Role of the [4.2.2] Bicyclic Unit in Bicyclomycin: Synthesis, Structure, Chemical, Biochemical, and Biological Properties.

Abstract

Twelve bicyclomycin derivatives were synthesized to determine the effect of modification of the [4.2.2] bicyclic unit in bicyclomycin (1) on drug function. Few bicyclomycin derivatives have been described in which the [4.2.2] ring system has been modified. The compounds evaluated were divided into two categories: the two N-methyl-modified bicyclomycins (2, 3) and the ten C(6)-substituted bicyclomycins (4-13). Substituents introduced at the C(6) site included alkoxy, thioalkoxy, thiophenoxy, anilino, and hydrogen. A procedure was developed to synthesize select C(6)-substituted bicyclomycins. Bicyclomycin was first converted to bicyclomycin C(2'),C(3')-acetonide (16) and then treated with methanesulfonyl chloride to give in situ the corresponding C(6) mesylate 17. Treatment of 17 with the appropriate nucleophile followed by removal 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-H(2)O mixtures of 4 with excess EtSH maintained at "pH" 8.0-9.0 led to no detectable reaction, while at more basic "pH" values 4 underwent stereospecific conversion to the bis-spiro derivative 33 and no appreciable EtSH addition to the C(5)-C(5a) 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 complete change (>75%) within 200 h. The [4.2.2] bicyclic-modified bicyclomycins were evaluated in the rho-dependent ATPase assay and their antimicrobial activities determined using a filter disc assay. Most of the compounds were also tested in the transcription termination assay. We observed that all structural modifications conducted within the [4.2.2] bicyclic unit led to a loss of rho-dependent ATPase (I(50) > 400 &mgr;M) and to transcription termination (I(50) > 100 &mgr;M) inhibitory activities, as well as a loss of antimicrobial activity (MIC > 32 mg/mL). Only N(10)-methylbicyclomycin (2) displayed moderate inhibitory activities in these assays. These findings indicated that the [4.2.2] bicyclic unit played an important role in the antibiotic-rho recognition process. Potential factors that govern this interaction are briefly discussed. 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 likely prohibit the bicyclomycin derivative from efficiently binding to rho.