Abstract
We found that the strain Aspergillus fumigatus var. fumigatus RK95-113 produced a new angiogenesis inhibitor, RK-95113. Spectroscopic analyses identified the structure of RK-95113 as a new fumagillin-related small molecule. RK-95113 preferentially inhibited the growth of human umbilical vein endothelial cells (HUVECs) rather than that of human normal fibroblasts in cell proliferation assays and blocked endothlial cell migration induced by vascular endothelial growth factor (VEGF).
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The culture broth was centrifuged and the supernatant was adjusted to pH 3.0. The supernatant was extracted with an equal volume of ethyl acetate. The organic layer was concentrated in vacuo to dryness. The concentrate material was applied to silica gel column chromatography. The materials were eluted with a stepwise gradient of CHCl3 - CH3OH (0∼100% MeOH in CHCl3). The active fraction was concentrated in vacuo to yield a crude material. Further purification of RK-95113 was carried out by HPLC (PEGASIL ODS, 20 i.d.×250 mm, Senshu Scientific Co. Ltd., Tokyo) with a linear gradient solvent system of CH3CN - water (from 20 : 80 to 0 : 100 for 40 minutes) at a flow rate of 9.0 ml/minute to give a yellow powder. Finally, 2.2 mg of RK-95113 was separated by preparative TLC with n-hexane - ethyl acetate (3 : 1). RK-95113 was obtained as a colorless amorphous powder, that is soluble in MeOH, acetone, and DMSO but not in n-hexane and H2O. The Rf value in the solvent system (CHCl3 - MeOH, 50 : 1) was 0.63 on a silica gel TLC (Merck 60 F254); [α]D23 −40.0° (c=0.035, EtOH); IR; KBr νmax 2920, 1700, 1610, 1265, 1225, 1125, 1005 cm−1; UV (MeOH) λmax nm (ε) 300 (47830).
1H NMR data of RK-95113: (500 MHz in acetone-d6), δH 1.15 (s, 3H, H-15), 1.29 (m, 2H, H-8), 1.64 (s, 3H, H-16), 1.72 (s, 3H, H-17), 1.79 (d, 3H, J=6.7 Hz, H-8′), 1.88 (m, 2H, H-7), 1.96 (d, 1H, J=10.40 Hz, H-4), 2.22 (m, 2H, H-12), 2.55 (m, 1H, H-11), 2.55 (m, 1H, H-2a), 2.96 (d, 1H, J=3.10 Hz, H-2b), 3.35 (s, 3H, H-18), 3.64 (d, 1H, J=10.40 Hz, H-5), 5.24 (br s, 1H, H-13), 5.64 (br s, 1H, H-6), 5.90 (d, 1H, J=15.2 Hz, H-2′), 6.00 (dq, 1H, J=14.4 Hz, 6.7 Hz, H-7′), 6.21 (dd, 1H, J=14.4 Hz, 11.1 Hz, H-6′), 6.32 (dd, 1H, J=14.2 Hz, 11.8 Hz, H-4′), 6.64 (dd, 1H, J=14.2 Hz, 11.1 Hz, H-5′), 7.31 (dd, 1H, J=15.2 Hz, 11.8 Hz, H-3′). 13C NMR data of RK-95113: (125 MHz in acetone-d6), δC 14.53 (q, C-15), 17.98 (q, C-16), 18.53 (q, C-8′), 25.80 (q, C-17), 26.43 (t, C-7), 28.11 (t, C-12), 30.10 (t, C-8), 49.08 (d, C-4), 51.57 (t, C-2), 56.64 (q, C-18), 58.88 (s, C-10), 60.14 (s, C-3), 60.75 (d, C-11), 67.37 (d, C-6), 80.31 (d, C-5), 120.34 (d, C-13), 121.21 (d, C-2′), 128.58 (d, C-4′), 132.27 (d, C-6′), 134.67 (s, C-14), 135.57 (d, C-7′), 141.98 (d, C-5′), 145.59 (d, C-3′), 166.48 (s, C-1′).
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Asami, Y., Kakeya, H., Okada, G. et al. RK-95113, a New Angiogenesis Inhibitor Produced by Aspergillus fumigatus. J Antibiot 59, 724–728 (2006). https://doi.org/10.1038/ja.2006.97
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DOI: https://doi.org/10.1038/ja.2006.97
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