Citreobenzofuran D–F and Phomenone A–B: Five Novel Sesquiterpenoids from the Mangrove-Derived Fungus Penicillium sp. HDN13-494

Five new sesquiterpenoids, citreobenzofuran D–F (1–3) and phomenone A–B (4–5), along with one known compound, xylarenone A (6), were isolated from the culture of the mangrove-derived fungus Penicillium sp. HDN13-494. Their structures were deduced from extensive spectroscopic data, high-resolution electrospray ionization mass spectrometry (HRESIMS), and electronic circular dichroism (ECD) calculations. Furthermore, the absolute structures of 1 were determined by single-crystal X-ray diffraction analysis. Citreobenzofuran E–F (2–3) are eremophilane-type sesquiterpenoids with rare benzofuran frameworks, while phomenone A (4) contains a rare thiomethyl group, which is the first report of this kind of sesquiterpene with sulfur elements in the skeleton. All the compounds were tested for their antimicrobial and antitumor activity, and phomenone B (5) showed moderate activity against Bacillus subtilis, with an MIC value of 6.25 μM.


Introduction
Natural products play an increasingly crucial role in drug discovery, owing to their great chemical and bioactive diversity [1]. As is well known, fungi are a rich source of novel natural products [2], and, in the last two decades, the fungal sources of new metabolites have been broadened from terrestrial strains to marine habitats [3]. The ocean turned out to be an attractive environment, since the search for new biomedicals from marine microorganisms resulted in the isolation of approximately 10,000 metabolites, many of which were endowed with pharmacodynamic properties [4].
Mangrove is a special marine ecosystem that occurs in tropical and subtropical intertidal estuarine zones, and is characterized by high salinity and rich organic matter [5], which makes it an extremely diverse microbial resource. Mangrove-associated fungi, as the second-largest ecological group of marine fungi [6], have proven to be a rich source of natural products, with unique chemical structures and diverse pharmacological activities. Up to now, a large number of metabolites produced by mangrove fungi have been reported, including alkaloids, macrolides, polyketides, quinones, terpenes, and so on [6], which displayed diverse bioactivity, such as antibacterial, insecticidal, antioxidant, and cytotoxic, etc. These findings indicate that mangrove-derived fungi may possess great potential to produce novel and active secondary metabolites. As part of our ongoing work searching for natural products from mangrove-derived fungi, one fungus, Penicillium sp. HDN13-494, from the root soil sample of a mangrove plant in Wenchang, Hainan, was selected for further chemical studies. Herein, the details of isolation, structural elucidation, and bioactivities of its metabolites are reported. selected for further chemical studies. Herein, the details of isolation, structural elucidation, and bioactivities of its metabolites are reported.

Results and Discussion
Chemical studies of Penicillium sp. HDN13-494 led to the isolation of five new sesquiterpenoids (1−5) and one known compound (6) ( Figure 1). Compound 4 represents the first example of eremophilane sesquiterpenoids containing sulfur elements. Citreobenzofuran D (1) and E (2) possess a rare cyclic system with 6,12-epoxy-eremophilane-type which has only been reported twice in natural products [7,8].  (Table 1 and 2) indicated that 1 shared the same skeleton with isoligularonic acid [7], except for the appearance of two hydroxyl groups at C-1 (δC 72.2) and C-13 (δC 55.2) (Table 1), which was supported by HMBC correlation from OH-1 to C-1, C-2, C-10 and from OH-13 to C-11 and C-13 ( Figure 2). In addition, the deshielded singlet at C-9 (δC 125.5) and C-10 (δC 163.5) indicated the occurrence of alkenylation between them ( Table  1). The relative configuration of 1 was elucidated as 1R*, 4S*, 5R* on the basis of NOESY correlations of H-1 with H-4, and H-14 with H-15 ( Figure 3). To determine the absolute configuration of 1, the ECD calculations of the optimized conformations of (1R, 4S, 5R)-1a and (1S, 4R, 5S)-1b were obtained at the B3LYP/6-31+G(d) level. The overall pattern of the experimental ECD spectrum was in reasonable agreement with the calculated ECD spectrum of 1a (Figure 4), which indicated the 1R, 4S, and 5R absolute configuration of 1. Single-crystal X-ray diffraction analysis by Cu Ka radiation further confirmed the absolute configuration of 1 ( Figure 5).  Figure S8). The 1D NMR data (Tables 1 and 2) indicated that 1 shared the same skeleton with isoligularonic acid [7], except for the appearance of two hydroxyl groups at C-1 (δ C 72.2) and C-13 (δ C 55.2) ( Table 1), which was supported by HMBC correlation from OH-1 to C-1, C-2, C-10, and from OH-13 to C-11 and C-13 ( Figure 2). In addition, the deshielded singlet at C-9 (δ C 125.5) and C-10 (δ C 163.5) indicated the occurrence of alkenylation between them ( Table 1). The relative configuration of 1 was elucidated as 1R*, 4S*, 5R* on the basis of NOESY correlations of H-1 with H-4, and H-14 with H-15 ( Figure 3). To determine the absolute configuration of 1, the ECD calculations of the optimized conformations of (1R, 4S, 5R)-1a and (1S, 4R, 5S)-1b were obtained at the B3LYP/6-31+G(d) level. The overall pattern of the experimental ECD spectrum was in reasonable agreement with the calculated ECD spectrum of 1a (Figure 4), which indicated the 1R, 4S, and 5R absolute configuration of 1. Single-crystal X-ray diffraction analysis by Cu Ka radiation further confirmed the absolute configuration of 1 ( Figure 5).
Citreobenzofuran E (2) was obtained as a green amorphous powder, with the molecular formula C 15 H 18 O 2 , based on the HRESIMS spectrum (m/z 229.1227 [M − H] − ) ( Figure S15), indicating seven degrees of unsaturation. Detailed analysis of the 1D NMR data of 2 (Tables 1 and 2) revealed highly structural similarities to the known compound citreobenzofuran B [8]. The difference was the disappearance of the hydroxyl group at C-3 (δ C 29.6), supported by the shielded singlet at δ C 29.6 (Table 1), and the HSQC correlation between H 2 -3 and C-3 ( Figure S12). The absolute configuration of 2 was assigned as 4R by comparing the experimental ECD with the calculated ECD ( Figure 4). Citreobenzofuran F (3) was obtained as a colorless oil, with the molecular formula C 15 H 16 O 3 , determined by the HRESIMS data (m/z 243.1032 [M − H] − ) ( Figure S22). The 1D NMR data (Tables 1 and 2) revealed partial structural similarity to the known compound 3-formyl-4,5-dimethyl-8-oxo-5H-6,7-dihydronaphtho[2,3-b] furan [9]. The difference was Mar. Drugs 2022, 20, 137 3 of 9 the replacement of carbonyl at C-13 with a hydroxyl group, which was supported by the chemical shift of C-13 (δ C 54.9) (Table 1), and the HSQC correlation between H 2 -13 and C-13 ( Figure S19). The absolute configuration of 3 was assigned as 4S by comparing the experimental ECD with the calculated ECD ( Figure 4).               Figure  S30). The 1 H and 13 C NMR spectra of 4 were similar to those of paraconiothin G [10], except for the oxidation of the hydroxyl group at C-12 (δ C 188.4) to an aldehyde group, and the appearance of the methylthio group at C-13 (δ C 158.0) ( Table 1). These changes were confirmed by the HMBC correlations from H-13 (δ H 8.01) to C-14 (δ C 17.5) and C-12(δ C 188.4), and from H-12 (δ H 9.28) to C-7(δ C 130.8) and C-11(δ C 134.9) (  Figure S38). According to the 1D NMR data, compound 5 (Tables 1 and 2) was similar to a known mycotoxic sesquiterpenoid, xylarenones A [11], except for the disappearance of the hydroxyl group at C-1, which was supported by the shielded singlets at δ C 32.6 ( Table 1). The H-6, 14-CH 3 , and 15-CH 3 were suggested to be located on the same face of the octahydronaphthalene ring, based on the NOESY correlation of H-6 (δ H 3.36) with H-15 (δ H 1.03), and H-14 (δ H 1.16) with H-15 ( Figure 3). Thus, the relative configuration was determined as 4S*, 5R*, 6R*, 7R*. The overall pattern of the experimental ECD spectrum was in reasonable agreement with the calculated ECD spectrum of (4S, 5R, 6R, 7R)-5a (Figure 4), which indicated the 4S, 5R, 6R and 7R absolute configuration of 5.
The known compound was identified as xylarenones A (6) [11] by comparison of the spectroscopic data with the literature values.
Compounds 1-6 were tested for their antimicrobial (Table 3) and antitumor activity against K562, MDA-MB-231, L-02, H69AR, and ASPC-1. Compound 5 showed promising antimicrobial activity against B. subtilis, with an MIC value of 6.25 µM, while the other compounds exerted no activity for all the tested strains. Moreover, none of the compounds displayed obvious anticancer activity.

General Experimental Procedures
Specific rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a HITACHI 5430. IR spectra were measured on a Bruker Tensor-27 spectrophotometer in KBr discs. NMR spectra were recorded on a JEOLJN M-ECP 600 spectrometer (JEOL, Tokyo, Japan) or an Agilent 500 MHz DD2 spectrometer using tetramethylsilane as an internal standard. HRMS were obtained on a Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) or Micromass Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer (Waters Corporation, Milford, MA, USA). Semipreparative HPLC was performed on an ODS column (YMC-Pack ODS-A, 10 × 250 mm, 5 µm, 3 mL/min).

Materials and Culture Conditions
The fungal strain, Penicillium sp. HDN13-494, was isolated from the root soil sample of a mangrove plant in Wenchang. The strain was identified by internal transcribed spacer (ITS) sequence (GenBank No. OM283301). The strain was incubated in media potato dextrose agar (PDA, 20% potato, 2% dextrose, and 2% agar) at 28 • C for 5 days for sporulation. For compound production, the strains were cultured in PDB (potato dextrose broth) at 28 • C, 180 rpm, for 9 days. The strain was deposited at the Marine Medicinal Bioresources Center, Ocean University of China, Qingdao, China.

Fermentation
The strain was grown on PDA plates for 5 days at 28 • C. The spores of Penicillium sp. HDN13-494 were inoculated into 500 mL Erlenmeyer flasks containing 150 mL of PDB medium, pH = 7.0 (in seawater collected from Huiquan Bay, Yellow Sea), and cultured at 28 • C for 9 days on a rotary shaker at 180 rpm. A total of 10 L of broth was extracted with EtOAc (4 × 10 L) to generate the extract (30 g).

Extraction and Purification
All the fermentation broth (10 L) was filtered through cheese cloth to separate the supernatant from the mycelia. The supernatant was extracted with EtOAc (4 × 10 L), and the mycelia was macerated and extracted with methanol (3 × 5 L). All extracts were evaporated under reduced pressure to give a crude gum. The extract was chromatographed over ODS, eluting with a gradient of increasing MeOH/H 2 O (30-100%) to afford twelve fractions (Fr.1 to Fr.12). Fr.

Computational Section
Conformational searches were performed by employing the systematic procedure implemented in Spartan 14, using the MMFF (Merck molecular force field). All MMFF minima were reoptimized with DFT calculations at the B3LYP/6-31+G(d) level using the Gaussian09 program. The geometry was optimized starting from various initial conformations, with vibrational frequency calculations confirming the presence of minima. Time-dependent DFT calculations were performed on lowest-energy conformations (>5% population) for each configuration using 20 excited states and using a polarizable continuum model for MeOH. ECD spectra were generated using the program SpecDis by applying a Gaussian band shape with a 0.30 eV width and 25 blue shifts to facilitate comparison to the experimental data.

Cytotoxicity Assay
Cytotoxic activities of compounds 1-6 were evaluated against the K562 (using the MTT method), MDA-MB-231, L-02, H69AR and ASPC-1 (using the SRB method) cell lines. Adriamycin (ADM) was used as a positive control. The detailed methodologies for biological testing have been described in previous reports [12,13].

Antimicrobial Activity
Antibacterial activities of compounds 1-6 were evaluated against B. subtilis, Proteus vulgaris, Acinetobacter baumannii, Candida. albicans, Escherichia. coli, and MRSA (Methicillinresistant Staphylococcus aureus) by using the agar dilution method as previously reported [14]. Ciprofloxacin was used as a positive control.

Conclusions
In summary, five new sesquiterpenoids, citreobenzofuran D-F (1-3) and phomenone A-B (4-5), along with one known compound, xylarenones A (6), were isolated from a mangrove-derived fungus, Penicillium sp. HDN13-494. Compounds 1, 4 and 5 are canonical eremophilane structures, while compounds 2 and 3 possess rare benzofuran structures in the skeletons. The biological activity assay showed that compound 5 possessed mild antimicrobial activity against Bacillus subtilis. Our research results expanded the diversity of the secondary metabolite in mangrove-derived fungi, and also enriched the diversity of terpenoids in natural products.