Meroterpenoids and Isocoumarinoids from a Myrothecium Fungus Associated with Apocynum venetum

Abstract

Four new meroterpenoids 1–4 and four new isocoumarinoids 5–8, along with five known isocoumarinoids (9–13), were isolated from the fungus Myrothecium sp. OUCMDZ-2784 associated with the salt-resistant medicinal plant, Apocynum venetum (Apocynaceae). Their structures were elucidated by means of spectroscopic analysis, X-ray crystallography, ECD spectra and quantum chemical calculations. Compounds 1–5, 7, 9 and 10 showed weak α-glucosidase inhibition with the IC₅₀ values of 0.50, 0.66, 0.058, 0.20, 0.32, 0.036, 0.026 and 0.37 mM, respectively.

1. Introduction

Since the discovery of penicillin, fungi have been an important source of lead compounds for drug development, which have provided a lot of attractive natural products (NPs) with different biological activities [1,2,3]. With the increase of study on the terrestrial fungal NPs, more and more known compounds were isolated repeatedly. Therefore, many researchers turned their attention to the fungi isolated from specific habitats, such as the marine-derived fungi [4,5,6,7] and the fungi associated with the plants or animals [8,9,10,11].

As part of our ongoing studies to search for bioactive NPs from fungi derived from special niche [12,13,14,15,16], we screened the fungus Myrothecium sp. OUCMDZ-2784 which is associated with the salt-resistant plant A. venetum (Apocynaceae) growing in the Yellow River Delta, a traditional Chinese medicine used for treatment of hypertension [17] and heart failure [18]. Myrothecium sp. has been reported to produce trichothecenes [19], sesquiterpenes [20,21], diterpenes [22] and cyclopeptides [23] with cytotoxic and antibacterial activities. The ethyl acetate (EtOAc) extract of the fermentation of Myrothecium sp. OUCMDZ-2784 showed 75% inhibition of α-glucosidase at 286 μg/mL. Chemical study resulted in the isolation and identification of four new meroterpenoids, myrothecisins A–D (1–4) and four new isocoumarinoids, myrothelactones A–D (5–8), together with five known isocoumarinoids that were identified as tubakialactone B (9) [24], acremonone G (10) [25], 6,8-dihydroxy-3-methylisocoumarin (11) [26], 3,4-dimethyl-6,8-dihydroxyisocoumarin (12) [27] and sescandelin B (13) [28], respectively by comparing ¹H and ¹³C NMR spectra (Figure S57, Table S1) as well as ESIMS spectra (Figure S59) with those reported.

2. Results and Discussion

Myrothecisin A (1) was isolated as a pale-yellow oil. Its molecular formula was assigned as C₂₅H₃₄O₇ by the HRESIMS peak at m/z 469.2188 [M + Na]⁺ (Figure S58A), indicating nine degrees of unsaturation. The ¹³C NMR (Figure S6) spectrum of 1 showed 25 signals that were classified by DEPT (Figure S7) and HSQC (Figure S8) as an aldehyde carbonyl carbon (δ_C 193.8), one acyl carbonyl carbon (δ_C 170.3), five sp² non-protonated carbons (δ_C 167.3, 159.8, 149.5, 112.3, 111.2) and three sp³ non-protonated carbons (δ_C 98.8, 42.8, 39.2), one sp² methine (δ_C 101.5) and four sp³ methines (δ_C 78.2, 71.4, 45.6, 36.0), five sp³ methylenes (δ_C 60.5, 35.1, 30.6, 30.4, 20.7) and five methyl carbons (δ_C 28.7, 21.3, 17.0, 16.6, 15.2) (Table 1). The ¹H (Figure S5) and HSQC NMR showed the singlet signals at δ_H 10.06 and 6.56 for an aldehyde proton and an aromatic proton, respectively. The key HMBC (Figure S10) correlations from H-7′ (δ_H 10.06) to C-2′/C-3′/C-4′, 2′-OH (δ_H 12.16) to C-1′/C-2′/C-3′, H-5′ (δ_H 6.56) to C-1′/C-4′/C-6′/C-8′, H-8′ (δ_H 4.74) to C-3′/C-4′/C-5′ and 8′-OH (δ_H 5.44) to C-4′/C-8′ suggested a penta-substituted benzene ring (Figure 2). The COSY (homonuclear correlation spectroscopy) correlations from H-1 through H-2 to H-3 and H-5 through H-6, H-7 and H-8 to H-12 (Figure 1 and Figure S9), along with the key HMBC correlations from H-2 to C-4/C-10/C-16, H-3 to C-5/C-13/C-14, 3-OH to C-2/C-3/C-4, H-1 to C-2/C-3/C-10/C-15, H-15 to C-1/C-5/C-9/C-10, H-5 to C-3/C-4/C-6/C-13, H-6 to C-5, H-7 to C-8, H-13 to C-3/C-5/C-14, H-14 to C-3/C-5/C-13, H-12 to C-7/C-8/C-9 and H-17 to C-16 revealed a sesquiterpene fragment (Figure 2). The connection of the above-mentioned two fragments were confirmed by the key HMBC correlations from H-11 to C-8/C-9/C-10/C-1′/C-2′/C-6′ (Figure 2) [29]. The relative configuration of 1 was determined by the NOESY correlations from H-8 to H-11 and H-15, H-3 to H-5 and H-13, H-2 to H-14 and H-15 and H-11 to H-15 (Figure 3 and Figure S11). The absolute configuration of 1 was determined by calculation of electronic circular dichroism (ECD) using time-dependent density functional theory (TDDFT) (Figure S1) [30,31] and the measured ECD spectrum of 1 matched well with the calculated ECD spectrum for (2R,3R,5S,8R,9R,10S)-1 (Figure 4).

Table 1.

¹H (600 MHz) and ¹³C (150 MHz) NMR data for 1–4 in DMSO-d₆.

No.	1		2		3		4
	δC, Type	δH, Mult. (J in Hz)	δC, Type	δH, Mult. (J in Hz)	δC, Type	δH, Mult. (J in Hz)	δC, Type	δH, Mult. (J in Hz)
1	35.1, CH2	1.16, m; 1.55 a	38.4, CH2	1.17, m; 1.57 a	114.7, CH	4.94, brs	116.3, CH	4.88, s
2	71.4, CH	4.83, ddd (11.5, 10.1, 4.1)	64.4, CH	3.65, m	28.0, CH2	1.89, m; 1.23, m	31.6, CH2	1.72, m
3	78.2, CH	2.96, dd (10.1, 4.8)	83.3, CH	4.29, d (9.9)	75.6, CH	4.62, dd (8.6, 6.5)	71.5, CH	3.30 a
4	39.2, C		39.0, C		35.6, C		36.9, C
5	45.6, CH	1.56 a	45.5, CH	1.63 a	43.1, CH	2.54, m	43.7, CH	2.45, m
6	20.7, CH2	1.54 a; 1.47, m	20.4, CH2	1.56 a; 1.48, m	26.7, CH2	1.79, m	26.7, CH2	1.77, m
7	30.6, CH2	1.55 a; 1.35, m	30.5, CH2	1.57 a; 1.38, m	30.2, CH2	1.54, m	30.3, CH2	1.52, m
8	36.0, CH	1.83, m	35.6, CH	1.85, m	43.3, CH	1.30, m	42.9, CH	1.29, m
9	98.8, C		98.8, C		43.7, C		43.7, C
10	42.8, C		42.5, C		143.9, C		143.0, C
11	30.4, CH2	3.03, d (16.1); 2.79, d (16.0)	30.2, CH2	3.11, d (16.5); 2.81, d (16.5)	23.5, CH2	2.68, d (12.9); 2.53, d (12.9)	23.3, CH2	2.63, d (12.7); 2.50, d (12.7)
12	15.2, CH3	0.64, d (6.3)	15.1, CH3	0.68, d (6.3)	17.1, CH3	1.03, d (6.6)	17.2, CH3	1.02, d (6.7)
13	28.7, CH3	0.97, s	28.4, CH3	0.79, s	25.0, CH3	0.88, s	25.2, CH3	0.95, s
14	17.0, CH3	0.78, s	17.5, CH3	0.81, s	17.1, CH3	0.73, s	14.8, CH3	0.59, s
15	16.6, CH3	1.04, s	16.6, CH3	1.03, s	23.0, CH3	0.96, s	22.9, CH3	0.91, s
16	170.3, C		170.3, C		169.8, C
17	21.3, CH3	1.91, s	21.0, CH3	2.02, s	21.0, CH3	1.98, s
1′	111.2, C		111.2, C		111.4, C		111.3, C
2′	159.8, C		159.8, C		164.5, C		164.8, C
3′	112.3, C		112.2, C		110.2, C		110.2, C
4′	149.5, C		149.4, C		145.4, C		145.4, C
5′	101.5, CH	6.56, s	101.6, CH	6.61, s	107.5, CH	6.51, s	107.3, CH	6.50, s
6′	167.3, C		167.3, C		164.6, C		164.6, C
7′	193.8, CH	10.06, s	193.8, CH	10.07, s	193.3, CH	9.96, s	193.4, CH	9.96, s
8′	60.5, CH2	4.74, s	60.4, CH2	4.74, s	59.9, CH2	4.70, s	59.8, CH2	4.70, s
2/3-OH		4.94, d (4.7)		4.62, d (4.9)				4.13, s
2′-OH		12.16, s		12.24, s		12.84, s		12.78, s
6′-OH								10.55, brs
8′-OH		5.44, s		5.39, s		5.35, s		5.36, s

^a Overlapped.

Figure 1.

Structures 1–13 isolated from Myrothecium sp. OUCMDZ-2784.

Figure 2.

Key homonuclear correlation spectroscopy (COSY) and key HMBC correlations for 1–8.

Figure 3.

NOESY correlations for 1–4.

Figure 4.

Measured and calculated ECD spectra for 1, 3, 5 and 6.

The molecular formula of 2 was also determined as C₂₅H₃₄O₇ by the HRESIMS peak at m/z 469.2200 [M + Na]⁺ (Figure S58B), implying that 2 is an isomer of 1. Comparison of its ¹H and ¹³C NMR spectra (Figures S12–S16) with those of 1 revealed that the acetyloxy group in 2 was on C-3. This was confirmed by the HMBC (Figure S17) correlation from H-3 (δ_H 4.29) to C-16 (δ_C 170.3) (Figure 2). The similar NOESY correlations (Figure 3 and Figure S18) suggested that 2 has the same relative configuration as 1. The similarity of ECD curves between 2 and 1 (Figure 5) indicated the same absolute configurations of its stereogenic carbons. Therefore 2 was named myrothecisin B.

Figure 5.

ECD spectra for 1−4.

Compound 3 was also obtained as a pale-yellow oil. Its molecular formula was determined as C₂₅H₃₄O₆ according to the HRESIMS peak at m/z 453.2238 [M + Na]⁺ (Figure S58C). The ¹³C NMR (Figure S20) spectrum of 3 showed one aldehyde carbonyl carbon (δ_C 193.3), one acyl carbonyl carbon (δ_C 169.8), six sp² non-protonated carbons (δ_C 164.6, 164.5, 145.4, 143.9, 111.4, 110.2) and two sp³ non-protonated carbons (δ_C 43.7, 35.6), two sp² methines (δ_C 114.7, 107.5) and three sp³ methines (δ_C 75.6, 43.3, 43.1), five sp³ methylenes (δ_C 59.9, 30.2, 28.0, 26.7, 23.5) and five methyl carbons (δ_C 25.0, 23.0, 21.0, 17.1, 17.1) (Table 1). Analysis of its 1D and 2D NMR (Figures S19–S24) data revealed the presence of a substituted benzene ring and a sesquiterpene unit, indicating 3 was an analogue of 1 and 2. Comparison of the ¹H and ¹³C NMR spectra with those of 1 and 2 suggested a same pentasubstituted benzene ring. The structure of the sesquiterpene unit was slightly modified and was determined by the COSY (Figure S23) correlations from H-1 through H-2 to H-3 and H-5 through H-6, H-7 and H-8 to H-12 and the key HMBC (Figure S24) correlations from H-3 to C-2/C-4/C-10/C-13/C-14, H-17 to C-16, H-1 to C-3/C-9, H-15 to C-10, H-8 to C-9, H-5 to C-9/C-13, H-12 to C-7 and H-2 to C-4/C-10 (Figure 2). The HMBC correlations from H₂-11 (δ_H 2.68/2.53) to C-8/C-10/C-15/C-2′/C-6′ (Figure 2) confirmed the connection between the sesquiterpene fragment and the benzene ring. The relative configuration of 3 was determined by the NOESY (Figure S25) correlations from H-13 to H-3 and H-5, H-8 to H-15, as well as H-5 to H-11 (Figure 3). The absolute configuration was determined as (3S,5R,8R,9R)- by comparison of the calculated and experimental ECD spectra (Figure 4 and Figure S2). Therefore 3 was named myrothecisin C.

The molecular formula of 4 was assigned as C₂₃H₃₂O₅ by the HRESIMS peak at m/z 411.2139 [M + Na]⁺ (Figure S58D), which was C₂H₂O less than that of 3. The similarity of the UV and NMR data between 3 and 4 (Table 1) suggested that 4 possesses the same skeleton as 3. Careful comparison of their ¹H and ¹³C NMR spectra (Figures S26–S31) showed that the acetyloxy group (δ_C 21.0/δ_H 1.98 and δ_C 169.8) in 3 was replaced by a hydroxy group (δ_H 4.13) in 4 (Table 1). The NOESY data (Figure 3 and Figure S32) suggested that 4 has the same relative configuration as 3. The ECD Cotton effects of 4 were nearly identical to those of 3 (Figure 5), indicating the same absolute configurations of the corresponding stereogenic carbons. Thus, 4 was named myrothecisin D.

Compound 5 was obtained as a colorless crystal with the molecular formula C₁₂H₁₂O₆ from the HRESIMS peak at m/z 251.0563 [M − H]⁻ (Figure S58E). The ¹H NMR spectrum showed two meta-coupled aromatic protons at δ_H 6.78 (d, J = 2.2 Hz) and δ_H 6.63 (d, J = 2.2 Hz) (Table 2, Figure S33), indicating the presence of a tetra-substituted benzene ring. The ¹³C (Figure S34) NMR spectrum showed 12 carbon signals that were classified by DEPT (Figure S35) and HSQC (Figure S36) spectra as six sp² non-protonated carbons (δ_C 166.4, 165.4, 163.4, 137.6, 118.6, 99.9), three sp² methines (δ_C 143.1, 100.5, 100.4) and one sp³ methine (δ_C 68.8), one sp³ methylene (δ_C 64.8) and one methoxy group (δ_C 56.0) (Table 2). The key HMBC correlations (Figure 2 and Figure S38) from CH₃O-6 to C-6, HO-11 to C-4/C-12, H-11 to C-3/C-10, H-3 to C-1/C-10, H-5 to C-4/C-7/C-9 and H-7 to C-9 along with the continuous COSY correlations of HO-11 (δ_H 5.50)/H-11 (δ_H 4.66)/H-12 (δ_H 3.51, 3.62)/HO-12 (δ_H 4.81) (Figure S37) revealed that 5 possesses a 4,6,8-trisubstituted isocoumarin skeleton with a hydroxy, a methoxy and a 1,2-dihydroxy ethyl at C-8, C-6 and C-4, respectively. The structure of 5 was further confirmed by X-ray crystallography (Figure 6). Because the value of the Flack parameter [−0.2(2)] was large, the absolute configuration determined by X-ray crystallography was not reliable. Thus, the ECD calculation method was used to further confirm the absolute configuration of C-11 of 5 as 11R- (Figure 4 and Figure S3). Consequently, 5 was named myrothelactone A.

Table 2.

¹H and ¹³C NMR data for 5–8 in DMSO-d₆.

No.	5 a		6 b		7 b		8 b
	δC, Type	δH, mult. (J in Hz)	δC, Type	δH, mult. (J in Hz)	δC, Type	δH, mult. (J in Hz)	δC, Type	δH, mult. (J in Hz)
1	165.4, C		164.9, C		163.4, C		165.2, C
3	143.1, CH	7.45, s	144.4, CH	7.62, s	153.1, CH	8.47, s	151.0, C
4	118.6, C		117.7, C		114.7, C		118.7, C
5	100.5, CH	6.78, d (2.2)	101.6, CH	6.84, s	102.3, CH	7.46, s	101.6, CH	6.86, s
6	166.4, C		166.4, C		166.8, C		166.3, C
7	100.4, CH	6.63 (d, 2.2)	100.6, CH	6.63, s	101.2, CH	6.68, s	99.8, CH	6.48, s
8	163.4, C		163.3, C		163.2, C		162.6, C
9	99.9, C		99.9, C		99.9, C		99.2, C
10	137.6, C		136.8, C		134.8, C		138.6, C
11	68.8, CH	4.66, td (5.2, 5.2)	68.5, CH	5.01, s	198.3, C		173.9, C
12	64.8, CH2	3.62, ddd (11.3, 5.3, 5.3); 3.51, ddd (11.3, 5.3, 5.3)	173.4, C		65.9, CH2	4.57, d (4.7)	17.9, CH3	2.29, s
6-OCH3	56.0, CH3	3.88, s	56.0, CH3	3.84, s	56.0, CH3	3.87, s	55.8, CH3	3.80, s
8-OH		11.37, s		11.22, s		11.03, s		11.21, s
11-OH		5.50, d (4.7)
12-OH		4.81, t (5.2)				5.28, t (5.0)

^a Data were measured at 600 MHz (¹H) and 150 MHz (¹³C). ^b Data were measured at 500 MHz (¹H) and 125 MHz (¹³C).

Figure 6.

ORTEP diagram of 5.

Compound 6 was obtained as a white powder. Its molecular formula was determined as C₁₂H₁₀O₇ based on the HRESIMS peak at m/z 265.0355 [M − H]⁻ (Figure S58F). The UV and ¹³C NMR data of 6 (Table 2) were similar to those of 5, indicating that they have the same isocoumarin scaffold. Comparison of their ¹H and ¹³C data (Figures S39–S43) indicated that the hydroxymethyl group (δ_C/H 64.8/3.62&3.51, δ_H 4.81) in 5 was replaced by the carboxyl group (δ_C 173.4). This change was verified by the key HMBC (Figure S44) correlations from H-11 to C-3/C-10/C-12. The absolute configuration of C-11 of 6 was determined as 11R- by comparison of the calculated and experimental ECD spectra (Figure 4 and Figure S4). Therefore, 6 was name myrothelactone B.

Compound 7 was obtained as a white powder. Its molecular formula was determined as C₁₂H₁₀O₆ according to its HRESIMS peak at m/z 249.0408 [M – H]⁻ (Figure S58G), which was only two hydrogen atoms less than that of 5. The difference observed in the NMR spectra of 7 and 5 was that the signals for hydroxymethine (δ_C/H 68.8/4.66) in 5 were replaced by the signal of a carbonyl group (δ_C-11 198.3) in 7 (Table 2, Figures S45–S49). The HMBC (Figure S50) correlations from H-3 (δ_H 8.47) and H-12 (δ_H 4.57) to C-11 further confirmed the structure of 7 which was name myrothelactone C (Figure 2).

The molecular formula of compound 8 was determined as C₁₂H₁₀O₆ on the basis of its HRESIMS peak at m/z 249.0407 [M − H]⁻ (Figure S58H), which is an isomer of 7. Analysis of its ¹H and ¹³C NMR spectra showed that 8 also had the same isocoumarin scaffold, whose difference is the replacement of carbonyl (δ_C 198.3), hydroxymethyl (δ_C 65.9, δ_H 4.57/5.28) and sp² methine (δ_C 153.1, δ_H 8.47) signals in 7 by two sp² non-protonated carbons (δ_C 173.9, 151.0) and methyl (δ_C 17.9, δ_H 2.29) signals in 8 (Table 2, Figures S51–S55). The HMBC correlations from H-12 (δ_H 2.29) to C-3 (δ_C 151.0) and C-4 (δ_C 118.7) suggested the methyl substitution at C-3 (Figure 2 and Figure S56). The chemical shift of the carboxyl signal (δ_C 173.9) together with 2D NMR data indicated the carboxyl substitution at C-4. The structure of myrothelactone D (8) was therefore determined (Figure 2).

The α-glucosidase inhibitory activity of 1–13 was preliminarily investigated. Compounds 1–5, 7, 9 and 10 exhibited inhibitory activity against the human-sourced α-glucosidase recombinant expressed in Saccharomyces cerevisiae [31,32,33] with IC₅₀ values of 0.50, 0.66, 0.058, 0.20, 0.32, 0.036, 0.026 and 0.37 mM, while the IC₅₀ value of positive control acarbose was 0.47 mM. Due to the low activity, the deeper investigation of the mechanism and type of enzymatic inhibition as well as the binding mode were not done.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were measured using a JASCO P-1020 digital polarimeter (JASCO Corporation, Tokyo, Japan). UV spectra were obtained on a Beckman DU 640 spectrophotometer (Beckman Coulter, Inc., Brea, CA, USA). CD data were performed using a JASCO J-815 spectropolarimeter (JASCO Corporation, Tokyo, Japan). IR spectra were obtained on a Nicolet Nexus 470 spectrophotometer (Thermo Nicolet Corporation, Madison, WI, USA) as KBr discs. NMR spectra were recorded on a Varian System 500 spectrometer (Varian, Palo Alto, CA, USA) or a Bruker Avance 600 spectrometer (Bruker, Fallanden, Switzerland) using residual solvent signals for referencing and chemical shifts were recorded as δ values. HRESIMS spectra were measured using the Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer (Waters Asia, Ltd., Singapore). Semi-preparative HPLC was performed using an ODS column (YMC-pack ODS-A, 10 mm × 250 mm, 5 μm, 4.0 mL/min, Kyoto, Japan). TLC and column chromatography (CC) were performed on plates pre-coated with silica gel GF₂₅₄ (10–40 µm, Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), respectively. Vacuum-liquid chromatography (VLC) utilized silica gel H (Qingdao Marine Chemical Factory).

3.2. Collection and Phylogenetic Analysis

The fungus OUCMDZ-2784 was isolated from Apocynum venetum (Apocynaceae) collected from the estuary of Yellow River, Dongying, China. The leaves of the plant were washed with tap water and sterile distilled water in sequence. Then, it was cut into small pieces, which were then put into a centrifuge tubes filled with different concentrations of sucrose solution. These tubes were centrifuged at 1200 rpm for 20 min. Four zones were separated by improved discontinuous sucrose gradient centrifugation. The interface between the third and the fourth bands was deposited on a PDA (200 g potato, 20 g glucose, 20 g agar per liter of sea water) plate containing chloramphenicol (100 μg/mL) as a bacterial inhibitor and was then cultured at 28 °C for 3 days. A single colony was transferred to PDA agar media and was identified as Myrothecium sp. according to its morphological characteristics and 18S rRNA gene sequences (GenBank accession No. KF977010).

3.3. Cultivation and Extraction

Fungus OUCMDZ-2784 was prepared on PDA agar medium. Spores were incubated at 28 °C for 48 h on a rotary shaker with shaking at 120 rpm in a 500 mL cylindrical flask containing 150 mL liquid medium (20 g maltose, 20 g mannitol, 10 g glucose, 3 g yeast extract, 10 g monosodium glutamate per liter of sea water). The cultures were transferred to 350 × 1000 mL Erlenmeyer flasks and each containing 300 mL liquid fermentation media (1 g peptone, 10g soluble starch per liter of sea water, pH 7.0). The flasks were incubated at room temperature under static conditions for 30 days. The cultures were extracted three times by EtOAc and the combined EtOAc extracts were dried in vacuo to yield 20.1 g of extract.

3.4. Purification

The extract (20.1 g) was fractionated by VLC, eluting with a step gradient of CH₂Cl₂-petroleum ether (50–100%) and MeOH-CH₂Cl₂ (0–50%) and five fractions (Fr.1–Fr.5) were collected. Fraction 2 (3.2 g) was subjected to Sephadex LH-20 chromatography eluting with CH₂Cl₂/MeOH (1:1) to afford three subfractions (Fr.2.1–Fr.2.3). Fr.2.1 (1.0 g) was further purified by HPLC on an ODS column (80% MeOH/H₂O) to give compounds 1 (25.2 mg, t_R 6.3 min) and 3 (30.1 mg, t_R 10.2 min). Fr.2.2 (50.2 mg) was purified by HPLC on an ODS column (60% MeOH/H₂O) to yield compounds 11 (3.5 mg, t_R 10.4 min) and 8 (3.3 mg, t_R 15.2 min). Fr.2.3 (46.3 mg) was purified by HPLC on an ODS column (60% MeOH/H₂O) to yield compounds 12 (5.1 mg, t_R 12.6 min) and 13 (12.0 mg, t_R 16.4 min). Fraction 3 (3.8 g) was separated into three subfractions (Fr.3.1–Fr.3.3) by Sephadex LH-20 eluting with MeOH-CH₂Cl₂ (1:1). Fr.3.1 (0.5 g) was purified by semi preparative HPLC on an ODS column (85% MeOH/H₂O) to yield compound 2 (36.2 mg, t_R 10.2 min). Fr.3.2 (1.1 g) was separated by silica gel VLC column eluting with CH₂Cl₂-petroleum (2:1) to yield compounds 10 (100.3 mg) and 5 (200.8 mg). Fr.3.3 (0.5 g) was further purified by Sephadex LH-20 eluting with MeOH to yield compound 9 (15.3 mg). Fraction 4 (1.8 g) was separated into three subfractions (Fr.4.1–Fr.4.3) by Sephadex LH-20 eluting with MeOH. Fr.4.1 (0.6 g) was further purified by semi preparative HPLC on an ODS column (85% MeOH/H₂O) to yield compound 4 (34.3 mg, t_R 12.2 min). Fr.4.2 (0.2 g) was further purified by semi preparative HPLC on an ODS column (50% MeOH/H₂O) to yield compound 7 (6.0 mg, t_R 12.7 min). Fr.4.3 (0.3 g) was purified by HPLC on an ODS column (40% MeOH/H₂O) to yield compound 6 (5.8 mg, t_R 10.6 min).

Myrothecisin A (1): pale yellow oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ + 9.0 (c 0.1, CHCl₃); UV (MeOH) λ_max (log ε) 212 (4.13), 237 (3.60), 305 (3.93) nm; ECD (0.002 M, MeOH) λ_max (Δε) 207 (+1.69), 244 (−0.51), 304 (+1.06) nm; IR (KBr) ν_max 3443, 2926, 1719, 1635, 1372, 1267 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 469.2188 [M + Na]⁺ (calcd for C₂₅H₃₄O₇Na, 469.2197).

Myrothecisin B (2): pale yellow oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ + 70.1 (c 0.1, CHCl₃); UV (MeOH) λ_max (log ε) 211 (4.10), 238 (3.62), 305 (3.88) nm; ECD (0.002 M, MeOH) λ_max (Δε) 207 (+1.62), 249 (−0.09), 305 (+0.49) nm; IR (KBr) ν_max 3424, 2940, 1733, 1638, 1460, 1371, 1263 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 469.2200 [M + Na]⁺ (calcd for C₂₅H₃₄O₇Na, 469.2197).

Myrothecisin C (3): pale yellow oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ + 63.5 (c 0.1, CHCl₃); UV (MeOH) λ_max (log ε) 212 (4.09), 239 (3.65), 307 (3.81) nm; ECD (0.002 M, MeOH) λ_max (Δε) 219 (+2.29), 293 (−0.49), 327 (+0.22) nm; IR (KBr) ν_max 3480, 2936, 1732, 1612, 1373, 1254 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 453.2238 [M + Na]⁺ (calcd for C₂₅H₃₄O₆Na, 453.2248).

Myrothecisin D (4): pale yellow oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ + 20.0 (c 0.1, CHCl₃); UV (MeOH) λ_max (log ε) 203 (4.16), 224 (3.56), 300 (3.86) nm; ECD (0.002 M, MeOH) λ_max (Δε) 220 (+1.57), 242 (+0.92), 295 (−0.27) nm; IR (KBr) ν_max 2928, 1718, 1621, 1370, 1264, 1027 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 411.2139 [M + Na]⁺ (calcd for C₂₃H₃₂O₅Na, 411.2142).

Myrothelactone A (5): colorless crystal; melting point (mp) 174–175 °C; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ − 36.0 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 246 (3.82), 328 (3.19) nm; ECD (0.004 M, MeOH) λ_max (Δε) 206.5 (−0.22), 241 (+0.24), 259 (−0.27) nm; IR (KBr) ν_max 3747, 3630, 3159, 2959, 1668, 1558, 1398, 1237 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 251.0563 [M − H]⁻ (calcd for C₁₂H₁₁O₆, 251.0561).

Myrothelactone B (6): white powder; mp 169–171 °C; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ − 30.0 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 245 (3.81), 328 (3.17) nm; ECD (0.004 M, MeOH) λ_max (Δε) 206.5 (−0.78), 241 (+0.24), 259 (−0.13) nm; IR (KBr) ν_max 3749, 2922, 1681, 1651, 1619, 1459, 1399 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 265.0355 [M − H]⁻ (calcd for C₁₂H₉O₇, 265.0354).

Myrothelactone C (7): white powder; mp 160–161 °C; UV (MeOH) λ_max (log ε) 228 (3.91), 263 (3.43), 325 (3.16) nm; IR (KBr) ν_max 3750, 3675, 3615, 1736, 1651, 1558, 1540, 1399 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 249.0408 [M − H]⁻ (calcd for C₁₂H₉O₆, 249.0405).

Myrothelactone D (8): white powder; mp 219–221 °C; UV (MeOH) λ_max (log ε) 231 (3.92), 263 (3.45), 325 (3.16) nm; IR (KBr) ν_max 3749, 3673, 3445, 3197, 1716, 1682, 1539, 1457, 1399 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 249.0407 [M − H]⁻ (calcd for C₁₂H₉O₆, 249.0405).

3.5. X-ray Structure Determination of Compound 5

Compound 5 was obtained as a colorless needles crystal with molecular formula C₁₂H₁₂O₆. Orthorhombic, space group P2₁2₁2₁, a = 4.9041(2) Å, b = 13.8470(5) Å, c = 15.7443(6) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1069.15(7) Å3, Z = 4, D_calcd = 1.567 Mg/m³, μ = 1.089 mm⁻¹, F(000) = 528, crystal size 0.30 mm × 0.18 mm × 0.15 mm, T = 293(2) K. A total of 1478 unique reflections (2θ < 50°) were collected on a CCD area detector diffractometer with graphite monochromated Cu Kα radiation (λ = 1.54178 Å). The structure was solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of full-matrix least squares refinement was based on 1478 unique reflections (2θ < 50°) and 165 variable parameters and converged with unweighted and weighted agreement factors of R₁ = 0.0326, wR₂ = 0.0885 and R = 0.0880 for I > 2sigma(I) data. Absolute structure parameter: −0.2(2). The deposited number of compound 5 in the Cambridge Crystallographic Data Centre is 980155.

3.6. α-Glucosidase Inhibitory Assays

The human-sourced α-glucosidase was recombinant expressed in the yeast Saccharomyces cerevisiae and the inhibitory effects of compounds 1–13 were tested using p-nitrophenyl-α-d-glucopyranoside (pNPG) as substrate [31,32,33]. The sample was dissolved in sodium phosphate buffer (PBS, pH 6.8) at three concentrations. 10 μL of the sample solution, 20 μL of 2.5 mM pNPG solution (in phosphate buffer) and 20 μL of PBS were mixed in a 96-well microplate and incubated at 37 °C for 5 min. A volume of 10 μL of α-glucosidase diluted to 0.2 U/mL by 0.01 M PBS was then added to each well. After incubating at 37 °C for 15 min, the absorbance at 405 nm was recorded by a Spectra max 190 micro plate reader (Molecular Devices Inc., San Jose, CA, USA). The blank was prepared by adding phosphate buffer instead of the α-glucosidase and the positive control was acarbose. Blank readings (no enzyme) were subtracted from each well and results were compared to the control. The inhibition (%) was calculated as [1 − (OD_drug/OD_blank)] × 100%. The IC₅₀ value was calculated as the compound concentration that is required for 50% inhibition and the IC₅₀ value of acarbose was 0.47 mM.

4. Conclusions

This study revealed eight new fungal NPs, meroterpenoids 1–4 and isocoumarinoids 5–8, from the culture of the salt-tolerant plant-associated fungus Myrothecium sp. OUCMDZ-2784. The new compounds 1–5 and 7 exhibited α-glucosidase inhibitory activity. Combined with bioactive NPs from mangrove-derived fungi [34,35,36,37,38], the results indicated that fungi living in the salt-tolerant plants are an important biological resources for new and bioactive NPs.

Supplementary Materials

The following are available online at http://www.mdpi.com/1660-3397/16/10/363/s1, Figures S1–S4: DFT-optimized structures for low-energy conformers of compounds 1, 3, 5 and 6, Figures S5–S11: NMR spectra of compound 1 in DMSO-d₆, Figures S12–S18: NMR spectra of compound 2 in DMSO-d₆, Figures S19–S25: NMR spectra of compound 3 in DMSO-d₆, Figures S26–S32: NMR spectra of compound 4 in DMSO-d₆, Figures S33–S38: NMR spectra of compound 5 in DMSO-d₆, Figures S39–S44: NMR spectra of compound 6 in DMSO-d₆, Figures S45–S50: NMR spectra of compound 7 in DMSO-d₆, Figures S51–S56: NMR spectra of compound 8 in DMSO-d₆, Figure S57: ¹H- and ¹³C-NMR spectra of compounds 9–13 in DMSO-d₆, Figure S58: HRESI-MS spectra of compounds 1–8, Figure S59: ESI-MS spectra of compounds 9–13, Table S1: ¹H and ¹³C NMR data for compounds 9–13 in DMSO-d₆.

Author Contributions

Y.X. performed the most experiments; C.W. prepared the draft of the manuscript; H.L. tested the α-glucosidase inhibitory activity; G.Z. performed the ECD calculations; P.F. checked the data; L.W. revised the manuscript; W.Z. designed and supervised the research and revised the final version.

Funding

This research was funded by the NSFC (Nos. 81561148012, U1501221, 81741150, U1606403), the 100 Leading Talents of Guizhou Province (fund for W. Zhu), the science and technology project of Guizhou (Grant No. QKHT Z-2014-4007) and the academician workstation of Guizhou (Grant No. QKH YSZ-2015-4009).

Conflicts of Interest

The authors declare no conflict of interest.