HETEROCYCLES

Short Paper | Regular issue | Vol. 83, No. 8, 2011, pp. 1867-1871
Received, 15th April, 2011, Accepted, 20th May, 2011, Published online, 26th May, 2011.
DOI: 10.3987/COM-11-12236

■ Two New Eujindoles from Eupenicillium javanicum

Shou Nakadate, Koohei Nozawa,* and Takashi Yaguchi

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

Abstract

Two new indole diterpenes, 8,21-dehydro-17-hydroxyeujindole (3) and 8,21-dehydro-17,20-epoxyeujindole (4) were isolated from the extract of Eupenicillium javanicum IFM 59075. The structures were determined by spectroscopic methods.

In view of structures, the indole diterpenes are one of large and diverse groups in natural products from fungi. Wicklow et al. found that sclerotia of Asperigillus and Penicillium spp. showed to be a rich source of secondary metabolites with insecticidal/antifeedant activity,1 such as the aflavinins2 and nominine,3 which were the indole diterpenes. Recently we reported the isolation and the structural elucidation of 17-hydroxyeujindole (1) and 17-ketoeujindole (2), new type indole diterpenes, which showed positive coloration (blue) with van Urk reagent,4 from Eupenicillium javanicum.5 During our research, we found yellow spots on TLC with van Urk reagent in the organic extract of E. javanicum IFM 59075. As the result of purification, the substance showing three yellow spots by spraying van Urk reagent were ergosta-4,6,8(14),22-tetraen-3-one6 and two new indole diterpenes, designated 8,21-dehydro-17- hydroxyeujindole (3) and 8,21-dehydro-17,21-epoxyeujindole (4). In this paper, we report the isolation and structure determination of 3 and 4.

The molecular formula of 3 was determined as C28H37NO by the HRFABMS, and that of 4 as C28H35NO by HRFABMS. The 13C NMR spectral data of 3 and 4 were similar to that of 1, except for the appearance of two sp2 carbons instead of that of two sp3 ones in 1, and that of a sp3 methyne carbon attached to an oxygen atom in 4 instead of that of one methylene carbon in 1.

Proton and carbon NMR data for 3 and 4 are provided in Table 1. In the HMBC of 3, the cross peaks from 9-H (δH 2.93) were observed to the two sp2 carbons at δC 124.2 and δC 135.6, along with C-10 (δC 30.6) and C-18 (δC 41.3). The cross peaks from the two tertiary methyl protons (δH 1.46 and 1.49) were observed to the carbon at δC 135.6, along with C-4 (δC 140.7) of indole moiety. Considering the above result, structure of 3 was assigned as 8,21-dehydro-17-hydroxy-eujindole. This conclusion was supported by other HMBC correlation peaks indicated in Table 1.
The NOESY correlations of 27-H3 (δH 0.92) with 12-Hax (δH 1.61) and 19-Hax (δH 1.92), and of 9-H with 11-Hax (δH 1.98), 14-H (δH 2.30) and 16-Hax (δH 1.83), of 14-H with 16-Hax, and even of 17-H (δH 4. 03) with 20-Hu (δH 2.43)7 suggested that both C and D rings were in a chair conformation and the B-C and C-D ring junctions were cis. The relative stereochemistry of 3 was thus assigned as depicted in structure 3. This conclusion was supported by other NOESY correlation peaks showed in Table 1.

In the HMBC of 4, the cross peaks from 9-H (δH 2.83) also were observed to the two sp2 carbons (δC 124.9 and δC 139.5) and C-19 (δC 32.9), along with C-10 (δC 30.7), C-17 (δC 84.5) and C-18 (δC 48.1). The cross peaks from the two tertiary methyl protons (δH 1.41 and 1.54) were observed to the carbon at δC 139.5, along with C-4 (δC 140.2) of indole moiety. Further HMBC correlations of 17-H (δH 3.57) with C-20 (δC 73.6) suggested that 4 possessed an ether ring. In the 1H-1H COSY spectrum of 4, the signal at δH 4.80 was connected with those of 19-H2 (δH 1.95 and 1.99). Considering the above result, structure of 4 was assigned as 8,21-dehydro-17,20-epoxyeujindole. This conclusion was supported by other HMBC correlation peaks indicated in Table 1. The relative stereochemistry of 4 also was established by the NOESY experiments. NOESY correlations of 9-H with 14-H (δH 2.05) and 17-H (δH 3.57), of 26-H3 (δH 0.92) with 11-H (δH 1.50), 16-Hu (δH 1.61) and 19-Hu (δH 1.95), and ever of 12-Hd (δH 1.79) with 9-H suggested that both C and D rings were in boat conformation, and B-C and C-D ring junctions were both cis. This conclusion also was supported by other NOESY correlation peaks.
The absolute configuration of 17-oxoeujindole was determined as depicted in 2 in the previous paper.5 The absolute configurations of 3 and 4 have not been determined, but those structures may be as shown in structures 3 and 4 containing the absolute configuration because of the co-occurrence of 2.
It seems that yellow spot on TLC by spraying van Urk reagent were derived from 8,21-dehydroeujindole derivatives. 8,21-Dehydro-17,20-epoxyeujindole (4) possessed a pentacyclic diterpene core fused to carbons at C-3 and C-4 in an indole moiety. The indole diterpene had the pentacyclic diterpene core is quite rare, except for indole diterpenes containing acetal moiety, which had diterpene core connected to carbons at C-2 and C-3 in indole, such as aflatrem8,9 and paspalitrem10 of the tremorgenic mycotoxin.
18 Strains of E. javanicum were examined for the capacity producing eujindole derivatives. TLC analysis of the CHCl3 extract of each strain grown on moist rice for 3 weeks revealed that two strains (strains IFM 60196 and 60197) possessed the producing capacity of eujindole derivatives. 1, 2, 4 and 10,23-Dihydro- 24,25-dehydroaflavinine (5) were detected in the CHCl3 extract of E. javanicum IFM 60196, and 1, 2 and 5 in that of E. javanicum IFM 60197.

EXPERIMENTAL
General Experimental Procedures. For general experimental details see ref. 5.
Fungal Material. The studied strains were 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 numbers IFM 59075, 60196 and 60197. 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. The 18 strains of E. javanicum were cultured at 25 °C for 21 days in Erlenmeyer flask (100 mL) containing 30 g of soaked rice in each flask.
Extraction and Isolation. The fermented rice was extracted with CHCl3-MeOH (6:4) 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 (14.4 g) was partitioned 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 (9:1) gave a fraction showing a positive coloration for van Urk reagent (blue). The positive fraction was purified by low-pressure liquid chromatography on a silica gel column using cyclohexane-acetone (12:1) to give ergosta-4,6,8(14),22-tetraen-3-one, which showed a positive coloration for van Urk reagent (yellow), and three fractions. The most polar fraction was purified by HPLC on silica gel column [cyclohexane-acetone (12:1)] to give 8,21-dehydro-17,21-epoxy-eujindole (4) (4 mg), 17-Hydroxy- eujindole (1) (35 mg) and 17-oxoeujindole (7 mg) in this elution order. The most polar fraction was also purified by HPLC on silica gel column [cyclohexane-acetone (12:1)] to give 17-oxoeujindole (7 mg) and 8,21-dehydro-17-hydroxyeujindole (3) (2 mg).
8,21-Dehydro-17-hydroxyeujindole (3): Colorless microcrystal (cyclohexane); mp 211.0 – 211.8 °C; [α]D24 -81° (c 0.07, CH2Cl2); UV (MeOH) λmax (log ε): 239 (4.19), 276 (3.86), 297 (3.81), 314 (sh 3.73) nm; IR νmax 3499 (NH), 3239 (OH), 1444, 749 cm-1; HRFAB(+)MS m/z : 404.2918 [M + H] + (calcd for 404.2953 for C28H38NO).
8,21-Dehydro-17,20-epoxyeujindole (4): Colorless leaves (acetone); mp(dec.) 257.3 – 258.3 °C; [α]D24 -332° (c 0.15, CH2Cl2); UV (MeOH) λmax (log ε): 234 (4.32), 278 (3.95), 298 (4.02), 308 (3.99), 317 (sh 3.84) nm; IR νmax 3228 (NH) cm-1; HRFAB(+)MS m/z : 402.2809 [M + H] + (calcd for 402.2797 for C28H36NO).
The assignments of 1H and 13C NMR signals for 3 and 4 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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