Synthesis and anticholinesterase activity of fumaramide derivatives

Article abstract of DOI:10.1007/s00044-013-0493-8

A series of fumaramide derivatives were synthesized from substituted benzanilines and their cholinesterase inhibitory activity was assayed according to Ellman's method using galanthamine-HBr as the reference compound. Most of the fumaramide compounds showed inhibitory activity of both cholinesterase enzymes. Compounds 29 (IC₅₀ = 0.14 μM) and 30 (IC₅₀ = 16.50 μM) were found to be the most active inhibitors on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes, respectively. Molecular docking studies were performed with Surflex-Dock programme to provide the possible interactions between compounds and enzymes. A Lineweaver-Burk plot and molecular modelling studies showed that fumaramide compounds targeted both the catalytic anionic site and the peripheral anionic site of AChE. It was revealed that the nature of α,β-unsaturated 1,4-diketone moiety in fumaramide compounds brought about useful and efficient modification especially on AChE inhibition.

Full text of DOI:10.1007/s00044-013-0493-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0493-8
ORIGINAL RESEARCH
Synthesis and anticholinesterase activity of fumaramide
derivatives
•
Kadir Ozden Yerdelen Halise Inci Gul
Received: 7 December 2012 / Accepted: 11 January 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of fumaramide derivatives were syn-
thesized from substituted benzanilines and their cholines-
terase inhibitory activity was assayed according to Ellman’s
method using galanthamine-HBr as the reference com-
pound. Most of the fumaramide compounds showed inhib-
itory activity of both cholinesterase enzymes. Compounds
29 (IC50 = 0.14 lM) and 30 (IC50 = 16.50 lM) were
found to be the most active inhibitors on acetylcholinester-
ase (AChE) and butyrylcholinesterase (BuChE) enzymes,
respectively. Molecular docking studies were performed
with Surflex-Dock programme to provide the possible
interactions between compounds and enzymes. A Lineweaver–
Burk plot and molecular modelling studies showed that
fumaramide compounds targeted both the catalytic anionic
site and the peripheral anionic site of AChE. It was revealed
that the nature of a,b-unsaturated 1,4-diketone moiety in
fumaramide compounds brought about useful and efficient
modification especially on AChE inhibition.
AD primarily affects the elderly section of the population,
specifically people aged 65 or older (Terry and Buccafusco,
2003). Due to its debilitating nature, this disease places an
enormous social and economic burden on society. The sig-
nificance of AD is further compounded, as it is estimated that
the number of identified cases will quadruple by the year
2050 (Brookmeyer et al., 2007). Medicines created to cure
AD have received significant attention for many years. Still,
the molecular causes of this condition remain unknown, in
spite of the existence of several theories regarding the
pathogenesis of AD (Castro et al., 2002; Doraiswamy, 2002;
Johannsen, 2004; Wilkinson et al., 2004).
Acetylcholinesterase (AChE) is a hydrolytic enzyme
that acts on ACh to terminate its actions in the synaptic
cleft by cleaving the neurotransmitter to choline and ace-
tate (Shen et al., 2002). Recent studies have shown that AD
pathogenesis is characterized by the rapid loss of AChE
activity in the early stages of the disease, along with the
increasing ratio of AChE as the disease progresses (Dar-
vesh et al., 2003; Greig et al., 2005). These results support
the need to control the activity of the AChE enzyme at
different stages of AD. On this basis, this hypothesis has
become the leading strategy for the development of AD
drugs. Up to now, AChE inhibition has represented a
purely palliative treatment for AD. However, the impor-
tance of AChE’s sister enzyme, butyrylcholinesterase
(BuChE), has also risen as a pharmacological target for AD
therapy in recent years. BuChE has been found to be
capable of compensating for the missing AChE catalytic
functions in the synaptic cleft (Li et al., 2000; Mesulam
et al., 2002) and that its activity significantly increases, by
30–60 %, during a time course in AD (Jhee et al., 2002;
Perry et al., 1978). Therefore, another promising approach
is the development of dual inhibitors using AChE and
BuChE (Fallarero et al., 2008), as BuChE activity seems to
Keywords Fumaramide ꢀ Benzanilines ꢀ
Acetylcholinesterase ꢀ Butyrylcholinesterase ꢀ
Molecular docking
Introduction
Alzheimer’s disease (AD), which is the most common form
of dementia, is a progressive and degenerative disorder. It
involves a decrease in cognitive functions such as altered
memory ability and learning and behavioural disturbances.
K. O. Yerdelen (&) ꢀ H. I. Gul
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Ataturk University, 25240 Erzurum, Turkey
e-mail: dadasozden@gmail.com
123

Med Chem Res
correlate with AChE activity in AD, such that a cognitive
improvement could be reached (Decker et al., 2008; Kamal
et al., 2008). Currently, several AChE inhibitors (such as
tacrine, galanthamine, donepezil and rivastigmine) are used
for the treatment of AD. However, they can only treat mild
to moderate levels of the disease (Grossberg, 2003) and
some of the drugs approved for therapeutic use show
hepatotoxicity (Knapp et al., 1994) and cause gastrointes-
tinal disturbances (Schulz, 2003). There is still a need to
develop more efficient drugs for AD.
as well as by high resolution mass spectra and reported for
the first time in this study. Amongst the fumaramide series,
compounds 23 (R1 = OC2H5; R2 = CH3) and 24
(R1 = OC2H5; R2 = H), containing the p-ethoxy substituted
aniline group, were obtained with the best yields in series
(72–71 %). The synthetic pathway is shown in Scheme 1.
In vitro inhibition study
The potency and selectivity of the synthesized compounds
1–34 were evaluated by their in vitro inhibitory effects on
AChE and BuChE. The inhibitory activities of the com-
pounds against freshly prepared AChE and BuChE were
investigated by determining the rate of hydrolysis of acet-
ylthiocholine and butyrylthiocholine in comparison with
reference compound galanthamine-HBr, by a modified
method of Ellman et al. (1961) as reported (Fawole et al.,
2010). Primarily, we have chosen to examine the possible
AChE inhibitory activity of compounds using thin-layer
choromotography/bioautography (TLC-B) assay which was
previously described (Marston et al., 2002). All of the
compounds especially fumaramide derivatives were visu-
alised as white spots on the TLC plates in comparison with
the reference compound. TLC-B assay played an important
role in monitoring the active compounds. However, this
qualitative evaluation gave a positive aspect in the course of
the biological study before applying the microplate assay.
Amongst the benzaniline series, 1 and 4 displayed a cer-
tain level of inhibitory activity against AChE (IC50 val-
ues = 1.33–13.01 lM range). N-benzylaniline (1) and
4-methoxy-N-(4-methylbenzyl)aniline (4) exhibited weak
(IC50 = 13.01 lM) and similar (IC50 = 1.33 lM) activities
on AChE inhibition, respectively, and no inhibition against
BuChE (IC50 values [100 lM). Merely, 4-ethoxy-N-(4-
methylbenzyl)aniline (6) exhibited potent BuChE inhibitor
activity (IC50 = 4.07 lM) in the benzaniline series. The
other benzaniline compounds in series exhibited no inhibiton
of both AChE and BuChE (IC50 values[100 lM).
The X-ray crystallographic structure of AChE has also
been reported (Harel et al., 1996). The active sites of AChE
comprise these binding sites: anionic substrate binding site,
such as Trp84, Glu199 and Phe330; an esteratic site that
contains the catalytic triad Ser200- His440- Glu327 (Giaco-
bini, 2003); acyl binding site, Phe288 and Phe299, which
binds to the acetyl group of ACh (Castro and Martinez, 2001).
Besides these, AChE also has a peripheral anionic site, such as
Trp279, Tyr70, Tyr121, Asp72, Glu199 and Phe290 (Harel
et al., 1996; Pang et al., 1996; Zhou et al., 2008).
It was shown that certain a,b-unsaturated compounds
exhibit good inhibition potency towards both AChE and
BuChE. These are chalcones (Hasan et al., 2005), which
show affinities in the micromolar range, as well as m- and
p-aminobenzoic acid maleamides and maleimides, which
are potent nanomolar cholinesterase inhibitors (Correa-
Basurto et al., 2005, 2006, 2007; Trujillo-Ferrara et al.,
2003). Some of these derivatives act as irreversible inhib-
itors, probably because of a Michael-type addition of
nucleophilic groups present in the active site gorge of
´
´
AChE (Vitorovic-Todorovic et al., 2010). To date, there
have been no reports regarding the cholinesterase inhibi-
tory activity of any fumaramide derivative. In this regard,
several new fumaramide compounds bearing a,b-unsatu-
rated 1,4-diketone moiety were constructed and their cho-
linesterase inhibiton capacities were evaluated using the
microplate inhibition assay.
Amongst the fumaramide series, N1,N4-bis(4-bromo-
phenyl)-N1,N4-bis(4-methylbenzyl) fumaramide (29) was
found to be the most potent inhibitor on AChE and pre-
sented an IC50 value of 0.14 lM and superior (169-fold)
AChE selectivity. Additionally, N1,N4-dibenzyl-N1,N4-
bis(4-bromophenyl) fumaramide (30), which has a similar
chemical structure to 29, was found to be the most potent
compound for BuChE inhibition and presented an IC50
value of 16.50 lM. Furthermore, N1,N4-dibenzyl-N1,N4-
bis(4-iodophenyl) fumaramide (34) was found to be the
second most effective inhibitor (IC50 = 0.97 lM) and
exhibited 24.07-fold selectivity towards AChE. Com-
pounds 23 (IC50 = 2.23 lM), 27 (IC50 = 2.42 lM), 28
(IC50 = 2.53 lM) and 32 (IC50 = 4.50 lM) showed
moderate inhibition activity toward AChE. Generally,
Results and discussion
Synthesis of targeting compounds
The target fumaramide compounds were synthesized in two
steps using the adequate benzanilines 1–17 as key interme-
diates. In the first step, benzanilines were synthesized using
microwave heating method under different conditions such
as phase-transfer catalysis and the solid supported solvent-
free. In the next step, N-Alkylation of benzaniline interme-
diates 1–17 with fumaryl chloride in dry ethylacetate in the
presence of triethylamine gave targeting fumaramide com-
pounds 18–34 in moderate yields (41–72 %). All new
fumaramides were fully characterized by 1H and 13C NMR,
123

Med Chem Res
R2
Scheme 1 Synthesis of the
compounds 2–17 and 18–34.
Reagents and conditions:
(i) TBAB, K2CO3, toluene,
microwave (90–360 Watt); (ii)
Al2O3 (basic), microwave
(90–360 Watt); (iii)
i
R2
NH2
H
N
+
R1
ClH2C
ii
R1
2-17
Triethylamine, fumaryl
chloride, 0–5 °C
R1
iii
R2
O
N
N
O
R2
18-34
R1
Docking study
fumaramide derivatives (except 23) exhibited slight inhi-
bition activity against BuChE (IC50 values = 16.50–
89.50 lM range). The IC50 values and selectivities of the
compounds investigated are summarized in Table 1.
According to the selectivity results, fumaramide com-
pounds have been found to be more selective against AChE
than BuChE. Any correlation between both cholinesterase
inhibitory activities and the log P values of the compounds
or Hammett (r), Hansch (p) and molar refractivity (MR)
values of the aryl substituents were investigated and no
correlations were noted (p B 0.05). Also, further studies
are needed to determine the structure activity relationships.
However, the activity results clearly show that almost all
of the fumaramides exhibited increased AChE and BuChE
inhibitory potency compared to their precursor benzanilines.
As an exception, compounds 21 (AChE, IC50 = 11.17 lM)
and 23 (BuChE, IC50 [ 100 lM) led the decrease of
cholinesterase inhibition compared with their starting
compounds 4 and 6, respectively. Although there are these
two exceptions, it can be easily said that a,b-unsaturated
1,4-diketone moiety plays a significant role on both cholin-
esterase inhibition.
To understand the binding interactions between compounds
29 and 30, the most potent inhibitors, molecular docking
simulations were performed using Surflex-Dock software.
Docking studies indicated that the complex of AChE (PDB
code: 1ACJ) and 29, compound 29 occupied the catalytic
anionic site (CAS) and peripheric anionic site (PAS). 29
was bound to CAS with a classic p–p stacking interaction
between Phe330 (3.0 A) and Trp84 (3.3 A). In the PAS,
two hydrogen bond interactions were observed between the
carbonyl group of the a,b-unsaturated moiety and the
´
´
˚
˚
´
˚
backbone hydroxyl groups of Tyr121 (2.3 A) and Ser122
´
˚
(1.6 A). Similar interactions were found in compound 30,
which was the most potent BuChE inhibitor in complex
with HuBuChE (PDB code: 1P0I). Because the crystal
structure of BuChE from equine serum had not been
reported and the sequences of BuChE from equine showed
high homology with human BuChE, the crystal structure of
HuBuChE was used in the docking study. A hydrogen bond
was found between compound 30 and hydroxyl group of
´
˚
Tyr332 (2.5 A) and p–p stacking interaction with Trp82
´
˚
(3.0 A). Docking models of the compounds are presented
in Fig. 2.
Kinetic study of AChE and BuChE
The most potent cholinesterase inhibitors, compounds 29
and 30, were chosen for kinetic studies with AChE and
BuChE to study the inhibitory mechanism of the synthesized
fumaramide compounds, respectively. Kinetic analysis of
Lineweaver–Burk plots of AChE and BuChE inhibitory
activity exhibited that lines crossing the x axis in the same
point (unchanged Km) and decreased Vmax with increasing
inhibitor concentrations. The results indicate non-competi-
tive inhibition and the inhibition data of the compounds are
shown in Fig. 1.
Experimental section
Chemistry
The 1H and 13C Nuclear Magnetic Resonance (NMR)
spectra were recorded with tetramethylsilane (TMS) as the
internal standard on a Bruker FT-400(100) MHz spec-
trometer using CDCl3 as the solvent. Coupling constants
were given in Hertz (Hz). TOF-Mass (TOF–MS) spectra
123

Med Chem Res
Table 1 In vitro inhibition IC50 (lM) and selectivity of compounds 1–34 on AChE and BuChE
Compound
R1
R2
IC50 (lM)
Selectivity for AChEc
AChEa
BuChEb
1
H
H
13.01 5.568
[100
[100
–
2
C2H5
C2H5
OCH3
OCH3
OC2H5
OC2H5
CH3
CH3
Cl
CH3
H
[100
–
3
[100
[100
–
4
CH3
H
1.33 0.577
[100
[100
–
5
[100
4.07 1.443
–
6
CH3
H
[100
–
7
[100
[100
–
8
CH3
H
[100
[100
–
9
[100
[100
–
10
CH3
H
[100
[100
–
11
Cl
[100
[100
–
12
Br
CH3
H
[100
[100
–
13
Br
[100
[100
–
14
F
CH3
H
[100
[100
–
15
F
[100
[100
–
16
I
CH3
H
[100
[100
–
17
I
[100
[100
–
18
H
H
11.81 0.850
29.38 1.214
32.16 1.285
11.17 0.870
13.95 0.221
2.23 0.197
35.90 0.865
43.08 0.981
12.54 0.758
2.42 1.027
2.53 0.766
0.14 0.168
8.06 0.661
23.90 2.886
4.50 0.971
17.72 2.434
0.97 1.312
1.02 0.275
52.37 0.289
44.73 4.571
49.27 7.211
40.97 1.780
43.60 9.781
[100
4.43
1.52
1.53
3.67
3.13
–
19
C2H5
C2H5
OCH3
OCH3
OC2H5
OC2H5
CH3
CH3
Cl
CH3
H
20
21
CH3
H
22
23
CH3
H
24
44.03 9.258
54.77 7.948
26.27 1.096
45.60 2.606
57.67 0.354
23.10 2.787
16.50 0.738
56.57 2.325
89.50 7.682
24.63 2.539
23.27 1.168
14.92 0.252
1.23
1.27
2.09
18.87
22.79
169.02
2.05
2.37
19.89
1.39
24.07
14.62
25
CH3
H
26
27
CH3
H
28
Cl
29
Br
CH3
H
30
Br
31
F
CH3
H
32
F
33
I
CH3
H
34
I
Galantamine-HBr
a
50 % inhibitory concentration (mean SD of three experiments) of AChE
50 % inhibitory concentration (mean SD of three experiments) of BuChE
Selectivity for AChE = IC50 (BuChE)/IC50 (AChE)
b
c
were recorded on VG Waters Micromass spectrometer at
Al2O3 (neutral). The chemical reagents and solvents used
in this study were purchased from Merck or Sigma Aldrich.
70 eV. Melting points of the compounds were obtained on
Electrothermal 9100 melting-point apparatus. Benzaniline
compounds were synthesized with CEM 3100 microwave
oven. Reaction progress and product mixtures were rou-
tinely checked by Thin-Layer Chromatography (TLC) on
Merck SilicaGel F254 aluminium plates. Column chroma-
tography was performed with SiO2 (70–230 mesh) and
General procedure for the synthesis of benzaniline
derivatives (2–17)
A mixture of substituted aniline (10 mmol), benzyl chlo-
ride or p-methylbenzyl chloride (5 mmol) was mixed with
123

Med Chem Res
Fig. 1 Lineweaver–Burk plots of the inhibition kinetics of the compounds 29 and 30
Fig. 2 Docking models
(a) 29-AChE complex;
(b) 30- HuBuChE complex.
Hydrogen bonds are represented
as dots. The residues are
rendered in orange sticks (Color
figure online)
Al2O3 (3 g) under the solvent-free conditions. The result-
ing fine powder was taken in a 10 ml glass tube with a
magnetic stirring bar and subjected to microwave irradia-
tion in microwave oven, in pulses (90–360 W) for a total
irradiation time ranging between 2 and 10 min. The reac-
tion was monitored by TLC. After complete conversion,
the mass was cooled to room temperature, extracted with
dichloromethane (2 9 10 ml) and the combined organic
extract was evaporated on rotary evaporator and the crude
product was purified by column chromotography on silica
gel with hexane:ethyl acetate (8:2) mobile phase. The other
synthesis procedure and details were reported in previous
study (Yerdelen, 2012).
(TEA) (1.3 mmol) and dry ethylacetate (5 ml). The mixture
was cooled with an ice bath to 0–5 °C and fumaryl chloride
(0.65 mmol) in 10 ml dry ethylacetate was added dropwise
by syringe over 30 min. The reaction mixture was stirred at
room temperature for 12 h. The reaction mixture was
quenched with 50 ml of water and the aqueous phase was
extracted with two portions of CH2Cl2. The combined
organic layers were dried over MgSO4, filtered and con-
centrated by rotary evaporation. The target compounds
were purified by SiO2 or Al2O3 (neutral) column chroma-
tography and recrystallized from ethanol.
N1,N4-Dibenzyl-N1,N4 diphenylfumaramide (18)
General procedure for the synthesis of fumaramide
This compound was obtained from N-benzylaniline 1
according to general procedure as white solid. Yield 70 %;
mp 189–191 °C. The crude compound was purified by col-
umn chromatography on SiO2 eluting with hexane/ethyl
acetate (6:4) and recrystallized from ethanol. 1H-NMR
(400 MHz, CDCl3) d: 4.91 (s, 4H, 29 CH2–N), 6.90 (s, 2H,
derivatives (18–34)
50 ml two-necked round-bottomed flask, which was
equipped with a magnetic stirring bar, was charged with
starting benzaniline compound (1.3 mmol), triethylamine
123

Med Chem Res
1
fumaryl CH=CH), 6.98 (d, J = 8.1 Hz, 4H, Ar–H), 7.14-
7.16 (dd, J = 2.6 and 1.5 Hz, 4H, Ar–H), 7.21–7.24 (m, 4H,
Ar–H), 7.31–7.36 (m, 8H, Ar–H). 13C-NMR (100 MHz,
CDCl3) 3.76 (CH2N), 127.72, 128.31, 128.49, 128.67,
128.88, 129.98, 132.37 (CH=CH), 137.19, 141.48, 164.61
(C=O). TOF–MS ES(?) m/z (M?H)? 447.2031; calcd. for
C30H26N2O2 = 447.1994.
recrystallized from ethanol. H-NMR (400 MHz, CDCl3)
d: 2.28 (s, 6H, 29 Ar–CH3), 3.80 (s, 6H, 29 OCH3), 4.81
(s, 4H, 29 CH2–N), 6.83–6.87 (m, 10H, Ar–H), 6.89 (s,
2H, fumaryl CH=CH), 7.03 (br s, 6H, Ar–H). 13C-NMR
(100 MHz, CDCl3) d: 25.72 (Ar–CH3), 48.40 (CH2–N),
58.49 (OCH3), 118.77, 121.64, 127.81, 128.53, 130.08,
132.74, 135.21 (CH=CH), 136.43, 155.81, 165.80 (C=O).
TOF–MS ES(?) m/z (M?H)? 535.2553; calcd. for
C34H34N2O4 = 535.2519.
N1,N4-Bis(4-methylbenzyl)-N1,N4-bis(4-
ethylphenyl)fumaramide (19)
N1,N4-Dibenzyl-N1,N4-bis(4-methoxyphenyl)fumar-
amide (22)
This compound was obtained from 4-ethyl-N-(4-methyl-
benzyl)aniline 2 according to general procedure as white
solid. Yield 58 %; mp 174–177 °C. The crude compound
was purified by column chromatography on SiO2 eluting
with hexane/ethyl acetate (6:4) and recrystallized from
ethanol. 1H-NMR (400 MHz, CDCl3) 1.24–1.26 (t,
J = 3.66, 6H, 29 CH2–CH3), 2.28 (s, 6H, 29 CH3-Ph),
2.62–2.65 (q, J = 3.7, 4H, 29 CH2–CH3), 4.83 (s, 4H 29
CH2–N), 6.86–6.90 (m, 4H, Ar–H), 6.91 (s, 2H, fumaryl
CH=CH), 7.04 (br s, 8H, Ar–H), 7.14 (d, J = 8.0 Hz, 4H,
Ar–H). 13C-NMR (100 MHz, CDCl3) 15.61 (CH2–CH3),
22.35 (Ar–CH3), 29.88 (CH2–CH3), 51.68 (CH2–N),
127.91, 128.34, 128.83, 132.11, 133.40, 134.51 (CH=CH),
136.28, 136.41, 143.85, 163.35 (C=O). TOF–MS ES(?) m/
z (M?H)? 531.2972; calcd. for C36H38N2O2 = 531.2933.
This compound was obtained from N-benzyl-4-methoxy-
aniline 5 according to general procedure as white solid.
Yield 70 %; mp 199–200 °C. The crude compound was
purified by column chromatography on SiO2 eluting with
hexane/ethyl acetate (6:4) and recrystallized from ethanol.
1H-NMR (400 MHz, CDCl3) d: 3.80 (s, 6H, 29 OCH3),
4.86 (s, 4H, 29 CH2–N), 6.81–6.83 (d, 4H, J = 7.8 Hz),
6.85 (s, 2H, fumaryl CH=CH), 6.86–6.88 (m, 6H, Ar–H),
7.13–7.15 (dd, 4H, J = 2.9 Hz and 1.5 Hz, Ar–H), 7.22 (d,
4H, J = 6.6 Hz, Ar–H). 13C-NMR (100 MHz, CDCl3) d:
46.21 (CH2–N), 55.84 (OCH3), 112.39, 120.19, 125.71,
126.80, 128.56, 132.41, 135.38 (CH=CH), 138.29, 159.11,
167.45 (C=O). TOF–MS ES(?) m/z (M?H)? 507.2245;
calcd. for C32H30N2O4 = 507.2206.
N1,N4-Dibenzyl-N1,N4-bis(4-ethylphenyl)fumaramide (20)
N1,N4-Bis(4-ethoxyphenyl)-N1,N4-bis(4-
methylbenzyl)fumaramide (23)
This compound was obtained from N-benzyl-4-ethylani-
line 3 according to general procedure as white solid. Yield
64 %; mp 151–155 °C. The crude compound was purified
by column chromatography on SiO2 eluting with hexane/
ethyl acetate (6:4) and recrystallized from ethanol. 1H-
NMR (400 MHz, CDCl3) d: 1.23-1.26 (t, 6H, 29 CH3,
J = 7.5 Hz), 2.62–2.67 (q, 4H, 29 CH2, J = 7.5 Hz), 4.88
(s, 4H, 29 CH2–N), 6.87–6.89 (d, 4H, J = 8.4 Hz, Ar–H),
6.92 (s, 2H, fumaryl CH=CH), 7.14–7.17 (m, 8H, Ar–H),
7.21-7.23 (m, 6H, Ar–H). 13C-NMR (100 MHz, CDCl3) d:
14.96 (CH2–CH3), 28.22 (CH2–CH3), 49.82 (CH2–N),
126.84, 126.97, 127.34, 127.93, 128.07, 132.24, 134.47
(CH=CH), 136.25, 142.81, 162.48 (C=O). TOF–MS ES(?)
m/z (M?H)? 503.2601; calcd. for C34H34N2O2 =
503.2620.
This compound was obtained from 4-ethoxy-N-(4-methyl-
benzyl)aniline 6 according to general procedure as white
solid. Yield 71 %; mp 182–184 °C. The crude compound
was purified by column chromatography on SiO2 eluting
with hexane/ethyl acetate (6:4) and recrystallized from
1
ethanol. H-NMR (400 MHz, CDCl3) d: 1.40–1.43 (t, 6H,
29 OCH2–CH3, J = 6.9 Hz), 2.28 (s, 6H, 29 CH3),
3.98–4.03 (q, 4H, 29 OCH2CH3, J = 6.9 Hz), 4.80 (s, 4H,
29 CH2–N), 6.78–6.87 (m, 12 H, Ar–H), 7.02 (s, 2H,
fumaryl CH=CH), 7.05 (d, 4H, J = 6.8 Hz, Ar–H). 13C-
NMR (100 MHz, CDCl3) d: 13.25 (OCH2CH3), 25.63 (Ar–
CH3), 41.38 (CH2–N), 63.25 (OCH2CH3), 108.34, 116.22,
123.49, 125.71, 130.52, 133.25, 135.41 (CH=CH), 137.63,
154.39, 165.21 (C=O). TOF–MS ES(?) m/z (M?H)?
563.2870; calcd. for C36H38N2O4 = 563.2832.
N1,N4-Bis(4-methoxyphenyl)-N1,N4-bis(4-
methylbenzyl)fumaramide (21)
N1,N4-Dibenzyl-N1,N4-bis(4-ethoxyphenyl)fumar-
amide (24)
This compound was obtained from 4-methoxy-N-(4-meth-
ylbenzyl)aniline 4 according to general procedure as bright
yellow solid. Yield 68 %; mp 190–193 °C. The crude
compound was purified by column chromatography on
SiO2 eluting with hexane/ethyl acetate (6:4) and
This compound was obtained from N-benzyl-4-ethoxyani-
line 7 according to general procedure as white solid. Yield
72 %; mp 175–178 °C. The crude compound was purified
123

Med Chem Res
by column chromatography on SiO2 eluting with hexane/
ethyl acetate (6:4) and recrystallized from ethanol. 1H-
NMR (400 MHz, CDCl3) d: 1.40–1.43 (t, 6H, 29 OCH2–
CH3, J = 6.9 Hz), 3.98–4.03 (q, 4H, 29 OCH2–CH3,
J = 6.9 Hz), 4.85 (s, 4H, 29 CH2–N), 6.88 (s, 2H, fumaryl
CH=CH), 6.79–6.85 (m, 8H, Ar–H), 7.13–7.15 (dd, 4H,
J = 2.6 Hz and 1.8 Hz, Ar–H), 7.21–7.24 (m, 6H, Ar–H).
13C-NMR (100 MHz, CDCl3) d: 14.10 (OCH2CH3), 42.49
(CH2–N), 64.01 (OCH2CH3), 110.84, 114.33, 122.21,
125.08, 131.47, 132.14, 135.95 (CH=CH), 137.88, 157.60,
163.49 (C=O). TOF–MS ES(?) m/z (M?H)? 535.2562;
calcd. for C34H34N2O4 = 535.2519.
solid. Yield 52 %; mp 226–229 °C. The crude compound
was purified by column chromatography on SiO2 eluting
with hexane/ethyl acetate (6:4) and recrystallized from
ethanol. 1H-NMR (400 MHz, CDCl3) d: 2.29 (s, 6H,
29CH3), 4.83 (s, 4H, 29 CH2–N), 6.83 (s, 2H, fumaryl
CH=CH), 6.88 (d, 4H, J = 8.4 Hz, Ar–H), 7.00 (d, 4H,
J = 8.0 Hz, Ar–H), 7.04 (d, 4H, J = 8.0 Hz, Ar–H), 7.30
(d, 4H, J = 8.4 Hz, Ar–H). 13C-NMR (100 MHz, CDCl3)
d: 26.83 (Ar–CH3), 47.31 (CH2–N), 122.09, 125.36,
128.98, 130.47, 132.11, 135.28 (CH=CH), 136.19, 137.55,
138.01, 165.14 (C=O). TOF–MS ES(?) m/z (M?H)?
543.1515; calcd. for C32H28Cl2N2O2 = 543.1528.
N1,N4-Bis(4-methylbenzyl)-N1,N4-bis(4-
methylphenyl)fumaramide (25)
N1,N4-Dibenzyl-N1,N4-bis(4-chlorophenyl)fumar-
amide (28)
This compound was obtained from 4-methyl-N-(4-methyl-
benzyl)aniline 8 according to general procedure as white
solid. Yield 66 %; mp 205–206 °C. The crude compound
was purified by column chromatography on SiO2 eluting
with hexane/ethyl acetate (6:4) and recrystallized from eth-
anol. dH (400 MHz, CDCl3) d: 2.28 (s, 6H, 29 Benzyl–CH3),
2.34 (s, 6H, 29 Ar–CH3), 4.83 (s, 4H, 29 CH2–N), 6.83 (d,
4H, J = 8.4 Hz, Ar–H), 6.85 (d, 4H, J = 8.4 Hz, Ar–H),
6.86 (s, 2H, fumaryl CH=CH), 7.03 (br s, 4H, Ar–H), 7.11 (d,
4H, J = 8.0 Hz, Ar–H). 13C-NMR (100 MHz, CDCl3) d:
19.64 (Benzyl–CH3), 21.63 (Ar–CH3), 45.21 (CH2–N),
127.65, 128.08, 130.05, 132.28, 134.95, 135.46 (CH=CH),
136.25, 137.12, 140.25, 163.91 (C=O). TOF–MS ES(?) m/z
(M?H)? 503.2634; calcd. for C34H34N2O2 = 503.2620.
This compound was obtained from N-benzyl-4-chloroani-
line 11 according to general procedure as white solid. Yield
69 %; mp 229–232 °C. The crude compound was purified
by column chromatography on SiO2 eluting with hexane/
ethyl acetate (6:4) and recrystallized from ethanol. dH
(400 MHz, CDCl3) d: 4.93 (s, 4H, 29 CH2–N), 6.90 (s, 2H,
fumaryl CH=CH), 6.94 (d, 4H, J = 8.5 Hz, Ar–H),
7.16–7.18 (dd, 4H, J = 3.7 Hz and 2.0 Hz, Ar–H), 7.27-
7.28 (m, 6H, Ar–H), 7.34-7.36 (d, 4H, J = 8.6 Hz, Ar–H).
13C-NMR (100 MHz, CDCl3) d: 46.99 (CH2–N), 123.18,
125.78, 126.93, 127.51, 129.25, 134.35, 135.47 (CH=CH),
137.89, 138.46, 164.98 (C=O). TOF–MS ES(?) m/z
(M?H)? 515.1218; calcd. for C32H24Cl2N2O2 = 515.1215.
N1,N4-Bis(4-bromophenyl)-N1,N4-bis(4-
methylbenzyl)fumaramide (29)
N1,N4-Dibenzyl-N1,N4-bis(4-methylphenyl)fumar-
amide (26)
This compound was obtained from 4-bromo-N-(4-methyl-
benzyl)aniline 12 according to general procedure as white
solid. Yield 56 %; mp 225–227 °C. The crude compound
was purified by column chromatography on SiO2 eluting
with hexane/ethyl acetate (6:4) and recrystallized from
This compound was obtained from N-benzyl-4-methylaniline
9 according to general procedure as white solid. Yield 60 %;
mp 198–200 °C. The crude compound was purified by col-
umn chromatography on SiO2 eluting with hexane/ethyl
acetate (6:4) and recrystallized from ethanol. 1H-NMR
(400 MHz, CDCl3) d: 2.34 (s, 6H, 29 CH3), 4.88 (s, 4H, 29
CH2–N), 6.83 (s, 2H, fumaryl CH=CH), 6.88 (d, 4H,
J = 8.4 Hz, Ar–H), 7.00 (d, 4H, J = 8.0 Hz, Ar–H), 7.04 (d,
4H, J = 8.0 Hz, Ar–H), 7.30(d, 4H, J = 8.4 Hz, Ar–H). 13C-
NMR (100 MHz, CDCl3) d: 22.04 (Ar–CH3), 45.34(CH2–N),
125.92, 126.51, 128.54, 129.48, 132.28, 134.98 (CH=CH),
135.47, 136.19, 140.11, 159.04 (C=O). TOF–MS ES(?) m/z
(M?H)? 475.2341; calcd. for C32H30N2O2 = 475.2307.
1
ethanol. H-NMR (400 MHz, CDCl3) d: 2.33 (s, 6H, 29
CH3), 4.88 (s, 4H, 29 CH2–N), 6.86 (s, 2H, fumaryl
CH=CH), 6.88 (d, 4H, J = 7.5 Hz, Ar–H), 7.05 (d, 4H,
J = 8.1 Hz, Ar–H), 7.08 (d, 4H, J = 8.1 Hz, Ar–H), 7.50
(d, 4H, J = 8.6 Hz, Ar–H). 13C-NMR (100 MHz, CDCl3)
d: 25.67 (Ar–CH3), 44.34 (CH2–N), 121.39, 125.64,
127.15, 131.01, 132.89, 134.52 (CH=CH), 135.91, 136.73,
138.44, 160.01 (C=O). TOF–MS ES(?) m/z (M?H)?
631.0515; calcd for C32H28Br2N2O2 = 631.0518.
N1,N4-Bis(4-chlorophenyl)-N1,N4-bis(4-
methylbenzyl)fumaramide (27)
N1,N4-Dibenzyl-N1,N4-bis(4-bromophenyl)fumar-
amide (30)
This compound was obtained from 4-chloro-N-(4-methyl-
benzyl)aniline 10 according to general procedure as white
This compound was obtained from N-benzyl-4-bromoani-
line 13 according to general procedure as white solid. Yield
123

Med Chem Res
1
52 %; mp 218–220 °C. The crude compound was purified by
column chromatography on Al2O3 (neutral) eluting with
hexane/ethyl acetate (1:1) and recrystallized from ethanol.
1H-NMR (400 MHz, CDCl3) d: 4.88 (s, 4H, 29 CH2–N),
6.83 (d, 4H, J = 8.4 Hz, Ar–H), 6.85 (s, 2H, fumaryl
CH=CH), 7.12–7.14 (dd, 4H, J = 3.3 Hz and 4.0 Hz, Ar–
H), 7.23–7.24 (m, 6H, Ar–H), 7.46 (d, 4H, J = 8.0 Hz, Ar–
H). 13C-NMR (100 MHz, CDCl3) d: 45.21 (CH2–N), 122.01,
126.14, 126.85, 128.33, 131.45, 133.21 (CH=CH), 137.08,
139.43, 165.38 (C=O). TOF–MS ES(?) m/z (M?H)?
603.0237; calcd. for C30H24Br2N2O2 = 603.0205.
from ethanol. H-NMR (400 MHz, CDCl3) d: 2.29 (s, 6H,
29 CH3), 4.83 (s, 4H, 29 CH2-N), 6.69 (d, 4H,
J = 8.4 Hz, Ar–H), 6.84 (s, 2H, fumaryl CH=CH), 7.00 (d,
4H, J = 8.4 Hz, Ar–H), 7.04 (d, 4H, J = 8.1 Hz, Ar–H),
7.65 (d, 4H, J = 8.4 Hz, Ar–H). 13C-NMR (100 MHz,
CDCl3) d: 27.34 (Ar–CH3), 45.69 (CH2–N), 105.27,
125.23, 127.65, 128.03, 132.14, 136.28 (CH=CH), 136.43,
137.21, 139.64, 162.39 (C=O). TOF–MS ES(?) m/z
(M?H)? 727.0255; calcd. for C32H28I2N2O2 = 727.0240.
N1,N4-Dibenzyl-N1,N4-bis(4-iodophenyl)fumaramide (34)
N1,N4-Bis(4-fluorophenyl)-N1,N4-bis(4-
methylbenzyl)fumaramide (31)
This compound was obtained from N-benzyl-4-iodoaniline
17 according to general procedure as white solid. Yield
41 %; mp 259–261 °C. The crude compound was purified
by column chromatography on Al2O3 (neutral) eluting with
hexane/ethyl acetate (1:1) and recrystallized from ethanol.
1H-NMR (400 MHz, CDCl3) d: 4.88 (s, 4H, 29 CH2–N),
6.70 (d, 4H, J = 8.1 Hz, Ar–H), 6.87 (s, 2H, fumaryl
CH=CH), 7.12–7.14 (m, 4H, Ar–H), 7.23–7.24 (m, 6H, Ar–
H), 7.66 (d, 4H, J = 8.4 Hz, Ar–H). 13C-NMR (100 MHz,
CDCl3) d: 44.86 (CH2–N), 101.47, 123.55, 125.12, 125.98,
128.51, 133.28 (CH=CH), 136.24, 136.98, 138.23, 161.95
(C=O). TOF–MS ES(?) m/z (M?H)? 698.9948; calcd. for
C30H24I2N2O2 = 698.9927.
This compound was obtained from 4-fluoro-N-(4-methyl-
benzyl)aniline 14 according to general procedure as white
solid. Yield 70 %; mp 189–201 °C. The crude compound was
purified by column chromatography on Al2O3 (neutral) elut-
ing with hexane/ethyl acetate (1:1) and recrystallized from
1
ethanol. H-NMR (400 MHz, CDCl3) d: 2.29 (s, 6H, 29
CH3), 4.82(s, 4H, 29 CH2–N), 6.89(s, 2H, fumarylCH=CH),
6.89–6.93 (dd, 4H, J = 4.8 Hz and 4.7 Hz, Ar–H), 7.00 (d,
4H, J = 6.9 Hz, Ar–H), 7.03–7.05 (m, 8H, Ar–H). 13C-NMR
(100 MHz, CDCl3) d: 21.06 (Ar–CH3), 48.39 (CH2–N),
113.27, 122.18, 127.81, 133.25, 133.76, 134.58 (CH=CH),
136.13, 159.18, 164.26 (C=O). TOF–MS ES(?) m/z (M?H)?
511.2142; calcd. for C32H28F2N2O2 = 511.2119.
Pharmacological activity
TLC-B assay for acetylcholinesterase inhibition
N1,N4-Dibenzyl-N1,N4-bis(4-fluorophenyl)fumaramide (32)
Benzaniline and fumaramide compounds (10 ll, 10 mg/
ml) and galanthamine-HBr (5 ll, 0.2 mM) were each
applied to Silica gel 60 F254 on a aluminium plate
(10 cm 9 20 cm) (Merck, Germany) and developed with
chloroform: methanol (80:20) in a pre-saturated chro-
matographic chamber. After drying, the TLC plate was
sprayed with 5 mM ATCI and 5 mM DTNB dissolved in
buffer until the TLC plate was saturated. The plates were
allowed to dry slightly and then sprayed with 3 U/ml
AChE dissolved in a buffer. After 2–5 min, a yellow
background appeared with white spots indicating the
presence of AChE inhibiting compounds. The white spots
were observed and recorded within 5 min.
This compound was obtained from N-benzyl-4-fluoroani-
line 15 according to general procedure as white solid. Yield
70 %; mp 201–203 °C. The crude compound was purified
by column chromatography on Al2O3 (neutral) eluting with
hexane/ethyl acetate (1:1) and recrystallized from ethanol.
1H-NMR (400 MHz, CDCl3) d: 4.87 (s, 4H, 29 CH2–N),
6.84 (s, 2H, fumaryl CH=CH), 6.90–6.94 (dd, 4H,
J = 4.7 Hz and 5.8 Hz, Ar–H), 7.03 (d, 4H, J = 8.4 Hz,
Ar–H), 7.13 (d, 4H, J = 6.9 Hz, Ar–H), 7.22–7.26 (m, 6H,
Ar–H). 13C-NMR (100 MHz, CDCl3) d: 47.23 (CH2–N),
114.33, 123.21, 125.92, 126.78, 128.19, 132.54 (CH=CH),
135.98, 134.75, 162.71, 168.18 (C=O). TOF–MS ES(?) m/z
(M?H)? 483.1853; calcd. for C30H24F2N2O2 = 483.1806.
Inhibition studies on AChE and BuChE
N1,N4-Bis(4-iodophenyl)-N1,N4-bis(4-
methylbenzyl)fumaramide (33)
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric
eel), butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from
equine serum), 5,50-dithiobis-(2-nitrobenzoic acid) DTNB,
acetylthiocholine iodide (ATCI) and butyrylthiocholine
iodide (BTCI) were purchased from Sigma Aldrich. Inhib-
itory activities of AChE and BuChE of the test compounds
were evaluated by colorimetric Ellman’s method with some
This compound was obtained from 4-iodo-N-(4-methyl-
benzyl)aniline 16 according to general procedure as white
solid. Yield 45 %; mp 247–250 °C. The crude compound
was purified by column chromatography on Al2O3 (neutral)
eluting with hexane/ethyl acetate (1:1) and recrystallized
123

Med Chem Res
modifications using commercially available galantamine-
HBr as the reference compound. The test compounds were
dissolved in ethanol/dimethylsulphoxide (1:1) and then
diluted in 50 mM KH2PO4/K2HPO4 phosphate buffer (pH
8.0) to provide a final concentration range. In a 96-well
plate, the assay medium in each well consisted of 50 ll of a
phosphate buffer, 125 ll of 3 mM DTNB (Ellman’s
reagent), 25 ll of 0.2 U/ml enzyme (AChE or BuChE) and
15 mM substrate (ATCI or BTCI). The assay mixture
containing enzyme, buffer, DTNB and 25 ll of inhibitor
compound was preincubated for 15 min at 37 °C, before the
substrate was added to begin the reaction. Galantamine-HBr
and all compounds were prepared at different concentra-
tions such as 0.195, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50
and 100 lg/ml. The absorbance of the reaction mixture was
then measured three times at 412 nm every 45 s using a
microplate reader (Bio-Tek ELx800, USA). Results are
presented as means standard errors of the experiment.
The IC50 values of the compounds showing percentage
inhibition[50 %; the measurements and calculations were
evaluated by non-linear regression analysis using GraphPad
Prism software programme.
through protomol generation by ligand. The parameters
used were threshold 0.5 and bloat 0.
Conclusion
In summary, seventeen new fumaramide derivatives (18–34)
were synthesized and tested towards AChE and BuChE.
Assuming biological data, all fumaramides have shown
moderate to high antiacetylcholinesterase activity in which
compounds 29 and 34 were the most potent inhibitors acting
in low micromolar concentrations. All synthetic fumaramide
compounds (except 23) had a very good selectivity for
AChE. Conversely, most of the fumaramide compounds
exhibited more powerful and selective inhibitory activity
than the precursor benzanilines against both the cholines-
terase enzymes.
Docking studies were performed with the Surflex-Dock
programme in order to clarify the cholinesterase inhibitory
activities of the compounds (29, 30) and to provide the
ideal interaction mode of the compounds in the binding
sites of the related enzymes. Accordingly, molecular
modelling simulations of AChE and BuChE inhibitor
complexes showed that the nature of a,b-unsaturated 1,4-
diketone moiety in fumaramide derivatives obviously
contributed to the inhibitory activities through interacting
with enzymes. In addition, kinetic studies indicated that
these inhibitory compounds exhibited non-competitive
inhibition against both types of cholinesterase enzymes. To
the best of our knowledge, cholinesterase inhibitors based
on fumaramide scaffold have not been previously reported,
and further investigations of these compounds are in
progress.
Kinetic characterization of AChE and BuChE inhibition
The mechanism of AChE and BuChE inhibition was
investigated in kinetic studies using Ellman’s test. Com-
pounds 29 and 30 were selected for kinetic measurements
because they were found to have the highest inhibitory
activity against AChE and BuChE, respectively. The test
was carried out without the inhibitor and in 0.1, 0.15 and
0.2 lM concentrations of the inhibitor for AChE and 10, 15
and 20 lM concentrations for BuChE. Substrate concen-
trations were varied from 0.1 to 1.5 mM. The obtained data
were used to create substrate-velocity curves, which were
transformed in GraphPad Prism programme to Linewe-
aver–Burk plots.
Acknowledgments This research work was supported by Ataturk
University Research Fund (Project No: 2011/294), Turkey.
Molecular modelling
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