Design, synthesis, and biological evaluation of selective and potent Carbazole-based butyrylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.08.035

Alzheimer's disease (AD) is the most common form of dementia. Inhibition of BChE might be a useful therapeutic target for AD. A new series of Carbazole-Benzyl Pyridine derivatives were designed synthesized and evaluated as butyrylcholinesterase (BChE) inhibitors. In vitro assay revealed that all of the derivatives had selective and potent anti- BChE activities. 3-((9H-Carbazol-9-yl)methyl)-1-(4-chlorobenzyl)pyridin-1-ium chloride (compound 8f) had the most potent anti-BChE activity (IC₅₀ value = 0.073 μM), the highest BChE selectivity and mixed-type inhibition. Docking study revealed that 8f interacted with the peripheral site, the choline binding site, catalytic site and the acyl pocket of BChE. Physicochemical properties were accurate to Lipinski's rule. In addition, compound 8f demonstrated neuroprotective activity at 10 μM. This compound could also inhibit AChE-induced and self-induced Aβ peptide aggregation at concentration of 100 μM and 10 μM respectively. The in-vivo study showed that compound 8f in 10 mg/kg increased the time spent in target quadrant in the probe day and decreased mean training period scape latency in rats. All results suggest that new sets of potent selective inhibitors of BChE have a therapeutic potential for the treatment of AD.

Full text of DOI:10.1016/j.bmc.2018.08.035

Accepted Manuscript
Design, Synthesis, and Biological Evaluation of selective and potent Carbazole-
Based Butyrylcholinesterase Inhibitors
Roshanak Ghobadian, Hamid Nadri, Alireza Moradi, Syed Nasir Abbas
Bukhari, Mohammad Mahdavi, Mehdi Asadi, Tahmineh Akbarzadeh, Hossein
Khaleghzadeh-Ahangar, Mohammad Sharifzadeh, Mohsen Amini
PII:
S0968-0896(18)30729-6
https://doi.org/10.1016/j.bmc.2018.08.035
BMC 14522
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 April 2018
20 August 2018
27 August 2018
Please cite this article as: Ghobadian, R., Nadri, H., Moradi, A., Bukhari, S.N.A., Mahdavi, M., Asadi, M.,
Akbarzadeh, T., Khaleghzadeh-Ahangar, H., Sharifzadeh, M., Amini, M., Design, Synthesis, and Biological
Evaluation of selective and potent Carbazole-Based Butyrylcholinesterase Inhibitors, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.08.035
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Design, Synthesis, and Biological Evaluation of selective and potent
Carbazole-Based Butyrylcholinesterase Inhibitors
Roshanak Ghobadian1, Hamid Nadri2, Alireza Moradi2, Syed Nasir Abbas Bukhari3, Mohammad
Mahdavi4, Mehdi Asadi1, Tahmineh Akbarzadeh1,5, Hossein Khaleghzadeh-Ahangar6,
Mohammad Sharifzadeh7, Mohsen Amini1
1Department of Medicinal Chemistry, Faculty of Pharmacy, and Drug Design & Development
Reseach Center, The institute of pharmaceutical Sciences (TIPS), Tehran University of Medical
Sciences, Tehran14176, Iran
2Pharmaceutical science research center and Faculty of Pharmacy, Shahid Sadoughi University
of Medical Sciences, Yazd 8915173143, Iran
3Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Al-jouf,
Sakaka 2014, Saudi Arabia.
4Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
5Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences,
Tehran, Iran
6Department of Physiology, School of Medicine, Babol University of Medical Sciences, Babol,
Iran
7Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran
Correspondence
Mohsen Amini (Ph.D.), Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran14176, Iran
Email: moamini@tums.ac.ir
ABSTRACT
1

Alzheimer’s disease (AD) is the most common form of dementia. Inhibition of BChE might be a
useful therapeutic target for AD. A new series of Carbazole-Benzyl Pyridine derivatives were
designed synthesized and evaluated as butyrylcholinesterase (BChE) inhibitors. In vitro assay
revealed that all of the derivatives had selective and potent anti- BChE activities. 3-((9H-
Carbazol-9-yl)methyl)-1-(4-chlorobenzyl)pyridin-1-ium chloride (compound 8f) had the most
potent anti-BChE activity (IC50 value = 0.073 μM), the highest BChE selectivity and mixed-type
inhibition. Docking study revealed that 8f interacted with the peripheral site, the choline
binding site, catalytic site and the acyl pocket of BChE. Physicochemical properties were
accurate to Lipinski's rule. In addition, compound 8f demonstrated neuroprotective activity at
10 µM. This compound could also inhibit AChE-induced and self-induced Aβ peptide
aggregation at concentration of 100 µM and 10 µM respectively. The in-vivo study showed that
compound 8f in 10 mg/kg increased the time spent in target quadrant in the probe day and
decreased mean training period scape latency in rats. All results suggest that new sets of potent
selective inhibitors of BChE have a therapeutic potential for the treatment of AD.
Key words: Butyrylcholinesterase; Alzheimer’s disease; docking study; Carbazole; In-vitro and
In-vivo assay
1. Introduction
AD is a progressive neurodegenerative disorder characterized by the loss of cognitive abilities,
with more than 47 million reported cases worldwide.1 Correspondingly, it is the most common
type of dementia accounting for about 50-60% of the overall cases of dementia among
individuals over 65 years old.2 Decreasing acetylcholine (Ach) in the cortex and hippocampus
regions of brain is the single most replicated neurotransmitter deficiency in AD.3 Furthermore,
in AD, there is an association between the deficiency of acetylcholine, the presence of plaques
of amyloid β-peptide (Aβ), neurofibrillary tangles and oxidative stress.
Both acetylcholinesterase (AChE) and BChE play important roles in the regulation of Ach level in
the nervous system.4 In a healthy brain, BChE seems only to play a supportive role, whereas,
AChE’s activity plays the main one. Nonetheless, it is reported that there was an enhancement
of BChE performance during the progression of AD.5 In vivo data supporting this hypothesis as
AChE knockout mouse model shows that the mice didn’t suffer from increasing ACh in the
absence of AChE, because of controlling the hydrolysis of ACh by BChE. Therefore, inhibition of
BChE might be a useful therapeutic target for the clinical use in AD.6
In recent years, there has been a switch of focus from AChE to BChE.3, 7-10 One of the benefits
for selective BChE inhibitors is that they don’t have the common side-effects of AChE inhibition,
such as, classical cholinergic toxicity.11
Both AChE and BChE have a similar overall structure, but some important differences exist in
the peripheral anionic site and the acyl binding pocket. The replacement between residues
2

Phe288 and Phe290 in AChE with Leu286 and Val288 in BChE leads to fit bulkier substrates in
the active site of BChE.7
Recently, it has been reported that selective BChE inhibitors such as N1-Phenetylnorcymserine
and Ethopropazine usually contain tri- or polycyclic structures.10, 12-14 On the other hand,
Carbazoles derivatives have shown beneficial effects in AD.15 They can, reportedly, inhibit Aβ
aggregation.16 For example, Carvedilol, a Carbazole derivative, is an effective inhibitor of Aβ
fibril formation.17 In recent studies by the D-ring opening of Galantamine, the series of
Carbazole derivatives have made.18-20 Subsequently, Carbazole derivatives could be a good
target for further studies.
SCHEME 1 Structures of Galantamine (A), Carbazole containing AChE inhibitors (B and C), benzyl
pyridine containing AChE inhibitor (D) and designed compounds 7a-j and 8a-j (E).
Secondly, Benzyl piperidine derivatives have been recently designed and synthesized, based on
the Donepezil structure, having cholinesterase inhibitory activity.21-24 It has been shown that
the designing of hybrids led to more potent cholinesterase inhibition.3, 4, 20, 25-28
Considering Carbazole and Benzyl piperidine as the object of further studies, the hypothesis of
the need for a bulkier compound to fit in the BChE active site and our interest to design of
hybrids, Carbazole-Benzyl piperidine hybriding might be a good strategy for designing a series
of potent BChE inhibitors as innovative anti-AD agents. Thus, the main objective of the present
study was to design and synthesize new Carbazole-Benzyl piperidine derivatives displaying anti-
Alzheimer activity through selective inhibition of BChE enzyme (Scheme 1 and 2).
3

SCHEME 2 Synthesis of compounds 7a-j and 8a-j.
2. Results and discussion
2.1. AChE and BChE inhibitory activity
The in vitro BChE and AChE inhibitory activities of the all compounds 7a-j and 8a-j were
calculated by the Ellman’s spectroscopic method and compared with Donepezil and
Galantamine as the reference drugs.24, 30 According to the Table 1, it is realized that some of the
synthesized derivatives (8c, 8e-j) had lower BChE IC50 values than Donepezil, while two of them
has equal values (7f and 7h). This is despite the fact that all of them were more potent BChE
inhibitor than Galantamine (BChE IC50 of Donepezil and Galantamine respectively: 0.35±0.02
μM, 9.4 ± 2.5 μM).
As it can be seen in Table 1, all derivatives show great anti-BChE activities (BChE IC50:0.073 -1.6
µM) and very excellent BChE selectivity index (SIBChE, IC50 of AChE /IC50 of BChE). Thus, all the
designed derivatives were selective for BChE inhibitory. These results could be a therapeutic
advantage for the treatment of AD, because in progressive form of AD, the loss of AChE-activity
is compensated by unaffected BChE.5 The best BChE IC50 was related to compound 8f (IC50
=
0.073±0.003μM) with great BChE selectivity (SIBChE= 273.97). BChE IC50 of 8f is 4.8 and 128.77-
times more than Donepezil and Galantamine respectively. It is obvious that the replacement of
4-(Chloromethyl) pyridine with 3-(Chloromethyl) pyridine, led to the reduction of anti-AChE
activity and the enhancement of anti-BChE activity in all derivatives (for AChE Ic50 8a-j> Ic50 7a-j
and for BChE Ic50 7a-j> Ic50 8a-j). Also, there is a great enhancement of SIBChE as a consequence
of the mentioned replacement. According to the Table 1, varying substituents on the phenyl
ring of the Benzyl-pyridine moiety led to changes of both anti-AChE and anti-BChE activity.
It seems that inclusion of methyl group in ortho position of benzyl moiety of derivatives (7h and
8j) led to the improvement of anti-BChE activities, but introduction of 2-Clor 2-F groups
diminished the anti-BChE activity (BChE Ic50 8d>8a>8j>8gandIc50 7a>7e>7d>7h). Thus, not only
substitution of 2-Me on benzyl moiety can be tolerated, but can also improve BChE inhibitory
activity. Also, inclusion of chlorine group in metha position of benzyl moieties in contrast to 3-F
4

and 3-Methyl, can improve inhibition of both enzymes (for both AChE and BChE: Ic50
8b>8h>8j>8e and Ic50 7b>7i>7d>7f). Furthermore, the introduction of substituent on the para
TABLE 1
AChE
inhibition
[IC50 (μM)]
BChE
inhibition
[IC50 (μM)]
SI BChE
Entry compoun
ds
x
y
z
z’
(IC50 AChE /IC50BuChE)
1
2
7a
Cl
Cl
Cl
Br
Cl
Br
Cl
Br
Cl
Cl
Cl
Br
Br
Cl
Br
Cl
Br
Cl
Cl
Br
-
H
F
H
H
F
F
H
12.7± 0.4
21.3±0.6
18.8±0.2
12.8±0.6
6.8±0.1
9.3±0.5
19.05±0.4
11.9±0.1
17.5±0.5
15.6±0.4
>100
0.67±0.04
1.10±0.011
0.89±0.018
0.58±0.006
0.59±0.023
0.35±0.017
1.60±0.048
0.35±0.007
0.73±0.029
18.95
19.32
21.12
22.06
11.52
26.57
11.9
7b
3
7c
H
H
H
Cl
H
H
Me
H
H
F
H
4
7d
H
H
H
Cl
H
H
Me
H
H
F
H
5
7e
Cl
H
6
7f
7
7g
H
8
7h
Me
H
34
9
7i
23.97
10
11
12
13
14
15
16
17
18
19
20
21
22
7j
H
0.81±0.016
0.37± 0.029
19.26
8a
F
>270.27
8b
H
>100
0.75±0.004
0.13±0.005
0.38 ±0.004
0.16±0.005
0.073±0.003
0.22±0.004
0.28±0.015
0.11±0.003
0.27
>133.34
225.15
>263.15
205
8c
H
H
Cl
H
H
Me
H
H
-
H
29.4±1.1
>100
8d
H
H
Cl
H
H
Me
H
-
Cl
H
8e
32.8±0.6
20.0±0.6
52.5±2.1
66.01
8f
H
273.97
238.63
235.75
210
8g
Me
H
8h
8i
8j
H
23.1±0.5
61.9
H
229.26
0.065
0.023±0.01
0.35±0.02
Donepezil
Galantam
in
-
-
-
-
-
1.7 ± 0.9
9.4 ± 2.5
0.18
aData are expressed as Mean ± SE (three independent experiments).
5

position of benzyl moiety in 7a-j derivatives diminished both anti-AChE and anti BChE activity as
observed in compounds 7c, 7g and 7j (for both AChE and BChE: Ic50 7c, 7g, 7j>7d). Although the
unsubstituted derivatives (7d and 8j) have shown good inhibitory activity against BChE (BChE
IC50: 0.58 and 0.27 µM respectively), substituent at para position of benzyl moieties of 8a-j
derivatives could slightly improve both AChE and BChE inhibitory activity. 4-Cl derivatives were
much more potent ChE inhibitors than 4-F and 4-Methyl ones (for both AChE and BChE: Ic50
8j>8c>8i>>8f). Overall, 8a-j derivatives were much more selective inhibitors of BChE than 7a-j
ones and the Chlorine substituent at para position of benzyl moieties due to its lipophilic
feature and bulkiness was the most favorable group for BChE inhibition in 8a-j derivatives (8f
has the best BChE IC50= 0.073 µM).
2.2. Inhibition of AChE-induced and self-induced Aβ aggregation
Compound 8f as a potent AChE inhibitor was selected to assess its ability to inhibit Aβ1-42
peptide self-aggregation employing the thioflavin T (ThT) fluorescence method and compared
with donepezil as reference compound. The obtained inhibition for compound 8f at 10 µM
concentration was 49.9 ± 1.7 % being higher than that of donepezil (16.4 ± 1.7 %) and Tacrine
(7.7 ± 1.5 %). To further explore the dual action of this compound, the capacity to inhibit the
AChE-induced Aβ1-40 peptide aggregation was examined employing the same ThT-based
fluorometric assay. Compound 8f in concentration of 100 µM exhibited a significant AChE-
induced Aβ aggregation inhibitory effect with percentage of inhibition 41.2 ± 3.5 more effective
than Tacrine and donepezil (7.2 ± 1.2 % and 25.2 ± 1.1, respectively), indicating that compound
8f binds to PAS of AChE.
2.3. Kinetic studies of AChE and BChE inhibition
As shown in Fig. 1, to realize the mechanism of BChE inhibition, Lineweaver-Burk curve was
drawn for the most potent derivative (8f) using three different concentrations of the inhibitor
(0, 0.042, 0.083 and 0.168 µM) and substrate (0.13, 0.32 and 0.69 mM).23 Fig. 1 clearly showed
a mixed-type inhibition for 8f derivative. Binding of compound 8f to BChE changed both Vmax
and Km values, a trend that is generally ascribed to mixed-type inhibition. In particular, a
decreased Vmax at increasing 8f concentrations and increasing intercepts (higher Km) with
higher 8f concentration were observed. According to Fig. 2, the inhibition constant (Ki) was
calculated from the plot of the slope versus the concentration of inhibitor and Ki value was
equal to 0.09 µM.
6

FIGURE 1 Kinetic study of compound 8f on the inhibition mechanism of BChE by Lineweaver-Burk plot
(1/V) sec.DA-1 versus (1/[S]) mM-1, where [I] and [S] mean concentrations of inhibitor and substrate
respectively.
FIGURE 2 The plot of the slope versus the concentration of inhibitor 8f (µM) for calculating Ki.
2.4. Docking studies
To obtain better concept on the binding to the active sites of BChE and the related inhibitory
activities, docking studies parallel to the design and synthesis of compounds were performed.
7

The mean binding energy scored (table 2) by Autodock4.2 ranged from -8.85 to -7.58 (kcal/mol)
and the lowest binding energy was selected for further analysis. Fig. 3a & b show position and
interactions of the most potent compound 8f using Pymol 2.1.1 and Discovery Studio Visualizer
v16.1.0.15350 software’s. The crystallographic structure of human BChE (1P0I) was chosen
from the RCSB protein data bank (http://www.rcsb.org) among other available one’s cause its
acceptable resolution (2.0 Å) and greater use of this pdb code in the articles.1, 7, 11, 34-36
As shown in the Fig. 3b, the binding mode of the compound 8f is characterized by the following
features: Aromatic planar properties of Carbazole moiety led to place over the choline binding
site, interact strongly with Trp82 by π-π stacking and hydrophobic π-alkyl interactions Also,
pyridine ring has two interactions with Leu286 and Tyr332 in the peripheral site.
Fig. 4 shows that the orientation of compound 8c, 8f and 8i as the three most potent
compounds (BChE IC50 values: 0.13, 0.073 and 0.11 µM respectively) in the active site of BChE is
similar.
FIGURE 3 a: Close-up view of the X-ray structure of huBChE in complex with compound 8f (PDB entry
1P0I; resolution 2.0 A), some important residues in the active site are labeled in black and 8f is shown as
green CPK, b: The binding mode of the most active compound 8f in the active site of BChE (carbons,
nitrogen and chlorine atoms of 8f are shown as brown, dark blue and light blue sticks, respectively).
Hydrogen bonds, Sulfur-π interaction, hydrophobic interactions, π-π T-shape and π-π stack interactions
are shown as red, yellow, green, blue and violet dotted lines, respectively.
8

FIGURE 4 The overlaid conformations of 8c, 8f and 8i as the three most potent compounds in the active
site of BChE. 8c, 8f, 8i and also their interactionsa are shown as orange, green and red dotted lines,
respectively.
TABLE 2. The mean binding energy of the compounds 7a-j and 8a-j.
Entry
compounds
Docking
energy
(kcal/mol)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
-7.76
-7.69
-7.63
-7.58
-7.63
-7.95
-7.72
-7.78
-8.08
-7.89
-8.09
-8.15
-8.25
-8.57
-8.45
-8.68
-8.44
-8.41
-8.85
-8.20
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
2.5. Screening of physicochemical properties
Lipinski's rule of five describes molecular
properties important
for
a
drug's pharmacokinetics and physicochemical properties in the human body.37 To ensure drug-
like physicochemical properties of 8f, a number of H-bond acceptors (HBA), a number of H-
bond donors (HBD), the polar surface area (tPSA), octanole/water partition coefficients (Clog P)
and a number of rotatable bonds (RBC) were calculated and shown in Table 3. According to the
table MW, HBD, HBA and ClogP values of 8f were accordant to Lipinski’s rule and would have
satisfactory physicochemical properties after the oral admission as a drug candidate.
TABLE 3 The mean binding energy of the compounds 7a-j and 8a-j.
Entry Compound HBD HBA ClogP tPSA[Å2] MW RBC
16
8f
0
2
3.05
6.25
383.90
4
a HBD: H-bond donors, HBA: H-bond acceptors, Clog P: Calculated octanol-water partition
coefficient, tPSA: topological polar surface area, RBC: Rotatable bond count.
9

2.6. Neuroprotective effect against H2O2-induced cell death in PC12 neurons
The MTT assay has been widely adopted and is a useful model for studying neuronal
differentiation. Thus, as an additional study neuroprotective activity for the most potent
compound (8f) against BChE was determined by in vitro MTT assay. As shown in fig. 5, data are
expressed as mean ± SEM (n = 8) and one-way analysis of variance (ANOVA) followed by
Newman-Keuls multiple comparisons test was carried out to determine the level of significance.
The percent of cell viability was calculated in comparison to the control group. Compound 8f
demonstrated good neuroprotective activity at 10 μM (% cell viability = 66.00and P<0.001
versus H2O2 treatment alone). In concentration of 1 and 100 µM there is no significant activity
that is referred to low concentration of drug in 1 µM and intrinsic toxicity of 8f at 100 µM.
FIGURE 5 Neuroprotectiveactivity of compound 8f basedon cell viability of PC12 cells in H2O2-induced
damage. Data are expressed as mean ± SEM (n = 8) and ANOVA followed by Newman-Keuls multiple
comparisons test was carried out to determine the level of significance. ****: P<0.0001 and ***:
P<0.001 versus H2O2 and +: P<0.05 versus H2O2 (negatively significant).
2.7. In vivo study
The 10 mg/kg dose of compound 8f significantly increased the time spent in target quadrant in
the probe day in compare with control and vehicle groups (p<0.05) but not as the donepezil
positive control group (fig. 6A). The 10 mg/kg dose of compound 8f significantly decreased the
10

mean training period scape latency (fig. 6B). The 10 mg/kg dose of compound 8f significantly
decreased the mean training period traveled distance to find the platform (p<0.05) but not as
the donepezil positive control group (fig. 6C). No significant difference was observed for
different treatments on swimming speed (fig. 6D).
FIGURE 6 Effects of i.p. injection of different doses of compound 8f, vehicle (DMSO), and donepezil as a
positive control group on the time spent in target quadrant in the probe day (A), scape latency average
during training (B), traveled distance average during training (C), and swimming speed average during
training (D). Each column shows mean±SEM. sco: scopolamine; don: donepezil (* P<0.05, and ***
P<0.001 different from the scopolamine 4mg/kg and scopolamine+DMSO 5% groups)
11

3. Conclusion
In conclusion, we reported design and synthesis of a new series of 9H-Carbazole-Benzyl
pyridinium hybrids as potent selective inhibitors of BChE. In vitro assay revealed that all of the
synthesized derivatives were more potent BChE inhibitor’s than Galantamine, while some of
them had lower BChE IC50 values than Donepezil. All derivatives show selective and potent anti-
BuChE activities ranging from 0.073 to 1.6 µM. This could be a therapeutic advantage for the
treatment of AD, because while AChE activity is strongly decreased in AD patients, BChE is
unaffected.5 Compound 8f was the most potent BChE inhibitor with excellent SI. Moreover,
compound 8f was found to be an inhibitor of AChE-induced Aβ aggregation, which can also
interfere with Aβ self-aggregation. Also, according to docking studies for 8f, aromatic planar
properties of Carbazole moiety led to place over the choline binding site of BChE active site. On
the other hand, while Benzyl pyridine moiety has some potent interactions with peripheral site,
the phenyl ring has an additional interaction with the acyl pocket. Screening of physicochemical
properties has revealed that MW, HBD, HBA and Clogp of 8f are accordant to Lipinski's rule and
thus would have satisfactory physicochemical properties after the oral admission as drug
candidate. Moreover, compound 8f demonstrated neuroprotective activity. All results suggest
that new potent selective inhibitors of BChE have a therapeutic potential for the treatment of
AD.
4. Experimental
4.1. Chemistry
1
All of Melting points were taken on a Kofler hot stage apparatus. Also, H and 13C nuclear
magnetic resonance (NMR) spectra were recorded on a Bruker FT-500, using TMS as an internal
standard and IR spectra were obtained on a Nicolet Magna FTIR 550 spectrophotometer (in
KBr). All chemicals were obtained from Merck and Sigma-Aldrich. Elemental analysis was
carried out by a Perkin Elmer 2400 CHN Elemental Analyzer using a tin capsule including 2-3 mg
of sample. The analysis was performed under excess oxygen exposure in the reactor chamber,
990 °C and tungsten trioxide catalyst for complete oxidation. The results of CHN analysis were
within ± 0.4 % of the calculated value.
General procedure for the synthesis of compound 4 and 5
According to scheme 2, to synthesize compound 4 or 5, TBAB (Tetrabutyl amonium bromide)
(2.73 mmol), was dissolved in an aqueous solution of NaOH 50% p/p (15 ml) and Toluene (15
ml) then stirred for 15 min. After a solution of compound 1 (5.45 mmol) in Toluene (15 ml) was
added. To this mixture, the compound 2 or 3 (16.3 mmol) was added and stirred at room
temperature for 12 hours.29 After completion of the reaction, the solvent was evaporated,
poured into water and extracted with Chloroform (50 ml). The organic layer was dried over
anhydrous Na2SO4, filtered and the solvent removed by evaporation. The resulting residue was
purified on a silica gel short column chromatography in Petroleum ether/Ethyl acetate (9/1) to
give a white/yellow solid (60% yield) (compound 4 or 5).
12

General procedure for the synthesis of compounds 7a-j and 8a-j
In order to synthesize 7a-j and 8a-j derivatives, compound 4 or 5 (1mmol) was dissolved in dry
Acetone (12mL) and collected with different Benzyl halides (1.5 mmol) (compounds 6a-j),
refluxing for 1-6 hours. The completion of each reaction was checked by thin layer
chromatography (TLC) and the precipitation was filtered off and washed with Petroleum
ether/Ethyl acetate solvent (4/1), to remove the remaining Benzyl halides. All products were
pure and did not need further purification.
9-(Pyridin-4-ylmethyl)-9H-carbazole (4)
Yellow solid; yield: 60%, melting point (mp) = 110-112°C. IR (KBr): 3109, 3043, 2925, 1672,
1638, 1448, 1375, 1327, 751 cm-1.1H NMR (DMSO-d6, 500 MHz): δ= 5.51 (s, 2H, N-CH2), 7.00 (d,
J=5.5 Hz, 2H, H3 ,5
՛
՛
), 7.26 (d, J= 7.0 Hz, 2H, H1,8), 7.28 (t, J= 7.5 Hz, 2H, H3,6), 7.43 (t, J= 8.0 Hz, 2H,
) ppm. 13C-NMR (DMSO-d6, 125
H2,7), 8.15 (d, J=8.0 Hz, 2H, H4,5), 8.48 (d, J=5.5 Hz, 2H, H2 ,6
՛
՛
MHz): δ= 43.1, 109.4, 119.2, 120.4, 122.3, 123.6, 125.9, 133.3, 148.3, 148.6 ppm.
9-(Pyridin-3-ylmethyl)-9H-carbazole (5)
1
Yellow solid; yield: 60%, mp = 115-117°C. IR (KBr): 3004, 3002, 1678, 1600, 1475 cm-1. H NMR
(DMSO-d6, 500 MHz): δ= 5.71 (s, 2H, N-CH2), 7.23(t, J=7.0 Hz, 2H, H3,6), 7.43 (d, J=8.5 Hz, 2H,
H2,7), 7.44-7.48 (m, 2H, H5 ,6
՛
՛
), 7.68 (d, J=8.5 Hz, 2H, H1,8), 8.18 (d, J=8.0 Hz, 2H, H4,5), 8.43 (dd,
) ppm.13C-NMR (DMSO-d6, 125 MHz): δ=
J1=5.5 Hz, J2=1.5 Hz, 1H, H4 ), 8.54 (d, J=1.5 Hz, 1H, H2
՛
՛
43.1, 109.4, 119.2, 120.4, 122.3, 123.6, 125.9, 133.3, 134.4, 139.9, 148.5, 148.6 ppm.
4-((9H-Carbazol-9-yl)methyl)-1-(2-fluorobenzyl)pyridin-1-ium chloride (7a)
Light brown solid; yield: 80%, mp = 116-120 °C. IR (KBr): 3041, 2919, 2852, 1639, 1617, 1488,
1455, 1342, 761 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ= 5.96 (s, 2H, N-CH2), 6.12 (s, 2H, N+-CH2),
7.25-7.29 (m, 4H, H3,6, 4˝,6˝), 7.42-7.48 (m, 3H, H2,7,3˝), 7.60 (d, J= 8.5 Hz, 2H, H1,8), 7.65 (t, J= 7.5
Hz, 1H, H5˝), 7.71(d, J= 6.0 Hz, 2H, H3 ,5
՛
՛
), 8.23 (d, J= 7.5 Hz, 2H, H4,5), 9.09 (d, J= 6.0 Hz, 2H, H2 ,6 )
՛
՛
ppm.13C-NMR (DMSO-d6, 125 MHz): δ= 44.9, 57.2, 109.3, 116.0 (JC-F= 20 Hz), 119.7, 120.5 (JC-F=
13 Hz), 121.0, 121.1, 122.5, 124.6, 125.2, 126.1, 126.8, 127.4, 131.7 (JC-F= 25 Hz), 132.0, 132.1,
139.8, 144.8, 145.0, 158.5, 160.4 (JC-F= 245 Hz) ppm. Mass, m/z (%): 471 (6), 350 (100), 274(10),
105 (7), 55(11) ppm. Anal. Calcd for C25H20ClFN2: C, 74.53; H, 5.00; N, 6.95. Found: C, 74.69; H,
5.30; N, 6.93.
4-((9H-Carbazol-9-yl)methyl)-1-(3-fluorobenzyl)pyridin-1-ium chloride (7b)
Dark yellow solid; yield: 81%, mp = 118-120 °C. IR (KBr): 3036, 2926, 1637, 1593, 1453, 1327,
1
754 cm-1. H NMR (DMSO-d6, 500 MHz): δ= 5.82 (s, 2H, N-CH2), 6.07 (s, 2H, N+-CH2), 7.25-7.29
(m, 3H, H3,6, 5˝), 7.35 (d, J= 7.5 Hz, 1H, H6 ˝), 7.42-7.49 (m, 4H, H2,7,2 ˝,4˝), 7.58 (d, J= 8.0 Hz, 2H,
13

H1,8), 7.69(d, J= 6.0 Hz, 2H, H3 ,5
՛
՛
), 8.23 (d, J= 7.5 Hz, 2H, H4,5), 9.07 (d, J= 6.0 Hz, 2H, H2 ,6 )
՛
՛
ppm.13C-NMR (DMSO-d6, 125 MHz): δ= 44.9, 61.8, 109.3, 113.0, 115, 119.7, 120.5, 121.0,
122.5, 124.6, 125.2, 126.1, 126.8, 127.4, 131.7 0, 132.0, 132.1, 139.8, 144.8, 145.0, 158.5, 160.4
(JC-F= 240 Hz) ppm. Anal. Calcd for C25H20ClFN2: C, 74.53; H, 5.00; N, 6.95. Found: C, 74. 39; H,
5.11; N, 6.70.
4-((9H-Carbazol-9-yl)methyl)-1-(4-fluorobenzyl)pyridin-1-ium chloride (7c)
Yellow solid; yield: 82%, mp = 236-238 °C. IR (KBr): 3093, 3035, 1675, 1603, 1513, 1444, 1374,
1
754 cm-1. H NMR (DMSO-d6, 500 MHz): δ= 5.72 (s, 2H, N-CH2), 6.03 (s, 2H, N+-CH2), 7.36 (t, J=
8.0 Hz, 2H, H3,6), 7.42 (d, J= 8.5 Hz, 2H, H1,8), 7.47 (t, J= 7.0 Hz, 2H, H2 ˝,6 ˝), 7.54 (t, J= 8.0, 2H,
H2,7), 7.77 (d, J= 8.0 Hz, 2H, H3 ˝,5 ˝), 8.23 (d, J= 7.5 Hz,2H, H4,5), 8.55 (d, J= 6.0 Hz, 2H, H3 ,5
՛
՛
), 9.51
(d, J= 6.0 Hz, 2H, H2 ,6
՛
՛
) ppm.13C-NMR (DMSO-d6, 125 MHz): δ= 44.9, 61.9, 116.1 (JC-F= 20 Hz),
120.5 (JC-F= 13 Hz), 124.6, 126.0, 126.8, 127.4, 130.0, 131.9 (JC-F= 8.5 Hz), 137.6, 146.5, 149.9,
161.7, 163.7 (JC-F= 250 Hz) ppm. Anal. Calcd for C25H20ClFN2: C, 74.53; H, 5.00; N, 6.95. Found: C,
74.72; H, 5.19; N, 7.08.
4-((9H-Carbazol-9-yl)methyl)-1-benzylpyridin-1-ium bromide (7d)
Cream solid; yield: 85%, mp = 160-162- °C. IR (KBr): 3109, 3043, 2925, 1672, 1638, 1448, 1375,
1327, 751 cm-1.1H NMR (DMSO-d6, 500 MHz): δ= 5.91 (s, 2H, N-CH2), 6.06 (s, 2H, N+-CH2), 7.42
(t, J= 8 Hz, 2H, H3,6), 7.44-7.52 (m, 5H, Ph), 7.59 (d, J= 8.5 Hz, 2H, H1,8), 7.68 (t, J= 8 Hz, 2H, H2,7),
8.02 (d, J= 6.0 Hz, 2H, H3 ,5
՛
՛
), 8.19 (d, J= 7.5 Hz, 2H, H4,5), 9.19 (d, J= 6.0 Hz, 2H, H2 ,6
) ppm.13C-
՛
՛
NMR (DMSO-d6, 125 MHz): δ= 44.6, 62.7, 109.3, 119.9, 120.3, 125.5, 127.4, 128.8, 129.3, 134.1,
137.7, 138.6, 145.0, 146.6, 159.3 ppm. Anal. Calcd for C25H21BrN2: C, 69.94; H, 4.93; N, 6.52.
Found: C, 69.81; H, 4.77; N, 6.77.
4-((9H-Carbazol-9-yl)methyl)-1-(2-chlorobenzyl)pyridin-1-ium chloride (7e)
Yellow solid; yield: 83%, mp = 170-172 °C. IR (KBr): 3003, 2924, 2639, 2742, 1736, 1456, 1375,
1
115, 747 cm-1. H NMR (DMSO-d6, 500 MHz): δ= 5.82 (s, 2H, N-CH2), 6.07 (s, 2H, N+-CH2), 7.28
(t, J= 8.0 Hz, 2H, H3,6), 7.42-7.50 (m, 3H, H4 ˝, 5 ˝, 6 ˝), 7.54-7.58 (m, 3H, H2,7,3 ˝), 7.61 (d, J= 8 Hz,
2H, H1,8), 7.71(d, J= 6 Hz, 2H, H3 ,5
՛
՛
), 8.24 (d, J= 7.5 Hz, 2H, H4,5), 9.03 (d, J= 6.0 Hz, 2H, H2 , 6 )
՛
՛
ppm.13C-NMR (DMSO-d6, 125 MHz): δ= 44.9, 60.5, 109.3, 116.0, 119.7, 120.5, 122.5, 124.6,
125.5, 126.1, 126.8, 127.3, 128.1, 130.0, 131.2, 131.5, 131.8, 132.1, 133.3, 139.8, 145.0, 145.1,
158.6 ppm. Anal. Calcd for C25H20Cl2N2: C, 71.61; H, 4.81; N, 6.68. Found: C, 71.69; H, 4.67; N,
6.87.
4-((9H-Carbazol-9-yl)methyl)-1-(3-chlorobenzyl)pyridin-1-ium bromide (7f)
Cream solid; yield: 81%, mp = 177-180 °C. IR (KBr): 3038, 2940, 1642, 1626, 1454, 1430, 1333,
1211, 753, 742, 686 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ= 5.82 (s, 2H, N-CH2), 6.08 (s, 2H, N+-
14

CH2), 7.29 (t, J= 8.0 Hz, 2H, H3,6), 7.43-7.50 (m, 5H, H2, 7,4˝, 5 ˝,6˝), 7.59 (d, J= 8.0 Hz, 2H, H1,8), 7.68
(s, 1H, H2 ˝), 7.69 (d, J= 6.5 Hz, 2H, H3 ,5 ), 8.23(d, J= 7.5 Hz, 2H, H4,5), 9.10 (d, J= 6.5 Hz, 2H, H2 ,6 )
՛
՛
՛
՛
ppm.13C-NMR (DMSO-d6, 125 MHz): δ= 44.9, 61.7, 109.3, 119.7, 120.5, 120.6, 122.5, 125.6,
126.1, 127.7, 128.4,129.3, 131.0, 133.6, 136.17,139.7, 144.7, 144.8, 158.3 ppm. Anal. Calcd for
C25H20BrClN2: C, 64.74; H, 4.35; N, 6.04. Found: C, 64.91; H, 4.44; N, 6.19.
4-((9H-Carbazol-9-yl)methyl)-1-(4-chlorobenzyl)pyridin-1-ium chloride (7g)
Yellow solid; yield: 84%, mpis higher than 250 °C (mp< 250°C). IR (KBr): 3098, 3022, 2981, 2928,
1
1662, 1643, 1448, 1368, 1332, 752 cm-1. H NMR (DMSO-d6, 500 MHz): δ= 5.98 (s, 2H, N-CH2),
6.13 (s, 2H, N+-CH2), 7.28 (t, J= 8.0 Hz, 2H, H3,6), 7.40-7.50 (m, 4H, H1,8,2˝,6 ˝), 7.55-7.62 (t, 2H, J=
8.0, H2,7), 7.71 (d, J= 8.0 Hz, 2H, H3 ˝,5 ˝ ), 8.23 (d, J= 8.0 Hz, 2H, H4,5), 8.55 (d, J= 6 Hz, 2H, H3 ,5
՛
՛),
9.08 (d, J= 6.0 Hz, 2H, H2 ,6
՛
՛
) ppm.13C- NMR (DMSO-d6, 500 MHz): δ= 44.9, 61.7, 109.3, 119.7,
120.6, 122.5, 125.6, 126.1, 127.7, 128.9, 131.0, 133.6, 139.8, 144.8, 158.4 ppm. Anal. Calcd for
C25H20Cl2N2: C, 71.61; H, 4.81; N, 6.68. Found: C, 71.55; H, 4.99; N, 6.79.
4-((9H-Carbazol-9-yl)methyl)-1-(2-methylbenzyl)pyridin-1-ium bromide (7h)
Cream solid; yield: 79%, mp = 145-146 °C. IR (KBr): 3042, 2966, 2926, 1645, 1597, 1455, 1420,
1326, 744 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ= 2.26 (s, 3H, CH3), 5.89 (s, 2H, N-CH2), 6.11 (s,
2H, N+-CH2), 7.17 (d, J= 7.5 Hz, 1H, H3˝), 7.24-7.32 (m, 5H, H3, 6, 4˝, 5 ˝, 6 ˝), 7.45 (t, J= 8.0 Hz, 2H,
H2,7), 7.61 (d, J= 8.0 Hz, 2H, H1,8), 7.72 (d, J= 6.5 Hz, 2H, H3 ,5
՛
՛
), 8.23 (d, J= 7.5 Hz, 2H, H4,5), 8.93
(d, J= 6.5 Hz, 2H, H2 ,6
՛
՛
) ppm.13C- NMR (DMSO-d6, 500 MHz): δ= 18.7, 44.9, 60.9, 109.3, 119.7,
120.5, 120.6, 122.5, 125.6, 126.1, 126.6, 129.3, 130.9, 132.0, 137.0, 139.8, 144.8, 158.2 ppm.
Anal. Calcd for C26H23BrN2: C, 70.43; H, 5.23; N, 6.32. Found: C, 70.19; H, 5.40; N, 6.29.
4-((9H-Carbazol-9-yl)methyl)-1-(3-methylbenzyl)pyridin-1-ium chloride (7i)
Dark yellow solid; yield: 80%, mp> 250°C. IR (KBr): 3314, 3006, 2924, 2858, 1637, 1598, 1454,
1327, 154 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ= 2.28 (s, 3H, CH3), 5.76 (s, 2H, N- CH2), 6.10 (s,
2H, N+- CH2), 7.22 (d, J= 7.0 Hz, 1H, H4˝), 7.26-7.33 (m, 5H, H3,6,1 ˝,5 ˝,6 ˝), 7.45 (t, J= 7.5 Hz, 2H,
H2,7), 7.59 (d, J= 7.5 Hz, 2H, H1,8), 7.68 (d, J= 6.0 Hz, 2H, H3 ,5
՛
՛
), 8.24 (d, J= 7.5 Hz, 2H, H4,5), 9.07
(d, J= 6.0 Hz, 2H, H2 ,6
՛
՛
) ppm.13C- NMR (DMSO-d6, 500 MHz): δ= 20.6, 20.8, 22.6, 44.9, 62.7,
109.2, 119.7, 120.6, 122.5, 125.6, 126.0, 126.3, 129.3, 133.9, 138.5, 139.8, 144.8, 158.2 ppm.
Anal. Calcd for C26H23ClN2: C, 78.28; H, 5.81; N, 7.02. Found: C, 78.18; H, 5.63; N, 6.83.
4-((9H-Carbazol-9-yl)methyl)-1-(4-methylbenzyl)pyridin-1-ium chloride (7j)
15

Cream solid; yield: 82%, mp> 250 °C. IR (KBr): 3037, 2922, 2854, 1639, 1616, 1594, 1454, 1352,
759 cm-1.1H NMR (DMSO-d6, 500 MHz): δ= 2.28 (s, 3H, CH3), 5.78 (s, 2H, N-CH2), 6.09 (s, 2H, N+-
CH2), 7.22 (d, J= 7.5 Hz, 2H, H3˝,5˝), 7.28 (t, J= 7.5 Hz, 2H, H3,6), 7.41-7.47 (m, 4H, H2, 7, 2˝, 6 ˝), 7.60
(d, J= 8.5 Hz, 2H, H1,8), 7.69 (d, J= 6.5 Hz, 2H, H3 ,5
՛
՛
), 8.24 (d, J= 7.5 Hz, 2H, H4,5), 9.09 (d, J= 6.5 Hz,
1H, H2 ,6
՛
՛
) ppm.13C-NMR (DMSO-d6, 125 MHz): δ= 20.6, 44.9, 62.5, 109.3, 119.7, 120.5, 122.4,
125.5, 126.1, 129.0, 129.6, 131.0, 138.9, 139.7, 144.5, 158.0 ppm. Anal. Calcd for C26H23ClN2: C,
78.28; H, 5.81; N, 7.02. Found: C, 78.39; H, 5.70; N, 7.17.
3-((9H-Carbazol-9-yl)methyl)-1-(2-fluorobenzyl)pyridin-1-ium chloride (8a)
Yellow solid; yield: 79%, mp> 250 °C. IR (KBr): 3040, 2926, 2852, 1620, 1488, 1454, 1328, 755
1
cm-1. H NMR (DMSO-d6, 500 MHz): δ= 5.95 (s, 4H, 2-(N-CH2)), 7.25-7.29 (m, 4H, H3,6, 4˝,6 ˝),
7.42-7.44 (m, 2H, H2,7), 7.52-7.54 (m, 2H, H3 ˝,5 ˝), 7.65 (d, J= 7.5 Hz, 2H, H1,8), 8.05-8.07 (m, 1H,
H5
՛
), 8.19-8.21(m, 3H, H4,5,6
՛
), 9.10-9.12 (m, 2H, H2 ,4
) ppm.13C-NMR (DMSO-d6, 125 MHz): δ=
՛
՛
42.7, 57.8, 110.6, 119.8, 120.5, 122.5, 123.6, 125.9, 126.2, 127.9, 128.5, 130.2, 131.0, 131.5,
135.8, 140.0, 142.6, 143.5, 162.5(JC-F= 235 Hz) ppm. Anal. Calcd for C25H20ClFN2: C, 74.53; H,
5.00; N, 6.95. Found: C, 74.44; H, 5.07; N, 7.09.
3-((9H-Carbazol-9-yl)methyl)-1-(3-fluorobenzyl)pyridin-1-ium bromide (8b)
Yellow solid; yield: 80%, mp> 250 °C. IR (KBr): 3012, 3002, 2928, 2877, 1640, 1626, 1481, 1454,
1327, 753 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ= 5.96 (s, 2H, N-CH2), 5.98 (s, 2H, N+-CH2), 7.24-
7.28 (m, 3H, H3,6,6˝), 7.40-7.50 (m, 5H, H2, 7, 3 ˝.4 ˝,5 ˝), 7.72 (d, J= 8.0 Hz, 2H, H1,8), 8.06 (t, J= 6.5
Hz, 1H, H5
՛
), 8.11 (d, J= 6.5 Hz, 1H, H6
՛
), 8.20 (d, J= 8.0 Hz, 2H, H4,5), 9.27 (d, J= 5.5 Hz, 1H, H4 ),
՛
9.47 (s, 1H, H2
՛
) ppm.13C-NMR (DMSO-d6, 125 MHz): δ= 42.7, 72.1, 110.1, 117.3, 120.5, 122.5,
126.1, 127.6, 128.8, 129.5, 130.8, 131.1, 133.5, 137.4, 139.7, 140.2, 143.1, 149.1, 162.2 (JC-F=
240 Hz) ppm. Anal. Calcd for C25H20BrFN2: C, 67.12; H, 4.51; N, 6.26. Found: C, 67.32; H, 4.64; N,
6.39.
3-((9H-Carbazol-9-yl)methyl)-1-(4-fluorobenzyl)pyridin-1-ium bromide (8c)
Cream solid; yield: 80%, mp> 250 °C. IR (KBr): 3045, 3004, 2932, 1627, 1598, 1435, 1432, 1323,
1222, 748 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ= 5.92 (s, 2H, N-CH2), 5.97 (s, 2H, N+-CH2), 7.26-
7.28 (m, 4H, H3,6, ,2 ˝,6 ˝), 7.45 (t, J= 7.0 Hz, 2H, H2,7), 7.64 (t, J= 7.0 Hz, 2H, H3 ˝,5 ˝), 7.71 (d, J= 8.0
Hz, 2H, H1,8), 8.05 (t, J= 6.5 Hz, 1H, H5
՛
), 8.12 (d, J= 6.5 Hz, 1H, H6
՛
), 8.20 (d, J= 7.5 Hz, 2H, H4,5),
9.23 (d, J= 5.5 Hz, 1H, H4 ), 9.36 (s, 1H, H2
՛
՛
) ppm. 13C-NMR (DMSO-d6, 125 MHz): δ= 42.7, 62.3,
109.5, 116.1 (JC-F= 23.5 Hz), 118.9, 119.7, 120.5(JC-F= 10 Hz), 122.5, 125.9, 126.2, 128.4, 130.2,
131.5, 139.0, 139.7, 142.6, 143.5, 143.9, 162.5 (JC-F= 250 Hz) ppm. Anal. Calcd for C25H20BrFN2:
C, 67.12; H, 4.51; N, 6.26. Found: C, 67.40; H, 4.59; N, 6.40.
3-((9H-Carbazol-9-yl)methyl)-1-(2-chlorobenzyl)pyridin-1-ium chloride (8d)
16

Light brown solid; yield: 80%, mp> 250 °C. IR (KBr): 3043, 3017, 2931, 1627, 1595, 1481, 1453,
1328 cm-1.1H NMR (DMSO-d6, 500 MHz): δ= 5.96 (s, 4H, 2-(N-CH2)), 7.25 (t, J= 7.0 Hz, 2H, H3,6),
7.41-7.51 (m, 6H, H2, 7, 3 ˝, 4 ˝,5 ˝,6 ˝), 7.66 (d, J= 8.0 Hz, 2H, H1,8), 8.10 (t, J= 6.5 Hz, 1H, H5
՛), 8.20 (d,
J= 7.5 Hz, 2H, H4,5), 8.30 (d, J= 6.5 Hz, 1H, H6 ), 8.99 (s, 1H, H2 ), 9.11 (d, J= 6.0 Hz, 1H, H4 )
՛
՛
՛
ppm.13C-NMR (DMSO-d6, 125 MHz): δ= 42.7, 61.3, 109.4, 119.6, 119.8, 120.5, 122.5, 125.9,
126.2, 127.9, 128.2, 128.3, 128.4, 129.9, 131.0, 131.3, 131.5, 131.8, 133.3, 139.0, 142.5, 144.1,
145, 150, 153 ppm. Anal. Calcd for C25H20Cl2N2: C, 71.61; H, 4.81; N, 6.68. Found: C, 71.49; H,
4.91; N, 6.48.
3-((9H-Carbazol-9-yl)methyl)-1-(3-chlorobenzyl)pyridin-1-ium bromide (8e)
Light brown solid; yield: 81%, mp> 250 °C. IR (KBr): 3011, 3004, 2926, 1677, 1630, 1480, 1428,
1
850, 735, 674 cm-1. H NMR (DMSO-d6, 500 MHz): δ= 5.99 (s, 4H, 2(N- CH2)), 7.27 (t, J= 7.5 Hz,
2H, H3,6), 7.43-7.50 (m, 4H, H2,7, 5˝,6˝), 7.55 (d, J= 7.5 Hz, 1H, H4 ˝), 7.73-7.74 (m, 3H, H1,8, 2 ˝), 8.06
(t, J= 6.5 Hz, 1H, H5
՛
), 8.12 (d, J= 6.5 Hz, 1H, H6
՛
), 8.20 (d, J= 8.0 Hz, 2H, H4,5), 9.28 (s, 1H, H2 ),
՛
9.48(d, J= 5.5 Hz, 1H, H4
՛
) ppm.13C- NMR (DMSO-d6, 500 MHz): δ= 42.7, 72.1, 109.5, 119.7,
120.4, 120.5, 122.5, 125.9, 126.2, 127.6, 128.5, 128.8, 129.2, 129.5, 130.8, 131.1, 133.5, 136.1,
139.9, 140.2, 143.1, 144.12, 145, 150, 153 ppm. Anal. Calcd for C25H20BrClN2: C, 64.74; H, 4.35;
N, 6.04. Found: C, 64.55; H, 4.19; N, 5.96.
3-((9H-Carbazol-9-yl)methyl)-1-(4-chlorobenzyl)pyridin-1-ium chloride (8f)
Yellow solid; yield: 82%, mp>250 °C. IR (KBr): 3038, 3007, 2929, 1626, 1594, 1489, 1452, 1320,
1
748 cm-1. H NMR (DMSO-d6, 500 MHz): δ= 5.86 (s, 2H, N-CH2), 5.94 (s, 2H, N+-CH2), 7.27 (t,
J=7.5 Hz, 2H, H3,6), 7.44 (t, J= 7.5 Hz, 2H, H2,7), 7.48 (d, J= 8.0 Hz, 2H, H2˝,6 ˝), 7.53 (d, J= 8.0 Hz,
2H, H3 ˝,5˝), 7.66 (d, J= 8.0 Hz, 2H, H1,8), 8.05 (t, J= 6.5 Hz, 1H, H5
՛
), 8.14 (d, J= 6.5 Hz, 1H, H6
՛
),
8.20 (d, J= 7.5 Hz, 2H, H4,5), 9.17 (d, J= 5.5 Hz, 1H, H4 ), 9.24 (s, 1H, H2
՛
՛
) ppm.13C-NMR (DMSO-d6,
125 MHz): δ= 42.7, 62.4, 109.4, 119.6, 120.5, 120.5, 122.6, 125.9, 126.2, 128.4, 129.0, 129.2,
130.8, 131.0, 132.8, 134.2, 139.1, 139.7, 142.7, 143.8, 144.2, 150, 153 ppm. Anal. Calcd for
C25H20Cl2N2: C, 71.61; H, 4.81; N, 6.68. Found: C, 71.79; H, 4.73; N, 6.41.
3-((9H-Carbazol-9-yl)methyl)-1-(2-methylbenzyl)pyridin-1-ium bromide (8g)
Light brown solid; yield: 80%, mp> 250 °C. IR (KBr): 3024, 2826, 2861, 1627, 1598, 1483, 1454,
1
1378 cm-1. H NMR (DMSO-d6, 500 MHz): δ= 2.17 (s, 3H, CH3), 5.99 (s, 2H, N-CH2), 6.02 (s, 2H,
N+-CH2),7.16-7.19 (m, 3H, H3˝,5 ˝,6 ˝), 7.24 (t, J=7.5 Hz, 2H, H3,6), 7.32 (t, J=7.0 Hz, 1H, H4 ˝), 7.43 (t,
J= 7.5 Hz, 2H, H2,7), 7.72 (d, J= 7.5 Hz, 2H, H1,8), 8.11 (t, J= 6.5 Hz, 1H, H5
՛
), 8.20 (d, J= 7.0 Hz, 2H,
) ppm.13C-NMR
H4,5), 8.24 (d, J= 6.5 Hz, 1H, H6 ), 9.04 (s, 1H, H2 ), 9.10(d, J= 5.5 Hz, 1H, H4
՛
՛
՛
(DMSO-d6, 125 MHz): δ= 18.7, 42.7, 61.3, 109.5, 119.6, 120.5, 122.5, 125.9, 126.5, 127.2, 128.2,
17

129.3, 130.8, 131.7, 136.9, 138.9, 139.6, 142.6, 143.8, 145 ppm. ESI-MS: m/z = 444.4 [M+1]+.
Anal. Calcd for C26H23BrN2: C, 70.43; H, 5.23; N, 6.32. Found: C, 70.29; H, 5.35; N, 6.43.
3-((9H-Carbazol-9-yl)methyl)-1-(3-methylbenzyl)pyridin-1-ium chloride (8h)
Light brown solid; yield: 81%, mp> 250 °C. IR (KBr): 3035, 3004, 2926, 1628, 1601, 1454, 1420,
1
1370, 747, 722, 676 cm-1. H NMR (DMSO-d6, 500 MHz): δ= 2.28 (s, 3H, CH3), 5.82 (s, 2H, N-
CH2), 5.94 (s, 2H, N+-CH2), 7.24-7.28 (m, 6H, H3, 6, 2 ˝, 4˝, 5 ˝,6 ˝), 7.45 (t, J= 7.5 Hz, 2H, H2,7), 7.68 (d,
J= 8.0 Hz, 2H, H1,8), 8.05 (t, J= 6.5 Hz, 1H, H5
՛
), 8.16 (d, J= 6.5 Hz, 1H, H6
՛
), 8.21 (d, J= 8.0 Hz, 2H,
H4,5), 9.16 (d, J= 6.0 Hz, 1H, H4 ), 9.47 (s, 1H, H2
՛
՛
) ppm.13C-NMR (DMSO-d6, 125 MHz): δ= 20.9,
42.7, 63.4, 109.4, 119.6, 119.7, 120.4, 120.5, 122.5, 125.9, 126.2, 128.5, 129.1, 129.3, 130.0,
133.9, 138.5, 139.1, 139.7, 142.6, 143.7, 143.8, 144.13, 144.79 ppm. Anal. Calcd for C26H23ClN2:
C, 78.28; H, 5.81; N, 7.02. Found: C, 78.40; H, 5.73; N, 6.96.
3-((9H-Carbazol-9-yl)methyl)-1-(4-methylbenzyl)pyridin-1-ium chloride (8i)
Yellow solid; yield: 82%, mp> 250 °C. IR (KBr): 3039, 3002, 2855, 1627, 1615, 1454, 1413, 1320,
754 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ= 2.31 (s, 3H, CH3), 5.80 (s, 2H, N-CH2), 5.92 (s, 2H, N+-
CH2), 7.21-7.26 (m, 4H, H3, 6, 3˝, 5 ˝), 7.35-7.37 (m, 2H, H2 ˝, 6 ˝), 7.43-7.45 (m, 2H, H2,7), 7.66 (d, J=
8.0 Hz, 2H, H1,8), 8.03 (t, J= 6.5 Hz, 1H, H5
H4,5), 9.12-9.14 (m, 1H, H4 ), 9.23 (s, 1H, H2
՛
), 8.12 (d, J= 6.5 Hz, 1H, H6
՛
), 8.20 (d, J= 7.5 Hz, 2H,
՛
՛
) ppm.13C- NMR (DMSO-d6, 500 MHz): δ= 20.7, 42.7,
64.3, 109.4, 119.6, 120.4, 122.5, 126.1, 127.2, 128.3, 128.9, 129.6, 132.8, 135.2, 139.0, 139.7,
143.0, 143.6 ppm. Anal. Calcd for C26H23ClN2: C, 78.28; H, 5.81; N, 7.02. Found: C, 78.39; H, 5.74;
N, 7.24.
3-((9H-Carbazol-9-yl)methyl)-1-benzylpyridin-1-ium bromide (8j)
Cream solid; yield: 80%, mp> 250 °C. IR (KBr): 3030, 3003, 22887, 1627, 1615, 1454, 1427, 1343,
1113 cm-1. 1H NMR (DMSO-d6, 500 MHz): δ= 5.96 (s, 2H, N-CH2), 6.00 (s, 2H, N+-CH2), 7.26 (t, J=
7.5 Hz, 2H, H3,6), 7.42-7.45 (m, 5H, H2, 7, 3˝, 4 ˝, 5 ˝), 7.56-7.58 (m, 2H, H2 ˝,6 ˝), 7.75 (d, J= 8.0 Hz, 2H,
H1,8), 8.06 (t, J= 5.5 Hz, 1H, H5
՛
), 8.11 (d, J= 5.5 Hz, 1H, H6 ), 8.20 (d, J= 7.5 Hz, 2H, H4, 5), 9.28-9.30
՛
(m, 1H, H4 ), 9.53 (s, 1H, H2
՛
՛
) ppm.13C- NMR (DMSO-d6, 500 MHz): δ= 42.7, 63.0, 109.5, 119.6,
120.4, 120.5, 122.5, 125.9, 126.2, 128.4, 128.7, 128.9, 129.1, 129.4, 134.0, 138.9, 139.6, 143.0,
143.5, 149.6 ppm. Anal. Calcd for C25H21BrN2: C, 69.94; H, 4.93; N, 6.52. Found: C, 69.76; H,
4.81; N, 6.69.
4.2. Cholinesterase inhibition assay
Electric eel (Torpedo californica) AChE (type VI-S), BChE (E.C.3.1.1.8, from equine serum),
Acetylthiocholine iodide, Butyrylthiocholine iodide and 5,5׳-Dithiobis[2-nitrobenzoic acid]
(DTNB) were purchased from Sigma–Aldrich (Steinheim, Germany). Potassium dihydrogen
phosphate, Dipotassium hydrogen phosphate, Potassium hydroxide and Sodium hydrogen
18

carbonate were obtained from Fluka. Donepezile hydrochloride and Galantamine hydro
bromide were used as reference drugs, which were purchased from Sigma-Aldrich. The AChE
and BChE inhibitory activity of derivatives were determined by the Ellman’s spectroscopic
method using Acetylthiocholine iodide or Butyrylthiocholine iodide respectively as substrate in
24-well plates.24, 30 The stock solutions of the derivatives were prepared by dissolving them in
Dimethyl sulfoxide (DMSO), then diluted in absolute Ethanol to three different concentrations
and done for each concentration in triplicate to obtain 20―80% enzyme inhibitions. The assay
solution consisted of 2 mL Phosphate buffer (0.1 M, pH = 8), 60 μL DTNB, 30 μL test compound
and 20 μL of 5 IU/mL AChE or BChE solution. Then, the above mixture was pre-incubated for 10
min at 25°C. The reaction was then initiated by adding 20 μL of Acetylthiocholine iodide or
Butyrylthiocholine iodide as substrate to each well. Blank assays were also carried out making
up all ingredients excluding the substrate. The absorbance changes were scored at 412 nm for 5
min using a Synergy HTX multimode plate reader. In order to calculate IC50 values the obtained
data were applied for drawing inhibition curves (log inhibitor concentration versus percent of
inhibition) by Microsoft Excel 2010.
4.3. Kinetic studies of BChE inhibition
In order to explore the inhibition mechanism of designed derivatives, reciprocal plots of1/V
versus 1/[S] were obtained using four different fixed concentrations of the most potent
derivative 8f(0, 0.042, 0.083 and 0.168 µM) and Butyrylthiocholine iodide (0.13, 0.32 and 0.69
mM) as inhibitor and substrate respectively.23 The experiments were performed similar to the
mentioned enzyme inhibition assay. The slopes of Lineweaver–Burk plots were then plotted
against the different concentrations of inhibitor 8f, and Ki was determined as the intercept on
the negative x-axis. All rate measurements were performed in triplicate and data analysis was
performed with Microsoft Excel 2010.
4.4. Determination of the inhibitory potency on Aβ1-42 self-aggregation and Aβ1-40 aggregatin
induced by AchE
38, 39
ThT-based fluorometric assay method
was performed for investigation of Aβ1-42 self-
aggregation. Pre-treated Aβ sample (Bachem company, Switzerland) with 1,1,1,3,3,3-
hexafluoro-2-propanol (HFIP) was diluted in assay buffer to have a stock solution ([Aβ1-42] 50
mM). Experiments were performed by incubating the peptide in phosphate buffer (pH 7.4), at
37 ˚C for 48 h (final Aβ concentration = 25 mM, 10 mL) with and without inhibitor. The inhibitor
(8f) was dissolved in DMSO and diluted in the assay buffer at a final concentration of 10 µM.
Blanks containing inhibitor and ThT were also prepared and evaluated to account for quenching
and fluorescence properties. After incubation, samples were diluted to a final volume of 200 µL
with 180 µL ThT (5 mM in 50 mM glycine-NaOH buffer, pH 8.5). The fluorescence intensity was
measured with Multi-mode plate reader (EnSpire, PerkinElmer Waltham, Massachusetts,
19

United States) at λex 446 nm and λem 490 nm, was used to measure the fluorescence intensity.
Each assay was run in triplicate and each reaction was repeated at least three independent
times, values at plateau were averaged. After subtracting the background fluorescence of 5 mM
ThT solution, the fluorescence intensities were compared and the percentage of inhibition was
calculated by the following equation: 100 - [(IFi - IFb)/(IFo - IFb) × 100] where IFi, IFo and IFb are
the fluorescence intensities obtained for Aβ aggregation in the presence of inhibitors, in the
absence of inhibitors and the blanks, respectively.
The potency of 8f in inhibition of Aβ1-40 aggregation was evaluated using thioflavin T (ThT)
method.40, 41 Aβ (1-40) as trifluoroacetate salt (Bachem Company, Switzerland) and AChE
(Sigma, Electrophorus electricus) were mixed in presence or absence of 8f (100µM) and were
incubated for 24 h at room temperature. The final concentrations of Aβ (dissolved in DMSO and
diluted 0.215 M sodium phosphate buffer, pH 8), AChE (dissolved in 0.215 M sodium phosphate
buffer, pH 8.0) and the tested compound are 200 µM, 2 µM and 100 µM respectively. The ThT
fluorescence method was used for measurement the fluorescence intensity by multi-mode
plate reader (EnSpire, PerkinElmer Waltham, Massachusetts, United States) at λex 446 nm and
λem 490 nm. After co-incubation, 20 µL of the mixture solutions was diluted to a final volume of
2 mL with ThT (1.5 µM in 50 mM glycine-NaOH buffer, pH 8.5). Blanks containing Ab, AChE, Ab
plus the tested compound, and AChE plus the tested compound in 0.215 M sodium phosphate
buffer (pH 8.0) were prepared. The percent inhibition of the AChE induced aggregation due to
the presence of inhibitor was calculated by using the above equation. Each assay was run in
triplicate and each reaction was repeated at least three independent times, values at plateau
were averaged.
4.5. Docking studies
In order to clarify the interaction mode of compound 8f in the active site of BChE, docking
studies were carried out using the autodock4.2.31, 42 The crystallographic structure of human
butyrylcholinesterase (1P0I) was retrieved from the RCSB protein data bank
(http://www.rcsb.org) and the water molecules were removed from the protein structure. To
validate our docking procedure, the co-crystallized ligand was re-docked into the active site of
the enzyme and the reasonable RMSD value of 1.948 Å was obtained. Subsequently the 3D
structure of the compound 8f was generated using Marvine Sketch 6.2.0, 2014
(http://www.chemaxon.com). The enzyme Pdbqt was provided using AutoDockTools-1.5.6,
2013 (http://mgltools.scripps.edu), polar hydrogen atoms were added to amino acid residues
and Koullman charges were assigned to all atoms of the enzyme. Also, maps were calculated
with 0.375 Ǻ spacing between grid points by Autogrid 4. The autodock4.2 parameters were set
as follow; box size: 60×60×60 Å, the center of box: x = 135.943, y = 112.748, z = 40.693. The
other parameters were left unchanged. Docking was performed with the Lamarckian genetic
20

algorithm (LGA) and the calculated geometries were ranked in terms of free energy of binding
(in kcal/mol). In all, 100 docking runs were performed and the lowest binding energy was
selected for further analysis. Graphic visualizations were performed by Discovery Studio
Visualizer v16.1.0.15350 (http://accelrys.com).
4.6. Computational methods
The ClogP, tPSA, HBD, and HBA values were calculated by the means of Marvine Sketch 6.2.0.
Also, RBC was calculated using AutoDockTools-1.5.6.
4.7. Cell culture and MTT assay
Neuroprotective activity for compound 8f was determined by MTT assay.32, 33 PC12 cell line was
obtained from Pasteur institute and all culture media and supplements were purchased from
Gibco (Europe). At first, cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 5% horse serum, 10% fetal calf serum and antibiotics (100 units/ml
Penicillin, 100 μg/ml streptomycin). In order to induce neuronal differentiation, PC12 cells were
re-suspended using Trypsin/EDTA (0.25%) and also seeded in 96 well culture plate
(4000cells/well) then cultured for 1 week in differentiation medium containing DMEM, 2%
horse serum, Nerve Growth Factor (NGF: 100 ng/ml), Penicillin and Streptomycin. After
changing of the culture medium to NGF free one, 10 μl of 8f (1, 10 and 100 μM) was added to
each well and incubated for 3 hours. Subsequently, 375 μM of H2O2 was added and incubation
was performed for 12 hours. To perform MTT assay, 10 μl of MTT solution (5 mg/ml) was added
and left for 3 hours. Then, 100 μl of the 10% SDS in 0.01 M HCl (w/v) as the solubilization
solution was added into each well to dissolve the insoluble formazan product into a colored
solution and the plates were incubated overnight. Finally, optical density (OD) was obtained at
570 nm on the ELISA reader and each experiment was performed in triplicate. Also, Quercetin
(10 μM) was used as a positive control. One-way analysis of variance (ANOVA) followed by
Newman-Keuls multiple comparisons test was carried out to determine the level of significance
using Graph Pad Prism 6.01, 2012 (www.graphpad.com).
4.8. In-vivo study
Subject
Thirty six adult male albino Wistar rats weighing 200–250 g were used in this experiment. All
experiments were executed in accordance with the Guide for the Care and Use of Laboratory
Animals (National Institutes of Health Publication No. 80-23, revised 1996) and were approved
by the Research and Ethics Committee of Tehran University of Medical Sciences.
Drug
21

The i.p. 4 mg/kg dose of scopolamine hydro bromide (Sigma-Aldrich, Steinheim, Germany) was
used to induce the AD. The i.p. 2.5mg/kg dose of Donepezile hydrochloride (Sigma–Aldrich) was
used to treat the AD as the positive control group. The 5% DMSO was used as vehicle.
Behavioral test: Morris water maze (MWM)
Apparatus
Briefly, the Morris water maze consisted of a dark circular pool (136 cm in diameter and 55 cm
high) filled with water (24±1°C) to a depth of 35 cm. A transparent Plexiglas platform (10 cm
diameter) was located 1 cm below the water surface in the center of one of designed north-
east (NE), south-east (SE), south-west (SW) or northwest (NW) quadrants.
Procedures
The single training session consisted of four trials with different starting positions that were
equally distributed around the maze. Each rat was placed in the water facing the wall of the
tank at one of the four designated starting points (north, east, south and west) and was allowed
to swim and find the hidden platform located in the target quadrant of the maze. Each of four
starting positions was used once in four training sessions; their order was randomized. During
each trial, each rat was given 90 s to find the hidden platform. If the rats could not find the
platform during this procedure, they were manually guided to the platform. After finding the
platform, the animals were allowed to remain there for 20 s; then they were placed in a holding
cage to pass a 30 s rest until the start of the next trial. These procedures were repeated in 4
consecutive days. After completion of training, the animals were returned to their home cages
until the probe trial 24 h later (on the test day). In the probe trial, the hidden platform was
removed and the animals were put in the counter side and allowed to swim freely for 90
second. After the probe trial, the platform was elevated above the water surface and placed in
the different position and the rats were allowed to swim freely for 120 second in order to test
their visual ability. Effects of i.p. infusion of 5, 10, and 20mg/kg doses of compound 8f on spatial
memory were investigated through measuring time spent on target quadrant, scape latency,
traveled distance, and swimming speed.
Statistical Analysis
To compare the differences between two or among several groups, t-test and one-way ANOVA
(followed by Newman-Keuls as) were used, respectively. P values less than 0.05 were
considered as significantly different.
ACKNOWLEDGMENT
22

The authors thank the equipment supports from the Department of Medicinal Chemistry,
Shahid Sadoughi University of Medical Sciences, Yazd, Iran and the Department of Cellular
Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR,
Isfahan, Iran. Also, we'd like to appreciate Miss Ronas Simaei for English language editing.
This work was financially supported by Tehran University of Medical Sciences and INSF (Iran
National Science foundation).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Design, Synthesis, and Biological Evaluation of selective and potent
Carbazole-Based Butyrylcholinesterase Inhibitors
Roshanak Ghobadian, Hamid Nadri, Alireza Moradi, Syed Nasir Abbas Bukhari, Mohammad
Mahdavi, Mehdi Asadi, Tahmineh Akbarzadeh, Hossein Khaleghzadeh-Ahangar, Mohammad
Sharifzadeh, Mohsen Amini
Anti-BChE, Neuroprotective
AChE-induced and self-induced Aβ aggregation inhibitor
Decrease mean training period-scape latency in rat
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