CN117440953A

CN117440953A - Substituted bicyclic heteroaryl compounds as KRAS G12D inhibitors

Info

Publication number: CN117440953A
Application number: CN202280040365.XA
Authority: CN
Inventors: 刘彬; 高峰; 张鹏志; 郭永起; 高宇; 吴卓
Original assignee: Suzhou Puhe Pharmaceutical Technology Co ltd
Current assignee: Suzhou Puhe Pharmaceutical Technology Co ltd
Priority date: 2021-12-09
Filing date: 2022-12-06
Publication date: 2024-01-23
Also published as: CN116253748A; WO2023104018A1; WO2023103523A1; TW202322797A

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

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 J Med.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 has not entered clinical research, and the oral absorption is weaker, the targeting KRAS G12D small molecule drug of the invention has excellent activity and higher in vivo exposure, and is expected to realize oral administration 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 cancer, AIDS-related cancers (e.g., lymphomas and kaposi's sarcoma), anal cancer, appendiceal cancer, astrocytomas, atypical teratoid, basal cell carcinoma, cholangiocarcinomas, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumor, burkitt's 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 disorders, colon cancer, colorectal cancer, craniopharyngeal tumor, skin T cell lymphoma, extrahepatic Duct Carcinoma In Situ (DCIS), embryonic tumor, CNS cancer, endometrial cancer ependymoma, esophageal carcinoma, 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, hypopharynx cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, renal cancer, laryngeal carcinoma, lip and oral cancer, liver cancer, lobular Carcinoma In Situ (LCIS), lung cancer, lymphoma, metastatic squamous neck cancer with occult primary foci, medium-line cancer, oral cancer, multiple endocrine tumor syndrome, multiple myeloma/plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplasia/myeloproliferative neoplasms, multiple myeloma, merkel cell carcinoma, malignant mesothelioma, malignant fibrous histiocytoma and osteosarcoma of the bone, nasal and sinus cancer, nasopharyngeal cancer, 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 cavity cancer, 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, cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymus cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastoma, cancer of children, cancer, uterine sarcoma, vaginal cancer, vulval cancer or virus-induced cancer, preferably cancer of the colon or non-small-cell carcinoma of the urinary tract.

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 pharmaceutical compositions 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

Preparation of key intermediates a1-a38

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) of the above step were dissolved in 15mL of phosphorus oxychloride, and the reaction was stopped by heating to 120℃for 8 hours. 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]Octane-8-carboxylic acidTert-butyl ester 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, 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. Slowly pouring 80mL of ice water into the reaction solution, and evaporating the organic solvent under reduced pressureAfter the reaction, ethyl acetate was extracted, dried over anhydrous sodium sulfate, filtered, concentrated, and flash column chromatographed to give intermediate a4 (900 mg,5.8 mmol). Yield: 44%. LC-MS: [ M+H ]] ⁺ ＝156。

Referring to the synthetic route for intermediate a4, 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: the intermediate a7-3 (19 g,63.1 mmol) of the above step was dissolved in 240mL of a mixed solution of ethanol and water (v/v, 6/1), and cyanidation was addedPotassium (4.49 g,69 mmol). 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. Cooled to room temperature, filtered, washed with saturated brine, extracted with dichloromethane, dried, concentrated, and flash column chromatographed to afford 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, the following intermediates were synthesized.

Synthesis of intermediates a15, a18, a33-a37

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 ℃ to react for 1 hour,the 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, 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. Dissolving the reaction solution in 50mL of water, extracting with ethyl acetate, drying with anhydrous sodium sulfate, concentrating, and passing the crude product through flash columnChromatography (PE/ea=1/1) gave a19 (200 mg,0.73 mmol) as a colourless oil, yield: 35, LC-MS: [ M+H ]] ⁺ ＝274。

Referring to the synthetic route for intermediate a19, 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 (800 mg,0.73 mmol) as a colorless oil, 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 intermediates a26, a38

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。

Referring to the synthetic route for intermediate a26, the following intermediates were synthesized.

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, and the reaction was stopped after heating to 60℃for 8 h. 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, 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. The reaction was slowly poured into ice and quenched, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the crude product was isolated by column chromatography (PE/EtOAc, 1/1) to give a29-4 (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。

Synthesis of intermediate a31

Step 1: under the protection of nitrogen, the raw material a31-1 (10.0 g,40.5 mmol) is dissolved in 100mL anhydrous tetrahydrofuran, and LiAlH is slowly added ₄ (2.3 g,60.7 mmol) was reacted at room temperature for 2 hours, and the reaction was stopped by monitoring the completion of the reaction by LC-MS. 300mL of ice water was added to the system, the organic solvent was distilled off under reduced pressure, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to give a pale yellow oil a31-2 (5.9 g,26.9 mmol), yield: 67%. LC-MS: [ M+H ] ]+＝220。

Step 2: in 50mL of anhydrous methylene chloride, the intermediate a31-2 (5.9 g,26.9 mmol) and imidazole (3.66 g,53.8 mmol) were dissolved in the above step under nitrogen protection, and tert-butyldiphenylchlorosilane TBDPSCl (7.4 g,26.92 mmol) was slowly added dropwise. The reaction was stirred at room temperature for 30 minutes, and the reaction was stopped by monitoring the completion of the reaction by LC-MS. To the reaction solution was added 100mL of ice water, extracted with methylene chloride, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to give a pale yellow solid a31-3 (11.4 g,24.9 mmol), yield: 93%. LC-MS: [ M+H ]] ⁺ ＝458。

Step 3: the intermediate a31-3 (11.4 g,24.93 mmol) of the above step was dissolved in a 1, 4-dioxane solution of hydrogen chloride (4M, 30 mL), stirred at room temperature for 1 hour, the reaction was stopped, suction filtration was performed, and the cake was dried to give hydrochloride a31-4 (7.0 g,17.8 mmol), yield: 71%. LC-MS: [ M+H ]] ⁺ ＝358。

Step 4: under nitrogen, the intermediate a31-4 (7.0 g,17.8 mmol), the starting cyclobutyl-1, 1-dicarboxylic acid monoethyl ester a31-5 (4.6 g,26.7 mmol) and DIEA (6.2 mL) were dissolved inHATU (10.2 g,26.7 mmol) was added to 60mL of DMF and the reaction was stopped at room temperature for 4 hours. 200mL of ice water was added to the system, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to give brown solid a31-6 (7.78 g,15.2 mmol), yield: 85%. LC-MS: [ M+H ] ] ⁺ ＝512。

Step 5: the intermediate a31-6 (7.78 g,15.2 mmol) of the above step and tetrabutylammonium fluoride TBAF (7.96 g,30.44 mmol) were dissolved in 80mL of tetrahydrofuran, and the reaction was stopped by heating to 50℃for 6 hours. 200mL of water was added to the system, the organic solvent was distilled off under reduced pressure, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to give a brown oil a31-7 (2.3 g,8.42 mmol), yield: 55%. LC-MS: [ M+H ]] ⁺ ＝274。

Step 6: the intermediates a31-7 (2.3 g,8.42 mmol), dihydropyran DHP (2.12 g,25.3 mmol) and p-toluenesulfonic acid TsOH (145 mg,0.84 mmol) of the above step were dissolved in 20mL of methylene chloride, and the reaction mixture was stirred at room temperature for 5 hours, and the reaction was monitored by LC-MS and stopped. 80mL of ice water was added to the system, the mixture was extracted with dichloromethane, the solvent was distilled off under reduced pressure, and the crude product was separated by column chromatography to give an oil a31-8 (1.5 g,4.2 mmol), yield: 50%. LC-MS: [ M+H ]] ⁺ ＝358。

Step 7: under the protection of nitrogen, the intermediate a31-8 (1.5 g,4.2 mmol) in the previous step is dissolved in 20mL anhydrous tetrahydrofuran, and LiAlH is slowly added ₄ (319 mg,8.40 mmol) was stirred at room temperature for 2 hours, the reaction was monitored by LC-MS and 100mL of ice-water was added to quench the reaction. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to give brown solid a31 (760 mg,2.5 mmol), yield: 60%. LC-MS: [ M+H ] ] ⁺ ＝302。

Synthesis of intermediate a32

Step 1: raw material 2-nitro-4-bromo-5-hydroxy-benzoic acid methyl ester a32-1 (25 g,90.6 mmol) and triethylamine (27.5 g,272 mmol) were dissolved in 250mL dichloromethane and acetyl chloride (10.66 g,135.9 mmol) was slowly added dropwise. After the completion of the dropping, the reaction was continued for 3 hours, and the reaction was stopped. 500mL of water was added to the system, extracted with methylene chloride, and the solvent was distilled off under reduced pressure to give crude intermediate a32-2.

Step 2: the crude product a32-2 and reduced iron powder (27.2 g,487.3 mmol) obtained in the above step were dissolved in 310mL of glacial acetic acid, and the reaction was stopped after heating to 40℃for 2 hours. The crude product was filtered through celite and the filtrate was concentrated under reduced pressure. To the filtrate was added a saturated aqueous sodium hydrogencarbonate solution to adjust the pH to about 8, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give crude intermediate a32-3 (16.7 g). LC-MS: [ M+H ]] ⁺ ＝290。

Step 3: the crude product a32-3 and 1-chloromethyl-4-fluoro-1, 4-diazabicyclo [2.2.2]Octane bis (tetrafluoroborate) (selectfluor, 30.8g,87.0 mmol) was dissolved in 170mL acetonitrile and reacted at room temperature for 15 hours, and the reaction was stopped. Saturated aqueous sodium bicarbonate solution was added to the reaction solution to adjust the pH to about 8, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to give compound a32-4 (1.6 g,5.2 mmol), three-step yield: 6%. LC-MS: [ M+H ] ] ⁺ ＝306。

Step 4: the intermediate a32-4 (1.6 g,5.2 mmol) and potassium carbonate (1.44 g,10.5 mmol) of the above step were dissolved in 16mL of methanol, and the reaction was stopped at room temperature for 2 hours. The reaction solution was adjusted to pH 5 with 1M dilute aqueous hydrochloric acid, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to give Compound a32-5 (1.2 g,4.54 mmol) in 87% yield. LC-MS: [ M+H ]] ⁺ ＝264。

Step 5: ice bath, intermediate a32-5 (1.2 g,4.54 mmol) and cesium carbonate (2.96 g,9.1 mmol) were dissolved in 12mL DMF, stirred for 5 min, methyl iodide (710 mg,5.0 mmol) was slowly added dropwise, and the reaction was continued for 2 h after the addition. 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 the crude product was separated by column chromatography to give Compound a32-6 (1.1 g,3.96 mmol) in 87% yield. LC-MS: [ M+H ]] ⁺ ＝278。

Step 6/7: the intermediate a32-6 (1.1 g,3.96 mmol) of the above step was dissolved in 11mL of methanol, and an aqueous NaOH solution (791 mg,19.8 mmol) was added dropwise thereto, and the reaction was stopped at room temperature for 6 hours. To the reaction mixture was added an aqueous potassium cyanate solution (963 mg,11.88 mmol), and the mixture was heated to 50℃and reacted for 2 hours, wherein the pH was adjusted to about 6.5 with 6M diluted hydrochloric acid. LC-MS monitored completion of the reaction, stopped the reaction, and suction filtered to give a32-8 (1.1 g,3.81 mmol) as a gray solid. LC-MS: [ M+H ] ] ⁺ ＝291。

Step 8: the above intermediate a32-8 (1.1 g,3.81 mmol) and DIEA (984 mg,7.62 mmol) were dissolved in 11mL phosphorus oxychloride, and the reaction was stopped by heating to 90℃for 15 hours. The reaction solution was concentrated under reduced pressure, the crude product was slowly poured into 100mL of ice water, a solid was precipitated, suction filtered, and the cake was washed with water and dried to give intermediate 11 (1.2 g,3.68 mmol). The yield thereof was found to be 96.6%. LC-MS: [ M+H ]] ⁺ ＝325。

Preparation of key intermediates b1-b10

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, and washed with dilute hydrochloric acid (2N), saturated aqueous sodium hydrogencarbonate solution, saturated brine, dried over anhydrous sodium sulfate and concentrated 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 in 100 mL DMF under nitrogen, and the reaction was stopped after heating to 100deg.C for 0.5 hours. 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: ice bath and nitrogen protection, the intermediateBodies b1-7 (21.0 g,77.5 mmol) and pyridine (18.38 g,232.41 mmol) were dissolved in 200mL of dichloromethane. 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 (74 mg,0.081 mmol) and tricyclohexylphosphine (45 mg,0.016 mmol) were 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 in 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 a pale yellow compound b2-5 (51.8 g,291.01 mmol), yield: 97%. LC-MS: [ M+H ]] ⁺ ＝179。

Step 4: 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-methylisopropenyl) ruthenium (II) (2.06 g3.4 mmol) was dissolved in 200mL1, 4-dioxane, the reaction was allowed to proceed to 100℃for 4 hours, monitored by LC-MS for completion, 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, drying gave compound b2 (45.9 g,73.4 mmol) as a white solid, yield: 77%. LC-MS: [ M+H ] ] ⁺ ＝625。

Synthesis of intermediate b3

Step 1: raw material b3-1 (6.0 g,15.5 mmol) and AIBN (1.27 g,7.75 mmol) were dissolved in 60mL of carbon tetrachloride and N-bromosuccinimide NBS (13.8 g,77.5 mmol) was slowly added. The reaction was stopped after heating to 60℃for 6 hours. The solvent was distilled off under reduced pressure, and the residue was chromatographed on a flash column (petroleum ether/ethyl acetate=10/1) to give b3-2 (7.6 g,14.0 mmol) as a pale yellow solid, yield: 90%. LC-MS: [ M+H ]] ⁺ ＝543。

Step 2: intermediate b3-2 (7.6 g,14 mmol) and DIEA (1.8 g,14.0 mmol) from the previous step were dissolved in 80mL of acetonitrile, and diethyl phosphite (1.95 g,14.0 mmol) was slowly added and reacted at room temperature for 1 hour to stop the reaction. The reaction solution was poured into 100mL ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=10/1) to give b3-3 (4.6 g,10.0 mmol) as a pale yellow solid, yield: 72%. LC-MS: [ M+H ]] ⁺ ＝465。

Step 3: the above intermediate b3-3 (4.6 g,10 mmol), potassium carbonate (2.8 g,20.0 mmol) and trimethylsilyl cyano TMSCN (1.5 g,15.0 mmol) were dissolved in 50mL acetonitrile and reacted at room temperature for 4 hours, stopping the reaction. The reaction solution was poured into 300mL ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=3/1) to give b3-4 (3.6 g,8.76 mmol) as a pale yellow solid, yield: 87%. LC-MS: [ M+H ] ] ⁺ ＝412。

Step 4: ice bath, under nitrogen protection, the intermediate b3-4 (3.6 g,8.76 mmol) in the previous step was dissolved in 40mL anhydrous tetrahydrofuran, naH (700 mg,17.5 mmol) was added, and after stirring for 10 minutes, 1, 2-dibromoethane (2.0 g,10.5 mmol) was added dropwise, after dropping, the reaction was stopped after warming to room temperature for 10 hours. The reaction solution was poured into 100mL ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=3/1) to give b3 (1.2 g,3.56 mmol) as a pale yellow solid, yield: 40%. LC-MS: [ M+H ]] ⁺ ＝338。

Synthesis of intermediates b4-b6

Step 1: the starting material 4-bromo-7-fluoro-1H-indole (3.5 g,16.4 mmol) was dissolved in 35mL DMF, N-iodosuccinimide NIS (3.69 g,16.4 mmol) was added, and the reaction was stopped by heating to 60℃for 1 hour. The reaction solution was poured into 150mL ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=3/1) to give b4-2 (2.0 g,5.9 mmol) as a pale yellow solid, yield: 36%. LC-MS: [ M+H ]] ⁺ ＝340。

Step 2: ice bath, under nitrogen protection, 3-iodine-4-bromine-7-fluorine-1H-indole b4-2 (2.0 g,5.9 mmol) as an intermediate in the previous step is dissolved in 20mL anhydrous tetrahydrofuran, naH (235 mg,8.9 mmol) and methyl iodide (92 mg,6.5 mmol) are added, and the reaction is stopped after the temperature is raised to room temperature for 2 hours. To the reaction solution was added 80mL of saturated aqueous ammonium chloride, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=3/1) to give b4-3 (1.1 g,3.12 mmol) as a pale yellow solid, yield: 53%. LC-MS: [ M+H ] ] ⁺ ＝354。

Step 3: under nitrogen protection, the intermediate b4-3 (1.1 g,3.12 mmol) and CuCN (1.1 g,12.4 mmol) were dissolved in 20mL DMSO, warmed to 100deg.C for 3 hours, and cooled to room temperature. The reaction solution was poured into 150mL ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=2/1) to give b4-4 (0.55 g,2.2 mmol) as a pale yellow solid, yield: 70%. LC-MS: [ M+H ]] ⁺ ＝253。

Step 4: under the protection of nitrogen, the intermediate b4-4 (350 mg,1.4 mmol), pinacol biborate (706 mg,2.8 mmol) and potassium acetate (408 mg,4.2 mmol) of the above step are dissolved in 10mL of 1, 4-dioxane, and the catalyst Pd (dppf) Cl is added ₂ (100 mg,0.14 mmol) and heating toThe reaction was carried out at 100℃for 8 hours and cooled to room temperature. The reaction solution was poured into 50mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=2/1) to give b4 (0.27 g,0.9 mmol) as a pale yellow solid, yield: 65%. LC-MS: [ M+H ]] ⁺ ＝301。

Referring to the synthetic route for intermediate b4, the following intermediates were synthesized.

Synthesis of intermediate b7

Step 1: raw material b7-1 (1.0 g,5.9 mmol) was dissolved in 10mL of DMF, N-iodosuccinimide NIS (1.99 g,8.85 mmol) was added, and the reaction was stopped by heating to 70℃for 1 hour. The reaction solution was poured into 50mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=9/1) to give b7-2 (800 mg,2.70 mmol) as a yellow solid, yield: 46%. LC-MS: [ M+H ] ] ⁺ ＝296。

Step 2: ice bath, under nitrogen protection, intermediate b7-2 (800 mg,2.70 mmol), anhydrous methanol (430 mg,13.6 mmol) and triphenylphosphine (1.07 g,4.1 mmol) were dissolved in 20mL DMF, diethyl azodicarboxylate (710 mg,4.1 mmol) was added to the reaction mixture, and the reaction was stopped at room temperature for 12 hours. 80mL of ice water was added to the reaction mixture, extraction was performed with ethyl acetate, washing was performed with saturated brine, drying was performed with anhydrous sodium sulfate, and concentrated, and the crude product was obtained byflash column chromatography (petroleum ether/ethyl acetate=5/1) gave b7-3 (530 mg,1.72 mmol) as a yellow solid in yield: 64%. LC-MS: [ M+H ]] ⁺ ＝310。

Step 3: under nitrogen protection, the intermediate b7-3 (530 mg,1.71 mmol) and CuCN (610 mg,5.84 mmol) of the above step were dissolved in 20mL of DMSO, heated to 100deg.C and reacted for 6 hours, and cooled to room temperature. The reaction solution was poured into 80mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=3/1) to give b7 (85 mg,0.41 mmol) as a yellow solid, yield: 24%. LC-MS: [ M+H ]] ⁺ ＝209。

Synthesis of intermediate b8

Step 1: compound b8-1 (300 mg,1.23 mmol) and ethyl isothiocyanato formate (240 mg,1.84 mmol) were dissolved in 15mL of 1, 4-dioxane under nitrogen, and the reaction was stopped after heating to 100℃for 4 hours. 50mL of ice water was slowly poured into the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (PE/EA=5/1) to give compound b8-2 (210 mg,0.59 mmol), yield: 48%. LC-MS: [ M+H ] ] ⁺ ＝356。

Step 2: the intermediate b8-2 (210 mg,0.59 mmol) obtained in the above step was dissolved in 8mL of DMSO, and 4mL of an aqueous sodium hydroxide solution (5N) was added thereto to react under reflux for 4 hours, and the reaction was stopped. Cooling to room temperature, slowly adding 50mL ice water into the reaction solution, extracting with ethyl acetate, washing with saturated saline water, drying with anhydrous sodium sulfate, concentrating, and separating the crude product by flash column chromatography (PE/EA=3/1) to obtain the compound b8-3, wherein the yield is quantitative. LC-MS: [ M+H ]] ⁺ ＝283。

Step 3: the intermediate b8-3 (175 mg,0.62 mmol), triethylamine (190 mg,1.86 mmol), 4-dimethylaminopyridine DMAP (15 mg,0.12 mmol) and di-tert-butyl dicarbonate (160 mg,0.74 mmol) were dissolved in 20mL dichloromethaneThe reaction was stopped after 4 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 b8-4.LC-MS: [ M+H ]] ⁺ ＝383。

Step 7: under the protection of nitrogen, crude b8-4 (100 mg,0.26 mmol), potassium acetate (77 mg,0.78 mmol) and pinacol biborate (79 mg,0.31 mmol) were dissolved in 7mL of 1, 4-dioxane, palladium acetate (12 mg,0.052 mmol) as a catalyst was added, and the reaction was stopped by heating to 100℃for 3 hours. Cooled to room temperature, filtered, the solvent was evaporated under reduced pressure and the crude product was isolated by flash column chromatography (PE/ea=5/1) to give intermediate b8 (60 mg,0.14 mmol), yield: 54%. LC-MS: [ M+H ] ] ⁺ ＝431。

Synthesis of intermediate b9

Step 1: the starting material 2-amino-3-cyano-4-bromoindole b9-1 (3.8 g,16.10 mmol) and 4-dimethylaminopyridine DMAP (196 mg,1.61 mmol) were dissolved in 50mL tetrahydrofuran, and di-tert-butyl dicarbonate (14.1 g,64.4 mmol) was slowly added and reacted at room temperature for 10 hours to stop the reaction. 200mL of ice water was added to the reaction mixture, extraction was performed with ethyl acetate, and concentration was performed to obtain crude b9-2.LC-MS: [ M+H ]] ⁺ ＝436。

Step 2: under the protection of nitrogen, the crude product b9-2 (600 mg,1.38 mmol), potassium acetate (405 mg,4.1 mmol) and pinacol biborate (4819 mg,1.93 mmol) were dissolved in 10mL of 1, 4-dioxane, and the catalyst Pd (dppf) Cl was added ₂ (102 mg,0.14 mmol), and the reaction was stopped after heating to 100℃for 2 hours. The solvent was distilled off under reduced pressure, and the crude product was separated by flash column chromatography to give intermediate b9 (190 mg,0.39 mmol). LCMS: [ M+1 ]] ⁺ ＝484。

Synthesis of intermediate b10

Step 1: compound b10-1 (5.5 g,23 mmol) and triethylamine (6.0 mL) were dissolved in 50mL of t-butanol under nitrogen, diphenyl azide phosphate DPPA (9.4 g,7.4 mL) was added, and after stirring for 10 minutes, the temperature was raised to 95℃for 3 hours, and the reaction was stopped. The solvent was distilled off under reduced pressure, and the crude product was dissolved in 50mL of methylene chloride, and di-tert-butyl dicarbonate (5.0 g,23 mmol) was added to react at room temperature for 2 hours to stop the reaction. The reaction solution was poured into 100mL of ice water, extracted with methylene chloride, the solvent was distilled off under reduced pressure, and the crude product was separated by flash column chromatography to give compound b10-2 (4.0 g,12.9 mmol), yield: 56%. LCMS: [ M+1 ] ] ⁺ ＝312。

Step 2: the intermediate b10-2 (1.0 g,3.2 mmol) of the previous step is dissolved in 10mL of anhydrous tetrahydrofuran under the protection of nitrogen at the temperature of-70 ℃, raw material chlorosulfonic acid isocyanate (0.42 mL) is added, and after stirring for 1 hour, chlorosulfonic acid isocyanate (0.42 mL) is added again, the reaction is continued for 1 hour, and the temperature is raised to the room temperature. To the reaction mixture was added 10mL of anhydrous DMF, and after stirring for 1 hour, the reaction was stopped. The reaction solution was poured into 100mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to give compound b10-3 (0.5 g,1.5 mmol) in 46% yield. LCMS: [ M+1 ]] ⁺ ＝337。

Step 3: under nitrogen, the intermediate b10-3 (0.5 g,1.5 mmol), potassium acetate (440 mg,4.5 mmol) and pinacol biborate (460 mg,1.8 mmol) were dissolved in 10mL 1, 4-dioxane and Pd (dppf) was added ₂ Cl ₂ (11 mg,0.15 mmol), and the reaction was stopped by heating to 100℃for 2 hours and filtering. 50mL of ice water was added to the reaction mixture, extraction was performed with ethyl acetate, drying was performed with anhydrous sodium sulfate, concentration was performed, and the crude product was separated by flash column chromatography to obtain compound b10 (270 mg,0.7 mmol) in 46% yield. LCMS: [ M+1 ]] ⁺ ＝385。

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) were dissolved in 25mL of methanol and reacted at room temperature for 16 hours to stop the reaction. 100mL of ice water was added to the system, extraction was performed with ethyl acetate, washing was performed with saturated brine, and anhydrous sodium sulfateDrying, concentrating 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, 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

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 added and the reaction was stopped at room temperature for 1 hour. 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。

Preparation of key intermediates d1-d5

Synthesis of intermediate d1

Step 1: in a 250mL reaction flask, the raw material d1-1 (5.0 g,18.6 mmol) was dissolved in 80mL pyridine, 20mL of oxalyl chloride monoethyl ester was slowly added dropwise to the system, and after the dropwise addition was completed, the mixture was stirred at room temperature for 1 hour, and then the temperature was raised to 50℃for 2 hours, and the reaction was stopped. The reaction solution was slowly poured into 300mL of ice water, suction filtration and cake drying to give intermediate d1-2 (5.2 g,14.8 mmol) in 80% yield. LC-MS: [ M+H ]] ⁺ ＝350。

Step 2: the intermediate d1-2 (5.2 g,14.8 mmol) of the above step was dissolved in 100mL of absolute ethanol, and 4.0g of ammonium acetate and1mL of acetic acid. The temperature was raised to reflux and reacted for 1 hour, and cooled to room temperature. Slowly dripping 1M diluted hydrochloric acid into the system to adjust the pH to about 5, precipitating solid, carrying out suction filtration, and drying a filter cake to obtain a crude product d1-3.LC-MS: [ M+H ]] ⁺ ＝349。

Step 3: the crude d1-3 from the previous step and DIEA (2.2 g,17.2 mmol) were dissolved in 20mL phosphorus oxychloride and reacted for 2 hours at 85℃with the reaction stopped. The solvent was distilled off under reduced pressure, the crude product was slowly poured into 100mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to give compound d1-4 (2.5 g,6.8 mmol), in 46% yield in two steps. LC-MS: [ M+H ]] ⁺ ＝367。

Step 4: ice bath, the intermediate d1-4 (2.5 g,6.8 mmol) and DIEA (1.75 g,13.6 mmol) were dissolved in 20mL anhydrous tetrahydrofuran, and a solution of a2-1 (1.44 g,6.8 mmol) prepared in advance in THF (3.0 mL) was slowly added dropwise to the reaction solution, and the reaction was stopped after the dropwise addition, and the temperature was raised to room temperature for 40 minutes. 100mL of water was added to the system, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give compound d1 (3.5 g,6.4 mmol) in 94% yield. LC-MS: [ M+H ] ] ⁺ ＝543。

Synthesis of intermediate d2

The steps are as follows: intermediate d1 (2.0 g,3.7 mmol) and intermediate a7 (2.1 g,1.02 mmol) were dissolved in 50mL of toluene under nitrogen, cesium carbonate (3.6 g,11.1 mmol) and catalyst Pd (DPEPhos) Cl were added sequentially ₂ (600 mg,1.1 mmol). The reaction mixture was heated to 105℃and allowed to react for 4 hours, and cooled to room temperature. Filtration, concentration of the filtrate under reduced pressure, and separation of the crude product by flash column chromatography gave compound d2 (1.4 g,1.85 mmol), yield: 50%. LC-MS: [ M+H ]] ⁺ ＝755。

Synthesis of intermediate d3

Step 1: raw material N-Boc-2-amino-2-methyl-1-propanol d3-1 (500 mg,2.64 mmol) was dissolved in 10mL of methylene chloride in an ice bath, dess Martin oxidant (1.68 g,3.96 mmol) was slowly added, and the reaction was stopped after heating to room temperature for 2 hours. The reaction solution was poured into 100mL of water, extracted with methylene chloride, and the solvent was distilled off under reduced pressure. The crude product was chromatographed on flash column (PE/ea=4/1) to give d3-2 (380 mg,2.02 mmol) as an oil, yield: 76%.

Step 2: under nitrogen, the intermediate d3-2 (380 mg,2.02 mmol) of the above step and the starting material (R) -3-fluoro-tetrahydropyrrole a5-1 (303 mg,2.42 mmol) were dissolved in 10mL of anhydrous THF and sodium triacetoxyborohydride (850 mg,4.04 mmol) was slowly added. The reaction mixture was reacted at room temperature for 10 hours, and the reaction was stopped. 50mL of ice water was added to the system, extracted with methylene chloride, and the solvent was distilled off under reduced pressure. The crude product was chromatographed on flash column (PE/EA=2:1) to give d3-3 (250 mg,0.96 mmol) as a white solid in yield: 48%. LC-MS: [ M+H ] ] ⁺ ＝261。

Step 3: the intermediate d3-3 (250 mg,0.96 mmol) of the above step was dissolved in 8mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under nitrogen. The reaction solution was reacted at room temperature for 1 hour, the reaction was stopped, a saturated sodium bicarbonate solution was slowly added and the pH was adjusted to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by flash column chromatography to give d3 (200 mg) as a yellow oil. LC-MS: [ M+H ]] ⁺ ＝161。

Referring to the synthetic route for intermediate d3, the following intermediates were synthesized.

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: 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 of compound P1, the following target molecule was synthesized using a similar backbone structure.

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 purified by TLC chromatography (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 4:

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 a14 (1.0 g,2.0 mmol) was dissolved in 10mL anhydrous THF, and the prepared solution S1 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 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). At the position ofUnder 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 of the compound H2a or H2b, the following target molecules were synthesized using a similar backbone structure.

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

example 5:

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. 50mL of water was added to the reaction mixture, extraction was performed with ethyl acetate, drying over anhydrous sodium sulfate, concentration was performed, and residueThe residue was chromatographed by flash column chromatography (petroleum ether: ethyl acetate=2:1) to give H11-2 (110 mg,0.17 mmol) as a yellow solid. 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 (DPEPhos) Cl was added sequentially 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 of compound H11, the following target molecule was synthesized using a similar backbone structure.

example 6:

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 a14 (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. Residue (C)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 of compound H13, the following target molecule was synthesized using a similar backbone structure.

example 7:

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 of compound P5, the following target molecule was synthesized using a similar backbone structure.

example 8:

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,0.34 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 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 9:

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 10:

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 6mL of a mixed solution of 1, 4-dioxane and water (v/v, 5/1) under nitrogen, and raw 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: iceUnder 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 target 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 of compound P9, the following target molecule was synthesized using a similar backbone structure.

Example 11:

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 temperature for 30 min to give 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 (a)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 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 of compound P12, the following target molecule was synthesized using a similar backbone structure.

Example 12:

step 1: in a high-pressure reactor, the raw material 4-bromo-2, 6-difluorobenzonitrile a5 (100 g,459 mmol) was dissolved in To 240mL of ethanol, 400mL of ammonia water was added, the temperature was raised to 90℃and the reaction was allowed to proceed for 16 hours, and the reaction was stopped after cooling to room temperature. 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: intermediate P14-4 (8.3 g,35.0 mmol) from the previous step was dissolved in 150mL of 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 the protection of nitrogen, the intermediate P14-5 (3.3 g,13.0 mmol) of the previous step is dissolved in 33mL of anhydrous THF, and tetrahydrofuran containing triphosgene (3.6 g,12.4 mmol) is slowly added dropwise at 0 ℃Solution (20 mL). 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 P14-6 (2.8 g) obtained in the previous step is dissolved in 50mL phosphorus oxychloride, 5mLN, 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 separation to give P14-8 (900 mg,1.82 mmol) as a white solid. 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) as a yellow solid mmol). 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 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 13:

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 of anhydrous tetrahydrofuran, and the reaction mixture was added and reacted at room temperature for 1 hour to stop the reaction. 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 2mL of a mixed solution 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 of compound P18, the following target molecule was synthesized using a similar backbone structure.

Example 14:

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, tetrabutylammonium fluoride (TBAF, 130mg,0.48 mmol) was added, and the reaction was continued at room temperature Should be 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 15:

step 1. Intermediate d2 (1.4 g,1.85 mmol) was dissolved in 10mL tetrahydrofuran, and a saturated aqueous lithium hydroxide solution (5.0 mL) was slowly added dropwise thereto, and the reaction was stopped at room temperature for 40 minutes. Slowly adding 1M diluted hydrochloric acid dropwise into the reaction to adjust the pH to about 5, extracting with ethyl acetate, concentrating to obtain crude product M1-1, and directly using in the next reaction. LC-MS: [ M+H ]] ⁺ ＝727。

Step 2: in an ice bath, the crude product M1-1 obtained in the above step was dissolved in a mixed solution of trifluoroacetic acid and methylene chloride (4.0 mL, 1/1), and the reaction was stopped after 1 hour in an ice bath. The solvent was distilled off under reduced pressure, and the crude product was subjected to preparative chromatography to give compound M1 (10 mg). LC-MS: [ M+H ] ] ⁺ ＝527。

1H NMR(400MHz,DMSO-d ₆ )δ8.10(s,2H),7.81(s,1H),7.32–7.21(m,1H),7.18–7.10(m,1H),4.27(d,J＝10.7Hz,2H),3.47(s,4H),1.58(s,4H).

Example 16:

step 1: compound M1-1 (400 mg,0.55 mmol), starting material M2-1 (109 mg,0.83 mmol) and DIEA (213 mg,1.65 mmol) were dissolved in 10mL of DMF and stirred for 5 min, HATU (315 mg,0.83 mmol) was added and reacted at room temperature for 2 h. The reaction solution was poured into 150mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to give M2-2 (393 mg,0.47 mmol) as a colorless oil in 85% yield. LC-MS: [ M+H ]] ⁺ ＝837

Step 2: the intermediate M2-2 (393 mg,0.47 mmol) of the above step was dissolved in 4.0mL of methylene chloride, and 1.0mL of trifluoroacetic acid was added thereto to react at room temperature for 1 hour, and the reaction was stopped. The solvent was distilled off under reduced pressure, the crude product was dissolved in 10mL of water, the pH was adjusted to about 8 with a saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to give Compound M2 (89 mg,0.14 mmol), yield: 30%. LC-MS: [ M+H ]] ⁺ ＝637。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.5(d,J＝8.4Hz,1H),8.12(s,2H),7.97(s,1H),7.33–7.25(m,1H),7.17(t,J＝8.9Hz,1H),4.49(t,J＝11.4Hz,1H),4.37(t,J＝10.4Hz,1H),4.17(t,J＝7.5Hz,1H),3.68-3.51(m,5H),2.86–2.80(m,1H),2.68–2.66(m,1H),2.34–2.31(m,1H),2.02–1.92(m,1H),1.84–1.75(m,1H),1.71–1.46(m,9H),1.25–1.22(m,2H).

Referring to the synthetic route of compound M2, the following target molecule was synthesized using a similar backbone structure.

* Indicates chiral molecules, and chiral resolution is not performed.

Example 17:

step 1: intermediate a31 (300 mg,1.0 mmol) was dissolved in 5mL dry THF under nitrogen in a 50mL reaction flask, potassium tert-butoxide (224 mg,2.0 mmol) was added and stirred at room temperature for 30 min to give solution S5. In another 50mL reaction flask, intermediate a14 (488 mg,1.0 mmol) was dissolved in 5mL anhydrous THF, and the prepared solution S5 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 H23-1 (240 mg,0.32 mmol) as a pale yellow solid. Yield: 31%, LC-MS: [ M+H ] ] ⁺ ＝757。

Step 2: compound H23-1 (240 mg,0.32 mmol) was dissolved in 6mL of anhydrous toluene under nitrogen, and intermediate a7 (188 mg,0.45 mmol) Pd (DPEPhos) Cl was added sequentially ₂ (46 mg,0.064 mmol) and cesium carbonate (209 mg,0.64 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 H23-2 (250 mg,0.26 mmol) as a yellow solid. Yield: 79%, LC-MS: [ M+H ]] ⁺ ＝982。

Step 3: compound H23-2 (250 mg,0.26 mmol) was dissolved in a mixed solution of 6mL 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, and the reaction solution is then reacted for 1 hourExtracting with ethyl acetate, and concentrating under reduced pressure. The residue was chromatographed on flash column to give H23 (10 mg) as a yellow solid, LC-MS: [ M+H ]] ⁺ ＝698。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.16(s,2H),7.87(s,1H),7.28(dd,J＝8.4,5.3Hz,1H),7.17(t,J＝8.9Hz,1H),5.19–5.05(m,1H),4.38–4.29(m,5H),3.68–3.33(m,9H),3.18–3.09(m,1H),3.01(d,J＝13.1Hz,1H),2.88-2.85(m,1H),2.67–2.56(m,2H),2.09-1.53(m,12H).

Example 18:

step 1: in a 50mL reaction flask, intermediate a18 (176 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 S6. 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 H24-1 (337 mg,0.45 mmol) as a pale yellow solid. Yield: 48%, LC-MS: [ M+H ] ] ⁺ ＝749。

Step 2: h24-1 (1.0 g,1.34 mmol), potassium carbonate and methylboronic acid (80 mg,1.34 mmol) were dissolved in 10mL anhydrous DMA under nitrogen, and Pd (PPh) was added ₃ )Cl ₂ (100 mg,0.13 mmol) and the reaction solution was reacted at 90℃for 2 hours, cooled to room temperature and 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=3:1) to give yellow solid H24-2 (340 mg,0.54 mmol). Yield: 40%, LC-MS: [ M+H ]] ⁺ ＝636。

Step 3: under the protection of nitrogen, the intermediate H24-2 (340 mg,0.54 mmol) in the previous step is dissolved in 10mL of anhydrous methylTo benzene was added in sequence intermediate a7 (900 mg,2.2 mmol), pd (DPEPhos) Cl ₂ (170 mg,0.24 mmol) and cesium carbonate (1.0 g,3.2 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 H24-3 (300 mg,0.35 mmol) as a yellow solid. Yield: 65%, LC-MS: [ M+H ]] ⁺ ＝848。

Step 4: h24-3 (300 mg,0.35 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 pressure. The residue was chromatographed by HPLC to give H24 (10 mg) as a yellow solid, LC-MS: [ M+H ] ] ⁺ ＝648。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.03(s,2H),7.61(s,1H),7.22–7.08(m,2H),5.24-5.10(m,1H),4.38(s,2H),4.27(dd,J＝25.6,12.1Hz,2H),3.53–3.42(m,5H),2.80–2.62(m,5H),2.40–2.30(m,1H),2.07(s,3H),2.01–1.66(m,12H).

Example 19:

step 1: compound H10-1 (1.1 g,1.68 mmol) obtained by the route of reference example 4 was dissolved in 20mL of anhydrous tetrahydrofuran at-25℃under nitrogen protection, and isopropyl magnesium bromide (5.0 mL,5.0 mmol) was slowly added dropwise, and after the addition, isopropyl pinacol borate (1.56 g,8.4 mmol) was added to the reaction solution to react at-25℃for 2 hours, and the reaction was stopped. To the mixture was added 20mL of saturated aqueous ammonium chloride to quench the reaction, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=2/1) to give H26-1 (800 mg,1.28 mmol) as a pale yellow solid, yield: 76%. LC-MS: [ M+H ]] ⁺ ＝622。

Step 2: compound H26-1 (250 mg,0.74 mmol), cesium carbonate (480 mg,1.48 mmol) and intermediate b3 (480 mg,1.11 mmol) were dissolved in 10mL of anhydrous toluene under nitrogen, and catalyst bis (diphenylphosphinophenyl ether) palladium dichloride (100 mg,0.15 mmol) was added to react at 105℃for 3 hours, the reaction was stopped, and the reaction was filtered. The solvent was distilled off under reduced pressure, and the crude product was chromatographed on a flash column (petroleum ether/ethyl acetate=1/2) to give H26-2 (160 mg,0.19 mmol) as a pale yellow solid, yield: 26%. LC-MS: [ M+H ]] ⁺ ＝835。

Step 3: compound H26-2 (160 mg,0.19 mmol) was dissolved in 5mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) in an ice bath, the reaction was stopped at room temperature for 1 hour, a 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 a flash column to give H26 (20 mg,0.032 mmol) as a yellow solid. LC-MS: [ M+H ] ] ⁺ ＝635。

¹ HNMR(400MHz,DMSO-d ₆ )δ7.85(s,1H),7.55(d,J＝8.6Hz,1H),6.55(d,J＝8.6Hz,1H),6.39–6.38(m,2H),5.11(dt,J＝9.4,5.1Hz,1H),4.42(s,2H),4.29(d,J＝12.3Hz,2H),3.70–3.62(m,6H),2.81–2.66(m,3H),2.65–2.57(m,3H),2.33(q,J＝8.2Hz,1H),2.10–1.80(m,9H),1.75–1.65(m,5H).

Example 20:

step 1: compound H10-1 (100 mg,0.15 mmol), hexamethylditin (74 mg,0.22 mmol), tricyclohexylphosphorus (8.4 mg,0.03 mmol) and lithium chloride (19 mg,0.45 mmol) obtained by the route of reference example 4 are dissolved in 10mL of 1, 4-dioxane under nitrogen protection, and the catalyst Pd is added ₂ dba ₃ (27 mg,0.03 mmol), and the reaction was stopped after heating to 95℃for 12 hours. Adding 40mL ice water, extracting with ethyl acetate, drying with anhydrous sodium sulfate, concentrating, and separating the crude product by flash column chromatography(petroleum ether/ethyl acetate=2/1) to give H32-1 (80 mg,0.11 mmol) as a yellow solid, yield: 72%. LC-MS: [ M+H ]] ⁺ ＝742。

Step 2: compound H32-1 (80 mg,0.11 mmol), cesium carbonate (110 mg,0.33 mmol) and intermediate b7 (25 mg,0.12 mmol) were dissolved in 10mL of 1, 4-dioxane under nitrogen, and catalyst bis (triphenylphosphine) palladium dichloride (15 mg,0.022 mmol) was added to react at 100℃for 4 hours, the reaction was stopped, and the mixture was filtered. The solvent was distilled off under reduced pressure, and the crude product was chromatographed on a flash column (petroleum ether/ethyl acetate=2/1) to give H32-2 (20 mg,0.03 mmol) as a pale yellow solid, yield: 24%. LC-MS: [ M+H ]] ⁺ ＝750。

Step 3: compound H32-2 (20 mg,0.03 mmol) was dissolved in 1mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) in an ice bath, the reaction was stopped at room temperature for 1 hour, a 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 separated by flash column chromatography to give H32 (2.1 mg) as a yellow solid. LC-MS: [ M+H ] ] ⁺ ＝650。

¹ H NMR(400MHz,DMSO-d ₆ )δ7.98(s,1H),6.67(s,2H),5.19–5.05(m,1H),4.42(s,2H),4.29(d,J＝12.3Hz,2H),3.70–3.62(m,6H),2.81–2.66(m,3H),2.65–2.57(m,3H),2.33–1.80(m,9H),1.75–1.65(m,5H).

Example 21:

step 1: compound H24-1 (1.0 g,1.34 mmol) obtained in the course of example 18 was dissolved in 20mL of anhydrous tetrahydrofuran at-60℃under nitrogen, and isopropyl magnesium bromide (1.3 mL,1.3 mmol) was slowly added dropwise, after stirring for 30 minutes, anhydrous DMF (196 mg,2.38 mmol) was added to the reaction solution, and after stirring again for 30 minutes, the reaction was stopped. To the mixture was added 20mL of saturated aqueous ammonium chloride solution to quench the reaction, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give crude H33-1.LC-MS:[M+H] ⁺ ＝650。

Step 2: in an ice bath, the crude product H33-1 obtained in the previous step was dissolved in 10mL of methanol, sodium borohydride (102 mg,2.68 mmol) was slowly added, and the reaction was stopped after heating to room temperature for 1 hour. To the reaction solution was added 30mL of saturated aqueous ammonium chloride, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was chromatographed by flash column chromatography (petroleum ether/ethyl acetate=2/1) to give H33-2 (200 mg,0.31 mmol) as a pale yellow solid, yield in two steps: 23%. LC-MS: [ M+H ]] ⁺ ＝652。

Step 3: under the protection of nitrogen, the intermediate H33-2 (200 mg,0.31 mmol), cesium carbonate (201 mg,0.62 mmol) and intermediate a7 (150 mg,0.50 mmol) in the previous step are dissolved in 10mL of anhydrous toluene, a catalyst bis (diphenylphosphinophenyl ether) palladium dichloride (44 mg,0.06 mmol) is added, the reaction solution is heated to 105 ℃ for 3 hours, the reaction is stopped, and the solvent is removed by filtration and reduced pressure evaporation. The crude product was chromatographed on flash column (petroleum ether/ethyl acetate=1/2) to give H33-3 (150 mg,0.17 mmol) as a pale yellow solid, yield: 56%. LC-MS: [ M+H ] ] ⁺ ＝864。

Step 4: compound H33-3 (150 mg,0.17 mmol) was dissolved in 3mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under nitrogen protection, the reaction was stopped at room temperature for 1 hour, a 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 H33 (30 mg) as a yellow solid, LC-MS: [ M+H ]] ⁺ ＝664。

¹ HNMR(400MHz,DMSO-d ₆ )δ8.01(s,2H),7.84(s,1H),7.21(dd,J＝8.3,5.4Hz,1H),7.12(t,J＝8.9Hz,1H),5.35–5.25(m,1H),5.13(dt,J＝56.1,5.6Hz,1H),4.40(s,2H),4.29–4.13(m,4H),3.52(s,2H),3.48–3.37(m,2H),2.81–2.66(m,3H),2.65–2.61(m,2H),2.36(dd,J＝15.3,8.2Hz,1H),2.10–1.80(m,9H),1.73–1.65(m,4H).

Example 22:

step 1: ice bath, raw material H40-1 (600 mg,2.54 mmol) and potassium tert-butoxide (404 mg,3.6 mmol) were dissolved in 5mL tetrahydrofuran and stirred for 10 min; the above mixture was poured into tetrahydrofuran (10 mL) containing intermediate a14 (882 mg,1.8 mmol). The reaction was continued under ice bath conditions for 1 hour, and stopped. 50mL of ice water was added to the system, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to give Compound H40-2 (500 mg,0.71 mmol) in 39% yield. LC-MS: [ M+H ]] ⁺ ＝705。

Step 2: under the protection of nitrogen, the compound H40-2 (100 mg,0.14 mmol), cesium carbonate (92 mg,0.28 mmol) and intermediate a7 (80 mg,0.20 mmol) of the above-mentioned compound were dissolved in 5mL of toluene, and bis (diphenylphosphinophenyl ether) palladium dichloride (10 mg,0.014 mmol) was added as a catalyst, and the reaction was stopped by heating to 105℃for 2 hours, followed by filtration. The solvent was distilled off under reduced pressure, and the crude product was separated by flash column chromatography to give Compound H40-3 (90 mg,0.098 mmol) in 69% yield. LC-MS: [ M+H ] ] ⁺ ＝917。

Step 3: the above compound H40-3 (90 mg) was dissolved in 4mL of a mixed solution of trifluoroacetic acid and methylene chloride (v/v, 1/1), reacted at room temperature for 1 hour, the reaction was stopped, a saturated sodium hydrogencarbonate solution was slowly added to the system and the pH was adjusted to about 8, extraction was performed with ethyl acetate, and concentration was performed under reduced pressure. The residue was separated by TLC thin layer chromatography to give H40 (10 mg) as a yellow solid, LC-MS: [ M+H ]] ⁺ ＝597。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.14(s,2H),7.89(s,1H),7.32(dd,J＝8.4,5.2Hz,1H),7.23–7.17(m,1H),4.73(t,J＝5.4Hz,1H),4.35–4.26(m,4H),3.64–3.35(m,7H),1.90–1.87(m,5H),1.73–1.60(m,5H).

Example 23:

step 1-intermediate d2 (75 mg,0.10 mmol) was dissolved inIn a mixed solution (3.0 mL, 1/1) of trifluoroacetic acid and dichloromethane, the reaction was stopped after 1 hour in an ice bath. The solvent was distilled off under reduced pressure, and the crude product was subjected to preparative chromatography to give compound M9 (30 mg). LC-MS: [ M+H ]] ⁺ ＝555。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.18(s,2H),8.07(s,1H),7.28(dd,J＝8.4,5.3Hz,1H),7.22–7.15(m,1H),4.64(d,J＝13.7Hz,1H),4.44(d,J＝13.7Hz,1H),4.38(q,J＝7.1Hz,2H),4.18(d,J＝27.8Hz,2H),4.03(d,J＝13.4Hz,1H),3.84(d,J＝13.6Hz,1H),1.95(m,J＝12.9Hz,2H),1.88–1.76(m,2H),1.34(m,J＝7.1Hz,4H).

Example 24:

step 1: compound M1-1 (35 mg,0.048 mmol), 4-dimethylaminopyridine DMAP (1.2 mg,0.09 mmol) and cyclopropyl alcohol (5.0 mg,0.072 mmol) were dissolved in 6mL of dichloromethane under nitrogen, N' -dicyclohexylcarbodiimide DCC (45 mg,0.096 mmol) was added and the reaction was stopped at room temperature for 12 hours. To the reaction solution was added 30mL of ice water, extracted with dichloromethane, the solvent was distilled off under reduced pressure, and the crude product was separated by flash column chromatography (PE/ea=3/1) to give compound M10-1 (38 mg), and the yield was quantified. LC-MS: [ M+H ] ] ⁺ ＝767。

Step 2: the above compound M10-1 (38 mg) was dissolved in a mixed solution of trifluoroacetic acid and methylene chloride (3.0 mL, 1/1), reacted in an ice bath for 1 hour, the solvent was distilled off under reduced pressure, and the crude product was separated by HPLC preparation chromatography to give compound M10 (8 mg). LC-MS: [ M+H ]] ⁺ ＝567。

¹ H NMR(400MHz,DMSO-d ₆ )δ9.54(s,1H),8.16(s,2H),8.04(s,1H),7.29(dd,J＝8.0,4.0Hz,1H),7.18(t,J＝8.0Hz,1H),4.53(dd,J＝76.0,12Hz,2H),4.38–4.34(m,1H),3.93–3.71(m,4H),3.73(d,J＝12Hz,1H),2.67(d,J＝20Hz,1H),2.33(d,J＝12Hz,1H),2.01–1.82(m,5H).

Referring to the synthetic route of compound M10, the following target molecule was synthesized using a similar backbone structure.

* Indicates chiral molecules, and chiral resolution is not performed.

Example 25:

step 1: in an ice bath, propionamide (1.0 g,2.05 mmol) was dissolved in 10mL of anhydrous tetrahydrofuran, naH (123 mg,3.07 mmol) was added, and after stirring for 30 minutes, intermediate a14 (976 mg,2.0 mmol) was added to the reaction system. After the reaction was warmed to room temperature for 2 hours, the completion of the reaction was monitored by LC-MS, 50mL of ice water was added to the reaction solution, extraction was performed with ethyl acetate, drying was performed with anhydrous sodium sulfate, concentration was performed, and the crude product was separated by flash column chromatography to obtain Compound M13-1 (663 mg,1.23 mmol), yield: 61%. LC-MS: [ M+H ]] ⁺ ＝542。

Step 2: under nitrogen, the above compound M13-1 (300 mg,0.55 mmol), cesium carbonate (360 mg,1.11 mmol) and intermediate a7 (264 mg,0.66 mmol) were dissolved in 5mL of toluene as a solvent, and palladium (40 mg,0.055 mmol) was added as a catalyst. The reaction was stopped after heating to 105℃for 12 hours, and the mixture was filtered. The solvent was distilled off under reduced pressure, and the crude product was separated by flash column chromatography to give Compound M13-2 (85 mg,0.11 mmol), yield: 21%. LC-MS: [ M+H ] ] ⁺ ＝754。

Step 3: the compound M13-2 (85 mg) of the above step was dissolved in a mixed solution of trifluoroacetic acid and methylene chloride (3.0 mL, 1/1), reacted in an ice bath for 1 hour, the solvent was distilled off under reduced pressure, and the crude product was separated by HPLC preparative chromatography to give the final productCompound M13 (15 mg). LC-MS: [ M+H ]] ⁺ ＝554。

¹ H NMR(400MHz,DMSO)δ10.53(s,1H),8.14(s,2H),7.89(s,1H),7.26(dd,J＝8.4,5.3Hz,1H),7.15(t,J＝8.9Hz,1H),4.51(dd,J＝79.5,13.4Hz,2H),4.10(d,J＝21.7Hz,2H),3.95(d,J＝13.1Hz,1H),3.76(d,J＝13.3Hz,1H),2.54(d,J＝7.4Hz,2H),2.05–1.76(m,5H),1.05(t,J＝7.5Hz,3H).

Example 26:

step 1: raw material 2-fluoro-5-methylaniline H44-1 (5.0 g,39.96 mmol), potassium carbonate (13.8 g,99.90 mmol) and potassium iodide (6.63 g,39.96 mmol) were dissolved in 50mL of N-methylpyrrolidone NMP under nitrogen atmosphere and stirred for 5 minutes; 4-methoxybenzyl chloride (12.83 g,81.92 mmol) was slowly added to the mixture, and the mixture was allowed to react at 65℃for 1 hour, and the reaction was stopped. 200mL of ice water was added to the system, extraction was performed with ethyl acetate, drying was performed with anhydrous sodium sulfate, and the crude product was separated by flash column chromatography to obtain Compound H44-2 (14.0 g), yield: 95%. LC-MS: [ M+H ]] ⁺ ＝366。

Step 2: 2, 6-tetramethylpiperidine (1.55 g,11.0 mmol) was dissolved in 10mL of anhydrous tetrahydrofuran at-5℃under nitrogen, n-butyllithium (4.38 mL, 2.5M) was slowly added dropwise, and after stirring for 10 minutes, the temperature was lowered to-60 ℃; the above compound H44-2 (1.0 g,2.74 mmol) was added and stirred for 30 minutes. Triisopropyl borate (620 mg,3.29 mmol) was added to the reaction solution, and the reaction was continued for 30 minutes, after which the reaction was stopped. 50mL of saturated ammonium chloride aqueous solution was slowly added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography to give Compound H44-3 (770 mg), yield: 68%. LC-MS: [ M+H ] ] ⁺ ＝:410。

Step 3: under nitrogen, the above compound H44-3 (209 mg,0.51 mmol), cesium carbonate (499 mg,1.53 mmol) and intermediate a14 (250 mg,0.51 mmol) were dissolved in 5mL of 1, 4-dioxane and the catalyst was addedPd (dppf) Cl as an agent ₂ (39 mg,0.052 mmol), was warmed to 105℃and reacted for 16 hours, the reaction was stopped, and filtered. The solvent was distilled off under reduced pressure, and the crude product was separated by flash column chromatography to give Compound H44-4 (190 mg), yield: 48%. LC-MS: [ M+H ]] ⁺ ＝774。

Step 4: the above compound H44-4 (190 mg,0.25 mmol) and N-iodosuccinimide NIS (62 mg,0.28 mmol) were dissolved in 2mL of acetonitrile, 2 drops of trifluoroacetic acid were added, and the reaction was stopped at room temperature for 0.5 hours. The reaction solution was poured into 20mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography to give Compound H44-5 (210 mg), yield: 95%. LC-MS: [ M+H ]] ⁺ ＝900。

Step 5: under nitrogen protection, the above compound H44-5 (140 mg,0.16 mmol), methyl fluorosulfonyl difluoroacetate (308 mg, 1.6 mmol) and CuI (152 mg,0.80 mmol) as a catalyst were dissolved in 3mL DMF and reacted at room temperature for 16 hours to stop the reaction. The reaction solution was poured into 30mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography to give Compound H44-6 (70 mg), yield: 53%. LC-MS: [ M+H ] ] ⁺ ＝842。

Step 6: intermediate a18 (78 mg,0.42 mmol) was dissolved in 4mL anhydrous tetrahydrofuran under nitrogen, potassium tert-butoxide (95 mg,0.84 mmol) was added and stirred at room temperature for 30 min to give solution S0. In another reaction flask, compound H44-6 (350 mg,0.42 mmol) was dissolved in 2mL anhydrous THF, and the prepared solution S0 was slowly added under ice bath, stirring was continued for 1 hour, and the reaction was stopped. The reaction solution was poured into 30mL of ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to give a pale yellow solid H44-7 (300 mg). Yield: 71%, LC-MS: [ M+H ]] ⁺ ＝1009。

Step 7: the above compound H44-7 (300 mg,0.30 mmol) was dissolved in 2mL of toluene, and 2mL of a mixed solution (v/v, 1/1) of trifluoroacetic acid and methanesulfonic acid was added thereto, and the reaction was stopped after heating to 50℃for 16 hours. The solvent was distilled off under reduced pressure, and the residue was chromatographed by HPLC to give H44 (20 mg) as a white solid. Yield: 9 percent,LC-MS:[M+H] ⁺ ＝669。

¹ H NMR(400MHz,DMSO-d ₆ )δ7.89(s,1H),6.84(d,J＝8.9Hz,1H),6.03(s,2H),5.11(dt,J＝10.9,5.8Hz,1H),4.40(s,2H),4.31–4.23(m,2H),3.64–3.46(m,5H),2.79–2.59(m,5H),2.38–2.32(m,4H),2.05–1.77(m,8H),1.67(s,4H).

example 27:

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-p44/42 MAPK (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 ]

Compounds of formula (I)	p-ERK/IC ₅₀ /nM	Compounds of formula (I)	p-ERK/IC ₅₀ /nM
H1a	0.29	P1	54
H1b	4.6	P2	534
H2a	42	P3	450
H2b	1.1	P4	21
H3a	40	P5	169
H3b	0.33	P6	117
H4	2.6	P7	>1000
H5a	>1000	P8	13
H5b	42	P9	0.88
H6	4.1	P10	1.9
H7	10	P11	2.8

H8	40	P12	259
H9	10	P13	18
H10a	98	P14	633
H10b	0.51	P15	158
H11	4.0	P16	94
H12a	112	P17	72
H12b	0.94	P18	7.9
H13	780	P19	3.6
H14	358	P20	0.77
H15	5.5	P21	13
H16	24	P22	3.0
H17	4.0	P23	13
H18	21	P24	0.42
H19a	>1000	P25	5.2
H19b	7.5	P26	1.4
H20	3.2	P27	1.2
H21	51	P28	1.9
H22	23	P29	105
H23	25	M1	>1000
H24	1.9	M2	117
H25	5.5	M3	112
H26	>1000	M4	28
H27	3.9	M5	84
H28a	42	M6	29
H28b	1.6	M7	19
H29	>1000	M8	204
H30	>1000	M9	48
H31	>1000	M10	401
H32	>1000	M11	97
H33	37	M12	165
H34	69	M13	193
H35a	184
H35b	2.8
H36a	261
H36b	3.6
H37a	716
H37b	2.0
H38a	384
H38b	2.0
H39	31
H40	239
H41a	167
H41b	10
H42a	613
H42b	3.3
H43	37
H44	277
MRTX1133	1.0

MRTX1133 structure:

example 28: 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 values; y: percent inhibition.

Inhibitory Activity Effect of the compounds of Table 2 on GTP-KRAS

Compounds of formula (I)	KRAS G12D-CRAF/IC ₅₀ /nM
H1a	5.1
H2b	9.2
H4	7.4
H10b	8.5
H19b	10
P4	16
P20	11
MRTX1133	11

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

Example 29:

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. Test compound (1. Mu.M as initial concentration, 3-fold dilution, total 10 concentrations) was added to each well after incubation with cells for 7 daysThe 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 3: 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 30:

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

cell culture: in T75 flasks (Corning, catalog No. 430641), PANC-1 pancreatic cancer cells were cultured in DMEM medium (GIBCO) containing 10% fetal bovine serum (Ausgenex, catalog No. FBS 500-S) and 1% green/streptomycin (Gibco, catalog No. 15140-122).

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

Table 4: antiproliferative effect of compounds on PANC-1 cell half-effective concentration

Compounds of formula (I)	IC ₅₀ /nM
H2b	1.3
H3b	1.0
H10b	1.8
H12b	2.4
H19b	7.7
P20	3.0
MRTX1133	3.6

Example 31:

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, H24, H25, H35b-H38b, P20, P24, etc.) have lower clearance and slower metabolism in humans than control MRTX 1133.

Compounds of formula (I)	Human Clint/(mL/min/kg)	MouseClint/(mL/min/kg)
H1a	27	451
H1b	9.7	456
H2b	40.4	156
H3b	44.9	229
H4	33.7	211.7
H5b	32.9	244
H10b	14.2	167
H11	39.6	298
H12b	24.1	182
H19b	32.9	177
H24	19.8	166
H25	10.3	N.D.
H35b	17.8	143
H36b	12.7	102
H37b	14.3	110
H38b	20.0	152
P4	40.8	595
P11	29.7	547
P20	16.2	334
P24	20.0	403
MRTX1133	28.7	260

N.d. =untested

Example 32:

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 33:

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.

The MRTX1133 intravenous administration solution and the compound P20 intravenous administration vehicle are: 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

Qualified healthy ICR mice (age 6-8 weeks, body weight 18-20 g) were selected, 3 in each group, each for single infusion administration. Starting with the maximum dose administered intravenously, if no death is seen, the dose is increased and if death occurs, the increase is stopped. MRTX1133 infusion dosing solution and compound P20 infusion dosing vehicle 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	Infusion of inf	4mpk	0/3
MRTX1133	Infusion of inf	8mpk	0/3
MRTX1133	Infusion of inf	16mpk	0/3
MRTX1133	Infusion of inf	32mpk	2/3
P20	Infusion of inf	16mpk	0/3
P20	Infusion of inf	32mpk	0/3
P20	Infusion of inf	96mpk	0/3
P20	Infusion of inf	150mpk	0/3

The results show that the molecule of the invention has better in vivo safety, and the safety is far better than MRTX1133 whether the molecule is administrated by vein or infusion.

Example 34:

in vivo pharmacokinetic experiments in mice of the compounds. The method comprises the following steps:

CD1 female mice are used as test animals, and are orally/intravenously administered (oral administration amount is 20mg/kg vehicle: DMSO-Solutol-H) ₂ O)。

Experimental protocol: the oral group had 3 animals per group. Plasma samples were collected before (0 h) and after (0.25, 0.5,1,2,4,8,24 h) dosing; the blood concentration of plasma after oral administration of mice was determined by LC/MS method, and the collected data were calculated by AB Sciex QTRAP 6500 software, and the experimental results were as follows:

*＝50mg/kg

The results show that the molecules of the invention have better in vivo absorption in mice, and part of the molecules have very high in vivo exposure in mice and are far higher than MRTX1133. The possibility of oral administration is expected in clinic.

Claims

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:
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.
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.
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.
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.
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, bronchial 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 disorders, colon cancer, colorectal cancer, craniopharyngeal tumor, colorectal cancer cutaneous T cell lymphoma, extrahepatic Ductal Carcinoma In Situ (DCIS), embryonic tumor, CNS cancer, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, skeletal fibroblastic tumor, gallbladder 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 carcinoma, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, renal cancer, laryngeal carcinoma, lip and oral cancer, liver cancer, lobular Carcinoma In Situ (LCIS), lung cancer, lymphoma, metastatic squamous neck cancer with hidden primary foci, mid-line cancer, oral cancer, multiple endocrine tumor syndrome, multiple myeloma/plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasms, 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, sinus and nasal cavity cancer, oral cavity cancer, malignant mesothelioma, malignant fibrous histiocytoma and osteosarcoma of the bone parathyroid cancer, penile cancer, pharyngeal cancer, pleural pneumoblastoma, 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 renal pelvis and ureter, trophoblastoma, rare childhood cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer or virus-induced cancer.
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.