Neuroprotective and Antioxidant Activities of 4-Methylcoumarins: Development of Structure–Activity Relationships

Abstract

Coumarins are a major class of polyphenols that are abundantly present in many dietary plants and possess different biological activities. Neuroprotective effect of 28 variously substituted 4-methylcoumarins was evaluated in a cell model of oxidative stress-induced neurodegeneration, which measures viability in PC12 cells challenged with hydrogen peroxide by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibitory activity of these compounds against intracellular reactive oxygen species (ROS) formation was also determined by 2′,7′-dichlorofluorescein diacetate method in the same cells. Chemical redox-based assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) tests were employed to explore structure–antioxidant activity relationships in a cell-free environment. The results demonstrated that 4-methylcoumarins containing ortho-dihydroxy or ortho-diacetoxy substituents on the benzenoid ring possess considerable neuroprotective effects. ortho-Dihydroxy compounds inhibited cytotoxicity (44.7–62.9%) and ROS formation (41.6–71.1%) at 50 µM and showed considerable antioxidant effects. We conclude that 4-methylcoumarins are promising neuroprotective and antioxidant scaffolds potentially usefull for management of neurodegenerative diseases.

Coumarin derivatives containing benzopyran-2-one central core comprise a large class of plant-derived phenolic compounds that have diverse pharmacological effects and are considered as promising scaffolds for drug discovery.^1–4) Among these compounds, 4-methylcoumarins have demonstrated potential antioxidant activity with less toxicity compared to other coumarin derivatives.^5,6) Antioxidant activities of various 4-methylcoumarin derivatives have been studied by our groups and other investigators^7–11) among which, 7,8-ortho-dihydroxy and 7,8-ortho-diacetoxy structures have shown promising effects in various test systems.^8,9,12,13) Since antioxidant agents may have great potential as neuroprotective compounds, in this study, we focused on the examination of the neuroprotective effect of previously synthesized 4-methylcoumarin derivatives in oxidative stress-induced neurotoxicity model in PC12 cells with a special focus on derivatives bearing 7,8-ortho-dihydroxy and 7,8-ortho-diacetoxy substitutions.

MATERIALS AND METHODS

Chemicals

RPMI1640, penicillin/streptomycin, sterile phosphate buffered saline (PBS) and trypsin ethylenediaminetetraacetic acid (EDTA) (0.25%) were purchased from Biosera (Ringmer, U.K.). Fetal bovine serum (FBS) and horse serum (HS) were acquired from Invitrogen (San Diego, CA, U.S.A.). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), hydrogen peroxide, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and N-acetyl-L-cysteine (NAC) were obtained from Sigma-Aldrich (San Louis, MO, U.S.A.). The rest of compounds were purchased from Merck (Darmstadt, Germany). The synthesis of 4-methylcoumarin derivatives MC1–22, MC24, MC26–28,⁴⁾ and MC23, MC25, MC29^10,11,14) containing different substituents on the benzenoid ring, were previously reported and they were synthesized according to the reported methods.

Cell Culture

PC12 cells (a generous gift of Professor Lloyd A. Greene, Department of Pathology and Cell Biology, Columbia University, New York, U.S.A.) were cultured in RPMI1640 supplemented with 10% HS, 5% FBS, 100 U/mL penicillin-G and 100 µg/mL streptomycin and grown at 37°C in humidified air containing 5% CO₂.

Cell Viability Assay

Cell viability after exposure to hydrogen peroxide was determined by MTT reduction assay.¹⁵⁾ Briefly, PC12 cells were initially plated in collagen-coated 96-well microplates at a density of 5×10⁵ cells/mL (100 µL per well). After 48 h incubation, derivatives were added for 3 h, followed by H₂O₂ 150 µM for 1 h. After 24 h, the cells were incubated in 0.5 mg/mL MTT dissolved in RPMI1640 for 1.5 h at 37°C. The generated formazan crystals were dissolved in 200 µL dimethyl sulfoxide (DMSO) and absorbance was measured at 570 nm. Experiments were performed in triplicate and were repeated 4–6 times. Viability was calculated as percentage compared to non-treated control cells. NAC, a known antioxidant and neuroprotective agent,¹⁶⁾ was used as a positive control.

Measurement of Intracellular Reactive Oxygen Species (ROS) by DCFH-DA

Intracellular accumulation of ROS was monitored using the fluorescent probe DCFH-DA.¹⁷⁾ PC12 cells (4×10⁵ cells/mL) were seeded in collagen-coated 24-well plates (500 µL per well) for 72 h. Test compounds were added for 3 h, followed by DCFH-DA 10 µM for 30 min at 37°C in the dark, afterwards, the media was replaced with PBS. Fluorescence intensity was quantified with a multi-mode microplate reader (BMG Labtech, Germany) at 485 and 520 nm excitation and emission filters (e₀), respectively. After 1 h of treatment with H₂O₂ 150 µM, fluorescence was measured again (e₆₀). ΔF was calculated as the differences between e₀ and e₆₀. ROS inhibition was determined as follows:   

DPPH Radical Scavenging Activity

Four different concentrations of test compounds were mixed with a methanolic solution of DPPH (100 µM) and incubated at room temperature for 30 min. Absorbance at 517 nm was measured by a spectrophotometer. IC₅₀ values were calculated by the software Curve-Expert (for Windows, version 1.34). Quercetin was used an standard antioxidant agent.¹⁸⁾

Ferric Reducing Antioxidant Power (FRAP) Assay

FRAP solution was freshly prepared by mixing 10 mL acetate buffer (300 mM) at pH 3.6, 1 mL ferric chloride (20 mM) and 1 mL 2,4,6-tris(2-pyridyl)-s-triazine (10 mM). Ten microliters of different concentrations of 4-methylcoumarin derivatives and quercetin dissolved in DMSO were mixed with 190 µL of FRAP solution in 96-well microplates and incubated at room temperature for 30 min. Absorbance at 595 nm was measured and FRAP values calculated.¹⁹⁾

RESULTS AND DISCUSSION

Antioxidant activities of synthetic 4-methylcoumarin derivatives were assessed using different cell-based and cell-free assays. Studied 4-methylcoumarins ate categorized into five groups (Fig. 1): 1) monohydroxy 4-methylcoumarins (MHMCs) containing hydroxyl group at the C-7 position (MC1–5); 2) dihydroxy 4-methylcoumarins with two hydroxyl groups at 7,8 positions (7,8-DHMC, MC6–11) or 5,7 positions of the coumarin ring (5,7-DHMC, MC12–13); 3) monoacetoxy 4-methylcoumarins (MAMCs) having acetoxy group at the C-7 position (MC14–15); 4) diacetoxy 4-methylcoumarins (DAMCs), including 7,8-DAMC (MC16–18), 5,7-DAMC (MC19) and 6,7-DAMC (MC20–21); 5) dimethoxy 4-methylcoumarins bearing two methoxy groups at the C-7 and C-8 positions (7,8-DMMC, M23–M25); 6) miscellaneous derivatives with different substitutions.

Fig. 1. General Structures of Synthesized 4-Methylcoumarins

Protective effect of 4-methylcoumarins against H₂O₂-induced cytotoxicity in PC12 cells was determined by the MTT reduction assay (Fig. 2, not-active compounds are not shown). Tested compounds’ inhibitory activity against ROS formation induced by H₂O₂ in PC12 cells was also measured by DCFH-DA assay (Table 1). The ortho disubstituted 4-methylcoumarins, including 7,8-DHMCs (MC6 to 9), 7,8-DAMCs (MC16 to 18) and 6,7-DAMCs (MC20, and 21, but not 22) demonstrated considerable inhibitory effects against neurotoxicity and ROS formation, which were for some of the derivative comparable to the effect of NAC, a reference antioxidant agent. 4-Methylcoumarins containing two hydroxy or two acetoxy moieties at meta position to each other including 5,7-DHMC (MC12–13) and 5,7-DAMC (MC19) were found to be devoid of the protective effect against H₂O₂-induced neurotoxicity, ROS formation and antioxidant activity (Tables 1, 2). Moreover, substitution by methoxy, amino or acetamido moieties at the benzenoid ring also eliminated the protective effects of compounds against oxidative stress. The superior activity of 7,8-DHMCs compared to monohydroxy compounds (7-MHMCs) and 5,7-DHMCs might be attributed to the increased stability of the catecholic radical as a result of electron donating characteristic of ortho-dihydroxy substituents.

Fig. 2. Inhibition of Hydrogen Peroxide-Induced Cytotoxicity in PC12 Neuronal Cells by Synthesized 4-Methylcoumarin Derivatives

Protective effects of 7,8-dihydroxymethylcoumarines (upper panel) and 7,8-diacetoxymethylcoumarines (lower panel) against H₂O₂ was determined in PC12 cells. Concentrations of tested methylcoumarins and N-acetylcysteine (NAC, positive control) are expressed as µM. * Significantly different from cells treated with hydrogen peroxide alone (p<0.05).

Table 1. Inhibition of Hydrogen Peroxide-Induced Cytotoxicity and Intracellular ROS Formation by 4-Methylcoumarin Derivatives in PC12 Neuronal Cells

Compound	X	Substitution on benzenoid ring	Substitution at C3 position	Inhibition of neurotoxicity induced by H2O2* (% Protection at 50 µM)	Intracellular ROS inhibition (% Protection at 50 µM)
MC1	CH3	7-Hydroxy	CH2CH3	NA**	NA
MC2	CH3	7-Hydroxy	n-C6H13	NA	NA
MC3	CH3	7-Hydroxy	n-C10H21	NA	NA
MC4	CH3	7-Hydroxy	CH2CO2C2H5	NA	NA
MC5	CH3	7-Hydroxy	CH2CH2CO2C2H5	NA	NA
MC6	CH3	7,8-Dihydroxy	H	56.4±8.3	41.6±13.2
MC7	CH3	7,8-Dihydroxy	C2H5	61.0±1.7	71.1±12.0
MC8	CH3	7,8-Dihydroxy	n-C6H13	62.9±4.2	69.1±7.5
MC9	CH3	7,8-Dihydroxy	n-C10H21	44.7±2.8	46.7±13.0
MC10	CH3	7,8-Dihydroxy	CH2CO2C2H5	58.6±5.8	63.5±13.4
MC11	CH3	7,8-Dihydroxy	CH2CH2CO2C2H5	53.7±18.0	46.8±8.7
MC12	CH3	5,7-Dihydroxy	CH2CO2C2H5	NA	NA
MC13	CH3	5,7-Dihydroxy	CH2CH2CO2C2H5	NA	NA
MC14	CH3	7-Acetoxy	CH2CO2C2H5	NA	NA
MC15	CH3	7-Acetoxy	CH2CH2CO2C2H5	NA	NA
MC16	CH3	7,8-Diacetoxy	H	51.3±4.0	35.4±8.4
MC17	CH3	7,8-Diacetoxy	CH2CO2C2H5	52.2±9.7	51.5±7.2
MC18	CH3	7,8-Diacetoxy	CH2CH2CO2C2H5	58.0±10.0	19.5±11.1
MC19	CH3	5,7-Diacetoxy	CH2CO2C2H5	NA	NA
MC20	CH3	6,7-Diacetoxy	H	74.9±3.5	37.0±14.0
MC21	CH3	6,7-Diacetoxy	CH2CO2C2H5	59.8±2.3	45.5±9.1
MC22	CH3	6,7-Diacetoxy	CH2CH2CO2C2H5	NA	NA
MC23	CH3	7,8-Dimethoxy	H	NA	NA
MC24	CH3	7,8-Dimethoxy	CH2CO2C2H5	NA	NA
MC25	CH3	7,8-Dimethoxy	CH2CH2CO2C2H5	NA	NA
MC26	CH3	7-NH2	H	NA	NA
MC27	CH2CH2CH3	7-NH2	H	NA	NA
MC28	CH2CH2CH3	7-NHAC	H	NA	NA

* Data are expressed as the mean±S.E.M. of 4–6 experiments. ** NA: Not active.

Table 2. Antioxidant Activity of 4-Methylcoumarin Derivatives Determined by DPPH and FRAP Assays

Compound	IC50 DPPH scavenging (µM)	FRAP value (mM Q/mol)*
MC1	3769.5±592.9	3.9±0.4
MC2	NA**	2.2±0.3
MC3	NA	4.3±0.6
MC4	NA	7.4±0.9
MC5	NA	0.6±0.0
MC6	ND	ND
MC7	15.8±3.3	964.0±51.1
MC8	18.0±3.0	779.5±30.7
MC9	17.5±3.1	667.4±82.8
MC10	15.4±3.4	660.0±136.3
MC11	12.1±0.6	1109.4±3.5
MC12	819.0±80.7	1.4±0.3
MC13	537.1±50.8	2.3±0.5
MC14	NA	0.3±0.0
MC15	ND	ND
MC16	ND	ND
MC17	97.8±4.8	35.8±1.9
MC18	37.9±7.7	119.3±17.6
MC19	3088.1±539.7	1.0±0.2
MC20	6.5±1.1	1489.2±100.3
MC21	23.3±9.3	499.0±10.5
MC22	NA	2.5±0.4
MC23	ND	ND
MC24	NA	1.2±0.1
MC25	ND	ND
MC26	NA	0.9±0.1
MC27	NA	1.4±0.3
MC28	NA	1.0±0.2
Quercetin	7.9±0.3	1000

* mM Equivalent of quercetin/mol. ** Not active (IC₅₀>5000 µM). Values represent the mean of 3–5 experiments±S.D.

Although it has been repeatedly shown that ortho-dihydroxy moiety confers a high antioxidant potential,¹³⁾ in our study the substitution of the catechol moiety with diacetoxy (as in 7,8-DAMCs and 6,7-DAMCs) did not considerably alter the activities. This particular phenomenon in 4-methylcoumarins has been previously reported in literature^20,21) and at least in biological systems may be ascribed to the production of deacetylated compounds through the activity of esterase enzymes.²¹⁾

Previous reports have shown that insertion of ester and other moieties at C3 position may alter the antioxidant capacities of 4-methylcoumarines,²¹⁾ therefore we examined the influence of these moieties on the activity of derivatives in our test systems; among 7,8-DHMCs, the presence of an alkyl chain such as ethyl (MC7), n-hexyl (MC8) and n-decyl (MC9) or ethoxycarbonyl methyl (MC10) and ethoxycarbonyl ethyl (MC11) moieties at C3 did not seem to considerably alter the neuroprotective, ROS inhibition or antioxidant activities in the active subset of 7,8-DHMCs. The same phenomenon was observed among the other two active subsets of 7,8-DAMCs and 6,7-DAMCs. The only exception seemed to be in the case of MC22 (a 6,7-DAMC) that the presence of an ethoxycarbonyl ethyl moiety completely abolished neuroprotective and antioxidant effects (Tables 1, 2). Similar to this finding, another study on galvynoxyl and DPPH radicals scavenging capacity of 4-methylcoumarins has found that the presence of ethoxycarbonyl ethyl moiety at C3 position increases the activity of meta- and ortho-dihydroxy coumarins, but not the activity of ortho-diacetoxy derivatives.²¹⁾

The findings of antioxidant assays (Table 2) closely resembled those obtained in cellular assays. Some of the compounds showed activities comparable to that of quercetin, a known antioxidant agent.

In conclusion, 4-methylcoumarins containing ortho-dihydroxy or ortho-diacetoxy substituents on the benzenoid ring showed considerable potential in inhibition of oxidative stress-induced neuronal damage in a cell model of neurodegeneration and also in cell-free antioxidant assays and represent promising scaffolds with antioxidnat and neuroprotective activities.

Acknowledgments

The authors wish to thank the financial support of the Vice-Chancellor for Research of the Shiraz University of Medical Sciences (Grant number: 88-4926) and the University of Delhi. SM thanks the Council of Scientific and Industrial Research (CSIR, India) for the award of a Senior Research Associateship.

Conflict of Interest

The authors declare no conflict of interest.

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