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Here, we show that two tautomers are produced by the protonation of the guanine-electron adduct. The fate of electron adducts of a variety of substituted guanosines was investigated by radiolytic methods and addressed computationally by means of time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations. The reaction of e(aq-) with guanosine and 1-methylguanosine produces two transient species, whereas the reaction with N2-ethylguanosine and N2,N2-diethylguanosine produces only one. The two short-lived intermediates, which show a substantial difference in their UV-visible spectra, are recognized to be two purine tautomers (i.e., iminic 18 and aminic 19 forms). The tautomerization 18 --> 19 occurs with a rate constant of ca. 1.5 x 106 s(-1) , and theory suggests that it is a water-assisted process.

Combretastatins are stilbene-based, tubulin depolymerization agents with selective activity against the tumor vasculature; two variants (A-1 and A-4) are currently undergoing clinical trials. Combretastatin A-1 (CA1) has a greater antitumor effect than combretastatin A-4 (CA4). We hypothesized that this reflects the enhanced reactivity conferred by the second (ortho) phenolic moiety in CA1. Oxidation of CA1 by peroxidase, tyrosinase, or Fe(III) generates a species with mass characteristics of the corresponding ortho-quinone Q1. After administration of CA1-bis(phosphate) to mice, the hydroquinone-thioether conjugate Q1H2-SG, formed from the nucleophilic addition of GSH to Q1, was detected in liver. In competition, electrocyclic ring closure of Q1, over a few minutes at pH 7.4, leads to a second ortho-quinone product Q2, characterized by exact mass and NMR. This product was also generated by human promyelocytic leukemia (HL-60) cells in vitro, provided that superoxide dismutase was added. Q2 is highly reactive toward glutathione (GSH) and ascorbate, stimulating oxygen consumption in a catalytic manner. Free radical intermediates formed during autoxidation of CA1 were characterized by EPR, and the effects of GSH and ascorbate on the signals were studied. Pulse radiolysis was used to initiate selective one-electron oxidation or reduction and provided further evidence, from the differing absorption spectra of the radicals formed on oxidation of CA1 or reduction of Q2, that two different quinones were formed on oxidation of CA1. The results demonstrate fundamental differences between the pharmacological properties of CA1 and CA4 that provide two possible explanations for their differential activities in vivo: oxidative activation to a quinone intermediate likely to bind to protein thiols and possibly to nucleic acids and stimulation of oxidative stress by enhancing superoxide/hydrogen peroxide production. The observation of the GSH conjugate Q1H2-SG in vivo provides a new marker for oxidative metabolism of relevance to current clinical trials of CA1-bis(phosphate) (OXi4503).

Oxidation of indole-3-acetic acid and its derivatives by peroxidases such as that from horseradish produces many products, including 3-methylene-2-oxindoles. These have long been associated with biological activity, but their reactivity has not been characterized. We have previously demonstrated the potential value of substituted indole acetic acids and horseradish peroxidase as the basis for targeted cancer therapy, since the compounds are of low cytotoxicity until oxidized, when high cytotoxicity is observed; the combination of prodrug and enzyme depletes intracellular thiols. In this study, 3-methylene-2-oxindole and derivatives substituted in the 4-, 5-, or 6-position with methyl, F, or Cl have been synthesized and their reactivity toward representative thiol nucleophiles (glutathione, cysteine, and a cysteinyl peptide) measured using stopped-flow kinetic spectrophotometry. Rate constants were in the range approximately 2 x 10(3) to 2 x 10(4) M(-)(1) s(-)(1) at pH 7.4, 25 degrees C, implying a lifetime of a few tens of milliseconds for these methylene oxindoles in the cellular environment and diffusion distances of a few micrometers. As expected, halogen substitution decreased the rate of production of the methylene oxindoles on treatment of horseradish peroxidase. The cytotoxicities of the compounds were measured using Chinese hamster V79 fibroblast-like cells in vitro. The halogen-substituted derivatives were much more cytotoxic than the 5-methyl analogue or the parent (unsubstituted) compound, consistent with the trends in rate constant for reaction with the thiols. The results show that the cytotoxic response in the prodrug (indole acetic acid) and enzyme (horseradish peroxidase) system reflects the reactivity of methylene oxindoles toward nucleophiles much more than the rate of generation of the oxindoles, and helps explain the possible advantages of 5-fluoroindole-3-acetic acid compared to IAA as a lead compound for investigation in targeted cancer therapy.

A series of indolequinones bearing a variety of leaving groups at the (indol-3-yl)methyl position was synthesized by functionalization of the corresponding 3-(hydroxymethyl)indolequinone, and the resulting compounds were evaluated in vitro as bioreductively activated cytotoxins. The elimination of a range of functional groups-carboxylate, phenol, and thiol-was demonstrated upon reductive activation under both chemical and quantitative radiolytic conditions. Only those compounds which eliminated such groups under both sets of conditions exhibited significant hypoxia selectivity, with anoxic:oxic toxicity ratios in the range 10-200. With the exception of the 3-hydroxymethyl derivative, radiolytic generation of semiquinone radicals and HPLC analysis indicated that efficient elimination of the leaving group occurred following one-electron reduction of the parent compound. The active species in leaving group elimination was predominantly the hydroquinone rather than the semiquinone radical. The resulting iminium derivative acted as an alkylating agent and was efficiently trapped by added thiol following chemical reduction and by either water or 2-propanol following radiolytic reduction. A chain reaction in the radical-initiated reduction of these indolequinones (not seen in a simpler benzoquinone) in the presence of a hydrogen donor (2-propanol) was observed. Compounds that were unsubstituted at C-2 were found to be up to 300 times more potent as cytotoxins than their 2-alkyl-substituted analogues in V79-379A cells, but with lower hypoxic cytotoxicity ratios.

The rates of oxidation of reducing substrates by heme peroxidases have previously been thought to be controlled only by their ease of oxidation. In the present study, we have compared the kinetics and thermodynamics of the oxidation of indole-3-acetic acid and derivatives and of phenols by horseradish peroxidase. Different dependencies of the reaction rates on the thermodynamic driving force reveal substrate specificity controlled by the enzyme-substrate complexes dissociation constants (Michaelis-Menten constants) and by the reorganization energies of electron-transfer within those complexes.

Studies have shown that 4-(alkylamino)-5-nitroquinolines possess high selectivity (20-60-fold) for hypoxic tumor cells in vitro, but are not active as hypoxia-selective cytotoxins (HSCs) in vivo. The compounds show inadequate rates of extravascular diffusion, likely due both to sequestration of the bisbasic compounds into lysosomes and rapid nitroreduction. A further series of analogues, designed to counteract these limitations, has been synthesized and evaluated. Analogues bearing one to three electron-donating substituents on the quinoline have one-electron reduction potentials up to 100 mV lower than that of the unsubstituted compound (5), but do not have improved biological activity. The relationship between hypoxic selectivity and rates of metabolic reduction suggests at least two mechanisms of cytotoxicity for this series of 5-nitroquinolines. Compounds with high rates of reduction are toxic via oxygen-sensitive net bioreduction, while compounds which are poor substrates for nitroreduction are toxic through an oxygen-insensitive non-bioreductive mechanism. As rates of metabolic reduction are lowered, the non-bioreductive mechanism of toxicity becomes dominant and hypoxic selectivity is lost. A small series of analogues bearing hydrophilic but neutral side chains were also prepared. Compounds with a dihydroxypropyl side chain retained cytotoxic potency and hypoxic cell selectivity in cell culture assays, and had lowered uptake into lysosomes, but none of three analogues evaluated against KHT tumors in mice showed activity as an HSC in vivo.

The rates of reaction of seven indole-3-acetic acid derivatives with horseradish peroxidase compound 1 at pH 5 were measured by stopped flow, and the reduction potentials and pKa of their radical cations were determined by pulse radiolysis. Reasonable correlation of these properties with Hammett substituent parameters was found, but not with Brown-Okamoto (theta +) parameters. The rates of reaction with compound I correlate well with the reduction potentials under the same conditions, with rates of reaction that increase by ca. 2.5 orders of magnitude with a 100 mV decrease in the reduction potential. This relationship is in agreement with that previously estimated for the reaction of compound I with phenols and anilines, suggesting that the rate of reaction depends solely on the reduction potential of the substrate radical, even for compounds of dissimilar structure.

The oxygen-sensitive bioreductive binding of 2-nitroimidazoles labeled with fluorescent side chains has been used to stain hypoxic mammalian cells selectively. Several novel compounds were synthesized with a 1-substituent containing a fluorescent, bicyclic system having a bridgehead-nitrogen atom. Additional amine and secondary alcohol substituents were also included in the link between the fluorophor and the nitroimidazole to improve water solubility. Their ability to discriminate between hypoxic and oxic cells was compared by flow cytometric analysis. A wide range of cellular fluorescence and hypoxic-oxic differentials in fluorescence was observed when compounds with indolizine fluorophors were incubated with cells, and one such compound was considered suitable for further evaluation in vivo. Two compounds with bimane fluorophors gave very little cellular fluorescence when incubated with hypoxic cells.

Several novel compounds having both a 2-nitroimidazole nucleus and a fluorescent ring system in their molecular structure were prepared and evaluated as potential fluorescent probes for hypoxia. Bioreduction of nitroimidazoles, which is inhibited by oxygen, is known to lead to binding of bioreductive metabolites to cellular macromolecules and this provides a mechanism for binding the fluorescent moiety to hypoxic cells. These compounds can incorporate a wide range of fluorophors and can therefore be designed to suit the laser-line wavelengths available for excitation of fluorescence in the flow cytometer. Several nitroimidazoles with naphthalimide side chains were rapidly taken up into cells and became concentrated in the cells, thus reducing their concentration in the extracellular medium. This suggests a potential microscopic bioavailability problem with probes of this type when used in vivo as they would become progressively depleted in the extracellular fluid as they diffused from blood vessels, through layers of packed cells in tumors, to the hypoxic cells where they could undergo hypoxia-specific metabolism. Synthesis of nitroimidazoles with coumarin fluorophors led to several potentially useful probes for hypoxia; substituents on the coumarin fluorophor had a marked effect on the cellular fluorescence of these compounds.

A series of eight novel nitropyrazines has been prepared by oxidation of sulfoximine intermediates. The partition coefficient, one-electron reduction potential, sensitizer enhancement ratio, and chronic and acute aerobic cytotoxicity have been measured for each. Two representatives of this series were tested in the Ames test and were not found to be mutagenic.

The semiquinones, Q.-, of derivatives of 2- and 6-methyl-1,4-naphthoquinones, some incorporating leaving groups with substituents such as CH2Br or CH2OCONHCH3, have been produced by radiolytic reduction of Q by (CH3)2COH radicals. The absorption spectra and decay kinetics of Q.- were all closely similar to that produced from 2-methyl-1,4-naphthoquinone, with no evidence for unimolecular elimination of a leaving group in the semiquinone form, but immediate loss of leaving group upon two-electron reduction of Q to the hydroquinone. The redox equilibria between Q/Q.- and O2/O2.- were characterized, and reduction potentials of the couples Q/Q.- in water at pH 7.6 were calculated. The implications of these observations for the use of these compounds as bioreductive alkylating agents or as radiosensitizers with potential selective activity toward hypoxic cells are discussed.