Species of Streptomyces are well known producers of diverse biological metabolites, and anthracyclines are one class of the typical antitumor antibiotics in drug studies. As one of the longest known antibiotics, anthracyclines have been received great attention frequently since its first discovery.1 Some of these anthracyclines have been used in medicine for the treatment of some cancers.2,3 Moreover, even in recent years, novel anthracyclines are constantly being discovered, such as aranciamycin anhydride.4 In our effort to search for new biological compounds from endophytes, a Streptomyces sp. YIM66403 producing special orange pigments caught our attention. Large-scale fermentation and subsequent fractionation of the extract of broth and mycelium proved that Streptomyces sp. YIM66403 was a strain yielding anthracyclines. As a result, we isolated a new anthracycline misamycin (1), together with η-pyrromycinone (2),5 4-Deoxy-ɛ-pyrromycinone (3)6 and other six known anthracyclines including cinerubin A (4),7 cinerubin B (5),7 antibiotic 80334 C (6),8 cinerubin X (7),9 collinemycin (8)10 and musettamycin (9)10 (Figure 1). Compound 1 showed cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) method and anti-Staphylococcus aureus activity in comparison with other antimicrobial activity.

Figure 1
figure 1

Structures of compounds 1–9.

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The structure of misamycin (1) was established by various spectroscopic methods, whereas the structures of other known compounds (2–9) were confirmed by comparing their 1H and 13C-NMR data with these of compounds in the literature. In this paper, we would like to report the isolation, structure determination and biological activity of 1.

The strain YIM66403 was isolated using glycerol-asparagine medium (1% glycerol, 0.1% asparagine, 0.1% dipotassium hydrogen phosphate, 1.2% agar, pH 7.2) from a healthy stem of the traditional Chinese medicinal plant Isodon eriocalyx obtained from Xishuangbanna, Yunnan, China, and was identified as Streptomyces sp. by 16S rRNA gene sequence. The seed medium consisted of 0.4% malt extract, 0.5% yeast extract, 0.5% glucose (adjusted to pH 7.2 before sterilization). Strain YIM66403 was cultured in 500 ml erlenmeyer flasks each containing 200 ml seed culture. After 4 days of incubation at 28 °C on a rotary shaker (125 r.p.m.), 10 ml portion of the culture was inoculated into 1000 ml erlenmeyer flasks each containing 250 ml of fermentation medium consisting of 0.5% yeast extract, 2.4% soluble starch, 0.1% glucose, 0.3% beef extract, 0.3% peptone, 0.4% calcium carbonate (adjusted to pH 7.0 before sterilization). The fermentation was carried out at 28 °C on a rotary shaker (125 r.p.m.) for 8 days. A total of 100 l of fermentation broth was collected, and the broth and mycelium were separated. The broth was extracted with ethyl acetate, and the combined extracts were concentrated under reduced pressure to give the crude residue (23 g). The mycelium was extracted with acetone for 30 min. The solvent was removed under vacuum and the remaining water phase was extracted with ethyl acetate; after concentration, 500 g oily crude extract was obtained. A flow diagram for the extraction of anthracyclines is shown in Scheme 1.

Compound 1 was isolated as amorphous powder. A molecular ion peak m/z 656.2480 was observed based on the high-resolution MS analysis of 1 (calcd for C34H40O13, 656.2469), consistent with a molecular formula of C34H40O13. The 1H-1H COSY correlations from H-2 to H-3, H-7 to H-8 and HMBC correlations from 1-OH to C-1/2/12a, H-2 to C-4, H-3 to C-1, 4-OH to C-3/4/4a, 6-OH to C-5a/6/6a, H-7 to C-6/9, H-8 to C-10, H-10 to C-13, H-11 to C-10/12, H-14 to C-13 drew the skeleton of anthracycline for us. Then the 13C-NMR data comparison with its analog musettamycin (9)10 confirmed our conclusion. Compared the NMR data of compound 1 with that of musettamycin (9), it was found that N-CH3 and two CH (bearing respectively amino-group and hydroxyl ) were missing in 1, but two CH2 were additional in structure of 1, although the structure of 1 was similar to that of musettamycin, the two CH were replaced by CH2 in the structure of 1. The correlations of COSY and HMBC in compound 1 were determined in this elucidation (Figure 2). The C-1′ of rhodinose connected to C-7 was confirmed by the correlations from H-7 to C-1′ in HMBC. The COSY correlations from H-1′ to H-6′, and the HMBC correlations from H-6′ to C-4′, H-4′ to C-2′ confirmed that the oxygen atom was connected to C-4′, together with the COSY correlations from H-1′′ to H-6′′, and the HMBC correlations from H-6′′ to C-4′′, H-4′′ to C-3′′ confirmed that the another OH was connected to C-4′′. The C-1′′ of rhodinose connected to C-4′ was confirmed by the correlation from H-1′′ to C-4′ in HMBC. By comparison with the NMR of known anthracyclines isolated from this strain, the configurations of compound 1 were determined having the same structure as musettamycin. The configurations of C-9,10 were determined as R by the chemical shift at C-16 (δ 6.66) compared with the NMR data of antibiotic 80334 B.8 The structure of compound 1 was described in PhD thesis of Balk-Bindseil.11 The anomeric configurations of two sugars were determined to be all α by the coupling constants of the anomeric protons of rhodinoses, H-1′ (d, J=2.8 Hz) and H-1′′ (d, J=3.4 Hz). The relative configurations between H-4′ and H-5′, H-4′′ and H-5′′, H-1′ and H-6′, H-1′′ and H-6′′ in the structures of sugars were also confirmed as cis-form by the NOESY correlations between them.

Figure 2
figure 2

Key HMBC and 1H-1H COSY correlations for the new anthracycline 1.

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Misamycin (1): amorphous powder;; UV (MeOH) λmax (log ɛ) 491(3.32), 256 (4.74), 232 (4.16) nm; IR (KBr) νmax 3441, 2931, 2354, 1732, 1602, 1449, 1213, 1004 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3), Table 1; HR-MS at m/z 656.2480 [M]+ (calcd for C34H40O13, 656.2469).

Table 1 1H (400 MHz) and 13C NMR (100 MHz) spectroscopic data for 1 (in CDCl3, J in Hz)
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Cytotoxicity of 1 was determined in vitro by the MTS method. Antimicrobial assays for 1 were performed in 96-well sterilized microplates using a microdilution method. The cytotoxicity of the misamycin (1) was tested by using human promyelocytic leukemia HL-60, human hepatoma SMMC-7721, non-small cell lung cancer A-549, breast cancer MCF-7 and human colorectal carcinoma SW4801 cell lines, it showed modest cytotoxicity against above tumor cells, generating IC50 values of 15.37, 16.34, 25.98, 20.71, 9.75 μM against HL-60, SMMC-7721, MCF-7, A-549, SW4801 cells, respectively. Cisplatin was used as a positive control, generating IC50 values of 1.18, 4.29, 5.25, 12.28 and 13.85 μM against HL-60, SMMC-7721, A-549, MCF-7, SW4801 cells, respectively.

In addition, misamycin (1) exhibited modest antibacterial activity against Gram- positive bacterium Staphylococcus aureus with an MIC of 64 μg ml−1, but it didn’t show antimicrobial activity against fungus Monilia albican and Gram-negative bacterium Escherichia coli with MICs >512 μg ml−1. Kanamycin showed antibacterial activity against S. aureus with an MIC of 4 μg ml−1 as a positive control.

scheme 1

Extraction of compounds 1–9 produced by Streptomyces sp. YIM66403. I: Recrystallized in CHCl3. II: Silica gel column eluted repeatedly with a CHCl3/MeOH gradient from CHCl3 to MeOH and Sephadex LH-20 (GE Healthcare Co., Buckinghamshire, UK) (MeOH). III: Silica gel column eluted with petroleum ether/ethyl acetate (10:1, 3:1, 1:1) followed by ethyl acetate and then MeOH, a CHCl3/MeOH gradient from CHCl3 to MeOH and Sephadex LH-20 (MeOH).