Synthesis, structure, cytotoxic and antioxidant properties of 6-ethoxy-4-methylcoumarin

Article abstract of DOI:10.1016/j.molstruc.2019.127577

Coumarins have a wide usage area in medicinal applications. It is known that the design and synthesis of new coumarin derivatives are performed to prepare new drugs in many laboratories. The aim of the study was to synthesize 6-ethoxy-4-methylcoumarin and detect its chemical and biological properties for the first time. 6-ethoxy-4-methylcoumarin was synthesized using Pechmann method. Atomic orbital (GIAO) ¹H and ¹³C NMR chemical shift values in the ground state were calculated and Density Functional Method (DFT) was applied with the 6–311++G (d, p) basis set to determine chemical properties. 6-ethoxy-4-methylcoumarin was applied to cultured blood samples at various concentrations (10–400 mg/L) for determining biological activity. 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays were used to evaluate cytotoxic effect. In addition, total antioxidant capacity (TAC) and total oxidative status (TOS) were examined to determine antioxidant properties. The results showed that theoretical vibrational frequencies and chemical shift values were close to experimental values. Moreover, 6-ethoxy-4-methylcoumarin showed a slight cytotoxic effect at dose of 200 mg/L and exhibited an antioxidant activity at 25 mg/L concentration without changing oxidative status at all of doses.

Full text of DOI:10.1016/j.molstruc.2019.127577

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Synthesis, Structure, Cytotoxic and Antioxidant Properties of 6-ethoxy-4-
methylcoumarin
F.C. Celikezen, C. Orek, A.E. Parlak, K. Sarac, H. Turkez, Özlem Özdemir TOZLU
PII:
S0022-2860(19)31686-2
DOI:
https://doi.org/10.1016/j.molstruc.2019.127577
MOLSTR 127577
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Accepted Date:
12 November 2019
11 December 2019
Please cite this article as: F.C. Celikezen, C. Orek, A.E. Parlak, K. Sarac, H. Turkez, Özlem
Özdemir TOZLU, Synthesis, Structure, Cytotoxic and Antioxidant Properties of 6-ethoxy-4-
methylcoumarin, Journal of Molecular Structure (2019), https://doi.org/10.1016/j.molstruc.
2019.127577
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Synthesis, Structure, Cytotoxic and Antioxidant Properties of
6-ethoxy-4-methylcoumarin
F. C. Celikezena, C. Orekb, A. E. Parlakc, K. Saracd, H. Turkeze, Özlem Özdemir TOZLU,
a
Department of Chemistry, Science Faculty, Bitlis Eren University, 13000 Bitlis, Turkey
b Research and Application Center, Kastamonu University, 37100 Kastamonu, Turkey
c Firat University, Keban Vocational School of Higher Education, Elazıg, Turkey
d Department of Chemistry, Science Faculty, Bitlis Eren University, 13000 Bitlis, Turkey
e Department of Molecular Biology and Genetics, Technical University, Erzurum Turkey
Abstract
Coumarins have a wide usage area in medicinal applications. It is known that the design and
synthesis of new coumarin derivatives are performed to prepare new drugs in many laboratories.
The aim of the study was to synthesize 6-ethoxy-4-methylcoumarin and detect its chemical and
biological properties for the first time. 6-ethoxy-4-methylcoumarin was synthesized using
Pechmann method. Atomic orbital (GIAO) 1H and 13C‒NMR chemical shift values in the ground
state were calculated and Density Functional Method (DFT) was applied with the 6-311++G (d,
p) basis set to determine chemical properties. 6-ethoxy-4-methylcoumarin was applied to cultured
blood samples at various concentrations (10-400 mg/L) for determining biological activity. 3-
(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) and lactate dehydrogenase
(LDH) release assays were used to evaluate cytotoxic effect. In addition, total antioxidant
capacity (TAC) and total oxidative status (TOS) were examined to determine antioxidant
properties. The results showed that theoretical vibrational frequencies and chemical shift values
were close to experimental values. Moreover, 6-ethoxy-4-methylcoumarin showed a slight
cytotoxic effect at dose of 200 mg/L and exhibited an antioxidant activity at 25 mg/L
concentration without changing oxidative status at all of doses.
Keywords: Coumarins, DFT, NMR, vibrational spectra, cytotoxic, antioxidant.
1

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1. Introduction
Natural products (NPs) are a very important source for drug development and design [1]. They
have been used to treat diseases for thousands of years [2]. Being secondary metabolites of
animals and plants, NPs have an important role in self-defense [3]. Coumarin and coumarin
derivatives are well known NPs containing benzene and pyrone rings found naturally in fruits,
seeds, leaves, and roots at high concentrations [5-8]. They can make hydrogen bonds,
hydrophobic bonds, electrostatic interactions, metal coordination, and van der Waals force with
proteins and enzymes [9]. Thus, they may indicate a wide range of biological activities including
antimicrobial, anti-inflammatory, antioxidant, and anti-cancer properties [10]. In addition,
coumarins have important roles in food [11], soap, perfumes, and detergent industries [12,13].
Moreover, they are also used in electronic applications including optical brightening agents,
fluorophores, and dyes [14].
Coumarins may be made synthetically. Various methods are employed to synthesize coumarins
such as Pechmann [15], Perkin and Knoevenagel [16,17] reactions. In general, Pechmann
condensation is used to synthesize 4-substituted coumarins. This reaction is based on
condensation of phenol or substituted phenol with β-ketoester and Lewis acids used as catalyst.
One of the methods used for the theorical calculation of the synthesized compounds is Density
Functional Theory (DFT). DFT method attracts an important interest due to its perfect analysis
capacity for structural, spectral, and electronic properties of many molecules and matches up with
the experimental data [18]. Being one of the DFT-functions, B3LYP method indicates an
effective precision in predicting the electronic, energetic, and spectral properties for many
molecules. So, it is called as brain of quantum chemistry [19-21].
Because coumarins have an effective biological activity, they have found a wide use in medical
chemistry. Today, it is known that the design and synthesis of new coumarin derivatives are
performed to prepare new drugs in many laboratories. In this study, a novel coumarin derivative
6-ethoxy-4-methylcoumarin was synthesized for the first time with the aim of preparing new
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functional compounds. After the structure of the compound was identified, DFT calculations
were performed. Besides, antioxidant and cytotoxic effects were determined.
2. Experimental
2.1.
Synthesis and physical properties of 6-ethoxy-4-methylcoumarin
All of the chemicals were purchased from Merck. For the synthesis of 6-ethoxy-4-
methylcoumarin, a mixture of 4-ethoxyphenol (I) (10 mmol) and ethyl acetoacetate in sulfuric
acid (6 ml) was refluxed for 2h. After reaching to room temperature, it was poured into ice-
cooled water (40 ml). The solid material obtained on cooling was filtered, washed with water,
dried and crystallized from ethanol [15]. Yield 29%; m.p. 161-162 ºC; IR (ν, cm-1): 1712 (C=O
lactone), 1629 (C=C aromatic), 1H NMR (600 MHz, DMSO-d6, ppm): δ 1.40 (t, 3H, CH3CH2 ),
13
2.35 (s, 3H, CH3), 4.10 (q, 2H, CH3CH2), 6.30 (s, 1H, CH), 6.95-7.40 (m, 3H, Ar-H); C NMR
(150 MHz, DMSO-d6, ppm): 161.0, 155.3, 152.0, 147.8, 120.4, 119.0, 117.9, 115.4, 108.5, 62.2,
18.7, 14.7
1
13
The H and C-NMR spectra were taken using Bruker ascend 600 NMR spectrometer, at
1
600 MHz for H and 150 MHz for 13C‒NMR. Compound was dissolved in DMSO-d6. The
infrared spectra were obtained by using Perkin-Elmer spectrum one FT-IR spectrophotometry.
Besides, melting point was determined by using an electrothermal 9100 apparatus. Figure 1
shows the synthesis of 6-ethoxy-4-methylcoumarin.
2.2.
Computational methods
The entire series of quantum calculations were performed on the basis of density functional
theory (DFT) with B3LYP levels employing the basis set of 6-311++G(d,p) and the operating
gradient geometry optimization based on the Gaussian 09W program package [22,23]. While the
optimized molecular structure was estimated by using the vibrational wave number, the B3LYP
protocol underpinned the performance of the calculations. Furthermore, to obtain the optimized
geometry associated with the minima on the potential, the self- consistent field equation was
3