Journal of Natural Products two hydrogen atoms. The UV spectrum of 2 also diﬀered signiﬁcantly from that of 1, with the low-energy λmax having shifted from 348 nm in 1 to 388 nm in 2. The NMR data obtained for 2 were very similar to that of 1, suggesting that the compounds were closely related. The NMR signals correspond- ing to the C-12 oxygenated methine present in 1 were absent from the NMR spectra of 2. Instead, the resonance at δH 7.62 (H-11) showed an HMBC to a carbon at δC 189.5 (C-12), typical of a quinone carbon, revealing that 2 is the quinone analogue of 1. Pseudonocardone C (3) gave a peak in the HRESI(+) MS consistent with a molecular formula of C26H22O11. The molecular formula of 3 diﬀered from the molecular formula of 2 by the addition of an oxygen atom. The UV and NMR data obtained for 3 were very similar to that of 2, suggesting that they are closely related. The aromatic singlet present in 2 at δH 7.25 (H-2) was absent in the NMR spectrum of 3, and the other aromatic singlet at δH 7.45 (H-4) was shifted upﬁeld to δH 6.95 (H-4) in 3. The methyl resonance at δH 2.50 showed an HMBC correlation to a downﬁeld carbon at δC 144.7 (C-2), suggesting that C-2 was substituted with an oxygen atom. An HMBC correlation from the anomeric proton at δH 4.76 (H-1′) to C-2 showed that the sugar moiety was attached to C-2 instead of to C-1 as found in 1 and 2. The lack of protons within three bonds from C-1 made the assignment of this position impossible to determine from the HMBC data. However, in order to satisfy the molecular formula of 3, C-1 must be oxygenated as it is in 1, 2, and 5. Compounds 1−5 were tested for antibiotic activity against Escherichia coli K12, Bacillus subtilis 3610, Candida albicans, and Saccharomyces cerevisiae. Compounds 4 and 5 were active against B. subtilis 3610 with MIC values of 25 and 3.13 μg/mL, respectively. None of the compounds showed any activity against E. coli, C. albicans, or S. cerevisiae at concentrations as high as 50 μg/mL. Compounds 1−5 were also tested in a liver- stage malaria assay recently developed in one of our laboratories.14 Compounds 4 and 5 were active against liver- stage Plasmodium berghei with IC50 values of 18.5 and 3.0 μM, respectively. Finally, compounds 1−5 were tested for cytotoxicity against HepG2 cells. Compound 5 was active against HepG2 cells with an IC50 value of 36.1 μM. The glycoside analogues (1−3) were completely inactive against B. subtilis, E. coli, C. albicans, S. cerevisiae, and HepG2 cells at concentrations as high as 50 μg/mL and showed only weak activity against liver-stage P. berghei with IC50 values of 38, 50, and >100 μM, respectively. The lack of activity for the glycosylated analogues provides insight into the structure− activity relationships of this family of compounds. A comparison of the activity of 2 with that of 5 shows that adding the glucuronic acid moiety at C-1 completely abolishes cytotoxic and antibiotic activity. Glycosylation of antibiotics has been proposed as one possible mechanism of self-resist- ance,15,16 and this might explain the lack of biological activity observed for 1−3. ■ EXPERIMENTAL SECTION General Experimental Procedures. An Agilent 1200 Series HPLC system equipped with a diode array detector and a Phenomenex C18 column (5 μm, 250 × 21.2 mm) was used for preparative HPLC. For HPLC-MS analysis, an Agilent HPLC system equipped with a diode array detector and a 6130 Series quadrupole mass spectrometer was used with a Phenomenex C18 (5 μm, 100 × 4.6 mm) column. The following gradient was used for HPLC-MS analysis: Note Table 2. Cytotoxic Activities of 1−5 against HepG2 Cells and Antibiotic Activities of 1−5 against P. berghei, E. coli, B. subtilis, C. albicans, and S. cerevisiaea P. HepG2 berghei E. coli B. subtilis C. albicans S. cerevisiae compound IC50 (μM) IC50 (μM) MIC MIC MIC MIC (μg/mL) (μg/mL) (μg/mL) (μg/mL) 1 >100 38 >50 >50 >50 >50 2 >100 50 >50 >50 >50 >50 3 >100 >100 >50 >50 >50 >50 4 >100 18.5 >50 25 >50 >50 5 36.1 3.0 >50 3.13 >50 >50 aData represent the average of two experiments each performed in triplicate. The MIC is deﬁned as the lowest concentration that gave less than 5% of the maximum growth. 0−5 min, isocratic 10% CH3CN + 0.1% formic acid; 5−25 min, linear gradient from 10% CH3CN + 0.1% formic acid to 100% CH3CN + 0.1% formic acid. NMR spectra were recorded in CD3OD (for compounds 1−4) or CD2Cl2 (for compound 5) at 600 MHz and referenced to the internal solvent peak at δH 3.30 and δC 49.0 or δH 5.32 and δC 53.8, respectively. High-resolution mass spectrometry (HR-MS) was performed at the University of Illinois Urbana− Champaign Mass Spectrometry Facility. Isolation of Pseudonocardia sp. EC080529-01. An ant colony of A. dentigerum was collected from Pipeline Road, Panama, on May 29, 2008, and placed in a sterile Petri dish with moist cotton. After allowing the nest to stabilize for a few days, the Pseudonocardia symbiont from this colony was isolated directly from the mesoternal lobe of a worker by scraping bacteria oﬀ the cuticle of the ant using a sterile scalpel and plating on chitin media following the methods of Caldera and Currie, and identiﬁed as Pseudonocardia sp. based on multilocus sequencing.10 Cultivation of Pseudonocardia sp. EC080529-01. Production cultures of Pseudonocardia sp. EC080529-01 were grown on solid ISP- 2 medium (per liter: yeast extract, 4 g; malt extract, 10 g; glucose, 4 g) in 12 Petri plates (150 × 20 mm, 1.2 L total) for 7 d at 30 °C. The solid agar was cut into small cubes and soaked in EtOAc (1.2 L) overnight. The EtOAc was ﬁltered and dried in vacuo to give the crude EtOAc extract. The solid agar was re-extracted overnight with MeOH (1.2 L), and the MeOH was ﬁltered and dried in vacuo to give the crude MeOH extract. The crude EtOAc extract was dissolved in 90% MeOH−H2O (20 mL) and passed through a C18 column, eluting with additional 90% MeOH−H2O, in order to remove nonpolar components. The eluent from this column was diluted with H2O to give a ﬁnal MeOH concentration of 60%. This solution was passed through another C18 column and washed with additional 60% MeOH−H2O solution, followed by 100% MeOH. The 60% MeOH−H2O fraction was puriﬁed by preparative HPLC using the following gradient: 0−5 min, isocratic 20% CH3CN−H2O; 5−60 min, linear gradient from 20% CH3CN−H2O to 100% CH3CN to give pure 4 (8.3 mg). The 100% MeOH fraction from this C18 column was puriﬁed by preparative HPLC using the following gradient: 0−10 min, isocratic 50% CH3CN−H2O; 10−60 min, linear gradient from 50% CH3CN−H2O to 100% CH3CN to give pure 5 (3.5 mg). The crude MeOH extract was dissolved in H2O and passed through an HP-20 column. The HP-20 column was washed with water to remove polar components, and the compounds of interest were then eluted with 100% MeOH. The 100% MeOH fraction was dried in vacuo, redissolved in 60% MeOH−H2O, and passed through a C18 column to remove nonpolar components. The eluent from this column was diluted with H2O to give a ﬁnal MeOH concentration of 30%. This solution was passed through another C18 column and washed with additional 30% MeOH−H2O, followed by 100% MeOH. The 100% MeOH fraction from this C18 column was puriﬁed by reversed-phase HPLC using the following gradient: 0−10 min, isocratic 10% CH3CN−H2O + 0.1% formic acid; 10−60 min linear gradient from 1808 dx.doi.org/10.1021/np300380t | J. Nat. Prod. 2012, 75, 1806−1809