New quinoxalinecarbonitrile 1,4-di-N-oxide derivatives as hypoxic-cytotoxic agents

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Abstract

We report the synthesis and biological in vitro activities of 16 new 2-quinoxalinecarbonitrile 1,4-di-N-oxides. These compounds present new basic lateral chains (piperazines and anilines) in the 3 position as well as different substituents in the 6 and/or 7 positions of the quinoxaline ring. Among piperazine derivatives, 4b (a 7-chloro-3-(4-methylpiperazin-1-yl) derivative) was the most potent (P = 0.5 ×10–6 M). In general, aniline derivatives were more potent and selective than the former, compound 12b (with a 4-(methylphenyl)amino moiety in the 3 position and a chlorine atom in the 7 position) being the best one (P = 3 × 10–6 M and HCR > 16).

Introduction

It has been proposed that the hypoxic cells in solid tumours play a negative role in the success of some human antitumour therapies because of their resistance to radiotherapy and conventional chemotherapeutic agents. The presence of hypoxic cells in solid tumours has been demonstrated both in clinical human tumours and human tumour xenografts in rodents by several techniques [1], [2], [3]. The hypoxic cellular population resistant to chemotherapy can firstly be explained because the location of these cells in poorly vascularized regions of the tumour renders the access of drugs difficult [4]. Also, many antineoplastic agents have a cytotoxicity dependent on oxygen [5]. Moreover, the hypoxic cells do not follow a normal cellular cycle and, therefore, a low response to cell cycle-specific agents can be expected. On the other hand, the microenvironment surrounding these hypoxic regions (low oxygen pressure, pH and nutrients) may favour the appearance of drug resistance through mutagenic mechanisms [6].

In 1972, Sartorelli et al. introduced the concept of bioreductive alkylation: the hypoxic cells in solid tumours, which are living in an environment more inclined towards reductive reactions than those which are well oxygenated, could transform some drugs into cytotoxic species capable of alkylating DNA. These prodrugs would be activated through a reductive mechanism in the absence of oxygen and, for this reason, be more toxic for hypoxic cells than for well-oxygenated ones [7]. Since then, a great number of hypoxic cell cytotoxins have been designed in order to take a therapeutic advantage of the hypoxia.

It has been well established that these selective cytotoxins for hypoxic cells, the so called bioreductive agents, undergo reductive activation by enzyme-catalysed reactions, which are inhibited or reverted by oxygen. Several reductases can mediate these reactions, including the NADPH microsomal enzymes cytochrome P450 reductase and cytochrome P450, as well as the cytosolic enzymes xanthine-oxidase, aldehyde oxidase and DT-diaphorase [8]. The ultimate cell cytotoxin is usually a DNA alkylating agent or, in the case of aromatic N-oxides, a hydrogen-abstracting radical.

Several kinds of compounds that are activated under hypoxic conditions are at various stages of development: nitroderivatives, including nitroimidazoles [9], 9-alkylamino-1-nitroacridines [10] and nitroquinolines [11], quinone derivatives [12] (the mitomycin C derivative, E09), and agents derived from 1,2,4-benzotriazine-1,4-di-N-oxide [13] and quinoxaline-1,4-di-N-oxide [14]. Taking the benzotriazine-3-amino-1,2,4-benzotriazine-1,4-dioxide (WIN 59075; Tirapazamine) as structural antecedent, our group is involved in the synthesis and biological evaluation of new agents derived from quinoxaline-1,4-di-N-oxide and related compounds that have proved to be efficient cytotoxic agents for hypoxic cells of solid tumours [14], [15], [16], [17]. The structure of WIN 59075 is represented in  figure 1.

In order to advance in the knowledge of structure–activity relationships, we have explored different amino substituents in position 3 of the quinoxaline ring together with electron-donating and electron-withdrawing groups in position 6 and/or 7. In this paper we report the synthesis and biological studies of 16 new compounds derived from 2-quinoxalinecarbonitrile-1,4-di-N-oxides with selective cytotoxic activity under hypoxic conditions. They are presented with their biological data in  table I.

Section snippets

Chemistry

Several ideas have been obtained from previous studies of structure–activity relationships: the cyano moiety in the 2 position seems to be necessary for the cytotoxic activity; the presence of an electron-withdrawing substituent in the 7(6) position, e.g. Cl, CF3 or F, increases the potency under hypoxic conditions of all of the series studied [17] and electron-donating substituents, such as CH3 and OCH3 in the 6 and/or 7 position decreased, in general, the potency but had a good effect on

Pharmacology

The quinoxaline derivatives were tested in a cloning assay using V79 cells. Suspension cultures were established from exponentially growing cells and gassed with pure air or nitrogen for 30 min before dosing with the compounds. Treatment lasted 2 h and gassing was continuous during this time. All of the compounds were tested at 20 μM in duplicate flasks in both air and nitrogen. The compounds that were more toxic in hypoxia than in air were tested at different doses to obtain a dose–response

Results and discussion

With the aim of improving the hypoxia-selective cytotoxicity of previous 2-quinoxalinecarbonitrile-1,4-di-N-oxides, 16 new quinoxaline derivatives have been prepared bearing 9 new amines (which had not been used to date) in the 3 position of the aromatic ring. Among the piperazine derivatives, compound 7e bearing 4-(4-nitrophenyl)piperazin-1-yl, a methyl group and a chlorine atom at the 3, 6 and 7 positions, respectively, showed the best in vitro profile: Potency = 2 × 10–6 M and HCR = 50 (only

Conclusions

With these results and the previously published in vitro profiles from many other quinoxalines [14], [15] we can conclude that these new 2-quinoxalinecarbonitrile-1,4-di-N-oxides are relatively potent but not as selective as they were expected to be. The low selectivity is thought to be due to the presence of aromatic rigid moieties (anilines and arylpiperazines) at the 3 position of the quinoxaline ring. This type of amine is quite different from the aliphatic (N,N-dialkylamino)alkylamino

General

Melting points were determined using a Mettler FP82+FP80 apparatus and are uncorrected. Elemental analyses were obtained from vacuum-dried samples (over phosphorus pentoxide at 3–4 mm Hg, 24 h, at ca. 80– 100 °C). Infrared spectra were recorded on a Perkin-Elmer 681 apparatus, using potassium bromide tablets for the preparation of the samples; the frequencies are expressed in cm–1. The 1H-NMR spectra were obtained on a Brucker AC-200E (200 MHz) instrument, using tetramethylsilane as the

Acknowledgements

The authors are grateful to Gobierno de Navarra for a predoctoral grant for Miguel Ángel Ortega, to I.C.I. (Instituto de Cooperación Iberoamericana) for a predoctoral grant for María Elena Montoya, and to Proyecto X-2 of CYTED (Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo). The excellent technical assistance of Ms. Elena Menéndez and Ms. Marta Ruiz is also gratefully acknowledged.

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