Secondary metabolites with chemical diversity from the marine-derived fungus Pseudallescheria boydii F19-1 and their cytotoxic activity

Abstract

Two aromadendrane-type sesquiterpene diastereomers pseuboydones A, B (1, 2), two diketopiperazines pseuboydones C, D (3, 4), and a cyclopiazonic acid analogue pseuboydone E (13) together with twenty known compounds (5–12, 14–25) were isolated from the culture broth of the marine-derived fungus associated with the soft coral Lobophytum crassum. The twenty-five compounds in total belong to diverse structural classes, including sesquiterpenoids, diketopiperazines, alkaloids, meroterpenoids, pyrazines, and polyketides. The structures of the new compounds were elucidated using HRMS, 1D and 2D NMR spectroscopic data, ECD calculations, and X-ray single crystal diffraction analysis. Compounds 3, 11, 16, 17, and 18 displayed significant cytotoxicity against the Sf9 cells from the fall armyworm Spodoptera frugiperda.

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1. Introduction

It is important to search for new environmentally friendly and biodegradable natural insecticides instead of using chemical pesticides which easily induce resistance and cause severe damage to the environment and humans.¹ Recently, we have initiated a program to screen and discover secondary metabolites with insecticidal activity from the marine fungi associated with invertebrates, such as sea stars and soft corals. In a previous study, several meroterpenoids, alkaloids, terpenoids, and polyketides from the fungus Neosartorya pseudofischeri associated with Acanthaster planci showed significant cytotoxicity against the Sf9 cell line derived from the fall armyworm Spodoptera frugiperda.² S. frugiperda is a major agricultural pest insect to economically important crops and forests and is used as a model organism to investigate insecticidal activity and develop pesticides.³ A cultured Sf9 cell line from S. frugiperda pupal ovarian tissue is commonly used for primary screenings to determine insecticidal activity.⁴

In our continued search for insecticidal compounds from marine-derived fungi, a fungus strain authenticated as Pseudallescheria boydii using an ITS sequence was isolated from the soft coral Lobophytum crassum collected in Hainan Sanya National Coral Reef Reserve, China. The ethyl acetate extract of the culture broth of this fungus cultivated in a GSY medium supplemented with amino acids, folic acid, and nicotinamide (1 L medium: glucose 15 g, starch 5 g, yeast extract 3 g, Phe 2 g, Met 2 g, Trp 2 g, Lys 2 g, Thr 2 g, Lys 2 g, folic acid 1 g, nicotinamide 1 g, sea salt 30 g, H₂O 1 L, and pH 7.5) showed significant insecticidal activity against the Sf9 cell line from the fall armyworm Spodoptera frugiperda with a 60% growth inhibition rate at 50 mg L⁻¹ concentration. The bioactivity-guided isolation resulted in the obtaining of five new compounds, including two aromadendrane-type sesquiterpene diastereomers pseuboydones A, B (1, 2), two diketopiperazines pseuboydones C, D (3, 4), and a cyclopiazonic acid analogue pseuboydone E (13) together with twenty known compounds (5–12, 14–25), see Fig. 1. Compounds 3, 11, 16, 17 and 18 displayed significant cytotoxicity against the Sf9 cells from S. frugiperda. Herein we report the isolation, structure determination, and cytotoxicity of these compounds.

Fig. 1 Chemical structures of compounds 1–25.

2. Results and discussion

2.1 Structural elucidation

Pseuboydone A (1) was afforded as colorless crystals. The HRESIMS spectrum displayed a strong quasi-molecular ion peak at m/z 235.16926 [M + H]⁺, corresponding to the molecular formula C₁₅H₂₂O₂, requiring five degrees of unsaturation. Absorption bands in the IR spectrum at 3471 and 1685 cm⁻¹ indicated the existence of a hydroxyl and an unsaturated carbonyl group. The ¹³C NMR and DEPT spectra displayed two sp² quaternary carbons, one sp³ quaternary carbon, one sp² methine, five sp³ methines, three sp³ methylenes and three methyls. The proton signal at δ_H 5.84 (d, 1.6), the olefinic methane at δ_C 125.3 and the olefinic quaternary carbonate δ_C 187.3 revealed the presence of a trisubstituted double bond conjugated with a carbonyl group. The NOE correlation between H-10 and H-2 suggested the Z geometry of Δ.¹ Two proton signals at δ_H 0.20 (dd, 9.6, 9.6) and 0.83 (ddd, 11.6, 9.6, 5.6) attributable to a cyclopropane were observed in the ¹H NMR spectrum. These signals as described above accounted for three unsaturation sites including an unsaturated carbonyl group. Thus the compound contained two additional rings. The proton network of the nonanyl partial structure was obviously detected in the ¹H–¹H COSY spectrum. The HMBC cross peaks from H-4 to C-3, from H-2 to C-3, C-1 and C-5, from H-10 to C-1 revealed α,β-unsaturated carbonyl was connected to the nonanyl moiety to form the five-membered and seven-membered fused ring system. The remaining methyl and oxygenated methylene must be attached to the C-11 position, which was further confirmed using the HMBC correlations from H₃-13 to C-11, C-6, and C-7, and from H₂-12 to C-7 (Fig. 2).

Fig. 2 ¹H–¹H COSY (bold line) and the key HMBC (arrows) of compounds 1, 3, 4, and 13.

The relative configuration was determined using a NOESY spectrum and X-ray single-crystal diffraction analysis. The couplings observed in the NOESY spectrum between H₃-15/H-6, H-6/H-12, H-12/H-7, H-7/H₃-14, H-5/H₃-13, H₃-5/H-10 indicated that 15-Me, H-6, H-7, H-12, and 14-Me were oriented on the same face, while H-5, 13-Me, and H-10 were placed on the opposite face (Fig. 3). The deduction was further confirmed using X-ray single-crystal diffraction analysis (Fig. 4) from the Flack parameter −0.4(3)⁵ using Cu Kα radiation. For the absolute configuration of 1, the experimental CD curve was consistent with the calculated ECD spectrum, supporting the 4R,5S,6S,7S,10S,11R-configuration of pseuboydone A (Fig. 5).

Fig. 3 Key NOESY correlations of compounds 1–2 and absolute configuration of compound 3.

Fig. 4 ORTEP drawing of compound 1.

Fig. 5 Comparison of the experimental ECD spectra of 1 and 2 with the calculated ECD spectra for four stereochemical options.

Pseuboydone B (2) was obtained as a pale yellow oil. The HRESIMS showed an [M + H]⁺ peak at m/z 235.1699. In conjunction with the ¹H and ¹³C NMR spectra, it was disclosed that the compound has the same molecular formula C₁₅H₂₂O₂ as pseuboydone A (1). Careful inspection of the NMR spectra of compound 2 revealed that the ¹H and ¹³C NMR data closely resembled those of compound 1. The only significant differences were that the chemical shifts of C-4, C-5, C-6 and C-15 at δ_C 46.7, 44.3, 25.3 and 10.0 in compound 1 were shifted downfield to δ_C 50.5, 48.9, 28.7 and 17.8 in compound 2. The NOE cross peaks of H-5 with H₃-13, H₃-13 with H-7 and H-10, and H-4 with H-6 indicated that H-4, H-5, H-6, H-7, H-10, and H₃-13 were placed on the same side and H-12, Me-13, and Me-15 were located on the opposite side. All of the above data indicated that compound 2 is the diastereomer of compound 1. With this assignment, pseuboydone B was demonstrated to have a (4R,5S,6R,7R,10S,11R)-configuration by comparing its CD spectrum with its calculated ECD spectrum. The positive effect about 217 nm and the negative one about 247 nm correspond to a 4R,5S,6R,7R,10S,11R-configuration, whereas the negative effect about 217 nm and the positive one about 247 nm reflect a 4S,5R,6S,7S,10R,11S-configuration. Therefore, the absolute configuration was determined to be (4R,5S,6R,7R,10S,11R) (Fig. 5).

Pseuboydone C (3) was obtained as a white solid. The molecular formula was deduced to be C₁₉H₂₀N₂O₃S using the HRESIMS quasi-molecular ion [M + Na]⁺ peak at m/z 379.10858. Careful analysis of the 1D NMR data disclosed that the compound was also a diketopiperazine with amino acid α-carbon substituted by heteroatoms, consisting of two amide carbonyls at δ_C 165.6, 166.5, two amide protons at δ_H 8.67, 8.58, two amide α-quaternary carbons at δ_C 82.2, 68.1, which was substituted by hydroxyl at δ_H 5.76 and methylthio at δ_H/δ_C 1.19/10.9. Ten aromatic protons in multiplicity in the region δ_H 7.16–7.24 in the ¹H NMR spectrum and ten aromatic methines in the region δ_C 126.7–130.8 and two aromatic quaternary carbons at δ_C 134.8, 135.3 in the ¹³C NMR and DEPT spectra indicated the presence of two monosubstituted phenyl rings. The HMBC cross peaks from two benzyl methylenes H₂-7 to C-6 and C-8, H₂-14 to C-3 and C-15 confirmed two phenyl alanine residues. Furthermore, the HMBC interrelations from OH-6 to C-6, C-8, and C-1, H₃-21 to C-3, NH-5 to C-4 and C-6 and from NH-2 to C-1 and C-3 established the planar structure as shown in Fig. 1.

The relative stereochemistry was determined using a NOESY spectrum. The NOE correlation between the hydroxyl and methylthio groups suggested that the hydroxyl and methylthio groups were located on the same face of the ring. The absolute stereochemistry of C-3 was proposed to be R which accounted for the uncommon upfield carbon shifts of the thiomethyl caused by the shielding effect of the phenyl ring, which was further confirmed by the NOESY cross-peaks between the methylthio group and H-18, H-19 and H-20. This deduction was consistent with the absolute configurations of the analogues, such as Sch 54794 and Sch 54796. Furthermore, it was reported that the NOESY correlations in the different solvent indicated the configurations of the compounds.⁶ Compound 3 exhibited the CD spectrum [CD (MeCN): 222.8 (Δε −11.7), 253 (Δε −3.2)], comparable to that of Sch 54794,⁷ of which the absolute configuration was determined as 3R,6R, although the ECD curves of the four configuration options (3R,6R)-, (3R,6S)-, (3S,6R)- and (3S,6S)- were identical. Therefore, the absolute configuration of compound 3 was established to be 3R,6R.

The molecular formula of pseuboydone D (4) was determined to be C₂₂H₂₄N₂O₅S₂ according to the HRESIMS pseudo-molecular ion peak at m/z 483.1019 [M + Na]⁺, requiring twelve double equivalents. The ¹H, ¹³C NMR and DEPT spectra displayed characteristic signals of methylthio diketopiperazines including two amide carbonyls at δ_C 167.9, 164.7, two amino acid α-quaternary carbons at δ_C 73.8, 73.3, and two methylthio groups substituted at α-quaternary carbons at δ_H/δ_C 2.28/15.0, 2.17/14.4. The remaining accounted for one acetyl group, four double bonds, two sp³ oxymethines, two sp³ methines, and two sp³ methylenes. One structural fragment on the one side of the diketopiperazine ring was deduced from the ¹H–¹H COSY correlations of H-7 with H-8, H-8 with H-9, and H-9 with H-10 in conjunction with the HMBC interactions from H-6, H-10, and H-12 to C-11, and from H₂-12 and H₃-21 to C-13. The acetoxyl group was located at the C-7 position based on the HMBC cross peaks from H-7 to C-23. The other structural fragment possessed the similar ¹H–¹H COSY and HMBC correlations. Actually, the chemical structure of compound 4 is very similar to that of compound 5. The only difference is that the acetyl group is at the C-7 position instead of the hydroxyl group at the C-7 position in compound 5. The large coupling constant 13.5 between H-6 and H-7 suggested that the protons at the C-6 and C-7 positions orient axially. During the isolation process, compounds 4–6 occurred in the same fraction. The absolute configurations of compounds 5 and 6 were determined as 3R,6S,7S,13R,14S,15S.^6,8 In view of the biosynthetic pathway, the absolute stereochemistry of pseuboydone D was proposed to be 3R,6S,7S,13R,14S,15S, agreeing with those of compounds 5 and 6.

Pseuboydone E (13) was afforded as a white solid. This compound had the molecular formula C₁₉H₂₀N₂O₃ as revealed using the HRESIMS peak at m/z 325.15467 [M + H]⁺. The ¹³C NMR and DEPT spectra displayed three carbonyls, eight olefinic/aromatic carbons, one sp³ quaternary carbon, two sp³ methines, two sp³ methylenes, and three methyls. The ¹H NMR spectrum displayed two methylene signals at δ_H 2.94 (17.2, 6.4), 3.21 (dd, 17.2, 6.4), 3.92 (d, 16.4), 4.02 (d, 16.4) and three singlet methyls at δ_H 1.69 (s), 1.25 (s), 2.24 (s). The HMBC cross peaks from H-11 and H-13 to C-15, from H-2 to C-3 and C-14 in conjunction with the chemical shift pattern and the amino proton at δ_H 8.10 suggested the existence of an indole ring. The ¹H–¹H COSY correlations between H-4 and H-8, H-8 and H-9 and the HMBC interactions of H-9 with C-10, H-4 with C-3, C-5, and C-7 indicated the presence of an additional fused six-membered and five-membered lactam. Two singlet methyls at δ_H 1.69 (s), 1.25 (s) were located at the C-7 positions based on the HMBC analysis of H₃-16 with C-7, C-17 and C-8, and H₃-17 with C-7 and C-8. The connection of the remaining side chain was established using the HMBC correlations from H₃-21 to C-20, H₂-19 to C-18 and C-20.

The NOE cross peaks between H-4 and H-8 in the NOESY spectrum disclosed that H-4 and H-8 were placed on the same face. Pseuboydone E (13), speradine B (14) and speradine C (15) belong to cyclopiazonic acid alkaloids. The stereochemistry of speradines B and C was assigned as 4R,8R. Furthermore, the ECD spectra with four configuration options (4R,8R)-, (4R,8S)-, (4S,8R)- and (4S,8S)- arisen from the two chiral centers in the structure were calculated and compared with the experimental CD curve. As shown in Fig. 6, the negative effect about 225 nm corresponds to a 4R,8R configuration, whereas the positive one about 227 nm reflects a 4S,8S configuration. Therefore, the absolute configuration of pseuboydone E was established as (4R,8R) by the negative cotton effect at 227.8 nm, which agreed with the experimental one.

Fig. 6 Comparison of the experimental ECD spectrum of 13 with the calculated ECD spectra for four stereochemical options.

The chemical structures of the known compounds 5–12 and 14–25 were elucidated as boydine A (5),⁸ haematocin (6),⁹ boydine B (7),⁸ phomazine B (8),¹⁰ bisdethiobis(methylthio)gliotoxin (9),¹¹ cyclo-(2,2′-dimethylthio-Phe-Phe) (10),¹² cyclo-(Phe-Phe) (11),¹³ ditryptophenaline (12),¹⁴ speradine B (15),^15,16 speradine C (16),¹⁵ cyclopiamide E (14),¹⁷ 24,25-dehydro-10,11-dihydro-20-hydro-xyaflavinin (17),¹⁸ aflavinine (18),¹⁸ β-aflatrem (19),^14,19,20 pyripyropene A (20),²¹ pseudofischerine (21),²² 4-(1-hydroxy-1-methylpropyl)-2-isobutyl-pyrazin-2(1H)-one (22),²³ 4-(1-hydroxy-1-methyl-propyl)-2-secbutylpyrazin-2(1H)-one (23),²⁴ O-methyl sterigmatocystin (24),²⁵ and asperfuran (25)²⁶ respectively by comparing their spectroscopic data with the literature values.

2.2 Cytotoxicity

Since compounds 1, 3, 11, 16, and 17–21 were isolated first in combination with the amount available, these compounds were evaluated for their cytotoxicity against the Sf9 cells (Table 3). After 48 h treatment, compounds 3, 11, 16, 17, and 18 displayed significant inhibition activity against the Sf9 cells with the mean IC₅₀ values of 0.7, 0.8, 0.9, 0.5, and 0.4 μM respectively. Especially, compounds 17 and 18 exhibited similar potent cytotoxicity to the positive control, rotenone. Further insecticidal activities in vivo and mechanism investigations are in progress.

Table 1 ¹³C NMR data for compounds 1–4 and 13^c

No.	1^a	2^a	3^b	4^a	13^a
a ^13C NMR data were measured in CDCl3.b ^13C NMR data were measured in DMSO-d6.c Recorded at 125 MHz. Others recorded at 100 MHz.
1	187.3, C	187.9, C	166.5, C	167.9, C	NH
2	125.3, CH	125.3, CH	NH	N	119.9, CH
3	211.1, C	212.8, C	68.1, C	73.8, C	106.5, C
4	46.7, CH	50.5, CH	165.6, C	164.7, C	40.4, CH
5	44.3, CH	48.9, CH	NH	N	176.3, C
6	25.3, CH	28.7, CH	82.2, C	64.2, CH	N
7	25.1, CH	25.8, CH	44.0, CH2	75.4, CH	66.1, C
8	23.2, CH2	23.6, CH2	134.8, C	128.4, CH	43.0, CH
9	35.8, CH2	35.4, CH2	130.6, CH	125.2, CH	25.2, CH2
10	40.2, CH	40.7, CH	126.7, CH	120.5, CH	128.2, C
11	26.8, C	27.4, C	127.9, CH	133.5, C	116.5, CH
12	72.5, CH2	72.7, CH2	126.7, CH	39.6, CH2	123.3, CH
13	11.5, CH3	11.5, CH3	130.6, CH	73.3, C	108.6, CH
14	19.7, CH3	20.0, CH3	43.8, CH2	69.1, CH	133.4, C
15	10.0, CH3	17.8, CH3	135.3, C	74.6, CH	126.0, C
16			130.8, CH	130.5, CH	26.5, CH3
17			126.8, CH	123.1, CH	22.1, CH3
18			127.9, CH	120.4, CH	168.5, C
19			126.8, CH	131.8, C	54.3, CH2
20			130.8, CH	38.9, CH2	201.6, C
21			10.9, CH3	15.0, CH3	30.2, CH3
22				14.4, CH3
23				170.6, C
24				21.5, CH3

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Table 2 ¹H NMR data of compounds 1–4 and 13 (J in Hz)

No.	1^a	2^a	3^b	4^a^,^c	13^a
a ^1H NMR data were measured in CDCl3.b ^1H NMR data were measured in DMSO-d6.c Recorded at 500 MHz. Others recorded at 400 MHz.
1					8.10 (brs)
2	5.84 (d, 1.6)	5.79 (d, 1.2)			7.14 (d, 1.6)
3			8.67, s
4	2.63 (m)	2.23 (q, 7.6)			4.11 (d, 7.6)
5	2.63 (m)	2.20 (d, 9.6)
6	0.20 (dd, 9.6, 9.6)	0.41 (dd, 9.6, 9.6)	8.58, s	5.14 (d, 13.5)
7	0.83 (ddd, 11.6, 9.6, 5.6)	0.85 (ddd, 11.2, 9.6, 6.4)	2.68 (d, 12.8); 3.27 (d, 12.8)	6.15 (d, 13.5)
8	1.24 (m); 2.06 (ddd, 14.0, 6.4, 5.6)	1.25 (m); 2.06 (ddd, 14.4, 6.4, 6.4)		5.60 (m)	2.70 (ddd, 7.6, 6.4, 6.4)
9	1.36 (ddd, 12.4, 12.0, 12.0); 2.00 (ddd, 12.4, 6.4, 5.2)	1.41 (d, 12.4, 12.4, 12.4); 1.96 (ddd, 12.4, 6.4, 6.0)		5.97 (m)	2.94 (dd, 17.2, 6.4); 3.21 (dd, 17.2, 6.4)
10	2.34 (m)	2.30 (m)		5.98 (m)
11			7.16–7.24 (m)		6.90 (d, 6.8)
12	3.43 (d, 11.2); 3.26 (d, 11.2)	3.38 (d, 10.8); 3.31 (d, 11.2)		3.08 (d, 16.0); 2.87 (d, 16.0)	7.16 (dd, 6.8, 6.4)
13	1.20 (s)	1.23 (s)			7.18 (d, 6.4)
14	1.24 (d, 6.8)	1.26 (d, 6.4)	2.86 (d, 13.2); 3.31 (d, 13.2)	4.84 (d, 13.0)
15	1.13 (d, 6.4)	1.14 (d, 7.6)		4.91 (d, 13.0)
16				5.74 (d, 10.0)	1.69 (s)
17				5.872 (m)	1.25 (s)
18				5.914 (m)
19			7.16–7.24 (m)		3.92 (d, 16.4); 4.02 (d, 16.4)
20				2.99 (d, 16.0); 2.88 (d, 16.0)
21			1.19, s	2.28 (s)	2.24 (s)
22				2.17 (s)
23
24				2.10 (s)
6-OH			5.76, s
12-OH	1.61 (brs)
14-SH

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Table 3 Cytotoxicity of compounds 1, 3, 11, 16–21 against insect cell line Sf9 after 48 h

Compounds	1	3	11	16	17	18	19	20	21	Rotenone
IC50 (μM)	2.2 ± 0.2	0.7 ± 0.1	0.8 ± 0.2	0.9 ± 0.1	0.5 ± 0.1	0.4 ± 0.1	>5	1.9 ± 0.3	2.1 ± 0.2	0.3 ± 0.1

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3. Experimental section

3.1 General procedures

Silica gel (SiO₂, 200–300 mesh) for the column chromatography (c.c) was purchased from Qingdao Marine Chemical Factory, Qingdao, China. SephadexLH-20 (g.c) was obtained from green herbs, Beijing, China. Optical rotations were measured on an Anton paar MCP500 polarimeter. IR spectra were acquired on a Fourier transform infrared (FT-IR) spectrophotometer (PerkinElmer Frontier) with an Ever-Glomid/near-IR source. 1D and 2D NMR spectra were recorded on a Bruker NMR spectrometer in CDCl₃ or acetone-d₆, DMSO-d₆ observed at 400 and 100 MHz, respectively. The chemical shifts are relative to the residual solvent signals (CDCl₃: δ_H 7.26 and δ_C 77.0; DMSO-d₆: δ_H 2.50 and δ_C 39.51). Preparative HPLC separation was carried out on a LC-20AT (Shimadzu) pump and a SPD-20A dual λ absorbance detector (Shimadzu) equipped with a Shiseido C18 reserved-phase column. The low- and high resolution ESI-MS spectra were measured with Thermo LCQ DECA XP liquid chromatography-mass spectrometry and Thermo Fisher LTQ Orbitrap Elite High Resolution liquid chromatography-mass spectrometry respectively. The high resolution EI mass spectra were obtained on Thermo MAT95XP mass spectrometers.

3.2 Fungal material

The marine fungus Pseudallescheria boydii was isolated from the inner tissue of the soft coral Lobophytum crassum collected from Hainan Sanya National Coral Reef Reserve, P. R. China. This fungal strain was maintained in 15% glycerol aqueous solution at −80 °C. A voucher specimen was deposited in the School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, P. R. China. Analysis of the ITS rDNA using BLAST database screening provided a 99.7% match to Pseudallescheria boydii (compared with KT223520).

3.3 Culture, extraction, and isolation

The fermentation medium was glucose 15 g L⁻¹, starch 5 g L⁻¹, yeast extract 3 g L⁻¹, Phe 2 g L⁻¹, Met 2 g L⁻¹, Trp 2 g L⁻¹, Lys 2 g L⁻¹, Thr 2 g L⁻¹, Lys 2 g L⁻¹, folic acid 1 g L⁻¹, nicotinamide 1 g L⁻¹, sea salt 30 g L⁻¹, H₂O 1 L, and pH 7.5. Fungal mycelia were cut and transferred separately to 1 L Erlenmeyer flasks, each containing 600 mL of sterilized liquid medium. The flasks were incubated at 28 °C for 40 days. 20 liters of liquid culture were filtered through cheesecloth. The culture broth was extracted three times with EtOAc and then was concentrated under reduced pressure to afford a crude extract (5.5 g). Fungal mycelia were extracted three times with MeOH and then were concentrated to afford a crude extract (7.0 g).

The culture broth extract was subjected to column chromatography over silica gel and eluted with a gradient of petroleum ether–EtOAc (10 : 0–0 : 10, v/v) followed by EtOAc–MeOH (10 : 0–0 : 10, v/v) to yield 10 fractions (Fr. 1–Fr. 10). Fr. 9 was recrystallized from MeOH to yield compound 3 (5.6 mg). The mother liquid of Fr. 9 was further purified using reversed phase semi-preparative HPLC using a mobile phase of MeOH–H₂O (65 : 35, v/v) to give compounds 9 (1.8 mg) and 11 (2.8 mg). Compound 10 (1.3 mg) was separated using a silica gel column and eluted with petroleum ether–EtOAc (10 : 3, v/v) in Fr. 6. Fr. 5 and Fr. 6 were loaded on a Sephadex LH-20 column and eluted with MeOH to afford three sub-fractions (Fr. 5.1–Fr. 5.3 and Fr. 6.1–Fr. 6.3) respectively. Then Fr. 5.3 and Fr. 6.3 were further purified respectively using reversed phase semi-preparative HPLC using a mobile phase of MeOH–H₂O (55 : 45, v/v) to obtain compounds 1 (4.3 mg) and 2 (0.7 mg). Then compounds 5 (0.9 mg), 4 (0.6 mg), and 6 (2.9 mg) were separated from Fr. 6.2 in the same manner, while compounds 22 (2.1 mg) and 23 (2.4 mg) were separated from Fr. 5.2 in another mobile phase of MeOH–H₂O (45 : 55, v/v). Further HPLC purification of Fr. 5.1 with MeOH–H₂O (66 : 34, v/v) gave compound 8 (2.2 mg). Followed by HPLC with MeOH–H₂O (55 : 45, v/v), Fr. 6.1 furnished compound 25 (2.5 mg). Fr. 7 was purified via reversed phase semi-preparative HPLC using a mobile phase of MeOH–H₂O (70 : 30, v/v) to give compound 7 (2.1 mg).

The mycelia extract was applied to a silica gel column and eluted with a gradient of petroleum ether–EtOAc (10 : 0–0 : 10, v/v) to yield 9 fractions (Fr. 1–Fr. 9). Fr. 8 was separated via a Sephadex LH-20 column and eluted with MeOH to give four sub-fractions (Fr. 8.1–Fr. 8.4). Fr. 8.3 was purified using reversed phase semi-preparative HPLC using a mobile phase of MeOH–H₂O (70 : 30, v/v) to obtain compounds 13 (0.8 mg) and 24 (2.4 mg). Further HPLC purification of Fr. 8.2 with MeOH–H₂O (65 : 35, v/v) produced compounds 12 (2.1 mg), 14 (1.8 mg), 16 (3.5 mg), and 20 (4.1 mg). Compound 19 (3.2 mg) was purified using a silica gel column and eluted with petroleum ether–EtOAc (10 : 2, v/v). Fr. 4 and Fr. 5 were separated on a Sephadex LH-20 column and eluted with MeOH to give three sub-fractions (Fr. 4.1–Fr. 4.3 and Fr. 5.1–Fr. 5.3) respectively. Then Fr. 4.3 was purified using reversed phase semi-preparative HPLC using a mobile phase of MeOH–H₂O (85 : 15, v/v) to afford compounds 17 (3.1 mg), 18 (3.4 mg), and 21 (3.2 mg). Compound 15 (2.0 mg) was separated from Fr. 5.3 in another mobile phase of MeOH–H₂O (60 : 40, v/v).

Pseuboydone A (1). Colorless crystals; [α]²⁵_D = −38.7 (c 0.1, MeOH). CD (MeCN): 191 (Δε 0), 211 (Δε +34.7), 226 (Δε 0); 240 (Δε −37.4). UV (MeOH) λ_max nm (log ε): 230 (3.97). IR (KBr) ν_max: 3471, 2957, 2900, 2875, 1685, 1593, 1404, 1381, 1275, 1213, 1178, 1111, 1034, 854, 728, 697 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 235.16926 [M + H]⁺ (calcd for C₁₅H₂₃O₂, 235.16980).

Pseuboydone B (2). Pale yellow oil; [α]²⁵_D = −44.2 (c 0.1, MeOH). CD (MeCN): 201 (Δε 0), 215 (Δε +36.7), 230 (Δε 0); 243 (Δε −28.3). ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 235.1699 [M + H]⁺ (calcd for C₁₅H₂₃O₂, 235.16980).

Pseuboydone C (3). White solid; [α]²⁵_D = −178.5 (c 0.1, MeOH). CD (MeCN): 203 (Δε 0), 235.8 (Δε −18.9), 260 (Δε 0); 276.6 (Δε +1.6). UV (MeOH) λ_max nm (log ε): 203 (4.13). IR (KBr) ν_max: 3264, 3204, 3066, 3033, 2869, 1666, 1438, 1321, 1301, 1085, 696 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 379.10858 [M + Na]⁺ (calcd for C₁₉H₂₀N₂O₃SNa, 379.10923).

Pseuboydone D (4). White amorphous powder; [α]²⁵_D = −32.7 (c 0.1, MeOH). ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 483.1019 [M + Na]⁺ (calcd for C₂₂H₂₄N₂O₅S₂Na, 483.1024).

Pseuboydone E (13). White solid; [α]²⁵_D = +46.7 (c 0.2, MeOH). CD (MeCN): 187 (Δε 0), 227.8 (Δε −11.7), 268 (Δε −3.2), 265 (Δε 0). UV (MeOH) λ_max nm (log ε): 270 (3.31), 220 (3.89). IR (KBr) ν_max: 3332, 1721, 1693, 1641, 1620, 1309, 1158, 751 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 2; HRESIMS m/z 325.15467 [M + H]⁺ (calcd for C₁₉H₂₁N₂O₃, 325.15522).

X-ray crystal data of pseuboydone A (1). Chemical_formula_weight 234.33, crystal dimensions 0.35 × 0.40 × 0.38 mm³, space group (No. 0), V = 1306.46(7) ų, Z = 4, D_c = 1.191 g cm⁻³, F₀₀₀ = 512, Bruker Smart Apex CCD, Cu Kα radiation, λ = 1.54178 Å, T = 173(2) K, 2θ_max = 67.09°, 9054 reflections collected, 2306 unique (R_int = 0.0292). The structure was solved and refined using the programs SHELXS-97 (Sheldrick, 1990) and SHELXL-97 (Sheldrick, 1997) respectively. The program X-Seed (Barbour, 1999) was used as an interface to the SHELX programs, and to prepare the figures. Final GooF = 1.074, R1 = 0.0387, wR2 = 0.0993, R indices based on 2148 reflections with I > 2 sigma(I) (refinement on F²), 154 parameters, 0 restraint. Crystallographic data (including structure factors) for pseuboydone A (1) (CCDC 1437851) reported in this paper have been deposited in the Cambridge Crystallographic Data.

3.4 Computational methods

The absolute configurations of compounds 1, 2 and 13 were determined using quantum chemical calculations of the ECD spectra using Gaussian 09 software.²⁷ Conformational analysis of the stereoisomers was performed by using the MMFF94 molecular mechanics force field.²⁸ The molecule lowest energy conformers were geometrically optimized using the DFT method at the B3LYP/6-31+G(d) level in acetonitrile. The ECD spectra of the different conformers were calculated using the TDDFT method at the PBE1PBE/6-311++G (d, p) level in acetonitrile.^29,30 The overall theoretical ECD spectra were obtained according to the Boltzmann weighting of each conformer. The ECD spectra were generated using the program SpecDis³¹ using a Gaussian band shape with a 0.3 eV exponential half-width from dipole-length dipolar and rotational strengths.

3.5 Cytotoxic activity assay

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 (vol/vol), 1% L-glutamine 200 mmol L⁻¹ and penicillin–streptomycin–neomycin solutions (vol/vol). The cell growth inhibition was measured using 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. And 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 an initial concentration of 500 μg mL⁻¹. After 48 h of treatment, 5 mg mL⁻¹ MTT was dissolved in PBS and 20 μL of this stock solution was 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 was added to dissolve the formazan crystals, and the absorbance was measured at 570 nm by using a microplate reader (Spectramex, 190 Molecular Devices Inc., USA). Each compound was evaluated in three wells in parallel and a mean IC₅₀ as well as the standard error was calculated. The data were analyzed with the SPSS 13.0 software package.

4. Conclusions

In summary, the fungus Pseudallescheria boydii F19-1 produced twenty-five compounds in total with diverse chemical structures comprising sesquiterpenoids, diketopiperazines, alkaloids, meroterpenoids, pyrazines and polyketides. The results demonstrated the enormous biosynthetic potential of this fungus strain. The richness of nitrogen-containing compounds may be caused by supplementing with amino acids. Pseuboydones A (1) and B (2) belong to a class of aromadendrane-type sesquiterpenes commonly discovered in plants and marine macroorganisms.³² This is the first reported isolation of aromadendranes from the marine-derived fungus. Pseuboydone C (3) displayed the unique hetero-atom substituted pattern. Usually, the α-positions of a diketopiperazine ring are both substituted by sulfur-containing groups. However, for this compound it is first reported that the hydroxyl and methylthio groups were substituted at the α-positions of the diketopiperazine ring respectively. Additionally, the significant cytotoxicity against the Sf9 cells of compounds 3, 11, 16, 17, and 18 indicated the potential of these compounds to develop as lead compounds for natural insecticidal agents.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

This project is financially supported by the Guangdong Provincial Science and Technology Research Program (No. 2013B021100010, 2013B021100012, 2014A020217004, 2015A020216007, and 2016A020222004), the Guangdong Department of Education Special and Innovative project for Natural Sciences (2015KTSCX073) and Innovative and Strong Project of Guangdong Pharmaceutical University (2015cxqx153), the Guangzhou Science and Technology Research Program (No. 2014J4100059), the Fundamental Research Funds for the Central Universities (No. 15ykpy05 and 14yksh01), and the instrumental analysis fund of Sun Yat-sen University (No. 0504015080000).

Notes and references

1. C. L. Cespedes, P. Torres, J. C. Marin, A. Arciniegas, A. R. de Vivar, A. L. Perez-Castorena and E. Aranda, Phytochemistry, 2004, 65, 1963–1975.
2. W. J. Lan, S. J. Fu, M. Y. Xu, W. L. Liang, C. K. Lam, G. H. Zhong, J. Xu, D. P. Yang and H. J. Li, Mar. Drugs, 2016, 14, 18–30.
3. Y. P. Deng, Y. H. Dong, V. Thodima, R. J. Clem and A. L. Passarelli, BMC Genomics, 2006, 7, 264–274.
4. Y. Zeng, Y. M. Zhang, Q. F. Weng, M. Y. Hu and G. H. Zhong, Molecules, 2010, 15, 7775–7791.
5. H. D. Flack, Acta Crystallogr., Sect. A: Found. Crystallogr., 1983, 39, 876–881.
6. M. Chu, R. Mierzwa, I. Truumees, F. Gentile, M. Patel, V. Gullo, T. M. Chan and M. S. Puar, Tetrahedron Lett., 1993, 34, 7537–7540.
7. Y. Usami, S. Aoki, T. Hara and A. Numata, J. Antibiot., 2002, 55, 655–659.
8. Q. Wu, N. Jiang, W. B. Han, Y. N. Mei, H. M. Ge, Z. K. Guo, N. S. Weng and R. X. Tan, Org. Biomol. Chem., 2014, 12, 9405–9412.
9. Y. Suzuki, Y. Esumi, T. Arie, T. Morita, H. Koshino, J. Uzawa, M. Uramoto, I. Yamaguchi and H. Takahashi, J. Antibiot., 2000, 53, 45–49.
10. F. D. Kong, Y. Wang, P. P. Liu, T. H. Dong and W. M. Zhu, J. Nat. Prod., 2013, 77, 132–137.
11. G. W. Kirby, D. J. Robins, M. A. Sefton and R. R. Talekar, J. Chem. Soc., Perkin Trans. 1, 1980, 119–121.
12. T. Rezanka, M. Sobotka, J. Spízek and K. Sigler, Anti-Infect. Agents Med. Chem., 2006, 5, 187–224.
13. K. B. Joshi and S. Verma, Tetrahedron Lett., 2008, 49, 4231–4234.
14. K. L. Sun, Y. Li, L. Guo, Y. Wang, P. P. Liu and W. M. Zhu, Mar. Drugs, 2014, 12, 3970–3981.
15. X. H. Ma, J. X. Peng, G. W. Wu and T. J. Zhu, Tetrahedron, 2015, 71, 3522–3527.
16. X. Hu, Q. W. Xia, Y. Y. Zhao, Q. H. Zheng, Q. Y. Liu, L. Chen and Q. Q. Zhang, Chem. Pharm. Bull., 2014, 62, 942–946.
17. X. Y. Xu, X. Y. Zhang, X. H. Nong, X. Y. Wei and S. H. Qi, Tetrahedron, 2015, 71, 610–615.
18. K. Nozawa, S. Sekita, M. Harada, S. I. Udagawa and K. I. Kawai, Chem. Pharm. Bull., 1989, 37, 626–630.
19. M. R. TePaske, J. B. Gloer, D. T. Wicklow and P. F. Dowd, J. Nat. Prod., 1992, 55, 1080–1086.
20. T. G. Rex, F. Janet, C. Jon, L. Albert, W. Franz and A. Werner, Tetrahedron Lett., 1980, 21, 235–238.
21. A. Odani, K. Ishihara, M. Ohtawa, H. Tomoda and S. Omura, Tetrahedron, 2011, 67, 8195–8203.
22. A. Eamvijarn, A. Kijjoa, C. Bruyère, V. Mathieu, L. Manich, F. Lefranc, A. Silva, R. Kiss and W. Herz, Planta Med., 2012, 78, 1767–1776.
23. T. Yokotsuka, M. Sasaki, T. Kikuchi and Y. Asao, J. Agric. Chem. Soc. Jpn., 1967, 41, 154–158.
24. T. Yokotsuka, Y. Asao, M. Sasaki and K. Oshita, Proceedings of the 1st US-Japan Conference on Toxic Microorganisms, Honolulu, Hawaii, Oct 7–10, 1968, pp. 133–142.
25. R. H. Cox and R. J. Cole, J. Org. Chem., 1977, 42, 112–114.
26. W. Pfefferle, H. Anke, M. Bross, B. Steffan, R. Vianden and W. Steglich, J. Antibiot., 1990, 43, 648–665.
27. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci and G. A. Petersson, Gaussian 09, Revision B.01, Gaussian, Pittsburgh, PA, 2009.
28. A. Schäfer, H. Horn and R. Ahlrichs, J. Chem. Phys., 1992, 97, 2571–2577.
29. J. P. Perdew, K. Burke and M. Ernzerhof, Phys. Rev. Lett., 1997, 78, 1396.
30. A. D. Rabuck and G. E. Scuseria, Chem. Phys. Lett., 1999, 309, 450–456.
31. T. Bruhn, A. Schaumloffel, Y. Hemberger and G. Bringmann, Chirality, 2013, 25, 243–249.
32. D. Stærk, B. Skole, F. S. Jørgensen, B. A. Budnik, P. Ekpe and J. W. Jaroszewski, J. Nat. Prod., 2004, 67, 799–805.

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Footnote

† Electronic supplementary information (ESI) available: The NMR spectra data of compounds 1–25 and crystallographic data in CIF of compound 1. See DOI: 10.1039/c6ra06661e

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