Inhibition of LPS-stimulated NO production in mouse macrophage-like cells by benzocycloheptoxazines

Twenty-six benzocycloheptoxazine derivatives were investigated for their effect on nitric oxide (NO) production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like RAW 264.7 cells. Benzo[b]cyclohepta[e][1,4]thiazine most effectively inhibited the LPS-stimulated NO production at noncytotoxic concentrations. 6H-Benzo[b]cyclohepta[e][1,4]-diazine cation, and benzo[b]cyclohepta[e][1,4]oxazine and its 6-bromo derivative also efficiently inhibited the LPS-stimulated NO production. Another sixteen benzo[b]cyclohepta[e]-[1,4]oxazine derivatives, 14H-[1,4]benzoxazino[3',2' :3,4]-cyclohepta[1,2-b][1,4]benzoxazine and its 7-bromo- and 7-isopropyl derivatives were slightly less active (selectivity index (SI)=83-66). Bromination of benzo[b]cyclohepta[e][1,4]-thiazine, benzo[b]cyclohepta[e][1,4]oxazine and 2-methylbenzo[b]cyclohepta[e][1,4]oxazine at C-6, C-8 or C-10 positions resulted in the significant reduction of the inhibitory activity. The observed inhibitory activity of benzo[b]cyclohepta-[e][1,4]thiazine and its 6,8-dibromo derivatives were not due to the reduction of the intracellular level of inducible NO synthase protein (based on Western blot analysis), nor to NO scavenging activity (based on ESR spectroscopy). These results suggest the possible anti-inflammatory action of benzocyclo-heptoxazines via inhibition of LPS-activated macrophages.