Five New Cytotoxic Metabolites from the Marine Fungus Neosartorya pseudofischeri

1. Introduction

The fall armyworm Spodoptera frugiperda is a pest feeding on the gramineous plants occurring worldwide and causing serious damage to several economically-important crops, such as the maize, sorghum, sugarcane, as well as the cotton, cruciferous and Cucurbitaceae plants. Although other alternative methods have been investigated on the biological control, as well as the development of resistant transgenic plants, the control of S. frugiperda still relies mainly on the use of chemical pesticides, easily inducing resistance and causing severe damage to the environment and consumers. However, some natural products with higher selectivity and safety become an important potential source of new biopesticides for the control of S. frugiperda [1,2,3,4,5]. Sf9 cells are clonal isolates of S. frugiperda Sf21 cells (IPLB-SF21-AE) [6]. A cultured Sf9 cell line is commonly used for primary screening to determine insecticidal activity [7] and in biomedical research for the purpose of recombinant protein expression using insect-specific viruses called baculoviruses [8].

In recent years, we conducted research on the metabolites of marine fungi and obtained a series of novel and/or bioactive metabolites [9,10,11,12,13]. The marine fungus Neosartorya pseudofischeri was isolated from the inner tissue of a starfish Acanthaster planci collected from the South China Sea. In the previous metabolites study, we obtained three novel compounds, neosartins A–C, as well as a series of known gliotoxin analogues and diketopiperazines with potent antitumor and antibacterial activities from the culture broth extract of GlyPY (glycerol-peptone-yeast extract) and GluPY (glucose-peptone-yeast extract) media [14]. During our insecticidal activity screening against the fall armyworm S. frugiperda, we found that the crude methanol extract of the mycelia of N. pseudofischeri cultivated in GluPY showed a 65% cell growth inhibition rate against the Sf9 cell line from S. frugiperda at a concentration of 50 mg/L. The bioactivity-guided metabolites isolation afforded five novel compounds, including 5-olefin phenylpyropene A (1), 13-dehydroxyl pyripyropene A (4), deacetylsesquiterpene (7), 5-formyl-6-hydroxy-8-isopropyl-2-naphthoic acid (9) and 6,8-dihydroxy-3-((1E,3E)-penta-1,3-dien-1-yl)isochroman-1-one (10), together with eleven known compounds, phenylpyropene A (2) and C (3), pyripyropene A (5), 7-deacetylpyripyropene A (6), (1S,2R,4aR,5R,8R,8aR)-1,8a-dihydroxy-2-acetoxy-3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,4a,5,6,7,8,8a-octahydronaphthalene (8), isochaetominine C (11), trichodermamide A (12), indolyl-3-acetic acid methyl ester (13), 1-acetyl-β-carboline (14), 1,2,3,4-tetrahydro-6-hydroxyl-2-methyl-l,3,4- trioxopyrazino[l,2-a]-indole (15) and fumiquinazoline F (16) (Figure 1). Compounds 1–11 and 15 showed significant cytotoxicity against the Sf9 cells from S. frugiperda. Herein, we report the isolation, structure elucidation and the bioactivity of these compounds.

Figure 1. Structures of Compounds 1–16.

Figure 1. Structures of Compounds 1–16.

2. Results and Discussion

2.1. Structural Elucidation

5-Olefin phenylpyropene A (1) was isolated as a yellowish oil. The molecular formula was established as C₃₂H₃₆O₉ based on the analysis of ¹³C-NMR data and HR(+)ESIMS m/z 565.2448 [M + H]⁺ (calcd. for C₃₂H₃₆O₉, 565.2432), implying fifteen degrees of unsaturation (Supplementary Figure S1). The IR spectrum indicated the presence of a carbonyl group (1680 cm⁻¹) and a benzene ring (3073, 1574 and 1507 cm⁻¹). UV maxima at 232, 278 and 322 nm supported the long conjugated system containing a benzene ring. The ¹³C-NMR and DEPT spectra displayed six methyls, four methylenes, ten methines and twelve quaternary carbons. The diagnostic aryl protons at δ_H 7.81 (m, 2H) and 7.44 (m, 3H) revealed a monosubstituted benzene ring. Two alkenyl protons at δ_H 6.46 (s) and 6.36 (s) indicated there were two trisubstituted double bonds in the molecule. Three methyl group singlets at δ_H 2.16, 2.10 and 2.04 showed HMBC correlations with the carbonyl groups at δ_C 169.9, 171.1 and 170.4, respectively, confirming there are three acyloxy groups. The other three methyl group singlets at δ_H 1.57, 1.25 and 0.87 are connected with the quaternary carbons at δ_C 21.2, 24.2 and 13.3, respectively. In the ¹H–¹H COSY spectrum, the cross peaks of H-1/H-2, H-2/H-3, H-7/H-8, H-8/H-9, H-2′′(H-6′′)/H-3′′(H-5′′) and H-3′′(H-5′′)/H-4′′ revealed the partial structure of –CHCH₂CH₂–, –CHCH₂CH– and –CHCHCHCHCH– in the molecule (Figure 2). The key HMBC correlations of H-1/C-10, H-11/C-10, H-15/C-10, H-12/C-3, H-12/C-4, H-12/C-5, H-13/C-4, H-13/C-5, H-13/C-6, H-7/C-6, H-14/C-6, H-12/C-9 and H-15/C-9 deduced the sesquiterpene moiety. The cross peaks of H-13 to C-2′, C-3′ and C-4′, H-5′ to C-4′, C-6′ and C-1′′ in the HMBC spectrum confirmed the connectivity of the sesquiterpene fragment, α-pyrone and the monosubstituted phenyl ring (Table 1 and Supplementary Figures S2–S7).

Figure 2. ¹H–¹H COSY (bold lines) and the main HMBC (arrows) correlations of Compounds 1, 4, 7, 9 and 10.

Figure 2. ¹H–¹H COSY (bold lines) and the main HMBC (arrows) correlations of Compounds 1, 4, 7, 9 and 10.

Table 1. ¹H and ¹³C-NMR data of 1, 4 and 6 at 400/100 MHz, respectively, δ in ppm.

Position	1 a		4 a		6 b
	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
1	73.3, CH	4.79, dd (11.6, 4.4)	73.5, CH	4.79, dd (11.6, 4.8)	74.4, CH	4.79, dd (11.2, 5.6)
2	23.2, CH2	1.98, m; 1.75, m	22.8, CH2	1.88, m; 1.70, m	23.6, CH2	1.84, m; 1.88, m
3	35.4, CH2	2.08, m; 1.62, m	36.6, CH2	1.83, m; 1.20, ddd (13.2, 13.2, 3.2)	36.8, CH2	1.43, td (12.8, 4.8); 2.15, td (12.8, 4.8)
4	38.7, C		36.6, C		38.8, C
5	143.7, C		50.3, CH	1.60, dd (12.4, 4.8)	55.0, CH	1.66, d (3.6)
6	83.5, C		81.9, C		86.2, C
7	77.7, CH	5.22, d (12.0, 5.2)	77.5, CH	5.02, dd (11.6, 4.8)	77.6, CH	4.97, dd (12.4, 5.2)
8	24.3, CH2	1.81, m; 1.63, m	24.9, CH2	1.78, m; 1.50, m	29.0, CH2	1.60, d (12.4); 1.82, m
9	41.0, CH	1.73, m	45.1, CH	1.66, d (12.0)	46.2, CH	1.53, d (3.6)
10	40.5, C		40.2, C		41.3, C
11	64.6, CH2	3.78, d (12.0); 3.74, d (12.0)	64.7, CH2	3.78, d (12.0); 3.73, d (12.0)	64.6, CH2	3.83, d (11.6); 3.77, d (11.6)
12	24.2, CH3	1.25, s	15.5, CH3	1.00, s	17.9, CH3	1.48, s
13	111.4, C	6.36, s	16.9, CH2	2.57, dd (17.2, 4.8); 2.33, dd (17.2, 12.4)	60.6, CH	4.95, d (3.6)
14	21.2, CH3	1.57, s	15.4, CH3	1.32, s	15.9, CH3	1.76, s
15	13.3, CH3	0.87, s	13.3, CH3	0.86, s	13.3, CH3	0.92, s
2′	162.1, C		163.7, C		163.5, C
3′	100.3, C		99.8, C		100.1, C
4′	160.2, C		162.2, C		157.9, C
5′	97.4, CH	6.46, s	99.2, CH	6.43, s	147.6, CH
6′	154.9, C		155.9, C		128.4, C
1′′	131.0, C
2′′	125.6, CH	7.81, m	146.7, CH	8.99, s	147.6, CH	9.07, dd (2.4, 0.8)
3′′	128.9, CH	7.44, m	127.3, C		127.5, C
4′′	131.0, CH	7.44, m	132.8, CH	8.09, brd (8.0)	133.6, CH	8.22, ddd (8.4, 2.4, 1.6)
5′′	128.9, CH	7.44, m	123.6, CH	7.39, dd (8.0, 4.8)	124.6, CH	7.53, ddd (8.4, 4.8, 0.8)
6′′	125.6, CH	7.81, m	151.2, CH	8.66, d (4.8)	152.3, CH	8.69, dd (4.8, 1.6)
1-OCOCH3	21.1, CH3	2.04, s	21.1, CH3	2.04, s	21.0, CH3	2.00, s
1-OCOCH3	170.0, C		170.5, C		170.6, C
7-OCOCH3	21.1, CH3	2.16, s	21.3, CH3	2.15, s
7-OCOCH3	169.9, C		170.1, C
11-OCOCH3	20.8, CH3	2.10, s	20.8, CH3	2.11, s	20.7, CH3	1.99, s
11-OCOCH3	171.1, C		171.0, C		170.8, C
13-OH						4.28, brs
7-OH						4.10, brs

^{a 1}H and ¹³C-NMR data were measured in CDCl₃; ^{b 1}H and ¹³C-NMR data were measured in acetone-d₆.

Table 1. ¹H and ¹³C-NMR data of 1, 4 and 6 at 400/100 MHz, respectively, δ in ppm.

Position	1 a		4 a		6 b
	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
1	73.3, CH	4.79, dd (11.6, 4.4)	73.5, CH	4.79, dd (11.6, 4.8)	74.4, CH	4.79, dd (11.2, 5.6)
2	23.2, CH2	1.98, m; 1.75, m	22.8, CH2	1.88, m; 1.70, m	23.6, CH2	1.84, m; 1.88, m
3	35.4, CH2	2.08, m; 1.62, m	36.6, CH2	1.83, m; 1.20, ddd (13.2, 13.2, 3.2)	36.8, CH2	1.43, td (12.8, 4.8); 2.15, td (12.8, 4.8)
4	38.7, C		36.6, C		38.8, C
5	143.7, C		50.3, CH	1.60, dd (12.4, 4.8)	55.0, CH	1.66, d (3.6)
6	83.5, C		81.9, C		86.2, C
7	77.7, CH	5.22, d (12.0, 5.2)	77.5, CH	5.02, dd (11.6, 4.8)	77.6, CH	4.97, dd (12.4, 5.2)
8	24.3, CH2	1.81, m; 1.63, m	24.9, CH2	1.78, m; 1.50, m	29.0, CH2	1.60, d (12.4); 1.82, m
9	41.0, CH	1.73, m	45.1, CH	1.66, d (12.0)	46.2, CH	1.53, d (3.6)
10	40.5, C		40.2, C		41.3, C
11	64.6, CH2	3.78, d (12.0); 3.74, d (12.0)	64.7, CH2	3.78, d (12.0); 3.73, d (12.0)	64.6, CH2	3.83, d (11.6); 3.77, d (11.6)
12	24.2, CH3	1.25, s	15.5, CH3	1.00, s	17.9, CH3	1.48, s
13	111.4, C	6.36, s	16.9, CH2	2.57, dd (17.2, 4.8); 2.33, dd (17.2, 12.4)	60.6, CH	4.95, d (3.6)
14	21.2, CH3	1.57, s	15.4, CH3	1.32, s	15.9, CH3	1.76, s
15	13.3, CH3	0.87, s	13.3, CH3	0.86, s	13.3, CH3	0.92, s
2′	162.1, C		163.7, C		163.5, C
3′	100.3, C		99.8, C		100.1, C
4′	160.2, C		162.2, C		157.9, C
5′	97.4, CH	6.46, s	99.2, CH	6.43, s	147.6, CH
6′	154.9, C		155.9, C		128.4, C
1′′	131.0, C
2′′	125.6, CH	7.81, m	146.7, CH	8.99, s	147.6, CH	9.07, dd (2.4, 0.8)
3′′	128.9, CH	7.44, m	127.3, C		127.5, C
4′′	131.0, CH	7.44, m	132.8, CH	8.09, brd (8.0)	133.6, CH	8.22, ddd (8.4, 2.4, 1.6)
5′′	128.9, CH	7.44, m	123.6, CH	7.39, dd (8.0, 4.8)	124.6, CH	7.53, ddd (8.4, 4.8, 0.8)
6′′	125.6, CH	7.81, m	151.2, CH	8.66, d (4.8)	152.3, CH	8.69, dd (4.8, 1.6)
1-OCOCH3	21.1, CH3	2.04, s	21.1, CH3	2.04, s	21.0, CH3	2.00, s
1-OCOCH3	170.0, C		170.5, C		170.6, C
7-OCOCH3	21.1, CH3	2.16, s	21.3, CH3	2.15, s
7-OCOCH3	169.9, C		170.1, C
11-OCOCH3	20.8, CH3	2.10, s	20.8, CH3	2.11, s	20.7, CH3	1.99, s
11-OCOCH3	171.1, C		171.0, C		170.8, C
13-OH						4.28, brs
7-OH						4.10, brs

^{a 1}H and ¹³C-NMR data were measured in CDCl₃; ^{b 1}H and ¹³C-NMR data were measured in acetone-d₆.

The relative configuration of 1 was determined by the analysis of the NOESY spectrum. The NOE correlations between H-12/H-14 and H-12/H-15, H-1/H-9, H-7/H-9 and H-7/H-11 were observed. Consequently, H-12, H-14 and H-15 are β-oriented, while H-1, H-7 and H-9 are α-oriented (Figure 3).

Figure 3. Key NOESY correlations of Compounds 1, 4 and 7.

Figure 3. Key NOESY correlations of Compounds 1, 4 and 7.

Compounds 2 and 3 were identified as phenylpyripyropene A and C [15,16], by comparing their NMR data to the reference values. The double bond between the C-5 and C-13 positions in 1 was replaced by the saturated carbon-carbon bond in 2. Compounds 1 and 2 have triacyloxy groups at the C-1, C-7 and C-11 positions; however, Compound 3 has only one acyloxy group at the C-1 position (Supplementary Figures S8–S11).

13-Dehydroxylpyripyropene A (4) was obtained as a white powder. It showed a molecular formula of C₃₁H₃₇NO₉ determined by the ¹³C-NMR data and HR(+)ESIMS peak at m/z 568.2549 [M + H]⁺ (calcd. for C₃₁H₃₇NO₉, 568.2541) (Supplementary Figure S12). The ¹³C-NMR and DEPT spectra (Table 1) displayed six methyls, five methylenes, nine methines and eleven quaternary carbons. By comparing the NMR data with pyripyropene A (5) (Supplementary Figures S13–S20) [17], a quick identification was made revealing that C-13 of 4 was a methylene (δ_C 16.9, δ_H 2.57, 2.33), corresponding to one methine group (δ_C 60.1, δ_H 4.99) connected to one hydroxyl group in pyripyropene A (Figure 2). The relative configuration of 4 was determined on the basis of NOESY data. The NOE correlations of H-12/H-15, H-14/H-15 supported these methyl groups on the β-position of the ring system; while those correlations of H-1/H-9, H-5/H-7, H-5/H-9, H-7/H-9 and H-9/H-11 were used to place these protons on the α-position (Figure 3).

Compound 6 was elucidated as 7-deacetyl pyripyropene A (Supplementary Figures S21 and S22). 6 was once obtained by hydrolysis of pyripyropene A with 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) in 80% methanol [18]. However, this is the first report including the detailed NMR data.

Deacetylsesquiterpene (7) was isolated as a yellowish oil. The molecular formula was determined as C₁₅H₂₄O₃ from the ¹³C-NMR data and the HR(+)ESIMS peak at m/z 275.1603 [M + Na]⁺ (calcd. for C₁₅H₂₄O₃Na, 275.1618), indicating four degrees of unsaturation (Supplementary Figure S23). The strong IR absorption at 3443 cm⁻¹ indicated the presence of the hydroxyl groups. The ¹³C-NMR and DEPT spectra displayed three methyls, three methylenes, six methines and three quaternary carbons (Table 2). The quaternary carbon (δ_C 132.3) and the methine (δ_C 124.4, δ_H 5.28) formed a trisubstituted double bond. The quaternary carbon (δ_C 147.2) and the methylene (δ_C 111.0, δ_H 4.94 and 4.72) constructed a terminal double bond. Thus, Compound 7 must be bicyclic to account for the four double bond equivalents required by the molecular formula. The ¹H–¹H COSY cross peaks of H-1/H-2, H-4/H-4a, H-4a/H-5, H-5/H-6, H-6/H-7, H-7/H-8 and H-8/H-13 established two partial structures, –CHCH– and –CHCHCHCH₂CH₂CHCH₃ (Figure 2). The HMBC correlations of H-1/C-8, H-2/C-3, H-5/C-10, H-8/C-8a, H-9/C-3 and H-11/C-10 established the skeleton; three hydroxyl groups were attached to C-1 (δ_C 74.6), C-2 (δ_C 74.2) and C-8a (δ_C 73.8). The NOESY correlations of H-1/H-12, H-7a/H-12, H-6e/H-12, H-2/H-4a, H-4a/H-5, H-4a/8a-OH and H-6a/H-13 revealing 1-OH, 8a-OH, H-5 and H-13 were α-oriented; however, 2-OH was β-oriented (Figure 3 and Supplementary Figures S24–S29).

Table 2. ¹H and ¹³C-NMR data of 7, 9 and 10, δ in ppm.

Position	7 a		9 b		10 c
	δC, Type	δH, Mult., (J in Hz)	δC, Type	δH, Mult., (J in Hz)	δC, Type	δH, Mult., (J in Hz)
1	74.6, CH	3.98, d (1.2)	127.9, CH	8.92, d (1.5)	169.1, C
2	74.2, CH	4.05, s	137.4, C
3	132.3, C		129.2, CH	8.22, dd (9.0,1.5)	78.4, CH	5.13, ddd (10.0, 6.8, 4.0)
4	124.4, CH	5.28, s	121.6, CH	8.77, d (9.0)	32.5, CH2	3.00, dd (16.4, 4.0); 2.93, dd (16.4, 10.0)
4a	38.1, CH	2.67, s	127.3, C		141.7, C
5	41.4, CH	2.38, brd (12.0)	111.4, C		107.1, CH	6.24, d (2.0)
6	25.9, CH2	1.56, m; 1.33, m	167.1, C		163.4, C
7	30.7, CH2	1.48, m; 1.40, m	117.0, CH	7.21, s	101.0, CH	6.18, d (2.0)
8	31.2, CH	1.99, m	159.5, C		164.8, C
8a	73.8, C		126.3, C		100.1, C
9	20.1, CH3	1.78, s	167.6, C		127.0, CH	5.71, dd (15.2, 6.8)
10	147.2, C		195.3, CH	11.42, brs	133.3, CH	6.35, dd (15.2, 10.0)
11	22.4, CH3	1.74, brs	30.1, CH	3.90, heptet (7.0)	130.4, CH	6.10, ddd (15.2, 10.0, 1.2)
12	111.0, CH2	4.94, q (1.2 ); 4.72, s	23.4, CH3	1.45, d (7.0)	131.7, CH	5.81, dq (15.2, 6.8)
13	15.0, CH3	0.93, d (6.8)	23.4, CH3	1.45, d (7.0)	18.0, CH3	1.74, dd (6.8, 1.2)
1-OH		2.00, s
2-OH		2.00, s
2-OCOCH3
2-OCOCH3
6-OH				13.42, s		11.06, brs
8-OH						11.06, brs
8a-OH		2.00, s
COOH				10.94, brs

^{a 1}H and ¹³C-NMR data were measured at 400/100 MHz, in CDCl₃; ^{b 1}H and ¹³C-NMR data were measured at 500/125 MHz, in acetone-d₆; ^{c 1}H and ¹³C-NMR data were measured at 400/100 MHz, in DMSO-d₆.

Table 2. ¹H and ¹³C-NMR data of 7, 9 and 10, δ in ppm.

Position	7 a		9 b		10 c
	δC, Type	δH, Mult., (J in Hz)	δC, Type	δH, Mult., (J in Hz)	δC, Type	δH, Mult., (J in Hz)
1	74.6, CH	3.98, d (1.2)	127.9, CH	8.92, d (1.5)	169.1, C
2	74.2, CH	4.05, s	137.4, C
3	132.3, C		129.2, CH	8.22, dd (9.0,1.5)	78.4, CH	5.13, ddd (10.0, 6.8, 4.0)
4	124.4, CH	5.28, s	121.6, CH	8.77, d (9.0)	32.5, CH2	3.00, dd (16.4, 4.0); 2.93, dd (16.4, 10.0)
4a	38.1, CH	2.67, s	127.3, C		141.7, C
5	41.4, CH	2.38, brd (12.0)	111.4, C		107.1, CH	6.24, d (2.0)
6	25.9, CH2	1.56, m; 1.33, m	167.1, C		163.4, C
7	30.7, CH2	1.48, m; 1.40, m	117.0, CH	7.21, s	101.0, CH	6.18, d (2.0)
8	31.2, CH	1.99, m	159.5, C		164.8, C
8a	73.8, C		126.3, C		100.1, C
9	20.1, CH3	1.78, s	167.6, C		127.0, CH	5.71, dd (15.2, 6.8)
10	147.2, C		195.3, CH	11.42, brs	133.3, CH	6.35, dd (15.2, 10.0)
11	22.4, CH3	1.74, brs	30.1, CH	3.90, heptet (7.0)	130.4, CH	6.10, ddd (15.2, 10.0, 1.2)
12	111.0, CH2	4.94, q (1.2 ); 4.72, s	23.4, CH3	1.45, d (7.0)	131.7, CH	5.81, dq (15.2, 6.8)
13	15.0, CH3	0.93, d (6.8)	23.4, CH3	1.45, d (7.0)	18.0, CH3	1.74, dd (6.8, 1.2)
1-OH		2.00, s
2-OH		2.00, s
2-OCOCH3
2-OCOCH3
6-OH				13.42, s		11.06, brs
8-OH						11.06, brs
8a-OH		2.00, s
COOH				10.94, brs

^{a 1}H and ¹³C-NMR data were measured at 400/100 MHz, in CDCl₃; ^{b 1}H and ¹³C-NMR data were measured at 500/125 MHz, in acetone-d₆; ^{c 1}H and ¹³C-NMR data were measured at 400/100 MHz, in DMSO-d₆.

The formula of Compound 8 was identified as C₁₇H₂₆O₄, based on the analysis of ¹³C-NMR data and HREIMS peak at m/z 294.1828 [M]⁺ (calcd. for C₁₇H₂₆O₄, 294.1826). The NMR spectroscopic data of Compound 8 were very similar to those of Compound 7, except that the C-2 hydroxyl group in 7 was replaced by an acetoxy group in 8 (Supplementary Figures S30 and S31). Fortunately, we obtained a single crystal of 8 from the MeOH solution. In the crystal, the adjacent molecules are interlinked by a pair of strong O1-H...O4 (hydroxyl) and O4-H...O3 (carbonyl) hydrogen bonds to form a zigzag chain running parallel to the b-axis. Neighboring chains of molecules are packed closely together with the hydrophobic methyl groups pointing towards each other. The absolute configuration of 8 was determined as 1S, 2R, 4aR, 5R, 8R, 8aR with the Flack parameter value −0.16(17) by single-crystal X-ray diffraction analysis (Figure 4 and Supplementary Crystallographic Information Framework (CIF)) using Cu Kα radiation. Compound 8, named (1S,2R,4aR,5R,8R,8aR)-1,8a-dihydroxy-2-acetoxy- 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,4a,5,6,7,8,8a-octahydronaphthalene, was previously obtained as the degradation product of the natural product CJ-12662, which was obtained from the fermentation broth of Aspergillus fischeri var. thermomutatus ATCC 18,618 [19] and later also isolated from fungi Neosartorya pseudofischeri and Eurotium chevalieri [20,21]. However, the single-crystal X-ray diffraction data were never reported.

Figure 4. ORTEP (Oak Ridge Thermal Ellipsoid Plot) drawing of Compound 8.

Figure 4. ORTEP (Oak Ridge Thermal Ellipsoid Plot) drawing of Compound 8.

Compound 9 was obtained as a white powder. Its molecular formula was deduced to be C15H14O4 by analysis of the ¹³C-NMR data and HR(-)ESIMS ion at m/z 257.0817 [M − H]⁻ (calcd. for C₁₅H₁₃O₄, 257.0819), which required nine degrees of unsaturation (Supplementary Figure S32). The IR spectrum revealed the presence of a carboxyl group (3283 and 1671 cm⁻¹) and an aldehyde group (2812 cm⁻¹). Two methyl groups at δ_H 1.45 (d, J = 7.0 Hz, 6H) showed ¹H–¹H COSY correlations with the methine group at δ_H 3.90 (seven peaks, J = 7.0 Hz) and formed an isopropyl fragment (Table 2, Figure 2 and Supplementary Figures S33–S36). According to the HMBC correlations of H-1/C-2, H-1/C-8a, H-3/C-2 and H-4/C-4a, the protons at δ_H 8.92 (d, J = 1.5 Hz, H-1), 8.22 (dd, J = 9.0, 1.5 Hz, H-3) and 8.77 (d, J = 9.0 Hz, H-4) were located at the 1,3,4-position on a phenyl ring. HMBC correlations of H-1/C-2, H-1/C-8a, H-3/C-2, H-4/C-4a, H-4/C-5, H-7/C-5, H-7/C-6, H-11/C-7 and H-11/C-8 indicated the existence of a naphthyl ring, accounting for seven degrees of unsaturation. The remaining carboxyl group (δ_C 167.6, δ_H 10.94), the aldehyde (δ_C 195.3, δ_H 11.42), phenolic hydroxyl group (δ_H 13.42, and the isopropyl fragment were located on C-2, C-5, C-6 and C-8, respectively, based on the HMBC cross peaks of H-1/C-9, H-3/C-9, H-10/C-6, 6-OH/C-6, 6-OH/C-7 and H-11/C-8. The aldehyde and hydroxyl groups formed an intramolecular hydrogen bond, which accounted for the downfield resonance of the phenolic hydroxyl group. Therefore, the structure of 9 was elucidated as 5-formyl-6-hydroxy-8-isopropyl-2-naphthoic acid.

Compound 10 was isolated as a white powder. The ¹³C-NMR data and HR(-)ESIMS peak at m/z 245.0815 [M − H]⁺ (calcd. for C₁₄H₁₃O₄, 245.0819), established the molecular formula as C₁₄H₁₄O₄ (Supplementary Figure S37). The ¹³C-NMR and DEPT spectra revealed fourteen carbon resonances, including one methyl group, one methylene group, seven methine groups and five quaternary carbons. The ¹H-NMR spectrum exhibited two meta-aromatic protons at δ_H 6.24 (d, J = 2.0 Hz) and 6.18 (d, J = 2.0 Hz), indicating the presence of a tetrasubstituted aromatic ring. Two phenolic hydroxyl groups at δ_H 11.06 (brs) were connected to C-6 (δ_C 163.4) and C-8 (δ_C 164.8). Additionally, two pairs of trans-oriented olefinic protons at δ_H 5.71 (dd, J = 15.2, 6.8 Hz), 6.35 (dd, J = 15.2, 10.0 Hz), 6.10 (ddd, J = 15.2, 10.0, 1.2 Hz), 5.81 (dq, J = 15.2, 6.8 Hz), one oxymethine at δ_H 5.13 (ddd, J = 10.0, 6.8, 4.0 Hz), one methylene at δ_H 3.00 (dd, J = 16.4, 4.0 Hz), 2.93 (dd, J = 16.4, 10.0 Hz) and one methyl group at δ_H 1.74 (dd, J = 6.8, 1.2 Hz) were connected in sequence and formed a pentadienyl group based on their ¹H–¹H COSY correlations. The remnant quaternary carbon (δ_C 169.1) connected with C-8a (δ_C 100.1) and the oxymethine and formed a lactone. The HMBC correlations of H-4/C-4a, H-4/C-8a, H-5/C-6, H-7/C-8 and H-7/C-8a revealed a 3,6,8-trisubstituted isocoumarin ring system (Table 2, Figure 2 and Supplementary Figures S38–S43). The absolute configuration at C-3 remains undetermined. Therefore, Compound 10 was elucidated as 6,8-dihydroxy-3-((1E,3E)-penta-1,3- dien-1-yl)isochroman-1-one.

The other known compounds, isochaetominine C (11) [22], trichodermamide A (12) [23], indolyl-3-acetic acid methyl ester (13) [24], 1-acetyl-β-carboline (14) [25], 1,2,3,4-tetrahydro- 6-hydroxy-2-methyl-l,3,4-trioxopyrazino[l,2-a]-indole (15) [26] and fumiquinazoline F (16) [27], were identified by comparing their spectroscopic data with the literature values (Supplementary Figures S44–S55).

2.2. Proposed Biosynthetic Pathways

Tomoda et al. described the biosynthetic origin of pyripyropene A (5), which was established by feeding experiments using various [¹³C] and [¹⁴C] precursors. Pyripyropene A (5) is derived from three mevalonates, five acetates and one nicotinic acid. The pyridino-α-pyrone moiety is produced via condensation of a primer nicotinic acid with two acetates in a “head-to-tail” fashion; an all-trans farnesyl pyrophosphate is produced via the mevalonate pathway; the two parts are linked and cyclized to form the core skeleton, and then three acetyl residues from the acetates are introduced into the skeleton to yield 5 [28]. Based on the above finding, we proposed that the phenyl-α-pyrones (1–3) shared a similar biosynthetic pathway (Figure 5). Benzoic acid, instead of nicotinic acid, is one of the precursors. It is very interesting that Compound 2 was observed slowly changing into Compound 1 when stored in DMSO-d₆. The change was recorded by ¹H-NMR spectra. After 18 days, Compound 2 was changed into Compound 1 thoroughly. However, the pure Compounds 1 and 2 are stable at the temperature range from −20 °C–90 °C.

Figure 5. Proposed biosynthetic pathways of 1–3.

Figure 5. Proposed biosynthetic pathways of 1–3.

2.3. Cytotoxicity

Compounds 1–11 and 15 showed significant cytotoxicity against the Sf9 cells (Table 3). After 48 h of treatment at the concentration of 50 mg/L, the cell growth inhibition rates of compounds 2, 3, 7–9, 11 and 15 were greater than 90%. Therefore, further in-depth studies to identify the insecticidal activities in vivo and the mechanism are warranted for their development as biorational pesticides.

Table 3. The death rate (%) of Compounds 1–11 and 15 against insect cell line Sf9 (n = 3, p ≤ 0.05).

Compounds	6 h	12 h	24 h	48 h
1	65.79	68.26	76.16	85.24
2	60.75	68.86	81.21	95.01
3	72.55	72.15	73.08	93.73
4	47.53	57.41	73.21	85.37
5	41.05	54.60	58.04	71.77
6	25.58	29.46	35.30	56.03
7	79.58	81.89	90.46	98.68
8	64.52	69.33	77.98	91.87
9	57.96	65.32	69.92	90.97
10	36.47	38.25	52.36	61.67
11	63.70	69.16	71.65	94.53
15	56.97	61.43	80.33	92.52
Rotenone	69.21	79.43	90.93	98.54

Table 3. The death rate (%) of Compounds 1–11 and 15 against insect cell line Sf9 (n = 3, p ≤ 0.05).

Compounds	6 h	12 h	24 h	48 h
1	65.79	68.26	76.16	85.24
2	60.75	68.86	81.21	95.01
3	72.55	72.15	73.08	93.73
4	47.53	57.41	73.21	85.37
5	41.05	54.60	58.04	71.77
6	25.58	29.46	35.30	56.03
7	79.58	81.89	90.46	98.68
8	64.52	69.33	77.98	91.87
9	57.96	65.32	69.92	90.97
10	36.47	38.25	52.36	61.67
11	63.70	69.16	71.65	94.53
15	56.97	61.43	80.33	92.52
Rotenone	69.21	79.43	90.93	98.54

3. Experimental Section

3.1. General Experimental Procedures

Preparative HPLC was performed using a Shimadzu LC-20AT HPLC pump (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) equipped with an SPD-20A dual λ absorbance detector (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) and a Shim-pack PRC-ODS HPLC column (250 mm × 20 mm, Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). Optical rotations were measured using a Schmidt and Haensch Polartronic HNQW5 optical rotation spectrometer (SCHMIDT + HAENSCH GmbH & Co., Berlin, Germany). UV spectra were recorded on a Shimadzu UV-VIS-NIR spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). IR spectra were recorded on a PerkinElmer Frontier FT-IR spectrophotometer (PerkinElmer Inc., Waltham, MA, USA). 1D and 2D NMR spectra were recorded on Bruker Avance II 400 spectrometers (Bruker BioSpin AG, Industriestrasse 26, Fällanden, Switzerland) and a Varian INOVA500NB spectrometer (Varian Medical Systems In., Salt Lake City, UT, USA). The chemical shifts are relative to the residual solvent signals (CDCl₃: δ_H 7.26 and δ_C 77.0; acetone-d₆: δ_H 2.05 and δ_C 29.92; DMSO-d₆: δ_H 2.50 and δ_C 39.51). The low- and high-resolution EI mass spectra were obtained on Thermo DSQ and Thermo MAT95XP mass spectrometers (Thermo Fisher Scientific, Waltham, MA, USA), respectively. The low- and high-resolution ESI-MS analyses were performed with a Thermo LCQ DECA XP liquid chromatography-mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA) and a Thermo Fisher LTQ Orbitrap Elite High Resolution liquid chromatography-mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA). X-ray diffraction data were acquired on a Bruker SMART APEX CCD X-ray single-crystal diffractometer (Bruker AXS GmbH, Karlsruhe, Germany).

3.2. Fungal Material

The marine fungus N. pseudofischeri was isolated from the inner tissue of the starfish A. planci collected from the Hainan Sanya National Coral Reef Reserve, China. The fungus was identified on the basis of the DNA sequences of the ITS1-5.8S-ITS2 (Internal Transcribed Spacer, ITS) regions of their rRNA gene. The ITS gene sequence showed 100% homology with that of the fungus N. pseudofischeri in GenBank (Accession Number KF999816).

3.3. Fermentation, Extraction and Isolation

The 20 L scale-up fermentation of N. pseudofischeri was carried out in 1000 mL Erlenmeyer flasks containing 500 mL liquid culture media, which was composed of glucose 10 g, peptone 5 g, yeast extract 2 g, CaCO₃ 1 g and sea water 1 L. After 30 days of growth, the mycelia of N. pseudofischeri were extracted with methanol, and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (5.36 g). Then, the extract was separated by flash silica gel column chromatography using petroleum ether-ethyl acetate in a gradient elution (100:0–0:100, v/v), followed by ethyl acetate-methanol in a gradient elution (100:0–0:100, v/v) to give 10 fractions (F1–F10). Subfractions F4.1–F4.6 were obtained from F4 (610 mg) by reverse phase silica gel using methanol-water (30:70–100:0, v/v) gradient elution. Then, F4.3 (44 mg) was further separated by RP-HPLC using methanol-water (65:35, v/v) as the eluent to obtain Compounds 1 (6.2 mg), 4 (4.1 mg) and 5 (3.4 mg). F4.4 (32 mg) was further separated by RP-HPLC eluted with methanol-water (68:32, v/v) to obtain Compounds 2 (10.1 mg), 3 (3.5 mg), 6 (1.7 mg) and 12 (2.4 mg). F5 (157 mg) was further separated by RP-HPLC eluted with methanol-water (70:30, v/v) to obtain Compound 8 (32.6 mg). F6 (113 mg) was further separated by RP-HPLC eluted with methanol-water (60:40, v/v) to obtain 7 (12.1 mg) and 9 (2.8 mg). F7 (201 mg) was separated by RP-HPLC eluted with MeCN-water (60:40, v/v) to get Compounds 10 (4.5 mg), 11 (3.1 mg) and 16 (2.3 mg). F8 (97 mg) was further separated by RP-HPLC eluted with MeCN-water (50:50, v/v) to obtain Compounds 13 (2.0 mg), 14 (2.9 mg) and 15 (23.2 mg).

5-Olefin phenylpyropene A (1): yellowish oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$: + 129° (c = 0.1, CHCl₃); UV (MeOH) λ_max (ɛ) 234 (13,810), 322 (10,108) nm; IR ν_max 2960, 2925, 1735, 1602, 1566, 1451, 1378, 1241, 1043, 758 cm⁻¹; for ¹H and ¹³C-NMR data, see Table 1; LR(+)ESIMS m/z 565.3 [M + H]⁺. HR(+)ESIMS m/z 565.2448 [M + H]⁺ (calcd. for C₃₂H₃₇O₉, 565.2432).

13-Dehydroxylpyripyropene A (4): white powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$: +20° (c = 0.1, CHCl₃); white powder; UV (MeOH) λ_max (ɛ) 216 (18,120), 246 (12,977), 278 (4038) nm; IR ν_max 2960, 2921, 2851, 1713, 1464, 1377, 1260, 1035, 810, 721 cm⁻¹. For ¹H and ¹³C-NMR data, see Table 1; LR(+)ESIMS m/z 568.2 [M + H]⁺. HR(+)ESIMS m/z 568.2549 [M + H]⁺ (calcd. for C₃₁H₃₈NO₉, 568.2541).

7-deacetylpyripyropene A (6): white powder; for ¹H and ¹³C-NMR data, see Table 1.

Deacetylsesquiterpene (7): yellowish oil; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$: +38° (c = 0.058, MeOH); UV (MeOH) λ_max (ɛ) 212 (136) nm; IR ν_max 3443, 2959, 2926, 2856 1721, 1675, 1609, 1455, 1376, 1260, 1095, 1029, 992, 887, 756 cm⁻¹. For ¹H and ¹³C-NMR data, see Table 2; HR(+)ESIMS: m/z 275.1603 [M + Na]⁺ (calcd. for C₁₅H₂₄O₃Na, 275.1618).

(1S,2R,4aR,5R,8R,8aR)-1,8a-dihydroxy-2-acetoxy-3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,4a,5,6,7,8, 8a-octahydronaphthalene (8): white crystal. ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$: −68° (c = 0.079, MeOH); IR (MeOH) ν_max 3677, 3436, 3317, 2928, 2862, 1731, 1642, 1444, 1405, 1369, 1238, 1030, 967, 945, 927, 885, 720, 609 cm⁻¹. For ¹H and ¹³C-NMR spectra, see Supplementary Figures S30 and S31; HREIMS: m/z 294.1828 [M]⁺ (calcd. for C₁₇H₂₆O₄, 294.1826). The crystal of 8 was obtained from MeOH solution. C₁₇H₂₆O₄, M = 294.38, colorless block, Orthorhombic system, P 2₁ 2₁ 2₁ (19), a = 8.3364 (1), b = 9.3709 (2), c = 21.3150 (5) Å. V = 1665.12 (6) ų, Z = 4. D_calcd = 1.174 g/cm³, crystal size 0.41 mm × 0.45 mm × 0.43 mm, F(000) = 640, T = 173(2) K.

5-formyl-6-hydroxy-8-isopropyl-2-naphthoic acid (9): white powder; IR ν_max 3283, 3020, 2953, 2928, 2867, 2812, 2720, 1671, 1616, 1601, 1512, 1458, 1422, 1393, 1289, 1026, 1208, 1148, 1109, 1080, 1008, 985, 921, 796 cm⁻¹; for ¹H and ¹³C-NMR data, see Table 2; HR(-)ESIMS m/z 257.0817 [M − H]⁻ (calcd. for C₁₅H₁₃O₄, 257.0819).

6,8-dihydroxy-3-((1E,3E)-penta-1,3-dien-1-yl)isochroman-1-one (10): white powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$: +40° (c = 0.1, MeOH); UV (MeOH) λ_max (ɛ) 232 (6312), 268 (3427), 302 (2060) nm; IR ν_max 3528, 3400, 2953, 2926, 2855, 1656, 1622, 1460, 1374, 1247, 1110, 1003, 772 cm⁻¹. For ¹H and ¹³C-NMR data, see Table 2; HR(-)ESIMS m/z 245.0815 [M − H]⁻ (calcd. for C₁₄H₁₃O₄, 245.0819).

3.4. Bioassay

To evaluate the biological activities of these compounds, cytotoxic assays were carried out with insect cultured cell line Sf9 from S. frugiperda. Sf9 cells were maintained at 27 °C in TC-199-MK medium supplemented with 10% FCS (v/v), 1% L-glutamine 200 mmol/L and penicillin-streptomycin-neomycin solutions (v/v). The cell growth inhibition was measured by the MTT method. Cells were seeded in 96-well microtitration plates at the exponential growth phase. Different concentrations of compounds diluted with medium were added at their log-phase growth stage. Additionally, 0.2 μL of solvent (DMSO) only was added as the control (CK). The final concentration of solvent in the cultures assayed was 1%. Compounds were solubilized with DMSO at concentrations of 10 and 50 mg/L. The rotenone was used as the positive control. After 6 h, 12 h, 24 h and 48 h of treatment, 5 mg/mL MTT were dissolved in PBS, and 20 μL of this stock solution were added to the culture cells. After an additional 3 h of incubation, the medium was discarded, and the 96-well plates were dried in the air. Then, 100 μL of DMSO were added to dissolve the formazan crystals, and the absorbance was measured at 570 nm by a microplate reader (Spectramex, 190 Molecular Devices Inc., Sunnyvale, CA, USA).

The cytotoxic effect was expressed as the relative percentage of inhibition calculated as follows: cell growth inhibition rate (%) = [(A control − A treatment)/A control] × 100 [29].

4. Conclusions

In summary, sixteen compounds, including five new compounds (1, 4, 7, 9 and 10), were obtained from the mycelium of marine fungus N. pseudofischeri. Their structure types include phenylpyripyropenes, pyripyropenes, sesquiterpenoids, isocoumarin and alkaloids. Our findings provide further evidence that the marine fungus N. pseudofischeri has the tremendous potential of biosynthesis. The potent cytotoxicity of these compounds against the Sf9 cells revealed the prospect to develop biorational pesticides.