4-Phenylcoumarins from Mesua ferrea with selective CYP1B1 inhibitory activity

Three new 4-phenylcoumarins, mesuaferlinns A–C (1–3), together with ten other known 4-phenylcoumarins, 4–13, were isolated from the branches and leaves of Mesua ferrea Linn. (Clusiaceae). The structure of compounds 1–3 were determined on the basis of spectroscopic methods including extensive analysis of NMR and mass spectroscopic data. Ten 4-phenylcoumarins were tested for their cytochrome P450 family 1 enzymes (CYP1A1, CYP1A2, and CYP1B1) inhibitory effects. Compounds 5 and 10 were found to be the most potent two CYP1B1 inhibitors with inhibitory viabilities values of 56.64% and 47.46%, respectively. Graphical abstract Graphical abstract


Introduction
P450 family 1 enzymes include CYP1A1, CYP1A2, and CYP1B1, which are important environmental xenobioticmetabolizing enzymes. Cytochrome P4501B1 (CYP1B1) is a heme-thiolate monooxygenase involved in NADPHdependent phase I metabolism of a variety of xenobiotics such as ethoxyresorufin, theophylline and caffeine, and shows activity toward activation of environmental carcinogens via the hydroxylation of procacinogens, including 27 polycyclic aromatic hydrocarbons and their derivatives, 17 heterocyclic and aryl amine and aminoazo dyes, 3 mycotoxins, 2 nitroaromatic hydrocarbons [1,2]. Different from P450s 1A1 and 1A2, CYP1B1 expression is not detected in human liver, but CYP1B1 is expressed in many extrahepatic tissues, such as the lung, colon, eye, and kidney, are also highly expressed in a wide of variety of cancers, such as prostate, uterus, and colon cancer. Therefore, selective inhibition of P450 1B1 is a potential molecular target for chemoprevention of environmental hydroxylation-caused These authors contributed equally: Fengxu Zhou, Ruoyue Huang DNA mutations and carcinogenesis. It has been reported that furanocoumarin could inhibit CYP1 enzyme and regulated the skin cancer induced by AHR in vitro [3]. AHR affects the major stages of tumorigenesis-initiation, promotion, progression and metastasis-and physiologically relevant AHR ligands are often formed during disease states or during heightened innate and adaptive immune responses, and studies of aggressive tumors and tumor cell lines have shown that AHR is chronically activated in tumors, thereby promoting tumor progression [4,5]. Coumarin is a kind of important compounds which have been shown to effectively inhibit CYP1B1, but coumarin has its potential ability to activate the aryl hydrocarbon receptor (AhR), which may lead to up-regulation of P450 [6,7]. In this work, to obtained new and selective P450 1B1 inhibitors, we performed a two-step design, isolation and evaluation on P450 family 1 enzymes.
The species Mesua ferrea Linn. belongs to the family Clusiaceae, which is a medium-to-large sized evergreen tree. Generally, it is widely distributed in Southeast Asia, India, and Sri Lanka. The dried floral bud of this plant is commonly known in India as Nagakesara and traditionally used as brain tonic appetizer, antiemetic, anthelmintic, aphrodisiac diuretic and antidote [8,9]. Previous studies on M. ferrea resulted in the isolation of xanthones, coumarins, triterpenoids, biflavones, cyclohexanedione derivatives, and an essential oil. Most of these isolated compounds were reported to show cytotoxic and antibacterial activities [10][11][12][13][14][15]. In our ongoing search for selective and inhibitory P450 1B1 coumarins from this plant, an investigation of the chemical constituents of Mesua ferrea Linn., which was collected in Gengma county, Yunnan Province, led to isolation of three new 4-phenylcoumarins, mesuaferlinns A-C (1-3), together with ten known 4-phenylcoumarins, 4-13. This report deals with the isolation and characterization of the new 4-phenylcoumarins. In addition, we describe the inhibitory effects of these 4-phenylcoumarins isolated in this study on P450 family 1 enzymes. And to identify the influence of tested inhibitors on CYP1B1 expression, an AhR-signaling assay was also performed.
This study was expected to study the inhibitory effects of these new compounds on CYP1B1, and provided evidences to be used as CYP1B1 enzyme inhibitors.
The characteristic singlet of H-3 of a 4-substituted coumarin was observed at δ(H) 6.07 ppm. And the presence of a monosubstituted phenyl group at C-4 was deduced by the presence of five aromatic proton signal at δ(H) 7  In the HMBC spectrum, C-OH (δ(H) 14.72) correlated with a carbonyl (C-1″′), together with the 1 H, 13 C NMR, UV, and IR spectra (Table 1) which indicated the presence of a coumarin skeleton with all positions substituted except C-3 and 8-acyl-5,7-dioxygenated-coumarin type, hence confirming the attachment of the 2-methylbutanoyl group at position C-8 in the coumarin skeleton. Assignments of the 1 H and 13 C NMR resonances of compound 1 (Table 1), which was named mesuaferlinn A, were determined through analysis of its COSY, HMQC, and HMBC data.
The IR spectrum exhibited strong absorptions at ν max 1371 cm −1 due to a gem-dimethyl, a peak at 1611 cm −1 belonging to a chelated acyl group and a broad peak at 3434 cm −1 due to a chelated hydroxyl group stretching [17].
All protons and carbons were assigned by analysis of 1D and 2D NMR spectroscopic data. The NMR spectroscopic data of 2 was quite close to those of 1, except that an isopropyl group attached to carbonyl C-1″′ (δ(C) 209.9) in place of a 2-methylbutanoyl moiety in 1. HMBC correlations clearly proved that the substituted dihydrofuran ring is located between δ(C)C-7 (s, 164.6) and C-8 (s, 106.4) by exhibiting the following correlations H-3″′ a, b/C-7, C-8, and H-2″′/C-8. A close comparison of the NMR data of 3 with those of the known compound mammea A/AA deshydrocyclo F indicated that the two compounds were identical except for the acyl substituents at C-6 [16]. Considering the structural resemblances of compound 3 and mammea A/AA deshydrocyclo F, the relative configuration of C-2 of 3 was suggested to be β due to the key ROESY correlations of H-2″′ (δ(H) 4.54) with H-3″′a (δ(H) 3.07) and H-3″′b (δ(H) 3.26), in combined with lacking direct ROESY correlations of H-5″′a (δ(H) 4.87), H-5″′b (δ(H) 5.06) and H-6″′ (δ(H) 1.90) with H-3″′a and H-3″′b (Fig. 2). So, the complete structural elucidation of 3 as mesuaferlinn C was made with the aid of COSY, HMQC, HMBC and ROESY spectra.

Effects on activation of AhR
The aryl hydrocarbon receptor (AhR) was a ligand-activated transcriptional factor that dimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT) [21]. And it was reported that CYP1A1 performs as target gene of AhR [22]. To determine whether the expression of AhR was functionally relevant, six compounds were given further QPCR experiment. The results of MTT assay showed that all compounds had no cytotoxicity (Fig. 3). And the results of the QPCR experiment supposed that at the gene level, all compounds could significantly activate the AhR target gene CYP1A1 (Fig. 4).

Evaluation of nicotinamide adenine dinucleotide phosphate (NADPH) dependent experiment
In order to further demonstrated the formation of inhibitory activity of tested compound, the reactive metabolites 4-Hydroxy-β-estradiol produced in mouse liver microsomes (MLMs) was determined. Respectively, estradiol was transformed to 4-Hydroxy-β-estradiol in the NADPHregenerating system, whereas the conjugates could not be detected in the MLMs incubation without NADPH (Fig. 5).

Molecular docking
Molecular docking results (Fig. 6) showed that compounds 1, 5 and 10 could bind to CYP1B1 enzyme compared with positive control resveratrol. There were conventional hydrogen bonding and pi-cation between resveratrol and CYP1B1 enzyme, conventional hydrogen bonding and pialkyl between the compound 1 and CYP1B1 enzyme, conventional hydrogen bonding and alkyl between the compound 5 and CYP1B1 enzyme, as well as conventional hydrogen bonding, pi-alkyl and pi-lone pair between the compound 10 and CYP1B1 enzyme. Therefore, it was speculated that the inhibition effect of these compounds on CYP1B1 is directly combined, rather than caused by AHR pathway.

Conclusions
The 13 coumarins isolated from M. ferrea Linn., including three new coumarins, showed similiarities of a phenyl group in C-4, an isopentenyl or acyl group in C-6 or C-8. Generally, these coumarins could be classified into three groups: simple coumarin, pyranocoumarin, and furanocoumarin. And ten of them was tested with mouse liver microsomes culture experiment of CYP1 enzymes, and exhibited varied levels of inhibitory potencies. Almost compounds displayed inhibitor activities to CYP1A1 except compound 4, which was totally different from the opposite off non inhibitor to CYP1A2. Compounds 5 and 10 were found to be the most potent two CYP1B1 inhibitors with inhibitory viabilities values of 56.64% and 47.46%. and as a general trend, furanocoumarin was more active than other coumarins in CYP1B1 inhibition. According to the result in Table 2, we could see that when the terminal hydrogen of the 6-position substitution was 3 atoms away from the parent nucleus, it tended to be lacking in inhibitory activity, even showing the opposite agonistic activity. This might result from the presence of a π-π conjugation effect that kept the corresponding binding site out of reach. Further AhR signaling experiment had taken in 3 compounds, and all of them could activate the AHR target gene CYP1A1. However, molecular docking results showed that they may directly bind CYP1B1 enzyme, rather than through the AHR pathway. NADPH dependent experiment indicated that the compounds could take CYP1B1 inhibitory activity in NADPH involved situation.
In conclusion, the potential use of furanocoumarin is as a selective inhibitor of P450 1B1 to prevent DNA mutations and carcinogenesis caused by environmental hydroxylation through the inhibition of P450 1B1.

Reagents
Nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse liver microsomes (MLMs) were obtained from Research Institute for Liver Diseases (Shanghai) Co., Ltd. The inhibitors resveratrol and ɑ-Naphthoflavone were purchased from Shanghai Maclin Biochemical Technology Co., LTD or Shanghai yuanye Bio-Technology Co., Ltd. β-estradiol were also purchased from Shanghai Maclin Biochemical Technology Co., LTD. 4-hydroxyestradiol were purchased from Cayman Chemical Company. All solvents (methanol and acetonitrile) were UPLC-MS (ultraperformance liquid chromatography-mass spectrometry) grade and reagents were analytical grade.

Plant material
The

Enzyme activity inhibition assays of CYP1A1, CYP1A2 and CYP1B1
The methods for measuring CYP enzyme activity were described in previous literature [23] and were briefly described as follows: the incubation system was carried out in 96-well plates, and the reaction system consists of β-estradiol (20 μM), the chemical inhibitor or the compound, which the concentration was the same as that of the inhibitor, MLM (0.5 mg/ml), and buffer solution (PBS, PH = 7.4). The chemical inhibitors of CYP1A1, CYP1A2 and CYP1B1 enzyme activity were α-naphthalenone (10.0 μM), α-naphthalenone (1.0 μM) and resveratrol (10.0 μM), respectively. After the reaction system was incubated, NADPH was added for reaction, the reaction was terminated and centrifuged, and the supernatant was taken for testing to determine the inhibitory effect of the compounds on CYP1s activity by UPLC-MS. Incubation system in triplicate. The groups were as follows: ①Positive group: the inhibitory, β-estradiol and NADPH. ②Negative group: no NADPH, replaced with equal volume PBS. ③Experimental group: β-estradiol, the compound and NADPH.
A series of compounds were added to the incubation system to test IC 50 , which was the same as that of CYPs inhibition assay (the final concentration in the system was 1, 5, 20, 50, 100 µM).

UPLC-ESI-QTOF-MS analysis
All samples were analyzed on a high-resolution Q Exactive Plus hybrid quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA). The drug metabolites were transported on an XDB-C18 column (2.1 × 100 mm, 1.8 mm, Agilent, Santa Clara, CA). The injection volume was 5ul, and the liquid flow was 0.3 ml/ min. Phase A was 0.01% formic acid aqueous solution, and phase B was acetonitrile containing 0.01% formic acid. The elution gradient was as follows: 0-12 min, 2-98%B; 12-14 min, 98%B; 14-16 min, 98%A. The column temperature was 45°C. The data were in positive ion mode. The flow rate of collision gas and dry gas was 9 L/min. Capillary voltage 3.5 kV, temperature 350°C, atomizer pressure 35 psi.

NADPH-dependency assay
The incubation system and the samples analysis of the assay was the same as that of CYPs inhibition assay. The groups were as follows: ①Sample group: β-estradiol, the compound and NADPH. ②N-NADPH group: β-estradiol and the compound, no NADPH, with equal volume of PBS instead. ③Control group: β-estradiol and NADPH.

Cell culture and cell activity assay
The human hepatoma cell line HepG2 were purchased from Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and tested by STR. The cells were preserved in Dulbecco's modified Eagle medium (DMEM), supplemented with 1% penicillin-streptomycin solution and 10% fetal bovine serum in a humidified environment of 37°C and 5% CO 2 . 2 × 10 4 cells/well were cultured on 96-well plates in 200 µL DMEM medium, and the cells were incubated with compounds (2.5, 12.5, 25 or 100 µM) for 24 h, then MTT solution was added to detect the cell activity [24].

AhR-signaling assay
Compounds or diosmine, the AHR agonist, were added to cells to determine the effect of compounds on AHR signaling pathways. 2 × 10 4 cells/well were cultured on 12-well plates in 2 mL DMEM medium, and the cells were incubated with compounds (25 µM) or diosmin (25 µM) for 24 h. RNA for the cells was extracted and QPCR was performed to determine the expressions of CYP1A1 mRNA.

Molecular docking
Discovery Studio 2020 software was used for molecular docking, and the experimental process was described in previous literature [25]. Briefly as follows: 3D structure of CYP1B1 enzyme (PDB ID: 6OYU) was downloaded from RCSB -PDB databases (http://www.rcsb.org/pdb/), processed the protein and defined the active site. After resveratrol and these compound molecules are processed, CDOCKER docking is performed.

Data and statistical analysis
Chromatographic and spectral data were analyzed using QE Plus data acquisition software. All values were expressed as mean ± SD, and Graphpad Prism software 9.0 (Inc., La Jolla, CA)) was used for statistical analysis to obtain IC 50 value. The Student's unpaired t-test was employed for statistical analysis. Compared with control group, p < 0.05 indicated a significant difference.

Data availability
The date that supports the findings of this study are available in the supplementary information of this article.