Two new lactam derivatives from a Sphagneticola trilobata derived fungus Penicillium rubens PQJ-2

Abstract A new bicyclic lactam derivatives penicilactam B (1) and a new monocyclic amide penicillamide D (2), along with four known compounds (3-6), were isolated from the fermentation broth of the derived fungus Penicillium rubens PQJ-2. Their structures and stereochemistry were elucidated by comprehensive spectroscopic analyses and quantum ECD calculations. All the compounds were evaluated for their antibacterial activities against Staphylococcus aureus subsp, Candida albicans, Escherichia coli and insecticidal activity against Helicoverpa armigera Hubner. Compounds 1-3 exhibited modest insecticidal activity against H. armigera Hubner. Graphical abstract


Introduction
Endophytic microorganisms are bacteria or fungi that live inside plant tissues without causing damage or disease symptoms to their hosts.They are promising sources of novel secondary metabolites, and the inspiration to drug discovery (Yang et al. 2022).
Fungal species belonging to the genus Penicillium represent a major source of novel compounds with diverse bioactive properties (Bladt et al. 2013;Kozlovski K % et al. 2013;Yang et al. 2021).For example, penicillin, the first broad-spectrum antibiotic, was isolated from Penicillium and saved countless lives in the past decades.So far, many novel secondary metabolites have been isolated from the Penicillium fungi, including alkaloids, fatty acids, terpenoids, polyketides, and so on (Zeng et al. 2018;Chen et al. 2019).These secondary metabolites have diverse biological activities, including antibacterial, anti-inflammatory, antitumor, antioxidant, enzyme inhibition, and cytotoxicity (Wang et al. 2010;Lee et al. 2013;Sun et al. 2013;Shah et al. 2014;Shuo et al. 2014).Therefore, the Penicillium genus plays a vital role in drug development.As part of our ongoing search for bioactive secondary metabolites from plantderived fungi, the fungi Penicillium rubens PQJ-2 was isolated from Sphagneticola trilobata.Subsequent chemical investigation of an EtOAc extract of the culture of this fungal strain led to the isolation of a new bicyclic lactam derivative penicilactam B (1) and a new monocyclic amide penicillamide D (2), together with four known compounds (3-6) (Figure .1).Herein, we described the isolation, structure identification, and bioassay of these compounds.160.6 and 161.8) and two olefinic carbons (d C 163.2 and 118.9) were observed.Comparison of 1 H-and 13 C-NMR combined with DEPT data of 1 with those of (þ) penicilactam A (3) indicated two differences in the structures of them.First, the 9-CH 2 in the (þ) penicilactam A (3) was replaced by a carbonyl carbon in 1.This hypothesis was confirmed by observing HMBC correlations from H-6 to C-7, C-8, C-9 , from H-7 to C-6, C-8, C-9, from H-8 to C-6, C-7, C-9.In addition, the six-membered oxygen-containing heterocyclic structure in (þ) penicilactam A (3) was substituted with a unique the six-membered nitrogen-containing heterocyclic.This hypothesis was confirmed by observing C-6 from (d H 5.20; d C 87.6) in 3 upfield to (d H 5.09; d C 72.1) in 1. Detailed analysis of 1 H-1 H COSY and HMBC spectra confirmed that the structure of the other parts of the molecule was the same as that of (þ) penicilactam A (3) (Figure . S1).

Result and discussion
In the low-frequency region of IR, the c ¼ C-H absorption band of alkenes is trans at 1000-950 cm À1 (s) and cis at 730-670 cm À1 (s) (Zhao et al. 2010).Therefore, the configuration of the double bond in the side chain of compound 1 was 15E by diagnostic absorption bands at 968 cm À1 in IR spectrum (Figure . S20).In addition, the 1 H NMR data characteristics of 1 (d H 5.41, m) was similar to (d H 5.41, m) of (þ)-Penicilactam A (Yang et al. 2018) , (d H 5.39, m) of 2-(hept-5-enyl)-3-methyl-4-oxo-6,7,8,8a-tetrahydro-4H-pyrrolo[2,1-b]-1,3-oxazine (Lai et al. 2013) of the side chain with E configuration for double bond in the references.Since compound 1 has one chiral center at C-6, to define the absolute configuration of 1, electronic circular dichroism (ECD) calculation was performed for the stereostructures (6S)-1 and (6 R)-1, using the time-dependent density functional theory (TDDFT) method at the B3LYP/6-31G (d, p) level by mean of the GAUSSIAN 09 program.The experimental ECD spectrum of 1 showed excellent consistency with the calculated ECD spectrum for (6S)-1 (Figure . S2).Thus, the structure of 1 was determined, and named as penicilactam B. To the best of our knowledge, this is the first example of oxygen-containing heterocycle being replaced by nitrogen-containing heterocycle in 6, 5-bicyclic lactam.
Compound 2 was obtained as a light-yellow oil, with the molecular formula C 15 H 23 NO 3 by HRESIMS (m/z 288.1576 for 2 [M þ Na] þ ; calcd 288.1576).The 1 H-and 13 C-NMR data were very similar to those of 1 except for the presence of the methylene group (d H 3.07, t, J ¼ 5.6 Hz; d C 32.6) in 2 and absence of the methyne group (d H 5.09, t, J ¼ 4.4 Hz; d C 72.1) in 1.These data indicated that the six-membered ring has been opened and amino group in 1 was replaced by a hydroxyl group in 2. This hypothesis was based on HRESIMS and confirmed by observing HMBC correlations from H-5 to C-2, C-6, C-7 and C-8, from H-6 to C-5, C-7, C-8, from H-7 to C-5, C-6, C-8.Therefore, the structure of 2 was identified and named as penicillamide D.
Compound 3 was obtained as a yellow oil, with the molecular formula C 15 H 23 NO 2 by HRESIMS (m/z 250.1789 for 3 [M þ H] þ ; calcd 250.1762).The 1 H-and 13 C-NMR data (Table S1) of 3 were the same as (þ) penicilactam A (Yang et al. 2018).Thus, the planar structure of 3 was identified.The absolute configuration at C-6 in the center of the palm of compound 3 was compared with the reference by testing the optical rotation, and the result [a] 24 D þ53.8 (c 0.08, CH 3 OH) of 3 was well consistent with the reference [a] 24 D þ55.6 (c 1.80, CH 3 OH) (Yang et al. 2018), so compound 3 was identified as (þ) penicilactam A.
All compounds were evaluated for antibacterial activity against Staphylococcus aureus subsp, Candida albicans, Escherichia coli (Table S2) by micro method, and ciprofloxacin was used as positive control.The results showed that compounds 5 and 6 had certain antibacterial activity against C. albicans, with MIC value of 50 lg/mL (the MIC value of positive control ciprofloxacin was 50 lg/mL).
The insecticidal activity of all compounds against H. armigera Hubner.(Table S3).Compounds 1-3 showed the modest lethal activity against H. armigera Hubner, with IC 50 value of 150 lg/mL, 200 lg/mL, and 100 lg/mL, respectively.The IC 50 value of positive control ciprofloxacin was 50 lg/mL.

General procedures
IR spectra were recorded on a Nicolet 6700 spectrophotometer.UV spectra were recorded on a Beckman DU 640 spectrophotometer.ECD spectra were measured by Biologic MOS450-SFM300 instrument.Optical rotation spectra were recorded on a Modular polarimeter MCP 5100.NMR spectra were recorded on a Bruker AV spectrometer (400 MHz for 1 H NMR and 100 MHz for 13 C NMR).TMS was used as an internal standard.HRESIMS spectrometer were measured on a Q-TOF Ultima Global GAA076 LC mass spectrometer.Semi-Preparative HPLC was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse XDBC18 column (9.4 Â 250 mm, 5 lm).Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200-300 mesh), octadecyl silyl silica gel (YMC; 12 nm-50 lm) were used for column chromatography (CC).Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin layer chromatography (TLC).

Fungal material
The fungal strain PQJ-2 was previously isolated from the leaf of invasive plants Sphagneticola trilobata collected in August 2020 from Haikou, China.This strain was identified as Penicillium rubens according to its morphological characteristics and sequencing of the ITS region (GenBank no.OM269040).The strain was deposited in the Key Laboratory of Tropical Medicinal Resources Chemistry of Ministry of Education, Hainan Normal University.
After cleaning the fresh plants of Sphagneticola trilobata, it is divided into four parts: leaf, stem, flower and root.Under sterile conditions, the four parts are cut into small sections respectively, and the surface is sterilised and disinfected with 75% alcohol for 3 minutes, rinsed 3 times with sterile water, soaked in 8% NaClO for 3 minutes, rinsed 4 times with sterile water, and dried the surface water with sterile filter paper.Then use a grinding pestle to smash the four parts in a mortar.When the leaching solution appears (too dry, add a small amount of sterile water), use a pipette to suck the leaching solution into a 1.5 mL centrifuge tube and add sterile water to dilute, and then transferred to potato dextrose agar (PDA) solid medium and spread evenly with a spreader.Finally, cover the lid and seal it with parafilm, and place it in a constant temperature incubator at 25 C for constant temperature incubation.The growth of the colony was observed every day, and when the mycelium grew on the plate, the fungus was transferred to a new medium by the tip mycelium picking method to continue culturing, and purified strains were obtained after 2 to 3 times of purification.In order to check whether it is endophytic fungi in plants, a blank PDA medium was placed in a sterile operating environment during the experiment, so as to ensure that the obtained fungi were endophytic fungi of plants rather than microorganisms in the air.Finally, we selected a green filamentous fungus rich in secondary metabolites from the leaf as the target strain and identified it as Penicillium rubens.

Antibacterial activity
The antibacterial activity of the three pathogens of Escherichia coli, Staphylococcus aureus subsp and Candida albicans was tested, and ciprofloxacin was used as the positive control (Pierce et al. 2008).

Insecticidal activity
The compounded artificial feed was sterilised at high temperature, and after cooling, 5 g was weighed with a balance and dispensed into a 6-well plate.Three parallel groups were set up.The concentration gradient of each compound was 200, 100, and 50 lg/mL.Mix well with the artificial diet in the 6-well plate, DMSO is the negative control, artificial diet is the blank control, and Azadirachtin is the positive control.Add a similar size of cotton bollworm larvae (usually 2-3 instars) to each well of the 6-well plate, and then place it in a constant temperature (25 ± 1 C) and constant humidity (80%) incubator for observation for about 8 days, and recorded on the 2th, 4th, 6th and 8th days of bollworm growth, death or growth inhibition (Guo et al. 2017).Compounds 1-3 showed modest insecticidal activity against H. armigera Hubner.

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