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

Article abstract of DOI:10.1016/j.molstruc.2018.02.080

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 B3L

Full text of DOI:10.1016/j.molstruc.2018.02.080

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
10.1016/j.molstruc.2018.02.080
MOLSTR 24902
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
18 October 2017
13 December 2017
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; erdem_ergan@hotmail.com
(E.E); yyuprojedestek@gmail.com (Y.K)
* Correspondences: esvakbas@hotmail.com ; suatekin@hotmail.com ; Tel.: +90
4322251701
OH
OH
O
O
S
OH
H
N
O
H
O
H
O
Ph
H
H
H
Ph
N
H2N
H2N
H
S
H
O
H
Ph
N
400
350
300
250
200
150
100
50
H
S
Ph
N
H
OH
Ph
N
+
HO
OH
HO
O
Ph
N
H
S
Ph
OH
H
H
Ph
O
O
O
H
Ph
N
CH3
H
Ph
N
Ph
N
OC2H5
OC2H5
S
Ph
N
O
0
1
2
3
4
5
-tocopherol Trolox


ACCEPTED MANUSCRIPT
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; erdem_ergan@hotmail.com (E.E);
yyuprojedestek@gmail.com (Y.K)
* Correspondences: esvakbas@hotmail.com ; suatekin@hotmail.com ; 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, anti-
inflammatory 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).
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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. 219-
1
220 ºC, IRKBr/cm-1: 1738, 1637 cm−1 (C=O). H-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,
13
d, CH2, J=17.1 Hz, B part of AB system). C-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.
1
250-251oC, IRKBr/cm-1: 1767, 1632 cm−1 (C=O), H-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.
1
194-195oC, IRKBr/cm-1: 1697, 1628 cm−1 (C=O), H-NMR (DMSOd6/ppm): δ 6.85-7.39 (m, 15H,
13
Harom. and OH), 2.22 (s,1H, CHpyrimidine), 3.05–3.25 (m, 4H, CH2-thiazine). C-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. 233-
1
234oC, IRKBr/cm-1: 3252, 3156 (NH), 3059, 2982 (CHarom.), 1632, 1612 (C=O); H 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-2-
picrylhydrazyl (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
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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 α-
tocopherol and trolox at same concentrations. The percentage of inhibition of ferrozine–Fe2+
complex formation was calculated by using the formula given below:
ATest
1-
Metal chelating effect (%) =
x100
Acontrol
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•+)
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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-6-
sulfonic 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.
AControl - ATest
Inhibition (%) =
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.
ATest
1-
Inhibition (%) =
x100
Acontrol
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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
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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 photo-
induced 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,
1
1637 cm-1 because of carbonyl and benzoyl absorption groups, respectively. In the H-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
1
disappearance of the NH peaks in H-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-(4-
hydroxyphenyl)-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-(4-
hydroxyphenyl)-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 β-
enaminocarbonyl 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
1
In H-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- picryl-
hydrazyl (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-6-
9

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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-6-
sulphonate) 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 hydroxyl-
substituted 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
H
N
O
H
S
OH
NH2
S
H
H
N
NH2
H
+
H2N
NH2
-H2O
H
HO
S
OH
HO
O
OH
Ph
O
H
H
Ph
+
H
Ph
N
N
NH2
H
Ph
N
H
1
S
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
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OH
OH
OH
OC2H5
O
H
N
O
S
OC2H5
Br
O
Ph
O
H
O
O
O
H
H
Ph
N
Ph
N
Ph
NH
C2H5O
H
k
e
OC2H5
t
o
-HBr
Ph
N
SH
OH
Ph
N
S
-
e
H
H
n
1
o
OH
l
H
O
H
O
H
O
OC2H5
O
O
Ph
N
OC2H5
O
Ph
NH
Ph
N
H
S
Ph
N
H
OC2H5
S
OC2H5
-S
OH
OH
OH
O
H
H
H
H
O
O
Ph
N
Ph
N
Ph
N
OC2H5
OC2H5
Ph
N
OC2H5
H
O
O
OC2H5
5
Scheme 3 Synthesis of compound 5.
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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).
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2
HOMO
LUMO
3
HOMO
LUMO
4
HOMO
LUMO
5
HOMO
LUMO
Fig. 3 The frontier orbitals of molecules 2-5.
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Fig. 4 1H-NMR spectra of compound 2
15

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2
3
5
4
Fig. 5 Molecular electrostatic potential maps of molecules 2-5
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400
350
300
250
200
150
100
50
DPPH
ABTS
Metal C.
PhNHNH2
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
3.73
2
3
4
5
-1696.89117097 -1696.88693998
-1736.21065639 -1736.20676064
-1736.20392745 -1736.18373175
-1720.76705759 -1720.93068581
-2,13
-2.11
-1.85
-1.64
-5.86
-5.84
-5.78
-5.13
3.73
3.93
3.49
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Table 2. Antioxidant properties of pyrimidine derivatives
Compounds
IC50 values (µM)
DPPH
r2
ABTS
r2
Metal C.
220.43
319.35
197.88
154.95
126.66
136.32
147.88
r2
PhNHNH2
120.43
273.53
223.33
116.44
150.40
135.63
101.49
r2
1
2
3
4
5
274.89 0.9607 237.01 0.9430
294.28 0.9668 296.57 0.9530
240.62 0.9593 234.44 0.9924
209.78 0.9707 170.19 0.9941
150.31 0.9604 133.84 0.9582
0.9784
0.9387
0.9869
0.9890
0.9804
0.9966
0.9908
0.9877
0.9785
0.9769
0.9384
0.9936
0.9834
0.9926
α-tocopherol 150.16 0.9943 109.28 0.9933
Trolox
156.46 0.9595 122.64 0.9252
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Highlights
 Synthesis of 4-hydroxyphenyl substituted thiopyrimidine derivatives
 Antioxidant activity of pyrimidine derivatives.
 DFT calculations.