Anti-Vibrio Indole-Diterpenoids and C-25 Epimeric Steroids From the Marine-Derived Fungus Penicillium janthinellum

A systematic chemical exploration of the marine-derived fungus Penicillium janthinellum led to the isolation of four indole-diterpenoid derivatives (1–4), including new penijanthines C and D (1 and 2), and a pair of new steroidal epimers, penijanthoids A and B (5 and 6). The calculated ECD spectra and Snatzke's method for the new compound 1 were carried out to determine its absolute configuration. The absolute configuration of 3 was established by X-ray diffraction and calculated ECD methods for the first time. DP4plus approach was used to elucidate the absolute configurations of the C-25 epimeric steroids 5 and 6. 25-Epimeric 5 and 6 represent the first examples of steroids forming a five-membered lactone between C-23 and C-27 from marine fungi. Compounds 1, 2, 5, and 6 displayed significant anti-Vibrio activity (Minimum inhibitory concentration, MIC values ranging from 3.1 to 50.0 μM) against three pathogenic Vibrio spp.


INTRODUCTION
Vibrio spp., such as Vibrio anguillarum, Vibrio parahemolyticus, and Vibrio alginolyticus, is a class of Gram-negative halophilic bacteria that occurs usually in marine and coastal environments throughout the world, which could lead vibriosis in crustaceans and cause serious damage to mariculture production (Vezzulli et al., 2015;Moreno et al., 2017). However, there was no effective vaccine to prevent vibriosis due to lacking adaptive immunity in crustacean species (Buchmann, 2014). In the past few decades, searching for marine-derived bioactive substances as anti-Vibrio agents has drawn the attention of chemists and pharmacologists (Meng et al., 2015;Wang et al., 2015). In our continuing efforts to explore anti-Vibrio natural products from marine-derived fungi (Xu et al., 2017;Yang et al., 2018), the Bohai Sea fungus Penicillium janthinellum was selected for further chemical exploration due to the anti-Vibrio activity of its EtOAc extract. As a result, two new indole-diterpenoids, penijanthines C and D (1 and 2), and two known analogs, PC-M6 (3) (Yamaguchi et al., 1993), 7-hydroxy-13-dehydroxypaxilline (4) (Mantle and Weedon, 1994), along with two new steroids, penijanthoids A and B (5 and 6), were obtained (Figure 1). Compounds 1-6 displayed anti-Vibrio activity against three pathogenic V. anguillarum, V. parahemolyticus, and V. alginolyticus.

General Experimental Procedures
Optical rotations (OR) values of the new compounds were determined using a JASCO-1020 polarimeter. Electronic circular dichroism (ECD) experiments, including Mo 2 (AcO) 4 ICD experiments, were carried out on a JASCO J-815 circular dichroism spectrometer. Ultraviolet-visible (UV) data were provided in MeOH by a Perkin-Elmer model 241 spectrophotometer. Infrared radiation (IR) data of the new compounds (using KBr pellets) were measured on a Nicolet NEXUS 470 spectrophotometer. 1D NMR ( 1 H NMR and 13 C NMR) and 2D NMR (HSQC, 1 H-1 H COZY, HMBC and NOESY) data were recorded on a Bruker AV-600 spectrometer. HR-ESI-MS spectra were performed with a Thermo Scientific LTQ Orbitrap XL spectrometer. Semi-preparation HPLC, which had the Shimadzu LC-20AT system with a SPD-M20A detector and a Waters RP-18 column, was used for chemical separation. Further chromatographic separation was taken on 200-300 mesh silica gel and 18-110 µm Sephadex LH-20 columns.

Isolation of the Fungal Material
The strain in our research, which was derived from the marine sediment collected from the Bohai Sea in June 2016, was deposited at Hebei University, China. According to its 16S rRNA amplification and sequencing of the ITS region, the strain was identified as Penicillium janthinellum (Gene Bank KY979507). The fungus Penicillium janthinellum was cultivated using solid medium in forty Erlenmeyer flasks (80 g raw rice, 60 mL H 2 O, 2.0 g sea salt in each Erlenmeyer flask) at 28 • C for 4 weeks. Mixed solvent of CH 2 Cl 2 -MeOH (v/v = 1:1) was used to extract fermented rice, and the solution was evaporated to give the crude extract, which was dissolved and extracted with EtOAc for five times to provide the EtOAc extract (12.0 g). The EtOAc extract, which was eluted with EtOAc-petroleum ether (PE) on the silica gel column chromatography (CC), was separated into different fractions ranged from Fr.1 to Fr.8. Fr.2 (1.46 g), which was eluted with 40% EtOAc in PE, was applied on a Sephadex LH-20 and waters RP-18 (XBridge OBD, 5 µm, 10 × 250 mm, 70%-MeOH in water) columns to produce 5 (10.5 mg) and 6 (7.6 mg). Fr.4 (4.34 g, 60% EtOAc in PE) was separated by repeatedly silica gel CC and HPLC to provide 1 (20.5 mg), 2 (5.8 mg), 3 (16.4 mg), and 4 (8.2 mg).

Computational Section
Conformational search of the new compounds 1, 5, and 6 for quantum calculations was taken using MMFF94S force field with low energetics from 0-10.0 kcal/mol. Optimization for these geometries were carried out in the gas phase at the B3LYP/6-311+G(d) level. The optimized conformations with the relative energy between 0 and 2.5 kcal/mol was selected for ECD calculations, which were computed at the B3LYP/6-311++G(2d,p) level (gas phase) (Zhu, 2009(Zhu, , 2015Zhu et al., 2014). For the DP4plus applications of 5 and 6, unshielding tensor values of the optimized conformers were computed at the mPW1PW91/6-311+G(d,p)//mPW1PW91/6-311+G(d,p) level in the gas phase. All of the quantum chemical calculations were performed using Gaussian 09 package (Frisch et al., 2009).

X-Ray Crystallographic Study of PC-M6 (3)
The crystal of 3 was acquired from a mixed solvent of methanol and dichlorine in a refrigerator for 14 days. The detail X-ray diffraction data of single-crystal 3 were collected by Bruker Smart APEXII with the crystal system of Mo target. The wavelength of radiation is 0.71073 Å. The block crystals of 3 are monoclinic, space group C2 with cell dimensions a = 19.2301 (8)

Anti-Vibrio Activity Assays
The conventional broth dilution assay described by on the related literature (Appendio et al., 2008) was used to test the anti-Vibrio activity of these compounds. Three pathogenic Vibrio strains, Vibrio anguillarum, Vibrio parahemolyticus and Vibrio alginolyticus were incubated about 16-18 h at 37 • C as the tested strains. The overnight cultures were used to prepare the turbidity of the bacterial suspensions, which had a concentration of 10 5 -10 6 colony formingunits/mL and had the absorbance of 0.4-0.6 at 600 nm. The 96-well plates, which contained 2 µL of test solutions/positive control ciprofloxacin and 198 µL of bacterial culture, were used to test the minimum inhibitory concentration (MIC) of anti-Vibrio activity for these compounds. Finally, the different concentrations of tested compounds from 25.0 to 0.195 µM were prepared and incubated overnight for 24 h at 37 • C to measure the MIC values of anti-Vibrio activity. Ciprofloxacin had the MIC values of 0.078, 0.312, and 0.625, respectively, against V. anguillarum, V. parahemolyticus, and V. alginolyticus.

RESULTS AND DISCUSSION
Penijanthine C (1) was isolated as a yellow amorphous powder. The molecular formula of C 28 H 41 NO 3 for 1 was determined Frontiers in Chemistry | www.frontiersin.org  (Figure S10), suggesting nine degrees of unsaturation in 1. In the 13 C NMR spectroscopic data (Table 1) of 1, 28 carbon signals which contain five methyls, seven methylenes, eight methines including four olefinic carbons, and two oxygen-bearing carbons, and eight quaternary carbons with four sp 2 and two oxygenated sp 3 were observed. The above 13 C NMR signals agreed well with the 1 H NMR spectroscopic data (  Penicillium camemberti (Fan et al., 2013), by careful comparison of their 1D NMR data. The structural difference between them was that the 26,27-trisubstituted double bond in emindole SB was replaced by a vic-diol moiety [δ H 3.00 (1H, t, J = 7.2 Hz, H-26); δ C 78.6 (CH, C-26) and 71.7 (C, C-27)] in 1 (Figures S4, S5). The long-range couplings of H 3 -28/C-26, H 3 -28/C-27, H 3 -29/C-26, H 3 -29/C-27, and H 2 -24/C-26 in the HMBC spectrum of 1, as well as the proton spin system of H 2 -25/H-26 from the 1 H-1 H COZY spectrum in 1, supported the above deduction (Figure 2). The assignment of the planar structure for 1 was consequently confirmed by the 2D NMR data of HSQC, 1 H-1 H COZY, and HMBC in 1 (Figures S6-S8).
The analysis of the NOESY data allowed the relative configuration of the five rings for the indole-diterpenoid nuclei in 1. The NOESY cross-peaks between the H-12 and H 3 -22, H 3 -22, and H 3 -23 as well as H-9 had NOE with both H 3 -21 and H-7 were observed in the NOESY experiment of 1, suggesting that H-12, H 3 -22, and H 3 -23 were place on the opposite direction to H-7, H-9, and H 3 -21 in the molecule of 1 (Figure 3). However, the NOESY experiment was unable to conclusively determine the configuration at C-26 in 1 (Figure S9).
Penijanthine D (2) had the molecular formula of C 30 H 43 NO 4 , which was determined by the high resolution mass data m/z = 504.3072 [M + H] + (calcd. 504.3084) of 2 ( Figure S17). Compound 2 was also defined as an indole-diterpenoid analog by the strikingly similar NMR data of 2 (Figures S11-S15) compared with those of 1 (Table 1), with the appearance of the additional acetoxy signals [δ H 1.94 (3H, s); δ C 169.9 and 21.0] in 2. This additional acetoxy group was connected at C-7 in 2 was through the key HMBC correlation between H-7 and C-COCH 3 . The NOESY ( Figure S16) and ECD (Figure 5) experiments of 2 indicated the stereochemistry of 2 was the same as 1. Therefore, compound 2 was assigned as the 7-acetylation derivative of 1.
The known PC-M6 (3) (Yamaguchi et al., 1993) and 7hydroxy-13-dehydroxypaxilline (4) (Mantle and Weedon, 1994) were determined by comparing their 1 H, 13 C NMR and positive Mass data with the corresponding data in the literature. The stereochemistry of PC-M6 (3) was further verified by the data of X-ray diffraction (Figure 6) and calculated ECD (Figure S1) for the first time. The present work affords four indole-diterpenoids (1-4), which consist of a common cyclic diterpene backbone and an indole moiety. According to the literature, over 100 indolediterpenoids with unique chemical scaffolds were produced by various fungal sources (Li et al., 2002;Zhao et al., 2018). The complexity of these intriguing structures may encourage further investigations on the chemistry and biological activity of this cluster of metabolites.
Vibriosis, which is also known as bacterial canker, is one of the bacterial diseases which cause serious damage and great losses to mariculture production (Vezzulli et al., 2015;Moreno et al., 2017). Research and development of effective anti-Vibrio drugs for controlling vibriosis is needed for mariculture. Thus, the anti-Vibrio activities against V. anguillarum, V. parahemolyticus, and V. alginolyticus of the new compounds 1, 2, 5, 6 were carried out. Compound 1 displayed strongest anti-Vibrio activity against V. Anguillarum (MIC = 3.1 µM), V. parahemolyticus (MIC = 6.3 µM), and V. Alginolyticus (MIC = 3.1 µM), respectively. Compound 2 showed moderate anti-Vibrio activity against three pathogenic Vibrio spp. with the same MIC values of 12.5 µM, suggesting that the presence of an acetoxy group at C-7 in 2 may decrease the anti-Vibrio activity. A literature survey showed that the other known indole diterpenoid analogs, such as 6hydroxylpaspalinine, paspalitrem C, emindole SB and so on, were also showed anti-Vibrio activity against three pathogenic Vibrio spp. (Hu et al., 2017). These finding suggested that it was worth ongoing to seek new anti-Vibrio compunds from indole diterpenoid derivatives. However, compounds 5 and 6 only exhibited weak anti-Vibrio activity against three pathogenic Vibrio spp. (MICs, 25.0-50.0 µM).

CONCLUSION
Four indole-diterpenoids and two steroidal epimers were isolated from the marine-derived fungus Penicillium janthinellum. Snatzke's, X-ray diffraction, and calculated ECD methods were used to assign the absolute configurations of these compounds.. The absolute configurations of steroidal epimers were suggested by DP4plus approach. Compounds 1 and 2 exhibited potential anti-Vibrio activity and represented a promising new class of anti-Vibrio agents.

AUTHOR CONTRIBUTIONS
L-LX and R-YY: contribute to fermentation, extraction, and isolation. X-CG: contribute to manuscript preparation. M-YY and L-DH: contribute to quantum chemistry calculation and bioactivities test. H-JZ and FC: were the project leaders organizing and guiding the experiments and manuscript writing.