Microwave-assisted synthesis, molecular docking, and cholinesterase inhibitory activities of new ethanediamide and 2-butenediamide analogues

Article abstract of DOI:10.1248/cpb.c14-00754

A novel series of meta -substituted ethanediamide and 2-butenediamide derivatives were synthesized and tested for their ability to inhibit electric eel acetylcholinesterase (AChE) and equine serum butyrylcholinesterase (BuChE). The synthesized compounds w

Full text of DOI:10.1248/cpb.c14-00754

210
Chem. Pharm. Bull. 63, 210–217 (2015)
Vol. 63, No. 3
Regular Article
Microwave-Assisted Synthesis, Molecular Docking, and Cholinesterase
Inhibitory Activities of New Ethanediamide and 2-Butenediamide
Analogues
,a
Mehmet Koca,a Kadir Ozden Yerdelen,* Baris Anil,b and Zeynep Kasapa
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University; Erzurum 25240, Turkey: and
b Department of Organic Chemistry, Faculty of Science, Ataturk University; Erzurum 25240, Turkey.
Received October 31, 2014; accepted December 25, 2014
A novel series of meta-substituted ethanediamide and 2-butenediamide derivatives were synthesized and
tested for their ability to inhibit electric eel acetylcholinesterase (AChE) and equine serum butyrylcholin-
esterase (BuChE). The synthesized compounds were evaluated against ChE enzymes using the colorimetric
method described by Ellman et al. (Biochem. Pharmacol., 7, 1961). It was revealed that some synthesized
compounds exhibited high anticholinesterase activity, among which compounds 1f and 2f were the most ac-
tive inhibitors against BuChE (IC50 value=1.47µM) and AChE (IC50 value=2.09µM), respectively. Docking
simulations revealed that the inhibitors 1f and 2f are capable of simultaneously binding the peripheral anion-
ic site as well as the catalytic anionic site of both ChE enzymes. These derivatives are considered interesting
candidates for Alzheimer’s disease treatment.
Key words ethanediamide; 2-butenediamide; anticholinesterase activity; molecular docking
Alzheimer’s disease (AD), the most common cause of de- metal ions (Fe2+, Cu2+, Zn2+) in AD patients is 3–7 times
mentia is an irreversible neurodegenerative disorder character- higher than the level in healthy individuals.20)
ized by the loss of memory, learning and inability to perform
In our previous studies, we determined the significant
routine activities.1–4) The progression and definite reasons effects of the synthesized compounds, which have different
of the AD are still mostly unknown, but the most favourite halogen atoms (F, Cl, Br, I) on meta- and para-positions of
hypotheses have been put forward on the basis of the various aniline rings, on acetyl- and butyryl-cholinesterase inhibi-
causative factors such as cholinergic, amyloid, tau and metal tion.21,22) In this context, we investigated some potential
hypothesis.5) The standard medical treatment for AD includes positive contributions to cholinesterase inhibitory activity by
cholinesterase inhibitors (ChEIs) and a partial N-methyl-D- addition a chloro atom to the para position of benzyl ring and
aspartate (NMDA) antagonists.6,7) The cholinergic hypothesis we report the synthesis of new ethanediamide and 2-butenedi-
is one of the oldest and most popular hypotheses outlining the amide analogues and their in vitro cholinesterases inhibitory
pathogenesis of AD. Acetylcholine (ACh) deficiency severely activities. Molecular docking and metal chelation studies were
affects the cognitive abilities, memory function and emotional also conducted for the most potent derivatives.
responses in AD patients.
According to the cholinergic hypothesis, the main approach
of the current pharmacotherapy for AD is increasing the lev-
Results and Discussion
Chemistry As depicted in Chart 1, intermediate starting
els of ACh through the inhibition of cholinesterase enzymes compounds,
4-chlorobenzyl-(3-substituted-phenyl)-amines,
(ChEs).8,9) Two major cholinesterases, acetylcholinesterase required for the synthesis of ethanediamide and 2-butenedi-
(AChE) and butyrylcholinesterase (BuChE), which are located amide derivatives were obtained by using microwave irra-
in the central nervous system (CNS), are able to hydrolyse the diation under solvent-free phase-transfer catalysis conditions
neurotransmitter ACh.10) Studies have indicated that AD is after a short reaction time (5min). A same synthetic procedure
defined by decrement of level of AChE in the early stages of was used for the synthesis of some meta-substituted benza-
the disease along with the increasing ratio of BuChE to AChE nilines which was reported in our previous study.21) 3-Substi-
in advanced stages of the disease.11) Furthermore, BuChE has tuted ethanediamides (1a–f) were synthesized by the reaction
been found capable of compensating for the missing AChE of oxalyl chloride with 4-chlorobenzyl-(3-substituted-phenyl)-
catalytic functions in the synaptic cleft and its activity in- amines in tetrahydrofuran in the presence of triethylamine
creases, 30–60%, during AD.12–15) Due to the role of BuChE (TEA) with moderate to good yields (14–81%). 2-Butenedi-
in the hydrolysis of ACh, the inhibition of both ChEs using amide derivatives (2a–f) were obtained in moderate yields
a dual inhibitor should result in increased levels of ACh in (13–66%) by following the same procedure described in the
the brain that provides more successful clinical efficacy of previously reported literature.22) The obtained spectroscopic
AD.16,17) High levels of metals like mercury cause severe and data are in accordance with the predicted structures. The
fatal neurologic diseases, as well as learning and memory synthesis scheme of the compounds is presented in Chart 1.
problems and movement disorders.18) Imbalances in the levels In the proton nuclear magnetic resonance (1H-NMR) spectra,
and distribution of these metals in the brain, especially zinc, the resonance signals of ethylene bridge protons are registered
copper and iron may play a role in dementias like Alzheimer’s as singlets in the range of 6.83 to 7.04ppm for the compounds
disease (the so-called “metal hypothesis”).19) The level of 2a–f. The signals for the aromatic protons were showed in
*To whom correspondence should be addressed. e-mail: dadasozden@gmail.com
© 2015 The Pharmaceutical Society of Japan

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
211
Chart 1. Synthesis of Ethanediamide (1a–f) and 2-Butenediamide (2a–f) Derivatives
Table 1. In Vitro Inhibitory Potential of Target Compounds 1a–f and 2a–f against AChE and BuChE
IC50 (µM)
Compound
R
Selectivity indexc)
AChEa)
BuChEb)
1a
H
CH3
C2H5
F
> 100
> 100
> 100
> 100
> 100
1.74 0.01
12.04 0.11
17.11 0.14
4.78 0.07
3.16 0.08
1.47 0.10
> 100
11.22 0.02
35.48 0.24
> 100
53.76
8.30
5.84
20.92
31.64
68.02
0.02
2.67
0.37
0.15
0.08
0.03
2.13
1.48
1b
1c
1d
1e
Cl
1f
Br
> 100
2a
H
3.51 0.03
30.00 0.13
19.18 0.02
15.13 0.13
7.94 0.02
2.09 0.18
6.76 0.01
4.07 0.06
2b
CH3
C2H5
F
2c
2d
2e
Cl
> 100
> 100
14.45 0.01
2f
Br
Neostigmin
Ambenonium
6.02 0.04
a) Fifty percent inhibitory concentration (means S.D. of three experiments) of AChE. b) Fifty percent inhibitory concentration (means S.D. of three experiments) of
BuChE. c) Selectivity for BuChE=IC50 (AChE)/IC50 (BuChE).
the range of δ 6.62–7.52ppm and δ 6.72–7.49ppm for the index for BuChE are summarized in Table 1. All butenedi-
compounds 1a–h and 2a–h, respectively. The N-CH2 protons amide derivatives 2a–f showed moderate to good inhibitory
appeared at δ 4.61–4.64ppm for the compounds 1a–h and δ activity on AChE with micromolar concentrations. The meta
4.83–4.85ppm for the compounds 2a–h. The carbon nuclear bromo-substituted compound among the butenediamide de-
magnetic resonance (13C-NMR) spectrum shows character- rivatives, 2f (IC50=2.09µM), showed the most potent inhibitory
istic carbon resonance frequencies of carbonyl atoms in the activity against AChE, being 3.23- and 1.94-fold stronger than
range of δ 163.77–164.44ppm for the compounds 1a–h and the reference compounds neostigmine bromide (IC50=6.76 µM)
δ 163.97–164.42ppm for the compounds 2a–h. The aromatic and ambenonium dichloride (IC50=4.07µM), respectively.
carbons appeared at δ 122.46–145.39ppm for the compounds The other compound in the series, 2a, was found the second
1a–h and δ 115.49–146.20ppm for the compounds 2a–h. most powerful compound with IC50 value of 3.51µM. Also,
N-CH2 groups appeared at δ 51.32–51.41ppm. In the positive the most potent compounds, 2a and 2f, were found more se-
electron impact (EI) mass spectra, the molecular ion peaks lective inhibitors on AChE than the other compounds with
[M+H] show that the predicted compound has formed.
the lowest selectivity index (SI) values against BuChE (0.02,
Cholinesterase Inhibitory Activity The inhibitory activ- 0.03, respectively). The compounds 2b–e showed a moder-
ity of the compounds against AChE and BuChE was measured ate inhibitory activity to the reference compounds. Only the
according to the colorimetric assay of Ellman et al.23) Neostig- compounds, (2b, 2c), showed a moderate inhibitory activity on
mine and ambenonium were used as the reference compounds. BuChE with IC50 values of 11.22 and 35.48µM.
The IC50 values of all tested compounds and their selectivity
All ethanediamide compounds showed moderate to high

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Chem. Pharm. Bull.
Vol. 63, No. 3 (2015)
Fig. 1. Binding Interactions of Compound 1f and HuBuChE
inhibitory activity on BuChE and among these, compound
Molecular Modeling Studies The binding of the most
1f, which have a bromo substituent group on meta position potent compounds to AChE and BuChE was performed be-
among the ethanediamide derivatives, exhibited the strongest tween the most active compounds 1f with BuChE and 2f with
inhibition to BuChE with an IC50 value of 1.47µM, which was AChE, using the SYBYL X 2.0. The docking results revealed
9.82- and 4.09-fold more potent than those of neostigmine that the BuChE inhibitor (1f) occurred a hydrogen bond inter-
(IC50=14.45µM) and ambenonium (IC50=6.02µM). In addition, action between the carbonyl group of the compound and OH
the compound 1f exhibited high BuChE inhibitor selectivity group of Thr120 (2.04Å) in oxyanion hole of the catalytic ac-
with the selectivity value of 68.02. The compound 1a, a non- tive site (CAS) of HuBuChE (1P0I). π–π Stacking interactions
substituted derivative, showed the similar potent inhibitory were occurred between the 3-bromo substituted phenyl ring of
activity on BuChE, with an IC50 of 53.76 µM. Also the other the compound 1f and indole moiety of Trp82 (3.02–3.65Å) of
meta-halogenated ethanediamide derivatives, 1d and e, exhib- the peripheral anionic site (PAS) of BuChE (Fig. 1).
ited high inhibitory activity (IC50=4.78 µM and IC50=3.16 µM,
The most potent AChE inhibitor, compound 2f, displayed
respectively) against BuChE (Table 1). Conversely, ethane- multiple binding patterns with Torpedo californica (TcAChE-
diamide compounds were found inactive against AChE with 1ACJ), as shown in Fig. 2. In the 1ACJ–2f complex, the oxy-
IC50 values of 100µM.
gen atom from the carbonyl group created a hydrogen bond
According to the activity results, the butenediamide deriva- with OH group of Ser200 (1.93Å) into the CAS and OH group
tives (2a–f) were found more effective than the ethanediamide of Ser122 (1.38Å) into the PAS. The chloro group in the para-
derivatives on AChE inhibition due to the ethylene bridge. positions on benzyl ring created H-bond such as the NH group
This finding suggests us that the AChE inhibitory activity of Trp84 (2.70Å) into the CAS. The p-chlorobenzyl moiety
is strongly dependent on the presence of ethylene moiety of of the 2f showed a π–π stacking interaction with Phe330
the chemical structure. In contrast, the ethanediamide deriva- (3.12–3.61Å) into the CAS.
tives (1a–f) were seemed as very effective inhibitors against
BuChE. This finding clearly indicates that ethylene moiety IC50 values with some scoring functions, the most potent six
especially plays an important role as AChE inhibition.
molecules in series and two reference compounds were con-
In order to evaluate the binding affinity and experimental
Also, the compounds were designed with different substitu- structed and docked into the active sites of the related cho-
ent groups (–H, –CH3, –C2H5, –F, –Cl, –Br) on meta position linesterase enzymes using the Surflex-dock software. Surflex-
of phenyl ring with varying electronic properties. The target Dock score (Tscore) and consensus score (CScore) modules,
compounds 1d–f were found as effective inhibitors against which provide multiple approaches to better evaluate ligand–
BuChE, which have electron-withdrawing halogen atoms receptor interactions, were also used. It is hoped that by using
(–F, –Cl, –Br). This approach, as shown in Table 1, 1b–c pos- different scoring functions, the limitations of one function
sessing an electron-donating group (–CH3, –C2H5) showed a may be overcome. Hence, Tscores and CScores were used as
decreased BuChE inhibitory activity. It can be seen as a very a basis to verify compounds that were expected to bind with
similar structure activity relationship on AChE inhibition a higher affinity. The docking scores revealed that the most
among the most potent 2-butenediamide compounds (2d–f).
potent ChE inhibitors, the compounds 1f (Tscores [5.13] and

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
213
Fig. 2. Binding Interactions of Compound 2f and TcAChE
Table 2. IC50 and Docking Scores of the Compounds
Docking scores of AChE
Compound
IC50 (µM)
TScore
D_Score
PMF_Score
G_Score
ChemScore
Cscore
2a
3.51
7.94
2.09
6.76
4.07
4.90
5.36
5.38
2.95
3.51
194.54
297.00
−84.07
−49.12
−84.26
−57.82
−45.16
−334.19
−129.18
−331.41
−140.16
−361.35
−58.65
−41.43
−59.57
−43.85
−49.36
3
3
4
3
3
2e
2f
151.86
Neostigmin
Ambenonium
−245.25
−2001
Docking scores of BuChE
1a
1.74
3.16
4.98
4.48
5.13
1.81
1.41
24.69
21.05
−39.08
−44.51
−65.00
−11.12
−179.55
−250.21
−220.34
−103.53
−152.10
−45.41
−41.45
−48.26
−13.27
−28.88
3
3
4
2
3
1e
1f
1.47
51.39
Neostigmin
Ambenonium
14.45
6.02
−383.72
−1325.36
−15.55
CScore [4]) and 2f (Tscores [5.38] and CScore [4]) have better the most potent compounds, 1f and 2f, for the metals such
scores than the reference molecules. As can be seen, high as Cu2+, Fe2+ and Zn2+ in dimethylsulphoxide were studied
Total scores and CScores parallels experimental IC50 quite ac- by using UV-VIS spectrophotometer. The UV absorption of
curately. The Tscores and CScores of all molecules are shown the compounds in dimethyl sulfoxide (DMSO) changed with
in Table 2.
the titration of Cu2+, Fe2+ and Zn2+. The absorption peaks of
Other scoring functions (potential of mean force (PMF), the compounds 1f (absorbance: 0.50 at 261nm) in DMSO in-
Chem, G_, and D_scores) were also used during the structure creased after the addition of Cu2+ (absorbance: 0.78 at 262nm)
verification. These scores estimate the binding of free energy: and Fe2+ (absorbance: 0.92 at 261nm) (Fig. 3). The change
the lower the value, the better the binding. A positive value in the absorption intensity, indicates the formation of metal
means an unfavourable binding. Similar results were obtained chelations with Cu2+ and Fe2+ for the compound 1f. The elec-
when comparing these scoring functions. Briefly, all the com- tronic spectra of the compound 2f exhibited a red shift after
pounds’ PMF, Chem, and G_scores (except compound 2e) re- adding Cu2+ (the peak at 264nm shifted to 285nm) and Zn2+
port lower binding energies than those found in the reference (the peak shifted to 268nm). The other biometal, Fe2+, also
compounds. However, examination of the other scoring func- expressed similar result as compound 1f–Fe2+ complex. The
tions revealed that all tested compounds, except the reference absorbance intensity of the compound 2f (absorbance: 1.19 at
compounds, possessed positive D_scores.
Metal Chelating Effect The complexation abilities of 263nm) (Fig. 4).
264nm) increased when Fe2+ was added (absorbance: 1.32 at

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Chem. Pharm. Bull.
Vol. 63, No. 3 (2015)
Fig. 3. (a) UV Spectrum of Compound 1f (100 µM); (b) Spectrum of a Mixture of 1f (100 µM) and ZnSO4 (100 µM); (c) Spectrum of a Mixture of 1f
(100 µM) and CuSO4; (d) Spectrum of a Mixture of 1f (100 µM) and FeSO4 (100 µM)
Fig. 4. (a) UV Spectrum of Compound 2f (100 µM); (b) Spectrum of a Mixture of 2f (100 µM) and ZnSO4 (100 µM); (c) Spectrum of a Mixture of 2f
(100 µM) and CuSO4; (d) Spectrum of a Mixture of 2f (100 µM) and FeSO4 (100 µM)
derivative, compound 1f, was found as the most effective
Conclusion
In our work, we investigated the influence of amide deriva- inhibitor against BuChE (IC50 value: 1.86µM) and good bio-
tives on AChE and BuChE inhibition activity. We explored metal (Cu2+, Fe2+) chelator. The docking analysis of the newly
the positive influence of the ethylene part of the 2-butene- synthesized compounds, 1f and 2f, showed hydrogen bonding
diamide derivatives (2a–f) on the AChE inhibition activity. interactions with the CAS and π–π stacking interactions with
Compound 2f, exhibited the most potent inhibition against the PAS of both ChEs.
AChE (IC50 value: 2.09µM) and good Cu2+, Fe2+ and Zn2+
chelating ability. Among the synthesized ethanediamide de-
Experimental
rivatives, 1a and 1d–f, non-substituted and meta-halogenated
Chemistry Melting points of the compounds were ob-
derivatives, inhibited BuChE in low micromolar range, were tained on Electrothermal 9100 melting-point apparatus.
inactive against AChE. Also the most potent ethanediamide The 1H- and 13C-NMR spectra were recorded with tetra-

Vol. 63, No. 3 (2015)
Chem. Pharm. Bull.
215
methylsilane (TMS) as the internal standard on a Bruker 123.97, 124.00, 128.74, 129.63, 130.41 (d, JC-F=9.4Hz), 133.69,
FT-400(100) MHz spectrometer using deuterated chloroform 134.33, 140.43, 140.52, 162.37 (d, JC-F=249.7Hz) 163.84
(CDCl3) as the solvent. Mass spectra were recorded on Agilent (C=O). LC-MS-ES (+) m/z [M+H] 525.3.
1200 mass spectrometer at 10eV. Silica gel-G plates (Merck)
were used for TLC analysis with a mixture of n-hexane–ethyl oxalamide (1e) was obtained from (4-chlorobenzyl)-(3-chloro-
N,N′-Bis-(4-chlorobenzyl)-N,N′-bis-(3-chloro-phenyl)-
acetate (9:1). The chemical reagents and solvents used in this phenyl)-amine according to general procedure as white solid.
study were purchased from Merck or Sigma-Aldrich.
Yield 81%; mp 113–116°C. The crude compound was purified
General Procedure for Synthesis of the Compounds 1(a–f) by column chromatography on SiO2 eluting with hexane/
1
To synthesis of the N,N′-bis-(4-chlorobenzyl)-N,N′-diaryl- ethyl acetate (6:4) and recrystallized from ethanol. H-NMR
oxalamide derivatives, a mixture of (4-chlorobenzyl)-(3- (400MHz, CDCl3) δ: 4.62 (s, 4H, 2×CH2-N), 6.78 (m, 6H,
substituted-phenyl)-amine (1.4mmol), triethylamine (TEA) Ar-H), 6.83 (s, 2H, Ar-H), 7.14 (d, 4H, J=8.41Hz, Ar-H), 7.23
(1.4mmol) and oxalyl chloride (0.7mmol) in tetrahydrofuran (d, 2H, J=8.05Hz, Ar-H), 7.36–7.34 (m, 2H, Ar-H). 13C-NMR
(10mL) was stirred at room temperature for 12h. The reaction (100MHz, CDCl3) δ: 51.41 (CH2-N), 126.65, 128.46, 128.77,
mixture was quenched with 15mL of distilled water and the 128.97, 129.63, 129.70, 130.20, 133.72, 134.72, 140.13, 163.79
aqueous phase was extracted with two portions of CH2Cl2. (C=O). LC-MS-ES (+) m/z [M+H] 558.2.
The combined organic layers were dried over Na2SO4, filtered
and concentrated. The obtained solid was recrystallized from oxalamide (1f) was obtained from (3-bromo-phenyl)-(4-
N,N′-Bis-(3-bromo-phenyl)-N,N′-bis-(4-chlorobenzyl)-
ethanol. Synthesis pathway is shown on Chart 1.
chlorobenzyl)-amine according to general procedure as white
N,N′-Bis-(4-chlorobenzyl)-N,N′-diphenyl-oxalamide
(1a) solid. Yield 69%; mp 103–106°C. The crude compound was
was obtained from (4-chlorobenzyl)-phenyl-amine accord- recrystallized from ethanol. 1H-NMR (400MHz, CDCl3) δ:
ing to general procedure as white solid. Yield 70%; mp 4.62 (s, 4H, 2×CH2-N), 6.77 (d, 4H, J=8.40Hz, Ar-H), 6.83
189–191°C. The crude compound was recrystallized from eth- (d, 2H, J=8.00Hz, Ar-H), 6.98 (s, 2H, Ar-H), 7.15 (d, 4H,
1
anol. H-NMR (400MHz, CDCl3) δ: 4.62 (s, 4H, 2×CH2-N), J=8.45Hz, Ar-H), 7.19 (d, 2H, J=8.05Hz, Ar-H), 7.52–7.49
6.66 (d, 4H, J=8.40Hz, Ar-H), 6.87 (d, 4H, J=8.56Hz, Ar-H), (m, 2H, Ar-H). 13C-NMR (100MHz, CDCl3) δ: 51.34 (CH2-N),
7.06 (d, 4H, J=8.40Hz, Ar-H), 7.31–7.27 (m, 4H, Ar-H), 122.46, 127.24, 128.80, 129.62, 130.47, 131.33, 131.92, 133.73,
7.37–7.35 (m, 2H, Ar-H). 13C-NMR (100MHz, CDCl3) δ: 134.23, 140.24, 163.77 (C=O). LC-MS-ES (+) m/z [M+H]
51.37 (CH2-N), 128.50, 128.54, 128.60, 129.14, 129.54, 133.23, 645.8.
134.81, 139.23, 164.34 (C=O). Liquid chromatography/mass
spectrometry/electrospray (LC-MS-ES) (+) m/z [M+H] 489.4.
General Procedure for Synthesis of the Compounds 2(a–f)
A mixture of 4-(chloro-benzyl)-(3-substituted-phenyl)-amine
N,N′-Bis-(4-chlorobenzyl)-N,N′-di-m-tolyl-oxalamide (1b) (1.4mmol), TEA (1.4mmol) and dry ethylacetate (5mL)
was obtained from (4-chlorobenzyl)-m-tolyl-amine accord- was cooled with an ice bath to 0–5°C and fumaryl chloride
ing to general procedure as white solid. Yield 24%; mp (0.65mmol) in 5mL dry ethylacetate was added dropwise by
113–115°C. The crude compound was recrystallized from syringe over 30min. And the reaction mixture was stirred
1
ethanol. H-NMR (400MHz, CDCl3) δ: 2.28 (s, 6H, 2×Ar- at room temperature for 12h. The reaction mixture was
CH3), 4.62 (s, 4H, 2×CH2-N), 6.67 (d, 4H, J=8.28Hz, Ar-H), quenched with 50mL of water and the aqueous phase was
6.68 (d, 2H, J=8.76Hz, Ar-H), 6.69 (s, 2H, Ar-H), 7.07 (d, 4H, extracted with two portions of CH2Cl2. The combined organic
J=8.40Hz, Ar-H), 7.18 (m, 4H, Ar-H). 13C-NMR (100MHz, layers were dried over MgSO4, filtered and concentrated by
CDCl3) δ: 21.25 (Ar-CH3), 51.39 (CH2-N), 125.44, 128.43, rotary evaporation. And the target compounds were recrystal-
128.47, 128.83, 128.90, 129.25, 129.42, 133.13, 134.99, 139.06, lized from ethanol.
164.44 (C=O). LC-MS-ES (+) m/z [M+H] 517.4.
But-2-enedioic Acid Bis-[(4-chlorobenzyl)-phenyl-amide]
N,N′-Bis-(4-chlorobenzyl)-N,N′-bis-(3-ethyl-phenyl)- (2a) was obtained from (4-chlorobenzyl)-phenyl-amine ac-
oxalamide (1c) was obtained from (4-chlorobenzyl)-(3-ethyl- cording to general procedure as bright yellow. Yield 52%; mp
phenyl)-amine according to general procedure as white solid. 203–206°C. The crude compound was recrystallized from eth-
1
Yield 14%; mp 120–123°C. The crude compound was recrys- anol. H-NMR (400MHz, CDCl3) δ: 4.85 (s, 4H, 2×CH2-N),
1
tallized from ethanol. H-NMR (400MHz, CDCl3) δ: 1.17–1.13 6.86 (s, 2H, fumaryl CH=CH), 6.96 (d, 4H, J=7.88Hz, Ar-H),
(t, 6H, 2×CH2-CH3), 2.59–2.53 (q, 4H, 2×CH2-CH3), 4.61 (s, 7.07 (d, 4H, J=8.32Hz, Ar-H), 7.19 (d, 4H, J=8.32Hz, Ar-H),
4H 2×CH2-N), 6.62 (d, 4H, J=8.40Hz, Ar-H), 6.70–6.68 (m, 7.36–7.33 (m, 6H, Ar-H). 13C-NMR (100MHz, CDCl3) δ:
4H, Ar-H), 7.05–7.03 (m, 6H, Ar-H), 7.21–7.19 (m, 2H, Ar-H). 52.90 (CH2-N), 128.01, 128.43, 128.61, 129,87 130.09, 132.06
13C-NMR (100MHz, CDCl3) δ: 15.26 (CH2–CH3), 28.53 (CH=CH), 133.40, 135.41, 140.97, 164.34 (C=O). LC-MS-ES
(CH2–CH3), 51.32 (CH2-N), 125.72, 127.95, 128.07, 128.47, (+) m/z [M+H] 515.7.
128.89, 129.47, 133.10, 135.08, 139.28, 145.39, 164.44 (C=O).
LC-MS-ES (+) m/z [M+H] 545.5.
But-2-enedioic Acid Bis-[(4-chlorobenzyl)-m-tolyl-amide]
(2b) was obtained from (4-chlorobenzyl)-m-tolyl-amine ac-
N,N′-Bis-(4-chlorobenzyl)-N,N′-bis-(3-fluoro-phenyl)- cording to general procedure as white solid. Yield 13%; mp
oxalamide (1d) was obtained from (4-chlorobenzyl)-(3-flu- 166–169°C. The crude compound was recrystallized from
1
oro-phenyl)-amine according to general procedure as white methanol. H-NMR (400MHz, CDCl3) δ: 2.32 (s, 6H, 2×Ar-
solid. Yield 34%; mp 134–137°C. The crude compound was CH3), 4.83 (s, 4H, 2×CH2-N), 6.72 (d, 2H, J=7.72Hz, Ar-H),
recrystallized from ethanol. 1H-NMR (400MHz, CDCl3) 6.80 (s, 2H, Ar-H), 6.86 (s, 2H, fumaryl CH=CH), 7.08 (d, 4H,
δ: 4.64 (s, 4H, 2×CH2-N), 6.68–6.66 (m, 4H, Ar-H), 6.79 J=8.36Hz, Ar-H), 7.13 (d, 2H, J=7.64Hz, Ar-H), 7.21–7.19
(d, 4H, J=8.36Hz, Ar-H), 7.11–7.07 (m, 2H, Ar-H), 7.13 (d, (m, 2H, Ar-H), 7.23 (d, 4H, J=7.72Hz, Ar-H). 13C-NMR
4H, J=8.36Hz, Ar-H), 7.29–7.26 (m, 2H, Ar-H). 13C-NMR (100MHz, CDCl3) δ: 21.31 (Ar-CH3), 52.95 (CH2-N), 125.22,
(100MHz, CDCl3) δ: 51.34 (CH2-N), 115.82 (d, JC-F=20.7Hz), 128.37, 128.55, 129.25, 129.55, 130.07, 132.01 (CH=CH),

216
Chem. Pharm. Bull.
Vol. 63, No. 3 (2015)
133.33, 135.53, 139.96, 140.96, 164.39 (C=O). LC-MS-ES (+) (pH 8.0) to provide a final concentration range. In a 96-well
m/z [M+H] 543.5. plate, the assay medium in each well consisted of 50µL of a
But-2-enedioic Acid Bis-[(4-chlorobenzyl)-(3-ethyl-phenyl)- Tris buffer, 125µL of 3mM DTNB (Ellman’s reagent), 25µL
amide] (2c) was obtained from (4-chlorobenzyl)-(3-ethyl- of 0.2U/mL enzyme (AChE or BuChE) and 15mM substrate
phenyl)-amine according to general procedure as white solid. (ATCI or BTCI). The assay mixture containing enzyme,
Yield 32%; mp 147–150°C. The crude compound was recrys- buffer, DTNB and 25µL of inhibitor compound was prein-
1
tallized from ethanol. H-NMR (400MHz, CDCl3) δ: 1.20–1.17 cubated for 15min at 37°C, before the substrate was added to
(t, 6H, 2×CH2–CH3), 2.64–2.58 (q, 4H, 2×CH2–CH3), 4.84 (s, begin the reaction. Neostigmine bromide, ambenonium dichlo-
4H 2×CH2-N), 6.75 (d, 2H, J=7.92Hz, Ar-H), 6.76 (s, 2H, ride and all test compounds were prepared at eleven different
Ar-H), 6.87 (s, 2H, fumaryl CH=CH), 7.08 (d, 4H, J=8.36Hz, concentrations such as 0.097, 0.195, 0.39, 0.78, 1.56, 3.13, 6.25,
Ar-H), 7.16 (d, 2H, J=7.70Hz, Ar-H), 7.21–7.18 (m, 2H, Ar-H), 12.5, 25, 50 and 100µg/mL. The absorbance of the reaction
7.24 (d, 4H, J=7.64Hz, Ar-H). 13C-NMR (100MHz, CDCl3) mixture was then measured three times at 412nm every 45s
δ: 15.26, 28.55, 52.89 (CH2-N), 125.31, 127.26, 128.00, 128.54, using a microplate reader (Bio-Tek ELx800, U.S.A.). Results
129.65, 130.12, 132.06 (CH=CH), 133.32, 135.58, 140.97, are presented as means standard errors (S.E.) of the experi-
146.21, 164.42 (C=O). LC-MS-ES (+) m/z [M+H] 571.6.
ment. The IC50 values of the compounds showing percentage
But-2-enedioic Acid Bis-[(4-chlorobenzyl)-(3-fluoro- inhibition, the measurements and calculations were evaluated
phenyl)-amide] (2d) was obtained from (4-chlorobenzyl)-(3- by non-linear regression analysis using GraphPad Prism soft-
fluoro-phenyl)-amine according to general procedure as white ware.
solid. Yield 34%; mp 196–199°C. The crude compound was
Chelation Capacity The metal chelation was performed
recrystallized from ethanol. 1H-NMR (400MHz, CDCl3) δ: in dimethylsulphoxide at room temperature using UV-Vis
4.85 (s, 4H, 2×CH2-N), 6.77–6.72 (m, 4H, Ar–H), 6.86 (s, 2H, spectrophotometer (Thermo Electron Heλios) with wavelength
fumaryl CH=CH), 7.08 (d, 4H, J=8.36Hz, Ar-H), 7.05–7.02 ranging from 190 to 380nm.25,26) The UV absorption of the
(m, 2H, Ar-H), 7.22 (d, 4H, J=8.36Hz, Ar-H), 7.36–7.31 (m, test compounds 1f and 2f, in the absence or presence of with
2H, Ar-H). 13C-NMR (100MHz, CDCl3) δ: 52.83 (CH2-N), CuSO4, FeSO4 and ZnSO4 was recorded in a 1cm quartz
115.70 (d, JC-F=20.7Hz), 124.05, 128.76, 130.04, 131.10 (d, cuvette after 20min at room temperature. The final concentra-
JC-F=9.3Hz), 132.12 (CH=CH), 133.66, 134.98, 142.30, 142.39, tions of the test compounds and metals were 100µM.
162.97 (d, JC-F=249.9Hz), 164.01 (C=O). LC-MS-ES (+) m/z
[M+H] 551.4.
Molecular Docking Study In order to find out the bind-
ing mode for the synthesized compounds, docking simulation
Bis-[(4-chlorobenzyl)-(3-chloro- studies were carried out with Surflex-Dock. 3D structures of
But-2-enedioic
Acid
phenyl)-amide] (2e) was obtained from (4-chlorobenzyl)-(3- the compounds 1f and 2f were constructed using the Sybyl
chloro-phenyl)-amine according to general procedure as white sketcher module. The structures were minimized using the
solid. Yield 55%; mp 203–205°C. The crude compound was Steepest descent conjugated gradient method until the gradi-
recrystallized from ethanol. 1H-NMR (400MHz, CDCl3) δ: ent was 0.05 kcal/mol, max iterations: 1000 with the Tripos
4.84 (s, 4H, 2×CH2-N), 6.83–6.81 (m, 4H, Ar-H), 7.04 (s, force field with the Gasteiger Huckel charge. The simulation
2H, fumaryl CH=CH), 7.08 (d, 4H, J=8.40Hz, Ar-H), 7.22 system was built on X-ray crystallographic structures of 1ACJ
(d, 4H, J=8.36Hz, Ar-H), 7.28 (d, 2H, J=8.00Hz, Ar-H), and 1P0I which were obtained from the Protein Data Bank.
7.35–7.31 (m, 2H, Ar-H). 13C-NMR (100MHz, CDCl3) δ: 52.89 At the commencement of docking, all the water and ligands
(CH2-N), 126.62, 128.10, 128.76, 128.90, 130.05, 130.82, 132.14 were removed and the random hydrogen atoms were added.
(CH=CH), 133.68, 134.89, 135.43, 142.05, 163.98 (C=O). LC- Docking calculations using Surflex-Dock for 1ACJ and 1P0I
MS-ES (+) m/z [M+H] 583.1.
But-2-enedioic Acid
benzyl)-amide] (2f) was obtained from (3-bromo-phenyl)-(4-
were performed through protomol generation by ligand. The
Bis-[(3-bromo-phenyl)-(4-chloro- parameters used were threshold 0.5 and bloat 0.
To evaluate the docking experiment, the Tscore,27) D_
chlorobenzyl)-amine according to general procedure as white score,28) PMF_Score,29) G_score,30) and Chem-Score31) values
solid. Yield 66%; mp 202–204°C. The crude compound was were estimated using the Cscore module of SYBYL X. Since
recrystallized from ethanol. 1H-NMR (400MHz, CDCl3) δ: Cscore is a consensus scoring function, the different scoring
4.84 (s, 4H, 2×CH2-N), 6.83 (s, 2H, fumaryl CH=CH), 6.85 functions in it provide multiple approaches to better evaluate
(d, 2H, J=8.44Hz, Ar-H), 7.08 (d, 4H, J=8.36Hz, Ar-H), ligand–receptor interactions. The higher CScore value is as-
7.23–7.21 (m, 6H, Ar-H), 7.26 (s, 2H, Ar-H), 7.49 (d, 2H, sociated with better promising hits.
J=8.08Hz, Ar-H). 13C-NMR (100MHz, CDCl3) δ: 52.93
(CH2-N), 123.28, 127.13, 128.77, 130.06, 130.94, 131.06, 131.81,
Acknowledgment This research work was supported by
132.15 (CH=CH), 133.68, 134.86, 142.17, 163.97 (C=O). LC- Ataturk University Research Fund (Project No. 2014/167),
MS-ES (+) m/z [M+H] 671.3.
Pharmacology AChE, BuChE, 5,5-dithiobis-(2-nitroben-
zoic acid) DTNB, acetylthiocholine iodide (ATCI) and
Turkey.
Conflict of Interest The authors declare no conflict of
butyrylthiocholine iodide (BTCI) were purchased from interest.
Sigma-Aldrich. Inhibitory activities of AChE and BuChE of
the test compounds were evaluated by colorimetric Ellman’s
Suppementary Materials The online version of this ar-
method23) with some modifications using commercially avail- ticle contains supplementary materials.
able neostigmine bromide10) and ambenonium dichloride24) as
the reference compounds. The test compounds were dissolved
in dimethylsulphoxide and then diluted in 50mM Tris buffer
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