In vitro and in silico biological studies of novel thiazolo[3,2-a] pyrimidine-6-carboxylate derivatives

Article abstract of DOI:10.1007/s00044-013-0606-4

In the present study, we report the synthesis, characterization of new series of thiazolo[3,2-a]pyrimidine-6-carboxylate derivatives 3a-f and 4a-f. The newly synthesized compounds were screened for in vitro antimicrobial and antiviral activities. The prob

Full text of DOI:10.1007/s00044-013-0606-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0606-4
ORIGINAL RESEARCH
In vitro and in silico biological studies of novel thiazolo
[
3,2-a]pyrimidine-6-carboxylate derivatives
Kundapura Umesha
V. Bhanuprakash R. Yogisharadhya
Mahmud T. H. Khan
•
Balladka K. Sarojini
•
Chenna G. Darshan Raj
•
•
•
•
R. Raghavendra
Received: 22 December 2012 / Accepted: 30 April 2013
Ó Springer Science+Business Media New York 2013
Abstract In the present study, we report the synthesis,
characterization of new series of thiazolo[3,2-a]pyrimi-
dine-6-carboxylate derivatives 3a–f and 4a–f. The newly
synthesized compounds were screened for in vitro antimi-
crobial and antiviral activities. The probable mode of
action of these active compounds was determined through
in silico docking study by docking the receptor methionyl-
along with the maximum hydrogen/hydrophobic interac-
tion with IMPDH. The study contributes towards identifi-
cation and screening of potential antimicrobial and
antiviral agent’s against the pathogens.
Keywords Thiazolo[3,2-a]pyrimidine-6-carboxylate ꢀ
In vitro antimicrobial activity ꢀ In vitro antiviral activity ꢀ
In silico docking studies
0
tRNA synthetase and human inosine-5 -monophosphate
dehydrogenase (IMPDH) for antibacterial and antiviral
activities, respectively. Among the compounds, 4c exhib-
ited excellent in vitro antimicrobial activity against all
tested strains with binding and docking energies -35.6 and
Introduction
-
12.4 kcal/mol, respectively. The antiviral studies were
Pyrimidine and its derivatives are widespread in nature and
found to be used in a wide range of application in thera-
peutically active compounds. Pyrimidines and fused
pyrimidines, being an integral part of DNA and RNA in it,
play an important role in several biological activities.
Nucleosides of pyrimidine bases have been extensively
used as antiviral and anticancer agents (Donald et al.,
carried out for the selected compounds in which 4a
exhibited 73.69 and 54.42 % of inhibition of buffalopox
and camelpox viruses, respectively. Furthermore, com-
pound 4a showed minimum docking and binding energy
K. Umesha ꢀ B. K. Sarojini (&) ꢀ C. G. Darshan Raj
Department of Chemistry, P.A. College of Engineering,
Nadupadavu, Mangalore 574153, Karnataka, India
e-mail: bksaroj35@gmail.com; bksaroj@yahoo.com
1
987). Recently, fluoropyrimidines and fluorouracil-based
combination therapy is used in the treatment of gastroin-
testinal cancer and solid tumors. Fluoropyrimidines are
found to be metabolites of dihydropyrimidinones that are
subtype-selective antagonists of the a -adreginic receptor
C. G. Darshan Raj
Department of Biomedicinal Research, Vidya Herbs Pvt. Ltd,
Bangalore 562106, Karnataka, India
1
a
antagonists (Lagu et al., 2000). Pyrimidine derivatives
found to possess wide range of pharmacological properties
such as analgesic, antiarrhythmic, anti-inflammatory, anti-
parkinsonian, and androgenic–anabolic activities (Nehad
et al., 2007; Amr et al., 2006). Various analogs of thio-
pyrimidones exhibited antibacterial, antifungal, antiviral,
and antileishmanial activities (Balalaie et al., 2006; Ram
et al., 1984, 1987, 1994). The partially hydrogenated
pyrimidine derivatives such as dihydropyrimidones and
thiodihydropyrimidones have been used as key substrates
to develop molecules as drug or potent leads in medicinal
V. Bhanuprakash ꢀ R. Yogisharadhya
Division of Virology, Indian Veterinary Research Institute,
Mukteswar, Uttarakhand, India
R. Raghavendra
Department of Microbiology and Cell Biology, Indian Institute
of Science, Bangalore 560012, Karnataka, India
M. T. H. Khan
Center for Pharmaceutical Biotechnology, University of Illinois
at Chicago, Chicago, IL, USA
123

Med Chem Res
chemistry. Among them thiazolo[3,2-a]pyrimidine deriva-
tives gained attention in drug discovery because of their
wide range of biological activity such as calcium channel
blocking (Kappe, 2003), antimalarial, and antitubercular
5-carboxylate (1 and 2) were synthesized through opti-
mized Biginelli reaction as shown in the Scheme 1. The
homogeneous solution of 4-methoxy/4-bromo benzalde-
hyde, methylacetoacetate and thiourea in mixture of ace-
tonitrile and dimethyl formamide was stirred in the
presence of catalytic amount of silicon tetrachloride
(Ramalingan and Kwak, 2008).
(
Geist et al., 2010), acetlylcholine esterase inhibitory (Liu
et al., 2009), glutamate receptor antagonistic (Wichmann
et al., 1999), 5-HT2a receptor antagonistic (Awadallah,
2
008) and anticancer activities (Rashad et al., 2010).
Fungal and viral infections are growing problems in
The structure of compounds 1 and 2 were confirmed by
1
13
IR, H, C NMR, LC–MS, and elemental analysis. The
absorption bands appeared at 3619, 3321, 3277, 2935,
contemporary medicine, yet only a few effective chemo-
therapeutic agents are used in clinical practice. There is
clearly a critical need for the development and evaluation
of novel-specific antifungal and antiviral agents. The
increasing need for novel antibiotics to overcome rapidly
developing resistance mechanisms observed in clinical
isolates of bacteria as well as virus has placed critical
emphasis on the search for unique antibacterial/viral
enzyme targets.
-
1
1648, 1556, 1420, 1170 cm were due to stretching of
NH, CH, C=O of ester moety, C=N, C–O, and C–Br
1
groups. The H NMR of compound 2 showed signals at d
2.28, a singlet for methyl protons and another singlet at d
3.54 for methyl protons of ester moiety, respectively. The
signal for hydrogen atom at the chiral carbon C-4 of di-
hydropyrimidone ring appeared as a doublet at d 5.15–5.14
(d, 1H, J = 3.72 Hz). Aromatic protons of phenyl ring
resonated at d 7.17–7.14 (d, 2H, J = 8.36 Hz Ar–H), and
7.55–7.53 (d, 2H, J = 8.28 Hz Ar–H). The signals for the
two NH protons of the pyrimidone ring appeared at 9.67 (d,
1H, J = 1.64 Hz –NH), and 10.39 (s, 1H, –NH) ppm
The most recent phase in the new drug discovery pro-
cess has utilized the knowledge of the three dimensional
structures of target macromolecules or of related proteins.
The same strategy was used in the present study for the
design of novel agents. The synthesis of lead compound by
systematic chemistry in conjunction with binding studies
was undertaken. From the experimental observation at the
atomic level we theoretically predicted that how inhibitors
bind the molecular targets, specific interactions that are
important in the molecular recognition (Krovat et al.,
respectively which disappeared on D O exchange. The
2
down field shift of the signal was due to thiol thione tau-
tomerism. Hence, the structure of the molecule 2 was
1
3
confirmed. It was further supported by recording C NMR
spectrum. The signals appeared in the spectrum account for
all the C-atoms present in molecule 2. LC–MS of the
compound 2 showed molecular ion peak at m/z, 341
(M?2), was in agreement with its molecular formula
C H BrN O S. Similarly structure of 1 was also con-
2
005). The structural and mechanistic investigation of
targets like to bacterial methionyl-tRNA synthetase (met-
0
RS) enzyme (Kim and Lee, 2003) and human Inosine-5 -
1
3
13
2 2
monophosphate dehydrogenase (IMPDH) enzyme were
used commonly to know the potential microbial inhibitors.
The present study describes the one pot synthesis and
biological evaluation of some novel thiazolo[3,2-a]pyrim-
idine-6-carboxylate derivatives. The molecules are
designed based on the preliminary docking studies by
choosing the appropriate pharmacophoric groups. We
herein report the synthesis of two different series of
thiazolo[3,2-a]pyrimidine-6-carboxylate derivatives (3a–f,
firmed by analytical and spectral data.
After confirming the structure of these two compounds
they were allowed to react with different aromatic aldehydes
and chloroacetic acid in the presence of sodium acetate,
acetic acid and acetic anhydride to afford the title
thiazolo[3,2-a]pyrimidine-6-carboxylate derivatives 3a–f
and 4a–f (Scheme 2).
The structure of 3a–f and 4a–f was confirmed by analyt-
ical and spectral data. The IR absorption bands appeared at
1.
3274, 2968, 1657, 1446, 1178, 1110, 690 cm The absence
-
4
a–f) and evaluation of their in vitro antibacterial, anti-
fungal, and antiviral activity against selected pathogens of
medical importance. Furthermore, in silico docking of the
newly synthesized compounds to targets such as metRS
and IMPDH enzymes are also presented.
of bands due NH in the compound confirmed the cyclization.
1
H NMR was recorded in the DMSO-d (400 MHz) for
6
1
compound 3a. The H NMR of compound 3a showed three
singlet signals at d 2.38, 3.59, and 3.70 for methyl, methyl
protons of ester moiety and methoxy protons, respectively.
The signal for hydrogen atom at C-4 appeared as a singlet at d
5.98. The aromatic proton of the methoxy phenyl ring
appeared as two doublets in the region d 6.89 (d, 2H,
J = 8.72 Hz, Ar–H), 7.21 (d, 2H, J = 8.68 Hz, Ar–H). The
signals for three protons of chloro fluoro substituted phenyl
appeared as multiplets at d 7.70–7.65 (m, 1H, Ar–H),
7.95–7.91 (m, 1H, Ar–H), 8.16–8.14 (m, 1H, Ar–H). This
Results and discussion
Chemistry
The key intermediates methyl-4-(4-methoxy/4-bromophe-
nyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-
1
23

Med Chem Res
Scheme 1 (i) Silicon
tetrachloride, DMF/ACN, 16 h
S
O
HN
O
NH
O
S
O
O
i
+
+
H N
2
NH2
O
R1
R1
1
: R = OCH
1
3
2
: R = Br
1
S
R1
O
HN
NH
O
R
2
O
i
+
O
R2
O
N
R1
O
S
N
H
(
1a-1f) and (2a-2f)
2
2
2
2
2
2
a: R =Br, R =2-Cl, 6-F
1 2
1 a: R =OMe, R =2-Cl, 6-F
1 b: R =OMe, R =3-CN, 4-F
1 2
1 c: R =OMe, R =3,4-difluoro
1 2
1 d: R =OMe, R =4-Cl, 3-CF
1 2 3
1 e: R =OMe, R =4-OMe
1 2
1 f: R =OMe, R =2-F, 4-OMe
1 2
1
2
b: R =Br, R =3-CN, 4-F
1
2
c: R =Br, R =3,4-difluoro
1
2
d: R =Br, R =4-Cl, 3-CF
1
2
3
e: R =Br, R =4-OMe
1
2
f: R =Br, R =2-F, 4-OMe
1
2
Scheme 2 (i) Chloro acetic acid, NaOAc, AcOH, Ac O, 130 °C, 3–5 h
2
complex pattern could be attributed to the H–F coupling. The
signal appeared at d 7.78 was due to exocyclic methylene
proton. The absence of the signals due to NH protons in this
product indicated the formation of thiazolopyrimidine ring.
The LC–MS spectrum showed a molecular ion peak at m/
z 473 (M?1), which corresponded to its molecular formula
C H ClFN O S. In addition, the molecular structure of the
Salmonella typhi bacterial strains in vitro. Antifungal
activity for the newly synthesized compounds was also
carried out against fungal strains Aspergillus niger and
Candida albicans by disk diffusion method (Cruickshank
et al., 1975; Warnock et al., 1998). The zone of inhibition
and MIC were noted and data are given in Table 1 and 2.
All the tested compounds (3a–f, 4a–f) showed comparable
activity with the standard compounds Ciprofloxacin and
Fluconazole for antibacterial and antifungal activities,
respectively. Among them 4c showed significant activity with
zone of inhibition 24 mm (MIC 26 lg/mL) for S. aureus,
26 mm (MIC 26 lg/mL) for B. subtilis, 23 mm (30 lg/mL)
S. typhiand 24 mm(30 lg/mL) for E. coli (24mm, 30lg/mL),
respectively. The zone of inhibition and MIC of 4c against
A. niger was found to be 21 mm and 25 lg/mL and against
C. albicans is 22 mm and 30 lg/mL. The compounds 3c, 3f,
and 4e were inactive against C. albicans even though they
exhibited good activity against other tested microbial strains.
2
3
18
2 4
compound 3f was established by single-crystal X-ray dif-
fraction studies (Fun et al., 2011). The ORTEP view of the
molecular structure showing the spatial atomic positions of
the compound 3f is given in Fig. 1. The crude product (3a–
f and 4a–f) was purified on a Biotage parallel column purifier
using EtOAc/Pet ether (3:2) as eluant. The purity of the final
compounds was determined by HPLC and all the compounds
possessed a purity of[95 %.
Biological activities
In vitro antibacterial and antifungal activities
Cytotoxicity and antiviral activity
The newly synthesized compounds 3a–f; 4a–f were
screened for their antibacterial activity against Escherichia
coli, Staphyllococcus aureus (Smith), Bacillus subtilis, and
Usually cell lines such as primary chick embryo, chick
kidney, calf kidney, Madin Darby Canine Kidney
123

Med Chem Res
Fig. 1 ORTEP diagram
showing the X-ray crystal
structure of 3f
Table 1 Antimicrobial activity of thiazolopyrimidine carboxylate derivatives 3a–f; 4a–f (zone of inhibition in mm)
Compd. conc. (40 lg) Bacterial strains
Fungal strains
Staphylococcus aureus Bacillus subtilis Salmonella typhi Escherichia coli Aspergillus niger Candida albicans
3
3
3
3
3
3
4
4
4
4
4
4
a
b
c
d
e
f
23
21
21
23
22
24
22
23
24
22
23
22
23
21
22
22
22
23
24
24
21
26
22
22
24
23
22
22
24
23
24
23
22
23
23
23
23
22
24
23
22
22
23
22
25
24
22
24
22
22
20
25
23
24
24
23
23
25
24
23
21
22
24
23
23
20
22
R
22
22
R
a
b
c
d
e
f
22
22
22
22
R
21
24
a
Standard
Compounds used: (40 lg/ml)
a
Standard drug used: bacteria (ciprofloxacin); fungal (fluconazole) (40 lg/ml)
(
MDCK), mink lung, and human respiratory epithelial cells
viruses grow well in this cell line. The effect of various
concentrations of DMSO on Vero cell monolayers was
tested by dye exclusion method assay, resulting in near to
could be used for in vitro antiviral assays (Sidwell and
Smee, 2000). But Vero cells are preferred because; pox
1
23

Med Chem Res
Table 2 Minimum inhibitory concentration (MIC in lg/ml)
Compd. conc.
1
Bacterial strains
Fungal strains
0–50 (lg/ml)
Staphylococcus
aureus
Bacillus
subtilis
Salmonella
typhi
Escherichia
coli
Aspergillus
niger
Candida
albicans
3
3
3
3
3
3
4
4
4
4
4
4
a
b
c
30
30
23
30
30
40
30
40
26
40
30
30
22
30
40
23
40
30
30
30
20
26
30
30
30
23
40
30
30
40
30
30
40
30
30
30
30
40
24
30
30
30
30
40
40
20
30
30
30
20
40
22
30
40
35
40
40
30
30
30
25
30
30
30
23
30
40
R
d
a
f
30
40
R
a
b
c
40
30
30
30
R
d
a
f
40
24
a
Standard
a
Standard drug used: bacteria (ciprofloxacin); fungal (fluconazole) (40 lg/ml)
1
00 % cell survival at more than 1 % DMSO after 48 h
by the tested compounds. The titers of both the viruses viz;
BPXV and CMLV both in control and treated
(virus?compound) wells are presented in Table 3. In this
investigation, among the tested compounds, 4a showed
in vitro inhibition of 73.29 and 54.42 % against two pox
viruses (DNA viruses) viz., BPXV and CMLV, respec-
tively at their MNTC.
incubation (data not shown). Moreover, potential interfer-
ence of DMSO in the assay throughout the screening
process was assessed for every plate with the addition of
0.2 lL of DMSO to three wells, volume that was equiva-
lent to those of compounds.
Selection of camelpox (CMLV) and buffalopox (BPXV)
inhibitors may be of value in obtaining antiviral agents
against pox viruses (De Clercq and Neyts, 2004) in general
and smallpox virus in particular, which has re-emerged as a
threat to use in bioterrorism (Breman and Henderson,
Docking studies
TM
Docking calculations with ICM (Internal Coordinate
Mechanics) dock All the docking calculations of com-
TM
pounds in this article were performed using the ICM
1
998).
Prior to evaluating antiviral activities of the compounds,
the cytotoxicity of the synthetic compounds on host cell
Vero cell) was carried out. The maximum non-toxic
docking module with the default setup as earlier mentioned
(
(Abagyan et al., 1994; Khan et al., 2009).
concentration/dose (MNTC/MNTD) for 3a, 3c, 4a, and 4c
was carried out and is shown in Table 3. There was no
visible toxicity to the cells at these concentrations. These
compounds at MNTCs were utilized for antiviral efficacy
testing against CMLV and BPXV. But there was no sig-
nificant inhibition in the replication of these viruses in vitro
Preparations of the inhibitors and target molecules The
2D structures of the compounds (in mol file formats) have
been converted to 3D and energy minimized at the 3D
space of ICM environment. The formal charges and 3D
topology were assigned by the atom types using local
Table 3 Cytotoxicity and inhibitory effects of different synthetic compounds on Vero cells, BPXV, and CMLV viruses
Compd.
Amount
mg)
DMSO
(ll)
MNTC
(lg/ml)
CMLV
control
Inhibitory titer
(virus?extract)
Percent of
inhibition
Buffalo pox
control
(TCID50/ml)
Inhibitory titer
(virus ? extract)
(TCID50/ml)
Percent of
inhibition
(PI)
(
(
PI)
6
6
6
6
6
.5
.5
.5
.5
.5
6.5
6.74
6.74
CR 22
3
2
2
4
2
200
200
200
300
300
250
10
10
10
10
10
10
0
10
10
0
6.16
10
6.74
10
6.74
10
3
3
4
4
a
c
a
c
31.2
31.2
20.8
05.2
54.29
0
0
6.5
10
6.74
10
6.74
10
0
6.16
10
6.74
10
6.16
10
54.42
0
73.69
0
6.5
10
6.74
10
6.74
10
123

Med Chem Res
chemical environment, Merck Molecular Force Field
MMFF) (Halgren, 1996a, b, c, d, e; Halgren and Nachbar,
996f).
residues of the active sites like Val140, Lys137, Pro145,
Leu139, Tyr134, Ile146, Arg132 and Thr55. Same S1 atom
of compound 3a showed strong hydrogen bonding inter-
actions with the CD1 atom of Ile146 and other atoms
showed hydrophobic interactions with Tyr134, Arg132,
Glu138, Pro145, and Val140. The S1 atom of compound
(
1
Docking process All the docking calculations were per-
formed using the ‘‘interactive docking’’ menu at the ICM
environment. After docking the stack of docking poses
were checked visually. Multiple stack conformations were
selected based on their docking energies, rmsd values
˚
3b showed long distance (3.19A) hydrogen bond with the
OH atom of Tyr134 and others showed hydrophobic
interactions with Val140, Pro145, Arg132, and Thr55. O4
and C14 atoms of compound 3c showed strong hydrogen
bonding interactions with the CG1 atom of Val140 and
others showed hydrophobic interactions with Glu138,
Pro145, Ile146, and Thr55. Compound 3d did not show any
hydrogen bonding with any residues of the active site of
metRS, but showed several hydrophobic interactions with
different residues of the active sites like Ile146, Thr55,
Val140, Pro145, Leu139, Glu138, Tyr134, and Arg132.
Again S1 atom of the compound 3e showed strong
hydrogen bonding interactions with the NE2 atom of
His147 and hydrophobic interactions with Ile146, Arg58,
Thr55, and Ala59. Compound 3f did not show any
hydrogen bonding, but showed some hydrophobic inter-
actions with Ile146, His147, Thr55, Arg132, and Glu138.
Compound 2 did not show any hydrogen bonding, but
showed some hydrophobic interactions with Ile146,
Pro145, Glu138, Tyr134, and Arg132. Compound 4a
showed hydrophobic interactions with only Pro145 and
Ile146. Compound 4b showed several hydrophobic inter-
actions with Thr55, Arg132, Tyr134, Ile146, Glu138,
Pro145, and Val140. Compound 4c showed hydrophobic
interactions with only Ile146, Arg132, and Thr55. Com-
pound 4d showed several hydrophobic interactions with
Thr55, Ile146, Pro145, Arg132, Val140, Tyr134, and
Glu138. Compound 4e also showed several hydrophobic
interactions with Thr55, Ile146, Pro145, and Val140.
Compound 4f showed hydrophobic interactions with only
Ile146, Arg132, Glu138, and Thr55. Docking energy gives
an idea about the energy required to cover the entire pro-
tein by a ligand molecule, whereas the binding energy
gives the information about the putative interaction of the
molecules at the active site of the enzyme. Lower the value
of these energies, efficient will be the molecule. All the
compounds showed minimum docking and binding energy,
among them compound 1, 2, 4d, and 4e could be effective
binders to metRS.
(
compared between the docked model and X-ray confor-
mation with similarities to closely related X-ray crystal
structures from PDB). Then the best conformations for
each of the compounds were finally chosen, and their
binding energies were calculated using ICM script.
Putative molecular interactions with metRS The com-
pounds 1, 3a–f, 2 and 4a–f have been docked into the
active site of the metRS (PDB ID: 1A8H).
The calculated docking and binding (DG) energies (in
Kcal/mol) of the compounds are shown in Table 4.
Docking energy gives an idea about the energy required to
cover the entire protein by a ligand molecule, whereas the
binding energy gives the information about the putative
interaction of the molecules at the active site of the
enzyme. The molecular interactions between the com-
pounds and the active site residues of metRS at 3D space
are shown in Fig. 2, different compounds in different
panels, accordingly. The S1 atom of the compound 1
showed strong hydrogen bonding interactions with O, C,
and CA atoms of Glu138 residue at the active site of metRS
and most of the other parts of the compounds showed short
and long distance hydrophobic interactions with different
Table 4 Calculated docking and binding (DG) energies of the com-
pounds against metRS
Compd.
Docking
Binding (DG)
1
-44.3
-13.8
-14.2
-22.1
-6.6
-10.9
-11.5
-12.4
-12.0
-11.0
-13.1
-7.2
3
3
3
3
3
3
2
4
4
4
4
4
4
a
b
c
d
e
f
-9.6
-36.3
-37.3
-15.9
-20.5
-35.6
-35.9
-23.4
-10.2
-7.2
a
b
c
d
e
f
-10.9
-11.9
-12.4
-13.2
-10.8
-11.6
Putative molecular interactions with the human IM-
PDH The compounds 3a and 4a have been docked into
the active site of the human IMPDH (PDB ID: 1NFB),
where Cys331 is the catalytic residue, based on their
in vitro antiviral results mentioned in Table 3 against
BPXV and CMLV. The calculated docking (DE) and
binding (DG) energies of 3a were -54.2 and -8.7 kcal/mol,
Calculated energies (in Kcal/mol)
1
23

Med Chem Res
Fig. 2 Molecular interactions with of the compounds 1 (A), 3a (B), 3b (C), 3c (D), 3d (E), 3e (F), 3f (G), 2 (H), 4a (I), 4b (J), 4c (K), 4d (L), 4e
(
M), and 4f (N) against methionyl-tRNA synthetase (metRS) (PDB ID: 1A8H)
respectively, whereas for compound 4a the calculated
energy values were -58.6 and -10.4 kcal/mol, respec-
tively. O4 oxygen atom of compound 3a exhibited long
intermolecular interactions are shown in the panel A of
Fig. 3. Whereas compound 4a exhibited at least 3 different
long distance hydrogen-bond interactions. Atom O1
˚
distance (3.24 A) hydrogen-bond interactions with the N
showed with NH atom of Arg259 with a distance of 3.06
2
˚
A, O3 showed with the NE2 atom of His93 with a distance
˚
of 2.87 A, and Cl atom showed with the O atom of Pro69
˚
with a distance of 2.87 A. Other atoms of the molecule
atom of the residue Met414 of human IMPDH and most of
the atoms of the molecule showed hydrophobic interactions
with Cys331, Met70, His93, Asn94, and Cys95. These
123

Med Chem Res
exhibited maximum anti-pox virus (BPXV and CMLV)
activity. The molecular docking studies of the compound
4c and 4a with target for metRS and human IMPDH
showed minimum binding and docking energies, respec-
tively coupled with more hydrogen bonding and hydro-
phobic interaction, which gives impetus for further work on
their antimicrobial/viral activities. This preliminary study
has opened up an interesting area to evaluate thiazolpyr-
imidine carboxylate derivatives as possible antimicrobial
and antiviral drug candidates.
Experimental
All the chemicals were analytical grade and purchased
from Sigma-Aldrich. Final purifications were carried out
using Quad biotage Flash purifier. TLC experiments were
performed on alumina-blocked silica gel 40 F254 plates.
Melting points were determined using Buchi-540 by open
capillary and are uncorrected. Elemental analysis was
carried out on an automatic Flash EA 1112 Series, CHNSO
Analyzer (Thermo). X-ray diffraction studies were carried
out on Oxaford Cryosystems Cobra open flow nitrogen
cryostat (Cosier and Glazer, 1986) operating at 100.0 (1) K
Fig. 3 Intermolecular interactions with of the compounds 3a (A) and
a (B) at the active site of human inosine monophosphate dehydro-
genase (IMPDH) type II (PDB ID: 1NFB), where Cys331 is the
catalytic residue. Both the compounds exhibited imperative hydrogen
bonding with a number of explicit residues of the active site of
IMPDH
4
1
13
(Universiti Sains Malaysia). H and C NMR spectra were
recorded on a Bruker AMX 400 or Bruker DPX 300
spectrometer, with 5 mm PABBO BB-1H TUBES LCMS
were obtained using Agilent 6200 series. The chemical
shifts are reported in ppm (d), the signals are designated as
follows: s, singlets; d, doublets; dd, doublet of doublets;
t, triplets; brs, broad singlet, m, and multiplet. LC and
Micromass zQ spectrometer.
showed several important hydrophobic interactions against
Asp256, Asn94, Met414, Asp71, and Tyr411 with different
distances. These intermolecular interactions are shown in
the panel B of Fig. 3. These results showed that the rela-
tively higher antiviral activity of compound 4a in com-
parison 3a may due to lower docking and binding energies
as well as higher hydrogen and hydrophobic interactions.
Earlier, in a review by Bhanuprakash et al. (2010) an
effective anti-poxvirus compounds which inhibited IMP
dehydrogenase, such as ribavarin, and tyrosine kinase
inhibitor, STI-571/imatinib mesylate, or Gleevec were
reported.
Synthesis of methyl-4-(4-methoxyphenyl)-6-methyl-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1)
To a homogeneous solution 4-methoxy/4-bromo benzal-
dehyde (5.0 g, 0.0367 mol) in mixture of dry acetonitrile
0
and N,N -dimethylformamide (2:1, 100 mL), were added
methylacetoacetate (4.26 g, 0.0367 mol) and thiourea
(
3.35 g, 0.0440 mol) at room temperature. The reaction
mixture was cooled to 0 °C and silicon tetrachloride
0.62 g, 0.00367 mol) was added in drops. The reaction
Conclusions
(
Two new series of thiazolopyrimidine carboxylate deriva-
tives 3a–f and 4a–f were synthesized with good yield by a
three component MCR reaction, characterized by spectral
and elemental analysis. All the tested compounds exhibited
promising antimicrobial (fungal and bacterial) activities.
However, compound methyl(2Z)-5-(4-bromophenyl)-2-
mass was stirred at 25–28 °C for 16 h. After the comple-
tion of the reaction, the reaction mass was quenched with
chilled water (100 mL) and stirred for 30 min. The solids
precipitated out were collected by filtration, washed with
water, and dried under suction to get the title compound
(8 g, 75 %) as a white solid.
[
(3,4-difluorophenyl)methylidene]-7-methyl-3-oxo-2,3-
dihydro-5H[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate
c exhibited excellent in vitro antimicrobial activity.
Among the tested compounds for antiviral activity, 4a
1 : Appearance: off white solid; yield 75 %; m.p:
1
-
1
180–182 °C; IR (KBr, cm ) vmax: 3318, 3270,1657; H
NMR (DMSO-d , 400 MHz), d: 2.06 (s, 3H, –CH ), 3.53
(s, 3H, –CO CH ), 3.71 (s, 3H, –OCH ), 5.10 (d, 1H,
4
6
3
2
3
3
1
23

Med Chem Res
J = 3.72 Hz, pyrimidine-CH–), 6.88 (dd, 2H, J = 2.00,
6
Methyl(2Z)-2-[(3-cyano-4-fluorophenyl)methylidene]-5-(4-
methoxyphenyl)-7-methyl-3-oxo-2,3-dihydro-5H-
[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (3b)
.88 Hz, Ar–H), 7.11 (dd, J = 1.92, 8.68 Hz, 2H, Ar–H),
.61 (d, 1H, J = 1.80 Hz, –NH), 10.31 (s, 1H, –NH) ppm;
9
1
3
C (DMSO-d , 100 MHz), 17.17, 51.05, 53.29, 55.07,
6
1
1
00.63, 113.9, 127.53, 135.47, 145.03, 158.73, 165.64,
74.01 ppm; Anal. for C H N O S; cal: C 57.52, H 5.52,
Appearance: yellow solid; yield 67 %; m.p: 172.3–173 °C;
-
1
IR (m cm ): 3278, 2976, 2364, 2189, 1656, 1448, 1175,
1
4 16 2 3
1
1107; H NMR (DMSO-d
N 9.58; found; C 57.47, H 5.55, N 9.50; LC/MS (ESI–MS)
6
, 400 MHz) d 2.36 (s, 3H,
), 3.71 (s, 3H, –OCH ), 5.98
m/z 293.0 (M?1).
2
–CH ), 3.58 (s, 3H, –CO CH
3
2
3
3
: Appearance: off white solid; yield 73 %; m.p:
-
(s, 1H pyrimidine-CH–), 6.91(d, 2H, J = 8.72 Hz, Ar–H),
7.22 (dd, 2H, J = 1.88, 8.72 Hz, Ar–H), 7.41–7.37 (m, 1H,
Ar–H), 7.48 (d, 1H, J = 7.44 Hz, Ar–H), 7.58–7.52 (m,
1H, Ar–H), 7.68 (s, 1H, Ar–CH=) ppm. Anal. for
1
1
1
68.3–170.7 °C; IR (m cm ): 3619, 3321, 3277, 2935,
1
648, 1556, 1420, 1170; H NMR (DMSO-d , 400 MHz),
6
d: 2.28 (s, 3H, –CH ), 3.54 (s, 3H, –CO CH ), 5.13 (d, 1H,
3
2
3
J = 3.72 Hz, pyrimidine CH–), 7.15 (d, 2H, J = 8.36 Hz,
Ar–H), 7.55 (d, 2H, J = 8.36 Hz, Ar–H), 9.69 (s, 1H,
C H18FN O S; cacld; C 62.19, H 3.91, N 9.07; found; C
24 3 4
62.26, H 3.87, N 9.02; LC/MS (ESI–MS) m/z, 464.0
(M?1).
1
NH), 10.41 (s, 1H, –NH) ppm; C (DMSO-d , 100 MHz),
3
–
1
1
4
6
7.21, 51.12, 53.36, 99.95, 120.84, 128.57, 131.54, 142.56,
45.62, 165.47, 174.26; Anal. for C H BrN O S; cal: C
1
3
13
2 2
Methyl(2Z)-2-[(3,4-difluorophenyl)methylidene]-5-(4-
methoxyphenyl)-7-methyl-3-oxo-2,3-dihydro-5H-
5.76, H 3.84, N 8.21; found; C 45.65, H 3.81, N 8.24; LC/
MS (ESI–MS) m/z 343 (M?2).
[
1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (3c)
General procedure for synthesis of methyl(2Z)-2-
Appearance: pale yellow solid; yield 68 %; m.p:
(
substituted benzylidene)-5-(4-methoxy/bromo-
-
1
1
1
–
97.5–198 °C; IR (m cm ): 3274, 2968, 1657, 1446, 1176,
phenyl)-7-methyl-3-oxo-2,3-dihydro-5H-[1,3]-
thiazolo[3,2-a]pyrimidine-6-carboxylates. 3a–3f
and 4a–4f
1
110, 690; H NMR (DMSO-d , 400 MHz), d: 2.37 (s, 3H,
6
CH ), 3.60 (s, 3H, –CO CH ), 3.71 (s, 3H, –OCH ), 5.99
3
2
3
3
(s, 1H pyrimidine-CH–), 6.87 (dd, 2H, J = 1.96, 8.72 Hz,
Ar–H), 7.20 (dd, 2H, J = 1.92, 8.76 Hz, Ar–H), 7.46–7.43
A mixture of methyl 4-(4-methoxyphenyl)/(4-bromophe-
nyl)-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-car-
boxylate 1/2 (2.59 mmol), chloro acetic acid (0.29 g,
(
m, 1H, Ar–H), 7.64–7.57 (m, 1H, Ar–H), 7.73–7.68 (m,
1
3
1H, Ar–H), 7.77 (s, 1H, Ar–CH=) ppm; C (DMSO-d6,
100 MHz), 22.33, 51.47, 54.29, 55.05, 108.90, 113.94,
114.03, 118.55, 118.72, 119.17, 119.35, 121.05, 121.07,
126.73, 126.76, 128.68, 130.52, 130.58, 130.62, 130.69,
132.08, 148.27, 148.40, 149.00, 149.12, 150.73, 150.87,
150.94, 151.63,154.88, 159.29, 164.01, 165.33 ppm; Anal.
3
.11 mmol), an aldehyde (3.11 mmol) and sodium acetate
(
10 mmol) in 1:1 mixture glacial acetic acid/acetic anhy-
dride (10 mL) were heated to 130 °C for 3–5 h. After the
completion of the reaction, the reaction mass was cooled to
room temperature and quenched to ice cooled water. The
solids precipitated out were collected by filtration and
purified on a Biotage parallel column purifier using EtOAc/
Pet ether (3:2) as eluant to afford title compounds
for C H F N O S; cacld; C 60.52, H 3.97, N 6.14; found;
23 18 2 2 4
C 60.46, H 3.94, N 6.18; LC/MS (ESI–MS) m/z, 457.0
M?1).
(
(
60–75 %).
Methyl(2Z)-2-[(4-chloro-3-trifluoromethane
phenyl)methylidene]-5-(4-methoxyphenyl)-7-methyl-3-oxo-
2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-
carboxylate (3d)
Methyl(2Z)-2-[(2-chloro-6-fluorophenyl)methylidene]-5-
4-methoxyphenyl)-7-methyl-3-oxo-2,3-dihydro-5H-
1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (3a)
(
[
Appearance: yellow solid; yield 70 %; m.p: 152.5–
Appearance: yellow solid; yield 65 %; m.p: 176.8–
-1
-
1
1
6
3
54.5 °C; IR (m cm ): 3274, 2968, 1657, 1446, 1178, 1110,
178.1 °C; IR (m cm ): 3287, 2986, 1657, 1450, 1175,
1
1
1104; H NMR (DMSO-d
90; H NMR (DMSO-d , 400 MHz), d: 2.38 (s, 3H, –CH ),
6
, 400 MHz), d: 2.38 (s, 3H,
CH ), 3.70 (s, 3H, –OCH ), 5.99
6
3
.59 (s, 3H, –CO CH ), 3.70 (s, 3H, –OCH ), 5.98 (s, 1H
3
–CH
3
), 3.59 (s, 3H, –CO
2
3
3
2
3
pyrimidine-CH–), 6.89 (d, 2H, J = 8.72 Hz, Ar–H), 7.21 (d,
H, 8.68 Hz, Ar–H), 7.70–7.65 (m, 1H, Ar–H), 7.78 (s, 1H
(s, 1H pyrimidine-CH–), 6.89 (d, 2H, J = 8.64 Hz, Ar–H),
7.22 (d, 2H, J = 8.64 Hz, Ar–H), 7.86–7.81 (m, 2H, Ar–
H), 7.88 (s, 1H, Ar–CH=), 8.09. (s, 1H, Ar–H) ppm. Anal.
2
Ar–CH=), 7.95–7.91 (m, 1H, Ar–H), 8.16–8.14 (m, 1H, Ar–
H) ppm; Anal. for C H ClFN O S; cal: C 58.41, H 3.84, N
for C24H18ClF N O S; cacld; C 55.12, H 3.47, N 5.36;
3 2 4
2
3
18
2 4
5
.92; found; C 58.36, H 3.82, N 5.96; LC/MS (ESI–MS)
found; C 55.23, H 3.40, N 5.33; LC/MS (ESI–MS) m/z,
523.0 (M?1).
m/z 473.0 (M?1).
123

Med Chem Res
Methyl(2Z)-2-[(4-methoxy phenyl)methylidene]-5-(4-
methoxyphenyl)-7-methyl-3-oxo-2,3-dihydro-5H-
3.59 (s, 3H, –CO CH ), 6.01 (s, 1H, pyrimidine-CH–), 7.26
2
3
(d, 2H, J = 8.4 Hz, Ar–H), 7.55 (d, 2H, J = 8.4 Hz),
7.76–7.66 (m, 1H, Ar–H), 7.79 (s, 1H, Ar–CH=), 7.97–7.93
[
1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (3e)
1
3
m, 1H, Ar–H), 8.18–8.16 (m, 3H, Ar–H) ppm; C (DMSO-
(
Appearance: yellow solid; yield 68 %; m.p: 188.1–189.3 °C;
IR (m cm ): 3275, 2975, 1658, 1449, 1172; H NMR
d6, 100 MHz), 22.45, 51.55, 54.51, 101.45, 101.60, 108.36,
113.26, 117.69, 117.89, 121.89, 129.67, 129.87, 130.49,
130.52, 131.69, 135.88, 136.17, 136.26, 139.23, 151.39,
155.00, 161.33, 163.88, 163.93, 165.14; Anal. for
C H BrFN O S; cacld; C 53.92, H 2.95, N 8.20; found; C
-1
1
(
DMSO-d , 400 MHz), d: 2.38 (s, 3H, –CH ), 3.60 (s, 3H,
6
3
–
CO CH ), 3.71 (s, 3H), 3.81 (s, 3H, –OCH ), 5.99 (s, 1H
2 3 3
pyrimidine-CH–), 6.89 (d, 2H, J = 8.64 Hz, Ar–H), 7.10 (d,
H, J = 8.8HZ), 7.21(d, 2H, J = 8.64 Hz, Ar–H), 7.86–7.81
m, 2H, Ar–H), 7.88 (s, 1H, Ar–CH=), 8.09. (s, 1H, Ar–H)
ppm. Anal. for C H N O S; cacld; C 63.98, H 4.92, N 6.22;
2
3
15
3 3
2
53.84, H 2.92, N 8.18; LC/MS (ESI–MS) m/z, 514.0 (M?2).
(
2
4 22 2 6
Methyl(2Z)-5-(4-bromophenyl)-2-[(3,4-
difluorophenyl)methylidene]-7-methyl-3-oxo-2,3-dihydro-
5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (4c)
found; C 63.90, H 4.90, N 6.19; LC/MS (ESI–MS) m/z, 451
M?1).
(
Methyl(2Z)-2-[(2-fluoro-4-methoxy phenyl)methylidene]-5-
4-methoxyphenyl)-7-methyl-3-oxo-2,3-dihydro-5H-
1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (3f)
Appearance: yellow solid; yield 72 %; m.p: 188.3–189.0 °C;
IR (m cm ): 2934, 2376, 1711, 1690, 1332, 1134, 788; H
-1
1
(
[
NMR (DMSO-d , 400 MHz), d: 2.38 (s, 3H, –CH ), 3.59 (s,
6
3
3H, –CO CH ), 6.01 (s, 1H, pyrimidine-CH–), 7.26 (d, 2H,
2 3
Appearance: yellow solid; yield 70 %; m.p: 204.9–205.5 °C;
IR (m cm ): 3278, 2980, 1657, 1444, 1173, 1107; H NMR
J = 8.4 Hz, Ar–H), 7.48–7.46 (m, 1H, Ar–H), 7.56 (d, 2H,
J = 8.4 Hz), 7.65–7.58 (m, 1H, Ar–H), 7.74–7.69 (m, 1H,
Ar–H), 7.78 (s, 1H, Ar–CH=) ppm. Anal.for C H BrF
-1
1
(
DMSO-d , 400 MHz), d: 2.39 (s, 3H, –CH ), 3.60 (s, 3H,
6
3
22 15
2
–
1
7
CO CH ), 3.71(s, 3H, –OCH ),3.84(s, 3H, –OCH ),6.00(s,
3
N O S; cacld; C 52.29, H 2.99, N 5.54; found; C 52.34, H
2 3
2
3
3
H pyrimidine-CH–), 6.90 (d, 2H, J = 8.6 Hz, Ar–H),
.07–6.98 (m, 1H), 7.23 (d, 2H, 8.6 Hz, Ar–H), 7.51 (t, 1H,
2.96, N 5.62; LC/MS (ESI–MS) m/z, 507.0 (M?2).
J = 8.76 Hz, Ar–H), 7.70 (s, 1H, Ar–CH=) ppm. Anal.for
C H FN O S; cacld; C 61.53, H 4.52, N 5.98; found; C
Methyl(2Z)-5-(4-bromophenyl)-2-{[4-chloro-3-
2
4
21
2 5
(
trifluoromethyl)phenyl]methylidene}-7-methyl-3-oxo-2,3-
dihydro-5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate
4d)
6
1.45, H 4.49, N 5.94; LC/MS (ESI–MS) m/z, 469.0 (M?1).
(
Methyl(2Z)-5-(4-bromophenyl)-2-[(2-chloro-6-
fluorophenyl)methylidene]-7-methyl-3-oxo-2,3-dihydro-
Appearance: yellow solid; yield 68 %; m.p: 186.3–187.5 °C;
5
H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (4a)
-1
1
IR (m cm ): 2942, 2353, 1714, 1687, 1316, 1132, 784; H
NMR (DMSO-d , 400 MHz), d: 2.38 (s, 3H, –CH ), 3.59 (s,
6
3
Appearance: brown solid; yield 75 %; m.p: 147.8–148.8 °C;
3H, –CO CH ), 6.03 (s, 1H, pyrimidine-CH–), 7.27 (d, 2H,
2 3
-1
IR (m cm ): 3092, 2917, 2358, 1724, 1689, 1342, 1163,784;
J = 8.4 Hz, Ar–H), 7.56 (d, 2H, J = 8.4 Hz), 7.91–7.84
m, 3H, Ar–H), 8.12 (s, 1H, Ar–H) ppm. Anal. for
C H BrClF N O S; cacld; C 48.31, H 2.64, N 4.90; found;
1
H NMR (DMSO-d ,400 MHz),d:2.36(s, 3H, –CH ),3.58(s,
6
3
(
3H, –CO CH ), 6.00 (s, 1H, pyrimidine-CH–), 7.28 (d, 2H,
2 3
2
2
15
3 2 3
J = 8.4 Hz, Ar–H), 7.42–7.38 (m, 1H, Ar–H), 7.48 (d, 1H,
J = 7.88 Hz, Ar–H), 7.59–7.53 (m, 3H, Ar–H), 7.69 (s, 1H,
C 48.25, H 2.63, N 4.95; LC/MS(ESI–MS)m/z, 574.0 (M?2).
1
3
Ar–CH=) ppm. C (DMSO-d , 100 MHz), 22.41, 51.54,
6
54.69, 108.52, 115.32, 115.53, 119.95, 121.91, 124.04, 126.26,
127.34, 129.80, 131.74, 132.90, 133.00, 134.16, 139.22,
151.09, 154.74, 157.92, 160.28, 163.28, 165.14.; Anal. for
Methyl(2Z)-5-(4-bromophenyl)-2-[(4-
methoxyphenyl)methylidene]-7-methyl-3-oxo-2,3-dihydro-
5H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (4e)
C H BrClFN O S; cacld; C 50.64, H 2.90, N 5.37; found; C
15
22
2 3
50.55, H 2.88, N 5.34; LC/MS (ESI–MS) m/z, 523.8 (M?2).
Appearance: yellow solid; yield 67 %;m.p: 173.3–174.1 °C;
IR (m cm ): 2918, 2364, 1719, 1688, 1316, 1109, 815; H
-1
1
Methyl(2Z)-5-(4-bromophenyl)-2-[(3-cyano-4-
fluorophenyl)methylidene]-7-methyl-3-oxo-2,3-dihydro-
NMR (DMSO-d , 400 MHz), d: 2.37 (s, 3H, –CH ), 3.59 (s,
3H, –CO CH ), 3.81 (s, 3H, –OCH ), 6.01 (s, 1H, pyrimidine-
2 3 3
6
3
5
H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (4b)
CH–), 7.11 (d, 2H, J = 8.84 Hz, Ar–H), 7.26 (d, 2H,
J = 1.84, 6.68 Hz, Ar–H), 7.58–7.52 (m, 4H), 7.75 (s, 1H,
Ar–CH=) ppm. Anal.for C H BrF N O S; cacld; C 55.32,
Appearance: yellow solid; yield 70 %; m.p: 152.3–153.3 °C;
2
3
19
2 2 4
-1
IR (m cm ): 3405, 2944, 2155, 1709, 1369, 1158, 821;
H 3.83, N 5.61; found; C 55.25, H 3.86, N 5.66; LC/MS (ESI–
1
H NMR (DMSO-d , 400 MHz), d: 2.38 (s, 3H, –CH ),
MS) m/z, 501.0 (M?2).
6
3
1
23

Med Chem Res
Methyl(2Z)-5-(4-bromophenyl)-2-[(2-fluoro-4-
methoxyphenyl)methylidene]-7-methyl-3-oxo-2,3-dihydro-
Fluconazole (50 lg in 100 lL) were used. The zone of
inhibition was compared with standard drug after 24 h of
incubation at 37°C for antibacterial activity and 72 h at
5
H-[1,3]thiazolo[3,2-a]pyrimidine-6-carboxylate (4f)
2
5 °C for antifungal activity. The data are given in Table 2.
Appearance: yellow solid; yield 72 %; m.p: 197.8–198.1 °C;
-
1
1
IR (m cm ): 2938, 2344, 1712, 1688, 1332, 1134, 790; H
NMR (DMSO-d , 400 MHz), d: 2.37 (s, 3H, –CH ), 3.58 (s,
Cytotoxicity assay: determination of the maximum non-
toxic concentration (MNTC)
6
3
3
H, –CO CH ), 3.83 (s, 3H, –OCH ), 6.01 (s, 1H, pyrimidine-
2 3 3
CH–), 7.06–6.97 (m, 2H, Ar–H), 7.26 (dd, 2H, J = 1.92,
.68 Hz, Ar–H), 7.55–7.48 (m, 3H, Ar–H), 7.69 (s, 1H, Ar–
The cytotoxicity of each compound to Vero cells was
determined using the method developed by Bhanuprakash
et al. (2008) with small modifications. In brief, initially the
compounds were dissolved in DMSO and different concen-
trations of compound with two-fold dilutions were prepared
in Eagle’s minimum maintenance medium (EMEM). Later,
added to the 24 h confluent Vero cell monolayer in 96-well
tissue culture plates and incubated in an atmosphere of 5 %
6
1
CH=) ppm; C (DMSO-d , 100 MHz), 22.49, 51.51, 54.41,
3
6
5
1
1
5
6.16, 102.14, 102.39, 108.04, 111.94, 118.70, 121.82,
24.26, 129.64, 129.92, 131.69, 139.46, 151.62, 155.48,
63.11, 164.16, 165.21; Anal. for C H BrFN O S; cacld; C
2
3
18
2 4
3.39, H 3.51, N 5.41; found; C 53.30, H 3.55, N 5.38; LC/MS
(
ESI–MS) m/z, (m/z, %), 519.0 (M?2).
CO at 37 °C for 4 days. Each concentration was tested in
2
triplicate along with controls. Cells were examined daily for
morphological changes (rounding, degeneration, cell lysis),
if any. Cells were observed at every 24 h interval for visible
morphological changes under inverted microscope (Leitz),
cell morphology was compared between treated and
untreated cultures (control). The highest concentration of the
synthetic compound that showed no cellular morphological
changes was considered as the maximum non-toxic con-
centration/dose (MNTC/MNTD). Furthermore, at the end of
incubation period, the test medium was removed from the
plate and washed gently with Phosphate buffered saline
Antimicrobial activity
The antimicrobial activity of synthesized compounds was
carried out using agar well-diffusion method. The bacterial
strains were collected from different infectious status of
patients who had not administered any antibacterial drugs
for at least two weeks with the suggestions of an authorized
physician, in Kiran diagnostic health centre of Chitradurga,
Karnataka state, India. Fungal strains were procured from
the culture maintained at National College of Pharmacy
Shimoga. The in vitro antimicrobial activity was carried
out against 24 h culture of four bacterial strains Gram-
positive S. aureus, B. subtilis, Gram-negative, S. typhi,
E. coli. Two fungal strains used were A. niger and
C. albicans. The stock solution of compounds was made at
(
(
0.1 M).Then, the wells were stained with crystal violet
1 %) in formalin (10 %) for 15 min. The cell viability was
evaluated as the percentage of the mean value of optical
density resulting from the 6 cell controls, which was set at
100 %. The 50 % cytotoxic concentration (CC ) was cal-
50
5
0 lg/100 lL and desired concentrations were prepared
culated from the mean dose-response of 3 independent
assays. The concentration of synthetic compounds at which
cell viability was above 80 % was further used in the study
for assessment of antiviral activity of the synthetic com-
pound against camelpox/buffalopox viruses in a dose-
dependent manner (Darshan Raj et al., 2012a, b).
using stock solution. DMSO was used as a solvent to dis-
solve compounds. Ciprofloxacin (40 lg in 100 lL) and
Fluconazole (50 lg in 100 lL) were used as standard drugs
for antibacterial and antifungal activities, respectively. The
zone of inhibition was compared with standard drug after
2
4 h of incubation at 37 °C for antibacterial activity and
2 h at 25 °C for antifungal activity. The zone of inhibition
7
was recorded in mm and given in Table 1.
Antiviral screening assay (cytopathic effect inhibitory
assay)
Minimum inhibitory concentrations (MIC)
Antiviral activity was determined for the compounds 3a,
3c, 4a, and 4c by reduction of virus titers using TCID50
determinations. Vero cells were grown in cell culture plates
containing 96 wells for 48 h. After decanting the growth
medium, the cells were treated with chemicals at their
respective MNTCs (50 lL/well) and immediately loga-
rithmic dilutions of virus (CMLV/BPXV) were added at
50 lL/well in treated and untreated cell cultures and
The MIC of all synthesized compounds 3a–f; 4a–f was
determined by a micro dilution method. The respective
clinical strain was spread separately on the medium. The
wells were created using a stainless steel sterilized cork
borer under aseptic conditions. The compounds at different
concentrations viz. 10, 20, 30, 40, and 50 lg in 100 lL of
DMSO and later was loaded into corresponding wells. The
standard drug Ciprofloxacin (50 lg in 100 lL) and
incubated at 37 °C in CO (5 %) for 24 h. After 24 h of
2
123

Med Chem Res
simultaneous incubation, both chemical and the virus were
discarded and the wells were replenished with fresh
maintenance medium. Subsequent change of medium was
done regularly at 48 h interval and the virus titers (TCID /
Fun HK, Loh WS, Sarojini BK, Umesha K, Narayana B (2011)
Methyl(2Z)-2-(2-fluoro-4-methoxybenzylidene)-5-(4-methoxy-
phenyl)-7-methyl-3-oxo-2,3-dihydro-5H-[1,3] thiazolo[3,2-a]
pyrimidine-6-carboxylate. Acta Crystallogr Sect E E67:o1913–
o1914
5
0
mL) were calculated after 6 days. The experiment was
carried out in triplicates. The antiviral activity was
expressed as percentage of inhibition (PI) using antiloga-
rithm values of TCID , as—PI = [1 - (T antilogarithm/C
Geist JG, Lauw S, Illarionova V, Illarionova B, Fischer M,
Grawert T, Rohdich F, Eisenreich W, Kaiser J, Groll M,
Scheurer C, Wittlin S, Alonso-Gomez JL, Schweizer WB,
Bacher A (2010) Thiazolopyrimidine inhibitors of 2-meth-
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MedChem 5:1092–1101
Halgren TA (1996a) Merck Molecular force field: I. Molecular
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Chem 17:553–586
5
0
antilogarithm)] 9 100. A chemical was considered active
when the PI is [80 % at its MNTC (Darshan Raj et al.,
2
012a early online).
Halgren TA (1996b) Merck molecular force field:II MMFF94Van der
Waals and electrostatic parameter for imtermolecular interac-
tion. J Comput Chem 17:520–552
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