The versatility of 4-(hydroxymethyl)-2(5H)-furanone as a starting point for the synthesis of several bromine and mixed halogen analogues of the potent water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) has been demonstrated. However, in some preparations the yields of desired products were lower for bromine- than chlorine-substituted counterparts. A total of 12 bromine-, chlorine-, and mixed halogen-substituted 4-methyl-2(5H)-furanones were tested repeatedly in 10 independent experiments for levels of Salmonella typhimurium (TA100) mutagenicities. The purpose of these experiments was to determine the mutagenic response to changing halogen content, type, and position as well as to learn the measure of these responses in the presence and absence of the C-5 OH group. Mutagenicities reached levels of 10(3) and 10(2) rev/nmol for all trihalo- and dihalo-4-methyl-5-hydroxy-2(5H)-furanones, respectively, notwithstanding substitutions by bromine or chlorine. Trihalides lacking the C-5 hydroxyl group possessed mutagenicities of the order of 10(2) rev/nmol, while hydroxyl group absence in the dihalides resulted in potency levels of slightly less than 10 rev/ nmol. Pairwise comparisons of compound mutagenicities showed that overall the C-5 H-by-OH replacement and, next in importance, increasing the number of C-6 halogens from one to two resulted in the greatest enhancements of mutagenicities. However, in comparing compound pairs within two different sets of four di- and trihalides, it was observed that replacement of a C-5 H by OH enhanced mutagenicity more for the dihalides than the trihalides indicating that increasing the C-6 halogen number simultaneously with replacing C-5 H by OH results in a nonlinear, additive enhancement. For fewer than half of the compound pairs compared, changing the C-6 halogen from chlorine to bromine resulted in small increases in mutagenicity, and for the remaining compound pairs, no increase could be discerned. This result points to the relative unimportance of only C-6 halogen type as a determinant of mutagenicity. Similarly, no impact on mutagenicity was observed for changing only the halogen type attached to C-3.

The Salmonella typhimurium (TA100) mutagenicities of six structural analogues of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) were determined and compared. These were also compared to previously determined mutagenicities for another four analogues. This study was conducted for the primary purpose of ascertaining the effect of C-6 chlorine-by-hydrogen replacement on mutagenicity. The compounds assayed were 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (3), 3-chloro-4-(chloromethyl)-2(5H)-furanone (4), 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (7), 3-chloro-4-methyl-2(5H)-furanone (8), 4-methyl-5-hydroxy-2(5H)-furanone (9), and 4-methyl-2(5H)-furanone (10). Compounds 3, 4, and 7 were mutagenic whereas 8-10 were not. All six compounds were stable under assay conditions. Mutagenicity data for the three active compounds were combined with data of another four active compounds studied previously to obtain an expanded data set. Mutagenicities of the seven compounds were compared, pairwise, in 21 comparisons and then by multiple regression analysis. On the average, chlorine-by-hydrogen replacement of a single chlorine located at a chloromethyl group (C-6) had a markedly greater effect in reducing mutagenicity than a similar replacement at C-3 or a hydroxyl-by-hydrogen replacement at C-5. The chlorine-by-hydrogen replacement at C-6 of compound 3 resulted in the greatest mutagenicity reduction of any single replacement and amounted to a 10(3)-fold diminished mutagenicity.

A difference in biological response to enantiomers is not an uncommon observation and is, therefore, to be expected in various manifestations of genotoxicity. The bacterial mutagen mucochloric acid (2,3-dichloro-5-hydroxy-2(5H)-furanone) has one chiral center, at C-5, but this mutagen exists in racemic form because of the facile stereoisomerization occurring by the mechanism of ring-chain tautomerism. Two readily synthesized enantiomeric analogs of mucochloric acid, as well as the racemic form of the two, were prepared from mucochloric acid and (R)-(+)-, (S)-(-)-, and (R,S)-(+/-)-cysteine. Using Salmonella typhimurium (TA100), the enantiomeric compounds were assayed together in four dose/response assays along with mucochloric acid, the reference mutagen. In three of the same four assays, the racemic form was also assayed. Neither statistically significant differences in mutagenicity, as determined in slope responses, nor distinctions from the plotted curves were observed among the two enantiomers and their racemic form. Therefore, no enantiospecific interaction between enantiomers and chiral DNA or enzymes involved in repair or replication could be concluded.