Substituent effects on the kinetics of reductively-initiated fragmentation of nitrobenzyl carbamates designed as triggers for bioreductive prodrugs

Abstract

4-Nitrobenzyl carbamates are of interest as triggers for bioreductive drugs, particularly in conjunction with the E. coli B nitroreductase, which efficiently reduces them to the corresponding hydroxylamines. These then fragment to release highly toxic amine-based toxins. While many 4-nitrobenzyl carbamate derivatives have been evaluated as bioreductive drugs, there has been no systematic study of substituent effects on the rate of this fragmentation (which should be as fast as possible following reduction). We therefore prepared a series of 2-, 3- and α-substituted 4-[N-methyl-N-(4-nitrobenzyloxycarbonyl)amino]phenylacetamides as model compounds to study these effects. The majority of the carbamates were prepared by in situ formation of the chloroformate of the appropriate 4-nitrobenzyl alcohol and reaction with methyl 4-(methylamino)phenylacetate, followed by ester hydrolysis and 1,1 ′-carbonyldiimidazole (CDI) mediated coupling with N,N-dimethylaminoethylamine. The hydroxylamines were generated by ⁶⁰Co γ-ray irradiation of the nitro compounds in aqueous phosphate-buffered-propan-2-ol. The reactions were analysed by reverse-phase HPLC to determine the maximum half-life (Mt_1/2) of the hydroxylamines generated, and the extent of release of amine from these after 10 half-lives (t_∞). The parent (unsubstituted) hydroxylaminobenzyl carbamate had a Mt_1/2 of 16 min under these conditions, while that of the corresponding α-methyl analogue was 9.5 min. Electron-donating substituents on the benzyl ring also accelerated fragmentation, with the data being fitted to the equation log(Mt_1/2) = 0.57σ + 1.30, where σ represents σ_pfor 2-substituents and σ_m for 3-substituents. The acceleration of fragmentation of the hydroxylamines with increasing substituent electron-donation is consistent with the proposed mechanism, and is presumably due to stabilisation of the developing positive charge on the benzylic carbon. The extent of release of amine (t_∞) also increased with increasing substituent electron-donation. These data suggest that the standard 4-nitrobenzyl carbamate trigger for nitroreductase enzyme (NTR) prodrugs can likely be improved on, by increasing the rate of fragmentation by the use of α-methyl and/or electron-donating benzyl substituents.

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