Structurally Diverse Polyketides From the Mangrove-Derived Fungus Diaporthe sp. SCSIO 41011 With Their Anti-influenza A Virus Activities

Influenza A virus (IAV) is a severe worldwide threat to public health and economic development due to its high morbidity and mortality. Marine-derived fungi have been evidenced as a prolific source for the discovery of pharmacologically-active lead compounds. During the course of our search for novel bioactive substances from marine microorganisms, six new polyketides, including two octaketides (1–2), one chromone derivative (13), two highly substituted phthalides (17–18), and one α-pyrone derivative (21) along with 22 known congeners were isolated from a mangrove-associated fungus Diaporthe sp. SCSIO 41011. Their structures were determined by spectroscopic analysis and by comparison with literature data. And the absolute configurations were established according to the specific rotation or electron circular dichroism method. Antiviral evaluation results revealed that compounds 14, 15, 26, and 5-chloroisorotiorin displayed significant anti-IAV activities against three influenza A virus subtypes, including A/Puerto Rico/8/34 H274Y (H1N1), A/FM-1/1/47 (H1N1), and A/Aichi/2/68 (H3N2), with IC50 values in the range of 2.52–39.97 μM. The preliminary structure-activity relationships (SARs) are also discussed. These findings expand the chemical and bioactive diversity of polyketides derived from the genus Diaporthe, and also provide a basis for further development and utilization of chromone, xanthone, and chloroazaphilone derivatives as source of potential anti-viral chemotherapy agents.


INTRODUCTION
Polyketides represent an important category of secondary metabolites with great structural diversity from simple aromatics to highly modified complex architectures, such as macrolides, polyphenols, polyethers, polyenes, and enediynes (Fujii, 2010;Zheng et al., 2015). Distributing broadly in microbial origins, they are constructed by combination of iterative polyketide synthases (PKSs) and multifunctional and iterative oxygenases (Hang et al., 2016). In addition, polyketides play a vital role in modern medicine due to their diverse pharmacological features, such as lovastatin, a wellknown fungal polyketide statins functioned as a cholesterollowering agent (Crawford and Townsend, 2010). Belonging to the family of octaketides, biosynthetically related cytosporones, dothiorelones and phomopsins are characterized with a di-/tri-hydroxybenzene lactone or a resorcinol scaffold harboring an n-heptane substituent, which were mainly encountered in endophytic fungi of several genera, such as Phomopsis (Kornsakulkarn et al., 2015;Kongprapan et al., 2017;Tan et al., 2017), Diaporthe (Brady et al., 2000;Liu et al., 2018), Cytospora (Brady et al., 2000;Abreu et al., 2010), Pestalotiopsis (Xu et al., 2009b). Of special note, cytosporone B (7) was reported as a nuclear orphan receptor Nur77 agonist as a promising therapeutic drug for cancers and hypoglycemia (Zhan et al., 2008), as well as the transcription factor NR4A1 agonist to control IAV infection and improve pulmonary function in infected mice (Xia et al., 2013;Egarnes et al., 2017), which had aroused a great interest for chemical synthesis study (Von Delius et al., 2017).
Influenza A virus (IAV), a negative sense RNA virus, is one of the main causes of human acute respiratory diseases characterized with high morbidity and mortality, posing a serious threat to public health and economic development . IAVs repeatedly circulate in many animal hosts, such as humans, birds, horses, dogs, and pigs, which can be subtyped to two envelope proteins: haemagglutinin (HA) and neuraminidase (NA) glycoproteins according to the antigenic properties (Medina and Garcia-Sastre, 2011). In 2009, the pandemic influenza H1N1 virus rapidly spread to 214 countries around the world, causing human infection and acute respiratory illness of more than 17,700 deaths (Bautista et al., 2010). As of 25th April 2018, there have been reported 1625 confirmed cases of human H7N9 infection and 623 deaths since 2013 according to the World Health Organization (http://www.fao. org/ag/againfo/programmes/en/empres/H7N9/situation_update.html). However, two families of antiviral drugs are hitherto currently used to treat human IAV infections, which are NA inhibitors, like zanamivir and oseltamivir, and inhibitors of the viral M2 protein exemplified by amantadine and rimantadine (Medina and Garcia-Sastre, 2011;Song et al., 2015). Due to the emergence of drug-resistant viral strains, there is an urgent development for novel classes of anti-IAV agents with new mode of action.
Marine-derived fungi are reported as a prodigious source of development for new antivirals against different important viruses (Moghadamtousi et al., 2015). In our continuing endeavor to discover biologically active compounds from marine microbes, a series of structurally interesting and biologically active natural products have been described (Luo et al., 2017;Tan et al., 2018). Recently, six new cytotoxic chloroazaphilone derivatives, isochromophilones A-F, have been isolated from the fungus Diaporthe sp. SCSIO 41011, an endophytic fungus obtained from the fresh tissue of the marine mangrove plant Rhizophora stylosa . Subsequent chemical investigations on the remaining fractions of the fungus led to the isolation of structurally diverse aromatic polyketides, including octaketides (dothiorelones or cytosporones), phthalides, chromones, xanthones, etc. (Figure 1). The structures of these compounds were determined by physicochemical properties and spectral data analysis as well as comparison with those reported in the literature. All the compounds were examined for anti-IAV activities against three influenza A virus subtypes, including A/Puerto Rico/8/34 H274Y (H1N1), A/FM-1/1/47 (H1N1), and A/Aichi/2/68 (H3N2). Details of the isolation, structure elucidation, and biological activity of these compounds, as well as preliminary SARs, are reported herein.

General Experimental Procedures
Semi-preparative HPLC was performed on a Hitachi Primaide apparatus using an ODS (octadecylsilanized silica) column (YMC-pack ODS-A, YMC Co. Ltd., 10 × 250 mm, 5 µm, 2.5 mL/min). Chiral HPLC separation was performed using CHIRALPAK IC column (250 × 4.6 mm, 5 µm). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10-40 µm) and over silica gel (200-300 mesh) (Qingdao Marine Chemical Factory), and Sephadex LH-20 (Amersham Biosciences), respectively. Spots were detected on TLC (Qingdao Marine Chemical Factory) under 254 nm UV light. All solvents employed were of analytical grade (Tianjin Fuyu Chemical and Industry Factory). The NMR spectra were obtained on a Bruker Avance spectrometer (Bruker) operating at 500 and 700 MHz for 1 HNMR, 125 MHz, and 175 MHz for 13 CNMR, using TMS as an internal standard. Optical rotations were acquired using a Perkin Elmer MPC 500 (Waltham) polarimeter. HRESIMS and ESIMS spectra data were recorded on a MaXis quadrupole-time-offlight mass spectrometer and an amaZon SL ion trap mass spectrometer (Bruker), respectively. UV spectra were recorded on a Shimadzu UV-2600 PC spectrometer (Shimadzu). ECD spectra were performed on a Chirascan circular dichroism spectrometer (Applied Photophysics). IR spectra were measured on an IR Affinity-1 spectrometer (Shimadzu). The artificial sea salt was a commercial product (Guangzhou Haili Aquarium Technology Company).

Fungal Material
The fungus Diaporthe sp. SCSIO 41011 had the same origination as that in our recent published paper . A voucher specimen was deposited in the CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.

Antiviral Activity
All the isolated compounds (1-28), along with recently reported co-isolated 5-chloroisorotiorin , were screened for their anti-IAV activities according to the previously reported 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, using ribavirin as a positive control (Li et al., 2017;Yang et al., 2018). In brief, Madin Derby canine kidney (MDCK) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Meanwhile, different influenza A virus subtypes, including A/Puerto Rico/8/34 H274Y (H1N1), A/FM-1/1/47 (H1N1), and A/Aichi/2/68 (H3N2), were multiplied in 10-day-old chick embryo at 37 • C. The cytotoxicity of the compounds was also evaluated by the MTT assay. Briefly, approximately 90% confluent cells in 96-well plates were exposed to the compounds at 2-fold serial dilutions. After 48 h of incubation, 100 µL of MTT solution, which was diluted by the medium to 0.5 mg/mL, was added and retained at 37 • C for 4 h. Then the supernatant was removed, followed by the addition of 150 µL of dimethyl sulfoxide (DMSO) to dissolve the formazan product. The optical density for each well was measured on the Tecan Genios Pro microplate reader (Bedford, MA, USA) at 570 nm. To determine the antiviral activities of the compounds, confluent MDCK cells were infected with the virus at multiplicity of infection (MOI) of 0.01 at 37 • C for 1 h. The compounds of non-cytotoxic concentrations were then added to the cells after washing away the unabsorbed virus with phosphate-buffered saline (PBS), and the cells were cultured for another 48 h. At the end of the culture, the MTT-based assay as previously described was assessed for the antiviral activity of the isolated compounds.

Statistical Analysis
All statistical analysis of the data were processed by GraphPad Prism. The results are presented as the mean ± standard deviation (SD) from experiments in triplicate. Student's t-test was used to analyze the statistical significance between two groups, more groups by one-way ANOVA with or without Tukey-Kramer multiple comparison. A p < 0.05 was regarded as statistically significant.

Identification of Compounds
The endophytic fungus Diaporthe sp. SCSIO 41011 was cultured on solid rice medium for 60 days. The EtOAc extract (150 g) of the fermentation was separated by continuously silica gel chromatography and semi-preparative HPLC chromatography to yield 28 aromatic polyketides (1-28). Their chemical structures were determined by comprehensive spectroscopic analyses or comparison with those reported data.
Compound 13 was also isolated as colorless oil and gave a molecular formula of C 18 H 22 O 5 relied on a deprotonated ion peak at m/z 317.1403 (calcd for C 18 H 21 O 5 , 317.1389) in the HRESIMS spectrum. The 1 H NMR data (Table 1)   and two methyls (δ C 14.2 and 14.5). The above mentioned spectral characteristics were closely consistent to those of the coisolated pestalotiopsone F (14) (Xu et al., 2009a), but suggested the appearance of an ethyl group (δ H/C 4.13/61.7 at C-1 ′ and 1.24/14.5 at C-2 ′ ) in 13 rather than a methyl group (δ H/C 3.66/52.2) at C-1 ′ in 14. These changes were also ascertained by the HMBC correlations from H 3 -1 ′ to C-1 and from H 3 -2 ′ to C-1 ′ , as well as the 1 H-1 H COSY correlation of H 3 -2 ′ and H 2 -1 ′ (Figure 2). Based on the above discussion, the structure of 13 was elucidated and the trivial name pestalotiopsone H was assigned.
Compound 17 was obtained as a white solid and its molecular formula was found to be C 14 H 18 O 7 on the basis of HRESIMS and NMR data. The 1 H NMR data ( Table 2) of 17 revealed four Omethyls at δ H 3.38, 3.38, 3.54, and 3.99, two singlet methylenes at δ H 4.60 and 4.65, and a hemiketal methine at δ H 6.29. In addition to the above seven corresponding hydrogen-bearing carbons, seven carbons remained in the 13 C NMR spectrum, including one carbonyl (δ C 169.6), six aromatic carbons [(δ C 111.4, 119.0, 121.4, 148.9), and two oxygenated ones at δ C 160.7 and 174.3]. The foregoing spectroscopic data showed great similarity to that of microsphaerophthalide F (Sommart et al., 2012) except that a methyl group (δ H/C 2.16/8.5) located at C-6 in microsphaerophthalide F was replaced by an ethoxyl group (δ H/C 4.60/65.3 at C-9 and 3.38/58.1 at C-10) anchored at C-6 in 17. This deduction was also supported by the HMBC correlations from H 3 -10 to C-9 and from H 2 -9 to C-5 and C-7. The barely measurable optical rotation value and quite weak Cotton effects in the ECD spectrum suggested 17 was racemic, which was also confirmed by the chiral HPLC analysis with two peaks (peak area ratio: 1:1) in the HPLC profile (Supplementary Materials). However, the quantity of 17 was too little to perform further resolution. Hence, the structure of compound 17 was elucidated and the trivial name (±)-microsphaerophthalide H was assigned.
Compound 18 was obtained as colorless needle crystals and had the molecular formula C 13 H 14 O 7 , as evidenced by HRESIMS ([M+H] + , 283.0809; calcd for C 13 H 15 O 7 , 283.0818) and the NMR data. The highly similar NMR spectroscopic data of 18 to those of 17 indicated that their structures were closely related, except for the presence of an aldehyde group (δ H/C 10.14/192.5) joined at C-4 in 18, rather than an ethoxyl group (δ H/C 4.65/67.2 and 3.38/58.4) anchored at C-4 in 17. The absolute configuration of C-3 of 18 was mainly determined by comparison of the specific rotations with those reported data, as well as comparison between  the computed and experimental ECD spectra. Among these reported 3-oxygenated phthalides, the 3S-and 3R-ones generally showed negative and positive specific rotations, respectively (Sommart et al., 2012). Thus, the positive sign of specific rotation [[α] 25 D = +25 (c 0.10, MeOH)] of 18 led to the deduction of 3R configuration in 18, which was also confirmed from experimental and calculated ECD spectra of 18 as shown in Figure 3. Further chiral HPLC analysis confirmed that 18 was single enantiomer. Consequently, the structure of 18 was determined as shown in Figure 1 and termed microsphaerophthalide I. Notably, the 3-oxygenated phthalides are uncommon in natural sources.
Compound 21 was obtained as a white solid and had the molecular formula C 12 H 14 O 5 , as determined from HRESIMS the NMR data. Detailed analyses of its NMR spectroscopic features implied that it was closely related structurally to convolvulopyrone (Tsantrizos et al., 1992), but for the presence of an additional O-methyl group (δ H/C 3.76/52.0) at C-10 in 21, indicating that 21 was a methyl derivative of convolvulopyrone. Besides, compound 21 and convolvulopyrone shared the same relative configuration of 7E due to the absence of a NOESY correlation between H-7 and H 3 -11. As a result, the structure of 21 was determined and given the trivial name methyl convolvulopyrone. However, compound 21 was likely obtained as an artifact formed in the process of extraction and purification using MeOH as a solvent.

AUTHOR CONTRIBUTIONS
XiaL: designed the experiments and performed the isolation and characterization of all the compounds and wrote the manuscript; JY and FC: performed the antiviral experiment; XiuL: performed the isolation and purification of the fungal strain; CC: contributed to isolation of the compounds; XZ: designed the research work and wrote the manuscript; SL and YL: contributed in project design and manuscript preparation. All authors reviewed the manuscript and approved for submission.