Eremophilane-Type Sesquiterpenoids from an Acremonium sp

Article pubs.acs.org/jnp

Eremophilane-Type Sesquiterpenoids from an Acremonium sp. Fungus Isolated from Deep-Sea Sediments Zhongbin Cheng,† Jingjun Zhao,‡ Dong Liu,† Peter Proksch,§ Zhimin Zhao,*,‡ and Wenhan Lin*,† †

State Key Laboratory of Natural and Biomimetic Drugs, Institute of Ocean Research, Peking University, Beijing, 100191, People’s Republic of China ‡ School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People’s Republic of China § Institute für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany S Supporting Information *

ABSTRACT: Chemical examination of an EtOAc extract of a cultured Acremonium sp. fungus from deep-sea sediments resulted in the isolation of 15 new eremophilane-type sesquiterpenoids, namely, acremeremophilanes A−O (1−15), together with seven known analogues. The structures of new compounds were determined through extensive spectroscopic analyses, in association with chemical conversions and ECD calculations for configurational assignments. The PKS-derived 4-hexenoic acid unit in 2−6 is rarely found in nature. All compounds were evaluated for inhibitory effects toward nitric oxide production induced by lipopolysaccharide in RAW 264.7 macrophage cells. Compounds 2−6 and 14 exhibited inhibitory effects with IC50 values ranging from 8 to 45 μM. with 52% inhibition at a single dose (50 μg/mL). Subsequent chromatography of the EtOAc fraction yielded 22 eremophilane-type sesquiterpenoids, including 15 new compounds, acremeremophilanes A−O (1−15), and known compounds 16−22. All of the isolated compounds were tested against LPSinduced NO production in RAW 264.7 macrophage cells.

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remophilane-type derivatives are structurally irregular and bicyclic natural products belonging to a small sesquiterpene family.1,2 These uncommon sesquiterpenes are biogenetically derived from farnesyl diphosphate in association with a methyl migration.3 The structural variety of eremophilane analogues is due to oxidation occurring at various sites along the bicyclic backbone and isopropyl side chain to generate alcohol, acid, lactone, furan, and lactone functionalities, with some of the alcohols further glycosylated. Moreover, some unusual derivatives such as nor-eremophilanes,4,5 secoeremophilanes,6,7 and eremophilane dimers8,9 have been reported. In addition to the related analogues obtained from terrestrial plants10−12 and plant-associated fungi,13−15 marinederived fungi are recognized as a new source of eremophilanebased derivatives.16,17 Eremophilane sesquiterpenoids have attracted much attention because many of them possess a range of biological or therapeutic activities, including cytotoxic and antitumor,18,19 antimicrobial,11 anti-inflammatory,20 antiviral (hepatitis B),21 and antiallergic effects.22 As part of our ongoing efforts to discover bioactive terpenoids derived from marine microorganisms, a chemical examination of our marine microorganism library resulted in an extract from an Acremonium sp. fungal strain, collected from the sediments at a depth of 2869 m in the South Atlantic Ocean (GPS 13.7501 W, 15.1668 S), displaying the HPLC fingerprint and 1H NMR resonances typical of terpenoids. A bioassay result revealed that the EtOAc extract of the fungal fermentation possessed inhibitory activity against the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Acremeremophilane A (1) had a molecular formula of C15H18O4, as established by the HRESIMS and NMR data, requiring seven degrees of unsaturation. The 1H NMR and HMQC spectra provided the resonances for two methyl groups, an olefinic methylene, three olefinic protons, a hydroxymethine, and four alkyl protons, while the 13C NMR spectrum exhibited six olefinic carbons for three double bonds and two carbonyl carbons for a ketone and a carboxylic acid group. The 1H and 13C NMR data in association with the HMBC correlations established an eremophilane-based sesquiterpene nucleus, structurally related to known eremophilan-12-oic acids.23 The vicinal coupling olefinic protons at δH 6.28 (d, J = 10.0 Hz) and 6.24 (dd, J = 3.6, 10.0 Hz) were assigned to H-1 and H-2, while the HMBC correlations from H-9 (δH 5.70, s) to C-1 (δC 127.2), C-5 (δC 35.9), C-7 (δC 46.5), and C-8 (δC 197.2) assigned an α,β-unsaturated ketone residing at C-8, C-9 (δC 123.7) and C-10 (δC 162.1). The exomethylene protons H2-12 (δH 5.68, 6.13) showed HMBC correlations with carboxylic acid carbon C-13 Received: December 13, 2015

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DOI: 10.1021/acs.jnatprod.5b01103 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

Figure 1. Key NOE correlations of 1, 7, 10, 12, 13, and 15.

and H-6a in an axial orientation. The NOE correlations from H3-14 (δH 1.24, s) to H3-15 (δH 1.01, d) and H-7 (δH 3.57, dd, J = 4.8, 12.8 Hz) and between H-6a (δH 2.05)/H-4, H3-15/H-6b (δH 1.90) (Figure 1) determined the same orientation of H3-14, H3-15, and H-7, while H-4 was in an axial orientation. Thus, the JH‑3/H‑4 value (4.8 Hz) reflected an equatorial−axial relationship of H-3 and H-4, respectively. Therefore, the relative configuration

(δC 167.2) and C-7, confirming an acrylic acid positioned at C-7. In addition, the location of a hydroxy group at C-3 (δC 65.9) was evident from the COSY relationship of H-3 (δH 4.00, dd, J = 3.6, 4.8 Hz) with H-2 and H-4 (δH 1.62, dq, J = 4.8, 7.2 Hz). The relative configuration of 1 was determined by NOESY correlations in association with J values. The coupling constant JH‑7/H‑6a (12.8 Hz) was indicative of the trans-relationship of H-7 B



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Figure 2. Experimental ECD spectra (200−400 nm) of 1−3 in MeOH and the calculated ECD spectra of 1 at the B3LYP/6-311++G(2d,2p) level.

Acremeremophilane C (3) had the same molecular formula as that of 2, as determined by the HRESIMS and NMR data. The similar NMR data of 3 and 2 indicated the structure of 3 to be related to 2. Analyses of 2D NMR data revealed 3 to be an olefinic isomer of 2 by the presence of a Δ7(11) instead of a Δ11(12) double bond, according to the HMBC correlations from the methylene protons H2-6 (δH 2.25, 3.24) to C-7 (δC 132.7) and C-11 (δC 138.4) as well as H3-12 (δH 2.22) to C-7, C-11, and C-13 (δC 171.9). The NOE relationships between H3-15/ H-6b, H-4/H-6a, H3-14/H3-15, and H-3/H-4 confirmed the same relative configurations of 3 as those of 2. The absence of a NOE correlation between H3-12 and H2-6 was indicative of an E configuration of the C-7/C-11 double bond. The positive CE at 374 nm of 3 for the n−π* transition with the same sign as that of 2 reflected the M-helicity of the cyclohexenone ring. In biogenetic consideration and in association with the NOE data, the structure of 3 was determined to be the E-Δ7(11) isomer of 2. Analyses of 2D NMR data revealed acremeremophilane D (4) to be an analogue of 2 with a different substitution at C-7, while the HRESIMS data provided its molecular formula to be C22H28O5. Apart from the closely similar NMR data of the bicyclic rings in 4 and 2, a formyl group (δH 9.23, δC 189.8), a methoxy group (δH 3.95, δC 62.5), and an olefinic methine (δH 7.03, δC 172.8) were observed in the HMQC spectrum of 4. The HMBC correlations from the formyl proton to the olefinic carbons C-11 (δC 123.0) and C-12 (δC 172.8) and from H-12 (δH 7.03, s) to the formyl carbon (δC 189.8, C-13) and methoxy carbon established a 3-methoxyacrylaldehyde moiety. The linkage of this moiety to C-7 (δC 38.4) was deduced by the HMBC relationship between H-12 and C-7. The 11E geometry was evident from the NOE relationship between H-12 and the formyl proton. The relative configuration of 4 was the same as that of 2 as determined by the similar NOE correlations. The absolute configuration of 4 can be determined by the ECD exciton chirality method.28,29 In the experimental ECD spectrum, the exciton split CD effects with a negative Cotton effect at λmax 280 nm (Δε +7.28) and a positive Cotton effect at λmax 246 nm

of 1 was assigned as 3S*, 4R*, 5R*, and 7S*. Based on Snatzke’s rule for a cyclohexenone unit,24,25 the positive Cotton effect (CE) at 350 nm in 1 as induced by the n−π* electronic transition was in agreement with M-helicity of the cyclohexenone ring and reflected a 7S configuration. On the basis of the relative configuration as determined by the NOE correlations, the configurations of the remaining stereogenic centers were assigned as 3S, 4R, and 5R. These assignments were supported by the ECD calculations, which were performed at the B3LYP/ 6-311++G(2d, p) level in the gas phase using the B3LYP/ 6-31G(d)-optimized geometries after conformational searches via the MMFF94S force field for the (S/R)-model molecules.26,27 The computed ECD spectra for the modeled (3S,4R,5R,7S)-1 and its enantiomeric counterpart were calculated by the TDDFT method. Comparison of the experimental ECD curve of 1 with the computed ECD curves (Figure 2) confirmed the configurational assignment. The molecular formula of acremeremophilane B (2) was determined to be C21H26O5, on the basis of the HRESIMS and NMR data. Analyses of 1D and 2D NMR data revealed that 2 contained the same eremophilane-based moiety as in 1. The difference was found by the presence of an additional moiety, which was assigned to a 4′-hexenoic acid group based on the COSY relationships from H2-2′ (δH 2.41, t, J = 6.8 Hz) to H3-6′ (δH 1.63, d, J = 5.6 Hz), in addition to the HMBC correlations from H3-6′ to the olefinic carbons C-4′ (δC 128.8) and C-5′ (δC 126.3) and from H2-2′ and H2-3′ to the carbonyl carbon C-1′ (δC 172.7). The JH‑4′‑H‑5′ value (16.0 Hz) was indicative of a 4′E configuration. The linkage of the 4′-hexenoic group to C-3 (δC 65.9) was deduced by the HMBC relationship between H-3 (δH 5.35) and the carbonyl carbon C-1′. The similar J values and NOE correlations of 1 and 2 determined the same relative configurations for both compounds. The positive Cotton effect at 343 nm for the n−π* electronic transition of the cyclohexenone ring reflected the 7S configuration (Figure 2), indicating the same absolute configuration as for 1. This assignment was further supported by the alkaline hydrolysis of 2 to produce 1. C



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Figure 3. Experimental ECD spectrum of 4 in MeOH and the calculated ECD spectra of the model molecules of 4 at the B3LYP/6-311++G(2d,2p) level.

chiral-phase HPLC chromatography. Analyses of 2D NMR and HRESIMS data revealed the structure of 5a to be a 7Z isomer of 3, based on the NOE correlation between H3-12 (δH 2.11, s) and H2-6 (δH 2.28, 2.94). Thus, the interconversion of 5 and 5a appears to occur in solution. The structure of acremeremophilane F (6) was determined to be a 12-hydroxylated analogue of 5, based on the close similarity of their NMR data (Tables 1 and 3) with the exception of a hydroxylmethyl group (δH 4.40, 4.36; δC 55.1) in 6 replacing the methyl resonances H3-12 (δH 1.88, s) of 5. The similar NOE correlations and ECD effects of both 6 and 5 (Figure 4) indicated both compounds share the same absolute configurations. In addition, the coexistence of 6 and its acyclic analogue 6a in a ratio of 6:1 was observed in the NMR spectrum, while both compounds are inseparable by chiral-phase HPLC column. Acremeremophilane G (7) has a molecular formula of C15H22O2, as determined by the HRESIMS and NMR data, requiring five degrees of unsaturation. Analyses of 1D and 2D NMR data established the gross structure to be the same as a known eremophilane-type analogue.32 The difference was attributed to the configuration of C-2, as recognized by the coupling constants of H-2 in 7 showing JH‑2/H‑1a (12.0 Hz) and JH‑2/H‑3a (10.0 Hz) for an axial orientation of H-2 instead of small coupling constants for equatorial H-2 of the known analogue. The NOE correlations between H-2 (δH 3.75)/H-4 (δH 1.50) and from H3-14 (δH 1.16) to H3-15 (δH 0.95) and H-7 (δH 3.15) confirmed the same orientation of H-2 and H-4, while H-7, H3-14, and H3-15 were on the opposite face relative to H-4. The absolute configuration of C-2 was determined to be R by the modified Mosher’s method (Figure 5).33 Therefore, the absolute configuration of the remaining stereogenic centers was assigned accordingly. The structure of acremeremophilane H (8) was determined as a 2-acetyl analogue of 7, based on the similar NMR data of both compounds (Tables 2 and 3), except for the presence of an acetyl group (δH 2.07; δC 21.2, 170.2) in 8. The deshielded H-2 (δH 4.80) showed an HMBC correlation with the acetyl carbonyl carbon, supporting the acetyl location at C-2. Acetylation of 7 generated a product whose NMR, MS, and specific rotation

(Δε +13.34) due to the interaction of the dienone and enal chromophores were observed. These findings indicated the chromophores of 4 to have negative chrality (Figure 3), suggesting the 7S configuration. In addition, the NOE correlations between H-7/H3-14, H3-14/H3-15, and H-3/H-4 determined the configurations of C-3, C-4, and C-5 to be the same as those of 3. The comparable experimental ECD curve with the computed ECD for the model molecule of 4 supported the stereogenic centers to be 3S, 4R, 5R, and 7S configured (Figure 3). The molecular formula of acremeremophilane E (5) was determined as C21H26O5 by the HRESIMS data, requiring nine degrees of unsaturation. The 2D NMR data provided the structure of 5 to be partially related to 3, possessing a bicyclic nucleus with two double bonds at C-1/C-2 and C-9/C-10 and the substitution of a 4′-hexenoic group at C-3. These functionalities accounted for six sites of unsaturation. The remaining NMR resonances were attributed to two olefinic carbons for a double bond, a carbonyl carbon, and a methyl group, providing two additional sites of unsaturation. The HMBC correlations from the methyl protons at δH 1.88 (s, H3-12) to the carbonyl carbon C-13 (δC 171.7) and the olefinic carbons C-7 (δC 157.4) and C-11 (δC 124.0) and from H2-6 (δH 2.47, 2.74) to C-7 and C-11 connected an α-methyl-α,β-unsaturated carbonyl group to C-7. Thus, the remaining one site of unsaturation accounted for an α,β-unsaturated γ-lactone, which was also supported by the IR absorption at 1735 cm−1. The 13C NMR resonance of C-8 (δC 99.8) was assigned to a hemiketal-type carbon, positioning a hydroxy group at C-8. The similar NOE data and J values of both 5 and 3 assigned the same relative configuration for both compounds. According to the empirical rule for α,β-unsaturated γ-lactones in the ECD spectrum,30,31 the positive Cotton effects at 221 nm for the π−π* transition and the negative CE at 253 nm for the n−π* transition reflected the absolute configuration at C-8 to be S. This assignment was supported by the experimental ECD curve of 5, which was comparable to the computed ECD data for (3S,4R,5R,8S)-5, whereas its enantiomeric counterpart showed a computed ECD curve with opposite phases (Figure 4). It is noted that a minor amount of compound 5a coexisted with 5 in a ratio of 0.4:1 in CDCl3, which was inseparable by D



Article

Figure 4. Experimental ECD spectra of 5 and 6 in MeOH and the calculated ECD spectra of the model molecules of 5 at the B3LYP/ 6-311++G(2d,2p) level.

Table 1. 1H NMR Data of 1−7 (400 MHz, δ in ppm, J in Hz) no.

1a

2b

H-1a H-1b H-2

6.28, d (10.0)

6.31, d (10.0)

6.34, d (10.0)

6.34, d (9.6)

6.24, dd (3.6, 10.0) 4.0, dd (3.6, 4.8)

6.20, dd (5.2, 10.0) 5.35, dd (4.8, 5.2)

6.26, dd (5.2, 10.0) 5.37, dd (4.8, 5.2)

6.22, dd (5.2, 9.6) 5.36, dd (4.8, 5.2)

1.62, dq (4.8, 7.2) 2.05, dd (12.8, 14.0) 1.90, dd (4.8, 14.0) 3.57, dd (4.8, 12.8) 5.70, s 6.13, brs 5.68, brs

1.97, dq (4.8, 7.2) 2.13, dd (12.8, 13.4) 2.02, dd (5.2, 12.8) 3.57, dd (5.2, 13.4) 5.81, s 6.40, s 5.68, s

2.02, dq (4.8, 7.2) 3.24, d (14.0)

1.98, dq (4.8, 7.2) 2.06, dd (12.7, 13.2) 1.89, dd (5.0, 13.2) 3.85, dd (5.0, 12.7) 5.86, s 7.03, s

1.24, s 1.01, d (7.2)

1.30, s 1.00, d (7.2) 2.41, t (6.8) 2.31, dt (5.6, 6.8) 5.42, dt (5.6, 16.0) 5.51, dq (5.6, 16.0) 1.63, d (5.6)

1.12, 0.97, 2.34, 2.25,

H-3a H-3b H-4 H-6a H-6b H-7 H-9 H-12a H-12b H-13 H-14 H-15 H-2′ H-3′ H-4′ H-5′ H-6′ OMe a

3b

2.25, d (14.0)

5.80, s 2.22, s

s (7.2) t (6.8) dt (6.0)

5.35, dt (6.0, 16.0) 5.50, dq (6.0, 16.0) 1.59, dd (6.0)

4b

9.23, s 1.32, s 0.98, d (7.2) 2.40, t (6.8) 2.32, dt (6.0, 16.0) 5.42, dt (6.0, 16.0) 5.49, dq (6.0, 16.0) 1.64, d (6.0) 3.95, s

5b

5ab

6c

6ac

7b

6.16, d (10.0) 6.36, d (10.0) 6.27, d (10.0) 6.48, d (10.0) 2.57, dd (4.8, 12.0) 2.32, t (12.0) 5.95, dd 6.29, dd 5.93, dd 6.26, dd 3.75,dddd (4.8, 10.0) (4.8, 10.0) (5.2, 10.0) (5.0, 10.0) (4.8, 6.0, 10.0, 12.0) 5.33, t (4.8) 5.38, t (4.8) 5.33, dd 5.38, dd 2.05, ddd (4.8, 5.2) (4.8, 5.0) (4.0, 6.0, 12.0) 1.56, dt (10.0, 12.0) 2.14, dq 2.04, dq 2.17, dq 2.12, dq 1.50, ddq (4.8, 7.0) (4.8, 7.2) (4.8, 6.8) (4.8, 7.0) (4.0, 7.0, 12.0) 2.74, d (12.4) 2.94, d (14.3) 3.22, d (12.0) 2.33, d (14.1) 2.00, dd (4.4, 12.0) 2.47, d (12.4) 2.28, d (14.3) 2.49, d (12.0) 3.12, d (14.1) 1.83, dd (12.0, 14.0) 3.15, dd (4.4, 14.0) 5.83, s 1.88, s

5.93, s 2.11, s

0.98, s 1.07, d (7.0) 2.37, t (7.0) 2.30, dt (6.0, 7.0) 5.42, dt (6.0, 16.0) 5.45, dq (6.0, 16.0) 1.64, d (6.0)

1.19, s 1.10, d (7.2) 2.37, t (7.0) 2.30, dt (6.0, 7.0) 5.42, dt (6.0, 16.0) 5.45, dq (6.0, 16.0) 1.63, d (6.0)

5.89, s 5.87, s 5.77, s 4.40, d (14.0) 4.52, d (14.0) 4.97, s 4.36, d (14.0) 4.38, d (14.0) 4.80, s 1.72, s 1.07, s 1.25, s 1.16, s 1.08, d (6.8) 1.11, d (7.0) 0.95, d (7.0) 2.38, t (6.5) 2.38, t (7.0) 2.29, dt 2.29, dt (6.0, 6.5) (6.0, 7.0) 5.43, dt 5.43, dt (6.0, 16.0) (6.0, 16.0) 5.48, dq 5.48, dq (6.0, 16.0) (6.0, 16.0) 1.60, d (6.0) 1.60, d (6.0)

In DMSO-d6. bIn CDCl3. cIn acetone-d6.

H2-13 (δH 3.91, 3.98) correlated to C-7 (δC 44.0), C-11, and C-12 (δC 108.3) in the HMBC spectrum. The NOE correlations from H3-14 to H3-15 and H-7 and between H-8 and H2-13 assigned the relative configurations of 9 to be the same as those of 17, in which H-8 and H-7 were in a trans orientation. On the basis of the Cotton effect rule for unsaturated ketones,25 the negative CE at 241 nm for the π−π* transition and a positive CE

were identical to those of 8, confirming 8 to be the 2-acetylated analogue of 7. The NMR data of acremeremophilane I (9) featured an eremophilane-type sesquiterpene, structurally related to guignarderemophilane E (17).15 The distinction was found by the presence of a hydroxymethyl instead of a methyl group located at C-11 (δC 151.5), based on the hydroxymethyl protons E



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Table 2. 1H NMR Data of 8−15 (400 MHz, δ in ppm, J in Hz) 8a H-1a H-1b H-2 H-3a H-3b H-4

2.60, dd (4.8, 14.0) 2.40, dd (12.0,14.0) 4.80, dddd (4.8, 6.0, 10.0, 12.0) 1.92, ddd (4.0, 6.0, 12.0) 1.57, dt (10.0, 12.0)

H-6a H-6b

1.55, ddq (4.0, 7.0, 12.0) 2.00, dd (4.5, 13.0) 1.83, dd (13.0, 14.0)

H-7

3.16, dd (4.5, 14.0)

H-8 H-9a H-9b

5.80, s

H-12a H-12b H-13a H-13b H-14 H-15

4.98, s 4.82, s 1.73, s

Ac OH-1 OH-10

2.07, s

a

1.19, s 0.96, d (7.0)

9b 5.69, s

10c

11a

12b

13b

14b

5.37, s

5.42, s

3.78, t (2.8)

4.13, brs

4.18, dd (5.6, 10.0)

4.26, dd (5.6, 10.0)

2.28, dd 1.72, ddd (5.6, 6.0, (12.0,16.0) 12.0) 2.04, dd (3.4, 16.0) 1.39, ddd (10.0, 11.0, 12.0) 1.90, ddq (3.4, 6.7, 1.48, ddq 12.0) (6.0, 6.7, 11.0) 1.82, dd (3.2, 13.6) 1.87, dd (3.2, 13.0) 1.24, dd 1.16, t (13.0) (12.0, 13.6) 2.23, ddd (3.2, 2.29, dt (3.2, 10.0) 11.0, 12.0) 3.43, ddd 3.47, dt (6.4, 10.0) (5.0, 11.0, 12.0) 2.50, dd (5.0, 12.0) 2.34, dd (6.4, 12.0) 2.44, t (12.0) 2.32, dd (10.0, 12.0) 5.05, s 5.16, s 4.86, s 4.96, s 3.91, d (14.6) 4.08, d (15.3) 3.98, d (14.6) 4.04, d (15.3) 1.07, s 1.03, s 0.88, d (6.7) 0.91, d (6.7)

1.79, ddd (5.6, 6.0, 12.0) 1.40, ddd (10.0, 11.0, 12.0) 1.50, ddq (6.0, 6.9, 11.0) 1.79, dd (3.6, 13.2) 1.14, dd (13.0, 13.2) 2.51, dt (3.6, 13.0)

5.61, dd (2.8, 10.0) 5.39, d (10.0)

5.43, brd (10.3) 5.25, d (10.3)

2.5, q (7.5)

1.96, q (7.2) 2.61, s

2.51, dq 1.84, dq (4.2, 6.9) (6.8, 11.4) 3.13, d (16.0) 2.96, d (16.0) 2.47, d (16.0) 2.26, d (16.0)

5.89, s

5.98, s

6.49, s

6.18, s

1.84, s

1.83, s

1.94, s

1.86, s

0.76, s 0.93, d (7.5)

0.92, s 0.87, d (7.2)

1.20, s 1.03, d (6.9)

0.88, s 1.00, d (6.8)

5.12, d (2.8) 4.94, s

5.07, br 5.07, s

4.76, ddd (5.2, 10.0, 13.0) 2.44, dd (5.2, 12.0) 2.38, dd (10.0, 12.0) 4.77, s 4.76, s 1.65, s 1.04, s 0.90, d (6.9)

2.62, d (16.0) 2.54, d (16.0)

2.00, s

6.17, s

15b 5.88, d (4.8) 3.96, t (4.8)

5.29, d (4.2)

3.42, dd (4.8, 11.4)

2.08, s

In CDCl3. bIn DMSO-d6. cIn CD3OD.

and the acetyl carbonyl carbon. The similar NOE relationships and J values of 11 and 18 confirmed both compounds possess the same relative configurations. Acetylation of 17 by Ac2O yielded an acetylated analogue of 17, and then reduction by NaBH4/MeOH35 provided a product that was identified as 11, based on the same NMR data and specific rotation of both compounds. In addition, reduction35 of 17 by NaBH4/MeOH produced 18, confirming the uncertain configuration of 18 in the literature33 to be 2R, 4S, 5R, 7S, and 8R. The molecular formula of acremeremophilane L (12) was determined to be C15H18O4 on the basis of the HRESIMS and NMR data. The NMR data featured an eremophilane-type sesquiterpene, structurally related to tsoongianolide A, an eremophilane lactone.36 The 2D NMR data assigned two double bonds at C-2 (δC 126.1)/C-3 (δC 132.5) and C-8 (δC 148.4)/ C-9 (δC 111.6). An α-methyl-α,β-unsaturated γ-lactone was fused to C-7 (δC 148.6) and C-8, based on the HMBC correlations from H3-12 (δH 1.84, s) to C-7, C-11 (δC 120.7), and C-13 (δC 170.5), H-9 (δH 5.89, s) to C-7, and H2-6 (δH 2.54, 2.62) to C-7 and C-11. In addition, C-1 (δC 70.4) and C-10 (δC 73.3) bearing hydroxy groups were evident from the COSY relationship of H-1 (δH 3.78, t, J = 2.8 Hz) with H-2 (δH 5.61, dd, J = 2.8, 10.0 Hz) and OH-1 (δH 5.12, d, J = 2.8 Hz) and the HMBC correlation of OH-10 (δH 4.94, s) with C-1, C-9, and C-10. The NOE correlations between H-1/H3-14, H3-14/H3-15, and OH-10/H-4 established the same orientation of H3-14, H3-15, and H-1, whereas H-4 and OH-10 were on the opposite face toward H3-14. Moreover, the experimental ECD spectrum exhibited a negative CE at 277 nm for the n−π* transition and a positive CE at 234 nm for the π−π* transition

at 316 nm for the n−π* transition of the cyclohexenone chromophore reflected the 5R configuration. Thus, the previously mentioned NOE correlations assigned the 4S, 7S, and 8R configurations for 9. In addition, the 8R configuration of guignarderemophilane E (17)15 was confirmed by the ΔδH (δR − δS) values of (R)- and (S)-MPA esters (Figure 5) using Mosher’s method. The similar Cotton effects of both 9 and 17 further supported the configurational assignment of 9. The structure of acremeremophilane J (10) was determined to be the 13-hydroxylated analogue of 18, based on the 2D NMR analyses and the comparison of the NMR data of both 10 and 18. The location of the hydroxymethyl group (δH 4.04, 4.08; δC 65.9) was deduced by the HMBC correlation of H2-13 (δH 4.04, 4.08) to C-7 (δC 46.3), C-11 (δC 152.1), and C-12 (δC 110.9). The NOE correlations between H-2/H-4, H3-14/ H3-15, H3-14/H-7, and H-8/H2-12 (Figure 2) in association with the J values JH‑2/H‑3a (10.0 Hz) and JH‑7/H‑8 (10.0 Hz) for the axial orientations of H-2 and H-8 established the relative configurations of 10 to be the same as those of 18. Reduction of 9 by NaBH4/MeOH35 yielded a product whose NMR data and specific rotation were identical to those of 10. These findings defined the 5R configuration of 10. Accordingly, the remaining stereogenic centers of 10 were assigned the 2R, 4S, 7S, and 8R configurations. Acremeremophilane K (11) had a molecular formula of C17H26O3 as determined by the HRESIMS and NMR data. Comparison of the NMR data revealed the structure of 11 to be closely related to 18 but having an acetyl group (δH 2.00; δC 21.1, 170.3). The location of the acetyl group at C-8 (δC 73.8) was deduced by the HMBC correlation between H-8 (δH 4.76) F


G

a

127.2, 138.7, 65.9, 41.5, 35.9, 40.4, 46.5, 197.2, 123.7, 162.1, 141.2, 126.7, 167.2, 19.0, 10.6,

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ OMe Ac

130.9, 132.2, 69.1, 40.6, 36.2, 40.6, 46.8, 197.8, 125.4, 160.5, 139.8, 127.9, 168.5, 18.4, 10.1, 172.7, 34.4, 27.8, 128.8, 126.3, 17.7,

CH CH CH CH C CH2 CH C CH C C CH2 C CH3 CH3 C CH2 CH2 CH CH CH3

2b

130.5, 132.7, 69.2, 39.8, 38.0, 41.5, 132.7, 191.2, 127.2, 160.5, 138.4, 17.3, 171.9, 18.9, 10.4, 172.7, 34.3, 27.8, 128.8, 126.2, 17.7,

CH CH CH CH C CH2 C C CH C C CH3 C CH3 CH3 C CH2 CH2 CH CH CH3

3b 131.2, 132.2, 69.3, 40.4, 36.1, 38.6, 38.4, 197.2, 125.7, 160.5, 123.0, 172.8, 189.8, 18.4, 10.2, 172.8, 34.5, 28.0, 129.0, 126.4, 17.8, 62.5,

CH CH CH CH C CH2 CH C CH C C CH CH CH3 CH3 C CH2 CH2 CH CH CH3 CH3

4b CH CH CH CH C CH2 C C CH C C CH3 C CH3 CH3 C CH2 CH2 CH CH CH3

5b 130.7, 127.7, 69.4, 41.1, 42.4, 34.8, 157.4, 99.8, 122.3, 146.2, 124.0, 8.3, 171.7, 19.8, 10.4, 172.8, 34.5, 28.0, 128.9, 126.4, 17.9,

In DMSO-d6. bIn CDCl3. cIn acetone-d6. dIn CD3OD.

CH CH CH CH C CH2 CH C CH C C CH2 C CH3 CH3

1a

no. CH CH CH CH C CH2 C C CH C C CH3 C CH3 CH2 C CH2 CH2 CH CH CH3

5ab 130.5, 133.6, 69.1, 39.9, 38.0, 39.2, 132.6, 188.5, 126.2, 162.2, 138.6, 17.6, 172.4, 20.0, 10.4, 172.8, 34.4, 28.0, 128.9, 126.5, 17.7,

Table 3. 13C NMR Data of 1−15 (100 MHz, δ in ppm) 131.7, 128.3, 70.2, 42.3, 42.9, 35.0, 160.8, 100.7, 124.1, 146.5, 127.5, 55.1, 170.8, 20.2, 10.8, 172.8, 35.1, 28.7, 130.3, 126.6, 18.0,

6c CH CH CH CH C CH2 C C CH C C CH2 C CH3 CH3 C CH3 CH3 CH CH CH3

131.4, 133.6, 69.9, 40.9, 38.7, 39.3, 132.9, 188.2, 126.8, 161.7, 142.9, 60.2, 170.5, 20.4, 10.7, 172.8, 34.9, 28.7, 130.3, 126.7, 18.0,

CH CH CH CH C CH2 C C CH C C CH2 C CH3 CH3 C CH2 CH2 CH CH CH3

6ac 42.0, 69.3, 39.5, 40.6, 38.5, 41.2, 50.8, 198.9, 125.6, 166.7, 143.4, 114.2, 20.0, 15.9, 14.7,

CH2 CH CH2 CH C CH2 CH C CH C C CH2 CH3 CH3 CH3

7b CH2 CH CH2 CH C CH2 CH C CH C C C CH3 CH3 CH3

21.2, CH3 170.2, C

38.0, 71.1, 35.4, 40.4, 38.6, 41.1, 50.9, 198.5, 126.3, 165.2, 143.4, 114.3, 20.0, 15.8, 14.7,

8b 124.6, 198.2, 41.5, 39.4, 38.6, 42.8, 44.0, 71.9, 41.9, 168.7, 151.5, 108.3, 63.7, 16.3, 14.9,

CH C CH2 CH C CH2 CH CH CH2 C C CH2 CH2 CH3 CH3

9a 127.6, 68.5, 37.6, 40.3, 39.0, 45.8, 46.3, 75.1, 42.4, 144.3, 152.1, 110.9, 65.9, 18.4, 16.0,

CH CH CH2 CH C CH2 CH CH CH2 C C CH2 CH2 CH3 CH3

10d CH CH CH2 CH C CH2 CH CH CH2 C C CH2 CH3 CH3 CH3

21.1, CH3 170.3, C

127.3, 67.9, 42.8, 38.9, 37.7, 42.8, 46.2, 73.8, 37.4, 142.4, 145.5, 112.2, 19.4, 18.0, 15.5,

11b 70.4, 126.1, 132.5, 35.3, 40.3, 31.4, 148.6, 148.4, 111.6, 73.3, 120.7, 8.2, 170.5, 15.8, 14.1,

CH CH CH CH C CH2 C C C C C CH3 C CH3 CH3

12a 69.7, 128.7, 132.4, 36.2, 42.8, 28.7, 148.2, 149.6, 107.1, 73.9, 121.2, 8.2, 170.4, 15.5, 14.7,

CH CH CH CH C CH2 C C C C CH2 CH3 C CH3 CH3

13a CH C CH CH C CH2 C C CH C C CH3 C CH3 CH3

20.4, CH3 169.4, C

125.7, 191.9, 72.6, 40.9, 37.9, 33.0, 145.9, 152.8, 106.2, 163.1, 124.4, 8.6, 169.5, 22.8, 10.1,

14a 130.2, 64.7, 69.3, 38.6, 39.4, 34.2, 146.8, 148.4, 108.2, 141.1, 120.8, 8.3, 170.4, 19.4, 10.1,

CH CH CH CH C CH2 C C CH C C CH3 C CH3 CH3

15a

Journal of Natural Products Article



Article

observed in the above analogues, confirmed the same orientation of H3-15 and H3-14. In addition, the absence of an NOE correlation between H-3 and H3-14 in association with the JH‑3/H‑4 value (4.2 Hz) for an equatorial−axial coupling deduced both H-3 and H-4 to be on the same face. The experimental ECD effects of 14 were comparable to the computed ECD data of the model molecule for the R configuration of C-3, C-4, and C-5, but in opposite phase to the computed ECD data of the model molecule for the S configuration of C-3, C-4, and C-5, supporting the configurational assignment (Figure 7). The structure of acremeremophilane O (15) was determined to be the C-3 epimer of 22, based on the close similarity of their NMR data and the 2D NMR data, with the exception of the JH‑2/H‑3 (4.8 Hz) and JH‑3/H‑4 (11.4 Hz) values, which were assigned to an axial−axial relationship between H-3 and H-4 and an equatorial−axial relationship between H-2 and H-3. The NOE correlations from H-3 to H3-14 and H3-15 further confirmed the β-orientation of H-3. The similar ECD effects of 15 compared with those for the model molecule with 2R, 3S, 4R, and 5R configurations allowed the assignment of the absolute configuration of 15 (Figure 8). Analyses of 1D and 2D NMR data assigned the gross structure of 16 to be the same as that of guignarderemophilane B.15 The NMR data of 16 as measured in acetone-d6 were nearly identical to those of guignarderemophilane B (Supporting Information). The J values of H-7 (JH‑7/H‑6a = 4.4 Hz and JH‑7/H‑6a = 14.6 Hz) in 16 were indicative of an axial orientation of H-7, while the NOE relationship between H-7 and H3-14 confirmed the same orientation of H-7 and H3-14. This finding was in contrast to the assignment of H-7 with α-orientation in guignarderemophilane B.15 However, the similar J values of H-7 in both 16 and guignarderemophilane B (JH‑7/H‑6a = 4.8 Hz and JH‑7/H‑6a = 15.0 Hz) indicated H-7 of the latter to be in an axial orientation rather than an equatorial orientation. In addition, the similar specific rotation of 16 ([α]20D +68 (c 0.10, MeOH)) in comparison with that of guignarderemophilane B ([α]20D +65 (c 0.03, MeOH)) and the similar Cotton effects of both compounds supported 16 being identical to guignarderemophilane B.

Figure 5. ΔδH (δR − δS) values of (R)- and (S)-MPA esters of 7 and 17.

(Figure 6A), which matched well with the computed ECD curves of the modeled 12 with the 10R configuration (Figure 6B). The calculated ECD curves of the model molecules (1S,10S; 1R,10R; 1S,10R; and 1R,10S isomers) revealed that the sign of the ECD effect at ca. 270 nm directly reflected the configuration of C-10, of which the negative CE for the 10R configuration and positive CE for the 10S configuration were recognized (Figure 6B and D). Thus, C-10 of 12 was assigned the R configuration. Accordingly, the remaining stereogenic centers were assigned 1S, 4S, and 5R configurations. The gross structure of acremeremophilane M (13) was determined to be the same as 12 based on the 2D NMR analysis. The similar NOE correlations of both 13 and 12 such as the NOE correlations between H-1/H3-14 and H3-14/H3-15 confirmed the same orientation of H-1, H3-14, and H3-15. In addition, the experimental ECD spectrum of 13 presented a positive CE at 270 nm for the n−π* transition and negative CE at 222 nm for the π−π* transition (Figure 6C), which was in accordance with the ECD data calculated for 10S isomers (Figure 6D). Thus, the structure of 13 was determined to be the C-10 epimer of 12. Analyses of the 2D NMR data indicated acremeremophilane N (14) to be an eremophilane-type analogue bearing an α-methyl-α,β-unsaturated γ-lactone, structurally related to PF1092 B.37 The distinction was attributed to the location of carbonyl C-2 (δC 191.9) in 14 replacing a hydroxymethine of PF1092 B, whereas the remaining NMR resonances were closely similar. The NOE correlations from H3-14 to H3-15 and H-6a (δH 3.13) and between H-4 and H-6b (δH 2.47), as

Figure 6. Experimental ECD spectra of 12 (A) and 13 (C) in MeOH and the calculated ECD spectra of model molecules at the B3LYP/6-311+ +G(2d,2p) level. H



Article

Figure 7. Experimental ECD spectrum of 14 in MeOH and the calculated ECD spectra of model molecules at the B3LYP/6-311++G(2d,2p) level.

Figure 8. Experimental ECD spectrum of 15 in MeOH and the calculated ECD spectra of model molecules at the B3LYP/6-311++G(2d,2p) level.

compounds. All compounds showed no or weak cytotoxic effects (over 90% cell survival) at a concentration of 50 μM on RAW 264.7 cells. Compounds showing inhibitory effects against NO production with values greater than 45% were further evaluated to calculate IC50 values. Compounds 2−5 and 14 exerted inhibitory effects with IC50 values ranging from 8 to 45 μM, with compounds 2 (IC50 = 8 μM) and 5 (IC50 = 15 μM) showing activity that is comparable to the positive control quercetin (IC50 = 15 μM) (Table 4). Preliminary structure−activity relationships revealed that the analogues with a 4-hexenoic group at C-3 (2−6) significantly enhanced the activity, as examplified by compound 2 showing 97% inhibition against NO production in RAW 264.7 macrophage cells at 50 μM, whereas compound 1 exerted 11% inhibition of NO production at the same dose. To explore the mechanism of these NO inhibitors, compounds 2 and 4−6 were selected to

Thus, the configuration of C-7 in guignarderemophilane B was revised to S. In addition, six additional known eremophilane-type sesquiterpenoids were identical to guignarderemophilane E (17),15 eremophil-1(10),11(12)-dien-2β,8β-diol (18),34 2-oxo-3hydroxyeremophila-1(10),3,7(11),8-tetraen-8,12-olide (19),38 the C-2 epimer of PF1092B (20),39 the C-3 epimer of PF1092C (21),39 and the C-2 epimer of PF1092C (22),39 based on the comparison of their NMR data and specific rotations with those reported in the literature. Compounds 1−22 were tested for inhibitory activity against NO production in LPS-activated RAW 264.7 macrophages,40 while natural NO inhibitor quercetin was selected as a positive control (IC50 = 15 ± 1 μM). The cell viability was determined first by the MTT method to determine whether the inhibition of NO production was due to the cytotoxicity of the tested I



Article

an internal standard. HRESIMS spectra were obtained on a Bruker APEX IV 70 eV FT-MS spectrometer and on a Thermo DFS spectrometer using a matrix of 3-nitrobenzyl alcohol. EIMS data (70 eV) were recorded on a Finnigan MAT 95 mass spectrometer. Column chromatography was carried by silica gel (200−300 mesh), and HF254 silica gel for TLC was obtained from Qingdao Marine Chemistry Co. Ltd. Sephadex LH-20 (18−110 μm) was obtained from Pharmacia. HPLC was performed with an Alltech 426 pump employing a UV detector, and a Prevail C18 column (semipreparative, 5 μm) was used for semipreparative HPLC separation. A chiral-phase column (Phenomenex Lux, cellulose-2, 250 × 10 mm, 5 μm) was used for enantioselective analyses. Fungal Strain and Identification. The fungus Acremonium sp. (TVG-S004-0211) was isolated from deep-sea sediment that was collected from the South Atlantic Ocean (GPS 13.7501 W, 15.1668 S) at a depth of 2869 m, in June 2011. The fungal strain was identified as Acremonium sp. based on microscopic examination and by internal transcribed spacer (ITS) sequencing. The sequence of the ITS of the fungus has been deposited in GenBank (http://www.ncbi.nlm.nih. gov) with accession number KU145487. The strain TVG-S004-0211 is deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China. Fermentation. The fermentation was carried out in 30 Fernbach flasks (500 mL), each containing 80 g of rice. Distilled H2O (100 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 40 days. Extraction and Isolation. The fermented material was extracted with petroleum ether (PE) (3 × 500 mL), EtOAc (3 × 500 mL), n-butanol (BuOH) (3 × 500 mL), and H2O (3 × 500 mL), successively. Each extract was evaporated to dryness under vacuum to afford a PE extract, an EtOAc extract, a BuOH extract, and an H2O extract. A bioassay for LPS-induced NO production in RAW 264.7 macrophage cells revealed that the EtOAc extract possessed antioxidative effects. The EtOAc extract (2.8 g) was then subjected to silica gel (200−300 mesh) vacuum liquid chromatography, eluting with PE/EtOAc (from 5:1 to 0:1, gradient) to obtain five fractions (F1 to F5). Fraction F2 (0.6 g) was chromatographed over C18 silica gel (ODS, MeOH/H2O = 75:25) to obtain three subfractions (SF2a− SF2c). SF2b (120 mg) was purified on a semipreparative reversedphase (RP) HPLC column using MeCN/H2O (3:2, 2 mL/min) as a mobile phase to obtain 2 (10.0 mg), 3 (14.5 mg), 4 (3.6 mg), 5 (5.2 mg), and 8 (4.8 mg). SF2a (126 mg) was subjected to RP-HPLC with a mobile phase of MeCN/H2O (1:1, 2 mL/min) to yield 6 (8 mg) and 18 (3 mg). Fraction F3 (0.68 g) was subjected to an ODS column eluting with a gradient of MeOH/H2O from 3:7 to 7:3, affording five subfractions (SF3a−SF3e). SF3a (20 mg) was purified on a silica gel column (CH2Cl2/MeOH, 80:1) to afford 10 (12.7 mg). SF3b (26 mg) was separated by RP-HPLC eluting with MeCN/H2O (1:1) to collect 1 (4.2 mg), 9 (2.1 mg), 12 (2.5 mg), 15 (4.3 mg), 21 (2.3 mg), and 22 (1.8 mg). SF3c (18 mg) was subjected to an RPHPLC column eluting with MeCN/H2O (1:1) to obtain 13 (4.6 mg) and 19 (2.5 mg). SF3d (55 mg) was chromatographed on an RPHPLC column with MeCN/H2O (3:2) as a mobile phase to obtain 7 (10.2 mg), 16 (2.3 mg), 11 (2.4 mg), 14 (1.5 mg), 17 (8 mg), and 20 (2.8 mg). Acremeremophilane A (1): colorless oil; [α]25D +244 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 280 (4.22); ECD (c 3.8 × 10−4 M, MeOH) λmax (Δε) 226 (+9.24), 277 (+7.04), 350 (+2.74); IR (KBr) νmax 3412, 2926, 1706, 1654, 1626, 1198 cm−1; 1H and 13C NMR data, Tables 1 and 3; HRESIMS m/z 261.1129 [M − H]− (calcd for C15H17O4, 261.1132), m/z 285.1103 [M + Na]+ (calcd for C15H18O4Na, 285.1097), m/z 245.1179 [MH − H2O]+ (calcd for C15H17O3, 245.1172). Acremeremophilane B (2): colorless oil; [α]25D +320 (c 0.1, CH2Cl2); UV (MeOH) λmax (log ε) 274 (4.27); ECD (c 4.2 × 10−4 M, MeOH) λmax (Δε) 239 (+6.82), 278 (+8.05), 343 (+4.96); IR (KBr) νmax 3435, 2935, 1729, 1664, 1630, 1444, 1375, 1247, 1163 cm−1; 1H and 13C NMR data, Tables 1 and 3; HRESIMS m/z 359.1858

Table 4. Inhibitory Effects of Compounds 1−22 on LPSActivated NO Production in RAW 264.7 Macrophage Cells no.

% inhibition (50 μM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 quercetin

11 97 46 66 92 63 16 29 7 15 30 7 12 68 23 22 4 24 16 36 21 11

IC50 (μM)a 8 45 25 15 26

± ± ± ± ±

2 1 1 1 2

22 ± 1

15 ± 1

CC50 (μM)a >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50

a

IC50: 50% inhibitory concentration, CC50: 50% cytotoxic concentration.

assay against NF-κB. However, the tested compounds showed weak inhibition toward NF-κB with IC50 values more than 72 μM (Table 5), whereas the positive control PDTC (pyrrolidine Table 5. Inhibitory Effects of 2 and 4−6 against NF-κB

a

no.

% inhibition (50 μM)

IC50 (μM)

2 4 5 6 PDTCa

40 38 42 20

78 ± 1 72 ± 1 37 ± 1

PDTC: pyrrolidine dithiocarbamate.

dithiocarbamate) inhibited NF-κB with an IC50 value of 37 μM. These data suggested that the active compounds targeted a protein other than NF-κB to inhibit the NO production. In summary, this work reports the structure elucidation of 15 new eremophilane-type sesquiterpenes, which provides additional evidence to support deep-sea-derived microorganisms as a new source of chemical diversity. The PKS-derived 4-hexenoic unit in 2−6 is rarely found in nature. Compounds 2 and 5 exhibited potent inhibition against NO production in LPS-activated RAW 264.7 macrophages, suggesting them to be a new chemical entity for anti-inflammatory effects.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on an Autopol III automatic polarimeter (Rudolph Research Co., Ltd.). UV spectra were measured on a Cary 300 spectrometer. ECD spectra were measured on a JASCO J-810/J-815 spectropolarimeter. IR spectra were measured on a Thermo Nicolet Nexus 470 FT-IR spectrometer. The 1H and 13C NMR spectra were recorded on a Bruker Avance-400FT NMR spectrometer using tetramethylsilane as J



Article

IR (KBr) νmax 2923, 2853, 1776, 1628, 1219 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 325.1051 [M + Na]+ (calcd for C17H18O5Na, 325.1046). Acremeremophilane O (15): colorless oil; [α]25D −290 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 319 (4.3); ECD (c 2.9 × 10−4 M, MeOH) λmax (Δε) 219 (−7.56), 253 (+1.55), 316 (−4.80); IR (KBr) νmax 3396, 2924, 1764, 1644, 1037 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 263.1285 [M + H]+ (calcd for C15H19O4, 263.1283), m/z 263.1285 [MH − H2O]+ (calcd for C15H19O4, 263.1283). Compound 16: [α]20D +68 (c 0.10, MeOH), [α]20D +65 (c 0.03, MeOH) for guignarderemophilane B in the literature.15 MPA Esterification of 7. To a CH2Cl2 solution (0.5 mL) of 7 (1.4 mg, 0.0051 mmol) were added (R)-MPA (2.0 mg, 0.012 mmol), DMAP (0.8 mg, 0.006 mmol), and N,N′-dicyclohexylcarbodiimide (DCC, 2.5 mg, 0.012 mmol). After reacting at room temperature (rt) for 1 h, the products were separated by silica gel column chromatography eluting with CH2Cl2/MeOH (100:1) to afford the (R)-MPA ester (1.3 mg). By the same protocol, the (S)-MPA ester (1.3 mg) was prepared from (S)-MPA. (R)-MPA ester of 7: 1H NMR (CDCl3, 400 MHz) δH 5.74 (1H, d, J = 1.9 Hz, H-9), 4.97 (1H, br s, H-12a), 4.84 (1H, m, H-2), 4.80 (1H, br s, H-12b), 3.11 (1H, dd, J = 14.5, 4.5 Hz, H-7), 2.44 (1H, ddd, J = 13.8, 4.8, 1.9 Hz, H-1α), 2.31 (1H, ddd, J = 13.8, 12.8, 1.9 Hz, H-1β), 1.98 (1H, dd, J = 13.1, 4.6 Hz, H-6β), 1.82 (1H, dd, J = 14.5, 13.1 Hz, H-6α), 1.91 (1H, m, H-3a), 1.72 (3H, s, CH3-13), 1.55 (2H, m, H-3b and H-4), 1.16 (3H, s, CH3-14), 0.94 (3H, d, J = 6.3 Hz, H-15); ESIMS m/z 383.33 [M + H]+, 405.24 [M + Na]+. (S)-MPA ester of 7: 1H NMR (CDCl3, 400 MHz) δH 5.79 (1H, d, J = 1.9 Hz, H-9), 4.98 (1H, s, H-12a), 4.84 (1H, m, H-2), 4.81 (1H, s, H-12b), 3.12 (1H, dd, J = 14.4, 4.5 Hz, H-7), 2.60 (1H, ddd, J = 13.7, 4.7, 1.9 Hz, H-1α), 2.44 (1H, ddd, J = 13.7, 12.8, 1.9 Hz, H-1β), 1.98 (1H, dd, J = 13.2, 4.5 Hz, H-6β), 1.82 (1H, dd, J = 14.4, 13.2 Hz, H6α), 1.74 (1H, m, H-3a), 1.72 (3H, s, CH3-13), 1.50 (1H, m, H-4), 1.48 (1H, m, H-3b), 1.16 (3H, s, CH3-14), 0.90 (3H, d, J = 6.3 Hz, H15); ESIMS m/z 383.33 [M + H]+, 405.43 [M + Na]+. Transformation of 2 to 1. Compound 2 (1.5 mg) was stirred with 0.5 mL of 0.1 M NaOH (MeOH/H2O, 5:1) for 2 h at rt; then the pH was adjusted to 8 with 0.1 M HCl. The mixture was subjected to Sephadex LH-20 chromatography using MeOH as eluent to afford a product that was identical to 1 by the comparable 1H NMR data and specific rotation. The specific rotation of 1 as derived from 2 was [α]25D +238 (c 0.10, MeOH). Acetylation of 7 to Generate 8. To a pyridine solution (0.5 mL) of compound 7 (2 mg) was added acetic anhydride (200 μL). The solution was stirred at rt for 12 h and quenched by adding 0.1 mL of H2O. After removing solvent under vacuum, the residue was purified on a silica gel column eluting with CH2Cl2 to afford a product (1.9 mg) that was identical to compound 8 by comparisons of the 1 H NMR data, Rf value in TLC, and specific rotations. The specific rotation of 8 as derived from 7 was [α]25D +91 (c 0.10, CH2Cl2). Reduction of 9 and 17. To a MeOH (0.5 mL) solution of 17 (2.0 mg) was added NaBH4 (1.0 mg), and the mixture was allowed to stir at rt for 15 min. The reacted mixture was purified on a Sephadex LH-20 column using MeOH as eluent to afford a product (1.5 mg) that was identical to compound 18 by the comparisons of the 1H NMR data, Rf value in TLC, and specific rotations. Following the same protocol as for the reduction of 17, compound 9 was converted to 10. The specific rotation of 10 as derived from 9 was [α]25D −52 (c 0.1, CH2Cl2). Compound 18: [α]25D −51.0 (c 0.10, CH2Cl2); [α]20D −49 (c 0.05, CHCl3) in the literature.32 Transformation of 17 to 11. Compound 17 (2.0 mg) was acetylated in pyridine solution (0.5 mL) by adding acetic anhydride (200 μL). The solution was stirred at rt for 12 h and quenched by adding 0.1 mL of H2O to obtain acetylated 17, which was then reduced by NaBH4. The mixture was then purified on a Sephadex LH-20 column eluting with MeOH to afford a product that was identical to 11 by the comparison of their 1H NMR data, Rf value, MS data, and specific rotation. The specific rotation of 11 as derived from 17 was [α]25D −18.0 (c 0.1, CH2Cl2).

[M + H]+ (calcd for C21H27O5, 359.1853), m/z 381.1674 [M + Na]+ (calcd for C21H26O5Na, 381.1672), m/z 341.1751 [MH − H2O]+ (calcd for C21H25O4, 341.1747). Acremeremophilane C (3): colorless oil; [α]25D +412 (c 0.1, CH2Cl2); UV (MeOH) λmax (log ε) 301 (4.16); ECD (c 3.6 × 10−4 M, MeOH) λmax (Δε) 224 (+11.37), 275 (+9.32), 374 (+2.24); IR (KBr) νmax 3430, 2924, 1729, 1656, 1625, 1239, 1163 cm−1; 1H and 13C NMR data, Tables 1 and 3; HRESIMS m/z 359.1856 [M + H]+ (calcd for C21H27O5, 359.1853), m/z 381.1670 [M + Na]+ (calcd for C21H26O5Na, 381.1672), m/z 341.1754 [MH − H2O]+ (calcd for C21H25O4, 341.1747). Acremeremophilane D (4): colorless oil; [α]25D +330 (c 0.1, CH2Cl2); UV (MeOH) λmax (log ε) 259 (4.23), 274 (4.02); ECD (c 2.7 × 10−4 M, MeOH) λmax (Δε) 246 (+13.34), 280 (−7.28), 344 (+4.42); IR (KBr) νmax 2926, 2850, 1732, 1664, 1631, 1243, 1161 cm−1; 1H and 13C NMR data, Tables 1 and 3; HRESIMS m/z 373.2015 [M + H]+ (calcd for C22H29O5, 373.2010), m/z 395.1832 [M + Na]+ (calcd for C22H28O5Na, 395.1829). Acremeremophilane E (5): colorless oil; [α]25D +170 (c 0.1, CH2Cl2); UV (MeOH) λmax (log ε) 224 (3.95), 244 (3.82), 300 (3.61); ECD (c 5.6 × 10−4 M, MeOH) λmax (Δε) 221 (+8.78), 253 (−2.46), 281 (+6.75); IR (KBr) νmax 3459, 2921, 2851, 1735, 1660, 1628, 1366, 1230, 1163 cm−1; 1H and 13C NMR data, Tables 1 and 3; HRESIMS m/z 359.1863 [M + H]+ (calcd for C21H27O5, 359.1858), m/z 381.1681 [M + Na]+ (calcd for C21H26O5Na, 381.1678). Acremeremophilane F (6): colorless oil; [α]25D +230 (c 0.2, CH2Cl2); UV (MeOH) λmax (log ε) 220 (3.93), 247 (3.47), 303 (3.73); ECD (c 5.3 × 10−4 M, MeOH) λmax (Δε) 222 (+8.56), 254 (−5.26), 281 (+6.74); IR (KBr) νmax 3449, 2968, 2936, 1732, 1657, 1618, 1165 cm−1; 1H and 13C NMR data, Tables 1 and 3; HRESIMS m/z 375.1816 [M + H]+ (calcd for C21H27O6, 375.1808), m/z 397.1632 [M + Na]+ (calcd for C21H26O6Na, 397.1630). Acremeremophilane G (7): colorless oil; [α]25D +120 (c 0.2, CH2Cl2); UV (MeOH) λmax (log ε) 241 (3.95); ECD (c 4.3 × 10−4 M, MeOH) λmax (Δε) 229 (+8.97), 324 (−1.17); IR (KBr) νmax 3425, 2926, 1665, 1196 cm−1; 1H and 13C NMR data, Tables 1 and 3; HRESIMS m/z 235.1698 [M + H]+ (calcd for C15H23O2, 235.1693). Acremeremophilane H (8): colorless oil; [α]25D +88 (c 0.3, CH2Cl2); UV (MeOH) λmax (log ε) 237; IR (KBr) νmax 2942, 1735, 1674, 1367, 1241, 1208 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 277.1808 [M + H]+ (calcd for C17H25O3, 277.1804). Acremeremophilane I (9): colorless oil; [α]25D −140 (c 0.04, CH2Cl2); UV (MeOH) λmax (log ε) 241 (3.94); ECD (c 4.0 × 10−4 M, MeOH) λmax (Δε) 241 (−6.76), 316 (+1.61); IR (KBr) νmax 3442, 2917, 1695, 1644, 1368 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 251.1644 [M + H]+ (calcd for C15H23O3, 251.1642), m/z 273.1463 [M + Na]+ (calcd for C15H22 O3Na, 273.1461). Acremeremophilane J (10): colorless oil; [α]25D −53 (c 0.2, CH2Cl2); IR (KBr) νmax 3328, 2927, 1456, 1298 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 297.1701 [M + HCOO]− (calcd for C16H25O5, 297.1707). Acremeremophilane K (11): colorless oil; [α]25D −19 (c 1.5, CH2Cl2); IR (KBr) νmax 3403, 2925, 2855, 1735, 1373, 1240 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 301.1780 [M + Na]+ (calcd for C17H26O3Na, 301.1775). Acremeremophilane L (12): colorless oil; [α]25D −102 (c 1.5, CH2Cl2); UV (MeOH) λmax (log ε) 274 (4.32); ECD (c 3.8 × 10−4 M, MeOH) λmax (Δε) 234 (+1.23), 277 (−3.37); IR (KBr) νmax 3432, 2965, 1761, 1651, 1373 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 263.1284 [M + H]+ (calcd for C15H19O4, 263.1278), m/z 245.1180 [MH − H2O]+ (calcd for C15H17O3, 245.1178). Acremeremophilane M (13): colorless oil; [α]25D +140 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 273 (4.30); ECD (c 1.1 × 10−4 M, MeOH) λmax (Δε) 222 (−1.56), 270 (+6.20); IR (KBr) νmax 3448, 1769, 2968, 1655, 1373 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 261.1131 [M − H]− (calcd for C15H17O4, 261.1132), m/z 307.1188 [M + HCOO]− (calcd for C16H19O6, 307.1182). Acremeremophilane N (14): colorless oil; [α]25D −86 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 340 (4.33); ECD (c 3.31 × 10−4 M, MeOH) λmax (Δε) 219 (−3.72), 247 (+3.76), 330 (+5.27), 383 (−9.28); K



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Cell Culture. The RAW 264.7 mouse macrophage cell line was purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), and was cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco Invitrogen Corp.), which was supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. The cells were placed at 37 °C in a humidified incubator containing 5% CO2. The inhibitory activity of the isolated compounds toward NO production was determined using the Griess reagent system (Beyotime). Cells were cultured in each well of 96-well plates at a density of 5 × 104 cells/well with 100 μL of DMEM for 24 h. Test samples dissolved in DMSO and diluted with DMEM were added as well as 2 μg/mL LPS as a stimulus. The final drug concentration was 50 μM with 1% DMSO. Wells treated with only LPS served as model controls, and wells treated with neither LPS nor test samples served as blank controls (all contained 1% DMSO). After 24 h incubation, half of the medium (50 μL) in each well was harvested. MTT Assay. The cytotoxicity of the isolated compounds toward RAW 264.7 cells was determined by the MTT assay. RAW264.7 cells were plated in 96-well plates (5 × 103/well) for 24 h. Then they were treated with test samples that were dissolved in DMSO and diluted in 100 μL of DMEM, making the final drug concentration 50 μM and 1% DMSO; 1% DMSO served as a vehicle control. Wells without cells containing only 100 μL of DMEM served as blank controls. MTT solution (20 μL) was added to each well after 24 h. After incubation for 4 h, the medium was removed and 100 μL of DMSO was added in each well; then the absorbance (A) was detected at 490 nm using a microplate reader. The inhibition of cell growth was calculated according to the following formula:

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01103. NMR spectra for acremeremophilanes A−O (1−15) and compound 16 including 1H, 13C, and 2D NMR, IR, and HRMS spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (Z.-M. Zhao): [email protected]. *Tel (W. Lin): +86-10-82806188. Fax: +86-10-82806188. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the 973 Program (2015CB755906), NSFC-Shangdong Join Fund for Marine Science (U1406402), the National Hi-Tech 863-Projects (2011AA090701, 2013AA092902), COMRA (DY125-15-T-01), and Sino-German Project GZ816.

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REFERENCES

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%Inhibition = [1 − (A sample − A blank )/(A solvent − A blank )] × 100 Inhibitory Activity against NO Production. Nitric oxide release was assessed by a colorimetric assay based on a diazotization reaction using the Griess reagent system. After 50 μL of Griess reagent I and 50 μL of Griess reagent II were added to each well, the absorbance was measured at 540 nm using a microplate reader. The inhibition of NO release was calculated according to the following formula: %Inhibition = [1 − (A sample − A blank )/(A model − A blank )] × 100 Inhibitory Activity toward NF-κB. The RAW264.7 cells (transfected with pNF-κB-Luc reporter plasmid presented by Professor Li Xiaojuan, Institute of Pharmacology, Southern Medical University) were cultured in DMEM, supplemented with 10% FBS, penicillin (100 units/mL), streptomycin (100 μg/mL), and geneticin (500 μg/mL) under a humidified 5% CO2 and 95% air atmosphere at 37 °C for 24 h. The medium was then removed and pretreated with different concentrations of samples (0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 μM) for 1 h prior to being stimulated with LPS (1 mg/mL, Escherichia coli 055:B5) for further incubation. After 6 h, the medium was removed and 200 μL of cold phosphate-buffered saline (PBS) was added to wash each well. The cold PBS was removed, cells were lysed with cell culture lysis reagent for 15 min, and then the cell lysis solution was added to 96-well white opaque plates and mixed with the same volume of luciferase assay system reagent. Pyrrolidinedithiocarbamate was used as a positive control. Finally the value of luminescence (LUM) in each well was measured by an automated microplate reader with 250 ms of integration time. The luciferase activity was evaluated with the following equation: % Inhibition = [1 − (LUMsample − LUMblank)/(LUMmodel − LUMblank)] × 100, where LUMLPS is the value of the cells stimulated with LPS, LUMsample is the value of a tested sample, and LUMblank is the value of the cell without LPS stimulation. It should be mentioned that no obvious cytotoxic effects were observed in the selected concentration range. The value was expressed as the mean ± standard deviation for triplicate experiments. Nonlinear regression (with sigmoidal dose response) was used to calculate the IC50 values using GraphPad Prism (GraphPad Software, Inc.). L



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Eremophilane-Type Sesquiterpenoids from an Acremonium sp

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