Novel tacrine-coumarin hybrids linked to 1,2,3-triazole as anti-Alzheimer's compounds: In vitro and in vivo biological evaluation and docking study

Article abstract of DOI:10.1016/j.bioorg.2018.10.056

A new series of tacrine-coumarin hybrids linked to 1,2,3-triazole were designed, synthesized, and tested as potent dual binding site cholinesterase inhibitors (ChEIs) for the treatment of Alzheimer's disease (AD). Among them, compound 8e was the most potent anti-AChE derivative (IC₅₀ = 27 nM) and compound 8m displayed the best anti-BChE activity (IC₅₀ = 6 nM) much more active than tacrine and donepezil as the reference drugs. Compound 8e was also evaluated for its BACE1 inhibitory activity and neuroprotectivity against PC12 cells exposed to Aβ_25-35 which indicated low activity. Finally, in vivo studies by Morris water maze task showed that compound 8e significantly reversed scopolamine-induced memory deficit in rats.

Full text of DOI:10.1016/j.bioorg.2018.10.056

Accepted Manuscript
Novel tacrine-coumarin hybrids linked to 1,2,3-triazole as anti-Alzheimer’s
compounds: in vitro and in vivo biological evaluation and docking study
Zahra Najafi, Mohammad Mahdavi, Mina Saeedi, Elahe Karimpour-Razkenari,
Najmeh Edraki, Mohammad Sharifzadeh, Mahnaz Khanavi, Tahmineh
Akbarzadeh
PII:
S0045-2068(18)30887-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.056
YBIOO 2590
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
16 August 2018
10 October 2018
27 October 2018
Please cite this article as: Z. Najafi, M. Mahdavi, M. Saeedi, E. Karimpour-Razkenari, N. Edraki, M. Sharifzadeh,
M. Khanavi, T. Akbarzadeh, Novel tacrine-coumarin hybrids linked to 1,2,3-triazole as anti-Alzheimer’s
compounds: in vitro and in vivo biological evaluation and docking study, Bioorganic Chemistry (2018), doi: https://
doi.org/10.1016/j.bioorg.2018.10.056
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Novel tacrine-coumarin hybrids linked to 1,2,3-triazole as anti-Alzheimer’s compounds:
in vitro and in vivo biological evaluation and docking study
a,b
c
d,e
e
Zahra Najafi , Mohammad Mahdavi , Mina Saeedi , Elahe Karimpour-Razkenari ,
f
g
h
a,e,
Najmeh Edraki , Mohammad Sharifzadeh , Mahnaz Khanavi , Tahmineh Akbarzadeh
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
b
Department of Medicinal Chemistry, School of Pharmacy, Hamadan University of Medical
Sciences, Hamadan, Iran
c
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
d
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
e
Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences,
Tehran, Iran
f
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
g
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran
h
Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
This paper is dedicated to our wonderful teacher in chemistry and medicinal chemistry,
Professor Abbas Shafiee (1937-2016).

Corresponding author. Tel.: +98-21-66122219; fax: +98-21-89785317.
E-mail address: akbarzad@tums.ac.ir (T.Akbarzadeh)
1

Abstract: A new series of tacrine-coumarin hybrids linked to 1,2,3-triazole were designed,
synthesized, and tested as potent dual binding site cholinesterase inhibitors (ChEIs) for the
treatment of Alzheimer’s disease (AD). Among them, compound 8e was the most potent anti-
AChE derivative (IC50 = 27 nM) and compound 8m displayed the best anti-BChE activity
(
IC50 = 6 nM) much more active than tacrine and donepezil as the reference drugs.
Compound 8e was also evaluated for its BACE1 inhibitory activity and neuroprotectivity
against PC12 cells exposed to Aβ25-35 which indicated low activity. Finally, in vivo studies by
Morris water maze task showed that compound 8e significantly reversed scopolamine-
induced memory deficit in rats.
Keywords: Alzheimer’s disease, Tacrine-Coumarins, 1,2,3-Triazole, Docking study, Dual
binding Site, Morris water maze
1
. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most
common form of dementia worldwide [1-2]. The cause of AD is still unclear and has been
described by the several reasons such as genetic factors [3-5], amyloid-β peptides aggregation
[
6], neurofibrillary tangles (NFTs) [7], cholinergic hypofunction, oxidative stress [8], metal
ion dyshomeostasis [9], and inflammation [10], etc. Among them, the cholinergic
hypofunction has been known as one of the significant causes of AD [11]. Acetylcholine
(
ACh) plays an important role in attention, learning, memory and motivation. One of the
main ways to increase ACh level in the brain is inhibition of cholinesterase enzymes (ChEs).
There are two ChEs known as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
that catalyze the hydrolysis of ACh. In the healthy brain, AChE has predominant activity in
terminating ACh-mediated neurotransmission whereas BChE acts as the secondary enzyme
[
12-13]. In patients with AD, a misbalance takes place between AChE and BChE so that the
2

activity of AChE does not change or decreases whereas BChE activity significantly increases.
Therefore, it seems that both enzymes are involved in regulating of the ACh level and
considered as valuable therapeutic targets against AD. As a result, dual inhibition of AChE
and BChE enzymes would be more useful for AD patients in the late stages of disease than
AChE selective inhibitors.
The crystal structural studies of both AChE and BChE enzymes revealed they possess
two binding sites including the catalytic site (CS) and peripheral anionic site (PAS) [14-15].
However, they have shown high binding affinity towards substrates and inhibitors. The main
difference between them is associated with acyl binding site (ABS) which that of BChE is
significantly bigger than of AChE. It seems that stimulatory inhibition of CS and PAS affords
inhibitors that are more potent. In addition, recent evidences suggested that the PAS of AChE
plays key roles in Aβ-aggregation [16-17]. Also, BChE knockout animal model showed
diminished fibrillar Aβ plaque deposition suggesting the lack of BChE reduces deposition of
fibrillar Aβ in AD [18]. Consequently, a great deal of attention has been paid to the
development of dual AChE and BChE inhibitors to increase inhibitory activity via increasing
the ligand-target interaction. According to the literature, tacrine has been found as a specific
inhibitor of the catalytic anionic site (CAS) of CS whereas coumarin displays affinity to the
PAS of ChEs [19-20]. All structures demonstrated in Fig. 1 (A-D) showed the importance of
coumarin and tacrine scaffolds in the development of ChEIs [20-22].
Based on the above mentioned reports and in continuation of our work on the
synthesis and evaluation of anti-Alzheimer’s compounds [23-24], we designed and
synthesized novel hybrid tacrine-coumarin-1,2,3-triazoles which prepared through click
chemistry approach (Scheme 1). All compounds 8a-r were evaluated for their in vitro and in
vivo anti-Alzheimer’s activity.
3

2
. Results and discussion
2
.1. Chemistry
The desired compounds 8a-r were obtained by the reaction sequence illustrated in
Scheme 1. The reaction of anthranilic acid derivative 1a-c and cyclohexanone 2 in refluxing
POCl for 3 h gave 1,2,3,4-tetrahydroacridine derivative 3a-c [25,26]. Compounds 3a-c were
3
treated with propargylamine in phenol at 100 °C for 2 h to afford propargylated acridine
derivatives 4a-c [27].Further reaction series is associated with preparation of desired azide
derivatives 7a-f. For this purpose, coumarin 5a was commercially obtained from Merck and
4
-methyl-7-hydroxycoumarin was prepared by Pechmann reaction via the condensation of
resorcinol and ethylacetoacetate in the presence of concentrated sulfuric acid [28].
Compounds 5a-b were reacted with different dibromoalkanes in the presence of anhydrous
◦
potassium carbonate in acetonitrile at 80 C for 4 h to obtain compounds 6a-f [29-31].
Compounds 7a-f [32-33] were obtained by the reaction of compounds 6a-f with sodium azide
in ethanol. After evaporation of solvent, the residue was used for the next step without
purification. Finally, desired products 8a-r were prepared by click reaction of compounds 4a-
c with azide derivatives 7a-f in H
2
O/t-BuOH (1:1) in the presence of Et
3
N along with a
catalytic amount of CuI at room temperature for 12-24 h [23].
1
13
The structure of all synthesized compounds was confirmed using HNMR and CNMR
spectroscopy. They were in good agreement with those expected chemical shifts and coupling
1
patterns. For example, HNMR spectrum of compound 8e showed eight protons belonging to
four methylene groups of 1,2,3,4-tetrahydroacridine moiety around 1.86, 2.69, and 3.01 ppm
as multiplet and triplet signals. The presence of methyl group and three methylene linker
protons were confirmed by multiplet signal for 2.36-2.41 ppm and triplet signals at 3.98 and
4
4
.54 ppm. Also, methylen group protons connected to NH was depicted as a singlet signal at
.68 ppm. Singlet proton related to proton at 3- position of coumarin was observed at 6.13
4

ppm and accordingly H
.74 ppm as doublet, doublet of doublet, and doublet, respectively with desired coupling
constants. The H5', H7', and H8' of 1,2,3,4-tetrahydroacridine moiety were distinguished at
.89, 7.27, and 7.90 ppm as singlet, doublet, and doublet signal, respectivley. Finally, the
existence of proton belonging to 1,2,3-triazole moiety was observed at 7.26 ppm as a singlet
5
, H
6
, and H
8
of coumarin moeity were observed at 7.47, 6.78, and
6
7
13
signal. Moreover, CNMR spectrum confirmed nine aliphatic, nineteen aromatic, and a
carbonyl group distinct resonances.
2
2
.2. Pharmacology
.2.1. AChE and BChEinhibitory activity
The in vitro AChE and BChE inhibitory activity of all synthesized compounds 8a-r
was evaluated using modified Ellman’s method [34] and compared with tacrine and
donepezil as the reference drugs (Table 1). All data were presented as the mean ± SD of three
independent experiments. Among the synthesized compounds 8a-r, the most potent anti-
AChE and anti-BChE activities were related to compounds 8e (IC50= 0.027 µM) and 8m
(
IC50= 0.006 µM), respectively.
In all designed compounds, 1,2,3,4-tetrahydroacridine and coumarin cores were
connected by methylene groups and 1,2,3-triazole moiety. Synthesized compounds 8a-r can
be divided into three series based on the length of methylene linker: 8a-f, 8g-l, and 8m-r
possessing three, four and five methylene groups, respectively.
In the case of AChEI activity, compound 8e belonging to the first series having Cl at
6
-position of 1,2,3,4-tetrahydroacridine and methyl at 4-position of coumarin exhibited the
best anti-AChE activity with IC50 value of 0.027 µM, more potent than tacrine and donepezil
with IC50 value of 0.048 and 0.039 µM, respectively. Changing the position of Cl
significantly decreased the anti-AChE activity in compound 8f (IC50 = 1.674 µM), however
deletion of Cl in compound 8d led to the decrease of activity in comparison to compound 8f
5

in such a manner that the order of activity was 8e>8d>8f. Considering the inhibitory activity
of compounds 8a, 8b, and 8c in the first series of compounds revealed that the presence of Cl
at 6-position of tetrahydroacridine moiety plays important role as compound 8b showed
AChEI activity with IC50 = 0.056 µM. Also, it was found that the presence of methyl group at
4
-positon of coumarin moiety is remarkable and similar order of activity was observed for
8
b>8a>8c. It was clear that the lack of methyl group in compounds 8a, 8b, and 8c led to the
decrease of activity comparing with their counterparts 8d, 8e, and 8f.
In the second serious of synthesized compounds (8g-l) having linker with four
methylen groups, compounds 8g, 8h, 8j, and 8k were found to be strong toward AChE since
the following IC50s = 0.046, 0.044, 0.066, and 0.068 µM were calculated, respectively. In this
series of compounds, the presence of Cl at 6-position of tetrahydroacridine moiety as well as
lack of Cl significantly affected anti-AChE activity. However, the presence of Cl at 7-
position of tetrahydroacridine moiety deteriorated AChEI activity in compounds 8i and 8l
(
IC50s = 1.829 and 0.614 µM). Also, the presence of methyl played no important role.
Considering our results obtained from the third series of compounds (8m-r)
confirmed similar anti-AChE activity as observed for the first series (8a-f). As expected the
presence of Cl at 6-position of tetrahydroacridine moiety and methyl at 4-position of
coumarin increased AChEI activity since compounds 8m, 8n, and 8o showed IC50s = 0.095,
0
0
.052, and 0.738 µM whereas their counterparts, compounds 8p, 8q, and 8r showed IC50s =
.050, 0.039, and 0.273 µM, respectively.
To sum up, SAR studies showed that anti-AChE activity was completely affected by
the absence and presence of Cl and Me groups as well as the length of methylene linker as
they can lead to different lipophilicity and spatial hindrance. Although there is no a definite
relation between these factor, generally compounds possessing Cl at 6- position of
tetrahydroacridine moiety showed better inhibitory activity comparing with those
6

counterparts in a category. Also, in each category compounds containing Cl at 7- position of
tetrahydroacridine depicted the weakest activity. Another point comes back to the presence of
Me group which has not demonstrated a certain role, however, its efficacy has been
confirmed in the third category of compounds as compounds 8p-r are more potent than 8m-o.
Our results in the case of BChEI activity (Table 1) also can be also discussed in three
categories according to the length of methylene linker. In the first series of compounds (8a-f),
compounds 8c and 8f possessing Cl at 7-position of tetrahydroacridine moiety depicted no
activity (IC50>10) and compound 8a lacking Cl and Me groups showed the best BChEI
activity (IC50 = 0.032 µM). Introduction of Cl into 6-position (compound 8b) and insertion of
Me at 4-position of coumarin (compound 8d) decreased activity with IC50s = 0.328 and 0.450
µM. However, introduction of Cl along with Me into the desired positions (compound 8e)
showed better inhibitory activity (IC50 = 0.104 µM) than compounds 8b and 8d.
In the second category of compounds 8 (8g-l), the best anti-BChE activity was
depicted by compound 8h having Cl at 6-position of tetrahydroacridine moiety, however, its
counterpart 8k having methyl group on the coumarin moiety demonstrated lower activity
(
IC50 = 0.320 µM). Also, compound 8j showed high activity with IC50 of 0.078 µM which
was more active than its counterpart 8g (IC50 = 0.133 µM). Instructive point refers to
compounds 8i and 8l which showed good activity (IC50s= 1.247 and 0.487 µM, respectively)
comparing with their analogs in the first series (8c and 8f) which were inactive toward BChE.
However, the presence of Cl at 6-position of tetrahydroacridine moiety depicted higher
BChEI activity than those compounds possessing Cl at 7-position.
In the third series of compounds (8m-r), compound 8m was the most potent anti-
BChE derivative even the most potent compound in the series 8. However, insertion of Cl at
6
- or 7-position of tetrahydroacridine moiety decreased the inhibitory of activity in
compounds 8n and 8o with IC50s of 0.070 and 1.455 µM, respectively. It should be noted that
7

compounds 8p-r showed lower activity than their counterparts of 8m-o, IC50s = 0.038, 0.123,
and 3.923 µM, respectively. Hence, derivatives lacking methyl group induced better
inhibitory activity than those having that group.
It can be concluded that anti-BChE activity in the first and second groups followed no
regularity. Absence and presence of Cl and Me groups as well as the length of methylene
linker play complex role in the inhibition of BChE and the effect of linker length can not be
considered as a single factor. It is only clear that chlorine-containing compounds at 7-position
of tacrine moiety induced less inhibitory activity than compounds possessing Cl at 6-position
or lacking substituent. However, substituents on the tacrine moiety in the third series of
compounds affected inhibitory activity in the following order: H> 6-Cl> 7-Cl. Also, the
presence of methyl group at 4-position of coumarin usually diminished anti-BChE activity
compared with unsubstituted coumarin.
Finally, the affinity of compounds 8b, 8e, 8k, 8q, and 8r for AChE was considered by
the selectivity index of 5.77, 3.85, 4.70, 2.35, and 14.36, respectively. All compounds had
chlorine atom at 6-position of 1,2,3,4-tetrahydroacridine moiety except compound 8r and
methyl group at 4-position of coumarin structure except compound 8b. Also, compounds 8a,
8
g, and 8m showed affinity to BChE by the selectivity index of 5.5, 2.87, and 15.83,
respectively. It should be noted that these compounds contained unsubstituted 1,2,3,4-
tetrahydroacridine with three, four, and five methylene linkers, respectively. In all series,
compounds bearing 7-Cl substituted 1,2,3,4-tetrahydroacridine ring displayed less inhibitory
activity against BChE.
2
.2.2. Kinetic studies of AChE and BChE inhibition
The most active compounds toward AChE (8e) and BChE (8m) were selected for
kinetic studies of enzymes inhibition using Lineweaver-Burkplots in the presence of
inhibitors (three concentrations) and their absence [24,34]. As can be seen in Fig. 2, graphical
8

analysis of the reciprocal Lineweaver-Burk plots showed both increasing slopes (decreased
max) and intercepts (increased Kmax) at increasing concentration of both compounds 8e and
m (Fig. 2A and 2B, respectively). The reciprocal plots confirmed mixed type of inhibition
V
8
for both AChE and BChE suggesting that compounds 8e and 8m were able to bind both the
CAS and PAS of the AChE and BChE. As seen in Fig. 3A and 3B, the inhibition constants Ki
were calculated for compounds 8e and 8m from the secondary plots of the slope versus the
concentration of inhibitors (0.061 µM and 0.010 µM, respectively).
2
.2.3. BACE1 enzyme inhibitory activity of compound 8e
BACE1(β-site APP-cleaving enzyme 1) is considered to be involved in the
construction of Aβ peptides. Thus, compound 8e was selected to be evaluated against BACE1
inhibition [35]. All experiments were repeated for three times and compared with OM99-2.
Table 2 indicated that compound 8e showed slight inhibitory activity against BACE1 with the
percent inhibition of 28.69 and 13.97 at 50 and 10 µM, respectively.
2
.2.4. Protection of neuronal PC12 cells against Aβ-induced damage
Neuroprotection effect of compound 8e against PC12 cells exposed to Aβ25-35 was
investigated by MTT assay to obtain insight about its neuroprotective properties [35].
Compound 8e with range concentration of 0.5 to 12.5 µM did not fully protect PC12 cells
from neurotoxic effect of Aβ25-35 at concentration of 5 µM.
2
.3. Docking studies
To investigate the possible interaction modes between active sites of ChEs and the
most active compounds, the molecular docking simulations were performed by AutoDock 4.2
and Autodock Vina (AV) 1.1.1 programs [36] with Discovery Studio 4.0 client as shown in
Fig. 4-8. The X-ray crystal structures of bifunctional inhibitor bis(7)-tacrine complexed with
Torpedo californica AChE (PDB Code: 2CKM) and human butyrylcholinesterase in
9

complex with tacrine (PDB Code: 4BDS) were selected to study the molecular modeling [37-
3
8].
The molecular docking studies were carried out for compounds 8e (IC50 = 0.027 µM),
h (IC50 = 0.044 µM), and 8q (IC50 =0.039 µM) as potent AChEIs and compounds 8a (IC50
8
=0.032µM), 8h (IC50 =0.060µM), and 8m (IC50 = 0.006 µM) as potent BChEIs (Fig.4-7).
In the case of AChEI activity, our results showed that the orientation of all three
compounds were completely similar to native ligand of crystallography (bis(7)-tacrine) with
docking score of -13.83, -13.32, and -13.71 ꢀkcal/mol for compounds 8e, 8h, and 8r,
respectively comparing with docking score of -13.79 kcal/mol for native ligand of
crystallography bis(7)-tacrine. Docking scoring showed a good correlation with experimental
results as docking score of the most active compound 8e (IC50 = 0.027 µM) is slightly more
negative than weaker inhibitors 8h (IC50 = 0.044 µM) and 8r (IC50 = 0.039 µM). All three
compounds 8e, 8h, and 8q were long enough to be accommodated in the PAS and CS of the
AChE. As can be seen in Fig.5, the 1,2,3,4-tetrahydoacridine moiety in compound 8e was
placed in the CAS and made a remarkable sandwich π-π stacking with Trp84 and Phe330.
Also, chlorine at 6-position of 1,2,3,4-tetrahydroacridine moiety showed a hydrophobic
interaction with Trp432, Tyr442, Ile439, Met436 and Phe330. The coumarin moiety was
located in the PAS and indicated a sandwich π-π interaction with Trp279 and Tyr70. 1,2,3-
Triazole ring was also located in the mid-gorge of AChE between the CAS and PAS and
created an anion-π interaction with Asp72.
In the case of BChEI activity, compounds 8a (IC50 = 0.032 µM), 8h (IC50 = 0.060
µM), and 8m (IC50 = 0.006 µM) were selected for the molecular docking studies and docking
scores were obtained -10.4, -10.5, and -10.5ꢀkcal/mol, respectively comparing with docking
score of -8.0 kcal/mol for native ligand of crystallography tacrine (IC50 = 0.010 µM). All
compounds were inserted into the active site of BChE and displayed similar interaction mode
10

in the active site of BChE. They demonstrated the same orientation of tacrine as the native
ligand (Fig. 6).
Fig. 7 displayed binding mode of the most potent anti-BChE compound 8m in the
active site cleft. The1,2,3,4-tetrahydroacridine core was located in hydrophobic cavity
interacting with amino acid residues Trp82 and Ala328. It made a remarkable π-π interaction
with the Trp82 of CAS. The coumarin core was fitted into ABS of CS. It displayed π-π and
hydrophobic interaction with Trp231 and Leu286. The 1,2,3-triazole ring was oriented
directly to the PAS and created the anion-π interaction with Asp70. Also, nitrogen of 1,2,3-
triazole ring formed hydrogen bonds with NH of backbone.
2
.4. Morris water maze test (MWM)
To evaluate the effect of compound 8e (the most potent in vitro anti-AChE) on
memory restoration in scopolamine-induced amnesia [39], the Morris water maze (MWM)
test was performed as described in our previous report [24, 40]. Scopolamine is a muscarinic
cholinergic receptor antagonist which is used to induce memory impairments in animal
models. Eleven groups were studied by MWM consisting of a group that received normal
saline 5 ml/kg, a vehicle group that received 20% polyethylene glycol 400 (PEG 400), a
group that received scopolamine 4 mg/kg, groups that received compound 8e with doses of
0
.3, 0.6, 1.25, 2.5, 5, 10 mg/kg, and two groups that received donepezil 1.25 and 2.5 mg/kg
(
positive control group). Each rat was subjected to the trial four times (each 90 sec) per day
for 4 consecutive days in the water maze task to find hidden platform (training days). As
shown in Fig. 8 (A-C), we evaluated results in terms of escape latency time in Fig. 8A (the
time required to find the platform), traveled distance in Fig. 8B (the distance required to find
the platform), and swimming speed in Fig. 8C. On the fifth day of the trial (post training
probe trial test), platform was removed from the pool and memory was assessed in term of
time spent in the target quadrant (Q ) (Fig. 9).
1
11

2
.4.1. Effects of compound 8e administration on escape latency, traveled distance and
swimming speed average for all training days
The escape latency (8A), traveled distance (8B) and swimming speed (8C) for all
groups in the first 4 days of trials has been shown in Fig. 8. The scopolamine injection
produced the significant increase of escape latency (P<0.01) (Fig. 8A) and traveled distance
(
P<0.001) (Fig. 8B) compared with the saline control. As seen in Fig. 8A and 8B, those
groups that received compound 8e plus scopolamine showed significant improvement in
escape latency and traveled distance in comparison to scopolamine administered group. The
groups treated with compound 8e displayed decrease of escape latency including compound
8
e (10 mg/kg) plus scopolamine: P<0.01; compound 8e (5 mg/kg) plus scopolamine:
P<0.001; compound 8e (2.5 mg/kg) plus scopolamine: P<0.01; compound 8e (1.25 mg/kg)
plus scopolamine: P<0.01; compound 8e (0.6 mg/kg) plus scopolamine: P<0.05; compound
8
e (0.3 mg/kg) plus scopolamine: P<0.05 in comparison to the scopolamine group (Fig. 8A).
The same results were obtained for the traveled distance. The administration of compound 8e
led to significantly decrease in the traveled distance in three groups compared to the
scopolamine group (Fig. 8B) (compound 8e (10 mg/kg) plus scopolamine: P<0.001;
compound 8e (5 mg/kg) plus scopolamine: P<0.01; compound 8e (2.5 mg/kg) plus
scopolamine: P<0.001 and compound 8e (1.25 mg/kg) plus scopolamine: P<0.05). However,
the compound 8e did not significantly restore the traveled distance with doses of 0.6 and 0.3
mg/kg. It should be noted that swimming speed did not show significant changes in all the
groups as shown in Fig. 8C.
2
.4.2.Effects of 8e administration on post training probe trial test
The post-training probe trial test was performed after training days on the fifth day.
We left the trained animals in the water for 90 seconds while the platform was removed and
memory was evaluated in term of time spent in the target quadrant (Q1). As seen in Fig. 9,
12

this parameter was significantly decreased for the group that received scopolamine in
comparison to the control group (P<0.001) and significantly increased for the groups that
received compound 8e (compound 8e (10 mg/kg) plus scopolamine: P<0.01; compound 8e (5
mg/kg) plus scopolamine: P<0.001, compound 8e (2.5 mg/kg) plus scopolamine: P<0.01),
compound 8e (1.25 mg/kg) plus scopolamine: P<0.001), compound 8e (0.6 mg/kg) plus
scopolamine: P<0.01) and compound 8e (0.3 mg/kg) plus scopolamine: P<0.05) in
comparison to scopolamine group. Also, donepezil plus scopolamine groups with doses of
2
.5 and 1.25 mg/kg were applied as positive control and compounds 8e created significant
memory improvement similar to donepezil groups.
3
. Conclusion
In conclusion, we reported design and synthesis of a novel series of tacrine-coumarin
hybrids linked to 1,2,3-triazoleas potent anti-ChEs. All synthesized compounds showed
remarkable inhibitory activity toward both AChE and BChE. Among them, compounds 8e
(
IC50= 0.027µM) and 8m (IC50= 0.006µM) displayed the best anti-AChE and anti-BChE
activity, respectively. In vivo evaluation of compound 8e using the Morris Water Maze test
showed valuable results based on induction of memory improvement in scopolamine-induced
impairment. Also, molecular modeling and kinetics studies showed that compounds 8e and
8
m bound to both the CS and PAS of AChE and BChE, respectively. Consequently, our
results indicated that these novel tacrine-coumarin hybrids may be have potential therapeutic
benefits for the future of AD treatment.
4
. Experimental
Melting points were determined on a Kofler hot stage apparatus and are uncorrected.
1
13
H and C NMR spectra were recorded on a Bruker FT-500, using TMS as an internal
13

standard. IR spectra were obtained on a Nicolet Magna FTIR 550 spectrophotometer (KBr
disks). MS were recorded on an Agilent Technology (HP) mass spectrometer operating at an
ionization potential of 70 eV. Elemental analysis was performed on an Elementar
Analysensystem GmbH VarioEL CHNS mode. All chemical were obtained from Merck and
Sigma. 4-Methyl-7-hydroxycoumarin was prepared by Pechmann reaction via the
condensation of resorcinol and ethylacetoacetate in the presence of concentrated sulfuric acid
[
28].
4
4
.1. Chemistry
.1.1. General procedure for the synthesis of compounds3, 4, 6, 7
All compounds 3, 4, 6, and 7 are known and were synthesized according to the
literature [25-33].
4
.1.2. General procedure for the synthesis of compounds 8
A solution of compounds 7a-f (0.06 g, 0.9 mmol), compound 4a-c (1 mmol) and CuI
(
7 mol%) and trimethylamine (0.13 g, 1.3 mmol) in water (4 mL) and tert-butyl alcohol (4
mL) was stirred at room temperature for 12-24 h. Upon completion of the reaction, monitored
by TLC, the mixture was diluted with H O, extracted with CH Cl, and dried over anhydrous
Na SO .After concentration, the resulting residue purified by flash chromatography on silica
gel using petroleum ether /ethyl acetate / methanol (1:8:1) as eluent in 48-68 % yield.
.1.2.1. 7-(3-(4-(((1,2,3,4-Tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-1-
2
3
2
4
4
yl)propoxy)-2H-chromen-2-one (8a)
White solid; yield: 68%, mp: 98-100 °C. IR (KBr): 3438, 3134, 2929, 2859, 1729, 1612,
-
1 1
1
561, 1502, 1408 cm . H NMR (500 MHz, CDCl
8.0 Hz, 1H, H5'), 7.61 (d, J = 9.5 Hz, 1H, H ), 7.61 (d, J = 8.0, 1.0 Hz, 1H, H6'), 7.42 (s,
H, triazole), 7.36-7.33 (m, 2H, H , H7'), 6.76 (dd, J = 8.5, 2.5 Hz, 1H, H ), 6.74 (d, J = 2.5
), 6.24 (d, J = 9.5 Hz, 1H, H ), 4.8 (s, 2H, NHCH ), 4.55 (t, J = 6.0 Hz, 2H, CH ),
3
): 8.02 (d, J = 8.0 Hz, 1H, H8'), 7.94 (d, J
=
4
1
5
6
Hz, 1H, H
8
3
2
2
14

3
2
1
1
.98 (t, J = 6.0 Hz, 2H, CH
2
), 3.05 (t, J = 5.5 Hz, 2H, CH
2
), 2.70 (t, J = 5.5 Hz, 2H, CH
2
),
1
3
.39 (quin, J = 6.0 Hz, 2H, CH
2
), 1.86 (m, 4H, 2×CH
2
). C NMR (125 MHz, CDCl ): 161.4,
3
60.9, 159.1, 157.4, 150.8, 147.1, 145.5, 143.2, 129.1, 128.9, 127.1, 124.4, 122.8, 121.8,
19.8, 116.9, 113.4, 112.9, 112.5, 101.6, 64.7, 47.7, 44.3, 32.8, 29.6, 24.5, 22.7, 22.3. Anal.
Calcd for C28
.1.2.2.
H
27
N
5
O
3
: C, 69.84; H, 5.65; N, 14.54. Found: C, 70.06; H, 5.88; N, 14.39.
4
7-(3-(4-(((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-
triazol-1-yl)propoxy)-2H-chromen-2-one(8b)
White solid, yield: 51%, mp: 100-102 °C. IR (KBr): 3388, 3171, 2943, 2861, 1724, 1609,
-
1 1
1
556, 1507, 1481 cm . H NMR (500 MHz, DMSO-d
d, J = 9.5 Hz, 1H, H ), 7.94 (s, 1H, triazole), 7.71 (s, 1H, H5'), 7.59 (d, J = 8.5 Hz,1H, H
.29 (d, J = 9.0 Hz,1H, H7'), 6.9 (s, 1H, H ), 6.87 (d, J = 8.5 Hz, 1H, H ), 6.27 (d, J = 9.5 Hz,
H, H ), 5.93 (t, J = 7.0 Hz, 1H, NH), 4.64 (d, J = 7.0 Hz, 2H, NHCH ), 4.49 (t, J = 6.0 Hz,
H, CH ), 3.98 (t, J = 6.0 Hz, 2H, CH ), 2.87 (t, J = 5.5 Hz, 2H, CH ), 2.71 (t, J = 5.5 Hz,
H, CH ), 2.22 (quin, J = 6.0 Hz, 2H, CH
): 161.3, 160.1, 159.5, 155.2, 149.8, 147.4, 145.7, 144.1, 132.3, 129.4, 129.3,
26.7, 125.3, 123.5, 122.8, 118.9, 117.2, 112.5, 112.4, 101.2, 65.0, 46.1, 42.9, 33.4, 29.1,
6
): 8.16 (d, J = 9.0 Hz, 1H, H8'), 7.96
(
4
5
),
7
1
2
2
8
6
3
2
2
2
2
1
3
2
2
), 1.76 (m, 4H, 2×CH ). C NMR (125 MHz,
2
DMSO-d
6
1
2
6
4
1
4.7, 22.4, 22.1. Anal. Calcd for C28
5.32; H, 5.21; N, 13.39.
H
26ClN
5
O : C, 65.18; H, 5.08; N, 13.57. Found: C,
3
.1.2.3. 7-(3-(4-(((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-
-yl)propoxy)-2H-chromen-2-one (8c)
White solid; yield: 59%, mp: 100-102 °C. IR (KBr): 3414, 3133, 2935, 2870, 1728, 1620,
-
1 1
1
555, 1503, 1468 cm . H NMR (500 MHz, CD
9.0 Hz, 1H, H5'), 7.60 (d, J = 9.5 Hz,1H, H ), 7.47 (d, J = 9.0, 2.0 Hz, 1H, H6'), 7.34 (d, J =
.5 Hz, 1H, H ), 7.31 (s, 1H, triazole), 6.78-6.75 (m, 2H, H , H ), 6.24 (d, J = 9.5 Hz, 1H,
), 4.66 (d, J = 7.0 Hz, 2H, NHCH ), 4.55 (t, J = 6.0 Hz, 2H, CH ), 3.99 (t, J = 6.0 Hz, 2H,
), 2.71 (t, J = 6.0 Hz, 2H, CH ), 2.40 (quin, J = 6.0 Hz, 2H,
). C NMR (125 MHz, CDCl ): 161.4, 160.8, 159.3, 155.8, 149.1,
3
Cl): 7.93 (d, J = 2.0 Hz, 1H, H8'), 7.84 (d, J
=
4
8
5
6
8
H
3
2
2
CH
2
2
), 3.01 (t, J = 6.0 Hz, 2H, CH
2
2
13
CH
), 1.86 (m, 4H, 2×CH
2
3
1
45.8, 145.6, 143.1, 130.6, 129.8, 129.1, 128.9, 122.9, 121.8, 121.5, 119.1, 113.5, 113.0,
15

1
6
4
12.4, 101.6, 64.6, 46.9, 44.3, 34.0, 29.6, 24.6, 22.8, 22.6. Anal. Calcd for C28
5.18; H, 5.08; N, 13.57. Found: C, 64.97; H, 4.92; N, 13.41.
H
26ClN
5
O : C,
3
.1.2.4. 4-Methyl-7-(3-(4-(((1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-1-
yl)propoxy)-2H-chromen-2-one (8d)
White solid; yield: 48%, mp: 98-100 °C.IR (KBr): 3411, 3134, 2932, 2855, 1708, 1617,
-
1 1
1
578, 1511, 1431 cm . H NMR (500 MHz, DMSO-d
6
): 8.12 (d, J = 8.5 Hz, 1H, H8'), 7.93
), 7.50 (t, J = 8.5,
), 6.89 (dd, J = 8.5,
), 5.77 (t, J = 7.0 Hz, 1H, NH), 4.60 (d, J = 7.0 Hz, 2H,
), 4.48 (t, J = 6.0 Hz, 2H, CH ), 3.98 (t, J = 6.0 Hz, 2H, CH ), 2.88 (t, J = 5.5 Hz, 2H,
), 2.72 (t, J = 5.5 Hz, 2H, CH ), 2.38 (s, 2H, CH ), 2.22 (quin, J = 6.0 Hz, 2H, CH ), 1.77
). C NMR (125 MHz, CDCl ): 161.2, 160.1, 158.0, 155.6, 152.3, 150.1,
47.2, 145.8, 128.6, 128.3, 125.7, 124.3, 122.6, 121.6, 120.3, 118.2, 114.0, 112.3, 112.1,
(
s, 1H, triazole), 7.70 (d, J = 8.5 Hz, 1H, H5'), 7.65 (d, J = 8.5 Hz, 1H, H
5
1
2
.0 Hz, 1H, H6'), 7.31 (t, J = 8.5 Hz, 1H, H7'), 6.91 (d, J = 2.5 Hz, 1H, H
8
.5 Hz, 1H, H ), 6.20 (s, 1H, H
6
3
NHCH
CH
m, 4H, 2×CH
2
2
2
2
2
3
2
13
(
2
3
1
1
7
4
01.6, 64.6, 46.9, 44.2, 33.6, 29.6, 24.7, 22.8, 22.6, 18.5. Anal. Calcd for C29
0.28; H, 5.90; N, 14.13. Found: C, 70.41; H, 6.08; N, 14.21.
H
29
N
5
O : C,
3
.1.2.5. 7-(3-(4-(((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-
1
-yl)propoxy)-4-methyl-2H-chromen-2-one (8e)
White solid; yield: 68%, mp 125-127 °C. IR (KBr): 3401, 3151, 2929, 2862, 1724, 1613,
-
1 1
1
7
8
4
2
559, 1488 cm . H NMR (500 MHz, CDCl
.47 (d, J = 8.5 Hz, 1H, H ), 7.27 (d, J = 9.0 Hz, 1H, H7'), 7.26 (s, 1H, triazole), 6.78 (dd, J =
.5, 2.0 Hz, 1H, H ), 6.74 (d, J = 2.0 Hz, 1H, H
.54 (t, J = 6.0 Hz, 2H, CH ), 3.98 (t, J = 6.0 Hz, 2H, CH
.69 (t, J = 5.5 Hz, 2H, CH ), 2.41-2.36 (m, 5H, CH , CH
125 MHz, CDCl
3
): 7.90 (d, J = 9.0 Hz, 1H, H8'), 7.89 (s, 1H, H5'),
5
6
8
), 6.13 (s, 1H, H
3
), 4.68 (s, 1H, NHCH
2
2
),
),
2
2
), 3.01 (t, J = 5.5 Hz, 2H, CH
1
3
2
3
2
), 1.86 (m, 4H, 2×CH ). C NMR
2
(
3
): 161.2, 160.9, 160.1, 157.1, 152.2, 149.9, 147.9, 145.6, 134.1, 127.7,
25.7, 124.9, 124.2, 121.5, 119.0, 118.2, 114.0, 112.3, 112.1, 101.6, 64.6, 46.9, 44.3, 34.0,
9.6, 24.6, 22.8, 22.6, 18.5. Anal. Calcd for C29 : C, 65.72; H, 5.32; N, 13.21.
1
2
H28ClN
5
O
3
Found: C, 65.58; H, 5.17; N, 13.06.
4
1
.1.2.6. 7-(3-(4-(((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-
-yl)propoxy)-4-methyl-2H-chromen-2-one (8f)
Cream solid; yield: 64%, mp 87-89 °C.IR (KBr): 3424, 3133, 2928, 2859, 1713, 1612, 1561,
-1 1
1
487 cm . H NMR (500 MHz, CDCl ): 7.92 (d, J = 2.0 Hz, 1H, H8'), 7.82 (d, J = 9.0 Hz,
3
16

1
6
H, H5'), 7.47-7.44 (m, 2H, H
.78 (d, J = 2.0 Hz, 1H, H ), 6.10 (s, 1H, H
), 3.99 (t, J = 6.0 Hz, 2H, CH ), 3.00 (t, J = 6.5 Hz, 2H, CH
), 2.41-2.33 (m, 2H, CH , CH
5
, H6'), 7.34 (s, 1H, triazole), 6.78 (dd, J = 9.0 , 2.0 Hz, 1H, H
), 4.65 (s, 2H, NHCH ), 4.56 (t, J = 6.0 Hz, 2H,
), 2.71 (t, J = 6.5 Hz, 2H,
). C NMR (125 MHz, CDCl ):
6
),
8
3
2
CH
2
2
2
2
1
3
CH
3
2
), 1.86 (m, 4H, 2×CH
2
3
1
1
1
61.2, 160.9, 159.3, 155.1, 152.2, 149.1, 145.8, 145.6, 130.6, 129.6, 129.0, 125.6, 121.7,
21.6, 121.5, 119.0, 113.9, 112.2, 112.0, 101.6, 64.6, 46.9, 44.3, 34.0, 29.6, 24.5, 22.7, 22.6,
8.51. Anal. Calcd for C29
H28ClN
5
3
O : C, 65.72; H, 5.32; N, 13.21. Found: C, 65.88; H, 5.50;
N, 13.37.
.1.2.7.
4
7-(4-(4-(((1,2,3,4-Tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-1-
yl)butoxy)-2H-chromen-2-one (8g)
cream solid; yield: 54%, mp 78-80 °C. IR (KBr): 3384, 3128, 2926, 2855, 1727, 1611, 1564,
-
1 1
1
504, 1411 cm . H NMR (500 MHz, CDCl
Hz, 1H, H5'), 7.60 (d, J = 9.5 Hz, 1H, H ), 7.55 (t, J = 8.0 Hz, 1H, H6'), 7.41 (s. 1H, triazole),
.36 (t, J = 8.0 Hz, 1H, H7'), 7.35 (d, J = 8.5 Hz, 1H, H ), 6.79 (dd, J = 8.5, 2.0 Hz, 1H, H ),
.75 (d, J = 2.0 Hz, 1H, H ), 6.23 (d, J = 9.5 Hz, 1H, H ), 4.40 (t, J =
.0 Hz, 2H, CH ), 4.00 (t, J = 7.0 Hz, 2H, CH ), 2.72 (t, J = 6.0
), 2.08 (quin, J = 6.0 Hz, 2H, CH
3
): 8.06 (d, J = 8.0 Hz, 1H, H8'), 7.97 (d, J = 8.0
4
7
6
6
5
6
8
3
), 4.82 (s, 2H, NHCH
2
2
2
2
), 3.06 (t, J = 7.0 Hz, 2H, CH
2
Hz, 2H, CH
2
), 1.86 (m, 4H, 2×CH ), 1.77 (quin, J = 7.0 Hz,
2
1
3
2
1
6
5
4
H, CH
2
). C NMR (125 MHz, CDCl ): 161.8, 160.9, 159.3, 154.5, 150.6, 148.1, 145.3,
3
43.2, 129.1, 128.8, 127.0, 124.8, 124.4, 123.0, 121.5, 119.7, 116.9, 113.3, 112.7, 101.4,
7.5, 49.9, 44.1, 32.8, 27.0, 25.9, 24.6, 22.7, 22.3. Anal. Calcd for C29
.90; N, 14.13. Found: C, 70.07; H, 5.76; N, 14.02.
H
29
N
5
3
O : C, 70.28; H,
.1.2.8. 7-(4-(4-(((6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-1-
yl)butoxy)-2H-chromen-2-one (8h)
Cream solid; yield: 49%, mp 121-124 °C. IR (KBr): 3314, 3147, 2931, 2869, 1730, 1617,
-
1 1
1
553, 1501, 1471 cm . H NMR (500 MHz, CDCl
2.0 Hz, 1H, H5'), 7.59 (d, J = 9.5 Hz, 1H, H ), 7.33 (d, J = 8.5 Hz,1H, H
triazole), 7.28 (dd, J = 9.0, 2 Hz,1H, H7'), 6.77 (d, J = 8.5, 2.0 Hz, 1H, H ), 6.72 (d, J = 8.5
Hz, 1H, H ), 6.21 (d, J = 9.5 Hz, 1H, H ), 4.67 (s, 2H, NHCH ), 4.39 (t, J = 7.0 Hz, 2H,
), 3.98 (t, J = 7.0 Hz, 2H, CH ), 2.99 (t, J = 6.0 Hz, 2H, CH ), 2.69 (t, J = 6.0 Hz, 2H,
), 2.06 (quin, J = 7.0 Hz, 2H, CH ), 1.86 (m, 4H, 2×CH ), 1.74 (quin, J = 7.0 Hz, 2H,
): 161.7, 160.9, 160.0, 155.7, 149.8, 147.9, 145.5, 143.1,
3
): 7.91 (d, J = 9.0 Hz, 1H, H8'), 7.86 (d, J
=
4
5
), 7.29 (s, 1H,
6
8
3
2
CH
CH
CH
2
2
2
2
2
2
2
1
3
). C NMR (125 MHz, CDCl
3
1
33.9, 128.7, 127.7, 124.7, 124.1, 121.1, 119.0, 118.2, 113.3, 113.0, 112.5, 101.3, 67.4, 49.8,
17

4
1
4
4.2, 34.0, 26.9, 25.8, 24.5, 22.7, 22.5. Anal. Calcd for C29
3.21. Found: C, 65.42; H, 5.21; N, 13.39.
H
28ClN
5
O : C, 65.72; H, 5.32; N,
3
.1.2.9. 7-(4-(4-(((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-
1
-yl)butoxy)-2H-chromen-2-one (8i)
Cream solid; yield: 62%, mp 70-72 °C. IR (KBr): 3382, 3141, 2933, 2860, 1728, 1612, 1556,
-
1 1
1
487 cm . H NMR (500 MHz, DMSO-d
5'), 7.90 (s, 1H, H8'), 7.71 (d, J = 9.5 Hz,1H, H
.0 Hz, 1H, H6'), 6.93 (s, 1H, H ), 6.24 (d, J = 8.5, 2.0 Hz, 1H, H
), 6.0 (t, J = 7.0 Hz, 1H, NHCH ), 4.60 (d, J = 7.0 Hz, 2H, NHCH
), 4.02 (t, J = 6.0 Hz, 2H, CH ), 2.89 (t, J = 6.0 Hz, 2H, CH
), 1.89 (quin, J = 6.0 Hz, 2H, CH ), 1.76 (m, 4H, 2×CH ), 1.57 (quin, J = 6.0 Hz, 2H,
): 161.6, 160.1, 158.8, 155.3, 148.9, 145.7, 145.1,
6
): 8.21 (s. 1H, triazole), 7.96 (d, J = 9.0 Hz, 1H,
), 7.60 (d, J = 8.5 Hz, 1H, H ), 7.49 (d, J =
), 6.27 (d, J = 9.5 Hz, 1H,
), 4.37 (t, J = 6.0 Hz,
), 2.76 (t, J = 6.0 Hz,
H
4
5
9
8
6
H
3
2
2
2
H, CH
H, CH
2
2
2
2
2
2
2
1
3
CH
2
). C NMR (125 MHz, DMSO-d
6
1
6
44.1, 130.3, 129.3, 129.2, 128.2, 128.0, 122.6, 121.8, 121.4, 118.2, 112.6, 112.4, 101.1,
7.4, 48.7, 42.6, 33.4, 26.3, 25.2, 25.1, 22.4, 22.1. Anal. Calcd for C29 : C, 65.72;
H28ClN
5
O
3
H, 5.32; N, 13.21. Found: C, 65.88; H, 5.50; N, 13.34.
4
1
.1.2.10. 4-Methyl-7-(4-(4-(((1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-
-yl)butoxy)-2H-chromen-2-one (8j)
Cream solid; yield: 61%, mp 60-62 °C. IR (KBr): 3390, 3139, 2926, 2855, 1716, 1613, 1577,
-
1 1
1
1
419 cm . H NMR (500 MHz, CDCl
H, H5'), 7.47 (t, J = 8.5 Hz, 1H, H6'), 7.39 (d, J = 8.5 Hz, 1H, H
7'), 7.19 (s, 1H, triazole), 6.73 (dd, J = 9.0, 2.5 Hz, 1H, H ), 6.67 (d, J = 2.5 Hz, 1H, H
.03 (s, 1H, H ), 4.63 (s, 2H, NHCH ), 4.30 (t, J = 6.0 Hz, 2H, CH ), 3.91 (t, J = 6.0 Hz, 2H,
), 2.96 (t, J = 6.0 Hz, 2H, CH ), 2.64 (t, J = 6.0 Hz, 2H, CH ), 2.29 (s, 2H, CH ), 1.98
), 1.78 (m, 4H, 2×CH ), 1.98 (quin, J = 6.0 Hz, 2H, CH
): 161.6, 161.0, 158.7, 155.2, 152.3, 149.8, 147.2, 145.8, 128.7,
28.4, 125.5, 124.2, 122.5, 121.1, 120.7, 118.1, 113.7, 112.3, 112.0, 101.4, 64.4, 49.8, 44.2,
3
): 7.91 (d, J = 8.5 Hz, 1H, H8'), 7.84 (d, J = 8.5 Hz,
), 7.29 (t, J = 8.5 Hz, 1H,
),
5
H
6
8
6
3
2
2
CH
2
2
2
3
1
3
(
quin, J = 6.0 Hz, 2H, CH
2
2
2
). C
NMR (125 MHz, CDCl
3
1
3
1
4
3.9, 27.0, 25.8, 24.7, 22.9, 22.7, 18.2. Anal. Calcd for C30
3.74. Found: C, 70.58; H, 6.01; N, 13.60.
H
30
N
5
3
O : C, 70.71; H, 6.13; N,
.1.2.11. 7-(4-(4-(((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-
1
-yl)butoxy)-4-methyl-2H-chromen-2-one (8k)
White solid; yield: 68%, mp 134-136 °C. IR (KBr): 3355, 3129, 2932, 1712, 1611, 1574,
-1
1
1
419 cm . H NMR (500 MHz, DMSO-d ): 8.17 (d, J = 9.0 Hz, 1H, H8'), 7.88 (s, H,
6
18

triazole), 7.70 (d, J = 2.0 Hz, 1H, H5'), 7.66 (d, J = 8.5 Hz, 1H, H
5
), 7.32 (d, J = 8.5 Hz, 1H,
), 5.99 (t, J = 7.0 Hz, 1H, NHCH ), 4.63 (d, J
), 2.87 (t, J
), 1.89 (quin, J = 6.0 Hz,
H
=
=
7'), 6.93-6.91 (m, 2H, H
7.0 Hz, 2H, NHCH ), 4.36 (t, J = 6.0 Hz, 2H, CH
6.0 Hz, 2H, CH ), 2.71 (t, J = 6.0 Hz, 2H, CH
), 1.716 (m, 4H, 2×CH
DMSO-d ): 161.5, 160.2, 159.5, 154.6, 153.2, 149.8, 147.4, 145.6, 132.3, 126.7, 126.3,
25.4, 123.6, 122.6, 119.0, 117.3, 113.1, 112.2, 111.1, 101.2, 64.4, 48.7, 42.8, 33.4, 26.3,
5.2, 24.8, 22.4, 22.1, 18.0. Anal. Calcd for C30 : C, 66.23; H, 5.56; N, 12.58.
6
, H
8
), 6.19 (s, 1H, H
3
2
2
2
), 4.02 (t, J = 6.0 Hz, 2H, CH
2
2
2
), 2.38 (s, 3H, CH
3
1
3
2
H, CH
2
2
), 1.58 (quin, J = 6.0 Hz, 2H, CH ). C NMR (125 MHz,
2
6
1
2
H30ClN
5
O
3
Found: C, 66.39; H, 5.71; N, 12.72.
4
1
.1.2.12. 7-(4-(4-(((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-
-yl)butoxy)-4-methyl-2H-chromen-2-one (8l)
White solid; yield: 67%, mp 122-124 °C.IR (KBr): 3388, 3144, 2930, 2862, 1713, 1610,
-
1 1
1
1
575, 1486, 1424 cm . H NMR (500 MHz, CDCl
H, H5'), 7.48-7.45 (m, 2H, H , H6'), 7.37 (s, 1H, triazole), 6.78 (d, J = 9.0 Hz, 1H, H
), 6.10 (s, 1H, H ), 4.70 (s, 2H, NHCH ), 4.42 (t, J = 6.0 Hz, 2H, CH ), 4.00 (t, J =
.0 Hz, 2H, CH ), 3.02 (t, J = 6.0 Hz, 2H, CH ), 2.71 (t, J = 6.0 Hz, 2H, CH ), 2.36 (s, 3H,
), 2.09 (quin, J = 6.0 Hz, 2H, CH ), 1.86 (m, 4H, 2×CH ), 1.77 (quin, J = 6.0 Hz, 2H,
): 161.6, 161.07, 158.8, 155.2, 149.4, 145.4, 145.2, 145.6,
3
): 7.97 (s, 1H, H8'), 7.86 (d, J = 8.5 Hz,
5
6
), 6.74
(
s, 1H, H
8
3
2
2
6
2
2
2
CH
3
2
2
2
1
3
CH
). C NMR (125 MHz, CDCl
3
1
4
5
4
30.0, 129.8, 129.3, 125.5, 121.9, 121.2, 120.5, 118.7, 113.7, 112.3, 112.0, 101.3, 64.4, 49.9,
4.2, 33.6, 27.0, 25.9, 24.5, 22.6, 22.4, 18.3. Anal. Calcd for C30
.32; N, 13.21. Found: C, 65.88; H, 5.50; N, 13.37.
H
30ClN
5
3
O : C, 65.72; H,
.1.2.13.
7-((5-(4-(((1,2,3,4-Tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-1-
yl)pentyl)oxy)-2H-chromen-2-one (8m)
Brown gum-solid; yield: 64%. IR (KBr): 3391, 3125, 2923, 2840, 1726, 1616, 1556, 1493
-
1 1
cm . H NMR (500 MHz, CDCl
.59 (d, J = 9.5 Hz, 1H, H ), 7.54 (t, J = 8.5 Hz, 1H, H6'), 7.35 (t, J = 8.5 Hz, 1H, H7'), 7.33
d, J = 8.5 Hz, 1H, H ), 7.28 (s, 1H, triazole), 6.77 (d, J = 8.5 Hz, 1H, H ), 6.74 (s, 1H, H ),
.23 (d, J = 9.5 Hz, 1H, H ), 4.71 (s, 2H, NHCH ), 4.32 (t, J = 6.0 Hz, 2H, CH ), 3.96 (t, J =
.0 Hz, 2H, CH ), 3.03 (t, J = 6.0 Hz, 2H, CH ), 2.71 (t, J = 6.0 Hz, 2H, CH ), 1.93-1.79 (m,
H, 4×CH ), 1.44 (quin, J = 6.0 Hz, 2H, CH ): 162.0, 160.9,
58.6, 155.8, 149.9, 147.0, 145.7, 143.2, 128.7, 128.5, 128.3, 124.1, 122.5, 121.0, 120.6,
3
): 7.99 (d, J = 8.5 Hz, 1H, H8'), 7.91 (d, J = 8.5 Hz, 1H, H5'),
7
4
(
5
6
8
6
6
8
1
3
2
2
2
2
2
1
3
2
2
). C NMR (125 MHz, CDCl
3
19

1
17.9, 112.9, 112.7, 112.5, 101.3, 67.9, 50.0, 44.2, 33.8, 29.8, 28.2, 24.6, 22.9, 22.8, 22.6.
Anal. Calcd for C30 : C, 70.71; H, 6.13; N, 13.74. Found: C, 70.57; H, 5.98; N, 13.57.
.1.2.14. 7-((5-(4-(((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-
H
31
N
5
O
3
4
triazol-1-yl)pentyl)oxy)-2H-chromen-2-one (8n)
Brown gum-solid; yield: 54%. IR (KBr): 3348, 3133, 2928, 2862, 1731, 1615, 1541, 1489
-
1 1
cm . H NMR (500 MHz, CDCl
.5 Hz, 1H, H ), 7.32-7.25 (m, 3H, H
), 6.19 (d, J = 9.5 Hz, 1H, H ), 4.68 (s, 2H, NHCH
J = 6.0 Hz, 2H, CH ), 2.99 (s, 2H, CH
3
): 7.92 (d, J = 9.0 Hz, 1H, H8'), 7.86 (s, 1H, H5'), 7.59 (d, J =
, H7', triazole), 6.77 (d, J = 8.5 Hz, 1H, H ), 6.72 (s, 1H,
), 4.34 (t, J = 6.0 Hz, 2H, CH ), 3.96 (t,
), 1.93-1.84 (m, 8H, 4×CH ), 1.45
): 161.9, 160.9, 159.9, 155.7, 149.8,
9
4
5
6
H
8
3
2
2
2
2
), 2.69 (s, 2H, CH
2
2
1
3
(
quin, J = 6.0 Hz, 2H, CH
2
). C NMR (125 MHz, CDCl
3
1
1
47.8, 145.4, 143.2, 133.9, 128.6, 127.6, 124.6, 124.1, 121.0, 118.9, 118.0, 112.9, 112.6,
12.4, 101.2, 67.8, 50.0, 44.2, 33.9, 29.7, 28.1, 24.5, 22.9, 22.7, 22.5. Anal. Calcd for
C
30
H30ClN
5
O
3
: C, 66.23; H, 5.56; N, 12.87. Found: C, 66.11; H, 5.39; N, 12.69.
4
.1.2.15.
7-((5-(4-(((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-
triazol-1-yl)pentyl)oxy)-2H-chromen-2-one (8o)
Brown gum-solid; yield: 60%. IR (KBr): 3359, 3127, 2941, 2852, 1720, 1619, 1503, 1487
-
1 1
cm . H NMR (500 MHz, CDCl
.5 Hz,1H, H ), 7.45 (d, J = 8.5 Hz, 1H, H
6'), 6.77 (d, J = 8.5 Hz, 1H, H ), 6.71 (s, 1H, H
), 4.35 (t, J = 6.0 Hz, 2H, CH ), 3.96 (t, J = 6.0 Hz, 2H, CH
), 1.94 (quin, J = 6.0 Hz, 2H, CH ), 1.84 (m, 8H, 4×CH
): 161.9, 160.9, 159.0, 155.7, 149.1, 145.6, 145.4,
43.2, 130.3, 129.5, 128.9, 128.6, 121.7, 121.3, 121.0, 118.8, 112.8, 112.6, 112.4, 101.2,
3
): 7.96 (s, 1H, H8'), 7.82 (d, J = 8.5 Hz, 1H, H5'), 7.58 (d, J =
), 7.36 (s, 1H, triazole), 7.31 (d, J = 8.5 Hz, 1H,
), 6.19 (d, J = 9.5 Hz, 1H, H ), 4.67 (s, 2H,
), 3.00 (s, 2H, CH ), 2.70
), 1.46 (quin, J = 6.0 Hz,
9
4
5
H
6
8
3
NHCH
2
2
2
2
(
s, 2H, CH
2
2
2
1
3
2
1
6
6
4
1
H, CH
2
). C NMR (125 MHz, CDCl
3
7.8, 49.9, 44.2, 33.8, 29.7, 28.1, 24.5, 22.9, 22.6, 22.4. Anal. Calcd for C30
6.23; H, 5.56; N, 12.87. Found: C, 66.08; H, 5.41; N, 12.71.
H30ClN
5
3
O : C,
.1.2.16. 4-Methyl-7-((5-(4-(((1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-triazol-
-yl)pentyl)oxy)-2H-chromen-2-one (8p)
Brown gum-solid; yield: 63%. IR (KBr): 3391, 3135, 2943, 2840, 1726, 1616, 1514, 1476
-1 1
cm . H NMR (500 MHz, CDCl
3
): 8.02 (d, J = 8.0 Hz, 1H, H8'), 7.92 (d, J = 8.0 Hz, 1H, H5'),
.54 (t, J = 8.0 Hz, 1H, H6'),7.45 (d, J = 9.0 Hz, 1H, H ),7.35 (t, J = 8.0 Hz, 1H, H7'),7.32 (s,
H, triazole), 6.80 (d, J = 9.0 Hz, 1H, H ), 6.74 (s, 1H, H ), 6.03 (s, 1H, H ), 4.74 (s, 2H,
), 4.33 (t, J = 6.0 Hz, 2H, CH ), 3.97 (t, J = 6.0 Hz, 2H, CH ), 3.04 (s, 2H, CH ), 2.71
t, 2H, CH ), 2.36 (s, 2H, CH ), 1.94-1.83 (m,8H, 4×CH ), 1.46 (quin, J = 6.0 Hz, 2H,
7
1
5
6
8
3
NHCH
2
2
2
2
(
2
3
2
20

1
3
CH
2
). C NMR (125 MHz, CDCl
3
): 161.6, 161.1, 158.1, 155.2, 152.4, 150.2, 146.5, 145.6,
1
4
6
4
28.6, 128.0, 125.5, 124.2, 122.7, 121.2, 120.3, 117.5, 113.5, 112.4, 111.9, 101.3, 67.9, 50.0,
4.2, 33.5, 29.8, 28.2, 24.6, 22.9, 22.8, 22.5, 18.4. Anal. Calcd for C31
.35; N, 13.37. Found: C, 71.27; H, 6.50; N, 13.53.
H
33
N
5
3
O : C, 71.11; H,
.1.2.17.
7-((5-(4-(((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-
triazol-1-yl)pentyl)oxy)-4-methyl-2H-chromen-2-one (8q)
Brown gum-solid; yield: 63 %. IR (KBr): 3320, 3139, 2934, 2862, 1731, 1618, 1553, 1493
-
1 1
cm . H NMR (500 MHz, CDCl
.0 Hz, 1H, H ), 7.31 (s, 1H, triazole), 7.26 (d, J = 8.5 Hz, 1H, H7'), 6.79 (d, J = 8.5 Hz, 1H,
), 6.72 (s, 1H, H ), 6.07 (s, 1H, H ), 4.68 (s, 2H, NHCH ), 4.33 (t, J = 6.0 Hz, 2H, CH ),
.96 (s, 2H, CH ), 2.99 (s, 2H, CH ), 2.69 (s, 2H, CH ), 2.35 (s, 3H, CH ), 1.93-1.84 (m, 8H,
×CH ), 1.58 (s, 2H, CH ). C NMR (125 MHz, CDCl ): 161.8, 161.0, 159.9, 155.1, 152.4,
3
): 7.92 (d, J = 8.5 Hz, 1H, H8'), 7.86 (s, 1H, H5'), 7.43 (d, J =
9
5
H
6
8
3
2
2
3
2
1
1
2
2
2
3
1
3
2
2
3
49.9, 147.8, 145.4, 133.9, 127.6, 125.4, 124.7, 124.3, 121.0, 118.9, 118.0, 113.4, 112.3,
11.7, 101.3, 67.8, 50.0, 44.3, 33.9, 29.8, 28.2, 24.5, 22.9, 22.7, 22.8, 18.2. Anal. Calcd for
C
31
H32ClN
5
O
3
: C, 66.72; H, 5.78; N, 12.55. Found: C, 66.58; H, 5.60; N, 12.37.
4
.1.2.18.
7-((5-(4-(((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)methyl)-1H-1,2,3-
triazol-1-yl)pentyl)oxy)-4-methyl-2H-chromen-2-one (8r)
Brown gum-solid; yield: 59%. IR (KBr): 3341, b3129, 2933, 2845, 1724, 1617, 1559, 1491
-
1 1
cm . H NMR (500 MHz, CDCl
m, 3H, H , H6', triazole), 6.78 (dd, J = 8.0, 2.0 Hz, 1H, H
.07 (s, 1H, H ), 4.67 (s, 2H, NHCH ), 4.37 (t, J = 6.0 Hz, 2H, CH
), 3.00 (s, 2H, CH ), 2.70 (s, 2H, CH ), 2.34 (s, 3H, CH ), 1.95 (quin, J = 6.0 Hz, 2H,
), 1.83-1.82 (m, 8H, 4×CH ), 1.47 (quin, J = 6.0 Hz, 2H, CH
): 161.7, 161.0, 158.5, 155.0, 152.3, 149.4, 145.3, 143.6, 129.7, 129.5, 129.1, 125.3,
21.7, 121.1, 120.1, 118.3, 113.3, 112.2, 111.6, 101.2, 67.8, 49.9, 44.0, 33.4, 29.7, 28.1, 24.6,
2.8, 22.6, 22.5, 18.6. C31 : C, 66.72; H, 5.78; N, 12.55. Found: C, 66.56; H, 5.58;
3
): 7.96 (s, 1H, H8'), 7.82 (d, J = 8.5 Hz, 1H, H5'), 7.48-7.31
), 6.74 (d, J = 2.0 Hz, 1H, H ),
), 3.96 (t, J = 6.0 Hz, 2H,
(
5
6
8
6
3
2
2
CH
2
2
2
2
3
13
CH
2
2
). C NMR (125 MHz,
CDCl
3
1
2
H
32ClN
5
O
3
N, 12.39.
.2. AChE and BChE inhibition assay
4
Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyophilized powder, from
electric eel), butylcholinesterase (BChE, E.C. 3.1.1.8, from equine serum), acetylthiocholine
iodide (ATCI), butyrylthiocholine iodide (BTCI) and 5,5-dithiobis-(2-nitrobenzoic acid)
(
DTNB) were purchased from Sigma-Aldrich. Potassium dihydrogen phosphate, dipotassium
21

hydrogen phosphate, potassium hydroxide, and sodium hydrogen carbonate were obtained
from Fluka. The solutions of the title compounds were prepared in a mixture of DMSO (5
mL) and methanol (5 mL) and diluted in 0.1 M KH
2
PO
4
/K
2
HPO buffer (pH 8.0) to obtain
4
final assay concentrations. All experiments were achieved at 25 °C. Four different
concentrations were tested for each compound in triplicate to obtain the range of 20%-80%
inhibition for AChE.
To measure in vitro AChE activity, modified Ellman’s method [34, 41] was
performed using a 96-well plate reader (BioTek ELx808). Each well contained 50 µL
potassium phosphate buffer (KH
2
PO
4
/ K
2
HPO
4
, 0.1 M, pH 8), 25 µL sample dissolved in
5
0% methanol and 50% DMSO and 25µL enzyme (final concentration 0.22 U/mL in buffer).
They were preincubated for 15 min at room temperature, then 125 µL DTNB (3 mM in
buffer) was added. Characterization of the hydrolysis of ATCI catalyzed by AChE was
performed spectrometrically at 405 nm followed by the addition of substrate (ATCI 3 mM in
water). The change in absorbance was measured at 405 nm after 20 min. The IC50 values
were determined graphically from inhibition curves (log inhibitor concentration vs. percent of
inhibition). A control experiment was performed under the same conditions without inhibitor
and the blank contained buffer, DMSO, DTNB, and substrate. The described method was
also used for BChE inhibition assay.
4
.3. Kinetic studies of AChE and BChE inhibition
For estimates of the inhibition model and inhibition constant Ki, reciprocal plots of
/V versus 1/[S] were obtained using different concentrations of the substrate. For this
1
purpose, experiments were performed similar to enzyme inhibition assay [34]. The rate of
enzymatic reaction was obtained with different concentrations of inhibitor and in the absence
of inhibitor. For each experiment, reaction was started by adding substrate and progress
curves were recorded at 405 nm over 2 min. Next, double reciprocal plots (1/v vs. 1/[s]) were
22

made using the slopes of progress curves to obtain the type of inhibition. Slopes of these
reciprocal plots were then plotted against the concentration of compound in a weighted
analysis, and Ki was determined as the intercept on the negative x-axis. All rate
measurements were performed in triplicate and data analysis was performed with Microsoft
Excel 2003.
4
.4. BACE1 enzymatic assay
The BACE1 enzyme inhibition assaywas carried out using a FRET (Forster resonance energy
transfer) kit, from Invitrogen (former Pan Vera corporation, Madison, WI) according to the
manufacturer instructions. BACE1 (purified baculovirus-expressed enzyme) was diluted with
the assay buffer (50 mM sodium acetate, pH 4.5) to make a 3X working solution of 1 U/mL.
The peptide substrate (Rh-EVNLDAEFK-Quencher) was also diluted with the same assay
buffer to provide the 3X stock solution (750 nM). The inhibitor stock solutions in DMSO
were diluted with buffer to provide 3X solution of test compounds at different concentrations.
The 3X solution of BACE1 enzyme (10 mL) and each inhibitor sample (10 mL) were placed
in 96-well plates and gently mixed. The substrate 3X solution (10 mL) was then added to this
mixture in each well to start the reaction at the final reaction volume of 30 mLthe final
concentration of DMSO in each sample and control wells was equal or less than 6%). The
reaction mixtures were incubated at 25 °C for 90 min in the dark and then the reaction was
stopped by adding 10 mL of 2.5 mM sodium acetate. Fluorescence was monitored at 544 nm
(
excitation wave length) and 590 nm (emission wavelength). OM99-2 was used as a reference
inhibitor compound. A multi-well spectrofluorometer instrument (BMG LABTECH, Polar
star, Germany) was used for measurements. IC50 values were calculated with Curve Expert
software version 1.34 for Windows. Each experiment was repeated for three to four times.
All data were presented as mean ± S.E.M.
23

4
.5. Neuroprotection assay against Aβ-induced damage
PC12Cells were grown in monolayer culture on collagen-coated plates at 37 °C in
humidified air containing 5% CO . Two-thirds of the growth medium was changed every 3-4
2
days and the cells were sub-cultured once a week. Evaluation of compound 8e for protecting
neuronal PC12 cells against Aβ25-35 induced damage was examined by the MTT assay [35].
Stock solution of Aβ25-35 (0.5 mM) was prepared in distilled waterand was diluted in media at
5
a concentration of 55 µM. PC12 cells at a density of 5 × 10 cells/ml (100 ml in each well)
were seeded in rat-tail collagen-coated 96-well plates and incubated for 48 h to adhere at 37
°C. Compound 8e at various concentrations was added in triplicate and incubated for 3 h.
Human Aβ25-35 wasthen added at a final concentration of 5 mM. After 24 h, 90 mL of the
medium was taken out and 20 mL of MTT (0.5 mg/ml dissolved in RPMI containing phenol
red) was added and incubated for an additional 2 h at 37 °C. Afterwards, formazan crystals
were solubilized in 200 mL DMSO. The optical density was measured at 570 nm with
background correction at 655 nm using a Bio-Rad microplate reader (Model 680, Bio-Rad).
Each experiment was repeated 3-5 times. The percentage of protection was calculated:
Protection of cells (%) = [(A
0
- A
b
)/(100- A
b
)] * 100. A is the viability% of the cell in the
0
presence of Aβ and compound 8e, Aβ is the viability % of cells in the presence of Aβ
regarded as 0% protection.
4
.6. Molecular docking studies
Molecular docking studies were performed using the Autodock 4.2 program for the
most potent anti-AChE compounds 8e, 8h, and 8q. For this purpose, the crystal structure of
torpedo californica acetylcholinesterase (2CKM) was retrieved from the Brookhaven protein
database (http://www.rcsb.org). Subsequently, the water molecules and the original inhibitors
were deleted from the protein structure. The 3D structure of the compounds was provided
using MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com) and converted to
24

pdbqt coordinate by Autodock Tools (ADT; version 1.5.4). To obtain enzyme. pdbqt, polar
hydrogen atoms were added to amino acid residues, Koullman charges were assigned to all
atoms of the enzymeusing Autodock Tools (ADT; version 1.5.4). All maps were calculated
with 0.375 Ǻ spacing between grid points by Autogrid. The center of the grid box was placed
at the center of donepezil with coordinates x = 3.771, y = 63.616, z = 63.737. The dimensions
of the active site box were set at 60×60×60Å.The docking was carried out with 100 runs
using the Lamarckian genetic algorithm (LGA). Other parameters were accepted as default. A
cluster analysis was performed on the docking results using a root mean square (RMS)
tolerance of 2.0 and the lowest energy conformation of the highest populated cluster was
selected for analysis. Graphic visualizations were done by Discovery Studio 4.0 client
software.
Moreover, docking simulations for the most potent anti-BChE compounds 8a, 8h, and
8
m was carried out by Autodock Vina (AV) 1.1.1 program [36]. The crystal structure of
human butyrylcholinesterase (4BDS) was retrieved from Protein Data Bank. Then, the water
molecules and inhibitor were removed and enzyme. pdbqt was prepared by Autodock Tools
(
ver 1.5.4) with default parameters. The selected compounds and their 3D structure were
generated by MarvineSketch 5.8.3, 2012 and then ligand.pdbqt was provided by Autodock
Tools (ver 1.5.4). The Autodock Vina parameters were set as follow; box size: 60×60×60 Å,
the center of box: x = 135.943, y = 112.748, z = 40.693 (geometrical center of co-crystallized
ligand), the exhaustiveness: 10 and the other parameters were left unchanged. The calculated
geometries were ranked in terms of free energy of binding and the best pose was selected for
further analysis. Molecular visualizations were performed by Discovery Studio 4.0 client
software (Accelrys, Inc.,San Diego, CA).
4
4
.7. Learning and memory evaluation by Morris water maze test (MWM)
.7.1. Drugs
25

All drugs including scopolamine hydrobromide and donepezil hydrochloride were
obtained from Sigma. They were dissolved in saline and the test compound 8e was dissolved
in 20% PEG 400 (Polyethylene glycol) as drug vehicle.
4
.7.2. Animals
Male albino Wistar rats (200-220g) were obtained from the Faculty of Pharmacy,
Tehran University of Medical Sciences. They were randomly selected to experimental
groups. A 12-hrs light/12-hrs dark cycle was kept and provided food and water ad libitum.
The room was maintained at the temperature 25 ± 2 °C. Our experiments were in agreement
with the guidelines of “Institutional Guide for the Care and Use of Laboratory Animals” and
were approved by the Ethical Committee for the Care and Use of Laboratory Animals at
Tehran University of Medical Sciences.
4
.7.3. Morris water maze test
Rats were trained for 4 successive days in a Morris water maze (MWM) apparatus
which was a black circular pool with a diameter of about 136 cm and a depth of 35 cm. The
pool was filled with water and the temperature was kept at 25 ±2 °C. Walls were covered by
different shaped visual cues. A circular-shaped invisible platform made of Plexiglass whose
diameter is 10 cm was submersed 1cm below the water surface in the northwest quadrant of
the pool (target quadrant). In each trial, rats were randomly released into the water at starting
point of a pool quadrant. A camera was installed above the pool and connected to a computer
equipped with Ethovision software (Noldus Information Technology, Wageningen,
Netherlands) to record the swimming pathway and obtain the related data. Rats were allowed
to swim freely maximum for 90 seconds. If rats did not find the platform during this period of
time, they were manually conducted to the platform. Then, they were allowed to stay on the
platform for 20 seconds. On the first 4 day, the rats were given daily sessions of 4 trials lasted
for 90 seconds. At this step, quantitative data was provided in terms of escape latency,
26

traveled distance, swimming speed for first 4 days of training and time spent in the target
quadrant (Q ) on fifth day (probe trial test). It should be noted that the platform in the target
1
quadrant was removed and animals did not receive treatment on test day. However, they were
treated for 4 days of training.
4
.7.4. In vivo Drug Treatment
In this study, eleven groups of rats were investigated. All drugs were administrated by
intraperitoneal injection (IP).The escape latency, traveled distance, swimming speed for first
days of trial (training days) and time spent in target quadranton fifth day of trial (probe trial
test) were evaluated for all groups.
4
4
.7.4.1. Control group.
The control group was given normal saline (5 mg/kg) 30 min prior to testing.
4
.7.4.2. Scopolamine Group.
Scopolamine hydrobromide (4 mg/kg) was administered 30 min before each trial for
first 4 days of trials.
4
.7.4.3. Vehicle Group.
2
0% PEG 400 (5 ml/kg) was administered to rats30 min before subjecting to the first
4
days of trials.
4
.7.4.4. The compound 8e groups (0.3, 0.6, 1.25, 2.5, 5, and 10 mg/kg).
The most potent AChEI activity compound (8e) and scopolamine (4 mg/kg) were
administered to rats 60 min and 30 min before subjecting to the first 4 days of trials,
respectively.
4
.7.4.5.Donepezil group (1.25, 2.5 mg/kg).
Donepezil and scopolamine (4 mg/kg) were administered torats60 min and 30 min
before subjecting to the first 4days of trials, respectively.
.7.5.Statistical analysis
4
27

One-way analysis of variance (ANOVA) followed by Bonferroni and Newman-Keuls
multiple comparison tests were used for the comparison of test groups, first 4 days of trials
(
training days), and post training probe trial test, respectively.
Acknowledgments
This work was supported by grants from the Research Council of Tehran University of
Medical Sciences with project No. 94-01-33-28713.
Conflict of interest
All authors declare no conflict of interest.
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