Abstract
Background/Aim: Coumarins are a broad class of naturally occurring oxygen-heterocyclic compounds found in plants with diverse biological properties, making them attractive for evaluation as novel therapeutic agents. We herein report the in vitro cytotoxic and monoamine oxidase (MAO) inhibitory activities of 3-acetylcoumarins (6a-e). Materials and Methods: The cytotoxic activity was evaluated using crystal violet dye binding assay, and those compounds unable to induce cytotoxicity were further tested for the monoamine oxidase (MAO) activity using the MAO-GloTM kit. Results: The 3-acetylcoumarins (6a-e) were non-cytotoxic (inactive) against MDA MB-231 (estrogen receptor-negative, ER-, highly invasive) and MCF-7 (estrogen receptor-positive, ER+, weakly invasive) breast cancer cell lines, but showed interesting MAOs inhibition activities. Among the synthesized compounds, 3-acetylcoumarin bearing dichloro (-diCl) (6d; IC50=0.31±0.04 μM) at Carbon-7, 8 positions showed higher inhibition, MAO B/A non-selectivity (selectivity index, SI=3.10), reversible inhibition against the hMAO-B enzyme, and neuroprotection against H2O2-treated human neuroblastoma (N2a) cells. Conclusion: Compound (6d) can be considered a promising scaffold for further investigation in developing hMAO-B inhibitors (MAOIs).
Coumarins (Benzopyran-2-one), a flavonoid group of plant secondary metabolite, belong to an essential group of naturally occurring oxygen-heterocyclic compounds consisting of fused benzene and α-pyrone rings (1). They exhibit diverse pharmacological properties such as anticoagulant, anticancer, anti-inflammatory, anti-HIV, antibacterial, antimicrobial, anti-Alzheimer, antioxidant, Central nervous system (CNS) stimulant, monoamine oxidase (MAO) inhibitory and scavenging of reactive oxygen species (ROS) (2-4).
Additionally, coumarins are used as additives in food, perfumes, agrochemicals, cosmetics, pharmaceuticals, insecticides, optical brightening agents, dispersed fluorescent, tunable laser dye, etc. (5). These diverse properties of coumarins are attributed to the nature of substituents and their pattern of substitution on the core coumarin molecule (6, 7). Given the above, coumarins have attracted intense interest in recent years, making them attractive for synthesis and further evaluation as novel therapeutic agents for medicinal applications. Some examples of coumarin-based drugs that are available in the market include warfarin (anticoagulant), tecarfarin (anticoagulant), ensaculin (KA-672) (NMDA antagonist and 5HT1A agonist), esculin (vasoprotective agent), carbochromen (coronary disease), scopoletin (anti-inflammation, anticancer, antioxidant, antibacterial and anti-fungal), and novobiocin (antibiotic) (5).
Among the diverse properties of coumarins, cytotoxic activity is the most extensively examined. Coumarin anti-tumor mechanisms are very diverse, such as inhibition of kinases, cell cycle phases, angiogenesis, heat shock protein 90 (HSP90), telomerase, antimitotic activity, carbonic anhydrase, monocarboxylate transporters, aromatase, and estrogen sulfatase (6). Recently, coumarin nucleus has emerged as a promising scaffold for MAO inhibitors (MAOIs), playing a critical role in activity and selectivity (8, 9). Their MAO activity is due to the inhibition of many signaling pathways, such as neurotransmitter (monoamine alterations), brain-derived neurotrophic factor (BDNF activation), inflammatory (cytokine modulation), and neuroinflammation (brain-gut-microbiome colony restoration) pathways (10). MAOIs, classified as MAO-A inhibitors (MAOAIs) and MAO-B inhibitors (MAOBIs), are drugs that are used in treating neurological disorders by inhibiting the activity of one or both MAO enzymes. The C-3, C-4, C-6, C-7, or C-8 substituted coumarins have been reported to modulate anticancer and MAO inhibitory activities depending on the nature of the substituent at these positions (2, 6, 8, 9, 11-19). For example, coumarin nuclei with acyl group at the C-3 position (1-5, Figure 1) have been reported as highly potent anti-MAO agents, reversible and selective MAO-B inhibitors (20-22). These findings aroused our interest in the present study involving 3-acetylcoumarin, a precursor in synthesizing different polyfunctional heterocyclic compounds (23-29). As part of our ongoing investigation on coumarins, we herein report the evaluation of the in vitro cytotoxicity of 3-acetylcoumarins (6a-e) against MDA-MB 231 and MCF-7 breast cancer cell lines and their hMAO inhibitory activities.
Materials and Methods
Chemicals. Cell lines and media were obtained from American Type Culture Collection (ATCC) (Rockville, MD USA). Penicillin-streptomycin anti-biotic solution (100×), fetal bovine serum (FBS), trypsin-EDTA solution (1×), phosphate buffered saline (PBS), 50% glutaraldehyde, crystal violet, clorgyline, pargyline, moclobemide, safinamide mesylate and tamoxifen were obtained from Sigma-Aldrich (St. Louis, MO, USA). MAO-Glo TM kit containing hMAO-A and MAO-B enzymes was obtained from Promega Corporation (Madison, WI, USA). The stock solutions of the compounds (6a-e) were made up in dimethyl sulfoxide (DMSO) and stored at 4°C.
Cell culture and cell viability assay. The MDA-MB-231 and MCF-7 breast cancer cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured as per the guidelines supplied according to our previous reported methods (11, 12). Cell viability assay was performed in DMEM/F12 (50:50) for MDA-MB-231, or DMEM (1X) with glutamax media for MCF-7 cells, and the cytotoxic concentration (CC50) was determined after 48 h of treatment with compounds (6a-e). All studies were repeated at least two times.
Inhibition studies of hMAO enzymes. The hMAO inhibitory activities of compounds (6a-e) and reference inhibitors (clorgyline, paragyline, moclobemide and safinamide mesylate) were assayed using MAO-Glo™ kit (Promega, Madison, WI, USA) according to our previous reported method (11). 12.5 μl of different concentrations (4×) of each test compound (diluted in water), 12.5 μl of substrate A or B (4×) and 25 μl of MAO-A (0.3 μg/reaction) or MAO-B (0.9 μg/reaction) enzyme solution were mixed in a white 96-well plate, followed by addition of 50 μl luciferin detection reagent. Luminescence was measured using the Promega Glomax Explorer GM3500 Microplate Reader (Promega).
Reversible inhibition assay. The reversibility of hMAO-B enzyme inhibition of the most active compound (6d) in comparison with reference inhibitors (pargyline and safinamide mesylate) was assayed using a time-dependent inhibition assay according to our previously reported method (11). Compound (6d), reference inhibitors (100× and 10× IC50), and MAO-B (9 μg/reaction) enzyme in eppendorf tubes were pre-incubated for 30 min at room temperature (RT). The compound (12.5 μl, 4× IC50) and MAO-B (0.9 μg/reaction) enzyme were concurrently pre-incubated along with controls (without inhibitor) in a white 96-well plate for 30 min at RT. The respective enzyme buffer (47.5 μl) was added to the concentration reactions (100× and 10× IC50), and 25 μl was transferred to the plate. Next, the respective enzyme buffer (87.5 μl) and respective enzyme substrate (12.5 μl, 4× IC50) were added (the compound concentration was diluted to 1× and 0.1×), followed by incubation for 1 h at RT. To the undiluted samples (4× inhibitor concentration reactions), the respective enzyme buffer (12.5 μl) and respective enzyme substrate (12.5 μl, 4×) were added, followed by incubation for 1 h at RT. Finally, 50 μl of constituted luciferin detection reagent was added to each well, and luminescence was measured using a Promega Glomax Explorer GM3500 Microplate Reader (Promega).
Neuroprotection assay. The neuroprotective effect of compound (6d) against Neuro 2A (N2a) cells was assayed using a crystal violet dye uptake assay according to our previous reported method (11) by pre-treating 5.0×105 N2a cells in RPMI 1640 complete medium with 1 or 10 μM of compound for 1 h (or same time treatment of cells), followed by 50 μM H2O2 for 24 h. Cell viability was then measured by crystal violet dye uptake assay.
Statistical analysis. The data were presented as mean±standard deviation (SD) (n=3). All treated cell data are presented as percentage values in comparison to the untreated control (100%). The data were analyzed for significance by one-way ANOVA, and then compared by Dunnett’s multiple comparison tests using the GraphPad Prism Software, version 5.00 (GraphPad Software, Inc, San Diego, CA, USA). Differences from the respective untreated control were considered statistically significant when p<0.05.
Results
Cytotoxic effect of compounds (7a-e) in cancer cell lines. The cytotoxic activity of 3-acetylcoumarins (6a-e) and tamoxifen (TAM, standard anticancer drug) was evaluated by a simple and reproducible crystal violet dye-staining assay at different concentrations (0, 25, 50, 75, and 100 μM) against the breast (MDA MB-231 and MCF-7) cancer cell lines. Interestingly, the CC50 values indicated that compounds (6a-e) were unable to induce any cytotoxicity (CC50 >100 μM; inactive) against the above-considered breast cancer cell lines with respect to untreated control cells (100%). Based on the in vitro cytotoxicity data, these compounds were selected for further evaluation as potential human MAO (hMAO) enzyme inhibitors.
Inhibition studies of hMAO enzymes. The ability of synthesized compounds (6a-e) to inhibit hMAO enzymes was evaluated using a MAO-Glo™ kit (Promega). The inhibition concentration (IC50) and MAO-B [Selectivity index, SI=[IC50 (MAO-A)]/[IC50 (MAO-B)]] of compounds (6a-e), and standard reference inhibitors are shown in Table I. According to the results, compounds (6a-d) showed higher MAO inhibitory activity against hMAO-A and B enzymes in comparison to moclobemide (IC50=238.3±0.25 μM) and pargyline (IC50=6.22±0.69μM) (reference inhibitors). Among the tested compounds, compound 6d (6,8-dichloro, IC50=0.31±0.04 μM) displayed the highest inhibitory activity against hMAO-A and B enzymes with 20-fold higher potency than pargyline, a known MAO-BI (Table I). Therefore, compound (6d) was selected for further study.
Inhibitory concentration (IC50) and selectivity index (SI) of 3-acetylcoumarins (6a-e) towards hMAO-A and B enzymes.
Reversible inhibition assay. The time-dependent inhibition assay was used to determine whether compound (6d) was a reversible or irreversible hMAO inhibitor compared to pargyline (irreversible MAO-B inhibitor drug) and safinamide mesylate (reversible MAO-B inhibitor drug). The results from this present study indicated that (i) compound (6d) showed a recovery percentage activity of 42.9%±1.82 at 1× IC50 and 119.48%±3.5 at 0.1× IC50, (ii) pargyline showed an activity of 7.85%±0.38 at 1× IC50 and 8.3%±0.44 at 0.1× IC50, and (iii) safinamide mesylate showed an activity of 72.3%±2.43 at 1× IC50 and 126.6%±7.25 at 0.1× IC50 against hMAO-B enzyme in comparison to the control reaction without the inhibitor (100%) (Figure 2a-b). The order of hMAO-B enzyme reversible inhibition is safinamide mesylate (highest reversibility) > compound (6d) > pargyline (lowest reversibility). Overall, compound (6d) showed higher recovery of hMBO-B enzyme (reversibility) compared to pargyline.
Reversibility assay of hMAO-B enzyme inhibition with (a) active compound (6d), (b) pargyline and safinamide mesylate. Data are represented as mean±standard deviation (SD), n=3 and #represents statistically significant difference compared to control (p<0.05) using Dunnett’s multiple comparison test.
Neuroprotection assay. The neuroprotective effect of compound (6d) against Neuro 2A (N2a) cells was investigated using a crystal violet dye uptake assay. Results showed that treatment of the cells with 50 μM H2O2 alone for 24 h decreased the cell viability to 20.8±0.19% compared to untreated control (100%) (Figure 2). The pre-treatment (PT) with compound for 2 h, followed by 50 μM H2O2 treatment for 24 h, showed an increase in the viability to 25.3%±0.77 (1 μM) and 38.66%±0.88 (10 μM) in comparison to H2O2-alone treated cells (20.8%±0.19) (Figure 2). Lastly, treatment with the compound, followed by 50 μM H2O2 for 24 h at same time (ST), showed an increase in the viability to 22.0%±0.66 (1 μM) and 25.8%±4.4 (10 μM) in comparison to H2O2-alone treated cells (20.8%±0.19).
Discussion
Coumarins are essential naturally occurring compounds used in drug discovery that have attracted considerable interest over the years due to their diverse pharmaceutical activities. In the present investigation involving the in vitro cytotoxic activity of 3-acetylcoumarins (6a-e), we herein report that these compounds could not induce cytotoxicity against MDA MB-231 and MCF-7 breast cancer cell lines with respect to the untreated control cells (100%). Recent studies have shown that the absence of any cytotoxicity is a mandatory requirement in the development of novel MAO-B inhibitors to target neurodegenerative diseases (30). Based on this finding, 3-acetylcoumarins (6a-e) were selected for further study as potential human MAO enzyme inhibitors. Interestingly, the tested 3-acetylcoumarins inhibit MAO-A and B enzymes. Among the tested compounds: (i) 6b (8-OCH3), 6c (8-OCH2CH3) and 6d (6,8-diCl) caused an increase in hMAO-A enzyme inhibition (exhibiting IC50 values between 0.96-2.31 μM) and (ii) 6d (6,8-diCl) and 6a (unsubstituted) caused an increase in hMAO-B enzyme inhibition (exhibiting IC50 values between 0.31-1.25 μM). Compound (6d) showed a 3-fold potency against MAO-B enzyme, higher potency than moclobemide (MAO-A inhibitor) and pargyline (MAO-B inhibitor), and lower potency than clorgyline (MAO-A inhibitor) and safinamide mesylate (MAO-B inhibitor) (Table I). This result supports previous reports indicating coumarins as emerging and promising MAOI drug candidates (31-33) and that the presence of chlorine and/or bromine groups at C-6,8 positions of 3-acetylcoumarin greatly enhanced MAO-B inhibitory potency and selectivity (20, 21, 24, 31, 34). Overall, compound (6d) showed the highest inhibition and non-selective activity against the MAO-B enzyme.
There are two types of MAO-B inhibitors. Reversible inhibitors bind covalently to the enzyme and can no longer dissociate from the binding site, whereas irreversible inhibitors bind non-covalently to the enzyme and dissociate over time. Our results showed that a higher degree of reversibility for hMAO-B enzyme inhibition was observed when treated with compound (6d), in comparison to pargyline (irreversible MAO-B inhibitor) (Figure 2a-b). Thus, the reversibility of compound (6d) for hMAO-B enzyme inhibition could eliminate the classical side effects associated with MAOIs irreversibilities, such as the cheese effect, psychosis, withdrawal, and possible drug interactions. Lastly, MAOIs possess neuroprotective activity by inhibiting H2O2, aldehyde, and substituted amine release, producing specific antioxidant actions (35-37). According to Figure 3, our result indicates that the compound (6d) protects cells from H2O2-induced cell death against N2a cells, thus exhibiting antioxidant properties by protecting against free radicals in the cells as previously reported (11, 38-40).
Neuroprotection against H2O2-induced neurotoxicity in N2a cells by compound (6d). Data are shown as mean±standard deviation (SD), n=3. #represents statistically significant difference compared to control and H2O2-alone treated cells (p<0.05) using Dunnett’s multiple comparison test.
Conclusion
In conclusion, the present study demonstrated that compounds (6a-e) caused an increase in MAO enzyme inhibition. However, compound (6d) showed higher inhibitory activity against the hMAO A and B enzymes in comparison to other tested compounds, paragyline (MAO-B inhibitor) and moclobemide (MAO-A inhibitor). It also displayed reversibility of MAO-B enzyme inhibition that is unlikely to provoke induced adverse effects associated with irreversible MAOIs and neuroprotection activity that may prevent neuronal loss in Alzheimer’s and Parkinson’s patients. Finally, this study provides new insight into how some functionalizing groups on the 3-acetylcoumarin ring could serve as a variable template for building new attractive and more promising MAOI drugs for treating neurodegenerative disorders.
Acknowledgements
The Authors gratefully acknowledge Florida A & M University TITLE III funding received from the U.S. Department of Education.
Footnotes
Authors’ Contributions
Monica O. Aghimien: supervision, formal analysis, writing – original draft, review, and editing. Musiliyu A. Musa: project administration, formal analysis, writing – review, and editing. Qudus Kolawole: conceptualization, investigation, methodology, writing – original draft. Lekan. Latinwo: funding acquisition, writing – editing. Philip O. Igbinoba: visualization, validation. All Authors approved the submitted version of the manuscript.
Conflicts of Interest
The Authors declare that they have no financial or non-financial competing interests.
- Received March 6, 2024.
- Revision received April 10, 2024.
- Accepted April 22, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
