A PAD4 inhibitor, its preparation method and its application in the preparation of anti-pulmonary fibrosis drugs

Abstract

The present invention provides a PAD4 inhibitor, a preparation method thereof, and an application thereof in the preparation of anti-pulmonary fibrosis drugs, belonging to the field of biomedicine technology. The present invention takes PAD4 as a new target for the treatment of pulmonary fibrosis, and performs structural modification on chloramidine to obtain a PAD4 inhibitor having a structure shown in Formula I. The small molecule inhibitor can inhibit PAD4 activity, reduce NETs formation, inhibit the activation of lung fibroblasts and their transformation into myofibroblasts, reduce lung collagen deposition, and slow down the progression of pulmonary fibrosis. In vitro, RC2-33 can reduce histone citrullination to inhibit NETs formation, inhibit TGF-β1-induced lung fibroblast proliferation, and downregulate interstitial marker proteins such as α-SMA, COL-I, FN-I, etc. Nebulized inhalation of RC2-33 can effectively reduce NETs formation in the lungs of mice, inhibit the transformation of epithelial cells to interstitial cells, reduce extracellular matrix deposition, and improve pulmonary fibrosis in mice.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a PAD4 inhibitor, a preparation method thereof, and application thereof in the preparation of anti-pulmonary fibrosis drugs. Background Art

[0002] Pulmonary fibrosis is an interstitial lung disease with a protracted and highly variable course. It typically begins with alveolar epithelial cell damage, which progresses to pulmonary inflammation. Ultimately, stimulated by various transforming and inflammatory factors, various alveolar epithelial cells or pulmonary fibroblasts transform into myofibroblasts. Neutrophil extracellular traps (NETs) are reticular or fibrillar structures released by neutrophils and composed of depolymerized chromatin filaments, myeloperoxidase (MPO), and elastase (NE). The central step in NET formation is the conversion of arginine on histones to citrulline catalyzed by PAD4. Excessive NET release has deleterious effects on lung disease, as NETs readily expand within the alveoli and cause lung damage. Furthermore, NETs and their related molecules can directly induce epithelial and endothelial cell death. Excessive NET formation has been found in several lung diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, respiratory syncytial virus bronchiolitis, influenza, bacterial pneumonia, and tuberculosis.

[0003] The pan-PAD inhibitor chloramidine has been shown to inhibit bleomycin (BLM)-induced NETs formation in vitro and in vivo. However, chloramidine has poor inhibitory activity against PAD4 enzyme, IC 50 is 150μM. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a PAD4 inhibitor and its preparation method and use in the preparation of anti-pulmonary fibrosis drugs. The PAD4 inhibitor provided by the present invention has good anti-pulmonary fibrosis ability.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a PAD4 inhibitor having a chemical structure shown in Formula I:

[0007]

[0008] The present invention provides a method for preparing the above-mentioned PAD4 inhibitor, comprising the following steps:

[0009] In the presence of a first condensing agent, biphenyl-3-carboxylic acid and the compound having the structure represented by Formula 1 undergo a first condensation reaction to obtain a compound having the structure represented by Formula 2;

[0010]

[0011] The compound having the structure shown in Formula 2 is subjected to a Bzl protecting group removal reaction to obtain a compound having the structure shown in Formula 3;

[0012]

[0013] In the presence of a second condensing agent, the compound having the structure shown in Formula 3 undergoes a second condensation reaction with indole-3-methylamine to obtain a compound having the structure shown in Formula 4;

[0014]

[0015] The compound having the structure shown in Formula 4 is subjected to a Boc protecting group removal reaction to obtain a compound having the structure shown in Formula 5;

[0016]

[0017] The compound having the structure shown in Formula 5 undergoes a substitution reaction with ethyl 2-chloroacetimidate hydrochloride to obtain a compound having the structure shown in Formula I.

[0018] Preferably, the first condensing agent and the second condensing agent include HOBt and DCC;

[0019] The first condensation reaction and the second condensation reaction are carried out under alkaline conditions; the time of the first condensation reaction and the second condensation reaction is independently 6 to 24 hours.

[0020] Preferably, the Bzl protection group removal reaction is carried out under conditions containing a Pd / C catalyst and H2;

[0021] The reaction time for removing the Bzl protecting group is 12 to 36 hours.

[0022] Preferably, the Boc protecting group removal reaction is carried out in an ethyl acetate solution of HCl, and the time for the Boc protecting group removal reaction is 1 to 5 hours.

[0023] Preferably, the molar ratio of the compound having the structure shown in Formula 5 to ethyl 2-chloroacetimidate hydrochloride is 1:1.5-6.

[0024] Preferably, the substitution reaction time is 8 to 24 hours.

[0025] The present invention provides use of the PAD4 inhibitor in the preparation of anti-pulmonary fibrosis drugs.

[0026] The present invention provides an anti-pulmonary fibrosis drug, comprising an active ingredient and a pharmaceutically acceptable carrier;

[0027] The active ingredient includes the above-mentioned PAD4 inhibitor.

[0028] Preferably, the dosage of the active ingredient is 1 to 10 mg / kg.

[0029] The present invention provides a PAD4 inhibitor having a structure shown in Formula I. The present invention uses PAD4 as a new target for the treatment of pulmonary fibrosis, and obtains a PAD4 inhibitor having a structure shown in Formula I by structurally modifying chloramidine, which is named RC2-33. The small molecule inhibitor can inhibit PAD4 activity, reduce NETs formation, inhibit the activation and transformation of lung fibroblasts into myofibroblasts, reduce lung collagen deposition, and slow down the progression of pulmonary fibrosis. The results of the examples show that in vitro, RC2-33 can reduce histone citrullination to inhibit NETs formation, inhibit TGF-β1-induced lung fibroblast proliferation, and downregulate interstitial marker proteins such as α-SMA, COL-I, FN-I, etc. The present invention established a bleomycin (BLM)-induced mouse pulmonary fibrosis model and found that aerosol inhalation of RC2-33 can effectively reduce NETs formation in mouse lungs, inhibit epithelial cell to mesenchymal cell transformation (EMT), reduce extracellular matrix deposition (ECM), and improve mouse pulmonary fibrosis.

[0030] The present invention provides a method for preparing the above-mentioned PAD4 inhibitor, which is simple to operate, low in cost, and easy to achieve industrial batch production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A synthetic route for the PAD4 inhibitor RC2-33;

[0032] Figure 2 PAD4 inhibitor RC2-33 1 H NMR spectrum;

[0033] Figure 3 is the mass spectrum of PAD4 inhibitor RC2-33;

[0034] Figure 4 The inhibitory effects of different compounds on the release of NETs from neutrophils;

[0035] Figure 5 This is a schematic diagram of the structure of the homemade mouse oral and nasal aerosol inhalation device;

[0036] Figure 6 The weight changes of mice in each group;

[0037] Figure 7 The results of H&E staining of the lungs of mice in each group;

[0038] Figure 8 The results of Masson staining of the lungs of mice in each group. DETAILED DESCRIPTION

[0039] The present invention provides a PAD4 inhibitor having the structure shown in Formula I:

[0040]

[0041] In the present invention, the particle size of the PAD4 inhibitor having the structure represented by Formula I is preferably 180 to 260 nm, more preferably 229.9±6.3 nm.

[0042] The present invention provides a method for preparing the above-mentioned PAD4 inhibitor, comprising the following steps:

[0043] In the presence of a first condensing agent, biphenyl-3-carboxylic acid and the compound having the structure represented by Formula 1 undergo a first condensation reaction to obtain a compound having the structure represented by Formula 2;

[0044]

[0045] The compound having the structure shown in Formula 2 is subjected to a Bzl protecting group removal reaction to obtain a compound having the structure shown in Formula 3;

[0046]

[0047] In the presence of a second condensing agent, the compound having the structure shown in Formula 3 undergoes a second condensation reaction with indole-3-methylamine to obtain a compound having the structure shown in Formula 4;

[0048]

[0049] The compound having the structure shown in Formula 4 is subjected to a Boc protecting group removal reaction to obtain a compound having the structure shown in Formula 5;

[0050]

[0051] The compound having the structure shown in Formula 5 undergoes a substitution reaction with ethyl 2-chloroacetimidate hydrochloride to obtain a compound having the structure shown in Formula I.

[0052] In the present invention, biphenyl-3-carboxylic acid and a compound having a structure represented by Formula 1 undergo a first condensation reaction in the presence of a first condensing agent to obtain a compound having a structure represented by Formula 2. In the present invention, the molar ratio of biphenyl-3-carboxylic acid to the compound having a structure represented by Formula 1 is preferably 1:1 to 1.5, more preferably 1:1.

[0053] In the present invention, the first condensing agent preferably includes HOBt and DCC, and the molar ratio of biphenyl-3-carboxylic acid to HOBt and DCC is preferably 1:1.05-1.4:1.05-1.5, more preferably 1:1.2:1.2.

[0054] In the present invention, the first condensation reaction is preferably carried out in an organic solvent, and the organic solvent is preferably one or more of tetrahydrofuran, dimethylformamide and acetonitrile.

[0055] In the present invention, the first condensing agent, biphenyl-3-carboxylic acid, the compound having the structure represented by Formula 1, and the organic solvent are preferably mixed by first mixing biphenyl-3-carboxylic acid with the organic solvent and the first condensing agent, stirring in an ice bath, and then adding the compound having the structure represented by Formula 1. In the present invention, the stirring time in the ice bath is preferably 30 minutes.

[0056] In the present invention, the first condensation reaction is preferably carried out under alkaline conditions, and the pH value of the alkaline conditions is preferably 7 to 8. In the present invention, the alkaline reagent providing the alkaline environment is preferably N-methylmorpholine.

[0057] In the present invention, the first condensation reaction is preferably carried out at room temperature with stirring, and the time of the first condensation reaction is preferably 6 to 24 hours, more preferably 8 hours.

[0058] After the first condensation reaction, the present invention preferably performs post-treatment on the obtained first condensation reaction liquid, and the post-treatment preferably includes the following steps:

[0059] The first condensation reaction liquid is subjected to solid-liquid separation to remove the organic solvent in the liquid phase, and the residue is dissolved with ethyl acetate. The obtained dissolved product is washed, dried and concentrated to obtain a pure compound having the structure shown in Formula 2.

[0060] In the present invention, the solid-liquid separation is preferably filtration, and the present invention removes dicyclohexylurea (DCU) produced during the reaction by the solid-liquid separation. In the present invention, the method for removing the organic solvent is preferably rotary evaporation. In the present invention, the washing reagents used in the washing are preferably saturated NaHCO3 aqueous solution, saturated NaCl aqueous solution, 5wt% KHSO4 aqueous solution, saturated NaCl aqueous solution, saturated NaHCO3 aqueous solution and saturated NaCl aqueous solution, and the present invention preferably uses the above-mentioned washing reagents to repeat washing three times in sequence. In the present invention, the drying is preferably anhydrous Na2SO4 drying, and after the drying, it is preferably to remove Na2SO4 by vacuum filtration. In the present invention, the concentration is preferably concentrated under reduced pressure.

[0061] After obtaining the compound having the structure shown in Formula 2, the compound having the structure shown in Formula 2 is subjected to a Bzl deprotection reaction to obtain a compound having the structure shown in Formula 3. In the present invention, the Bzl deprotection reaction is preferably carried out in the presence of a Pd / C catalyst and H2; the mass of the Pd / C catalyst is preferably 5 to 15% of the mass of the compound having the structure shown in Formula 2, more preferably 10%.

[0062] In the present invention, the Bzl protecting group removal reaction is preferably carried out in an organic solvent, and the organic solvent is preferably methanol, ethanol, acetonitrile and dioxane.

[0063] In the present invention, the Bzl deprotection reaction is preferably carried out at room temperature with stirring; the Bzl deprotection reaction time is preferably 12 to 36 hours, more preferably 24 hours.

[0064] After the Bzl protection group removal reaction, the present invention preferably performs post-treatment on the obtained Bzl protection group removal reaction solution, and the post-treatment preferably includes the following steps:

[0065] The Bzl protection group removal reaction solution is subjected to solid-liquid separation, and the resulting liquid phase is concentrated to obtain a pure compound having the structure shown in Formula 3.

[0066] In the present invention, the solid-liquid separation method is preferably vacuum filtration, and the present invention removes the Pd / C catalyst through the solid-liquid separation. In the present invention, the concentration is preferably vacuum concentration to dryness.

[0067] After obtaining the compound having the structure represented by Formula 3, the compound having the structure represented by Formula 3 is subjected to a second condensation reaction with indole-3-methylamine in the presence of a second condensing agent to obtain a compound having the structure represented by Formula 4. In the present invention, the molar ratio of the compound having the structure represented by Formula 3 to indole-3-methylamine is preferably 1:1.

[0068] In the present invention, the second condensing agent preferably includes HOBt and DCC, and the molar ratio of the compound having the structure represented by Formula 3 to HOBt and DCC is preferably 1:1.05-1.4:1.05-1.5, more preferably 1:1.2:1.2.

[0069] In the present invention, the second condensation reaction is preferably carried out in an organic solvent, and the organic solvent is preferably one or more of tetrahydrofuran, dimethylformamide and acetonitrile.

[0070] In the present invention, the second condensing agent, the compound having the structure represented by Formula 3, indole-3-methylamine, and the organic solvent are preferably mixed by first mixing the compound having the structure represented by Formula 3 with the organic solvent and the condensing agent, stirring in an ice bath, and then adding indole-3-methylamine. In the present invention, the stirring time in the ice bath is preferably 30 minutes.

[0071] In the present invention, the second condensation reaction is preferably carried out under alkaline conditions, and the pH value of the alkaline conditions is preferably 7 to 8. In the present invention, the alkaline reagent providing the alkaline environment is preferably N-methylmorpholine.

[0072] In the present invention, the second condensation reaction is preferably carried out at room temperature with stirring, and the time of the second condensation reaction is preferably 6 to 24 hours, more preferably 8 hours.

[0073] After the second condensation reaction, the present invention preferably performs post-treatment on the obtained second condensation reaction liquid, and the post-treatment preferably includes the following steps:

[0074] The second condensation reaction liquid is subjected to solid-liquid separation to remove the organic solvent in the liquid phase, and the residue is dissolved with ethyl acetate. The obtained dissolved product is washed, dried and concentrated to obtain a pure compound having the structure shown in Formula 4.

[0075] In the present invention, the solid-liquid separation is preferably filtration. In the present invention, the method for removing the organic solvent is preferably rotary evaporation. In the present invention, the washing reagents used in the washing are preferably saturated NaHCO3 aqueous solution, saturated NaCl aqueous solution, 5wt% KHSO4 aqueous solution, saturated NaCl aqueous solution, saturated NaHCO3 aqueous solution and saturated NaCl aqueous solution, and the present invention preferably uses the above washing reagents to repeat washing three times in sequence. In the present invention, the drying is preferably drying with anhydrous Na2SO4, and after the drying, Na2SO4 is preferably removed by filtration under reduced pressure. In the present invention, the concentration is preferably concentrated under reduced pressure.

[0076] After obtaining the compound having the structure shown in Formula 4, the compound having the structure shown in Formula 4 is subjected to a Boc-protecting group removal reaction to obtain a compound having the structure shown in Formula 5. In the present invention, the Boc-protecting group removal reaction is preferably carried out in an ethyl acetate solution of HCl, wherein the concentration of HCl in the ethyl acetate solution is preferably 2 to 5 mol / L, more preferably 4 mol / L. In the present invention, the Boc-protecting group removal reaction is preferably carried out in an ice bath, and the Boc-protecting group removal reaction time is preferably 1 to 5 hours, more preferably 4 hours.

[0077] After the Boc protection group removal reaction, the present invention preferably performs post-treatment on the obtained Boc protection group removal reaction solution, and the post-treatment preferably includes the following steps:

[0078] The Boc protection group removal reaction solution was drained, and the obtained residue was added to dry ethyl acetate. The process of draining and adding dry ethyl acetate was repeated three times, and finally drained to obtain a drained residue;

[0079] Dry petroleum ether was added to the obtained drained residue, and the mixture was drained. The process of adding dry petroleum ether and draining was repeated three times to obtain a pure compound having the structure represented by Formula 5.

[0080] After obtaining the compound having the structure shown in Formula 5, the compound having the structure shown in Formula 5 is subjected to a substitution reaction with ethyl 2-chloroacetimidate hydrochloride to obtain a compound having the structure shown in Formula I. In the present invention, the molar ratio of the compound having the structure shown in Formula 5 to ethyl 2-chloroacetimidate hydrochloride is preferably 1:5.

[0081] In the present invention, the substitution reaction is preferably carried out in an organic solvent, and the organic solvent is preferably one or more of methanol, ethanol and acetonitrile.

[0082] In the present invention, the compound having the structure shown in Formula 5, ethyl 2-chloroacetimidate hydrochloride and the organic solvent are preferably mixed in the following manner: firstly mixing the compound having the structure shown in Formula 5 with the organic solvent, and then adding ethyl 2-chloroacetimidate hydrochloride under ice bath conditions.

[0083] In the present invention, the substitution reaction is preferably carried out under alkaline conditions, and the alkaline pH value is preferably 10. In the present invention, the alkaline agent used to adjust the pH value is preferably N,N-diisopropylethylamine (DIPEA).

[0084] In the present invention, the substitution reaction is preferably carried out at room temperature with stirring; the substitution reaction time is preferably 8 to 24 hours, more preferably 12 hours.

[0085] After the substitution reaction, the present invention preferably performs post-treatment on the obtained substitution reaction solution, and the post-treatment preferably includes the following steps:

[0086] The substitution reaction solution was concentrated, and the obtained concentrate was dissolved in water and purified by C18 column chromatography to obtain a pure product of the compound with the structure shown in Formula I.

[0087] In the present invention, the concentration is preferably concentrated under reduced pressure, and the conditions for the C18 column chromatography purification include methanol: dichloromethane: acetic acid = 1: 5-15: 0.1-0.5.

[0088] The present invention provides use of the PAD4 inhibitor in the preparation of anti-pulmonary fibrosis drugs.

[0089] The present invention provides an anti-pulmonary fibrosis drug comprising an active ingredient and a pharmaceutically acceptable carrier. The present invention has no particular requirements for the carrier; any pharmaceutically acceptable carrier known in the art may be used. In the present invention, the carrier is preferably one or more of lipid nanoparticles, lipid-polymer hybrid nanoparticles, nanostructured lipid carriers, and cationic polymers.

[0090] The active ingredient includes the above-mentioned anti-pulmonary fibrosis drug.

[0091] In the present invention, the dosage form of the anti-pulmonary fibrosis drug is preferably an aerosol.

[0092] In the present invention, the dosage of the active ingredient is preferably 1 to 10 mg / kg.

[0093] The PAD4 inhibitor provided by the present invention, its preparation method, and its application in the preparation of anti-pulmonary fibrosis drugs are described in detail below with reference to the examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0094] Example 1 Synthesis of PAD4 Inhibitor RC2-33

[0095] according to Figure 1 The PAD4 inhibitor RC2-33 was synthesized using the following method:

[0096] 1) Preparation of (S)-2-([1,1'-biphenyl]-3-formamido)-5-((tert-butyloxycarbonyl)amino)pentanoic acid benzyl ester:

[0097] Dissolve 10 mmol of biphenyl-3-carboxylic acid in 20 mL of anhydrous tetrahydrofuran (THF). Add 12 mmol of N-hydroxybenzotriazole (HOBt) and allow to dissolve completely. Slowly add 12 mmol of dicyclohexylcarbodiimide (DCC) and stir in an ice bath for 30 min. Then, add 10 mmol of (S)-2-amino-5-((tert-butoxycarbonyl)amino)pentanoic acid benzyl ester hydrochloride. Add N-methylmorpholine (NMM) dropwise and adjust the pH to 7-8. Stir at room temperature for 8 h. TLC (dichloromethane:methanol = 25:1) indicates the disappearance of the starting material. Remove dicyclohexylurea (DCU) by filtration, and remove the THF by rotary evaporation. The residue was dissolved in 20 mL of ethyl acetate (EA), and the resulting solution was washed three times with saturated aqueous NaHCO3, saturated aqueous NaCl, 5% aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The ethyl acetate layer was dried over anhydrous Na2SO4, and the Na2SO4 was removed by filtration under reduced pressure. The filtrate was concentrated to dryness under reduced pressure to obtain compound (S)-2-([1,1'-biphenyl]-3-formamido)-5-((tert-butoxycarbonyl)amino)benzyl pentanoate.

[0098] ESI-MS (m / z): 503.31 [M+H] + . 1 H NMR (500MHz, DMSO-d6) δ8.87(d,J=7.5Hz,1H),8.14(t,J=1.9Hz,1H),7.84(ddt,J=17.3,7.8,1.4Hz ,2H),7.74-7.68(m,2H),7.56(t,J=7.7Hz,1H),7.49(dd,J=8.4,7.0Hz,2H),7.43-7.36(m,1H),7.34 (dddd,J=11.5,7.3,5.9,1.7Hz,4H),7.33-7.25(m,1H),6.78(t,J=5.6Hz,1H),5.15(s,2H),4.53(dd d,J=9.8,7.3,5.0Hz,1H),2.95(q,J=6.5Hz,2H),1.93-1.74(m,2H),1.60-1.43(m,2H),1.34(s,9H).

[0099] 2) Preparation of (S)-2-([1,1'-biphenyl]-3-formamido)-5-((tert-butyloxycarbonyl)amino)pentanoic acid:

[0100] 10 mmol of benzyl (S)-2-([1,1'-biphenyl]-3-formamido)-5-((tert-butyloxycarbonyl)amino)pentanoate was dissolved in an appropriate amount of methanol with stirring. 10 wt% Pd / C was added. The reaction system was sealed, air was evacuated using a tee, hydrogen was introduced, and evacuation was repeated using a tee. The reaction system was stirred at room temperature for 24 hours while maintaining a hydrogen atmosphere. TLC (dichloromethane:methanol = 20:1) monitored the disappearance of the starting material spot. Pd / C was removed by filtration under reduced pressure, and the filtrate was concentrated to dryness under reduced pressure to obtain (S)-2-([1,1'-biphenyl]-3-formamido)-5-((tert-butyloxycarbonyl)amino)pentanoic acid. ESI-MS (m / z): 411.89 [MH] - .

[0101] 3) Preparation of tert-butyl (S)-(5-(((1H-indol-3-yl)methyl)amino)-4-([1,1'-biphenyl]-3-formamido)-5-oxopentyl)carbamate:

[0102] Dissolve 10 mmol of (S)-2-([1,1'-biphenyl]-3-carboxamido)-5-((tert-butyloxycarbonyl)amino)pentanoic acid in 20 mL of anhydrous tetrahydrofuran (THF). Add 12 mmol of N-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC). Stir for 30 minutes in an ice bath. Then, add 10 mmol of indole-3-methylamine. Add N-methylmorpholine (NMM) dropwise, adjust the pH to 7-8, and stir at room temperature for 8 hours. TLC (dichloromethane:methanol = 20:1) indicates the disappearance of benzoic acid. Remove dicyclohexylurea (DCU) by filtration, and remove THF by rotary evaporation. The residue was dissolved in 20 mL of ethyl acetate (EA), and the resulting solution was washed three times with saturated aqueous NaHCO3 solution, saturated aqueous NaCl solution, 5% aqueous KHSO4 solution, saturated aqueous NaCl solution, saturated aqueous NaHCO3 solution, and saturated aqueous NaCl solution. The ethyl acetate layer was dried over anhydrous Na2SO4, and the desiccant was removed by filtration under reduced pressure. The filtrate was concentrated to dryness under reduced pressure to obtain compound (S)-tert-butyl(5-(((1H-indol-3-yl)methyl)amino)-4-([1,1'-biphenyl]-3-formamido)-5-oxopentyl)carbamate.

[0103] ESI-MS (m / z): 541.35 [M+H] + . 1 H NMR(300MHz,DMSO-d6)δ10.92(d,J=

[0104] 2.5Hz,1H),8.61(d,J=8.1Hz,1H),8.28-8.14(m,2H),7.85(ddt,J=15.6,7.8,1.3Hz,2H),7 .79-7.69(m,2H),7.62-7.46(m,4H),7.46-7.30(m,2H),7.25(d,J=2.4Hz,1H),7.07(ddd,J= 8.2,7.0,1.2Hz,1H),6.95(ddd,J=7.9,7.0,1.1Hz,1H),6.79(t,J=5.6Hz,1H),5.76(s,0H), 4.47(dd,J=15.7,5.4Hz,3H),2.92(q,J=6.5Hz,2H),1.74(s,2H),1.47(s,2H),1.35(s,9H).

[0105] 4) Preparation of (S)-N-(1-(((1H-indol-3-yl)methyl)amino)-5-amino-1-oxopentan-2-yl)-[1,1'-biphenyl]-3-carboxamide:

[0106] To tert-butyl (S)-(5-(((1H-indol-3-yl)methyl)amino)-4-([1,1'-biphenyl]-3-carboxamido)-5-oxopentyl)carbamate was added 4N HCl / EtOAc solution in an ice bath and stirred for 4 h. TLC (DCM:MeOH:HAc=25:1:2d) showed the disappearance of the starting material spot, and ninhydrin showed a purple color at the origin. The solution was pumped dry with a circulating water pump, and dry ethyl acetate was added. The reaction solution was pumped dry again with a water pump three times. Dry petroleum ether was then added and pumped dry again with a water pump. This was repeated three times to obtain compound (S)-N-(1-(((1H-indol-3-yl)methyl)amino)-5-amino-1-oxopentan-2-yl)-[1,1'-biphenyl]-3-carboxamide. ESI-MS (m / z): 441.31 [M+H] + .

[0107] 5) Preparation of (S)-N-(1-(((1H-indol-3-yl)methyl)amino)-5-(2-chloroacetimidino)-1-oxopentan-2-yl)-[1,1'-biphenyl]-3-carboxamide (RC2-33):

[0108] 1 mmol of (S)-N-(1-(((1H-indol-3-yl)methyl)amino)-5-amino-1-oxopentan-2-yl)-[1,1'-biphenyl]-3-carboxamide was dissolved in an appropriate amount of anhydrous methanol with stirring. 5 mmol of ethyl 2-chloroacetimidate hydrochloride was added under ice-water bath, and the pH was adjusted to 10 with N,N-diisopropylethylamine (DIPEA). Stirring was performed at room temperature for 12 hours. TLC (EA:H2O:HAc = 4:1:1) indicated the disappearance of the starting material spot. The mixture was concentrated to dryness under reduced pressure, dissolved in ultrapure water, and purified by C18 column chromatography to obtain the target product RC2-33.

[0109] ESI-MS (m / z): 516.17 [M+H] + . 1 H NMR (300MHz, DMSO-d6) δ10.95(d,J=2.5Hz,1H),9.60(s,1H),9.17(s,1H),8.74(d,J=8.1Hz,1H),8.36(t,J=5.5Hz,1H ),8.21(d,J=1.9Hz,1H),7.91(dt,J=7.7,1.4Hz,1H),7.84(dt,J=7.8,1.4Hz,1H),7.80-7.70(m,2H),7.62-7.46(m,4 H),7.46-7.30(m,2H),7.25(d,J=2.4Hz,1H),7.06(ddd,J=8.1,6.9,1.2Hz,1H),6.94(ddd,J=7.9,7.0,1.1Hz,1H),4. 56(dt,J=8.5,4.3Hz,1H),4.55-4.38(m,4H),3.27(t,J=6.5Hz,2H),1.91-1.76(m,2H),1.64(dq,J=15.7,8.0Hz,2H).

[0110] The obtained PAD4 inhibitor RC2-33 1 H NMR spectrum (300 MHz, DMSO-d6) Figure 2 shown.

[0111] The mass spectrum of the obtained PAD4 inhibitor RC2-33 is shown in FIG. Figure 3 shown.

[0112] The melting point, particle size, and zeta potential of the obtained PAD4 inhibitor are shown in Table 1. The particle size was measured using a nanoparticle size analyzer (Nano-ZS90, Malvern, UK); the zeta potential was measured using a zeta potential analyzer; and the melting point was measured using a melting point instrument (Shanghai Shenguang WRR visual melting point instrument).

[0113] Table 1 Melting point, particle size and zeta potential of PAD4 inhibitor RC2-33

[0114] Compound Melting point (℃) Particle size (nm) Zeta potential (mV) RC2-33 132.4～133.9 229.9±6.3 12.3±0.4

[0115] As can be seen from Table 1, the nanoparticle size distribution of RC2-33 is reasonable, which can reduce the influence of gas shear force (<250 nm) and is suitable for inhalation administration.

[0116] Test Example 1 In vitro anti-pulmonary fibrosis evaluation

[0117] 1. Anti-proliferation of lung fibroblasts

[0118] HLF (human lung fibroblast) cells were seeded at 5000 cells per well in a 96-well plate and cultured overnight in F12K medium supplemented with 10% FBS. Compounds at different concentrations were then added and incubated for 24 h. 5 mg / mL MTT was then added and incubated for another 4 h. The absorbance at 490 and 570 nm was measured using a multifunctional microplate reader. BEAS-2B (human lung epithelial cells) were treated in the same manner. The data were analyzed using the one-way ANOVA method using Graphpad Prism 9.4.0 software. The results are shown in Table 2.

[0119] Table 2 IC of the test compounds against HLF and BEAS-2B in vitro 50 value

[0120]

[0121] As shown in Table 2, compound RC2-33 inhibited the proliferation of HLF and BEAS-2B cells in a dose-dependent manner within the concentration range of 0 to 100 μM. 50 It is 12.44±1.19.

[0122] 2. Anti-lung fibroblast metastasis

[0123] 50,000 HLF cells suspended in serum-free F12K medium were seeded in the upper chamber of a transwell. 600 μL of culture medium containing 10% fetal bovine serum and TGF-β1 (5 ng / mL) was added to the upper chamber. The cells were then divided into 1) a control group (no treatment) and 2) an RC2-33 group. After adding 5 μM of drug to the upper chamber and incubating for 24 hours, the upper chamber fluid was aspirated, and the cells were fixed with 4% paraformaldehyde at 4°C for 30 minutes and stained with crystal violet for 15 minutes at room temperature. The crystal violet was washed off, and unmigrated cells on the upper membrane were wiped clean with a cotton swab. After drying, the cells were photographed using a light microscope (Zeiss, Germany). Migration rates were expressed as area and expressed as mean ± SD. The results are shown in Table 3.

[0124] Table 3 In vitro anti-HLF metastasis activity of the test compounds

[0125] Group Dosage (μM) Relative mobility (%) Control / 100±8.45 RC2-33 5 50.83±6.31****

[0126] In Table 3, compared with the Control group, ****p<0.0001.

[0127] As shown in Table 3, the number of cells passing through the Transwell chamber membrane in the RC2-33-treated group was significantly reduced compared with the control group without any treatment, indicating that the RC2-33-treated group can inhibit the activity of TGF-β1-induced HLF cell migration in vitro.

[0128] 3. Inhibit pulmonary fibroblast activation

[0129] HLF cells were cultured at 5 × 10 5 The cells were inoculated into confocal microplates and cultured overnight in F12K medium supplemented with 10% FBS. The medium was then replaced with serum-free medium for starvation for 12 h. 5 μM drugs were added for incubation for 12 h, and then TGF-β1 (5 ng / mL) was added. After incubation for 48 h, the medium was removed and the cells were fixed with 4% paraformaldehyde for 30 min at 4°C. The cells were washed with PBST and PBS, respectively, and then blocked with 5% BSA for 30 min. Rabbit polyclonal antibodies against smooth actin (α-SMA), fibronectin (FN-I), and collagen (COL1) were added. The cells were incubated overnight at 4°C, washed again with PBST and PBS, and goat anti-rabbit secondary antibodies labeled with AlexaFluor488 and AlexaFluor568 were added, respectively. The cells were incubated in the dark for 2 h at room temperature. After washing with PBST and PBS, the nuclei were stained with DAPI for 3 min. After washing with PBST and PBS, anti-fluorescence quenching mounting medium was added. The cells were examined using a laser confocal microscope (TCS) Images were taken using SP8 STED (Leica, Germany), and mean fluorescence intensity was analyzed using Image J. Protein expression is expressed as mean fluorescence intensity (mean ± SD), and the results are shown in Table 4. RC2-33 was also processed and tested using the same method.

[0130] Table 4 Inhibitory effect of test compounds on TGF-β-induced HLF fibrosis

[0131] Group α-SMA FN-I COL1 Control 6.20±1.45 25.41±5.23 7.66±0.86 TGF-β1 31.34±5.70 68.31±6.99 44.12±1.83 RC2-33 16.48±1.54**** 20.66±5.29**** 8.12±1.23****

[0132] In Table 4, ****p<0.0001 compared with the TGF-β1 group.

[0133] As shown in Table 4, compared with the control group without any treatment, the expression of fibrosis marker proteins α-SMA, FN-I, and COL1 in HLF cells treated with TGF-β1 increased, while compound RC2-33 treatment could downregulate the expression of these fibrosis marker proteins, indicating that compound RC2-33 can inhibit the fibrosis of HLF cells.

[0134] 4. Inhibition of NETs Release from Neutrophils in Vitro

[0135] ICR mice were sacrificed by cervical dislocation, and their hind legs were removed. The bone ends were cut to expose the cavity, and the bone marrow suspension was flushed with PBS. The bone marrow single cell suspension was filtered through a 70 μm nylon filter. Neutrophils were isolated using a mouse neutrophil isolation kit (TBDSceicge, LZS1100) and seeded into confocal microplates at 5 × 10 cells per dish. 5 The compound RC2-33 (5 μM) was immediately added and incubated for 2 hours. The cells were then induced with 5 μmol / L calcium ionophore A23187 for 2 hours. The medium was then removed by centrifugation and the cells were washed with PBST and PBS, fixed with 4% paraformaldehyde for 15 minutes, washed with PBST and PBS, and blocked with 5% BSA for 30 minutes. After washing with PBST and PBS, H3cit antibody (ABCAM: ab5103) was added and incubated overnight at 4°C. The primary antibody was recovered by centrifugation, washed with PBST and PBS, and stained with DAPI for 3 minutes. After washing with PBST and PBS, anti-fading mounting medium was added and the cells were observed using a Leica STED super-resolution confocal microscope (TCS SP8 STED, Leica, Germany).

[0136] The inhibitory effect of the compounds on NETs release from neutrophils is as follows Figure 4 As shown in the figure, after induction with phorbol methyl parathionate (PMA), neutrophils release filaments composed of DNA (blue fluorescence) and H3cit (red), which are neutrophil extracellular traps (NETs). In comparison, after treatment with compound RC2-33, these filaments are significantly reduced and the red fluorescence intensity is weakened, indicating that compound RC2-33 can inhibit the release of NETs from neutrophils. Scanning electron microscopy also confirms this result.

[0137] Test Example 2 Evaluation of Anti-fibrotic Activity in Vivo

[0138] 1. Experimental Methods

[0139] Male C57 / BL6 mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. All animal experiments were performed in accordance with the protocols approved by the Animal Care and Use Committee of Capital Medical University. Mice were randomly divided into 1) Normal (n = 6), 2) Model (n = 10), 3) Compound RC2-33 group (5 mg / kg, n = 10), 4) Positive drug pirfenidone group (PFD) (100 mg / kg, n = 10). After 3 days of adaptive feeding, the mice were anesthetized with isoflurane, and bleomycin (BLM, 5 mg / kg) was instilled intratracheally to induce mouse pulmonary fibrosis model except for the Normal group. Ten days after the instillation, the mice were treated with the following: Figure 5 The mice were treated with drugs using a homemade oral and nasal aerosol inhalation device. The model group received normal saline, while the compound group received the compound according to body weight for 10 consecutive days. Mouse weights were recorded at intervals. At the end of the experiment, the lungs were removed and weighed, and the lung weight to body weight ratio was calculated as the lung coefficient. Blood was collected from the mouse eyeballs and centrifuged to obtain serum, which was then assayed with ELISA kits for various inflammatory factors and fibrosis markers. Lung tissue was fixed, sectioned, and stained with HE and Masson staining. The degree of fibrosis was scored according to the ASHCROFT method. Lung sections were also subjected to immunohistochemistry to examine the expression of fibrosis-related proteins.

[0140] 2. Statistical methods

[0141] The data were expressed as mean ± standard deviation (x ± s) and statistically analyzed using Graphpadprism software. One-way analysis of variance (ANOVA) was used for comparison among multiple groups. p < 0.05 indicated statistically significant differences.

[0142] 3. Results and Analysis

[0143] 3.1 Effects of compounds on body weight in mice with pulmonary fibrosis

[0144] The body weight of mice in each group was weighed and recorded before modeling and 7, 14, and 21 days after modeling by intratracheal instillation of bleomycin (BLM). The body weight of mice generally decreases rapidly after pulmonary fibrosis.

[0145] The changes in body weight of mice in each group were as follows Figure 6 As shown in the figure, the weight of mice in the Normal group increased steadily, while the weight of mice in the Model group decreased sharply 7 days after modeling. The weight of mice in the other groups decreased within 7 days of modeling, which is also an argument for the establishment of the model. After the start of treatment, the weight of mice in the positive control PFD group and the compound RC2-33 group began to recover. The weight of the compound RC2-33 group had returned to the pre-modeling level by day 21. The above shows that RC2-33 is beneficial for the recovery of the physical condition of mice with pulmonary fibrosis.

[0146] 3.2 Effects of compounds on lung coefficients in mice with pulmonary fibrosis

[0147] The lungs of mice in each group were removed and weighed, and the lung coefficient was calculated according to the following formula: lung coefficient = lung weight (mg) / body weight (g). Because of the large infiltration of inflammatory cells in the early stages of pulmonary fibrosis, and the increase in fibroblasts and collagen deposition in the later stages, which leads to the formation of extracellular matrix (ECM) in the lungs, lung weight and lung coefficient increase are increased. Lung weight, body weight, and lung coefficient for each group of mice are shown in Table 5.

[0148] Table 5 Lung weight, body weight and lung coefficient of mice in each group

[0149] Group Lung weight (mg) Weight (g) Lung coefficient (mg / g) Normal 147.75±11.85 21.72±0.50 6.80±0.46 Model 235.14±19.74 15.11±1.65 15.78±2.36 PFD 152.18±33.5 20.36±2.64 7.77±2.76**** RC2-33 200.08±39.43 20.33±1.34 9.90±2.11****

[0150] In Table 5, compared with the Model group, ***p<0.001, ****p<0.0001.

[0151] 3.3 Effects of compounds on lung tissue morphology in mice with pulmonary fibrosis

[0152] The lungs of mice in each group were removed, fixed with tissue fixative, and then embedded in paraffin and sectioned for H&E and Masson staining. Figure 7 、 Figure 8 and as shown in Table 6.

[0153] Table 6 Effects of compound RC2-33 on BLM-induced pulmonary fibrosis in mice

[0154] Group Dosage (mg / kg) Ashcroft scoring Normal / 0.54±0.18 Model / 4.29±0.58 PFD 100 2.30±0.42*** RC2-33 5 2.57±0.64***

[0155] In Table 6, compared with the Model group, ***p<0.001.

[0156] It can be seen that the lung tissue structure of mice in the Normal group was clear, the alveoli were intact, the alveolar cavities were large and abundant, and the alveolar septa were not thickened. After BLM induction, the lung structure of mice in the Model group changed, specifically manifested as thickening of the alveolar wall, narrowing or even disappearance of the alveolar cavity, severe inflammatory infiltration, and Masson staining showed a large amount of fibrosis and collagen deposition in the pulmonary interstitium (p<0.0001); the lung tissue structure of mice in the positive drug PFD group and RC2-33 group was significantly improved (p<0.001), and the alveolar structure was restored, which indicates that compound RC2-33 can improve BLM-induced pulmonary fibrosis in mice.

[0157] 3.4 Effects of compounds on serum-related factors in mice with pulmonary fibrosis

[0158] Blood was collected from the eyeballs of mice and placed at room temperature for 2 hours. The blood was then centrifuged at 3000 rpm for 10 minutes. The supernatant was removed and the contents of SP-A, Hyp, KL-6, TNF-α, IL-17A, and MMP-7 factors were detected using ELISA kits. The results are shown in Table 7.

[0159] Table 7 Effects of compounds on the expression of fibrosis-related factors in the serum of mice with pulmonary fibrosis

[0160] Normal Model PFD RC2-33 SP-A 86.19±15.70 <![CDATA[155.73±14.89 #### ]]> 98.94±8.57 102.98±4.66**** Hyp 84.56±7.75 <![CDATA[143.56±11.16 #### ]]> 103.14±4.56 115.49±4.65**** KL-6 4.16±0.26 <![CDATA[6.23±0.38 #### ]]> 4.60±0.33 4.58±0.37**** TNF-α 273.68±12.13 <![CDATA[682.68±35.81 #### ]]> 426.32±60.88 527.21±7.15**** IL-17A 9.58±0.72 <![CDATA[31.32±3.05 #### ]]> 16.01±1.13 19.59±2.02**** MMP-7 31.47±1.85 <![CDATA[47.29±2.38 #### ]]> 31.53±2.33 34.51±1.61****

[0161] In Table 7, compared with the Normal group, #### p<0.0001, **p<0.01, ****p<0.0001 compared with the Model group.

[0162] It can be seen that compared with the Normal group mice, the levels of SP-A, Hyp, KL-6, TNF-α, IL-17A and MMP-7 in the serum of mice with BLM-induced pulmonary fibrosis increased, which indicates that the inflammatory response of mice with pulmonary fibrosis increased and collagen was deposited in large quantities. Compared with the Model group, the levels of related factors in the serum of the RC2-33 group decreased, and the differences were statistically significant.

[0163] In summary, the PAD4 inhibitor RC2-33 provided by the present invention has good anti-pulmonary fibrosis ability.

[0164] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A PAD4 inhibitor having the chemical structure shown in Formula I:

2. The method for preparing the PAD4 inhibitor according to claim 1, comprising the following steps: In the presence of a first condensing agent, biphenyl-3-carboxylic acid and the compound having the structure represented by Formula 1 undergo a first condensation reaction to obtain a compound having the structure represented by Formula 2; The compound having the structure shown in Formula 2 is subjected to a Bzl protecting group removal reaction to obtain a compound having the structure shown in Formula 3; In the presence of a second condensing agent, the compound having the structure shown in Formula 3 undergoes a second condensation reaction with indole-3-methylamine to obtain a compound having the structure shown in Formula 4; The compound having the structure shown in Formula 4 is subjected to a Boc protecting group removal reaction to obtain a compound having the structure shown in Formula 5; The compound having the structure shown in Formula 5 undergoes a substitution reaction with ethyl 2-chloroacetimidate hydrochloride to obtain a compound having the structure shown in Formula I.

3. The preparation method according to claim 2, characterized in that The first condensing agent and the second condensing agent include HOBt and DCC; The first condensation reaction and the second condensation reaction are carried out under alkaline conditions; the time of the first condensation reaction and the second condensation reaction is independently 6 to 24 hours.

4. The preparation method according to claim 2, characterized in that The Bzl protection group removal reaction is carried out under conditions of Pd / C catalyst and H2; The reaction time for removing the Bzl protecting group is 12 to 36 hours.

5. The preparation method according to claim 2, characterized in that The Boc protection group removal reaction is carried out in an ethyl acetate solution of HCl, and the reaction time for the Boc protection group removal reaction is 1 to 5 hours.

6. The preparation method according to claim 2, characterized in that The molar ratio of the compound having the structure shown in Formula 5 to ethyl 2-chloroacetimidate hydrochloride is 1:1.5-6.

7. The preparation method according to claim 2 or 6, characterized in that The substitution reaction time is 8 to 24 hours.

8. Use of the PAD4 inhibitor according to claim 1 in the preparation of anti-pulmonary fibrosis drugs.

9. An anti-pulmonary fibrosis drug comprising an active ingredient and a pharmaceutically acceptable carrier; The active ingredient comprises the PAD4 inhibitor according to claim 1.

10. The anti-pulmonary fibrosis drug according to claim 9, characterized in that The dosage of the active ingredient is 1 to 10 mg / kg.

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