Characterization of Cladosporols from the Marine ... - ACS Publications

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Characterization of Cladosporols from the Marine Algal-Derived Endophytic Fungus Cladosporium cladosporioides EN-399 and Configurational Revision of the Previously Reported Cladosporol Derivatives Hong-Lei Li,†,‡ Xiao-Ming Li,† Attila Mándi,§ Sándor Antus,§ Xin Li,† Peng Zhang,†,‡ Yang Liu,†,‡ Tibor Kurtán,*,§ and Bin-Gui Wang*,† †

Laboratory of Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao 266071, People’s Republic of China ‡ University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, People’s Republic of China § Department of Organic Chemistry, University of Debrecen, P.O. Box 400, 4002 Debrecen, Hungary S Supporting Information *

ABSTRACT: Four new cladosporol derivatives, cladosporols F−I (1−4), the known cladosporol C (5), and its new epimer, cladosporol J (6), were isolated and identified from the marine algal-derived endophytic fungus Cladosporium cladosporioides EN399. Their structures were determined by detailed interpretation of NMR and MS data, and the absolute configurations were established on the basis of TDDFT-ECD and OR calculations. The configurational assignment of cladosporols F (1) and G (2) showed that the previously reported absolute configuration of cladosporol A and all the related cladosporols need to be revised from (4′R) to (4′S). Compounds 1−6 showed antibacterial activity against Escherichia coli, Micrococcus luteus, and Vibrio harveyi with MIC values ranging from 4 to 128 μg/mL. Compound 3 showed significant cytotoxicity against A549, Huh7, and LM3 cell lines with IC50 values of 5.0, 1.0, and 4.1 μM, respectively, and compound 5 showed activity against H446 cell line with IC50 value of 4.0 μM.

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antifungal activity.4 Cladosporols have been attracting attention for the remarkable antitumor activity, stimulating G1-phase arrest of the cell cycle in human colon carcinoma HT-29 cells.5−7 In the course of our research exploring multiple bioactive natural products from marine-derived fungi,8−12 we performed a chemical investigation of C. cladosporioides EN399, an endophytic fungus isolated from the marine red alga Laurencia okamurai, resulting in the isolation of the new cladosporol derivatives, cladosporols F−I (1−4), the known cladosporol C (5), and its new epimer, cladosporol J (6). The structures and absolute configurations of compounds 1−6 were elucidated primarily by NMR and ECD spectroscopic analysis. On the basis of the configurational assignment of cladosporols F (1) and G (2) by TDDFT-ECD calculations, the absolute configurations in the whole cladosporol class need to be revised. Details of the isolation, structure elucidation, and biological activities of compounds 1−6 as well as the

INTRODUCTION Cladosporols are known as unique metabolites of the fungal genus Cladosporium. To date, a total of eight cladosporol derivatives were discovered as natural products which exhibited β-l,3-glucan biosynthesis inhibitory,1,2 antimicrobial,3,4 and antitumor activities.5−7 Cladosporol A, isolated from C. cladosporioides in 1995,1−3 was reported as a β-1,3-glucan biosynthesis inhibitor. The (2R,3R,4R)-absolute configuration of cladosporol A was determined by comparing its positive 340 nm Cotton effect (CE) with those of the known (+)-epoxydon and (+)-isoepoxydon,2 while the (4′R) absolute configuration was deduced from the negative exciton couplet centered at 216 nm.2 The absolute configuration and ECD spectrum of cladosporol A was then used as a reference for the determination of absolute configuration in all of the other cladosporol derivatives.3,4 Cladosporols B−E, isolated in 2004 from C. tenuissimum (a hyperparasite of rust fungi), were active in inhibiting the urediniospore germination of the bean rust agent Uromyces appendiculatus.3 In 2009, Chen et al. reported a stereoisomer of cladosporol A and three other derivatives with © 2017 American Chemical Society

Received: May 24, 2017 Published: August 30, 2017 9946

DOI: 10.1021/acs.joc.7b01277 J. Org. Chem. 2017, 82, 9946−9954

Article

The Journal of Organic Chemistry

Table 2. 13C NMR Data for Compounds 1−6 (125 MHz)

configurational revision of the previously reported cladosporols are discussed herein.

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1a

2a

3a

4b

5a

6a

199.3, C 37.2, CH2 26.1, CH2 76.2, CH 127.5, C 154.5, C 121.6, CH 131.7, CH 137.1, C 131.2, C 205.4, C 36.8, CH2 30.6, CH2 39.9, CH 148.5, C 120.0, CH 136.4, CH 115.6, CH 162.7, C 117.7, C 55.2, CH3

199.1, C 37.6, CH2 26.5, CH2 76.0, CH 127.5, C 154.6, C 121.4, CH 131.3, CH 137.2, C 131.3, C 205.5, C 36.5, CH2 30.7, CH2 40.1, CH 148.5, C 120.0, CH 136.4, CH 115.7, CH 162.8, C 117.8, C 54.9, CH3

198.4, C 39.8, CH2 37.4, CH2 202.9, C 117.6, C 161.4, C 123.6, CH 139.1, CH 135.6, C 133.8, C 204.8, C 37.1, CH2 30.9, CH2 39.7, CH 147.4, C 119.7, CH 136.5, CH 116.1, CH 163.0, C 118.0, C

61.8, CH 30.2, CH2 32.5, CH2 206.7, C 115.4, C 161.1, C 118.0, CH 137.8, CH 133.2, C 143.6, C 205.9, C 38.3, CH2 30.7, CH2 48.7, CH 149.2, C 119.7, CH 137.0, CH 115.8, CH 162.6, C 117.4, C

199.7, C 37.5, CH2 31.4, CH2 67.4, CH 129.8, C 154.4, C 121.3, CH 131.5, CH 137.1, C 130.6, C 205.6, C 36.6, CH2 29.7, CH2 40.0, CH 148.5, C 120.0, CH 136.5, CH 115.7, CH 162.7, C 117.7, C

199.3, C 36.4, CH2 31.9, CH2 68.0, CH 129.6, C 154.6, C 121.3, CH 131.6, CH 137.2, C 130.7, C 205.6, C 36.4, CH2 30.6, CH2 40.0, CH 148.4, C 120.0, CH 136.5, CH 115.7, CH 162.7, C 117.8, C

position 1 2

RESULTS AND DISCUSSION The EtOAc extracts of C. cladosporioides EN-399 were separated and purified by repeated column chromatography (CC) on Si gel, Sephadex LH-20, and Lobar LiChroprep RP-18 as well as by prep. TLC to yield compounds 1−6 (Figure 1).

3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 4′a 5′ 6′ 7′ 8′ 8′a 4-OMe

Figure 1. Structures of the isolated compounds 1−6 and cladosporol A (7).

Compound 1 was obtained as a yellowish solid, and its formula was determined as C21H20O5 on the basis of HRESIMS data, indicating 12 degrees of unsaturation. Its 1H and 13C NMR data (Tables 1 and 2) indicated the presence of a 1,2,3trisubstituted and a 1,2,3,4-tetrasubstituted benzene rings, one methoxy, four methylenes, two methines, and two carbonyl as well as two exchangeable protons resonating at δH 12.59 (8′OH) and 8.20 (5-OH). The COSY correlations of H-3 with H-

a

Measured in CDCl3. bMeasured in DMSO-d6.

2 and H-4 along with the HMBC correlations from H-2 to C-4 and C-8a and from H-3 to C-1 and C-4a indicated the presence of a substituted tetralone moiety. The observed COSY correlations of H-3′ with H-2′ and H-4′ as well as the HMBC correlations from H-3′ to C-1′ and C-4′a and from H-

Table 1. 1H NMR Data for Compounds 1−6 (500 MHz, J in Hz) position 1 2 3 4 6 7 2′ 3′ 4′ 5′ 6′ 7′ 4-OMe 5-OH 8′-OH a

1a

2a

2.61, m 2.90, m 2.32, m 2.51, m 4.99, br s 6.97, d (8.5) 6.86, d (8.5) 2.68, m 2.68, m 2.15, m 2.37, m 5.50, br s 6.35, d (7.9) 7.31, t (7.9) 6.82, d (7.9) 3.58, s 8.20, s 12.59, s

2.67, m 2.87, m 2.30, m 2.57, m 5.05, br s 6.96, d (8.5) 6.83, d (8.5) 2.68, m 2.68, m 2.20, m 2.44, m 5.40, br s 6.33, d (7.9) 7.30, t (7.9) 6.81, d (7.9) 3.57, s 8.17, s 12.61, s

3a

4b

3.09, 3.16, 2.76, 3.17,

m m m m

5.07, 2.18, 2.26, 2.51, 3.12,

m m m m m

7.14, 7.24, 2.73, 3.16, 2.20, 2.42, 5.35, 6.27, 7.31, 6.84,

d (8.9) d (8.9) m m m m dd (4.8, 8.1) d (7.9) t (7.9) d (7.9)

6.89, 7.25, 2.72, 2.91, 2.22, 2.39, 4.70, 6.18, 7.38, 6.80,

d (8.8) d (8.8) m m m m dd (3.7, 10.5) d (8.0) t (8.0) d (8.0)

12.69, s 12.58, s

12.69, s 12.69, s

5a 2.65, 2.91, 2.29, 2.51, 5.34, 6.96, 6.82, 2.66, 2.66, 2.14, 2.38, 5.50, 6.34, 7.29, 6.81,

6a

m m m m dd (5.1, 7.7) d (8.6) d (8.6) m m m m br s d (8.0) t (8.0) d (8.0)

8.29, s 12.57, s

2.72, 2.87, 2.27, 2.58, 5.44, 6.97, 6.84, 2.65, 2.65, 2.17, 2.43, 5.37, 6.34, 7.31, 6.81,

m m m m br s d (8.4) d (8.4) m m m m br s d (7.9) t (7.9) d (7.9)

8.28, s 12.60, s

Measured in CDCl3. bMeasured in DMSO-d6. 9947


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Figure 2. Key HMBC (arrows) and COSY (bold lines) correlations of compounds 1−6.

Figure 3. HPLC chromatograms of compounds 1 (a), 2 (b), and the mixture of 1 and 2 (c), as well as their UV spectra, over a CHIRALPAK AD-H column (eluant: isopropanol−MeOH 9:1, flow rate: 1.0 mL/min), detected at UV 235 nm.

Figure 4. Structure and population of the low-energy B97D/TZVP PCM/MeCN conformers (>2%) of (4S,4′R)-2 with the orientation of the 4′-aryl and 4-OMe groups indicated.

them. The intense high-energy ECD transitions below 260 nm were attributed to the interaction of the two tetralone chromophores, and thus they reflected the absolute configuration of the C-4′ chirality center determining the relative orientation of the two interacting chromophores.1 According to the ECD study of cladosporol A, the positive ECD couplet centered around 216 nm was assigned to (4′R) absolute configuration,1 which was used as a reference for the configurational assignment of all the cladosporols. Since compounds 1 and 2 had a similar ECD pattern in this region, their C-4′ absolute configuration is expected to be the same, while the ECD differences above 300 nm could derive from the different absolute configuration of C-4. In order to elucidate the absolute configuration of the stereoisomeric metabolites 1 and 2, TDDFT-ECD and optical rotation (OR) calculations were performed on the arbitrarily chosen (4S,4′S)-1 and (4S,4′R)-2 stereoisomers. MMFF conformational search of the (4S,4′R) diastereomer resulted in 13 conformers in a 21 kJ/mol energy window, while 17 lowenergy conformers were obtained for the (4S,4′S) diastereomer. These conformers were reoptimized at four different levels: B3LYP/6-31G(d) in vacuo, B3LYP/TZVP PCM/ MeOH, B97D/TZVP PCM/MeCN, and B97D/TZVP PCM/ MeOH levels.13,14 The fused carbocyclic ring of the tetralone

2′ to C-1′, C-4′, and C-8′a implied the presence of another substituted tetralone moiety (Figure 2). The observed HMBC correlations from H-4′ to C-8a and from H-5′ and H-7 to C-4′ suggested that there is a C-8−C-4′ linkage between the two tetralone moieties. Comprehensive analysis of the NMR data revealed that compound 1 was very similar to cladosporol C,3 except for an additional methoxy group (δH 3.58, δC 55.2) in compound 1. The HMBC correlation from 4-OMe to C-4 disclosed the planar structure of compound 1. Compound 2 was obtained as a yellowish solid and was determined to have a molecular formula C21H20O5 based on HRESIMS analysis, same as that of 1. The NMR data of 2 was almost identical to that of 1 (Tables 1 and 2), indicating that they are diastereoisomers and share the same planar structure. This was confirmed by HMBC and COSY correlations (Figure 2) as well as the HPLC analysis of 1 and 2, in which they exhibited two partially overlapped peaks over RP-18 column and two baseline separated peaks on a chiral column, with virtually identical UV spectra (Figure 3). The three intense high-energy Cotton effects (CEs) of 1 and 2 showed the same positive/negative/positive pattern at about 254, 231, and 209 nm, and the 338 nm positive CE was also produced by both isomers. However, compound 1 showed additional negative CEs at 313 and 372 nm, which could be used to distinguish 9948


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Figure 5. Structure and population of the low-energy B97D/TZVP PCM/MeCN conformers (>2%) of (4S,4′S)-1.

chromophores can adopt P or M helicity conformations, which can flip the C-4 and C-4′ substituents to axial or equatorial orientations. The B3LYP/6-31G(d) in vacuo reoptimization of (4S,4′R)-2 (Figure S36 in the SI) resulted in four low-energy conformers, and the lowest-energy conformer had equatorial 4′-aryl and 4-OMe groups (44.9%). However, conformer B had axial 4′-aryl group with comparable population (44.5%). B97D/ TZVP PCM/MeCN reoptimization of (4S,4′R)-2 produced a lowest-energy conformer (64.6%) with axial 4′-aryl and equatorial 4-OMe groups (Figure 4). The total population of conformers with axial 4′-aryl substituent was 72.7% represented by conformers A and C. The Boltzmann-weighted ECD spectra computed for the different sets of conformers of (4S,4′R)-2 gave mirror image ECD curves of the experimental curve of cladosporol G (2), suggesting its (4R,4′S) absolute configuration (Figures 6 and 7). This (4′S) absolute configuration of cladosporol G (2) is opposite to the published (4′R) absolute configuration of cladosporol C,3 which was determined on the basis of

Figure 7. Experimental ECD spectra of cladosporol F [(4S,4′S)-1] and cladosporol G [(4R,4′S)-2] in MeCN compared with the Boltzmannweighted BH&HLYP/TZVP PCM/MeCN//B97D/TZVP PCM/ MeCN spectra of (4S,4′R)-2 and (4S,4′S)-1.

cladosporol A.1 All of the different conformers and different methods gave mirror-image CEs of the major experimental ECD bands regardless of the axial or equatorial orientation of the C-4 and C-4′ substituents, which confirmed that the (4′S) assignment is solid. The B3LYP/6-31G(d) in vacuo reoptimization of (4S,4′S)-1 (Figure S34) resulted in seven low-energy conformers, and the lowest-energy conformer had equatorial 4′-aryl and 4-OMe groups (27.8%). The conformers with equatorial 4′-aryl group had a total population of 58.6% represented by four conformers, while those with axial 4′-aryl group had 39.9% total population from three conformers. B97D/TZVP PCM/ MeCN reoptimization of (4S,4′S)-1 increased the contribution of the conformers with axial 4′-aryl group, which had a total of 70.7% including the lowest-energy conformer (44.9%) (Figure 5). The ECD spectra computed for the B3LYP/6-31G(d) in vacuo and B97D/TZVP PCM/MeCN conformers of (4S,4′S)-1 reproduced well the experimental ECD of cladosporol F (1), confirming again the (4′S) absolute configuration. The (4S) absolute configuration of cladosporol F (1) could be tentatively

Figure 6. Experimental ECD spectra of cladosporol F [(4S,4′S)-1] and cladosporol G [(4R,4′S)-2] in MeCN compared with the Boltzmannweighted BH&HLYP/TZVP//B3LYP/6-31G(d) spectra of (4S,4′R)2 and (4S,4′S)-1. 9949


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OR calculations for the revised absolute configurations of 1 and 2 could reproduce the large positive experimental OR values, the (4′S) absolute configuration was confirmed. Based on the ECD and OR calculations of cladosporol F (1) and G (2), structurally closely related to cladosporol C, the C4′ absolute configuration of the cladosporol family, relying on the ECD analysis of cladosporol A, needs to be revised to (4′S). In order to confirm this revision of absolute configuration independently, the conformational and TDDFT studies were also performed for the known cladosporol A (7), and the computational results were compared with the literature data measured in EtOH.1,3 Cladosporol A (7) has two weak highwavelength positive CEs [340 nm (5.02), 260 nm (8.91)], an intense negative CE [230 nm (−51.6)], and a positive lowwavelength CE [206 nm (25.1)]. MMFF conformational searches of (2R,3R,4R,4′R)-7 and (2R,3R,4R,4′S)-7 resulted in 15 and 13 conformers in a 21 kJ/mol energy window, respectively. Boltzmann-averaged ECD spectra of the two epimers computed at various levels for the B3LYP/6-31G(d) in vacuo, the B97D/TZVP PCM/EtOH and the CAM-B3LYP/ TZVP PCM/EtOH low-energy conformers suggested consistently (2R,3R,4R,4′S) absolute configuration by reproducing the two relatively weak positive transitions at higher wavelengths, the large negative one at ca. 230 nm, and the large positive transition at ca. 205 nm (Figure S42). The Boltzmannaveraged ECD spectra of the (2R,3R,4R,4′R) isomer always showed negative CEs above 250 nm with all of the applied methods contradicting the experimental ECD data (Figure S40). Different conformers of the (2R,3R,4R,4′R) isomer exhibited opposite CEs in this region, which also confirmed that the 340 nm positive CE of 7 cannot be used safely to determine the absolute configuration of C-2, C-3, and C-4 by simple comparison with analogues as suggested by the literature. 2 Sodium D line OR calculations of the (2R,3R,4R,4′S) isomer performed at various levels for the PCM conformers verified the ECD results (+174.9, +139.2, +149.3, and +168.9 at various levels for the CAM-B3LYP/ TZVP PCM/EtOH conformers vs +157 experimental value,1 while small negative values were obtained for the (2R,3R,4R,4′R) stereoisomer at all the applied levels) and allowed unambiguous elucidation of the absolute configuration of cladosporol A as (2R,3R,4R,4′S) (Tables S11−S14). Cladosporol H (3) was obtained as a yellowish solid with the molecular formula C20H16O5 as established by HRESIMS data, indicating 13 degrees of unsaturation. Analysis of the 1H and 13 C NMR data (Tables 1 and 2) of 3 suggested that it was an analogue of cladosporols F (1) and G (2). The signals for methoxy group (4-OMe) in the NMR spectra of 1 and 2 disappeared, and an additional carbonyl (C-4) was observed in

assigned on the basis of BH&HLYP/TZVP ECD spectrum of (4S,4′S)-1 computed for the B97D/TZVP PCM/MeCN conformers (Figure 7), which could reproduce the alternating signs of CEs above 290 nm characteristic of cladosporol F (1). In contrast, the same ECD calculation method of (4S,4′R)-2 produced a single broad negative ECD band which was observed in the experimental ECD of cladosporol G (2). In order to verify the revision of the C-4′ absolute configuration independently, optical rotation (OR) calculations were also performed15,16 at the same levels applied for the ECD calculations. OR calculations with B3LYP/TZVP, BH&HLYP/ TZVP, and PBE0/TZVP using PCM for MeOH for the B3LYP/TZVP PCM/MeOH and B97D/TZVP PCM/MeOH conformers gave large positive optical rotation values for all the conformers of the (4S,4′S)-1 [experimental value: [α]25 D +93 (c 0.14, MeOH)] (Tables S1 and S2). In accordance, the Boltzmann-averaged optical rotation of (4S,4′R)-2 was found negative at all three applied levels for both sets of conformers [experimental value: [α]25 D +100 (c 0.07, MeOH)] (Tables S3 and S4). Optical rotations of 1 and 2 were also measured and computed at 578, 546, and 436 nm, which in accordance with the expectation, showed positive signs increasing with the decreasing wavelength (Figure 8, Tables S1-S4). Since all the

Figure 8. Experimental OR values of 1 (black) and 2 (gray) measured at 4 points (589.3, 578, 546, and 436 nm) compared with the computed values for the B97D/TZVP PCM/MeOH conformers (red: B3LYP/TZVP PCM/MeOH, blue: BH&HLYP/TZVP PCM/MeOH, and purple: PBE0/TZVP PCM/MeOH computed for (4S,4′R)-2; orange: B3LYP/TZVP PCM/MeOH, light blue: BH&HLYP/TZVP PCM/MeOH, and magenta: PBE0/TZVP PCM/MeOH computed for (4S,4′S)-1).

Figure 9. Structure and population of the low-energy B3LYP/6-31G(d) conformers (>2%) of (R)-3. 9950


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conformers in a 21 kJ/mol energy window, respectively, the B3LYP/6-31G(d) in vacuo reoptimization of which produced 7 and 5 low-energy conformers for ECD calculations (Figures S38 and S39). The Boltzmann-weighted B3LYP/TZVP ECD spectra of the (1R,4′S)-4 and (1S,4′S)-4 diastereomers computed for the B3LYP/6-31G(d) in vacuo and CAMB3LYP/TZVP PCM/MeCN conformers are shown in Figures 11 and 12. The better agreement of computed ECD of the

that of 3. The HMBC correlations from H-2 to C-1, C-3, and C-4 and from H-3 to C-1, C-2, and C-4 implied that C-4 was oxidized to carbonyl in 3 (Figure 2). Compound 3 had a single chirality center (C-4′) attaching the 1-tetralone chromophore with a dihydro-1,4-naphthoquinone moiety. Due to the different chromophore, the experimental ECD spectrum of 3 was markedly different from those of 1 and 2. Thus, the solution TDDFT-ECD protocol was carried out on the arbitrarily chosen (R) enantiomer to determine the absolute configuration. The MMFF conformational search yielded 9 conformers in a 21 kJ/mol energy window, the reoptimization of which resulted in 4−5 low-energy (≥2%) conformers at all of the applied levels. The B3LYP/6-31G(d) conformers are shown at Figure 9, the population of which showed a preference for the conformers with equatorial C-4′ aryl group (60.1% versus 39.2%). ECD spectra computed at all the applied levels gave nice mirrorimage curves of the experimental ECD with BH&HLYP/TZVP level of the gas-phase conformers, providing the best agreement (Figure 10). PCM model OR calculations were in line with the

Figure 11. Experimental ECD spectrum of cladosporol I [(1S,4′S)-4] in MeCN compared with the Boltzmann-weighted BH&HLYP/ TZVP//B3LYP/6-31G(d) spectra of (1R,4′S)-4 and (1S,4′S)-4.

Figure 10. Experimental ECD spectrum of 3 in MeCN compared with the Boltzmann-weighted BH&HLYP/TZVP // B3LYP/6-31G(d) spectra of (R)-3.

ECD results (Tables S5 and S6). Thus, the absolute configuration of 3 could be unambiguously determined as (S) in accordance with the corresponding chirality center of 1 and 2. Cladosporol I (4) was also obtained as yellowish solid, and its formula was assigned as C20H18O5 on the basis of HRESIMS data, indicating 12 degrees of unsaturation. The 1H and 13C NMR data (Tables 1 and 2) of 4 was very similar to those of cladosporol H (3).3 However, the signal for carbonyl (C-1) disappeared in the NMR spectra of 3, and the resonances of a methine (δH 5.07, δC 61.8) were observed in that of 4. The HMBC correlations from H-1 to C-3, C-4a, and C-8 confirmed the structure of 4 (Figure 2). Although NOE correlation was observed between H-1 and H-4′, the DFT conformational analysis of the (1R,4′S)-4 and (1S,4′S)-4 diastereomers revealed that both diastereomers can give this correlation, and hence it is not suitable to determine the relative configuration by NOESY experiment. Thus, TDDFT-ECD calculations were performed on the (1R,4′S)-4 and (1S,4′S)-4 diastereomers to determine both the relative and absolute configurations. The initial MMFF conformational search of (1R,4′S)-4 and (1S,4′S)-4 resulted in 10 and 9

Figure 12. Experimental ECD spectrum of cladosporol I [(1S,4′S)-4] in MeCN compared with the Boltzmann-weighted B3LYP/TZVP PCM/MeCN//CAM-B3LYP/TZVP PCM/MeCN spectra of (1R,4′S)-4 and (1S,4′S)-4.

(1S,4′S)-4 diastereomer allowed the unambiguous assignment of the (4′S) absolute configuration, and it also suggests (1S) absolute configuration, which is based on the better reproduction of the intense 224 nm positive CE compared to that of the (1R,4′S)-4 isomer. OR calculations performed with PCM solvent model verified the 4′S absolute configuration (see Tables S7−S10). Compounds 5 and 6 were found to be C-4 epimers as well, isolated as yellowish solids, exhibiting two partially overlapped peaks over RP-18 column and two independent peaks over a chiral column, with virtually identical UV spectra (Figure 13). Their formulas were confirmed as C20H18O5 on the basis of HRESIMS, indicating 12 degrees of unsaturation. The NMR spectra for 5 and 6 were almost identical with that of 9951


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Figure 13. HPLC chromatograms of compounds 5 (a), 6 (b), and the mixture of 5 and 6 (c), as well as their UV spectra, over a CHIRALPAK AD-H column (eluant: isopropanol−MeOH 9:1, flow rate: 1.0 mL/min), detected at UV 235 nm.

In summary, six secondary metabolites including four new cladosporol derivatives, cladosporols F−I (1−4), the known cladosporol C (5), and its new epimer, cladosporol J (6), were isolated and identified from the marine algal-derived endophytic fungus C. cladosporioides EN-399. The TDDFTECD and OR calculations of 1−4 allowed determining the absolute configuration, and it also facilitated the revision of absolute configuration from (4′R) to (4′S) in the cladosporol family. Compounds 1−6 showed inhibitory activity against E. coli, M. luteus, and V. harveyi with MIC values of 4−128 μg/mL, while compounds 2, 3, and 5 showed cytotoxicity against some of the tested tumor cell lines. These results prove that compounds 1−6 might be used as potential molecules in the development of drug leads or being modified to find more active derivatives for the treatments of microbial infection or cancer diseases.

cladosporol C, suggesting the same planar structure with cladosporol C. The ECD behavior of 5 and 6 was similar to that of 1 and 2, respectively, which show high-energy positive/ negative/positive CEs at about 254, 231, and 209 nm as well as positive CE at 338 nm. Compound 5 was identified as cladosporol C, having (4S,4′S) absolute configuration (revised), which was determined by the negative CEs at 313 and 372 nm. Compound 6 was the C-4 epimer of cladosporol C with (4R,4′S) absolute configuration, and it was named cladosporol J. This was confirmed by the HPLC analysis over the chiral column, in which (4R,4′S)-epimer possesses the longer retention time than (4S,4′S)-epimer, same as that of 1 and 2 (Figure 13). Compounds 1−6 were assayed for their antimicrobial activities against two human pathogens (Escherichia coli and Staphylococcus aureus), seven aquatic bacteria (Aeromonas hydrophila, Edwardsiella tarda, Micrococcus luteus, Pseudomonas aeruginosa, Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahemolyticus), and five plant-pathogenic fungi (Alternaria brassicae, Colletotrichum gloeosporioides, Fusarium oxysporum, Gaeumannomyces graminis, and Physalospora piricolav). Compounds 1−6 showed inhibitory activity against E. coli, M. luteus, and V. harveyi with MIC values of 4−128 μg/mL. None of them showed activity against other tested microbes (MIC > 128 μg/mL, Table 3). Cytotoxic activities of compounds 1−6

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General Experimental Procedures. Optical rotations were measured on an Optical Activity AA-55 and a Perkin Elmer 241 polarimeter. UV spectra were recorded on a Gold Spectrumlab 54 UV−vis spectrophotometer. ECD spectra were acquired on a Chirascan spectropolarimeter. Experimental ECD spectra of 1 and 2 were scaled to the computed spectra. NMR spectra were recorded on a Bruker Avance 500 spectrometer (500 MHz for 1H and 125 MHz for 13 C) with TMS as an internal standard. ESIMS and HRESIMS data were obtained on a Waters Micromass Q-TOF Premier and a Thermo Fisher Scientific LTQ Orbitrap XL spectrometers, respectively. HPLC analysis was performed on a Dionex HPLC system equipped with a P680 pump, an ASI-100 automated sample injector, a TCC-100 column oven, a UV-DAD 340U detector, a Dionex Acclaim ODS column (4.6 × 250 mm, 5 μm), and a CHIRALPAK AD-H column (4.6 × 250 mm, 5 μm). Semipreparative HPLC was conducted on a Dionex UltiMate U3000 system using an Agilent Prep. RP-18 column (21.2 × 250 mm, 10 μm) with UV detection. Silica gel (SiO2: 100− 200 mesh, 200−300 mesh, and GF254) for column chromatography and preparative thin-layer chromatography were produced by Qingdao Haiyang Chemical Group Corporation. RP-18 reverse-phase Si gel (40−63 μm) and Sephadex LH-20 were purchased from the Merck Corporation. Solvents were distilled prior to use for extraction and purification procedures. Fungal Material. The fungus C. cladosporioides EN-399 was isolated from the marine red alga L. okamurai collected in Qingdao, China, in September 2013. The fungus was identified using a molecular biological protocol by DNA amplification and sequencing of the ITS region, as described previously.17 The sequence derived from the fungus, which was the same (100%) to the sequence of C. cladosporioides H4242 (compared with GU595035.1), has been deposited at GenBank (with accession no. KM103661). The fungal strain EN-399 is preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS).

Table 3. Antibacterial Activity of Compounds 1−6 (MIC, μg/mL) compd

Escherichia coli

Micrococcus luteus

Vibrio harveyi

1 2 3 4 5 6 chloramphenicol

32 64 32 64 8 16 0.25

64 128 64 64 32 64 0.5

32 64 4 16 16 32 2

EXPERIMENTAL SECTION

were also evaluated against eight tumor cell lines including A549, H446, HeLa, Huh7, L02, LM3, MCF-7, and SW1990 (Table 4). Compounds 2, 3, and 5 displayed cytotoxic activities against most of the tested cell lines, with IC50 values ranging from 1.0 to 20.0 μM. Notably, compound 3 showed cytotoxicity against A549, Huh7, and LM3 cell lines with IC50 values of 5.0, 1.0, and 4.1 μM, respectively, and compound 5 exhibited cytotoxic activity against H446 cell line with IC50 value of 4.0 μM. These results indicated that methoxylation of C-4 strengthened the cytotoxic activity (2 vs 6), and the presence of dihydro-1,4-naphthoquinone moiety was important for the cytotoxicity (3 vs 1, 2, and 4−6). 9952


Article

The Journal of Organic Chemistry Table 4. Cytotoxicity of Compounds 1−6 against Eight Tumor Cell Lines (IC50, μM) compd

A549a

H446b

HeLac

Huh7d

L02e

LM3f

MCF-7g

SW1990h

1 2 3 4 5 6 positive control

15.0 13.0 5.0 na 14.0 15.0 1.3i

na 11.0 10.0 na 4.0 11.0 4.0j

10.0 na na 10.8 na 15.0 4.9k

na 10.0 1.0 na na 20.0 6.2l

na 11.0 na na na na 13.0i

na 14.0 4.1 na na na 9.1i

na na 10.0 na na 12.0 1.8k

na 15.0 14.0 na na na 2.2m

a

Human lung adenocarcinoma. bHuman small cell lung cancer. cHuman cervix carcinoma. dHuman hepatocarcinoma. eHuman normal hepatocyte. Human liver cancer. gHuman breast carcinoma. hHuman pancreatic cancer. Positive controls used. iCisplatin. jAdriamycin. kPaclitaxel. lFluorouracil. m Gemcitabine; na: no activity. f

Cladosporol I [(1S,4′S)-4]. Yellowish solid; [α]25 D = +150 (c 0.08, MeCN); UV (MeOH) λmax (log ε) 337 (4.02), 259 (4.36), 219 (4.69) nm; ECD (0.96 mM, MeOH) λmax (Φ) 346 (−7.09), 332 sh (−4.53), 308 sh (+3.77), 279 (+5.41), 267 (−7.96), 252 sh (+21.33), 224 (+76.81), 207 (−22.89) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z: [M − H]− 337.11; HRMS (ESI-LTQ Orbitrap XL) m/ z: [M − H]− Calcd for C20H17O5 0.337.1071; Found 337.1079. Cladosporol C [(4S,4′S)-5]. Yellowish solid; [α]25 D = +108 (c 0.37, MeOH); UV (MeOH) λmax (log ε) 336 (3.78), 259 (4.06), 216 (4.38) nm; ECD (MeCN) λmax (Φ) 372 (−0.3), 350 sh (+2.1), 339 (+3.3), 313 (−1.5), 272 sh (18.5), 256 (+27.4), 232 (−62.4), 208 (+48.8), 195 sh (+29.6) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z: [M + H]+ 339.12; HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ Calcd for C20H19O5 339.1232; Found 339.1227. Cladosporol J [(4R,4′S)-6]. Yellowish solid; [α]25 D = +73 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 336 (3.97), 259 (4.25), 216 (4.58) nm; ECD (MeCN) λmax (Φ) 342 (+4.15), 259 (+15.7), 232 (−41.5), 207 (+28.2), 195 sh (+13.2) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z: [M + H]+ 339.12; HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ Calcd for C20H19O5 339.1232; Found 339.1229. Computational Methods. Mixed torsional/low-mode conformational searches were carried out by means of the Macromodel 9.9.223 software18 using the Merck Molecular Force Field (MMFF) with an implicit solvent model for CHCl3 applying a 21 kJ/mol energy window. Geometry reoptimizations of the resultant conformers [B3LYP/6-31G(d) level in vacuo, B3LYP/TZVP with PCM solvent model for MeCN and MeOH, B97D/TZVP with PCM solvent model for MeCN, MeOH, or EtOH, and CAM-B3LYP/TZVP with PCM solvent model for MeCN or EtOH], OR, and ECD calculations were performed with Gaussian 09.19 Chiroptical values were computed using various functionals (B3LYP, BH&HLYP, CAM-B3LYP, PBE0) and the TZVP basis set. ECD spectra were generated as the sum of Gaussians20 with 3000 and 1800 cm−1 half-height width (corresponding to ca. 16 and 11 at 250 nm), using dipole-velocity-computed rotational strengths. Boltzmann distributions were estimated from the ZPVE-corrected B3LYP/6-31G(d) energies in the gas-phase calculations and from the B3LYP/TZVP, B97D/TZVP, and CAM-B3LYP/ TZVP energies in the PCM model ones. The MOLEKEL21 software package was used for visualization of the results. Antimicrobial Assay. Antimicrobial evaluation against two human pathogens (E. coli EMBLC-1 and S. aureus EMBLC-2), seven aquatic bacteria (A. hydrophila QDIO-1, E. tarda QDIO-2, M. luteus QDIO-3, P. aeruginosa QDIO-4, V. alginolyticus QDIO-5, V. harveyi QDIO-7, and V. parahemolyticus QDIO-8), and five plant-pathogenic fungi (A. brassicae QDAU-1, C. gloeosprioides QDAU-2, F. oxysporum QDAU-5, G. graminis QDAU-3, and P. piricolav QDAU-6) was carried out by the microplate assay.22 The human and aquatic pathogens were obtained from the Institute of Oceanology, Chinese Academy of Sciences, while the plant pathogens were provided by the Qingdao Agricultural University. Chloramphenicol and amphotericin B were used as positive control against bacteria and fungi, respectively. Cytotoxicity Assay. Evaluations for the cytotoxic activity of compounds 1−6 were performed as previously reported.23

Fermentation, Extraction, and Isolation. For chemical investigations, the fermentation was statically carried out on a rice solid medium (70 g rice, 0.3 g peptone, 0.1 g corn syrup, and 100 mL naturally sourced and filtered seawater, which was obtained from the Huiquan Gulf of the Yellow Sea near the campus of IOCAS, pH 6.5− 7.0) in 1000 mL conical flasks for 30 days at room temperature. The fermented medium (64 flasks) was exhaustively extracted three times with EtOAc, which was evaporated under reduced pressure to afford an extract (18.0 g). The extract was fractionated by Si gel vacuum liquid chromatography (VLC) using different solvents of increasing polarity from petroleum ether (PE) to MeOH, to yield eight fractions (Frs.1−8), based on TLC and HPLC analysis. Fr.3 (2.0 g) was separated by column chromatography (CC) on Lobar LiChroprep C18 eluting with MeOH−H2O gradient to give seven subfractions (Frs.3.1−3.7). Further purification of Fr.3.3 (80.2 mg) by CC on Sephadex LH-20 (MeOH) and then by prep. TLC (plate: 20 × 20 cm, developing solvents: CHCl3/MeOH, 30:1) yielded compound 3 (9.2 mg). Fr.4 (0.9 g) was separated by CC over LiChroprep RP-18 eluting with a MeOH−H2O gradient (from 20:80 to 100:0) to afford five subfractions (Frs.4.1−4.5). Fr.4.2 (154.7 mg) was subjected to CC on Sephadex LH-20 (MeOH) and then further purified by semiprep. HPLC (Elite ODS-BP column, 10 μm; 10 mm × 300 mm; 65% MeOH−H2O, 16 mL/min) obtained compounds 1 (8.0 mg, tR 32.8 min) and 2 (7.9 mg, tR 33.6 min). Fr.4.3 (50 mg) was subjected to CC on Sephadex LH-20 (MeOH) and then further purified by prep. TLC (plate: 20 × 20 cm, developing solvents: CHCl3−MeOH, 20:1) yielded compound 4 (7.9 mg). Fr.4.5 (89 mg) was fractioned by CC on Sephadex LH-20 (MeOH) and further purified by prep. TLC (plate: 20 × 20 cm, developing solvents: CHCl3−MeOH, 20:1) and semiprep. HPLC (Elite ODS-BP column, 10 μm; 10 mm × 300 mm; 62% MeOH−H2O, 16 mL/min) afforded compounds 5 (13.2 mg, tR 31.4 min) and 6 (9.4 mg, tR 32.0 min). Cladosporol F [(4S,4′S)-1]. Yellowish solid; [α]25 D = +93 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 333 (3.90), 258 (4.78), 219 (4.53) nm; UV (MeCN) λmax (log ε) 330 (3.52), 258 (3.85), 218 (4.33) nm; ECD (MeCN) λmax (Φ) 372 (−0.5), 350 sh (+1.8), 338 (+2.8), 313 (−2.1), 272 sh (+6.5), 254 (+15.2), 231 (−51.0), 209 (+39.2), 195 sh (+25.7) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z: [M + H]+ 353.14; HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ Calcd for C21H21O5 353.1384; Found 353.1387. Cladosporol G [(4R,4′S)-2]. Yellowish solid; [α]25 D = +100 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 334 (4.00), 258 (4.28), 216 (4.64) nm; UV (MeCN) λmax (log ε) 331 (3.52), 257 (3.85), 217 (4.35) nm; ECD (MeCN) λmax (Φ) 336 (+3.4), 259 (+11.3), 230 (−49.6), 205 (+26.2), 195 sh (+20.3) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z: [M + H]+ 353.14, [M + Na]+ 375.12; HRMS (ESILTQ Orbitrap XL) m/z: [M + H]+ Calcd for C21H21O5 353.1384; Found 353.1389. Cladosporol H [(4′S)-3]. Yellowish solid; [α]25 D = +83 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 347 (4.06), 226 (4.53) nm; ECD (1.12 mM, MeCN) λmax (Δε) 335 (−1.65), 305 sh (+3.92), 266 (+23.79), 238 (−28.07), 228 (+10.11), 216 (−49.98), 201 (+57.59) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z: [M − H]− 335.09; HRMS (ESI-LTQ Orbitrap XL) m/z: [M − H]− Calcd for C20H15O5 335.0914; Found 335.0919. 9953


Article


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(14) Sun, P.; Xu, D. X.; Mándi, A.; Kurtán, T.; Li, T. J.; Schulz, B.; Zhang, W. J. Org. Chem. 2013, 78, 7030−7047. (15) Polavarapu, P. L. Chirality 2008, 20, 664−672. (16) Sun, P.; Yu, Q.; Li, J.; Riccio, R.; Lauro, G.; Bifulco, G.; Kurtán, T.; Mándi, A.; Tang, H.; Zhuang, C. L.; Gerwick, W. H.; Zhang, W. J. Nat. Prod. 2016, 79, 2552−2558. (17) Wang, S.; Li, X. M.; Teuscher, F.; Li, D. L.; Diesel, A.; Ebel, R.; Proksch, P.; Wang, B. G. J. Nat. Prod. 2006, 69, 1622−1625. (18) MacroModel; Schrödinger LLC: New York, 2012, https://www. schrodinger.com/macromodel. (19) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision B.01; Gaussian, Inc.: Wallingford, CT, 2010. (20) Stephens, P. J.; Harada, N. Chirality 2010, 22, 229−233. (21) Varetto, U. MOLEKEL 5.4; Swiss National Supercomputing Centre: Manno, Switzerland, 2009. (22) Pierce, C. G.; Uppuluri, P.; Tristan, A. R.; Wormley, F. L., Jr; Mowat, E.; Ramage, G.; Lopez-Ribot, J. L. Nat. Protoc. 2008, 3, 1494− 1500. (23) Engelke, L. H.; Hamacher, A.; Proksch, P.; Kassack, M. U. J. Cancer 2016, 7, 353−363.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b01277. Selected 1D and 2D NMR spectra of compounds 1−6, conformers, ECD spectra, optical rotation values, atomic coordinates and energies of the computed structures of 1−4 and 7 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Attila Mándi: 0000-0002-7867-7084 Tibor Kurtán: 0000-0002-8831-8499 Bin-Gui Wang: 0000-0003-0116-6195 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (NSFC grant no. 31330009) and the NSFC-Shandong Joint Fund for Marine Science Research Centers (U1406402) is gratefully acknowledged. T.K., S.A., and A.M. thank the National Research, Development and Innovation Office (NKFI K120181, K112951, and PD121020) for financial support and the Governmental Information-Technology Development Agency (KIFÜ ) for CPU time. B.-G.W. appreciates the support of Taishan Scholar Program from Shandong Province of China.

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DEDICATION Dedicated to the memory of a good friend and mentor, Dr. István Komáromi. REFERENCES

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