Computer simulation of the in vitro and in vivo anti-inflammatory activities of dihydropyrimidines acid derivatives through the inhibition of cyclooxygenase-2

Article abstract of DOI:10.1007/s00044-012-0244-2

Simulation of virtually designed 20 compounds as COX-2 inhibitors using molecular modelling of protein-ligand interactions to predict drug structure-activity relationship was performed in this study. A synthetic route with a rational chemical approach to (E)-2-oxo-(thio)-4-substituted phenyl-6-styryl-1,2,3,4-tetrahydro-pyrimidine-5-caboxylic acid was designed and demonstrated. A comparative analysis of antimetabolite drug and corresponding metabolites (virtually designed compounds) provided a better understanding of rational drug design. COX-1(pdb entry: 1eqg) and COX-2(pdb entry: 6cox) enzymes docked with novel ligands were evaluated for binding energies. Lead optimization was performed by computational simulation: methoxy-substituted analogues displayed the highest negative ligand-protein-binding energies. These results prompted us to evaluate in vivo anti-inflammatory activity by carrageenan-induced paw oedema test in rats at a dose of 100 mg/kg. Ibuprofen was administered as standard drug. Lead compounds having significant activity were tested for in vitro cyclooxygenase isoenzyme inhibition assay and found to be more selective towards COX-2 as indicated by COX-2 selective index. The objective of our research is to accept the challenge of discovery of new drug. To ensure the desired target specificity and potency, bioavailability and lack of toxicity, our approach stems out lead generation from virtual screening to their synthesis and ends up with biological assays.

Full text of DOI:10.1007/s00044-012-0244-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2493–2504
DOI 10.1007/s00044-012-0244-2
ORIGINAL RESEARCH
Computer simulation of the in vitro and in vivo anti-inflammatory
activities of dihydropyrimidines acid derivatives through
the inhibition of cyclooxygenase-2
•
Raksha Dhankar Anjali M. Rahatgaonkar
•
•
Rakesh Shukla Mukund Chorghade
•
Ashutosh Tiwari
Received: 7 May 2012 / Accepted: 14 September 2012 / Published online: 27 September 2012
Ó Springer Science+Business Media New York 2012
Abstract Simulation of virtually designed 20 compounds
as COX-2 inhibitors using molecular modelling of protein–
ligand interactions to predict drug structure–activity rela-
tionship was performed in this study. A synthetic route with a
rational chemical approach to (E)-2-oxo-(thio)-4-substituted
phenyl-6-styryl-1,2,3,4-tetrahydro-pyrimidine-5-caboxylic
acid was designed and demonstrated. A comparative analy-
sis of antimetabolite drug and corresponding metabolites
(virtually designed compounds) provided a better under-
standing of rational drug design. COX-1(pdb entry: 1eqg)
and COX-2(pdb entry: 6cox) enzymes docked with novel
ligands were evaluated for binding energies. Lead optimi-
zation was performed by computational simulation: meth-
oxy-substituted analogues displayed the highest negative
ligand–protein-binding energies. These results prompted
us to evaluate in vivo anti-inflammatory activity by
carrageenan-induced paw oedema test in rats at a dose of
100 mg/kg. Ibuprofen was administered as standard drug.
Lead compounds having significant activity were tested for
in vitro cyclooxygenase isoenzyme inhibition assay and
found to be more selective towards COX-2 as indicated by
COX-2 selective index. The objective of our research is to
accept the challenge of discovery of new drug. To ensure the
desired target specificity and potency, bioavailability and
lack of toxicity, our approach stems out lead generation from
virtual screening to their synthesis and ends up with bio-
logical assays.
Keywords Cyclooxygenase enzyme I and II ꢀ
Anti-inflammatory inhibitors ꢀ Dihydropyrimidines ꢀ
Computational simulation ꢀ Selective index
Introduction
R. Dhankar
Department of Chemistry, Sardar Patel Mahavidyalaya,
Ganjward, Chandrapur 442402, Maharashtra, India
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the
enzymatic mechanism of cyclooxygenase (COX) enzyme
responsible for transformation of arachidonic acid to pros-
taglandin H2. Prostaglandins are recognized for inflamma-
tion, fever and pain (Dannhart and Kiefer, 2001). In recent
years, two forms of COX enzymes have been recognized as
COX-1 and COX-2. The COX-1 is constitutive and regulates
the platelet aggregation and homeostasis of gastrointestinal
tract whereas COX-2, the inducible form, is associated with
acute and chronic inflammation (Kurumbail et al., 1996).
The inhibitory activity of generally prescribed NSAID drugs
is targeted on both COX-1 and COX-2 enzymes. The major
drawback of these drugs is their selective inhibitory potency.
Some NSAID are non selective and inhibit both isoforms
thereby leading to life threatening gastrointestinal ulcers;
others like celecoxib and rofecoxib as presented in Fig. 1 are
A. M. Rahatgaonkar (&)
Department of Chemistry, Institute of Science, Civil Lines,
Nagpur 440001, Maharashtra, India
e-mail: anjali_rahatgaonkar@yahoo.com
R. Shukla
Central Drug Research Institute, Lucknow 226001,
Uttar Pradesh, India
M. Chorghade
Chorghade Enterprises, 14 Carlson Circle, Natick, MA, USA
A. Tiwari (&)
Biosensors and Bioelectronics Centre, Institute of Physics,
¨
Chemistry and Biology, Linkoping University,
¨
58183 Linkoping, Sweden
e-mail: ashutosh.tiwari@liu.se
123

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Med Chem Res (2013) 22:2493–2504
Fig. 1 Selective COX-2
inhibitors
SO2CH3
SO2NH2
SO2NH2
CH3
Br
N
N
N
N
O
O
F3C
Celecoxib
F3C
Rofecoxib
SC 558
COX-2 selective drugs. SC558 is another important inhibi-
tor. Cardiovascular risks associated with Rofecoxib COX-2
inhibitor prompted researchers to explore other specific,
advanced and targeted small molecules with efficacy (Prasit
et al., 1999).
The anti-inflammatory activity exhibited by acid deriv-
atives of pyrimidines prompted us to design, synthesize and
evaluate these scaffolds as COX-1 and COX-2 inhibitors
and anti-inflammatory agents. Although traditional selective
COX-2 inhibitors contain two vicinal diaryl moieties
attached with 5-membered pyrazole or furanone rings, we
herein report the acid and styryl derivatives of dihydropyr-
imidines lacking the traditional heterocyclic or carbocyclic
ring. The COX enzymes as COX-1(pdb entry: 1eqg) and
COX-2(pdb entry: 6cox) were downloaded from (http://
www.rscb.org/) as PDB files. A good inhibitor must possess
significant structural and chemical complimentarily to their
target receptors. The X-ray crystal structure analysis of
murin COX-2(pdb entry: 6cox) with selective inhibitor as
SC558 (1 phenyl sulphonamide-3-triflouromethyl-5-bro-
mophenylpyrazol) acquire three distinct anchoring sites.
The inhibitors were completely enclosed within the main
channel of COX active site of COX-2. The good inhibitors
were analysed in vivo as anti-inflammatory agents by car-
rageenan-induced paw oedema method. Further enzymatic
COX inhibition assay was carried out to strategically design
a better COX-2 inhibitor. The cardiovascular activity is still
unexplored for our compounds.
The demand for diverse chemical entities for screening
in search of potent drugs is the need of day. Molecular
modelling study on diverse building blocks has become a
facile approach for simulation of compounds with struc-
tural variations. A major challenge to organic synthesis
researchers and drug designers is in recognizing the
appropriate ligand with diverse functioning and to match it
with specific protein sequence.
Pyrimidine nucleus is the essential building block of
DNA; RNA encodes the genetic information of an organ-
ism. Current emphasis has been placed on synthesis of
dihydropyrimidines (DHPMs) condensed with other het-
erocycles and investigation of their biological activity.
Pyrimidines are also found to be calcium channel blocker
(Kappe, 2000), antihypertensive (Atwal et al., 1989),
antiviral, antibacterial and anticarcinogenic (Mukinsty and
Reading 1994), alpha1a antagonists (Atwal et al., 1991).
Naturally occurring marine alkaloids, batzalladines are
found to inhibit the binding of HIV gp-120-CD4 cell and
provide an opportunity to develop as anticancer agents
(Franklin et al., 1999). Pyrimidine derivatives are reported
to be anti-inflammatory and analgesic (Hill et al., 2004;
Carter et al., 2003; Agarwal et al., 2003; Liu et al., 2005;
Clare et al., 2005). Acid derivatives of pyrimidines [1, 2
and 3] as shown in Fig. 2 are characterized by potent anti-
inflammatory activity with high degree of selectivity
(Kalgutkar et al., 2000; Bahekar and Shinde, 2003; Bruno
et al., 2001; Sondhi et al., 2005; Mokale et al., 2010).
Literature precedence revealed that many selective
COX-1 and COX-2 inhibitors belong to tricyclic group of
compounds with central five membered ring of pyrazole,
furanone, isoxazole or oxazole possessing a diaryl-stillbene
like structure with sulphonyl group at para position of one
of the aryl rings. A few references (Kalgutkar et al., 2000;
Bahekar and Shinde, 2003; Bruno et al., 2001; Sondhi et al.,
2005; Mokale et al., 2010) are available in support of anti-
inflammatory activity of pyrimidines. The suggested spec-
ificity and selectivity of pyrimidine derivatives towards
Fig. 2 Acid derivatives of
pyrimidines as COX-2
inhibitors
NHCH2COOH
O
R
R1
N
N
N
N
HO
NH
CH3
N
N
S
H
COOH
R2
O
R1
1
2
3
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Med Chem Res (2013) 22:2493–2504
2495
COX-1 and COX-2 prompted us to focus our efforts on
evaluating on the mechanism of binding of our molecules to
COX-1 and COX-2.)
carboxylic ethyl ester referred as Biginelli compound
(Biginelli, 1893) is its hydrolysis. Currently available
experimental data of hydrolysis in mild alkaline conditions
show the adaptability of Biginelli framework leading to
decomposition into benzaldehyde, ammonia and potassium
carbonate (Zigeuner et al., 1976). The hydrolysis in strongly
alkaline conditions has not been much investigated (Steel
et al., 1998; Shutalev and Aksionov, 2005). We focussed our
investigation on the hydrolysis in strong alkaline condition in
10 % NaOH to secure 2a–i. A mixture of 6 methyl-2-oxo-4-
phenyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic ethyl
ester (1.9 mmol, 500 mg), and 25 mL of 10 % NaOH were
refluxed for 4 h. It was cooled and poured in 20 % HCl
solution, filtered and washed with water. The solid obtained
was recrystallized from ethanol (Scheme 1). Yield, 70 %;
mp 220 °C; IR (KBr), t (cm-1): 3409 (O–H), 3114 (N–H),
We have virtually designed the acid derivatives of
pyrimidines for evaluation as potential COX-2 inhibitors
by docking simulation, pursuing their synthesis. In vivo
and in vitro COX assay was achieved that could be bene-
ficial in the development of anti-inflammatory therapies.
The virtual screening paradigm represents a rich array of
opportunities that assist in expediting the discovery of new
targets and ultimately lead to compounds with predicted
biological activities of the novel targets.
Materials and methods
1
Melting points were determined by open capillary method
and are uncorrected. Chemicals were obtained from S.D.
Fine of AR Grade Analytical. TLCs were carried out on
0.25-mm layer silica gel plates, and single spots were
obtained. Column chromatography was performed with
silica gel of 120 mesh (Merk).1H NMR, and 13CNMR
spectra were recorded from CDCl3/DMSO-d6 solution on a
Brucker Avance II 400 (400 MHz) NMR Spectrometer.
Chemical shifts were reported as in d values (ppm) down-
field relative to TMS (0.00 ppm) as an internal standard. IR
spectra were obtained on a Perkin Elmer 1800 spectro-
photometer using KBr discs. Elemental analyses were car-
ried out using Perkin Elmer 2400 elemental analyser. Mass
spectra were measured with a GC–MS (70 eV). The purity
of the samples was checked by TLC and spectroscopic data.
1685 (C=O), 970 (CH=CH). H NMR (CDCl3), d (ppm):
11.5 (s, 1H, OH), 8.5 (s, 1H, N1H), 7.9 (s, 1H, N3H), 7.4–7.1
(m, 10H, ArH), 6.9 (d, 1H, J = 16.4, CH=CH), 6.4 (d, 1H,
J = 16.5, CH=CH), 5.4 (s, 1H, methine). 13C NMR
(200 MHz CDCl3), d 50.03, 106.4, 126.1, 126.4, 126.9,
127.1, 127.3, 127.4, 127.6, 127.7, 128.0, 128.3, 132.6, 135.1,
143.0, 150.4, 171.0. Mass (m/z): 320 M?; Elemental analy-
sis: Calculated for (C19H16N2O3): C 71.24, H 5.0, N 8.74;
Found: C 71.56, H 5.10, N 8.76.
(E)-4-Phenyl-6-styryl-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine-5-caboxylic acid (2b)
Yield, 67 % mp, 179 °C; IR (KBr), t (cm-1): 3410 (O–H),
3116 (N–H), 1689 (C=O), 986 (CH=CH). 1H NMR
(CDCl3), d (ppm): 11.1 (s, 1H, OH), 8.35 (s, 1H, N1H),
7.7–7.5 (m, 10Hs, ArH), 7.2 (d, 1H, J = 16.4, CH=CH),
6.7 (d, 1H, J = 16.5, CH=CH), 6.35 (s, 1H, N3H), 5.7 (s,
1H, methine). 13C NMR (200 MHz, CDCl3), d: 54.0,
103.5, 126.0, 126.1, 126.3, 127.3, 127.33, 127.6, 127.7,
127.9, 128.4, 128.6, 128.66, 131.4, 135.0, 143.0, 159.2,
171.3, and 174.4. Mass (m/z): 336 M?; Elemental analysis:
Calculated for (C19H16N2O2S): C 67.84, H 4.79, N 8.33;
Found: C 68.04, H 4.83, N 8.85.
General synthetic procedure for (E)-2-oxo-4-phenyl-6-
styryl-1,2,3,4-tetrahydro-pyrimidine-5-caboxylic
acid (2a)
6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydro-pyrimidine-5-
carboxylicethylesterwhenrefluxedinstrongalkaline(NaOH)
initiated hydrolysis with gas release and resultant copious froth
formation to furnish 2a–i. The most interesting feature of
6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydro-pyrimidine-5-
Scheme 1 Synthesis of 2-oxo/
thio-4-substituted phenyl-6-
styryl -1, 2, 3, 4-tetrahydro-
pyrimidine-5-carboxylic acid
R
R
O
O
C2H5O
H3C
10% NaOH Reflux
HO
NH
NH
4 hours
N
X
N
H
X
H
R
2a-i
R = H, OH, OCH3, N(CH3)2, NO2
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Med Chem Res (2013) 22:2493–2504
(E)-4-(4-Methoxyphenyl)-6-(4-methoxystyryl)-2-oxo-
J = 16.2, CH=CH), 5.5 (s, 1H, methine), 5.4 (d, 1H,
J = 16.5, CH=CH). 13C NMR (200 MHz, CDCl3), d: 54.0,
103.4, 115.2, 116.3, 123.6, 127.1, 127.2, 127.4, 128.6,
131.5, 135.1, 156.0, 157.6, 159.0, and 174.1. Mass (m/z):
368 M?. Elemental analysis: Calculated for (C19H16N2O4S):
C 61.94, H 4.38, N 7.60; Found: C 62.03, H 5.09,
N 7.51.
1,2,3,4-tetrahydro pyrimidine-5-carboxylic acid (2c)
Yield, 69 % mp, 256 °C; IR (KBr), t (cm-1): 3425 (O–H),
3209 (N–H), 1683 (C=O), 1284 (C–O–C), 972 (CH=CH). 1H
NMR (CDCl3), d (ppm): 11.63 (s, 1H, OH), 7.9 (s, 1H, N1H),
7.6–7.3 (m, 8H, ArH), 6.9 (d, 1H, J = 16.4, CH=CH), 6.5 (d,
1H, J = 16.2, CH=CH), 6.2 (s, 1H, N3H), 5.7 (s, 1H,
methine), 3.7 (s, 3H, methoxy), 3.8 (s, 3H, methoxy). 13C
NMR (200 MHz, CDCl3), d: 50.3, 56.0, 106.6, 124.6, 127.2,
128.8, 131.1, 135.0, 146.6, 150.8, 159.0, and 171.8. Mass (m/
z):380M?. Elementalanalysis:Calculatedfor(C20H18N2O4):
C 66, H 5.30, N 7.36; Found: C 67.01, H 5.34, N 7.40.
(E)-4-(4-Dimethylaminophenyl)-6-(4-
dimethylaminostyryl)-2-oxo-1,2,3,4-tetrahydro
pyrimidine-5-carboxylic acid (2g)
Yield, 67 %; mp 228 °C; IR (KBr) t (cm-1): 3406 (O–H),
3223 (N–H), 1684 (C=O), 968 (CH=CH). 1H NMR
(CDCl3), d (ppm): 11.4 (s, 1H, OH), 8.6–8.0 (m, 8H, ArH),
7.5 (s, 1H, N1H), 7.1 (s, 1H, N3H), 6.7 (d, 1H, J = 16.4,
CH=CH), 6.3 (d, 1H, J = 16.5, CH=CH), 5.6 (s, 1H,
methine), 3.2 (s, 6H, N-dimethyl), 2.8 (s, 6H, N-dimethyl).
13C NMR (200 MHz, CDCl3), d: 40.1, 50.0, 106.6, 114.2,
115.2, 124.0, 127.3, 128.0, 131.9, 132.0, 147.9, 148.0,
159.4, 171.0, and 174.9. Mass (m/z): 406 M?. Elemental
analysis: Calculated for (C23H26N4O3): C 67.96, H 6.45, N
13.78; Found: C 68.09, H 6.50, N 13.89.
(E)-4-(4-Methoxyphenyl)-6-(4-methoxystyryl)-2-
thioxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylic acid
(2d)
Yield, 71 %; mp, 122 °C, IR (KBr), t (cm-1): 3426 (O–H),
3210 (N–H), 1686 (C=O), 1285 (C–O–C), 975 (CH=CH). 1H
NMR (CDCl3), d (ppm): 11.98 (s, 1H, OH), 8.5 (s, 1H, N1H),
8.1–7.9 (m, 8H, ArH), 7.5 (d, 1H, J = 16.6, CH=CH), 7.1 (d,
1H, J = 16.5, CH=CH), 6.8 (s, 1H, N3H), 5.5 (s, 1H, methine),
3.4 (s, 3H, methoxy), 3.7 (s, 3H, methoxy). 13C NMR
(200 MHz, CDCl3), d: 54.7, 55.0, 103.1, 114.2, 114.8, 124.5,
127.0, 128.2, 131.1, 135.1, 158.0, 159.0, 171.6, and 174.3.
Mass (m/z): 396 M?. Elemental analysis: Calculated for
(C21H20N2O4S): C 63.62, H 5.08, N 7.07; Found: C 63.43, H
5.10, N 7.09.
(E)-4-(4-Dimethylaminophenyl)-6-(4-
dimethylaminostyryl)-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylic acid (2h)
Yield, 73 %; mp 174 °C; IR (KBr) t (cm-1): 3433 (O–H),
3216 (N–H), 1689 (C=O), 973 (CH= CH). 1H NMR
(CDCl3), d (ppm): 11.32 (s, 1H, OH), 8.3 (s, 1H, N1H), 7.1
(s, 1H N3H), 6.8–6.6 (m, 8H, ArH), 6.2 (d, 1H, J = 16.2,
CH=CH), 5.8 (d, 1H, J = 16.4, CH=CH), 5.4 (s, 1H,
methine), 3.4 (s, 6H, N-dimethyl). 3.1 (s, 6H, N-dimethyl).
13C NMR (200 MHz, CDCl3), d: 40.3, 54.4, 103.9, 114.2,
115.2, 124.0, 127.3, 128.0, 131.9, 132.0, 147.9, 148.0,
159.4, 171.0, and 174.9. Mass (m/z): 422 M?. Elemental
analysis: Calculated for (C23H26N4O2S): C 65.38, H 6.20,
N 13.26; Found: C 65.50, H 6.39, N 13.56.
(E)-4-(4-Hydroxy-phenyl)-6-(4-hydroxystyryl)-2-oxo-
1,2,3,4-tetrahydro pyrimidine-5-carboxylic acid (2e)
Yield, 68 %; mp 224 °C; IR (KBr) t (cm-1): 3419 (O–H),
3215 (N–H), 1689 (C=O), 986 (CH=CH).1H NMR (CDCl3), d
(ppm):11.3 (s, 1H, acidic OH), 10.2 (s, 1H, subs. OH), 10.4 (s,
1H, subs. OH), 8.0 (s, 1H, N1H), (s, 1H, N3H), 7.2–6.7 (m, 8H,
ArH), 6.1 (d, 1H, J = 16.9, CH=CH), 5.7 (d, 1H, J = 16.5,
CH=CH), 5.3 (s, 1H, methine). 13C NMR (200 MHz, CDCl3),
d: 49.0, 106.4, 115.1, 116.1, 127.1, 127.3, 127.4, 128.6, 128.7,
131.6, 135.1, 146.2, 150.4, 156.0, 157.6, and 171.0. Mass (m/
z):352 M?. Elementalanalysis:Calculatedfor(C19H16N2O5):
C 64.77, H 4.58, N 7.95; Found: C 64.89, H 4.60, N 8.12.
(E)-4-(4-Nitrophenyl)-6-(4-nitrostyryl)-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylic acid (2i)
Yield, 72 %; mp 240 °C; IR (KBr) t (cm-1): 3429 (O–H),
3215 (N–H), 1686 (C=O), 986 (CH=CH). 1H NMR
(CDCl3), d (ppm): 11.23 (s, 1H, OH), 8.2 (s, 1H, N1H), 7.7
(s, 1H, N3H), 7.4–6.8 (m, 8H, ArH), 6.4 (d, 1H, J = 16.1,
CH=CH), 5.7 (s, 1H, methine), 5.2 (d, 1H, J = 16.4,
CH=CH). 13C NMR (200 MHz, CDCl3), d: 49.6, 106.7,
120.7, 121.1, 124.1, 127.1, 127.4, 131.0, 141.1, 146.2,
147.3, 149.1, 150.4, and 171.0. Mass (m/z): 410 M?. Ele-
mental analysis: Calculated for (C19H14N4O7): C 55.61, H
3.44, N 13.65; Found: C 56.32, H 3.56, N 13.67.
(E)-4-(4-Hydroxyphenyl)-6-(4-hydroxystyryl)-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid
(2f)
Yield, 64 %; mp 217 °C; IR (KBr) t (cm-1): 3418 (O–H),
3218 (N–H), 1670 (C=O), 984 (CH=CH). 1HNMR
(CDCl3), d (ppm): 11.62 (s, 1H, acidic OH), 10.3 (s, 1H,
subs. OH), 10.6 (s, 1H, subs. OH), 8.2 (s, 1H, N1H),
7.6–7.0 (m, 8H, ArH), 6.7 (s, 1H, N3H), 6.4 (d, 1H,
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Med Chem Res (2013) 22:2493–2504
Docking study
2497
based on XScore of Wang and coworkers (Wang et al.,
2004). The docking is carried with flexible ligand into a rigid
protein-active site. The general procedure for docking pro-
cess starts with the addition of energy-minimized target
ligand on the enzyme. The active site and the ligand were
specified in the program. The different starting parameters
were optimized by using 15 9 15 9 15 box located at the
centre of the target active site using a united atom (explicit
hydrogen are not considered) potential of mean force (PMF)
with a docking algorithm that has a population of 50 chro-
mosomes and runs for 6,000 generations. The process of
docking is repeated until a constant value of docking score is
reached. This takes about 12,000–18,000 generations. The
final results are parameterized in terms of docking score in
kcal/mole, which are depicted in Tables 2 and 3. The
docked ligand–active site complex is interpreted by looking
at the H-bonding or hydrophobic interaction of the ligand
with the amino acid residues in the active site. The same
procedure was followed for docking different substituted
acid derivatives of dihydropyrimidines into the active site of
COX-1 and COX-2 enzymes.
COX-1
The file containing crystal structure of COX-1 complexed
with ibuprofen (PDB entry 1EQG) was downloaded. It was
having the dimeric structure with two chains, each having
551 residues, seven molecules of N-acetyl-D-glucosamine
(NAG), two molecules of B-octylglucoside, one heme
group and a molecule of ibuprofen. The monomer within
˚
the 5 A (Angstrom) radius of chain A was refined and
cleaned by checking the hybridization, valence of ligand,
and introducing the H- atoms to the protein residues.
COX-1 carries a net charge of ?2 and 9,680 atoms.
COX-2
For the COX-2complex, pdb code 6coxwas selected and used
for docking study. The motif of the complex clarifies the three
type of ligands as HEM (protoporphyrin IX Fe[Heme], NAG
and SC 558 (1-phenylsulphonamide-3-trifluoromethyl-5-
bromophenylpyrazole). As SC558 plays the role of inhibitor,
the vicinity where this SC 558 is situated should be an active
site. Therefore, SC558 was taken out from pdb file of this
COX-2 and treated as the so-called model inhibitor for the
docking study. The ligand and water molecules were
removed, and all missing hydrogens were added. The active
In vivo anti-inflammatory activity
The method of Winter et al. (1962) was employed with some
modifications. The protocol of animal experiments has been
approved by the Institutional Animal Ethics Committee
(IAEC). All test samples were administered to animals at a
100 mg/kg dosage, as suspension in DMSO and adminis-
tered orally. After 60 min of drug dose, the injection of
0.1 mL of solution of carrageenan (0.5 mg/25 mL) was
injected into the sub-plantar tissue of the left hind paw of
each rat. Out of this, one group was treated with ibuprofen as
standard (100 mg/kg).The initial volume of paw was mea-
sured within 30 s after carrageenan injection. Later on, paw
volume was measured after 1–5 h. The relative increase in
the paw volume was calculated in the individual animal of
the control, test, and standard groups, respectively. The %
inhibition of oedema was calculated as follows:
˚
site radius was taken to be 8 A from the centroid of the ligand,
so that the amino acid residues (namely ARG120, GLN192,
HIS90, LEU352, LEU359, PHE518, TRP387, SER353,
TYR355, TYR385, ARG513, VAL523 and VAL349) could
be included.
Docking and binding evaluation
In the automated module of Argus Lab ver. 4.0 work sys-
tems (2010) (http://www.arguslab.com), the ligand was
docked into the active site of COX-1 and COX-2 proteins
using Argus dock with a fast, simplified potential of mean
force (PMF). In Argus Lab, ligand’s root node (group of
bonded atoms that do not have rotatable bonds) is placed on
a search point in the binding site, and a set of diverse and
energetically favourable rotations is created. For each
rotation, torsions in breadth-first order are constructed, and
those poses that survive the torsion search are scored. The
N-lowest energy poses are retained, and the final set of poses
undergoes coarse minimization, re-clustering and ranking.
The first step was the construction of ligands in Chemdraw
3D Ultra 8.047 (2008) (www.cambridgesoft.com) followed
by their optimization. The Universal Force Field a molec-
ular mechanics method was used initially for cleaning up
structures sketched in the builder, and for refining initial
geometries. An empirical scoring function was AScore
Anti-inflammatory activity ð% inhibitionÞ
¼ ½1 ꢁ ðDt=DcÞ ꢂ 100ꢃ
where Dt is the mean relative change in a paw volume in
test group, and Dc is mean relative change in paw volume
in control group and is summarized in Table 4.
Enzyme assay and inhibition study
The ability of the test compounds to inhibit ovine COX-1 and
COX-2 (IC50 value, mM) was determined using an enzyme
immunoassay (EIA) kit (catalogue no. 560101, Cayman
Chemical, Ann Arbor, MI, USA) according to the method-
ology described by Jashim Uddin et al. (2004). COX catalyses
123

2498
Med Chem Res (2013) 22:2493–2504
Table 1 List of virtually designed analogues of acid derivatives of pyrimidines
S. no.
Compounds 2a–t
1
(E)-2-oxo-4-phenyl-6-styryl-1,2,3,4-tetrahydro-pyrimidine-5-caboxylic acid 2a
(E)-4-phenyl-6-styryl-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-caboxylic acid 2b
2
3
(E)-4-(4-methoxyphenyl)-6-(4-methoxystyryl)-2-oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylic acid 2c
(E)-4-(4-methoxyphenyl)-6-(4-methoxystyryl)-2-thioxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylic acid 2d
(E)-4-(4-hydroxy-phenyl)-6-(4-hydroxystyryl)-2-oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylic acid 2e
(E)-4-(4-hydroxyphenyl)-6-(4-hydroxystyryl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2f
(E)-4-(4-dimethylaminophenyl)-6-(4-dimethylaminostyryl)-2-oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylic acid 2g
(E)-4-(4-dimethylaminophenyl)-6-(4-dimethylaminostyryl)-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2h
(E)-4-(4-nitrophenyl)-6-(4-nitrostyryl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2i
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
(E)-4-(4-nitrophenyl)-6-(4-nitrostyryl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2j
(E)-6-(2,4,6-trichlorostyryl)-4-(2,4,6-trichlorophenyl-1,2,3,4-tetrahydro-2-oxo-pyrimidine-5-carboxylic acid 2k
(E)-6-(2,4,6-trichlorostyryl)-4-(2,4,6-trichlorophenyl-1,2,3,4-tetrahydro-2-thioxo-pyrimidine-5carboxylicacid 2l
(E)-4-(3-nitrophenyl)-6-(3-nitrostyryl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2m
(E)-4-(3-nitrophenyl)-6-(3-nitrostyryl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2n
(E)-4-(3,4,5 rimethoxyphenyl)-6-(4-3,4,5 trimethoxystyryl)-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2o
(E)-4-(3,4,5 rimethoxyphenyl)-6-(4-3,4,5 trimethoxystyryl)-thiooxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2p
(E)-4-(2-chlorophenyl)-6-(2-chlorostyryl)-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2q
(E)-4-(2-chlorophenyl)-6-(2-chlorostyryl)-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2r
(E)-4-(4-chlorophenyl)-6-(4-chlorostyryl)-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2s
(E)-4-(4-chlorophenyl)-6-(4-chlorostyryl)-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid 2t
the first step in the biosynthesis of arachidonic acid (AA) to
PGH2. PGF2a produced from PGH2 by reduction with stan-
nous chloride is measured by enzyme immunoassay
(ACE_competitive EIA). Stock solution of test compounds
was dissolved in a minimum volume of DMSO. In brief, to a
series of supplied reaction buffer solutions (960 mL, 0.1 M
Tris–HCl pH 8.0 containing 5 mM EDTA and 2 mM phenol)
with either COX-1 orCOX-2 (10 mL) enzyme in the presence
of heme (10 mL) were added 10 mL each of various con-
centrations of test drug solutions (0.01, 0.1, 1, 10, 50 and
100 mM in a final volume of 1 mL). These solutions were
incubated for a period of 5 min at 37 °C after which 10 mL of
AA (100 mM) solution was added, and the COX reaction was
stopped by the addition of 50 mL of 1 M HCl after 2 min.
PGF2a produced from PGH2 by reduction with stannous
chloride was measured by enzyme immunoassay. This assay
was determined spectrophotometrically, and the concentra-
tion of test compound causing 50 % inhibition (IC50, mM)
was calculated from the concentration–inhibition response
curve (duplicate determinations) as presented in Table 5.
studies. Overall, amongst 20 virtual molecules as depicted
in Table 1, 13 molecules displayed the highest negative
binding scores (2a–m), and the other 7 (2n–t) molecules
exhibited negligible results and were thus discarded.
Chemistry
We encountered some challenges in synthesizing the entire
virtually designed library of molecules: of the 13 molecules
that displayed ligand–protein interactions, we succeeded in
synthesizing only 9 (2a–i) molecules.
Shutalev and Aksionov (2005) reported the exciting
results towards the hydrolytic behaviour of Biginelli
compound 1a in alkaline conditions. They have found that
the reaction proceeded via C4-substituted ring cleavage
afforded
4-aryl-6-styryl-1,2,3,4tetrahydropyrimidin-2-
ones. The principal feature of the reactivity of Biginelli
compounds is their behaviour towards hydrolysis. Biginelli
(1893) demonstrated that the boiling of 1a (ethyl 6-methyl
4-phenyl-2-oxo-1,2,3,4
tetrahydropyrimidine-5-carbox-
ylate) in aq. KOH led to extensive decomposition into
benzaldehyde, ammonia and potassium carbonate. Zigeu-
ner (1976) explored that N1 (methyl)-substituted underwent
easy hydrolysis in 5 % alcoholic KOH to form corre-
sponding tetrahydropyrimidine-5-carboxylic acids than the
unsubstituted ones. Kappe (1993) discussed poor reactivity
ester group of Biginelli compounds towards hydrolysis.
However, it was reported (Steel et al., 1998) that
Result and discussion
Virtual designing of library of compounds
A library of 20 molecules was designed. The newly tailored
molecules were considered as standard templates for docking
123

Med Chem Res (2013) 22:2493–2504
2499
N(1)-unsubstituted compounds, when refluxed in methanol
in presence of an aqueous 1 M NaOH solution, were sub-
jected to hydrolysis and decarboxylation to produce the
mixtures of 5-unsubstituted 4-hydroxyhexahydro,1,2,3,4
tetrahydro and 1,2,5,6 tetrahydropyrimidin-2-ones. This
exceptional behaviour of Biginelli compounds towards
hydrolysis prompted us to explore the variance in reactiv-
ity. Our initial efforts were focussed on delineating the
hydrolysis of Biginelli compounds by means of various
metal catalysts and catalytic solvents systems. All permu-
tations generated by varying parameters such as solvents
(methanol, toluene and xylene) and sodium as a metal
catalyst, reaction time for (1–48 h), stoichiometry of both
the reactant and metal catalyst (0.012 mmol to
0.025 mmol), work up at 3.2–7 pH did not lead to higher
yields. Numerous experiments aimed at efficient synthesis
of 2a–i were frustratingly unsuccessful. The yields were
drastically low, and the isolation procedures were tedious
and cumbersome. After extensive experimentation, we
developed a facile synthesis of (E)-2-oxo-4-phenyl-6-
styryl-1,2,3,4-tetrahydro-pyrimidine-5-caboxylic acid 2a as
revealed in Scheme 1. The essential feature of our syn-
thesis targeted the ester group at C5 as well as methyl
group at C6 of Biginelli compound. We succeeded in pre-
dicting the hydrolytic behaviour of Biginelli compounds in
strong alkaline aqueous media. The reaction, after suitable
modification of other parameters, has been fairly well
optimized.
We initiated alkaline hydrolysis of 1a (0.19 mmol,
500 mg) and 25 mL of 5 % NaOH (1.25 g, 31.25 mmol) in
aqueous medium under reflux for 4 h. Interestingly, this led
to formation of 2a with 48 % yields. We were surprised to
note that both alkaline hydrolysis and styryl group forma-
tion progressed at the same time. We proceeded the
hydrolysis with 1a (500 mg, 0.19 mmol) and 25 mL of
10 % NaOH (2.5 g, 62.5 mmol) under reflux resulting to
70 % (0.352 mg) yield. We rationalize the overall trans-
formation in strong alkaline condition proceeded in two
steps. The plausible mechanism proceeds as in step I
hydroxide anion is attracted to electropositive carbonyl
carbon atom and pi bond localized at the oxygen atom of
the carbonyl group with the formation of an intermediate,
elimination of ethoxide ion and deprotonation leading to
the formation of a carboxylic acid functionality. In step II,
there was attachment of second molecule of 1a with the
formation of styryl group. The course of reaction was
followed by TLC and it was revealed that the reaction
mixture analysed at a point of time is a mixture of starting
material and three other compounds. As reaction proceeds
further, ratio of all components of mixture changes, and
most chromatographically, mobile compound accumulates
and in 4 h, this material practically becomes single.
R
R
O
O
Alkaline Hydrolysis
Step I
C2H5O
NH
HO
H3C
NH
2
OH
H3C
N
X
N
X
H
H
Alkaline Condition
Step II
R
O
HO
NH
N
X
H
R
123

2500
Med Chem Res (2013) 22:2493–2504
Computational simulation of anti-inflammatory activity
(2d) to both COX-1 and COX-2 compared with the
hydroxyl derivative (2e) may be explained by considering a
higher number of interacting conformations of the former
with the enzyme.
COX-1 and COX-2 are two identified COX isoenzymes
that share 60 % homology and have the same affinity to
convert arachidonic acid to prostaglandin. While COX-1 is
constitutive and important for maintenance of physiologi-
cal homeostasis, COX-2 is inducible upon inflammation. In
this context, it is, therefore, preferable that any inhibitors
targeted to COX-2 show minimum COX-1 inhibitory
activity. Hence, the compounds showing COX-1 and COX-
2 inhibitions were further evaluated for their inhibitory
activity.
The compounds 2a–i have been categorized on the basis
of structural features into three main segments for better
understanding of binding in the cavity: C5-acidic pyrimidine
ring, C6-styryl phenyl ring and C4-substituted pyrimidine
ring as explicated in Fig. 3. These compounds contain a
central core pyrimidine ring with –COOH group which is
characteristic of vital anti-inflammatory agents. Previous
studies have shown the importance of ionic interactions
between the NSAID COOH group and ARG120 and its
critical role in COX-1 inhibition (Greig et al., 1997; Selinsky
et al., 2001). On the other hand, it is interesting to note that
the COOH group undergoes both electrostatic and hydrogen-
bonding interactions with polar amino acid residues (Moreau
et al., 2006). Two aryl rings at C4 and C6 carbon positions on
opposite faces of nearly planar pyrimidine ring result in
decrease in steric repulsion on opposite faces of pyrimidine
ring providing the free rotation of two aryl rings.
Free binding energies for all COX-compound com-
plexes were obtained. The periphery of the study was
extended from non-selective behaviour of ibuprofen, in-
domethacine and diclofenac to selective behaviour of
celecoxib, rofecoxib and SC558 towards COX-1 and COX-
2 enzyme. Further, all of the complexes analysed showed a
value close to that we obtained of celecoxib (-10.7 kcal/
mol, COX-1 and -11.3 kcal/mol, COX-2). This suggests
that
(E)-2-(thio)-oxo-4-(substituted)
phenyl-6-styryl-
1,2,3,4-tetrahydro-pyrimidine-5-caboxylic acid derivatives
should have a high anti-inflammatory activity and a
reduced toxicity in in vivo models. These binding energies
are presented in Table 2.
All molecules docked freely and showed negative
binding scores. Ligands 2a, 2b, 2c, 2d and 2e show the
highest binding scores with 6cox binding site in compari-
son with the rofecoxib, celecoxib and SC558 as standard
NSAIDs. The inhibitory scores of all ligands with COX-1
(1eqg) has displayed less binding energies compared with
COX-2 enzyme indicating that compounds have more
inhibitory activity for COX-2 than COX-1. These results
also clearly points out that the nature of substituents at C4
significantly affect their binding in COX-1/COX-2 active
sites. The presence of –COOH moiety at C5 position
enhances the binding capacity and selectivity and results in
better discrimination between COX-1 and COX-2 active
site. Further rationalization of modes of 6cox and 1eqg
enzymes had based upon amino acid residues present
around the ligands in the active site pocket of proteins.
The X-ray crystal structures of ovine PGHS-1 and murine
PGHS-2 reveal that the positioning of carboxylate-
A possible relationship between the affinity and struc-
tural features of the ligand may be established. For
example, the higher affinity shown by methoxy derivative
Table 2 Binding energies (kcal/mol) of acidic analogues of pyrimi-
dines 2a–i with COX-1 and COX-2 compared with standard drugs
S. no.
Ligand
COX-1 (kcal/mol)
COX-2 (kcal/mol)
1.
Celecoxib
-10.07
-10.3
-9.6
-9.0
-8.6
-9.9
-10.0
-9.9
-9.0
-8.8
-9.3
-9.5
-9.2
-8.9
-9.0
-8.3
-8.9
-8.4
-8.2
-11.3
-12.4
-10.71
-10.6
-9.5
2.
Rofecoxib
3.
Indomethacin
4.
Diclofenac
5.
SC 558
6.
Ibuprofen
-11.1
-12.32
-12.29
-12.39
-12.33
-12.26
-11.34
-10.99
-10.31
-10.59
-9.3
7.
2a
2b
2c
2d
2e
2f
8.
R
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
C
O
2g
2h
2i
HO
NH
A
N
H
X
B
2j
R
2k
2l
-9.4
-9.2
2m
-9.5
Fig. 3 Structure–activity relationship
123

Med Chem Res (2013) 22:2493–2504
2501
containing inhibitors (S-flurbiprofen, indomethacin) in the
COX active site channel of the PGHS isoforms is such that
the carboxylate group interacts with the guanidinium group
of Arg120 of PGHS-1 (Ouellet and Percival, 1995) and the
analogous Arg120 of PGHS-2 (Picot et al., 1994; Loll et al.,
1995). Other charged residues in the COX active site include
Glu524 in PGHS-1, Glu524 and Arg513 in PGHS-2. The
phenolic side chain of Tyr355 in PGHS-1 and PGHS-2 is
present near the mouth of the channel opposite Arg120. The
biochemical, biophysical and molecular modelling studies
(Loll et al., 1996; Pouplana et al., 1999; Lozano et al., 1997)
indicate that in PGHS-1, Arg120 is a key in the interaction
with arachidonic acid and NSAIDs containing carboxylic
acid groups. Inspection of the crystal structure of the ovine
PGHS-1/S-flurbiprofen complex suggests that Arg120 forms
a salt bridge with Glu524 and that Tyr355, which is in close
proximity to Arg120, might determine the stereochemical
specificity of PGHS-1 towards 2-phenylpropionic acid
inhibitors (Bhattacharyya et al., 1996).
centrally placed characteristic ring A is enveloped into
pocket of GLY526, SER359 and LEU352. Ring B of styryl
substituted C6 carbon adheres at the entry gate of MET522,
PHE518, TRP387. Ring C of substituted phenyl ring is at a
˚
distance of 2.89 A of two NH of ARG120. Table 3 eluci-
date the various amino acids around the different segments
of compounds.
These observations were in close proximity to the inter-
actions of amino acid residues and synthesized inhibitors.
The most prominent binding was observed in ligands 2a,
2b, 2c, 2d and 2e. The residues in terms of protein–ligand
interactions are located in three regions. One region is
located in cavity of TRP387, LEU352 and MET522. Sec-
ond region encompasses the binding site of phenyl ring
which involves the cavity of ALA527, LEU359, VAL349
and GLY526. Finally, the third region is at the mouth of
COX active site and mediates the binding inhibitors with
residues of ARG120 and TYR355. For ligands 2a and 2b,
Fig. 4 Ligand 2d docked in active site of 6 COX enzyme
Table 3 Amino acid residues around all segment of styryl derivatives of pyrimidines (2a–i) docked with 6 COX active site
S.
no.
Ligand Central pyrimidine ring with acidic group Styryl ring
Phenyl ring
1.
2.
3.
4.
5.
6.
7.
8.
9.
2a
2b
2c
2d
2e
2f
SER353, VAL349, VAL523, LEU352,
GLY526
MET522, TRP387, PHE518, TYR385 ALA527, ARG120, LEU359, SER530
SER359, VAL352, TYR355, GLY526
VAL523, TYR355, PHE518, GLN192
TYR355, VAL523, SER353, ARG120
VAL523, GLY526, TYR353, MET522
TYR355, ARG120, LEU359, ALA526
ARG120, VAL116, LEU531, LEU359 LEU352, VAL523, MET522, PHE518,
TRP387
LEU352, MET522, GLY526, TRP387, ARG120, ALA527, TYR355, LEU359
TYR385
TRP387, LEU352, MET522
ALA527, LEU359, VAL349, GLY526,
LEU531
LEU359, LEU531, SER530, ARG120 LEU352, MET522, SER353, VAL523,
VAL349,
HIS90, ARG513, ALA516, GLN192,
PHE518, MET522,
GLY526, VAL349, SER530, LEU359
2g
2h
2i
GLN192, PHE518, MET522, ALA516, GLY526, TYR355, LEU352
VAL523, GLY526
SER530, LEU531, ALA349, VAL116,
ARG120, TYR355
TRP387, LEU352, TYR355, ARG120
VAL349, LEU359, VAL116, LEU531, HIS90, ALA16, ARG513, PHE518
SER530 VAL523, TYR355, GLN192
SER530, GLN192, VAL349, LEU531
ALA527, VAL523, ARG120, ARG513, VAL349, LEU359, VAL116, TYR355
GLY526
123

2502
Med Chem Res (2013) 22:2493–2504
Ligands 2c and 2d as revealed in Fig. 4 also show
Table 5 In vitro COX-1 and COX-2 enzyme inhibition assay data for
compounds 2c and 2d
prominent binding activities. Both ligands exhibit methoxy
group (OCH3) at C4 ring. Centrally placed C5 substituted
ring A is stabilized by hydrophobic interactions amongst
MET522, TYR385 and TRP387 in such a way that –COOH
group is oriented in secondary pocket of amino acids. The
methoxy group is sterically fitted into pocket of ARG120,
ALA527, TYR355 and LEU359. Ligand 2e shows signif-
icant docking inhibition. C5 ring is surrounded by
VAL523, GLY526, TYR353, while C6 ring is engrossed
into LEU359, SER530 and LEU531. Ring C of C4 carbon
is enveloped into MET522, SER353 and VAL523.
Compound COX-1a (IC50 lM) COX-2 (IC50 lM) COX-2 SIb
2c
14.7
14.3
23
0.048
0.041
0.057
306
348
403
2d
Celecoxib
a
Values are means of two determinations acquired using an ovine
COX-1/COX-2 assay kit
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50
)
306 and 348 for COX-2 and COX-1, respectively. These
compounds 2c and 2d have IC50 values (COX-2) of
0.048 lM and 0.041 lM and IC50 values (COX-1) of 14.7
and 14.3 lM, respectively, suggesting that methoxy group
helps in orienting the compound.
In vivo anti-inflammatory activity
Compounds 2a–i were evaluated for their in vivo anti-
inflammatory screening by carrageenan-induced paw
oedema method with reference to ibuprofen as a standard
drug. The results obtained led us to the conclusion that
methoxy group attached to para position of both aryl rings
enhanced the anti-inflammatory activity (compounds 2c
and 2d). The data showed considerable anti-inflammatory
action. 2a and 2e displayed reduced activity explained in
Table 4. Rest of the compounds were found to be inactive.
Structure–activity relationship
Structure–activity relationship is the relationship between
the chemical and three-dimensional structures of a mole-
cule and its biological activity. The analysis of SAR
enables the determination of the chemical groups respon-
sible for evoking a target biological effect in the organism.
This allows modification of the effect or the potency of a
bioactive compound (typically a drug) by changing its
chemical structure. Medicinal chemists insert new chemi-
cal groups into the biomedical compound and test the
modifications for their biological effects. Our hybrid mol-
ecule may be considered as a template scaffold in which
one can insert substituent at different positions to enhance
the specificity towards microorganisms as pharmacological
activities.
In vitro COX inhibition assay
Compound 2d displayed[55 %, while 2c displayed &41 %
of inhibition in oedema compared with standard drug; these
were further evaluated for their ability to inhibit COX
according to the method described by Jashim Uddin et al.
In vitro COX-1 and COX-2 enzyme inhibition data showed
that the compounds were more selective towards COX-2
than COX-1 as presented in Table 5. Compound 2c and 2d
with 4-methoxy substitution showed selectivity indexes of
The computational simulation exhibited very good
inhibitory binding energies for 2a, 2b, 2c, 2d and 2e
compared with most potent standard drug i.e. celecoxib.
The molecules 2c, 2d, 2e and 2f were established as target
hits because of the presence of electron-donating groups.
Both hydroxyl and methoxy moieties donate the electrons
which affects the electronics of pi system of aromatic ring.
On the other hand, –COOH group as a strong electron-
withdrawing set destabilizes the pyrimidine moiety. Alto-
gether, the effects stabilize the ring, and strain is negligible.
The compounds 2g, 2h and 2i possess electron-withdraw-
ing groups and thus altogether destabilizes the ring. In vivo
analysis of compounds as anti-inflammatory agents dis-
played that 2d and 2c with methoxy (an electron-donating
group) substituent showed appreciable % of inhibition.
This implies the possibility that other substituted groups
are responsible for strain during the inhibitor and ligand
interaction thus leading towards lesser binding energies
and no significant % inhibition.
Table 4 Anti-inflammatory effect of acid derivatives against carra-
geenan-induced rat paw oedema model in rats
Compound
R
X
% Inhibition
16
NIa
2a
H
O
S
2b
H
2c
OCH3
OCH3
OH
O
S
23
2d
38
2e
O
S
12
2f
OH
NIa
NIa
NIa
NIa
56
2g
N(CH3)2
N(CH3)2
NO2
O
S
2h
2i
O
Ibuprofen
a
No inhibition
123

Med Chem Res (2013) 22:2493–2504
2503
rational drug design approach. In vivo anti-inflammatory
screening of compounds revealed 2c and 2d to exhibit
potent inhibitory activity. In vitro enzyme inhibition assays
with selective indexes of 306 and 348, respectively, for 2c
and 2d were promising.
Acknowledgments RPD is grateful to the UGC, New Delhi for
financial assistance under Faculty Improvement Program of XI plan.
The authors are thankful to the SAIF, Chandigarh for 1HNMR,
13CNMR, CHN, Mass spectral analysis and also thankful to the
Department of Pharmacy, Nagpur for IR spectral analysis. The
authors also thank the Director, Institute of Science, Nagpur for
providing lab facilities, and the CDRI Lucknow for in vivo anti-
inflammatory activity.
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