A new diketopiperazine-type alkaloid from the endophytic fungus Penicillium expansum

Abstract A chemical investigation on an endophytic fungus Penicillium expansum isolated from the medicinal plant Plantago depressa Willd. (Plantaginaceae) afforded one new diketopiperazine-type alkaloid, namely penicimine A (1), as well as two known congeners (2 and 3). Their structures were elucidated by widespread spectroscopic data, and the absolute configurations of 1 and 2 were further confirmed by single-crystal X-ray diffraction analyses. Compound 1 represented the first example of benzyl-containing diketopiperazine-type alkaloid bearing a methyl group attached at C-15 position. Compound 1 showed anti-inflammatory activity against LPS-induced nitric oxide (NO) production in RAW264.7 mouse macrophages with an IC50 value of 25.65 μM. Graphical Abstract


Introduction
Alkaloids represent one of the largest groups of secondary metabolites widely found in plants and microorganisms and have been demonstrated to be an important resource for drug discovery (Liu and Qin 2019).Among them, the 2,5-diketopiperazine (DKP)-containing secondary metabolites possessed complex chemical architectures involving fused heterocycles, polythiobridging, prenylation, dimerization, and oxidation (Borgman et al. 2019;Jiang et al. 2021;Lin et al. 2020), and showed attractive bioactivities (Cimmino et al. 2021;Huang et al. 2021;Ma et al. 2021;Song et al. 2021), including anti-inflammatory, antimicrobial, antiplasmodial, and cytotoxic activities, which have attracted much attention from natural product researchers for a long time.In the process of searching for structurally complex and bioactive secondary metabolites from natural resources, an endophytic fungus Penicillium expansum isolated from the medicinal plant Plantago depressa Willd.was obtained.A chemical investigation on rice cultures of the title fungus led to the discovery of three diketopiperazine-type alkaloids (1-3), including a new one, namely penicimine A (1), which represented the first example of benzyl-containing diketopiperazine-type alkaloid bearing a methyl group attached at C-15 position.The structure including absolute configuration of 1 was determined via extensive spectroscopic data and single-crystal X-ray diffraction analysis.Herein, we report the isolation, structure elucidation, and anti-inflammatory activity evaluation of these compounds (Figure 1).

Results and discussion
Compound 1 was determined to have a molecular formula of C 22 H 21 N 3 O 2 , as deduced from a sodium adduct ion at m/z 382.1545 ([M þ Na] þ , calcd for 382.1531) from the HRESIMS data (14 degrees of unsaturation).The 1 H and 13 C NMR data (Table S1) of 1 were similar to those of compound 2, whose absolute structure was completely defined by crystallography experiment (Figure S2), indicating that both compounds were DKP-containing indole alkaloids (Liu et al. 2007;Li et al. 2019).The obvious differences were that an isopropyl group in 2 was replaced by a benzyl group in 1 at the same position and an additional methyl group was attached at C-15 in 1, which were further confirmed by the detailed 2 D NMR data analyses (Figure S1).Fortunately, a suitable crystal of 1 was obtained at room temperature, which was then subjected to a single-crystal X-ray diffraction analysis (Figure S2), allowing an explicit assignment of its absolute configuration as 9S,12S,15R according to the Flack parameter of 0.14(7).Hence, the absolute structure of 1 was defined and named as penicimine A.

General experimental procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter (in MeOH).UV and IR data were collected on a Shimadzu UV2401PC spectrophotometer and a Tenor 27 FT-IR spectrometer with KBr pellets, respectively.NMR data were obtained on a Bruker AM-400 NMR spectrometer, and the 1 H and 13 C NMR chemical shifts were referenced to the solvent peaks for CD 3 OD (d H 3.31/d C 49.0).High-resolution electrospray ionization mass spectrometry (HRESIMS) data were acquired in the positive ion mode with a Thermo Fisher LC-LTQ-Orbitrap XL spectrometer.Compounds were purified by an Agilent 1100 liquid chromatography using an RP-C 18 column (5 lm, 9.4 Â 250 mm, Welch Ultimate XB-C 18 ).Column chromatography (CC) was performed with Silica gel (200-300 mesh, Qingdao Marine Chemical Inc., China), octadecylsilyl (ODS, 50 lm, YMC Co. Ltd., Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden).Fractions were monitored by TLC and spots were visualized by spraying heated silica gel plates with 10% H 2 SO 4 in EtOH.

Fungal material
Strain Penicillium expansum was separated from an inner part of the medicinal plant Plantago depressa Willd.(Plantaginaceae) which was collected in Tongshan County of Xianning City, Hubei Province in July 2018.The sequence data for this strain have been submitted to GenBank under accession no.OL958430.The fungus was deposited in the culture collection of the College of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology.

Fermentation, extraction, and isolation
The strain was cultured on potato dextrose agar at 26 C for one week and inoculated into 50 sterilized 1000 mL Erlenmeyer flasks (components: 200 g of rice and 200 mL of H 2 O).After incubating for 28 days at 26 C, 300 mL of EtOH was appended to each flask to restraint the growth of fungal cells, and the fermented rice media of Penicillium expansum were extracted with ethyl acetate for five times.The concentrated dry extract (50 g) was subjected to silica gel CC eluted with petroleum ether-ethyl acetate (10:1-0:1) to yield five main fractions (A-E).

X-Ray crystallographic data analysis
Compounds 1 and 2 were recrystallized from CH 3 OH with two drops of water at room temperature to obtain colorless crystals.The intensity data for 1 and 2 were recorded on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Cu Ka radiation at 100 K. Refinements were performed with SHELXL-97 using full-matrix leastsquares, with anisotropic displacement parameters used for all non-hydrogen atoms (Liu et al. 2019).Crystallographic data for the reported structures (excluding structure factor tables) have been deposited in the Cambridge Crystallographic Data Center as supplementary publication No. CCDC 2126501 for 1 and No. CCDC 2177159 for 2.
3.6.Anti-inflammatory activity against the NO production in RAW264.7 macrophages The RAW264.7 cells were seeded into the 96-well cell culture plates (2 Â 10 5 cells/ well), each of which contained RPMI-1640 (Hyclone).After preincubation for 24 h, the seeded cells were treated with gradient dilutions of all test compounds with a maximum concentration of 100 lM, followed by stimulation with LPS (1 lg/mL) for about 24 h.Nitric oxide (NO) production in the supernatant was assessed by the Griess reagent (Sigma).After incubation for 5 min, the absorbance at 570 nm was measured with a 2104 Envision multilabel plate reader (PerkinElmer Life Sciences, Inc., Boston, MA, USA).The inhibitor of proteasome (MG132) was selected as the positive control (Hu et al. 2017).

Conclusions
To sum up, one new diketopiperazine-type alkaloid, namely penicimine A (1), and two known congeners (2 and 3) were isolated from a plant endophytic fungus Penicillium expansum.Their structures were elucidated by widespread spectroscopic data, and the absolute configurations of 1 and 2 were further confirmed by single-crystal X-ray diffraction analyses.Compound 1 represented the first example of benzyl-containing diketopiperazine-type alkaloid bearing a methyl group attached at C-15 position.Compound 1 showed anti-inflammatory activity against LPS-induced NO production in RAW264.7 mouse macrophages with an IC 50 value of 25.65 lM.Our new findings in this research greatly enrich the chemical and pharmacological knowledges on the species of Penicillium genus.