HETEROCYCLES

Short Paper | Regular issue | Vol. 83, No. 2, 2011, pp. 351-356
Received, 29th October, 2010, Accepted, 2nd December, 2010, Published online, 10th December, 2010.
DOI: 10.3987/COM-10-12094

■ New Type Indole Diterpene, Eujindoles, from Eupenicillium javanicum

Shou Nakadate, Koohei Nozawa,* Hitoshi Horie, Yuichi Fujii, and Takashi Yaguchi

School of Pharmaceutical Sciences, Ohu University, 31-1 Misumido, Tomita-machi, Koriyama, Fukushima 963-8611, Japan

Abstract

Two new indole diterpenes, 17-hydroxyeujindole (1) and 17-oxoeujindole (2), were isolated from the extract of Eupenicillium javanicum IFM 59075 (UC62). The structures containing the absolute configuration were determined by spectroscopic methods.

We have been searching fungal metabolites which showed antifungal activity against pathogenic filamentous fungi, Aspergillus fumigatus and A. niger, and/or pathogenic yeasts, Candida albicans and Cryptococcus neoformans. During our research,1 we found that the organic extract of Eupenicillium javanicum IFM 59075 showed a positive coloration with van Urk reagent.2 Further fractionation of the extract led to the isolation of two new indole diterpenes, designated 17-hydroxyeujindole (1) and 17-oxoeujindole (2), along with 10,23-dihydro-24,25-dehydroaflavinine (3),3 nominine (4)4 and 2,3-anhydromevalonic acid δ-lactone.5 In this paper, we report the isolation and structure determination of 17-hydroxyeujindole (1) and 17-oxoeujindole (2) containing their absolute configurations.

The molecular formula of 1 was determined as C28H39NO by the HRFABMS, and that of 2 as C28H37NO by HRFABMS. A positive coloration (blue) with van Urk reagent and the UV spectra suggested the presence of an indole moiety in 1 and 2.
Proton and carbon NMR data for 1 and 2 are provided in Table 1. The 13C NMR spectra of 1 and 2 revealed the presence of 8 and 9 sp2 carbons, respectively. Considering the index of hydrogen deficiency (1; 10, 2; 11), both 1 and 2 are hexacyclic containing indole ring, as 2 has one carbonyl group.

Three proton spin systems observed at 6-H and 7-H (δ 7.16, 2H, m) and 5-H (7.01, 1H, br t) in 1 and at 6-H (δ 7.16, 1H, m), 7-H (7.15, 1H, m) and 5-H (6.99, 1H, dd) in 2 may be assigned to the adjacent protons of the benzene ring of the indole moiety, whereas the protons at 2-H (δ 6.81 in 1 and 6.90 in 2) correlated with NH protons (δ 7.88 and 7.91, respectively) in 1H-1H COSY spectra should be assigned to the proton at C-2 in the indole moiety. The 13C NMR spectrum of 1 was similar to that of aflavinine (6),6 except for the appearance of one quaternary carbon, two methynes and two tertiary methyls instead of that of two olefinic carbons and isopropyl group in 6. The cross peaks between two tertiary methyl protons at 23-H3 (δH 1.04) and 24-H3 (δH 1.46) and two carbons at C-22 (quaternary carbon at δC 37.8) and at C-4 (δC 142.3) of the indole moiety in the HMBC spectrum of 1 suggested that the C-22 was attached to C-4 in the indole moiety. Considering the above result, structures of 17-hydroxyeujindole was assigned as 1. This conclusion was supported by other HMBC correlation peaks indicated in Table 1.

The relative stereochemistry of 1 was assigned by analogy to that of aflavinines and supported by the results of NOESY experiments showed in Table 1. The NOESY correlations of 23-H3 (δH 1.04) with 8-H (δH 3.43) and 20-Hax (δH 1.67), of 17-H (δH 4.61) with 8-H, of 27-H3 (δH 0.86) with 12-Hax (δH 1.57), 19-Hax (δH 1.84) and 21-H (δH 2.31), and even of 9-H (δH 2.79) with 11-Hax (δH 1.99) suggested that both B and C rings were in a chair conformation and A-B and B-C ring junctions were tans and cis, respectively. Further correlations of 14-H (δH 2.31) with 9-H and 16-Hax (δH 2.00), and of 26-H3 (δH 1.01) with 15-Hax (δH 1.75) indicated that D ring also was in a chair conformation and C-D ring junction was cis. Above results revealed relative stereochemistry of 1 corresponding to that of aflavinines. The structure of 17-hydroxyeujindole was thus assigned as depicted in structure 1. This conclusion was supported by other NOESY correlation peaks and the result of NOESY experiment in acetone-d6.
The 13C NMR spectrum of 2 was similar to that of 1, except for the presence of the carbonyl carbon (δC 217.0) instead of one of the carbon bearing a hydroxyl group in 1. From 1H-1H COSY, HMQC and HMBC spectra, the plane structure of 2 was assigned as 17-oxoeujindole oxidized at C-17 of 1. The relative stereochemistry of 2 also was established by the NOESY experiments. NOESY correlations of 23-H3 (δH 0.93) with 8-H (δH 2.64) and 20-Hax (δH 2.01), of 19-Hax (δH 1.70) with 21-H (δH 2.31), of 27-H3 (δH 0.94) with 12-Hax (δH 1.57) and 19-Hax, of 9-H (δH 3.43) with 11-Hax (δH 2.18), 14-H (δH 2.83) and 16Hax (δH 2.97), and even of 26-H3 (δH 0.65) with 15-Hax (δH 1.64) suggested that all B, C and D rings were in a chair conformation and A-B, B-C and C-D ring junctions were tans, cis, and cis, respectively. Consequently, the relative configuration of 2 also has as same as that of 1. This conclusion also was supported by other NOESY correlation peaks.
Because 2 possess substituted cyclohexanone moiety having a chair form, the CD spectrum was examined to propose the absolute stereochemistry. Kirk and Klyne proposed the empirical calculation of CD contribution of the cis-decalons from experimental data.7 According to their conclusion, cis-decalone in 2 was in Class c2ax. The CD spectrum of 2 indicated a negative Cotton effect at 292 nm (Δε -1.4). The absolute configuration of 17-oxoeujindole (2) was thus determined as depicted in 2. The absolute configuration of 1 has not been determined, but it was assumed to be as shown in structure (1) because of the co-occurrence of 17-oxoeujindole (2). 10,23-Dihydro-24,25-dehydroaflavinine (3) and nominine (4) were isolated from the extract of the strain used in this study and identified by comparison with published data.4,5 Though the absolute configurations of 3 and 4 have not yet been determined, those structures also may be assigned as depicted in structures 3 and 4 with the absolute stereochemistry shown because of the co-occurrence of 2.
In view of structures, the indole diterpenes are one of large and diverse groups in natural products from fungi. A number of indole diterpenes have the common tetracyclic diterpene core connected to carbons at C-2 and C-3 in an indole moiety, such as aflatrem,6,8 penitrems9 and paxilline,10 which are the tremorgenic mycotoxin. However, indole diterpenes possessed the tetracyclic diterpene core fused to carbons at C-3 and C-4 in an indole moiety is quiet rare. Petromindole (5)11 from Petromyces muricatus is only known as naturally occurring compound, though the ring formation of the diterpene moiety differs from that of 1 and 2.
Aflavinines and petromindole were isolated from sclerotia and ascostromata, respectively. 17-Hydoxyeujindole (1) also was detected mainly in the CHCl3 extract of the ascomata of the strain IMF 59075 grown on potato dextrose agar (PDA), along with 2 and 3. We have now an interest that E. javanicum may have the capability producing indole diterpenes such as aflavinines and eujindoles.

EXPERIMENTAL
General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 photopolarimeter. The UV and IR spectra were recorded on a JASCO V-560 and a JASCO FT/IR-4100 spectrophotometer, respectively. CD curves were recorded on a JASCO J-820 spectropolarimeter. 1H and 13C NMR spectra were recorded on a JEOL JNM-ECA500 (1H, 500.16 MHz; 13C, 125.77 MHz) spectrometer, using CDCl3 or acetone-d6 solution containing TMS as an internal standard. FABMS was taken with a JEOL JMS-MS700V spectrometer.
Fungal Material. The studied strain was isolated from a cultivated soil in Chiba, Japan, identified as Eupenicillium javanicum based on morphology (by T. Y.), and deposited at the Medical Mycology Research Center, Chiba University, under the accession number IFM 59075 (UC 62). The strain IFM 59075 was cultured at 25 °C for 21 days in 10 Roux flasks containing 250 g of soaked rice in each flask.
Extraction and Isolation. The fermented rice was extracted with CHCl3-MeOH (1:1) and the organic layer was evaporated in vacuo. The resultant extract was suspended in H2O and extracted with EtOAc, and then the organic layer was evaporated in vacuo. The EtOAc extract (13.4 g) was portioned between MeCN and hexane. The MeCN soluble portion (3.6 g) was separated by column chromatography on silica gel (80 g), eluting with CHCl3 containing increasing amounts of acetone. Elution with CHCl3-acetone (20:1) gave a fraction showing a positive coloration for van Urk reagent (blue). The positive fraction was purified by LPLC on a silica gel column using cyclohexane-EtOAc (8:1) followed by the further purification of HPLC on silica gel column [cyclohexane-EtOAc (6:1)] to give 17-Hydroxyeujindole (1) (35 mg), nominine (4) (2 mg), 17-oxoeujindole (2) (7 mg), 10,23-dihydro-24,25-dehydroaflavinine (3) (2 mg) and 2,3-anhydromevalonic acid δ-lactone (6 mg)5 in this elution order.
17-Hydroxyeujindole (1): Colorless needles (hexane-acetone); mp 129.3-130.2 °C; [α]D22 -43.7 ° (c 0.40, MeOH); UV (MeOH) λmax (log ε): 226 (4.51), 283 (3.73), 293 (3.66) nm; IR νmax 3480 (NH), 3287 (OH) cm-1; HRFAB(-)MS m/z : 404.2925 [M - H] + (calcd for 404.2953 for C28H38NO); 1H NMR (acetone-d6) δ 9.75 (NH) 7.08 (7-H, d, J=8.0), 6.99 (2-H, m), 6.98 (6-H, m), 6.87 (5-H, d, J=6.9), 4.59 (17-H, brs), 3.42 (8-H, dd, J=12.0, 5.2), 2.85 (9-H, m), 2.78 (10-H, m), 2.29 (19-Heq, m), 2.27 (14-H, m), 2.24 (21-H, m), 2.02 (11-Hax, m), 1.94 (16-Hax, m), 1.81 (15-Hax, ddd, J=15.1, 12.6, 3.2), 1.8-1.7 (20-H2, m), 1.67 (19-Hax, m), 1.59 (16-Heq, m), 1.55 (12-Hax, td, J=14.3, 2.8), 1.39 (24-H3, s), 1.20 (15-Heq, m), 1.16 (12-Heq, dt, J=14.3, 3.4), 1.04 (11-Heq, brd, J=13.1), 1.00 (26-H3, s), 0.95 (23-H3, s), 0.81 (27-H3, d, J=7.4), 0.79 (25-H3, d, J=6.9); 13C NMR (acetone-d6) δ 142.5(C-4), 135.3(C-7a), 126.8 (C-3a), 122.7 (C-6), 118.1 (C-2), 115.3 (C-3), 112.3 (C-5), 108.8 (C-7), 68.3 (C-17), 44.6 (C-21), 44.3 (C-18), 39.9 (C-13), 38.3 (C-22), 37.2 (C-9), 33.8 (C-8), 32.0 (C-14), 30.6 (C-16), 30.0 (C-10), 29.4 (C-11), 28.7 (C-12), 26.4 (C-15), 26.1 (C-19), 25.2 (C-24), 24.5 (C-23), 23.1 (C-27), 21.6 (C-20), 18.8 (C-26), 16.2 (C-25); NOESY data (H↔H#) 14-H↔9-H, 16-Hax; 21-H↔19-Hax, 24-H3, 27-H3. The other NOESY data were the same as the results in CDCl3 in Table 1. The assignments of 1H and 13C NMR signals for 1 in CDCl3 are summarized in Table 1.
17-Oxoeujindole (2): Colorless amorphous; [α]D22 -28.7 ° (c 0.40, MeOH); UV (MeOH) λmax (log ε): 225 (4.51), 282 (3.83), 292 (3.76) nm; IR νmax 3406 (NH), 1683 (C=O) cm-1; CD (MeOH) Δε (nm) -1.4 (292); HRFAB(+)MS m/z : 404.2930 [M + H] + (calcd for 404.2953 for C28H38NO). The assignments of 1H and 13C NMR signals for 2 in CDCl3 are summarized in Table 1.

ACKNOWLEDGEMENTS
This study was supported in part by an Ohu University Joint Research Fund.

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