Seven New Drimane-Type Sesquiterpenoids from a Marine-Derived Fungus Paraconiothyrium sporulosum YK-03

Seven new drimane-type sesquiterpenoids, namely the sporulositols A–D (1–4), 6-hydroxydiaporol (5), seco-sporulositol (6) and sporuloside (7) were isolated from the ethyl acetate extract of fermentation broth for a marine-derived fungus Paraconiothyrium sporulosum YK-03. Their structures were elucidated by analysis of extensive spectroscopic data, and the absolute configurations were established by crystal X-ray diffraction analysis and comparisons of circular dichroism data. Among them, sporulositols A–E (1–4) and seco-sporulositol (6) represent the first five examples of a unique class of drimanic mannitol derivatives, while compounds 6 and 7 may represent two new series of natural drimanes, possessing an aromatic ring with a rare 4,5-secodrimanic skeleton and an unusual CH3-15 rearranged drimanic α-D-glucopyranside, respectively. Furthermore, the origin of mannitol moiety was investigated by reliable HPLC and NMR analyses.


Results and Discussion
The fermentation broth of P. sporulosum YK-03 was concentrated and extracted with ethyl acetate and n-butanol, successively. Then the ethyl acetate extract of the fermentation broth of P. sporulosum YK-03 was subjected to various modern chromatographic isolation methods (including silica gel/Sephadex LH-20 column chromatography and reversed-phase C18 preparative high performance liquid chromatography) to give seven new compounds 1−7 (4.3 mg, 93.3 mg, 89.8 mg, 2.6 mg, 2.3 mg, 2.5 mg and 2.0 mg). Their structures and the absolute configurations were elucidated by analysis of HRESIMS, 1D/2D NMR, circular dichroism (CD) and X-ray diffraction analyses.

Results and Discussion
The fermentation broth of P. sporulosum YK-03 was concentrated and extracted with ethyl acetate and n-butanol, successively. Then the ethyl acetate extract of the fermentation broth of P. sporulosum YK-03 was subjected to various modern chromatographic isolation methods (including silica gel/Sephadex LH-20 column chromatography and reversed-phase C 18 preparative high performance liquid chromatography) to give seven new compounds 1−7 (4.3 mg, 93.3 mg, 89.8 mg, 2.6 mg, 2.3 mg, 2.5 mg and 2.0 mg). Their structures and the absolute configurations were elucidated by analysis of HRESIMS, 1D/2D NMR, circular dichroism (CD) and X-ray diffraction analyses.  (Tables 1  and 2), indicating three, three, three, three, and five indices of hydrogen deficiency, respectively, for 1-4 and 6. The IR spectra of sporulositols A-D (1-4) and seco-sporulositol (6) showed the presence of hydroxyl (ν max 3384.7-3416.6 cm −1 ) and olefinic (ν max 1632.0-1659.0 cm −1 ) groups in their structures, and beyond these, there was an aromatic (ν max 1597.8, 1554.4, and 1432.7 cm −1 ) group in 6.
6-Hydroxydiaporol (5)   The relative configurations of compounds 1-5 were assigned by 1 H-NMR J-values and NOE correlations (Figure 3). In these drimane-type sesquiterpenoids, the doublet J values (11.1-13.2 Hz) of H-5 suggested a trans-junction of the two cyclohexatomic ring system [35], H 3 -15 and H 3 -13 (or H 2 -13) adopt the same β-orientation, whereas H 3 -14, H-5 and HO-6 (or HO-7) oriented in the opposite α-direction. To determine the absolute configurations of 1-5, compounds 2 and 3 were selected and subjected to acid hydrolysis (5% trifluoroacetic acid in methanol; Figure 4), due to their abundant amounts.     [44], which was also isolated from the same strain P. sporulosum YK-03. NOESY spectra of 2a and 3a ( Figure 3) indicated that they share the same relative configurations as compounds 1-5. After many attempts, crystal of 2a suitable for single-crystal X-ray diffraction (Cu Kα) analysis ( Figure 5) was successfully obtained upon slow evaporation of the solvent mixture (methanol-water, 20:1) by keeping the sample at room temperature for nearly one month. Thus, the absolute configuration of 2a was unambiguously determined as 5S,6S,10S. Based on the fact that the CD patterns of 1-5 and 3a ( Figure 6) were identical to that of 2a, the absolute configurations of drimane-type sesquiterpenoid were assigned as 5S,10S in 1, 5S,6S,10S in 2, 4S,5R,10S in 3a, 4S,5R,10S in 3, 4S,5R,7R,10S in 4, and 4S,5R,6S,10S in 5. Acid hydrolysis of 2 and 3 afforded a hexitol, together with 2a and 3a, respectively, whose structures were elucidated by extensive NMR spectroscopic data (Figure 2, Figure 3 and Figures S19-S29, S38-S45 in supplementary materials). Furthermore, NMR ( Figure S28 and S29) and optical rotation data ([α] 25 D +135.5 (c 0.380, CH 3 OH)) of the hexitol were quite similar as those reported of D-mannitol [44], which was also isolated from the same strain P. sporulosum YK-03. NOESY spectra of 2a and 3a (Figure 3) indicated that they share the same relative configurations as compounds 1-5. After many attempts, crystal of 2a suitable for single-crystal X-ray diffraction (Cu Kα) analysis ( Figure 5) was successfully obtained upon slow evaporation of the solvent mixture (methanol-water, 20:1) by keeping the sample at room temperature for nearly one month. Thus, the absolute configuration of 2a was unambiguously determined as 5S,6S,10S. Based on the fact that the CD patterns of 1-5 and 3a ( Figure 6) were identical to that of 2a, the absolute configurations of drimane-type sesquiterpenoid were assigned as 5S,10S in 1, 5S,6S,10S in 2, 4S,5R,10S in 3a, 4S,5R,10S in 3, 4S,5R,7R,10S in 4, and 4S,5R,6S,10S in 5.       Besides the NMR data of the hexitol, the remaining NMR signals of 6 ( Table 2)  2 Hz) to δ C 133.2 (C-10), 135.6 (C-9) and 140.2, and from δ H 2.71 to δ C 133.2 and 134.9 led to the assignment of the tetrasubstituted benzene with two methyls located at C-5 and C-8, an oxygenated methyl located at C-9, and a prenyl methyl located at C-10. Further, the HMBC correlation of δ H 3.76 (m, H-3 ) to δ C 67.4 (C-11) indicated C-3' was connected to C-11 by an ether O to afford the structure of 6. Based on the NMR data and biosynthetic homology, the hexitol moiety of 6 was presumed to be D-mannitol, the same as that of 1-5.
Sporuloside (7) -1 ) combined the abovementioned groups to afford the planar structure of 7, in which C-13 was glycosidated by a α-glucose. Luckily, crystals of 7 suitable for single-crystal X-ray diffraction (Cu Kα) analysis ( Figure 5) were successfully obtained upon slow evaporation of the solvent mixture (methanol-water, 20:1) by keeping the sample at room temperature for nearly one month, so the absolute configuration of C-4 was assigned as S, and α-glucosyl group as D-form. from the medium, the normal medium (mannitol-contained, control group), modified medium No.1 (no mannitol, blank group) and modified medium No.2 (mannitol replaced by sorbitol, experimental group) were included for simultaneous cultivation of P. sporulosum YK-03 and HPLC analysis of the metabolites. Compounds 1-3 could be detected in all the three groups ( Figure 7A), and compound 2 was isolated from the extract of experimental group ( Figure 7B,C), revealing that the fungus can produce the mannitol moiety intrinsically, no matter whether the medium contains mannitol or not.

Investigation on the Origin of Mannitol Moiety
Molecules 2019, 24, x FOR PEER REVIEW 10 of 16 or derived from the medium, the normal medium (mannitol-contained, control group), modified medium No.1 (no mannitol, blank group) and modified medium No.2 (mannitol replaced by sorbitol, experimental group) were included for simultaneous cultivation of P. sporulosum YK-03 and HPLC analysis of the metabolites. Compounds 1-3 could be detected in all the three groups ( Figure 7A), and compound 2 was isolated from the extract of experimental group (Figures 7B and  C), revealing that the fungus can produce the mannitol moiety intrinsically, no matter whether the medium contains mannitol or not. Compounds 1-7 were tested for cytotoxicity against two cell lines A549 (human lung adenocarcinoma cells) and MCF-7 (human breast cancer cells). Unfortunately, compounds 1-7 did not show any detectable cytotoxicity.

General Experimental Procedures
Optical rotations were measured using a Perkin-Elmer Model 241 polarimeter (Perkin Elmer, Inc. Waltham, MA, USA). UV spectra were obtained on a Shimadzu UV-1601 (Shimadzu Corp., Kyoto, Japan). IR spectra were taken on a Bruker IFS-55 infrared spectrophotometer (Bruker Optik BmbH, Ettlingen, Germany) with KBr disks. The HRESIMS data were obtained on a microTOF-Q Bruker mass instrument (Bruker Daltonics, Billerica, MA, USA). CD spectra were recorded with a Compounds 1-7 were tested for cytotoxicity against two cell lines A549 (human lung adenocarcinoma cells) and MCF-7 (human breast cancer cells). Unfortunately, compounds 1-7 did not show any detectable cytotoxicity.

Fungal Material
Paraconiothyrium sporulosum YK-03 was isolated from the sea mud collected from the intertidal zone of Bohai Bay in Liaoning Province of China. It was identified based on the analysis of ITS sequence (GenBank accession No. KC416199) and has been deposited in the School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University.

Cytotoxic Activity Assay
The cytotoxicity was evaluated by using the MTT assay as described previously [45]. Doxorubicin hydrochloride was used as a positive control. The A549 and MCF-7 Cells (China Infrastructure of Cell Lines Resources, Beijing, China were cultured in McCoy's 5A medium and DMEM basic medium (1×) at 37 • C under an atmosphere of 5% CO 2 , and were seeded on each well of 96-well plates containing 200 µL of tumor cell suspension (1 × 10 4 cells). After 24 h, each well was added 2 µL of test solution and incubated for another 72 h. 50 µL of MTT solution (1 mg/mL, Beijing Cellchip Biotechnology Co., Ltd., Beijing, China was added to each well, and the plate was incubated for 3h under the same condition. Then, the plate was centrifuged and the supernatants were removed and cells were dissolved in 150 µL of DMSO to determine the IC 50 values.