COMPARE analysis of the toxicity of an iminoquinone derivative of the imidazo[5,4-f]benzimidazoles with NAD(P)H:quinone oxidoreductase 1 (NQO1) activity and computational docking of quinones as NQO1 substrates

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Abstract

Synthesis and cytotoxicity of imidazo[5,4-f]benzimidazolequinones and iminoquinone derivatives is described, enabling structure–activity relationships to be obtained. The most promising compound (an iminoquinone derivative) has undergone National Cancer Institute (NCI) 60 cell line (single and five dose) screening, and using the NCI COMPARE program, has shown correlation to NQO1 activity and to other NQO1 substrates. Common structural features suggest that the iminoquinone moiety is significant with regard to NQO1 specificity. Computational docking into the active site of NQO1 was performed, and the first comprehensive mitomycin C (MMC)-NQO1 docking study is presented. Small distances for hydride reduction and high binding affinities are characteristic of MMC and of iminoquinones showing correlations with NQO1 via COMPARE analysis. Docking also indicated that the presence of a substituent capable of hydrogen bonding to the His194 residue is important in influencing the orientation of the substrate in the NQO1 active site, leading to more efficient reduction.

Introduction

NAD(P)H:quinone oxidoreductase 1 (NQO1 and also known as DT-diaphorase) is an enzyme which possesses a flavin prosthetic group, and uses NADH or NADPH coenzymes with equal efficiency.1, 2 NQO1 is responsible for obligatory two-electron reduction of quinones to hydroquinones, as well as the reduction of many other substrates including iminoquinones, nitro and azo-compounds.3 It is often described as a detoxification enzyme since it can override one-electron reductions that generate damaging oxygen radicals by redox recycling,4 and has been linked with superoxide scavenging (O2radical dot).5 Conversely, NQO1 bioreduction can lead to activation of prodrugs, and can induce a cytotoxic or cytostatic effect. Although NQO1 is expressed in normal tissues, elevated levels are known to occur in many human tumor cell lines.6, 7, 8 The cytotoxicity of well-known anti-tumor agents, mitomycin C (MMC) and EO9, show correlation to the levels of NQO1 in the cell lines used at the National Cancer Institute (NCI-60 tumor cell line panel).6

Imidazobenzimidazolequinones exist in two heterocyclic arrangements: imidazo[4,5-f]benzimidazolequinones and imidazo[5,4-f]benzimidazolequinones. Examples of the former were first reported by Schulz and Skibo. Dipyrrolo ring-fused compound 1a was shown to be an excellent substrate for rat liver NQO1, with high specificity towards melanoma cell lines (Fig. 1).9 In a subsequent article, molecular modeling studies showed that dipyrido ring-fused analog 1b was capable of forming stable substrate complexes with human NQO1.10 More recently, Fagan et al., reported the synthesis and cytotoxicity evaluation of imidazo[5,4-f]benzimidazolequinones, containing alicyclic and [1,4]oxazino fused rings (Fig. 2).11, 12 Cytotoxicity was assessed using two human cancer cell lines reported to possess high NQO1 activity; cervical (HeLa),7 and prostate (DU-145)6 cancer cells, and a human normal fibroblast cell line (GM00637).

The dipyridoimidazo[5,4-f]benzimidazolequinone 2b was marginally more potent towards the two human cancer cell lines than its isomeric [4,5-f] analog 1b (Table 1). Iminoquinone 2a was the most potent imidazobenzimidazole evaluated towards the prostate cancer DU-145 cell line, being about 12 times more toxic towards this cell line than towards the normal cell line.11 The DU-145 cell line is one of the 60 cell lines in the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI).13 One quinone and two iminoquinones, including compound 2a, were submitted for this more detailed in vitro cytotoxicity analysis by the DTP NCI-60 screen, and we now present this data along with the results of COMPARE analysis13 revealing interesting structure–activity relationships (SARs) with regard to specificity towards NQO1. We also establish SARs for the toxicity of fused alicyclic and nonfused compounds, including the synthesis and evaluation of novel quinones 3a, 3c, 4 and 5. The first comprehensive computational docking study of the clinical drug mitomycin C (MMC) into the human NQO1 active site is provided, along with that of the imidazobenzimidazoles, in order to determine the substrate structural requirements for efficient NQO1 reduction, and to assess the relationship between cytotoxicity and protein–ligand interactions.

Section snippets

Synthesis

Dipyrrolo- and diazepino-fused quinones 3a and 3c were prepared from previously reported dipyrrolo- and diazepino-imidazo[5,4-f]benzimidazoles, respectively,11 according to Scheme 1. Nitro compounds 6 and 7 were isolated in high yields (85% and 92%) by treatment with concentrated nitric and sulfuric acid. Nitro compounds 6 and 7 were catalytically hydrogenated, and Frémy oxidation of the resultant amine intermediates performed under acidic conditions (pH 4), gave mixtures of iminoquinone

Conclusions

Cytotoxicity evaluations have shown that the expansion of the fused alicyclic rings in imidazo[5,4-f]benzimidazolequinones and iminoquinones decreases potency, while potency is increased by replacing the fused pyrido rings by n-butyl substituents. The DTP NCI-60 cell line screening results of dipyridoiminoquinone 2a supported the exceptionally high cytotoxicity towards the prostate DU-145 cancer cell line, previously reported using the MTT assay.11 DTP NCI-60 screening also revealed high

General

All materials were obtained commercially, and the synthesis of 2,3,8,9-tetrahydro-1H,7H-pyrrolo[1,2-a]pyrrolo[1′,2′:1,2]imidazo[5,4-f]benzimidazole and 2,3,4,5,10,11,12,13-octahydro-1H,9H-azepino[1,2-a]azepino[1′,2′:1,2]imidazo[5,4-f]benzimidazole has been reported using a one-pot double radical cyclization protocol.11 Thin layer chromatography (TLC) was carried out on aluminium-backed plates coated with silica gel (Merck Kieselgel 60 F254). Flash chromatography and dry column vacuum

Acknowledgements

We thank the Irish Research Council for Science Engineering and Technology funded by the National Development Plan for awarding an Embark Scholar Award to Vincent Fagan. We also gratefully acknowledge the financial support from National University of Ireland Galway and Science Foundation Ireland for a Research Frontiers Programme Award (07/RFP/CHEF227). We are grateful to Professor William Watson (School of Medicine & Medical Sciences, University College Dublin, Ireland) for the DU-145 cell

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