In recent years, the malfunction of vacuolar-type ATPases (V-ATPases), which are ubiquitous proton pumps present in the endomembrane system of all eukaryotic cells and in the plasma membranes of many animal cells, has been correlated with an increasing number of diseases such as osteopetrosis, male infertility and renal acidosis.1 In addition, V-ATPase has come into the focus of biomedical research and has even been considered to be an attractive target for cancer chemotherapy and osteoporosis treatment.1 To understand the development of these diseases and to design efficient drugs for their therapy, it is necessary to identify substances that satisfy specific therapeutic requirements. In the course of our screening for V-ATPase inhibitors by using acridine orange staining of V-ATPase-dependent acidic organelles,2 we isolated a new bafilomycin analogue termed JBIR-100 (1) from a culture of Streptomyces sp. IR027-SDHV6 (Figure 1a). In this article, we describe the fermentation, isolation, structure elucidation, and in brief, the biological activity of 1.

Figure 1
figure 1

(a) Structure of 1. (b) Key correlations of 1H-1H DQF-COSY (bold lines) and HMBC (solid arrows).

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Streptomyces sp. IR027-SDHV6 was isolated from a soil sample collected at Iriomote Island, Okinawa Prefecture, Japan, by the sodium dodecyl sulfate-yeast extract method.3 Using the basic local alignment search tool (BLAST), we compared the 16S rRNA gene sequence (AB572859) of IR027-SDHV6 with 16S rRNA gene sequences available in the EzTaxon server to identify the species of strain IR027-SDHV6. Streptomyces sp. IR027-SDHV6 was expected as a new species of the genus Streptomyces; the 16S rRNA gene sequence comparison showed a low sequence similarity of 98.6% with S. capoamus JCM 4734 (AB045877). The detailed identification of this strain will be reported elsewhere.

The strain was cultured on a rotary shaker (220 r.p.m.) at 27 °C for 5 days in 500-ml Erlenmeyer flasks (20 flasks) containing 100 ml of a production medium consisting of 2.5% starch (Kosokagaku, Tokyo, Japan), 1.5% soybean meal (Nisshin Oillio Group, Tokyo, Japan), 0.2% dry yeast (Mitsubishi Tanabe Pharma, Osaka, Japan), 0.4% CaCO3 (Kozakai Pharmaceutical, Tokyo, Japan) and 1% Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), pH 7.0 (before sterilization). The mycelia from the culture broth (100 ml × 20 flasks) were treated with Me2CO (400 ml). After in vacuo concentration, the residue was extracted with EtOAc. The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness in vacuo. The residue (312 mg) was purified using normal-phase medium-pressure liquid chromatography (Purif-Pack, SI 30 μm; Shoko Scientific, Yokohama, Japan) and developed with hexane–EtOAc (1:1). The active fraction was further purified by preparative reversed-phase HPLC using a CAPCELL PAK C18 MG II column (5.0 μm, 20 i.d. × 150 mm; Shiseido, Tokyo, Japan) and developed with 85% MeOH–H2O containing 0.4% formic acid (flow rate, 10 ml min−1) to yield 1 (20.0 mg, retention time=32.5 min).

Compound 1 was obtained as a colorless amorphous solid ([α]D25–15.7, c 0.1, UV λmax(ɛ) 244 (28700), 272 (sh, 13400) nm, in MeOH). The IR spectrum (CHCl3) of 1 revealed the characteristic absorptions of carbonyl groups (νmax: 1720, 1700, 1680 cm−1), suggesting the presence of conjugated esters and/or carboxylic acids. The molecular formula of 1 was determined to be C39H60O11 (found: m/z 703.4082 [M−H]−, calcd: m/z 703.4057 for C39H59O11) by HR-electrospray ionization MS. The direct bonds between each proton and carbon were established by the heteronuclear single quantum coherence spectrum. The 13C and 1H NMR spectral data for 1 are shown in Table 1. Analysis of double-quantum filtered (DQF)-COSY and constant time heteronuclear multiple bond correlation4 spectra revealed the structure of 1 as follows.

Table 1 13C and 1H NMR data for 1
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In the DQF-COSY spectrum, we observed four allylic couplings between a methyl proton 2-CH3 (δH 2.03) and an olefinic proton 3-H (δH 7.22), between a methyl proton 4-CH3 (δH 1.99) and an olefinic proton 5-H (δH 5.85), between methylene protons 9-H (δH 2.16, 1.97) and an olefinic proton 11-H (δH 5.85) and between a methyl proton 10-CH3 (δH 1.94) and the olefinic proton 11-H (Figure 1b). In addition, we determined the sequences from 5-H to 9-H through a methine proton 6-H (δH 2.56), which was in turn coupled to a methyl proton 6-CH3 (δH 1.08), an oxymethine proton 7-H (δH 3.30, δC 81.3) and a methine proton 8-H (δH 1.95), which showed 1H spin coupling with a methyl proton 8-CH3 (δH 0.94). The sequences from an olefinic proton 11-H to an oxymethine proton 15-H (δH 4.96) through olefinic protons 12-H (δH 6.50) and 13-H (δH 5.19) and an oxymethine proton 14-H (δH 3.91) were also determined. Furthermore, on the basis of the HMBC correlations from 2-CH3 and 15-H to a carbonyl carbon C-1 (δC 172.0), from 4-CH3 to C-3 (δC 146.4) and from a methoxyl proton (δH 3.25) to C-14 (δC 82.6), we found that the structure of 1 was that of a 16-membered macrolide.

Three other partial structures were elucidated as follows. The sequences from a methyl proton 16-CH3 (δH 0.81) to 18-CH3 (δH 1.02) through methine protons 16-H (δH 2.08), 17-H (δH 4.08), which was in turn coupled to a hydroxyl proton 17-OH (δH 4.85), and 18-H (δH 1.74) indicated a pentan-3-ol substructure. The sequence from methylene protons 20-H (δH 2.37, 1.24) to 25-H (δH 0.91) through an oxymethine proton 21-H (δH 5.10), 22-H (δH 1.61), which was also coupled to a methyl proton 22-CH3 (δH 0.83) and oxymethine proton 23-H (δH 3.62) and 24-H (δH 1.90), was established. Additional spin couplings between 22-H and a methyl proton 22-CH3 (δH 0.83) and between 24-H and a methyl proton 26-H (δH 0.78) determined the substituted positions of these methyl residues. In addition, 1H-13C long-range couplings from 18-H, 18-Me, a hydroxyl proton 19-OH (δH 5.67), 20-H and 23-H to an acetal carbon C-19 (δC 98.8) determined a pyran ring substructure. Finally, 1H-13C long-range couplings from 15-H to C-16 (δC 37.6) indicated that this side chain substructure was substituted at the position of C-15 in the macrolide unit.

The remaining fumaric acid unit was deduced on the basis of proton coupling between two olefinic protons 2′-H (δH 6.84) and 3′-H (δH 6.92), and long-range couplings of 2′-H and 3′-H to two carbonyl carbons C-1′ (δC 164.1) and C-4′ (δC 169.0). A long-range coupling from oxymethine proton 21-H to C-1′ proved that the substitution position of the fumaric acid unit was at the C-21 position, as shown in Figure 1b.

The four olefins at C-2, C-4, C-12 and C-2′ were all determined to be E-configuration on the basis of their high-field 13C chemical shifts of the methyl residues 2-CH3 (δC 13.7) and 4-CH3 (δC 15.2), and the coupling constants between 12-H/13-H (J12-H–13-H,-15.0 Hz) and 2′-H/3′-H (J2′-H–3′-H,-15.5 Hz). Another olefin at C-10 also appeared to possess the E-configuration when the 13C chemical shifts at 10-CH3 (δC 20.1) were compared with those of known analogues.5 Thus, the structure of 1 was determined to be that of a new 16-membered macrolide, as shown in Figure 1a. The structure of 1 was found to be structurally related to bafilomycin C1, which shows V-ATPase inhibitory activity, and differs from that of bafilomycin C1 in the presence of the methyl group at the C-2 position.

The cytotoxic activity of 1 against human cervical carcinoma HeLa cells 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). Compound 1 exhibited cytotoxic effect against HeLa cells (IC50=72.6 nM) at 48 h after administration. Next, the V-ATPase inhibitory activity of 1 against the accumulation of intracellular acidic organelles, which is strongly linked to V-ATPase inhibition in HeLa cells, was evaluated by acridine orange staining.2 Dye accumulation (orange dots) was found to be inhibited by the administration of 1 (100 nM) (Figure 2), indicating the inhibitory property of 1 against V-ATPase.

Figure 2
figure 2

Inhibition of V-ATPase activity by 1 in HeLa cells. Cells were treated with 100 nM of 1 for an hour. Then, the cells were stained with acridine orange (Sigma, St Louis, MO, USA) at a concentration of 0.5 μg ml−1, incubated for 10 min, and washed twice with phosphate-buffered saline. The images were visualized under an LSM 510 Confocal Laser Scanning microscope (Carl Zeiss MicroImaging, Thornwood, NY, USA). Acridine orange emits red fluorescence in acidic vesicular organelles and green fluorescence in the cytoplasm and nucleus.

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