| Objective:A new type of N-Alkyl-4-Sulfonoxy substituted Benzoxazolone derivatives were designed and synthesized,and the iNOS inhibitory and anti-inflammatory activity were evaluated to obtain new iNOS inhibitors with high anti-inflammatory activity.Methods:The intermedium 4-hydroxy-benzoxazolone was synthesized by the condensation reaction using 2-aminoresorcinol as the starting material,and then the intermedium was reacted with sulfonyl chloride and halogenated hydrocarbon to gain the target compounds.The structure of the target compound was confirmed by ESI-MS,1H-NMR and 13C-NMR.The effects of NO inhibitory on LPS induced mouse RAW264.7 macrophage were determined by Griess method,and the effects of the compounds on iNOS were determined by ELISA method.Then,the PCR method was used to test the mRNA expression of iNOS.In order to research the interaction between compounds and iNOS protein,the molecular docking method was established,and the structure-activity relationship was obtained.Afterwards,the effects of the compounds on TNF-?,IL-6,IL-1?inhibitions in vitro were measured using ELISA methods,and the anti-inflammatory activities in vivo were evaluated by Xylene induced mouse ear edema.Finally,the expression of TNF-?,IL-6,IL-1?,iNOS in serum,immunohistochemical expression of iNOS and histopathological section of lung tissues in LPS induced mouse acute lung injury models were measured to evaluate the anti-inflammatory effect of the target compound.Results:1.A total of 21 compounds were synthesized,and 18 target compounds were not reported in the literature.All the compounds were confirmed by ESI-MS,1H-NMR and 13C-NMR.2.All the target compounds showed certain inhibitory effect against NO in LPS induced RAW264.7 cell at 25?M.The compounds 2c,2d and 3d presented higher inhibitory activity than that of positive drug L-NNA(IC500 27.55±0.19?M)with IC500 values16.43±0.23?M,14.72±0.34?M and 13.44±0.16?M,respectively.The IC50-iNOS0-iNOS values of the compounds 2c,2d and 3d were 4.605±0.30?M,3.342±0.17?M and 9.733±0.77?M,which were equivalent to the positive drug L-NNA(IC500 2.918±0.12?M).Meanwhile,the compounds 2c,2d and 3d also showed significant iNOS inhibition effects on the mRNA expression.3.The compounds 2c,2d and 3d could significantly inhibit the mouse ear edema induced by Xylene at 12.5mg/kg,which were better than those of the positive drugs chlorzoxazone and L-NNA.4.The compound 2d could reduce the expression of iNOS,TNF-?,IL-6 and IL-1?in serum of LPS induced acute lung injury mice with a dose-dependent manner.Meanwhile,the inflammatory infiltration and pulmonary congestion in the lung tissue were decreased after the treatment with compound 2d,and the level of iNOS in lung tissue were also decreased significantly in a dose-dependent manner in compound 2d treatment group.Conclusions:1.A total of 21 compounds were synthesized,and 18 target compounds were not reported in the literature.2.N-alkyl-4-sulfonyloxybenzoxazolone compounds had certain inhibitory effect against NO,and the structure-activity relationship presented that most of the N,4-disubstituted compounds were more effective than the 4-mono substituted compounds.The NO inhibition activity was increased for the introduction of benzene ring in N-substituted group,and the introduction of electron donating group or the electron withdrawing group into benzene ring could reduce the NO inhibition activity.3.N-alkyl-4-sulfonyloxy benzoxazolones compounds could inhibit the release of iNOS,down-regulate the mRNA expression of iNOS in LPS-induced RAW264.7 cells.And the compounds could be better embedded in the cavity structure of iNOS protein to form hydrogen bond with amino acid residue.Therefore,these compounds might be new potential iNOS inhibitors.4.The compound 2d showed better anti-inflammatory effect on the treatment of LPS induced acute lung injury,and our results would provide experimental basis for the therapeutic effect of iNOS inhibitor in acute lung injury. |
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