Preparation, anticholinesterase activity, and docking study of new 2-butenediamide and oxalamide derivatives

Article abstract of DOI:10.3109/14756366.2014.959947

Several new oxalamide and 2-butenediamide derivatives have been designed, synthesized and evaluated as the acetyl- and butyryl-cholinesterase inhibitors for Alzheimer's disease. The enzyme inhibitory activity of the synthesized compounds was measured using Ellman's colorimetric method. It was revealed that compound 1a (N,N′-bis-(4-chloro-benzyl)-N,N′-diphenyl-oxalamide) showed maximum activity against BuChE with a half maximal inhibitory concentration (IC₅₀) = 1.86 μM and compound 2a (but-2-enedioic acid bis-[(4-chloro-benzyl)-phenyl-amide]) exhibited optimum AChE (IC₅₀ = 1.51 μM) inhibition with a high-selectivity index. To better understand the enzyme-inhibitor interaction of the most active compounds towards cholinesterase, molecular modelling studies were carried out. Docking simulations revealed that inhibitors 1a and 2a targeted both the catalytic active site and the peripheral anionic site of 1ACJ and 1P0I.

Full text of DOI:10.3109/14756366.2014.959947

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.959947
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RESEARCH ARTICLE
Preparation, anticholinesterase activity, and docking study of new
2-butenediamide and oxalamide derivatives
Kadir Ozden Yerdelen1, Mehmet Koca1, Zeynep Kasap1, and Baris Anil2
2
1Department of Pharmaceutical Chemistry and Department of Organic Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey
Abstract
Keywords
Several new oxalamide and 2-butenediamide derivatives have been designed, synthesized and
evaluated as the acetyl- and butyryl-cholinesterase inhibitors for Alzheimer’s disease. The
enzyme inhibitory activity of the synthesized compounds was measured using Ellman’s
colorimetric method. It was revealed that compound 1a (N,N0-bis-(4-chloro-benzyl)-N,N0-
diphenyl-oxalamide) showed maximum activity against BuChE with a half maximal inhibitory
concentration (IC50) ¼ 1.86 mM and compound 2a (but-2-enedioic acid bis-[(4-chloro-benzyl)-
phenyl-amide]) exhibited optimum AChE (IC50 ¼ 1.51 mM) inhibition with a high-selectivity
index. To better understand the enzyme–inhibitor interaction of the most active compounds
towards cholinesterase, molecular modelling studies were carried out. Docking simulations
revealed that inhibitors 1a and 2a targeted both the catalytic active site and the peripheral
anionic site of 1ACJ and 1P0I.
2-butenediamide, Alzheimer, metal chelation,
molecular modelling, oxalamide
History
Received 6 August 2014
Revised 27 August 2014
Accepted 27 August 2014
Published online 27 November 2014
Introduction
High levels of metals like mercury cause severe and fatal
neurologic diseases, as well as learning and memory problems
and movement disorders20. However, some metals are essential
for brain function. Imbalances in the level and distribution of
these metals in the brain, especially zinc, copper and iron, may
play a role in diseases like AD (this pertains to the so-called
‘‘metal hypothesis’’)21. The level of metal ions (Fe2+, Cu2+ and
Zn2+) in AD patients is 3–7 times higher than it is in healthy
individuals22. For example, abnormally high levels of iron are
often found in the brains of people who die of Alzheimer’s
disease23. In addition, copper, iron and zinc are all associated with
abnormal clumps of brain proteins (plaques), which are a
Dementia is a loss of brain function that occurs with certain
diseases. Alzheimer’s disease (AD) is one form of dementia that
gradually gets worse over time and it can cause symptoms such as
the loss of memory, learning and language skills1–4. The progres-
sion and definite causes of AD are still mostly unknown, but the
most common hypotheses to have been put forward are based on
causative factors and include the cholinergic, amyloid, tau and
metal hypotheses5. Standard medical treatment for AD includes
cholinesterase inhibitors (ChEIs) and partial N-methyl-D-aspartate
(NMDA) antagonists6,7. The cholinergic hypothesis is one of the
oldest and most popular hypotheses outlining the pathogenesis of
AD. According to the cholinergic hypothesis, the main approach of
the current pharmacotherapy for AD is to increase the levels of
acetylcholine (ACh) through the inhibition of cholinesterase
enzymes (ChEs)8–10. Acetylcholinesterase (AChE) and butyryl-
cholinesterase (BuChE), which are located in the central nervous
system (CNS), are able to hydrolyse the neurotransmitter ACh11.
hallmark of Alzheimer’s disease24–26
.
The aim of this study was to synthesize and develop new
oxalamide and 2-butenediamide derivatives as potential antic-
holinesterase and chelator agents.
Materials and methods
Studies have shown that AD is defined by the rapid loss of AChE Experimental
activity in the early stages of the disease, along with an increasing
The melting points were measured on an Electrothermal 9100
1
ratio of AChE as the disease progresses12,13. Furthermore, BuChE
has been found to be capable of compensating for the missing
AChE catalytic functions in the synaptic cleft, and its activity
increases by 30–60% during AD14–17. Due to the role of BuChE in
the hydrolysis of ACh, the inhibition of both ChEs using a dual
inhibitor should result in increased levels of ACh in the brain,
(Thomas Hoover, USA) melting-point apparatus. The H and 13C
NMR spectra were recorded with a Bruker FT-400(100) MHz
(Rheinstetten, Germany) spectrometer using deuterated chloro-
form (CDCl3) as the solvent. Mass spectra were recorded on an
Agilent 1200 (Waldbronn, Germany) mass spectrometer at 10 eV.
Benzaniline compounds were synthesized with a CEM 3100
(Matthews, NC) microwave oven. Reaction progress and product
mixtures were routinely checked by thin-layer chromatography
(TLC) on Merck SilicaGel F254 (Darmstadt, Germany) alumin-
ium plates. The chemical reagents and solvents used in this study
were purchased from Merck (Darmstadt, Germany) or Sigma
Aldrich (Munich, Germany).
which provide more successful clinical efficacy of AD18,19
.
Address for correspondence: Kadir Ozden Yerdelen, Assist. Prof.,
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk
University, 25240 Erzurum, Turkey. Tel: +90-442-2315220.
Fax: +90-442-2360962. E-mail: dadasozden@gmail.com

2
K. O. Yerdelen et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
A general procedure for the synthesis of oxalamide
compounds (1a–h)
ꢀ: 21.19 (Ar-CH3), 51.41 (CH2–N), 128.37, 129.61, 129.71,
133.13, 135.03, 136.70, 138.65, 164.52 (C¼O). ESI-MS m/z
[M+H] 517.5.
(4-Chloro-benzyl)-(4-substituted-phenyl)-amine derivatives were
prepared with microwave (150 W, 120 ꢀC) irradiation under
solvent-free phase transfer catalysis conditions. To synthesise
the oxalamide derivatives, a mixture of (4-chloro-benzyl)-(4-
substituted-phenyl)-amine (1.4 mmol), triethylamine (1.4 mmol)
and oxalyl chloride (0.7 mmol) in tetrahydrofuran (10 ml) was
stirred at room temperature for 12 h. The reaction mixture was
quenched with 15 ml of distilled water, and the aqueous phase
was extracted with two portions of CH2Cl2. The combined
organic layers were dried over Na2SO4, filtered and concentrated.
The obtained solid was recrystallized from ethanol. The synthesis
pathway is shown in Scheme 1.
N,N0-Bis-(4-chloro-benzyl)-N,N0-bis-(4-ethyl-phenyl)-oxala-
mide (1c) was obtained from (4-chloro-benzyl)-(4-ethyl-phenyl)-
amine according to the general procedure as white solid. Yield
59%; m.p. 192–195 ꢀC. The crude compound was recrystallized
1
from ethanol. H-NMR (400 MHz, CDCl3) ꢀ: 1.28–1.25 (t, 6H,
2ꢁ CH2–CH3), 2.72–2.66 (q, 4H, 2ꢁ CH2–CH3), 4.59 (s, 4H 2ꢁ
CH2–N), 6.71 (d, 4H, J ¼ 8.40 Hz, Ar-H), 6.77 (d, 4H,
J ¼ 8.32 Hz, Ar-H), 7.05 (d, 4H, J ¼ 8.40 Hz, Ar-H), 7.09 (d,
4H, J ¼ 8.30 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3) ꢀ: 15.18
(CH2–CH3), 28.41 (CH2–CH3), 51.46 (CH2–N), 128.36, 128.40,
129.67, 129.71, 133.12, 135.07, 136.85, 144.79, 164.55 (C¼O).
ESI-MS m/z [M+H] 545.7.
N,N0-Bis-(4-chloro-benzyl)-N,N0-diphenyl-oxalamide (1a) was
obtained from (4-chloro-benzyl)-phenyl-amine according to the
general procedure as white solid. Yield 68%; m.p. 102–105 ꢀC.
N,N0-Bis-(4-chloro-benzyl)-N,N0-bis-(4-methoxy-phenyl)-oxa-
lamide (1d) was obtained from (4-chloro-benzyl)-(4-methoxy-
phenyl)-amine according to the general procedure as white solid.
Yield 44%; m.p. 187–190 ꢀC. The crude compound was
1
The crude compound was recrystallized from ethanol. H-NMR
(400 MHz, CDCl3) ꢀ: 4.62 (s, 4H, 2ꢁ CH2-N), 6.66 (d, 4H,
J ¼ 8.40 Hz, Ar-H), 6.87 (d, 4H, J ¼ 8.56 Hz, Ar-H), 7.06 (d, 4H,
J ¼ 8.40 Hz, Ar-H), 7.31–7.27 (m, 2H, Ar-H), 7.37–7.35 (m, 2H,
Ar-H). 13C-NMR (100 MHz, CDCl3) ꢀ: 51.37 (CH2–N), 128.50,
128.60, 129.14, 129, 17 129.54, 133.23, 134.81, 139.23, 164.34
(C¼O). ESI-MS m/z [M+H] 489.4.
1
recrystallized from ethanol. H-NMR (400 MHz, CDCl3) ꢀ: 3.83
(s, 6H, 2ꢁ OCH3), 4.58 (s, 4H, 2ꢁ CH2–N), 6.66 (d, 4H,
J ¼ 8.30 Hz, Ar-H), 6.77 (d, 4H, J ¼ 9.30 Hz, Ar-H), 6.81 (d, 4H,
J ¼ 9.28 Hz, Ar-H), 7.06 (d, 4H, J ¼ 8.30 Hz, Ar-H). 13C-NMR
(100 MHz, CDCl3) ꢀ: 51.40 (CH2-N), 51.42 (OCH3), 114.02,
128.43, 129.73, 130.08, 131.87, 133.18, 135.10, 159.59, 164.73
(C¼O). ESI-MS m/z [M + H] 549.3.
N,N0-Bis-(4-chloro-benzyl)-N,N0-di-p-tolyl-oxalamide (1b) was
obtained from (4-chloro-benzyl)-p-tolyl-amine according to the
general procedure as white solid. Yield 64%; m.p. 175–178 ꢀC.
N,N0-Bis-(4-chloro-benzyl)-N,N0-bis-(4-ethoxy-phenyl)-oxala-
mide (1e) was obtained from (4-chloro-benzyl)-(4-ethoxy-
phenyl)-amine according to the general procedure as white
solid. Yield 26%; m.p. 137–140 ꢀC. The crude compound was
recrystallized from ethanol. 1H-NMR (400 MHz, CDCl3) ꢀ: 1.49–
1.46 (t, 6H, 2ꢁ OCH2–CH3), 4.05–4.00 (q, 4H, 2ꢁ OCH2CH3),
1
The crude compound was recrystallized from ethanol. H-NMR
(400 MHz, CDCl3) ꢀ: 2.45 (s, 6H, 2ꢁ Ar-CH3), 4.58 (s, 4H, 2ꢁ
CH2–N), 6.69 (d, 4H, J ¼ 8.36 Hz, Ar-H), 6.75 (d, 4H,
J ¼ 8.28 Hz, Ar-H), 7.05 (d, 4H, J ¼ 8.36 Hz, Ar-H), 7.07
(d, 4H, J ¼ 8.28 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3)
Scheme 1. Synthesis of the target compounds 1a–h and 2a–h.

DOI: 10.3109/14756366.2014.959947
2-Butenediamide and oxalamide derivatives
3
4.57 (s, 4H, 2ꢁ CH2-N), 6.65 (d, 4H, J ¼ 8.28 Hz, Ar-H), 6.75 1H-NMR (400 MHz, CDCl3) ꢀ: 2.35 (s, 6H, 2ꢁ Ar-CH3), 4.83
(d, 4H, J ¼ 9.24 Hz, Ar-H), 6.78 (d, 4H, J ¼ 9.32 Hz, Ar-H), 7.06 (s, 4H, 2ꢁ CH2–N), 6.82 (d, 4H, J ¼ 8.20 Hz, Ar-H), 6.86 (s, 2H,
(d, 4H, J ¼ 8.32 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3) ꢀ: 14.81 fumaryl CH¼CH), 7.08 (d, 4H, J ¼ 8.40 Hz, Ar-H), 7.13 (d, 4H,
(OCH2CH3), 51.40 (CH2–N), 63.74 (OCH2CH3), 114.41, 128.42, J ¼ 8.08 Hz, Ar-H), 7.18 (d, 4H, J ¼ 8.02 Hz, Ar-H). 13C-NMR
129.69, 130.04, 131.68, 133.13, 135.14, 159.64, 164.82 (C¼O). (100 MHz, CDCl3) ꢀ: 21.11 (Ar-CH3), 52.87 (CH2–N), 127.75,
ESI-MS m/z [M+H] 577.5.
128.56, 130.12, 130.47, 131.97 (CH¼CH), 133.32, 135.54,
138.32, 138.41, 164.50 (C¼O). ESI-MS m/z [M + H] 543.2.
But-2-enedioic acid bis-[(4-chloro-benzyl)-(4-ethyl-phenyl)-
N,N0-Bis-(4-chloro-benzyl)-N,N0-bis-(4-fluoro-phenyl)-oxala-
mide (1f) was obtained from (4-chloro-benzyl)-(4-fluoro-phenyl)-
amine according to the general procedure as white solid. Yield amide] (2c) was obtained from (4-chloro-benzyl)-(4-ethyl-
52%; m.p. 147–150 ꢀC. The crude compound was recrystallized phenyl)-amine according to general procedure as bright yellow.
from ethanol. 1H-NMR (400 MHz, CDCl3) ꢀ: 4.59 (s, 4H, 2ꢁ Yield 55%; m.p. 194–197 ꢀC. The crude compound was
CH2-N), 6.85 (d, 4H, J ¼ 8.88 Hz, Ar-H), 6.86 (d, 4H, recrystallized from ethanol. 1H-NMR (400 MHz, CDCl3)
J ¼ 8.88 Hz, Ar-H), 6.98 (d, 4H, J ¼ 8.20 Hz, Ar-H), 7.12 ꢀ: 1.27–1.23 (t, 6H, 2ꢁ CH2–CH3), 2.68–2.62 (q, 4H, 2ꢁ CH2–
(d, 4H, J ¼ 8.36 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3) ꢀ: CH3), 4.83 (s, 4H 2ꢁ CH2–N), 6.86 (d, 4H, J ¼ 8.24 Hz, Ar-H),
51.37 (CH2–N), 115.98, 116.20, 128.66, 129.68, 130.59, 130.67, 6.89 (s, 2H, fumaryl CH¼CH), 7.09 (d, 4H, J ¼ 8.40 Hz, Ar-H),
133.67, 134.46 (C-F), 164.12 (C¼O). ESI-MS m/z [M+H] 525.3. 7.16 (d, 4H, J ¼ 8.24 Hz, Ar-H), 7.19 (d, 4H, J ¼ 8.40 Hz, Ar-H).
N,N0-Bis-(4-chloro-benzyl)-N,N0-bis-(4-chloro-phenyl)-oxala-
13C-NMR (100 MHz, CDCl3) ꢀ: 24.35 (CH2–CH3), 29.89 (CH2–
mide (1g) was obtained from (4-chloro-benzyl)-(4-chloro-phenyl)- CH3), 52.39 (CH2–N), 127.91, 128.36, 129.67, 129.71, 134.42
amine according to the general procedure as white solid. Yield (CH¼CH), 135.07, 136.45, 143.85, 163.35 (C¼O). ESI-MS m/z
42%; m.p. 182–185 ꢀC. The crude compound was recrystallized [M + H] 571.4.
from ethanol. 1H-NMR (400 MHz, CDCl3) ꢀ: 4.59 (s, 4H, 2ꢁ
But-2-enedioic
acid
bis-[(4-chloro-benzyl)-(4-methoxy-
CH2–N), 6.69 (d, 4H, J ¼ 8.40 Hz, Ar-H), 6.80 (d, 4H, phenyl)-amide] (2d) was obtained from (4-chloro-benzyl)-(4-
J ¼ 8.68 Hz, Ar-H), 7.14 (d, 4H, J ¼ 8.40 Hz, Ar-H), 7.27 (d, methoxy-phenyl)-amine according to general procedure as bright
4H, J ¼ 8.84 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3) ꢀ: 51.31 yellow solid. Yield 52%; m.p. 160–163 ꢀC. The crude compound
(CH2–N), 128.73, 129.40, 129.66, 129.95, 133.77, 134.33, was recrystallized from ethanol. 1H-NMR (400 MHz, CDCl3)
134.91, 137.53, 163.89 (C¼O). ESI-MS m/z [M + H] 557.4.
ꢀ: 3.80 (s, 6H, 2ꢁ OCH3), 4.81 (s, 4H, 2ꢁ CH2-N), 6.84 (s, 2H,
N,N0-Bis-(4-bromo-phenyl)-N,N0-bis-(4-chloro-benzyl)-oxala-
fumaryl CH¼CH), 6.85 (br s, 8H, Ar-H), 7.08 (d, 4H, J ¼ 8.24 Hz,
mide (1h) was obtained from (4-bromo-phenyl)-(4-chloro- Ar-H), 7.19 (d, 4H, J ¼ 8.28 Hz, Ar-H). 13C-NMR (100 MHz,
benzyl)-amine according to the general procedure as white CDCl3) ꢀ: 52.95 (CH2–N), 55.44 (OCH3), 114.97, 128.58, 129.17,
solid. Yield 66%; m.p. 217–220 ꢀC. The crude compound was 130.24, 131.94 (CH¼CH), 133.35, 133.52, 135.51, 159.29,
1
recrystallized from ethanol. H-NMR (400 MHz, CDCl3) ꢀ: 4.59 164.64 (C¼O). ESI-MS m/z [M + H] 575.6.
(s, 4H, 2ꢁ CH2–N), 6.69 (d, 4H, J ¼ 8.40 Hz, Ar-H), 6.73 (d, 4H,
But-2-enedioic acid bis-[(4-chloro-benzyl)-(4-ethoxy-phenyl)-
J ¼ 8.64 Hz, Ar-H), 7.16 (d, 4H, J ¼ 8.40 Hz, Ar-H), 7.42 (d, 4H, amide] (2e) was obtained from (4-chloro-benzyl)-(4-ethoxy-
J ¼ 8.64 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3) ꢀ: 51.28 (CH2– phenyl)-amine according to general procedure as white solid.
N), 128.78, 128.82, 129.65, 132.42, 133.78, 134.32, 138.05, Yield 45%; m.p. 167–170 ꢀC. The crude compound was
163.80 (C¼O). ESI-MS m/z [M + H] 645.7.
recrystallized from ethanol. 1H-NMR (400 MHz, CDCl3)
ꢀ: 1.44–1.41 (t, 6H, 2ꢁ OCH2–CH3), 4.04–3.98 (q, 4H, 2ꢁ
OCH2CH3), 4.80 (s, 4H, 2ꢁ CH2–N), 6.82 (s, 2H, fumaryl
CH¼CH), 7.07 (d, 4H, J ¼ 6.56 Hz, Ar-H), 7.08 (d, 4H,
J ¼ 6.64 Hz, Ar-H), 7.19 (d, 4H, J ¼ 6.52 Hz, Ar-H), 7.20 (d,
General procedure for the synthesis of 2-butenediamide
derivatives (2a–h)
The mixture of (4-chloro-benzyl)-(4-substituted-phenyl)-amine 4H, J ¼ 6.48 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3) ꢀ: 14.80
(1.4 mmol), triethylamine (TEA) (1.4 mmol) and dry ethylacetate (OCH2CH3), 52.94 (CH2-N), 63.70 (OCH2CH3), 115.39, 128.56,
(5 ml) was cooled with an ice bath to 0–5 ꢀC, and fumaryl chloride 129.14, 130.25, 131.93 (CH¼CH), 133.34, 135.54, 158.73,
(0.65 mmol) in 5 ml dry ethylacetate was added dropwise by 165.21 (C¼O). ESI-MS m/z [M + H] 603.5.
syringe over 30 min. The reaction mixture was then stirred at
But-2-enedioic acid bis-[(4-chloro-benzyl)-(4-fluoro-phenyl)-
room temperature for 12 h. The reaction mixture was quenched amide] (2f) was obtained from (4-chloro-benzyl)-(4-fluoro-
with 50 ml of water, and the aqueous phase was extracted with two phenyl)-amine according to general procedure as white solid.
portions of CH2Cl2. The combined organic layers were dried over Yield 27%; m.p. 194–197 ꢀC. The crude compound was
1
MgSO4, filtered and concentrated by rotary evaporation. The recrystallized from ethanol. H-NMR (400 MHz, CDCl3) ꢀ: 4.82
target compounds were recrystallized from ethanol. The synthesis (s, 4H, 2ꢁ CH2-N), 6.82 (s, 2H, fumaryl CH¼CH), 6.92 (d, 4H,
procedure is described in our previously reported study27.
J ¼ 8.84 Hz, Ar-H), 7.03 (d, 4H, J ¼ 8.24 Hz, Ar-H), 7.06 (d, 4H,
But-2-enedioic acid bis-[(4-chloro-benzyl)-phenyl-amide] J ¼ 8.36 Hz, Ar-H), 7.21 (d, 4H, J ¼ 8.40 Hz, Ar-H). 13C-NMR
(2a) was obtained from (4-chloro-benzyl)-phenyl-amine accord- (100 MHz, CDCl3) ꢀ: 52.92 (CH2–N), 128.72, 129.92, 130.14,
ing to general procedure as bright yellow. Yield 52%; m.p. 130.17, 130.20, 131.99 (CH¼CH), 133.63, 135.07 (C–F), 164.20
203–206 ꢀC. The crude compound was recrystallized from etha- (C¼O). ESI-MS m/z [M + H] 551.2.
nol. 1H-NMR (400 MHz, CDCl3) ꢀ: 4.85 (s, 4H, 2ꢁ CH2–N), 6.86
But-2-enedioic acid bis-[(4-chloro-benzyl)-(4-chloro-phenyl)-
(s, 2H, fumaryl CH¼CH), 6.96 (d, 4H, J ¼ 7.88 Hz, Ar-H), 7.07 amide] (2g) was obtained from (4-chloro-benzyl)-(4-chloro-
(d, 4H, J ¼ 8.32 Hz, Ar-H), 7.19 (d, 4H, J ¼ 8.32 Hz, Ar-H), phenyl)-amine according to general procedure as white solid.
7.35 (d, 4H, J ¼ 7.88 Hz, Ar-H). 13C-NMR (100 MHz, CDCl3) ꢀ: Yield 57%; m.p. 216–219 ꢀC. The crude compound was
1
52.90 (CH2–N), 128.01, 128.43, 128.61, 129.87, 130.09, 132.06 recrystallized from ethanol. H-NMR (400 MHz, CDCl3) ꢀ: 4.83
(CH¼CH), 133.40, 135.41, 140.97, 164.34 (C¼O). ESI-MS m/z (s, 4H, 2ꢁ CH2–N), 6.82 (s, 2H, fumaryl CH¼CH), 6.89 (d, 4H,
[M + H] 515.7.
J ¼ 8.60 Hz, Ar-H), 7.07 (d, 4H, J ¼ 8.31 Hz, Ar-H), 7.21 (d, 4H,
But-2-enedioic acid bis-[(4-chloro-benzyl)-p-tolyl-amide] (2b) J ¼ 8.30 Hz, Ar-H), 7.33 (d, 4H, J ¼ 8.60 Hz, Ar-H). 13C-NMR
was obtained from (4-chloro-benzyl)-p-tolyl-amine according to (100 MHz, CDCl3) ꢀ: 52.83 (CH2-N), 128.76, 129.34, 130.15,
general procedure as white solid. Yield 45%; m.p. 201–204 ꢀC. 132,06 (CH¼CH), 133.68, 134.96, 139.31, 164.02 (C¼O).
The crude compound was recrystallized from ethanol. ESI-MS m/z [M + H] 583.4.

4
K. O. Yerdelen et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
But-2-enedioic acid bis-[(4-bromo-phenyl)-(4-chloro-benzyl)- performed through protomol generation by ligand. The parameters
amide] (2h) was obtained from (4-bromo-phenyl)-(4-chloro- used were threshold 0.5 and bloat 0.
benzyl)-amine according to general procedure as bright yellow.
Yield 27%; m.p. 213–216 ꢀC. The crude compound was Result and discussion
recrystallized from ethanol. H-NMR (400 MHz, CDCl3) ꢀ: 4.83
1
Chemistry
(s, 4H, 2ꢁ CH2–N), 6.82 (d, 4H, J ¼ 8.52 Hz, Ar-H), 6.83 (s, 2H,
fumaryl CH¼CH), 7.07 (d, 4H, J ¼ 8.40 Hz, Ar-H), 7.21 (d, 4H, Compounds 1a–h were synthesized by the reaction of oxalyl
J ¼ 8.36 Hz, Ar-H), 7.48 (d, 4H, J ¼ 8.60 Hz, Ar-H). 13C-NMR chloride with para-(4-chloro-benzyl)-(4-substituted-phenyl)-
(100 MHz, CDCl3) ꢀ: 52.79 (CH2–N), 122.55, 128.77, 129.64, amines in tetrahydrofuran in the presence of triethylamine with
130.13, 132.07 (CH¼CH), 133.69, 134.94, 139.83, 163.95 moderate to good yields (26–68%). 2-Butenediamide derivatives
(C¼O). ESI-MS m/z [M + H] 670.8.
(2a–h) were obtained in moderate yields (27–57%). The obtained
spectroscopic data are in accordance with the predicted structures.
The synthesis scheme of the compounds is presented in Scheme 1.
In the proton nuclear magnetic resonance (1H-NMR) spectra, the
resonance signals of ethylene bridge protons are registered as
singlets in the range of 6.82 to 6.89 ppm for compounds 2a–f. The
signals for aromatic protons were showed in the range of ꢀ 6.65–
7.42 and ꢀ 6.82–7.48 ppm for compounds 1a–h and 2a–h,
respectively. The N-CH2 protons appeared at ꢀ 4.57–4.62 ppm
for compounds 1a–h and ꢀ 4.80–4.85 ppm for compounds 2a–h.
The carbon nuclear magnetic resonance (13C-NMR) spectrum
shows characteristic carbon resonance frequencies of carbonyl
atoms in the range of 163.80–164.82 ppm for compounds 1a–h
and 163.35–165.21 ppm for compounds 2a–h. The aromatic
carbons appeared at ꢀ 114.97–159.64 ppm and N–CH2 groups
appeared at ꢀ 51.28–52.90 ppm. In the positive electron impact
(EI) mass spectra, the molecular ion peaks [M + H] show that the
predicted compound has formed. Spectral data of the compounds
are documented in Supplementary file.
Inhibition studies on AChE and BuChE
AChE, BuChE, 5,5-dithiobis-(2-nitrobenzoic acid) DTNB, acet-
ylthiocholine iodide (ATCI) and butyrylthiocholine iodide (BTCI)
were purchased from Sigma Aldrich. The inhibitory activities of
the AChE and BuChE test compounds were evaluated using
Ellman’s colorimetric method28 with some modifications using
commercially available neostigmine bromide11 and ambenonium
dichloride29 as the reference compounds. The test compounds
were dissolved in dimethylsulfoxide and then diluted in a
50-mM Tris buffer (pH 8.0) to provide a final concentration
range. In a 96-well plate, the assay medium in each well consisted
of 50 ml of a Tris buffer, 125 ml of 3 mM DTNB (Ellman’s
reagent), 25 ml of 0.2 U/ml enzyme (AChE or BuChE) and a
15-mM substrate (ATCI or BTCI). The assay mixture containing
the enzyme, buffer, DTNB and 25 ml of the inhibitor com-
pound was preincubated for 15 min at 37 ꢀC before the substrate
was added to begin the reaction. Neostigmine bromide,
ambenonium dichloride and all test compounds were prepared
at 11 different concentrations: 0.09, 0.195, 0.39, 0.78, 1.56, 3.13, In vitro cholinesterase enzymes inhibitory activity
6.25, 12.5, 25, 50 and 100 mg/ml. The absorbance of the reaction
The inhibitory activity of the compounds against AChE and
mixture was then measured three times at 412 nm every 45 s
BuChE was measured according to the colorimetric assay of
using a microplate reader (Bio-Tek ELx800, USA). The results
Ellman28. Neostigmine and ambenonium were used as the
are presented as mean standard errors of the experiment.
reference compounds. The IC50 values of all tested compounds
The IC50 values of the compounds showing percentage
and their selectivity index for AChE are summarized in Table 1. In
inhibition, the measurements and the calculations were
general, butenediamide derivatives 2a–h showed moderate to good
evaluated by non-linear regression analysis using GraphPad
inhibitory activity on AChE with micromolar concentrations. The
Prism software.
non-substituted compound among the butenediamide derivatives,
2a (IC50 ¼ 1.51 mM), showed the most potent inhibitory activity
Spectrophotometric measurement of chelation capacity
against AChE, being 4.47- and 2.69-fold stronger than the
reference compounds neostigmine bromide (IC50 ¼ 6.76 mM) and
The ability of metal chelation was determined by measuring the
formation of metal complexes in dimethylsulfoxide (DMSO) at
room temperature using a UV–visible spectrophotometer (Thermo
ambenonium dichloride (IC50 ¼ 4.07 mM), respectively. The other
compound in the series, 2f, was found to be the second most
powerful compound, with an IC50 value of 2.47 mM. In addition,
the most potent compounds, 2a and 2f, were found to be more
selective inhibitors on AChE than the other compounds with 62.11
and 40.48 selectivity index (SI) values, respectively. At the same
time, compounds 2g and 2h showed similar activity to the reference
compounds. Compounds 2b and 2c were found to have moderate
inhibitory activity on AChE and BuChE, with an IC50 of 87.09 and
26.30 mM, respectively. A similar situation was seen on BuChE
inhibition with IC50 values of 11.22 and 35.48 mM for the same
compounds (2b and 2c). Compound 2g exhibited the strongest
Electron Heꢁios) with a wavelength ranging from 190 to
380 nm30,31. The UV absorption of test compounds 1a and 2a,
in the absence or presence of CuSO4, FeSO4 and ZnSO4, was
recorded in a 1-cm quartz cuvette after 20 min at room
temperature. The final volume of the reaction mixture was 3 ml,
and the final concentrations of the test compounds and metals
were 100 mM.
Docking procedure
The docking study was performed using Surflex-Dock in Sybyl-X inhibition to BuChE, with an IC50 value of 7.07 mM. In addition,
2.0 by Tripos Associates. 3D structures of compounds 1a and 2a compound 2g exhibited 2.04-fold stronger inhibition against
were constructed using the Sybyl sketcher module. The structures BuChE than the reference compound neostigmine bromide
were minimized using the steepest descent conjugated gradient (IC50 ¼ 14.45 mM).
method until the gradient was 0.001 kcal/mol and max iterations:
On the other hand, 1a exhibited the strongest inhibition to
1000 with the Tripos force field with the Gasteiger–Huckel BuChE, with an IC50 value of 1.86 mM, which was 7.76- and 3.23-
charge. The simulation system was built on the crystal structures fold more potent than those of neostigmine (IC50 ¼ 14.45 mM) and
of 1ACJ and 1P0I, which were obtained from the Protein Data ambenonium (IC50 ¼ 6.02 mM). In addition, compound 1a had a
Bank. At the commencement of docking, all the water and ligands high level of BuChE inhibitor selectivity. Compound 1b was
were removed and the random hydrogen atoms were added. found to have moderate inhibitory activity on BuChE, with an
Docking calculations using Surflex-Dock for 1ACJ and 1P0I were IC50 of 72.02 mM. The other oxalamide derivatives did not exhibit

DOI: 10.3109/14756366.2014.959947
2-Butenediamide and oxalamide derivatives
5
inhibitory activity against both ChEs (Table 1). These compounds oxalamide derivatives on AChE inhibition due to the ethylene
in the series were considered to be inactive since their IC50 values bridge for the linker between the two carbonyl groups. This
were more than 100 mM.
finding suggests that acetylcholinesterase’s inhibitory activity is
From the IC50 values, the butenediamide derivatives strongly dependent on the presence of an ethylene moiety in the
(except for 2d and 2e) seemed to be more effective than the inhibitors. In our previously reported study, the results of
anticholinesterase activity showed that the a,b-unsaturated
Table 1. Anticholinesterase activity of IC50 (mM) and selectivity of
compounds 1a–h and 2a–h on AChE and BuChE.
moiety contributed to the interaction with cholinesterase
enzymes27.
The target compounds (1a–h and 2a–h) were synthesized
using different para-substituted anilines with varying electronic
properties. The target compounds 2f–h were found to be effective
inhibitors against AChE, which have various electron-drawing
halogen groups (F, Cl and Br) at the para-position of the phenyl
ring. To support this approach, as shown in Table 1, 2b–d
possessing an electron-donating group (–CH3, –C2H5, –OCH3,
–OC2H5) at the para-position of the aniline ring moiety showed a
decreased AChE inhibitory activity. These results imply that an
electron-drawing group at the para-position of the aniline ring is
beneficial to AChE inhibitory activity.
IC50 (mM)
Selectivity
indexc
Compounds
R1
R2
AChEa
BuChEb
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
2d
2e
2f
2g
H
CH3
C2H5 Cl
OCH3 Cl
OC2H5 Cl
F
Cl
Br
Cl
Cl
4100
4100
4100
4100
4100
4100
4100
1.86 0.029
72.02 0.058
4100
0.02
0.72
–
–
–
–
–
–
62.11
0.12
1.35
–
4100
4100
4100
Cl
Cl
Cl
Cl
4100
4100
4100
4100
1.51 0.121
H
CH3
Docking simulation
Cl 87.09 0.011 11.22 0.021
C2H5 Cl 26.30 0.045 35.48 0.24
To explore the possible interaction mode of the most potent
compounds with AChE and BuChE, molecular modelling simu-
lations were performed between the most active compounds, 2a
with AChE and 1a with BuChE, using SYBYL X 2.0 Surflex-
Dock software. The enzyme structures were obtained from the
Protein Data Bank. The docking results showed that compound 2a
displayed multiple binding patterns with Torpedo californica
(TcAChE-1ACJ), as shown in Figure 1. In the 1ACJ-2a complex,
the oxygen atom from the carbonyl group created two hydrogen
OCH3 Cl
OC2H5 Cl
F
Cl
Br
4100
4100
2.47 0.18
4100
4100
4100
–
Cl
Cl
Cl
40.48
0.85
18.21
2.13
1.48
8.31 0.154 7.07 0.065
5.49 0.32 4100
6.76 0.012 14.45 0.015
4.07 0.068 6.02 0.045
2h
Neostigmin
Ambenonium
a50% inhibitory concentration (means SD of three experiments)
of AChE.
b50% inhibitory concentration (means SD of three experiments)
of BuChE.
˚
bonds with an OH group of Ser200 (2.54 A) and an NH group of
˚
Gly118 (2.16 A) CAS. The chloro group in the para-positions on
benzyl rings created an H-bond, such as the OH group of Tyr334
˚
(2.55 A) into the PAS. The benzyl moiety of compound 2a was
cSelectivity for AChE ¼ IC50 (BuChE)/IC50 (AChE).
Figure 1. Docking model of compound 2a and TcAChE complex.

6
K. O. Yerdelen et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Figure 2. Docking model of compound 1a and HuBuChE complex.
Figure 3. (a) UV spectrum of compound 1a
(100 mM). (b) Spectrum of a mixture of 1a
(100 mM) and ZnSO4 (100 mM). (c) Spectrum
of a mixture of 1a (100 mM) and CuSO4.
(d) Spectrum of a mixture of 1a (100 mM)
and FeSO4 (100 mM).
located on the residues of Trp84 and Phe330 in a ‘‘sandwich’’
form. The p-chlorobenzyl moiety of 2a was observed to bind to
Metal chelating effect
CAS via parallel ꢂ–ꢂ stacking interactions between Trp84 The complexation abilities of the most potent compounds, 1a and
2a, for metals such as Cu2+, Fe2+ and Zn2+ in DMSO were
˚
(3.75 A) and Phe330 (3.23 A) in a ‘‘sandwich’’ form.
˚
The most potent BuChE inhibitor, 1a, showed interactions with studied using a UV–visible spectrophotometer. The UV absorp-
Gly116, Gly117, Ala199 and Trp82 residues of human butyr- tion of the compounds in DMSO changed with the titration of
ylcholinesterase enzyme (HuBuChE-1P0I) (Figure 2). Hydrogen Cu2+, Fe2+ and Zn2+, and the results are shown in Figures 3 and 4.
bond interactions occurred between the carbonyl group of The absorption peaks of compounds 1a and 2a in DMSO
compound 1a and the NH groups of Gly116 (1.92 A), Gly117 increased after the addition of Cu2+ and Fe2+. Only compound 1a
˚
˚
˚
(2.06 A) and Ala199 (2.51 A) in an oxyanion hole of the CAS of showed an increased absorption peak after adding ZnSO4.
HuBuChE. In addition, one of the phenyl rings entered the PAS of A change in the absorption intensity was observed after adding
BuChE and ꢂ–ꢂ stacking interactions occurred with the indole CuSO4, FeSO4 and ZnSO4, indicating the formation of all
biometal complexes for compound 1a (Cu2+, Fe2+ and Zn2+).
˚
moiety of Trp82 (3.30–5.25 A).

DOI: 10.3109/14756366.2014.959947
2-Butenediamide and oxalamide derivatives
7
Figure 4. (a) UV spectrum of compound 2a
(100 mM). (b) Spectrum of a mixture of 2a
(100 mM) and ZnSO4 (100 mM). (c) Spectrum
of a mixture of 2a (100 mM) and CuSO4.
(d) Spectrum of a mixture of 2a (100 mM)
and FeSO4 (100 mM).
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Conclusion
In conclusion, our study involved the synthesis of new oxalamide
and 2-butenediamide compounds. These compounds were eval-
uated as potential inhibitors of ChE and as chelators of biometals.
Among the synthesized 2-butenediamide derivatives, compound
2a exhibited the greatest inhibitory potency towards AChE
(IC50 value: 1.51 mM) and good Fe2+ and Cu2+ chelating ability.
In addition, the most potent oxalamide derivative, compound 1a,
showed inhibitory activity towards BuChE (IC50 value: 1.86 mM).
In addition, compound 1a exhibited high-metal chelation ability
with all of the biometals. Docking simulations showed that
compounds 1a and 2a were bound mainly with the catalytic active
sites of AChE and BuChE, respectively. Modelled derivatives
created lots of hydrogen bonding interactions with the CAS and
ꢂ–ꢂ stacking interactions with the PAS of both ChEs, which
confirms their high-inhibitory potency. As a result, further
investigations of AD candidates based on these results are in
progress.
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mitigate nausea and vomiting in patients with Alzheimer’s
disease who receive rivastigmine. Clin Neuropharmacol 2002;25:
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butyrylcholinesterase inhibition by a novel experimental Alzheimer
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2008;33:745–53.
Declaration of interest
This research work was supported by Ataturk University Research Fund
(Project No: 2013/271), Turkey. The authors have declared no conflicts of
interest with the presented data from this article.
17. Mesulam MM, Guillozet A, Show P, et al. Acetylcholinesterase
knockouts establish central cholinergic pathways and can use
butyrylcholinesterase to hydrolyze acetylcholine. Neuroscience
2002;110:627–39.
18. Basiri A, Murugaiyaha V, Osman H, et al. An expedient, ionic liquid
mediated multi-component synthesis of novel piperidone grafted
cholinesterase enzymes inhibitors and their molecular modeling
study. Eur J Med Chem 2013;67:221–9.
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Supplementary material available online