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

Journal Pre-proof Synthesis, Structure, Cytotoxic and Antioxidant Properties of 6-ethoxy-4methylcoumarin

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

Reference:

MOLSTR 127577

To appear in:

Journal of Molecular Structure

Received Date:

12 November 2019

Accepted Date:

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-4methylcoumarin, 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 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 b

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.

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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-4methylcoumarin, 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 icecooled 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 ), 2.35 (s, 3H, CH3), 4.10 (q, 2H, CH3CH2), 6.30 (s, 1H, CH), 6.95-7.40 (m, 3H, Ar-H); 13C 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 The 1H and

13C-NMR

spectra were taken using Bruker ascend 600 NMR spectrometer, at

600 MHz for 1H 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

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solved in an iterative way. The same theoretical principles were applied in the analysis of the harmonic vibration frequencies for the optimized structure. Vibrational band assignments were carried out with Gauss-View molecular visualization program [24]. Polarizable Continuum Model (PCM) technique [25,26] was employed to assess the energetic and atomic charge behavior of 6-ethoxy-4-methylcoumarin in solvent media at the level of B3LYP/6-311++G(d,p) in optimized determinations of the solvents ε = 4.81, chloroform (CHCl3); ε = 24.5, ethanol (C2H5OH); ε = 80.1 and water (H2O). Moreover, the reactive sites of 6-ethoxy-4-methylcoumarin were examined by assessing the molecular electrostatic potentials via the B3LYP/6-311++G(d,p) technique. The thermodynamic parameters and frontier molecular orbitals (FMO) were also determined by employing the optimized structure (B3LYP/6311++G(d,p)). 2.3.

Cell cultures

Human peripheral whole blood cultures were prepared according to slightly modified version of Evans and O'Riordan protocol [27]. The samples were collected from four males aged 26-28 years who were healthy, had no smoking or alcohol addiction, was not receiving any drug therapy and had no recent history of exposure to mutagens. The heparinized blood (0.4 mL) was cultured in 6.0 mL of culture medium (PB-MAX(tm) Karyotyping Medium, Gibco / Spain) with 5.0 mg/mL of phytohemagglutinin (Sigma Aldrich-Steinheim /Germany) [28]. The 6-ethoxy-4methylcoumarin was dissolved in ethanol (40 µL) and added into the cultures just before the incubation. The concentrations (10, 25, 50, 100, 200 and 400 µg/mL) were specified according to Çelik’s study [29]. Triton-X (%1, Sigma-Aldrich) was used as the positive control in the MTT and LDH tests. Ascorbic acid (10 μM, Sigma-Aldrich) and hydrogen peroxide (25 μM, SigmaAldrich) were also used as a positive control in TAC and TOS analysis, respectively. 2.4.

MTT assay

The viability of the cells was determined by the formazan formation from MTT spectrophotometrically (MTT cell proliferation kit Cayman Chemical Company(r), USA). The human blood samples were added in 96-well plates, and then incubated at 37°C in a humidified 5% CO2 + 95% air mixture. Afterwards, the samples were treated with 6-ethoxy-4methylcoumarin at varied concentrations (0-400 µg mL-1) for 48 hours. Briefly, MTT was added

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to the cell cultures for 3 h and formazan crystals formed were dissolved in dimethyl sulfoxide (Sigma-Aldrich(r)). Then, the plates were analyzed by using Elisa reader (Bio-Tek, USA) at 570 nm wavelength [30,31]. 1.1.

LDH release assay

LDH activity was measured using an LDH kit, Cayman Chemical (USA), to determine cytotoxicity of culture medium. In brief, 104-105 µL-1 cells/well were added in 96-well plates and exposed to 6-ethoxy-4-methylcoumarin at varied concentrations (0-400 µg mL-1) for 48 h. Afterwards, 96-well plate was centrifuged at 400g for 5 min to settle down the present 6-ethoxy4-methylcoumarin in the solution. Then, a 100-µL-1 supernatant was added to a fresh well of 96well plate, which contained 100 µL of reaction mixture from the kit, and was incubated at room temperature for 30 min. Then, the absorbance of solution was evaluated at 490 nm using a microplate reader (Elisa reader Bio-Tek, USA). LDH levels in the media versus the cells were quantified and compared with the control values according to the instruction of kit [32]. 1.2.

TAC and TOS analysis

The assay was calibrated with H2O2 and the results were expressed in terms of micromolar H2O2 equivalent per liter (μmol H2O2Equiv. L-1). The automated TAC and TOS tests were performed using commercially available kits (Rel Assay Diagnostics, Gaziantep, Turkey). Plasma samples were treated with 6-ethoxy-4-methylcoumarin for 48 h. 2. Results and discussion 2.1.

Molecular geometry

The best steady optimal geometry acquired from B3LYP/6-311++G(d,p) method. Fig. 2b shows the numbering scheme of atoms of the 6-ethoxy-4-methylcoumarin molecules. Molecular symmetry can be utilized to prescribe numerous molecular features such as its dipole moment and spectroscopic transitions. The 6-ethoxy-4-methylcoumarin is planar, aromatic and cyclic in nature due to the continual delocalization of electrons in the coumarin ring pattern. From the DFT calculations of our compound, C4-O19 and C11‒O19 bound distances were determined as 1.382 Å and 1.391Å respectively, using B3LYP/6-311++G(d,p) method. It has 5

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been reported that actual bond length of C‒O in heterocyclic ring is 1.43 Å [33]. The results showed that C4-O19 bond length significantly reduced due to the fusion of benzene ring with the α-pyrone ring through O18 atom and these data were compatible with previous studies [34, 35]. In addition, the C11═O18 bond distance was compatible with the results for coumarin structures [36]. The optimized geometrical parameters of 6-ethoxy-4-methylcoumarin were compared with XRD data of previous studies [34]. Table 1 shows the values of the geometric parameters. The Root mean square error (RMSE) was used to compare experimental values with the theoretical data. The obtained RMSE results for bond angles and bond lengths were 0.23° and 0.016Å for B3LYP/6-311++G(d,p), respectively. The difference between experimental and calculated geometrical parameters may be caused by the environment of the compound. It was clear that while theoretical calculations belonged to gaseous phase, experimental results belonged to solid phase. A logical method for global comparison of the structures obtained with the theoretical calculations is superimposing the molecular skeleton with that obtained from X-ray diffraction [34] and giving a RMSE of 0.042Å for B3LYP (Fig. 2c). According to these results, it may be asserted that the DFT calculation well reproduced the geometry of the 6-ethoxy-4methylcoumarin. 2.2.

Nuclear Magnetic Resonance (NMR) spectra

The GIAO (Gauge Including Atomic Orbital) method is one of the most common approaches for calculating isotropic nuclear magnetic shielding tensors [37,38]. In the present study, the 1H and

13C

NMR chemical shifts of the 6-ethoxy-4-methylcoumarin were calculated by using

B3LYP functional method with 6-311++G(d,p), 6-311G(d,p) and cc-pVTZ basis sets. The calculations were performed in dimethyl sulfoxide (DMSO) solution using IEF-PCM method. The results obtained in gas phase were compatible with experimental data. Table 2 shows the 13C and 1H theoretical and experimental chemical shifts, isotropic shielding constants, and the assignments of the 6-ethoxy-4-methylcoumarin. Chemical shift of all carbons in heterocyclic ring was set to downfield because of the existence of electronegative oxygen atom in pyrone ring. Therefore, C8, C11, and C12 chemical shifts were obtained at the downfield of 152.0, 161.0, and 115.4 ppm. Aromatic carbons signal

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with chemical shift values between 100 and 200 ppm [39]. The chemical shifts of the benzene ring carbon atoms C1, C2, C3, C4, C5 and C6 were 155.3, 108.5, 120.4, 147.8, 119.0, and 117.9 ppm, respectively. Because of electronegative atoms (O19 and O27) in the ring, the chemical shift of the C1 and C4 carbon atoms in benzene ring was set to downfield. The C12 atom was highly shielded than C8 owing to the delocalization of the lone pair electrons from C11=O18 group the adjacent C8=C12; therefore, the chemical shift of the C8 was observed at the downfield at 152.05 ppm; whereas, the chemical shift of C12 was observed at 115.45 ppm. Moreover, the above observed peak at the δ 18.72 ppm was assigned to methyl group. The proton (H13) in pyrone ring and the chemical shifts of aromatic protons (H7, H9 and H10) were detect at 6.30, 7.00, 7.10, and 7.30 ppm respectively. These protons appeared in upper area due to the increase in the electron density by giving electron to ring of methyl group. In addition, H16, H17 and H18 chemical shifts were determined at 2.35 ppm. The protons in the methyl group were equivalent and produced one signal. The results are parallel with the previous reports [40,41]. As seen in Table 2, theoretical 1H and

13C

chemical shift outcomes of 6-ethoxy-4-

methylcoumarin were generally closer to the experimental chemical shift data. 3.3 Vibrational assignments While the harmonic vibrational frequencies of 6-ethoxy-4-methylcoumarin were determined by applying the DFT/B3LYP technique with 6-311++G(d,p) basis set, the vibrational band assignment was carried out based on the Gauss-View molecular visualization program. The harmonic vibrational frequencies associated with the optimized structure was evaluated according to the same theoretical principles and the scaling of the resulting frequencies was done by 0.9684. The vibrational frequencies were analyzed in order to make the assignment of the visible peaks easier and the calculation outcomes for 6-ethoxy-4-methylcoumarin were compared with the experimental results (Table 3). The coherence of the experimental results with our calculation gave good conclusions in general. The plots in Figure 3 show the correspondence between the experimental and calculated frequencies. 3.4.

CO vibrations

Coumarins were observed at two types of CO stretching vibrations; the first one was C‒O and the second one was C=O stretching vibrations. The C=O stretching frequency was observed in

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the range of 1600-1850 cm-1 owing to its large change in dipole moment and its characteristic frequency used to study a wide range of the compounds [42]. A very strong infrared absorption band at 1712 cm-1 was readily assigned to the carbonyl vibration in 6-ethoxy-4-methylcoumarin and calculated as 1753 cm-1 for B3LYP. The C11‒O19 and C4‒O19 vibrations of pyrone rings of other coumarins were observed in the 1250-850 cm-1 region [43]. CO vibrations values for structural study of 6-ethoxy-4methylcoumarin compound, the O19‒C11 and C4‒O19 stretching vibrations were observed at 786 cm-1 and 1243 cm-1 experimentally and calculated at 835 cm-1 and 1265 cm-1 for B3LYP/6311++G(d,p) level. 3.5.

CH and CH3 vibrations

The aromatic structures commonly show multiple bands between 3000 and 3100 cm-1 which is the characteristic region for the C‒H stretching vibrations [44]. In the present study, the experimental C‒H stretching vibration was observed in the FTIR spectrum at 3023–3099 cm-1. The same vibration was calculated theoretically in the range of 3065–3131 cm-1 using B3LYP method and it showed a good correlation with the experimental data. The C‒H out-of- plane bending vibrations appeared in the region of 1000-675 cm-1 and C‒H in-plane bending vibrations appeared in the region of 1400-1050 cm-1 [45,46]. The C‒H in-plane bending vibrations of ring appeared at 1277 cm-1 and 1116 cm-1 experimentally and calculated as 1283 cm-1 and 1160 cm-1 for B3LYP method, respectively. The C‒H out-of-plane bending vibrations of ring appeared at 889 cm-1 experimentally and calculated as 923 cm-1 for B3LYP method. The CH3 group usually gives rise to nine fundamental vibrations such as three bending modes, three stretching modes, two rocking modes, and a single torsional mode. The symmetric and asymmetric stretching vibrations of methyl group normally appear at 2850-3000 cm-1 [47]. The calculated wave numbers of CH3 group vibrations were observed at 3015 cm-1 (vas, ethoxy), 2983 cm-1 (vas, pyron), 2943 cm-1 (vs, ethoxy) and 2927 cm-1 (vs, pyron) for B3LYP method. 3.6.

CC vibrations

The 6-ethoxy-4-methylcoumarin was composed of hetero aromatic ring fused with aromatic benzene ring. The aromatic ring carbon-carbon stretching modes appeared in the region of 16501200 cm-1 [42]. Therefore, the stretching vibrations were observed at 1629, 1583 and 1323 cm-1

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experimentally and calculated as 1650 cm-1, 1616 cm-1 and 1351 cm-1 for B3LYP method respectively. Table 3 shows the other values. 3.7.

Frontier molecular orbitals

As application of a molecular orbital theory that characterizes HOMO-LUMO interactions, the frontier molecular orbital theory is critical in quantum chemistry. The most straight forward of such interactions, which contributes to the identification of molecular attributes, is the one related to the discrepancy between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of a neutral system [48]. The LUMO energy is associated with the electron affinity and defines how sensitive the molecule is to a nucleophilic attack. The HOMO energy is associated with the ionization potential and defines how sensitive the molecule is to an electrophilic attack. The compound’s chemical activity is usually indicated by the energy values of HOMO and LUMO and the difference of energy between them. While a robust interaction and quick reaction is denoted by a small energy difference between HOMO and LUMO, a weak interaction and slow reaction is denoted by a large energy difference between HOMO and LUMO [49]. For this study, there was a very robust interaction between donor and acceptor because the reaction of 4-ethoxyphenol (I) and ethyl acetoacetate is extremely fast. Figure 4 shows arrangement manners of HOMO-1, HOMO, LUMO and LUMO+1 orbitals and their levels of energy, as determined at the B3LYP/6-311++G(d,p) level for 6-ethoxy-4methylcoumarin. It clearly reflected the delocalization of the HOMO, HOMO-1, LUMO and LUMO+1 electrons on the benzene and pyrone ring, as well. The HOMO-LUMO energy difference was 4.210 eV. Thus, the energy difference was an indicative of the molecule’s chemical activity. Several molecular parameters can be determined based on the energy values of HOMO and LUMO. The minimum amount of energy necessary to eliminate an electron from the atom or molecule in gaseous state is known as the ionization potential (I = - EHOMO). The amount of energy discharged as a result of the addition of one electron to a gaseous molecule is called as the electron affinity (A = - ELUMO). An atom’s predilection to attracting electrons is known as electronegativity (χ). Weight transfer prevention in a molecule is denoted by chemical hardness (η). The weight transfer of a molecule with higher chemical hardness is either minimal or non-

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existent [50]. Table 4 shows the parameters of the electronic structure determined based on the B3LYP/6-311++G(d,p) technique. 3.8.

UV–Vis spectral studies

Equivalent to the transition from the ground to the first excited state, the electronic absorption is primarily defined by one electron excitation from HOMO to LUMO. Categorization of electronic transitions is typically based on the orbitals involved or certain sections of the molecule in question. The electronic transitions most frequently encountered in organic molecules include π–π*, n–π* and π* (acceptor)–π (donor). Figure 5a shows the experimental UV-Vis absorption spectra of 6-ethoxy-4-methylcoumarin that were documented in the range of 200-400 nm in ethanol. The electronic absorption spectrum in ethanol was subjected to DFT calculation to gain an insight into the title compound’s electronic transitions. Owing to the fact that line shape and relative strength are qualitatively consistent, this calculation can characterize the spectral attributes of 6-ethoxy-4-methylcoumarin. The vibrational transition of electrons from ground to excited state is the source of the absorption spectra of organic compounds. The title molecule’s low-lying excited states were determined through B3LYP/6-311++G(d,p) calculation underpinned by a fully optimized ground state structure. Figure 5b shows the UV spectrum that was visualized. In an UV spectrum, the maximum absorption peak (λmax) is equivalent to vertical excitation according to the Frank-Condon principle. Consistent with the experimental values of 330 nm in ethanol, the calculation anticipated one intense electronic transition at 3.6642 eV (335 nm) with an oscillator strength of f = 0.0856. The molecular orbital coefficients and their plots show that major electronic excitations arise from π–π* transitions. The transition originates from H‒1/L with oscillator strength 0.1623 corresponding to second absorption maxima at 275 nm. The third band at 225 nm corresponds to H‒2/L transition calculated at 215 nm. These values are similar to those found in related compounds [35,36,40]. 3.9.

Molecular electrostatic potential

The charge distributions of molecules are shown in 3D by electrostatic potential maps, otherwise called electrostatic potential energy maps, which enable observation of charge variations in the different areas of a molecule. The nature of the interaction between molecules

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can be elucidated if the charge distributions are known. Furthermore, the analysis and anticipation of the reactive behavior of a molecule can be effectively undertaken based on the electrostatic potential map (MEP) that the nuclei and electrons of molecule, which are considered as charge distributions, generate in the space surrounding the molecule [51]. Different colors denote different electrostatic potential values of the surface, with red and blue which indicates the most negative and most positive potential, respectively. Colors to intermediate potentials is allocated in accordance with the color spectrum: red< orange < yellow < green < blue. At this point, the red areas on the map correspond to the molecular areas with the highest abundance of electrons; whereas, the blue areas correspond to the molecular areas with the fewest electrons [52]. For the compound, the color code of the MEP map ranged from -0.0425 a.u. (deepest red) to 0.0425 a.u. (deepest blue), the strongest attraction and strongest repulsion are denoted by the blue area and the red area, respectively. Figure 6a and Figure 6b show the mapping of the electrostatic potential surface for the compound. According to the results, the electronegative oxygen atoms and the nucleophilic reactive hydrogen atoms are respectively covered by the negative potential areas typically related to the single pair of electronegative atoms and by the positive potential areas. The strongest repulsion and the strongest attraction are denoted by red and blue, respectively. It was observed that the strongest repulsion was exhibited by the oxygen atoms. The compound negative areas were primarily located over the carbonyl (C=O) group, as indicated by the MEP map; whereas, the maximum positive areas were located over the methyl (CH3) groups. This result is also informative about the area where intermolecular interaction between compounds is possible. 3.10.

Atomic charge distributions in gas and solution phases

The electronic charge distribution in a molecule as well as the nature of the molecular orbitals (i.e. antibonding, bonding or nonbonding) for certain atom pairs can be characterized using the technique of Mulliken charge distribution [53]. The formation of pairs of donors and acceptors including the charge transfer in the molecule is indicated by the charge distribution over the atoms. Table 5 shows the Mulliken atomic charges for carbon and oxygen atoms of 6-ethoxy-4methylcoumarin determined at the B3LYP/6-311++G(d,p) level with the molecule in gas phase. Additionally, Table 5 also shows the obtained values of the three solvents (chloroform, water and ethanol) which were chosen to determine the solvent effect on the atomic charge distributions of

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6-ethoxy-4-methylcoumarin on the basis of the B3LYP/6-311++G(d,p) model and PCM technique. According to the Mulliken technique, the negative atomic charges of the O18 and O19 atoms of pyrone ring, C14 atom of methyl and C23 atom of ethoxy are larger compared to the gas phase. On the other hand, the O18, O19, and O27 atoms have larger atomic charge values in the solution phase compared to the gas phase. These data increase with increasing solvent polarity. Thermodynamic properties Several thermodynamic parameters of 6-ethoxy-4-methylcoumarin such as thermal energy, rotational temperatures, rotational constants, zero-point vibrational energy, specific heat capacity and entropy were calculated by B3LYP/6-311++G(d,p) basis set at 1.00 atm pressure and 298.150 K as seen in Table 6. 3.11.

Biological Activities

Coumarins are well known natural compounds possessing a system of benzene and pyrone rings [54]. Several studies have investigated great biologic potentials of the members of these heterocyclic compounds [55-57]. The cytotoxic properties of 6-ethoxy-4-methylcoumarin compound were examined. MTT analysis was used to determine the number of living cells in the proliferation. According to the obtain results, 6-ethoxy-4-methylcoumarin showed a cytotoxic effect at 200 mg/L in peripheral human blood lymphocytes (Table 7). These results are compatible with previous data. In a previous study, the cytotoxic effects of 7-hydroxy-4-methylcoumarin were evaluated using the MTT assay in Hep2 (human epithelioid larynx carcinoma) cell lines and coumarin was shown to reduce cell viability with an IC50 of 62.5 mg/ml. [58] In another study, it was stated that 7 - [(E) -3 ', 7'-dimethyl-6'-oxo-2', 7'-octadienyl] oxy coumarin had a cytotoxic effect (IC50 8.10 μM) [59]. Furthermore, it was confirmed that many coumarin derivatives had a significant cytotoxic activity in both in vitro and in vivo [60]. Besides, LDH activity was also measured spectrophotometrically to determine the cytotoxicity of the culture medium. The cytotoxic effect of 6-ethoxy-4-methylcoumarin was determined as 400 mg/L. With the parallel the study results, Ilić et al. (2014) showed that there

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was an increase in LDH release in mouse fibrosarcoma L929, human glioma U251 and mouse melanoma B16 cell lines after exposure to a coumarin-derived ligand [61]. It is well known that oxidative stress originates from imbalance between oxidant species and antioxidant status [62]. The decreased TAC levels and increased TOS levels may cause reactive oxygen species that lead to cytotoxic and genotoxic injury [63,64]. Nowadays, there is a growing interest among scientists to synthesize new antioxidant molecules to be used in the treatment of diseases associated with oxidative stress. By this way, we evaluated TAC and TOS parameters as oxidative stress indicators for determining biological efficacy of 6-ethoxy-4-methylcoumarin. TAC and TOS levels were detected by colorimetric methods to evaluate antioxidant/oxidant potentials. The obtained data indicated that 6-ethoxy-4-methylcoumarin had an antioxidant activity at a dose of 25 mg /L. Moreover, 6-ethoxy-4-methylcoumarin did not change oxidative status at used doses as seen in Table 7. Parallel to the study results, Al-Majedy et al., (2016) reported that different coumarin derivatives had antioxidant activity [65]. In another study, Datta et al., (2011) showed that new synthesized 6-coumarin compound had antioxidant effects [66]. 4. Conclusion In this study, a new 6-ethoxy-4-methylcoumarin was synthesized by reaction of 4ethoxyphenol and ethyl acetoacetate. Characterization of 6-ethoxy-4-methylcoumarin was confirmed by UV, FT‒IR, 1H‒NMR and 13C‒NMR. As a conclusion:

1. The structural data of 6-ethoxy-4-methylcoumarin calculated at B3LYP/6-311++G(d,p) and B3LYP/G-311G(d,p) were in very good correspondence with X-ray experimental values. 2. The best geometrical parameters were obtained for (C4-O19, C11-O19 and C11=O18) bond distance with a RMSE overlay errors of 0.016 A⸰. 3. An agreement was determined between the theoretical and experimental results with regard to the accurate allocation of the vibrational frequencies to the molecular structure based on the theoretical calculations. Furthermore, the positive and negative potential sites surrounded the hydrogen atoms and the electronegative atoms, respectively.

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4. The basic transition in the spectrum of 6-ethoxy-4-methylcoumarin was found to be π–π*. 5. The title compound had an antioxidant effect and a low toxicity on cultured human blood cells. 6-ethoxy-4-methylcoumarin may be used in pharmaceutical and medical applications but further related studies are needed. Conflict of interest No potential conflict of interest was reported by the authors. Acknowledgements: This research was supported by Kastamonu University Scientific Research Projects Coordination Department under the Grant no. KU-BAP01/2019-63. Computational resources were provided by the FeynmanGrid HPC Cluster at the CompecTA The HPC Company.

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Manuscript title: Synthesis, Structure, Cytotoxic and Antioxidant Properties of 6-ethoxy-

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Figure Captions Fig. 1. The reaction of the synthesis 6-ethoxy-4-methylcoumarin Fig. 2. (a) Molecular structure of 6-ethoxy-4-methylcoumarin (b) The theoretical geometric structure of the title compound (with B3LYP/6-311++G(d,p) level). (c) Atom-by-atom superimposition of the structures calculated (green) over the X-ray structure (black) for the title compound. For the sake of clarity, H atoms not involved in the motifs shown were omitted. Fig. 3. (a) FT-IR spectrum of the title compound. (b) Simulated B3LYP/6-311++G(d,p) level IR spectra. Fig. 4. Molecular orbital surfaces and energy levels given in parentheses for the HOMO, HOMO1, LUMO and LUMO+1 of the title compound computed at B3LYP/6-311++G(d,p) level. Fig. 5. Experimental UV spectrum (a) and TD-DFT//B3LYP/6-311++G(d,p) level UV spectrum of the title compound (b). Fig. 6. (a) Molecular electrostatic potential map calculated at B3LYP/6-311++G(d,p) level. (b) Contour shape of the title compound.

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Fig. 1. The reaction of the synthesis 6-ethoxy-4-methylcoumarin

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Fig. 2. (a) Molecular structure of 6-ethoxy-4-methylcoumarin (b) The theoretical geometric structure of the title compound (with B3LYP/6-311++G(d,p) level). (c) Atom-by-atom superimposition of the structures calculated (green) over the X-ray structure (black) for the title compound. For the sake of clarity, H atoms not involved in the motifs shown were omitted.

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Fig. 3. (a) FT-IR spectrum of the title compound. (b) Simulated B3LYP/6311++G(d,p) level IR spectra.

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Fig. 4. Molecular orbital surfaces and energy levels given in parentheses for the HOMO, HOMO-1, LUMO and LUMO+1 of the title compound computed at B3LYP/6-311++G(d,p) level.

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Fig. 5. Experimental UV spectrum (a) and TD-DFT//B3LYP/6-311++G(d,p) level UV spectrum of the title compound (b).

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Fig. 6. (a) Molecular electrostatic potential map calculated at B3LYP/6311++G(d,p) level. (b) Contour shape of the title compound.

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Journal Pre-proof Highlights 1) The structural data of 6-ethoxy-4-methylcoumarin calculated at B3LYP/6311++G(d,p) and B3LYP/G-311G(d,p) were in very good correspondence with X-ray experimental values. 2) Experimental data are in agreement with theoritical data 3) Biological results are agreement with the literature

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Table Captions Table 1. Selected molecular structure parameters. Table 2. Theoretical and experimental 1H and 13C isotropic chemical shifts (with respect to TMS, all values in ppm) for the title compound. Table 3. Comparison of the experimental and calculated vibrational frequencies. (cm-1) Table 4. Electronic structure parameters of the title compound. Table 5. Atomic charges (e) of the title compound in gas and solution phases. Table 6. Thermodynamic properties of the title compound. Table 7. The results of cytotoxicity TAC and TOS of 6-Ethoxy-4-methyl coumarin in cultured human blood cells for 48h. Values are expressed as mean ±SD for four cultures in each group. The mean values are shown by different letters.

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Table 1. Selected molecular structure parameters. Parameters

6-311++G(d,p)

6-311G(d,p)[a]

Bond lengths (Å) O(18)-C(11) 1.220 O(19)-C(11) 1.391 C(4)-O(19) 1.382 C(1)-O(27) 1.386 C(20)-O(27) 1.469 C(11)-C(12) 1.455 C(1)-C(2) 1.394 C(8)-C(14) 1.511 C(3)-C(4) 1.408 C(1)-C(6) 1.405 Bond Angles (°) C(12)-C(11)-O(18) 127.3 C(12)-C(11)-O(19) 114.9 C(4)-O(19)-C(11) 121.9 C(3)-C(4)-O(19) 122.0 C(12)-C(8)-C(14) 121.1 C(3)-C(8)-C(14) 119.5 C(1)-O(27)-C(20) 119.6 C(2)-C(1)-O(27) 115.9 C(6)-C(1)-O(27) 125.4 C(2)-C(1)-C(6) 119.0 Dihedral Angles (°) C(4)-C(3)-C(8)-C(14) 179.9 C(1)-C(2)-C(3)-C(8) -179.8 C(3)-C(8)-C(12)-C(11) 0.04 a Values are taken from Ref. [35]. b Values are taken from Ref. [34].

2

Experimental results[b]

1.201 1.409 1.354 1.350 1.426 1.462 1.413 1.405 1.394

1.206 1.376 1.381 1.367 1.417 1.439 1.376 1.500 1.394 1.400

128.3 115.7 123.7 120.7 118.9 115.2 124.2 120.5

126.3 116.9 123.5 121.5 121.5 119.9 117.8 115.6 124.2 120.2

-

179.4 -179.8 -179.0

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Table 2. Theoretical and experimental 1H and 13C isotropic chemical shifts (with respect to TMS, all values in ppm) for the title compound.

C2

Theoretical (B3LYP) 6-311++G(d,p) cc-pVTZ 151.9 162.4 114.5 108.6

C3

121.2

126.3

125.1

120.5

C4

146.8

155.8

154.9

147.8

C5

117.5

122.1

122.3

119.1

C6

115.4

126.6

126.7

117.9

C8

148.9

163.1

161.3

152.1

C11

158.5

166.1

162.9

161.1

C12

114.9

118.3

118.1

115.5

C14

33.3

20.7

20.3

18.7

C20

73.6

66.3

65.6

62.2

C23

25.8

15.7

15.4

14.8

2H(CH3CH2)

5.04 and 5.06

4.36

3.97

4.10

3H(CH3CH2)

1.60, 1.80 and 2.01

1.72, 1.79 and 1.79

1.38, 1.54 and 1.54

1.40

3H(CH3)

2.70, 3.21 and 3.22

2.64, 2.97 and 2.97

2.22, 2.55 and 2.55

2.35

3H(Ar-H)

7.20, 7.30 and 7.40

7.53, 7.70 and 7.74

6.95, 7.38 and 7.44

6.95-7.40

1H(CH)

6.42

6.66

6.25

6.30

Atom C1

161.6

Experimental New exp. 155.3

107.5

108.5

6-311G(d,p)

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Table 3. Comparison of the experimental and calculated vibrational frequencies. (cm-1) Assignments ʋ aromatic ring CH ʋas aromatic ring CH ʋ pyron CH ʋas aromatic ring CH ʋas ethoxy (CH3 + CH2) ʋas methyl CH3 ʋas ethoxy CH3 ʋas ethoxy CH2 ʋas pyron CH3 ʋs ethoxy CH3 ʋs ethoxy CH2 ʋs pyron CH3 ʋ pyron C=O ʋ pyron C=C ʋ aromatic ring CC ʋ aromatic ring CC δ pyron CH3 δ ethoxy CH3 δ pyron CH3 ʋ aromatic ring CC β ethoxy CH2 δ aromatic ring C-H ʋ pyron C4-O19 + δ aromatic ring

Experimental FT-IR (cm-1) with KBr 3099 3082 3023 2984 2932 2885 1712 1629 1583 1497 1480 1381 1323 1277 -

CH

δ aromatic ring CH 1243 ʋ ethoxy C20-O27 1010 α aromatic ring CH 889 α (aromatic ring + pyron) CH ʋ pyron O19-C11 786 ʋ, stretching; δ, in-plane bending; α, out of-plane bending β, rocking; s, symmetric; as, asymmetric

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Calculated B3LYP 3131 3130 3122 3118 3029 3021 3015 3007 2983 2943 2941 2927 1753 1669 1650 1616 1499 1497 1418 1351 1298 1283 1265 1160 1002 923 871 835

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Table 4. Electronic structure parameters of the title compound. Parameters

B3LYP 6-311++G(d,p)

EHOMO (eV)

-5.884

ELUMO (eV)

-1.735

∆E= ELUMO − EHOMO (eV)

4.149

I (eV)

5.884

A (eV)

1.735

X (eV)

3.809

𝑛 (eV)

2.075

S (eV-1)

0.241

Dipole moment (D) μtotal

6.4363

μx

-5.5833

μy

3.2019

μz

0.0002

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Table 5. Atomic charges (e) of the title compound in gas and solution phases. Atom

C1 C2 C3 C4 C5 C6 C8 C11 C12 C14 C20 C23 O18 O19 O27

In gas phase B3LYP/6-311++G(d,p) 0.400 −0.274 0.090 0.318 −0.182 −0.191 0.195 0.612 −0.295 −0.531 −0.042 −0.463 −0.475 −0.542 −0.546

In solution phase B3LYP/6-311++G(d,p) Chloroform Ethanol Water (Ꜫ=4.90)

0.292 −0.171 0.059 0.234 −0.124 −0.132 0.142 0.501 −0.217 −0.488 −0.026 −0.416 −0.470 −0.564 −0.583

6

(Ꜫ=24.3)

(Ꜫ=78.39)

0.292 −0.171 0.057 0.235 −0.126 −0.135 0.144 0.501 −0.218 −0.489 −0.029 −0.419 −0.490 −0.569 −0.589

0.292 −0.171 0.057 0.235 −0.127 −0.135 0.144 0.502 −0.218 −0.489 −0.029 −0.419 −0.493 −0.570 −0.590

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Table 6. Thermodynamic properties of the title compound. Parameters

B3LYP 6-311++G(d,p)

Zero-point vibrational energy(kcal mol-1)

135.77913

Total energy (a.u.)

-690.1791

Rotational constants (GHz) 137957 0.32553 0.27185 Rotational temperatures (K) 0.06621 0.01562 0.01562 Entropy (cal mol-1 K-1) Rotational

32.243

Translational

41.844

Vibrational

37.400

Total

111.488

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Journal Pre-proof Table 7. The results of cytotoxicity TAC and TOS of 6-Ethoxy-4-methyl coumarin in cultured human blood cells for 48h. Values are expressed as mean ±SD for four cultures in each group. The mean values are shown by different letters. Dose Control (-) Control (+) 10 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L 400 mg/L

MTT (Cell viability %)

LDH release (%)

TAC level

TOS level

100d

100a

6.2±0.7a

11.6±2.3a

40.3±4.7a

245.4±25.8c

12.5±1.5c

39.1±3.2b

100.2±4.5d 99.7±4.3d 96.6±4.7d 93.4±4.4cd 88.2±5.0c 79.5±4.3b

97.9±5.1a 97.6±4.2a 100.9±4.4a 101.5±4.7a 102.6±5.1a 119.8±5.4b

6.4±0.8a 7.3±0.6b 6.5±0.7a 6.1± 0.6a 6.1±0.7a 5.9±0.8a

11.0±2.4a 11.3±2.3a 11.4±2.6a 11.5±2.3a 11.9±2.2a 12.2±2.1a

The different letters are significantly different from each other at level of 5 %

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