Oxidation of
indole-3-acetic acid and its derivatives by
peroxidases such as that from horseradish produces many products, including 3-methylene-2-oxindoles. These have long been associated with
biological activity, but their reactivity has not been characterized. We have previously demonstrated the potential value of substituted
indole acetic acids and
horseradish peroxidase as the basis for targeted
cancer therapy, since the compounds are of low cytotoxicity until oxidized, when high cytotoxicity is observed; the combination of
prodrug and
enzyme depletes intracellular
thiols. In this study, 3-methylene-2-oxindole and derivatives substituted in the 4-, 5-, or 6-position with methyl, F, or Cl have been synthesized and their reactivity toward representative
thiol nucleophiles (
glutathione,
cysteine, and a cysteinyl
peptide) measured using stopped-flow kinetic spectrophotometry. Rate constants were in the range approximately 2 x 10(3) to 2 x 10(4) M(-)(1) s(-)(1) at pH 7.4, 25 degrees C, implying a lifetime of a few
tens of milliseconds for these methylene
oxindoles in the cellular environment and diffusion distances of a few micrometers. As expected,
halogen substitution decreased the rate of production of the methylene
oxindoles on treatment of
horseradish peroxidase. The cytotoxicities of the compounds were measured using Chinese hamster V79 fibroblast-like cells in vitro. The
halogen-substituted derivatives were much more cytotoxic than the 5-methyl analogue or the parent (unsubstituted) compound, consistent with the trends in rate constant for reaction with the
thiols. The results show that the cytotoxic response in the
prodrug (
indole acetic acid) and
enzyme (
horseradish peroxidase) system reflects the reactivity of methylene
oxindoles toward nucleophiles much more than the rate of generation of the
oxindoles, and helps explain the possible advantages of
5-fluoroindole-3-acetic acid compared to IAA as a lead compound for investigation in targeted
cancer therapy.