A mycophenolic acid derivative from the fungus Penicillium sp. SCSIO sof101

Abstract Mycophenolic acid (MPA) is a group of metabolite derived from several species of Penicillium, which shows potent bioactivity. In this study, a new derivative of MPA compound named penicacid D (1), was isolated from the marine derived fungus Penicillium sp. SCSIO sof101, along with seven known compounds (2-8). Their structures were elucidated based on the HR-ESI-MS and NMR data. Moreover, the 1H and 13C NMR data of compound 2 and the 13C NMR data of compound 3 are reported. Compounds 1, 4 and 6 exhibited weak activities against Escherichia coli (clinical isolation number 100385570) and Acinetobacter baumannii (clinical isolation number 100069). Graphical Abstract


Introduction
Two-thirds of clinical drugs are directly or indirectly derived from natural products, and approximately 10% of bioactive natural products are derived from microbes (Blunt et al. 2018;Blunt et al. 2017;Demain and Sanchez 2009). Microbes are easy to be manipulated in order to obtain their metabolites, and they play a prominent role in the development of drugs for the treatment of serious diseases (Demain and Sanchez 2009). Thus, we have focused our efforts on screening for novel or bioactive metabolites from microorganisms, and have reported several families of natural products derived from microorganisms such as halogenated anthraquinones , cyclohexadepsipeptides , diketopiperazine derivatives (Luo et al. 2016), and equisetin .
The fungi of the Penicillium genus have the ability to produce diverse antibiotics, such as penicillin (anti-gram-positive bacteria activity) (Leclercq et al. 2017) and griseofulvin (antifungal activity) (Marto et al. 2016), which are prolific source for the discovery of drug leads. Mycophenolic acid (MPA) is a kind of representative metabolite of Penicillium. Due to the potent bioactivity, many derivatives of MPA have been investigated for drug development, and one derivative mycophenolate mofetil has been approved as an immunosuppressive drug (Bentley 2000;Regueira et al. 2011). The fungus Penicillium sp. SCSIO sof101 was isolated from a marine sediment sample from the South China Sea. Our previous chemical investigation of this strain led to the isolation of eight amino acid conjugated anthraquinones with immunosuppressive activity (Luo et al. 2017). The crude fermentation extracts of SCSIO sof101 showed antibacterial activities against Escherichia coli and Acinetobacter baumannii. In this study, we report the isolation, spectral data, structural elucidation, and antibacterial activities of the new mycophenolic acid derivative (1) as well as seven known compounds (2 À 8) from the antibacterial agents of SCSIO sof101. Additionally, we report of the 1 H and 13 C NMR data of compound 2 and the 13 C NMR data of compound 3 for reference.
Compound 3 was identified as a known compound. Although the 1 H NMR data in pyridine-d 5 for compound 3 has been reported, it still lacked the 13 C NMR data and structural elucidation (Jones et al. 1970). We found that the 1 H and 13 C NMR data of compounds 1 and 3 were nearly identical, with the exception that the chemical shifts of the atoms at position 3 were different in compound 1 (d H 6.49 (s, 1H), d C 99.1) compared with those in compound 3 (d H 5.2 (s, 2H), d C 70.8). Therefore, compound 3 was designated as 2-(4-methoxy-5-methyl-8-oxo-2,3,6,8-tetrahydrobenzo[1,2-b:5,6-c']difuran-2yl)propanoic acid. Compound 3 was presumed to be a degradation product of dihydromycophenolic acid (Jones, Moore and Crawley 1970). Although the biosynthesis of mycophenolic acid had been studied, the enzymes which are responsible the ring-opening/ closing of compound 3 have not been identified (Bentley 2000, Jones et al. 1970, Regueira et al. 2011). The fact that compound 1 is a 3-hydroxy derivative compound of 3 suggests there may be a hydroxylase which is responsible for this reaction.
Compounds 1-8 were evaluated for their antibacterial activities against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii. Compounds 1 and 4 showed antibacterial activities against Escherichia coli with a MIC value of 64 lg/mL, and compound 6 showed antibacterial activity against Acinetobacter baumannii with a MIC value of 64 lg/mL. Kanamycin and ampicillin were used as positive controls against Acinetobacter baumannii (MIC values of 4.0 and 2.0 lg/mL, respectively) and Escherichia coli (MIC values of 2.0 and 1.0 lg/mL, respectively). The other compounds, however, have no antimicrobial activities against these strains at a concentration of 128 lg/mL.

General experimental procedures
Column chromatography separation used 200-300 mesh silica gel from Jiangyou Silica Gel Development, Inc. Medium-pressure liquid chromatography (MPLC) was performed on a CHEETAH 100 automatic flash chromatography system (Bonna-Agela) with an ODS-A flash column (S-50 lm, 12 nm; 100 Â 20 mm, YMC). Semi-preparative HPLC was performed with a YMC-Pack ODS-A column (250 Â 10 mm, 5 lm) on a Hitachi L-2000 system. High resolution mass spectra were obtained on a Bruker MaXis quadrupole-TOF mass spectrometer. Optical rotations were obtained with a Rudolph Autopol I polarimeter. UV spectra were performed on an Agilent Cary60 spectrometer (Agilent). IR spectra were recorded on an IRAffinity-1 spectrometer (Shimadzu). NMR spectra were obtained with a Bruker Avance 500 spectrometer.

Antibacterial activities
Knowing that the crude fermentation extracts of Penicillium sp. SCSIO sof101 have antibacterial activities against Escherichia coli and Acinetobacter baumannii, compounds 1 À 8 were further evaluated for their antibacterial activities against Staphylococcus aureus (clinical isolation number 1312230073), Escherichia coli (clinical isolation number 100385570), Pseudomonas aeruginosa (clinical isolation number 202225634), and Acinetobacter baumannii (clinical isolation number 100069), which were provided by Department of Clinical Laboratory, Institute of Surgery Research, Daping Hospital, Third Military Medical University. The evaluations were performed according to the published protocols using a broth dilution method (Zhou et al. 2014). Compounds 1 À 8 were each dissolved in DMSO, serially diluted to concentrations of 1 À 128 lg/mL, and tested in triplicate in a 96-well plate using Mueller-Hinton broth (Zhou et al. 2014). Kanamycin and ampicillin were used as positive controls, while DMSO in M-H broth was used as blank control.

Conclusions
During our chemical investigation of antibacterial agents of Penicillium sp. SCSIO sof101, we isolated the new MPA derivative 1, together with the seven known compounds 2 À 8. The structure of 1 was determined from its HR-ESI-MS and NMR data, and, based on the structure, 1 was identified as 2-(4-methoxy-5-methyl-6-hydroxy-8-oxo-2,3,6,8-tetrahydrobenzo[1,2-b:5,6-c']difuran-2-yl)propanoic acid. Moreover, the 1 H and 13 C NMR data of compound 2 and the 13 C NMR data of compound 3 were reported for the first time. We found that compounds 1, 4, and 6 showed weak antibacterial activities, indicating that they may be responsible for the antibacterial activities of the fungus Penicillium sp. SCSIO sof101.

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

Funding
This study was supported in part by Chongqing Programs for Science and Technology Development (cstc2018jxjl-jbky0002) and Chongqing Municipal Bureau of Health Science and technology of traditional Chinese medicine project (ZY20170380).