HETEROCYCLES

Short Paper | Regular issue | Vol. 87, No. 12, 2013, pp. 2651-2657
Received, 3rd October, 2013, Accepted, 21st October, 2013, Published online, 23rd October, 2013.
DOI: 10.3987/COM-13-12853

■ 3,4-Dihydro-2H,5H-pyrano[3,2-c]chromene and Benzophenone Derivatives from Cultured Lichen Mycobionts of Vietnamese Graphis sp.

Yukiko Takenaka,* Shota Taguchi, Duy Hoang Le, Nobuo Hamada, and Takao Tanahashi

Organic chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan

Abstract

Spore-derived mycobionts of the lichen Graphis sp. collected in Vietnam were cultivated on a malt-yeast extract medium supplemented with 10% sucrose and their metabolites were investigated. Two new phenolic compounds 2 and 3 were isolated along with proserin A (1). Their structures were determined by spectroscopic methods.

Lichens are distinct symbiotic entities composed of an algal or cyanobacterial photobiont and a fungal mycobiont. Lichen thalli are well known to produce a great variety of compounds with different structures, that is, lichen substances, which are considered to have potential biological and ecological functions, such as antimicrobial and antiherbivore activities.1-3 Most of these metabolites are produced by the fungal partner in symbiosis or in the aposymbiotic state. On the other hand, aposymbiotically cultured mycobionts under stressed conditions frequently biosynthesize unique substances that are not detectable in the lichen thalli but are structurally related to fungal metabolites.4,5 From our interest in the metabolic capability of the isolated lichen mycobionts, we previously cultivated the mycobionts of many lichen species collected in Japan and other countries and isolated diverse metabolites from the cultures. In the course of our studies on cultured mycobionts of Vietnamese lichens,6,7 we cultivated the spore-derived mycobionts of an unidentified Graphis sp., and isolated new benzophenone and 3,4-dihydro-2H,5H-pyrano[3,2-c]chromene derivatives from their cultures. We report here the isolation and characterization of these compounds.
Specimens of Graphis sp. were collected from tree bark in Ma Da Forest, Dong Nai Province in Sept. 2008. The polyspore-derived mycobionts were cultivated on a malt-yeast extract medium supplemented with 10% sucrose at 18 oC in the dark. After 14 months, the cultures were harvested and extracted with ether, acetone and then with MeOH. The MeOH extract was partitioned between H2O and n-BuOH. Subsequent purification of the n-BuOH extract by a combination of column chromatography, preparative TLC and preparative HPLC gave three metabolites 1—3.
Compound 1 was identified as a 7-oxo-5,7-dihydrooxepino[4,3,2-de]isochromene derivative, proserin A, which has been isolated from the cultured mycobionts of Graphis proserpens Vain. collected in Japan.8

Compound 2, obtained as a colorless solid, was assigned the molecular formula of C15H14O6 by HR-ESIMS. Its 1H-NMR spectrum showed signals for a methyl group at δH 1.51 (d), an oxygenated methine proton at δH 4.68 (dqd) and methylene protons at δH 2.49 and 2.54 (each dd). The sequence from the methyl group to methylene protons was revealed by a series of 1H-1H COSY correlations and was defined by the NOESY cross peak between the methyl and a proton signal at δH 2.49. The 1H-NMR spectrum, furthermore, revealed a methoxyl at δH 3.82 (s), two oxygenated methylene protons, and a pair of meta-coupled aromatic protons at δH 6.43 and 6.61 (each d, J = 2.5 Hz). The 13C-NMR, DEPT and HSQC spectra of 2 showed the presence of a total of 15 carbon atoms, including a carbonyl at δC 191.6, a carboxyl at δH 176.0 and a methoxyl at δH 56.3, as well as one CH3, two CH2, and one CH sp3 carbons and two CH and six quaternary sp2 carbons. These spectral features were similar to those of (–)-2,3-dihydrocitromycetin (4)9 isolated from a marine-derived isolate of Penicillium bilaii and (–)-neuchromentin (5)10,11 from the cultured broth of Eupenicillium javanicum var. meloforme PF1181. The 1H-NMR spectral data of 2 differed from those of 4 in terms of the presence of an additional methoxy signal and two doublets due to meta-coupled aromatic protons instead of a singlet of aromatic proton. In the 13C-NMR spectrum of 2, an oxygenated aromatic carbon observed in 4 was replaced by an aromatic CH carbon. These findings suggested 2 to be a methylated and deoxygenated derivative of 4. Detailed analysis of the HMBC spectrum of 2 showed the correlations of oxygenated methylene protons at δH 4.69 and 5.07 and an aromatic proton at δH 6.43 with a carbon at δC 162.4 (C-9) and another aromatic proton at δH 6.61 with carbons at δC 101.9 (C-10), 108.1 (C-13) and 176.0 (carboxyl), which revealed the location of the aromatic protons at C-10 and C-12, the methoxyl at C-11, and the carboxyl group at C-13. This substitution pattern was further supported by NOESY interactions between the methoxyl and two aromatic protons (Figure 2). The absolute configuration of C-2 in 2 could be tentatively assigned to S from the same negative sign of its specific optical rotation as that of 4 and 5. Thus, compound 2 was elucidated to be (S)-(–)-8-methoxy-2-methyl-4-oxo-3,4-dihydro-2H,5H-pyrano[3,2-c]chromene-10- carboxylic acid.

Compound 3 was isolated as a colorless solid. Its molecular formula was established as C28H24O14 based on the [M + H]+ ion peak in the HR-ESIMS. Its IR absorptions at 3384, 1711, 1686, 1612 and 1581 cm-1 showed the presence of hydroxyl, aromatic ketone and aromatic ester groups. Its 1H-NMR spectrum revealed signals for a methyl at δH 2.72, four methoxyls at δH 3.58, 3.66, 3.93 and 3.96 and four aromatic protons at δH 7.14 (s), 7.19 (s), 7.79 (d, J = 2.0 Hz) and 8.35 (d, J = 2.0 Hz), the latter two of which showed meta-coupling. The 13C-NMR and DEPT spectra of 3 (Table 1) showed a total of 28 carbon signals. Of these, 18 carbon resonances were observed as duplicate signals, indicating the presence of two sets of the same partial structure. The partial structure consisted of two methoxyls (δC 56.80, 52.6/δC 56.75, 52.4), an ester carbonyl (δC 167.40/δC 167.45), and six aromatic carbons due to a pentasubstituted benzene ring. The chemical shift of three aromatic carbons in the region of δC 140-150 implied a 1,2,3-trioxygenated benzene core. The aromatic proton singlet at δH 7.19 showed an HSQC correlation with a carbon signal at δC 106.1 (C-5) and HMBC correlations with two quaternary carbons at δC 119.3 and 123.2 (C-1 and C-6), two oxygenated quaternary carbons at δC 140.5 (C-3) and 149.5 (C-4), and a carbonyl at 167.4 (C-7). The methoxyl signal at δH 3.96 showed an HMBC correlation with C-4, while the methoxyl signal at δH 3.66 showed an HMBC correlation with the carbonyl carbon C-7, confirming a methoxycarbonyl function. Furthermore, the NOESY interactions of the aromatic proton with the signals of the methoxyl and carbomethoxyl indicated both substituents to be situated at ortho positions of the aromatic proton. The other two oxygenated carbons were deduced to possess hydroxyl groups from the molecular formula of 3. Another set of the signals of aromatic proton at δH 7.14, methoxyl at δH 3.93 and carbomethoxyl at δH 3.58 showed similar HSQC, HMBC and NOESY correlations to those described above (Figure 2). These findings suggested the partial structure to be a 2,3-dihydroxy-4-methoxy-6-methoxycarbonylphenyl group.

The remaining structure of 3 corresponded to C10H6O4. The 13C-NMR and DEPT spectra revealed that ten carbons comprised a methyl (δC 31.1), two aromatic CH carbons, four aromatic quaternary carbons including a hydroxylated carbon, and three carbonyls. These carbon signals were assigned by correlations in the HSQC, HMBC, and NOESY spectra as follows: the methyl signal at δH 2.72 showed HMBC correlations with a carbonyl at δH 200.8 and a quaternary carbon at δH 127.1, indicating substitution of an acetyl group on the benzene ring. A meta-coupled aromatic proton at δH 8.35 showed an HSQC correlation with a carbon signal at δC 137.6 (C-5') and HMBC correlations with another CH carbon at δC 139.3 (C-7'), two carbonyl carbons at δC 200.8 (C-8') and δC 195.9 (C-10'), and an oxygenated aromatic carbon at δC 166.1 (C-3') and NOESY correlation with the methyl signal at δH 2.72; another meta-coupled methine proton at δH 7.79 showed an HSQC correlation with C-7' and HMBC correlations with C-3', C-5', and two carbonyl carbons at δC 195.9 (C-10') and 202.6 (C-1'), which confirmed the substitution pattern of the benzene ring. Each of two carbonyl groups C-1' and C-10' could be connected to the partial structure, 2,3-dihydroxy-4-methoxy-6-methoxycarbonylphenyl group by significant 4-bond HMBC correlations from H-5 to C-1' and from H-5" to C-10'. Thus, the structure of compound 3 was determined as shown and designated graphisidin. The related metabolites with benzophenone moieties, disulochrin12 and acremonidin E13 were isolated from the fermentation of fungi.
For the genus Graphis, our previous cultivation and chemical investigations have been carried out on G. scripta var. pulverulenta,5 G. scripta,14,15 G. prunicola,14 G. cognata,14 G. scripta var. serpentina,16 G. rikuzensis,16 G. apriens,17 G. handelii,17 G. awaensis,17 G. proserpens,8 G. vestitoides18 and unidentified Graphis sp.,19 resulting in the isolation of 6H-dihydro[b,d]pyran-6-one derivatives, isocoumarins, thiophene derivative, chromones, phenyl ethers, 7-oxo-5,7-dihydrooxepino[4,3,2-de]isochromenes, and a 14-membered macrolide. In this study, we have cultivated the spore-derived mycobionts of an unidentified Graphis sp. collected in Vietnam and isolated two new compounds, 3,4-dihydro-2H,5H-pyrano[3,2-c]chromene derivative 2 and benzophenone 3. These types of metabolites have never been detected in the thalli of lichens and lichen mycobionts. The present study constitutes an example to show the diversity of metabolites produced by the cultured mycobionts of Graphis sp.

EXPERIMENTAL
General Procedures. The UV spectra were recorded on a Shimadzu UV-240 spectrophotometer and the IR spectra on a Shimadzu FTIR-8200 infrared spectrophotometer. The optical rotation was measured on a Jasco DIP-370 digital polarimeter. HR-ESIMS were obtained with a Thermo Scientific Q Exactive. The NMR experiments were performed with Varian NMR System-500 and Varian UNITY INOVA (500 MHz) spectrometers with tetramethylsilane as an internal standard. HPLC was performed using a Shimadzu system (LC-10AD VP Liquid Chromatograph, SPD-10A VP UV-VIS Detector). Thin-layer chromatography was performed on pre-coated Kieselgel 60F254 plates (Merck), and spots were visualized under UV light.
Plant Material. Specimens of Graphis sp. were collected from the bark of trees in Ma Da Forest, Dong Nai Province (ca 90 m alt.) in Sept. 2008. The voucher specimens were identified by Prof. H. Miyawaki, Saga University, and were deposited at Saga University, Japan, with the registration No. V62. Mycobionts were obtained from spores discharged from apothecia of a thallus and were cultivated in test tubes containing modified MY10 medium (malt extract 10 g, yeast extract 4 g, sucrose 100 g, agar 15 g, H2O 1 l, pH 7) at 18 ˚C in the dark for 14 months.
Isolation of Metabolites. The harvested colonies (131 test tubes, freeze-dried weight 68 g) were extracted with Et2O, acetone and then with MeOH at room temperature. The Et2O extract (72 mg) and acetone extract (404 mg) were mainly mixtures of fatty acids and glycerides. After concentration, the MeOH extract was suspended in H2O and extracted with n-BuOH. The n-BuOH extract (1.838 g) was chromatographed on a Wakosil 40C18 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) column (H2O-MeOH). The eluate with 30% MeOH was concentrated and the residue was then repeatedly subjected to preparative TLC (CHCl3-MeOH, 4:1) and preparative HPLC (µBondasphere 5µC18-100Å, H2O-MeCN, 4:1, 13:7 or 3:2), giving rise to 1 (36.6 mg), 2 (5.8 mg) and 3 (20.9 mg).
(S)-(–)-8-Methoxy-2-methyl-4-oxo-3,4-dihydro-2H,5H-pyrano[3,2-c]chromene-10-carboxylic acid (2): [α]D29 -18 (c 0.57, MeOH); UV (MeOH) λmax nm (log ε): 211 (3.96), 230 sh (3.86), 250 (3.76), 309 (3.64), 357 (3.75); IR (KBr) νmax cm-1: 3451, 1711, 1642, 1598; 1H-NMR (CD3OD): δ 1.51 (3H, d, J = 6.0 Hz, H3-1), 2.49 (1H, dd, J = 17.0, 4.5 Hz, H-3), 2.54 (1H, dd, J = 17.0, 12.5 Hz, H-3), 3.82 (3H, s, 11-OCH3), 4.68 (1H, dqd, J = 12.5, 6.0, 4.5 Hz, H-2), 4.69, 5.07 (each 1H, d, J = 12.5 Hz, H2-6), 6.43 (1H, d, J = 2.5 Hz, H-10), 6.61 (1H, d, J = 2.5 Hz, H-12); 13C-NMR (CD3OD): δ 20.4 (C-1), 43.7 (C-3), 56.3 (OCH3), 63.9 (C-6), 77.5 (C-2), 101.9 (C-10), 103.7 (C-5), 108.1 (C-13), 109.2 (C-12), 143.1 (C-8), 162.4 (C-9), 165.7 (C-11), 166.3 (C-7), 176.0 (COOH), 191.6 (C-4); HR-ESIMS m/z: Calcd for C15H15O6 [M+H]+: 291.0869. Found : 291.0867.
Graphisidin (3): UV (EtOH) λmax nm (log ε): 217 (4.66), 238 (4.57), 276 (4.33), 340 (3.88); IR (KBr) νmax cm-1: 3384, 1711, 1686, 1612, 1581; 1H- and 13C-NMR: spectroscopic data see Table 1; HR-ESIMS m/z: Calcd for C28H25O14 [M+H]+: 585.1245. Found: 585.1233.

ACKNOWLEDGEMENTS
We are grateful to the Vietnamese Government (Project 322, Ministry of Education and Training) for the fellowship to D. H. Le. We thank Prof. H. Miyawaki (Saga University, Japan) for identification of the lichen specimen. Thanks are also due to Dr. C. Tode (Kobe Pharmaceutical University) for 1H- and 13C-NMR spectra, and to Dr. A. Takeuchi (Kobe Pharmaceutical University) for MS spectral measurements. This research was financially supported in part by Kobe Pharmaceutical University Collaboration Fund.

References

1. S. Huneck, Naturwissenshaften, 1999, 86, 559. CrossRef
2. S. Huneck, Progress in the Chemistry of Organic Natural Products, ed. by W. Herz, H. Falk, G. W. Kirby, and R. E. Moore, Vol. 81, pp. 1-313, Springer Verlag, Wien, New York, 2001.
3. K. Molnár and E. Frakas, Z. Naturforsch., 2010, 65c, 157.
4. H. Miyagawa, N. Hamada, M. Sato, and T. Ueno, Phytochemistry, 1994, 36, 1319. CrossRef
5. T. Tanahashi, M. Kuroishi, A. Kuwahara, N. Nagakura, and N. Hamada, Chem. Pharm. Bull., 1997, 45, 1183. CrossRef
6. D. H. Le, Y. Takenaka, N. Hamada, and T. Tanahashi, Phytochemistry, 2013, 91, 242. CrossRef
7. Y. Takenaka, Y. Naito, D. H. Le, N. Hamada, and T. Tanahashi, Heterocycles, 2013, 87, 1897. CrossRef
8. Y. Takenaka, N. Morimoto, N. Hamada, and T. Tanahashi, Phytochemistry, 2011, 72, 1431. CrossRef
9. R. J. Capon, M. Stewart, R. Ratnayake, E. Lacey, and J. H. Gill, J. Nat. Prod., 2007, 70, 1746. CrossRef
10. Y. Hayakawa, H. Yamamoto, N. Tsuge, and H. Seto, Tetrahedron Lett., 1996, 37, 6363. CrossRef
11. Y. Tanada and K. Mori, Eur. J. Org. Chem., 2001, 1963. CrossRef
12. A. L. Gerea, K. M. Branscum, J. B. King, J. You, D. R. Powell, A. N. Miller, J. R. Spear, and R. H. Cichewicz, Tetrahedron Lett., 2012, 53, 4202. CrossRef
13. H. He, R. Bigelis, E. H. Solum, M. Greenstein, and G. T. Carter, J. Antibiot., 2003, 56, 923. CrossRef
14. T. Tanahashi, Y. Takenaka, N. Nagakura, and N. Hamada, Phytochemistry, 2003, 62, 71. CrossRef
15. Y. Takenaka, T. Tanahashi, N. Nagakura, and N. Hamada, Heterocycles, 2000, 53, 1589. CrossRef
16. Y. Takenaka, T. Tanahashi, N. Nagakura, and N. Hamada, Chem. Pharm. Bull., 2003, 51, 794. CrossRef
17. Y. Takenaka, N. Hamada, and T. Tanahashi, Heterocycles, 2011, 83, 2157. CrossRef
18. D. H. Le, Y. Takenaka, N. Hamada, H. Miyawaki, and T. Tanahashi, Chem. Pharm. Bull., 2013, 61, 358. CrossRef
19. T. Tanahashi, Y. Takenaka, N. Nagakura, N. Hamada, and H. Miyawaki, Heterocycles, 2000, 53, 723. CrossRef