Neutrophil elastase inhibitor and application thereof

Abstract

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

Description

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 that is mainly distributed in the azure granules of neutrophils. In the acute phase of inflammation, neutrophils are the first cells to be 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 neutrophils recruit to the site of inflammation by degrading bacterial structural proteins, reduce bacterial infection, and participate in inflammation regulation.

[0003] However, excessive release and accumulation of NE can damage normal physiological tissue structures, and also induce increased microvascular permeability and excessive mucus secretion. In addition, excessive release of NE can also lead to some serious cardiopulmonary diseases. Diseases associated with excessive expression of NE include acute lung injury (ALI), acute respiratory distress syndrome (ARDS), acute lung injury / acute respiratory distress syndrome with systemic inflammatory response syndrome (SIRS), etc.

[0004] In the past years of research, researchers have designed and developed a variety of small molecule NE inhibitor compounds of different structural types. Including pivalate ester derivatives, such as Sivelestat (Sivelestat, which has an inhibitory activity of 44nM on neutrophil elastase) disclosed in Document 1, which is the world's first drug used to treat acute lung injury (ALI) / acute respiratory distress syndrome (ARDS) to improve systemic inflammatory response syndrome (SIRS). In addition, many research reports have also verified that Sivelestat has therapeutic and protective effects in the fields of sepsis, cystic fibrosis, chronic obstructive pulmonary disease (COPD) and ischemia-reperfusion injury of various tissues. Other types of small molecule NE inhibitors also include pyridone derivatives (such as Alvelestat), dihydropyrimidone derivatives (such as BAY 85-8501), small molecule peptides (such as POL6014), etc.

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

[0006] Reference 1: EP 0347168 B1 Summary of the invention

[0007] The first aspect of the present invention provides a compound represented by formula I

[0008]

[0009] or a pharmaceutically acceptable salt thereof, wherein

[0010] R 1 Selected from (1) R 6 -C(=O)-; or (2) R 1 is connected to the benzene ring to form a five-membered or six-membered ring, wherein the five-membered or six-membered ring is optionally 6 replace;

[0011] R 6 Selected from tert-butyl, C 3 -C 6 Cycloalkyl, C 3 -C 6 Heterocycloalkyl;

[0012] Ring A is selected from 5-10 membered cycloalkyl, heterocycloalkyl, heteroaryl, aryl, and the ring A is optionally substituted by one or more R 5 replace;

[0013] R 2 , R 5 Each independently selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 3 -C 6 Cycloalkyl, C 3 -C 6 Heterocycloalkyl, C 3 -C 6 heteroaryl;

[0014] R 3 Selected from hydrogen, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 3 -C 6 Cycloalkyl, C 1 -C6 Haloalkyl;

[0015] R 4 Optional from -COOH, -SO 3 H, -PO 3 H 2 ;

[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, C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, C 1 -C 3 Deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, amino, oxo, -CONH 2 、-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, cyclohexyl, and the ring A is unsubstituted or optionally substituted with one or more R 5 Substitution; said R 5 Optionally selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 3 -C 6 Cycloalkyl, C 3 -C 6 Heterocycloalkyl, C 3 -C 6 Heteroaryl.

[0020] In some embodiments of the present invention, R 5 It can be 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, cyclohexyl, and the ring A is unsubstituted or optionally substituted with one or more R 5 Substitution; said R 5Optionally selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, methyl, methoxy, unsubstituted or methyl-substituted imidazolyl, pyrazolyl, isoxazolyl.

[0022] In some embodiments of the present invention, R 6 It is tert-butyl.

[0023] In some embodiments of the present invention, R 6 It is cyclopropyl, cyclobutyl, azetidinyl, and the cyclopropyl, cyclobutyl, azetidinyl is optionally substituted by one or more halogens or methyl.

[0024] In some embodiments of the present invention, R 6 Cyclopropyl, oxetanyl, azetidinyl may be substituted by 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]

[0027] in,

[0028] Ring A is a 5- to 6-membered ring optionally containing 1 to 3 heteroatoms, and the ring A is optionally substituted by one or more R 5 replace;

[0029] R 4 Selected from -COOH, -SO 3 H, -PO 3 H 2 ;

[0030] R 2 , R 5 Each independently selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 3 -C 6 Cycloalkyl, C 3 -C 6 Heterocycloalkyl, C 3 -C 6 heteroaryl;

[0031] R 3 Selected from hydrogen, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 3 -C 6 Cycloalkyl, C1 -C 6 Haloalkyl;

[0032] m, n are independently 0, 1, 2, 3;

[0033] The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, and heteroaryl groups may be unsubstituted or optionally substituted with one or more of the following substituents: deuterium, C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, C 1 -C 3 Deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, amino, -CONH 2 、-COOH.

[0034] In some embodiments of the present invention, in the formula Ia, ring A is phenyl, pyrazolyl, imidazolyl, pyrazinyl, cyclohexyl, and ring A is optionally replaced by one or more R 5 replace.

[0035] 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:

[0036]

[0037] R 2 , R 5 Each independently selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 3 -C 6 Cycloalkyl, C 3 -C 6 Heterocycloalkyl, C 3 -C 6 heteroaryl;

[0038] R 3 From hydrogen, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 3 -C 6 Cycloalkyl, C 1 -C 6 Haloalkyl;

[0039] m, p are independently 0, 1 or 2;

[0040] The premise is R 2 , R3 , R 5 Not hydrogen at the same time.

[0041] The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, and heteroaryl groups may be unsubstituted or optionally substituted with one or more of the following substituents: deuterium, C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, C 1 -C 3 Deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, amino.

[0042] In some embodiments of the present invention, R 2 Optionally selected from hydrogen and fluorine.

[0043] In some embodiments of the present invention, R 3 Optionally selected from hydrogen, methyl, fluorine-substituted methyl, methoxymethyl, deuterated methoxymethyl.

[0044] In some embodiments of the present invention, R 5 Optionally selected from hydrogen, fluorine, methyl, cyano, methoxy, unsubstituted or alkyl-substituted imidazolyl, pyrazolyl, oxazolyl, isoxazolyl.

[0045] 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:

[0046]

[0047] In the formula Ic,

[0048] R 4 Optional from -SO 3 H, -PO 3 H 2 ;

[0049] R 2 , R 5 Each independently selected from hydrogen, halogen, cyano, trifluoromethyl, nitro, amino, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 3 -C 6 Cycloalkyl, C 3 -C 6 Heterocycloalkyl, C 3 -C 6 heteroaryl;

[0050] R 3From hydrogen, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, C 3 -C 6 Cycloalkyl, C 1 -C 6 Haloalkyl;

[0051] n, m, p are independently 0, 1 or 2;

[0052] The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, and heteroaryl groups may be unsubstituted or optionally substituted with one or more of the following substituents: deuterium, C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, C 1 -C 3 Deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, amino, -CONH 2 、-COOH.

[0053] In some embodiments of the present invention, in Formula I, Formula Ia, Formula Ic, R 4 Optional from -SO 3 H, -PO 3 H 2 .

[0054] 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 R 5 replace.

[0055] In some embodiments of the present invention, in Formula I, Formula Ia, Formula Ib, Formula Ic, R 3 It is hydrogen, methyl, fluoromethyl, methoxymethyl, or deuterated methoxymethyl.

[0056] In some embodiments of the present invention, in Formula I, Formula Ia, Formula Ib, Formula Ic, R 2 It is hydrogen, fluorine, cyano, trifluoromethyl, nitro, and amino.

[0057] In some embodiments of the present invention, in Formula I, Formula Ia, Formula Ib, Formula Ic, R 5 It is hydrogen, fluorine, cyano, trifluoromethyl, nitro, amino, unsubstituted or methyl-substituted imidazolyl, pyrazolyl, oxazolyl or isoxazolyl.

[0058] In some embodiments of the present invention, in Formula I, Formula Ia, Formula Ib, Formula Ic, when a chiral center exists, its configuration may be R configuration, S configuration, or racemic form.

[0059] The compound represented by formula I or a pharmaceutically acceptable salt thereof of the present invention has the following structure:

[0060]

[0061]

[0062] In another aspect of the present invention, a pharmaceutical composition is provided, 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.

[0063] 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.

[0064] 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 a tablet, capsule, granule, etc.

[0065] The compound of the present invention can inhibit the activity of neutrophil elastase, is a NE inhibitor, and can be used to prevent or treat related diseases caused by abnormal increase of neutrophil elastase.

[0066] 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 drug for treating or preventing related diseases caused by an abnormal increase in neutrophil elastase.

[0067] The diseases include autoimmune inflammation, infectious inflammation, traumatic inflammation, allergic inflammation, systemic inflammation, respiratory system inflammation, lung inflammation, and pain.

[0068] 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

[0069] Explanation of terms

[0070] "Halogen" includes fluorine, chlorine, bromine and iodine.

[0071] "Alkyl" refers to a saturated aliphatic hydrocarbon group, which may be a straight chain or branched chain alkyl group, preferably C 1 -C 6 Alkyl, more preferably C 1 -C 3 Alkyl. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, etc. Alkyl can be unsubstituted or substituted. When substituted, the substituent can be in any position that can be substituted.

[0072] "Alkoxy" is -O-alkyl or -O-cycloalkyl, non-limiting examples of -O-alkyl include: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, etc. Non-limiting examples of -O-cycloalkyl include: cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, etc. The alkoxy group may be further substituted on the alkyl or cycloalkyl group.

[0073] "Cycloalkyl" refers to a saturated or partially saturated monocyclic or polycyclic alkane substituent, preferably C 3 -C 6 Cycloalkyl, non-limiting examples include: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, etc. The cycloalkyl may be unsubstituted or further substituted with a substituent. When substituted, the substituent may be at any position on the ring that may be substituted.

[0074] "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. 3 -C 6 Heterocycloalkyl. Non-limiting examples include: oxetanyl, azetidinyl, oxirane, etc. The heterocycloalkyl may be unsubstituted or further substituted with a substituent. When substituted, the substituent may be at any position on the ring that can be substituted.

[0075] "Heteroaryl" refers to a 5 to 10 membered heteroaromatic group containing 1 to 4 heteroatoms. Heteroatoms include nitrogen, oxygen and sulfur atoms. Non-limiting examples include imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridyl, pyrimidyl, pyrazinyl, etc. The heteroaryl may be attached to a ring carbon atom or to a heteroatom. The heteroaryl may be unsubstituted or further substituted with a substituent. When substituted, the substituent may be at any position on the ring that may be substituted.

[0076] "Aryl" refers to a 6- to 10-membered all-carbon monocyclic or fused polycyclic ring with a conjugated π electron system. For example, 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.

[0077] The alkyl, alkoxy, cycloalkyl, heterocycloalkyl and heteroaryl groups described in the present invention, when substituted, are preferably substituted by one or more of the following substituents: deuterium, C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, C 1 -C 3 deuterated alkoxy, halogen, cyano, trifluoromethyl, nitro, amino, oxo (=O), -CONH 2 、-COOH.

[0078] "Oxo" refers to a =0 substituent.

[0079] "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 hydrates of salts.

[0080] 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.

[0081]

[0082] Where R 1 , R 2 , R 3 , R 5 , m, and p have the same definition as in Formula I, and Pg is a protecting group, such as benzyl, tert-butyl, and the like.

[0083] In some embodiments of the present invention, the compounds of the present invention can be prepared by the following method of Scheme 2: 1 The substituted carboxylic acid compound reacts with the compound of formula M4 in the presence of a condensing agent, and then the protecting group Pg is removed to obtain a compound.

[0084]

[0085] Where R 1 , R 2 , R 3 , R 5 , m, and p have the same definition as in Formula I, and Pg is a protecting group, such as benzyl, tert-butyl, and the like.

[0086] In some embodiments of the present invention, the compound of the present invention can be prepared by the method of the following route 3:

[0087] Alternatively, replace Equation M7 with Equation Reaction preparation.

[0088] Where R 1 , R 2 , R 3 , R 5 , m, and p have the same definition as in Formula I, and Pg is a protecting group, such as benzyl, tert-butyl, and the like.

[0089] In a specific embodiment of the present invention, when the ring A of the compound is a non-benzene ring, it can be prepared by methods similar to those in Schemes 1 to 3.

[0090] 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 from the market or prepared by methods known in the art. Unless otherwise specified, the drug configuration, biological activity test, various index detection and animal modeling methods related to the biological activity test and animal test of this patent can be carried out by conventional methods in the art.

[0091] Intermediate preparation

[0092] Preparation of intermediate 1

[0093]

[0094] Step 1: 4-(Benzylthio)-2-fluorophenol

[0095]

[0096] 4-Bromo-2-fluorophenol (2 g, 10.5 mmol), phenylmethyl mercaptan (2 g, 15.8 mmol), DIPEA (2.7 g, 21 mmol), Pd 2 dba 3 A mixture of 4-(phenylmethylthio)-2-fluorophenol (1 g, 42% yield) 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).

[0097] MS (ESI): m / z = 235.1 [M + H] + .

[0098] Step 2: 4-(Benzylthio)-2-fluorophenyl pivalate

[0099]

[0100] 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 h. H 2 O (2 mL). The aqueous phase was separated and extracted with EtOAc (5 mL×3). The combined organic layers were washed with anhydrous Na 2 SO 4 Dry, filter and evaporate. 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] + .

[0101] Step 3: 4-(Chlorosulfonyl)-2-fluorophenyl pivalate

[0102] 4-(Benzylthio)-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) / H 2 O (0.5 mL) in CH 3 The solution in CN (12 mL) was stirred at room temperature for 15 min. EA (100 mL) was added to the reaction mixture and H 2 O (50 mL) and brine (50 mL). The organic layer was washed with Na 2 SO 4 Dry, filter and evaporate. 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] + .

[0103] Preparation of intermediate 2

[0104]

[0105] Step 1: 4-Bromo-2-pivalamidophenyl pivalate

[0106]

[0107] Pivaloyl chloride (1.5 mL, 12.2 mmol) was added dropwise to a solution of 2-amino-4-bromophenol (2.5 g, 13.3 mmol) / DIPEA (3.3 mL, 20 mmol) in DCM (50 mL) at 0 ° C. The reaction mixture was then stirred at room temperature for 2 hours. The reaction mixture was evaporated. The residue was purified by SGC (0-20% EA / PE, Rf=0.7 in PE / EA 4 / 1) to give 4-bromo-2-pivalamidophenyl pivalate (1.6 g, 33% yield) as a light brown solid. MS (ESI): m / z=356.2 [M+H] + .

[0108] Step 2: 5-Bromo-2-(tert-butyl)benzo[d]oxazole

[0109]

[0110] 4-Bromo-2-pivalamidophenyl ester (1.6 g, 4.5 mmol) / p-TsOH H 2 A mixture of 2-(tert-butyl)-1-( ... + .

[0111] Step 3: 5-(Benzylthio)-2-(tert-butyl)benzo[d]oxazole

[0112]

[0113] 5-Bromo-2-(tert-butyl)benzo[d]oxazole (550 mg, 2.17 mmol) / phenylmethyl mercaptan (280 μL, 2.39 mmol) / DIPEA (720 μL, 4.4 mmol) / Pd 2 dba 3A mixture of (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, yield 93%) as a yellow oil. MS (ESI): m / z=298.2[M+H] + .

[0114] Step 4: 2-(tert-Butyl)benzo[d]oxazole-5-sulfonyl chloride

[0115] 5-(Benzylthio)-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) / H 2 O (0.5 mL) in CH 3 The solution in CN (12 mL) was stirred at room temperature for 15 min. The reaction mixture was dissolved in EA (100 mL) and precipitated with H 2 O (50 mL) and brine (50 mL). The EA layer was washed with Na 2 SO 4 Dry, filter and evaporate. 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] + .

[0116] Preparation of intermediate 3

[0117] (2-Aminobenzoyl)glycine benzyl ester

[0118]

[0119] 2H-Benzo[d][1,3]oxazine-2,4(1H)-dione (5 g, 30.7 mmol), glycine benzoate hydrochloride (6.8 g, 33.7 mmol), and TEA (10.5 mL, 75.5 mmol) were dissolved in CH 3 The mixture in CN (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 (grey solid / oil) and extracted with EA (500 mL, 100 mL×2). The EA layers were combined, washed with brine (100 mL), and purified by Na2 SO 4 Dry, filter and evaporate. The residue was washed with EA (50 mL) to give (2-aminobenzoyl)glycine benzyl ester (2.39 g, yield 27%) as a white solid. MS (ESI): m / z=285.1 [M+H] + .

[0120] Preparation of intermediate 4

[0121] (4-Amino-1-methyl-1H-pyrazole-3-carbonyl)glycine benzyl ester

[0122]

[0123] Step 1: 1-Methyl-4-nitro-1H-pyrazole-3-carboxylic acid

[0124]

[0125] 1-Methyl-4-nitro-1H-pyrazole-3-carboxylic acid methyl ester (1800 mg, 9.72 mmol), LiOH·H 2 O (815 mg, 19.44 mmol) in THF (10 mL) and H 2 The mixture in 4% 4-nitro-1H-pyrazole-3-carboxylic acid (1.6 g, yield 96%) was stirred at room temperature for 2 hours. The pH of the reaction mixture was adjusted to 4-5 with Amberlyst 15 (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, yield 96%) as a yellow solid. MS (ESI) m / z=172.1 [M+H] + .

[0126] Step 2: (1-Methyl-4-nitro-1H-pyrazole-3-carbonyl)glycine benzyl ester

[0127]

[0128] 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, yield 70%) as a white solid. MS (ESI) m / z=319.2 [M+H] + .

[0129] Step 3: (4-amino-1-methyl-1H-pyrazole-3-carbonyl)glycine benzyl ester

[0130] (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) were dissolved in MeOH (4 mL) and saturated NH 4 The solution was stirred at 80 °C for 2 h in 4% Cl (20 mL). The reaction mixture was filtered and washed with EA (100 mL) and H 2 O (20 mL). 3 The filtrate was neutralized to pH 7. 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, yield 52%) as a white solid. MS (ESI) m / z=289.3 [M+H] + .

[0131] Preparation of intermediate 5

[0132] 2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoic acid

[0133]

[0134] Step 1: Benzyl 2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoate

[0135]

[0136] 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, yield 97%). MS (ESI): m / z=468.1[M+H] + .

[0137] Step 2: 2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoic acid

[0138] 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 a crude product (690 mg, 90% yield) as a white solid. MS (ESI): m / z=378.1 [M+H] + .

[0139] Preparation of intermediate 6

[0140] (2-Amino-5-fluorobenzoyl)-D-alanine tert-butyl ester

[0141]

[0142] 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 h. The reaction mixture was washed with H 2 O (2 mL) and extracted with EtOAc (5 mL×3). The combined organic layers were washed with brine (10 mL) and dried over anhydrous Na 2 SO 4 Dry, filter and concentrate. 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] + .

[0143] Preparation of intermediate 7

[0144] (2-Amino-5-cyanobenzoyl)-D-alanine tert-butyl ester

[0145]

[0146] 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 h. H 2 O (2 mL). The aqueous phase was separated and extracted with EtOAc (5 mL×3). The combined organic layers were washed with anhydrous Na 2 SO 4 Dry, filter and concentrate. 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, yield 80%). MS (ESI): m / z=312.2 [M+Na] + .

[0147] Preparation of intermediate 8

[0148] (2-Amino-5-methoxybenzoyl)glycine benzyl ester

[0149]

[0150] 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 washed with H 2 O (10 mL) and extracted with DCM (30 mL × 3). The organic phases were combined, washed with brine, and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate. The residue was purified by silica gel column (15% EtOAc / PE) to give (2-amino-5-methoxybenzoyl)glycine benzyl ester (800 mg, yield 85%) as a yellow oil. MS (ESI): m / z 315.1 [M+H] + .

[0151] Preparation of intermediate 9

[0152] (S)-2-Amino-3-fluoropropionic acid benzyl ester 2,2,2-trifluoroacetate

[0153]

[0154] Step 1: (R)-1,2,3-oxathiazolidine-3,4-dicarboxylic acid 4-benzyl 3-(tert-butyl ester) 2,2-dioxide

[0155]

[0156] A solution of (tert-butoxycarbonyl)-D-serine benzyl ester (5 g, 16.9 mmol) in DCM (170 mL) was added dropwise to SOCl at -40 °C under Ar. 2 The mixture was stirred for 30 minutes at -35 °C to -40 °C. Py (7.1 mL, 87.9 mmol) was slowly added to the reaction mixture. The reaction mixture was warmed to room temperature and stirred for 1 hour. The reaction mixture was quenched with ice water (110 mL) and saturated with NaHCO 3 (110 mL) and washed with Na 2 SO 4 Drying, filtration and evaporation gave the intermediate (7 g) as a colorless oil. 4 (6 g, 28 mmol) of water (110 mL) was added dropwise to the intermediate (7 g) / RuCl 3 (80 mg, 0.39 mmol) in CH 3 CN (110 mL). The reaction mixture was then stirred at 0 °C for 1 h. The reaction mixture was washed with saturated NaHCO 3 The mixture was quenched with 1% paraformaldehyde (10% ethanol) (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, yield 69%). MS (ESI): m / z=380.3 [M+Na] + .

[0157] Step 2: (S)-2-((tert-Butyloxycarbonyl)amino)-3-fluoropropionic acid benzyl ester

[0158]

[0159] KF (3.9 g, 10.9 mmol) and 18-crown-6 (1.9 g, 32.8 mmol) were suspended in anhydrous CH 3 CN (100 mL) and stirred at room temperature for 15 minutes. Then, the compound of step 1 (3.9 g, 10.9 mmol) was added to the reaction mixture and the mixture was stirred at room temperature overnight. The reaction mixture was evaporated. DCM (60 mL) / 20% H 2 SO 4 (20 mL) and stirred at room temperature for 1.5 hours. The reaction mixture was washed with saturated NaHCO 3 The mixture was neutralized to pH 7 and extracted with DCM (100 mL x 2). The DCM layers were combined and purified by Na 2 SO 4 Dry, filter and evaporate. The residue was purified by SGC (0-25% EA / PE, Rf of the product = 0.5 in PE / EA 4 / 1) to give the compound as a colorless oil (130 mg, yield 4%). MS (ESI): m / z = 320.2 [M+Na] + .

[0160] Step 3: (S)-2-amino-3-fluoropropionic acid benzyl ester 2,2,2-trifluoroacetate

[0161] 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 will be used directly in the next step. MS (ESI): m / z=198.2 [M+H] + .

[0162] Preparation of intermediate 10

[0163] N-(2-Aminobenzoyl)-O-methyl-D-serine benzyl ester

[0164]

[0165] Step 1: N-(tert-Butyloxycarbonyl)-O-methyl-D-serine benzyl ester

[0166]

[0167] 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 washed with H 2 O (10 mL) and extracted with DCM (30 mL × 3). The organic phases were combined, washed with brine, and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate. 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, yield 92%) as a colorless oil. MS (ESI): m / z 210.1 [M-99] + .

[0168] Step 2: O-methyl-D-serine benzyl ester

[0169]

[0170] 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 in vacuo 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] + .

[0171] Step 3: N-(2-aminobenzoyl)-O-methyl-D-serine benzyl ester

[0172] 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] + .

[0173] Preparation of intermediate 11

[0174] ((1R,2R)-2-Amino-5,5-difluorocyclohexane-1-carbonyl)glycine tert-butyl ester

[0175]

[0176] Step 1: ((1R,2R)-2-(((benzyloxy)carbonyl)amino)-5,5-difluorocyclohexane-1-carbonyl)glycine tert-butyl ester

[0177]

[0178] 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 then 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), and purified by Na 2 SO 4 Dry, filter and concentrate. The residue was purified by silica gel column chromatography (eluted with EtOAc / PE=1 / 3 to 1 / 2, V / V) to give the product as a white solid (1 g, yield 82%). MS (ESI): m / z=449.2 [M+Na] + .

[0179] Step 2: ((1R,2R)-2-amino-5,5-difluorocyclohexane-1-carbonyl)glycine tert-butyl ester

[0180] 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] + .

[0181] Example 1

[0182]

[0183] Step 1: (2-amino-5-bromobenzoyl)glycine benzyl ester

[0184]

[0185] 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 and purified by Na 2 SO 4 Dry, filter and concentrate 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, yield 76%) as a brown solid. MS (ESI): m / z=365.0 [M+H] + .

[0186] Step 2: (2-amino-5-(1-methyl-1H-pyrazol-5-yl)benzoyl)glycine benzyl ester

[0187]

[0188] (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)Cl 2 (60 mg, 0.08 mmol) and potassium acetate (485 mg, 4.95 mmol) in dioxane / H 2 The mixture in 2% 2-HNO3 O (30 mL, 5:1) was stirred at 80 °C under Ar for 16 h. The reaction was monitored by LCMS. The resulting mixture was extracted with EA (30 mL×3). The organic layers were combined, washed with brine, and purified by Na 2 SO 4 Dry, filter and concentrate in vacuo. The residue was purified by SGC (0-30% EA / PE, Rf of the product = 0.5 in PE / EA 2 / 1) to give (2-amino-5-(1-methyl-1H-pyrazol-5-yl)benzoyl)glycine benzyl ester (340 mg, yield 68%) as a light brown gum. MS (ESI): m / z = 365.1 [M+H] + .

[0189] Step 3: 4-(N-(2-((2-(benzyloxy)-2-oxoethyl)carbamoyl)-4-(1-methyl-1H-pyrazol-5-yl)phenyl)sulfamoyl)phenyl pivalate

[0190]

[0191] 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), and washed with anhydrous Na 2 SO 4 Dry, filter and evaporate. The residue was purified by silica gel column chromatography (EtOAC / petroleum ether, 0% to 80%) to give a brown gum (240 mg, yield 41%). MS (ESI): m / z=605.1 [M+H] + .

[0192] Step 4: (5-(1-methyl-1H-pyrazol-5-yl)-2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoyl)glycine

[0193] 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 at room temperature under H 2 Stir overnight under atmosphere. Monitor the reaction by LCMS. After completion, filter the mixture. Concentrate the filtrate to give the crude product, which is purified by preparative HPLC (using NH 4 HCO 3 Buffer A: 10 mM NH 4 HCO 3 / water; B: acetonitrile; column: Waters XBridge Peptide BEH C18, 19×250 mm, 10 μm, ) was purified to obtain the target compound (138 mg, yield 73%) as a white solid. MS (ESI) m / z: 515.3 [M+H] + LCMS purity: 100.00% (214 nm), RT = 1.749 min.

[0194] 1 H NMR (400 MHz, DMSO-d 6): 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).

[0195] Using a method similar to Example 1, the following compounds were prepared using different intermediates:

[0196]

[0197]

[0198]

[0199]

[0200]

[0201] Example 12

[0202]

[0203] Step 1: ((Tritylamino)methyl)phosphonic acid diphenylmethyl ester

[0204]

[0205] Aqueous formaldehyde solution (37%, 3.8mL, 46.3mmol) was added to a solution of tritylamine (6g, 23.2mmol) in toluene (100mL), and the reaction mixture was stirred at room temperature for 20 hours. Dean-Stark was then used to remove water and add the corresponding hydrogen phosphate (7.28g, 27.8mmol) and triethylamine (234mg, 2.3mmol). The reactant was stirred overnight under reflux in toluene. 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 as a white solid (10g, yield 82%). MS (ESI): m / z=1089.2[2M+Na] + .

[0206] Step 2: (Aminomethyl)phosphonic acid diphenylmethyl ester hydrochloride

[0207]

[0208] 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 a crude product (2.1 g, yield 90%). The crude product was used directly in the next step. MS (ESI): m / z=292.2 [M+H] + .

[0209] Step 5: 4-(N-(2-(((bis(benzyloxy)phosphoryl)methyl)carbamoyl)phenyl)sulfamoyl)phenyl pivalate

[0210]

[0211] 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, yield 53%). MS (ESI): m / z=651.3 [M+H] + .

[0212] 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: CH 3 CN; column: Xbridge BEH peptide C18, 19 mm×250 mm) and lyophilized to obtain compound 12 (210 mg, 86%) as a yellow solid. MS (ESI): m / z=471.1 [M+H] + .

[0213] 1 H NMR (400 MHz, DMSO-d 6): 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).

[0214] Embodiment 13

[0215]

[0216] Step 1: 2-(Tritylthio)ethan-1-amine

[0217]

[0218] 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 and washed with water (200 mL) and saturated NaCl solution (150 mL). The organic layer was purified by MgSO 4 Drying, filtration and concentration gave crude 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] + .

[0219] Step 2: 2-amino-N-(2-(tritylthio)ethyl)benzamide

[0220]

[0221] 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] + .

[0222] Step 3: 4-(N-(2-((2-(tritylthio)ethyl)carbamoyl)phenyl)sulfamoyl)phenyl pivalate

[0223]

[0224] To a solution of 2-amino-N-(2-(tritylthio)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: CH 3 CN; 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, yield 91%) as a white solid. MS (ESI): m / z=701.1 [M+Na] + .

[0225] Step 4: 2-(2-((4-(pivaloyloxy)phenyl)sulfonamido)benzamido)ethane-1-sulfonic acid A mixture of 4-(N-(2-((2-(tritylthio)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: CH 3 CN; column: Xbridge BEH peptide C18, 19 mm×250 mm) and lyophilized to obtain the target compound 13 (110 mg, yield 24%) as a white solid. MS (ESI): m / z=485.1 [M+H] + .

[0226] 1 H NMR (400 MHz, DMSO-d 6 ): δ11.77(s,1H),8.83(t,J=5.0Hz,1H),7.81-7.78(m,2H),7.62(d,J=7.6Hz,1H),7.50-7.43(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).

[0227] Example 14: Preparation of O-(methyl-d3)-N-(2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoyl)-D-serine (25)

[0228]

[0229] a) Under nitrogen protection at room temperature, to a solution of benzyl(tert-butyloxycarbonyl)-D-serine (500.0 mg, 1.7 mmol) in MeCN (8 mL) were added in sequence: Ag; 2 O (588.5 mg, 2.5 mmol) and CD 3 I (0.3 mL, 5.0 mmol). Stirring was continued for 18 hours under light-shielding conditions. The reaction mixture was filtered, the filtrate was concentrated and purified by silica gel column chromatography (eluted with 10% EtOAc / PE, V / V) to obtain a colorless liquid product 25a (400 mg, 76%). MS (ESI): m / z 212.7 [M-100] + .

[0230] b) Under nitrogen protection at room temperature, add HCl / Dioxane (4M, 3 mL) to 25a (0.4 g, 1.3 mmol). Continue stirring for 2 hours and concentrate to obtain a white solid product 25b (300 mg, crude). MS (ESI): m / z = 212.7 [M+H] + The compound was used directly in the next reaction without purification.

[0231] c) Under nitrogen protection at room temperature, 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.) were added 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) in sequence. The mixture was stirred for 20 hours, and the reaction solution was concentrated and purified by silica gel column chromatography (eluted with PE / EA 2:1, V / V) to obtain a colorless oily product 25c (560 mg, 100%).

[0232] d) To a solution of 25c (740 mg, 0.92 mmol, 1.0 eq.) in EtOAc (20 mL) was added 5% Pd / C (70 mg) and Pd(OH) at room temperature. 2(70 mg). At 40°C, hydrogen was added and stirred for 16 hours. Cooled to room temperature, the reaction mixture was filtered, the filtrate was concentrated and purified by reverse phase column to obtain a white solid product 25 (450 mg, 78%). (ESI): m / z = 481.75 [M+H] + . 1 HNMR (400 MHz, DMSO-d 6 ): 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).

[0233] Biological activity test

[0234] Compound preparation: All compounds were dissolved in DMSO at a concentration of 10 mM and stored at -20°C.

[0235] Prepare 1x assay buffer, dilute the positive compound (Sivelestat) and the test compound 3 times in DMSO, and dilute 10 concentration points in total. Add 1 μL of the serially diluted compound and 65.67 μL of assay buffer to the well plate to obtain 3X positive compound and test compound working solution (DMSO final concentration is 1.5%). Seal the well plate with a sealing film and shake on an oscillator for 15 minutes.

[0236] 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 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 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.

[0237] Prepare 3x substrate solution in 1x assay buffer. Add 5 μl of 3x substrate working solution to a 384-well plate. Centrifuge the 384-well plate at 1000 rpm for 1 minute. Incubate at room temperature in the dark for 1 hour. Read the fluorescence value FLU at 360nm / 450nm on a plate reader.

[0238] Calculate the inhibition rate, % inhibition rate = (1-(FLU compound-FLU positive well) / (FLU reference well-FLU positive well)). FLU reference wells are wells with only enzyme added without inhibitor, and FLU positive wells are wells with enzyme and 3000nM Sivelestat-2 added. Then use Graphpad 8.0 software Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X)*HillSlope)) to calculate the IC50 value.

[0239] Compound No. IC50(nM) 1 20.42 2 30.46 3 23.6 6 32.8 11 27.04 12 5.69 13 4.22 17 15.35 19 2.25 20 2.47 21 2.26 22 2.2 23 3.69 24 1.5 25 2.54 26 8.49 27 130.5

[0240] 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.

[0241] Animal testing 1

[0242] Experimental animals: ICR mice, SPF grade, male, body weight range at the time of administration: 25-28 g.

[0243] 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).

[0244] Establishment of LPS-induced acute lung injury mouse model: intramuscular injection of mice The animals were anesthetized with 50% paraformaldehyde (0.05 mL / animal). The animals were fixed in the supine position, the trachea in the neck was separated, 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.

[0245] 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 for each administration was 25 mg / kg, and the total daily dose was 75 mg / kg.

[0246] 24h after LPS airway spraying, the mice were killed, the trachea was separated, and the tube was intubated. The left lung lobe was ligated, and the lungs were lavaged with 0.5mL of normal saline (containing 1% serum and 2U / mL heparin), which was repeated twice. The lavage fluid mixed three times was placed on ice for later use. For animals 1 to 8 in the blank control group, model group, experimental group, and positive control group, the ligated left lung lobe 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.

[0247]

[0248] Note: Compared with the blank control group: ## P<0.01, ### P<0.001; compared with the model group: * P<0.05, ** P<0.01.

[0249] 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.

[0250] Animal testing 2

[0251] 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).

[0252] Establishment of the rat model of acute lung injury induced by hydrochloric acid: 0.1 mL / rat of 0.1 N HCL was sprayed into the airways.

[0253] Dosage: The experimental group was administered with compound 20, and the positive control group was administered with sivelestat sodium. Both groups were injected twice through the tail vein, 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.

[0254] The rats were killed 5 hours after modeling, and alveolar lavage was performed to determine the inflammatory cells, protein exudation, pulmonary hemorrhage and NE enzyme activity in the lavage fluid. The results of each group are shown in the following table.

[0255]

[0256]

[0257] 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.

[0258] In the hydrochloric acid-induced acute lung injury rat model, compound 20 improved pulmonary hemorrhage and reduced protein infiltration 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 or a pharmaceutically acceptable salt thereof according to claim 1, 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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