Synthesis of 1,2,3,4-tetrazino[5,6-f]benzo-1,2,3,4-tetrazine 1,3,7,9-tetra-N-oxides.

[reaction: see text] The novel heterocyclic system 10 with the phenanthrene type skeleton, in which the benzene ring is annulated with two 1,2,3,4-tetrazine 1,3-di-N-oxide rings, is of considerable interest in the context of the high nitrogen system stability and from a heteroaromaticity standpoint. The step-by-step synthetic approach to this system involves treatment of 5 with N2O5, resulting in the first 1,2,3,4-tetrazine 1,3-dioxide ring formation. The following displacement of the chlorine atom at the 6-position with ammonia and subsequent repeated treatment with N2O5 results in the second 1,2,3,4-tetrazine 1,3-dioxide ring formation. The structure of 10 was confirmed by a 13C and 14N NMR study.

Derivatives of benzotetrazine-1,3-dioxide are new NO-donors, activators of soluble guanylate cyclase, and inhibitors of platelet aggregation.

The ability of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides to generate nitric oxide (NO) and activate soluble guanylate cyclase was investigated. All of these compounds were found to be thiol-dependent NO-donors and guanylate cyclase activators. The maximal stimulatory effect of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides was observed at 10 microM concentration and the activity increase was 4.5-, 15.0-, and 8.2-fold in the presence of 20 microM dithiothreitol and 11.3-, 31.6-, and 20.5-fold, respectively, in the presence of added glutathione (100 microM). The NO-dependent mechanism of benzotetrazine-1,3-dioxide nitroderivative-induced activation of soluble guanylate cyclase (in the presence of 100 microM glutathione) was confirmed by the inhibition (by 78%) of 7-nitrobenzotetrazine-1,3-dioxide (10 microM)-stimulated guanylate cyclase activity in the presence of the NO-scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Carboxy-PTIO, 50 microM) and by the inhibition with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 0.3 microM) of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides (10 microM)-stimulated guanylate cyclase by 34, 69, and 39%, respectively. All compounds used inhibited ADP-induced aggregation of human platelets with IC50 of 10.0, 1.3, and 2.0 microM for 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides, respectively. A clearly defined correlation was established between the ability of the compounds to generate NO, activate soluble guanylate cyclase, and inhibit platelet aggregation.