A small molecule compound and its application in the preparation of anti-pancreatitis drugs

Abstract

The present invention discloses a small molecule compound and its application in the preparation of anti-pancreatitis drugs. The molecular formula of the small molecule compound is C 18 H 21 FN4O2, with a molecular weight of 344. The structural formula is shown in Formula I, specifically (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide. The small molecule compound is obtained by acylating (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol and (E)-4-(4-fluorophenyl)but-3-enoic acid. Experimental results show that the small molecule compound has strong anti-acute pancreatitis activity and is expected to be applied to the preparation of various anti-acute pancreatitis drugs. The method for preparing the small molecule compound in the present invention has a simple synthesis process, is easy to operate, has low cost, and is suitable for popularization.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and particularly relates to a small molecule compound and its application in the preparation of anti-pancreatitis drugs. Background Art

[0002] Acute pancreatitis is a common acute abdominal disease, which is a serious disease caused by the abnormal activation of pancreatic enzymes in the pancreas, resulting in digestion of the pancreas. Local and even systemic inflammatory reactions occur during the progression of acute pancreatitis. Pancreatitis has symptoms such as abdominal pain, abdominal distension, nausea, vomiting, fever, shock, and organ dysfunction, and can be fatal in severe cases. Therefore, the development of effective drugs for the treatment of pancreatitis is of great significance.

[0003] It has been found that in the innate immune response of acute pancreatitis, macrophage infiltration in the pancreas can be observed. The cytokines secreted by macrophages play an important role in the occurrence and development of acute pancreatitis. Therefore, a cell model of acute pancreatitis can be established by co-culturing primary pancreatic acinar cells and peritoneal macrophages. In this co-culture system, cerulein and lipopolysaccharide (LPS) are used simultaneously to induce the activation of digestive enzymes and the secretion of cytokines. Therefore, the above-established cell model of acute pancreatitis can be used as a model for screening small molecule compounds with anti-acute pancreatitis activity.

[0004] Currently, there is no report on the anti-acute pancreatitis activity of small molecule compounds, specifically (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide. Summary of the Invention

[0005] Aiming at the technical problems existing in the background art, the purpose of the present invention is to provide a small molecule compound and its application in the preparation of anti-pancreatitis drugs. This small molecule compound has strong anti-acute pancreatitis activity and is expected to be applied in the preparation of various anti-acute pancreatitis drugs.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a small molecule compound is provided. The molecular formula of the small molecule compound is C 18 H 21 FN4O2, with a molecular weight of 344, and the structural formula is shown in Formula I:

[0008]

[0009] Preferably, the small molecule compound is obtained by carrying out an acylation reaction with (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl) cyclopentanol and (E)-4-(4-fluorophenyl)-3-butenoic acid as raw materials under the action of a catalyst. The specific steps are as follows:

[0010] Dissolve (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl) cyclopentanol and (E)-4-(4-fluorophenyl)-3-butenoic acid in an organic solvent, then add a catalyst and carry out the acylation reaction. After the reaction is completed, purify the obtained crude product by silica gel column chromatography to obtain the small molecule compound.

[0011] Preferably, the molar ratio of (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl) cyclopentanol to (E)-4-(4-fluorophenyl)-3-butenoic acid is 1-10:10-1.

[0012] Preferably, the dosage ratio of the organic solvent to (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl) cyclopentanol is 10-500 mL:1 mmol.

[0013] Preferably, the organic solvent is one or a combination of two or more of DCM, chloroform, DMSO, and DMF, and further preferably DCM (dichloromethane).

[0014] Preferably, the molar ratio of the catalyst to (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl) cyclopentanol is 0.01-5:1.

[0015] Preferably, the catalyst is one or a combination of two or more of HATU (2-(7-azabenzotriazol)-N,N,N',N'-tetramethylurea hexafluorophosphate), HBTU (benzotriazol-N,N,N',N'-tetramethylurea hexafluorophosphate), HCTU (6-chlorobenzotriazol-1,1,3,3-tetramethylurea hexafluorophosphate), DIEA (N,N-diisopropylethylamine), DCC (dicyclohexylcarbodiimide), and DMAP (4-dimethylaminopyridine), and further preferably a combination of HATU and DIEA.

[0016] Preferably, the acylation reaction is carried out at room temperature, and the reaction time is 4-24 h.

[0017] Preferably, when the crude product is purified by silica gel column chromatography, the eluent used is a mixed solvent composed of any one of dichloromethane, ethyl acetate and chloroform and petroleum ether, and in the composition of the mixed solvent, the volume ratio of the petroleum ether to any one of dichloromethane, ethyl acetate and chloroform is 10 to 1:1 to 10, and more preferably 1:1.

[0018] The third aspect of the present invention provides the use of the above-mentioned small molecule compound in the preparation of drugs for treating acute pancreatitis.

[0019] The fourth aspect of the present invention provides a pharmaceutical composition which contains a therapeutically effective dose of the above-mentioned small molecule compound and a pharmaceutically acceptable carrier.

[0020] The present invention has the following beneficial effects:

[0021] The present invention uses (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol and (E)-4-(4-fluorophenyl)-3-butenoic acid as raw materials, and carries out an acylation reaction under the action of a catalyst to prepare a small molecule compound with a novel structure, specifically (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide. And the effects on the pancreatitis cell model and animal model were investigated. The experimental results show that the small molecule compound has strong anti-acute pancreatitis activity and is expected to be applied to the preparation of various anti-acute pancreatitis drugs.

[0022] (2) The method for preparing the small molecule compound of the present invention has a simple synthesis process, is easy to operate, has low cost, and is suitable for popularization. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is the structural formula of the small molecule compound, specifically (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide;

[0025] Figure 2Synthetic route diagram for the preparation of (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide in Example 1;

[0026] Figure 3 Mass spectrum of (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide prepared in Example 1;

[0027] Figure 4 Results diagram of the in vitro acute pancreatitis experiment of (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide prepared in Example 1: Among them, Figure A represents the lipase activity result; Figure B represents the amylase activity result; Figure C represents the TNFα level result; Figure D represents the MCP-1 level result; Figure E represents the IL-6 level result.

[0028] Figure 5 Results diagram of the in vivo acute pancreatitis experiment of (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide prepared in Example 1: Among them, Figure A represents the lipase activity result in serum; Figure B represents the amylase activity result in serum, Figure C represents the IL-6 level result of cytokines in serum; Figure D represents the MCP-1 level result of cytokines in serum; Figure E represents the TNFα level result of cytokines in serum; Figure F represents the picture of mouse pancreatic pathological section. Detailed implementation mode

[0029] In the following description, for the purpose of illustration rather than limitation, specific details such as specific system structures and technologies are presented to thoroughly understand the embodiments of the present invention. However, those skilled in the art should clearly understand that the present invention can also be implemented in other embodiments without these specific details.

[0030] Example 1

[0031] Prepare (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide, and its synthetic route is as Figure 2 shown:

[0032]

[0033] The specific steps are as follows: (1) Weigh 0.11 mmol of (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol, 0.1 mmol of (E)-4-(4-fluorophenyl)-3-butenoic acid, 0.12 mmol of HATU and 15 ml of DCM, add them to a 50 ml round-bottom flask, stir in an ice-water bath for 5 min, then add 0.3 mmol of DIEA, stir the mixed solution at room temperature for 10 h. After the reaction is completed, the solvent is dissolved and evaporated to obtain the crude product; (2) The obtained crude product is purified by silica gel column chromatography (the elution solvent is a mixed solvent composed of petroleum ether and dichloromethane mixed in a volume ratio of 1:1) to obtain a white solid product, which is the target substance (29 mg, yield 84.3%).

[0034] The mass spectrometry characterization of the above white solid product is as Figure 3 shown. From Figure 3 the results, it can be seen that in Example 1, (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide was successfully synthesized, and its structural formula is as Figure 1 shown.

[0035] Example 2

[0036] Weigh 0.10 mmol of (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol, 0.12 mmol of (E)-4-(4-fluorophenyl)-3-butenoic acid, 0.11 mmol of HCTU and 18 ml of chloroform, add them to a 50 ml round-bottom flask, stir in an ice-water bath for 5 min, then add 0.5 mmol of DIEA, stir the mixed solution at room temperature for 8 h. After the reaction is completed, the solvent is dissolved and evaporated to obtain the crude product; (2) The obtained crude product is purified by silica gel column chromatography (the elution solvent is a mixed solvent composed of petroleum ether and ethyl acetate mixed in a volume ratio of 1:1) to obtain a white solid product, which is the target substance (26 mg, yield 75.6%).

[0037] Example 3

[0038] Weigh 0.12 mmol of (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol, 0.1 mmol of (E)-4-(4-fluorophenyl)-3-butenoic acid, 0.2 mmol of DCC and 15 ml of dichloromethane and add them to a 50 ml round-bottom flask. Stir for 5 min in an ice-water bath, then add 0.3 mmol of DMAP. Stir the mixed solution at room temperature for 6 h. After the reaction is completed, dissolve and evaporate the solvent to obtain the crude product; (2) Purify the obtained crude product by silica gel column chromatography (the elution solvent is a mixed solvent composed of petroleum ether, dichloromethane and ethyl acetate in a volume ratio of 2:1:1) to obtain a white solid product, which is the target substance (23 mg, yield 66.9%).

[0039] Example 4

[0040] Weigh 0.13 mmol of (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol, 0.10 mmol of (E)-4-(4-fluorophenyl)-3-butenoic acid, 0.15 mmol of HBTU and 16 ml of chloroform and add them to a 50 ml round-bottom flask. Stir for 5 min in an ice-water bath, then add 0.5 mmol of DIEA. Stir the mixed solution at room temperature for 9 h. After the reaction is completed, dissolve and evaporate the solvent to obtain the crude product; (2) Purify the obtained crude product by silica gel column chromatography (the elution solvent is a mixed solvent composed of petroleum ether and dichloromethane in a volume ratio of 2:1) to obtain a white solid product, which is the target substance (24 mg, yield 69.8%).

[0041] Example 5

[0042] Weigh 0.1 mmol of (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol, 0.1 mmol of (E)-4-(4-fluorophenyl)-3-butenoic acid, 0.15 mmol of HATU, 0.2 mmol of DCC and 15 ml of dichloromethane and add them to a 50 ml round-bottom flask. Stir for 5 min in an ice-water bath, then add 0.3 mmol of DIEA and 0.3 mmol of DMAP. Stir the mixed solution at room temperature for 10 h; After the reaction is completed, dissolve and evaporate the solvent to obtain the crude product; (2) Purify the obtained crude product by silica gel column chromatography (the elution solvent is a mixed solvent composed of petroleum ether and dichloromethane in a volume ratio of 1:1) to obtain a white solid product, which is the target substance (27 mg, yield 78.5%).

[0043] Example 6

[0044] Weigh 0.1 mmol of (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol, 0.1 mmol of (E)-4-(4-fluorophenyl)-3-butenoic acid, 0.5 mmol of HATU and 15 ml of DCM and add them to a 50-ml round-bottom flask. Stir for 5 min in an ice-water bath, then add 0.5 mmol of DIEA. Stir the mixed solution at 35 °C for 6 h. After the reaction is completed, dissolve and evaporate the solvent to dryness to obtain the crude product; (2) Purify the obtained crude product by silica gel column chromatography (the elution solvent is a mixed solvent composed of petroleum ether and dichloromethane mixed in a volume ratio of 1:1) to obtain a white solid product, which is the target substance (27 mg, yield 78.4%).

[0045] Example 7

[0046] Weigh 0.1 mmol of (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol, 0.1 mmol of (E)-4-(4-fluorophenyl)-3-butenoic acid, 0.1 mmol of HCTU, 8 ml of chloroform and 8 ml of DCM and add them to a 50-ml round-bottom flask. Stir for 5 min in an ice-water bath, then add 0.1 mmol of DIEA. Stir the mixed solution at room temperature for 10 h. After the reaction is completed, dissolve and evaporate the solvent to dryness to obtain the crude product; (2) Purify the obtained crude product by silica gel column chromatography (the elution solvent is a mixed solvent composed of petroleum ether and ethyl acetate mixed in a volume ratio of 1:1) to obtain a white solid product, which is the target substance (20 mg, yield 58.1%).

[0047] Example 8

[0048] Weigh 0.1 mmol of (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol, 1 mmol of (E)-4-(4-fluorophenyl)-3-butenoic acid, 0.6 mmol of DCC, 25 ml of dichloromethane and 25 ml of chloroform and add them to a 100-ml round-bottom flask. Stir for 5 min in an ice-water bath, then add 0.5 mmol of DMAP. Stir the mixed solution at room temperature for 9 h. After the reaction is completed, dissolve and evaporate the solvent to dryness to obtain the crude product; (2) Purify the obtained crude product by silica gel column chromatography (the elution solvent is a mixed solvent composed of petroleum ether, dichloromethane and ethyl acetate mixed in a volume ratio of 2:1:1) to obtain a white solid product, which is the target substance (25 mg, yield 72.7%).

[0049] Example 9

[0050] Weigh 1 mmol of (1S,2S,4R)-2-(aminomethyl)-4-(4H-1,2,4-triazol-3-yl)cyclopentanol, 0.1 mmol of (E)-4-(4-fluorophenyl)-3-butenoic acid, 0.15 mmol of HBTU, 5 ml of chloroform and 5 ml of DCM, add them to a 50 ml round-bottom flask, stir in an ice-water bath for 5 min, then add 0.5 mmol of DIEA, stir the mixed solution at room temperature for 9 h. After the reaction is completed, the solvent is dissolved and evaporated to dryness to obtain the crude product; (2) The obtained crude product is purified by silica gel column chromatography (the elution solvent is a mixed solvent composed of petroleum ether and dichloromethane mixed in a volume ratio of 2:1) to obtain a white solid product, which is the target substance (20 mg, yield 58.1%).

[0051] Cell experiment on anti-acute pancreatitis activity

[0052] In this invention, a cell model of acute pancreatitis is established by co-culturing primary pancreatic acinar cells and peritoneal macrophages. The specific detection steps are as follows:

[0053] (1) Solution preparation

[0054] Dissolve the (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide prepared in Example 1 with DMSO to prepare a sample stock solution with a concentration of 10 mM.

[0055] (2) Isolation of primary cells

[0056] To isolate primary pancreatic acinar cells, the pancreatic tissue is cut into small pieces and soaked in a collagenase solution at 37 °C. After digestion and filtration, pancreatic acinar cells are obtained and cultured in DMEM medium containing 20% fetal bovine serum.

[0057] To isolate peritoneal macrophages, 4 ml of 3% sodium thioglycollate solution is injected into the peritoneal cavity of C57BL / 7 mice. After 3 days, the mice are sacrificed, pre-cooled PBS solution is injected into the peritoneal cavity, the PBS solution is aspirated, and then peritoneal macrophages are separated by centrifugation. The separated cells are cultured in DMEM medium containing 10% fetal bovine serum.

[0058] (3) Construction of a co-culture system of primary pancreatic acinar cells and peritoneal macrophages

[0059] Inoculate the isolated pancreatic acinar cells (about 2×10 5 cells) on a 24-well plate. After the cells adhere, peritoneal macrophages (about 2×10 5(s) were inoculated into the well plates. Then, 200 nM cerulein and 10 ng / ml LPS were added and incubated for 6 h.

[0060] (4) In vitro anti - acute pancreatitis activity detection

[0061] (E) - 4 - (4 - fluorophenyl) - N - (((1S,2S,4R) - 2 - hydroxy - 4 - (4H - 1,2,4 - triazol - 3 - yl)cyclopentyl)methyl)but - 3 - enamide (10 μM, 6 h) prepared in Example 1 was added to the co - culture model of primary pancreatic acinar cells and peritoneal macrophages. Then, the levels of cytokines IL - 6, TNFα, and MCP - 1 in the culture medium were detected by flow cytometry using a cytokine CBA kit.

[0062] The culture medium of the co - culture model after drug treatment was collected, diluted, and the activities of amylase and lipase in it were detected by an automatic biochemical analyzer. The experimental results are shown in Figure 4 , where the abscissa in the figure represents each experimental group. Among them, Cerulein represents cerulein, LPS represents lipopolysaccharide, and Z10 represents the small - molecule compound (E) - 4 - (4 - fluorophenyl) - N - (((1S,2S,4R) - 2 - hydroxy - 4 - (4H - 1,2,4 - triazol - 3 - yl)cyclopentyl)methyl)but - 3 - enamide.

[0063] Figure 4 The results showed that under the stimulation of cerulein combined with LPS, the activities of amylase and lipase in the culture medium increased, and the secretion of cytokines (IL - 6, TNFα, and MCP - 1) increased. However, after treatment with the small - molecule compound, it could reverse the increase in amylase and lipase activities and cytokine release caused by the stimulation of cerulein and LPS, indicating that the small - molecule compound (E) - 4 - (4 - fluorophenyl) - N - (((1S,2S,4R) - 2 - hydroxy - 4 - (4H - 1,2,4 - triazol - 3 - yl)cyclopentyl)methyl)but - 3 - enamide of the present invention has good in vitro anti - acute pancreatitis activity.

[0064] (5) In vivo anti - acute pancreatitis activity detection

[0065] Specific detection steps: Inject cerulein (50 μg / kg) into the peritoneal cavity of C57BL / 6 mice once every hour for a total of 7 times. Add (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide (10 mg / kg) during the first injection, and add LPS (10 mg / kg) simultaneously during the last injection. Detect the lipase, amylase activities and the levels of cytokines IL-6, TNFα and MCP-1 in the serum 3 hours after injection. And perform HE staining pathological analysis on pancreatic tissues. The experimental results are shown in Figure 5 . The abscissa in the figure represents the experimental groups. Among them, Cerulein represents cerulein, LPS represents lipopolysaccharide, and Z10 represents (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide.

[0066] Figure 5 The results showed that under the stimulation of cerulein combined with LPS, the activities of amylase and lipase in the serum of mice increased, and the secretion of cytokines (IL-6, TNFα and MCP-1) increased. After treatment with the small molecule compound, the increased activities of amylase and lipase and the cytokine release caused by the stimulation of cerulein combined with LPS could be reversed, indicating that (E)-4-(4-fluorophenyl)-N-(((1S,2S,4R)-2-hydroxy-4-(4H-1,2,4-triazol-3-yl)cyclopentyl)methyl)but-3-enamide has good anti-acute pancreatitis activity. The further results of pancreatic HE staining pathological sections also showed that this small molecule compound could reduce the pathological damage of the acute pancreatitis model, indicating good in vivo anti-acute pancreatitis activity.

[0067] The present invention is not limited to the above specific embodiments. Those of ordinary skill in the art starting from the above concepts and making various changes without creative labor fall within the protection scope of the present invention.

Claims

1. Use of a small molecule compound in the preparation of a drug for treating acute pancreatitis, characterized in that, The molecular formula of the small molecule compound is C 18 H 21 FN4O2, with a molecular weight of 344, and the structural formula is shown in Formula I: （I）。 2. A pharmaceutical composition, characterized in that, Comprising a small molecule compound as described in claim 1 and a pharmaceutically acceptable carrier.

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