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Alkyl N-acyloxybenzohydroxamates are relatively stable compounds which are readily synthesised from inexpensive starting materials and provide a convenient source of alkoxy stabilised nitrenium ions under acidic conditions.
In aqueous acetonitrile, butyl N-acetoxybenzohydroxamate 100a decomposes via an AAl1 mechanism to a nitrenium ion and follows pseudo-unimolecular kinetics. The progress of the reaction was monitored by 1H NMR across the temperature range 298-338K. The solvolysis was repeated with a number of electron-donating and withdrawing para substituents and the Arrhenius data was obtained which determined that nitrenium ion formation proceeds with positive DS and modest activation energies. The Hammett correlation revealed an excellent s+ relationship at 308K indicating that the para substituents had some degree of interaction with a developing positive charge tempered by the intermediate carbonyl moiety. The moderate slope reflected this separation between the ring and nitrenium ion.
The extent to which electronic factors on the alkoxy side chain influence the formation of nitrenium ion during acid-catalysed solvolysis was measured by synthesis of a series of para-substituted benzyl N-acetoxybenzohydroxamates 151. Hammett and Arrhenius relationships as well as isotope studies have been presented that indicate that nitrenium ion formation is modified by electronic effects exerted by the benzyloxy side chain and in cases where positive mesomerism is possible, elimination to give para-substituted benzyl cations is found.
The formation of the nitrenium ion was also investigated as a function of the differing electronic effects on the leaving group by synthesis of a series of benzyl N-(para-substituted benzoyloxy) benzohydroxamates 172. Under acid-catalysis, increased electronegativity of the para substituent lowered the energy required for separation of the leaving group from the precursor and favoured nitrenium ion formation.
Analysis of the acid-catalysed solvolysis products revealed a number of complex solvolysis pathways. Across the three series, most products resulted from acid-catalysed decomposition of the transient intermediate N-alkoxybenzohydroxamic acids formed by water capture of N-acyl-N-alkoxynitrenium ions or through uncatalysed HERON reactions which afford esters.
Alkyl N-acetoxybenzohydroxamate 100 also react with hydroxide ion in a rare BAl2 fashion. Attack at nitrogen was confirmed by measuring the rate of nitrenium ion formation by altering the electronic effects on the leaving group. The rate of reaction of benzyl N-(para-substituted benzoyloxy) benzohydroxamates 172 with hydroxide was determined at 275.4K by HPLC analysis. Hammett data are in accord with a BAl2 reaction mechanism rather than the normal BAc2 solvolysis. Under basic conditions the products were esters formed by HERON rearrangement of N-alkoxy benzohydroxamate anion 177.
The mutagenicity of alkyl N-acyloxybenzohydroxamates has been measured by the Ames test using TA100 salmonella typhimurium bacteria. While no conclusive relationship between electronic effects and mutagenicity levels was detected for the benzoyl 100 and benzyloxy series 151, in the benzoyloxy series 172, a correlation with stability appears likely. In addition, an increase in the potency of the precursor was evident with an increase in the number of aromatic rings flanking the central nitrogen, or where biphenyl rather than phenyl groups were present. The significantly higher mutagenicity levels may be due to more efficient transportation across cellular membranes or, more likely, enhanced hydrophobic associations or intercalation with DNA.
These studies have indicated that while nitrenium ion formation is the likely course of reaction where acid-catalysis is possible, the mutagens can be expected to react readily with nucleophilic species. Recent evidence has indicated that they interact with N-7 of guanine, the most nucleophilic centre in DNA. Thus alkyl N-acyloxybenzohydroxamates are either source of the electrophilic nitrenium ions or may behave as electrophilic molecules. Interaction with DNA is mandatory for mutagenesis and results contained in this thesis indicate that activity may be enhanced where mutagens are least reactivate and can survive the environment long enough to allow them encounter nucleic acids. Furthermore hydrophobic substructure may in fact facilitate the association with DNA.
Studies in these laboratories now centre upon utilising the chemical and mutagenic results described herein to design anti-cancer agents based upon the alkyl N-acyloxybenzohydroxamate structure. To that end, substrates bearing DNA intercalators tethered to low reactivity mutagenic centres are being designed as part of a drug development program.