Hydrolytic reactivity trends among potential prodrugs of the O2-glycosylated diazeniumdiolate family. Targeting nitric oxide to macrophages for antileishmanial activity.

Glycosylated diazeniumdiolates of structure R 2NN(O)NO-R' (R' = a saccharide residue) are potential prodrugs of the nitric oxide (NO)-releasing but acid-sensitive R 2NN(O)NO (-) ion. Moreover, cleaving the acid-stable glycosides under alkaline conditions provides a convenient protecting group strategy for diazeniumdiolate ions. Here, we report comparative hydrolysis rate data for five representative glycosylated diazeniumdiolates at pH 14, 7.4, and 3.8-4.6 as background for further developing both the protecting group application and the ability to target NO pharmacologically to macrophages harboring intracellular pathogens. Confirming the potential in the latter application, adding R 2NN(O)NO-GlcNAc (where R 2N = diethylamino or pyrrolidin-l-yl and GlcNAc = N-acetylglucosamin-l-yl) to cultures of infected mouse macrophages that were deficient in inducible NO synthase caused rapid death of the intracellular protozoan parasite Leishmania major with no host cell toxicity.

Experimental evidence for enzyme-enhanced coupled motion/quantum mechanical hydrogen tunneling by ketosteroid isomerase.

Although there are considerable data demonstrating that quantum mechanical hydrogen tunneling (HT) occurs in both enzymatic and nonenzymatic systems, little data exist that address the question of whether enzymes enhance the amount of HT relative to the corresponding nonenzymatic reactions. To investigate whether 3-oxo-Delta (5)-steroid isomerase (ketosteroid isomerase, KSI) enhances HT relative to the nonenzymatic (acetate-catalyzed) isomerization of Delta (5)-androstene-3,17-dione ( 1) to Delta (4)-androstene-3,17-dione ( 3), alpha-secondary deuterium kinetic isotope effects (KIE) at C-6 of the steroid were determined for both the KSI- and acetate-catalyzed isomerizations. The normal intrinsic secondary KIE for both wild type (WT) KSI (1.073 +/- 0.023) and acetate (1.031 +/- 0.010) suggest the possibility of coupled motion (CM)/HT in both the enzymatic and nonenzymatic systems. To assess the contribution of CM/HT in these reactions, the secondary KIE were also measured under conditions in which deuterium instead of hydrogen is transferred. The decrease in secondary KIE for WT (1.035 +/- 0.011) indicates the presence of CM/HT in the enzymatic reaction, whereas the acetate reaction shows no change in secondary KIE for deuterium transfer (1.030 +/- 0.009) and therefore no evidence for CM/HT. On the basis of these experiments, we propose that KSI enhances the CM/HT contribution to the rate acceleration over the solution reaction. Active site mutants of KSI (Y14F and D99A) yield secondary KIEs similar to that of WT, indicating that mutations at the hydrogen-bonding residues do not significantly decrease the contribution of CM/HT to the KSI reaction.

Synthesis, nitric oxide release, and anti-leukemic activity of glutathione-activated nitric oxide prodrugs: Structural analogues of PABA/NO, an anti-cancer lead compound.

Diazeniumdiolate anions and their prodrug forms are reliable sources of nitric oxide (NO) that have generated interest as promising therapeutic agents. A number of structural analogues of O(2)-(2,4-dinitro-5-(4-(N-methylamino)benzoyloxy)phenyl) 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), an anti-cancer lead compound that is designed to release NO upon activation by glutathione, were prepared. The nitric oxide release patterns of these O(2)-(2,4-dinitrophenyl) diazeniumdiolates in the presence of glutathione were tested and it was found that in the absence of competing pathways, these compounds release nearly quantitative amounts of NO. The ability of PABA/NO and its structural analogues to inhibit human leukemia cell proliferation was determined and it was found that compounds releasing elevated amounts of NO displayed superior cytotoxic effects.

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.