2754A: a new inhibitor of VEGFR-2 isolated from the Streptomyces sp. 2754

Abstract

Angiogenesis has an important role in solid tumor growth and tumor metastasis. VEGF functions as a highly specific mitogen directly involving in the process of angiogenesis, which induces endothelial cell division and proliferation and also increases vascular permeability.1,2 VEGF performs its function through binding to special receptors. Studies have showed that VEGFR-2 is the major signal transducer for the differentiation and proliferation of endothelial cells. Consequently, inhibition of VEGFR-2 signal is an effective way to block tumor angiogenesis.3,4 In the course of screening for VEGFR-2 inhibitor, a new compound, 2754A (1, Figure 1) was isolated from the Streptomyces sp. 2754. In this paper, we report the fermentation, isolation, physico-chemical properties, structure elucidation and bioactivities of 2754A. A stock culture of the strain Streptomyces sp. 2754 was maintained on yeast and malt extract with glucose International Streptomyces Projects Medium 2 agar slant consisting of 0.4% yeast extract (Beijing Aoboxing Biotechnology, Beijing, China), 1.0% malt extract (Beijing Aoboxing Biotechnology), 0.4% glucose and 1.2% agar (pH 7.1). The stock culture was inoculated into 250-ml Erlenmeyer flasks containing 50 ml of medium consisting of 0.5% yeast extract (Beijing Aoboxing Biotechnology), 0.5% glucose, 0.5% tryptone, 0.5% beef extract, 0.4% corn steep liquor (North China Pharmaceutical Corporation, Hebei, China), 1.0% soybean extract (Beijing Comwin Pharm-Culture, Haidian, Beijing, China), 2.0% starch and 0.4% CaCO3 (pH 7.1). The flask culture was incubated on a rotary shaker (250 r.p.m.) at 28 1C for 48 h. The seed culture (5 ml) was transferred into each of the 100, 500-ml Erlenmeyer flasks containing 100 ml of the same medium. The fermentation was carried out at 28 1C for 96 h on a rotary shaker (250 r.p.m.). The fermentation broth (10 l) was filtered and the filtrate was absorbed on a column of Amberlite XAD-5 (Amberlite XAD-5 Macroporous Resin, Sigma, St Louis, MO, USA) (1 l), after washing with 4 l of water, 3 l of 30% aqueous acetone and 3 l of 50% aqueous acetone, successively, the absorbed materials were eluted with 3 l of 80% aqueous acetone. The fraction eluted with 80% aqueous acetone showed the VEGFR-2 antagonistic activity. The active fraction was concentrated in vacuo and lyophilized to obtain a crude powder (2.8 g). It was then chromatographed on a column of Sephadex LH-20 (Pharmacia, Sweden, 1.5 100 cm), developed with methanol. The first yellow fraction showing the activity was concentrated to give a yellow material (12 mg). This material containing enriched 1 was finally purified by medium-pressure liquid chromatography (Yamazen FMI-C pump, Yamazen, Japan; column: Ultra-pack ODS, 15 300 mm, 30 per 50mm; mobile phase: 30% aqueous acetonitrile; flow rate: 1 ml min 1) to yield 1 (1.5 mg) and 1 of 14 mg was obtained from 100 l of the fermentation broth. Compound 1 was obtained as pale yellow amorphous powder and is soluble in methanol, acetonitrile and dimethyl sulfoxide, but insoluble in n-hexane and water. The other physico-chemical properties of 1 are as follows: HR-ESI-MS (M+Na)+ (m/z) 373.0829, calcd. 373.0899 for C17H18O8; lMeOH max nm (log e) 202 (4.13), 231 (4.35) and 345 (3.73); IR nmax (KBr) cm 1 3422, 2951, 1709, 1656, 1605, 1578, 1266, 1037 and 758. The direct connectivity between protons and carbons was established by the heteronuclear single quantum coherence. The 1H(nuclear magnetic resonance) NMR and 13C-NMR spectral data of 1 are shown in Table 1. Three carbonyl carbon signals (d 198.97, 193.19 and 171.49) and six aromatic carbon signals (d 162.21, 137.00, 132.84, 124.17, 119.33 and 115.78) were readily observed by analysis of 13C-NMR and DEPT of 1. Further analysis of the six aromatic carbon signals through heteronuclear signal quantum coherence together with 1H-1H COSY and HMBC (Figure 2) revealed that three quaternary carbon signals at d 132.84 (C-5a), d 115.78 (C-9a) and d 162.21 (C-9), and three tertiary carbon signals at d 119.33 (C-6), d 137.00 (C-7) and d 124.17 (C-8) formed a 1, 2 and 3-trisubstituted benzene ring, as proton signal at d 7.64 (1H, t, J1⁄47.8 Hz, 7-H) was coupled with proton signals at d 7.57 (1H, d, J1⁄47.8 Hz, 6-H) and d 7.27 (1H, d, J1⁄47.8 Hz, 8-H) in 1H-1H COSY. Both 8-H and 6-H were long-range correlated with C-9a, and 7-H was long-range correlated with C-9 and C-5a in HMBC.