Seven new prenylated indole diketopiperazine alkaloids from holothurian- derived fungus Aspergillus fumigatus Fazuo Wang, Yuchun Fang, Tianjiao Zhu, Min Zhang, Aiqun Lin, Qianqun Gu * , Weiming Zhu * Key Laboratory of Marine Drugs, Chinese Ministry of Education, Institute of Marine Drugs and Food, Ocean University of China, Qingdao 266003, PR China article info Article history: Received 18 February 2008 Received in revised form 11 May 2008 Accepted 3 June 2008 Available online 10 June 2008 Keywords: Alkaloids Marine-derived fungi Aspergillus fumigatus Structure elucidation Cytotoxicity abstract Seven new prenylated indole diketopiperazine alkaloids, including compound 1 , 3 spirotryprostatins C–E (2–4), 2 derivatives of fumitremorgin B (5 and 6), and 13-oxoverruculogen (7), have been isolated from the holothurian-derived fungus Aspergillus fumigatus, along with 12 known ones (8–19). The structures of the new compounds were determined on the basis of extensive spectroscopic data and amino acid analysis. All new compounds were evaluated for their cytotoxic activities on MOLT-4, A549, HL-60, and BEL-7420 cell lines by the MTT and SRB methods. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction In our ongoing search for antitumor metabolites from marine microorganisms, we found that ethyl acetate extracts of marine- derived fungus Aspergillus fumigatus Fres., isolated from holothurian Stichopus japonicus, contained different cytotoxic alkaloids constit- uents that were found from our previous works on the same genera strain by active and chemical screening. 1,2 Therefore, investigations of the active constituents of this fungus led to the isolation of 7 new prenylated indole diketopiperazine alkaloids, including 4 spiro- oxindole diketopiperazines, 2 derivatives of fumitremorgin B, and 1 derivative of verruculogen, along with 13 known ones. A series of indole diketopiperazine alkaloids, biosynthetically originated from L-tryptophan and L-proline, were discovered with cell cycle inhibitory activity from the broth of A. fumigatus. 3–11 Among them, the spirotryprostatins showed the most attention due to their interesting intriguing molecular structures, 12–17 al- though their biological activity is relatively modest. To the best of our knowledge, only two compounds with this spiro-oxindole skeleton were reported from nature. In this paper we report the isolation, structure elucidation, and cytotoxic activities of these new compounds (1–7). 2. Results and discussion A. fumigatus was cultured in liquid medium for nine days and the metabolites were extracted with EtOAc. The crude extract was then subjected to repeated column chromatography and followed by semi-preparative HPLC separation to obtain 7 new indole diketopiperazine alkaloids (1–7), along with 12 known ones (8–19). The known compounds 8–19 were identified as spirotryprostatin A(8), 10 13-oxofumitremorgin B (9), 18–20 fumitremorgin B (10) 3,4 verruculogen (11), 21 3-b hydroxy cyclo-L-tryptophyl-L-proline (12), 22,23 cyclo-L-tryptophyl-L-proline (13), 24 tryprostatin B (14), 7,8 tryprostatin A (15), 7,8 N-prenyl-cyclo-L-tryptophyl-L-proline (16), 25 fumitremorgin C (17), 8 12,13-dihydroxyfumitremorgin C (18), 8 and cyclotryprostatins A (19), 11 respectively. 13-Oxofumitremorgin (9) was first reported from the Aspergillus group and its stereochem- istry was verified by X-ray diffraction analysis. 20 Compound 1 was isolated as pale yellow amorphous solid. Its molecular formula was determined as C 22 H 25 N 3 O 6 according to HRESIMS (found m/z 450.1620 [MþNa] þ , calcd 450.1641). In the UV spectrum, compound 1 showed characteristic absorption curve suggestive of a 6-O-methylindole chromophore with absorption maxima at 220 (log 3 4.29), 251 (4.24), 286 (4.09), and 392 (3.53) nm, 7–11 and the IR spectrum of 1 showed absorption bands at 3281 (OH), 1652 (C]O), 1619 (C]C) in the functional group region. Comparisons of the 1D NMR spectra of 1 with those of 8 suggested that 1 showed the similar oxindole diketopiperazine skeleton as 8, 10 and the differences were presence of two hydroxyl groups [8- OH (d H 5.38, br s, d C 73.5, C-8) and 9-OH (d H 7.42, br s, d C 86.3, C-9)] * Corresponding authors. Tel.: þ86 532 82032065; fax: þ86 532 82033054 (Q.G.); tel.: þ86 532 82032065; fax: þ86 532 82033054 (W.Z.). E-mail addresses: guqianq@ouc.edu.cn (Q. Gu), weimingzhu@ouc.edu.cn (W. Zhu). Contents lists available at ScienceDirect Tetrahedron journal homepage: www.elsevier.com/locate/tet 0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2008.06.013 Tetrahedron 64 (2008) 7986–7991