Anuruddha Rajapakse

Generation of DNA-damaging reactive oxygen species via the autoxidation of hydrogen sulfide under physiologically relevant conditions: chemistry relevant to both the genotoxic and cell signaling properties of H(2)S.

Hydrogen sulfide (H(2)S) has long been known for its toxic properties; however, in recent years, evidence has emerged that this small, gaseous molecule may serve as an endogenous cell-signaling agent. Though perhaps surprising in light of its potential role as an endogenous signaling agent, a number of studies have provided evidence that H(2)S is a DNA-damaging mutagen. In the work reported here, the chemical mechanisms of DNA damage by H(2)S were examined. Using a plasmid-based DNA strand cleavage assay, we found that micromolar concentrations of H(2)S generated single-strand DNA cleavage. Mechanistic studies indicate that this process involved autoxidation of H(2)S to generate superoxide, hydrogen peroxide, and, ultimately, the well-known DNA-damaging agent hydroxyl radical via a trace metal-mediated Fenton-type reaction. Strand cleavage by H(2)S proceeded in the presence of physiological thiol concentrations, and the known byproducts of H(2)S oxidation such as thiosulfate, sulfite, and sulfate do not contribute to the strand cleavage process. However, initially generated oxidation products such as persulfide (S(2)(2-)) likely undergo rapid autoxidation reactions that contribute to the generation of superoxide. The potential relevance of autoxidation processes to the genotoxic and cell signaling properties of H(2)S is discussed.

Hypoxia-Selective, Enzymatic Conversion of 6-Nitroquinoline into a Fluorescent Helicene: Pyrido(3,2-f)quinolino(6,5-c)cinnoline 3-Oxide

Regions of low oxygen concentration (hypoxia) occur in both normal human physiology and under pathophysiological conditions. Fluorescent probes for the direct imaging of cellular hypoxia could be useful tools that complement radiochemical imaging and immunohistochemical staining methods. In this work, we set out to characterize the hypoxia-selective enzymatic metabolism of a simple nitroaryl probe, 6-nitroquinoline (1). We envisioned that this compound might undergo hypoxia-selective, bioreductive conversion to the fluorescent product, 6-aminoquinoline (2). The probe 1 was, indeed, converted to a fluorescent product selectively under hypoxic conditions by the one-electron reducing enzyme NADPH:cytochrome P450 reductase. However, inspection of the fluorescence spectrum and LC-MS analysis of the reaction mixture revealed that the expected product 2 was not formed. Rather, the 63-fold increase in fluorescence emission at 445 nm resulting from the hypoxic metabolism of 1 was due to formation of the azoxy-helicene product, pyrido[3,2-f]quinolino[6,5-c]cinnoline 3-oxide (4). The generation of 4 involves an unusual biaryl bond formation under reductive conditions. The mechanism of this process remains uncertain but could proceed via combination of a nitroaryl radical anion with a neutral nitrosoaryl radical, followed by tautomerization and intramolecular condensation between the resulting hydroxylamine and nitroso functional groups. Bioreductive metabolism of nitroaryl compounds represents a promising strategy for the selective delivery of cytotoxic agents and fluorescent markers to hypoxic tissue, but the results described here provide an important glimpse of the chemical complexity that can be associated with the enzymatic one-electron reduction of nitroaryl compounds.

DNA strand cleaving properties and hypoxia-selective cytotoxicity of 7-chloro-2-thienylcarbonyl-3-trifluoromethylquinoxaline 1,4-dioxide

The heterocyclic N-oxide, 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine, 1), shows promising antitumor activity in preclinical studies, but there is a continuing need to explore new compounds in this general structural category. In the work described here, we examined the properties of 7-chloro-2-thienylcarbonyl-3-trifluoromethylquinoxaline 1,4-dioxide (9h). We find that 9h causes redox-activated, hypoxia-selective DNA cleavage that mirrors the lead compound, tirapazamine, in both mechanism and potency. Furthermore, we find that 9h displays hypoxia-selective cytotoxicity against human cancer cell lines.

Enzymatic conversion of 6-nitroquinoline to the fluorophore 6-aminoquinoline selectively under hypoxic conditions.

There is substantial interest in small molecules that can be used to detect or kill the hypoxic (low oxygen) cells found in solid tumors. Nitroaryl moieties are useful components in the design of hypoxia-selective imaging agents and prodrugs because one-electron reductases can convert the nitroaryl group to nitroso, hydroxylamino, and amino metabolites selectively under low oxygen conditions. Here, we describe the in vitro, cell free metabolism of a pro-fluorescent substrate, 6-nitroquinoline (1) under both aerobic and hypoxic conditions. Both LC-MS and fluorescence spectroscopic analyses provided evidence that the one-electron reducing enzyme system, xanthine/xanthine oxidase, converted the nonfluorescent parent compound 1 to the known fluorophore 6-aminoquinoline (2) selectively under hypoxic conditions. The presumed intermediate in this reduction process, 6-hydroxylaminoquinoline (6), is fluorescent and can be efficiently converted by xanthine/xanthine oxidase to 2 only under hypoxic conditions. This finding provides evidence for multiple oxygen-sensitive steps in the enzymatic conversion of nitroaryl compounds to the corresponding amino derivatives. In a side reaction that is separate from the bioreductive metabolism of 1, xanthine oxidase converted 1 to 6-nitroquinolin-2(1H)-one (5). These studies may enable the use of 1 as a fluorescent substrate for the detection and profiling of one-electron reductases in cell culture or biopsy samples. In addition, the compound may find use as a fluorogenic probe for the detection of hypoxia in tumor models. The occurrence of side products such as 5 in the enzymatic bioreduction of 1 underscores the importance of metabolite identification in the characterization of hypoxia-selective probes and drugs that employ nitroaryl units as oxygen sensors.

Isotopic Labeling Experiments That Elucidate the Mechanism of DNA Strand Cleavage by the Hypoxia-Selective Antitumor Agent 1,2,4-Benzotriazine 1,4-Di-N-oxide

The 1,2,4-benzotriazine 1,4-dioxides are an important class of potential anticancer drugs that selectively kill the low-oxygen (hypoxic) cells found in solid tumors. These compounds undergo intracellular one-electron enzymatic reduction to yield an oxygen-sensitive drug radical intermediate that partitions forward, under hypoxic conditions, to generate a highly reactive secondary radical that causes cell killing DNA damage. Here, we characterized bioreductively activated, hypoxia-selective DNA-strand cleavage by 1,2,4-benzotriazine 1,4-dioxide. We found that one-electron enzymatic activation of 1,2,4-benzotriazine 1,4-dioxide under hypoxic conditions in the presence of the deuterium atom donor methanol-d4 produced nondeuterated mono-N-oxide metabolites. This and the results of other isotopic labeling studies provided evidence against the generation of atom-abstracting drug radical intermediates and are consistent with a DNA-damage mechanism involving the release of hydroxyl radical from enzymatically activated 1,2,4-benzotriazine 1,4-dioxides.

Application of Suzuki–Miyaura and Buchwald–Hartwig Cross‐coupling Reactions to the Preparation of Substituted 1,2,4‐Benzotriazine 1‐Oxides Related to the Antitumor Agent Tirapazamine

Many 1,2,4-benzotriazine 1,4-dioxides display the ability to selectively kill the oxygen-poor cells found in solid tumors. As a result, there is a desire for synthetic routes that afford access to substituted 1,2,4-benzotriazine 1-oxides that can be used as direct precursors in the synthesis of 1,2,4-benzotriazine 1,4-dioxides. Here we describe the use of Suzuki-Miyaura and Buchwald-Hartwig cross-coupling reactions for the construction of various 1,2,4-benzotriazine 1-oxide analogs bearing substituents at the 3-, 6-, and 7-positions.

Crystal structure of N-(quinolin-6-yl)hydroxyl-amine.

The title compound crystallized with four independent molecules in the asymmetric unit. They are linked via two N—H⋯O and one O—H⋯N hydrogen bond, forming a tetramer-like unit.

Abstract 4558: 6-Nitroquinoline as a metabolically-activated probe for direct fluorescence detection of hypoxia

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Hypoxic (oxygen poor) cells in tumors play an important role in cancer biology. For example, the hypoxic environment may select for cells that are incapable of undergoing apoptosis. In addition, there is recent speculation that cancer stem cells that may be responsible for metastases are harbored in the hypoxic niche of tumors. Indeed, tumor hypoxia correlates with poor patient prognosis. Accordingly, detection of tumor hypoxia may soon become an important part of the clinical characterization of human cancers. Fluorescent probes for the direct detection of hypoxia in biological systems could provide a useful complement to the radiochemical imaging and immunohistochemical staining methods that are commonly used in this field. Building on our longstanding interest in metabolically-activated, hypoxia-selective antitumor agents, we set out to develop a probe that would be metabolized under hypoxic conditions into a product with known utility as a fluorescent reporter. We selected the molecule 6-nitroquinoline as a forum to explore the fundament chemistry, biochemistry and biology underlying the development of hypoxia-selective, bioreductively-activated fluorophores. We expected that hypoxia-selective reduction of the nitro group in this probe would generate the fluorophore 6-aminoquinoline. First, we confirmed that 6-nitroquinoline is not fluorescent. We next examined whether enzymatic metabolism converted this probe to a fluorescent product. The xanthine/xanthine oxidase enzyme system was employed for the one-electron reductive activation of the probe. We find that incubation of the probe with xanthine oxidase in the presence of a stoichiometric excess (5 equiv) of the substrate xanthine generates a substantial (approximately 30 fold) increase in fluorescence at 530 nm, as expected for generation of the 6-aminoquinoline product. Mass spectrometric analysis confirms that 6-aminoquinoline is generated under these conditions. In contrast, only a small increase in fluorescence at 530 nm is observed under aerobic conditions. Experiments that varied the amount of xanthine substrate used to “fuel” the reduction reaction revealed the presence of intermediate fluorescent products that generate an emission maximum at about 430 nm. This is likely due to 6-hydroxylamino and dimerized azoxy metabolites. The enzyme NADPH: cytochrome P450 reductase similarly converts 6-nitroquinoline to fluorescent products in a hypoxia-selective manner. In conclusion, we have established that 6-nitroquinoline can serve as a direct-light-up fluorescent probe for the detection of hypoxia and we have characterized the mechanisms by which this agent is converted to fluorescent products. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4558.