CHEMICAL, BIOCHEMICAL, AND BIOLOGICAL STUDIES ON SELECT C(1) TRIOL MODIFIED BICYCLOMYCINS

To determine the importance of the C(1) triol group to bicyclomycin (1)-mediated transformations we prepared the bicyclomycin diastereomers 6 (C(1')-R, C(2')-S) and 7 (C(1')-S, C(2')-R), in which the stereochemical configuration at C(1') and C(2') in the triol group in 1 (C(1')-S, C(2')-S) was reversed, and the C(1') ketone analogue 8 (C(2')-S), in which the stereogenic center at C(1') in 1 was removed. Synthesis of 6 and 8 proceeded from C(1') ketobicyclomycin C(2'), C(3') acetonide (10). Reduction (NaBH4, CeCl3) of 10 produced a diastereomeric mixture, that, after separation and removal of the acetonide protecting group, gave 6. Correspondingly, deprotection of 10 gave 8. Bicyclomycin analogue 7 was prepared by dissolving the known bicyclomycin C(2'), C(3') epoxide (13) in dilute methanolic sulfuric acid; this process produced the novel [O(9)-C(2')]cyclized bicyclomycin (14). Compound 14 formed with inversion of the C(2') center. Subsequent aqueous acid hydrolysis yielded 7. Data documenting the proposed reaction pathways and structures for compounds 6-8 are presented. The stability of bicyclomycin analogues 6-8 and 1 in deuterium oxide (pD 5.6-5.8, 7.4, 9.2-9.4) and in DMF-d(7) solutions were examined. Compounds 7 and 8 were stable under these conditions (room temperature, 14 days), whereas bicyclomycin underwent noticeable change only in basic deuterium oxide. Correspondingly, 6 was rapidly converted (t(1/2) < 30 h) to a new set of products in both acidic and basic deuterium oxide as well as in DMF-d(7). The facility of these conversions have been attributed in part to the role of the C(1) triol substituent in the ring opening of the C(6) hemiketal group in 6. All three bicyclomycin analogues reacted with ethanethiol at the C(5)-C(5a) exomethylene unit at rates comparable to 1 in buffered (''pH'' 8.0-8.5) THF-H2O (3:1) mixtures. The products generated from 6 and 7 were similar to those previously determined for 1, except for the configuration of the C(1') and C(2') substituents, whereas 8 yielded the novel piperidine adduct 33. The ethanethiol-8 reaction proceeded easily in spite of earlier projections that the C(1') hydroxyl group in bicyclomycin was required for exomethylene modification. Similarly the corresponding C(2'), C(3') acetonide of 8, 10, readily underwent reaction with ethanethiol. Significantly, compounds 6 and 7 only partially (25-35%) inhibited rho-dependent hydrolysis of ATP at the concentration levels observed to block ATPase activity by 1, and no inhibition of ATP hydrolysis was detected for 8. Our previous studies established that the primary site of bicyclomycin action in Escherichia coli is the cellular protein transcription termination factor rho. Similarly, none of the three compounds exhibited antibiotic activity at a concentration of 1200 mu g/mL, using a filter disc assay. These cumulative results suggested that key interactions existed between the C(1) triol group in bicyclomycin and the antibiotic binding site in rho, which are necessary for drug utilization and function.