Homo/Hetero-Dimers of Aromatic Bisabolane Sesquiterpenoids with Neuroprotective Activity from the Fungus Aspergillus versicolor A18 from South China Sea

Chromatographic fractionation of the EtOH extracts of the marine-derived fungus Aspergillus versicolor A18 has led to the isolation of 11 homo/hetero-dimers of aromatic bisabolane sesquiterpenoids including eight diphenyl ether-coupled aromatic bisabolanes (1a/1b and 5–10) and three homodimers (2–4), together with their monomers including three aromatic bisabolanes (11–13) and two diphenyl ethers (14 and 15). Their structures and absolute configurations were elucidated by extensive spectroscopic analysis including HRESIMS, 1D/2D NMR, calculated ECD, and the optical rotatory data. Among the four new compounds, (+/−)-asperbisabol A (1a/1b), asperbisabol B (2), and asperbisabol C (3), the enantiomers 1a and 1b represent an unprecedented skeleton of diphenyl ether-coupled aromatic bisabolane sesquiterpenoids with a spiroketal core moiety. The neuroprotective effects of selected compounds against sodium nitroprusside (SNP)-induced injury were evaluated in PC12 cells by the MTT assay. Five compounds (1a, 6, and 8–10) showed remarkable neuroprotective activities at 10 μM, being more active than the positive control edaravone.

Within the past decade, our group has reported a series of compounds with neuroprotective effects, such as polyhydroxypregnane glycosides from Cynanchum otophyllum [10], neolignans from Aristolochia fordiana [11], mulberry Diels-Alder-type adducts from Morus alba [12], and prenylated xanthones from Garcinia mangostana [13]. To obtain natural neuroprotective agents, we expanded the coverage of the sources of lead compounds through the in-depth study of marine-derived fungi.
In our ongoing research on novel neuroprotective metabolites from the fungus Aspergillus versicolor A18 from South China Sea, a pair of undescribed enantiomeric spiroketal diphenyl ether-coupled aromatic bisabolane sesquiterpenoids (1a/1b), two new aromatic bisabolane homodimers (2 and 3), as well as 12 known analogues (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) (Figure 1), were isolated from the rice media of the strain A18. It is noteworthy that compound 12 was first isolated as a natural product and its absolute configuration was also assigned. The structures of those compounds were determined by comprehensive spectroscopic data, and the absolute configurations were elucidated by ECD calculations or comparing the ECD spectra with those of correlative known analogues. The neuroprotective effects of selected compounds were evaluated. Herein, we report the isolation, structures elucidation, and neuroprotective activities of those compounds.
to build a spiroketal core moiety. Thus, part b of 1 was a spiroketal skeleton derived from a diphenyl ether. The HMBC correlations from H2-15 to C-3′, C-4′, and C-5′ and from 4′-OH to C-15 established that the aromatic bisabolane unit (part a) and highly variable diphenyl ether moiety (part b) were linked by the C-15-C-4′ bond. Finally, the planar structure of asperbisabol A (1) was elucidated.
In the NOESY spectrum of 1, the observed NOESY correlations of H-15β/H-6′β and 2-OH/10′-OH suggested that the relative configurations of C-4′ and C-5′ were defined as shown in Figure 3. The optical rotatory data of 1 was zero, which indicated that asperbisabol A (1) may be a racemate. Subsequent chiral resolution of 1 by semipreparative HPLC afforded the corresponding enantiomers 1a ( α = +37.00) and 1b ( α = −26.00). The absolute configurations of this pair of enantiomers were determined by comparison of their experimental ECD spectra with the calculated ECD spectra of (4′R,5′R)-1 and (4'S,5′S)-1. As shown in Figure 4, the experimental ECD curves of 1a and 1b matched well with the calculated ECD spectra of (4′R,5′R)-1 and (4'S,5′S)-1, respectively. Therefore, the absolute configurations of 1a (4′R,5′R) and 1b (4'S,5′S) were unambiguously determined, and given their trivial names (+)-asperbisabol A and (−)-asperbisabol A, respectively.   Compound 2 was obtained as a colorless oil. The molecular formula of 2 was settled as C 31 H 46 O 6 with 9 IHDs by the HRESIMS ion at m/z 537.3210 [M + Na] + (calcd. for C 31 H 46 O 6 Na 537.3187). The 1 H NMR data (Table 1) displayed the characteristic signals assigned to seven methyls (including one oxygenated), two 1,2,4-trisubstitutedphenyls, and two hydroxyl groups. The 13 C NMR data (Table 1) in combination with DEPT and HSQC spectra confirmed seven sp 2 quaternary carbons (including one carbonyl and two other oxygenated ones), two oxygenated sp 3 quaternary carbons, six sp 2 methines, two sp 3 methines, seven sp 3 methylenes (including an oxygenated), and seven sp 3 methyls (including a methoxyl). The 1 H NMR and 13 C NMR data of 2 closely resembled those of peniciaculin B (4), except for the presence of an additional methoxyl (δ H 3.13, δ C 50.4). Key HMBC correlation from 7-OMe to C-7 confirmed the position of the methoyl group. The ECD spectrum of compound 2 was similar to that of 4 in terms of positive Cotton effect (CE) from 260 nm to 320 nm and negative CE around 210 nm ( Figure S1) [3]. Therefore, the absolute configuration of 2 was also logically established as 7S,7 S and given the trivial name asperbisabol B.
Compound 3 was isolated as a colorless oil and assigned a positive HRESIMS ion at m/z 521.2689 [M + K] + (calcd. for C 30 H 42 O 5 K 521.2664), which well matched a molecular formula of C 30 H 42 O 5 with 10 IHDs. Through cumulative analysis of the 1 H NMR and 13 C NMR data of compounds 3 and 4, it made sense that 3 was a dehydration product of 4. This speculation was supported by the presence of a double bond (δ H 5.56; δ C 131.6 and 132.3) in 3 rather than an oxygenated sp 3 quaternary carbon (δ C 78.9) and a sp 3 methylene (δ H 1.80 and 1.89; δ C 43.0) in 4. The HMBC correlations from H 3 -14 to C-1, C-7, and C-8 further confirmed the location of the double bond. The positive CE from 260 nm to 320 nm and negative CE around 210 nm ( Figure S1) [3] unequivocally established the absolute configuration of 3 as 7 S and given the trivial name asperbisabol C.
The neuroprotective effects of 1a, 1b, and 2-14 against sodium nitrosprusside (SNP, 700 µM) induced injury were evaluated by the MTT assay in PC12 cells. The results of preliminary screening at a concentration of 10 µM ( Figure 5A) showed that compounds 1a, 6, and 8-10 exhibited more neuroprotective activities than that of the positive control edaravone (Eda, a free radical scavenger). Furthermore, these five active compounds have potent effects in a concentration-dependent manner in the range of 2.5-10 µM ( Figure 5B).

General Experimental Procedures
Optical rotations were measured on an Anton Paar MCP200 polarimeter (Graz, Austria). Circular dichroism spectra and UV spectra were obtained on an Applied Photophysics Chirascan spectrometer (Surrey, UK). HRESIMS were performed on a Shimadzu LCMS-IT-TOF spectrometer (Kyoto, Japan). NMR spectra were measured on

Fungal Material
The fungal strain Aspergillus versicolor A18 was isolated from a surface water sample collected in South China Sea and identified as Aspergillus versicolor on the base of the ITS region (GenBank MT5827511) [22]. The voucher specimen is deposited in East China Sea Fisheries Research Institute.

Fermentation and Extraction
The strain A. versicolor A18 was cultured on PDA plates (PDA media 24.0 g, agar 18.0 g and sea salt 30.0 g in 1.0 L H 2 O) at 28 • C for 7 days. The seed medium (PDB media 24.0 g and sea salt 30.0 g in 1.0 L H 2 O) was inoculated with strain A. versicolor A18 and incubated at 28.0 • C for 3 days on a rotating shaker (180 rpm). For chemical investigations, a large-scale fermentation of A. versicolor A18 was incubated for 28 days at 28 • C in 1.5 L × 40 conical flasks (each flask contained 450.0 g rice and 300.0 mL H 2 O with 3% salinity). After incubating, every flask was ultrasonically extracted with 4 × 0.4 L 95% EtOH for 30 min. The combined extract was subjected to nanofiltration membrane (300 D) for desalination and concentration. Then total 30 L concentrated solution was evaporated under reduced pressure to yield a dark brown gum, which was redissolved in 2 L water and subsequently extracted three times with petroleum ether (PE, 3 L each time) and five times with EtOAc (3 L each time) to afford PE fraction and EtOAc fraction.

ECD Calculation for Assigning the Absolute Configurations of 1a and 1b
The absolute configurations of 1a and 1b were determined by quantum chemical calculations of their theoretical ECD spectra. (4 R,5 R)-1, one of the enantiomers for 1, was arbitrarily chosen for theoretical studies. Conformational analyses were first carried out via Monte Carlo searching using molecular mechanism with MMFF force field in the Spartan 18 program. The results showed 20 lowest energy conformers for 1 within an energy window of 2.0 Kcal/mol. These conformers were reoptimized using DFT at the B3LYP/6-31G(d) level in gas phase using the Gaussian 09 program. 11 conformers of 1 ( Figure S51) with the relative Gibbs free energies (∆G) in the range of 0-1.5 Kcal/mol were refined and considered for next step. All the reoptimized conformers were applied for theoretical ECD calculation. The energies, oscillator strengths, and rotational strengths of the first 30 electronic excitations were calculated using the TD-DFT methodology at the M062X/TZVP level in PCM (acetonitrile). The ECD spectra were simulated by the overlapping Gaussian function (σ = 0.40 eV), in which velocity rotatory strengths of the first 18 exited states for 1 were adopted. To get the final ECD spectrum of each compound, the simulated spectra of the lowest energy conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (∆G). The theoretical ECD curve of (4 S,5 S)-1 was obtained by directly reversing that of (4 R,5 R)-1.

Neuroprotective Bioassays
Compounds 1a, 1b, and 2-14 as well as the positive control edaravone (Aladdin, Shanghai, China) were dissolved in DMSO (Sigma-Aldrich, Shanghai, China) as a stock, and the tested compounds was further diluted by DMEM medium (Gibco, Beijing, China) into three gradient concentrations (2.5, 5, and 10 µM). PC12 cells were digested and seeded into 96-well plates at a density of 5 ×10 3 cells per well and cultured in DMEM medium with 5% CO 2 for 24 h. Then the cell culture medium was replaced by DMEM medium containing different concentrations of compounds for pretreatment for 2 h and then treated with 700 µM SNP (Sigma-Aldrich, Shanghai, China) for another 24 h. About 10 µL of MTT (Beyotime Institute of Biotechnology, Shanghai, China) (5 mg/mL) was added into each well and incubated at 37 • C for 3.5 h. Afterwards, the supernatant was removed and the crystals were dissolved in 100 µL DMSO. The optical absorbance at 570 nm was read with an EPOCH 2 microplate reader (BioTek Devices, San Mateo, CA, USA). The experiments were repeated three times.