Herbarone, a Rearranged Heptaketide Derivative from the Sea Hare

Note pubs.acs.org/jnp

Herbarone, a Rearranged Heptaketide Derivative from the Sea Hare Associated Fungus Torula herbarum Wan-Li Geng,†,‡ Xian-You Wang,†,‡ Tibor Kurtán,§ Attila Mándi,§ Hua Tang,† Barbara Schulz,⊥ Peng Sun,*,† and Wen Zhang*,† †

Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, 325 Guo-He Road, Shanghai 200433, People’s Republic of China ‡ Pharmaceutical College of Henan University, Kaifeng 475001, People’s Republic of China § Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary ⊥ Institut für Mikrobiologie, Technische Universität Braunschweig, 31806 Braunschweig, Germany S Supporting Information *

ABSTRACT: Herbarone (1), a novel heptaketide with a tetrahydro-5,9methanobenzo[8]annulen-10(5H)-one skeleton, together with the new ent-astropaquinones B (2) and C (3) and four known pyranonaphthoquinones (4−7), was isolated from the sea hare associated fungus Torula herbarum. The structures of the new compounds were elucidated by detailed spectroscopic analysis, and the absolute configurations were determined by solution TDDFT/ECD calculations. Absolute configurations of the known compounds were studied by ECD measurements and calculations. The isolation of heptaketide 1 suggests that an intramolecular aldol reaction takes place to form the tricyclic scaffold.

A

intramolecular aldol reaction might occur to form the tricyclic scaffold.

romatic polyketides comprise a structurally diverse and pharmacologically important group of natural products obtained from microbes and plants. Most aromatic polyketides are highly substituted, fused-ring polyphenols.1 The aromatic polyketides from fungi are biosynthesized by nonreducing iterative polyketide synthases (NR-PKS).2 In particular, fungal heptaketides are supposed to originate from a similar poly-βketo precursor, and their structural diversity arises from regioselective cyclization and subsequent post-PKS modifications.1,2 To date, a great variety of fungi-derived heptaketides have been discovered, including pyranonaphthoquinones,3−8 naphthoquinones,8,9 and other derivatives formed by ringopening,4,10 rearrangement,11 decarboxylation, and dimerization.11 These metabolites display a broad spectrum of biological activities, including phytotoxic, insecticidal, antibacterial, and fungicidal properties.8,9 As part of our ongoing search for bioactive compounds from symbiotic fungi,12 a dematiaceous fungus, Torula herbarum, was isolated from the viscera of the sea hare Notarchus leachii cirrosus Stimpson, collected from the South China Sea. The fungus T. herbarum is well known to produce herbarin (5) and its derivatives, O-methylherbarin (4) and dehydroherbarin (7).5−7 Our investigation on the EtOAc extract of this fungus resulted in the discovery of a novel heptaketide derivative with a rearranged carbon skeleton, herbarone (1), together with entastropaquinones B (2) and C (3), enantiomers of the known astropaquinones B and C, and four known compounds (4−7). We herein report the isolation, structure determination, and biological evaluation of these metabolites. A plausible biosynthetic pathway to 1 is proposed, which suggests an © 2012 American Chemical Society and American Society of Pharmacognosy

Herbarone (1) was obtained as an optically active, off-white, amorphous powder. The molecular formula of C16H20O6 was established by HRESIMS, indicating seven double-bond equivalents. The IR spectrum of 1 showed bands for hydroxy groups (3315 cm−1), an α,β-unsaturated ketone functionality (1680 cm−1), and a substituted aromatic ring (1603, 1454, 870 cm−1). These observations were in agreement with the appearance of signals in the 13C NMR and DEPT spectra Received: September 19, 2012 Published: October 8, 2012 1828

dx.doi.org/10.1021/np300642t | J. Nat. Prod. 2012, 75, 1828−1832

Journal of Natural Products

Note

(Table 1) for two methylene groups (δC 38.1, 48.0 ppm), two methine groups (δC 47.4, 51.0 ppm), five oxygenated carbons Table 1. 1H and 13C NMR Spectroscopic Data for 1a position 1 2 3 4 4a 5 6eq 6ax 7 8eq 8ax 9 10 10a 11 12a 12b 2-OCH3 4-OCH3 5-OH 7-OH 12-OH

δC, type 101.2, 160.1, 106.3, 158.3, 124.8, 75.6, 48.0,

CH C CH C C C CH2

66.3, CH 38.1, CH2 47.4, 197.7, 133.7, 51.0, 63.9,

CH C C CH CH2

55.7, CH3 56.2, CH3

δH (J in Hz) 7.16, d (2.5)

2.52, 1.82, 3.31, 2.17, 1.72, 2.92,

ddd (11.5, 3.5, 1.5) dd (11.5, 11.0) m brd (12.5) ddd (12.5, 12.5, 5.0) m

2.39, 3.85, 3.48, 3.87, 3.96, 5.50, 3.48, 3.48,

ddd (9.5, 3.0, 3.0) dd (9.5, 9.5) ovb s s s ovb ovb

δ in ppm, in CDCl3, at 500 MHz for 1H and 125 MHz for experiments. bov = overlapped. a

Figure 2. Key NOE correlations for 1.

6.78, d (2.5)

13

between H-6ax, H-8ax, and H-11 indicated the cis 1,3-diaxial arrangement of these protons. The axial orientation of H-7 was deduced from its NOE effects with the equatorial hydrogens H6eq and H-8eq. The large coupling constants between H-7ax and the adjacent axial protons (3J7,8ax = 12.5 Hz, 3J6ax,7 = 11.5 Hz) further confirmed the above conclusion. The cis equatorial orientation of H-9 and 5-OH is a consequence of the bridged skeleton, which corroborated the NOE effects between H-9 and both H-11 and H-12b and between 5-OH and both H-6ax and H-6eq, respectively. The small proton coupling constant between H-9eq and H-11ax (3J9,11 = 3.0 Hz) further supported the equatorial orientation of H-9. The absolute configuration of herbarone (1), containing a tetralone chromophore,13 was assigned by the solution TDDFT ECD calculation protocol, a powerful method for the configurational assignment of natural products. The 69 Merck molecular force field (MMFF) conformers of (5S,7S,9S,11R)-1 were reduced to six conformers with P helicity above a 1% population by DFT reoptimization at the B3LYP/6-31G(d) level (Figure S1). All of the Boltzmann-averaged ECD spectra obtained with the six low-energy conformers with various functionals (B3LYP, BH&HLYP, PBE0) gave mirror image curves compared to the experimental ECD spectrum, allowing the determination of the absolute configuration as (+)-(5R,7R,9R,11S)-1 (Figure 3). The ECD calculations also

C

(δC 56.2, and 57.7, CH3; 63.6, CH2; 66.3, CH; 75.6 ppm, C), one conjugated ketone carbonyl atom (δC 197.7 ppm, C), and six aromatic atoms (δC 101.2, CH; 106.3, C; 124.8, C; 133.7, C; 158.3, C; 160.1 ppm, C), accounting for five double-bond equivalents. The remaining double-bond equivalents were attributed to the presence of two more rings in the molecule. Analysis of the COSY spectrum readily established the proton sequence from H2-6 to H2-12, as shown in Figure 1.

Figure 1. Key HMBC (arrow) and COSY (bold) correlations for 1. Figure 3. Solution ECD spectrum of herbarone (1) in acetonitrile compared with the B3LYP/TZVP Boltzmann-weighted ECD spectrum of (5S,7S,9S,11R)-1. Bars represent the rotatory strengths of the lowest energy conformer.

The significant HMBC cross-peaks of H2-6 with C-5 and C-11 indicated the linkage from C-6 to C-11, through the oxygenated tertiary carbon of C-5, to form a six-membered ring. The HMBC correlations from both H-1 and H-8ax to C-10 established the presence of a carbonyl group at C-10 between C-9 and C-10a. Finally, C-5 was found to be linked to C-4a by observation of the diagnostic HMBC correlation from H-6eq to C-4a, affording the 5,7-dihydroxy-11-(hydroxymethyl)-2,4dimethoxy-6,7,8,9-tetrahydro-5,9-methanobenzo[8]annulen10(5H)-one structure of 1. The relative configuration of 1 was deduced from a NOESY experiment (Figure 2) in combination with the analysis of 1 H−1H coupling constants. The distinct NOE cross-peaks

revealed that the 351 nm negative Cotton effect (CE) is of pure n−π* origin, while the 317 nm positive CE derives from two π−π* transitions. In this case, the n−π* transition can be safely used for the configurational assignment, and its positive CE derives from P helicity of the fused carbocyclic ring. ent-Astropaquinone B (2) was isolated as an optically active, yellow solid. The planar and relative structure of 2 was determined on the basis of detailed spectroscopic analysis, 1829



Note

which has not been reported yet,5,16 showed a distinct ECD spectrum, and TDDFT ECD calculations (Figures 5 and S4)

including 1D (1H, 13C, and DEPT) and 2D NMR (COSY, HSQC, HMBC, NOESY) spectra and ESIMS. A search of the literature revealed that 2 was identical in all aspects, expect for the specific rotation {[α]18D −45 (c 0.04, CHCl3)}, to astropaquinone B, previously isolated from the freshwater fungus Astrosphaeriella papuana.10 The absolute configuration of astropaquinone B was assigned as (1R,3S) by total synthesis,14 showing consistency in the specific rotations {[α]19D +35.0 (c 0.14, CHCl3)} with that of the natural isolate {[α]27.8D +45.1 (c 0.12, CHCl3)}. Because the sign of the specific rotation of 2 was opposite that of astropaquinone B, 2 is the enantiomer of astropaquinone B. Similarly, entastropaquinone C (3), the demethyl analogue of 2, was found to be the enantiomer of astropaquinone C. The optical activity and absolute configuration of entastropaquinones B and C (2, 3) were further studied by solution ECD measurement and TDDFT/ECD calculations. The ent-astropaquinones B and C had near identical ECD spectra, with bands around 290 and 250 nm. The DFT optimization of the MMFF conformers with the arbitrarily chosen (1R,3S) absolute configuration of 2 produced six conformers with an equatorial C-3 methyl, an axial 1-OH, and P helicity of ring C (Figure S2). The computed Boltzmannweighted ECD curves were mirror images of the experimental one, which proved that ent-astropaquinone B (2) has the (1S,3R) absolute configuration (Figure 4) as indicated above. Because 2 and 3 had near identical ECD spectra, the (1S,3R) absolute configuration of 3 was also confirmed by the ECD measurement.

Figure 5. Solution ECD spectrum of O-methlyherbarin (4) in acetonitrile compared with the PBE0/TZVP-computed Boltzmannweighted ECD spectrum of (R)-4. Bars represent the rotatory strength of the lowest energy conformer.

determined its absolute configuration as R. The ECD studies of 2−4 showed the relationship among the characteristic ECD bands (290 and 250 nm) of the 3,4-dihydro-1H-benzo[g]isochromene-5,10-dione chromophore and configuration (helicity of ring C and absolute configuration), which may serve as a general and convenient method for the configurational assignment of related derivatives. The biological activity of 1 was evaluated in cell-based cytotoxicity assays, in which no significant inhibition against the tumor cells MG-63, LoVo, and A549 was observed (IC50 > 97.4 μM). The discovery of herbarone (1) not only extends the heptaketide family to include a herbarone-type scaffold but also gives insight into parallel or competing biosynthetic processes. The loading of an acetyl-CoA starter unit and Claisen condensation with six malonyl-CoA units generates a common heptaketide precusor, which undergoes aldol cyclization and reductive release to furnish the hemiacetals1 astropaquinone C (3) and herbarin (5). These two metabolites can be further converted to astropaquinone B (2), O-methylherbarin (4), 8-Omethylfusarubrin (6), and dehydroherbarin (7). Herbarone (1) presumably derives from herbaridine A, a C4a−C10a hydrogenation product of herbarin previously isolated from the fungus IBWF79B-90A,16 which can exist in both hemiacetal (8a) and ketone forms (8b). The ketone form of 8 can spontaneously convert to an enol form, which can cyclize by an intramolecular aldol reaction with the C-5 carbonyl group to afford a ketone precursor that is reduced to herbarone (1). Similar reactions have been observed in the Michael reaction of diterpenoids with acetone under basic conditions.17 Further studies should be conducted to confirm the biogenesis of the metabolite 1.

Figure 4. Solution ECD spectrum of ent-astropaquinone B (2) in acetonitrile compared with the PBE0/TZVP-computed Boltzmannweighted ECD spectrum of (1R,3S)-astropaquinone B. Bars represent the rotatory strength of the lowest energy conformer.

As the stereochemical studies of the known compounds are either contradicting or missing and their chiroptical properties have not been reported, their absolute configurations were also studied by ECD measurements and TDDFT ECD calculations. For herbarin (5), the absolute configuration and specific rotation were usually not reported without any indication whether it is racemic or optically active.3,15 Gunatilaka and coworkers reported a low specific rotation for herbarin {[α]15D +4.8 (c 0.06, CHCl3)}.4 The ECD measurement of our isolates of herbarin (5) and 8-O-methylfusarubrin (6) showed a baseline curve, which suggested that these samples are racemic due to the labile cyclic hemiacetal moiety (Figure S3). In contrast, O-methylherbarin (4), the absolute configuration of

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Perkin-Elmer 241 polarimeter. UV absorption spectra were recorded on a Varian Cary 100 UV−vis spectrophotometer; wavelengths are reported in nm. ECD spectra were recorded with a Jasco-715 spectropolarimeter. IR spectra were recorded in thin polymer films on a Nexus 470 FT-IR spectrophotometer (Nicolet,

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Note

Scheme 1. Putative Rearrangement Pathway to 1

USA); peaks are reported in cm−1. The NMR spectra were recorded at 300 K on Bruker Avance 400 and DRX-500 NMR spectrometers. Chemical shifts are reported in parts per million (δ), using the residual CDCl3 signal (δH 7.26 ppm; δC 77.0 ppm) as an internal standard; assignments were supported by COSY, HMQC, HMBC, and NOESY experiments. The mass spectra and high-resolution mass spectra were performed on a Q-TOF Micro mass spectrometer in m/z, resolution 5000; an isopropyl alcohol solution of sodium iodide (2 mg/mL) was used as a reference compound. Semipreparative HPLC was performed on an Agilent-1100 system equipped with a refractive-index detector using a YMC-Pack-ODS-A column (250 × 10 mm, 5 μm). Commercial silica gel (200−300 and 400−500 mesh; Yantai, China) was used for column chromatography. Precoated silica gel plates (HSGF-254, Yantai, China) were used for analytical TLC. Spots were detected on TLC under UV light or by heating after spraying with anisaldehyde H2SO4 reagent. Fungal Material. The fungus Torula herbarum was isolated following surface sterilization from the viscera of sea hare Notarchus leachii cirrosus Stimpson collected from Beihai, Guangxi Province, at a depth of 20 m, in July 2008. The fungus was identified by Dr. Siegfried Draeger (Institut für Mikrobiologie, Technische Universität Braunschweig, Germany). A voucher strain of this fungus (internal strain no.10081) was deposited at the Second Military Medical University. Fermentation and Isolation. The fungus T. herbarum was incubated on biomalt (5% w/v; Villa Natura Gesundprodukte GmbH, Kirn, Germany) solid agar medium at 28 °C for 28 days. After fermentation, the culture medium was extracted with EtOAc (3 × 3 L). The combined extracts were concentrated under vacuum to afford a residue (5.4 g). The residue was fractionated by column chromatography (CC) on silica and eluted with a gradient of acetone in petroleum ether (0:100 → 5:1) to give 27 subfractions based on their TLC profiles. Fraction 13 was subjected to semipreparative HPLC (82% MeOH/H2O; 1.5 mL/min) to yield 7 (5.2 mg, tR 31.6 min). Fraction 14 was purified by semipreparative HPLC (74% MeOH/H2O; 1.5 mL/min), yielding 4 (45.0 mg, tR 24.6 min) and 2 (2.7 mg, tR 43.7 min). Fraction 22 was purified by semipreparative HPLC (50% MeOH/H2O; 1.5 mL/min), yielding 6 (30.3 mg, tR 30.1 min), 3 (10.0 mg, tR 46.7 min), and 5 (170.5 mg, tR 53.8 min). Fraction 26 was purified by semipreparative HPLC (40% MeOH/ H2O; 1.5 mL/min) to give 1 (1.2 mg, tR 22.8 min). Herbarone (1): off-white, amorphous powder; [α]15D +10 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 218 (3.48), 263 (3.01) nm; ECD (CH3CN, c = 6.5 × 10−4) λmax (Δε) 365sh (−0.40), 351 (−1.02), 340sh (−0.63), 329sh (0.16), 317 (0.57), 307sh (0.49), 264 (−0.18), 228 (−1.46), 207sh (2.32), 197 (2.76) nm; IR (film) νmax 3315, 2292, 2851, 1680, 1603, 1454, 1033, 870 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; ESIMS m/ z 309 [M + H]+, 331 [M + Na]+; HRESIMS m/z 331.1160 [M + Na]+ (calcd for C16H20O6Na, 331.1158). ent-Astropaquinone B (2): yellow, amorphous powder; [α]18D −45 (c 0.04, CHCl3); UV (MeOH) λmax (log ε) 216 (4.01), 264 (3.70) nm; ECD (CH3CN, c = 6.3 × 10−4) λmax (Δε) 467 (−0.03), 404 (0.09), 339 (−0.07), 284 (1.48), 251 (−1.78), 214 (−0.78) nm; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table S1; ESIMS m/z 341 [M + Na]+; HRESIMS m/z 341.0990 [M + Na]+ (calcd for C17H18O6Na, 341.0996).

ent-Astropaquinone C (3): yellow, amorphous powder; [α]19D −27 (c 0.04, CHCl3); UV (MeOH) λmax (log ε) 216 (3.61), 258 (3.34) nm; ECD (CH3CN, c = 1.1 × 10−3) λmax (Δε) 462 (−0.07), 403 (0.10), 339 (−0.08), 289 (1.54), 254 (−1.82), 217 (−0.78); 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table S1; ESIMS m/z 327 [M + Na]+; HRESIMS m/z 305.1020 [M + H]+ (calcd for C16H17O6, 305.1025). O-Methylherbarin (4): yellow, amorphous powder; [α]17D +37 (c 0.51, CHCl3); UV (MeOH) λmax (log ε) 215 (4.29), 265 (3.97) nm; ECD (CH3CN, c = 1.0 × 10−3) λmax (Δε) 412 (0.13), 328 (−0.08), 290 (1.10), 287 (−0.93), 229 (−0.09), 206 (1.37) nm; ESIMS m/z 341 [M + Na]+. Computational Section. Conformational searches were carried out by means of the Macromodel 9.7.211 software18 using the Merck molecular force field with an implicit solvent model for chloroform. Geometry reoptimizations at B3LYP/6-31G(d) in vacuo followed by TDDFT ECD calculations using various functionals (B3LYP, BH&HLYP, PBE0) and the TZVP basis set were performed by the Gaussian 0919 package. Boltzmann distributions were estimated from the ZPVE-corrected B3LYP/6-31G(d) energies. ECD spectra were generated as the sum of Gaussians with 2400 cm−1 half-height width (corresponding to ca. 14 at 240 nm), using dipole-velocity computed rotational strengths for conformers above 3%. The MOLEKEL software package was used for visualization of the results. Cytotoxicity Assay. The metabolite 1 was evaluated for cytotoxic activity against human osteosarcoma MG-63 cells, colon cancer LoVo cells, and A-549 lung cancer cells, using a modification of the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method.20 Adriamycin was used as positive control, IC50 = 2.7 μM.

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ASSOCIATED CONTENT

* Supporting Information S

DFT-optimized geometries of the lowest energy conformers of 1, 2, 4, and 5, NMR data for 2 and 3, and HRESIMS and NMR spectra of 1 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86 21 81871257. Fax: 86 21 81871257. E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research work was financially supported by NSFC (Nos. 30873200, 41076082, 81202453), the HURO/0901/274/2.2.2 project, and the NIIDI (10038).

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REFERENCES

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Herbarone, a Rearranged Heptaketide Derivative from the Sea Hare

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