Synthesis and anti-inflammatory activity of 2-(2-aroylaroxy)-4,6-dimethoxy pyrimidines

Article abstract of DOI:10.1016/j.bmcl.2008.06.061

Abstract-Reaction of 6a-f individually with 2-methylsulfonyl-4,6-dimethoxypyrimidine yielded 7a-f in excellent yield. The newly synthesized heterocycles were characterized by IR, ¹H NMR, and mass spectral data. Compounds 7a-f was screened for their anti-inflammatory activity and were compared with standard drugs. Of the compounds studied, the compound 7e showed more potent activity than the standard drugs at all doses tested.

Full text of DOI:10.1016/j.bmcl.2008.06.061

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4409–4412
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-inflammatory activity of 2-(2-aroylaroxy)-4,6-dimethoxy
pyrimidines
b
T. D. Venu a, S. A. Khanum c, Aiysha Firdouse c, B. K. Manuprasad a, Sheena Shashikanth a, , Riyaz Mohamed ,
*
Bannikuppe Sannanaik Vishwanth b
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, Karnataka 570 006, India
b Department of Biochemistry, University of Mysore, Manasagangotri, Mysore, Karnataka 570 006, India
c Department of Chemistry/Biochemisty, Yuvaraja college, University of Mysore, Karnataka 570005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Abstract-Reaction of 6a–f individually with 2-methylsulfonyl-4,6-dimethoxypyrimidine yielded 7a–f in
excellent yield. The newly synthesized heterocycles were characterized by IR, 1H NMR, and mass spectral
data. Compounds 7a–f was screened for their anti-inflammatory activity and were compared with stan-
dard drugs. Of the compounds studied, the compound 7e showed more potent activity than the standard
drugs at all doses tested.
Received 1 December 2007
Revised 13 May 2008
Accepted 17 June 2008
Available online 20 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Pyrimidines
Anti-inflammatory activity
The competence of benzophenone analogues as chemothera-
peutic agents, especially as anti-inflammatory, is well recog-
nized.1,2 Some of these analogues were synthesized by several
scientists of the world and have been reported as potent anti-
inflammatory agents.3–5 Recently Ottosen et al. have reported syn-
thesis and structure–activity relationship of benzophenones,
which are novel class of p38 MAP kinase inhibitors with high
anti-inflammatory activity.6
Condensation of thiourea 1 with diethylmalonate 2 generated
the starting material, 2-mercapto-pyrimidine-4,6-diol 3. Alkylation
with methyl iodide gave 2-methylmercapto-4,6-dimethoxypyrim-
idine 4, which further on perborate oxidation converted the meth-
ylmercapto group to a sulfone 5, a better leaving group for
displacement by nucleophiles. Reaction of 6a–f individually with
2-methylsulfonyl-4,6-dimethoxypyrimidine yielded 7a–f in good
to excellent yield. The newly synthesized compounds 7a–f17 were
characterized by IR, 1H NMR and mass spectrophotometer. The
synthetic maneuvering leading to 7a–f has been represented in
Scheme 1.
Anti-inflammatory activity: Albino rats were used to perform
paw edema inhibition test adopting Winter et al.18 method. Groups
of five rats (body weight 125–160 g), were given a dose of a test
compound. After 30 min, 0.2 mL of 1% carrageenan suspension in
0.9% sodium chloride solution was injected subcutaneously, into
planter aponeurosis of the hind paw and the paw volume was mea-
sured by a water plethysmometer socrel and then measured again
after a time span of 3 h. The mean increase of paw volume at each
time interval was compared with that of control group (five rats
treated with carrageenan, but not with test compounds at the
same time intervals). The percentage inhibition values were calcu-
lated using the formula
In our continuing work on benzophenones,7,8 we are interested
to incorporate a pyrimidine group in one of the phenyl rings. The
reason for this is that pyrimidine derivatives comprise a diverse
and interesting group of drugs.9 The subject has been discussed re-
cently.10 Earlier, a comprehensive review concerning pyrimidines
had been published by Brown.11 Pyrimidines in general are extre-
mely important for their biological activities. For example, some
are antiviral agents,12 the others are selective cholecystokinin sub-
type 1 (CCK1) receptor antagonists,13 and a few are anti-inflamma-
tory.14–16 In fact, there are so many pyrimidine derivatives with
pharmacological activities.
Since many benzophenone analogues have presented anti-
inflammatory activities,14 we perceived that when two moieties,
such as benzophenone and pyrimidine are joined the new mole-
cules might exhibit superior anti-inflammatory activity. With this
idea in mind the present work was undertaken.
% anti-inflammatory activity ¼ ð1 ꢀ Gt=GcÞ ꢁ 100
where Gt and Gc represent tested and control groups, respectively.
Ulcerogenic activity: Group of 10 rats (body weight 200–230 g),
fasted for 24 h. were treated with an oral dose of test compound,
* Corresponding author.
E-mail address: shashisheena@yahoo.com (S. Shashikanth).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.061

4410
T. D. Venu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4409–4412
HO
OH
NH2
NH2
O
OEt
CH3I
+
NaOC2H5
N
N
S
OEt
O
Anhy. K2CO3
Acetone
SH
1
2
3
O
OH
R1
H3CO
OCH3
H3CO
OCH3
NaBO3
N
N
N
N
+
R2
SO2CH3
S
H3C
4
5
6a-f
Anhy. K2CO3
MIBK
OCH3
N
a: R1 = R2 = CH3
b: R1 = CH3, R2 = Cl
c: R1 = R2 = Cl
O
O
N
OCH3
d: R1 = F, R2 = CH3
e: R1 = F, R2 = Cl
f: R1 = Br, R2 = Cl
R2
R1
7a-f
Scheme 1.
except the control group. All animals were sacrificed 5 h after the
completion of dosing. With the aid of a microscope, the stomach
and small intestine of the rats were examined to find incidence
of hyperaemia, shedding of epithelium, petechial, frank haemor-
rhages and erosion or discrete ulceration with or without perfora-
tion. The presence of any of these criteria was considered to be an
evidence of ulcerogenic activity.19
standard. PG chromatographic profile was obtained by isocratic
elution with 150 mM H3PO4 in water, pH 3.5, containing 30% ace-
tonitrile, at a flow rate of 1 mL/min monitoring the UV absorption
at 214 nm. Radioactivity that co-eluted with authentic PGE2 was
quantified by liquid scintillation spectrometry. Test samples were
compared to paired control incubations. The percentage of inhibi-
tion was calculated as follows
Acute toxicity study: Nearly 50% lethal dose (ALD50) of the com-
pounds was determined in albino mice (body weight 25–30 g). The
test compounds were injected intraperitoneally at different dose
levels in groups of 10 animals. After 24 h of drug administration,
percent mortality in each group was observed from the data ob-
tained. ALD50 was calculated by adopting Smith20 method.
Cyclooxygenase activity: The in vitro test on microsomal fraction
of mucosal preparation of rabbit distal colon was carried out in or-
der to search out the plausible mechanism of the compounds. By
adopting Calderano et al.21 procedure the preparation was carried
out. About 2-3 g of stripped colonic mucosa was minced and
homogenized in 3 vols of tris buffer 0.1, pH 8.0 and the homoge-
nized was centrifuged. The precipitate was suspended in tris buffer
0.1 M, pH 8.0, and recentrifuged. For enzyme assay cyclooxygenase
activity, the microsomal pellet was used immediately. By measur-
ing the rate of conversion of arachidonic acid to PGE2, cyclooxygen-
ase activity was assayed. About 50 mL of microsomal fractions was
½ðcpm control-cpm test=ðcpm controlÞÞ ꢁ 100ꢂ
Phospholipase A2 (PLA2) activity: Assayed with [14C] oleate-labeled
autoclaved Escherichia coli as the substrate.22 The reaction mixture,
350 l
l, contained 100 mM Tris/HCl, pH 8.0, 5 mM Ca2+ and
3.15 ꢁ 109 autoclaved E. coli cells (corresponding to 10,000 c.p.m.
and 60 nmol of lipid phosphorous). The amount of enzyme protein
was chosen such that 10–15% hydrolysis of substrate was obtained
when incubated at 37 °C for 60 min. The reaction components were
mixed in the following order: buffer, calcium, water, and benzoyl
phenyl benzoates. Adding labeled E. coli substrate started the reac-
tion. The reaction was terminated by adding 100
ll of 2.0 N HCl and
100 l of fatty acid free BSA (100 mg/ml). The tubes were vortex-
l
mixed and centrifuged at 20,000g for 5 min. Aliquot (140 ll) of
the supernatant containing released [14C] Oleic acid was mixed with
scintillation cocktail and counted in a Hewlett Packard liquid Scin-
tillation Analyzer TRI CARB 2100 TR.
All the 2-(2-aroylaroxy)-4,6-dimethoxy pyrimidines 7a–f have
shown superior anti-inflammatory activity in the range 21.5–
48.6% at a dose of 40 mg/kg po. Among 7a–f, the compound 7e
with chloro and fluoro groups at para position in benzoyl and
phenoxy moieties of benzophenone elicited maximum inhibition
of edema (48.6%). Compound 7a with two methyl groups at the
para position in benzoyl and phenoxy moieties of benzophenone
displayed more activity (45.8%) compared to 7f (33.2%) in which
para position of benzoyl and phenoxy moiety of benzophenone
carries chloro and bromo groups, respectively. Nevertheless com-
pound 7b having methyl and chloro substituents at para position
incubated with test agents for 10 min at 37 °C in 30
pH 8.0, containing 2 mM reduced glutathione, 5 mM
1 lM hematin. The substrate, 20 lM arachidonic acid with trace
l
L Tris–HCl,
L-trytophan,
amount of [1- 14C] arachidonic acid [approximately 200 (xx)
cpm] was added, and the reaction proceeded for 5 min at 37 °C.
The reaction was stopped by addition of 0.2 mL of ether/metha-
nol/citric acid 0.2 M (30:4:1), which was precooled at ꢀ25 °C.
PGE2 was extracted twice into the same mixture. The solvent
was evaporated under nitrogen stream and radiolabeled arachi-
donic acid was separated, and from this radiolabeled PGE3 was sep-
arated by RP-HBLC with 2 nmol unlabeled PGE2 as an internal

T. D. Venu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4409–4412
4411
Table 1
Anti-inflammatroy, ulcerogenic, cyclooxygenase, PLA2, and toxicity data of compounds 7a–f
Compound
Dose
Anti-inflammatory Dose
Ulcerogenic
Activity
Cyclooxygenase activity ED50
PLA2 activity
(mg/kg po) assay inhibitory
action % inhibition
ALD50
(mg/kg po)
(mg/kg po) activity % edema
inhibition relative
to control
(mg/kg po) % of animal
% of animal assay inhibitory action
with hyperemia with ulcer
of some selected
compound % inhibition
10
10
80
40
20
60
30
90
lg
10
lM
7a
20
40
80
20.2
45.8
55.3
100
200
400
50
70
100
20
30
40
>1000
>1000
>1000
>1000
>1000
>1000
—
87
60.2
77.5
78.3
62.5
51.2
77.2
98.3
7b
20
40
80
11.5
21.5
45.5
100
200
400
20
40
60
40
50
80
ni
20
40
ni
7c
20
40
80
13.4
29.5
58.7
100
200
400
40
60
100
10
20
40
7d
20
40
80
19.4
29.1
58.2
100
200
400
70
90
100
10
20
40
7e
20
40
80
29.9
48.6
88.6
100
200
400
30
60
90
10
20
12
7f
20
40
80
13.7
33.2
57.7
100
200
400
50
70
90
5
10
15
70
99
Aspirin
20
40
80
29.9
38.8
61.4
100
200
400
30
60
90
80
90
90
Phenyl
20
29.6
100
30
30
—
butazone
40
80
34.1
51.9
200
400
60
90
60
90
89
ni
—
—
85
ni
Control
20
40
80
–
30
60
90
—
—
—
ni, no inhibition.
of phenoxy and benzoyl moieties, respectively, of benzophenone
displayed least activity (22.2%) at a dose of 40 mg/kg po. On the
contrary compounds 7c (29.5%) and 7d (29.1%) in which benzoyl
moiety carries chloro and methyl groups, respectively, and phen-
oxy moiety carries chloro and fluoro groups, respectively, have
shown less activity compared to compounds 7a, 7e and 7f. Based
on the above results, title compounds have been tested at three
graded doses (20, 40, and 80 mg/kg po) and compared with stan-
dard drugs aspirin and phenyl butazone. The comparison results
with standard drugs such as are listed in Table 1.
From the results of the biological activities, it appears that
introduction of pyrimidine moiety in 6a–f is successful as it was
found that 7a–f showed good anti-inflammatory with reduced ul-
cer production activity.
Acknowledgment
The authors express their sincere gratitude to the University of
Mysore, Mysore for providing laboratory facilities.
Ulcerogenic activity: The title compounds 7a–f exhibited low de-
gree of ulcer production activity (10–50%) at 200 mg/kg po. Among
7a–f, compound 7e with chloro and fluoro groups at para position
in benzophenone moiety exhibited lesser ulcerogenic activity
(20%) compared to standard drugs such as aspirin and
phenylbutazone.
Cyclooxygenase assay activity: Compounds 7a, 7c, 7d and 7f
showed good cyclooxygenase activity indicating that these com-
pounds reduce inflammatory response by inhibition of Prostaglan-
dins. The other compounds 7b and 7e do not inhibit the
cyclooxygenase activity. PLA2 assay activity: In addition, com-
pounds 7a–f were tested for PLA2 activity. The PLA2 class of en-
zymes catalyzes hydrolysis of the 2-acyl ester of 3-Sn
phosphoglycerides to yield arachidonic acid which is responsible
for the production of pro-inflammatory lipid mediators such as
PGs.23–25 Among compounds 7a–f, 7b and 7e have exhibited more
inhibition and compound 7c has exhibited moderate activity.
ALD50 studies: The toxicity study of these compounds indicates
their good safety margin.
References and notes
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4412
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C, 56.27; H, 3.42; Cl, 17.46; N, 6.86%.7d: Yield 0.6 g (82%). M.p. 105–107 °C; IR
(Nujol):) 1559 (pyrimidin-skeletal), 1660 cmꢀ1 (C@O); 1H NMR (CDCl3): d 2.42
(s, 3H, CH3), 3.71 (s, 6H, 2OCH3), 5.72 (s, 1H), 7.18–7.62 (m, 7H, Ar–H); LC-MS:
m/z 369 (M++1, 95); Anal. calcd for C20H17FN2O4: C, 65.20; H, 4.65; F, 5.16; N,
7.60. Found: C, 65.16; H, 4.60; F, 5.12; N, 7.54%.7e: Yield 0.61 g (79%). M.p. 89–
91 °C; IR (Nujol):) 1565 (pyrimidin-skeletal), 1668 cmꢀ1 (C@O); 1H NMR
(CDCl3): d 3.72 (s, 6H, 2OCH3), 5.75 (s, 1H), 7.2–7.65 (m, 7H, Ar–H); LC-MS: m/z
389 (M++1, 96); Anal. calcd for C19H14ClFN2O4: C, 58.70; H, 3.62; Cl, 9.12; F,
4.89; N, 7.20. Found: C, 58.65; H, 3.57; Cl, 9.08; F, 4.82; N, 7.14%.7f: Yield 0.61 g
14. Santagati, A.; Granata, G.; Santagati, M.; Cutuli, V.; Mangano, M. G.; Caruso, A.
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(79%).
M.p.
109–111 °C; IR
(Nujol):) 1570
(pyrimidin-skeletal),
1661 cmꢀ1(C@O); 1H NMR (CDCl3): d 3.74 (s, 6H, 2OCH3), 5.78 (s, 1H), 7.25–
16. Tozkoparan, B.; Ertan, M.; Kelicen, P.; Demirdamar, R. Il Farmaco 1999, 54, 588.
17. Compound 7a: Yield 0.59 g (82%). M.p. 124–126°C; IR (Nujol):) 1569
(pyrimidin-skeletal), 1663 cmꢀ1 (C@O); 1H NMR (CDCl3): d 2.36 (s, 3H, CH3),
2.41 (s, 3H, CH3), 3.7 (s, 6H, 2OCH3), 5.64 (s, 1H), 7.15–7.52 (m, 3H, Ar–H), 7.32
(d, J = 8.3 Hz, 2H, Ar–H), 7.58 (d, J = 8.5 Hz, 2H, Ar–H); LC-MS: m/z 365 (M++1,
95); Anal. calcd. for C21H20N2O4: C, 69.21; H, 5.53; N, 7.69. Found: C, 69.17; H,
5.48; N, 7.65%.7b: Yield 0.6 g (79%). M.p. 114–116 °C; IR (Nujol):) 1560
(pyrimidin-skeletal), 1665 cmꢀ1 (C@O); 1H NMR (CDCl3): d 2.36 (s, 3H, CH3),
3.7 (s, 6H, 2OCH3), 5.7 (s, 1H), 7.16–7.62 (m, 7H, Ar–H); LC-MS: m/z 385 (M++1,
93); Anal. calcd for C20H17ClN2O4: C, 62.42; H, 4.45; Cl, 9.21; N, 7.25. Found: C,
62.37; H, 4.40; Cl, 9.17; N, 7.21%.7c: Yield 0.63 g (78%). M.p. 99–101 °C; IR
(Nujol):) 1567 (pyrimidin-skeletal), 1670 cmꢀ1 (C@O); 1H NMR (CDCl3): d 3.72
(s, 6H, 2OCH3), 5.68 (s, 1H), 7.21–7.64 (m, 7H, Ar–H); LC-MS: m/z 406 (M++1,
96); Anal. calcd for C19H14Cl2N2O4: C, 56.30; H, 3.48; Cl, 17.50; N, 6.90. Found:
7.75 (m, 7H, Ar–H); LC-MS: m/z 450 (M++1, 91); Anal. calcd for
C
19H14BrClN2O4: C, 50.75; H, 3.14; Br, 17.77; Cl, 7.88; N, 6.23. Found: C,
50.68; H, 3.07; Br, 17.69; Cl, 7.80; N, 6.18%.
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