New classes of carbazoles as potential multi-functional anti-Alzheimer's agents

Article abstract of DOI:10.1016/j.bioorg.2019.103164

Multi-Target approach is particularly promising way to drug discovery against Alzheimer's disease. In the present study, we synthesized a series of compounds comprising the carbazole backbone linked to the benzyl piperazine, benzyl piperidine, pyridine, quinoline, or isoquinoline moiety through an aliphatic linker and evaluated as cholinesterase inhibitors. The synthesized compounds showed IC₅₀ values of 0.11–36.5 μM and 0.02–98.6 μM against acetyl- and butyrylcholinesterase (AChE and BuChE), respectively. The ligand-protein docking simulations and kinetic studies revealed that compound 3s could bind effectively to the peripheral anionic binding site (PAS) and anionic site of the enzyme with mixed-type inhibition. Compound 3s was the most potent compound against AChE and BuChE and showed acceptable inhibition potency for self- and AChE-induced Aβ_1-42 aggregation. Moreover, compound 3s could significantly protect PC12 cells against H₂O₂-induced toxicity. The results suggested that the compounds 3s could be considered as a promising multi-functional agent for further drug discovery development against Alzheimer's disease.

Full text of DOI:10.1016/j.bioorg.2019.103164

Journal Pre-Proof
New classes of carbazoles as potential multi-functional anti-Alzheimer’s agents
Niloufar Choubdar, Mostafa Golshani, Leili Jalili-Baleh, Hamid Nadri, Tuba
Tüylü Kü ç ükkilinç, Beyza Ayazgök, Alireza Moradi, Farshad Homayouni
Moghadam, Zahra Abdolahi, Alieh Ameri, Fatemeh Salehian, Alireza
Foroumadi, Mehdi Khoobi
PII:
S0045-2068(19)30555-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.103164
YBIOO 103164
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 April 2019
20 July 2019
29 July 2019
Please cite this article as: N. Choubdar, M. Golshani, L. Jalili-Baleh, H. Nadri, T. Tüylü Kü ç ükkilinç, B.
Ayazgök, A. Moradi, F. Homayouni Moghadam, Z. Abdolahi, A. Ameri, F. Salehian, A. Foroumadi, M. Khoobi,
New classes of carbazoles as potential multi-functional anti-Alzheimer’s agents, Bioorganic Chemistry (2019), doi:
https://doi.org/10.1016/j.bioorg.2019.103164
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New classes of carbazoles as potential multi-functional anti-Alzheimer’s agents
Niloufar Choubdara, Mostafa Golshanib, Leili Jalili-Balehb, Hamid Nadric, Tuba Tüylü Küçükkilinçd,
Beyza Ayazgökd, Alireza Moradic, Farshad Homayouni Moghadame, Zahra Abdolahic, Alieh Amerif,
Fatemeh Salehianb, Alireza Foroumadib, Mehdi Khoobib,*
aDepartment of Organic Chemistry, Faculty of Pharmaceutical Chemistry, Tehran Medical Sciences, Islamic Azad
University, Tehran, Iran
bBiomaterials Group, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran,
1417614411, Iran
cDepartment of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shahid
Sadoughi University of Medical Sciences, Yazd, Iran
dHacettepe University, Faculty of Pharmacy, Department of Biochemistry, Ankara, Turkey
eDepartment of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR,
Isfahan, Iran
fDepartment of Medicinal Chemistry, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran
Abstract
Multi-Target approach is particularly promising way to drug discovery against Alzheimer's
disease. In the present study, we synthesized a series of compounds comprising the carbazole
backbone linked to the benzyl piperazine, benzyl piperidine, pyridine, quinoline, or isoquinoline
moiety through an aliphatic linker and evaluated as cholinesterase inhibitors. The synthesized
compounds showed IC50 values of 0.11-36.5 µM and 0.02-98.6 µM against acetyl- and
butyrylcholinesterase (AChE and BuChE), respectively. The ligand-protein docking simulations and
kinetic studies revealed that compound 3s could bind effectively to the peripheral anionic binding site
(PAS) and anionic site of the enzyme with mixed-type inhibition. Compound 3s was the most potent
compound against AChE and BuChE and showed acceptable inhibition potency for self- and AChE-
induced Aβ1-42 aggregation. Moreover, compound 3s could significantly protect PC12 cells against
H2O2-induced toxicity. The results suggested that the compounds 3s could be considered as a
promising multi-functional agent for further drug discovery development against Alzheimer's disease.
Keywords: Alzheimer's disease, acetylcholinesterase, butyrylcholinesterase, carbazoles, docking
study, β-amyloid aggregation.
* Corresponding author: The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, P.O. Box:
14155-6451, Tehran, Iran; Tel/Fax: ++98-21-64121510; E-mails: mehdi.khoobi@gmail.com
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1. Introduction
Alzheimer's disease (AD), the most common cause of dementia, is considered as a progressive and
degenerative disorder of the brain which affects approximately 10% of the population over the age of
65 [1,2]. A complex series of neurochemical factors involve in the initiation and progression of the
disease. It is associated with loss of presynaptic markers of the cholinergic system such as
acetylcholine (ACh) and other related neurotransmitters presenting in hippocampus. The presence of
neurofibrillary tangles and amyloid depositions in the brain are also some important signs of the
disease [3,4].
Over the past two decades, several hypotheses have been proposed for AD mechanism including
cholinergic hypothesis, amyloid hypothesis, and oxidative hypothesis leading to the generation of
different therapeutic approaches [5-7].
Although there have been several theories concerning the pathogenesis of AD, the molecular cause
of the disease has remained unknown. Cholinergic hypothesis suggests that the decline in cognitive
and mental functions is associated with the loss of cortical cholinergic neurotransmission. Therefore,
inhibition of acetylcholinesterase (AChE) activity could be helpful to increase the cholinergic
neurotransmission. According to the cholinergic hypothesis, AChE inhibitors (AChEIs) increase the
level of synaptic ACh, leading to improve the cholinergic function in the central nervous system [8-
10]. Consequently, AChEIs slow down the progression of AD and therefore postpone the cognitive
deterioration of patients suffering from AD [11].
Galantamine, rivastigmine, donepezil, and tacrine are four clinically approved AChEIs for the
treatment of AD. These drugs are able to enhance cholinergic neurotransmission by increasing
acetylcholine in the synaptic cleft [12]. Although, tacrine a former AChE inhibitor has been
discontinued due to the high incidence of hepatoxicity [13, 14], attempts have been continuing to
prepare modified tacrine analogues without hepatoxicity [15, 16].
The X-ray analysis of AChE reported by Sussman et al. has revealed that the active site of AChE
comprises at least four active sites including peripheral anionic binding site (PAS), esteratic site (ES),
aromatic cation-binding site (ACS) and anionic substrate binding site (AS) [17-19]. AChEIs with the
ability to interact exclusively with PAS or with both catalytic and peripheral binding sites can prevent
the AChE-induced amyloid-beta (Aβ) aggregation [20]. Dual catalytic site and PAS binding
inhibitors control the disease through cholinesterase inhibition as well as the prevention of AChE-Aβ
complex formation, which is more toxic than self-induced Aβ aggregation. Several studies have
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shown that some AChEIs have a dual action thereby facilitate the cholinergic transmissions and
interfere in the deposition and aggregation of amyloid peptides [21, 22].
Carbazoles are naturally occurring phytochemicals in many plants with a wide range of biological
activities. Naturally occurring carbazoles and their synthetic derivatives have demonstrated wide
range of pharmacological activities [23-25]. Carbazole derivatives have increasingly gained interest
as lead scaffold in the design of anti-AD agents. Carbazole-tacrine hybrids could interact
simultaneously with the CAS and PAS of AChE and showed appropriate antioxidant activity. They
could efficiently protect neuroblastoma cells against toxicity induced by Aβ1-40 and H2O2 (compound
a, Fig. 1) [26]. Additionally, carbazole derivatives bearing pyridinium or quinolinium salts showed
perfect inhibitory activity against self-induced and AChE-induced Aβ aggregation [27] and potent
neuroprotective activity against Aβ in human neuronal-like cells (compound b, Fig. 1) [25]. β-
carbolinium salts also showed potent inhibition activity against both AChE/BChE. The most potent
derivative showed IC50 values of 0.5 µM for AChE and 5.7 µM for BChE, respectively (compound c,
Fig. 1) [28].
Figure 1
In the light of the above findings and in pursuit of searching for multifunctional drug candidates as
potent anti-AD [29-33], in the present study we introduce a new class of carbazole derivatives bearing
benzyl piperazine, piperidine, pyridinium, quinolinium, or isoquinolinium part as well-known
pharmacophores inhibiting CAS of AChE linked through aliphatic carbon chain length together. The
synthesized compounds were evaluated as multifunctional agents against AChE and BuChE, self- and
AChE-induced amyloid-beta protein 1-42 (Aβ1-42) peptide aggregating, and hydrogen peroxide-
induced neurotoxicity. Kinetic study and ligand-AChE docking simulation were also done to
elucidate the mechanism of the ligand interaction with enzyme.
2. Results and Discussion
2.1. Chemistry
Target compounds were easily synthesized from commercially available carbazole. Compound 1
was synthesized via the reaction of carbazole with different alkyl dibromides in the presence of NaH
in DMF. The intermediate 2 was then reacted with excess amount of piperazine, KI in EtOH under
reflux condition followed by reaction with different substituted benzyl halides in the presence of

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catalytic amounts of K2CO3 in dry acetonitrile under reflux condition to afford the desired final
products 3a-r (Scheme 1). Also, products 3s-w were directly synthesized from the reaction of
compound 1 with quinoline, isoquinoline, pyridine, piperidine or 4-benzylpiperidine in dry
acetonitrile under reflux condition.
All reactions were monitored by TLC and the products were purified by flash column
1
13
chromatography using petroleum ether/ethyl acetate (10:1- 3:1) as the eluent. H, C NMR and MS
spectroscopy were utilized for products characterization.
Scheme 1
2.2. Anti-cholinesterase activity
The inhibitory activities of the synthesized compounds 3a-w were evaluated in vitro against AChE
and BuChE enzymes based on Ellman procedure [34]. The results are presented as IC50 values in
Table 1 comparing to donepezil as a standard drug. IC50 values for the tested compounds were in the
range of 0.11-36.5 µM against AChE and in the range of 0.02-98.6 µM against BuChE indicating the
potential anti-cholinesterase activity of the designed compounds (Table 1).
Based on the results, it has been concluded that different factors such as type and position of the
substituents with the length of the linker could modulate the activity of the compounds. In the series
of the compounds bearing N-benzylpiperazine, five carbon chain length as cross-linker demonstrated
better inhibitory activity comparing to the corresponding linker with three carbon chain length. All
the tested compounds showed activity in the range of micro to nano molar. Compounds 3s-u showed
higher activity than donepezil as the reference drug against BuChE (IC50 = 0.02-1.4 µM).
Comparing the inhibitory activity of N-benzylpiperazine 3f with 4-benzylpiperidine 3w,
demonstrated that replacing the piperazine ring with piperidine, could decrease the activity against
both AChE and BuChE. According to this finding, it could be suggested that the nitrogen atom at
position 4 of the piperazine ring play an important role in interaction with the enzyme. By removing
the benzyl moiety in compound 3w, the anticholinesterase activity improved (see the IC50 value of
compound 3v).
The N-benzylpiperazine derivatives with five carbon chain lengths (3f-r) exhibited inhibitory
activities in the range of 1.4 to 22.0 µM. The meta-brominated N-benzylpiperazine derivative 3k was
the most active compound against both AChE and BuChE with the IC50 value of 1.4 µM and 5.1 µM,
respectively. 3,4-Dichlorobenzyl substituted derivative 3m showed the least activity against both
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enzymes. Considering the potency of meta-methoxy substituted compound 3l (IC50 AChE=19.0, IC50
BuChE=67.9), it has been revealed that the more lipophilic substituent such as Br is necessary for
optimum activity.
Among fluorinated derivatives, compound with 4-F substituent 3r showed a better inhibitory
activity comparing to its 2-F analogue 3h. In contrast, among chlorinated benzyl derivatives, 2-
chlorobenzylpiperazine derivative 3g showed better inhibition comparing to its para congener 3p. In
another analogy, the order of the activity for 4-substituted benzyl derivatives having five carbon chain
length was 4-Br > 4-F > 4-OMe > H > 4-Me > 4-Cl (IC50 values of 1.7, 4.4, 9.4, 10.1, 10.7, 10.9 µM,
respectively). Considering these results, it could be concluded that the inhibitory activities of the 4-
substituted benzylpiperazine derivatives are not significantly affected by the electron withdrawing or
electron donating properties of the substituents at the para position. Alternatively, the ortho-
substituted benzyl piperazine derivatives with five carbon chain linker showed relative inhibitory
activity of 2-Cl~2-Me > 2-NO2 > 2-F with IC50 values of 5.8, 5.9, 9.8, 11.5 µM, respectively (Table
1). Among compounds 3a-3e except for 3b, by shortening the linker to three atoms the activity
against acetylcholinesterase diminished due to the shrinking of the molecule. Replacing the cyclic
aliphatic amine with the aromatic amines such as pyridine or quinoline led to the superior activity
implying the fact that aromatic stacking interactions has crucial role in the enzyme inhibition.
Furthermore, bicyclic aromatic amines (quinoline and isoquinoline) expressed higher activity owing
to the extra π-stacking interactions. According to the calculated BuChE/AChE selectivity, the best
selectivity (4.00) is related to the compound 3q possessing 4-bromo-benzylpiperazine group
connected to carbazolzole moiety via 5 carbon spacer.
Table 1
2.3. Kinetic study of AChE inhibition
To determine the inhibition type and steady-state inhibition constant, enzyme kinetic studies were
carried out on compound 3s as the most active compound using AChE and BuChE. The results
proposed that compound 3s exhibited mixed type-inhibition on AChE and BuChE with Ki values of
48 and 6.6 µM, respectively. The dual mode of inhibition (competitive and uncompetitive) for
compound 3s advocates the fact that compound 3s could simultaneously bind to both catalytic active
site (CAS) and peripheral anionic site (PAS). The Lineweaver–Burk double reciprocal plot and
secondary plot of compound 3s for both AChE and BuChE are represented in Figure 2. The kinetic

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studies were carried out as described above using different concentrations of the substrate and the
compounds. Compound 3s with concentrations of 216, 108, 54 and 0 µM were used for AChE kinetic
analysis while the concentrations of 48, 24, 12 and 0 µM were used for BuChE kinetic analysis.
Figure 2
2.4. Inhibition of self- and AChE-induced Aβ1-42 aggregation
Inhibitory properties of compound 3s as the most active compound and 3k as the best active
compound among carbazole derivatives conjugated to the benzylpiperazine moiety on self-induced
and AChE-induced aggregation of Aβ1-42 were determined using a thioflavin T (ThT)-based
fluorescence assay and compared with donepezil and rifampicin as reference compounds [35,36].
Based on the results (Table 2), both compounds 3s and 3k showed acceptable potency for inhibition
of self-induced Aβ aggregation of 27.4 and 19.5%, respectively, relative to the reference compounds
rifampicin (27.5%) and donepezil (22%). Compound 3s bearing quinoline moiety was more active to
inhibit Aβ self-aggregation in comparison to compound 3k with benzylpiperazine. Moreover, both
compounds showed moderate inhibitory activity toward AChE-induced Aβ aggregation at 100 μM
(24.9% and 19.2% respectively) which are less than their Aβ self-aggregation inhibitory activity,
while biologic data analysis and molecular docking study confirmed the binding affinity of the
compounds to PAS as a dual AChE inhibitor. This result indicated that there was no correlation
between AChE-induced Aβ aggregation inhibitory activity of the compounds and their high binding
affinity to PAS.
Table 2
2.5. Neuroprotection against H2O2-induced cell death in PC12 cells
The neuroprotective effect of compound 3s against H2O2-induced PC12 cell injury at the
concentrations of 1, 10, and 100 µM, prior to treatment with H2O2 (375 µM) was evaluated via MTT-
based colorimetric assay. Compound 3s showed significant effect on cell viability at a concentration
of 10 µM (Cell violability = 1.054, P < 0.0098) which is comparable to the Quercetin (Cell violability
= 1.092, P < 0.001) as standard control suggesting acceptable neuroprotective activity of compound
3s (Fig. 3).
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Figure 3
2.6. Ligand-protein docking simulation
According to the IC5o values, compound 3s demonstrated the highest potency for interaction with
the active site of the enzyme. Based on the docking data, compound 3s binds effectively to the PAS
and anionic site of the enzyme with the assistance of hydrophobic and π stacking interactions. π-
Stacking interaction is formed between Tyr-332 and quinoline fragment of the compound (Fig. 4). As
a result, compound 3s is fitted quite well into the PAS and anionic site of the AChE demonstrating
higher therapeutic activity.
Figure 4
3. Conclusion
We have designed and synthesized a new class of carbazole derivatives bearing benzyl piperazine,
benzyl piperidine, pyridinium, quinolinium, or isoquinolinium salts linked through a 3 or 5 carbon
atom chain length. All of the tested compounds showed reasonable activity in the range of micro to
nano molar; among them the best candidate was quinolinium salt derivative 3s with IC50 = 0.11 µM
and 0.02 µM against AChE and BuChE, respectively. Replacing the benzyl piperazine moiety with
quinolinium or isoquinolinium salts (3s and 3t) improved the enzyme inhibition quite remarkably.
Two factors might have been contributed in the high inhibitory activity of these compounds including
formation of the stable cationic part and the improved π-stacking interactions of the quinoline or
isoquinoline moieties with the PAS of the enzyme. Kinetic studies for compound 3s exhibited mixed-
type inhibition on AChE and BuChE. Docking data also confirmed the dual mode of inhibition for
compound 3s advocating the fact that compound 3s could simultaneously bind to both CAS and PAS.
Above all, compound 3s demonstrated good potency for inhibition of self-induced Aβ aggregation
and revealed acceptable neuroprotective activity against PC12 cells injury induced by H2O2. Owing
to the high AChE and BuChE inhibition activity, acceptable neuroprotective effects, satisfactory self-
and AChE-induced Aβ aggregation inhibition, compound 3s could be suggested as a promising
multifunctional compound against Alzheimer’s disease.
4. Materiarls and Methods
4.1. Chemistry

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All commercially available chemicals were purchased from Merck, Sigma, and across without
1
further purification. H NMR and 13C NMR were recorded on Brucker 500 MHz instrument with
frequencies of 500 and 125 MHz, respectively. Solvents used for NMR analyses were CDCl3 and
DMSO and tetramethylsilane were used as an internal standard. The chemical shifts (δ) and coupling
constants (J) were expressed in parts per million and Hertz, respectively. Mass spectra were obtained
by HP Agilent Technologies 5937 at ionization potential of 70 eV. Elemental analyses were
performed with CHN-Rapid Heraeus. Melting points were measured by Electrothermal 1A9100.
Elemental Analyzer and the results (C, H, N) were within ± 0.4% of the calculated values.
Analytical thin-layer chromatography (TLC) was performed on aluminum plates precoated with silica
gel 60F-254 as the adsorbent. Column chromatography was performed using silica gel 60 (230-400
mesh).
4.1.1. General procedure for the synthesis of compound 1
The commercially available carbazole (10 mmol) was dissolved in dry DMF (15 mL). The mixture
was cooled to 0 °C and NaH (10 equiv.) was added followed by dropwise addition of the appropriate
dibromoalkane (15 equiv.). The mixture was stirred at room temperature for 15 h and quenched by
addition of ice. The crude product was diluted with ethyl acetate (25 mL) and washed with brine (100
mL). The organic phase was dried over Na2SO4 and filtered. The concentrated crude product was
purified by flash chromatography using petroleum ether to afford compound 1 as a colorless oil [37].
1
4.1.1.1. 9-(3-bromopropyl)-9H-carbazole 1a (n =3) ) Yield 85%; H NMR (500 MHz, DMSO-d6) δ
8.13 (d, 2H, J = 7.5 Hz), 7.58 (d, 2H, J = 8.0 Hz), 7.44 (t, 2H, J = 7.0 Hz), 7.19 (t, 2H, J = 7.0 Hz),
4.38 (t, 2H, J = 7.0 Hz), 3.40 (t, 2H, J = 6.0 Hz), 1.88 (m, 2H).
1
4.1.1.2. 9-(5-bromopentyl)-9H-carbazole 1b (n = 5) Yield 82%; H NMR (500 MHz, DMSO-d6) δ
8.13 (d, 2H, J = 7.7 Hz), 7.57 (m, 2H), 7.44 (m, 2H), 7.19 (m, 2H), 4.36 (t, 2H, J = 7.0 Hz), 3.42 (m,
2H), 1.76 (m, 4H), 1.43 (m, 2H); 13C NMR (DMSO- d6, 125 MHz) δ 139.9, 125.6, 122.0, 120.2,
118.6, 109.2, 42.0, 34.8, 31.9, 28.3, 25.2.
4.1.2. General procedure for the synthesis of piperazine intermediate 2
Compound 1 (5 mmol) was dissolved in anhydrous ethanol (30 mL). Anhydrous piperazine (75
mmol) and KI (2.86 mmol) were added to the mixture and refluxed for 1h. The solvent was removed
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under reduced pressure and the crude product was diluted with ethyl acetate (25 mL) and washed with
brine (100 mL). The organic phase was dried over Na2SO4 and filtered. The concentrated crude
product was purified by flash chromatography (petroleum ether/EtOAc 3:1) to afford piperazine
intermediate 2 as a pale yellow oil [38].
1
4.1.2.1. 9-(3-(piperazin-1-yl)propyl)-9H-carbazole 2a (n = 3) Yield 85%; H NMR (500 MHz,
DMSO-d6) δ 8.13 (d, 2H, J = 8.0 Hz), 7.59-7.56 (m, 2H), 7.43 (t, 2H, J = 7.5 Hz), 7.18 (t, 2H, J =
13
7.5 Hz), 4.38 (t, 2H, J = 7.0 Hz), 2.73 (bs, 4H), 2.16-2.13 (m, 6H), 1.90-1.86 (m, 2H); C NMR
(DMSO- d6, 125 MHz) δ 140.0, 125.4, 122.0, 120.0, 118.5, 109.1, 56.0, 54.9, 52.8, 45.0, 25.1.
1
4.1.2.1. 9-(5-(piperazin-1-yl)pentyl)-9H-carbazole 2b (n = 5) Yield 80%; H NMR (500 MHz,
DMSO-d6) δ 8.11 (d, 2H, J = 7.0 Hz), 7.57 (d, 2H, J = 8.5 Hz), 7.43 (t, 2H, J = 7.5 Hz), 7.20-7.17
(m, 2H), 4.38 (t, 2H, J = 6.5 Hz), 2.73 (bs, 2H), 2.26 (bs, 4H), 2.16-2.14 (m, 2H), 1.78-1.76 (m, 2H),
1.42-1.27 (m, 4H).
4.1.3. General procedure for the synthesis of compound 3a-r
A mixture of piperazine intermediate 2 synthesized above (1 mmol), anhydrous K2CO3 (1.1 mmol)
and appropriate benzyl bromide (1.2 mmol) was refluxed in dry acetonitrile (25 mL) for 3 h. The
mixture was cooled to room temperature and filtered. The solvent was removed under reduced
pressure and the crude product was purified by flash chromatography (petroleum ether/EtOAc 5:1) to
afford compound 3a-r [38].
4.1.3.1. 9-(3-(4-benzylpiperazin-1-yl)propyl)-9H-carbazole 3a: [39] Yield 80%; Amber oil; 1H NMR
(DMSO-d6, 500 MHz) δ 8.08 (d, 1H, J = 7.6 Hz), 7.79 (d, 1H, J = 8.0 Hz), 7.64 (d, 1H, J = 8.1 Hz),
7.57-7.47 (m, 6H) 7.36-7.11 (m, 4H), 4.63-4.34 (m, 2H), 3.62 (m, 2H), 2.41-2.22 (m, 10H), 1.93 (m,
2H); 13C NMR (DMSO-d6, 125 MHz) δ 139.9, 137.4, 133.7, 132.6, 130.4, 129.0, 128.9, 128.4, 127.5,
127.3, 125.9, 122.3, 119.1, 118.6, 109.3, 60.3, 57.2, 54.4, 53.1, 45.1, 30.7. Anal. Calcd. for
C26H29N3(383.53): C, 81.42; H, 7.62; N, 10.96. Found: C, 81.40; H, 7.78; N, 11.02.
1
4.1.3.2. 9-(3-(2-(2-fluorobenzyl)piperazin-1-yl)propyl)-9H-carbazole 3b: Yield 70%; Amber oil; H
NMR (DMSO-d6, 500 MHz) δ 8.14 (m, 2H), 7.60 (m, 2H), 7.44-7.35 (m, 2H), 7.30 (m, 2H), 7.15-
7.12 (m, 4H), 4.39 (t, 1H, J = 6.5 Hz), 3.50 (bs, 2H), 2.51-2.15 (m, 10H), 1.89 (t, 2H, J = 6.5 Hz); 13C

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NMR (DMSO-d6, 125 MHz) δ161.8 and 159.8 (C-F, J = 242.5 Hz), 140.0, 131.6, 131.6, 129.1, 129.0,
125.6, 124.8, 124.6, 122.0, 120.2, 118.6, 115.2, 115.0, 109.3, 54.5, 54.4, 52.6, 52.5, 25.4. Anal.
Calcd. for C26H28FN3(401.53): C, 77.77; H, 7.03; N, 10.47. Found: C, 77.54; H, 7.32; N, 10.33.
1
4.1.3.3. 9-(3-(4-fluorobenzyl)piperazin-1-yl)propyl)-9H-carbazole 3c: Yield 60%; Amber oil; H
NMR (DMSO-d6, 500 MHz) δ 8.14 (d, 2H, J = 7.5 Hz), 7.59 (d, 2H, J = 8.0 Hz), 7.45-7.42 (m, 2H),
7.31-7.29 (m, 2H), 7.28-7.11 (m, 4H), 4.40 (t, 2H, J = 7.0 Hz), 3.40 (s, 2H), 2.34-2.17 (m, 8H), 2.17-
2.15 (m, 2H), 1.98 (t, 2H, J = 5.0 Hz); 13C NMR (DMSO-d6, 125 MHz) δ 162.2 and 160.3 (C-F, J =
237.5 Hz), 140.1, 135.2, 134.4, 130.7, 130.6, 125.8, 125.4, 122.2, 122.1, 120.2, 118.9, 118.8, 115.2,
114.9, 114.8, 109.3, 61.2, 54.4, 52.6, 25.5. Anal. Calcd. for C26H28FN3(401.53): C, 77.77; H, 7.03; N,
10.47. Found: C, 77.38; H, 7.32; N, 10.67.
1
4.1.3.4. 9-(3-(4-bromobenzyl)piperazin-1-yl)propyl)-9H-carbazole 3d: Yield 63%; Amber oil; H
NMR (DMSO-d6, 500 MHz) δ 8.14 (d, 2H, J = 8.0 Hz), 7.58-7.42 (m, 6H), 7.29-7.17 (m, 4H), 4.39
13
(t, 2H, J = 6.5 Hz), 3.45 (s, 2H), 2.35-2.17 (m, 10H), 1.92-1.88 (m, 2H); C NMR (DMSO-d6, 125
MHz) δ 140.0, 137.7, 131.0, 130.9, 128.6, 125.6, 122.0, 120.2, 119.9, 118.6, 109.2, 61.2, 54.4, 54.3,
50.0, 25.4. Anal. Calcd. for C26H28BrN3(462.44): C, 67.53; H, 6.10; N, 9.09. Found: C, 67.81; H,
6.38; N, 9.33.
1
4.1.3.5. 9-(3-(4-methoxybenzy)lpiperazin-1-yl)propyl)-9H-carbazole 3e: Yield 61%; Amber oil; H
NMR (DMSO-d6, 500 MHz) δ 8.14 (d, 2H, J = 7.7 Hz), 7.60 (d, 2H, J = 8.5 Hz), 7.43 (t, 2H, J = 8.0
Hz), 7.19-7.16 (m, 4H), 6.87 (d, 2H, J = 8.5 Hz), 4.40 (t, 2H, J = 7.0 Hz), 3.72 (s, 3H, OMe), 3.37
13
(bs, 2H), 2.35-2.19 (m, 10H), 1.91-1.89 (m, 2H); C NMR (DMSO-d6, 125 MHz) δ 158.4, 130.2,
129.5, 129.4, 129.2, 129.2, 127.9, 125.6, 122.0, 118.7, 113.7, 113.6, 113.6, 113.4, 109.3, 73.3, 70.8,
62.5, 61.2, 55.0, 54.3, 52.2, 25.3. Anal. Calcd. for C27H31N3O(413.57): C, 78.42; H, 7.56; N, 10.16.
Found: C, 78.15; H, 7.46; N, 10.32.
4.1.3.6. 9-(5-(4-benzylpiperazin-1-yl)pentyl)-9H-carbazole 3f: Yield 78%; White solid; m.p. 108-110
1
°C; H NMR (DMSO-d6, 500 MHz) δ 8.15 (d, 1H, J = 7.6 Hz), 7.64 (d, 1H, J = 8.1 Hz), 7.55-7.44
(m, 5 H) 7.32-7.18 (m, 6H), 4.59-4.45 (m, 2H), 3.55 (bs, 2H), 3.34 (bs, 2H), 3.09 (bs, 3H), 2.83-2.79
13
(m, 2H), 2.63 (bs, 3H), 1.90-1.80 (m, 4H), 1.29 (bs, 2H); C NMR (DMSO-d6, 125 MHz) δ 140.0,
137.3, 133.7, 132.9, 130.3, 129.0, 128.8, 128.4, 127.4, 127.3, 125.7, 122.0, 120.3, 118.7, 109.3, 60.4,
10

JOURNAL PRE-PROOF
56.8, 45.4, 40.8, 28.2, 24.2, 20.6. Anal. Calcd. for C27H31N3O(413.57): C, 78.42; H, 7.56; N, 10.16.
Found: C, 78.15; H, 7.46; N, 10.32.
1
4.1.3.7. 9-(5-(4-(2-chlorobenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3g: Yield 80%; Amber oil; H
NMR (DMSO-d6, 500 MHz) δ 8.14 (d, 2H, J = 7.5 Hz), 7.58 (d, 2H, J = 8.5 Hz), 7.44-7.41 (m, 4H),
7.30-7.28 (m, 2H), 7.17 (t, 2H, J = 7.5 Hz), 4.37 (t, 2H, J = 7.0 Hz), 3.50 (s, 2H), 2.49-2.15 (m, 10H),
13
1.76 (bs,2H), 1.39 (bs, 2H), 1.23 (bs, 2H); C NMR (DMSO-d6, 125 MHz) δ 140.0, 135.6, 133.2,
130.8, 129.2, 128.5, 127.0, 125.6, 122.0, 120.2, 118.6, 109.2, 58.6, 57.7, 52.7, 42.2, 28.3, 26.0, 24.4.
Anal. Calcd. for C28H32ClN3(446.04): C, 75.40; H, 7.23; N, 9.42. Found: C, 75.33; H, 7.40; N, 9.21.
1
4.1.3.8. 9-(5-(4-(2-fluorobenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3h: Yield 60%; Amber oil; H
NMR (DMSO-d6, 500 MHz) δ 8.14 (d, 2H, J = 8.0 Hz), 7.57 (d, 2H, J = 8.0 Hz), 7.43 (t, 2H, J = 7.0
Hz), 7.42-731 (m, 2H), 7.20-7.15 (m, 4H), 4.35 (t , 2H, J = 7.0 Hz), 3.51 (bs, 2H), 2.37-2.31 (m, 8H),
13
2.17-2.14 (m, 2H), 1.77-1.74 (m, 2H), 1.39-1.36 (m, 2H), 1.26-1.23 (m, 2H); C NMR (DMSO-d6,
125 MHz) δ 161.8 and 159.9 (C-F, J = 242.5 Hz), 140.0, 131.6, 129.3, 129.1, 129.1, 125.7, 124.6,
124.4, 124.2, 122.1, 120.3, 118.7, 115.3, 115.1, 109.3, 57.6, 54.5, 52.6, 52.3, 42.2, 28.0, 25.0, 25.9,
24.4. Anal. Calcd. for C28H32FN3(429.58): C, 78.29; H, 7.51; N, 9.78. Found: C, 78.13; H, 7.20; N,
9.41.
4.1.3.9. 9-(5-(4-(2-methylbenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3i: Yield 81%; White solid;
m.p. 130-132 °C; 1H NMR (DMSO-d6, 500 MHz) δ 8.17 (d, 2H, J = 5 Hz), 7.65 (d, 2H, J = 8.0 Hz),
7.47-7.37 (m, 6H), 7.31-7.29 (m, 2H), 7.25-7.21 (m, 2H), 4.61-4.47 (m, 2H), 3.52 (bs, 2H), 3.15-3.12
(m, 2H), 2.73-2.66 (m, 8H), 2.37 (s, 3H, Me), 1.91-1.76 (m, 4H), 1.36 (bs, 2H); 13C NMR (DMSO-d6,
125 MHz) δ 140.3, 140.0, 137.1, 135.1, 134.5, 131.6, 130.3, 130.1, 129.6, 127.2, 126.0, 125.9, 125.6,
125.4, 122.0, 120.2, 118.6, 109.2, 62.8, 58.3, 55.8, 54.6, 45.3, 41.9, 28.1, 23.4, 20.9, 19.7, 18.7. Anal.
Calcd. for C29H35N3(425.62): C, 81.84; H, 8.29; N, 9.87. Found: C, 82.15; H, 8.02; N, 9.51.
1
4.1.3.10. 9-(5-(4-(2-nitrobenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3j: Yield 70%; Amber oil; H
NMR (DMSO-d6, 500 MHz) δ 8.12 (d, 2H, J = 8.0 Hz), 7.83 (d, 1H, J = 5.0 Hz), 7.63-7.40 (m, 7H),
7.17 (t, 2H, J = 7.5 Hz), 4.34 (t, 2H, J = 7.0 Hz), 3.72 (t, 2H, J = 7.5 Hz ), 2.25-2.11 (m, 10H), 1.76-
1.73 (m, 2H), 1.37-1.24 (m, 4H); 13C NMR (DMSO-d6, 125 MHz) δ 149.6, 139.9, 132.7, 132.5,
131.0, 128.5, 125.5, 124.0, 122.0, 120.1, 118.5, 109.1, 58.1, 57.4, 52.5, 52.4, 42.1, 28.2, 25.8, 24.3,

JOURNAL PRE-PROOF
21.0. Anal. Calcd. for C28H32N4O2(456.59): C, 73.66; H, 7.06; N, 12.27. Found: C, 73.43; H, 7.36; N,
12.31.
1
4.1.3.11. 9-(5-(4-(3-bromobenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3k: Yield 88%; Amber oil; H
NMR (DMSO-d6, 500 MHz) δ 8.14 (bs, 2H), 7.60 (bs, 2H), 7.48-7.43 (m, 4H), 7.29 (bs, 2H), 7.19
(bs, 2H), 4.40 (bs, 2H), 3.58 (m, 2H), 3.47-3.35 (m, 4H), 3.25 (m, 1H), 2.66 (bs, 2H), 1.98 (bs, 2H),
13
1.58 (bs, 2H), 1.29 (bs, 2H); C NMR (DMSO-d6, 125 MHz) δ 139.9, 131.3, 130.5, 130.4, 130.1,
130.0, 127.8, 125.6, 122.0, 121.7, 121.6, 120.3, 118.7, 118.6, 109.2, 59.5, 57.6, 45.2, 42.0, 28.2, 23.4,
20.5. Anal. Calcd. for C28H32BrN3(490.49): C, 68.57; H, 6.58; N, 8.57. Found: C, 68.33; H, 6.87; N,
8.65.
4.1.3.12. 9-(5-(4-(3-methoxybenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3l: Yield 73%; Amber oil;
1H NMR (DMSO-d6, 500 MHz) δ 8.14 (d, 2H, J = 7.7 Hz), 7.58 (d, 2H, J = 8.2 Hz), 7.43 (t, 2H, J =
7.0 Hz), 7.20-7.16 (m, 3H), 6.84-6.79 (m, 2H), 4.37 (t, 2H, J = 7.0 Hz), 3.72 (s, 3H, OMe), 3.38 (s,
13
2H), 2.30-2.18 (m, 5H), 1.78-1.75 (m, 2H), 1.40 (bs, 2H), 1.27 (bs, 2H); C NMR (DMSO-d6, 125
MHz) δ 159.2, 139.9, 129.1, 125.6, 122.0, 120.9, 120.2, 118.6, 114.2, 112.2, 109.2, 61.9, 57.6, 54.9,
52.7, 52.5, 42.1, 28.3, 25.9, 24.4. Anal. Calcd. for C29H35N3O(441.62): C, 78.87; H, 7.99; N, 9.52.
Found: C, 79.12; H, 6.97; N, 9.35.
4.1.3.13. 9-(5-(4-(3,4-dichlorobenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3m: Yield 76%; Amber
oil; 1H NMR (DMSO-d6, 500 MHz) δ 8.13 (d, 2H, J = 7.6 Hz), 7.58-7.47 (m, 6H), 7.27-7.16 (m, 3H),
4.37 (t, 2H, J = 6.5 Hz), 3.41 (s, 2H), 2.29-2.17 (m, 8H), 1.90 (bs, 2H), 1.39 (bs, 2H), 1.26 (bs, 2H);
13C NMR (DMSO-d6, 125 MHz) δ 140.0, 139.7, 130.4, 130.3, 129.3, 129.0, 125.6, 122.0, 120.2, 60.4,
57.6, 52.6, 52.3, 42.2, 28.3, 25.9, 24.4, 21.0. Anal. Calcd. for C28H31Cl2N3(480.48): C, 69.99; H,
6.50; N, 8.75. Found: C, 70.22; H, 6.67; N, 8.95.
4.1.3.14. 9-(5-(4-(4-methoxybenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3n: Yield 65%; Amber oil;
1H NMR (DMSO-d6, 500 MHz) δ 8.13 (d, 2H, J = 8.0 Hz), 7.58 (d, 2H, J = 8.0 Hz), 7.43 (t, 2H, J =
7.5 Hz), 6.86-6.84 (m, 6H), 4.36 (bs, 2H), 3.71 (s, 3H, OMe), 2.34 (bs, 2H), 2.30-2.10 (m, 10H), 1.76
(bs, 2H), 1.39 (bs, 2H), 1.26 (bs, 2H); 13C NMR (DMSO-d6, 125 MHz) δ 158.2, 139.9, 130.0, 125.6,
122.0, 120.2, 118.6, 113.5, 109.2, 61.4, 57.6, 55.0, 52.6, 52.4, 52.3, 42.1, 28.3, 25.8, 24.4. Anal.
Calcd. for C29H35N3O(441.62): C, 78.87; H, 7.99; N, 9.52. Found: C, 78.77; H, 7.71; N, 9.81.
12

JOURNAL PRE-PROOF
4.1.3.15. 9-(5-(4-(4-methylbenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3o: Yield 67%; Amber oil; 1H
NMR (DMSO-d6, 500 MHz) δ 8.13 (d, 2H, J = 7.6 Hz), 7.57 (d, 2H, J = 8.0 Hz), 7.42 (t, 2H, J = 8.0
Hz), 7.19-7.09 (m, 6H), 4.35 (t, 2H, J = 7.0 Hz), 3.37 (s, 2H), 2.25-2.21 (m, 8H), 1.95 (bs, 2H), 1.90
(3H, Me), 1.77 (bs, 2H), 1.39 (bs, 2H), 1.25 (bs, 2H); 13C NMR (DMSO-d6, 125 MHz) δ 140.0,
136.0, 134.8, 128.8, 128.7, 125.6, 122.0, 120.2, 118.6, 109.2, 61.7, 57.4, 52.5, 52.1, 42.1, 28.3, 25.7,
24.3, 21.1, 20.7. Anal. Calcd. for C29H35N3(425.62): C, 81.84; H, 8.29; N, 9.87. Found: C, 81.98; H,
8.46; N, 9.65.
1
4.1.3.16. 9-(5-(4-(4-chlorobenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3p: Yield 73%; Amber oil; H
NMR (DMSO-d6, 500 MHz) δ 8.13 (bs, 2H), 7.59 (bs, 2H), 7.43 (bs, 2H), 7.37 (bs, 2H), 7.30 (bs,
2H), 7.19 (bs, 2H), 4.37 (bs, 2H), 3.44 (bs, 2H), 2.45-2.25 (bs, 8H), 1.17 (bs, 2H), 1.47 (bs, 2H),
1.22-1.16 (m, 4H), 1.16 (m, 1H); 13C NMR (DMSO-d6, 125 MHz) δ 140.0, 139.7, 130.8, 130.4,
130.3, 129.3, 129.0, 125.6, 122.0, 120.2, 118.6, 109.2, 60.4, 57.6, 52.6, 52.3, 54.1, 28.3, 25.9, 24.4,
•
•
21.0; MS m/z: 445.60 (M-H)+, 265.15 (C15H22ClN2 ), 223.10 (C12H16ClN2 ), 180.08 (C13H10N•),
125.02 (C7H6Cl•); Anal. Calcd. for C28H32ClN3(446.04): C, 75.40; H, 7.23; N, 9.42. Found: C, 75.43;
H, 7.16; N, 9.33.
4.1.3.17. 9-(5-(4-(4-bromobenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3q: Yield 93%; White solid;
m.p. 128-129 °C; 1H NMR (DMSO-d6, 500 MHz) δ 8.16 (bs, 2H), 7.64-7.39 (m, 10H), 4.60-4.45 (m,
2H), 3.53 (bs, 2H), 2.95 (bs, 2H), 2.74 (bs, 2H), 2.63 (bs, 2H), 1.98-1.79 (m, 4H), 1.28-1.17 (m, 2H);
13C NMR (DMSO-d6, 125 MHz) δ 140.0, 136.9, 135.0, 132.0, 131.2, 131.0, 126.7, 125.7, 124.2,
122.0, 120.3, 118.7, 109.3, 59.5, 56.9, 45.2, 42.0, 28.2, 23.2, 20.5.
Anal. Calcd. for
C28H32BrN3(490.49): C, 68.57; H, 6.58; N, 8.57. Found: C, 68.33; H, 6.24; N, 8.21.
1
4.1.3.18. 9-(5-(4-(4-fluorobenzyl)piperazin-1-yl)pentyl)-9H-carbazole 3r: Yield 86%; Amber oil; H
NMR (DMSO-d6, 500 MHz) δ 8.13 (d, 2H, J = 7.6 Hz), 7.57 (d, 2H, J = 8.02 Hz), 7.42 (t, 2H, J = 7.0
Hz), 7.28 (t, 2H, J = 6.0 Hz), 7.18-7.09 (m, 4H), 4.36 (t, 2H, J = 7.0 Hz), 3.49 (bs, 2H), 2.30-2.11 (m,
13
10H), 1.75 (bs, 2H), 1.40 (bs, 2H), 1.25-1.16 (m, 4H); C NMR (DMSO-d6, 125 MHz) δ 162.2 and
160.2 (C-F, J = 240.85 Hz) , 139.9, 134.2, 130.6, 125.6, 122.0, 120.2, 118.6, 114.9, 114.7, 109.2,
61.0, 57.4, 52.5, 52.1, 42.1, 29.0, 25.7, 24.3. Anal. Calcd. for C28H32FN3(429.58): C, 78.29; H, 7.51;
N, 9.78. Found: C, 78.41; H, 7.67; N, 9.87.

JOURNAL PRE-PROOF
4.1.4. General procedure for the synthesis of compound 3s-w
Compound 1 (1 mmol) was dissolved in dry acetonitrile (20 mL). The desired amine 2 (1.2 equiv.)
was added to the mixture and refluxed for 6-8 h. The solvent was removed under reduced pressure
and the crude product was purified by flash chromatography (petroleum ether/EtOAc 5:1) to afford
compound 3s-w [38].
1
4.1.4.1. 9-(5-(quinolin-1(2H)-yl)pentyl)-9H-carbazole 3s: Yield 61%; Amber oil; H NMR (CDCl3,
500 MHz) δ 10.18 (d, 1H, J = 5.5 Hz), 8.87 (d, 1H, J = 8.5 Hz), 8.15 (d, 1H, J = 8.5 Hz), 8.10 (d, 1H,
J = 9.0 Hz), 7.99-7.99 (m, 5H), 7.37-7.15 (m, 3H), 7.15 (t, 2H, J = 7.0 Hz), 5.18 (t, 2H, J = 7.5 Hz),
4.25 (t, 2H, J = 7.0 Hz), 2.03 (bs, 2H), 1.96-1.87 (m, 4H), 1.52 (m, 2H); 13C NMR (CDCl3, 125 MHz)
δ 149.9, 147.0, 140.1, 137.3, 135.8, 131.0, 129.8, 125.7, 122.5, 122.1, 120.1, 118.8, 118.0, 108.8,
57.4, 42.6, 29.8, 28.2, 24.0; MS m/z: 235.14 (C17H17N•), 180.08 (C13H10N•), 166.07 (C12H8N•),
129.07 (C9H7N•); Anal. Calcd. for C25H26N2(366.50): C, 85.21; H, 7.15; N, 7.64. Found: C, 85.45; H,
7.53; N, 7.71.
1
4.1.4.2. 9-(5-(isoquinolin-2(3H)-yl)pentyl)-9H-carbazole 3t: Yield 60%; Amber oil; H NMR
(CDCl3, 500 MHz) δ 10.68 (s, 1H), 8.49 (d, 2H, J = 9.0 Hz), 8.02-7.88 (m, 5H), 7.74 (t, 1H, J = 7.0
Hz), 7.34-31 (m, 3H), 7.09 (d, 2H, J = 7.5 Hz), 4.79 (t, 2H, J = 7.0 Hz), 4.21(t, 2H, J = 7.0 Hz), 2.45
13
(bs, 2H), 2.01-1.98 (m, 2H), 1.82-1.79 (m, 2H), 1.41-1.40 (m, 2H); C NMR (CDCl3, 125 MHz) δ
149.6, 140.0, 136.9, 136.6, 134.2, 130.8, 127.4, 126.7, 125.8, 125.6, 122.4, 120.0, 118.7, 108.7, 60.7,
42.4, 31.0, 27.9, 23.5. Anal. Calcd. for C26H26N2(366.51): C, 85.21; H, 7.15; N, 7.64. Found: C,
85.33; H, 7.16; N, 7.81.
1
4.1.4.3. 9-(5-(pyridin-1(2H)-yl)pentyl)-9H-carbazole 3u: Yield 55%; Amber oil; H NMR (CDCl3,
500 MHz) δ 9.11 (d, 2H, J = 6.0 Hz), 8.18 (t, 1H, J = 8.0 Hz), 8.00 (d, 2H, J = 7.5 Hz), 7.75 (t, 2H),
7.41-7.28 (m, 4H), 7.17 (t, 1H, J = 7.5 Hz), 4.65 (t, 2H, J = 7.5 Hz), 4.24 (t, 1H, J = 7.0 Hz), 2.40
13
(bs, 2H), 1.90-1.81 (m, 4H), 1.34-1.28 (m, 2H); C NMR (CDCl3, 125 MHz) δ 144.7, 144.6, 140.1,
128.2, 125.8, 122.5, 120.2, 118.9, 108.8, 61.1, 42.4, 31.0, 27.8, 23.3. Anal. Calcd. for
C22H24N2(316.45): C, 83.50; H, 7.64; N, 8.85. Found: C, 85.67; H, 7.82; N, 9.22.
14

JOURNAL PRE-PROOF
1
4.1.4.4. 9-(5-(piperidin-1-yl)pentyl)-9H-carbazole 3v: Yield 52%; Amber oil; H NMR (DMSO-d6,
500 MHz) δ 8.14 (d, 2H, J = 8.0 Hz), 7.63 (d, 2H, J = 8.5 Hz), 7.45 (t, 2H, J = 7.5 Hz), 7.19 (t, 2H, J
13
= 7.0 Hz), 4.41 (t, 2H, J = 6.5 Hz), 2.98-2.75 (m, 6H), 180-1.55 (m, 8H), 1.31 (bs, 4H); C NMR
(DMSO-d6, 125 MHz) δ 139.8, 125.5, 121.9, 120.0, 118.5, 109.1, 55.3, 51.6, 43.3, 41.8, 27.8, 22.5,
22.0, 21.9, 21.5, 21.2. Anal. Calcd. for C22H28N2(320.48): C, 82.45; H, 8.81; N, 8.74. Found: C,
82.21; H, 8.91; N, 8.66.
1
4.1.4.5. 9-(5-(4-benzylpiperidin-1-yl)pentyl)-9H-carbazole 3w: Yield 61%; Amber oil; H NMR
(DMSO-d6, 500 MHz) δ 8.14 (d, 2H, J = 7.5 Hz), 7.57 (d, 2H, J = 8.0 Hz), 7.44 (t, 2H, , J = 7.0 Hz),
7.28-7.25 (m, 2H), 7.20-7.12 (m, 5H), 4.37 (t, 2H, J = 7.0 Hz), 2.96-2.93 (m, 2H), 2.50-2.40 (m, 4H),
13
2.14-2.10 (m, 2H), 1.91 (s, 1H), 1.79-1.76 (m, 2H), 1.55-148 (m, 4H), 1.29-1.22 (m, 4H); C NMR
(DMSO-d6, 125 MHz) δ 139.9, 128.9, 128.0, 125.7, 125.5, 122.0, 120.1, 118.5, 109.1, 56.8, 52.4,
42.0, 41.8, 36.2, 30.2, 28.8, 28.1, 24.8, 24.1, 21.1. Anal. Calcd. for C29H34N2(410.60): C, 84.83; H,
8.35; N, 6.82. Found: C, 85.11; H, 8.13; N, 6.54.
4.1.5. In vitro AChE/BuChE inhibition assay
The AChE and BuChE inhibitory activities were determined by Ellman’s method [34]. AChE
(electric eel), BuChE (equine serum), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), acetyl- and
butyrylthiocholine iodides were purchased from Sigma-Aldrich. Five different concentrations of the
corresponding compounds in Ethanol-DMSO (9:1) were prepared. For assay, 20 μL of the
corresponding enzyme (0.2 units/mL in Phosphate-pH 8.0 containing 25% v/v glycerol) was added in
a 24-well plate containing 2000 μL of PBS, 30 μL of the tested compound solution and 60 μL of
DTNB. After 3 min of incubation, 20 μL of the substrate solution (ATC/BTC) was added and then
incubated for at least 1 min at 25 °C. The absorbance was measured at 412 µM using a microplate
reader (BioTek synergy HT). The inhibition curve was obtained by plotting percentage of enzyme
activity (100% for the reference) versus logarithm of the tested compound concentration. Results are
reported as the mean ± SD for at least three different experiments.
For the kinetic analysis, the rate of ChE inhibition by different concentrations of the potent inhibitor
3s was measured in the presence of substrate (ATCh and/or BTCh). The cholinesterase inhibition was
determined as describedin the experimental section. For each inhibitor concentration, the initial
velocity was measured at different substrate concentrations (S) and the reciprocal of the initial velocity
(1/v) was plotted respect to the reciprocal of substrate concentration (1/[S]). Also the Lineweavere-

JOURNAL PRE-PROOF
Burk secondary plot for the inhibition of AChE/BuChE through re-plotting of slope versus at different
concentrations of the potent inhibitor 3s was measured in the presence of substrate (ATCh and/or
BTCh) for calculation of steady-state inhibition constant (Ki).
4.1.6. Determination of the inhibitory potency on Aβ1-42 self-induced and AChE-induced aggregation
Inhibitory activity of our compounds on self-induced and AChE induced aggregation of Aβ1-42 were
measured using a thioflavin T (ThT)-based fluorescence assay [36]. Aβ1-42 (Sigma A9810) was
dissolved in Phosphate Buffer Saline (PBS, pH 7.4, HyClone Thermo Scientific) containing 1%
ammonium hydroxide. This solution (50 µM) was incubated for 72 h at 37 oC for pre-fibrillation. For
the assay, Aβ1-42 (10 µL) ± human recombinant AChE (0.01 u/mL, Sigma C1682) was added to 0.05
M KP buffer (pH 7.4) and incubated at 37 oC for 48 h in the absence and presence of 100 μM solution
of the target compound. 100 µL of incubated mixture was mixed with 50 μL of thioflavin T (ThT,
200 μM) in 50 mM glycine-NaOH buffer (pH 8.5). The ThT excitation and emission were measured
at 448 µM and 490 µM, respectively (SpectraMax® Microplate Reader). Self or AChE-induced
aggregation percentages due to the presence of target compounds were calculated by the following
equation:
[(IFi/IFo) × 100] where IFi and IFo are the fluorescence intensities for Aβ ± AChE in the presence
and in the absence of inhibitors, respectively. Rifampicin (100 μM, Sigma R-3501) and donepezil
(100 μM, Sigma D-6821) were used as the reference agents.
4.1.7. Neuroprotection assay against H2O2-induced cell death in PC12 cells
Cell viability of rat differentiated PC12 cells (provided by the Pasteur Institute, Tehran, Iran)
was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay
[40]. All cultured media and supplements were purchased from Gibco. Cells were cultivated in
DMEM supplemented with 10% fetal calf serum, 5% horse serum and antibiotics (100 units/mL
penicillin, 100 μg/mL streptomycin). To induce neuronal differentiation, PC12 cells were re-
suspended using trypsin/EDTA (0.25%) and seeded in 96 well culture plate (4000 cells/well) and
cultured for 1 week in differentiation medium (DMEM + 2% horse serum + NGF (100 ng/mL) +
penicillin & streptomycin). To evaluate the effect of the compounds on survival rate of neurons,
the cultured medium was changed to NGF free medium and different concentrations of
compound 3s (1, 10, 100 μM) were applied on the cells. Quercetin (10 μM) was used as a
positive control. Compound 3s was diluted in DMEM and added to each well in the volume of
16

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10 μL. Induction of ROS mediated apoptosis was initiated by adding H2O2 (375 μM) to their
medium after three hours and MTT assay was performed after 12 hours. MTT solution (5
mg/mL) was added to each well in a volume of 10 μL, and after 3 h 100 μL of the solution (10%
SDS in 0.01 M HCl (v/v)) was added into each well. The plates were allowed to stand overnight
in the incubator in a humidified atmosphere. Using an ELISA reader, absorbance at 570 µM was
recorded for each well. Each experiment was performed in triplicate.
4.1.8. Docking simulation
Ligand-protein docking was performed using Autodock Vina (1.1.2) [41]. The binding poses of the
ligand to the active site of AChE was retrieved from the earlier mentioned application. 3D
coordination of the AChE was obtained from Protein Data Bank (PDB). The crystal structure of
AChE (PDB ID: 1eve) in complex with donepezil was chosen. All of non-protein atoms were omitted
during the protein. (RMSD = 0.3 Å) the protein was minimized. A grid box with the size of
252525 was determined and the center of box was fixed on the co-crystal ligand. After docking the
best pose was selected for further analysis.
Conflict of interest
The authors confirm that this article content has no conflict of interest.
Acknowledgments
This work was supported by a grant from the Research Council of Tehran University of Medical
Sciences and National Institute for Medical Research Development (NIMAD, grant number:
971370).
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N
K2CO3, benzyl bromides
CH3CN reflux, 3 h
Piperazine, KI
n
EtOH, reflux, 1 h
N
HN
2a, n=3
2b, n=5
NaH, Br(CH)nBr (n = 3 or 5)
DMF
N
N
n
N
H
n
R2R1N
Br
quinoline, isoquinoline
pyridine
piperidine and 4-benzylpiperidine
Carbazole
3a-w
1a, n=3
1b, n=5
CH3CN, reflux, 6-8 h
Scheme 1. Synthesis of target compounds 3a-w.
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Table 1. Inhibitory activity of the target compounds 3a-w against AChE and BuChE.
N
n
R2R1N
3a-w
AChE IC50
(µM)a
BuChE IC50
Compound
Nc
NR1R2
SId
(µM)b
3a
3b
3
3
3
3
3
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
benzylpiperazine
2-F-benzylpiperazine
4-F-benzylpiperazine
4-Br-benzylpiperazine
4-OMe-benzylpiperazine
benzylpiperazine
15.9±0.12
6.8±0.12
36.5±0.11
11.8±0.15
23.3±0.13
10.1±0.12
5.8±0.11
11.5±1.02
5.9±0.16
9.8±1.01
1.4±0.19
19.0±0.13
22.0±1.00
9.4±0.15
10.7±0.12
10.9±0.11
1.7±0.14
4.4±0.13
0.11±0.18
0.44±0.12
6.4±0.11
11.9±0.12
24.2±0.19
0.016±0.12
32.1±0.11
17.1±0.13
98.6±0.11
27.2±0.15
58.3±0.17
31.2±0.12
24.5±0.11
36.4±0.15
22.5±0.15
38.3±0.14
5.1±1.02
2.01
2.51
2.70
2.30
2.50
3.08
4.22
3.16
3.81
3.91
3.64
3.57
3.66
3.21
4.32
2.94
4.00
2.68
0.18
0.18
0.22
3.88
3.02
281.25
3c
3d
3e
3f
3g
2-Cl-benzylpiperazine
2-F-benzylpiperazine
2-Me-benzylpiperazine
2-NO2-benzylpiperazine
3-Br-benzylpiperazine
3-OMe-benzylpiperazine
3,4-dichlorobenzylpiperazine
4-OMe-benzylpiperazine
4-Me-benzylpiperazine
4-Cl-benzylpiperazine
4-Br-benzylpiperazine
4-F-benzylpiperazine
quinoline
3h
3i
3j
3k
3l
67.9±0.16
80.6±0.13
30.2±0.11
46.3±0.13
32.1±0.12
6.8±1.02
3m
3n
3o
3p
3q
3r
11.8±0.15
0.02±0.11
0.08±0.15
1.4±0.15
3s
3t
isoquinoline
3u
pyridine
3v
piperidine
46.2±0.18
73.2±0.15
4.5±0.11
3w
Donepezil
4-benzylpiperidine
-
a Inhibitor concentration (mean ± SEM of three experiments) required for 50% inactivation of AChE (electric eel).
b Inhibitor concentration (mean ± SEM of three experiments) required for 50% inactivation of BuChE (equine serum).
c Length of linker (n = 3 or 5).
d Selectivity index = IC50 (BChE)/IC50 (AChE).

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Table
2
. Inhibition of AChE-induced and self-induced Aβ aggregation by the compounds 3k and 3s.
Inhibition of Aβ aggregation (%)
Compound
Self-induceda
AChE-inducedb
3k
3s
19.5±1.3
27.4±1.8
27.5±4.3
22.0±5.4
19.2±3.4
24.9±6.4
12.2±3.0
26.1±2.5
Rifampicin
Donepezil
aInhibition of self-induced Aβ1-42 aggregation (10 µM) produced by the tested compound at 100
µM concentration. Values are expressed as means ± SEM of three experiments.
b Co-aggregation inhibition of Aβ1-42 and AChE (0.01 u/mL) by the tested compound at 100 µM
concentration was detected by ThT assay. Values are expressed as means ± SEM of three
experiments.
c Not Active
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R
N
I
H
N
N
4
N
OH
O
N
O
H
O
N
a
b
N
CH3
N
I
(CH2)9
N
H3C
I
N
c
Figure 1. Biologically active carbazoles.

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3
2.5
2
0.4
0.3
0.2
0.1
1.5
1
0.5
0
0
-6
-4
-2
0
2
4
6
8
-0.5
-1
-50
0
50
100 150 200 250
[I] (µM)
1/[S] (mM-1)
2-1: Lineweaver-Burk plot for the inhibition of
AChE by compound 3s.: control, ▄ : 216 µM,
▲ 108 µM, ● 54 µM.
2-2: Lineweaver-Burk secondary plot
of compound 3s for AChE Ki
calculation.
1.5
0.25
0.2
1
0.15
0.1
0.5
0
0.05
0
-6
-4
-2
0
2
4
6
8
-10
0
10
20
30
40
50
1/[S] (mM-1)
[I] (µM)
-0.5
2-3: Lineweaver-Burk plot for the inhibition
of BuChE by compound 3s.  : control,
▄: 48 µM, ▲: 24 µM, ●: 12 µM.
2-4: Lineweaver-Burk secondary plot of
compound 3s for BuChE Ki calculation.
Figure 2. Lineweaver-Burk plot for the inhibition of AChE and BuChE with compound 3s.
26

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1.5
1.0
0.5
****
***
**
Norm
Ctrl Querc10 M 1 M
10 M

100 M



compound 3s
Figure 3. The protective effect of selected compound 3s against H2O2 induced apoptosis in PC12 cells at
different concentrations. ****: P<0.0001, ***: P<0.001, **: P<0.0098 all versus control (Ctrl) group.
Quercetin (Querc) 10 µM was used as a positive control group.

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Figure 4. Binding modes of compound 3s with AChE.
28

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Highlights
 Novel carbazoles were introduced as multifunctional anti-Alzheimer’s disease
 Compound 3s having quinolinium part showed high AChE and BuChE inhibition activity
 Compound 3s showed significant anti-Aβ aggregation activity
 Compound 3s exhibited high neuroprotective activity
Graphical Abstract