LAUSR

2,5-Diketopiperazines are cyclodipeptides obtained by the condensation of two α-amino acids. They not only abound in nature but are often produced as degradation products of polypeptides, especially in processed food and beverages [1]. The 2,5-diketopiperazines occur in numerous natural products, and this subunit is often found alone or embedded in larger, more complex architectures in a variety of natural products from fungi, bacteria, plant, and mammals [2]. Their peculiar heterocyclic system found in several natural products constitutes a rich source of new biologically active compounds. The wide spectrum of their biological properties points to various therapeutic possibilities [3]. In our ongoing screening for biologically active secondary metabolites from endophytic fungi, we investigated metabolites produced by the endophytic fungus Aspergillus sp. isolated from roots of Astragalus membranaceus, a Chinese medicinal plant of the loess plateau of China. We have continued investigating endophytes from this biotope, since previous investigations have shown that a particularly high proportion of endophytes from plants from the Hengshan Mountains, China, were biologically active. The fungus was cultivated on PDA agar medium. The crude ethyl acetate extract of the culture showed very good anticancer activity and moderate antifungal activity. This prompted us to carry out a secondary metabolites studies on this fungus, which resulted in the isolation of eight 2,5-diketopiperazines. We herein report on the isolation, structure, and anticancer properties of these compounds. The fungal strain AR-17-2 was isolated from the roots of A. membranaceus collected in the Hengshan Mountains, China, on August, 2017. By classical microscopic analysis and a molecular biological protocol by DNA amplification and sequencing of the ITS region, the fungus was identified as Aspergillus sp. The fungal strain has been preserved at the Applied Biotechnology Institute of Shanxi Datong University, Shanxi Province, China. The endophytic fungus AR-17-2 was fermented as described in the literature [4]. The culture was filtered, concentrated, and extracted with ethyl acetate three times. The EtOAc extract was separated on a silica gel column eluted with ethyl acetate–methanol gradient. The collected material was combined into nine fractions (Frs. A–I) based on TLC analysis. The bioactive Fr. B was further separated on a silica gel column (200 g) with a gradient of ethyl acetate in petroleum ether to give four subfractions (Subfrs. B1–4). Subfraction B2 was further separated by HPLC with H2O–MeOH (2:5) to give compounds 1 (24.1 mg) and 2 (12.6 mg). Subfraction B3 was chromatographed on a Sephadex LH-20 column eluting with methanol to afford compound 3 (38 mg). Subfraction B4 was chromatographed over a silica gel column eluting with petroleum ether–ethyl acetate (50:1) and then subjected to PTLC to yield compound 4 (15.5 mg). The bioactive Fr. C was separated by column chromatography on silica gel with a gradient of ethyl acetate in petroleum ether to give five subfractions (Subfrs. C1–5). Subfraction C2 that eluted with methanol was separated by semipreparative reversed-phase HPLC (H2O–MeOH, 1:4, 2.5 mL/min) to yield compounds 5 (9 mg), 6 (10.7 mg) and 7 (8.3 mg). Compound 8 (12 mg) were obtained from Subfr. C3 after separation with Sephadex LH-20 by MeOH–CHCl3 (1:1). These eight compounds were elucidated respectively as cyclo-(Pro-Gly) (1) [5], cyclo-(Pro-Leu) (2) [6], cyclo-(Pro-Ile) (3) [7], cyclo-(Pro-Vla) (4) [7], cyclo-(Phe-Gly) (5) [8], cyclo-(Phe-Ala) (6) [9], cyclo-(Phe-Tyr) (7) [10], and cyclo-(Phe-Pro) (8) [11] by comparison of their spectral data with the reported data in the literature. Cyclo-(Pro-Gly) (1). Colorless square crystals, mp 205–208°C. ESI-MS m/z 155.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ, ppm, J/Hz): 8.01 (1H, s, 2-NH), 4.12 (1H, d, J = 15.0, H-3a), 4.05 (1H, d, J = 8.2, H-9), 3.57 (1H, d, J = 15.0, H-3b), 3.35 (2H, m, H-6), 2.18 (1H, m, H-8a), 1.99 (1H, m, H-8b), 1.95 (2H, m, H-7). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 170.4 (C-1), 45.3 (C-3), 165.1 (C-4), 46.0 (C-6), 22.1 (C-7), 27.9 (C-8), 59.2 (C-9).