Acetylcholinesterase Inhibitory Meroterpenoid from a Mangrove Endophytic Fungus Aspergillus sp. 16-5c

Abstract

One new meroterpenoid, named 2-hydroacetoxydehydroaustin (1), together with nine known meroterpenoids, 11-acetoxyisoaustinone (2), isoaustinol (3), austin (4), austinol (5), acetoxydehydroaustin (6), dehydroaustin (7), dehydroaustinol (8), preaustinoid A2 (9), and 1,2-dihydro-acetoxydehydroaustin B (10), were isolated from the mangrove endophytic fungus, Aspergillus sp. 16-5c. These structures were characterized by spectroscopic analysis, further the absolute configurations of stereogenic carbons for Compounds 1, 3, 4, 6, 7, 8, 9, and 10 were determined by single crystal X-ray diffraction analysis using Cu Kα radiation. Moreover, the absolute configurations of stereogenic carbons for Known Compounds 3, 7, 8, and 9 are identified here for the first time. Compounds 3, 7, and 8 showed acetylcholinesterase (AchE) inhibitory activity with IC₅₀ values of 2.50, 0.40, and 3.00 μM, respectively.

1. Introduction

Meroterpenoids are natural products derived from terpenoids mixed biosynthetic origin [1]. Meroterpenoids display structural diversity with widespread biological activities including antimicrobial, antiviral, and antitumoral activities and enzyme inhibition. Despite the structural diversity, austin and austin analogues are mixed polyketide–terpenoid meroterpenoids derived from 3,5-dimethylorsellinic acid, which were most often isolated from fungi [2]. Austin was first isolated as a novel polyisoprenoid mycotoxin from Aspergillus ustus in 1976 [3]. Subsequently, some austin analogues from Aspergillus sp. and Penicillium sp. have been reported [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. As part of our ongoing investigations on novel bioactive compounds from mangrove endophytic-derived fungi [17,18,19,20], ten meroterpenoids, including one new meroterpenoids named 2-hydro-acetoxydehydroaustin (1), together with nine known meroterpenoids, 11-acetoxyisoaustinone (2), isoaustinol (3), austin (4), austinol (5), acetoxydehydroaustin (6), dehydroaustin (7), dehydroaustinol (8), preaustinoid A2 (9), and 1,2-dihydro-acetoxydehydroaustin B (10), were isolated from the fungus Aspergillus 16-5c, which was isolated from the leaves of the mangrove plant Sonneratia apetala collected on the coastal saltmarsh of the South China Sea (Figure 1). Compound 10 was reported as a mixture previously [16], but it is reported as a single compound here. The absolute configurations of stereogenic carbons for Compounds 3, 7, 8, and 9 are determined here for the first time (Figure 2). Herein, we report the isolation, structural elucidation, acetylcholinesterase (AChE) inhibitory activity, and cytotoxicity of these meroterpenoids.

2. Results and Discussion

Compound 1 was obtained as colorless crystal, m.p. >300 °C. The HRESIMS result (m/z 575.2124 [M + H]⁺) suggested the molecular formula of Compound 1 as C₂₉H₃₄O₁₂ with 13 degrees of unsaturation. The data of ¹D-NMR (

Table 1. ¹H (400 MHz) and ¹³C (100 MHz) NMR data for 1 and 2 (CDCl₃, in ppm).

Position	1		2
	δH (J in Hz)	δC	δH (J in Hz)	δC
1	2.23, dd (11.5, 13.4)	37.1, CH2	6.52, d (9.9)	146.7, CH
	2.98, dd (7.4, 13.4)
2	4.28, dd (7.4, 11.5)	64.8, CH	6.03, d (9.9)	120.0, CH
3		174.9, C		163.7, C
4		90.0, C		85.6, C
5		46.1, C		46.3, C
6	1.76, dd (11.9, 13.0)	36.1, CH2	2.66, td (13.5, 3.6)	27.1, CH2
	1.90, dd (3.9, 13.0)		1.79, dt (13.5, 3.6)
7	5.35, dd (3.9, 11.9)	67.8, CH	1.67,dd (12.2, 3.6)	27.0, CH2
			1.61,dd (12.2, 3.6)
8		56.6, C		41.3, C
9		93.2, C		134.8, C
10		138.1, C		138.1, C
11	5.70, s	74.3, CH	5.74, s	74.1, CH
12	1.37, s	12.0, CH3	1.57, s	23.0, CH3
13	5.81, d (1.5)	124.4, CH2	1.75, s	15.2, CH3
	5.85, d (1.5)
14	1.43, s	23.5, CH3	1.37, s	25.9, CH3
15	1.47, s	27.4, CH3	1.20, s	22.8, CH3
16		170.6, C	2.04, s	20.7, CH3
17	2.07, s	20.8, CH3		171.5, C
18		168.9, C
19	2.05, s	21.1, CH3
1′	5.73, s	116.9, CH2	5.41, d (0.9)	111.7, CH
	6.14, s		5.33, d (0.9)
2′		136.9, C		142.6, C
3′		82.5, C		59.2, C
4′		168.9, C		209.5, C
5′	5.26, q (6.9)	76.6 d	4.35, q (6.4)	76.2, CH
6′		85.5, C		91.2, C
7′		61.6, C		66.4, C
8′		167.3, C		169.1, C
9′	1.58, s	19.6, CH3	1.25, s	12.9, CH3
10′	1.70, d (6.9)	13.8, CH3	1.30, d (6.4)	12.7, CH3

) combined with HSQC spectrum showed that 1 has seven methyl groups [δ_H 1.37, 1.43, 1.47, 1.58, 1.70, 2.05, 2.07 (each 3H, s)], two methylene groups, four oxygenated methines (δ_H 4.28/δ_C 64.8, δ_H 5.35/δ_C 67.8, δ_H 5.70/δ_C 74.3, and δ_H 5.26/δ_C 76.6), one terminal olefin moiety [δ_H 5.81, 5.85 (each 1H, J = 1.5 Hz) and δ_H 5.73, 6.14 (each 1H, s)], two acetyl groups (δ_H 2.05/δ_C 20.8, 170.6 and δ_H 2.07/δ_C 21.1, 168.9), and three others.

The above spectroscopic features suggested that 1 belonged to the austin analogue [16], whose planar structure is similar to the 1,2-dihydro-acetoxydehydroaustin B. However, the carbon chemical shifts values of C-1 (δ_C 37.1) and C-2 (δ_C 64.8) were different from those of 1,2-dihydro-acetoxydehydroaustin B. The detailed comparison of the NMR data of 1 with those of 1,2-dihydro-acetoxydehydroaustin B clearly confirmed that 1 was a new structure (Figure 3). The absolute configurations of strereogenic carbons for 1 were determined by single-crystal X-ray diffraction using Cu Kα radiation [21]. Therefore, Compound 1 was named 2-hydroacetoxydehydroaustin. Its absolute configurations were 1S, 7R, 8S, 9R, 11S, 3′R, 5′R, 7′R.

Compound 2 was obtained as a colorless powder. Its molecular formula was assigned to be C₂₇H₃₂O₈ from the HRESIMS molecular ion peak at m/z 507.1988 [M + Na]⁺. Comparison of the NMR data revealed that the structure of 2 resembled that of 3 except for the presence of an acetyl group. A carbon signal at δ_C 74.1 (C-11) was observed in 2 compared to that of 3 [7]. It was deduced that the acetyl group was connected to C-11 via the hydroxyl group in 2 supported by the HMBC correlation from H-11 (δ_H 5.74, 1H, s) to 11-CH₃CO (δ_C 171.5). The relative configuration was confirmed by the NOE correlations, and the correlation signals between 9-CH₃ (δ_H 1.25), 11-OCOCH₃ (δ_H 2.04), and 12-CH₃ (δ_H 1.57) showed that the 11-acetoxyl, 12 and 9’-CH₃ were oriented at the same side. Furthermore, the NOE correlations between 10’-CH₃ (δ_H 1.30) and 6’-OH (δ_H 2.88) supported that the methyl and hydroxyl groups were at the same side. The NOE correlations of 2 were the same as 3, in addition to their positive optical rotation values. Finally, the absolute configurations of stereogenic carbons for 2 were assigned as 5R, 8S, 11S, 3′R, 5′R, 6′R, 7′R. These results confirmed that 2 was consistent with 11-acetoxylisoaustinone [22].

Austin was first isolated from Aspergillus ustus in 1976 before the Flack constant (the absolute configurations were determined) reported in 1983, and its absolute configuration was not definite. Herein, the absolute configurations of stereogenic carbons for Known Compounds 3, 7, 8, and 9 are identified for the first time by single crystal X-ray diffraction analysis using Cu Kα radiation. The absolute configurations were shown as follows: 3 is 5R, 8S, 3′R, 5′R, 6′R, 7′R, 7 is 5R, 8R, 11S, 3′S, 5′R, 6′R, 7′R, 8 is 5R, 8R, 11S, 3′S, 5′R, 6′R, 7′R, and 9 is 5S, 8S, 9S, 10S, 3′R, 5′S, 7′R (see the Supplementary Materials).

Although meroterpenoids have a wide range of biological activities [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16], few biological activities have been reported for austin and austin analogues. In this work, Compounds 1–10 were evaluated for their acetylcholinesterase (AChE) inhibitory activity using Huperzine A as a control (

Table 2. Acetylcholinesterase (AChE) inhibitory activity of Compounds 1–10 ^a.

Compound	1	2	3	4	5	6	7	8	9	10	HUP b
	>50	>50	2.50	>50	>50	>50	0.40	3.00	>50	>50	0.07

^a Data are expressed in IC₅₀ values (μmol/L). ^b HUP (Huperzine A) used as positive control.

). The results showed Compounds 3, 7, and 8 were potential AChE inhibitors with IC₅₀ values of 2.50, 0.40, and 3.00 μM, respectively.

In addition, four human cancer cell lines (human breast cancer cell lines MCF-7 and MDA-MB-435, human hepatoma cell line HepG2, and human prostatic cancer cell line PC-3) were used in the in vitro cytotoxicity bioassay. The results showed that Compounds 1–10 have no cytotoxicity (>50 μM) against human cancer cell lines.

3. Experimental Section

3.1. General

Melting points were determined on an X-4 micromelting point apparatus and are uncorrected. Optical rotations were measured on a Polartronic HHW5 digital polarimeter. IR spectra were measured on a Bruker Vector 22 spectrophotometer (Bruker, Billerica, MA, USA) using KBr pellets. The NMR spectra were recorded on a Bruker Avance 400 spectrometer at 400 MHz for ¹H and 100 MHz for ¹³C in CDCl₃. All chemical shifts (δ) are given in ppm with reference to the solvent signal (CDCl₃, δ_H 7.26 for ¹H, δ_C 77.23 for ¹³C; DMSO, δ_H 2.50 for ¹H, δ_C 39.52 for ¹³C), and coupling constants (J) are given in Hz. HRESIMS spectra were recorded on a Finnigan LCQ-DECA mass spectrometer (Thermo Scientific, shanghai, China). ESIMS spectra were recorded on a Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu, Taiwan). Single-crystal data were measured on an Oxford Gemini S Ultra diffractometer (Oxford Instrument, Oxfordshire, UK). Column chromatography (CC) was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Pharmacia, Piscataway, NJ, USA).

3.2. Fungal Material

The fungus used in this study was isolated from a mangrove, leaves of S. apetala, which were collected in Hainan Island, China. The fungus was identified as Aspergillus sp. by the ITS region (deposited in GenBank, accession number JX993829). A voucher strain was deposited in the China Center for Type Culture Collection under patent depository number CCTCC M 2012358.

3.3. Extraction and Isolation

The fungus Aspergillus sp. 16-5c was fermented on autoclaved rice solid-substrate medium for 28 days at room temperature. The mycelia and solid rice medium were extracted with MeOH. Then, the MeOH layer was dried in vacuo to yield 6.8 g of organic extract. The extract was separated by column chromatography (CC) over silica gel eluting with a gradient of CHCl₃/MeOH from 1:0 to 1:45 to yield five fractions (Fractions 1–5). Fraction 3 (120 mg) was applied to Sephadex LH-20 CC, eluting with CHCl₃/MeOH (1:1) to obtain Compound 1 (0.8 mg), 2 (1.8 mg), and 3 (2.0 mg). Fraction 4 (65 mg) was applied to Sephadex LH-20, eluting with CHCl₃/MeOH (1:1) to obtain Compound 4 (1.2 mg), 5 (1.6 mg), and 6 (2.6 mg). Fraction 5 (30 mg) was applied to Sephadex LH-20 CC, eluting with CHCl₃/MeOH (1:1) to obtain Compound 7 (1.4 mg), 8 (5.6 mg), 9 (2.4 mg), and 10 (4.6 mg).

2-Hydroacetoxydehydroaustin (1): colorless crystals (MeOH/CHCl₃); m.p. >300 °C; [α${]}_{\mathrm{D}}^{20}$ = + 60 (c 0.01, MeOH); UV (MeOH): λ_max 210, 236 nm; IR (KBr): ν_max 3528, 2998, 1748, 1431, 1384, 1214 and 1040 cm⁻¹; ¹H-NMR(CDCl₃, 400 MHz), ¹³C-NMR(CDCl₃, 100 MHz), see Table 1; ESIMS 575[M + H]⁺; HRESIMS m/z = 575.2124, [M + H]⁺ (calcd for C₂₇H₃₂O₈, 574.2050).

11-Acetoxyisoaustinone (2): colorless crystals (MeOH/CHCl₃); m.p. >300 °C; [α${]}_{\mathrm{D}}^{20}$ +150 (c 0.1 MeOH); IR (KBr) ν_max 3466, 3108, 2988, 2944, 2890, 1754, 1705, 1450, 1377, 1287, and 1226 cm⁻¹; ¹H-NMR (CDCl₃, 400 MHz) and ¹³C-NMR (CDCl₃, 100 MHz), see Table 1; ESIMS m/z 507 [M + Na]⁺; HRESIMS m/z 507.1988 [M + Na]⁺ (calcd for C₂₇H₃₂O₈Na, 507.1994).

3.4. X-Ray Crystallographic Analysis of 1, 3, 4, 6, 7, 8, 9 and 10

All single crystal X-ray diffraction data were collected at 150(2) K on an Oxford Gemini S Ultra diffractometer with Cu Kα radiation (λ = 1.54178 Å). The structures were solved by direct methods (SHELXS-97) and refined using full-matrix least-squares difference Fourier techniques. Hydrogen atoms bonded to carbons were placed on the geometrically ideal positions by the “ride on” method. Hydrogen atoms bonded to oxygen were located by the difference Fourier method and were included in the calculation of structure factors with isotropic temperature factors. Crystallographic data for 1, 3, 4, 6–10 have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC 1533328, 12 Union Road, Cambridge CB2 1EZ, UK (fax: t44-(0)1223-336033, or e-mail: [email protected]).

Crystal data of 1: C₂₉H₃₄O₁₂, Mr = 574.56, Orthorhombic, a = 8.8330(2) Å, b = 12.2482(2) Å, c = 12.3432(2) Å, α = 90°, β = 97.342(2)°, γ = 90°, V =1324.44(4) Å3, space group P21, Z = 2, Dx = 1.363 mg/m³, μ (Cu Kα) = 0.850 mm⁻¹, and F (000) = 1064. Crystal dimensions: 0.40 × 0.33 × 0.29 mm. Independent reflections: 4336 (Rint = 0.0392). The final R1 values were 0.0258, wR2 = 0.0659 (I > 2σ(I)). Flack parameter = 0.08 (8). CCDC number: 1533329.

Crystal data of 3: C₂₅H₃₀O₆, Mr = 426.49, Monoclinic, a = 11.5899(2) Å, b = 7.4432(2) Å, c = 12.0804(2) Å, α = 90°, β = 92.060(2)°, γ = 90°, V = 1041.45(4) Å3, space group P21, Z = 2, Dx = 1.360 mg/m³, μ(Cu Kα) = 0.786 mm⁻¹, and F (000) = 456. Crystal dimensions: 0.30 × 0.23 × 0.22 mm. Independent reflections: 3696 (Rint = 0.0549). The final R1 values were 0.0476, wR2 = 0.1215 (I > 2σ(I)). Flack parameter = 0.01 (2). CCDC number: 921711.

Crystal data of 4: C₂₇H₃₂O₉, Mr = 500.53, Orthorhombic, a = 7.71560(10) Å, b = 14.9523(2) Å, c = 21.1376(3) Å, α = 90°, β = 90°, γ = 90°, V = 2438.56(6) Å3, space group P212121, Z = 4, Dx = 1.363 mg/m³, μ (Cu Kα) = 0.850 mm⁻¹, and F (000) = 1064. Crystal dimensions: 0.40 mm × 0.33 mm × 0.29 mm. Independent reflections: 4336 (Rint = 0.0392). The final R1 values were 0.0258, wR2 = 0.0659 (I > 2σ(I)). Flack parameter = 0.08 (12). CCDC number: 921707.

Crystal data of 6: C₂₉H₃₂O₁₁, Mr = 556.55, Monoclinic, a = 8.8841 (3) Å, b = 12.5166 (4) Å, c = 12.1254 (5) Å, α = 90°, β = 99.052 (4) °, γ = 90°, V = 1331.54 (8) Å3, space group P21, Z = 2, Dx = 1.388 mg/m³, μ (Cu Kα) = 0.897 mm⁻¹, and F (000) = 588. Crystal dimensions: 0.44 mm × 0.35 mm × 0.21 mm. Independent reflections: 4474 (Rint = 0.0620). The final R1 values were 0.0326, wR2 = 0.0740 (I > 2σ (I)). Flack parameter = 0.01 (1). CCDC number: 921708.

Crystal data of 7: C₂₉H₃₃NO₉, Mr = 539.56, Hexagonal, a = 11.20730 (10) Å, b = 11.20730 (10) Å, c = 36.3299(5) Å, α = 90°, β = 90°, γ = 120°, V = 3951.82(7) Å3, space group P65, Z = 6, Dx = 1.360 mg/m³, μ(Cu Kα) = 0.841 mm⁻¹, and F (000) = 1716. Crystal dimensions: 0.44 mm × 0.35 mm × 0.32 mm. Independent reflections: 4596 (Rint = 0.0815). The final R1 values were 0.0597, wR2 = 0.1612 (I > 2σ(I)). Flack parameter = 0.01 (1). CCDC number: 921709.

Crystal data of 8: C₂₅H₂₈O₈, Mr = 456.47, Orthorhombic, a = 7.8409(2) Å, b = 11.6017(2) Å, c = 23.9529(4) Å, α = 90°, β = 90°, γ = 90°, V = 2178.94(8) Å3, space group P212121, Z = 4, Dx = 1.391 mg/m³, μ (Cu Kα) = 0.862 mm⁻¹, and F (000) = 968. Crystal dimensions: 0.37 mm × 0.31 mm × 0.22 mm. Independent reflections: 3880 (Rint = 0.0455). The final R1 values were 0.0277, wR2 = 0.0684 (I > 2σ(I)). Flack parameter = 0.01 (2). CCDC number: 921710.

Crystal data of 9: C₂₆H₃₄O₇, Mr = 458.53, Orthorhombic, a = 8.29600(10) Å, b = 13.9817(2) Å, c = 19.5230(2) Å, α = 90°, β = 90°, γ = 90°, V = 2264.52(5) Å3, space group P212121, Z = 4, Dx = 1.345 mg/m³, μ (Cu Kα) = 0.793 mm⁻¹, and F (000) = 984. Crystal dimensions: 0.33 mm × 0.30 mm × 0.25 mm. Independent reflections: 4027 (Rint = 0.0434). The final R1 values were 0.0280, wR2 = 0.0728 (I > 2σ (I)). Flack parameter = 0.08 (12). CCDC number: 921712.

Crystal data of 10: C₂₉H₃₄O₁₁, Mr = 558.56, Orthorhombic, a = 8.29600(10) Å, b = 13.9817(2) Å, c = 19.5230(2) Å, α = 90°, β = 98.7090(10)°, γ = 90°, V = 2264.52(5) Å3, space group P21, Z = 2, Dx = 1.345 mg/m³, μ (Cu Kα) = 0.793 mm⁻¹, and F (000) = 984. Crystal dimensions: 0.33 mm × 0.30 mm × 0.25 mm. Independent reflections: 4027 (Rint = 0.0434). The final R1 values were 0.0280, wR2 = 0.0728 (I > 2σ (I)). Flack parameter = 0.04 (11). CCDC number: 1533328.

3.5. Assays for Enzyme Inhibiting Activities and Cytotoxic Activities

These two experiments were conducted according to reference procedures [14,23].

4. Conclusions

One new and nine known meroterpenoids (1–10) were isolated and identified from the culture of the endophytic fungus Aspergillus sp. 16-5c. The absolute configurations of Known Compounds 3, 7, 8, and 9 were first identified. Compounds 3, 7, and 8 showed acetylcholinesterase (AchE) inhibitory activity with IC₅₀ values of 2.50, 0.40, and 3.00 μM, respectively.