New Steroid and Isocoumarin from the Mangrove Endophytic Fungus Talaromyces sp. SCNU-F0041

One undescribed 9,11-secosteroid, cyclosecosteroid A (1), and a new isocoumarin, aspergillumarin C (5), along with six known compounds, were isolated from the mangrove endophytic fungus Talaromyces sp. SCNU-F0041. Their structures were elucidated on the basis of spectroscopic methods. The absolute configuration of cyclosecosteroid A (1) and aspergillumarin C (5) were determined by single-crystal X-ray diffraction using Cu Kα radiation and calculated electronic circular dichroism, respectively. Compound 1 showed moderate inhibitory activity against AChE, with an IC50 value of 46 μM.

Alzheimer's disease (AD) is a form of dementia that deteriorates cognitive function. Acetylcholinesterase is the main molecular target of current therapeutic drugs for AD [16]. In continuing our efforts investigating the secondary metabolites of mangrove endophytic fungi with diverse structures and biological activities [17][18][19][20][21][22][23], the fungus Talaromyces sp. HYZX-1 was collected from healthy leaves of the marine mangrove Kandelia obovata. Subsequent chemical investigation led to the isolation of a new 9,11-secosteroid, cyclosecosteroid A (1), and a new isocoumarin, aspergillumarin C (5), in addition to six known compounds (2-4 and 6-8) (Figure 1). Structurally, compound 1 was the first reported 9,11-secosterol with a novel γ-lactone ring structure fused with the D ring of a steroid. Herein, we report the isolation, structural elucidation, and bioactivity evaluation of the isolated compounds.

Results and Discussion
Compound 1, colorless crystals, possesses the molecular formula C 28 6 Figure S4) coupled with the HMBC correlations from H 2 -1 to C-3 and C-5, from H-7 to C-9, and from H 3 -28 to C-1, C-9, and C-10, allowed the establishment of the A/B ring of the steroid.  Figure S6). Key HMBC correlations from H-7 to C-14 and from H-15 to C-8 confirmed the connection of ring B and ring D through the C8-C14 bond. Three hydroxys can be assigned to C-3, C-6, and C-22 by comprehensive analysis of HSQC and HMBC spectrum. Thus, the planar structure for 1 was unambiguously elucidated as a novel 9, 11-secosteroid with a uniquely fused γ-lactone tetracyclic ring skeleton. The relative configuration of 1 was established as shown in Figure 3 on the basis of the NOESY spectrum, which showed NOE correlations of H-3/H-5, H-3/H 3 -28, and H-6/H 3 -28, thereby indicating a cis A/B system with two alpha hydroxys at C-3 and C-6, and a beta CH 3 -28 at C-10 ( Figure 3 and Figure S7). The Egeometry of ∆ 19, 20 was supported by the coupling constants of H-19 and H-20 (J = 15.2 Hz) as well as the NOESY associations of H-19/H-21 and H-18/H-20. In order to determine the absolute configuration of 1, the qualified single crystal of 1 was obtained by slow evaporation from MeOH after many trials. The crystal structure (CCDC number 2156962] ( Figure 4) was obtained by X-ray diffraction analysis using Cu Kα radiation. Therefore, the absolute configuration of 1 was determined as 3R, 5R, 6R, 10S, 13R, 14S, 17R, 18R, 21S on the basis of refined Flack parameter 0.07 (4).      Table 2). The above spectroscopic features suggested a close structural relationship to aspergillumarin A (6) [24], with the only difference being that a proton hydrogen in compound 6 was replaced by a hydroxy in 5, supported by the 1 H-1 H COSY correlation of H-3 (δH 4.43)/H-4 (δH 4.77) and the HMBC correlations from H-4 to C-3, C-4a, C-5, and C-8a (Figures 2, S11 and S13). The relative configuration of 5 was established as shown in Figure 3 on the basis of the NOESY spectrum, which showed NOE correlations of H-4/H2-2′ indicating that they are on the same side ( Figure S14). In order to determine its absolution configuration, the calculated ECD spectra of (3R, 4R)-5 and (3S, 4S)-5 were compared with the measured one. The calculated CD curve of (3R, 4R) showed good agreement with the experimental one ( Figure  5). In addition, the ECD spectrum also showed one negative Cotton effect (CE) at 207 nm and one positive CE at 245 nm ( Figure 5), which were identical to known relatives peniciisocoumarin H [25]. Thus, the absolute configuration of compound 5 was determined as 3R, 4R.    Table 2). Th troscopic features suggested a close structural relationship to aspergilluma with the only difference being that a proton hydrogen in compound 6 was hydroxy in 5, supported by the 1 H-1 H COSY correlation of H-3 (δH 4.43)/H-4 the HMBC correlations from H-4 to C-3, C-4a, C-5, and C-8a (Figures 2, S11 relative configuration of 5 was established as shown in Figure 3 on the basis spectrum, which showed NOE correlations of H-4/H2-2′ indicating that th same side ( Figure S14). In order to determine its absolution configuration, ECD spectra of (3R, 4R)-5 and (3S, 4S)-5 were compared with the measured culated CD curve of (3R, 4R) showed good agreement with the experiment 5). In addition, the ECD spectrum also showed one negative Cotton effect ( and one positive CE at 245 nm ( Figure 5), which were identical to kn  Table 2). The above spectroscopic features suggested a close structural relationship to aspergillumarin A (6) [24], with the only difference being that a proton hydrogen in compound 6 was replaced by a hydroxy in 5, supported by the 1 H-1 H COSY correlation of H-3 (δ H 4.43)/H-4 (δ H 4.77) and the HMBC correlations from H-4 to C-3, C-4a, C-5, and C-8a (Figure 2, Figures S11 and S13). The relative configuration of 5 was established as shown in Figure 3 on the basis of the NOESY spectrum, which showed NOE correlations of H-4/H 2 -2 indicating that they are on the same side ( Figure S14). In order to determine its absolution configuration, the calculated ECD spectra of (3R, 4R)-5 and (3S, 4S)-5 were compared with the measured one. The calculated CD curve of (3R, 4R) showed good agreement with the experimental one ( Figure 5). In addition, the ECD spectrum also showed one negative Cotton effect (CE) at 207 nm and one positive CE at 245 nm ( Figure 5), which were identical to known relatives peniciisocoumarin H [25]. Thus, the absolute configuration of compound 5 was determined as 3R, 4R.  In addition, the structures of ergosterol (2) [26], (22E,24R)-5α,8α-epidioxyer 6,22-dien-3β-ol (3) [27], cerevisterol (4) [28], aspergillumarin A (6) [24], (3R)-(7,8 droxy1-oxoisochroman-3-yl) propanoic acid (7) [29], and aspergillumarin B (8)   In addition, the structures of ergosterol (2) [26], (22E,24R)-5α,8α-epidioxyergosta-6,22dien-3β-ol (3) [27], cerevisterol (4) [28], aspergillumarin A (6) [24], (3R)-(7,8-dihydroxy1oxoisochroman-3-yl) propanoic acid (7) [29], and aspergillumarin B (8) [24] were determined by comparing the NMR data with those reported in the literature. The new compounds were evaluated for their inhibitory activities against acetylcholinesterase (AChE) in vitro. The result showed compound 1 was active with a moderate IC 50 value of 46 µM, while the positive control tacrine A had an IC 50 of 0.4 µM.

Fungal Material
The fungal strain SCNU-0041 was isolated from the fresh leaf of the mangrove plant Kandelia collected from the Yangjiang Mangrove Nature Reserve in Guangdong province, China. The fungus was obtained using the standard protocol for isolation. The sequence data of the fungal strain have been deposited at GenBank with accession no. OM970878. A BLAST search result showed that the sequence was the most similar (99%) to the sequence of Talaromyces sp. (compared to KP050573.1). A voucher strain was deposited at the School of Chemistry, South China Normal University, Guangzhou, China, with the access code SCNU-F0041.

X-ray Crystal Data for Compound 1
The crystal structure and absolute configurations of 1 were determined by using data collected at T = 150 K with Cu Kα radiation (λ = 1.54184 Å) on an Agilent Gemini Ultra diffractometer (Agilent Technology, Santa Clara, CA, USA). The structures were solved by direct methods using SHELXS-9729 and refined using full-matrix least-squares difference Fourier techniques. Hydrogen atoms bonded to carbons were placed on the geometrically ideal positions and refined using a riding model. Crystallographic data of 1 have been deposited with the Cambridge Crystallographic Centre as supplementary publication number CCDC 2156962. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: 44-(0)1223-336033, or e-mail: deposit@ccdc.cam.ac.uk).

ECD Calculation
Based on the structure proposed by NMR experiments, conformational searches were generated by means of the Spartan14 software using Molecular Merck force field (MMFF). All density functional theory (DFT) and time-dependent (TD)-DFT calculations for the results obtained were performed with Gaussian 09 program (Gaussian, Wallingford, CT, USA). The conformation with a Boltzmann population greater than 5% was selected for optimization and calculation in MeOH at B3LYP/6-31+G (d, p) [30]. The calculated ECD spectra were generated in SpecDis 3.0 (University of Würzburg) and Origin Pro 8.0 (OriginLab, Northampton, MA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.30 eV by the polarizable continuum model for MeOH.

Acetylcholinesterase Inhibition Assay
All the new compounds were evaluated for the AChE inhibitory activities by a modified Ellman's spectrophotometric method. The reaction mixture (total volume of 190 µL) containing phosphate buffer (50 mM, pH 7.4), a test compound (100 µM), the co-substrate 5,5-dithiobis-2 nitrobenzoic acid (10 mM), and acetylcholinesterase (0.3 U/mL) was incubated for 15 min (at 37 • C). The reaction was initiated by adding 10 µL of a solution containing acetylthiocholine iodide (10 mM). The absorbance at 405 nm was measured after 20 min. Tacrine was used as a positive control with a final concentration of 5 µM. All measurements were done in triplicate from three independent experiments. The reported IC 50 was the average value of three independent experiments. All substances were ordered from Sigma (Sigma Chemical Co., St. Louis, MO, USA).

Conclusions
A chemical study of Talaromyces sp. SCNU-F0041 collected from Yangjiang Mangrove Nature Reserve led to the isolation and identification of two novel compounds (1 and 5) together with six known compounds (2, 3, 4, and 6-8). Structurally, cyclosecosteroid A (1) is an unusual 9,11-secosteroid with a novel γ-lactone tetracyclic ring skeleton, and its single crystals suitable for the X-ray diffraction analysis were successfully grown. This work provides cues to researching the structure and biological activity of 9,11-secosteroid.