Effects of alkylating metabolites of ifosfamide and its bromo analogues on DNA of HeLa cells

The in vitro cytotoxicity and mechanism of action of three alkylating compounds: an active metabolite of ifosfamide ( 1, isophosphoramide mustard, N, N'-bis(2-chloroethyl)phosphorodiamidic acid) and its bromo substituted analogues, 2 (one chlorine atom replaced by bromine atom) and 3 (two chlorine atoms replaced by bromine atoms), were studied in cultured HeLa cells. Alkaline elution analysis of cellular DNA demonstrated the presence of concentration- and time-dependent interstrand crosslinks, DNA-protein crosslinks and alkali-labile sites (ALSs) in HeLa cells following a 1 hr exposure to the compounds. The bromo analogues were more cytotoxic than 1 and exhibited higher crosslinking potency. The time-course of crosslink formation and removal for the three compounds was similar. ALSs in DNA were produced by all tested drugs but 3 exhibited exceptionally high activity and was able to induce two kinds of alkali-labile lesion (fast- and slow-appearing) whereas 1 and 2 generated only slow-appearing ones. The results suggest that 1 and 2 are more specific in their reaction with DNA in that they produced a lesser variety of lesions than 3. A potential advantage of 2 over 1 seems to be its higher DNA interstrand crosslinking activity.