阅读说明：本技术 (4-(1,2,4-恶二唑-5-基)苯基)甲酰胺衍生物及其在抗关节炎药物中的应用 ((4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivatives and application thereof in anti-arthritis drugs ) 是由 李增 李俊 吕熊文 刘彤彤 陈世明 黄成� 孟晓明 于 2021-07-19 设计创作，主要内容包括：本发明属于药物治疗学领域,具体涉及(4-(1,2,4-恶二唑-5-基)苯基)甲酰胺衍生物的医药学用途,特别是其在制备预防或治疗关节炎药物中的应用。该化合物的结构式如式A所示：研究表明,本发明所涉及的(4-(1,2,4-恶二唑-5-基)苯基)甲酰胺衍生物能够抑制NO,IL-1β和TNF-α的释放,减轻佐剂型关节炎大鼠模型的症状,具有发展成为抗关节炎药物的潜力。(The invention belongs to the field of pharmacotherapeutics, and particularly relates to a medical application of a (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative, in particular to an application of the derivative in preparing a medicament for preventing or treating arthritis. The structural formula of the compound is shown as a formula A: research shows that the (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative provided by the invention can inhibit the release of NO, IL-1 beta and TNF-alpha, relieve the symptoms of an adjuvant arthritis rat model, and has the potential of developing into an anti-arthritis drug.)

1. A (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative is characterized in that the structural formula is shown as a formula (A):

2. use of a (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative according to claim 1 in a medicament for the inhibition of nitric oxide, NO, production.

3. Use of a (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative according to claim 1 in a medicament for the inhibition of the production of TNF- α.

4. Use of a (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative as claimed in claim 1 in a medicament for the inhibition of interleukin IL-1 β production.

5. Use of the (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative according to claim 1 in the manufacture of a medicament for the prevention or treatment of arthritis.

6. The use of claim 5, wherein the arthritis is rheumatoid arthritis.

7. The use of claim 2 or 3 or 4 or 5, wherein the medicament is any one of an injection, a tablet, a pill, a capsule, a suspension or an emulsion.

Technical Field

The invention belongs to the field of pharmacotherapeutics, and particularly relates to a 4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative and application thereof in preparing a medicament for preventing or treating arthritis.

Background

Inflammation is a common pathological process in the body caused by various injurious factors, and is mediated by a variety of inflammatory mediators, mainly including Prostanoids (PGs) and Leukotrienes (LTs). The immunity is a specific physiological reaction of the body contacting antigenic foreign matters or foreign components, and the function of the immunity is to identify and eliminate the antigenic foreign matters so as to maintain the physiological balance of the body. Both immune and inflammatory responses are protective defense responses of the body, however, excessive immunity and inflammation are harmful to the human body. Immunity and inflammation are two different responses of the body to foreign bodies, but by their very nature, immunity and inflammation are two sides of a problem, overlapping each other, inseparable. The anti-inflammatory immune drugs which are most widely used in clinic are non-steroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs, but the drugs have serious adverse reactions while achieving good anti-inflammatory effects, so that people pay more attention, and how to reduce the adverse reactions or find alternative drugs is one of the most important research tasks in pharmacy.

Arthritis is one of many autoimmune diseases, and the presence of autoantibodies triggers immune cells to release inflammatory molecules, causing damage to joints and other organ systems. Anti-cytokine therapies such as tumor necrosis factor (TNF- α), interleukin (IL-1 β) are currently used in the treatment of autoimmune diseases. Therefore, in the process of designing and developing a new drug, a compound with anti-arthritis potential can be screened according to whether the drug activity can inhibit the release of inflammatory factors NO, IL-1 beta and TNF-alpha. The research of analyzing the treatment condition of the compound on an adjuvant-induced arthritis model and the like is combined, so that an experimental basis can be provided for the treatment development of arthritis in the field of medicine.

Disclosure of Invention

In order to solve the above technical problems, an object of the present invention is to provide a (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative, which adopts the following technical scheme: a (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative, which has the structural formula shown in formula A below and is named f 9:

the preparation route of the (4- (1,2, 4-oxadiazol-5-yl) phenyl) formamide derivative f9 is as follows:

the preparation routes are mainly intended to illustrate the invention without imposing any limitation thereon.

i. Performing nucleophilic substitution reaction on 2-chloro-4-cyanopyridine and various anilines according to a method reported by a literature, and purifying a crude product by column chromatography to obtain a compound a;

reaction of compound a with hydroxylamine hydrochloride in K₂CO₃Reacting under the action of the catalyst to generate a compound b;

iii, reacting the compound b with 4-nitrobenzoyl chloride to obtain a compound c;

the compound c is subjected to reduction reaction to obtain an intermediate d;

v, carrying out substitution reaction on the compound d and chloroacetyl chloride to obtain a compound e;

and vi, dissolving the intermediate e in absolute ethyl alcohol, reacting with para-fluoroaniline (75 ℃), and purifying by column chromatography to obtain the (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative f 9.

The second purpose of the invention is to provide the application of the (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative in preparing medicines for inhibiting the generation of nitric oxide NO and/or tumor necrosis factor TNF-alpha and/or interleukin IL-1 beta.

Further, the medicine is any one of injection, tablet, pill, capsule, suspending agent or emulsion.

The invention also aims to provide the application of the (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative in medicaments for treating arthritis diseases.

Further, the arthritis is rheumatoid arthritis.

Further, the medicine is any one of injection, tablet, pill, capsule, suspending agent or emulsion.

The invention has the advantages that:

1. by adding the (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative f9 into RAW264.7 cells induced by LPS, experimental determination results show that the (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative f9 provided by the invention can well inhibit secretion and release of NO, IL-1 beta and TNF-alpha.

2. Through in vivo experiments, (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivatives f9 can obviously treat rats with adjuvant-induced arthritis, and the (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivatives f9 are shown to have potential to be developed into drugs for treating arthritis diseases.

Drawings

FIG. 1 is a graph showing the effect of varying concentrations of (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 on NO release when RAW264.7 cells are stimulated by LPS;

FIG. 2 Effect of different concentrations of (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 on IL-1. beta. and TNF-. alpha.release when RAW264.7 cells were stimulated by LPS;

FIG. 3 is a graph showing a comparison of the right hind paw of different groups of rats in an adjuvant-induced arthritis model test;

FIG. 4 is the swelling change of the feet of different groups of rats in an adjuvant-induced arthritis model test, wherein the abscissa is time and the ordinate is the volume of the feet;

FIG. 5 is an index of arthritis for different groups in an adjuvant-induced arthritis model test, where the abscissa is time and the ordinate is the arthritis index;

FIG. 6 is a graph of the body weight change of rats in different groups in an adjuvant-induced arthritis model test, in which the abscissa is time;

FIG. 7 is a graph showing the change in IL-1 β and TNF- α levels in different groups of rats in an adjuvant-induced arthritis model test, in which FIG. 7A is IL-1 β, FIG. 7B is TNF- α, and the abscissa of the graph is time.

Detailed Description

Unless otherwise indicated, the terms used herein have the meanings that are conventionally understood by those skilled in the art.

The technical scheme of the invention is more specifically explained by combining the following embodiments:

example 1

Synthesis of (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f 9:

compound e (294mg, 0.6mM) was dissolved in 80mL of anhydrous ethanol, followed by addition of para-fluoroaniline (889mg, 4.8mM) and triethylamine (101mg, 1.2mM) and refluxing at 75 ℃ for 8 h. The progress of the reaction was checked by TLC. After the reaction, the reaction solvent was removed. The compound f9 is obtained by purification in column chromatography with ethyl acetate and petroleum ether (1:4), and the compound f9 is a white solid with the yield of 56%.

¹H NMR(500MHz,DMSO-d₆)δ10.48(s,1H),9.67(s,1H),8.37(d,J＝5.3Hz, 1H),8.14(d,J＝8.8Hz,2H),7.93(d,J＝8.8Hz,2H),7.86(d,J＝9.2Hz,2H),7.57 (s,1H),7.35(dd,J＝5.2,1.4Hz,1H),7.30(d,J＝8.6Hz,2H),6.97(t,J＝8.9Hz, 2H),6.63(dd,J＝9.0,4.5Hz,2H),6.03(t,J＝6.1Hz,1H),3.94(d,J＝5.8Hz,2H)；¹³C NMR(126MHz,DMSO)δ48.28,109.26,113.53,113.59,115.67,115.85,118.00, 119.56,119.71,119.87,121.73,122.07,129.54,135.11,141.02,142.23,143.91, 145.42,149.03,154.24,156.08,156.68,167.50,170.62,176.05。

HRMS(ESI)m/z[M+H]⁺:565.15666calcd for C₂₈H₂₀F₄N₆O₃:565.15985。

Example 2

Anti-inflammatory Activity test of (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9

1) Experimental materials: (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 (synthesized by design in the laboratory), NO detection kit (Biyuntian), RAW264.7 (college of medicine of Anhui medical university), ELASA kit (Gentamei, Wuhan), enzyme-linked immunosorbent assay (BIO-RAD corporation), inverted biomicroscope (Chongqing optical instruments factory); reagent: cell culture medium, DMEM (high Glucose) (GIBCO), fetal bovine serum (hangzhou siji qing ltd), DMSO (Sigma).

2) Cell culture:

RAW264.7 macrophage used in the experiment is an adherent growth cell strain, and DMEM culture solution containing 10% fetal calf serum is adopted for culture. The culture environment is 37 ℃ and 5% CO₂The culture medium is replaced once a day in the incubator, and the cells in the logarithmic growth phase are taken for experiment.

3) Inhibition of NO release experiments:

the anti-inflammatory capacity of the compounds was assessed by their ability to inhibit the release of NO in the LPS-induced RAW264.7 cell model.

RAW264.7 cells were cultured in DMEM high-glucose medium additionally supplemented with 10% fetal bovine serum, penicillin 100U/mL and streptomycin 100U/mL. RAW264.7 cells were plated at 6X 10 per well⁴After 24h of culture in 48-well plates, the old medium was discarded. Then pretreated with pre-prepared media containing (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 at various concentrations for 1 hour and incubated with LPS at a concentration of 1. mu.g/ml for 24 hours. 50 μ L of the supernatant was put in a 96-well plate, mixed with the same volume of Griess assay agent, incubated at room temperature for 10 minutes, and the absorbance was measured at 540nm with a multifunctional microplate reader. As shown in FIG. 1, it can be seen from FIG. 1 that the concentration of NO gradually decreased as the amount of (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 increased from 0.78125. mu.M to 12.5. mu.M, indicating that (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 inhibited LPS-induced NO release (IC) in RAW264.7 cells in a dose-dependent manner (IC)₅₀＝1.981μM)。

4) Inhibition of TNF-alpha and IL-1 beta inflammatory factors

The potential anti-inflammatory capacity of the (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 was further assessed by testing its inhibitory effect on the inflammatory factors TNF-. alpha.and IL-1. beta.in the LPS-induced RAW264.7 cell model.

RAW264.7 cells were plated at 6X 10 per well⁴After each seed was cultured in 48-well plates for 24h, media containing (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 at various concentrations were added. After 1 hour, 30. mu.L LPS (1. mu.g/mL) was added and incubated for 24 hours. The supernatant was then aspirated and assayed according to the instructions of the ELASA kit. As shown in FIG. 2, it can be seen from FIG. 2 that (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 inhibits the release of IL-1. beta. and TNF-. alpha.inflammatory factors and is active against IL-1. beta. (IC)₅₀Greater than TNF-alpha (IC) for inhibition of 5.94 ± 0.35 μ M)₅₀＝10.23±1.04μM)。

Example 3

Adjuvant-induced arthritis model test

Experimental materials: Sprague-Dawley female rats weighing 160-. The temperature, relative humidity and dark light circulation of the animal room are respectively kept at 23-25 ℃ and 40-60 percent.

2. Induction and experimental design of adjuvant arthritis

0.1mL of Freund's Complete Adjuvant (FCA) was injected intradermally into the left hind paw of the rat to cause inflammation. Normal group rats were injected with an equal amount of physiological saline at the same site.

10 days after FCA injection, rats were randomized into four groups. Two groups were drug groups treated with (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9(10mg/kg and 30mg/kg) for 14 days, positive control rats treated with indomethacin (10mg/kg) for 14 days, and the other group was Model group and was given daily saline.

The results are shown in fig. 3-6, fig. 3 is a comparison of the feet of rats in different treatment groups, and fig. 4 is a graph of the change in volume of the feet of rats in different groups. It can be seen from fig. 3 and 4 that the drug group reduced the swelling of the feet of the rats in a concentration-dependent manner as compared to the model group. Furthermore, as can be seen from FIG. 5, 30mg/kg of the (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 significantly reduced the arthritic index in rats. FIG. 6 shows that the body weight of the rats after the administration of the drug is improved compared to that of the model group.

3. In vivo IL-1 beta and TNF-alpha level assays

After the rats were anesthetized, blood was taken from the cardiac artery. After 30 minutes of standing, the blood sample was centrifuged at 3000r/min at 4 ℃ for 10 minutes to collect serum. The levels of IL-1. beta. and TNF-. alpha.in the serum were determined by ELISA. As shown in FIGS. 7A and 7B, it can be seen that the (4- (1,2, 4-oxadiazol-5-yl) phenyl) carboxamide derivative f9 can reduce the levels of the inflammatory factors IL-1. beta. and TNF-. alpha.in rat serum in a dose-dependent manner as compared with the model group.

Experiments prove that the (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative f9 can inhibit the release and secretion of cell NO, IL-1 beta and TNF-alpha, and obviously treat rats with adjuvant-induced arthritis, and the (4- (1,2, 4-oxadiazole-5-yl) phenyl) formamide derivative f9 provided by the invention has potential to be developed into a medicament for treating arthritis diseases.

The above is only a preferred embodiment of the present invention, and is not intended to limit the invention; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that: any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.