5-Demethoxy-10′-ethoxyexotimarin F, a New Coumarin with MAO-B Inhibitory Potential from Murraya exotica L.

Rutaceae plants are known for being a rich source of coumarins. Preliminary molecular docking showed that there was no significant difference for coumarins in Clausena and Murraya, both of which had high scoring values and showed good potential inhibitory activity to the MAO-B enzyme. Overall, 32 coumarins were isolated from Murraya exotica L., including a new coumarin 5-demethoxy-10′-ethoxyexotimarin F (1). Their structures were elucidated on the basis of a comprehensive analysis of 1D and 2D NMR and HRMS spectroscopic data, and the absolute configurations were assigned via a comparison of the specific rotations and the ECD exciton coupling method. The potential of new coumarin (1) as a selective inhibitor of MAO-B was initially evaluated through molecular docking and pharmacophore studies. Compound (1) showed selectivity for the MAO-B isoenzyme and inhibitory activity in the sub-micromolar range with an IC50 value of 153.25 ± 1.58 nM (MAO-B selectivity index > 172).


Introduction
Depression, Alzheimer's disease, and Parkinson's disease, as three major neuropsychiatric diseases, have seriously affected human health and quality of life. Monoamine oxidase (MAO, EC 1.4.3.4) is a flavoenzyme bound to the mitochondrial outer membranes of the cells, which is responsible for the oxidative deamination of neurotransmitters and dietary amines. It exists in two isoforms, MAO-A and MAO-B. Although sharing 70% sequence identity, MAO-A and B displayed different substrate and inhibitor specificities; serotonin and norepinephrine are preferentially metabolized by MAO-A and phenylethylamine, benzylamine, and dopamine by MAO-B, whereas clorgyline and L-deprenyl selectively inhibit MAO-A and B, respectively. They are the well-known target for antidepressants, Parkinson's disease, and neuroprotective drugs [1][2][3][4]. Monoamine oxidase inhibitors (MAOIs) were used for the treatment of various neurodegenerative disorders. However, they are associated with serious side effects and lack efficacy and selectivity for a single MAO isoform. It is commonly accepted that the discovery of MAOIs from herbal sources is an important strategy for drug design and development to treat various neurodegenerative diseases such as depression, anxiety, Parkinson's disease, and Alzheimer's disease [5].
Plant-derived coumarins are an important class of compounds due to their significant biological activities. Coumarins are abundantly found in species of the Rutaceae family, such as the genus Clausena and Murraya [6][7][8][9][10][11][12]. Our previous studies obtained a novel coumarin, anisucoumaramide, from Clausena anisum-olens, which was found to exhibit high

Molecular Docking of Coumarins in Clausena and Murraya
Through a previous literature collection, 141 known coumarins were found in Clausena plants and 177 coumarins found in Murraya plants. Molecular docking studies on coumarins from Clausena and Murraya were carried out, and the potential inhibitory activity of monoamine oxidase B of the two was compared. The docking results are shown in the following table (Table 1). A negative scoring value indicated that they could bind to the receptor protein. A smaller scoring value indicated better binding [14]. The results showed that only the compounds had better scores, those with scores below −85. The preliminary screening results showed that 40 of the coumarins in Clausena had the better scoring value, accounting for 28.4% of the total number, and 48 of the coumarins in Murraya had the better scoring value, accounting for 27.1% of the total number. The coumarins of Murraya and Clausena have similar molecular docking scores; there was no significant difference between the two. Therefore, Murraya could be a highly valuable research resource for its coumarins that have MAO-B inhibition potential. The powdered leaves and twigs of Murraya exotica L. (2.5 kg) were repeatedly extracted with 95% EtOH at room temperature. The extract was then concentrated under reduced pressure to give a brown syrup, which was suspended in water and successively partitioned with petroleum ether, ethyl acetate (EtOAc) and n-butanol (n-BuOH). The EtOAc fraction was subjected to a multi-step chromatography procedure to yield 32 coumarins (Figure 1), including a new coumarin (1).

Virtual Screen of Coumarins Isolate from Murraya exotica L.
A total of 32 coumarin molecules from Murraya exotica L. were screened by molecular docking with the ligand C18_1503 in the target protein as the reference and the coumarin anisucoumaramide as the positive control. The results are shown in the following Table 3. The scores showed that nine coumarins were better, accounting for 28.1% of the total number, which was similar to the results in the previous study. In addition, the new coumarin 5-demethoxy-10′-ethoxyexotimarin F (1) had a higher scoring value and a higher docking advantage than the positive control anisucoumaramide and the original ligand C18_1503. Therefore, 2D diagrams of the interactions between the new coumarin (1), anisucoumaramide and C18_1503 and protein crystals were displayed, respectively (Figure 4). It could be found that all three had hydrogen bond interaction, spatial interaction with residue Ile 199, and hydrogen bond interaction with residue Tyr 326, which was consistent with the action reported in the literature [32,33]. Therefore, the new compound 5-demethoxy-10′ethoxyexotimarin F (1) had a certain selective inhibition potential for MAO-B.   In the pharmacophore study of MAO-B selective inhibitor, the pharmacophore model 01 had the highest CAI value (see Table 4). Moreover, its other indicators were also high. Therefore, the pharmacophore model 01 was determined as that optimal pharmacophore. According to the obtained optimal pharmacophore, 32 coumarins isolated from Murraya exotica L. were virtually screened. The screening results are shown in Table 5. Overall, 27 molecules could be matched with the pharmacophore, indicating that the coumarins in Murraya exotica L. had high overall matching with the pharmacophore model of MAO-B selective inhibitor. The matching of each molecule was evaluated based on the Fit Value. A higher value indicated a higher degree of matching between the molecule and the pharmacophore model. As can be seen from the table, the new compound 5-demethoxy-10 -ethoxyexotimarin F (1) has a high matching property and, therefore, a high potential as a selective inhibitor of MAO-B.   (7) 1.51473 auraptenol (18) 1.23251 pranferin (31) 1.2031 murranganon (19) 1.14868 isomeranzin (10) 0.912292 murrangatin (28) 0.79533 murrangatin acetate (21) 0.734945 minumicrolin (30) 0.725833 meranzin (5) 0.536345 albiflorin-3 (22) 0.476371 osthol (4) 0.316498 isomurralonginol (24) 0.265571 phebalosin (6) 0.244483 isomurralonginol acetate (9) 0.209117 trans-dehydroosthol (3) 0.104663 7-methoxy-8-(2-formyl-2-methylpropyl)coumarin (12) 0.0957507

Biological Activity of Coumarin 1
The potential inhibitory effects of the new coumarin (1) were evaluated on human recombinant monoamine oxidase (hMAO) isoforms. The inhibitory effects were assessed by measuring the production of H 2 O 2 from p-tyramine using the Amplex Red MAO assay kit with selegiline and iproniazide as reference drugs. The IC 50 values and MAO-B selectivity indices for the inhibitory effects of both the new compound and reference inhibitors were calculated (  (1) shows selectivity for the MAO-B isoenzyme and inhibitory activity in the sub-micromolar range. The anti-inflammatory activity of (1) was evaluated by measuring the inhibition against LPS-induced NO production in RAW264.7 cells. Compound (1) displayed no inhibitory effect, showing only a 34.59% inhibition, less than 50%, compared with NGmonomethyl-L-arginine (L-NMMA) with an inhibition rate of 56.13%. Meanwhile, the acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity of compound (1) was assayed. (1) did not show any inhibitory activity at a concentration of 50 µM. Tacrine (0.33 µM) was used as the positive control and showed 50.1% inhibition. The α-glucosidase enzymatic activity of compound (1) was investigated and found to be inactive since its percentage of inhibition was less than 50% at a concentration of 50 µM compared to 53.17 % inhibition of quercetin (positive control). Compound (1) did not lead to a significant inhibitory effect against four bacterial pathogens, Salmonella enterica (ATCC14028), Staphylococcus aureus (ATCC29213), Escherichia coli (ATCC25922), and Pseudomonas aeruginosa (ATCC27853), and fungal pathogen Candida albicans (ATCC10231) at a concentration of 100 µM.

General Experimental Procedures
The specific optical rotation data were acquired on a Rudolph Autopol III automatic polarimeter (Rudolph Research, Fairfield, NJ, USA). The UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer (Thermo Nicolet, Vernon Hills, IL, USA). The ECD data were acquired on a JASCO 810 CD spectrophotometer (Jasco Corporation, Tokyo, Japan). NMR spectra were recorded on a Brucker-400 and 600 NMR spectrometer (Bruker Corp. Billerica, MA, USA), with tetramethylsilane as an internal standard. HRESIMS experiments were measured on a Waters Xevo G2 Q-TOF mass spectrometer (Waters MS Technologies, Manchester, UK). Silica gel (100-200 mesh or 200-300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) were used for open CC. TLC analyses were carried out on the pre-coated silica gel GF254 plates (Qingdao Marine Chemical Co. Ltd., Qingdao, China). The spots were visualized under the UV lights (254 nm and 365 nm). All of the solvents were distilled prior to use.

Plant Materials
The leaves and twigs of Murraya exotica L. were collected in Hekou County, Honghe Prefecture, Yunnan Province in May 2019 and identified by Professor Chen Yu. The sample was deposited in the School of Chemical Science and Technology, Yunnan University.

Molecular Docking
Molegro Virtual Docker 6.0 (MVD 6.0, 2013, Molexus, Odder, Denmark) semi-flexible docking software is mainly used, which is higher than the docking accuracy of most software on the market. The key is that it comes with a number of different scoring functions and search algorithms. The combination of the two can obtain the docking algorithm for the receptor protein. After repeated attempts using different scoring functions and search algorithms in the software, a docking method with good repeatability was finally obtained [34]. The RMSD value when reproducing the original protein-ligand was 1.066, less than 2, indicating that the docking method was available. In the finally determined docking method, the PLANTS Score [GRID] is used for the scoring function, and Iterated Simplex is used for the search algorithm. In this study [35], the protein crystal 2V61 obtained from the PDB database was combined with the selective inhibitor 7-(3-chlorobenzyloxy)-4-(methylamino) methyl-coumarin, providing more reference value for the docking analysis of coumarin compounds [36].

Pharmacophore Model
The pharmacophore model was constructed based on the common pharmacodynamic characteristics [37], and the active compounds related to MAO-B selective inhibitor were obtained by using the protein code P27338 of protein crystal 2V61 and the Binding Data Base database. The training set consisted of 17 active compounds. The specific structure and IC 50 activity values are shown in Figure 5. The test set consisted of 50 active compounds and 100 inactive compounds [38][39][40][41]. Pharmacophore model evaluation is based on the CAI system, and the evaluation results are shown in Table 4 [42][43][44].
The pharmacophore model was constructed based on the common pharmacodynamic characteristics [37], and the active compounds related to MAO-B selective inhibitor were obtained by using the protein code P27338 of protein crystal 2V61 and the Binding Data Base database. The training set consisted of 17 active compounds. The specific structure and IC50 activity values are shown in Figure 5. The test set consisted of 50 active compounds and 100 inactive compounds [38][39][40][41]. Pharmacophore model evaluation is based on the CAI system, and the evaluation results are shown in Table 4 [42][43][44].

In Vitro MAO Inhibitory Assay
The effects of (1) on the hMAO isoform enzymatic activity were evaluated by measuring the effects on the production of H2O2 from p-tyramine using a fluorimetric method The effects of (1) on the hMAO isoform enzymatic activity were evaluated by measuring the effects on the production of H 2 O 2 from p-tyramine using a fluorimetric method following the experimental protocol previously described [6]. Selegiline and iproniazide served as reference inhibitors.

Anti-Inflammatory Assay
The Nitric oxide (NO) production inhibition of the compounds (1) was determined using a procedure described before [45].

Acetylcholinesterase/Butrylcholinesterase Inhibitory Activity
Acetylcholinesterase/butyrylcholinesterase (AChE/BuChE) inhibitory activity of the isolated compound (1) was assayed by the spectrophotometric method developed by Ellman's method with slight modification [46]. S-Acetylthiocholine iodide, S-butyrylthiocholineiodide, 5,5 -dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman's reagent), acetylcholinesterase and butyrylcholinesterase derived from human erythrocytes were purchased from Sigma Chemical (St. Louis, MO, USA). The compounds were dissolved in DMSO. The reaction mixture (totally 200 µL) containing phosphate buffer (pH 8.0), test compound (50 µM), and acetyl cholinesterase (0.02 U/mL) or butyrylcholinesterase (0.016 U/mL), was incubated for 20 min (37 • C). Then, the reaction was initiated by the addition of 40 µL of the solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM) or butyrylthiocholine iodide (0.625 mM) for AChE or BuChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholineorbutyrylthiocholine was monitored at 405 nm every 30 s for one hour. Tacrine was used as a positive control with a final concentration of 0.333 µM. All of the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E − S)/E × 100 (E is the activity of the enzyme without test compound, and S is the activity of the enzyme with test compound).

α-Glucosidase Inhibition Assay
An enzyme inhibitor screening model was chosen using 4-nitro-phenol-α-D-glucopyranoside (4-NPGP) and slightly modified [47]. Briefly, the test compound (50 µM), αglucosidase solution (0.025 U/mL), phosphate buffer (pH 6.9), and (4-NPGP) (1 µM) were incubated in 96-well plates at 37 • C for 50 min. Absorbance at 405 nm was recorded on a microplate reader. Blank readings (no enzyme) were subtracted from each well, and the results were compared to the control. Quercetin was selected as the positive control. All of the reactions were repeated three times. The inhibition rate (%) was calculated as (1 − OD sample )/OD control blank × 100%.

Antimicrobial Assay
The broth dilution method and Oxford cup method were used to detect antimicrobial activity against reference strains, including Salmonella enterica subsp. enterica (ATCC14028), Staphylococcus aureus subsp. aureus (ATCC29213), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), and Candida albicans (ATCC10231). Ceftazidime and penicillin G sodium and amphotericin B served as an antibacterial and antifungal positive control, respectively. The percentage inhibition of cell growth below 50% was regarded as inactive.

Conclusions
A new coumarin 5-demethoxy-10 -ethoxyexotimarin F (1) was obtained from Murraya exotica L. (1) could be a new potential MAO-B selective inhibitor, which showed better than the known positive control coumarin anisucoumaramide and the original ligand reference C18_1503 through molecular docking and pharmacophore model evaluation. Compound (1) showed selectivity for the MAO-B isoenzyme and inhibitory activity in the sub-micromolar range with an IC 50 value of 153.25 ± 1.58 nM (MAO-B selectivity index > 172), of 26.3 ± 1.03 µM to the MAO-A. The exploration and discovery of bioactive components from medicinal herbs is one of the most important approaches for developing new drugs and improving the therapeutic properties in drug discovery. This study enriches the chemical diversity of coumarins in Murraya species and provides a theoretical basis for the traditional usage of Murraya exotica L. The results encourage us to further explore the potential of this family of derivatives as potential lead candidates for the treatment of neurodegenerative disorders.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.