Marine microorganisms, particularly, marine actinomycetes, are one of the most important resources for new biologically active metabolites,1 and in fact, many new compounds have been isolated from sponge-derived actinomycetes.2, 3, 4 We have recently discovered novel compounds, namely the anthracyclines tetracenoquinocin and 5-iminoaranciamycin,5 a teleocidin JBIR-31,6 the tetrapeptides JBIR-34 and JBIR-35,7 the isoprenoids JBIR-46, JBIR-47, JBIR488, 9 and a new salicylamide JBIR-58.10 Our intention was to support the idea that new species are capable of producing unique metabolites. For this purpose, we isolated new species of actinomycetes from marine sponges and then searched for secondary metabolites in the culture of isolated strains. In this study, we isolated a new species (SpB081030SC-15) of Actinomadura from an unidentified marine sponge and purified a new diterpene compound designated as JBIR-65 (1, Figure 1a) from the fermentation broth of Actinomadura sp. SpB081030SC-15. In this study, we report the fermentation procedure to obtain 1 and its subsequent isolation, structure elucidation and, in brief, its biological activities.

Figure 1
figure 1

(a) Structure of JBIR-65 (1). (b) Key correlations in double-quantum filtered-COSY (bold lines) and heteronuclear multiple-bond correlation (HMBC) (arrows) spectra of 1.

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Actinomadura sp. SpB081030SC-15 was isolated from an unidentified marine sponge collected offshore of Ishigaki Island, Okinawa Prefecture, Japan. To identify the species of the strain SpB081030SC-15, we compared its 16S ribosomal RNA gene sequences (Accession No. AB123566) with those available in the DNA Data Bank of Japan using a basic local alignment search tool.11 The strain was identified as a new species of the genus Actinomadura because comparison of its 16S ribosomal RNA gene sequence revealed a low sequence similarity of 98% with Actinomadura nitritigenes (AB364595).

The strain SpB081030SC-15 was cultivated in 50-ml test tubes each containing 15 ml of a seed medium consisting of starch (Kosokagaku, Tokyo, Japan) 1.0%, polypeptone (Nihon Pharmaceutical, Tokyo, Japan) 1.0%, molasses (Dai-Nippon Meiji Sugar, Tokyo, Japan) 1.0% and meat extract 1.0% (Extract Ehlrich, Wako Pure Chemical Industry, Osaka, Japan), pH 7.2 (adjusted before sterilization). The seed culture in test tubes was agitated on a reciprocal shaker (355 r.p.m.) at 27 °C for 2 days. Aliquots (2.5 ml) of the broth were transferred to 500-ml baffled Erlenmeyer flasks containing 100 ml of a production medium consisting of glycerin 2.0%, molasses 1.0%, casein 0.5% and CaCO3 0.4%, pH 7.2 (adjusted before sterilization) and cultured on a rotary shaker (180 r.p.m.) at 27 °C for 5 days.

The mycelia in the fermentation broth (2 l) was separated by centrifugation and then extracted with 80% acetone. The extract was evaporated in vacuo to remove the acetone, and the aqueous residue was extracted with EtOAc. The organic layer was dried over Na2SO4 and evaporated to dryness. The residue (700 mg) was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI-60, Moritex, Tokyo, Japan) and eluted with a gradient system of n-hexane–EtOAc (0–30% EtOAc) and CHCl3-MeOH (0–50% MeOH), successively. The 3% MeOH-eluted fraction (50.1 mg) was further purified by preparative reverse-phase HPLC using an L-column 2 column (20 i.d. × 150 mm; Chemical Evaluation and Research Institute, Tokyo, Japan) with 60% MeOH-H2O containing 0.1% formic acid (flow rate: 10 ml·min−1) to yield 1 (0.22 mg, retention time 15.5 min).

Compound 1 was isolated as a colorless oil ([α]D25 −90.0, c 0.1, MeOH) that yielded an [M+H] + ion at m/z 317.1736 in HR electrospray ionization-MS spectra consistent with the molecular formula C19H24O4 (calcd for C19H25O4, 317.1753). The IR absorption (KBr) vmax at 3360 and 1660 cm−1 showed the presence of hydroxyl and quinone functional groups in 1. Direct connectivity between protons and carbons was established by analysis of the heteronuclear single quantum coherence spectrum, and the tabulated 13C and 1H NMR spectral data for 1 are shown in Table 1. The analyses of double-quantum filtered (DQF)-COSY and heteronuclear multiple-bond correlation spectra revealed the structure of 1 (Figure 1b). Correlations between olefinic protons 15-H (δH 5.75) and 16-H (δH 4.85 and 4.92) and between methylene protons 11-H (δH 2.71) and 12-H (δH 1.52), respectively, were observed in the DQF-COSY spectrum. The 1H-13C long-range couplings between a singlet methyl proton 17-H (δH 1.02) and a quaternary carbon C-13 (δC 33.9), methylene carbons C-12 (δC 33.2) and C-14 (δC 33.2) and an olefinic carbon C-15 (δC 146.4) revealed the connectivity between C-11 (δC 27.0), C-12, C-13, C-14, C-15 and C-17 (δC 25.2). Furthermore, 1H-13C long-range couplings between 11-H and methylene proton 14-H (δH 1.91 and 2.46) and olefinic quaternary carbons C-8 (δC 130.1) and C-9 (δC 165.4) and homoallylic coupling between 11-H and 14-H proved the existence of a 4-methyl-4-vinylcyclohex-1-ene moiety.

Table 1 1H and 13C NMR spectral data for JBIR-65 (1)
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A correlation between a methine proton 5-H (δH 3.39) and methylene protons 6-H (δH 2.61 and 2.78) was observed in the DQF-COSY spectrum. The 1H-13C long-range couplings from a singlet methyl proton 18-H (δH 0.98) to an oxymethine carbon C-1 (δC 78.2), a quaternary carbon C-10 (δC 49.0), C-9 and a methine carbon C-5 (δC 42.0), whose methine proton 5-H was in turn long-range coupled to an α,β-unsaturated ketone carbonyl carbon C-7 (δC 197.4) and C-8, revealed the presence of an octalone substructure. A hydroxymethyl proton 19-H (δH 4.25) was 1H-13C long-range coupled to an olefinic methine carbon C-3 (δC 120.4), olefinic quaternary carbon C-4 (δC 165.1) and C-5. The 1H-13C long-range couplings between an olefinic methine proton 3-H (δH 6.45) and C-1 and between an oxymethine proton 1-H (δH 4.28) and an α,β-unsaturated ketone carbonyl carbon C-2 (δC 199.2) established the presence of a six-membered moiety, as shown in Figure 1b. The 13C NMR chemical shifts in the case of 1 resembled those in the case of 15-isopimaradien-7-one12 and 2-oxo-18-hydroxy-10α,17α,19α,20β-(−)-cleroda-3,13(16),14-triene,13 supporting the planar structure of 1, as shown in Figure 1a.

To evaluate the biological activity of 1, we conducted tests to evaluate its cytotoxic, antimicrobial and radical scavenging activities. The results showed that 1 protects neuronal hybridoma N18-RE-105 cells14, 15, 16 from L-glutamate toxicity with an EC50 value of 31 μM. However, this protective effect of 1 was weaker than that of the representative antioxidant α-tocopherol (EC50 value, 6.3 μM). It would be interesting to clarify the mode of action of 1 because its radical scavenging activity could not be determined on the basis of its chemical structure. The cytotoxic activity of 1 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 showed no cytotoxicity even at a concentration of 50 μg ml−1 for 48 h. In addition, 1 showed no antimicrobial activity against Micrococcus luteus, Escherichia coli and Candida albicans.

In conclusion, we isolated a new diterpene, JBIR-65 (1), from the strain SpB081030SC-15. Diterpenes have been isolated from many fungi and plants, however, few reports of the isolation of diterpenes from actinobacteria are available. To our knowledge, this is the first report of a diterpene compound isolated from the genus Actinomadura. This study suggests that new species of actinomycetes isolated from marine organisms have the potential to produce new bioactive compounds.