Synthesis and evaluation of 7-substituted coumarin derivatives as multimodal monoamine oxidase-B and cholinesterase inhibitors for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2016.09.041

A series of 7-substituted coumarin derivatives were designed and synthesised to display ChE and MAO-B inhibitory activity. The compounds consisted out of a coumarin structure (MAO-B inhibitor) and benzyl-, piperidine-, N-benzylpiperidine- or p-bromo-N-benzylpiperizine moiety, resembling the N-benzylpiperidine function of donepezil (ChE inhibitor), connected via an alkyl ether linkage at the 7 position. The biological assay results indicated that all the compounds (1–25) displayed selective inhibition to hMAO-B over hMAO-A, with the benzyloxy series (1–8, 10–13) showing nano-molar hMAO-B inhibition (IC₅₀: 0.5–73?nM). Limited ChE inhibitory activity was however observed for the benzyloxy series with the exception of 2 and especially 3 showing selective BuChE inhibition. From this series 3 showed the best multifunctional activity (eqBuChE IC₅₀?=?0.96?μM, hMAO-A IC₅₀?=?2.13?μM, hMAO-B IC₅₀?=?0.0021?μM). Within the N-benzylpiperidine (16–19) and p-bromo-N-benzylpiperizine (21–24) series the compounds in general showed moderate ChE and MAO-B inhibitory activity. Of these compounds 19 was the most potent multifunctional agent showing good eeAChE and eqBuChE inhibition (IC₅₀?=?9.10?μM and 5.90?μM, respectively), and relatively potent and selective hMAO-B inhibition (IC₅₀?=?0.30?μM, SI?= >33). Molecular modeling revealed that 19 was able to bind simultaneously to the CAS, mid-gorge and PAS sites of AChE and BuChE suggesting that it will be able to inhibit AChE induced Aβ aggregation. From this study, compounds that 3 and 19 can be considered as promising multifunctional lead compounds.

Full text of DOI:10.1016/j.ejmech.2016.09.041

Accepted Manuscript
Synthesis and evaluation of 7-substituted coumarin derivatives as multimodal
monoamine oxidase-B and cholinesterase inhibitors for the treatment of Alzheimer's
disease
Jacques Joubert, Germaine B. Foka, Benjamin P. Repsold, Douglas W. Oliver, Erika
Kapp, Sarel F. Malan
PII:
S0223-5234(16)30772-3
10.1016/j.ejmech.2016.09.041
EJMECH 8907
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 July 2016
Revised Date: 12 September 2016
Accepted Date: 13 September 2016
Please cite this article as: J. Joubert, G.B. Foka, B.P. Repsold, D.W. Oliver, E. Kapp, S.F. Malan,
Synthesis and evaluation of 7-substituted coumarin derivatives as multimodal monoamine oxidase-B
and cholinesterase inhibitors for the treatment of Alzheimer's disease, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.041.
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ACCEPTED MANUSCRIPT
Synthesis and evaluation of 7-substituted coumarin derivatives as multimodal
monoamine oxidase-B and cholinesterase inhibitors for the treatment of
Alzheimer’s disease
Jacques Jouberta,*, Germaine B. Fokaa, Benjamin P. Repsoldb, Douglas W. Oliverb, Erika Kappa,
Sarel F. Malana
aPharmaceutical Chemistry , School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville, South
Africa. bSchool of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, South Africa
*Corresponding author at present address: School of Pharmacy, University of the Western Cape, Private Bag X17,
Bellville 7535, South Africa. Tel: +27 21959 2195; e-mail: jjoubert@uwc.ac.za
Abstract:
A series of 7-substituted coumarin derivatives were designed and synthesised to display ChE and MAO-B inhibitory
activity. The compounds consisted out of a coumarin structure (MAO-B inhibitor) and benzyl-, piperidine-, N-
benzylpiperidine- or p-bromo-N-benzylpiperizine moieties, resembling the N-benzylpiperidine function of donepezil
(ChE inhibitor), connected via an alkyl ether linkage at the 7 position. The biological assay results indicated that all the
compounds (1-25) displayed selective inhibition to hMAO-B over hMAO-A, with the benzyloxy series (1-8, 10-13)
showing nano-molar hMAO-B inhibition (IC50: 0.5 – 73 nM). Limited ChE inhibitory activity was however observed for
the benzyloxy series with the exception of 2 and especially 3 showing selective BuChE inhibition. From this series 3
showed the best multifunctional activity (eqBuChE IC50 = 0.96 ꢀM, hMAO-A IC50 = 2.13 ꢀM, hMAO-B IC50 = 0.0021
ꢀM). Within the N-benzylpiperidine (16-19) and p-bromo-N-benzylpiperizine (21-24) series the compounds in general
showed moderate ChE and MAO-B inhibitory activity. Of these compounds 19 was the most potent multifunctional
agent showing good eeAChE and eqBuChE inhibition (IC50 = 9.10 ꢀM and 5.90 ꢀM, respectively), and relatively potent
and selective hMAO-B inhibition (IC50 = 0.30 ꢀM, SI = >33). Molecular modeling revealed that 19 was able to bind
simultaneously to the CAS, mid-gorge and PAS sites of AChE and BuChE suggesting that it will be able to inhibit
AChE induced Aß aggregation. From this study, compounds that 3 and 19 can be considered as promising
multifunctional lead compounds.
Keywords: Alzheimer’s disease, Coumarin, Donepezil, MAO-B, Cholinesterase, Molecular Modeling
1. Introduction
Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterised by progressive
memory loss and decline in language skills, and other cognitive impairments [1]. Although the
etiology of AD is not fully understood several factors, such as cholinergic dysfunction [2], τ-protein
aggregation [3], amyloid-β (Aβ) deposits [4] and oxidative stress [5,6] are considered to play
important roles in the pathophysiology of AD. The selective loss of cholinergic neurons in AD
results in a deficit of acetylcholine (ACh) in specific regions of the brain that mediate learning and
memory functions [7]. Consequently, AD patients have been treated with acetylcholinesterase
(AChE) inhibitors [8-10] but unfortunately with limited therapeutic success, mainly because of the
multifactorial nature of AD. Recent studies have also shown that compounds that are able to inhibit
butyrylcholinesterase (BuChE) may be of value in the treatment of AD [11]. In AD the ratio of
BuChE/AChE gradually increases as the disease progresses, partially as a consequence of the
1

progressive loss of the cholinergic synapses where AChE enzymes are located [12]. A compound
ACCEPTED MANUSCRIPT
able to inhibit both AChE and BuChE may thus be of more therapeutic value in AD. Alterations in
other neurotransmitter systems, especially dopaminergic and serotoninergic [13,14] are also thought
to be responsible for the behavioral disturbances observed in AD [15]. This evidence has led to the
suggestion that inhibitors of monoamine oxidase might also be of value in the treatment of AD
[16,17].
Monoamine oxidase (MAO) is the enzyme that is responsible for the oxidative deamination of
various biogenic and xenobiotic amines [18,19]. MAO exists as two isoforms, MAO-A and MAO-
B, with MAO B being abundant in the brain [20]. The oxidative deamination catalysed by MAO-B
leads to the production of neurotoxic producs, such as hydrogen peroxide and aldehydes, which
promote the formation of reactive oxygen species that cause oxidative stress and increased neuronal
damage [21,22]. Studies have shown that the MAO-B activity in the brain of AD patients increases
with time [23,24], thus increasing the rate of neuronal damage in the already diseased brain. MAO-
A is more widely distributed peripherally than centrally, and as such, many side effects could occur
if inhibited [25]. Therefore selective MAO-B inhibitors are better suited for the treatment of AD.
Selegiline is a selective MAO-B inhibitor that has shown evidence of neuronal cell protection due to
oxidative stress in AD patients [26], thereby confirming the important role of MAO-B in the cascade
of events leading to neuronal cells death in AD.
The above observations has prompted the search for Multi-Target-Directed-Ligands (MTDLs),
based on the “one molecule, multiple target” paradigm [27-29]. Thus, in this context, MTDLs able
to simultaneously inhibit both cholinesterases and monoamine oxidases have been designed and
investigated [30-39]. Among these MTDLs, Ladostigil has been approved for phase IIb clinical
trials [31], which prompted us to search for new MTDLs with ChE and MAO-B inhibitory activity.
In this work we report the design, synthesis, biochemical evaluation and molecular modeling of
coumarin structures conjugated with a benzyl-, piperidine-, N-benzylpiperidine- or p-bromo-N-
benzylpiperizine moiety via a flexible alkyl ether linkage at the 7 coumarin position as MTDLs for
the potential treatment of AD (Figure 1). Compounds were designed to incorporate coumarin
structures known to show MAO-B inhibition [40] and selected structural elements of donepezil a
selective AChE inhibitor currently used in the pharmacological treatment of AD [8]. Studies suggest
that simultaneous inhibition of MAO-B and ChE does not only improve the level of ACh and reduce
oxidative stress in the brain but also decreases Aß deposition which may make such a compound
more effective against AD [34]. We expected that the coumarin moiety would be able to occupy the
substrate cavity of MAO-B thereby conferring MAO-B inhibitory activity to the designed
compounds. Substitutions were made on the 7-position of the coumarin moiety as previous studies
showed that substitutions at this position lead to increased MAO-B activity and selectivity [40-43].

In addition to the above, the series of compounds were also designed to allow for exploration of the
ACCEPTED MANUSCRIPT
influence of various distinct functional group modifications on the 3- and/or 4-position of the
coumarin structure. In order to maximize ChE activity, the coumarin and the N-benzylpiperidine
derivatives/portions were connected by a flexible alkyl chain. This linkage is expected to allow the
N-benzylpiperidine derivative to interact with the catalytic anionic site (CAS), in a similar manner
as donepezil, and the coumarin moiety to reach and interact with the peripheral anionic site (PAS) of
AChE. Targeting both the CAS and PAS should result in significant AChE inhibitory activity while
possibly inhibiting the Aβ pro-agreggating action of AChE via the PAS interaction [44-47]. All
designed compounds (1-25) were synthesized and evaluated for their ability to inhibit ChEs and
MAOs. The data of the structurally related compounds 26-29, recently reported by Sai-Sai et al.,
2016 [39], were included in this report in order to compare certain structural elements with
compounds 1-25 and draw up a comprehensive list of structure activity relationships.
R2
O
R3
&
N
O
HO
O
O
O
Coumarin
Donepezil
R2
O
R1 =
16-20
R1 =
R3
O
N
R1O
2
1-4
R = H/CH /CF
3
3
3
Br
R = H/Cl/CN
N
N
X
21-25
5-13, X = Br/F/Cl
NH
N
N
39
26-29
14-15
Figure 1: Design strategy for the 7-substituted coumarins incorporating different portions and distinct
variations of the N-benzylpiperidine moiety of donepezil.
2. Results and discussion
2.1. Chemistry
Compound 1–15 were synthesised (Figure 2) by conjugating the commercially available 7-
hydroxylcoumarin derivatives (1a–5a) to a benzylbromide- (1–4), halogen substituted
benzylbromide- (5–13) or 1-(2-chloroethyl)piperidine (14 and 15) moiety through an SN2
nucleophilic substitution reaction using ethanol (1-13) or acetonitrile (14 and 15) as solvent in the

presence of potassium carbonate. The synthesis of compounds 16-25 (Figure 3) commenced from
ACCEPTED MANUSCRIPT
coumarins 1–5a which where conjugated with an excess of a 1,2-dibromoethane linker to afford the
key intermediates 1b–5b. These intermediates were then treated with N-benzylpiperidine or 1-(4-
bromobenzyl)piperazine in the presence of potassium carbonate in acetonitrile to give the target
compounds 16-25.
R1
O
O
O
R1
Br
R3
O
O
OH
R2
K2CO3
1 - 13
or
N
+
R3
EtOH or CH3CN,
2 - 5 hr, 60-80 oC
O
O
O
Cl
R2
N
1a - 5a
R3
R1
R1
R2
14 - 15
1a R2 = H, R3 = H
2a R2 = CH3, R3 = H
3a R2 = CH3, R3 = Cl
4a R2 = CH3, R3 = CN
5a R2 = H, R3 = H
1 R1 = H, R2 = H, R3 = H
2 R1 = H, R2 = CH3, R3 = H
3 R1 = H, R2 = CH3, R3 = Cl
4 R1 = H, R2 = CH3, R3 = CN
5 R1 = 3-Br, R2 = H, R3 = H
6 R1 = 3-Br, R2 = CH3, R3 = H
7 R1 = 3-Br, R2 = CH3, R3 = Cl
8
9
R1 = 3-Br, R2 = CH3, R3 = CN
R1 = 3-Br, R2 = CF3, R3 = H
10 R1 = 3-F,
R2 = CH3, R3 = CN
11 R1 = 3-Cl, R2 = CH3, R3 = CN
12 R1 = 4-Br, R2 = CH3, R3 = CN
13 R1 = 3,4-Br, R2 = CH3, R3 = CN
14 R1 = H,
15 R1 = H,
R2 = H,
R3 = H
R2 = CH3, R3 = H
Figure 2: Synthetic pathway for the synthesis of compounds 1 – 15.
R1
O
O
OH
O
O
O
K2CO3
Br
K2CO3
HN
1,2-dibromoethane
+
R4
CH3CN, 8 hr, 60oC
Acetone, 4 hr, 60oC
R3
R3
R2
R2
1a - 5a
1b - 5b
16 R1 = H, R2 = H, R3 = H, R4 = CH
17 R1 = H, R2 = CH3, R3 = H, R4 = CH
18 R1 = H, R2 = CH3, R3 = Cl, R4 = CH
19 R1 = H, R2 = CH3, R3 = CN, R4 = CH
20 R1 = H, R2 = CF3, R3 = H, R4 = CH
21 R1 = Br, R2 = H, R3 = H, R4 = N
22 R1 = Br, R2 = CH3, R3 = H, R4 = N
23 R1 = Br, R2 = CH3, R3 = Cl, R4 = N
24 R1 = Br, R2 = CH3, R3 = CN, R4 = N
25 R1 = Br, R2 = CF3, R3 = H, R4 = N
O
O
O
R1
N
R4
R3
R2
16 - 25
Figure 3: Synthetic pathway for the synthesis of compounds 16 - 25.
2.2. Monoamine oxidase inhibition studies
The target compounds (1-25) were investigated for their inhibitory activity against hMAO by
measuring the extent by which the test inhibitor reduces the MAO-catalysed oxidation of
kynuramine, a mixed MAO-A/B substrate [48,49]. The inhibition potencies of the test inhibitors (1–
25, 26–29 [39]) and reference compounds, selegiline and clorgyline, were expressed as IC50 values
and are presented in Table 1. The results indicate that all the test compounds have better inhibitory
activity for MAO-B over MAO-A with SI values ranging from >2 – 1310. This corresponds well to
previous studies where it was found that coumarin structures substituted at the 7-position with
various distinct functional moieties show selective MAO-B inhibition [40-43].

Compounds 1-8 showed highly potent activity (IC50: 0.5 – 3.8 nM) that was better than the positive
ACCEPTED MANUSCRIPT
control selegiline (IC50 = 8 nM). The bromobenzyl-coumarin moieties (5-8) in general have slightly
better inhibitory activity for MAO than the benzyl-coumarin moieties (1-4). This corresponds to the
observations made by Gnerre et al [41] wherein they noted that an increase in activity toward MAO
is seen when a halogen is substituted on the para-position of the benzyl ring. The same para-bromine
substitution however decreased the degree of MAO-B selectivity of the compounds (Table 1). Other
halogen substitutions on the para and/or metha position(s) on the benzyl ring (10–13) did not
improve the inhibitory activity of compounds 4 and 8 indicating that the bulk of the para-bromine
substitution is favoured. Replacing the benzyl- with an ethylpiperidine moiety (14-15) lead to a
drastic decrease in MAO-B activity, signifying the importance of the aromatic benzyl function. The
substituents at the 3- and/or 4-position of the coumarin moiety did not, in general, influence the
activities on MAOs with the exception of compound 9 where the CF3 substitution at the 4 position
lead to a significant decrease in activity. This could indicate that electron withdrawing hydrophobic
substitutions on position 4 is not tolerated in the active site cavity of MAOs.
The addition of the piperidine (16-20 and 26-29 [39]) or piperizine (21-25) function between the
benzyl and coumarin moieties lead to a decrease in MAO activity compared to compounds 1-13
(Table 1). This decrease in activity could be attributed to the larger substitutions on position 7 of the
coumarin moiety as suggested by Brühlmann et al., 2001 [30]. The compounds, as expected, did not
show any activity towards MAO-A, but did show promising activity towards MAO B inhibition
(IC50: 0.29-5.64 µM). Unlike what was observed with compounds 5-8, the para-bromine substitution
on the benzyl moiety (21-25) reduced the inhibitory capacity of these compounds. A reason for the
decrease in activity observed could be the presence of the piperazine moiety instead of the
piperidine moiety. When the piperidine-containing compounds 16-20 are compared to the
piperidine-containing compounds (26-29 [39]), it is noticeable that the position of the nitrogen in the
piperidine moiety influences the MAO-B activity. When the piperidine nitrogen is connected to the
coumarin (16-20) a 5–50 fold increase in MAO-B activity is observed compared to when the
piperidine nitrogen is connected to the benzyl moiety (26-29). The linker, containing an additional
amine function, between the coumarin and piperidine moiety may also have influenced the activity
of these compounds. When compounds 16–17 are compared to their counterparts lacking the benzyl
moiety (14-15) a definite increase in activity is observed signifying the importance of the benzyl
moiety in these structures. As observed with compounds 1-13, substituents at the 3- and/or 4-
position did not, in general, influence the MAO-B activities.
Table 1: In vitro IC50 values of test compounds 1-15 for hMAO-A, hMAO-B, eeAChE and eqBuChE.

ACCEPTED MANUSCRIPT
R1
O
O
O
O
O
O
N
R3
R1
R3
R2
R2
Compounds 1 - 13
Compounds14 - 15
N
O
O
O
R1
O
O
O
N
NH
R1
R4
R3
R3
R2
R2
Compounds 26 - 29
Compounds 16 - 25
Compound
R1
R2
R3
R4
MAO-A
MAO-B
SI
AChE
BuChE
SI
AChEb
IC50 µM
IC50 µM MAO-Ba IC50 µM IC50 µM
H
H
H
H
H
H
H
-
3.49
2.62
2.13
1.38
0.24
0.05
0.37
0.05
>10
0.60
1.13
>10
0.72
n.d.
0.0038
0.0020
0.0021
0.0019
0.0005
0.0009
0.0008
0.0013
0.104
0.0022
0.013
0.073
0.018
9.21
930
1310
1024
700
480
56
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
54.90
66.43
36.70
29.40
31.30
9.10
>100
9.60
-
1
CH3
CH3
CH3
-
<0.10
2
Cl
CN
H
-
0.96
<0.01
3
-
21.80
>100
>100
>100
>100
>100
>100
>100
>100
>100
n.d.
<0.22
4
p-Br
p-Br
p-Br
p-Br
p-Br
H
-
-
5
CH3
CH3
CH3
CF3
CH3
CH3
CH3
CH3
H
H
-
-
6
Cl
CN
H
-
-
463
38
-
-
7
8
-
>96
273
87
-
9
p-F
p-Cl
o-Br
o, p-Br
H
CN
CN
CN
CN
H
-
-
10
-
-
11
-
>137
40
-
12
-
-
13
-
-
-
14
H
CH3
H
H
-
n.d.
3.09
-
n.d.
-
15
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>100
>100
>100
>100
H
H
CH
CH
CH
CH
CH
N
N
N
N
N
-
0.47
>21
>19
>34
>33
>2
20.30
5.19
0.47
0.18
0.04
0.67
-
16
H
CH3
CH3
CH3
CF3
H
H
0.53
17
H
Cl
CN
H
0.29
1.27
18
H
0.30
5.90
19
H
5.33
>100
>100
12.8
>100
50.10
9.70
20
21
Br
Br
Br
Br
Br
H
H
1.70
>6
0.53
0.76
<0.03
0.35
-
CH3
CH3
CH3
CF3
H
H
3.60
>3
22
Cl
CN
H
1.55
>7
>100
38.5
3.12
23
1.41
>7
13.30
>100
5.34
24
5.64
>2
>100
4.42
25
26c
27c
28c
H
8.39
>12
>36
>7
1.21
0.78
0.90
1.11
-
H
CH3
CH3
CF3
-
H
-
2.75
5.62
4.38
H
Cl
H
-
14.7
1.58
1.42
29c
H
-
28.5
>4
9.18
10.22
n.d.
-
-
-
n.d.
0.001
n.d.
0.008
n.d.
-
n.d.
Selegiline
Clorgyline
Donepezil
Tacrine
-
-
-
-
-
n.d.
n.d.
-
-
-
-
-
n.d.
-
0.007
0.102
2.87
428
0.17
-
-
-
-
n.d.
n.d.
-
0.017
ahMAO-B selectivity index = IC50(hMAO-A)/IC50(hMAO-B). bAChE selectivity index = IC50(eqBuChE)/IC50(eeAChE).
cData taken from Sai-Sai et al [39]. n.d. = not determined.
6

2.3. Cholinesterase inhibition studies
ACCEPTED MANUSCRIPT
The inhibitory activity of the target compounds 1-25 against eeAChE (from electric eel) and
eqBuChE (from equine serum) were measured according to the method of Ellman et al., using
tacrine and donepezil as reference compounds [50,51]. The IC50 values of the test compounds and
reference compounds are given in Table 1 (1–25, 26-29 [39]). Compounds 1, 5-13 showed little to
no inhibitory activity towards either of the cholinesterases at a 100 µM concentration. These results
were as expected because these compounds lack the N-benzylpiperidine or similar moiety of
donepezil. However, an unexpected result was observed for compounds 2-4 were selective and
moderately potent BuChE inhibitory activity, especially for compound 3 (IC50 = 0.96 µM, SI > 104)
was observed. The addition of the para-bromine moiety to the benzyl function (6-8) abolished the
cholinesterase inhibitory activity of these compounds (2-4). In contrast to what was observed in the
MAO studies the substituents at the 3- and/or 4-position of the coumarin moiety did influence the
ChE activities of 1-5. The addition of a methyl at position 4 (2) increased the BuChE inhibitory
activity (IC50 = 9.60 µM) compared to compound 1 (IC50 > 100 µM). Adding a chlorine on position
3 while retaining the methyl at position 4 (3) increased the BuChE activity and selectivity even
further as seen by the IC50 values. Replacing the chlorine with a nitrile (4) decreased the activity
(IC50 = 21.80 µM) and a CF3 substitution at position 4 (5) rendered the compound inactive.
Compounds (16-19) containing the N-benzylpiperidine moiety showed moderate inhibitory activity
against both ChEs with some slight selectivity towards BuChE. When these compounds are
compared to their reported counterparts (26-29) [39] it is evident that they show similar BuChE
activity, however the AChE activity for 16-19 decreased. The substituents at the 3- and/or 4-position
did not influence the ChE activities of 26-29 as previously reported [39], but did influence the ChE
activity of 16-20. Especially compound 19 with a methyl substituent at the 4 position and a nitrile
substituent at the 3 position showed a marked improvement in AChE activity. This implies that
compounds 16-20 can be further explored to improve their AChE activity based on distinct
substitutions on the 3- and/or 4-position without affecting MAO-B activity. Sugimoto et al., 2000
[52] demonstrated that replacement of the piperidine with a piperazine moiety, as is the case with
compounds 20-25, drastically decreases AChE activity of donepezil. This same effect, although not
as pronounced as in Sugimoto’s study, was observed with compounds 21-25.
2.4. Molecular modeling
The enzyme assays point to 3 and 19 as promising multitarget inhibitors. To provide insight, the
binding mode of 3 and 19 in hMAO-A, hMAO-B, eeAChE and eqBuChE were examined using
molecular docking. The structures of human MAO-A co-crystallized with harmine (PDB entry:
2Z5X) [53], human MAO-B co-crystallized with 7-(3-chlorobenzyloxy)-4-(methylamino)methyl-

coumarin (PDB entry: 2V61) [43] and eeAChE co-crystallized with donepezil (PDB entry: 1EVE)
ACCEPTED MANUSCRIPT
[54] were retrieved from the Brookhaven Protein Data Bank (www.rcsb.org/pdb) and the docking
was carried out with the Dock application of the Molecular Operating Environment (MOE) software
[55]. With respect to BuChE, in the absence of a X-ray structure of eqBuChE, a homology model
was used to rationalize the experimental data. The modeling of the 3D structure was performed by
an automated homology-modeling program (SWISS-MODEL) [56-57]. This putative three-
dimensional structure of eqBuChE has been created based on the crystal structure of hBuChE (PDB:
2PM8) as these two enzymes exhibited 89% sequence identity. The docking of the compounds into
this homology model of eqBuChE was carried out with Autodock Vina software [58] using a grid
box that was placed over the active site. The docking results generated were directly loaded into and
analyzed with MOE.
2.4.1. MAO molecular modeling studies
The MAO inhibition studies show that all the test compounds (1-25) showed selective MAO-B
activity over MAO-A. To provide additional insight into the selective MAO-B inhibitory activity,
the binding modes of compound 3 and 19 in MAO-A and MAO-B were examined using molecular
docking. The best-ranked docking solutions of compounds 3 and 19 within the active site of MAO-
B shows that the coumarin moiety of both compounds bind in the polar region of the substrate cavity
in the vicinity of the FAD co-factor and the ‘aromatic sandwich’ defined by Tyr398 and Tyr435
(Figure 4). The binding orientation of the coumarin moiety of 3 and 19 are similar to that observed
for the co-crystallized coumarin within the active site of MAO-B [43]. The carbonyl moiety of 3
showed a hydrogen bond interaction with Cys172 similar to the co-crystallized coumarin. Docking
simulations of compounds 1-2 and 4-8 also showed this hydrogen bond interaction with Cys172
(data not shown). The nitrile on the 3 position of 19 exhibited a hydrogen bond interaction with N5
of FAD but was lacking the interaction with Cys172. As indicated by the biological results the
substitutions at the 3- and/or 4 position did not largely influence the MAO-B activity of compounds
1-8 and 16-19. Therefore the interaction of the nitrile of 19 with FAD seems to be less important
than the interaction with Cys172. This may explain why 19 had lower MAO-B activity compared to
3. The benzyl- and N-benzylpiperidine side chain of 3 and 19 extends past Ile199 which is situated
in the entrance cavity of the enzyme. Within the hydrophobic environment of the entrance cavity,
the benzyl- and N-benzylpiperidine side chain is most likely stabilized by Van der Waals
interactions. These favorable binding orientations and interactions of 3 and 19 may explain the
potent and moderate MAO-B activity, respectively, observed for these compounds. The docking
results of 3 and 19 in complex with MAO-A (data not shown) showed that in both cases the
coumarin moiety was stabilized in the entrance cavity compared to the substrate cavity as observed

for these compounds in MAO-B. This observation may explain the selective MAO-B activity of the
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test compounds.
Figure 4: The hMAO-B active site cavity (left) and interaction maps (right) displaying the binding and
interactions of compounds 3 (top) and 19 (bottom). FAD is shown in red and the compounds are shown in
green in the active site cavity.
2.4.2. Cholinesterase molecular modeling studies
As indicated by the biological results, compound 3 showed selective BuChE inhibitory activity over
AChE and compound 19 presented with both AChE and BuChE inhibitory activity. Molecular
docking was thus employed to gain some insight into the binding modes of these compounds. The
docking results of compounds 3 and 19 docked within the active site of eeAChE are shown in Figure
5. Compound 3 binds to AChE with its coumarin moiety sitting in the area of the PAS and its benzyl
moiety reaching the mid-gorge [54,59]. No interactions with any of the surrounding residues were
predicted by the software. Therefore the inability of 3 to reach the CAS and no interactions with
surrounding residues may explain the lack of AChE inhibitory activity. The docking results of 19
showed that the coumarin moiety binds to the PAS site of the enzyme, establishing a π-π stacking
interaction between its phenyl ring and the indole ring of Trp279. The piperidine moiety of 19
interacts with Tyr324 in the mid-gorge and similar to donepezil, the N-benzylpiperidine moiety is
located at the CAS, which showed a π-π stacking interaction with Trp84 [54,59]. The ability of 19 to

interact with both the CAS and PAS sites of AChE could explain the inhibitory activity observed
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and indicate that 19 could have the ability to inhibit the Aβ pro-agreggating action of AChE [44-47].
PAS
CAS
PAS
CAS
Figure 5: The eeAChE active site cavity (left) and interaction map (right) displaying the binding and
interactions of compounds of 3 (top) and 19 (bottom).
The binding mode of 3 and 19 at the active site of the eqBuChE homology build model is illustrated
in Figure 6. The docking results of 3 showed that the benzyl- and coumarin moieties bind in the
CAS and PAS region of the enzyme respectively [59], establishing π-π stacking interactions with
Trp82 and Tyr332. This favourable position of 3 within the active site of eqBuChE may account for
the higher affinity and selectivity observed for BuChE over AChE. As depicted in Figure 7, 19 was
well accommodated inside the active site gorge and showed a binding mode with a U-shaped
conformation. The lowest energy binding orientation of 19 enabled the benzyl moiety to interact
with Trp82 in the CAS allowing a π-π stacking interaction. A hydrogen bond between the piperidine
NH+ and Asp70 is observed in the PAS region of the enzyme. The carbonyl of the coumarin moiety
interacted though a hydrogen bond with the catalytic-triad residue Ser198 [59]. These important
interactions of 3 and 19 may explain the good BuChE activities observed for these compounds.

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CAS
PAS
CAS
PAS
Figure 6: Complex of compound 3 (top) and 19 (bottom) with the eqBuChE homology model (left) and the
interaction maps (right).
3. Conclusion
A series of 7-substituted coumarin derivatives was designed and synthesized as multipotent
inhibitors able to selectively inhibit hMAO-B, as well as eeAChE and eqBuChE. Compound 3 can
be considered as a new multifunctional lead compound because of its potent MAO-B (IC50 = 0.0021
µM) and moderate BuChE (IC50 = 0.96 µM) inhibitory activities. Molecular modeling revealed the
favourable binding modes and important interactions of 3 in the active sites of BuChE and MAO-B.
Considering the increasing attention given to BuChE [11,60,61] and the ability of 3 to inhibit
MAOs, this compound might be advantageous in AD treatment and should be investigated further.
Compound 19 was also identified as a MTDL being able to selectively inhibit hMAO-B (IC50 = 0.30
µM, SI >33) and inhibit both eeAChE (IC50 = 9.10 µM) and eqBuChE (IC50 = 5.90 µM). Docking
studies indicated that the promising hMAO-B inhibitory activity of 19 may be ascribed to the
position of the coumarin in the substrate cavity and the ability of the benzyl moiety to occupy the
11

entrance cavity. Further in silico studies showed that 19 is able to bind to both the CAS and PAS of
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AChE and BuChE. Based on the role played by PAS in the induction of Aß aggregation by AChE it
is expected that 19 would show inhibition of hAChE induced Aß aggregation. This study has thus
provided new insights into the structure activity relationships of these compounds and could
possibly afford new attractive and more promising drugs for the treatment of AD.
4. Experimental section
4.1. Chemistry
Unless otherwise specified, materials were obtained from commercial suppliers and used without
further purification. All reactions were monitored by thin-layer chromatography on 0.20 mm thick
aluminium silica gel sheets (Alugram® SIL G/UV254, Kieselgel 60, Macherey-Nagel, Düren,
Germany). Visualisation was achieved using UV light (254 nm and 366 nm), an ethanol solution of
ninhydrin or iodine vapours, with mobile phases prepared on a volume-to-volume basis.
Chromatographic purifications were performed on silica gel (0.063–0.2 mm, Sigma Aldrich) except
when otherwise stated. The MS spectra were recorded on a Perkin Elmer Flexar SQ 300 mass
spectrometer by means of direct injection with a syringe pump. High resolution electron spray
ionisation (HREI) mass spectra for all compounds were recorded on a Waters API Q-Tof Ultima
mass spectrometer at 70 eV and 100 °C. The IR spectra were recorded on a Perkin Elmer Spectrum
400 spectrometer, fitted with a diamond attenuated total reflectance (ATR) attachment. Melting
points were determined using a Stuart SMP-300 melting point apparatus and capillary tubes. All the
1
melting points determined were recorded uncorrected. H and 13C NMR spectra were determined
using a Bruker Avance III HD spectrometer at a frequency of 400 MHz and 100 MHz, respectively.
Tetramethylsilane (TMS) was used as internal standard. All chemical shifts are reported in parts per
million (ppm), relative to the internal standard. The following abbreviations are used to indicate the
multiplicities of the respective signals: s - singlet; s - broad singlet; d - doublet; dd - doublet of
doublets; t – triplet and m - multiplet.
4.2. General procedure for the synthesis of compounds 1-13
The appropriate commercially available 7-hydroxylcoumarin (1 mmol, 1a–5a) and K2CO3 (1.1
mmol) were dissolved in 10 ml ethanol. To the mixture, benzylbromide (1 mmol for compounds 1 -
4) or the appropriate bromobenzyl derivative (1 mmol for compounds 5-13) was added depending
on the compound to be synthesised. The mixture was stirred under reflux conditions until the
reaction reached completion (monitored by TLC, mobile phase = EtOAc). Once the reaction was
complete, 20 ml of distilled water was added and the mixture was allowed to cool to room
temperature. This resulted in the formation of a precipitate that was filtered of and allowed to dry

overnight in a fume cupboard rendering the pure 7-(benzyloxy)coumarin derivatives (1–13) as off-
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white amorphous solids.
4.2.1. 7-(Benzyloxy)-2H-chromen-2-one (1)
Yield: 56 % mp: 155 °C; 1H NMR: (400 MHz, CDCl3) δH: 7.64–7.60 (d, 1H, J = 9.2 Hz), 7.42–7.35
(m, 5H), 6.92–6.87 (m, 2H), 6.26–6.22 (d, 1H, J = 9.2 Hz), 5.11 (s, 2 H); 13C NMR (100 MHz,
CDCl3): 161.84, 161.13, 155.78, 143.33, 135.72, 128.72, 128.35, 127.47, 113.21, 112.69, 101.88,
70.48; IR (ATR, cm-1): 1707, 1110, 693; MS (EI, 70 eV) m/z: 253.10 [M+H]+; HR-ESI [M+H]+:
calcd. 259.0859, found. 259.0862.
4.2.2. 7-(Benzyloxy)-4-methyl-2H-chromen-2-one (2)
Yield: 52 % mp: 130 °C; 1H NMR: (400 MHz, CDCl3) δH: 7.49–7.41 (d, 1H, J = 8.8 Hz), 7.42–7.33
13
(m, 5H), 6.94–6.90 (d, 1H, J = 8.8 Hz), 6.88 (s, 1H), 6.12 (s, 1H), 5.12 (s, 2H), 2.38 (s, 3H); C
NMR (100 MHz, CDCl3): 161.71, 161.29, 155.23, 152.53, 135.85, 128.77, 128.38, 127.53, 125.58,
113.80, 112.95, 112.09, 101.96, 70.49, 18.68; IR (ATR, cm-1): 3032, 1706, 699; MS (EI, 70 eV)
m/z: 267.14 [M+H]+; HR-ESI [M+H]+: calcd. 267.1016, found. 267.1018.
4.2.3. 7-(Benzyloxy)-3-chloro-4-methyl-2H-chromen-2-one (3)
Yield: 49 % mp: 145-146 °C; 1H NMR: (400 MHz, CDCl3) δH: 7.53–7.50 (d, 1H, J = 8.8 Hz), 7.41–
7.34 (m, 5 H), 6.98–6.95 (d, 1H, J = 8.8 Hz), 6.88 (s, 1H), 5.12 (s, 2H), 2.53 (s, 3H); 13C NMR (100
MHz, CDCl3): 161.52, 157.34, 152.98, 147.84, 135.57, 128.71, 128.37, 127.44, 125.83, 117.85,
113.55, 113.41, 101.78, 70.51, 16.08; IR (ATR, cm-1): 3057, 1707, 750, 696; MS (EI, 70 eV) m/z:
301.05 [M+H]+; HR-ESI [M+H]+: calcd. 301.0626, found. 301.0621.
4.2.4. 7-(Benzyloxy)-4-methyl-2-oxo-2H-chromene-3-carbonitrile (4)
Yield: 12 % mp: 136 °C; 1H NMR: (400 MHz, CDCl3) δH: 7.64–7.61 (d, 1H, J = 8.8 Hz), 7.41–7.37
(m, 5H), 7.03–7.00 (d, 1H, J = 8.8 Hz), 6.89 (s, 1H), 5.16 (s, 2H), 2.70 (s, 3H); 13C NMR (100
MHz, CDCl3): 164.43, 162.06, 157.33, 155.54, 135.06, 128.85, 128.68, 127.56, 127.31, 114.63,
113.90, 112.10, 102.11, 98.84, 70.93, 18.14; IR (ATR, cm-1): 3079, 1722, 1611, 682; MS (EI, 70
eV) m/z: 292.14 [M+H]+; HR-ESI [M+H]+: calcd. 292.0968, found. 292.0976.
4.2.5. 7-[(4-Bromobenzyl)oxy]-2H-chromen-2-one (5)
1
Yield: 68%; mp: 160 °C; H NMR: (400 MHz, CDCl3) δH: 7.65 – 7.61 (d, 1H, J = 9.6 Hz), 7.60 –
7.50 (d, 2H, J = 8.4 Hz), 7.40–7.35 (d, 1H, J = 8.4 Hz), 7.32 – 7.28 (d, 2H, J = 8.4 Hz), 6.95–6.88
(dd, 1H, J = 8.8, 2.4 Hz), 6.87–6.84 (m, 1H), 6.28–6.24 (d, 1H, J = 9.6 Hz), 5.11 (s, 2H); 13C NMR
(100 MHz, CDCl3): 161.51, 161.04, 155.77, 143.28, 134.75, 131.89, 129.09, 128.84, 122.34,
113.39, 113.13, 112.86, 101.87, 69.69; IR (ATR, cm-1): 3068, 2257, 1705, 1128, 686; MS (EI, 70
eV) m/z: 330.93 [M+H]+; HR-ESI [M+H]+: calcd. 330.9964, found. 330.9959.

4.2.6. 7-[(4-Bromobenzyl)oxy]-4-methyl-2H-chromen-2-one (6)
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Yield: 68%; mp: 153 °C; 1H NMR: (400 MHz, CDCl3) δH: 7.54–7.49 (m, 3H), 7.32–7.29 (d, 2 H, J
= 8.4 Hz), 6.93–6.88 (dd, 1H, J = 8.8, 2.4 Hz), 6.87–6.85 (m, 1H), 6.14 (s, 1H), 5.07 (s, 2H), 2.39 (s,
3H): 13C NMR: (100 MHz, CDCl3): 161.35, 161.19, 155.18, 152.46, 134.84, 131.90, 129.12,
125.63, 122.34, 113.95, 112.85, 112.23, 101.91, 69.67, 18.68; IR (ATR, cm-1): 3074, 1707. 1070,
680; MS (EI, 70 eV) m/z: 344.94 [M+H]+; HR-ESI [M+H]+: calcd. 345.0121, found. 345.0118.
4.2.7. 7-[(4-Bromobenzyl)oxy]-3-chloro-4-methyl-2H-chromen-2-one (7)
Yield: 61 %; mp: 191 – 192 ºC; 1H NMR: (400 MHz, CDCl3) δH: 7.55–7.51(m, 3H), 7.32–7.28 (d, 2
H, J = 8.4 Hz), 6.98–6.95 (dd, 1 H, J = 8.8, 2.4 Hz), 6.88–6.85 (m, 1H), 5.08 (s, 2 H), 2.26 (s, 3 H);
13C NMR (100 MHz, CDCl3): 161.26, 157.36, 153.04, 147.87, 134.67, 131.96, 129.15, 126.01,
122.45, 118.12, 113.68, 113.55, 101.85, 69.79, 16.20; IR (ATR, cm-1): 3074, 1725, 1600, 801, 683;
MS (EI, 70 eV) m/z: 379.18 [M+H]+; HR-ESI [M+H]+: calcd. 378.9131, found. 378.9136.
4.2.8. 7-[(4-Bromobenzyl)oxy]-4-methyl-2-oxo-2H-chromene-3-carbonitrile (8)
1
Yield: 55%; mp: 186 – 187 ºC; H NMR: (400 MHz, CDCl3) δH: 7.66–7.63 (d, 1H, J = 9.2 Hz),
7.56–7.53 (d, 2H, J = 8.0 Hz), 7.32–7.28 (d, 2H, J = 8.0 Hz), 7.03–6.99 (dd, 1H, J = 8.8, 2.4 Hz),
6.88 (s, 1H), 5.12 (s, 2H), 2.71 (s, 3H); 13C NMR (100 MHz, CDCl3): 164.07, 162.04, 157.24,
155.51, 134.07, 132.06, 129.19, 127.40, 122.72, 114.54, 113.85, 112.27, 102.09, 99.06, 70.13,
18.18; IR (ATR, cm-1): 3079, 2226, 1710, 1244, 657; MS (EI, 70 eV) m/z: 369.97 [M+H]+; HR-
ESI [M+H]+: calcd. 370.0073, found. 370.0072.
4.2.9. 7-[(4-Bromobenzyl)oxy]-4-(trifluoromethyl)-2H-chromen-2-one (9)
Yield: 65 % mp: 136 ºC; 1H NMR: (400 MHz, CDCl3) δH: 7.66–7.64 (d, 1H, J = 8.8 Hz), 7.56–7.52
(d, 2 H, J = 8.4 Hz), 7.32–7.28 (d, 2H, J = 8.4 Hz), 7.00–6.96 (dd, 1H, J = 8.8, 2.4 Hz), 6.92–6.91
(m, 1H, H–8), 6.63 (s, 1H, H–3), 5.11 (s, 2H, H–15); 13C NMR (100 MHz, CDCl3): 162.22, 159.29,
156.23, 134.37, 132.00, 129.12, 126.49, 122.56, 113.91, 112.58, 112.52, 107.41, 102.42, 69.87; IR
(ATR, cm-1): 3080, 1724, 1130, 657; MS (EI, 70 eV) m/z: 398.99 [M+H]+; HR-ESI [M+H]+: calcd.
398.9838, found. 398.9839.
4.2.10. 7-[(4-Fluorobenzyl)oxy]-4-methyl-2-oxo-2H-chromene-3-carbonitrile (10)
1
Yield: 57%; H NMR: (400 MHz, CDCl3) δH: 7.68–7.66 (d, 1H, J = 9.1 Hz), 7.56–7.43 (m, 2H),
7.15–7.10 (m, 2H), 7.05–7.00 (dd, 1H, J = 8.8, 2.4 Hz), 6.90 (s, 1H), 5.15 (s, 2H), 2.74 (s, 3H); MS
(EI, 70 eV) m/z: 310.10 [M+H]+; HR-ESI [M+H]+: calcd. 310.0874, found. 310.0876.
4.2.11. 7-[(4-Chlorobenzyl)oxy]-4-methyl-2-oxo-2H-chromene-3-carbonitrile (11)
Yield: 82%; 1H NMR: (400 MHz, CDCl3) δH: 7.68–7.66 (d, 1H, J = 9.2 Hz), 7.57–7.55 (d, 2H, J =
8.0 Hz), 7.34–7.32 (d, 2H, J = 8.0 Hz), 7.02–6.98 (dd, 1H, J = 8.8, 2.4 Hz), 6.90 (s, 1H), 5.15 (s,

2H,), 2.74 (s, 3H); MS (EI, 70 eV) m/z: 326.10 [M+H]+; HR-ESI [M+H]+: calcd. 326.0578, found.
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326.0579.
4.2.12. 7-[(3-Bromobenzyl)oxy]-4-methyl-2-oxo-2H-chromene-3-carbonitrile (12)
Yield: 76%; 1H NMR: (400 MHz, CDCl3) δH: 7.69–7.66 (d, 1H, J = 9.2 Hz), 7.61 (s, 1H) 7.57–7.54
(d, 1H, J = 8.0 Hz), 7.33–7.28 (m, 2H), 7.05–7.00 (dd, 1H, J = 8.8, 2.4 Hz), 6.90 (s, 1H), 5.12 (s,
2H), 2.71 (s, 3H); MS (EI, 70 eV) m/z: 369.98 [M+H]+; HR-ESI [M+H]+: calcd. 370.0073, found.
370.0074.
4.2.13. 7-[(3,4-Dibromobenzyl)oxy]-4-methyl-2-oxo-2H-chromene-3-carbonitrile (13)
Yield: 76%; 1H NMR: (400 MHz, DMSO) δH: 7.95-7.92 (d, 1H, J = 9.3 Hz), 7.84–7.81 (d, 1H, J =
9.2 Hz), 7.77 (s, 1H), 7.42-7.38 (d, 1H, J = 8.0 Hz), 7.22–7.14 (m, 2H), 5.30 (s, 2H), 2.69 (s, 3H);
MS (EI, 70 eV) m/z: 369.98 [M+H]+; HR-ESI [M+H]+: calcd. 370.0073, found. 370.0074.
4.2. General procedure for the synthesis of compounds 14 and 15
The appropriate commercially available 7-hydroxylcoumarin (6.17 mmol, 1a or 2a) and K2CO3
(12.33 mmol) were dissolved in 50 ml acetonitrile. To the mixture 1-(2-chloroethyl)piperidine
hydrochloride (6.17 mmol) was added and stirred under reflux conditions at 80 °C for 3 hours,
cooled down, filtered and evaporated to dryness. The remaining residue was extracted with 100 mL
brine and 2 x 30 ml ethyl acetate. The combined organic fractions were dried over MgSO4, filtered
and concentrated. Recrystallisation from EtOAc rendered compound 14 or 15 as yellow crystals.
4.2.14. 7-[2-(Piperidin-1-yl)ethoxy]-2H-chromen-2-one (14)
Yield: 18.15 %; mp: 90 °C; 1H NMR (600 MHz, CDCl3) δH: 7.60 (d, 1H J = 9.5 Hz); 7.33 (d, 1H, J
= 8.6 Hz); 6.82 (dd, 1H, J = 8.6, 2.4 Hz); 6.79 (d, 1H, J = 2.4 Hz); 6.21 (d, 1H, J = 9.5 Hz); 4.12 (t,
13
2H); 2.76 (t, 2H); 2.47 (s, 4H); 1.60 – 1.55 (m, 4H); 1.42 (s, 2H). C NMR (150 MHz, CDCl3):
162.05; 161.21; 155.81; 143.38; 128.67; 113.03; 112.98; 112.50; 101.48; 66.63; 57.58; 55.06;
25.88; 24.09; HR-ESI [M+H]+: calcd. 274.1438, found. 274.1434.
4.2.15. 4-Methyl-7-[2-(piperidin-1-yl)ethoxy]-2H-chromen-2-one (15)
1
Yield: 82.70 %; mp: 102–104 °C; H NMR (600 MHz, CDCl3) δH: 7.44 (d, 1H, J = 8.8 Hz), 6.82
(dd, 1H, J = 8.8, 2.4 Hz), 6.76 (d, 1H, J = 8.4 Hz), 6.08 (d, 1H, J = 8.4 Hz), 4.12 (t, 2H), 2.76 (t,
13
2H), 2.48 (s, 4H), 2.35 (m, 3H), 1.58 (m, 4H), 1.41 (s, 2H); C NMR (150 MHz, CDCl3) 161.77;
161.23; 155.13; 152.48, 125.42, 113.52, 112.56, 111.85, 101.48, 66.44, 57.52, 55.00, 25.76, 24.00,
18.60; HR-ESI [M+H]+: calcd. 288.1594, found. 288.1593.
4.4. General procedure for the synthesis of compounds 16 – 25

A mixture of 1b–5b (1 mmol) and 4-benzylpiperidine (1 mmol for compounds 16-20) or 1-(4-
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bromobenzyl)piperazine (1 mmol for compounds 21–25) in acetonitrile (10 mL) was refluxed in the
presence of anhydrous K2CO3 (1.1 mmol) for 8 h or until completion (monitored by TLC, mobile
phase = CH3Cl/MeOH, 50:1). After completion the reaction mixture was dried in vacuo, and the
residue was purified by silica gel chromatography using CHCl3/MeOH (50:1) as eluent to obtain
compounds 16-25 as yellow oils.
4.4.1. 7-[2-(4-Benzylpiperidin-1-yl)ethoxy]-2H-chromen-2-one (16)
Yield: 34 %; 1H NMR (400 MHz, CDCl3) δH: 7.67–7.63 (d, 1H, J = 9.6 Hz), 7.39–7.36 (d, 1H, J =
8.4 Hz), 7.32–7.28 (m, 1H), 7.23–7.15 (m, 4H) 6.88–6.84 (d, 1H, J = 8.8), 6.83 (s, 1 H), 6.29–6.25
(d, 1 H, J = 9.5 Hz), 4.17 (s, 2 H), 3.02 – 2.99 (d, 2 H, J = 10.6 Hz), 2.83 (s, 2 H), 2.58 – 1.27 (m, 9
H): 13C NMR (100 MHz, CDCl3): 162.02, 161.24, 155.83, 143.40, 140.54, 129.11, 128.67, 128.18,
125.82, 113.12, 112.99, 112.59, 101.52, 66.62, 57.18, 54.46, 43.12, 37.66, 32.03; IR (ATR, cm-1):
2922, 1729, 1610, 1124; MS (EI, 70 eV) m/z: 364.15 [M+H]+; HR-ESI [M+H]+: calcd. 364.1907,
found. 364.1910.
4.4.2. 4-Methyl-7-[2-(benzylpiperidine-1-yl)ethoxy]-2H-chromen-2-one (17)
1
Yield: 62 % H NMR: (400 MHz, CDCl3) δH: 7.48–7.45 (d, 1H, J = 8.8 Hz), 7.27 – 7.11 (m, 5H),
6.85–6.81 (dd, 1H, J = 8.8, 2.4 Hz), 6.79–6.77 (m, 1H), 6.12–6.11 (s, 1H), 4.15–4.12 (t, 2H), 3.00–
2.96 (m, 2H), 2.82–2.78 (t, 2H), 2.54 – 2.51 (d, 2H, J = 7.04 Hz), 2.38–2.37 (s, 3H), 2.09–2.03 (t,
13
2H), 1.67–1.63 (d, 2H, J = 12.8 Hz), 1.57–1.50 (m, 1H), 1.40–1.29 (m, 2H); C NMR (100 MHz,
CDCl3): 161.83, 155.23, 152.59, 140.56, 129.13, 128.20, 125.85, 125.50, 113.65, 112.69, 111.99,
101.55, 66.51, 57.23, 54.49, 43.13, 37.68, 32.01, 18.69; IR (ATR, cm-1): 3025, 2922, 1715, 1605,
1135; MS (EI, 70 eV) m/z: 378.19 [M+H]+; HR-ESI [M+H]+: calcd. 378.2064, found. 378.2059.
4.2.3. 3-Chloro-4-methyl-7-[2-(benzylpiperidin-1-yl)ethoxy]-2H-chromen-2-one (18)
1
Yield: 30 %; H NMR (400 MHz, CDCl3) δH: 7.52–7.49 (d, 1H, J = 8.8 Hz), 7.29–7.25 (t, 2H),
7.21–7.18 (t, 1H, H–27), 7.15–7.12 (d, 2H, J = 7.2 Hz), 6.92–6.88 (dd, 1H, J = 9.2, 2.8 Hz), 6.82–
6.80 (m, 1H), 4.16–4.12 (t, 2H), 2.99–2.95 (d, 2H, J = 12.8), 2.82–2.78 (t, 2H), 2.56–2.52 (d, 5H, J =
4.8 Hz), 2.10–2.02 (m, 2H), 1.67–1.63 (d, 2H, J = 12.8), 1.58–1.49 (m, 1H), 1.40–1.29 (m, 2H) 13C
NMR (100 MHz, CDCl3): 161.82, 157.33, 753.10, 148.08, 140.41, 129.11, 128.17, 125.82, 113.37,
101.45, 66.68, 57.16, 54.49, 43.15, 37.66, 32.05, 16.16; IR (ATR, cm-1): 3070, 2930, 1719, 1600,
811; MS (EI, 70 eV) m/z: 412.16 [M+H]+; HR-ESI [M+H]+: calcd. 412.1674, found. 412.1673.
4.2.4. 4-Methyl-2-oxo-7-[2-(benzylpiperidin-1-yl)-ethoxy]-2H-chromene-3-carbonitrile (19)
1
Yield: 56 %; H NMR (400 MHz, CDCl3) δH: 7.71–7.68 (d, 1H, J = 8.8 Hz), 7.38–7.20 (m, 5H),
7.05–7.02 (d, 1H, J = 8.8 Hz), 6.90 (s, 1H), 4.27 (s, 2H), 3.07–3.03 (d, 2H, J = 10.7 Hz), 2.89 (s,
2H), 2.80–2.79 (s, 3H), 2.64–2.61 (d, 2H, J = 6.7), 2.26–2.12 (t, 2H), 1.76–1.72 (d, 2H, J = 13.2 Hz),

1.63–1.62 (m, 1H), 1.47–1.33 (m, 2H): 13C NMR: (100 MHz, CDCl3): 164.63, 162.07, 157.38,
ACCEPTED MANUSCRIPT
155.56, 140.51, 129.10, 128.18, 127.20, 125.83, 114.41, 113.94, 111.93, 101.65, 98.69, 67.14,
57.05, 54.51, 43.09, 37.65, 32.03, 18.17; IR (ATR, cm-1): 3070, 2917, 1714, 1598; MS (EI, 70 eV)
m/z: 403.21 [M+H]+; HR-ESI [M+H]+: calcd. 403.2016, found. 403.2020.
4.2.5. 7-[2-(Benzylpiperidin-1-yl)ethoxy]-4-(trifluoromethyl)-2H-chromen-2-one (20)
1
Yield: 74 %; H NMR (400 MHz, CDCl3) δH: 7.26–7.12 (m, 6H), 6.94-6.90 (dd, 1H, J = 8.8, 2.4
Hz), 6.88–6.86 (m, 1H), 6.61 (s, 1H), 4.18–4.14 (t, 2H), 2.98–2.94 (d, 2H, J = 2.4 Hz), 2.82–2.79 (t,
2), 2.55–2.53 (d, 2H, J = 6.8 Hz), 2.09–2.02 (t, 2H), 1.68–1.64 (d, 2H), 1.63–1.40 (m, 1H), 1.41–
1.33 (m, 2H); 13C NMR (100 MHz, CDCl3): 140.53, 129.19, 128.11, 125.81, 113.71, 102.06, 66.96,
57.15, 54.54, 43.20, 37.68, 32.01; IR (ATR, cm-1): 3070, 1713, 1614, 1132; MS (EI, 70 eV) m/z:
432.23 [M+H]+; HR-ESI [M+H]+: calcd. 432.1781, found. 432.1785.
4.2.6. 7-[2-[4-(4-Bromobenzyl)piperazin-1-yl]ethoxy]-2H-chromen-2-one (21)
1
Yield: 32 %; H NMR (400 MHz, CDCl3) δH: 7.63–7.60 (d, 1H, J = 9.6 Hz), 7.43-7.40 (d, 2H, J =
8.4 Hz), 7.36–7.33 (d, 1H, J = 8.6 Hz), 7.20–7.17 (d, 2H, J = 8.4 Hz), 6.84–6.81 (dd, 1H, J = 8.4, 2.4
Hz), 6.80– 6.79 (m, 1H), 6.25 – 6.22 (d, 1H, J = 9.6 Hz), 4.16–4.12 (t, 2H), 3.44 (s, 2H), 2.85–2.81
(t, 2H, Hz), 2.61–2.48 (d, 8H, J = 48.0 Hz); 13C NMR (100 MHz, CDCl3): 161.93, 161.17, 155.83,
143.34, 137.07, 131.32, 130.79, 128.71, 120.87, 113.17, 112.98, 112.61, 101.49, 66.52, 62.20,
56.81, 53.58, 52.86; IR (ATR, cm-1): 3072, 2944, 1729, 1614, 695; MS (EI, 70 eV) m/z: 443.15
[M+H]+; HR-ESI [M+H]+: calcd. 443.0965, found. 443.0971.
4.2.7. 4-Methyl-7-[2-[4-(4-bromobenzyl)piperazin-1-yl]ethoxy]-2H-chromen-2-one (22)
Yield: 11 %; 1H NMR (400 MHz, CDCl3) δH: 7.49–7.46 (d, 1H, J = 8.8 Hz), 7.44–7.41 (d, 2H, J =
8.4 Hz), 7.21–7.18 (d, 2H, J = 8.4 Hz), 6.87–6.84 (dd, 1H, J = 8.8, 2.4 Hz), 6.81–6.80 (m, 1H), 6.12
(s, 1H), 4.17–4.13 (t, 2H), 3.45 (s, 2H), 2.86–2.82 (t, 2H, J = 5.6 Hz), 2.62–2.49 (d, 7H, J = 47.2
13
Hz), 7.38 (s, 3H): C NMR: (100 MHz, CDCl3): 161.78, 161.34, 155.19, 152.55, 137.10, 131.33,
130.80, 125.54, 120.85, 113.68, 112.72, 111.99, 101.52, 66.48, 56.85, 53.62, 52.88, 18.68; IR
(ATR, cm-1): 2938, 1610, 705; MS (EI, 70 eV) m/z: 457.17 [M+H]+; HR-ESI [M+H]+: calcd.
457.1121, found. 457.1121.
4.2.8. 3-Chloro-4-methyl-7-[2-[4-(4-bromobenzyl)piperazine-1-yl]ethoxy]-2H-chromen-2-one
(23)
Yield: 15 %; 1H NMR (400 MHz, CDCl3) δH: 7.52–7.48 (d, 1H, J = 8.8 Hz), 7.44–7.41 (d, 2H, J =
8.4 Hz), 7.21–7.17 (d, 2 H, J = 8.4 Hz), 6.90–6.86 (dd, 1H, J = 9.2, 2.8 Hz), 6.79–6.77 (m, 1H),
4.17–4.13 (t, 2H), 3.46 (s, 2H), 2.88–2.84 (t, 2H), 2.64–2.52 (m, 11H); 13C NMR (100 MHz,
CDCl3): 161.77, 131.37, 130.86, 125.86, 113.22, 101.42, 66.81, 62.22, 56.80, 53.55, 52.73, 29.74;

IR (ATR, cm-1): 3395, 2923, 1601, 1002, 684; MS (EI, 70 eV) m/z: 491.14 [M+H]+; HR-ESI
ACCEPTED MANUSCRIPT
[M+H]+: calcd. 491.0732, found. 491.0733.
4.2.9. 4-Methyl-2-oxo-7-[2-[4-(4-bromobenzyl)piperazin-1-yl]ethoxy]-2H-chromene-3-
carbonitrile (24)
Yield: 40 %; 1H NMR (400 MHz, CDCl3) δH: 7.62–7.58 (d, 1H, J = 8.8 Hz), 7.43–7.40 (d, 2H, J =
8.4 Hz), 7.20–7.17 (d, 2H, J = 8.4 Hz), 6.96–6.92 (dd, 1H, J = 8.8, 2.4 Hz), 6.82–6.80 (m, 1H),
4.19–4.16 (t, 2H), 3.44 (s, 2H), 2.89–2.75 (t, 2H), 2.69 (s, 3H), 2.54–2.47 (d, 8H, J = 23.2 Hz); 13C
NMR (100 MHz, CDCl3): 164.56, 162.04, 157.33, 155.59, 137.10, 131.35, 130.80, 127.23, 120.91,
114.40, 113.92, 112.00, 101.65, 98.80, 67.04, 62.22, 56.69, 53.65, 52.87, 18.14; IR (ATR, cm-1):
3071, 1720, 1007, 735; MS (EI, 70 eV) m/z: 482.20 [M+H]+; HR-ESI [M+H]+: calcd. 482.1074,
found. 482.1079.
4.2.10. 7-[2-[4-(4-Bromobenzyl)piperazin-1-yl]ethoxy]-4-(trifluoromethyl)-2H-chromen-2-one
(25)
Yield: 21 %; 1H NMR: (400 MHz, CDCl3) δH: 7.62–7.59 (d, 1 H, J = 9.2 Hz), 7.44–7.41 (d, 2 H, J =
8.4 Hz), 7.21–7.18 (d, 2H, J = 8.4 Hz), 6.93–6.89 (dd, 1H, J = 9.2, 2.4 Hz), 6.87–6.86 (m, 1H), 6.61
(s, 1H), 4.19–4.15 (t, 2H), 3.45 (s, 2H), 2.87–2.83 (t, 2H), 2.62–2.49 (m, 8H); 13C NMR (100 MHz,
CDCl3): 131.35, 130.81, 113.73, 107.12, 102.10, 66.67, 62.19, 56.64, 53.61, 52.86: IR (ATR, cm-1):
2936, 2809, 1609, 1125, 1008; MS (EI, 70 eV) m/z: 511.14 [M+H]+; HR-ESI [M+H]+: calcd.
511.0839, found. 511.0840.
4.3. Recombinant human MAO-A and MAO-B inhibition studies
The MAO assays were conducted using a similar procedure as previously described [48,49].
Recombinant human MAO-A and -B (5 mg/mL) were obtained from Sigma–Aldrich and were pre-
aliquoted and stored at -70 °C. All enzymatic reactions were carried out to a final volume of 250 µL
in potassium phosphate buffer (100 mM, pH 7.4, made isotonic with KCl, 20.2 mM) and contained
kynuramine as substrate, MAO-A or MAO-B (0.0075 mg/mL) and various concentrations of the test
inhibitor (0–1000 µM). The final concentrations of kynuramine in the reactions were 45 µM and 30
µM for MAO-A and -B, respectively. Stock solutions of the test inhibitors were prepared in DMSO
and added to the reactions to yield a final concentration of 2% (v/v) DMSO. The reactions were
incubated for 20 min at 37 °C and were terminated with the addition of 100 µL NaOH (2 N). Finally
250 µL from each eppendorf vial was added to individual black plate wells. The fluorescence of the
MAO generated 4-hydroxyquinoline in the supernatant fractions were measured using a Biotek
fluorescent microplate reader at an excitation wavelength of 310 nm and an emission wavelength of
400 nm. IC50 values were calculated as concentration of the compound that produces 50% enzyme
activity inhibition, using the Graph Pad Prism 6 software (San Diego, CA, USA).

4.4. Cholinesterase inhibition studies
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AChE from electric eel, BuChE from equine serum, S-butylthiocholine iodide (BTCI),
acetylthiocholine iodide (ATCI), 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB),
donepezil and tarcine hydrochloride were purchased from Sigma-Aldrich. The inhibitory activities
of test compounds 1 - 25 were evaluated by Ellman’s method [50]. The compounds were dissolved
in DMSO and diluted with the buffer solution (50 mM Tris–HCl, pH = 8.0) to yield the
corresponding test concentrations (DMSO less than 0.01%). In each well of the plate, 160 ꢀL of 1.5
mM DTNB, 50 ꢀL of AChE (0.22 U/mL eeAChE) or 50 ꢀL of BuChE (0.12 U/mL eqBuChE) were
incubated with 10 ꢀL of different concentrations of test compounds (0.01–100 ꢀM) at 37°C for 10
min. After this period, acetylthiocholine iodide (15 mM) or S-butyrylthiocholine iodide (15 mM) as
the substrate (30 ꢀL) was added, incubated for a further 10 minutes, and thereafter the absorbance
was measured at a wavelength of 405 nm at different time intervals (0, 60, 120, and 180 s). IC50
values were calculated as concentration of the compound that produces 50% enzyme activity
inhibition, using the Graph Pad Prism 6 software (San Diego, CA, USA).
4.5. Molecular modeling studies
4.5.1. MAO docking studies
Computer-assisted docking was carried out using the CHARMm force field and hMAO-A co-
crystallized with harmine (PDB ID: 2Z5X) [53] and hMAO-B co-crystallized with 7-(3-
chlorobenzyloxy)-4-(methylamino)methyl-coumarin (PDB ID: 2V61) [43], which were recovered
from the Brookhaven Protein Database (www.rcsb.org/pdb). Docking simulations were performed on
the test compounds using Molecular Operating Environment (MOE) [55] with the following protocol.
(1) Enzyme structures were checked for missing atoms, bonds and contacts. (2) Hydrogens and
partial charges were added using the protonate 3D application in MOE. (3) The ligands were
constructed using the builder module and were energy minimized. (4) Ligands were docked within
the MAO-A or MAO-B active sites using MOEDock application, the poses were generated by the
Triangle Matcher placement method and were rescored using the ASE scoring function. (5) The
retained best poses were visually inspected and the interactions with binding pocket residues were
analyzed. To determine the accuracy of this docking protocol, the co-crystallised ligand, harmine
(PDB ID: 2Z5X), was re-docked into the MAO-A active site and the co-crystallised ligand, 7-(3-
chlorobenzyloxy)-4-(methylamino)methyl-coumarin (PDB ID: 2V5Z), was re-docked into the MAO-
B active site. This procedure was repeated three times and the best ranked solutions of harmine and
7-(3-chlorobenzyloxy)-4-(methylamino)methyl-coumarin exhibited RMSD values of 0.42 Å and 0.62
Å from the position of the co-crystallised ligand for MAO-A and MAO-B, respectively. In general,
RMSD values smaller than 2.0 Å, indicate that the docking protocol is capable of accurately
predicting the binding orientation of the co-crystallised ligand [62,63]. These protocols were thus

deemed to be suitable for the docking of inhibitors into the active site models of MAO-A and MAO-
ACCEPTED MANUSCRIPT
B.
4.5.2. Cholinesterase docking studies
4.5.2.1. Acetylcholinesterase
The dock function of MOE [55] was used to predict the interactions and binding modes of the
synthesized inhibitors in the eeAChE active site. The crystal structure of the eeAChE protein co-
crystallized with donepezil was acquired from the Protein Data Bank (PDB ID: 1EVE) [54]. The
docking procedure followed the same protocol as described for the MAO docking. To determine the
accuracy of this docking protocol, the co-crystallised ligand, was re-docked into the AChE active
site. This procedure was repeated three times and the best ranked solution exhibited an RMSD value
of 1.15 Å from the position of the co-crystallised ligand. The RMSD value in this case is smaller than
2.0 Å indicating that the docking protocol is capable of accurately predicting the binding orientation
of the co-crystallised ligand [62,63]. This protocol was thus deemed to be suitable for the docking of
inhibitors into the active site model of AChE.
4.5.2.2. Butyrylcholinesterase
Because no X-ray structure exists for eqBuChE, a homology model was used to rationalize the
experimental data. The eqBuChE model was retrieved from the SWISS-MODEL Repository. This is
a database of annotated three-dimensional comparative protein structure models generated by the
fully automated homology-modeling pipeline SWISS-MODEL. A putative three-dimensional
structure of eqBuChE has been created based on the crystal structure of hBuChE (PDB ID: 2PM8),
these two enzyme exhibited 89% sequence identity. Proper bonds, bond orders, hybridization and
charges were assigned and CHARMm force field was applied using MOE [55]. The prepared protein
was directly loaded into AutoDockTools (ADT; version 1.5.4), hydrogens were added and partial
charges for proteins and ligands were calculated using Gasteiger charges. Flexible torsions in the
ligands were assigned with the AutoTors module, and the acyclic dihedral angles were allowed to
rotate freely. Docking calculations were performed with the program Autodock Vina [58]. Because
VINA uses rectangular boxes for the binding site, the box center was defined and the docking box
was displayed using ADT. All dockings were performed where a cube of 75 Ǻ with grid points
separated by 1 Ǻ, was positioned over the active site of the protein (x = 29.885; y = -54.992; z =
58.141). Default parameters were used except num_modes, which was set to 40. The lowest docking-
energy conformation was considered as the most stable orientation. Finally, the docking results
generated were analysed using MOE.
Acknowledgements

We are grateful to the University of the Western Cape, North-West University and the National
ACCEPTED MANUSCRIPT
Research Foundation of South Africa for financial support.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
Synthesis and evaluation of 7-substituted coumarin derivatives as multimodal
monoamine oxidase-B and cholinesterase inhibitors for the treatment of
Alzheimer’s disease
Jacques Jouberta,*, Germaine B. Fokaa, Benjamin P. Repsoldb, Douglas W. Oliverb, Erika Kappa,
Sarel Malana
aPharmaceutical Chemistry , School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville, South
Africa. bSchool of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, South Africa
CH3
CH3
N
Cl
O
N
O
O
O
O
O
19
3
Compound
IC50 (µM)
hMAO-A
IC50 (µM)
hMAO-B
IC50 (µM)
eeAChE
IC50 (µM)
eqBuChE
2.13
>10
0.0021
0.30
>100
9.10
0.96
5.90
3
19

HIGHLIGHTS
ACCEPTED MANUSCRIPT
•
•
•
•
•
Twenty-five 7-substituted coumarin derivatives were successfully synthesised and evaluated.
Compound 3 showed potent and selective MAO-B and BuChE inhibitory activity.
Compound 19 was able to inhibit MAO-B, AChE and BuChE in the µm range.
Docking revealed that 19 binds simultaneously to the CAS and PAS sites of AChE.
It is suggested that 19 will be able to inhibit AChE induced Aß aggregation.

ACCEPTED MANUSCRIPT
List of captions
Figure 1: Design strategy for the 7-substituted coumarins incorporating different portions and
distinct variations of the N-benzylpiperidine moiety of donepezil.
Figure 2: Synthetic pathway for the synthesis of compounds 1 – 15.
Figure 3: Synthetic pathway for the synthesis of compounds 16 - 25.
Figure 4: The hMAO-B active site cavity (left) and interaction maps (right) displaying the binding
and interactions of compounds 3 (top) and 19 (bottom). FAD is shown in red and the compounds are
shown in green in the active site cavity.
Figure 5: The eeAChE active site cavity (left) and interaction map (right) displaying the binding
and interactions of compounds of 3 (top) and 19 (bottom).
Figure 6: Complex of compound 3 (top) and 19 (bottom) with the eqBuChE homology model (left)
and the interaction maps (right).
Table 1: In vitro IC50 values of test compounds 1-15 for hMAO-A, hMAO-B, eeAChE and
eqBuChE.