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.

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

“…Aliquots (2 l) were removed at various times (15,30,45, 60, and 75 s) during the reaction and spotted onto polyethyleneimine TLC sheets that had been prerun in water and dried. [␥- 32 P]ATP and 32 P i were separated by chromatography on the polyethyleneimine sheets using 0.75 M KH 2 PO 4 , pH 3.5, as the mobile phase. The developed TLC plates were used to expose PhosphorImager plates (10 -20 min) and scanned using a Fuji BAS 1000 BioImaging Analyzer; the radioactive spots were analyzed using the Macintosh BAS analysis program.…”

“…2 The use of analogues acting as irreversible inactivators has proven to be a useful approach for the identification of substrate-binding sites in enzymes (30). A recent structure-activity relationship study of Bcm (31)(32)(33) has identified 5a-(3-formylanilino)dihydrobicyclomycin (FD-Bcm) as a potential reductive amination probe that warranted further study (34). FD-Bcm has Rho inhibitory properties comparable with Bcm.…”

The Molecular Basis for the Mode of Action of Bicyclomycin

“…In the first, we determined the structureactivity relationship (SAR) for bicyclomycin inhibition of rho [119][120][121][122][123][124][125][126][127][128][129][130][131]. In the second, we identified key amino acid residues in rho necessary for rho binding [74].…”

“…The second sector modified in 1 was the central bicyclic unit [123,124]. The 11 compounds prepared were divided into two categories: the amide-modified bicyclomycins (A) and the C(6)-substituted bicyclomycins (B) (Fig.…”

“…The first group included 28 and 29, and the second included 30-38. Necessary for this second set of compounds was our development of a chemoselective method to replace the hindered C(6) hydroxyl group in 1 with a wide range of nucleophilic substituents [123]. Biochemical assays demonstrated that of the two amide-modified bicyclomycins only 28 displayed moderate inhibitory activity [123].…”