Design, synthesis and biological evaluation of novel coumarin-N-benzyl pyridinium hybrids as multi-target agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2017.07.055

Combining N-benzyl pyridinium moiety and coumarin into in a single molecule, novel hybrids with ChE and MAO-B inhibitory activities were designed and synthesized. The biological screening results indicated that most of compounds displayed potent inhibitory activity for ChE and Aβ (1–42) self-aggregation, and clearly selective inhibition to MAO-B over MAO-A. Of these compounds, compound 7f was the most potent inhibitor for hMAO-B, and it was also a good and balanced inhibitor to ChEs and hMAO-B (0.0373 μM for eeAChE; 2.32 μM for eqBuChE; 1.57 μM for hMAO-B). Molecular modeling and kinetic studies revealed that compound 7f was a mixed-type inhibitor, which bond simultaneously to CAS and PAS of AChE, and it was also a competitive inhibitor, which occupied the active site of MAO-B. In addition, compound 7f with no toxicity on PC12 neuroblastoma cells, showed good ability to inhibit Aβ (1–42) self-aggregation and cross the BBB. Collectively, all these results suggested that compound 7f might be a promising multi-target lead candidate worthy of further pursuit.

Full text of DOI:10.1016/j.ejmech.2017.07.055

Accepted Manuscript
Design, synthesis and biological evaluation of novel coumarin-N-benzyl pyridinium
hybrids as multi-target agents for the treatment of Alzheimer's disease
Jin-Shuai Lan, Yue Ding, Yun Liu, Ping Kang, Jian-Wei Hou, Xin-Yu Zhang, Sai-Sai
Xie, Tong Zhang
PII:
S0223-5234(17)30577-9
10.1016/j.ejmech.2017.07.055
EJMECH 9618
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 February 2017
Revised Date: 28 April 2017
Accepted Date: 23 July 2017
Please cite this article as: J.-S. Lan, Y. Ding, Y. Liu, P. Kang, J.-W. Hou, X.-Y. Zhang, S.-S. Xie, T.
Zhang, Design, synthesis and biological evaluation of novel coumarin-N-benzyl pyridinium hybrids as
multi-target agents for the treatment of Alzheimer's disease, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.07.055.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, synthesis and biological evaluation of novel coumarin-N-benzyl
pyridinium hybrids as multi-target agents for the treatment of Alzheimer’s
disease

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel coumarin-N-benzyl
pyridinium hybrids as multi-target agents for the treatment of Alzheimer’s
disease
1
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1#
1
1
1
1
Jin-Shuai Lan , Yue Ding , Yun Liu , Ping Kang , Jian-Wei Hou , Xin-Yu Zhang ,
3
2
Sai-Sai Xie *, Tong Zhang *
1
Experiment Center of Teaching & Learning, Shanghai University of Traditional
Chinese Medicine, Shanghai 201203, China;
School of Pharmacy, Shanghai University of Traditional Chinese Medicine,
Shanghai 201203, China;
National Pharmaceutical Engineering Center for Solid Preparation in Chinese
Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang
2
3
3
30006, China.
*
*
#
Corresponding author. Sai-Sai Xie, Tong Zhang
E-mail: zhangtongshutcm@hotmail.com (TZ); xiesaisainanchang@hotmail.com
These authors contributed equally to this work.
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Abstract
Combining N-benzyl pyridinium moiety and coumarin into in a single molecule,
novel hybrids with ChE and MAO-B inhibitory activities were designed and
synthesized. The biological screening results indicated that most of compounds
displayed potent inhibitory activity for ChE and Aβ (1-42) self-aggregation, and
clearly selective inhibition to MAO-B over MAO-A. Of these compounds, compound
7
f was the most potent inhibitor for hMAO-B, and it was also a good and balanced
inhibitor to ChEs and hMAO-B (0.0373 µM for eeAChE; 2.32 µM for eqBuChE; 1.57
µM for hMAO-B). Molecular modeling and kinetic studies revealed that compound 7f
was a mixed-type inhibitor, which bond simultaneously to CAS and PAS of AChE,
and it was also a competitive inhibitor, which occupied the active site of MAO-B. In
addition, compound 7f with no toxicity on PC12 neuroblastoma cells, showed good
ability to inhibit Aβ (1-42) self-aggregation and cross the BBB. Collectively, all these
results suggested that compound 7f might be a promising multi-target lead candidate
worthy of further pursuit.
Keywords: monoamine oxidase inhibitors; acetylcholinesterase inhibitors; multi-target
ligand design; Alzheimer’s disease; coumarin derivatives.
Abbreviations
AD, Alzheimer’s disease; ChEs, Cholinesterases; AChE, Acetylcholinesterase;
BuChE, Butyrylcholinesterase; MAO, Monoamine oxidase; CAS, Catalytic active site;
PAS, Peripheral anionic site; MTDL, Multi-target-directed ligand; SI, Selectivity
index; Th-T, Thioflavin-T; CNS, Central nervous system; BBB, Blood-brain barrier;
PAMPA-BBB, Parallel artificial membrane permeation assay for BBB; MTT, Methyl
thiazolyl tetrazolium.
2

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1
. Introduction
Alzheimer’s disease (AD), that disrupts an individual’s language, behavioral,
thinking, and cognitive skills, is a neurodegenerative disorder of the brain [1]. To date
almost 48 million elderly people are affected by AD, and the number is estimated to
spread 131.5 million by 2050 with an unparalleled growth [2]. Although several
factors including low levels of acetylcholine (ACh), the formation of β-amyloid (Aβ)
deposits, oxidative stress and dyshomeostasis of biometals have been demonstrated to
play significant roles in the pathogenesis of AD, the exact etiology is still not fully
understood [3-7]. The current therapeutic options against AD are composed by one
N-methyl-D-aspartate (NMDA) receptor antagonist and three acetylcholinesterase
(AChE) inhibitors, namely memantine (NMDA), donepezil, rivastigmine, and
galantamine (AChE) [8]. Nevertheless, these marketed drugs modestly alleviate the
symptoms but cannot cure brain damage or stop neuronal degeneration.
The primary approach of AD therapy is mainly based on cholinergic
hypothesis[3],which suggests that a decline of ACh levels in specific brain regions
leads to cognitive and memory deficits, and sustaining or recovering cholinergic
function can alleviate these symptoms. Furthermore, studies have illustrated that
AChE interacts with Aβ through the peripheral anionic site (PAS) promoting the
formation of steady AChE-Aβ complexes, which are more toxic than single Aβ
peptides [10]. However, due to the complex nature of AD, these AChE inhibitors
drugs can only reverse the symptoms for a short period of time instead of halting or
curing the neurodegeneration [11]. Thus, a more appropriate approach termed the
Multi-Target Directed Ligands (MTDLs) strategy has been proposed to face this
disease [12-14]. MTDLs mean a single compound that can simultaneously modulate
different targets involved in the neurodegenerative AD cascade [15-16]. One of the
most widely adopted approaches for designing MTDLs is to modify a ChEI and
render it exert other biological properties useful for treating AD.
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In addition to the ChEs, monoamine oxidases (MAOs) have also received
increasing attention in recent years due to their roles in the treatment of AD [17].
MAOs are flavin adenine dinucleotide (FAD) containing enzymes, which exist as two
different isoforms: MAO-A and MAO-B [18]. They were located in the mitochondrial
outer membrane of glial, neuronal and other cells, and have been classified based on
inhibitor sensitivity and substrate specificity [19-20]. MAO-A is involved in
psychiatric conditions and depression, whereas MAO-B accelerates the oxidative
deamination of neurotransmitters, resulting in enhanced production of free radicals
thus causing oxidative stress [21]. MAO-B increases with age and its activity is found
elevated in AD patients, leading to an enhanced metabolism of dopamine and to the
production of large amounts of hydrogen peroxide, which ultimately give rise to
neuronal damage [22]. Inhibition of MAO-B can improve the symptoms of AD, and
several selective MAO-B inhibitors like selegiline and rasagiline have been
demonstrated to retard the further neurodegeneration in AD[23,24]. Therefore,
selective MAO-B inhibitors are useful in the treatment of neurodegenerative disorders,
such as AD.
In recent years, many MTDLs have been designed and synthesized by combining
the ChEI and MAO-B inhibitor into one molecule [25-27]. Because studies suggest
that simultaneous inhibition of MAO-B and ChE can not only improve the level of
ACh and reduce oxidative stress in brain but also can decrease β-amyloid deposition,
another hallmark in AD pathogenesis, which will be more effective against AD [28].
Among all these MTDLs, TV2236 (Ladostigil) has been approved for phase IIb
clinical trials, which prompt us to search new multi-target compounds with ChE, Aβ
aggregation and MAO-B inhibitory activity.
Very recently, we have reported a series of coumarin hybrids as multi-target
compounds for treatment of AD (Fig. 1) [29,30]. Among these compounds, the
coumarin moiety was chosen to inhibit MAOs: compound T1 displayed potent
inhibitory activity toward AChE and BuChE, and clearly selective inhibition for
MAO-B. In order to continue our work to discovery new multi-target compounds with
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both ChE and MAO-B inhibitory activity and to optimize the coumarin hybrids. In
this study, we focused on the structure of benzyl pyridinium salts (Fig. 1), which may
represent a privileged scaffold that can be used to develop new acetylcholinesterase
inhibitors [31,32]. And docking studies have shown that N-benzyl pyridinium moiety
interacted with the CAS of AChE, and the heterocyclic interacting with the PAS of
AChE might contribute to encouraging stacking [33]. The design strategy of new
compounds is shown in Figure 1: the N-benzyl pyridinium moiety, like tacrine, could
inhibit the ChEs through binding to the catalytic anionic site (CAS) of ChEs, and the
coumarin scaffold was used for MAO-B inhibition. Besides, due to the aromatic
character of the coumarin, it might have potential interaction with the PAS of AChE.
All designed compounds were synthesized and evaluated for their ability to inhibit
ChEs, Aβ and MAOs. The kinetic and molecular modeling studies were carried out to
investigate the interaction mechanism of selected compounds with ChE and MAO-B.
In vitro the blood–brain barrier (BBB) permeation and the PC12 neuroblastoma cell
toxicity assays were also performed to test the preliminary drug-like properties of
selected compounds. Herein, we report the design, synthesis and evaluation of a series
of novel coumarin-N-benzyl pyridinium hybrids as multi-target compounds for AD
treatment.
2
2
. Results and discussion
.1. Chemistry
As shown in Scheme 1, the target compounds 7a-l were synthesized. The coumarin
derivatives 3 were obtained under relatively mild condition by condensing resorcinol
with ethyl acetoacetate in the presence of a catalytic amount of concentrated sulfuric
acid in 1,4-dioxane at 60 °C according to the previous reported methods [29].
Reacting the compounds 3 with the 4-(chloromethyl) pyridine hydrochloride in the
presence of K CO as a base afforded the key intermediates 5. Finally, the benzyl
2
3
pyridinium bromide salts 7 were efficiently obtained by refluxing proper benzyl
bromides 6 with the intermediates 5 in dry acetonitrile in good yields.
2
.2. Pharmacological evaluation
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2
.2.1. Cholinesterase inhibitory activity and SAR discussion.
The inhibitory activities of the newly synthesized compounds against both AChE
from electric eel) and BuChE (from equine serum) were examined using the
(
spectrophotometric method of Ellman et al [34]. Donepezil was used as a standard
compound for comparison. The IC values of all test compounds and their selectivity
5
0
index for eeAChE over eqBuChE were summarized in Table 1. The novel coumarin
derivatives showed high activity towards AChE with IC values in the nanomolar
5
0
range, and high selectivity for AChE over BuChE, indicating that these derivatives are
selective AChE inhibitors. Among the coumarin derivatives, compound 7c (IC =
5
0
2
4.9 nM) and 7e (IC = 25.9 nM) showed the most potent inhibitory activity against
50
AChE, which was 1.9-fold higher than that of donepezil (IC = 47.4 nM). By contrast,
5
0
compound 7h exhibited the highest inhibitory activity against BuChE (IC = 0.48
50
µM), resulting 9.5-times more potent than that of donepezil (IC = 4.6 µM). However,
5
0
the ChE inhibitory activities of the coumarin were remarkably low (IC > 100 µM),
5
0
suggesting that the N-benzyl pyridinium moiety was unavoidably required for higher
activity.
To improve the inhibitory activity of the compounds against ChEs, we introduced
substituents with different sizes and electronic properties on the benzyl group of the
N-benzyl pyridinium moiety. The IC value of compounds 7a-l indicated that ChE
5
0
inhibition was affected by the substituents on the benzyl group. Compared with
compound 7a, the introduction of fluorine, methyl, bromine and nitro the benzyl
groups increased AChE inhibitory activity (except compound 7l). For example,
compound 7b-d (IC = 29.5, 25.9 and 29.1 nM for AChE) possessing F on the benzyl
5
0
group were more potent than compound 7a (IC = 380 nM for AChE). Moreover, the
5
0
AChE inhibitory activity was also influenced by the type and position of the
substituents on the benzyl group. Firstly, the substituents with different sizes (fluorine,
bromine and nitro) were introduced to the benzyl groups, which resulted in increasing
the inhibitory activity. And the replacement of the F atoms on benzyl group with
bromine or nitro group slightly decreased the inhibitory activity. Furthermore,
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substituents with varying electronic properties were introduced to the benzyl groups.
Compared with compounds 7h-j, compounds 7e-g bearing a methyl group exhibited a
slight increase in AChE inhibition. These results implied that electron-donating
groups on the benzyl group seemed beneficial to AChE inhibitory activity. Lastly in
compounds 7b-7l, 2- or 3-position substitutions were similar in AChE inhibition.
Interestingly, the substituents on the para-position had the most impact on the AChE
activity. Any group except the fluorine group introduced on the para-position of the
benzyl group remarkably diminished the inhibitory activity. It was well known that
the size of fluorine was similar to that of hydrogen, far less than bromine, methoxy
and nitro group. Thus, it was reasonable to presume that the size of substituent on
para-position, regardless of its electronic properties, was important to the activity of
the target compounds.
Compared with AChE inhibition, all test compounds on BuChE inhibition were
lower with IC50 values in the micromolar range. When substituents with different
sizes and electronic properties on the benzyl group were changed, there was no clear
trend for the eqBuChE inhibitory activities. However, it was worth noting that
2
-position substituents showed more potent BuChE inhibition, such as 7b (IC = 1.64
50
µM), 7e (IC = 1.61 µM) and 7h (IC = 0.48 µM). Among compounds 7b, 7e and 7h,
5
0
50
compound 7h exhibited the highest inhibitory activity against BuChE, and we may
rationally deduce that the large size of a 2-position substituent on the benzyl group of
the N-benzyl pyridinium moiety increase BuChE inhibition.
In next step, the inhibitory activities of the newly synthesized compounds were
examined by using human AChE and BuChE. All tested derivatives gave IC values
50
in the nanomolar range, which were slightly less potent inhibition for hAChE than for
eeAChE and showed the same tendency as that of eeAChE (Table 2). Among the
tested compounds, compound 7e (IC = 35.4 nM) showed the most potent inhibitory
5
0
activity for hAChE, which was similar with that of the reference compound donepizil.
However, the hBuChE inhibition of the tested derivatives was remarkably low (IC >
5
0
1
0 µM), which showed high selectivity for hAChE over hBuChE.
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2
.2.2. In vitro inhibition of human MAOs
All compounds tested for ChEs were also evaluated for their ability to inhibit MAO,
and a nonselective MAO inhibitor, iproniazide, was used as a reference compound
35]. As shown in Table 3, all compounds can effectively inhibit MAO-B at
[
micromolar range, while no compound (except compound 7d) show significantly
inhibitory activity for MAO-A at 10 µM, indicating all compounds are clearly
selective MAO-B inhibitors. Among these compounds, compound 7f (IC = 1.57 µM)
5
0
showed the most potent inhibitor activity for MAO-B with IC value 4.1-fold more
5
0
potent than that of iproniazide (IC = 7.89 µM). Different from AChE inhibition,
5
0
3
-position substituents on the benzyl group of the N-benzyl pyridinium moiety
showed more potent MAO-B inhibition, such as compound 7c, compound 7f and
compound 7i. However the present results did not give a clear structure-activity
relationship (SAR) on MAO inhibition. Because of our previous conclusion in
tacrine-coumarin hybrids that substituents at 3- and/or 4-position of coumarin moiety
could largely influence the inhibitory activity on MAOs, different substituents at 3-
and/or 4-position of coumarin moiety in hybrids was selected for further study.
2
.2.3. Inhibition of self-induced Aβ (1-42) aggregation.
To complete the multi-target biological profile of the target compounds, all
compounds tested for ChEs and MAOs inhibition were also evaluated by a thioflavin
T-based fluorometric assay for their ability to inhibit self-induced Aβ (1-42)
aggregation [36,37]. Curcumin (Cur) was used as a reference, because of its known
inhibitory activity against Aβ (1-42) self-aggregation. The results were gathered in
Table 4 and Figure 2. Compounds 7a-l exhibited moderate to good potencies
(39.3-67.6% at 20 µM) compared with curcumin (65.2% at 20 µM). Notably,
compounds 7j (67.6% at 20 µM) showed the highest potency. Interestingly,
compounds 7h-j with the Br substituent on the benzyl group exhibited high inhibition
against self-induced Aβ (1-42) aggregation with inhibition ranging from 62.7 to 67.6%
at 20 µM. However, the F substituent on the benzyl group (see compounds 7b-d)
decreased the inhibition against self-induced Aβ (1-42) aggregation. In view of all
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above results, compound 7f, presenting the most potent inhibition for hMAO-B and
efficient inhibition for ChEs/Aβ aggregation, was selected as a promising compound
for further study.
2
.2.4. Kinetic study of ChE inhibition.
To explore the AChE inhibitory mechanism of action of the coumarin derivatives,
the potent inhibitor, compound 7f, was selected for a kinetic study using
Lineweaver-Burk plots [38]. Graphical analysis (Figure 3) revealed both increasing
slopes and increasing intercepts with increasing inhibitor concentration. According to
this pattern, compound 7f is a mixed-type inhibitor for AChE and might be able to
bind to the CAS and the PAS of AChE.
In contrast, a different plot for BuChE was obtained (Figure 4), showing increasing
slopes and constant intercepts in different inhibitor concentrations. This suggested a
competitive inhibition, which revealed that compound 7f might compete for the same
binding site as the substrate acetylcholine。
2
.2.5. Reversibility and kinetic study of hMAO-B inhibition.
As we know, MAO-B inhibitors could be classified as irreversible and reversible.
Compound 7f was selected as representative inhibitors since it displayed the most
potent MAO-B inhibitory activity. Therefore, to investigate whether compound 7f is
a/an reversible or irreversible inhibitor of MAO-B, the recovery of enzymatic activity
after dilution of the enzyme-inhibitor complexes was evaluated, and an irreversible
inhibitor, pargyline, was used as a reference compound [29]. MAO-B was
preincubated with compounds 7f at concentrations of 0, 10 and 100 * IC for 30 min
5
0
and then diluted 100-fold to yield concentrations of 0, 0.1 and 1 * IC . For reversible
5
0
inhibition, enzymatic activity is expected to recover to approximately 90% after
dilution to 0.1 * IC , and 50% after dilution to 1 * IC . For an irreversible inhibitor,
5
0
50
enzyme activity is expected not to recover after diluting the enzyme–inhibitor
complex. From the Figure 5, it can be seen that, after the dilution of 7f to 0.1 * IC ,
5
0
the MAO-B catalytic activities are recovered to levels of 86% of the control value
recorded in absence of inhibitor). After dilution to 1 * IC , the MAO-B catalytic
(
5
0
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activities are recovered to levels of 47%. This behavior is consistent with a reversible
interaction of the compound with MAO-B. After similar incubation of MAO-B with
the irreversible inhibitor pargyline at 10 * IC , and dilution of the enzyme–inhibitor
5
0
complex to 0.1 * IC , the MAO-B activities were not fully recovered (less than 10%
5
0
of control).
2
.2.6. Kinetic study of hMAO-B inhibition.
Compound 7f was also used to further investigate the mode of MAO-B inhibition.
The type of MAO-B inhibition was determined by Michaelis-Menten kinetic
experiments [38]. The catalytic rates were measured at five different p-tyramine
concentrations (50-3000 µM), and each plot was constructed at four different
concentrations of compound 7f (0, 0.75, 1.50 and 3.00 µM). The overlaid reciprocal
Lineweaver-Burk plots (Fig. 6) showed that the plots for different concentrations of
compound 7f were linear and intersected at the y-axis. This pattern indicated that
compound 7f was a competitive MAO-B inhibitor, and these results further proved
that the coumarin derivatives were reversible MAO-B inhibitors.
2
2
.3. Docking study [39].
.3.1. Docking study of compounds 7f with AChE.
To further study the dual-site mode of compound 7f for AChE, a molecular docking
study was performed using the software package MOE 2008.10.43. The X-ray crystal
structure of the TcAChE complex with donepezil (hAChE, PDB code 1EVE) was
applied to establish the starting model of AChE. As shown in Figure 7, the N-benzyl
pyridinium moiety of compound 7f was bound to the CAS of AChE, via an aromatic
π-π stacking interaction with the phenyl ring from Trp 84 with the ring-to-ring
distance of 3.68 Å. Moreover, the charged nitrogen of the pyridine ring was bound to
the CAS via a cation–π interaction with Phe 330 with the distance of 4.77 Å. The
coumarin moiety occupied the PAS and interacted with Arg 289 via a hydrogen bond.
All these results obviously indicated that compound 7f could simultaneously bind to
the PAS and CAS of AChE. Moreover, the representative compounds 7a, 7f and
Donepezil were compared by the molecular docking study to explain the differences
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in AChE activity (in Supplementary data).
2
.3.2. Docking study of compounds 7f with MAO-B.
The binding mode of compound 7f with respect to MAO-B was investigated based
on the X-ray crystal structure of the human monoamine oxidase B in complex with
7
-(3-chlorobenzyloxy)-4-(methylamino)methyl-coumarin (PDB code 2V61). And the
3
5
molecular docking study was performed using software package MOE 2008.10.23.
The 3D and 2D pictures of binding were illustrated in Figure 8. As shown in Fig. 8A
and 8B, compound 7f located in the well-known binding pocket of MAO-B [40,41],
with the coumarin ring (A) interacting with FAD via a hydrogen bond at bottom of the
substrate cavity; The N-benzyl pyridinium occupied the entrance cavity which was a
hydrophobic subpocket existing only in the MAO-B isoform and constituted by Leu
1
71, Ile 316, Tyr 326, Ile 199, Trp 199, Pro 104, Leu 164 and Phe 168. Furthermore,
we have explained the selectivity of compounds in relation to MAO-B (in
Supplementary data).
2
.3.3. Docking study of compound 7f with Aβ (1-42) peptide.
To further study the interaction mode with compound 7f for Aβ (1-42), a molecular
docking study was performed using the software package MOE 2008.10.43. The
X-ray crystal structure of the protein Aβ structure (PDB 1IYT) from the Protein Data
Bank was used. As revealed in Figure 9, the coumarin interacted with the Tyr 10 via a
π–π stacking interaction with the ring-to-ring distance of 3.01 Å. These results
indicated that the π–π stacking interaction played crucial roles in the stability of the
7
f/Aβ (1-42) complex.
2
.4. ADMET prediction.
High pharmacological activity and low toxicological effects are not enough for a
compound to become a drug candidate. Pharmacokinetic profiles of the new drug
candidates should be evaluated as early as possible in the drug development process.
In recent years, significant developments in combinatorial chemistry have made the
estimation of absorption, distribution, metabolism and excretion (ADME) relatively
easy [42]. Hence, ADME properties of the synthesized compounds 7a-l were
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calculated by the online Molinspiration property program [43]. The rule requires that
an orally active drug has no more than one violation. With the aim of developing the
central nervous system (CNS) drugs, the ability of compounds to cross the
blood-brain barrier (BBB) is also very important [44]. Calculated log BB for potential
applications in brains and defined by the restrictive terms of Lipinski’s rules.
The theoretical calculations of ADME parameters (molecular weight, log P,
topological polar surface area (tPSA), number of hydrogen donors, number of
hydrogen acceptors, number of rotatable bonds and volume) are presented in Table 5
along with the violations of Lipinski’s rule [45]. According to these data, all the
compounds 7a-l follow Lipinski’s rule by causing no more than one violation and
satisfied possible brain penetration (except compounds 7k and 7l). This suggests that
newly synthesized compounds may have a good pharmacokinetics profile, which
further strengthens their biological importance.
2
.5. In vitro blood-brain barrier permeation assay.
For successful central nervous system (CNS) drugs, the first requirement is
crossing the blood-brain barrier (BBB) to reach brain. The potential ability of these
compounds to penetrate into the brain was further evaluated using a parallel
artificial membrane permeation assay as described by Di et al [46]. Assay
validation was completed by comparing the experimental permeabilities of 9
commercial drugs with previously reported values (Table 6) [46]. A plot of the
experimental data versus bibliographic values gave a good linear correlation, Pe
2
(
exp.) = 1.0121 P (bibl.) - 0.5774 (R = 0.9441). From this equation and taking
e
into account the limit established by Di et al. For blood-brain barrier permeation,
we classified the compounds as follows:
-
6
-1
(
(
(
a) ‘ CNS + ’ (high BBB permeation predicted): P (10 cm s ) > 3.47.
e
-
6
-1
b) ‘ CNS - ’ (low BBB permeation predicted): P (10 cm s ) < 1.45.
e
-
6
-1
c) ‘ CNS +/- ’ (BBB permeation uncertain): P (10 cm s ) from 3.47 to 1.45.
e
Compound 7f with good activities against MAO-B, Aβ (1-42) aggregation and
AChE was selected. The P value of compound 7f was 4.28 ± 0.37 (CNS +). It
e
1
2

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indicated that that compound 7f might be able to penetrate the BBB.
2
.6. PC12 neuroblastoma cell toxicity.
To gain insight into the therapeutic potential of the target compounds, cell viability
was assayed using the neuroblastoma cell line PC12. Treatment of PC12 cells with
compounds 7a-l at 100 µM did not show modified cell viability (table 4), which
suggested that the coumarin derivatives might be suitable multi-target agents for the
treatment of AD. .
3
. Conclusions
In conclusion, a new series of coumarin derivatives were designed, synthesized and
evaluated as multitarget-directed anti-AD agents for their inhibitory activities on
ChEs, MAOs, and Aβ (1-42) aggregation. According to our screening results, most of
these compounds could effectively and selectively inhibit AChE and MAO-B.
Enzyme kinetics and molecular modeling studies revealed that the compounds could
bind simultaneously to the PAS and CAS of AChE, as well as occupying the substrate
binding site in hMAO-B. Compound 7f also exhibited inhibitory percentages similar
to the reference compound curcumin in the inhibition of Aβ (1-42) self-aggregation
assay. Besides, these compounds showed no toxicity to PC12 cells at 100 µM with
brain penetration capacity for CNS activity. Altogether, the multifunctional effects of
these coumarin-N-benzyl pyridinium hybrids qualify them as potential anti-AD agents,
and compound 7f might be a promising drug candidate for further research.
4
4
. Experimental section
.1. Chemistry.
All chemicals (reagent grade) used were purchased from Sino pharm Chemical
Reagent Co., Ltd. (China). Reaction progress was monitored using analytical thin
layer chromatography (TLC) on precoated silica gel GF254 (Qingdao Haiyang
Chemical Plant, Qing-Dao, China) plates and the spots were detected under UV light
(254 nm). Melting point was measured on an XT-4 micromelting point instrument and
uncorrected. IR (KBr-disc) spectra were recorded by Bruker Tensor 27 spectrometer.
1
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1
13
H NMR and C NMR spectra were measured on a BRUKER AVANCE III
spectrometer at 25°C and referenced to TMS. Chemical shifts are reported in ppm (δ)
using the residual solvent line as internal standard. Splitting patterns are designed as s,
singlet; d, doublet; t, triplet; m, multiplet. Mass spectra were obtained on a MS
Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS).
4
.1.1. 7-hydroxy-4-methyl-2H-chromen-2-one (3)
The coumarin derivatives 3 were obtained accordingto the previous reported
methods.
4
.1.2. 4-methyl-7-(pyridin-4-ylmethoxy)-2H-chromen-2-one (5)
A mixture of compound 3 (2.60 mmol) and K CO (10.4 mmol) in
2
3
N,N-dimethylformamide (15 ml) was stirred at r.t for 30 min. A solution of
-(chloromethyl) pyridine hydrochloride (3.90 mmol) in N,N-dimethylformamide (10
4
ml) was then added to the reaction mixture, and the mixture was refluxed further for 3
h. After cooling to the ambient temperature, the reaction mixture was diluted with
water and extracted with ethyl acetate. The combined organic layer was washed with
water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure. The residue was purified by silica gel chromatography using CH Cl /MeOH
2
2
as eluent to obtain target compounds 5 as red solid, Yield 75%; ESI/MS m/z: 268.2
+
[
M+H] .
4
.1.3. General procedure for the preparation of compounds 7
Compound 5 (10 mmol), and appropriate benzyl bromide (12 mmol) in dry
acetonitrile (5 ml) was refluxed for 1-2 h. When the reaction was completed as
indicated by TLC, the mixture was then concentrated under reduced pressure, and 20
ml of diethyl ether was added. On cooling, the precipitates was filtered and washed
with diethyl ether to get the target compounds 7a-l with high yields.
1
-benzyl-4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)pyridin-1-ium bromide
7a)
Yield 87%; red solid; IR νmax/cm (KBr) 1716 (C=O), 1607 (C=C alkene); m.p.
(
-1
+
1
2
05.1-205.4 °C; ESI/MS m/z: 358.2 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.22 (d,
6
1
4

ACCEPTED MANUSCRIPT
J = 6.8 Hz, 2H), 8.20 (d, J = 6.7 Hz, 2H), 7.77 (d, J = 9.5 Hz, 1H), 7.56 (d, J = 1.9 Hz,
1
H), 7.54 (d, J = 1.5 Hz, 1H), 7.48 – 7.44 (m, 3H), 7.14 (dd, J = 7.2, 2.4 Hz, 2H), 6.27
1
3
(
d, J = 1.2 Hz, 1H), 5.87 (s, 2H), 5.64 (s, 2H), 2.42 (s, 3H). C NMR (100 MHz,
DMSO-d ) δ 160.62, 160.43, 157.27, 155.06, 153.81, 145.17, 134.75, 129.87, 129.73,
6
1
29.27, 127.34, 125.93, 114.50, 113.06, 112.21, 102.42, 67.86, 63.40, 18.62.
1
-(2-fluorobenzyl)-4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)pyridin-1-ium
bromide (7b)
Yield 91%; red solid; IR νmax/cm (KBr) 1708 (C=O), 1609 (C=C alkene); m.p.
-1
+
1
2
23.5-225.2 °C; ESI/MS m/z: 376.2 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.14 (d,
6
J = 6.6 Hz, 2H), 8.20 (d, J = 6.6 Hz, 2H), 7.77 (d, J = 9.5 Hz, 1H), 7.62 (t, J = 7.1 Hz,
1
H), 7.54 (m, 1H), 7.34 (m, 2H), 7.14 (dd, J = 7.1, 2.3 Hz, 2H), 6.28 (d, J = 0.9 Hz,
1
3
1
H), 5.97 (s, 2H), 5.66 (s, 2H), 2.42 (s, 3H). C NMR (100 MHz, DMSO-d ) δ
6
1
1
63.50(d, J = 256.06 Hz), 160.61, 160.43, 157.63, 155.07, 153.81, 145.39, 132.65(d,
CF
3
2
J = 9.12 Hz), 131.99, 127.35, 125.89, 125.82, 121.64, 116.51(d, J = 21.08 Hz),
CF
CF
1
1
14.51, 113.07, 112.22, 102.41, 67.87, 58.09, 18.63.
-(3-fluorobenzyl)-4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)pyridin-1-ium
bromide (7c)
Yield 92%; red solid; IR νmax/cm (KBr) 1711 (C=O), 1609 (C=C alkene); m.p.
-1
+
1
1
92.4-193.7 °C; ESI/MS m/z: 376.2 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.22 (d,
6
J = 6.7 Hz, 2H), 8.20 (d, J = 6.6 Hz, 2H), 7.80 – 7.74 (m, 1H), 7.51 (m, 3H), 7.40 (d,
J = 7.7 Hz, 1H), 7.30 (td, J = 8.4, 1.9 Hz, 1H), 7.14 (dd, J = 7.5, 2.2 Hz, 2H), 6.27 (d,
1
3
J = 1.1 Hz, 1H), 5.89 (s, 2H), 5.65 (s, 2H), 2.42 (s, 3H). C NMR (100 MHz,
1
DMSO-d ) δ 162.68(d, J = 243.15 Hz), 160.62, 160.43, 157.41, 155.06, 153.81,
6
CF
3
3
1
1
6
1
45.27, 137.12(d, J = 8.24 Hz), 131.86(d, J = 9.28 Hz), 127.34, 125.96, 125.50,
CF CF
2
2
16.80(d, J = 20.73 Hz), 116.38(d, J = 22.26 Hz), 114.50, 113.07, 112.21, 102.42,
CF
CF
7.88, 62.59, 18.63.
-(4-fluorobenzyl)-4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)pyridin-1-ium
bromide (7d)
1
5

ACCEPTED MANUSCRIPT
-1
Yield 85%; red solid; IR νmax/cm (KBr) 1713 (C=O), 1614 (C=C alkene); m.p.
+
1
2
17.3-218.5 °C; ESI/MS m/z: 376.2 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.24 (d,
6
J = 5.8 Hz, 2H), 8.20 (d, J = 5.7 Hz, 2H), 7.77 (d, J = 8.5 Hz, 1H), 7.71 – 7.62 (m,
H), 7.32 (t, J = 8.5 Hz, 2H), 7.13 (d, J = 7.7 Hz, 2H), 6.27 (s, 1H), 5.89 (s, 2H), 5.65
2
1
3
1
(
s, 2H), 2.42 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 163.07(d, J = 244.06 Hz),
6 CF
3
1
1
6
4
60.62, 160.43, 157.26, 155.05, 153.80, 145.09, 131.98(d, J = 8.19 Hz), 130.96,
CF
2
27.33, 125.92, 116.63(d, J = 22.18 Hz), 114.49, 113.06, 112.20, 102.42, 67.86,
CF
2.47, 18.63.
-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-1-(2-methylbenzyl)pyridin-1-ium
bromide (7e)
Yield 90%; red solid; IR νmax/cm (KBr) 1709 (C=O), 1606 (C=C alkene); m.p.
-1
+
1
1
2
17.4-217.9 °C; ESI/MS m/z: 372.2 [M] ; H NMR (400 MHz, DMSO-d ) δ H NMR
6
(400 MHz, DMSO) δ 9.05 (d, J = 6.8 Hz, 2H), 8.20 (d, J = 6.7 Hz, 2H), 7.80 – 7.73
(m, 1H), 7.33 (tt, J = 19.8, 7.1 Hz, 3H), 7.19 – 7.10 (m, 3H), 6.28 (d, J = 1.1 Hz, 1H),
1
3
5
.93 (s, 2H), 5.67 (s, 2H), 2.42 (s, 3H), 2.31 (s, 3H). C NMR (100 MHz, DMSO-d )
6
δ 160.63, 160.44, 157.36, 155.07, 153.82, 145.33, 137.47, 132.72, 131.44, 129.93,
1
1
4
29.68, 127.35, 127.21, 125.85, 114.51, 113.09, 112.22, 102.42, 67.90, 61.64, 19.24,
8.63.
-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-1-(3-methylbenzyl)pyridin-1-ium
bromide (7f)
Yield 93%; red solid; IR νmax/cm (KBr) 1711 (C=O), 1606 (C=C alkene); m.p.
-1
+
1
2
17.4-217.6 °C; ESI/MS m/z: 372.2 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.21 (d,
6
J = 6.8 Hz, 2H), 8.19 (d, J = 6.7 Hz, 2H), 7.80 – 7.73 (m, 1H), 7.34 (dd, J = 12.1, 6.0
Hz, 3H), 7.25 (d, J = 6.1 Hz, 1H), 7.13 (dd, J = 7.5, 2.2 Hz, 2H), 6.27 (d, J = 1.2 Hz,
1
3
1
H), 5.83 (s, 2H), 5.64 (s, 2H), 2.42 (s, 3H), 2.32 (s, 3H). C NMR (100 MHz,
DMSO-d ) δ 160.62, 160.43, 157.22, 155.06, 153.81, 145.14, 139.11, 134.66, 130.49,
6
1
29.78, 129.64, 127.34, 126.35, 125.91, 114.50, 113.07, 112.22, 102.42, 67.86, 63.41,
1.37, 18.63.
2
1
6

ACCEPTED MANUSCRIPT
4
-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-1-(4-methylbenzyl)pyridin-1-ium
bromide (7g)
Yield 91%; red solid; IR νmax/cm (KBr) 1709 (C=O), 1613 (C=C alkene); m.p.
-1
+
1
2
17.5-218.9 °C; ESI/MS m/z: 372.2 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.18 (d,
6
J = 6.8 Hz, 2H), 8.18 (d, J = 6.7 Hz, 2H), 7.77 (d, J = 9.5 Hz, 1H), 7.45 (d, J = 8.0 Hz,
2
H), 7.27 (d, J = 7.9 Hz, 2H), 7.13 (dd, J = 7.3, 2.3 Hz, 2H), 6.27 (d, J = 1.2 Hz, 1H),
1
3
5
.81 (s, 2H), 5.63 (s, 2H), 2.42 (s, 3H), 2.31 (s, 3H). C NMR (100 MHz, DMSO-d )
6
δ 160.62, 160.43, 157.17, 155.06, 153.81, 145.04, 139.52, 131.76, 130.25, 129.36,
1
1
27.35, 125.89, 114.50, 113.07, 112.22, 102.42, 67.84, 63.26, 21.21, 18.62.
-(2-bromobenzyl)-4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)pyridin-1-ium
bromide (7h)
Yield 89%; red solid; IR νmax/cm (KBr) 1708 (C=O), 1607 (C=C alkene); m.p.
-1
+
1
1
92.7-193.9 °C; ESI/MS m/z: 436.0 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.10 (d,
6
J = 6.0 Hz, 2H), 8.23 (d, J = 5.9 Hz, 2H), 7.79 (d, J = 7.1 Hz, 2H), 7.52 (t, J = 7.0 Hz,
1
2
1
H), 7.47 – 7.38 (m, 2H), 7.15 (d, J = 6.6 Hz, 2H), 6.28 (s, 1H), 5.99 (s, 2H), 5.68 (s,
1
3
H), 2.43 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 160.61, 160.43, 157.76, 155.07,
6
53.82, 145.63, 133.91, 133.51, 132.07, 131.98, 129.19, 127.36, 125.80, 123.96,
1
14.51, 113.09, 112.22, 102.42, 67.90, 63.42, 18.63.
-(3-bromobenzyl)-4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)pyridin-1-ium
bromide (7i)
Yield 92%; red solid; IR νmax/cm (KBr) 1696 (C=O), 1609 (C=C alkene); m.p.
1
-1
+
1
2
07.0-209.3 °C; ESI/MS m/z: 436.0 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.21 (d,
6
J = 6.8 Hz, 2H), 8.20 (d, J = 6.7 Hz, 2H), 7.85 (t, J = 1.7 Hz, 1H), 7.79 – 7.74 (m, 1H),
7
7
.68 – 7.63 (m, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.14 (dd, J =
13
.2, 2.4 Hz, 2H), 6.28 (d, J = 1.2 Hz, 1H), 5.85 (s, 2H), 5.64 (s, 2H), 2.42 (s, 3H). C
NMR (100 MHz, DMSO-d ) δ 160.62, 160.43, 157.42, 155.07, 153.81, 145.25,
6
1
37.11, 132.77, 132.18, 131.85, 128.49, 127.34, 125.98, 122.73, 114.51, 113.08,
112.22, 102.41, 67.88, 62.51, 18.63.
1
7

ACCEPTED MANUSCRIPT
1
-(4-bromobenzyl)-4-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)pyridin-1-ium
bromide (7j)
Yield 87%; red solid; IR νmax/cm (KBr) 1728 (C=O), 1614 (C=C alkene); m.p.
-1
+
1
2
16.5-217.8 °C; ESI/MS m/z: 436.0 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.20 (d,
6
J = 6.7 Hz, 2H), 8.20 (d, J = 6.5 Hz, 2H), 7.77 (d, J = 9.5 Hz, 1H), 7.68 (d, J = 8.4 Hz,
2
2
1
H), 7.52 (d, J = 8.4 Hz, 2H), 7.17 – 7.06 (m, 2H), 6.27 (d, J = 0.9 Hz, 1H), 5.86 (s,
1
3
H), 5.64 (s, 2H), 2.42 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 160.62, 160.42,
6
57.36, 155.06, 153.80, 145.21, 133.99, 132.64, 131.64, 127.34, 125.94, 123.39,
1
14.50, 113.06, 112.22, 102.42, 67.86, 62.57, 18.63.
-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-1-(3-nitrobenzyl)pyridin-1-ium
bromide (7k)
Yield 91%; red solid; IR νmax/cm (KBr) 1717 (C=O), 1612 (C=C alkene); m.p.
4
-1
+
1
2
34.9-236.1 °C; ESI/MS m/z: 430.1 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.27 (d,
6
J = 6.8 Hz, 2H), 8.55 (t, J = 1.9 Hz, 1H), 8.30 (dd, J = 8.3, 1.4 Hz, 1H), 8.22 (d, J =
6
.8 Hz, 2H), 8.04 (d, J = 7.9 Hz, 1H), 7.81 – 7.74 (m, 2H), 7.14 (dd, J = 7.9, 2.1 Hz,
13
2
H), 6.27 (d, J = 1.2 Hz, 1H), 6.02 (s, 2H), 5.65 (s, 2H), 2.42 (s, 3H). C NMR (100
MHz, DMSO-d ) δ 160.62, 160.42, 157.50, 155.06, 153.80, 148.54, 145.36, 136.37,
6
1
1
4
36.24, 131.31, 127.34, 126.02, 124.76, 114.50, 113.08, 112.22, 102.42, 67.87, 62.17,
8.62.
-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-1-(4-nitrobenzyl)pyridin-1-ium
bromide (7l)
Yield 90%; red solid; IR νmax/cm (KBr) 1724 (C=O), 1614 (C=C alkene); m.p.
-1
+
1
2
17.2-219.7 °C; ESI/MS m/z: 430.1 [M] ; H NMR (400 MHz, DMSO-d ) δ 9.22 (d,
6
J = 6.5 Hz, 2H), 8.35 – 8.28 (m, 2H), 8.23 (d, J = 6.7 Hz, 2H), 7.78 (dd, J = 9.2, 2.7
Hz, 3H), 7.14 (dd, J = 7.1, 2.4 Hz, 2H), 6.28 (d, J = 1.2 Hz, 1H), 6.03 (s, 2H), 5.66 (s,
1
3
2
1
1
H), 2.42 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 160.61, 157.66, 155.07, 153.81,
6
48.42, 145.55, 141.65, 130.58, 127.34, 126.06, 124.67, 114.52, 113.09, 112.24,
09.65, 102.42, 67.88, 62.29, 18.63.
1
8

ACCEPTED MANUSCRIPT
4
4
.2. Biological activity
.2.1. In vitro inhibition studies on AChE and BuChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7) from electric eel and
butyrylcholinesterase (BuChE, E.C. 3.1.1.8) from equine serum and human serum, 5,
’-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), S-butyrylthiocholine
5
iodide (BTCI), acetylthiocholine iodide (ATCI), and donepezil hydrochloride were
purchased from Sigma-Aldrich. The capacity of the test compounds 7a-l to inhibit
3
4
AChE and BuChE activities were assessed by Ellman’s method. Test compounds
was dissolved in a minimum volume of DMSO (1%) and was diluted using the buffer
.
2
solution (50 mM Tris-HCl, pH= 8.0, 0.1 M NaCl, 0.02 M MgCl 6H O). In 96-well
2
plates, 160 µL of 1.5 mM DTNB, 50 µL of AChE (0.22 U/mL prepared in 50 mM
Tris-HCl, pH =8.0, 0.1% w/v bovine serum albumin, BSA) or 50 µL of BuChE (0.12
U/mL prepared in 50 mM Tris-HCl, pH=8.0, 0.1% w/v BSA) were incubated with 10
µL of various concentrations of test compounds (0.001-100 µM) at 37 °C for 6 min
followed by the addition of the substrate (30 µL) acetylthiocholine iodide (15 mM) or
S-butyrylthiocholine iodide (15 mM) and the absorbance was measured at different
time intervals (0, 60, 120, and 180 s) at a wavelength of 405 nm. The concentration of
compound producing 50% of enzyme activity inhibition (IC ) was calculated by
5
0
nonlinear regression analysis of the response-concentration (log) curve, using the
Graph-Pad Prism program package (Graph Pad Software; San Diego, CA). Results
are expressed as the mean ± SEM of at least three different experiments performed in
triplicate.
4
.2.2. Kinetic analysis of ChE inhibition
To obtain the mechanism of action 7f, reciprocal plots of 1/velocity versus
/substrate were constructed at different concentrations of the substrate thiocholine
1
iodide (0.05-0.5mM) by using Ellman’s method. Three concentrations of 7f were
selected for the studies: 20, 40 and 80 nM for the kinetic analysis of AChE inhibition.
The plots were assessed by a weighted least-squares analysis that assumed the
variance of velocity (v) to be a constant percentage of v for the entire data set. Slopes
1
9

ACCEPTED MANUSCRIPT
of these reciprocal plots were then plotted against the concentration of 7f in a
weighted analysis. Data analysis was performed with Graph Pad Prism 4.03 software
(Graph Pad Software Inc.).
4
.2.3. In vitro inhibition of Monoamine oxidase
Human MAO-A and MAO-B were purchased from Sigma-Aldrich. The capacity of
the test compounds to inhibit MAO-A and MAO-B activities was assessed by Amplex
3
5
Red MAO assay. Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4)
containing the test drugs at various concentrations and adequate amounts of
recombinant hMAO-A or hMAO-B required and adjusted to obtain in our
experimental conditions the same reaction velocity, i.e., to oxidize (in the control
group) the same concentration of substrate: 165 pmol of p-tyramine/min (hMAO-A:
1
.1 µg protein; specific activity: 150nmol of p-tyramine oxidized to
p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5 µg protein; specific
activity: 22 nmol of p-tyramine transformed/min/mg protein) were incubated for 15
min at 37 °C in a flat-black-bottom 96-well microtest plate placed in a dark
fluorimeter chamber. After this incubation period, the reaction was started by adding
2
00 µM (final concentrations) Amplex Red reagent, 1 U/mL horseradish peroxidase,
and 1 mM p-tyramine. The production of H O and consequently, of resorufin, was
2
2
quantified at 37 °C in a SpectraMax Paradigm (Molecular Devices, Sunnyvale, CA)
muti-mode detection platform reader based on the fluorescence generated (excitation,
5
45 nm; emission, 590 nm). The specific fluorescence emission was calculated after
subtraction of the background activity. The background activity was determined from
wells containing all components except the hMAO isoforms, which were replaced by
a sodium phosphate buffer solution (0.05 M, pH 7.4). The percent inhibition was
calculated by the following expression: (1 − IFi/IFc) × 100 in which IFi and IFc are
the fluorescence intensities obtained for hMAO in the presence and absence of
inhibitors after subtracting the respective background.
4
.2.4. Reversibility and irreversibility study
The reversibility of MAO-B inhibition was determined by dilution assay. At
2
9
2
0

ACCEPTED MANUSCRIPT
concentrations equal to 10 * IC and 100 * IC for hMAO-B inhibition, compound
5
0
50
5
m was incubated with the enzyme (0.75 mg/ml) for 30 min at 37 °C in PBS (0.05 M,
pH 7.4). The parallel control was conducted by replacing the compound with buffer,
and the corresponding amount of DMSO was added as co-solvent to all incubations.
After the incubation period, the complex was diluted 100-fold to obtain final
concentrations of compound 7f equal to 0.1 * IC and 1 * IC . For comparison,
50
50
pargyline were incubated with hMAO-B at concentrations of 10 * IC in similar
5
0
manner and diluted to 0.1 * IC . The residual enzyme catalytic rates were determined
5
0
following the method for the IC determination and all results were expressed as
5
0
mean ± SD.
4
.2.5. Kinetic study of MAO-B inhibition
To obtain of the mechanism of action 7f, reciprocal plots of 1/velocity versus
/substrate were constructed at different concentrations of the substrate p-tyramine
1
(50–3000 µM). Four different concentrations of 7f (0, 0.75, 1.50 and 300 µM) were
selected for the kinetic analysis of MAO-B inhibition. The plots were assessed by a
weighted least-squares analysis that assumed the variance of velocity (v) to be a
constant percentage of v for the entire data set. Slopes of these reciprocal plots were
then plotted against the concentration of 7f in a weighted analysis. Data analysis was
performed with Graph Pad Prism 4.03 software (Graph Pad Software Inc.).
4
.2.6. Molecular modeling studies
Molecular modeling calculations and docking studies were performed using
Molecular Operating Environment (MOE) software version 2008.10 (Chemical
3
9
Computing Group, Montreal, Canada). The X-ray crystallographic structures of
AChE (PDB code 1EVE), hMAO-B (PDB code 2V61) and Aβ (1-42) (PDB code
PDB 1IYT) were obtained from the Protein Data Bank. All water molecules in PDB
files were removed and hydrogen atoms were subsequently added to the protein. The
compound 7f was built using the builder interface of the MOE program and energy
minimized using MMFF94x force field. Then the 7f was docked into the active site of
the protein by the “Triangle Matcher” method, which generated poses by aligning the
2
1

ACCEPTED MANUSCRIPT
ligand triplet of atoms with the triplet of alpha spheres in cavities of tight atomic
packing. The Dock scoring in MOE software was done using ASE scoring function
and Force field was selected as the refinement method. The best 10 poses of
molecules were retained and scored. After docking, the geometry of resulting complex
was studied using the MOE’s pose viewer utility.
4
.2.7. Inhibition of Aβ (1-42) self-induced aggregation
Inhibition of self-induced Aβ (1-42) aggregation was measured using a Thioflavin
3
6,37
T (ThT)-binding assay.
HFIP pretreated Aβ (1-42) samples (Anaspec Inc) were
resolubilized with a 50 mM phosphate buffer (pH 7.4) to give a 25 µM solution. Each
tested compound was firstly prepared in dimethyl sulfoxide (DMSO) at a
concentration of 10 mM and 1 µL of each was added to the well of black, opaque
Corning 96-well plates such that the final solvent concentration was 10%. The final
concentration of each compound was 20 µM and was prepared in independent
triplicates. The solvent control was also included. Then, 9 µL of 25 mM Aβ (1-42)
sample was added to each well and the samples mixed by gentle trapping. Plates were
covered to minimize evaporation and incubated in dark at room temperature for 46-48
h with no agitation.
After the incubation period, 200 µL of 5 µM ThT in 50 mM glycine-NaOH buffer
(
pH 8.0) was added to each well. Fluorescence was measured on a SpectraMax M5
Molecular Devices, Sunnyvale, CA, USA) multi-mode plate reader with excitation
(
and emission wavelengths at 446 nm and 490 nm, respectively. The fluorescence
intensities were compared and the percent inhibition due to the presence of the
inhibitor was calculated by the following formula: 100 - (IF / IF × 100) where IF and
i
o
i
IF are the fluorescence intensities obtained for Aβ (1-42) in the presence and in the
o
absence of inhibitor, respectively.
4
.2.8. Cell culture and measurement of cell viability
The toxicity effect of the tested compounds on the rat pheochromocytoma (PC12)
cells was examined according to previously reported methods. PC 12 cells was
obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China)
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and routinely grown at 37 °C in a humidified incubator with 5% CO in Dulbecco’s
2
modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum, 100
units per mL penicillin, and 100 units per mL of streptomycin. Cells were
sub-cultured in 96-well plates at a seeding density of 5000 cells per well and allowed
to adhere and grow. When cells reached the required confluence, they were placed
into serum-free medium and treated with compound 5l. Twenty-four hours later the
survival of cells was determined by MTT assay. Briefly, after incubation with 20 µL
of MTT at 37 °C for 4 h, living cells containing MTT formazan crystals were
solubilized in 200 µL DMSO. The absorbance of each well was measured using a
microculture plate reader with a test wavelength of 570 nm and a reference
wavelength of 630 nm. Results are expressed as the mean ± SD of three independent
experiments.
4
.2.9. In vitro blood–brain barrier permeation assay
Brain penetration of compounds was evaluated using a parallel artificial membrane
4
6
permeation assay (PAMPA) in a similar manner as described by Di et al.
Commercial drugs were purchased from Sigma and Alfa Aesar. The porcine brain
lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate (PVDF
membrane, pore size 0.45 mm) and the acceptor microplate were both from Millipore.
@
The 96-well UV plate (COSTAR ) was from Corning Incorporated. The acceptor
9
6-well microplate was filled with 300 µL of PBS/EtOH (7 : 3), and the filter
membrane was impregnated with 4 µL of PBL in dodecane (20 mg/mL). Compounds
were dissolved in DMSO at 5 mg/mL and diluted 50-fold in PBS/EtOH (7 : 3) to
achieve a concentration of 100 mg/mL, 200 µL of which was added to the donor wells.
The acceptor filter plate was carefully placed on the donor plate to form a sandwich,
which was left undisturbed for 16 h at 25 °C. After incubation, the donor plate was
carefully removed and the concentration of compound in the acceptor wells was
@
determined using an UV plate reader (Flexsta-tion 3). Every sample was analyzed at
five wavelengths, in four wells, in at least three independent runs, and the results are
given as the mean ± SD. In each experiment, 9 quality control standards of known
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BBB permeability were included to validate the analysis set.
Acknowledgments
This work was supported by the programs of the National Natural Science
Foundation of China [Grant No. 81403175, 81274200]; Youth Talent Sail Plan from
Shanghai Committee of Science and Technology [Grant No. 14YF1411300]; Project
from Shanghai Municipal Commission of Health and Family planning [Grant No.
2
0134Y053]; Project from Health and Family planning Commission of Jiangxi
province [Grant No. 2016A048]; Research Fund of Shanghai University of
Traditional Chinese Medicine [Grant No. 2016YSN21] and Research Fund for the
Docoral Program of Shanghai [Grant No. B201703]
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Figure
Figure 1. Drug design strategy for multi-target-directed ligands.
Figure 2. Inhibition of self-induced Aβ (1-42) aggregation by compounds 7a-l
comparing with that of curcumin.
Figure 3. Kinetic study on the mechanism of EeAChE inhibition by compound 7f.
Figure 4. Kinetic study on the mechanism of eqBuChE inhibition by compound 7f.
Figure 5. Recovery of enzyme activity after dilution.
Figure 6. Kinetic study on the mechanism of human MAO-B inhibition by compound
7
f.
Figure 7. (A) 3D docking model of compound 7f with TcAChE. (B) 2D schematic
diagram of docking model of compound 7f with TcAChE.
Figure 8. (A) 3D docking model of compound 7f with hMAO-B. (B) 2D schematic
diagram of docking model of compound 7f with hMAO-B.
Figure 9. (A) 3D docking model of compound 7l with Aβ (1-42). (B) 2D schematic
diagram of docking model of compound 7l with Aβ (1-42).
Scheme
Scheme 1. Synthesis of coumarin derivatives 7.
Table
Table 1. Inhibition of ChEs activity and selectivity index of compounds 7a-l
.
Table 2. Inhibition of MAO activity and selectivity index of compounds 7a-l
.
Table 3. Inhibition of Aβ (1-42) self-induced aggregation and cell viability on PC12
by target compounds.
Table 4. Some physicochemical parameters of the compounds 7a-l used in prediction
of ADME profiles.
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Table 5. Permeability (Pe × 10 cm s ) in the PAMPA-BBB assay for 9 commercial
drugs, used in the experiment validation.
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