Design,synthesis And Evaluation Of Anti-inflammatory Activity Of A Novel NLRP3 Inflammasome Inhibitor: A Pterostilbene Derivative

Inflammatory bowel disease（inflammatory bowel disease,IBD）is a group of chronic intestinal diseases that includes Crohn’s disease（crohn disease,CD）and ulcerative colitis（ulcerative colitis,UC）,with clinical symptoms including diarrhoea,abdominal pain,and bloody stools.The incidence of IBD is currently at the top of the list worldwide,and the number of IBD patients in China is increasing by the year.More seriously,IBD greatly increases the risk of cancer,with clinical statistics finding that about 20%of UC patients develop colorectal cancer within 30 years and the death rate is as high as 50%.Therefore,there is an urgent clinical need to find effective drugs to treat IBD.According to some studies,NLRP3 inflammasome play an important role in the pathogenesis of IBD and may become a new drug target for the treatment of IBD.As a natural product containing a stilbene and two methoxy structures,also known as 4’-hydroxy-3,5-dimethoxyastragalus,pterostilbene has a variety of biological activities such as anti-inflammatory,anti-bacterial and anti-oxidant.However,the weak activity,low bioavailability and unclear targets of pterostilbene have severely limited its medicinal value.The group has been working on pterostilbene for many years and found that pterostilbene has potential inhibitory activity against the activation of NLRP3 inflammasome.While the amide group has a key role in anti-inflammatory activity,we have therefore designed and synthesised 41 pterostilbene amide derivatives D1-D41.The toxic effects of D1-D41 on Hepa RG cells were investigated by MTT assay.The results showed that all compounds had no significant effect on the survival of Hepa RG at a concentration of 20μM,and the IC₅₀ was greater than 50μM,indicating that these compounds have a good safety profile.The effect of D1-D41 on LPS/Nigericin-induced IL-1βrelease in BMDMs cells was examined by Elisa method,and the results showed that at a concentration of 20μM,D20,D22,D23,D26 and D38had a good inhibitory effect on IL-1βrelease,among them,D20 had the best inhibitory effect with 52±0.9%,but it was weaker than that of the positive drugs.Western blot assays showed that D20 was able to inhibit the secretion of IL-1βand caspase-1 in a concentration-dependent manner without affecting the expression of pro-caspase-1（p45）and pro-IL-1β.D20 was also found to significantly inhibit the expression of NLRP3 protein in a significant concentration-dependent relationship.We therefore speculate that D20 may inhibit the activation of NLRP3 inflammasome by suppressing the activation of NF-κB/MAPK pathway.To confirm our speculation,we conducted further exploratory experiments.The effect of the target compounds on NO release from LPS-treated RAW264.7 cells was first verified.The Griess assay revealed that most of the compounds had an inhibitory effect,with D20 showing the most significant inhibition of NO release with an IC₅₀ of 3.72±0.18μM.The effect of the compounds on RAW264.7 cells was also investigated by MTT,and the results showed that the drug concentration at 20μM was not significantly toxic to RAW264.7 cells,and the IC₅₀was greater than 50μM.The Western blot assay also showed that D20 significantly inhibited the phosphorylation of IκB protein in a concentration dependent manner,and inhibited the phosphorylation of p65 protein,which prevented its nuclear translocation,indicating that D20 inhibited the activation of NF-κB pathway.At the same time,D20also significantly inhibited the phosphorylation of JNK,ERK1/2 and p38 proteins in a concentration dependent manner,which suggested that D20 inhibited the activation of MAPK signalling pathway.In vivo activity assays showed that compound D20 was able to alleviate DSS-induced acute colitis in mice.The MTT method explored the effect of the compound on the repair of oxidative damage caused by H₂O₂ in Hepa RG cells,and the results showed that compound D20 at a concentration of 10μM strongly repaired the oxidative damage in Hepa RG cells.In summary,compound D20 is a low-toxic,highly potent anti-inflammatory small molecule that works primarily by inhibiting the activation of the NF-κB pathway in the first phase（initiation phase）of the inflammasome activation and ROS in the second phase（activation phase）,acting together to inhibit the expression of NLRP3 protein and further inhibit the activation of NLRP3 inflammasome,thereby attenuating the inflammatory response.The implementation of this project will provide a reference for the development of drugs for NLRP3 inflammatory vesicle-related diseases.