Derivatives of Benzotetrazine-1,3-dioxide Are New NO-donors, Activators of Soluble Guanylate Cyclase, and Inhibitors of Platelet Aggregation

N. V. Pyatakova¹, Yu. V. Khropov², A. M. Churakov³, N. I. Tarasova⁴, V. A. Serezhenkov⁴, A. F. Vanin⁴, V. A. Tartakovsky³, and I. S. Severina^1*

¹Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya ul. 10, Moscow, 119992 Russia; fax: (095) 245-0857; E-mail: khropov_y@pochtamt.ru

²Department of Bioorganic Chemistry, School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-2788

³Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky pr. 47, Moscow, 117913 Russia; E-mail: churakov@carc.ioc.ras.ru

⁴Institute of Chemical Physics, Russian Academy of Sciences, ul. Kosygina 4, Moscow, 119991 Russia; E-mail: mikoyan@center.chph.ras.ru

^* To whom correspondence should be addressed.

Received October 29, 2001

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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 µM concentration and the activity increase was 4.5-, 15.0-, and 8.2-fold in the presence of 20 µM dithiothreitol and 11.3-, 31.6-, and 20.5-fold, respectively, in the presence of added glutathione (100 µM). The NO-dependent mechanism of benzotetrazine-1,3-dioxide nitroderivative-induced activation of soluble guanylate cyclase (in the presence of 100 µM glutathione) was confirmed by the inhibition (by 78%) of 7-nitrobenzotetrazine-1,3-dioxide (10 µM)-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 µM) and by the inhibition with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 0.3 µM) of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides (10 µM)-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 µM 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.

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KEY WORDS: soluble guanylate cyclase, nitric oxide, derivatives of benzotetrazine-1,3-dioxide, platelet aggregation

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