Design,Synthesis And Evaluation Of Anti-inflammatory And Analgesic Activity Of Coumarin Derivatives

Chronic inflammatory reactions would lead to vascular proliferation,fibrosis,and tissue damage,causing metabolic disorders of the body,and the emergence of rheumatoid arthritis,atherosclerosis and other complications,and then lead to cancer and other major diseases,endanger people's health seriously.Pain accompanied with inflammatory reaction also affects the feelings of patients and the quality of life greatly.In the daily training,military personnel often suffer from different degrees of training injuries,which in turn cause inflammation and pain and affect the military capabilities of the commanders and fighters,lead to decline capabilities of the army in the combat derectly.Anti-inflammatory and analgesic drugs are one of the effective measures for this issue.Glucocorticoids used in clinical have a wide range of anti-inflammatory effects,However,they would have side effects such as cardiovascular diseases and water-sodium retention in long-term use,and replaced by non-steroidal anti-inflammatory drugs gradually,such as aspirin,ibuprofen,and coxibine.Their main mechanism is inhibition of cyclooxygenase?COX?in the body to interfere with the synthesis of prostaglandins?PGs?.The three subtypes of COX,COX-1,COX-2,and COX-3,are expressed in tissues of the body widely,it is difficult to inhibit the inducible COX-2 selectively besides the COX-1 and COX-2 involved in the normal metabolism of the organism within non-steroidal anti-inflammatory drugs on the market or the investigational selective inhibitors of cyclooxygenase.Therefore NSAIDs have unavoidable side effects of gastrointestinal and cardiovascular.Coumarin derivatives have various activities such as anti-inflammatory,anti-oxidation and anti-cancer.Studies on the mechanism of coumarin derivatives show that some kinds of Coumarin derivatives can inhibit the 5-lipoxygenases?5-LOXs?pathway to reduce the level of leukotrienes to relieve inflammation;some kinds of coumarin derivatives can act on nitric oxide synthase?NOS?,COX,etc.,and can inhibit the release of inflammatory factors such as TNF-?,IL-1,IL-6,etc.During the research of analgesic candidate drugs,it has been found that TRV130 and PZM21 are the?-opioid-receptor-biased agonists which specific to the?-opioid-receptor and biased towards the Gi signaling pathway and minimal of?-arrestin-2 recruitment,with good analgesic efficacy and lower side effects.Both PZM21 and traditional opioid analgesics act on?-opioid-receptors,the difference is that PZM21 is a novel,small-molecule structure-based opioid analgesics with reduced side effects such as respiratory depression,addiction,constipation,etc.which the traditional opioid analgesics does.In this paper,Based on the structure-activity relationship of the anti-inflammatory activity of coumarin derivatives and the analgesic activity of PZM21,we prepared to design,synthesis and study on the activity of novel active coumarin molecule with anti-inflammatory and analgesic.It is expected to find the novel active molecule with anti-inflammatory and analgesic activity,which lays the foundation for the study of coumarin derivatives with anti-inflammatory and analgesic activity.We have completed the following work primarily:1.Designed a series of novel urea-containing coumarin derivatives PaX1⁵,PbX1⁵,MaX1⁵,MbX1⁵.There are four design ideas:1)The basic structure of simple coumarin was retained,and the arylaminourea side chain of PZM21 was introduced on the 7-position hydroxyl group;2)The substituents at the 3,4,6-position on the benzoheterocycle in the coumarin structure was changed to investigate the structure-activity relationship;3)The substituent of para-aromatic ring on the side chain of arylaminourea was replaced with methoxy to study the influence of the group on analgesic activity;4)The chiral constitution of the side chain of arylaminourea was changed,we designed the compounds of?S?and?S,S?configuration to study the effect of molecular chiral configuration on the activity;2.Twenty novel urea-containing coumarin derivatives have been synthesized and identified with ¹H-NMR,¹³C-NMR,MS,The synthesis methods of compound PZM21was improved successfully,the purification and identification of PZM21 have accomplished.3.Twoanionic?-cyclodextrinsweredesignedandsynthesized:mono-6-taurine-?-cyclodextrin,mono-6-L-aspartic acid-?-cyclodextrin,in order to solve the problem of possible water solubility of the target product,and studied its solubilization application preliminary.In the step of post-processing,the methods of protection of excess amino acids with Boc₂O and extraction with organic solvent replaced the separation with Sephadex column chromatography reported in the literature,which simplified the experimental operation and lower the cost and could be more suitable for industrial production.Since the 20 hydrochlorides derivatives of coumarins are with good water solublility,we have not study their inclusion behaviors with?-cyclodextrin derivatives,it could provide a kind of methods to increase the solubility of poorly soluble compounds in the future.We studied the inclusion of two anionic?-cyclodextrins with the insoluble drug indomethacin with the method of phase solubility.The hydroxypropyl-?-cyclodextrin was used as the control group.The results showed that the hydroxypropyl-?-cyclodextrin and indomethacin could form clathrates with an inclusion ratio of 1:1,and the phase dissolution curve is a typical A_L;two anionic?-cyclodextrins failed to form clathrates with indomethacin.Failure to achieve solubilization may be due to the fact that indomethacin is more soluble in alkaline aqueous solutions than acidic or neutral environments.4.In vitro anti-inflammatory activity evaluations have been performed on the target compounds.We have studied the effects of 20 compounds on cell viability of macrophage RAW264.7 at concentrations of 40,20,10,5,1,0.2?mol/L with CCK-8 cytotoxicity assay.Tests showed that those compounds have a smaller toxicity to the cell and the cell viability are higher than 80%within the concentration of 10umol/L,the concentration of10?mol/L could be selected as the maximum concentration of active screen.Twenty compounds have been slected for the inhibitory activity of TNF-?released by lipopolysaccharide?LPS?-stimulated RAW264.7 cells,Dexamethasone was regarded as a positive control drug.Studies have shown that the compounds of PaX1,PbX1,PaX2,PbX2,PaX3,PbX3,PaX4,PbX4,PbX5,MaX2,MbX2,MbX3 MaX4,MbX4,MaX5,MbX5 have obvious inhibitory activity compared to the negative control group,of which compounds PbX2,MbX2,MaX5,MbX5 have prominent inhibitory activity.Two concentrations of 10?mol/L and 5?mol/L were set respectively in the active rescreening experiment.The experimental results showed that the four compounds have good inhibitory activity compared with the negative control group at the two concentrations levels.In vivo evaluation of anti-inflammatory activity:we established a mouse ear-swelling model induced by xylene to study the in vivo anti-inflammatory activity of the four compounds PbX2,MbX2,MaX5,and MbX5.Dexamethasone was set as a positive control drug.High and low concentrations were set at 10 mg/kg and 5 mg/kg,respectively;four compounds were set at high,medium,and low concentrations of 50mg/kg,25 mg/kg,5 mg/kg,respectively.The results showed that the inhibition rate of high-dose dexamethasone group?10 mg/kg?and low-dose group?5 mg/kg?was 64.3%and 54.9%?^*P?27?0.05?,indicating a good swelling inhibitory effect.The inhibitory rates of compound PbX2,MbX2,MaX5,and MbX5 were 40.3%,27.0%,37.2%,and 34.7%,at high concentration levels?50 mg/kg?respectively?^*P?27?0.05?,indicating inhibitory effects.The inhibitory effects of PbX2,MaX5,and MbX5 were more prominent;however,there was no statistically significant difference in the inhibitory rates of medium?25 mg/kg?and low?5 mg/kg?concentrations?^#P?29?0.05?.Based on the experimental results of anti-inflammatory activity in vivo and in vitro,compounds PbX2,MaX5,and MbX5 were selected for evaluation of analgesic activity in vivo.In vivo evaluation of analgesic activity:a rat model of acetic acid writhing was established to evaluate the analgesic activity of PbX2,MaX5,and MbX5 in vivo.Indomethacin?10 mg/kg?and PZM21?40 mg/kg?were set as positive control groups;PbX2,MaX5,and MbX5 were administered at a concentration of 50 mg/kg.There were6,4,6,4,3,4 animals showed with writhing response in the blank group,indomethacin group,and groups of PZM21 PbX2,MaX5,MbX5 and writhing frequency of 27.00?10.65?3.75?1.26?5.00?3.10?5.00?3.10?6.25?2.50?4.00?2.65?5.00?2.16,respectively,and the pain thresholds were 3.03?0.75min?5.26?1.22min?5.15?1.70min?5.08?1.30 min?10.32?1.51 min?5.48?1.39 min,respectively.Compared with the blank group,the administration group showed a good analgesic effect,the number of animals exhibiting a writhing response was reduced,the number of writhing was also significantly reduced,and the threshold of the writhing response was prolonged,which was statistically significant.The results of anti-inflammatory and analgesic activity showed that compounds PbX2,MbX2,MaX5,and MbX5 showed good inhibitory activity anti-inflammatory activity evaluation experiments both in vivo and in vitro;Compounds PbX2,MaX5,and MbX5 showed significant analgesic activity in the evaluation of acetic acid writhing analgesic activity in vivo,which had outstanding anti-inflammatory activity,too.The acetic acid writhing model is a pathological level pain model generated by acute inflammatory lesions caused by non-external stimulation.The anti-inflammatory compounds PbX2,MaX5,and MbX5 also exhibited good analgesic activity at the same time,and the experimental results were consistent with the theoretical predictions.5.The preliminary structure-activity relationship analysis was performed on the anti-inflammatory and analgesic activities of the target compounds.The results showed that:the substituent group of R² is methyl and the target compounds have?S,S?configuration can enhance anti-inflammatory analgesic activity;the substituent group of R⁴ is methyl and R⁵ is an electron-withdrawing group--cyano in coumarin nucleus can enhance the inhibitory activity of TNF-?,the substituent group of R³ is an electron-donating group methoxy in coumarin nucleus may exhibit weaker inhibitory activity of TNF-?,and the substituent group of R¹ is an electron-donating group such as the methoxy group may enhance its inhibitory activity;It can enhance the inhibitory activity of TNF-?significantly when R³ is a chlorine atom coupled with the?S,S?configuration of the molecule,which can confirm the prediction of the structure-activity relationship partially.We combined the structural characteristics and structure-activity relationship of the coumarin derivatives and the analgesic active molecule PZM21,aimed to discover novel coumarin derivatives with anti-inflammatory and analgesic activities and their structure-activity relationship rules through structure design,synthesis,preliminary anti-inflammatory and analgesic activity screening and summary of the structure-activity relationship.which lays the foundation for the further study of new anti-inflammatory analgesics.