7-Methoxy-13-dehydroxypaxilline: New indole diterpenoid from an endophytic fungus Penicillium sp. Nb 19

ABSTACT A chemical investigation of the endophyte Penicillium sp. Nb 19, isolated from leaves of the traditionally medical plant Baphicacanthus cusia (Nees) Bremek., yielded one new indole diterpenoid, 7-methoxy-13-dehydroxypaxilline (1) together with seven known metabolites (2-8). The obtained structure of compound 1 was elucidated by its spectroscopic data. In addition, the absolute configuration of compound 6 was confirmed by ECD for the first time. Compounds 1-6 were evaluated for antitumor activity against MCF-7, HepG2, and HCCC-9810 cell lines. GRAPHICAL ABSTRACT


Introduction
Endophytic fungi are significant sources of a wide variety of active secondary metabolites with pharmaceutical potential (Feng et al. 2014).For instance, anti-inflammatory compound brevione A was isolated from P. bialowiezense (Kwon et al. 2021), the compound penipyrol C with antidiabetic activity was discovered from Penicillium sp.HDN-11-131 (Wang et al. 2021).Moreover, mycophenolic acid and mycophenolate methyl ester with potent cytotoxicity were obtained from P. parvum HDN17-478 (Chen et al. 2020), and the immunosuppressant compounds were obtained from P. griseofulvum (Zang et al. 2021).The aforementioned results exhibited the extremely potential value of endophytic Penicillium sp.Recently, an endophytic fungus Penicillium sp.Nb 19 has been investigated due to its intense antioxidant activity, which is isolated from the medicinal plant of Baphicacanthus cusia grown in China and widely used in numerous diseases (for example cough, common cold, antiphlogosis, fever, and influenza) (Zhu et al. 2020).
Indole diterpenoids, as a large class of diverse fungal secondary metabolites, typically have an indole moiety attached to diterpene skeleton (Xu et al. 2014).It is formed by the conversion of geranylgeranyl pyrophosphate and indole-3-glycerophosphate (Byrne et al. 2002).Up to now, all reported indole diterpenoids compounds were mainly obtained from the secondary metabolites of fungi such as Penicillium, Aspergillus, Dichotomomyces, Epichloe, Nodulisporium, Emericella, and Claviceps (Ariantari et al. 2019).Especially, some of them, known as tremorgenic mycotoxins, could result in neurological disorders for farm animals, for instance, penitrems A-F (Gonzalez et al. 2003), lolitrem B , aflatrem (Gallagher et al. 1980), and paspalitrems A and B (Cole et al. 1977).Apart from the toxicity, the indole diterpenoids were also invested to have antibacterial activity (Ogata et al. 2007), antiviral activity (Fan et al. 2013), antiproliferative activity (against breast cancer cell lines and human glioblastoma) (Sallam et al. 2013), and insecticidal activity .Moreover, paxilline has proved to be anticonvulsant effect for inhibiting the channels of high-conductance Ca 2þ -activated K þ (Sanchez et al. 1996).Due to the significant role of active indole diterpenes, some of them have already been synthesized by chemists, for instance, paspalinine, paspaline, and nodulisporic acids (Enomoto. 2021).Indeed, it is very significant to study indole diterpenes because of their novel, complexity structure, and prominent bioactivities.
In this study, a new indole diterpenoid analogue, 7-methoxy-13-dehydroxypaxilline (1), together with seven known compounds (2-8) have been successfully isolated from endophytic fungus Penicillium sp.Nb 19 (Figure 1).The absolute configuration of the new compound 1 was confirmed via NMR spectra and ECD calculation.In addition, the absolute configuration of compound 6 was illuminated for the first time by ECD test.Furthermore, the cytotoxicity of the compounds 1-6 were also investigated in this study.
The relative configuration of 1 was supported by the ROESY experiment.The ROESY correlation of H-5a and H-16/H 3 -26 indicated the C-26, H-16 and H-5a were b oriented.In addition, the ROESY correlations of H-13/H-5b; H 3 -25 and H 3 -7 0 '/H-9 and H-5b illuminated that these protons were a oriented.These data combined with the consideration of biogenesis suggested that compound 1 has two isomers (7 R and 7S).Therefore, the absolute configuration of compound 1 was confirmed by comparing the experimental and calculated ECD spectrum of its isomers (Figure S2).Obviously, there is a good coincidence between the ECD spectra of calculation for isomers (7 R)-1 and experimental ECD spectra.Furthermore, the two possible absolute configurations of (7 R)-1 (A), (7S)-1 (B) was assigned to perform the 1 H and 13 C NMR chemical shift calculations.The gauge independent atomic orbital (GIAO) method (Wang et al. 2020;Shu et al. 2020) at the PCM/mPW1PW91/6-311 þ G(d,p) level of theory was employed for chemical shift calculations and DP4þ probability analyses.The result of 1 H, 13 C data and all data ( 1 H þ 13 C) of DP4þ method (Figure S5) showed that (7 R)-1 (A) was the most possible stereoisomer.Consequently, based on the above analysis results, the structure of compound 1 was identified as shown in Figure 1.Compound 1 was named 7-methoxy-13-dehydroxypaxilline.
Their cytotoxic activities of compounds 1-6 against three human tumor cell lines (breast cancer MCF-7, liver cancer HepG2, and HCCC-9810) were evaluated by CCK-8 assay in comparison with cisplatin as positive control.As the obtained results, compound 4 exhibited moderate cytotoxic activities against HCCC-9810 with IC 50 value of 4.37 lM, compared to cisplatin (IC 50 value of 2.53 lM).The remaining compounds performed no obvious cytotoxic activity against the three tumor cells, and the IC 50 values were all greater than 10 lM (Table S5).

General experimental procedures
The NMR data ( 1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC, and ROESY) were recorded on a Bruker Avance-500 spectrometer with using tetramethylsilane as the internal standard (Bruker, Germany).The chemical shifts (d) were referred to the residual solvent peaks at d H 3.31 (CD 3 OD) and d H 7.27 (CDCl 3 ) ppm for 1 H, including d C 49.15 (CD 3 OD) and d C 77.23 (CDCl 3 ) ppm for 13 C. High resolution electrospray ionization mass spectroscopy (HRESIMS) analyses were obtained by an Agilent Accurate-Mass TOF LC/MS 6230 instrument in positive ion mode (Agilent, USA).Optical rotations were performed on a Jasco P-1020 digital polarimeter (Jasco Co., Japan), while UV spectra were detected by a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Japan).Electronic circular dichroism (ECD) curves were measured on an Applied Photophysics Chirascan spectrometer (Applied Photophysics Ltd., United Kingdom).IR spectra were obtained by a Shimadzu IRAffinity-1S FTIR spectropolarimeter (Shimadzu, Japan).Semi-preparative high performance liquid chromatography (HPLC) was conducted via using an HPLC system equipped with a Waters 2535 (Waters, USA) quaternary gradient module, 2707 autosampler, and 2998 photodiode array detector.Column chromatography (CC) was performed on Sephadex LH-20 gel (solarbio, China) and Silica gel (Qindao Haiyang chemical Factory, China).TLC plates silica gel GF 254 was used for analysis.The absorbance was measured by SpectraMax i3 microplate reader (Molecular Devices, USA).The cell density was determined with Countstar IC1000 automatic cell counting apparatus (Countstar, USA).

Fungal material
The Penicillium sp.Nb 19 fungal strain was isolated from healthy and fresh leaves of Baphicacanthus cusia, which was collected in January 2019 at Honghe Autonomous prefecture, Yunnan province, China.Penicillium sp.Nb 19 was identified by DNA amplification and internal transcribed space (ITS) analysis, while PCR amplification was performed with ITS1 (5 0 -TCCGTAGGTGAACCTGCGG-3 0 ) and ITS4 (5 0 -TCCTCCGCTTA TTGATATGC-3 0 ).Moreover, a Blast search result illuminated that there is 100% similarity between the sequence and that of Penicillium Paxilli (accession number: MK120566).The strain has been kept in the Yunnan Key Laboratory of Southern Medicine Utilization.Penicillium sp.Nb 19 was grown on solid rice medium each 500milliliter conical flasks containing 80 grams of rice and 100 milliliters ultra-pure water.
The 25 conical flasks were fermented at room temperature under static conditions for 30 days.

Cytotoxicity assay
Compounds 1-6 were evaluated for cytotoxicity by cell counting Kit-8 (CCK-8) method against MCF-7, HepG2, and HCCC-9810 cell lines.Three cell lines were purchased from Procell Life Science and Technology Co. Ltd (Hubei, China).The in vitro cytotoxicity tests were performed according to a previously reported method (Wang et al. 2020).MCF-7 and HepG2 cells were grown in DMEM medium, and HCCC-9810 cells were grown in RPMI-1640 medium.When the cells at logarithmic growth phase were digested and collected, the cell density was measured by Countstar automatic cell counter.The cell concentration was adjusted to 3 Â 10 5 /mL with serum-free medium according to the measured density value, and inoculated into 96-well culture plates with 200 lL per well.The cells were cultured at 37 C and 5% CO 2 for 12 h, then the medium was sucked out and adding drugs and step by step dilution make final concentration 100, 50, 25, 12.5, 6.25 and 3.125 lM, respectively.After incubation for 24 h, CCK-8 (Biyuntian Biotechnology Co., Ltd, Shanghai, China) was added to each well and incubated at 37 C for another 4 h.Finally, the absorbance of each well at 450 nm was measured with microplate reader (A).Cisplatin as the positive control in this assay.