Discovery Of INOS Dimerization Inhibitors:Design,Synthesis And Anti-inflammatory Activity Of Novel 5,7-Substituted Pyrazolo[4,3-d]Pyrimidine Derivatives

NO is an important inflammatory factor in human body.Inducible nitric oxide synthase?iNOS?is mainly responsible for the synthesis and release of NO in inflammation.iNOS over expresses NO in many acute and chronic inflammatory diseases such as rheumatoid arthritis,septic shock,organ transplant rejection,etc.,therefore,research and development of small molecule inhibitors with high inhibitory of iNOS can brings new hope for the treatment of these diseases.According to the literature report and the previous experimental research of the research group,we determined that the pyrazolo[4,3-d]pyrimidine nucleus is an active medicinal small molecule skeleton structure,The C5 and C7 positions of the pyrazolo[4,3-d]pyrimidine nucleus were modified to obtain four series of pyrazolo[4,3-d]pyrimidine structural derivatives of A,B,C and D series.The structures of all compounds were verified by ¹H-NMR and ¹³C-NMR and HR-MS.First,we connected the 3,4,5-trimethoxyphenyl group to the core structure at the C5 position,and then substituted the aromatic amines at the C7 position,and substituted the aliphatic amines to obtain the A series compounds,according to the NO release inhibition experiment.As a result,it was found that the A series compounds generally have a low NO release inhibitory activity.Then we modified the C5 position of the nucleus with styryl,2-furanyl and 3,4,5-trimethoxystyryl,respectively,to obtain B,C,D series compounds,the structure-activity relationship analysis showed that the anti-inflammatory activity of the compound was enhanced by the introduction of vinyl group to increase the distance between the C5 substituent of the mother nucleus and the pyridazo[4,3-d]pyrimidine nucleus.respectively,and tested for their inhibition of NO release.The activity test gave the most active compound D27 in this series.By in vitro enzyme activity experiments,we verified that the inhibition of NO release by the compound is positively correlated with the inhibition of iNOS activity,indicating that the pyrazolo[4,3-d]pyrimidine structural derivative exerts anti-inflammatory by inhibiting the activity of iNOS enzyme.Then,the effect of compound D27 on the expression of iNOS in cells showed that the pyrazolo[4,3-d]pyrimidine structural derivative play a role in inhibiting NO synthesis and release did not affect the expression of iNOS protein in cells,but inhibited the formation of dimers of iNOS activity.Finally,we used an adjuvant arthritis rat model to verify the in vivo anti-inflammatory activity of pyrazolo[4,3-d]pyrimidine structural derivatives,from rat paw swelling,weight loss,serum related inflammatory factors and joint tissues,the results of HE staining showed that the pyrazolo[4,3-d]pyrimidine structural derivative had good anti-inflammatory activity in vivo.In this study,the most inhibitory activity of compound D27 was screened by NO release inhibition assay,and its anti-inflammatory activity was verified by a series of experiments,which provided a good research basis for our next study.D27 can be used as a molecular compound guiding compound for the design and synthesis of more efficient small molecule inhibitors of iNOS.