New tensidols, JBIR-86 and JBIR-87, isolated from Aspergillus sp. fJ80

Aspergillus is a member of the phylum Ascomycota. There are over 185 known species of ascomycetes, about 20 of which are known to be harmful to humans and other animals. The Aspergillus species is known to produce more than 1000 secondary metabolites containing bioactive compounds, such as mevinolin, aflatoxin and citrinin.¹ In a previous study, we reported that a fungus belonging to the Aspergillus species produces a novel secondary metabolite, JBIR-15.² Therefore, in this study, we screened for novel secondary metabolites from cultures of Aspergillus species and succeeded in discovering novel furopyrrole compounds, JBIR-86 (1) and JBIR-87 (2), in addition to the known compounds, tensidols A and B (Figure 1a). In this study, we report the fermentation, isolation and structural determination of 1 and 2, and briefly describe their biological activities.

Figure 1

(a) Structures of tensidols, JBIR-86 (1) and JBIR-87 (2). (b) Key correlations in DQF-COSY (bold lines) and HMBC (arrows) spectra of 1. (c) Key correlations in DQF-COSY (bold lines) and HMBC (arrows) spectra of 2. (d) Key correlations in DQF-COSY (bold lines) and HMBC (arrows) spectra of tensidol B.

Aspergillus sp. fJ80 was isolated from a soil sample collected in Akita Prefecture, Japan. The strain was cultivated in 50 ml test tubes containing 15 ml of the seed medium (24 g l⁻¹ potato dextrose broth; BD Biosciences, San Jose, CA, USA). The test tubes were shaken on a reciprocal shaker (355 r.p.m.) at 27 °C for 3 days. Aliquots (5 ml) of the seed culture were inoculated into 500 ml Erlenmeyer flasks containing the production medium (15 g brown rice and 45 ml solution containing 0.67 g l⁻¹ yeast extract, 0.33 g l⁻¹ sodium tartrate dihydrate and 0.33 g l⁻¹ K₂HPO₄) and incubated in static culture at 27 °C for 14 days.

The production culture (10 flasks) was extracted with 80% aqueous Me₂CO (1 l) and concentrated in vacuo. The aqueous concentrate (200 ml) was extracted with EtOAc (200 ml × 5). After drying over Na₂SO₄, the organic layer was evaporated to dryness. The residue (0.52 g) was subjected to normal-phase, medium-pressure liquid chromatography (Purif-Pack SI-60; Moritex, Tokyo, Japan) and successively eluted with gradient systems of n-hexane–EtOAc (0–30% EtOAc) and CHCl₃–MeOH (0–50% MeOH). The 1% MeOH-eluted fraction (70.0 mg) was purified by preparative reversed-phase HPLC using an L-column2 ODS column (20 i.d. × 150 mm; Chemical Evaluation and Research Institute, Tokyo, Japan) with 55% MeOH–H₂O containing 0.1% formic acid (flow rate 10 ml min⁻¹) to yield 2 (0.99 mg, retention time (Rt) 12.6 min), tensidol B (3.22 mg, Rt 9.1 min) and a crude fraction of 1 (7.25 mg, Rt 4.3 min). The eluate containing 1 was further purified by preparative reversed-phase HPLC using an L-column2 ODS column (20 i.d. × 150 mm) with 50% MeOH–H₂O containing 0.1% formic acid (flow rate 10 ml min⁻¹) to yield 1 (1.02 mg, Rt 13.0 min) and tensidol A (3.81 mg, Rt 11.0 min).

Compound 1 was isolated as a colorless oil that yielded an [M + H]⁺ ion at m/z 244.0986 in the HR-ESI-MS corresponding to a molecular formula of C₁₄H₁₂NO₃ (calcd for C₁₄H₁₃NO₃, 244.0974). Furthermore, 1 displayed the following UV and IR spectra: UV (CHCl₃) λ_max(ɛ) 241 (11 750), IR (KBr) ν_max 1670 cm⁻¹. The ¹H and ¹³C NMR spectral data for 1 are listed in Table 1. The structural information on 1 was obtained by a series of 2D NMR analyses such as heteronuclear single-quantum coherence, HMBC and double-quantum filtered (DQF)-COSY spectra (Figure 1b). The spin couplings observed in the DQF-COSY spectrum among aromatic protons 9/13-H (δ_H 7.24), 10/12-H (δ_H 7.32) and 11-H (δ_H 7.32) revealed the existence of a phenyl residue. In the HMBC spectrum, a singlet methylene proton 7-H (δ_H 3.83) was ¹H–¹³C long-range coupled to aromatic methine carbons C-9/13 (δ_C 129.4). The ¹H–¹³C long-range couplings from 9/13-H to aromatic carbon C-11 (δ_C 128.0) and to methylene carbon C-7 (δ_C 40.0) established the presence of a benzyl moiety, as shown in Figure 1b. In addition to the resonances ascribed to the benzyl moiety, 1 showed seven additional ¹³C resonances. Long-range couplings from aromatic protons 2-H (δ_H 8.43) and 5-H (δ_H 6.21) to each of the aromatic carbons C-3 (δ_C 163.9), C-3a (δ_C 119.8), C-4 (δ_C 175.0) and C-6a (δ_C 167.6) were observed. Moreover, the long-range coupling between the methoxyl proton 3-OMe (δ_H 3.87) and C-3 revealed that the methoxyl group is substituted at the C-3 position. A comparison of the ¹³C chemical shifts with those of tensidol A suggested the existence of a 4-methoxy-6H-furo[2,3-b]pyrrol-3-ol moiety³ (Figure 1b). Finally, the ¹H–¹³C long-range couplings from 7-H to C-5 and C-6a and from 5-H to C-7 established a relation between these partial structures. Although four-bond HMBC correlations from H-2 to C-4 and from H-5 to C-3 were observed, and the ¹³C chemical shifts were identical to the tensidol A chemical shifts, we could not determine the exact assignment of the C-3 and C-4 signals.

Table 1 ¹H and ¹³C NMR spectral data for JBIR-86 (1), JBIR-87 (2) and tensidol B

Compound 2 was obtained as a colorless oil ([α]_D + 6.0, c 0.1, MeOH), and it displayed a UV spectrum (λ_max(ɛ) 239 (14 050), in CHCl₃) and an IR spectrum (KBr, ν_max 1730 and 1670 cm⁻¹). The molecular formula of 2 was established as C₁₉H₁₈NO₆ by HR-ESI-MS (m/z 358.1286 [M + H]⁺, calcd for C₁₉H₁₉NO₆, 358.1291). The NMR data were similar to those of 1 except for the appearance of two carbonyl carbons C-1′ (δ_C 174.4) and C-4′ (δ_C 177.8), a methine carbon C-3′ (δ_C 36.3), a methylene carbon C-2′ (δ_C 42.6) and a methyl carbon C-5′ (δ_C 17.4), as listed in Table 1. The sequence from doublet methyl protons H-5′ (δ_H 1.22) to methylene protons H-2′ (δ_H 2.90, 3.16) through a methine proton 3′-H (δ_H 2.98) was observed in the DQF-COSY spectrum. The long-range couplings from methoxyl protons 4′-OMe (δ_H 3.66) to C-4′; from 5′-H to C-2′, C-3′ and C-4′; and from 2′-H and 3′-H to C-1′ established the presence of a 4-methoxy-3-methyl-4-oxobutanoate moiety, as shown in Figure 1c. Because the long-range couplings from 3-OH (δ_H 11.71) were not the same as those observed in 1, we could not determine the substituted position of the side-chain moiety. To determine the assignment of C-3 and C-4, which would establish the substituted positions of the methyl residue and the side-chain moiety in 1 and 2, respectively, detailed NMR analyses of tensidol B isolated from Aspergillus sp. fJ80 were carried out (Figure 1d). Using the analysis results, we could confirm that the long-range couplings from a hydrogen-bonded phenolic hydroxy proton 3-OH (δ_H 11.72) to C-2 (δ_C 163.4), C-3 (δ_C 161.5) and C-3a (δ_C 118.8) established the exact assignment of all the signals of tensidol B and 3-OH was found to be substituted at the C-3 position. Taking into consideration the ¹³C chemical shifts at C-3 and C-4 in both 1 and 2, we conclude that the methyl residue and 4-methoxy-3-methyl-4-oxobutanoate moiety of 1 and 2 are substituted at the C-3 and C-4 positions, respectively. Note that the assignment of the ¹H and ¹³C NMR signals published by Fukuda et al.³ should be corrected as per the results of this study. The identical ¹J_C–H values at C-2 and C-5 in tensidol B, 1 and 2 (¹J_2C–2H: tensidol B 200.6 Hz, 1 197.4 Hz, 2 198.0 Hz; ¹J_5C–5H: tensidol B 167.5 Hz, 1 165.4 Hz, 2 168.0 Hz) provided further evidence in support of the proposed structures of 1 and 2. Thus, 1 and 2 were established as 3-O-methyl tensidol A and 4′-O-methyl tensidol B, respectively.

Because it has been reported that tensidols A and B potentiate miconazole activity against Candida albicans,³ we tested the miconazole-potentiating activities of 1 and 2 using the previously reported paper disk method.^{3, 4} As in the case of the tensidols, 1 and 2 did not show an inhibition zone against C. albicans at 50 μg per disk (plate A) and exhibited miconazole-potentiating activity in a dose-dependent manner on plate B (plate A containing 0.06 μM miconazole), as listed in Table 2. However, the activity of 1 was weaker than that of tensidol A. In addition, the cytotoxic activity of 1 and 2 against several cancer cell lines was tested by the WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) colorimetric assay (Cell Counting Kit; Dojindo, Kumamoto, Japan). However, 1 and 2 showed no cytotoxicity even at a concentration of 50 μg ml⁻¹ for 48 h.

Table 2 Miconazole-potentiating activity of tensidols, JBIR-86 (1) and JBIR-87 (2)