Anti-IAV indole-diterpenoids from the marine-derived fungus Penicillium citrinum

Abstract A new indole-diterpenoid, penijanthine E (1), and a known analogue (2), were obtained from the PDB culture of the marine-derived fungus Penicillium citrinum ZSS-9. The absolute configuration of 1 was elucidated by calculated TDDFT ECD and DP4plus calculations. The absolute configuration of 2 was confirmed by single-crystal X-ray diffraction analysis and TDDFT ECD calculations. Compounds 1 and 2 showed antiviral activity against influenza A virus (IAV) of A/WSN/33(H1N1) and A/PR/8/34(H1N1) strains with IC50 values ranging from 12.6 to 46.8 μM. Graphical Abstract


Introduction
Influenza A virus (IAV), which could cause zoonotic infections and adapt to humans, leading to sustained transmission and emergence of novel virus, is a major threat to human health (Rambaut et al. 2008). For example, the outbreak of H1N1 virus in 2009 has rapidly swept across 214 countries around the world, causing human infection and acute respiratory illness of more than 17,700 deaths worldwide (Baccam et al. 2006). Due to the changes in the climate and ecology, it is highly likely that new IAV outbreaks are unavoidable in the future. However, resistance has been found in many clinical anti-IAV drugs, such as oseltamivir, zanamivir, and amantadine (Ison 2011). Developing a safe and effective agent with a unique structure is urgently needed to combat IAV infection. In the past decades, hundreds of new natural products with antiviral activities have been isolated from various marine sources (Gogineni et al. 2015;Sun et al. 2021), indicating that seeking anti-IAV compounds from marine was worth to explore.
Marine-derived fungi of the genus Penicillium have been reported to produce unique and biologically active secondary metabolites (Sun et al. 2014;Zheng et al. 2016, Zheng et al. 2019Salendra et al. 2021). For instance, a series of indole-diterpenes, penijanthines A-D, have been obtained from the fungi Penicillium spp. exhibited significant activity (Su et al. 2014;Guo et al. 2019;Itabashi et al. 2009). During our ongoing search for bioactive metabolites from marine-derived fungi Guo et al. 2019;Cao et al. 2020), a marine sediment-derived fungus Penicillium citrinum ZSS-9, collected from the Bohai Sea, attracted our attention because an EtOAc extract of the fungal culture exhibited anti-IAV activity. Subsequent chemical investigation of the fermentation broth of this strain led to the isolation of one new indole-diterpenoid, penijanthine E (1), and one known analogue, 4a-demethylpaspaline-4a-carboxylic acid (2) (Figure 1). In this paper, we report the fermentation, isolation, structure elucidation, as well as antiviral activity of 1 and 2.

Results and discussion
Penijanthine E (1) was isolated as a light yellow, amorphous powder. The HRESIMS spectrum of 1 gave the [M -H]ion peak at m/z 466.2604, which indicated the molecular formula of C 28 H 37 NO 5 , accounting for 11 degrees of unsaturation. The 1 H NMR data of 1 (Table S1)  ]. These characteristic data implied the structure of 1 to be closely related to 4a-demethylpaspaline-4a-carboxylic acid (2) (Fan et al. 2013) and that they potentially share an identical indole-diterpenoid skeleton. While one of the four CH 3 protons observed in the 1 H NMR of 2 was missing in 1, the presence of two geminally coupled proton signals observed at 3.32 (1H, d, J ¼ 10.8, Hz) and 3.24 (1H, d, J ¼ 10.8, Hz) were indicative of the presence of an oxygen-bearing CH 2 group. The 13 C NMR and HSQC spectra of 1 indicated the presence of 28 carbons and also revealed its close structural similarity to 2. Especially, the oxygen-bearing methylene [d C 67.0 (C-3 0 )] in 1 suggested that the 3 0 -CH 3 in 2 was replaced by 3 0 -CH 2 -OH in 1, which was confirmed by the key HMBC correlations from H 2 -3 0 to C-1 0 and C-2 of 1 ( Figure S11). Detailed analysis of the HSQC, COSY, and HMBC spectra allowed the assignment of all proton and carbon resonances. Thus, the planar structure of 1 was established.
Compound 2, named 4a-demethylpaspaline-4a-carboxylic acid, has been previously obtained from the marine-derived fungus P. camemberti (Fan et al. 2013). Our study was the first time to verify the planar structure and relative configuration of 2 by single-crystal X-ray diffraction with Mo Ka radiation ( Figure S15). Finally, the absolute configuration of 2 was determined as 2S,4aR,4bR,6aS,12bS,12cS,14aS by using quantum chemical calculations of the ECD spectra ( Figure S16).
Compounds 1 and 2 were evaluated for their antiviral activity against influenza A virus of A/WSN/33(H1N1) and A/PR/8/34(H1N1) strains using a cytopathogenic effect assay. The concentrations of 1 and 2 that inhibited 50% of virus-induced cytopathic effect in MDCK cells were determined as IC 50 values. While, the concentrations that caused 50% cytotoxic effect toward MDCK cells were denoted as CC 50 values. It was showed that 1 could inhibit the two strains with IC 50 values of 12.6 and 18.9, lM, respectively, stronger than that of the positive control T-705 (IC 50 , 30.6 and 37.2 lM, respectively). However, compound 2 only displayed moderate activity against A/WSN/33(H1N1) and A/PR/8/ 34(H1N1) with IC 50 values of 42.5 and 46.8 lM, respectively, suggesting that the 3 0 -CH 2 -OH in 1 may play an important role for the activity. Furthermore, 1 and 2 had the similar toxicity to MDCK cells with CC 50 values of 62.8 and 68.7 lM, respectively.

General experimental procedures
Optical rotations, ECD, and UV spectra were acquired using a JASCO P-2000, a MOS-450/SFM300, and a Shimadzu UV-3600 spectrophotometers, respectively. 1 D and 2 D NMR data (TMS as the internal standard) were recorded on a Bruker AV-600 spectrometer. HRESIMS spectrum was obtained from a Thermo Scientific LTQ Orbitrap XL spectrometer. HPLC separation was performed with a C 18 HPLC column (Waters, 10 Â 250 mm, 5 lm) on the Shimadzu LC-20AT system containing a SPD-M20A photodiode array detector. Column chromatography was performed on Silica gel 200-300 mesh and Sephadex LH-20 18 À 110 lm.

Fungal material
The fungal strain Penicillium citrinum ZSS-9 was isolated from the Bohai Sea (Huanghua, Hebei Province, China, June 2016), and it was deposited in the College of Pharmaceutical Sciences, Hebei University, China, with a Genbank accession number MH377076.

Fermentation and purification
The fungal strain ZSS-9 was cultivated using liquid medium with 400 mL of PDB in each 1 L Erlenmeyer flask (total 20 flasks) at 28 C for 4 weeks. The fermentation broth was extracted exhaustively with EtOAc, while the mycelia were extracted by 95% methanol. The combined organic phases were evaporated to dryness to provide the crude extract (2.1 g). The extract was subjected to silica gel column chromatography (CC) using a stepwise gradient of petroleum ether (PE)/EtOAc (90:10, 50:50, 10:90) to give three fractions: Fr.1-Fr.3. Fr.2 was separated by repeated silica gel, Sephadex LH-20 ODS silica gel CC, and finally purified by semi-preparative HPLC using a Waters RP-18 column (5 lm, 10 Â 250 mm) at a flow rate of 2.0 mL/min (MeOH À H 2 O, 80:20) to yield 1 (6.2 mg) and 2 (8.1 mg).

Anti-IAV activity assays
The anti-IAV activities of 1 and 2 were determined by the CPE inhibition assay (Xiao et al. 2022). Monolayer of MDCK cells on 96-well plates was washed with PBS and then infected with influenza A virus of 100 TCID 50 in FBS-free MEM. After incubating for 2 h, the medium was removed and 100 lL of compound in FBS-free MEM at twofold serial dilution and 1 lg/mL TPCK treated-trypsin (not for WSN infection) were added. After 48 h, cell viability was evaluated with MTT assay. The concentration of compound that inhibited 50% of virus-induced cytopathic effect was determined as IC 50 (50% inhibitory concentration). T-705 were used as positive controls. Three parallel experiments were performed for confirmation.

Conclusion
Two indole-diterpenoids, including the new penijanthine E, were obtained from the marine-derived fungus Penicillium citrinum ZSS-9. Single-crystal X-ray diffraction analysis, TDDFT ECD, and DP4plus probability were used to establish their configurations. Two indole-diterpenoids showed anti-IAV activity, suggesting their application prospects in the pharmaceutical field.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This research was funded by the Science and technology project of Henan Province (No. 212102310243), the Natural Science Foundation of Hebei Province of China (No. H2020201298), and the High Performance Computer Center of Hebei University.