WO2024088307A1

WO2024088307A1 - Novel peptidyl nitrile compound and use thereof

Info

Publication number: WO2024088307A1
Application number: PCT/CN2023/126526
Authority: WO
Inventors: 叶斌
Original assignee: 上海壹迪生物技术有限公司
Priority date: 2022-10-26
Filing date: 2023-10-25
Publication date: 2024-05-02

Abstract

Disclosed are a novel peptidyl nitrile compound and a use thereof, and specifically disclosed are a compound of formula (I) and/or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising the compound and/or the pharmaceutically acceptable salt thereof, a method for preparing the compound, and a use of the compound for treating diseases caused by cathepsin C and a downstream serine protease thereof.

Description

A novel peptide-based nitrile compound and its application

This application claims the priority of Chinese Patent Application No. 2022113165806 filed on October 26, 2022 and Chinese Patent Application No. 2023103201603 filed on March 29, 2023. This application cites the full text of the above Chinese patent application.

Technical Field

The present invention relates to the field of medical technology, in particular to a novel peptide-based nitrile compound and application thereof.

Background technique

Inflammatory diseases are currently an important area of drug research and development. Although interleukin antibody drugs and small molecule drugs such as JAK inhibitors have been used clinically, they all have defects to varying degrees. For example, antibody drugs can only be administered by injection and have low clinical compliance. JAK inhibitors are used for inflammatory diseases and have potential adverse cardiovascular reactions due to their target mechanism (Norman P, Expert Opinion on Investigational Drugs. 2014, 23(8):1067–77). Therefore, it is still necessary to explore new anti-inflammatory drugs.

Cathepsin C (CTSC), also known as dipeptidyl peptidase I (DPP-1), is a lysosomal cysteine protease belonging to the papain family with a molecular weight of 200 KDa. Cathepsin C acts as a key enzyme that activates neutrophil and mast cell granule serine peptidases in inflammatory cells (such as four neutrophil proteases, elastase (NE), cathepsin G (CatG), proteinase 3 (PR3) and neutrophil serine protease (NSP4), as well as mast cell-related chymase, tryptase and serine protease, etc.) (Guay, D. et al, Curr. Top. Med. Chem. 2010, 10, 708-716; Korkmaz, B. et al, Pharmacol. Ther. 2018, 190, 202-236). Once activated by cathepsin C, these proteases are able to degrade various extracellular matrix components, which leads to tissue damage and chronic inflammation. Therefore, inhibitors of cathepsin C can be used as potential therapeutic drugs for the treatment of neutrophil-dominated inflammatory diseases, including chronic obstructive pulmonary disease (COPD), emphysema, asthma, multiple sclerosis, idiopathic pneumonia, cystic fibrosis, etc. (Laine et al, Expert Opin. Ther. Patents 2010, 20, 497).

Other immune diseases such as inflammatory bowel disease, rheumatoid arthritis, anti-neutrophil cytoplasmic antibody-associated necrotizing crescentic glomerulonephritis, ANCA-mediated vasculitis, and more serious systemic inflammatory diseases such as sepsis, acute lung injury (acute respiratory distress syndrome), acute pancreatitis, etc., a large part of their pathogenesis is caused by the increased activity of some of these inflammatory proteases, so cathepsin C inhibitors can also be used as potential drugs for the treatment of these diseases. In addition, cathepsin downstream serine protease elastase plays a very important role in the occurrence and metastasis of cancer, as well as cardiovascular and cerebrovascular diseases such as myocardial infarction. Therefore, theoretically, inhibiting cathepsin also has the same pharmacological effect as inhibiting elastase. Therefore, theoretically, cathepsin inhibitors can also be used for the treatment of cancer diseases and cardiovascular and cerebrovascular diseases. (Pharmacol Ther. 2018 Oct; 190: 202-236; Int J Mol Sci. 2021 Jan 13; 22(2): 722.).

It has been more than 70 years since the discovery of cathepsin C, but clinical drugs for cathepsin C inhibitors are still very limited (Korkmaz B. et al, J. Med. Chem., 2020, 63, 13258; Shen, XB et al, EJ Med. Chem., 2021, 225-113818). It was not until June 2020 that the U.S. Food and Drug Administration granted breakthrough drug qualification to the cathepsin C inhibitor Brenocatib, which had just completed Phase II clinical trials, for the treatment of non-cystic fibrosis bronchiectasis (NCFBE) in adults (Doyle K. et al, J. Med.Chem., 2016, 59, 9457). There is still a large unmet demand for cathepsin C inhibitors. Existing compounds are difficult to inhibit the activity of cathepsin C and downstream serine proteases, so it is necessary to provide new peptide-based nitrile compounds and their applications.

Summary of the invention

One object of the present invention is to provide a compound of formula (I) or a pharmaceutically acceptable salt thereof,

and/or a pharmaceutically acceptable prodrug thereof, and/or a solvate, hydrate, metabolite, nitrogen oxide, racemic mixture, enantiomer, diastereomer and tautomer thereof or a mixture thereof in any ratio including a racemic mixture, wherein:

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are each independently selected from H, cyano, halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy; or R ₁ , R ₂ , R ₄ and R ₅ are connected to form a 3-6 membered ring, the ring atoms of the 3-6 membered ring can be arbitrarily substituted by 1-2 S, O, N, C=O; the 3-6 membered ring is optionally substituted by R ₆ ;

X, Y are each independently selected from O, -NH, -NMe, -CH ₂ -, -C(O)-;

_R6 is optionally selected from H, =O, _C1 - _C4 alkyl, _C1 - _C4 deuterated alkyl, _C1 - _C4 cycloalkyl.

In a preferred embodiment of the present invention, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently selected from H, cyano, fluorine, C ₁ -C ₃ alkyl or C ₁ -C ₃ alkoxy.

In a preferred embodiment of the present invention, R ₄ and R ₅ are each independently selected from H and cyano.

In a preferred embodiment of the present invention, R ₁ , R ₂ , and R ₃ are each independently selected from H, fluorine, C ₁ -C ₃ alkyl, and C ₁ -C ₃ alkoxy.

In a preferred embodiment of the present invention, R ₁ and R ₂ are linked to form a cycloalkyl group.

In a preferred embodiment of the present invention, R ₄ and R ₅ are connected to form a five-membered ring, and the ring atoms of the five-membered ring can be arbitrarily substituted by S, O, N, or C=O; the five-membered ring can be unsubstituted or substituted by R ₆ , and R ₆ can be selected from H, =O, C ₁ -C ₄ alkyl, C ₁ -C ₄ deuterated alkyl, and C ₁ -C ₄ cycloalkyl.

In a preferred embodiment of the present invention, R ₄ and R ₅ are connected to form the following structure:

Wherein, R ₆ is selected from H, =O, C ₁ -C ₄ alkyl, C ₁ -C ₄ deuterated alkyl, C ₁ -C ₄ cycloalkyl. Preferably, R ₆ is selected from H, methyl, ethyl, cyclopropyl, propyl, isopropyl, deuterated methyl.

In a preferred embodiment of the present invention, when R ₄ and R ₅ are connected to form a five-membered ring, the compound of formula (I) has a structure of formula (II), formula (III), or formula (IV):

Wherein, Z is O or S; R ₃ is selected from H, cyano, fluorine, C ₁ -C ₃ alkyl or C ₁ -C ₃ alkoxy.

In a preferred embodiment of the present invention, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds:

The beneficial effects of the present invention relate to the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and preventing diseases of cathepsin C and its downstream serine proteases NE, PR3, CaTG, and NSP4.

Further, the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the preparation of a pharmaceutical composition for use in the treatment of patients with respiratory diseases, metabolic diseases, cardiovascular and cerebrovascular diseases, autoimmune diseases, cancers, infectious diseases and other inflammatory diseases, such as asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary hypertension, pulmonary arterial hypertension, non-cystic fibrosis, cystic fibrosis, bronchiectasis, bronchitis, pneumonia, emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), sepsis, allergic diseases, immune inflammatory bowel disease, rheumatoid arthritis, nephrotic syndrome, Use of the present invention in a drug for treating glomerulonephritis, eosinophilic diseases, neutrophilic diseases, ANCA-related inflammation, anti-neutrophil cytoplasmic antibody-related necrotizing crescentic glomerulonephritis, acute brain trauma, acute myocarditis, acute kidney injury, α-1-antitrypsin deficiency (AATD) and related inflammation, liver fibrosis, fatty liver and hepatic steatosis, obesity, insulin resistance, diabetes, pathogenic microbial infection, infectious gastrointestinal inflammatory disease, lung cancer and/or radiation injury syndrome or in patients at risk of the above diseases.

The present invention also relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

Furthermore, the pharmaceutical composition contains one or more compounds of formula (I) and a pharmaceutically active compound selected from a group consisting of other compounds, wherein the other compounds include but are not limited to: b mimetics, anticholinergic drugs, corticosteroids, PDE4 inhibitors, LTD4 antagonists, EGFR inhibitors, CRTH2 inhibitors, 5-LO inhibitors, histamine receptor antagonists, CCR9 antagonists and SYK inhibitors, NE inhibitors, MMP9 inhibitors, MMP12 inhibitors and a combination of two or three active substances.

Furthermore, the pharmaceutical composition also includes combined use with small molecule compounds and/or macromolecular antibodies to treat cancer, inflammation, bone marrow-related diseases and autoimmune diseases, and the small molecule compounds and/or macromolecular antibodies include but are not limited to glucocorticoids, adrenergic agonists, cholinergic receptor antagonists, theophylline drugs, antioxidants, elastase inhibitors, metalloproteinase inhibitors, PDE4 inhibitors, LTD4 antagonists, EGFR inhibitors, CRTH2 inhibitors, 5-LO inhibitors, histamine receptor antagonists, CCR9 antagonists and SYK inhibitors, chemokine receptor inhibitors, interleukin antibodies such as IL-6 antibodies, IL-23 antibodies, targeted anti-thymic stromal lymphopoietin (TSLP) antibodies such as tezepelumab, and complement inhibitors.

The beneficial effects of the present invention relate to the use of a composition of the compound of formula (I) in a drug for treating and preventing diseases caused by cathepsin C and its downstream serine proteases NE, PR3, CaTG, and NSP4, wherein the disease is selected from respiratory diseases, metabolic diseases, cardiovascular and cerebrovascular diseases, autoimmune diseases, cancer, infectious diseases, or inflammatory infectious diseases.

The compounds of the present invention may be asymmetric, for example, having one or more stereocenters. Unless otherwise specified, all stereoisomers, for example, enantiomers and diastereomers. Containing asymmetrically substituted carbon atoms. The compounds of the present invention may be isolated into optically pure or racemic forms. Optically pure forms may be prepared by resolution of racemates, or by using chiral synthons or chiral reagents.

The compounds of the present invention may also include tautomeric forms. Tautomeric forms are generated by the interchange of a single bond with an adjacent double bond accompanied by the migration of a proton.

The compounds of the present invention may also include all isotopic forms of atoms present in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include deuterium and tritium.

The present invention also includes pharmaceutically acceptable salts of compounds of formula (I). Pharmaceutically acceptable salts refer to derivatives of compounds of formula (I) wherein the parent compound is modified by conversion of the base moiety present into its salt form, or derivatives of compounds of formula (I) wherein the parent compound is modified by conversion of the acid moiety present into its salt form.

Specifically, examples of pharmaceutically acceptable salts include, but are not limited to, salts of inorganic or organic acids of basic groups (such as amines), or salts of inorganic or organic bases of acidic groups (such as carboxylic acids). The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds of formula (I) by reacting the free base forms of these compounds with 1-4 equivalents of an appropriate acid in a solvent system. Suitable salts are listed in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977).

Compounds of the present invention and pharmaceutically acceptable salts thereof also include solvate forms or hydrate forms. Generally speaking, solvate forms or hydrate forms are equivalent to non-solvate forms or non-hydrate forms, and are all included within the scope of the present invention. Some compounds of the present invention may exist in multiple crystal forms or amorphous forms. Generally speaking, all physical forms of compounds are included within the scope of the present invention.

The present invention also includes prodrugs of compounds of formula (I). A prodrug is a pharmacological substance (i.e., a drug) derived from a parent drug. Once administered, the prodrug is metabolized in vivo to become the parent drug. Prodrugs can be prepared by substituting one or more functional groups present in a compound, wherein the substituents in the prodrug are removed in vivo in such a way as to be converted into the parent compound. The preparation and use of prodrugs are described in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems" Vol. 14 of the A.C.S. Symposium Series and Bioreversible.

Definition and Description

The following terms, phrases and symbols used in the present invention have the meanings described below unless otherwise indicated in the context in which they are used.

The term "alkyl" used in the present invention refers to a straight or branched saturated hydrocarbon group containing 1 to 18 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 6 carbon atoms, and such as 1 to 4 carbon atoms. For example, "C1-C6 alkyl" is within the scope of "alkyl" and represents the alkyl group having 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl ("Me"), ethyl ("Et"), n-propyl ("n-Pr"), isopropyl ("i-Pr"), n-butyl ("n-Bu"), isobutyl ("i-Bu"), sec-butyl ("sBu"), and tert-butyl ("t-Bu").

The term "halo" as used herein refers to fluoro, chloro, bromo and iodo, and "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "cycloalkyl" used in the present invention refers to a saturated or partially unsaturated cyclic hydrocarbon group containing 3-12 ring carbon atoms, for example 3-8 ring carbon atoms, and for example 3-6 ring carbon atoms, which may have one or more rings, for example 1 or 2 rings. For example, "C3-8 cycloalkyl" represents the cycloalkyl group with 3-8 ring carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, and similar groups.

The term "cycloalkoxy" as used herein refers to the group -O-cycloalkyl, wherein cycloalkyl is as defined above. Examples of cycloalkoxy include, but are not limited to, cyclopropyloxy, cyclobutyloxy, including their isomers.

As used herein, "group" and "radical" are synonymous and are used to indicate a functional group or a molecular fragment that can be connected to other molecular fragments.

If a certain structural formula of the present invention contains an asterisk "*", the compound represented by the structural formula is a chiral compound, that is, the compound is in R-configuration or S-configuration. The configuration of the compound can be determined by a person skilled in the art using a variety of analytical techniques, such as single crystal X-ray crystallography and/or optical polarimetry and according to conventional protocols.

The terms "optional", "optional", "optionally", "optionally", "optionally" or "optionally" as used herein mean The substitution pattern, event or situation described herein may occur once or multiple times, or may not occur, and the description includes situations where the substitution pattern occurs and situations where the substitution pattern does not occur. For example, "optionally substituted alkyl" includes "unsubstituted alkyl" and "substituted alkyl" as defined herein. It should be understood by those skilled in the art that for any group containing one or more substituents, the group does not include any sterically impractical, chemically incorrect, synthetically infeasible and/or inherently unstable substitution pattern.

The term "substituted" or "substituted by..." as used herein means that one or more hydrogen atoms on a given atom or group are replaced by one or more substituents selected from a given substituent group, provided that the normal valence of the given atom is not exceeded. When the substituent is oxo (i.e., =O), two hydrogen atoms on a single atom are replaced by oxygen. Such combinations are permitted only if the combination of substituents and/or variables results in a chemically correct and stable compound. A chemically correct and stable compound means that the compound is stable enough to be isolated from a reaction mixture and the chemical structure of the compound can be determined, and then can be formulated into a formulation that at least has practical utility.

Unless otherwise indicated, substituents are named into the core structure. For example, it should be understood that when (cycloalkyl)alkyl is listed as a possible substituent, it means that the point of attachment of the substituent to the core structure is at the alkyl portion.

It will be appreciated by those skilled in the art that some compounds of formula (I) may contain one or more chiral centers, and therefore there are two or more stereoisomers. Racemic mixtures of these isomers, single isomers and mixtures enriched in one enantiomer, and diastereomers and mixtures partially enriched in specific diastereomers when there are two chiral centers are within the scope of the present invention. It will also be appreciated by those skilled in the art that the present invention includes all single stereoisomers (e.g., enantiomers), racemic mixtures or partially resolved mixtures of compounds of formula (I), and, where appropriate, single tautomers thereof.

In other words, in some embodiments, the present invention provides compounds having various degrees of stereoisomeric purity, i.e., diastereomeric or enantiomeric purity expressed as different "ee" or "de" values. In some embodiments, the compounds of formula (I) (e.g., as described herein) have an enantiomeric purity of at least 60% ee (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% ee, or any value between these recited values). In some embodiments, the compounds of formula (I) (e.g., as described herein) have an enantiomeric purity of greater than 99.9% ee, up to 100% ee. In some embodiments, the compound of formula (I) (e.g., as described herein) has a diastereomeric purity of at least 60% de (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% de, or any value between these recited values). In some embodiments, the compound of formula (I) (e.g., as described herein) has a diastereomeric purity of greater than 99.9% de.

The term "enantiomeric excess" or "ee" refers to the amount of one enantiomer relative to the other. For a mixture of R and S enantiomers, the percentage of enantiomeric excess is defined as |R-S|*100, where R and S are the mole or weight fractions of each enantiomer in the mixture, and R+S=1. If the optical rotation of a chiral substance is known, the percentage of enantiomeric excess is defined as ([a]obs/[a]max)*100, where [a]obs is the optical rotation of the enantiomeric mixture and [a]max is the optical rotation of the pure enantiomer.

Determination of diastereomers and/or enantiomeric excess may be accomplished using a variety of analytical techniques, including nuclear magnetic resonance spectroscopy, chiral column chromatography, and/or optical polarimetry, and according to conventional protocols familiar to those skilled in the art.

The racemic mixture can be used as such or resolved into the individual isomers. The method of separating isomers is well known, including physical methods, such as chromatography using chiral adsorbents. A single isomer in chiral form can be prepared from a chiral precursor. Alternatively, a single isomer can be chemically separated from a mixture by forming a diastereomeric salt with a chiral acid (e.g., a single enantiomer of 10-camphorsulfonic acid, camphoric acid, α-bromocamphoric acid, tartaric acid, diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid, etc.), fractionally crystallizing the salt, and then freeing one or two of the resolved bases, optionally repeating this process, thereby obtaining one or two isomers that are substantially free of another isomer, that is, an optical purity of, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% by weight of the desired stereoisomer. Alternatively, as is well known to those skilled in the art, the racemate can be covalently linked to a chiral compound (auxiliary) to provide diastereomers which can be separated by chromatography or fractional crystallization, followed by chemical removal of the chiral auxiliary to provide the pure enantiomers.

The term "pharmaceutically acceptable salt" as used herein refers to a non-toxic, biologically tolerable or other biologically suitable salt of a free acid or base of a compound of formula (I) for administration to a subject for treatment.

"Pharmaceutically acceptable salts" include, but are not limited to, acid addition salts formed by compounds of formula (I) with inorganic acids, such as hydrochlorides, hydrobromides, carbonates, bicarbonates, phosphates, sulfates, sulfites, nitrates, etc.; and acid addition salts formed by compounds of formula (I) with organic acids, such as formate, acetate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate, and salts formed with alkanedicarboxylic acids of the formula HOOC-(CH ₂ )n-COOH (wherein n is 0-4), etc. "Pharmaceutically acceptable salts" also include base addition salts formed by compounds of formula (I) with acidic groups and pharmaceutically acceptable cations such as sodium, potassium, calcium, aluminum, lithium and ammonium. The molar ratio of the compound of formula (I) to the acid or cation in the resulting pharmaceutically acceptable salts includes, but is not limited to, 1:1, 1:2, 1:3 and 1:4.

The "prodrug" of the present invention refers to a pharmacological substance (i.e., drug) derived from a parent drug. Once administered, the prodrug is metabolized into the parent drug in the body. The prodrug can be prepared by replacing one or more functional groups present in the compound, wherein the substituent in the prodrug is removed in the body in such a way as to be converted into the parent compound. The preparation and use of prodrugs can be found in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series and Bioreversible. "Prodrugs" include, but are not limited to: esters of compounds of formula (I) such as phosphates, formates, and carbamates; amides such as formamide and acetamide.

In addition, if the compounds of the present invention are obtained in the form of acid addition salts, their free base forms can be obtained by alkalizing a solution of the acid addition salt. Conversely, if the product is in the form of a free base, its acid addition salt, particularly a pharmaceutically acceptable acid addition salt, can be obtained by dissolving the free base in a suitable solvent and treating the solution with an acid in accordance with conventional procedures for preparing acid addition salts from basic compounds. Those skilled in the art can determine various synthetic methods that can be used to prepare non-toxic pharmaceutically acceptable acid addition salts without undue experimentation.

The term "solvate" means a solvent addition form containing a stoichiometric or non-stoichiometric amount of solvent. Some compounds have a tendency to entrain a fixed molar ratio of solvent molecules in the solid state, thereby forming a solvate. If the solvent is water, the solvate formed is a hydrate, and when the solvent is ethanol, the solvate formed is an ethanolate. Hydrates are formed by one or more molecules of water with one molecule of the substance, wherein the water retains its molecular state of H ₂ O, and such a combination can form one or more hydrates, such as hemihydrates, monohydrates and dihydrates, as well as variable hydrates.

The terms "group" and "radical" used in the present invention are synonymous and are used to indicate a functional group or a molecular fragment that can be connected to other molecular fragments.

The term "active ingredient" is used to refer to a chemical substance with biological activity. In some embodiments, an "active ingredient" is a chemical substance with pharmaceutical use. In the United States, actual drug activity can be determined by appropriate preclinical tests, whether in vitro or in vivo. However, drug activity that is sufficient to be accepted by regulatory agencies (such as the FDA in the United States) must have higher standards than preclinical tests. Whether such a higher standard of drug activity can be successfully obtained generally cannot be reasonably expected from preclinical test results, but can be established through appropriate and effective randomized, double-blind, controlled clinical trials conducted in humans.

The term "effective amount" as used herein refers to an amount or dosage of a cathepsin C inhibitor that is generally sufficient to produce a beneficial therapeutic effect in patients who need treatment for a disease or disorder mediated by cathepsin C and downstream serine protease activity. The effective amount or dosage of the active ingredient in the present invention can be determined by conventional methods (e.g., modeling, dose escalation studies, or clinical trials) in combination with conventional influencing factors (e.g., the mode or route of administration or administration, the pharmacokinetics of the drug ingredient, the severity and course of the disease or disorder, the individual's previous or ongoing treatment, the individual's health status and response to the drug, and the judgment of the attending physician). In the United States, the determination of an effective dose is generally difficult to predict from preclinical trials. In fact, the dose is completely unpredictable, and new and unpredictable dosage regimens will develop after the dose is originally used in randomized, double-blind, controlled clinical trials.

Typical dosage range is from about 0.0001 to about 200 mg of active ingredient per kg of individual body weight per day, for example, from about 0.001 to 100 mg/kg/day, or about 0.01 to 35 mg/kg/day, or about 0.1 to 10 mg/kg, once a day or in divided dose units (for example, twice a day, three times a day, four times a day). For a 70 kg person, a suitable dosage range can be about 0.05 to about 7 grams/day, or about 0.2 to about 5 grams/day. Once the patient's disease or disorder improves, the dosage can be adjusted to maintain treatment. For example, the dosage or the number of administrations can be adjusted according to the change of symptoms, or the dosage and the number of administrations can be reduced to the level of the desired therapeutic effect. Of course, if the symptoms are alleviated to an appropriate level, treatment can be stopped. However, for the recurrence of symptoms, the patient may need intermittent long-term treatment.

The term "subject" as used herein refers to mammals and non-mammals. The term "subject" is not limited to a specific age or gender. In some embodiments, the subject is a human.

Detailed ways

The following examples are illustrative of the present invention and are not intended to limit the present invention in any way. Unless otherwise stated, all fractions are weight fractions and temperatures are in degrees Celsius. Pressure is atmospheric pressure or close to atmospheric pressure. All data are measured by Agilent (Agilent 6120 and/or 1100). Except for synthetic intermediates, all reagents used in the present invention are obtained from commercial channels. The names of all compounds except reagents are generated by ChemDrew 20.0. The abbreviations of the present invention have the conventional meanings in the art.

The meanings of some abbreviations are as follows:
Boc tert-Butyloxycarbonyl
(Boc) ₂ O tert-Butyl pyrocarbonate
BH ₃ Borane
DIEPA N,N-Diisopropylethylamine
EDCI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
HATU 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate
HBTU O-Benzotriazole-tetramethyluronium hexafluorophosphate
HOBt 1-Hydroxybenzotriazole
ee enantiomeric excess
NCS N-chlorosuccinimide
PE Petroleum Ether
Pd(dppf) ₂ Cl ₂ [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride
Pd ₂ (dba) ₃ -trisdibenzylideneacetone dipalladium
Pd(PPh ₃ ) ₄ -Tetrakis(triphenylphosphine)palladium
PMB p-Methoxybenzyl cyanide
Pin ₂ B ₂ -Pinacol Bisboronic Acid Ester
TFA Trifluoroacetic acid
TsOH 4-Toluenesulfonic acid
Xphos 2-Dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl
Burgess reagent Methyl N-(triethylammoniumsulfonyl)carbamate

Example 1: Synthesis of Compound 1

1) Preparation of 4-bromo-3-(methoxymethyl)benzonitrile (1-b):

At 0°C, (1-a, 4 g, 20.2 mmol) was dissolved in dichloromethane (40 mL), and then DIPEA (5.47 g, 42.4 mmol) and MOMBr (3.03 g, 24.2 mmol) were slowly added dropwise. The reaction solution was allowed to warm to room temperature naturally and stirred for 16 hours. Water (150 mL) was added to quench the mixture and dichloromethane was used for extraction (2 x 200 mL). The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography (eluent: PE/DCM=1:2, V/V) to obtain a white solid product (1-b, 4.5 g, 92%). MS (ESI): m/z=242.1[M+H] ⁺ .

2) Preparation of 3-(methoxymethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (1-c):

At room temperature, 1-b (5.7 g, 23.6 mmol), Pin ₂ B ₂ (11.97 g, 47.1 mmol), Pd(dppf)Cl ₂ (861 mg, 1.2 mmol) and KOAc (6.92 g, 70.7 mmol) were added to dioxane (100 mL) in sequence, and the air was removed, and then stirred at 90°C under argon protection for 16 hours. The reaction solution was cooled to room temperature and purified by column chromatography (eluent: PE/EtOAc=10:1, V/V) to obtain a brown oil product (1-c, 6.4 g, 92%). MS (ESI): m/z=290.3[M+H] ⁺ .

3) Preparation of 5-bromo-4-fluoro-2-iodo-benzoic acid (1-e):

At 0°C, slowly add a solution of NaNO ₂ (10.6 g, 153.8 mmol) in HCl (conc., 106.8 mL, 1282 mmol) and H ₂ O (220 mL) in water (60 mL) over 30 minutes. After stirring for 30 minutes at 0°C, slowly add a solution of KI (31.9 g, 32.1 mmol) in water (60 mL) over 20 minutes. After stirring for 1 hour at 0°C, heat to 90°C and continue stirring for 30 minutes. Cool to room temperature and add a saturated aqueous solution of Na ₂ S ₂ O ₃ (150 mL). The reaction solution is extracted with EtOAc (2 x 500 mL), the organic phases are combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography to obtain a yellow solid product (1-e, 40 g, 91%). MS (ESI): m/z = 345.1 [M + H] ⁺ .

4) Preparation of 5-bromo-4-fluoro-2-iodo-benzyl alcohol (1-f):

At 0°C, slowly add borane/THF (13.5 mL, 13.5 mmol, 1 mol/L) to a solution of 1-e (3 g, 12.2 mmol) in THF (20 mL) over 15-20 minutes, and then stir at 65°C for 6 hours. The excess boron reagent is carefully quenched with a solution of THF/H ₂ O (1:1), and then a saturated K ₂ CO ₃ solution is added. The reaction solution is extracted with ethyl acetate EtOAc (2 x 200 mL), the organic phases are combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography (eluent: PE/EtOAc=4:1, V/V) to obtain a white solid product (1-f, 2.6 g, 92%). MS (ESI): m/z = 312.9 [M-17] ⁺ . ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.66 (d, J = 6.8 Hz, 1H); 7.59-7.57 (m, 1H); 4.62 (s, 2H); 2.24 (s, 1H).

5) Preparation of 4'-bromo-5'-fluoro-2'-(hydroxymethyl)-2-(methoxymethyl)-[1,1'-biphenyl]-4-carbonitrile (1-g):

At room temperature, 1-c (5.1 g, 17.6 mmol), 1f (7.01 g, 21.2 mmol), Pd(PPh ₃ ) ₄ (2.04 g, 1.8 mmol) and K ₂ CO ₃ (4.87 g, 35.3 mmol) were added to a solution of dioxane (100 mL) and water (10 mL) in sequence. The air was removed and the mixture was stirred at 60°C for 48 hours under argon. The reaction solution was cooled to room temperature, concentrated, and purified by column chromatography (eluent: PE/EtOAc=4:1, V/V) to obtain a white solid product (1-g, 3.4 g, 53%). MS (ESI): m/z=388.0[M+Na] ⁺ .

6) Preparation of 4'-bromo-5'-fluoro-2-hydroxy-2'-(hydroxymethyl)-[1,1'-biphenyl]-4-carbonitrile (1-h):

At room temperature, 1-g (3.4 g, 9.3 mmol) and TsOH (320 mg, 1.9 mmol) were added to dioxane (60 mL) and water (60 mL). The reaction solution was stirred at 100°C under nitrogen for 48 hours. Cooled to room temperature, concentrated, and purified by column chromatography (eluent: PE/EtOAc=2:3, V/V) to obtain a brown solid (1-h, 2.4 g, 80%). MS (ESI): m/z=322.0[M+H] ⁺ .

7) Preparation of 8-bromo-9-fluoro-6H-benzo[c]chromene-3-carbonitrile (1-i):

At 0°C, a solution of DIAD (2.45 g, 12.1 mmol) in THF (10 mL) was added dropwise to a solution of 1-h (2.6 g, 3.19 mmol) and PPh ₃ (3.19 g, 12.1 mmol) in THF (90 mL). After 2 hours, the reaction solution was concentrated, and water (100 mL) was added, and EtOAc (2 x 200 mL) was extracted. The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and recrystallized (50% PE in DCM) to obtain a yellow solid product (1-i, 2.1 g, 86%). MS (ESI): m/z=304.1[M+H] ⁺ .

8) Preparation of (S)-2-((tert-butyloxycarbonyl)amino)-3-(3-cyano-9-fluoro-6H-benzo[c]chromone-8-yl)propanoic acid methyl ester (1-j):

Trimethylsilyl chloride (2.57 g, 23.7 mmol) was slowly added dropwise to DMF (40 mL) containing zinc powder (3.59 g, 55.3 mmol) at room temperature. After stirring for 1.5 hours, iodine (468 mg, 1.8 mmol) was added and stirring was continued for 15 minutes. Then, a DMF solution (8 mL) of methyl(R)-2-((tert-butoxycarbonyl)amino)-3-iodopropanoate ((R)-2-((tert-butoxycarbonyl)amino)-3-iodopropanoate, 2.6 g, 7.9 mmol) was slowly added dropwise over 20 minutes. After stirring for 20 minutes, the reaction solution was cooled to room temperature, and a DMF solution (25 mL) of 1-i (800 mg, 2.6 mmol) was added dropwise over 20 minutes. Then, Pd ₂ (dba) ₃ (722 mg, 0.79 mmol) and S-Phos (647 mg, 1.58 mmol) were added in sequence. The reaction mixture was stirred at 50°C overnight. After cooling to room temperature, the solid was removed by filtration. The filtrate was diluted with water (200 mL), extracted with EtOAc (3 x 200 mL), the organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography (eluent: 25% ethyl acetate in hexane) to obtain a yellow solid product (1-j, 850 mg, 76%). MS (ESI): m/z = 449.2 [M + Na] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ):δ8.07(d,J＝8.4Hz,1H);7.81(d,J＝11.2Hz,1H);7.53-7.48(m,2H);7.37(d,J＝8.0Hz,1H);7.24(d,J＝8.0Hz,1H);5.20-5.12(m,2H);4.28-4.22(m,1H);3.61(s,3H);3.15-3.10(m,1H);2.93-2.87(m,1H);1.30(s,9H).

9) Preparation of tert-butyl (S)-(1-amino-3-(3-cyano-9-fluoro-6H-benzo[c]chromone-8-yl)-1-oxopropane-2-yl)carbamate (1-k):

At room temperature, 1-j (300 mg, 0.7 mmol) was added to NH ₃ /MeOH (25 mL, 7 mol/L) and stirred overnight. Concentrated and purified by column chromatography to obtain a white solid product (1-k, 289 mg, 86%). MS (ESI): m/z = 356.1 [M-55] ⁺ .

10) Preparation of (S)-2-amino-3-(3-cyano-9-fluoro-6H-benzo[c]chromone-8-yl)propionamide TFA salt (1-1):

At room temperature, TFA (2 mL) was added dropwise to a solution of 1-k (289 mg, 0.7 mmol) in DCM (20 mL), and stirring was continued for 2 hours. Concentration gave a brown oily product (1-1, 300 mg, 99%). MS (ESI): m/z = 312.3 [M+H] ⁺ .

11) Preparation of (S)-2-((S)-1-amino-3-(3-cyano-9-fluoro-6H-benzo[c]chromone-8-yl)-1-oxopropane-2-yl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylic acid tert-butyl ester (1-m):

At room temperature, DIPEA (546 mg, 4.23 mmol), HOBt (114 mg, 0.85 mmol) and HBTU (321 mg, 0.85 mmol) were added to a solution of 1-1 (300 mg, 0.71 mmol) and (S)-4-(tert-butoxycarbonyl)-1,4-oxazepane-2-carboxylic acid (173 mg, 0.71 mmol) in DCM (10 mL) in sequence. After stirring for 4 hours, the reaction solution was concentrated and purified by reverse phase column chromatography (C18, CH ₃ CN, 10 mM NH ₄ HCO ₃ aqueous solution) to obtain a white solid product (1-m, 300 mg, 79%). MS (ESI): m/z = 561.3 [M + Na] ⁺ .

12) Preparation of (S)-N-((S)-1-cyano-2-(3-cyano-9-fluoro-6H-benzo[c]chromon-8-yl)ethyl)-1,4-oxazaheterocycle-2-carboxamide (1-n):

At room temperature, Burgess reagent (652 mg, 2.74 mmol) was added to a solution of 1-m (295 mg, 0.55 mmol) in DCM (10 mL), and stirring was continued for 3 hours. The reaction solution was concentrated and purified by HPLC (NH ₄ HCO ₃ buffer: A: 10 mM NH ₄ HCO ₃ aqueous solution; B: acetonitrile; Column: Waters XBridge Peptide BEH C18, 19×250 mm, 10 μm, ) to obtain a white solid product (1n, 251 mg, 88%). MS (ESI): m/z = 543.3 [M + Na] ⁺ .

13) Preparation of (S)-N-((S)-1-cyano-2-(3-cyano-9-fluoro-6H-benzo[c]chromon-8-yl)ethyl)-1,4-oxazaheterocycle-2-carboxamide (1):

At room temperature, TFA (1 mL) was added dropwise to a solution of 1-n (246 mg, 0.47 mmol) in DCM (10 mL), and stirring was continued for 1 hour. Saturated NaHCO ₃ (150 mL) was added, and the mixture was extracted with DCM (3×150 mL). The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by HPLC (A: 0.8% NH ₄ HCO ₃ aqueous solution, B: CH ₃ CN; Column: Xbridge BEH peptide C18, 19 mm×250 mm) to obtain a white solid product (1, 130 mg, 65%). MS (ESI): m/z=421.2 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ7.73 (d, J=8.8 Hz, 1H); 8.09 (d, J=8.4 Hz, 1H); 7.85 (d, J=10.4 Hz, 1H); 7.54-7.50 (m, 2H); 7.31 (d, J=7.2 Hz, 1H); 5.21-5.14 (m, 2H); 5.08-5.02 (m, 1H); 4.01-3.9 8 (m, 1H); 3.88-3.82 (m, 1H); 3.75-3.69 (m, 1H); 3.31-3.28 (m, 1H); 3.20-3.15 (m, 1H); 3.05-3.00 (m, 1H); 2.79-2.72 (m, 1H); 2.63-2.52 (m, 2H); 1.77-1.67 (m, 2H).

According to the above method, the following compounds were synthesized

Example 2: Synthesis of Compound 2

1) Preparation of 4-bromo-5-methyl-2-nitrophenol:

Under nitrogen at 0°C, a solution of bromine (27 ml) in acetic acid (70 ml) was added dropwise to a solution of 5-methyl-2-nitrophenol (2-a, 40 g, 0.26 mol) in acetic acid (400 mL) within 30 minutes. After stirring at room temperature for 2 hours, most of the acetic acid was removed under reduced pressure, the residue was diluted with water, and then extracted with ethyl acetate, the organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography (petroleum ether/ethyl acetate = 10:1, v/v) to obtain a yellow solid product (2-b, 55 g, 91.4%).

2) Preparation of 2-amino-4-bromo-5-methylphenol (2-c):

At room temperature, SnCl ₂ (214.95 g, 0.95 mol) and hydrochloric acid (138.91 mL, 1.67 mol) were added to a solution of 2-b (55 g, 0.24 mol) in methanol (825 mL). The reaction mixture was stirred at 75°C for 2 hours. Most of the methanol was removed under reduced pressure, and the residue was diluted with water and ethyl acetate, and the pH was adjusted to pH 14 with ammonia. The solid was filtered off, and the organic phase was washed with saturated NaCl water and dried over anhydrous Na ₂ SO _4. The residue was dried, filtered and concentrated to obtain a yellow solid product (2-c, 43.5 g, 90.1%).

3) Preparation of 5-bromo-6-methylbenzo[d]oxazol-2(3H)-one (2-d):

At room temperature, CDI (42.11 g, 0.22 mol) was added to a solution of 2-c (43.5 g, 0.22 mol) in THF (450 mL). The reaction solution was stirred at 80°C for 2 hours. The product was cooled to room temperature, washed with water, extracted with ethyl acetate, and the organic phases were combined, dried over _anhydrous _Na2SO4 , filtered, concentrated, and purified by column chromatography (petroleum ether/ethyl acetate = 3:1, v/v) to obtain a yellow solid product (2-d, 37 g, 91.36%).

4) Preparation of 5-bromo-3,6-dimethylbenzo[d]oxazol-2(3H)-one (2-e):

At room temperature, K ₂ CO ₃ (33.79 g, 0.24 mol) and iodomethane (34.71 g, 0.24 mol) were added to a DMF (260 mL) solution of 2-d (37 g, 0.16 mol) in sequence. After reacting at room temperature for 16 hours, the reaction solution was poured into water, filtered and washed with water, and air-dried for one day to obtain a yellow solid product (2-e, 21.5 g, 54.74%). 1H NMR (400 MHz, DMSO) δ7.53 (s, 1H), 7.35 (s, 1H), 3.30 (s, 3H), 2.34 (s, 3H).

5) Preparation of 5-bromo-6-(bromomethyl)-3-methylbenzo[d]oxazol-2(3H)-one (2-f):

At room temperature, AIBN (164 mg, 1 mmol, 0.05 eq.) and NBS (4.04 g, 22.7 mmol, 1.1 eq.) were added to a solution of 2-d (5.0 g, 20.6 mmol, 1.0 eq.) in dichloroethane (50 mL) in sequence. The reaction solution was stirred at 80 °C for 2 hours. Cooled to room temperature and diluted with water (100 mL). Extracted with ethyl acetate (3 x 50 mL), the organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography (PE/DCM: 20/1) to obtain a yellow solid product (2-f, 5.3 g, 80%). MS (ESI): m/z = 322.0 [M + H] ⁺ .

6) Preparation of 5-bromo-6-(hydroxymethyl)-3-methylbenzo[d]oxazol-2(3H)-one (2-g):

At room temperature, acetone/H ₂ O (200 mL, 1:1) and Na ₂ CO ₃ (2.6 g, 24.8 mmol, 1.5 eq.) were added to 2-f (5.3 g, 16.5 mmol) in sequence. After the reaction solution was refluxed for 5 hours, it was cooled to room temperature and most of the acetone was removed under reduced pressure. The product was filtered, washed with water, and dried to obtain a yellow solid product (2-g, 3.8 g, 89%). MS (ESI): m/z = 280.0 [M + H] ⁺ .

7) Preparation of 4-bromo-2-fluoro-5-hydroxybenzoic acid (2-i):

Under nitrogen at 0℃, bromine (2mL, 38.4mmol, 3.0eq.) was added dropwise to a solution of 2h (2.0g, 12.8mmol, 1.0eq.) in CHCl ₃ (15mL)/acetic acid (15mL). After stirring at room temperature for 20 hours, the reaction was quenched with saturated Na ₂ SO ₃ (100mL) solution, extracted with ethyl acetate (3x150mL), and the organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated to obtain the initial product. The white solid product (2-i, 2.1g, yield 70%) was obtained by slurrying with n-hexane. MS (ESI): m/z=235.0[M+H] ⁺ .

8) Preparation of methoxymethyl 4-bromo-2-fluoro-5-(methoxymethyl)benzoate (2-j):

At 0°C under nitrogen, MOMBr (685 μL, 8.4 mmol, 3.0 eq.) and DIPEA (1.95 mL, 11.2 mmol, 4.0 eq.) were added dropwise to a solution of 2-I (660 mg, 2.8 mmol, 1.0 eq.) in DCM/DMF (25 mL, 4:1) in sequence. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and saturated NH ₄ Cl solution was added. The mixture was extracted with ethyl acetate (3x50 mL). The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography (PE/EA: 9/1) to obtain an oily product 2-j (820 mg, yield 91%). MS (ESI): m/z=263.0 [M-MOMO+H] ⁺ .

9) Preparation of (4-bromo-2-fluoro-5-(methoxymethyl)phenyl)methanol (2-k):

At room temperature, LiOH·H ₂ O (12.6 mg, 0.30 mmol, 2.0 eq.) was added to a solution of 2-j (50 mg, 0.15 mmol, 1.0 eq.) in THF/H ₂ O (6 mL, 1:1), and stirring was continued for 2 hours. The pH of the reaction solution was adjusted to pH < 3 with 1N HCl. The mixture was extracted with ethyl acetate (3x5 mL), the organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated to obtain the intermediate (42 mg, 99%). The intermediate was dissolved in anhydrous THF (5 mL) at 0°C under nitrogen, and then BH ₃ ·THF (1 M THF solution, 2.15 mL) was added dropwise. After reacting at room temperature for 2 hours, the reaction solution was quenched with alcohol and concentrated to obtain an oily product 2-k (40 mg, purity: 78%). MS (ESI): m/z = 249.1 [M-OH] ⁺ .

10) Preparation of 1-bromo-4-(chloromethyl)-5-fluoro-2-(methoxymethyl)benzene (2-1):

At room temperature, SOCl ₂ (558 μL, 7.7 mmol, 1.2 eq.) and DMF (10 μL) were added dropwise to a solution of 2-k (1.7 g, 6.4 mmol, 1.0 eq.) in DCM (50 mL), and the mixture was stirred for 2 hours. The reaction solution was concentrated and dissolved in DCM (50 mL). MOMBr (522 μL, 6.4 mmol, 1.0 eq.) and DIPEA (1.67 mL, 9.6 mmol, 1.5 eq.) were then added dropwise. After stirring for 2 hours, saturated NH ₄ Cl aq. (50 mL) was added. The mixture was extracted with DCM (30 mL*3), the organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography (PE/EA (9/1) to obtain a white solid product 2-1 (1.72 g, 95%).

11) Preparation of (2S, 5R)-2-(4-bromo-2-fluoro-5-(methoxymethyl)benzyl)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (2-n) Preparation:

At -78℃ under argon, n-buthyl lithium 2.5M in hexane (3.6mL containing 2.5M n-butyl lithium) was added dropwise to a solution of (R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (2-m, 1.66g, 9.0mmol) in THF (50ml). The reaction solution was stirred at -78℃ for 2 hours, and then 2-l (1.7g, 6.0mmol) was added all at once. The reaction was allowed to slowly warm to room temperature and stirred overnight. Saturated NH ₄ Cl (30mL) was added, and ethyl acetate was extracted (3x50mL). The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography (PE/EA: 10/1, v/v) to obtain an oily product 2-n (2.4g, 93%). MS (ESI): m/z = 433.1 [M + H] ⁺ .

12) Preparation of (5-fluoro-4-(((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine-2-yl)methyl)-2-(methoxymethyl)phenyl)boronic acid:

At -78℃ under argon, n-buthyl lithium 2.5M in hexane (2.5M hexane solution of n-butyl lithium, 1.34mL) was added dropwise to a solution of 2-n (1.2g, 2.8mmol, 1.0eq.) in THF (30mL). The reaction solution was stirred at -78℃ for 30 minutes, and B(OiPr) ₃ (1.3mL, 5.6mmol, 2.0eq.) was added. The reaction was allowed to slowly warm to room temperature and stirred overnight. Saturated NH ₄ Cl (30mL) solution was added, and the mixture was extracted with ethyl acetate (3x50mL). The organic phases were combined, dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography (eluent: DCM/MeOH=95/5, v/v) to obtain an oily product 2-o (960mg, 86%). MS (ESI): m/z=397.2[M+H] ⁺ .

13) Preparation of 5-(5-fluoro-4-(((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)methyl)-2-(methoxymethyl)phenyl)-6-(hydroxymethyl)-3-methylbenzo[d]oxazol-2(3H)-one (2-p):

At room temperature, ₂ -o (844 mg, 2.13 mmol, 1.1 eq.), 2-g (500 mg, 1.94 mmol, 1.0 eq.), XPhos Pd G3 (84 mg, 0.1 mmol, 0.05 eq.) and K ₃ PO ₄ (849 mg, 4.0 mmol, 2.0 eq.) were added to dioxane/H 2 O (22 mL, 10:1) in sequence. The reaction solution was stirred at 80°C under argon for 2 hours, cooled to room temperature, concentrated, and purified by reverse phase column with NH ₄ HCO ₃ buffer to obtain a yellow foam product 2-p (680 mg, 66%). MS (ESI): m/z = 530.2 [M+H] ⁺ .

14) Preparation of methyl 2-amino-3-(2-fluoro-5-hydroxy-4-(6-(hydroxymethyl)-3-methyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)propanoate (2-q):

At room temperature, add 2 mL of concentrated hydrochloric acid to a solution of 2-p (430 mg, 0.81 mmol, 1.0 eq.) in 20 mL of MeCN/H ₂ O (3/7). Stir overnight at room temperature and directly purify with a C18 column to obtain a white foam product 2-q (387 mg). MS (ESI): m/z = 391.0 [M+H] ⁺ .

15) Preparation of methyl (S)-2-amino-3-(2-fluoro-5-hydroxy-4-(6-(hydroxymethyl)-3-methyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)propanoate (2-q):

16) Preparation of (S)-tert-butyl 2-(((S)-3-(2-fluoro-5-hydroxy-4-(6-(hydroxymethyl)-3-methyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)-1-methoxy-1-oxopropane-2-yl)carbamoyl)-1,4-oxazacyclo-4-carboxylate (2-r):

At room temperature, 2-p (387 mg, 1.0 mmol, 1.0 eq.), DIPEA (871 μL, 5.0 mmol, 5.0 eq.), HOBt (270 mg, 2.0 mmol, 2.0 eq.) and PyBOP (1.04 g, 2.0 mmol, 2.0 eq.) were added to a DMF (10 mL) solution of (S)-4-(tert-butoxycarbonyl)-1,4-oxazepane-2-carboxylic acid (490 mg, 2.0 mmol, 2.0 eq.) in sequence. After stirring at room temperature for 2 hours, the reaction solution was directly purified by reverse phase column to obtain a white foam product 2-r (324 mg, 52%). MS (ESI): m/z = 640.3 [M + Na] ⁺ .

17) Preparation of (S)-tert-butyl 2-(((S)-3-(2-fluoro-10-methyl-9-oxo-9,10-dihydro-6H-benzo[3,4]isochromeno[6,7-d]oxazol-3-yl)-1-methoxy-1-oxopropane-2-yl)carbamoyl)-1,4-oxazacyclo-4-carboxylate (2-s):

At 0°C under argon, PPh ₃ (204 mg, 0.78 mmol, 1.5 eq.) and DIAD (154 μL, 0.78 mmol, 1.5 eq.) were added to a solution of 2-r (324 mg, 0.52 mmol, 1.0 eq.) in THF (30 mL) in sequence. Stir at room temperature overnight, concentrate and purify with a reverse phase column to obtain a white solid product 2-s (280 mg, 90%). MS (ESI): m/z = 544.3 [M-55] ⁺ .

18) Preparation of (S)-tert-butyl 2-((S)-1-amino-3-(2-fluoro-10-methyl-9-oxo-9,10-dihydro-6H-benzo[3,4]isochromeno[6,7-d]oxazol-3-yl)-1-oxopropane-2-yl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylate (2-t):

At room temperature, 2-s (280 mg, 0.47 mmol) was added to 30 mL of NH ₃ aq. (25-28%)/MeCN (1:2) solution. Stirred at room temperature overnight, and lyophilized to obtain a crude product. Reverse phase column purification gave a white solid product (2-t, 110 mg, 40%). MS (ESI): m/z = 529.0 [M-55] ⁺ .

19) Preparation of tert-butyl (S)-2-((S)-1-cyano-2-(2-fluoro-10-methyl-9-oxy-9,10-dihydro-6H-benzo[3,4]isochromeno[6,7-d]oxazol-3-yl)ethyl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylate:

At room temperature, add Burgess reagent (304 mg, 1.28 mmol, 3.0 eq.) to a solution of 2-t (249 mg, 0.42 mmol, 1.0 eq.) in DCM (10 mL). Stir at room temperature for 3 hours, dilute with DCM (20 mL), wash with water (20 mL) and brine (5 mL x 2). Combine the organic phases, dry over anhydrous Na ₂ SO ₄ , filter, and concentrate. Purify with a reverse phase column to obtain a white solid product (2-u, 155 mg, 64%). MS (ESI): m/z = 589.2 [M + Na] ⁺ .

20) Preparation of (S)-N-((S)-1-cyano-2-(2-fluoro-10-methyl-9-oxy-9,10-dihydro-6H-benzo[3,4]isochromeno[6,7-d]oxazol-3-yl)ethyl)-1,4-oxazaheterocycle-2-carboxamide (2):

At room temperature, 2-u (155 mg, 0.27 mmol, 1.0 eq.) was dissolved in TFA/DCM (1:10, 5.5 mL). The mixture was stirred at room temperature for 2 hours, concentrated, and purified by reverse phase column to obtain a white solid product (2, 73.6 mg, 58%). MS (ESI): m/z = 467.1 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO) δ8.76 (d, J = 8.5 Hz, 1H), 7.83 (s, 1H), 7.81 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 6.98 (d, J = 6.5 Hz, 1H), 5.10 (s, 2H), 5.05 (dd, J = 15.8, 8.6 Hz, 1H), 4.06 (dd, J = 8.3, 3.5 Hz, 1H), 3.91– 3.82 (m, 1H), 3.74 (ddd, J = 12.0, 7.4, 4.2 Hz, 1H), 3.39 (s, 3H), 3.24 (dd, J = 13.7, 6.9 Hz, 2H), 3.17 (d, J = 9.1 Hz, 1H), 3.10 (dd, J = 14.2, 3.6 Hz, 1H), 2.90–2.79 (m, 1H), 2.73–2.63 (m, 1H), 2.60 (dd, J = 14.2, 8.4 Hz, 1H), 1.89–1.66 (m, 2H).

The following compounds were synthesized by similar methods:

Example 3: Synthesis of Compound 3

1) Preparation of 2-amino-5-methoxyphenol (3-b)

At room temperature, a mixture of 3-a (25 g, 148 mmol) and 10% Pd-C (3 g) in MeOH (400 mL) was placed in a hydrogen atmosphere and stirred overnight. The solid was filtered off, concentrated, and purified by column chromatography to obtain a brown solid product (3-b, 18 g, 87%). MS (ESI): m/z = 140.1 [M + H] ⁺ .

2) Preparation of 6-methoxybenzo[d]oxazol-2(3H)-one (3-c):

At room temperature, a mixture of 3-b (18 g, 129 mmol) and CDI (21 g, 129 mmol) in THF (600 mL) was heated to reflux overnight. After cooling to room temperature, the mixture was washed with water (150 mL x 2) and saturated brine (150 mL) respectively. The organic phase was dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography with EA/PE 1/1 (100 mL) to obtain a brown solid product (3-c, 7.2 g, 33%). MS (ESI): m/z = 166.0 [M+H] ⁺ .

3) Step 3: Preparation of 5-bromo-6-methoxybenzo[d]oxazol-2(3H)-one (3-d)

At room temperature, drop bromine Br ₂ (3.4 mL, 66.3 mmol) into a solution of 3-c (7.2 g, 43.6 mmol) in AcOH (40 mL) and water (40 mL). Stir overnight at room temperature, add water (200 mL), and filter to obtain a brown solid. Dissolve the solid in ethyl acetate (200 mL) and wash with saturated NaHCO ₃ (100 mL). Dry the organic phase with Na ₂ SO ₄ , concentrate, and purify by column chromatography to obtain a brown solid product (3-d, 8.6 g, 80%). MS (ESI): m/z = 243.9 [M + H] ⁺ .

4) Preparation of 5-bromo-6-methoxy-3-methylbenzo[d]oxazol-2(3H)-one (3-e):

At 0°C, MeI (6.6 mL, 106 mmol) was added dropwise to a mixture of 3-d (10 g, 41 mmol) and Cs ₂ CO ₃ (17.5 g, 53.7 mmol) in DMF (120 mL). Stir at room temperature for 2 hours, add water (600 mL), and filter to obtain a solid. The solid was further dissolved in ethyl acetate (800 mL) and washed with saturated brine (150 mL x 2). The organic phase was dried over Na ₂ SO ₄ , concentrated, and purified by column chromatography to obtain a brown solid product (3-e, 8.7 g, 82%). MS (ESI): m/z = 258.1 [M+H] ⁺ .

5) Preparation of 6-methoxy-3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-2(3H)-one (3-f)

Under argon, a mixture of 3-e (8.5 g, 32.9 mmol), Pin ₂ B ₂ (10 g, 39.4 mmol), Pd(OAc) ₂ (740 mg, 3.3 mmol), XPhos (3.15 g, 6.6 mmol) and KOAc (9.7 g, 99 mmol) in dioxane (200 mL) was stirred at 100°C for 2 hours. The mixture was cooled to room temperature, the solid was filtered off, the filtrate was concentrated, and the brown solid product (3-f, 5 g, 37%) was purified by column chromatography. MS (ESI): m/z = 306.3 [M+H] ⁺ .

6) Preparation of 5-(4-bromo-5-fluoro-2-(hydroxymethyl)phenyl)-6-methoxy-3-methylbenzo[d]oxazol-2(3H)-one (3-g):

Under argon, 3-f (5 g, purity: 76%, 12.5 mmol), 1-f (5.4 g, 16.4 mmol), PdCl ₂ dppf (920 mg, 1.26 mmol), K ₃ PO ₄ (8 g, 37.7 mmol) in dioxane (100 mL) and H ₂ O (10 mL) were stirred at 50°C for 4.5 hours. The mixture was cooled to room temperature, concentrated under reduced pressure, diluted with ethyl acetate (300 mL), washed with saturated brine (50 mL), dried over Na ₂ SO ₄ , concentrated, and purified by column chromatography to obtain a light brown solid product (3-g, 3.2 g, 67%). MS (ESI): m/z = 384.1 [M+H] ⁺ .

7) Preparation of 5-(4-bromo-2-(bromomethyl)-5-fluorophenyl)-6-hydroxy-3-methylbenzo[d]oxazol-2(3H)-one (3-h):

At 0°C, slowly drop BBr ₃ (42 mL, 17% in DCM, 28.6 mmol) into a solution of 3-g (3.2 g, 8.37 mmol) in DCM (100 mL). Warm to room temperature and stir overnight. The reaction solution was diluted with DCM (100 mL), cooled to 0°C, and treated with saturated NaHCO ₃ to pH 7. Dry over _{Na 2} SO ₄ and concentrate to obtain a light brown solid product (3-h, 2.5 g, 69%). MS (ESI): m/z = 432.0 [M+H] ⁺ .

8) Preparation of 3-bromo-2-fluoro-10-methyl-5H-benzo[3,4]chromeno[6,7-d]oxazol-9(10H)-one (3-i):

At room temperature, K ₂ CO ₃ (800 mg, 5.8 mmol) was added to a DMF (40 mL) solution of 3-h (2.5 g, 5.8 mmol). After stirring for 1 hour, water (100 mL) was added and filtered to obtain a brown solid. The solid was dissolved in ethyl acetate (300 mL) and washed with saturated brine (100 mL). Drying with Na ₂ SO ₄ , concentration and column chromatography purification gave a light brown solid product (3-i, 1.9 g, 93%). MS (ESI): m/z＝350.0[M+H] ⁺ .

9) Preparation of methyl (S)-2-((tert-butylcarbonyl)amino)-3-(2-fluoro-10-methyl-9-oxo-9,10-dihydro-5H-benzo[3,4]chromeno[6,7-d]oxazol-3-yl)propanoate (3-j):

At room temperature, TMSCl (8.5 mL, 67.1 mmol) was added dropwise to a DMF (150 mL) solution containing Zn (10 g, 154 mmol) over 20 minutes. After stirring for 1.5 hours, I ₂ (1.2 g, 4.7 mmol) was added. After stirring for 15 minutes, a DMF (30 mL) solution of methyl (R)-2-((tert-butoxycarbonyl)amino)-3-iodopropanoate ((R)-2-((tert-butoxycarbonyl)amino)-3-iodopropanoate, 8.9 g, 27 mmol) was added dropwise over 20 minutes. After stirring for 20 minutes, a DMF (70 mL) solution of 3-i (2.36 g, 6.74 mmol) was added dropwise over 20 minutes. Then Pd ₂ dba ₃ (1.85 g, 2 mmol) and S-Phos (1.66 g, 4 mmol) were added. Stirred at 50°C overnight, cooled to room temperature, added ethyl acetate (1 L), washed with water (300 mL x 3) and saturated salt (300 mL x 2), dried over Na ₂ SO ₄ , concentrated, and purified by column chromatography to obtain a light brown solid product (3-j, 3 g, 94%). MS (ESI): m/z = 495.2 [M + Na] ⁺ .

10) Preparation of (S)-2-amino-3-(2-fluoro-10-methyl-9-oxo-9,10-dihydro-5H-benzo[3,4]chromeno[6,7-d]oxazol-3-yl)propanoic acid methyl ester (3-k):

At room temperature, 4N HCl-dioxane (10 mL) was added dropwise to a solution of 3-j (1 g, 2.1 mmol) in CH ₃ CN (40 mL). After stirring for 1 hour, Et ₂ O (150 mL) was added, and then centrifuged (3200 r/m) for 2 minutes to remove the solvent and dry to obtain a white solid product (3-k, 800 mg, 93%). MS (ESI): m/z = 373.1 [M+H] ⁺ .

11) Preparation of (S)-tert-butyl 2-((S)-3-(2-fluoro-10-methyl-9-oxo-9,10-dihydro-5H-benzo[3,4]chromeno[6,7-d]oxazol-3-yl)-1-methoxy-1-oxopropane-2-yl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylate (3-1):

At room temperature, DIPEA was added to a suspension of 3-k (800 mg, 1.96 mmol) in DCM (50 mL) until the pH was 7-8. Then, (S)-4-(tert-butoxycarbonyl)-1,4-oxazepane-2-carboxylic acid (500 mg, 2.04 mmol), HBTU (820 mg, 2.16 mmol), and HOBt (292 mg, 2.16 mmol) were added in sequence. and DIPEA (1 mL, 6 mmol). After stirring for 1 hour, the reaction solution was concentrated and purified by reverse phase column chromatography to obtain a white solid product (3-1, 650 mg, 55%). MS (ESI): m/z = 622.2 [M + Na] ⁺ .

12) Preparation of (S)-tert-butyl 2-((S)-1-amino-3-(2-fluoro-10-methyl-9-oxo-9,10-dihydro-5H-benzo[3,4]chromeno[6,7-d]oxazol-3-yl)-1-oxopropan-2-yl)carbamoyl)-1,4-oxazacyclo-4-carboxylate (3-m);

At room temperature, 25% aqueous ammonia (50 mL) was added dropwise to a solution of 3-1 (625 mg, 1.04 mmol) in CH ₃ CN (50 mL). After stirring for 1 hour, the reaction solution was concentrated and saturated brine (50 mL) was added. The mixture was extracted with ethyl acetate (50 mL x 3), dried over Na ₂ SO ₄ , concentrated, and purified by column chromatography to obtain a white solid product (3-m, 580 mg, 95%). MS (ESI): m/z = 529.2 [M-55] ⁺ .

13) Preparation of (S)-tert-butyl 2-((S)-1-cyano-2-(2-fluoro-10-methyl-9-oxo-9,10-dihydro-5H-benzo[3,4]chromeno[6,7-d]oxazol-3-yl)ethyl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylate (3-n):

At room temperature, add Burgess reagent (950 mg, 4 mmol) to 3-m (580 mg, 0.99 mmol) in DCM (20 mL). After stirring for 2 hours, concentrate and purify by column chromatography to obtain a white solid product (3-n, 400 mg, 71%). MS (ESI): m/z = 589.2 [M + Na] ⁺ .

14) Preparation of (S)-N-((S)-1-cyano-2-(2-fluoro-10-methyl-9-oxo-9,10-dihydro-5H-benzo[3,4]chromeno[6,7-d]oxazol-3-yl)ethyl)-1,4-oxazaheterocycle-2-carboxamide (3):

At room temperature, 4N HCl in dioxane (5 mL) was added dropwise to a solution of 3-n (400 mg, 0.71 mmol) in CH ₃ CN (20 mL). After stirring for 1 hour, MTBE (80 mL) was added and the mixture was centrifuged (3200 r/m) for 2 minutes. The solvent was removed and DMF (10 mL) was added to the obtained solid, which was neutralized to pH 7-8 with saturated NaHCO 3. The product was directly separated and purified by reverse phase column (C18, CH ₃ CN, NH ₄ HCO ₃ aqueous solution) to obtain a white solid product (3, 90 mg, 27%). MS (ESI): m/z = 467.2 [M+H] +. ¹ H NMR (400 MHz, DMSO-d ₆ ):δ8.73(d,J＝8.4Hz,1H),7.84(s,1H),7.76(d,J＝11.2Hz,1H),7.26(d,J＝7.6Hz,1H),7.08(s,1H),5.10-5.01(m,3H),3.99(dd,J＝8,3.6Hz,1H),3.88-3.82(m,1H),3.75-3.69(m,1H),3.37(s,3H),3.27-3.25(m,1H),3.18-3.13(m,1H),3.03(dd,J＝14,3.6Hz,1H),2.79-2.73(m,1H),2.63-2.53(m, 2H),1.78-1.68(m,2H)

According to the above method, the following compounds were synthesized

Example 4: Synthesis of Compound 4:

1) Preparation of 4-iodo-3-nitrobenzonitrile (4-b):

At 5-10°C, add 30% H ₂ SO ₄ (20 mL) to a solution of 4-a (4.5 g, 27.6 mmol) in DMSO (15 mL). Then, dropwise add an aqueous solution (3 mL) of NaNO2 (2.09 g, 30.3 mmol). After stirring for 1 hour, add NaI (2.09 g, 30.3 mmol). After stirring for another 1 hour, add water (150 mL) and EA (150 mL). Extract with ethyl acetate (50 mL x 3), dry with Na ₂ SO ₄ , concentrate, and purify by column chromatography to obtain a yellow solid product 4-b (6 g, 79%).

2) Preparation of 3-amino-4-iodobenzonitrile 4-c:

At room temperature, NH ₄ Cl (5.86 g, 110 mmol) and Fe (3.68 g, 65.7 mmol) were added to a solution of 4-b in 30% EtOH (60 mL). After stirring at 50°C for 2 hours, the mixture was cooled to room temperature and water (250 mL) and EA (250 mL) were added. The solid was filtered off, and the filtrate was extracted with ethyl acetate (30 mL x 3), dried over Na ₂ SO ₄ , concentrated, and purified by column chromatography to obtain a yellow solid product 4-c (5.1 g, 95%).

3) Preparation of 4-fluoro-5-methyl-2-nitrobenzoic acid 4-e:

At 0°C, KNO ₃ (49.1 g, 487.01 mmol) was added in batches to a solution of 4-d (50 g, 324.7 mmol) in concentrated H ₂ SO ₄ (675 mL). After stirring at room temperature for 2 hours, the reaction solution was poured into 1 L of ice water, extracted with dichloromethane (300 mL x 3), dried over Na ₂ SO ₄ , and concentrated to give a white solid product 4-e (58 g, 95%).

4) Preparation of methyl 4-fluoro-5-methyl-2-nitrobenzoate 4-f

At room temperature, SOC1 ₂ (69.3 g, 582.9 mmol) was added dropwise to a solution of 4-e in MeOH (500 mL). The mixture was heated under reflux overnight, cooled to room temperature, concentrated under reduced pressure to remove methanol, added with water (200 mL), extracted with ethyl acetate (100 mL x 3), dried over Na ₂ SO ₄ , and concentrated to give a white solid product 4-f (58 g).

5) Preparation of methyl 2-amino-4-fluoro-5-methylbenzoate 4-g

At room temperature, a mixture of 4-f (58 g) and 5% Pd/C (5.8 g) in MeOH (500 mL) was subjected to hydrogen absorption at normal pressure overnight, concentrated, and purified by column chromatography to obtain a white solid product 4-g (22.2 g, 95%).

6) Preparation of methyl 2-bromo-4-fluoro-5-methylbenzoate 4-h

At 0°C, to a suspension of 4-g (15 g, 81.9 mmol) in 40% HBr (600 mL), an aqueous solution of NaNO ₂ (5.6 g, 81.9 mmol) (600 mL) was added dropwise. Then, CuBr (11.9 g, 81.9 mmol) was added. After stirring for 1 hour, water was added. The mixture was extracted with ethyl acetate, dried over Na ₂ SO ₄ , concentrated, and purified by column chromatography to obtain a white solid product 4-h (58 g, 50%).

7) Preparation of methyl 2-bromo-5-(bromomethyl)-4-fluorobenzoate 4-i

At room temperature, NBS (10.8 g, 60.7 mmol) and AIBN (1.33 g, 8.1 mmol) were added to a solution of 4-h (10 g, 40.5 mmol) in CCl ₄ (100 mL). The mixture was heated under reflux for 6 hours, cooled to room temperature, and water was added. The mixture was extracted with ethyl acetate, dried over Na ₂ SO ₄ , concentrated, and purified by column chromatography to obtain a white solid product 4-h (2.9 g, 22%).

8) Preparation of methyl 2-bromo-4-fluoro-5-(((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine-2-yl)methyl)benzoate (4-j):

At -78 °C in argon, (R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine To a THF (40ml) solution of 4-i (3.2g, 9.8mmol), add n-buthyl lithium (3.9ml, 10.8mmol, 2.5M hexane solution of n-butyl lithium) slowly. After stirring for 1 hour, add a THF (10ml) solution of 4-i (3.2g, 9.8mmol). After stirring at -78℃ for 3 hours, slowly warm to room temperature and continue stirring for 1 hour. Add saturated NaHCO ₃ (200ml) solvent, extract with ethyl acetate (300mLx3), dry with Na ₂ SO ₄ , concentrate, and purify by column chromatography to obtain a yellow oily product (4-j, 2.8g, 67%). MS (ESI): m/z＝429.1[M+H] ⁺ .

9) Preparation of (S)-2-bromo-5-(2-(tert-butylcarbonyl)amino)-3-methoxy-3-oxopropyl)-4-fluorobenzoic acid methyl ester (4-k):

At room temperature, HCl (0.2 mol/L, 14 mmol, 70 mL) was added dropwise to a solution of 4-j (3 g, 7 mmol) in MeCN (70 mL). After stirring for 16 hours, the mixture was concentrated and neutralized to pH 8 with a saturated NaHCO ₃ solution. Then, (Boc) ₂ O (3.05 g, 4 mmol) and MeCN (70 mL) were added in sequence. After stirring for 3 hours, the mixture was concentrated and extracted with ethyl acetate (200 mL x 3), dried over Na ₂ SO ₄ , concentrated, and purified by column chromatography to obtain a white solid product (4-k, 680 mg, 23%). MS (ESI): m/z = 456.0 [M + Na] ⁺ .

10) Preparation of (S)-2-((tert-butylcarbonyl)amino)-3-(3-cyano-9-fluoro-6-oxo-5,6-dihydrophenanthridin-8-yl)propanoic acid methyl ester (4-1):

Under argon protection at room temperature, 4-k (660 mg, 1.52 mmol), 4-c (408 mg, 1.67 mmol), Pd(dppf)Cl ₂ (111 mg, 0.15 mmol) and potassium acetate (447 mg, 4.56 mmol) were added to dioxane (20 mL) and water (2 mL) in sequence. After reacting at 85°C for 7 hours, the mixture was cooled to room temperature, concentrated, and directly purified by column chromatography to obtain a white solid product (4-1, 450 mg, 67%). MS (ESI): m/z = 384.0 [M-55] ⁺ .

11) Preparation of methyl 2-amino-3-(3-cyano-9-fluoro-6-oxo-5,6-dihydrophenanthridin-8-yl)propanoate TFA salt (4-m):

At room temperature, TFA (4 mL) was added dropwise to a solution of 4-1 (200 mg, 0.46 mmol) in DCM (16 mL). After stirring for 1 hour, the mixture was concentrated to obtain a brown oily crude product (4-m, 210 mg, 90%. MS (ESI): m/z = 340.1 [M+H] ⁺ .

12) Preparation of (S)-2-((S)-3-(3-cyano-9-fluoro-6-oxo-5,6-dihydro-8-yl)-1-methoxy-1-oxopropane-2-yl)carbamoyl)-1,4-oxazaheterocyclic-4-carboxylic acid tert-butyl ester (4-n):

At room temperature, a mixture of 4-m (210 mg, 0.45 mmol), (S)-4-(tert-butoxycarbonyl)-1,4-oxazepane-2-carboxylic acid ((S)-4-(tert-butoxycarbonyl)-1,4-oxazepane-2-carboxylic acid, 123 mg, 0.50 mmol), DIPEA (353 mg, 2.73 mmol), HOBt (74 mg, 0.55 mmol) and HBTU (207 mg, 0.55 mmol) in DCM (20 mL) was stirred for 2 hours, concentrated, and directly separated by reverse phase column C18, CH ₃ CN, 10 mM NH ₄ HCO ₃ aqueous solution) to obtain a brown solid product (4-n, 240 mg, 93%. MS (ESI): m/z = 467.2 [M-99] ⁺ .

13) Preparation of (S)-2-((S)-1-amino-3-(3-cyano-9-fluoro-6-oxo-5,6-dihydrophenanthridin-8-yl)-1-oxopropane-2-yl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylic acid tert-butyl ester (4-o):

At room temperature, 4-n (280 mg, 0.49 mmol) was added to a solution of NH ₃ /MeOH (20 mL, 7 mol/L). The mixture was stirred overnight at room temperature and concentrated to obtain a brown solid crude product (4-o, 270 mg, 90%). MS (ESI): m/z = 452.1 [M-99] ⁺ .

14) Preparation of tert-butyl (S)-2-((S)-1-cyano-2-(3-cyano-9-fluoro-6-oxo-5,6-dihydrophenanthridin-8-yl)ethyl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylate (4-p)

At room temperature, add Burgess reagent (648 mg, 2.72 mmol) to a solution of 4-o (250 mg, 0.45 mmol) in DMF (14 mL). After stirring at room temperature for 4 hours, concentrate and directly purify by HPLC to obtain a white solid product (4-p, 190 mg, 79%). m/z = 434.2 [M-99] ⁺ .

15) Preparation of (S)-N-((S)-1-cyano-2-(3-cyano-9-fluoro-6-oxo-5,6-dihydrophenanthridin-8-yl)ethyl)-1,4-oxazaheterocycle-2-carboxamide (4):

To a solution of 4-p (185 mg, 0.35 mmol) in DCM (20 mL) was added TFA (2 mL) at room temperature. After stirring at room temperature for 1 hour, a saturated NaHCO ₃ solution (150 mL) was added. The mixture was extracted with DCM (5×250 mL), dried over Na ₂ SO ₄ , concentrated, and purified by HPLC to obtain a white solid product (4, 45 mg, 30%). MS (ESI): m/z = 434.1 [M + H] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.96 (s, 1H); 8.78-8.72 (m, 1H); 8.58 (d, J = 8.8 Hz, 1H); 8.49-8.45 (m, 1H); 8.34 (d, J = 7.6 Hz, 1H); 7.70-7.66 (m, 2H); 5.17-5.04 (m, 1H); 3.96-3.81 (m, 2H); 3.74-3.65 (m, 1H); 3.47-3.39 (m, 2H); 3.12-2.89 (m, 1H); 2.82-2.34 (m, 3H); 1.78-1.64 (m, 2H).

Example 5: Synthesis of Compound 22:

1) 2-Bromo-5-(hydroxymethyl)phenol (22-b)

At 0°C, BH ₃ ·THF (1M THF solution, 62.2 mL) was added dropwise to a solution of 4-bromo-3-hydroxybenzoic acid (22-a, 15 g, 81.9 mmol) in anhydrous THF (100 mL) at 0°C, then the mixture was warmed to room temperature and stirred overnight. After the reaction was completed by LCMS, methanol (50 mL) was added to quench the reaction, and the mixture was concentrated to obtain a colorless oily product (22-b, 5.1 g, 100%).

2) 2-Bromo-5-(chloromethyl)phenol (22-c)

At room temperature, SOCl ₂ (3.6 mL, 50 mmol) and DMF (0.5 mL) were added to a solution of 22-b (5.1 g crude, 24.88 mmol) in dichloromethane (150 mL), stirred overnight, then saturated NaHCO ₃ aq. (150 mL) was added, extracted with dichloromethane (20 mL x 3), dried over Na ₂ SO ₄ , filtered, and concentrated. Column chromatography with DCM/MeOH (95/5, v/v) gave a yellow oily product 22-c (3.68 g, 67% yield in 2 steps). MS (ESI): m/z=218.9 [MH] ^- .

3) 1-Bromo-4-(chloromethyl)-2-(methoxymethoxy)benzene (22-d)

At room temperature, to a solution of 22-c (3.68 g, 16.6 mmol) in dichloromethane (50 mL) were added MOMBr (1.5 mL, 18.3 mmol) and DIPEA (4.3 mL, 24.9 mmol). After stirring at room temperature for 2 hours, saturated NH ₄ Cl aq. (50 mL) solution was added, extracted with dichloromethane (20 mL x 3), dried with Na ₂ SO ₄ , filtered and concentrated. Column chromatography with PE/EA (9/1) was used to obtain a yellow oily product 22-d (3.3 g, 75% yield). MS (ESI): m/z = 231.1 [M-Cl] ⁺ .

4) 4-(((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)methyl)-2-(methoxymethoxy)phenyl)boronic acid (22-e)

Under argon protection at -78°C, n-butyllithium solution (2.5M n-butyllithium in hexane, 6mL, 15mmol) was added dropwise to a solution of (R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (2.7g, 15mmol) in THF (50mL). After stirring at -78°C for 30 minutes, 22-d (2.6g, 10mmol) was added in one portion. The mixture was warmed to room temperature and stirred for 1 hour. The mixture was cooled to -78°C again and n-butyllithium solution (2.5M n-butyllithium in hexane, 6mL, 15mmol) was added dropwise. After stirring at -78°C for 30 minutes, B(OiPr) ₃ (4.6mL, 20mmol) was added dropwise. The mixture was warmed to room temperature and stirred overnight, and then a mixture of ethyl acetate and water (1:1, 200mL) was added. The mixture was further extracted with ethyl acetate (30mL x 3), and the organic phases were combined, dried over Na ₂ SO ₄ , filtered, and concentrated. Column chromatography purification with DCM/MeOH (95/5) gave a yellow oily product 22-e (2.7 g, 71%). MS (ESI): m/z = 379.2 [M+H] ⁺ .

5) Ethyl 3-(4-bromo-3-chlorophenyl)acrylate (22-g)

Under argon protection at 0°C, NaH (3.3 g, 60% Wt in mineral oil, 82.5 mmol) was added in batches to a solution of ethyl diethylphosphonoacetate (16.4 mL, 82.2 mmol) in THF (200 mL). After stirring at 0°C for 30 minutes, 4-bromo-3-chloro-benzaldehyde (15 g, 68.5 mmol) was added, then the mixture was warmed to room temperature and refluxed for 1 hour. After cooling to room temperature, saturated NH ₄ Cl aq. (300 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (150 mL x 3). The organic phases were combined, dried over Na ₂ SO ₄ , filtered, and concentrated to obtain a colorless oily product 22-g (17.5 g, 88%). The crude product was used directly in the next step without purification. MS (ESI): m/z=291.1[M+H] ⁺ .

6) Ethyl 3-(4-bromo-3-chlorophenyl)propionate (22-h)

A mixture of 22-g (17.5 g, 60.4 mmol) and PtO ₂ (400 mg) in EtOH (250 mL) was subjected to hydrogen absorption at room temperature overnight. The solid was filtered off and concentrated to give 22-h (17.6 g, 100%). MS (ESI): m/z = 293.1 [M+H] ⁺ .

7) 3-(4-bromo-3-chlorophenyl)propanoic acid (22-i)

At room temperature, LiOH H ₂ O (5.1 g, 120.7 mmol) was added to a solution of 22-h (17.6 g, 60.36 mmol) in THF/H ₂ O (200 mL/200 mL). After stirring at room temperature for 2 hours, EtOAc/H ₂ O (200 mL/200 mL) was added. The aqueous phase was washed with ethyl acetate twice, and then treated with 4N HCl until pH <3. The aqueous phase was extracted with dichloromethane (200 mL x 3, the organic phases were combined, dried over Na ₂ SO ₄ , filtered, concentrated, and slurried with n-hexane to obtain colorless crystals 22-i (14.1 g, 88%). MS (ESI): m/z = 264.9 [M+H] ⁺ .

8) 6-Bromo-5-chloro-2,3-dihydro-1H-inden-1-one (22-j)

Add SOCl ₂ (200 mL) to 22-h (14.1 g, 53.23 mmol) and heat to reflux for 3 hours. Concentrate to remove most of SOCl ₂ , add dichloromethane (250 mL) and AlCl ₃ (8.5 g, 63.87 mmol). After stirring at 40°C for 2 hours, add DCM/H ₂ O (200 mL/200 mL). The aqueous phase is extracted with dichloromethane (200 mL*3), the organic phases are combined, dried over Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography to obtain a white solid 22-j (3.5 g, 27%). MS (ESI): m/z=246.9[M+H] ⁺ .

9) 6-Bromo-5-chloro-2,3-dihydrospiro[indene-1,2'-[1,3]dithiolane](22-k)

22-j (3.5 g, 14.29 mmol) 1,2-ethanedithiol (1.6 g, 17.14 mmol), boron trifluoride etherate (7.1 g, 50.01 mmol) and molecular( A mixture of _2-(4- ( _2- methyl-1-oxo _- 1-yl)-2-nitrogen ( ₂ -nitrogen ^...

10) 5-Chloro-2,3-dihydrospiro[indene-1,2'-[1,3]dithiolane]-6-carbaldehyde (22-1)

Under argon protection at -78℃, n-BuLi (2.5M hexane solution of n-butyl lithium, 3.5mL, 8.75mmol) was added dropwise to a solution of 22-k (1g, 3.46mmol) in THF (20mL). After stirring for 30 minutes, DMF (1mL, 13.84mmol) was added dropwise. The temperature was raised to 0℃ and stirring was continued for 2 hours. Water (50mL) was added to quench the reaction and dichloromethane was extracted (30mL x 3). Drying with Na ₂ SO ₄ , filtration, concentration and column chromatography purification were performed to obtain a yellow solid 22-l (0.4g, 43%). MS (ESI): m/z = 271.1 [M + H] ⁺ .

11)(5-chloro-2,3-dihydrospiro[indene-1,2'-[1,3]dithiopentyl]-6-yl)methanol(22-m)

Under argon protection at -78°C, NaBH ₄ (1.55 g, 40.98 mmol) was added to a THF (30 mL) solution of 22-1 (3.7 g, 13.66 mmol), and then stirred at room temperature for 30 minutes. Water (30 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (20 mL*3), dried with Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography to obtain a yellow solid 22-m (2.5 g, 67%). MS (ESI): m/z=273.1[M+H] ⁺ .

12)(5-(5-fluoro-4-(((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)methyl)-2-(methoxymethoxy)phenyl)-2,3-dihydrospiro[indene-1,2'-[1,3]dithiopentyl]-6-yl)methanol(22-n)

Under argon protection, a mixture of 22-e (1.36 g, 3.43 mmol), 22-m (1.2 g, 4.12 mmol), XPhos Pd G3 (290 mg, 0.343 mmol)/, K ₃ PO ₄ (1.5 g, 6.86 mmol) in dioxane (40 mL) and H ₂ O (4 mL) was stirred at 70°C for 4 hours. The mixture was cooled to room temperature, concentrated to remove the reaction solvent, and purified by column chromatography (0-30% EA in PE) to obtain Y22-n (950 mg, 47%). MS (ESI): m/z = 589.3 [M+H] ⁺ .

13) (S)-2-amino-3-(2-fluoro-5-hydroxy-4-(6-(hydroxymethyl)-2,3-dihydrospiro[indene-1,2'-[1,3]dithiopentyl]-5-yl)phenyl)propanoate (22-o)

At room temperature, 3N HCl (2 mL) was added to a mixed solution of 22-n (730 mg, 1.24 mmol) in CH ₃ CN (100 mL) and H ₂ O (75 mL). After stirring for 1 hour, concentrated HCl (4 mL) was added. After stirring for 1 hour, concentrated HCl (40 mL) was added. After stirring for 1 hour, the mixture was neutralized to pH 7 with saturated NaHCO ₃ aqueous solution. CH ₃ CN was removed by concentration, and the aqueous phase was extracted with EA (100 mL x 3) with ethyl acetate, dried over Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography to obtain a brown solid 22-o (250 mg, 44%). MS (ESI): m/z = 450.0 [M + H] ⁺ .

14)(S)-2-(((S)-3-(2-fluoro-5-hydroxy-4-(6-(hydroxymethyl)-2,3-dihydrospiro[indene-1,2'-[1,3]dithiopentyl-5-yl)phenyl)-1-methoxy-1-oxopropan-2-yl)carbamoyl)-1,4-oxazepane-4-carboxylic acid butyl ester (22-p)

At room temperature, 22-o (210 mg, 0.47 mmol), (S)-4-(tert-butyloxycarbonyl)-1,4-oxazepane-2-carboxylic acid (92 mg, 0.376 mmol), EDCI HCl (81 mg, 0.42 mmol), HOBt (58 mg, 0.43 mmol), and DIPEA (235 uL, 1.42 mmol) were added to DCM (5 mL) in sequence. After stirring for 2 hours, the mixture was concentrated and purified by reverse phase column (C18, NH ₄ HCO ₃ aqueous solution) to obtain a white solid product 22- p(140mg,44%). MS (ESI): m/z = 698.8 [M + Na] ⁺ .

15) (S)-2-(((S)-3-(2-fluoro-9,10-dihydro-6H-spiro[indeno[5,6-c]benzopyran-8,2'-[1,3]dithiolane]-3-yl)-1-methoxy-1-oxopropan-2-yl)carbamoyl)-1,4-oxopropane-4-carboxylic acid butyl ester (22-q)

At room temperature, PPh ₃ (80 mg, 0.3 mmol) and DIAD (70 uL, 0.36 mmol) were added to a THF solution of 22-p (170 mg, 0.25 mmol) in sequence. After stirring for 2 h, the mixture was concentrated and purified by reverse phase column (C18, NH ₄ HCO ₃ aqueous solution) to obtain a white solid 22-q (120 mg, 72%). MS (ESI): m/z = 681.3 [M + Na] ⁺ .

16) (S)-2-(((S)-3-(2-fluoro-8-oxo-6,8,9,10-tetrahydroindeno[5,6-c]benzopyran-3-yl)-1-methoxy-1-oxopropyl-2-yl)carbamoyl)-1,4-oxazepane-4-carboxylic acid butyl ester (22-r)

AgNO ₃ (65 mg, 0.38 mmol) was added to a solution of 22-q (115 mg, 0.175 mmol) in EtOH (10 mL) at room temperature. The mixture was stirred overnight, and then AgNO ₃ (70 mg, 0.41 mmol) was added. The mixture was stirred for 3 hours, and the solid was filtered off. The filtrate was concentrated and purified by SGC (0-60% EA in PE, Rf = 0.2 in PE/EA 1/1) to obtain a white solid product 22-r (30 mg, 29%). MS (ESI): m/z = 527.3 [M-55] ⁺ .

17) (S)-2-(((S)-1-amino-3-(2-fluoro-8-oxo-6,8,9,10-tetrahydroindeno[5,6-c]benzopyran-3-yl)-1-oxopropyl-2-yl)carbamoyl)-1,4-oxazepane-4-carboxylic acid butyl ester (22-s)

At room temperature, 25% ammonia (3 mL) was added to a solution of 22-r (28 mg, 0.048 mmol) in CH ₃ CN (2 mL), stirred for 1 hour, concentrated, and freeze-dried to obtain a white solid product 22-s (25 mg, 91%). MS (ESI): m/z = 590.3 [M + Na] ⁺ .

18) (S)-2-(((S)-1-cyano-2-(2-fluoro-8-oxo-6,8,9,10-tetrahydroindeno[5,6-c]benzopyran-3-yl)ethyl)carbamoyl)-1,4-oxazepane-4-carboxylic acid butyl ester (22-t)

At room temperature, add Burgess reagent (100 mg, 0.42 mmol) to a dichloromethane solution (4 mL) of 2-s (24 mg, 0.042 mmol). After stirring for 3 hours, add Burgess reagent (100 mg, 0.42 mmol) and continue stirring for 2 hours. Concentrate and purify by TLC to obtain a brown oily product 22-t (20 mg, 86%). MS (ESI): m/z = 572.3 [M + Na] ⁺ .

19) (S)-N-((S)-1-Cyano-2-(2-fluoro-8-oxo-6,8,9,10-tetrahydroindeno[5,6-c]benzopyran-3-yl)ethyl)-1,4-oxazepane-2-carboxamide hydrochloride (22)

At room temperature, add 4N HCl in dioxane (0.75 mL) to a solution of 22-t (19 mg, 0.0346 mmol) in CH ₃ CN (3 mL). After stirring for 15 minutes, add ether Et ₂ O (40 mL). Centrifuge (3200 r/m) for 2 minutes, remove the solvent, wash the solid with Et ₂ O (40 mL), and centrifuge again (3200 r/m) for 2 minutes to obtain a white solid product 22 (hydrochloride, 7.7 mg, yield 45%).

MS (ESI): m/z = 450.2 [M + H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ):δ9.14-9.10(m,1H),9.04(brs,2H),8.08(s,1H),7.90(d,J＝10.4Hz,1H),7.59(s,1H),7.07-7.04(m,1H),5.19(s,2H),5.10-5.03(m,1H),4.47(dd,J＝3.2,10.4Hz,1H),3.98-3.88(m,1H),3.81-3.74(m,1H),3.53-3.48(m,1H),3.29-3.23(m,2H),3.19-3.07(m,4H),3.00-2.94(m,1H),2.69-2.65(m,2H),2.04-1.98(m,2H).

According to the above method, the following compounds were synthesized

Example 6: Synthesis of Compound 23:

1) 3-(3-bromo-4-methoxyphenyl)propanoic acid (23-b)

At 10°C, add liquid bromine (3.2 ml, 61.04 mmol) dropwise to a solution of 3-(4-methoxyphenyl)propionic acid (10.0 g, 55.49 mmol) in acetic acid (50 mL). After stirring at room temperature for 30 minutes, add water (200 mL). Extract with ethyl acetate (200 mL x 3), wash with water (150 mL) and saturated brine (150 mL) in turn, dry with Na ₂ SO ₄ , filter, and concentrate to obtain a crude product. Further wash with EA:PE=1:2 (100 ml) to obtain a white solid product 23-b (yield 13.9 g, 97%). MS (ESI): m/z=281.1[M+Na] ⁺ .

2) 5-Bromo-6-methoxy-2,3-dihydro-1H-inden-1-one (23-c)

At room temperature, SOCl ₂ (30 mL) was added to 23-b (13.9 g, 53.7 mmol) and refluxed for 1 hour. Cooled to room temperature, concentrated, and then CH ₂ Cl (150 mL) was added. Cooled to 0°C. _{AlCl 3} ₍ 7.9 g, 59.2 mmol) was added in batches within 10 minutes. Warmed to room temperature, stirred for 1 hour, and the reaction solution was poured into ice water (200 mL). Extracted with dichloromethane (150 mL x 2), dried with Na ₂ SO ₄ , filtered, concentrated, and purified by column chromatography to obtain a white solid 23-c (6.23 g, 48%). MS (ESI): m/z = 243.1 [M + H] ⁺ .

3) 6-Methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-1-one (23-d)

Under argon protection at room temperature, 23-c (6.2 g, 25.7 mmol), Pin ₂ B ₂ (7.9 g, 31.1 mmol), PdCl ₂ dppf (1 g, 1.37 mmol), KOAc (5 g, 51 mmol) were added to a flask containing dioxane (200 mL) in sequence. The mixture was heated to 100°C and stirred for 3 hours. The mixture was cooled to room temperature, the solid was filtered off, and the filtrate was concentrated. SGC purification (0-25% EA in PE, RF = 0.3 in PE/EA 4/1) gave a brown solid 23-d (7.2 g, 97%). MS (ESI): m/z = 289.3 [M+H] ⁺ .

4) 5-(4-bromo-5-fluoro-2-(hydroxymethyl)phenyl)-6-methoxy-2,3-dihydro-1H-inden-1-one (23-e)

A mixture of 22-d (7.2 g, 25 mmol), (5-bromo-4-fluoro-2-iodophenyl)methanol (9.2 g, 27.8 mmol), PdCl ₂ dppf (1 g, 1.4 mmol) and K ₃ PO ₄ (16 g, 75 mmol) in dioxane (200 mL)/H ₂ O (20 mL) was stirred at 50° C. under argon protection. 12 hours. Cool to room temperature, add EA (500 mL), wash with water. Dry over Na ₂ SO ₄ , filter, concentrate, and purify by SGC (5-30% EA in PE, Rf=0.6 in EA/PE 1/1, weak UV 254 nm) to obtain white solid 23-e (6.2 g, 67%). MS (ESI): m/z=365.1 [M+H] ⁺ .

5) 5-(4-bromo-5-fluoro-2-(hydroxymethyl)phenyl)-6-hydroxy-2,3-dihydro-1H-indene-1-one (23-f)

At room temperature, BF ₃ CS ₂ (11 mL, 104.5 mmol) was added dropwise to a dichloromethane (100 mL) solution of 23-e (6.2 g, 17 mmol). After 2 hours of reaction, water (50 mL) was added, dichloromethane was extracted (100 mL x 2), saturated NaHCO ₃ (100 mL x 2) was washed dropwise, Na ₂ SO ₄ was dried, filtered, concentrated, and purified by SGC (0-60% EA in PE, Rf = 0.4 in PE/EA 1/1) to obtain a solid product 23-f (3.9 g, 65%). MS (ESI): m/z = 353.1 [M+H] ⁺ .

6) 3-Bromo-2-fluoro-9,10-dihydrobenzo[c]cyclopenta[g]benzopyran-8(5H)-one (23-g)

At room temperature, PPh ₃ (3.2 g, 12.2 mmol) and DIAD (2.6 mL, 13.1 mmol) were added to a solution of 23-f (3.88 g, 11 mmol) in THF (100 mL) in sequence. After stirring for 30 minutes, the mixture was concentrated and then washed with ethyl acetate to obtain a light yellow solid product 23-g (2 g, 54%). MS (ESI): m/z = 333.1 [M+H] ⁺ .

7) Methyl (S)-2-((tert-butyloxycarbonyl)amino)-3-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopenta[g]benzopyran-3-yl)propanoate (23-h)

At room temperature, TMSCl (2.7 mL, 21.3 mmol) was added dropwise to a suspension of Zn (2.7 g, 41.5 mmol) in DMF (50 mL). After stirring for 1.5 hours, I ₂ (370 mg, 1.46 mmol) was added and stirred for another 15 minutes. A solution of (R)-2-((tert-butyloxycarbonyl)amino)-3-iodopropionic acid methyl ester (2.8 g, 8.5 mmol) in DMF (10 mL) was added dropwise over 20 minutes. After stirring for 20 minutes, a solution of 23-g (0.7 g, 2.1 mmol) in DMF (20 mL) was added dropwise. Pd ₂ dba ₃ (576 mg, 0.63 mmol) and S-Phos (517 mg, 1.26 mmol) were added in sequence. The reaction solution was stirred at 50°C overnight. Cool to room temperature, add ethyl acetate (500 mL), filter out the solid, and wash the filtrate with water (150 mL x 3) and saturated sodium chloride solution (150 mL x 2) in turn. Dry with Na ₂ SO ₄ , filter, After concentration, SGC purification gave a light brown solid 23-h (0.48 g, purity: 65%, yield 32%). MS (ESI): m/z = 456.3 [M+H] ⁺ .

8)(S)-2-amino-3-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopentane[g]benzopyran-3-yl)propanoic acid methyl ester hydrochloride (23-i)

At room temperature, 4N HCl dioxane solution (4 mL) was added dropwise to 23-h (0.47 g, purity: 65%, 0.67 mmol) in CH ₃ CN (16 mL). After stirring for 30 minutes, ether Et ₂ O (80 mL) was added. Centrifugation (3200 r/m) for 2 minutes was performed, and the upper liquid was removed to obtain a solid product 23-i (400 mg). MS (ESI): m/z = 356.0 [M+H] ⁺ .

9) (S)-2-(((S)-3-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopentane[g]benzopyran-3-yl)-1-methoxy-1-oxopropan-2-yl)carbamoyl)-1,4-oxazepane-4-carboxylic acid butyl ester (23-j)

At room temperature, DIPEA was added to a suspension of 23-i (crude product 400 mg, 0.67 mmol) in DCM (30 mL) to pH 7-8. Then, (S)-4-(tert-butyloxycarbonyl)-1,4-oxazepane-2-carboxylic acid (197 mg, 0.8 mmol), HBTU (303 mg, 0.8 mmol), HOBt (108 mg, 0.8 mmol) and DIPEA (0.4 mL, 2.4 mmol) were added in sequence. The mixture was stirred at room temperature for 1 hour and concentrated. The white solid product 23-j (260 mg, 2-step yield 66%) was purified by reverse phase column (C18, CH ₃ CN, NH ₄ HCO ₃ aqueous solution). MS (ESI): m/z=527.0[M-55] ⁺ .

10) (S)-2-(((S)-1-amino-3-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopentane[g]benzopyran-3-yl)-1-oxopropan-2-yl)carbamoyl)-1,4-oxazepane-4-carboxylic acid butyl ester (23-k)

At room temperature, 25% aqueous ammonia (30 mL) was added to a solution of YD02-214-i (250 mg, 0.43 mmol) in CH ₃ CN (20 mL). After stirring at room temperature for 1 hour, the mixture was concentrated, brine (30 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 3), dried over Na ₂ SO ₄ , filtered, and concentrated to give a light yellow solid 23-k (300 mg). The crude product was not further purified and was directly used in the next step. MS (ESI): m/z＝590.0[M+Na] ⁺ .

11) (S)-2-(((S)-1-cyano-2-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopentane[g]benzopyran-3-yl)ethyl)carbamoyl)-1,4-oxazepane-4-carboxylic acid butyl ester (23-1)

At room temperature, Burgess reagent (615 mg, 2.58 mmol) was added to a dichloromethane solution (2 mL) of 23-k (crude product 300 mg, 0.43 mmol). After stirring for 2 hours, Burgess reagent (300 mg, 1.26 mmol) was added. After stirring for 30 minutes, the mixture was concentrated and purified by SGC (0-60% EA in PE, Rf = 0.5 in PE/EA 1/2) to obtain a solid product 23l (190 mg, two-step yield 80%). MS (ESI): m/z = 572.4 [M + Na] ⁺ .

12) (S)-N-((S)-1-cyano-2-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopentane[g]benzopyran-3-yl)ethyl)-1,4-oxazepane-2-carboxamide (23)

At room temperature, 4N HCl dioxane solution (2.5 mL) was added to a solution of 23-1 (190 mg, 0.346 mmol) in CH ₃ CN (10 mL). After stirring for 30 minutes, ether (40 mL) was added, centrifuged (3200 r/m) for 2 minutes, the upper solvent was removed, ethyl acetate (100 mL) was added, and the mixture was neutralized to pH 7 with saturated NaHCO ₃ . Further ethyl acetate extraction (50 mL*2) was performed, the organic phases were combined, washed with brine (50 mL), dried over Na ₂ SO ₄ , filtered, and concentrated. Purification by preparative HPLC (NH ₄ HCO ₃ buffer: A: 10 mM NH ₄ HCO ₃ aqueous solution; B: acetonitrile; Column: Waters XBridge Peptide BEH C18, 19×250 mm, 10 μm, ) to give a white solid mixture 23 (45 mg, 28%).

MS (ESI): m/z = 450.3 [M + H] ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ):δ8.74(d,J＝8.8Hz,1H),8.14(s,1H),7.89-7.85(m,2H),7.35-7.31(m,1H),7.14(s,1H),5.15-5.03(m,3H),3.99(dd,J＝4.0,8.0Hz,1H),3.88-3.81(m,1H),3.75-3.67(m,1H),3.30-3.28(m,1H),3.21-3.13(m,1H),3.10-3.00(m,3H),2.82-2.71(m,1H),2.68-2.65(m,2H),2.63-2.53(m,2H),1.78-1.67(m,2H)

According to the above method, the following compounds were synthesized

Example 7: Activity Test

A. U937 cell inhibition of DPP1 activity test

The intracellular enzyme activity assay was performed in a 384-well plate, with cell culture medium: 1640, 10% FBS, 1*PS. 30 μL of cell culture medium cell suspension containing U937 cells was added to the 384-well plate, so that each well contained 2×10 ⁴ cells; 30 nL of AZD7986, vehicle control (100% DMSO) or serial dilutions of the test compound were added to the wells by Echo, and after incubation at 37°C for 1 hour, h-Gly-phe-AFC (10 μL) was added to each well and the reaction was started, and further incubated at 37°C for 1 hour, and then the fluorescence absorption was read by EXλ400nm and EMλ505nm. The above test results were calculated using Graphpad 8.0 for IC ₅₀ values, and the results are listed in Table 1.

B. Inhibition effect test of DPP1 enzyme activity

Experimental Materials:

Recombinant human cathepsin rhCathepsin C/DPP1, purchased from R&D systems

Recombinant human cathepsin rhCathepsin L, purchased from R&D systems

AZD7986, purchased from MCE

Gly-Arg-AMC (hydrochloride) was purchased from Cayman Chemical

DMSO was purchased from Sigma-Aldrich

experimental method:

1) Compound preparation and treatment: Weigh the exact amount of compound and dissolve it in 10 mL of DMSO to prepare 10 mL of stock solution, which is further diluted as required.

2) Screening test: Dilute recombinant human cathepsin rhCathepsin C/DPP1 and recombinant human cathepsin rhCathepsin L with activation buffer solution and incubate at 37°C for another 60 minutes. Add 4uL of test compound to the white microwells of the plate containing 384 wells. Take 4uL of enzyme mixture after incubation and add it to the white microwells. Block the 384-well plate and place it at room temperature for 30 minutes, add 8uL of 2-fold diluted Gly-Arg-AMC (hydrochloride). Block the 384-well plate and place it at room temperature for 2 hours. Prepare 4x stop solution and add 4uL stop solution to the white microwells. Read the fluorescence data on the fluorimeter Victor Nivo35.

3) Data analysis:

All _IC50 values were converted to inhibition percentage using Prism Graphpad 8.0. The results are listed in Table 1:

Table 1: In vitro activities of compounds

*N/A: Not tested

The examples of this patent are provided by way of illustration only and not limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially similar results.

Although the technical solutions of the present invention have been described and listed in detail, it should be understood that it is obvious to those skilled in the art to make modifications to the above embodiments or adopt equivalent alternatives. These modifications or improvements made without departing from the spirit of the present invention are within the scope of protection required by the present invention.

Claims

A compound of formula (I) or a pharmaceutically acceptable salt thereof,

in:

R 1 , R 2 , R 3 , R 4 , R 5 are each independently selected from H, cyano, halogen, C 1 -C 3 alkyl, C 1 -C 3 alkoxy; or R 1 , R 2 , R 4 and R 5 are connected to form a 3-6 membered ring, the ring atoms of the 3-6 membered ring can be arbitrarily substituted by S, O, N, C=O; the 3-6 membered ring is optionally substituted by R 6 ;

X, Y are each independently selected from O, -NH, -NMe, -CH 2 -, -C(O)-;

R6 is optionally selected from H, =O, C1 - C4 alkyl, C1 - C4 deuterated alkyl, C1 - C4 cycloalkyl.
The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from H, cyano, fluorine, C 1 -C 3 alkyl or C 1 -C 3 alkoxy.
The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 4 and R 5 are each independently selected from H and cyano.
The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 , R 2 , and R 3 are each independently selected from H, fluorine, C 1 -C 3 alkyl, and C 1 -C 3 alkoxy.
The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 and R 2 are connected to form a cycloalkyl group.
According to the compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, R 4 and R 5 are connected to form the following structure:

Wherein, R 6 is selected from H, =O, C 1 -C 4 alkyl, C 1 -C 4 deuterated alkyl, C 1 -C 4 cycloalkyl.
According to claim 1, the compound of formula (I) or a pharmaceutically acceptable salt thereof, the compound of formula (I) has the following structure:

wherein R 1 , R 2 , R 3 , R 6 , X and Y are as defined in claim 1 ; and Z is O or S.
According to claim 1, the compound of formula (I) or a pharmaceutically acceptable salt thereof, the compound of formula (I) has the following structure:
Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 in the preparation of a medicament for treating and preventing diseases of cathepsin C and its downstream serine proteases NE, PR3, CaTG, and NSP4.
According to any one of claims 1 to 8, the compound of formula (I) or a pharmaceutically acceptable salt thereof is used in the preparation of a pharmaceutical composition for use in the treatment of patients with respiratory diseases, metabolic diseases, cardiovascular and cerebrovascular diseases, autoimmune diseases, cancers, infectious diseases and other inflammatory-related diseases, such as asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary hypertension, pulmonary arterial hypertension, non-cystic fibrosis, cystic fibrosis, bronchiectasis, bronchitis, pneumonia, emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), sepsis, allergic diseases, immune inflammatory bowel disease, rheumatoid arthritis. The invention relates to a method for treating inflammation, glomerulonephritis, eosinophilic diseases, neutrophilic diseases, ANCA-related inflammation, anti-neutrophil cytoplasmic antibody-related necrotizing crescentic glomerulonephritis, acute brain trauma, acute myocarditis, acute kidney injury, α-1-antitrypsin deficiency (AATD) and related inflammation, liver fibrosis, fatty liver and hepatic steatosis, obesity, insulin resistance, diabetes, pathogenic microbial infection, infectious gastrointestinal inflammatory disease, lung cancer and/or radiation injury syndrome or a patient at risk of the disease.
The use according to claim 9 can be further combined with one or more drugs selected from the following: b-mimetic agents, anticholinergic drugs, corticosteroids, PDE4 inhibitors, LTD4 antagonists, EGFR inhibitors, CRTH2 inhibitors, 5-LO inhibitors, histamine receptor antagonists, CCR9 antagonists and SYK inhibitors, NE inhibitors, MMP9 inhibitors, MMP12 inhibitors.
A pharmaceutical composition comprising a compound of formula (I) as claimed in any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.