Analgesic, anti-pyretic and DNA cleavage studies of novel pyrimidine derivatives of coumarin moiety

Article abstract of DOI:10.1016/j.ejmech.2010.02.048

A novel series of 4-[4-(6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-chromen-2-ones [5-7(a-e)] were synthesized from various 4-bromomethyl coumarins 1(a-e). The synthesized compounds were screened for in-vivo analgesic and anti-pyretic activities at a dose of

Full text of DOI:10.1016/j.ejmech.2010.02.048

European Journal of Medicinal Chemistry 45 (2010) 2597e2605
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Analgesic, anti-pyretic and DNA cleavage studies of novel pyrimidine derivatives
of coumarin moiety
*
Rangappa S. Keri, Kallappa M. Hosamani , Ramya V. Shingalapur, Mallinath H. Hugar
P.G. Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad 580 003, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 4-[4-(6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-chromen-2-ones [5e7(aee)] were
synthesized from various 4-bromomethyl coumarins 1(aee). The synthesized compounds were screened
for in-vivo analgesic and anti-pyretic activities at a dose of 25 and 100 mg/kg body weight (b.w),
respectively. Among them, compounds 5(d), 6(c) and 7(d) exhibited significant analgesic activity
comparable with standard drug analgin using Tail-flick model. Compounds 5(a) and 7(aed) showed
significant anti-pyretic activities comparable with standard drug aspirin using yeast-induced pyrexia
model. DNA cleavage study by agarose gel electrophoresis method was also studied. Qualitative SAR
studies indicate that, compounds with amino group at 2-position of pyrimidine ring enhances analgesic
and anti-pyretic activities and compounds with hydroxyl and thio group at 2-position of pyrimidine ring
increase DNA cleavage activities.
Received 4 October 2009
Received in revised form
22 February 2010
Accepted 22 February 2010
Available online 1 March 2010
Keywords:
Pyrimidine
Coumarin
Analgesic
Ó 2010 Elsevier Masson SAS. All rights reserved.
Anti-pyretic
DNA cleavage
1. Introduction
The pyrimidine entity is one of the most prominent structures
found in nucleic acid chemistry. Pyrimidine derivatives including
Pain is an unpleasant and subjective sensation resulting from
a harmful sensorial stimulation that alerts the body about a current
or potential damage to its tissues and organs [1]. It is estimated that
more than 75 million people refer to health services annually,
presenting some form of recurrent or persistent pain [2]. In spite of
painful sensation that can be solved most efficiently by removal of
the underlying cause, the pain-causing stimulus cannot always be
either easily defined or quickly removed. Therefore, the health
professionals are usually faced with the necessity to manage the
symptomatology of the pain [1].
Feveris a complex physiologic responsetriggered byinfections or
aseptic stimuli. Elevation in body temperature occurs when the
concentration of prostaglandin E2 (PGE2) increases within parts of
the brain. Such an elevation contributes to a considerable alteration
in the firing rate of neurons that control the thermoregulation
process in the hypothalamus. It is now evident that most anti-
pyretics exert their action by inhibiting the enzymatic activity of
cyclooxygenase and consequently reducingthe levels of PGE2 within
the hypothalamic region. In order to combat these diseases caused
by pathogens, it is usual that chemotherapeutic, analgesic and anti-
pyretic agents are prescribed separately in clinical practices [3].
uracil, thymine, cytosine, adenine, and guanine are fundamental
building blocks for deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA). Vitamin B1 (thiamine) is a well-known example of
a naturally occurring pyrimidine that is encountered in our daily
lives. They also play an essential role in several biological
processes, found in nucleoside antibiotics, anti-bacterials, cardio-
vascular as well as considerable chemical reactions and also
occupy a prominent place in the pharmaceutical arena [4].
Pyrimidine derivatives form a component in a number of useful
drugs and are associated with many biological, pharmaceutical
and therapeutical activities [5]. Condensed pyrimidine derivatives
have been reported as anti-microbial [6], analgesic, anti-viral, anti-
inflammatory [7], anti-HIV [8], anti-tubercular [9], anti-tumor [10],
anti-neoplastic [11], anti-malarial [12], diuretic [13], cardiovas-
cular [14] agents. Pyrimidine compounds are also used as hypnotic
drugs for the nervous system [15], calcium-sensing receptor
antagonists [16] and also for antagonists of the human A2A
adenosine receptor [17]. Like pyrimidine, coumarin also exhibits
diverse biological properties [18].
It was envisaged that these two active pharmacophores, if
linked together would generate novel molecular templates which
are likely to exhibit interesting biological properties in animal
models. The above-cited applications prompted us to synthesize
a series of new compounds reported in this article. Owing to the
importance and in continuation of our work on synthesis of
* Corresponding author. Tel.: þ91 836 2215286; fax: þ91 836 2747884/2771275.
E-mail address: dr_hosamani@yahoo.com (K.M. Hosamani).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.048

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R.S. Keri et al. / European Journal of Medicinal Chemistry 45 (2010) 2597e2605
biologically active compounds [19e22], now we wish to describe
the synthesis of new pyrimidine derivatives from 4-arylox-
ymethylcoumarins (Scheme 1). The compounds were screened for
their in-vivo analgesic, anti-pyretic and DNA cleavage study. Thus,
we have created new avenues to explore the potent heterocyclic
moieties for the pharmacological activities in medicinal chemistry.
for this could be competition between the activated C4eCH2 and
COCH3 group for carbanion formation. Compounds 4(aee) were
prepared by a different method, in which the chalcone (3) was
prepared separately by the reaction of p-hydroxy benzaldehyde
and acetophenone in presence of 20% alcoholic NaOH solution. This
was then treated with 4-bromomethyl coumarins 1(aee) in the
presence of anhydrous K2CO3 in dry acetone at room temperature.
In the IR spectrum of 6-methyl-4-[4-3-oxo-3-phenyl-propenyl)-
phenoxymethyl]-chromem-2-one 4(a), the lactone carbonyl
2. Chemistry
stretching frequency was observed at 1722 cmꢀ1 and
rated keto group at 1660 cmꢀ1. In the 1H NMR spectrum of
compound 4(a), the olefinic protons (cis) were observed at 7.43
a, b-unsatu-
For the synthesis of target compounds, the reaction sequence
outlined in Scheme 1, were followed. Synthesis of various 4-bro-
momethyl coumarins (1aee) was brought about by the Pechmann
cyclisation of phenols with 4-bromoethylacetoacetate. 4-bromo-
methyl coumarins 1(aee) were reacted with 4-hydroxy benzalde-
hyde in the presence of anhydrous K2CO3 in dry acetone at room
temperature to give the corresponding ethers 2(aee). In the IR
spectrum of 4-(2-oxo-2H-chromen-4-yl-methoxy)-benzaldehyde 2
(a), the lactone carbonyl stretching frequency was observed at
1718 cmꢀ1, whereas the aldehydic carbonyl stretching appeared at
1690 cmꢀ1. In the 1H NMR spectrum of compound 2(a), a singlet
d
and 7.92 ppm as two separate doublets. Further the compounds 4
(aee) were treated with urea, thiourea and guanidine hydrochlo-
ride in DMF to yield the corresponding pyrimidine derivatives. The
IR spectrum of 4-[4-2-hydroxy-6-phenyl-pyrimidin-4-yl-phenox-
ymethyl]-6-methyl-chromem-2-one 5(a) shows lactone carbonyl
stretching frequency at 1718 cmꢀ1 and hydroxyl group of pyrimi-
dine at 3498 cmꢀ1. In the PMR spectrum of compound 5(a),
a singlet was observed at
C4-CH2 protons were observed downfield as a singlet at
The pyrimidine proton was observed downfield as a singlet at
7.03 ppm and hydroxyl proton at 10.7 ppm. The aromatic
protons resonated as multiplet in the region of 6.88e8.15 ppm. All
d
2.16 ppm due to C6eCH3 protons. The
d
5.47 ppm.
was observed at
protons were observed downfield as a singlet at
C3eH of coumarin appeared at 6.64 ppm. The aldehydic proton
appeared as a singlet in the downfield at 9.95 ppm. Attempted
d
2.38 ppm due to C6eCH3 protons. The C4eCH2
d
5.31 ppm. The
d
d
d
d
d
the physical and analytical data of the synthesized compounds are
presented in Table 1.
condensation of 2(aee) with acetophenone in 40% NaOH did not
result in the formation of chalcones 4(aee). A plausible explanation
Scheme 1. Schematic representation for the synthesis of pyrimidines derivatives from 4-aryloxymethylcoumarin.

R.S. Keri et al. / European Journal of Medicinal Chemistry 45 (2010) 2597e2605
2599
Table 1
Physical and analytical data of pyrimidine derivatives [5e7(aee)].
O
N
N
5
6
4
3
R
7
R'
O
O
2
8
1
Entry
Producta
R
R0
Yield (%)b
m. p (ꢁ C)c
Mol. Formula/Mol. Wt
Elem. Analysis (Cal./Found)
C
H
N
1
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
6-CH3
OH
OH
OH
OH
OH
SH
61.0
57.0
53.0
64.0
58.0
73.2
61.0
64.0
58.0
68.0
64.0
73.0
68.0
67.0
59.0
265e266
203e204
176e177
165e166
210e211
216e218
168e169
137e138
243e244
221e222
274e275
227e228
176e177
142e144
227e229
C27H20N2O4/436
C27H20N2O4/436
C26H17ClN2O4/457
C30H20N2O4/472
74.30
74.32
74.30
74.29
68.35
68.37
76.26
76.30
76.26
76.39
71.66
71.68
71.66
71.69
66.03
66.06
73.75
73.78
73.75
73.73
74.47
74.51
74.47
74.45
68.50
68.53
70.42
70.45
70.42
70.46
4.62
4.65
4.62
4.60
3.75
3.78
4.27
4.30
4.27
4.25
4.45
4.47
4.45
4.43
3.62
3.66
4.13
4.16
4.13
4.10
4.86
4.89
4.86
4.85
3.98
3.96
4.49
4.52
4.49
4.50
6.42
6.45
6.42
6.45
6.13
6.10
5.93
5.97
5.93
5.95
6.19
6.20
6.19
6.17
5.92
5.96
5.73
5.70
5.73
5.75
9.65
9.62
9.65
9.67
9.22
9.19
8.91
8.93
8.91
8.88
2
7-CH3
3
6-Cl
4
5,6-Benzo
7,8-Benzo
6-CH3
5
C30H20N2O4/472
6
C27H20N2O3S/452
C27H20N2O3S/452
C26H17ClN2O3S/473
7
7-CH3
SH
8
6-Cl
SH
9
5,6-Benzo
7,8-Benzo
6-CH3
SH
C30H20N2O3S/488
10
11
12
13
14
15
SH
C30H20N2O3S/488
C27H21N2O3/435
C27H21N2O3/435
C26H18ClN3O3/456
C30H22ClNO5/471
C30H22ClNO5/471
NH2
NH2
NH2
NH2
NH2
7-CH3
6-Cl
5,6-Benzo
7,8-Benzo
a
Products were characterized by IR, NMR, MS and elemental analysis.
Isolated yields.
Melting points are uncorrected.
b
c
3. Pharmacology
rats up to 3 g kgꢀ1, p.o. during the 24 h observation period. There
was no sedation, convulsions and tremors upon inspection, no
ulceration and no haemorrhagic spots were observed. Postmortem
examination of the stomach, and intestine did not reveal any ulcer-
haemorrhagic spots.
All the synthesized compounds [5e7(aee)] were screened for
analgesic activity by tail-flick method in rats, used by D'Amour and
Smith [23]. The reaction time was measured at the end of 0, 30, 60
and 90 min after the administration of the compound and the
standard drug employed was analgin. The anti-pyretic activity of
the newly synthesized compounds [5e7(aee)] was screened by
using the yeast-induced pyrexia method [24]. Rectal temperature
was recorded by using clinical thermometer before (ꢀ18 h) and
18 h after (0 h) brewer's yeast injection. Aspirin (300 mg/kg, p.o.)
was used as standard drug for comparing the anti-pyretic activity of
the synthesized compounds. The DNA cleavage of newly synthe-
sized compounds [5e6(aee)] and 7(aeb) pyrimidine derivatives
were studied by agarose gel electrophoresis method [25].
4.2. Analgesic activity
All the synthesized compounds were screened for analgesic
activity by tail-flick method used by D'Amour and Smith [23]. The
reaction time was measured at the end of 0, 30, 60 and 90 min after
the administration of the compound and the standard drug
employed was analgin. The analgesic screening results revealed
that the entire tested compounds exhibited moderate to good
analgesic activity (Table 2) compared to the reference drug. The
compounds 5(b), 5(d), 5(e), 6(b), 6(c), 7(c) and 7(d) showed
excellent analgesic activity at 60 and 90 min. Whereas compounds
5(a), 6(a), 6(e), 7(a) and 7(b) showed moderate to good analgesic
activity comparable with standard drug analgin at 60 and 90 min.
Compounds 5(c), 6(d) and 7(e) showed lesser degree of activity.
Compounds 5(d), 6(c) and 7(d) showed nearly comparable activity
to that of reference drug analgin in peripheral analgesic activity.
4. Results and discussion
4.1. Acute toxicity studies
All the compounds have shown good safety profile till the
highest dose. No adverse effect or mortality was detected in albino

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R.S. Keri et al. / European Journal of Medicinal Chemistry 45 (2010) 2597e2605
Table 2
Analgesic activity of synthesized compounds [5e7(aee)] by tail-flick method in rats.
Compound
Dose (mg/kg)
Body weight
Average (ꢂ) reaction time (s). Time after drugs treatment (min)
0 (min)
30 (min)
60 (min)
90 (min)
Control 1% Tween 80
Standard Analgin
e
3.72 (ꢂ 0.358)
4.22 (ꢂ 0.410)
3.94 (ꢂ 0.410)
4.00(ꢂ 0.408)
3.80(ꢂ 0.000)
3.74(ꢂ 0.249)
4.00(ꢂ 0.408)
3.85(ꢂ 0.249)
3.80(ꢂ 0.408)
4.00(ꢂ 0.353)
3.76(ꢂ 0.540)
3.84(ꢂ 0.353)
3.94(ꢂ 0.249)
4.00(ꢂ 0.353)
3.80(ꢂ 0.408)
3.28(ꢂ 0.353)
3.90(ꢂ 0.408)
3.97 (ꢂ 0.287)
5.77 (ꢂ 0.250)*
4.48 (ꢂ 0.409)
4.50 (ꢂ 0.577)
4.02(ꢂ 0.108)
5.00(ꢂ 0.517)
4.75(ꢂ 0.50)
4.00 (ꢂ 0.360)
8.76 (ꢂ 0.252)**
5.46 (ꢂ 0.577)*
6.25 (ꢂ 0.410)**
4.04(ꢂ 0.208)
8.25(ꢂ 0.456)**
6.25(ꢂ 0.353)**
6.50(ꢂ 0.108)*
7.25(ꢂ 0.050)**
7.54(ꢂ 0.408)**
4.50(ꢂ 0.208)
5.25(ꢂ 0.300)*
6.75(ꢂ 0.500)*
6.75(ꢂ 0.500)*
7.00(ꢂ 0.572)*
7.25(ꢂ 0.500)*
4.75(ꢂ 0.249)
3.87 (ꢂ 0.355)
9.02(ꢂ 0.000)**
6.20 (ꢂ 0.252)*
7.75 (ꢂ 0.248)**
4.25(ꢂ 0.200)
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8.87(ꢂ 0.450)**
7.25(ꢂ 0.55)**
7.45(ꢂ 0.330)**
7.50(ꢂ 0.408)**
8.50(ꢂ 0.408)**
4.50(ꢂ 0.20)
4.50(ꢂ 0.219)
4.50(ꢂ 0.409)
5.10(ꢂ 0.00)
4.25(ꢂ 0.118)
4.52(ꢂ 0.408)
4.75(ꢂ 0.353)
5.10(ꢂ 0.000)
5.02(ꢂ 0.408)
4.00(ꢂ 0.201)
4.04(ꢂ 0.408)
6.25(ꢂ 0.408)**
7.25(ꢂ 0.288)**
7.50(ꢂ 0.408)**
7.10(ꢂ 0.354)**
8.00(ꢂ 0.408)**
5.00(ꢂ 0.577)**
Method: tail-flick method; test animals: albino rats; number of animals per group: 6; route of administration: oral; standard: Analgin (25 mg/kg); *P < 0.05, **P < 0.01, when
compared to control.
Statistical analysis: the statistical analysis was performed by one-way ANOVA followed Dunnet's test.
4.3. Anti-pyretic activity
4.4. Electrophoretic analysis
All the synthesized compounds [5e7(aee)] were screened for
anti-pyretic activity by using the brewer's yeast-induced pyrexia
method [24]. Aspirin is used as a reference drug. Fever was induced
by injecting 20 ml/kg (s.c.) of 20% aqueous suspension of brewer's
yeast in normal saline below the nape of the neck and rectal
temperature was recorded by clinical thermometer immediately
before (ꢀ18 h) and 18 h after (0 h). The anti-pyretic screening
results revealed that (Table 3), the compounds 5(a), 5(c), 6(c), 6(d),
7(a), 7(b), 7(c) and 7(d) significantly decrease the temperature of
pyretic rats at 1, 3 and 6 h after compound administration (Table 3).
The maximum mean rectal temperatures produced by brewer's
yeast in the presence of compounds 7(a) and 7(c) were (31.68,
32.45 ꢁC), respectively. In addition, compounds 6(c), 6(d), 6(e), 7(b)
and 7(c), showed a decrease in the rectal temperature after 3 h
were (32.61, 31.98, 32.00, 32.60, 32.45 ꢁC), respectively compared
to 34.68 ꢁC in the control group.
The DNA cleavage of some pyrimidine derivatives [5e6(aee)]
and 7(aeb) were studied by agarose gel electrophoresis method
and are presented in Figs. 1 and 2. The gel after electrophoresis
clearly revealed that, the compound 5(a) and 5(d) shown more
intense streak, indicating the activity. Significant activity was also
seen in compound 5(b), 5(c), 5(e), 6(b), 6(d), 6(e) and 7(b). The
difference was observed in the bands of compounds (Lanes 1e12)
compared to the control DNA of E. coli. This shows that the control
DNA alone does not show any apparent cleavage as the compounds
did. However, the nature of reactive intermediates involved in the
DNA cleavage by the compounds has not been clear. The results
indicate the importance of pyrimidine derivatives in these isolated
DNA cleavage reactions. As the compounds 5(a) and 5(d) were
observed to cleave the DNA, it can be concluded that the compound
inhibits the growth of the pathogenic organism by cleaving the
genome.
Table 3
Anti-pyretic activity of synthesized compounds [5e7(aee)] on brewer's yeast-induced pyrexia in rats.
Compound
Dose (mg/kg)
Body weight
Rectal temperature in ꢁC at time (h)
ꢀ18a
0b
1
3
5
6
Control malsaline
Standard Aspirin
e
34.68(ꢂ 0.18)
34.70(ꢂ 0.12)
34.40(ꢂ 0.18)
34.71(ꢂ 0.15)
34.52(ꢂ 0.11)
34.68(ꢂ 0.09)
34.75(ꢂ 0.06)
34.80(ꢂ 0.11)
34.82(ꢂ 0.09)
34.44(ꢂ 0.12)
34.46(ꢂ 0.18)
34.68(ꢂ 0.22)
34.36(ꢂ 0.36)
34.54(ꢂ 0.32)
34.72(ꢂ 0.18)
34.48(ꢂ 0.14)
34.82(ꢂ 0.15)
33 ꢂ 0.08 (ꢂ 0.68)c
33.78 (ꢂ 0.18)
32.82 (ꢂ 0.14)***
33.18 (ꢂ 0.16)*
32.40 (ꢂ 0.15)
33.18 (ꢂ 0.17)**
32.22 (ꢂ 0.18)
32.25 (ꢂ 0.20)
32.55 (ꢂ 0.22)*
32.81 (ꢂ 0.26)*
32.50 (ꢂ 0.32)*
32.92 (ꢂ 0.18)*
33.01 (ꢂ 0.21)*
32.66 (ꢂ 0.35)*
32.74 (ꢂ 0.33)*
32.74 (ꢂ 0.33)*
32.80 (ꢂ 0.26)
32.68 (ꢂ 0.30)*
32.80(ꢂ 0.09)
34.36(ꢂ 0.14)
32.30(ꢂ 0.11)***
33.12(ꢂ 0.26)**
33.10(ꢂ 0.26)
33.28(ꢂ 0.29)
32.82(ꢂ 0.24)
33.44(ꢂ 0.11)**
32.80(ꢂ 0.08)**
32.90(ꢂ 0.12)**
32.84(ꢂ 0.36)
33.01(ꢂ 0.14)**
33.09(ꢂ 0.19)
33.30(ꢂ 0.18)
33.80(ꢂ 0.13)**
32.09(ꢂ 0.15)
32.22(ꢂ 0.17)
32.51(ꢂ 0.23)
33.44(ꢂ 0.18)
32.38(ꢂ 0.12) ***
32.97(ꢂ 0.24)
32.40(ꢂ 0.24)
32.61(ꢂ 0.28)
32.68(ꢂ 0.08)***
32.74(ꢂ 0.21)
31.98(ꢂ 0.18)
31.82(ꢂ 0.20)
32.84(ꢂ 0.22)**
32.66(ꢂ 0.24)
32.50(ꢂ 0.32)
33.00(ꢂ 0.08)***
32.80(ꢂ 0.11)**
32.54(ꢂ 0.12)
31.88(ꢂ 0.14)
32.09(ꢂ 0.18)**
300
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
33.08 ꢂ 0.26 (ꢂ 0.84)c
33.14 ꢂ 0.32 (ꢂ 0.90)c
33.08 ꢂ 0.44 (ꢂ 0.84)c
33.82 ꢂ 0.60 (ꢂ 0.95)c
33.43 ꢂ 0.52 (ꢂ 0.83)c
33.54 ꢂ 0.44 (ꢂ 0.75)c
33.64 ꢂ 0.20 (ꢂ 0.95)c
33.78 ꢂ 0.36 (ꢂ 0.88)c
33.80 ꢂ 0.50 (ꢂ 0.82)c
33.78 ꢂ 0.62 (ꢂ 0.91)c
33.88 ꢂ 0.58 (ꢂ 0.83)c
33.44 ꢂ 0.66 (ꢂ 0.82)c
33.38 ꢂ 0.55 (ꢂ 0.80)c
33.62 ꢂ 0.44 (ꢂ 0.88)c
33.77 ꢂ 0.36 (ꢂ 0.75)c
33.64 ꢂ 0.55 (ꢂ 0.72)c
32.00 (ꢂ 0.11)***
32.31(ꢂ 0.22)
32.84(ꢂ 0.08)**
32.64(ꢂ 0.21)*
32.72(ꢂ 0.18)
32.68(ꢂ 0.20)*
32.77(ꢂ 0.24)
32.66(ꢂ 0.26)
32.61(ꢂ 0.28)**
31.98(ꢂ 0.32)
32.00(ꢂ 0.16)**
31.68 (ꢂ 0.07)***
32.60(ꢂ 0.09)**
32.45 (ꢂ0.08)***
31.84 (ꢂ 0.11)**
31.40 (ꢂ 0.18)
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
n ¼ Six animals in each group, values are mean ꢂ SEM, *P < 0.05, **P < 0.01, ***P < 0.001 when compared to control.
a
Temperature just before yeast injection.
Temperature just before drug administration.
Change in temperature following yeast injection.
b
c

R.S. Keri et al. / European Journal of Medicinal Chemistry 45 (2010) 2597e2605
2601
(d) exhibited significant and the remaining compounds showed
good to moderate analgesic activity comparable to that of standard
drug analgin in the tail-flick model at 25 mg/kg body weights of the
animals. The compounds 5(a), 7(a), 7(b), 7(c) and 7(d) have
significant anti-pyretic activity comparable with standard drug
aspirin in yeast-induced pyrexia model at 100 mg/kg. The DNA
cleavage studies revealed that, pyrimidine derivatives 5(a) and 5(d)
showed non-specific cleavage of DNA. In general, test compounds
are non-toxic up to 3000 mg kgꢀ1 body weights of the animals and
showed significant in-vivo analgesic and anti-pyretic activities.
Qualitative SAR studies indicate that the compounds containing
amino group at the 2-position of pyrimidine ring increases their
analgesic and anti-pyretic activities and the compounds with
hydroxyl and thio group at the 2-position of pyrimidine ring
increases DNA cleavage activities.
Fig. 1. DNA cleavage analysis of pyrimidine derivatives [5(aee) and 6a].
6. Experimental protocols
4.5. Structure activity relationship (SAR) study
6.1. Chemistry
The effect of varying and introducing new substituents in
pyrimidine moiety has been explored in synthetic organic chem-
istry for significant biological activity in medicinal chemistry
(Table 1). Thus, a variety of 2-substituted pyrimidine analogues
containing eOH, eSH and eNH2 groups have been exploited for
their analgesic and anti-pyretic activities. These compounds with
eNH2 functional group at 2-position of the pyrimidine moiety have
shown significant analgesic and anti-pyretic activities compared to
the eSH and eOH groups. The synthesized compounds with eSH
functional group at 2-position of pyrimidine ring showed moderate
analgesic and anti-pyretic activities compared to that of -OH
functional group. Whereas, the title compounds 5(b), 5(c), 5(e), 6
(b), 6(d) and 6(e) showed significant DNA cleavage activity.
Therefore, such compounds containing eOH and eSH substitutents
at 2-position of the pyrimidine moiety enhance the DNA cleavage
which could be exploited in medicinal chemistry for significant
biological activities. Hence, we concluded that amongst the tested
compounds, those containing eNH2 functional group at 2-position
of pyrimidine ring increases their analgesic and anti-pyretic activ-
ities. But, in case of DNA cleavage study, it is regarded as exactly
reverse of analgesic and anti-pyretic activities.
All reagents and solvents were used as obtained from the
supplier or recrystalized/ redistilled as necessary. The melting
points of the products were determined by open capillaries on
a Buchi apparatus and are uncorrected. The IR spectra were recor-
ded on a Nicolet Impact-410 FT-IR spectrophotometer, using KBr
pellets. 1H NMR and 13C NMR spectra were recorded on a Bruker
AC-300F 300 MHz spectrometer in DMSO/CDCl3 using TMS as an
internal standard with 1H resonant frequency of 300 MHz and 13C
resonant frequency of 75 MHz. D2O exchange was applied to
confirm the assignment of the signals of NH/OH protons. Mass
spectra were recorded on an Autospec EI-MS. The elemental
analysis was carried out using Heraus CHN rapid analyzer. All the
compounds gave C, H and N analysis within ꢂ0.4% of the theoretical
values. The homogeneity of the compounds was described by TLC
on alumina silica gel 60 F254 (Merck) detected by U.V light (254 nm)
and iodine vapours. The in-vivo analgesic and anti-pyretic activities
were screened at Luqman Pharmacy College, Gulbarga, Rajiv
Gandhi University, India.
6.2. General procedure for the preparation of compounds
5. Conclusion
6.2.1. Preparation of 4-bromomethyl coumarins 1(aee)
The present synthetic strategy begins with the generation of the
required4-bromomethylcoumarins. These compounds are prepared
by the Pechmann cyclisation of phenols with 4-bromoethylacetoa-
cetate [26].
A
new series of 4-[4-(6-phenyl-pyrimidin-4-yl)-phenox-
ymethyl]-chromen-2-ones [5e7(aee)] analogues were designed,
synthesized and characterized. The synthesized compounds
screened for their in-vivo analgesic, anti-pyretic and DNA cleavage
studies. Some of the synthesized compounds viz., 5(d), 6(c) and 7
6.2.2. Preparation of 2-oxo-2H-chromen-4-ylmethoxy-
benzaldehydes 2(aee)
p-Hydroxy benzaldehyde (10 mmol) and anhydrous K2CO3
(1.38 g, 10 mmol) were stirred in dry acetone (25 ml) for 30 min.
4-Bromomethylcoumarins (2.52 g, 10 mmol) were added and stir-
ring was continued for 24 h. The reaction mixture was concentrated
to one-fourth of the original volume and poured onto ice-cold
water. The solid separated was filtered and washed with 5% HCl
(10 ml) to neutralize excess of K2CO3, then washed with 100 ml of
cold water and with dilute ethanol. The crude product was dried
and recrystalized from DMF.
6.2.2.1. 4-(6-Methyl-2-oxo-2H-chromen-4-ylmethoxy)-benzalde-
hyde 2(a). Colourless solid, m.p. 218e219 ꢁC, IR (KBr, cmꢀ1): 3046
y
(]CHe), 2778 (CH of CHO), 1718 (C]O of coumarin), 1690 (C]O of
aldehyde), 1534 (C]C) cmꢀ1,1022 (CeOeC); 1H NMR (300 MHz,
CDCl3,
d ppm): 2.38 (s, 3H, CH3), 5.31 (s, 2H, CH2O), 6.64 (s, 1H, C3H),
Fig. 2. DNA cleavage analysis of pyrimidine derivatives [6(bee) and 7(aeb)].
7.12e7.97 (m, 7H, AreH), 9.95 (s, 1H, CHO); 13C NMR (75 MHz,

2602
R.S. Keri et al. / European Journal of Medicinal Chemistry 45 (2010) 2597e2605
CDCl3,
d
ppm): 21.1, 72.1, 109.5, 112.7, 114.0, 118.0, 123.0, 128.5, 133.1,
147.2, 157.7, 160.2, 162.3, 179.0; ESI-MS: 396 (Mþ); Anal. Calcd for
C26H20O4: C, 78.77; H, 5.09; Found C, 78.79; H, 5.12%.
133.3, 136.5, 148.2, 152.4, 158.2, 161.0, 192.0; ESI-MS: 294 (Mþ);
Anal. Calcd for C18H14O4: C, 73.45; H, 4.80; Found C, 73.46; H, 4.83%.
6.2.3.2. 7-Methyl-4-[4-(3-Oxo-3-phenyl-propenyl)-phenoxymethyl]-
6.2.2.2. 4-(7-Methyl-2-oxo-2H-chromen-4-ylmethoxy)-benzalde-
chromen-2-one 4(b). Colourless shiny crystals, Yield 58%, m.
hyde 2(b). Colourless solid, m.p.223e225 ꢁC, IR (KBr,
y
cmꢀ1): 3038
p.205e206 ꢁC, IR (KBr, cmꢀ1): 3079 (]CHe), 1717 (C]O of
y
(]CHe), 2788 (CH of CHO), 1707 (C]O of coumarin), 1697 (C]O of
coumarin), 1655 (
a,
b
-unsaturated keto group), 1520 (C]C) cmꢀ1
ppm): 2.15 (s, 3H, CH3), 5.33 (s, 2H,
;
aldehyde), 1562 (C]C) cmꢀ1, 1016 (CeOeC); 1H NMR (300 MHz,
1H NMR (300 MHz, CDCl3,
d
CDCl3,
7.15e7.91 (m, 7H, AreH), 9.91 (s, 1H, CHO); 13C NMR (75 MHz,
CDCl3, ppm): 20.6, 78.0, 108.6, 113.5, 115.6, 117.8, 121.3, 124.6,
d
ppm): 2.48 (s, 3H, CH3), 5.33 (s, 2H, CH2O), 6.60 (s,1H, C3H),
CH2O), 6.67 (s, 1H, C3H), 7.47 (d, J ¼ 15.5 Hz, 1H, eCH]CH), 7.87
(d, J ¼ 15.5 Hz 1H,eCH]CH), 6.88e8.13 (m, 12H, AreH); 13C NMR
d
(75 MHz, CDCl3, d ppm): 22.6, 79.3, 106.3, 110.3, 112.6, 117.8, 120.7,
128.4, 130.3, 132.6, 134.1, 148.2, 155.4, 158.6, 167.6, 188.0; ESI-MS:
294 (Mþ); Anal. Calcd for C18H14O4: C, 73.44; H, 4.81; Found C,
73.47; H, 4.85%.
122.0, 125.9, 126.9, 128.3, 129.8, 130.6, 131.1, 131.9, 132.8, 134.0,
135.8, 139.8, 145.6, 149.0, 162.3, 165.1, 175.7; ESI-MS: 396 (Mþ);
Anal. Calcd for C26H20O4: C, 78.77; H, 5.09; Found C, 78.80; H, 5.10%.
6.2.2.3. 4-(6-Chloro-2-oxo-2H-chromen-4-ylmethoxy)-benzaldehyde
6.2.3.3. 6-Chloro-4-[4-(3-Oxo-3-phenyl-propenyl)-phenoxymethyl]-
2(c). Colourless solid, m.p.220e222 ꢁC, IR (KBr,
y
cmꢀ1) : 3062
chromen-2-one 4(c). Shiny pale yellow crystals, Yield 63%, m.
(]CHe), 2808 (CH of CHO), 1722 (C]O of coumarin), 1698 (C]O of
p.145e147 ꢁC, IR (KBr,
y
cmꢀ1): 3088 ( ¼ CH-), 1715 (C]O of
aldehyde), 1571 cmꢀ1 (C]C), 1021 (CeOeC); 1H NMR (300 MHz,
coumarin), 1647 (
a, b ;
-unsaturated keto group), 1532 (C]C) cmꢀ1
CDCl3,
d
ppm): 5.25 (s, 2H, CH2O), 6.73 (s, 1H, C3eH), 7.21- 7.91
ppm):
1H NMR (300 MHz, CDCl3,
d ppm): 5.38 (s, 2H, CH2O), 6.53 (s, 1H,
(m, 7H, AreH), 9.90 (s,1H, CHO); 13C NMR (75 MHz, CDCl3,
d
C3H), 7.32 (d, J ¼ 15.9 Hz, 1H, eCH]CH), 15.6 (d, J ¼ 15.8 Hz,
1H,eCH]CH), 6.76e7.98 (m, 12H, AreH); 13C NMR (75 MHz, CDCl3,
83.4, 110.0, 113.5, 116.0, 119.8, 126.0, 127.9, 130.1, 131.6, 135.1, 138.3,
148.2, 151.6, 161.0, 167.7, 192.0; ESI-MS: 315 [M þ 1]þ; Anal. Calcd
for C17H11ClO4: C, 64.87; H, 3.57; Found C, 64.90; H, 3.60%.
d
ppm): 75.8, 104.6, 112.6, 113.6, 117.5, 119.4, 122.0, 123.6, 125.3,
127.2, 129.6, 131.3, 132.5, 132.9, 134.3, 135.2, 136.3, 141.2, 148.3,
156.8, 159.1, 161.5, 184.4; ESI-MS: 418 [M þ 1]þ; Anal. Calcd for
C26H17ClO4: C, 72.03; H, 4.11; Found C, 72.00; H, 4.14%.
6.2.2.4. 4-(3-Oxo-3H-benzochromen-1-ylmethoxy)-benzaldehyde
2
(d). Pale yellow solid, m.p.222e224 ꢁC, IR (KBr, cmꢀ1): 3080
y
(]CHe), 2836 (CH of CHO), 1712 (C]O of coumarin), 1695(C]O of
6.2.3.4. 1-[4-(3-Oxo-3-phenyl-propenyl)-phenoxymethyl]-benzo[f]
aldehyde), 1562 cmꢀ1 (C]C), 1031 (CeOeC); 1H NMR (300 MHz,
chromen-3-one 4(d). Yellow crystals, Yield 73%, m.p.242e243 ꢁC,
CDCl3,
d
ppm): 5.75 (s, 2H, CH2O), 6.94 (s, 1H, C3eH), 7.13e8.15
ppm):
IR (KBr,
y
cmꢀ1): 3103 ( ¼ CH-), 1728 (C]O of coumarin), 1664
(m, 10H, AreH), 9.96 (s,1H, CHO); 13C NMR (75 MHz, CDCl3,
d
(a, b ;
-unsaturated keto group), 1542 (C]C) cmꢀ1 1H NMR
81.4, 106.0, 113.5, 114.0, 117.8, 120.5, 121.6, 123.0, 126.8, 129.1, 129.9,
130.3, 133.5, 135.2, 148.7, 149.6, 163.5, 169.3, 193.2.; ESI-MS: 330
(Mþ); Anal. Calcd for C21H14O4: C, 76.38; H, 4.27; Found C, 76.41; H,
4.30%.
(300 MHz, CDCl3,
(d, J ¼ 15.4 Hz, 1H, eCH]CH), 7.83 (d, J ¼ 15.5 Hz 1H,eCH]CH),
7.02- 8.41 (m, 15H, AreH); 13C NMR (75 MHz, CDCl3,
ppm): 83.5,
106.3, 112.6, 113.3, 115.2, 116.7, 121.6, 123.3, 124.7, 126.5, 127.6,
128.3, 129.3, 130.2, 131.3, 133.1, 136.5, 140.8, 150.2, 158.3, 160.4,
163.3, 177.2; ESI-MS: 433[M þ 1]þ; Anal. Calcd for C29H20O4: C,
80.54; H, 4.66; Found C, 80.53; H, 4.69%.
d ppm): 5.13 (s, 2H, CH2O), 6.37 (s, 1H, C3H), 7.54
d
6.2.2.5. 4-(2-Oxo-2H-benzochromen-4-ylmethoxy)-benzaldehyde
2
(e). Pale red solid, m.p.248e249 ꢁC, IR (KBr, cmꢀ1): 3052 (]CHe),
y
2842 (eCH of CHO), 1718 (C]O of coumarin), 1707 (C]O of alde-
hyde), 1569 cmꢀ1 (C]C), 1028 (CeOeC); 1H NMR (300 MHz, CDCl3,
6.2.3.5. 4-[4-(3-Oxo-3-phenyl-propenyl)-phenoxymethyl]-benzo[h]
d
ppm): 5.48 (s, 2H, CH2O), 6.80 (s, 1H, C3eH), 7.08e8.89 (m, 10H,
chromen-2-one 4(e). Reddish crystals, Yield 58%, m.p.221e222 ꢁC,
AreH), 9.92 (s,1H, CHO); 13C NMR (75 MHz, CDCl3,
d
ppm): 80.8,
IR (KBr,
y
cmꢀ1): 3093 ( ¼ CH-), 1725 (C]O of coumarin), 1655
107.5, 112.6, 113.1, 116.3, 122.1, 125.6, 127.6, 128.2, 129.8, 131.6, 132.6,
133.0, 135.3, 146.6, 149.6, 164.4, 168.8, 191.5.; ESI-MS: 330 (Mþ);
Anal. Calcd for C21H14O4: C, 76.39; H, 4.26; Found C, 76.42; H, 4.23%.
(a, b ;
-unsaturated keto group), 1526 (C]C) cmꢀ1 1H NMR
(300 MHz, CDCl3,
(d, J ¼ 16.2 Hz, 1H, eCH]CH), 7.92 (d, J ¼ 16.3 Hz 1H, eCH]CH),
6.89e8.23 (m, 15H, AreH); 13C NMR (75 MHz, CDCl3,
ppm): 81.6,
d ppm): 5.36 (s, 2H, CH2O), 6.42 (s, 1H, C3H), 7.36
d
6.2.3. Preparation of 4-[4-3-Oxo-3-phenyl-propenyl-
phenoxymethyl]-chromen-2-one 4(aee)
104.8, 111.6, 114.5, 115.6, 118.3, 120.4, 122.3, 123.6, 125.9, 128.1,
128.9, 129.5, 130.5, 131.0, 132.3, 135.3, 138.7, 144.9, 148.7, 160.0,
161.3, 162.8, 180.0; ESI-MS: 433 [M þ 1]þ; Anal. Calcd for C29H20O4:
C, 80.54; H, 4.66; Found C, 80.57; H, 4.69%.
Compound (3) (0.01 mol) and anhydrous K2CO3 (0.01 mol) were
stirred in anhydrous acetone for half an hour. To this substituted
4-bromomethyl coumarins 1(aee) (0.01 mol) were added and the
stirring was continued for 24 h. The reaction mixture was added to
the crused ice and neutralized with 1:1 HCl. The solid separated
was filtered and washed with water, dried and recrystalized from
dioxan-ethanol mixture.
6.2.4. Preparation of 4-[4-(6-phenyl-pyrimidin-4-yl)-
phenoxymethyl]-chromen-2-one [(5e7)aee]
Chalcone 4(aee) (0.01 mol) and urea, thiourea or guanidine
hydrochloride (0.01 mol) were dissolved in DMF (20 ml). Few drops
of concentrated HCl were added and the reaction mixture was
refluxed and the reaction was monitored by TLC. After completion of
reaction, the reaction mixture was poured onto 250 ml of ice-cold
water and kept aside for some time. The crude solid was filtered and
subjected to column chromatography. Elution with solvent system
ethyl acetate/petroleum ether (60e80 ꢁC) gave pure compound.
6.2.3.1. 6-Methyl-4-[4-(3-Oxo-3-phenyl-propenyl)-phenoxymethyl]-
chromen-2-one 4(a). Colourless shiny crystals, yield 70%, m.
p.232e233 ꢁC, IR (KBr,
y
cmꢀ1): 3059 (]CHe), 1722 (C]O of
coumarin), 1660 (
a
,
b
-unsaturated keto group), 1527 (C]C) cmꢀ1
ppm): 2.26 (s, 3H, CH3), 5.42 (s, 2H,
;
1H NMR (300 MHz, CDCl3,
d
CH2O), 6.60 (s, 1H, C3H), 7.43 (d, J ¼ 15.7 Hz, 1H, eCH]CH), 7.92
(d, J ¼ 15.8 Hz, 1H, eCH]CH), 6.76e7.81 (m, 12H, AreH); 13C NMR
6.2.4.1. 4-[4-(2-Hydroxy-6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-
(75 MHz, CDCl3,
d
ppm): 20.0, 74.6, 103.8, 112.6, 113.9, 116.8, 119.2,
6-methyl- chromen-2-one 5(a). Colourless shiny crystals, IR (KBr,
122.5, 126.5, 127.6, 128.3, 129.8, 130.6, 131.6, 132.9, 135.0, 140.1,
y
cmꢀ1): 3498 (eOH), 3055 (AreH), 1718 (C]O of coumarin), 1603

R.S. Keri et al. / European Journal of Medicinal Chemistry 45 (2010) 2597e2605
2603
(C]N) cmꢀ1
;
1H NMR (300 MHz, CDCl3,
d
ppm): 2.16 (s, 3H,
1613 (C]N) cmꢀ1
;
1H NMR (300 MHz, DMSO-d6,
d
ppm): 2.13
C6eCH3), 5.47 (s, 2H, CH2O), 6.46 (s, 1H, C3H), 6.61 (s, 1H, pyrimi-
dine proton), 6.88e8.15 (m, 12H, AreH), 10.7 (br s, OH, D2O
(s, 3H, C6eCH3), 5.46 (s, 2H, CH2O), 6.43 (s, 1H, C3eH), 6.65 (s, 1H,
pyrimidine proton), 7.06e8.34 (m, 12H, AreH), 11.87 (s, SH, D2O
exchangeable); 13C NMR (75 MHz, CDCl3,
d
ppm): 21.5, 79.3, 101.0,
exchangeable); 13C NMR (75 MHz, DMSO-d6,
d ppm): 17.9, 81.2,
107.4, 113.2,115.2,117.2,120.2,124.0,125.4,126.2, 127.0, 127.8,128.3,
128.6, 129.0, 130.8, 133.4, 134.6, 136.3, 145.0, 147.8, 155.3, 161.3,
164.6 171.3; ESI-MS: 437 [M þ 1]þ.
104.6, 110.6, 112.9, 114.6, 115.8, 121.7, 126.5, 126.9, 127.9, 128.2, 128.7,
129.2,129.8,130.7,133.7,138.3,145.8,149.7,157.8,158.9,161.7,163.9,
184.3; ESI-MS: 453 [M þ 1]þ.
6.2.4.2. 4-[4-(2-Hydroxy-6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-
6.2.4.8. 6-Chloro-4-[4-(2-Mercapto-6-phenyl-pyrimidin-4-yl)-phe-
7-methyl-chromen-2- one 5(b). Colourless shiny crystals, IR (KBr,
noxymethyl]-chromen-2-one 6(c). Colourless shiny crystals, IR (KBr,
y
cmꢀ1): 3486 (eOH), 3073 (AreH), 1721 (C]O of coumarin), 1589
y
cmꢀ1): 3078 (AreH), 2587 (SH), 1719 (C]O of coumarin), 1609
(C]N) cmꢀ1
;
1H NMR (300 MHz, CDCl3,
d
ppm): 2.25 (s, 3H,
(C]N) cmꢀ1; 1H NMR (300 MHz, CDCl3,
d ppm): 5.29 (s, 2H, CH2O),
C7eCH3), 5.53 (s, 2H, CH2O), 6.33 (s, 1H, C3H), 6.67 (s, 1H, pyrimi-
dine proton), 7.05e8.44 (m, 12H, AreH), 11.6 (br s, OH, D2O
6.36 (s, 1H,C3H), 6.89 (s, 1H, pyrimidine proton), 7.04e8.43 (m, 12H,
AreH), 11.78 (s, SH, D2O exchangeable); 13C NMR (75 MHz, CDCl3,
exchangeable); 13C NMR (75 MHz, CDCl3,
d
ppm): 19.4, 80.3, 104.6,
d ppm): 82.1,104.6,107.1,111.9,115.6,118.2,121.5,123.6,125.8,126.8,
108.5, 112.6, 115.8, 117.8, 121.7, 125.3, 126.6, 127.7, 128.9, 129.3, 130.4,
131.6, 132.9, 136.8, 138.7, 147.6, 150.8, 157.3, 159.2, 161.7, 176.8;
ESI-MS: 437 [M þ 1]þ.
127.5, 127.9, 128.2, 128.6, 129.9, 132.8, 136.9, 137.3, 144.9, 146.7,
157.8, 159.7, 161.8, 162.4, 179.6; ESI-MS: 474 [M þ 1]þ.
6.2.4.9. 1-[4-(2-Mercapto-6-phenyl-pyrimidin-4-yl)-phenox-
6.2.4.3. 6-Chloro-4-[4-(2-hydroxy-6-phenyl-pyrimidin-4-yl)-phe-
ymethyl]-benzo[f]chromen-3- one 6(d). Yellow shiny crystals, IR
noxymethyl]-chromen-2- one 5(c). Colourless shiny crystals, IR (KBr,
(KBr,
n
cmꢀ1): 3093 (AreH), 2599 (SH), 1726 (C]O of coumarin),
y
cmꢀ1): 3508 (eOH), 3067 (AreH), 1725 (C]O of coumarin), 1612
1603 (C]N) cmꢀ1 1H NMR (300 MHz, CDCl3,
; d ppm): 4.88 (s, 2H,
(C]N) cmꢀ1; 1H NMR (300 MHz, CDCl3,
d
ppm): 5.53 (s, 2H, CH2O),
CH2O), 6.27 (s, 1H, C3-H), 6.88 (s, 1H, pyrimidine proton), 7.12e8.51
(m, 15H, AreH), 12.13 (s, SH, D2O exchangeable); 13C NMR (75 MHz,
6.41 (s, 1H, C3H), 6.68 (s, 1H, pyrimidine proton), 6.91e8.33 (m, 12H,
AreH), 10.87 (br s, OH, D2O exchangeable); 13C NMR (75 MHz,
CDCl3,
d ppm): 79.3, 106.7, 110.5, 113.2, 115.3, 117.2, 121.6, 123.6,
CDCl3,
d
ppm): 77.7, 103.6, 109.3, 111.5, 113.1, 115.9, 119.3, 125.7,
126.2, 126.7, 127.4, 128.0, 128.3, 128.9, 129.8, 130.2, 132.8, 134.9,
136.9, 149.4, 153.2, 157.4, 161.7, 163.0, 165.1, 184.6; ESI-MS: 489
[M þ 1]þ.
126.8, 127.2, 127.6, 128.4, 128.9, 130.5, 134.2, 135.5, 146.3, 147.9,
155.8,160.1, 163.8 176.2; ESI-MS: 458 [M þ 1]þ.
6.2.4.4. 1-[4-(2-Hydroxy-6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-
6.2.4.10. 4-[4-(2-Mercapto-6-phenyl-pyrimidin-4-yl)-phenox-
benzo[f]chromen-3- one 5(d). Yellow shiny crystals, IR (KBr,
ymethyl]-benzo[h]chromen-2-one 6(e). Red shiny crystals, IR (KBr,
cmꢀ1): 3087 (AreH), 2607 (SH), 1732 (C]O of coumarin), 1596 (C]
N) cmꢀ1; 1H NMR (300 MHz, CDCl3,
ppm): 5.12 (s, 2H, CH2O), 6.42
y
y
cmꢀ1): 3517 (-OH), 3088 (AreH), 1731 (C]O of coumarin), 1606
(C]N) cmꢀ1; 1H NMR (300 MHz, CDCl3,
d
ppm): 5.43 (s, 2H, CH2O),
d
6.35 (s, 1H, C3H), 6.71 (s, 1H, pyrimidine proton), 7.03e8.42 (m, 15H,
(s, 1H,C3eH), 6.79 (s, 1H, pyrimidine proton), 7.07e8.36 (m, 15H,
AreH), 11.04 (br s, OH, D2O exchangeable); 13C NMR (75 MHz,
AreH), 11.76 (s, SH, D2O exchangeable); 13C NMR (75 MHz, CDCl3,
CDCl3,
d
ppm): 80.8, 102.3, 108.0, 114.8, 115.8, 116.0, 117.3, 120.1,
d ppm): 80.0,107.5,112.8,113.3,116.1,117.5,119.9,122.9,125.9,125.9,
122.3, 124.5, 126.3, 127.1, 127.7, 128.3, 128.9, 129.2, 130.6, 131.8,
133.8, 135.3, 136.9, 144.3, 151.3, 156.9, 158.6, 162.3, 164.2 172.1;
ESI-MS: 473 [M þ 1]þ.
126.4, 127.8,128.1, 128.7, 129.0, 129.3,130.6, 131.5, 135.2,138.7, 150.1,
159.2, 160.6, 164.2, 166.6, 182.2; ESI-MS: 489 [M þ 1]þ.
6.2.4.11. 4-[4-(2-Amino-6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-
6.2.4.5. 4-[4-(2-Hydroxy-6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-
6-methyl-chromen-2-one 7(a). Colourless crystals, IR (KBr, n
cmꢀ1):
benzo [h]chromen-2-one 5(e). Reddish shiny crystals, IR (KBr,
3433 and 3316 (NH2), 3065 (AreH), 1722 (C]O of coumarin), 1601
(C]N) cmꢀ1; 1H NMR (300 MHz, DMSO-d6, d ppm): 2.31 (s, 3H, C6-
CH3), 5.07 (s, 2H, CH2O), 5.75 (s, 2H,NH2, D2O exchangeable), 6.88 (s,
1H, pyrimidine proton), 7.09e8.32 (m, 12H, AreH); 13C NMR
y
cmꢀ1): 3503 (-OH), 3065 (AreH), 1726 (C]O of coumarin), 1589
(C]N) cmꢀ1; 1H NMR (300 MHz, CDCl3,
d ppm): 5.53 (s, 2H, CH2O),
6.35 (s, 1H,C3eH), 6.88 (s, 1H, pyrimidine proton), 6.97e8.13
(m, 15H, AreH), 10.65 (br s, OH, D2O exchangeable); 13C NMR
(75 MHz, DMSO-d6,
d ppm): 17.9, 80.3, 107.3, 112.8, 113.2, 114.5,
(75 MHz, CDCl3,
d
ppm): 77.5, 99.4, , 111.0, 113.4, 115.2, 115.9, 116.7,
117.0, 120.3, 125.7, 126.8, 127.3, 128.0, 128.4, 128.9, 129.3, 129.9,
133.2, 134.8, 135.3, 147.6, 152.2, 158.0, 160.3, 161.3, 167.7, 170.3;
ESI-MS: 436 [M þ 1]þ.
119.6, 122.0, 124.1, 126.3, 127.3, 127.9, 128.1, 128.8, 129.3, 129.9,
130.5, 131.6, 134.6, 138.5, 147.7, 154.3, 159.5, 160.4, 161.6, 175.5;
ESI-MS: 473 [M þ 1]þ.
6.2.4.12. 4-[4-(2-Amino-6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-
7-methyl-chromen-2-one 7(b). Colourless crystals, IR (KBr, v cmꢀ1):
3427 and 3299 (NH2), 3042 (AreH), 1719 (C]O of coumarin), 1597
6.2.4.6. 4-[4-(2-Mercapto-6-phenyl-pyrimidin-4-yl)-phenox-
ymethyl]-6-methyl-chromen-2-one 6(a). Colourless shiny crystals,
IR (KBr,
y
cmꢀ1): 3048 (AreH), 2592 (SH), 1719 (C]O of coumarin),
(C]N) cmꢀ1 1H NMR (300 MHz, DMSO-d6,
; d ppm): 2.25 (s, 3H,
1609 (C]N) cmꢀ1
;
1H NMR (300 MHz, DMSO-d6,
d
ppm): 2.22
C7eCH3), 5.56 (s, 2H, CH2O), 5.85 (s, 2H,NH2, D2O exchangeable),
(s, 3H, C6eCH3), 5.52 (s, 2H, CH2O), 6.38 (s, 1H, C3eH), 6.70 (s, 1H,
pyrimidine proton), 6.76e8.04 (m, 12H, AreH), 12.02 (s, SH, D2O
6.76 (s, 1H, pyrimidine proton), 7.01e8.42 (m, 12H, AreH); 13C NMR
(75 MHz, DMSO-d6,
d ppm): 18.6, 78.5, 106.8, 110.2, 113.7, 114.3,
exchangeable); 13C NMR (75 MHz, DMSO-d6,
d
ppm): 20.1, 79.7,
116.7, 119.6, 124.3, 127.0, 127.5, 128.1, 128.4, 128.9, 129.1, 129.7, 133.7,
134.0, 135.8, 137.7, 145.3,149.8, 157.5, 160.1, 161.7, 164.3, 168.1, 169.2;
ESI-MS: 436 [M þ 1]þ.
103.5,109.3,112.7,113.3,115.3,122.3,125.8,126.3,127.5,128.1,128.8,
129.6, 129.9, 130.5, 132.7, 135.7, 136.8, 146.0, 148.7, 155.8, 158.8,
159.8, 162.3, 166.2, 187.5; ESI-MS: 453 [M þ 1]þ.
6.2.4.13. 4-[4-(2-Amino-6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-
6.2.4.7. 4-[4-(2-Mercapto-6-phenyl-pyrimidin-4-yl)-phenox-
6-chloro-chromen-2-one 7(c). Colourless crystals, IR (KBr,
3408 and 3319 (NH2), 3032 (AreH), 1726 (C]O of coumarin), 1614
(C]N) cmꢀ1 1H NMR (300 MHz, DMSO-d6,
ppm): 5.21 (s, 2H,
y
cmꢀ1):
ymethyl]-7-methyl-chromen-2-one 6(b). Colourless shiny crystals,
IR (KBr,
y
cmꢀ1): 3053 (AreH), 2603 (SH), 1722 (C]O of coumarin),
;
d

2604
R.S. Keri et al. / European Journal of Medicinal Chemistry 45 (2010) 2597e2605
CH2O), 5.47 (s, 2H,NH2, D2O exchangeable), 6.57 (s, 1H, pyrimidine
6.3.4. Anti-pyretic activity
proton), 6.97e8.24 (m, 12H, AreH); 13C NMR (75 MHz, DMSO-d6,
The anti-pyretic activity was evaluated using brewer's yeast-
induced pyrexia in rats [24]. Fever was induced by injecting
20 ml/kg (s.c.) of 20% aqueous suspension of brewer's yeast in
normal saline below the nape of the neck and rectal temperature
was recorded by clinical thermometer immediately before
(andminus;18 h) and 18 h after (0 h) brewer's yeast injection.
Prior to the experiment, the rats were maintained in separate
cages for 7 days and the animals with approximately constant
rectal temperature were selected for the study. Aspirin (300 mg/
kg, p.o.) was used as standard drug for comparing the anti-
pyretic action of compounds. The experimental rats showed
a mean increase of about 0.86 ꢁC in rectal temperature, 18 h after
brewer's yeast injection. Compounds at 100 mg/kg produced
significant (P < 0.05 and P < 0.01, respectively) anti-pyretic
activity at 1 h and 3 h after drug administration.
d
ppm): 82.6,102.5,107.2,113.4,115.8,116.7,121.8,127.1,127.8,128.0,
128.6, 129.1, 129.8, 131.5, 135.9, 147.3, 151.2, 157.8, 162.6, 167.4, 168.1;
ESI-MS: 457 [M þ 1]þ.
6.2.4.14. 1-[4-(2-Amino-6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-
benzo[f]chromen-3- one 7(d). Yellow shiny crystals, IR (KBr,
y
cmꢀ1): 3423 and 3335 (NH2), 3025 (AreH), 1731 (C]O of
coumarin), 1607 (C]N) cmꢀ1
ppm): 5.43 (s, 2H, CH2O), 5.68(s, 2H,NH2, D2O exchangeable), 6.75
(s, 1H, pyrimidine proton), 7.07e8.36 (m, 15H, AreH); 13C NMR
(75 MHz, DMSO-d6, ppm): 81.7, 107.2, 112.5, 114.8, 115.6, 116.0,
;
1H NMR (300 MHz, DMSO-d6,
d
d
117.7,121.0,123.9,126.0,127.2,127.6,127.9,128.5,128.9,129.4,129.9,
131.6, 135.3, 145.9, 150.2, 153.8, 159.2, 162.1, 163.1, 166.6, 168.1;
ESI-MS: 472 [M þ 1]þ.
6.2.4.15. 4-[4-(2-Amino-6-phenyl-pyrimidin-4-yl)-phenoxymethyl]-
6.3.5. Statistical analysis
benzo[h]chromen-2-one 7(e). Red shiny crystals, IR (KBr,
3417 and 3326 (NH2), 3020 (AreH), 1726 (C]O of coumarin), 1600
(C]N) cmꢀ1 1H NMR (300 MHz, DMSO-d6,
ppm): 5.31 (s, 2H,
y
cmꢀ1):
Statistical analysis was performed by one-way analysis of vari-
ance (ANOVA) followed by Dunnett's t-test for multiple compari-
sons of all compounds in various pharmacological assays. Data are
expressed as mean ꢂ SEM.
;
d
CH2O), 5.54 (s, 2H,NH2, D2O exchangeable), 6.69 (s, 1H, pyrimidine
proton), 6.92e8.26 (m, 15H, AreH); 13C NMR (75 MHz, DMSO-d6,
d
ppm): 78.9, 106.7, 110.4, 113.2, 114.7, 115.7, 117.0, 118.4, 120.7, 122.4,
6.3.6. DNA cleavage experiment
125.7, 126.8, 127.0, 127.9, 128.1, 128.7, 129.3, 129.9, 130.5, 131.6,
6.3.6.1. Preparation of culture media. DNA cleavage experiments
were done according to the literature [25]. Nutrient broth [peptone,
10; yeast extract, 5; NaCl, 10; ins (g/l)] was used for culturing of
Escherichia coli. 50 ml media was prepared, autoclaved for 15 min
at 121 ꢁC under 15 lb pressure. The autoclaved media were inocu-
lated for 24 h at 37 ꢁC.
134.6, 148.7, 157.9, 160.3, 162.4, 165.1, 166.3; ESI-MS: 472 [M þ 1]þ.
6.3. Pharmacology
6.3.1. Animals
Albino-Swiss mice weighing (20e25 g) and albino Wistar rats
weighing (150e200 g) were used for studying in-vivo analgesic and
anti-pyretic activities. Animals were maintained under standard
laboratory conditions (24 ꢂ 2 ꢁC; relative humidity 60e70%). Study
protocol was approved by the institutional Animal Ethics
Committee (IAEC, Reg. No. 346/ CPCSEA: Dated. 03-01-2001) before
experiment. Albino-Swiss mice and albino Wistar rats from Labo-
ratory Animal Resource Section, Department of Pharmacy, Luqman
College of pharmacy, Gulbarga were used in the study. The animals
were kept in polypropylene cages and maintained on balanced
ration with free access to clean drinking water. All experimental
procedures were conducted in accordance with the guide for Care
and use of laboratory animals and in accordance with the Local
animal care and use committee.
6.3.6.2. Isolation of DNA. The fresh bacterial culture (1.5 ml) is
centrifuged to obtain the pellet which is then dissolved in 0.5 ml of
lysis buffer (100 mM tris pH 8.0, 50 mM EDTA, 10% SDS). To this
0.5 mL of saturated phenol was added and incubated at 55 ꢁC for
10 min, then centrifuged at 10,000 rpm for 10 min and to the
supernatant, equal volume of chloroform: isoamyl alcohol (24:1)
and 1/20th volume of 3 M sodium acetate (pH 4.8) was added.
Again centrifuging at 10,000 rpm for 10 min and to the superna-
tant, 3 volumes of chilled absolute alcohol were added. The
precipitated DNA was separated by centrifugation and the pellet
was dried and dissolved in TAE buffer (10 mM Tris pH 8.0, 1 mM
EDTA) and stored in cold condition.
6.3.6.3. Agarose gel electrophoresis. Cleavage products were
analyzed by agarose gel electrophoresis method [25]. Test
samples (1 mg/ml) were prepared in DMF. The samples (25 mg)
were added to the isolated DNA of E. coli. The samples were
incubated for 2 h at 37 ꢁC and then 20 ml of DNA sample (mixed
with bromophenol blue dye at 1:1 ratio) was loaded carefully
into the electrophoresis chamber wells along with standard DNA
marker containing TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M
EDTA/1 L) and finally loaded on agarose gel and passed the
constant 50 V of electricity for around 30 min. Removing the gel
and being stained with 10.0 mg/ml ethidium bromide for
10e15 min, the bands were observed under Vilberlourmate Gel
documentation system and then photographed to determine the
extent of DNA cleavage. Henceforth the results were compared
with standard DNA marker.
6.3.2. Acute toxicity studies
For testing the acute toxicity potential of the test compounds,
albino rats of either sex weighing 25e300 g were selected, sepa-
rated into groups each containing six rats. The dosage was varied
from 100 up to 3000 mg kgꢀ1 body weight. The rats were contin-
uously observed for 8 h for any signs of acute toxicity such as
increasededecreased motor activity, ataxia, tremors, convulsions,
sedation, lacrimation, etc. After 24 h the rats were sacrificed,
stomach, intestine, and liver were inspected under the magnifying
lenses for any ulcer-haemorrhagic spots.
6.3.3. Analgesic activity
The animals were divided into groups as shown in Table 2. The
reaction time was measured at the end of 0, 30, 60 and 90 min after
the administration of the compound and the standard employed
was analgin. The drugs were administered orally. The tail-flick
latency was assessed by the time taken by the rat to withdraw its
tail from the organ bath containing hot water (temperature
55 ꢂ 0.5 ꢁC). The tail-flick latency of treated animals was compared
with control and standard.
Acknowledgment
This research work is financially supported by the Council of
Scientific and Industrial Research (CSIR), New Delhi 110 012. (Ref:
No. 01(2301)/09/EMR-II dated 19-03-2009).

R.S. Keri et al. / European Journal of Medicinal Chemistry 45 (2010) 2597e2605
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