Spin Trapping Hydroxyl and Aryl Radicals of One-Electron Reduced Anticancer Benzotriazine 1,4-Dioxides.

Wen Qi,Michael P Hay,Cho R Hong,Robert F Anderson,Pooja Yadav,Ralph J Stevenson

doi:10.3390/molecules27030812

Abstract

Hypoxia in tumors results in resistance to both chemotherapy and radiotherapy treatments but affords an environment in which hypoxia-activated prodrugs (HAP) are activated upon bioreduction to release targeted cytotoxins. The benzotriazine 1,4-di-N-oxide (BTO) HAP, tirapazamine (TPZ, 1), has undergone extensive clinical evaluation in combination with radiotherapy to assist in the killing of hypoxic tumor cells. Although compound 1 did not gain approval for clinical use, it has spurred on the development of other BTOs, such as the 3-alkyl analogue, SN30000, 2. There is general agreement that the cytotoxin(s) from BTOs arise from the one-electron reduced form of the compounds. Identifying the cytotoxic radicals, and whether they play a role in the selective killing of hypoxic tumor cells, is important for continued development of the BTO class of anticancer prodrugs. In this study, nitrone spin-traps, combined with electron spin resonance, give evidence for the formation of aryl radicals from compounds 1, 2 and 3-phenyl analogues, compounds 3 and 4, which form carbon C-centered radicals. In addition, high concentrations of DEPMPO (5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide) spin-trap the •OH radical. The combination of spin-traps with high concentrations of DMSO and methanol also give evidence for the involvement of strongly oxidizing radicals. The failure to spin-trap methyl radicals with PBN (N-tert-butylphenylnitrone) on the bioreduction of compound 2, in the presence of DMSO, implies that free •OH radicals are not released from the protonated radical anions of compound 2. The spin-trapping of •OH radicals by high concentrations of DEPMPO, and the radical species arising from DMSO and methanol give both direct and indirect evidence for the scavenging of •OH radicals that are involved in an intramolecular process. Hypoxia-selective cytotoxicity is not related to the formation of aryl radicals from the BTO compounds as they are associated with high aerobic cytotoxicity.

Highlights

IntroductionTirapazamine, TPZ, (3-amino-1,2,4-benzotriazine-1,4 dioxide, 1) (Figure 1A), is the most investigated example of the 1,2,4-benzotriazine 1,4-N-dioxide (BTO) class of bioreductive drugs, which are selectively cytotoxic against hypoxic tumor cells [1,2,3]
We investigate the two possible mechanisms for aryl radical formation in compounds 1, 2 and the water soluble 3-phenyl substituted
The one-electron reduction potential, E0’, of compound 3 was measured at pH 7.0 using pulse radiolysis and found to be ca. 18 mV lower than that of compound 4, Table 1, reflecting the electron-donating property of the 7-OR substituent

Summary

Introduction

Tirapazamine, TPZ, (3-amino-1,2,4-benzotriazine-1,4 dioxide, 1) (Figure 1A), is the most investigated example of the 1,2,4-benzotriazine 1,4-N-dioxide (BTO) class of bioreductive drugs, which are selectively cytotoxic against hypoxic tumor cells [1,2,3]. The mechanism by which BTO compounds are activated to cause oxidative damage to DNA continues to be the subject of considerable research. The hypoxic cytotoxicity ratio (HCR) is defined as the ratio of drug concentrations giving equitoxic effects in an in vitro assay under normoxia and anoxia: HCR = IC50oxic/IC50anoxic. The HCR values for compound 1 typically fall in the range of 50–300 [7,8] depending on cell type, culture conditions and drug exposure, and compound 2 was shown to be consistently more selective across a panel of cell lines [9]

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Results

Conclusion