Synthesis and molecular modeling study of Cu(II) complexes derived from 2-(diphenylmethylene)hydrazinecarbothioamide derivatives with cholinesterase inhibitory activities

Article abstract of DOI:10.1016/j.cclet.2013.04.013

Thiosemicarbazones of 2-amino-5-chlorobenzophenone and 3-aminobenzophenone (L¹-L⁴) have been synthesized and their Cu(II) complexes (1-4) were afforded via coordination with cupric chloride. All these compounds were characterized by

Full text of DOI:10.1016/j.cclet.2013.04.013

Chinese Chemical Letters 24 (2013) 609–612
Contents lists available at SciVerse ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Synthesis and molecular modeling study of Cu(II) complexes derived from
2-(diphenylmethylene)hydrazinecarbothioamide derivatives with
cholinesterase inhibitory activities
a
b
Yi Chen Chan a, Abdussalam Salhin Mohamed Ali a, , Melati Khairuddean , Kooi Yeong Khaw ,
*
Vikneswaran Murugaiyah b, Alireza Basiri b
a School of Chemical Sciences, Universiti Sains Malaysia, Penang 11800, Malaysia
b Discipline of Pharmacology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang 11800, Malaysia
A R T I C L E I N F O
A B S T R A C T
Thiosemicarbazones of 2-amino-5-chlorobenzophenone and 3-aminobenzophenone (L1–L4) have been
synthesized and their Cu(II) complexes (1–4) were afforded via coordination with cupric chloride. All
these compounds were characterized by UV–vis and IR spectroscopy together with CHN elemental
analysis. NMR spectroscopy was also applied to characterize the ligands. In vitro cholinesterase
Article history:
Received 29 January 2013
Received in revised form 22 March 2013
Accepted 1 April 2013
Available online 17 May 2013
inhibitory assays for the complexes (1–4) showed IC50 values less than 10
mmol/L, with complex
1 exhibiting the most activity, IC50 = 2.15 mol/L and 2.16 mol/L for AChE and BuChE, respectively.
m
m
Keywords:
Molecular modeling simulation revealed the binding interaction template for complex 1 with the AChE
and BuChE receptors. In DPPH assay, the complexes also showed more in vitro antioxidant activities in
comparison to their parent ligands.
Thiosemicarbazone
Cu(II) complex
Cholinesterase
Molecular modeling
ß 2013 Abdussalam Salhin Mohamed Ali. Published by Elsevier B.V. on behalf of Chinese Chemical
Society. All rights reserved.
1. Introduction
level [6]. Four cholinesterase inhibitors (tacrine, donepezil,
rivastigmine, and galantamine) are approved by the US Food
Alzheimer’s disease (AD) is the most common cause of
dementia among people over the age of 65 years. 25 million
individuals are now estimated to suffer from this disease
throughout the world [1]. Due to a lack of significant advances
in the treatment of AD, the number of symptomatic AD cases is
predicted to increase in the following years [2].
One of the leading therapeutic strategies in AD treatment is the
use of cholinesterase inhibitors which were shown to ameliorate
the cognitive function and activities of daily living of patients with
AD [3]. According to the cholinergic hypothesis, the pathogenesis
of AD has been linked to a deficit of acetylcholine (ACh), a key
neurotransmitter in learning and memory [4]. In the brain,
acetylcholinesterase (AChE) terminates the activity of ACh by
hydrolyzing it into acetal and choline while butyrylcholinesterase
(BuChE) plays a secondary role by regulating the ACh level. It was
demonstrated that as AD progresses, BuChE activity increases
while AChE activity remain unchanged or decreases [5]. Hence,
either selective BuChE inhibition or dual inhibition of these
enzymes constitutes a promising approach to increase the ACh
and Drug Administration [7]. Nevertheless, the applicability of
these drugs is limited due to their adverse side-effects [8].
Consequently, there is still a need to develop new drugs to
antagonize AD.
Transition metal ions such as Cu(II), Zn(II) and Fe(III) accom-
plish a wide range of biological tasks in the brain due to their
involvement in redox reactions. However, in AD patients, these
metal ions were found to be involved in amyloid-b aggregation and
oxidative stress which are the two major pathogenesis of
Alzheimer’s disease [9]. Accordingly, metal chelators which bind
and deactivate transition metal ions in the brain are a feasible
alternative to treat AD patients [10]. Our interest to apply metal
chelator in AD therapy comes from the study by Ikram et al. [11]
where metal based drugs are suggested as potential inhibitors of
AChE and BuChE. Thiosemicarbazones are an important class of
multidentate ligands which provide potential binding sites for a
variety of transition metal ions [12]. Due to their remarkable
biological properties, thiosemicarbazones and their metal com-
plexes have been extensively studied. Cu(II) complexes of
thiosemicrabazones exhibited more significant biological proper-
ties rather than the free ligands [13]. Thiosemicarbazones were
also reported as potent cholinesterase inhibitor [14]. Herein, we
report the synthesis and characterization of thiosemicarbazide
* Corresponding author.
E-mail address: abdussalam@usm.my (A.S. Mohamed Ali).
1001-8417/$ – see front matter ß 2013 Abdussalam Salhin Mohamed Ali. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.04.013

[
Y.C. Chan et al. / Chinese Chemical Letters 24 (2013) 609–612
Scheme 1. The reaction scheme for the synthesis of L1–L4.
[F(ig._1)TD$FIG]
substituted benzophenone derivatives and their respective Cu(II)
complexes. These compounds were subsequently evaluated for the
cholinesterase inhibitory activity and antioxidant activity. A
molecular docking simulation was applied to reveal possible
ligand–receptor interactions which give rise to high inhibitory
activity in the most potent compound.
2. Experimental
2.1. Synthesis of thiosemicarbazones
To an ethanolic solution (20 mL) of thiosemicarbazide or 4-
ethyl-3-thiosemicarbazide (3 mmol) was added the respective
benzophenone (3 mmol) dissolved in the same solvent (5 mL). The
mixture was refluxed for 7–8 h in the presence of 2–3 drops of
concentrated H2SO4. This was then cooled to room temperature
and slowly evaporated until sufficient solid was formed.
Fig. 1. The proposed structure of complex 1.
solution. As a representative case, Fig. 1 displays the proposed
structure of complex 1. All of the synthesized compounds were
obtained in satisfactory yields and had been properly characterized
by 1H NMR and 13C NMR, FTIR, UV–vis, and elemental analysis
(CHN) [15]. Since the Cu(II) complexes are paramagnetic, their 1H
NMR spectra could not be obtained.
2.2. Synthesis of Cu(II) complexes
Into a hot ethanolic solution (10 mL) of the respective ligand
(1 mmol) was added cupric chloride (1 mmol) dissolved in a
minimum quantity of ethanol. The mixture was refluxed for 2–3 h.
After keeping the mixture at room temperature for overnight, the
colored solid was filtered from the solution, washed with small
quantity of ethanol and then air dried.
Physical properties and analytical data, IR and electronic
spectral data of the studied ligands and Cu(II) complexes are
showed in supporting information (Table S1–3).
The inhibitory activities of the test compounds against AChE
and BuChE enzymes were evaluated in vitro by means of modified
Ellman’s method [16] (See supporting information). Results with
physostigmine as the positive control drug show IC50 values of
(0.18 Æ 0.005) mmol/L and (0.29 Æ 0.02) mmol/L for AChE and BuChE,
respectively. Meanwhile, IC50 data of the synthesized compounds
against both enzymes are summarized in Table 1. Due to the low
percentage of inhibition (<50%) exhibited by L1 at 50 mg/mL, its IC50
value was not further assessed. Cu(II) complexesgenerallyshowhigher
inhibitory potency against the cholinesterase enzymes as compared to
the parent ligands, as previously reported by Ikram et al. [11].
Complex 1 shows identical selectivity for AChE and BuChE.
Other compounds display more selectivity on AChE rather than
BuChE, except for L3 and complex 2, which exhibit more selectivity
toward BuChE. Recent studies had clarified that BuChE activity
3. Results and discussion
Thiosemicarbazones (L1–L4) were afforded using acid-catalyzed
condensation between 2-amino-5-chlorobenzophenone/3-amino-
benzophenone and thiosemicarbazide analogs in 1:1 molar ratio
(Scheme 1).
Cu(II) complexes (1–4) were obtained through the interaction
of the ligands with CuCl2 in 1:1 or 2:1 molar ratio in ethanolic
Table 1
IC50 values of the cholinesterase inhibitory activities and radical-scavenging assays for the test samples.
Compound
AChE
BuChE
Selectivity
AChE
DPPH assay
IC50 mol/L)
IC50
(
m
mol/L)
S.D.a
IC50
(
m
mol/L)
S.D.a
BuChE
(
m
S.D.a
L1
L2
L3
L4
1
–
–
–
–
–
–
964.59
505.46
–
6.76
6.71
–
38.09
183.93
115.98
2.15
5.28
11.11
15.51
0.18
0.26
0.27
0.11
122.23
171.07
148.21
2.16
10.82
11.71
10.62
0.46
0.19
0.34
0.35
3.21
0.93
1.28
1.00
0.80
1.23
1.47
0.31
1.08
0.78
1.00
1.25
0.81
0.68
–
–
33.90
35.69
40.19
237.79
0.04
0.33
0.40
1.05
2
3.22
2.57
3
4.48
5.52
4
5.32
7.82
a
Standard deviation.

[
Y.C. Chan et al. / Chinese Chemical Letters 24 (2013) 609–612
611
Fig. 2. Complex 1 docked into (a) active site of TcAChE (b) active site of hBuChE.
raises drastically as AD progresses. BuChE are also found at high
concentrations in amyloid plaques and neurofibrillary tangles [17].
Thus, dual inhibitors or selective BuChE inhibitors such as
compounds L3, 1 and 2 may enhance ACh availability in AD
patients.
thiosemicarbazones in combination with copper are useful in the
inhibition of the cholinesterase enzymes, which is in accordance
with the relative abundance of copper in the brain. Furthermore,
thiosemicarbazones with characteristic chelating properties are
expected to cause a reduction of Ab deposits by removing excess
Oxidative stress was extensively reported as one of the main
symptoms of AD [18], thus the antioxidant capabilities of the
synthesized compounds were also evaluated using DPPH assay
(See supporting information). Complex 1 again shows the most
deregulated metal ions accumulate in the brain.
Acknowledgments
promising antioxidant effect with IC50 value of 33.90
mmol/L
The authors would like to thank Universiti Sains Malaysia –
Science Fund Grant No. 1001/PKIMIA/823003 and RU Grant No.
1001/PKIMIA/811196 for the financial support of this work.
(Table 1) that is approximately two-fold less effective than
quercetin [IC50 = (14.73 Æ 0.07) mmol/L] and almost as active as
morin [IC50 = (24.81 Æ 0.08) mmol/L].
The molecular modeling simulation were undertaken for the
most active compound, complex 1, to investigate and disclose
feasible binding interactions with AChE and BuChE receptors
ensuing its high dual cholinesterase inhibitory properties (See
supporting information).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2013.04.013.
For AChE receptor, mild polar, hydrophobic and
are the dominant interactions observed with the active site
residues. The stacking interaction with Trp84 at choline
p–p stacking
References
p–p
binding site is assumed to be a major factor in appropriate
accommodation of this compound inside the gorge. Tyr70, Tyr121
and Tyr334 at peripheral anionic and Phe330 at choline binding
site are other residues which are possibly assisted docking of
complex 1 to the bottom of the gorge (Fig. 2(a)).
[1] G. Small, R. Bullock, Defining optimal treatment with cholinesterase inhibitors in
Alzheimer’s disease, Alzheimers Dement. 7 (2011) 177–184.
[2] M.A. Kamal, P. Klein, Q.S. Yu, et al., Kinetics of human serum butyrylcholinesterase
and its inhibition by a novel experimental Alzheimer therapeutic, bisnorcymser-
ine, J. Alzheimers Dis. 10 (2006) 43–51.
[3] H.J. Han, J.J. Lee, S.A. Park, et al., Efficacy and safety of switching from oral
cholinesterase inhibitors to the rivastigmine transdermal patch in patients with
probable Alzheimer’s disease, J. Clin. Neurol. 7 (2011) 137–142.
[4] A. Adsersen, A. Kjølbye, O. Dall, et al., Acetylcholinesterase and butyrylcholines-
terase inhibitory compounds from Corydalis cava schweigg. & kort, J. Ethnophar-
macol. 113 (2007) 179–182.
[5] N.H. Greig, D.K. Lahiri, K. Sambamurti, Butyrylcholinesterase: an important new
target in Alzheimer’s disease therapy, Int. Psychogeriatr. 14 (2002) 77–91.
[6] A. Fallarero, P. Oinonen, S. Gupta, et al., Inhibition of acetylcholinesterase by
coumarins: the case of coumarin 106, Pharmacol. Res. 58 (2008) 215–221.
[7] Z. Yang, X. Zhang, D. Duan, et al., Modified TLC bioautographic method for
screening acetylcholinesterase inhibitors from plant extracts, J. Sep. Sci. 32
(2009) 3257–3259.
For BuChE, docking simulations revealed that aromatic rings
existing in complex 1 are totally involved in the
p–p stacking
interaction with indole moiety of Trp82 at choline binding site.
This interaction along with hydrophobic interaction with the
backbone residues such as Tyr128 and Tyr332 completely
anchored this compound to the bottom of active site gorge
(Fig. 2(b)). It can be proposed that a similar interaction template
of complex 1 with choline binding site residues (Trp84/Phe330
in AChE and Trp82 in BuChE) in both receptors is the major
factor which leads to its high/dual cholinesterase inhibitory
activity.
[8] P.K. Mukherjee, V. Kumar, M. Mal, et al., Acetylcholinesterase inhibitors from
plants, Phytomedicine 14 (2007) 289–300.
[9] A. Budimir, N. Humbert, M. Elhabiri, et al., Hydroxyquinoline based binders:
promising ligands for chelatotherapy? J. Inorg. Biochem. 105 (2011) 490–496.
[10] D.J. Bonda, G. Liu, P. Men, et al., Nanoparticle delivery of transition-metal
chelators to the brain: oxidative stress will never see it coming, CNS Neurol.
Disord. Drug Targets 11 (2012) 81–85.
4. Conclusion
In conclusion, complex 1 which is the most active compound in
AChE and BuChE inhibitory activities, shows equal selectivity for
[11] M. Ikram, R. Saeed Ur, S. Rehman, et al., Synthesis, characterization and distinct
butyrylcholinesterase activities of transition metal complexes of 2-[(E)-(quino-
lin-3-ylimino)methyl]phenol, Inorg. Chim. Acta 390 (2012) 210–216.
[12] M.C. Rodriguez-Argu¨elles, M.B. Ferrari, G.G. Fava, et al., Acenaphthenequinone
thiosemicarbazone and its transition metal complexes: synthesis, structure, and
biological activity, J. Inorg. Biochem. 66 (1997) 7–17.
both enzymes with IC50 = 2.15
AChE and BuChE, respectively. In addition, complex 1 also acts as a
potent antioxidant with an IC50 value of 33.90 mol/L. Therefore,
mmol/L and 2.16 mmol/L against
m

[13] S. Sharma, F. Athar, M.R. Maurya, et al., Novel bidentate complexes of Cu(II)
derived from 5-nitrofuran-2-carboxaldehyde thiosemicarbazones with antia-
moebic activity against E. histolytica, Eur. J. Med. Chem. 40 (2005) 557–562.
[14] G. Vatsa, O.P. Pandey, S.K. Sengupta, Synthesis, spectroscopic and toxicity studies
of titanocene chelates of isatin-3-thiosemicarbazones, Bioinorg. Chem. Appl. 3
(2005) 151–160.
612
Y.C. Chan et al. / Chinese Chemical Letters 24 (2013) 609–612
(C5S). Complex 1: army green, 60% yield, mp: 186–187 8C. Anal. Calcd. for
[Cu(L1)Cl]ClÁH2O (%): C, 36.76; H, 3.28; N, 12.25. Found: C, 36.84; H, 3.22; N,
12.18. IR (KBr, cmÀ1): n (NH2)aromatic 1635, (C5N) 1580, n (C5S) 801. UV–vis (DMF,
nm): 272, 293, 364, 433, 622. m/z (ESIÀMS): 367.8 (M+ – 2Cl – H2O, 62%). meff:
1.78 B.M.
[16] G.L. Ellman, D. Courtney, V.J. Andres, et al.,
A new and rapid colorimetric
[15] The characteristic data for representative compounds L1: pale yellow solid, 78%
yield, mp: 200–202 8C. Anal. Calcd. for C14H13ClN4S (%): C, 55.17; H, 4.27; N, 18.39.
Found: C, 55.13; H, 4.18; N, 18.45. IR (KBr, cmÀ1): n (NH2)aromatic 1622, n (C5N)
1591, n (C5S) 818. UV–vis (DMF, nm): 267, 319. 1H NMR (500 MHz, DMSO-d6): d
5.29 (s, 2H, Ar–NH2), 6.91 (d, 1H, J = 9.0 Hz, Ar–H), 6.92 (d, 1H, J = 2.5 Hz, Ar–H),
7.27–7.30 (dd, 1H, J = 2.5, 9.0 Hz, Ar–H), 7.36–7.41 (m, 3H, Ar–H), 7.70–7.71 (m,
2H, Ar–H), 8.28 (s, 1H, CSNH2), 8.61 (s, 1H, CSNH2), 8.61 (s, 1H, NH). 13C NMR
(125 MHz, DMSO-d6): d 119.68 (C–Cl), 144.62 (C–NH2), 146.43 (C5N), 178.07
determination of acetylcholinesterase activity, BioChem. Pharmacol. 7 (1961)
88–95.
´
[17] A. Zawadzka, I. Łozinska, Z. Mole˛da, et al., Highly selective inhibition of butyr-
ylcholinesterase by a novel melatonin–tacrine heterodimers, J. Pineal Res. (2012).
[18] S.M. Salga, H.M. Ali, M.A. Abdullah, et al., Synthesis, characterization, acetylcho-
linesterase inhibition, molecular modeling and antioxidant activities of some
novel Schiff bases derived from 1-(2-ketoiminoethyl)piperazines, Molecules 16
(2011) 9316–9330.