Marine microorganisms, particularly marine actinomycetes, have attracted considerable attention as one of the most important resources for new biologically active metabolites.1 For example, new compounds have been isolated from actinomycetes of sponge origin.2, 3, 4 Our group was engaged in the isolation of actinobacteria from marine sponges. We have recently discovered novel compounds, namely the anthracyclines tetracenoquinocin and 5-imino aranciamycin,5 a teleocidin JBIR-31,6 the tetrapeptides JBIR-34 and -35,7 and the isoprenoids JBIR-46–48.8, 9 Our intention was to support the idea that new species are capable of producing unique metabolites. For this purpose, we isolated new species of Streptomyces from marine sponges and then searched for secondary metabolites in the cultures of isolated strains. In this study, we isolated a new species (SpD081030ME-02) of Streptomyces from a Demospongiae class of marine sponge, and purified a new compound termed JBIR-58 (1, Figure 1a) from the fermentation broth of Streptomyces sp. SpD081030ME-02. We report herein the fermentation, isolation, structure elucidation and, in brief, the biological activity of 1.

Figure 1
figure 1

(a) Structure of JBIR-58 (1) and (b) key correlations in double-quantum filtered-COSY (bold lines) and constant time-HMBC (arrows) spectra of 1. (c) NOE observation for 1 (dashed arrows).

Full size image

Streptomyces sp. SpD081030ME-02 was isolated from a Demospongiae class of marine sponge, collected offshore of Ishigaki City, Okinawa Prefecture, Japan. To identify the species of the strain SpD081030ME-02, we compared the 16S rRNA gene sequences (accession no. AB539864) with those available in the DNA Data Bank of Japan using the basic local alignment search tool.10 The strain was identified as a new species of the genus Streptomyces, as the 16S rRNA gene sequence comparison exhibited a low sequence similarity of 98% with Streptomyces chromofuscus. SpD081030ME-02 was cultured at 27 °C for 5 days in 500-ml Erlenmeyer flasks containing 100 ml of a medium consisting 2.5% starch (Kosokagaku, Tokyo, Japan), 1.5% soybean meal (Nisshin Oillio, Tokyo, Japan), 0.2% dry yeast (Mitsubishi Tanabe Pharma, Osaka, Japan), 0.4% CaCO3 (Kozaki Pharmaceutical, Tokyo, Japan), and Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), with pH adjusted to 6.2 before sterilization. The supernatant of whole broth (2 l) collected by centrifugation was acidified to pH 2–3 and extracted with EtOAc. The EtOAc layer was dried over anhydrous Na2SO4 and evaporated to dryness in vacuo. The residue (2.03 g) was dissolved with n-hexane–CHCl3 (6:1), CHCl3, CHCl3–MeOH (1:1) and MeOH, successively. The CHCl3-soluble fraction (487 mg) was purified using normal-phase medium-pressure liquid chromatography (Purif-Pack SI 30 μm, Moritex, Tokyo, Japan; CHCl3–MeOH, 99:1) and preparative TLC (SiO2; CHCl3–MeOH, 19:1) to yield 1 (9.8 mg, Rf=0.43).

Compound 1 was obtained as a yellow powder ([α]24D +72.3, c. 0.1 (CHCl3), UV (MeOH) λmax (log ɛ) 226 (4.23), 286 (4.18), 297 (4.10, sh) and 412 (3.57) nm). The IR spectrum (CHCl3) of 1 revealed the characteristic absorptions of a ketone (νmax 1662 cm−1) and an amide (1662, 1624 and 1431 cm−1) groups. The HR-ESI-MS data of 1 gave the sodium-adduct ion at m/z 332.0746 (calcd. for C14H15NO7Na, 332.0753). The direct connectivity between protons and carbons was established by the heteronuclear single-quantum coherence spectrum, and the 13C and 1H NMR spectral data for 1 are shown in Table 1. The analyses of double-quantum filtered-COSY and constant time-HMBC11 spectra revealed the structure of 1 as follows.

Table 1 13C and 1H NMR data for 1
Full size table

The proton spin coupling between an oxymethine proton 5-H (δH 4.57) and a methyl proton 11-H (δH 1.57), and the sequence from an oxymethine proton 6a-H (δH 5.09) through methylene proton 7-H (δH 2.41, 2.03) to an acetal proton 8-H (δH 5.42) were observed in the double-quantum filtered-COSY spectrum (Figure 1b). A methoxyl proton (δH 3.52) and 5-H were long-range coupled to C-8 (δC 100.2) and C-6a (δC 57.6), respectively, indicating the 3,7-dioxaoctane structure. The HMBCs from 5-H to a carbonyl carbon C-4 (δC 202.9) and an aromatic carbon C-3a (δC 112.6), from 6a-H to three aromatic carbons C-3a, C-3b (δC 112.0) and C-9a (δC 139.0), and from 8-H to C-9a established a pyranopyran structure.

The long-range coupling from an amide proton 1-NH (δH 6.48) to a carbonyl carbon C-10 (δC 171.6) and an aromatic proton C-1 (δC 108.0), from a hydroxyl proton 2-OH (δH 13.80) to three aromatic carbons C-1, C-2 (δC 153.7) and C-3 (δC 146.8), and from a hydroxyl proton 3-OH (δH 11.20) to three aromatic carbons C-2, C-3 and C-3a indicated that a hydroxysalicylamide unit conjugated to the pyranopyran unit (Figure 1b). Although direct connectivity between C-1 and C-9a was not observed in COSY and HMBC, the NOEs among 8-H,8-O-CH3 and an amide proton 1-NH (δH 8.21) suggested the conjugation between C-1 and C-9a (Figure 1c). Thus, the planar structure of 1 was determined as shown in Figure 1a.

The relative configurations were assigned on the basis of coupling constants and the analysis of NOE experiments. The large coupling constant (J=12.5 Hz) between 6a-H and 7-H (axial, δH 2.03) indicated that 6a-H is in axial location. The NOEs between 6a-H and 11-H, and between 6a-H and 8-O-CH3, also revealed that both the methyl group on C-5 and the methoxyl group on C-8 are in the same location as 6a-H (Figure 1c).

To evaluate the cytotoxic activity of 1 against human cervical carcinoma HeLa cells, the activity was tested by 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). As a result, 1 showed cytotoxic effect against HeLa cells with an IC50 value of 28 μ M for 48 h.

We have isolated a new salicylamide derivative having pyranoisochromone structure from the culture of a new species of Streptomyces. The pyranoisochromone derivatives have been reported to have been isolated as berkelic acid12 from extremophile Penicillium sp. and as perinadine A13 from marine-derived Penicillium citrinum. The compounds show cytotoxicity against several cancer cell lines. However, this is the first report of the pyranoisochromone derivative isolated from Streptomyces. These results confirm that this sponge contains undiscovered microorganisms that possess the ability to produce new substances. We anticipate that this study will convince chemists that new species of Streptomyces can produce compounds containing unique skeletal structures and also encourage them to investigate such species.