HNO and NO release from a primary amine-based diazeniumdiolate as a function of pH.

The growing evidence that nitroxyl (HNO) has a rich pharmacological potential that differs from that of nitric oxide (NO) has intensified interest in HNO donors. Recently, the diazeniumdiolate (NONOate) based on isopropylamine (IPA/NO; Na[(CH(3))(2)CHNH(N(O)NO)]) was demonstrated to function under physiological conditions as an organic analogue to the commonly used HNO donor Angeli's salt (Na(2)N(2)O(3)). The decomposition mechanism of Angeli's salt is dependent on pH, with transition from an HNO to an NO donor occurring abruptly near pH 3. Here, pH is shown to also affect product formation from IPA/NO. Chemical analysis of HNO and NO production led to refinement of an earlier, quantum mechanically based prediction of the pH-dependent decomposition mechanisms of primary amine NONOates such as IPA/NO. Under basic conditions, the amine proton of IPA/NO is able to initiate decomposition to HNO by tautomerization to the nitroso nitrogen (N(2)). At lower pH, protonation activates a competing pathway to NO production. At pH 8, the donor properties of IPA/NO and Angeli's salt are demonstrated to be comparable, suggesting that at or above this pH, IPA/NO is primarily an HNO donor. Below pH 5, NO is the major product, while IPA/NO functions as a dual donor of HNO and NO at intermediate pH. This pH-dependent variability in product formation may prove useful in examination of the chemistry of NO and HNO. Furthermore, primary amine NONOates may serve as a tunable class of nitrogen oxide donor.

PABA/NO as an anticancer lead: analogue synthesis, structure revision, solution chemistry, reactivity toward glutathione, and in vitro activity.

PABA/NO is a diazeniumdiolate of structure Me(2)NN(O)=NOAr (where Ar is a 5-substituted-2,4-dinitrophenyl ring whose 5-substituent is N-methyl-p-aminobenzoic acid). It has shown activity against human ovarian cancer xenografts in mice rivaling that of cisplatin, but it is poorly soluble and relatively unstable in water. Here we report structure-based optimization efforts resulting in three analogues with improved solubility and stability in aqueous solution. We sought to explain PABA/NO's physicochemical uniqueness among these four compounds, whose aminobenzoic acid precursors differ structurally only in the presence or absence of the N-methyl group and/or the position of the carboxyl moiety (meta or para). Studies revealed that PABA/NO's N-methyl-p-aminobenzoic acid substituent is bound to the dinitrobenzene ring via its carboxyl oxygen while the other three are linked through the aniline nitrogen. This constitutes a revision of the previously published PABA/NO structure. All four analogues reacted with GSH to produce bioactive nitric oxide (NO), but PABA/NO was the most reactive. Consistent with PABA/NO's potent suppression of A2780 human ovarian cancer xenograft growth in mice, it was the most potent of the four in the OVCAR-3 cell line.

Structure-Based Design of Anticancer Prodrug PABA/NO.

Glutathione S-transferase (GST) is a superfamily of detoxification enzymes, represented by GSTalpha, GSTmu, GSTpi, etc. GSTalpha is the predominant isoform of GST in human liver, playing important roles for our well being. GSTpi is overexpressed in many forms of cancer, thus presenting an opportunity for selective targeting of cancer cells. Our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GSTpi-overexpressing cancer cells yielded PABA/NO, which exhibited anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin (Findlay et al. Mol. Pharmacol. 2004, 65, 1070-1079). Here, we present the details on structural modification, molecular modeling, and enzymatic characterization for the design of PABA/NO. The design was efficient because it was on the basis of the reaction mechanism and the structures of related GST isozymes at both the ground state and the transition state. The ground-state structures outlined the shape and property of the substrate-binding site in different isozymes, and the structural information at the transition-state indicated distinct conformations of the Meisenheimer complex of prodrugs in the active site of different isozymes, providing guidance for the modifications of the molecular structure of the prodrug molecules. Two key alterations of a GSTalpha -selective compound led to the GSTpi-selective PABA/NO.

Metabolism of a liver-selective nitric oxide-releasing agent, V-PYRRO/NO, by human microsomal cytochromes P450.

Endogenously generated nitric oxide (NO) mediates a host of important physiological functions, playing roles in the vascular, immunological, and neurological systems. As a result, exogenous agents that release NO have become important therapeutic interventions and research tools. O(2)-Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a prodrug designed with the hypothesis that it might release nitric oxide via epoxidation of the vinyl group by cytochrome P450, followed by enzymatic and/or spontaneous epoxide hydration to release the ultimate NO-donating moiety, 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO) ion. In this study, we investigated this hypothetical activation mechanism quantitatively for V-PYRRO/NO using cDNA-expressed human cytochrome P450 (CYP)2E1. Incubation with CYP2E1 and an NADPH-regenerating system resulted in a time-dependent decomposition of V-PYRRO/NO, with a turnover rate of 2.0 nmol/min/pmol CYP2E1. Nitrate and nitrite were detected in high yield as metabolites of NO. The predicted organic metabolites pyrrolidine and glycolaldehyde were also detected in near-quantitative yields. The enzymatic decomposition of V-PYRRO/NO was also catalyzed, albeit at lower rates, by CYP2A6 and CYP2B6. We conclude that the initial step in the metabolism of V-PYRRO/NO to NO in the liver is catalyzed efficiently but not exclusively by the alcohol-inducible form of cytochrome P450 (CYP2E1). The results confirm the proposed activation mechanism involving enzymatic oxidation of the vinyl group in V-PYRRO/NO followed by epoxide hydration and hydrolytic decomposition of the resulting PYRRO/NO ion to generate nitric oxide.