Design,Synthesis And Anti-inflammatory Evaluation Of Paeonol Derivatives

Background: Inflammation is regarded as one of three major diseases in the world,the most common is rheumatoid arthritis(RA)and osteoarthritis(OA).RA is a chronic autoimmune inflammatory disease characterized by multiple joint involvements,high morbidity and patient disability.Now treating RA drugs are including steroidal anti-inflammatory drugs(SAIDs)and non-steroidal anti-inflammatory drugs(NSAIDs),although the two kinds of drugs have apprent anti-arthritic effects,the side effects would occur on the damage of gastrointestinal,liver,kidney and cardiovascular system after long time administration.Therefore,finding anti-inflammatory drug with safty,effectiveness,low side effect has become a hot research topic.Paeonol is a major component in Cortex Moutan which as a traditional Chinese herbal medicine.It exerts a broad pharmacological activity such as anti-inflammatory and antipyretic effects.There are paeonol table and ointment saling on the market with the key function of anti-allergy for skin.According to the literatures,little information about antiarthritic activities of paeonol and its derivatives,relationship of its structure and activity,and anti-inflammatory mechanism are available.There are only a few literatures reported the anti-arthritic effect of paeonol,especially on the strong inhibition of mice paw edema induced by complete adjuvent agent.So the research content of this paper is based on the anti-inflammatory activity of Paeonol,design and synthesis of new Paeonol derivatives through Paeonol as lead compound,than explore the anti-inflammatory activity and the anti-inflammatory mechanism of derivatives.Objective: Based on the receptor structure of cyclooxygenase-2 enzyme(4ux6),the different ligands of paeonol derivatives were designed.After that,the best anti-arthritic activity of paeonol derivatives in theory were synthesized and evaluated by the in vitro and in vivo parameters of physic,chemistry and biology.These results not only revealed anti-arthritic mechanism of paeonol derivatives,but also estimated arelationship between those structures and activities.The results always provided the theoretical and technical support for the development of paeonol derivatives as antiarthritic candidate drug in the future.Method: Pure paeonol was obtained from raw materials Cortex Mudan in Dianjiang(Chongqing,China)with the methods of ethanol extraction and crystallization.Based on the structure of enzyme(4ux6)under computer-assisted drug design,the molecular docking was carried out after the ligands of paeonol derivatives were selected.Those compounds of gained high scores by lib-dock calculation were synthesized and confirmed with the spectra of ultraviolent(UV),infrared spectroscopy(IR),high resolution mass spectrometry(HRMS)and nuclear magnetic resonace spectorscopy(NMR).And then,the pharmacological activities of those synthetic compounds were performed.In vitro,physical and chemical analysis was used to evaluate scavenging abilities of compounds on free radicals(DPPH,·OH,O2-·,ABTS+·);Spectrofluorimetry was adopted to study quenching effects of derivatives on human serum albumin(HSA);In vivo,an arthritic murine model was established induced by complete Freund's adjuvant,the inhibition of paeonol derivatives on mice paw edema were determined use mouse foot tester,the concentration of free radicals and inflammatory cytokines in mice serum were analyzed by enzyme-linked immunosorbent assay(ELISA).The results not only revealed anti-arthritic mechanism of paeonol derivatives,but also established a relationship of their structure-activity.Result:(1)Based on the receptor structure of cyclooxygenase-2 enzyme(4ux6),different ligands of paeonol derivatives were designed and synthesizied by molecular docking,sixteen new compounds(7a-h,8a-h)were confirmed and they had higher binding scores than paeonol in molecular docking,especially compound 7h and 8h obtained the highest scores.(2)In the in vitro experiment of paeonol derivatives on HSA and scavenging free radicals,we found that the compounds 7a-h and 8a-h strongly scavenged free radicals and displayed a regular change that scavenging ability increased with the length elongating of carbon chain.Compound 8h showed the stongest ability among synthetic compounds and better than Vitamin C.And all derivatives have quenched effects on HSA,compounds 7,7h and 8 displayed dynamic quenching on HSA,but compound 8h showed that static quenching resulted from the formation of a new complex compound.According to the thermodynamic parameters,it was determined that the temperature had a great effect on the binding ability between the drug and the protein.And at 31?,the main binding force of the compounds 7,7h and 8h with HSA was hydrogen bond or van der Waals force.This results are consistent with the protein binding form of docking.(3)In the model of mice paw edema induced by complete Freund's adjuvant,the inhibitory rate of these synthetic compounds on mice paw edema displayed dosedependent relation.The inhibitory rate of paw swelling was 7h-H > 8h-H > Cel ?7-H > 8h-L > 7h-L > 7-L > 8-H > 8-L > Mod(H is a high dose,L is a low dose,Cel is an abbrivation of Celecoxib);In serum of arthritic mice,7,7h,8 and 8h increased the content of T-AOC and SOD,7h-H and 8h-H showed better than others.In addition,the concentration of COX-2,PGE2 and MDA also inhibited by 7,7h,8 and 8h,especially7-H and 8-H showed better inhibition,7h and 8h showed less.8h-H indicated the strongest inhibition.Conclusion: In this study,two series of paeonol derivatives(7a-h and 8a-h)were designed and synthesized based on the receptor structure of COX-2 enzyme.8h and 7h had better antiarthritic activities were in consistence to the predict result of molecular docking.As for the modification of paeonol,the substitution of bromine and alkoxy on phenyl increased antiarthritic activities and the length of carbon chain had some positive influences in biological activity.There were two major aspects to revealed antiarthritic activities of paeonol derivatives.One was antioxidant ability increased through the scavenging on free radicals;the other was the inhibitory rate on mice paw edema promoted with the inhibition of COX-2 and PGE2 activities.From the above results,a relationship of structure and activity of paeonol derivatives was established and it provided the theoretical and technical support for the development of paeonol derivatives as antiarthritic candidant drug in the future.