International Journal of Applied Engineering Research ISSN 0973-4562 Volume 11, Number 20 (2016) pp. 10075-10078

© Research India Publications. http://www.ripublication.com

10075

4-Hydroxycoumarins: Synthesis, Characterization and Antioxidant Activities

Y. Al-Majedy a,c*, A. Kadhuma, A. Mohamada,b, A. Al-Amieryc

aDepartment of Chemical and Process Engineering, Universiti Kebangsaan Malaysia, Bangi, Selangor 43000, Malaysia. bFuel Cell Institute, Universiti Kebangsaan Malaysia, 43600 Bangi Selangor, Malaysia.

cUniversity of Technology (UOT), Baghdad 10001, Iraq.

Abstract

4-hydroxycoumarins were synthesized and characterized by the

Fourier transformation infrared (FT-IR) and Nuclear magnetic

resonance (1H-NMR) spectroscopies. The scavenging activities

were evaluated by various in vitro antioxidant assays, 1, 1-

diphenyl-2-picrul-hydrazil free radical (DPPH•) and hydrogen

peroxide and we were using (vitamin C) as a reference. All of the

synthesized compounds have shown excellent radical scavenging

activities.

Keywords: Antioxidant activity, 4-hydroxycoumarins,

DDPH, H2O2.

INTRODUCTION

Antioxidants in food play an important role as a health-protecting

factor. Scientific evidence suggests that antioxidants reduce the

risk for chronic diseases including cancer and heart disease.

Primary sources of naturally occurring antioxidants are whole

grains, fruits and vegetables. Plant sourced food antioxidants like

vitamin C, vitamin E, carotenes, phenolic acids, phytate and

phytoestrogens have been recognized as having the potential to

reduce disease risk [1] Coumarins are plant flavonoids widely

distributed in nature, Coumarin (2-oxo-2H-chromene) and its

derivatives represent one of the most important classes of

compounds possessing numerous biological activities [2-4] Some

of these compounds have proven to be active as antibacterial [5-7]

antifungal [8] anti-inflammatory [9], anticoagulant [10], anti-HIV

[11] and antitumor agents[12]. Coumarin derivatives are widely

used as additives in food and cosmetics [13], pharmaceuticals and

optical brighteners [14] and laser dyes [15].Coumarins have also

proven to be an excellent antioxidants as well as antifungal agents

[16]. In continuation of previous studies [17–26], we have

focused on synthesis of new heterocyclic compounds, and testing

their free radical scavenging activity by using DPPH and

hydrogen peroxide free radical scavenging methods.

MATERIALS AND METHODS

All chemicals were supplied by Sigma-Aldrich (Selangor,

Malaysia). FT-IR spectra were obtained on a Nicolet 6700 FT-IR

spectrophotometer (Thermo-Nicolet-Corp., Madison, WI, USA),

in cm−1. NMR spectra were recorded using an AVANCE III 600

MHz spectrometer (Bruker, Billerica, MA, USA), using DMSO

and expressed in δ ppm. CHN microanalysis was performed on an

Elementar Vario El III Carlo Erba 1108 elemental analyzer

(Carlo Erba Reagenti SpA, Rodano, Italy).

Synthesis of compounds 1-11.

All compounds were synthesized according to Al-Amiery

2014 [27].

Antioxidant Activity

DPPH free radical scavenging activity

The antioxidant activity of synthesis compounds and the

standard was assessed on the basis of the radical scavenging

effect of the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-

free radical activity by modified method (Braca et al .2002).

The diluted working solutions of the test compound were

prepared in methanol. Ascorbic acid was used as standard in

1-100 µg/ml solution. 0.002% of DPPH was prepared in

methanol and 1 ml of this solution was mixed with 1 ml of

sample solution and standard solution separately. These

solution mixtures were kept in dark for 30 min and optical

density was measured at 517 nm using Spectrophotometer.

Methanol (1 ml) with DPPH solution (0.002%, 1 ml) was

used as blank. The optical density was recorded and %

inhibition was calculated using the formula given below [28]:

(1)

Where Ao is the absorbance of the control reaction and A is

the absorbance in the presence of the samples or standards.

Hydrogen Peroxide Scavenging Activity

A solution of hydrogen peroxide (40mM) was prepared in

phosphate buffer (pH 7.4). Different concentrations (250,

500, and 1000 µg/mL) of synthesized compounds (or

ascorbic acid as control) were added to a hydrogen peroxide

solution (0.6 mL, 40 mM). Absorbance of hydrogen peroxide

at 230 nm was determined after 10min. against a blank

solution containing phosphate buffer without hydrogen

Peroxide [29, 30]. Hydrogen peroxide percentage scavenging

activity was then calculated using the equation 1:

International Journal of Applied Engineering Research ISSN 0973-4562 Volume 11, Number 20 (2016) pp. 10075-10078

© Research India Publications. http://www.ripublication.com

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RESULTS AND DISCUSSION

Chemistry

Methyl 2-(coumarin-4-yloxy) acetate (1) was obtained by

refluxing methyl bromoacetate with 4-hydroxycoumarin in

anhydrous acetone in the presence of anhydrous potassium

carbonate. The FT-IR spectrum of this compound showed an

absorption band at 1,723.1 cm-1 (ester C=O carbonyl stretching).

The 1H-NMR spectrum exhibited a singlet at δ 3.63 ppm due to

the three (CH) protons. The reaction of 1 with hydrazine hydrate

afforded hydrazide 2 in good yield. The FT-IR spectrum of

compound 2 showed absorption bands at 3,233.3 and 3,210.0cm-1. 1H-NMR spectrum exhibited a singlet at δ 4.45 ppm due to the

two (CH2) (hydrazide (NH-NH) protons and a singlet due to the

single NH proton at δ 8.21 ppm. The reaction of compound 2 with

various carbonyl compounds (2-methylbenzaldehyde, 4-

aminoantipyrine and ethyl methyl ketone) yielded the new Schiff

bases N'-(2-methylbenzylidene)-2-[(coumarin-4-yl)oxy]aceto

hydrazide (3), N'-[4-amino-1,5-dimethyl-2-phenyl1H-pyrazol-

3(2H)-ylidene]-2-[(coumarin-4-yl)oxy]acetohydrazide (4) and N'-

(butan-2-ylidene)-2[(coumarin-4-yl)oxy]acetohydrazide (5),

respectively. Schiff bases 3, 4 and 5 were cyclized by reaction

with mercaptoacetic acid to yield novel coumarin compounds N-

[4-oxo-2-(O-tolyl)thiazolidin3-yl]-2-[(coumarin-4-

yl)oxy]acetamide (6), N-(4-amino-2,3-dimethyl-8-oxo-1-phenyl-

6-thia-1,2,9triazaspiro[4.4]non-3-en-9-yl)-2-[(coumarin-4-

yl)oxy]acetamide (7) and N-(2-ethyl-2-methyl-4oxothiazolidin-3-

yl)-2-[(coumarin-4-yl)oxy]acetamide (8), respectively. The FT-IR

spectrum of compound 6 showed absorption bands at 3,191.4

cm−1 (NH) and 1,715.6 cm−1 (C=O, lactone) as well as 1,695 and

1,677 cm−1 (C=O, amide). The 1H-NMR spectrum exhibited a

singlet due to the (S-CH) proton at δ 4.88 ppm and another singlet

due to the (O-CH) proton at δ 5.11 ppm (2H). For compound 7,

the FT-IR spectrum showed absorption bands at 3,388.3 cm−1for

NH2 and 3,189.1 cm-1 for (N-H) and the( C=N) bands disappeared

due to cyclization. The 1H-NMR spectrum exhibited a doublet

due to the two protons at δ 4.89 and δ 4.12 ppm and singlet due to

the single S-CH proton at δ 3.89 ppm. For compound 8, the FT-IR

spectrum showed absorption bands at 3,199.0 cm−1 (NH) as well

as 1,691 and 1,685 cm−1 (C=O, amide); the (C=N) bands

disappeared due to cyclization. The 1H-NMR spectrum exhibited

a doublet due to the(S-CH2). Proton at δ 3.86 and δ 8.02 ppm, and

a singlet due to the (NH) proton. Another three novel compounds

were synthesized by the cyclization of Schiff bases 3, 4 and 5

with maleic anhydride to yield N-[4,7-dioxo-2-(O-tolyl)-1,3-

oxazepin-3(2H,4H,7H)-yl]-2-[(coumarin4-yl)oxy] acetamide (9),

N-[2-(4-amino-1,5-dimethyl-2-phenyl-2,3-dihydro-1H-pyrazol-3-

yl)4,7dioxo-1,3-oxazepin-3(2H,4H,7H)-yl]-2-[(coumarin-4-

yl)oxy] acetamide (10) and N-[2-ethylmethyl4,7-dioxo-1,3-

oxazepin-3(2H,4H,7H)-yl]-2-[(coumarin-4-yl)oxy] acetamide

(11), respectively. For compound 9, the FT-IR spectrum showed

absorption bands at 3,196.2 cm−1(NH) and 1,667.9 cm-1 for the

amide as well as 1,755 and 1,745 cm−1 for carbonyls. The 1H-

NMR spectrum exhibited a singlet at δ 5.38 ppm due to the two

O-CH protons. For compound 10, the FT-IR spectrum of showed

absorption bands at 2,981.4 cm−1. (C-H, aliphatic), 1,765 and

1,734 cm−1 for the carbonyls, and 1,678 cm−1 for the amide. The 1H-NMR spectrum exhibited a singlet δ 4.89 ppm due to the two

(O-CH) protons at. For compound 11, the FT-IR spectrum of

showed absorption bands at 3,199 cm-1. The (NH) and 1,669.2

cm−1 for the amides as well as 1,758 and 1,733 cm−1 for the

carbonyls. The 1H-NMR spectrum exhibited a singlet due to

the two (O-CH2) protons at δ 4.65 ppm.

Antioxidant Activities

Free radicals have been a subject of critical interest among

researchers in the previous decade. The wide range of free

radical effects in biological systems has garnered interest

from many specialists. It has been demonstrated that free

radicals assume an important role in the pathogenesis of

specific diseases and aging [31-33]. Numerous synthetic

cancer prevention agents have presented toxic and/or

mutagenic effects; thus, naturally occurring antioxidants have

been considered. Synthesized coumarins 6–11 were screened

for in vitro scavenging activity utilizing DPPH and hydrogen

peroxide. These tested coumarins showed high scavenging

activity (Figure 1 and 2).

Figure 1. Showed that the six synthesized compounds (6-11)

demonstrated a strong scavenging activity against DPPH.

0

10

20

30

40

50

60

70

80

90

100%

inh

ibit

ion

concentration

250 μg/mL500 μg/mL750 μg/mL1000 μg/mL

Figure 1.

Figure 2. Showed that the six synthesized compounds (6-11)

demonstrated a strong scavenging activity against H2O2.

Figure 2.

International Journal of Applied Engineering Research ISSN 0973-4562 Volume 11, Number 20 (2016) pp. 10075-10078

© Research India Publications. http://www.ripublication.com

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ACKNOWLEDGEMENTS

The acknowledgement for funding to Universiti Kebangsaan Malaysia.

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