Novel peptidyl nitrile compound and application thereof

Abstract

The invention discloses a novel peptidyl nitrile compound and application thereof, and particularly discloses a compound shown in a formula (I) and / or pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the compound and / or pharmaceutically acceptable salt thereof, a method for preparing the compound, and a preparation method of the compound. And the use of the compounds for the treatment of diseases caused by cathepsin C and its downstream serine protease.

Description

A novel peptide-based nitrile compound and its application

[0001] This application claims priority to Chinese Patent Application No. 2022113165806, filed on October 26, 2022, and Chinese Patent Application No. 2023103201603, filed on March 29, 2023. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[0002] The present invention relates to the field of medical technology, in particular to a novel peptide-based nitrile compound and application thereof. Background Art

[0003] 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, but due to their target mechanism, they have potential adverse cardiovascular reactions (Norman P, Expert Opinion on Investigational Drugs. 2014, 23(8):1067–77). Therefore, it is still very necessary to explore new anti-inflammatory drugs.

[0004] Cathepsin C (CTSC), also known as dipeptidyl peptidase I (DPP-1), is a lysosomal cysteine ​​protease with a molecular weight of 200KDa belonging to the papain family. Cathepsin C acts as a key enzyme in activating neutrophil and mast cell granule serine peptidases (such as four neutrophil proteases, elastase (NE), cathepsin G (CatG), proteinase 3 (PR3) and neutrophil serine protease (NSP4), as well as mast cell-associated 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 these proteases are activated by cathepsin C, they are able to degrade various extracellular matrix components, which leads to tissue damage and chronic inflammation. Therefore, cathepsin C inhibitors may 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).

[0005] Other immune diseases such as inflammatory bowel disease, rheumatoid arthritis, antineutrophil 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), and acute pancreatitis, a large part of their pathogenesis is caused by the increased activity of some of these inflammatory proteases. Therefore, cathepsin C inhibitors can also be used as potential drugs for the treatment of these diseases. In addition, the serine protease elastase downstream of cathepsin plays a very important role in the occurrence and metastasis of cancer and 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 to treat cancer diseases and cardiovascular and cerebrovascular diseases. (Pharmacol Ther. 2018 Oct; 190: 202-236; Int J Mol Sci. 2021 Jan 13; 22(2): 722.).

[0006] Cathepsin C has a history of over 70 years since its discovery, 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 Brensocatib, 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 need for cathepsin C inhibitors. Existing compounds are difficult to inhibit the activity of cathepsin C and downstream serine proteases, so there is a need to provide new peptide-based nitrile compounds and their applications.

[0007] Summary of the Invention

[0008] One object of the present invention is to provide a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0009] 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:

[0010] R1, R2, R3, R4, and R5 are each independently selected from H, cyano, halogen, C1-C3 alkyl, and C1-C3 alkoxy; or R1, R2, R4, and R5 are linked to form a 3-6 membered ring, wherein the ring atoms of the 3-6 membered ring may be arbitrarily substituted with 1-2 S, O, N, or C=O; and the 3-6 membered ring may be optionally substituted with R6;

[0011] X, Y are each independently selected from O, -NH, -NMe, -CH2-, -C(O)-;

[0012] R6 is optionally selected from H, =O, C1-C4 alkyl, C1-C4 deuterated alkyl, C1-C4 cycloalkyl.

[0013] In a preferred embodiment of the present invention, R1, R2, R3, R4, and R5 are each independently selected from H, cyano, fluorine, C1-C3 alkyl, or C1-C3 alkoxy.

[0014] In a preferred embodiment of the present invention, R4 and R5 are each independently selected from H and cyano.

[0015] In a preferred embodiment of the present invention, R1, R2, and R3 are each independently selected from H, fluorine, C1-C3 alkyl, and C1-C3 alkoxy.

[0016] In a preferred embodiment of the present invention, R1 and R2 are linked to form a cycloalkyl group.

[0017] In a preferred embodiment of the present invention, R4 and R5 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 R6, and R6 is optionally selected from H, =O, C1-C4 alkyl, C1-C4 deuterated alkyl, and C1-C4 cycloalkyl.

[0018] In a preferred embodiment of the present invention, R4 and R5 are connected to form the following structure:

[0019] Wherein, R6 is selected from H, =O, C1-C4 alkyl, C1-C4 deuterated alkyl, C1-C4 cycloalkyl. Preferably, R6 is selected from H, methyl, ethyl, cyclopropyl, propyl, isopropyl, deuterated methyl.

[0020] In a preferred embodiment of the present invention, when R4 and R5 are connected to form a five-membered ring, the compound of formula (I) has a structure such as formula (II), formula (III), or formula (IV):

[0021] Wherein, Z is O or S; R3 is selected from H, cyano, fluorine, C1-C3 alkyl or C1-C3 alkoxy.

[0022] 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:

[0023] 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.

[0024] Further relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for the treatment of respiratory diseases, metabolic diseases, cardiovascular and cerebrovascular diseases, autoimmune diseases, cancer, 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, nephrotic syndrome, Use of the present invention in a drug for treating glomerulonephritis, eosinophilic diseases, neutrophilic diseases, ANCA-related inflammation, anti-neutrophil cytoplasmic antibody-associated 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 diseases.

[0025] 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.

[0026] 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.

[0027] Furthermore, the pharmaceutical composition also includes use in combination with small molecule compounds and / or large molecule antibodies to treat cancer, inflammation, bone marrow-related diseases and autoimmune diseases, and the small molecule compounds and / or large molecule 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.

[0028] The beneficial effects of the present invention relate to the use of a composition of a 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.

[0029] The compounds of the present invention may be asymmetric, e.g., have one or more stereocenters. Unless otherwise specified, all stereoisomers, e.g., enantiomers and diastereomers, are described. They may contain 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.

[0030] The compounds of the present invention may also include tautomeric forms.Tautomeric forms are generated by the interchange of a single bond and an adjacent double bond accompanied by the migration of a proton.

[0031] 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.

[0032] 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 converting the base moiety present into its salt form, or derivatives of compounds of formula (I) wherein the parent compound is modified by converting the acid moiety present into its salt form.

[0033] 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 compound of formula (I) by reacting the free base form 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).

[0034] The compounds of the present invention and their pharmaceutically acceptable salts 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 can exist in multiple crystal forms or amorphous forms. Generally speaking, all physical forms of the compound are included within the scope of the present invention.

[0035] The present invention also includes prodrugs of the 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 the parent drug. Prodrugs can be prepared by substituting one or more functional groups present in the compound, wherein the substituents in the prodrug are removed in vivo in such a manner that the prodrug is converted to 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 ACS Symposium Series and Bioreversible.

[0036] Definition and Description

[0037] The following terms, phrases and symbols used in the present invention have the meanings described below unless the context indicates otherwise.

[0038] The term "alkyl" as used herein refers to a straight or branched saturated hydrocarbon group containing 1 to 18 carbon atoms, for example, 1 to 12 carbon atoms, further for example, 1 to 6 carbon atoms, and further for example, 1 to 4 carbon atoms. For example, "C1-C6 alkyl" within the scope of "alkyl" refers to an 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").

[0039] As used herein, the term "halo" refers to fluoro, chloro, bromo and iodo, and "halogen" refers to fluorine, chlorine, bromine and iodine.

[0040] The term "cycloalkyl" as used herein refers to a saturated or partially unsaturated cyclic hydrocarbon group containing 3-12 ring carbon atoms, for example 3-8 ring carbon atoms, and further 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" refers to a cycloalkyl group having 3-8 ring carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, and similar groups.

[0041] 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, and their isomers.

[0042] As used herein, "group" and "radical" are synonymous and are used to indicate a functional group or a molecular fragment that can be linked to other molecular fragments.

[0043] If a 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 the R-configuration or the S-configuration. The configuration of the compound can be determined by a person skilled in the art using various analytical techniques, such as single crystal X-ray crystallography and / or optical polarimetry, according to conventional protocols.

[0044] The terms "selective", "selective", "selectively", "optional", "optional" or "optionally" as used herein mean that the subsequently described substitution pattern, event or situation may occur once or more than once, or may not occur, and that the description includes instances where the substitution pattern occurs as well as instances where the substitution pattern does not occur. For example, "optionally substituted alkyl" includes "unsubstituted alkyl" and "substituted alkyl" as defined herein. It will be understood by those skilled in the art that, for any group containing one or more substituents, the group does not include any substitution pattern that is sterically impractical, chemically incorrect, synthetically infeasible and / or inherently unstable.

[0045] As used herein, the term "substituted" or "substituted with" means that one or more hydrogen atoms on a given atom or group are replaced with one or more substituents selected from a given group of substituents, provided that the normal valence of the given atom is not exceeded. When the substituent is oxo (i.e., =0), then two hydrogen atoms on a single atom are replaced with oxygen. Combinations of substituents and / or variables are permissible only if such combinations result in chemically correct and stable compounds. A chemically correct and stable compound means a compound that is sufficiently stable to be isolated from a reaction mixture and to determine the chemical structure of the compound, and subsequently formulated into a formulation that at least has practical utility.

[0046] 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.

[0047] It will be appreciated by those skilled in the art that some compounds of formula (I) may comprise one or more chiral centers, and therefore have two or more stereoisomers. Racemic mixtures of these isomers, single isomers, and a mixture enriched in enantiomers, as well as diastereomers when there are two chiral centers, and mixtures partially enriched in specific diastereomers 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, includes its single tautomers.

[0048] In other words, in some embodiments, the present invention provides compounds having various degrees of stereoisomeric purity, i.e., diastereomeric or enantiomeric purity, as expressed in 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 compounds of formula (I) (e.g., as described herein) have 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 compounds of formula (I) (e.g., as described herein) have a diastereomeric purity of greater than 99.9% de.

[0049] The term "enantiomeric excess" or "ee" indicates the amount of one enantiomer relative to the other. For a mixture of R and S enantiomers, the percent enantiomeric excess is defined as |RS|*100, where R and S are the molar or weight fractions of their respective enantiomers in the mixture, and R+S=1. If the optical rotation of a monochiral substance is known, the percent 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.

[0050] Determination of diastereomers and / or enantiomeric excess can be accomplished using a variety of analytical techniques, including nuclear magnetic resonance spectroscopy, chiral column chromatography, and / or optical polarimetry, according to conventional protocols familiar to those skilled in the art.

[0051] The racemic mixture can be used as is or resolved into its individual isomers. Resolution can yield a stereochemically pure compound or a mixture enriched in one or more isomers. Methods for separating isomers are well known and include physical methods, such as chromatography using chiral adsorbents. Individual isomers can be prepared in chiral form from chiral precursors. Alternatively, the individual isomers can be chemically separated from the mixture by forming diastereomeric salts with chiral acids (e.g., individual enantiomers of 10-camphorsulfonic acid, camphoric acid, α-bromocamphoric acid, tartaric acid, diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid, etc.), fractionally crystallizing the salts, then liberating one or both of the resolved bases, and optionally repeating this process to obtain one or both isomers substantially free of the other isomer, i.e., the desired stereoisomer having an optical purity of, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% by weight. Alternatively, as is well known to those skilled in the art, the racemate can be covalently linked to a chiral compound (auxiliary) to obtain diastereomers, which can be separated by chromatography or fractional crystallization, followed by chemical removal of the chiral auxiliary to obtain the pure enantiomers.

[0052] The term "pharmaceutically acceptable salt" as used herein refers to a salt of a free acid or base of a compound of formula (I) that is non-toxic, biologically tolerable, or other biologically suitable for administration to a subject for treatment.

[0053] "Pharmaceutically acceptable salts" include, but are not limited to, acid addition salts formed with inorganic acids such as hydrochlorides, hydrobromides, carbonates, bicarbonates, phosphates, sulfates, sulfites, nitrates, and the like; and acid addition salts formed 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-(CH2)n-COOH (wherein n is 0-4). "Pharmaceutically acceptable salts" also include base addition salts formed with compounds of formula (I) having an acidic group 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.

[0054] The term "prodrug" as used herein refers to a pharmacological substance (i.e., a drug) derived from a parent drug. Once administered, the prodrug is metabolized in vivo to 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 and converted to 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 ACSSymposium Series and Bioreversible. "Prodrugs" include, but are not limited to, esters of compounds of formula (I), such as phosphates, formates, and carbamates; and amides, such as formamide and acetamide.

[0055] Furthermore, 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 according to conventional procedures for preparing acid addition salts from basic compounds. Those skilled in the art can readily identify various synthetic methods for preparing non-toxic pharmaceutically acceptable acid addition salts without undue experimentation.

[0056] The term "solvate" refers to a solvent addition form containing either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to entrain fixed molar ratios of solvent molecules in the solid state, thereby forming solvates. 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 form of HO. Such a combination can form one or more hydrates, such as hemihydrates, monohydrates, and dihydrates, as well as variable hydrates.

[0057] 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 linked to other molecular fragments.

[0058] The term "active ingredient" is used to refer to a chemical substance with biological activity. In some embodiments, the "active ingredient" is a chemical substance with pharmaceutical use. In the United States, actual drug activity can be determined by appropriate preclinical trials, 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 a higher standard than preclinical trials. Whether such a higher standard of drug activity can be successfully achieved generally cannot be reasonably expected from the results of preclinical trials, but can be established through appropriate and effective randomized, double-blind, controlled clinical trials conducted in humans.

[0059] 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 a patient in need of treatment for a disease or disorder mediated by cathepsin C and downstream serine protease activity. The effective amount or dosage of the active ingredient of 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 pharmaceutical 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 a randomized, double-blind, controlled clinical trial.

[0060] Typical dosage range is from about 0.0001 to about 200 milligrams of active ingredient per kilogram of individual body weight every 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 divided dose unit taking (for example, twice a day, three times a day, four times a day). For a 70 kilogram people, 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 obstacle show improvement, dosage can be adjusted to maintain treatment. For example, according to the variation of symptoms, dosage or administration frequency can be, or dosage and administration frequency can be reduced to the level of maintaining desired therapeutic effect. Of course, if the symptoms are alleviated to appropriate level, treatment can be stopped. However, for the recurrence of symptoms, the patient may need intermittent long-term treatment.

[0061] 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 DESCRIPTION

[0062] 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 by weight and temperatures are in degrees Celsius. Pressure is atmospheric or near atmospheric. All data were obtained by Agilent (Agilent 6120 and / or 1100). Except for synthetic intermediates, all reagents used in the present invention were obtained from commercial sources. All compound names, except for reagents, were generated using ChemDrew 20.0. Abbreviations herein have conventional meanings in the art.

[0063] The meanings of some abbreviations are as follows: Boc tert-butoxycarbonyl (Boc)2O tert-butyl pyrocarbonate BH3 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)2Cl2 [1,1'-Bis(diphenylphosphino)ferrocene]palladium dichloride Pd2(dba)3 Trisdibenzylideneacetone dipalladium Pd(PPh3)4 Tetrakis(triphenylphosphine)palladium PMB p-Methoxybenzylcyanide Pin2B2 Pinacol borate TFA Trifluoroacetic acid (TsOH), 4-toluenesulfonic acid (Xphos), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl, Burgess reagent, methyl N-(triethylammoniumsulfonyl)carbamate

[0064] Example 1: Synthesis of Compound 1

[0065] 1) Preparation of 4-bromo-3-(methoxymethyl)benzonitrile (1-b):

[0066] At 0°C, (1-a, 4 g, 20.2 mmol) was dissolved in dichloromethane (40 mL), followed by the slow dropwise addition of DIPEA (5.47 g, 42.4 mmol) and MOMBr (3.03 g, 24.2 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The mixture was quenched with water (150 mL) and extracted with dichloromethane (2 x 200 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (eluent: PE / DCM = 1:2, v / v) to afford the product (1-b, 4.5 g, 92%) as a white solid. MS (ESI): m / z = 242.1 [M+H] + .

[0067] 2) Preparation of 3-(methoxymethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)benzonitrile (1-c):

[0068] At room temperature, 1-b (5.7 g, 23.6 mmol), Pin2B2 (11.97 g, 47.1 mmol), Pd(dppf)Cl2 (861 mg, 1.2 mmol), and KOAc (6.92 g, 70.7 mmol) were added sequentially to dioxane (100 mL). The mixture was deaerated and stirred at 90°C under argon for 16 hours. The reaction mixture was cooled to room temperature and purified by column chromatography (eluent: PE / EtOAc = 10:1, V / V) to afford the product (1-c, 6.4 g, 92%) as a brown oil. MS (ESI): m / z = 290.3 [M+H] + .

[0069] 3) Preparation of 5-bromo-4-fluoro-2-iodo-benzoic acid (1-e):

[0070] To a solution of 1-d (30 g, 128.2 mmol) in HCl (conc., 106.8 mL, 1282 mmol) and H₂O (220 mL) at 0°C, a solution of NaNO₂ (10.6 g, 153.8 mmol) in water (60 mL) was slowly added dropwise over 30 minutes. Stirring was continued at 0°C for 30 minutes, followed by a solution of KI (31.9 g, 32.1 mmol) in water (60 mL) added dropwise over 20 minutes. Stirring was continued at 0°C for 1 hour, then heated to 90°C and stirred for another 30 minutes. Cooled to room temperature, saturated aqueous Na₂S₂O₃ (150 mL) was added. The reaction mixture was extracted with EtOAc (2 x 500 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by column chromatography to afford the product (1-e, 40 g, 91%) as a yellow solid. MS (ESI): m / z = 345.1 [M+H] + .

[0071] 4) Preparation of 5-bromo-4-fluoro-2-iodo-benzyl alcohol (1-f):

[0072] To a solution of 1-e (3 g, 12.2 mmol) in THF (20 mL) at 0°C, borane / THF (13.5 mL, 13.5 mmol, 1 mol / L) was slowly added dropwise over 15-20 minutes, followed by stirring at 65°C for 6 hours. Excess boron was carefully quenched with THF / H₂O (1:1) solution, followed by the addition of saturated K₂CO₃ solution. The reaction mixture was extracted with ethyl acetate (EtOAc) (2 x 200 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by column chromatography (eluent: PE / EtOAc = 4:1, v / v) to afford the product (1-f, 2.6 g, 92%) as a white solid. MS (ESI): m / z = 312.9 [M-17] + . 1 H NMR (400MHz, CDCl3): δ7.66 (d, J=6.8Hz, 1H); 7.59-7.57 (m, 1H); 4.62 (s, 2H); 2.24 (s, 1H).

[0073] 5) Preparation of 4'-bromo-5'-fluoro-2'-(hydroxymethyl)-2-(methoxymethyl)-[1,1'-biphenyl]-4-carbonitrile (1-g):

[0074] To a solution of dioxane (100 mL) and water (10 mL) at room temperature were added 1-c (5.1 g, 17.6 mmol), 1f (7.01 g, 21.2 mmol), Pd(PPh3)4 (2.04 g, 1.8 mmol), and K2CO3 (4.87 g, 35.3 mmol), in that order. Air was removed and the mixture was stirred at 60°C under argon for 48 hours. The reaction mixture was cooled to room temperature, concentrated, and purified by column chromatography (eluent: PE / EtOAc = 4:1, V / V) to give the product (1-g, 3.4 g, 53%) as a white solid. MS (ESI): m / z = 388.0 [M+Na] + .

[0075] 6) Preparation of 4'-bromo-5'-fluoro-2-hydroxy-2'-(hydroxymethyl)-[1,1'-biphenyl]-4-carbonitrile (1-h):

[0076] 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 mixture was stirred at 100°C under nitrogen for 48 hours. After cooling to room temperature, it was concentrated and purified by column chromatography (eluent: PE / EtOAc = 2:3, V / V) to give a brown solid (1-h, 2.4 g, 80%). MS (ESI): m / z = 322.0 [M+H] + .

[0077] 7) Preparation of 8-bromo-9-fluoro-6H-benzo[c]chromene-3-carbonitrile (1-i):

[0078] To a solution of 1-h (2.6 g, 3.19 mmol) and PPh3 (3.19 g, 12.1 mmol) in THF (10 mL) was added dropwise at 0°C. After 2 hours, the reaction mixture was concentrated and water (100 mL) was added. The mixture was extracted with EtOAc (2 x 200 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated, and recrystallized (50% PE in DCM) to afford the product (1-i, 2.1 g, 86%) as a yellow solid. MS (ESI): m / z = 304.1 [M+H] + .

[0079] 8) Preparation of methyl (S)-2-((tert-butyloxycarbonyl)amino)-3-(3-cyano-9-fluoro-6H-benzo[c]chromon-8-yl)propionate (1-j):

[0080] At room temperature, trimethylsilyl chloride (2.57 g, 23.7 mmol) was slowly added dropwise to a solution of zinc powder (3.59 g, 55.3 mmol) in DMF (40 mL). After stirring for 1.5 hours, iodine (468 mg, 1.8 mmol) was added, and stirring continued for 15 minutes. Then, a solution of methyl(R)-2-((tert-butoxycarbonyl)amino)-3-iodopropanoate ((R)-2-((tert-butoxycarbonyl)amino)-3-iodopropanoate, 2.6 g, 7.9 mmol) in DMF (8 mL) was slowly added dropwise over 20 minutes. After stirring for 20 minutes, the reaction mixture was cooled to room temperature, and a solution of 1-i (800 mg, 2.6 mmol) in DMF (25 mL) was added dropwise over 20 minutes. Subsequently, Pd2(dba)3 (722 mg, 0.79 mmol) and S-Phos (647 mg, 1.58 mmol) were added sequentially. 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) and extracted with EtOAc (3 x 200 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (eluent: 25% ethyl acetate in hexane) to afford the product (1-j, 850 mg, 76%) as a yellow solid. MS (ESI): m / z = 449.2 [M+Na] + . 1 H NMR (400MHz, DMSO-d6): 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).

[0081] 9) Preparation of tert-butyl (S)-(1-amino-3-(3-cyano-9-fluoro-6H-benzo[c]chromon-8-yl)-1-oxopropane-2-yl)carbamate (1-k):

[0082] 1-j (300 mg, 0.7 mmol) was added to NH3 / MeOH (25 mL, 7 mol / L) at room temperature and stirred overnight. The solution was concentrated and purified by column chromatography to give the product as a white solid (1-k, 289 mg, 86%). MS (ESI): m / z = 356.1 [M-55] + .

[0083] 10) Preparation of (S)-2-amino-3-(3-cyano-9-fluoro-6H-benzo[c]chromon-8-yl)propionamide TFA salt (1-1):

[0084] To a solution of 1-k (289 mg, 0.7 mmol) in DCM (20 mL) was added dropwise TFA (2 mL) at room temperature and stirred for 2 hours. Concentration afforded the product as a brown oil (1-1, 300 mg, 99%). MS (ESI): m / z = 312.3 [M+H] + .

[0085] 11) Preparation of (S)-tert-butyl 2-((S)-1-amino-3-(3-cyano-9-fluoro-6H-benzo[c]chromon-8-yl)-1-oxopropane-2-yl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylate (1-m):

[0086] To a solution of 1-1 (300 mg, 0.71 mmol) and (S)-4-(tert-butoxycarbonyl)-1,4-oxazepane-2-carboxylic acid ((S)-4-(tert-butoxycarbonyl)-1,4-oxazepane-2-carboxylic acid, 173 mg, 0.71 mmol) in DCM (10 mL) was added DIPEA (546 mg, 4.23 mmol), HOBt (114 mg, 0.85 mmol), and HBTU (321 mg, 0.85 mmol) in sequence at room temperature. After stirring for 4 hours, the reaction mixture was concentrated and purified by reverse phase column chromatography (C18, CH3CN, 10 mM NH4HCO3 in water) to afford the product (1-m, 300 mg, 79%) as a white solid. MS (ESI): m / z = 561.3 [M+Na] + .

[0087] 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):

[0088] To a solution of 1-m (295 mg, 0.55 mmol) in DCM (10 mL) was added Burgess reagent (652 mg, 2.74 mmol) at room temperature and stirring was continued for 3 hours. The reaction solution was concentrated and purified by HPLC (NH4HCO3 buffer: A: 10 mM NH4HCO3 aqueous solution; B: acetonitrile; Column: Waters XBridge Peptide BEH C18, 19×250 mm, 10 μm, ) to give a white solid product (1n, 251 mg, 88%). MS (ESI): m / z = 543.3 [M+Na] + .

[0089] 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):

[0090] To a solution of 1-n (246 mg, 0.47 mmol) in DCM (10 mL) was added dropwise TFA (1 mL) at room temperature and stirring continued for 1 hour. Saturated NaHCO₃ (150 mL) was added, and the mixture was extracted with DCM (3 × 150 mL). The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by HPLC (A: 0.8% aqueous NH₄HCO₃, B: CH₃CN; Column: Xbridge BEH peptide C18, 19 mm × 250 mm) to afford the product (1, 130 mg, 65%) as a white solid. MS (ESI): m / z = 421.2 [M+H] + .

[0091] 1 H NMR (400MHz, DMSO-d6): 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=7.2Hz,1H);5.21-5.14(m,2H);5.08-5.02(m,1H) ;4.01-3.98(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).

[0092] Referring to the above method, the following compounds were synthesized

[0093] Example 2: Synthesis of Compound 2

[0094] 1) Preparation of 4-bromo-5-methyl-2-nitrophenol:

[0095] To a solution of 5-methyl-2-nitrophenol (2-a, 40 g, 0.26 mol) in acetic acid (400 mL) was added a solution of bromine (27 mL) in acetic acid (70 mL) dropwise over 30 minutes at 0°C under nitrogen. 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 extracted with ethyl acetate. The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 10:1, v / v) to afford the product (2-b, 55 g, 91.4%) as a yellow solid.

[0096] 2) Preparation of 2-amino-4-bromo-5-methylphenol (2-c):

[0097] To a solution of 2-b (55 g, 0.24 mol) in methanol (825 mL) at room temperature were added SnCl₂ (214.95 g, 0.95 mol) and hydrochloric acid (138.91 mL, 1.67 mol) in sequence. 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, then adjusted to pH 14 with aqueous ammonia. The solid was filtered off, and the organic phase was washed with saturated NaCl solution, dried over anhydrous Na₂SO₄, filtered, and concentrated to yield the product (2-c, 43.5 g, 90.1%) as a yellow solid.

[0098] 3) Preparation of 5-bromo-6-methylbenzo[d]oxazol-2(3H)-one (2-d):

[0099] To a solution of 2-c (43.5 g, 0.22 mol) in THF (450 mL) was added CDI (42.11 g, 0.22 mol) at room temperature. The reaction mixture was stirred at 80°C for 2 hours. After cooling to room temperature, the mixture was washed with water and extracted with ethyl acetate. The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 3:1, v / v) to give the product (2-d, 37 g, 91.36%) as a yellow solid.

[0100] 4) Preparation of 5-bromo-3,6-dimethylbenzo[d]oxazol-2(3H)-one (2-e):

[0101] To a solution of 2-d (37 g, 0.16 mol) in DMF (260 mL) at room temperature were added K2CO3 (33.79 g, 0.24 mol) and iodomethane (34.71 g, 0.24 mol) in sequence. After 16 hours of reaction at room temperature, the reaction solution was poured into water, filtered, washed with water, and air-dried for one day to afford the product (2-e, 21.5 g, 54.74%) as a yellow solid. 1H NMR (400 MHz, DMSO) δ 7.53 (s, 1H), 7.35 (s, 1H), 3.30 (s, 3H), 2.34 (s, 3H).

[0102] 5) Preparation of 5-bromo-6-(bromomethyl)-3-methylbenzo[d]oxazol-2(3H)-one (2-f):

[0103] To a solution of 2-d (5.0 g, 20.6 mmol, 1.0 eq.) in dichloroethane (50 mL) at room temperature were added AIBN (164 mg, 1 mmol, 0.05 eq.) and NBS (4.04 g, 22.7 mmol, 1.1 eq.) in sequence. The reaction mixture was stirred at 80°C for 2 hours. The mixture was cooled to room temperature and diluted with water (100 mL). Extraction was performed with ethyl acetate (3 x 50 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (PE / DCM: 20 / 1) to afford the product (2-f, 5.3 g, 80%) as a yellow solid. MS (ESI): m / z = 322.0 [M+H] + .

[0104] 6) Preparation of 5-bromo-6-(hydroxymethyl)-3-methylbenzo[d]oxazol-2(3H)-one (2-g):

[0105] To 2-f (5.3 g, 16.5 mmol) was added acetone / H₂O (200 mL, 1:1) and Na₂CO₃ (2.6 g, 24.8 mmol, 1.5 eq.) at room temperature. After reflux for 5 hours, the reaction mixture 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 (2-g, 3.8 g, 89%). MS (ESI): m / z = 280.0 [M+H] + .

[0106] 7) Preparation of 4-bromo-2-fluoro-5-hydroxybenzoic acid (2-i):

[0107] To a solution of 2h (2.0 g, 12.8 mmol, 1.0 eq.) in CHCl₃ (15 mL) / acetic acid (15 mL) at 0°C under nitrogen was added dropwise bromine (2 mL, 38.4 mmol, 3.0 eq.). After stirring at room temperature for 20 hours, the reaction was quenched with saturated Na₂SO₃ (100 mL) and extracted with ethyl acetate (3 x 150 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated to afford the crude product. The product (2-i, 2.1 g, 70% yield) was obtained by slurrying with n-hexane. MS (ESI): m / z = 235.0 [M+H] + .

[0108] 8) Preparation of methoxymethyl 4-bromo-2-fluoro-5-(methoxymethyl)benzoate (2-j):

[0109] To a solution of 2-I (660 mg, 2.8 mmol, 1.0 eq.) in DCM / DMF (25 mL, 4:1) at 0°C under nitrogen was added MOMBr (685 μL, 8.4 mmol, 3.0 eq.) and DIPEA (1.95 mL, 11.2 mmol, 4.0 eq.) dropwise in sequence. The mixture was stirred at room temperature overnight, concentrated under reduced pressure, and saturated NH4Cl solution was added. The mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (PE / EA:9 / 1) to afford the oily product 2-j (820 mg, 91% yield). MS (ESI): m / z = 263.0 [M-MOMO + H] + .

[0110] Preparation of (9) (4-bromo-2-fluoro-5-(methoxymethyl)phenyl)methanol (2-k):

[0111] To a solution of 2-j (50 mg, 0.15 mmol, 1.0 eq.) in THF / H₂O (6 mL, 1:1) was added LiOH·H₂O (12.6 mg, 0.30 mmol, 2.0 eq.) at room temperature and stirred for 2 hours. The reaction mixture was adjusted to pH <3 with 1N HCl. Extraction was performed with ethyl acetate (3 x 5 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated to afford the intermediate (42 mg, 99%). This intermediate was dissolved in anhydrous THF (5 mL) at 0°C under nitrogen, followed by the dropwise addition of BH₃·THF (1 M in THF, 2.15 mL). After 2 hours at room temperature, the reaction mixture was quenched with alcohol and concentrated to afford the oily product 2-k (40 mg, 78% purity). MS (ESI): m / z = 249.1 [M-OH] + .

[0112] 10) Preparation of 1-bromo-4-(chloromethyl)-5-fluoro-2-(methoxymethyl)benzene (2-1):

[0113] To a solution of 2-k (1.7 g, 6.4 mmol, 1.0 eq.) in DCM (50 mL) at room temperature, SOCl2 (558 μL, 7.7 mmol, 1.2 eq.) and DMF (10 μL) were added dropwise, and stirring continued for 2 hours. The reaction mixture 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 NH4Cl aq. (50 mL) was added. The mixture was extracted with DCM (30 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (PE / EA (9 / 1)) to afford 2-l (1.72 g, 95%) as a white solid.

[0114] 11) Preparation of (2S, 5R)-2-(4-bromo-2-fluoro-5-(methoxymethyl)benzyl)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (2-n):

[0115] To a solution of (R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (2-m, 1.66 g, 9.0 mmol) in THF (50 mL) at -78°C under argon was added dropwise 2.5M n-buthyl lithium in hexane (3.6 mL containing 2.5M n-butyl lithium in hexane). The reaction mixture was stirred at -78°C for 2 hours, after which 2-l (1.7 g, 6.0 mmol) was added all at once. The reaction mixture was allowed to slowly warm to room temperature and stirred overnight. Saturated NH4Cl (30 mL) was added, and the mixture was extracted with ethyl acetate (3 x 50 mL). The organic phases were combined, dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (PE / EA: 10 / 1, v / v) to afford 2-n as an oil (2.4 g, 93%). MS (ESI): m / z = 433.1 [M+H] + .

[0116] Preparation of (12) (5-fluoro-4-(((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)methyl)-2-(methoxymethyl)phenyl)boronic acid:

[0117] To a solution of 2-n (1.2 g, 2.8 mmol, 1.0 eq.) in THF (30 mL) at -78°C under argon was added dropwise 2.5 M n-buthyl lithium in hexane (1.34 mL of 2.5 M n-butyl lithium in hexane). The reaction mixture was stirred at -78°C for 30 minutes before the addition of B(OiPr)3 (1.3 mL, 5.6 mmol, 2.0 eq.). The reaction mixture was allowed to slowly warm to room temperature and stirred overnight. Saturated NH4Cl (30 mL) was added, and the mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography (eluent: DCM / MeOH = 95 / 5, v / v) to afford the product 2-o as an oil (960 mg, 86%). MS (ESI): m / z = 397.2 [M+H] + .

[0118] 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):

[0119] To dioxane / H₂O (22 mL, 10:1) were added 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 K₃PO₄ (849 mg, 4.0 mmol, 2.0 eq.) at room temperature. The reaction mixture was stirred at 80°C under argon for 2 hours, cooled to room temperature, concentrated, and purified on a reverse phase column using NH₄HCO₃ buffer to afford the yellow foam product 2-p (680 mg, 66%). MS (ESI): m / z = 530.2 [M+H] + .

[0120] 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):

[0121] To a solution of 2-p (430 mg, 0.81 mmol, 1.0 eq.) in 20 mL of MeCN / H2O (3 / 7) was added 2 mL of concentrated hydrochloric acid at room temperature. The mixture was stirred overnight at room temperature and directly purified on a C18 column to afford the product 2-q (387 mg) as a white foam. MS (ESI): m / z = 391.0 [M+H]+ .

[0122] 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):

[0123] To a solution of 2-p (430 mg, 0.81 mmol, 1.0 eq.) in 20 mL of MeCN / H2O (3 / 7) was added 2 mL of concentrated hydrochloric acid at room temperature. The mixture was stirred overnight at room temperature and directly purified on a C18 column to afford the white foam product 2-q (387 mg). MS (ESI): m / z = 391.0 [M+H] + .

[0124] 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-oxopropan-2-yl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylate (2-r):

[0125] To a solution of (S)-4-(tert-butoxycarbonyl)-1,4-oxazepane-2-carboxylic acid ((S)-4-(tert-butoxycarbonyl)-1,4-oxazepane-2-carboxylic acid, 490 mg, 2.0 mmol, 2.0 eq.) in DMF (10 mL) at room temperature were added 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.). After stirring at room temperature for 2 hours, the reaction mixture was directly purified using a reverse phase column to afford the product 2-r (324 mg, 52%) as a white foam. MS (ESI): m / z = 640.3 [M+Na] + .

[0126] 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-oxopropan-2-yl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylate (2-s):

[0127] To a solution of 2-r (324 mg, 0.52 mmol, 1.0 eq.) in THF (30 mL) at 0°C under argon was added PPh3 (204 mg, 0.78 mmol, 1.5 eq.) and DIAD (154 μL, 0.78 mmol, 1.5 eq.) in sequence. Stir at room temperature overnight, concentrate, and purify with a reverse-phase column to afford the product 2-s (280 mg, 90%) as a white solid. MS (ESI): m / z = 544.3 [M-55] + .

[0128] 18) Preparation of tert-butyl (S)-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-oxopropan-2-yl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylate (2-t):

[0129] At room temperature, 2-s (280 mg, 0.47 mmol) was added to 30 mL of a 25-28% NH3aq. / MeCN (1:2) solution. The mixture was stirred overnight and lyophilized to obtain the crude product. Purification by reverse phase column gave the product as a white solid (2-t, 110 mg, 40%). MS (ESI): m / z = 529.0 [M-55] + .

[0130] 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:

[0131] To a solution of 2-t (249 mg, 0.42 mmol, 1.0 eq.) in DCM (10 mL) was added Burgess reagent (304 mg, 1.28 mmol, 3.0 eq.) at room temperature. The mixture was stirred at room temperature for 3 hours, diluted with DCM (20 mL), and washed with water (20 mL) and brine (5 mL x 2). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated. Purification by reverse phase column afforded the product (2-u, 155 mg, 64%) as a white solid. MS (ESI): m / z = 589.2 [M+Na] + .

[0132] 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):

[0133] At room temperature, 2-u (155 mg, 0.27 mmol, 1.0 eq.) was dissolved in TFA / DCM (1:10, 5.5 mL). 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] + .

[0134] 1 H NMR (400MHz, DMSO) δ8.76(d,J=8.5Hz,1H),7.83(s,1H),7.81(d,J=10.8Hz,1H),7.31(s,1H),6.9 8(d,J=6.5Hz,1H),5.10(s,2H),5.05(dd,J=15.8,8.6Hz,1H),4.06(dd,J=8.3,3.5Hz,1H),3.91– 3.82(m,1H),3.74(ddd,J=12.0,7.4,4.2Hz,1H),3.39(s,3H),3.24(dd,J=13.7,6.9Hz,2H),3.17(d,J=9.1Hz,1H),3 .10(dd,J=14.2,3.6Hz,1H),2.90–2.79(m,1H),2.73–2.63(m,1H),2.60(dd,J=14.2,8.4Hz,1H),1.89–1.66(m,2H).

[0135] The following compounds were synthesized by similar methods:

[0136] Example 3: Synthesis of Compound 3

[0137] 1) Preparation of 2-amino-5-methoxyphenol (3-b)

[0138] A mixture of 3-a (25 g, 148 mmol) and 10% Pd-C (3 g) in MeOH (400 mL) was placed under a hydrogen atmosphere and stirred overnight at room temperature. The solid was filtered off, concentrated, and purified by column chromatography to give the product (3-b, 18 g, 87%) as a brown solid. MS (ESI): m / z = 140.1 [M+H] + .

[0139] 2) Preparation of 6-methoxybenzo[d]oxazol-2(3H)-one (3-c):

[0140] A mixture of 3-b (18 g, 129 mmol) and CDI (21 g, 129 mmol) in THF (600 mL) was heated under reflux overnight at room temperature. The mixture was cooled to room temperature and washed with water (150 mL x 2) and saturated brine (150 mL). The organic phase was dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography with EA / PE 1 / 1 (100 mL) to afford a brown solid (3-c, 7.2 g, 33%). MS (ESI): m / z = 166.0 [M+H] + .

[0141] 3) Step 3: Preparation of 5-bromo-6-methoxybenzo[d]oxazol-2(3H)-one (3-d)

[0142] At room temperature, liquid bromine (Br2) (3.4 mL, 66.3 mmol) was added dropwise to a solution of 3-c (7.2 g, 43.6 mmol) in AcOH (40 mL) and water (40 mL). The mixture was stirred overnight at room temperature, and water (200 mL) was added. The mixture was filtered to obtain a brown solid. The solid was dissolved in ethyl acetate (200 mL) and washed with saturated NaHCO3 (100 mL). The organic phase was dried over Na2SO4, concentrated, and purified by column chromatography to obtain the product as a brown solid (3-d, 8.6 g, 80%). MS (ESI): m / z = 243.9 [M+H] + .

[0143] 4) Preparation of 5-bromo-6-methoxy-3-methylbenzo[d]oxazol-2(3H)-one (3-e):

[0144] At 0°C, MeI (6.6 mL, 106 mmol) was added dropwise to a mixture of 3-d (10 g, 41 mmol) and Cs2CO3 (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. This solid was further dissolved in ethyl acetate (800 mL) and washed with saturated brine (150 mL x 2). The organic phase was dried over Na2SO4, concentrated, and purified by column chromatography to obtain the product as a brown solid (3-e, 8.7 g, 82%). MS (ESI): m / z = 258.1 [M+H] + .

[0145] 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)

[0146] Under argon, a mixture of 3-e (8.5 g, 32.9 mmol), Pin2B2 (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, and the filtrate was concentrated and purified by column chromatography to give the product (3-f, 5 g, 37%) as a brown solid. MS (ESI): m / z = 306.3 [M+H] + .

[0147] 6) Preparation of 5-(4-bromo-5-fluoro-2-(hydroxymethyl)phenyl)-6-methoxy-3-methylbenzo[d]oxazol-2(3H)-one (3-g):

[0148] Under argon, a mixture of 3-f (5 g, purity: 76%, 12.5 mmol), 1-f (5.4 g, 16.4 mmol), PdCl2dppf (920 mg, 1.26 mmol), and K3PO4 (8 g, 37.7 mmol) in dioxane (100 mL) and H2O (10 mL) was stirred at 50°C for 4.5 hours. The mixture was cooled to room temperature, concentrated under reduced pressure, and diluted with ethyl acetate (300 mL). The mixture was washed with saturated brine (50 mL), dried over Na2SO4, concentrated, and purified by column chromatography to give the product as a light brown solid (3-g, 3.2 g, 67%). MS (ESI): m / z = 384.1 [M+H] + .

[0149] 7) Preparation of 5-(4-bromo-2-(bromomethyl)-5-fluorophenyl)-6-hydroxy-3-methylbenzo[d]oxazol-2(3H)-one (3-h):

[0150] At 0°C, BBr3 (42 mL, 17% in DCM, 28.6 mmol) was slowly added dropwise to a solution of 3-g (3.2 g, 8.37 mmol) in DCM (100 mL). The mixture was warmed to room temperature and stirred overnight. The reaction mixture was diluted with DCM (100 mL), cooled to 0°C, and treated with saturated NaHCO3 to pH 7. The mixture was dried over Na2SO4 and concentrated to afford the product (3-h, 2.5 g, 69%) as a light brown solid. MS (ESI): m / z = 432.0 [M+H] + .

[0151] 8) Preparation of 3-bromo-2-fluoro-10-methyl-5H-benzo[3,4]chromeno[6,7-d]oxazol-9(10H)-one (3-i):

[0152] At room temperature, K2CO3 (800 mg, 5.8 mmol) was added to a solution of 3-h (2.5 g, 5.8 mmol) in DMF (40 mL). After stirring for 1 hour, water (100 mL) was added and filtered to obtain a brown solid. This solid was dissolved in ethyl acetate (300 mL) and washed with saturated brine (100 mL). Drying over Na2SO4, concentration, and column chromatography purification afforded the product (3-i, 1.9 g, 93%) as a light brown solid. MS (ESI): m / z = 350.0 [M+H] + .

[0153] Preparation of methyl 9)(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):

[0154] At room temperature, TMSCl (8.5 mL, 67.1 mmol) was added dropwise to a solution of Zn (10 g, 154 mmol) in DMF (150 mL) over 20 minutes. After stirring for 1.5 hours, I2 (1.2 g, 4.7 mmol) was added. After stirring for 15 minutes, a solution of methyl(R)-2-((tert-butoxycarbonyl)amino)-3-iodopropanoate ((R)-methyl 2-((tert-butoxycarbonyl)amino)-3-iodopropanoate, 8.9 g, 27 mmol) in DMF (30 mL) was added dropwise over 20 minutes. After stirring for 20 minutes, a solution of 3-i (2.36 g, 6.74 mmol) in DMF (70 mL) was added dropwise over 20 minutes. Pd2dba3 (1.85 g, 2 mmol) and S-Phos (1.66 g, 4 mmol) were then added. Stirring was continued at 50°C overnight, the mixture was cooled to room temperature, and ethyl acetate (1 L) was added. The mixture was washed with water (300 mL x 3) and saturated sodium chloride (300 mL x 2), respectively. The mixture was dried over Na2SO4, concentrated, and purified by column chromatography to afford the product (3-j, 3 g, 94%) as a light brown solid. MS (ESI): m / z = 495.2 [M+Na] + .

[0155] 10) Preparation of methyl (S)-2-amino-3-(2-fluoro-10-methyl-9-oxo-9,10-dihydro-5H-benzo[3,4]chromeno[6,7-d]oxazol-3-yl)propanoate (3-k):

[0156] To a solution of 3-j (1 g, 2.1 mmol) in CH3CN (40 mL) at room temperature was added dropwise 4N HCl-dioxane (10 mL). After stirring for 1 hour, Et2O (150 mL) was added. The mixture was then centrifuged (3200 rpm) 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] + .

[0157] 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-oxopropan-2-yl)carbamoyl)-1,4-oxazaheterocycle-4-carboxylate (3-1):

[0158] To a suspension of 3-k (800 mg, 1.96 mmol) in DCM (50 mL) at room temperature was added DIPEA to a pH of 7-8. Then, (S)-4-(tert-butoxycarbonyl)-1,4-oxazepane-2-carboxylic acid (500 mg, 2.04 mmol), HBTU (820 mg, 2.16 mmol), HOBt (292 mg, 2.16 mmol), and DIPEA (1 mL, 6 mmol) were added sequentially. After stirring for 1 hour, the reaction mixture was concentrated and purified by reverse phase column chromatography to afford the product as a white solid (3-l, 650 mg, 55%). MS (ESI): m / z = 622.2 [M+Na] + .

[0159] 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);

[0160] To a solution of 3-1 (625 mg, 1.04 mmol) in CH3CN (50 mL) was added dropwise 25% aqueous ammonia (50 mL) at room temperature. After stirring for 1 hour, the reaction mixture was concentrated and saturated brine (50 mL) was added. The mixture was extracted with ethyl acetate (50 mL x 3), dried over Na2SO4, concentrated, and purified by column chromatography to afford the product as a white solid (3-m, 580 mg, 95%). MS (ESI): m / z = 529.2 [M-55] + .

[0161] 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):

[0162] To a solution of 3-m (580 mg, 0.99 mmol) in DCM (20 mL) was added Burgess reagent (950 mg, 4 mmol) at room temperature. After stirring for 2 hours, the mixture was concentrated and purified by column chromatography to afford the product as a white solid (3-n, 400 mg, 71%). MS (ESI): m / z = 589.2 [M+Na] + .

[0163] 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):

[0164] To a solution of 3-n (400 mg, 0.71 mmol) in CH3CN (20 mL) at room temperature was added dropwise 4N HCl in dioxane (5 mL). After stirring for 1 hour, MTBE (80 mL) was added and the mixture was centrifuged (3200 rpm) for 2 minutes. The solvent was removed, and the resulting solid was added to DMF (10 mL). The mixture was neutralized with saturated NaHCO3 to pH 7-8. The product was directly separated and purified on a reverse-phase column (C18, CH3CN, NH4HCO3 aqueous solution) to obtain the product as a white solid (90 mg, 27%). MS (ESI): m / z = 467.2 [M+H]+. 1H NMR (400MHz, DMSO-d6): 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=8,3.6 Hz,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)

[0165] Referring to the above method, the following compounds were synthesized

[0166] Example 4: Synthesis of Compound 4:

[0167] 1) Preparation of 4-iodo-3-nitrobenzonitrile (4-b):

[0168] To a solution of 4-a (4.5 g, 27.6 mmol) in DMSO (15 mL) at 5-10°C was added 30% H₂SO₄ (20 mL). Then, a solution of NaNO₂ (2.09 g, 30.3 mmol) in water (3 mL) was added dropwise. After stirring for 1 hour, NaI (2.09 g, 30.3 mmol) was added. Stirring was continued for 1 hour, followed by the addition of water (150 mL) and EA (150 mL). The product was extracted with ethyl acetate (50 mL x 3), dried over Na₂SO₄, concentrated, and purified by column chromatography to afford the product 4-b (6 g, 79%) as a yellow solid.

[0169] 2) Preparation of 3-amino-4-iodobenzonitrile 4-c:

[0170] To a solution of 4-b in 30% EtOH (60 mL) at room temperature were added NH4Cl (5.86 g, 110 mmol) and Fe (3.68 g, 65.7 mmol). 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 Na2SO4, concentrated, and purified by column chromatography to afford the product 4-c (5.1 g, 95%) as a yellow solid.

[0171] 3) Preparation of 4-fluoro-5-methyl-2-nitrobenzoic acid 4-e:

[0172] To a solution of 4-d (50 g, 324.7 mmol) in concentrated H₂SO₄ (675 mL) at 0°C, KNO₃ (49.1 g, 487.01 mmol) was added portionwise. After stirring at room temperature for 2 hours, the reaction mixture was poured into 1 L of ice water. Extraction with dichloromethane (300 mL x 3), drying over Na₂SO₄, and concentration afforded the product 4-e (58 g, 95%) as a white solid.

[0173] 4) Preparation of methyl 4-fluoro-5-methyl-2-nitrobenzoate 4-f

[0174] To a solution of 4-e in MeOH (500 mL) at room temperature, SOC12 (69.3 g, 582.9 mmol) was added dropwise. The mixture was heated under reflux overnight, cooled to room temperature, and concentrated under reduced pressure to remove methanol. Water (200 mL) was added, and the mixture was extracted with ethyl acetate (100 mL x 3). The mixture was dried over Na2SO4 and concentrated to give the product 4-f (58 g) as a white solid.

[0175] 5) Preparation of methyl 2-amino-4-fluoro-5-methylbenzoate 4-g

[0176] A mixture of 4-f (58 g) and 5% Pd / C (5.8 g) in MeOH (500 mL) was subjected to hydrogen absorption at room temperature overnight. The mixture was concentrated and purified by column chromatography to obtain the white solid product 4-g (22.2 g, 95%).

[0177] 6) Preparation of methyl 2-bromo-4-fluoro-5-methylbenzoate 4-h

[0178] To a suspension of 4-g (15 g, 81.9 mmol) in 40% HBr (600 mL) at 0°C was added dropwise a solution of NaNO2 (5.6 g, 81.9 mmol) in water (600 mL). 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 Na2SO4, concentrated, and purified by column chromatography to afford the product 4-h (58 g, 50%) as a white solid.

[0179] 7) Preparation of methyl 2-bromo-5-(bromomethyl)-4-fluorobenzoate 4-i

[0180] To a solution of 4-h (10 g, 40.5 mmol) in CCl₄ (100 mL) at room temperature were added NBS (10.8 g, 60.7 mmol) and AIBN (1.33 g, 8.1 mmol). The mixture was heated at reflux for 6 hours, cooled to room temperature, and water was added. The mixture was extracted with ethyl acetate, dried over Na₂SO₄, concentrated, and purified by column chromatography to afford the product 4-h (2.9 g, 22%) as a white solid.

[0181] 8) Preparation of methyl 2-bromo-4-fluoro-5-(((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)methyl)benzoate (4-j):

[0182] To a solution of (R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (1.8 g, 9.8 mmol) in THF (40 ml) at -78°C under argon was slowly added n-buthyl lithium (3.9 ml, 10.8 mmol, 2.5 M n-butyl lithium in hexane) dropwise. After stirring for 1 hour, a solution of 4-i (3.2 g, 9.8 mmol) in THF (10 ml) was added dropwise. After stirring at -78°C for 3 hours, the mixture was slowly warmed to room temperature and stirred for another 1 hour. Saturated NaHCO₃ (200 ml) was added, and the mixture was extracted with ethyl acetate (300 mL x 3), dried over Na₂SO₄, concentrated, and purified by column chromatography to afford the product (4-j, 2.8 g, 67%) as a yellow oil. MS (ESI): m / z = 429.1 [M+H] + .

[0183] 9) Preparation of (S)-2-bromo-5-(2-(tert-butylcarbonyl)amino)-3-methoxy-3-oxopropyl)-4-fluorobenzoic acid methyl ester (4-k):

[0184] To a solution of 4-j (3 g, 7 mmol) in MeCN (70 mL) at room temperature was added dropwise HCl (0.2 mol / L, 14 mmol, 70 mL). After stirring for 16 hours, the mixture was concentrated and neutralized to pH 8 with saturated NaHCO₃ solution. (Boc)₂O (3.05 g, 4 mmol) and MeCN (70 mL) were then added sequentially. Stirring was continued for 3 hours, the mixture was concentrated, extracted with ethyl acetate (200 mL x 3), dried over Na₂SO₄, concentrated, and purified by column chromatography to afford the product as a white solid (4-k, 680 mg, 23%). MS (ESI): m / z = 456.0 [M+Na] + .

[0185] 10) Preparation of methyl (S)-2-((tert-butylcarbonyl)amino)-3-(3-cyano-9-fluoro-6-oxo-5,6-dihydrophenanthridin-8-yl)propanoate (4-1):

[0186] Under argon at room temperature, 4-k (660 mg, 1.52 mmol), 4-c (408 mg, 1.67 mmol), Pd(dppf)Cl2 (111 mg, 0.15 mmol), and potassium acetate (447 mg, 4.56 mmol) were added sequentially to dioxane (20 mL) and water (2 mL). After reacting at 85°C for 7 hours, the mixture was cooled to room temperature, concentrated, and directly purified by column chromatography to obtain the product as a white solid (4-1, 450 mg, 67%). MS (ESI): m / z = 384.0 [M-55]. + .

[0187] 11) Preparation of methyl 2-amino-3-(3-cyano-9-fluoro-6-oxo-5,6-dihydrophenanthridin-8-yl)propanoate TFA salt (4-m):

[0188] To a solution of 4-1 (200 mg, 0.46 mmol) in DCM (16 mL) was added TFA (4 mL) dropwise at room temperature. After stirring for 1 hour, the mixture was concentrated to give a brown oily crude product (4-m, 210 mg, 90%. MS (ESI): m / z = 340.1 [M+H] + .

[0189] 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-oxazaheterocycle-4-carboxylic acid tert-butyl ester (4-n):

[0190] 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 at room temperature for 2 hours, concentrated, and directly separated on a reverse phase column (C18, CH3CN, 10 mM NH4HCO3 aqueous solution) to give the product (4-n, 240 mg, 93%) as a brown solid. MS (ESI): m / z = 467.2 [M-99]+ .

[0191] 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):

[0192] At room temperature, 4-n (280 mg, 0.49 mmol) was added to a solution of NH3 / MeOH (20 mL, 7 mol / L). The mixture was stirred overnight at room temperature and concentrated to give a crude brown solid (4-o, 270 mg, 90%). MS (ESI): m / z = 452.1 [M-99] + .

[0193] 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)

[0194] To a solution of 4-o (250 mg, 0.45 mmol) in DMF (14 mL) at room temperature was added Burgess reagent (648 mg, 2.72 mmol). After stirring at room temperature for 4 hours, the mixture was concentrated and directly purified by HPLC to give the product as a white solid (4-p, 190 mg, 79%). m / z = 434.2 [M-99] + .

[0195] 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):

[0196] 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 NaHCO₃ solution (150 mL) was added. The product was extracted with DCM (5 × 250 mL), dried over Na₂SO₄, concentrated, and purified by HPLC to give the product as a white solid (4, 45 mg, 30%). MS (ESI): m / z = 434.1 [M+H] + . 1H NMR (400MHz, DMSO-d6): δ11.96 (s, 1H); 8.78-8.72 (m, 1H); 8.58 (d, J = 8.8Hz, 1H); 8.49-8.45 (m, 1H); 8.34 (d, J = 7.6Hz, 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).

[0197] Example 5: Synthesis of Compound 22:

[0198] 1) 2-Bromo-5-(hydroxymethyl)phenol (22-b)

[0199] To a solution of 4-bromo-3-hydroxybenzoic acid (22-a, 15 g, 81.9 mmol) in anhydrous THF (100 mL) at 0°C was added dropwise H₃·THF (1 M solution in THF, 62.2 mL). The mixture was then allowed to warm to room temperature and stirred overnight. After completion of the reaction, methanol (50 mL) was added to quench the reaction and the mixture was concentrated to afford the product (22-b, 5.1 g, 100%) as a colorless oil.

[0200] 2) 2-Bromo-5-(chloromethyl)phenol (22-c)

[0201] To a solution of 22-b (5.1 g crude, 24.88 mmol) in dichloromethane (150 mL) at room temperature were added SOCl2 (3.6 mL, 50 mmol) and DMF (0.5 mL). The mixture was stirred overnight, followed by the addition of saturated NaHCO3 aq. (150 mL). The mixture was extracted with dichloromethane (20 mL x 3), dried over Na2SO4, filtered, and concentrated. Purification by column chromatography using DCM / MeOH (95 / 5, v / v) afforded the product 22-c (3.68 g, 67% yield over two steps) as a yellow oil. MS (ESI): m / z = 218.9 [MH] - .

[0202] 3) 1-Bromo-4-(chloromethyl)-2-(methoxymethoxy)benzene (22-d)

[0203] To a solution of 22-c (3.68 g, 16.6 mmol) in dichloromethane (50 mL) at room temperature were added MOMBr (1.5 mL, 18.3 mmol) and DIPEA (4.3 mL, 24.9 mmol) in sequence. After stirring at room temperature for 2 hours, a saturated NH4Cl solution (50 mL) was added. The mixture was extracted with dichloromethane (20 mL x 3), dried over Na2SO4, filtered, and concentrated. Purification by column chromatography with PE / EA (9 / 1) afforded the product 22-d (3.3 g, 75% yield) as a yellow oil. MS (ESI): m / z = 231.1 [M-Cl] + .

[0204] 4) 4-(((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)methyl)-2-(methoxymethoxy)phenyl)boronic acid (22-e)

[0205] To a solution of (R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (2.7 g, 15 mmol) in THF (50 mL) at -78°C under argon was added n-butyllithium solution (2.5 M n-butyllithium in hexane, 6 mL, 15 mmol) dropwise. After stirring at -78°C for 30 minutes, 22-d (2.6 g, 10 mmol) was added in one portion. The mixture was allowed to warm to room temperature and stirred for another hour. The mixture was cooled again to -78°C, and n-butyllithium solution (2.5 M n-butyllithium in hexane, 6 mL, 15 mmol) was added dropwise. After stirring at -78°C for 30 minutes, B(OiPr)3 (4.6 mL, 20 mmol) was added dropwise. The mixture was allowed to warm to room temperature and stirred overnight, followed by the addition of a mixture of ethyl acetate and water (1:1, 200 mL). The mixture was further extracted with ethyl acetate (30 mL x 3). The combined organic phases were dried over Na2SO4, filtered, and concentrated. Purification by column chromatography with DCM / MeOH (95 / 5) gave the yellow oily product 22-e (2.7 g, 71%). MS (ESI): m / z = 379.2 [M+H] + .

[0206] 5) Ethyl 3-(4-bromo-3-chlorophenyl)acrylate (22 g)

[0207] Under argon at 0°C, NaH (3.3 g, 60% Wt in mineral oil, 82.5 mmol) was added portionwise 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-chlorobenzaldehyde (15 g, 68.5 mmol) was added. The mixture was then warmed to room temperature and refluxed for 1 hour. After cooling to room temperature, the reaction was quenched with saturated NH4Cl aq. (300 mL) and extracted with ethyl acetate (150 mL x 3). The organic phases were combined, dried over Na2SO4, filtered, and concentrated to yield the product 22-g (17.5 g, 88%) as a colorless oil. The crude product was used directly in the next step without purification. MS (ESI): m / z = 291.1 [M+H] + .

[0208] 6) Ethyl 3-(4-bromo-3-chlorophenyl)propionate (22-h)

[0209] A mixture of 22-g (17.5 g, 60.4 mmol) and PtO2 (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] + .

[0210] 7) 3-(4-Bromo-3-chlorophenyl)propionic acid (22-i)

[0211] To a solution of 22-h (17.6 g, 60.36 mmol) in THF / H2O (200 mL / 200 mL) at room temperature was added LiOH / H2O (5.1 g, 120.7 mmol). After stirring at room temperature for 2 hours, EtOAc / H2O (200 mL / 200 mL) was added. The aqueous phase was washed dropwise 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 Na2SO4, filtered, concentrated, and slurried with n-hexane to afford colorless crystals of 22-i (14.1 g, 88%). MS (ESI): m / z = 264.9 [M+H] + .

[0212] 8) 6-Bromo-5-chloro-2,3-dihydro-1H-inden-1-one (22-j)

[0213] To 22-h (14.1 g, 53.23 mmol) was added SOCl2 (200 mL) and heated at reflux for 3 hours. Most of the SOCl2 was removed by concentration, and dichloromethane (250 mL) and AlCl3 (8.5 g, 63.87 mmol) were added. After stirring at 40°C for 2 hours, DCM / H2O (200 mL / 200 mL) was added. The aqueous phase was extracted with dichloromethane (200 mL*3). The combined organic phases were dried over Na2SO4, filtered, concentrated, and purified by column chromatography to afford 22-j (3.5 g, 27%) as a white solid. MS (ESI): m / z = 246.9 [M+H] + .

[0214] 9) 6-Bromo-5-chloro-2,3-dihydrospiro[indene-1,2'-[1,3]dithiolane](22-k)

[0215] 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-naphthenate (2-naphthenate-1-molecular sieves, 3.5 g) in chloroform (250 mL) was heated at reflux for 16 hours. The mixture was cooled to room temperature, the solid was filtered off, and the filtrate was washed sequentially with saturated Na2CO3 (300 mL x 2) and brine (200 mL), dried over Na2SO4, filtered, concentrated, and purified by column chromatography to give 22-k (4.5 g, 95%) as a yellow solid. MS (ESI): m / z = 322.1 [M+H] + .

[0216] 10) 5-chloro-2,3-dihydrospiro[indene-1,2'-[1,3]dithiolane]-6-carbaldehyde (22-1)

[0217] To a solution of 22-k (1 g, 3.46 mmol) in THF (20 mL) at -78°C under argon was added dropwise n-BuLi (2.5 M n-butyllithium in hexane, 3.5 mL, 8.75 mmol). Stirring was continued for 30 minutes, followed by the dropwise addition of DMF (1 mL, 13.84 mmol). The temperature was raised to 0°C, and stirring was continued for 2 hours. Water (50 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (30 mL x 3). Drying over Na2SO4, filtration, concentration, and column chromatography were performed to obtain 22-l (0.4 g, 43%) as a yellow solid. MS (ESI): m / z = 271.1 [M+H] + .

[0218] 11)(5-chloro-2,3-dihydrospiro[indene-1,2'-[1,3]dithiopentyl]-6-yl)methanol(22-m)

[0219] To a solution of 22-1 (3.7 g, 13.66 mmol) in THF (30 mL) was added NaBH4 (1.55 g, 40.98 mmol) at -78°C under argon. The mixture was then stirred at room temperature for 30 minutes. The reaction was quenched with water (30 mL), extracted with dichloromethane (20 mL x 3), dried over Na2SO4, filtered, concentrated, and purified by column chromatography to afford 22-m (2.5 g, 67%) as a yellow solid. MS (ESI): m / z = 273.1 [M+H] + .

[0220] 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)

[0221] Under argon, 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) / K3PO4 (1.5 g, 6.86 mmol) in dioxane (40 mL) and H2O (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 afford Y22-n (950 mg, 47%). MS (ESI): m / z = 589.3 [M+H] + .

[0222] 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)

[0223] At room temperature, 3N HCl (2 mL) was added to a mixture of 22-n (730 mg, 1.24 mmol) in CH3CN (100 mL) and H2O (75 mL). Stirring was continued for 1 hour, and then 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 with saturated aqueous NaHCO3 to pH 7. The CH3CN was removed by concentration, and the aqueous phase was extracted with ethyl acetate (100 mL x 3), dried over Na2SO4, filtered, concentrated, and purified by column chromatography to afford 22-o (250 mg, 44%) as a brown solid. MS (ESI): m / z = 450.0 [M+H] + .

[0224] 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)

[0225] 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 sequentially to DCM (5 mL). After stirring for 2 hours, the mixture was concentrated and purified on a reverse phase column (C18, aqueous NH4HCO3) to give 22-p (140 mg, 44%) as a white solid. MS (ESI): m / z = 698.8 [M+Na] + .

[0226] 15)(S)-2-(((S)-3-(2-fluoro-9,10-dihydro-6H-spiro[indeno[5,6-c]chromen-8,2'-[1,3]dithiolane]-3-yl)-1-methoxy-1-oxopropan-2-yl)carbamoyl)-1,4-oxopropane-4-carboxylic acid butyl ester (22-q)

[0227] To a THF solution of 22-p (170 mg, 0.25 mmol) at room temperature were added PPh3 (80 mg, 0.3 mmol) and DIAD (70 μL, 0.36 mmol). After stirring for 2 h, the mixture was concentrated and purified on a reverse phase column (C18, aqueous NH4HCO3) to afford 22-q (120 mg, 72%) as a white solid. MS (ESI): m / z = 681.3 [M+Na] + .

[0228] 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)

[0229] To a solution of 22-q (115 mg, 0.175 mmol) in EtOH (10 mL) at room temperature was added AgNO₃ (65 mg, 0.38 mmol). The mixture was stirred overnight, followed by the addition of AgNO₃ (70 mg, 0.41 mmol) and continued stirring for 3 hours. The solid was filtered off, and the filtrate was concentrated and purified by SGC (0-60% EA in PE, Rf = 0.2 in PE / EA 1 / 1) to afford the product 22-r (30 mg, 29%) as a white solid. MS (ESI): m / z = 527.3 [M-55] + .

[0230] 17)(S)-2-(((S)-1-amino-3-(2-fluoro-8-oxo-6,8,9,10-tetrahydroindeno[5,6-c]chromen-3-yl)-1-oxopropyl-2-yl)carbamoyl)-1,4-oxazepane-4-carboxylic acid butyl ester (22-s)

[0231] To a solution of 22-r (28 mg, 0.048 mmol) in CH3CN (2 mL) was added 25% aqueous ammonia (3 mL) at room temperature. The mixture was stirred for 1 hour, concentrated, and lyophilized to give the white solid product 22-s (25 mg, 91%). MS (ESI): m / z = 590.3 [M+Na] + .

[0232] 18) (S)-butyl 2-(((S)-1-cyano-2-(2-fluoro-8-oxo-6,8,9,10-tetrahydroindeno[5,6-c]chromen-3-yl)ethyl)carbamoyl)-1,4-oxazepane-4-carboxylate (22-t)

[0233] To a solution of 2-s (24 mg, 0.042 mmol) in dichloromethane (4 mL) at room temperature was added Burgess reagent (100 mg, 0.42 mmol). After stirring for 3 hours, an additional Burgess reagent (100 mg, 0.42 mmol) was added and stirring continued for 2 hours. The product 22-t was obtained by concentration and TLC purification as a brown oil (20 mg, 86%). MS (ESI): m / z = 572.3 [M+Na] + .

[0234] 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)

[0235] To a solution of 22-t (19 mg, 0.0346 mmol) in CH3CN (3 mL) at room temperature was added a 4N HCl solution in dioxane (0.75 mL). After stirring for 15 minutes, diethyl ether (Et2O) (40 mL) was added. The mixture was centrifuged (3200 rpm) for 2 minutes, the solvent removed, and the solid was washed with Et2O (40 mL). The mixture was centrifuged again (3200 rpm) for 2 minutes to afford the product 22 (hydrochloride salt, 7.7 mg, 45% yield) as a white solid.

[0236] MS (ESI): m / z = 450.2 [M+H] + .

[0237] 1 H NMR (400MHz, DMSO-d6): 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),3.29-3.2 3(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).

[0238] Referring to the above method, the following compounds were synthesized

[0239] Example 6: Synthesis of Compound 23:

[0240] 1) 3-(3-Bromo-4-methoxyphenyl)propionic acid (23-b)

[0241] To a solution of 3-(4-methoxyphenyl)propanoic acid (10.0 g, 55.49 mmol) in acetic acid (50 mL) at 10°C was added dropwise bromine (3.2 mL, 61.04 mmol). After stirring at room temperature for 30 minutes, water (200 mL) was added. Extraction was performed with ethyl acetate (200 mL x 3), followed by sequential washing with water (150 mL) and saturated brine (150 mL), drying over Na2SO4, filtration, and concentration to obtain the crude product. Further washing with EA:PE = 1:2 (100 mL) afforded the product 23-b as a white solid (yield 13.9 g, 97%). MS (ESI): m / z = 281.1 [M+Na] + .

[0242] 2) 5-Bromo-6-methoxy-2,3-dihydro-1H-inden-1-one (23-c)

[0243] To 23-b (13.9 g, 53.7 mmol) was added SOCl2 (30 mL) at room temperature and refluxed for 1 hour. The mixture was cooled to room temperature, concentrated, and then CH2ClCH2Cl (150 mL) was added. The mixture was cooled to 0°C. AlCl3 (7.9 g, 59.2 mmol) was added portionwise over 10 minutes. The mixture was warmed to room temperature and stirred for 1 hour. The reaction mixture was then poured into ice water (200 mL). The mixture was extracted with dichloromethane (150 mL x 2), dried over Na2SO4, filtered, concentrated, and purified by column chromatography to afford 23-c (6.23 g, 48%) as a white solid. MS (ESI): m / z = 243.1 [M+H] + .

[0244] 3) 6-Methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-1-one (23-d)

[0245] Under argon at room temperature, 23-c (6.2 g, 25.7 mmol), Pin2B2 (7.9 g, 31.1 mmol), PdCl2dppf (1 g, 1.37 mmol), and KOAc (5 g, 51 mmol) were added sequentially to a flask containing dioxane (200 mL). 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. Purification by SGC (0-25% EA in PE, RF = 0.3 in PE / EA 4 / 1) afforded 23-d (7.2 g, 97%) as a brown solid. MS (ESI): m / z = 289.3 [M+H]+ .

[0246] 4) 5-(4-bromo-5-fluoro-2-(hydroxymethyl)phenyl)-6-methoxy-2,3-dihydro-1H-inden-1-one (23-e)

[0247] A mixture of 22-d (7.2 g, 25 mmol), (5-bromo-4-fluoro-2-iodophenyl)methanol (9.2 g, 27.8 mmol), PdCl2dppf (1 g, 1.4 mmol), and K3PO4 (16 g, 75 mmol) in dioxane (200 mL) / H2O (20 mL) was stirred at 50°C under argon for 12 hours. The mixture was cooled to room temperature, EA (500 mL) was added, and the mixture was washed with water. The mixture was dried over Na2SO4, filtered, concentrated, and purified by SGC (5-30% EA in PE, Rf = 0.6 in EA / PE 1 / 1, weak UV 254 nm) to afford 23-e (6.2 g, 67%) as a white solid. MS (ESI): m / z = 365.1 [M+H] + .

[0248] 5) 5-(4-bromo-5-fluoro-2-(hydroxymethyl)phenyl)-6-hydroxy-2,3-dihydro-1H-inden-1-one (23-f)

[0249] At room temperature, BF3CS2 (11 mL, 104.5 mmol) was added dropwise to a solution of 23-e (6.2 g, 17 mmol) in dichloromethane (100 mL). After 2 hours of reaction, water (50 mL) was added, and the mixture was extracted with dichloromethane (100 mL x 2). The mixture was washed dropwise with saturated NaHCO3 (100 mL x 2), dried over Na2SO4, filtered, concentrated, and purified by SGC (0-60% EA in PE, Rf = 0.4 in PE / EA 1 / 1) to give the solid product 23-f (3.9 g, 65%). MS (ESI): m / z = 353.1 [M+H] + .

[0250] 6) 3-Bromo-2-fluoro-9,10-dihydrobenzo[c]cyclopenta[g]chromen-8(5H)-one (23-g)

[0251] To a solution of 23-f (3.88 g, 11 mmol) in THF (100 mL) at room temperature were added PPh3 (3.2 g, 12.2 mmol) and DIAD (2.6 mL, 13.1 mmol) in sequence. After stirring for 30 minutes, the mixture was concentrated and then washed dropwise with ethyl acetate to give the product 23-g as a light yellow solid (2 g, 54%). MS (ESI): m / z = 333.1 [M+H] + .

[0252] 7)(S)-2-((tert-Butyloxycarbonyl)amino)-3-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopenta[g]chromen-3-yl)propanoic acid methyl ester (23-h)

[0253] 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) at room temperature. After stirring for 1.5 hours, I2 (370 mg, 1.46 mmol) was added, and the mixture was stirred for an additional 15 minutes. A solution of (R)-methyl 2-((tert-butoxycarbonyl)amino)-3-iodopropanoate (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. Pd2dba3 (576 mg, 0.63 mmol) and S-Phos (517 mg, 1.26 mmol) were then added sequentially. The reaction was stirred at 50°C overnight. Cool to room temperature, add ethyl acetate (500 mL), filter out the solid, and wash the filtrate dropwise with water (150 mL x 3) and saturated sodium chloride solution (150 mL x 2). Dry over Na2SO4, filter, concentrate, and purify by SGC to afford 23-h (0.48 g, purity: 65%, yield: 32%) as a light brown solid. MS (ESI): m / z = 456.3 [M+H] + .

[0254] 8)(S)-2-amino-3-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopenta[g]chromen-3-yl)propanoic acid methyl ester hydrochloride (23-i)

[0255] To a solution of 23-h (0.47 g, purity: 65%, 0.67 mmol) in CH3CN (16 mL) was added dropwise a 4N HCl solution in dioxane (4 mL) at room temperature. After stirring for 30 minutes, ether (Et2O) (80 mL) was added. The mixture was centrifuged (3200 rpm) for 2 minutes, and the supernatant was removed to obtain a solid product 23-i (400 mg). MS (ESI): m / z = 356.0 [M+H] + .

[0256] 9)(S)-2-(((S)-3-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopenta[g]chromen-3-yl)-1-methoxy-1-oxopropan-2-yl)carbamoyl)-1,4-oxazepane-4-carboxylic acid butyl ester (23-j)

[0257] To a suspension of 23-i (crude product 400 mg, 0.67 mmol) in DCM (30 mL) at room temperature was added DIPEA to a pH of 7-8. (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 then added sequentially. The mixture was stirred at room temperature for 1 hour and concentrated. The product 23-j (260 mg, 66% yield over two steps) was obtained by purification on a reverse phase column (C18, CH3CN, NH4HCO3 aqueous solution) as a white solid. MS (ESI): m / z = 527.0 [M-55] + .

[0258] 10) Butyl (S)-2-(((S)-1-amino-3-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopenta[g]chromen-3-yl)-1-oxopropan-2-yl)carbamoyl)-1,4-oxazepane-4-carboxylate (23-k)

[0259] To a solution of YD02-214-i (250 mg, 0.43 mmol) in CH3CN (20 mL) at room temperature was added 25% aqueous ammonia (30 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). The mixture was dried over Na2SO4, filtered, and concentrated to afford a light yellow solid 23-k (300 mg). The crude product was used directly in the next reaction without further purification. MS (ESI): m / z = 590.0 [M+Na] + .

[0260] 11)(S)-2-(((S)-1-cyano-2-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopenta[g]chromen-3-yl)ethyl)carbamoyl)-1,4-oxazepane-4-carboxylic acid butyl ester (23-1)

[0261] To a solution of 23-k (crude product 300 mg, 0.43 mmol) in dichloromethane (2 mL) was added Burgess reagent (615 mg, 2.58 mmol) at room temperature. After stirring for 2 hours, an additional Burgess reagent (300 mg, 1.26 mmol) was added. Stirring was continued for 30 minutes, followed by concentration and purification by SGC (0-60% EA in PE, Rf = 0.5 in PE / EA 1 / 2) to afford the solid product 23l (190 mg, 80% yield over two steps). MS (ESI): m / z = 572.4 [M+Na] + .

[0262] 12)(S)-N-((S)-1-Cyano-2-(2-fluoro-8-oxo-5,8,9,10-tetrahydrobenzo[c]cyclopenta[g]chromen-3-yl)ethyl)-1,4-oxazepane-2-carboxamide (23)

[0263] At room temperature, a 4N HCl dioxane solution (2.5 mL) was added to a solution of 23-1 (190 mg, 0.346 mmol) in CH3CN (10 mL). After stirring for 30 minutes, ether (40 mL) was added and the mixture was centrifuged (3200 r / m) for 2 minutes. The upper layer of solvent was removed, ethyl acetate (100 mL) was added, and the mixture was neutralized to pH 7 with saturated NaHCO3. Further ethyl acetate extraction (50 mL*2) was performed, and the organic phases were combined, washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated. Purification was performed by preparative HPLC (NH4HCO3 buffer: A: 10 mM NH4HCO3 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%).

[0264] MS (ESI): m / z = 450.3 [M+H] + .

[0265] 1H NMR (400MHz, DMSO-d6): 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)

[0266] Referring to the above method, the following compounds were synthesized

[0267] Example 7: Activity Test

[0268] A. U937 cell inhibition of DPP1 activity test

[0269] The intracellular enzyme activity assay was performed in a 384-well plate. The cell culture medium was 1640, 10% FBS, and 1*PS. 30 μL of cell culture medium 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 via Echo. After incubation at 37°C for 1 hour, h-Gly-phe-AFC (10 μL) was added to each well to initiate the reaction. The cells were further incubated at 37°C for 1 hour, and fluorescence absorption was read at EXλ400 nm and EMλ505 nm. The above test results were calculated using Graphpad 8.0. 50 The results are listed in Table 1.

[0270] B. Inhibitory effect test of DPP1 enzyme activity

[0271] Experimental Materials:

[0272] Recombinant human cathepsin rhCathepsin C / DPP1 was purchased from R&D systems

[0273] Recombinant human cathepsin rhCathepsin L was purchased from R&D systems

[0274] AZD7986, purchased from MCE

[0275] Gly-Arg-AMC (hydrochloride) was purchased from Cayman Chemical

[0276] DMSO was purchased from Sigma-Aldrich

[0277] Experimental methods:

[0278] 1) Compound preparation and treatment: The exact amount of compound was weighed and dissolved in 10 mL of DMSO to prepare 10 mL of stock solution, which was further diluted as required.

[0279] 2) Screening: Dilute recombinant human cathepsin rhCathepsin C / DPP1 and recombinant human cathepsin rhCathepsin L in activation buffer and incubate at 37°C for 60 minutes. Add 4 μL of test compound to the white wells of a 384-well plate. Add 4 μL of the enzyme mix after incubation to the white wells. Block the 384-well plate and incubate at room temperature for 30 minutes. Add 8 μL of a 2x diluted Gly-Arg-AMC (hydrochloride). Block the 384-well plate and incubate at room temperature for 2 hours. Prepare a 4x stop solution and add 4 μL of the stop solution to the white wells. Read fluorescence data on a Victor Nivo35 fluorimeter.

[0280] 3) Data Analysis

[0281] All ICs 50 The values ​​were converted to percentage inhibition using Prism Graphpad 8.0. The results are listed in Table 1:

[0282] Table 1: In vitro activity of compounds *N / A: Not tested

[0283] 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.

[0284] 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 claimed by the present invention.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, in: R1, R2, R3, R4, R5 are each independently selected from H, cyano, halogen, C1-C3 alkyl, C1-C3 alkoxy; or R1, R2, R4 and R5 are linked to form a 3-6 membered ring, wherein the ring atoms of the 3-6 membered ring may be arbitrarily substituted by S, O, N, or C=O; and the 3-6 membered ring may be optionally substituted by R6; X, Y are each independently selected from O, -NH, -NMe, -CH2-, -C(O)-; R6 is optionally selected from H, =O, C1-C4 alkyl, C1-C4 deuterated alkyl, C1-C4 cycloalkyl.

2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R1, R2, R3, R4, and R5 are each independently selected from H, cyano, fluorine, C1-C3 alkyl, or C1-C3 alkoxy.

3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R4 and R5 are each independently selected from H and cyano.

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R1, R2, and R3 are each independently selected from H, fluorine, C1-C3 alkyl, and C1-C3 alkoxy.

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R1 and R2 are linked to form a cycloalkyl group.

6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R4 and R5 are linked to form the following structure: in, R6 is optionally selected from H, =O, C1-C4 alkyl, C1-C4 deuterated alkyl, C1-C4 cycloalkyl.

7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) has the following structure: wherein R1, R2, R3, R6, X, and Y are as defined in claim 1; and Z is O or S.

8. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) has the following structure:

9. Use of the 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.

10. The compound of formula (I) according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof is used in the preparation of a pharmaceutical composition for 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 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, Arthritis, glomerulonephritis, eosinophilic diseases, neutrophilic diseases, ANCA-related inflammation, antineutrophil cytoplasmic antibody-associated 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 use in drugs for treating the diseases in patients at risk of the diseases.

11. The use according to claim 9, which can be further combined with one or more drugs selected from the following: beta-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.

12. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.