CN116554208A - Substituted bicyclic heteroaryl compounds as KRAS G12D inhibitors - Google Patents

Abstract

The present invention provides a substituted bicyclic heteroaryl compound as a KRAS G12D inhibitor. The invention also provides pharmaceutical compositions comprising the compounds and their use in the treatment of cancer.

Description

Substituted bicyclic heteroaryl compounds as KRAS G12D inhibitors

The invention requires the following priorities:

CN202211529648.9, application day 2022, 12 months 2; CN202310503687.X, application day 2023, 5, 6.

Technical Field

The invention belongs to the field of medicines, and particularly relates to a substituted bicyclic heteroaryl compound which can be used as a KRAS G12D inhibitor.

Background

The human RAS gene family comprises 3 classes of RAS genes KRAS, NRAS and HRAS, encoding four different RAS proteins (KRAS-4 a, KRAS-4b, NRAS and HRAS). RAS proteins belong to the GTPase protein family, which is inactive when bound to GDP, but active when bound to GTP, and can lead to activation of signaling pathways such as downstream RAF-MAPK, PI3K-Akt, etc., leading to anti-apoptotic and proliferative effects of cells (Cell, 2017;170 (1): 17-33; cell,2020;183 (4): 850-859;Nat Rev Drug Discov,2020;19 (8): 533-552.). Activating mutations in the RAS gene are the most common oncogene driver in human cancers, with KRAS most frequently undergoing oncogenic activating mutations. For example, KRAS has a mutation rate of 86-96% in pancreatic cancer, 40-54% in colorectal cancer, and 27-39% in lung cancer (PNAS, 2019;116 (32): 15823-15829;Pathol Res Pract,2009;205,858-862; nature,2012;491,399-405; nature,2014;511, 543-550).

Oncogenic driving mutations can occur at various sites in the KRAS gene, most commonly at the G12 site, including G12C, G12D, G V, and these mutations can reduce the GTPase activity of the KRAS protein, thereby resulting in the KRAS protein being active for a long period of time, resulting in malignant transformation of the Cell and carcinogenesis (Cell, 2017;170 (1): 17-33;Nat Rev Drug Discov,2020;19 (8): 533-552). The frequently occurring KRAS mutation types vary among different cancer types, e.g., about 13% of lung cancer patients have G12C mutations (N Engl Med J,2021;384 (25): 2382-2393); whereas in pancreatic cancer, 33.8% of patients develop G12D mutations, but only 1.7% of patients develop G12C mutations; in colorectal Cancer, about 10-12% of patients develop G12D mutations, while the incidence of G12C mutations is less than 3% (Nat Rev Cancer 2018;18 (12): 767-777).

Unlike ATP-dependent protein kinases (affinity of proteins to ATP at the micromolar level), KRAS proteins have affinities to GDP/GTP at the picomolar level, compound molecules have difficulty competing effectively with GDP/GTP production to inhibit KRAS signaling pathways, which severely hampers the development of KRAS inhibitors (Cell, 2017;170 (1): 17-33; cell,2020;183 (4): 850-859;Nat Rev Drug Discov,2020;19 (8): 533-552). In recent years, "allosteric" binding cavities have been found on KRAS proteins that compete with non-GDP/GTP for efficient binding to small molecules, which greatly facilitate the development of targeted drugs that target KRAS mutation-driven tumors. Currently, MRTX-849 (Adagrasib) and AMG510 (Sotorasib) targeting KRAS G12C have demonstrated excellent efficacy in clinical studies (N Engl JMED.2020;383 (13): 1207-1217;Cancer Discov.2020;10 (1): 54-71), and AMG510 has been successfully marketed under FDA approval at month 5 of 2021. Drug development targeting KRAS G12D mutations is also in an early stage relative to KRAS G12C drug development; however, as indicated above, there is a very high KRAS G12D mutation rate in many tumors and the development of KRAS G12D inhibitors is of great importance.

The U.S. Mirati company reports that the KRAS G12D small molecule MRTX1133 is administrated intravenously, but the invention does not enter clinical research, and the targeting KRAS G12D small molecule drug has excellent in vivo efficacy and greater safety, so as to solve the clinical unmet needs.

Disclosure of Invention

In one aspect, the invention provides a compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, prodrug, polymorph, hydrate, or solvate thereof, wherein the compound is selected from the group consisting of:

in another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention, and optionally a pharmaceutically acceptable excipient.

In another aspect, the invention provides pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable excipient, which also contains an additional therapeutic agent.

In another aspect, the invention provides the use of a compound of the invention in the manufacture of a medicament for the treatment and/or prophylaxis of KRAS G12D mutein mediated diseases.

In another aspect, the invention provides a method of treating and/or preventing KRAS G12D mutein-mediated diseases in a subject comprising administering to the subject a compound of the invention or a composition of the invention.

In another aspect, the invention provides a compound of the invention or a composition of the invention for use in the treatment and/or prevention of KRAS G12D mutein-mediated diseases.

In particular embodiments, the disease treated by the present invention includes a cancer selected from the group consisting of: acute myeloid leukemia, juvenile cancer, childhood adrenocortical carcinoma, AIDS-related cancers (e.g., lymphoma and kaposi's sarcoma), anal carcinoma, appendicular carcinoma, astrocytoma, atypical teratoid, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone carcinoma, brain stem glioma, brain tumor, breast carcinoma, bronchial tumor, burkitt lymphoma, carcinoid tumor, atypical teratoid, embryo tumor, germ cell tumor, primary lymphoma, cervical cancer, childhood cancer, chordoma, heart tumor, chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngeal tumor, cutaneous T cell lymphoma, extrahepatic Duct Carcinoma (DCIS), embryo tumor CNS cancers, endometrial cancers, ependymomas, esophageal cancers, olfactory neuroblastomas, ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, eye cancers, skeletal fibroblastic tumors, gall bladder cancers, stomach cancers, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastoma, hairy cell leukemia, head and neck cancers, heart cancers, liver cancers, hodgkin's lymphoma, hypopharynx cancers, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancers, laryngeal cancers, lip and oral cancers, liver cancers, lobular Carcinoma In Situ (LCIS), lung cancers, lymphomas, metastatic squamous neck cancers with harboring primary foci, mesogenic cancers, oral cancers, multiple endocrine tumor syndromes, multiple myeloma/plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasms, multiple myeloma, merkel cell carcinoma, malignant mesothelioma, malignant fibrous histiocytoma and osteosarcoma of the bones, nasal and sinus cancers, nasopharyngeal carcinoma, neuroblastoma, non-hodgkin's lymphoma, non-small cell lung cancer (NSCLC), oral cancer, lip and oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pleural-lung blastoma, primary Central Nervous System (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, gastric cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymus cancer, thyroid cancer, transitional cell carcinoma of the kidney and ureter, cell tumor, rare cancer, vaginal cancer, uterine sarcoma, cancer, vulval cancer or virus-induced cancer, preferably cancer of the urinary tract or non-small intestine, colorectal cancer, pancreatic cancer or non-small cell carcinoma.

Other objects and advantages of the present invention will be apparent to those skilled in the art from the detailed description, examples, and claims that follow.

Definition of the definition

The term "KRAS G12D" refers to a mutant form of a mammalian KRAS protein that contains an aspartic acid to glycine amino acid substitution at amino acid position 12.

The term "pharmaceutically acceptable salts" as used herein means those carboxylate salts, amino acid addition salts of the compounds of the invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response and the like commensurate with a reasonable benefit/risk ratio, and effective for their intended use, including (if possible) zwitterionic forms of the compounds of the invention.

The "subject" to be administered includes, but is not limited to: a human (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle aged adults, or senior adults)) and/or a non-human animal, e.g., a mammal, e.g., a primate (e.g., cynomolgus monkey, rhesus monkey), cow, pig, horse, sheep, goat, rodent, cat, and/or dog. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human", "patient" and "subject" are used interchangeably herein.

"disease," "disorder," and "condition" are used interchangeably herein.

In general, an "effective amount" of a compound refers to an amount sufficient to elicit a biological response of interest. As will be appreciated by those of ordinary skill in the art, the effective amount of the compounds of the present invention may vary depending on the following factors: for example, biological targets, pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age health and symptoms of the subject. The effective amount includes a therapeutically effective amount and a prophylactically effective amount.

"combination" and related terms refer to the simultaneous or sequential administration of a compound of the invention and another therapeutic agent. For example, the compounds of the invention may be administered simultaneously or sequentially with other therapeutic agents in separate unit dosage forms, or simultaneously with other therapeutic agents in a single unit dosage form.

Detailed description of the preferred embodiments

Herein, "the compounds of the present invention" refers to the following compounds, pharmaceutically acceptable salts, enantiomers, diastereomers, solvates, hydrates or isotopic variants thereof, and mixtures thereof.

In one embodiment, the invention relates to a compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, prodrug, polymorph, hydrate, or solvate thereof, wherein the compound is selected from the group consisting of:

The compounds of the invention may include one or more asymmetric centers and thus may exist in a variety of stereoisomeric forms, for example, enantiomeric and/or diastereomeric forms. For example, the compounds of the invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. The isomers may be separated from the mixtures by methods known to those skilled in the art, including: chiral High Pressure Liquid Chromatography (HPLC), formation and crystallization of chiral salts; alternatively, preferred isomers may be prepared by asymmetric synthesis.

The compounds of the invention may also exist as tautomers. Compounds that exist in different tautomeric forms, one of the compounds is not limited to any particular tautomer, but is intended to encompass all tautomeric forms.

Those skilled in the art will appreciate that the organic compound may form a complex with a solvent in or from which it reacts or from which it precipitates or crystallizes. These complexes are referred to as "solvates". When the solvent is water, the complex is referred to as a "hydrate". The present invention encompasses all solvates of the compounds of the present invention.

The term "solvate" refers to a form of a compound or salt thereof that is bound to a solvent, typically formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, for example, in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "solvate" includes both solvates in solution and separable solvates. Representative solvates include hydrates, ethanolates and methanolates.

The term "hydrate" refers to a compound that binds to water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Thus, the hydrates of the compounds can be used, for example, of the formula R x H ₂ O represents, wherein R is the compound, and x is a number greater than 0. A given compound may form more than one hydrate type, including, for example, monohydrate (x is 1), lower hydrate (x is a number greater than 0 and less than 1, e.g., hemihydrate (r.0.5H) ₂ O)) and polyhydrates (x is a number greater than 1, e.g., dihydrate (r.2h) ₂ O) and hexahydrate (R.6H) ₂ O))。

The compounds of the present invention may be in amorphous or crystalline form (polymorphs). Furthermore, the compounds of the present invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the present invention. The term "polymorph" refers to a crystalline form (or salt, hydrate or solvate thereof) of a compound of a particular crystal stacking arrangement. All polymorphs have the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can lead to a crystalline form predominating. Various polymorphs of a compound can be prepared by crystallization under different conditions.

The invention also includes isotopically-labelled compounds (isotopically-variant) which are identical to those recited in formula (a), but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively, for example ² H、 ³ H、 ¹³ C、 ¹¹ C、 ¹⁴ C、 ¹⁵ N、 ¹⁸ O、 ¹⁷ O、 ³¹ P、 ³² P、 ³⁵ S、 ¹⁸ F and F ³⁶ Cl. The compounds of the invention, prodrugs thereof, and pharmaceutically acceptable salts of the compounds or prodrugs thereof, which contain the isotopes described above and/or other isotopes of other atoms, are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, e.g., for incorporation of a radioisotope (e.g. ³ H and ¹⁴ c) Those useful in drug and/or substrate tissue distribution assays. Tritium, i.e. tritium ³ H and carbon-14 ¹⁴ The C isotopes are particularly preferred because they are easy to prepare and detect. Further, substitution by heavier isotopes, e.g. deuterium, i.e ² H may be preferred in some cases because higher metabolic stability may provide therapeutic benefits, such as extended in vivo half-life or reduced dosage requirements. Isotopically-labeled compounds of formula (a) of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes and/or examples and preparations below by substituting a readily available isotopically-labeled reagent for a non-isotopically-labeled reagent.

In addition, prodrugs are also included within the context of the present invention. The term "prodrug" as used herein refers to a compound that is converted in vivo by hydrolysis, e.g. in blood, into its active form having a medical effect. Pharmaceutically acceptable prodrugs are described in t.higuchi and v.stilla, prodrugs as Novel Delivery Systems, a.c. s.symposium Series vol.14, edward b.roche, ed., bioreversible Carriers in Drug Design, american Pharmaceutical Association and Pergamon Press,1987, and d.fleisher, s.ramon and h.barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs ", advanced Drug Delivery Reviews (1996) 19 (2) 115-130, each of which is incorporated herein by reference.

Pharmaceutical compositions and kits

In another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a compound of the present invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a compound of the invention. In some embodiments, the pharmaceutical composition comprises a prophylactically effective amount of a compound of the present invention.

Pharmaceutically acceptable excipients for use in the present invention refer to non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the co-formulated compounds. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

The invention also includes kits (e.g., pharmaceutical packages). Kits provided can include a compound of the invention, other therapeutic agent, and first and second containers (e.g., vials, ampoules, bottles, syringes, and/or dispersible packages or other suitable containers) containing a compound of the invention, other therapeutic agent. In some embodiments, the provided kits may also optionally include a third container containing pharmaceutically acceptable excipients for diluting or suspending the compounds of the invention and/or other therapeutic agents. In some embodiments, the compounds of the invention and other therapeutic agents provided in the first and second containers are combined to form one unit dosage form.

Administration of drugs

The pharmaceutical compositions provided herein may be administered by a number of routes including, but not limited to: oral, parenteral, inhalation, topical, rectal, nasal, buccal, vaginal, by implantation or other means of administration. For example, parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration, intramuscularly administration, intralesional administration, and intracranial injection or infusion techniques, preferably by intravenous administration.

Typically, an effective amount of a compound provided herein is administered. The amount of the compound actually administered may be determined by a physician, according to the circumstances involved, including the condition being treated, the route of administration selected, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, and the like.

When used to prevent a disorder of the present invention, a subject at risk of developing the disorder is administered a compound provided herein, typically based on physician recommendations and administered under the supervision of a physician, at a dosage level as described above. Subjects at risk for developing a particular disorder generally include subjects having a family history of the disorder, or those subjects determined by genetic testing or screening to be particularly susceptible to developing the disorder.

The pharmaceutical compositions provided herein may also be administered chronically ("chronically"). Chronic administration refers to administration of a compound or pharmaceutical composition thereof over a prolonged period of time, e.g., 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may continue administration indefinitely, e.g., for the remainder of the subject's life. In some embodiments, chronic administration is intended to provide a constant level of the compound in the blood over a prolonged period of time, e.g., within a therapeutic window.

Various methods of administration may be used to further deliver the pharmaceutical compositions of the present invention. For example, in some embodiments, the pharmaceutical composition may be administered as a bolus, e.g., in order to increase the concentration of the compound in the blood to an effective level. Bolus doses depend on the targeted systemic level of active ingredient through the body, e.g., intramuscular or subcutaneous bolus doses cause slow release of the active ingredient, whereas bolus injections delivered directly to veins (e.g., by IV intravenous drip) can be delivered more rapidly, causing the concentration of the active ingredient in the blood to rise rapidly to effective levels. In other embodiments, the pharmaceutical composition may be administered in the form of a continuous infusion, for example, by IV intravenous drip, thereby providing a steady state concentration of the active ingredient in the subject's body. Furthermore, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by continuous infusion.

Oral compositions may take the form of bulk liquid solutions or suspensions or bulk powders. More typically, however, the compositions are provided in unit dosage form in order to facilitate accurate dosing. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human patients and other mammals, each unit containing a predetermined quantity of active material suitable for producing the desired therapeutic effect in association with a suitable pharmaceutical excipient. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions, or in the case of solid compositions, pills, tablets, capsules and the like. In such compositions, the compound is typically a minor component (about 0.1 to about 50 wt.%, or preferably about 1 to about 40 wt.%) with the remainder being various carriers or excipients and processing aids useful for forming the desired administration form.

For oral doses, a typical regimen is one to five oral doses per day, especially two to four oral doses, typically three oral doses. Using these modes of dosing, each dose provides from about 0.01 to about 20mg/kg of a compound of the invention, with preferred doses each providing from about 0.1 to about 10mg/kg, especially from about 1 to about 5mg/kg.

In order to provide similar blood levels to, or lower than, the use of an injected dose, a transdermal dose is typically selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably about 0.5 to about 15% by weight.

From about 1 to about 120 hours, especially 24 to 96 hours, the injection dosage level is in the range of about 0.1 mg/kg/hour to at least 10 mg/kg/hour. To achieve adequate steady state levels, a preloaded bolus of about 0.1mg/kg to about 10mg/kg or more may also be administered. For human patients of 40 to 80kg, the maximum total dose cannot exceed about 2 g/day.

Liquid forms suitable for oral administration may include suitable aqueous or nonaqueous carriers, buffers, suspending and dispersing agents, colorants, flavors, and the like. Solid forms may include, for example, any of the following components, or compounds having similar properties: binders, for example microcrystalline cellulose, gum tragacanth or gelatin; excipients, for example starch or lactose, disintegrants, for example alginic acid, primogel or corn starch; lubricants, for example, magnesium stearate; glidants, for example, colloidal silicon dioxide; sweeteners, for example, sucrose or saccharin; or a flavoring agent, for example, peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based on sterile saline or phosphate buffered saline for injectable use, or other injectable excipients known in the art. As previously mentioned, in such compositions, the active compound is typically a minor component, often about 0.05 to 10% by weight, the remainder being an injectable excipient or the like.

Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredient. When formulated as ointments, the active ingredients are typically combined with a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art and typically include other components for enhancing stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and compositions are included within the scope provided by the present invention.

The compounds of the invention may also be administered via a transdermal device. Transdermal administration may thus be achieved using a reservoir (reservoir) or porous membrane type, or a variety of solid matrix patches.

The above components of the compositions for oral administration, injection or topical administration are merely representative. Other materials and processing techniques, etc. are set forth in Remington's Pharmaceutical Sciences,17th edition,1985,Mack Publishing Company,Easton,Pennsylvania, section 8, incorporated herein by reference.

The compounds of the present invention may also be administered in sustained release form, or from a sustained release delivery system. A description of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

The invention also relates to pharmaceutically acceptable formulations of the compounds of the invention. In one embodiment, the formulation comprises water. In another embodiment, the formulation comprises a cyclodextrin derivative. The most common cyclodextrins are α -, β -and γ -cyclodextrins consisting of 6, 7 and 8 α -1, 4-linked glucose units, respectively, optionally including one or more substituents on the linked sugar moiety, including but not limited to: methylated, hydroxyalkylated, acylated and sulfoalkyl ether substitutions. In some embodiments, the cyclodextrin is a sulfoalkyl ether β -cyclodextrin, e.g., sulfobutyl ether β -cyclodextrin, also known as Captisol. See, for example, U.S.5,376,645. In some embodiments, the formulation comprises hexapropyl- β -cyclodextrin (e.g., 10-50% in water).

Examples

The reagents employed in the present invention are commercial reagents purchased directly or synthesized by conventional methods well known in the art.

The specific reaction schemes or steps illustrated below are for use in the present invention and are as follows:

example 1

Commonly used abbreviation notes:

abbreviations: PE = petroleum ether; EA = ethyl acetate; meoh=methanol; DCM = dichloromethane; DCE = dichloroethane; CH (CH) ₃ Cn=acetonitrile; 1,4-dioxane = 1, 4-dioxane; DMSO = dimethyl sulfoxide; HFIP = hexafluoroisopropanol; DMF = N, N-dimethylformamide; hex = n-hexane; ipa=isopropanol; nmp=n-methylpyrrolidone; nmo=n-methylmorpholine-N-oxide; TEA = triethylamine; DIEA = diisopropylethylamine; cuI = cuprous iodide; cuCN = cuprous cyanide; triphosgene = triphosgene; p-TsOH = p-toluenesulfonic acid; t (T) ₃ P=1-propylphosphoric acid cyclic anhydride; tsN ₃ P-toluenesulfonyl azide; PPA = polyphosphoric acid; SEM-cl=2- (trisilyl) ethoxymethyl chloride; MOMCl = chloromethyl methyl ether.

Preparation of key intermediates a1-a30

Synthesis of intermediate a1

Step 1: raw material 2-amino-3-fluoro-4-bromobenzoic acid a1-1 (15 g,64.1 mmol) was dissolved in 100mL DMF, N-chlorosuccinimide (8.56 g,64.1 mmol) was slowly added, and the reaction was stopped by heating to 80℃for 16 hours. The reaction solution was poured into 500mL of ice water, suction filtration was performed to obtain a cake, and drying was performed to obtain intermediate a1-2 (13.2 g,49.2 mmol), yield: 77%. LC-MS [ M-H ] ] ^- ＝267。

Step 2: to a 200mL round bottom flask was added urea (35.3 g,588 mmol) and the previous intermediate a1-2 (10.5 g,39.2 mmol), and the reaction was warmed to 200℃for 12 hours. After cooling to 80℃100mL of water was added to the system, refluxed for 10 minutes, cooled to room temperature, filtered to give a cake, washed with water, and dried in an oven to give intermediate a1-3 (4.0 g,13.7 mmol). Yield: 35%. LC-MS: [ M+H ]] ⁺ ＝294。

Step 3: the intermediates a1-3 (4.0 g,13.7 mmol) and N, N-diisopropylethylamine (5.3 g,41.1 mmol) were dissolved in 15mL phosphorus oxychloride, and the reaction was stopped after heating to 120℃for 8 hoursStopping the reaction. The solvent was distilled off under reduced pressure, and column chromatography was carried out to give intermediate a1 (1.9 g,5.8 mmol). Yield: 42%. LC-MS: [ M+H ]] ⁺ ＝331。

Synthesis of intermediates a2, a3, a16, a30

The steps are as follows: intermediate a1 (1.9 g,5.8 mmol) and starting 3, 8-diazabicyclo [3.2.1]Tert-butyl octane-8-carboxylate a2-1 (1.85 g,8.7 mmol) was dissolved in 20mL of methylene chloride, and N, N-diisopropylethylamine (2.3 g,17.4 mmol) was added thereto and reacted at room temperature for 8 hours. 60mL of water was added to the system, extraction was performed with methylene chloride, drying over anhydrous sodium sulfate, filtration, concentration and column chromatography were performed to obtain a pale yellow solid a2 (1.2 g,2.4 mmol). Yield: 41%. LC-MS: [ M+H ] ] ⁺ ＝507。

Referring to the synthetic route for intermediate a2, using a similar starting material/framework structure, the following intermediates were synthesized:

synthesis of intermediates a4-a6, a8-a13

Step 1: 1- (methoxycarbonyl) cyclopropane-1-carboxylic acid a4-2 (3.0 g,20.8 mmol) was dissolved in 60mL of methylene chloride in ice bath, oxalyl chloride (10.5 g,83.3 mmol) was slowly added, and 5 drops of anhydrous DMF was added to react at 0℃for 20 minutes. The ice bath was removed, warmed to room temperature and stirring continued for 1 hour, and the solvent was distilled off under reduced pressure. The mixture was dissolved in 40mL of methylene chloride, and N, N-diisopropylethylamine (5.4 g,41.2 mmol) and tetrahydropyrrole a4-1 (1.78 g,25.0 mmol) were added and reacted at room temperature for 3 hours. The reaction was stopped, the solvent was distilled off under reduced pressure, and flash column chromatography was carried out to give intermediate a4-3 (2.6 g,13.2 mmol). Yield: 64%. LC-MS: [ M+H ]] ⁺ ＝198。

Step 2: intermediate a4-3 (2.6 g,13.2 mmol) was dissolved in 30mL anhydrous tetrahydrofuran at-78deg.C and lithium aluminum hydride (26.4 mL, 1M) was slowly added. The reaction was stopped after 2 hours at room temperature. The reaction solution was slowly poured into 80mL of ice water, and after the organic solvent was distilled off under reduced pressure, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by flash column chromatography to give intermediate a4 (900 mg,5.8 mmol). Yield: 44%. LC-MS: [ M+H ] ] ⁺ ＝156。

Referring to the synthetic route for intermediate a4, using a similar starting material/framework structure, the following intermediates were synthesized:

synthesis of intermediate a7

Step 1: 6-bromo-2, 3-difluorobenzaldehyde a7-1 (25 g,113 mmol) was dissolved in 250mL of methanol in an ice bath, sodium borohydride (8.54 g,226 mmol) was slowly added, and after stirring for 20 minutes, the reaction was continued at room temperature for 1 hour, and stopped. The reaction solution was slowly poured into saturated NH ₄ Aqueous Cl solution, dichloromethane extraction, drying over anhydrous sodium sulfate, filtration and concentration gave a7-2 (23.9 g,107 mmol) as a white solid. Yield: 95%.

Step 2: intermediate a7-2 (23.9 g,107 mmol) and N, N-diisopropylethylamine (20.7 g,161 mmol) were dissolved in 200mL anhydrous tetrahydrofuran in an ice bath, methylsulfonic anhydride (20.5 g,118 mmol) was slowly added, after stirring for 20 minutes, the ice bath was removed and the reaction was continued at room temperature for 18 hours, stopping the reaction. The reaction solution was slowly poured into ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered and concentrated to give an oil a7-3 (19 g,63.1 mmol). Yield: 59%.

Step 3: to a solution of the above intermediate a7-3 (19 g,63.1 mmol) in 240mL of a mixed solution of ethanol and water (v/v, 6/1), potassium cyanide (4.49 g,69 mmol) was added. The reaction was stopped by reacting at reflux for 1 hour. The organic solvent was distilled off under reduced pressure, and the reaction solution was poured into a saturated sodium carbonate solution, stirred for 20 minutes, extracted with methylene chloride, concentrated, and separated by column chromatography to give intermediate a7-4 (10.4 g,44.8 mmol). Yield: 71%. LC-MS: [ M+H ] ] ⁺ ＝233。

Step 4: under ice bath, the intermediate a7-4 (10.4 g,44.8 mmol) of the above step was dissolved in 80mL of DMF, potassium tert-butoxide (5.3 g,47.2 mmol) was slowly added, after stirring for 20 minutes, the solution became red, and a solution of ethyl isothiocyanate in DMF (6.2 g,47.2mmol,5 mL) prepared in advance was slowly added dropwise. The reaction mixture was stirred for 1 hour, and then heated to 100℃for 30 minutes. Cooled to room temperature, the reaction mixture was slowly poured into ice water to quench the reaction, filtered to give a cake, washed with n-hexane and dried in a vacuum oven to give intermediate a7-5 (13.7 g,40.0 mmol). Yield: 89%. LC-MS: [ M+H ]] ⁺ ＝344。

Step 5: the intermediate a7-5 (6.7 g,19.5 mmol) of the above step was dissolved in 30mL of DMSO and 30mL of aqueous sodium hydroxide (5M) was added. The reaction was stopped after 4 hours under reflux. Cooling to room temperature, slowly adding 100mL of ice water to quench the reaction, filtering to obtain a filter cake, washing with water, and drying in a vacuum drying oven to obtain a crude intermediate a7-6 (3.8 g). LC-MS: [ M+H ]] ⁺ ＝272。

Step 6: the crude product of the above step a7-6 (3.8 g) and DMAP (122 mg,1 mmol) were dissolved in 30mL of a mixed solution of THF/DMF (v/v, 1/1), and Boc anhydride (4.3 g,19.5 mmol) was added. The reaction was stopped after 12 hours at room temperature. 80mL of water was added to the system, extracted with methylene chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to give crude intermediate a7-7 (3.3 g).

Step 7: under nitrogen, crude a7-7 (3.3 g) and raw a7-8 (6.01 g,26.7 mmol) were dissolved in 30mL 1, 4-dioxane, and potassium acetate (2.62 g,26.7 mmol) and Pd (DPEphos) Cl were slowly added ₂ (643 mg,0.9 mmol), the temperature was raised to 100℃and the reaction was stopped for 1 hour. Cooling to room temperature, filtering, washing with saturated salineDichloromethane extraction, drying, concentration, flash column chromatography gave intermediate a7 (2.5 g,6.2 mmol). LC-MS: [ M+H ]] ⁺ ＝405。

Synthesis of intermediates a14, a17

The steps are as follows: intermediate a2 (50 g,98.77 mmol) was dissolved in 750mL anhydrous DMF under nitrogen, csF (45 g,29.63 mmol) was added, and the reaction was stopped after heating to 60℃for 8 h. The reaction solution was added to 1L of water, the mixture was extracted 3 times with 750mL of ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to obtain intermediate a14 in a yield of 90%. LC-MS: [ M+H ]] ⁺ ＝489。

Referring to the synthetic route for intermediate a14, using a similar starting material/framework structure, the following intermediates were synthesized:

synthesis of intermediates a15, a18

Step 1: raw material 3-methoxy-2, 2-dimethyl-3-oxomalonic acid a15-1 (1.0 g,6.8 mmol) and raw material a5-1 (850 mg,6.8 mmol) were dissolved in 20mL of anhydrous dichloromethane, EDCI (1.56 g,8.2 mmol), N-diisopropylethylamine (1.77 g,13.6 mmol) and HOBt (1.1 g,8.2 mmol) were added, and the reaction was stopped at room temperature for 16 h. To the reaction mixture was added 60mL of ice water, extracted with methylene chloride, and dried over anhydrous sodium sulfate. The mixture was separated by Flash column chromatography to give intermediate a15-2 (1.04 g,4.6 mmol) in 68% yield. LC-MS: [ M+H ] ] ⁺ ＝218。

Step 2: intermediate a15-2 (1.04 g,4.6 mmol) was dissolved in 15mL anhydrous tetrahydrofuran at-78deg.C, and lithium aluminum hydride (350 mg,9.2 mmol) was slowly added. Heating to 0 ℃ for reaction for 1 hourThe reaction was stopped. To the reaction solution was slowly added 2mL of 10% aqueous NaOH solution to precipitate floccules, and the filtrate was concentrated under reduced pressure and flash column chromatographed to give intermediate a15 (700 mg,4.0 mmol). Yield: 87%. LC-MS: [ M+H ]] ⁺ ＝176。

Referring to the synthetic route for intermediate a15, using a similar starting material/framework structure, the following intermediates were synthesized:

synthesis of intermediates a19-a23

Step 1: in a 50mL reaction flask, (3S, 4R) -4-fluoro-3-hydroxypyrrolidine a19-1 (300 mg,2.86 mmol) and intermediate a19-2 (1.06 g,3.14 mmol) were added and dissolved in 5mL anhydrous THF. After stirring for 5 minutes, naBH (OAc) was added to the reaction mixture ₃ (1.81 g,8.58 mmol) and 5 drops of acetic acid were reacted at room temperature for 10 hours, the reaction was stopped, the reaction solution was poured into 150mL of ice water, extracted with ethyl acetate, the organic phase was washed with saturated saline, dried over anhydrous sodium sulfate and concentrated to give 1.1g of crude intermediate a19-3 as colorless oil, LC-MS: [ M+H] ⁺ ＝428。

Step 2: 1.1g of the crude product of the above step, intermediate a19-3 (1.1 g), was dissolved in 15mL of anhydrous THF, 3, 4-dihydro-2H-pyran (390 mg) and p-toluenesulfonic acid (200 mg) were added, and stirred at room temperature for 1 hour, and TLC showed completion of the reaction. The reaction solution was poured into 80mL of ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate and concentrated to give 1.5g of crude intermediate a19-4, LC-MS: [ M+H ] ] ⁺ ＝514。

Step 3: the intermediate a19-4 (1.5 g) of the above step was dissolved in 15mL of tetrahydrofuran, and tetrabutylammonium fluoride (1.5 g) was added. The reaction was allowed to react at room temperature for 3 hours, and TLC monitored the reaction was complete. The reaction solution was dissolved in 50mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (PE/ea=1/1) to give a19 (200 mg,0.73 mmol) as a colorless oil) Yield: 35, LC-MS: [ M+H ]] ⁺ ＝274。

Referring to the synthetic route for intermediate a19, using a similar starting material/framework structure, the following intermediates were synthesized:

synthesis of intermediate a24

Step 1: a100 mL reaction flask was charged with starting 3, 3-difluorocyclobutan-1-amine a24-1 (1.0 g,9.34 mmol) and intermediate a19-2 (3.16 g,9.34 mmol) and 40mL anhydrous THF was dissolved. After stirring for 5 minutes, naBH (OAc) was added to the reaction mixture ₃ (2.57 g,12.34 mmol) and 10 drops of acetic acid, reacted at room temperature for 10 hours, the reaction was stopped, the reaction solution was poured into 200mL of ice water, extracted with ethyl acetate, the organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, concentrated, and flash column chromatography (PE/EA, 1/1) to give 2.62g of intermediate a24-2, LC-MS: [ M+H ] as a colorless oil] ⁺ ＝430。

Step 2: in a 100mL reaction flask were added intermediate a24-2 (2.62 g,5.91 mmol) and aqueous formaldehyde (0.26 mL), and 40mL of tetrahydrofuran was dissolved. After stirring for 5 minutes, naBH (OAc) was added to the reaction mixture ₃ (1.63 g,7.68 mmol) and 10 drops of acetic acid, reacted at room temperature for 2 hours, the reaction was stopped, the reaction solution was poured into 100mL of ice water, extracted with ethyl acetate, the organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, concentrated, and flash column chromatography (PE/EA, 10/1) to give 2.0g of intermediate a24-3 as a colorless oil, LC-MS: [ M+H ]] ⁺ ＝444。

Step 3: the intermediate a24-3 (2.0 g,4.51 mmol) of the above step was dissolved in 20mL of tetrahydrofuran, and tetrabutylammonium fluoride (2.2 g,9.0 mmol) was added. The reaction was allowed to proceed at room temperature for 12 hours, and TLC monitored the reaction was complete. The reaction was dissolved in 50mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (DCM/MeOH, 1/1) to give a24 (8) as a colorless oil00mg,0.73 mmol), yield: 87%, LC-MS: [ M+H ]] ⁺ ＝206。

Synthesis of intermediate a25

Step 1: raw material a25-1 (1.0 g,4.5 mmol) and raw material a5-1 (680 mg,5.4 mmol) were dissolved in 50mL of anhydrous dichloromethane, EDCI (1.29 g,6.75 mmol), N-diisopropylethylamine (1.74 g,13.5 mmol) and HOBt (910 mg,6.75 mmol) were added, and the reaction was stopped at room temperature for 4 hours. The reaction mixture was added to 80mL of ice water, extracted with methylene chloride, dried over anhydrous sodium sulfate, and concentrated to give crude intermediate a25-2 (1.21 g), LC-MS: [ M+H ] ] ⁺ ＝294。

Step 2: intermediate a25-2 (1.21 g,4.13 mmol) was dissolved in 25mL anhydrous tetrahydrofuran under ice-bath, and lithium aluminum hydride (310 mg,8.3 mmol) was slowly added. The reaction was stopped after 2 hours at room temperature. To the reaction solution was slowly added 2mL of 10% aqueous NaOH solution to precipitate floccules, the filtrate was suction-filtered, concentrated under reduced pressure, and flash column chromatographed to give intermediate a25 (180 mg,0.76 mmol). Yield: 18%. LC-MS: [ M+H ]] ⁺ ＝238。

Synthesis of intermediate a26

Step 1: an oxetane-3, 3-diyldimethanol a26-1 (4.1 g,33.9 mmol) and triethylamine (4.11 g,40.63 mmol) were charged in a 50mL reaction flask, and 40mL of anhydrous dichloromethane was dissolved. After stirring for 5 minutes, t-butyldiphenylchlorosilane (7.18 g,33.86 mmol) was added to the reaction mixture, the reaction was stopped at room temperature for 2 hours, the reaction mixture was poured into 200mL of ice water, extracted with methylene chloride, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 12g of crude intermediate a26-2.

Step 2: the crude intermediate a26-2 (12.0 g) of the above step was dissolved in 40mL of tetrahydrofuran at-10℃and the reaction was stopped by adding pyridine sulfur trioxide (12.8 g,81.0 mmol) and reacting for 3 hours in ice bath, pouring the reaction solution into 100mL of ice water, extracting with methylene chloride, washing the organic phase with saturated brine, drying over anhydrous sodium sulfate, concentrating, separating by column chromatography to obtain intermediate a26-3 (8.0 g,22.7 mmol) in two steps of yield: 67%.

Step 3: a100 mL reaction flask was charged with the above intermediate a26-3 (8.0 g,22.7 mmol) and intermediate a5-1 (3.4 g,27.1 mmol) and 40mL anhydrous THF was dissolved. After stirring for 5 minutes, naBH (OAc) was added to the reaction mixture ₃ (7.18 g,33.86 mmol) and 10 drops of acetic acid were reacted at room temperature for 10 hours, the reaction was stopped, the reaction solution was poured into 200mL of ice water, extracted with ethyl acetate, the organic phase was washed with saturated saline, dried over anhydrous sodium sulfate and concentrated to give 5.0g of crude intermediate a26-4, LC-MS: [ M+H] ⁺ ＝428。

Step 4: the crude product a26-4 (5.0 g,11.69 mmol) from the previous step was dissolved in 30mL of tetrahydrofuran and tetrabutylammonium fluoride (4.58 g,17.5 mmol) was added. The reaction was warmed to 40 ℃ for 12 hours and monitored by TLC to complete the reaction. The reaction was dissolved in 150mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (DCM/MeOH, 1/1) to give a colorless oil a26 (1.9 g,10.2 mmol), yield: 88%, LC-MS: [ M+H ]] ⁺ ＝190。

Synthesis of intermediates a27-a28

Step 1: intermediate a1 (3.0 g,9.15 mmol) and starting 4, 7-diazaspiro [2.5 ] at room temperature]Tert-butyl octane-4-carboxylate a27-1 (2.12 g,10.1 mmol) was dissolved in 30mL of tetrahydrofuran, and N, N-diisopropylethylamine (2.3 g,17.4 mmol) was added thereto and reacted at room temperature for 8 hours. 100mL of water was added to the system, extraction was performed with methylene chloride, drying over anhydrous sodium sulfate, filtration, concentration and column chromatography were performed to obtain a pale yellow solid a27-2 (3.74 g,7.4 mmol). Yield: 81%. LC-MS: [ M+H ] ] ⁺ ＝505。

Step 2: intermediate a27-2 (3.74 g,7.4 mmol) was dissolved in 75mL anhydrous DMF under nitrogen, csF (3.4 g,22.2 mmol) was added, the temperature was raised to 60℃for reaction for 8h,the reaction was stopped. The reaction solution was added to 300mL of water, the mixture was extracted with ethyl acetate 3 times, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to obtain intermediate a27 in a yield of 90%. LC-MS: [ M+H ]] ⁺ ＝489。

Referring to the synthetic route for intermediate a27, using a similar starting material/framework structure, the following intermediates were synthesized:

synthesis of intermediate a29

Step 1: 2-fluoro-3-methyl-4-bromopyridine a29-1 (4.5 g,23.9 mmol) was dissolved in 25mL of carbon tetrachloride, N-bromosuccinimide NBS (6.35 g,35.8 mmol) and azobisisobutyronitrile AIBN (390 mg,2.3 mmol) were slowly added and reacted at room temperature for 3 hours to stop the reaction. The reaction solution was slowly poured into 150mL of ice water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (PE/EtOAc, 5/1) to give a29-2 (6.1 g,22.9 mmol) as an oil. Yield: 94%.

Step 2: intermediate a29-2 (5.2 g,3.7 mmol) and trimethylcyano silane TMSCN (2.9 g,5.6 mmol) were dissolved in 20mL acetonitrile, and a solution of TBAF (5.5 mmol) in tetrahydrofuran (29 mL) was added and reacted at room temperature for 16 hours to stop the reaction. The solvent was evaporated under reduced pressure and column chromatographed (PE/EtOAc, 5/1) to give a29-3 (3.4 g,15.9 mmol) as an oil. Yield: 81%.

Step 3: under ice bath, intermediate a29-3 (3.4 g,15.9 mmol) of the above step was dissolved in 20mL of DMF, naH (2.9 g,19.1 mmol) was slowly added, after stirring for 30 min, the solution became red, and a previously prepared solution of ethyl isothiocyanamide in DMF (1.8 g,15.9mmol,5 mL) was slowly added dropwise. The reaction mixture was heated to 100℃and allowed to react for 1 hour, and cooled to room temperature. Slowly pouring ice water into the reaction solution for quenching reaction, extracting with ethyl acetate, drying with anhydrous sodium sulfate, and subjecting the crude product to column chromatographyIsolation (PE/EtOAc, 1/1) afforded a29-4 (1.05 g,3.2 mmol) as a pale yellow solid. Yield: 20%. LC-MS: [ M+H ]] ⁺ ＝325。

Step 4: the intermediate a29-4 (1.0 g,3.1 mmol) of the above step was dissolved in 10mL of DMSO, and 10mL of aqueous sodium hydroxide (5M) was added. The reaction was stopped after 4 hours under reflux. Cooled to room temperature, the reaction was quenched by slowly adding 100mL of ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the crude product was isolated by column chromatography (PE/EtOAc, 1/1) to give a29-5 (450 mg,1.8 mmol) as a pale yellow solid. Yield: 20%. LC-MS: [ M+H ]] ⁺ ＝254。

Step 5: to a mixed solution (v/v, 1/1) of the above intermediate a29-5 (450 mg,1.8 mmol) and DMAP (5 mg,0.04 mmol) in 20mL THF/DMF was added Boc anhydride (460 mg,2.16 mmol). The reaction was stopped after 12 hours at room temperature. 50mL of water was added to the system, extracted with methylene chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to give crude intermediate a29-6 (760 mg).

Step 6: under nitrogen, crude a29-6 (760 mg) and starting a7-8 (560 mg,2.6 mmol) were dissolved in 10mL 1, 4-dioxane, and potassium acetate (433 mg,4.32 mmol) and Pd (DPEphos) Cl were slowly added ₂ (46 mg,0.065 mmol), and the reaction was stopped after heating to 100℃for 1 hour. Cooled to room temperature, filtered, washed with saturated brine, extracted with dichloromethane, dried, concentrated, and flash column chromatographed to afford intermediate a29 (320 mg,0.82 mmol). LC-MS: [ M+H ]] ⁺ ＝388。

Preparation of key intermediates b1-b2

Synthesis of intermediate b1

Step 1: raw material b1-1 (10.00 g,56.8 mmol), methoxyamine hydrochloride (6.73 g,83.2 mmol) and pyridine (5.69 g,68.10 mmol) were dissolved in 10mL absolute ethanol at room temperature. The reaction solution was reacted at room temperature for 2 hours, the reaction was stopped, and concentrated under reduced pressure. The residue was dissolved in methylene chloride, washed with dilute hydrochloric acid (2N), saturated aqueous sodium bicarbonate solution and saturated brine, and dried over anhydrous sodium sulfateConcentrated under reduced pressure to give crude colorless oil b1-2 (11.30 g,55.1 mmol). LC-MS: [ M+H ]] ⁺ ＝206。

Step 2: the intermediate b1-2 (1.0 g,4.9 mmol), palladium acetate (55 mg,0.24 mmol) and NBS (0.87 g,4.87 mmol) of the above step were dissolved in 10mL of anhydrous acetic acid, the reaction was stopped after the temperature of the reaction solution was raised to 80℃for 1 hour, the reaction was cooled to room temperature, the reaction solution was poured into water, filtered, and the cake was dried to give a brown solid b1-3 (1.03 g,3.6 mmol). LC-MS: [ M+H ] ] ⁺ ＝284。

Step 3: the intermediate b1-3 (12.5 g,43.99 mmol) of the above step was dissolved in a mixed solution of 60mL of concentrated hydrochloric acid and 100mL of 1, 4-dioxane. The reaction solution was stirred under reflux for 1 hour, the reaction was stopped, and concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed with aqueous sodium hydroxide (1N), saturated brine, dried over anhydrous sodium sulfate, concentrated, and flash column chromatographed (PE/ea=4/1) to give b1-4 (10.9 g,42.7 mmol) as a yellow solid, yield: 97%. LC-MS: [ M+H ]] ⁺ ＝255。

Step 4: intermediate b1-4 (7.90 g,30.97 mmol) and 1-chloromethyl-4-fluoro-1, 4-diazabicyclo [2.2.2 ] under nitrogen]Octane bis (tetrafluoroborate) (Selectfluor, 16.46g,46.5 mmol) was dissolved in 80mL methanol and 0.3mL concentrated sulfuric acid was slowly added dropwise. The reaction mixture was heated to 50℃and reacted for 5 hours, the reaction was stopped, and the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and flash column chromatographed (PE/ea=10/1) to give b1-5 (6.37 g,23.35 mmol) as a red solid, yield: 75%. LC-MS: [ M+H ]] ⁺ ＝273。

Step 5: intermediate b1-5 (32.0 g,117.2 mmol) and pyridinium tribromide (41.22 g,128.89 mmol) were dissolved in 300mL acetonitrile under nitrogen, the reaction was stopped by heating to 60℃for 0.5 h, and the solvent was distilled off under reduced pressure. Saturated brine, ethyl acetate extraction, drying over anhydrous sodium sulfate, concentration, and flash column chromatography (PE/ea=10/1) of the crude product gave yellow solid b1-6 (36.0 g,102.3 mmol), yield: 87%. LC-MS: [ M+H ] ] ⁺ ＝350。

Step 6: intermediate b1-6 (36.0 g,102.3 mmol) and lithium bromide (19.5 g,225 mmol) were dissolved under nitrogenThe reaction was stopped by heating to 100℃in 100mL of DMF for 0.5 hour. To the system was added 300mL of water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was isolated by flash column chromatography (PE/ea=10/1) to give b1-7 (21.0 g,77.5 mmol) as a pale yellow solid, yield: 75%. LC-MS: [ M+H ]] ⁺ ＝271。

Step 7: intermediate b1-7 (21.0 g,77.5 mmol) and pyridine (18.38 g,232.41 mmol) were dissolved in 200mL of dichloromethane under an ice bath and nitrogen blanket. To the reaction solution was slowly added dropwise trifluoromethanesulfonic anhydride (26.2 g,92.96 mmol), and the reaction was slowly warmed to room temperature for 1 hour, stopped, and the solvent was distilled off under reduced pressure. The crude product was washed with saturated brine, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and separated by flash column chromatography (PE/ea=8/1) to give yellow solid b1-8 (27.50 g,68.2 mmol), yield: 88%. LC-MS: [ M+H ]] ⁺ ＝403。

Step 8: intermediate b1-8 (1.0 g,2.48 mmol), zinc cyanide (146 mg,1.24 mmol), pd under nitrogen ₂ (dba) ₃ (114 mg,0.12 mmol) and 1,1' -bis (diphenylphosphine) ferrocene (dppf, 137mg,0.25 mmol) were dissolved in 10mL anhydrous DMF. The reaction mixture was warmed to 70℃and allowed to react for 3 hours, and cooled to room temperature. The crude product was poured into 50mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was chromatographed on flash column (PE/ea=5/1) to give b1-9 (270 mg,0.96 mmol) as a white solid in yield: 38%. LC-MS: [ M+H ] ] ⁺ ＝208。

Step 9: intermediate b1-9 (50 mg,0.18 mmol) was dissolved in 2mL of dichloromethane at-78℃under nitrogen, and 0.44mL of boron tribromide dichloromethane solution (concentration 2M) was slowly added dropwise. The system was slowly warmed to 0 ℃ and reacted for 16 hours, and quenched by adding 10mL of methanol. The solvent was evaporated under reduced pressure and the crude product was chromatographed on flash column (PE/ea=3/1) to give b1-10 (20 mg,0.075 mmol) as a white solid, yield: 42%. LC-MS: [ M+H ]] ⁺ ＝266。

Step 10: intermediate b1-10 (430 mg,26.7 mmol), pinacol biborate (823 mg,3.24 mmol), potassium acetate (477 mg,4.86 mmol), pd under nitrogen ₂ (dba) ₃ (74mg,0.081mmol)And tricyclohexylphosphine (45 mg,0.016 mmol) was dissolved in 8mL of 1, 4-dioxane, and the reaction was stopped after heating to 105℃for 10 hours. Cooled to room temperature, filtered, the system was poured into 30mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was chromatographed on flash column (PE/ea=3/1) to give b1 (350 mg,6.2 mmol) as a pale yellow solid, yield: 69%. LC-MS: [ M+H ]]+＝314。

Synthesis of intermediate b2

Step 1: raw material 4-fluorophenylacetic acid b2-1 (50.0 g,324.4 mmol) and malonic acid cyclo (isopropylidene) ester b2-2 (51.4 g,356.8 mmol) were dissolved in 500mL acetonitrile, and 4-dimethylaminopyridine (DMAP, 3.57g,29.2 mmol) and DIEA (88.0 g,681.2 mmol) were added. After stirring for 5 minutes, pivaloyl chloride (43.0 g,356.8 mmol) was slowly added dropwise. The reaction mixture was stirred at 45℃for 3 hours, and then cooled to room temperature. The reaction solution was placed in an ice bath, and a 4N aqueous hydrochloric acid solution was added dropwise to adjust the pH to about 5, and after stirring was continued for 1 hour, the reaction solution was diluted with water, and again, the pH of the reaction solution was adjusted to about 2 with 4N hydrochloric acid, a large amount of solids were precipitated, suction filtration, washing of the cake with water, and drying to give a white solid b2-3 (104 g,371.1 mmol), and yield was quantitative. LC-MS: [ M+H ] ] ⁺ ＝281。

Step 2: the above intermediate b2-3 (54.0 g,192.7 mmol) was slowly added to trifluoromethanesulfonic acid (228.5 g,1.5 mol). The reaction was stirred at room temperature for 2 hours and monitored by LC-MS for completion. The reaction solution was slowly poured into 500mL of ice water, and a solid was precipitated, suction-filtered, and the cake was washed with water and dried to give b2-4 (66.0 g,295.96 mmol) as a brown solid. Yield: 93%, LC-MS: [ M+H ]] ⁺ ＝223。

Step 3: the intermediate b2-4 (66.0 g,295.96 mmol) of the above step was dissolved in 660mL of a mixed solution of acetonitrile and water (v/1, 1/1), and the reaction was stopped after heating to 80℃for 13 hours. The solvent was distilled off under reduced pressure, washed with a saturated aqueous sodium hydrogencarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give pale yellow compound b2-5 (51.8 g,291.01 mmol),yield: 97%. LC-MS: [ M+H ]] ⁺ ＝179。

Step 4: under nitrogen, crude b2-5 (20.0 g,112.3 mmol), (2-bromoethynyl) triisopropylsilane (30.8 g,112.3 mmol), potassium acetate (22.0 g,224.5 mmol) and dichlorobis (4-cymene) ruthenium (II) (2.06 g,3.4 mmol) were dissolved in 200mL of 1, 4-dioxane, reacted at 100℃for 4 hours, monitored by LC-MS and filtered. The solvent was distilled off under reduced pressure, 100mL of water was added to the system, extraction was performed with ethyl acetate, drying was performed with anhydrous sodium sulfate, and concentration was performed. The crude product was chromatographed on flash column (PE/ea=10/1) to give b2-6 (28.0 g,78.2 mmol) as a pale yellow solid, yield: 69%. LC-MS: [ M+H ] ] ⁺ ＝359。

Step 5: the intermediates b2-6 (28 g,78.2 mmol) and DIEA (20.19 g,156.20 mmol) were dissolved in 300mL of dichloromethane, triisopropylchlorosilane TIPSCl (18.1 g,93.7 mmol) was slowly added dropwise, the reaction solution was stirred at room temperature for 1 hour, and LC-MS monitored that the reaction was complete. The reaction solution was poured into 500mL of ice water, extracted with methylene chloride, dried over anhydrous sodium sulfate, and concentrated to give crude fuchsin oily compound b2-7.LC-MS: [ M+H ]] ⁺ ＝515。

Step 6: under the protection of nitrogen, crude b2-7 (58.4 g,113.43 mmol) and DIEA (51.3 g,397.0 mmol) were dissolved in 300mL of dichloromethane at-40 ℃ and trifluoromethanesulfonic anhydride (54.4 g,192.8 mmol) was slowly added dropwise, after 3 hours, stirring was continued for 0.5 hour, and the reaction was stopped. The reaction solution was poured into 500mL of ice water, extracted with methylene chloride, dried over anhydrous sodium sulfate, and concentrated. The crude product was chromatographed on flash column (PE/ea=10/1) to give compound b2-8 (57.7 g,89.2 mmol) as a pale red oil, yield: 78%. LC-MS: [ M+H ]] ⁺ ＝647。

Step 7: intermediate b2-8 (61.6 g,95.2 mmol), triethylamine (38.5 g,380.9 mmol) and pinacol borane (48.7 g,380.9 mmol) were dissolved in 600mL acetonitrile under nitrogen and stirred for 5 min before adding catalyst Pd (dppf) Cl ₂ (4.2 g,5.7 mmol). The reaction solution was stirred at 80℃for 4 hours and cooled to room temperature. The mixture was slowly quenched with MeOH, kept below 25 ℃ and solids precipitated. Suction filtration, meOH washing of the filter cake and drying gave compound b2 (45.9 g,73.4 mmo) as a white solid l), yield: 77%. LC-MS: [ M+H ]] ⁺ ＝625。

Referring to the synthetic route for intermediate b2, using a similar starting material/framework structure, the following intermediates were synthesized:

synthesis of intermediate b3

Step 1: intermediate b2-6 (28.0 g,78.2 mmol) and DIEA (20.2 g,156.2 mmol) were dissolved in 280mL anhydrous dichloromethane, MOMCl (18.1 g,93.7 mmol) was added and the reaction stopped at room temperature for 1 hour. To the mixture was added 500mL of water, extracted with methylene chloride, dried over anhydrous sodium sulfate, and concentrated. Oily matter b3-1 (47.7 g) was obtained. LC-MS: [ M+H ]] ⁺ ＝403。

Step 2: in an ice bath, crude b3-1 (400 mg,0.99 mmol) from the previous step and DIEA (300 mg,2.97 mmol) were dissolved in 5mL of anhydrous dichloromethane, DMAP (121 mg,0.99 mmol) and pivaloyl chloride PivCl (358 mg,2.97 mmol) were added and the reaction was stirred at room temperature for 2 hours and monitored by LC-MS for completion. The reaction solution was slowly poured into 20mL of ice water, extracted with methylene chloride, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash column chromatography (PE/EA, 10/1) to give b3-2 (240 mg) as a pale yellow solid. Yield: 49%, LC-MS: [ M+H ]] ⁺ ＝487。

Step 3: the intermediate b3-2 (190 mg,0.39 mmol) of the above step was dissolved in 2mL of DMF, csF (355 mg,2.34 mmol) was added, and the reaction was stopped at room temperature for 1 hour. To the mixture was added 10mL of water, extracted with methylene chloride, dried over anhydrous sodium sulfate, and concentrated. Oil b3-3 (200 mg) was obtained. LC-MS: [ M+H ] ] ⁺ ＝331。

Step 4: the crude product b3-3 (2)00mg, crude) and catalyst Pd/C (20 mg) were dissolved in 2mL of methanol, reacted under a hydrogen atmosphere (4 atm) at room temperature for 16 hours, the reaction was stopped, filtered, and the solvent was distilled off under reduced pressure. The crude product was separated by flash column chromatography (PE/EA, 5/1) to give oil b3-4 (85 mg). Yield in two steps: 65%, LC-MS: [ M+H ]] ⁺ ＝335。

Step 5: repeat the amplification reaction as above. Intermediate b3-4 (5.0 g,14.97 mmol) was dissolved in 52mL of methanol, KOH (2.5 g,44.8 mmol) was added, the reaction was stopped at room temperature for 1 hour, and the reaction was filtered. The reaction solution was slowly poured into 200mL of ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash column chromatography (PE/EA, 2/1) to give oil b3-5 (1.8 g). Yield: 48%, LC-MS: [ M+H ]] ⁺ ＝251。

Step 6: intermediate b3-5 (1.4 g,6.0 mmol) and DIEA (2.34 g,9.0 mmol) were dissolved in 140mL of anhydrous dichloromethane at-40 ℃ under nitrogen, and triflic anhydride (2.3 g,18.1 mmol) was added and the reaction stopped at this temperature for 1 hour after dropping. The reaction solution was slowly poured into 200mL of ice water, extracted with methylene chloride, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash column chromatography to give oil b3-6 (1.9 g). Yield: 88%, LC-MS: [ M+H ] ] ⁺ ＝383。

Step 7: under nitrogen, the intermediates b3-6 (1.87 g,4.89 mmol), TEA (1.98 g,19.6 mmol) and pinacolborane (2.5 g,19.6 mmol) were dissolved in 20mL acetonitrile and the catalyst Pd (dppf) Cl was added ₂ (399 mg,0.49 mmol) was reacted at 80℃for 4 hours, and the reaction was monitored by LC-MS and filtered. The solvent was distilled off under reduced pressure, 100mL of water was added to the system, extraction was performed with ethyl acetate, drying was performed with anhydrous sodium sulfate, and concentration was performed. The crude product was chromatographed on flash column (PE/ea=10/1) to give b3 (1.3 g) as a pale red oil, yield: 66%. LC-MS: [ M+H ]] ⁺ ＝361。

Referring to the synthetic route for intermediate b3, using a similar starting material/framework structure, the following intermediates were synthesized:

preparation of key intermediates c1-c12

Synthesis of intermediates c1-c8

Step 1: raw material c1-1 (20.0 g,81.5 mmol) and TEA (16.5 g,163.1 mmol) were dissolved in 250mL of methylene chloride in an ice bath, methanesulfonic anhydride (15.6 g,89.7 mmol) was slowly added to the system, and after the dropwise addition, the reaction was stopped by heating to room temperature and reacting for 2 hours. 300mL of ice water is added into the system, dichloromethane extraction, anhydrous sodium sulfate drying and concentration are carried out, thus obtaining crude product c1-2, LC-MS: [ M+H ]] ⁺ ＝324。

Step 2: crude c1-2 (25 g,77.3 mmol) was dissolved in 300mL DMF and sodium methyl mercaptide (6.5 g,92.8 mmol) was added at room temperature and stirred for 16 hours at 90℃and the reaction stopped. The reaction solution was poured into 500mL of ice-water, extracted with ethyl acetate, washed with saturated brine, concentrated, and the crude product was separated by column chromatography to give compound c1-3 (10 g,36.4 mmol) in 47% yield. LC-MS: [ M+H ] ] ⁺ ＝275。

Step 3: intermediate c1-3 (16.5 g,51.0 mmol) was dissolved in 200mL anhydrous tetrahydrofuran at-78deg.C under nitrogen, LDA (12.8 g,76.6 mmol) was slowly added dropwise, and stirring was continued for 0.5 h. 1-bromo-3-chloropropane (40.2 g,255.2 mmol) was slowly added dropwise to the system, and after the completion of the dropwise addition, the reaction was continued at room temperature for 1 hour by heating, and 100mL of ice water was added to the reaction solution for quenching. Extraction with ethyl acetate, washing with saturated saline, drying with anhydrous sodium sulfate, and concentrating to obtain crude product c1-4, LC-MS: [ M+H ]] ⁺ ＝352。

Step 4: the crude product c1-4 obtained in the previous step was dissolved in 50mL of methylene chloride, 150mL of trifluoroacetic acid was added, and the reaction was stopped after stirring at room temperature for 1 hour. The solvent was distilled off under reduced pressure, the mixture was made weakly basic with saturated aqueous sodium hydrogencarbonate, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by column chromatography to give compounds c1-5 (2.5 g) and c1-6 (1.3 g), LC-MS: [ M+H ]] ⁺ ＝252。

Step 5: intermediate c1-5 (2.5 g,10.5 mmol), potassium iodide (0.17 g,1.1 mmol) and potassium carbonate (6.9 g, 21).0 mmol) was dissolved in 25mL of methanol and reacted at room temperature for 16 hours, and the reaction was stopped. 100mL of ice water was added to the system, extraction was performed with ethyl acetate, washing was performed with saturated brine, drying was performed with anhydrous sodium sulfate, and concentration was performed to obtain crude product c1-7, LC-MS: [ M+H ] ] ⁺ ＝216。

Step 6: the crude product c1-7 (2.1 g,9.8 mmol) was dissolved in 20mL tetrahydrofuran in ice bath, lithium aluminum hydride (750 mg,19.5 mmol) was added and stirring was continued for 1 hour, stopping the reaction. 10mL of methanol was added to the system to quench it, filtered, concentrated under reduced pressure, and the mixture was added with 50mL of water, extracted with methylene chloride, dried over anhydrous sodium sulfate, and concentrated to give crude product c1 (directly used in the next reaction). LC-MS: [ M+H ]] ⁺ ＝188。

Step 7: intermediate c1-5 is replaced with c1-6 to afford intermediate c2.

Referring to the synthetic route for intermediate c1, using a similar starting material/framework structure, the following intermediates were synthesized:

synthesis of intermediate c9

Step 1: raw material c9-1 (15.0 g,71.0 mmol) was dissolved in 150mL of anhydrous tetrahydrofuran (150 mL) in an ice bath, sodium borohydride (806 mg,21.3 mmol) was slowly added, and the reaction was stopped under the ice bath for 3 hours. The solvent was distilled off under reduced pressure, 50mL of ice water was added to the mixture, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was isolated by column chromatography to give intermediate c9-2 (12.0 g,56.3 mmol). LC-MS: [ M+H ]] ⁺ ＝214。

Step 2: ice bath, intermediate c9-2 (12.0 g,56.3 mmol) and triethylamine (17.3 g,170.5 mmol) from the previous step were dissolved in 120mL of dichloromethane. After stirring for 5 minutes, a methylene chloride solution (20 mL) of methanesulfonic anhydride (14.9 g,85.2 mmol) was added dropwise thereto, and stirring was continued for 1 hour after the completion of the addition. The reaction was stopped, 200mL of ice water was added to the system, extracted with dichloromethane, and concentrated under reduced pressure to give crude product to c9-3.

Step 3: the crude c9-3 and potassium thioacetate (9.4 g,82.4 mmol) were dissolved in 150mL DMF and reacted at 60℃for 15 hours, stopping the reaction. 300mL of ice water was added to the system, extraction was performed 3 times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was separated by column chromatography to give intermediate c9-4 (15 g,55.4 mmol), yield in two steps: 10%. LC-MS: [ M+H ]] ⁺ ＝272。

Step 4: ice bath, intermediate c9-4 (15 g,55.4 mmol) from the previous step was dissolved in 300mL anhydrous tetrahydrofuran and lithium aluminum hydride (5.2 g,138 mmol) was added. After stirring for 5 minutes, the ice bath was removed and the reaction was allowed to proceed at 60℃for 2 hours. The reaction was stopped. Adding dilute hydrochloric acid into the system to quench the reaction, adjusting the pH to about 7, precipitating solid, carrying out suction filtration, and washing a filter cake with ethyl acetate to obtain an intermediate c9.LC-MS: [ M+H ]] ⁺ ＝174。

Synthesis of intermediate c10

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The steps are as follows: intermediate c9 (2.0 g,11.5 mmol) was dissolved in 20mL DMF and NaH (920 mg,23 mmol) was added. The reaction was carried out at room temperature for 30 minutes. A solution of trifluoroiodomethane (3.4 g,17.3 mmol) in DMF (5 mL) was added to the system at-40 ℃. The reaction was stopped by slowly warming to room temperature and stirring continued for 1 hour. 50mL of ice-water was added to the reaction mixture, which was extracted with ethyl acetate, washed with saturated brine, concentrated under reduced pressure, and separated by flash column chromatography to give intermediate c10 (1.3 g,5.4 mmol) in 47% yield. LC-MS: [ M+H ] ] ⁺ ＝242。

Synthesis of intermediate c11

The steps are as follows: in an ice bath, intermediate c9 (2.2 g,12.7 mmol) was dissolved in 30mL DMSO and sodium hydride (1.02 g,25.9 mmol) was added. After stirring for 30 minutes, bromocyclopropane (2.3 g,19.0 mmol) was addedThe reaction was stopped after 1 hour at room temperature. To the system was added 100mL of ice water, extracted with ethyl acetate, washed with saturated brine, and concentrated under reduced pressure to give intermediate c11.LC-MS: [ M+H ]] ⁺ ＝214。

Synthesis of intermediate c12

The steps are as follows: ice bath, intermediate c9 (1.6 g,9.2 mmol) was dissolved in 30mL DMSO and sodium hydride (378 mg,18.4 mmol) was added. After stirring for 30 minutes, bromocyclobutane (1.87 g,13.9 mmol) was added and the reaction was stopped at room temperature for 1 hour. The reaction was stopped after 1 hour at room temperature. To the system was added 100mL of ice water, extracted with ethyl acetate, washed with saturated brine, and concentrated under reduced pressure to give intermediate c12.LC-MS: [ M+H ]] ⁺ ＝228。

Example 2:

step 1: intermediate a3 (2.4 g,5.61 mmol) was dissolved in 50mL dioxane under nitrogen and starting material P1-1 (1.34 g,8.42 mmol) and N, N-diisopropylethylamine DIEA (1.45 g,11.2 mmol) were added. The reaction solution was stirred at 80℃for 12 hours and cooled to room temperature. 100mL of water was added to the system, extracted with ethyl acetate, dried, filtered, the solvent was distilled off under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate=3:1) to give compound P1-2 (2.7 g,4.9 mmol). Yield: 87%, LC-MS: [ M+H ] ] ⁺ ＝552。

Step 2: under nitrogen, the above compound P1-2 (300 mg,0.54 mmol) and potassium phosphate (229 mg,1.08 mmol) were mixed in 6mL of anhydrous toluene, followed by the sequential addition of intermediate a7 (262 mg,0.65 mmol), xphos Pd G3 (93 mg,0.11 mmol) and Xphos (53 mg,0.11 mmol). The reaction solution was reacted at 100℃for 1 hour under nitrogen protection. The reaction was stopped, cooled to room temperature, filtered, and the solvent was distilled off under reduced pressure. The residue was chromatographed by TLC to give compound P1-3 (120 mg,0.15 mmol). Yield is good：28％，LC-MS:[M+H] ⁺ ＝807。

Step 3: p1-3 (120 mg,0.15 mmol) was dissolved in 4mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice bath. The reaction was continued for 1 hour under nitrogen protection, and stopped. Saturated sodium bicarbonate solution was slowly added to the system and adjusted to around pH 8. Ethyl acetate extraction, evaporation of the solvent under reduced pressure and purification of the residue by preparative HPLC gave the title compound P1 (10.2 mg). LC-MS: [ M+H ]] ⁺ ＝607。

¹ H NMR(400MHz,DMSO-d ₆ )δ9.07(s,1H),8.08(s,2H),7.41(dd,J＝8.4,5.3Hz,1H),7.14(dd,J＝9.5,8.4Hz,1H),5.27(br,J＝72Hz,1H),4.42(d,J＝12.3Hz,2H),4.13(d,J＝10.4Hz,1H),4.03(d,J＝10.3Hz,1H),3.71–3.52(m,4H),3.13–3.06(m,2H),3.01(s,1H),2.83(q,J＝8.5Hz,1H),2.35–2.27(m,1H),2.17–2.10(m,1H),2.08–1.98(m,2H),1.88–1.74(m,3H),1.65(d,J＝6.6Hz,2H),1.56(d,J＝7.5Hz,2H).

Referring to the synthetic route for compound P1, using a similar intermediate/backbone structure, the following target molecules were synthesized:

example 3:

step 1: intermediate a2 (500 mg,0.99 mmol) was dissolved in 6mL of anhydrous DMF under nitrogen, followed by addition of starting material P1-1 (314 mg,2.0 mmol) and cesium carbonate (966 mg,2.96 mmol), and the reaction was stopped at 140℃for 2 hours under nitrogen, and cooled to room temperature. 30mL of water was added to the system, extraction was performed with ethyl acetate, and the solvent was distilled off under reduced pressure. The residue was chromatographed by TLC (petroleum ether: ethyl acetate=1:4) to give H1-1 (110 mg,0.18 mmol) as a pale yellow solid. Yield: 18%, LC-MS: [ M+H ]] ⁺ ＝630。

Step 2: h1-1 (210 mg,0.34 mmol) was dissolved in 5mL of anhydrous toluene under nitrogen, and intermediate a7 (189 mg,0.47 mmol) Pd (DPEPhos) Cl was added sequentially ₂ (72 mg,0.10 mmol) and anhydrous cesium carbonate (273 mg,0.84 mmol). Under the protection of nitrogen, the reaction solution reacts for 6 hours at 105 ℃, the reaction is stopped, the reaction solution is cooled to room temperature, and the solvent is distilled off under reduced pressure. The residue was chromatographed by TLC (petroleum ether: ethyl acetate=1:2) to give H1-2 (80 mg,0.10 mmol) as a yellow solid. Yield: 28%, LC-MS: [ M+H ]] ⁺ ＝841。

Step 3: h1-2 (80 mg,0.10 mmol) was dissolved in 3mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice bath. Under the protection of nitrogen, the reaction solution is reacted for 0.5 hour at room temperature, the reaction is stopped, saturated sodium bicarbonate solution is slowly added into the system, the pH is regulated to about 8, the ethyl acetate is used for extraction, and the concentration is reduced pressure. The residue was purified by preparative SFC (Xselect CSH C18 OBD) to give the title compound H1a (4.0 mg) and H1b (4.1 mg).

H1a: ¹ H NMR(300MHz,DMSO-d ₆ )δ8.23(s,1H),8.11(s,1H),7.85(s,1H),7.26(dd,J＝8.4,5.3Hz,1H),7.15(dd,J＝9.5,8.4Hz,1H),5.27(br,J＝72Hz,1H),4.29(dd,J＝20.9,12.2Hz,3H),4.14–3.91(m,2H),3.67–3.39(m,5H),3.21–2.95(m,3H),2.92–2.70(m,1H),2.14(d,J＝6.8Hz,1H),2.09–1.93(m,2H),1.88–1.52(m,6H).

H1b: ¹ H NMR(400MHz,DMSO-d ₆ )δ8.21(s,1H),8.09(s,2H),7.84(s,1H),7.26(dd,J＝8.4,5.3Hz,1H),7.18–7.11(m,1H),5.27(br,J＝72Hz,1H),4.27(dd,J＝19.6,12.0Hz,2H),4.08(d,J＝10.3Hz,1H),3.99(d,J＝10.3Hz,1H),3.49–3.55(m,3H),3.08(d,J＝9.5Hz,3H),3.02(d,J＝11.5Hz,2H),2.81(s,1H),2.14–2.12(m,1H),2.02(d,J＝18.1Hz,2H),1.91–1.71(m,4H),1.66–1.54(m,4H).

Example 3:

step 1: in a 50mL reaction flask, intermediate a5 (420 mg,2.42 mmol) was dissolved in 10mL anhydrous THF, potassium tert-butoxide (340 mg,3.64 mmol) was added and stirred at room temperature for 30 min to give solution S1. In another 50mL reaction flask, intermediate a2 (1.0 g,2.0 mmol) was dissolved in 10mL anhydrous THF, and the prepared solution S1 was slowly added under ice bath, and stirring was continued for 1 hour, to stop the reaction. The reaction solution was poured into 100mL of ice water, extracted with ethyl acetate, and the solvent was distilled off under reduced pressure. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate=1:1) to give H2-1 (916 mg,1.42 mmol) as a pale yellow solid. Yield: 71%, LC-MS: [ M+H ] ] ⁺ ＝644。

Step 2: h2-1 (480 mg,0.75 mmol) was dissolved in 10mL of anhydrous toluene under nitrogen, and intermediate a7 (414 mg,1.02 mmol), pd (DPEPhos) Cl was added sequentially ₂ (22 mg,0.03 mmol) and cesium carbonate (438 mg,1.35 mmol). Under the protection of nitrogen, the reaction liquid reacts for 10 hours at 105 ℃, the reaction is stopped, the reaction liquid is cooled to room temperature, and the solvent is distilled off under reduced pressure. The residue was chromatographed by flash column chromatography (petroleum ether: ethyl acetate=1:2) to give H2-2 (450 mg,0.53 mmol) as a yellow solid. Yield: 70%, LC-MS: [ M+H ]] ⁺ ＝854。

Step 3: h2-2 (600 mg,0.70 mmol) was dissolved in a mixed solution of 10mL trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice bath. Under the protection of nitrogen, the reaction solution is reacted for 1 hour at room temperature, the reaction is stopped, saturated sodium bicarbonate solution is slowly added into the system, the pH is regulated to about 8, the ethyl acetate is used for extraction, and the concentration is reduced pressure. The residue was chromatographed on a flash column to give H2 (320 mg,0.49 mmol) as a yellow solid, LC-MS: [ M+H ]] ⁺ =654. Yield: 70%.

H2 was purified by chiral column: unichiral CMD-5H was purified to give the target compounds H2a (peak 1) and H2b (peak 2).

Referring to the synthetic route for compounds H2a or H2b, using similar intermediate/backbone structures, the following target molecules were synthesized:

* Or (b)Represents a chiral site; * Representing that chiral resolution is not performed;

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step 1: in a 50mL reaction flask, intermediate a5 (170 mg,0.94 mmol) was dissolved in 10mL anhydrous THF, potassium tert-butoxide (180 mg,1.56 mmol) was added and stirred at room temperature for 30 min to give solution S2. In another 50mL reaction flask, intermediate a17 (400 mg,0.78 mmol) was dissolved in 10mL anhydrous THF, and the prepared solution S2 was slowly added under ice bath, stirring was continued for 1 hour, and the reaction was stopped. The reaction solution was poured into 100mL of ice water, extracted with ethyl acetate, and the solvent was distilled off under reduced pressure. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate=1:1) to give H11-1 (300 mg,0.41 mmol) as a pale yellow solid. Yield: 44, LC-MS: [ M+H ]] ⁺ ＝734。

Step 2: h11-1 (300 mg,0.41 mmol) and CuCN (150 mg,1.64 mmol) were dissolved in 8mL anhydrous DMF under nitrogen, and the reaction was allowed to react at 100deg.C for 6 hours, cooled to room temperature, and the reaction was stopped. To the reaction solution was added 50mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the residue was separated by flash column chromatography (petroleum ether: ethyl acetate=2:1) to give yellow solid H11-2 (110 mg,0.17 mmol). Yield: 42, LC-MS: [ M+H ]] ⁺ ＝633。

Step 3: h11-2 (110 mg,0.17 mmol) was dissolved in 10mL of anhydrous toluene under nitrogen, and intermediate a7 (94 mg,0.22 mmol), pd was added sequentially (DPEPhos)Cl ₂ (22 mg,0.03 mmol) and cesium carbonate (160 mg,0.48 mmol). Under the protection of nitrogen, the reaction liquid reacts for 6 hours at 110 ℃, the reaction is stopped, the reaction liquid is cooled to room temperature, and the solvent is distilled off under reduced pressure. The residue was chromatographed by flash column chromatography (petroleum ether: ethyl acetate=1:1) to give H11-3 (90 mg,0.11 mmol) as a yellow solid. Yield: 63%, LC-MS: [ M+H ]] ⁺ ＝845。

Step 4: h11-3 (90 mg,0.11 mmol) was dissolved in a mixed solution of 8mL trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice bath. Under the protection of nitrogen, the reaction solution is reacted for 1 hour at room temperature, the reaction is stopped, saturated sodium bicarbonate solution is slowly added into the system, the pH is regulated to about 8, the ethyl acetate is used for extraction, and the concentration is reduced pressure. The residue was chromatographed on a flash column to give H11 (30 mg,0.05 mmol) as a yellow solid, LC-MS: [ M+H ]] ⁺ =645. Yield: 43%.

¹ H NMR(400MHz,DMSO-d ₆ )δ8.45(s,1H),8.26(s,2H),7.35(dd,J＝8.4,5.2Hz,1H),7.21(t,J＝8.8Hz,1H),4.58(d,J＝13.6Hz,1H),4.47(d,J＝12.8Hz,1H),4.34(s,2H),4.15(d,J＝16.8Hz,2H),4.02(d,J＝13.6Hz,2H),3.88(d,J＝12.8Hz,2H),3.52-3.16(m,4H),1.96-1.83(m,6H),1.23(s,2H),0.85–0.74(m,4H).

Referring to the synthetic route for compound H11, using a similar intermediate/backbone structure, the following target molecule was synthesized:

* Or (b)Represents a chiral site; * Representing that chiral resolution is not performed;

example 5:

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step 1: in a 50mL reaction flask, intermediate a19 (150 mg,0.55 mmol) was dissolved in 5mL anhydrous THF, potassium tert-butoxide (93 mg,0.82 mmol) was added and stirred at room temperature for 30 min to give solution S3. In another 50mL reaction flask, intermediate a2 (280 mg,0.58 mmol) was dissolved in 5mL anhydrous THF, and the prepared solution S3 was slowly added under ice bath, stirring was continued for 1 hour, and the reaction was stopped. The reaction solution was poured into 50mL of ice water, extracted with ethyl acetate, and the solvent was distilled off under reduced pressure. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate=1:1) to give H13-1 (205 mg,0.28 mmol) as a pale yellow solid. Yield: 51%, LC-MS:

[M+H] ⁺ ＝744。

Step 2: h13-1 (205 mg,0.28 mmol) was dissolved in 6mL dry toluene under nitrogen, and intermediate a7 (200 mg,0.50 mmol), pd (DPEPhos) Cl was added sequentially ₂ (13 mg,0.015 mmol) and cesium carbonate (175 mg,0.54 mmol). Under the protection of nitrogen, the reaction liquid reacts for 10 hours at 105 ℃, the reaction is stopped, the reaction liquid is cooled to room temperature, and the solvent is distilled off under reduced pressure. The residue was chromatographed by flash column chromatography (petroleum ether: ethyl acetate=1:2) to give H13-2 (120 mg,0.13 mmol) as a yellow solid. Yield: 45%, LC-MS: [ M+H ]] ⁺ ＝955。

Step 3: h13-2 (120 mg,0.13 mmol) was dissolved in 6mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice bath. Under the protection of nitrogen, the reaction solution is reacted for 1 hour at room temperature, the reaction is stopped, saturated sodium bicarbonate solution is slowly added into the system, the pH is regulated to about 8, the ethyl acetate is used for extraction, and the concentration is reduced pressure. The residue was chromatographed on a flash column to give H13 (32 mg) as a yellow solid, LC-MS: [ M+H ]] ⁺ ＝671。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.26(s,2H),7.97(d,J＝11.2Hz,1H),7.38–7.09(m,2H),5.88–5.43(m,2H),4.70–4.37(m,2H),4.29–4.00(m,6H),3.82-3.64(m,,4H),3.74–3.36(m,4H),1.93(m,4H),1.60-1.50(m,2H),1.40-1.20(m,2H).

Referring to the synthetic route for compound H13, using a similar intermediate/backbone structure, the following target molecule was synthesized:

* Or (b)Represents a chiral site; * Representing that chiral resolution is not performed;

example 6:

step 1: intermediate H1-1 (1.0 g,1.59 mmol) was dissolved in 12mL of methanol under nitrogen, followed by addition of sodium methoxide (102 mg,4.77 mmol), and the reaction was warmed to 60℃for 2 hours, stopped, and cooled to room temperature. 40mL of water was added to the system, extraction was performed with ethyl acetate, and the solvent was distilled off under reduced pressure. The residue was chromatographed by flash column chromatography to give P5-1 (305 mg,0.48 mmol) as a pale yellow solid. Yield: 30%, LC-MS: [ M+H ] ] ⁺ ＝640。

Step 2: p5-1 (305 mg,0.48 mmol) was dissolved in 5mL of anhydrous toluene under nitrogen, and intermediate a7 (270 mg,0.67 mmol), pd (DPEPhos) Cl was added sequentially ₂ (90 mg,0.14 mmol) and anhydrous cesium carbonate (330 mg,0.96 mmol). Under the protection of nitrogen, the reaction solution reacts for 8 hours at 105 ℃, the reaction is stopped, the reaction solution is cooled to room temperature, and the solvent is distilled off under reduced pressure. The residue was chromatographed by column chromatography (PE/EA, 1/2) to give P5-2 (327 mg,0.38 mmol) as a yellow solid. Yield: 80%, LC-MS: [ M+H ]] ⁺ ＝853。

Step 3: p5-2 (327 mg,0.38 mmol) was dissolved in 4mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice-bath. Under the protection of nitrogen, the reaction solution is reacted for 1 hour at room temperature, the reaction is stopped, saturated sodium bicarbonate solution is slowly added into the system, the pH is regulated to about 8, the ethyl acetate is used for extraction, and the concentration is reduced pressure. The residue was purified by HPLC preparative chromatography to give the title compound P5 (74 mg,0.11 mmol). Yield: 30%, LC-MS: [ M+H ]] ⁺ ＝653。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.16(s,2H),7.87(d,J＝0.8Hz,1H),7.29(dd,J＝8.4,5.3Hz,1H),7.22–7.11(m,1H),5.68-5.54(m,1H),4.28(dt,J＝35.0,17.4Hz,2H),4.06(dd,J＝35.9,10.3Hz,2H),3.56(t,J＝18.0Hz,4H),3.21(s,3H)3.18–3.08(m,2H),3.03(s,1H),2.90–2.76(m,1H),2.19–1.75(m,6H),1.63(dd,J＝20.6,9.9Hz,4H).

Referring to the synthetic route for compound P5, using a similar intermediate/backbone structure, the following target molecule was synthesized:

* Or (b)Represents a chiral site; * Representing that chiral resolution is not performed;

example 7:

step 1: intermediate H2-1 (500 mg,0.78 mmol) and trifluoroethanol (156 mg,1.56 mmol) were dissolved in 10mL of tetrahydrofuran in an ice bath under nitrogen protection, then NaH (94 mg,2.34 mmol) was added, the reaction was allowed to react at room temperature for 3 hours, the reaction was stopped, and the reaction was cooled to room temperature. 30mL of water was added to the system, extraction was performed with ethyl acetate, and the solvent was distilled off under reduced pressure. The residue was chromatographed by flash column chromatography to give P7-1 (337 mg,0.47 mmol) as a pale yellow solid. Yield: 60%, LC-MS: [ M+H ] ] ⁺ ＝722。

Step 2: p7-1 (305 mg,0.42 mmol) was dissolved in 5mL of anhydrous toluene under nitrogen, and intermediate a7 (270 mg,0.67 mmol), pd (DPEPhos) Cl was added sequentially ₂ (90 mg,0.14 mmol) and anhydrous cesium carbonate (330 mg,0.96 mmol). Under the protection of nitrogen, the reaction solution reacts for 8 hours at 105 ℃, the reaction is stopped, the reaction solution is cooled to room temperature, and the solvent is distilled off under reduced pressure. The residue was chromatographed by column chromatography (PE/EA, 1/2) to give P7-2 (313 mg,0.34 mmol) as a yellow solid. Yield: 80%, LC-MS: [ M+H ]] ⁺ ＝934。

Step 3: p7-2 (313 mg, 0) was added under ice bath.34 mmol) was dissolved in 5mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1). Under the protection of nitrogen, the reaction solution is reacted for 1 hour at room temperature, the reaction is stopped, saturated sodium bicarbonate solution is slowly added into the system, the pH is regulated to about 8, the ethyl acetate is used for extraction, and the concentration is reduced pressure. The residue was purified by HPLC preparative chromatography to give the title compound P7 (82 mg,0.11 mmol). Yield: 33, LC-MS: [ M+H ]] ⁺ ＝734。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.02(s,2H),7.82(s,1H),7.20(dd,J＝8.3,5.4Hz,1H),7.12(t,J＝8.9Hz,1H),5.24-5.10(m,1H),4.96–4.75(m,2H),4.24(dd,J＝10.8,4.4Hz,2H),3.65(d,J＝13.2Hz,4H),3.51(d,J＝12.1Hz,2H),2.84(dd,J＝23.1,10.0Hz,2H),2.72–2.58(m,1H),2.48(s,1H),2.36(dt,J＝15.9,9.7Hz,2H),2.21–1.76(m,2H),1.76–1.60(m,4H),0.66–0.60(m,2H),0.46–0.41(m,2H).

Example 8:

step 1: intermediate H2-1 (500 mg,0.78 mmol) and 4-methoxybenzyl alcohol (215 mg,1.56 mmol) were dissolved in 10mL tetrahydrofuran in an ice bath under nitrogen, naH (94 mg,2.34 mmol) was then added, the reaction was allowed to react at room temperature for 3 hours, the reaction was stopped, and the reaction solution was cooled to room temperature. 30mL of water was added to the system, extraction was performed with ethyl acetate, and the solvent was distilled off under reduced pressure. The residue was chromatographed on flash column to give P8-1 (385 mg,0.51 mmol) as a pale yellow solid. Yield: 65%, LC-MS: [ M+H ] ] ⁺ ＝760。

Step 2: p8-1 (319 mg,0.42 mmol) was dissolved in 5mL of anhydrous toluene under nitrogen, and intermediate a7 (270 mg,0.67 mmol), pd (DPEPhos) Cl, was added sequentially ₂ (90 mg,0.14 mmol) and anhydrous cesium carbonate (330 mg,0.96 mmol). Under the protection of nitrogen, the reaction solution reacts for 8 hours at 105 ℃, the reaction is stopped, the reaction solution is cooled to room temperature, and the solvent is distilled off under reduced pressure. The residue was chromatographed by column chromatography (PE/EA, 1/2) to give P8-2 (122 mg,0.13 mmol) as a yellow solid. Yield: 30%, LC-MS: [ M+H ]] ⁺ ＝972。

Step 3: p8-2 (122 mg,0.13 mmol) was dissolved in 5mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice bath. Under the protection of nitrogen, the reaction solution is reacted for 1 hour at room temperature, the reaction is stopped, saturated sodium bicarbonate solution is slowly added into the system, the pH is regulated to about 8, the ethyl acetate is used for extraction, and the concentration is reduced pressure. The residue was purified by HPLC preparative chromatography to give the title compound P8 (19 mg,0.03 mmol). Yield: 23, LC-MS: [ M+H ]] ⁺ ＝653。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.02(s,2H),7.82(s,1H),7.20(dd,J＝8.3,5.4Hz,1H),7.12(t,J＝8.9Hz,1H),5.32-5.18(m,1H),4.24(dd,J＝10.8,4.4Hz,2H),3.65(d,J＝13.2Hz,4H),3.51(d,J＝12.1Hz,2H),2.84(dd,J＝23.1,10.0Hz,2H),2.72–2.58(m,1H),2.48(s,1H),2.36(dt,J＝15.9,9.7Hz,2H),2.21–1.76(m,2H),1.76–1.60(m,4H),0.68–0.60(m,2H),0.48–0.41(m,2H).

Example 9:

step 1: intermediate a3 (900 mg,2.1 mmol) and intermediate a5 (360 mg,2.1 mmol) were dissolved in 15mL of tetrahydrofuran in an ice bath under nitrogen, then NaH (100 mg,4.2 mmol) was added, the reaction mixture was warmed to 40℃and allowed to react for 3 hours, the reaction was stopped, and the mixture was cooled to room temperature. 30mL of water was added to the system, extraction was performed with ethyl acetate, and the solvent was distilled off under reduced pressure. The residue was chromatographed on a flash column to give P9-2 (490 mg,0.81 mmol) as a yellow solid. Yield: 38, LC-MS: [ M+H ] ] ⁺ ＝565。

Step 2: p9-2 (440 mg,0.78 mmol) was dissolved in a mixed solution of 6mL1, 4-dioxane and water (v/v, 5/1) under nitrogen, and starting material P9-1 (480 mg,0.94 mmol), xphos Pd G1 (180 mg,0.23 mmol) and anhydrous cesium carbonate (1.02G, 3.12 mmol) were added sequentially. Under the protection of nitrogen, the reaction solution reacts for 3 hours at 95 ℃, the reaction is stopped, the reaction solution is cooled to room temperature, and the solvent is distilled off under reduced pressure. The residue was chromatographed by column chromatography (PE/EA, 1/2) to give P9-3 (500 mg,0.55 mmol) as a yellow solid. Yield: 70%, LC-MS: [ M+H ]] ⁺ ＝915。

Step 3: intermediate P9-3 (500 mg,0.55 mmol) of the above step was dissolved in 20mL of tetrahydrofuran, and tetrabutylammonium fluoride (220 mg,0.9 mmol) was added. The reaction was allowed to proceed at room temperature for 12 hours, and TLC monitored the reaction was complete. The reaction solution was dissolved in 50mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give crude P9-4 (400 mg), LC-MS: [ M+H ]] ⁺ ＝759。

Step 4: under ice bath, crude P9-4 (400 mg,0.53 mmol) was dissolved in 5mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1). Under the protection of nitrogen, the reaction solution is reacted for 1 hour at room temperature, the reaction is stopped, saturated sodium bicarbonate solution is slowly added into the system, the pH is regulated to about 8, the ethyl acetate is used for extraction, and the concentration is reduced pressure. The residue was purified by HPLC prep chromatography to give the title compound P9 (45 mg,0.07 mmol). Yield: 14, LC-MS: [ M+H ] ] ⁺ ＝615。

¹ H NMR(400MHz,DMSO-d ₆ )δ10.30(s,1H),9.10(s,1H),7.98(dd,J＝9.2,6.0Hz,1H),7.47(t,J＝9.0Hz,1H),7.41(d,J＝2.4Hz,2H),7.37(s,1H),7.24(s,1H),7.22(s,2H),7.11(s,1H),4.65(d,J＝13.7Hz,1H),4.49(s,1H),4.35(s,3H),4.18(s,4H),4.0-3.90(m,6H),1.97(m,7H).

Referring to the synthetic route for compound P9, using a similar intermediate/backbone structure, the following target molecule was synthesized:

example 10:

step 1: in a 20mL reaction flask, intermediate a5 (142 mg,0.82 mmol) was dissolved in 5mL anhydrous THF, potassium tert-butoxide (138 mg,2.34 mmol) was added and stirred at room temperatureMix for 30 minutes to obtain solution S4. In another 20mL reaction flask, intermediate a27 (400 mg,0.82 mmol) was dissolved in 5mL anhydrous THF, and the prepared solution S4 was slowly added under ice bath, stirring was continued for 1 hour, and the reaction was stopped. The reaction solution was poured into 100mL of ice water, extracted with ethyl acetate, and the solvent was distilled off under reduced pressure. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate=1:1) to give P12-1 (420 mg,0.66 mmol) as a pale yellow solid. Yield: 80%, LC-MS: [ M+H ]] ⁺ ＝642。

Step 2: p12-1 (420 mg,0.66 mmol) was dissolved in 5mL of anhydrous toluene under nitrogen, and intermediate a7 (352 mg,0.87 mmol), pd (DPEPhos) Cl, was added sequentially ₂ (22 mg,0.03 mmol) and cesium carbonate (438 mg,1.35 mmol). Under the protection of nitrogen, the reaction liquid reacts for 10 hours at 105 ℃, the reaction is stopped, the reaction liquid is cooled to room temperature, and the solvent is distilled off under reduced pressure. The residue was chromatographed by flash column chromatography (petroleum ether: ethyl acetate=1:2) to give yellow solid P12-2 (450 mg,0.53 mmol). Yield: 80%, LC-MS: [ M+H ] ] ⁺ ＝854。

Step 3: p12-2 (450 mg,0.53 mmol) was dissolved in 6mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice bath. Under the protection of nitrogen, the reaction solution is reacted for 1 hour at room temperature, the reaction is stopped, saturated sodium bicarbonate solution is slowly added into the system, the pH is regulated to about 8, the ethyl acetate is used for extraction, and the concentration is reduced in pressure. The residue was chromatographed on a flash column to give P12 (104 mg,0.16 mmol) as a yellow solid, LC-MS: [ M+H ]] ⁺ =654. Yield: 30%.

¹ H NMR(400MHz,DMSO-d ₆ )δ8.15(s,2H),7.81(s,1H),7.26(dd,J＝8.4,5.3Hz,1H),7.15(t,J＝8.9Hz,1H),5.24–5.10(m,1H),4.22(dt,J＝19.4,10.7Hz,2H),3.86–3.62(m,4H),3.01(s,2H),2.87–2.75(m,2H),2.42-2.33(m,3H),2.20–1.95(m,2H),1.91–1.75(m,1H),0.67–0.38(m,8H).

Referring to the synthetic route for compound P12, using a similar intermediate/backbone structure, the following target molecule was synthesized:

example 11:

step 1: in a high-pressure reaction vessel, the raw material 4-bromo-2, 6-difluorobenzonitrile a5 (100 g,459 mmol) was dissolved in 240mL of ethanol, 400mL of aqueous ammonia was added, the reaction was allowed to proceed at 90℃for 16 hours, and the reaction was allowed to cool to room temperature, to stop the reaction. The solvent was distilled off under reduced pressure, 100mL of ice water was added to the reaction solution, extraction was performed with ethyl acetate, and the solvent was distilled off under reduced pressure. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate, 9/1) to give P14-2 (74.9 g,352 mmol) as a yellow solid. Yield: 77, LC-MS: [ M+H ]] ⁺ ＝215。

Step 2: 2, 2-diethoxyethanol (33.8 g,252 mmol) was dissolved in 450mL anhydrous DMF under nitrogen, naH (10.08 g,252 mmol) was slowly added at 0deg.C, and after stirring for 1 hour, the last step intermediate P14-2 (45 g,210 mmol) was added. Removing ice bath, heating to 50deg.C, reacting for 2 hr, stopping reaction, cooling to room temperature, adding 1L water, extracting with ethyl acetate, and evaporating under reduced pressure to remove solvent. The residue was chromatographed by flash column chromatography (petroleum ether/ethyl acetate, 10/1) to give P14-3 (25.3 g,77.1 mmol) as a yellow solid. Yield: 37%, LC-MS: [ M+H ] ] ⁺ ＝329。

Step 3: 100mL of toluene was added to polyphosphoric acid (10.42 g), the temperature was raised to 100℃and the reaction was continued at that temperature for 2 hours by adding the intermediate P14-3 (10.0 g,30.4 mmol) in the previous step, and the reaction was stopped. The reaction solution was slowly poured into a large amount of ice water, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was chromatographed by flash column chromatography (petroleum ether/ethyl acetate, 10/1) to give P14-4 (1.04 g,4.4 mmol) as a yellow solid, yield: 14%. LC-MS: [ M+H ]] ⁺ ＝236。

Step 4: the intermediate P14-4 (8.3 g,35.0 mmol) of the above step was dissolved in 150mTo L ethanol, aqueous KOH (7.94 g,50 mL) was added, and the reaction was stopped after heating to 90℃for 4 hours. The organic solvent was distilled off under reduced pressure, 100mL of ice water was added to the reaction solution, extraction was performed with methylene chloride, and concentration was performed under reduced pressure. The residue was chromatographed by flash column chromatography (petroleum ether/dichloromethane, 2/1) to give P14-5 (4.0 g,15.7 mmol) as a yellow solid, yield: 45%. LC-MS: [ M+H ]] ⁺ ＝256。

Step 5: under nitrogen, the intermediate P14-5 (3.3 g,13.0 mmol) from the previous step was dissolved in 33mL dry THF and a tetrahydrofuran solution (20 mL) containing triphosgene (3.6 g,12.4 mmol) was slowly added dropwise at 0deg.C. The reaction was stopped after the reaction was warmed to room temperature for 2 hours, 100mL of water was added to the system, extraction was performed with ethyl acetate, and the solvent was distilled off under reduced pressure to obtain crude P14-6 (2.8 g). LC-MS: [ M+H ] ] ⁺ ＝282。

Step 6: under the protection of nitrogen, the crude product P14-6 (2.8 g) in the previous step is dissolved in 50mL phosphorus oxychloride, 5mL N, N-diisopropylethylamine is added, and the temperature is raised to 100 ℃ for reaction for 2 hours. The solvent was distilled off under reduced pressure, 100mL of water was added to the system, extracted with ethyl acetate, concentrated, and the residue was chromatographed by flash column chromatography (petroleum ether/ethyl acetate, 3/1) to give P14-7 (1.1 g,3.5 mmol) as a yellow solid, yield in two steps: 27%. LC-MS: [ M+H ]] ⁺ ＝319。

Step 7: intermediate P14-7 (1.0 g,3.15 mmol) and starting 3, 8-diazabicyclo [3.2.1]Tert-butyl octane-8-carboxylate a2-1 (640 mg,3.15 mmol) was dissolved in 20mL of 1, 4-dioxane, N-diisopropylethylamine (1.7 mL,9.5 mmol) was added, and the mixture was heated to 50℃and reacted for 2 hours. 60mL of water was added to the system, extraction was performed with methylene chloride, drying over anhydrous sodium sulfate, filtration, concentration and column chromatography were performed to obtain white solid P14-8 (900 mg,1.82 mmol). Yield: 58%. LC-MS: [ M+H ]] ⁺ ＝495。

Step 8: intermediate P14-8 (520 mg,1.05 mmol) and cesium carbonate (684 mg,2.10 mmol) were dissolved in 5mL DMF under nitrogen, intermediate a5 (803 mg,2.10 mmol) was added and the reaction was allowed to proceed at 140℃for 2 hours. Cooled to room temperature, the system was added with 60mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (petroleum ether/ethyl acetate, 1/1) to give P14-9 (155 mg,0.25 mmol) as a yellow solid ). Yield: 23%. LC-MS: [ M+H ]] ⁺ ＝630。

Step 9: intermediate P14-9 (155 mg,0.25 mmol) and cesium carbonate (200 mg,0.62 mmol) were dissolved in 5mL of toluene under nitrogen, and intermediate a7 (139 mg,0.34 mmol) and Pd (DPEPhos) Cl were added ₂ (52 mg,0.074 mmol) was reacted at 105℃for 3 hours. Cooled to room temperature, the system was added with 30mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (petroleum ether/ethyl acetate, 1/4) to give P14-10 (85 mg,0.25 mmol) as a yellow solid. Yield: 41%. LC-MS: [ M+H ]] ⁺ ＝842。

Step 10: p14-10 (65 mg,0.077 mmol) was dissolved in 3mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice bath. Under the protection of nitrogen, the reaction solution is reacted for 1 hour at room temperature, the reaction is stopped, saturated sodium bicarbonate solution is slowly added into the system, the pH is regulated to about 8, the ethyl acetate is used for extraction, and the concentration is reduced in pressure. The residue was chromatographed by HPLC (X Bridge Shield RP OBD column, 19X 150mm,5 μm; mobile phase A: water (10 mmol/L NH) ₄ HCO ₃ ) Mobile phase B is acetonitrile; flow rate: 25 mL/min) to give P14 (9.2 mg) as a white solid, LC-MS: [ M+H ]] ⁺ ＝642。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.11(d,J＝4.0Hz,1H),7.97(brs,2H),7.36-7.31(m,2H),7.17-7.11(m,1H),6.57(d,J＝4.0Hz,1H),5.17(d,J＝56.0Hz,1H),4.31-4.18(m,2H),4.07-4.03(m,1H),3.93-3.89(m,1H),3.49-3.45(m,2H),2.89-2.73(m,3H),2.41-2.27(m,3H),2.17-2.08(m,2H),1.93-1.79(m,3H),1.66-1.62(m,2H),1.32(s,1H),0.64-0.61(m,2H),0.45-0.42(m,2H).

Example 12:

step 1: intermediate c1 (330 mg,1.76 mmol) was dissolved in 1mL of anhydrous tetrahydrofuran in an ice bath, naH (85 mg,3.52 mmol) was added, and the mixture was stirred at room temperature for 0.5 hour. Intermediate a3 (350 mg,0.82 mmol) was dissolved in 1mL anhydrous tetrahydrofuran, and the reaction mixture was added at room temperature The reaction was stopped after 1 hour. Concentrating under reduced pressure, and separating the crude product by flash column chromatography to obtain compound P18-1 (240 mg,0.42 mmol) with a yield of 24%. LC-MS: [ M+H ]] ⁺ ＝579。

Step 2: under nitrogen, compound P18-1 (240 mg,0.42 mmol), intermediate b2 (270 mg,0.44 mmol), cesium carbonate (270 mg,0.83 mmol) and methanesulfonic acid [ n-butylbis (1-adamantyl) phosphine](2-amino-1, 1' -biphenyl-2-yl) palladium (Pd-G3, 10mg,0.014 mmol) was dissolved in a mixed solution of 2mL of 1, 4-dioxane and water (v/v, 5/1), and the reaction was stopped after heating to 95℃for 1 hour. The solvent was distilled off under reduced pressure, and the crude product was separated by flash column chromatography to give Compound P18-2 (300 mg,0.29 mmol) in a yield of 70%. LC-MS: [ M+H ]] ⁺ ＝1041。

Step 3: compound P18-2 (270 mg,0.26 mmol) and cesium fluoride (80 mg,0.52 mmol) were dissolved in 1mL DMF, and the reaction was stopped after heating to 50℃for 1 hour. 5mL of water is added into the system, extraction is carried out by ethyl acetate, drying is carried out by anhydrous sodium sulfate, and decompression concentration is carried out, thus obtaining crude P18-3.LC-MS: [ M+H ]] ⁺ ＝729。

Step 4: the crude product P18-3 obtained in the above step was dissolved in 1mL of 1, 4-dioxane solution of hydrogen chloride (4M concentration), reacted at room temperature for 1 hour, and a saturated aqueous sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, concentrated under reduced pressure, and purified by TLC thin layer chromatography to give Compound P18 (50 mg). LC-MS: [ M+H ] ] ⁺ ＝629。

¹ H NMR(400MHz,DMSO-d ₆ )δ10.17(s,1H),9.06(s,1H),7.99(dd,J＝9.2,5.9Hz,1H),7.48(t,J＝9.0Hz,1H),7.41(d,J＝2.5Hz,1H),7.19(t,J＝2.2Hz,1H),4.49(d,J＝11.4Hz,1H),4.33(d,J＝12.6Hz,1H),4.12(dd,J＝10.5,3.4Hz,1H),4.02(dd,J＝10.6,2.7Hz,1H),3.95(s,1H),3.69–3.53(m,4H),3.45–3.25(m,2H),2.95–2.85(m,1H),2.72–2.64(m,1H),2.57–2.52(m,1H),2.47–2.32(m,2H),2.09(s,3H),1.95–1.72(m,4H),1.90–1.60(m,4H),1.51(t,J＝11.7Hz,1H).

Referring to the synthetic route for compound P18, using a similar intermediate/backbone structure, the following target molecule was synthesized:

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example 13:

raw material P29-1 (305 mg,2.4 mmol) and intermediate a3 (500 mg,1.98 mmol) were dissolved in 10mL of methylene chloride under ice bath, and triethylamine (301 mg,2.97 mmol) was added thereto to react at room temperature for 2 hours. To the system was added 30mL of water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to give P29-2 (650 mg,1.9 mmol) as a white solid in yield: 95%. LC-MS: [ M+H ]] ⁺ ＝344。

In a 50mL reaction flask, compound P29-2 (300 mg,0.87 mmol) was dissolved in 6mL anhydrous tetrahydrofuran, sodium hydride (70 mg,1.74 mmol) was added, and the mixture was stirred for 30 minutes to obtain a solution S1. Intermediate a18 was added to solution S1, and the reaction was stopped after the reaction was continued at 70 ℃ for 16 hours and cooled to room temperature. The reaction was quenched by adding 30mL of ice water, extracted with ethyl acetate, and the solvent was removed by concentrating under reduced pressure. The residue was purified by flash column chromatography (DCM/meoh=10/1) to give P29-3 (200 mg,0.40 mmol) as a white solid, yield: 46%. LC-MS: [ M+H ]] ⁺ ＝495。

Compound P29-3 (450 mg,0.91 mmol) and starting material P9-1 (560 mg,1.1 mmol) were dissolved in 10mL of 1, 4-dioxane and 2mL of water under nitrogen, and the catalyst XPhos Pd G2 (215 mg,0.3 mmol) and cesium carbonate (1.18G, 3.64 mmol) were added sequentially. The reaction mixture was reacted at 95℃for 2 hours, and the reaction was stopped. The solvent was removed by concentration under reduced pressure and the residue was chromatographed on flash column (DCM/meoh=20/1) to give P29-4 (500 mg,0.59 mmol) as a yellow solid. Yield: 65%, LC-MS: [ M+H ] +=845.

Compound P29-4 (200 mg,0.24 mmol) was dissolved in 4mL of tetrahydrofuran at room temperature, and tetrabutylammonium fluoride (TBAF, 130mg,0.48 mmol) was added thereto and reacted at room temperature for 1 hour. To the system was added 20mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated to give crude yellow oil P29-5 (160 mg,0.23 mmol). Yield: 98%. LC-MS: [ M+H ]] ⁺ ＝689。

The crude P29-5 (163 mg,0.24 mmol) obtained in the above step was dissolved in 2mL of methylene chloride, and 1mL of a 1, 4-dioxane solution (4N concentration) of hydrogen chloride was added dropwise to react at room temperature for 1 hour to stop the reaction. Saturated sodium bicarbonate solution was slowly added to the system and the pH was adjusted to about 8, extracted with ethyl acetate and concentrated under reduced pressure. The residue was chromatographed on flash column to give 10mg of P29 as a yellow solid. LC-MS: [ M+H ]] ⁺ ＝645。

¹ H NMR(400MHz,DMSO-d ₆ )δ10.16(s,1H),9.34(s,1H),8.77(s,1H),8.01(dd,J＝9.2,5.9Hz,1H),7.49(t,J＝9.0Hz,1H),7.42(d,J＝2.4Hz,1H),7.19(t,J＝3.5Hz,1H),5.30–5.02(m,1H),4.54–4.41(m,2H),4.22-4.05(m,1H),4.00-3.92(m,1H),3.87-3.68(m,1H),2.85–2.60(m,5H),2.42-2.20(m,7H),2.16–1.67(m,14H).

Example 14:

the compounds were tested for KRAS G12D-mediated inhibition of p-ERK (directly reflecting the cellular level inhibitory effect of the test compounds). The method comprises the following steps:

AGS cells cultured in F-12K medium (Gibco, cat. No. 30-2004) containing 10% fetal bovine serum and 1% penicillin were inoculated onto 384-well microplates and incubated overnight at 37℃under 5% carbon dioxide. 200. Mu.l of the compound (final concentration of dimethyl sulfoxide 0.5%) was added to each well and incubated at 37℃for 3 hours. Cells were then fixed in 8% fixative (Solarbio, cat. No. p 1112) and washed once with Phosphate Buffer (PBS). After washing, blocking solution (LI-COR, cat. No. 927-40000) was added to each well and blocked for 1 hour at room temperature. After removal of blocking solution, phospho-p44/42MAPK (T202/Y204) Rabbit mAb (CST, cat.No.97166S) and GAPDH (D4℃ 6R) Mouse mAb (CST, cat.No.4370S) antibody working solution were added to each well and incubated overnight at 4 ℃. The microwell plates were washed three times with PBS solution (PBST) containing 0.1% Tween-80, IRDye 800CW gold anti-Rabbit IgG (H+L) (LI-COR, cat. No. 926-32211) and IRDye 680RD Goat anti Mouse IgG (H+L) (LI-COR, cat. No. 926-68070) antibody working solutions were added and the microwell plates were incubated at room temperature in the absence of light. After washing the microwell plates three times using PBST, the microwell plates were centrifuged at 1000rpm for 1 min, the read plates were scanned using an Odyssey CLx (LI-COR) instrument and signal values were recorded.

IC ₅₀ Is calculated by the formula of (2)

Calculation of compound IC using nonlinear regression equation ₅₀ Value: y=lower plateau signal + (upper plateau signal-lower plateau signal)/(1+10 ((log ic) ₅₀ -X) hill slope); x = log compound concentration.

Table 1: half-effective concentration inhibitory Effect of Compounds on p-ERK [ MRTX1133 as a Positive control ]

N.d. =untested

MRTX1133 structure:

example 15: inhibitory Activity of Compounds on GTP-KRAS

Diluting the test compound with 4-fold concentration gradient, transferring 0.1 μl of different concentrations of test compound to each well of 384-well microplate using ECHO (Labcyte), sequentially adding 5 μl of diluted Tag2-KRAS G12D & GTP or Tag2-KRAS WT & GTP to each well, and centrifuging at 1000RPM for 1 min; then 5. Mu.L of diluted Tag1-cRAF was added to each well, centrifuged at 1000RPM for 1 min and incubated at 25℃for 15 min; mu.L of a mixture of anti-Tag1-Tb3 and anti-Tag2-XL665 was added to each well, centrifuged at 1000RPM for 1 minute and incubated at 4℃for 3 hours; the plate was scanned at 665/615nm using Envision and signal values were recorded.

In the above analysis, the relevant detection reagents for KRAS-G12D were all derived from the commercial kit KRAS-G12D/cRAF BINDING ASSAY KITS (Cisbio, cat. No.63ADK000CB21 PEG); in KRAS-WT assays, GTP was purchased from Sigma (Cat. No. V900868), GST-cRAF was prepared by Beijing-Kaglon formation (Cat. No. 20190718), MAb Anti GST-Tb cryptate was purchased from Cisbio (Cat. No.61 GSTTLA), and other key reagents were obtained from the commercial kit KRAS-WT/SOS1 BINDING ASSAY KITS (Cisbio, cat. No.63ADK000CB15 PEH).

The calculation results were analyzed according to the following formula:

relative Ratio (RR) = (Ratio) _665/615 -Ratio _Background )

Percent inhibition= [1- (RR) _{Compounds of formula (I)} -RR _{Positive control well} Average value)/(RR _{Negative control well} Average value-RR _{Positive control well} Average value of]×100

IC ₅₀ And (3) calculating: y=lower plateau signal + (upper plateau signal-lower plateau signal)/(1+10 ≡log ic ₅₀ -X) X hill slope). X, compound concentration log; y: percent inhibition.

The results indicate that the molecules of the invention have excellent inhibitory effect on KRAS G12D protein activation.

Example 16:

3D antiproliferative effect of compounds on KRAS G12D mutated pancreatic cancer AsPC-1 cell lines. The method comprises the following steps:

cell culture: in T75 flasks (Corning, cat. No. 430641), asPC-1 pancreatic cancer cells were cultured in RPMI 1640 medium (Hyclone, cat. No. SH 3080901B) containing 10% fetal bovine serum (Ausgreex, cat. No. FBS 500-S) and 1% green/streptomycin (Gibco, cat. No. 15140-122).

The test process comprises the following steps: the diluted test compound was added to 384-well low adsorption cell culture plate (Labcytoe, catalog number PP-0200) using nanoliter pipetting system (LABCYTE, catalog number Echo 550), and after plating into cells, the culture plate was placed at 37℃with 5% CO ₂ Culturing in a constant temperature incubator. After incubation of test compounds (1. Mu.M as initial concentration, 3-fold dilution, total of 10 concentrations) with cells for 7 days, cellTiter-The 3D reagent (Promega, catalog number G9683) was read with an Envision multifunctional enzyme-labeled instrument (Perkin Elmer, catalog number Envision 2104) and the light signal was proportional to the amount of ATP in the system, while the ATP content directly characterizes the number of viable cells in the system. Finally, using XLFIT software to obtain IC of compound by using nonlinear fitting formula ₅₀ (half inhibition concentration).

Inhibition (%) =100× (negative control mean-compound read)/(negative control mean-positive control mean)

Negative control: DMSO. Positive control: a culture medium.

Table 2: antiproliferative effect of compounds on AsPC-1 cells at half-effective concentrations

Compounds of formula (I)	IC 50 /nM
		H1a	4.2
H1b	49
		H2b	23
P4	851
		P11	50
P20	47

The result shows that the molecules of the invention have good antiproliferative effect on KRAS G12D mutant tumor cell lines.

Example 17:

liver microsomal stability assay of the compounds. The method comprises the following steps:

the compound of the invention is subjected to liver microsome stability test research, the compound to be tested is incubated with liver microsomes of different species with or without NADPH, the final concentration of the compound to be tested in a test system is 1 mu M, the final concentration of NADPH is 1mM, and the final concentration of the liver microsome is 0.5mg/ml. The compound concentrations in the supernatants of the incubations at different time points over 60 minutes were measured and pharmacokinetic parameters (e.g. clearance Clint) were calculated.

The results indicate that the molecules of the invention have better metabolic stability (especially in humans).

Some molecules (e.g., H1b, H10b, P20, P24, M1, etc.) have lower clearance and slower metabolism in humans than the control MRTX 1133.

N.d. =untested.

Example 18:

in vivo efficacy experiment of BALB/c nude mice. The method comprises the following steps:

colorectal cancer tumor cells GP2D mutated in KRAS G12D were cultured and inoculated into 6-8 week old female BALB/c nude mice (weighing about 20G) and all mice were inoculated subcutaneously. Mice were grown in an SPF-grade experimental environment and all mice were free to receive a commercially certified standard diet. When the average tumor volume of the mice grows to 150mm ³ On the left and right, the test compounds were started daily intraperitoneal (ip) administration. The dosage is as follows: blank vehicle (10% Captisol in 50mM citrate buffer pH 5.0). The dose of the administration group is 10mg/kg twice daily. Tumor volumes were measured three times a week with two-dimensional calipers and animals were weighed daily. After 10 days of continuous dosing, the inhibition (TGI/100%) was calculated from the final tumor volume. The volume calculation formula is: v=1/2 a×b ² A represents the long diameter of the tumor, and b represents the short diameter of the tumor.

Test agent	Dosage for administration	TGI
			Blank group	0	0％
MRTX1133	10mg/kg,BID	186％
			P20	10mg/kg,BID	187％

The result shows that the molecule of the invention has better in vivo efficacy, can inhibit the growth of KRAS G12D mutant tumor, and has better effect than MRTX1133.

Example 19:

animal in vivo safety experiments of the compounds of the invention. The method comprises the following steps:

qualified healthy ICR mice (age 6-8 weeks, body weight 18-20 g) were selected, 3 in each group, each for single intravenous administration. A single intravenous pre-trial was performed and the dose was fuelled from 2mg/kg, with dose escalation if no mortality was seen and discontinuation if mortality occurred.

MRTX1133, compound M1 and Compound P20 intravenous vehicles were: DMSO/Tween 80/Solutol/physiological saline (the volume ratio of the four is 5/3/10/82), and after vortex ultrasonic treatment to dissolve the materials sufficiently, administration treatment is carried out.

Compounds of formula (I)	Administration mode	Dosage for administration	Mortality rate/3
				MRTX1133	Vein iv	2mpk	0/3
MRTX1133	Vein iv	4mpk	1/3
				P20	Vein iv	2mpk	0/3
P20	Vein iv	4mpk	0/3
				P20	Vein iv	8mpk	0/3
P20	Vein iv	16mpk	2/3
				M1	Vein iv	2mpk	0/3
M1	Vein iv	4mpk	0/3
				M1	Vein iv	8mpk	0/3
M1	Vein iv	16mpk	0/3
				M1	Vein iv	32mpk	1/3

The result shows that the molecule of the invention has better in vivo safety, the safety of P20 and M1 is far better than MRTX1133 after intravenous administration, the safety benefit brought by replacing F with methyl thio is suggested, and the molecule of the invention is expected to have better clinical safety.

Claims (7)

1. A compound, or a tautomer, stereoisomer, prodrug, crystal form, pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the compound is selected from the group consisting of:

2. a pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, hydrate, or isotopic variant thereof, and a pharmaceutically acceptable excipient; preferably, it also contains other therapeutic agents.

3. Use of a compound of claim 1, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, hydrate or isotopic variant thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of KRAS G12D mutein mediated diseases.

4. A method of treating and/or preventing KRAS G12D mutein-mediated diseases in a subject, the method comprising administering to the subject the compound of claim 1 or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, hydrate or isotopic variant thereof or the pharmaceutical composition of claim 2.

5. The compound of claim 1 or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, hydrate or isotopic variant thereof or the pharmaceutical composition of claim 2 for use in the treatment and/or prevention of KRAS G12D mutein-mediated diseases.

6. The use of claim 3 or the use of the method of claim 4 or the compound or composition of claim 5, wherein the KRAS G12D mutein-mediated disease is selected from acute myeloid leukemia, juvenile cancer, childhood adrenocortical cancer, AIDS-related cancers (e.g., lymphoma and kaposi's sarcoma), anal cancer, appendiceal cancer, astrocytoma, atypical teratoid, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchogenic tumor, burkitt lymphoma, carcinoid tumor, atypical teratoid, embryonic tumor, germ cell tumor, primary lymphoma, cervical cancer, childhood cancer, chordoma, cardiac tumor, chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), chronic myeloproliferative disorder, colon cancer colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic Ductal Carcinoma In Situ (DCIS), embryonic tumors, CNS cancers, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, skeletal fibroblastic tumor, gall bladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastoma, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, renal cancer, laryngeal cancer, lip and oral cancer, liver cancer, small Leaf Carcinoma In Situ (LCIS), lung cancer, lymphoma, metastatic squamous neck cancer with suppressed primary focus, mesogenic carcinoma, oral cancer, multiple endocrine tumor syndrome, multiple myeloma/plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm, multiple myeloma, merkel cell carcinoma, malignant mesothelioma, malignant fibrous histiocytoma and osteosarcoma of the bone, nasal and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-hodgkin's lymphoma, non-small cell lung cancer (NSCLC), oral cancer, lip and oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, paranasal and nasal cancers, parathyroid cancer, penile cancer, pharyngeal cancer, pleural-pulmonary blastoma, primary Central Nervous System (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, gastric cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, cellular lymphoma, testicular cancer, laryngeal cancer, thymoma and thymus cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastoma, rare childhood cancer, urinary tract cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or virus-induced cancer.

7. The use of claim 3 or the method of claim 4 or the use of the compound or composition of claim 5, wherein the KRAS G12D mutein mediated disease is selected from pancreatic cancer, colorectal cancer or non-small cell lung cancer.