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Bicyclomycin (1) is a novel antibiotic that targets rho transcription termination factor in Escherichia coli. We have demonstrated that retention of the C(5)-C(5a) exomethylene unit in 1 is not essential for inhibition. In a recent paper we proposed a working model for 1 and rho function and suggested that 1 binds in a cleft with the C(5)-C(5a) exomethylene unit directed toward the dimeric interface of two rho monomers. This report examines the bicyclomycin C(5)-C(5a) structural constraints necessary for retention of rho inhibitory activity. Three classes of C(5)-C(5a)-modified bicyclomycins have been prepared and their inhibitory activities evaluated in the poly C-dependent ATPase and filter disk antimicrobial assays. The first series consisted of 12 analogues (8-19) that contained a C(5a)-unsaturated substituent and possessed C(5E)-geometry. The second set were a pair of C(5a)-substituted C(5E)- and C(5Z)-geometrical isomers (21 and 23). The final group of compounds consisted of six C(5)-C(5a)-dihydrobicyclomycins (24-28, 34) where the terminal substituent was systematically varied. We find that extending the C(5)-C(5a) double bond with unsaturated substituents provides bicyclomycin derivatives with excellent inhibitory activities in the biochemical assay, and that enhanced inhibitory activity is observed for the C(5E) geometrical isomer compared with its C(5Z) counterpart. Finally, C(5a)-substituted dihydrobicyclomycin inhibitory activity appears to be tightly regulated by the nature and spatial placement of the C(5a)-terminal substituent with respect to the [4.2.2]-bicyclic ring system. The observed biochemical activities for the C(5a)-extended conjugated bicyclomycin derivatives and the (5E) and (5Z) isomers were correlated with a structural model for the 1-rho complex.

The synthesis of 1,2,8,8a-tetrahydrocyclopropa[c]pyrrolo[3, 2-e]indol-4(5H)-one (CPI), the parent CC-1065 and duocarmycin SA alkylation subunit, is detailed. The parent CPI alkylation subunit lacks the C7 methyl substituent of the CC-1065 alkylation subunit and the C6 methoxycarbonyl group of duocarmycin SA, and their examination permitted the establishment of the impact of these natural product substituents. The studies revealed a CPI stability comparable to the CC-1065 alkylation subunit but which was 6x more reactive than the (+)-duocarmycin SA alkylation subunit, and it displayed the inherent reaction regioselectivity (4:1) of the natural products. The single-crystal X-ray structure of (+)-N-BOC-CPI depicts a near identical stereoelectronic alignment of the cyclopropane accounting for the identical reaction regioselectivity and a slightly diminished vinylogous amide conjugation relative to (+)-N-BOC-DSA suggesting that the stability distinctions stem in part from this difference in the vinylogous amide as well as alterations in the electronic nature of the fused pyrrole. Establishment of the DNA binding properties revealed that the CPI-based agents retain the identical DNA alkylation selectivities of the natural products. More importantly, the C6 methoxycarbonyl group of duocarmycin SA was found to increase the rate (12-13x) and efficiency (10x) of DNA alkylation despite its intrinsic lower reactivity while the CC-1065 C7 methyl group was found to slow the DNA alkylation rate (4x) and lower the alkylation efficiency (ca. 4x). The greater DNA alkylation rate and efficiency for duocarmycin SA and related analogues containing the C6 methoxycarbonyl is proposed to be derived from the extended length that the rigid C6 methoxycarbonyl provides and the resulting increase in the DNA binding-induced conformational change which serves to deconjugate the vinylogous amide and activate the alkylation subunit for nucleophilic attack. The diminished properties resulting from the CC-1065 C7 methyl group may be attributed to the steric impediment this substituent introduces to DNA minor groove binding and alkylation. Consistent with this behavior, the duocarmycin SA C6 methoxycarbonyl group increases biological potency while the CC-1065 C7 methyl group diminishes it.

Bicyclomycin (1) is a commercial antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. A recent structure-activity relationship study of 1 showed that replacing the C(6)-hydroxy group with alkoxy and thioalkoxy substituents led to dramatic losses of inhibitory activity in rho biochemical assays. The origin for this structural specificity has been explored by the synthesis and chemical, biochemical, and biological evaluation of C(6)-amino- (13), C(6)-(hydroxylamino)-(14), and C(6)-mercaptobicyclomycin (15). These compounds, like 1, are capable of entering into hydrogen bond donor interactions with rho and are capable of undergoing C(6) ring opening to generate alpha, beta-unsaturated carbonyl, imine, or thione systems. The chemical reactivity of 13-15 was compared with that of 1. We observed that 1, upon treatment with EtSH under moderate and basic conditions, readily underwent C(6) hemiaminal bond cleavage followed by conjugate addition to beta-methylene-alpha-ketoamide 2 to give Michael addition adducts whereas 13-15 reacted by initial cleavage of the C(1)-O(2) bond. Biochemical and biological assays of 13-15 and related analogues demonstrated that the C(6) hydroxy group in 1 was essential for activity. We found that replacing the C(6)-hydroxy group in 1 with weaker hydrogen bond donors led to low inhibitory activities in the rho-dependent ATPase and transcription termination assays. None of the bicyclomycin derivatives exhibited antibiotic activity against E. coli W3350 cells at a 32 mg/mL concentration. The apparent specificity for the C(6)-hydroxy group in 1 suggests that an efficient hydrogen bond donor interaction from the C(6)-hydroxy group to rho or the C(6) hemiaminal bond cleavage to 2 or both is necessary for drug function.

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.