ROLE OF THE [4.2.2]-BICYCLIC UNIT IN BICYCLOMYCIN - SYNTHESIS, STRUCTURE, CHEMICAL, BIOCHEMICAL, AND BIOLOGICAL PROPERTIES

Twelve bicyclomycin derivatives were synthesized to determine the effe ct of modification of the [4.2.2] bicyclic unit in bicyclomycin (1) on drug function. Few bicyclomycin derivatives have been described in wh ich the [4.2.2] ring system has been modified. The compounds evaluated were divided into two categories: the two N-methyl-modified bicyclomy cins (2, 3) and the ten C(6)-substituted bicyclomycins (4-13). Substit uents introduced at the C(6) site included alkoxy, thioalkoxy, thiophe noxy, anilino, and hydrogen. A procedure was developed to synthesize s elect C(6)-substituted bicyclomycins. Bicyclomycin was first converted to bicyclomycin C(2'),C(3')-acetonide (16) and then treated with meth anesulfonyl chloride to give in situ the corresponding C(6) mesylate 1 7. Treatment of 17 with the appropriate nucleophile followed by remova l of the C(2'),C(3')-acetonide group gave the desired C(6)-substituted bicyclomycin. The chemical properties of C(6) O-methylbicyclomycin (4 ) were examined. Treatment of THF-H2O mixtures of 4 with excess EtSH m aintained at ''pH'' 8.0-9.0 led to no detectable reaction, while at mo re basic ''pH'' values 4 underwent stereospecific conversion to the bi s-spiro derivative 33 and no appreciable EtSH addition to the C(5)-C(5 a) exomethylene unit. These results were compared to the reactivity of 1 with EtSH. The stability (pH 7.4, 37 degrees C) of C(6)-substituted bicyclomycins 4, 6, and 10-13 in aqueous solutions were examined. We observed that most of these compounds (4, 6, 10-12) underwent near com plete change (>75%) within 200 h. The [4.2.2] bicyclic-modified bicycl omycins were evaluated in the rho-dependent ATPase assay and their ant imicrobial activities determined using a filter disc assay. Most of th e compounds were also tested in the transcription termination assay. W e observed that all structural modifications conducted within the [4.2 .2] bicyclic unit led to a loss of rho-dependent ATPase (I-50 > 400 mu M) and to transcription termination (I-50 > 100 mu M) inhibitory acti vities, as well as a loss of antimicrobial activity (MIC > 32 mg/mL). Only N(10)-methylbicyclomycin (2) displayed moderate inhibitory activi ties in these assays. These findings indicated that the [4.2.2] bicycl ic unit played an important role in the antibiotic-rho recognition pro cess. Potential factors that govern this interaction are briefly discu ssed. We concluded that placement of an irreversible inactivating unit at the N- and O-sites within the [4.2.2] bicyclic unit in 1 would lik ely prohibit the bicyclomycin derivative from efficiently binding to r ho.