Synthesis of novel paclitaxel prodrugs designed for bioreductive activation in hypoxic tumour tissue

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Abstract

The syntheses and preliminary evaluation of the first potential bioreductive paclitaxel prodrugs are described. These prodrugs were designed as potential candidates in more selective chemotherapy by targeting hypoxic tumour tissue. Aromatic nitro and azide groups were used as the bioreductive trigger. Generation of paclitaxel occurs after reduction and subsequent 1,6-elimination or 1,8-elimination. All prodrugs are stable in buffer and indeed give paclitaxel after chemical reduction of the aromatic nitro or azide functionality. In aerobic cytotoxicity assays several prodrugs exhibit diminished cytotoxicity. These compounds are interesting candidates for further biological evaluation.

Introduction

Many solid tumours are inefficient in the development of their own blood supply, resulting in hypoxic regions within the tumour cell population.1 In recent years, a number of low toxic (pro)drugs have been developed, capable of selective activation in hypoxic tissue.2 Many of these compounds use the reduction of an aromatic nitro compound as a bioreductive switch to generate the active species.3 Among these, analogues of nitroimidazoles,4 N-oxides5 and nitrobenzyl carbamates6 have been used as reductive trigger. We now report the first synthesis of bioreductive prodrugs of paclitaxel Scheme 1, Scheme 2, Scheme 3, employing the aromatic nitro and azido group as the bioreductive trigger. Paclitaxel is one of the most promising anti-cancer agents used in the clinic today. However, also paclitaxel exhibits dose limiting side effects, a common problem with cytotoxic agents. Prodrug strategies can reduce these side effects by selective release of the active drug in tumour tissue. Because of the potency of the parent drug, paclitaxel prodrugs can be very powerful chemotherapeutic agents. Low toxicity of paclitaxel prodrugs can be achieved by blocking the C2′–OH group, which is important for activity.7 The paclitaxel prodrugs are designed to release paclitaxel after reduction of the nitro group to a hydroxylamine or amine group and subsequent 1,6-elimination of a 4-hydroxylamino or 4-amino benzyloxycarbonyl moiety, a concept that has been used successfully in prodrug chemistry.8

Section snippets

Synthesis

In a proof of principal study, prodrug 1a was prepared via reaction of paclitaxel with 4-nitrobenzyl chloroformate (PNBC). The compound was stable in buffer solution (pH=7.4, 37 °C) and upon chemical reduction of the nitro group formation of paclitaxel was indeed observed. Next, a series of prodrugs was prepared with additional electron withdrawing substituents on the aromatic ring and with nitro heteroaromatic moieties (which both increase the reduction potential of the nitro group). Finally

Biological Activity

All prodrugs proved to be stable in Tris–buffer (pH=7.4, 37 °C) for at least 24 h. Cytotoxicity assays against seven well defined human tumour cell lines (Table 1) showed that prodrugs 1ab and 1di possess only a slightly reduced cytotoxicity. This is probably due to enzymatic hydrolysis of the benzyl carbonate by esterases present in the assay. This results in nonspecific release of paclitaxel. Lack of stability is a major problem for many paclitaxel prodrugs.15

Prodrug 1c displayed a

Conclusions

In conclusion, we have prepared the first bioreductive paclitaxel prodrugs. All prodrugs were stable in buffer and released paclitaxel after reduction. Unfortunately, benzyl carbonate prodrugs 1ab and 1di did not show a much reduced cytotoxicity under aerobic conditions. Prodrugs 1c and 1k did display a lower cytotoxicity and these are selected for further evaluation in anaerobic environment.

Experimental

1H NMR spectra were recorded on a Bruker AM-300 (300 MHz) or on a Bruker AC100 (100 MHz) spectrometer. Chemical shift values are given in ppm (δ) relative to TMS as internal standard. For numbering of the atoms in paclitaxel, see Scheme 1. Mass spectra were obtained with VG Micromass 7070E spectrometer. Elemental analyses were carried out in triplicate on a Carlo Erba EA 1108 element analyzer. Melting points were measured on a Reichert Thermopan microscope and are uncorrected. TLC analysis was

Acknowledgements

We gratefully acknowledge the financial support from Pharmachemie B.V. Haarlem and from The Academy of Finland.

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