Sarkomycin is an antitumor antibiotic isolated from the culture filtrate of Streptomyces erythrochromogenes.1, 2, 3 Until 1960s, sarkomycin was used as a prescription drug to treat cancer in Japan. Although sarkomycin exhibits strong inhibitory effect on several cancer cell lines, the activity is not effective enough.3, 4, 5 A great effort has been extended to isolate or synthesize the analogous compounds of sarkomycin (Figure 1) expecting for more intensive antitumor chemicals with lower toxicity.6, 7, 8, 9, 10 During the course of searching for novel microorganism-derived antitumor secondary metabolites, we investigated the chemical constituents of a strain Streptomyces sp. HS-HY-144. As a result, a new sarkomycin analog (1, Figure 1) was isolated from the fermentation broth of the strain. In this paper, the details of isolation and structure characterization of compound 1 are described.

Figure 1
figure 1

Structures of 1 and sarkomycin, and the key 1H-1H COSY and HMBC correlations of 1.

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Strain Streptomyces sp. HS-HY-144 was isolated from a soil sample collected from a farmland located in Tengchong, Yunnan province, China. The strain was identified as the genus Streptomyces because its 16S rDNA sequence (accession no: KR232515) exhibited a high sequence similarity of 100% with that of S. mirabilis strain 3662 (accession no: EF371431.1).

The strain Streptomyces sp. HS-HY-144 was maintained on the medium containing glucose (Sinopharm Chemical Reagent Co, Ltd, Shanghai, China) 10 g, maltose (Sinopharm Chemical Reagent Co, Ltd) 3 g, yeast extract (Oxoid Ltd, Basingstoke, UK) 3 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, KNO3 1 g and agar (Becton Dickinson and company, Franklin Lake, NJ, USA) 20 g in 1.0 l of tap water, pH 7.0. The seed medium consisted of glucose 4 g, maltodextrin (Shandong Xiwang Group Ltd, Binzhou, Shandong, China) 10 g, yeast extract 4 g and CaCO3 2 g in 1.0 l water at pH 7.2–7.4. All the media were sterilized at 121 °C for 20 min. Slant culture was incubated for 6–7 days at 28 °C. Fermentation was carried out in a 50-l fermentor (containing 30 l of production medium; Shanghai Guoqiang Bioengineering Equipment, Shanghai, China; THS-50 l), tank pressure control at 0.05 MPa. The producing medium was composed of glucose 1%, soluble amylum (Haiyan Liuhe Starch Chemical Co, Ltd, Haiyan, China) 4%, yeast extract 0.5%, soybean powder (Ningbo Beilun Jiangnan Grease Co, Ltd, Ningbo, China) 2.5%, peptone (Sincere, Shanghai Sincere Biotech Co, Ltd, Shanghai, China) 0.5%, CaCO3 0.2%, MgSO4·7H2O 0.8%, FeSO4·7H2O 0.6%, ZnSO4·7H2O 0.2%, MnSO4·H2O 0.2% and CoCl2·6H2O 0.05%, Na2MoO4·2H2O 0.2% at pH 7.0 before sterilization. The fermentation was conducted at 28 °C for 7 days by stirring at 100 r min−1 with an aeration rate of 900 l of air per hour.

The final 30 l of broth from the 50-l fermentor was filtered, and the resulting cake was washed with water (3 l) and subsequently extracted with MeOH (3 l). The supernatant and the wash water were subjected to a Diaion HP-20 resin column eluting with 95% EtOH (5 l). The MeOH extract and the EtOH eluents were evaporated under reduced pressure to 1 l at 50 °C, and the resulting concentrate was extracted three times using an equal volume of EtOAc. The combined EtOAc phase was concentrated under reduced pressure to yield a mixture (23 g). The mixture was chromatographed on a silica gel (Qingdao Haiyang Chemical Group, Qingdao, Shandong, China; 100–200 mesh) column and successively eluted with a stepwise gradient of CHCl3/MeOH (100:0–50:50, v/v) to obtain three fractions Fr.1–Fr.3 based on the TLC profiles. TLC was performed on silica-gel plates (HSGF254, Yantai Chemical Industry Research Institute, Yantai, China), with solvent system of CHCl3/MeOH (9:1). The developed TLC plates were observed under a UV lamp at 254 nm or by heating after spraying with sulfuric acid/ethanol, 5:95 (v/v). The Fr.2 was subjected to a Sephadex LH-20 gel (GE Healthcare, Glies, UK) column eluted with CHCl3/MeOH (1:1, v/v) and detected using TLC to give two fractions (Fr.2-1 to Fr.2-2). The Fr.2-1 was further isolated with semi-preparative HPLC (Agilent 1100, Zorbax SB-C18, 5 μm, 250 × 9.4 mm inner diameter; 1.5 ml min−1; 220 nm; Agilent, Palo Alto, CA, USA) eluting with CH3CN/0.1% formic acid (30:70, v/v) to give compound 1 (tR 8.2 min, 83 mg). 1H and 13C NMR spectra were measured with a Bruker DRX-400 (400 MHz for 1H and 100 MHz for 13C) spectrometer (Bruker, Rheinstetten, Germany). The electrospray ionization mass spectrometry (ESIMS) and high resolution electrospray ionization mass spectroscopy (HRESIMS) spectra were taken on a Q-TOF Micro LC-MS-MS mass spectrometer (Waters Co, Milford, MA, USA).

Compound 1 was isolated as a colorless crystal (in MeOH) with m.p. 237–239 °C, ([α]25D +19.4(c 0.2, EtOH) and UV (EtOH) λmax nm (log ɛ): 284 (2.12). Its molecular formula C13H16O6 with 6 °C of unsaturation was determined using HRESIMS at m/z 269.1015 [M+H]+ (calculated as 269.1020 for C13H17O6). The IR spectrum displayed absorption band for carbonyl (at 1725 cm−1) functionality. Analysis of the 1H and 13C NMR data (Table 1) with the aid of DEPT experiment revealed the presence of three methylenes (δC 24.2, 27.7, 36.3), two aliphatic methines (δC 46.4, 48.6), one carboxyl carbonyl carbon (δC 175.3) and one carbonyl group (δC 217.3). The 1H-1H COSY correlations of H-2A/H-6A/H-5A/H-4A (Figure 1) established the C-2A-C-4A structural fragment. The observed HMBC correlation networks of H-2A, H2-5A and H2-4A/C-3A; H-2A, H-6A and H2-5A/C-7A revealed the presence of a 3-oxocyclopentanecarboxylic acid moiety. The methylene group at δC 27.7 was connected to C-2A by the 1H-1H COSY correlation of H2-1 and H-2A and the HMBC correlations from H2-1 to C-3A and C-6A. Taking the molecular formula and 13C NMR of 1 into account, 1 was probably included two 3-oxocyclopentanecarboxylic acid substructures. The linkage of the two 3-oxocyclopentanecarboxylic acid moieties via C-1 was corroborated by the HMBC correlations between H-2A/C-2 and H-2/C-2A. To secure the assigned structure and also to determine its absolute configuration, a suitable crystal of 1 obtained from MeOH was subjected to X-ray diffraction analysis (Figure 2). The X-ray result not only completely agreed with the proposed structure but also allowed unambiguous assignment of its absolute configuration with absolute structure parameter 0.10(13) based on the final refinement on the Cu Kα data. Thus, the structure of 1, including the absolute configuration, was confidently assigned.

Table 1 1H and 13C NMR dataa for compound 1
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Figure 2
figure 2

The X-ray crystallographic structure for 1. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Crystal data of compound 1 were collected using a Bruker APEX-II CCD with a graphite monochromated Cu Kα radiation, λ=1.54184 Å at 140(2) K. Crystal data: C13H16O6, M=268.26, monoclinic, space group C2; unit cell dimensions were determined to be a=18.7920(4) Å, b=5.82020(10) Å, c=5.56990(10) Å, α=90°, β=103.1570(10)°, γ=90°, V=593.21(2) Å3, Z=2, Dx=1.502 Mg m−3, F (000)=284, μ (Cu Kα)=1.013 mm−1. Overall, 2485 reflections were collected until θmax=69.346°, in which independent unique 992 reflections were observed [R(int)=0.0421]. The structure was solved by direct methods using the SHELXS-2013 program (Sheldrick, 2013), and refined by the SHELXL-2013 program and full-matrix least-squares calculations. In the structure refinements, non-hydrogen atoms were placed on the geometrically ideal positions using the ‘ride on’ method. Hydrogen atoms bonded to oxygen were located by the structure factors with isotropic temperature factors. The final refinement gave R=0.0434, weighted residual factors (RW)=0.1119, Flack=0.10(13). Crystal data of 1 were deposited in the Cambridge Crystallographic Data Centre (CCDC 1054654).

The cytotoxicity of 1 was assayed for growth-inhibition activity in vitro against human cervical carcinoma HeLa cells using the sulforhodamine B (SRB) method.11 As a result, 1 showed poor cytotoxicity against the HeLa cells (7.3% inhibition at the dose of 100 μM), whereas the IC50 value of the positive control (doxorubicin) was 0.234±0.015 μM.

The antimicrobial activity of 1 was assessed against the pathogenic bacterium Staphylococcus epidermidis with the broth microdilution MIC method recommended by the Clinical and Laboratory Standards Institute Standards12 using kanamycin monosulfate as a positive control. Compound 1 showed weak antibacterial activity (MICs: 1, 10 mg ml−1; kanamycin monosulfate, 0.08 mg ml−1).

In previous report, sarkomycin exhibited strong cytotoxicity against HeLa cells.5 In this research, the bioassay showed that 1 exerted unfavorable cytotoxic activity on this cell line. This may be due to the reduction of the α, β-unsaturated ketone moiety in 1. This structural subunit existing in many natural products was demonstrated to be the key factor responsible for their antitumor activities and it was weak or inactive when the α, β-unsaturated ketone moiety was absent.13 Studies on the biosynthetic pathway and other bioactivities of 1 are currently underway.