Members of the class Actinobacteria are known to produce pharmaceutically important compounds and have been extensively studied. Thus far, soil has been the primary source of Actinobacteria. However, the rate of discovery of novel compounds from soil Actinobacteria strains has decreased significantly. Therefore, we explored the possibility of isolating unusual Actinobacteria capable of producing new metabolites by employing specially devised methods instead of the conventional selection methods using antibiotics, and attempted to isolate rare Actinobacteria from soil samples using the membrane filter (MF) method.1 In fact, we have already isolated a new Streptomyces sp. using this method,2 and have described new promothiocin derivatives from the isolated Streptomyces.3 In the course of searching for metabolites from cultures of Actinobacteria isolated using the MF method, we isolated novel compounds designated JBIR-73 (1) and JBIR-67 (2), which possess unique structures, as well as the known benzastatin derivatives, virantmycin4 and 7-hydroxyl benzastatin D,5 from Streptomyces sp. RI18 (Figure 1a).

Figure 1
figure 1

(a) Structures of JBIR-73 (1) and JBIR-67 (2). (b) Key correlations observed in 1 by 1H–1H COSY (bold lines) and HMBC (solid arrows). (c) Key correlations observed in 2 by 1H–1H COSY (bold lines) and HMBC (solid arrows).

Full size image

Streptomyces sp. RI18 was isolated from a soil sample collected in Shuri, Okinawa Prefecture, Japan, using the MF method.1, 2 To identify the genus of the strain RI18, we compared the 16S rRNA gene sequence of RI18 with those available in the DNA Data Bank of Japan using the basic local alignment search tool. The strain was identified as the genus Streptomyces.

The strain was cultivated in 50-ml test tubes containing 15 ml of a seed medium consisting of starch (Kosokagaku, Tokyo, Japan) 1.0%, polypepton (Nihon Pharmaceutical, Tokyo, Japan) 1.0%, molasses (Dai-Nippon Meiji Sugar, Tokyo, Japan) 1.0% and meat extract (Extract Ehlrich, Wako Pure Chemical Industry, Osaka, Japan) 1.0%, pH 7.2 (before sterilization). The test tubes were shaken 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 (20 flasks) containing 100 ml of a production medium consisting of starch 1.0%, glucose 1.0%, glycerol 1.0%, polypepton 0.5%, yeast extract (BD Biosciences, San Jose, CA, USA) 0.2%, corn steep liquor (Oriental Yeast, Tokyo, Japan) 1 ml, NaCl 0.1% and CaCO3 0.32%, pH 7.4 (before sterilization), and cultured on a rotary shaker (180 r.p.m.) at 27 °C for 5 days.

The fermentation broth (2 l) was centrifuged, and the mycelial cake was extracted in Me2CO (500 ml). After in vacuo concentration, the aqueous concentrate was extracted with EtOAc (100 ml × 3). The extract was dried over Na2SO4 and evaporated to dryness. The residue (0.79 g) was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI-60; Shoko Scientific, Yokohama, Japan), and successively eluted with a gradient system of n-hexane-EtOAc (0–30% EtOAc) and CHCl3-MeOH (0–50% MeOH). Virantmycin was isolated from the fraction eluted with 2% MeOH. The fraction eluted with 3% MeOH (57.2 mg) was purified by preparative reverse-phase high-performance liquid chromatography using an L-column2 ODS column (20 i.d. × 150 mm; Chemical Evaluation and Research Institute, Tokyo, Japan), with 65% MeOH-H2O containing 0.1% formic acid (flow rate 10 ml min−1) to yield 2 (0.88 mg; retention time 11.3 min) and 7-hydroxyl benzastatin D. The supernatant of the fermentation broth was applied to a Diaion HP-20 column (Mitsubishi Chemical., Tokyo, Japan). The column was washed with 30% aqueous MeOH and eluted with 100% MeOH. The 100% MeOH eluate was evaporated in vacuo, and the residue (0.99 g) was subjected to reverse-phase medium pressure-liquid chromatography (Purif-Pack ODS-100; Shoko Scientific.) with an aqueous MeOH linear gradient system (20–100% MeOH). The 70% MeOH eluate (85.1 mg) was subjected to preparative reverse-phase high-performance liquid chromatography using an L-column2 ODS column developed with 55% aqueous MeOH containing 0.1% formic acid to give 1 (1.94 mg; retention time 22.2 min).

Compound 1 was isolated as a colorless oil ([α]D +12.0, c 0.1, MeOH) that gave an [M+H]+ ion at m/z 545.2797 on HR-ESI-MS. This spectrum was consistent with a molecular formula of C28H40N4O5S (calcd for C28H41N4O5S, 545.2798). Compound 1 displayed the following IR and UV spectra: IR (KBr) νmax=3300 and 1670 cm−1; UV λmax (ɛ)=306 nm (14 900) in MeOH. The 1H and 13C NMR spectral data for 1 are shown in Table 1. The structure of 1 was elucidated by a series of 2D NMR analyses, including HSQC, field-gradient 1H–1H COSY and HMBC (Figure 1b). In the HMBC spectrum, a singlet methyl proton H-16 (δH 1.50) was long-range coupled to a methylene carbon C-12 (δC 28.1), olefinic quaternary carbons C-13 (δC 127.4) and C-14 (δC 124.4), which, in turn, was coupled from singlet methyl protons H-15 (δH 1.51) and H-17 (δH 1.50). The singlet methyl protons H-15 and H-17 were 1H–13C long-range coupled to each other. Thus, the assignments of these three methyl signals were established, although the chemical shifts of H-16 and H-17 overlapped. In addition to these HMBC correlations, the spin coupling between methylene protons H-11 (δH 1.53) and H-12 (δH 2.08 and 1.95) observed in the 1H–1H COSY spectrum revealed a 2,3-dimethylpent-2-ene moiety.

Table 1 1H and 13C NMR spectral data for JBIR-73 (1) and JBIR-67 (2)
Full size table

An ortho-coupling between the aromatic protons H-5 (δH 6.66) and H-6 (δH 7.53), which meta-coupled to the aromatic proton H-2, indicated the presence of a 1,3,4-trisubstituted benzene ring moiety. In addition, 1H–13C long-range couplings from H-2 to aromatic quaternary carbons C-4 (δC 147.6), C-6 (δC 129.8) and the carbonyl carbon C-7 (δC 168.0), from H-5 to quaternary carbons C-1 (δC 118.8) and C-3 (δC 115.8), and from H-6 to C-2 (δC 132.3), C-4 and C-7 established the assignments of the benzene ring, and revealed that the substitution in the carbonyl functional group was at C-1.

An oxymethylene proton H-18 (δH 3.52, 3.40) was 1H–13C long-range coupled to a methylene carbon C-11 (δC 34.1), a quaternary carbon C-10 (δC 57.3) and a methylene carbon C-9 (δC 49.0). The correlation between the methylene protons H-8 (δH 3.21 and 2.80) and the methine proton H-9 (δH 4.08) was observed in the 1H–1H COSY spectrum. The HMBC correlation between the methoxyl proton H-19 (δH 3.24) and the methylene carbon C-18 (δC 74.4) revealed that the methoxyl group was substituted at C-18. 1H–13C long-range couplings from H-2 to C-8 (δC 31.4) and from H-8 to C-2, C-3 and C-4 proved the connectivity of these partial structures.

The correlation between methylene protons H-23 (δH 3.56 and 3.29) and a methine proton H-24 (δH 4.35) was determined by 1H–1H COSY. 1H–13C long-range coupling from equivalent singlet methyl protons H-26, H-27 and H-28 (δH 3.25) to each other (δC 52.6) and the methine carbon C-24 (δC 73.0) was observed. H-24 was long-range coupled to the methyl carbons C-26, C-27 and C-28. Although the methyl carbons C-26, C-27 and C-28, and the methine carbon C-24 are thought to be connected through an oxygen atom according to the 13C chemical shift values of C-26, C-27 and C-28 (δC 52.6), and of C-24 (δC 73.0), these carbons are connected through an ammonium ion because C-26, C-27 and C-28 are equivalent. A trimethyl ammonium functional group with identical 13C chemical shift values was also found in ergothioneine6 and clithioneine.7 Both H-23 and H-24 were long-range coupled to a carbonyl carbon C-25 (δC 168.1), suggesting the existence of an amino-acid derivative. Moreover, the methylene proton H-23 was 1H–13C long-range coupled to the aromatic carbons C-22 (δC 129.5) and C-21 (δC 121.0), while the proton H-21 (δH 7.58) was long-range coupled to aromatic carbons C-20 (δC 140.3) and C-22. These correlations indicated the presence of a hercynine moiety in 1. The 1H–13C long-range coupling from H-9 to C-20 established the connectivity between two partial structures. The remaining nitrogen and sulfur atoms were assigned according to the 13C chemical shift values of C-4, C-10, C-9 and C-20 as shown in Figure 1b. Thus, the planar structure of 1 was determined to be a derivative of 7-hydroxyl benzastatin D,5 in which the hydroxyl residue at C-9 is replaced by an ergothioneine moiety.

Compound 2 was obtained as a colorless oil ([α]D +19.0, c 0.1, MeOH) and displayed the following IR and UV spectra: IR (KBr) νmax=1720 cm−1; UV λmax (ɛ)=302 (8500) in MeOH. The molecular formula of 2 was determined to be C17H23NO3 by HR-ESI-MS (m/z 290.1763 [M+H]+, calcd for C17H24NO3, 290.1756). The structural information on 2 was obtained by a series of 2D NMR analyses, including HSQC, field-gradient 1H–1H COSY and HMBC (Figure 1c). The NMR data for 2 were similar to those of benzastatin G8 (Table 1). On the basis of the differences between the molecular formulae of 2 (C17H23NO3) and benzastatin G (C17H23N2O2), we conclude that 2 is a carboxylic acid form of benzastatin G, with the substitution at position of C-7 (1-carboxyl benzastatin G). In addition, the specific rotation value of 2 is closely similar to the specific rotation value ([α]D +23.0 c 0.1, MeOH)8 reported for benzastatin G. Thus, the absolute configuration of 2 was deduced to be the same as that of benzastatin G.

We evaluated the cytotoxic and antimicrobial activities of 1 and 2, and found that these compounds were weakly cytotoxic to human cervical carcinoma HeLa cell lines (IC50>50 μM), but did not show antimicrobial activity against Candida albicans, Micrococcus luteus or Escherichia coli.

In conclusion, we isolated two novel benzastatin derivatives, 1 and 2, from a culture of Streptomyces sp. RI18 selected with the MF method. The structures of 1 and 2 were found to be structurally related to benzastatins isolated from Streptomyces spp.5, 8, 9 In addition, although a compound possessing an S-hercynine moiety, like the one found in 1, has been isolated from Clitocybe acromelalga,7 there have been no reports of a compound that, like 1, possesses both a benzastatin skeleton and an S-hercynine moiety. We anticipate that this study will convince chemists that Actinobacteria isolated using the MF method may be a source of new compounds containing unique skeletal structures, and encourage others to investigate new methods of isolating Actinobacteria.