QSARs in prooxidant aerobic cytotoxicity of heteroaromatic N-oxides: Role of flavoenzyme-catalyzed single-electron reduction

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit antitumour, antibacterial, and antiprotozoal activities, which are attributed to reductive activation and free radical generation ([1], and references therein). Although ArN→O are typically considered as potential hypoxia-selective antitumour agents, in some cases they possess high anticancer activity under oxic conditions. Besides, their oxidative stress-type mammalian cell cytotoxicity may be important side effect in antimicrobial and antiparasitic action Extending our previous studies [2], we aimed to clarify the role of flavoenzyme-catalyzed single-electron reduction of ArN→O in their aerobic cytotoxicity. We synthesized a series of ArN→O wavailable and unavailable values of single-electron reduction potential (E¹₇) (n = 18). Their reactivity (k_cat/K_m) towards single-electron transferring flavoenzymes NADPH:cytochrome P-450 reductase, ferredoxin:NADP+ oxidoreductase, and NO synthase (nNOS) increased with their E¹₇ (∆log k_cat/K_m/∆E¹₇ ≈ 10 V^-1). The reactivity of ArN→O with unavailable E¹₇ values correlated with the σ values of their substituents. The reactivity of ArN→O was similar to that of quinones with the same E¹₇ values. Their toxicity in murine hepatoma MH22a and human carcinoma HCT-116 cells increased with their E¹₇ and/or the average of their log k_cat/K_m in single-electron enzymatic reduction. However, it exceeded the cytotoxicity of quinones with the same redox activity by one order of magnitude. The protective effects of antioxidants demonstrated the prooxidant character of cytotoxicity. The enhanced cytotoxicity of ArN→O was partly attributed to the action of cytochromes P-450, because their inhibitors protected against the toxicity, but potentiated the cytotoxicity of quinones. Another factor is the action of NAD(P)H:quinone oxidoreductase (NQO1), which reduces quinones in two-electron way and contributes to their detoxification. Its inhibitor, dicoumarol, enhanced the cytotoxicity of quinones and protected against the cytotoxicity of ArN→O. We found that NQO1 atypically reduces ArN→O in mixed single- and two-electron way, and possibly contributes to their redox cycling. The multiparameter regression analysis shows that there exists a statistically significant relationship between k_cat/K_m ArN→O towards NQO1 and their cytotoxicity. These data show that apart from the typical representatives of single-electron transferring flavoenzymes, NQO1 is another important target of ArN→O, and provide certain guidelines for their rational synthesis. (author)