Dihydroisocoumarins from the Mangrove-Derived Fungus Penicillium citrinum

Three new dihydroisocoumarin penicimarins G–I (1–3), together with one known dihydroisocoumarin (4) and three known meroterpenoids (5–7), were obtained from a fungus Penicillium citrinum isolated from the mangrove Bruguiera sexangula var. rhynchopetala collected in the South China Sea. Their structures were elucidated by the detailed analysis of spectroscopic data. The absolute configuration of 1 was determined by the X-ray diffraction analysis using Cu Kα radiation. The absolute configurations of 2 and 3 were determined by comparison of their circular dichroism (CD) spectra with the literature. All compounds were evaluated for their antibacterial activities and cytotoxic activities.


Results and Discussion
Compound 1 was obtained as colorless crystals. Its molecular formula of C15H20O5 (six degrees of unsaturation) was determined by HR-ESI-MS and combined with 1 H and 13 C NMR spectroscopic data. In the 1 H NMR spectrum (Table 1), the signals and the coupling constants at δH 7.05 (d, J = 8.4 Hz) and 6.90 (d, J = 8.4 Hz) indicated the presence of a 1,2,3,4-tetrasubstituted benzene system. Two oxygenated methine proton signals at δH 4.39 (m) and 3.75 (m), one methoxyl group at δH 3.82 (s), one methyl group at δH 1.17 (d, J = 6.2 Hz), and four methylene groups at δH (3.16 (dd, J = 16.8, 3.0 Hz) and 2.58 (dd, J = 16.8, 11.6 Hz)), (1.84 (m) and 1.70 (m)), 1.59 (m) and 1.55 (m) were also observed. The 1 H NMR and 13 C NMR spectra indicated that this compound included four methylenes, two methines, one methoxy, and one methyl. The 13 C NMR spectrum revealed the signals for six aromatic carbons and one carbonyl carbon, including one lactone carbonyl carbon at δC 165.8. Thus, these spectroscopic features suggested that 1 was very similar to penicimarin B [17]. The obvious difference in the 1 H NMR spectrum was the absence of one aromatic proton signal at δH 6.79 (d, J = 7.2 Hz, H-5) in 1. Furthermore, in the 13 C NMR spectra, the C-5 signal moved downfield significantly [δC 148.2 (C) in 1 vs 119.3 (CH)] in penicimarin B, indicating an aromatic proton is replaced by a hydroxyl group. The 1 H, 1 H-COSY, HMQC, and HMBC spectra allowed the complete assignment for 1 ( Figure 2). The relative configuration of 1 could, however, not be confirmed unambiguously. Crystallization of 1 from MeOH afforded colorless crystals, which gave an X-ray crystal structure with a Flack parameter of 0.0 (3) (Figure 3). The absolute configuration of C-3 was also determined by circular dichroism (CD) spectroscopy ( Figure 4). The negative circular dichroism at 259 nm ( Figure 4) suggested the R configuration at C-3, by comparison with data for dihydroisocoumarins described in the literature [18]. Thus, compound 1 was named as penicimarin G, and the absolute configuration of 1 was defined as (3R, 4′R).

Results and Discussion
Compound 1 was obtained as colorless crystals. Its molecular formula of C 15 H 20 O 5 (six degrees of unsaturation) was determined by HR-ESI-MS and combined with 1 H and 13 C NMR spectroscopic data. In the 1 H NMR spectrum (Table 1), the signals and the coupling constants at δ H 7.05 (d, J = 8.4 Hz) and 6.90 (d, J = 8.4 Hz) indicated the presence of a 1,2,3,4-tetrasubstituted benzene system. Two oxygenated methine proton signals at δ H 4.39 (m) and 3.75 (m), one methoxyl group at δ H 3.82 (s), one methyl group at δ H 1.17 (d, J = 6.2 Hz), and four methylene groups at δ H (3.16 (dd, J = 16.8, 3.0 Hz) and 2.58 (dd, J = 16.8, 11.6 Hz)), (1.84 (m) and 1.70 (m)), 1.59 (m) and 1.55 (m) were also observed. The 1 H NMR and 13 C NMR spectra indicated that this compound included four methylenes, two methines, one methoxy, and one methyl. The 13 C NMR spectrum revealed the signals for six aromatic carbons and one carbonyl carbon, including one lactone carbonyl carbon at δ C 165.8. Thus, these spectroscopic features suggested that 1 was very similar to penicimarin B [17]. The obvious difference in the 1 H NMR spectrum was the absence of one aromatic proton signal at δ H 6.79 (d, J = 7.2 Hz, H-5) in 1. Furthermore, in the 13 C NMR spectra, the C-5 signal moved downfield significantly [δ C 148.2 (C) in 1 vs 119.3 (CH)] in penicimarin B, indicating an aromatic proton is replaced by a hydroxyl group. The 1 H, 1 H-COSY, HMQC, and HMBC spectra allowed the complete assignment for 1 ( Figure 2). The relative configuration of 1 could, however, not be confirmed unambiguously. Crystallization of 1 from MeOH afforded colorless crystals, which gave an X-ray crystal structure with a Flack parameter of 0.0 (3) (Figure 3). The absolute configuration of C-3 was also determined by circular dichroism (CD) spectroscopy ( Figure 4). The negative circular dichroism at 259 nm ( Figure 4) suggested the R configuration at C-3, by comparison with data for dihydroisocoumarins described in the literature [18]. Thus, compound 1 was named as penicimarin G, and the absolute configuration of 1 was defined as (3R, 4 R).    Compound 2 was isolated as colorless crystals with the molecular formula assigned as C15H18O5 (seven degrees of unsaturation) on the basis of its HR-ESI-MS and combined with 1 H and 13 C NMR spectroscopic data. The 1D NMR data of 2 was very similar with that of 1, except for the presence of a carbonyl signal (δC 211.4) for C-4′ in 2 and the absence of one oxygenated methine proton signal (δC     Compound 2 was isolated as colorless crystals with the molecular formula assigned as C15H18O5 (seven degrees of unsaturation) on the basis of its HR-ESI-MS and combined with 1 H and 13 C NMR spectroscopic data. The 1D NMR data of 2 was very similar with that of 1, except for the presence of a carbonyl signal (δC 211.4) for C-4′ in 2 and the absence of one oxygenated methine proton signal (δC     Compound 2 was isolated as colorless crystals with the molecular formula assigned as C15H18O5 (seven degrees of unsaturation) on the basis of its HR-ESI-MS and combined with 1 H and 13 C NMR spectroscopic data. The 1D NMR data of 2 was very similar with that of 1, except for the presence of a carbonyl signal (δC 211.4) for C-4′ in 2 and the absence of one oxygenated methine proton signal (δC Compound 2 was isolated as colorless crystals with the molecular formula assigned as C 15 H 18 O 5 (seven degrees of unsaturation) on the basis of its HR-ESI-MS and combined with 1 H and 13 C NMR spectroscopic data. The 1D NMR data of 2 was very similar with that of 1, except for the presence of a carbonyl signal (δ C 211.4) for C-4 in 2 and the absence of one oxygenated methine proton signal (δ C 68.3) for C-4 in 1. This was corroborated by the HMBC correlations from H-3 and H-5 to C-4 ( Figure 2). The 1 H-1 H-COSY, HMQC, and HMBC spectra allowed the complete assignment for 1. Thus, compound 2 was named as penicimarin H, and the absolute configuration of C-3 was also determined to be R by CD spectra (Figure 4) [18].
Compound 3 was obtained as a colorless oil. Its molecular formula of C 13 H 14 O 5 (seven degrees of unsaturation) was determined by HR-ESI-MS and combined with 1 H and 13 C NMR spectroscopic data. In the 1 H NMR spectrum (Table 1), the proton signals and the coupling constants at δ H 7.42 (dd, J = 8.0, 7.6 Hz), 6.89 (d, J = 8.0 Hz), and 6.70 (d, J = 7.6 Hz) indicated the presence of an 1,2,3-trisubstituted benzene system. One hydrogen-bonded hydroxyl group at δ H 10.9 (s), one oxygenated methine proton signal at δ H 4.65 (m), one methoxyl group at δ H 3.70 (s), and three methylene groups at δ H 2.92 (m), 2.62 (m), and 2.13 (m) were also observed. The 1 H NMR and 13 C NMR spectra included three methylenes, one methine, and one methoxy. The 13 C NMR data exhibited the presence of six aromatic carbons and two carbonyl carbons, including two lactone carbonyl carbons at δ C 173.2 (C) and 169.7 (C). Thus, these spectroscopic features suggested that 3 was very similar to aspergillumarin A (4) [14]. The obvious differences in the 1 H NMR spectra were the absence of one methylene signal at δ H 1.76 (m), a singlet methyl signal at δ H 2.16 (s), and the presence of a methoxy signal at δ H 3.70 (s) in 3. The position of this methoxy group was confirmed by the HMBC correlation from 4 -OMe to C-3 ( Figure 1). The 1 H, 1 H-COSY, HMQC, and HMBC spectra allowed the complete assignment for 3. Thus, compound 3 was named as penicimarin I, and the absolute configuration of C-3 was also determined to be R by CD spectra (Figure 4) [18].
By comparing physical and spectroscopic data with literature values, the four known compounds were identified as aspergillumarin A (4) [14] and three meroterpenoids: dehydroaustin (5) [15], 11β-acetoxyisoaustinone (6) [15], and austinol (7) [16]. The absolute configuration of C-3 in compound 4 was determined to be R by comparison with the CD spectrum and the optical rotation data of those reported in the literature [14,18].
More detailed spectra of new compounds are available in the Supplementary Materials (Figures S1-S18).
All the isolated compounds were evaluated for their antibacterial activities against five terrestrial pathogenic bacteria and two marine pathogenic bacteria. Compounds 1, 2, and 5-7 exhibited selective antibacterial activity (Table 2). Compounds 2 and 7 showed moderate activity against Staphylococcus epidermidis and S. aureus with the same minimum inhibitory concentration (MIC) values of 10 µM. Compounds 1 and 2 showed a broad spectrum of antibacterial activity against the five pathogenic bacteria S. epidermidis, S. aureus, Escherichia coli, Bacillus cereus, and Vibrio alginolyticus. All compounds were also tested for cytotoxic activity against HeLa, MCF-7, and A549 cells, however, these compounds showed no cytotoxic activity (IC 50 > 50 µM).

General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a Thermo Nicolet 6700 (using KBr disks) spectrophotometer (Thermo Scientific, Madison, WI, USA). Both 1D and 2D NMR spectra were measured on a Bruker AV-400 (Bruker Corporation, Switzerland) instrument with TMS as the internal standard. HR-ESI-MS spectra were obtained on the Bruker Daltonics Apex-Ultra 7.0 T (Bruker Corporation, Billerica, MA, USA) and the Q-TOF Ultima Global GAA076 LC mass spectrometer. Single-crystal data: Agilent Gemini Ultra X-ray single crystal diffractometer (Cu Kα radiation). CD spectra were recorded on a MOS-450 spectrometer. Prep. HPLC were used for Agilent 1260 prep-HPLC system with an Agilent C18 analytical HPLC column (4.6 × 250 mm, 5 µm) and semi-preparative column (9.4 × 250 mm, 7 µm). Sephadex LH-20 (Pharmacia Co. Ltd, Sandwich, UK) and Silica gel (200-300 mesh, 300-400 mesh Qingdao Marine Chemical Factory, Qingdao, China) were used for column chromatography (CC). All solvents were purchased from Xilong Chemical Reagent Factory (Guangzhou, China).

Fungal Materials
The fungal strain Penicillium citrinum HL-5126 was isolated from the mangrove Bruguiera sexangula

Identification of Fungus
The fungus was identified according to its morphological characteristics and by comparison of the ITS sequence amplification, primer pair ITS1F and ITS4 and sequencing of the internal transcribed spacer (ITS) region. The sequence data has been submitted to GenBank, with an accession number KJ466981, and the fungal strain was identified as Penicillium citrinum.

Extraction and Isolation
The fungal cultures were filtered through cheesecloth, and the filtrate was extracted with EtOAc (3 × 20 L, 24 h each). The organic extracts were concentrated in vacuo to yield an oily residue (10.2 g), which was subjected to silica gel column chromatography (CC) (petroleum ether, EtOAc v/v, gradient 100:0-0:100) to generate six fractions (Fr. 1-Fr. 5). Fr  X-ray Crystallographic Analysis of 1. Colorless crystals of 1 were obtained from MeOH. Single-crystal X-ray diffraction data were collected on a Xcalibur, Atlas, Gemini ultra diffractometer with Cu Kα radiation (λ = 1.54180 Å) at 120.01(10) K, respectively. The structure was solved by direct methods (ShelXS) and refined with the ShelXL refinement package using least squares minimization. All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined relatively isotropically with a riding model. Crystallographic data for 1 has been deposited in the Cambridge Crystallographic Data Centre with the deposition number CCDC 1489691, respectively. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK (Fax: +44-(0)1223-336033, or e-mail: deposit@ccdc.cam.ac.uk).
Crystal data for 1:

Biological Assays
Antibacterial activity was determined against five terrestrial pathogenic bacteria, including Staphylococcus epidermidis, S. aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus, and two marine pathogenic bacteria Vibrio parahaemolyticus and V. alginolyticus by the microtitre plate assay method [19]. Ciprofloxacin was used as the positive control. Cytotoxic activities of all compounds against HeLa, MCF-7 and A549 cell lines were evaluated by the MTT method [20]. Epirubicin was used as a positive control.

Conclusions
Seven secondary metabolites, including three new dihydroisocoumarin penicimarins G-I (1-3), together with one known dihydroisocoumarin (4) and three known meroterpenoids (5-7), were obtained from a fungus P. citrinum isolated from the mangrove B. sexangula var. rhynchopetala collected in the South China Sea. Their structures were elucidated by the detailed analysis of spectroscopic data. The absolute configuration of 1 was determined by the X-ray diffraction analysis using Cu Kα radiation. The absolute configurations of 2-3 were determined by comparison of their CD spectra with literature. All compounds were evaluated for their antibacterial activities and cytotoxic activities.   Figure S14: 13C NMR (100 MHz, CDCl3) spectrum of Compound 3, Figure S15: HMQC (CDCl3) spectrum of Compound 3, Figure S16: 1H-1H COSY (CDCl3) spectrum of Compound 3, Figure S17: HMBC (CDCl3) spectrum of Compound 3, Figure S18: HRESIMS spectrum of Compound 3.