Neutrophil elastase inhibitor and use thereof

Abstract

The present invention relates to a neutrophil elastase inhibitor and the use thereof, a preparation method therefor, a pharmaceutical composition, and the use thereof in the treatment of related diseases caused by an abnormal increase of neutrophil elastase. The compound of the present invention has a good inhibitory activity against neutrophil elastase.

Description

Neutrophil elastase inhibitor and its application Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a neutrophil elastase inhibitor and its application, a preparation method and a pharmaceutical composition thereof, as well as its application in the treatment of related diseases caused by abnormal increase of neutrophil elastase. Background Art

[0002] Neutrophil elastase (NE) is a serine protease primarily found in the azure granules of neutrophils. During the acute phase of inflammation, neutrophils are the first cells recruited to the site of inflammation and form the earliest line of defense against invasion. NE can degrade a variety of extracellular matrix proteins, such as elastin, proteoglycans, laminin, and fibronectin. In normal physiological processes, NE can degrade or repair damaged tissues by degrading these tissue structural proteins to maintain tissue homeostasis. NE can also help recruit neutrophils to sites of inflammation by degrading bacterial structural proteins, reducing bacterial infection and participating in the regulation of inflammation.

[0003] However, excessive release and accumulation of NE can disrupt normal physiological tissue structure, increase microvascular permeability, and induce excessive mucus secretion. Excessive NE release can also lead to serious cardiopulmonary diseases. Diseases associated with NE overexpression include acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and acute lung injury / ARDS with systemic inflammatory response syndrome (SIRS).

[0004] Over the past years, researchers have designed and developed a variety of small-molecule NE inhibitor compounds with diverse structural types. These include pivalate ester derivatives, such as Sivelestat (44 nM inhibitory activity against neutrophil elastase), disclosed in Reference 1. Sivelestat is the world's first drug used to treat and improve systemic inflammatory response syndrome (SIRS)-related acute lung injury (ALI) / acute respiratory distress syndrome (ARDS). Furthermore, numerous studies have confirmed the therapeutic and protective effects of Sivelestat in sepsis, cystic fibrosis, chronic obstructive pulmonary disease (COPD), and ischemia-reperfusion injury in various tissues. Other small-molecule NE inhibitors include pyridone derivatives (such as Alvelestat), dihydropyrimidone derivatives (such as BAY 85-8501), and small peptides (such as POL6014).

[0005] It is well known in the art that slight differences in the structures of compounds may lead to huge differences in activity. The present invention develops new compounds based on the existing technology, which have excellent NE inhibitory activity and can be used to treat related diseases caused by abnormal increase of neutrophil elastase.

[0006] Document 1: EP 0347168B1

[0007] Summary of the Invention

[0008] The first aspect of the present invention provides a compound represented by formula I

[0009] or a pharmaceutically acceptable salt thereof, wherein

[0010] R1 is selected from (1) R6-C(=O)-; or (2) R1 is connected to a benzene ring to form a five-membered or six-membered ring, and the five-membered or six-membered ring is optionally substituted by R6;

[0011] R6 is selected from tert-butyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl;

[0012] Ring A is selected from 5-10 membered cycloalkyl, heterocycloalkyl, heteroaryl, aryl, and is optionally substituted with one or more R5;

[0013] R2 and R5 are each independently selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 heteroaryl;

[0014] R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C1-C6 haloalkyl;

[0015] R4 is selected from -COOH, -SO3H, -PO3H2;

[0016] m, n are independently 0, 1, 2, 3;

[0017] The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, and heteroaryl groups may be unsubstituted or substituted with one or more of the following substituents: deuterium, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, amino, oxo, -CONH2, and -COOH.

[0018] In some embodiments of the present invention, Ring A is a 5- to 6-membered ring optionally containing 1 to 3 heteroatoms; the heteroatoms include nitrogen atoms, oxygen atoms, and sulfur atoms.

[0019] In some embodiments of the present invention, ring A is phenyl, pyrazolyl, imidazolyl, pyrazinyl, or cyclohexyl, and the ring A is unsubstituted or optionally substituted with one or more R5; the R5 is optionally selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, or C3-C6 heteroaryl.

[0020] In some embodiments of the present invention, R5 is selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, unsubstituted or alkyl-substituted imidazolyl, pyrazolyl, oxazolyl, isoxazolyl.

[0021] In some embodiments of the present invention, ring A is phenyl, pyrazolyl, imidazolyl, pyrazinyl, or cyclohexyl, and the ring A is unsubstituted or optionally substituted with one or more R5; the R5 is optionally selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, methyl, methoxy, unsubstituted or substituted with methyl imidazolyl, pyrazolyl, or isoxazolyl.

[0022] In some embodiments of the present invention, R6 is tert-butyl.

[0023] In some embodiments of the present invention, R6 is cyclopropyl, cyclobutyl, or azetidinyl, and the cyclopropyl, cyclobutyl, or azetidinyl is optionally substituted with one or more halogen or methyl.

[0024] In some embodiments of the present invention, R6 is cyclopropyl, oxetanyl, azetidinyl, optionally substituted with one or more fluorine or methyl groups.

[0025] In another aspect of the present invention, a compound of Formula I or a pharmaceutically acceptable salt thereof is provided, wherein Formula I has a structure as shown in Formula Ia:

[0026] in,

[0027] Ring A is a 5- to 6-membered ring optionally containing 1 to 3 heteroatoms, and said ring A is optionally substituted with one or more R5;

[0028] R4 is selected from -COOH, -SO3H, -PO3H2;

[0029] R2 and R5 are each independently selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 heteroaryl;

[0030] R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C1-C6 haloalkyl;

[0031] m, n are independently 0, 1, 2, 3;

[0032] The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, and heteroaryl groups may be unsubstituted or optionally substituted with one or more of the following substituents: deuterium, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, amino, -CONH2, and -COOH.

[0033] In some embodiments of the present invention, in Formula Ia, Ring A is phenyl, pyrazolyl, imidazolyl, pyrazinyl, or cyclohexyl, and Ring A is optionally substituted by one or more R5.

[0034] In another aspect of the present invention, a compound of Formula I or a pharmaceutically acceptable salt thereof is provided, wherein Formula I has a structure as shown in Formula Ib:

[0035] R2 and R5 are each independently selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 heteroaryl;

[0036] R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C1-C6 haloalkyl;

[0037] m, p are independently 0, 1 or 2;

[0038] The premise is that R2, R3, and R5 are not hydrogen at the same time.

[0039] The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, and heteroaryl groups may be unsubstituted or optionally substituted with one or more of the following substituents: deuterium, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, and amino.

[0040] In some embodiments of the present invention, R2 in Formula Ib is optionally selected from hydrogen and fluorine.

[0041] In some embodiments of the present invention, R3 in Formula Ib is selected from hydrogen, methyl, fluorine-substituted methyl, methoxymethyl, and deuterated methoxymethyl.

[0042] In some embodiments of the present invention, R5 in Formula Ib is optionally selected from hydrogen, fluorine, methyl, cyano, methoxy, unsubstituted or alkyl-substituted imidazolyl, pyrazolyl, oxazolyl, isoxazolyl.

[0043] In another aspect of the present invention, a compound of Formula I or a pharmaceutically acceptable salt thereof is provided, wherein Formula I has a structure as shown in Formula Ic:

[0044] In the formula Ic,

[0045] R4 is optionally selected from -SO3H, -PO3H2;

[0046] R2 and R5 are each independently selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 heteroaryl;

[0047] R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C1-C6 haloalkyl;

[0048] n, m, p are independently 0, 1 or 2;

[0049] The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, and heteroaryl groups may be unsubstituted or optionally substituted with one or more of the following substituents: deuterium, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, amino, -CONH2, and -COOH.

[0050] In some embodiments of the present invention, in Formula I, Formula Ia, and Formula Ic, R4 is optionally selected from -SO3H and -PO3H2.

[0051] In some embodiments of the present invention, in Formula I and Formula Ia, Ring A is phenyl, pyrazolyl, imidazolyl, pyrazinyl, or cyclohexyl, and Ring A is unsubstituted or optionally substituted with one or more R5.

[0052] In some embodiments of the present invention, in Formula I, Formula Ia, Formula Ib, and Formula Ic, R3 is hydrogen, methyl, fluoromethyl, methoxymethyl, or deuterated methoxymethyl.

[0053] In some embodiments of the present invention, in Formula I, Formula Ia, Formula Ib, and Formula Ic, R2 is hydrogen, fluorine, cyano, trifluoromethyl, nitro, or amino.

[0054] In some embodiments of the present invention, in Formula I, Formula Ia, Formula Ib, and Formula Ic, R5 is hydrogen, fluorine, cyano, trifluoromethyl, nitro, amino, unsubstituted or methyl-substituted imidazolyl, pyrazolyl, oxazolyl, or isoxazolyl.

[0055] In some embodiments of the present invention, in Formula I, Formula Ia, Formula Ib, and Formula Ic, when a chiral center exists, its configuration can be R configuration, S configuration, or racemate.

[0056] The compound represented by Formula I or a pharmaceutically acceptable salt thereof of the present invention has the following structure:

[0057] In another aspect, the present invention provides a pharmaceutical composition comprising a compound selected from any one of Formula I, Formula Ia, Formula Ib, and Formula Ic, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0058] The pharmaceutically acceptable excipients include one or more of excipients, surfactants, pH regulators, osmotic pressure regulators, lubricants, disintegrants, stabilizers, suspending agents, flavoring agents, solvents, emulsifiers, dispersants, solubilizers, and the like.

[0059] The pharmaceutical composition can be administered by inhalation in the form of a solution, suspension, aerosol, dry powder, etc.; or administered by drip after reconstitution in the form of a lyophilized powder, etc.; or administered by injection in the form of an injection solution, etc.; or administered orally in the form of tablets, capsules, granules, etc.

[0060] The compound of the present invention can inhibit the activity of neutrophil elastase and is a NE inhibitor, which can be used to prevent or treat related diseases caused by abnormal increase of neutrophil elastase.

[0061] In another aspect, the present invention provides a use of a compound represented by any one of Formula I, Formula Ia, Formula Ib, and Formula Ic, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for treating or preventing diseases caused by an abnormal increase in neutrophil elastase.

[0062] The diseases include autoimmune inflammation, infectious inflammation, traumatic inflammation, allergic inflammation, systemic inflammation, respiratory system inflammation, lung inflammation, and pain.

[0063] The diseases include inflammatory diseases, including acute lung injury (ALI), acute respiratory distress syndrome (ARDS), acute lung injury / acute respiratory distress syndrome with systemic inflammatory response syndrome (SIRS), etc. DETAILED DESCRIPTION

[0064] Explanation of terms

[0065] "Halogen" includes fluorine, chlorine, bromine and iodine.

[0066] "Alkyl" refers to a saturated aliphatic hydrocarbon group, which can be a straight chain or branched chain alkyl group, preferably a C1-C6 alkyl group, more preferably a C 1- C3 alkyl. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, etc. The alkyl group can be unsubstituted or substituted. When substituted, the substituent can be in any substituted position.

[0067] "Alkoxy" refers to -O-alkyl or -O-cycloalkyl groups. Non-limiting examples of -O-alkyl groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like. Non-limiting examples of -O-cycloalkyl groups include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. The alkoxy group may be further substituted on the alkyl or cycloalkyl group.

[0068] "Cycloalkyl" refers to a saturated or partially saturated monocyclic or polycyclic alkane substituent, preferably a C3-C6 cycloalkyl. Non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and the like. The cycloalkyl group may be unsubstituted or further substituted with a substituent. When substituted, the substituent may be at any substitutable position on the ring.

[0069] "Heterocycloalkyl" refers to a saturated or partially saturated monocyclic or polycyclic hydrocarbon radical containing 3 to 10 ring atoms, one or more of which are selected from nitrogen, oxygen, or sulfur. Preferred heterocycloalkyls are C3-C6 heterocycloalkyls. Non-limiting examples include oxetanyl, azetidinyl, oxirane, and the like. The heterocycloalkyl radical may be unsubstituted or further substituted with a substituent. When substituted, the substituent may be at any substitutable position on the ring.

[0070] "Heteroaryl" refers to a 5- to 10-membered heteroaromatic group containing 1-4 heteroatoms. Heteroatoms include nitrogen, oxygen, and sulfur atoms. Non-limiting examples include imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, and the like. The heteroaryl group may be attached to a ring carbon atom or to a heteroatom. The heteroaryl group may be unsubstituted or further substituted with a substituent. When substituted, the substituent may be located at any substitutable position on the ring.

[0071] "Aryl" refers to a 6- to 10-membered, all-carbon monocyclic or fused polycyclic ring with a conjugated π electron system. Examples include phenyl and naphthyl. The aryl group may also be fused with a heteroaryl group, a heterocycloalkyl group, or a cycloalkyl group. The aryl group may be unsubstituted or further substituted with a substituent.

[0072] When substituted, the alkyl, alkoxy, cycloalkyl, heterocycloalkyl, and heteroaryl groups described in the present invention are preferably substituted by one or more of the following substituents: deuterium, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, amino, oxo (=O), -CONH2, and -COOH.

[0073] "Oxo" refers to a =0 substituent.

[0074] "Pharmaceutically acceptable salts" include alkali metal salts and salts formed with organic bases, such as sodium salts, potassium salts, amine salts, etc., and also include salt hydrates.

[0075] In some embodiments of the present invention, the compound of the present invention is prepared by the method of the following route 1, wherein formula M1 and formula M2 are reacted under alkaline conditions, and then the protecting group Pg is removed to obtain the compound.

[0076] wherein R1, R2, R3, R5, m, and p have the same definitions as those in Formula I, and Pg is a protecting group, such as benzyl, tert-butyl, and the like.

[0077] In some embodiments of the present invention, the compound of the present invention can be prepared by the method of the following route 2, wherein a carboxylic acid compound substituted with R1 is reacted with a compound of formula M4 in the presence of a condensing agent, and then the protecting group Pg is removed to obtain the compound.

[0078] wherein R1, R2, R3, R5, m, and p have the same definitions as those in Formula I, and Pg is a protecting group, such as benzyl, tert-butyl, and the like.

[0079] In some embodiments of the present invention, the compound of the present invention can be prepared by the method of the following route 3:

[0080] Alternatively, combine Equation M7 with Equation Reaction preparation.

[0081] wherein R1, R2, R3, R5, m, and p have the same definitions as those in Formula I, and Pg is a protecting group, such as benzyl, tert-butyl, and the like.

[0082] In a specific embodiment of the present invention, when Ring A of the compound is a non-benzene ring, it can be prepared by methods similar to those in Schemes 1 to 3.

[0083] The following specific examples are used to further illustrate the inventive concept of the present invention and should not be construed as limiting the present invention. Unless otherwise specified, the reagents or intermediates used in this patent can be obtained commercially or prepared by methods known in the art. Unless otherwise specified, the drug formulation, biological activity testing, various index detection, and animal modeling methods related to the biological activity testing and animal experiments in this patent can be carried out by conventional methods in the art.

[0084] Intermediate preparation

[0085] Preparation of intermediate 1

[0086] Step 1: 4-(phenylmethylthio)-2-fluorophenol

[0087] A mixture of 4-bromo-2-fluorophenol (2 g, 10.5 mmol), phenylmethylthiol (2 g, 15.8 mmol), DIPEA (2.7 g, 21 mmol), Pd2dba3 (916 mg, 1 mmol) and Xant-phos (991 mg, 2.1 mmol) in dioxane (20 mL) was stirred overnight at 110 ° C under argon protection. The reaction mixture was evaporated and the residue was purified by silica gel column (25% EtOAc / PE) to give 4- (phenylmethylthio) -2-fluorophenol (1 g, 42% yield).

[0088] MS (ESI): m / z = 235.1 [M+H] + .

[0089] Step 2: 4-(phenylmethylthio)-2-fluorophenyl pivalate

[0090] A mixture of 4-(phenylmethylthio)-2-fluorophenol (1 g, 4.3 mmol), pivaloyl chloride (564 mg, 4.7 mmol) and pyridine (2.7 g, 34.4 mmol) in DCM (10 mL) was stirred at room temperature for 3 hours. H2O (2 mL) was added to the reaction mixture. The aqueous phase was separated and extracted with EtOAc (5 mL×3). The combined organic layer was dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified by silica gel chromatography (10-60% EtOAc / petroleum ether) to give 4-(phenylmethylthio)-2-fluorophenyl pivalate (700 mg, 51% yield). MS (ESI): m / z=319.1[M+H] + .

[0091] Step 3: 4-(chlorosulfonyl)-2-fluorophenyl pivalate

[0092] A solution of 4-(phenylmethylthio)-2-fluorophenyl pivalate (450 mg, 1.52 mmol) / 1,3-dichloro-5,5-dimethylimidazolidine-2,4-dione (600 mg, 3.05 mmol) / AcOH (0.8 mL) / H2O (0.5 mL) in CH3CN (12 mL) was stirred at room temperature for 15 minutes. EA (100 mL) was added to the reaction mixture and washed with H2O (50 mL) and brine (50 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified by silica gel chromatography (10-60% EtOAc / petroleum ether) to give 4-(chlorosulfonyl)-2-fluorophenyl pivalate (290 mg, 69% yield) as an orange / brown solid. MS (ESI): m / z=295.0[M+H] + .

[0093] Preparation of intermediate 2

[0094] Step 1: 4-Bromo-2-pivalamidophenyl pivalate

[0095] Pivaloyl chloride (1.5mL, 12.2mmol) is added dropwise to a solution of 2-amino-4-bromophenol (2.5g, 13.3mmol) / DIPEA (3.3mL, 20mmol) in DCM (50mL) at 0°C. The reaction mixture is then stirred at room temperature for 2 hours. The reaction mixture is evaporated. The residue is purified by SGC (0-20% EA / PE, Rf=0.7 in PE / EA 4 / 1) to give 4-bromo-2-pivalamidophenyl pivalate (1.6g, 33% yield) as a light brown solid.

[0096] MS (ESI): m / z = 356.2 [M+H] + .

[0097] Step 2: 5-Bromo-2-(tert-butyl)benzo[d]oxazole

[0098] A mixture of 4-bromo-2-pivalamidophenyl ester (1.6 g, 4.5 mmol) / p-TsOH H2O (1.7 g, 8.9 mmol) in toluene (80 mL) was refluxed at 130 ° C for 1.5 hours using a Dean-Stark distillation system. The reaction mixture was cooled to room temperature and evaporated. The residue was purified by SGC (0-20% EA / PE, 0-10% MeOH / DCM, Rf=0.4 in PE / EA 6 / 1) to give 5-bromo-2-(tert-butyl)benzo[d]oxazole (0.58 g, 50% yield) as a brown / orange oil. MS (ESI): m / z=255.9[M+H] + .

[0099] Step 3: 5-(Benzylthio)-2-(tert-butyl)benzo[d]oxazole

[0100] A mixture of 5-bromo-2-(tert-butyl)benzo[d]oxazole (550 mg, 2.17 mmol) / phenylmethanethiol (280 μL, 2.39 mmol) / DIPEA (720 μL, 4.4 mmol) / Pd2dba3 (200 mg, 0.22 mmol) / Xantphos (253 mg, 0.44 mmol) in toluene (10 mL) was stirred overnight at 110 ° C under Ar. The reaction mixture was evaporated. The residue was purified by SGC (0-10% EA / PE, Rf=0.6 in PE / EA 8 / 1) to give 5-(phenylmethylthio)-2-(tert-butyl)benzo[d]oxazole (0.6 g, 93% yield) as a yellow oil. MS (ESI): m / z=298.2[M+H] + .

[0101] Step 4: 2-(tert-Butyl)benzo[d]oxazole-5-sulfonyl chloride

[0102] A solution of 5-(phenylmethylthio)-2-(tert-butyl)benzo[d]oxazole (450 mg, 1.52 mmol) / 1,3-dichloro-5,5-dimethylimidazolidine-2,4-dione (600 mg, 3.05 mmol) / AcOH (0.8 mL) / H2O (0.5 mL) in CH3CN (12 mL) was stirred at room temperature for 15 minutes. The reaction mixture was dissolved in EA (100 mL), washed with H2O (50 mL) and brine (50 mL). The EA layer was dried over Na2SO4, filtered and evaporated. The residue was purified by SGC (PE:EA=10:1, Rf=0.6 in PE / EA 10 / 1) to give 2-(tert-butyl)benzo[d]oxazole-5-sulfonyl chloride (290 mg, 69% yield) as an orange / brown solid. MS (ESI): m / z = 274.1 [M+H] + .

[0103] Preparation of intermediate 3

[0104] (2-Aminobenzoyl)glycine benzyl ester

[0105] A mixture of 2H-benzo[d][1,3]oxazine-2,4(1H)-dione (5 g, 30.7 mmol), glycine benzyl ester hydrochloride (6.8 g, 33.7 mmol), TEA (10.5 mL, 75.5 mmol) in CH3CN (100 mL) was stirred at 70 ° C for 4 hours. The reaction mixture was cooled to room temperature and evaporated. Water (100 mL) was added to the residue (gray solid / oil) and extracted with EA (500 mL, 100 mL×2). The EA layers were combined, washed with brine (100 mL), dried over Na2SO4, filtered and evaporated. The residue was washed with EA (50 mL) to give (2-aminobenzoyl)glycine benzyl ester (2.39 g, 27% yield) as a white solid. MS (ESI): m / z=285.1[M+H] + .

[0106] Preparation of intermediate 4

[0107] (4-Amino-1-methyl-1H-pyrazole-3-carbonyl)glycine benzyl ester

[0108] Step 1: 1-Methyl-4-nitro-1H-pyrazole-3-carboxylic acid

[0109] A mixture of 1-methyl-4-nitro-1H-pyrazole-3-carboxylic acid methyl ester (1800 mg, 9.72 mmol), LiOH·H2O (815 mg, 19.44 mmol) in THF (10 mL) and H2O (10 mL) was stirred at room temperature for 2 hours. The pH of the reaction mixture was adjusted to 4-5 using Amberlyst15 (H) ion exchange resin. The reaction mixture was filtered and the filtrate was evaporated to give 1-methyl-4-nitro-1H-pyrazole-3-carboxylic acid (1.6 g, 96% yield) as a yellow solid. MS (ESI) m / z=172.1[M+H] + .

[0110] Step 2: (1-Methyl-4-nitro-1H-pyrazole-3-carbonyl)glycine benzyl ester

[0111] A mixture of 1-methyl-4-nitro-1H-pyrazole-3-carboxylic acid (1.6 g, 9.36 mmol), glycine benzyl ester hydrochloride (2.8 g, 14.04 mmol), HATU (4.3 g, 11.23 mmol), HOAt (1.5 g, 11.23 mmol) and TEA (4 mL, 28.80 mmol) in DMF (20 mL) was stirred at room temperature for 1 hour. EA (100 mL) was added to the mixture and washed with brine (3 × 100 mL). The EA layer was separated and evaporated. The residue was purified by silica gel column chromatography (eluted with EA) to give (1-methyl-4-nitro-1H-pyrazole-3-carbonyl)glycine benzyl ester (2.1 g, 70% yield) as a white solid. MS (ESI) m / z=319.2[M+H] + .

[0112] Step 3: (4-Amino-1-methyl-1H-pyrazole-3-carbonyl)glycine benzyl ester

[0113] A solution of (1-methyl-4-nitro-1H-pyrazole-3-carbonyl)glycine benzyl ester (2.1 g, 6.60 mmol) and Fe (2.2 g, 39.62 mmol) in MeOH (4 mL) and saturated NH4Cl (20 mL) was stirred at 80 ° C for 2 hours. The reaction mixture was filtered and the filter cake was washed with EA (100 mL) and H2O (20 mL). The filtrate was neutralized to pH 7 with saturated NaHCO3. The EA layer was separated and evaporated. The residue was purified by SGC (EA=100%, Rf=0.4 in EA) to give (4-amino-1-methyl-1H-pyrazole-3-carbonyl)glycine benzyl ester (1 g, 52% yield) as a white solid. MS (ESI) m / z=289.3[M+H] + .

[0114] Preparation of intermediate 5

[0115] 2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoic acid

[0116] Step 1: Benzyl 2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoate

[0117] To a solution of 2-amino-benzoic acid benzyl ester (500 mg, 2.2 mmol) and pyridine (2 mL) in DCM (15 mL) was added 4-(pivaloyloxy)phenyl)sulfonyl chloride (670 mg, 2.4 mmol) and the resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated and the residue was purified by silica gel column chromatography (eluted with PE / EtOAc=4 / 1, V / V) to give the product as a white solid (1 g, 97% yield). MS (ESI): m / z=468.1[M+H] + .

[0118] Step 2: 2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoic acid

[0119] Benzyl 2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoate (1 g, 2.1 mmol) and Pd / C (250 mg, 10% on C) were mixed with THF (30 mL) and connected to a hydrogenation apparatus. The system was evacuated and then refilled with hydrogen 4 times. The mixture was stirred at room temperature for 3 hours. The mixture was filtered and the filtrate was concentrated to give the crude product (690 mg, 90% yield) as a white solid. MS (ESI): m / z=378.1[M+H] + .

[0120] Preparation of intermediate 6

[0121] (2-Amino-5-fluorobenzoyl)-D-alanine tert-butyl ester

[0122] A mixture of 6-fluoro-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (1 g, 5.5 mmol), D-alanine tert-butyl ester hydrochloride (880 mg, 6 mmol) and TEA (1.6 g, 16.5 mmol) in THF (10 mL) was refluxed for 3 hours. The reaction mixture was quenched with H2O (2 mL) and extracted with EtOAc (5 mL×3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (10-60% EtOAc / petroleum ether) to give (2-amino-5-fluorobenzoyl)-D-alanine tert-butyl ester (850 mg, yield: 89%). MS (ESI): m / z=283.1[M+H] + .

[0123] Preparation of intermediate 7

[0124] (2-Amino-5-cyanobenzoyl)-D-alanine tert-butyl ester

[0125] A mixture of 2-amino-5-cyanobenzoic acid (1 g, 6 mmol), D-alanine tert-butyl ester hydrochloride (1.2 g, 6.6 mmol), HOBt (891 mg, 6.6 mmol), EDCI HCl (1 g, 6.6 mmol) and DIPEA (2.3 g, 18 mmol) in DCM (10 mL) was stirred at room temperature for 3 hours. H2O (2 mL) was added to the reaction mixture. The aqueous phase was separated and extracted with EtOAc (5 mL×3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (10-60% EtOAc / petroleum ether) to give (2-amino-5-cyanobenzoyl)-D-alanine tert-butyl ester (1.3 g, 80% yield). MS (ESI): m / z=312.2 [M+Na] + .

[0126] Preparation of intermediate 8

[0127] (2-Amino-5-methoxybenzoyl)glycine benzyl ester

[0128] A mixture of 2-amino-5-methoxybenzoic acid (500 mg, 3 mmol), glycine benzyl ester hydrochloride (600 mg, 3 mmol), EDCI HCl (634 mg, 3.3 mmol), HOBt (446 mg, 3.3 mmol) and DIPEA (1.2 g, 9 mmol) in DCM (10 mL) was stirred at room temperature overnight. The mixture was quenched with H2O (10 mL) and extracted with DCM (30 mL × 3). The organic phases were combined, washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel column (15% EtOAc / PE) to give (2-amino-5-methoxybenzoyl) glycine benzyl ester (800 mg, 85% yield) as a yellow oil. MS (ESI): m / z 315.1[M+H] + .

[0129] Preparation of intermediate 9

[0130] (S)-2-Amino-3-fluoropropionic acid benzyl ester 2,2,2-trifluoroacetate

[0131] Step 1: (R)-4-Benzyl 3-(tert-butyl)-1,2,3-oxathiazolidine-3,4-dicarboxylate 2,2-dioxide

[0132] Under Ar at -40 DEG C, a solution of (tert-butoxycarbonyl)-D-serine benzyl ester (5g, 16.9mmol) in DCM (170mL) is added dropwise to SOCl(3.1mL, 42.7mmol) in DCM (85mL). Temperature is maintained at <-35 DEG C. Subsequently, the reaction mixture is stirred at -35 DEG C to -40 DEG C for 30 minutes. Py (7.1mL, 87.9mmol) is slowly added to the reaction mixture. The reaction mixture is warmed to room temperature and stirred for 1 hour. The reaction mixture is quenched with frozen water (110mL), washed with saturated NaHCO(110mL), dried over NaSO, filtered and evaporated to obtain the intermediate (7g) in a colorless oil. In a 0 ° C solution, water (110 mL) containing NaIO4 (6 g, 28 mmol) was added dropwise to a solution of intermediate (7 g) / RuCl3 (80 mg, 0.39 mmol) in CH3CN (110 mL). The reaction mixture was then stirred at 0 ° C for 1 hour. The reaction mixture was quenched with saturated NaHCO3 (110 mL) and extracted with EA (150 mL × 3). The organic layers were combined, washed with brine (150 mL), filtered and evaporated. The residue was purified by SGC (0-25% EA / PE, Rf = 0.4 in PE / EA 4 / 1, weak UV, ninhydrin) to give a white solid compound (4.2 g, 69% yield). MS (ESI): m / z = 380.3 [M + Na] + .

[0133] Step 2: (S)-benzyl 2-((tert-butoxycarbonyl)amino)-3-fluoropropionate

[0134] KF (3.9 g, 10.9 mmol), 18-crown ether -6 (1.9 g, 32.8 mmol) are suspended in anhydrous CH3CN (100 mL) and stirred at room temperature for 15 minutes. Subsequently, step 1 compound (3.9 g, 10.9 mmol) is added to the reaction mixture and the mixture is stirred at room temperature overnight. The reaction mixture is evaporated. DCM (60 mL) / 20% H2SO4 (20 mL) is added to the residue and stirred at room temperature for 1.5 hours. The reaction mixture is neutralized to pH 7 with saturated NaHCO3 and extracted with DCM (100 mL × 2). The DCM layers are combined, dried over Na2SO4, filtered and evaporated. The residue is purified by SGC (0-25% EA / PE, Rf=0.5 of the product in PE / EA 4 / 1) to obtain a colorless oily compound (130 mg, 4% yield). MS (ESI): m / z=320.2[M+Na] + .

[0135] Step 3: (S)-2-amino-3-fluoropropionic acid benzyl ester 2,2,2-trifluoroacetate

[0136] A mixture of the compound of step 2 (130 mg, 0.44 mmol) in TFA (1 mL) / DCM (4 mL) was stirred at room temperature for 1.5 hours. DCM (20 mL) was added to the reaction mixture and evaporated to dryness. The residue was dissolved in DCM (20 mL) and evaporated to dryness again to give a crude product of (S)-2-amino-3-fluoropropionic acid benzyl ester 2,2,2-trifluoroacetate (200 mg) as a colorless oil. This crude product was used directly in the next step. MS (ESI): m / z=198.2[M+H] + .

[0137] Preparation of intermediate 10

[0138] N-(2-Aminobenzoyl)-O-methyl-D-serine benzyl ester

[0139] Step 1: N-(tert-Butoxycarbonyl)-O-methyl-D-serine benzyl ester

[0140] A mixture of N-(tert-butoxycarbonyl)-O-methyl-D-serine (1 g, 4.6 mmol), CbzCl (1.56 g, 9.2 mmol), TEA (0.9 g, 9.2 mmol) and DMAP (112 mg, 0.92 mmol) in DCM (10 mL) was stirred at 0 ° C overnight. The mixture was quenched with H2O (10 mL) and extracted with DCM (30 mL×3). The organic phases were combined, washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel column (13% EtOAc / PE) to give the product N-(tert-butoxycarbonyl)-O-methyl-D-serine benzyl ester (1.3 g, 92% yield) as a colorless oil. MS (ESI): m / z 210.1[M-99] + .

[0141] Step 2: O-methyl-D-serine benzyl ester

[0142] A mixture of N-(tert-butoxycarbonyl)-O-methyl-D-serine benzyl ester (1.3 g, 4.2 mmol) and TFA (5 mL) in DCM (50 mL) was stirred at room temperature for 2 hours. The mixture was dried under vacuum to give the product as a white solid (800 mg, crude material). The crude product was used directly in the next step. MS (ESI): m / z = 210.2 [M+H] + .

[0143] Step 3: N-(2-aminobenzoyl)-O-methyl-D-serine benzyl ester

[0144] A mixture of the product of step 2 (200 mg, 0.62 mmol), 2H-benzo[d][1,3]oxazine-2,4(1H)-dione (186 mg, 1.14 mmol) and TEA (192 mg, 1.9 mmol) in THF (5 mL) was stirred at room temperature overnight. The reaction mixture was concentrated and purified by silica gel column (25% EtOAc / PE) to give N-(2-aminobenzoyl)-O-methyl-D-serine benzyl ester (100 mg, 49% yield) as a colorless oil. MS (ESI): m / z 329.1 [M+H] + .

[0145] Preparation of intermediate 11

[0146] ((1R,2R)-2-Amino-5,5-difluorocyclohexane-1-carbonyl)glycine tert-butyl ester

[0147] Step 1: ((1R,2R)-2-(((benzyloxy)carbonyl)amino)-5,5-difluorocyclohexane-1-carbonyl)glycine tert-butyl ester

[0148] A mixture of (1R, 2R)-2-(((benzyloxy)carbonyl)amino)-5,5-difluorocyclohexane-1-carboxylic acid (900 mg, 2.88 mmol), HOBt (388 mg, 2.88 mmol), DIPEA (1.11 g, 8.63 mmol) and HBTU (1.42 g, 3.74 mmol) in THF (30 mL) was stirred at room temperature for 5 minutes. Glycine tert-butyl ester (565 mg, 4.31 mmol) was subsequently added and the mixture was stirred at room temperature for 2 hours. Water (100 mL) was added to the reaction mixture and extracted with EtOAc (200 mL×3). The organic layers were combined, washed with brine (150 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (eluted with EtOAc / PE=1 / 3 to 1 / 2, V / V) to give the product (1 g, 82% yield) as a white solid. MS (ESI): m / z = 449.2 [M + Na] + .

[0149] Step 2: ((1R,2R)-2-amino-5,5-difluorocyclohexane-1-carbonyl)glycine tert-butyl ester

[0150] Step 1 compound (1 g, 2.3 mmol) and Pd / C (250 mg, 10% on C) were mixed with THF (30 mL) and connected to a hydrogenation apparatus. The system was evacuated and then refilled with hydrogen 4 times. The mixture was stirred at room temperature overnight. The mixture was filtered and the filtrate was concentrated to give a crude product (690 mg, 90% yield) as a white solid. MS (ESI): m / z=293.1[M+H] + .

[0151] Example 1

[0152] Step 1: (2-Amino-5-bromobenzoyl)glycine benzyl ester

[0153] To a solution of 6-bromo-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (2 g, 8.26 mmol) in MeCN (50 mL) was added glycine benzyl ester hydrochloride (1.83 g, 9.08 mmol) and TEA (1.25 g, 12.39 mmol). The mixture was heated to 60 ° C and stirred for 16 hours and monitored by LCMS. The resulting mixture was extracted with EA (30 mL×3). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with PE / EA (1 / 9) to give (2-amino-5-bromobenzoyl)glycine benzyl ester (2.3 g, 76% yield) as a brown solid. MS (ESI): m / z=365.0[M+H] + .

[0154] Step 2: (2-Amino-5-(1-methyl-1H-pyrazol-5-yl)benzoyl)glycine benzyl ester

[0155] A mixture of (2-amino-5-bromobenzoyl)glycine benzyl ester (600 mg, 1.65 mmol), 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (412 mg, 1.98 mmol), Pd(dppf)Cl2 (60 mg, 0.08 mmol) and potassium acetate (485 mg, 4.95 mmol) in dioxane / HO (30 mL, 5:1) was stirred at 80 ° C for 16 hours under Ar. The reaction was monitored by LCMS. The resulting mixture was extracted with EA (30 mL×3). The organic layers were combined, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by SGC (0-30% EA / PE, product Rf = 0.5 in PE / EA 2 / 1) to give (2-amino-5-(1-methyl-1H-pyrazol-5-yl)benzoyl)glycine benzyl ester (340 mg, 68% yield) as a light brown gum. MS (ESI): m / z = 365.1 [M+H] + .

[0156] Step 3: 4-(N-(2-((2-(benzyloxy)-2-oxoethyl)carbamoyl)-4-(1-methyl-1H-pyrazol-5-yl)phenyl)sulfamoyl)phenyl pivalate

[0157] To a solution of (2-amino-5-(1-methyl-1H-pyrazol-5-yl)benzoyl)glycine benzyl ester (350 mg, 0.96 mmol) in DCM (20 mL) was added 4-(chlorosulfonyl)phenyl pivalate (397 mg, 1.44 mmol) and Py (1137 mg, 14.4 mmol). The mixture was stirred at room temperature for 16 hours. Water (30 mL) was added to the reaction mixture and extracted with DCM (30 mL×3). The organic layers were combined, washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and evaporated. The residue was purified by silica gel column chromatography (EtOAC / petroleum ether, 0% to 80%) to give a brown gum (240 mg, 41% yield). MS (ESI): m / z=605.1[M+H] + .

[0158] Step 4: (5-(1-methyl-1H-pyrazol-5-yl)-2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoyl)glycine

[0159] To a suspension of step 3 compound (200 mg, 0.33 mmol) in THF (30 mL) was added 10% Pd / C (50 mg) at room temperature. The resulting mixture was stirred overnight at room temperature under an H2 atmosphere. The reaction was monitored by LCMS. After completion, the mixture was filtered. The filtrate was concentrated to give a crude product, which was purified by preparative HPLC (using NH4HCO3 buffer: A: 10 mM NH4HCO3 / water; B: acetonitrile; column: Waters XBridge Peptide BEH C18, 19 × 250 mm, 10 μm, ) was purified to obtain the title compound (138 mg, 73% yield) as a white solid. MS (ESI) m / z: 515.3 [M+H] + LCMS purity: 100.00% (214 nm), RT = 1.749 min.

[0160] 1 H NMR (400MHz, DMSO-d6): δ12.81(s,1H),11.72(s,1H),9.35(s,1H),7.91-7.80(m,3H),7.67(d,J=8Hz,1H),7.60(d,J=8Hz ,1H),7.46(d,J=1.6Hz,1H),7.32(d,J=8Hz,2H),6.42(d,J=2.0Hz,1H), δ3.94(d,J=5.6Hz,2H),3.80(s,3H),1.27(s,9H).

[0161] The following compounds were prepared using a method similar to Example 1 using different intermediates:

[0162] Example 12

[0163] Step 1: Diphenylmethyl ((tritylamino)methyl)phosphonate

[0164] The mixture was concentrated and the residue was purified by silica gel column chromatography (eluted with EtOAc / PE=1 / 1, V / V) to give the product (10 g, 82% yield) as a white solid. MS (ESI): m / z=1089.2[2M+Na] + .

[0165] Step 2: (Aminomethyl)phosphonic acid diphenylmethyl ester hydrochloride

[0166] A mixture of diphenylmethyl ((tritylamino)methyl)phosphonate (2 g, 3.75 mmol) and HCl / dioxane (4 mol / L, 3.8 mL) in THF (40 mL) was stirred at room temperature overnight. The mixture was concentrated to give the crude product (2.1 g, 90% yield). The crude product was used directly in the next step. MS (ESI): m / z = 292.2 [M+H] + .

[0167] Step 5: 4-(N-(2-(((bis(benzyloxy)phosphoryl)methyl)carbamoyl)phenyl)sulfamoyl)phenyl pivalate

[0168] A mixture of 2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoic acid (350 mg, 0.93 mmol), diphenylmethyl (aminomethyl)phosphonate hydrochloride (crude material 693 mg, 1.3 mmol), HOBt (125 mg, 0.93 mmol), DIPEA (479 mg, 3.7 mmol) and HBTU (457 mg, 1.2 mmol) in DCM (20 mL) was stirred at room temperature for 2 hours. The mixture was concentrated and the residue was purified by silica gel column chromatography (eluted with PE / EtOAc=1 / 1, V / V) to give the product as a white solid (550 mg, 53% yield). MS (ESI): m / z=651.3 [M+H] + .

[0169] Step 6: ((2-((4-(pivaloyloxy)phenyl)sulfonamido)benzamido)methyl)phosphonic acid Step 5 compound (520 mg, 0.8 mmol) and Pd / C (150 mg, 10%) were mixed with THF (20 mL) and connected to a hydrogenation apparatus. The system was evacuated and then refilled with hydrogen 4 times. The mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered. The filtrate was concentrated and directly purified by preparative HPLC (A: 0.1% TFA / water, B: CH3CN; column: Xbridge BEH peptide C18, 19 mm × 250 mm) and lyophilized to give compound 12 (210 mg, 86%) as a yellow solid. MS (ESI): m / z=471.1 [M+H] + .

[0170] 1 H NMR (400MHz, DMSO-d6): δ11.70(s,1H),9.02(t,J=5.8Hz,1H),8.70(s,2H),7.84(d,J=8.8Hz,2H),7.80(d ,J=7.6Hz,1H),7.50-7.43(m,2H),7.30-7.28(m,2H),7.14-7.10(m,1H),3.58-3.53(m,2H),1.27(s,9H).

[0171] Example 13

[0172] Step 1: 2-(Tritylthio)ethan-1-amine

[0173] A mixture of 2-aminoethane-1-thiol hydrochloride (2 g, 17.6 mmol) and triphenylmethanol (4.58 g, 17.6 mmol) in TFA (15 mL) was stirred at room temperature for 1 hour. The solution was co-evaporated with toluene and the residue was dissolved in ethyl acetate (300 mL). The resulting mixture was adjusted to pH 8 with 1 M NaOH aqueous solution, washed with water (200 mL) and saturated NaCl solution (150 mL). The organic layer was dried over MgSO4, filtered and concentrated to give the crude product of 2-(tritylthio)ethan-1-amine (5.7 g, 99% yield) as a brown solid. The crude product was used directly in the next step. MS (ESI): m / z=342.0 [M+Na] + .

[0174] Step 2: 2-amino-N-(2-(tritylthio)ethyl)benzamide

[0175] A mixture of 2-(tritylthio)ethan-1-amine (2.94 g, 9.2 mmol), 2H-benzo[d][1,3]oxazine-2,4(1H)-dione (1.5 g, 9.2 mmol) and TEA (1.86 g, 18.4 mmol) in MeCN (30 mL) was heated to reflux for 4 hours. The mixture was concentrated and purified by silica gel column chromatography (eluted with EtOAc / PE=1 / 3, V / V) to give the product 2-amino-N-(2-(tritylthio)ethyl)benzamide (2 g, 50% yield) as a white solid. MS (ESI): m / z=461.0 [M+Na] + .

[0176] Step 3: 4-(N-(2-((2-(tritylthio)ethyl)carbamoyl)phenyl)sulfamoyl)phenyl pivalate

[0177] To a solution of 2-amino-N-(2-(tritylsulfanyl)ethyl)benzamide (500 mg, 1.14 mmol) and pyridine (2 mL) in DCM (15 mL) was added 4-(chlorosulfonyl)phenyl pivalate (379 mg, 1.37 mmol) and stirred at room temperature overnight. The mixture was concentrated and purified by preparative HPLC (A: 0.1% TFA / water, B: CH3CN; column: Xbridge BEH peptide C18, 19 mm × 250 mm) to give the product, 4-(N-(2-((2-(tritylsulfanyl)ethyl)carbamoyl)phenyl)sulfamoyl)phenyl pivalate (700 mg, 91% yield) as a white solid. MS (ESI): m / z = 701.1 [M+Na] + .

[0178] Step 4: 2-(2-((4-(pivaloyloxy)phenyl)sulfonamido)benzamido)ethane-1-sulfonic acid A mixture of 4-(N-(2-((2-(tritylsulfanyl)ethyl)carbamoyl)phenyl)sulfamoyl)phenyl pivalate (650 mg, 0.96 mmol), m-CPBA (1.65 g, 9.6 mmol) and NaOAc (197 mg, 2.4 mmol) in DCM (20 mL) was stirred at room temperature for 2 hours. The mixture was concentrated and directly purified by preparative HPLC (A: 0.1% TFA / water, B: CH3CN; column: Xbridge BEH Peptide C18, 19 mm×250 mm) and lyophilized to give the product, target compound 13 (110 mg, 24% yield), as a white solid. MS (ESI): m / z=485.1 [M+H] + .

[0179] 1 H NMR (400MHz, DMSO-d6): δ11.77(s,1H),8.83(t,J=5.0Hz,1H),7.81-7.78(m,2H),7.62(d,J=7.6Hz,1H),7.5 0-7.43(m,2H),7.30-7.27(m,2H),7.16-7.09(m,1H),3.50-3.46(m,2H),2.67(t,J=7.2Hz,2H),1.27(s,9H).

[0180] Example 14: Preparation of O-(methyl-d3)-N-(2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoyl)-D-serine (25)

[0181] a) To a solution of benzyl(tert-butyloxycarbonyl)-D-serine (500.0 mg, 1.7 mmol) in MeCN (8 mL) at room temperature under nitrogen was added AgO (588.5 mg, 2.5 mmol) and CDI (0.3 mL, 5.0 mmol) in sequence. Stirring was continued for 18 hours in the dark. The reaction mixture was filtered, and the filtrate was concentrated and purified by silica gel column chromatography (eluted with 10% EtOAc / PE, v / v) to give the product 25a (400 mg, 76%) as a colorless liquid. MS (ESI): m / z 212.7 [M-100] + .

[0182] b) Under nitrogen at room temperature, HCl / Dioxane (4M, 3 mL) was added to 25a (0.4 g, 1.3 mmol). Stirring was continued for 2 hours, and the mixture was concentrated to afford the product 25b (300 mg, crude) as a white solid. MS (ESI): m / z = 212.7 [M+H] + The compound was used directly in the next reaction without purification.

[0183] c) To a solution of intermediate 5 (346 mg, 0.92 mmol, 1.0 eq.) and 25b (228 mg, 0.92 mmol, 1.0 eq.) in DCM (10 mL) at room temperature under nitrogen was added HBTU (452 ​​mg, 1.19 mmol, 1.3 eq.), HOBT (124 mg, 0.92 mmol, 1.0 eq.), and DIEA (1.18 g, 9.17 mmol, 10.0 eq.). Stirring was continued for 20 hours. The reaction solution was concentrated and purified by silica gel column chromatography (eluted with PE / EA 2:1, v / v) to afford the product 25c (560 mg, 100%) as a colorless oil.

[0184] d) To a solution of 25c (740 mg, 0.92 mmol, 1.0 eq.) in EtOAc (20 mL) at room temperature were added 5% Pd / C (70 mg) and Pd(OH)2 (70 mg). The mixture was stirred at 40°C for 16 hours under hydrogenation. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated and purified by reverse phase column chromatography to afford the product 25 (450 mg, 78%) as a white solid. (ESI): m / z = 481.75 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ12.98(s,1H),11.38(s,1H),9.04(d,J=7.2Hz,1H),7.83-7.78(m,3H),7.51- 7.48(m,2H),7.30-7.27(m,2H),7.19-7.15(m,1H),4.62-4.58(m,1H),3.77-3.66(m,2H),1.27(s,9H).

[0185] Biological activity test

[0186] For compound preparation, all compounds were dissolved in DMSO at a concentration of 10 mM and stored at -20°C.

[0187] Prepare 1x assay buffer and serially dilute the positive compound (Sivelestat) and the test compound in DMSO 3-fold for a total of 10 dilutions. Add 1 μL of the serially diluted compound and 65.67 μL of assay buffer to the plate to obtain a 3x working solution of the positive compound and the test compound (final DMSO concentration of 1.5%). Seal the plate with film and shake on a shaker for 15 minutes.

[0188] Prepare 1x activation buffer and dilute rhELA2 in activation buffer containing DPPI. Incubate at 37°C for 2 hours to activate rhELA2. Add 5 μl of the 3x diluted compound working solution to a 384-well plate. Dilute rhELA2 with 1x assay buffer to make a 3x rhELA2 working solution. Add 5 μl of the 3x rhELA2 working solution to each well of the 384-well plate. Seal the plate and incubate at room temperature in the dark for 30 minutes.

[0189] Prepare a 3x substrate solution in 1x assay buffer. Add 5 μl of the 3x substrate working solution to a 384-well plate. Centrifuge the plate at 1000 rpm for 1 minute. Incubate at room temperature in the dark for 1 hour. Read the fluorescence (FLU) at 360 nm / 450 nm on a plate reader.

[0190] Inhibition was calculated as % Inhibition = (1 - (FLU compound - FLU-positive wells) / (FLU reference wells - FLU-positive wells)). FLU reference wells were treated with enzyme alone without inhibitor, while FLU-positive wells were treated with enzyme and 3000 nM Sivelestat-2. IC50 values ​​were then calculated using GraphPad 8.0 software using the formula Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)).

[0191] The compounds of the present invention have an IC50 of less than 100 nM; in particular, they have an IC50 of less than 50 nM; more preferably, the compounds have an IC50 of less than 10 nM; the compounds of the present invention show excellent inhibitory activity.

[0192] Animal testing 1

[0193] Experimental animals: ICR mice, SPF grade, male, body weight range at the time of administration: 25-28 g.

[0194] Animal grouping: divided into 4 groups, 10 animals in each group, namely blank control group, model group, experimental group (Compound 20) and positive control group (Sivelestat sodium).

[0195] Establishment of LPS-induced acute lung injury mouse model: intramuscular injection of mice The animals were anesthetized with 50 μg of ethanol (0.05 mL / animal). The animals were fixed in the supine position, the trachea in the neck was isolated, and lipopolysaccharide (LPS) (1 mg / mL, 35 μL / animal) was sprayed with a high-pressure atomizer. The skin of the neck was sutured and disinfected with iodine.

[0196] Dosage: The experimental group and the positive control group were intravenously injected with compound 20 or sivelestat sodium once at 0.5h, 4h and 12h after LPS modeling, respectively. The dose of each administration was 25 mg / kg, and the total daily dose was 75 mg / kg.

[0197] 24 hours after LPS airway inhalation in mice, the mice were killed, the trachea was isolated, and the tube was intubated. The left lung lobe was ligated, and the lungs were lavaged with 0.5 mL of normal saline (containing 1% serum and 2 U / mL heparin), repeated twice, and the lavage fluid mixed three times was placed on ice for later use. The left lung lobe of animals 1 to 8 in the blank control group, model group, experimental group, and positive control group was taken, weighed, and 0.9% NaCl was added at a ratio of 1:6. After homogenization, the supernatant was centrifuged at 4°C and 10000g for 10 minutes, and the supernatant was used to determine the content of inflammatory factors IL-1β and TNF-α. The results of each group are shown in the following table. Note: Compared with the blank control group: ## P<0.01,### P<0.001; compared with the model group: * P<0.05, ** P<0.01.

[0198] In the LPS-induced acute lung injury mouse model, compound 20 was significantly better than sivelestat sodium in inhibiting the inflammatory factors IL-1β and TNF-α.

[0199] Animal testing 2

[0200] Animal grouping: divided into 4 groups, 8 animals in each group, namely blank control group, model group, experimental group (Compound 20) and positive control group (Sivelestat sodium).

[0201] The rat model of acute lung injury induced by hydrochloric acid was established: 0.1 mL of 0.1 N HCL was inhaled into the airways of rats.

[0202] Dosage: The experimental group was administered with compound 20, and the positive control group was administered with sivelestat sodium. Both groups were administered twice by tail vein injection, immediately after modeling and again 2.5 hours after modeling. The total dose of compound 20 administered to the experimental group was 120 mg / kg, and the total dose of sivelestat sodium administered to the positive control group was 30 mg / kg.

[0203] Five hours after modeling, the rats were sacrificed and alveolar lavage was performed. The inflammatory cells, protein exudate, pulmonary hemorrhage, and NE enzyme activity in the lavage fluid were measured. The results of each group are shown in the table below. Note: Compared with the blank control group: ## P<0.01, ### P<0.001; compared with the model group: * P<0.05, ** P<0.01, *** P<0.001.

[0204] In the hydrochloric acid-induced acute lung injury rat model, compound 20 improved pulmonary hemorrhage and reduced protein exudate and white blood cell count in bronchoalveolar lavage fluid more significantly than sivelestat sodium.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof in, R1 is selected from R6-C(=O)-, or R1 is connected to a benzene ring to form a five-membered or six-membered ring, and the five-membered or six-membered ring is optionally substituted by R6; R6 is selected from tert-butyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl; Ring A is selected from 5-10 membered cycloalkyl, heterocycloalkyl, heteroaryl, aryl, and the ring A is optionally substituted by one or more R5; R2 and R5 are each independently selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 heteroaryl; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C1-C6 haloalkyl; R4 is selected from -COOH, -SO3H, -PO3H2; m, n are independently 0, 1, 2, 3; The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, heteroaryl groups are unsubstituted or optionally substituted with one or more deuterium, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, amino, -CONH2, -COOH.

2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein the formula I has a structure as shown in formula Ia, in, Ring A is phenyl, pyrazolyl, imidazolyl, pyrazinyl, cyclohexyl, optionally substituted by one or more R5; R4 is selected from -COOH, -SO3H, -PO3H2; R2, R3, R5, m, and n are as defined in claim 1.

3. The compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the formula I has a structure as shown in formula Ib, R2, R3, R5, m are as defined in claim 1, p is 0, 1 or 2; The prerequisite is that in formula Ib, R2, R3, and R5 are not hydrogen at the same time.

4. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein the formula I has a structure as shown in formula Ic, R4 is selected from -SO3H, -PO3H2; R2 and R5 are each independently selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 heteroaryl; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C1-C6 haloalkyl; n, m, p are independently 0, 1 or 2.

5. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein R2 is optionally selected from hydrogen and fluorine.

6. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein R3 is selected from hydrogen, methyl, fluoro-substituted methyl, methoxymethyl, and deuterated methoxymethyl.

7. The compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R5 is selected from hydrogen, fluorine, methyl, cyano, methoxy, unsubstituted or methyl-substituted imidazolyl, pyrazolyl, oxazolyl, isoxazolyl.

8. The compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof, which is selected from the following compounds:

9. A pharmaceutical composition comprising a compound represented by formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

10. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating or preventing diseases related to abnormal increase of neutrophil elastase.

11. The use according to claim 10, wherein the disease comprises autoimmune inflammation, infectious inflammation, traumatic inflammation, allergic inflammation, systemic inflammation, respiratory system inflammation, lung inflammation, and pain.

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