Antibacterials and modulators of bacterial resistance from the immature cones of Chamaecyparis lawsoniana

Several anti-staphylococcal diterpenes were isolated from the immature cones of the conifer Chamaecyparis lawsoniana. The most active, ferruginol, caused an 80-fold potentiation of oxacillin activity against EMRSA-15, with more modest potentiation of antibiotic activity against effluxing clinical isolates of Staphylococcus aureus. The results of an efflux inhibition assay suggest that ferruginol is a weak efflux pump inhibitor. As part of an on-going project to characterize compounds from immature conifer cones with antibacterial or modulatory activity against multidrug-resistant (MDR) strains of Staphylococcus aureus, eight compounds were isolated from the cones of Chamaecyparis lawsoniana. The active compounds were mainly diterpenes, with minimum inhibitory concentrations ranging from 4 to 128 μg/ml against MDR effluxing S. aureus strains and two epidemic methicillin-resistant (EMRSA) clinical isolates. The compounds extracted were the diterpenes ferruginol, pisiferol and its epimer 5-epipisiferol, formosanoxide, trans-communic acid and torulosal, the sesquiterpene oplopanonyl acetate and the germacrane 4β-hydroxygermacra-1(10)-5-diene. Some of these compounds also exhibited modulatory activity in potentiating antibiotic activity against effluxing strains and ferruginol, used at a sub-inhibitory concentration, resulted in an 80-fold potentiation of oxacillin activity against strain EMRSA-15. An efflux inhibition assay using an S. aureus strain possessing the MDR NorA efflux pump resulted in 40% inhibition of ethidium bromide efflux at 10 μM ferruginol (2.86 μg/ml). We report the 1H and 13C NMR data for the cis A/B ring junction epimer of pisiferol which we have named 5-epipisiferol. We also unambiguously assign all 1H and 13C NMR resonances for trans-communic acid.