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Polyketides from the Mangrove-Derived Endophytic Fungus Cladosporium cladosporioides

Five new polyketides, namely, 5R-hydroxyrecifeiolide (1), 5S-hydroxyrecifeiolide (2), ent-cladospolide F (3), cladospolide G (4), and cladospolide H (5), along with two known compounds (6 and 7), were isolated from the endophytic fungal strain Cladosporium cladosporioides MA-299 that was obtained from the leaves of the mangrove plant Bruguiera gymnorrhiza. The structures of these compounds were established by extensive analysis of 1D/2D NMR data, mass spectrometric data, ECDs and optical rotations, and modified Mosher’s method. The structures of 3 and 6 were confirmed by single-crystal X-ray diffraction analysis and this is the first time for reporting the crystal structures of these two compounds. All of the isolated compounds were examined for antimicrobial activities against human and aquatic bacteria and plant pathogenic fungi as well as enzymatic inhibitory activities against acetylcholinesterase. Compounds 1–4, 6, and 7 exhibited antimicrobial activity against some of the tested strains with MIC values ranging from 1.0 to 64 μg/mL, while 3 exhibited enzymatic inhibitory activity against acetylcholinesterase with the IC50 value of 40.26 μM.


Structure Elucidation of the New Compounds
5R-Hydroxyrecifeiolide (1) was isolated as a colorless oil and the molecular formula C12H20O3 was deduced from the (+)-HRESIMS data, indicating three degrees of unsaturation. The 1 H and 13 C NMR spectra of 1 showed the signals for one ester/lactone carbonyl, two olefinic and two oxygenated sp 3 methines, six sp 3 methylenes, and one methyl group (Table 1). In addition, the 1 H NMR data of 1 were quite similar to those of recifeiolide (11-hydroxy-trans-8-dodecenoic acid lactone) [16,17], except that one methylene (δH 1.5-2.3 ppm) in recifeiolide was replaced by an oxygenated methine (δH 3.51 ppm) in 1. The key COSY correlations elucidated the connectivity from H-2 through H-12 ( Figure 2). Key HMBC correlations from H-2 to C-1 and C-4, from H-3 to C-1 and C-5, and from H-11 to C-1, connected C-1 and C-2 and determined the 12-membered macrolide skeleton of 1 ( Figure 2). The relative configuration at C-5 and C-11 for 1 was established by the NOESY experiment ( Figure S8). The NOESY correlations ( Figure 3) from H-2β to H-5 and H-11 revealed a β orientation of these protons [18]. The coupling constants between H-8 and H-9 (JH-8/H-9 = 15.3 Hz) suggested the E-configuration of the C-8/C-9 double bond. The absolute configuration of C-5 of 1 was assigned by application of the modified Mosher's method [19]. The ∆δ values obtained for the (S)-and (R)-MTPA esters (1a and 1b, respectively) of 1 ( Figure 4) suggested that the absolute configuration of C-5 is R. Furthermore, the electronic circular dichroism (ECD) spectrum of 1 was recorded and then computed with the time-dependent density function theory (TD-DFT) method at the gas-phase B3LYP/6-31G (d) level [20,21]. The calculated ECD spectra were produced by SpecDis software [22]. The experimental ECD spectrum for 1 matched well with the calculated spectrum for 11R ( Figure 5). Therefore, the 5R, 11R configuration of 1 was established, and the trivial name 5R-hydroxyrecifeiolide was assigned.

Structure Elucidation of the New Compounds
5R-Hydroxyrecifeiolide (1) was isolated as a colorless oil and the molecular formula C 12 H 20 O 3 was deduced from the (+)-HRESIMS data, indicating three degrees of unsaturation. The 1 H and 13 C NMR spectra of 1 showed the signals for one ester/lactone carbonyl, two olefinic and two oxygenated sp 3 methines, six sp 3 methylenes, and one methyl group (Table 1). In addition, the 1 H NMR data of 1 were quite similar to those of recifeiolide (11-hydroxy-trans-8-dodecenoic acid lactone) [16,17], except that one methylene (δ H 1.5-2.3 ppm) in recifeiolide was replaced by an oxygenated methine (δ H 3.51 ppm) in 1. The key COSY correlations elucidated the connectivity from H-2 through H-12 ( Figure 2). Key HMBC correlations from H-2 to C-1 and C-4, from H-3 to C-1 and C-5, and from H-11 to C-1, connected C-1 and C-2 and determined the 12-membered macrolide skeleton of 1 ( Figure 2). The relative configuration at C-5 and C-11 for 1 was established by the NOESY experiment ( Figure  S8). The NOESY correlations ( Figure 3) from H-2β to H-5 and H-11 revealed a β orientation of these protons [18]. The coupling constants between H-8 and H-9 (J H-8/H-9 = 15.3 Hz) suggested the E-configuration of the C-8/C-9 double bond. The absolute configuration of C-5 of 1 was assigned by application of the modified Mosher's method [19]. The ∆δ values obtained for the (S)-and (R)-MTPA esters (1a and 1b, respectively) of 1 ( Figure 4) suggested that the absolute configuration of C-5 is R. Furthermore, the electronic circular dichroism (ECD) spectrum of 1 was recorded and then computed with the time-dependent density function theory (TD-DFT) method at the gas-phase B3LYP/6-31G (d) level [20,21]. The calculated ECD spectra were produced by SpecDis software [22]. The experimental ECD spectrum for 1 matched well with the calculated spectrum for 11R ( Figure 5). Therefore, the 5R, 11R configuration of 1 was established, and the trivial name 5R-hydroxyrecifeiolide was assigned. for the (S)-and (R)-MTPA esters (1a and 1b, respectively) of 1 ( Figure 4) suggested that the absolute configuration of C-5 is R. Furthermore, the electronic circular dichroism (ECD) spectrum of 1 was recorded and then computed with the time-dependent density function theory (TD-DFT) method at the gas-phase B3LYP/6-31G (d) level [20,21]. The calculated ECD spectra were produced by SpecDis software [22]. The experimental ECD spectrum for 1 matched well with the calculated spectrum for 11R ( Figure 5). Therefore, the 5R, 11R configuration of 1 was established, and the trivial name 5R-hydroxyrecifeiolide was assigned.   The molecular formula of 2 was determined as C12H20O3, which was the same as that of 1, according to its (+)-HRESIMS data. The 1 H and 13 C NMR spectra (Table 1) of 2 were similar to those of 1, except for the different 13 C chemical shifts at C-5 (δC 70.2 in 1, and δC 65.4 in 2) and its adjacent positions (C-2-C-4 and C-6-C-8), which indicated that compound 2 was the 5-epimer of 1. The  The molecular formula of 2 was determined as C12H20O3, which was the same as that of 1, according to its (+)-HRESIMS data. The 1 H and 13 C NMR spectra (Table 1) of 2 were similar to those of 1, except for the different 13 C chemical shifts at C-5 (δC 70.2 in 1, and δC 65.4 in 2) and its adjacent positions (C-2-C-4 and C-6-C-8), which indicated that compound 2 was the 5-epimer of 1. The chemical shifts at C-2 and C-8 (γ-position of C-5) exhibited obvious difference in 2 and 1 probably  The molecular formula of 2 was determined as C12H20O3, which was the same as that of 1, according to its (+)-HRESIMS data. The 1 H and 13 C NMR spectra (Table 1) of 2 were similar to those of 1, except for the different 13 C chemical shifts at C-5 (δC 70.2 in 1, and δC 65.4 in 2) and its adjacent positions (C-2-C-4 and C-6-C-8), which indicated that compound 2 was the 5-epimer of 1. The chemical shifts at C-2 and C-8 (γ-position of C-5) exhibited obvious difference in 2 and 1 probably due to the space effect. As expected, the experimental ECD spectrum of 2 matched well with the The molecular formula of 2 was determined as C 12 H 20 O 3 , which was the same as that of 1, according to its (+)-HRESIMS data. The 1 H and 13 C NMR spectra (Table 1) of 2 were similar to those of 1, except for the different 13 C chemical shifts at C-5 (δ C 70.2 in 1, and δ C 65.4 in 2) and its adjacent positions (C-2-C-4 and C-6-C-8), which indicated that compound 2 was the 5-epimer of 1. The chemical shifts at C-2 and C-8 (γ-position of C-5) exhibited obvious difference in 2 and 1 probably due to the space effect. As expected, the experimental ECD spectrum of 2 matched well with the calculated spectrum of 11R ( Figure 5). The trivial name 5S-hydroxyrecifeiolide was assigned to 2.
Compound 3 was initially obtained as pale yellow powder and possessed a molecular formula C 12 H 22 O 4 by (+)-HRESIMS, implying two degrees of unsaturation. The 1 H and 13 C NMR data ( Table 2) exhibited signals attributed to one ester carbonyl, three oxygenated sp 3 methines, seven sp 3 methylenes, and one methyl group. These data were very similar to those of cladospolide F [12], suggesting that they had the same planar structure, which was also confirmed by the COSY and HMBC correlations ( Figure 2). However, the signs of the optical rotations of 3 (−29.41, MeOH) and cladospolide F (+15.7, MeOH) were opposite, indicating that the absolute configurations of their stereogenic carbons were different. The relative configuration at C-3, C-4, and C-11 could not be concluded by NOESY experiment. Nevertheless, suitable crystals were obtained for X-ray diffraction analysis using Cu Kα radiation which confirmed the absolute configuration of C-3, C-4 and C-11 as 3R, 4S, and 11R ( Figure 6). The ent-cladospolide F was therefore assigned as a trivial name for 3.
Mar. Drugs 2019, 17 FOR PEER REVIEW 4 stereogenic carbons were different. The relative configuration at C-3, C-4, and C-11 could not be concluded by NOESY experiment. Nevertheless, suitable crystals were obtained for X-ray diffraction analysis using Cu Kα radiation which confirmed the absolute configuration of C-3, C-4 and C-11 as 3R, 4S, and 11R ( Figure 6). The ent-cladospolide F was therefore assigned as a trivial name for 3. Compound 4 was obtained as a pale yellow oil and its molecular formula was determined as C14H24O5 on the basis of (+)-HRESIMS, requiring three degrees of unsaturation. The 1 H and 13 C NMR data for 4 ( Table 2) were quite similar to those of 3, except for the presence of additional ester carbonyl (C-13) and methyl (C-14) groups, which indicated the replacement of 11-OH group in 3 by an OAc group in 4, and thus caused the down-field shift of 11-H from δH 3.55 in 3 to δH 4.78 in 4. Detailed interpretation of the COSY and HMBC spectra revealed that 4 was an analogue of 3, with the hydroxyl group at C-11 in 3 being replaced by an acetoxyl group in 4. The HMBC correlation from H-11 to C-13 established the presence of an acetoxyl group at C-11, and the planar structure of 4 was hence confirmed as shown (Figure 2). In a biogenetic perspective, it was tentatively assigned the same relative configuration as that of 3. The similar optical rotations of 4 (−24.56, MeOH) and 3 (−29.41, MeOH) also supported that the absolute configurations of the stereogenic carbons in 4 were the same as those in 3. Therefore, the absolute configurations of the stereogenic carbons in 4 were tentatively assigned as 3R, 4S, and 11R, and the trivial name cladospolide G was assigned. Acetylation of compounds 3 and 4 using acetyl chloride yielded the same diacetylated derivative, which further correlated the structure relationship of compounds 3 and 4.
Compound  [13] suggested that they may have the same relative and absolute stereochemistry. However, neither the relative nor the absolute configuration was determined [13]. In 2001, Franck et al. carried out the first synthesis of iso-cladospolide B and proposed that it has the 4S, 5S, and 11R configuration [14]. Later, in 2005, the absolute configuration Compound 4 was obtained as a pale yellow oil and its molecular formula was determined as C 14 H 24 O 5 on the basis of (+)-HRESIMS, requiring three degrees of unsaturation. The 1 H and 13 C NMR data for 4 ( Table 2) were quite similar to those of 3, except for the presence of additional ester carbonyl (C-13) and methyl (C-14) groups, which indicated the replacement of 11-OH group in 3 by an OAc group in 4, and thus caused the down-field shift of 11-H from δ H 3.55 in 3 to δ H 4.78 in 4. Detailed interpretation of the COSY and HMBC spectra revealed that 4 was an analogue of 3, with the hydroxyl group at C-11 in 3 being replaced by an acetoxyl group in 4. The HMBC correlation from H-11 to C-13 established the presence of an acetoxyl group at C-11, and the planar structure of 4 was hence confirmed as shown (Figure 2). In a biogenetic perspective, it was tentatively assigned the same relative configuration as that of 3. The similar optical rotations of 4 (−24.56, MeOH) and 3 (−29.41, MeOH) also supported that the absolute configurations of the stereogenic carbons in 4 were the same as those in 3. Therefore, the absolute configurations of the stereogenic carbons in 4 were tentatively assigned as 3R, 4S, and 11R, and the trivial name cladospolide G was assigned. Acetylation of compounds 3 and 4 using acetyl chloride yielded the same diacetylated derivative, which further correlated the structure relationship of compounds 3 and 4. Compound 6 was isolated as colorless crystals and gave ion peaks at m/z 229.1432 [M + H] + and 246.1699 [M + NH 4 ] + in the (+)-HRESIMS, corresponding to a molecular formula C 12 H 20 O 4 , indicating three degrees of unsaturation. All the 1 H and 13 C NMR data of 6 were quite similar to those of the previously reported polyketide metabolite iso-cladospolide B [13]. The COSY and HMBC correlations (Figure 2) confirmed that the planar structure of 6 was the same as that of iso-cladospolide B. The high similarity of specific rotations of 6 ([α] 25 D = −90.91 (c 0.11, MeOH) ) and iso-cladospolide B ([α] 25 D = −90 (c 0.23, MeOH)) [13] suggested that they may have the same relative and absolute stereochemistry. However, neither the relative nor the absolute configuration was determined [13]. In 2001, Franck et al. carried out the first synthesis of iso-cladospolide B and proposed that it has the 4S, 5S, and 11R configuration [14]. Later, in 2005, the absolute configuration of iso-cladospolide B, isolated from Cladosporium sp. isolated from the Red Sea sponge Niphates rowi, was assigned to be 4S, 5S, and 11S ([α] 28 D = −61 (c 16.6, MeOH)) [15]. It was later stated that both diastereomers appear to be natural products and (4S, 5S, 11S)-isomer referred to as 11-epi-iso-cladospolide B [23,24]. The relative configuration of 6 could not be assigned by NOESY experiments but the coupling constant for C-4 (J = 1.5 Hz) confirmed the threo relative configuration [14]. Upon slow evaporation of the solvent (MeOH-H 2 O), compound 6 was crystallized and the X-ray analysis was carried out, which was first reported for iso-cladospolide B (Figure 4). The Cu Kα Flack parameter 0.5 (7) allowed preliminary confirmation of the relative configurations of 6 as 4S*, 5S*, 11R*.
Compound 5 was obtained as a pale yellow oil and possessed a molecular formula of C 12 H 18 O 3 by (+)-HRESIMS, implying four degrees of unsaturation. The 1D NMR data ( Table 2) and HSQC spectrum ( Figure S38) suggested signals attributed to one ester and one olefinic quaternary carbons, one oxygenated and three olefinic methines, five sp 3 methylenes, and one methyl group. These NMR data were similar to those of iso-cladospolide B (6) [13]. However, resonances for two oxygenated methines (C-4 and C-5) in 6 were not detected in the NMR spectra of 5. Instead, two additional olefinic signals including one quaternary sp 2 (C-4, δ C 149.4) and one methine sp 2 (C-5, δ C 117.1/δ H 5.53) carbons were observed in the NMR spectra of 5 (Table 2). These data indicated that 5 was a reduced analogue of 6, and this deduction was supported by the molecular formula. The COSY and HMBC spectra established the structure of 5 as shown in Figure 1. In the NOESY experiment, the correlation between H-3 and H-5 indicated the Z-conformation of the double bond between C-4 and C-5 ( Figure 3). The absolute stereochemistry of 5 could not be determined by Mosher's method because of the limited amount of material available. From a biogenetic point of view, 5 was putatively produced by reduction of 6. Therefore, it was tentatively assigned the absolute configuration of C-11 of 5 as 11R. From these data, the name cladospolide H was assigned for 5.
Compound 7 was acquired as white powder and showed ion peaks at m/z 267.1197 [M + Na] + in the positive HRESIMS, corresponding to a molecular formula of C 12 H 20 O 5 . A literature search indicated that all the 1 H and 13 C NMR data of 7 were almost the same as those of previously reported compound pandangolide 1 [13,15] [15] revealed that they may have the same relative and absolute configurations.

Fungal Material
The fungal strain Cladosporium cladosporioides MA-299 was isolated from the leaves of the mangrove plant Bruguiera gymnorrhiza, collected in Hainan Island, China, in March 2015. The strain was identified as Cladosporium cladosporioides by analysis of its ITS region of the rDNA, which is the same (100%) as that of C. cladosporioides DCF-1 (accession no. MG208055). The sequence data were deposited in GenBank with the accession number MH822624. The strain is preserved at Key Laboratory of Experimental Marine Biology, Institute of Oceanology of the Chinese Academy of Sciences (IOCAS).

Fermentation
For chemical investigations, the strain of C. cladosporioides MA-299 was cultured on PDA (Potato Dextrose Agar) medium at 28 • C for six days and then inoculated into 100 × 1 L flasks, each containing 70 g of rice, 0.1 g corn syrup, 0.3 g peptone, 0.1 g methionine and 100 mL seawater that was obtained from the Huiquan Gulf of the Yellow Sea near the campus of IOCAS, statically cultured for 48 days at room temperature.

Extraction and Isolation
After 48 days, the fermented rice substrate was mechanically fragmented and then extracted three times with 300 mL EtOAc every flask. All of the EtOAc extracts were filtered and evaporated under reduced pressure to yield a crude extract (52.3 g).

X-Ray Crystallographic Analysis of Compounds 3 and 6
All crystallographic data were collected on an Agilent Xcalibur Eos Gemini CCD plate diffractometer, using graphite monochromatized Cu/Kα radiation (λ= 1.54178 Å) [25]. The data were corrected for absorption by using the program SADABS [26]. The structures were solved by direct methods with the SHELXTL software package [27]. All nonhydrogen atoms were refined anisotropically. The H atoms were located by geometrical calculations, and their positions and thermal parameters were fixed during the structure refinement. The structure was refined by full-matrix least-squares techniques [28].
Crystal data for compound 3: The 5282 measurements yielded 2081 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R 1 = 0.0727 and wR 2 = 0.1620 (I > 2σ(I)). The Flack parameter was 0.5 (7) in the final refinement for all 2081 reflections with 150 Friedel pairs.

Acetylation of Compounds 3 and 4
To 5 µmol samples of compound 3 or 4 in glass-stoppered flask were added 400 µL dichloromethane, then excess amount of triethylamine was added. Drip 20 µmol of acetylchloride slowly into the flask in ice bath and keeping the reaction for 12 h. Then stop the reaction by adding 20 µL of water into the flask. The progress of the reaction was monitored by TLC analysis. The resulting reaction mixture was extracted with dichloromethane (2 × 400 µL), dried with Na 2 SO 4 , and concentrated in vacuo to obtain the product.