Analgesic study of novel pyrimidine derivatives linked with coumarin moiety

Article abstract of DOI:10.1007/s00044-011-9675-4

A novel series of 2-amino-4-(coumarin-3-yl)-6-substituted phenyl pyrimidines (5a-h) were synthesized from 3-acetylcoumarin (3). The structures of the synthesized compounds were elucidated by I.R., ¹H NMR, ¹³C NMR, and Mass spectroscopic techniques. The synthesized compounds were screened for in vivo analgesic activities at a dose of 20 mg/kg body weight (b.w). Among them, compounds 5b and 5h exhibited significant analgesic activity comparable with control as well as standard drug diclofenac sodium using acetic acid-induced writhing model. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9675-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1625–1632
DOI 10.1007/s00044-011-9675-4
ORIGINAL RESEARCH
Analgesic study of novel pyrimidine derivatives linked
with coumarin moiety
•
Jitendra Kumar Gupta Pramod K. Sharma Rupesh Dudhe
•
•
•
•
•
Anshu Chaudhary Avnesh Singh P. K. Verma Sambhu C. Mondal
•
•
Rakesh Kumar Yadav Shivjee Kashyap
Received: 13 May 2010 / Accepted: 16 May 2011 / Published online: 3 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A novel series of 2-amino-4-(coumarin-3-yl)-6-
substituted phenyl pyrimidines (5a–h) were synthesized
from 3-acetylcoumarin (3). The structures of the synthe-
Keywords Coumarin ꢀ Pyrimidines ꢀ
Knoevenagel reaction ꢀ Claisen–Schmidt condensation ꢀ
Acetic acid-induced writhing test ꢀ Analgesic
1
sized compounds were elucidated by I.R., H NMR, 13C
NMR, and Mass spectroscopic techniques. The synthesized
compounds were screened for in vivo analgesic activities at
a dose of 20 mg/kg body weight (b.w). Among them,
compounds 5b and 5h exhibited significant analgesic
activity comparable with control as well as standard drug
diclofenac sodium using acetic acid-induced writhing
model.
Introduction
Inflammation is a complex defensive mechanism of the
body to any noxious stimulus; this process may vary from a
localized to a generalized response characterized by the
accumulation of fluids and leukocytes leading to edema
and pain (Black, 2005). This inflammatory response seems
to be mediated by different physiological and immuno-
logical mediators that play a role in acute and chronic
inflammation. The acute inflammation occurs as the initial
response to tissue injury, being mediated by the release
of autocoids, for example, histamine, bradykinin, prosta-
glandins, and leukotrienes (Black, 2005; Sherwood and
Toliver-Kinsky, 2004). On the other hand, the chronic
inflammatory process involves the release of diverse
mediators, as interleukins, interferon and tumor necrosis
factor a (TNF-a), a cytokine that plays a major role in
this kind of inflammatory process and whose production
is associated with some inflammatory diseases such as
rheumatoid arthritis (Black, 2005; Sherwood and Toliver-
Kinsky, 2004). Non-steroidal anti-inflammatory drugs
(NSAIDs) are commonly used for the treatment of pain and
inflammation associated with different diseases particularly
rheumatoid arthritis (Sorbera et al., 2001).
J. K. Gupta (&) ꢀ P. K. Sharma ꢀ R. Dudhe ꢀ A. Singh ꢀ
S. C. Mondal ꢀ R. K. Yadav ꢀ S. Kashyap
Department of Pharmaceutical Technology, Meerut Institute of
Engineering & Technology, NH-58, Baghpat Bypass Crossing,
Meerut 250005, UP, India
e-mail: jitendraeishwer@yahoo.co.in;
jitendraeishwer@gmail.com
R. Dudhe
Uttarakhand Technical University Government Girls
Polytechnic, Post Office, Chandanwadi, Prem Nagar,
Sudhowalam, Dehradun 248007, Uttarakhand, India
A. Chaudhary
Vishveshwarya Institute of Medical Science, Gautambudh
Nagar, Dadri, UP 203207, India
Coumarins have a variety of bioactivities including
analgesic, antimicrobial (Radulovic et al., 2006), antico-
agulant, vasodilator, anthelmintic, sedative and hypnotics,
and antipyretic activity (Soine, 1964; O’Kennedy and
Thornes, 1997). In addition to this, studies are performed
on coumarin derivatives for their therapeutic role in the
A. Chaudhary
NIMS University, Shobha Nagar, Jaipur, Rajasthan 303001,
India
P. K. Verma
M.D. University, Rohtak, Haryana, India
123

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Med Chem Res (2012) 21:1625–1632
treatment of cancer (Lacy and O’Kennedy, 2004). Various
pyrimidine derivatives have been reported as anti-micro-
bial (El-Sayed Ali, 2009), analgesic and anti-inflammatory
(Amr et al., 2007; Abu-Hashem et al., 2010; El-Gazzar
et al., 2009; Chikhale et al., 2009), anticonvulsants (Gupta
et al., 2009), anti-HIV (Fujiwara et al., 2008), antiviral
(Hockov et al., 2004), anti-tubercular (Ballell et al., 2007),
anti-tumor (Wagner et al., 2008), anti-neoplastic (Xie
et al., 2009), antioxidant, and radioprotective agents
(Ghorab et al., 2010).
newly synthesized compounds were confirmed by their
spectral analysis. The physical data of these compounds are
summarized in Tables 1 and 2.
When salicylaldehyde (1) is treated with ethyl acetoac-
etate (2) in refluxing ethanol in the presence of piperidine,
it afforded a single precipitated product that was analyzed
correctly for C11H8O3. The reaction takes 2–3 h for com-
pletion. The structure of the precipitated product was
identified as 3-acetyl coumarin (3) based on its spectral
data. For example, IR spectrum of the isolated compound
Both the pyrimidines and coumarins exhibit diverse
biological properties (Kulkarni et al., 2006; Shafiee et al.,
2010). It was observed that linking of these two active
pharmacophores would generate novel molecular templates
which are likely to exhibit interesting biological properties
in animal models (Keri et al., 2010). Owing to the
importance, we wish to describe the synthesis of new
pyrimidine derivatives linked with coumarin moiety from
3-acetylcoumarins (Reaction Scheme 1). The compounds
were screened for their in vivo analgesic activity.
showed the characteristic band at 1,275 cm-1 13C NMR
,
spectrum revealed the presence of 11 carbon atoms in the
form of 11 peak signal.
In the second step, treatment of 3-acetyl coumarin (3)
with different types of benzaldehydes in the presence of
piperidine for 8–10 h using ethanol as a solvent yielded
one precipitated product (4a–h) respective of their benz-
aldehydes. The isolated product was identified as 3-
substituted phenyl-1-(coumarin-3-yl) prop-2-ene-1-ones
(4a–h). The IR spectrum of the isolated product revealed in
each case bands due to carbonyl function and aryl ethers
1
functions. Moreover, the H NMR spectrum of compound
Chemistry
(4a–h), taken as an example, revealed two doublet signals
between d 6–8 ppm, and one multiplet at d 7–8 ppm due to
methylene protons and aromatic protons, respectively.
The formation of compounds (4a–h) can be explained
on the basis of ‘‘Claisen–Schmidt condensation’’.
For the synthesis of target compounds, the reaction
sequence outline in Scheme 1, were followed. Salicylal-
dehyde (1) is reflux with ethyl acetoacetate (2) in the
presence of piperidine which gave 3-acetyl coumarin (3).
This synthesized compound condensed with different
substituted benzaldehydes using piperidine as a catalyst
yielded 3-substituted phenyl-1-(coumarin-3-yl) prop-2-
ene-1-ones (4a–h). 2-amino-4-(coumarin-3-yl)-6-substi-
tuted phenyl pyrimidines (5a–h) were prepared by treating
compounds (4a–h) with guanidine HCl. The structures of
In the third step, compound (4a–h) is refluxed with
guanidine HCl, in equimolar quantity in ethanol as a sol-
vent for 8–10 h, which afforded the final compounds (5a–
h). The structure of the final compounds was assigned
based on their elemental analysis and spectral data. For
example IR spectrum of the compounds (5a–h) revealed
the broad band between 3,300 and 3,400 cm-1 due to
O
O
OH
Scheme 1 Schematic
representation for the synthesis
of pyrimidine derivatives
COCH3
Piperidine
CH2
+
Ethanol
2-3 hrs
COOC2H5
CHO
(3)
(2)
(1)
O
8-10 hrs
H
C
Piperidine
Ethanol
O
X
O
O
O
O
N
NH2
a= 2-Chloro
b= 3-Chloro
c= 4-Chloro
d= 2-Bromo
e= 3-Bromo
f= 4-Bromo
g= 2-methoxy
h= 3-methoxy
O
Ethanol
Guanidine HCl
N
8-10 hrs
(5a-5h)
(4a-4h)
X
X
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Med Chem Res (2012) 21:1625–1632
1627
Table 1 Physical data of the synthesized compounds (4a–h)
Physical data of 3-substituted phenyl-1-(coumarin-3-yl) prop-2-ene-1-one (4a–h)
O
O
H
C
Ar
O
(4a-4h)
Compounda
-Ar
Yield (%)b
65
m.p. (°C)c
Rf value
Molecular formula
C18H11O3Cl
Compound 4a
156–158
0.73
Cl
Compound 4b
70
165–167
0.75
C18H11O3Cl
Cl
Compound 4c
Compound 4d
60
70
162–165
185–188
0.71
0.77
C18H11O3Cl
Cl
C18H11O3Br
Br
Compound 4e
75
185–187
0.76
C18H11O3Br
Br
Compound 4f
75
75
190–192
173–175
0.69
0.72
C18H11O3Br
Br
Compound 4g
C19H14O4
O
O
Compound 4h
75
177–179
0.76
C19H14O4
a
Products were characterized by IR, NMR, MS, and elemental analysis
Isolated yields
b
c
Melting points are uncorrected
amino group. In addition to this 1H NMR spectrum showed
the singlet signal d 4–5 ppm due to the amino group. These
signals were absent in the spectral data of compounds (4a–
h). Moreover, mass spectrum and elemental analysis data
also supported the structure assigned to the final com-
pounds (5a–h).
Result and discussion
Analgesic activity
Analgesic activity was performed according to Turner
(1965) technique using Swiss albino mice. The results of
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Med Chem Res (2012) 21:1625–1632
Table 2 Physical data of the synthesized compounds (5a–h)
Physical data of 2-amino-4-(coumarin-3-yl)-6-substituted phenyl pyrimidines (5a–h)
O
O
N
NH2
N
(5a-5h)
Ar
Compounda
-Ar
Yield (%)b
65
m.p. (°C)c
Rf value
Molecular formula
C19H12N3O2Cl
Compound 5a
145–147
0.62
Cl
Compound 5b
60
170–172
0.74
C19H12N3O2Cl
Cl
Compound 5c
Compound 5d
70
65
175–177
142–145
0.70
0.75
C19H12N3O2Cl
Cl
C19H12N3O2Br
Br
Compound 5e
50
133–135
0.72
C19H12N3O2Br
Br
Compound 5f
Compound 5g
60
65
170–173
190–191
0.68
0.67
C19H12N3O2Br
Br
C20H15N3O3
O
O
Compound 5h
65
173–175
0.65
C20H15N3O3
a
Products were characterized by IR, NMR, MS, and elemental analysis
Isolated yields
b
c
Melting points are uncorrected
analgesic activity indicated that all test compounds, except
5a and 5c, exhibited significant analgesic activity. Com-
pounds 5b and 5h have approximately half of the analgesic
activity as that of reference drug, and the remaining com-
pounds had moderate analgesic activity (Table 3).
Conclusion
A novel series of 2-amino-4-(coumarin-3-yl)-6-(substituted
phenyl) pyrimidines analogues were synthesized and
characterized. The synthesized compounds screened for
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Med Chem Res (2012) 21:1625–1632
1629
UV light (254 nm) and iodine vapors. The in vivo anal-
gesic activity was performed at Meerut Institute of Engi-
neering and Technology, Meerut, India.
Table 3 Analgesic activity of synthesized compounds (5a–h) by
acetic acid-induced writhing response model
Compounds
tested
Percent protection
0.5 h
1 h
2 h
General procedures for the preparation of compounds
Diclofenac
sodium
91.13 0.33
96.76 0.47
93.35 0.57
Synthesis of 3-acetyl coumarin (3): general procedure
Compound 5a 8.22 2.77
12.05 2.67
11.63 2.62
Compound 5b 52.21 4.20*a,b 60.58 3.95*a,b 56.50 3.90*a,b
A mixture of salicylaldehyde (1) (0.02 mol) and ethyl
acetoacetate (2) (0.03 mol) in ethanol were taken in round
bottom flask. To this mixture, few drops of piperidine were
added and refluxed for 2–3 h. After completion of reaction,
the content was poured on crushed ice. The solid separated
was filtered, dried, and recrystallized from ethanol. The
purity of compound was established on the basis of TLC.
M. P. 111–113°C, I. R. (KBr, cm-1): 1741.60 and 1677.95
(C=O), 1275 (aryl ethers, C–O–C); 1H NMR (CDCl3-d6, d,
ppm): 2.58 (s, 3H, CH3), 7.25–7.98 (m, 5H, Ar-H); 13C
NMR (CDCl3-d6, d, ppm): 35.50, 120.9, 123.8, 126.6,
127.3, 130.5, 132.5, 139.8, 155.7, 163, 200.6; MS, [M?],
m/z 188 (100%), [M? ?2], m/z 190 (15%), [M? ?4], m/z
192 (2%); Anal. Calcd for C11H8O3 (188.18): C, 70.21; H,
4.29. Found: C, 70.15; H, 4.25.
Compound 5c 9.49 2.38
Compound 5d 23.41 3.13
Compound 5e 25.94 1.65
Compound 5f 28.16 3.84
Compound 5g 35.44 3.05
12.35 2.33
30.58 3.40
33.52 1.62
29.41 3.77
35.58 3.03
10.80 2.49
27.70 3.39
31.57 1.77
26.59 3.54
33.79 2.99
Compound 5h 46.51 2.42*a,b 53.52 2.48*a,b 49.30 2.07*a,b
Method: acetic acid-induced writhing response model; test animals:
albino mice; number of animals per group: 6; route of administration:
oral; standard: Diclofenac sodium (20 mg/kg)
Statistical analysis: the statistical analysis was performed by one-way
ANOVA followed Dunnet’s test
* P B 0.05, *awhen compared to control, and *bstandard drug
Values are expressed as mean
SEM and *P C 0.05, indicates the
level of statistical significance as compared with control (*a) as well
as standard drug (*b)
Synthesis of 3-substituted phenyl-1-(coumarin-3-yl) prop-
2-ene-1-ones (4a–h): general procedure
their in vivo analgesic activity. Some of the synthesized
compounds viz., 5b and 5h exhibited good analgesic
activity in comparison to that of standard drug diclofenac
sodium in the acetic acid-induced writhing response model
at 20 mg/kg body weights of the animals.
Equimolar quantities of 3-acetyl coumarin (3) and different
substituted benzaldehydes were refluxed in absolute etha-
nol using piperidine as a catalyst for 8–10 h. The solution
mixture was concentrated and poured on to crushed ice.
The precipitate was filtered, dried, and recrystallized from
ethanol to give pure crystalline solid.
Experimental section
Synthesis of 3-(2-chlorophenyl)-1-(coumarin-3-yl) prop-2-
ene-1-one (4a) It was obtained from reaction of com-
pound (3) with 2-chlorobenzaldehyde. IR (KBr, cm-1):
Chemistry
1
1724.24 and 1662.52 (C=O), 1184.21 (C–O–C); HNMR
All reagents and solvents were used as obtained from the
supplier or recrystallized/redistilled as necessary. The
melting points of the products were determined by open
capillary method and are uncorrected. I.R. Spectra (KBr)
were recorded on FTIR Spectrophotometer (Shimadzu
(CDCl3-d6, d, ppm): 6.02 (d, 1H, CH), 7.11–7.93 (m, 9H,
Ar-H), 8.03 (d, 1H, CH); 13C NMR (CDCl3-d6, d, ppm):
120.3, 124.2, 125.3, 125.9, 129.1, 129.9, 130, 131.9, 132.5,
133, 138.9, 142.6, 143.9, 145.2, 147.6, 157.8, 159.6, 180.5;
MS, [M?], m/z 310 (100%), [M? ?2], m/z 312 (35%), [M?
?4], m/z 314 (10%); Anal. Calcd for C18H11ClO3 (310.73):
C, 69.58; H, 3.57. Found: C, 69.52; H, 3.52.
1
FTIR 84005, 4,000–400 cm-1). H NMR and 13C NMR
spectra were recorded on a JEOL AL300 FTNMR
300 MHz spectrometer in CDCl3 using TMS as an internal
1
standard, with H resonance frequency of 300 MHz and
Synthesis of 3-(3-chlorophenyl)-1-(coumarin-3-yl) prop-2-
ene-1-one (4b) It was obtained from reaction of compound
(3) with 3-chlorobenzaldehyde. IR (KBr, cm-1): 1728.10
and 1685.67 (C=O), 1107.06 (C–O–C); 1HNMR (CDCl3-d6,
d, ppm): 7.03 (d, 1H, CH), 7.15–8.02 (m, 9H, Ar-H), 8.66 (d,
1H, CH); 13C NMR (CDCl3-d6, d, ppm): 120.9, 122.9, 124.6,
125.9, 127.6, 128.9, 130.2, 130.9 131.5, 132.7, 133, 135.7,
13C resonance frequency of 75 MHz. Chemical shift values
are expressed in d ppm. Mass spectra were recorded on a
70 eV EI-MS-QP 1000 EX (Schimadzu). The elemental
analysis was carried out using Heraus CHN rapid analyzer.
The homogeneity of the compounds was determined by
TLC on alumina silica gel 60 F254 (Merck) detected by
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Med Chem Res (2012) 21:1625–1632
138.9, 144.9, 148.2, 158.3, 160.5, 178.6; MS, [M?], m/z 309
(100%), [M? ?2], m/z 311 (30%), [M? ?4], m/z 313 (5%);
Anal. Calcd for C18H11ClO3 (310.73): C, 69.58; H, 3.57.
Found: C, 69.62; H, 3.52.
Synthesis of 3-(2-methoxyphenyl)-1-(coumarin-3-yl) prop-
2-ene-1-one (4g) It was obtained from reaction of com-
pound (3) with 2-methoxybenzaldehyde. IR (KBr, cm-1):
1
1728.10 (C=O), 1164.92 (C–O–C). HNMR (CDCl3-d6, d,
ppm): 3.56 (s, 3H, CH3), 6.86 (d, 1H, CH), 7.02–7.96 (m,
9H, Ar-H), 8.09 (d, 1H, CH); 13C NMR (CDCl3-d6, d,
ppm): 62.7, 113.5, 118.6, 120.3, 121.6, 123.6, 125.9, 127.6,
128, 128.9, 129, 129.9, 143.9, 150, 155.6, 160.3, 163.5,
163.9, 179; MS, [M?], m/z 305 (100%), [M? ?2], m/z 307
(25%), [M? ?4], m/z 309 (2%); Anal. Calcd for C19H14O4
(306.31): C, 74.50; H, 4.61. Found: C, 74.54; H, 4.57.
Synthesis of 3-(4-chlorophenyl)-1-(coumarin-3-yl) prop-2-
ene-1-one (4c) It was obtained from reaction of compound
(3) with 4-chlorobenzaldehyde. IR (KBr, cm-1): 1728.10
and 1685.67 (C=O), 1107.06 (C–O–C). 1HNMR (CDCl3-d6,
d, ppm): 6.36 (d, 1H, CH), 6.90 (d, 1H, CH), 7.02–8.48 (m,
9H, Ar-H); 13C NMR (CDCl3-d6, d, ppm): 120.5, 123.4,
124.6, 127.5, 128.4, 128.6, 128.9, 130.5, 130.9, 131.5, 131.7,
132.6, 132.9, 144.4, 145.6, 157.2, 158.6, 182.9; MS, [M?],
m/z 309 (100%), [M? ?2], m/z 311 (33%), [M? ?4], m/z 313
(3%); Anal. Calcd for C18H11ClO3 (310.73): C, 69.58; H,
3.57. Found: C, 69.55; H, 3.51.
Synthesis of 3-(3-methoxyphenyl)-1-(coumarin-3-yl) prop-
2-ene-1-one (4h) It was obtained from reaction of com-
pound (3) with 3-methoxybenzaldehyde. IR (KBr, cm-1):
1
1735.81 (C=O), 1137.92 (C–O–C). HNMR (CDCl3-d6, d,
ppm): 3.90 (s, 3H, CH3), 6.98 (d, 1H, Ar-H), 7.00–7.85 (m,
9H, Ar-H), 8.10 (d, 1H, CH); 13C NMR (CDCl3-d6, d,
ppm): 63.2, 112.5, 118.2, 120.9, 122.9, 122.5, 126.9, 127.9,
128, 128.6, 129.3, 129.9, 142.6, 150.3, 154.6, 160.8, 163.6,
165.9, 182.3; MS, [M?], m/z 305 (100%), [M? ?2], m/z
307 (20%), [M? ?4], m/z 309 (1.5%); Anal. Calcd for
C19H14O4 (306.31): C, 74.50; H, 4.61. Found: C, 74.45; H,
4.56.
Synthesis of 3-(2-bromophenyl)-1-(coumarin-3-yl) prop-2-
ene-1-one (4d) It was obtained from reaction of com-
pound (3) with 2-bromobenzaldehyde. IR (KBr, cm-1):
1
1724.24 and 1683.74 (C=O), 1184.21 (C–O–C); HNMR
(CDCl3-d6, d, ppm): 6.86 (d, 1H, CH), 7.02–7.93 (m, 9H, Ar-
H), 8.00 (d, 1H, CH); 13C NMR (CDCl3-d6, d, ppm): 120.1,
120.9, 121.5, 121.9, 124.6, 125.6, 127.6, 127.9, 128.6, 128.9,
129.4, 129.9, 130.9, 145.6, 149.3, 159.6, 161.9, 178.5; MS,
[M?], m/z 354 (100%), [M? ?2], m/z 356 (25%), [M? ?4],
m/z 358 (2%); Anal. Calcd for C18H11BrO3 (355.18): C,
60.87; H, 3.12. Found: C, 60.81; H, 3.10.
Synthesis of 2-amino-4-(7-substituted coumarin-3-yl)-6-
substituted phenyl pyrimidines-(5a–h): general procedure
A mixture of 3-substituted phenyl-1-(coumarin-3-yl) prop-
2-ene-1-ones (0.01 mol) and guanidine HCl (0.02 mol) was
refluxed in ethanol for 8–10 h. The content was evaporated
to dryness and the product so obtained was washed with
water repeatedly and recrystallized from ethanol.
Synthesis of 3-(3-bromophenyl)-1-(coumarin-3-yl) prop-2-
ene-1-one (4e) It was obtained from reaction of com-
pound (3) with 3-bromobenzaldehyde. IR (KBr, cm-1):
1
1728.10 and 1685.67 (C=O), 1107.06 (C–O–C). HNMR
(CDCl3-d6, d, ppm): 7.08 (d, 1H, CH), 7.11–7.99 (m, 9H,
Ar-H), 8.05 (d, 1H, CH); 13C NMR (CDCl3-d6, d, ppm):
1209, 123.5, 124.6, 125.9, 126.9, 127.8, 128.7, 129, 129.4,
130, 131.5, 131.6, 134.6, 140, 147.3, 150.6, 158.3, 179.2;
MS, [M?], m/z 354 (100%), [M? ?2], m/z 356 (20%), [M?
?4], m/z 358 (1.6%); Anal. Calcd for C18H11BrO3
(355.18): C, 60.87; H, 3.12. Found: C, 60.81; H, 3.09.
Synthesis of 2-amino-4-(coumarin-3yl)-6-(2-chlorophenyl)
pyrimidines (5a) It was obtained from reaction of gua-
nidine HCl with compound (4a). IR (KBr, cm-1): 3151.47
(br, NH), 1758.96 (C=O), 1666.38 (C=N), 1114.78 (C–O–
C); 1HNMR (CDCl3-d6, d, ppm): 4.256 (s, 2H, NH2), 6.85–
7.72 (m, 10H, Ar-H); 13C NMR (CDCl3-d6, d, ppm):110.1,
124.2, 125.3, 128.6, 129.1, 129.9, 130, 131.9, 132.5, 135.5,
138.9, 142.6, 143.9, 145.2, 147.6, 157.8, 165.6, 168.5,
170.5; MS, [M?], m/z 348 (100%), [M? ?2], m/z 350
(40%), [M? ?4], m/z 352 (10%); Anal. Calcd for
C19H12ClN3O2 (349.77): C, 65.24; H, 3.46; N, 12.01.
Found: C, 65.30; H, 3.48; N, 12.06.
Synthesis of 3-(4-bromophenyl)-1-(coumarin-3-yl) prop-2-
ene-1-one (4f) It was obtained from reaction of compound
(3) with 4-bromobenzaldehyde. IR (KBr, cm-1): 1739.67
and 1677.95 (C=O), 1107.06 (C–O–C). 1HNMR (CDCl3-d6,
d, ppm): 7.03 (d, 1H, CH), 7.11–7.94 (m, 9H, Ar-H), 8.23
(d, 1H, CH); 13C NMR (CDCl3-d6, d, ppm): 121.9, 122.3,
123.6, 124.6, 125.3, 125.9, 128.6, 128.9, 129.5, 129.9,
130.5, 132.3, 135, 145.6, 150, 160.3, 164.2, 165.1, 180; MS,
[M?], m/z 354 (100%), [M? ?2], m/z 356 (18%), [M? ?4],
m/z 358 (2.5%); Anal. Calcd for C18H11BrO3 (355.18): C,
60.87; H, 3.12. Found: C, 60.90; H, 3.14.
Synthesis of 2-amino-4-(coumarin-3yl)-6-(3-chlorophenyl)
pyrimidines (5b) It was obtained from reaction of guani-
dine HCl with compound (4b). IR (KBr, cm-1): 3352.05
(br, NH), 1758.96 (C=O), 1635.52 (C=N), 1242.07
(C–O–C); 1HNMR (CDCl3-d6, d, ppm): 4.25 (s, 2H, NH2),
6.92–7.36 (m, 10H, Ar-H); 13C NMR (CDCl3-d6, d, ppm):
123

Med Chem Res (2012) 21:1625–1632
1631
109.2, 122.9, 124.6, 125.9, 127.6, 128.9, 130.2, 131.5, 132.7,
133, 135.7, 138.9, 144.9, 148.2, 158.3, 160.5, 161.4, 163.4;
MS, [M?], m/z 348 (100%), [M? ?2], m/z 350 (45%),
[M? ?4], m/z 352 (15%); Anal. Calcd for C19H12ClN3O2
(349.77): C, 65.24; H, 3.46; N, 12.01. Found: C, 65.20; H,
3.40; N, 12.0.
122.3, 123.6, 124.6, 125.3, 125.9, 128.6, 128.9, 129.5,
129.9, 130.5, 132.3, 135, 145.6, 150, 160.3, 164.2, 165.1,
167; MS, [M?], m/z 393 (100%), [M? ?2], m/z 395 (55%),
[M? ?4], m/z 397 (15%); Anal. Calcd for C19H12BrN3O2
(394.22): C, 57.89; H, 3.07; N, 10.66. Found: C, 57.85; H,
3.01; N, 10.60.
Synthesis of 2-amino-4-(coumarin-3yl)-6-(2-methoxyphenyl)
pyrimidines (5g) It was obtained from reaction of gua-
nidine HCl with compound (4g). IR (KBr, cm-1): 3421.48
(N–H), 1635.52 (C=O), 1600.81 (C=N), 1164.92 (C–O–C);
1HNMR (CDCl3-d6, d, ppm): 3.87 (s, 3H, CH3), 4.25 (s,
2H, NH2), 6.92–8.00 (m, 10H, Ar-H); 13C NMR (CDCl3-
d6, d, ppm): 63.7, 106.3, 113.5, 118.6, 120.3, 121.6, 123.6,
125.9, 127.6, 128, 128.9, 129, 129.9, 143.9, 150, 155.6,
160.3, 163.5, 163.9, 166.3; MS, [M?], m/z 344 (100%),
[M? ?2], m/z 346 (25%), [M? ?4], m/z 348 (5%); Anal.
Calcd for C20H15N3O3 (345.35): C, 69.56; H, 4.38; N,
12.17. Found: C, 69.62; H, 4.35; N, 12.11.
Synthesis of 2-amino-4-(coumarin-3yl)-6-(4-chlorophenyl)
pyrimidines (5c) It was obtained from reaction of gua-
nidine HCl with compound (4c). IR (KBr, cm-1): 3367.48
(br, NH), 1662.52 (C=O), 1585.38 (C=N), 1226.64 (C–O–
1
C); HNMR (CDCl3-d6, d, ppm): 4.25 (s, 2H, NH2), 7.02–
7.50 (m, 10H, Ar-H); 13C NMR (CDCl3-d6, d, ppm): 110.5,
123.4, 124.6, 127.5, 128.4, 128.6, 128.9, 130.5, 130.9,
131.5, 131.7, 132.6, 132.9, 144.4, 145.6, 157.2, 158.6,
160.9, 163.7; MS, [M?], m/z 348 (100%), [M? ?2], m/z
350 (47%), [M? ?4], m/z 352 (17%); Anal. Calcd for
C19H12ClN3O2 (349.77): C, 65.24; H, 3.46; N, 12.01.
Found: C, 65.19; H, 3.50; N, 12.02.
Synthesis of 2-amino-4-(coumarin-3yl)-6-(3-methoxyphenyl)
pyrimidines (5h) It was obtained from reaction of gua-
nidine HCl with compound (4h). IR (KBr, cm-1): 3413.77
(N–H), 1639.38 (C=O), 1585.38 (C=N), 1157.21 (C–O–C);
1HNMR (CDCl3-d6, d, ppm): 3.81 (s, 3H, CH3), 4.04(s, 2H,
NH2), 6.86–7.25 (m, 10H, Ar-H); 13C NMR (CDCl3-d6, d,
ppm): 63.2, 106.6, 112.5, 118.2, 120.9, 122.9, 122.5, 126.9,
127.9, 128, 128.6, 129.3, 129.9, 142.6, 150.3, 154.6, 160.8,
163.6, 165.9, 167.5; MS, [M?], m/z 344 (100%), [M? ?2],
m/z 346 (20%), [M? ?4], m/z 348 (8%); Anal. Calcd for
C20H15N3O3 (345.35): C, 69.56; H, 4.38; N, 12.17. Found:
C, 69.50; H, 4.34; N, 12.15.
Synthesis of 2-amino-4-(coumarin-3yl)-6-(2-bromophenyl)
pyrimidines (5d) It was obtained from reaction of gua-
nidine HCl with compound (4d). IR (KBr, cm-1): 3402.20
(N–H), 1674.10 (C=O), 1546.80 (C=N), 1110.92 (C–O–C);
1HNMR (CDCl3-d6, d, ppm): 4.96 (s, 2H, NH2), 7.25–7.63
(m, 10H, Ar-H); 13C NMR (CDCl3-d6, d, ppm): 107.9,
120.5, 121.5, 121.9, 124.6, 125.6, 127.6, 127.9, 128.6,
128.9, 129.4, 129.9, 130.9, 145.6, 149.3, 159.6, 161.9,
162.8, 164.9; MS, [M?], m/z 393 (100%), [M? ?2], m/z
395 (50%), [M? ?4], m/z 397 (20%); Anal. Calcd for
C19H12BrN3O2 (394.22): C, 57.89; H, 3.07; N, 10.66.
Found: C, 57.85; H, 3.02; N, 10.60.
Pharmacological screening
Synthesis of 2-amino-4-(coumarin-3yl)-6-(3-bromophenyl)
pyrimidines (5e) It was obtained from reaction of gua-
nidine HCl with compound (4e). IR (KBr, cm-1): 3421.48
(N–H), 1654.81 (C=O), 1488.94 (C=N), 1110.92 (C–O–C);
1HNMR (CDCl3-d6, d, ppm): 4.27 (s, 2H, NH2), 6.93–7.63
(m, 10H, Ar-H); 13C NMR (CDCl3-d6, d, ppm):109.9,
123.5, 124.6, 125.9, 126.9, 127.8, 128.7, 129, 129.4, 130,
131.5, 131.6, 134.6, 140, 147.3, 150.6, 158.3, 160, 165.8;
MS, [M?], m/z 393 (100%), [M? ?2], m/z 395 (45%), [M?
?4], m/z 397 (15%); Anal. Calcd for C19H12BrN3O2
(394.22): C, 57.89; H, 3.07; N, 10.66. Found: C, 57.92; H,
3.05; N, 10.60.
Animals
Albino-Swiss mice weighing (20–25 g) were used for
studying in vivo analgesic activity. Animals were main-
tained under standard laboratory conditions (24 2°C
relative humidity 60–70%). Study protocol was approved
by the institutional Animal Ethics Committee for the Pur-
pose of Control and Supervision on Experiments on Ani-
mals (IAEC, Approval No. 711/02/a/CPCSEA) before
experiment. Albino-Swiss mice from Laboratory Animal
House Section, Department of Pharmaceutical Technology,
Meerut Institute of Engineering & Technology, Meerut
were used in the study. The animals were kept in poly-
propylene cages and maintained on balanced ration with
free access to clean drinking water. All experimental pro-
cedures 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.
Synthesis of 2-amino-4-(coumarin-3yl)-6-(4-bromophenyl)
pyrimidines (5f) It was obtained from reaction of guani-
dine HCl with compound (4f). IR (KBr, cm-1): 3348.19
(N–H), 1674.10 (C=O), 1542.95 (C=N), 1107.06 (C–O–C);
1HNMR (CDCl3-d6, d, ppm): 4.16 (s, 2H, NH2), 6.90–7.73
(m, 10H, Ar-H); 13C NMR (CDCl3-d6, d, ppm): 109.3,
123

1632
Med Chem Res (2012) 21:1625–1632
El-Gazzar ABA, Youssef MM, Youssef AMS, Abu-Hashem AA,
Badria FA (2009) Design and synthesis of azolopyrimidoquin-
olines, pyrimidoquinazolines as anti-oxidant, anti-inflammatory
and analgesic activities. Eur J Med Chem 44:609–624
Analgesic activity (acetic acid-induced writhing response
model)
The compounds were selected for screening of their anal-
gesic activity in acetic acid-induced writhing response in
Swiss albino mice following the method of Turner (Turner,
1965). Sixty mice were selected and divided into 10 groups
(six in each group), starved for 16 h and pretreated as
follows, the first group which served as control positive
was orally received distilled water in appropriate volumes.
The second to ninth groups were received the aqueous
suspension of synthesized compounds orally in a dose of
20 mg/kg. The last group was orally received diclofenac
sodium in a dose of 20 mg/kg. After 30 min, each mice was
administered 1% of an aqueous solution of acetic acid
(10 ml/kg) and the mice were then placed in transparent
boxes for observation. The number of writhes was counted
for 30 min after acetic acid injection. The number of writhes
in each treated group was compared to that of a control
group. The number of writhing was recorded and the per-
centage protection was calculated using the following ratio:
El-Sayed Ali T (2009) Synthesis of some novel pyrazolo[3,4-
b]pyridine and pyrazolo[3,4-d]pyrimidine derivatives bearing
5,6-diphenyl-1,2,4-triazine moiety as potential antimicrobial
agents. Eur J Med Chem 44:4385–4392
Fujiwara N, Nakajima T, Ueda Y, Fujita H, Kawakami H (2008)
Novel piperidinylpyrimidine derivatives as inhibitors of HIV-1
LTR activation. Bioorg Med Chem 16:9804–9816
Ghorab MM, Ragab FA, Alqasoumi SI, Alafeefy AM, Aboulmagd SA
(2010) Synthesis of some new pyrazolo[3,4-d]pyrimidine deriv-
atives of expected anticancer and radioprotective activity. Eur J
Med Chem 45:171–178
Gupta SV, Baheti K, Bora R, Dekhane D, Chhabrya M, Shingare M,
Pawar S, Shishoo CJ, Thore SN (2009) Synthesis of novel
bioactive derivatives of 3-(4-chlorophenyl)-2-hydrazino-5,6,7,8-
tetrahydrobenzo(b)thieno[2,3-d]pyrimidine-4(3H)-ones. Eur
Med Chem 44:4721–4725
J
Hockov D, Holy A, Masojidkova M, Andrei G, Snoeck R, Clercq ED,
Balzarini J (2004) Synthesis and antiviral activity of 2,4-
diamino-5-cyano-6-[2-(phosphonomethoxy)ethoxy]pyrimidine
and related compounds. Bioorg Med Chem 12:3197–3202
Keri RS, Hosamani KM, Shingalapur RV, Hugar MH (2010)
Analgesic, anti-pyretic and DNA cleavage studies of novel
pyrimidine derivatives of coumarin moiety. Eur J Med Chem
45:2597–2605
Kulkarni MV, Kulkarni GM, Lin CH, Sun CM (2006) Synthesis of
novel triheterocyclic thiazoles as anti-inflammatory and analge-
sic agents. Curr Med Chem 13:2795–2818
Lacy A, O’Kennedy R (2004) Studies on coumarins and coumarin-
related compounds to determine their therapeutic role in the
treatment of cancer. Curr Pharm Des 10:3797–3811
O’Kennedy R, Thornes RD (eds) (1997) Coumarins—biology,
applications and mode of action. Wiley, Chichester
Radulovic N, Stojanovic G, Vukicevic R, Dekic V, Dekic B, Palic R
(2006) New 3,4-annelated coumarin derivatives: synthesis,
antimicrobial activity, antioxidant capacity and molecular mod-
eling. Monatsh Chem 137:1477–1486
Shafiee A, Motamedi R, Firuzi O, Meili S, Mehdipour AR, Miri R
(2010) Synthesis and cytotoxic activity of novel benzopyr-
ano[3,2-c]chromene-6,8-dione derivatives. Med Chem Res
20:466–474
% Protection ¼ ðControl mean ꢁ Treated mean=
Control meanÞ ꢂ 100
Statistical analysis
Statistical analysis was performed by one-way analysis of
variance (ANOVA) followed by Dunnett’s t test for mul-
tiple comparisons of all compounds in various pharmaco-
logical assays. Data are expressed as mean SEM.
Acknowledgment This research work is financially supported by
the Meerut Institute of Engineering & Technology, Meerut, India-
250005.
Sherwood ER, Toliver-Kinsky T (2004) Mechanisms of the inflam-
matory response. Best Pract Res Clin Anaesthesiol 18:385–405
Soine TO (1964) Naturally occurring coumarins and related physi-
ological activities. J Pharm Sci 53:231
Sorbera LA, Lesson PA, Castanar J, Castanar RM (2001) Valdecoxib
and parecoxib sodium. Drugs Fut 26:133–140
References
Abu-Hashem AA, Gouda MA, Badria FA (2010) Synthesis of some
new pyrimido[20,10:2,3]thiazolo[4,5-b]quinoxaline derivatives as
anti-inflammatory and analgesic agents. Eur J Med Chem
45:1976–1981
Amr AE, Nermien MS, Abdulla MM (2007) Synthesis, reactions, and
anti-inflammatory activity of heterocyclic systems fused to a
thiophene moiety using citrazinic acid as synthon. Monatsh
Chem 138:699–707
Ballell L, Field RA, Chung GAC, Young RJ (2007) New thiopyrazolo
[3,4d] pyrimidine derivatives as antimycobacterial agents. Bio-
org Med Chem Lett 17:1736–1740
Black JG (2005) Microbiology principles and exploration, 6th edn.
Wiley, US
Turner RA (1965) Analgesic. In: Turner RA (ed) Screening methods
in pharmacology. Academic Press, London
Wagner E, Al-Kadasi K, Zimecki M, Sawka-Dobrowolska W (2008)
Synthesis and pharmacological screening of derivatives of
isoxazolo[4,5-d]pyrimidine. Eur J Med Chem 43:2498–2504
Xie F, Zhao H, Zhao L, Lou L, Hu Y (2009) Synthesis and biological
evaluation of novel 2,4,5-substituted pyrimidine derivatives for
anticancer activity. Bioorg Med Chem Lett 19:275–278
Chikhale RV, Bhole RP, Khedekar PB, Bhusari KP (2009) Synthesis
and pharmacological investigation of 3-(substituted 1-phenyleth-
anone)-4-(substituted phenyl)-1,2,3,4-tetrahydropyrimidine-5-
carboxylates. Eur J Med Chem 44:3645–3653
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