New α-glucosidase inhibitors from a marine sponge-derived fungus, Aspergillus sp. OUCMDZ-1583

Abstract

Eighteen new compounds named aspergones A–Q (1–17) and 6-O-demethylmonocerin (18), along with five known analogues (19–23), were isolated from the fermentation broth of Aspergillus sp. OUCMDZ-1583 associated with an unidentified marine sponge (XD10410) from the Xisha Islands of China. The structures, including the absolute configurations, were unambiguously elucidated by spectroscopic, X-ray crystallographic, chemical, and Mosher’s methods along with quantum ECD calculations. Compounds 1, 2, 5, 10, 11, 14–18, and 21–23 showed α-glucosidase inhibition with IC₅₀ values of 2.36, 1.65, 1.30, 2.37, 2.70, 1.36, 1.54, 2.21, 2.26, 0.027, 1.65, 1.19 and 1.74 mM, respectively (with acarbose as the positive control; IC₅₀ = 0.95 mM), among which compound 18 is 35 times more potent than acarbose. In addition, compounds 18 and 21 exhibited inhibitory activity against the influenza A (H1N1) virus with IC₅₀ values of 172.4 and 175.5 μM, respectively (with ribavirin as the positive control; IC₅₀ = 137.3 μM).

---

Introduction

Microorganisms continue to play an important role in the search for novel and bioactive compounds for drug development.^1,2 However, with the deepening of research on terrestrial microbial natural products, the discovery of new entities from these microorganisms is increasingly difficult due to chemical redundancy.³ As a result, many natural product chemists turned their attention to marine counterparts, especially marine fungi, which are supposed to be a tremendous resource for drug discovery with their specialised niches.^4,5 With this trend in mind, and as a continuation of our investigations on structurally new and bioactive natural products of marine fungal origin,^6–9 an endozoic fungus, Aspergillus sp. OUCMDZ-1583, was isolated from an unidentified marine sponge (XD10410) from the Xisha Islands of China. The EtOAc extract of the fermentation broth showed α-glucosidase inhibition with an IC₅₀ value of 0.97 mg mL⁻¹, while the IC₅₀ value of acarbose (the positive control) was 0.61 mg mL⁻¹. Chemical examination of the fermentation broth resulted in the isolation and identification of eighteen new compounds that we named aspergones A–Q (1–17) and 6-O-demethylmonocerin (18), as well as five known compounds: epoxyquinol (19),¹⁰ 7-O-demethylmonocerin (20),¹¹ (+)-monocerin (21),^11,12 fusarentin 6-methyl ether (22),¹¹ and 6,7-O-dimethyl-4R-hydroxy-10-epifusarentin (23)¹³ (Table S1, ESI†).

Results and discussion

Aspergone A (1) was obtained as a brown oil, with a molecular formula of C₁₁H₁₄O₃, which was established from an HRESIMS peak of m/z 195.1014 [M + H]⁺. The IR spectrum of 1 showed the presence of a lactone carbonyl (1777 cm⁻¹), and a hydroxy group (3377 cm⁻¹). The ¹H and ¹³C NMR data (Table 1 and 2) along with the HSQC spectrum showed the presence of one triplet methyl (δ_C/H 13.7/0.91), three methylenes including an oxygenated one, six olefinic carbons (where four were protonated), and one lactone carbonyl (δ_C 170.1). These data were similar to the known 5-(E)-but-2-enylidene-3-propyl-5H-furan-2-one (24)¹⁴ except for an oxygenated methylene (δ_C/H 63.3/4.24) replacing the corresponding methyl signals, indicating the hydroxylation of C-8 in 1. This deduction was further evidenced by the COSY cross-peaks from H-5 (δ_H 5.67) to H₂-8 (δ_H 4.24) through H-6 (δ_H 6.74) and H-7 (δ_H 6.04) and from H₂-9 (δ_H 2.31) to H₃-11 (δ_H 0.91) through H₂-10 (δ_H 1.57) as well as the HMBC correlations of H-3 (δ_H 6.96) to C-1 (δ_C 170.1), C-2 (δ_C 134.4), C-4 (δ_C 148.0), and C-9 (δ_C 27.4), H-6 to C-4, H-5 to C-3 (δ_C 136.6) and C-4, and of H-9 to C-1, C-2, and C-3 (Fig. 1). The E- and Z-geometries of the Δ⁶- and Δ⁴-double bonds could be deduced from the large J_H-6,H-7 value (16.3 Hz) (Table 2) and the NOE difference experiment (Fig. 2), respectively. The H-3 signal (δ_H 6.96) was significantly enhanced when the H-5 signal (δ_H 5.67) was irradiated.

Table 1 ¹³C NMR data for 1–18 (δ_C ppm)

Position	1^b^,^c	2^b^,^d	3 and 4^b^,^c	5^b^,^d	6^a^,^d	7 and 8^b^,^c	9^a^,^c	10^a^,^c	11^a^,^c	12^a^,^c	13^a^,^c	14^a^,^c	15^a^,^d	16^a^,^c	17^a^,^c	18^b^,^c
a Recorded in DMSO-d6.b Recorded in CDCl3.c Measured using a JEOL JNM-ECP 600 spectrometer, and the δC values of C-4a, C-8a, C-12, C-13 and 7-OMe for 18 were 131.4 (C), 101.3 (C), 19.2 (CH2), 14.1 (CH3) and 60.9 (CH3), respectively.d Measured using an Agilent 500 MHz DD2 spectrometer.
1	170.1, C	171.1, C	170.8, C	175.9, C	70.1, CH2	206.9, C	68.7, CH2	69.0, CH2	68.9, CH2	62.4, CH2	65.0, CH2	133.4, C	134.8, C	131.2, C	126.0, C	168.2, C
2	134.4, C	129.7, C	134.1, C	47.6, CH	140.2, C	128.9, CH	75.0, C	75.2, C	75.3, C	135.7, C	140.9, C	135.8, C	135.4, C	133.2, C	140.2, C
3	136.6, CH	139.3, CH	137.0, CH	78.4, CH	74.1, CH	180.7, C	41.0, CH2	140.8, CH	141.6, CH	126.7, CH	124.3, CH	68.5, CH	73.3, CH	65.5, CH	67.4, CH	81.5, CH
4	148.0, C	146.2, C	149.8, C	79.4, CH	86.6, CH	76.7, CH	125.1, CH	132.2, CH	123.6, CH	127.6, CH	128.1, CH	68.7, CH	69.0, CH	54.5, CH	69.8, CH	74.3, CH
5	111.2, CH	113.8, CH	110.4, CH	126.0, CH	132.9, CH	55.3, CH	134.7, CH	134.2, CH	128.8, CH	132.6, CH	130.8, CH	68.3, CH	75.6, CH	54.3, CH	77.9, CH	108.1, CH
6	123.8, CH	124.9, CH	35.8, CH2	135.0, CH	131.8, CH	31.7, CH2	131.3, CH	128.3, CH	130.8, CH	43.1, CH2	43.2, CH2	67.9, CH	73.4, CH	62.7, CH	75.5, CH	155.4, C
7	137.1, CH	136.5, CH	67.6, CH	131.1, CH	136.9, CH	135.3, CH	128.6, CH	137.8, CH	132.9, CH	66.6, CH	66.7, CH	127.7, CH	126.9, CH	128.3, CH	126.0, CH	134.9, C
8	63.3, CH2	19.0, CH3	23.4, CH3	121.7, CH2	118.2, CH2	117.4, CH2	18.1, CH3	117.4, CH2	118.5, CH2	23.6, CH3	23.6, CH3	128.0, CH	129.6, CH	127.0, CH	130.2, CH	155.6, C
9	27.4, CH2	35.0, CH2	27.2, CH2	30.4, CH2	125.7, CH	32.9, CH2	133.3, CH	40.0, CH2	40.0, CH2	125.8, CH	30.4, CH2	19.3, CH3	19.3, CH3	19.2, CH3	19.2, CH3	39.1, CH2
10	21.0, CH2	66.3, CH	21.0, CH2	19.9, CH2	21.9, CH2	20.2, CH2	135.2, CH	16.8, CH2	16.8, CH2	125.7, CH	22.1, CH2	58.1, CH2	57.5, CH2	56.8, CH2	58.2, CH2	78.7, CH
11	13.7, CH3	23.3, CH3	13.8, CH3	13.9, CH3	14.6, CH3	14.0, CH3	17.9, CH3	15.2, CH3	15.2, CH3	19.3, CH3	14.6, CH3				154.8, C	38.1, CH2

---

Table 2 ¹H NMR data for 1–18 (δ_H ppm, J in Hz)

Position	1^b^,^c	2^b^,^d	3 and 4 ^b^,^c	5^b^,^d	6^a^,^d	7 and 8^b^,^c	9^a^,^c	10^a^,^c	11^a^,^c	12^a^,^c	13^a^,^c	14^a^,^c	15^a^,^d	16^a^,^c	17^a^,^c	18^b^,^c
a Recorded in DMSO-d6.b Recorded in CDCl3.c Measured using a JEOL JNM-ECP 600 spectrometer, and the δH values of H-12, H-13, HO-8 and 7-OMe for 18 were 1.41 (m)/1.33 (m), 0.90 (t, J = 7.5 Hz, 3H), 11.5 (s) and 3.97 (s3H), respectively.d Measured using an Agilent 500 MHz DD2 spectrometer.
1					4.27, d (12.5)		3.45, d (11.7)	3.25, d (12.0)	3.26, d (12.5)	4.07, s	3.87, s
					4.18, d (12.5)		3.43, d (11.7)	3.21, d (12.0)	3.22, d (12.5)
2				2.62, m		5.87, s
3	6.96, s	7.15, s	6.99, s	3.97 dd (7.8, 8.9)	4.22, dd (5.5, 5.4)		2.31, m; 2.32, m	5.79, d (14.8)	5.82, d (15.0)	6.03, d (11.6)	5.97, d (11.0)	4.17, dd (5.3, 4.9)	4.24, dd (10.6, 7.5)	4.52, dd (5.1, 3.2)	4.24, dd (4.8, 3.6)	5.02, m
4				4.98, dd (7.8, 7.8)	4.09, dd (5.4, 5.4)	4.45, d (3.0)	5.48, dt (14.5, 7.4)	6.23, dd (14.8, 10.7)	6.68, dd (15.0, 12.4)	6.51, dd (15.0, 11.2)	6.26, dd (15.4, 11.0)	3.60, m	3.66, dd (10.6, 10.6)	3.35, dd (3.2, 4.0)	3.55, m	4.49, d (3.0)
5	5.67, d (11.6)	5.71, d (11.3)	5.26, t (8.2)	5.41, dd (10.2, 7.8)	5.71, dd (15.7, 6.7)	2.37, m	6.08, dd (14.5, 11.0)	6.28, dd (14.8, 10.7)	6.03, dd (11.3, 12.4)	5.70, m	5.62, dt (15.4, 7.5)	3.55, m	3.34, m	3.28, dd (4.0, 1.9)	4.81, dd (9.6, 7.6)	6.60, s
6	6.74, dd (16.3, 11.6)	6.52, dd (15.6, 11.3)	2.55, m; 2.49, m	6.31, dd (10.2, 11.3)	6.21, dd (15.7, 10.2)	2.56, ddd (14.6, 6.0, 6.2) 2.21, ddd (14.6, 7.9, 7.4)	6.02, dd (14.4, 10.2)	6.19, dd (14.8, 10.2)	5.93, dd (11.2, 11.3)	2.24, m; 2.14, m	2.19, m; 2.10, m	4.23, dd (4.0, 4.0)	3.99, dd (6.5, 6.5)	4.45, dd (7.7, 1.9)	5.60, d (7.6)
7	6.04, dt (16.3, 4.9)	5.99, dq (15.6, 6.8)	3.96, m	6.68, ddd (16.6, 11.3, 10.3)	6.32, ddd (17.0, 10.2, 10.2)	5.75, m	5.62, dq (14.4, 6.9)	6.36, ddd, (17.1, 10.2, 10.2)	6.82, ddd (16.7, 11.2, 10.4)	3.65, m	3.62, m	6.34, dd (15.7, 1.6)	6.15, d (16.2)	6.36, d (15.8)	6.42, br.d (16.3)
8	4.24, d (4.9)	1.84, d (7.1)	1.23, d (6.1)	5.37, br.d (16.6); 5.30, br.d (10.3)	5.21 br.d (17.0); 5.07 br.d (10.2)	5.11, br.d (17.0); 5.03, br.d (10.5)	1.73, d (6.9)	5.20, d (17.1); 5.06, d (10.2)	5.24, d (16.7); 5.15, (10.4)	1.03, d (6.1)	1.02, d (6.2)	5.94, dq (15.7, 6.7)	5.82, m	5.91, dq (15.8, 6.7)	5.93, dq (16.3, 6.8)
9	2.31, t (7.5)	2.53, dd (15.0, 7.9); 2.46, dd (15.0, 3.9)	2.32, t (7.6)	1.84, m; 1.58, m	5.36, t (7.3)	2.49, m; 2.36, m	5.44, d (15.5)	1.41, m; 1.42, m	1.43, m; 1.41, m	6.49, dd (16.6, 1.5)	2.07, t (7.3)	1.77, dd (1.6, 6.7)	1.73, d (5.5)	1.76, d (6.7)	1.80, dd (6.6, 1.5)	2.56, ddd, (14.6, 8.7, 6.4); 2.13, ddd (14.6, 6.0, 1.4)
10	1.57, m	4.04, m	1.59, m	1.57, m; 1.51, m	2.12, m	1.56, m; 1.62, m	5.73, dq (15.5, 6.6)	1.30, m; 1.18, m	1.30, m; 1.18, m	5.72, m	1.37, m	4.18, dd (12.0, 5.8); 3.92, dd (12.0, 5.8)	4.11, s	4.16, dd (6.0, 11.7); 4.13, dd (6.0, 11.7)	4.31, dd (12.4, 4.7); 3.95, dd (12.4, 4.7)	4.10, m
11	0.91, t (7.6)	1.21, d (6.4)	0.95, t (7.8)	0.96, t (7.3)	0.91, t (7.7)	0.95, t (7.4)	1.72, d (6.6)	0.83, t (7.3)	0.83, t (6.7)	1.76, dd (6.8, 1.5)	0.86, t (7.6)					1.68, m; 1.55, m
3-OH					5.27, d (5.5)							4.51, d (5.3)	5.39, d (7.5)	5.13, d (5.1)	5.14, d (4.8)
4-OH												4.27, d (5.9)			5.47, d (5.9)
5-OH												4.41, d (5.4)	5.41, d (5.6)
6-OH												4.44, d (4.0)	5.09, d (6.5)	5.16, d (7.7)
10-OH												4.64, t (5.8)	4.37, t (5.4)	4.35, t (6.)	4.91, t (4.9)

---

Fig. 1 Key COSY and HMBC correlations of 1–18.

Fig. 2 Key NOE correlations of compounds 1–7, 12, 13 and 18.

Aspergone B (2) has the same molecular formula as 1, with an HRESIMS peak at m/z 217.0832 [M + Na]⁺, and similar ¹³C NMR data (Table 1). The difference was that hydroxymethine (δ_C/H 66.3/4.04) and methyl (δ_C/H 19.0/1.84) signals replaced the corresponding hydroxymethylene and methylene signals of 1. HMBC correlations from H-3 (δ_H 7.15) to C-1 (δ_C 171.1), C-2 (δ_C 129.7) and C-4 (δ_C 146.2), H₂-9 (δ_H 2.46, 2.53) to C-1, C-2 and C-3 (δ_C 139.3), and from H₃-8 (δ_H 1.84) to C-6 (δ_C 124.9) and C-7 (δ_C 136.5) along with the ¹H–¹H COSY data of H₂-9/H-10 (δ_H 4.04)/H₃-11 (δ_H 1.21) suggested that the hydroxy group was located at C-10 in 2. The E- and Z-geometries of the Δ⁶- and Δ⁴-double bonds could be deduced from the large J_H-6,H-7 value (15.6 Hz) (Table 1) and the NOE difference experiment (Fig. 2), respectively. The H-5 signal (δ_H 5.71) was significantly enhanced when the H-3 signal (δ_H 7.15) was irradiated. The absolute configuration of C-10 was determined by the modified Mosher’s method.¹⁵ The Δδ values between the (S)-MTPA ester (2b) and (R)-MTPA ester (2a) clearly indicated the 10S-configuration (Fig. 3).

Fig. 3 Δδ (= δ_S − δ_R) values for (S)- and (R)-MTPA esters of 2, 3, 5–7 and 12a.

Aspergones C (3) and D (4) were initially obtained as a racemic mixture, as shown by the zero value of the specific rotation, and with the same molecular formula of C₁₁H₁₆O₃ as established from an HRESIMS peak at m/z 219.0988 [M + Na]⁺. Although the ¹H and ¹³C NMR data revealed the presence of the same furan-2(5H)-one nucleus as 1, the remaining portion was slightly different. ¹H–¹H COSY data (ESI†) and HMBC correlations of H-7 (δ_H 3.96) to C-5 (δ_C 110.4) and H-5 (δ_H 5.26) to C-3 (δ_C 137.0) and C-4 (δ_C 149.8) revealed that a 3-hydroxybutylidene group in 3 and 4 replaced the corresponding 4-hydroxybutenylidene group in 1. The Z-geometry of the Δ⁴-double bond was deduced from the NOE enhancements of the H-5 signal after the irradiation of H-3 (δ_H 6.99) (Fig. 2). Upon chiral chromatography on a CHIRAPAK IA HPLC column, optically pure 3 and 4 were obtained. The distribution of Δδ values between the (S)- and (R)-MTPA esters (3a and 3b) indicated the 7R-configuration of 3 (Fig. 3). Therefore, the absolute configuration of 4 was determined to be 7S.

The molecular formula of aspergone E (5) was established as C₁₁H₁₆O₃ from an HRESIMS peak at m/z 219.0989 [M + Na]⁺, with four degrees of unsaturation. Strong IR absorptions at 1769 and 1645 cm⁻¹ implied the presence of lactone carbonyl and double bond functional groups. ¹H–¹H COSY cross-peaks from H₂-8 (δ_H 5.30, 5.37) through H-7 (δ_H 6.68), H-6 (δ_H 6.31), H-5 (δ_H 5.41), H-4 (δ_H 4.98), H-3 (δ_H 3.97), H-2 (δ_H 2.62), H₂-9 (δ_H 1.84, 1.58) and H₂-10 (δ_H 1.57, 1.51) to H₃-11 (δ_H 0.96), and HMBC correlations from H-2 and H₂-9 to C-1 (δ_C 175.9), indicated a 4-(but-1,3-dienyl)-3-hydroxy-2-propylbutyrrolactone structure (Fig. 2). The Z-geometry of the Δ⁵-double bond could be determined by the J_H-5,H-6 value (10.2 Hz, Table 2) and the NOE enhancement of H-7 after the irradiation of H-4. Furthermore, NOE enhancements of H-5 and H-9 were observed when H-3 was irradiated, while the H-2 signal was enhanced after the irradiation of H-4, indicating the trans-orientations of both H-2 and H-4 with H-3 (Fig. 2). The distribution of Δδ values between the (S)- and (R)-MTPA esters (5a and 5b) indicated the 3S-configuration (Fig. 3). Thus, the absolute configuration of 5 was determined to be 2S, 3S, and 4R.

The molecular formula of aspergone F (6) was established as C₁₁H₁₆O₂ from an HRESIMS peak at m/z 203.1037 [M + Na]⁺, with one oxygen less than that of 5. Comparison of the ¹³C NMR data between 6 and 5 (Table 1) revealed that an olefinic quaternary carbon, an olefinic methine and an oxygenated methylene signal in 6 replaced the corresponding sp³-methine, sp³-methylene and ester carbonyl signals in 5. Two separate ¹H–¹H COSY systems of H-3/H-4/H-5/H-6/H-7/H-8 and H-9/H-10/H-11 (ESI†) were observed in 6, indicating that a propylidene group replaced the propyl group in 5. The key HMBC data from H₂-1 (δ_H 4.27, 4.18) to C-2 (δ_C 140.2), C-4 (δ_C 86.6) and C-9 (δ_C 125.7) and from H-9 (δ_H 5.36) to C-1 (δ_C 70.1) confirmed the replacement of the carbonyl group in 5 by a –CH₂– group in 6. The E-geometry of the Δ⁵-double bond was deduced from the large J_H-5,H-6 value (15.7 Hz, Table 2) and the NOE enhancement of H-6 (δ_H 6.21) after the irradiation of H-4 (δ_H 4.09) (Fig. 2). The NOE enhancements of H-5 (δ_H 5.71) and H-9 after the irradiation of H-3 (δ_H 4.22) (Fig. 2) indicated the trans-orientation of H-3 and H-4 and the E-geometry of the Δ²⁽⁹⁾-double bond. The distribution of Δδ values between the (S)- and (R)-MTPA esters (6a and 6b) indicated the 3S-configuration (Fig. 3). Thus, the absolute configuration of 6 was determined as 3S and 4R.

Aspergones G (7) and H (8) were initially isolated as a racemic mixture and the molecular formula was established as C₁₁H₁₆O₂ on the basis of an HRESIMS peak at m/z 203.1038 [M + Na]⁺. The strong UV and IR absorptions at a λ_max value of 223 nm and ν_max values of 1703 and 1614 cm⁻¹, respectively, indicated the presence of a conjugated enone moiety, which was further supported by the HMBC correlations from an olefinic proton (δ_H-2 5.87) to a sp²-quaternary carbon (δ_C-3 180.7) and a carbonyl carbon (δ_C-1 206.9). ¹H–¹H COSY (Fig. 1) and HSQC data indicated the presence of two isolated spin systems, CH(4)–CH(5)–CH₂(6)–CH(7)–CH₂(8) and CH₃(11)–CH₂(10)–CH₂(9). The key HMBC correlations of H-4 (δ_H 4.45) to C-1, C-2 (δ_C 128.9), C-5 (δ_C 55.3), C-6 (δ_C 31.7) and C-9 (δ_C 32.9) linked these three moieties as 5-allyl-4-hydroxy-3-propylcyclopent-2-en-1-one. When H-4 was irradiated, the H₂-6 (δ_H 2.56, 2.21) signals were enhanced, indicating a trans-orientation between H-4 and H-5 (δ_H 2.37). Chiral HPLC separation on a CHIRAPAK IA column afforded the optically pure D-isomer (7) and L-isomer (8). The distribution of Δδ values between the (S)- and (R)-MTPA esters (7a and 7b) indicated the 4R-configuration (Fig. 3). Thus, the absolute configuration of 7 was determined to be 4R and 5R. As a consequence, the absolute configuration of 8 was determined to be 4S and 5S.

Aspergone I (9) had an HRESIMS peak at m/z 205.1195 [M + Na]⁺, corresponding to a molecular formula of C₁₁H₁₈O₂. Its 1D NMR revealed two methyls (δ_C 17.9 & 18.1), an oxygenated methylene (δ_C 68.7), six olefinic methines and an oxygenated quaternary carbon (δ_C 75.0) (Table 1). ¹H–¹H COSY and HSQC data indicated the presence of CH₃(8)–CH(7)–CH(6)–CH(5)–CH(4)–CH₂(3) and CH(9)–CH(10)–CH₃(11) units (Fig. 1). HMBC correlations (Fig. 1) from H₂-1 (δ_H 3.45, 3.43) to C-2 (δ_C 75.0), C-3 (δ_C 41.0) and C-9 (δ_C 133.3) and from H₂-3 (δ_H 2.31, 2.32) to C-1 (δ_C 68.7), C-2 and C-9 connected these structural moieties as 2-propenylocta-4,6-diene-1,2-diol (Fig. 1). The large J values of H-4/H-5 (14.5 Hz), H-6/H-7 (14.4 Hz) and H-9/H-10 (15.5 Hz) (Table 2) indicated that all the Δ^4,6,9-double bonds have an E-geometry. The absolute configuration was assigned using the in situ dimolybdenum CD method.^16,17 After the addition of Mo₂(OAc)₄ to a DMSO solution of 9, a metal complex auxiliary chromophore was generated. Because the contribution from the inherent CD was subtracted, the Cotton effect observed in the induced CD spectrum originates solely from the chirality of the vic-diol moiety. The negative Cotton effect observed at 320 (Δε −0.64) and 400 (Δε −0.10) nm in the induced CD spectrum (Fig. 5) revealed the 2S-configuration according to Snatzke’s empirical rule.¹⁸ The structure of 9 was therefore elucidated as (2S,4E,6E)-2-(E-propenyl)octa-4,6-diene-1,2-diol.

Fig. 4 ORTEP drawings of 14 and 15.

Fig. 5 CD curve for the complex of 9 with Mo₂(OAc)₄ subtracted from the inherent CD.

Aspergone J (10) has the same molecular formula of C₁₁H₁₈O₂ as 9, with an HRESIMS peak at m/z 205.1196 [M + Na]⁺. NMR comparison revealed that a methylene and an olefinic methylene of 10 replaced the methyl and olefinic methine of 9. The ¹H–¹H COSY spectrum of 10 further indicated a hexatrienyl group and propyl group in place of the hexadienyl and propenyl groups in 9, which was also supported by the key HMBC correlations from H-3 (δ_H 5.79) to C-1 (δ_C 69.0), C-2 (δ_C 75.2) and C-9 (δ_C 40.0). The E-geometries of the Δ^3,5-double bonds could be deduced from the J values of H-3/H-4 (14.8 Hz) and H-5/H-6 (14.8 Hz) (Table 2), and the same sign of the [α]_D value (−55.3) as that of 9 (−34.8) implied the same 2R-configuration; therefore the structure is (2R,3E,5E)-2-propylocta-3,5,7-triene-1,2-diol.

Aspergone K (11) also has the molecular formula C₁₁H₁₈O₂, based on an HRESIMS peak at m/z 205.1195 [M + Na]⁺. The NMR data and ¹H–¹H COSY and HMBC coupling modes, along with the [α]_D value (−64.5), were very similar to those of 10, indicating almost the same structure. The only difference was found to be the Z-geometry of the Δ⁵-double bond, as deduced from the relatively small J_H-5,H-6 value (11.3 Hz). Thus, the structure of 11 was determined as (2R,3E,5Z)-2-propylocta-3,5,7-triene-1,2-diol.

The molecular formula of aspergone L (12) was also established as C₁₁H₁₈O₂ from an HRESIMS peak at m/z 205.1196 [M + H]⁺. 1D NMR, ¹H–¹H COSY and HSQC data indicated an oxygenated methylene, a 5-hydroxyhex-2-en-1-ylidene (CH₃(8)–CH(7)–CH₂(6)–CH(5)–CH(4)–CH(3)), a propenyl (CH(9)–CH(10)–CH₃(11)), and an olefinic quaternary carbon. The key HMBC correlations of H-3 (δ_H 6.03) to C-1 (δ_C 62.4), C-2 (δ_C 135.7) and C-9 (δ_C 125.8) along with H₂-1 (δ_H 4.07) to C-2, C-3 (δ_C 126.7) and C-9 connected the above structural moieties as 2-propenylocta-2,4-diene-1,7-diol (Fig. 2). All the geometries of the Δ^2,4,9-double bonds were assigned as E according to the large J values of H-4/H-5 (15.0 Hz) and H-9/H-10 (16.6 Hz) (Table 2) and the NOESY cross-peak between H-3 and H₂-1 (Fig. 2). To determine the absolute configuration of 12, the 1-O-t-butyldimethylsilyl (TBDMS) derivative (12a) and the (R)- and (S)-MTPA esters of 12a were prepared. The distribution of Δδ values between the (S)- and (R)-MTPA esters (12aa and 12ab) indicated the 7R-configuration (Fig. 3). Thus, compound 12 was determined to be (7R,2E,4E)-2-(E-propenyl)octa-2,4-diene-1,7-diol.

The molecular formula of aspergone M (13) was established as C₁₁H₂₀O₂ from an HRESIMS peak at m/z 207.1350 [M + Na]⁺, equivalent to 12 with an additional H₂ unit. Comparison of the ¹H and ¹³C NMR data between 13 and 12 revealed that their structures were only different in the replacement of the propenyl group in 12 with a propyl group in 13. This deduction was further supported by the ¹H–¹H COSY connectivity of H₃-11/H₂-10/H₂-9 and the key HMBC correlations of H₂-1 (δ_H 3.87) to C-2 (δ_C 140.9), C-3 (δ_C 124.3) and C-9 (δ_C 30.4). The E-geometries of both the Δ²- and Δ⁴-double bonds were assigned from the large J_H-4,H-5 (15.4 Hz) and the NOESY cross-peak of H-3 (δ_H 5.97) with H₂-1 (Fig. 2). The close specific rotation value of 13 to that of 12 (−36.7 vs. −30.5) implied the same 7R-configuration. Thus, compound 13 was elucidated as (7R,2E,4E)-2-propylocta-2,4-diene-1,7-diol.

Aspergone N (14) had a molecular formula of C₁₀H₁₆O₅, as determined from an HRESIMS peak at m/z 239.0888 [M + Na]⁺, with three degrees of unsaturation. The IR spectrum of 14 showed the absorption of the double bonds (1645 cm⁻¹) and the hydroxy groups (3287 cm⁻¹). The ¹H and ¹³C NMR along with the HSQC data revealed the presence of a doublet methyl, an oxygenated methylene, four oxygenated methines, two vicinal olefinic methines, two olefinic quaternary carbons, and five hydroxy groups. This accounted for two degrees of unsaturation, indicating the presence of a cyclic nucleus in 14. ¹H–¹H COSY correlations reveal the presence of CH(–OH)(3)–CH(–OH)(4)–CH(–OH)(5)–CH(–OH)(6), CH₃(9)–CH(8)–CH(7), and CH₂(–OH)(10) moieties. HMBC correlations from H-7 (δ_H 6.34) to C-1 (δ_C 133.4), C-2 (δ_C 135.8) and C-6 (δ_C 67.9) and from H₂-10 (δ_H 4.18, 3.92) to C-1, C-2, and C-3 (δ_C 68.5) connected these moieties as 2-hydroxymethyl-1-propenyl-1-cyclohexene-3,4,5,6-tetraol. The large J_H-7,H-8 value (15.7 Hz) suggested an E-Δ⁷-double bond. The relative configurations of C-3–C-6 and the geometry of the Δ⁷-double bond were supported by X-ray single crystal diffraction (Fig. 4). The absolute configurations of all the chiral centers were assigned as R by ECD calculations of 14 and ent-14 using the time-dependent density functional theory (TD-DFT) method at the B3LYP/6-31G(d) level.¹⁹ The results showed that the measured CD curve matches well with the calculated ECD curve for 14 and is the opposite to that of ent-14 (Fig. 6).

Fig. 6 CD curves of 14, 15, 18, 20 and the calculated ECD curves of 14 and ent-14.

Aspergone O (15) was isolated as a colorless orthorhombic crystal with a molecular formula of C₁₀H₁₅ClO₄ on the basis of an HRESIMS peak at m/z 257.0548 [M + Na]⁺, indicating the substitution of –Cl for –OH relative to 14. In addition, we noted that there was one hydroxy signal less, and the ¹H–¹H COSY and HMBC coupling patterns were similar to 14 except for the lack of a COSY correlation between 4-OH and 4-CH, supporting the substitution of 4-Cl in 15 for 4-OH in 14. The large J_H-7,H-8 value (16.2 Hz) indicated an E-geometry of the Δ⁷-double bond. The similar CD data to that of 14 (Fig. 6) indicated a (3R,4S,5S,6R)-configuration of 15 that was further confirmed by single-crystal X-ray diffraction (Fig. 4) with a small Flack parameter (0.05(7)) and a heavy chlorine atom in the molecule.^20,21 Thus, aspergone O (15) was identified as (3R,4S,5S,6R,7E)-4-chloro-2-hydroxymethyl-1-propenylcyclohex-1-ene-3,5,6-triol.

Aspergone P (16) had a molecular formula of C₁₀H₁₄O₄ as determined from its HRESIMS peak at m/z 221.0780 [M + Na]⁺, which is equivalent to 14 minus a H₂O unit. The absence of two hydroxy proton signals and the upfield oxygenated methine carbon signals (δ_C 54.5, 54.3) indicated the replacement of two hydroxy groups by an epoxy group. This epoxidation was deduced to occur at C-4 and C-5 according to the ¹H–¹H COSY of OH-3/H-3/H-4/H/5/H-6/OH-6 and the HMBC correlations from H-3 (δ_H 4.52) to C-1 (δ_C 131.2) along with H-6 (δ_H 4.45) to C-2 (δ_C 133.2), C-4 (δ_C 54.5) and C-7 (δ_C 128.3). The large J_H-7,H-8 value (15.8 Hz) suggested an E-geometry of the Δ⁷-double bond. The absolute configuration of 16 was defined by its chemical transformation into 15. After adding hydrochloric acid to a methanol solution of 16, compound 15 was yielded as the major product (Fig. S120†) which showed identical [α]_D, NMR and MS data to those of the natural product, indicating a (3R,4R,5S,6R)-configuration of 16. This result also indicated that compound 15 might be an artifact formed under acidic conditions in the fermentation process.

The molecular formula of aspergone Q (17) was established as C₁₁H₁₄O₆ from an HRESIMS peak at m/z 265.0680 [M + Na]⁺. The NMR data (Table 1 and 2) of 17 are similar to those of 14, except for the absence of two hydroxy proton signals and the presence of an additional carbonate carbonyl signal (δ_C 154.8), suggesting that two hydroxy groups of 14 were esterified to form a cyclic carbonate. HMBC correlations of H-5 (δ_H 4.81) and H-6 (δ_H 5.60) to C-11 (δ_C 154.8) and H-7 (δ_H 6.42) to C-6 (δ_C 75.5) supported the theory that the 5,6-dihydroxy group formed a cyclic carbonate in 17 (Fig. 2). Treatment of 17 in 1% aqueous sodium hydroxide yielded compound 14 which showed identical [α]_D, NMR and MS data to the natural product, indicating a (3R,4R,5S,6R)-configuration of 17.

Compound 18 was found to have the molecular formula C₁₅H₁₈O₆, as established from an HRESIMS peak at m/z 295.1171 [M + H]⁺. Apart from the phenyl motif, the ¹H and ¹³C NMR data (Table 1 and 2) were nearly identical to the known compound 20,¹¹ indicating a similar structure which is isomeric in the phenyl motif. The HSQC data revealed the presence of five aromatic quaternary carbons, one aromatic methine (δ_C/H 108.1/6.60), three oxygenated methines, three methylenes, one methyl (δ_C/H 14.1/0.90), one methoxyl (δ_C/H 60.9/3.97), and one ester carbonyl group (δ_C 168.2). The key HMBC correlations from a phenolic hydroxy proton (δ_HO-8 11.5) to three aromatic quaternary carbons (δ_C-7,8,8a 134.9, 155.6, 101.3) and the methoxy protons (δ_H 3.97) to a relative upfield oxygenated aromatic quaternary carbon (δ_C-7 134.9) revealed that O-methylation occurred at 7-OH in 18 as opposed to 6-OH in 20. The relative configuration was established from the NOE data and the comparison of NMR data with 20, especially the vicinal coupling constants. The small J_H-3,H-4 value (3.0 Hz) (Table 2) along with the NOESY correlations from H-10 (δ_H 4.10) to H-3 (δ_H 5.02) and H-4 (δ_H 4.49) indicated the same cis-orientation of H-3, H-4 and H-10 as in 20. The similar CD spectra of 18 and 20 (Fig. 5) revealed the same (3R,4R,10S)-configuration. Because the absolute configuration of 20 has been resolved by chemical synthesis¹¹ and furthermore by Cu-Kα radiated X-ray single crystal diffraction (Fig. S107†) with a small Flack parameter (0.0(3)) in this paper, the structure of compound 18 could be clearly elucidated as 6-O-demethylmonocerin.

All the isolated compounds (1–23) were evaluated for antiviral activity against the H1N1 flu virus, as well as α-glucosidase inhibition, by previously described methods.^6,22 Only compounds 18 and 21 exhibited anti-H1N1 activities, with IC₅₀ values of 172.4 μM and 175.5 μM, respectively (with ribavirin as the positive control; IC₅₀ = 137.3 μM), while the other compounds were not active against H1N1. These data suggested that the tetrahydrofuran moiety and the methoxy group located at C-7 in the isocoumarins were required for antiviral activity against the influenza A H1N1 virus. In addition, compounds 1, 2, 5, 10, 11, 14–18, and 21–23 showed α-glucosidase inhibition with IC₅₀ values of 2.36, 1.65, 1.30, 2.37, 2.70, 1.36, 1.54, 2.21, 2.26, 0.027, 1.65, 1.19 and 1.74 mM, respectively (with acarbose as the positive control; IC₅₀ = 0.95 mM) (Table 3). The results showed that compound 18 is 35 times more potent than the positive control, which supports the idea that marine fungi might be a promising source for drug discovery. Compounds 1–23 were also tested for their cytotoxicities against A549 and K562 tumor cells, and against MCF-7 cells, by the MTT²³ and CCK-8 (ref. 24) methods, respectively. Their antimicrobial activities against Escherichia coli, Enterobacter aerogenes, Bacillus subtilis, Pseudomonas aeruginosa and Candida albicans were also evaluated, by an agar dilution method.²⁵ However, none of the compounds showed any activity against the tested tumor cells and pathogens.

Table 3 α-Glucosidase inhibitions of compounds 1, 2, 5, 10, 11, 14–18, and 21–23

Compound	Acarbose	1	2	5	10	11	14	15	16	17	18	21	22	23
IC50 (mM)	0.95	2.36	1.65	1.30	2.37	2.70	1.36	1.54	2.21	2.26	0.027	1.65	1.19	1.74

---

Experimental section

General experimental procedures

Optical rotations were measured using a JASCO P-1020 digital polarimeter, and UV spectra were measured using a Beckman DU 640 spectrophotometer. CD data were collected using a JASCO J-715 spectropolarimeter. IR spectra were taken using a Nicolet Nexus 470 spectrophotometer with KBr discs. ¹H NMR, ¹³C NMR, DEPT, HMQC, HSQC, HMBC, COSY and NOESY spectra were acquired using a JEOL JNM-ECP 600 spectrometer or an Agilent 500 MHz DD2 spectrometer using TMS as an internal standard or residual solvent signals for referencing. HR-ESI-MS spectra were determined using a Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer. Semi-preparative HPLC was carried out using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 4 mL min⁻¹) and a πNAP column (COSMOSIL-pack, 10 × 250 mm, 5 μm, 4 mL min⁻¹). Sea salt used was made by the evaporation of seawater collected in Laizhou Bay (Weifang Haisheng Chemical Factory). Thin layer chromatography (TLC) and column chromatography (CC) were performed on plates precoated with silica gel GF₂₅₄ (10–40 μm, Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Biosciences), respectively. Vacuum-liquid chromatography (VLC) utilized silica gel H (Qingdao Marine Chemical Factory).

Fungal material and fermentation

The fungus Aspergillus sp. OUCMDZ-1583 was isolated from a piece of sponge (XD10410) from the Xisha Islands of China in August 2010. After it was ground into a powder, the sample (1 g) was diluted to 10⁻² g mL⁻¹ with sterile water, 100 μL of which was deposited on a PDA (200 g potato, 20 g glucose, and 20 g agar per liter of tap water) plate containing chloramphenicol (100 μg mL⁻¹) as a bacterial inhibitor. A single colony was transferred onto another PDA plate and was identified according to its morphological characteristics and ITS gene sequences (GenBank accession no. KM056275, ESI†). A reference culture of Aspergillus sp. OUCMDZ-1583 maintained at −80 °C is deposited in our laboratory. The isolate was cultured on slants of PDA medium at 28 °C for 5 days. Plugs of agar supporting the mycelium growth were cut and transferred aseptically to 200 × 1000 mL Erlenmeyer flasks each containing 300 mL of liquid medium (2% mannitol, 2% maltose, 1% glucose, 1% monosodium glutamate, 0.3% yeast extract, 0.05% corn meal, 0.05% KH₂PO₄, 0.03% MgSO₄, 1.75% Na₂HPO₄·2H₂O, 1.05% C₄H₂O₇·H₂O, and 3.3% sea salt; pH 7.0). The flasks were incubated at room temperature under static conditions for 30 days.

Extraction and isolation

The cultures (50 L) were filtered through cheesecloth to separate the mycelial mass from the aqueous layer. The filtrate was then extracted three times with 3-fold volumes of EtOAc, while the mycelium was extracted with acetone. After removing some of the acetone by evaporation under vacuum, the obtained aqueous acetone solution was extracted three times with equal volumes of EtOAc. The combined EtOAc extracts were dried under vacuum to produce 28.7 g of extract. The EtOAc extract was subjected to chromatography with a silica gel VLC column, eluting with a stepwise gradient of 0%, 20%, 40%, 60%, 80% and 100% MeOH in CH₂Cl₂ (v/v), to give 20 fractions (fractions 1–20). Fraction 1 (1.2 g) was subjected to Sephadex LH-20 chromatography (5 × 200 cm) with CH₂Cl₂–MeOH (1 : 1) to afford two subfractions (1.1 and 1.2). Fraction 1.1 (106 mg) was further purified by HPLC on a πNAP column (70% MeOH/H₂O, v/v) to give compounds 23 (t_R 11.8 min; 20 mg) and 21 (t_R 16.0 min; 24 mg). Fraction 1.2 (234 mg) was subjected to HPLC on an ODS column (55% MeOH/H₂O, v/v) to yield 20 (t_R 9.5 min, 16.3 mg), 2 (t_R 9.7 min, 10.2 mg), 1 (t_R 12.6 min, 7.6 mg) and 18 (t_R 13.8 min, 7.6 mg). Fraction 2 (3.4 g) was eluted with CH₂Cl₂–MeOH (100 : 1) and was subjected to reversed-phase C18 silica column chromatography eluting with a stepwise gradient of 30% to 100% MeOH in H₂O to obtain four subfractions (2.1–2.4). Fraction 2.1 (247 mg) was further separated into three subfractions (2.1.1–2.1.3) by Sephadex LH-20 chromatography (5 × 200 cm) eluting with CH₂Cl₂–MeOH (1 : 1, v/v). Fraction 2.1.1 (56 mg) was subjected to HPLC on an ODS column (55% MeOH/H₂O, v/v) to yield fraction 2.1.1.1, which was further purified by HPLC on a πNAP column (50% MeOH/H₂O, v/v) to give compound 5 (t_R 17.0 min; 8.2 mg) and a racemic mixture of 7 and 8 (t_R 14.7 min; 16 mg) that was further subjected to HPLC on a CHIRAPAK IA column (75% n-C₆H₁₄/EtOH, v/v) to give the optically pure 7 (t_R 5.7 min; 7.1 mg) and 8 (t_R 5.9 min; 8.3 mg). Fraction 2.1.2 (78 mg) was subjected to HPLC on an ODS column (35% MeCN/H₂O, v/v) to yield a racemic mixture of 3 and 4 (t_R 7.7 min; 24 mg) that was further purified on a CHIRAPAK IA column (75% n-C₆H₁₄/EtOH, v/v) to give optically pure 3 (t_R 6.9 min; 11.3 mg) and 4 (t_R 7.8 min; 10.9 mg). Fraction 2.1.3 (39 mg) was subjected to HPLC on an ODS column (50% MeOH/H₂O, v/v) to yield compound 6 (t_R 21.3 min; 11.3 mg). Fractions 15–20 (4.2 g) were combined and re-chromatographed on Sephadex LH-20 (5 × 200 cm, MeOH) to afford four subfractions (Fr. 15.1–Fr. 15.4). Fraction 15.4 (706 mg) was subjected to HPLC on an ODS column (25% MeOH/H₂O, v/v) to give 17 (t_R 6.2 min; 76 mg), 14 (t_R 4.0 min; 22 mg) and subfraction 15.4.1. Fraction 15.4.1 (215 mg) was fractionated by silica gel CC using petroleum ether–EtOAc (4 : 6, v/v) to afford 15 (35.5 mg), 16 (106.5 mg) and 19 (5.4 mg). Fractions 6 and 7 (1.3 g) were combined and chromatographed on Sephadex LH-20 (5 × 200 cm, MeOH) to afford three subfractions (Fr. 6.1–Fr. 6.3). Fraction 6.1 (78 mg) was subjected to HPLC on an ODS column (55% MeOH/H₂O, v/v) to yield compounds 24 (t_R 6.7 min; 24.3 mg) and 22 (t_R 9.9 min; 14.7 mg). Fraction 6.2 (108 mg) was subjected to HPLC on an ODS column (50% MeOH/H₂O, v/v) to yield compounds 9 (t_R 26.6 min; 18.3 mg), 10 (t_R 27.9 min; 3.7 mg) and 11 (t_R 30.5 min; 4.5 mg). Fraction 6.3 (94 mg) was separated by HPLC on an ODS column (50% MeOH/H₂O, v/v) to yield compounds 12 (t_R 11.7 min; 18.3 mg) and 13 (t_R 13.9 min; 3.5 mg).

Aspergone A (1). Brown oil; UV (MeOH) λ_max (log ε) 325 (3.67) and 208 (3.09) nm; IR (KBr) ν_max 3377, 2984, 1777, 1715 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 195.1014 [M + H]⁺ (calcd for C₁₁H₁₅O₃, 195.1016).

Aspergone B (2). Brown oil; [α]²⁵_D −27.8 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 324.8 (3.63) and 208.6 (3.11) nm; IR (KBr) ν_max 3416, 2972, 1766, 1641 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 217.0832 [M + Na]⁺ (calcd for C₁₁H₁₄O₃Na, 217.0832).

Aspergone C (3). Brown oil; [α]²⁵_D −20.6 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 275.2 (3.80) and 201.6 (3.10) nm; IR (KBr) ν_max 3420, 2918, 1676, 1645 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 219.0988 [M + Na]⁺ (calcd for C₁₁H₁₆O₃Na, 219.0992).

Aspergone D (4). Brown oil; [α]²⁵_D +21.8 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 275.2 (3.80) and 201.6 (3.10) nm; IR (KBr) ν_max 3420, 2918, 1676, 1645 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 219.0988 [M + Na]⁺ (calcd for C₁₁H₁₆O₃Na, 219.0992).

Aspergone E (5). Brown oil; [α]²⁵_D −16.8 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 225.2 (3.78) and 204.3 (3.17) nm; CD (c 0.1, MeOH) λ_max (Δε) 226.5 (+1.05) and 209 (+2.35) nm; IR (KBr) ν_max 3420, 2966, 1769, 1645 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 219.0989 [M + Na]⁺ (calcd for C₁₁H₁₆O₃Na, 219.0992).

Aspergone F (6). Brown oil; [α]²⁵_D +18.7 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 225.5 (3.75) and 203.6 (3.50) nm; CD (c 0.1, MeOH) λ_max (Δε) 217.5 (+3.20) and 210.5 (−0.87) nm; IR (KBr) ν_max 3443, 2966, 1680, 1645 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 203.1037 [M + Na]⁺ (calcd for C₁₁H₁₆O₂Na, 203.1043).

Aspergone G (7). Brown oil; [α]²⁵_D +10.2 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 223 (3.38) and 203.5 (2.99) nm; CD (c 0.05, MeOH) λ_max (Δε) 235 (+1.96) and 207 (−0.51) nm; IR (KBr) ν_max 3404, 2960, 1703, 1614 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 203.1038 [M + Na]⁺ (calcd for C₁₁H₁₆O₂Na, 203.1043).

Aspergone H (8). Brown oil; [α]²⁵_D −9.1 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 223 (3.38) and 203.5 (2.99) nm; CD (c 0.05, MeOH) λ_max (Δε) 235 (−2.06) and 207 (+0.54) nm; IR (KBr) ν_max 3404, 2960, 1703, 1614 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 203.1038 [M + Na]⁺ (calcd for C₁₁H₁₆O₂Na, 203.1043).

Aspergone I (9). Brown oil; [α]²⁵_D −34.8 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 229.5 (3.45) and 204 (2.94) nm; CD (c 0.05, MeOH) λ_max (Δε) 233.5 (+0.99) and 209 (+0.40) nm; IR (KBr) ν_max 3420, 2918, 1676, 1645 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 205.1195 [M + Na]⁺ (calcd for C₁₁H₁₈O₂Na, 205.1199).

Aspergone J (10). Brown oil; [α]²⁵_D −55.3 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 253 (3.34), 263 (3.57) and 274 (3.38) nm; CD (c 0.1, MeOH) λ_max (Δε) 273 (+2.23) and 258.5 (−0.51) nm; IR (KBr) ν_max 3420, 2949, 1680, 1641 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 205.1196 [M + Na]⁺ (calcd for C₁₁H₁₈O₂Na, 205.1199).

Aspergone K (11). Brown oil; [α]²⁵_D −64.5 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 253 (3.44), 263 (3.48) and 274 (3.38) nm; CD (c 0.05, MeOH) λ_max (Δε) 265 (+2.54) and 249 (−0.70) nm; IR (KBr) ν_max 3392, 2964, 1676, 1645 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 205.1195 [M + Na]⁺ (calcd for C₁₁H₁₈O₂Na, 205.1199).

Aspergone L (12). Brown oil; [α]²⁵_D −30.5 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 258.5 (3.79), 269.5 (3.87) and 280 (3.77) nm; CD (c 0.1, MeOH) λ_max (Δε) 276 (+2.43) and 261.5 (−1.10) nm; IR (KBr) ν_max 3389, 2918, 1649, 1614 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 205.1196 [M + Na]⁺ (calcd for C₁₁H₁₈O₂Na, 205.1199).

Aspergone M (13). Brown oil; [α]²⁵_D −36.7 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 233 (3.72), 239 (3.74) and 248.5 (3.57) nm; CD (c 0.1, MeOH) λ_max (Δε) 251.5 (+0.26) and 234 (−0.55) nm; IR (KBr) ν_max 3389, 2918, 1649, 1614 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 207.1350 [M + Na]⁺ (calcd for C₁₁H₂₀O₂Na, 207.1356).

Aspergone N (14). Colorless orthorhombic crystal; mp 123–125; [α]²⁵_D −73.5 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 239.5 (3.39), 206 (3.03) nm; CD (c 0.05, MeOH) λ_max (Δε) 230.5 (−1.02) nm; IR (KBr) ν_max 3287, 2910, 1645, 1443 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 239.0888 [M + Na]⁺ (calcd for C₁₀H₁₆O₅Na, 239.0890).

Aspergone O (15). Colorless orthorhombic crystal; mp 117–119; [α]²⁵_D −12.7 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 238 (3.58), 206 (3.18) nm; CD (c 0.05, MeOH) λ_max (Δε) 236.5 (−1.67) nm; IR (KBr) ν_max 3392, 2925, 1657, 1443 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 257.0548 and 259.0518 [M + Na]⁺ (calcd for C₁₀H₁₅O₄ClNa, 257.0551 and 257.0522).

Aspergone P (16). White amorphous powder; [α]²⁵_D +81.0 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 238 (3.48), 206.5 (2.90) nm; CD (c 0.1, MeOH) λ_max (Δε) 232 (−1.54) nm; IR (KBr) ν_max 3350, 2921, 1660, 1446 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 221.0780 [M + Na]⁺ (calcd for C₁₀H₁₄O₄Na, 221.0784).

Aspergone Q (17). Colorless oil; [α]²⁵_D −11.6 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 238 (3.36), 205.5 (2.97) nm; CD (c 0.05, MeOH) λ_max (Δε) 231 (−1.29) nm; IR (KBr) ν_max 3350, 2921, 1660, 1446 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 265.0680 [M + Na]⁺ (calcd for C₁₁H₁₄O₆Na, 265.0683).

6-O-Demethylmonocerin (18). Brown oil; [α]²⁵_D +23.3 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 219 (3.58), 232.5 (3.53), 274 (3.37), 309 (3.02) nm; CD (c 0.1, MeOH) λ_max (Δε) 274 (−2.23), 242 (+0.62), and 212.5 (+2.68) nm; IR (KBr) ν_max 3335, 2956, 1660, 1468, 1376 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 295.1171 [M + H]⁺ (calcd for C₁₅H₁₉O₆, 295.1176).

Preparation of 12-O-tert-butyldimethylsilylaspergone L (12a)

tert-Butyldimethylsilyl chloride (TBDMSCl) (6.7 mg) was added to a mixture of compound 12 (4.1 mg) and imidazole (10 mg) in DMF (1.5 mL). The reaction mixture was stirred at r.t. for 2 h. Then the reaction mixture was quenched with H₂O and extracted three times with EtOAc. The EtOAc layers were combined and separated by semipreparative HPLC on an ODS column (100% MeOH) to afford 12a (t_R 4.4 min, 3.4 mg) as one product (Fig. S119†).

12-O-tert-Butyldimethylsilylaspergone L (12a). ¹H NMR (CDCl₃, 500 MHz) δ 6.59 (dd, J = 15.4, 11.8, 1H, H-4), 6.48 (d, J = 16.2, 1H, H-9), 6.14 (d, J = 11.2, 1H, H-3), 5.73 (m, 1H, H-10), 5.69 (m, 1H, H-5), 4.32 (s, 2H, H-1), 3.86 (m, 1H, H-7), 2.34 (m, 1H, H-6a), 2.25 (m, 1H, H-6b), 1.81 (d, J = 6.6, 3H, H-11), 1.21 (d, J = 6.3, 3H, H-8), 0.92 (s, 9H, H-TBDMS), 0.08 (s, 6H, H-TBDMS); ¹³C NMR (CDCl₃, 125 MHz) δ 135.1 (C, C-2), 130.3 (CH, C-5), 128.9 (CH, C-4), 125.7 (CH, C-9), 125.6 (CH, C-10), 124.5 (CH, C-3), 67.3 (CH, C-7), 63.8 (CH₂, C-1), 43.0 (CH₂, C-6), 26.0 (3 × CH₃, C-TBDMS), 22.8 (CH₃, C-8), 19.0 (CH₃, C-11), 18.4 (C, C-TBDMS), −5.3 (2 × CH₃, C-TBDMS); ESIMS m/z 297.2 [M + H]⁺.

Preparation of MTPA esters

Compound 2 (1 mg for each) was reacted with either R-(−)- or S-(+)-MTPA chloride (10 μL) in anhydrous pyridine (500 μL) for 6 h. The reaction mixture was quenched with H₂O and extracted three times with CH₂Cl₂. The organic layers were combined and separated by semipreparative HPLC on an ODS column (80% MeOH/H₂O, v/v) to afford the S-MTPA ester 2a (1.2 mg, t_R 10.2 min) and R-MTPA ester 2b (0.8 mg, t_R 10.3 min), respectively. By the same procedure, the S-MTPA esters 3a, 5a, 6a, 7a, and 12aa and R-MTPA esters 3b, 5b, 6b, 7b, and 12ab were also prepared.

Compound 2a. ¹H NMR (CDCl₃, 500 MHz) δ 6.93 (s, 1H, H-3), 6.57 (m, 1H, H-6), 6.04 (m, 1H, H-7), 5.65 (d, J = 11.3 Hz, 1H, H-5), 5.38 (m, 1H, H-10), 2.74 (dd, J = 16.0, 8.3 Hz, 1H, H_a-9), 2.67 (dd, J = 16.0, 4.8 Hz, 1H, H_b-9), 1.90 (d, J = 7.2 Hz, 3H, H-8), 1.35 (d, J = 6.7 Hz, 3H, H-11); ESIMS m/z 411.1 [M + H]⁺.

Compound 2b. ¹H NMR (CDCl₃, 500 MHz) δ 6.62 (s, 1H, H-3), 6.54 (m, 1H, H-6), 6.03 (m, 1H, H-7), 5.53 (d, J = 11.3 Hz, 1H, H-5), 5.37 (m, 1H, H-10), 2.67 (dd, J = 16.2, 8.5 Hz, 1H, H_a-9), 2.62 (dd, J = 16.2, 5.4 Hz, 1H, H_b-9), 1.89 (d, J = 7.0 Hz, 3H, H-8), 1.40 (d, J = 6.3 Hz, 3H, H-11); ESIMS m/z 411.1 [M + H]⁺.

Compound 3a. ¹H NMR (CDCl₃, 500 MHz) δ 6.85 (s, 1H, H-3), 5.26 (m, 1H, H-7), 4.89 (t, J = 10.2 Hz, H-5), 2.67 (m, 1H, H_a-6), 2.64 (m, 1H, H_b-6), 2.32 (t, J = 9.0 Hz, 2H, H-9), 1.56 (m, 2H, H-10), 1.38 (d, J = 8.3, 3H, H-8), 0.97 (t, J = 9.2 Hz, 3H, H-11); ESIMS m/z 413.1 [M + H]⁺.

Compound 3b. ¹H NMR (CDCl₃, 500 MHz) δ 6.94 (s, 1H, H-3), 5.28 (m, 1H, H-7), 5.07 (t, J = 10.3 Hz, H-5), 2.74 (m, 1H, H_a-6), 2.71 (m, 1H, H_b-6), 2.34 (t, J = 9.0 Hz, 2H, H-9), 1.60 (m, 2H, H-10), 1.31 (d, J = 8.3, 3H, H-8), 0.97 (t, J = 9.2 Hz, 3H, H-11); ESIMS m/z 413.1 [M + H]⁺.

Compound 5a. ¹H NMR (CDCl₃, 500 MHz) δ 6.57 (m, 1H, H-7), 6.35 (t, J = 11.0 Hz, 1H, H-6), 5.45 (t, J = 10.7 Hz, H-5), 5.42 (d, J = 16.7 Hz, 1H, H_a-8), 5.34 (d, J = 9.8 Hz, 1H, H_b-8), 5.32 (m, 1H, H-4), 5.18 (dd, J = 9.0, 6.8 Hz, 1H, H-3), 2.70 (m, 1H, H-2), 1.79 (m, 1H, H_a-9), 1.66 (m, 1H, H_b-9), 1.61 (m, 1H, H_a-10), 1.40 (m, 1H, H_b-10), 0.91 (t, J = 7.2 Hz, 3H, H-11); ESIMS m/z 571.1 [M + H]⁺.

Compound 5b. ¹H NMR (CDCl₃, 500 MHz) δ 6.45 (m, 1H, H-7), 6.29 (t, J = 11.0 Hz, 1H, H-6), 5.43 (t, J = 10.1 Hz, H-5), 5.37 (d, J = 16.7 Hz, 1H, H_a-8), 5.29 (d, J = 9.8 Hz, 1H, H_b-8), 5.29 (m, 1H, H-4), 5.18 (dd, J = 9.2, 5.8 Hz, 1H, H-3), 2.78 (m, 1H, H-2), 1.84 (m, 1H, H_a-9), 1.72 (m, 1H, H_b-9), 1.63 (m, 1H, H_a-10), 1.48 (m, 1H, H_b-10), 0.95 (t, J = 7.2 Hz, 3H, H-11); ESIMS m/z 571.1 [M + H]⁺.

Compound 6a. ¹H NMR (CDCl₃, 500 MHz) δ 6.38 (m, 1H, H-7), 6.35 (m, 1H, H-6), 5.74 (dd, J = 14.5, 5.9 Hz, 1H, H-5), 5.69 (s, H-3), 5.60 (m, 1H, H-9), 5.29 (d, J = 15.7 Hz, 1H, H_a-8), 5.17 (d, J = 9.0 Hz, 1H, H_b-8), 4.66 (d, J = 6.9 Hz, 1H, H-4), 4.50 (d, J = 12.6 Hz, 1H, H_a-1), 4.38 (d, J = 12.6 Hz, 1H, H_b-1), 2.34 (m, 1H, H_a-10), 2.06 (m, 1H, H_b-10), 0.87 (t, J = 7.7 Hz, 3H, H-11); ESIMS m/z 397.1 [M + H]⁺.

Compound 6b. ¹H NMR (CDCl₃, 500 MHz) δ 6.37 (m, 1H, H-7), 6.32 (m, 1H, H-6), 5.76 (s, H-3), 5.73 (dd, J = 14.5, 6.3 Hz, 1H, H-5), 5.66 (m, 1H, H-9), 5.28 (d, J = 15.7 Hz, 1H, H_a-8), 5.18 (d, J = 9.0 Hz, 1H, H_b-8), 4.50 (d, J = 6.3 Hz, 1H, H-4), 4.50 (d, J = 12.3 Hz, 1H, H_a-1), 4.39 (d, J = 12.3 Hz, 1H, H_b-1), 2.34 (m, 1H, H_a-10), 2.16 (m, 1H, H_b-10), 1.0 (t, J = 7.7 Hz, 3H, H-11); ESIMS m/z 397.1 [M + H]⁺.

Compound 7a. ¹H NMR (CDCl₃, 500 MHz) δ 6.07 (s, 1H, H-2), 5.90 (s, 1H, H-4), 5.69 (m, H-7), 5.25 (m, 1H, H_a-8), 5.12 (m, 1H, H_b-8), 2.63 (m, 1H, H_a-6), 2.53 (s, 1H, H-5), 2.52 (m, 1H, H_b-6), 2.14 (m, 1H, H_a-9), 2.11 (m, 1H, H_b-9), 1.56 (m, 1H, H_a-10), 1.48 (m, 1H, H_b-10), 0.90 (t, J = 6.5 Hz, 3H, H-11); ESIMS m/z 397.1 [M + H]⁺.

Compound 7b. ¹H NMR (CDCl₃, 500 MHz) δ 6.10 (s, 1H, H-2), 5.87 (s, 1H, H-4), 5.67 (m, H-7), 5.23 (m, 1H, H_a-8), 5.10 (m, 1H, H_b-8), 2.61 (m, 1H, H_a-6), 2.47 (m, 1H, H_b-6), 2.42 (s, 1H, H-5), 2.34 (m, 1H, H_a-9), 2.27 (m, 1H, H_b-9), 1.64 (m, 1H, H_a-10), 1.58 (m, 1H, H_b-10), 0.97 (t, J = 6.5 Hz, 3H, H-11); ESIMS m/z 397.1 [M + H]⁺.

Compound 12aa. ¹H NMR (CDCl₃, 500 MHz) δ 6.49 (dd, J = 15.5, 11.2 Hz, 1H, H-4), 6.41 (d, J = 16.0 Hz, 1H, H-9), 6.05 (d, J = 11.7 Hz, H-3), 5.71 (m, 1H, H-10), 5.52 (m, 1H, H-5), 5.22 (m, 1H, H-7), 4.30 (s, 2H, H-1), 2.45 (m, 1H, H_a-6), 2.40 (m, 1H, H_b-6), 1.80 (d, J = 6.6 Hz, 3H, H-11), 1.35 (d, J = 6.2 Hz, 3H, H-8), 0.92 (s, 9H, H-TBDMS), 0.08 (s, 6H, H-TBDMS); ESIMS m/z 513.1 [M + H]⁺.

Compound 12ab. ¹H NMR (CDCl₃, 500 MHz) δ 6.57 (dd, J = 15.5, 11.2 Hz, 1H, H-4), 6.44 (d, J = 16.1 Hz, 1H, H-9), 6.11 (d, J = 11.7 Hz, H-3), 5.71 (m, 1H, H-10), 5.63 (m, 1H, H-5), 5.21 (m, 1H, H-7), 4.32 (s, 2H, H-1), 2.51 (m, 1H, H_a-6), 2.44 (m, 1H, H_b-6), 1.80 (d, J = 6.6 Hz, 3H, H-11), 1.28 (d, J = 6.2 Hz, 3H, H-8), 0.92 (s, 9H, H-TBDMS), 0.08 (s, 6H, H-TBDMS); ESIMS m/z 513.1 [M + H]⁺.

Chemical transformation of 16 to 15

To a solution of compound 16 (2 mg) in MeOH (1 mL) was added 15 μL concentrated HCl (37%). After stirring at r.t. for 1 h, the reaction mixture was subjected to HPLC separation on an ODS column (10% MeOH/H₂O, v/v) to yield 15 (t_R 15.5 min, 1.2 mg).

Chemical transformation of 17 to 14

To a solution of compound 17 (2 mg) in methanol (1 mL) was added 500 μL NaOH solution (2%). After stirring at r.t. for 5 h, the reaction was neutralized to pH 7 using 1 M HCl. The obtained mixture was concentrated and then added to 1.5 mL H₂O. The H₂O layer was extracted twice with 3 mL EtOAc and the combined EtOAc extracts were concentrated and purified by HPLC on an ODS column (20% MeOH/H₂O, v/v) to yield 14 (t_R 4.6 min, 1.5 mg).

X-ray crystal data for 14 and 15

Colorless crystals of 14 and 15 were obtained in MeOH–H₂O (1 : 1, v/v). Crystal data of 14 were obtained using a Bruker APEX DUO area detector diffractometer with graphite monochromatic Cu-Kα radiation (λ = 1.54178 Å). Crystal data of 15 were obtained using a Bruker Smart CCD area detector diffractometer with graphite monochromatic Mo-Kα radiation (λ = 0.71073 Å) (ESI†).

Crystal data for aspergone N (14). Monoclinic, C₁₀H₁₆O₅·H₂O; space group P2(1) with a = 15.2823(12) Å, b = 4.5964(3) Å, c = 16.3515(14) Å, V = 1143.39(15) ų, Z = 4, D_calcd = 1.361 Mg m⁻³, μ = 0.957 mm⁻¹, and F(000) = 504; unique cell angle (β) = 95.4540(10); crystal size: 0.21 × 0.14 × 0.08 mm³. T = 293(2) K. The structure was solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of the full-matrix least-squares refinement was based on 3891 unique reflections (2θ < 50°) and 292 variable parameters and converged with unweighted and weighted agreement factors of R₁ = 0.1339, wR₂ = 0.3268, and R = 0.2463 for I > 2σ(I) data. The absolute structure parameter is 0.0(12) and it could not be used to determine the absolute configuration.

Crystal data for aspergone O (15). Trigonal, C₁₀H₁₅ClO₄; space group R3 with a = 24.752(2) Å, b = 24.752(2) Å, c = 5.0591(6) Å, V = 2684.3(4) ų, Z = 1, D_calcd = 1.307 Mg m⁻³, μ = 0.313 mm⁻¹, and F(000) = 1116; crystal size: 0.48 × 0.37 × 0.35 mm³. T = 298(2) K. The structure was solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of the full-matrix least-squares refinement was based on 2066 unique reflections (2θ < 50°) and 137 variable parameters and converged with unweighted and weighted agreement factors of R₁ = 0.0378, wR₂ = 0.0787, and R = 0.0532 for I > 2σ(I) data. The absolute structure parameter is 0.05(7).

Conclusion

In conclusion, eighteen new compounds (1–18) were isolated from the marine sponge-derived Aspergillus sp. strain OUCMDZ-1583. Compounds 5, 14 and 18 displayed comparable or stronger α-glucosidase inhibition than acarbose (IC₅₀ = 0.95 mM) with IC₅₀ values of 1.30, 1.37 and 0.027 mM, respectively. In addition, the α-glucosidase inhibition of fusarentin 6-methyl ether (22) was reported here for the first time with an IC₅₀ value of 1.19 mM.

Acknowledgements

This work was supported by grants from NSFC (No. 21172204, 41376148 & 81373298), from 863 programs of China (No. 2012AA092104 & 2013AA092901), from NSFC-Shandong Joint Fund for Marine Science Research Centers (No. U1406402), and from the Special Fund for Marine Scientific Research in the Public Interest of China (No. 201405038).

References

1. F. Pietra, Nat. Prod. Rep., 1997, 14, 453–464.
2. J. W. Blunt, B. R. Copp, R. A. Keyzers, M. H. Munro and M. R. Prinsep, Nat. Prod. Rep., 2013, 30, 237–323.
3. R. K. Pettit, Appl. Microbiol. Biotechnol., 2009, 83, 19–25.
4. M. E. Rateb and R. R. Ebel, Nat. Prod. Rep., 2011, 28, 290–344.
5. M. Saleem, M. S. Ali, S. Hussain, A. Jabbar, M. Ashraf and Y. S. Lee, Nat. Prod. Rep., 2007, 24, 1142–1152.
6. Y. Q. Fan, Y. Wang, P. P. Liu, P. Fu, T. H. Zhu, W. Wang and W. M. Zhu, J. Nat. Prod., 2013, 76, 1328–1336.
7. J. K. Zheng, Z. H. Xu, Y. Wang, K. Hong, P. P. Liu and W. M. Zhu, J. Nat. Prod., 2010, 73, 1133–1137.
8. Y. B. Zhuang, X. C. Teng, Y. Wang, P. P. Liu, G. Q. Li and W. M. Zhu, Org. Lett., 2011, 13, 1130–1133.
9. F. Kong, Y. Wang, P. Liu, T. Dong and W. M. Zhu, J. Nat. Prod., 2014, 77, 132–137.
10. C. Li and J. A. Porco, J. Org. Chem., 2005, 70, 6053–6065.
11. B. Fang, X. Xie, C. Zhao, P. Jing, H. Li, Z. Wang, J. Gu and X. She, J. Org. Chem., 2013, 78, 6338–6343.
12. H. K. Kwon, Y. E. Lee and E. Lee, Org. Lett., 2008, 10, 2995–2996.
13. W. Zhang, K. Krohn, S. Draeger and B. Schulz, J. Nat. Prod., 2008, 71, 1078–1081.
14. M. Johansson, B. Köpcke, H. Anke and O. Sterner, J. Antibiot., 2002, 55, 104–106.
15. R. Ueoka, Y. Nakao, S. Kawatsu, J. Yaegashi, Y. Matsumoto, S. Matsunaga, K. Furihata, R. W. van Soest and N. Fusetani, J. Org. Chem., 2009, 74, 4203–4207.
16. L. Di Bari, G. Pescitelli, C. Pratelli, D. Pini and P. Salvadori, J. Org. Chem., 2001, 66, 4819–4825.
17. M. Górecki, E. Jabłońska, A. Kruszewska, A. Suszczyńska, Z. Urbańczyk-Lipkowska, M. Gerards, J. W. Morzycki, W. J. Szczepek and J. Frelek, J. Org. Chem., 2007, 72, 2906–2916.
18. S. Chen, F. Ren, S. Niu, X. Liu and Y. Che, J. Nat. Prod., 2014, 77, 9–14.
19. P. J. Stephens, J. J. Pan and K. J. Krohn, J. Org. Chem., 2007, 72, 7641–7649.
20. J. A. Beutler, K. L. McCall, K. Herbert, D. L. Herald, G. R. Pettit, T. Johnson, R. H. Shoemaker and M. R. Boyd, J. Nat. Prod., 2000, 63, 657–661.
21. H. D. Flack, Acta Crystallogr., Sect. A: Found. Crystallogr., 1983, 39, 876–881.
22. S. V. Nampoothiri, A. Prathapan, O. L. Cherian, K. G. Raghu, V. V. Venugopalan and A. Sundaresan, Food Chem. Toxicol., 2011, 49, 125–131.
23. T. Mosmann, Immunol. Methods, 1983, 65, 55–63.
24. T. Xiong, X. Q. Chen, H. Wei and H. Xiao, Arch. Med. Sci., 2015, 11, 301–306.
25. L. L. Zaika, J. Food Saf., 1988, 9, 97–118.

---

Footnote

† Electronic supplementary information (ESI) available: Bioassay protocols used, the NMR spectra of compounds 19–23, the ITS gene sequences of Aspergillus sp. OUCMDZ-1583. CCDC 995363 and 995361. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra11185d

---