FLU (Sequoia Study Products, UK), dissolved in DMSO was added to the FLU + inocula at a concentration of ~1/10th (strains, T

FLU (Sequoia Study Products, UK), dissolved in DMSO was added to the FLU + inocula at a concentration of ~1/10th (strains, T. efflux pump-containing strains (little to no synergy with FLU in the null pump strain). The marine sponge sp. was therefore selected for bioassay-guided fractionation due to significant enhancement of FLU activity ( 9) in the genetically-altered strain overexpressing the MDR1 efflux pump. Subsequent bioassay-guided fractionation of the draw out led to the isolation and recognition of two fresh sulfated sterols, geodisterol-3-sp. was carried out by using a Supelco LC-18 cartridge eluted with aqueous MeOH. The actives that shown a synergistic effect with FLU against the MDR1 strain were located in the two fractions (60 and 80% aq. MeOH). Subsequent scale-up reversed-phase chromatography of the extract, monitored by bioassay and TLC comparison with the active fractions mentioned above, yielded compounds 1 and 2. HRESIMS data for compound 1 contained a pseudomolecular ion at 551.2397 corresponding to a molecular formula of C28H41Na2O6S. The presence of sulfur was supported by the intensity of the [M + 2]+ peak which was about 7.7% of the pseudomolecular ion peak. Evidence for the sulfate group came from the strong asymmetric S=O stretch absorption at 1226 cm?1 in the IR spectrum of 1.10 The 13C NMR data of 1 1 indicated the presence of 28 carbons comprising five methyl, seven methylene, 10 methine, and six quaternary carbons (C28H39). Thus, the remaining two hydrogens were assigned to two hydroxy groups attached to Gata3 the carbons with resonances at 74.5 (CH) and 78.0 (qC). The five methyls resonated at 1.04 (6H, d, = 7.2 Hz), 1.19 (3H, s), 1.37 (3H, s), 1.39 (1H, d, = 7.8 Hz), and 1.63 (3H, d, = 6.6 Hz) in the 1H NMR spectrum. This indicated that compound 1 might possess a steroidal skeleton as in the sulfated sterols isolated from the same genus.11 A distinguishing feature of the 1H NMR spectrum Fluzinamide of 1 was the presence of the resonances at 7.02 (brs), 7.05 (d, = 8.4 Hz), and 7.23 (d, = 8.4 Hz) attributable to a 1,2,4-trisubstituted aromatic system. The six aromatic carbon resonances were identified by HMQC and HMBC at 119.7 (CH), 122.4 (CH), 126.8 (CH), 138.1 (C), 138.6 (C), and 151.6 (C) from the eight aromatic/olefinic carbon resonances in the downfield region of the 13C NMR spectrum. Naturally occurring sterols with an aromatic ring system from marine organisms are rare. A literature survey showed that there was only one compound, geodisterol (3), that was isolated from the tropical marine sponge sp. and possessed an aromatic A ring.12 On comparison of the NMR data of 1 1 with those of 3, the close resemblance indicated that 1 was an in Hz)in Hz)by Moshers method, while the absolute configuration of C-20 was assigned as on the basis of the NOE correlation between Me-21 and H-12.12 We have further examined the issue concerning the C-20 configuration in 1. According to the results reported by Nes and Varkey,14,15 free rotation around the C-17 ? C-20 bond in 20-hydroxysteroids is usually hindered when C-20 is usually tetrahedral but is usually unhindered when C-20 is usually trigonal planar. Reasons for Fluzinamide these observations were based on steric interactions between the hydrogens of Me-21 and C-22 with the hydrogens of C-16 and Me-18, which in the case of 20by comparison of the chemical shift for Me-21 of 1 1 ( 1.37) Fluzinamide with that of similar 20537.2252 corresponding to the molecular formula C27H39Na2O6S. As for 1, the presence of sulfur was indicated from the intensity of the [M + 2]+ ion (8%). The IR spectrum also showed the same strong asymmetric stretch absorption of the S=O bond at 1229 cm?1 seen in 1. Analysis of the 1H NMR spectrum of 2 showed it to have marked similarities with that of 1 1 except for the distinct absence of the methyl doublet at 1.63 present in the 1H NMR spectrum of 1. Instead, the presence of a 1,1-disubstituted double bond was evident from the proton resonances at 4.74 (brs) and 4.77 (brs). Further analysis of the 13C, COSY, HMQC, and HMBC NMR data verified the structural similarities and difference between 1 and 2, leading to the proposed structure.