| Inflammation is a protective response of the body,but a continuous inflammatory response can damage tissues and even lead to loss of function,tumors and death.At present,people mainly rely on drug treatment to relieve inflammation.Anti-inflammatory drugs can be divided into steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs（NSAIDs）.Complications caused by the use of steroidal anti-inflammatory drugs have limited the clinical use of such drugs.Steroid anti-inflammatory drugs have been shown to cause suppression of the hypothalamic-pituitary-adrenal axis and immune system.The possible side effects that exacerbate diabetes,high blood pressure,osteoporosis,and growth retardation in children have limited their therapeutic use.Most of the anti-inflammatory drugs currently in use are non-steroidal anti-inflammatory drugs.Non-steroidal anti-inflammatory drugs are mainly used to relieve symptoms,whether it is traumatic,infectious,episodic or rheumatic pain.In recent years,some adverse reactions have led to the discontinuation of some NSAIDs.The main mechanism of NSAID action is the inhibition of cyclooxygenase（COX）,and the adverse reactions are mainly related to the relative inhibition of COX.Therefore,it is still of great significance to find new anti-inflammatory drugs with improved activity and reduced toxicity.The active ingredients of natural products have become an important source of drug research and development due to their diverse framework structures and extensive biological activities.At present,there are more than 40 new drugs developed from natural products in my country,such as Huperzine A,Helicobacter,Artemisinin,etc.These innovative drugs discovered from natural products have been widely used in clinical practice due to the characteristics of definite curative effect and low side effects.Therefore,it is a feasible way to find and develop innovative drugs by modifying the structure of active natural products.In the early stage,through literature research,glycyrrhetinic acid,limonin and bakuchiol were selected as anti-inflammatory lead compounds among many natural products,because these three natural products have definite anti-inflammatory activities,low prices,and easy availability,and the structure is easy to be modified.In this paper,three natural products were modified and designed to synthesize 87 novel derivatives.The structures of all synthetic derivatives were verified by 1H,13C-NMR and HRMS spectra,and all derivatives were screened for anti-inflammatory activity.In the first part,in order to find new anti-inflammatory lead compound,a series of new glycyrrhetinic acid derivatives were designed and synthesized through structural modifications such as molecular splicing.The cytotoxicity of 34 target compounds on RAW264.7 cells was screened by MTT method at a concentration of 25μg/m L.Due to the low cytotoxicity,these 14 target compounds were selected for further anti-inflammatory evaluation.The enzyme-linked immunosorbent assay（Elisa）was used to further test the effects of 14 low-toxicity compounds on the secretion of TNF-αand IL-6 in RAW264.7 cells induced by LPS.The screening results show that compound 5b has a significant inhibitory effect on the secretion of TNF-αand IL-6 in RAW264.7 cells induced by LPS.Therefore,the best active compound in this series,compound5b（（2S,4a S,6a S,6b R,11E,12a S）-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14 bicosahydro-2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxo-11-（（1-phenyl-1H-1,2,3-triazol-4-yl）methylene）picene-2-carboxylicacid）,was selected for further evaluation.The Griess method was used to measure the effect of compound 5b on the expression of NO in RAW264.7 cells induced by LPS,and the results indicate that compound5b showed a positive effect in inhibiting LPS-induced NO synthesis in RAW264.7 cells.In addition,the effect of compound 5b on the expression of i NOS and COX-2 was tested by Western blot（WB）.The WB experiment was used to test the effect of compound 5b on the expression of proteins related to the activation of NF-κB signaling pathway in RAW264.7 cells induced by LPS.In order to further study the anti-inflammatory mechanism of compound 5b,the effect of compound 5b on the phosphorylation level of p38 MAPK and JNK was tested.Further studies have shown that compound 5b can inhibit the expression of pro-inflammatory cytokines in RAW264.7 cells induced by LPS in a dose-dependent manner,including IL-6,TNF-αand NO,and can also inhibit the expression of i NOS and COX-2.In addition,WB results indicate that the inhibitory effect of compound 5b on pro-inflammatory cytokines is related to the NF-κB and MAPK signal pathways.The results of animal experiments confirmed that compound 5b can reduce LPS-induced acute lung injury in mice.The preliminary molecular docking results can well explain the selectivity of compound 5b to COX-2 enzyme.In summary,the experimental results obtained indicate that compound 5b may be developed into an anti-inflammatory lead compound,which can be used to prevent and treat inflammatory diseases.In the second part,in order to find anti-inflammatory lead compound with good activity,30 new bakuchiol derivatives were designed and synthesized,and their anti-inflammatory activities were screened.First,in order to ensure the safety of the compound and avoid false positive results during the anti-inflammatory evaluation process,we used the MTT method to detect the inherent cytotoxicity of all synthetic compounds to RAW264.7 cells.Then,to screen the anti-inflammatory activity of all synthetic derivatives,the effects of 30 compounds on the secretion of NO,TNF-αand IL-6 in RAW264.7 cells induced by LPS were further studied by Griess method and Elisa analysis.The results show that compound 13a（2-（4-（（R,E）-3,7-dimethyl-3-vinylocta-1,6-dienyl）phenoxy）-1-（pyrrolidin-1-yl）ethanone）has a significant inhibitory effect on the secretion of NO,TNF-αand IL-6 in RAW264.7cells induced by LPS,so compound 13a is regarded as the best compound in this series for further evaluation.Subsequent determination of anti-inflammatory activity shows that compound 13a can inhibit the production of pro-inflammatory cytokines,including IL-6,TNF-αand NO in RAW 264.7 cells induced by LPS at a certain dose,and inhibit i NOS and COX-2 expression in a dose-dependent manner.In addition,WB results showed that compound 13a inhibited the release of pro-inflammatory cytokines,and the inhibitory effect was related to the activation of the Nrf2/HO-1 signaling pathway and the NF-κB and MAPK signaling pathways.The in vivo results show that compound 13a inhibits the production of NO and ROS in a dose-dependent manner on LPS-induced zebrafish.The results obtained in this study indicate that compound13a has the potential to be further studied for the prevention and treatment of anti-inflammatory diseases.The third part is to search for new anti-inflammatory lead compound as the research purpose.This paper designed limonin derivatives and synthesized a series of 23 new limonin derivatives.All compounds were confirmed by 1H,13C NMR and HR-MS.Subsequently,17 other compounds with almost no cytotoxicity at 40（?）M were screened through the MTT experiment to further evaluate the anti-inflammatory effects.To evaluate the anti-inflammatory activity,we measured the production of NO.Based on the results of the screening,we conclude that,compared with limonin and celecoxib,compound f4（（12S,12a S,Z）-8-（（（1-（4-methoxyphenyl）-1H-1,2,3-triazol-4-yl）methoxy）imino）-12-（furan-3-yl）-6,6,8a,12a-tetramethyldodecahydro-1H,3H-oxireno[2,3-d]pyrano[4′,3′:3,3a]isobenzofuro[5,4-f]isochromene-3,10（9a H）-dione）is the most active derivative among the limonin derivatives synthesized in this article.Subsequently,the study of pharmacological mechanism found that compound f4 significantly inhibited the production of TNF-α,IL-6 and NO in LPS-induced RAW264.7 cells and mouse acute lung injury experiments,and compound f4 significantly inhibited the production of TNF-α,IL-6 and NO in a dose-dependent manner,and inhibited the expression of i NOS and COX2 in acute lung injury induced by LPS.It can block the NF-κB/MAPK signaling pathway.The effect of compound f4on zebrafish mortality and heart rate showed that the toxicity of compound f4 was less than that of limonin and celecoxib.These results indicate that compound f4 will be a promising lead compound candidate for treating inflammatory diseases. |
