Diketopiperazines from the Cordyceps-Colonizing Fungus Epicoccum nigrum

Abstract

New diketopiperazines, epicoccins E-H (1-4) and diphenylalazines A (5) and B (6), have been isolated from the solid-substrate fermentation culture of the Cordyceps-colonizing fungus Epicoccum nigrum. The structures of 1-6 were determined primarily by NMR experiments, and the structure of 1 was confirmed by X-ray crystallography. The absolute configurations of 3 and 5 were assigned using the modified Mosher (3) and Marfey's (5) methods, respectively. Compounds 3-5 showed inhibitory effects on HIV-1 replication in C8166 cells. Diketopiperazines (DKPs) are an important class of compounds displaying a variety of biological effects, such as antimicorbial, herbicidal, antiviral, immunosuppressive, and antitumor activities. 1-3 Naturally occurring DKPs have frequently been isolated from fungal sources. Examples include golmaenone, a radical scavenger from a marine-derived Aspergillus sp., 4 the rostratins, cytotoxic DKP disulfides from a marine-derived Exserohilum rostratum, 5 the epicorazines, antibacterial metabolites from a basidiomycete Stereum hirsutum, 6 and the verticillins, dimeric DKPs from a marine-derived Penicillium sp. 7 Cordyceps sinensis (Berk.) Sacc. (Anamorph: Hirsutella sinensis; Clavicipataceae), 8 known as Chinese caterpillar fungus or "Dong Chong Xia Cao" (caterpillar-in-winter, herb-in-summer), is the combination of the fungus and the dead caterpillar larva of the moth Hepilus spp. It has been known and used for centuries in traditional Chinese medicine (TCM). 9 In nature, it is found only at high altitudes on the Himalayan Plateau and is therefore difficult to harvest. Chemical investigations of the fungal species that colonize the fruiting body of C. sinensis (Cordyceps-colonizing fungi) in our laboratory have led to the isolation of structurally diverse and biologically active metabolites. [10][11][12] In a search for anti-HIV-1 agents from this unique fungal source, the fungus Epicoccum nigrum (XZC04-CS-302) isolated from a sample of C. sinensis collected in Linzhi, Tibet, People's Republic of China, was grown in a solidsubstrate fermentation culture. Its organic solvent extract showed inhibitory effects on HIV-1 replication in C8166 cells. Fractionation of the extract afforded six new DKPs, named epicoccins E-H (1-4) and diphenylalazines A (5) and B (6), together with the known epicoccins A (7), B (8), and D (9), which were previously isolated by us from a different strain of Cordyceps-colonizing E. nigrum. 11 Details of the isolation, structure elucidation, and anti-HIV-1 activity of these metabolites are reported herein. Results and DiscussionEpicoccin E (1) was assigned the molecular formula C 18 H 18 N 2 O 7 S 2 (11 degrees of unsaturation) on the basis of its HRESIMS (m/z 461.0448 [M + Na] + ; ∆ +0.5 mmu). The 1 H and 13 C NMR data of 1 (Table 1) suggested the presence of three exchangeable protons (δ 5.56, 5.74, and 6.24, respectively), three methylenes, nine methines (five bonded to oxygen or nitrogen), two heteroatom-bonded sp 3 quaternary carbons, and four carbonyl carbons including ...

Two New Imidazolone‐Containing Alkaloids and Further Metabolites from the Ascomycete Fungus Tricladium sp.

“…Further correlations from CH 2 (6) to C(4) and C(2) indicated that C(2), C(4), and C (6) were all connected to the same N-atom, N(3), which is consistent with the NMR chemical shifts of these C-atoms. Considering the 13 C-NMR chemical shift of C(2) (d(C) 86.8) and unsaturation requirement for 1, C(2) was connected to the remaining N-atom in 1 by default to complete a 2,2,3,5-tetrasubstituted imidazol-4-one moiety.…”

“…), 20-hydroxyaflavinine (5), N-(2-phenylethyl)acetamide (6), and N b -acetyltryptamine (7) by comparison of their NMR and MS data with those reported in [13 -17]. Bacillamide A (3) was initially isolated from the marine bacterium Bacillus sp.…”

Polyphasic Analysis of Intraspecific Diversity in Epicoccum nigrum Warrants Reclassification into Separate Species

“…Many studies have focused on the ability of this fungus to produce antimicrobial compounds such as epicorazins A–B [12], epicoccins A–D [13], epicoccarines A–B and epipyridone [14], flavipin [15] and epirodins [16]. The production of novel bioactive chemical compounds including siderophores [17], antioxidants [18], inhibitors of HIV-1 replication [19], [20], inhibitors of leucemic cells [21], inhibitors of protease [22], inhibitors of telomerase [23] and fluorescent compounds with biotechnological applications [24], [25] have also been described in Epicoccum isolates.…”

Cordyceps industry in China

“…Except for the 22 fungal species reported to be associated with natural Chinese cordyceps during the 1980s and 1990s (Jiang and Yao 2002), mycoflora investigation indicated the richness of fungal diversity on Chinese cordyceps based on culture-dependent and culture-independent methods (Zhang et al 2010). Many new novel secondary metabolites have been identified from the cordyceps-colonizing fungi (Zhang et al 2007, 2009; Guo et al 2007; Guo, Sun, Gao, Chen, et al 2009; Guo, Sun, Gao, Niu, et al 2009; Ma et al 2011). There is no doubt that the cordyceps-colonizing fungi are important resources for the discovery of novel compounds and for developing medicinal and healthy products.…”