TWI754474B - Selective cyclooxygenase-2 inhibitory 1,2,4-triazole derivatives substance and use thereof - Google Patents

Abstract

The patent discloses that 1,2,4-triazole derivatives are novel selective cyclooxygenase-2 inhibitors, which exhibit an excellent inhibitory effect on cyclooxygenase-2 based on in vitro study. The 1,2,4-triazole derivatives exhibited better in vivo anti-inflammation and gastric protection results compared to the marketed anti-inflammatory drug, Indomethacin.

Description

1,2,4-Triazole derivatives with selective type II cyclooxygenase inhibitory activity and their uses

The present invention relates to a triazole derivative; in detail, the present invention relates to the use of a 1,2,4-triazole ring derivative with a novel selective second type cyclooxygenase inhibitory activity .

Most clinically used non-steroidal anti-inflammatory drugs (NSAIDs) are known to inhibit two isoforms of cyclooxygenase: COX-1 (constitutive) and COX-2 (inducible). The use of non-selective NSAIDs acts on both cyclooxygenase isoforms and increases the risk of gastrointestinal (GI) complications (C. Sostres, Best Pract. Res. Clin. Gastroenterol, 2010, 24, 121-132) . Selective type II cyclooxygenase inhibitors show potent anti-inflammatory activity without causing significant gastrointestinal damage (P.McGettigan, J.Am.Med.Assoc., 2006, 296, 1633-1644) . Besides, type II cyclooxygenase inhibitors have also been reported to have anticancer activity (C.G.Crosby, Expert Opinion on Emerging Drugs, 2003, 8, 1-7). Therefore, the research on modification of selective type II cyclooxygenase inhibitors has attracted people's attention and has become a major challenge.

According to literature reports, 1,2,4-triazole ring derivatives have a wide range of biological activities (Q. Zhang, J Med Chem. 2006, 49, 4044-4047). 1,2,4-Triazole ring derivatives have significant anti-inflammatory effects on the inflammatory response induced by carrageenans in rats, Its anti-inflammatory effect is comparable to that of the marketed drug Indomethacin (A.M.Megee, European Journal of Medicinal Chemistry. 2009, 44, 117-123.).

Our laboratory synthesized 1,2,4-triazole ring derivatives as anti-inflammatory drugs (S.M., Li, Bioorganic Chemistry, 2020, 104, 104333). In this patent application, a novel selective type II cyclooxygenase inhibitor is disclosed, which has an inhibitory effect on type II cyclooxygenase in vitro and is more anti-inflammatory than the commercially available drug Indomthacin in mice. active and without the side effects of stomach damage.

The present invention discloses a novel selective type II cyclooxygenase inhibitor, which comprises administering a therapeutically effective amount of a compound of formula (I), its stereochemical isomer or a pharmaceutically acceptable salt thereof to a need of the individual:

wherein, R ¹ is independently an aryl group, a heteroaryl group, or a heterocyclic group. ^R2 is independently a methylene halogen which is fluorine, chlorine, bromine or iodine.

The following description is merely illustrative of specific embodiments of the present invention. Therefore, specific embodiments or modifications discussed herein should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various modifications or equivalents can be made without departing from the scope of the present invention.

In order to make the present invention clearer and easier to understand, certain terms must first be defined. Forehead Definitions other than those are listed in the full text of the embodiments. Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art of the present invention.

As used herein, the articles "a" and "an" refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element.

1. Materials and Methods

1.1 Materials

The purity of the tested drugs used in the present invention are all above 95%, synthesized according to literature methods (SM, Li, Bioorganic Chemistry, 2020, 104, 104333), and the reference drugs indomethacin and celecoxib (celecoxib) were purchased from Sigma Chemical Co. COX Inhibition Screening Assay Kit (catalog no. 560131) was purchased from Cayman Chemical. Male ICR mice were purchased from BioLASCO Taiwan Co., Ltd.

1.2 In vitro COX-1 and COX-2 inhibition assays

Test compounds were tested for their ability to inhibit COX-2 and COX-1 ( _IC50 values, μM) using an enzyme immunoassay (EIA) kit (code 560131 ) following the manufacturer's recommended procedures. The half-maximal inhibitor concentration _IC50 ( μM ) was calculated by the Excel forecast function, and the selectivity index (SI) value was calculated by dividing the _IC50 (COX-1) by the _IC50 (COX-2).

1.3 Carrageenan-induced mouse foot edema and inflammation test

Mice (about 29-39g) were randomly divided into 6 groups (n=6 mice/group) and treated with low dose (1.25mg/kg), medium dose (2.5mg/kg) and high dose (5mg/kg) kg) of the test compound added to 1% CMC as the test group. The positive control group and the vehicle control group were orally administered the same volume of 1% CMC and the same volume of 1% CMC containing indomethacin 10 mg/kg, respectively. dosing Thirty minutes later, acute foot edema was induced by injecting 50 μL of 1% carrageenan in 0.9% normal saline through the right sole. The volume of the soles of the feet was measured using a Plethysmometer before injection and at intervals of 1, 2, 3, 4 and 5 hours after injection.

1.4 Measurement of nitric oxide (NO) concentration in mouse serum

After the carrageenan-induced mouse foot edema and inflammation test, the blood of the mice was drawn, and the production of the inflammation-inducing substance NO was evaluated by the colorimetric measurement of the Griess reaction.

1.5 Determination of serum cytokines by ELISA

Serum TNF- α and IL-6 concentrations in mice were determined using commercially available ELISA kits (Biosource International Inc.) according to the manufacturer's instructions. Concentrations of TNF- α and IL-6 were expressed as pg/mL.

1.6 Tissue Sections

After the mice were bled, foot tissue sections were fixed in 1.85% formaldehyde and 1% acetic acid for 1 week at room temperature, dehydrated with ethanol and embedded in paraffin. Tissue sections were dewaxed with xylene and stained with hematoxylin and eosin for cell counts. All samples were observed and photographed with a BH-2 Olympus microscope.

1.7 Gastric ulcer protective activity

After the mice were sacrificed, the stomachs were removed, incised along the curve, washed with distilled water, and gently washed by immersion in saline, and histopathological examinations were performed to confirm the degree of inflammation in the gastric mucosa of the treated mice's stomachs.

2. The examples listed below are examples related to the selective inhibition of COX-2 by 1,2,4-triazole ring derivatives and the anti-inflammation in mice to explain the present invention in more detail; but the present invention Not limited to these examples.

3. Implementation Cases 1 to 11

In Examples 1 to 11 of the present invention (Table 1, columns 1-11), 1,2,4-triazole ring derivatives 1a - 1k were subjected to in vitro COX inhibition assay to explore COX-1/COX-2 selection The study of sex. The COX-2 inhibitory effect has a trend of substitution positions on the benzene ring as meta>para>ortho (Table 1, columns 1-6). Compounds 1b , 1e , 1g and 1h have meta substituents on the benzene ring on nitrogen No. 1, including fluorine, trifluoromethyl, chlorine and methyl, and have significant COX-2 inhibitory ability (0.00712, 1.58, 0.773 and 2.69 μM, Table 1, columns 2, 5, 7 and 8). The results show that meta-halogenated aryl compounds are more favorable for inhibiting COX-2 activity than meta-electron donating groups (Table 8, column 8). Fluorine atoms have more significant COX-2 inhibitory activity. Compounds 1b (0.00712 μM) and 1c (0.0179 μM) with meta- and para-fluorines were 0.05 to 7-fold more active against COX-2 than the marketed drug celecoxib. The tendency of substitution on the No. 1 nitrogen benzene ring to inhibit COX-2 is: 2,4-dichloro<bromo<cyanide<methyl<trifluoromethyl<chlorine<fluorine.

1,2,4-Triazole ring derivatives generally showed stronger inhibition of COX-2 than COX-1 (Table 1), showing a lower incidence of gastrointestinal toxicity (S. Kawai , Inflammation Research, 1998, 47, 102-106). According to the selectivity index results, 1,2,4-triazole ring derivatives 1c have excellent selectivity (COX-1/COX-2=1080, Table 1, column 3) and inhibitory activity against COX-2 . Therefore, 1,2,4-triazole ring derivatives 1c are safe and effective selective COX-2 inhibitors in vitro.

Based on the in vitro COX-2 screening results, Example 3 of this case was selected for in vivo anti-inflammatory activity test. Using carrageenan-induced mouse foot edema as an acute inflammation model, all groups reached a peak of foot edema 3 hours after carrageenan stimulation (Figure 1). The negative control group not injected with compound had an increase in foot volume by 0.18 ml by injection of carrageenan. In the positive control group, indomethacin was effective in reducing foot edema 3 hours after carrageenan stimulation. The 1,2,4-triazole ring derivative 1c showed a dose-dependent reduction in foot edema in mice (Figure 1). The 1,2,4-triazole ring derivative 1c at a concentration of 5 mg/kg exhibited more anti-inflammatory activity than the control indomethacin at 10 mg/kg (Figure 1).

In Example 3, after the 1,2,4-triazole ring derivative 1c was subjected to the mouse foot edema experiment, the mice were sacrificed, the blood of the mice was drawn and the supernatant was removed by centrifugation. Nitric oxide release was measured by Griess reagent. In Figure 2A, 5 hours after carrageenan injection, the serum nitric oxide concentration was significantly increased, representing an inflammatory phenomenon, whereas the 1,2,4-triazole ring derivative 1c could significantly reverse the nitric oxide concentration, and Dose-dependent. Treatment with the 1,2,4-triazole ring derivative 1c at a dose of 5 mg/kg significantly reduced plasma concentrations of nitric oxide to 86.4%, which was similar to the indomethacin group (10 mg/kg). , Figure 2A). Similarly, 5 hours after carrageenan injection, both TNF- α and IL-6 concentrations in aqueous blood were significantly increased (p<0.001), given the 1,2,4-triazole ring derivative 1c group, presenting the dose TNF- α and IL-6 concentrations were decreased in a dependent manner (Figures 2B and 2C). Both foot edema and inflammatory chemokine experiments in mice showed that half-dose of 5 mg/kg of 1,2,4-triazole ring derivatives 1c showed similar performance to normal dose of 10 mg/kg of indomethacin Anti-inflammatory results. Therefore, using a lower dose of 1,2,4-triazole ring derivative 1c has the same anti-inflammatory activity as the marketed drug, and because of the lower dose, the risk of side effects is lower.

The sacrificed mice were subjected to foot slices to confirm the inflammation status of the control group and the control group. There was no cellular infiltration in normal foot connective tissue (Fig. 3A). In contrast, in the carrageenan-induced group of mice, cellular infiltration was evident in the connective tissue of the foot, and the infiltration accumulated in collagen fibers and intercellular spaces (Fig. 3B). The marketed drug indomethacin at 10 mg/kg significantly prevented tissue infiltrates accumulation (Fig. 3C). Foot biopsies from 1,2,4-triazole derivatives 1c -treated mice showed a dose-dependent reduction in carrageenan-induced inflammation (Figure 3D-F). In the 5 mg/kg compound 1c group, inflammatory cells were significantly reduced, and almost no cellular infiltration was observed in the intercellular space, confined to the vascular area (Fig. 3F). In addition, many COX-2 immunoreactive cells were observed in the brown portion of the foot tissue section (Fig. 3B-F). However, administration of 2.5 and 5 mg/kg of the 1,2,4-triazole ring derivative 1c and 10 mg/kg of indomethacin both significantly reduced the increase in COX-2 immunoreactive cells in the foot (Fig. 3C and 3E-F).

To further demonstrate whether the triazole derivatives 1c have side effects of causing gastric ulcers, 1.25, 2.5 and 5 mg/kg of 1,2,4-triazole derivatives 1c and 10 mg/kg indole were orally administered For the group of mexin, the histopathological observation of gastric mucosa in mice was used to determine the ulcer-causing ability of the compound. Epithelial shedding and bacterial shedding were not observed in normal gastric mucosa (Fig. 4A). Several epithelial shedding and bacterial adhesion were found in the carrageenan-induced inflammation group (Fig. 4B). Sections from the indomethacin group, although similar to the carrageenan-induced group, had mild to moderate gastric mucosal erosions (Fig. 4C). The degree of inflammation in the mouse stomach, indicating that the triazole derivatives 1c group did not have ulcers, gastric mucosal maceration, necrosis or lymphocyte infiltration, indicating that the gastric mucosal side effects of the triazole derivatives 1c were much lower than The marketed drug indomethacin (Figure 4D-F).

The first (1) figure shows the time of carrageenan (Carr)-induced inflammation and the change of edema volume in mice.

The second graph shows the effects of compound (Comp.) 1c and indomethacin (Indo) on the production of (A) nitric oxide (Nitrite), (B) TNF-α and (C) IL-6 in mouse serum influence. *, ** and *** indicate p<0.05, p<0.01 and p<0.001, respectively, compared to the control group.

The third picture is the pathological examination of the tissue section of the foot edema induced by carrageenan (Carr) inflammation. (A) control group (saline only; Control), (B) induction group (Carr), (C) indomethacin group (Indo), (D) 1c group at 1.25 mg/kg (E) 2.5 mg Group 1c per kg (F) Group 1c at 5 mg per kg.

The picture (4) is the pathological examination of gastric mucosa tissue sections induced by carrageenan (Carr) inflammation (A) control group (saline only; Control), (B) induced group (Carr), (C) indomethacin Group (Indo), (D) 1.25 mg/kg 1c group (E) 2.5 mg/kg 1c group (F) 5 mg/kg 1c group.

Claims (4)

A kind of use such as formula (I) 1,2,4-triazole-3-carboxylate methyl ester heterocyclic compound or its pharmaceutically acceptable salt is used as selective second type cyclooxygenase inhibition Use of the agent:

Wherein, R ¹ is p-fluorophenyl; R ² is independently a methylene halogen, and the halogen is fluorine, chlorine, bromine or iodine. A kind of use containing the compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a drug with selective inhibition of type II cyclooxygenase. A use of the compound of formula (I) as claimed in claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical active ingredient that can effectively treat diseases mediated by type II cyclooxygenase. A kind of medical composition containing the compound of formula (I) as described in claim 1 or a pharmaceutically acceptable salt thereof for the manufacture and treatment of type 2 cyclooxygenase-mediated inflammation, arthritis or fever, etc. use.

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