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

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.

Figure 1

Structure of 2754A (1).

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 °C 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 °C 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 50 μm; 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; λ^MeOH_max nm (log ɛ) 202 (4.13), 231 (4.35) and 345 (3.73); IR ν_max (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.

Table 1 NMR data of 2754A (1) in CDCl₃

Three carbonyl carbon signals (δ 198.97, 193.19 and 171.49) and six aromatic carbon signals (δ 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 hetero-nuclear signal quantum coherence together with ¹H-¹H COSY and HMBC (Figure 2) revealed that three quaternary carbon signals at δ 132.84 (C-5a), δ 115.78 (C-9a) and δ 162.21 (C-9), and three tertiary carbon signals at δ 119.33 (C-6), δ 137.00 (C-7) and δ 124.17 (C-8) formed a 1, 2 and 3-trisubstituted benzene ring, as proton signal at δ 7.64 (1H, t, J=7.8 Hz, 7-H) was coupled with proton signals at δ 7.57 (1H, d, J=7.8 Hz, 6-H) and δ 7.27 (1H, d, J=7.8 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.

Figure 2

(a) Summary of ¹H-¹H COSY and selected HMBC correlations of 1. (b) ROESY spectrum of 1.

By tracing cross peaks from the aromatic proton (6-H) in HMBC, the carbonyl carbon of δ 193.19 (C-5) was assigned. The proton signals at δ 11.61 was also readily observed and assigned to a phenolic hydroxyl proton (9-OH), according to the chemical shift of 162.21 (C-9), a phenolic hydroxyl was substituted at C-9, and according to the chemical shift of 11.61 (9-OH), it should form an intramolecular hydrogen bond with a carbonyl group,^{5, 6} so another carbonyl carbon of δ 198.97 (C-10) was assigned. It led to the unambiguous assignments of NMR data in the substructure I (Figure 2).

As the three-carbonyl carbons and one benzene ring in 1 accounted for seven of the nine degrees of unstauration required for the molecular formula, 1 should have another two rings. This was confirmed by tracing cross peaks in the ¹H-¹H COSY and HMBC from the two oxy-methine protons at δ 4.32(1H, q, J=7.2 Hz, 1-H), δ 4.59 (1H, m, 3-H) and the two methene protons at δ 1.95 (1H, dd, J=2.4, 12.6 Hz, 4-H), δ 2.28 (1H, dd, J=15.0, 12.6 Hz, 4-H) observed in ¹H-NMR. The cross peaks in ¹H-¹H COSY between the protons at 3-H and δ 2.28 (1H, dd, J=15.0, 12.6 Hz 4-H), together with the cross peaks in HMBC between 1-H and C-4a (δ 75.95), 1-H and C-3 (δ 62.38), 4-H and C-10a (δ 74.90) established the structure of the six-membered tetrahydropyrane ring. The methyl proton signals at δ 1.74 (3H, d, J=7.2 Hz, 11-CH₃) observed in ¹H-NMR were assigned by ¹H-¹H COSY, and long-range coupling between 11-H and C-1 (74.47), C-10a in HMBC further confirmed its substitute position in the ring. A methyl acetate side chain linked with the ring at C-3 was identified by the contiguous correlation from 12-H to 3-H in ¹H-¹H COSY and correlations from proton signals at δ 2.62 (1H, dd, J=15.6, 9.7 Hz, 12-H), δ 2.66 (1H, dd, J=15.6, 3.3 Hz, 12-H) to C-4 (δ 32.93) and C-13 (δ 171.49), δ 3.72 (3H, s, 14-H) to C-13 in HMBC. The cross peaks observed in HMBC between hydroxyl proton signals at δ 6.53 (4a-OH) and C-10a, δ 6.71 (10a-OH) and C-4a, 10a-OH and C-10, as well as 11-H and C-10a, indicated that 4a-OH and 10a-OH were linked to the tetrahydropyrane ring at C-4a and C-10a, respectively. These results indicated the presence of substructure II in 1 (Figure 2).

To satisfy nine degrees of unstauration required for the molecular formula, 1 must have another ring, and the linkage between the two substructures (I and II) was established by HMBC. The long-range coupling between 10a-OH and C-10 was observed in HMBC. The data above revealed the linkage of two substructures through 10a-C with 10-C and 4a-C with 5-C. Rotating frame overhauser effect spectroscopy (ROESY) spectrum of 1 further confirmed the result. In the ROESY experiment, the correlation peaks were not observed between 4-H (δ 1.95, 2.28) and 10a-OH, the correlation peaks were observed between 4-H (δ 1.95) and 4a-OH. Thus, the planar structure of 1 was determined.

The relative stereochemistry of 1 was determined by the analysis of ROESY (in dimethyl-d6 sulfoxide (DMSO-d₆)) spectra. In the ROESY experiment, the correlation peaks were observed between 1-H and 10a-OH, between 3-H and 11-H, 4a-OH, 4-H (δ 1.95) and between 4a-OH and 4-H (δ 1.95), 11-H. However, the correlation peaks were not observed between 10a-OH and 4-H (δ 2.28) (Figure 2). On the basis of the above results, the chemical structure of 1 was determined as shown in Figure 1. It was a new member of nanaomycins group of antibiotics and was named 2754A.

1 belongs to benzoisochromane-quinone class of antibiotics and is most closely related to nanaomycin αB produced by Streptomyres sp. OM-173, which possess a hydrogen rather than a hydroxyl substituent on the 4a-position of the tetrahydropyrane ring.⁷ It was reported that nanaomycin-type antibiotics showed inhibitory activities against Gram-positive bacterias and fungi.^{5, 7, 8, 9, 10, 11} However, 1 has no inhibitory activity against Candida albicans, Piricularia oryzae, Staphylococcus aureus, Bacillus subtilis, Sarcina lutea and Escherichia coli at 100 μg ml⁻¹ using the paper (6 mm i.d.) disc method and does not have any cytotoxicities on the HCT-8 (human coloncancer), Bel-7402 (human hepatocarcinoma) and BGC-823 (human gastric carcinoma) cell lines at 10 μmol l⁻¹ by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays,¹² but was detected for the VEGFR-2 antagonistic activity using an ELISA assay method.¹³ In addition, nanaomycin αA and nanaomycin βA isolated from Streptomyces sp. 2754 also were detected for the VEGFR-2 antagonistic activity.

The experimental procedures were described as follows: A random co-polymer of glutamic acid and tyrosine (poly (Glu, Tyr) 4:1) (Sigma) was coated on to a 96-well immunoplate (Nunc) at 37 °C overnight (2 μg/well). After washing the plate with phosphate-buffered saline (PBS) (0.024% KH₂PO₄, 0.144% Na₂HPO₄, 0.02% KCl and 0.8% NaCl, pH 7.4) three times and blotting dry, the purified recombinant VEGFR-2-CD (the catalytic core in intracellular tyrosine kinase domain) protein was dissolved in TK buffer (50 mM Hepes, pH 7.4, 20 mM MgCl₂, 1.0 mM MnCl₂, 0.2 mM Na₃VO₄ and 1 mM dithiothreitol). Then, 2 μl of VEGFR-2-CD (0.5 μg), 50 μl of ATP solution (12.5 μM ATP in TK buffer) and 50 μl sample (dissolved in 10% DMSO) were added to each well. After being allowed to react at 37 °C for 1 h and washing the plate five times with PBST (PBS with 0.1% Tween 20), 200 μl of 5% (w/v) bovine serum albumin in PBS was added to each well. After incubation for 1 h at 25 °C, the plate was washed five times with PBST and dried. Then, 100 μl of mouse monoclonal antibody against phosphotyrosine (PY99) (1:1000 (v/v) dilution in PBS) was added to each well, and the plate was left at 25 °C for 2 h. The plate was washed again as described above before reaction with 100 μl of secondary antibody, namely horseradish-peroxidaseconjugated horse anti-mouse polyclonal antibody IgG (H+L) (1:2000 (v/v) dilution; Zhongshan), at 25 °C for 1 h. After final washing as described above, 3, 3′, 5, 5′-tetramethylbenzidine solution (You Yi Biotechnology Company, Beijing, China) and H₂O₂ each 50 μl was added to each well, and the reaction was allowed to proceed at 25 °C for 1 h (during which the solution should turn blue), before the reaction was stopped by the addition of 100 μl of 1 M HCl. The reaction was monitored by measuring the optical density at A₄₅₀. DMSO (10%) was used as the negative control, The inhibition rate was calculated using the following equation:

The VEGFR-2 antagonistic activities of 2754A, nanaomycin αA and nanaomycin βA are shown in Figure 3.

Figure 3

Inhibition of VEGFR-2/KDR by 2754A, nanaomycin αA and nanaomycin βA. The VEGFR-2 antagonistic activities were determined in an ELISA assay method, each assay point was determined in duplicate. 2754A, nanaomycin αA and nanaomycin βA were isolated from Streptomyces sp. 2754.