at 0 °C, and the mixture was heated to 25 °C and reacted for 2 hours. LC / MS indicated that the reaction was complete. After the reaction was complete, the mixture was quenched with an appropriate amount of water, then extracted with EtOAc (15 mL x 3). The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified by column chromatography to obtain intermediate c13 (391.4 mg, 84% yield).

[0206] MS-ESI (m / z): 466.3 [M+l] + .

[0207] Step 2: Synthesis of 2S)-2-cyclopentyl-2-((5S)-7-((3R)-3-cyclopropyl-1-methylaziridin-2-carbonyl)-2,7-diazaspiro[4.4]non-2-yl)acetic acid (c14)

[0208] A solution of (2S)-2-cyclopentyl-2-((5S)-7-((3R)-3-cyclopropyl-1-methylaziridin-2-carbonyl)-2,7-diazaspiro[4.4]non-2-yl)acetic acid benzyl ester (933.5 mg, 2.0 mmol) in THF (10 mL) was added to a solution of LiOH●H2O (168.0 mg, 4.0 mmol) in H2O (5 mL). The reaction mixture was stirred for 2 hours at room temperature. The pH was adjusted to about 1 with 0.5 N HCl at 0 °C, and then extracted with EtOAc (20 mL x 3). The organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified by column chromatography to give intermediate C14 (609.1 mg, 81% yield).

[0209] MS-ESI (m / z): 376.3 [M+l] + .

[0210] Example 1

[0211] (2S)-2-cyclopentyl-2-((S)-7-((2R,3R)-3-cyclopropyl-1-methylaziridin-2-carbonyl)-2,7-diazaspiro[4.4]nonane-2-yl)-N-((2 2 S, 6 3 S, 4S)-1 2 -(5-(4-cyclopropyl-5,6-dihydro-1,2,4-triazin-1(4H)-yl)-2-((S-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxa-1 1 -(2,2,2-trifluoroethyl)-6 1 6 2 6 3 6 4 6 5 6 6 Preparation of hexahydro-11H-8-oxa2-(4,2)-morpholin-1-(5,3)-indole-6-(1,3-pyridazinylcycloundecane-4-yl)acetamide

[0212]

[0213] Intermediate c-14 (93.8 mg, 0.25 mmol), DIEA (130 mg, 1.0 mmol), and HATU (228 mg, 0.6 mmol) were added sequentially to a DMF (5 mL) solution of intermediate b-7 (162.0 mg, 0.2 mmol) at 0 °C, and the mixture was heated to 25 °C and reacted for 3 hours. LC / MS indicated that the reaction was complete. After the reaction was complete, the mixture was quenched with an appropriate amount of water, then acidified to pH 6.0 with 1 M HCl aqueous solution, extracted with EtOAc (10 mL x 3), and the organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the residue. The residue was further purified by preparative HPLC to give compound 1 (165.7 mg, 71% yield).

[0214] MS-ESI (m / z): 1167.69 [M+l] + .

[0215] 1H NMR(400MHz,DMSO-d6)δ8.74(d,J=2.0Hz,1H),7.58(m,1H),7.22-6.95(m,4H),6.80(s, 1H),5.30-5.01(m,5H),4.91(s,2H),4.87-4.02(m,12H),3.76(m,5H),3.73-3.64(m,4H) ,3.42(m,6H),3.25(m,4H),3.06-2.97(m,5H),2.81(m,3H),2.71(m,4H),2.30(m,2H),2 .22-1.86(m,4H),1.42(m,3H),1.11-1.05(m,4H),1.03-0.82(m,7H),0.59-0.32(m,8H).

[0216] Referring to the route method of Example 1, Examples 2-4 were synthesized accordingly, and the specific structural information is shown in Table 1.

[0217] Table 1

[0218]

[0219] Example 5 Biological Test

[0220] Biological screening and results of RAS inhibitors

[0221] Test 1: In vitro cell proliferation inhibition assay

[0222] Due to the diversity of RAS mutations, and in order to evaluate the activity of the test compound in different RAS mutant cell lines, KRAS was selected. WT and KRAS G12D The cell lines used for mutation assays (see Table 2 below, all provided by the Cell Bank of the Chinese Academy of Sciences) were used for in vitro activity evaluation and screening of the compounds.

[0223] Cell phosphorylation assay

[0224] Experimental Protocol: Ceirnter-Gkrli Cell Luminescent Viability Assay (Promega)

[0225] Preparation of compounds: Each accurately weighed test compound was completely dissolved in 100% DMSO and prepared into a 20 mM stock solution for storage in a refrigerator at 4°C in the dark.

[0226] Preparation of complete culture medium: Add 45 mL of RPMI-1640 (Gibco) basal medium, 5 mL of fetal bovine serum and 0.5 mL of penicillin / streptomycin solution to a 50 mL centrifuge tube, mix well to prepare complete culture medium, and store at 4 °C for cell culture.

[0227] 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 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 test 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, with 10 concentration gradients of 3-fold dilutions. After co-incubation of cells and compounds for 72 h, the 96-well plates were removed from the incubator and allowed to equilibrate at room temperature for 30 min. Then, 25 μL of CellTiter 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), fluorescence signal values ​​were read using a multi-functional microplate reader. 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. 50 This was used to evaluate the antiproliferative activity of the compounds of the present invention against KRAS cell mutants.

[0228] Table 2 shows the activity assay data for the compounds and reference compounds of the embodiments of the present invention.

[0229] Table 2. Data on the activity assay of the test compounds (examples and reference compounds of the present invention) IC 50 (nM)

[0230]

[0231] The structure of the reference compound RMC-9805 is as follows:

[0232] The data in the table show that Examples 1-4 have superior KRAS compared to RMC-9805. G12D Inhibitory activity, and IC 50 Both decreased by more than 29%.

[0233] Test 2: KRAS G12DAssay of AsPC-1pERK activity in cell line

[0234] AsPC-1 cells were cultured in 1640 medium containing 10% fetal bovine serum and double antibiotics (streptomycin 100 μg / ml and penicillin 100 μg / ml) in a cell culture incubator at 37°C, 5% CO2, and 90% humidity. The cell culture medium was changed approximately every 72-96 hours. When the AsPC-1 cells reached the logarithmic growth phase, they were passaged using trypsin digestion (trypsin treatment time should not exceed 6 minutes to avoid cell damage) for further culture or experiments. For testing, cells were cultured at a density of 20,000 cells / well in 96-well plates with a black transparent bottom and allowed to adhere for 12-14 hours. Subsequently, they were treated with the test compound, serially diluted 3-fold, with a maximum concentration of 10 μM, for a total of 10 concentration gradients, including a DMSO control. After 3 hours of treatment, the cells were fixed by incubation in 50 μl of 4.0% formaldehyde for 20 minutes at room temperature. The formaldehyde was then removed, and 150 μl of ice-cold methanol was added for 10 minutes to allow cell permeation, followed by removal of the methanol. 100 μl of blocking buffer (Licor) was added at room temperature and reacted for 1 hour to inhibit the binding of nonspecific antibodies. The amount of phosphorylated ERK (pERK) was determined using an antibody specific to the phosphorylated form of ERK and compared with the amount of GAPDH. The primary antibodies used for detection were added as follows: phosphorylated ERK (Cell signaling) diluted 1:500, and GAPDH (Abcam) diluted 1:800. The secondary antibodies used for visualization were added as follows: goat anti-rabbit-800 (Licor) and goat anti-mouse-680 (Licor) were both diluted 1:800 in Odyssey blocking buffer + 0.05% Tween 20 and incubated at room temperature for 1 hour. The plate was washed three times with 150 μl of PBS + 0.1% Tween 20. The plate was dried and imaged using a Li-COR Odyssey CLX dual-color infrared laser imaging system. The plate was analyzed by normalizing the phosphorylated ERK (Thr202 / Tyr204) signal of each well to a GAPDH signal, and the percentage of DMSO control values ​​was calculated. IC50 was generated using a four-parameter logistic fitting method of the dose-response curve. 50 value.

[0235] Table 3 shows the activity assay data for the compounds and reference compounds of the embodiments of the present invention.

[0236] Table 3. Data on the activity assay of the test compounds (examples and reference compounds of the present invention) IC 50 (nM)

[0237] Test compound AsPC-1pERK IC50(nM) Example 1 11 Example 2 10 Example 3 9 Example 4 7 RMC-9805 19

[0238] The data in the table show that compounds 1-4 in the embodiments of the present invention have significantly improved inhibitory activity compared with RMC-9805, and their IC50 values ​​are all reduced by more than 42%.

[0239] Test 3: Evaluation of compound stability using human liver microsomes

[0240] The liver microsomal enzyme stability of the compounds in the examples was compared with that of RMC-9805.

[0241] Assay System: The metabolic stability of the test compounds was tested using liver microsomes from mixed male and female samples with 1.0 mM NADPH. Samples were analyzed using mass spectrometry. HRMS was used to determine the peak area response ratio (corresponding to the peak area of ​​the test compound or control divided by the peak area of ​​the analytical internal standard) without running a standard curve. HRMS scans were performed within an appropriate m / z range to detect all possible metabolites.

[0242] Assay conditions: This assay was performed using a single incubation (N=1). The test compound was incubated at 37°C in a buffer containing 0.5 mg / mL liver microsomal protein. The reaction was initiated by adding a cofactor, and samples were taken at 0, 2, 4, 8, 16, and 24 hours. The positive control (5 μM testosterone) was incubated in parallel, and samples were taken at 0, 2, 4, 8, 16, and 24 hours.

[0243] Quality control assays: Testosterone, a control compound, was performed in parallel to confirm the enzyme activity in (liver) microsomes. After the final time point, NADPH was added to the reaction mixture using fluorescence assay. The T1 / 2 of the control compound met acceptable internal standard requirements.

[0244] Analysis method:

[0245] Liquid chromatography column: Thermo BDS Hypersil C18 30X2.0mm, 3μm, with guard column MP; buffer: 25mM formic acid buffer, pH 3.5;

[0246] Aqueous phase (A): 90% water, 10% buffer solution;

[0247] Organic phase (B): 90% acetonitrile, 10% buffer solution;

[0248] Flow rate: 300 microliters / minute

[0249] Automated sampler: injection volume 10 μL

[0250] See Table 4 for the gradient procedure.

[0251] Table 4. Gradient Procedure

[0252] Time (minutes) ％A ％B 0.0 100 0 1.5 0 100 2.0 0 100 2.1 100 0 3.5 100 0

[0253] The measured stability results are shown in Table 5.

[0254] Table 5. Stability results of compounds in human liver microsomes

[0255]

[0256] Using human liver microsomes, the results showed that Examples 1-4 had better metabolic stability than RMC-9805, and their relatively long metabolic half-life gave them the potential to reduce medical dosage and extend dosing intervals.

[0257] Although the invention has been described through specific embodiments above, it should not be construed as being limited thereto; rather, the invention encompasses the general aspects previously disclosed. Various modifications and embodiments are possible without departing from the spirit and scope of the invention.

Claims

1. A compound having the structure shown in general formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug thereof: In the formula: X is CR3, Y is N; or X is N, Y is CR3; R1, R2, and R3 are independently selected from H and C, respectively. 1-3 Alkyl, Halogenated C 1-3 Alkyl, or C 3-6 cycloalkyl; L is selected from -CH2- or empty bond.

2. The compound according to claim 1 and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope-labeled compounds, and prodrugs, wherein the compound is selected from:

3. The compounds according to claims 1-2 and their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope-labeled substances, and prodrugs, characterized in that, The pharmaceutically acceptable salt is an inorganic or organic salt. The inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, nitrate, phosphate, and acid phosphate. The organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, pyruvic acid, acetic acid, succinic acid, phenylacetic acid, mandelic acid, and ferulic acid.

4. A pharmaceutical composition, characterized in that, It comprises the compound of any one of claims 1-3 or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label or prodrug, and pharmaceutical excipients.

5. The composition according to claim 4, characterized in that, The pharmaceutical excipients include pharmaceutically acceptable carriers, excipients, or diluents.

6. The composition according to claim 5, characterized in that, Pharmaceutically acceptable carriers include microspheres, nanoparticles, and liposomes.

7. The composition according to claim 4, characterized in that, The dosage forms of the pharmaceutical composition include injections, lyophilized powder for injection, suspensions, implants, embolic agents, capsules, tablets, pills, and oral solutions.

8. Use of the compound of any one of claims 1-3 or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug in the preparation of a drug for inhibiting RAS protein.

9. Use of the compound of any one of claims 1-3 or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug in a medicament for the prevention or treatment of RAS-mutant and RAS-dependent tumors.

10. The use according to claim 9, characterized in that, The RAS mutations and RAS-dependent tumors are solid tumors.

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