Synthesis and biological evaluation of novel pyrimidine-2(1H)-ones/thiones as potent anti-inflammatory and anticancer agents

Article abstract of DOI:10.1007/s00044-011-9909-5

Recently there has been an increased interest in the synthesis of pyrimidinone and their applications in medicinal chemistry. Pyrimidinone derivatives represent one of the active classes of the compounds possessing a wide spectrum of biological activities

Full text of DOI:10.1007/s00044-011-9909-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3598–3606
DOI 10.1007/s00044-011-9909-5
ORIGINAL RESEARCH
Synthesis and biological evaluation of novel pyrimidine-2(1H)-
ones/thiones as potent anti-inflammatory and anticancer agents
•
Vachala Seekarajapuram Dinakaran
•
Divya Jacob Jessy Elizabeth Mathew
Received: 5 July 2011 / Accepted: 16 November 2011 / Published online: 25 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Recently there has been an increased interest in
the synthesis of pyrimidinone and their applications in
medicinal chemistry. Pyrimidinone derivatives represent
one of the active classes of the compounds possessing a
wide spectrum of biological activities such as anti-tumour,
anti-fungal, anti-inflammatory, anti-bacterial, etc. This
study utilises chalcones as starting compounds that are
obtained by Claisen-Schmidt condensation. These chal-
cones underwent cyclisation upon treatment with urea and
thiourea in the presence of sodium hydroxide to obtain the
corresponding pyrimidinones and pyrimidinethiones. The
structures of the synthesized compounds were investigated
Introduction
A large number of compounds containing pyrimidines are
known for their varied biological properties. Brugnatelli
was the first scientist to isolate Alloxan, a pyrimidine
derivative in 1818, and later this compound was found to
possess antineoplastic properties. Nucleosides of pyrimi-
dine bases have been used extensively as antiviral and
anticancer agents. Recently, fluoropyrimidines and fluoro-
uracil-based combination therapy is used in the treatment
of gastrointestinal cancer and solid tumours (Sun and
Halller, 2001). Fluoropyrimidinones are found to be
metabolites of dihydropyrimidinones that are subtype-
selective antagonists of the a1a-adrenergic receptor antag-
onists. The pyrimidine entity is one of the most prominent
structures found in nucleic acid chemistry. Pyrimidines
play a vital role in many biological processes since their
ring system is present in several vitamins, coenzymes, and
nucleic acids. Several pyrimidine derivatives have wide
varieties of usages and its nucleus is also present in vitamin
B2 and folic acid. As pyrimidine is a basic nucleus in DNA
and RNA, it has been found to be associated with diverse
biological activities (Shivarema Holla et al., 2004). The
biological significance of the pyrimidine derivatives has
led us to the synthesis of substituted pyrimidine. Pyrimi-
dine heterocycles possessing hydroxyl group has a unique
place in medicinal chemistry, and also plays a vital role in
biological processes as well as synthetic drugs (Baddiley
et al., 1944). Pyrimidine derivatives are an important class
of heterocyclic chemistry and are associated with various
biological, pharmaceutical and therapeutical activities like
anticancer (Kenner et al., 1943), anti-inflammatory (Lang
and Cohen, 1975), anti-HIV (Fujiwara et al., 2008), etc.
It is evident from the literature that pyrimidinones
played an essential role in several biological processes and
1
by means of UV, IR, H NMR and Mass spectral analysis.
The anti-oxidant activity of the compounds was measured
by standard free radical scavenging assays (DPPH, Nitric
oxide radicals). The compounds with significant anti-oxi-
dant activity were then screened for the in vivo anti-
inflammatory activity and it showed that the compounds
ETT and DMT possess good activity as comparable to the
standard. All the synthesized compounds were evaluated
for their in vitro anti-cancer activity by MTT assay among
which compounds CLU and ETU showed significant
activity against HeLa and HepG2 cell lines, respectively.
Keywords Pyrimidinones ꢀ Pyrimidine-thiones ꢀ
Anti-oxidant ꢀ Anti-inflammatory ꢀ Anti-cancer
V. S. Dinakaran (&) ꢀ D. Jacob ꢀ J. E. Mathew
Department of Pharmaceutical Chemistry,
College of Pharmaceutical Sciences, MCOPS,
Manipal University, MadavNagar, Manipal 576 104,
Karnataka, India
e-mail: sdvachu@yahoo.com
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Med Chem Res (2012) 21:3598–3606
3599
have considerable chemical and pharmacological impor-
tance. One of the methods for the synthesis of such com-
pound is from a,b-unsaturated carbonyls (chalcone) by the
cyclisation with urea and thiourea. The synthesis of sulphur
and oxygen containing heterocycles has been explored for
their therapeutic activity. Chalcone has been proved to be
an important intermediate for the synthesis of many het-
erocyclic compounds. Chalcones and its derivatives pos-
sess some interesting biological properties such as
antibacterial (Hugo et al., 2008), antifungal (Batovska
et al., 2007), insecticidal (Mudaliar and Joshi, 1995), anti-
inflammatory (Zdizislawa Nowakowska, 2007), analgesic,
anticancer (Nicholas et al., 2006), etc. Chalcones possess
conjugated double bonds and a completely delocalized
p-electron system on both benzene rings. Molecules pos-
sessing such system have relatively low redox potentials
and have a greater probability of undergoing electron
transfer reactions (Chetana et al., 2009). Chalcones exist as
either E or Z isomers. E isomer is the most stable form and
consequently majority of chalcones are isolated as E iso-
mer (Neeraj et al., 2009).
Results and discussion
Chemistry
Totally sixteen novel arylpyrimidin-2(1H)-ones and aryl-
pyrimidin-2(1H)-thiones were synthesized from chalcones
according to the scheme given in Fig. 1. In the first step,
the reaction of 20-hydroxy acetophenone with different
aromatic aldehydes in the presence of strong aqueous
solution of sodium hydroxide resulted in the formation of
bright coloured chalcones (1-{20-hydroxy}-3-arylidene-2-
propen-1-ones). In the second step the reaction of chal-
cones with urea in anhydrous ethanol at reflux temperature
gave the corresponding 4-(20-hydroxyphenyl)-6-arylpyr-
imidin-2(1H)-ones. Similarly, the treatment of chalcones
with thiourea furnished 4-(20-hydroxyphenyl)-6-arylpyr-
imidine-2(1H)-thiones. The completion of the reaction was
monitored by TLC analysis. The final compounds were
column chromatographed using gradient elution technique.
The elution of pure compound could be achieved using
petroleum ether and ethyl acetate (7:3). The melting points
of the synthesized compounds were taken in open capillary
tubes and were uncorrected.
The highly polarised pyrimidinones can add a carbon
nucleophile at either C-4 or C-6 to form the corre-
sponding 3,4- or 3,6-dihydro derivatives. Dihydropyri-
midinones (DHPM’s) have always remained in the
forefront due to their therapeutic and pharmacological
properties. As early as in 1930 simple derivatives were
patented for the protection of wool moths. Parlato et al.
synthesized various dihydropyrimidinones derivatives by
modification of the substituents in virtually all the six
positions of the pyrimidine nucleus which provided with
interesting activity against HIV, Sendai virus and rubella
virus (Maria et al., 2004). Besides these, substituted
pyrimidine derivatives have been used as antihypertensive
agents, anticancer agents (Ducki et al., 1998), antimi-
crobial agents (Wageeh et al., 2011), antifungal agents
(Singh et al., 2008), antiinflammatory agents (Rajesh
et al., 2004), calcium channel blockers (Yang et al.,
2009), neuropeptide c-antagonists and a-1a-antagonists
(Dipti et al., 2010).
OH
O
2-hydroxy acetophenone
R-CHO, EtOH, NaOH
0-5 oC
OH
R
O
1-(2'-hydroxy)-3-arylidene-2-propen-1-ones
It was recently found that some pyrimidinones carrying
an arylidene moiety have potential as antitumor agents,
some of these analogues also showed, besides broad-
spectrum antitumor activity, a distinctive pattern of selec-
tivity towards individual cell lines, such as that of leukemia
(Singh et al., 2007). Based on the above observation it is
worthwhile to prepare newer compounds for their antimi-
crobial, anti-inflammatory and anticancer activities. In the
view of varied biological and pharmacological application,
we synthesized some new pyrimidine derivatives from
chalcones and evaluated for anti-oxidant, anti-inflamma-
tory and anti-cancer activities.
Thiourea, EtOH, NaOH
Reflux
Urea, EtOH, NaOH
Reflux
OH
OH
R
R
N
NH
N
NH
S
4-(2'-hydroxy)-6-arylpyrimidine-2(1H)-thiones
O
4-(2'-hydroxy)-6-arylpyrimidine-2(1H)-ones
Fig. 1 Synthesis of 4-(20-hydroxy)-6-aryl pyrimidine-2(1H)-ones/
thiones
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Med Chem Res (2012) 21:3598–3606
Characterization of the synthesized compounds
The structure of the titled compounds has been confirmed
1
on the basis of UV, IR, H NMR and Mass spectral data.
Table 1 List of pyrimidine derivatives synthesized
R2
OH
R1
R3
The IR spectra of the compounds were recorded in the
500–4000 cm-1 range on Shimadzu FT-IR 8310 JAPAN
using KBr pellets. The IR spectra of these pyrimidine deriv-
atives showed the presence of absorption bands in the region
1570–1678 cm-1 due to C=N and at 3100–3500 cm-1 due to
NH group, which indicated the transformation of a,b-unsat-
urated carbonyl functionality into cyclic pyrimidines. The IR
spectrum also exhibited a characteristic absorptionbandin the
region 3100–3600 cm-1 due to phenolic hydroxyl group,
absorption bands at 1480–1590 cm-1 due to aromatic C=C
stretching, 1600–1690 cm-1 due to C=O stretching and
1025–1100 cm-1 due to C=S stretching.
N
NH
X
Compound Mol. formula
code
Mol. wt. R1
R2
R3
X
CLT
CLU
FLT
C16H11ClN2OS
16H11ClN2O2
314.79 H
298.73 H
298.34 H
282.27 H
349.24 Cl
H
H
H
H
H
H
H
H
H
H
Cl
Cl
F
S
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
O
S
16H11FN2OS
16H11FN2O2
16H10Cl2N2OS
FLU
DCT
DCU
BRT
BRU
MET
MEU
DMT
DMU
EMT
EMU
ETT
F
O
S
1
The H NMR spectra were recorded on Bruker Biospin
Cl
Cl
Br
Br
H
Avance-300 MHz in CDCl3/DMSO-d6 as solvent. The
chemical shifts were reported as parts per million down-
field from tetramethylsilane (Me4Si). The 1H NMR spectra
revealed a singlet at 8.9 ppm due to NH proton, a singlet at
10.2 ppm due to hydroxyl proton and a multiplet observed
in the region 6.7–7.8 ppm was due to aromatic protons.
Mass spectra were recorded on the Shimadzu MS–MS
QP5050. The mass spectral data of the compounds showed
the presence of molecular ion peaks in reasonable inten-
sities Table 1.
16H10Cl2N2O2 333,17
Cl
O
S
16H11BrN2OS
16H11BrN2O2
17H14N2O2S
17H14N2O3
18H16N2O3S
18H16N2O4
19H18N2O3S
19H18N2O4
18H16N2O2S
18H16N2O3
359.25 H
343.18 H
310.38 OCH3
294.31 OCH3
340.40 H
324.34 H
354.43 H
338.36 H
324.40 H
308.34 H
O
S
H
O
S
OCH3 OCH3
OCH3 OCH3
OCH3 OC2H5
OCH3 OC2H5
O
S
O
S
H
H
OC2H5
OC2H5
ETU
O
Biological evaluation
13.87 lg/ml. The results of this study revealed that the
most of the pyrimidine-2(1H)-thiones were showing good
antioxidant activity whereas pyrimidine-2(1H)-ones deriv-
atives did not show any activity even at 500 lg/ml. Among
the pyrimidine-2(1H)-thione derivatives, 4-ethoxyphenyl
substituted pyrimidine-2(1H)-thione (ETT) showed very
good antioxidant activity at low concentration.
In vitro anti-oxidant activity-DPPH method
All the synthesized compounds with concentration ranging
from 7.825 to 500 lg/ml were tested for their antioxidant
activity by DPPH method. Ascorbic acid was used as a
standard. The % DPPH Radical Scavenging activity of the
test compounds were given in Table 2. Compounds ETT,
EMT and DMT were showing very good free radical
scavenging activity. Their percentage scavenging of
DPPH radical was found to be 81.26, 71.90 and 70.46 at
500 lg/ml. At 7.8125 lg/ml concentration, all these three
compounds showed moderate activity when compared to
the standard. All other compounds were showing less
antioxidant activity.
In vitro anti-oxidant activity-nitric oxide scavenging
method
All the synthesized compounds with concentration ranging
from 7.825 to 500 lg/ml were tested for their antioxidant
activity by nitric oxide scavenging method and the results
were summarized in Table 3. The IC50 value of ascorbic
acid was found to be 60.29 lg/ml. All the test compounds
showed their IC50 above 500 lg/ml.
The antioxidant activity of the test compounds was
estimated by their IC50 value. The IC50 values of the
compounds were given in Fig. 2. In DPPH method, com-
pounds ETT and DMT showed antioxidant activity with
IC50 values as 108.13 and 115.49 lg/ml, respectively. The
compounds FLT, EMT and BRT showed moderate activity
with IC50 values as 163.71, 173.50 and 451.83 lg/ml,
respectively. All other compounds were having IC50 above
500 lg/ml. The standard drug’s IC50 was found to be
In vitro anti-cancer activity-MTT assay
All the sixteen synthesized compounds were screened for
their in vitro anticancer activity by MTT assay method.
Hep G2 and HeLa cell lines were selected to determine
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Med Chem Res (2012) 21:3598–3606
3601
Table 2 Free radical scavenging activity by DPPH method
Compound code
% DPPH radical scavenging at different concentrations (lg/ml)
500
250
125
62.5
31.25
15.625
7.8125
IC50
CLT
53.02
11.95
68.58
22.33
46.97
13.54
48.12
14.12
51.72
16.13
70.46
18.44
71.90
18.87
81.26
19.59
58.82
47.69
11.38
67.72
16.28
43.94
12.39
44.81
8.64
45.38
5.76
41.06
5.47
37.75
4.46
31.26
4.51
17.14
4.42
[500
[500
CLU
FLT
55.9
50.28
11.23
30.11
11.23
25.21
7.49
40.63
8.64
32.56
8.06
22.7
163.71
[500
469.32
[500
451.83
[500
497.14
[500
115.49
[500
173.5
[500
108.13
[500
13.87
FLU
14.12
36.02
11.67
31.7
6.19
DCT
21.75
11.09
19.88
7.2
20.02
9.79
16.42
6.77
DCU
BRT
15.56
6.34
11.52
6.05
BRU
MET
MEU
DMT
DMU
EMT
EMU
ETT
8.21
45.24
14.12
67.14
16.28
60.95
17.86
74.63
14.69
57.93
44.23
12.24
61.23
15.12
56.05
13.54
66.85
13.83
56.45
42.79
9.22
36.74
8.78
25.93
8.50
17.00
7.06
55.47
14.40
47.98
13.40
49.27
13.40
54.53
43.94
11.81
42.21
12.53
44.23
13.08
53.34
36.31
9.22
28.96
8.50
31.98
12.24
32.99
11.81
50.38
21.32
8.21
25.21
9.36
ETU
Ascorbic acid
39.57
the 50% growth inhibition at very low concentrations.
Their IC50 value was found to be 20.26, 25.83 and
27.97 lM, respectively. Compounds MEU, CLT, EMT,
ETT and FLT showed 50% inhibition of cell growth at
41.72, 49.33, 74.18, 80.52 and 83.06 lM, respectively.
Remaining all other compounds was having higher IC50
values. The results showed that the p-fluorophenyl and
p-ethoxy phenyl substituted pyrimidine-2(1H)-ones
showed good activity with low IC50 values.
In vivo anti-inflammatory activity—carrageenan-induced
paw oedema model
Fig. 2 Antioxidant activity by DPPH method: IC50 of selected
compounds
The compounds with significant antioxidant activity were
selected and evaluated for the in vivo anti-inflammatory
activity in Wistar rats by Carrageenan-induced hind paw
oedema method. The mean increase in paw volume was
given in Table 6. Ibuprofen was used as standard. The
percentage inhibition of inflammation at 1st h was given in
Fig. 3. Compound DMT, EMT and ETT showed very good
anti-inflammatory activity (60, 57.5 and 55%, respec-
tively). The standard drug showed 62.5% inhibition of
inflammation. The percentage inhibition of inflammation at
3rd h was given in Fig. 4. Compounds ETT, DMT and
EMT showed very good anti-inflammatory activity and
their percentage activity was found to be 69.23, 66.15 and
60%, respectively. At 5th h Compound ETT, with an
p-ethoxyphenyl substitution was found to be the more
active with 73.52% inhibition of oedema as compared to
76.47% of the standard drug (Fig. 5). Compounds DMT
their efficacy to inhibit the growth of cancer cells. The IC50
values of the test compounds against HeLa cell line was
given in the Table 4. Compounds CLU and BRU showed
the cell growth inhibition at low concentration. Their IC50
values against HeLa cell line were found to be 30.09 and
49.83 lM, respectively. The compounds CLT, ETT, FLU
and BRT showed moderate inhibition at 69.06, 73.77,
79.21 and 87.93 lM, respectively. All other compounds
were showing the antiproliferative activity at higher con-
centrations. Out of all the sixteen compounds, p-chloro-
phenyl and p-bromophenyl substituted pyrimidine-2(1H)-
ones showed good activity with low IC50 values.
All the compounds were tested against Hep G2 cell line.
The IC50 values of the test compounds were given in
Table 5. The standard drug cisplatin showed the IC50 at
25.27 lM. Compounds ETU, FLU and DMT were showing
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Med Chem Res (2012) 21:3598–3606
Table 3 Nitric oxide radical scavenging activity
Compound code
% Nitric oxide radical scavenging at different concentrations (lg/ml)
500
250
125
62.5
31.25
15.625
7.8125
IC50
CLT
25.29
25.60
29.44
29.22
30.05
19.58
23.32
31.92
31.97
25.34
28.64
25.99
30.05
25.58
26.62
24.02
58.16
24.95
24.27
26.19
28.05
25.75
19.09
22.81
31.04
26.06
25.31
27.96
25.02
29.36
25.31
25.36
23.32
57.14
23.59
23.68
22.47
26.94
18.90
17.17
22.74
27.11
25.97
23.73
27.52
24.19
27.82
25.19
24.07
22.13
55.06
23.08
23.56
21.52
26.48
16.59
17.07
22.49
26.23
24.44
23.22
25.17
23.59
27.05
24.87
23.63
21.50
54.14
22.23
23.51
21.13
26.04
15.93
16.86
22.40
23.80
23.17
22.54
24.51
22.86
26.46
24.51
23.49
19.21
52.56
22.03
22.69
21.03
25.63
15.91
16.25
20.43
22.83
21.59
22.27
24.48
22.40
25.79
23.83
22.71
18.31
49.91
22.01
21.35
20.06
25.31
13.43
15.01
19.04
20.43
19.84
21.45
23.34
16.15
24.68
23.00
22.69
14.55
49.46
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
CLU
FLT
FLU
DCT
DCU
BRT
BRU
MET
MEU
DMT
DMU
EMT
EMU
ETT
ETU
Ascorbic acid
60.29
Table 4 In-vitro anti-cancer
activity of synthesized
compounds on HeLa cell lines
(cervical cancer) by MTT assay
Table 5 In-vitro anti-cancer
activity of synthesized com-
pounds on HepG2 cell lines
(Human liver carcinoma) by
MTT assay
Compound
code
IC50 values
(lM)
Compound
code
IC50 values
(lM)
ETU
MET
MEU
DMT
BRU
EMT
DCT
BRT
CLU
CLT
307.55
228.43
117.33
187.83
49.83
ETU
MET
MEU
DMT
BRU
EMT
DCT
BRT
CLU
CLT
20.26
164.44
41.72
27.97
197.91
74.18
262.08
225.43
87.93
182.85
261.68
126.27
49.33
30.09
69.06
ETT
73.77
ETT
80.52
FLU
79.21
FLU
25.83
FLT
486.69
1818.55
223.27
21.13
FLT
83.06
DMU
DCU
Cisplatin
DMU
DCU
Cisplatin
259.67
167.66
25.27
and FLT have shown percentage activity as 72.05 and
66.17%, respectively.
were made to react with urea and thiourea to form pyri-
midinones and pyrimidine thiones, respectively. The
application of this method provides a simple, powerful tool
for the synthesis of a large number of multifunctional
pyrimidine-2-ones/thiones. All the synthesized compounds
were evaluated for antioxidant, antiinflammatory and
anticancer activities.
Conclusion
Based on the literature survey an attempt was done to
synthesise some pyrimidine derivatives through chalcones
by conventional method. The various substituted chalcones
From the biological studies, it was understood that, most
of the pyrimidine-2(1H)-thiones were showing good
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Med Chem Res (2012) 21:3598–3606
3603
Table 6 In-vivo anti-inflammatory activity of the selected
compounds
90
80
70
60
50
40
30
20
10
0
Group
Dose (mg/kg Mean increase in paw volume
body weight) (Mean SEM)
1st h
3rd h
5th h
Control
0.5% CMC 0.40 0.05
0.65 0.03
0.68 0.04
Ibuprofen 100
0.15 0.03 0.18 0.04** 0.16 0.02*
0.22 0.02** 0.27 0.02* 0.23 0.05
FLT
100
100
100
100
100
Ibuprofen
FLT
DMT
EMT
ETT
BRT
DMT
EMT
ETT
BRT
0.16 0.03
0.17 0.02
0.22 0.03
0.19 0.02*
Compounds
0.26 0.02* 0.33 0.03**
Fig. 5 The percentage inhibition of inflammation at 5th h
0.18 0.02** 0.20 0.02
0.18 0.04
0.20 0.04
0.31 0.03
0.29 0.03*
substituted pyrimidine-2(1H)-ones showed good anticancer
activity with low IC50 values against Hep G2 cell line.
The compounds with significant antioxidant activity
were considered for in vivo anti-inflammatory activity by
Carrageenan induced paw oedema method in the view that
it could be useful in quenching overproduced reactive
oxygen species and in reducing inflammation. Compound
ETT, with p-ethoxyphenyl substituted pyrimidine-2(1H)-
thione was found to be the most active with 73.52% inhi-
bition of oedema. This study shows that this class of
pyrimidinones can potentially serve as lead structure for
further optimization because of their dual action as anti-
oxidants and antiinflammatory agents.
* P\0.05; ** P\0.01
70
60
50
40
30
20
10
0
Ibuprofen
FLT
DMT
EMT
ETT
BRT
Compounds
The above activities are quite interesting and further
study in the molecule is essential. Thus, from the above
discussion it may be concluded that it is worthwhile to
pursue further investigation by manipulating the above
novel pyrimidine derivatives.
Fig. 3 The percentage inhibition of inflammation at 1st h
80
60
40
20
0
Materials and methods
The melting point of the synthesized compounds was
determined using open capillary tubes in scientific melting
point apparatus and was uncorrected. UV values were
obtained by using Shimadzu UV 4650 JAPAN, at MCOPS,
Manipal. IR spectra of the synthesized compounds were
scanned using Shimadzu FT-IR 8310 JAPAN and KBr
press at MCOPS, Manipal. Proton NMR spectra of the
synthesized compounds were recorded on Bruker Biospin
Avance-300 MHz at IICT, Hyderabad. Mass spectra of the
synthesized compounds were recorded on Shimadzu MS–
MS QP5050 at IISC, Bangalore. The progress, purity of the
reaction and the intermediates were analysed using pre-
coated TLC plates and using UV chamber. Thin layer
chromatography was carried out using silica gel G (Merck)
as stationary phase in the solvent system petroleum ether:
ethyl acetate (7:3). Female Wistar rats were procured and
the anti-inflammatory activity was carried out on approval
Ibuprofen
FLT
DMT
EMT
ETT
BRT
Compounds
Fig. 4 The percentage inhibition of inflammation at 3rd h
antioxidant activity whereas pyrimidine-2(1H)-ones deriv-
atives did not show any activity even at 500 lg/ml. Among
the pyrimidine-2(1H)-thione derivatives, 4-ethoxyphenyl
substituted pyrimidine-2(1H)-thione (ETT) showed very
good antioxidant activity at low concentration.
Out of all the sixteen compounds, p-chlorophenyl and p-
bromophenyl substituted pyrimidine-2(1H)-ones showed
good antiproliferative activity with low IC50 values against
HeLa cell line and p-fluorophenyl and p-ethoxy phenyl
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Med Chem Res (2012) 21:3598–3606
4-(2-hydroxyphenyl)-6-(2-methoxyphenyl)pyrimidine-
2(1H)-thione (MET)
of Institutional Animal Ethics Committee, KMC, Manipal
(No. IAEC/KMC/OS/2011-2012).
Yield 80%; m.p. 182°C; IR (KBr)cm-1: 3416 (OH), 3312
(NH), 1635 (C=N), 1552 (C=C), 1022(C=S); 1H NMR
(DMSO) d ppm: 10.1 (s, 1H, OH), 8.7 (s, 1H, NH), 6.7–7.4
(m, Ar–H); MS: m/z 310 (M?), 311, 312; C17H14N2O2S; Calc.
C 65.79, H 4.55, N 9.03; Found. C 65.73, H 4.61, N 9.10.
Synthesis of 4-(20-hydroxy)-6-aryl pyrimidine-2(1H)-
ones/thiones
Synthesis of 1-(20-hydroxy)-3-arylidene-2-propen-1-ones
To a solution of 20-hydroxy acetophenone (0.01 mol) in
anhydrous ethanol (10 ml), freshly distilled aromatic
aldehyde (0.01 mol) was added slowly, the reaction mix-
ture was cooled to 0–58C. A solution of 70% sodium
hydroxide (5 ml) was added portion wise while stirring.
The reaction mixture was stirred at room temperature for
24–36 h. The reaction mass was quenched into crushed ice
and neutralized with dilute hydrochloric acid. The yellow
solid thus obtained was filtered, washed with ice cold water
followed by recrystallization using absolute ethanol.
4-(2-hydroxyphenyl)-6-(3,4-dimethoxyphenyl)pyrimidine-
2(1H)-thione (DMT)
Yield 78%; m.p. 174°C; IR (KBr)cm-1: 3416 (OH),
3381(NH), 1572 (C=N), 1512 (C=C), 1024(C=S); 1H NMR
(DMSO)d ppm:8.8 (s, 1H, NH), 10.1(s, 1H, OH), 6.6–7.4(m,
Ar–H); MS: m/z 340 (M?), 341, 342; C18H16N2O3S; Calc.
C 63.51, H 4.74, N 8.23; Found. C 63.48, H 4.67, N 8.19.
6-(4-ethoxyphenyl)-4-(2-hydroxyphenyl)pyrimidine-2(1H)-
thione (ETT)
Synthesis of 4-(2-hydroxyphenyl)-6-substituted pyrimidin-
2(1H)-one
Yield 75%; m.p. 165°C; IR (KBr)cm-1: 3360 (NH), 3223
(OH), 1637 (C=N), 1566 (C=C), 1041 (C=S); H NMR
1
To a solution of chalcone (0.01 mol) and urea (0.01 mol)
in anhydrous ethanol (50 ml), 10% sodium hydroxide
(6 ml) was added. The reaction mixture was refluxed for
12 h and poured into ice cold water. The product thus
obtained was filtered, washed with cold water and purified
by column chromatography (petroleum ether: ethyl acetate
[7:3]).
(DMSO) d ppm: 8.8 (s, 1H, NH), 10.2 (s, 1H, OH), 6.8–7.7
(m, Ar–H); MS: m/z 324 (M?), 325, 326; C18H16N2O2S; Calc.
C 61.64, H 4.97, N 8.64; Found. C 61.58, H 4.91, N 8.70.
6-(4-fluorophenyl)-4-(2-hydroxyphenyl)pyrimidin-2(1H)-
one (FLU)
Yield 60%; m.p. 134°C; IR (KBr)cm-1: 3404 (OH), 3232
(NH), 1637 (C=O), 1604 (C=N), 1566 (C=C), 1255 (C–
F);1H NMR (DMSO) d ppm: 8.0 (s, 1H, NH), 10.1 (s, 1H,
OH), 6.8–7.2 (m, Ar–H); MS: m/z 282 (M?), 283, 284;
C16H11FN2O2; Calc. C 68.08, H 3.93, N 9.92; Found. C
68.12, H 3.88, N 9.95.
Synthesis of 4-(2-hydroxyphenyl)-6-substituted pyrimidine-
2(1H)-thiones
To a mixture of chalcone (0.01 mol) and thiourea
(0.01 mol) in anhydrous ethanol (50 ml), 10% sodium
hydroxide (6 ml) was added. The reaction mixture was
refluxed for 12 h and poured into ice cold water. The solid
thus separated was filtered, washed with cold water and
purified by column chromatography (petroleum ether: ethyl
acetate [7:3]).
Biological evaluation
Antioxidant activity-DPPH radical scavenging assay
The assay was carried out in a 96-well micro titre plate. To
100 ll of DPPH solution, 100 ll of each of the test sample
or the standard solution was added separately in wells of
the microtitre plate. The final concentrations of the test and
standard solutions used were 500 to 7.8125 lg/ml. The
plates were incubated at 37°C for 20 min and the absor-
bance of each solution was measured at 540 nm, using
ELISA micro titre plate reader. The experiment was per-
formed in triplicate and % scavenging activity was calcu-
lated using the formula given below. IC50 (Inhibitory
Concentration) is the concentration of the sample required
Spectral data of the representative compounds
6-(4-chlorophenyl)-4-(2-hydroxyphenyl) pyrimidine-2(1H)-
thione (CLT)
Yield 80%; m.p. 198°C; IR (KBr)cm-1: 3385 (OH), 3178
(NH), 1635 (C=N), 1548 (C=C), 1087 (C=S), 831 (C–Cl);
1H NMR (DMSO) d ppm: 10.2 (s, 1H, OH), 8.1 (s, 1H,
NH), 6.8–7.2 (m, Ar–H); MS: m/z 314 (M?), 316, 315;
C16H11ClN2OS; Calc. C 61.05, H 3.52, N 8.9; Found. C
61.11, H 3.07, N 8.89.
123

Med Chem Res (2012) 21:3598–3606
3605
to scavenge 50% of DPPH free radicals and it was calcu-
lated from the graph, % scavenging versus concentration
(Azam et al., 2008).
trypsinization. From a 60–70% confluent flask, the culture
media was aspirated out using a micropipette. Cells were
washed with 3 ml of PBS to remove trace amount of
media. To each culture flask 2 ml of trypsin–EDTA was
added and after few seconds it was aspirated and the flask
was kept in the incubator for 3–4 min for cell detachment.
Culture flasks were observed under an inverted microscope
(Nikon Eclipse, Japan) to ensure that cells were completely
dislodged. Trypsin activity was stopped by adding 2–3 ml
media containing 10% FBS.
Control ꢁ Test
% Scavenging =
ꢂ 100
Control
In-vitro anticancer activity—MTT assay
Maintenance of cell lines
HepG2 and Hela cells procured from NCCS Pune were
grown in 25 cm2 tissue culture flasks containing DMEM
media supplemented with 10% FBS, 1% L-glutamine and
50 lg/ml Gentamycin sulphate at 37°C in CO2 incubator in
an atmosphere of humidified 5% CO2 and 95% air. The
cells were maintained by routine sub culturing in 25 cm2
tissue culture flasks.
Method
Exponentially growing cells were harvested from 25 ml
flask and a stock cell suspension was prepared. A 96-well
flat bottom tissue culture plate was seeded with 5 9 104
cells/ml in medium and supplemented with 10% FBS and
incubated at 37°C for 24 h in 5% CO2 atmosphere. A
partial monolayer was formed after 24 h; the supernatant
was flicked off and to this 100 lL of different drug con-
centrations diluted in the medium to get 100, 50, 25, 12.5,
6.25, 3.125, 1.5625 lg/ml were added to the cells in the
microtitre plate. The cells in the control group received no
treatment. The plates were then incubated at 37°C for
3 days in 5% CO2 atmosphere. After the treatment for
72 h, drug containing media was removed and the plates
were washed twice with 100 ll of PBS. To each well of the
96-well plate, 100 ll of MTT reagent (stock: 2 mg/ml) was
added and incubated for 4 h at 37°C. Plates were centri-
fuged at 2000 RPM for 10 min and inverted on tissue paper
to remove the media. To solubilize formazan crystals in the
wells, 100 ll of isopropanol was added to each well and
incubated at 37°C for 30 min. The optical density (OD)
was measured by an enzyme linked immune sorbent assay
(ELISA) plate reader at 540 nm (Mosmann, 1983). The
percentage growth inhibition was calculated using the
following formula.
Sub culturing process of cell lines
The culture media from the flasks containing monolayer
culture was aspirated and washed with sterile phosphate
buffered saline (PBS).To the flasks, 2 ml of 0.1% trypsin–
EDTA solution was added and after few seconds it was
aspirated and flask was kept in incubator 2–3 min for
detachment. The flasks were removed from the incubator
and gently tapped to detach all the adhering cells. The cell
detachment was confirmed by observing under an inverted
microscope (Nikon Eclipse TE 2000-5, Japan). Once the
cells were completely detached from the flasks, 2–3 mL of
media containing 10% FBS was added and mixed well.
Cell viability was checked with a small sample of the
suspension by trypan blue dye exclusion test. From the
stock cell suspension, 1 9 104 viable cells/ml suspended in
media were seeded in 25 cm2 tissue culture flask contain-
ing about 4 mL of fresh media and incubated until the
flasks attained 60–70% confluence.
Percentage growth inhibition =
ꢀ
Preservation of the tumour cells
ꢁ
Mean OD of individual test group
Mean OD of control group
1 ꢁ
ꢂ 100
Tumour cells from the first and second passage of trans-
plantation were stored in liquid nitrogen in cryovials con-
taining respective medium supplemented with 10% FCS
and 10% DMSO as preservative at a concentration of
1 9 106 cells/ml. This constituted the tumour bank. After
every ten passages, that tumour cell line was discarded and
new passage was started using the original tumour cells
from the tumour bank.
The IC50 value obtained was the concentration of sample
required to inhibit the growth of 50% of viable cell
population.
In vivo anti-inflammatory activity
Carrageenan-induced rat hind paw oedema model Car-
boxymethylcellulose (0.5% w/v) was selected as vehicle to
suspend the standard drug and test compounds. The rats
were starved for 18 h prior to the experiment. The animals
were weighed, marked for identification and divided into
seven groups; each containing of six animals. Oedema was
Trypsinization
To obtain a single cell suspension from a monolayer cul-
ture, cells were dislodged from the culture flasks by
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3606
Med Chem Res (2012) 21:3598–3606
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induced in the left hind paw of all rats by subcutaneous
injection of 0.1 ml of 1% (w/v) carrageenan in 0.9% saline
into their footpads. The first group was kept as control and
was given 0.5% CMC. The second to sixth groups were
given an aqueous suspension of the synthesized compounds
at 100 mg kg-1 body mass. And seventh group (standard)
was administered Ibuprofen (100 mg kg-1 body mass, sus-
pension in 0.5% CMC). All the test compounds and the
standard drug were administered orally, 1 h before the car-
rageenan injection. The paw volume of each rat was mea-
sured using a digital plethysmometer (UGO Basil, Italy), just
before the carrageenan injection (0 h) and then at 1st, 3rd
and 5th h post administration of the carrageenan injection.
The increase in paw volume at 1st, 3rd and 5th h was cal-
culated for each group and the results were expressed as
mean increase in paw volume SEM (Winter et al., 1962).
Results were analysed statistically using one-way ANOVA
followed by multiple comparison by post hoc Dunnett’s test.
The minimum level of significance was fixed at P \ 0.01.
The percentage inhibition of paw volume for each test group
was calculated using the following equation.
Percentage of inhibition ð%Þ ¼ 100 ð1 ꢁ ðx=yÞÞ
where, x is the mean increase in paw volume of treated
animals; y is the mean increase in paw volume of control
animals
Acknowledgments The authors (Vachala SD and Divya J) are
thankful to AICTE and DST for providing the instrumental facilities
(FTIR and UV). The authors are also thankful to Manipal University,
Manipal, and Dr. N. Udupa, Principal, Manipal College of Pharma-
ceutical Sciences, for providing all necessary facilities to carry out
this study.
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