Synthesis, DFT calculations, spectroscopy and in vitro antioxidant activity studies on 4-hydroxyphenyl substituted thiopyrimidine derivatives

Accepted Manuscript Synthesis, DFT calculations, spectroscopy and in vitro antioxidant activity studies on 4-hydroxyphenyl substituted thiopyrimidine derivatives

Esvet Akbas, Suat Ekin, Erdem Ergan, Yagmur Karakus PII:

S0022-2860(18)30234-5

DOI:

10.1016/j.molstruc.2018.02.080

Reference:

MOLSTR 24902

To appear in:

Journal of Molecular Structure

Received Date:

18 October 2017

Revised Date:

13 December 2017

Accepted Date:

20 February 2018

Please cite this article as: Esvet Akbas, Suat Ekin, Erdem Ergan, Yagmur Karakus, Synthesis, DFT calculations, spectroscopy and in vitro antioxidant activity studies on 4-hydroxyphenyl substituted thiopyrimidine derivatives, Journal of Molecular Structure (2018), doi: 10.1016/j.molstruc. 2018.02.080

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ACCEPTED MANUSCRIPT

Synthesis, DFT calculations, spectroscopy and in vitro antioxidant activity studies on 4-hydroxyphenyl substituted thiopyrimidine derivatives Esvet Akbas1*, Suat Ekin1**, Erdem Ergan2 and Yagmur Karakus1 1

Van YuzuncuYil University, Department of Chemistry, 65080 Van, Turkey.

2

Van YuzuncuYil University, Van Vocational School Of Security, Department of

property protection and security, 65080 Van, Turkey; [email protected] (E.E); [email protected] (Y.K) * Correspondences: [email protected] ; [email protected] ; Tel.: +90 4322251701 OH

OH

OH

O HHN 2

400 350 300

+

HO

H2N

O HO Ph

Ph

S

O

N Ph

N H

Ph

N OH

OH O

H

O CH3 H

N Ph

N

H H

Ph

N Ph

S

N O

150 100 50 0 3

4

5

-tocopherol

O OC2H5

200

2

S

H H H H

OH

250

1

O

S

Ph IC5 0 values (µM)

H S

N

H N

Ph

H

N Ph

H

O

H

Ph

H

Ph

O

O

Trolox

OC2H5

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Synthesis, DFT calculations, spectroscopy and in vitro antioxidant activity studies on 4-hydroxyphenyl substituted thiopyrimidine derivatives Esvet Akbasa*, Suat Ekina**, Erdem Erganb and Yagmur Karakusa aVan

YuzuncuYil University, Department of Chemistry, 65080 Van, Turkey.

bVan

YuzuncuYil University, Van Vocational School of Security, Department of property

protection and security, 65080 Van, Turkey; [email protected] (E.E); [email protected] (Y.K) * Correspondences: [email protected] ; [email protected] ; Tel.: +90 4322251701

Abstract 4-Hydroxyphenyl substituted thiopyrimidine derivatives have been synthesized with the starting

compound

5-benzoyl-6-phenyl-4-(4-hydroxyphenyl)-1,2,3,4-tetrahydro-2-

thioxopyrimidine (1). The compounds optimized geometrically with DFT in Gaussian at the B3LYP/6-31G (d, p) level in order to obtain information about the 3D geometries and electronic structures. The structures were characterized on the basis of 1H NMR, 13C NMR, FT-IR, and elemental analysis. All compounds tested in vitro in order to assess their antioxidant activity. On the biological properties including of free radical scavenging ability (DPPH•), ABTS•+ assay, PhNHNH2 (phenyl hydrazine) induced haemolysis of erythrocytes and metal chelating activities were performed. The results were compared to standard antioxidants, such as -tocopherol and trolox. The conclusion shows that the compounds thioxopyrimidine (1), dihydropyrimido[2,1-b][1,3]thiazin-4(6H)-one (4) and dihydropyrimidin-2(1H)-ylidene) malonate (5) exhibit a stronger antioxidant activity than the other derivatives. Keywords Synthesis Thiopyrimidines DFT calculations Antioxidant activity

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1. Introduction Due to activity of various substituted pyrimidines, such as anti-inflammatory, analgesic, antipyretic, antihypertensive, antagonistic, herbicides, plant growth regulators and calcium ion modulators considerable attention has focused on these compounds [1-4]. In particular, thiazolo[3,2-a]pyrimidine derivatives have been the focus of intense research and several members of this class have been reported to possess analgesic, anti-cancer, anti-microbial, antiinflammatory and anti-hypertensive activities, as well as inotropic activity; other possible applications include their use as antimalarial, HIV reverse transcriptase inhibitors, and therapeutic agents for neurodegenerative diseases [5-10]. The combination of potentially significant therapeutic value with simple and efficient synthetic procedures makes this class of compounds very appealing for biological testing. In order to modulate the affinity of these compounds to specific biological targets (e.g., receptors) by rational design it is of utmost importance to obtain a full characterization of their spectroscopic and electronic characteristics. So in continuation of our previous work [11], and our long term interest on synthesis of biologically important pyrimidine derivatives, herein we report the synthesis of 4-hydroxyphenyl substituted pyrimidine derivatives and their antioxidant activities have also been studied. Moreover, the density functional theory (DFT) calculations have been performed to obtain various molecular properties of the present compounds. DFT/ B3LYP method has been commonly preferred to study structure and various properties of organic molecules because this method is efficient and offers an excellent trade-off between chemical accuracy and computational cost [12-15]. 2. Experimental 2.1. Synthetic procedures Melting points were determined on an Electrothermal Gallenkamp apparatus and are uncorrected. Microanalyses were performed on LECO CHNS 932 Elemental Analyzer. The FTIR spectra were obtained in as potassium bromide pellets using a Mattson 1000 FTIR spectrometer. The 1H and 13C NMR spectra were recorded on Bruker 300 MHz spectrometers, using TMS as an internal standard. All experiments were followed by TLC using DC Alufolien Kieselgel 60 F 254 Merck and Camag TLC lamp (254/365 nm).

2

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2.1.1. 5-Benzoyl-6-phenyl-4-(4-hydroxyphenyl)-1,2,3,4-tetrahydro-2-thioxopyrimidine (1) This compound was synthesized according to reference [11]. A mixture of dibenzoylmethane (1.6 mmol), 4-hydroxybenzaldehyde (1.1 mmol), thiourea (1.1 mmol) and 10 mL of glacial acetic acid containing a few drops concentrated hydrochloric acid was heated under reflux in condition for 8 h. The solution was allowed to stand for about 4 hours to yield of compound 1. Mp, 258-259oC. 2.1.2. 6-benzoyl-5-(4-hydroxyphenyl)-7-phenyl-5H-thiazolo[3,2-a]pyrimidin-3(2H)-one(2) A mixture of 1 (1mmol), 2-bromoacetic acid (1.1 mmol), and anhydrous sodium acetate (2 mmol), acetic anhydride (1.2 ml) were heated under reflux for 1 h in acetic acid (20 ml). The residue was treated with water (100 ml), the precipitate filtered off, and the formed crude product was recrystallized from methanol. Compound 2 was obtained in yield 0.195 g (45.7%). mp. 219220 ºC, IRKBr/cm-1: 1738, 1637 cm−1 (C=O). 1H-NMR (DMSOd6/ppm): δ 6.97-7.50 (m, 15H, Harom and OH), 6.36 (s, 1H, CHpyrimidine), 3.95 (2H, d, CH2, J=17.4 Hz, A part of AB system), 3.84 (2H, d, CH2, J=17.1 Hz, B part of AB system).

13C-NMR

(DMSOd6/ppm) δ 196.3 (C=Obenzoyl), 170.2

(C=O), 159.2, 150.9, 148.5, 137.7, 137.2, 137.0, 131.9, 129.5, 129.4, 128.9, 128.8, 127.9, 127.7, 122.1, 116.4, 56.8, 32.5. Anal. Calc. for C25H18N2O3S (426.49). C, 70.40; H, 4.25; N, 6.57; Found: C, 70.04; H, 4.74; N, 6.50. 2.1.3. 6-Benzoyl-5-(4-hydroxyphenyl)-2-methyl-7-phenyl-5H-thiazolo[3,2-a]pyrimidin-3(2H)-one (3) A mixture of 1 (1mmol) and 2-bromopropionic acid (1 mmol) were refluxed 2 h in dioxane (5 ml). The reaction mixture was cooled and the precipitate filtered off and then washed with water. The crude product was recrystallized from methanol. Yield 0.156 g (35.2%). mp. 250-251oC, IRKBr/cm-1: 1767, 1632 cm−1 (C=O), 1H-NMR (DMSOd6/ppm): δ 6.68-7.36 (m, 15H, Harom and OH), 6.03 (s, 1H, CHpyrimidine), 4.50 (q, 1H, CHthiazole J=2.7 Hz), 1.40 (d, 3H, CH3 J=7.2 Hz).

13C-NMR

(DMSOd6/ppm) δ 196.0 (C=Obenzoyl), 174.2(C=O), 159.2, 157.5, 145.7, 137.3,

136.9, 132.2, 130.1, 129.9, 129.0, 128.8, 128.6, 128.2, 127.8, 127.7, 117.0, 56.5, 42.3, 18.8. Anal. Calc. for C26H20N2O3S (440.51). C, 70.89; H, 4.58; N, 6.36; Found: C, 70.90; H, 4.60; N, 6.30.

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2.1.4.

7-Benzoyl-6-(4-hydroxyphenyl)-8-phenyl-2,3-dihydropyrimido[2,1-b][1,3]thiazin-4(6H)-

one (4) A mixture of 1 (1 mmol), 3-bromopropionic acid (1.1 mmol), anhydrous sodium acetate (2 mmol), and acetic anhydride (2 ml) was heated under reflux for 2 h in acetic acid (20 ml). The residue was treated with water (100 ml), the precipitate filtered off, and the formed crude product was recrystallized from 2-propanol. The compound 4 was obtained in yield 0.27 g (61.3%). Mp. 194-195oC, IRKBr/cm-1: 1697, 1628 cm−1 (C=O), 1H-NMR (DMSOd6/ppm): δ 6.85-7.39 (m, 15H, Harom. and OH), 2.22 (s,1H, CHpyrimidine), 3.05–3.25 (m, 4H, CH2-thiazine). 13C-NMR (DMSOd6/ppm) δ 196.4 (C=Obenzoyl), 169.4 (C=Othiazine), 156.5, 148.1, 140.4, 138.2, 137.0, 132.7, 129.9, 129.7, 129.6, 129.4, 129.1, 128.5, 128.3, 127.1, 119.3, 52.8, 35.9, 21.9. Anal. Calc. for C26H20N2O3S (440.51). C, 70.89; H, 4.58; N, 6.36; Found: C, 70.87; H, 4.62; N, 6.33. 2.1.5. Diethyl 2-(5-benzoyl-4-(4-hydroxyphenyl)-6-phenyl-3,4-dihydropyrimidin-2(1H)-ylidene) malonate (5) A mixture of 1 (1 mmol), and diethyl 2-bromomalonate (1 mmol) dissolved in dioxane/pyridine (10 ml/1 ml). The solution was heated under reflux for 3 h. After evaporated of solvent recovered oil substance was treated with 10 ml mixture of water and HCl (37%) (1:1) and washed with water. The resulting oil substance dissolved ethyl alcohol and precipitated with water. The separated solid filtered off and recrystallized methanol Yield 0.33 g (65%). Mp. 233234oC, IRKBr/cm-1: 3252, 3156 (NH), 3059, 2982 (CHarom.), 1632, 1612 (C=O); 1H NMR (DMSOd6/ppm): δ 12.05 (s, 1H, N1H), 10.64 (s, 1H, N3H), 7.36-6.97 (m, 13H, Harom and OH), 6.62-6.66 (m, 2H, Harom.), 5.66 (d, 1H, Cpyrimidine J= 3.6 Hz), 4.12-4.26 (m, 4H, CH2), 1.31 (t, 6H, CH3);

13C

NMR (DMSOd6/ppm): δ 195.7 (C=Obenzoyl), 171.1 (C=C) and 170.7 (C=O), 157.3,

156.1, 143.6, 138.7, 134.1, 133.7, 131.6, 130.3, 129.0, 128.9, 128.6, 128.0, 127.7, 115.8, 111.4, 60.2 (OCH2), 53.8 and 14.3 (CH3). Anal. Calcd. for C30H28N2O6 (512.55) C, 70.30; H, 5.51; N, 5.47; Found: C, 70.47 H, 5.44; N, 5.38. 2.2. Biological assay (Antioxidant Properties) 2.2.1. DPPH DPPH assay the hydrogen atoms or electrons donation ability of the corresponding hydroxyl-substituted pyrimidine compounds were measured from the bleaching of purple colored methanol solution of DPPH. This spectrophotometric assay uses stable radical 1,1-diphenyl-2picrylhydrazyl (DPPH) as a reagent. Briefly, 5 µL of various methanol concentrations of pyrimidine compounds (30-300 µM) were added to 5 mL of a 0.004 % methanol solution of 4

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DPPH. After a 30 min incubation period at room temperature in dark at room, the absorbance was read against a blank at 517 nm. The DPPH test is very useful in the micromolar range. Antioxidant activity of the pyrimidine compound samples were expressed in term of IC50 values (µM) required to inhibit DPPH radical formation by 50 %), which was calculated form the graph plotted inhibition percentage against extract concentration. Three replicates for each sample concentration were tested and compared with the appropriate standard α-tocopherol and trolox. Inhibition of free radical DPPH in percent was obtained by the following equation. AControl - Atest

Inhibition (%) =

X 100

AControl

Where control is the absorbance of the control (DPPH solution without test sample) and Atest is the absorbance of the test sample (DPPH solution plus scavenger). The control contains all reagents except the scavenger [12, 13]. 2.2.2. Metal chelating activity assay The chelating activity of pyrimidine compounds on ferrous ions was measured according to the method of Decker and Welch, Dinis et.al,. [14, 15] Aliquots of 0.2 mL of different methanol concentrations of the samples (30-300 µM) were mixed with 3.7 mL of ethanol. The mixture was incubated with FeCl2 (2 mM, 0.1 mL) for 30 min. The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL). Then, the mixture was shaken vigorously and left at room temperature

for

10

minutes.

Absorbance

of

the

solution

was

then

measured

spectrophotometrically at 562 nm against a blank. A lower absorbance indicates a higher chelating power. The chelating activity of the compound on Fe2+ was compared with αtocopherol and trolox at same concentrations. The percentage of inhibition of ferrozine–Fe2+ complex formation was calculated by using the formula given below:

Metal chelating effect (%) = 1-

ATest Acontrol

x100

2.2.3. ABTS assay Radical scavenging activities of the pyrimidine compounds were carried out using the method recommended by Arnao et al. (2001) [16] and with some modifications. The method is based on the capacity of different components to scavenge the ABTS radical cation (ABTS•+) 5

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compared to a standard antioxidant (α-tocopherol, trolox). General procedure for evaluation of ABTS radical activity: stock solution of 2 mM ABTS (2.2-azinobis-(3-ethylbenzothiazoline-6sulfonic acid) was prepared by dissolving in phosphate buffered saline (PBS, 100 ml, pH: 7.4) and 2.45 mM K2S2O8 water solutions were prepared as stock solution. The working solution was then prepared by mixing the two stock solutions in equal quantities. The mixture was left to stand in the dark at room temperature for 14–16 h before use. The solution was then diluted to obtain the absorbance of 0.700 ± 0.020 at 734 nm using the UV–Vis spectrophotometer. ABTS•+ Solution was prepared for each assay. 0.5 mL of different methanol concentrations of each compound (30-300 µM) was added to 0.3 mL of the ABTS•+ solution after vortexes allow to react for 1 h in the dark, and then, the absorbance was read at 734 nm. PBS solution was used as a blank sample. Free radical scavenging activity as percentage of the samples and the standard, positive control were calculated using the following formula. Inhibition (%) =

AControl - ATest

X 100

AControl

2.2.4. Assay for phenyl hydrazine induced haemolysis of erythrocytes Assay for phenyl hydrazine induced haemolysis of erythrocytes [17]. The phenyl hydrazine was used to induce hemolysis and radical scavenging activities of pyrimidine compounds were compared with a known antioxidant, α-tocopherol, trolox. The erythrocyte suspension (20% PCV) was prepared from rat blood. To the different sample methanol concentrations (30-300 µM) 1 ml of 0.5 mM phenyl hydrazine, 0.1 ml of 20% erythrocyte suspension were added and made up the volume to 3 ml with phosphate buffer saline. This was incubated at 37 °C for 1 h and centrifuged at 1000 g for 10 min. The supernatant was transferred to fresh test tubes and the absorbance was recorded at 540 nm. Percentage of anti-hemolysis was obtained by the following equation.

Inhibition (%) =

6

1-

ATest Acontrol

x100

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3. Results and discussion 3.1. DFT calculations and Chemistry The compound 1 was obtained according to literature 11. The spectral characteristics of compound 1 are in good agreement with the literature data. The reaction pathway and mechanism for compound (1) was given in scheme 1. Scheme 1 The theoretical analysis was carry out in order to obtain some structural and physicochemical data via by the quantum chemical methods with the density functional theory (DFT) in the gas phase using the 6-31G (d, p) method for the compounds 1-5 [11,22-24]. The values of the highest occupied molecular orbital (EHOMO), energy of the lowest unoccupied molecular orbital (ELUMO) and the energy gap (ELUMO-HOMO) were obtained. The frontier molecular orbital energies (EHOMO and ELUMO) are significant parameters for the prediction of the reactivity of a chemical species. The EHOMO is often associated with the electron donating ability of a molecule. Therefore, increasing values of EHOMO indicates higher tendency for the donation of electrons to the appropriate acceptor molecule with low energy and empty molecular orbital. Similarly the ELUMO indicates the ability of the molecule to accept electrons. The lower value of ELUMO means that the molecule would accept electrons. Literature reveals that a larger value of the energy gap indicates low reactivity to a chemical species because the energy gap is related to the softness or hardness of a molecule. A soft molecule is more reactive than a hard molecule because a hard molecule has a larger energy gap [25, 26]. As a result, this thioxopyrimidine derivative was found reactive for electrophilic attracts. Because it has lower ELUMO level and the narrow ELUMO-HOMO gap (Fig. 1) [11]. Fig. 1 The geometry optimizations of molecules 2-5 were carried out in gas phase (Fig.2). Fig 2 There is a stereogenic center in all synthesized compounds. Because of that the total energies of the R and S enantiomers are different from each other (Table 1). The calculated total energies (zero point corrected), frontier molecular orbital energies (EHOMO and ELUMO) and interfrontier energy gap (ELUMO-HOMO) data have been given in Table 1. Table 1 7

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The frontier orbital HOMO and LUMO of a chemical species are very important in defining its reactivity. High value of EHOMO of the molecules shows its tendency to donate electrons to appropriate acceptor molecules with low energy empty molecular orbitals. The energy of the lowest unoccupied molecular orbital indicates the ability of the molecule to accept electrons. The lower the value of ELUMO, the more probable the molecule would accept electrons. Consequently, concerning the value of the energy gap ΔE, larger values of the energy difference will provide low reactivity to a chemical species. The EHOMO and ELUMO energy of compound 5 is greater than the other derivatives. The narrow ELUMO-HOMO gap which will cause readily photoinduced excitation that may lead subsequent chemical reactions. The higher occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) diagrams are illustrated in Fig.3. Fig 3 According to these results the compound 1 with various electrophiles were studied and achieved synthesis of compound 2-4 (Scheme 2). The compound 2 was synthesized reactions of the starting material 1 and 2-bromoacetic acid in acetic acid. The IR spectrum of compound 2 showed that the absorption band at 1738, 1637 cm-1 because of carbonyl and benzoyl absorption groups, respectively. In the 1H-NMR (DMSOd6/ppm) appeared δ 6.36 (s, 1H, CHpyrimidine), 3.95 (2H, d, CH2thiazole, J=17.4 Hz, A part of AB system), 3.84 (2H, d, CH2thiazole, J=17.1 Hz, B part of AB system). On the other hand disappearance of the NH peaks in 1H-NMR spectra of compound 2 is a good evidence of the expected reaction (Fig. 4). Fig 4 Scheme 2 The reaction of 1 and 2-bromopropionic acid as a cyclocondensation reagent in dioxane under reflux condition gave compound 3 (Scheme 2). In 1H NMR spectra, the singlet at 6.03 ppm is due to the resonance of the CHpyrimidine. The quartet and doublet at 4.50 and 1.40 ppm (J = 7.2 Hz) are assigned to CHthiazole and to the methyl protons, respectively. When the starting compound 1 and 3-bromopropionic acid was heated under reflux condition with anhydrous sodium acetate/acetic anhydride in acetic acid lead to 7-benzoyl-6-(4hydroxyphenyl)-8-phenyl-2,3-dihydropyrimido[2,1-b][1,3]thiazin-4(6H)-one (4). 8

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In the 1H-NMR spectra of compound 4 absences of the characteristic absorption signals of the NH group in position 1 and 3 of starting material, is good evidence of the expected reactions. The 5-benzoyl-6-phenyl-4-(4-hydroxyphenyl)-1,2,3,4-tetrahydro-2-thioxopyrimidine (1) and diethyl 2-bromomalonate were performed in dioxane and diethyl 2-(5-benzoyl-4-(4hydroxyphenyl)-6-phenyl-3,4-dihydropyrimidin-2(1H)-ylidene) malonate (5) was obtained. This reaction is similar to the Eschenmoser sulfide contraction. The Eschenmoser reaction yields βenaminocarbonyl derivatives by the elimination of sulfur from an episulfide intermediate. This method requires a base and a tertiary phosphine. In this study, pyridine was used instead of phosphine in Eschenmoser sulphide contraction. It is proposed that initially, selective alkylation of the sulfur occurs in the thioxopyrimidine ring system, followed by elimination of the bridged sulfur by addition of a thiophilic agent (Scheme 3). Scheme 3 In 1H-NMR spectra of compound 5 appeared the signals of the δ 12.05 and 10.64 (s, 1H, NH), 7.36-6.66 (m, 15H, Harom and OH.), 5.66 (s, 1H, CHpyrimidine, J= 3.6 Hz), 4.12-4.26 (m, 4H, CH2), 1.31 (t, 6H, CH3). 3.2. Molecular electrostatic potential (MEP) It is well known that the molecular electrostatic potential (MEP) provides information about reactive sites for electrophilic and nucleophilic attack as well as hydrogen-bonding interactions in the molecules. The MEP obtained at the B3LYP method with 6-31G (d, p) basis set in gas phase for molecules 2-5 (Fig. 5). In figure, the negative (red) regions of MEP were related to electrophilic reactivity and the positive (blue) regions to nucleophilic reactivity. Fig 5 3.2. Biological assay (Antioxidant Properties) Table 2 It can be seen from Table 2 that pyrimidine compounds 4 and 5 showed radical scavenging activities in DPPH assay. It was important to note that compound 5 (IC50: 150.31 µM) showed close DPPH radical scavenging activity to the synthetic antioxidant α-tocopherol and trolox with IC50 values of 150.16 and 156.46 µM, respectively. The 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activities of tested was found to be in the order of -tocopherol>5 >trolox>4>3>1>2. Powerful antioxidant activity is associated with a lower IC50 value. Compounds 5 (IC50: 133.84 µM) exhibited close 2,2-azinobis- (3-ethylbenzthiazoline-69

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sulphonate) cation (ABTS+) to the synthetic antioxidant α-tocopherol and trolox with IC50 values of 109.28 and 122.64 µM, respectively. IC50 values of the 2,2-azinobis- (3-ethylbenzthiazoline-6sulphonate) cation (ABTS+) scavenging activities of pyrimidine compounds was found to be in the order of α-tocopherol > trolox > 5 > 4 > 3 > 1 > 2. Pyrimidine compound 5 (IC50: 126.66 µM) showed better metal chelating effect than the synthetic antioxidants α-tocopherol and trolox with IC50 values of 136.32 and 147.88 µM, respectively. The metal chelating effect of tested was found to be in the order of 5 > α-tocopherol > trolox > 4 > 3 > 1 > 2. Pyrimidine compounds 4 and 1 (IC50: 116.44 and 120.43 µM) exhibited better PhNHNH2 (phenyl hydrazine) induced haemolysis of erythrocytes than the synthetic antioxidant α-tocopherol, with IC50 value of 135.63 µM. The phenyl hydrazine induced haemolysis of erythrocytes of tested was found to be in the order of trolox >4 > 1 > α-tocopherol > 5 > 3 > 2 (Fig. 6). Fig 6 4. Conclusions This article deals with synthesis, structure and antioxidant activities of hydroxylsubstituted pyrimidine compounds. All synthesized compounds structure was determined IR, 1H/13C-NMR

and elemental analysis.

The calculation data show that the total energies of the R and S enantiomers are different from each other. The EHOMO and ELUMO energy of compound 5 is greater in absolute value than the other derivatives. The compound 5 has the narrow ELUMO-HOMO gap which will cause readily photo-induced excitation that may lead subsequent chemical reactions. Antioxidant activities of the compounds were evaluated by DPPH•, ABTS•+, Metal Chelating Effect and phenyl hydrazine induced haemolysis of erythrocytes assays (Table 1). The compounds 4 and 5 showed superior activity over other synthesized molecules in DPPH and ABTS+. On the other hand, the compounds 4 and 1 inhibited phenyl hydrazine induced haemolysis of erythrocytes revealing their ability to scavenge most of the free radicals generated. The pyrimidine compound 4 (IC50: 116.44) exhibited better PhNHNH2 (phenyl hydrazine) induced haemolysis of erythrocytes than the synthetic antioxidant α-tocopherol and other synthesized compounds. When a carbon atom in an organic compound loses a bond to hydrogen and gains a new bond to a heteroatom (or to another carbon), the compound has been dehydrogenated, or oxidized. A very common biochemical example is the oxidation of an alcohol to a ketone or 10

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aldehyde. When a carbon atom loses a bond to hydrogen and gains a bond to a heteroatom (or to another carbon atom), it is considered to be an oxidative process because hydrogen, of all the elements, is the least electronegative. Thus, in the process of dehydrogenation the carbon atom undergoes an overall loss of electron density and loss of electrons is oxidation. The compounds 1, 4 and 5 contained electron-rich ester, ketone, and sulfur groups, respectively. Therefore the compounds 1, 4 and 5 showed highest antioxidant effect.

HO

HO O

H

S H +

HO

H2N

NH2

H

OH

N

H

H

NH2 -H O 2

H

N

NH2 S

S OH HO

O H +

H

N

OH

Ph

Ph

NH2

O

H

Ph

N Ph

S

N H 1

H S

Scheme 1 Formation reaction of compound 1 ELUMO= -2.13eV electron acceptor

EHOMO= -5.72eV electron donor

ELUMO-HOMO= 3.59 eV

Fig. 1 EHOMO and ELUMO and the energy gap for compound 1

11

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OH OH

OH

O

OC2H5 O

H

Ph

Ph

NH N H

Ph

S

N H

1

C2H5O

H

Ph N H

OH

OH

O Ph

O

NH Ph

O

N H

S OH

O

-S

N Ph

Ph

N H

N H

O OC2H5

OH

OH O

H H

Ph

N Ph

OC2H5 O

OC2H5

S

OC2H5

H H

Ph

OC2H5

H O N

Ph

OC2H5

O

H

O

H

H S

-HBr

SH

OC2H5

N Ph

O

O

keto-enol

Ph

Br

O

H N

O

N

OC2H5 O

OC2H5

O

OC2H5

5

Scheme 3 Synthesis of compound 5.

12

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R

S

2

3

4

5

Fig. 2 The optimized structures of molecules 2-5 obtained at B3LYP/6-31G (d,p). 13

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2

4

HOMO

HOMO

LUMO

3

HOMO

LUMO

LUMO

5

HOMO

LUMO

Fig. 3 The frontier orbitals of molecules 2-5.

14

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Fig. 4 1H-NMR spectra of compound 2

15

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2

3

4

5

Fig. 5 Molecular electrostatic potential maps of molecules 2-5

16

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400 DPPH ABTS Metal C. PhNHNH2

350

IC5 0 values (µM)

300 250 200 150 100 50 0 1

2

3

4

5

-tocopherol

Trolox

Fig. 6 IC50 values of DPPH, ABTS, Metal Chelating Effect and PhNHNH2 (phenyl hydrazine) induced haemolysis of erythrocytes in pyrimidine compounds (1, 2, 3, 4, 5) and compared standard antioxidants, -tocopherol and trolox. Table 1. Total energy and frontier molecular orbital energies (EHOMO and ELUMO) and energy gap (E) Total Energy Molecules

R

S

ELUMO

EHOMO

ELUMO-HOMO

2

-1696.89117097

-1696.88693998

-2,13

-5.86

3.73

3

-1736.21065639

-1736.20676064

-2.11

-5.84

3.73

4

-1736.20392745

-1736.18373175

-1.85

-5.78

3.93

5

-1720.76705759

-1720.93068581

-1.64

-5.13

3.49

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Table 2. Antioxidant properties of pyrimidine derivatives Compounds 1 2 3 4 5 α-tocopherol Trolox

DPPH 274.89 294.28 240.62 209.78 150.31 150.16 156.46

r2 0.9607 0.9668 0.9593 0.9707 0.9604 0.9943 0.9595

ABTS 237.01 296.57 234.44 170.19 133.84 109.28 122.64

IC50 values (µM) r2 Metal C. 0.9430 220.43 0.9530 319.35 0.9924 197.88 0.9941 154.95 0.9582 126.66 0.9933 136.32 0.9252 147.88

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r2 0.9784 0.9387 0.9869 0.9890 0.9804 0.9966 0.9908

PhNHNH2 120.43 273.53 223.33 116.44 150.40 135.63 101.49

r2 0.9877 0.9785 0.9769 0.9384 0.9936 0.9834 0.9926

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References [1] S.M. Sondhi, N. Singh, M. Johar , A. Kumar, Bioorg Med Chem. 15 (2005) 6158. [2] O. Alam, S.A. Khan, N. Siddiqui, W. Ahsan, S.P. Verma, S.J. Gilani, Eur. J. Med. Chem. 45 (2010) 5113. [3] I. V. Gemisheva, I. Sergiev, D. Todorova, V. Alexieva, E. Stanoeva, V. Lachkova, E. Karanov, Plant Growth Regulation. 46 (2005) 193. [4] C.O. Kappe, Tetrahedron. 49 (1993) 6937. [5] T.P. Selvam, V. Karthick, P.V. Kumar, M.A. Ali, Drug Disc. Ther. 6 (2012) 198. [6] B. Pan, R. Huang, L. Zheng, C. Chen, S. Han, D. Qu, M. Zhu, P. Wei, Eur. J. Med. Chem. 46 (2011) 819. [7] N.A. Abdel-Latif, N.M. Sabry, A.M. Mohamed, M.M. Abdulla, Monatsh. Chem. 138 (2007) 715. [8] C.O. Kappe, W.M.F. Fabian, M.A. Semones, Tetrahedron. 53 (1997) 2803. [9] K.S. Atwal, B.N. Swanson, S.E. Unger, D.M. Floyd, S. Moreland, A. Hedberg, B.C. O'Reilly, J. Med. Chem. 34 (1991) 806. [10] H. Nagarajaiah, N.S. Begum, J. Chem. Sci. 126 (2014) 1347. [11] E. Akbas, A. Levent, S. Gumus, M. R. Sumer, I. Akyazi, Bull. Korean Chem. Soc. 31 (2010) 3632. [12] M. Mroueh, C. Daher, E. Hariri, S. Demirdjian, S. Isber, E. S. Choi, B. Mirtamizdoust, H.H. Hammud, Chemico-Biological Interactions 231 (2015) 53. [13] H. Chermette, Coord. Chem. Rev. 178 (1998) 699. [14] B. Shaabani, B. Mirtamizdoust, D. Viterbo, G. Croce, H. Hammud, ZAAC J. Inorg. Gen. Chem. 636 (8) (2010) 1596. [15] B. Shaabani, B. Mirtamizdoust, D. Viterbo, G. Croce, H.Hammud,P. Hojati-lalemi, A. Khandar, ZAAC J. Inorg. Gen. Chem. 637 (6) (2011) 713. [16] Y. Chen, H. Chang, C. Wang, F. Cheng, The Asian-Australian. J. Ani. Sci. 22 (2009) 1587. [17] M. Cuendet, K. Hostettmann, O. Potterat, W. Dyatmiko, Helv. Chim. Acta. 80 (1997) 1144. [18] T.C.P. Dinis, V.M.C. Madeira, L.M. Almeida, Arc. Biochem. Biophys. 315 (1994) 161. [19] E.A. Decker, B. Welch, J. Agric Food Chem. 38 (1990) 674. [20] M.B. Arnao, A. Cano, M. Acosta, Food Chemistry. 73 (2001) 239. [21] A. Valenzuela, T. Barría, R. Guerra, A. Garrido, Biochem. Biophys. Res. Commun. 126 (1985) 712. [22] M.J. Frisch, et al. Gaussian 09, Revision A.02. Gaussian, Inc. Wallingford, C.T. (2009). [23] A.D. Becke, J. Chem. Phys. 98 (1993) 5648. [24] C.T. Lee, W. Yang, R.G. Parr, Phys. Rev. B. 37 (1988) 785. [25] R. M. Issa, M. K. Awad, F. M. Atlam, Appl. Surf. Sci. 255 (2008) 2433. [26] H. Wang, X. Wang, H. Wang, L. Wang, A. Liu, J. Mol. Model. 13 (2007) 147.

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ACCEPTED MANUSCRIPT Highlights  Synthesis of 4-hydroxyphenyl substituted thiopyrimidine derivatives  Antioxidant activity of pyrimidine derivatives.  DFT calculations.