Novel tacrine-1,2,3-triazole hybrids: In vitro, in vivo biological evaluation and docking study of cholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.11.008

A new series of tacrine-1,2,3-triazole hybrids were designed, synthesized, and evaluated as potent dual cholinesterase inhibitors. Most of synthesized compounds showed good in vitro inhibitory activities toward both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Among them, 7-chloro-N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-tetrahydroacridin-9-amine (5l) was found to be the most potent anti-AChE derivative (IC₅₀= 0.521 μM) and N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-tetrahydroacridin-9-amine (5j) demonstrated the best anti-BChE activity (IC₅₀= 0.055 μM). In vivo studies of compound 5l in Morris water maze task confirmed memory improvement in scopolamine-induced impairment. Also, molecular modeling and kinetic studies showed that compounds 5l and 5j bound simultaneously to the peripheral anionic site (PAS) and catalytic sites (CS) of the AChE and BChE.

Full text of DOI:10.1016/j.ejmech.2016.11.008

Accepted Manuscript
Novel tacrine-1,2,3-triazole hybrids: In vitro, in vivo biological evaluation and docking
study of cholinesterase inhibitors
Zahra Najafi, Mohammad Mahdavi, Mina Saeedi, Elahe Karimpour-Razkenari,
Raymond Asatouri, Fahimeh Vafadarnejad, Farshad Homayouni Moghadam, Mahnaz
Khanavi, Mohammad Sharifzadeh, Tahmineh Akbarzadeh
PII:
S0223-5234(16)30946-1
10.1016/j.ejmech.2016.11.008
EJMECH 9041
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 August 2016
Revised Date: 1 November 2016
Accepted Date: 4 November 2016
Please cite this article as: Z. Najafi, M. Mahdavi, M. Saeedi, E. Karimpour-Razkenari, R. Asatouri, F.
Vafadarnejad, F.H. Moghadam, M. Khanavi, M. Sharifzadeh, T. Akbarzadeh, Novel tacrine-1,2,3-triazole
hybrids: In vitro, in vivo biological evaluation and docking study of cholinesterase inhibitors, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.008.
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ACCEPTED MANUSCRIPT
Novel tacrine-1,2,3-triazole hybrids: in vitro, in vivo biological evaluation and docking
study of cholinesterase inhibitors
Zahra Najafi, Mohammad Mahdavi, Mina Saeedi, Elahe Karimpour-Razkenari, Raymond
Asatouri, Fahimeh Vafadarnejad, Farshad Homayouni Moghadam, Mahnaz Khanavi, Mohammad
Sharifzadeh, Tahmineh Akbarzadeh
A new series of tacrine-1,2,3-triazole hybrids were designed, synthesized, and evaluated as
potent dual cholinesterase inhibitors. Most of synthesized compounds showed good in vitro
inhibitory activities toward both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE).
In Vivo study of the most potent anti-AChE compound 5l in Morris water maze task confirmed
memory improvement in scopolamine-induced impairment
.

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Novel tacrine-1,2,3-triazole hybrids: in vitro, in vivo biological evaluation and docking
study of cholinesterase inhibitors
a
b
d
Zahra Najafi , Mohammad Mahdavi , Mina Saeedi c,d, Elahe Karimpour-Razkenari ,
e
a
f
Raymond Asatouri , Fahimeh Vafadarnejad , Farshad Homayouni Moghadam , Mahnaz
Khanavi g, Mohammad Sharifzadeh h*, Tahmineh Akbarzadeh a,d,
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
b
Drug Design and Development Research Center, Tehran University of Medical Sciences,
Tehran, Iran
c
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
d
Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences,
Tehran, Iran
e Department of Biology, Science and Research Branch Islamic Azad, Tehran, Iran
f Department of Cellular Biotechnology at Cell Science Research Center, Royan Institute for
Biotechnology, ACECR, Isfahan, Iran
g
Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
h
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran
This paper is dedicated to the memory of our unique teacher in Chemistry and
Medicinal Chemistry, Professor Abbas Shafiee.
Corresponding author. Tel.: +98-21-66419413; fax: +98-21-66461178.
E-mail address: akbarzad@tums.ac.ir (T. Akbarzadeh), msharifzadeh@tums.ac.ir (M. Sharifzadeh).
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Abstract
A new series of tacrine-1,2,3-triazole hybrids were designed, synthesized, and evaluated as
potent dual cholinesterase inhibitors. Most of synthesized compounds showed good in vitro
inhibitory activities toward both acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE). Among them, 7-chloro-N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-
1,2,3,4-tetrahydroacridin-9-amine (5l) was found to be the most potent anti-AChE derivative
(IC50 = 0.521 µM) and N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-
tetrahydroacridin-9-amine (5j) demonstrated the best anti-BChE activity (IC50 = 0.055 µM).
In Vivo studies of compound 5l in Morris water maze task confirmed memory improvement
in scopolamine-induced impairment. Also, molecular modeling and kinetic studies showed
that compounds 5l and 5j bound simultaneously to the peripheral anionic site (PAS) and
catalytic sites (CS) of the AChE and BChE.
Keywords: Alzheimer’s disease, Cholinesterase, Docking study, Dual binding, Morris water
maze, Tacrine-1,2,3-Triazole
1. Introduction
Alzheimer’s disease (AD), the most common form of dementia in the elderly is
described by a progressive loss of cognitive abilities, such as memory, language skills,
attention, disorientation, and depression [1]. Although the etiology of AD is unknown, a wide
range of factors seem to play significant roles in the onset and progression of AD. They
usually include plaque deposits in the brain due to the accumulation of the amyloid-beta
peptide (Aβ), cytoskeletal changes owing to the hyperphosphorylation of microtubule-
associated tau protein, oxidative stress, inflammation and decreased cholinergic transmission
and acetylcholine (ACh) level [2,3]. Up to now, the cholinergic hypothesis has been known
as one of the major causes of AD. Cholinesterase (ChE) is an esterase characterized by
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hydrolyzing choline-based esters such as acetylcholine (ACh) which acts as a
neurotransmitter. There are two enzymes known as AChE and BChE that catalyze the
hydrolysis of cholinergic neurotransmitters. In the healthy brain, AChE activity is
predominant whereas supportive role of BChE which acts as a co-regulator of cholinergic
neurotransmission has been recognized [4]. In patients with AD, AChE activity remains
unchanged or declines whereas BChE activity progressively increases and results in
imbalance between AChE and BChE [5]. Therefore, both enzymes are likely to be involved
in the regulation of the ACh level and considered as valuable therapeutic targets to improve
the cholinergic deficit [6]. Accordingly, simultaneous inhibition of AChE and BChE may be
useful in the later stages of the disease and selective AChE/BChE inhibitory activity has
played a significant role [7]. Also, the key role of AChE and BChE in Aβ-aggregation has
been well documented in the literature [8-9]. Hence, AChE and BChE inhibitory drugs have
emerged as efficient symptomatic treatments of AD. To date, four cholinesterase inhibitors
(AChEIs) including tacrine, donepezil, rivastigmine, and galantamine have been approved for
commercial use. However, tacrine was removed from the pharmaceutical market owing to
severe adverse effects associated with hepatotoxicity [10].
AChE possesses two binding sites including the catalytic site (CS) and the peripheral
anionic site (PAS) [11]. AChE can be inhibited by AChE inhibitors (AChEI) through the
interaction of the inhibitor and amino acid residues in the CS (competitive inhibitors) and
PAS (non-competitive inhibitors). However, dual interaction with two binding sites (CS and
PAS) leads to much better inhibitory activity. Also, it has been demonstrated that the
formation of stable Aβ-aggregates are accelerated through interactions with the PAS of AChE
[12]. In this respect, dual binding site AChEIs have been currently recognized as a new
strategy to increase inhibitory activity through the enhancement of number of drug-target
interactions and prevent from Aβ-aggregation. However, several dual-binding inhibitors have
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been synthesized, evaluated, and special attention has been paid to the tacrine-based
derivatives to profit from anti-cholinesterase activity of tacrine and elimination of the
corresponding side effects (A-D) (Fig. 1) [13-18].
In this work, in continuation of work on the synthesis and evaluation of anti-
cholinesterase compounds [19-24], we studied anti-cholinesterase activity of novel tacrine-
1,2,3-triazole hybrids as well as Morris water maze task and docking study of the related
compounds 5a-5o (Scheme 1).
2. Results and discussion
2.1. Chemistry
The route for the synthesis of the desired compounds 5a-5o is illustrated in Scheme 1.
The reaction of anthranilic acid derivatives 1 and cyclohexanone 2 in refluxing POCl3 for 3 h
gave acridine derivatives 3 [25]. Compound 3 was treated with propargylamine in phenol at
100 °C for 2 h to afford propargylated derivatives 4 [25]. Finally, click reaction of
compounds 4 with in situ prepared azide derivatives gave the desired product 5. For this
purpose, a benzyl chloride/bromide derivative reacted with sodium azide in H2O/t-BuOH
(1:1) in the presence of Et₃N and then, compound 4 along with a catalytic amount of CuI
were added to the freshly prepared organic azide derivative and the reaction was continued
for further 24 h [26].
2.2. Pharmacology
2.2.1. AChE and BChE inhibitory activity
The in vitro AChE and BChE inhibitory activity of the all compounds 5a-5o was
evaluated using modified Ellman’s method [19,27] and compared with tacrine and
rivastigmine as the reference drugs (Table 1). All data were presented as mean ± SEM of
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three independent experiments. It should be noted that the anti-AChE and anti-BChE
activities of all synthesized compounds 5a-5o were respectively 3-21 and 2-140 times more
potent than rivastigmine.
As can be seen in Table 1, the best anti-AChE was related to compound 5l possessing
chlorine at 7-position of acridine moiety and 4-methoxyphenyl connected to 1,2,3-triazole
ring (IC50 = 0.521 µM). Varying substituents on the aryl group connected to 1,2,3-triazole
moiety (replacement of methoxy group by methyl, chlorine, fluorine, and hydrogen) led to
the reduction of anti-AChE activity and IC50 values were calculated as 0.768, 1.440, 1.960,
and 2.394 µM for compounds 5i, 5o, 5f, and 5c, respectively (5i > 5o > 5f > 5c). Compound
5k having chlorine at 6-position of acridine ring and 4-methoxyphenyl connected to 1,2,3-
triazole moeity showed less AChEI activity (IC50 = 0.737 µM) compared with compound 5l.
It should be noted that replacement of methoxy group by chlorine, methyl, fluorine, and
hydrogen in the counterparts of compound 5k (compounds 5e, 5h, 5n, and 5b) led to the
reduction of anti-AChE activity with IC50s = 0.983, 1.113, 2.283, and 2.320 µM (5e > 5h >
5n > 5b). Another instructive point comes from compounds 5g, 5d, 5m, and 5a which have
un-substituents acridine moiety. Compound 5g containing 4-methylphenyl connected to
1,2,3-triazole ring showed the highest activity in this group (IC50 = 0.621 µM). Replacement
of methyl group by chlorine, methoxy, fluorine, and hydrogen led to the reduction of
inhibitory activity with IC50s = 1.966, 2.000, 2.382, and 3.301 µM, respectively (5g > 5d > 5j
> 5m> 5a).
It is clear that AChEI activity of the synthesized compounds was completely
influenced by the electronic properties of substituents on the aromatic ring connected to
1,2,3-triazole and acridine moieties. It seems that methoxy and methyl groups on the aryl ring
connected to 1,2,3-triazole induced better anti-AChE activity compared with fluorine,
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chlorine, and hydrogen. Among them, inhibitory activity was in the order of Cl>F>H which
may be related to the hydrophobic property of chlorine.
According to our results, compound 5j depicted the best BChEI activity (IC50 = 0.055
µM) and compound 5m showed very similar inhibitory activity toward BChE with negligible
difference in comparison to compound 5j (IC50 = 0.059 µM). It should be noted that both
compounds 5j and 5m have un-substituents acridine moiety. Compounds 5h and 5g with
IC50s = 0.065 and 0.072 µM, showed high anti-BChE activity and both of them beard 4-
methylphenyl group connected to 1,2,3-triazole ring. Also, compounds 5k, 5d, 5a, 5b, and 5n
showed good inhibitory activity against BChE as IC50s = 0.115, 0.151, 0.180, 0.197, and
0.216 µM, respectively. Compounds 5o and 5i depicted good activity with IC50s = 0.721 and
0.447 µM, however relative good inhibitory activity was obtained by compounds 5l and 5f,
(IC50s = 1.853 and 3.936 µM, respectively). Compounds 5c and 5e showed less anti-BChE
activity compared with other derivatives (IC50 > 5 µM).
It is obvious that the anti-AChE activity does not follow anti-BChE activity as
compound 5l with the most potent AChEI activity displayed moderate BChEI activity (IC50 =
1.853 µM). According to the calculated BChE/AChE selectivity, the best selectivity (3.55) is
related to the most active anti-AChE compound 5l possessing 4-methoxyphenyl group
connected to 1,2,3-triazole and chlorine at 7-position of acridine moiety. Also, derivatives
with no substituents on the acridine ring 5a, 5d, 5g, 5j, and 5m showed the lowest selectivity.
2.2.2. Kinetic studies of AChE and BChE inhibition
To determine the kinetic type of AChE and BChE inhibition, the most active
compounds toward AChE (5l) and BChE (5j) were selected for kinetic studies of inhibition
of enzymes using Lineweaver-Burk secondary plots [19, 27]. The graphical analysis of
steady-state inhibition data for compounds 5l and 5j are shown in Fig. 2A and B,
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respectively. The reciprocal plots confirmed mixed type of inhibition for both AChE and
BChE suggesting that compounds 5l and 5j were able to bind both the CAS and PAS of
AChE and BChE. As can be seen in Fig. 3A and B, the inhibition constants Ki were
calculated for 5l and 5j from the plots of the slope versus the concentration of inhibitors (0.94
µM and 0.121 µM, respectively).
2.2.3. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity
Compounds 5d, 5j and 5l were randomly selected to be evaluated for their 1,1-
diphenyl-2-picrylhydrazyl radical scavenging activity (DPPH) compared with hydroxyanisole
(BHA) as a standard antioxidant [28]. All the tested compounds showed no notable
antioxidant activities.
2.2.4. Neuroprotective effect against H2O2-induced cell death in PC12 neurons
The PC12 cell line is a useful model for assessing neuronal differentiation and other
neurobiochemical and neurobiological studies. Hence, neuroprotective activity of the most
potent compound 5l against AChE was evaluated by in vitro neuroprotective assay against
H2O2-induced damage in PC12 cells. As depicted in Fig. 4, the percent of cell viability was
calculated in comparison to control group. Compound 5l demonstrated remarkable
neuroprotective activity at 50 and 100 µM (cell viability = 73.45 and 74.76 %, respectively
and P <0.001 vs. H2O2 treatment alone). However, compound 5l showed moderate
neuroprotective activity at 10 µM (cell viability = 65.40 % and P <0.05 vs. H2O2 treatment
alone).
2.2.5. Cytotoxic activity
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Cytotoxicity of compounds 5a, 5b, 5j, 5l, 5m and 5n were evaluated against MCF-7,
T-47D, and MDA-MB-231cell lines through MTT assay (Table 2). As it is clear in Table 2,
they showed no significant cytotoxic effect.
2.3. Docking studies
Docking study of the most active anti-AChE (5l) and anti-BChE (5j) compounds were
conducted to identify the possible interactions between that compounds and enzyme active
sites. It was performed using the docking program, AutoDock 4.2 and Autodock Vina (AV)
1.1.1 [29] with Discovery Studio 4.0 client as shown in Fig. 5-8. Our results showed that the
orientation of compound 5l and 5j in the active site of AChE was completely similar to native
ligand of crystallography (bis(5)-tacrine) (Fig. 5). Compound 5l is long enough to
accommodate in the peripheral anionic site (PAS) and the catalytic site (CS) of AChE.
However, methoxy oxygen atom on the aryl group connected to 1,2,3-triazole ring was
located at the same position of tacrine nitrogen atom. As can be seen in Fig. 6, there is a
hydrogen bonding between oxygen of methoxy and indole nitrogen of Trp279. There is also a
π-π interaction between aromatic ring of 4-methoxyphenyl and Tyr334. Moreover, a
remarkable sandwich π-π stacking is observed between acridine moiety and two amino acids
residue (Trp84 and Phe330) of CAS. It is clear that there is a hydrophobic interaction
between chlorine present on the acridine ring and Tyr334, Phe330, and Trp432. Another
hydrophobic interaction is related to the interaction of cyclohexyl moiety of acridine moiety
and Trp84. 1,2,3-Triazole ring located between acridine moiety and aryl group depicted
important hydrogen bonding with Tyr121.
On the other hand, molecular docking of compound 5j in the active site of BChE has
been shown in Fig. 7. Acridine moiety of compound 5j makes π-π stacking interaction with
Trp82 of choline (cation-π) binding site of CS. Also, there is hydrophobic interaction
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between Ala328 with acridine moiety of compound 5j. 1,2,3-triazole linker placed in the
vicinity of Asp70 of PAS and establishes π-anion interaction. Also, the methoxy group of
benzyl moiety orients to Ser72 and makes hydrogen binding interaction.
2.4. Morris water maze test
In order to evaluate the effect of compound 5l (the most potent in vitro anti-AChE) on
memory restoration in scopolamine-induced amnesia, the Morris water maze (MWM) test
was performed using Male Albino-Wistar rats [30-33]. Scopolamine is an anti-cholinergic
agent which is experimentally used to induce memory impairments in animal models. Our
results are shown in Fig. 8 and 9 for seven groups consisting of group that received normal
saline 5 ml/kg, vehicle group that received PEG 400 (20%), group that received scopolamine
4 mg/kg, groups that received compound 5l with doses of 5, 10 and 20 mg/kg and group that
received donepezil 2.5 mg//kg (positive control group). Each animal was subjected to the trial
4 times (each 90 sec) per day in the water maze task for 4 consecutive days to find hidden
platform (training days). The results were evaluated in terms of escape latency time (the time
required to find the platform), traveled distance (the distance required to find the platform),
and swimming speed. 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
(Q1).
2.4.1. Effects of compound 5l administration on escape latency, traveled distance and
swimming speed average for all training days.
Fig. 8 displays escape latency (8A), traveled distance (8B) and swimming speed (8C) for all
groups for the first 4 days of trials. The significant increase of escape latency (P<0.001) (Fig.
8A) and traveled distance (P<0.001) (Fig. 8B) was observed compared with the control group
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produced by scopolamine injection. It is clear that escape latency and traveled distance have
improved in groups that received compound 5l plus scopolamine in comparison to
scopolamine administered group. All compound 5l treated groups displayed decreased escape
latency (for compound 5l (5 mg/kg) plus scopolamine: P<0.05; for compound 5l (10 mg/kg)
plus scopolamine: P<0.01 and compound 5l (20 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 traveled distance was significantly decreased in all groups that
received compound 5l (for compound 5l (5 mg/kg) plus scopolamine: P<0.01; for compound
5l (10 mg/kg) plus scopolamine: P<0.01 and compound 5l (20 mg/kg) plus scopolamine:
P<0.05) in comparison to the scopolamine group (Fig. 8B). It is noticeable that no significant
changes were observed in swimming speed between the groups, as shown in Fig. 8C.
2.4.2. Effects of 5l administration on post training probe trial test
Fig. 9 shows the results related to post-training probe trial test. On the fifth day, we left the
trained animals in the water for 90 seconds while the platform was removed and the time
spent in the target quadrant (Q1) was measured. Our results showed that 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 5l (for
compound 5l (5 mg/kg) plus scopolamine: P<0.01; and for both compound 5l (10 mg/kg)
plus scopolamine and compound 5l (20 mg/kg) plus scopolamine: P<0.001) in comparison to
scopolamine group.
3. Conclusion
In conclusion, we reported design and synthesis of a novel series of tacrine-1,2,3-
triazole hybrids which were screened for their in vitro anticholinesterase activities and in vivo
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evaluation using the Morris water maze AD animal model. In vitro assay revealed that
compounds 5l (IC50= 0.521 µM) and 5j (IC50= 0.055 µM) were the most anti-AChE and anti-
BChE derivatives, respectively. In vivo evaluation of compounds 5l confirmed memory
improvement in scopolamine-induced impairment. Also, molecular modeling and kinetics
studies showed that compounds 5l and 5j bound to the catalytic site (CS) and peripheral
anionic site (PAS) of AChE and BChE, respectively. All results suggest that new dual
binding site AChE inhibitors have a therapeutic potential for the treatment of AD.
4. Experimental
Melting points were determined on a Kofler hot stage apparatus and are uncorrected.
1H and 13C NMR spectra were recorded on a Bruker FT-500, using TMS as an internal
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.
4.1. General procedure for the synthesis of acridine derivatives 3
POCl3 (20 mL) was added to a mixture of anthranilic acid derivative 1a-c (23 mmol) and
cyclohexanone 2 (25 mmol) in an ice bath [25]. Then, the mixture was heated at reflux for 3
h. It was cooled to room temperature and concentrated to give slurry. The residue was diluted
with ethyl acetate, neutralized with aqueous K2CO3, the organic layer was dried over
anhydrous Na
2
SO and concentrated and purified by flash chromatography on silica gel using
4
petroleum ether/ ethyl acetate (9:1) to give 3a-c as yellow solid in 70-80 % yield.
4.2. General procedure for the synthesis of compounds 4
A mixture of acridine deravatives 3a-b (1 mmol), propargylamine (4 mmol) and phenol (0.5
g) were heated at 100 °C under argon for 2 h, and then cooled to room temperature [25]. The
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mixture was diluted with CH3Cl and made basic with 10% KOH solution. The organic layer
was washed with water and brine, and then dried over anhydrous Na
2
SO . After
4
concentration, the resulting residue was purified by column chromatography on silica gel
using petroleum ether/ ethyl acetate (1:9) to give compounds 4a-c in 60-75 % yield on silica
gel as yellow oil.
4.3. General procedure for the synthesis of compounds 5a-5o
A solution of benzyl chloride/bromide derivative (1.1 mmol), sodium azide (0.06 g, 0.9
mmol), and trimethylamine (0.13 g, 1.3 mmol) in water (4 mL) and tert-butyl alcohol (4 mL)
was stirred at room temperature for 30 min. Then, compound 4a-c (1 mmol) and CuI (7
mol%) were added to the reaction mixture and it was continued for 24 h [24,26]. Upon
completion of the reaction, monitored by TLC, the mixture was diluted with H2O, extracted
with CH3Cl, and dried over anhydrous Na2SO4. After concentration, the resulting residue
purified by flash chromatography on silica gel using ethyl acetate / methanol (9:1) as eluent
in 48-68 % yield.
4.3.1. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-tetrahydroacridin-9-amine (5a)
Cream solid; yield: 48%, mp 65-67 °C. IR (KBr): 3344, 3138, 3061, 2919, 2848, 1610, 1560
1
cm-1. H NMR (500 MHz, DMSO): 8.20 (d, J = 8.0 Hz, 1H, H8), 7.96 (s, 1H, triazole), 7.77
(d, J = 8.0 Hz, 1H, H5), 7.59 (t, J = 8.0 Hz, 1H, H6), 7.36 (t, J = 8.0 Hz, 1H, H7), 7.30 (m,
3H, Ph), 7.14 (m, 2H, Ph), 6.34 (bs, 1H, NH), 5.53 (s, 2H, CH2), 4.73 (d, J = 5.0 Hz, 2H,
13
NHCH2), 2.91 (bs, 2H, CH2), 2.71 (bs, 2H, CH2), 1.76 (m, 4H, 2 × CH2). C NMR (125
MHz, DMSO): 156.5 (NCC4), 151.1 (C9), 146.0 (NCC5), 144.8 (C1'), 136.2 (C-triazole),
129.0 (C3', C5'), 128.7 (C6), 128.0 (C5), 127.6 (C2', C6'), 126.3 (C4'), 123.9 (C7), 123.5 (C8),
123.2 (C8-C-C9), 119.5 (C-triazole), 116.1 (C9-C-C1), 52.7 (CH2), 42.8 (NCH2), 32.3 (C4),
24.8 (C1), 22.4 (C3), 21.9 (C2). Anal. Calcd for C23H23N5: C, 74.77; H, 6.27; N, 18.96. Found:
C, 74.90; H, 6.08; N, 18.80.
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4.1.2. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-6-chloro-1,2,3,4-tetrahydroacridin-9-
amine (5b)
Cream solid; yield: 57%, mp 135-137 °C. IR (KBr): 3329, 3119, 3061, 2926, 2853, 1605,
1
1582, 1554 cm-1. H NMR (500 MHz, DMSO): 8.15 (d, J = 9.0 Hz, 1H, H8), 7.87 (s, 1H,
triazole), 7.71 (s, 1H, H5), 7.32-7.30 (m, 4H, H7, Ph), 7.09 (m, 2H, Ph), 5.96 (t, J = 7.0 Hz,
1H, NH), 5.50 (s, 2H, CH2), 4.75 (d, J = 7.0 Hz, 2H, NHCH2), 2.87 (t, J = 6.0 Hz, 2H, CH2),
13
2.68 (t, J = 6.0 Hz, 2H, CH2), 1.75 (m, 4H, 2 × CH2). C NMR (125 MHz, DMSO): 159.6
(NCC4), 149.9 (C9), 147.4 (NCC5), 146.0 (C1'), 136.2 (C6), 132.5 (C-triazole), 128.6 (C3', C5'),
128.0 (C5), 127.5 (C2', C6'), 126.7 (C4'), 125.4 (C8), 123.8 (C-triazole), 123.0 (C7), 119.1 (C8-
C-C9), 117.6 (C9-C-C1), 52.5 (CH2), 42.8 (NCH2), 33.5 (C4), 25.0 (C1), 22.5 (C2), 22.2 (C3).
Anal. Calcd for C23H22ClN5: C, 68.39; H, 5.49; N, 17.34. Found: C, 68.22; H, 5.36; N, 17.19.
4.1.3.
N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-7-chloro-1,2,3,4-tetrahydroacridin-9-
amine (5c)
Cream solid; yield: 51%, mp 127-129 °C. IR (KBr): 3322, 3130, 3065, 2928, 2853, 1581,
1551 cm-1. 1H NMR (500 MHz, CD3Cl): 7.94 (d= J = 2.0 Hz, H8), 7.86 (d, J = 8.5 Hz, H5),
7.49 (dd= J = 8.5, 2.0 Hz, H6), 7.36-7.35 (m, 3H, Ph), 7.19-7.17 (m, 3H, triazole, Ph), 5.48 (s,
2H, CH2), 4.65 (s, 2H, NHCH2), 3.01 (t, J = 6.0 Hz, 2H, CH2), 2.66 (t, J = 6.0 Hz, 2H, CH2),
13
1.82 (m, 4H, 2 × CH2). C NMR (125 MHz, DMSO): 159.0 (NCC4), 149.1 (C9), 146.2
(NCC5), 145.2 (C1'), 136.2 (C-triazole), 130.4 (C5), 128.7 (C7), 128.5 (C6), 128.2 (C3', C5'),
128.0 (C2', C6'), 127.4 (C4'), 123.1 (C-triazole), 122.0 (C8-C-C9), 121.6 (C8), 118.3 (C9-C-C1),
52.6 (CH2), 42.5 (NCH2), 33.4 (C4), 25.3 (C1), 22.5 (C2), 22.3 (C3). Anal. Calcd for
C23H22ClN5: C, 68.39; H, 5.49; N, 17.34. Found: C, 68.53; H, 5.65; N, 17.20.
4.1.4.
N-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-tetrahydroacridin-9-
amine (5d)
13

ACCEPTED MANUSCRIPT
Cream solid; yield: 60%, mp 105-107 °C. IR (KBr): 3340, 3137, 3060, 2945, 2923, 1646,
1566 cm-1. 1H NMR (500 MHz, DMSO): 8.11 (d, J = 8.2 Hz, 1H, H8), 7.90 (s, 1H, triazole),
7.71 (d, J = 8.2 Hz, 1H, H5), 7.51 (t, J = 8.2 Hz, 1H, H6), 7.36 (d, J = 8.0 Hz, 2H, H3′, H5′),
7.32 (t, J = 8.2 Hz, 1H, H7), 7.13 (d, J = 8.0 Hz, 2H, H2′, H6′), 5.85 ( t, J = 7.0 Hz, 1H, NH),
5.51 (s, 2H, CH2), 4.59 (d, J = 7.0 Hz, 2H, NHCH2), 2.87 (t, J = 6.0 Hz, 2H, CH2), 2.69 (t, J
13
= 6.0 Hz, 2H, CH2), 1.72 (m, 4H, 2 × CH2). C NMR (125 MHz, CD3Cl): 158.2 (NCC4),
149.8 (C9), 146.8 (NCC5), 146.4 (C1'), 135.2 (C4'), 132.8 (C-triazole), 129.5 (C3', C5'), 128.9
(C2', C6'), 128.7 (C6), 128.3 (C5), 128.0 (C7), 123.7 (C8), 123.0 (C-triazole), 120.7 (C8-C-C9),
117.4 (C9-C-C1), 53.5 (CH2), 43.7 (NCH2), 31.3 (C4), 24.4 (C1), 23.4 (C2), 22.4 (C3). Anal.
Calcd for C23H22ClN5: C, 68.39; H, 5.49; N, 17.34. Found: C, 68.52; H, 5.60; N, 17.49.
4.1.5.
6-Chloro-N-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-
tetrahydroacridin-9-amine (5e)
Cream solid; yield: 58%, mp 118-120 °C. IR (KBr): 3334, 3124, 3066, 2941, 2860, 1604,
1583, 1553 cm-1. 1H NMR (500 MHz, CD3Cl): 7.92 (d, J = 9.0 Hz, 1H, H8), 7.90 (s, 1H, H5),
7.33 (d, J = 8.5 Hz, 2H, H3′, H5′), 7.30 (d, J = 9.0 Hz, 1H, H7), 7.15 (s, 1H, triazole), 7.11 (d,
J = 8.5 Hz, 2H, H2′, H6′), 5.43 (s, 2H, CH2), 4.68 (s, 2H, NHCH2), 3.02 (t, , J = 6.0 Hz, 2H,
CH2), 2.65 (t, , J = 6.0 Hz, 2H, CH2), 1.83 (m, 4H, 2 × CH2). 13C NMR (125 MHz, DMSO):
158.1 (NCC4), 149.6 (C9), 146.8 (NCC5), 146.4 (C6), 135.2 (C1'), 132.7 (C4'), 132.3 (C-
triazole), 129.5 (C3', C5'), 128.6 (C2', C6'), 128.3 (C5), 127.9 (C8), 123.6 (C-triazole), 123.0
(C7), 120.7 (C8-C-C9), 117.4 (C9-C-C1), 51.8 (CH2), 42.9 (NCH2), 33.5 (C4), 25.0 (C1), 22.6
(C2), 22.4 (C3). Anal. Calcd for C23H21Cl2N5: C, 63.02; H, 4.83; N, 15.98. Found: C, 62.18;
H, 4.69; N, 16.13.
4.1.6.
7-Chloro-N-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-
tetrahydroacridin-9-amine (5f)
14

ACCEPTED MANUSCRIPT
Off-cream solid; yield: 64%, mp 117-119 °C. IR (KBr): 3324, 3130, 3062, 2933, 2853, 1637,
1582, 1557 cm-1. 1H NMR (500 MHz, CD3Cl): 7.95 (s, 1H, H8), 7.90 (d= J = 9.0 Hz, 1H, H5),
7.50 (d, J = 9.0 Hz, 1H, H6), 7.33 (d, J = 8.5 Hz, 2H, H3′, H5′), 7.21 (s, 1H, triazole), 7.13 (d,
J = 8.5 Hz, 2H, H2′, H6′), 5.45 (s, 2H, CH2), 4.69 (s, 2H, NHCH2), 3.03 (t, J = 6.0 Hz, 2H,
13
CH2), 2.66 (t, J = 6.0 Hz, 2H, CH2), 1.84 (m, 4H, 2 × CH2). C NMR (125 MHz, DMSO):
158.7 (NCC4), 149.0 (C9), 146.1 (NCC5), 145.1 (C1'), 135.1 (C4'), 132.7 (C-triazole), 130.3
(C6), 129.4 (C7), 128.6 (C5), 128.4 (C3', C5'), 128.2 (C2', C6'), 123.0 (C-triazole), 121.9 (C8-C-
C9), 121.5 (C8), 118.3 (C9-C-C1), 51.8 (CH2), 42.5 (NCH2), 33.3 (C4), 25.3 (C1), 22.5 (C2),
22.2 (C3). Anal. Calcd for C23H21Cl2N5: C, 63.02; H, 4.83; N, 15.98. Found: C, 63.21; H,
4.68; N, 15.76.
4.1.7.
N-((1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-tetrahydroacridin-9-
amine (5g)
Off-cream solid; yield: 54%, mp 100-102 °C. IR (KBr): 3296, 3126, 3061, 2926, 2860, 1656,
1
1613, 1562 cm1. H NMR (500 MHz, DMSO): 8.13 (d, J = 8.5 Hz, 1H, H8), 7.87 (s, 1H,
triazole), 7.73 (d, J = 8.5 Hz, 1H, H5), 7.52 (t, J = 8.5 Hz, 1H, H6), 7.32 (t, J = 8.5 Hz, 1H,
H7), 7.10 (d, J = 7.5 Hz, 2H, H2′, H6′), 7.03 (d, J = 7.5 Hz, 2H, H3′, H5′), 5.84 ( t, J = 7.0 Hz,
1H, NH), 5.45 (s, 2H, CH2), 4.60 (d, J = 7.0 Hz, 2H, NHCH2), 2.88 (t, J = 6.0 Hz, 2H, CH2),
13
2.69 (t, J = 6.0 Hz, 2H, CH2), 2.25 (s, 3H, CH3), 1.73 (m, 4H, 2 × CH2). C NMR (125
MHz, DMSO): 158.1 (NCC4), 149.7 (C9), 146.9 (NCC5), 146.3 (C4'), 137.3 (C1'), 133.1 (C-
triazole), 129.2 (C3', C5'), 128.3 (C6), 127.9 (C5), 127.6 (C2', C6'), 123.6 (C7), 123.1 (C-
triazole), 122.8 (C8), 120.7 (C8-C-C9), 117.4 (C9-C-C1), 52.5 (CH2), 43.0 (NCH2), 33.5 (C4),
25.0 (C1), 22.6 (C2), 22.4 (C3), 20.7 (Me). Anal. Calcd for C24H25N5: C, 75.17; H, 6.57; N,
18.26. Found: C, 75.31; H, 6.46; N, 18.09.
4.1.8.
6-Chloro-N-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-
tetrahydroacridin-9-amine (5h)
15

ACCEPTED MANUSCRIPT
Off-cream solid; yield: 55%, mp 121-123 °C. IR (KBr): 3333, 3122, 3062, 2940, 2858, 1604,
1
1583, 1552 cm-1. H NMR (500 MHz, DMSO): 8.15 (d, J = 9.0 Hz, 1H, H8), 7.83 (s, 1H,
triazole), 7.72 (s, 1H, H5), 7.32 (d, J = 9.0 Hz, 1H, H7), 7.11 (d, J = 7.5 Hz, 2H, H2′, H6′),
7.01 (d, J = 7.5 Hz, 2H, H3′, H5′), 6.03 ( bs, 1H, NH), 5.44 (s, 2H, CH2), 4.62 (d, J = 6.5 Hz,
2H, NHCH2), 2.86 (t, J = 6.0 Hz, 2H, CH2), 2.67 (t, J = 6.0 Hz, 2H, CH2), 2.27 (s, 3H, CH3),
13
1.73 (m, 4H, 2 × CH2). C NMR (125 MHz, DMSO): 158.8 (NCC4), 150.4 (C9), 146.6
(NCC5), 145.9 (C4'), 137.3 (C6), 133.1 (C1'), 132.9 (C-triazole), 129.2 (C3', C5'), 127.6 (C5),
125.9 (C2', C6'), 125.6 (C8), 123.9 (C-triazole), 122.9 (C7), 118.7 (C8-C-C9), 117.1 (C9-C-C1),
52.4 (CH2), 42.7 (NCH2), 33.0 (C4), 24.8 (C1), 22.3 (C2), 22.0 (C3), 20.7 (Me). Anal. Calcd
for C24H24ClN5: C, 68.97; H, 5.79; N, 16.76. Found: C, 68.79; H, 5.62; N, 16.80.
4.1.9. 7-Chloro-N-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-
tetrahydroacridin-9-amine (5i)
Off-cream solid; yield: 62%, mp 118-120 °C. IR (KBr): 3338, 3142, 3068, 2955, 2851, 1610,
1
1572, 1545 cm-1. H NMR (500 MHz, CD3Cl): 7.86 (d, J = 2.5 Hz, 1H, H8), 7.76 (d, J = 9.0
Hz, 1H, H5), 7.40 (dd, J = 9.0, 2.5 Hz, 1H, H6), 7.08 (d, J = 8.0 Hz, 2H, H2′, H6′), 7.06 (s, 1H,
triazole), 7.02 (d, J = 8.0 Hz, 2H, H3′, H5′), 5.35 (s, 2H, CH2), 4.55 (s, 2H, NHCH2), 2.92 (t, J
= 6.0 Hz, 2H, CH2), 2.58 (t, J = 6.0 Hz, 2H, CH2), 2.26 (s, 3H, CH3), 1.74 (m, 4H, 2 × CH2).
13C NMR (125 MHz, DMSO): 158.8 (NCC4), 148.8 (C9), 146.0 (NCC5), 145.1 (C4'), 137.9
(C1'), 133.0 (C-triazole), 130.3 (C6), 129.0 (C7), 128.2 (C5), 128.0 (C3', C5'), 127.4 (C2', C6'),
122.6 (C-triazole), 121.8 (C8-C-C9), 121.4 (C8), 118.2 (C9-C-C1), 52.3 (CH2), 42.5 (NHCH2),
33.6 (C4), 25.0 (C1), 22.4 (C3), 22.1 (C2), 20.5 (Me). Anal. Calcd for C24H24ClN5: C, 68.97;
H, 5.79; N, 16.76. Found: C, 69.10; H, 5.60; N, 16.58.
4.1.10. N-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-tetrahydroacridin-9-
amine (5j)
16

ACCEPTED MANUSCRIPT
White solid; yield: 57%, mp 75-78 °C. IR (KBr): 3343, 3123, 3061, 2931, 2861, 1612, 1583,
1562 cm-1. 1H NMR (500 MHz, DMSO): 8.10 (d, J = 8.0 Hz, 1H, H8), 7.85 (s, 1H, triazole),
7.71 (d, J = 8.0 Hz, 1H, H5), 7.52 (d, J = 8.0 Hz, 1H, H6), 7.32 (d, J = 8.0 Hz, 1H, H7), 7.11
(d, J = 8.5 Hz, 2H, H2′, H6′), 6.86 (d, J = 8.5 Hz, 2H, H3′, H5′), 5.83 (t, J = 6.5 Hz, 1H, NH),
5.42 (s, 2H, CH2), 4.58 (d, J = 6.5 Hz, 2H, NHCH2), 3.73 (s, 3H, OCH3), 2.88 (t, J = 6.0 Hz,
13
2H, CH2), 2.68 (t, J = 6.0 Hz, 2H, CH2), 1.74 (m, 4H, 2 × CH2). C NMR (125 MHz,
DMSO): 159.0 (NCC4), 158.0 (C4'), 149.7 (C9), 146.7 (NCC5), 146.2 (C-triazole), 129.2 (C2',
C6'), 128.3 (C1'), 128.0 (C5), 127.9 (C6), 123.6 (C7), 123.0 (C-triazole), 122.6 (C8), 120.6 (C8-
C-C9), 117.2 (C9-C-C1), 114.0 (C3', C5'), 55.1 (OCH3), 52.1 (CH2), 42.9 (NCH2), 33.4 (C4),
24.9 (C1), 22.6 (C2), 22.4 (C3). MS (m/z, %): 399 ([M]+., 13), 197 (100), 182 (13), 121 (93),
77 (16). Anal. Calcd for C24H25N5O: C, 72.16; H, 6.31; N, 17.53. Found: C, 72.29; H, 6.17;
N, 17.42.
4.1.11.
6-Chloro-N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-
tetrahydroacridin-9-amine (5k)
White solid; yield: 68%, mp 98-100 °C. IR (KBr): 3335, 3122, 3066, 2935, 2860, 1608,
1
1582, 1554 cm-1. H NMR (500 MHz, DMSO): 8.44 (d, J = 8.5 Hz, 1H, H8), 8.06 (s, 1H,
triazole), 7.91 (s, 1H, H5), 7.49 (d, J = 8.5 Hz, 1H, H7), 7.17 (d, J = 8.0 Hz, 2H, H2′, H6′), 6.88
(d, J = 8.0 Hz, 2H, H3′, H5′), 5.47 (s, 2H, CH2), 5.03 (s, 2H, NHCH2), 3.73 (s, 3H, OCH3),
2.97 (bs, 2H, CH2), 2.68 (bs, 2H, CH2), 1.80 (bs, 4H, 2 × CH2). 13C NMR (125 MHz,
DMSO): 159.1 (NCC4), 155.1 (C4'), 151.9 (C9), 144.2 (NCC5), 139.0 (C6), 136.8 (C-triazole),
129.4 (C2', C6'), 127.9 (C1'), 127.4 (C5), 125.1 (C8), 123.4 (C-triazole), 118.4 (C7), 114.4 (C8-
C-C9), 114.0 (C9-C-C1), 112.4 (C3', C5'), 55.1 (OCH3), 52.3 (CH2), 42.3 (NCH2), 28.3 (C4),
23.9 (C1), 21.3 (C2), 20.9 (C3). Anal. Calcd for C24H24ClN5O: C, 66.43; H, 5.57; N, 16.14.
Found: C, 66.29; H, 5.65; N, 16.02.
17

ACCEPTED MANUSCRIPT
4.1.12.
7-Chloro-N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-
tetrahydroacridin-9-amine (5l)
White solid yield: 65%, mp 84-86 °C. IR (KBr): 3339, 3144, 2929, 2856, 1611, 1584, 1559
1
cm-1. H NMR (500 MHz, DMSO): 8.24 (s, 1H, H8), 7.88 (s, 1H, triazole), 7.73 (d, J = 8.5
Hz, 1H, H5), 7.56 (d, J = 8.5 Hz, 1H, H6), 7.11 (d, J = 8.0 Hz, 2H, H2′, H6′), 6.85 (d, J = 8.0
Hz, 2H, H3′, H5′), 6.73 (bs, 1H, NH), 5.43 (s, 2H, CH2), 4.63 (d, J = 5.0 Hz, 2H, NHCH2),
13
3.72 (s, 3H, OCH3), 2.88 (s, 2H, CH2), 2.70 (s, 2H, CH2), 1.73 (m, 4H, 2 × CH2). C NMR
(125 MHz, CDCl3): 159.0 (NCC4), 157.9 (C4'), 149.9 (C9), 145.9 (NCC5), 143.9 (C-triazole),
129.2 (C2', C6'), 129.1 (C6), 128.4 (C7), 128.0 (C5), 122.7 (C1'), 122.2 (C-triazole), 120.9 (C8-
C-C9), 117.5 (C8), 114.2 (C9-C-C1), 114.0 (C3', C5'), 55.1 (OCH3), 52.3 (CH2), 42.3 (NCH2),
28.3 (C4), 23.9 (C1), 21.3 (C2), 20.9 (C3). MS (m/z, %): 435 ([M+2]+., 3), 433 (M+, 9), 399
(12), 231 (45), 197 (75), 121 (100), 47 (25). Anal. Calcd for C24H24ClN5O: C, 66.43; H, 5.57;
N, 16.14. Found: C, 66.59; H, 5.75; N, 15.98. M+
4.1.13.
N-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-tetrahydroacridin-9-
amine (5m)
Off-cream; yield: 51%, mp 78-80 °C. IR (KBr): 3340, 3125, 2925, 2864, 1617, 1580, 1559
cm-1. 1H NMR (500 MHz, CDCl3): 7.98 (d, J = 8.0 Hz, 1H, H8), 7.91 (d, J = 8.0 Hz, 1H, H5),
7.53 (t, J = 8.0 Hz, 1H, H6), 7.31 (t, J = 8.0 Hz, 1H, H7), 7.22 (s, 1H, triazole), 7.15 (m, 2H,
H2', H6'), 7.01 (t, J = 8.5 Hz, 2H, H3', H5'), 5.41 (s, 2H, CH2), 4.71 (s, 2H, NHCH2), 3.02 (s,
2H, CH2), 2.66 (s, 2H, CH2), 1.81 (m, 4H, 2 × CH2). 13C NMR (125 MHz, CDCl3): 162.8 (d,
JC-F = 247.5 Hz, C4'), 157.9 (NCC4), 150.3 (C9), 146.2 (NCC5), 146.0 (C1'), 130.3 (C-
triazole), 129.7(d, JC-F = 8.8 Hz, C2', C6'), 128.7 (C6), 127.8 (C5), 124.3 (C7), 122.7 (C8), 121.2
(C-triazole), 120.2 (C8-C-C9), 117.5 (C9-C-C1), 116.0 (d, JC-F = 22.5 Hz, C3', C5'), 53.3 (CH2),
44.1 (NCH2), 33.3 (C4), 24.5 (C1), 22.7 (C2), 22.4 (C3). Anal. Calcd for C23H22FN5: C, 71.30;
H, 5.72; N, 18.08. Found: C, 71.13; H, 5.85; N, 17.89.
18

ACCEPTED MANUSCRIPT
4.1.14.
6-Chloro-N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-
tetrahydroacridin-9-amine (5n)
Off-cream solid; yield: 52% mp, 84-86 °C. IR (KBr): 3377, 3129, 3071, 2942, 2857, 1627,
1
1581, 1509 cm-1. H NMR (500 MHz, DMSO): 8.39 (d, J = 8.5 Hz, 1H, H8), 8.07 (s, 1H,
triazole), 7.78 (s, 1H, H5), 7.48 (d, J = 8.5 Hz, 1H, H7), 7.26 (m, 2H, H2′, H6′), 7.17 (t, J = 8.0
Hz, 2H, H3′, H5′), 5.53 (s, 2H, CH2), 4.95 (s, 2H, NHCH2), 2.94 (bs, 2H, CH2), 2.68 (bs, 2H,
CH2), 1.80 (bs, 4H, 2 × CH2). 13C NMR (125 MHz, DMSO): 162.3 (d, JC-F = 238.8 Hz, C4'),
155.8 (NCC4), 149.9 (C9), 146.3 (NCC5), 144.6 (C6), 132.1 (C1'), 129.9 (d, JC-F = 7.5 Hz, C2',
C6'), 127.8 (C5), 127.7 (C-triazole), 126.7 (C8), 124.7 (C-triazole), 121.8 (C7), 120.1 (C8-C-
C9), 117.8 (C9-C-C1), 115.3 (d, JC-F = 21.2 Hz, C3', C5'), 51.9 (CH2), 42.4 (NCH2), 29.7 (C4),
24.0 (C1), 21.5 (C2), 20.7 (C3). Anal. Calcd for C23H21ClFN5: C, 65.48; H, 5.02; N, 16.60.
Found: C, 65.31; H, 4.88; N, 16.42.
4.1.15.
7-Chloro-N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,2,3,4-
tetrahydroacridin-9-amine (5o)
Off-cream solid; yield: 59%, mp 75-78 °C. IR (KBr): 3335, 3125, 3060, 2925, 2848, 1610,
1581, 1561 cm-1. 1H NMR (500 MHz, DMSO): 7.93 (s, 1H, H8), 7.85 (d, J = 8.5 Hz, 1H, H5),
7.47 (d, J = 8.5 Hz, 1H, H6) , 7.19-7.18 (m, 3H, triazole, H2′, H6′), 7.03 (t, J = 8.0 Hz, 2H, H3′,
H5′), 5.43 (s, 2H, CH2), 4.65 (s, 2H, NHCH2), 3.00 (bs, 2H, CH2), 2.66 (bs, 2H, CH2), 1.82
13
(bs, 4H, 2 × CH2). C NMR (125 MHz, CHCl3): 162.9 (d, JC-F = 242.5 Hz, C4'), 159.0
(NCC4), 149.2 (C9), 145.9 (NCC5), 146.5 (C1'), 130.3 (C-triazole), 129.7(d, JC-F = 7.5 Hz, C2',
C6'), 129.2 (C5), 127.3 (C7), 121.8 (C6), 121.4 (C-triazole), 121.0 (C8-C-C9), 119.0 (C8), 117.6
(C9-C-C1), 116.0 (d, JC-F = 22.5 Hz, C3', C5'), 53.4 (CH2), 44.3 (NCH2), 33.7 (C4), 24.5 (C1),
22.7 (C2), 22.5 (C3). Anal. Calcd for C23H21ClFN5: C, 65.48; H, 5.02; N, 16.60. Found: C,
65.31; H, 5.15; N, 16.79.
19

ACCEPTED MANUSCRIPT
4.2. AChE and BChE inhibition assay
Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyophilized powder, from
electric eel, 1000 unit), 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 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 KH2PO4/K2HPO4 buffer
(pH 8.0) to obtain 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 was performed [19-27]
using a 96-well plate reader (BioTek ELx808). Each well contained 50 µL potassium
phosphate buffer (KH2PO4/ K2HPO4, 0.1 M, pH 8), 25µL sample dissolved in 50% 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
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For estimates of the inhibition model and inhibition constant Ki, reciprocal plots of
1/V versus 1/[S] were obtained using different concentrations of the substrate (0.187- 3 mM).
For this purpose, experiments were performed similar to enzyme inhibition assay [19-27].
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 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. Neuroprotection assay
Neuroprotective activity was evaluated as described in the literature [23].
Differentiated PC12 cells were incubated with different concentrations of the compound 5l
for 3 h before treatment with H2O2 (300 mM). Cell death by apoptosis was established after
staining with DAPI, and cell viability was measured after 24 h by using the MTT assay.
Briefly, 10 mL of MTT solution (5 mg/mL, Sigma) was added to the cell culture media (150
mL) and incubated in a CO2 incubator for 3.5 h. Then, medium was removed and DMSO
(150 mL) was added into the each well and the formazan precipitates were dissolved by
shaking the plate for 10 min at speed of 120 rpm. Finally, optical density (OD) was
determined at 560 nm on the microplate reader (BioTek synergy HT). Results were adjusted
considering OD measured in the blank.
4.5. DPPH radical scavenging activity (DPPH)
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Antioxidant activity of compounds 5d, 5j and 5l were evaluated using DPPH (1,1-diphenyl-
2-picrylhydrazyl) [28]. Different concentrations of test compounds in DMSO were prepared
and the compound solution (0.5 mL) was added to the methanolic DPPH solution (1.0 mL,
0.1 mM). The mixture was kept in the dark for 30 min. Then, the absorbance at 517 nm was
measured by an UV⁄visible spectrophotometer. The percent scavenging activity was
calculated using the following formula: inhibition (%) = 100× [(Abscontrol – Abssample) ⁄
Abscontrol] since Abscontrol = AbsDPPH + Abssolvent
.
4.6. Cytotoxicity
The in vitro cytotoxicity of compounds 5a, 5b, 5j, 5l, 5m and 5n were evaluated against
MCF-7, T-47D, and MDA-MB-231using MTT colorimetric assay comparing with etoposide
[34]. Cancer cell lines were grown in RPMI-1640 medium supplemented with 10% heat-
inactivated fetal calf serum (Gibco BRL), 100 µg/mL streptomycin and 100 U/mL penicillin
at 37º in a humidified atmosphere with 5% CO2 in air. Cells were harvested by Trypsin-
EDTA in the exponential growth phase and diluted in complete growth medium to give a
total cell count of 5* 104 cells/mL. 195 µL of the cell suspension was seeded into the wells of
96-well plates (Nunc, Denmark). The plates were incubated overnight in a humidified air
atmosphere at 37° with 5% CO2. After overnight incubation, 5 µL of the media containing
various concentrations of the compounds was added per well in triplicate (final concentration
1, 5, 10 and 20 µg/mL). The plates were incubated for further 72 h. The final concentration of
DMSO was 0.1%. In each plate, there were three control wells (cells without test compounds)
and three blank wells (the medium with 0.1% DMSO) for cell viability. After treatment, the
medium was removed and 200 µL phenol red-free medium containing MTT (1 mg/mL) was
added to the wells, followed by 4 h incubation. After incubation, the culture medium was
replaced with 100 µL of DMSO and the absorbance of each well was measured by using a
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microplate reader at 492 nm. For each compound, the concentration causing 50% cell growth
inhibition (IC50) compared with the control (etoposide) was calculated from concentration
response curves by non-linear regression analysis.
4.7. Molecular docking study
Docking studies were carried out using the Autodock 4.2 and program for compound
5l. For this purpose, the pdb structure of 2CMF 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 compound 5l was provided
using MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com) and converted to
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 enzyme using 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 = 3.386, y = 65.434, z = 63.34. The dimensions
of the active site box were set at 60×60×60 Å. 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 compound 5j was carried out by Autodock Vina
(AV) 1.1.1 program [29]. The crystal structure of human butyrylcholinesterase (1P0I) 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
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compound 5j and its 3D structure were generated by MarvineSketch 5.8.3, 2012 and then
ligand.pdbqt provided by Autodock Tools (ver 1.5.4). The Autodock Vina parameters were
set as follow; box size: 50×50×50 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).
4.8. Learning and memory evaluation by Morris water maze test (MWM)
4.8.1. Drugs
Required drugs, scopolamine hydrobromide and donepezil hydrochloride were
obtained from Sigma. They were dissolved in saline and the test compound 5l was dissolved
in 20% PEG 400 (Polyethylene glycol) as drug vehicle.
4.8.2. Animals
Male albino Wistar rats (200-220 g) 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.
4.8.3. Morris water maze test
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Rats were trained for four 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 1 cm 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 four trials
lasted for 90 seconds. At this step, quantitative data was provided in terms of escape latency,
traveled distance, swimming speed for first 4 days of training and time spent in the target
quadrant (Q1) on fifth day (probe trial test). It should be noted that the platform in the target
quadrant was removed and animals did not receive treatment on test day. However, they were
treated for four days of training.
4.8.4. In Vivo Drug Treatment
In this study, seven groups of rats were investigated. All drugs were administrated by
intraperitoneal injection (IP).
4.8.4.1. Group I: normal control.
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The control group was given normal saline (5 mg/kg) 30 min prior to testing. Also,
the escape latency, traveled distance, swimming speed for first 4 days of trial (training days)
and time spent in target quadrant on fifth day of trial (probe trial test) were evaluated.
4.8.4.2. Group II: scopolamine.
Scopolamine hydrobromide (4 mg/kg) was administered 30 min before each trial for
first 4 days of trials. In scopolamine treated rats, the escape latency, traveled distance,
swimming speed for first 4 days of trial (training days) and time spent in target quadrant on
fifth day of trial (probe trial test) were evaluated as described for group I.
4.8.4.3. Group III: vehicle.
20% PEG 400 (5 ml/kg) was administered to rats 30 min before subjecting to the first
4 days of trials. Then, it was continued as described for group I.
4.8.4.4. Group IV, V and VI: The compound 5l (5, 10, and 20 mg/kg).
The most potent AChEI activity compound (5l) and scopolamine (4mg/kg) were
administered to rats 60 min and 30 min before subjecting to the first 4 days of trials,
respectively. Then, it was continued as described for group I.
4.8.4.5. Group VII: donepezil.
Donepezil (2.5 mg/kg) and scopolamine (4 mg/kg) were administered to rats 60 min
and 30 min before subjecting to the first 4 days of trials, respectively. Then, it was continued
as described for group I.
4.8.5. Statistical analysis
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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. 94-03-33-30167.
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