Polyketides and Alkaloids from the Marine-Derived Fungus Dichotomomyces cejpii F31-1 and the Antiviral Activity of Scequinadoline A against Dengue Virus

In our continuous chemical investigation on the marine-derived fungus Dichotomomyces cejpii F31-1, two new polyketides dichocetides B-C (1, 2), two new alkaloids dichotomocejs E-F (3, 4), and three known fumiquinozalines: scequinadoline A (5), quinadoline A (6), and scequinadoline E (7) were discovered from the culture broth and the mycelium in the culture medium, by the addition of l-tryptophan and l-phenylalanine. Their chemical structures were established by one dimensional (1D), two dimensional (2D) nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HR-MS) data. Among them, scequinadoline A (5) exhibited significant inhibitory activity against dengue virus serotype 2 production by standard plaque assay, equivalent to the positive control andrographlide. Scequinadoline A (5) possesses the potential for further development as a dengue virus inhibitor.


Introduction
Dengue, the most prevalent mosquito-borne infection, is induced by four genetically similar, however, antigenically distinct serotypes of dengue virus (DENV)-DENV 1-4 [1,2]. DENV is rapidly spreading throughout the tropical and subtropical areas worldwide [3]. It is also an epidemic in provinces in Southern China, such as Guangdong, Fujian, and Jiangxi. Dengue has emerged as a severe public health problem and has imposed a heavy burden on both the infected individuals and the healthcare systems [4]. However, no licensed vaccines or specific antiviral drugs have been successfully developed for this disease [1]. The discovery and development of safe and effective antiviral drugs have therefore become imperative [3]. Natural products from marine microorganisms are chemically antiviral drugs have therefore become imperative [3]. Natural products from marine microorganisms are chemically unique and exhibit a variety of interesting pharmaceutical properties [5]; indeed several compounds with anti-DENV activity have been isolated from the marine environment [6]. Among the compounds isolated from marine organisms, small metabolites of fungal origin represent a very important source for drug discovery [7,8]. Therefore, the search for small molecular dengue virus inhibitors from marine-derived fungi represents an attractive approach to treat the disease.
The fungal strain Dichotomomyces cejpii F31-1 was isolated from the soft coral Lobophytum crassum. Previously, through the addition of amino acids, twenty-eight diverse compounds were obtained from the culture broth [9]. When searching for dengue virus inhibitors, a methanol extract of the mycelium displayed inhibitory activity against DENV 2. By bioactivity-guided isolation and purification, three known fumiquinozalines: scequinadoline A (5), quinadoline A (6), and scequinadoline E (7) have been obtained. Scequinadoline A (5) inhibited DENV 2 production by standard plaque assay, equivalent to the positive control andrographlide [10]. Furthermore, an additional two new polyketides dichocetides B-C (1, 2) and two new alkaloids dichotomocejs E-F (3,4) were discovered from the culture broth ( Figure 1).

Structural Elucidation
Dichotomocej F (4) was isolated as a yellow powder with a molecular formula of C 21 H 20 N 2 O 5 based on the negative HR-ESI-MS [M − H] − peak at m/z 379.1299 (calcd. for 379.1299), implying thirteen degrees of unsaturation. Inspection of the 1D NMR spectra recorded for 4 in CDCl 3 (Table 2 and Figures S40-S43) indicated resonances that could be assigned to seventeen sp 2 carbons and four methoxy groups. Careful analysis of the 1D and 2D NMR spectra of 4 displayed that an AA BB spin system of a para-substituted aromatic ring was present from the key 1 3.88, s). In addition to the four degrees of unsaturation, a further seven sites of unsaturation are attributed to a naphthalene ring built on the HMBC spectrum from H-4 (δ H 7.24, s) to C-3 (δ C 128.4), C-6 (δ C 102.9) and C-10 (δ C 119.1), from H-6 (δ H 6.94, s) to C-7 (δ C 151.7), C-8 (δ C 141.2) and C-10 (δ C 119.1). The HMBC correlations of H-20 (δ H 3.96, s) to C-7 (δ C 151.7), from H-21 (δ H 3.88, s) to C-8 (δ C 141.2) and H-22 (δ H 3.28, s) to C-9 (δ C 149.8) showed that three methoxyl groups were attached to C-7, C-8 and C-9 separately. The existence of an imidazolidin was verified by the chemical signals of two active protons [(δ H 7.34, brs), (δ H 8.58, brs)] and the pivotal HMBC correlations from NH-17 (δ H 7.34, brs) to C-2 (δ C 128.7) and C-18 (δ C 155.9), matching with the remaining two sites of unsaturation ( Figure 2). The strict comparison of the NMR spectra with a synthetic compound named 1,3-Dihydro-5,6,7-trimethoxy-4-(4-methoxyphenyl)-1-methyl-2H-naphth [2,3-d]imidazole-2-one highlighted that the only subtle difference between the known compound and 4 was a methoxyl group in N-19 being replaced by a hydrogen in 4 [11]. Therefore, the chemical structure of 4 was elucidated as shown in Figure 1.

Effect of Compounds 5-7 on DENV Infection
The levels of DENV 2 infection in cells treated with compounds 5-7 were not significantly different from control (DMSO) treated cells, while cells treated with andrographolide showed a significant reduction in DENV 2 infection levels, in agreement with our earlier observation [10]. While quinadoline A (6) and scequinadoline E (7) showed no effect on DENV virus production as assessed by standard plaque assay, a significant reduction in virus output was observed upon treatment with scequinadoline A (5) as compared to control (DMSO) treated cells (Figure 4).  5), quinadoline A (6) and scequinadoline E (7), in parallel with 50 μM of andrographlide and DMSO as a control. After 24 h of incubation, the treated cells were collected for evaluation of the percentage of infection by flow cytometry (A) and the supernatant was collected to determine the viral production by plaque assay (B). All experiments were undertaken as independent triplicates. Data is shown normalized against the control (0.5% DMSO). Error bars represent SD (* p < 0.05, ** p < 0.01, *** p < 0.001).

General Procedures
The Shimadzu UV-Vis-NIR spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) was used for UV spectra analysis. The Fourier transform infrared (FT-IR) spectrophotometer (PerkinElmer Frontier, Waltham, MA, USA) with an Ever-Glomid/near-IR source was applied for IR spectra measurement. 1D and 2D NMR spectra were recorded on the Bruker Avance II 400 and 500 spectrometers (Bruker Bio Spin AG, Industriestrasse 26, Fällanden, Switzerland) in CDCl3, and the chemical shifts are corresponding to the residual solvent signals (CDCl3: δH 7.260 and δC 77.160). The low and high resolution ESI-MS spectra were obtained with Thermo LCQ DECA XP (San

Effect of Compounds 5-7 on DENV Infection
The levels of DENV 2 infection in cells treated with compounds 5-7 were not significantly different from control (DMSO) treated cells, while cells treated with andrographolide showed a significant reduction in DENV 2 infection levels, in agreement with our earlier observation [10]. While quinadoline A (6) and scequinadoline E (7) showed no effect on DENV virus production as assessed by standard plaque assay, a significant reduction in virus output was observed upon treatment with scequinadoline A (5) as compared to control (DMSO) treated cells (Figure 4).

Effect of Compounds 5-7 on DENV Infection
The levels of DENV 2 infection in cells treated with compounds 5-7 were not significantly different from control (DMSO) treated cells, while cells treated with andrographolide showed a significant reduction in DENV 2 infection levels, in agreement with our earlier observation [10]. While quinadoline A (6) and scequinadoline E (7) showed no effect on DENV virus production as assessed by standard plaque assay, a significant reduction in virus output was observed upon treatment with scequinadoline A (5) as compared to control (DMSO) treated cells (Figure 4).  (6) and scequinadoline E (7), in parallel with 50 μM of andrographlide and DMSO as a control. After 24 h of incubation, the treated cells were collected for evaluation of the percentage of infection by flow cytometry (A) and the supernatant was collected to determine the viral production by plaque assay (B). All experiments were undertaken as independent triplicates. Data is shown normalized against the control (0.5% DMSO). Error bars represent SD (* p < 0.05, ** p < 0.01, *** p < 0.001).

General Procedures
The Shimadzu UV-Vis-NIR spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) was used for UV spectra analysis. The Fourier transform infrared (FT-IR) spectrophotometer (PerkinElmer Frontier, Waltham, MA, USA) with an Ever-Glomid/near-IR source was applied for IR spectra measurement. 1D and 2D NMR spectra were recorded on the Bruker Avance II 400 and 500 spectrometers (Bruker Bio Spin AG, Industriestrasse 26, Fällanden, Switzerland) in CDCl3, and the chemical shifts are corresponding to the residual solvent signals (CDCl3: δH 7.260 and δC 77.160). The low and high resolution ESI-MS spectra were obtained with Thermo LCQ DECA XP (San  (6) and scequinadoline E (7), in parallel with 50 µM of andrographlide and DMSO as a control. After 24 h of incubation, the treated cells were collected for evaluation of the percentage of infection by flow cytometry (A) and the supernatant was collected to determine the viral production by plaque assay (B). All experiments were undertaken as independent triplicates. Data is shown normalized against the control (0.5% DMSO). Error bars represent SD (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fungal Material
Dichotomomyces cejpii F31-1, the marine fungus, was isolated from the inner tissue of the soft coral Lobophytum crassum which was acquired from Hainan Sanya National Coral Reef Reserve, P. R. China. The fungal strain was preserved in 15.0% (v/v) glycerol aqueous solution at −80 • C. A voucher specimen was stored in the School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, P. R. China. The ITS rDNA (GenBank EF669956) analysis by BLAST database screening furnished a hundred percent match to Dichotomomyces cejpii.

Culture, Extraction, and Isolation
Fungus strain Dichotomomyces cejpii F31-1 was cultured in the medium (yeast extract 2 g/L, sea salt 30 g/L, glucose 20 g/L, peptone 5 g/L, L-Phe 2 g/L, D, L-Trp 2 g/L, H 2 O 1 L, pH 7.5). Fungal mycelia were cut uniformly and transferred aseptically to 1 L Erlenmeyer flasks with each containing 600 mL sterilized liquid medium. The flasks were incubated for 60 days at 25 • C. Cheesecloth was used for filtration of 90 liters of liquid culture. The culture was extracted three times by using EtOAc and then was concentrated under reduced pressure to provide crude extract (35 g) when the mycelium was extracted three times by using MeOH and then was concentrated under reduced pressure to furnish a crude extract (15 g).

Compounds Preparation
Scequinadoline A (5), quinadoline A (6) and scequinadoline E (7) were dissolved in 100% DMSO to a final concentration of 10 mM, and stored at −80 • C. Compounds were diluted with complete DMEM. Andrographolide (365645, Sigma, St. Louis, MO, USA) was used as a positive control for compound screening. Final concentration of andrographolide at 10 mM was prepared by dissolving with 100% DMSO. Control of experiments contained 0.5% DMSO.

Cytotoxicity Assay
Cytotoxicity of the compounds was initially determined by observation of alterations of cell morphology. HEK293T/17 cells were seeded on six well plates under standard growth condition. Cells were incubated with 1, 10 and 50 µM of scequinadoline A (5), quinadoline A (6) and scequinadoline E (7). At 24 h post treatment, cell morphology was directly observed under inverted light microscope (Nikon EclipseTS100, Nikon Instruments Inc., Melville, NY, USA). The treated cells were compared with individual DMSO concentrations and various concentrations of andrographolide were used as a positive control.

Antiviral Activity against DENV 2
HEK293T/17 cells were cultured in six well plates until they reached approximately 90% confluency. Cells were mock infected or infected with DENV 2 at 5 p.f.u/mL at 37 • C with 5% CO 2 for 2 h. Then virus was removed and the 50 µM of scequinadoline A (5), quinadoline A (6), scequinadoline E (7) and andrographolide added to the cells at 0 h after infection, together with DMSO control. For further experiments, supernatant and cells were collected at 24 h post treatment.
Mock and DENV 2 infected cells from compound treatments were collected at 24 h.p.i and incubated with 100 µL of 10% normal goat serum (NGS; Gibco TM Invitrogen) in PBS/IFA on ice for 30 min. Then, cells were washed with 800 µL of 1% BSA in PBS/IFA followed by fixing with 200 µL of 4% paraformaldehyde in PBS/IFA in dark at room temperature for 20 min. After washing cells twice with 1% BSA in PBS/IFA, cells were permeabilized with 200 µL of 0.2% Triton X in PBS/IFA at room temperature in dark for 10 min. The cell were washed twice with 1% BSA in PBS/IFA and incubated with 50 µL of a mouse monoclonal anti-DENV E protein antibody (HB114) with dilution 1:150 at 4 • C for overnight. Subsequently, cells were washed twice with 1% BSA in PBS/IFA, followed by incubation with 50 µL goat anti-mouse IgG antibody conjugated with FITC (Milipore Corp., Burlington, MA, USA) at room temperature for 1 h in dark at room temperature. Finally, cells were washed twice with 1% BSA in PBS/IFA and analyzed by flow cytometry on BD FACSCalibur cytometer (Becton Dickinson, San Jose, CA, USA) using CELLQuest™ software. All experiments were performed as independent triplicates.
All data were analyzed using GraphPad Prism program (GraphPad Software). Statistical analysis was performed using the PASW statistics 18 (SPSS Inc., Chicago, IL, USA). The percentage of infection Mar. Drugs 2018, 16, 229 9 of 10 and viral production from triplicate value of independent experiments are shown as mean ± S.D. A p value less than 0.05 was considered as statistically significant.

Conclusions
Overall, an amino acid strategy is effective for eliciting the fungus Dichotomomyces cejpii F31-1 to produce alkaloids with chemical diversity. Among the thirty-five compounds produced by the fungus in total, twenty-six compounds are alkaloids. The fungus showed amazing potential for the biosynthesis of alkaloids and other types of natural products. Additionally, scequinadoline A (5) exhibited a significant inhibitory activity against DENV 2 production as assessed by standard plaque assay. The inhibition of virus production but not infection level suggests that this compound inhibits virus formation or virus release from the cell. Scequinadoline A (5) displays the potential for development as a dengue virus inhibitor. Dichocetide C (2) and dichotomocejs E-F (3, 4) were assayed for cytotoxic activity against a macrophage cell line (RAW264.7) and displayed no significant inhibitory effect.