Broad-Spectrum Antiviral Natural Products from the Marine-Derived Penicillium sp. IMB17-046

A new pyrazine derivative, trypilepyrazinol (1), a new α-pyrone polyketide, (+)-neocitreoviridin (2), and a new ergostane analogue, 3β-hydroxyergosta-8,14,24(28)-trien-7-one (3), were isolated and characterized along with five known compounds from the marine-derived fungus Penicillium sp. IMB17-046. The structures of these new compounds were determined using spectroscopic data analyses (HRESIMS, 1D- and 2D-NMR), X-ray crystallography analysis, and TDDFT ECD calculation. Compounds 1 and 3 exhibited broad-spectrum antiviral activities against different types of viruses, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza A virus (IAV), with IC50 values ranging from 0.5 to 7.7 μM. Compounds 1 and 2 showed antibacterial activities against Helicobacter pylori, a causative pathogen of various gastric diseases, with minimum inhibitory concentration (MIC) values of 1–16 μg/mL.


Introduction
Viruses such as the human immunodeficiency virus (HIV), influenza virus, and hepatitis B and C viruses (HBV and HCV) are great global threats to public health. According to the recent World Health Organization (WHO) reports, 36.7 million people were living with HIV in 2015, among which approximately 2.7 million and 2.3 million had a chronic HBV and HCV coinfection, respectively [1,2]. It is estimated that hepatitis viruses caused 1.34 million deaths in 2015 while influenza viruses are responsible for 250,000 to 500,000 death globally every year. Despite the great achievements made in antiviral drug discovery during the past 50 years, there is still no effective antiviral drug for over 200 infectious diseases [2]. The increasing emergence of drug-resistant viral strains urgently requires the development of new antiviral drugs with novel action mechanisms. Particularly, broad-spectrum antiviral drugs are needed to combat multiple viral infectious diseases since most of the current antiviral drugs are effective to only certain viral strains [2].
Marine fungi are prolific sources of new structurally diverse compounds, and have yielded more than 1000 new metabolites, many of them displaying interesting biological activities, such as antibacterial, antifungal, antiviral, cytotoxic, and antiprotozoal properties [3,4]. During our ongoing program screening for new bioactive natural products from marine-derived microorganisms [5][6][7][8][9], the extracts from the solid cultures of the marine-derived strain Penicillium sp. IMB17-046 were shown to

Results and Discussion
Compound 1 was isolated as a colorless plate crystal. Its molecular formula was determined to be C 18 H 21 N 3 O 2 by HRESIMS, indicating 10 degrees of unsaturation. The IR spectrum showed the presence of the hydroxy or amino (3421 cm −1 ) and the aromatic ring (1615 cm −1 ) groups. The 1 H-NMR spectrum in CDCl 3 (Table 1) revealed three methyl signals comprising a methoxy (δ H 3.98) and two aliphatic methyls (δ H 1.20, d; 0.87, t), two methylenes (δ H 4.13/4.10; 1.81/1.55), a methine (δ H 3.15), four protons between δ H 7.09 and 7.68 ascribed to an ortho-disubstituted aromatic ring, an isolated olefinic proton (δ H 7.22), as well as an exchangeable proton (δ H 8.04, H-1 ). The 13 C-NMR and DEPT spectra showed 18 carbon signals, including three methyls, two methylenes, five sp 2 one aliphatic methine, and seven non-protonated sp 2 carbons. The COSY correlations of H-4 /H-5 /H-6 /H-7 and H 3 -4 /H-1 /H-2 a/H-2 b/H 3 -3 confirmed the presence of the ortho-disubstituted aromatic ring and a sec-butyl fragment (Figure 2a). The HMBC cross-peaks of H 2 -8 with C-2 , C-3 , and C-3a ; NH-1 with C-3 , C-7 , and C-7a ; and H-4 with C-3 and C-3a suggested that the aromatic ring was present as an (indole-3-yl)methyl unit. In the 13 C-NMR spectrum, four unassigned quaternary carbons resonated at δ C 153.3, 149.8, 148.7, and 128.7. In addition, the remaining atoms required by the molecular formula included a hydrogen, an oxygen, and two nitrogens. By taking into consideration the unsaturation requirement, these data suggested the presence of a pyrazine ring in the molecule [15,16]. The HMBC correlations of H-1 with the carbons at δ C 153.3 (C-2) and 149.8 (C-3) along with the correlation of H-4 with the carbon at δ C 149.8 (C-3) located the sec-butyl at C-3 of the pyrazine ring. The HMBC correlations of H 2 -8 with the carbons at δ C 128.7 and δ C 148.7 along with the correlation of the methoxy protons (δ H 3.98) with the carbon at δ C 148.7 suggested that the (indole-3-yl)methyl unit and the methoxy group were attached to the carbons at δ C 128.7 and δ C 148.7, respectively. However, these correlations did not allow for distinction of 2,5-versus 2,6-dioxy substitutions and thus could not define the location of the methoxy group (C-5 or C-6). Furthermore, it was not possible to determine whether the C-2 of the pyrazine ring was enolized based on the NMR data [15,16]. Therefore, compound 1 was subjected to X-ray crystallographic analysis using Cu Kα radiation (Figure 2b), which unambiguously established the structure as 1 with an enol group at C-2. The small values of the Flack parameter 0.02 (8) [17] and the Hooft parameter 0.04 (7) [18] in the final refinement of the crystallographic data allowed for assignment of the absolute configuration of 1 as 1 S. Therefore, the structure of 1 was determined as (S)-6-((1H-indol-3-yl)methyl)-3-(sec-butyl)-5-methoxypyrazin-2-ol. According to the proposed biosynthetic pathway for natural pyrazines [19,20], compound 1 was probably derived from the precursors tryptophan and isoleucine and was thereby assigned the trivial name trypilepyrazinol. which unambiguously established the structure as 1 with an enol group at C-2. The small values of the Flack parameter 0.02 (8) [17] and the Hooft parameter 0.04 (7) [18] in the final refinement of the crystallographic data allowed for assignment of the absolute configuration of 1 as 1′′S. Therefore, the structure of 1 was determined as (S)-6-((1H-indol-3-yl)methyl)-3-(sec-butyl)-5-methoxypyrazin-2-ol. According to the proposed biosynthetic pathway for natural pyrazines [19,20], compound 1 was probably derived from the precursors tryptophan and isoleucine and was thereby assigned the trivial name trypilepyrazinol.    Compound 2 was obtained as yellow needles with a molecular formula C 23 H 30 O 6 as suggested by HRESIMS. The 1 H-NMR spectrum of 2 displayed the signals for six coupled olefinic protons at δ H 6.35-7.18, two isolated olefinic proton singlets at δ H 5.51 (H-2) and 5.75 (H-13), two oxygenated methine protons at δ H 4.02 (s, H-15) and 3,84 (q, H-17), five methyls at δ H 1.20-1.96, and a methoxy group at δ H 3.83 (Table 1). The 13 C-NMR and DEPT spectra in CDCl 3 showed the presence of one ester carbonyl, four nonprotonated sp 2 , and two nonprotonated oxygen-bearing sp 3 carbons in addition to the methyl and methine signals mentioned above. The molecular formula, UV, and NMR data of 2 were similar to those of citreoviridin obtained from Aspergillus terreus [21] and other Penicillium strains (Table S1) [11,22,23]. Interestingly, compound 2 has a positive optical rotation ([α] 20 D +67.3 in MeOH), opposite to that of citreoviridin ([α] 20 D −105 in MeOH and −107.8 in CHCl 3 ) [24,25]. In addition, the resonances for H-11 and C-21 in 2 were deshielded by ∆δ H +0.77 and ∆δ C +9.2 ppm, respectively, in comparison with those of citreoviridin recorded in CDCl 3 , whereas H-13, H 3 -21, C-11, and C-13 in 2 were shielded by ∆δ H −0.16 and −0.08 and ∆δ C −7.5 and −2.3 ppm, respectively, indicating that compound 2 was the ∆ 12 (13)  In addition, the relative configuration of the tetrahydrofuran ring was determined to be identical with that of citreoviridin by ROESY correlations. The calculated ECD spectrum for 14S,15R,16R,17R-2 matched well with the experimental curve, indicating that 2 possessed the identical absolute configuration with citreoviridin ( Figure 4). Consequently, the structure of 2 was determined as 12Z-citreoviridin and named (+)-neocitreoviridin. Compound 2 was obtained as yellow needles with a molecular formula C23H30O6 as suggested by HRESIMS. The 1 H-NMR spectrum of 2 displayed the signals for six coupled olefinic protons at δH 6.35-7.18, two isolated olefinic proton singlets at δH 5.51 (H-2) and 5.75 (H-13), two oxygenated methine protons at δH 4.02 (s, H-15) and 3,84 (q, H-17), five methyls at δH 1.20-1.96, and a methoxy group at δH 3.83 (Table 1). The 13 C-NMR and DEPT spectra in CDCl3 showed the presence of one ester carbonyl, four nonprotonated sp 2 , and two nonprotonated oxygen-bearing sp 3 carbons in addition to the methyl and methine signals mentioned above. The molecular formula, UV, and NMR data of 2 were similar to those of citreoviridin obtained from Aspergillus terreus [21] and other Penicillium strains (Table S1) [11,22,23]. Interestingly, compound 2 has a positive optical rotation ([α] 20 D +67.3 in MeOH), opposite to that of citreoviridin ([α] 20 D −105 in MeOH and −107.8 in CHCl3) [24,25]. In addition, the resonances for H-11 and C-21 in 2 were deshielded by ΔδH +0.77 and ΔδC +9.2 ppm, respectively, in comparison with those of citreoviridin recorded in CDCl3, whereas H-13, H3-21, C-11, and C-13 in 2 were shielded by ΔδH −0.16 and −0.08 and ΔδC −7.5 and −2.3 ppm, respectively, indicating that compound 2 was the Δ 12 (13)  In addition, the relative configuration of the tetrahydrofuran ring was determined to be identical with that of citreoviridin by ROESY correlations. The calculated ECD spectrum for 14S,15R,16R,17R-2 matched well with the experimental curve, indicating that 2 possessed the identical absolute configuration with citreoviridin ( Figure 4). Consequently, the structure of 2 was determined as 12Z-citreoviridin and named (+)-neocitreoviridin.   Compound 2 was obtained as yellow needles with a molecular formula C23H30O6 as suggested by HRESIMS. The 1 H-NMR spectrum of 2 displayed the signals for six coupled olefinic protons at δH 6.35-7.18, two isolated olefinic proton singlets at δH 5.51 (H-2) and 5.75 (H-13), two oxygenated methine protons at δH 4.02 (s, H-15) and 3,84 (q, H-17), five methyls at δH 1.20-1.96, and a methoxy group at δH 3.83 (Table 1). The 13 C-NMR and DEPT spectra in CDCl3 showed the presence of one ester carbonyl, four nonprotonated sp 2 , and two nonprotonated oxygen-bearing sp 3 carbons in addition to the methyl and methine signals mentioned above. The molecular formula, UV, and NMR data of 2 were similar to those of citreoviridin obtained from Aspergillus terreus [21] and other Penicillium strains (Table S1) [11,22,23]. Interestingly, compound 2 has a positive optical rotation ([α] 20 D +67.3 in MeOH), opposite to that of citreoviridin ([α] 20 D −105 in MeOH and −107.8 in CHCl3) [24,25]. In addition, the resonances for H-11 and C-21 in 2 were deshielded by ΔδH +0.77 and ΔδC +9.2 ppm, respectively, in comparison with those of citreoviridin recorded in CDCl3, whereas H-13, H3-21, C-11, and C-13 in 2 were shielded by ΔδH −0.16 and −0.08 and ΔδC −7.5 and −2.3 ppm, respectively, indicating that compound 2 was the Δ 12 (13)  In addition, the relative configuration of the tetrahydrofuran ring was determined to be identical with that of citreoviridin by ROESY correlations. The calculated ECD spectrum for 14S,15R,16R,17R-2 matched well with the experimental curve, indicating that 2 possessed the identical absolute configuration with citreoviridin ( Figure 4). Consequently, the structure of 2 was determined as 12Z-citreoviridin and named (+)-neocitreoviridin.   The molecular formula of compound 3 was determined as C 28 H 42 O 2 by HRESIMS. The IR spectrum displayed the absorption bands for the hydroxy (3394 cm −1 ) and the unsaturated ketone  H-3). The 13 C-NMR and DEPT spectra revealed 28 carbon resonances that were ascribed to five methyls, 10 methylenes (with one olefinic carbon at δ C 106.1), six methines (including a cone oxygenated carbon at δ C 69.9 and one sp 2 carbon at δ C 126.6), and seven nonprotonated carbons (with one carbonyl at δ C 197.5 and four sp 2 carbons at δ C 127.1, 141.3, 156.7, and 165.2). These NMR data were similar to those of ergosta-24(28)-ene-3-ol derivatives [13], suggesting an ergostane skeleton for 3.
Analysis of the COSY data revealed the presence of four partial structures as illustrated by the bold lines in Figure 5. These partial structures were connected by the HMBC correlations from H3-19 to C-1, C-5, C-9, and C-10; from H3-18 to C-12, C-13, C-14, and C-17; and from H-15 to C-8 and C-13.    (Table 2). Of the tested compounds, compound 1 exhibited inhibitory activities against HIV-1 and HCV with IC50 values of 4.6 and 7.7 μM, respectively. Compound 2 showed significant inhibitory activity against IAV with an IC50 value of 3.6 μM as compared to 15.4 μM of the positive control ribavirin. Compound 3 showed anti-HIV activity with an IC50 of 3.5 μM and potent anti-IAV activity with an IC50 of 0.5 μM, 300-fold stronger than ribavirin. Compounds 4-6 were inactive against the above viruses at the concentration of 10 μM. It is interesting to note that the other ergostane derivatives, 7 and 8, showed no detectable inhibitory activity against IAV (IC50 > 10 μM). A recent study by Ge et al. [26] showed that the ergostane derivative, (20S,24R)-3β,20dihydroxyergostan-5(6)-en-7,16-dione (amotsterol D), which was effective against wild-type and multi-drug resistant HIV-1 in the low micromolar range, might target the host cell kinases PKM2, a   (Table 2). Of the tested compounds, compound 1 exhibited inhibitory activities against HIV-1 and HCV with IC 50 values of 4.6 and 7.7 µM, respectively. Compound 2 showed significant inhibitory activity against IAV with an IC 50 value of 3.6 µM as compared to 15.4 µM of the positive control ribavirin. Compound 3 showed anti-HIV activity with an IC 50 of 3.5 µM and potent anti-IAV activity with an IC 50 of 0.5 µM, 300-fold stronger than ribavirin. Compounds 4-6 were inactive against the above viruses at the concentration of 10 µM. It is interesting to note that the other ergostane derivatives, 7 and 8, showed no detectable inhibitory activity against IAV (IC 50 > 10 µM). A recent study by Ge et al. [26] showed that the ergostane derivative, (20S,24R)-3β,20-dihydroxyergostan-5(6)-en-7,16-dione (amotsterol D), which was effective against wild-type and multi-drug resistant HIV-1 in the low micromolar range, might target the host cell kinases PKM2, a rate-limiting enzyme of glycolysis, to inhibit replication of HIV-1. Host cell kinases are vital for the replication of a number of viruses and might be targets for broad-spectrum antivirals [27,28]. Due to the close structural similarity with amotsterol D, it was likely that compound 3 exerted broad-spectrum antiviral activity by the same target. Helicobacter pylori is a Gram-negative pathogen whose infection has been recognized as the causative factor of chronic gastritis, peptic ulceration, and gastric malignancies [29]. In the antibacterial assay, compounds 1 and 2 showed significant antibacterial activities against clinically isolated H. pylori (including the drug-sensitive strain G27 and the drug-resistant strain 159) with minimum inhibitory concentrations (MICs) of 1-16 µg/mL, whereas they were inactive against Gram-positive Staphylococcus aureus and Bacillus subtilis and Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae (MIC > 128 µg/mL).

Fungal Material
The fungus Penicillium sp. IMB17-046 was isolated from marine sediments collected from a mangrove swamp in Sanya, Hainan province, China. The strain was identified as a member of the genus Penicillium by morphological characteristics and sequence analysis. Its 18S and ITS-5.8S rDNA gene sequences (GenBank accession no. MK720046 and MK720045) showed the closest match with P. decumbens (GenBank KX553859) and P. manginii (GenBank MH858641) with 99.09% and 99.48% sequence similarities, respectively. The strain was deposited in the National Laboratory for Screening Microbial Drug, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences.

Crystallographic Analysis of 1
A colorless plate crystal of 1 was obtained from CHCl 3 -MeOH (9:1 v/v) via slow evaporation. The crystal data were obtained at 293 K using Cu K radiation (1.54184 Å) on an Oxford Diffraction Gemini Ultra CCD diffractometer. The structure was solved by direct methods (SHELXS-97) and refined with the SHELXL-97 refinement package using Least Squares minimization [30]. Crystal data of 1 was deposited in the Cambridge Crystallographic Data Centre with the deposition number CCDC 1935589. CCDC 1935589 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk).
Crystal data of 1: 2(C 18  3.5. ECD Calculation of (14S,15R,16R,17R)-2 A conformation search was performed on Spartan 14 software (version 1.1.0, Wavefunction Inc. 2014, Irvine, CA, USA) using the MMFF94 molecular mechanics force field. The obtained conformers within a 4 kcal/mol upper energy limit were geometry optimized at the B3LYP/6-31+G (d,p) level in gas using the Gaussian 09 program [31]. The harmonic vibrational frequencies were calculated at the same level to provide their free energy values (∆G). The low-energy conformers with ∆G ≤ 4.0 kcal/mol were subjected to further geometry optimization and frequency calculation at the M06-2X/6-311+G(d,p) level with the polarizable continuum model (PCM) in MeOH. The equilibrium population of each conformer at 298.15 K was calculated according to the ∆G obtained by the second-round optimization at the m06-2x/6-311+G(d,p) level. Energies of the conformers with Boltzmann distribution (>1%) were calculated using the TDDFT methodology (NStates = 65) at the CAM-B3LYP/TZVP level. The ECD spectrum of each conformer was generated by the SpecDis program [32] using a Gaussian function band width σ = 0.30 eV. The calculated ECD spectrum for (14S,15R,16R,17R)-2 was obtained by averaging the calculated data of each conformer according to their Boltzmann population.

Anti-HIV Assay
Anti-HIV activities were evaluated according to a procedure described previously [33]. Briefly, 293T cells were co-transfected with the vector pNL-luc-E-containing a full-length HIV-1 proviral DNA with a firefly luciferase gene and the vesicular stomatitis virus glycoprotein-expressing vector pHCMV-G. After cultivation for 48 h, the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped HIV-1 viruses were harvested by filtration. SupT1 cells (1 × 10 5 ) were infected with the VSV-G-HIV virus (multiplicity of infection, MOI = 1) and treated with the test compounds in triplicate. Efavirenz was used as a positive control. After 48 h inoculation, the SupT1 cells were lysed and the inhibition rate was determined using a firefly Luciferase Assay System (Promega). The concentration of compounds inhibiting 50% viral replication (IC 50 ) was calculated by Origin 8.0 (OriginLab Co. Northampton, MA, USA).

Anti-Influenza A Virus Assay
Anti-influenza A virus assays were performed by using the cell-based high-throughput approach [34]. Briefly, 293T-Gluc cells were treated with test compounds and incubated for 2 h prior to infection. Then, the cells were infected with influenza A/WSN/33 (H1N1) viruses with an MOI of 0.3. After a further 24 h incubation, the cell supernatant was collected and measured for Gaussia luciferase activity. Ribavirin was used as a positive and negative control.

Anti-HCV Assay
Anti-HCV assays were carried out as described previously [5].

Cytotoxicity Assay
Cytotoxicity of compounds were assayed for the uninfected 293T-Gluc, SupT1, and Huh7.5 cells. 293T-Gluc and Huh7.5 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS, Invitrogen). SupT1 cells were cultured in RPMI1640 medium supplemented with 10% FBS. Various concentrations of test compounds (1 µL each well) were added to the 293T-Gluc and SupT1 cells (1 × 10 5 cells/well) in 96-well plates. After incubation at 37 • C for 48 h, 10 µL of CCK-8 reagent was added to the cells and they were incubated for another 4 h. Then, the absorbance of each well was recorded at 450 nm. A cytotoxicity assay for Huh7.5 cells was carried out using the MTT method as described previously [5]. The 50% cytotoxicity concentration (CC 50 ) was calculated by Origin 8.0 software.

Antibacterial Assay
The antibacterial activities against H. pylori, S. aureus, B. subtilis, P. aeruginosa, and K. pneumonia were assayed by examining the MIC using the broth micro-dilution method as previously described [5,35].

Conclusions
In summary, three new natural products, trypilepyrazinol (1), (+)-neocitreoviridin (2), and 3β-hydroxyergosta-8,14,24(28)-trien-7-one (3) were identified from the solid cultures of the mangrove-derived Penicillium sp. IMB17-046. Compounds 1 and 3 showed broad-spectrum antiviral properties against different types of viruses while 2 displayed significant antibacterial activity against H. pylori as well as an anti-IAV effect. Trypilepyrazinol (1) is characterized by a pyrazine motif. Pyrazine heterocycle is an important pharmacophore present as a basic scaffold in various clinical drugs with a wide range of pharmacological and therapeutic activities, such as antitumor, anti-inflammatory, antithrombotic, anti-diabetic, and anti-tubercular [36,37]. However, natural products containing a pyrazine moiety are relatively rare [19]. To the best of our knowledge, trypilepyrazinol (1) is the first example of natural pyrazines exhibiting broad-spectrum antiviral activities and antibacterial activity against H. pylori.