Synthesis And Immunosuppressive Activity Of Nitidine Derivatives

At present,Searching new immunosuppressive agents with high effect and low toxicity has become a research hotspot.Our research group had studied that nitidine chloride could promote the secretion of IL-10 by inhibiting topoisomerase Ι(Topo I),which had potential immunosuppressive effect and could be used for treating AD.However,the application of nitidine chloride is limited due to its poor solubility and low bioavailability.In this paper,we expect to find one or more derivatives with higher activity through structure modification of nitidine chloride.First of all,with the contributions of the related literature,we designed a synthetic route of nitidine,with 5-nitro-2,3-dinaphthyl phenol as a starting material,and eventually got nitidine by seven steps in an overall yield of about 40%.In order to improve the poor solubility of nitidine chloride,we modified the structure of nitidine:(1)Introducing methylol group into C-12 of position nitidine,and we got compound 3-4 in yields of 16%;(2)According to previous structure-activity relationship(SAR),we designed and synthesized three kinds of simplified structures,including: compound 3-22(the naphthalene ring was simplified into benzene ring),compound 3-28(removing methylenedioxy)and compound 3-34(removing pepper ring);(3)Based on compound 3-34,we introduced different oxygen-containing groups into p-N-position,obtained compounds(3-42,3-48,3-52,3-58 and 3-59).Eventually,we synthesized more than 40 intermediates and target derivatives by 8 synthetic routes.Structures of all intermediates and target derivatives were confirmed by HRMS,1H-NMR and 13C-NMR.Secondly,the target derivatives and some intermediates were analyzed by biographic evaluation,including promoting secretion of IL-10,inhibition of Topo I and cytotoxicities.1.At 2 μM concentration,the target derivatives and some intermediates were evaluated their activities for promoting the secretion of IL-10 on BMDCs and RAW 264.7 cells.Preliminary results showed that,all derivatives exhibited lower activities than nitidine chloride on BMDCs.While on RAW 264.7 cells,activities of all derivatives were better than those on BMDCs.Among them,the activity of compound 3-15 was equivalent to nitidine chloride,the activities of compounds 3-14,3-16 and 3-4 were approximately 80% and 73% of nitidine chlorine.We summarized the SARs on RAW 264.7 cell as following:(1)Methoxy groups at C-8 and C-9 positions are active groups;(2)The anion can influence the activity of IL-10 production and iodide ion may reduce the activity;(3)The derivatives with ring-opening of isoquinolinium also have activities of promoting secretion of IL-10,revealing that isoquinolinium ring may not be necessary;(4)As for derivatives with oxygen-containing substituents at para-N-position,the analogues with aldehyde group has higher activity than those with hydroxymethyl substituent.For evaluating cytotoxicities of derivatives,some analogues were tested on RAW 264.7 cells and L-02 cells.The results showed that all the derivatives exhibited lower cytotoxicities than that of nitidine chloride.2.At several different concentrations(2 ? 20 μM),all the derivatives were evaluated by topo Ι inhibitory activity.The results showed that compounds 2-13,3-4,3-16,3-22,3-28,3-34,3-48,3-51 and 3-58 had inhibitory activities on Topo I at 20 μM concentration.Especially,compounds 2-13,3-4,3-16 and 3-22 still possessed equivalent inhibitory activities to nitidine chloride and camptothecin as low as 2 μM concentration.These results indicated that there was a certain consistency between Topo I inhibitory activity and IL-10 secretion promotion for derivatives 2-13,3-4,3-16 and 3-22.Further docking studies also explain the action mode of these selected compounds with Topo I inhibitory activity.Based on the results above,compounds 3-22 and 3-48 were studied about survival rates in Sepsis Mice.The results showed that compounds 3-22 and 3-48 could not improve the survival rates of septic mice at 10 mg/kg.However,at 3 mg/kg,compound 3-48 could improve the survival rates.Compared to nitidine chloride,3-48 has the similar effects at 3 mg/kg.Considering cytotoxicity and other biological data,compound 3-48 can be used as an optimization compound for further expeiments.In summary,this paper has finished studying structural modification of nitidine chloride(a kind of Topo I inhibitor)and testing these derivatives’ activity for promoting the secretion of IL-10 on RAW 264.7 cells.Finally,we found four compounds showed better water solubility and lower cytotoxicities than those of nitidine chloride.This study hopes to provide new direction for searching for novel immunosuppressive molecules with proper druggability and better activities through inhibiting Topo I.