Meroterpenoids with Diverse Ring Systems from the Sponge

Article pubs.acs.org/jnp

Meroterpenoids with Diverse Ring Systems from the SpongeAssociated Fungus Alternaria sp. JJY-32 Guojian Zhang,† Guangwei Wu,† Tianjiao Zhu,† Tibor Kurtán,‡ Attila Mándi,‡ Jieying Jiao,† Jing Li,† Xin Qi,† Qianqun Gu,† and Dehai Li*,† †

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ Department of Organic Chemistry, University of Debrecen, POB 20, 4010 Debrecen, Hungary S Supporting Information *

ABSTRACT: Fifteen meroterpenoids (1−15) with diverse ring systems including an unprecedented oxaspiro[5.5]nonane-fused cyclohexenone (1), hydrogenated benzofurans (2−5), hydrogenated chromans (6, 7), hydrogenated cyclopenta[b]chromans (8−11), and four monocyclic structures (12−15) were isolated from the sponge-associated fungus Alternaria sp. JJY-32. The structures were elucidated by detailed spectroscopic analysis, including 2D NMR and electronic circular dichroism (ECD) calculations, and assisted by chemical derivatizations. On the basis of supplementation experiments with specific enzyme inhibitors and putative precursors, a shikimate−isoprenoid hybrid biosynthetic pathway is proposed. The NF-κB inhibitory activities of 1−15 were tested, and all of them, except 6 and 7 (IC50 > 100 μM), showed activities with IC50 values ranging from 39 to 85 μM in RAW264.7 cells.

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led to the isolation of nine new meroterpenoids (1−9) and two known compounds, ACTG-toxins D and H (10, 11). The diverse structures and their relationships among the aforementioned compounds inspired us to pursue their biosynthetic pathways. By feeding specific inhibitors of the polyketide pathway (cerulenin) or the shikimate pathway (1,10phenanthroline)7 and subsequently adding putative precursors into the liquid culture, the meroterpenoids are proposed to be generated from a hybrid shikimate−isoprenoid route. Furthermore, from the cultures with additions of the putative precursor sodium 3,4-dihydroxybenzoic acid or shikimic acid (5 L for each batch of fermentation), four additional new monocyclic meroterpenoids (12−15) were obtained. Herein, we report the details of isolation and structure elucidation, bioactivity evaluation, and biosynthetic pathway investigation of compounds 1−15.

arine-derived microorganisms are versatile producers of natural products with unusual and diverse structural features.1 Such chemical entities not only provide insight into the biogenetic landscape, revealing new structure classes and new biosynthetic pathways, but also enlighten the approach to biomimetic synthesis and further bioactivity investigations, suggesting potential candidates for the development of new pharmaceutical agents.2 Our ongoing search for new bioactive compounds from marine-derived microorganisms has afforded a variety of structures,3,4 such as novel sorbicillin dimer and trimer derivatives,3a−d diketopiperazine dimers,3e aspergiolide polyketides with cytotoxic and tyrosine kinase inhibitory activities,3f−h hybrid isoprenoids,4a,b and anti-inflammatory pyronepolyene-glucosides.4c During a recent project, we screened extracts from South China Sea-derived fungal strains for their inhibitory activities against LPS-induced NF-κB activation in RAW264.7 cells.5 From our previous research, one active meroterpenoid, ACTGtoxin H, was discovered from the fungal strain Alternaria sp. tzp-11.6 In the present work, an extract of the fungal strain JJY32 identified as an Alternaria sp., which was isolated from a Callyspongia sp. sponge collected offshore at Sanya, also exhibited NF-κB inhibitory activity with an IC50 value of 30 μg/mL. The HPLC-UV profiles (Figure S3 in Supporting Information) of the extract showed abundant peaks with similar UV absorptions to ACTG-toxin H, which indicated the potential for numerous analogues. Studies on the active constituents of the extract from scale-up fermentation (40 L) © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 was obtained as a colorless oil. The HRESIMS [M + H]+ peak at m/z 349.2365 indicated a molecular formula of C21H32O4, requiring six double-bond equivalents (DBEs). The 1H and 13C NMR spectra indicated four methyls, seven sp3 methylenes, three methines, two oxygenated sp3 quaternary carbons, one olefinic methine, and four sp2 quaternary carbons including a conjugated carbonyl carbon (Tables 1 and 3). The above data suggested the presence of a cyclohexanone and a Received: July 16, 2013

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dx.doi.org/10.1021/np4005757 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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structure from C-4 to C-6, which was extended to form the cyclohexenone based on the HMBC correlations from OH-4 to C-3 and C-4, from H-5 to C-1 and C-3, and from H-6 to C-2 (Figure 1A). The sesquiterpene unit from C-1′ to C-15′ was deduced by the COSY correlations and the HMBC correlations from H-1′ to C-2′ and C-6′, from H-13′ to C-2′, C-3′, and C4′, and from H3-12′ and H3-15′ to C-10′ and C-11′. The two moieties were connected based on the HMBC correlations from H-1′ to C-2 and C-3 (Figure 1A). Finally, the planar structure of 1 was completed by the attachment of OH-3′ to C3′, evidenced from the HMBC correlation from OH-3′ to C-4′, and the connection from C-3 to C-2′ via an ether bridge accounting for the molecular formula and the chemical shift of C-2′. The relative configuration was assigned by NOESY experiments (Figure 1B). The key NOE correlations from H-1′a (δH 2.44) to H-7′/H-14′ and from H-1′b (δH 2.83) to OH-3′ indicated the cofacial orientation of C-1′, C-7′, and OH-3′. The correlation between H-13′ and OH-4 suggested that OH-4 is oriented to the same side as C-3′. The relative configuration of C-6′ and C-7′ was tentatively proposed based on detailed conformational analysis (Figure 1C). In the 2D NOESY spectrum, consistent NOE correlations were observed from H314′ to H-6′ and H-1′a, from H-1′a to H-7′, and from H-6′ to H-8′. However, there was no apparent NOESY correlation between H-6′ and H-7′. The above data indicated the 2′S* 3′S* relative configuration. The absolute configuration of 1 was determined by comparison of the experimental ECD data to the TDDFT ECD calculated data of the model compound 1a, in which the C-6′ substituent of 1 was replaced by an isopropyl group. This

sesquiterpene unit in 1 (Figure 1). The COSY correlations (OH-4/H-4/H-5/H-6) (Figure 1A) constructed the partial Table 1. 1H NMR Data for Compounds 1−7 1b

no.

3a

4b

4 5

4.36, dd (5.5, 11.0) 1.80, m; 2.09, m

2.57, brd (7.3) 1.89, m; 2.42, m

6

2.17, m; 2.33, m

4.12, dd (12.4, 5.0)

1′

2.44, d (15.4); 2.83, dd (15.4, 2.2)

2.74, dd (14.2, 9.2); 2.87, dd (14.2, 8.0) 4.70, t (9.6)

2.58, brd (7.0) 1.89, m; 2.42, m 4.12, dd (12.1, 4.4) 2.73, m; 2.85, m 4.71, t (9.4)

6b

7b

4′

1.52, m

1.57, m; 1.78, m

1.50, m

5′

1.22, m; 1.74, m

1.59, m; 1.65, m

2.04, m

6′ 7′ 8′

2.01, m 1.42, m 0.99, m; 1.38, m

3.37, d (9.2)

2.09, m; 2.17, m 5.20, t (6.6)

5.14, t (6.6)

5.13, t (6.6)

5.14, m

5.14, t (6.6)

2.06, m

2.05, m

2.05, m

2.06, m

9′

1.85, m; 1.97, m

2.05, m; 2.15, m

2.23, m

2.23, m

2.23, m

2.23, m

10′

5.07, dd (7.7, 6.6)

5.14, t (7.1)

2.07, m; 2.24, m 1.41, m; 1.58, m 3.33, d (11.0)

6.61, dd (7.7, 5.5)

6.61, dd (7.7, 6.6)

6.61, dd (8.8, 7.7)

6.62, dd (7.7, 5.5)

12′ 13′ 14′ 15′ 4-OH 6-OH 2′-OH 3′-OH

1.64, 1.19, 0.80, 1.56, 5.46,

1.68, 1.25, 1.19, 1.63,

1.25, 1.27, 1.64, 1.15,

1.08, 1.59, 1.72, 5.60,

1.08, s 1.59, s 1.72, s

1.08, s 1.59, s 1.72, s

1.08, 1.60, 1.72, 5.37,

s s d (6.6) s d (5.5)

1.40, m; 1.72, m

s s s s

s s s s

4.38, t (5.5) 1.82, m; 2.08, m 2.20, m; 2.33, m 2.60, m

5b 2.52, m 1.75, m; 2.12, m 3.94, dd (11.0, 4.4) 2.46, m; 2.68, m 4.64, dd (9.7, 8.8) 1.36, dd (8.8, 7.7) 2.02, m

2′

a

2a

4.62, dd (9.8, 7.7) 1.37, t (8.8)

s s s s

2.31, m; 2.41, m 1.68, m; 2.07, m

4.10, dd (6.5, 4.7) 1.83, m; 2.11, m

3.89, ddd (11.0, 4.4, 3.6) 1.90, m; 2.46, m

2.15, m; 2.33, m

3.57, ddd (7.7, 5.5, 5.4) 1.57, m; 1.60, m

3.56, ddd (7.6, 5.5, 4.4) 1.61, m

2.04, m

1.99, m; 2.04, m

4.93, d (3.6) 5.13, d (5.5) 4.66, s

1.90, m; 2.47, m

s s s d (5.5)

5.13, d (4.4)

4.54, s

Recorded in CDCl3. bRecorded in DMSO-d6. B



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Table 2. 1H NMR Data for Compounds 8, 9, and 12−15 8a

no. 1 4 5 6 1′ 2′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 1-OH 3-OH 4-OH 7′-OH 4-OCH3 6-OCH3 10′-OH 11′-OH a

9a

12b

13b

14a

15a

7.19, d (2.0) 4.27, 2.24, 3.72, 2.25, 2.11, 1.64, 1.53, 1.65,

t (4.5) m; 2.40, m dd (6.9, 3.2) m; 2.68 (d, 17.4) m m; 2.01, m m; 1.79, m m

4.41, 2.06, 2.36, 2.21, 2.11, 1.65, 1.55, 1.67,

t (5.9) m; 2.27, m m; 2.64, m dd (17.4, 6.0); 2.69, d (17.4) dt (16.7, 4.7) m; 1.99, m m; 1.80, m m

6.56, 6.18, 3.19, 5.18, 1.89, 1.99, 5.08,

d (8.7) d (8.7) d (5.9) t (5.9) m m t (6.4)

7.24, d (2.0) 3.23, 5.28, 1.98, 1.17, 5.12,

d (7.7) dd (7.7, 6.6) m m; 2.05, m t (6.6)

1.43, m 2.03, m 5.13, t (5.9)

1.48, m 2.01, m 5.12, t (6.9)

1.85, m; 2.14, m 1.15, m; 1.60, m 3.02, dd (9.1, 5.9)

1.88, m; 2.15, m 1.20, m; 1.60, m 3.03, d (9.9)

1.69, 1.34, 1.15, 1.61,

1.68, 1.33, 1.15, 1.61,

1.03, 1.70, 1.53, 0.97, 8.68, 8.25,

1.03, 1.66, 1.55, 0.98,

s s s s

s s s s

s s s s s s

s s s s

4.05, 1.76, 4.19, 2.99, 5.05, 1.94, 2.06, 5.06,

dd (12.3, 4.5) m; 2.82, m dd (11.0, 4.5) m; 3.05, m t (7.3) m m t (5.9)

4.05, 1.76, 3.09, 2.99, 5.00, 2.02, 2.20, 5.09,

dd (12.3, 4.5) m; 2.82, m dd (11.0, 4.5) m; 3.05, m t (7.3) m m t (5.9)

1.95, 1.42, 1.40, 2.45,

m m; 1.97, m m; 1.63, m m

2.06, m 1.58, m; 1.89, m 6.61, t (6.0)

1.70, s 1.54, s 1.16, d (6.8)

1.70, s 1.54, s 1.64, s

3.52, s

3.36, s

3.50, s 2.90, brs 3.68, s 3.49, s 4.28, d (5.9) 4.05, s

Recorded in CDCl3. bRecorded in DMSO-d6.

Table 3. 13C NMR Data for Compounds 1−9 and 12−15 (150 MHz, TMS, δ in ppm)

a

no.

1b

2a

3a

4b

5b

6b

7b

8a

9a

12b

13b

14a

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 4-OCH3 6-OCH3 6-COOH

194.2 113.6 176.5 62.3 31.9 34.6 26.4 102.5 79.9 34.7 23.4 49.2 35.1 34.5 25.1 125.2 131.2 26.1 23.6 18.1 18.0

195.4 111.5 178.9 22.8 30.1 71.4 26.6 93.2 73.0 34.5 24.3 79.0 74.9 36.0 22.1 124.6 132.0 25.8 22.1 23.5 17.8

195.2 111.6 178.2 22.9 30.1 71.4 26.6 92.7 73.5 36.8 21.9 124.5 135.9 36.8 29.7 78.2 73.2 26.5 22.6 16.1 23.3

194.3 113.1 177.4 62.4 32.0 34.7 26.8 90.8 72.2 38.7 21.9 125.6 134.2 38.3 27.2 141.7 128.6 169.4 22.1 16.2 12.8

195.2 111.8 177.3 22.5 30.8 71.2 26.7 91.7 72.1 38.7 21.9 125.6 134.2 38.2 27.2 141.8 128.3 169.4 22.2 16.2 12.8

197.3 106.0 168.6 26.7 29.3 70.3 24.6 65.6 81.2 37.6 20.9 124.5 133.9 37.2 26.6 141.1 127.7 168.7 17.6 15.6 12.2

196.3 107.9 168.4 64.2 29.4 32.1 24.6 65.7 80.8 37.6 20.9 124.6 133.8 37.2 26.6 141.1 127.7 168.8 17.5 15.6 12.2

195.0 105.8 168.4 65.9 34.7 77.3 18.8 41.0 88.9 38.3 24.7 50.1 75.2 40.9 22.4 124.3 132.2 25.8 22.3 23.7 17.8

198.1 107.5 168.6 66.2 29.0 33.4 18.8 41.0 89.1 38.5 24.7 50.1 75.3 40.9 22.4 124.3 132.2 25.8 22.5 23.6 17.8

150.3 115.9 145.4 141.1 110.4 104.9 23.0 123.6 133.8 39.8 26.9 124.0 135.6 37.2 30.0 77.6 72.2 26.9 16.5 16.6 25.1 57.1

122.7 128.1 148.2 144.8 114.5 121.3 28.3 122.6 136.0 39.9 26.9 123.9 135.8 37.1 30.0 77.6 72.2 26.9 16.5 16.6 25.1

193.3 113.2 171.5 69.7 35.9 74.6 21.4 121.5 135.6 39.6 25.5 124.2 134.6 39.4 26.1 33.4 39.4 181.8 16.0 15.9 17.1

69.2 45.7 74.6 21.1 122.6 133.6 39.1 26.1 124.4 133.5 37.4 26.7 141.1 127.6 168.7 15.8 15.7 12.1

56.8

56.0

58.5

15a 112.0

168.0

Recorded in CDCl3. bRecorded in DMSO-d6.

cyclohexenone ring. The solution ECD spectrum of compound 1 showed positive Cotton effects (CEs) at 322, 266, and 194 nm and negative ones at 293 and 211 nm. The initial MMFF

simplification is justified, as the ECD features of 1 are governed by the α,β-unsaturated ketone chromophore of ring A, which in turn reflects the conformation and configuration of the C



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and the relative configuration, the absolute configuration of 1 was determined as 4R,2′S,3′S,6′S,7′S, and 1 was named tricycloalternarene A. Tricycloalternarene A (1) is the first example of a meroterpenoid skeleton with the unique fusion of an oxaspiro[5.5]nonane and a cyclohexenone ring. Moreover, compounds with tetrahydrobenzofuran-4-one structures are relatively rare. Apart from several synthetic precedents,8 the reported natural examples are the octaketides trichodermaketones A−D (from Trichoderma koningii), the prenylated butylphloroglucinols takaneols A−C (from Hypericum spp.), and the flavanone bonannione B (from Bonannia graeca).9 Bicycloalternarene A (2) was obtained as a pale yellow oil and analyzed for the molecular formula C21H34O6 by HRESIMS and 1D NMR data (Tables 1 and 3). The UV and IR data indicated 2 possessed the same chromophore as 1. The COSY experiment (Figure 3) offered four spin systems, H4/H-5/H-6, H-1′/H-2′, H2-4′/H-5′/H-6′, and H-8′/H-9′/H10′, and one dimethyl-substituted alkene group (H-12′/H-15′). The HMBC correlations (Figure 3) enabled the connections of the above fragments as well as the ether bridge from C-2′ to C3. Finally, the planar structure of 2 was completed by connecting the carbonyl carbon (C-1) to C-2 based on the molecular formula and chemical shifts. To establish the relative configuration of the bicyclic ring, hydrogenated derivative 2a was prepared by Pd/C catalysis.10 The NOESY correlations (H-6/H-2/H-3/H-2′) in 2a indicated the 2′S*, 6R* relative configuration in 2 (Figure 3). The relative configuration between C-2′ and C-3′ was tentatively assigned by detailed conformational analysis for 2. In the NOESY spectrum, consistent correlations of H-1′/H-13′/H2′/H-4′ were observed, while there was no apparent NOESY correlation between H2-1′ and H2-4′ (Figure 3). The above data indicated a 2′S*, 3′S* relative configuration. The syn configuration of the vicinal diol at C-6′ and C-7′ was deduced by the 13C chemical shifts11 (δC 78.3, C-6′ and 74.6, C-7′) and the observation of NOE enhancement from H-6′ to H3-14′/16′ and from H3-14′ to H3-16′ in the acetonide derivative 2b (Figure 3). The absolute configurations of C-6 and C-6′ were determined as 6R and 6′R by interpretation of the 1H NMR chemical shift differences (ΔδS − δR) between Mosher esters (2c vs 2e, 2d vs 2f) (Figure 4).12 Therefore, the absolute configuration of 2 was deduced as 6R, 2′S, 3′S, 6′R,7′S, which was also confirmed by TDDFT ECD calculations of the truncated model compound (6R,2′S)-2g (the C-4′ side-chain in 2 was replaced by a methyl group) (Figure 2B and Figure S5). The conformational analysis of (6R,2′S)-2g provided seven conformers above 2% population, in which the hydrogenbonded 6-OH group had an equatorial orientation in accordance with the large value of the coupling constant for 3 J6Hax,5Hax. The Boltzmann-averaged TDDFT ECD spectra of (6R,2′S)-2g resembled the experimental ECD curve (Figure 2B), which confirmed the 6R absolute configuration of 2. Bicycloalternarenes B−D (3−5) were obtained as pale yellow oils. Examination of their 1D NMR spectra (Tables 1 and 3) indicated that they shared the same carbon skeleton as 2, but differed in the degree of oxygenation. HRESIMS data of 3 revealed the same molecular formula as 2. Comparison of 1D-NMR data (Tables 1 and 3) indicated different locations for the olefinic bond and diol groups in the isoprenoid tail moiety. Comprehensive analysis of the 2D NMR data (Figure 3) led to the confident construction of the planar

Figure 1. Key COSY (), HMBC (→), and NOESY (↔) correlations of compound 1.

conformational search and subsequent DFT reoptimization of the truncated model compound (4R,2′S,3′S,6′S)-1a provided 13 conformers above 2.0% of the total conformer population (Figure S4). The conformers differed in the conformations of rings A and C and the orientation of the C-6′ substituent. By flipping ring A or C, the 4-OH or 3′-OH can adopt either an equatorial or an axial position. In the lowest energy conformer, both 4-OH and 3′-OH adopt equatorial orientations, and this conformation is represented by three slightly different conformers totaling 42.2% of the total population. The 4-OH was also found to be equatorial in the four 4-OHeq,3′-OHax conformers, totaling 24.5%. In the four 4-OHax,3′-OHeq conformers, totaling 23.0% of the population, the conformation of the cyclohexenone ring was inverted, moving the 4-OH to an axial orientation. The 4-OH adopted an equatorial orientation in conformers totaling 66.7% of the population and an axial one in 25.6%. This is in agreement with the measured 11.0 Hz coupling constant for the 3JH‑4,H‑5, suggesting a trans-diaxial arrangement of these protons. The (4R,2′S,3′S,6′S)-1a has a planar enone chromophore with a 179.7° ωOC1,C2,C3 torsional angle in the lowest energy conformer and with C-5 out of plane. The TDDFT ECD calculation of (4R,2′S,3′S,6′S)-1a using various functionals (B3LYP, BH&HLYP, PBE0) and the TZVP basis set reproduced well the main transitions of the experimental ECD spectrum (Figure 2A) except for the weak lowest energy positive band at 322 nm, which presumably derives from minor conformers. On the basis of the agreement

Figure 2. Experimental/calculated ECD spectra for 1/1a (A) and 2/ 2g (B); CD spectra of 1, 2, 4, and 6 (C). D



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Figure 3. Key COSY (―), HMBC (→), and NOESY (↔) correlations of 2, 2a, 2b, 3, and 4.

Figure 4. Δδ values [Δδ = δs − δR] obtained for (S)- and (R)-MTPA esters of 2−4 and 8.

structure for 3. The E geometry for the Δ6′ double bond was identified by the upfield chemical shift of C-14′ (δC 16.1 ppm).13 The similar 1H and 13C NMR data for the signals around the bicyclic ring in 2 and 3 indicated the same relative configurations for C-6, C-2′, and C-3′. Similarities of CD curves between 2 and 3 suggested the same 6R absolute configuration. Further comparison of 1H NMR data of the MTPA ester derivatives (Figure 4) determined the absolute configuration of C-10′ as R. Therefore, the absolute configuration of 3 was assigned as 2′S, 3′S, 6R, 10′R. According to the HRESIMS data, compounds 4 and 5 were deduced to possess the same molecular formula of C21H30O6, requiring two more DBEs than 3. Comparison of the 1D NMR spectra of 4 with those of 3 revealed an extra double bond at C10′, a carboxy group at the end of the isoprenoid tail, and a 4hydroxy to replace a 6-hydroxy group in the cyclohexanone ring. Further 2D NMR correlations (Figure 3) confirmed the planar structure of 4. Comparison of the 13C NMR data (Tables 1 and 3) between 5 and 4 revealed a noticeable upfield shift by 39.9 ppm at C-4 and a downfield shift by 36.5 ppm at C-6, indicating that instead of the 4-hydroxy in 4, the hydroxy group was at C-6 in compound 5. The E configurations of the two side-chain double bonds in 4 and 5 were supported by the NOESY correlations between H-5′/H3-14′, H-6′/H-8′, and H9′/H3-15′. Differences between the 1H NMR chemical shifts for the MTPA ester derivatives 4a and 4b (Figure 4) led to the

assignment of the R configuration at C-4 in 4. The CD data as well as the splitting pattern of H-6 [δH 3.94 (dd, J = 11.0, 4.4 Hz)] of 5 showed considerable similarities to those of 2 and 3, indicating the same 6R absolute configuration (Figure 2C). On the basis of biogenetic considerations, the configurations at C2′ and C-3′ for compounds 4 and 5 were presumed to be identical to those in 2. As revealed by the HRESIMS data, compounds 6 and 7 are isomers of 4 and 5. Apart from the signals for the C3−C2− C1′−C3′ segment, most of the 1D NMR data of 6 (Tables 1 and 3) revealed considerable similarity to those of 5, leading to the inference that in 6 C-3 was linked to C-3′ rather than C-2′. Moreover, the presence of the 3,3′-ether bridge was confirmed by the splitting patterns of H-2′ [δH 3.57 (ddd, J = 7.7, 5.5, 5.4 Hz)] and 2′-OH [δH 5.13 (d, J = 5.5 Hz)] (Table 1). NOESY cross-peaks from H-2′ to H3-13′ (δH 1.08)/H-1′b (δH 1.90)/H4′a (δH 1.57) and from H3-13′ to H-1′b (δH 1.90) were consistent with the 2′S*, 3′S* relative configuration (Figure 5).

Figure 5. Selected NOESY (↔) correlations for 6. E



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(cerulenin)7c or shikimate pathway (1,10-phenanthroline, PA)7d−f to the culture of JJY-32 were carried out. The results showed that cerulenin exhibited no distinct influence on either biomass accumulation or the meroterpenoid profile, while PA exhibited concentration-dependent effects on the meroterpenoid production (Table S1 and Figure S3). This indicated that 1−15 were likely to be synthesized via a hybrid route combining the shikimate and isoprenoid pathways. Feeding experiments with the shikimate derivative 3,4-dihydroxybenzoic acid (DBA) and shikimic acid were performed to give further evidence. As shown in Figure 7, production of meroterpenoids that were completely inhibited by 80 μM PA (Figure 7e) could be partially recovered by the addition of DBA (Figure 7c) or shikimic acid (Figure 7d) both at a final concentration of 5 mM. These results implied that DBA and shikimic acid, shunt products of the shikimate pathway, could be incorporated into the meroterpenoids, further providing evidence for the involvement of the shikimate pathway. During the successive chemical studies, we obtained compounds 12 and 13 from a DBA-supplemented culture and 14 and 15 from a shikimic acid-enriched culture. Monocycloalternarene A (12) was isolated as a dark yellow oil. A molecular formula of C22H34O5 was established by HRESIMS and supported by 1H and 13C NMR data. Interpretation of the COSY, HMQC, and HMBC data assigned the skeleton of 12 as a farnesylated 1,3-dihydroxy-4methoxybenzene (Figure 8). A 1,2-diol was positioned at C10′ and C-11′ by the chemical shifts of C-10′ (δC 77.6) and C11′ (δC 77.2), together with the crucial HMBC correlations from H3-12′ and H3-15′ to C-10′ and C-11′. Examination of 1D NMR data for monocycloalternarene B (13) (Tables 2 and 3) revealed an analogue of 12 with a similar triprenylated benzene skeleton, but the aromatic head moiety was changed to a hydroxy protocatechuic acid group. Analysis of the MTPA ester derivatives 12a and 12b (Figure 8) allowed the assignment of the 10′R absolute configuration for 12. The specific rotation ([α]25D +10.7, c 0.1, MeOH) for 13 suggested the same 10′R absolute configuration as 12 ([α]25D +13.5, c 0.1, MeOH). Monocycloalternarene C (14) was obtained as a dark yellow oil. An [M + Na]+ ion at m/z 417.2250 indicated a molecular formula of C22H34O6. Comparison of its IR and NMR spectroscopic data (Tables 2 and 3) with those of compounds 1−13 indicated a carbon framework with a cyclohexenone ring and a modified linear farnesyl side chain (Figure 9). The 2D NMR data, especially the COSY and HMBC correlations, revealed that the terminal isoprene unit of the farnesyl chain was saturated and one of the geminal methyls had been oxidized to a carboxylic acid. The structure of 14 was completed by connecting the two fragments on the basis of the key HMBC correlations from H2-1′ to C-1, C-2, and C-3. A NOESY correlation from H-4 to H-6 gave evidence for the cofacial relationship for these two protons. The CD data showed similarities to those of 8, revealing the same 4S, 6R absolute configurations as in 8. The configuration at C-11′ was not assigned. HRESIMS of monocycloalternarene D (15) gave an exact mass of m/z 391.2123 [M − H]−, suggesting a molecular formula of C22H32O6, which was two H atoms less than that of 14. The 1D NMR spectroscopic data (Tables 2 and 3) were in striking accordance with those of 14 except for the substitution of a double bond resonating at δC 141.1 and δC 127.6 for a saturated bond resonating at δC 39.4 and δC 33.4, revealing that

The 6R configuration was proposed based on the similar CD data to those of 2, 3, and 5. A 2′S, 3′S absolute configuration is tentatively suggested according to the proposed biogenesis (discussed later in the Biogenesis section). Careful comparison of 1D NMR data of 7 with those of 6 (Tables 1 and 3) indicated the 6-hydroxy group was replaced by a 4-hydroxy in 7. The configurations at C-2′ and C-3′ were proposed to be in agreement with those of 6. The CD curves and the 1H NMR signals of H-4 (Table 1) showed considerable similarities to those of 4, thus indicating the same 4R absolute configuration. Compounds 6 and 7 were named bicycloalternarenes E and F, respectively. Tricycloalternarenes B (8) and C (9) were also obtained as colorless oils. Their molecular compositions were determined to be C22H34O5 and C21H32O4, respectively, by HRESIMS. Their 1H and 13C NMR data (Tables 2 and 3) showed considerable resemblance to the data of ACTG-toxin D (10),14e−g a phytopathotoxin previously isolated from cultures of A. citri and A. alternata, indicating the same ring scaffold as 10, but with different modifications to the core structure. Comprehensive analysis of 2D NMR data revealed the exact planar structures of 8 and 9. The only difference between them is the presence of an additional 6-OMe group in 8. The NOESY correlations between H3-13′ and H-2′ in both 8 and 9 revealed the cis fusion between rings B and C, which was in accordance with that of 10. The NOESY correlation from H314′ to H-2′ indicated a 2′S*, 3′R*, 6′R* relative configuration (Figure 6). Rotation about the C-6′/C-7′ bond and only weak

Figure 6. Key COSY (―), HMBC (→), and NOESY (↔) correlations of 8 and 9.

NOE correlations from H3-14′ and H2-8′ to protons in the ring system prevented the assignment of the relative configuration at C-6′/C-7′. Further NOESY correlations from H-4 to H-6 in 8 established the 4S*, 6R* configuration (Figure 6). For compound 8 the absolute configuration at C-4 was determined to be S by the modified Mosher’s method (Figure 4). Evidence from the CD spectrum and the 1H NMR data of H-4 [δH 4.41, (t, J = 5.9 Hz)] indicated the 4R absolute configuration of 9. It is generally accepted that the C15 skeleton (farnesyl) is derived from the isoprenoid pathway, while the six-membered nonterpenoid moiety possibly arises from the polyketide pathway or the shikimate pathway.15 In order to clarify which pathway is involved in the biosynthesis of alternarenes 1−15, additions of specific inhibitors of the polyketide pathway F



Article

Figure 7. HPLC profiles of (a) standard culture, (b) standard compounds, (c) culture with 80 μM PA and 5 mM DBA, (d) culture with additions of 80 μM PA and 5 mM shikimic acid, and (e) culture with 80 μM PA.

Figure 9. Key COSY (―), HMBC (→), and NOESY (↔) correlations of 14.

Figure 8. Key COSY (―), HMBC (→), and NOESY (↔) correlations and Δδ values [Δδ = δs − δR] obtained for (S)- and (R)MTPA esters of 12.

intermediates permits the establishment of an advanced biogenetic proposal that bridges the monocycloalternarenes, bicycloalternarenes, and tricycloalternarenes. Biogenesis. Scheme 1 depicts a plausible biogenetic pathway to compounds 1−15 starting from shikimate derivatives as preliminary precursors. The aromatic precursor DBA can be transformed into 12 and 13 through a series of reactions including farnesylation, hydroxylation, and decarboxylation or methylation. Shikimic acid can be converted into M1 by decarboxylation, hydroxylation, and farnesylation. The farnesyl unit in M1 can be modified and further folded in multiple ways, thus generating the bicyclo- and tricycloskeletons leading to compounds 1−11 and 14 and 15. The existence of the key intermediate M1 skeleton was supported

15 was a 10′,11′-dehydro derivative of 14. Evidenced by the shielded 13C chemical shifts for CH3-13′, CH3-14′, and CH315′ groups, E configurations were assigned for the three double bonds at C-2′, C-6′, and C-10′.13 Compounds 1−15 belong to a family of meroterpenoids derived from a farnesyl chain linked to a six-membered ring system. The farnesyl unit may exist in acyclic, monocyclic, or bicyclic forms, and the structural variations can also focus around the degree of oxygenation and the substitutions to the six-membered nonterpenoid moiety. Numerous examples of this meroterpenoid family have so far emerged from various origins including fungi, plants, and especially marine invertebrates.14 The discovery of 14 and 15 as plausible biosynthetic G



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Scheme 1. Plausible Biogenetic Pathway to Meroterpenoids 1−15

by the isolation of monocycloalternarenes C and D (14 and 15), and the presence of the B3 backbone is rationalized by related compounds previously reported.14i The keto−enol tautomer or the diketone form in the nonterpenoid part makes an important contribution to the structural diversity.14i,j,16 Specifically, four pairs of prototype intermediates for the above meroterpenoids are proposed in Scheme 1 (B1/B1′; B2/B2′; T1/T1′; T2/T2′). In each pair, the two isomeric structures are distinguished from each other by the location of the hydroxy substituent on the nonterpenoid ring (C-4 or C-6). The prototype intermediates are presumed to be derived from the corresponding upstream 1,3-diketone precursors (M2 and B3), in which the two ketones can be attacked by a hydroxy group with equal chance, thus affording the diverse structures with 6R (2, 3, 5, 6, 8, 10, 11, 14, and 15) or 4R (1, 4, 7, and 9) absolute configurations (Figure 10). According to the above pathway, the skeletons of 2−5, 6, and 7 are generated from the same intermediate M2 by nucleophilic attack either from the 2′-OH or the 3′-OH. On the basis of this biogenetic relationship, the absolute configurations at C-2′ and C-3′ in 6 and 7 were presumed to be the same as those in compounds 1−5. NF-κB, a key transcription factor involved in the inflammatory pathway, is constitutively active in most cancers, and suppression of this factor inhibits the growth of tumor cells, leading to the concept of “NF-κB addiction” in cancer cells.17

Figure 10. Stereochemistry details in B-ring formation.

By means of transient transfection and reporter gene expression assay, compounds 1−15 were tested for their NF-κB inhibitory activities in RAW264.7 cells.5 They showed moderate to weak H



Article

of the seed culture was inoculated into a 500 mL flask with 150 mL of production PDB medium and cultured at 28 °C on a rotary platform shaker at 170 rpm for 7 days. For supplementation experiments, cerulenin (Sigma−Aldrich) and shikimic acid (Sangon Biotech Shanghai, Co., Ltd.) were dissolved in distilled H2O, while 3,4dihydroxybenzoic acid (Sangon Biotech Shanghai, Co., Ltd.) and 1,10phenanthroline (Sangon Biotech Shanghai, Co., Ltd.) were dissolved in 50% DMSO. All solutions were filter-sterilized. According to the set final concentrations, each additive was divided into three equal parts and added into the cultures at one-day intervals. The addition experiments were started 40 h after seeding the flasks. All of the experiments were carried out in triplicate and performed in shake flasks. Extraction and Isolation. The fermented materials were extracted with EtOAc, and the organic solvent was evaporated to dryness under vacuum to afford the corresponding extracts. The extract (40.0 g) from a 40 L fermentation was separated into five fractions by a silica gel chromatography column (CHCl3/MeOH, 100:0 → 0:100, v/v). Fr. 2 (2.0 g) was further fractionated on a Sephadex LH-20 column (CHCl3/MeOH, 50:50, v/v) to give Fr. 2-1 and Fr. 2-2. Fr. 2-1 and Fr. 2-2 were subjected to semipreparative HPLC (MeOH/H2O, 70:30) to afford compounds 1 (12.0 mg, tR 28.4 min), 9 (8.0 mg, tR 29.8 min), 10 (21.0 mg, tR 24.3 min), and 11 (32.0 mg, tR 25.8 min), and compound 8 (9.0 mg, tR 23.2 min), respectively. Fr. 3 (3.0g) was subjected to a silica gel column (petroleum ether/EtOAc, 70:30 → 30:70, v/v) to give six subfractions (Fr. 3-1 to Fr. 3-6). Subfraction 3-3 was further purified with a Sephadex LH-20 column (MeOH) and semipreparative HPLC (MeOH/H 2 O, 60:40, v/v) to afford compounds 2 (13.0 mg, tR 36.5 min), 3 (10.0 mg, tR 38.2 min), 4 (9.0 mg, tR 35.4 min), and 5 (11.0 mg, tR 33.3 min). Subfraction 3-4 was subjected to RP-18 chromatography (MeOH/H2O, 30:70 → 80:20) to give Fr. 3-4-1 to Fr. 3-4-4. Fr. 3-4-3 was further fractionated on Sephadex LH-20 (CHCl3/MeOH, 50:50, v/v) and finally purified by semipreparative HPLC (MeOH/H2O, 60:40, v/v) to yield 6 (2.0 mg, tR 35.2 min) and 7 (1.5 mg, tR 37.0 min). The extract (6.0 g) derived from a moderate scale fermentation (5 L) with addition of 5 mM DBA was fractionated by RP-18 CC (MeOH/H2O, 20:80 → 80:20) to give Fr. 1 to Fr. 4. Fr. 3 (800 mg) was chromatographed by Sephadex LH-20 (MeOH) and semipreparative HPLC (MeOH/H2O, 60:40, v/v) to give compounds 12 (10.0 mg, tR 32.7 min) and 13 (12.0 mg, tR 34.5 min). The extract (5.0 g) from a 5 L fermentation with addition of 5 mM shikimic acid was first subjected to RP-18 CC (MeOH/H2O, 20:80 → 80:20); then the subfraction (900 mg) was further purified by Sephadex LH-20 (CHCl3/MeOH, 50:50). Finally compounds 14 (7.0 mg, tR 38.1 min) and 15 (1.5 mg, tR 39.5 min) were obtained using semipreparative HPLC eluting with 65% MeOH. Tricycloalternarene A (1): colorless oil; [α]25D +62.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 263 (4.07) nm; CD (c 2.87 mM, MeOH) λmax (Δε) 322 (+0.12), 293 (−0.59), 266 (+0.30), 211 (−0.23), 194 (+2.72) nm; IR (KBr) νmax 3364, 1613, 1448, 1408, 1361, 1235, 1070, 1023, 950 cm−1; 1H NMR data (DMSO-d6, 600 MHz, TMS, δ ppm) in Tables 1 and 3; HRESIMS m/z 349.2365 [M + H]+ (calcd for C21H33O4, 349.2379). Bicycloalternarene A (2): pale yellow oil; [α]25D +79.6 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 265 (4.12) nm; CD (c 2.62 mM, MeOH) λmax (Δε) 267 (+2.54) 193 (−1.73) nm; IR (KBr) νmax 3311, 1633, 1627, 1375, 1235, 1196, 1129, 1063, 911 cm−1; 1H NMR and 13 C NMR data in Tables 1 and 3; HRESIMS m/z 383.2442 [M + H]+ (calcd for C21H35O6, 383.2434). Hydrogenated derivative 2a: colorless oil; 1H NMR (400 MHz, CD3OD TMS, δ ppm) δ 4.25 (1H, dd, J = 8.0, 12.4 Hz, H-6), 4.17 (1H, m, H-3), 3.70 (1H, t, J = 12.4 Hz, H-2′), 2.92 (1H, t, J = 11.0 Hz, H-2), 2.69 (2H, m, H-1′), 1.59 (2H, m, H-5), 1.5−0.8 (overlap, m); ESIMS m/z 409.1 [M + Na]+, 795.3 [2 M + Na]+. Acetonide derivative 2b: pale yellow oil; 1H NMR (600 MHz, CDCl3 TMS, δ ppm) δ 5.12 (1H, t, J = 7.1 Hz, H-10′), 4.70 (1H, t, J = 9.6 Hz, H-2′), 4.12 (1H, dd, J = 5.0, 12.4 Hz, H-6), 3.69 (1H, d, J = 11.0, H-6′), 2.87 (1H, dd, J = 10.5, 14.2 Hz Ha-1′), 2.75 (1H, dd, J = 9.2, 14.2 Hz Hb-1′), 2.57 (2H, brs, H2-4), 2.43 (1H, m, H-5a), 2.19 (1H, m, Ha-9′), 2.01 (1H, m, Hb-9′), 1.89 (1H, m, Hb-5), 1.75

bioactivities, with IC50 values ranging from 39 to 85 μM (PDTC as a positive control with an IC50 value of 3 μM) (Table 4). In addition, compounds 1−15 were also evaluated Table 4. NF-κB Inhibitory Activities of Compounds 1−15 in the RAW264.7 Cell Linea 1

2

3

4

5

IC50 (μM)

52 6

62 7

70 8

67 9

85 10

IC50 (μM)

>100 11

>100 12

76 13

75 14

50 15

IC50 (μM)

39

45

47

42

58

a

PDTC (pyrrolidine dithiocarbamic acid, ammonium salt) as positive control with IC50 value of 3 μM.

for their cytotoxicities against two cancer cell lines, A549 and HL-60, using the MTT18 or SRB19 methods. However, no obvious activity was observed. In summary, as a result of chemical investigation on the sponge-associated fungal strain Alternaria sp. JJY-32, we obtained 13 new structurally related meroterpenoids including four monocyclic (12−15), six bicyclic (2−7), and three tricyclic (1, 8, 9) meroterpenoids. On the basis of supplementation experiments with enzyme inhibitors and biosynthetic precursors, these compounds are proposed to be derived from a shikimate−isoprenoid mixed biosynthetic pathway. The compounds showed weak to moderate NF-κB inhibitory activities.

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EXPERIMENTAL SECTION

General Experimental Procedures. Specific rotations were obtained on a JASCO P-1020 digital polarimeter. CD spectra were recorded on a JASCO J-815 spectropolarimeter. UV spectra were recorded on Beckman DU 640 spectrophotometer. IR (KBr) spectra were recorded with a Nicolet NEXUS 470 spectrophotometer. 1D and 2D NMR spectra were recorded on a JEOL JNM-ECP 600 spectrometer and Bruker AM-400 using TMS as internal standard, and chemical shifts were recorded as δ values. ESI-MS were measured on a Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer (Waters). 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) and Sephadex LH-20 (Amersham Biosciences), respectively. Vacuum liquid chromatography (VLC) was carried out over silica gel H (Qingdao Marine Chemical Factory). Analytical HPLC was performed with a C18 column [YMC-pak ODS-AM, 4.6 × 250 mm, 5 μm, 1 mL/mim]; semiprepartive HPLC, with an ODS column [YMC-pak ODS-A, 10 × 250 mm, 5 μm, 4 mL/min]. Fungal Material and Storage. The strain of Alternaria sp. JJY-32 was isolated from a sponge Callyspongia sp. collected off the coast of Hainan Island, China, in September 2009 and identified based on the analysis of morphology and ITS DNA sequence (Gene Bank Accession: HQ317498). A single colony was subcultured onto potato dextrose agar (PDA) plates. After incubation for 9 days at 28 °C, spores were harvested by flooding with sterile distilled H2O and agitating with a sterile glass spreader. The resulting spore suspension was filtered through four thicknesses of sterile muslin, and the spores were centrifuged and washed in sterile distilled H2O three times. Finally, the spores were resuspended in 10% glycerol at a density of 4.0 × 107 per mL and stored at −80 °C. Fermentation and Addition of Inhibitors and Putative Precursors. For the inoculum, 2.0 mL of the spore suspension stock was transferred aseptically to 500 mL Erlenmeyer flasks containing 100 mL of potato dextrose broth (PDB) medium. The cultivation was performed at 28 °C and 165 rpm for 36 h to produce the fermenter inoculums. In the standard shake flask cultures, 3.0 mL I



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13′), 1.19 (3H, s, H3-12′), 1.13 (3H, s, H3-15′), 7.20−7.80 (m, Ph-H); ESIMS m/z 621.4 [M + Na]+. (R)-MTPA ester derivative 3d: 1H NMR data (600 MHz, CDCl3 TMS, δ ppm) δ 5.61 (1H, dd, J = 5.0, 12.0 Hz, H-6), 5.19 (1H, t, J = 7.1 Hz, H-6′), 4.97 (1H, d, J = 11.0, H-10′), 4.72 (1H, t, J = 9.6 Hz, H2′), 2.85 (1H, m, Ha-1′), 2.76 (1H, m, Hb-1′), 2.63 (1H, m, H-4), 2.55 (1H, m, H-4), 2.23 (2H, m, H2-5), 2.12 (1H, m, Ha-5′), 2.06 (1H, m, Hb-5′), 1.85 (2H,m, H2-8′), 1.68 (1H, m, Ha-9′), 1.57 (1H, m, Hb-9′), 1.46 (2H, m, H2-8′), 1.54 (3H, s, H3-14′), 1.25 (3H, s, H313′), 1.19 (3H, s, H3-12′), 1.13 (3H, s, H3-15′), 7.20−7.80 (m, Ph-H); ESIMS m/z 815.6 [M + H]+. Bicycloalternarene C (4): pale yellow oil; [α]25D +76.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 270 (4.13) nm; CD (c 2.65 mM, MeOH) λmax (Δε) 323 (−0.18), 268 (+1.00), 201 (−0.27), 191 (+0.30) nm; IR (KBr) νmax 3337, 1693, 1613, 1600, 1509, 1388, 1222, 1182, 1129, 924 cm−1; 1H NMR and 13C NMR data in Tables 1 and 3; HRESIMS m/z 401.1954 [M + Na]+ (calcd for C21H30O6Na, 401.1940). (S)-MTPA ester derivative 4a: 1H NMR data (600 MHz, CDCl3 TMS, δ ppm) δ 6.83 (1H, t, J = 11.0, H-10′), 5.97 (1H, m, H-4), 5.15 (1H, t, J = 7.0, Hz, H-6′), 4.57 (1H, d, J = 6.6, H-2′), 2.78 (2H, m, H21′), 2.53 (1H, m, Ha-6), 2.45 (1H, m, Hb-6), 2.38 (1H, m, Ha-5), 2.25 (1H, m, Hb-5), 1.43 (2H, m, H2-4′), 2.32 (1H, m, Ha-5′), 2.13 (1H, m, Hb-5′), 2.13 (2H, m, H2-8′), 2.05 (2H, m, H2-9′), 1.82 (3H, s, H315′), 1.60 (3H, s, H3-14′), 1.25 (3H, s, H3-13′), 7.20−7.80 (m, Ph-H); ESIMS m/z 595.4 [M + H]+. (R)-MTPA ester derivative 4b: 1H NMR data (600 MHz, CDCl3 TMS, δ ppm) δ 6.84 (1H, dd, J = 5.0, 12.0 Hz, H-10′), 5.97 (1H, 1H, m, H-4), 5.16 (1H, d, J = 11.0, H-6′), 4.69 (1H, t, J = 7.0 Hz, H-2′), 2.83 (1H, m, H2-1′), 2.43 (1H, m, Ha-6), 2.35 (1H, m, Hb-6), 2.28 (1H, m, Ha-5), 2.15 (1H, m, Hb-5), 1.48 (2H, m, H2-4′), 2.30 (1H, m, Ha-5′), 2.14 (1H, m, Hb-5′), 2.13 (2H, m, H2-8′), 2.05 (2H, m, H29′), 1.82 (3H, s, H3-15′), 1.60 (3H, s, H3-14′), 1.26 (3H, s, H3-13′), 7.20−7.80 (m, Ph-H); ESIMS m/z 595.4 [M + H]+. Bicycloalternarene D (5): pale yellow oil; [α]25D +63.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 270 (4.09) nm; CD (c 2.65 mM, MeOH) λmax (Δε) 269 (+2.24), 194 (−1.90) nm; IR (KBr) νmax 3337, 1693, 1613, 1600, 1509, 1401, 1235, 1129, 1023, 944 cm−1; 1H NMR and 13C NMR data in Tables 1 and 3; HRESIMS m/z 401.1952 [M + Na]+ (calcd for C21H30O6Na, 401.1940). Bicycloalternarene E (6): pale yellow oil; [α]25D +93.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 264 (4.13) nm; CD (c 2.65 mM, MeOH) λmax (Δε) 269 (+1.97), 195 (−1.85) nm; IR (KBr) νmax 1699, 1653, 1593, 1527 cm−1; 1H NMR data (DMSO, 600 MHz, TMS, δ ppm) in Tables 1 and 3; HRESIMS m/z 401.1958 [M + Na]+ (calcd for C21H30O6Na, 401.1940). Bicycloalternarene F (7): pale yellow oil; [α]25D +37.8 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 265 (4.06) nm; CD (c 2.65 mM, MeOH) λmax (Δε) 318 (−0.30), 262 (+1.19), 208 (−1.37) nm; IR (KBr) νmax 1699, 1613, 1388, 1242, 1017, 977 cm−1; 1H NMR data (DMSO, 600 MHz, TMS, δ ppm) in Tables 1 and 3; HRESIMS m/z 401.1922 [M + Na]+ (calcd for C21H30O6Na, 401.1940). Tricycloalternarene B (8): colorless oil; [α]25D +66.5 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 264 (4.08) nm; CD (c 2.65 mM, MeOH) λmax (Δε) 300 (−0.11), 262 (−1.14), 203 (−0.18), 185 (+2.52) nm; IR (KBr) νmax 3750, 3673, 3576, 1704, 1652, 1540, 1454, 1015, 509, 449 cm−1; 1H and 13C NMR in Tables 2 and 3; HRESIMS m/z 401.2315 [M + Na]+ (calcd for C22H34O5Na, 401.2304). (S)-MTPA ester derivative 8a: 1H NMR data (600 MHz, CDCl3 TMS, δ ppm) δ 5.94 (1H, t, J = 4.5 Hz, H-4), 5.12 (1H, t, J = 6.0 Hz, H-10′), 3.77 (1H, dd, J = 7.0, 3.0 Hz, H-6), 3.51 (3H, s, 6-OCH3), 2.67 (1H, d, J = 18.0 Hz, Ha-1′), 2.57 (1H, m, Ha-5), 2.31 (1H, dd, J = 18.0, 6.0 Hz, Hb-1′), 2.10 (1H, m, Hb-5), 2.07 (1H, m, Ha-4′), 2.03 (1H, m, Ha-9′), 1.80 (1H, m, Ha-5′), 1.69 (3H, s, H3-12′), 1.61 (3H, m, H3-15′), 1.52 (1H, m, Hb-5′), 1.48 (1H, m, Hb-4′), 1.48 (1H, m, Hb-9′), 1.46 (2H, m, H2-8′), 1.27 (3H, s, H3-13′), 1.14 (3H, s, H314′), 7.25−7.61 (20H, m, Ph-H); ESIMS m/z 595.4 [M + H]+. (R)-MTPA ester derivative 8b: 1H NMR data (600 MHz, CDCl3 TMS, δ ppm) δ 5.90 (1H, t, J = 4.5 Hz, H-4), 5.11 (1H, t, J = 6.0 Hz, H-10′), 3.77 (1H, dd, J = 7.0, 3.0 Hz, H-6), 3.55 (3H, s, 6-OCH3),

(1H,m, Ha-4′), 1.72 (1H, m, Ha-8′), 1.69 (3H, s, H3-12′), 1.65 (1H, m, Ha-5′), 1.61 (3H, s, H3-15′), 1.57 (1H, m, Hb-5′), 1.55 (1H, m, Hb-4′), 1.53 (1H, m, Hb-8′), 1.42 (3H, s, acetonide), 1.36 (3H, s, acetonide), 1.23 (3H, s, H3-13′), 1.22 (3H, s, H3-14′); ESIMS m/z 445.2 [M + Na]+; HRESIMS m/z 445.2563 [M + Na]+ (calcd for C24H38O6Na, 445.2566). (S)-MTPA ester derivative 2c: 1H NMR (600 MHz, CDCl3 TMS, δ ppm) δ 5.57 (1H, dd, J = 5.0, 12.4 Hz, H-6), 5.14 (1H, t, J = 7.1 Hz, H-10′), 4.70 (1H, t, J = 9.6 Hz, H-2′), 3.37 (1H, d, J = 11.0, H-6′), 2.84 (1H, m, Ha-1′), 2.76 (1H, m, Hb-1′), 2.65 (2H, brs, H2-4), 2.36 (2H, m, H2-5), 2.15 (1H, m, Ha-9′), 2.06 (1H, m, Hb-9′), 1.69 (3H, s, H3-12′), 1.65 (1H, m, Ha-5′), 1.63 (3H, s, H3-15′), 1.59 (1H, m, Hb5′), 1.57 (1H, m, Hb-4′), 1.40 (1H, m, Hb-8′), 1.25 (3H, s, H3-13′), 1.19 (3H, s, H3-14′), 7.20−7.80 (m, Ph-H); ESIMS m/z 621.4 [M + Na]+. (S)-MTPA ester derivative 2d: 1H NMR (600 MHz, CDCl3 TMS, δ ppm) δ 5.57 (1H, m, H-6), 5.02 (1H, H-10′), 5.01 (1H, H-6′), 4.70 (1H, t, J = 9.6 Hz, H-2′), 2.72 (2H, d, J = 9.6 Hz, H-1′), 2.62 (1H, m, Ha-4), 2.50 (1H, m, Hb-4), 2.34 (2H, m, H-5), 2.01 (2H, m, H2-9′), 1.88 (1H, m, Ha-5′), 1.78 (1H, m, Hb-5′), 1.69 (3H, s, H3-12′), 1.63 (3H, s, H3-15′), 1.46 (2H, m, H2-4′), 1.36 (1H, m, Ha-8′), 1.35 (1H, m, Hb-8′), 1.18 (3H, s, H3-13′), 1.14 (3H, s, H3-14′), 7.20−7.80 (m, Ph-H); ESIMS m/z 837.5 [M + Na]+. (R)-MTPA ester derivative 2e: 1H NMR (600 MHz, CDCl3 TMS, δ ppm) δ 5.61 (1H, dd, J = 5.0, 12.4 Hz, H-6), 5.14 (1H, t, J = 7.1 Hz, H-10′), 4.70 (1H, t, J = 9.6 Hz, H-2′), 3.37 (1H, d, J = 11.0, H-6′), 2.86 (1H, m, Ha-1′), 2.77 (1H, m, Hb-1′), 2.58 (2H, brs, H2-4), 2.24 (1H, m, Ha-5), 2.16 (1H, m, Hb-5), 2.15 (1H, m, Ha-9′), 2.06 (1H, m, Hb-9′), 1.69 (3H, s, H3-12′), 1.65 (1H, m, Ha-5′), 1.63 (3H, s, H315′), 1.59 (1H, m, Hb-5′), 1.57 (1H, m, Hb-4′), 1.40 (1H, m, Hb-8′), 1.25 (3H, s, H3-13′), 1.19 (3H, s, H3-14′), 7.20−7.80 (m, Ph-H); ESIMS m/z 599.4 [M + H]+. (R)-MTPA ester derivative 2f: 1H NMR (600 MHz, CDCl3 TMS, δ ppm) δ 5.61 (1H, m, H-6), 5.05 (1H, m, H-10′), 5.01 (1H, H-6′), 4.70 (1H, t, J = 9.6 Hz, H-2′), 2.72 (2H, d, J = 9.6 Hz, H-1′), 2.61 (1H, m, Ha-4), 2.07 (2H, m, Ha-9′), 2.01 (1H, m, Hb-9′), 1.78 (1H, m, Ha5′), 1.68 (1H, m, Hb-5′), 1.69 (3H, s, H3-12′), 1.50 (1H, m, Ha-8′), 1.40 (1H, m, Hb-8′), 1.37 (2H, m, H2-4′), 1.63 (3H, s, H3-15′), 1.59 (1H, m, Hb-5′), 1.16 (3H, s, H3-13′), 1.14 (3H, s, H3-14′), 7.20−7.80 (m, Ph-H); ESIMS m/z 815.5 [M + H]+. Bicycloalternarene B (3): pale yellow oil; [α]25D +78.6 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 263 (4.11) nm; CD (c 2.87 mM, MeOH) λmax (Δε) 266 (+2.30), 194 (−1.58) nm; IR (KBr) νmax 3417, 1633, 1381, 1235, 1189, 1129, 1023, 917 cm−1; 1H NMR and 13C NMR data in Tables 1 and 3; HRESIMS m/z 383.2435 [M + H]+ (calcd for C21H35O6, 383.2434). (S)-MTPA ester derivative 3a: 1H NMR data (600 MHz, CDCl3 TMS, δ ppm) δ 5.57 (1H, t, J = 11.0, H-6), 5.19 (1H, t, J = 7.1 Hz, H6′), 4.70 (1H, t, J = 9.6 Hz, H-2′), 3.33 (1H, d, J = 11.0, H-10′), 2.84 (1H, m, Ha-1′), 2.74 (1H, m, Hb-1′), 2.65 (2H, brs, H2-4), 2.36 (2H, m, H2-5), 2.15 (2H, m, H2-5′), 2.06 (2H, m, H2-4′), 1.64 (3H, s, H314′), 1.59 (1H, m, Hb-9′), 1.50 (1H, m, Hb-9′), 1.49 (1H, m, Ha-8′), 1.40 (1H, m, Hb-8′), 1.25 (3H, s, H3-13′), 1.19 (3H, s, H3-12′), 1.13 (3H, s, H3-15′), 7.20−7.80 (m, Ph-H); ESIMS m/z 599.4 [M + H]+. (S)-MTPA ester derivative 3b: 1H NMR data (600 MHz, CDCl3 TMS, δ ppm) δ 5.57 (1H, t, J = 11.0, H-6), 5.19 (1H, t, J = 7.1 Hz, H6′), 4.97 (1H, d, J = 11.0, H-10′), 4.72 (1H, t, J = 9.6 Hz, H-2′), 2.85 (1H, m, Ha-1′), 2.75 (1H, m, Hb-1′), 2.65 (2H, m, H2-4), 2.35 (2H, m, H2-5), 2.12 (1H, m, Ha-5′), 2.06 (1H, m, Hb-5′), 1.97 (2H, m, H28′), 1.78 (1H, m, Ha-9′), 1.65 (1H, m, Hb-9′), 1.46 (2H, m, H2-8′), 1.58 (3H, s, H3-14′), 1.40 (1H, m, Hb-8′), 1.25 (3H, s, H3-13′), 1.19 (3H, s, H3-12′), 1.13 (3H, s, H3-15′), 7.20−7.80 (m, Ph-H); ESIMS m/z 837.5 [M + Na]+. (R)-MTPA ester derivative 3c: 1H NMR data (600 MHz, CDCl3 TMS, δ ppm) δ 5.61 (1H, dd, J = 5.0, 12.0 Hz, H-6), 5.19 (1H, t, J = 7.1 Hz, H-6′), 4.72 (1H, t, J = 9.6 Hz, H-2′), 3.33 (1H, d, J = 11.0, H10′), 2.85 (1H, m, Ha-1′), 2.76 (1H, m, Hb-1′), 2.55 (2H, brs, H2-4), 2.24 (2H, m, H2-5), 2.15 (2H, m, H2-5′), 2.06 (2H, m, Ha-4′), 1.64 (3H, s, H3-14′), 1.59 (1H, m, Ha-9′), 1.50 (1H, m, Hb-9′), 1.57 (1H, m, Hb-4′), 1.49 (1H, m, Ha-8′), 1.40 (1H, m, Hb-8′), 1.25 (3H, s, H3J



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2.64 (1H, d, J = 18.0 Hz, Ha-1′), 2.60 (1H, m, Ha-5), 2.27 (1H, dd, J = 18.0, 6.0 Hz, Hb-1′), 2.06 (1H, m, Hb-5), 2.06 (1H, m, Ha-9′), 2.05 (1H, m, Ha-4′), 1.80 (1H, m, Ha-5′), 1.68 (3H, s, H3-12′), 1.61 (3H, m, H3-15′), 1.52 (1H, m, Hb-5′), 1.48 (1H, m, Hb-4′), 1.48 (1H, m, Hb-9′), 1.46 (2H, m, H2-8′), 1.12 (3H, s, H3-13′), 1.03 (3H, s, H314′), 7.25−7.60 (20H, m, Ph-H); ESIMS m/z 595.4 [M + H]+. Tricycloalternarene C (9): colorless oil; [α]25D +67.5 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 265 (4.10) nm; CD (c 2.87 mM, MeOH) λmax (Δε) 318 (+0.30), 263 (+1.22), 194 (−0.55) nm; IR (KBr) νmax 3837, 3748, 3673, 3613, 3558, 1750, 1700, 1540, 1515, 1457, 504, 418 cm−1; 1H and 13C NMR in Tables 2 and 3; HRESIMS m/z 371.2204 [M + Na]+ (calcd for C21H32O4Na, 371.2198). Monocycloalternarene A (12): dark yellow oil; [α]25D +13.5 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 272 (3.88) nm; IR (KBr) νmax 3850, 3747, 3671, 3612, 3564, 1741, 1696, 1649, 1539, 1506, 1451, 1012, 421 cm−1; 1H and 13C NMR in Tables 2 and 3; HRESIMS m/z 401.2296 [M + Na]+ (calcd for C22H34O5Na, 401.2304). (S)-MTPA ester derivative 12a: 1H NMR data (600 MHz, CDCl3 TMS, δ ppm) δ 7.02 (1H, d, J = 8.0, H-5), 6.86 (1H, d, J = 8.0, H-6), 4.96 (1H, H-2′), 4.95 (1H, m, H-10′), 4.82 (1H, m, H-6′), 3.55 (3H, s, 4-OCH3), 3.00 (1H, m, H-1′), 1.91 (1H, m, Ha-8′), 1.91 (2H, m, H25′), 1.82 (2H, m, H2-4′), 1.80 (1H, m, Hb-8′), 1.74 (1H, m, Ha-9′), 1.62 (1H, m, Hb-9′), 1.45 (3H, s, H3-13′), 1.26 (3H, s, H3-14′), 1.20 (3H, s, H3-12′), 1.14 (3H, s, H3-15′), 7.4−7.8 (m, Ph-H); ESIMS m/z 833.4 [M + Na]+. (R)-MTPA ester derivative 12b: 1H NMR data (600 MHz, CDCl3 TMS, δ ppm) δ 7.02 (1H, d, J = 8.0, H-5), 6.86 (1H, d, J = 8.0, H-6), 5.03 (1H, t, J = 6.0 Hz, H-2′), 4.96 (1H, dd, J = 5.9, 9.1, H-10′), 4.82 (1H, m, H-6′), 3.55 (3H, s, 4-OCH3), 3.00 (1H, m, H-1′), 1.92 (1H, m, Ha-8′), 1.91 (2H, m, H2-5′), 1.82 (2H, m, H2-4′), 1.81 (1H, m, Hb8′), 1.62 (1H, m, Ha-9′), 1.53 (1H, m, Hb-9′), 1.45 (3H, s, H3-13′), 1.26 (3H, s, H3-14′), 1.14 (3H, s, H3-12′) 1.10 (3H, s, H3-15′), 7.4− 7.8 (m, Ph-H); ESIMS m/z 833.4 [M + Na]+. Monocycloalternarene B (13): dark yellow oil; [α]25D +10.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 265 (4.08) nm; IR (KBr) νmax 1693, 1620, 1509, 1441, 1308, 1222, 1017, 990, 505, 417 cm−1; 1 H and 13C NMR data in Tables 2 and 3; HRESIMS m/z 415.2101 [M + Na]+ (calcd for C22H32O6Na, 415.2097). Monocycloalternarene C (14): dark yellow oil; [α]25D +2.5 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 272 (3.93) nm; CD (c 2.53 mM, MeOH) λmax (Δε) 316 (−0.19), 275 (−0.60), 202 (−0.19) nm; IR (KBr) νmax 3856, 3743, 3679, 3640, 3615, 1738, 1704, 1640, 1546, 1503, 507, 488, 429 cm−1; 1H and 13C NMR data in Tables 2 and 3; HRESIMS m/z 417.2250 [M + Na]+ (calcd for C22H34O6Na, 417.2253). Monocycloalternarene D (15): dark yellow oil; [α]25D +2.0 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 272 (3.93) nm; IR (KBr) νmax 3870, 3754, 3680, 3615, 1756, 1700, 1640, 1545, 1501, 507, 429 cm−1; 1 H and13C NMR data in Tables 2 and 3; ESIMS m/z 391.2123 [M − H]− (calcd for C22H31O6, 391.2121). Computational Section. Mixed torsional/low-mode conformational searches were carried out with the Macromodel 9.9.223 software20 using the Merck molecular force field (MMFF) with an implicit solvent model for chloroform. Geometry reoptimizations at B3LYP/6-31G(d) in vacuo and B3LYP/TZVP with the PCM solvent model for acetonitrile followed by TDDFT calculations using various functionals (B3LYP, BH&HLYP, PBE0) and the TZVP basis set were performed with the Gaussian 09 package.21 ECD spectra were generated as the sum of Gaussians with 2400 and 4200 cm−1 halfheight widths (corresponding to ca. 17 and 31 at 210 nm) using dipole-velocity-computed rotational strengths.22 Boltzmann distributions were estimated from the ZPVE-corrected B3LYP/6-31G(d) energies in vacuo and from the B3LYP/TZVP energies in the solvent model calculations. The MOLEKEL software package was used for visualization of the results.23 Transient Transfection and NF-κB-Dependent Reporter Gene Expression Assay. RAW 264.7 cells (2.5 × 105 cells/well) were placed in 24-well plates at 37 °C. After 24 h incubation, the cells were then transiently transfected with the pNF-κB-Luc expression plasmid (0.5 μg/well). Transfections were performed using lipofect-

amine 2000 in accordance with the instructions of the manufacturer (Invitrogen). After 24 h, the cells were incubated with the test compounds for 2 h and subsequently treated with LPS (0.1 μg/mL) for an additional 4 h. The luciferase assay was performed with the aid of a Steady-Glo luciferase assay system in accordance with the instructions of the manufacturer (Promega). PDTC (pyrrolidinedithiocarbamic acid, ammonium salt) was used as the positive control in the inhibition assay. Cytotoxicity Bioassays. Cytotoxicities of the new compounds against A549 and HL-60 human tumor cells were determined by the MTT18 and SRB19 methods. ADR (adriamycin) was used as the positive control with IC50 values of 0.35 and 0.09 μM on A549 and HL-60 cancer cells, respectively.

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ASSOCIATED CONTENT

S Supporting Information *

Details for chemical derivatizations, culture with additions of DBA and PA; NMR and MS spectra for new compounds 1−9 and 12−15, CD spectra for compounds 3, 5, 7−9, and 14. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Fundation of China (No. 41176120), the National High Technology Research and Development Program of China (No. 2013AA092901), the Program for New Century Excellent Talents in University (No. NCET-12-0499), the Public Projects of State Oceanic Administration (No. 2010418022-3), the Promotive Research Fund for Excellent Young and Middle-aged Scientisits of Shandong Province (No. BS2010HZ027), the State Key Laboratory of Bioorganic and Natural Products Chemistry (SKLBNPC12331), and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0944). The authors thank the National Development Agency for financial support in the frame of the TÉT-12_CN-1-2012-0023 Chinese-Hungrian Bilateral Project. T.K. and A.M. thank the Hungarian National Research Foundation (OTKA K105871) and the TÉT-12_CN Programme for financial support and the National Information Infrastructure Development Institute (NIIFI 10038) for CPU time.

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REFERENCES

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