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Identification and Bioactivity of 3-epi-Xestoaminol C Isolated from the New Zealand Brown Alga Xiphophora chondrophylla Nathaniel Dasyam,† Andrew B. Munkacsi,† Nazmi H. Fadzilah,† Dinindu S. Senanayake,† Ronan F. O’Toole,‡ and Robert A. Keyzers*,§ †
Center for Biodiscovery and School of Biological Sciences, Victoria University of Wellington, PO Box 600, Kelburn, Wellington 6140, New Zealand ‡ Department of Clinical Microbiology, Trinity College Dublin, Sir Patrick Dun Laboratory, Central Pathology Laboratory, St. James’s Hospital, Dublin 8, Ireland § Center for Biodiscovery and School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600, Kelburn, Wellington 6140, New Zealand S Supporting Information *
ABSTRACT: We report here the bioassay-guided isolation of a new 1-deoxysphingoid, 3-epi-xestoaminol C (1), isolated from the New Zealand brown alga Xiphophora chondrophylla. This is the first report of a 1-deoxysphingoid from a brown alga. We describe the isolation and full structure elucidation of this compound, including its absolute configuration, along with its bioactivity against mycobacteria and mammalian cell lines and preliminary mechanism of action studies using yeast chemical genomics.
isolation, full structure elucidation, and biological characterization of 1. Displaying an MIC of 200 μg/mL against M. tuberculosis H37Ra, a MeOH extract of X. chondrophylla collected from the Hen and Chicken Islands, New Zealand, underwent bioassayguided fractionation using reversed-phase (polystyrene− divinylbenzene copolymer) and repetitive stages of normal phase (diol) benchtop chromatography that led to the isolation of 3-epi-xestoaminol C (1) (58 mg, MIC 65 μM). Isolated as a white solid, the molecular formula of 1 was deduced as C14H31NO from detection of a protonated adduct by HRESIMS, requiring no double-bond equivalents. Analysis of the 1H, 13C, and multiplicity-edited HSQC NMR spectra in CD3OD (Table 1; Supporting Information S4, S5, and S7) identified all 14 carbons, including two methyls (δC 16.1 and 14.5), an oxymethine (δC 73.2), an aminomethine (δC 53.5), and a number of methylenes. A 2,3-amino alcohol substructure was identified using a contiguous sequence of COSY
Mycobacterium tuberculosis is the causative agent for tuberculosis (TB), with 8.6 million new cases and 1.3 million deaths due to this disease reported by the World Health Organization in 2012.1 Treatment for drug-susceptible TB consists of a combination of rifampicin, isoniazid, ethambutol, and pyrazinamide taken over a six-month period and a minimum of 18 months for resistant forms of TB using second-line drugs, which are more frequently associated with adverse reactions in patients.2,3 There is therefore a pressing need for the development of new drugs to counter drug resistance and to shorten treatment duration in the control of TB. In our aim to identify marine natural products with antitubercular activity from New Zealand marine organisms, a screen of a library containing 288 marine extracts against M. smegmatis and validated against M. tuberculosis H37Ra was performed. Following the screen, the extract of a brown alga was identified as having anti-TB activity and was pursued further. Bioassayguided fractionation of the algal extract led to the isolation and identification of the 1-deoxysphingoid, 3-epi-xestoaminol C (1), a stereoisomer of xestoaminol C (2).4 Here we report the © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 23, 2014
A
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Table 1. CD3OD 1H (600 MHz) and 13C (150 MHz) NMR Data of 3-epi-Xestoaminol C (1) δC 1 2 3 4a 4b 5a 5b 6−11 12 13 14
16.1, 53.5, 73.2, 34.7,
CH3 CH CH CH2
26.4, CH2 30.5−30.8, CH2 33.1, CH2 23.8,CH2 14.5, CH3
δH (J in Hz) 1.27, 3.09, 3.44, 1.40, 1.56, 1.39, 1.54, 1.30, 1.29, 1.31, 0.91,
d (6.7) quin (6.8) ddd (8.5, 7.0, 3.0) m m m m m m m t (7.0)
formed by treating 1 in a 1:1 mix of pyridine and acetic anhydride. The ester was deprotected in basic MeOH to afford N-acetyl-3-epi-xestoaminol C (5), which was subsequently treated with either R- or S-MTPA acids to yield Mosher’s esters 6a and 6b (Figure 1). Analyzing the chemical shifts of the Mosher’s esters in CDCl3 resulted in intramolecular hydrogen bonding, which led to a mixture of both positive and negative δΔ shifts flanking both sides of the oxymethine and preventing accurate interpretation. Use of a polar NMR solvent (CD3OD) disrupted the hydrogen bonding and resulted in consistent shifts with either solely positive or negative Δδ values on resonances adjacent to the ester (Figure 2). On the basis of the Δδ shifts, the absolute configuration of compound 1 was established as 2S-aminotetradecan-3S-ol (1), epimeric to 2 at C-3.
HMBC 2,3,7 1,3,4 1,4,5 3,5 5,9 2,3 6 13 12,14 12,13
correlations from the terminal methyl doublet (C-1: δC 16.1, δH 1.27) to C-4 (δC 34.7, δH 1.40, 1.56) via the amino- (C-2: δC 53.5, δH 3.09) and oxymethines (C-3: δC 73.2, δH 3.44), supported by HMBC correlations further linking to C-5 (δC 26.4, δH 1.39, 1.54). Similarly COSY correlations from the methyl triplet (C-14: δC 14.5, δH 0.91) to methylenes C-13 (δC 23.8, δH 1.31) and C-12 (δC 33.1, δH 1.29) established the other chain terminus. The termini were linked together based upon 1D-TOCSY experiments obtained in CDCl3. Irradiation of H-3 (δH 3.65) and H-4/5 (δH 1.56/1.53) resulted in TOCSY transfer to H3-14 (δH 0.88), while irradiation of H3-14 resulted in TOCSY transfer to H-4/5. This provided unequivocal evidence for the connectivity of the proposed planar structure (1) of a 1-deoxysphingosine (Supporting Information S9). Ascidians and sponges are common sources of such compounds,5 with an early report of a 1-deoxysphingoid from a Papua-New Guinean Xestospongia sp.6 The planar structure of our isolated compound was the same as the known compound xestoaminol C (2), first reported by Jiménez and Crews from a Fijian Xestospongia.4 Further analysis of our NMR data (CDCl3) indicated the shifts δH 1.38 (H-1) and δH 3.65 (H3) of 1 did not match those reported for 2 (δH 1.15 and 3.95, respectively),4 suggesting a stereoisomer. Consequently oxazolidinone 3 was formed to elucidate the relative configuration of 1. A coupling constant of approximately 6.5 Hz was measured between H-2 and H-3, diagnostic of a trans relationship between the protons of the oxazolidinone ring,7 supported by 1D-NOE correlations between H-3 and H3-1, and H-2 and H-4a and 4b (Figure 1; Supporting Information S14). Identification of the absolute configuration of 1 was achieved using Mosher’s method. Diacetyl-3-epi-xestoaminol C (4) was
Figure 2. Δδ shifts between MTPA esters of N-acetyl-3-epixestoaminol C (6a and 6b).
3-epi-Xestoaminol C was evaluated using a variety of assays (Table 2), with IC50 values of 19.4, 8.8, and 18.0 μM against M. tuberculosis H37Ra, HL-60 cells, and HEK cells, respectively, indicating that the potential of this compound as an antitubercular agent is overshadowed by its antitumor activity. 1-Deoxysphingosines have displayed a range of activities from antimicrobial to broad cytotoxic effects;5c,e however within this class one particular compound has drawn much interest, spisulosine or ES-285 (7), originally isolated from the bivalve mollusk Spisula polynyma (surf clam).8 Due to the initially promising anticancer activity displayed by 7, it progressed to phase 1 clinical trials, although it was subsequently dropped due to nonspecific cytotoxic effects.9 The mode of action of 7 and other 1-deoxysphingoids has not been fully identified although multiple pathways such as disruption of actin stress fibers,10 activation of caspases 3 and 12, phosphorylation of p53,11 or the activation of protein kinase C ζ up-regulating ceramide levels,12 all leading to cell death, have been implicated. More recently, N-acylation of 7 and its derivatives significantly increased N-C18 1-deoxyceramide levels in cells treated with compounds that have the natural 2S configuration.13 The literature to date may allude to the mode of activity of 1; however the most effective way to study the global effects of this compound within a cell is to utilize an assay that can observe the effects on a genome wide scale. To do this, we utilized a genome-wide screen against a yeast deletion library. Chemical genetics employs a library containing 4800 nonessential gene deletion mutants of Saccharomyces cerevisiae (baker’s yeast).14 The library was screened at the IC20 concentration, as this ensures bioactivity for the identification of mutants that are the most sensitive to drug treatment. A dose−response assay in S. cerevisiae identified an IC50 of 17 μM and an IC20 of 3.5 μM. The library of nonessential mutants was grown for 16 h in the presence of 1 at the IC20 concentration, which identified 805 hits. Hits were selected using a t test with a Bonferroni correction adjusting the p value to 0.017. Using this
Figure 1. Diagnostic NOE correlations of oxazolidinone 3. B
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Table 2. Bioactivity Profile of 3-epi-Xestoaminol C (1)a M. tuberculosis H37Ra MIC 1 3 4 5 Str Tet Cyc
65.4 ≥260 NA NA 3.1
IC50 19.4 114 NA NA 12.5
S. aureus MIC
IC50
E. coli
P. aeruginosa
S. cerevisiae
HL60
HEK
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
NA
NA
NA
17.1
32.7
8.8
32.7
18.0
>500
143.3 1.0
0.04
65.1
17.0
32.6
b
62.5
5.7
≥500
26.4
All concentrations listed are μM. MIC is defined as less than 2% growth compared to uninhibited growth controls. NA is defined as no observed activity at the highest concentrations tested (260). Samples not tested in this model. Str: streptomycin, Tet: tetracycline, Cyc: cycloheximide. bGraph too steep to calculate IC50 value. a
only in marine sponges,4 ascidians,5b and mollusks,8 this study is the first report of a 1-deoxysphingoid, 3-epi-xestoaminol C (1), from a marine alga. Intriguingly this may suggest that the sessile invertebrate origins of this compound class may lie in microalgal dietary sources and that 1-deoxysphingoids are an often overlooked but important class of alleopathic compounds in marine alga.
selection criterion, sensitive strains that displayed less than 70% residual growth and resistant strains that had greater than 130% growth in comparison to DMSO controls were chosen for validation. Using the same selection criterion these hits were validated, which identified 28 mutants of significance (Figure 3). This analysis indicated the cellular processes that 1 may
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were determined using a Rudolph Autopol II polarimeter, with IR spectra obtained using a Bruker Tensor-27 spectrometer. 1H, 13C, and 2D NMR spectra were acquired using a Varian 600 NMR DirectDrive spectrometer with spectra referenced to residual solvent peaks (CDCl3: δC 77.0, δH 7.26; CD3OD: δC 49.0, δH 3.31).16 HRESIMS data were acquired using an Agilent 6530 Q-TOF system. Separation utilized a C18 Phenomenex Prodigy column (4.6 × 250 mm, 5 μm particle size) using a 1 mL/min flow rate. All solvents used were HPLC grade, purchased form Fisher Scientific. Bench-top chromatography was performed using Supleco Diaion HP20 polystyrenedivinylbenzene and Supelco Discovery DSC-Diol functionalized silica 3-(2,3-dihydroxypropoxy)propylsilica (Diol) resins. Fractions were monitored using TLC plates using a 4:1:2 butanol/acetic acid/H2O mix for the running solvent. TLC plates were visualized with 5% H2SO4/MeOH solutions followed by 0.1% vanillin before charring. Algal Material. Xiphophora chondrophylla was collected off the Hen and Chicken Islands using scuba at a depth of 10−15 m in 2003. Algal identification was performed by Dr. Joe Zuccarello (Victoria University of Wellington). A voucher sample (PTN2_93D) is held at the School of Chemical and Physical Sciences, Victoria University of Wellington. The sample was kept frozen until use. Extraction and Isolation. X. chondrophylla (300 g) was extracted twice using MeOH (400 mL) and loaded onto HP20 reversed-phase polymeric resin (120 mL), which was eluted with 20%, 40%, 60%, and 80% acetone in H2O and 100% acetone fractions (360 mL). On the basis of bioassay results the 40% fraction was further fractionated three times using Diol columns (30 mL) eluting with solvent mixtures of 0%, 50%, and 100% EtOAc (90 mL) in hexanes (collected in bulk) followed by 25%, 50%, and 100% MeOH in EtOAc (collected in test tubes); finally the column was stripped with 50% H2O in MeOH. Test tube fractions 5−7 were combined based on TLC, affording 3-epixestoaminol C (1, 58 mg). 3-epi-Xestoaminol C (1): amorphous, white solid; [α]25D −6.19 (c 0.42, CHCl3); IR νmax 3367, 2954, 2871, 1602 cm−1; 1H and 13C NMR data (CD3OD) see Table 1; 1H NMR (CDCl3, 600 MHz) δ 3.65 (1H, t, J = 8.5 Hz, H-3), 3.32 (1H, m, H-2), 1.56 (1H, m, H-4b), 1.53 (1H, m, H5-b), 1.40 (1H, m, H-4a), 1.38 (3H, d, J = 6.6 Hz, H-1), 1.35 (1H, m, H-5a), 1.27 (2H, m, H-13), 1.25 (14H, m, H-6 to H-12), 0.88 (3H, t, J = 7.0 Hz, H-14); 13C NMR (CDCl3, 150 MHz) δ 73.0 (C-3), 53.3 (C-2), 33.6 (C-4), 32.1 (C-12), 29.88−29.52 (C-6 to C-11), 25.5
Figure 3. Genetic network identifying processes affected by 3-epixestoaminol C (1).
interfere with. The largest number are genes targeting unknown processes such as PST2 and TDA8, followed by genes involved in lipid metabolism (AIM6, PLB3, CRC1, SPS19, and OPI1) and actin cytoskeleton organization (AKL1, RHO2, and SLA1) (Figure 3). Targets of interest include those involved in lipid metabolism, which is unsurprising as sphingolipids are known to be involved in this process.15 Actin cytoskeleton organization was another process of interest identified, as it has been previously suggested that the 1-deoxysphingoid 7 prevents the formation of actin stress fibers by decreasing the activity of Rho proteins.10 This study has also identified potential new processes that 1deoxysphingoids may interact with, such as RNA catabolism, protein targeting, and phosphorylation (Figure 3). Studying how 1-deoxysphingoids interact with these new and unknown pathways may allow for a better understanding of the mechanism of action of these compounds. Compound 1 is a moderately cytotoxic bioactive with broad spectrum antimicrobial activity. Using the powerful tool of chemical genetics, we have identified multiple pathways of interest to be further investigated in order to gain a better understanding of the mechanism of action of 1-deoxysphingoids. Further studies to delineate the mechanism of action of 1 are under way. Given 1-deoxysphingoids were historically found C
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(C-5), 22.9 (C-13), 15.8 (C-1), 14.3 (C-14); HRESIMS m/z 230.2467 [M + H]+ (calcd for C14H32NO, 230.2478). Oxazolidinone (3). 3-epi-Xestoaminol C (1) (3 mg) was reacted with 1,1′-carbonyldiimidiazole (CDI; 3 mg, 1.4 equiv) for 24 h in CH2Cl2, after which a further 7 mg of CDI was added to the reaction. After 2 h, the reaction was partitioned against H2O with the organic layer dried and purified using a 1 mL Diol column eluted with 3 mL volumes of 100% hexanes, 50% and 75% mixtures of hexanes and EtOAc, 100% EtOAc, followed by 50% EtOAc and MeOH to give 3 (2.8 mg): colorless film; [α]25D −43.3 (c 0.18, CHCl3); IR νmax 3285, 2922, 2853, 1747, 1541 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.18 (1H, s, NH), 4.09 (1H, ddd, J = 7.9, 6.4, 4.8 Hz, H-3), 3.57 (1H, quin, J = 6.2 Hz, H-2), 1.71 (1H, m, H-4b), 1.62 (1H, m, H-4a), 1.48 (1H, m, H-5b), 1.37 (1H, m, H-5a), 1.28 (2H, m, H-13), 1.27 (3H, d, J = 6.2 Hz, H-1), 1.26 (12H, m, H-6 to H-11), 1.25 (2H, m, H-12), 0.88 (3H, t, J = 7.0 Hz, H-14); 13C NMR (CDCl3, 150 MHz) δ 158.97 (C15), 84.4 (C-3) 53.7 (C-2), 34.3 (C-4), 32.1 (C-12), 29.76−29.46 (C6 to C-11), 25.0 (C-5), 22.84 (C-13), 20.82 (C-1) 14.3 (C-14); HRESIMS m/z 256.2264 [M + H]+ (calcd for C15H30NO2, 256.2271) Diacetyl-3-epi-xestoaminol C (4). 3-epi-Xestoaminol C (1) (10 mg) was reacted with a 1:1 mixture of pyridine and acetic anhydride for 3 h at room temperature (rt). The reaction mixture was evaporated and dissolved in 10 mL of CH2Cl2 followed by successive partitioning against 1 M HCl (3 × 10 mL), 1 M NaOH (3 × 10 mL), and H2O (3 × 10 mL). The organic layer was dried to give 4 (7 mg): colorless film; [α]25D −23 (c 0.1, CHCl3); IR νmax 3297, 2923, 2854, 1740, 1652, 1460, 1373 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.53 (1H, d, J = 9.3 Hz, NH), 4.85 (1H, ddd, J = 7.6, 5.9, 4.1 Hz, H-3), 4.20 (1H, dqd, J = 9.3, 6.8, 4.1 Hz, H-2), 2.09 (3H, s, OAc, H-18), 1.99 (3H, s, NAc, H16), 1.52 (2H, m, H-4), 1.28 (2H, m, H-13), 1.27 (2H, m, H-5) 1.25 (2H, m, H-12) 1.24 (12H, m, H-6 to H-11), 1.10 (3H, d, J = 6.8 Hz, H-1), 0.87 (3H, t, J = 7.0 Hz, H-14); 13C NMR (CDCl3, 150 MHz) δ 171.1 (C-17), 169.6 (C-15), 76.6 (C-3), 47.4 (C-2), 32.1 (C-12), 31.7 (C-4), 29.78−29.5 (C-6 to C-11), 25.4 (C-5), 23.6 (C-16), 22.8 (C13), 21.2 (C-18), 18.7 (C-1), 14.3 (C-14); HRESIMS m/z 314.2693 [M + H]+ (calcd for C18H36NO3, 314.2690). N-Acetyl-3-epi-xestoaminol C (5). Diacetyl-3-epi-xestoaminol C (4) was reacted with 5 mg of KCO3 in MeOH at 0 °C for 30 min followed by warming to rt for 16 h. The reaction mixture was dried down and redissolved in EtOAc. This was partitioned against H2O three times to yield N-acetyl-3-epi-xestoaminol C (5) (4.7 mg): colorless film; [α]25D −6.3 (c 0.16, CHCl3); IR νmax 3477, 2924, 2854, 1652 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.68 (1H, d, J = 8.54 Hz, NH), 3.97 (1H, dqd, J = 8.6, 6.8, 3.2 Hz, H-2), 3.53 (1H, q, J = 4.4, 3.7 Hz, H-3), 2.00 (3H, s, NAc, H-16), 1.44 (1H, m, H-4), 1.41 (1H, m, H-5b), 1.34 (1H, m, H-5a), 1.29 (2H, m, H-13), 1.25 (14H, m, H-6 to H-12), 1.19 (3H, d, J = 6.5 Hz, H-1), 0.88 (3H, t, J = 7 Hz, H-14); 13C NMR (CDCl3, 150 MHz) δ 170.3 (C-15), 75.0 (C-3), 49.1 (C-2), 34.6 (C-4), 32.1 (C-12), 29.8−29.5 (C-6 to C-11), 25.8 (C-5), 23.7 (C-16), 22.9 (C-13), 18.5 (C-1), 14.3 (C-12); HRESIMS m/z 272.2581 [M + H]+ (calcd for C16H34NO2, 272.2584) R- and S-Mosher’s Esters (6a, 6b). N-Acetyl-3-epi-xestoaminol C (5) (1.5 mg) was treated with EDCI (7.4 mg, 8.6 equiv) and DMAP (1.4 mg, 2.1 equiv) dissolved in dry CH2Cl2 under argon, after which R-MTPA acid (9.0 mg, 7 equiv) was added. After 22 h reaction the mixture (2 mL) was washed with 0.01 M HCl (3 × 2 mL) followed by 0.01 NaOH (3 × 2 mL) and finally H2O (3 × 2 mL) before performing HPLC purification of the R ester. The same process was performed with the S-MTPA acid to give the S-Mosher’s ester. Compound 6a (0.6 mg): colorless film; [α]25D 0 (c 0.04, CHCl3); IR νmax 3378, 2924, 2854, 1746, 1651 cm−1; 1H NMR (CD3OD, 600 MHz) δ 7.55 (2H, m, H-24), 7.45 (2H, m, H-22), 7.44 (1H, m, H-23), 5.5 (1H, s, NH), 5.11 (1H, ddd, J = 7.6, 5.6, 4.6 Hz, H-3), 4.17 (1H, qd, J = 6.9, 5.5 Hz, H-2), 3.54 (3H, s, OMe, H-19), 1.83 (3H, s, NAc, H-16), 1.64 (2H, m, H-4), 1.34 (3H, m, H-5b, H-6), 1.31 (2H, m, H5a, H-13b), 1.29 (1H, m, H-13a), 1.28 (12H, m, H-7 to H-12), 0.99 (3H, J = d, 6.9 Hz), 0.90 (3H, t, J = 7 Hz, H-14); 13C NMR (CD3OD, 150 MHz) δ 172.7 (C-15), 167.5 (C-17), 133.4 (C-18), 130.9 (C-23/ 25), 129.5 (C-24), 128.7 (C-22/26), 125.8 (C-21), 86.1 (C-20), 79.7 (C-3), 56.1 (C-19), 47.7 (C-2), 33.1 (C-12), 31.5 (C-4), 30.75−30.4,
26.1 (C-6 to C-11), 30.48 (C-5), 23.8 (C-13), 22.6 (C-16), 16.8 (C1), 14.5 (C-14); HRESIMS m/z 488.2979 [M + H]+ (calcd for C26H41F3NO4, 488.2982). Compound 6b (1.2 mg): colorless film; [α]25D −17.5 (c 0.13, CHCl3); IR νmax 3335, 2925, 2854, 1746, 1651 cm−1; 1H NMR (CD3OD, 600 MHz) δ 7.55 (2H, m, H-24), 7.44 (3H, m, H-22, H23), 5.5 (1H, s, NH), 5.11 (1H, ddd, J = 8.0, 6.4, 4.0 Hz, H-3), 4.2 (1H, quin, J = 6.8 Hz, H-2), 3.55 (3H, s, OMe H-19), 1.9 (3H, s, NAc, H-16), 1.59 (1H, m, H-4b), 1.54 (1H, m, H-4a), 1.32 (1H, m, H-13b), 1.29 (1H, m, H-13a), 1.28 (2H, m, H-12), 1.27−1.19 (12H, m, H-6 to H-11), 1.15 (2H, m, H-5), 1.13 (3H, d, J = 6.9 Hz, H-1), 0.90 (3H, t, J = 6.9 Hz, H-14); 13C NMR (CD3OD, 150 MHz) δ 172.7 (C-15), 167.5 (C-17), 133.5 (C-18), 130.9 (C-23/25), 129.5 (C-24), 128.5 (C-22/26), 125.8 (C-21), 85.9 (C-20), 79.5 (C-3), 56.2 (C-19), 48.1 (C-2), 33.1 (C-12), 31.3 (C-4), 30.74−30.3 (C-6 to C-11), 25.7 (C-5), 23.8 (C-13), 22.7 (C-16), 17.0 (C-1), 14.5 (C-14); HRESIMS m/z 488.2979 [M + H]+ (calcd for C26H41F3NO4, 488.2982). Bacteria and Cell Line Culturing and Biological Assays. Full experimental details describing bacterial strains, cell line culturing, and yeast chemical genetic profiling are provided in the Supporting Information.
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ASSOCIATED CONTENT
* Supporting Information S
Full details describing the bacterial and mammalian cell culturing, yeast chemical genomics, and 1D and 2D NMR spectra for 1−5 are provided. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel: +64 4 463 5117. Fax: +64 4 463 5241. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Dr. J. Harvey is thanked for providing reagents and assistance with derivatization reactions. We gratefully acknowledge Associate Professor J. Zuccarello for algal identification and Associate Professor P. Northcote for algal collection and for assistance with NMR experiments.
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REFERENCES
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(8) Faircloth, G.; Cuevas, C. Prog. Mol. Subcell. Biol. 2006, 43, 363− 379. (9) (a) Vilar, E.; Grünwald, V.; Schöffski, P.; Singer, H.; Salazar, R.; Iglesias, J.; Casado, E.; Cullell-Young, M.; Baselga, J.; Tabernero, J. Invest. New Drugs 2012, 30, 299−305. (b) Massard, C.; Salazar, R.; Armand, J. P.; Majem, M.; Deutsch, E.; García, M.; Oaknin, A.; Fernández-García, E. M.; Soto, A.; Soria, J. C. Invest. New Drugs 2012, 30, 2318−2326. (c) Schöffski, P.; Dumez, H.; Ruijter, R.; Miguel-Lillo, B.; Soto-Matos, A.; Alfaro, V.; Giaccone, G. Cancer Chemother. Pharmacol. 2011, 68, 1397−1403. (d) Baird, R. D.; Kitzen, J.; Clarke, P. A.; Planting, A.; Reade, S.; Reid, A.; Welsh, L.; López Lázaro, L.; de las Heras, B.; Judson, I. R.; Kaye, S. B.; Eskens, F.; Workman, P.; deBono, J. S.; Verweij, J. Mol. Cancer Ther. 2009, 8, 1430−1437. (10) Cuadros, R.; Montejo de Garcini, E.; Wandosell, F.; Faircloth, G.; Fernández-Sousa, J. M.; Avila, J. Cancer Lett. 2000, 152, 23−29. (11) Salcedo, M.; Cuevas, C.; Alonso, J. L.; Otero, G.; Faircloth, G.; Fernandez-Sousa, J. M.; Avila, J.; Wandosell, F. Apoptosis 2007, 12, 395−409. (12) Sánchez, A. M.; Malagarie-Cazenave, S.; Olea, N.; Vara, D.; Cuevas, C.; Díaz-Laviada, I. Eur. J. Pharmacol. 2008, 584, 237−245. (13) Abad, J. L.; Nieves, I.; Rayo, P.; Casas, J.; Fabriàs, G.; Delgado, A. J. Org. Chem. 2013, 78, 5858−5866. (14) Winzeler, E. A.; Shoemaker, D. D.; Astromoff, A.; Liang, H.; Anderson, K.; Andre, B.; Bangham, R.; Benito, R.; Boeke, J. D.; Bussey, H.; Chu, A. M.; Connelly, C.; Davis, K.; Dietrich, F.; Dow, S. W.; El Bakkoury, M.; Foury, F.; Friend, S. H.; Gentalen, E.; Giaever, G.; Hegemann, J. H.; Jones, T.; Laub, M.; Liao, H.; Liebundguth, N.; Lockhart, D. J.; Lucau-Danila, A.; Lussier, M.; M’Rabet, N.; Menard, P.; Mittmann, M.; Pai, C.; Rebischung, C.; Revuelta, J. L.; Riles, L.; Roberts, C. J.; Ross-MacDonald, P.; Scherens, B.; Snyder, M.; SookhaiMahadeo, S.; Storms, R. K.; Veronneau, S.; Voet, M.; Volckaert, G.; Ward, T. R.; Wysocki, R.; Yen, G. S.; Yu, K.; Zimmermann, K.; Philippsen, P.; Johnston, M.; Davis, R. W. Science 1999, 285, 901−906. (15) Worgall, T. S. Curr. Opin. Clin. Nutr. Metab. Care 2007, 10, 149−155. (16) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512−7515.
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dx.doi.org/10.1021/np500171z | J. Nat. Prod. XXXX, XXX, XXX−XXX
Identification and Bioactivity of 3-epi-Xestoaminol C Isolated from the New Zealand Brown Alga Xiphophora chondrophylla Nathaniel Dasyam,† Andrew B. Munkacsi,† Nazmi H. Fadzilah,† Dinindu S. Senanayake,† Ronan F. O’Toole,‡ and Robert A. Keyzers*,§ †
Center for Biodiscovery and School of Biological Sciences, Victoria University of Wellington, PO Box 600, Kelburn, Wellington 6140, New Zealand ‡ Department of Clinical Microbiology, Trinity College Dublin, Sir Patrick Dun Laboratory, Central Pathology Laboratory, St. James’s Hospital, Dublin 8, Ireland § Center for Biodiscovery and School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600, Kelburn, Wellington 6140, New Zealand S Supporting Information *
ABSTRACT: We report here the bioassay-guided isolation of a new 1-deoxysphingoid, 3-epi-xestoaminol C (1), isolated from the New Zealand brown alga Xiphophora chondrophylla. This is the first report of a 1-deoxysphingoid from a brown alga. We describe the isolation and full structure elucidation of this compound, including its absolute configuration, along with its bioactivity against mycobacteria and mammalian cell lines and preliminary mechanism of action studies using yeast chemical genomics.
isolation, full structure elucidation, and biological characterization of 1. Displaying an MIC of 200 μg/mL against M. tuberculosis H37Ra, a MeOH extract of X. chondrophylla collected from the Hen and Chicken Islands, New Zealand, underwent bioassayguided fractionation using reversed-phase (polystyrene− divinylbenzene copolymer) and repetitive stages of normal phase (diol) benchtop chromatography that led to the isolation of 3-epi-xestoaminol C (1) (58 mg, MIC 65 μM). Isolated as a white solid, the molecular formula of 1 was deduced as C14H31NO from detection of a protonated adduct by HRESIMS, requiring no double-bond equivalents. Analysis of the 1H, 13C, and multiplicity-edited HSQC NMR spectra in CD3OD (Table 1; Supporting Information S4, S5, and S7) identified all 14 carbons, including two methyls (δC 16.1 and 14.5), an oxymethine (δC 73.2), an aminomethine (δC 53.5), and a number of methylenes. A 2,3-amino alcohol substructure was identified using a contiguous sequence of COSY
Mycobacterium tuberculosis is the causative agent for tuberculosis (TB), with 8.6 million new cases and 1.3 million deaths due to this disease reported by the World Health Organization in 2012.1 Treatment for drug-susceptible TB consists of a combination of rifampicin, isoniazid, ethambutol, and pyrazinamide taken over a six-month period and a minimum of 18 months for resistant forms of TB using second-line drugs, which are more frequently associated with adverse reactions in patients.2,3 There is therefore a pressing need for the development of new drugs to counter drug resistance and to shorten treatment duration in the control of TB. In our aim to identify marine natural products with antitubercular activity from New Zealand marine organisms, a screen of a library containing 288 marine extracts against M. smegmatis and validated against M. tuberculosis H37Ra was performed. Following the screen, the extract of a brown alga was identified as having anti-TB activity and was pursued further. Bioassayguided fractionation of the algal extract led to the isolation and identification of the 1-deoxysphingoid, 3-epi-xestoaminol C (1), a stereoisomer of xestoaminol C (2).4 Here we report the © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 23, 2014
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Table 1. CD3OD 1H (600 MHz) and 13C (150 MHz) NMR Data of 3-epi-Xestoaminol C (1) δC 1 2 3 4a 4b 5a 5b 6−11 12 13 14
16.1, 53.5, 73.2, 34.7,
CH3 CH CH CH2
26.4, CH2 30.5−30.8, CH2 33.1, CH2 23.8,CH2 14.5, CH3
δH (J in Hz) 1.27, 3.09, 3.44, 1.40, 1.56, 1.39, 1.54, 1.30, 1.29, 1.31, 0.91,
d (6.7) quin (6.8) ddd (8.5, 7.0, 3.0) m m m m m m m t (7.0)
formed by treating 1 in a 1:1 mix of pyridine and acetic anhydride. The ester was deprotected in basic MeOH to afford N-acetyl-3-epi-xestoaminol C (5), which was subsequently treated with either R- or S-MTPA acids to yield Mosher’s esters 6a and 6b (Figure 1). Analyzing the chemical shifts of the Mosher’s esters in CDCl3 resulted in intramolecular hydrogen bonding, which led to a mixture of both positive and negative δΔ shifts flanking both sides of the oxymethine and preventing accurate interpretation. Use of a polar NMR solvent (CD3OD) disrupted the hydrogen bonding and resulted in consistent shifts with either solely positive or negative Δδ values on resonances adjacent to the ester (Figure 2). On the basis of the Δδ shifts, the absolute configuration of compound 1 was established as 2S-aminotetradecan-3S-ol (1), epimeric to 2 at C-3.
HMBC 2,3,7 1,3,4 1,4,5 3,5 5,9 2,3 6 13 12,14 12,13
correlations from the terminal methyl doublet (C-1: δC 16.1, δH 1.27) to C-4 (δC 34.7, δH 1.40, 1.56) via the amino- (C-2: δC 53.5, δH 3.09) and oxymethines (C-3: δC 73.2, δH 3.44), supported by HMBC correlations further linking to C-5 (δC 26.4, δH 1.39, 1.54). Similarly COSY correlations from the methyl triplet (C-14: δC 14.5, δH 0.91) to methylenes C-13 (δC 23.8, δH 1.31) and C-12 (δC 33.1, δH 1.29) established the other chain terminus. The termini were linked together based upon 1D-TOCSY experiments obtained in CDCl3. Irradiation of H-3 (δH 3.65) and H-4/5 (δH 1.56/1.53) resulted in TOCSY transfer to H3-14 (δH 0.88), while irradiation of H3-14 resulted in TOCSY transfer to H-4/5. This provided unequivocal evidence for the connectivity of the proposed planar structure (1) of a 1-deoxysphingosine (Supporting Information S9). Ascidians and sponges are common sources of such compounds,5 with an early report of a 1-deoxysphingoid from a Papua-New Guinean Xestospongia sp.6 The planar structure of our isolated compound was the same as the known compound xestoaminol C (2), first reported by Jiménez and Crews from a Fijian Xestospongia.4 Further analysis of our NMR data (CDCl3) indicated the shifts δH 1.38 (H-1) and δH 3.65 (H3) of 1 did not match those reported for 2 (δH 1.15 and 3.95, respectively),4 suggesting a stereoisomer. Consequently oxazolidinone 3 was formed to elucidate the relative configuration of 1. A coupling constant of approximately 6.5 Hz was measured between H-2 and H-3, diagnostic of a trans relationship between the protons of the oxazolidinone ring,7 supported by 1D-NOE correlations between H-3 and H3-1, and H-2 and H-4a and 4b (Figure 1; Supporting Information S14). Identification of the absolute configuration of 1 was achieved using Mosher’s method. Diacetyl-3-epi-xestoaminol C (4) was
Figure 2. Δδ shifts between MTPA esters of N-acetyl-3-epixestoaminol C (6a and 6b).
3-epi-Xestoaminol C was evaluated using a variety of assays (Table 2), with IC50 values of 19.4, 8.8, and 18.0 μM against M. tuberculosis H37Ra, HL-60 cells, and HEK cells, respectively, indicating that the potential of this compound as an antitubercular agent is overshadowed by its antitumor activity. 1-Deoxysphingosines have displayed a range of activities from antimicrobial to broad cytotoxic effects;5c,e however within this class one particular compound has drawn much interest, spisulosine or ES-285 (7), originally isolated from the bivalve mollusk Spisula polynyma (surf clam).8 Due to the initially promising anticancer activity displayed by 7, it progressed to phase 1 clinical trials, although it was subsequently dropped due to nonspecific cytotoxic effects.9 The mode of action of 7 and other 1-deoxysphingoids has not been fully identified although multiple pathways such as disruption of actin stress fibers,10 activation of caspases 3 and 12, phosphorylation of p53,11 or the activation of protein kinase C ζ up-regulating ceramide levels,12 all leading to cell death, have been implicated. More recently, N-acylation of 7 and its derivatives significantly increased N-C18 1-deoxyceramide levels in cells treated with compounds that have the natural 2S configuration.13 The literature to date may allude to the mode of activity of 1; however the most effective way to study the global effects of this compound within a cell is to utilize an assay that can observe the effects on a genome wide scale. To do this, we utilized a genome-wide screen against a yeast deletion library. Chemical genetics employs a library containing 4800 nonessential gene deletion mutants of Saccharomyces cerevisiae (baker’s yeast).14 The library was screened at the IC20 concentration, as this ensures bioactivity for the identification of mutants that are the most sensitive to drug treatment. A dose−response assay in S. cerevisiae identified an IC50 of 17 μM and an IC20 of 3.5 μM. The library of nonessential mutants was grown for 16 h in the presence of 1 at the IC20 concentration, which identified 805 hits. Hits were selected using a t test with a Bonferroni correction adjusting the p value to 0.017. Using this
Figure 1. Diagnostic NOE correlations of oxazolidinone 3. B
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Table 2. Bioactivity Profile of 3-epi-Xestoaminol C (1)a M. tuberculosis H37Ra MIC 1 3 4 5 Str Tet Cyc
65.4 ≥260 NA NA 3.1
IC50 19.4 114 NA NA 12.5
S. aureus MIC
IC50
E. coli
P. aeruginosa
S. cerevisiae
HL60
HEK
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
NA
NA
NA
17.1
32.7
8.8
32.7
18.0
>500
143.3 1.0
0.04
65.1
17.0
32.6
b
62.5
5.7
≥500
26.4
All concentrations listed are μM. MIC is defined as less than 2% growth compared to uninhibited growth controls. NA is defined as no observed activity at the highest concentrations tested (260). Samples not tested in this model. Str: streptomycin, Tet: tetracycline, Cyc: cycloheximide. bGraph too steep to calculate IC50 value. a
only in marine sponges,4 ascidians,5b and mollusks,8 this study is the first report of a 1-deoxysphingoid, 3-epi-xestoaminol C (1), from a marine alga. Intriguingly this may suggest that the sessile invertebrate origins of this compound class may lie in microalgal dietary sources and that 1-deoxysphingoids are an often overlooked but important class of alleopathic compounds in marine alga.
selection criterion, sensitive strains that displayed less than 70% residual growth and resistant strains that had greater than 130% growth in comparison to DMSO controls were chosen for validation. Using the same selection criterion these hits were validated, which identified 28 mutants of significance (Figure 3). This analysis indicated the cellular processes that 1 may
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were determined using a Rudolph Autopol II polarimeter, with IR spectra obtained using a Bruker Tensor-27 spectrometer. 1H, 13C, and 2D NMR spectra were acquired using a Varian 600 NMR DirectDrive spectrometer with spectra referenced to residual solvent peaks (CDCl3: δC 77.0, δH 7.26; CD3OD: δC 49.0, δH 3.31).16 HRESIMS data were acquired using an Agilent 6530 Q-TOF system. Separation utilized a C18 Phenomenex Prodigy column (4.6 × 250 mm, 5 μm particle size) using a 1 mL/min flow rate. All solvents used were HPLC grade, purchased form Fisher Scientific. Bench-top chromatography was performed using Supleco Diaion HP20 polystyrenedivinylbenzene and Supelco Discovery DSC-Diol functionalized silica 3-(2,3-dihydroxypropoxy)propylsilica (Diol) resins. Fractions were monitored using TLC plates using a 4:1:2 butanol/acetic acid/H2O mix for the running solvent. TLC plates were visualized with 5% H2SO4/MeOH solutions followed by 0.1% vanillin before charring. Algal Material. Xiphophora chondrophylla was collected off the Hen and Chicken Islands using scuba at a depth of 10−15 m in 2003. Algal identification was performed by Dr. Joe Zuccarello (Victoria University of Wellington). A voucher sample (PTN2_93D) is held at the School of Chemical and Physical Sciences, Victoria University of Wellington. The sample was kept frozen until use. Extraction and Isolation. X. chondrophylla (300 g) was extracted twice using MeOH (400 mL) and loaded onto HP20 reversed-phase polymeric resin (120 mL), which was eluted with 20%, 40%, 60%, and 80% acetone in H2O and 100% acetone fractions (360 mL). On the basis of bioassay results the 40% fraction was further fractionated three times using Diol columns (30 mL) eluting with solvent mixtures of 0%, 50%, and 100% EtOAc (90 mL) in hexanes (collected in bulk) followed by 25%, 50%, and 100% MeOH in EtOAc (collected in test tubes); finally the column was stripped with 50% H2O in MeOH. Test tube fractions 5−7 were combined based on TLC, affording 3-epixestoaminol C (1, 58 mg). 3-epi-Xestoaminol C (1): amorphous, white solid; [α]25D −6.19 (c 0.42, CHCl3); IR νmax 3367, 2954, 2871, 1602 cm−1; 1H and 13C NMR data (CD3OD) see Table 1; 1H NMR (CDCl3, 600 MHz) δ 3.65 (1H, t, J = 8.5 Hz, H-3), 3.32 (1H, m, H-2), 1.56 (1H, m, H-4b), 1.53 (1H, m, H5-b), 1.40 (1H, m, H-4a), 1.38 (3H, d, J = 6.6 Hz, H-1), 1.35 (1H, m, H-5a), 1.27 (2H, m, H-13), 1.25 (14H, m, H-6 to H-12), 0.88 (3H, t, J = 7.0 Hz, H-14); 13C NMR (CDCl3, 150 MHz) δ 73.0 (C-3), 53.3 (C-2), 33.6 (C-4), 32.1 (C-12), 29.88−29.52 (C-6 to C-11), 25.5
Figure 3. Genetic network identifying processes affected by 3-epixestoaminol C (1).
interfere with. The largest number are genes targeting unknown processes such as PST2 and TDA8, followed by genes involved in lipid metabolism (AIM6, PLB3, CRC1, SPS19, and OPI1) and actin cytoskeleton organization (AKL1, RHO2, and SLA1) (Figure 3). Targets of interest include those involved in lipid metabolism, which is unsurprising as sphingolipids are known to be involved in this process.15 Actin cytoskeleton organization was another process of interest identified, as it has been previously suggested that the 1-deoxysphingoid 7 prevents the formation of actin stress fibers by decreasing the activity of Rho proteins.10 This study has also identified potential new processes that 1deoxysphingoids may interact with, such as RNA catabolism, protein targeting, and phosphorylation (Figure 3). Studying how 1-deoxysphingoids interact with these new and unknown pathways may allow for a better understanding of the mechanism of action of these compounds. Compound 1 is a moderately cytotoxic bioactive with broad spectrum antimicrobial activity. Using the powerful tool of chemical genetics, we have identified multiple pathways of interest to be further investigated in order to gain a better understanding of the mechanism of action of 1-deoxysphingoids. Further studies to delineate the mechanism of action of 1 are under way. Given 1-deoxysphingoids were historically found C
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(C-5), 22.9 (C-13), 15.8 (C-1), 14.3 (C-14); HRESIMS m/z 230.2467 [M + H]+ (calcd for C14H32NO, 230.2478). Oxazolidinone (3). 3-epi-Xestoaminol C (1) (3 mg) was reacted with 1,1′-carbonyldiimidiazole (CDI; 3 mg, 1.4 equiv) for 24 h in CH2Cl2, after which a further 7 mg of CDI was added to the reaction. After 2 h, the reaction was partitioned against H2O with the organic layer dried and purified using a 1 mL Diol column eluted with 3 mL volumes of 100% hexanes, 50% and 75% mixtures of hexanes and EtOAc, 100% EtOAc, followed by 50% EtOAc and MeOH to give 3 (2.8 mg): colorless film; [α]25D −43.3 (c 0.18, CHCl3); IR νmax 3285, 2922, 2853, 1747, 1541 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.18 (1H, s, NH), 4.09 (1H, ddd, J = 7.9, 6.4, 4.8 Hz, H-3), 3.57 (1H, quin, J = 6.2 Hz, H-2), 1.71 (1H, m, H-4b), 1.62 (1H, m, H-4a), 1.48 (1H, m, H-5b), 1.37 (1H, m, H-5a), 1.28 (2H, m, H-13), 1.27 (3H, d, J = 6.2 Hz, H-1), 1.26 (12H, m, H-6 to H-11), 1.25 (2H, m, H-12), 0.88 (3H, t, J = 7.0 Hz, H-14); 13C NMR (CDCl3, 150 MHz) δ 158.97 (C15), 84.4 (C-3) 53.7 (C-2), 34.3 (C-4), 32.1 (C-12), 29.76−29.46 (C6 to C-11), 25.0 (C-5), 22.84 (C-13), 20.82 (C-1) 14.3 (C-14); HRESIMS m/z 256.2264 [M + H]+ (calcd for C15H30NO2, 256.2271) Diacetyl-3-epi-xestoaminol C (4). 3-epi-Xestoaminol C (1) (10 mg) was reacted with a 1:1 mixture of pyridine and acetic anhydride for 3 h at room temperature (rt). The reaction mixture was evaporated and dissolved in 10 mL of CH2Cl2 followed by successive partitioning against 1 M HCl (3 × 10 mL), 1 M NaOH (3 × 10 mL), and H2O (3 × 10 mL). The organic layer was dried to give 4 (7 mg): colorless film; [α]25D −23 (c 0.1, CHCl3); IR νmax 3297, 2923, 2854, 1740, 1652, 1460, 1373 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.53 (1H, d, J = 9.3 Hz, NH), 4.85 (1H, ddd, J = 7.6, 5.9, 4.1 Hz, H-3), 4.20 (1H, dqd, J = 9.3, 6.8, 4.1 Hz, H-2), 2.09 (3H, s, OAc, H-18), 1.99 (3H, s, NAc, H16), 1.52 (2H, m, H-4), 1.28 (2H, m, H-13), 1.27 (2H, m, H-5) 1.25 (2H, m, H-12) 1.24 (12H, m, H-6 to H-11), 1.10 (3H, d, J = 6.8 Hz, H-1), 0.87 (3H, t, J = 7.0 Hz, H-14); 13C NMR (CDCl3, 150 MHz) δ 171.1 (C-17), 169.6 (C-15), 76.6 (C-3), 47.4 (C-2), 32.1 (C-12), 31.7 (C-4), 29.78−29.5 (C-6 to C-11), 25.4 (C-5), 23.6 (C-16), 22.8 (C13), 21.2 (C-18), 18.7 (C-1), 14.3 (C-14); HRESIMS m/z 314.2693 [M + H]+ (calcd for C18H36NO3, 314.2690). N-Acetyl-3-epi-xestoaminol C (5). Diacetyl-3-epi-xestoaminol C (4) was reacted with 5 mg of KCO3 in MeOH at 0 °C for 30 min followed by warming to rt for 16 h. The reaction mixture was dried down and redissolved in EtOAc. This was partitioned against H2O three times to yield N-acetyl-3-epi-xestoaminol C (5) (4.7 mg): colorless film; [α]25D −6.3 (c 0.16, CHCl3); IR νmax 3477, 2924, 2854, 1652 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.68 (1H, d, J = 8.54 Hz, NH), 3.97 (1H, dqd, J = 8.6, 6.8, 3.2 Hz, H-2), 3.53 (1H, q, J = 4.4, 3.7 Hz, H-3), 2.00 (3H, s, NAc, H-16), 1.44 (1H, m, H-4), 1.41 (1H, m, H-5b), 1.34 (1H, m, H-5a), 1.29 (2H, m, H-13), 1.25 (14H, m, H-6 to H-12), 1.19 (3H, d, J = 6.5 Hz, H-1), 0.88 (3H, t, J = 7 Hz, H-14); 13C NMR (CDCl3, 150 MHz) δ 170.3 (C-15), 75.0 (C-3), 49.1 (C-2), 34.6 (C-4), 32.1 (C-12), 29.8−29.5 (C-6 to C-11), 25.8 (C-5), 23.7 (C-16), 22.9 (C-13), 18.5 (C-1), 14.3 (C-12); HRESIMS m/z 272.2581 [M + H]+ (calcd for C16H34NO2, 272.2584) R- and S-Mosher’s Esters (6a, 6b). N-Acetyl-3-epi-xestoaminol C (5) (1.5 mg) was treated with EDCI (7.4 mg, 8.6 equiv) and DMAP (1.4 mg, 2.1 equiv) dissolved in dry CH2Cl2 under argon, after which R-MTPA acid (9.0 mg, 7 equiv) was added. After 22 h reaction the mixture (2 mL) was washed with 0.01 M HCl (3 × 2 mL) followed by 0.01 NaOH (3 × 2 mL) and finally H2O (3 × 2 mL) before performing HPLC purification of the R ester. The same process was performed with the S-MTPA acid to give the S-Mosher’s ester. Compound 6a (0.6 mg): colorless film; [α]25D 0 (c 0.04, CHCl3); IR νmax 3378, 2924, 2854, 1746, 1651 cm−1; 1H NMR (CD3OD, 600 MHz) δ 7.55 (2H, m, H-24), 7.45 (2H, m, H-22), 7.44 (1H, m, H-23), 5.5 (1H, s, NH), 5.11 (1H, ddd, J = 7.6, 5.6, 4.6 Hz, H-3), 4.17 (1H, qd, J = 6.9, 5.5 Hz, H-2), 3.54 (3H, s, OMe, H-19), 1.83 (3H, s, NAc, H-16), 1.64 (2H, m, H-4), 1.34 (3H, m, H-5b, H-6), 1.31 (2H, m, H5a, H-13b), 1.29 (1H, m, H-13a), 1.28 (12H, m, H-7 to H-12), 0.99 (3H, J = d, 6.9 Hz), 0.90 (3H, t, J = 7 Hz, H-14); 13C NMR (CD3OD, 150 MHz) δ 172.7 (C-15), 167.5 (C-17), 133.4 (C-18), 130.9 (C-23/ 25), 129.5 (C-24), 128.7 (C-22/26), 125.8 (C-21), 86.1 (C-20), 79.7 (C-3), 56.1 (C-19), 47.7 (C-2), 33.1 (C-12), 31.5 (C-4), 30.75−30.4,
26.1 (C-6 to C-11), 30.48 (C-5), 23.8 (C-13), 22.6 (C-16), 16.8 (C1), 14.5 (C-14); HRESIMS m/z 488.2979 [M + H]+ (calcd for C26H41F3NO4, 488.2982). Compound 6b (1.2 mg): colorless film; [α]25D −17.5 (c 0.13, CHCl3); IR νmax 3335, 2925, 2854, 1746, 1651 cm−1; 1H NMR (CD3OD, 600 MHz) δ 7.55 (2H, m, H-24), 7.44 (3H, m, H-22, H23), 5.5 (1H, s, NH), 5.11 (1H, ddd, J = 8.0, 6.4, 4.0 Hz, H-3), 4.2 (1H, quin, J = 6.8 Hz, H-2), 3.55 (3H, s, OMe H-19), 1.9 (3H, s, NAc, H-16), 1.59 (1H, m, H-4b), 1.54 (1H, m, H-4a), 1.32 (1H, m, H-13b), 1.29 (1H, m, H-13a), 1.28 (2H, m, H-12), 1.27−1.19 (12H, m, H-6 to H-11), 1.15 (2H, m, H-5), 1.13 (3H, d, J = 6.9 Hz, H-1), 0.90 (3H, t, J = 6.9 Hz, H-14); 13C NMR (CD3OD, 150 MHz) δ 172.7 (C-15), 167.5 (C-17), 133.5 (C-18), 130.9 (C-23/25), 129.5 (C-24), 128.5 (C-22/26), 125.8 (C-21), 85.9 (C-20), 79.5 (C-3), 56.2 (C-19), 48.1 (C-2), 33.1 (C-12), 31.3 (C-4), 30.74−30.3 (C-6 to C-11), 25.7 (C-5), 23.8 (C-13), 22.7 (C-16), 17.0 (C-1), 14.5 (C-14); HRESIMS m/z 488.2979 [M + H]+ (calcd for C26H41F3NO4, 488.2982). Bacteria and Cell Line Culturing and Biological Assays. Full experimental details describing bacterial strains, cell line culturing, and yeast chemical genetic profiling are provided in the Supporting Information.
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ASSOCIATED CONTENT
* Supporting Information S
Full details describing the bacterial and mammalian cell culturing, yeast chemical genomics, and 1D and 2D NMR spectra for 1−5 are provided. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel: +64 4 463 5117. Fax: +64 4 463 5241. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Dr. J. Harvey is thanked for providing reagents and assistance with derivatization reactions. We gratefully acknowledge Associate Professor J. Zuccarello for algal identification and Associate Professor P. Northcote for algal collection and for assistance with NMR experiments.
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REFERENCES
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