Asperversins A and B, Two Novel Meroterpenoids with an Unusual 5/6/6/6 Ring from the Marine-Derived Fungus Aspergillus versicolor

Abstract

Asperversins A (1) and B (2), two novel meroterpenoids featuring an uncommon 5/6/6/6 ring system, along with five new analogues (3–7) and a known compound asperdemin (8), were obtained from the marine-derived fungus Aspergillus versicolor. Their structures and absolute configurations were confirmed by extensive spectroscopic analyses, single-crystal X-ray diffraction studies, and electronic circular dichroism (ECD) calculation. All new compounds were tested for their acetylcholinesterase enzyme (AChE) inhibitory activities and cytotoxic activities, of which compound 7 displayed moderate inhibitory activity against the AChE with an IC₅₀ value of 13.6 μM.

1. Introduction

Meroterpenoids are hybrid secondary metabolites that partially derive from the terpenoid pathways [1]. Naturally occurring meroterpenoids have been isolated from a variety of sources including animals, plants, bacteria, and fungi, [2] and are exemplified by ubiquinone-10 (coenzyme Q10) [3], α-tocopherol (vitamin E) [4], vinblastine [5], merochlorin A [6,7], and teleocidin B-4 [8].

Biosynthetically, the complex structures of fungal meroterpenoids are mostly derived from simple precursors like a linear isoprenoid or the C-2 carbon unit acetyl-CoA, via a series of chemical transformations catalyzed by two enzyme families, terpene cyclases and polyketide synthases (PKSs) [9,10]. In addition to the enormous structural diversity, fungal meroterpenoids have attracted wide interest from the scientific community due to their broad spectrum of pharmacological activities [11,12,13,14].

During our investigation of bioactive metabolites from fungi [15,16,17,18,19], many novel meroterpenoids were isolated from various fungi. Asperterpene A, obtained from Aspergillus terreus, represents a potential lead compound and a versatile scaffold for the development of drugs for the treatment of Alzheimer’s disease [15]. Apiroaspertrione A, bearing a unique spiro[bicyclo[3.2.2]nonane-2,1′-cyclohexane] carbocyclic skeleton, shows an effective activity against methicillin-resistant Staphylococcus aureus (MRSA) [16]. The metabolites of the fungus Aspergillus versicolor, that were obtained from mud from the South China Sea, are reported here. This endeavor resulted in the isolation of seven undescribed meroterpenoids, called as asperversins A–G (1–7), and a structurally related known compound, asperdemin (8) [20]. Notably, asperversins A (1) and B (2) have a unique 5/6/6/6 ring system that features a tetrahydrofuran ring. Herein, the isolation, structure elucidation, plausible biosynthetic pathway, and the biological evaluations of compounds 1–7 are presented.

2. Results and Discussion

Asperversin A (1), a white amorphous powder, was established to have a molecular formula of C₂₄H₃₂O₈ by HRESIMS ion at m/z 449.2173 ([M + H]⁺, calcd. for C₂₄H₃₃O₈, 449.2175), indicating 9 degrees of unsaturation. The strong IR absorptions at 1741 and 1703 cm⁻¹ suggested the presence of ester and unsaturated ester groups. The ¹H NMR data (in methanol-d₄,

Table 1. ¹H NMR Data for Compounds 1–7 (δ in ppm, J in Hz).

No.	1 a	2 a	3 a	4 b	5 b	6 a	7 a
1	3.98, dd (9.6, 2.3)	3.98, dd (9.7, 2.0)	5.42, dd (11.0, 1.8)	3.87, t (7.0)	3.79, dd (7.0, 5.2)	4.96, d (7.3)	7.08, d (10.2)
2	2.46, dd (15.7, 9.6) 2.68, m	2.39, m 2.63, dd (15.7, 2.0)	2.43, dd (14.8, 3.9) 2.88, dd (14.8, 1.8)	2.84, m 3.33, m	2.79, dd (16.0, 7.0) 3.32, d (16.0)	3.10, dd (16.2, 7.3) 3.55, d (16.2)	5.87, d (10.2)
5	2.13, d (2.3)	2.12, d (2.5)	2.49, d (3.0)	2.21, dd (12.1, 2.4)	2.38, d (2.3)	2.49, d (2.6)	1.98, d (1.8)
6	5.48, dd (4.5, 2.3)	5.48, m	5.27, m	1.67, m 1.92, m	5.52, dd (6.3, 3.0)	5.64, m	5.60, m
7	2.11, m 2.35, m	2.11, m 2.35, m	2.04, m 2.30, dd (14.4, 2.9)	1.72, m 2.06, m	1.92, dd, (14.6, 6.3) 2.26, dd, (14.6, 3.0)	2.05, m 2.40, dd (14.7, 3.0)	1.91, dd (14.7, 3.0) 2.42, m
9	1.97, dd (12.6, 5.2)	1.98, dd (11.3, 6.4)	1.90, dd (12.7, 4.6)	2.32, dd (12.5, 4.6)	2.34, m	2.15, m	1.81, dd (12.9, 4.3)
11	2.33, m 2.41, m	2.40, m 2.41, m	2.40, m 2.80, dd (17.4, 4.6)	2.08, m 2.43, dd (16.2, 4.6)	2.09, m 2.35, m	2.29, m 2.30, m	2.42, dd (16.5, 12.9) 2.76, dd (16.5, 4.3)
15	5.91, d (1.0)	5.92, d (1.1)	5.91, d (1.1)	5.81, d (1.1)	5.72, d (0.6)	5.91, d (1.1)	5.70, d (1.1)
17	2.20, d (1.0)	2.21, d (1.1)	2.21, d (1.1)	2.15, d (1.1)	2.05, d (0.6)	2.20, d (1.1)	2.18, d (1.1)
18	1.43, s	1.43, s	1.42, s	1.30, s	1.32, s	1.41, s	1.40, s
19	1.31, s	1.32, s	1.33, s	1.14, s	1.35, s	1.53, s	1.46, s
20	1.26, s	1.26, s	5.05, m 5.08, m	1.41, s	1.31, s	1.49, s	1.23, s
21	1.18, s	1.18, s	1.94, s	1.50, s	1.57, s	1.73, s	1.19, s
23	2.07, s	2.07, s	2.07, s		2.06, s	2.14, s	2.09, s
24	3.70, s		2.08, s
25						1.99, s

^a Recorded at 400 MHz in methanol-d₄; ^b recorded at 600 MHz in acetone-d₆.

) displayed an olefinic proton at δ_H 5.91 (d, J = 1.0 Hz, H-15), a methoxy group at δ_H 3.70 (s, OMe-24) and six methyl groups at δ_H 2.20 (d, J = 1.0 Hz, H₃-17), 2.07 (s, H₃-23), 1.43 (H₃-18), 1.31 (H₃-19), 1.26 (H₃-20), and 1.18 (H₃-21). The ¹³C and DEPT spectra revealed the presence of three carbonyl carbons (δ_C 173.6, 171.6, and 167.1) and two enol systems (δ_C 165.1, 162.2, 102.2, and 98.5), three quaternary sp³ carbons (two oxygenated), four methines (two oxygenated), three methylene carbons, and seven methyl groups.

Comparison of the ¹H and ¹³C NMR data (Table 1 and

Table 2. ¹³C and DEPT NMR Data of Compounds 1–7 (δ in ppm, J in Hz).

No.	1 a	2 a	3 a	4 b	5 b	6 a	7 a
1	84.0, CH	84.5, CH	77.2, CH	68.8, CH	69.3, CH	72.6, CH	155.1, CH
2	37.3, CH2	37.9, CH2	36.2, CH2	39.8, CH2	39.9, CH2	36.3, CH2	125.1, CH
3	173.6, C	176.7, C	173.2, C	171.2, C	171.0, C	172.9, C	203.2, C
4	79.7, C	79.6, C	144.4, C	84.8, C	84.1, C	86.7, C	45.1, C
5	61.4, CH	61.5, CH	51.3, CH	50.2, CH	49.5, CH	51.3, CH	53.0, CH
6	68.3, CH	68.3, CH	73.4, CH	23.3, CH2	72.6, CH	72.7, CH	68.8, CH
7	45.3, CH2	45.3, CH2	43.3, CH2	40.6, CH2	43.3, CH2	43.3, CH2	43.4, CH2
8	81.5, C	81.6, C	81.2, C	80.9, C	79.9, C	80.4, C	79.3, C
9	48.1, CH	48.1, CH	44.2, CH	44.8, CH	44.4, CH	45.1, CH	46.9, CH
10	46.5, C	46.4, C	45.6, C	45.3, C	45.4, C	45.1, C	39.4, C
11	19.4, CH2	19.4, CH2	19.7, CH2	17.5, CH2	17.2, CH2	17.6, CH2	16.9, CH2
12	98.5, C	98.6, C	99.0, C	98.2, C	98.3, C	98.3, C	97.3, C
13	167.1, C	167.2, C	167.1, C	164.5, C	164.5, C	164.6, C	164.8, C
14	162.2, C	162.2, C	162.2, C	160.8, C	161.0, C	162.3, C	160.5, C
15	102.2, CH	102.0, CH	101.8, CH	100.6, CH	100.6, CH	101.7, CH	100.3, CH
16	165.1, C	165.2, C	164.8, C	163.3, C	162.8, C	166.8, C	162.7, C
17	19.5, CH3	19.5, CH3	19.5, CH3	19.5, CH3	19.5, CH3	19.5, CH3	19.7, CH3
18	23.0, CH3	23.0, CH3	22.1, CH3	20.9, CH3	21.9, CH3	22.0, CH3	22.0, CH3
19	12.4, CH3	12.5, CH3	15.3, CH3	15.2, CH3	16.2, CH3	16.0, CH3	18.1, CH3
20	30.5, CH3	30.6, CH3	117.7, CH2	34.9, CH3	33.9, CH3	33.9, CH3	26.6, CH3
21	25.5, CH3	25.6, CH3	26.5, CH3	24.0, CH3	26.4, CH3	26.2, CH3	22.8, CH3
22	171.6, C	171.7, C	171.5, C		170.2, C	171.2, C	169.7, C
23	21.3, CH3	21.3, CH3	20.9, CH3		21.4, CH3	21.3, CH3	21.5, CH3
24	52.3, OCH3		172.2, C			171.1, C
25			21.4, CH3			20.6, CH3

^a Recorded at 100 MHz in methanol-d₄; ^b recorded at 150 MHz in acetone-d₆.

) of 1 with the known compound asperdemin (8) [20] revealed their close similarities, except for the presence of an additional methoxy group (δ_H 3.70, δ_C 52.3) and an acetoxyl unit. Further analyses of the ¹H–¹H COSY and HMBC spectra of 1 indicated that 1 shared the same B/C/D ring system as that of 8 (Figure 1). The key HMBC correlation from H-6 to C-22 (δ_C 171.6) indicated the acetoxyl group at C-6. Furthermore, the ¹H–¹H COSY cross-peaks of H-1/H₂-2 and the HMBC connections from H-1 to C-3, from H₂-2 to C-3, and from OMe to C-3 confirmed the existence of a methyl ester group in 1, rather than a seven-membered lactone. Although there was no obvious HMBC correlation to connect C-1 and C-4, the absence of hydroxyl at C-1 confirmed by the 1D and 2D NMR data in DMSO-d₆ and the chemical shifts of C-1 (δ_C 81.9 for 1; 68.0 for 8) and C-4 (δ_C 77.7 for 1; 84.8 for 8) indicated that C-1 and C-4 were connected with an ether bond to establish a tetrahydrofuran ring, which was consistent with the unsaturation degrees deduced by HRESIMS. Thus, the unusual tetrahydrofuran A ring replaced the seven-membered lactone ring, which distinguished compound 1 from other metabolites reported in this compound class.

The relative configuration of 1 was partially elucidated by the examination of its Nuclear Overhauser Effect SpectrocopY (NOESY) spectrum (Figure 2). The configuration of Me-19 was arbitrarily assigned as same as that of 8 in a β-orientation. Furthermore, the NOESY correlations of H₃-18/H-11b and H-11b/H₃-19 suggested their cofacial and β-orientation. In contrast, H-9 should be α-oriented. The NOESY correlations of H-1/H-9, H-9/H-5, and H-1/H-5 indicated their α-orientation. The spin-coupling constant (J = 2.3 Hz) of H-5 and H-6 suggested H-6 was equatorial. To confirmed the absolute configuration, the ECD calculation of 1 [1S,5S,6R,8R,9R,10R-1 (1a) and 1R,5R,6S,8S,9S,10S-1 (1b)] was conducted in MeOH using the time-dependent density functional theory (TD-DFT) method at the B3LYP/6-311+g (d, p) level. The experimental ECD of 1 matched well with the calculated ECD curve of 1a, indicating the absolute configuration of 1 as 1S,5S,6R,8R,9R,10R (Figure 3).

The HRESIMS spectrum suggested a molecular formula of C₂₃H₃₀O₈ for compound 2, indicating a molecular weight of 14 mass units lower than 1. The ¹H and ¹³C NMR data of 2 (Table 1 and Table 2) closely resembled those of compound 1, except for the difference of chemical shift of carbonyl (C-3: δ_C 176.7 for 2; 173.6 for 1), and the absence of the methoxy group. Compound 2 was speculated to be the carboxylic acid analogue of 1. The structure of 2 was confirmed by extensive analyses of its ¹H–¹H COSY and HMBC spectra. Compound 2 was confirmed to have the same relative and absolute configurations as 1 by the NOESY spectra and their similar ECD curves (Figure 1, Figure 3 and Figure 4).

Asperversin C (3), a white amorphous powder, has a molecular formula of C₂₆H₃₄O₉, which was determined on the basis of the HRESIMS ion peak at m/z 491.2269 ([M + H]⁺, calcd. for C₂₆H₃₅O₉, 491.2281). The similar IR, UV, 1D, and 2D NMR spectra of 3 and 1 suggested both compounds shared the same B/C/D rings. A comparison of their 1D and 2D NMR data indicated the presence of a double bond (δ_H 5.05 and 5.08, δ_C 117.7, CH₂-20; δ_C 144.4, C-4) and the absence of a methyl group in compound 3. The location of the double bond between C-4 and C-20 was confirmed by the HMBC correlations from H₂-20 to C-4, C-5, and C-21, and from H₃-21 to C-4 and C-20. Moreover, the HMBC correlations from H-1 to C-24 and H₃-25 to C-24 defined the location of an additional acetoxyl unit at C-1. Finally, the planar structure of 3 with the A ring opened was confirmed as shown (Figure 1). The NOE correlations from H-1/H₃-19, H₃-18/H₃-19 suggested H-1, Me-18, and Me-19 were β-oriented. In contrary, H-5, H-6 and H-9 were α-oriented based on the correlations of H-5/H-6 and H-5/H-9. In consideration of the similar specific rotation and ECD spectra (Figure 4), the absolute configuration of 3 was assigned as 1R,5S,6R,8R,9R,10R.

Asperversin D (4) possesses a molecular formula of C₂₁H₂₈O₆ with 16 units less than that of compound 8 on the basis of HRESIMS data. Comparison of the ¹H and ¹³C NMR data of 4 with those of 8 revealed the presence of an additional methylene group and the absence of an oxygenated methine group in 4. Inspection of the 2D NMR of 4 indicated this methylene group (δ_C 23.3; δ_H 1.67 and 1.92) should be assigned to C-6 by the ¹H–¹H COSY correlations of H-5/H₂-6/H-7. The gross structure of 4 and its relative configuration were then confirmed by comprehensive analyses of the ¹H–¹H COSY, HMBC, and NOESY spectra. Finally, the absolute configuration of 4 was ascertained as 1R,5S,8R,9R,10R by a similar ECD spectrum to that of 1 (Figure 4).

Asperversin E (5), colorless crystals, exhibits a molecular formula of C₂₃H₃₀O₈ with nine degrees of unsaturation from a HRESIMS ion peak at m/z 457.1830 ([M + Na]⁺, calcd. for 457.1838). Close similarities were observed in the 13C NMR data (Table 2) between compounds 5 and 4. Inspection of the 1D NMR spectra, HSQC, and HMBC spectra revealed that 5 contained an additional acetoxyl group (δ_H 2.06, δ_C 21.4, and 170.2). Detailed HMBC correlations from H₃-23 (δ_H 2.06) to C-22 (δ_C 170.2) and from H-6 (δ_H 5.52) to C-22 confirmed the location. The X-ray crystallographic diffraction (Figure 5) revealed the configuration of 5 was 1R,5S,6R,8R,9R,10R.

A protonated molecular ion peak at m/z 477.2098 ([M + H]⁺, calcd. 477.2125) was obtained from the HRESIMS experiment and indicated asperversin F (6) has the molecular formula C₂₅H₃₂O₉. The ¹H and ¹³C NMR spectra of 6 showed there were two more acetoxyl groups (δ_H 2.14, δ_C 21.3, and 171.2; δ_H 1.99, δ_C 20.6, and 171.1) than 4. The locations of two acetoxyls were assigned to C-1 and C-6 by the HMBC correlations from H-1 (δ_H 4.96) to C-24 and from H-6 (δ_H 5.64) to C-22, respectively. The relative configuration and absolute configuration of 6 were determined by analyzing the NOESY spectrum and by comparing its ECD spectrum with that of 5 (Figure 4).

Asperversin G (7), colorless cubic crystals, has a molecular formula of C₂₃H₂₈O₆ based on the HRESIMS spectrum. The ¹H and ¹³C NMR data (Table 1 and Table 2) of 7 resembled those of 5, with the main difference being the presence of an α,β-unsaturated ketone group (δ_H 7.08, d, J = 10.2 Hz and 5.87, d, J = 10.2 Hz; δ_C 203.2, 155.1, and 125.1). The α,β-unsaturated ketone further constructed an 2-cyclohexen-1-one (ring A), as ascertained by the HMBC correlations from H₃-19 to C-1, C-5, C-9, and C-10, from H-1 to C-3, from H-2 to C-3 and C-4, and from H₃-20 and H₃-21 to C-3, C-4, and C-5. The remainder of 7 was identical to 5 as elucidated by 2D NMR spectra. The absolute configuration of 7 was established unambiguously as 5S,6R,8R,9R,10R by a single-crystal X-ray diffraction (Figure 5).

Asperversins A (1) and B (2) were found to be the first examples of pyrone meroterpenoids featuring an exclusive 5/6/6/6 ring system compared to all known meroterpenoids [2]. Therefore, a plausible biosynthetic pathway is proposed to illustrate the generation of 1 and 2 (Scheme 1). Asperversins A (1) and B (2) are probably biosynthesized via a polyketide and mevalonate hybrid biogenetic pathway [2,21,22]. After a series of oxidations, the A ring of I was opened, and then, followed by the key electrophilic addition of OH-4 with the α,β-unsaturated carboxylic acid bond, the intermediate III, featuring a tetrahydrofuran ring, was derived from II. Compounds 2 and 1 were produced subsequently by an additional acetylation and methylation.

All compounds (1–8) were inactive for cytotoxicity against A549, MCF-7, HepG2, and HL-60 cells, and did not have any antimicrobial effects against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), Escherichia coli, or Pseudomonas aeruginosa. The acetylcholinesterase (AChE) inhibition effects of these compounds (1–8) were assessed by Ellman’s spectrophotometric method [23] using human recombinant AChE with galanthamine as control compound (

Table 3. The inhibitory value and docking score of compounds 1–8 with AChE.

Compounds	Inhibitory Ratio at 100 μM	IC50 Value (μM) a	Docking Score b
1	35.7%	>40	3.25
2	36.2%	>40	3.89
3	27.6%	>40	2.76
4	50.2%	>40	5.32
5	48.7%	>40	4.98
6	62.9%	>40	5.87
7	92.4%	13.6	7.85
8	25.3%	>40	2.58
Galanthamine		3.57

^a IC₅₀ value of compounds against AChE (μM); ^b Docking score/interaction potential of compounds with target protein.

). Compound 7 exhibited the strongest inhibition to AChE with an IC₅₀ value of 13.6 μM, and compounds 1–6 and 8 showed no activities up to a concentration of 40 μM.

The molecular docking studies were conducted in order to get an insight into the binding pattern and extent of binding of compound 7 with the target enzyme (Figure 6). This was mainly attributed to the basic skeleton of compound 7 which provided better binding prospects with the formation of hydrogen bonding interactions to the amino acid residues Glu 291 and Tyr 341 of the protein. So we speculated the α,β-unsaturated ketone group was the key pharmacophore for the acetylcholinesterase (AChE) inhibition effect.

3. Experimental Section

3.1. General Experimental Procedures

The NMR spectra were recorded on Bruker AM-400 and 600 spectrometers (Bruker, Karlsruhe, Germany). The ¹H and ¹³C NMR chemical shifts were referenced to the solvent or solvent impurity peaks for methanol-d₄ (δ_H 3.31 and δ_C 49.0), and acetone-d₆ (δ_H 2.05 and δ_C 206.3 and 29.8). The FT-IR spectra were determined with a Bruker Vertex 70 instrument (Bruker, Karlsruhe, Germany). The UV and ECD spectra were measured using a Perkin Elmer Lambda 35 UV spectrophotometer (PerkinElmer, Inc., Fremont, CA, USA) and a JASCO-810 ECD spectrometer (JASCO Co., Ltd., Tokyo, Japan), respectively. Optical rotations were measured by an AUTOPOL IV-T Automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). HRESIMS data were obtained in the positive ion mode on a Thermo Fisher LTQ XL spectrometer (Thermo Fisher, Palo Alto, CA, USA). Crystal X-ray diffraction data were measured on a Rigaku XtaLAB PRO MM007HF (Rigaku, Tokyo, Japan). Semipreparative HPLC was carried out using an Agilent 1200 quaternary system with a UV detector using a reversed-phase C₁₈ column (5 μm, 10 × 250 mm, Welch Ultimate XB-C18). Column chromatography (CC) was performed using silica gel (100–200 and 200–300 mesh; Qingdao Marine Chemical Inc., Qingdao, China), ODS (50 μm, YMC, Kyoto, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Thin-layer chromatography (TLC) was performed on silica gel 60 F254 (Yantai Chemical Industry Research Institute, Yantai, China).

3.2. Fungi Material

The fungus Aspergillus versicolor was isolated from mud of the South China Sea. The sequence data for this strain have been submitted to DDBJ/EMBL/GenBank under accession no. 2081031. A voucher sample has been deposited in the culture collection of Tongji Medical College, Huazhong University of Science and Technology, P. R. China.

3.3. Fermentation and Extraction

The Aspergillus versicolor strain was cultured on potato dextrose agar (PDA) at 28 °C for 7 days to prepare the seed culture and inoculated into 100 sterilized Erlenmeyer flasks (1 L), which contained 200.0 g of rice and 160.0 mL of distilled water, then incubated at 28 °C for 28 days. Following incubation, the rice was soaked with 95% EtOH many times until the solvent was near-colorless at room temperature. Finally, solvent removal afforded a brown extract that was suspended in water (2 L) and extracted with EtOAc (1:1, v/v) three times. After removal of EtOAc, a dark brown gum (170 g) was obtained.

3.4. Isolation

The extracts (170.0 g) were subjected to a silica gel chromatography column (CC) eluting with PE/ EtOAc (20:1–0:1) progressively to obtain six fractions (Fr. 1–Fr. 6). Fr.3 was further separated with silica gel CC to yield four subfractions (Fr. 3.1–Fr. 3.4). The subfraction Fr. 3.1 was subjected to a Sephadex LH-20 (MeOH) to afford three parts (Fr. 3.1a–Fr. 3.1c). After crude purification of Fr. 3.1a by ODS column (MeOH-H₂O, 20:80–70:30, v/v), the second part of Fr. 3.1a (Fr. 3.1a-2, MeOH–H₂O, 60:40, v/v) was purified by repeated semi-preparative HPLC (MeCN–H₂O, 55:45, 2 mL/min) to yield 1 (9.3 mg, t_R 25.2 min), 2 (6.2 mg, t_R 28.5 min), 7 (23.6 mg, t_R 30.1 min).

Three subfractions (Frs. 3.2a–3.2c) were obtained from Fr. 3.2 by chromatography on an ODS column (MeOH–H₂O, 20:80–70:30, v/v). Fr.3.2b was chromatographed on ODS (MeOH–H₂O, 40:60–70:30) to afford a mixture (4, 5, 6, 8) in 40% yield. The mixture was purified by semi-preparative HPLC (MeCN–H₂O, 40:60–60:40, 2 mL/min) to yield 8 (16.3 mg, t_R 25.5 min, 45:55), 5 (200.0 mg, t_R 27.2 min, 45:55), 4 (3.7 mg, t_R 21.0 min, 40:60), and 6 (10.2 mg, t_R 21.0 min, 40:60). Fr. 3.2c was chromatographed on ODS (MeOH–H₂O, 30:70–80:20, v/v) to yield four parts. Fr. 3.2c-2 (the second part of Fr. 3.2c, MeOH–H₂O, 50:50–60:40, v/v) was purified by repeated semi-preparative HPLC (MeCN–H₂O, 46:54, 2 mL/min) to yield 3 (2.78 mg, t_R 15.1 min).

3.4.1. Asperversin A (1)

C₂₄H₃₂O₈; a white amorphous powder; [α]²⁵_D +46.7 (c 0.34, MeOH); UV (MeOH) λ_max (log ε) 205 (4.51) and 286 (3.84) nm; IR (KBr) ν_max 3445, 2958, 1741, 1710, 1654, 1588, 1444, 1403, 1384, 1237, 1194, 996 cm⁻¹; ECD (MeOH) λ_max (Δε) = 205 (+25.02) and 286 (+1.86) nm; For ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) data, see Table 1 and Table 2; HRESIMS 449.2173 ([M + H]⁺, calcd. for C₂₄H₃₃O₈, 449.2175).

3.4.2. Asperversin B (2)

C₂₃H₃₀O₈; a white amorphous powder; [α]²⁵_D +100.3 (c 0.29, MeOH); UV (MeOH) λ_max (log ε) 202 (4.78) and 285 (4.14) nm; IR (KBr) ν_max 3433, 2963, 2929, 1740, 1710, 1652, 1584, 1446, 1407, 1235, 1194, 1028 cm⁻¹; ECD (MeOH) λ_max (Δε) = 207 (+21.37) and 286 (+0.11) nm; For ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) data, see Table 1 and Table 2; HRESIMS m/z 457.1846 ([M + Na]⁺, calcd. for C₂₃H₃₀NaO₈, 457.1838).

3.4.3. Asperversin C (3)

C₂₅H₃₂O₉; a white amorphous powder; [α]²⁵_D +100.9 (c 0.23, MeOH); UV (MeOH) λ_max (log ε) 205 (4.51) and 284 (3.87) nm; IR (KBr) ν_max 3425, 2922, 1743, 1711, 1655, 1591, 1233, 1192, 1032 cm⁻¹; ECD (MeOH) λ_max (Δε) = 203 (+19.88) and 286 (+2.34) nm; For ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) data, see Table 1 and Table 2; HRESIMS m/z 491.2269 ([M + H]⁺, calcd. for C₂₆H₃₅O₉, 491.2281).

3.4.4. Asperversin D (4)

C₂₁H₂₈O₆; a white amorphous powder; [α]²⁵_D +98.4 (c 0.25, MeOH); UV (MeOH) λ_max (log ε) 205 (4.77) and 286 (3.93) nm; IR (KBr) ν_max 3429, 2928, 1702, 1653, 1586, 1449, 1408, 1386, 1313, 1274, 1154, 1113 cm⁻¹; ECD (MeOH) λ_max (Δε) = 205 (+23.13) and 285 (+2.24) nm; For ¹H NMR (600 MHz) and ¹³C NMR (150 MHz) data, see Table 1 and Table 2; HRESIMS 377.1919 ([M + H]⁺, calcd. for C₂₂H₂₉O₈, 377.1964).

3.4.5. Asperversin E (5)

C₂₃H₃₀O₈; colorless crystals; [α]²⁵_D +75.5 (c 0.20, MeOH); UV (MeOH) λ_max (log ε) 205 (4.57) and 286 (3.89) nm; IR (KBr) ν_max 3431, 2988, 2931, 1714, 1653, 1588, 1447, 1402, 1233, 1128, 1086 cm⁻¹; ECD (MeOH) λ_max (Δε) = 208 (+28.96) and 281 (+1.86) nm; For ¹H NMR (600 MHz) and ¹³C NMR (150 MHz) data, see Table 1 and Table 2; HRESIMS 457.1830 ([M + Na]⁺, calcd. for C₂₃H₃₀O₈Na, 457.1838).

3.4.6. Asperversin F (6)

C₂₅H₃₂O₉; a white amorphous powder; [α]²⁵_D +100.6 (c 0.48, MeOH); UV (MeOH) λ_max (log ε) 205 (4.61) and 286 (3.77) nm; IR (KBr) ν_max 3462, 2992, 2934, 1736, 1655, 1590, 1448, 1409, 1374, 1230, 1202, 1131, 1024 cm⁻¹; ECD (MeOH) λ_max (Δε) = 215 (+23.68) and 282 (+0.34) nm; For ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) data, see Table 1 and Table 2; HRESIMS 477.2143 ([M + H]⁺, calcd. for C₂₅H₃₃O₉, 477.2098).

3.4.7. Asperversin G (7)

C₂₃H₂₈O₆; colorless cubic crystals; [α]²⁵_D +110.2 (c 0.21, MeOH); UV (MeOH) λ_max (log ε) 205 (4.60), 228 (4.18) and 285 (3.88) nm; IR (KBr) ν_max 3434, 2975, 2927, 2862, 1793, 1706, 1674, 1587, 1448, 1408, 1257, 1232, 1141 cm⁻¹; ECD (MeOH) λ_max (Δε) = 215 (+16.60), 291 (+1.11) and 342 (–1.01) nm; For ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) data, see Table 1 and Table 2; HRESIMS [M + H]⁺ m/z 401.1977 (calcd. for C₂₃H₂₉O₆, 401.1964).

3.5. X-ray Crystal Structure Analysis

The crystallographic data for 5 (deposition no. CCDC 1816743), and 7 (deposition no. CCDC 1813401) have been deposited in the Cambridge Crystallographic Data Centre. Copies of the data can be obtained free of charge from the Cambridge Crystallographic Data Centre.

3.5.1. Crystal Data for Asperversin E (5)

C₂₃H₃₀O₈∙H₂O (M = 452.48 g/mol), orthorhombic, space group P2₁2₁2₁ (no. 19), a = 9.95215(4) Å, b = 12.89469(4) Å, c = 17.07108(6) Å, V = 2190.729(13) ų, Z = 4, T = 100.01(10) K, μ (CuKα) = 0.881 mm⁻¹, Dcalc = 1.372 g/cm³, 23,143 reflections measured (8.594 ≤ 2Θ ≤ 147.672), 4396 unique (R_int = 0.0181, R_sigma = 0.0097) which were used in all calculations. The final R₁ was 0.0274 (I > 2σ(I)) and wR₂ was 0.0772. The Flack parameter was 0.039(16).

3.5.2. Crystal Data for Asperversin G (7)

C₂₃H₂₈O₆ (M = 400.45 g/mol), monoclinic, space group P2₁, a = 8.77354(16) Å, b = 13.09891(17) Å, c = 9.64200(17) Å, α = 90.0°, β = 116.686(2)°, γ = 90.0°, V = 990.06(3) ų, Z = 2, T = 100.01(10) K, μ (CuKα) = 0.790 mm⁻¹, Dcalc = 1.541 g/cm³, 23,143 reflections measured (10.268 ≤ 2Θ ≤ 147.12), 3462 unique (R_int = 0.0195, R_sigma = 0.0162) which were used in all calculations. The final R₁ was 0.0276 (I > 2σ(I)) and wR₂ was 0.0738. The Flack parameter was 0.11(7).

3.6. Computational Methods

Monte Carlo conformational searches were carried out by the Spartan’s 10 software using the Merck Molecular Force Field (MMFF). The conformers with a Boltzmann-population of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31+g (d, p) level in MeOH using the CPCM polarizable conductor calculation model. The theoretical calculation of ECD was conducted in MeOH using Time-dependent Density functional theory (TD-DFT) at the B3LYP/6-311+g (d, p) level for all conformers of compounds 1a and 1b. Rotatory strengths for a total of 50 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV [24].

3.7. Acetylcholinesterase Inhibitory Activities Assay

The inhibitory activities of compounds 1–8 against AChE were evaluated by Ellman’s method with slight modification [25]. Briefly, 20 µL AChE (0.05 U/mL) and 20 µL different concentrations of compounds (0.1 M phosphate buffer, pH 8.0) containing 0.1% Triton X-100) were pre-incubated at 37 °C for 30 min, then 160 µL of the assay solution which consisted of a 0.1 M phosphate buffer pH 8.0, with the addition of 600 µM 5,5’-dithio-bis (2-nitrobenzoic acid) and 550 µM acetylthiocholine iodide (ATCh) were added in the 96-well plates. The absorbance value at 425 nm was recorded for 15 min and enzyme activity was calculated from the slope of the obtained linear trend. The reaction rates were compared and the % inhibition due to the presence of tested inhibitors was calculated. Each concentration was analyzed in triplicate, and IC₅₀ values were determined graphically from log concentration inhibition curves (Graphpad Prism 5, GraphPad Software Inc., San Diego, CA, USA). Galanthamine was used as a standard inhibitor.

3.8. Docking Studies

Cocrystal structure of human AChE with compound 1 (PDB code: 4EY7) was obtained from the Protein Data Bank for docking tests (http://www.rcsb.org) [26]. The virtual screening was implemented in the Surflex-Dock module of the Sybyl software. Molecules were built with Chemdraw and optimized at molecular mechanical and semiempirical level by using Open Babel GUI. The crystallographic ligand was extracted from the active site and the designed ligands were modelled. All the hydrogen atoms were added to define the correct ionization and tautomeric states, and the carboxylate, phosphonate and sulfonate groups were considered in their charged form. In the docking calculation, the default FlexX scoring function was used for exhaustive searching, solid body optimization, and interaction scoring. Finally, the ligands with the lowest-energy and the most favorable orientation were selected [27].

4. Conclusions

In conclusion, eight meroterpenoids were isolated from the marine derived fungus Aspergillus versicolor, including seven new ones, asperversins A–E (1–7), and a known one, asperdemin (8). Notably, asperversins A (1) and B (2) possess a unique 5/6/6/6 ring system with a constructed tetrahydrofuran ring. The absolute structures of all new compounds were confirmed by comprehensive spectroscopic analyses: compound 1 was elucidated by experimental and calculated ECD, and compounds 5 and 7 were ascertained by the X-ray crystallographic diffractions. All isolated meroterpenoids were tested for their acetylcholinesterase enzyme (AChE) inhibitory activities, and compound 7 displayed moderate inhibitory activity against the AChE with an IC₅₀ value of 13.6 μM.