Antitumor Benzothiazoles. 8.<SUP>1</SUP><BBR RID="jm990104ob00001"> Synthesis, Metabolic Formation, and Biological Properties of the C- and N-Oxidation Products of Antitumor 2-(4-Aminophenyl)benzothiazoles

2-(4-Aminophenyl)benzothiazoles <BO>1 </BO>and their N-acetylated forms have been converted to C- and N-hydroxylated derivatives to investigate the role of metabolic oxidation in the mode of action of this series of compounds. 2-(4-Amino-3-methylphenyl)benzothiazole (<BO>1a</BO>, DF 203, NSC 674495) is a novel and potent antitumor agent with selective growth inhibitory properties against human cancer cell lines. Very low IC<INF>50</INF> values (&lt;0.1 μM) were encountered in the most sensitive breast cancer cell lines, MCF-7 and T-47D, whereas renal cell line TK-10 was weakly inhibited by <BO>1a</BO>. Cell lines from the same tissue origin, MDA-MB-435 (breast), CAKI-1 (renal), and A498 (renal), were insensitive to <BO>1a</BO>. Accumulation and metabolism of <BO>1a</BO> were observed in sensitive cell lines only, with the highest rate of metabolism occurring in the most sensitive MCF-7 and T-47D cells. Thus, differential uptake and metabolism of <BO>1a</BO> by cancer cell lines may underlie its selective profile of anticancer activity. A major metabolite in these sensitive cell lines has been identified as 2-(4-amino-3-methylphenyl)-6-hydroxybenzothiazole (<BO>6c</BO>). Hydroxylation of <BO>1a</BO> was not detected in the homogenate of previously untreated MCF-7, T-47D, and TK-10 cells but was readily observed in homogenates of sensitive cells that were pretreated with <BO>1a</BO>. Accumulation and covalent binding of [¹⁴C]<BO>1a</BO> derived radioactivity was observed in the sensitive MCF-7 cell line but not in the insensitive MDA-MB-435 cell line. The mechanism of growth inhibition by <BO>1a</BO>, which is unknown, may be dependent on the differential metabolism of the drug to an activated form by sensitive cell lines only and its covalent binding to an intracellular protein. However, the 6-hydroxy derivative <BO>6c</BO> is not the ‘active' metabolite since, like all other C- and N-hydroxylated benzothiazoles examined in this study, it is devoid of antitumor properties in vitro.