New anti-inflammatory guaianes from the Atlantic hydrotherm-derived fungus Graphostroma sp. MCCC 3A00421

Abstract

Nine new guaianes (graphostromanes A–I, 1–9) were isolated from the deep-sea-derived fungus Graphostroma sp. MCCC 3A00421, along with four known ones (10–13). The relative configurations were established mainly by detailed analysis of the NMR and HRESIMS data, while the absolute configurations were assigned using the X-ray crystallography and modified Mosher’s method. All isolates were evaluated for their inhibitory effects against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages. Graphostromanes F (6) showed remarkable inhibitory effect with an IC₅₀ value of 14.2 μM, which was even stronger than that of aminoguanidine, a positive control with an IC₅₀ value of 23.4 μM.

Introduction

Guaianes are sesquiterpenoids bearing a bicyclo[5.3.0]-decane skeleton with enormously structural diversities, including nor-guaianes¹, guaiane alkaloids^2,3, guaiane dimers^4,5, etc. They possessed a variety of intriguingly biological activities, such as antioxidant², antimalarial^6,7, antinociceptive⁷, anti-emetic⁸, antitumor^9–11, anti-inflammatory^12,13, and antibacteria^14,15. Guaianes occur mainly in terrestrial plants¹⁶. Very rarely they were found from terrestrial microorganisms and corals^17–19. However, they have never been isolated from marine microbes. In our continuing investigation on the deep-sea-derived Graphostroma sp. MCCC 3A00421²⁰, nine new (1–9) and four known (10–13) guaianes were obtained (Fig. 1). Herein, we report the isolation, structure elucidation, and anti-inflammatory activities of these compounds.

Figure 1.

Chemical structures of 1–13.

Results and Discussion

Compound 1 was isolated as a colorless oil. The molecular formula C₁₅H₂₆O₂ was established on the basis of the [M + H]⁺ ionic peak at m/z 239.2044 in its positive HRESIMS, requiring three indices of hydrogen deficiency. The ¹H NMR spectroscopic data (Table 1) showed two singlet (δ_H 1.40 and 1.76) and one doublet (δ_H 1.08) methyls, one exomethylene (δ_H 4.83, 4.72), and one oxygenated methine (δ_H 4.22). These signals were resonance in the ¹³C NMR data (Table 2) as three methyls (δ_C 14.4, 19.9, and 29.4), one exocyclic methylene (δ_C 107.8), one oxygenated methine (δ_C 77.4). Altogether, the ¹H and ¹³C NMR exhibited 15 carbons attributing to three methyls, five methylenes, five methines, and two nonprotonated carbons (one sp² at δ_C 152.7 and one oxygenated sp³ at δ_C 73.0). Since an olefinic bond accounted for one unsaturation degree, a bicyclic framework was required for 1. In the COSY spectrum, one spin coupling system from H-1 through H₂-2 to H-3/H-4/H-5/H₂-6/H-7/H₂-8/H₂-9, from H-5 to H-1, and from H-4 to H₃-15 constructed a long fragment. In the HMBC spectrum, correlations from 14-Me (δ_H 1.40) to C-1 (δ_C 53.0), C-9 (δ_C 37.8), and C-10 (δ_C 73.0) and from 12-Me to C-7, C-11 (δ_C 152.7), and C-13 (δ_C 107.8) established 1 as a guaiane sesquiterpene (Fig. 2).

Table 1.

¹H NMR spectroscopic data of 1–9 recorded at 400 MHz (δ in ppm, J in Hz within the parenthesis).

no.	1 a	2 b	3 a	4 a	5 b	6 a	7 a	8 a	9 b
1	2.93, dt (8.3, 9.9)	2.17, m	2.61, m	2.71, m	2.07, dd (9.9, 7.6)	2.51, m	2.55, m	3.20, m	2.12, m
2	2.22, m	1.73, m; 1.58, m	2.67, m; 2.26, m	3.18, ddd (19.7, 4.2, 1.3); 2.51, dd (19.4, 9.7)	4.27, q (7.2)	4.70, q (7.3)	4.64, q (6.9)	2.10, m; 1.76, m	1.72, m; 1.54, m
3	4.22, dt (9.5, 3.4)	1.73, m; 1.28, m	3.87, dt (6.8, 9.1)		1.70, m	1.96, m; 1.91, m	1.95, m	2.04, m; 1.92, td (12.8, 3.7)	1.72, m; 1.25, m
4	2.29, m	2.03, m	1.67, m	2.04, m	2.21, m	2.15, m	2.28, m		2.02, m
5	2.66, m	2.04, m	1.69, m	2.04, m	2.38, m	2.51, m	2.53, m	2.54, m	1.96, m
6	1.63, m; 1.47, td (13.3, 2.8)	1.40, m; 1.27, m	1.84, br d (12.9); 1.57, br t (11.5)	1.99, m; 1.48, m	1.42, m; 1.38, m	1.64, m	2.22, m; 1.29, m	2.63, dd (13.5, 3.2); 1.36, m	1.44, m; 1.08, m
7	2.59, td (10.3, 4.9)	2.34, m	2.10, br t (11.0)	2.29, td (10.9, 2.2)	2.19, m	2.39, m	2.27, m	2.51, m	2.05, m
8	2.09, m; 1.57, m	1.87, m; 1.43, m	1.76, m; 1.64, m	1.87, m; 1.54, m	1.75, m; 1.47, m	1.98, m; 1.68, m	2.16, m; 1.43, m	2.28, m; 1.49, m	1.87, m; 1.35, m
9	2.11, m; 1.80, m	1.91, m; 1.55, m	2.24, m; 1.98, td (13.5, 3.2)	2.19, m; 1.91, m	1.85, m; 1.69, m	2.15, m; 1.95, m	2.17, m; 1.91, m	2.21, m; 1.83, m	1.90, m; 1.51, m
11									2.30, m
12	1.76, s	1.70, s	1.76, s	1.75, s	1.70, s	4.47, t (1.4)	4.42, s	3.99, d (10.5); 3.89, d (10.5)
13	4.83, d (1.7); 4.72, m	4.66, m; 4.58, m	4.84, d (1.5); 4.75, m	4.83, m; 4.74, m	4.65, m; 4.59, m	5.47, q (1.8); 5.08, m	1.36, s	1.41, s	1.10, d (7.0)
14	1.40, s	1.18, s	1.50, s	1.30, s	1.33, s	1.61, s	1.68, s	1.46, s	1.15, s
15	1.08, d (7.2)	0.92, d (6.8)	1.20, d (6.0)	1.08, d (6.4)	0.89, d (7.2)	0.83, d (7.3)	0.93, d (7.3)	1.54, s	0.92, d (7.0)
Ac							1.99, s
2-OH							5.75, br s
10-OH				5.78, br s			5.63, br s
11-OH							5.96, br s

^aMeasured in pyridine-d₅. ^bMeasured in CD₃OD.

Table 2.

¹³C NMR spectroscopic data of compounds 1–9.

no.	1 a	2 b	3 a	4 a	5 b	6 a	7 a	8 a	9 b
1	53.0, CH	56.0, CH	49.6, CH	47.3, CH	62.7, CH	61.2, CH	62.6, CH	52.7, CH	56.1, CH
2	36.8, CH2	27.1, CH2	37.4, CH2	40.2, CH2	75.2, CH	74.1, CH	74.0, CH	25.9, CH2	27.0, CH2
3	77.4, CH	32.1, CH2	77.7, CH	220.6, C	43.1, CH2	42.5, CH2	43.0, CH2	39.6, CH2	31.8, CH2
4	48.0, CH	40.1, CH	49.0, CH	49.7, CH	37.0, CH	35.8, CH	36.3, CH	80.9, C	40.3, CH
5	44.9, CH	47.0, CH	46.0, CH	45.5, CH	46.2, CH	44.4, CH	46.0, CH	54.5, CH	47.3, CH
6	30.4, CH2	29.6, CH2	37.0, CH2	38.3, CH2	33.0, CH2	34.0, CH2	26.2, CH2	26.0, CH2	26.1, CH2
7	46.7, CH	47.1, CH	47.9, CH	44.9, CH	48.6, CH	43.7, CH	46.4, CH	45.5, CH	41.4, CH
8	29.7, CH2	29.7, CH2	32.0, CH2	32.5, CH2	31.1, CH2	31.6, CH2	26.2, CH2	25.9, CH2	28.0, CH2
9	37.8, CH2	36.8, CH2	46.2, CH2	45.0, CH2	42.0, CH2	43.8, CH2	40.7, CH2	38.0, CH2	35.4, CH2
10	73.0, C	75.2, C	73.9, C	73.3, C	75.7, C	74.3, C	73.9, C	73.4, C	75.3, C
11	152.7, C	153.4, C	152.5, C	152.1, C	153.5, C	157.9, C	73.4, C	75.3, C	47.0, CH
12	19.9, CH3	20.3, CH3	20.1, CH3	19.9, CH3	20.3, CH3	64.1, CH2	70.6, CH2	68.8, CH2	180.2, C
13	107.8, CH2	108.4, CH2	107.8, CH2	108.0, CH2	108.4, CH2	105.8, CH2	20.3, CH3	19.7, CH3	13.5, CH3
14	29.4, CH3	30.1, CH3	24.1, CH3	24.4, CH3	28.0, CH3	26.9, CH3	29.1, CH3	29.6, CH3	30.5, CH3
15	14.4, CH3	16.8, CH3	16.4, CH3	13.9, CH3	16.8, CH3	16.7, CH3	16.6, CH3	25.0, CH3	16.6, CH3
Ac							20.6, CH3 170.7, C

^aMeasured in pyridine-d₅ at 100 MHz. ^bMeasured in CD₃OD at 100 MHz.

Figure 2.

Selected COSY (), HMBC (), and NOESY () correlations of 1.

In the NOESY spectrum, correlations from H-5 (δ_H 2.66) to H-1/H-4/H-7 (δ_H 2.59) and from Me-15 to Me-14 and H-3 disclosed that H-1, H-4, H-5, and H-7 were on the same face, whereas H-3, 14-Me, and 15-Me were on the opposite side (Fig. 2). This was unambiguously confirmed by the Cu-Kα X-ray crystallography (Fig. 3). Therefore, 1 was determined to be (1R,3R,4R,5S,7R,10R)-11(13)-en-3,10-dihydroxyguaiene, and named graphostromane A.

Figure 3.

X-ray crystallographic structure of 1.

Compound 2 showed the formula molecular of C₁₅H₂₆O as established by the positive HRESIMS at m/z 223.2057 [M + H]⁺. The ¹H and ¹³C NMR spectra were nearly identical to those of 1 except that a methylene (δ_C 32.1) instead of an oxygenated methine was located at C-3 position. This observation was evidenced by the HMBC correlation of 15-Me (δ_H 0.92) to the methylene unit. On the basis of its key NOESY correlations (Fig. 4) and the similar optical rotation value (${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −28.5 for 2, while −22.0 for 1), 2 was therefore deduced to be (1R,4S,5S,7R,10R)-11(13)-en-10-hydroxyguaiene, and named graphostromane B.

Figure 4.

Key NOESY correlations of 2, 3, and 5.

Compound 3 showed the same molecule formula as that of  1 by the positive HRESIMS at m/z 261.1829 (calcd for C₁₅H₂₆O₂Na, 261.1830). Interestingly, it also exhibited almost the same ¹H and ¹³C NMR spectra, except for the shielded chemical shift of H-3 (δ_H 3.87) and its peak pattern, indicating 3 and 1 might be C-3 stereoisomers. The NOESY correlations between H-3/H-1 (δ_H 2.61), H-1/H-9a (δ_H 1.98), H-9a/H-7, and H-9b (δ_H 2.24) to H₃-14 (δ_H 1.50) deduced H-1, H-3, and H-7 were in the α orientations, while H₃-14 was β-oriented. However, it is difficult to establish the relative configurations of C-4 and C-5 positions by the NOESY correlations because of the overlap signals of H-4 (δ_H 1.67) and H-5 (δ_H 1.69). Therefore, all four possible stereoisomers including a pair of cis-fused C-4 epimers [(1R*,3S*,4R*,5S*,7R*,10R*)-3a and (1R*,3S*,4S*,5S*,7R*,10R*)-3b] and a pair of trans-fused C-4 epimers [(1R*,3S*,4R*,5R*,7R*,10R*)-3c and (1R*,3S*,4S*,5R*,7R*,10R*)-3d] were subjected for the theoretical calculation of the CMR data at mPW1PW91/6-311 + G(2d,p) level using the IEFPCM model in pyridine-d₅ by Gaussian 09. As shown in  Fig. 5, 3c displayed the smallest deviation, suggesting the relative configuration of 3 to be 1R*,3S*,4R*,5R*,7R*, and 10R*. By the modified Mosher’s method, C-3 was determined to be S configuration (Fig. 6)²¹. Based on the above evidences, 3 was then established to be (1R,3S,4R,5R,7R,10R)-11(13)-en-3,10-dihydroxyguaiene, and named graphostromane C.

Figure 5.

Calculated ¹³C NMR spectroscopic data of four possible stereoisomers of 3 (3a, 3b, 3c, and 3d) at mPW1PW91/6-311 + G(2d,p) level in pyridine-d₅.

Figure 6.

Δδ_H (δ_H^R ‒ δ_H^S) values of (R)- and (S)-MPA esters of 3, 5, and 7 in CDCl₃.

Compound 4 was established the molecular formula C₁₅H₂₄O₂ on the basis of its HRESIMS. The ¹H and ¹³C NMR spectra were very similar to those of 3 except that a ketone group (δ_C 220.6) rather than a hydroxy unit was located at C-3. The assumption was corroborated by the HMBC relationships from 15-Me (δ_H 1.08) to the carbonyl carbon. Therefore, 4 was assigned as (1R,4S,5S,7R,10R)-3-oxo-11(13)-en-10-hydroxyguaiene, and named graphostromane D.

Compound 5 exhibited the same molecular formula as that of 1 according to its positive HRESIMS at m/z 261.1833 [M + Na]⁺. Close comparison of its ¹H and ¹³C NMR spectra to those of 1 revealed a general similarity except that the hydroxy unit should be attached to C-2 instead of C-3 in 5. This was confirmed by the HMBC correlations of 15-Me to the methylene unit at δ_C 43.1 of the C-3 position. In the NOESY spectrum, cross-peaks from H-9a (δ_H 1.85) to Me-14 (δ_H 1.33) and H-2 revealed H-2 was in β orientation (Fig. 4). By the modified Mosher’s method, C-2 was determined as S-configuration (Fig. 6). Accordingly, 5 was established to be (1S,2S,4S,5S,7R,10R)-11(13)-en-2,10-dihydroxyguaiene, and named graphostromane E.

Compound 6 was assigned the molecular formula C₁₅H₂₆O₃ by the HRESIMS at m/z 277.1767 [M + Na]⁺. The ¹H and ¹³C NMR spectra were nearly identical to those of 5, except for an additional hydroxy unit at C-12 (δ_C 64.1). The assumption was evidenced by the HMBC correlations from H₂-13 (δ_H 5.47, 5.08) to C-7 (δ_C 43.7), C-11 (δ_C 157.9), and C-12. Accordingly, 6 was determined to be (1S,2S,4S,5S,7R,10R)-11(13)-en-2,10,12-trihydroxyguaiene, and named graphostromane F.

Compound 7 gave a molecular formula C₁₇H₃₀O₅ from its positive HRESIMS at m/z 337.1979 [M + Na]⁺. Its ¹H and ¹³C NMR spectra were close to those of (1S,2S,4S,5S,7R,10R)-guaiane-2,10,11,12-tetraol (10)²², except for an additional acetyl group (δ_H 1.99; δ_C 20.6, 170.7) at C-12. This was evidenced by the HMBC cross-peaks from H₂-12 (δ_H 4.42) to the carbonyl (δ_C 170.7) of the acetyl group. The absolute configuration of C-2 was established to be S on the basis of the modified Mosher’s method (Fig. 6), which was further corroborated by the X-ray single-crystal experiment (Fig. 7). Therefore, 7 was unambiguously determined to be (1S,2S,4S,5S,7R,10R,11R)-12-acetyl-2,10,11-trihydroxyguaiane, and named graphostromane G.

Figure 7.

X-ray crystallographic structure of 7.

Compound 8 had the molecular formula of C₁₅H₂₈O₄ as established by its HRESIMS. Analysis of the 1D and 2D NMR spectra established the structure of 8 was closely related to 10, except that the hydroxy group was located at C-4 instead of C-2. This was evidenced by the HMBC correlations from 15-Me (δ_H 1.54) to C-3 (δ_C 39.6), C-4 (δ_C 80.9), and C-5 (δ_C 54.5). The NOE cross-peaks from H-2a (δ_H 2.10) to 14-Me (δ_H 1.46) and 15-Me revealed that 4-OH was in α-oriented. Therefore, 8 was identified to be (1R,4R,5R,7R,10R)-4,10,11-trihydroxyguaiane, and named graphostromane H.

Compound 9 was assigned the molecular formula C₁₅H₂₆O₃ on the basis of the HRESIMS at m/z 277.1783 [M + Na]⁺. Its ¹H and ¹³C NMR spectra were very similar to those of (1R,4S,5S,7R,10R,11S)-guaiane-10,11,12-triol (11)¹⁵ except that a hydroxy group was absent at C-11, while a carboxyl moiety (δ_C 180.2) instead of an oxymethylene was attached to C–11. This was corroborated by the HMBC correlations form 13-Me (δ_H 1.10) to C-7 (δ_C 41.4), C-11 (δ_C 47.0), and C–12 (δ_C 180.2). By the theoretical calculation of CMR spectrum, C-11 was assigned as R-configuration (Fig. 8). Therefore, 9 was established to be (1R,4S,5S,7R,10R,11R)-10-hydroxyguai-12-oic acid, and named graphostromane I.

Figure 8.

Calculated ¹³C NMR spectroscopic data of a pair of C-11 epimers of (1R,4S,5S,7R,10R,11S)-9a and (1R,4S,5S,7R,10R,11R)-9b at mPW1PW91/6-311 + G(2d,p) level in CD₃OD.

By comparison of the NMR, MS, and OR data with those published in the literature, four known guaianes were identified as (1S,2S,4S,5S,7R,10R)-guaiane-2,10,11,12-tetraol (10)²², (1R,4S,5S,7R,10R,11S)-guaiane-10,11,12-triol (11)¹⁵, (1R,3S,4R,5S,7R,10R,11S)-guaiane-3,10,11,12-tetraol (12)¹⁵, and (1R,4S,5S,7S,9R,10S,11R)-guaiane-9,10,11,12-tetraol (13)¹⁵.

All isolates were evaluated for their anti-inflammatory activities against LPS-induced NO production in RAW264.7 macrophages (Table 3). Compound 6 exhibited remarkable anti-inflammatory activity with an IC₅₀ value of 14.2 μM, which was stronger than that of the positive control, aminoguanidine, with an IC₅₀ of 23.4 μM. In addition, 4, 9, and 13 showed weak anti-inflammatory activities with IC₅₀ values of 72.9, 79.1, and 88.2 μM, respectively.

Table 3.

Anti-inflammatory activities of compounds 1–13 against LPS-stimulated NO production by in RAW 264.7 macrophages.

Compounds	IC50 (μM)	CC50 (μM)
1	310.1	>50
2	103.2	>50
3	112.6	>50
4	72.9	>50
5	138.2	>50
6	14.2	>50
7	101.0	>50
8	165.8	>50
9	79.1	>50
10	150.4	>50
11	141.0	>50
12	122.4	>50
13	88.2	>50
Aminoguanidinea	23.4	>50

^aPositive control.

In conclusion, chemical investigation on the deep-sea-derived fungus Graphostroma sp. MCCC 3A00421 led to the isolation of 9 new (graphostromanes A–I, 1–9) and 4 known (10–13) guaianes. They are first examples of guaiane sesquiterpenoids reported from the marine-derived fungi. Additionally, 6 showed potent anti-inflammatory activity against LPS-induced NO production in RAW264.7 macrophages, indicating its potential usage for anti-inflammatory drugs.

Materials and Methods

General Experimental Procedures

An automatic polarimeter Rudolph IV Autopol was used for recording optical rotation data at 25 °C. A Xevo G2 Q-TOF mass spectrometer was used for measuring HRESIMS. A Bruker Avance 400 MHz NMR spectrometer was used for measuring ¹H, ¹³C, HSQC, COSY, HMBC, and NOESY spectra. Chemical shifts (δ) were expressed in ppm referring to the solvent peaks, and coupling constants are in Hz. A Bruker D8 Advance X-ray single-crystal diffractometer was used for measuring X-ray data with Cu Kα radiation. Column chromatography (CC) were performed on Sephadex LH-20 (18–110 μm, Pharmacia, Uppsala, Sweden), silica gel (100–200 or 200–300 mush, Qingdao Marine Chemistry Co. Ltd, Qingdao, China), and ODS (50 μm, Daiso, Japan). TLC precoated silica gel plates (GF254, Qingdao Marine Chemistry Co. Ltd, Qingdao, China) were used for TLC detection. All chemical reagents used were analytical grade.

Fungal Identification and Fermentation

The fungus Graphostroma sp. MCCC 3A00421 was isolated from a hydrothermal sulfide deposit in August 2012 from the Atlantic Ocean (W 13.36°, S 15.17°) at a depth of −2721 m. It was identified to be Graphostroma genus on the basis of comparison of its ITS1-5.8S-ITS2 rRNA gene sequence (KM190888) with those deposited in GenBank of the NCBI using a BLAST searching tool. The voucher strain was deposited at the Marine Culture Collection of China (MCCC) with the accession number MCCC 3A00421.

The working strain was cultured on a PDA plate medium under 25 °C for 3 days. Then the fresh mycelia were inoculated into 30 Erlenmeyer flasks (1 L), each containing 120 mL distilled water and 80 g rice, and then statically fermented for 28 days at 25 °C.

Extraction, Isolation, and Purification

After 28 days, the fermented cultures were extracted with EtOAc for three times. The EtOAc solution was evaporated under reduced pressure to get an organic extract, which was then partitioned between MeOH and petroleum ether (PE). The MeOH fraction was evaporated to get the defatted extract (7.0 g), which was separated by column chromatography (CC) over ODS eluting with gradient MeOH-H₂O (5→100%) to yield 24 fractions (F1–F24). Fraction F7 (624 mg) was subjected to CC over Sephadex LH-20 (MeOH), followed by repeated CC on silica gel (CHCl₃-MeOH, 15:1; EtOAc-MeOH, 50:1) to yield 10 (48.4 mg) and 12 (10.2 mg). Fraction F9 (420 mg) was purified by CC over Sephadex LH-20 (MeOH) to get three subfractions (SF9-1-9-3). SF9-1 was purified by CC over silica gel using PE-acetone (5:1), followed by Prep. TLC (CHCl₃-MeOH, 8:1) to provide 7 (12.6 mg). SF9-3 was subjected to CC over silica gel (CHCl₃-MeOH, 15:1) to afford 8 (8.2 mg). Compounds 6 (18.8 mg) and 13 (24.2 mg) were isolated from fraction F10 (603 mg) by CC over Sephadex LH-20 (MeOH) and silica gel (PE-acetone, 3:1; PE-EtOAc, 3:1→1:1). Fraction F14 (389 mg) was subjected to CC over silica gel using gradient PE-EtOAc to give seven subfractions (SF14-1‒SF14-7). SF14-2 was chromatographed on Sephadex LH-20 (MeOH) to yield 4 (26.0 mg). SF14-3 was purified by Prep. TLC (PE-acetone, 2:1) to get 1 (14.7 mg). Compound 3 (10.2 mg) was isolated from SF14-5 by Prep. TLC (EtOAc-acetone, 30:1). SF14-6 was separated by CC over Sephadex LH-20 (MeOH) and silica gel (CHCl₃-MeOH, 20:1) to yield 11 (39.2 mg). Fraction F16 (462 mg) was subjected to CC on Sephadex LH-20 (MeOH) and silica gel (CHCl₃-MeOH, 100:1→1:1; EtOAc-MeOH, 200:1→10:1; and PE-acetone, 5:1) to yield 2 (23.0 mg), 5 (15.5 mg), and 9 (11.6 mg).

Graphostromane A (1): colorless oil; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −22.0 (c 0.58, MeOH); ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 239.2044 [M + H]⁺ (calcd for C₁₅H₂₇O₂, 239.2011).

Graphostromane B (2): colorless oil; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −28.5 (c 2.16, MeOH); ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 223.2057 [M + H]⁺ (calcd for C₁₅H₂₇O, 223.2062).

Graphostromane C (3): colorless oil; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −8.5 (c 0.25, MeOH); ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 261.1829 [M + Na]⁺ (calcd for C₁₅H₂₆O₂Na, 261.1830).

Graphostromane D (4): colorless oil; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +15.4 (c 0.73, MeOH); ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 237.1851 [M + H]⁺ (calcd for C₁₅H₂₅O₂, 237.1855).

Graphostromane E (5): colorless oil; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +8.13 (c 0.39, MeOH); ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 261.1833 [M + Na]⁺ (calcd for C₁₅H₂₆O₂Na, 261.1830).

Graphostromane F (6): colorless oil; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +3.41 (c 0.44, MeOH); ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 277.1767 [M + Na]⁺ (calcd for C₁₅H₂₆O₃Na, 277.1780).

Graphostromane G (7): colorless oil; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ +6.0 (c 0.56, MeOH); ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 337.1979 [M + Na]⁺ (calcd for C₁₇H₃₀O₅Na, 337.1991).

Graphostromane H (8): colorless oil; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −21.0 (c 0.25, MeOH); ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 295.1878 [M + Na]⁺ (calcd for C₁₅H₂₈O₄Na, 295.1885).

Graphostromane I (9): colorless oil; ${\left[\alpha \right]}_{\mathrm{D}}^{25}$ −10.3 (c 0.12, MeOH); ¹H and ¹³C NMR data, Tables 1 and 2; HRESIMS m/z 277.1783 [M + Na]⁺ (calcd for C₁₅H₂₆O₃Na, 277.1780).

Compound 10: ¹³C NMR (CD₃OD, 100 MHz) δ_C 63.4 (CH-1), 75.1 (CH-2), 43.3 (CH₂-3), 37.3 (CH-4), 47.3 (CH-5), 25.8 (CH₂-6), 45.7 (CH-7), 26.4 (CH₂-8), 39.5 (CH₂-9), 75.4 (C-10), 76.6 (C-11), 69.2 (CH₂-12), 19.0 (Me-13), 29.6 (Me-14), 16.7 (Me-15).

Preparation of (R)- and (S)-MPA Esters of Compounds 3, 5, and 7

Compound 3 (2.0 mg) was dissolved in CHCl₃ (600 µL). Then (R)-MPA (2.5 mg), DCC (2.5 mg), and DMAP (2.5 mg) were added. After stirred 16 h at room temperature, the reactive products were subjected to CC over silica gel (PE-acetone, 3:1) to give the R-MPA ester 3a (1.7 mg). Similarly, the S-MPA ester 3b (1.9 mg) was obtained from (S)-MPA. Analogue treatment of compounds 5 and 7 separately with (R)-MPA and (S)-MPA obtained (R)-MPA esters (5a and 7a) and (S)-MPA esters (5b and 7b), respectively.

(R)-MPA ester of 3 (3a): ¹H NMR (CDCl₃, 400 MHz) δ_H 7.32–7.47 (5 H, m, phenyl protons), 4.78 (1 H, s, CH of MPA), 3.43 (3 H, s, OMe of MPA), 2.25 (1 H, m, H-1), 1.44 (1 H, m, H-2a), 2.22 (1 H, m, H-2b), 4.68 (1 H, m, H-3), 1.61 (1 H, m, H-4), 1.66 (1 H, m, H-5), 1.36 (1 H, m, H-6a), 1.69 (1 H, m, H-6b), 1.96 (1 H, dt, J = 2.2, 11.5 Hz, H-7), 1.46 (1 H, m, H-8a), 1.71 (1 H, m, H-8b), 1.63 (1 H, m, H-9a), 1.85 (1 H, ddd, J = 2.6, 4.7, 8.1 Hz, H-9b), 1.70 (3 H, s, Me-12), 4.62 (1 H, br s, H-13a), 4.65 (1 H, br s, H-13b), 1.18 (3 H, s, Me-14), 0.91 (3 H, d, J = 6.2 Hz, Me-15).

(S)-MPA ester of 3 (3b): ¹H NMR (CDCl₃, 400 MHz) δ_H 7.33–7.47 (5 H, m, phenyl protons), 4.77 (1 H, s, CH of MPA), 3.44 (3 H, s, OMe of MPA), 2.28 (1 H, m, H-1), 1.64 (1 H, m, H-2a), 2.34 (1 H, m, H-2b), 4.63 (1 H, m, H-3), 1.51 (1 H, m, H-4), 1.61 (1 H, m, H-5), 1.34 (1 H, m, H-6a), 1.65 (1 H, m, H-6b), 1.93 (1 H, t, J = 10.8 Hz, H-7), 1.48 (1 H, m, H-8a), 1.71 (1 H, m, H-8b), 1.64 (1 H, m, H-9a), 1.89 (1 H, m, H-9b), 1.69 (3 H, s, Me-12), 4.61 (1 H, br s, H-13a), 4.64 (1 H, br s, H-13b), 1.26 (3 H, s, Me-14), 0.69 (3 H, d, J = 6.5 Hz, Me-15).

(R)-MPA ester of 5 (5a): ¹H NMR (CDCl₃, 400 MHz) δ_H 7.32–7.47 (5 H, m, phenyl protons), 4.78 (1 H, s, CH of MPA), 3.43 (3 H, s, OMe of MPA), 2.35 (1 H, m, H-1), 5.25 (1 H, m, H-2), 1.51 (1 H, m, H-3a), 1.64 (1 H, m, H-3b), 2.16 (1 H, m, H-4), 2.30 (1 H, m, H-5), 1.23 (1 H, m, H-6a), 1.45 (1 H, m, H-6b), 2.11 (1 H, m, H-7), 1.45 (1 H, m, H-8a), 1.75 (1 H, m, H-8b), 1.67 (1 H, m, H-9a), 1.82 (1 H, m, H-9b), 1.69 (3 H, s, Me-12), 4.61 (1 H, br s, H-13a), 4.65 (1 H, br s, H-13b), 1.22 (3 H, s, Me-14), 0.87 (3 H, d, J = 7.0 Hz, Me-15).

(S)-MPA ester of 5 (5b): ¹H NMR (CDCl₃, 400 MHz) δ_H 7.31–7.44 (5 H, m, phenyl protons), 4.73 (1 H, s, CH of MPA), 3.41 (3 H, s, OMe of MPA), 2.21 (1 H, m, H-1), 5.15 (1 H, m, H-2), 1.74 (2 H, m, H-3), 2.22 (2 H, m, H-4 and H-5), 1.17 (1 H, m, H-6a), 1.44 (1 H, m, H-6b), 2.10 (1 H, dt, J = 4.1, 11.4 Hz, H-7), 1.38 (1 H, m, H-8a), 1.73 (2 H, m, H-8b and H-9b), 1.55 (1 H, t, J = 10.1 Hz, H-9a), 1.67 (3 H, s, Me-12), 4.60 (1 H, br s, H-13a), 4.63 (1 H, br s, H-13b), 0.90 (3 H, s, Me-14), 0.91 (3 H, d, J = 7.0 Hz, Me-15).

(R)-MPA ester of 7 (7a): ¹H NMR (CDCl₃, 400 MHz) δ_H 7.32–7.44 (5 H, m, phenyl protons), 4.74 (1 H, s, CH of MPA), 3.41 (3 H, s, OMe of MPA), 2.31 (1 H, t, J = 6.9 Hz, H-1), 5.17 (1 H, dt, J = 2.9, 7.6 Hz, H-2), 1.45 (1 H, m, H-3a), 1.68 (1 H, m, H-3b), 2.16 (1 H, m, H-4), 2.18 (1 H, m, H-5), 0.91 (1 H, m, H-6a), 1.81 (1 H, m, H-6b), 1.77 (1 H, m, H-7), 1.19 (1 H, m, H-8a), 1.76 (1 H, m, H-8b), 1.59 (1 H, m, H-9a), 1.82 (1 H, m, H-9b), 1.06 (3 H, s, Me-12), 3.97 (1 H, d, J = 11.5 Hz, H-13a), 4.06 (1 H, d, J = 11.5 Hz, H-13b), 1.14 (3 H, s, Me-14), 0.90 (3 H, d, J = 6.5 Hz, Me-15).

(S)-MPA ester of 7 (7b): ¹H NMR (CDCl₃, 400 MHz) δ_H 7.32–7.42 (5 H, m, phenyl protons), 4.71 (1 H, s, CH of MPA), 3.40 (3 H, s, OMe of MPA), 2.16 (1 H, m, H-1), 5.05 (1 H, dt, J = 2.8, 7.7 Hz, H-2), 1.68 (1 H, m, H-3a), 1.80 (1 H, m, H-3b), 2.23 (1 H, m, H-4), 2.21 (1 H, m, H-5), 0.86 (1 H, m, H-6a), 1.71 (2 H, m, H-6b and H-8b), 1.77 (1 H, m, H-7), 1.15 (1 H, m, H-8a), 1.46 (1 H, m, H-9a), 1.72 (1 H, m, H-9b), 1.05 (3 H, s, Me-12), 3.95 (1 H, d, J = 11.5 Hz, H-13a), 4.04 (1 H, d, J = 11.5 Hz, H-13b), 0.71 (3 H, s, Me-14), 0.96 (3 H, d, J = 6.8 Hz, Me-15).

X-ray Crystal Data of Compounds 1 and 7

Graphostromane A (1) was obtained as colorless crystals. The monoclinic crystals (0.1 × 0.2 × 0.8 mm³) was recorded on a Bruker D8 Advance X-ray single-crystal diffractometer with Cu Kα radiation. Crystal data of 1: empirical formula C₁₅H₂₆O₂, M = 236.36; space group P2₁, unit cell dimensions a = 6.3808 (4) Å, b = 7.7943 (4) Å, c = 13.9771 (8) Å, α = 90.00°, β = 101.329 (6)°, γ = 90.00°, V = 681.59 (7) ų, Z = 2, D_calcd = 1.1516 g/cm³, µ = 0.579 mm⁻¹, F (000) = 260.8; A total of 6871 reflections were collected in the range of 6.44° < 2θ < 124.16°, of which 2128 independent reflections [R_int = 0.0548, R_sigma = 0.0552] were used for the analysis. The structure was solved by the direct methods with the SHELXL-97 program and refined using full-matrix least-squares difference Fourier techniques. The final R indexes [all data] gave R₁ = 0.0642, wR₂ = 0.1712 and the Flack parameter = −0.1 (3). Crystallographic data of 1 have been deposited in the Cambridge Crystallographic Data Center (deposition number CCDC 1529237).

Colorless crystals of 7 were obtained from MeOH. The monoclinic crystals (0.3 × 0.4 × 0.8 mm³) was measured on a Bruker D8 Advance X-ray single-crystal diffractometer with Cu Kα radiation. Crystal data of 7: empirical formula C₁₇H₃₀O₅, M = 314.42; space group C2, unit cell dimensions a = 12.2072 (4) Å, b = 14.3161 (3) Å, c = 13.4457 (4) Å, α = γ = 90.00°, β = 114.561 (4)°, Volume = 2137.16 (12) ų, Z = 9, D_calcd = 1.173 g/cm³, µ = 0.774 mm⁻¹, F (000) = 828.0; A total of 11239 reflections were collected in the range of 7.228° < 2θ < 123.85°, of which 3337 independent reflections [R_int = 0.0330, R_sigma = 0.0280] were used for the analysis. The final R indexes [all data] gave R₁ = 0.0368, wR₂ = 0.1006 and the Flack parameter = 0.04 (7). Crystallographic data of 7 have been deposited in the Cambridge Crystallographic Data Center (deposition number CCDC 1577811).

Theoretical Calculation of CMR Data

The calculated CMR data of 3 and 9 were carried out by Gaussian 09²³. Conformational analyses were initially performed using Confab²⁴ with MMFF94 force field for configurations of both compounds. The conformers, which were chosen for CMR calculations with Boltzmann-population over 1%, were firstly optimized at PM6 by semi-empirical theory method to filter some conformers with low Boltzmann-populations. Then, the remaining conformers were optimized at B3LYP/6-31 + G(d,p) in gas phase. The CMR calculation was conducted by the Gauge-Including Atomic Orbitals (GIAO) method at mPW1PW91/6-311 + G(2d,p) level using the IEFPCM model in pyridine for 3, whereas in MeOH for 9, respectively. Finally, the TMS-corrected ¹³C NMR chemical shift values were averaged according to Boltzmann distribution for each conformer and fitting to the experimental values by linear regression. The calculated CMR chemical shift values of TMS in pyridine and in MeOH were 187.3194 ppm and 187.3772 ppm, respectively.

Anti-Inflammatory Assay

This experiment was conducted according the reported procedure²⁵.

Author Contributions

Xian-Wen Yang designed the project; Siwen Niu isolated compounds and determined their structures; Chun-Lan Xie and Jin-Mei Xia conducted the anti-inflammatory experiments; Zhu-Hua Luo and Zongze Shao isolated and identified the fungus; Siwen Niu and Xian-Wen Yang wrote and revised the paper. All authors reviewed the manuscript.

Competing Interests

The authors declare that they have no competing interests.