Identification of substituted [3, 2-a] pyrimidines as selective antiviral agents: Molecular modeling study

Article abstract of DOI:10.1016/j.antiviral.2012.05.010

A series of novel substituted dihydropyrimidine and 5H-thiazolo [3, 2-a] pyrimidine derivatives were designed and synthesized as a potential target to discover drugs fighting against the viral diseases. The main objective of the present work is to carry out the QSAR studies for all the series of the compounds starting from 4a to 6j to find out their molecular descriptors and predict the biological properties. All of them are showing the best QSAR descriptors, hence chosen for the prediction of anti-viral activity against Newcastle disease virus (NDV). Initially their inhibitory activity was predicted by molecular docking of these compounds against haemaglutinin-neuraminidase (HN) protein using molecular operating environment (MOE) software. Based on the best affinity and highest docking scores 4b, 5b and 6b were assayed in vivo on NDV infected chicks and it was found that there is significant improvement in the survival of the chicks with the treatment (P<0.05). 4b and 6b showed better curative effect than 5b at the dose concentration of 40. mg/kg body weight of chicks. The results from molecular docking study and biological assays can be inferred to consider these molecules as potential antiviral drugs.

Full text of DOI:10.1016/j.antiviral.2012.05.010

Antiviral Research 95 (2012) 118–127
Contents lists available at SciVerse ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
Identification of substituted [3, 2-a] pyrimidines as selective antiviral agents:
Molecular modeling study
Kilaru Ravendra Babu a,f, Valasani Koteswara Rao a,e, Yellapu Nanda Kumar b, Kishore Polireddy c,
Kadiam Venkata Subbaiah d, Matcha Bhaskar b, Valluru Lokanatha d, Chamarthi Naga Raju a,
⇑
a Department of Chemistry, Sri Venkateswara University, Tirupati 517502, India
b Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati 517502, India
c Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA
d Department of Biotechnology, Dravidian University, Kuppam 517 425, India
e Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66047, USA
f Mylan Laboratories Ltd., CRD, Anrich Industrial estate, Bollaram, Jinnaram, Medak 502325, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel substituted dihydropyrimidine and 5H-thiazolo [3, 2-a] pyrimidine derivatives were
designed and synthesized as a potential target to discover drugs fighting against the viral diseases. The
main objective of the present work is to carry out the QSAR studies for all the series of the compounds
starting from 4a to 6j to find out their molecular descriptors and predict the biological properties. All
of them are showing the best QSAR descriptors, hence chosen for the prediction of anti-viral activity
against Newcastle disease virus (NDV). Initially their inhibitory activity was predicted by molecular dock-
ing of these compounds against haemaglutinin–neuraminidase (HN) protein using molecular operating
environment (MOE) software. Based on the best affinity and highest docking scores 4b, 5b and 6b were
assayed in vivo on NDV infected chicks and it was found that there is significant improvement in the sur-
vival of the chicks with the treatment (P < 0.05). 4b and 6b showed better curative effect than 5b at the
dose concentration of 40 mg/kg body weight of chicks. The results from molecular docking study and bio-
logical assays can be inferred to consider these molecules as potential antiviral drugs.
Received 29 January 2012
Revised 20 May 2012
Accepted 22 May 2012
Available online 29 May 2012
Keywords:
Antiviral inhibitors
Newcastle disease virus
Dihydropyrimidine
Haemaglutinin–neuraminidase
Molecular modeling
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
trum of biological activities like antiviral, anticancer (Focher et al.,
2000; Sherman and Kaplan, 1975), calcium channel blockers
Viruses are the small infectious nucleoprotein particles that can
replicate and survive only inside the living cells of organisms and
can spread many ways. They can infect all types of organisms
including humans and are responsible for a wide range of diseases
(Breitbart and Rohwer, 2005). Although a number of anti-viral
drugs are available, still virus infections remain the major problem
to the world due to increasing resistance. Multiple factors such as
mutations in the genome, adaptation to new environments, etc.,
are responsible for the resistance of viruses (Edwards and Rohwer,
2005). Considerable efforts should be devoted to new anti-viral
drug discovery for the purpose of improved therapeutic option.
Development of new agents with novel structure and mode of ac-
tion has been particularly hastened providing for emergence of
mutant viral antigens and drug resistance. In this context, we have
reported a series of dihydropyrimidinone (DHPMs) derivatives
which can become promising candidates for anti-viral agents hav-
ing unique chemical structure. The DHPMs show such broad spec-
(Eisenberg et al., 2004), antihypertensive agents (Cho et al.,
1989), -1a-antagonists (Bossert and Vater, 1989) and neuropep-
tide Y (NPY) antagonists (Kappe, 1998, 2000). This activity is due
a
to the presence of dihydropyrimidine core unit.
Herein, we report a simple protocol for the parallel synthesis of
DHPMs using appropriate acid catalyst and also the above-cited
applications prompted us to synthesize a series of novel pyrimi-
dine derivatives through the reaction of 3,4-dihydropyrimidin-
2(1H)-ones (4) with 1-bromo-4-phenyl butan-2-ones (5) to form
the title compounds 5H-thiazolo [3, 2-a] pyrimidine derivatives
and evaluation of anti-viral activity of the title compounds. The
structures of the newly synthesized compounds (4a–j), (5a–j)
and (6a–j) have been established by the elemental analysis and
spectral data (IR, 1H, 13C NMR and mass) (Scheme 1).
A QSAR study was carried out for all the series of molecules (4a–
6j) which can help in early preclinical development and avoid
costly late-stage preclinical and clinical failures (Santos et al.,
2009; Walters et al., 1999). Three dimensional models can be con-
structed to analyze the structure based interaction with other bio-
logical macromolecules like proteins. The stable confirmation of
⇑
Corresponding author.
E-mail address: rajuchamarthi10@gmail.com (C. Naga Raju).
0166-3542/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.antiviral.2012.05.010

K. Ravendra Babu et al. / Antiviral Research 95 (2012) 118–127
119
R
R
S
CHO
H+
PPA
O
O
O
O
(5),
THF
Ethyl Acetate
NH2
H N
+
+
2
EtO
NH
EtO
R
N
O
N
H
S
O
(3)
N
H
(1a-j)
S
(2)
(4a-l)
Ethanol
4
8
%
A
q
u
e
o
u
s
H
B
r
,
(5a-j)
O
Br
(5)
R
O
EtO
N
S
N
H
(6a-j)
Scheme 1. Synthetic route for the designed target molecules.
the structures can be obtained by molecular dynamics simulations
which is an important task to know the behavior of molecule in the
system whether in isolated or solvated environment (Kokhan and
Shinkarev, 2011).
values of hemagglutination and the activity of antioxidant metab-
olism related enzymes SOD and CAT and lipid peroxidation levels
in the liver tissue of NDV infected chicks. The influence of the ini-
tial three new drug molecules on the pathogenicity of NDV was
studied.
Three representative molecules were tested for their inhibitory
activity against Newcastle disease virus (NDV) in chicks which is
highly contagious viral disease affecting wild and domestic avian
species (Seal et al., 2000). The nucleoprotein (NP), the phosphopro-
tein (P) and the large polymerase protein (P) form the nucleocap-
sid, the haemaglutinin–neuraminidase (HN) and fusion (F)
proteins constitute the external envelope, and the matrix protein
(M) forms the inner layer of the virion. The pathogenicity of NDV
is mainly due to the presence of multiple basic amino acids at
the cleavage site (Collins et al., 1993). Initially a molecular docking
study was carried out on the HN protein of NDV which is the key
pathogenic factor (Huang et al., 2004; Ravindra et al., 2008). This
study can help to predict the molecular interaction of the com-
pounds with the HN protein, so that their inhibitory activity on
NDV could be determined.
2. Materials and methods
2.1. Chemistry
2.1.1. Synthesis of ethyl 6-methyl-4-(3-nitrophenyl)-2-thioxo-1, 2, 3,
4-tetrahydro- pyrimidine-5-carboxylate (4b)
A solution of ethyl acetoacetate (510 mg, 3.9 mmol), 3-nitro
benzaldehyde (500 mg, 3.3 mmol) and thiourea (300 mg,
3.9 mmol) in ethanol (5 mL) was heated under reflux (78–80 °C)
in the presence of poly phosphoric acid (1110 mg, 3.3 mol%) for
12 h under nitrogen atmosphere. The progress of the reaction
was monitored by TLC (hexane: ethyl acetate, 1:1 v/v). The reaction
mixture, after being concentrated under vacuum at 50 °C, cooled to
room temperature, it was poured into crushed ice (10 g) and stir-
red for 5–10 min. The solid separated was filtered under suction,
washed with ice-cold water (20 mL) and then recrystallized from
hot ethanol to afford pure product (900 mg, 90%), mp 206–207 °C
(lit. mp 206–207 °C). Rf 0.37; (hexane: ethyl acetate, 1:1 v/v). Yield:
82%; IR: 3179, 2988, 2676, 1715, 1660, 1595, 1532, 1475, 1376,
1344, 1325, 1275, 1190, 1130, 1102, 1038, 1015, 906, 889, 871,
To determine the real efficacy of the compounds, an in vivo
study was conducted for the three compounds (4b, 5b and 6b) that
have shown the best less docking score in chick models which are
infected with NDV. In general the entry of virus into the host sys-
tem results in the activation of phagocytes, it may also release pro-
oxidant cytokines such as tumor necrosis factor alpha (TNF-a),
interleukin I, soluble neurotoxins, excitatory neurotransmitters
and reactive oxygen species (ROS) and reactive nitrogen species
(RNS) (Peterhans, 1997). Production of ROS which are free radical
derivatives of molecular oxygen and hydrogen peroxide are in-
volved in the damage of various components such as lipids, pro-
teins, carbohydrates, nucleic acids leading to oxidative stress
often accompanied by loss of cell function, apoptosis and/or necro-
sis (Nordberg and Arner, 2001). The ROS are primarily generated by
mitochondria, which possess an antioxidant system capable of
scavenging by natural cellular defense mechanisms. The antioxi-
dant system comprises of enzymes such as superoxide dismutase
(SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione
peroxidase (GPx) and glutathione molecule under normal condi-
tions (Yu, 1994). ROS have been suggested to be a mediator of cell
death induced by a variety of triggers, including viral infection
(Schwarz, 1996). In the present study aimed at the mean titer
808, 786, 737, 689, 650, 508 cmÀ1 1H NMR (DMSO–d6) d 1.12 (t,
;
3H), 2.36 (s, 3H), 3.98–4.14 (m, 2H), 5.40 (d, J = 7.4 1H), 7.69–
7.76 (m, 2H), 8.13–8.21(m, 2H), 9.83 (s, 1H), 10.5 (s, 1H); 13C
NMR (DMSO–d6) d 13.87, 17.18, 53.4, 59.7, 99.8, 121.1, 122.6,
130.3, 132.9, 145.4, 145.9, 147.7, 164.8, 174.5; LCMS m/z (%):
322 [MH+ 100%]; Anal. Calcd. C14H15N3O4S: C, 52.33; H, 4.70; N,
13.08; Found: C, 52.26; H, 4.67; N, 13.04.
2.1.2. Synthesis of ethyl 6-methyl-4-(3-nitrophenyl)-3-(2-oxo-4-
phenylbutyl)-2-thioxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate
(5b)
Ethyl 6-methyl-4-(3-nitrophenyl)-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine-5-carboxylate (500 mg, 1.5 mmol), ethyl acetate
(2.5 mL), and tetrahydrofuran (2.5 mL)were taken into three

120
K. Ravendra Babu et al. / Antiviral Research 95 (2012) 118–127
necked round bottom flask and cooled to 0–5 °C, then 1-bromo-4-
phenylbutan-2-one (370 mg, 1.6 mmol) was added, and the reac-
tion mixture was stirred for 20 h. Progress of the reaction was
monitored by TLC (hexane: ethylacetate1:1), and the white solid
formed was filtered off and washed with chilled ethyl acetate, solid
was dried under vacuum at 70 °C to get the pure product (600 mg,
83%). Rf 0.42; (hexanes: ethyl acetate, 1:1 v/v). Yield: 78%;
m.p.196–198 °C; IR: 3235, 3108, 2796, 1715, 1668, 1569, 1527,
1348, 1322, 1301, 1278, 1254, 1195, 1146, 1092, 1055, 1030,
conformation. The active site was identified from PDBSum and also
correlated with ‘Site Finder’ module of MOE to define the docking
site for the ligands. A ligand data base has been developed for all
the compounds to proceed for docking. Docking procedure was fol-
lowed using the standard protocol implemented in MOE 2008.10.
After the successful docking process, the best energy conforma-
tions of receptor–ligand complexes were studied and evaluated
to infer the affinity levels of all the ligands.
963, 873, 740, 699, 639, 609, 588, 520 cmÀ1 1H NMR (DMSO–d6)
;
2.3. Pharmacology
d 1.08 (t, 3H), 2.23 (s, 3H), 2.26–2.77 (m, 4H), 3.34–3.56 (m, 2H),
3.92–4.10 (m, 3H), 6.87–6.90 (m, 1H), 7.04–7.38 (m, 5H), 7.59–
7.85 (m, 2H), 8.08–8.15 (m, 2H); 13C NMR (DMSO–d6) d 14.2,
17.3, 28.9, 36.7, 37.9, 54.5, 60.7, 66.9, 99.5, 100.5, 122.2, 123.4,
126.5, 128.3, 130.7, 133.8, 140.0, 142.8, 143.6, 147.4, 163.4,
166.6. LCMS m/z (%): 468 [MH+ 100%]; Anal. Calcd. C24H25N3O5S:
C, 61.65; H, 5.39; N, 8.99; Found: C, 61.59; H, 5.34; N, 8.95.
2.3.1. Procurement and maintenance of experimental animals
Day old male layer specific pathogen free (SPF) chicks (35 5 g)
were selected as experimental animals. The chicks were purchased
from Balaji Hatcheries Pvt. Ltd., Chittoor, AP. Animals were held in
separate isolators with free access to food and water in an air-con-
ditioned environment (25 2 °C) with a 12 h light and 12 h dark
cycle. The experiments were carried out in accordance with the
guidelines of the Committee for the Purpose of Control and Super-
vision on Experiments on Animals, Government of India (CPCSEA,
2003) and approved by the Animal Ethical Committee at Sri Venk-
ateswara University, Tirupati, India (vide No. 04/a/CPCSEA/IAEC/
08–09/ZOOL/WR/dated 01.09.2009) (Schwarz, 1996).
2.1.3. Synthesis of ethyl 7-methyl-5-(3-nitrophenyl)-2-phenethyl-5H-
thiazolo [3, 2-a] pyrimidine-6-carboxylate (6b)
Ethyl 6-methyl-4-(3-nitrophenyl)-3-(2-oxo-4-phenylbutyl)-2-
thioxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate 5b (500
mg,1.5 mmol), ethyl acetate (2.5 mL), 1-bromo-4-phenylbutan-2-
one (370 mg, 1.6 mmol), 48% aqueous hydrobromic acid (155 mg,
1.5 mmol) and ethanol (5 mL) were taken into three necked round
bottom flask and refluxed with stirring for 3 h, the progress of the
reaction was monitored by TLC, solvent was removed under vac-
uum at 50 °C, solid obtained was taken into distilled water
(10 mL) extracted with dichloro methane (20 mL), solvent was re-
moved under vacuum at 50 °C, solid was recrystallized from etha-
nol to get white solid (600 mg, 85%). m.p. 182–184 °C. Rf 0.5;
(hexane: ethyl acetate, 1:1 v/v). Yield: 71%; m.p.182–184 °C; IR:
3432, 2975, 2939, 2879, 2802, 2739, 2677, 2602, 2529, 2491,
2238, 2134, 1902, 1687, 1596, 1531, 1476, 1434, 1397, 1384,
1289, 1272, 1256, 1186, 1171, 1070, 1036, 904, 849, 805,
2.3.2. Virus
The mesogenic strain of Newcastle disease virus (NDV) was ob-
tained from Department of Microbiology, Sri Venkateswara Veter-
inary University, Tirupati; A.P. Virus titers of the NDV were
determined by inoculating chick embryonating eggs and calculated
median egg infectious (EID50) by the method of Armitage (Armit-
age and Allen, 1950) EID50 was found to be 10À2 (Ohkawa et al.,
1979). This virus stock was stored at À40 °C until use.
2.3.3. Haemagglutination test
Serial twofold dilutions of each viral preparation were made in
759 cmÀ1 1H NMR (DMSO–d6) d 1.04 (t, 3H), 2.21 (s, 3H), 2.42–
;
0.01 M PBS, using 50
lL volumes in V-shaped 96-well micro titer
2.64 (m, 3H), 2.89–2.98 (m, 1H), 3.85–3.99 (m, 2H), 6.46 (s, 1H),
6.85–7.09 (m, 6H), 7.45–7.66 (m, 2H), 7.99–8.14 (m, 2H); 13C
NMR (DMSO–d6) d 13.8, 15.1, 23.1, 27.2, 32.5, 57.6, 60.7, 61.2,
101.1, 109.1, 122.1, 124.1, 126.2, 128.3, 131.6, 139.3, 140.6,
141.5, 143.3, 147.7, 161.1, 163.6. LCMS m/z (%): 450 [MH+ 92%];
Anal. Calcd.C24H23ClN2O2S: C, 65.67; H, 5.28; N, 6.38; Found: C,
65.62; H, 5.23; N, 6.34.
plates. 50 L of 0.5% suspension of chick red blood cells (RBCs)
l
were added to each well of the plates. The contents in plates were
mixed with a mechanical vibrator. The plates were incubated at
room temperature (22–25 oC) until the cell control showed com-
plete setting of RBCs. The haemagglutination titer (HA titer) was
the reciprocal of the dilution of virus in the last well with complete
haemagglutination.
The same experimental procedure was adopted for the prepara-
tion of the remaining title compounds (6a, 6c–j).
2.3.4. Assay of antioxidants and antioxidant enzymes
Selected biochemical constituents and enzymes representing
antioxidant metabolism were estimated in selected tissues of con-
trol and experimental chicks. MDA levels were estimated accord-
ing to the method of Hiroshi et al. (Misra and Fridovich, 1972;
Ohkawa et al., 1979). The activity of superoxide dismutase (SOD)
was assayed by the method of (Misra and Fridovich, 1972; Ohkawa
et al., 1979) catalase (CAT) activity was estimated by the method of
Kar (Kar and Mishra, 1976).
2.2. Molecular docking of HN protein
The ligand study was carried out by HyperChem software which
is a sophisticated molecular modeling environment that uniting
with quantum chemical calculations, molecular mechanics, and
dynamics (Dastmalchi et al., 2008). Three-dimensional structures
were constructed and optimized for all the molecules. QSAR
descriptors were studied followed by Lipinski rule filtration.
Molecular dynamics simulations were carried out by solvating
the structures with water molecules in AMBER force field and
the stabilized confirmations were saved (Sippl, 2002).
Docking studies have been performed using MOE 2008.10. The
crystal structure of HN protein (PDB ID: 1USX) was retrieved from
Protein Data Bank (http://www.rcsb.org/pdb/home/home.do)
(Kumar et al., 2005). In order to prepare the protein for docking
studies, it is loaded into MOE and all water molecules and hetero
atoms were removed. As the protein is a homo trimer consisting
of A, B and C chains, chain A was considered for docking process.
The structure is protonated, polar hydrogens were added and
energy minimization was carried out to get the stabilized
2.3.5. Histopathological examination
At the time of slaughtering, liver tissue was isolated and imme-
diately fixed in 10% buffered formalin. Fixed tissues were pro-
cessed by dehydration and paraffin embedding. Five micrometer
sections were stained with hematoxylin and eosin (H&E) and his-
tological changes were observed under the light microscope.
2.3.6. Protein estimation
Protein content was determined following the method of Lowry
(Morton and Evans, 1992; Ohkawa et al., 1979) using bovine serum
albumin as a reference standard.

K. Ravendra Babu et al. / Antiviral Research 95 (2012) 118–127
121
Table 1
Representative compounds.
Entry
R
Time (min)/Yield (%)a
M.P. (°C)b
Found
Reported
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
17/95
17/95
17/95
15/92
16/82
20/90
18/87
18/87
18/87
18/87
204–205
206–207
133–134
128–130
197–199
183–184
164–165
172–174
185–187
239–240
205–206 (Walters et al., 1999)
206–207 (Santos et al., 2009)
-
150–152 (Kokhan and Shinkarev, 2011)
196–198 (Seal et al., 2000)
184 (Collins et al., 1993)
165–166 (Huang et al., 2004)
172–174 (Ravindra et al., 2008)
184–186 (Peterhans, 1997)
240–242 (Santos et al., 2009)
3-No2
2-No2
3-OMe
3-Cl
4-F
3,4-OMe
4-Me
3-OH
4-OMe
4j
a
Isolated yields.
b
All the melting points were matched with the reported data.
541.54 D and 4a has the lowest molecular weight of 276.35 Da. All
the compounds are showing hydrogen bond donors and hydrogen
bond acceptors less than 5 and 10 respectively and finally the log P
values of these compounds are superior to act as drug which is less
than 5. Molar refractivity is also in the range of 40–130. QSAR
study is a powerful lead optimization tool that can quantitatively
relate variations in biological activity to changes in molecular
properties. The molecular descriptors of the present molecules
are in optimal ranges and log P value that indicates the absorption
and solubility were not exceeded the limited value of 5.
3. Results and discussion
3.1. Chemistry
3-Nitro benzaldehyde 1b, ethyl acetoacetate 2 and thiourea 3
were taken in ethanol and heated under reflux in the presence of
poly phosphoric acid (3.3 mol%) for 12 h to yield dihydropyrimi-
dine derivative 4b. The compound 4b was taken in an equal molar
ratio of ethylacetate in THF and cooled to 0–5 °C, then 1-bromo-4-
phenylbutan-2-one 5, was added and the reaction mixture was
stirred for 20 h to obtain the title compound 5b, where as the com-
pound 4b when treated with 1-bromo-4-phenylbutan-2-one in
presence of aqueous HBr (48%) in ethanol under reflux for 3 h to
obtain the title compound 6b (Scheme 1, Tables 1 and 2).
3.3. Molecular docking of HN protein
The crystal structure of HN protein was loaded into MOE
(Molecular Operating Environment (MOE, version 2002.03)
(2002) with a resolution of 2.70 Å and a library was constructed
for all the lead molecules (Davies et al., 2011). The binding site of
2-deoxy-2,3-dehydro-n-acetyl-neuraminic acid (DAN) on HN pro-
tein was identified from PDBSum (Laskowski, 2001) and the resi-
dues Arg 174, Glu 258, Tyr 299, Tyr 317, Arg 363, Glu 401, Arg
416, Val 466, Arg 498 and Tyr 526 were found to be interacting
with DAN. The site finder module of the MOE was used to identify
the additional ligand binding sites within the protein structure.
Hydrophobic and hydrophilic spheres are used to identify the
interactive positions which will be potential ligand binding sites
in the each possible position. Among the group of binding sites
generated by site finder module, the centroid sphere of site 4
was selected which also includes the residues of DAN binding site.
All the dummy atoms were matched for centroid sphere of site 4
during the characterization of ligand binding site.
The chemical structures of all the title compounds 4a–j, 5a–j
and 6a–j were characterized by IR, 1H, 13C NMR and APCI–MS stud-
ies and their data are presented in the experimental section. Char-
acteristic IR stretching absorptions were observed in the regions
3144–3275 cmÀ1 and 3568–3365 cmÀ1 for N–H (Kumar et al.,
2006), O–H (Khandazhinskaya et al., 2002) respectively. The ester
carbonyl stretching frequency was observed in the range of
1715–1745 cmÀ1 (Rao et al., 2011). In the 1H NMR spectra of title
compounds 4d, 4e, 5d and 5e, the chemical shifts of aromatic
hydrogens of the phenyl ring appeared as multiplets in the region
d 7.54–7.88 (Han et al., 1992; Rao et al., 2010, 2011). The N–H pro-
ton resonated as a broad singlet in the region d 9.25–10.52 (Rajan-
arendar et al., 2010).The OH protons were observed as singlets in
the region 8.52–11.34 ppm. 13C NMR chemical shifts for title com-
pounds were observed in their expected regions.
3.2. QSAR study
Docking simulations were performed with MOE-dock system
using the ligand data base generated for all the series of leads. A to-
tal of 30 most favorable binding sites and orientations were gener-
ated for each lead molecule. The affinity scoring function dG was
used to assess and rank the receptor–ligand complexes. The ligand
poses were also scored within the centroid sphere of binding pock-
et. The docking scores of all the lead molecules and their hydrogen
bonding strength were tabulated and shown in Table 4.
Three-dimensional structures were built for all the compounds
ranging from 4a to 6j and optimized by using Hyper Chem soft-
ware. Molecular dynamics simulations were carried out and
molecular descriptors were determined by QSAR study and Lipin-
ski rule and the results showing that all molecules have drug like
properties (Table 3). All the compounds have the molecular weight
less than 500 Da except 6c which is showing a molecular weight of
Among all docking conformations, 5b is showing best least
docking score of À20.471 and the next best least docking score is
found with 4b followed by 6b. 5b is found to be forming two
hydrogen bonds of lengths 2.6 and 2.7 Å each with Asp278 and
Gln280 respectively. The –OH group of the ligand is interacting
with the –COOH group of Gln280 and amino nitrogen of heterocy-
clic ring of ligand is interacting with –COOH group of the Asp278
(Fig. 2). 4b is forming only a single hydrogen bond of 2.7 Å using
double bonded oxygen atom with –OH group of hydroxy benzene
ring of Tyr262. This ligand is also making a solvent contact with
Table 2
Representative compounds.
Entry
R
Entry
R
5a and 6a
5b and 6b
5c and 6c
5d and 6d
5e and 6e
H
5f and 6f
5g and 6g
5h and 6h
5i and 6i
5j and 6j
4-F
3,4-OMe
4-Me
3-OH
4-OMe
3-No2
2-No2
3-OMe
3-Cl

Table 3
Molecular descriptors of substituted thiazolo [3, 2-a] pyrimidine derivatives from QSAR study.
Ligand Molecular weight Hydrogen bond
Hydrogen bond
acceptors
Log
P
Molar refractivity
(Ao3
Surface area
(Ao2
Volume
(Ao3
Hydration energy
(K.cal/mol)
Polarizability
(Ao3
Gradient energy (K.cal/
molAo)
Total energy
(K.cal/mol)
(Da)
donors
)
)
)
)
4a
4b
4c
4d
4e
4f
4 g
4 h
4i
276.35
321.35
321.35
306.38
287.36
294.34
336.41
290.38
292.35
306.38
422.54
467.54
467.54
452.57
433.54
440.53
482.59
436.57
438.54
452.57
406.54
451.54
541.54
436.57
417.55
424.53
466.59
420.57
422.54
436.57
2
2
2
2
2
2
2
2
3
2
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
2
1
4
6
6
5
4
5
6
4
5
5
5
7
7
6
5
6
7
5
6
6
4
6
6
5
4
5
6
4
5
5
1.73 43.64
453.14
481.02
442.48
521.30
370.48
427.73
509.33
436.40
402.84
502.75
618.60
597.82
647.96
640.44
489.42
612.29
643.33
553.88
558.24
675.02
570.31
631.07
464.77
613.42
567.85
623.86
691.73
640.25
559.01
657.39
837.28
887.67
868.73
887.45
832.60
825.48
957.20
839.09
À8.51
À12.77
À10.06
À8.60
À8.01
À7.74
À10.18
À7.66
À13.55
À9.41
À6.91
À13.66
À10.98
À8.82
À8.11
À7.02
À9.96
À7.39
À11.58
À8.54
À5.47
À11.54
À5.79
À6.07
À4.86
À5.38
À7.85
À4.62
À10.72
À6.85
30.82
32.53
49.64
33.29
30.43
30.73
35.76
32.65
31.46
33.29
47.91
49.62
49.62
50.38
47.52
47.81
52.85
49.74
48.54
50.38
46.61
48.32
48.32
49.08
46.22
46.52
51.55
48.44
47.24
49.08
0.097
0.096
0.096
63.964
66.525
67.727
72.303
60.2325
59.298
78.592
1.7
2
49.64
42.53
1.70 50.06
1.98 42.75
2.02 43.55
3.65 56.49
2.12 48.25
1.75 45.03
1.07 50.06
3.64 63.72
2.99 69.72
3.82 69.72
3.82 70.14
3.98 62.83
4.14 63.63
4.83 76.57
3.88 68.33
2.69 65.11
4.17 70.14
4.86 59.65
4.84 65.64
4.46 65.64
4.84 66.07
4.82 58.75
4.16 59.55
4.62 72.49
4.25 64.25
4.88 61.01
4.64 66.07
0.0988
0.0874
0.08745
0.0977
0.09917
0.0995
0.0959
0.0972
0.3311
0.0984
0.0960
0.0953
0.09243
0.2088
0.2818
0.0984
0.0894
0.0939
0.09237
0.09106
0.0963
0.0995
0.0948
0.0922
0.0906
0.0908
0.0943
69.3137
64.0868
71.443
101.788
1236.99
110.224
111.233
1234.9
99.5773
1251.36
1238.49
98.773
111.891
114.416
117.088
119.364
116.287
114.011
112.613
132.001
120.196
113.005
122.092
818.60
908.98
4j
5a
5b
5c
5d
5e
5f
5 g
5 h
5i
1234.43
1242.03
1273.74
1272.70
1192.10
1228.04
1349.29
1236.09
1202.15
1309.31
1174.20
1258.98
1148.48
1251.22
1236.81
1220.50
1351.47
1244.63
1201.10
1283.93
5j
6a
6b
6c
6d
6e
6f
6 g
6 h
6i
6j

K. Ravendra Babu et al. / Antiviral Research 95 (2012) 118–127
123
Table 4
best docking scores among all the leads. In vivo assays were con-
ducted and evaluated to check whether these three compounds
have therapeutic potential or not. To achieve this task, two studies
were performed. Firstly, in vivo screening of these compounds in
NDV infected chickens for antiviral activity and secondly, pro-
and anti-oxidant enzyme status in the liver of the same. Hemag-
glutination (HA) titers were significantly increased in NDV-in-
fected animals as compared to control animals on 10 days of post
infection on the other hand the HA titers were significantly
(P < 0.001) decreased in NDV+compound 4b and NDV+compound
6b treated group animals and slightly in NDV+compound 5b trea-
ted animals (Fig. 1). A significant (P < 0.05) increase was observed
in the survival rates of NDV infected and compounds (4b and 5b)
treated animals when compared to NDV-infected animals (Fig. 5).
A significant increase was observed in the levels of lipid peroxida-
tion (Fig. 6) and depletion in the activity levels of catalase (Fig. 7)
and superoxide dismutase (Fig. 8) in the liver tissue of NDV in-
fected chicks. Interestingly the chicks treated with compounds
4b and 6b significantly (P < 0.001) decreased MDA levels and im-
proved the antioxidant enzyme activities in liver tissue of chicks
compared to NDV infected animals.
Docking interactions substituted thiazolo [3, 2-a] pyrimidine derivatives.
Liganda
Docking scoreb
No. of hydrogen bonds with binding sitec
4a
4b
4c
4d
4e
4f
4g
4h
4i
À12.378
À19.271
À12.212
À14.060
À14.404
À14.205
À15.491
À13.489
À18.817
À16.005
À9.216
0
1
4
0
0
0
1
0
0
2
2
2
1
0
0
0
0
1
0
1
1
1
4
1
0
1
3
1
0
1
4j
5a
5b
5c
5d
5e
5f
5g
5h
5i
À20.471
À10.478
À12.612
À11.280
À10.068
À12.201
À11.605
À10.459
À9.766
5j
6a
6b
6c
6d
6e
6f
6g
6h
6i
À11.729
À19.195
À18.202
À12.826
À12.541
À12.585
À13.131
À11.338
À12.931
À9.460
The histological alterations like intense vacuolar degeneration
of hepatocytes and focal coagulation necrosis of parenchyma and
relapse of hepatocytes were observed in the liver of NDV infected
chicks. On the other hand, the lesions in the liver of NDV infected
chickens were reduced after injection of drugs 4b and 6b over virus
alone infected chickens (Fig. 9). Whereas, injection of 5b into NDV
infected chicks showed mild necrosis in the liver.
6j
The results clearly demonstrated that the therapeutic concen-
trations of compounds 4b and 6b at 20 or 40 mg/kg body weight
inhibited the viral concentrations as evidenced by significant
(P < 0.05) decrease in the mean titers of the hemagglutination. Fur-
ther, an inverse correlation was observed between the concentra-
tion of the compounds (4b and 6b) and the mean titers of
hemagglutination, suggesting that higher concentrations of the
compounds 4b and 6b (40 mg/kg bw) were very effective in inhib-
iting the virus concentrations. However, injection of 5b at 20 or
40 mg/kg bw was unable to elicit such antiviral response.
The content of malondialdehyde levels, an indicator of lipid per-
oxidation was significantly increased in the liver of chicks exposed
to NDV. This increase in the lipid peroxidation products were
accompanied by a significant decrease in the activity levels of
superoxide dismutase and catalase. This clearly indicates that
NDV-mediated infection affects pro-and anti-oxidant balance in
the liver of chicks. Earlier, it has been reported that influenza virus
induces oxidative stress as evidenced by accumulation of lipid
a
The novel thiazolo [3, 2-a] pyrimidine derivatives.
Docking scores generated during MOE docking between the novel leads and HN
b
protein binding domain.
c
Number of hydrogen bonds formed between the HN protein binding domain
and the novel leads.
Arg 363 (Fig. 3). 6b is also forming a single hydrogen bond of 2.6 Å
with the help of one of the nitrogen atom of hetero cyclic ring of
ligand with –COOH group of Asp143 (Fig. 4). These three ligands
are showing first best least docking scores with stable ligand pose
interactions in the docking sphere followed by the remaining lead
compounds.
3.4. Biological activity
After synthesis, QSAR and molecular docking studies, further
experiments were carried out with 4b, 5b and 6b as they showed
Fig. 1. Mean of haemagglutination titers after the inoculation of chicks with Newcastle disease virus and the administration of synthesized chemical candidates (4b, 5b and
6b) after virus infection. ⁄Indicates significantly different from virus control group (P < 0.05).

124
K. Ravendra Babu et al. / Antiviral Research 95 (2012) 118–127
Fig. 2. Interaction of 5b with binding site of HN protein.
Fig. 3. Interaction of 4b with binding site of HN protein.
Fig. 4. Interaction of 6b with binding site of HN protein.
peroxidation products in the blood and lung of animal models
leading to tissue injury (Kornbrust and Mavis, 1980).
It is well established that an intrinsic antioxidant mechanism is
involved to protect the cells/tissues from ROS-induced damage or
to check their levels within normal limits. Under physiological con-
ditions, superoxide dismutase (SOD) is believed to be the first line
of antioxidant enzyme which converts ROS, the superoxide anion,
into hydrogen peroxide and molecular oxygen and the resulting

K. Ravendra Babu et al. / Antiviral Research 95 (2012) 118–127
125
Fig. 8. Bar graph showing activity levels of superoxide dismutase in liver of control,
NDV-infected and NDV+compounds (4b, 5b and 6b) treated chicks. All values are
expressed as mean S.D of six individual observations. ⁄P < 0.05 between control
group and NDV-infected group, ⁄⁄P < 0.05 between NDV-infected group and
NDV+4b treated group, ⁄⁄⁄P < 0.001 between NDV-infected group and 6b treated
group, ns = non significant.
decline in the activity of SOD observed in the present study. Con-
sistent with our results, it also reported (Kumar et al., 2005) that
decreased SOD activity levels in mice infected with influenza virus.
Further, the decreased activities of catalase in virus infected tissues
of birds over control chicks may indicate the accumulation of
hydrogen peroxide. However, treatment of compounds 4b and 6b
were significantly (P < 0.05) increased the activity levels of antiox-
idant enzymes like SOD and CAT in the liver tissue. Since, catalase
protects SOD inactivation by H2O2, while the SOD reciprocally pro-
tects catalase against inhibition by superoxide anion. Thus, a bal-
ance of the activities of these enzyme systems may be essential
to eliminate superoxide and peroxide radicals generated in the tis-
sues. The treatment of 4b and 6b to NDV infected chicks sustains
the balanced circuit of SOD/catalase in the liver and as a result in-
creases the activity levels of both enzymes.
Fig. 5. Survival analysis of NDV infected chicks after treatment with the title
compounds 4b, 5b, 6b: Kaplan–Meier estimates of survival between healthy, NDV
infected, and treatment groups. Log-rank comparisons revealed that title com-
pounds 4b and 6b increased the survival time of NDV infected chicks (log-rank;
v2 = 27.7, P = 1.47E–0.5).
The histological alterations like intense vacuolar degeneration
of hepatocytes and also in the live parenchyma in the liver was ob-
served in the NDV infected tissues over a period of 10 days. On the
other hand, these histological alterations were restored to normal
status in compounds like 4b and 6b treated virus infected liver of
chicks (Fig. 8). It is evident from these studies that increased gen-
eration of free radicals are responsible for tissue damage (Machlin
and Bendich, 1987) during virus infection as evidenced by in-
creased lipid peroxidation products accompanied by poor antioxi-
dant enzyme status. Whereas, compounds like 4b and 6b
treatment in virus infected chicks alleviates the activity levels of
antioxidant enzymes which in turn combat the lipid peroxidation
products to protect tissues from free-radical injury. Nevertheless,
further experiments are required to elucidate the mechanism of
action for these compounds.
Fig. 6. Bar graph showing malonaldehyde levels in liver of control, NDV-infected
and NDV+compounds (4b, 5b and 6b) treated chicks. All values are expressed as
mean S.D of six individual observations. ⁄P < 0.05 between control group and NDV-
infected group, ⁄⁄P < 0.05 between NDV-infected group and NDV+4b treated group,
⁄⁄⁄P < 0.001 between NDV-infected group and 6b treated group, ns = non significant.
In conclusion the molecular descriptors of three molecules ex-
plained their drug likeliness and their usefulness as drugs. The
molecules with the optimum parameter values obtained in QSAR
study will not affect the host system, but acts against pathogens
like virus. All the docking scores and hydrogen bond interactions
of three compounds with HN protein encouraging them as good
antiviral drugs which in turn strengthen by their QSAR study. In
addition, the results from biological assay of three molecules in
NDV infected chicks giving experimental support for their anti-vir-
al activity. Thus with the best binding energies, good hydrogen
bond interactions and less toxicity ensures 4b, 5b and 6b mole-
cules as antiviral agents and can be promoted for further drug
development stages.
Fig. 7. Bar graph showing activity levels of catalase in liver of control, NDV-infected
and NDV+compounds (4b, 5b and 6b) treated chicks. All values are expressed as
mean S.D of six individual observations. ⁄P < 0.05 between control group and NDV-
infected group, ⁄⁄P < 0.05 between NDV-infected group and NDV+4b treated group,
⁄⁄⁄P < 0.001 between NDV-infected group and 6b treated group, ns = non significant.
hydrogen peroxide is converted to water by the enzyme catalase
(Halliwell and Gutteridge, 1985). In the present study, the activity
of SOD was significantly lowered in liver of NDV-infected chicks
when compared with the control animals. This may lead to the
accumulation of singlet oxygen and hydroxyl radicals in the imme-
diate environment which in turn may be responsible for the
3.5. Statistical analysis
The data were analyzed using a statistical package for social sci-
ences (SPSS, 11.5 versions). A one way ANOVA was performed fol-
lowed by Tukey multiple comparisons test, for comparison of

126
K. Ravendra Babu et al. / Antiviral Research 95 (2012) 118–127
Fig. 9. (A) Photomicrograph of liver of control chicks showing normal histology. H and E: Lens 40Â, (B) photomicrograph of liver of NDV infected chicks on 10 dpi, showing
focal coagulation necrosis of parenchyma and degeneration of hepatocytes (arrows). H&E: Lens 40Â, (C) photomicrograph of liver of NDV+4b treated chicks, showing cell
regenerative process. H&E: Lens 40Â, (D) photomicrograph of liver of NDV+5b treated chicks, showing necrosis and slight recovery process. H&E: Lens 40Â, (E)
photomicrograph of liver NDV+6b treated chicks, showing cells regenerative process as normalcy. H&E: Lens 40Â. Scale bar = 10
lm.
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.antiviral.2012.05.
010.
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