New azaphilones from mangrove endophytic fungus Penicillium sclerotiorin SCNU-F0040

Abstract Two new sclerotioramines (1 and 2) and a new natural product of sclerotioramine analog (3), together with seven known compounds have been isolated from the mangrove endophytic fungus Penicillium sclerotiorin SCNU-F0040. Their structures were identified based on the 1 D, 2 D NMR and HRESIM spectra. The absolute configurations of new compounds were deduced by specific rotation data and electronic circular dichroism spectra. All the isolated new compounds were tested on anti-diabetes activity by using a-glucosidase inhibition assay and anti-inflammatory activity by using cyclooxygenase inhibition assay, respectively. Compounds 1 and 2 have a-glucosidase inhibition activity with IC50 values of 102.3 and 217.5 μM. Compound 2 shows a moderate cyclooxygenase-2 inhibitory activity with an IC50 value of 47.8 μM. Graphical Abstract


Introduction
Azaphilones are a class of polyketides mainly derived from fungi and structurally characterized with a highly oxygenated pyrone-quinone bicyclic core and a chiral quaternary centre. Up to now, over six hundred azaphilones of 18 categories have been reported from different fungi, remarkably the Penicillium (Gao et al. 2013;Chen et al. 2020). Sclerotiorin is one of the most important structural types of azaphilones. Based on the pyrone-quinone motif of azaphilones, sclerotiorin possesses an additional side chain at C 3 of pyrone skeleton, namely, 3, 5-dimethyl-1, 3-heptadiene (Gao et al. 2013). Sclerotioramines were generated by sclerotiorin reacting with endogenous ammonia or exogenous diverse amino compounds during the process of fungal culture (Lin et al. 1992;Jung et al. 2003). Sclerotiorin and sclerotioramines were found to have a variety of biological activities, such as anti-microbial (Zhou et al. 2016), cytotoxic (Luo et al. 2018), anti-malarial, anti-acetylcholinesterase (Hemtasin et al. 2016) and anti-inflammatory activities (Liu et al. 2019). Due to their structural diversity and promising bioactivity, researchers are interested in the total synthesis and derivatization of azaphilones, such as the total synthesis of chaetoglobin A by Kang et al and the synthesis of sclerotiorins derivatives by Fern andez et al. These synthesized azaphilone analogs were found to have cytotoxic activity and fungicidal activity (Ge et al. 2008;Lin et al. 2012;Kang et al. 2018). Investigation on the metabolites of the mangrove endophytic fungus Penicillium sclerotiorin SCNU-F0040 led to the isolation of two new sclerotioramines named sclerketide E (1) and sclerketide F (2), one new natural product of sclerotioramine analog sclerketide G (3) together with seven known compounds (4-10) ( Figure 1). Furthermore, the cytotoxicity, a-glucosidase and cyclooxygenase inhibitory activities have been screened for compounds 1 and 2. Details of the isolation, structure elucidation and biological activities of the metabolites from Penicillium sclerotiorin SCNU-F0040 are described herein.
Compound 3 was obtained as red powder and its molecular formula was deduced to be C 21 H 26 ClNO 4 based on the HRESIMS positive ion peak at m/z 392.1618 [M þ H] þ (calcd for C 21 H 27 ClNO 4 , 392.1550), suggesting 9 degrees of unsaturation. The 1 H and 13 C NMR spectra of compound 3 were very similar to those of isochromophilone VI (6) (Arai et al. 1995), except for an acetyl group less in compound 3. The mass spectrometry data also identified the missing of 42 mass unit comparing with isochromophilone VI (6). The above results revealed that compound 3 was the hydrolysis product of isochromophilone VI (6). Furthermore, the absolute configuration at C-7 of compound 3 was biogenetically expected to be R based on the same positive specific rotation value ([þ315 (c 0.05, methanol)]) as in compounds 1 and 2. Interpretation of the HRESIMS and NMR data for 3 revealed that our metabolite matched the structure of 5-chloro-3-[(1E, 3E)-3, 5-dimethylhepta-1, 3-dienyl]-7-hydroxy-2-(2-hydroxyethy-l)-7-methylisoquinoline-6, 8-dione, which had been previously sold as commercial source. However, we are unable to find any structural data published for this substance, so this is the first report on the planar structural data and absolute configurations of 3.
In this study, the a-glucosidase inhibitory activity of new compounds 1 and 2 was assayed and the acarbose was selected as a positive control. Compounds 1 and 2 displayed a slightly more potent inhibitory activity than acarbose with IC 50 values of 102.3 and 217.5 lM, respectively (acarbose, 396.3 lM) (Table S3). Moreover, compounds 1 and 2 were first assayed for their anti-inflammatory property by cyclooxygenase COX-1 and COX-2. The result disclosed compound 2 shows a moderate cyclooxygenase-2 inhibitory activity with an IC50 value of 47.8 lM. Furthermore, compounds 1 and 2 were evaluated for their cytotoxic activity in vitro against A549 and MDA-MB-435 human cell lines and exhibited no obvious activity at 50 lM (Table S3).

Fungal material
The fungal strain SCNU-0040 was isolated from the fresh leaf of the mangrove plant Bruguiera gymnorhiza collected from Zhanjiang Mangrove Nature Reserve in Guangdong province, China. The fungus was obtained using the standard protocol for isolation. The sequence data of the fungal strain have been deposited at Gen Bank with accession no. MW-541637. A BLAST search result showed that the sequence was the most similar (100%) to the sequence of P. sclerotiorum. A voucher strain was deposited in School of Chemistry, South China Normal University, Guangzhou, China, with the access code SCNU-F0040.

Biological assay
3.4.1. Cytotoxic activity Cytotoxic activity was determined according to the MTT method as described in our previous report (Chen et al. 1975). The cytotoxicity of compound 1 and 2 at a serial final concentration of 50, 25, 12.5, 6.25, and 3.125 lM were evaluated against A549 (human lung cancer cells) and MDA-MB-435 (breast cancer cells) (Chen et al. 2016).

In vitro inhibition studies on a-glucosidase
An assay of a-glucosidase inhibitory activity by the enzyme-inhibitor approach using p-nitrophenyl-a-D-glucopyranoside (pNPG) as a substrate was based on our previous work (Yan et al. 2020). All the assays were carried out under 0.01 M potassium phosphate buffer (PH ¼ 6.8), using a Bio-Rad iMark microplate reader (Bio-Rad Laboratories, Inc., Kyoto, Japan). Enzyme solution was prepared to give 1.25 units per mL in 1 mL buffer solution. Diluted enzyme solution (20 lL), test samples (10 lL, in DMSO) and buffer solution (160 lL) were mixed in each well of a 96-well microtiter plate. After pre-incubated for 20 min at 37 C, p-Nitrophenyl-b-D-glucoside (p-NPG, 10 lL, 1.5 mg/mL) was added to start the enzymatic reaction measured by a Bio-Rad iMark microplate reader at 405 nm at 37 C immediately. Calculation was performed according to the equation: g (%) ¼ [(B-S)/B] Â 100% (B stands for the assay medium with DMSO; S stands for the assay medium with inhibitor). All measurements were done in triplicate from three independent experiments. The reported IC 50 was the average value of three independent experiments.

Cyclooxygenase inhibitory activity
Compounds were evaluated for cyclooxygenase inhibitory activity in vitro by using COX-1 and COX-2 (human) inhibitor screening assay kits (Item NO:701070 and 701080) supplied by Cayman chemicals USA, respectively. The assay is based on the competition between prostaglandins (PGs) and a PGacetylcholinesterase (ACHE) conjugate (PG tracer) for a limited amount of PG antiserum. The cyclooxygenase of percent inhibition in test compound was inversely proportional to the amount of PGs produced by each wells. Diclofenac sodium was used as a positive control and the tested compounds was dissolved in DMSO. The amount of PGs produced by enzyme in the presence of the test compounds was measured and compared with the control experiments. Finally, the calculations were performed as per the kit guidelines. All measurements were done in triplicate from three independent experiments. The reported IC 50 was the average value of three independent experiments. The experimental method of COX-2 inhibitory activity was also described in our previous work (Chen et al. 1975).

Conclusions
Chemical study of Penicillium sclerotiorin SCNU-F0040 collected from Zhanjiang Mangrove Nature Reserve led to the isolation and identification of two novel compounds (1 and 2) together with a new natural product (3) and seven known compounds (4-10). The biological evaluation for compounds 1 and 2 were carried out, and the results disclosed two new compounds showed a moderate inhibitory activity against a-glucosidase and compound 2 showed moderate inhibitory activity COX-2.

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

Funding
The authors gratefully acknowledge grants from the National Natural Science Foundation of China, and the Natural Science Foundation of Guangdong Province.