New Isocoumarin and Pyrone Derivatives from the Chinese Mangrove Plant Rhizophora mangle-Associated Fungus Phomopsis sp. DHS-11

Mangrove-associated fungi are important sources for the discovery of new bioactive natural products. Three new isocoumarins (1–3) and one new pyrone derivative (4) were isolated from the ethyl acetate extract of the fermentation broth of the mangrove endophytic fungus Phomopsis sp. DHS-11. Nuclear magnetic resonance (NMR) spectroscopy (one-dimensional and two-dimensional) and mass spectrometry were used to determine the structures of these new compounds. The absolute configurations for the new isocoumarins 1–3 were determined by comparing their experimental and calculated electronic circular dichroism (ECD) spectra, while the configuration for the new pyrone-derivative 4 was tentatively solved by comparison of its 13C NMR data with reported data. In the biological activity test, compounds 1 and 3 showed cytotoxic activity against HeLa cells with IC50 values of 11.49 ± 1.64 µM and 8.70 ± 0.94 µM, respectively. The initial structure and activity relationship (SAR) analysis revealed that the length of the side chain at C-3 for isocoumarin-type compounds 1–3 could affect the cytotoxicity against HeLa cells. Compound 4 exhibited cytotoxic activities against human hepatoma cells HepG2 with an IC50 value of 34.10 ± 2.92 µM. All compounds have no immunosuppressive activity.


Introduction
Marine-derived fungi are important sources for the discovery of natural products with unique structures and significant pharmacological activities [1][2][3][4]. Mangrove plants grow in saline-alkali habitats at the junction of tropical and subtropical climates, land and ocean, and endophytic fungi inhabiting them are the second-largest ecological group among marine fungi [5][6][7][8]. Endophytes are a group of parasitic species living in the tissue of plants without causing any obvious pathogenic symptoms [9]. Mangrove endophytic fungi form prolific metabolic pathways and adaptive mechanisms in unique environments, which produces a large number of natural secondary metabolites with novel structures and biological significance, such as terpenes, polyketides, alkaloids, etc., attracting extensive attention in drug mining and agrochemical applications [10][11][12][13]. Phomopsis sp. is a ubiquitous fungus and research on the secondary metabolites of this genus from a chemical point of view has shown that a variety of biologically active products have been found [14,15], such as lung cancer prevention potential drug cytochalasin, antibacterial chromones, antifungal lactones, and cytotoxic sesquiterpenes, etc., [16][17][18][19][20].
Previously, our research group has found many bioactive secondary metabolites from mangrove-plant-derived endophytic fungi from the Dong Zhai Gang mangrove garden in Hainan [21][22][23]. Recently, the fungus Phomopsis sp. DHS-11 was isolated from the root of freshly harvested mangrove plant, Rhizophora mangle. Preliminary phytochemical investigation on the fermentation products of the mangrove endophyte Phomopsis sp. DHS-11 led to the discovery of norpestaphthalides and a cerebroside-type natural compound, alternaroside B; however, biological tests showed that none of them had cytotoxic or immunosuppressive activities [24]. As part of our ongoing exploration of new and bioactive natural product targets in endophytic fungi of Hainan mangrove plants, one new pyronetype and three new isocoumarin-type polyketides were isolated from the fermentation broth of Phomopsis sp. DHS-11, including phomoisocoumarin E (1), phomodihydroisocoumarin A (2), phomoisocoumarin F (3), and phomopyrone D (4) (Figure 1). Herein, we report the isolation of these compounds, the elucidation of their structures, and their biological activities.
Previously, our research group has found many bioactive secondary met from mangrove-plant-derived endophytic fungi from the Dong Zhai Gang ma garden in Hainan [21][22][23]. Recently, the fungus Phomopsis sp. DHS-11 was isolate the root of freshly harvested mangrove plant, Rhizophora mangle. Prel phytochemical investigation on the fermentation products of the mangrove end Phomopsis sp. DHS-11 led to the discovery of norpestaphthalides and a cerebrosi natural compound, alternaroside B; however, biological tests showed that none had cytotoxic or immunosuppressive activities [24]. As part of our ongoing explor new and bioactive natural product targets in endophytic fungi of Hainan mangrov one new pyrone-type and three new isocoumarin-type polyketides were isolate the fermentation broth of Phomopsis sp. DHS-11, including phomoisocoumari phomodihydroisocoumarin A (2), phomoisocoumarin F (3), and phomopyron ( Figure 1). Herein, we report the isolation of these compounds, the elucidation structures, and their biological activities.

Structure Elucidation of the New Compounds
Compound 1 was isolated as an amorphous powder. Its molecular form C13H14O5 was determined by high-resolution electrospray ionization mass spect (HRESIMS) [m/z 249.0774 [M − H] − (calcd for C13H13O5, 249.0768], possessing seven of unsaturation. According to the 1 H NMR ( Figure S1) data of compound 1 (Table 1 were three aromatic proton signals at δH 6.25 (1H, s, H-4), 6.31 (1H, s, H-5), and 6 s, H-7). In addition, two methylene proton signals at δH 2.45 (1H, dd, J = 14.2, 7.1 9a) and 2.41 (1H, dd, J = 14.2, 5.6 Hz, H-9b), an oxygenated methine proton signa (1H, m, H-10), a methyl proton signal at δH 1.12 (3H, d, J = 6.2 Hz, H-11), a methoxy signal at δH 3.80 (3H, s, H-12), and a hydroxyl group signal at δH 4.78 (1H, brs, were present. The 13 C NMR ( Figure S2) and DEPT135 ( Figure S3) data ( Table 2) that compound 1 contained thirteen carbons, including an ester carbonyl carbo aromatic or olefinic carbons (three of which are protonated), a methylene car oxygenated methine carbon, and two methyl carbons. The data for compound similar to those of pestalotiorin, which were reported in the literature [25]. Th difference was the absence of a methyl group at C-7 in compound 1. The 1 H-1 H   Figure S1) data of compound 1 (Table 1), there were three aromatic proton signals at δ H 6.25 (1H, s, H-4), 6.31 (1H, s, H-5), and 6.40 (1H, s, H-7). In addition, two methylene proton signals at δ H 2.45 (1H, dd, J = 14.2, 7.1 Hz, H-9a) and 2.41 (1H, dd, J = 14.2, 5.6 Hz, H-9b), an oxygenated methine proton signal at 3.96 (1H, m, H-10), a methyl proton signal at δ H 1.12 (3H, d, J = 6.2 Hz, H-11), a methoxyl proton signal at δ H 3.80 (3H, s, H-12), and a hydroxyl group signal at δ H 4.78 (1H, brs, OH-10) were present. The 13 C NMR ( Figure S2) and DEPT135 ( Figure S3) data ( Table 2) showed that compound 1 contained thirteen carbons, including an ester carbonyl carbon, eight aromatic or olefinic carbons (three of which are protonated), a methylene carbon, an oxygenated methine carbon, and two methyl carbons. The data for compound 1 were similar to those of pestalotiorin, which were reported in the literature [25]. The major difference was the absence of a methyl group at C-7 in compound 1. The 1 H-1 H COSY correlations from H-10 to H 2 -9 and H 3 -11 and key HMBC correlations from H-4 to C-5, C-4a, C-8a, C-3, and C-9, from H-7 to C-1, C-5, C-6, C-8, and C-8a, and from H 3 -12 to C-8 were observed. Additionally, the methoxyl protons (H 3 -12) only showed NOE correlation with H-7, permitting the location of the methoxyl group at C-8. The overall analysis of the HSQC, HMBC, NOESY, and MS spectra ( Figure 2 and Figures S4-S8) led to the full assignment of the structure as shown in Figure 1. We tried to use the modified Mosher's method to determine the absolute configuration of C-10 in compound 1 but without success. Thus, the absolute configuration of C-10 was tentatively determined to be 10S based on its specific rotation and calculated ECD spectrum ( Figure 3). Therefore, the structure of compound 1 was identified and named phomoisocoumarin E.  with H-7, permitting the location of the methoxyl group at C-8. The overall analysis of the HSQC, HMBC, NOESY, and MS spectra (Figures 2 and S4-S8) led to the full assignment of the structure as shown in Figure 1. We tried to use the modified Mosher's method to determine the absolute configuration of C-10 in compound 1 but without success. Thus, the absolute configuration of C-10 was tentatively determined to be 10S based on its specific rotation and calculated ECD spectrum ( Figure 3). Therefore, the structure of compound 1 was identified and named phomoisocoumarin E.      (Table 2 and Figure S10), DEPT135 (Figure S11), and HSQC ( Figure S12) NMR data revealed the presence of a methyl group, a methylene group, three oxygenated methine groups, eight aromatic or olefinic carbons (four of which are protonated), and an ester carbonyl group. Additionally, three hydroxyl proton signals at δ H 11.09 (1H, s, 8-OH), 4.89 (1H, s, 11-OH or 12-OH), and 4.52 (1H, s, 11-OH or 12-OH) were observed. The 1 H-1 H COSY (Figure 2 and Figure S14) spectrum showed correlations from H-12 to H 3 -13 and H-11, from H-10 to H-9 and H-11, from H-9 to H-3 and H-10, and from H-3 to H-4 and H-9 ( Figure 2). The HMBC spectra ( Figure S13) correlations of H-9 with C-3 and C-4, H-7 with C-1, C-5, C-6, C-8, and C-8a, H-5 with C-4, C-7, and C-8a, and H 2 -4 with C-3, C-4a, C-5, C-8a, and C-9 revealed an isocoumarin ring system ( Figure 2). Comprehensive NMR analysis of HSQC, HMBC, NOESY, and MS data allowed the assignment of structure as shown in Figure 2. The E configuration of the olefinic bond C9-C10 was deduced by the large coupling constant (J H-9 / H-10 = 15.6 Hz). The absolute configuration at C-3, C-11, and C-12 was determined to be 3S, 11R, and 12R by comparing the calculated ECD spectrum with the experimental ECD spectrum (Figure 3). Therefore, the structure of compound 2 was determined and named phomodihydroisocoumarin A.

Structure Elucidation of the New Compounds
Compound 3 was isolated as a viscous oil. It has a molecular formula of C 12 H 12 O 6 as determined by HRESIMS at m/z 275.0520 (calculated for C 12 H 12 NaO 6 , 275.0526 [M + Na] + ) ( Figure S24) and NMR spectrum ( Figures S17-S23). Analysis of the 1 H NMR data (Table 1) revealed one methyl group signal at δ H 1.12 (1H, d, J = 6.2 Hz, H 3 -11), three aromatic proton signals at δ H 6.59 (1H, s, H-4), 6.42 (1H, d, J = 1.9 Hz, H-5) and 6.33 (1H, d, J = 1.9 Hz, H-7), two oxygenated methine proton signals at δ H 3.98 (1H, d, J = 6.5 Hz, H-9) and 3.80 (1H, qui, J = 6.3 Hz, H-10), and three hydroxyl proton signals at δ H 11.00, 5.65, and 4.78. The 13 C NMR combined with DEPT135 spectra (Table 2) showed a total of 12 carbon signals, including one methyl group, two oxygenated methine groups, eight aromatic or olefinic carbons (three of which are protonated), and one ester carbonyl group. The 1 H and 13 C NMR data of 3 were very similar to those of compound 1. The distinction was attributed to the replacement of the methoxyl group at C-8 by a new phenolic hydroxyl group and the presence of a hydroxyl group at C-9 in 3. The HMBC, 1 H-1 H COSY, and NOESY experiments (Figure 2) confirmed the deduction and allowed the assignment of structure as shown in Figure 1. Based on the analysis of the chemical shifts of C-9 (δ C 74.8) and C-10 (δ C 67.5) and the small coupling constant (J H-11/H-12 = 6.5 Hz), suggesting a cis configuration of C9 and C10 [26,27]. The relative configurations of C9 and C10 were also determined by NOESY correlations from H-4 to H-9 and H-10 ( Figure 2). The absolute configurations of C9 and C10 were established as 9S,10S by comparison of their experimental ECD spectrum with the calculated ECD curves (Figure 3). Thus, compound 3 was identified as a new compound and named phomoisocoumarin F. Compound 4 was obtained as an amorphous powder. Its molecular formula was deduced as C 13 Table 1). The 13 C NMR spectrum ( Figure S26) showed the presence of one methyl carbon signal at δ C 23.5 (C-13) and one methoxyl carbon signal at δ C 57.0 (C-14) ( Table 2). The 1 H-1 H COSY spectrum ( Figure S30 [28]. In the HMBC spectrum, key correlations from H-8 to C-6, from H-7 to C-5 and C-6, from H-5 to C-4 and C-6, and from H-3 to C-2 and C-4 allowed the connection of C-7 with C-6 on the 2-pyrone moiety (Figure 2). The methoxyl group positioned at C-4 of 2-pyrone moiety was secured by the observation of key HMBC correlations of H 3 -14 with C-4 and NOE correlations ( Figure S31) of H 3 -14 with H-3 and H-5. The modified Mosher's method was applied to determine the absolute configuration of C-12 in 4 but without success. The planar structure of 4 was very similar to scirpyrone D, possessing the same stereocenter with one hydroxyl group. Due to almost the same chemical shifts of the chiral carbons (δ C 68.3 for C-12 in 4 and δ C 68.1 for C-4 in scirpyrone D) and opposite optical rotation values [−11.0 (c 0.10, MeOH) for 4 and 2.8 (c 0.60, MeOH) for scirpyrone D] [28], the absolute configuration of C-12 in 4 was determined to be 12S. Thus, the structure of 4 was elucidated as shown in Figure 1 and named phomopyrone D.

Biological Evaluation
The immunosuppressive and cytotoxic activities of compounds 1-4 were screened. For the cytotoxic activity test, all of these compounds (1)(2)(3)(4) were first evaluated at the concentrations of 10 µg/mL. The result showed that only compounds 1, 3, and 4 had activity at this concentration. So the cytotoxic activities of these three compounds (1, 3, and 4) were evaluated in depth. The experimental results indicated that compounds 1 and 3 showed moderate inhibitory activities against human cervical cancer cells HeLa with IC 50 values of 11.49 ± 1.64 µM and 8.70 ± 0.94 µM, respectively, which is less active than the positive drug doxorubicin with IC 50 values of 0.95 ± 0.61 µM. The initial SAR analysis revealed that the length of the side chain at C-3 for isocoumarin-type compounds 1-3 could affect their cytotoxicity against HeLa cells, while the methoxyl modification at C-8 on the isocoumarin ring and the hydroxylation at C-9 on the side chain did not affect the activity. Simultaneously, only the pyrone derivative 4 exhibited significant cytotoxic activities against human hepatoma cells HepG2 with an IC 50 value of 34.10 ± 2.92 µM, comparable with the positive drug 5-fluorouracil with an IC 50 value of 21.69 ± 9.11 µM (Table 3), while all isolated isocoumarins 1-3 did not show activity when tested at the concentration of 10 µg/mL. However, all compounds have no immunosuppressive activity at the concentration of 10 µg/mL. "-" no activity at the concentration of 10 µg/mL; "-" no data.

Fungal Material and Culture Conditions
The endophytic fungus strain Phomopsis sp. DHS-11 was isolated from the living root of the mangrove plant Rhizophora mangle collected in Dong Zhai Gang mangrove garden on Hainan Island, China, in October 2015. It was identified as Phomopsis sp. by ITS gene sequence (GenBank Accession no. MT126606) analysis [29]. This strain was deposited and maintained in the research group of one of the authors, J.X. The strain Phomopsis sp. DHS-11 was cultivated on PDA medium (potato extract 200 g/L, glucose 20 g/L, agar 15 g/L, chloramphenicol 0.1 g/L) at 28 • C for 6 days. Then the agar blocks with mycelia were added into 130 × 1000 mL Erlenmeyer flasks containing 100 g rice and 100 mL of seawater (1000 mL conical flask with 100 mL seawater), then fermented for 35 days.

Extraction and Isolation
After fermentation, the whole fermentation mixtures of Phomopsis sp. DHS-11 were collected and mashed with a glass rod and extracted three times with ethyl acetate at room temperature. Then, the whole organic solvent was concentrated in vacuo to obtain 80 g of crude extract. The crude extract was fully mixed and ground with silica gel (60-80 mesh), then subjected to silica gel (200-300 mesh) CC eluted by gradient elution of

Electronic Circular Dichroism (ECD) Calculation Details
Monte Carlo conformational searches were conducted by means of Spartan's 14 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmannpopulation of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31g level in gas. The theoretical calculations of ECD were carried out in MeOH using time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d, p) level for all conformers of compounds 1-3. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, San Diego, CA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.

Cell Viability Assay
The antitumor activity of all isolated compounds 1-4 was evaluated as previously reported by using the MTT assay method [30]. At first, the antitumor activity of all of these isolated compounds (1)(2)(3)(4) was evaluated at the concentrations of 10 µg/mL. Doxorubicin and 5-fluorouracil were used as positive control for human cervical cancer cells HeLa and human hepatoma cells HepG2, respectively. The prepared concentrations for each of compounds 1, 3, 4, and positive drugs in tests were 10, 5, 2.5, 1, 0.5, 0.1, 0.05, and 0.01 µg/mL, while compound 2 was only evaluated at the concentration of 10 µg/mL as it displayed no activity. All cell lines were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. The IC 50 values of these compounds and positive controls were calculated after 24 h for HeLa cells and 48 h for HepG2 cells.

Immunosuppressive Assay
The immunosuppressive activity testing of compounds 1-4 was conducted with the previously reported CCK-8 assay method [30]. In the test, we set up three parallel trials, using cyclosporin A as the positive control, experiments of concanavalin A (ConA)-induced T cells, and lipopolysaccharide (LPS)-induced B cells. The concentrations of compounds 1-4, and cyclosporin A, ConA, and LPS were 10, 5, and 10 µg/mL, respectively. All mice were donated by the Hainan Medical College.

Conclusions
To summarize, four previously undescribed polyketides, including three new isocoumarins (1-3) and one new pyrone derivative (4), were obtained from the mangrovederived fungus Phomopsis sp. DHS-11. The structures of these isolated compounds were determined by analysis of HRESIMS, 1D-and 2D-NMR, and ECD data. The antitumor activity assay suggested that the new pyrone compound 4 exhibited cytotoxic activities against human hepatoma cells HepG2 with an IC 50 value of 34.10 ± 2.92 µM, comparable with the positive drug 5-fluorouracil, indicating that this new compound has the potential to develop novel anti-hepatoma drugs and deserves further study. In addition, two new isocoumarin-type compounds 1 and 3 showed inhibitory activities against human cervical cancer cells HeLa with IC 50 values of 11.49 ± 1.64 µM and 8.70 ± 0.94 µM, respectively. In general, the results of this study expand the diversity of chemical constituents and biological activities isolated from the secondary metabolites of Phomopsis sp. DHS-11, and may provide new potential molecules for antitumor drug discovery. These results also prove that mangrove-associated fungi are still pools for mining new bioactive natural molecules.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The authors declare that all relevant data supporting the results of this study are available within the article and its Supplementary Materials file, or from the corresponding authors upon request.