Synthesis and biological evaluation of a series of dithiocarbamates as new cholinesterase inhibitors

Article abstract of DOI:10.1002/ardp.201300045

In the present paper, a novel series of dithiocarbamates was synthesized via the treatment of 4-(trifluoromethyl)benzyl chloride with appropriate sodium salts of N,N-disubstituted dithiocarbamic acids. The chemical structures of the compounds were elucidated by ¹H NMR, mass spectral data, and elemental analyses. Each derivative was evaluated for its ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) using a modification of Ellman's spectrophotometric method. The most potent AChE inhibitor was found as compound 2g (IC₅₀ = 0.53 ± 0.001 μM) followed by compounds 2f (IC₅₀ = 0.74 ± 0.001 μM) and 2j (IC₅₀ = 0.89 ± 0.002 μM) when compared with donepezil (IC₅₀ = 0.048 ± 0.001 μM). Compounds 2f and 2g were more effective than donepezil (IC₅₀ = 7.88 ± 0.52 μM) on BuChE inhibition. Compounds 2f and 2g exhibited the inhibitory effect on BuChE with IC₅₀ values of 1.39 ± 0.041 and 3.64 ± 0.072 μM, respectively.

Full text of DOI:10.1002/ardp.201300045

Arch. Pharm. Chem. Life Sci. 2013, 346, 571–576
571
Full Paper
Synthesis and Biological Evaluation of a Series of
Dithiocarbamates as New Cholinesterase Inhibitors
Mehlika D. Altıntop1, A. Selen Gurkan-Alp2, Yusuf Özkay1, and Zafer A. Kaplancıklı1
1
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Anadolu University, Eskişehir, Turkey
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara University, Tandogan, Ankara, Turkey
2
In the present paper, a novel series of dithiocarbamates was synthesized via the treatment of
4-(trifluoromethyl)benzyl chloride with appropriate sodium salts of N,N-disubstituted dithiocarbamic
acids. The chemical structures of the compounds were elucidated by 1H NMR, mass spectral data, and
elemental analyses. Each derivative was evaluated for its ability to inhibit acetylcholinesterase (AChE)
and butyrylcholinesterase (BuChE) using a modification of Ellman’s spectrophotometric method. The
most potent AChE inhibitor was found as compound 2g (IC50 ¼ 0.53 ꢀ 0.001 mM) followed by
compounds 2f (IC50 ¼ 0.74 ꢀ 0.001 mM) and 2j (IC50 ¼ 0.89 ꢀ 0.002 mM) when compared with
donepezil (IC50 ¼ 0.048 ꢀ 0.001 mM). Compounds 2f and 2g were more effective than donepezil
(IC50 ¼ 7.88 ꢀ 0.52 mM) on BuChE inhibition. Compounds 2f and 2g exhibited the inhibitory effect
on BuChE with IC50 values of 1.39 ꢀ 0.041 and 3.64 ꢀ 0.072 mM, respectively.
Keywords: Acetylcholinesterase / Anticholinesterase / Butyrylcholinesterase / Dithiocarbamate
Received: February 6, 2013; Revised: June 7, 2013; Accepted: June 12, 2013
DOI 10.1002/ardp.201300045
Introduction
parasympathic synapses in the periphery, and in the
neuromuscular junction. Whereas AChE is selective for
ACh hydrolysis, BuChE hydrolyses acetylcholine and other
choline esters as a non-specific cholinesterase [1–9].
Carbamates are the most widely studied class of anticho-
linesterase agents and considerable research on them in
relation to Alzheimer’s disease has been accomplished.
Rivastigmine, a dual AChE and BuChE inhibitor, is one of
the most widely used anticholinesterase agents bearing
carbamate group, which resembles the ester linkage of
acetylcholine [3–10].
Dithiocarbamates have attracted a great deal of interest
in medicinal chemistry due to the fact that new effective
compounds can be obtained by the bioisosteric replace-
ment of carbamate moiety with dithiocarbamate moiety.
They are also important pharmacophores due to their
lipophilicity, which is crucial for the delivery of CNS
drugs to their site of action through the blood–brain barrier
[11–18].
Inhibition of disease-associated enzymes by small molecule
drugs is a promising approach for pharmacologic interven-
tion in human disease. The catalytic activity of specific
enzymes is often critical to the pathophysiology of disease,
such that inhibition of catalysis is disease modifying. The
binding pockets for natural ligands of enzymes are often
uniquely well-suited for interactions with small molecule
drugs. Thus, the very nature of the chemistry of enzyme
catalysis makes these proteins amenable to inhibition by
small molecular weight, drug-like molecules [1, 2].
Cholinesterases (ChEs) remain a major focus of pharma-
ceutical research for the treatment of some of the symptoms
of Alzheimer’s disease (AD) owing to the fundamental roles
of ChEs in normal brain structure and function and in the
initiation and development of AD. Two ChEs are present in
humans: acetylcholinesterase (AChE) and butyrylcholines-
terase (BuChE). Both ChEs are present in cholinergic
synapses in the central nervous system (CNS), in the
On the basis of these findings and in the continuation of our
ongoing research program in the field of synthesis and
biological evaluation of heterocyclic compounds as cholines-
terase inhibitors [19, 20], herein we report the synthesis and
biological evaluation of some dithiocarbamate derivatives as
new anticholinesterase agents.
Correspondence: Dr. Mehlika D. Altıntop, Faculty of Pharmacy, Depart-
ment of Pharmaceutical Chemistry, Anadolu University, 26470 Eskişehir,
Turkey.
E-mail: mdaltintop@anadolu.edu.tr
Fax: þ90 222 3350750
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

572
M. D. Altıntop et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 571–576
Results and discussion
S
NaOH
+ CS2
N
H
N
SNa
The synthesis of dithiocarbamate derivatives 2a–j was carried
out according to the steps shown in Scheme 1. Sodium salts of
N,N-disubstituted dithiocarbamic acids were obtained by the
reaction of secondary amine with carbon disulfide in the
presence of sodium hydroxide.
R
R
_
1a j
The reaction of 4-(trifluoromethyl)benzyl chloride with
sodium salts of N,N-disubstituted dithiocarbamic acids 1a–j
afforded dithiocarbamate derivatives 2a–j. Some properties of
the compounds are given in Table 1.
Cl
F3C
The anticholinesterase effects of the compounds 2a–j on
AChE and BuChE were determined by a modification of
Ellman’s spectrophotometric method (Table 2). Donepezil, a
selective AChE inhibitor, was used as the reference drug [9].
The enzymatic assay indicated that piperazine derivatives
were more effective than other derivatives on AChE inhibi-
tion. Among piperazine derivatives, compound 2g can be
identified as the most promising anticholinesterase agent
due to its inhibitory effect on AChE with an IC50 value of
0.53 ꢀ 0.001 mM when compared with donepezil (IC50
¼ 0.048 ꢀ 0.001 mM). Compounds 2f and 2j exhibited AChE
S
S
N
R
F3C
_
2a j
Scheme 1. The synthesis of the compounds 2a–j.
Table 1. Some properties of the compounds 2a–j.
Compound
Ring
R
Yield (%)
m.p. (°C)
Molecular formula
Molecular weight
2a
2b
2c
2d
2e
2f
2g
2h
2i
Thiomorpholinyl
Morpholinyl
Pyrrolidinyl
Piperidinyl
Piperidinyl
Piperazinyl
Piperazinyl
Piperazinyl
Piperazinyl
Piperazinyl
H
H
H
87
85
76
79
79
80
78
82
80
83
68
72
73
52
69
C13H14F3NS3
C13H14F3NOS2
C13H14F3NS2
C14H16F3NS2
C15H18F3NS2
C14H17F3N2S2
C15H19F3N2S2
C19H19F3N2S2
C20H21F3N2OS2
C17H17F3N4S2
337.45
321.38
305.38
319.41
333.44
334.42
348.45
396.49
426.52
398.47
H
4-Methyl
4-Methyl
4-Ethyl
75
73.5
113
87
4-Phenyl
4-(4-Methoxyphenyl)
4-(2-Pyrimidinyl)
2j
140
Table 2. AChE/BuChE % inhibition of the compounds 2a–j and IC50 values.
AChE inhibition (%)
BuChE inhibition (%)
Compound
100 (mM)
1 (mM)
0.01 (mM)
IC50 (mM)
100 (mM)
1 (mM)
IC50 (mM)
2a
2b
2c
2d
2e
2f
2g
2h
30.59 ꢀ 3.21
30.51 ꢀ 5.16
34.38 ꢀ 5.15
39.81 ꢀ 4.37
24.80 ꢀ 3.64
85.92 ꢀ 4.71
87.30 ꢀ 4.42
37.51 ꢀ 4.46
25.84 ꢀ 5.37
79.73 ꢀ 6.28
97.23 ꢀ 4.27
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
>100
>100
>100
>100
>100
32.23 ꢀ 4.16
24.57 ꢀ 3.41
21.93 ꢀ 4.38
36.09 ꢀ 5.38
28.82 ꢀ 3.27
74.63 ꢀ 3.67
72.24 ꢀ 4.50
29.34 ꢀ 1.73
29.47 ꢀ 2.09
14.35 ꢀ 1.43
70.62 ꢀ 4.26
NC
NC
NC
NC
NC
>100
>100
>100
>100
>100
58.26 ꢀ 2.73
62.75 ꢀ 2.56
NC
14.54 ꢀ 1.12
36.27 ꢀ 3.83
NC
0.74 ꢀ 0.001
0.53 ꢀ 0.001
>100
38.71 ꢀ 2.59
23.64 ꢀ 3.93
NC
1.39 ꢀ 0.041
3.64 ꢀ 0.072
>100
>100
>100
7.88 ꢀ 0.52
2i
2j
NC
NC
>100
NC
NC
56.61 ꢀ 3.19
15.37 ꢀ 1.23
0.89 ꢀ 0.002
Donepezil
81.69 ꢀ 3.36
36.42 ꢀ 5.41
0.048 ꢀ 0.001
38.29 ꢀ 2.81
NC, not calculated.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 571–576
Dithiocarbamates as New Cholinesterase Inhibitors
573
inhibitory activity with IC50 values of 0.74 ꢀ 0.001 and
0.89 ꢀ 0.002 mM, respectively. On the other hand, com-
pounds 2h and 2i bearing a piperazine moiety showed weak
inhibition on AChE (IC50 > 100 mM). This outcome confirms
that the substituents at the 4th position of the piperazine ring
may have a considerable influence on AChE inhibition.
Compounds 2f and 2g were more effective than donepezil
on BuChE inhibition. Compound 2f exhibited the highest
inhibitory effect on BuChE with an IC50 value of 1.39 ꢀ
0.041 mM when compared with donepezil (IC50 ¼ 7.88 ꢀ
0.52 mM). Compound 2g exhibited BuChE inhibitory activity
with an IC50 value of 3.64 ꢀ 0.072 mM. Although compound
2j bearing a pyrimidine moiety at the 4th position of the
piperazine ring showed AChE inhibitory activity with an IC50
value of 0.89 ꢀ 0.002 mM, it showed weak inhibition on
BuChE (IC50 > 100 mM). Other derivatives showed weak
inhibition on BuChE (IC50 > 100 mM). It is apparent that
there is a positive correlation between BuChE inhibitory
activity and the 4-alkylpiperazine moiety.
studies [22]. Novel compound 2g was docked into the binding
site of the X-ray structure of TcAChE. Low energy docked
coordinates was selected to analyze the most likely interac-
tion with the receptor, based on the thiocarbamate pharma-
cophore showing the best superposition with that of the X-ray
coordinates of N-saccharinohexyl-galanthamine. Amino acids
in the active site of TcAchE and the interaction of the best
docking pose of compound 2g are shown in Fig. 2. In the
molecular docking study, it was observed that the molecule
was compatible with the active site thanks to the residues
Phe330, Phe331, Tyr334 at the bottom and Trp279 at the
entrance of the binding pocket. It was shown that compound
2g was settled down with the formation of p–p interaction
between phenyl ring of 4-(trifluoromethyl)benzyl moiety and
phenyl ring of the Phe331 residue at the bottom of the active
site. The formation of an interaction between piperazine
moiety and phenyl ring of Trp279 residue was also observed at
the entrance of the active site of the enzyme (Fig. 2A). These
interactions stabilize the ligand in the binding pocket of the
enzyme (Fig. 2B), but no hydrogen bonding was observed in
the active site.
The kinetics of this new class of AChE inhibitors were
studied in detail using the most active compound 2g. The
nature of AChE inhibition, caused by this compound, was
investigated by the graphical analysis of steady-state inhibi-
tion data (Fig. 1). Reciprocal plots (Lineweaver–Burk plots) Conclusion
described compound 2g as a mixed type inhibitor, due to
different intercepts on both the y- and x-axes. The values of Km
and Vmax were calculated by nonlinear regression according
to Heng et al. [21]. For compound 2g, the Km and Vmax values
were 3.18 and 3.23, respectively.
In order to gain more insight into whether the ligands
inhibit AChE, docking study was implemented using the X-ray
structure of Torpedo californica acetylcholinesterase (TcAChE;
PDB ID: 3I6Z), which was previously used by Razavi et al. [22]
for the development of some AChE inhibitors. As the structure
of TcAChE is similar to Electrophorus electricus AChE (electric eel
AChE), it was used as the receptor model for docking
In the present work, we described the synthesis of a series of
dithiocarbamate derivatives, which were evaluated for their
anticholinesterase effects on AChE and BuChE.
Piperazine derivatives were more effective than other
derivatives on AChE inhibition. The most potent AChE
inhibitor was found as compound 2g (IC50 ¼ 0.53 ꢀ
0.001 mM) followed by compounds 2f (IC50 ¼ 0.74 ꢀ
0.001 mM) and 2j (IC50 ¼ 0.89 ꢀ 0.002 mM) when compared
with donepezil (IC50 ¼ 0.048 ꢀ 0.001 mM). Although com-
pounds 2h and 2i carry a piperazine moiety, they showed
weak inhibition on AChE (IC50 > 100 mM). The substituents at
Figure 1. Lineweaver–Burk plots for com-
pound 2g (IC50 ¼ 0.53 mM). Substrate (ATCI)
concentrations used: 0.5, 0.25, 0.125, 0.0625,
0.03125 mM. 1/V: 1/velocity of reaction [1/
(absorbance/1 min)], 1/S: 1/substrate concen-
tration (1/mM ATCI).
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

574
M. D. Altıntop et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 571–576
Experimental
Chemistry
All reagents were purchased from commercial suppliers and
used without further purification. Melting points (m.p.) were
determined on a Electrothermal 9100 melting point apparatus
(Weiss-Gallenkamp, Loughborough, UK) and are uncorrected. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded on a
Bruker 500 MHz spectrometer (Bruker, Billerica, MA, USA). Chemical
shifts were expressed in parts per million (ppm) and tetramethyl-
silane was used as an internal standard. Mass spectra were recorded
on a VG Quattro mass spectrometer (Agilent, Minnesota, USA).
Elemental analyses were performed on a Perkin Elmer EAL 240
elemental analyser (Perkin-Elmer, Norwalk, CT, USA).
General procedure for the synthesis of the compounds
Sodium salts of N,N-disubstituted dithiocarbamic acids
(1a–j)
Sodium hydroxide (10 mmol) was dissolved in ethanol (80 mL) with
constant stirring. After addition of the secondary amine (10 mmol)
the mixture was cooled in an ice bath and carbon disulfide
(100 mmol) was added dropwise with stirring. The reaction
mixture was stirred for 1 h at room temperature. The products
were afforded by filtration and washed with diethyl ether [18].
Dithiocarbamate derivatives (2a–j)
A mixture of 4-(trifluoromethyl)benzyl chloride (0.01 mol) and
appropriate sodium salts of N,N-disubstituted dithiocarbamic
acids (0.01 mol) was treated in acetone (15 mL) at room
temperature for 3 h. The solvent was evaporated, the resulting
solid was washed with water, and recrystallized from ethanol.
4-(Trifluoromethyl)benzyl thiomorpholine-4-carbodithioate
Figure 2. View of the active site of TcAChE (PDB ID: 3I6Z) in
complex with compound 2g constructed by UCSF Chimera
package [29]. (A) Amino acid residues in the active site; (B) ligand
binding pocket of the enzyme.
(2a)
1H NMR (d ppm) (DMSO-d6): 2.70 (4H, t, C3 and C5 protons of
thiomorpholine), 4.22 and 4.53 (4H, two brs, C2 and C6 protons of
thiomorpholine), 4.68 (2H, s, COCH2), 7.62 and 7.68 (4H, two d
(J ¼ 8 and 8 Hz), 1,4-disubstituted phenyl protons).
For C13H14F3NS3, calcd.: C, 46.27; H, 4.18; N, 4.15; Found: C,
46.28; H, 4.15; N, 4.12.
the 4th position of the piperazine ring also have
a
MS (ES): [Mþ1]þ: 338.
considerable influence on AChE inhibition.
Compounds 2f and 2g were found to be more effective than
donepezil (IC50 ¼ 7.88 ꢀ 0.52 mM) on BuChE inhibition.
Compounds 2f and 2g exhibited the inhibitory effect on
BuChE with IC50 values of 1.39 ꢀ 0.041 and 3.64 ꢀ 0.072 mM,
respectively. It is clear that there is a positive correlation
between BuChE inhibitory activity and the 4-alkylpiperazine
moiety.
The kinetics of this new class of AChE inhibitors were
studied in detail using the most active compound 2g.
According to the results, compound 2g was described as a
mixed type inhibitor. Molecular docking study of compound
2g was also carried out. Based on the molecular docking
study, compound 2g might be involved in the interaction with
TcAChE (PDB ID: 3I6Z) and considered as an AChE inhibitor.
4-(Trifluoromethyl)benzyl morpholine-4-carbodithioate
(2b)
1H NMR (d ppm) (DMSO-d6): 3.67 (4H, s, C3 and C5 protons of
morpholine), 3.92 and 4.24 (4H, two brs, C2 and C6 protons of
morpholine), 4.69 (2H, s, COCH2), 7.62 and 7.67 (4H, two d (J ¼ 8.5
and 8.5 Hz), 1,4-disubstituted phenyl protons).
For C13H14F3NOS2, calcd.: C, 48.58; H, 4.39; N, 4.36; Found: C,
48.60; H, 4.37; N, 4.33.
MS (ES): [Mþ1]þ: 322.
4-(Trifluoromethyl)benzyl pyrrolidine-1-carbodithioate
(2c)
1H NMR (d ppm) (DMSO-d6): 1.90 and 2.01 (4H, two p, C3 and C4
protons of pyrrolidine), 3.61 and 3.79 (4H, two t, C2 and C5 protons
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 571–576
Dithiocarbamates as New Cholinesterase Inhibitors
575
of pyrrolidine), 4.68 (2H, s, COCH2), 7.62 and 7.67 (4H, two d (J ¼ 7
and 8 Hz), 1,4-disubstituted phenyl protons).
For C13H14F3NS2, calcd.: C, 51.13; H, 4.62; N, 4.59; Found: C,
51.15; H, 4.60; N, 4.58.
For C19H19F3N2S2, calcd.: C, 57.56; H, 4.83; N, 7.07; Found: C,
57.55; H, 4.85; N, 7.07.
MS (ES): [Mþ1]þ: 397.
MS (ES): [Mþ1]þ: 306.
4-(Trifluoromethyl)benzyl 4-(4-methoxyphenyl)piperazine-
1-carbodithioate (2i)
1H NMR (d ppm) (DMSO-d6): 3.13 (4H, brs, C3 and C5 protons of
piperazine), 3.69 (3H, s, OCH3), 4.06 and 4.39 (4H, two brs, C2 and
C6 protons of piperazine), 4.70 (2H, s, COCH2), 6.84 and 6.92 (4H,
two d (J ¼ 9 and 9 Hz), 1,4-disubstituted N-phenyl protons), 7.62
and 7.68 (4H, two d (J ¼ 8 and 8.5 Hz), 1,4-disubstituted phenyl
protons).
4-(Trifluoromethyl)benzyl piperidine-1-carbodithioate
(2d)
1H NMR (d ppm) (DMSO-d6): 1.53–1.61 (4H, m, C3 and C5 protons of
piperidine), 1.62–1.68 (2H, m, C4 protons of piperidine), 3.88 and
4.23 (4H, two s, C2 and C6 protons of piperidine), 4.66 (2H, s,
COCH2), 7.61 and 7.67 (4H, two d (J ¼ 8 and 8 Hz), 1,4-
disubstituted phenyl protons).
For C20H21F3N2OS2, calcd.: C, 56.32; H, 4.96; N, 6.57; Found: C,
56.30; H, 4.97; N, 6.55.
For C14H16F3NS2, calcd.: C, 52.64; H, 5.05; N, 4.39; Found: C,
52.63; H, 5.07; N, 4.37.
MS (ES): [Mþ1]þ: 427.
MS (ES): [Mþ1]þ: 320.
4-(Trifluoromethyl)benzyl 4-(2-pyrimidinyl)piperazine-1-
4-(Trifluoromethyl)benzyl 4-methylpiperidine-1-
carbodithioate (2e)
carbodithioate (2j)
1H NMR (d ppm) (DMSO-d6): 3.85–3.91 (4H, m, C3 and C5 protons of
piperazine), 4.04 and 4.35 (4H, two brs, C2 and C6 protons of
piperazine), 4.70 (2H, s, COCH2), 6. 69 (1H, t (J ¼ 5 and 4.5 Hz) C4
proton of pyrimidine), 7.64 and 7.68 (4H, two d (J ¼ 8.5 and
8.5 Hz), 1,4-disubstituted phenyl protons), 8.40 (2H, d (J ¼ 5 Hz),
C1 and C3 protons of pyrimidine).
1H NMR (d ppm) (DMSO-d6): 0.90 (3H, d (J ¼ 6 Hz), CH3), 1.02–1.15
(2H, m, piperidine protons), 1.68–1.77 (3H, m, piperidine
protons), 3.15–3.35 (2H, m, piperidine protons), 4.38–4.49
(1H, m, piperidine proton), 4.66 (2H, d (J ¼ 10 Hz), COCH2),
5.22–5.33 (1H, m, piperidine proton), 7.60 and 7.66 (4H, two d
(J ¼ 8 and 8.5 Hz), 1,4-disubstituted phenyl protons).
For C15H18F3NS2, calcd.: C, 54.03; H, 5.44; N, 4.20; Found: C,
54.05; H, 5.43; N, 4.19.
For C17H17F3N4S2, calcd.: C, 51.24; H, 4.30; N, 14.06; Found: C,
51.25; H, 4.29; N, 14.07.
MS (ES): [Mþ1]þ: 399.
MS (ES): [Mþ1]þ: 334.
AChE/BuChE inhibition
4-(Trifluoromethyl)benzyl 4-methylpiperazine-1-
All compounds were subjected to a slightly modified method of
Ellman’s test [23] in order to evaluate their potency to inhibit AChE
and BuChE. The spectrophotometric method is based on the
reaction of released thiocholine to give a colored product with a
chromogenic reagent 5,50-dithio-bis(2-nitrobenzoic acid) (DTNB).
AChE, (E.C.3.1.1.7 from electric eel, 500 units), BuChE (E.C. 3.1.1.8,
from horse serum, 1000 units), and donepezil hydrochloride were
purchased from Sigma–Aldrich (Steinheim, Germany). Potassium
dihydrogen phosphate, DTNB, potassium hydroxide, sodium
hydrogen carbonate, gelatine, acetylthiocholine iodide (ATC), and
butyrylthiocholine iodide (BTC) were obtained from Fluka (Buchs,
Switzerland). Spectrophotometric measurements were performed
on a 1700 Shimadzu UV-1700 UV–Vis spectrophotometer.
The anticholinesterase activity of the compounds 2a–j was
measured in 100 mM phosphate buffer (pH 8.0) at 25°C, using
ATC and BTC (75 mM) as substrates. In both cases, DTNB (10 mM)
was used in order to observe absorbance changes at 412 nm.
Donepezil hydrochloride was used as a positive control [24].
carbodithioate (2f)
1H NMR (d ppm) (DMSO-d6): 2.19 (3H, s, CH3), 2.33–2.42 (4H, m, C3
and C5 protons of piperazine), 3.90 and 4.24 (4H, two brs, C2 and
C6 protons of piperazine), 4.67 (2H, s, COCH2), 7.61 and 7.65 (4H,
two d (J ¼ 8.5 and 8 Hz), 1,4-disubstituted phenyl protons).
For C14H17F3N2S2, calcd.: C, 50.28; H, 5.12; N, 8.38; Found: C,
50.30; H, 5.11; N, 8.36.
MS (ES): [Mþ1]þ: 335.
4-(Trifluoromethyl)benzyl 4-ethylpiperazine-1-
carbodithioate (2g)
1H NMR (d ppm) (DMSO-d6): 1.00 (3H, t, CH3), 2.34 (2H, q, N–CH2),
2.42 (4H, brs, C3 and C5 protons of piperazine), 3.89 and 4.24 (4H,
two brs, C2 and C6 protons of piperazine), 4.67 (2H, s, COCH2), 7.61
and 7.65 (4H, two d (J ¼ 8 and 8.5 Hz), 1,4-disubstituted phenyl
protons).
For C15H19F3N2S2, calcd.: C, 51.70; H, 5.50; N, 8.04; Found: C,
51.72; H, 5.49; N, 8.03.
Enzymatic assay
MS (ES): [Mþ1]þ: 349.
Enzyme solutions were prepared in gelatine solution (1%), at a
concentration of 2.5 units/mL. AChE or BuChE solution (50 mL)
and compound solution (50 mL), which is prepared in 2% DMSO at
a concentration range of 10ꢁ1–10ꢁ6 mM, were added to 3.0 mL
phosphate buffer (pH 8 ꢀ 0.1) and incubated at 25°C for 5 min.
The reaction was started by adding DTNB (50 mL) and ATC (10 mL)
to the enzyme-inhibitor mixture. The production of the yellow
anion was recorded for 10 min at 412 nm. As a control, an
identical solution of the enzyme without the inhibitor was
processed following the same protocol. The blank reading
contained 3.0 mL buffer, 50 mL 2% DMSO, 50 mL DTNB, and
4-(Trifluoromethyl)benzyl 4-phenylpiperazine-1-
carbodithioate (2h)
1H NMR (d ppm) (DMSO-d6): 3.26–3.32 (4H, m, C3 and C5 protons of
piperazine), 4.07 and 4.39 (4H, two brs, C2 and C6 protons of
piperazine), 4.71 (2H, s, COCH2), 6.81 (1H, t, (J ¼ 7 and 7.5 Hz), C4
proton of N-phenyl), 6.94 (2H, d (J ¼ 8 Hz), C3 and C5 protons of
N-phenyl), 7.24 (2H, t (J ¼ 8 and 8 Hz), C2 and C6 protons of
N-phenyl), 7.62 and 7.68 (4H, two d (J ¼ 8 and 8 Hz), 1,4-
disubstituted phenyl protons).
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

576
M. D. Altıntop et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 571–576
10 mL substrate. All processes were assayed in triplicate. The
[7] P. Johannsen, CNS Drugs 2004, 18, 757–768.
inhibition rate (%) was calculated by the following equation:
[8] A. Martinez, A. Castro, Expert Opin. Investig. Drugs 2006, 15, 1–
12.
ðAC ꢁ AIÞ
Inhibition %=
ꢂ 100
[9] G. Pepeu, M. G. Giovannini, Curr. Alzheimer Res. 2009, 6,
86–96.
AC
where AI is the absorbance in the presence of the inhibitor, AC is
the absorbance of the control, and AB is the absorbance of blank
reading. Both of the values were corrected with blank-reading
value. SPSS for Windows 15.0 was used for statistical analysis.
Data were expressed as mean ꢀ SD.
[10] T. L. Lemke, D. A. Williams, in Foye’s Principles of Medicinal
Chemistry, Lippincott Williams & Wilkins, Baltimore and
Philadelphia, USA 2008, chapter 12.
[11] P. M. Madalageri, O. Kotresh, J. Chem. Pharm. Res. 2012, 4 (5),
2697–2703.
[12] R. B. Silverman, in The Organic Chemistry of Drug Design and
Drug Action, Elsevier Academic Press, Burlington, USA 2004,
chapter 2.
Kinetic characterization of AChE inhibition
Enzyme kinetic characterization study for compound 2g was
performed under same incubation conditions as described above
using acetylthiocholine as substrate at various concentrations
(0.5, 0.25, 0.125, 0.0625, and 0.03125 mM ATCI) and DTNB was
used as chromophoric reagent [25]. A parallel control with no
inhibitor in the mixture was used for comparison. Compound 2g
(IC50 ¼ 0.53 mM) was analyzed in triplicate; and Lineweaver–
Burk (1/V vs. 1/[S]) plot was constructed.
[13] H. Waterbeemd, R. Mannhold, Lipophilicity descriptors for
structure–property correlation studies: Overview of experi-
mental and theoretical methods and a benchmark of log P
calculations. Lipophilicity in Drug Action and Toxicology, in: R.
Mannhold, H. Kubinyi, H. Timmerman (Series Eds.), V. Pliška,
B. Testa, H. Waterbeemd (Vol. Eds.), VCH Publishers, New
York, USA 1996, chapter 23.
[14] R. Tokuyama, Y. Takahashi, Y. Tomita, M. Tsubouchi, T.
Yoshida, N. Iwasaki, N. Kado, E. Okezaki, O. Nagata, Chem.
Pharm. Bull. 2001, 49 (4), 353–360.
Molecular docking study
Docking studies were carried out by Autodock_vina v.1.1 [26]. The
X-ray structure of TcAChE in complex with N-saccharinohexyl-
galanthamine, a derivative showing more than 40 times AChE
inhibitory activity higher than that of a known AChE inhibitor,
galanthamine [27], was obtained from the Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do), PDB ID: 3I6Z. All non-
protein molecules including water, ions, and the ligand N-
saccharinohexyl-galanthamine were removed from the pdb
structure before docking. MGL Tools v.1.5.4. [28] was used to
prepare the ligand and the receptor, which were saved in pdbqt
format. Autodock_vina v.1.1 [26] was used to dock the ligand into
the binding site of TcAChE and the docking parameters were set as
follows: center_x ¼ 1.292, center_y ¼ 65.557, center_z ¼ 65.639,
size_x ¼ 40, size_y ¼ 40, size_z ¼ 40, exhaustiveness ¼ 8.
Docking of the compound 2g was performed in a confined grid
box defined by MGL Tools v.1.5.4 [28]. Docked ligand were
analyzed with MGL Tools v.1.5.4 [28] and the UCSF Chimera-1.7
package [29].
[15] G. A. Patani, E. J. LaVoie, Chem. Rev. 1996, 96, 3147–3176.
[16] X.-J. Wang, H.-W. Xu, L.-L. Guo, J.-X. Zheng, B. Xu, X. Guo, C.-
X. Zheng, H.-M. Liu, Bioorg. Med. Chem. Lett. 2011, 21, 3074–
3077.
[17] K. Bacharaju, S. R. Jambula, S. Sivan, S. Jyostnatangeda, V.
Manga, Bioorg. Med. Chem. Lett. 2012, 22, 3274–3277.
[18] G. Turan-Zitouni, A. Özdemir, K. Güven, Arch. Pharm. Chem.
Life Sci. 2005, 338, 96–104.
[19] M. D. Altintop, Z. A. Kaplancikli, A. Ozdemir, G. Turan-
Zitouni, H. E. Temel, G. Akalın, Arch. Pharm. Chem. Life Sci.
2012, 345 (2), 112–116.
[20] G. Turan-Zitouni, A. Ozdemir, Z. A. Kaplancikli, M. D.
Altintop, H. E. Temel, G. Akalın Çiftçi, J. Enzyme Inhib. Med.
Chem. 2013, 28 (3), 509–514.
[21] S. Heng, W. Tieu, S. Hautmann, K. Kuan, D. S. Pedersen, M.
Pietsch, M. Gütschow, A. D. Abell, Bioorg. Med. Chem. 2011, 19,
7453–7463.
The authors have declared no conflict of interest.
[22] S. F. Razavi, M. Khoobi, H. Nadri, A. Sakhteman, A. Moradi, S.
Emami, A. Foroumadi, A. Shafiee, Eur. J. Med. Chem. 2013, 64,
252–259.
References
[23] N. S. L. Perry, P. J. Houghton, A. E. Theobald, P. Jenner, E. K.
[1] R. A. Copeland, in: Evaluation of Enzyme Inhibitors in Drug
Discovery: A Guide for Medicinal Chemists and Pharmacologists,
Wiley-Interscience, New Jersey, USA 2005, chapter 1.
Perry, J. Pharm. Pharmacol. 2000, 52, 895–902.
[24] G. L. Ellman, K. D. Courtney, V. Andres, R. M. Feather-Stone,
Biochem. Pharmacol. 1961, 7, 88–95.
[2] R. A. Copeland, R. R. Gontarek, L. Luo, Enzyme Inhibitors:
Biostructure-Based and Mechanism-Based Designs. in: (Eds.:
P. Krogsgaard-Larsen, K. Strømgaard, U. Madsen) Textbook of
Drug Design and Discovery, CRC Press, Boca Raton, USA 2010,
chapter 11.
[25] T. Palmer, Understanding Enzymes, Prentice Hall/Ellis Hor-
wood, London 1995.
[26] O. Trott, A. J. Olson, J. Comput. Chem. 2010, 31, 455–461.
[27] C. Bartolucci, L. A. Haller, U. Jordis, G. Fels, D. Lamba, J. Med.
Chem. 2010, 53, 745–751.
[3] Z.-X. Shen, Med. Hypotheses 2004, 63, 298–307.
[4] D. G. Wilkinson, P. T. Francis, E. Schwam, J. Payne-Parrish,
[28] M. F. Sanner, J. Mol. Graph. Mod. 1999, 17, 57–61.
Drugs Aging 2004, 21, 453–478.
[29] E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M.
Greenblatt, E. C. Meng, T. E. Ferrin, J. Comput. Chem. 2004, 25,
1605–1612.
[5] J. Grutzendler, J. C. Morris, Drugs 2001, 61, 41–52.
[6] E. Giacobini, Neurochem. Res. 2003, 28, 515–522.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com