Design, synthesis and biological activity evaluation of novel carbazole-benzylpiperidine hybrids as potential anti Alzheimer agents

Article abstract of DOI:10.1016/j.molstruc.2020.128793

Alzheimer's disease (AD) as the most common form of dementia in aged people, is an intricate neurodegenerative disease. Therefore, a novel strategy so-called multi-target-directed ligand has received much attention for the effective treatment of AD. In this study a series of novel carbazole-benzylpiperidine hybrids 9a-m was designed, synthesized and evaluated as acetylcholinesterase and butyrylcholinesterase inhibitors. Moreover, some of these compounds were evaluated for anti β-secretase (BACE1) activity and metal chelation properties. Among the synthesized compounds, compounds 9b (IC₅₀ = 16.5 μM for AChE and IC₅₀ = 0.59 μM for BuChE) and 9c (IC₅₀ = 26.5 μM for AChE and IC₅₀ = 0.18 μM for BuChE) showed the highest inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Furthermore, these compounds (9b and 9c) displayed interaction with Zn²⁺ ion and compound 9c showed moderate inhibitory activity against BACE1 (24.5% at 50 μM). Kinetic and docking studies exhibited that these compounds likely act as a non-competitive inhibitor able to interact with the catalytic active site (CAS) and peripheral anionic site (PAS) of acetylcholinesterase simultaneously.

Full text of DOI:10.1016/j.molstruc.2020.128793

Journal Pre-proof
Design, synthesis and biological activity evaluation of novel carbazole-
benzylpiperidine hybrids as potential anti Alzheimer agents
Batool Sadeghian, Amirhossein Sakhteman, Zeinab Faghih, Hamid Nadri, Najmeh
Edraki, Aida Iraji, Issa Sadeghian, Zahra Rezaei
PII:
S0022-2860(20)31118-2
DOI:
https://doi.org/10.1016/j.molstruc.2020.128793
MOLSTR 128793
Reference:
To appear in: Journal of Molecular Structure
Received Date: 20 May 2020
Revised Date: 18 June 2020
Accepted Date: 28 June 2020
Please cite this article as: B. Sadeghian, A. Sakhteman, Z. Faghih, H. Nadri, N. Edraki, A. Iraji,
I. Sadeghian, Z. Rezaei, Design, synthesis and biological activity evaluation of novel carbazole-
benzylpiperidine hybrids as potential anti Alzheimer agents, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.128793.
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Credit Author Statement
Design, synthesis and biological activity evaluation of novel carbazole-
benzylpiperidine hybrids as potential anti Alzheimer agents
a
a
b
c
Batool Sadeghian , Amirhossein Sakhteman , Zeinab Faghih , Hamid Nadri , Najmeh
d
d
b
a
Edraki , Aida Iraji , Issa Sadeghian , Zahra Rezaei *
a
Department of Medicinal Chemistry, School of Pharmacy, Shiraz University of Medical
Sciences, Shiraz, Iran.
b
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
c
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
d
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran.
Batool Sadeghian: PhD student and writing Original draft preparation
Amirhossein Sakhteman: Conceptualization, Supervisor, molecular modeling
Zeinab Faghih: Assistant, synthesis of compounds
Hamid Nadri: Assistant, biological assay
Najmeh Edraki: Data curation
Aida Iraji: Assistant, metal chelation assay
,
Issa Sadeghian: Writing- Reviewing and Editing,
Zahra Rezaei: Conceptualization, Methodology, Supervisor

Design, synthesis and biological activity evaluation of novel carbazole-
benzylpiperidine hybrids as potential anti Alzheimer agents
a
a
b
c
Batool Sadeghian , Amirhossein Sakhteman , Zeinab Faghih , Hamid Nadri , Najmeh
d
d
b
a
Edraki , Aida Iraji , Issa Sadeghian , Zahra Rezaei *
a
Department of Medicinal Chemistry, School of Pharmacy, Shiraz University of Medical
Sciences, Shiraz, Iran.
b
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
c
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
d
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran.
*
Corresponding author. Tel.: +98 713 2425305; Fax: +98 7132424128; Email: rezaeiza@sums.ac.ir

Abstract
Alzheimer’s disease (AD) as the most common form of dementia in aged people, is an intricate
neurodegenerative disease. Therefore, a novel strategy so-called multi-target-directed ligand has
received much attention for the effective treatment of AD. In this study a series of novel
carbazole-benzylpiperidine hybrids 9a-m was designed, synthesized and evaluated as
acetylcholinesterase and butyrylcholinesterase inhibitors. Moreover, some of these compounds
evaluated for anti β-secretase (BACE1) activity and metal chelation properties. Among the
synthesized compounds, compounds 9b (IC50 = 16.5 µM for AChE and IC50 = 0.59 µM for
BuChE) and 9c (IC = 26.5 µM for AChE and IC = 0.18 µM for BuChE) showed the highest
5
0
50
inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Furthermore, these
2
+
compounds (9b and 9c) displayed interaction with Zn ion and compound 9c showed moderate
inhibitory activity against BACE1 (24.5% at 50 µM). Kinetic and docking studies exhibited that
these compounds likely act as a non-competitive inhibitor able to interact with the catalytic
active site (CAS) and peripheral anionic site (PAS) of acetylcholinesterase simultaneously.
Keywords: Alzheimer’s disease, Carbazole, Benzylpiperidine, Acetylcholinesterase,
Butyrylcholinesterase, Docking study.

1
. Introduction
Alzheimer’s disease (AD) as the most common form of dementia in aged people, is an intricate
neurodegenerative disease characterized by the irreversible loss of cognitive ability, severe
behavioral abnormalities, decreased language skills and ultimately death [1, 2]. According to the
World Alzheimer Report 2018, around 50 million people are living with dementia in the world.
This number is expected to raise to 152 million people in 2050 [3]. Huge financial and human
resources annually are devoted to the care and treatment of these patients. The estimated
worldwide funding devoted to the therapy of AD is US$818 billion, and expected to rise to one
trillion by 2018 [4].
Although numerous research studies have been focused on understanding the pathophysiology of
AD, its etiology is not comprehensively perceived. However, several factors including low levels
of acetylcholine (ACh), amyloid-β (Aβ) deposits, τ-protein aggregation, oxidative stress and
dyshomeostasis of biometals have been identified which involved in the pathogenesis of AD.
According to these factors several hypotheses have been suggested to describe the mechanism of
AD pathology [4-9].
The cholinergic hypothesis describes that memory impairments in AD are caused by the drastic
decline of acetylcholine (ACh) in the brain [10, 11]. Thus, cholinesterase inhibitors (AChEIs)
such as donepezil, tacrine, rivastigmine and galantamine have been major agents in AD therapy.
However, these treatments are only a symptomatic treatment that is not able to prevent the
progression of the disorder [12-15]. Therefore, further research is needed to develop more
effective treatment strategies in AD.
ACh can be degraded by two types of cholinesterases termed acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) [1]. Since the AChE is the major ChE in the normal brain, it has
received much attention as a target in the treatment of AD [16]. But in recent years BuChE
received much attention because of its key modulatory role in regulating the ACh level in AD
patients [17]. Although BuChE only plays a secondary role in the healthy brains, some new
evidence indicates that by decreasing AChE activity during disease progression, BuChE acts as a
key degradation enzyme [10, 18]. It is reported that BuChE inhibition is not associated with
notable adverse complications and may show efficacy without remarkable side effects.
Therefore, BuChE can be considered as an ideal drug target for the treatment of AD [19, 20].

Consequently, simultaneous inhibition of AChE and BuChE can be an appropriate strategy in the
treatment of AD.
Based on the crystal structure of AChE, two ligand-binding sites have been identified: 1) a
catalytic active site (CAS) located at the bottom, and 2) a peripheral cationic site (PAS) located
at the entrance. It is suggested that the interaction of AChE inhibitors to either of these sites
(CAS and PAS) could inhibit the enzyme activity [13, 21, 22]. In addition, studies also show that
AChE can promote the formation of Aβ fibrils and Aβ plaques through the interaction between
Aβ and PAS site of the enzyme. Accordingly, the development of dual binding site inhibitors of
both CAS and PAS could be a promising strategy for the treatment of AD [23].
Another hypothesis called the amyloid hypothesis implies that aggregation of plaques composed
of β-amyloid peptides (Aβ) in the brain is the major factor in the pathogenesis of AD [24]. Aβ1–40
and Aβ1–42 are two major isoforms that derived from cleavage of APP (amyloid precursor
protein) by two proteases named β-site amyloid precursor protein cleaving enzyme-1 (BACE1)
and δ-secretase. Aβ1–42 is the predominant form in senile plaques, having the high aggregative
capability and stronger neurotoxicity than Aβ1–40. BACE1 is considered as the rate-limiting
enzyme for the production of Aβ. Therefore, BACE1 inhibitors can be considered as good
candidates for preventing Aβ aggregation to treat and/or prevent AD [25, 26].
Furthermore, several pieces of evidence have indicated that metal ions such as zinc, iron and
copper may be involved in the AD pathology. It is suggested that Aβ as a metalloprotein exhibits
2
+
2+
a high affinity for Cu and Zn , which might cause its accumulation and toxicity. In addition,
the binding of these metal ions to Aβ may also lead to the formation of ROS species that have
neurodegenerative effects. Therefore, preventing metal ions from interacting with Aβ may be an
effective strategy for Alzheimer's treatment [4, 27].
Due to the multifactorial nature of AD and the involvement of various enzymes and protein in its
development, it is indicated that the classic ‘one molecule, one target’ approach may not have
enough effectiveness for the treatment of AD [28, 29]. Therefore, a novel strategy so-called
multi-target-directed ligand (MTDL) strategy has received much attention for the effective
treatment of AD. In MTDL a single compound can simultaneously interact with multiple targets,
thus may provide better therapeutic effects compared to those of single targeted drugs [22, 30].
Donepezil is the most effective AChE inhibitor currently used in clinical context for the

treatment of AD [31]. However, it can only reduce the symptoms of this disease and has very
limited potential in treatment of moderate to severe AD [2, 32]. Hence, in recent years, rational
modification of donepezil scaffold has attracted a lot of attention [2]. Accordingly, various
donepezil derivatives have been designed and synthesized as MTDLs agents by replacements of
the benzylpiperidine or indanone moieties of this drug [33]. For example, Donepezil-coumarin
hybrids [34], donepezil-ebselen hybrids [35], donepezil-melatonin hybrids [13], donepezil-
curcumin hybrids [5] and donepezil-indolyl hybrids [36] have been reported as MTDLs agents.
In this study, a series of novel carbazole-benzylpiperidine hybrids as multifunctional agents for
the treatment of AD was designed and synthesized. To provide the novel hybrids with increased
biological properties, the N-benzylpiperidine fragment of the donepezil as a recognized AChE
inhibitor, attached to the carbazole fragment with various biological activities related to
Alzheimer's disease, using various linkers. The in vitro activity of these compounds to inhibition
of AChE, BuChE and BACE1, as well as metal chelation activity were evaluated. Finally,
molecular modelling and kinetic studies of the most active compounds were performed to find
the possible interaction mode with AChE and BuChE.
2
. Experimental
2
.1. General methods
All reagents and analytical grade solvents were purchased from Sigma-Aldrich and Merck
Chemical Company and were used without additional purification. The chemical reactions were
routinely followed by thin-layer chromatography (TLC) on commercially available alumina
plates coated with silica gel 60 F , and the spots were detected using a UV light. The
2
54
purification of synthesized compounds was performed by recrystallization or column
1
chromatography. The chemical structure of synthesized compounds was confirmed by H-NMR,
1
3
1
13
C-NMR and Mass spectroscopy. H NMR and C NMR spectra were recorded in DMSO or
CDCl solutions using TMS as the internal standard on a Brucker Avance DPX 300, 400 or 500
3
MHz instrument. Chemical shift values were reported in ppm (δ). The coupling constants (J
values) were expressed in hertz (Hz) and proton coupling patterns were expressed as s (singlet),
d (doublet), t (triplet), dd (doublet of doublet), m (multiplet) and br (broad singlet). Mass spectra

(MS) were recorded on Agilent Technologies (HP) MS. Melting points were measured in
capillary tubes with an electrothermal 9200 instrument. Intermediate compounds 2-8 were
synthesized according to previously reported methods with some modification [30, 37].
2
.2. General procedure for preparation of intermediates 2-4, 5-6 and 7-8
.2.1. General procedure for preparation of intermediates 2-4
2
To a solution of 9H-carbazole 1a (1 eq, 18 mmol) and NaH (3eq, 54 mmol) in dry DMF (30
mL), corresponding di-bromoalkane derivatives (3eq, 54 mmol) was added dropwise and the
mixture was stirred in an ice bath for 30 min. Then, the mixture was extracted with
dichloromethane (DCM) and water. The organic layer was dried over anhydrous sodium sulfate
(
Na SO ) and evaporated. The obtained product was purified by column chromatography on
2 4
silica gel using n-Hexane/Ethyl acetate (20/2) as eluent to provide the intermediates 2-4 as white
crystals in 67-73 % yields.
2
.2.2. General procedure for preparation of intermediates 5-6
A mixture of 9H-carbazole 1a (1 eq, 24 mmol) and triethylamine (TEA) (1.2 eq, 28.8 mmol) was
dissolved in acetonitrile. Then 3-chloropropionyl chloride or 4-chlorobutyryl chloride (3 eq, 72
mmol) was added dropwise to the stirred solution. The reaction mixture refluxed at 85 ˚C for 48
h. Then, the solvent was evaporated with a rotatory evaporator and extracted using
2 4
dichloromethane (DCM) and water. The organic layer was dried on Na SO and then the solvent
was evaporated. The crude product was purified by column chromatography on silica gel using n-
Hexane/Ethyl acetate (20/1) as eluent to give the intermediates 5 and 6 as white solids in 60-65
%
yields.
2
.2.3. General procedure for preparation of intermediates 7-8
In a round-bottomed flask (100mL), 9H-carbazole 1a or 3,6-dibromocarbazole 1b (1eq, 1.5
mmol) was treated with KOH (1.2 eq, 1.8 mmol) in dry DMF at room temperature for 1 h. Then
epichlorohydrin (2.5 eq, 4 mmol) was added dropwise to the solution mixture. After stirring at
room temperature for 24 h, the mixture was poured into crushed ice and the residue precipitate
was purified by plate chromatography using n-Hexane/Ethyl acetate (20/4) as eluent to give
intermediate 7 and 8 in 52-55 % yields.

2
2
.3. The general method for preparation of compounds 9a-m
.3.1. The general method for preparation of compounds 9a-i
A mixture of intermediates 2-6 (1 eq, 1 mmol), corresponding amines (4-amino-N-
benzylpiperidine or 4-benzylpiperidine) (1 eq, 1 mmol) and TEA (1.2 eq, 1.2 mmol) were
dissolved in 30 mL dry acetonitrile and refluxed at 85 ˚C for 24 h (compound 9a-f) or stirred at
room temperature for 1h (compound 9g-i). Then, the resulting precipitated was filtered, washed
with acetonitrile and dried at room temperature. The resulting product was purified by
recrystallization or column chromatography on silica gel to afford pure compounds 9a-i.
2
.3.2. The general method for preparation of compounds 9j-m
In a round-bottomed flask (50 mL), a mixture of intermediate 7 or 8 (1eq, 1 mmol), appropriate
amin such as 4-amino-N-benzylpiperidine or 4-benzylpiperidine (1 eq, 1 mmol) in EtOH (20
mL) was refluxed at 65 ˚C for 24 h. Then, the resulting products were purified by
recrystallization with ethanol or column chromatography on silica gel to afford pure compounds
9
j-m.
2
.3.1.1. N-(4-(9H-carbazol-9-yl) butyl)-1-benzylpiperidin-4-amine (9a)
According to the general method, a mixture of intermediate 2 (0.30 g, 1mmol), 4-amino-N-
benzylpiperidine (0.19 g, 1 mmol) and TEA (0.12g, 1.2 mmol) were dissolved in 30 mL
acetonitrile and refluxed at 85 ˚C for 24 h. After the reaction completed, the resulting
precipitated was filtered, washed with acetonitrile and dried at room temperature. The resulting
product was purified by recrystallization in n-hexane and DCM to afford compound 9a. White
1
solid; yield: 69.3 %, mp: 278-280 ˚C. H NMR (DMSO, 400 MHz): δ (ppm): 1.60-1.65 (m, 2H),
1
.72-1.74 (m, 2H), 1.80-1.84 (m, 3H), 1.92-1.94 (br, 3H), 2.78-2.84 (m, 5H), 3.42 (s, 2H), 4.40
(t, J = 6.8 Hz, 2H), 7.18 (t, J = 7.6 Hz, 2H), 7.23-7.32 (m, 5H), 7.43 (t, J = 7.6 Hz, 2H), 7.62 (d, J
1
3
=
8.0 Hz, 2H), 8.14 (d, J = 7.6 Hz, 2H), NH signal not detected. C NMR (DMSO, 400 MHz): δ
(ppm): 139.84, 138.06, 128.75, 128.14, 126.93, 125.65, 122.00, 120.22, 118.67, 109.29, 61.54,
+
5
3
4.32, 50.99, 43.28, 41.80, 27.56, 25.80, 23.29. Formula: C H N ; MS: m/z 411.4 [M ] (5.93),
2
8
33
3
20.4 (9.70), 222.2 (7.50), 193.2 (4.22), 180.2 (29.56), 173.3 (100), 91.2 (68.27), 56.2 (8.09).

2
.3.1.2. N-(5-(9H-carbazol-9-yl) pentyl)-1-benzylpiperidin-4-amine (9b)
According to the general method, a mixture of intermediate 3 (0.315g, 1mmol), 4-amino-N-
benzylpiperidine (0.19 g, 1mmol,) and TEA (0.12g, 1.2 mmol) were dissolved in 30 mL
acetonitrile and refluxed at 85 ˚C for 24 h. After the reaction completed, the resulting
precipitated was filtered, washed with acetonitrile and dried at room temperature. The resulting
product was purified by recrystallization in n-hexane and DCM to afford compound 9b. White
1
solid; yield: 72.9 %, mp: 158-160 ˚C. H NMR (CDCl 400 MHz): δ ppm: 1.32-1.39 (m, 2H),
3
,
1
3
7
.80-1.84 (m, 2H), 1.94-1.96 (br, 5H), 2.11-2.13 (br, 3H), 2.81 (t, J = 7.6 Hz, 2H), 2.92-3.01 (m,
H), 3.51 (s, 2H), 4.22 (t, J = 6.8 Hz, 2H), 7.22 (t, J = 7.2 Hz, 2H), 7.25-7.31 (m, 5H + CDCl ),
.36 (d, J = 8.0 Hz, 2H), 7.45 (t, J=8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), NH signal not detected.
3
1
3
3
C NMR (CDCl , 400 MHz): δ ppm: 139.92, 137.84, 128.87, 128.18, 127.06, 125.65, 122.01,
1
20.25, 118.64, 109.24, 61.39, 54.12, 50.77, 43.46, 41.99, 28.00, 27.71, 25.35, 23.51. Formula:
+
C H N ; MS: m/z 425.4 [M ] (6.18), 334.3 (12.49), 279.3 (8.72), 180.2 (25.56), 173.3 (100),
29
35
3
1
46.2 (11.22), 91.2 (59.05), 56.2 (8.85).
2
.3.1.3. N-(6-(9H-carbazol-9-yl) hexyl)-1-benzylpiperidin-4-amine (9c)
According to the general method, a mixture of intermediate 4 (0.330g, 1mmol), 4-amino-N-
benzylpiperidine (0.19 g, 1mmol) and TEA (0.12g, 1.2 mmol) were dissolved in 30 mL
acetonitrile and refluxed at 85 ˚C for 24 h. After the reaction completed, the resulting
precipitated was filtered, washed with acetonitrile and dried at room temperature. The resulting
product was purified by recrystallization in n-hexane and DCM to afford compound 9c. White
1
solid; yield: 75.9 %, mp: 205-208 ˚C. H NMR (CDCl ,300 MHz) δ ppm: 0.84-0.91 (m, 2H),
3
1
2
.83-2.13 (m, 12H), 2.79 (t, J = 7.2 Hz, 2H), 2.93-3.03 (m, 3H), 3.54 (s, 2H), 4.24 (t, J = 6.6 Hz,
H), 7.21 (t, J = 7.5 Hz, 2H), 7.25-7.32 (m, 5H+ CDCl ), 7.37 (d, J = 8.1 Hz, 2H), 7.46 (t, J = 7.8
3
1
3
Hz, 2H), 8.08 (d, J = 7.5 Hz, 2H), NH signal not detected. C NMR (DMSO, 100 MHz): δ ppm:
1
4
40.31, 139.91, 128.92, 128.04, 125.65, 125.55, 121.96, 120.21, 118.55, 109.21, 57.34, 53.20,
+
2.40, 41.95, 37.46, 31.70, 26.17, 23.73. Formula: C H N ; MS: m/z 439.4 [M ] (0.75), 291.1
3
0
37
3
(
5.79), 236.3 (11.13), 188.2 (39.99), 167.1 (26.86), 127.0 (60.97), 91.1 (48.60), 74.1 (8.37), 57.1
100).
(
2
.3.1.4. 9-(4-(4-benzylpiperidin-1-yl) butyl)-9H-carbazole (9d)

According to the general method, a mixture of intermediate 2 (0.30 g, 1mmol), 4-
benzylpiperidine (0.175 g, 1mmol) and TEA (0.12g, 1.2 mmol) were dissolved in 30 mL
acetonitrile and refluxed for 24 h. After the completion of reaction, the mixture was dried with a
rotatory evaporator and then extracted with dichloromethane (DCM) and water. The organic
fraction was dried with anhydrous sodium sulfate (Na SO ), evaporated and purified by column
2
4
chromatography using n-Hexane/Ethyl acetate (20/1) as eluent to afford compound 9d. White
1
solid; yield: 78.6 %, mp: 71-73˚C. H NMR (DMSO, 400 MHz) δ ppm: 1.06-1.15 (m, 2H), 1.21-
1
2
3
.25 (m, 1H), 1.39-1.47 (m, 4H), 1.66-1.78 (m, 4H), 2.20 (t, J = 7.2 Hz, 2H), 2.44 (d, J = 6.8 Hz,
H), 2.69-2.72 (br, 2H), 4.37 (t, J = 7.2 Hz, 2H), 7.12 (d, J = 7.2 Hz, 2H), 7.18 (t, J = 7.6 Hz,
H), 7.25 (t, J = 7.6 Hz, 2H), 7.43 (t, J = 7.2 Hz, 2H), 7.60 (d, J = 8.0 Hz, 2H), 8.14 (d, J = 8.0
1
3
Hz, 2H). C NMR (DMSO, 400 MHz): δ ppm: 140.81, 140.40, 129.42, 128.54, 126.15, 126.05,
1
22.46, 120.71, 119.04, 109.71, 57.84, 53.70, 42.89, 42.45, 37.96, 32.20, 26.67, 24.23. Formula:
+
C H N ; MS: m/z 396.4 [M ] (10.41), 222.2 (2.64), 188.3 (100), 180.2 (14.42), 91.2 (11.80),
28
32
2
5
7.2 (5.76).
2
.3.1.5. 9-(5-(4-benzylpiperidin-1-yl) pentyl)-9H-carbazole (9e)
According to the general method, a mixture of intermediate 3 (0.315g, 1mmol), 4-
benzylpiperidine (0.175 g, 1mmol) and TEA (0.12g, 1.2 mmol) were dissolved in 30 mL
acetonitrile and refluxed at 85 ˚C for 24 h. After the reaction completed, the mixture was dried
with a rotatory evaporator and then extracted with dichloromethane (DCM) and water. The
2 4
organic fraction was dried with anhydrous sodium sulfate (Na SO ), evaporated and purified by
column chromatography using n-Hexane/Ethyl acetate (20/1) as eluent to afford 9e. White solid;
1
yield: 80.4 %, mp: 53-55˚C. H NMR (DMSO,400 MHz) δ ppm: 1.03-1.11 (m, 2H), 1.21-1.29
(m, 2H), 1.35-1.45 (m, 5H), 1.66 (t, J = 10.8 Hz, 2H), 1.71-1.79 (m, 2H), 2.09 (t, J = 7.2 Hz, 2H),
2
7
2
1
2
2
9
.44 (d, J = 6.8 Hz, 2H), 2.68-2.71 (br, 2H), 4.36 (t, J = 7.2 Hz, 2H), 7.12 (d, J = 7.2 Hz, 2H),
.18 (t, J = 7.6 Hz, 3H), 7.25 (t, J = 7.6 Hz, 2H), 7.43 (t, J = 8.0 Hz, 2H), 7.57 (d, J = 8.4 Hz,
1
3
H), 8.14 (d, J = 7.6 Hz, 2H). C NMR (DMSO, 400 MHz): δ ppm: 140.36, 139.92, 128.91,
28.04, 125.64, 125.57, 121.97, 120.20, 118.54, 109.18, 58.04, 53.32, 42.39, 42.14, 37.49, 31.70,
+
8.33, 26.20, 24.46. Formula: C29
H
34
N
2
; MS: m/z 410.4 [M ] (8.09), 319.3 (0.48), 244.3 (2.85),
30.3 (3.63), 217.2 (0.47), 203.2 (2.46), 188.3 (100), 180.2 (15.57), 167.2 (4.06), 152.2 (4.24),
1.2 (14.63), 69.2 (12.68).

2
.3.1.6. 9-(6-(4-benzylpiperidin-1-yl) hexyl)-9H-carbazole (9f)
According to the general method, a mixture of intermediate 4 (0.330g, 1mmol), 4-
benzylpiperidine (0.175 g, 1mmol,) and TEA (0.12g, 1.2 mmol) were dissolved in 30 mL
acetonitrile and refluxed at 85 ˚C for 24 h. After the reaction completed, the resulting
precipitated was filtered, washed with acetonitrile and dried at room temperature. The resulting
product was purified by recrystallization in n-hexane and DCM to afford 9f. White solid; yield:
1
8
5
2
7
3.8 %, mp: 82-84˚C. H NMR (CDCl
3
, 500 MHz): δ ppm: 1.31-1.36 (m, 4H), 1.41-1.53 (m,
H), 1.63-1.65 (br, 2H), 1.82 (t, J = 12.0 Hz, 2H), 1.87-1.93 (m, 2H), 2.26 (t, J = 7.5 Hz, 2H),
.55 (d, J= 7.0 Hz, 2H), 2.87-2.89 (br, 2H), 4.32 (t, J = 7.0 Hz, 2H), 7.17 (d, J = 7.5 Hz, 2H),
.21-7.28 (m, 3H), 7.30 (t, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H) 7.49 (t, J = 7.5 Hz, 2H), 8.13
1
3
(
d, J = 8.0 Hz, 2H). C NMR (CDCl , 300 MHz): δ ppm: 140.80, 140.43, 129.17, 128.18,
3
1
2
1
25.79, 125.61, 122.83, 120.37, 118.74, 108.68, 59.06, 54.02, 43.29, 43.02, 38.03, 32.23, 28.96,
+
7.52, 27.29, 26.99. Formula: C H N ; MS: m/z 424.4 [M ] (7.75), 331.2 (4.33), 244.3 (6.08),
3
0
36
2
88.3 (100), 180.2 (46.18), 91.2 (11.07).
2
.3.1.7. 3-((1-benzylpiperidin-4-yl) amino)-1-(9H-carbazol-9-yl) propan-1-one (9g)
According to the general method, a mixture of intermediate 5 (0.257g, 1mmol), 4-amino-N-
benzylpiperidine (0.190 g, 1mmol) and TEA (0.12g, 1.2 mmol) were dissolved in 30 mL
acetonitrile and stirred at 0˚C for 1h. After the reaction completed, the resulting precipitated was
filtered, washed with acetonitrile and dried at room temperature to afford compound 9g. White
1
solid; yield: 82.7 %, mp: 206-208˚C. H NMR (DMSO, 400 MHz) δ ppm: 1.64-1.67 (m, 2H),
2
3
.01-2.08 (m, 4H), 2.88-2.90 (br, 2H), 3.10-3.15 (m, 1H), 3.41 (t, J = 6.4 Hz, 2H), 3.50 (s, 2H),
.77 (t, J = 6.4 Hz, 2H), 7.26-7.34 (m, 5H), 7.46 (t, J = 7.2 Hz, 2H), 7.55 (t, J = 8.4 Hz, 2H), 8.23
1
3
(
d, J = 7.2 Hz, 2H), 8.28 (d, J = 8.4 Hz, 2H), 9.11 (br, 0.98H), NH signal not detected. C NMR
(DMSO, 400 MHz): δ ppm: 171.01, 138.27, 129.35, 128.68, 128.02, 127.53, 126.26, 124.38,
1
4
20.77, 116.91, 116.02, 61.94, 54.88, 51.36, 36.08, 31.15, 28.49. Formula: C H N O; MS: m/z
27 29 3
+
11 [M ] (1.75), 320.3 (1.08), 221.2 (26.79), 188.2 (10.39), 167.2 (90.29), 91.1 (100), 55.1
(36.03).

2
.3.1.8. 3-(4-benzylpiperidin-1-yl)-1-(9H-carbazol-9-yl) propan-1-one (9h)
According to the general method, a mixture of intermediate 5 (0.257g, 1mmol), 4-
benzylpiperidine (0.175 g, 1 mmol) and TEA (0.12g, 1.2 mmol) were dissolved in 30 mL
acetonitrile and stirred at 0˚C for 1 h. After the reaction completed, the resulting precipitated was
filtered, washed with acetonitrile and dried at room temperature to afford 9h. White solid; yield:
1
6
3
3.1 %, mp: 218-220˚C. H NMR (DMSO, 400 MHz) δ ppm: 1.51-1.65 (m, 2H), 1.76-1.84 (m,
H), 2.56 (d, J = 6.8 Hz, 2H), 2.98-3.06 (m, 2H), 3.50-3.54 (m, 2H), 3.60-3.63 (br, 2H), 3.87 (t, J
=
=
7.2 Hz, 2H), 7.22 (t, J = 7.2 Hz, 3H), 7.31 (t, J = 7.6 Hz ,2H), 7.46 (t, J = 7.6 Hz, 2H), 7.55 (t, J
1
3
8.4 Hz, 2H), 8.23 (d, J = 7.2 Hz, 2H), 8.32 (d, J = 8.4 Hz, 2H), 10.24 (br, 0.89H). C NMR
(DMSO, 400 MHz): δ ppm: 170.40, 139.45, 137.75, 128.99, 128.26, 127.57, 126.03, 125.77,
1
3
28 2
23.92, 120.24, 116.58, 52.21, 51.83, 41.46, 34.89, 33.31, 28.82. Formula: C27H N O; MS: m/z
+
96.4 [M ] (8.15), 221.2 (14.45), 188.3 (100), 167.2 (49.39), 140.1 (6.27), 91.2 (19.37), 55.2
(17.57).
2
.3.1.9. 4-(4-benzylpiperidin-1-yl)-1-(9H-carbazol-9-yl) butan-1-one (9i)
According to the general method, a mixture of intermediate 6 (0.271g, 1mmol), 4-
benzylpiperidine (0.175 g, 1mmol) and TEA (0.12g, 1.2 mmol) were dissolved in 30 mL
acetonitrile and stirred at 80˚C for 24 h. After the reaction completed, the resulting precipitated
was filtered, washed with acetonitrile and dried at room temperature and recrystallization in
1
acetonitrile to afford 9i. White solid; yield: 78.5 %, mp: 199-204˚C. H NMR (DMSO, 400
MHz): δ ppm: 1.60.1.66 (m, 2H), 1.72-1.80 (m, 3H), 2.22 (br, 2H), 2.53 (d, J = 6.4 Hz, 2H),
2
2
.86-2.89 (br, 2H), 3.17 (br, 2H), 3.41-3.50 (m, 4H), 7.20 (t, J = 7.2 Hz, 3H), 7.31 (t, J = 6.8 Hz,
H), 7.43 (t, J = 6.8 Hz, 2H), 7.52 (t, J = 7.2 Hz, 2H), 8.21 (d, J = 7.2 Hz, 2H), 8.34 (d, J = 8.4
1
3
Hz, 2H), 10.53 (br, 0.89H). C NMR (DMSO, 400 MHz): δ ppm: 172.48, 139.49, 137.89,
1
3
28.97, 128.27, 127.45, 126.02, 125.66, 123.64, 120.14, 116.65, 55.27, 51.66, 41.51, 35.71,
+
5.04, 28.63, 18.68. Formula: C28
H
30
N
2
O; MS: m/z 410.4 [M ] (7.29), 244.3 (23.93), 215.3
(2.41), 202.3 (5.68), 188.3 (100), 167.2 (15.11), 91.2 (18.85), 70.2 (6.37).
2
.3.2.1. 1-((1-benzylpiperidin-4-yl) amino)-3-(9H-carbazol-9-yl) propan-2-ol (9j)
According to the general method, in a round-bottomed flask (100 mL), a mixture of intermediate
(0.223 g, 1 mmol), appropriate 4-amino-N-benzylpiperidine (0.190 g, 1 mmol) in EtOH
7

(30mL), was refluxed at 65 ˚C for 24 h. Then, the resulting products were purified by plate
1
chromatography to afford compound 9j. yellow oil; yield: 56.7 %. H NMR (DMSO, 400 MHz):
δ ppm: 1.21-1.32 (m, 2H), 1.72-1.77 (m, 2H), 1.89 (t, J = 11.2 Hz, 2H), 2.32-2.38 (m, 1H), 2.56-
2
.74 (m, 5H), 3.40 (s, 2H), 3.99-4.04 (m, 1H), 4.27-4.33 (dd, J = 14.8, 6.8 Hz, 1H), 4.44-4.49
(dd, J = 14.4, 5.2 Hz, 1H), 7.18 (t, J = 7.2 Hz, 2H ), 7.24-7.32 (m, 5H), 7.43 (t, J = 7.2 Hz,
1
3
2
4
1
H),7.66 (d, J=8.4 Hz, 2H ), 8.13 (d, J = 8.0 Hz, 2H), NH signal not detected . C NMR (DMSO,
00 MHz): δ ppm: 140.55, 138.61, 128.69, 128.07, 126.75, 125.42, 121.97, 119.99, 118.58,
09.79, 68.75, 62.17, 55.98, 54.70, 51.81, 50.02, 47.03, 31.87, 18.54. Formula: C H N O.
2
7
31
3
2
.3.2.2. 1-((1-benzylpiperidin-4-yl) amino)-3-(3,6-dibromo-9H-carbazol-9-yl) propan-2-ol (9k)
According to the general method, in a round-bottomed flask (100 mL), a mixture of intermediate
(0.381 g, 1 mmol), appropriate 4-amino-N-benzylpiperidine (0.190 g, 1 mmol) in EtOH
30mL) was refluxed at 65 ˚C for 24 h. Then, the resulting product was purified by
8
(
recrystallization with ethanol to afford compounds 9k. white solid; yield: 55.7 %, mp: 92-94˚C.
1
H NMR (DMSO, 400 MHz): δ ppm: 1.20-1.28 (m, 2H), 1.73 (br, 2H), 1.91 (t, J = 10.8 Hz, 2H),
2
1
7
4
1
.27-2.32 (m, 1H), 2.50-2.62 (m, 2H), 2.72 (d, J = 10.8 Hz, 2H), 3.41 (s, 2H+ DMSO), 3.90 (br,
H), 4.23-4.28 (dd, J = 14.8, 7.2 Hz, 1H), 4.42-4.46 (dd, J = 14.8, 4.0 Hz, 1H), 5.04 (br, 1H),
1
3
.21-7.32 (m, 5H), 7.57-7.64 (m, 4H), 8.45 (s, 2H), NH signal not detected. C NMR (DMSO,
00 MHz) δ ppm: 139.71, 138.68, 128.68, 128.54, 128.07, 126.74, 123.12, 122.86, 112.23,
2 3
11.14, 69.11, 62.23, 56.00, 54.70, 51.91, 50.13, 47.34, 32.34, 18.54. Formula: C27H29Br N O.
2
.3.2.3. 1-(4-benzylpiperidin-1-yl)-3-(9H-carbazol-9-yl) propan-2-ol (9l)
According to the general method, in a round-bottomed flask (100 mL), a mixture of intermediate
(0.223 g, 1 mmol), appropriate 4-benzylpiperidine (0.175 g, 1 mmol) in EtOH (30mL) was
7
refluxed at 65 ˚C for 24 h. Then, the resulting products were purified by column chromatography
1
on silica gel to afford compounds 9l. white solid; yield: 59.12 %, mp: 88-90˚C. H NMR
(DMSO, 500 MHz): δ ppm: 1.21-1.33 (m, 2H), 1.51-1.52 (m, 1H), 1.62 (br, 2H), 1.83 (t, J = 11.5
Hz, 1H), 2.19 (t, J = 11.5 Hz, 1H), 2.38-2.47 (m, 2H), 2.53 (d, J = 7.0 Hz, 2H), 2.75 (d, J = 11.0
Hz, 1H), 2.90 (d, J = 10.5 Hz, 1H), 3.71-3.72 (m, 1H), 4.21-4.24 (m, 1H), 4.35-4.44 (m, 2H),
7
.15 (d, J = 7.5 Hz, 2H), 7.22 (t, J = 7.0 Hz, 1H), 7.26-7.32 (m, 4H), 7.48-7.53 (m, 4H), 8.13 (d,

1
3
J = 7.5 Hz, 2H). C NMR (DMSO, 500 MHz): δ ppm: 141.08, 140.52, 129.12, 128.23, 125.89,
1
3
25.75, 123.00, 120.28, 119.12, 109.22, 66.28, 62.10, 55.58, 52.33, 47.30, 43.08, 37.73, 32.41,
2.12. Formula: C H N O.
2
7
30
2
2
.3.2.4. 1-(4-benzylpiperidin-1-yl)-3-(3,6-dibromo-9H-carbazol-9-yl) propan-2-ol (9m)
According to the general method, in a round-bottomed flask (100 mL), a mixture of intermediate
(0.381, 1 mmol), appropriate 4-benzylpiperidine (0.175 g, 1 mmol) in EtOH (30mL) was
8
refluxed at 65 ˚C for 24 h. After the reaction completed, the resulting products were purified by
recrystallization with ethanol afford desired compounds 9m. white solid; yield: 53.1 %, mp: 134-
1
1
1
36 ˚C. H NMR (CDCl , 300 MHz): δ ppm: 1.16-1.34 (m, 2H), 1.49-1.64 (m, 3H), 1.87 (t, J =
3
1.4 Hz, 1H), 2.20 (t, J = 11.7 Hz, 1H), 2.36 (d, J = 6.6 Hz, 2H), 2.51 (d, J = 6.3 Hz, 2H), 2.73
(d, J = 10.5 Hz, 1H), 2.89 (d, J = 10.5 Hz, 1H), 4.15-4.17 (m, 1H), 4.29 (br, 2H), 7.13 (d, J = 7.2
Hz, 2H), 7.20 (t, J = 6.9 Hz, 1H), 7.28 (t, J= 8.1 Hz, 2H), 7.37 (d, J= 8.7 Hz, 2H), 7.56 (d, J = 8.4
1
3
Hz, 2H), 8.13 (s, 2H). C NMR (CDCl
3
, 400 MHz): δ ppm: 140.35, 139.98, 129.12, 129.06,
1
3
28.22, 125.91, 123.58, 123.12, 112.33, 111.06, 66.22, 61.71, 55.56, 52.38, 47.37, 42.97, 37.61,
2 2
2.25, 31.95. Formula: C27H28Br N O.
2
2
.4. Biological activity
.4.1. In vitro inhibition studies on AChE
The inhibitory activity of compounds 9a-m was investigated against AChE using the Ellman’s
method. Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), 5, 5′-dithiobis-( 2-
nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATCI) and donepezil were purchased from
Sigma Aldrich company. For the assay, donepezil was used as the reference compound. The
stock solutions of the synthesized compounds were prepared in DMSO (20 mM). The assay
solution consisted of 2000 μl of phosphate buffer (0.1 M, pH = 8.0), 60 μl of DTNB (0.01 M), 20
μl of AChE (1ku prepared in phosphate buffer (0.1 M) and glycerine 25%, pH=8.0, 2 units/mL in
0
.5 mL aliquots) and 30 μl of various concentrations of tested compounds were incubated 15 min
at room temperature. Then, 20 μl of the substrate, acetylthiocholine iodide (0.15 M), was added
and the changes in absorbance were measured at the wavelength of 412 nm in the 2 min intervals
using a Synergy HTX Multi-Mode Reader-BioTek. The IC50 values for compounds with
percentage of inhibition higher than 50% at the screening concentration (20 mM) were calculated

using nonlinear regression analysis of the response–concentration (log) curve. Results are
presented as the mean ± standard deviation (SD) at least three different experiments performed in
triplicate.
2
.4.2. In vitro inhibition studies on BuChE
The BuChE inhibition activity of compounds 9a-m was performed using the Ellman’s method in
9
2
6-well plates. Butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum), 5, 5′-dithiobis-(
-nitrobenzoic acid) (DTNB) and butylthiocholine iodide (BTCI) were obtained from Sigma
Aldrich company. The stock solutions of testing compounds were prepared in DMSO (20 mM)
and diluted in MeOH to obtain final concentrations. The assay solution consisted of 200 μl of
phosphate buffer (0.1 M, pH = 7.4), 20 μl of DTNB (0.01 M), 20 μl of BuChE and 10 μl of
various concentrations of tested compounds (0.1-30 µM) were incubated 15 min at room
temperature. Then, 20 μl of the substrate, butylthiocholine iodide (0.15 M), was added and the
changes in absorbance were measured at the wavelength of 412 nm in the 2 min intervals using a
Synergy HTX Multi-Mode Reader-BioTek. The IC50 values for compounds with percentage of
inhibition higher than 50% at the screening concentration (20 mM) were calculated using
nonlinear regression analysis of the response–concentration (log) curve. Results are presented as
the mean ± standard deviation (SD) at least three different experiments performed in triplicate.
2
.4.3. Kinetic study of AChE inhibition
The mechanism of AChE inhibition for compound 9g was investigated at different
concentrations, 14.1, 28.2, and 56.3 µM using Ellman’s method. The reciprocal plot of 1/velocity
versus 1/[substrate] was plotted at different concentrations of the substrate acetylthiocholine
(0.47, 0.94 and 2.31 mM). The plots were assessed by a weighted least-squares analysis that
assumed the variance of velocity (v) to be a constant percentage of v for the entire data set.
Slopes of these reciprocal plots were then plotted against the concentration of 9g in a weighted
analysis, and the inhibition constant (K ) was determined as the intercept on the negative x-axis.
i
Data analysis was performed using Microsoft Excel 2016.
2
.4.4. Kinetic study of BuChE inhibition

The mechanism of BuChE inhibition for compound 9c was investigated at different
concentrations, 0.18, and 0.36 µM using Ellman’s method. The reciprocal plot of 1/velocity
versus 1/[substrate] was plotted at different concentrations of the substrate butyrylcholinesterase
(0.05, 0.17, 0.25, 0.37 and 0.56 mM). The plots were assessed by a weighted least-squares
analysis that assumed the variance of velocity (v) to be a constant percentage of v for the entire
data set. Slopes of these reciprocal plots were then plotted against the concentration of 9c in a
weighted analysis, and the inhibition constant (Ki) was determined as the intercept on the
negative x-axis. Data analysis was performed using Microsoft Excel 2016.
2
.4.5. BACE1 assay
BACE1 fluorescence resonance energy transfer (FRET) assay kit of PanVera was used following
the supplied protocol with small modifications. The kit containing BACE1 enzyme (purified
baculovirus-expressed BACE1), the substrate (Rh-EVNLDAEFK-quencher, based on the
Swedish” mutant of APP), reaction buffer (50 mM sodium acetate, pH = 4.5) and stop solution.
The test compounds stock solutions were prepared in DMSO and further diluted with assay
buffer (50 mM sodium acetate; pH = 4.5) to prepare a 3X concentration. The final concentration
of DMSO was 6% (v/v). 10 μL of 3X concentration BACE1 substrate was added to 10 μL of
each test compound (3X concentration) in separate wells of a black 96-well microplate and
gently mixed. Then 10 μL of 3X BACE1 enzyme was added to each well to start the reaction.
The reaction mixtures were incubated at 25 °C for 90 min in the dark and to stop the reaction, 10
µL of the stop solution (2.5 M sodium acetate) was added. Finally, the fluorescent intensity of
the enzymatic product was measured using a multiwell spectrofluorometer instrument (BMG
LABTECH, Polar star, Germany) capable of measuring at 544 nm excitation and 590 nm
emission wavelengths. Each experiment was repeated 3–5 times. The IC values were
5
0
determined using nonlinear regression (GraphPad Prism 5) by plotting the residual enzyme
activities against the applied inhibitor concentration [38].
2
.4.6. Metal chelating assay

The study of metal chelation was performed in methanol using UV–vis spectrophotometer
Agilent 100, in a 1ꢀcm quartz) with wavelength ranging from 200 to 600ꢀnm. A fixed amount of
compounds 9b and 9c (25ꢀμM) were mixed with increasing amounts of FeSO and ZnCl (0–
(
4
2
8
0ꢀμM) for 30ꢀmin at room temperature [38]. The stoichiometry of the ligands : Zn(II) was
determined by employing the mole ratio method, through titration of the methanol solution of the
selected compounds with ascending concentrations of mentioned metal [39].
2
.4.7. Docking study method
The co-crystal structures of AChE (PDB code: 1EVE) and BuChE (PDB code: 5K5E) were
obtained from the RCSB Protein Data Bank (www.rcsb.org). Docking studies were carried out
using the Autodock Vina (1.1.2) with the docking parameters set at default values. The docking
procedure was validated by re-docking the original ligands into the AChE and BuChE proteins.
For docking studies, the structure of compounds was drawn, optimized and saved in mol format
2
which then converted into the pdbqt format using Auto Dock Tools package (1.5.6). To prepare
protein, cognate ligand and water molecules were removed and hydrogen atoms were
subsequently added to the protein. Non-polar hydrogens were merged with their corresponding
carbons and then the protein was converted into the required pdbqt format using Auto Dock
Tools package (1.5.6). The size of grid box was set at 30× 30 × 30 and the center of grid box
was placed at the co-crystalized ligand with coordinates x = 5.16, y = 66.91, z = 66.42 for 1EVE
and x = 2.35, y = 12.53, z = 15.40 for 5K5E. The exhaustiveness was set to 100 and the other
docking parameters were set as default. Finally, the conformations with the most favorable free
energy of binding were selected for analyzing the enzyme-inhibitor interactions.
3
. Results and discussion
3
.1. Design

The purpose of this study was the design and synthesis of a novel series of carbazole-
benzylpiperidine hybrid with increased biological properties, such as anti-BuChE, anti-BACE1
and metal chelating properties. Donepezil (a), an FDA-approved drug for AD treatment, is a
potent and selective AChE inhibitor [30]. The main pharmacophoric part of this drug, N-
benzylpiperidine moiety that interacts with the CAS of AChE [28], incorporated in the design of
the final scaffold of target compounds. While the indanone fragment that interacts with PAS of
AChE [28], replaced with carbazole group in order to provide novel carbazole-benzylpiperidine
hybrids. Carbazole is a tricyclic structure with various biological activities related to Alzheimer's
disease that previously some of its derivatives have been reported with anti-BACE1 (b),
neuroprotective (c), anti-AChE (d), anti-Aβ-aggregation (e), anti BuChE and antioxidative
activity [37, 40, 41] (Fig.1). Since, benzylpiperidine derivatives (4-amino-N-benzylpiperidine
and 4-benzylpiperidine) attached to the carbazole moiety by various linkers to produce the target
hybrids with cholinesterase and BACE1 inhibitory potential.

Fig. 1. The design strategy of novel carbazole-benzylpiperidine hybrids.
3
.2. Chemistry
The general method for the synthesis of the novel carbazole-benzylpiperidine hybrids 9a-m is
shown in Scheme 1. These novel hybrids were synthesized in a two-step route. The intermediates
were synthesized according to the previous methods by some modifications [30, 37]. To prepare
compounds 9a-f, in the first step, intermediates 2-4 bearing different alkyl chains were
synthesized via N-alkylation of carbazole (1a) using different di-bromoalkane derivatives in dry
DMF and NaH as a base (67-73% yields). In the next step, the resulting intermediates (2-4)

reacted with corresponding amines in acetonitrile and triethylamine (TEA) in order to obtain
desire compounds. Compounds 9g-i were prepared by N-acylation of carbazole using different
acyl-chlorides in acetonitrile and TEA under reflux conditions in order to afford compounds 5
and 6 (60-65% yields). Afterward, the further reaction of these intermediates (5, 6) with
corresponding amines in acetonitrile and TEA resulted in compounds 9g-i. Compounds 9j-m
were obtained via N-alkylation of carbazole (1a) or 3,6-dibromocarbazole (1b) with
epichlorohydrin in dry DMF and KOH with 52-55% yields and subsequent reaction of these
resulting epoxide intermediates (7, 8) with corresponding amines in EtOH. Different
1
13
spectroscopic techniques, such as Mass, H NMR and C NMR were used to confirmed
chemical structures of the synthesized compounds.

Scheme 1. Synthesis of carbazole-benzylpiperidine hybrids 9a-m. Reagents and conditions: a) di-
bromoalkanes, NaH, DMF, r.t., 30 min; b) 4-amino-N-benzylpiperidine, TEA, acetonitrile, reflux 24 h; c)
4
-benzylpiperidine, TEA, acetonitrile, reflux 24 h; d) 3-chloropropionyl chloride or 4-chlorobutyryl
chloride, TEA, acetonitrile, reflux 48 h; e) 4-amino-N-benzylpiperidine, TEA, acetonitrile, r.t., 1 h; f) 4-
benzylpiperidine, TEA, acetonitrile, r.t., 1 h; g) epichlorohydrin, KOH, DMF, r.t., 24 h; h) 4-amino-N-
benzylpiperidine, EtOH, 65 ˚C, 24 h; i) 4-benzylpiperidine, EtOH, 65 ˚C, 24 h.

3
3
.3. Biological activity
.3.1. AChE and BuChE inhibitory activity
The AChE (from electric eel) and BuChE (from equine serum) inhibitory activities of the
synthesized compounds 9a-m were evaluated by Ellman’s assay [42]. In the first step, the
inhibition percentage of synthesized compounds was determined at 20 mM for eelAChE and
eqBuChE. In the following, the IC50 values of the synthesized compounds that displayed
inhibitory potency higher than 50% were determined in comparison with donepezil as a control.
The results of the biological evaluation are shown in Table 1.
According to the results, synthesized compounds exhibited moderate eelAChE inhibitory activity
with IC50 values in the micromolar range. Among synthesized derivatives, the most active
compounds against eelAChE, 9b, 9c, 9g and 9i demonstrated IC values of 16.5, 26.5, 13.4 and
5
0
2
6.9 µM respectively.
Investigation of the results on eqBuChE showed that all the synthesized compounds except 9k,
m and 9h were potent for inhibition of eqBuChE with IC values of 0.18-12.63 µM (Table 1).
9
5
0
The most active derivative for inhibition of eqBuChE was compound 9c bearing C6 alkyl linker
with IC value of 0.18 µM.
5
0
3
.3.2. Structure activity relationship (SAR)
According to the biological results, N-alkyl-carbazole derivatives that attached to the 4-amino-N-
benzylpiperidine fragment with C5 and C6 alkyl linkers (9b with IC = 16.5 ± 0.21 µM and 9c
5
0
with IC = 26.5 ± 0.17 µM), as well as the N-acyl-carbazole derivative with C3 and C4 spacers
5
0
that attached to the 4-amino-N-benzylpiperidine and 4-benzylpiperidine respectively (9g with
IC50 = 13.4 ± 0.08 µM and 9i with IC50 = 26.9 ± 0.26 µM), were active for inhibition of
eelAChE. These results indicated that the inhibitory activity against eelAChE significantly
affected by the length and nature of the linker between carbazole and benzylpiperidine fragment.
In general, derivatives containing alkyl linker were more potent than derivatives containing acyl
and 2-hydroxypropyl linkers. Among the compounds with alkyl linkers (9a-f), 4-amino-N-
benzylpiperidine derivatives were more active than 4-benzylpiperidine derivatives that indicated
the nitrogen atom in the linker may form an effective hydrogen bond with the active site of
eelAChE. In addition, for 4-amino-N-benzylpiperidine derivatives, the inhibitory potency against

eelAChE increased in the order of C5 > C6 > C4 linker length. Unexpectedly, compound 9g
IC50 = 13.4 ± 0.08 µM) containing acyl linker with C3 spacer, displayed higher activity than
(
other compounds. This higher activity might be due to the more favorable orientation of
compound 9g inside the active site of eelAChE.
According to the results, the modification of the linker between carbazole and benzylpiperidine
fragment significantly influenced the eqBuChE inhibitory activity. Among synthesized
compounds, 9a-f derivatives that in which N-benzylpiperidine moiety were attached to the
carbazole fragment through four-, five-, and six-membered alkyl spacers, respectively, displayed
superior inhibitory potential against eqBuChE compared to other derivatives. It was also
observed that replacement of 4-amino-N-benzylpiperidine fragment with 4-benzylpiperidine has
slightly influence on eqBuChE inhibitory activity (9b with IC50 = 0.59 ± 0.17 µM vs 9e with 1.39
±
0.51 µM and 9c with IC = 0.18 ± 0.08µM vs 9f with 0.81 ± 0.17 µM). Results showed that
50
replacement of 9H-carbazole moiety with 3,6-dibromocarbazole in 4-benzylpiperidine
derivatives that bearing 2-hydroxypropyl linker resulted in reduced inhibitory potential against
eqBuChE, 9l with 2.4 ± 0.85 µM vs 9m (inactive) (Table 1).

Table 1. In vitro inhibition of eelAChE and eqBuChE of compounds 9a-m and donepezil.
a
a
Compounds
Linker
R
H
H
eelAChE IC50 (µM) ,
eqBuChE IC (µM) ,
50
%inhibition
b
b
%
inhibition
9
9
a
33.18%
5.4 ± 0.70
b
16.5 ± 0.21
26.5 ± 0.17
0.59 ± 0.17
9c
H
0.18 ± 0.08
9
d
H
H
H
45.02%
46.18%
47.79%
0.90 ± 0.24
1.39 ± 0.51
0.81 ± 0.17
9
e
9
f
9
9
g
H
H
H
13.4 ± 0.08
36.56%
12.6 ± 0.30
35.59%
h
9
i
26.9 ± 0.26
10.3 ± 0.12
9
j
H
23.70%
21.94%
6.4 ± 0.47
39.26 %
9
k
Br
9
l
H
16.35%
4.9%
2.4 ± 0.85
c
9
m
Br
Inactive

donepezil
IC50: 50% inhibitory concentration (mean ± SD of three independent experiments).
inhibition at 20mM concentration.
inactive at 20mM concentration.
-
-
0.027 ± 0.01
10.6 ± 2.1
a
b
c
%
3
.3.3. Kinetic study of AChE inhibition
The kinetic studies for the most active inhibitor of eelAChE (9g), was performed to elucidate the
involved mechanism of eelAChE inhibition. For this purpose, three fixed inhibitory
concentrations of test compound 9g (14.1 µM, 28.2 µM and 56.3 µM) were used and for each
concentration, the initial velocity (V) of the substrate hydrolysis was measured in the range of
0
.47-2.31 mM. The data was collected and used for drawing Lineweaver-Burk double reciprocal
plots (Fig. 2) which showed increased slopes (decreased Vmax) and preserved intercepts
unchanged K ) at higher concentrations for compound 9g as an inhibitor. This pattern
(
m
demonstrated a linear non-competitive inhibitory effect of 9g on the enzyme, which showed
compound 9g might be able to interact with the peripheral anionic site (PAS) rather than the
catalytic active site (CAS) of eelAChE. The inhibition constant (Ki) of 27.57 μM was estimated
using the slopes of double reciprocal plots versus the different concentrations of compound 9g
(
Fig. 2).

(
A)
(B)
Fig. 2. Kinetic study of eelAChE inhibition by compounds 9g. A) Lineweaver-Burk double reciprocal
plot of eelAChE initial velocity at increasing substrate concentration (0.47–2.31 mM) in the absence and
presence of 9g, B) Steady-state inhibition constant (Ki) of compound 9g.
It has been reported that AChE could promote the generation of Aβ fibrils and Aβ plaques. This
process was assumed to be mediated by an interaction between Aβ and peripheral anionic site of
the enzyme (PAS) [43-45]. That finding provided a new opportunity for the development of
AChE inhibitors that capable to block both CAS and the PAS site of the enzyme. This strategy
may provide a better therapeutic effect for the treatment of AD, since it enables simultaneous
inhibition of AChE activity and Aβ plaques generation.
3
.3.4. Kinetic study of BuChE inhibition
To obtain insight into the eqBuChE inhibition mechanism, kinetic studies were performed at
three concentrations of compound 9c. As shown in Fig. 3 graphical analysis of a Lineweaver-
m
Burk reciprocal plot demonstrated both increasing slopes (decreasing Vmax) and intercepts (K )
at increasing concentration of inhibitor 9c. This pattern revealed a mixed-type inhibitory effect of
compound 9c on eqBuChE, which is indicated that compound 9c may interact with both sites of
the enzyme (CAS and PAS). The Ki= 0.11 μM was calculated from the secondary plots of the
slope versus the concentration of inhibitors 9c (Fig. 3).

(
A)
(B)
Fig. 3. Kinetic study of eqBuChE inhibition by compounds 9c. A) Lineweaver-Burk double reciprocal
plot of eqBuChE initial velocity at increasing substrate concentration (0.05-0.56 mM) in the absence and
presence of 9c, B) Steady-state inhibition constant (Ki) of compound 9c.
3
.3.5. Molecular modelling study on AChE
In order to find the possible interaction mode of the most active derivative 9g with AChE the
docking study was carried out. For this purpose, the co-crystal structure of AChE was obtained
from the Protein Data Bank. Due to the structural similarity of synthesized compounds to
donepezil, PDB code 1EVE which AChE complexed with donepezil was selected. Docking
studies were done using Auto Dock Vina (1.1.2) program [46]. In the first step, re-docking of
ligand E20 (original ligand) into the active site of AChE was done to confirm the reliability of
the docking protocol (RMSD = 0.80). Then the most active derivative 9g was docked using
optimized parameters. The binding mode of compound 9g into AChE active site is shown in Fig.
4
. The docking results revealed that the benzyl fragment interacted with Trp84 in the CAS of the
enzyme through π-π stacking interaction. The protonated piperidine ring interacted with Asp 72.
The protonated nitrogen atom in linker forms cation-π interactions with Phe331 and hydrogen
interaction with Phe330 in the middle of the active gorge. The carbazole fragment interacted
with Trp279 in the PAS of the enzyme through π-π stacking interaction. The docking results
showed that compound 9g accommodated in the gorge of the enzyme and occupied the cavity
between CAS and PAS. The interactions of ligand 9g were similar to other ligands, but these
interactions were more effective. According to the results of the docking study, this compound

interacted with CAS and PAS of AChE simultaneously. It seems that π−π stacking, cation-π and
hydrogen bond interactions play an important role in the stability of complex which lead to the
lower binding free energy (∆G) and consequently, better biological effect of compound 9g.
Fig. 4. Binding mode of compound 9g in the active site of AChE, PDB code: 1EVE
.
3
.3.6. Molecular modelling study on BuChE
Due to the potency of synthesized compounds to inhibit the BuChE, the molecular modelling
study was also performed on BuChE. The most potent derivative 9c was selected to find the
possible interaction mode (PDB ID: 5K5E). Docking study was done according to the same
protocol used for AChE (RMSD = 0.96 Aº). The binding mode of compound 9c into the BuChE
active site is shown in Fig. 5. The docking results revealed that the ligand 9c bound to the active
site of BuChE with a folded conformation. The carbazole fragment interacted with Trp82 in the
CAS of the enzyme through π-π stacking interaction. The benzyl fragment interacted with
Trp231 in the PAS of the enzyme through H-π interaction. The protonated piperidine ring
interacted with Pro285 through hydrogen interaction. According to the results, this compound
accommodated in CAS and PAS of BuChE enzyme simultaneously.