Synthesis, molecular docking studies, and in vitro screening of barbiturates/thiobarbiturates as antibacterial and cholinesterase inhibitors

Article abstract of DOI:10.1007/s00044-013-0847-2

On the basis of observed biological activity of barbiturates/ thiobarbiturates, a set of 13 hydrazinecarboxamide/hydrazinecarbothioamides derivatives were designed and synthesized in good to excellent yield with extensive structural characterization. These compounds were screened for antibacterial and cholinesterase inhibitory activities. Two of the compounds 1 and 2 showed moderate bactericidal activity. Compounds 10 and 4 were found to be the most active acetyl/butyryl cholinesterase inhibitor, respectively (AChEI; 10; IC₅₀ = 40.78 μM and BChEI; 4; IC₅₀ = 3.31 μM). In silico molecular docking studies were carried out to identify active interacting sites of drug and enzyme and to establish structure-Activity relationships. When predicted cholinesterase binding energies were compared with the experimentally determined inhibitory concentrations (IC₅₀), most active compounds were also found to be the most favorable for binding. The binding scores of compounds 10 and 4 were -10.2 and -9.3 kcal/mol, respectively.

Full text of DOI:10.1007/s00044-013-0847-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0847-2
ORIGINAL RESEARCH
Synthesis, molecular docking studies, and in vitro screening
of barbiturates/thiobarbiturates as antibacterial
and cholinesterase inhibitors
•
Saira Mumtaz Rashad Hussain Abdul Rauf
•
•
•
•
•
¨
M. Q. Fatmi H. Bokhari M. Oelgemoller
A. M. Qureshi
Received: 9 July 2013 / Accepted: 14 October 2013
Ó Springer Science+Business Media New York 2013
Abstract On the basis of observed biological activity of
barbiturates/thiobarbiturates, a set of 13 hydrazinecarbox-
amide/hydrazinecarbothioamides derivatives were designed
and synthesized in good to excellent yield with extensive
structural characterization. These compounds were screened
for antibacterial and cholinesterase inhibitory activities. Two
of the compounds 1 and 2 showed moderate bactericidal
activity. Compounds 10 and 4 were found to be the most
active acetyl/butyryl cholinesterase inhibitor, respectively
Keywords Thiobarbituric acid ꢀ Thiosemicarbazone ꢀ
Hydrazinecarboxamide/hydrazinecarbothioamides ꢀ
Molecular modeling ꢀ Acetyl cholinesterase ꢀ
Butyryl cholinesterase
Introduction
One of the most desirable and excruciating process, in
pharmacy, is to develop new drugs with potential thera-
peutic applications. Recent technological advancements in
computational biology/chemistry have made possible that
the rational drug designing become more viable and less
tedious. For the target-oriented drug synthesis, mechanism
of action at molecular level is required. Molecular modeling
is one of the computational tools that can assist chemists by
establishing such structure activity relationships (SAR).
Molecular modeling gives valuable insight into the
mode of action of barbituric acid derivatives as hypnotic,
sedative, anti-urease, and antiepileptic therapeutic drugs
(Khan et al., 2011; Mihai et al., 2008). HIV-1 protease
inhibition by barbiturates was also subjected to predict new
active pharmacophores (Santos-Filho and Hopfinger, 2006;
Andrade et al., 2010; Pan et al., 2003).
(AChEI; 10; IC50 = 40.78 lM and BChEI; 4; IC50
=
3.31 lM). In silico molecular docking studies were carried
out to identify active interacting sites of drug and enzyme
and to establish structure–activity relationships. When pre-
dicted cholinesterase binding energies were compared with
the experimentally determined inhibitory concentrations
(IC50), most active compounds were also found to be the
most favorable for binding. The binding scores of compounds
10 and 4 were -10.2 and -9.3 kcal/mol, respectively.
S. Mumtaz ꢀ A. M. Qureshi (&)
Institute of Chemical Sciences, Bahauddin Zakariya University,
Multan 60800, Pakistan
e-mail: shamashfaq@yahoo.com; amqureshi@bzu.edu.pk
Moreover barbiturates are found to be active against
cholinesterase. On the account of their behavior and speci-
ficity to inhibitors, animal cholinesterase is classified into
acetyl cholinesterase (AChE) and butyryl cholinesterase
(BChE). Basically, AChE and BChE hydrolyzes two dif-
ferent classes of choline, i.e., acetylcholine and butyrylch-
R. Hussain ꢀ M. Q. Fatmi ꢀ H. Bokhari
Department of Biosciences, COMSATS Institute of Information
Technology, Chak Shahzad, Islamabad, Pakistan
A. Rauf
Department of Chemistry, The Islamia University, Bahawalpur,
Pakistan
¨
oline (Chatonnet and Lockridge, 1989; Ryhanen, 1983;
Ekholm, 2001). Besides sharing 65 % homology in amino
acid sequence, AChE and BChE, are encoded by the corre-
sponding genes located on different chromosomes. The
active center of AChE mainly consists of esteric site and
¨
M. Oelgemoller
James Cook University, School of Pharmacy and Molecular
Sciences, Townsville, QLD 4811, Australia
123

Med Chem Res
O
O
R
CH(OEt)3
2-Butanol,
HN
HN
NH
+
H2N
R
reflux for 3 hours
X
N
H
O
X
N
H
O
Scheme 1 X = O, R = COCH3, (2) X = O, R = NH–C=S–NH2,
(3) X = O, R = NH–C=ONHPh, (4) X = S, R = NH–C=ONHPh,
(5) X = S, R = NH–C=SNHPh, (6) X = S, R = NH–C=SNH-p-F-
Ph, (7) X = S, R = NH–C=SNH-p-Cl–Ph, (8) X = O, R = NH–
C=SNH–o–CH3, p-Cl–Ph, (9) X = S, R = NH–C=SNH-p-Br–Ph,
(10) X = O, R = NH–C=SNH-m-CF3–Ph, (11) X = S, R = NH–
C=SNH-p-CF3–Ph, (12) X = S, R = NH–C=SNH-p-OCH3–Ph, (13)
X = O, R = NH–C=SNH-p-CN–Ph
anionic site (Hucho et al., 1991; Quinn, 1987). AChE func-
tions in rapid hydrolysis of acetylcholine at cholinergic
synapses (Allderdice et al., 1991), while BChE is important
for detoxification and hydrolysis of ester-containing drugs,
as well as, scavenging of cholinesterase inhibitors, prior
reaching their synaptic targets. Cholinesterase inhibitors are
important for treatment of various diseases like organo-
phosphate poisoning (Sussman et al., 1991), myasthenia
gravis, glaucoma, and promisingly Alzhemier disease (AD)
(Gilman et al., 1990; Hallak and Giacobini, 1989).
DMSO. Of these, only 1 and 2 displayed moderate activity
against three strains, i.e., E. coli, P. aeruginosa, and S.
aureus (Tables 1, 2).
In vitro cholinesterase activity
Bioassay results for enzyme inhibition are represented in
Fig. 1 and Table 3. Nineteen compounds showed report-
able IC50 value against AChE of which 10 appeared as a
most potent inhibitor with IC50 value 40.78 lM at 0.5 mM.
On the whole all such compounds are mostly, excellent
BChE inhibitors. Compound 4 showed maximum inhibi-
tion, thus acts as the most potent inhibitor with IC50 value
of 3.31 0.08 lM at 0.125 mM followed by 11 and 8
with IC50 values of 8.11 and 8.81 lM at 0.5 mM, respec-
tively. Structure–activity relationship studies revealed that
electrostatic effects of the substituents play an important
role in the enzyme inhibitory potential of the synthetic
compounds.
In present study, anticholinesterase activity of 13 bar-
biturates/thiobarbiturates was evaluated. All synthesized
compounds were docked into active sites of AChE and
BChE and binding models were constructed.
Results and discussion
Chemistry
For synthesis, to a refluxing solution of thio/barbituric acid
and triethylorthoformate in 2-butanol was added respective
amine (Scheme 1). Precipitates formed were collected and
washed with hot ethanol offering pure products in excellent
yields (88–94 %) (Rauf et al., 2012). Structures of the
synthesized compounds were deduced by analytical and
spectroscopic (1H NMR, 13C NMR, and EIMS) data. Sat-
isfactory elemental analyses ( 0.4 % of calculated values)
were obtained for all the compounds. In EI mass spectra,
all the compounds showed molecular ions of different
intensity, which confirmed their molecular weights. Few of
the compounds did not show the molecular ion peaks in
their spectra. However, the fragments corresponding to
thiosemicarbazone moiety, formed by the cleavage of N–N
and NH–CS bonds confirmed their structures.
Docking
Biological assay and docking analysis
In order to rationalize ligand–protein interaction for
establishing SAR, molecular docking studies of all syn-
thesized barbiturate or thiobarbiturates were performed
against AChE and BChE. Compound 10 indicated a better
docking score, -10.20 kcal/mol, against AChE as com-
pared to the reference inhibitor, eserine, -7.10 kcal/mol.
Likewise, compounds 4, 11, and 8 exhibited better binding
energy values of -9.30, -9.50, and -8.50 kcal/mol,
respectively, against BChE as compared to that of the
reference inhibitor, eserine, i.e., -7.50 kcal/mol.
Binding model of compound 10 in the active site of
AChE is depicted in Fig. 2a, which clearly demonstrates
that both amino and amido groups of the ligand form
H-bonds with the carboxylic acid group of Tyr124 (bond
Antibacterial activity (in vitro)
˚
˚
distance 3.26 A) and Tyr337 (bond distance 3.26 A).
Another H-bond with comparatively short distance is
formed by the nitrogen of pyrimidinetrione ring with the
The compounds were subjected for bacterial activity against
three Gram-negative (Escherichia coli, Pseudomonas
aeruginosa, and Salmonella typhi) and one Gram-positive
(Staphylococcus aureus) bacterial strains at 1 mg/mL in
˚
oxygen of Ser203 (bond distance 2.97 A). Compound 10
123

Med Chem Res
Table 1 Antibacterial activity (1)–(13)
Compound (1 mg/mL)
Zone of inhibition in ‘‘mm’’
E.coli
P. aerginosa
S. typhi
V. cheolera
S. aureous
DH5a
DG2
5604
1
15
14
25
–
–
–
–
–
–
18
–
–
–
–
–
–
–
18
15
25
7
Chloramphenicol
25
– No activity
Table 2 Minimum inhibitory concentration of the active compounds
interactions with pyrimidinetrione ring of the inhibitor,
which clearly demonstrate that a significant contribution
for ligand binding has come through van der Waal inter-
actions. The order of activity among 13 compounds as
determined experimentally is 10 [ 4 [ 6 [ 9 [ 1 [
11 [ 5 [ 13 [ 2 [ 7. The binding models of these com-
pounds with AChE indicate Ser203, Tyr124, Tyr337,
Tyr341, Trp86, Trp286, Phe297, Phe338, Glu202, and
His447 as common residues, involved in ligand interac-
tions, and therefore, indicate their importance in ligand
binding as well as in possible catalytic activities. These
residues may also play a vital role in lead optimization, and
thereby, aid in improving the ligand affinity with the pro-
tein. Other residues that interact with receptor site but not
present commonly are Ala204, Phe295, Asp74, Tyr72, and
Gly122 (Table 4).
Compound
MIC (lg/mL)
E. coli (DH5 a)
P. aerginosa
S. aureous
1
100
200
1.56
200
400
1.56
100
7
100
Chloramphenicol
\0.78
Inhibition (%)
100
90
80
70
60
50
40
30
20
10
0
AChE
BChE
Binding models of 4, 11, and 8 in the active site of
BChE are depicted in Fig. 2b–d, respectively. Compound 4
exhibits H-bond with the active site residue, Gly117, of
BChE through the oxygen of acetamide group with a dis-
1
2
3
4
5
6
7
8
9
10 11 12 13
˚
tance of 3.39 A. A comparatively weak H-bond can also be
observed between the nitrogen of acetamide group and the
˚
carboxylic acid moiety of Ser198 (bond distance 4.00 A).
IC 50 Value (µM)
250
Both nitrogen atoms of thiopyrimidinedione ring are
involved in H-bonding with the same oxygen of Gly115
200
150
100
50
˚
˚
(bond distance 3.15 A) and Thr120 (bond distance 3.39 A).
Compound 4 also contribute in binding through p–p
interaction with Trp231, and interact electrostatically with
Glu197 and Ala199. Whereas, residues Tyr114, Ile69,
Leu125, Phe329, Phe398, and Trp82 are involved in van
der Waal interactions with thiopyrimidinedione ring of the
inhibitor (Fig. 2b). The nitrogen of hydrazine group in
compound 11 displays a moderately strong H-bond with
AChE
BChE
0
1
2
3
4
5
6
7
8
9
10 11 12 13
˚
the hydroxyl group of Tyr332 (bond distance 2.87 A).
Furthermore, both nitrogen atoms of thiopyrimidinedione
ring show H-bond interactions with hydroxyl groups of
˚
Asn83 (bond distance 3.02 A) and Thr120 (bond distance
Fig. 1 Graphical representation of compounds (1–13) activity in
terms of % inhibition (a) and IC50 values (b), where series1: Acetyl
cholinesterase; and series2: Butyryl cholinesterase
˚
2.93 A). A p–p interaction can also be observed in com-
also exhibits p–p interactions by each Trp286 and Tyr341
(Fig. 2a). Most of the residues such as Ala204, Trp86,
Phe297, and Phe338 are involved in van der Waal
pound 11, where Trp82 of BChE is involved with triflo-
urobenzene ring of the ligand. Residues His438 and Tyr128
are interacting electrostatically with the compound 11,
123

Med Chem Res
Table 3 Acetly and butryl cholinesterase inhibition with their IC50 values
Compound structure and code
Inhibition %
AChE
IC50 (lmol)
Binding energy (kcal/mol)
BChE
AChE
BChE
AChE
BChE
O
77.65 0.72a 88.26 0.71a 90.91 0.33a
116.61 0.24a
-7.90
-7.10
O
HN
N
H
1
O
N
H
O
65.75 0.71a 87.52 0.92a 119.11 0.24a
56.51 0.18a
38.51 0.01b
3.31 0.08b
57.11 0.36a
57.81 0.14a
75.41 0.28a
8.81 0.02
-7.60
-10.20
-9.40
-8.80
-8.80
-9.40
-9.90
-6.40
-9.60
-9.30
-8.50
-8.60
-8.20
-8.50
O
H
N
NH2
HN
N
H
S
2
O
N
H
O
39.61 0.12b 79.21 0.11b NILb
O
H
N
H
N
HN
N
H
O
O
N
H
O
3
66.80 0.28b 85.12 0.06b 74.21 0.01b
73.23 0.44a 83.18 0.14a 115.11 0.17a
86.93 0.64a 88.82 0.24a 78.41 0.25a
61.96 0.14a 81.61 0.42a 121.11 0.01a
O
H
H
N
N
HN
N
H
O
S
N
H
O
4
O
H
H
N
N
HN
N
H
S
S
N
H
O
5
O
H
H
N
N
HN
N
H
S
S
N
H
O
6
F
O
H
H
N
N
HN
N
H
S
S
N
H
O
7
Cl
49.41 0.56
75.79 0.64
NIL
O
H
N
H
N
HN
N
H
S
O
N
H
O
8
Cl
80.39 0.17a 84.47 0.74a 79.21 0.08a
68.41 0.21a
136.91 0.08
8.11 0.24a
-9.40
-10.20
-9.90
-8.10
-9.30
-9.50
O
H
H
N
N
HN
N
H
S
S
N
H
O
9
Br
83.26 0.35
75.88 0.63
40.78 0.35
O
H
N
H
N
CF3
HN
HN
HN
N
H
S
O
S
S
N
H
O
10
75.56 0.11a 78.19 0.45a 99.41 0.31a
O
H
N
H
N
N
H
S
N
H
O
11
CF3
O
59.08 0.63a 85.41 0.44a 206.31 0.06a
56.81 0.02a
-8.80
-8.10
H
H
N
N
N
H
S
N
H
O
12
OCH3
123

Med Chem Res
Table 3 continued
Compound structure and code
Inhibition %
AChE
IC50 (lmol)
Binding energy (kcal/mol)
BChE
AChE
BChE
AChE
BChE
O
11.37 0.62a 66.82 0.28a NILa
231.61 0.55a
-10.10
-8.80
H
N
H
N
HN
N
H
S
O
N
O
13
H
CN
REserine
92.54a
92.54a
0.04 0.001a
0.85 0.001a -7.10
-7.50
Methanol was used as solvent and concentration of all the samples was 0.5 mM
R reference inhibitor of the enzyme
a
b
Stands for 0.25 mM concentration of sample and for 0.125 mM
whereas, Pro84 and Gly121 are involved in van der Waal
interactions with thiopyrimidinedione ring of the inhibitor
(Fig. 2c). Compound 8 binds to BChE through nitrogen as
well as oxygen atoms of pyrimidinetrione ring, and forms
H-bonds with hydroxyl group of Pro285 (bond distance
Results from in vitro and docking studies revealed similar
outcomes, regarding inhibitory activity on AChE, and
BChE. Present work will add in existing knowledge about
inhibition mechanism of AChEI and BChEI, such as, a
better understanding of interaction of active sites of
enzyme with barbiturates or thiobarbiturattes.
˚
3.38 A) and amino group of Gly117 (bond distance
˚
2.88 A), respectively. One comparatively weaker H-bond
between the nitrogen of hydrazine group of ligand and the
˚
hydroxyl group of Ser198 (bond distance 4.24 A) can also
Experimental
be observed. Compound 8 is further involved in p–p
interaction with Trp82 through its chloromethyl benzene
ring. BChE residues Glu197, His438, Tyr332, and Tyr440
are interacting electrostatically with the ligand, whereas
Ala199, Phe329, and Phe398 are involved in van der Waal
interactions with pyrimidinetrione ring and Trp430 with
chloromethyl benzene ring of the inhibitor (Fig. 2d). On
the basis of experimental IC50 values, the activity of
compounds is sorted as 4 [ 11 [ 8 [ 3 [ 12 [ 5 [ 6 [
9 [ 2. With few exceptions, this trend in activity gets
support from docking studies as well (Table 3). Common
residues which are involved in complex interaction are:
Trp82, His438, Trp231, Ala199, Phe398, Tyr332, Tyr128,
Pro285, and Ser198. Other residues that interact with the
receptor site but not present commonly are Gly115,
Gly117, Asn83, Thr120, Glu197, Tyr430, and Tyr440
(Table 5). These common interacting residues in BChE
highlight the role of these residues in ligand binding as well
as in their catalytic activities, which can further be
explored by mutational dynamics simulation studies, and
verified by experiments.
Melting points were taken on a Fisher-Johns melting point
apparatus and are uncorrected. Elemental analyses were
1
performed on a Leco CHNS-9320 elemental analyzer. H
and 13C NMR spectra were recorded in DMSO-d6 on
Bruker (Rhenistetten-Forchheim, Germany) NMR spec-
trometer at 300 and 75.4 MHz respectively. The electron
impact mass spectra (EIMS) were determined with a
Finnigan MAT-312 and
spectrometer.
a JEOL MS Route mass
General procedure for the preparation of compounds
(1)–(13)
To a hot stirred solution of thio/barbituric acid (2.00 mmol)
and ethylorthoformate (2.02 mmol) in 2-butanol (10 mL)
was added the respective amine (2.00 mmol). Then the
reaction mixture was refluxed till the completion of the
reaction (for &3 h). The precipitates formed were col-
lected by suction filtration. Washing with hot ethanol
afforded pure products in good to excellent yield.
N-[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]
acetamide (1) Yield 88 % as yellow solid; m.p. 240 °C;
1H NMR (300 MHz, DMSO-d6, d, ppm): 2.83 (3H, s,
CH3), 4.51 (1H, s, NHCOCH3), 7.82 (1H, s, =CH), 10.51
(2H, s, NHBA); 13C NMR (300 MHz, DMSO-d6, d, ppm):
170 (C=O amide), 150 (C=O amide), 145 (CH ethylene),
95 (C ethylene), 25 (CH3); EIMS (70 eV) m/z (%): ([M?],
Conclusions
In conclusion, several barbiturates and thiobarbiturates
1
were synthesized and characterized through H NMR, 13C
NMR, EIMS, and elemental analysis. Antibacterial assay
showed that two of the compounds were found to be
moderately active against three different bacterial strains.
123

Med Chem Res
Fig. 2 Complex binding models of (a) 32 with AChE and (b–d) 10, 34, and 24, respectively, with BChE. Protons are omitted for clarity
N-phenyl-2-[(2,4,6-trioxotetrahydropyrimidin-5(2H)-yli-
dene)methyl]hydrazinecarbox amide (3) Yield 90 % as
yellow solid; m.p. 220 °C; 1H NMR (300 MHz, DMSO-d6,
d, ppm): 5.65 (2H, s, NHAr), 7.04 (1H, m, ArH), 7.26 (2H,
m, ArH), 7.48 (1H, m, ArH), 7.96 (1H, d, J 9, ArH), 8.77
(1H, s, =CH), 10.50 (2H, s, NH NH),), 11.00 (2H, s,
NHBA); 13C NMR (300 MHz, DMSO-d6, d, ppm), 158
(C=O amide), 154 (C=O amide), 150 (CH ethylene), 139
(C benzene), 128(CH benzene), 121(CH benzene), 118
(CH benzene), 89 (C ethylene); EIMS (70 eV) m/z (%):
([M?], 289 (2)), 261 (5), 231 (3), 199 (3), 170 (22), 154
(15), 128 (5), 119 (100), 93 (94), 77 (20), 66 (34), 51 (8),
43 (14); (Found: C, 50.01; H, 3.45; N, 24.29, O %. Calc.
For C12H10N5O4: C, 50.00; H, 3.47; N, 24.31 %).
197 (100)), 183 (61), 155 (48), 140 (22), 127 (6), 77 (11),
63 (14), 50 (8), 44 (17); (Found: C, 42.66; H, 3.53;
N, 21.33 %. Calc. For C7H7N3O4: C, 42.64; H, 3.55; N, 21.
32 %).
2-[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]
hydrazinecarbothioamide (2) Yield 90 % as orange yel-
low solid; m.p. 210 °C; 1H NMR (300 MHz, DMSO-d6, d,
ppm): 7.81 (3H, s, NH2), 9.14 (1H, s, =CH), 10.12 (2H, s,
NH NH), 11.11 (2H, s, NHBA); 13C NMR (300 MHz,
DMSO-d6, d, ppm) 181(C=S thiamide), 164 (C=O amide),
163 (C=O amide), 151 (C=O amide), 116 (CH ethylene),
96 (C ethylene); EIMS (70 eV) m/z (%): ([M?], 229
(100)), 186 (61), 158 (48), 144 (22), 136 (22), 127 (6), 115
(20), 101 (5), 89 (25), 77 (11), 63 (14), 50 (8), 44 (17);
(Found: C, 31.45; H, 3.08; N, 30.55; S, 13.
96 %. Calc. For C6H7N5O3S: C, 31.44; H, 3.06; N, 30.57;
S, 13.97 %).
2-[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)
methyl]-N-phenylhydrazine carboxamide (4) Yield 92 %
1
as yellow solid; m.p. 274 (dec.) °C; H NMR (300 MHz,
123

Med Chem Res
Table 4 Interactions of AChE against selected top 10 active inhibitors based on IC50 value
CN H-bonding
Interacting residues
p–p Interactions
Electrostatic interactions VDW
˚
Distance (A)
10 Ser203:OH_N:C4H2N2O3
Tyr337:OH_N:NHNH2
2.97
3.26
3.26
Trp286 with C6H4–CF3
Tyr341 with C6H4–CF3
*Ala204 with C4H2N2O3
Trp86 with C4H2N2O3
Phe338 with C4H2N2O3 ring
Phe297 C4H2N2O3
Tyr124:OH_N:NHNH2
Gly122 C4H2N2O3
4
Gly120:b OH_N:C4H2N2O2S 3.76
Tyr341 with C6H5–NH Glu202 with C6H5–NH
His447 with NHNH2
Trp86 with C4H2N2O2S
Ala204 with C4H2N2O2S
Trp286 with C6H5–NH
Phe295 with C6H5–NH
Phe297 with C6H5–NH
Phe338 with C6H5–NH
Ser203:OH_N:C4H2N2O2S
Tyr337:OH_N:NHNH2
Tyr124:OH_N:NHNH2
3.18
2.80
2.77
6
Tyr124:OH_N:NHNH2
3.34
Tyr341 with C6H4F
Glu202 with C4H2N2O2S Trp86 withC4H2N2O2S
Gly120:b OH_N:C4H2N2O2S 3.54
His447 with NHNH2
Ala204 with C4H2N2O2S
Phe297 with C6H4F
Phe338 with C6H4F
Phe295 with C6H4F
Trp286 with C6H4F
Tyr337:OH_N:NHNH2
2.99
9
1
Tyr124:OH_N:NHNH2
Ser203:OH_N:C4H2N2O2S
Tyr337:OH_N:NHNH2
2.72
3.16
3.15
Tyr341 with C6H4Br
Trp286 with C6H4Br
Glu202 with C4H2N2O2S Phe297 with NHNH2
His447 with C4H2N2O2S Phe295 C4H2N2O2S
Phe338 with C4H2N2O2S & NHNH2
Tyr72 withC6H4Br
Trp86 with C4H2N2O2S
Gly126:b NH_O:CONH
Tyr337:OH_N:C4H2N2O3
His447:b OH_N:C4H2N2O3
3.32
3.34
3.36
3.19
3.14
3.19
Glu202 with C4H2N2O3 Trp86 with C4H2N2O3
His447 with C4H2N2O3
Trp439 with C4H2N2O3 (weak)
Phe338 with C4H2N2O3
11 Tyr337:OH_N:NHNH2
Ser203:OH_N:C4H2N2O2S
Tyr124:OH_N:NHNH2
Tyr341 with C6H4–CF3 Glu202 with C4H2N2O2S Phe297 with NHNH2
Trp286 with C6H4–CF3 His447 with C4H2N2O2S Phe295 C4H2N2O2S
Phe338 with C4H2N2O2S & NHNH2
Tyr72 with C6H4–CF3
Trp86 with C4H2N2O2S
Trp86 with C4H2N2O2S
5
Tyr124:OH_N:NHNH2
2.81, 3.26
3.03
Trp286 with C6H5–NH ASP74 with C6H5–NH
Ser203:OH_N:C4H2N2O2S
Tyr341 with C6H5–NH Glu202 with C4H2N2O2S Ala204 with C4H2N2O2S
His447 with C4H2N2O2S Phe297 with NHNH2
Tyr72 with C6H5–NH
Phe338 with NHNH2
13 Ser203:OH_N:C4H2N2O3
Tyr337:OH_N:NHNH2
3.00
3.32
2.80
2.96
3.27
3.08
2.82
3.14
3.23
2.71
Tyr341 with C6H4–CN ASP74 with C6H4–CN
Phe338 with NHNH2
Trp286 with C6H4–CN Glu202 with C4H2N2O3 Phe297 with C4H2N2O3
Tyr124:OH_N:NHNH2
His447 with C4H2N2O3
ASP74 with CSNH
Tyr72 with C6H4–CN
Trp86 with NHNH2
2
Tyr337:OH_N:NHNH2
Tyr341:OH_N:CSNH
Asp74:O–N:CSNH
Glu202 with C4H2N2O3 Ala204 with C4H2N2O3
His447 with C4H2N2O3
Phe297 with C4H2N2O3
Phe295 with C4H2N2O3
Trp86 with C4H2N2O2S
Ser203:OH_N:C4H2N2O3
Ser203:OH_N:C4H2N2O2S
Tyr337:OH_N:NHNH2
Tyr124:OH_N:NHNH2
7
Tyr341 with C6H4Cl
Trp286 with C6H4Cl
ASP74 with C6H4Cl
Glu202 with C4H2N2O2S Ala204 with C4H2N2O2S
His447 with C4H2N2O2S Phe297 with NHNH2
Phe295 with C4H2N2O2S
123

Med Chem Res
Table 5 Interactions of BChE selected top 9 active inhibitors based on IC50 value
CN H-bonding
Interacting residues
p–p Interactions
Electrostatic interactions VDW
˚
Distance (A)
4
Thr120:O_N:C4H2N2O2S
Gly117:b NH_O:CONH
Ser198:OH_N:CONH
3.39
3.39
4
Glu197 with NHNH2
Ala199 with CO
Tyr114 with C4H2N2O2S
Ile69 with C4H2N2O2S
Leu125 with C4H2N2O2S
Phe398 with C6H5–NH
Trp231 with C6H5–NH
Phe329 with C6H5–NH
Trp82 with C4H2N2O2S
Pro84 with C4H2N2O2S
Gly121 with C4H2N2O2S
Gly115:b OH_N:NHNH2
3.15
11 Asn83:b OH_N:C4H2N2O2S 3.02
Trp82 with C6H4–CF3
Trp82 with C7H6Cl
His438 with CONH
Tyr332:OH_N:NHNH2
Thr120:O_N:C4H2N2O2S
Pro285:O_N:C4H2N2O3
Gly117:N_O: C4H2N2O3
Ser198:OH_N:NHNH2
2.87
2.93
3.38
2.881
4.24
Tyr128 with C6H4–CF3
8
3
Glu197 with CSNH
His438 with NHNH2
Tyr440 with C7H6Cl
Tyr332 with C7H6Cl
His438 with C4H2N2O3
Ala199 with CO
Phe329 with C4H2N2O3 and NHNH2
Phe398 with C4H2N2O3
Ala199 with C4H2N2O3
Trp430 with C7H6Cl
Ser198:OH_O:CO
3.03
3.22
3.62
Trp82 with C4H2N2O3
Tyr440 with C4H2N2O3
Trp231 with C6H5–NH
Gly439 with C4H2N2O3
Gly117 with NHNH2
His438:OH_N:C4H2N2O3
Trp82:NH_N:C4H2N2O3
Gly115 with NHNH2
Phe329 with C6H5–NH
Phe398 with C6H5–NH
12 Ser287:b OH_O:C4H2N2O2S 3.46
Trp82 with C6H4–OCH3 Tyr332 with C6H4–OCH3 Trp231 with C4H2N2O2S
Ser287:OH_O:C4H2N2O2S
His438:N_N:NHNH2
2.77
3.21
Glu197 with NHNH2
Trp430 with C6H4–OCH3
Tyr128 with CSNH
Gly78 with C6H4–OCH3
Gly439 with C6H4–OCH3
Ala199 with C4H2N2O2S
Pro285 with C4H2N2O2S
Pro285:b OH_N:CSNH
His438:NH_O:CSNH
3.27
2.99
Glu197 with C4H2N2O2S Phe398 with C6H5–NH
5
6
–
Ala199 with C6H5–NH
Phe329 with C6H5–NH
Phe398 with C6H5–NH
Trp231 with C6H5–NH
Trp82 with C4H2N2O2S
Tyr128:OH_O:C4H2N2O2S 2.78
Pro285:b OH_N:CSNH
Glu197:O_N:C4H2N2O2S
3.24
3.31
His438 with C4H2N2O2S Trp231 with C6H4F
Tyr128 with C4H2N2O2S Phe329 with C6H4F
Glu197 with C4H2N2O2S Phe398 with C6H4F
Ala199 with C6H4F
Gly115:b N_O:C4H2N2O2S 3.68
9
2
Asn83:b OH_N:C4H2N2O2S 2.92
Trp82 with C6H4Br
Glu197 with C6H4Br
His438 with C6H4Br
Asp70:O_N:NHNH2
3.04
2.87
3.18
3.29
3.01
Phe329 with CSNH
Tyr332:OH_N:NHNH2
Ser198:OH_N:C4H2N2O3
Trp231:N_O:C4H2N2O3
His438:NH_O:C4H2N2O3
Trp82 with C6H4Br
123

Med Chem Res
DMSO-d6, d, ppm): 4.59 (1H, s, NHAr), 7.00 (1H, m,
ArH), 7.20 (2H, m, ArH), 7.40 (2H, m, ArH), 8.77 (1H, s,
=CH), 9.34 (2H, s, NHNH), 12.01 (2H, s, NHBA); 13C
NMR (300 MHz, DMSO-d6, d, ppm) 197 (C=S thiamide),
177 (C=O amide), 161 (C=O amide), 157 (CH ethylene),
155 (C benzene), 128 (CH benzene), 122 (CH benzene),
118 (CH benzene), 91 (C ethylene); EIMS (70 eV) m/z (%):
([M?], 305 (3)), 274 (2), 247 (5), 199 (3), 177 (35), 151
(51), 119 (100), 93 (89), 77 (54), 64 (48), 51 (16), 44 (51);
(Found: C, 47.38; H, 3.27; N, 23.04; S, 10.55 %. Calc. For
C12H10N5O3S: C, 47.37; H, 3.29; N, 23.03; S, 10.53 %).
197 (C=S thiamide), 167 (C=O amide), 163 (C=O amide),
151 (CCl benzene), 148 (CH ethylene), 138 (C benzene),
127 (CH benzene), 118 (CH benzene); EIMS (70 eV) m/z
(%): ([M?], 355 (2)), 339(3), 304 (4), 288 (3), 272 (3),
242(2), 211 (9), 201(11), 184 (13), 169(100), 141 (5), 111
(67), 101 (6), 90 (16), 75 (32), 65 (14), 44 (47); (Found: C,
40.50; H, 2.82; N, 12.69; S, 18.01 %. Calc. For
C12H10N5O2S2Cl: C, 40.51; H, 2.81; N, 12.69; S, 18.00 %).
N-(2-chloro-6-methylphenyl)-2-[(2,4,6-trioxotetrahydropy-
rimidin-5(2H)-ylidene)methyl] hydrazinecarbothioamide
1
(8) Yield 91 % as yellow solid; m.p. 310 (dec.) °C; H
2-[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)
methyl]-N-phenylhydrazine carbothioamide (5) Yield
93 % as yellow solid; m.p. 180 (dec.) °C; 1H NMR
(300 MHz, DMSO-d6, d, ppm): 4.28 (1H, s, NHAr), 6.97
(1H, m, ArH), 7.01 (1H, m, ArH), 7.13 (1H, t, J 9,15,
ArH), 7.33 (1H, t, J 9,15, ArH), 7.55 (1H, d, J 6, ArH), 8.
03 (1H, s, =CH), 9.76 (2H, s, NHNH), 11.41 (1H, s,
NHBA), 11.76 (1H, s, NHBA); 13C NMR (300 MHz,
DMSO-d6, d, ppm) 179 (C=S thiamide), 167 (C=O amide),
160 (C=O amide), 153 (CH ethylene), 139 (C benzene),
129 (CH benzene), 128 (CH benzene), 124 (CH benzene),
113 (CH benzene), 99 (C ethylene); EIMS (70 eV) m/z (%):
([M?], 321 (3)), 268 (27), 247 (75), 185 (2), 167 (20), 143
(5), 135 (74), 116 (54), 104 (12), 93 (33), 77 (71), 69 (36),
59 (21), 43 (45); (Found: C, 44.87; H, 3.42; N, 21.80; S, 19.
93 %. Calc. For C12H11N5O2S2: C, 44.86; H, 3.43; N, 21.
81; S, 19.94 %).
NMR (300 MHz, DMSO-d6, d, ppm): 3.86 (3H, s, CH3), 4.
38 (1H, s, NHAr), 7.37 (1H, m, ArH), 7.58 (2H, m, ArH),
8.50 (1H, s, =CH), 9.89 (2H, s, NHNH), 11.03 (2H, s,
NHBA); 13C NMR (300 MHz, DMSO-d6, d, ppm) 197 (C=
S thiamide), 168 (C=O amide), 163 (C=O amide), 151 (CCl
benzene), 137 (CH ethylene), 126 (C benzene), 117 (CH
benzene), 91 (CH benzene), 87 (C ethylene), 18 (CH3);
EIMS (70 eV) m/z (%): ([M?], 353 (2)), 339 (3), 304 (78),
288 (3), 279 (46), 229 (2), 211 (9), 201 (11), 184 (13),
169(100), 141 (5), 128 (67), 106 (6), 85 (16), 69 (32), 51
(14), 42 (47); (Found: C, 44.02; H, 3.68; N, 19.74; S, 9.
02 %. Calc. For C13H13N5O3SCl: C, 44.01; H, 3.67; N, 19.
75; S, 9.03 %).
N-(4-bromophenyl)-2-[(4,6-dioxo-2-thioxotetrahydropyrim-
idin-5(2H)-ylidene)methyl] hydrazinecarbothioamide
(9) Yield 89 % as yellowish orange solid; m.p. 220 (dec.)
1
°C; H NMR (300 MHz, DMSO-d6, d, ppm): 4.60 (1H, s,
2-[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)
methyl]-N-(4-fluorophenyl) hydrazinecarbothioamide
NHAr), 7.25 (4H, s, ArH), 8.01 (1H, s, =CH), 10.07 (2H, s,
NHNH), 11.12 (2H, s, NHBA); 13C NMR (300 MHz,
DMSO-d6, d, ppm) 177 (C=S thiamide), 163 (C=O amide),
140 (CBr benzene), 138 (CH ethylene), 131 (C benzene),
130 (CH benzene), 125 (CH benzene), 118 (CH benzene),
113 (CH benzene), 79 (C ethylene); EIMS (70 eV) m/z
(%): ([M?/M?2], 400/402 (4/4)), 352 (61), 287 (10), 247
(4), 230 (6), 213/215 (100/94), 198 (18), 171 (48), 155
(22), 134 (22), 117 (20), 102 (5), 75 (11), 44 (17);
(Found: C, 36.08; H, 2.53; N, 17.53; S, 16.03 %. Calc.
For C12H10N5O2S2Br: C, 36.09; H, 2.51; N, 17.54;
S, 16.04 %).
1
(6) Yield 92 % as yellow solid; m.p. 218 °C; H NMR
(300 MHz, DMSO-d6, d, ppm): 5.00 (1H, s, NHAr), 7.49
(2H, m, ArH), 7.17 (2H, m, ArH), 8.13 (1H, s, =CH), 9.91
(2H, s, NHNH), 11.99 (2H, s, NHBA); 13C NMR
(300 MHz, DMSO-d6, d, ppm) 177 (C=S thiamide), 161
(C=O amide), 153 (CF benzene), 135 (CH ethylene), 128
(C benzene), 125 (CH benzene), 115 (CH benzene), 114
(CH benzene), 90 (C ethylene); EIMS (70 eV) m/z (%):
304 (13), 265 (76), 227 (14), 207 (4), 195 (15), 186 (81),
171 (24), 153 (87), 144 (49), 126 (67), 116 (18), 111 (100),
98 (2), 95 (37), 84 (42), 69 (17), 59 (24), 43 (13); (Found:
C, 42.49; H, 2.94; N, 20.66; S, 18.89 %. Calc. For
C12H10N5O2S2F: C, 42.48; H, 2.95; N, 20.65; S, 18.88 %).
N-[3-(trifluoromethyl)phenyl]-2-[(2,4,6-trioxotetrahydropy-
rimidin-5(2H)-ylidene)methyl] hydrazinecarbothioamide
(10) Yield 88 % as mustard solid; m.p. 238 °C; 1H NMR
(300 MHz, DMSO-d6, d, ppm): 4.41 (1H, s, NHAr), 7.38
(1H, s, ArH), 7.51 (3H, m, ArH), 8.12 (1H, s, =CH), 10.31
(2H, s, NHNH), 11.00 (2H, s, NHBA); 13C NMR
(300 MHz, DMSO-d6, d, ppm) 197 (C=S thiamide), 167
(C=O amide), 154 (CH ethylene), 151(C benzene), 150
(CH benzene), 131 (CH benzene), 123 (CF3), 99 (C
N-(4-chlorophenyl)-2-[(4,6-dioxo-2-thioxotetrahydropy-
rimidin-5(2H)-ylidene)methyl] hydrazinecarbothioamide
1
(7) Yield 90 % as yellow solid; m.p. 220 °C; H NMR
(300 MHz, DMSO-d6, d, ppm): 4.38 (1H, s, NHAr), 7.37
(2H, d, J 9, ArH), 7.58 (2H, d, J 9, ArH), 8.04 (1H, s, =
CH), 9.84 (2H, s, NHNH), 11.39 (1H, s, NHBA), 11.76
(1H, s, NHBA); 13C NMR (300 MHz, DMSO-d6, d, ppm)
123

Med Chem Res
ethylene); EIMS (70 eV) m/z (%): ([M?], 373 (2)), 345
(5), 293 (20), 251 (5), 234 (10), 218 (2), 202 (20), 188 (23),
177 (23), 150 (31), 128 (15), 100 (15), 85 (9), 69 (17), 42
(14); (Found: C, 41.81; H, 2.67; N, 18.78, S, 8.59 %. Calc.
For C13H10N5O3SF3: C, 41.82; H, 2.68; N, 18.77; S, 8.
58 %).
(20), 64 (7), 51 (7); (Found: C, 47.26; H, 3.04; N, 25.46; S,
9.72 %. Calc. For C13H10N6O3S: C, 47.27; H, 3.03; N, 25.
45; S, 9.70 %).
Antibacterial activity (in vitro)
All the synthesized compounds were screened for their
antibacterial activity against E. coli, P. aeruginosa, S. typhi,
and S. aureus, by agar-well diffusion method (Olaleye,
2007). Nutrient agar plates were prepared by pouring
20–25 mL autoclaved nutrient agar into sterile plates and
allowed to solidify. The plates were swabbed with 2–8-h-
old bacterial inoculums containing approximately 104–106
colony forming units (CFU/mL) (already prepared and kept
at 37 °C). The wells (6 mm in diameter) were dug in media
with sterile metallic borer followed by the addition of 50 lL
of test solution (1 mg/mL in DMSO) into the respective
wells. Other wells supplemented with DMSO and reference
antibacterial drug, chloramphenicol, served as negative and
positive controls, respectively. The plates were incubated at
37 °C for 24 h, after that the zone of inhibition around each
well was measured using a transparent scale.
2-[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)
methyl]-N-[4-(trifluoromethyl)
phenyl]hydrazinecarbo-
thioamide (11) Yield 91 % as yellow solid; m.p. 280
1
(dec.) °C; H NMR (300 MHz, DMSO-d6, d, ppm): 4.37
(1H, s, NHAr), 7.37 (2H, d, J 9, ArH), 7.75 (2H, d, J 9,
ArH), 8.00 (1H, s, =CH), 9.70 (2H, s, NHNH), 11.15 (1H,
s, NHBA), 11.40 (1H, s, NHBA); 13C NMR (300 MHz,
DMSO-d6, d, ppm) 192 (C=S thiamide), 163 (C=O amide),
121 (CH benzene), 120 (CH benzene), 118 (CF3), 99 (C
ethylene); EIMS (70 eV) m/z (%): ([M?], 389 (2)), 367
(5), 345 (5), 293 (20), 251 (5), 234 (10), 218 (2), 202 (20),
188 (23), 177 (23), 150 (31), 122 (15), 108 (15), 95 (9), 69
(17), 44 (14); (Found: C, 40.11; H, 2.58; N, 17.99; S, 16.
47 %. Calc. For C13H10N5O2S2F3: C, 40.10; H, 2.59; N,
17.99; S, 16.45 %).
Minimum inhibitory concentration (MIC) values of
these test samples were determined by ‘‘Micro-titer assay’’
as reported by (Sarker et al., 2007) using 2,3,5-triphenyl-
tetrazolium chloride (TTC) as indicator solution. For this a
stock solution of concentrations 4,000, 2,000, 1,000, 500,
250, 125, 62.5, and 31.25 lg/mL of each test compound
and positive control (Chloramphenicol) was prepared in
DMSO by serial dilution, while the bacterial culture was
maintained in nutrient broth. Twenty microliters from each
of the above-mentioned stock solutions was added into the
respective well of 96-well plate followed by the addition of
180 lL of bacterial suspension (108 CFU/mL) such that
each test compound was checked at the final concentration
of 400, 200, 100, 50, 25, 12.5, 6.25, and 3.12 lg/mL.
Controls were used on each plate, a broad spectrum anti-
biotic as positive control (Chloramphenicol), negative
control (DMSO), and blank (bacterial suspension in nutri-
ent broth). The plates were prepared in triplicate and
incubated at 37 °C for 18–24 h. After incubation, to each
well 20 lL of TTC indicator solution (prepared as
5 mg/mL DꢀH2O) was added, and plates were incubated
again for 15–20 min. The change in color was then
observed visually. Any color change toward pink indicated
the presence of viable bacteria, while no change in color
was considered as dead bacteria. The MIC values were
taken as lowest concentration at which no color change
appeared. Experiment was repeated three times and the
average of three readings was taken as MIC for the test
compound against a specific bacteria.
2-[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)
methyl]-N-(4-methoxyphenyl) hydrazinecarbothioamide
1
(12) Yield 91 % as yellow solid; m.p. 230 (dec.) °C; H
NMR (300 MHz, DMSO-d6, d, ppm): 3.75 (3H, s, OCH3),
4.60 (1H, s, NHAr), 6.90 (2H, dd, J 3,9, ArH), 7.38 (2H, d,
J 9, ArH), 9.53 (1H, s, =CH), 9.73 (2H, s, NHNH), 11.57
(2H, s, NHBA); 13C NMR (300 MHz, DMSO-d6, d, ppm)
198 (C=S thiamide), 163 (C=O amide), 156 (C benzene),
132 (CH ethylene), 131 (C benzene), 126 (CH benzene),
125 (CH benzene), 113 (C ethylene), 55 (OCH3); EIMS
(70 eV) m/z (%): ([M?], 351 (2)), 328 (70), 313 (27), 277
(3), 239 (48), 224 (8), 207 (2), 197 (61), 180 (6), 165 (100),
150 (90), 133 (22), 122 (52), 108 (46), 92 (8), 77 (11), 63
(8), 44 (4); (Found: C, 44.43; H, 3.73; N, 19.92; S, 18.
22 %. Calc. For C13H13N5O3S2: C, 44.44; H, 3.70; N, 19.
94; S, 18.23 %).
N-(4-cyanophenyl)-2-[(2,4,6-trioxotetrahydropyrimidin-
5(2H)-ylidene)methyl]
hydrazine
carbothioamide
(13) Yield 94 % as whitish yellow solid; m.p. 228 °C; 1H
NMR (300 MHz, DMSO-d6, d, ppm): 4.91 (1H, s, NHAr),
7.32 (2H, d, J 9, ArH), 7.69 (2H, d, J 9, ArH), 8.24 (1H, s,
=CH), 9.50 (2H, s, NHNH), 11.12 (2H, s, NHBA); 13C
NMR (300 MHz, DMSO-d6, d, ppm): 179 (C=S thiamide),
167 (C=O amide), 151 (CH ethylene), 135 (C benzene),
128 (CH benzene), 126 (CN), 86 (C ethylene); EIMS
(70 eV) m/z (%): ([M?], 330 (1)), 316 (18), 192 (41), 160
(100), 143 (42), 129 (28), 118 (33), 102 (25), 90 (11), 75
123

Med Chem Res
˚
of 120.491, 106.059, and -136.443 A, respectively. All the
Cholinesterase inhibition assay (in vitro)
docking runs were performed on Intel(R) Core(TM) i5-
2410M CPU @ 2.30 GHz of Sony origin, with 6.0 GB DDR
RAM. AutoDock Vina was compiled and run under Windows
7 Professional 64-bit operating system.
AChE and BChE inhibition activities were performed, with
slight modifications, according to Ellman et al., (1961).
Total volume of the reaction mixture was raised to 100 by
60 lL Na2HPO4 buffer (of concentration 50 mM and pH
7.7) and 10 lL test compound (0.5 mM well-1), followed
by the addition of 10 lL (0.005 unit well-1) enzyme. After
mixing the contents were pre-read at 405 nm and pre-
incubated for 10 min at 37 °C. The addition of 10 lL of
0.5 mM well-1 substrate (acetyl thiocholine iodide/butyryl
thiocholine iodide) initiated the reaction, followed by the
addition of 10 lL DTNB (0.5 mM well-1). Absorbance
was measured, after 30 min of incubation at 405 nm and
37 °C, using Eserine (0.5 mM well-1) as a positive control.
Statistical analysis was performed using Microsoft Excel
2003, presenting results as mean SEM. The formula
used for calculating percent inhibition is as follows:
Acknowledgments One of the authors Saira Mumtaz likes to thanks
Higher Education Commission of Pakistan for providing funding and
support throughout this study.
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Search space was restricted to a grid box size of
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