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 stereochem ical 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 anal ogue 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 struct ures for compounds 6-8 are presented. The stability of bicyclomycin an alogues 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 un derwent noticeable change only in basic deuterium oxide. Corresponding ly, 6 was rapidly converted (t(1/2) < 30 h) to a new set of products i n both acidic and basic deuterium oxide as well as in DMF-d(7). The fa cility of these conversions have been attributed in part to the role o f 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 buffe red (''pH'' 8.0-8.5) THF-H2O (3:1) mixtures. The products generated fr om 6 and 7 were similar to those previously determined for 1, except f or the configuration of the C(1') and C(2') substituents, whereas 8 yi elded the novel piperidine adduct 33. The ethanethiol-8 reaction proce eded easily in spite of earlier projections that the C(1') hydroxyl gr oup in bicyclomycin was required for exomethylene modification. Simila rly the corresponding C(2'), C(3') acetonide of 8, 10, readily underwe nt reaction with ethanethiol. Significantly, compounds 6 and 7 only pa rtially (25-35%) inhibited rho-dependent hydrolysis of ATP at the conc entration levels observed to block ATPase activity by 1, and no inhibi tion of ATP hydrolysis was detected for 8. Our previous studies establ ished that the primary site of bicyclomycin action in Escherichia coli is the cellular protein transcription termination factor rho. Similar ly, none of the three compounds exhibited antibiotic activity at a con centration of 1200 mu g/mL, using a filter disc assay. These cumulativ e results suggested that key interactions existed between the C(1) tri ol group in bicyclomycin and the antibiotic binding site in rho, which are necessary for drug utilization and function.