Indole-Diterpenoids with Anti-H1N1 Activity from the Aciduric Fungus

Article pubs.acs.org/jnp

Indole-Diterpenoids with Anti-H1N1 Activity from the Aciduric Fungus Penicillium camemberti OUCMDZ-1492 Yaqin Fan,† Yi Wang,† Peipei Liu, Peng Fu, Tonghan Zhu, Wei Wang, and Weiming Zhu* Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China S Supporting Information *

ABSTRACT: An aciduric fungal strain, Penicillium camemberti OUCMDZ-1492, was isolated from an acidic marine niche, mangrove soil and mud, around the roots of Rhizophora apiculata. Six new indole-diterpenoids (1−6), along with five known analogues, emindole SB (7), 21-isopentenylpaxilline (8), paspaline (9), paxilline (10), and dehydroxypaxilline (11), were isolated from the fermentation broth of P. camemberti OUCMDZ-1492 grown at pH 5.0. On the basis of spectroscopic analyses, CD spectra, quantum ECD calculations, and chemical methods, new structures 1−6 were established as 3-deoxo-4b-deoxypaxilline, 4a-demethylpaspaline-4a-carboxylic acid, 4a-demethylpaspaline-3,4,4a-triol, 2′hydroxypaxilline, 9,10-diisopentenylpaxilline, and (6S,7R,10E,14E)-16-(1H-indol-3-yl)-2,6,10,14-tetramethylhexadeca-2,10,14triene-6,7-diol, respectively. Compounds 1−3 and 5−10 exhibited significant activity against the H1N1 virus with IC50 values of 28.3, 38.9, 32.2, 73.3, 34.1, 26.2, 6.6, 77.9, and 17.7 μM, respectively. The results showed that 3-oxo, 4b-hydroxy, and 9isopentenyl substitutions tend to increase the anti-H1N1 activity of hexacyclic indole-diterpenoids.

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antibiotic activities.16,17 Six new indole-diterpenoid alkaloids (1−6), as well as five known analogues, emindole SB (7),6 21isopentenylpaxilline (8),7 paspaline (9),8 paxilline (10),6 and dehydroxypaxilline (11),9 were isolated and identified. Compared with ribavirin (IC50 113.1 μM), compounds 1−3 and 5−10 exhibited significant activity in vitro against the H1N1 virus with IC50 values of 28.3, 38.9, 32.2, 73.3, 34.1, 26.2, 6.6, 77.9, and 17.7 μM, respectively.

he influenza A virus subtype H1N1, which was last involved in an outbreak in 2009, generated widespread concern about dire consequences posed by a familiar strain of influenza virus in mammals.1 A strain of the same H1N1 subtype caused the pandemic Spanish flu in 1918, which infected 5% of the world population and led to 20−50 million deaths worldwide.2 Only a few drugs are available to treat H1N1 viral infections, and most are expensive. In addition, a case of Tamiflu-resistant H1N1 flu has been reported in Denmark.3 Thus, it has become imperative to develop new and effective antiviral drugs to combat H1N1 influenza A virus infections. In our continuing efforts to discover new antiH1N1-active compounds from aciduric fungi,4,5 we investigated acidic marine niches including mangrove soil and mud. The aciduric fungal strain OUCMDZ-1492 identified as Penicillium camemberti was isolated from the mud (pH 5.0) around the roots of Rhizophora apiculata (Rhizophoraceae), which was collected from the Wenchang mangrove natural reserve area of China in June 2010. The EtOAc extract of the fermentation broth of P. camemberti OUCMDZ-1492 grown at pH 5 showed richer chemical diversity than those grown at pHs 2.5, 3, 4, 6, 7, and 8 (Figure S58) and displayed a series of peaks with similar UV absorptions to those of indole-diterpenoids at 228 and 280 nm in the HPLC-UV profile.6−9 Indole-diterpenoids are known as a large and structurally diverse group of fungal secondary metabolites10 that possess a common cyclic diterpene backbone derived from geranylgeranyl diphosphate and an indole group derived from indole-3-glycerol phosphate.11 They are known as tremorgenic mycotoxins12,13 and display anti-insectan14,15 and © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The bioactive EtOAc extract of the fermentation broth of P. camemberti OUCMDZ-1492 was chromatographed on silica gel, Sephadex LH-20, and preparative HPLC columns to give compounds 1−11. Compound 1 was obtained as a colorless crystal. The molecular formula was C27H35NO2 on the basis of a HRESIMS peak at m/z 406.2741 [M + H]+, indicating 11 degrees of unsaturation. Its UV spectrum showed characteristic peaks of an indole chromophore at λmax 229 and 280 nm.6−9 The IR spectrum further indicated the presence of an indole nucleus at 3397, 1454, 1099, and 750 cm−1.18 The 1H and 13C NMR spectra of 1 are very similar to those of dehydroxypaxilline (11),6 indicating that it was an indole-diterpenoid. The detailed comparison of 1D NMR data between 1 and 11 revealed a replacement of the carbonyl (δC 196.5 in 11) by the −CH2− Received: April 10, 2013 Published: July 2, 2013 1328

dx.doi.org/10.1021/np400304q | J. Nat. Prod. 2013, 76, 1328−1336

Journal of Natural Products

Article

Table 1. 1H and 13C NMR Data for Compounds 1−5 (600, 150 MHz, DMSO-d6, TMS, δ ppm) 1 position

δC

2

79.6, CH

3

29.5, CH2

4

115.7, CH

2 δH (J in Hz) 3.15, t (11.3) 2.00, m 1.94, d (12.3) 5.36, d (5.4)

3

δC

δH (J in Hz)

δC

84.3, CH

3.12, dd (11.1, 2.2) α 1.65, m

82.6, CH

3.04, s

71.5, CH

3.93, dd (3.2, 2.8)

198.8, C

73.5, CH

3.35, dd (8.7, 2.8)

119.3, CH

22.8, CH2

5a

4 δH (J in Hz)

δC 78.7, CH

δH (J in Hz) 3.95, s

δC 83.5, CH

δH (J in Hz) 3.66, d (1.9)

197.6, C

β 1.18, m 35.3, CH2

α 1.23, m

5.79, s

118.9, CH

5.75, d (1.9)

β 2.35, d (11.4) 4a 4b

140.8, C 40.8, CH

5

24.4, CH2

6

25.8, CH2

6a 7

49.1, CH 27.0, CH2

7a 7b 8

115.7, C 124.4, C 117.7, CH

9

118.5, CH

10

119.4, CH

11

111.9, CH

11a 12a 12b 12c 13

140.1, 151.0, 49.8, 40.2, 31.4,

C C C C CH2

2.16, d (12.3) 1.68, m 1.59, m 1.53, m 1.44, m 2.66, m 2.59, dd (6.2, 13.4) 2.29, t (11.2)

7.25, d (7.6) 6.89, dd (7.6, 6.8) 6.92, dd (7.6, 6.8) 7.25, d (7.6)

1.96, m

47.3, C 46.1, CH

1.67, m

76.1, C 44.4, CH

1.63, m

21.6, CH2

α 1.61, m

24.4, CH2

α 1.66, m

32.6, CH2

25.1, CH2

β 2.53, m 1.53, m

20.9, CH2

24.8, CH2 48.7, CH 27.0, CH2

25.1, CH2

14a

76.0, CH

1.94, m 2.02, m 3.97, m

1′ 2′ 3′

70.3, C 24.9, CH3 26.7, CH3

1.10, s 1.05, s

12b-Me 12c-Me 1′-OH 3′-OH 3-OH 4-OH 4a-OH 4b-OH 4a-CO2H NH

14.7, CH3 15.6, CH3

0.97, s 0.80, s 4.20, s

48.1, CH 27.1, CH2

β 2.24, dd (12.9, 10.6) 115.9, C 124.4, C 117.7, CH

7.24, d (7.9)

118.5, CH

6.89, t (7.9)

119.4, CH 111.9, CH 140.3, 151.0, 51.7, 39.9, 32.4,

C C C C CH2

25.2, CH2 83.7, CH

2.68, m α 2.55, dd (13.0, 6.2)

49.4, CH 26.9, CH2

β 2.21, dd (13.0, 10.9) 115.8, C 124.5, C 117.6, CH

1.80, m 14

β 1.81, m α 1.50, m β 1.69, m 2.61, m α 2.57, dd (12.9, 6.4)

168.8, C 75.8, C

168.9, C 75.7, C α 1.67, d (12.4) β 1.89, m α 1.63, m β 1.92, m 2.72, m α 2.62, dd (5.9, 12.8)

32.6, CH2

20.9, CH2 49.3, CH 27.0, CH2

β 2.32, t (11.7)

α 1.87, m β 1.64, m α 1.58, m β 1.90, m 2.68, m α 2.56, m β 2.26, m

7.24, d (6.9)

115.0, C 124.5, C 117.7, CH

7.27, d (7.6)

114.4, C 123.0, C 117.6, CH

118.4, CH

6.88, t (7.1)

118.5, CH

6.90, t (7.6)

129.6, C

6.93, t (7.9)

119.3, CH

6.91, t (7.1)

119.1, CH

6.93, t (7.6)

130.9, C

7.26, d (7.9)

111.9, CH

7.25, d (7.0)

111.9, CH

7.26, d (7.6)

111.6, CH

7.00, s

α 1.83, m

140.3, 151.5, 51.8, 39.9, 32.1,

α 2.57, m

138.9, 152.2, 50.2, 42.5, 26.0,

α 1.77, m

β 1.97, dt (12.6, 3.1) α 1.61, m β 2.54, m 3.10, dd (11.0, 2.3)

C C C C CH2

23.7, CH2 82.4, CH

70.4, C 24.5, CH3 27.0, CH3

0.99, s 1.09, s

71.7, C 26.6, CH3 27.3, CH3

14.5, CH3 16.2, CH3

0.92, s 0.97, s

14.6, CH3 18.7, CH3

α 1.77, td (13.2, 3.7) β 1.90, dt (13.2, 3.1) α 2.06, m β 1.65, m 3.07, dd (3.8,11.7) 1.18, s 1.19, s

0.91, s 1.12, s 4.83, s

139.9, 152.7, 50.3, 42.5, 26.1,

C C C C CH2

C C C C CH2

β 1.82, m 28.4, CH2 72.4, CH 74.2, C 20.5, CH3 66.1, CH2

16.3, CH3 18.8, CH3

α 2.22, m β 1.78, m 4.80, dd (9.3, 8.2) 1.08, s 3.42, dd (6.4, 16.1) 3.35, dd (6.4, 16.1) 1.25, s 0.90, s 4.35, s 4.58, t (6.4)

7.01, s

β 2.54, m 28.4, CH2 72.5, CH 71.0, C 26.0, CH3 26.1, CH3

16.2, CH3 18.8, CH3

α 2.22, m β 1.75, m 4.81, t (8.5) 1.20, s 1.16, s

1.21, s 0.87, s 4.35, s

5.58, d (3.2) 4.31, d (8.6) 4.03, s 4.99, s

4.97, s

10.73, br s

10.46, br s

175.4 10.70, br s

10.59, br s

10.55, br s

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Table 1. continued The NMR data for prenyl moieties: δ 31.3 (CH2, C-1″), 124.1 (CH, C-2″), 130.7 (C, C-3″), 17.8 (CH3, C-4″), 25.9 (CH3, C-5″), 31.7 (CH2,C1′″), 124.6 (CH,C-2′″), 130.3 (C,C-3′″), 17.7 (CH3, C-4′″), and 25.6 (CH3, C-5′″), and 3.29 (d, 4H, J = 6.8 Hz, H-1″/1′″), 5.24 (t, J = 7.0 Hz, H2″), 1.67 (s,3H, H-4″), 1.71 (s, 3H, H-5″), 5.19 (t, J = 7.0 Hz, H-2′″), and 1.68 (s, 6H, H-4′″/5′″), respectively. a

Figure 1. Key COSY and HMBC correlations of compounds 1−6.

Compound 2 was obtained as a white, amorphous powder, and the molecular formula C28H37NO4 was assigned on the basis of the HRESIMS peak at m/z 474.2613 [M + Na]+. The similar IR and UV absorptions to those of 1 implied that it was an analogue. Except for a carboxy signal (δC 175.4) substitution of methyl signals (δC/H 14.0/0.94), the 1D NMR spectra were very similar to those of paspaline (9).8 Careful comparison of the NMR data between 2 and 9 revealed that C-4 and C-14a of 2 shifted upfield, while C-4a shifted downfield (Tables 1 and S1). In addition, the HMBC experiment exhibited long-rang correlations of H-4b (δH 1.67) and H-14a (δH 3.10) to the carboxy carbon (δC 175.4), of H2-4 (δH 2.35/1.23) and H-2 (δH 3.12) to C-14a (δC 83.7), and of H-4b to C-4a (δC 47.3). These data indicated that the 4a-methyl of 9 has been oxidized in compound 2 to yield a carboxylic acid. The NOESY spectrum

group (δC/H 29.5/1.94 and 2.00 in 1). In addition, obvious upfield shifts for C-2, C-4, C-4a, and C-1′ were observed (Tables 1 and S1). These data suggested compound 1 as the deoxo derivative of 11 at C-3, which was further verified by the key COSY correlations of H-2/H2-3/H-4 and the key HMBC correlations from H-4 (δH 5.36) to C-4b (δC 40.8), from H-4 and H-2 (δH 3.15) to C-14a (δC 76.0), and from H-2′ (δH 1.10) and H-3′ (δH 1.05) to C-2 (δC 79.6) (Figure 1). The relative configuration was determined by the single-crystal X-ray diffraction analysis (Figure S60). The final refinement of the Cu Kα data resulted in a −0.1(2) Flack parameter, allowing an unambiguous assignment of the absolute configuration as 2S, 4bR, 6aS, 12bS, 12cS, and 14aS (Figure 3). Thus, compound 1 was elucidated as (2S,4bR,6aS,12bS,12cS,14aS)-3-deoxo-4bdeoxypaxilline. 1330



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Figure 2. Key NOESY correlations of compounds 2−5 and 6a.

UV and 1D NMR spectra to those of 1 suggested the same indole-diterpenoid skeleton. The differences in the 1D NMR data between 3 and 1 are that two oxygenated methine signals (δC/H 71.5/3.93 and 73.5/3.35) and an oxygenated quaternary carbon (δC 76.1) signal replace methylene (δC/H 29.5/2.00 and 1.94), vinyl methine (δC/H 115.7/5.36), and vinyl quaternary carbon (δC 140.8) signals (Table 1). The COSY correlations of H-2/H-3/H-4 along with HMBC (Figure 1) correlations from 3-OH (δH 5.58) to C-2 (δC 82.6), C-3 (δC 71.5), and C-4 (δC 73.5), from 4-OH (δH 4.31) to C-4 and C-4a (δC 76.1), and from 4a-OH (δH 4.03) to C-4 and C-4a indicated that C-3, C-4, and C-4a were all hydroxylated in compound 3. The NOESY spectrum showed correlations of H-4 (δ 3.35)/H-5β (δ 2.53)/ H-6a (δ 2.68)/H3-12c (δ 1.12)/H-13β (δ 1.90), H-4b (δ 1.63)/H-14a (δ 3.07)/H-13α (δ 1.77)/H3-12b (δ 0.91), H14a/H-2 (δ 3.04), and 4a-OH (δ 4.03)/3-OH (δ 5.58)/1′-OH (δ 4.83) (Figure 2), indicating the same relative configuration as 1 and a 3β,4α,4aβ-trihydroxy substitution. Additionally, the NOESY correlation between H-14a and H-2 suggested H-2 to be axial. Thus, small values of 3JH‑2,H‑3 (3.0 Hz) and 3JH‑4,H‑3 (3.0 Hz) implied that H-3 was equatorial, which further supported the β-orientation of 3-OH. The CD curve of 3 showed a negative Cotton effect at 240 (Δε −1.3) nm (Figure S2) similar to that of 1 (238 (Δε −3.7) nm), suggesting the same 4bR, 6aS, 12bS, and 12cS configurations. Thus, compound 3 was elucidated as (2S,3R,4R,4aS,4bR,6aS,12bS,12cS,14aS)-4a-demethylpaspaline-3,4,4a-triol. Compound 4 was obtained as a white, amorphous powder. The molecular formula of 4 was determined to be C27H33NO5 by the HRESIMS peak at m/z 474.2253 [M + Na]+, indicating 12 degrees of unsaturation. 1H and 13C NMR (Table 1) data analysis showed that 4 was an analogue of paxilline (10), which differs from compound 10 in the replacement of the methyl signals (δC/H 26.0/1.20) by hydroxymethyl signals (δC/H 66.1/ 3.42 and 3.35, 4.58). In addition, C-3/C-1′ and C-2/C-2′ of 4 showed obvious downfield and upfield shifts, respectively (Tables 1 and S1). These data implied that compound 4 is the 3′-hydroxy derivative of paxilline (10), which was further

Figure 3. ORTEP drawing of 1 (Cu Kα).

of 2 displayed a similar correlative pattern to those of 1 and 9 (Figure 2), indicating the same relative orientations of H-2α, H4bα, H-6aβ, H-14aα, H3-12bα, and H3-12cβ. In addition, the key NOESY correlation between H-4b (δ 1.63) and H-4α (δ 1.23) and no NOESY correlation between H3-12c (δ 0.97) and H3-2′ (δ 0.99) or H3-3′ (δ 1.09) suggested the β-orientation of CO2H-4a. This deduction was further supported by 13C NMR quantum chemical calculations of model compounds at the B3LYP/6-311++G(2d,p)//B3LYP/6-31G(d) level (Table S2).19 The magnetic shielding values were converted into chemical shifts after the corrections using the slope and intercept of the linear-square functions, and the relative errors of chemical shifts were computed by subtracting the calculated 13 C NMR from the measured shifts.19 The relative errors between the measured 13C shifts and the calculated 13C shifts are less than 7.1 ppm in 4aβ-2, while the maximum error at C14a reached 11.2 ppm in 4aα-2 (Table S2). The absolute configuration of 2 was determined by ECD calculations of 2 and ent-2 at the B3LYP/6-31G(d) level in Gaussian 03.20 The result showed that the CD curve of 2 is consistent with the calculated ECD curve of 2 but opposite that of ent-2 (Figure S4). Thus, the structure of compound 2 was elucidated as (2S,4aR,4bR,6aS,12bS,12cS,14aS)-4a-demethylpaspaline-4a-carboxylic acid. Compound 3 was isolated as a white, amorphous powder. The molecular formula of 3 was determined to be C27H37NO5 by analysis of 1H, 13C, and DEPT NMR data and was verified by a HRESIMS peak at m/z 478.2560 [M + Na]+. The similar 1331



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of H-10 (δ 3.37) to H-11 (δ 5.38), H-13b (δ 2.02) to H-14a (δ 2.07), H-17a (δ 1.86) to H-18b (δ 1.64), H-18a (δ 1.22) to 19OH (δ 4.30) through H-19 (δ 3.08), and H-21b (δ 1.42) to H23 (δ 5.07) through H-22b (δ 2.00) also supported the supposition. The key HMBC correlation between H-10 (δ 3.37) and C-3 (δ 113.8) connected the indole and diterpenoid alcohol moieties into 16-(1H-indol-3-yl)-2,6,10,14-tetramethylhexadeca-2,10,14-triene-6,7-diol. The NOESY correlations of H-11 (δ 5.38) with H-13a (δ 2.02) and H-15 (δ 5.12) with H17a (δ 2.16) indicated E-configurations of both Δ11 and Δ15 double bonds. To elucidate the relative configuration of 19,20diol, the acetonide (6a) was prepared. The NOESY correlations of H-19 (δ 3.61)/H3-27 (δ 1.11)/H3-2′ (δ 1.23) in 6a (Figure 2) clearly suggested an erythro-configuration of the 19,20-diol in 6. In addition, the NOESY correlations of H-10 (δ 3.37) with H3-29 (δ 1.72) and Hb-14 (δ 2.07) with H3-28 (δ 1.54) in 6a also indicated 11E- and 15E-configurations. The absolute configuration of the 19,20-diol in 6 was assigned by a dimolybdenum CD method. 21, 22 Upon addition of Mo2(OAc)4 to a solution of compound 6 in DMSO, a chiral dimolybdenum complex was generated in situ as an auxiliary chromophore. Because the contribution from the inherent CD was subtracted to give the induced CD of the complex, the observed sign of the Cotton effect in the induced spectrum originates solely from the chirality of the vic-diol moiety expressed by the sign of the O−C−C−O torsion angle. The negative CD Cotton effects at 299 (Δε −0.6) and 346 (Δε −0.3) nm (Figure S57) permitted us to assign the (19R, 20S)configurations on the basis of Snatzke’s empirical rule,21 with the large group pointing away from the remaining portion of the complex (Figure S57). Thus, the structure of 6 was unambiguously determined as (6S,7R,10E,14E)-16-(1H-indol3-yl)-2,6,10,14-tetramethylhexadeca-2,10,14-triene-6,7-diol. Comparison of the CD curves between the closely related sets of compounds 1 and 3, 5 and 8, and 4, 10, and 11 revealed that all compounds showed a strong negative Cotton effect at short wavelength (λmax 210−250 nm) that arose from the π−π* transitions of the indole ring, which could correspond to (6aS,12bS,12cR,4bS)- and (6aS,12bS,12cS,4bR)-configurations of hexacyclic indole-diterpenoids with and without 4b-OH substitutions, respectively.6,23,24 This deduction is further supported by the ECD calculations of two simplified analogues, (4aS,10bS,1S,2R)-model I and (4aR,10bR,1R,2S)-model II, at B3LYP/6-31G(d) level in Gaussian 03.20 The calculated ECD curve (Figures 6 and S4) of (4aS,10bS,1S,2R)-model I showed a similar Cotton effect at λmax 225 nm to the indolediterpenoids. These data combined with the comparisons of the CD curves of 1 and 9 with the calculated ECD curves of

confirmed by HMBC correlations from H-3′ (δH 3.42 and 3.35) to C-2 (δC 78.7), C-1′ (δC 74.2), and C-2′ (δC 20.5), from H-2 (δH 3.95) to C-3 (δC 198.8) and C-14a (δC 72.4), and from HO-3′ (δH 4.58) to C-3′ (δC 66.1). The same relative configuration as 1 and 10 could be deduced from the NOESY data of H-5β (δ 1.89)/H-6a (δ 2.72)/H3-12c (δ 0.90)/H-13β (δ 1.82), H-2 (δ 3.95)/H-14a (δ 4.80)/H-13α (δ 2.57)/H3-12b (δ 1.25), H3-12c/H-14β (δ 1.78), and H-5α (δ 1.67)/4b-HO (δ 4.99)/H-14a (Figure 2). Compound 4 displayed a similar CD Cotton effect at 242 (Δε −14.7) nm (Figure S2) and specific rotation ([α]25D −10.3) to those of 10 (240 (Δε −17.3) nm, [α]25D −21.6), indicating the same absolute configuration. Thus, compound 4 was identified as (2R,4bS,6aS,12bS,12cR,14aS)-2′-hydroxypaxilline. Compound 5 was isolated as a white, amorphous powder. The molecular formula of 5 was determined to be C37H49NO4 by the HRESIMS peak at m/z 594.3546 [M + Na]+, indicating 14 degrees of unsaturation. Analysis of 1D and 2D NMR spectra revealed that compound 5 is also an indole-diterpene with two additional isopentenyl substituents (Table 1). When compared with those of the monoisopentenyl-substituted indole-diterpene, 21-isopentenylpaxilline (8),7 the 1H NMR spectrum of 5 at δH 7.00 (s)/7.01 (s) suggested a 1,2,4,5tetrasubstituted phenyl nucleus rather than a 1,2,4-trisubstituted one (Tables 1 and S1), indicating an additional isopentenyl group located at position 10 of the indolediterpenoid skeleton. The HMBC correlations of H-11 (δH 7.00) to C-1′″ (δC 31.7), of H-1′″ (δH 3.29) to C-3′″ (δC 130.3), and of H-4′″ (δH 1.68) to C-2′″ (δC 124.6), C-3′″ and C-5′″ (δC 25.6) confirmed this conclusion. The NOESY spectrum of 5 showed correlations of H-2 (δ 3.66)/H-14a (δ 4.81)/H-13α (δ 1.77)/H3-12b (δ 1.21), H-6a (δ 2.68)/H3-12c (δ 0.87)/H-13β (δ 2.54), and H-14a/4b-OH (δ 4.97)/H3-12b, indicating trans-H-6a and Me-12b, trans-Me-12c and Me-12b, trans-Me-12c and H-14a, cis-H-2 and H-14a, and cis-H-14a and 4b-OH (Figure 2). The NOESY spectrum of 5 showed a similar correlation pattern to that of 4 (Figure 2), indicating the same relative configurations as those of 4 and 8. The similar CD Cotton effect at 249 (Δε −13.7) nm (Figure S2) and specific rotation ([α]25D −44.5) to those of 8 (242 (Δε −9.0) nm, [α]25D −12)7 indicated the same absolute configuration. The structure of compound 5 was thus elucidated as (2R,4bS,6aS,12bS,12cR,14aS)-9,10-diisopentenylpaxilline. Compound 6 was obtained as a brown oil with the molecular formula of C28H41NO2 based on a HREIMS peak at m/z 446.3027 [M + Na]+. The UV spectrum also showed the characteristic peak of an indole chromophore at λmax 228 and 280 nm. The 1H, 13C, and DEPT NMR spectra along with HMQC data displayed seven sp3 methylenes, three trisubstituted vinyl moieties, one oxymethine, one oxygenated quaternary carbon, and one β-substituted indole nucleus. The presence of β-substituted indole nucleus was further affirmed by the COSY correlations of H-5/H-6/H-7/H-8 and HMBC correlations of NH (δ 10.72), H-2 (δ 7.02), and H-5 (δ 7.45) to C-3 (δ 113.8), of NH and H-5 to C-4 (δ 127.1), and of H-8 (δ 7.32) to C-9 (δ 136.5). The remaining 20 carbons were speculated to be part of a diterpenoid alcohol. This supposition was confirmed by HMBC correlations from H3-29 (δ 1.72) to C-11 (δ 123.5), C-12 (δ 134.5), and C-13 (δ 39.8), from H3-28 (δ 1.56) to C-15 (δ 123.4), C-16 (δ 135.3), and C-17 (δ 36.8), from H3-27 (δ 0.94) to C-19 (δ 75.9), C-20 (δ 73.1), and C-21 (δ 38.8), and from H3-25 (δ 1.55) to C-23 (δ 125.5), C-24 (δ 130.0), and C-26 (δ 25.6). Furthermore, the COSY correlations

Figure 4. ORTEP drawing of 7 (Cu Kα). 1332



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epoxide 6b followed by cyclization and rearrangement produces 7. Compound 7 can be subjected to epoxidation, and then successive nucleophilic cleavage of the epoxide forms 9, which can undergo oxidation to produce 2. Formal elimination of the carboxylic acid of 2 generates compound 1, which can undergo oxidation steps to give compounds 3 and 11. Hydroxylation of 11 produces compounds 4 and 10. Isoprenylation of 10 then yields 5 and 8 (Scheme 1). The antiviral activities of compounds 1−11 against the H1N1 virus were evaluated by the CPE inhibition assay.27 When compared with ribavirin (IC50 113.1 μM), compounds 1−3 and 5−10 exhibited significant protection against H1N1 virus-induced cytopathogenicity in MDCK cells with IC50 values of 28.3, 38.9, 32.2, 73.3, 34.1, 26.2, 6.6, 77.9, and 17.7 μM, respectively (Table 2). The results showed that a C-4b hydroxy (10), a C-4a carboxy (2), a C-4a hydroxy (3), a methylene at C-3 (1), and a C-9 isopentenyl group (8) tended to increase the anti-H1N1 activity of hexacyclic indolediterpenoids. However, further hydroxylation at C-2′ (4) and isoprenylations at the phenyl (5) tended to decrease the activity. The open-chain indole-diterpene 6 and pentacyclic indole-diterpene 7 also displayed significant activity, indicating that the cyclic diterpenoid moiety is not an essential core for the anti-H1N1 activity of indole-diterpenoids. However, the monoisopentenyl-substituted hexacyclic indole-diterpene (8) with 4b-hydroxy and 3-oxo groups showed the most powerful anti-H1N1 activity among the 11 tested indole-diterpenoids. Although these kinds of indole-diterpenoids have been reported to display anti-insectan and antibiotic activities, the anti-H1N1 activity is reported here for the first time, indicating that indolediterpenoids might be used for screening as new natural antiH1N1 virus candidates.

Figure 5. Δδ (=δS − δR) values for (S)- and (R)-MTPA esters of 7.

Figure 6. CD curve of compound 1 and the calculated ECD curves of models I and II.

models I and II, and the CD spectrum of 2 with the calculated ECD curves of 2 and ent-2, revealed that the strong negative CD Cotton effects at λmax 210−250 nm could be used to determine the absolute configurations of C-4b, C-6a, C-12b, and C-12c in the hexacyclic indole-diterpenoids. Although the relative configuration of the known emindole SB (7) has been proposed,6 the absolute configuration has not yet been firmly established. The relative configuration of 7 was confirmed by X-ray diffraction as trans-H-6a and CH3-12b, trans-CH3-12b and CH3-12c, trans- H-4b and CH3-12c, and erythro-CH3-4a and 14a-OH (Figure S60). The absolute configuration at C-14a was determined by the modified Mosher’s method (Supporting Information).25 The chemical shift differences between the (S)- and (R)-MTPA esters (ΔδS−R, Figure 5) clearly defined the S-configuration at C14a. The absolute configuration of 7 was further established by the single-crystal X-ray diffraction analysis (Figure 4). The final refinement of the Cu Kα data resulted in a −0.01(2) Flack parameter, allowing an unambiguous assignment of the absolute configurations of all stereogenic centers of emindole SB (7) as (4aS, 4bR, 6aS, 12bS, 12cS, 14aS). The identification of compounds 6 and 7 supports the postulation that the epoxide 6b is a key intermediate in the biosynthesis of pentacyclic and hexacyclic indole-diterpenoids.26 Hydration of epoxide 6b forms 6, while opening of

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV data were recorded with a Beckman DU 640 spectrophotometer, and CD data were collected using a JASCO J-715 spectropolarimeter. IR spectra were taken on a Nicolet NEXUS 470 spectrophotometer as KBr disks. 1 H NMR, 13C NMR, DEPT, HMQC, HMBC, COSY, and NOESY spectra were recorded using a JEOL JNM-ECP 600 spectrometer or Bruker Avance 500 spectrometer using TMS as an internal standard, and chemical shifts were recorded as δ values. Chemical shift values were referenced to residual solvent signals for DMSO (δH/δC, 2.50/ 39.5). HRESIMS data were recorded using a Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer. Semipreparative HPLC were performed using an ODS column [YMC-pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min]. TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10−40 μm) and over silica gel (200−300 mesh, Qingdao Marine Chemical Factory)

Scheme 1. Postulated biosynthetic pathway of the new compounds 1−6

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124.7 ± 9.4

73.3 ± 2.1

34.1 ± 2.4

26.2 ± 0.3

6.6 ± 0.3

77.9 ± 8.2

17.7 ± 0.9

>150

113.1 ± 5.0

and Sephadex LH-20 (Amersham Biosciences). Vacuum-liquid chromatography (VLC) used silica gel H (Qingdao Marine Chemical Factory). Sea salt was made by evaporation of seawater collected in Laizhou Bay (Weifang Haisheng Chemical Factory). Fungal Material. The fungus Penicillium camemberti OUCMDZ1492 was originally obtained from sediment around the mangrove roots of Rhizophora apiculata collected from Wenchang, Hainan Province, China, in June 2010. The sediments (1 g) were air-dried for 24 h, and then the dried sediments were diluted to 10−2 g/mL with sterile water, 100 μL of which was deposited on a potato dextrose agar (200 g potato, 20 g glucose, 20 g agar per liter of tap water) plate containing chloramphenicol (100 μg/mL) as a bacterial inhibitor. A single colony was transferred onto another PDA plate and was identified according to its morphological characteristics and 18S rRNA gene sequences (GenBank access No. JX910356, Supporting Information). A reference culture is maintained in our laboratory at −80 °C. Working stocks were prepared on PDA slants stored at 4 °C. Fermentation and Extraction. The fungus P. camemberti OUCMDZ-1492 was cultured in 300 mL of fermentation medium in a 1 L conical flask, containing 20 g of mannitol, 20 g of maltose, 10 g of glucose, 10 g of monosodium glutamate, 3 g of yeast extract, 0.5 g of corn meal, 0.5 g of KH2PO4, 0.3 g of MgSO4, 17.5 g of Na2HPO4·2H2O, 10.5 g of citric acid monohydrate, and 33 g of sea salt per liter of tap water (pH 5.0), at 25 °C for 30 days. The whole fermentation broth (66 L) of cultivated medium was extracted exhaustively with EtOAc, yielding 18.2 g of extract; the mycelia were extracted by 80% volume aqueous acetone, yielding 64.5 g of extract. The extracts of the fermentation broth and the mycelia were combined after HPLC analysis demonstrated they were of similar chemical composition. Purification. The combined extract (82.7 g) from P. camemberti OUCMDZ-1492 was subjected to silica gel chromatography using a VLC column, eluting with a stepwise gradient of a mixture of petroleum ether−CH2Cl2 (2:1 and 0:1) and then of CH2Cl2−MeOH (100:1, 50:1, 30:1, 10:1, 1:1, and 0:1) to yield eight major primary fractions (Fr.1−Fr.8). Fraction 3 (6.4 g) eluted with CH2Cl2−MeOH (100:1) and was further resolved into four fractions (Fr.3.1−Fr.3.4) following reversed-phase C18 silica column chromatography eluting with a stepwise gradient of 30% to 100% MeOH in H2O. Fraction 3.1 was further purified by Sephadex LH-20 eluting with MeOH to obtain Fr.3.1.1−Fr.3.1.3. Fraction 3.1.1 was purified by semipreparative HPLC eluting isocratically with 88% MeOH−H2O to yield compound 1 (4.0 mg, tR 15.2 min). Similarly, fraction 3.1.2 and fraction 3.1.3 were purified to yield compounds 5 (9.0 mg, tR 10.0 min) and 6 (8.0 mg, tR 7.9 min) eluting with 80% and 95% MeOH−H2O, respectively. Fraction 3.2 was purified by Sephadex LH-20 eluting with CH2Cl2− MeOH (1:1) and then further separated by HPLC (88% MeOH− H2O) to yield 8 (3.7 mg, tR 13.0 min). Compounds 9 (53.2 mg, tR 14.0 min) and 10 (13.0 mg, tR 7.7 min) were purified from Fr.3.3 by HPLC (90% MeOH−H2O). Fraction 4 (1.3g) was subjected to SiO2 CC eluting with CH2Cl2−MeOH (50:1−0:100) to afford four subfractions (Fr.4.1−Fr. 4.4). Fractions 4.1 and 4.2 were separated by Sephadex LH-20 eluting with MeOH to provide five subfractions (Fr.4.1.1− Fr.4.1.5 and Fr.4.2.1−Fr.4.2.5), respectively. Fraction 4.1.3 was further isolated by Sephadex LH-20 eluting with CH2Cl2−MeOH (1:1) to provide four subfractions (Fr.4.1.3.1−Fr.4.1.3.4). Compounds 3 (2.1 mg, tR 4.6 min) and 4 (15.3 mg, tR 4.8 min) were purified from Fr.4.1.3.4 and Fr.4.2.4 by semipreparative HPLC eluting with 90% and 88% MeOH−H2O, respectively. Fraction 5 (2.1 g) obtained from the elution with CH2Cl2−MeOH (30:1) was further subjected to a Sephadex LH-20 column (20 × 800 mm, MeOH) to obtain Fr.5.1 and Fr.5.2. Fraction 5.1 was then purified by semipreparative HPLC (75% MeOH−H2O), leading to pure compound 2 (9.5 mg, tR = 6.7 min). Fraction 5.2 was subjected to semipreparative HPLC (80% MeOH− H2O), yielding 11 (13.4 mg, tR = 12.0 min). 3-Deoxo-4b-deoxypaxilline (1): colorless crystals; mp 296−298 °C; [α]25D −59 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 229 (4.27) and 280 (3.88) nm; CD (c 0.1, MeOH) λmax (Δε) 218 (+1.8), 226 (−1.3), 232 (+1.8), 238 (−3.7), 258 (+1.1), and 276 (+1.2) nm; IR (KBr) νmax 3397, 2976, 2926, 2857, 1658, 1454, 1373, 1099, 750, and

38.9 ± 1.3 IC50 (μM)

28.3 ± 1.0

32.2 ± 3.1

4 3 2 1

Table 2. Antiviral Activities against Influenza A H1N1 Virus for 1−11

5

6

7

8

9

10

11

Ribavirin


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669 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 406.2741 [M + H]+ (calcd for C27H36 NO2, 406.2741). 4a-Demethylpaspaline-4a-carboxylic acid (2): white, amorphous powder; [α]25D −54 (c 0.1, CHCl3); UV (MeOH) λmax 225 (4.30) and 280 (3.87) nm; CD (c 0.1, MeOH) λmax (Δε) 209 (+0.6), 229 (−0.5), 237 (+0.1), 244 (−0.3), and 273 (+0.3) nm; IR (KBr) νmax 3419, 2971, 2937, 2861, 1712, 1455, 1375, 1302, 1127, 1090, 1051, and 744 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 474.2613 [M + Na]+ (calcd for C28H37NO4Na, 474.2615). 4a-Demethylpaspaline-3,4,4a-triol (3): white, amorphous powder; [α]25D −91 (c 0.2, CHCl3); UV (MeOH) λmax 225 (4.39) and 280 (4.29) nm; CD (c 0.1, MeOH) λmax (Δε) 216 (−1.2), 231 (+2.6), 240 (−1.3), 256 (+0.3), and 286 (−0.9) nm; IR (KBr) νmax 3423, 2980, 2935, 1658, 1451, 1383, 1261, 1162, 1085, 1023, 800, 744, and 669 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 478.2560 [M + Na]+ (calcd for C27H37NO5Na, 478.2564). 2′-Hydroxypaxilline (4): white, amorphous powder; [α]25D −10 (c 0.3, CHCl3); UV (MeOH) λmax 237 (4.28) and 278 (4.70) nm; CD (c 0.1, MeOH) λmax (Δε) 212 (−2.9), 220 (+1.0), 242 (−14.7), and 304 (+1.7) nm; IR (KBr) νmax 3393, 2979, 2936, 1669, 1617, 1455, 1376, 1304, 1167, 1126, 1025, and 750 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 474.2253 [M + Na] + (calcd for C27H33NO5Na, 474.2251). 9,10-Diisopentenylpaxilline (5): white, amorphous powder; [α]25D −44(c 0.2, CHCl3); UV (MeOH) λmax 237 (4.80) and 284 (4.36) nm; CD (c 0.1, MeOH) λmax (Δε) 216 (−2.3), 234 (+0.1), 249 (−13.7), and 310 (+1.2) nm; IR (KBr) νmax 3358, 2974, 2930, 1660, 1453, 1376, 1181, 1126, 1025, 941, and 855 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 594.3546 [M + Na]+ (calcd for C37H49NO4Na, 594.3554). (6S,7R,10E,14E)-16-(1H-Indol-3-yl)-2,6,10,14-tetramethylhexadeca-2,10,14-triene-6,7-diol (6): brown oil; [α]25D −9.7 (c 0.5, CHCl3); UV (MeOH) λmax 228 (3.76) and 280 (4.20) nm; IR (KBr) νmax 2996, 2922, 2848, 1455, 1373, and 672 cm−1. 1H NMR (DMSO-d6, 600 MHz) δ 10.72 (br s, 1H, NH), 7.02 (s, 1H, H-2), 7.45 (d, J = 7.9 Hz, 1H, H-5), 7.04 (t, J = 7.4 Hz, 1H, H-6), 7.05 (t, J = 7.4 Hz, 1H, H-7), 7.32 (d, J = 7.9 Hz, 1H, H-8), 3.37 (d, J = 7.1 Hz, 2H, H2-10), 5.38 (t, J = 7.5 Hz, 1H, H-11), 1.94 (m, 1H, H-13a), 2.02 (m, 1H, H-13b), 2.07 (m, 1H, H-14a), 2.10 (m, 1H, H-14b), 5.12 (t, J = 6.6 Hz, 1H, H15), 2.16 (m, 1H, H-17a), 1.86 (m, 1H, H-17b), 1.22 (m, 1H, H-18a), 1.64 (m, 1H, H-18b), 3.08 (m, 1H, H-19), 4.30 (d, J = 6.2 Hz, 1H, OH-19), 3.96 (s, 1H, OH-20), 1.31 (dt, J = 5.0 Hz, J = 13.1 Hz, 1H, H-21a), 1.42 (dt, J = 5.0 Hz, J = 13.2 Hz, 1H, H-21b), 1.21 (m, 1H, H22a), 2.00 (m, 1H, H-22b), 5.07 (t, J = 7.2 Hz, 1H, H-23), 1.55 (s, 3H, H3-25), 1.62 (s, 3H, H3-26), 0.94 (s, 3H, H3-27), 1.56 (s, 3H, H3-28), 1.72 (s, 3H, H3-29); 13C NMR (600 MHz, DMSO) δ 122.1 (CH, C2), 113.8 (C, C-3), 127.1 (C, C-4), 118.4 (CH, C-5), 120.9 (CH, C6), 118.2 (CH, C-7), 111.4 (CH, C-8), 136.5 (C, C-9), 23.7 (CH2, C10), 123.5 (CH, C-11), 134.5 (C, C-12), 39.8 (CH2, C-13), 26.2 (CH2, C-14), 123.4 (CH, C-15), 135.3 (C, C-16), 36.8 (CH2, C-17), 29.4 (CH2, C-18), 75.9 (CH, C-19), 73.1 (C, C-20), 38.8 (CH2, C21), 21.6 (CH2, C-22), 125.5 (CH, C-23), 130.0 (C, C-24), 17.6 (CH3, C-25), 25.6 (CH3, C-26), 21.8 (CH3, C-27), 15.9 (CH3, C-28), 16.1 (CH3, C-29); HRESIMS m/z 446.3027 [M + Na]+ (calcd for C28H41NO2Na, 446.3030). Emindole SB (7): colorless crystals; mp 68−70 °C; [α]25D −19 (c 0.2, CHCl3); CD (c 0.1, MeOH) λmax (Δε) 207 (−1.4), 231 (+1.6), 246 (−0.4), and 285 (−0.6) nm; HRESIMS m/z 406.3101 [M + H]+ (calcd for C28H40NO, 406.3104). Relative Configuration of the 1,2-Diol Moiety in Compound 6 (ref 28). Compound 6 (1 mg) in acetone (500 μL) was added to 2,2-dimethoxypropane (200 μL) and pyridinium p-toluenesulfonate (PPTS, 3 mg), and the resulting solution was stirred at room temperature (rt) for 7 h. Then 1 mL of H2O was added. The solution was extracted by 10 mL of CH2Cl2, and the organic phase was concentrated under reduced pressure. The residue was purified by semipreparative HPLC (95% MeOH−H2O) to yield the acetonide (6a) (0.5 mg, tR 5.7 min). Its structure was identified by 1D and 2D NMR analysis (Figures S38−44) and HRESIMS.

Compound 6a: 1H NMR (DMSO-d6, 600 MHz) δ 10.71 (s, 1H, NH), 7.02 (s, 1H, H-2), 7.44 (d, J = 8.0 Hz, 1H, H-5), 7.04 (t, J = 7.3 Hz, 1H, H-6), 6.95 (t, J = 7.2 Hz, 1H, H-7), 7.31 (d, J = 8.0 Hz, 1H, H-8), 3.37 (d, J = 6.3 Hz, 2H, H2-10), 5.37 (t, J = 7.3 Hz, 1H, H-11), 2.02−1.99 (m, 2H, Ha-13, Ha-22), 1.94−1.90 (m, 3H, Hb-13, Hb-17, Hb-22), 2.05 (m, 1H, Ha-14), 2.07 (m, 1H, Hb-14), 2.09 (m, 1H, Ha17), 5.12 (t, J = 6.7 Hz, 1H, H-15), 1.47−1.44 (m, 2H, H2-18), 3.61 (dd, J = 8.6, 4.1 Hz, 1H, H-19), 1.39−1.35 (m, 2H, H2-21), 5.09 (t, J = 6.4 Hz, 1H, H-23), 1.56 (s, 3H, H-25), 1.62 (s, 3H, H-26), 1.11 (s, 3H, H3-27), 1.54 (s, 3H, H-28), 1.72 (s, 3H, H-29), 1.23 (s, 3H, H3-2′), 1.29 (s, 3H, H3-3′); 13C NMR (600 MHz, DMSO) δ 122.1 (CH, C2), 113.8 (C, C-3), 127.1 (C, C-4), 118.4 (CH, C-5), 120.9 (CH, C6), 118.2 (CH, C-7), 111.4 (CH, C-8), 136.5 (C, C-9), 23.7 (CH2, C10), 124.2 (CH, C-11), 134.2 (C, C-12), 39.9 (CH2, C-13), 26.1 (CH2, C-14), 123.6 (CH, C-15), 134.3 (C, C-16), 34.7 (CH2, C-17), 26.6 (CH2, C-18), 83.2 (CH, C-19), 81.1 (C, C-20), 36.4 (CH2, C21), 21.7 (CH2, C-22), 125.6 (CH, C-23), 130.5 (C, C-24), 17.5 (CH3, C-25), 25.5 (CH3, C-26), 22.6 (CH3, C-27), 15.9 (CH3, C-28), 15.9 (CH3, C-29), 110.2 (C, C-1′), 26.9 (CH3, C-2′), 28.4 (CH3, C3′); HRESIMS m/z 464.3514 [M + H]+ (calcd for C31H46NO2, 464.3523). Absolute Configuration of the 1,2-Diol Moiety in 6. HPLC grade DMSO was dried with 4 Å molecular sieves. According to a published procedure,29,30 a mixture of the 1:2 diol−Mo2(OAc)4 of 6 was subjected to CD measurements at a concentration of 0.125 mg/ mL. The first CD spectrum was recorded immediately after mixing, and its time evolution was monitored until stationary (about 10 min after mixing). The inherent CD spectrum was subtracted. The observed signs of the diagnostic bands in the region of 300−400 nm in the induced CD spectrum were correlated to the absolute configuration of the 1,2-diol moiety. ECD Calculations. The ECD spectra of compound 2 and the models I and II were calculated by using the software package Gaussian 03.20 The preliminary conformational distribution search was performed with HyperChem 7.5 software. All ground-state geometries were optimized at the B3LYP/6-31G(d) level. Solvent effects of the MeOH solution were evaluated at the same DFT level by using the SCRF/PCM method.31 X-ray Crystal Data for 1 and 7. Colorless crystals of 1 and 7 were obtained from MeOH and DMSO, respectively. Crystal data of 1 and 7 were obtained on Bruker APEX DUO and Bruker Smart CCD area detector diffractometers with graphite-monochromated Cu Kα radiation (λ = 1.54178 Å) and Mo Kα radiation (λ = 0.71073 Å), respectively. Crystallographic data for 1 (Mo Kα and Cu Kα) and 7 (Mo Kα and Cu Kα) have been deposited with the Cambridge Crystallographic Data Centre as supplementary publications Nos. CCDC 929738, 916196 and 916197, 941273, respectively. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Crystal Data for 1 (Mo Kα radiation): 298 K, orthorhombic, C27H35NO2, space group P2(1)2(1)2 with a = 11.6639(11) Å, b = 24.764(2) Å, c = 7.7832(5) Å, V = 2248.2(3) Å3, Z = 4, Dcalcd = 1.198 mg/m3, μ = 0.074 mm−1, and F(000) = 880. Crystal size: 0.17 × 0.10 × 0.07 mm3. Independent reflections: 2290 with Rint = 0.0519. The structure was solved by direct methods (SHELXTL) and refined using full-matrix least-squares difference Fourier techniques.32 The final agreement factors are R1 = 0.0380 and wR2 = 0.0794 [I > 2σ(I)]. Crystal Data for 1 (Cu Kα radiation): 100 K, orthorhombic, C27H35NO2, space group P2(1)2(1)2 with a = 11.5420(5) Å, b = 24.4903(12) Å, c = 7.7765(4) Å, V = 2198.16(18) Å3, Z = 4, Dcalcd = 1.225 mg/m3, μ = 0.588 mm−1, and F(000) = 880. Crystal size: 0.84 × 0.20 × 0.05 mm3. Independent reflections: 3862 with Rint = 0.0396. Absolute structure parameter: −0.1(2). The structure was solved by direct methods (SHELXS-97) and refined using full matrix leastsquares difference Fourier techniques.32 The final agreement factors are R1 = 0.0312 and wR2 = 0.0835 [I > 2σ(I)]. Crystal Data for 7 (Mo Kα radiation): 298 K, orthorhombic, C19H21N3O7, space group P2(1)2(1)2(1) with a = 8.1634(5) Å, b = 12.7824(8) Å, c = 26.7737(17) Å, V = 2793.8(3) Å3, Z = 4, Dcalcd = 1.152 mg/m3, μ = 0.142 mm−1, and F(000) = 1060. Crystal size: 0.45 1335



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× 0.21 × 0.20 mm3. Independent reflections: 2845 with Rint = 0.0684. The structure was solved by direct methods (SHELXTL) and refined using full-matrix least-squares difference Fourier techniques.32 The final agreement factors are R1 = 0.0539 and wR2 = 0.1073 [I > 2σ(I)]. Crystal Data for 7 (Cu Kα radiation): 293 K, orthorhombic, C19H21N3O7, space group P2(1)2(1)2(1) with a = 8.1683(5) Å, b = 12.8017(8) Å, c = 26.8302(17) Å, V = 2805.58(14) Å3, Z = 4, Dcalcd = 1.145 mg/m3, μ = 1.209 mm−1, and F(000) = 1056. Crystal size: 0.45 × 0.30 × 0.28 mm3. Independent reflections: 4564 with Rint = 0.0328. Absolute structure parameter: −0.01(2). The structure was solved by direct methods (SHELXS-97) and refined using full matrix leastsquares difference Fourier techniques.32 The final agreement factors are R1 = 0.0456 and wR2 = 0.1183 [I > 2σ(I)].

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ASSOCIATED CONTENT

S Supporting Information *

Bioassay protocols used, NMR and CD spectra of compounds 1−11, crystal structures of 1 and 7 (Mo Kα radiation), and the18S rRNA gene sequences of P. camemberti OUCMDZ1492. These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-532-82031268. Fax: +86-532-82031268. E-mail: [email protected]. Author Contributions †

Y. Fan and Y. Wang contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from the NSFC (Nos. 21172204 and 21002094), the 973 Program of China (No. 2010CB833804), the 863 Program of China (Nos. 2013AA092901, 2012AA092104, and 2011AA09070106), and the Special Fund for Marine Scientific Research in the Public Interest of China (No. 2010418022-3).

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