Pharmacological and biological evaluation of a series of substituted 1,4-naphthoquinone bioreductive drugs Roger M. Phillips a, * , Mohammed Jaffar b , Derek J. Maitland c , Paul M. Loadman a , Steven D. Shnyder c , Gillian Steans c , Patricia A. Cooper c , Amanda Race c , Adam V. Patterson b , Ian J. Stratford b a Cancer Research Unit, Tom Connors Cancer Research Centre, University of Bradford, Bradford BD71DP, UK b School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, UK c School of Pharmacy, University of Bradford, Bradford BD71DP, UK Received 14 April 2004; accepted 3 August 2004 Abstract The indolequinone compound EO9 has good pharmacodynamic properties in terms of bioreductive activation and selectivity for either NAD(P)H:quinone oxidoreductase-1 (NQO1)-rich aerobic or NQO1-deficient hypoxic cells. However, its pharmacokinetic properties are poor and this fact is believed to be a major reason for EO9’s lack of clinical efficacy. The purpose of this study was to develop quinone-based bioreductive drugs that retained EO9’s good properties, in terms of bioreductive activation, but have improved pharmacokinetic properties. Out of 11 naphthoquinone compounds evaluated, 2-aziridinyl-5-hydroxy-1,4-naphthoquinone (com- pound 2), 2,3-bis(aziridinyl)-5-hydroxy-1,4-naphthoquinone (compound 3), and 2-aziridinyl-6-hydroxymethyl-1,4-naphthoquinone (compound 11) were selected for further evaluation based on good substrate specificity for NQO1 and selectivity towards NQO1-rich cells in vitro. Compound 3 was of particular interest as it also demonstrated selectivity for NQO1-rich cells under hypoxic conditions. Compound 3 was not metabolised by murine whole blood in vitro (in contrast to compounds 2, 11 and EO9) and pharmacokinetic studies in non-tumour-bearing mice in vivo (at the maximum soluble dose of 60 mg kg À1 administered intraperitoneally) demon- strated significant improvements in plasma half-life (16.2 min) and AUC values (22.5 mM h) compared to EO9 (T 1/2 = 1.8 min, AUC = 0.184 mM h). Compound 3 also demonstrated significant anti-tumour activity against H460 and HCT-116 human tumour xenografts in vivo, whereas EO9 was inactive against these tumours. In conclusion, compound 3 is a promising lead compound that may target both aerobic and hypoxic fractions of NQO1-rich tumours and further studies to elucidate its mechanism of action and improve solubility are warranted. # 2004 Elsevier Inc. All rights reserved. Keywords: NQO1; Hypoxia; Bioreductive drugs; Naphthoquinones 1. Introduction The indolequinone compound EO9 (3-hydroxy-5-azir- idinyl-1-methyl-2[indole-4,7-dione]-prop-b-en-a-ol) is a bioreductive drug that was selected for clinical evaluation in the early 1990s on the basis of a novel mechanism of action and promising preclinical activity [1,2]. The enzyme NAD(P)H:Quinone oxidoreductase-1 (NQO1, EC 1.6.99.2) plays a central role in bioreductively activat- ing EO9 to DNA-damaging species [3,4] and good corre- lations between NQO1 activity and chemosensitivity in vitro under aerobic conditions have been reported [5–8]. EO9 is also selectively toxic to hypoxic cells, although good hypoxic cytotoxicity ratios (HCR) are only obtained in cells that have low NQO1 activity [5,9,10]. Despite evidence of activity (albeit modest) against a range of solid tumour models in vivo [1], EO9 failed to show activity in phase II clinical trials [11,12]. Several possible explana- tions for EO9’s lack of clinical efficacy have been sug- gested [13], although the major causative factor is likely to be poor drug delivery to tumours as a result of rapid pharmacokinetic elimination and poor penetration through www.elsevier.com/locate/biochempharm Biochemical Pharmacology 68 (2004) 2107–2116 Abbreviations: DMSO, dimethylsulphoxide; NQO1, NAD(P)H:Qui- none oxidoreductase 1; P450R, NADPH cytochrome P450 reductase; NSCLC, non small cell lung cancer; MTD, maximum tolerated dose; MMC, mitomycin C * Corresponding author. Tel.: +44 1274 233226; fax: +44 1274 233234. E-mail address: r.m.phillips@bradford.ac.uk (R.M. Phillips). 0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2004.08.007