Polyhydroxy Cyclohexanols from a Dendrodochium sp. Fungus

Article pubs.acs.org/jnp

Polyhydroxy Cyclohexanols from a Dendrodochium sp. Fungus Associated with the Sea Cucumber Holothuria nobilis Selenka Dong-Xiao Xu,†,‡ Peng Sun,†,‡ Tibor Kurtán,§ Attila Mándi,§ Hua Tang,† Baoshu Liu,† William H. Gerwick,⊥ Zhi-Wei Wang,∥,* and Wen Zhang†,* †

Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, 325 Guo-He Road, Shanghai 200433, People’s Republic of China § Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary ⊥ Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, United States ∥ Department of Orthopaedics, Changhai Hospital, Second Military Medical University, 168 Chang-Hai Road, Shanghai 200003, People’s Republic of China S Supporting Information *

ABSTRACT: Four cyclohexanol analogues, dendrodochols A−D (1− 4), were isolated from a Dendrodochium sp. fungus associated with the sea cucumber Holothuria nobilis Selenka collected from the South China Sea. The structures were elucidated by means of detailed spectroscopic analysis. The absolute configurations were assigned using a solution TDDFT/ECD calculation approach and the modified Mosher’s method. In an in vitro bioassay, these compounds exhibited no growth inhibition activity against the A549 and MG63 cell lines. Dendrodochols 1 and 3 exhibited modest antifungal activity against Candida strains, Cryptococcus neoformans, and Trichophyton rubrum, whereas 2 and 4 showed no activity against the tested strains.

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dendrodochols A−D (1−4). We herein report the isolation, structure elucidation, and bioactivity of four new representatives of these small highly oxidized polyketides, dendrodochols A−D (1−4).

arine-derived fungi are a rich source of structurally novel metabolites with interesting biological activities and are attracting increasing attention as potential sources of new pharmaceutical leads.1 A sharp increase in the number of published structures from marine fungi occurred in the early 1990s and developed quickly in the years that followed. In this regard, nearly 700 new secondary metabolites were reported from marine fungi during the period from 2006 to mid-2010.2 Roughly two-thirds of all new compounds reported from marine fungi were derived from isolates coming from living matter, mainly algae, mangrove habitats, sponges, and mollusks. Only nine new compounds were obtained from the sea cucumber-associated fungus Acremonium striatisporum, which was isolated from Eupentacta f raudatrix.3−6 During our ongoing screening for biologically active secondary metabolites from fungi,7−11 we isolated a Dendrodochium sp. fungus from the sea cucumber Holothuria nobilis Selenka collected from the South China Sea. Previous investigation of another Dendrodochium sp. resulted in the discovery of two novel 16-mer linear peptides as inhibitors of HIV-1 integrase, namely, integramides A and B.12 The investigation of the EtOAc extract of Dendrodochium sp. in our laboratory led to the discovery of 13 new 12-membered macrolides, dendrodolides A−M.13 It was the first report of 12membered macrolides from fungi of the genus Dendrodochium. Our continuing investigation of this fungus has now resulted in the isolation of four new polyhydroxy cyclohexanols, © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The Dendrodochium sp. fungus was cultivated on biomalt agar medium for 28 days and then extracted with EtOAc. The EtOAc extract was subjected to repeated column chromatography on silica gel, Sephadex LH-20, and RP-HPLC to yield the pure dendrodochols A−D (1−4).

Dendrodochol A (1) was isolated as an optically active, amorphous solid. The molecular formula C14H20O4 was established by HRESIMS, indicating five double-bond equivalents. The IR spectrum of 1 showed absorptions for hydroxy groups (3373 cm−1), a ketone group (1716 cm−1), and an Received: January 11, 2014 Published: April 21, 2014 1179

dx.doi.org/10.1021/np500024r | J. Nat. Prod. 2014, 77, 1179−1184

Journal of Natural Products

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Table 1. 13C NMR Data for Dendrodochols A−D (1−4) 1a

no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 a

18.5, 130.7, 133.1, 133.5, 134.0, 131.0, 135.0, 40.0, 39.9, 75.3, 82.8, 209.4, 55.8, 59.1,

1b

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

18.5, 129.9, 132.0, 132.4, 132.8, 130.7, 135.8, 39.4, 39.7, 75.0, 83.4, 209.7, 55.6, 58.3,

2c

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

18.2, 129.0, 132.4, 131.7, 131.1, 130.9, 138.0, 37.0, 41.0, 70.0, 78.7, 72.0, 48.4, 62.4,

3c CH3 CH CH CH CH CH CH CH CH2 CH CH CH CH CH2

18.7, 129.4, 132.8, 132.1, 131.6, 131.1, 139.0, 37.5, 44.3, 65.6, 44.4, 69.4, 48.7, 63.4,

4c CH3 CH CH CH CH CH CH CH CH2 CH CH2 CH CH CH2

24.5, 67.8, 140.4, 128.4, 132.0, 134.1, 84.1, 43.6, 34.8, 71.6, 79.5, 68.6, 50.3, 67.7,

CH3 CH CH CH CH CH CH CH CH2 CH CH CH CH CH2

In CD3OD, measured at 150 MHz. bIn C5D5N, measured at 100 MHz. cIn C5D5N, measured at 150 MHz.

Table 2. 1H NMR Data for Dendrodochols A−D (1−4) 1a

no. 1 2 3 4 5 6 7 8 9α 9β 10 11α 11β 12 13 14a 14b

1.80, 5.74, 6.10, 6.13, 6.17, 6.21, 5.59, 2.35, 1.85, 2,08, 3.54, 4.08,

d (6.6) dq (15.2, 6.6) ovd ov ov ov dd (15.0, 8.4) m q (12.0) ddd (12.0, 4.4, 3.6) ddd (12.0, 10.0, 4.4) dd (10.0, 1.2)

2.45, m 3.70, dd (11.4, 6.0) 3.80, dd (11.4, 2.4)

1b 1.66, 5.67, 6.13, 6.21, 6.19, 6.29, 5.72, 2.64, 2.15, 2.38, 4.05, 4.59,

d (6.8) dq (15.0, 6.9) m ov ov ddd (15.0, 7.0, 3.3) dd (15.0, 8.0) m q (12.0) ddd (12.0, 4.3, 3.6) ddd (12.0, 11.0, 4.3) d (9.5)

2.66, m 4.07, dd (11.0, 4.9) 4.19, dd (11.0, 1.6)

2c 1.63, 5.63, 6.10, 6.14, 6.20, 6.23, 5.69, 2.94, 1.70, 2.29, 4.55, 3.84,

d (6.6) qd, (13.8, 6.6) ov ov ov ov dd (15.0, 9.0) ddd (15.0, 12.0, 3.6) q (12.0) dt (12.0, 4.2) ddd (13.8, 11.4, 4.8) dd (9.0, 2.4)

4.90, 1.78, 4.05, 4.20,

br s dt (8.4, 2.4) dd (10.2, 3.0) dd (10.2, 8.4)

3c

4c

1.61, d (6.6) 5.65, qd (13.8, 6.6) 6.13, ov 6.16, ov 6.25, ov 6.29, ov 5..77, dd (14.4, 9.0) 3.02, ddd (15.0, 12.0, 3.0) 1.63, q (12.0) 2.40, dd (12.0, 2.4) 4.76, ddd (12.0, 9.0, 4.2) 1.82, dt (12.0, 2.4) 2.73, dd (12.6, 1.8) 4.88, d (2.4) 1.60, ov 4.12, dd (10.4, 3.0) 4.21, dd (10.4, 7.2)

1.42, d (6.0) 4.60, q (6.0) 6.01, dd (14.4, 5.4) 6.52, dd (14.4, 10.8) 6.47, dd (14.4, 7.2) 5.89, dd (14.4, 7.2) 4.17, dd (9.6, 7.2) 2.50, ddd (16.2, 12.6, 3.6) 1.58, q (12.0) 2..38, ddd (15.6, 12.0, 4.2) 4.46, dt (10.8, 4.8) 3.90, dd (9.0, 3.0) 4.49, br s 2.15, dd (19.8, 10.8) 4.06,e t (7.2) 4.25,f dd (10.8, 7.2)

a In CD3OD, measured at 600 MHz. bIn C5D5N, measured at 400 MHz. cIn C5D5N, measured at 600 MHz. dov = overlapped. eα-Orientation. fβOrientation.

alkene function (3012, 1638, 995 cm−1). This observation was in agreement with the signals in the 13C NMR and DEPT spectra (Table 1) for one carbonyl carbon and three double bonds at lower field and seven sp3 carbon atoms at higher field (2 × OCH, 1 × OCH2, 2 × CH, 1 × CH2, 1 × CH3), accounting for four double-bond equivalents. The remaining double-bond equivalent was due to the presence of one ring in the molecule. The HSQC spectrum facilitated the assignment of all of the protonated carbons (Tables 1 and 2). The COSY spectrum established the proton sequences from H3-1 to H-11 and from H-8 to H-13 and then to H2-14 (Figure 1). The observation of HMBC correlations from both H-11 and H2-14 to C-12 and from H-13 to C-11 led to the formation of a cyclohexanone and the planar structure of 1 (Figure 1). The relative configuration of 1 was deduced from the 1H−1H coupling constants and NOESY data. The obvious NOE crosspeak between H-8 and H-10 indicated a 1,3-diaxial relationship of the two protons, which were arbitrarily assigned the αorientation. In contrast, the distinct NOE effect between H-9β, H-11, and H-13 indicated axial positions for these protons, and

Figure 1. COSY (bold) and key HMBC (arrow) correlations of 1.

they were accordingly assigned the β-orientation. The large coupling constants between H-10 and both H-9β and H-11 (3JH9β,H10 = 12.0, 3JH10,H11 = 10.0, Hz) further supported this conclusion (Figure 2). The geometries of the Δ2 and Δ6 double bonds were assigned as E based on the proton coupling constants (3JH2,H3 = 15.2, 3JH6,H7 = 15.0, Hz). The overlapped proton signals for H-4 and H-5 prevented the configurational assignment by proton coupling constants. Careful comparison of the 13C NMR shift values of the conjugated double bonds with literature data14,15 led to the determination of the geometry of Δ4 as E. The 13C NMR shifts of C-3 and C-6 would be markedly upfield shifted for a Z-double bond due to the γ-gauche effect.16−19 These NMR-based structural assignments were strongly supported by comparison with the reported data of its 10-epimer, namely, 2,3-didehydropalitantin, 1180



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Figure 2. Key NOESY correlations of 1 indicated on the lowest-energy DFT solution conformer.

recently obtained from a Paraphaeosphaeria sp. fungus.20 The relative configuration of 1 was thus determined as (2E,4E,6E,8R*,10R*,11S*,13S*). The absolute configuration of 1 was determined by the solution time-dependent density functional theory/electronic circular dichroism (TDDFT-ECD) approach. The solution ECD spectrum of 1 showed a negative n−π* Cotton effect (CE) at 287 nm and positive CEs at 262, 232, and 194 nm. The DFT optimization of the initial Merck molecular force field (MMFF) conformers of the arbitrarily chosen (8R,10R,11S,13S) enantiomer afforded four conformers above a 1% population, which differed only in the orientation of the C-13 hydroxymethyl and C-8 heptatrienyl groups (Figure 3). In

Figure 4. Experimental solution spectrum (black) of dendrodochol A (1) compared with BH&HLYP/TZVP-calculated ECD spectra of (8R,10R,11S,13S)-1 (blue) and (8S,10S,11R,13R)-1 (red). Bars represent the calculated rotational strength values of the lowestenergy conformer of (8R,10R,11S,13S)-1.

alcohol in 2 (δH 4.90, δC 72.0). The secondary alcohol was readily assigned at C-12 due to the proton connectivity from H3-1 to H2-14 as deduced from the COSY data. A β-orientation of H-12 was deduced from its NOE effect with both H-11 and H-13 and further confirmed by the small coupling constant between H-12 (br s) and both H-11 and H-13. The relative configurations of 2 at C-8, C-10, C-11, and C-13 were shown to be the same as those of 1 due to the observation of similar NOE patterns as shown in Figure 5. Thus, the relative

Figure 5. Key NOESY correlations of 2 indicated on the lowest-energy conformer.

Figure 3. Structure and population of the low-energy DFT conformers (>1%) of (8R,10R,11S,13S)-1.

configuration of 2 was determined as (8R*,10R*,11S*,12R*,13S*). On the basis of the common biogenetic origin of 1 and 3, we propose the absolute configuration of 2 as (8R,10R,11S,12R,13S). Dendrodochol C (3) was also isolated as an optically active, amorphous solid. Its molecular formula of C14H22O3 was deduced from the HRESIMS data. The IR and UV spectra of 3 closely resembled those of 2, showing similar functionalities in the molecule. Analysis of the 1H and 13C NMR spectra of 3 also revealed similarities to those of 2 (Tables 1 and 2) except for the absence of the secondary hydroxy group at C-11 (δH 3.84, δC 78.7). In combination with the appearance of a methylene group (δH 1.82 and 2.73, δC 44.4), compound 3 was determined as the 11-deoxy analogue of 2. The assignment of H2-11 was further confirmed by the proton sequence from H3-1 to H2-14, established by COSY data. The 11-deoxy structure of 3 was further confirmed by detailed analysis of its 2D NMR data, and thus the relative configuration of 3 was assigned as (8R*,10R*,12S*,13S*). Dendrodochol C (3) contains a conjugated triene chromophore with an adjacent stereogenic

all of the computed conformers, the four substituents had equatorial orientations in accordance with the measured NOE effects. The ECD spectra of the conformers of (8R,10R,11S,13S)-1 were calculated with B3LYP, BH&HLYP, and PBE0 functionals and the TZVP basis set, and the Boltzmann-weighted ECD spectra corroborated well the experimental one, with BH&HLYP/TZVP giving the best agreement (Figure 4). Thus, the absolute configuration of 1 was determined as (8R,10R,11S,13S), which was also in accordance with the octant rule of the substituted cyclohexanone ring.21 The negative n−π* CE derived mainly from the negative contribution of the C-13 heptatrienyl group located in the right upper or left lower back octant. Dendrodochol B (2) was obtained as an optically active, amorphous solid. Its molecular formula was determined as C14H22O4 by HRESIMS, thus possessing two mass units more than 1. The absorption band of the ketone group was absent in the IR spectrum of 2. The 1H and 13C NMR spectra of compound 2 closely correlated to those of 1, except that the ketone group (δC 209.4) in 1 was replaced by a secondary 1181



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Figure 6. Structure and population of the low-energy DFT conformers (>1%) of (8R,10R,12S,13S)-3.

condensed tetrahydrofuran ring (Figure 8) and consequently established the planar structure of 4.

center at C-8, and hence it has a weak ECD spectrum with positive CEs at 308 and 226 nm and a negative one at 264 nm. In order to determine its absolute configuration independently from that of 1, the solution TDDFT-ECD calculation method was applied. The DFT optimization of the MMFF conformers of (8R,10R,12S,13S)-3 produced a major conformer as 93.6% of the population and a minor one as 3.0%, with the remaining conformers being below 1% of the population (Figure 6). The two conformers differed in the orientation of their hydroxy protons and the heptatrienyl side-chain by rotation about the C-7−C-8 bond. The Boltzmann-averaged BH&HLYP/TZVP ECD spectrum of (8R,10R,12S,13S)-3 (Figure 7) reproduced

Figure 8. COSY and key HMBC correlations of 4.

The relative configuration of the cyclohexane subunit of 4 was proven to be the same as that of 2 due to the similar NOE interactions between H-8 and H-10, and between H-9β, H-11, and H-13. The β-orientation of H-7 was deduced from its NOE interaction with H-13 (Figure 9), while the geometries of both

Figure 7. Experimental solution ECD spectrum (black) of dendrodochol C (3) compared with the BH&HLYP/TZVP-calculated ECD spectra of (8R,10R,12S,13S)-3 (blue) and (8S,10S,12R,13R)-3 (red). Bars represent the calculated rotational strength values of the low-energy conformer of (8R,10R,12S,13S)-3.

Figure 9. Key NOESY correlations of 4 indicated on the lowest-energy MMFF solution conformer.

the Δ3 and Δ5 double bonds were assigned as E based on the proton coupling constants (3JH3,H4 = 3JH5,H6 = 14.4 Hz). On the basis of the same biosynthetic origin of 2 and 4, the absolute configuration of the trans-fused octahydro-2-benzofuran is proposed as (7R,8S,10R,11S,12R,13S). The absolute configuration at C-2 was determined by the modified Mosher’s method.22 The (R)- and (S)-MPA esters of dendrodochol D (4) were prepared by treatment at room temperature (rt) with (R)- and (S)-α-methoxyphenylacetic acid (MPA), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and 4-(dimethylamino)pyridine (DMAP) in dry CH2Cl2. Significant ΔδR−S values for the protons near the asymmetric center C-2 were observed as shown in Figure 10. According to Mosher’s rule, the absolute configuration at C2 was determined as R. The absolute stereostructure of 4 was thus determined as (2R,7R,8S,10R,11S,12R,13S). The isolated dendrodochols A−D (1−4) were tested for their antifungal (Table S1) and tumor cell growth inhibition

well the experimental solution ECD spectrum of dendrodochol C, allowing assignment of its absolute configuration as (8R,10R,12S,13S). Thus, the corresponding asymmetric centers of 1 and 3 were found to be homochiral, as expected from their common biogenetic origin. Dendrodochol D (4) was also isolated as an optically active, amorphous solid. The HRESIMS data of 4 established the molecular formula as C14H22O5, having one more oxygen atom than 2. The 1H and 13C NMR spectra of 4 resembled those of 2. However, two sp2 carbons of the conjugated double bonds in 2 were replaced by two oxygenated methines (δH 4.60, δC 67.8; δH 4.17, δC 84.1) in 4 (Tables 1 and 2), suggesting an additional ring in molecule 4 with respect to 2. The two oxygenated methines were assigned at C-2 and C-7, respectively, due to the proton sequence from H3-1 to H2-14 as established by the COSY experiment. The diagnostic HMBC correlation from H-7 to C-14 indicated the presence of a 1182



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45:55; 1.5 mL/min) to yield 1 (7.3 mg, 32 min). Compounds 2−4 were obtained by open column chromatography. Fraction 18 gave 3 (3.4 mg) after successive Sephadex LH-20 (CH2Cl2/MeOH, 2:1) and silica gel (CH2Cl2/MeOH, 30:1) column chromatography. Similarly, fraction 22 was subjected to Sephadex LH-20 (CH2Cl2/MeOH, 2:1) and silica gel (CH2Cl2/MeOH, 20:1) chromatography to yield 2 (16.0 mg) and 4 (6.4 mg). Dendrodochol A (1): amorphous solid; [α]20 D +67 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 278 (3.31), 266 (3.37), 261 (3.33), 258 (3.35), 218 (2.66), 204 (3.03) nm; ECD (c 1.3 × 10−3, MeCN) λmax (Δε) 287 (−0.25), 262 (0.14), 232 (0.19), 194 (0.33) nm; IR (film) νmax 3373, 3012, 2944, 2877, 1716, 1638, 995 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 275.1260 [M + Na]+ (calcd for C14H20O4Na, 275.1259). Dendrodochol B (2): amorphous solid; [α]20 D +72 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 277 (3.21), 274 (3.19), 267 (3.27), 260 (3.19), 258 (3.20), 252 (3.17), 234 (3.40), 215 (3.26), 210 (3.29) nm; IR (film) νmax 3390, 2928, 1400, 1052 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 277.1418 [M + Na]+ (calcd for C14H22O4Na, 277.1416). Dendrodochol C (3): amorphous solid; [α]20 D +13 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 278 (3.47), 267 (3.59), 261 (3.47), 258 (3.49), 230 (3.19), 203 (3.55) nm; ECD (c 4.6 × 10−4, MeCN) λmax (Δε) 308 (0.06), 264 (−0.06), 226 (0.08) nm; IR (film) νmax 3368, 2926, 2854, 1652, 1052 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 261.1464 [M + Na]+ (calcd for C14H22O3Na, 261.1467). Dendrodochol D (4): amorphous solid; [α]20 D −21 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 233 (3.85), 215 (3.73), 209 (3.83) nm; ECD (c 4.6 × 10−4, MeCN) λmax (Δε) 243 (0.13), 227 (−0.11), 195 (−0.66) nm; IR (film) νmax 3373, 2923, 1602, 1378, 1067, 987, 698 cm−1; 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 269.1390 [M − H]− (calcd for C14H21O5, 269.1389). Preparation of (R)-MPA Ester 4. To a solution of 4 (0.5 mg, 2 μmol) with EDC (4.0 mg, 0.02 mmol) and DMAP (1 crystal) in dry CH2Cl2 (1.0 mL) was added (R)-MPA (0.4 mg, 2.5 μmol) for 24 h at rt. The reaction solvent was removed under reduced pressure, and the residue was subjected to mini open column chromatography on silica (gradient CH2Cl2/MeOH from 40:1 to 20:1) to afford the (R)-MPA ester (0.30 mg, yield 38.8%): 1H NMR (600 MHz, CD3OD) δ 6.08 (1H, m, H-5), 5.82 (1H, m, H-4), 5.53 (1H, m, H-3), 5.45 (1H, m, H2), 5.41 (1H, m, H-6), 3.84 (1H, m, H-7), 3.83 (1H, m, H-14b), 3.73 (1H, m, H-14a), 3.71 (1H, m, H-12), 3.61 (1H, m, H-10), 3.28 (1H, m, H-11), 2.02 (1H, m, H-13), 1.97 (1H, m, H-9β), 1.91 (1H, m, H8), 1.17 (1H, m, H-9α), 1.31 (3H, d, J = 6.1 Hz, H-1); ESIMS m/z 441 [M + Na]+. Preparation of (S)-MPA Ester 4. The same reaction of 4 (0.5 mg, 2 μmol) with EDC (4.0 mg, 0.02 mmol), DMAP (1 crystal) in CH2Cl2 (1.0 mL), and (S)-MPA (0.4 mg, 2.5 μmol) afforded the (S)-MPA ester of 4 (0.23 mg, yield 30.2%): 1H NMR (600 MHz, CD3OD) δ 6.22 (1H, m, H-4), 6.14 (1H, m, H-5), 5.70 (1H, m, H-3), 5.57 (1H, m, H-6), 5.39 (1H, m, H-2), 3.84 (1H, m, H-7), 3.83 (1H, m, H-14b), 3.74 (1H, m, H-14a), 3.70 (1H, m, H-12), 3.60 (1H, m, H-10), 3.31 (1H, m, H-11), 2.02 (1H, m, H-13), 2.00 (1H, m, H-9β), 1.98 (1H, m, H-8), 1.17 (1H, m, H-9α), 1.22 (3H, d, J = 6.0 Hz, H-1); ESIMS m/z 441 [M + Na]+. Cytotoxicity Assay. Cytotoxicity was tested against human lung adenocarcinoma (A549) and human osteosarcoma (MG63) cell lines, using a modification of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] colorimetric method.32 Adriamycin was used as positive control, IC50 = 5.7 μM. Antimicrobial Bioassay. The antifungal activities of the dendrodochols 1−4 were tested against eight strains: Candida albicans Y0109, C. albicans SC5314, C. parapsilosis 22019, C. glabrata 537, Cryptococcus neoformans 32609, Aspergillus f umigatus 07544, Trichophyton rubrum Cmccftla, and Microsporum gypseum Cmccfmza. The data of the antifungal activities were evaluated by measuring optical density at 630 nm using an automatic microplate reader. The MIC80 was defined as the first well with an approximate 80% reduction in growth compared to the growth of the drug-free well. The data

Figure 10. ΔδR−S values (in ppm) for the MPA esters of 4.

activities. In the in vitro bioassay, these compounds exhibited no growth inhibition activity against the A549 and MG63 cell lines. Dendrodochols 1 and 3 exhibited modest antifungal activity against several Candida strains, Cryptococcus neoformans, and Trichophyton rubrum (MIC80 = 8−16 μg/mL), whereas 2 and 4 showed no activity against the tested strains. This group of new metabolites is typical of fungal polyketide metabolites. The first example of such metabolites, palitantin, was obtained from culture filtrates of Penicillium palitans Westling in 1936,23 and its structure was determined in 1959.24 Its structure closely resembled that of frequentin, an antifungal and antibiotic agent.25 In 1997, a reinvestigation of a Penicillium sp. fungus led to the isolation of two additional analogues, penienone and penihydrone, as plant growth regulators.26 Recently, a new member of the family, 2,3-didehydropalitantin, was isolated from a Paraphaeosphaeria sp. fungus.20 The interesting biological activities and unique structural features of these molecules have attracted considerable attention as targets for total synthesis.27−31 The discovery of dendrodochols demonstrates the productivity of this fungus and enriches the structural diversity of this family of cyclohexanols.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in MeOH on an Autopol IV polarimeter at the sodium D line (589 nm). UV absorption spectra were recorded on a Varian Cary 100 UV−vis spectrophotometer. Electronic circular dichroism spectra were recorded on a JASCO J-810 spectropolarimeter. Infrared spectra were recorded in thin polymer films on a Nexus 470 FT-IR spectrophotometer (Nicolet). The NMR spectra were recorded at 300 K on Bruker DRX 400 and Avance 600 spectrometers. Chemical shifts are reported in parts per million (δ), using the residual CH3OH signal (δH = 3.31 ppm), C5H5N (δH = 7.20, 7.57, 8.73 ppm) as an internal standard for 1H NMR and CD3OD (δC = 49.5 ppm), C5D5N (δC = 123.6, 135.8, 150.0 ppm) for 13C NMR; coupling constants (J) are in Hz. 1H NMR and 13C NMR assignments were supported by COSY, HSQC, HMBC, and NOESY experiments. The mass spectra and high-resolution mass spectra were obtained on a Q-TOF Micro mass spectrometer, resolution 5000. Semipreparative RP-HPLC was performed on an Agilent1100 system equipped with a refractive index detector using a YMC Pack ODS-A column (particle size 5 μm, 250 × 10 mm). Commercial silica gel (Yantai, P. R. China, 200−300, 400− 500 mesh) was used for column chromatography. Precoated silica gel plates (Yantai, P. R. China, HSGF-254) were used for analytical thinlayer chromatography (TLC). Spots were detected on TLC under UV or by heating after spraying with anisaldehyde/sulfuric acid reagent. Culture, Extraction, and Isolation. The associated Dendrodochium sp. fungus (GenBank accession number KJ589419), internal strain no. 10087, was isolated following surface sterilization from the sea cucumber Holothuria nobilis Selenka, collected from the South China Sea, and was cultivated on biomalt (5% w/v; Villa Natura Gesundprodukte GmbH) solid agar medium at rt for 28 days.7−11 The culture medium (12 L) was then extracted with EtOAc to afford a residue (8.0 g) after removal of the solvent under reduced pressure. The extract was subjected to column chromatography (CC) on silica gel to give 25 fractions, eluting with a gradient of MeOH in CH2Cl2 (0 to 20%). Fraction 16 was subjected to Sephadex LH-20 (CH2Cl2/ MeOH, 2:1) and silica gel (CH 2 Cl 2 /MeOH 30:1) column chromatography, followed by HPLC purification (MeOH/H2O, 1183



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represented the means of three independent experiments in which each compound concentration was tested in three replicate wells. Fluconazole (FCZ), itraconazole (ICZ), voriconazole (VCZ), ketoconazole (KCZ), and terbinafine (TBF) were used as positive controls. Computational Section. Conformational searches were carried out by means of the Macromodel 9.7.21133 software using the MMFF with an implicit solvent model for chloroform. Geometry reoptimizations at B3LYP/6-31G(d) in vacuo followed by TDDFT ECD calculations using various functionals (B3LYP, BH&HLYP, PBE0) and the TZVP basis set were performed with the Gaussian 0934 package. Boltzmann distributions were estimated from the ZPVE-corrected B3LYP/6-31G(d) energies. ECD spectra were generated as the sum of Gaussians35 with 3000 cm−1 half-height width (corresponding to ca. 20 at 260 nm), using dipole-velocity-computed rotational strengths for conformers above 1%. The MOLEKEL36 software package was used for visualization of the results.

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ASSOCIATED CONTENT

S Supporting Information *

HRESIMS and NMR spectra of 1−4 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(W. Zhang) Tel/Fax: 86 21 81871257. E-mail: [email protected]. *(Z.-W. Wang) E-mail: [email protected]. Author Contributions ‡

D.-X. Xu and P. Sun contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research work was financially supported by the Natural Science Foundation of China (Nos. 30873200, 41176125), the International S&T Cooperation Program of China (No. 0S2014GR0014), the Hundred Talents Program of SMCH (No. XBR2013111), and the Shanghai Pujiang Program (No. PJ2008). T.K. and A.M. thank the Hungarian National Research Foundation (OTKA K105871) for financial support and the National Information Infrastructure Development Institute (NIIFI 10038) for computer time.

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REFERENCES

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Polyhydroxy Cyclohexanols from a Dendrodochium sp. Fungus

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