Discovery of aromadendrane anologues from the marine-derived fungus Scedosporium dehoogii F41-4 by NMR-guided isolation

Abstract

Eight closely biogenetically-related aromadendrane-type sesquiterpenoids, six of them new, scedogiines A–F (1–3, 6–8), together with a new polyketide scedogiine G (9) were discovered from the culture broth of the marine-derived fungus Scedosporium dehoogii F41-4 by NMR-guided isolation. Their structures were determined from extensive NMR and HR-ESI-MS data. The absolute configurations of the new sesquiterpenoids were elucidated by ECD calculation. Plausible biogenetic pathways of the aromadendrane analogues were postulated.

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1. Introduction

Aromadendranes possess a carbon skeleton of a ger-dimethylcyclopropane fused to a hydroazulene, and have been discovered in a variety of organisms, such as plants, liverworts, microorganisms, corals and sponges.¹ This group of sesquiterpenoinds has attracted much attention for their structural uniqueness and diversity as well as their wide range of biological properties including cytotoxic,² antifungal,³ antibacterial,⁴ antiviral,⁵ neuroprotective,⁶ antifouling,⁷ and other activities.^8,9 Synthetic chemists have developed various routes to construct these molecular scaffolds.^10–13 Microbiologists have utilized fungal strains as cell factories to produce these sesquiterpenoids.^14,15 This family of compounds displayed highly shielded protons in the range of δ_H −0.2–0.8 with a doublet of doublets (dd) or a doublet of doublets of doublets (ddd) splitting pattern with at least a coupling constant of about 9.6. Previously, we isolated a pair of aromadendrane diastereoisomers from a marine-derived fungus Pseudallescheria boydii F19-1.¹⁶ In our continuous screening of the fungi associated with invertebrates such as soft corals, starfishes, and sponges to obtain new and bioactive metabolites, we have isolated and purified a fungal strain authenticated as Scedosporium dehoogii F41-4 from the sponge Phyllospongia foliascens collected in the Hainan Sanya National Coral Reef Reserve, China. Scedosporium dehoogii is a member of Pseudallescheria/Scedosporium species complex, opportunistic fungal pathogens which can cause potentially fatal invasive infections in immunocompromised patients and immunocompetent individuals.¹⁷ A literature search revealed the research about the fungus Scedosporium dehoogii focusing on the identification and distribution^17–19 and none of any report on the chemical investigation up to date. The ethyl acetate extraction of the culture broth in a small-scale cultivation displayed several characteristic resonance signals of cylopropanes in the range of δ_H −0.2–0.8 in the ¹H NMR spectrum, implying the possible presence of aromadendrane sesquiterpenoids. Therefore, we determined to investigate the secondary metabolites of the fungus Scedosporium dehoogii F41-4. By tracking characteristic ¹H NMR signals in the high shield region, six new aromadendrane-type sesquiterpenoids scedogiines A–F (1–3, 6–8), two known analogues pseuboydones A, and B (4, 5), were isolated, along with a new polyketide scedogiine G (9). Here, we report the isolation, structure determination and proposed biosynthetic pathways of these compounds.

2. Results and discussion

2.1 Structural elucidation

Scedogiine A (1) showed a molecular ion peak at m/z 235.1684 [M + H]⁺ in the HRESIMS spectrum, corresponding to a molecular formula of C₁₅H₂₂O₂, indicating five degrees of unsaturation. The ¹³C NMR and DEPT spectra showed four methyls, two methylenes, five methines, and four quaternary carbons. The ¹H NMR spectrum displayed two characteristic signals of a cyclopropane ring at δ_H −0.04 (dd, 10.0, 10.0), and 0.64 (ddd, 10.0, 12.0, 5.6) for H-6 and H-7. A sp² methine at δ_H/δ_C 5.97(s)/128.4, a sp² quaternary carbon at δ_C 181.8 and a carbonyl group at δ_C 211.7 revealed the existence of an α,β-unsaturated ketone, which was confirmed by the HMBC correlations from H-2 to C-1 and C-3. Apart from these signals as described above, the remaining two sites of unsaturation are attributed to two additional rings in 1. The COSY correlations of H₃-15/H-4, H-4/H-5, H-5/H-6, H-6/H-7, H-7/H-8, H-8/H-9 revealed the heptyl moiety (C15–C4–C9). Further analysis of the HMBC correlations of H-4/C-3, H₃-15/C-3, H-5/C-3, H-5/C-1, H-5/C-1, H-9/C-10, H-9/C-1 deduced a five- and seven-membered fused ring system. The singlet methyl-14 at δ_H 1.58 was connected to the quaternary carbon C-10 from the HMBC interaction between H₃-14 and C-10. The geminal methyls were attached to the quaternary carbon C-11 position based on the HMBC correlations of H₃-12/C-11 and H₃-13/C-11. The remaining hydroxyl group was ambiguously linked to C-10 matching well with the chemical shift at δ 83.1 for the oxygenated quaternary carbon. It was supposed that cooperative influence of the electron-donating effect of two methyls, α-orientation of H-6 and steric effect of the C-10 substituents caused the highly shielded signal of H-6 at δ −0.04 (Fig. 1).

Fig. 1 Chemical structures of compounds 1–9.

The relative configuration was determined by the NOESY experiment. The NOE cross peaks of H₃-15 with H-6, H₃-12 with H-6 and H-7, H₃-14 with H-7 implied that Me-15, H-6, H-7, Me-12, and Me-14 were oriented to the same side, the NOE interaction between H-5 and H₃-13 suggested H-5 and Me-13 were placed on the other side. The absolute stereochemistry of compound 1 was achieved by ECD computed method. The ECD curve for (4R,5S,6S,7S,10R)-1 matched well with the experimental one. Therefore, the absolute configuration of compound 1 was determined as 4R, 5S, 6S, 7S, 10R.

The molecular formula of scedogiine B (2) was deduced to be C₁₅H₂₂O₂, which is the same molecular formula as 1, from the molecular ion peak at m/z 235.1688 [M + H]⁺ in the HRESIMS spectrum. Careful inspection of 1D NMR spectra of compound 2 revealed that the splitting patterns of protons and types of carbons are identical to those in 1. The only difference is the chemical shift between them, which suggested that compound 2 is a stereoisomer of 1. The planar structure of 2 was further confirmed by the ¹H-¹H COSY and HMBC spectra data. The cross-peaks of H-4 with H-6 and H₃-13, H-5 with H₃-13 and H₃-14, and H-7 with H₃-14 inferred that H-4, H-5, H-6, H-7, Me-13, and Me-14 were oriented on the same side, whereas Me-15, Me-12, and OH-10 were located on the other side. Finally, the calculated ECD spectrum for (4R,5S,6R,7R,10S)-2 had an excellent fit with the experimental one. Therefore, the absolute configuration of 2 was assigned as 4R, 5S, 6R, 7R, 10S.

The HRESIMS spectrum of scedogiine C (3) gave a molecular ion peak at m/z 273.1455 [M + Na]⁺, indicating a molecular formula of C₁₅H₂₂O₃, one more oxygen atom than that of 2. The NMR spectra of compound 3 are similar to those of compound 2. The significant difference is that the hydroxylated methylene at C-12 in 3 in place of methyl-12 in 2. The NOESY spectrum of 3 also displayed the similar relative configuration with that of 2. By ECD calculation, the absolute stereochemistry of compound 3 was assigned as 4R, 5S, 6R, 7R, 10S, 11R.

Scedogiine D (6) demonstrated a molecular ion peak at m/z 299.1615 [M + Na]⁺. From its HRESIMS spectrum, a molecular formula of C₁₇H₂₄O₃ was inferred, requiring six degrees of unsaturation. Its NMR data pattern resembles that of compound 4. The main difference is that an acetoxy group in 6 instead of a hydroxyl group in 4, in accordance with the molecular formula of 6. The NOE cross peaks of H₃-15 with H-6, H₂-12 with H-6 and H-7, H-5 with H-10 and H₃-13 suggested that Me-15, H-6, H-7, CH₂-12, and Me-14 were placed on the same orientation, whereas H-4 and Me-13 were located on the opposite direction. The absolute configuration was unambiguously elucidated as 4R, 5S, 6S, 7S, 10S, 11R by comparing the computed ECD spectrum with the experimental CD curve.

From the molecular ion peak at m/z 235.1689 [M + H]⁺ in the HRESIMS spectrum, scedogiine E (7) was found to have the same molecular formula of C₁₅H₂₂O₂ as compound 1. The NMR data are similar to those of 1. The difference is the hydroxyl group substituted at the C-8 position at δ_H/δ_C 3.74/69.4 in 7 instead of the C-10 position at δ_C 83.1 in 1. This was supported by the ¹H-¹H correlations between H-7 and H-8, and between H-8 and H₂-9. In the NOESY spectrum, the interactions of H₃-15 with H-6, H₃-12 with H-6 and H-7, H-5 with H₃-13, H₃-13 with H-8, and H-8 with H-10 indicated that Me-15, H-6, H-7, Me-12, and Me-14 were syn-oriented, while H-4, H-5, Me-13, H-8, and H-10 were on the opposite side. The absolute configuration was assigned as 4R, 5S, 6R, 7S, 8R, 10S by comparing the calculated ECD spectrum with the experimental one (Fig. 2).

Fig. 2 ¹H-¹H COSY (bold line) and the key HMBC correlations (arrows) of compounds 1, 8 and 9.

The molecular formula of scedogiine F (8) was established as C₁₅H₂₂O₂ on the analysis of the HRESIMS peak at m/z 235.1682 [M + H]⁺, implying five degrees of unsaturation. Two highly shielded proton signals at δ_H 1.00 (dd, 9.6, 11.2), and 0.78 (ddd, 9.6, 5.6, 11.6) inferred the presence of a cyclopropane ring. The ¹³C and DEPT NMR spectra displayed signals at δ_C 133.7, 164.1, and 201.9 of an α,β-unsaturated ketone. Above spectroscopic data indicated compound 8 also possessed an aromadendrane skeleton as compounds 1–7. The ¹H-¹H COSY couplings between H-2 and H₂-3 and HMBC correlations from H₃-14 to C-10, C-1, and C-9, from H₂-8 to C-9, from H-2, H-3, and H-5 to C-1 undoubtedly determined the substituted positions of the hydroxyl and α,β-unsaturated ketone groups. The NOE correlations of Me-12 with H-6 and H-7, H-6 with Me-15, H-2 with H-4, H-5 with Me-13 revealed that Me-15, Me-12, H-6, and H-7 were β-oriented, while H-2, H-4, H-5 and Me-13 were α-positioned. The calculated ECD spectrum for (2R,4S,5R,6S,7S)-8 showed an excellent fit with the experimental one. Therefore, the absolute configuration of compound 8 was determined as 2R, 4S, 5R, 6S, 7S (Fig. 3).

Fig. 3 Key NOESY correlations of compounds 1–3 and 6–8.

The molecular formula of compound 9 was established as C₂₀H₃₄O₃ based on the molecular ion peak at m/z 321.2429 [M − H]⁻ in the HRESIMS spectrum, requiring four double bond equivalents. The ¹³C NMR and DEPT spectra revealed five sp² quaternary carbon resonances at δ_C 166.2, 164.9, 162.2, 106.2, 98.1 due to the typical α-pyrone carbons in the range of unsaturated carbon, accounting for the sites of unsaturation. Thus the remaining moiety was aliphatic. The ¹H NMR spectrum showed two olefinic methyl singlets substituted on the α-pyrone ring, four aliphatic methyl doublets, and one aliphatic methyl triplet. Additionally, the ¹H NMR spectrum also showed a series of methylene and/or methine protons with close chemical shifts, which resulted in the indistinctive ¹H-¹H COSY couplings. However, a 4,6,8-trimethyldecanyl side chain was clearly inferred based on the HMBC couplings of H₃-18 with C-7 and C-8, H₃-19 with C-9, C-8 and C-10, H₃-20 with C-11, C-10, and C-12, H₃-21 with C-13, C-12 and C-14, H₃-15 with C-14 and C-13. The connection of the alkyl chain with the α-pyrone ring was confirmed from the HMBC interactions from H-8, and H₃-18 to C-6, and the weak correlation from H-7 to C-6. Unfortunately, the sample was changed into a mixture with very intricate signals, which prevented us to further stereochemistry elucidation (Fig. 4).

Fig. 4 Comparison of the experimental ECD and the calculated ECD spectra of 1 and 8.

The chemical structures of two known sesquiterpenoids were determined as pseuboydones A (4), and B (5) by comparing their spectroscopic data with the literature values (Fig. 5).

Fig. 5 Proposed biosynthetic pathways of 1–3 and 6–8.

2.2 The proposed biosynthetic pathways

Obviously, compounds 1–8 are derived from the melavonic acid pathway. Therefore, the biosynthetic pathways of these compounds were proposed as the following. The process initiated with the conversion of acetyl-coenzyme A (CoA) to the isopentenyl pyrophosphate (IPP), which generated the sesquiterpenoid precursor farnesyl pyrophosphate (FPP) catalyzed by various prenyltransferases.²⁰ The elimination of the pyrophosphate group resulted in the formation of the carbocation B, which performed dehydration, followed by cyclization to form stereoisomers D₁ and D₂. Then dehydrogenation of D₁ at C-1/C-2 and C-1/C-10 positions and D₂ at C-1/C-2 position furnished the intermediates E_1-1, E_1-2, and E₂ respectively, which were oxygenated to yield compound 8 and the intermediates F₁ and F₂ separately. Further oxygenation of F₁ at C-10, C-12, or C-8 positions and oxygenation of F₂ at C-10 or C-12 positions individually generated compounds 1, 4, 7, 2, and 5 individually. Finally, esterization of 4 and further oxygenation of 3 at C-12 position to obtain the products 6 and 3 separately. Thus it can be seen that the cyclization of carbon cation B can simultaneously occur the configuration conversion of C-6/C-7. Additionally, the generation of compounds 2 and 3 indicated that oxygenation of C-10 can also accompany the configuration alteration.

3. Experimental

3.1 General procedures

1D and 2D NMR spectra were generated on Bruker Avance II 400 spectrometers (Bruker BioSpin AG, Industriestrasse 26, Fällanden, Switzerland). The chemical shifts are corresponding to the residual solvent signals (CDCl₃: δ_H 7.26 and δ_C 77.0, acetone-d₆: δ_H 2.05, CD₃OD δ_H 3.31). The low- and high resolution ESI-MS spectra were obtained with Thermo LCQ DECA XP liquid chromatography-mass spectrometry and Thermo Fisher LTQ Orbitrap Elite High Resolution liquid chromatography-mass spectrometry respectively. Optical rotations were measured on an Anton paar MCP500 polarimeter. CD spectra were performed on a JASCO J-810 circular dichroism spectrometer (JASCO International Co. Ltd., Hachioji, Tokyo, Japan). UV spectra were recorded on a Shimadzu UV-VIS-NIR spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). IR spectra were acquired on a Fourier transform infrared (FT-IR) spectrophotometer (PerkinElmer Frontier) with an Ever-Glomid/near-IR source. Preparative HPLC was carried on a Shimadzu LC-20AT HPLC pump (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) outfitted with an SPD-20A dual λ absorbance detector (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) and a Shim-pack PRC-ODS HPLC column I(250 × 20 mm i.d., 5 μm, Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan), a Capcell pak C-18 HPLC column II(250 × 20 mm i.d., 5 μm, Shiseido Company, Limited, Nihonbashi, Japan), using a flow rate of 4.0 ml min⁻¹ at room temperature. Silica gel (SiO₂, 100–200 mesh and 200–300 mesh) for column chromatography (c.c) was purchased from Qingdao Marine Chemical Factory, Qingdao, China. Sephadex LH-20 (GE healthcare Bio-Sciences Corp., Piscataway, USA) was obtained from green herbs, Beijing, China.

3.2 Fungal material

The marine fungal Scedosporium dehoogii F41-4 was isolated from the inner tissue of the sponge Phyllospongia foliascens 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 (GenBank KP132700) by BLAST database screening provided a 99.9% match to Scedosporium dehoogii.

3.3 Culture, extraction, and isolation

This fungal strain was cultured in 500 ml Erlenmeyer flasks containing 250 ml sterile liquid nutrient media, which consisted of glucose 10 g, peptone 5 g, yeast extract 2 g, CaCO₃ 1 g and sea water 1 L. The flasks were incubated at 28 °C for 40 days. Then the fermentation broth was carried out pretreatment to sweep impurity through the filtration of cheesecloth. The fermentation broth was extracted three times with EtOAc at room temperature, and the organic solvent was evaporated in vacuum to afford the crude extract (10.32 g). The crude extract was chromatographed on silica gel (200–300 mesh) with a gradient solvent system of petroleum ether–EtOAc–MeOH (50 : 1 : 0–0 : 1 : 10, v/v/v) to give twelve major fractions (1–12). Fraction 2 (0.82 g) was subjected to a Sephadex LH-20 column and eluted with MeOH to gain three fractions (2.1, 2.2, 2.3). Compound 1 (1.30 mg) was obtained from fraction 2.3 by preparative reversed phase HPLC (MeOH–H₂O, 58 : 42, v/v, t_R = 108 min, column II). Fraction 3 (1.52 g) was chromatographed on SiO₂ with a gradient solvent system of petroleum ether-EtOAc (10 : 1–1 : 3, v/v, 100–200 mesh), yielding four fractions (3.1, 3.2, 3.3, 3.4). The compounds 8 (1.10 mg) and 9 (5.20 mg) were obtained from fraction 3.2 by the further purification using Sephadex LH-20 and preparative reversed phase HPLC (MeOH–H₂O, 80 : 20, v/v, t_R8 = 37.5 min, t_R9 = 41 min, column II). Fraction 3.3 was subjected to preparative reversed phased HPLC (MeOH–H₂O, 56 : 44, v/v, t_R = 20 min, column I) to furnish compound 3 (0.90 mg), fraction 3.4 was subjected to Sephadex LH-20 and preparative reversed phased HPLC (MeOH–H₂O, 75 : 25, v/v, t_R = 40.5 min, column I) to yield compound 6 (1.33 mg). Fraction 4 (0.56 g) was subjected to column chromatography over ODS (MeOH–H₂O, 9 : 1–1 : 9, v/v) to yield six major subfractions (4.1, 4.2, 4.3, 4.4, 4.5, 4.6), fraction 4.3 were further purified via preparative reversed phase HPLC (MeOH–H₂O, 70 : 30, v/v, t_R4 = 24 min, t_R5 = 26.5 min, column I) to give compound 4 (1.53 mg) and 5 (1.40 mg). Fraction 6 (0.52 g) was subjected to chromatographed on Sephadex LH-20 preparative reversed phase HPLC (MeOH–H₂O, 68 : 32, v/v, t_R = 20.5 min, column I) to give compound 7 (1.08 mg). Fraction 9 (1.15 g) was subjected to Sephadex LH-20 column using MeOH to afford four fractions, the second of which was chromatographed by preparative reversed phase HPLC (MeOH–H₂O, 58 : 42, v/v, t_R = 18 min, column I) to obtain compound 2 (0.95 mg).

Scedogiine A (1). Sticky liquid; [α]²⁵_D = −111.8 (c 0.1, CHCl₃). CD (MeCN): 212 (Δε +23.4), 226.5 (Δε 0), 241 (Δε −23.8), 277.2 (Δε 0). UV (MeOH) λ_max nm (log ε): 227 (3.78). IR (KBr)ν_max: 3384, 2972, 2926, 2869, 1705, 1600, 1456, 1404, 1377, 1262, 1090, 1051, 881, 802 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 2. HRESIMS m/z 235.1684 [M + H]⁺ (calcd for C₁₅H₂₃O₂, 235.1693).

Table 1 ¹³C NMR data for compounds 1–3 and 6–9 (100 MHz, CDCl₃)

No.	1	2	3	6	7	8	9
1	181.8, C	185.8, C	188.6, C	187.0, C	185.9, C	164.1, C
2	128.4, CH	124.6, CH	124.5, CH	125.7, CH	126.0, CH	75.1, CH	166.2, C
3	211.7, C	214.1, C	212.3, C	211.1, C	211.1, C	42.5, CH2	98.1, C
4	47.4, CH	50.7, CH	50.6, CH	46.9, CH	46.9, CH	32.3, CH	164.9, C
5	41.7, CH	45.6, CH	45.4, CH	44.5, CH	45.9, CH	45.4, CH	106.2, C
6	29.7, CH	32.2, CH	28.4, CH	25.8, CH	28.9, CH	31.8, CH	162.2, C
7	27.9, CH	28.7, CH	25.3, CH	25.6, CH	35.1, CH	22.9, CH	32.2, CH
8	18.0, CH2	18.6, CH2	19.7, CH2	23.1, CH2	69.4, CH	42.3, CH2	41.5, CH2
9	38.6, CH2	41.1, CH2	42.2, CH2	35.7, CH2	45.7, CH2	201.9, C	28.0, CH
10	83.1, C	71.5, C	75.0, C	40.3, CH	36.5, CH	133.9, C	46.2, CH2
11	20.8, C	20.9. C	26.7, C	24.0, C	20.4, C	26.0, C	27.3, CH
12	28.3, CH3	28.7, CH3	72.5, CH2	73.8, CH2	28.1, CH3	28.3, CH3	44.6, CH2
13	15.9, CH3	15.9, CH3	11.7, CH3	12.1, CH3	15.5, CH3	16.0, CH3	31.8, CH
14	23.7, CH3	30.0, CH3	28.0, CH3	19.9, CH3	19.5, CH3	14.5, CH3	30.6, CH2
15	10.0, CH3	17.8, CH3	18.1, CH3	10.0, CH3	9.8, CH3	16.5, CH3	11.6, CH3
16				171.3, C			8.7, CH3
17				21.0, CH3			9.8, CH3
18							19.3, CH3
19							20.7, CH3
20							19.9, CH3
21							19.1, CH3

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Table 2 ¹H NMR data for compounds 1–3 (100 MHz, CDCl₃)

No.	1	2	3
1
2	5.97 (s)	5.84 (s)	6.09 (s)
3
4	2.66 (qd, 7.2, 6.4)	2.30 (q, 7.2)	2.31 (q, 7.6)
5	2.94 (dd, 10.0, 6.4)	2.56 (d, 9.6)	2.10 (d, 9.6)
6	−0.04 (dd, 10.0, 10.0)	0.22 (dd, 9.6, 9.6)	0.45 (dd, 9.6, 9.6)
7	0.64 (ddd, 10.0, 12.0, 5.6)	0.66 (ddd, 9.6, 10.8, 6.4)	0.88 (ddd, 9.6, 5.6, 11.6)
8	1.75 (dddd, 14.8, 5.6, 5.6, 1.2)	1.79 (ddd, 14.4, 6.4, 6.4)	1.10 (m)
	1.48 (dddd, 14.8, 12.0, 12.0, 1.2)	1.55 (ddd, 14.4, 11.6, 11.6)	2.00 (m)
9	2.11 (ddd, 14.8, 5.6, 1.2)	1.98 (dd, 12.4, 6.4)	1.83 (dd, 12.0, 12.0)
	1.65 (ddd, 14.8, 12.0, 1.2)	1.69 (dd, 12.4, 12.4)	2.00 (m)
10
11
12	1.03 (s)	1.04 (s)	3.34 (s)
13	1.09 (s)	1.14 (s)	1.21 (s)
14	1.58 (s)	1.56 (s)	1.40 (s)
15	1.15 (d, 7.2)	1.14 (d, 7.2)	1.15 (d, 7.6)
16			1.94 (brs)

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Scedogiine B (2). Sticky liquid; [α]²⁵_D = −93.0 (c 0.1, CHCl₃). CD (MeCN): 200.7 (Δε 0), 214 (Δε +33.0), 228.4 (Δε 0), 245 (Δε −29.6). UV (MeOH) λ_max nm (log ε): 229 (3.85). IR (KBr)ν_max: 3403, 2959, 2926, 2869, 1690, 1601, 1568, 1457, 1374, 1265, 1174, 1150, 869, 810, 681 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 2 HRESIMS m/z 235.1688 [M + H]⁺ (calcd for C₁₅H₂₃O₂, 235.1693).

Scedogiine C (3). Sticky liquid; [α]²⁵_D = −28.8 (c 0.1, CHCl₃). CD (MeCN): 204 (Δε 0), 216 (Δε +16.7), 228.2 (Δε 0), 241 (Δε −18.8). UV (MeOH) λ_max nm (log ε): 231 (4.03). IR (KBr)ν_max: 3378, 2972, 2925, 2870, 1688, 1600, 1459, 1409, 1375, 1172, 1086, 1049, 1031, 881, 870 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 2 HRESIMS m/z 273.1455 [M + Na]⁺ (calcd for C₁₅H₂₂O₃Na, 273.1461).

Scedogiine D (6). Sticky liquid; [α]²⁵_D = −44.2 (c 0.1, CHCl₃). CD (MeCN): 190.4 (Δε 0), 210 (Δε +29.4), 223.7 (Δε 0), 238 (Δε −40.0). UV (MeOH) λ_max nm (log ε): 230 (3.84). IR (KBr)ν_max: 3418, 2956, 2927, 2871, 1738, 1705, 1601, 1457, 1379, 1246, 1229, 1048, 1028, 982 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 3. HRESIMS m/z 299.1615 [M + Na]⁺ (calcd for C₁₇H₂₄O₃Na, 299.1618).

Table 3 ¹H NMR data for compounds 6–9 (100 MHz, CDCl₃)

No.	6	7	8	9
1
2	5.84 (d, 1.6)	5.84 (d, 1.6)	4.80 (dd, 6.8, 6.8)
3			2.25 (ddd, 12.8, 6.8, 6.8)
			1.62 (ddd, 12.8, 9.6, 7.6)
4	2.63 (qd, 6.8, 6.8)	2.58 (qd, 6.8, 6.8)	2.14 (m)
5	2.62 (dd, 6.8, 10.4)	2.49 (dd, 10.0, 6.8)	2.62 (dd, 9.6, 9.6)
6	0.29 (dd, 9.6, 9.6)	0.22 (dd, 10.0, 10.0)	1.00 (dd, 9.6, 11.2)
7	0.89 (ddd, 9.6, 11.2, 5.6)	0.76 (dd, 10.0, 10.0)	0.78 (ddd, 9.6, 5.6, 11.6)	3.01 (m)
8	2.06 (m)	3.74 (ddd, 10.0, 10.0, 1.2)	2.87 (dd, 14.8, 5.6)	1.83 (ddd, 14.0, 10.0, 4.0)
	1.24 (m)		2.33 (dd, 14.8, 11.6)	1.14 (dd, 4.0, 4.0)
9	1.99 (m)	2.06 (ddd, 12.4, 4.8, 1.2)		1.32 (ddddd, 6.8)
	1.37 (ddd, 12.4, 12.4, 12.4)	1.72 (dd, 12.4, 12.4, 10.0)
10	2.34 (m)	2.46 (m)		1.11 (dddd, 6.8, 6.8, 6.8, 6.8)
				0.91 (dd, 6.8, 6.8)
11				1.55 (ddddd, 6.8)
12	3.89 (d, 11.2)	1.10 (s)	1.08 (s)	0.95 (dd, 6.8)
	3.74 (d, 11.2)
13	1.18 (s)	1.19 (s)	1.17 (s)	1.35 (ddddd, 6.8)
14	1.24 (d, 7.6)	1.26 (d, 6.8)	1.95 (dd, 1.2, 1.2)	1.09 (dddd, 6.8, 6.8, 6.8, 6.8)
				1.22 (dd, 7.6, 6.0)
15	1.14 (d, 7.6)	1.11 (d, 6.8)	1.10 (d, 7.2)	0.84 (t, 7.2)
16				2.00 (s)
17	2.04 (s)			2.00 (s)
18				1.17 (d, 6.8)
19				0.83 (d, 7.2)
20				0.73 (d, 6.4)
21				0.79 (d, 6.4)

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Scedogiine E (7). Sticky liquid; [α]²⁵_D = −35.6 (c 0.1, CHCl₃). CD (MeCN): 193.9 (Δε 0), 212 (Δε +22.5), 225.4 (Δε 0), 238 (Δε −27). UV (MeOH) λ_max nm (log ε): 224.5 (3.99), 281 (3.27). IR (KBr)ν_max: 3359, 2959, 2926, 2871, 2856, 1693, 1601, 1571, 1458, 1414, 1377, 1310, 1263, 1177, 1089, 1049, 881 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 3 HRESIMS m/z 235.1689 [M + H]⁺ (calcd for C₁₅H₂₃O₂, 235.1693).

Scedogiine F (8). Sticky liquid; [α]²⁵_D = +128.0 (c 0.1, CHCl₃). CD (MeCN): 206 (Δε 0), 228 (Δε −29.6), 245.4 (Δε 0), 262 (Δε +23.1). UV (MeOH) λ_max nm (log ε): 243 (3.63). IR (KBr)ν_max: 3393, 2957, 2926, 2871, 2856, 1711, 1645, 1567, 1455, 1415, 1377, 1303, 1091, 1052, 989, 979, 883 cm⁻¹. ¹H and ¹³C NMR data see Tables 1 and 3 HRESIMS m/z 235.1682 [M + H]⁺ (calcd for C₁₅H₂₃O₂, 235.1693).

3.4 Computational methods

The absolute configurations of compounds 1–3 and 6–8 were determined by quantum chemical calculation of ECD spectra using Gaussian 09 software.²¹ Conformational searches were performed by the MOE software using the MMFF94 molecular mechanics force field.²² Within a 2 kcal mol⁻¹ energy window. After the conformational search, 3 conformations with the lowest energy were found for 1, 2, 7 and 8, 4 for 3, 6 for 6 (Fig. S1†). 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 for the optimized conformers were calculated using the TDDFT method at the PBE1PBE/6-311++G (d, p) level in acetonitrile.^23,24 The overall theoretical ECD spectra were obtained according to the Boltzmann weighting of each conformer. The ECD spectra were generated by the SpecDis 1.6 software²⁵ using a Gaussian band shape with a 0.3 eV exponential half-width from dipole-length dipolar and rotational strengths.

4. Conclusion

Although numerous novel natural products have been obtained from a large number of marine-derived fungi, actually the discovering rate of the new entitles is still not high. Developing technologies for efficient dereplication of known compounds is still one of the major challenges for chemical investigation of fungal origin. In this paper we illustrated an example to effectively increase the discovery rate of new compounds by tracking the diagnostic resonance signals.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

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

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: The NMR spectra data of compounds 1–9. See DOI: 10.1039/c6ra21142a

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