CN114591254B - 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative and preparation method and application thereof - Google Patents

3, 5-disubstituted phenyl-1, 2, 4-triazole derivative and preparation method and application thereof Download PDF

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CN114591254B
CN114591254B CN202210337261.7A CN202210337261A CN114591254B CN 114591254 B CN114591254 B CN 114591254B CN 202210337261 A CN202210337261 A CN 202210337261A CN 114591254 B CN114591254 B CN 114591254B
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CN114591254A (en
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张静夏
陈健文
黄萍
劳尧强
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Sun Yat Sen University
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Abstract

The invention discloses a 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative, a preparation method and application thereof, belongs to the technical field of biological medicines, and belongs to the technical field of biological medicines, wherein the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a formula I, has a good anti-inflammatory effect at a cellular level, and a preferable compound has an effect of preventing and/or treating ulcerative colitis on mice, and is characterized by reducing hematochezia degree, improving fecal characteristics, reducing weight reduction and increasing DSS-induced colon length;

Description

3, 5-disubstituted phenyl-1, 2, 4-triazole derivative and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative, and a preparation method and application thereof.
Background
Ulcerative colitis is a chronic, non-specific inflammatory disease of the intestinal tract. It presents chronic disease course and recurrent disease throughout the life, the good sites are rectum and colon, and chronic or subacute diarrhea, mucopurulent bloody stool, abdominal pain and the like are manifested, often accompanied by systemic symptoms of different degrees. The risk of patient cancer increases due to the long course of the disease. It is currently believed that the multi-factor interactions result in, for example, genetic, environmental, gut microbiota disorders, inflammatory processes resulting from an imbalance in the gut mucosal immune system, and the like. Recent studies have shown that oxidative stress is also an important mechanism for inducing ulcerative colitis.
At present, clinical medication is mainly to inhibit inflammatory reactions, such as aminosalicylic acid preparations, glucocorticoids and immunosuppressants. The long-term application of the treatment schemes has larger side effect and is easy to be repeated, and the invention provides a medicament for effectively preventing and treating the ulcerative colitis with smaller toxicity, which is a technical problem to be solved by pharmaceutical researchers.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative, a preparation method and application thereof, wherein the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has good effect of preventing and/or treating ulcerative colitis and has small toxic and side effects.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in formula I:
wherein R is 1 Is CF (CF) 3 、H、Cl、C 1 -C 4 One of the alkoxy groups;
R 2 is H;
R 3 is C 1 -C 6 Alkyl, C 1 -C 6 Hydroxy, C 1 -C 6 Halo, C 1 -C 6 Polyhaloalkyl, C 1 -C 6 Nitro, C 1 -C 6 Carboxyl, C 1 -C 6 Amino, C 1 -C 6 Hydrazino radicals C 1 -C 4 Substituted amino, C 1 -C 4 Ether group, C 1 -C 4 Keto, C 1 -C 4 One of ester group and heteroaryl group.
The inventor of the present invention found in a great deal of triazole derivative researches that the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative shown in the structural formula has good effect of preventing and/or treating ulcerative colitis, and the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative shows the effects of reducing hematochezia, improving fecal characteristics, reducing weight loss, increasing DSS-induced colon length and reducing DSS-induced spleen enlargement.
The inventor finds that compared with the existing clinical medicines, the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has the advantage of small toxic and side effects, and the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has small dosage, and the effective dosage is 15mg/kg, so that the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has obvious improvement effect.
As a preferred embodiment of the present invention, the heteroaryl group is one of pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, triazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl, quinolinyl, naphthyl, benzothiazolyl, benzothienyl, benzofuranyl, purinyl.
As a preferred embodiment of the present invention, the R 1 Is CF (CF) 3 、H、Cl、OCH 3 One of the following; the R is 2 Is H; the R is 3 Is CH (CH) 3 ) 2 、C(CH 3 ) 3 、CH(CH 2 ) 4 、(CH 2 ) 2 OH、CH 2 CF 3 、CH 2 COOH、CH 2 COOCH 2 CH 3 One of pyridyl, pyrazinyl, naphthyl and benzothiazolyl.
Especially when R 1 、R 2 、R 3 The above groups can be used for preventing and/or treating ulcerUlcerative colitis has effects.
The invention also provides a preparation method of the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative, which comprises the following steps:
adding an o-hydroxybenzoic acid derivative and an o-hydroxybenzoamide derivative into a solvent A, adding pyridine, adding thionyl chloride while stirring, heating and refluxing to remove the solvent and residual thionyl chloride, and recrystallizing to obtain an oxazine intermediate;
and adding the oxazine intermediate and the hydrazine derivative into the solvent B, removing part of the solvent after heating and refluxing, crystallizing, and recrystallizing to obtain the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative.
The 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative capable of preventing and/or treating ulcerative colitis is prepared by taking the o-hydroxybenzoic acid derivative, the o-hydroxybenzoamide derivative and the hydrazine derivative as raw materials.
As a preferred embodiment of the present invention, the o-hydroxybenzoic acid derivative is 2-hydroxy-4- (trifluoromethyl) benzoic acid, and the o-hydroxybenzoamide derivative is 2-hydroxybenzoamide.
As a preferred embodiment of the present invention, the hydrazine derivative is at least one of isopropyl hydrazine, t-butyl hydrazine, p-fluorobenzene hydrazine hydrochloride, cyclopentane hydrazine, hydroxyethyl hydrazine, trifluoroethyl hydrazine, ethyl hydrazinoacetate, hydrazinoacetic acid, hydrazinopyridine, hydrazinopyrazine, hydrazinonaphthalene, hydrazinobenzothiazole.
As a preferred embodiment of the present invention, the solvent a is at least one of toluene, xylene, diphenyl ether, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide; the solvent B is at least one of ethanol, propanol, isopropanol, tertiary butanol, tetrahydrofuran, dioxane, butanone, acetonitrile, ethyl acetate, chloroform, 1-dichloroethane and pyridine.
As a preferred embodiment of the invention, the material ratio of the o-hydroxybenzoic acid derivative to thionyl chloride is 1g: (1-5) ml; the ratio of the amounts of the substances of the o-hydroxybenzoic acid derivative to the o-hydroxybenzoamide derivative is 1: (0.9-3). The ratio of the amounts of oxazine intermediate to hydrazine derivative material is 1: (0.9-3).
The invention also provides an application of the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative or the pharmaceutically acceptable salt thereof in preparing medicines for preventing and/or treating ulcerative colitis.
The invention also provides a medicament for preventing and/or treating ulcerative colitis, which takes the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative or pharmaceutically acceptable salt thereof as a main active substance.
As a preferred embodiment of the present invention, the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative or pharmaceutically acceptable salt thereof is effective in an amount of 1 to 30mg/kg.
The invention has the beneficial effects that: the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has good effect of preventing and/or treating ulcerative colitis, and the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative is capable of reducing hematochezia, improving fecal characteristics, reducing weight loss and increasing DSS-induced colon length.
Drawings
FIG. 1 is a graph showing the effect of 3, 5-disubstituted phenyl-1, 2, 4-triazole derivatives described in examples 1 to 19 on the activity of RAW cells.
FIG. 2 is a graph showing the effect of 3, 5-disubstituted phenyl-1, 2, 4-triazole derivatives described in examples 2, 5, 8, 10, 11, 12, 14, 15, 16, 17, 19 on LPS stimulation of NO by RAW cells.
FIG. 3 is a graph showing weight change of mice on DSS model with 3, 5-disubstituted phenyl-1, 2, 4-triazole derivatives described in example 5.
FIG. 4 is a graph showing the daily food intake of mice in DSS model with 3, 5-disubstituted phenyl-1, 2, 4-triazole derivatives described in example 5.
FIG. 5 shows the colon length change of the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative described in example 5 versus a DSS model mouse.
FIG. 6 is a graph showing colon length statistics of mice with DSS model based on 3, 5-disubstituted phenyl-1, 2, 4-triazole derivatives described in example 5.
FIG. 7 is a graph showing the variation of DAI scores of mice with DSS model for 3, 5-disubstituted phenyl-1, 2, 4-triazole derivatives described in example 5.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 1:
synthesis of Compound 1
(1) Synthesis of oxazine intermediates
3.09g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.92g of 2-hydroxybenzoamide were added to the flask, 25mL of xylene and 100. Mu.L of pyridine were added, 3mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 5 hours. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with absolute ethyl alcohol to obtain the oxazine intermediate.
(2) Synthesis of Compound 1
0.9g of oxazine intermediate and 0.1g of isopropyl hydrazine were added to the flask, 10mL of absolute ethanol was added, the reaction was heated under reflux for 9 hours, and the reaction was monitored by thin layer chromatography. And naturally cooling the reaction solution to room temperature after the reaction is finished, and recrystallizing with absolute ethyl alcohol to obtain the compound 1.
Structure corroboration data for compound 1: a white solid was used as a solid, 1 H NMR(400MHz,DMSO-d6)δ11.51(s,1H),10.39(s,1H),8.20(d,J＝8.3Hz,1H),7.49–7.41(m,2H),7.36–7.29(m,2H),7.07(d,J＝8.4Hz,1H),7.01(t,J＝7.5Hz,1H),4.48(p,J＝6.5Hz,1H),1.47(s,3H),1.45(s,3H)。
example 2
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 2:
synthesis of Compound 2
(1) Synthesis of oxazine intermediates
2.0g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.3g of 2-hydroxybenzoamide were added to the flask, 25mL of DMF and 150. Mu.L of pyridine were added, and 2.5mL of thionyl chloride was added at room temperature with stirring, heated under reflux for 7 hours, and the reaction was monitored by thin layer chromatography. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with absolute ethyl alcohol to obtain the oxazine intermediate.
(2) Synthesis of Compound 2
1.0g of oxazine intermediate and 0.1g of t-butylhydrazine were added to the flask, 10mL of absolute ethanol was added, the reaction was heated under reflux for 7 hours, and the reaction was monitored by thin layer chromatography. After the reaction, naturally cooling the reaction solution to room temperature, and recrystallizing with methanol to obtain compound 2 (R) 1 ＝CF 3 ,R 2 ＝H,R 3 ＝C(CH 3 ) 3 )。
Structure corroboration data for compound 2: a white solid was used as a solid, 1 H NMR(400MHz,Chloroform-d)δ8.12(d,J＝8.2Hz,1H),7.36–7.28(m,3H),7.17(d,J＝8.2Hz,1H),7.02(t,J＝7.5Hz,1H),6.89(d,J＝8.1Hz,1H),1.61(s,9H)。
example 3
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 3:
synthesis of Compound 3
(1) Synthesis of oxazine intermediates
1.9g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.2g of 2-hydroxybenzoamide were added to the flask, 25mL of DMSO and 160. Mu.L of pyridine were added, and 1.8mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 5 hours. And after the reaction is finished, naturally cooling the reaction solution to room temperature, precipitating solids, filtering, taking a filter cake, and recrystallizing with acetonitrile to obtain the oxazine intermediate.
(2) Synthesis of Compound 3
1.0g of oxazine intermediate and 0.1g of p-fluorobenzenehydrazine hydrochloride were added to the flask, 10mL of acetonitrile was added, and the reaction was heated under reflux for 8 hours, and the reaction was monitored by thin layer chromatography. After the reaction, the reaction mixture was cooled to room temperature and recrystallized from acetonitrile to give compound 3 (r1=cf) 3 ,R 2 ＝H,R 3 ＝CH(CH 2 ) 4 )。
Structure corroboration data for compound 3: 1 H NMR(400MHz,Chloroform-d)δ10.28(s,2H),8.21(d,J＝8.1Hz,1H),7.59(d,J＝7.9Hz,1H),7.48(t,J＝7.8Hz,1H),7.34(s,1H),7.28(d,J＝4.4Hz,1H),7.21(d,J＝8.3Hz,1H),7.07(t,J＝7.6Hz,1H),5.16(p,J＝6.9Hz,1H),2.32–2.24(m,4H),2.15–2.07(m,2H),1.89–1.80(m,2H)。
example 4
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 4:
synthesis of Compound 4
(1) Synthesis of oxazine intermediates
2.2g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.6g of 2-hydroxybenzoamide were added to the flask, 25mL of diphenyl ether and 150. Mu.L of pyridine were added, and 2.2mL of thionyl chloride was added at room temperature with stirring, heated under reflux for 4 hours, and the reaction was monitored by thin layer chromatography. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with ethyl acetate to obtain the oxazine intermediate.
(2) Synthesis of Compound 4
1.0g of oxazine intermediate and 0.2g of hydroxyethylhydrazine were added to the flask, 10mL of ethyl acetate was added, and the reaction was heated under reflux for 8 hours, and monitored by thin layer chromatography. After the reaction, the reaction mixture was cooled to room temperature, and recrystallized from ethyl acetate to give Compound 4 (R) 1 ＝CF 3 ,R 2 ＝H,R 3 ＝CH 2 CH 2 OH)。
Structure corroboration data for compound 4: a white solid was used as a solid, 1 H NMR(500MHz,DMSO-d6)δ11.52(s,1H),10.37(s,1H),8.19(d,J＝8.3Hz,1H),7.49(d,J＝8.0Hz,1H),7.44(t,J＝8.6Hz,1H),7.35–7.29(m,2H),7.07(d,J＝8.3Hz,1H),7.01(d,J＝7.5Hz,1H),4.89(s,1H),4.22(t,J＝5.7Hz,2H),3.79(t,J＝5.7Hz,2H)。
example 5
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 5:
synthesis of Compound 5
(1) Synthesis of oxazine intermediates
2.1g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.7g of 2-hydroxybenzoamide were added to the flask, 25mL of xylene and 100. Mu.L of pyridine were added, and 2mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 6 hours. And naturally cooling the reaction liquid to room temperature after the reaction is finished, separating out solids, filtering, taking a filter cake, and recrystallizing with tertiary butanol to obtain the oxazine intermediate.
(2) Synthesis of Compound 5
1.0g of oxazine intermediate and 0.1g of trifluoroethylhydrazine were added to the flask, 10mL of t-butanol was added, and the reaction was heated under reflux for 10 hours, and the reaction was monitored by thin layer chromatography. After the reaction, naturally cooling the reaction solution to room temperature, and recrystallizing with tert-butanol to obtain the compound 5 (R) 1 ＝CF 3 ,R 2 ＝H,R 3 ＝CH 2 CF 3 )。
Structure corroboration data for compound 5: a white solid was used as a solid, 1 H NMR(500MHz,Chloroform-d)δ9.80(s,2H),8.19(d,J＝8.1Hz,1H),7.54(d,J＝7.7Hz,1H),7.50(t,J＝7.7Hz,1H),7.33(s,1H),7.24(d,J＝6.9Hz,1H),7.17(d,J＝8.0Hz,1H),7.08(t,J＝7.5Hz,1H),5.01(q,J＝7.7Hz,2H)。
example 6
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 6:
synthesis of Compound 6
(1) Synthesis of oxazine intermediates
2.0g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.5g of 2-hydroxybenzoamide were added to the flask, 25mL of DMF and 300. Mu.L of pyridine were added, and 2.4mL of thionyl chloride was added at room temperature with stirring, heated under reflux for 8 hours, and the reaction was monitored by thin layer chromatography. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with isopropanol to obtain the oxazine intermediate.
(2) Synthesis of Compound 6
1.0g of oxazine intermediate and 0.2g of hydrazinoacetic acid were added to the flask, 10mL of isopropanol was added, and the reaction was heated under reflux for 10 hours, and monitored by thin layer chromatography. After the reaction, naturally cooling the reaction solution to room temperature, and recrystallizing with acetone to obtain a compound 6 (R) 1 ＝CF 3 ,R 2 ＝H,R 3 ＝CH 2 COOH)。
Structure corroboration data for compound 6: a white solid was used as a solid, 1 H NMR(500MHz,DMSO-d6)δ13.24(s,1H),11.34(s,1H),10.62(s,1H),8.18(d,J＝8.0Hz,1H),7.47(d,J＝7.2Hz,1H),7.44(d,J＝7.3Hz,1H),7.37–7.28(m,2H),7.08(d,J＝8.2Hz,1H),7.01(t,J＝7.5Hz,1H),5.13(s,2H)。
example 7
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 7:
synthesis of Compound 7
(1) Synthesis of oxazine intermediates
1.9g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.4g of 2-hydroxybenzoamide were added to the flask, 25mL of N-methylpyrrolidone and 150. Mu.L of pyridine were added, and 1.9mL of thionyl chloride was added at room temperature with stirring, heated to reflux for 6 hours, and the reaction was monitored by thin layer chromatography. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with pyridine to obtain the oxazine intermediate.
(2) Synthesis of Compound 7
1.0g of oxazine intermediate and 0.15g of ethyl hydrazinoacetate were added to the flask, 10mL of pyridine was added, and the reaction was heated under reflux for 8 hours, and monitored by thin layer chromatography. After the reaction, the reaction mixture was cooled to room temperature, and recrystallized from ethyl acetate to give Compound 7 (R) 1 ＝CF 3 ,R 2 ＝H,R 3 ＝
CH 2 COOCH 2 CH 3 )。
Structure corroboration data for compound 7: white solid, 1H NMR (400 mhz, dmso-d 6) δ11.29 (s, 1H), 10.61 (s, 1H), 8.17 (d, j=8.2 hz, 1H), 7.51-7.43 (m, 2H), 7.37-7.31 (m, 2H), 7.07 (d, j=8.2 hz, 1H), 7.01 (t, j=7.5 hz, 1H), 5.21 (s, 2H), 4.10 (q, j=7.1 hz, 2H), 1.13 (t, j=7.1 hz, 3H).
Example 8
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 8:
synthesis of Compound 8
(1) Synthesis of oxazine intermediates
2.0g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.9g of 2-hydroxybenzoamide were added to the flask, 25mL of DMF and 200. Mu.L of pyridine were added, and 2.3mL of thionyl chloride was added at room temperature with stirring, heated under reflux for 5 hours, and the reaction was monitored by thin layer chromatography. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with isopropanol to obtain the oxazine intermediate.
(2) Synthesis of Compound 8
1.0g of oxazine intermediate and 0.3g of 2-hydrazinopyridine were added to the flask, 10mL of isopropyl alcohol was added, and the reaction was heated under reflux for 10 hours, and monitored by thin layer chromatography. After the reaction, the reaction mixture was cooled to room temperature and recrystallized from methanol to give Compound 8 (R) 1 ＝CF 3 ,R 2 ＝H,R 3 =pyridinyl).
Structure corroboration data for compound 8: a white solid was used as a solid, 1 H NMR(500MHz,Chloroform-d)δ10.79(s,1H),10.08(s,1H),8.62(dd,J＝4.9,1.8Hz,1H),8.25(d,J＝8.1Hz,1H),8.03(td,J＝7.8,1.8Hz,1H),7.70(d,J＝8.0Hz,1H),7.55(dd,J＝7.5,4.8Hz,1H),7.38(td,J＝7.8,1.5Hz,1H),7.34(d,J＝1.6Hz,1H),7.29–7.25(m,1H),7.14(d,J＝8.1Hz,1H),6.98(dd,J＝8.0,1.6Hz,1H),6.75(t,J＝7.5Hz,1H)。
example 9
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 9:
synthesis of Compound 9
(1) Synthesis of oxazine intermediates
2.1g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.7g of 2-hydroxybenzoamide were added to the flask, 20mL of xylene and 180. Mu.L of pyridine were added, and 2.5mL of thionyl chloride was added at room temperature with stirring, heated under reflux for 7 hours, and the reaction was monitored by thin layer chromatography. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with tetrahydrofuran to obtain the oxazine intermediate.
(2) Synthesis of Compound 9
1.0g of oxazine intermediate and 0.4g of 2-hydrazinopyrazine were added to the flask, 10mL of tetrahydrofuran was added, and the reaction was heated under reflux for 6 hours, and monitored by thin layer chromatography. After the reaction, the reaction mixture was cooled to room temperature and recrystallized from tetrahydrofuran to give Compound 9 (R) 1 ＝CF 3 ,R 2 ＝H,R 3 =pyrazinyl).
Structure corroboration data for compound 9: a white solid was used as a solid, 1 H NMR(500MHz,Chloroform-d)δ10.05(s,2H),9.03(s,1H),8.82(d,J＝2.3Hz,1H),8.59(s,1H),8.25(d,J＝8.1Hz,1H),7.40(t,J＝8.0Hz,1H),7.35(s,1H),7.28(d,J＝8.2Hz,1H),7.13(d,J＝8.3Hz,1H),6.99(d,J＝8.1Hz,1H),6.78(t,J＝7.6Hz,1H)。
example 10
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown as a compound 10:
synthesis of Compound 10
(1) Synthesis of oxazine intermediates
2.1g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.7g of 2-hydroxybenzoamide were added to the flask, 25mL of DMSO and 240. Mu.L of pyridine were added, 3mL of thionyl chloride was added with stirring at room temperature, and the reaction was monitored by thin layer chromatography under reflux for 6 hours. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with isopropanol to obtain the oxazine intermediate.
(2) Synthesis of Compound 10
1.0g of oxazine intermediate and 0.5g of 2-hydrazinonaphthalene were added to the flask, 10mL of isopropyl alcohol was added, and the reaction was heated under reflux for 11 hours, and monitored by thin layer chromatography. Naturally cooling the reaction solution to room temperature after the reaction is finished, and recrystallizing with propanol to obtain a target product (R) 1 ＝CF 3 ,R 2 ＝H,R 3 =naphthyl).
Structure corroboration data for compound 10: a white solid was used as a solid, 1 H NMR(500MHz,Chloroform-d)δ11.40(s,1H),9.95(s,1H),8.25(d,J＝8.2Hz,1H),8.04(d,J＝8.4Hz,2H),7.99(d,J＝8.0Hz,1H),7.92(d,J＝7.9Hz,1H),7.70–7.60(m,2H),7.52(dd,J＝8.6,2.1Hz,1H),7.36–7.24(m,3H),7.14(d,J＝8.3Hz,1H),6.95(d,J＝8.1Hz,1H),6.56(t,J＝7.7Hz,1H)。
example 11
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 11:
synthesis of Compound 11
(1) Synthesis of oxazine intermediates
2.1g of 2-hydroxy-4- (trifluoromethyl) benzoic acid and 1.8g of 2-hydroxybenzoamide were added to the flask, 20mL of xylene and 170. Mu.L of pyridine were added, and 2.3mL of thionyl chloride was added at room temperature with stirring, heated to reflux for 9 hours, and the reaction was monitored by thin layer chromatography. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with dioxane to obtain the oxazine intermediate.
(2) Synthesis of Compound 11
1.0g of the oxazine intermediate and 0.8g of 2-hydrazinobenzothiazole were charged into a flask, 10mL of dioxane was added, and the reaction was heated under reflux for 8 hours, and monitored by thin layer chromatography. After the reaction, naturally cooling the reaction solution to room temperature, and recrystallizing with dioxane to obtain the target product (R) 1 ＝CF 3 ,R 2 ＝H,R 3 Benzothiazolyl).
Compound 11 constructs confirmation data: a white solid was used as a solid, 1 H NMR(500MHz,Chloroform-d)δ10.24(s,1H),9.86(s,1H),8.23(d,J＝8.1Hz,1H),8.00(d,J＝8.1Hz,1H),7.93(d,J＝8.0Hz,1H),7.70(d,J＝8.1Hz,1H),7.58(t,J＝7.7Hz,1H),7.52(t,J＝7.6Hz,1H),7.46(t,J＝7.4Hz,1H),7.35(s,1H),7.27(d,J＝8.0Hz,1H),7.17(d,J＝8.3Hz,1H),6.91(t,J＝7.6Hz,1H)。
example 12
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 12:
synthesis of Compound 12
(1) Synthesis of oxazine intermediates
1.7g of 2-hydroxy-4-benzoic acid and 1.8g of 2-hydroxybenzoamide were added to the flask, 20mL of xylene and 170. Mu.L of pyridine were added, 2.3mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 9 hours. And after the reaction is finished, naturally cooling the reaction solution to room temperature, precipitating solids, filtering, taking a filter cake, and recrystallizing with methanol to obtain the oxazine intermediate.
(2) Synthesis of Compound 12
1.0g of oxazine intermediate and 0.5g of trifluoroethylhydrazine were added to the flask, 10mL of methanol was added, and the reaction was heated under reflux for 8 hours, and the reaction was monitored by thin layer chromatography. After the reaction, the reaction mixture was cooled to room temperature and recrystallized from methanol to give Compound 12 (R) 1 ＝H,R 2 ＝H,R 3 ＝CH 2 CF 3 )。
Structure corroboration data for compound 12: a white solid was used as a solid, 1 H NMR(400MHz,DMSO-d6)δ10.68(s,1H),10.66(s,1H),7.98(d,J＝7.7Hz,1H),7.54–7.44(m,2H),7.37(t,J＝7.7Hz,0H),7.09(d,J＝8.2Hz,1H),7.05–6.94(m,3H),5.26(q,J＝8.8Hz,2H)。
example 13
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 13:
synthesis of Compound 13
(1) Synthesis of oxazine intermediates
1.8g of 2-hydroxy-4-benzoic acid and 1.8g of 2-hydroxybenzoamide were added to the flask, 20mL of DMSO and 170. Mu.L of pyridine were added, and 2.3mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 9 hours. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with ethyl acetate to obtain the oxazine intermediate.
(2) Synthesis of Compound 13
1.0g of oxazine intermediate and 1.0g of 2-hydrazinopyridine were added to the flask, 10mL of ethyl acetate was added, and the reaction was heated under reflux for 8 hours, and monitored by thin layer chromatography. After the reaction, the reaction mixture was cooled to room temperature, and recrystallized from ethyl acetate to give Compound 13 (R) 1 ＝H,R 2 ＝H,R 3 =pyridinyl).
Structure corroboration data for compound 13: a white solid was used as a solid, 1 H NMR(400MHz,DMSO-d6)δ10.83(s,1H),9.88(s,1H),8.36(d,J＝5.4Hz,1H),8.13–8.01(m,2H),7.79(d,J＝8.1Hz,1H),7.57(dd,J＝7.7,1.7Hz,1H),7.48(dd,J＝7.5,4.8Hz,1H),7.42–7.32(m,2H),7.07–6.94(m,3H),6.82(d,J＝8.2Hz,1H)。
example 14
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 14:
synthesis of Compound 14
(1) Synthesis of oxazine intermediates
2.1g of 2-hydroxy-4- (methoxy) benzoic acid and 1.8g of 2-hydroxybenzoamide were added to the flask, 20mL of DMF and 170. Mu.L of pyridine were added, and 2.3mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 9 h. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with dioxane to obtain the oxazine intermediate.
(2) Synthesis of Compound 14
1.0g of oxazine intermediate and 0.6g of trifluoroethylhydrazine were added to the flask, 10mL of dioxane was added, and the reaction was heated under reflux for 8 hours, and monitored by thin layer chromatography. After the reaction, naturally cooling the reaction solution to room temperature, and recrystallizing with dioxane to obtain the compound 14 (R) 1 ＝OCH 3 ,R 2 ＝H,R 3 ＝CH 2 CF 3 )。
Structure corroboration data for compound 14: white solid, 1H NMR (400 mhz, dmso-d 6) δ10.80 (s, 1H), 10.64 (s, 1H), 7.88 (d, j=8.6 hz, 1H), 7.52-7.43 (m, 2H), 7.09 (d, j=8.1 hz, 1H), 7.02 (t, j=8.0 hz, 1H), 6.65-6.51 (m, 2H), 5.23 (q, j=8.8 hz, 2H), 3.80 (s, 3H).
Example 15
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 15:
synthesis of Compound 15
(1) Synthesis of oxazine intermediates
2.1g of 2-hydroxy-4- (methoxy) benzoic acid and 1.9g of 2-hydroxybenzoamide were added to the flask, 20mL of xylene and 170. Mu.L of pyridine were added, and 2.3mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 9 hours. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with tetrahydrofuran to obtain the oxazine intermediate.
(2) Synthesis of Compound 15
1.0g of oxazine intermediate and 0.7g of isopropyl hydrazine were added to the flask, 10mL of tetrahydrofuran was added, and the reaction was heated under reflux for 8 hours, and monitored by thin layer chromatography. After the reaction, the reaction mixture was cooled to room temperature and recrystallized from tetrahydrofuran to give Compound 15 (R) 1 ＝OCH 3 ,R 2 ＝H,R 3 ＝CH(CH 3 ) 2 )。
Structure corroboration data for compound 15: a white solid was used as a solid, 1 H NMR(500MHz,Chloroform-d)δ10.77(s,1H),10.02(s,1H),7.97(d,J＝8.5Hz,1H),7.50(d,J＝8.1Hz,1H),7.42(t,J＝7.7Hz,1H),7.16(d,J＝8.4Hz,1H),7.01(t,J＝7.8Hz,1H),6.62–6.54(m,2H),4.99(p,J＝6.6Hz,1H),3.84(s,3H),1.65(s,3H),1.63(s,3H)。
example 16
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 16:
synthesis of Compound 16
(1) Synthesis of oxazine intermediates
2.1g of 2-hydroxy-4- (methoxy) benzoic acid and 1.8g of 2-hydroxybenzoamide were added to the flask, 20mL of xylene and 170. Mu.L of pyridine were added, and 2.3mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 9 hours. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with isopropanol to obtain the oxazine intermediate.
(2) Synthesis of Compound 16
1.0g of oxazine intermediate and 0.6g of t-butylhydrazine were added to the flask, 10mL of isopropanol was added, and the reaction was heated under reflux for 8 hours, and monitored by thin layer chromatography. After the reaction, the reaction mixture was cooled to room temperature and recrystallized from isopropanol to give Compound 16 (R) 1 ＝OCH 3 ,R 2 ＝H,R 3 ＝C(CH 3 ) 3 )。
Structure corroboration data for compound 16: a white solid was used as a solid, 1 H NMR(500MHz,Chloroform-d)δ7.87(d,J＝8.7Hz,1H),7.31–7.19(m,2H),6.93(t,J＝7.6Hz,1H),6.81(d,J＝8.2Hz,1H),6.54(s,1H),6.48(d,J＝8.7Hz,1H),3.82(s,3H),1.56(s,9H).13C NMR(126MHz,Chloroform-d)δ161.96,158.41,157.99,154.84,150.46,131.87,130.43,127.90,119.78,117.53,117.36,107.44,106.76,101.29,61.90,55.34,30.27。
example 17
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 17:
synthesis of Compound 17
(1) Synthesis of oxazine intermediates
2.1g of 2-hydroxy-4- (methoxy) benzoic acid and 2.0g of 2-hydroxybenzoamide were added to the flask, 20mL of toluene and 170. Mu.L of pyridine were added, and 2.3mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 9 hours. And after the reaction is finished, naturally cooling the reaction solution to room temperature, precipitating solids, filtering, taking a filter cake, and recrystallizing with acetonitrile to obtain the oxazine intermediate.
(2) Synthesis of Compound 17
1.0g of oxazine intermediate and 0.7g of 1-hydrazinocyclopentane were added to the flask, 10mL of acetonitrile was added, and the reaction was heated under reflux for 8 hours, and the reaction was monitored by thin layer chromatography. After the reaction, naturally cooling the reaction solution to room temperature, and recrystallizing with acetonitrile to obtain a compound 17 (R) 1 ＝OCH 3 ,R 2 ＝H,R 3 ＝CH(CH 2 ) 4 )。
Structure corroboration data for compound 17: a white solid was used as a solid, 1 H NMR(500MHz,Chloroform-d)δ10.69(s,2H),7.97(d,J＝8.3Hz,1H),7.56(d,J＝7.7Hz,1H),7.42(t,J＝8.0Hz,1H),7.16(d,J＝8.4Hz,1H),7.01(t,J＝7.8Hz,1H),6.62–6.54(m,2H),5.10(p,J＝6.9Hz,1H),3.84(s,3H),2.27–2.19(m,4H),2.11–2.02(m,2H),1.84–1.73(m,2H。
example 18
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 18:
synthesis of Compound 18
(1) Synthesis of oxazine intermediates
2.1g of 2-hydroxy-4- (methoxy) benzoic acid and 1.8g of 2-hydroxybenzoamide were added to the flask, 20mL of DMSO and 170. Mu.L of pyridine were added, and 2.3mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 9 hours. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with ethyl acetate to obtain the oxazine intermediate.
(2) Synthesis of Compound 18
1.0g of oxazine intermediate and 0.8g of trifluoroethylhydrazine were added to the flask, 10mL of dioxane was added, and the reaction was heated under reflux for 8 hours, and monitored by thin layer chromatography. After the reaction, naturally cooling the reaction solution to room temperature, and recrystallizing with dioxane to obtain a compound 18 (R) 1 ＝Cl,R 2 ＝H,R 3 ＝CHCF 3 )。
Structure corroboration data for compound 18: white solid, 1H NMR (400 MHz, DMSO-d 6) δ10.95 (s, 1H), 10.67 (s, 1H), 7.97 (d, J=8.4 Hz, 1H), 7.53-7.44 (m, 2H), 7.14-
6.99(m,4H),5.27(q,J＝8.8Hz,2H)。
Example 19
A3, 5-disubstituted phenyl-1, 2, 4-triazole derivative has a structural formula shown in a compound 19:
synthesis of Compound 19
(1) Synthesis of oxazine intermediates
2.1g of 2-hydroxy-4-chlorobenzoic acid and 1.9g of 2-hydroxybenzoamide were put into a flask, 20mL of DMF and 170. Mu.L of pyridine were added, 2.3mL of thionyl chloride was added at room temperature with stirring, and the reaction was monitored by thin layer chromatography under reflux for 9 hours. And naturally cooling the reaction liquid to room temperature after the reaction is finished, precipitating solids, filtering, taking a filter cake, and recrystallizing with ethanol to obtain the oxazine intermediate.
(2) Synthesis of Compound 19
1.0g of oxazine intermediate and 0.8g of isopropylHydrazine was added to the flask, 10mL of ethanol was added, the reaction was heated at reflux for 8h, and the reaction was monitored by thin layer chromatography. After the reaction, naturally cooling the reaction solution to room temperature, and recrystallizing with ethanol to obtain compound 19 (R) 1 ＝Cl,R 2 ＝H,R 3 ＝CH(CH 3 ) 2 )。
Structure corroboration data for compound 19: white solid, 1H NMR (400 MHz, DMSO-d 6) δ11.41 (s, 1H), 10.37 (s, 1H), 7.99 (d, J=8.4 Hz, 1H), 7.49-7.38 (m, 2H), 7.10-
6.98(m,4H),4.53–4.35(m,1H),1.45(s,3H),1.43(s,3H)。
To further demonstrate the effect of the present invention, the following test methods were provided:
1. the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivatives described in examples 1 to 19 act on LPS-induced RAW cell inflammation models.
1.1 experimental materials and reagents: RAW264.7 cells (Shanghai national academy of sciences), lipopolysaccharide (sigma), cck8 (Biyun), 15ml centrifuge tube, 96-well plate, DMEM medium, fetal bovine serum (Gibco, usa), trypsin, 1% diabody, DMSO, 0.22 μm microporous filter, drug addition tank, PBS buffer, celecoxib.
1.2 laboratory apparatus: centrifuge (Thermo Fisher, model microST 40), microplate reader (Thermo Fisher), microscope (Nikon eclipse TS 100).
1.3 experimental method:
1.3.1 preparing 100ml of DMEM cell culture medium containing 10% of fetal bovine serum and 1% of diabodies (penicillin and streptomycin);
1.3.2 preparation of experimental liquid medicine: dissolving a certain amount of compound sample with a certain amount of solvent to prepare mother solution (if the dissolution effect is poor, a small amount of DMSO can be added for dissolution assisting), and placing the mother solution in a 4-degree refrigerator for standby, and diluting the medicine to a desired concentration before use;
1.3.3 Preparation of 5mg/ml MTT: 100mgMTT is dissolved in 20ml PBS, and after being fully and evenly mixed, the solution is filtered by a microporous filter membrane with the thickness of 0.22 mu m to remove bacteria in the solution, and the solution is packaged and can be stored at the temperature of minus 20 ℃ for a long time in a dark place.
Culture of 1.4RAW264.7 macrophages
1.4.1 passage of cells and seed plates
Taking well-grown RAW264.7 cells (when the cells are added to more than 80% of the area of a culture flask), discarding the original culture medium, adding 3ml of PBS solution to wash the cells twice, adding 4ml of complete culture medium to blow the cells, and centrifuging at 1000rpm for 3min. The supernatant was discarded and 3ml of complete medium was added for resuspension. The concentration was adjusted after cell counting for passaging or plating, and the number of cell plating was about 1×10 4 Number of plates per well (varying depending on cell number).
1.4.2MTT method for detecting influence of each compound on RAW264.7 cell viability
Mu.l of RAW264.7 cell suspension was inoculated into each well of a 96-well plate, and the wells were placed at 37℃in 5% CO 2 After incubation for 24h, administration was performed. The control group and the experimental group are 3 compound holes, 5 sample solutions with different concentrations are added into the experimental group (DMEM culture medium is added into the control group), 10 μl of MTT solution is added into each hole after continuous culture is carried out for 20 hours, the supernatant is discarded after culture is carried out for 4 hours, 100 μl of DMSO is added for light-shielding oscillation, after crystallization is fully dissolved, OD value of each hole is measured at 570nm on an enzyme labeling instrument, the survival rate of cells is calculated, and the result is expressed as (mean+/-SD), wherein the cell survival rate (%) = absorbance of the experimental group/absorbance of the blank group x100%.
1.4.3Griess assay for detecting the amount of NO released by each Compound on LPS-induced RAW264.7 cells
Mu.l of RAW264.7 cell suspension was inoculated into each well of a 96-well plate, and the wells were placed at 37℃in 5% CO 2 The culture in the incubator is used for 24 hours and then the medicine is taken. 100 mu L of LPS solution containing 1000ng/mL is added into the administration group and the LPS group, the DMEM culture solution with the same volume as that of the control group is repeatedly used for 3 holes, the culture is continued for 18 hours, a 96-hole plate is taken out, a shake plate is adopted, 50 mu L of cell supernatant is transferred into another 96-hole cell culture plate, 100 mu L of Griess A and B mixed reagent (1:1) is added, the reaction is carried out in dark for 10 minutes, and OD value is measured at 540 nm. The concentration of NO was calculated from the standard curve of NO production, and the results are expressed as (mean±sd).
The experiments set 3 wells each for control group (DMEM medium added without LPS stimulation), LPS model group (DMEM medium added with LPS stimulation), positive drug group (0.625, 1.25, 2.5 μm celecoxib with LPS stimulation), experimental group (0.625, 1.25, 2.5 μm compound with LPS stimulation).
2.3, 5-disubstituted phenyl-1, 2, 4-triazole derivative (Compound 5) as described in example 5 was used to induce ulcerative colitis in mice.
2.1 Experimental materials
Experimental animals: SPF C57BL/6 male mice were purchased from Jiangsu Jiyaokang biotechnology Co., ltd, and animal pass certificate: no.44824700007301, 6-8 weeks old, 18-22 g.
The main instrument is as follows: stopwatch (Shangzhixing diamond stopwatch Co., ltd.), ACS-3H electronic balance (Kiril electronic weighing apparatus Co., ltd., zhongshan), 1mL syringe (Jiangsu Zhengkang medical instruments Co., ltd.), surgical instruments (Beijing Sichuan technologies Co., ltd.).
The main reagent comprises: compound 5, salazosulfapyridine colonolytic capsule (guangdong strong base pharmaceutical company limited).
2.2 test method: DSS mouse model preparation: 30 SPF-class C57BL/6 male mice are selected, and are randomly divided into a blank control group, a model group, a compound 5 intervention group and a positive medicine group, wherein each group comprises 7 mice, 6-8 weeks old, 18-22 g, the temperature of 22+/-2 ℃ and the humidity of 40-60%, the bright and dark alternation is realized in 12 hours, animals eat freely, drink water and feed adaptively for 7 days. The treatment of each group of mice is shown in table 1:
TABLE 1 grouping and treatment of animals
2.2.1 index determination
2.2.1.1 overall condition record of mice: from day 0, mice were scored daily for weight change, food intake, and macroscale.
2.2.1.2 Disease Activity Index (DAI), DAI = body weight change + hematochezia degree + stool shape.
TABLE 2 mouse DAI scoring rules
2.2.2 colon length: the mice of each group were dissected for 7 days, the complete colon parts of the mice were taken separately, the length from the cecum end to the colon end was measured with a ruler after the water was blotted with filter paper, and the readings were recorded and photographed.
2.2.3 spleen organ coefficients: spleen morphology was also observed, called spleen weight and recorded.
2.3 the experimental results are shown in FIGS. 1-7.
FIG. 1 shows the effect of 3, 5-disubstituted phenyl-1, 2, 4-triazole derivatives described in examples 1 to 19 on the activity of RAW cells. As shown in the figure, the compound is in the range of 0.625 μm-2.51 μm, and the compound 3,4,6,7,9,13 has a certain influence on RAW activity. Compounds 2, 5, 8, 10, 11, 12, 14, 15, 16, 17, 18, 19 had a relatively small effect on RAW activity. Compounds 2, 5, 8, 10, 11, 12, 14, 15, 16, 17, 18, 19 were taken for NO detection.
FIG. 2 effect of compounds on LPS-stimulated NO production by RAW cells. As shown, 0.625 μm compound 2, 5, 8, 10, 11, 12, 14, 15, 17 can significantly reduce NO production; 1.25 μm compounds 2, 5, 8, 10, 11, 12, 14, 15, 16, 17, 19 can significantly reduce NO production; compounds 2, 5, 8, 10, 11, 12, 14, 15, 16, 17, 18, 19 of 2.5 μm significantly reduce NO production. Compound 5 has less effect on RAW activity and can significantly reduce NO production. Compound 5 was selected for in vivo model validation.
Wherein fig. 3 is a graph of Compound 5 (Compound 5) versus DSS model mice weight change, as can be seen from fig. 3, mice in the model group (DSS) showed a continuous decrease in weight change, starting at 95.64% on day three and decreasing to 80.92% on day seven; the weight loss of the dosing group was not significant and was 94.36% of the seventh day. The above data indicate that compound 5 can improve the mice body weight loss trend caused by DSS.
FIG. 4 is a graph of Compound 5 (Compound 5) versus daily food intake for mice in the DSS model, as can be seen from FIG. 4, the food intake for the DSS model group was significantly reduced from that for the normal group, and only 7.9g per 7 mice were ingested on the seventh day, starting from the first day of modeling; the first day diet of the group to which the compound 5 was administered was not significantly changed from that of the normal group, and the intake of 14g per 7 mice on the seventh day was reduced from that of the normal group by 23g, but significantly improved from that of the model group.
Fig. 5 is a graph of Compound 5 (Compound 5) versus DSS model mice colon length, and fig. 6 is a graph of Compound 5 (Compound 5) versus DSS model mice colon length statistics, as can be seen from fig. 5 and 6, on day 7, experimental results show that the colon length of model group (DSS) mice is reduced and has significant bloody stool, the length is only 4.8cm, the length is significantly reduced compared with the normal group of 7.0cm, the length of Compound 5 administration group is 6.2cm, the colon length of positive control group is 6.1cm, the colon length of administration group and the symptom improvement such as bleeding of colon content are significant, which indicates that Compound 5 can prevent intestinal shortening and improve hematochezia of UC mice.
FIG. 7 is a graph of Compound 5 (Compound 5) versus DSS model mouse DAI score. As can be seen from FIG. 5, on day 7, the results of the experiment revealed that the faeces of the mice in the model group (DSS) were relatively weak and had significant bloody stool, and that the mice in the dosing group had different levels of faecal occult blood. But the blood level is reduced and the weight is recovered. The data show that the administration of the compound 5 can relieve the symptoms of diseases such as hematochezia, fecal property softening, weight reduction and the like of mice caused by DSS.
In conclusion, the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative disclosed by the invention can effectively prevent and treat ulcerative colitis. The ulcerative colitis mice model was successfully established from the above experiments, and compound 5 had a certain recovery effect on the intestinal tract of the ulcerative colitis mice relative to the model group after 7 days of administration. The specific expression is that the weight drop is slowed down, the colon length is increased, and the disease score DAI is obviously recovered.
3. Acute toxicity test of 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative (Compound 5) described in example 5.
3.1 limit test: the administration mode of each sample is intraperitoneal injection, the administration is carried out for 1 time, and the maximum administration concentration is 1000mg/kg. The test subjects were administered to 1 animal. If the animal dies, a main trial is performed; if the animal survives, the test subjects are given to another 4 animals in sequence, with a total of 5 animals. If 1 animal dies at the end of the trial and the other animals survive, dosing of the other animals should be stopped, all animals should be observed, and if death also occurs during similar observations. Later dead animals should be counted as other dead animals and the results evaluated as follows: when 3 or more animals die, the LD50 is less than 1000mg/kg; when 3 or more animals survive, LD50 is greater than 1000mg/kg.
TABLE 3 Compound 5 Limit test results
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Note that: o stands for survival and X stands for death.
3.2 limit test results of Compound 5: the limit test results of Compound 5 are shown in Table 3.3 or more animals survived, and their LD50 was judged to be >1000mg/kg.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (6)

1. The application of 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative or pharmaceutically acceptable salt thereof in preparing medicaments for preventing and/or treating ulcerative colitis is characterized in that the structural formula is shown as formula I:
wherein R is 1 Is CF (CF) 3 ；
R 2 Is H;
R 3 is CH 2 CF 3 。
2. The use according to claim 1, characterized in that the process for the preparation of 3, 5-disubstituted phenyl-1, 2, 4-triazole derivatives comprises the steps of:
adding an o-hydroxybenzoic acid derivative and an o-hydroxybenzoamide derivative into a solvent A, adding pyridine, adding thionyl chloride while stirring, heating and refluxing to remove the solvent and residual thionyl chloride, and recrystallizing to obtain an oxazine intermediate;
adding an oxazine intermediate and a hydrazine derivative into a solvent B, removing part of the solvent after heating and refluxing, crystallizing, and recrystallizing to obtain a 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative;
the o-hydroxybenzoic acid derivative is 2-hydroxy-4- (trifluoromethyl) benzoic acid, and the o-hydroxybenzoamide derivative is 2-hydroxybenzoamide;
the hydrazine derivative is trifluoroethyl hydrazine.
3. The use according to claim 2, wherein the solvent a is at least one of toluene, xylene, diphenyl ether, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide; the solvent B is at least one of ethanol, propanol, isopropanol, tertiary butanol, tetrahydrofuran, dioxane, butanone, acetonitrile, ethyl acetate, chloroform, 1-dichloroethane and pyridine.
4. The use according to claim 2, wherein the material ratio of the o-hydroxybenzoic acid derivative to thionyl chloride is 1g: (1-5) ml; the ratio of the amounts of the substances of the o-hydroxybenzoic acid derivative to the o-hydroxybenzoamide derivative is 1: (0.9-3); the ratio of the amounts of oxazine intermediate to hydrazine derivative material is 1: (0.9-3).
5. A medicament for preventing and/or treating ulcerative colitis, characterized in that a 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative or a pharmaceutically acceptable salt thereof according to claim 1 is used as a main active material.
6. The agent for preventing and/or treating ulcerative colitis according to claim 5, wherein the effective dose of the 3, 5-disubstituted phenyl-1, 2, 4-triazole derivative or a pharmaceutically acceptable salt thereof is 1-30mg/kg.
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