Novel tetrahydrocarbazole benzyl pyridine hybrids as potent and selective butryl cholinesterase inhibitors with neuroprotective and β-secretase inhibition activities

Article abstract of DOI:10.1016/j.ejmech.2018.05.031

Butyrylcholinesterase (BuChE) inhibitors have become interesting target for treatment of Alzheimer's disease (AD). A series of dual binding site BuChE inhibitors were designed and synthesized based on 2,3,4,9-tetrahydro-1H-carbazole attached benzyl pyridine moieties. In-vitro assay revealed that all of the designed compounds were selective and potent BuChE inhibitors. The most potent BuChE inhibitor was compound 6i (IC₅₀ = 0.088 ± 0.0009 μM) with the mixed-type inhibition. Docking study revealed that 6i is a dual binding site BuChE inhibitor. Also, Pharmacokinetic properties for 6i were accurate to Lipinski's rule. In addition, compound 6i demonstrated neuroprotective and β-secretase (BACE1) inhibition activities. This compound could also inhibit AChE-induced and self-induced Aβ peptide aggregation at concentration of 100 μM and 10 μM respectively. Generally, the results are presented as new potent selective BuChE inhibitors with a therapeutic potential for the treatment of AD.

Full text of DOI:10.1016/j.ejmech.2018.05.031

Accepted Manuscript
Novel tetrahydrocarbazole benzyl pyridine hybrids as potent and selective butryl
cholinesterase inhibitors with neuroprotective and β-secretase inhibition activities
Roshanak Ghobadian, Mohammad Mahdavi, Hamid Nadri, Alireza Moradi, Najmeh
Edraki, Tahmineh Akbarzadeh, Mohammad Sharifzadeh, Syed Nasir Abbas Bukhari,
Mohsen Amini
PII:
S0223-5234(18)30444-6
10.1016/j.ejmech.2018.05.031
EJMECH 10442
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 March 2018
Revised Date: 17 May 2018
Accepted Date: 20 May 2018
Please cite this article as: R. Ghobadian, M. Mahdavi, H. Nadri, A. Moradi, N. Edraki, T. Akbarzadeh,
M. Sharifzadeh, S.N.A. Bukhari, M. Amini, Novel tetrahydrocarbazole benzyl pyridine hybrids as potent
and selective butryl cholinesterase inhibitors with neuroprotective and β-secretase inhibition activities,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.031.
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ACCEPTED MANUSCRIPT
Novel tetrahydrocarbazole -benzyl pyridine hybrids as potent and selective butryl
cholinesterase inhibitors with neuroprotective and β-secretase inhibition activities
Roshanak Ghobadian, Mohammad Mahdavi, Hamid Nadri, Alireza Moradi, Najmeh Edraki,
Tahmineh Akbarzadeh, Mohammad Sharifzadeh, Mohsen Amini1
A series of dual binding site BuChE inhibitors were designed and synthesized based on 2,3,4,9-
tetrahydro-1H-carbazole attached benzyl pyridine moieties. The most potent BuChE inhibitor
was compound 6i (IC50 = 0.088 µM). In addition, compound 6i demonstrated neuroprotective
and β-secretase inhibition activities.

ACCEPTED MANUSCRIPT
Novel tetrahydrocarbazole benzyl pyridine hybrids as potent and selective butryl
cholinesterase inhibitors with neuroprotective and β-secretase inhibition activities
Roshanak Ghobadian1, Mohammad Mahdavi2, Hamid Nadri3, Alireza Moradi3, Najmeh Edraki4,
Tahmineh Akbarzadeh1,5, Mohammad Sharifzadeh6, Syed Nasir Abbas Bukhari7, Mohsen Amini*1
1Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran14176, Iran
2Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
3Pharmaceutical science research center and Faculty of Pharmacy, Shahid Sadoughi University
of Medical Sciences, Yazd 8915173143, Iran
4Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
5Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences,
Tehran, Iran
6Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran
7Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Aljouf, Sakaka
2014, Saudi Arabia
* Corresponding author. Tel.:+989122182581
E-mail address: moamini@tums.ac.ir
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ABSTRACT
Butyrylcholinesterase (BuChE) inhibitors have become interesting target for treatment of
Alzheimer’s disease (AD). A series of dual binding site BuChE inhibitors were designed and
synthesized based on 2,3,4,9-tetrahydro-1H-carbazole attached benzyl pyridine moieties. In-
vitro assay revealed that all of the designed compounds were selective and potent BuChE
inhibitors. The most potent BuChE inhibitor was compound 6i (IC50 = 0.088±0.0009μM) with the
mixed-type inhibition. Docking study revealed that 6i is a dual binding site BuChE inhibitor. Also,
Pharmacokinetic properties for 6i were accurate to Lipinski's rule. In addition, compound 6i
demonstrated neuroprotective and β-secretase (BACE1) inhibition activities. This compound
could also inhibit AChE-induced and self-induced Aβ peptide aggregation at concentration of
100 µM and 10 µM respectively. Generally, the results are presented as new potent selective
BuChE inhibitors with a therapeutic potential for the treatment of AD.
Key words: Alzheimer’s disease, butyrylcholinesterase, docking study, 2,3,4,9-tetrahydro-1H-
carbazole, in-vitro assay
1. Introduction
Alzheimer’s disease (AD) is the most common form of dementia leading to psychiatric and
behavioral disturbances, cognitive dysfunction and difficulties in performing daily activities [1].
Correspondingly, it has shown surge in the overall mortality rate by over 66% [2]. Decreasing
Acetylcholine (Ach) in the cortex and hippocampus regions of brain is the most simulated
neurotransmitter deficiency in AD [3]. Thus, the most promising approach to alleviate the
symptoms of AD is the inhibition of cholinestrase (ChE) [4]. Though in the normal healthy brain,
AChE has the main role in the regulation of ACh, It seems that there is a significant increase in
BuChE activity in the most affected areas of the brain during the progression of AD [5, 6].
According to a recent study, selective BuChE inhibitors increase brain ACh and enhance learning
in rodents [7]. Moreover, selective BuChE inhibitors might not have common side-effects as
AChE inhibitors such as classical cholinergic toxicity [8].
Thus, in recent years, BuChE inhibitors have become more interesting targets for the treatment
of AD [3, 6, 7, 9, 10].
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Although both ChE have almost similar anionic site and oxyanion hole in their active sites,
spatial capacity of the Acyl pocket is the most prominent difference between the structures of
AChE and BuChE. The replacement of Phe288 and Phe290 residues (in AChE) with Leu286 and
Val288 (in BuChE) enables BuChE to hydrolyze bulkier substrates [6, 11].
Selective BuChE inhibitors usually contain tri- or polycyclic structures (N1-
phenetylnorcymserine and ethopropazine) [5, 7, 11-13]. Especially, indole containing tricycle
derivatives based on Latrepirdine have recently shown selective BuChE inhibition [12, 14, 15].
All in all, to obtain more information on BuChE’s selectivity of indole containing tricycle
derivatives, we have designed and synthesized a novel series of 2,3,4,9-tetrahydro-1H-
carbazole containing derivatives.
On the other hand, the design of dual binding site ChE inhibitors seems to be a promising
strategy to increase inhibitory activity through the enhancement of many drug-targeted
interactions [16-20].
Recently, benzyl pyridine derivatives have been designed and synthesized, according to the
donepezil structure, to have the cholinesterase inhibitory effect [21-24].
Therefore, in this study, a series of dual binding site BuChE inhibitors were designed and
synthesized according to 2,3,4,9-tetrahydro-1H-carbazole attached benzyl pyridine moieties.
Moreover, in-vitro and MTT assay, study of β-secretase inhibition activity, pharmacokinetic and
docking studies were performed.
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Scheme1. A): The structure of some recently reported AChE inhibitors B): new designed
compounds 5a-j and 6a-j [15, 17, 22].
2. Results and discussion
2.1. Chemistry
According to scheme 2, compound 1 was prepared as the literature reported [25]. Synthesis of
3a/b was carried out in a two phasic condition using TBAB as phase transfer catalyst agent.
Purification of the product was carried out using column chromatography method.
Condensation of 3a (or 3b) with different benzyl halides (4a-j) gave the final compounds 5 (or
6)-a-j in reflux condition using dry acetone as solvent.
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Scheme2. Synthesis of compounds 5a-j and 6a-j.
2.2. AChE and BuChE inhibitory activity
The in-vitro IC50 values of the all derivatives 5a-j and 6a-j were determined according to the
Ellman’s method and compared with Donepezil as the reference drug (Table1) [24, 27]. All
derivatives have much more potent anti-BuChE activities than anti-AChE activities (BuChE IC50:
0.088 -2.1 µM, AChE IC50: 4.8-77.1 µM). So, according to the BuChE selectivity indexes of
derivatives (SIBuChE: IC50 of AChE /IC50 of BuChE), all the designed compounds were selective for
BuChE’s inhibition. During AD’s progression, the loss of AChE-activity is compensated by
unaffected BuChE [28]. Thus, the above result might have a therapeutic advantage for the
treatment of AD. Among all the derivatives, the most potent BuChE inhibitor was compound 6i
(IC50 = 0.088±0.0009μM) with great BuChE selectivity (SIBuChE= 180.68). According to the Table1,
anti-BuChE activities of eight of the synthesized derivatives (5a, b, e and 6a-c, h, i) were more
potent than Donepezil (BuChE IC50 of donepezil: 0.35±0.02 μM). Particularly, BuChE IC50 of 6i
was 3.97 times less than that of donepezil.
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It is understood that derivatives having 3-(Chloromethyl) pyridine moiety in comparison to
those having 4-(Chloromethyl) pyridine, show lower anti-AChE activity and higher anti-BuChE
activity (for AChE Ic50 6a-6j > Ic50 5a-5j and for BuChE Ic50 5a-5j > Ic50 6a-6j). Thus, the previously
mentioned replacement led to a great enhancement of SIBuChE. It seems that both anti-AChE and
anti-BuChE activities can be changed by varying substituents on the benzyl group connected to
3 or 4-(Chloromethyl) pyridine moieties (replacement of benzyl bromide with different chlorine,
fluorine and methyl benzyl halides). The introduction of fluorine in benzyl moiety diminished
both anti-BuChEand anti-AChE activities. So, the fluorine especially in metha position of the
benzyl moiety couldn’t be tolerated. However, the insertion of chlorine or a methyl group
increases both anti-BuChE and anti-AChE activities in all derivatives (except for anti-BuChE
activity of 5f). Furthermore, it seems that among derivatives with ortho substitutions, 2-Cl
derivatives (5a, 6a) led to the best enhancement of ChE’s inhibition (AChE and BuChE IC50 5d,
5g, 6d, 6g>5a, 6a). Similarly, among derivatives with para substitutions, 4-methyl derivatives (6i,
5f) showed the most improvement of ChE’s inhibition (AChE and BuChE IC50 5c, 5i, 6c, 6f > 6i,
5f).
Overall, derivatives having 3-(Chloromethyl) pyridine moiety were much more selective
inhibitors of BuChE than those having 4-(Chloromethyl) pyridine moiety and also the methyl
substituent at para position of benzyl moieties due to its bulkiness was the most favorable
group for BuChE’s inhibition in derivatives having 3-(Chloromethyl) pyridine moiety (6i has the
best BuChE IC50= 0.088 µM).
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Table 1. The IC50 values of the compounds 5a-j and 6a-j against AChE and BuChEa.
AChE
BChE
SI BChE
Entry compounds
x
y
z
z’
inhibition
[IC50 (μM)]
inhibition
[IC50 (μM)]
(IC50 AChE /IC50 BuChE)
1
2
5a
5b
Cl
Br
Cl
Cl
H
H
H
Cl
H
H
H
6.5±0.10
4.8±0.10
0.29±0.009
0.26±0.015
2.1±0.044
0.40±0.004
0.24±0.005
0.7±0.08
22.41
18.46
12.19
21
3
5c
Cl
H
25.6±1.40
8.4±0.20
4
5d
Br Me
H
5
5e
Cl
Cl
Cl
Br
Cl
Br
Cl
Cl
Cl
Cl
Cl
Cl
H
H
F
Me
H
H
5.1±0.20
21.25
20
6
5f
Me
H
14±0.90
7
5g
H
30±0.40
47.61
35
0.63±0.003
0.98±0.010
0.73±0.012
8
5h
H
H
H
Cl
H
H
F
F
H
34.30±0.60
32.8±0.20
30.05±0.60
36.1±0.40
30.04±0.007
27.3±0.80
67.69±0.012
77.1±1.50
71.75±0.009
47±1.20
9
5i
H
F
44.93
62.60
180.50
120.16
124.09
132.72
137.68
137.98
123.68
152.73
180.68
126.31
0.065
10
11
12
13
14
15
16
17
18
19
20
21
5j
H
H
0.48±0.006
0.20±0.004
6a
H
H
6b
Cl
H
H
0.25±0.001
0.22±0.013
0.51±0.4
6c
Cl
H
6d
H
6e
H
H
F
H
0.56±0.028
0.52±0.015
0.38±0.023
0.11±0.001
0.088±0.0009
0.38±0.007
0.35±0.02
6f
H
F
6g
Cl Me
H
H
6h
Cl
Cl
Br
-
H
H
H
-
Me
H
H
16.8±0.80
15.90±0.10
48.1±0.50
0.023±0.01
6i
Me
H
6j
H
Donepezil
-
-
aData are expressed as Mean ± SE (three independent experiments).
During the last two decades, few AChEIs have been introduced to clinic for treatment of mild
and moderate stage of AD, such as tacrine, donepezil, rivastigmine and galanthamine.
Limitation of oral bioavailability and other pharmacokinetic parameters of rivastigmine, and
hepatoxicity of tacrine led to decrease their application in AD. Important of donepezil has been
referred to good potency and low side effects. Two major interaction binding sites with AChE
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have been suggested in the structure of donepezil, N-benzyl piperidine and indanone moiety.
Consequently the efforts for introducing new AChEIs have been focoused to preparation of the
hybrid molecules with keeping the important pharmacophoric groups from two active
molecules. A hybrid derivative of donepezil-rivastigmine and donepezil-tacrine showed
remarkable activity in inhibition of AChE and BChE [34, 35]. Furthermore, some derivatives
related to those hybrids showed significant β-amyloid anti-aggregation property [36].
Development of donepezil derivatives conduct ed to introduce ligand AP 2238 in which
indanone moiety subunit has been substituted by dimethoxy coumarin, with an IC50 =44.5 nM
for AChE and also a potency better than donepezil in inhibition of β–amyloid aggregation [35].
Recently, some potent AChE inhibitor were reported in which, N-benzylpyridinum salt group
was introduced into a series of aromatic scaffold with capability interact with catalytic active
site (CAS) and peripheral anionic site (PAS). The activities of most potent compounds in each
series are presented in table 2.
Table 2. The biological data for reported compounds, benzylpyridinum derivative
Ar
R1
-
R2, R3
AChE
(IC50, µM)
7.19
BChE
(IC50, µM)
45.61
SI
Ref
37
R2=H,
6.34
R3= 3-F
-
R2=H,
0.77
8.71
11.31
37
R3= 2-F
-
-
R2=H,
R3= 2-Cl
0.0004
0.004
1370
3113
17
21
R2=3-
Me, R3=
3-Me
-
-
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6-Br
R2=H,
0.00011
0.489
4445
38
R3= 2-F
-
-
R2=H,
R3= 3-F
10.14
0.008
12.16
2.07
0.83
232
39
40
R2=H,
R3= 4-F
-
R2=H,
0.038
1.84
48.4
19
R3= 4-F
Comparison between their activity in inhibition of AChE and BChE (table 2) and the synthesized
compounds in the current study clearly shows that potency of compounds 5, 6(a-j) in inhibition
of AChE is reduced. A significant increasing in activity of our compound and SI could be
observed in inhibition of BChE. Apparently, substitution of tetrahydrocarbazole group as an aryl
nonpolar ring in N-benzylpyridinum analogous changes the activity in favor of BChE inhibition
and increases the selectivity index.
However, there are some reports that emphasize in the positive role of BChE inhibitors in AD [3,
6, 7, 9, 10], the usefulness of BChE inhibition in AD needs more studies. Our findings could be
considered in design of new compounds and have been used for regulation of balance between
AChE and BChE inhibition activity.
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2.3. Kinetic studies of AChE and BuChE inhibition
To investigate the mechanism of the BuChE’s inhibition, the most potent derivative (6i, BuChE
IC50= 0.088 µM) was chosen and Lineweaver-Burk curves were outlined (Fig. 1) [21]. Three
different concentrations of the chosen derivative (0, 0.043, 0.087 and 0.174 µM) and
butrylcholine iodide (0.13, 0.32 and 0.69 µM) were used as an inhibitor and a substrate
respectively. According to Fig.1, it was comprehended that a mixed-type inhibition could be
attributed to the compound 6i. Binding of compound 6i to BuChE changed both Vmax and Km
values, a trend that is generally attributed to a mixed-type inhibition. Thus, in accordance with
the docking studies, a dual binding site interaction with the receptor is expected.
Fig.1. Kinetic study of compound 6i on the inhibition mechanism of BuChE by Lineweaver-Burk
plot (1/v) sec.DA-1 vs. (1/[s]) mM-1.
Furthermore, the inhibition constant Ki for compound 6i was calculated (Ki =0.09 µM) using a
plot of slope versus the inhibitor’s concentration (Fig. 2).
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Fig.2. The plot of the slope versus the concentration of inhibitor 6i (µM) for calculating Ki.
2.4. Docking studies
Due to dynamic movement of some resides in the active site of cholinesterase that is crucial for
accommodation of various ligands in the active site; the molecular dynamics study has been
done. To estimate stability of the trajectories, their total energy was calculated. An illustration
of total system energy changes during the evolution of trajectories (fig 3) clearly displays the
stability of the system. The molecular plasticity of the ligand-enzyme complex was estimated
based on RMSD calculations. The RMSD of the complex system was shown in fig 4. According to
the results the complex system was equilibrated around 3 ns. The RMSD remained constant
until 10 ns. Finally the best frame of the trajectories was selected and further analyzed to
finding the key interactions. The ligand-enzyme schematic interactions were shown in fig 5. The
plain structure of carbazole moiety contributes for making a π-π staking with Tyr332. The
central pyridinium ring was attracted by the negative charge (made by Trp82 and Glu197) that
deposited at the bottom of the active site. Moreover the extra charge transfer binding with
Trp82 may be beneficial for better interaction. The rest of the molecule was fitted in the
hydrophobic cavity made by Ile62, Pro84, Thr120 and Gly 121.
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-1484000
-1485000
-1486000
-1487000
-1488000
-1489000
-1490000
-1491000
-1492000
-1493000
0
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
Time (ps)
Fig.3. The change of total potential energy of the complex system during 10 ns simulation.
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0.3
0.25
0.2
0.15
0.1
0.05
0
0
1
2
3
4
5
6
7
8
9
10
Time (ns)
Fig. 4. The calculated RMSD of the complex system during 10 ns simulation.
Fig.5. The binding pattern of the ligand (6i) in the active site of the enzyme. For simplification
only the key residues were shown.
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2.5. Screening of physicochemical properties
Some of physicochemical properties of compound 6i including the polar surface area (tPSA),
octanole/water partition coefficients (Clog P), a number of H-bond acceptors (HBA), a number
of H-bond donors (HBD) and a number of rotatable bonds (RBC) were calculated and shown in
Table 3. It is obvious that mentioned properties of 6i were confirmed by Lipinski rules of 5 [30].
Thus, 6i tends to have lower attrition rates during clinical trials and hence has an increased
chance of reaching the market.
Table 3. Molecular descriptorsa of compound 6i.
Entry Compound HBD HBA ClogP tPSA[Å2]
19 6i 3.024 6.25
MW
RBC
4
0
2
402.97
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.
2.6. Neuroprotective effect against H2O2-induced cell death in PC12 neurons
The MTT assay is a useful model for assessing neuronal differentiation and has been widely
adopted. Hence, neuroprotective activity for the most potent compound (6i) against BuChE was
determined by in-vitro MTT assay. The data is expressed as followed ± SEM (n = 8) and the one-
way analysis of the variance (ANOVA) continued by Newman-Keuls multiple comparisons test
were carried out to determine the level of significance. According to Fig 4, compound 6i
demonstrated good neuroprotective activity at 10 μM (cell viability = 65 and P <0.01 vs. H2O2).
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Fig.6. Neuroprotective activity of compound 6i based on cell viability of PC12 cells in H2O2-
induced damage. Data is expressed as followed ± SEM (n = 8) and ANOVA proceeded by
Newman-Keuls multiple comparisons test was carried out to determine the level of significance.
****: P<0.0001 and **: P<0.01 versus H2O2.
2.7. BACE1 enzyme inhibitory activity of compound 6i
BACE-1 inhibitory activity of the most potent BuChE inhibitor (6i) was evaluated via a
fluorescence resonance energy transfer (FRET) based BACE-1 kit including BACE-1 enzyme and
specific APP based peptide substrate (Rh-EVNLDAEFK-quencher). Also experiments were
repeated for three times and compared with OM99-2 as the reference compound. According to
Table 4 compound 6i possesses moderate inhibition against BACE1 at the concentration of 50
µM. The non-peptide structure of compound 6i is desired and acts as dual inhibitor of BuChE
and BACE1.
Table 4. BACE1 inhibitory activitya of compound 6i.
Compound
6i
Inhibitionb at 50 μM (%)
30±0.1
Inhibitionb at 10 μM (%)
10±0.2
a The IC50 of standard drug OM99-2 was 3 nM.
b Values represent means ± standard error (S.E.) of three independent experiments.
2.8. Inhibition of AChE-induced and self-induced Aβ aggregation
Compound 6i 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 mM
concentration was 55.7 ± 2.9 % 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 6i exhibited a significant AChE-induced Aβ aggregation
inhibitory effect with percentage of inhibition 51.8 ± 1.5 more effective than Tacrine and
donepezil (7.2 ± 1.2 % and 25.2 ± 1.1, respectively), indicating that compound 8i binds to PAS of
AChE.
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3. Conclusion
In summary, a new series of potent selective BuChE inhibitors were designed. All of the
compounds have shown selective inhibition of BuChE. They might have a therapeutic advantage
for the treatment of AD as during AD’s progression the loss of AChE-activity is compensated by
unaffected BuChE. Also, eight of them had lower BuChE IC50 values than donepezil as the
reference drug. In-vitro assay revealed that compound 6i indicated the most potent anti-BuChE
activity (IC50 = 0.088±0.0009μM). Moreover, compound 6i was found to be an inhibitor of
AChE-induced Aβ aggregation, which can also interfere with Aβ self-aggregation. It was
understood from molecular modeling studies that compound 6i had important interactions
with choline’s binding site, peripheral binding site and catalytic site of BuChE. Furthermore, it
was long enough to interact with the acyl pocket. Also, compound 6i demonstrated
neuroprotective and BACE1 inhibition activities. Additionally, the pharmacokinetic properties of
6i were confirmed by Lipinski rules of 5. Generally, our study has presented new potent
selective inhibitors of BuChE with a therapeutic potential for the treatment of AD.
4. Experimental
4.1. General chemistry
All reagents were purchased from Sigma Aldrich, Merck and Fluka and were used as provided.
1
For all the derivatives, H and 13C nuclear magnetic resonance (NMR) spectra’s were recorded
on a Bruker FT-500, using TMS as an internal standard and the IR spectra’s were recorded with
KBr disks using the Nicolet Magna FTIR 550 spectrophotometer. Furthermore, all of melting
points were taken on a Kofler hot stage apparatus.
Synthesis of 2,3,4,9-tetrahydro-1H-carbazole (1)
Synthesis of 2,3,4,9-tetrahydro-1H-carbazol (1) was prepared according to the published
procedure [25]. To a mixture of cyclohexanone (10 mmol), acetic acid (60 mmol) and phenyl
hydrazine (10 mmol) were added and reflux for 1h. The progress of reaction was check by TLC.
The mixture was kept in room temperature overnight and then white solid crude was filtered
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and washed with water and ethanol (75%) respectively. The solid material was crystalized in
methanol to yield compound 1.
Synthesis of 9-(2-(pyridin-4-yl) ethyl)-2,3,4,9-tetrahydro-1H-carbazole and 9-(2-(pyridin-3-
yl)ethyl)-2,3,4,9-tetrahydro-1H-carbazole (3a or 3b)
To solution of 2.73 mmol tetrabuthyl ammonium bromide (TBAB) in 15 mL of sodium hydroxide
solution (50%) was added toluene (15 mL) and stirred for 15 min. Five mmol of compound 1 in
toluene (15 mL) and 16.3 mmol of compound 2 (a or b) were added and stirred at room
temperature for 12h. the solvent was removed under reduced pressure. To the residue 25 mL
of water and chloroform (50 mL) were added and mixed. The organic layer was separated, dried
over anhydrous sodium sulphate and filtered. The solvent was removed under reduced
pressure and the residue was purified on a silica short column with using a mixture of
petroleum ether: ETOAc (9:1) to give a yellow solid of 3a (or 3b) [26].
General procedure for the synthesis of compounds 5a-j and 6a-j
To a solution 1 mmol of 3a (or 3b) in dry acetone (12 mL) was added 1.5 mmol of different
benzyl halides (4a-j) and reflux for 1- 6h, checked by TLC. The solid material was filtered and
washed with a mixture of petroleum ether/ETOAc (4:1) to give 5a-j and 6a-j.
2,3,4,9-tetrahydro-1H-carbazole (1)
White solid; yield: 70% m.p. = 132 °C. IR (KBr): 3400, 3025, 2852, 1670, 1603, 1475,.1H NMR
(CDCl3, 500 MHz): δ = 1.85-1.92 (m, 4H, tetrahydro-1Hcarbazole-CH2), 2.69-2.72 (m, 4H,
tetrahydrocarbazole-CH2), 7.05-7.09 (t, 2H, H6, 7), 7.11 (d, J= 7 Hz, 1H, H8), 7.24 (d, J= 8 Hz, 1H,
H5), 7.44 (s, 1H, NH) ppm.
9-(pyridin-4-ylmethyl)-2,3,4,9-tetrahydro-1H-carbazole (2a)
Yellow solid; yield: 58%, m.p. = 215-217 °C. IR (KBr): 2996, 2842, 1675, 1608, 1465 cm1-, 1H NMR
(CDCl3, 500 MHz): δ = 1.86-1.93 (m, 4H, tetrahydrocarbazole-CH2), 2.59 (t, J= 6.5 Hz, 2H, H4),
2.76 (t, J= 6 Hz, 2H, H1), 5.23 (s, 2H, N-CH2), 6.86 (d, J=5.5 Hz 2H, H3 ,5
(d, J= 7.0 Hz, 1H, H5), 8.48 (d, J=5.5 Hz, 2H, H2 ,6 ) ppm.
9-(pyridin-3-ylmethyl)-2,3,4,9-tetrahydro-1H-carbazole (2b)
՛
՛
), 7.11 (m, 3H, H6,7,8), 7.24
՛
՛
Yellow solid; yield: 60%, m.p.= 208-210 °C. IR (KBr): 2980, 2845, 1678, 1600, 1475 cm-1. 1H NMR
(CDCl3, 500 MHz): δ = 1.77-1.83 (m, 4H, tetrahydrocarbazole-CH2). 2.64 (m, 2H, H4), 3.35 (m, 2H,
17

ACCEPTED MANUSCRIPT
H1), 5.34 (s, 2H, N- CH2), 6.99 (t, J=7.0 Hz, 1H, H5), 7.04 (t, J= 7.0 Hz, 1H, H8), 7.26 (d, J= 7.0 Hz,
1H, H6), 7.35 (d, J= 8.0 Hz, 1H, H7), 7.39-7.41 (m, 2H, H4 , 5
՛
՛
), 8.3 (s, 1H, H2 ), 8.43 (d, J= 6.0 Hz, 1H,
՛
H6
՛
) ppm. 13C- NMR (CDCl3, 500 MHz): δ = 20.6, 21.6 (C4), 22.6 (C1), 22.7 (C2), 43.1(C3), 109.1
(NCH2), 109.3 (NCH), 117.4 (CH), 118.6 (C8), 120.6 (C6
՛
), 123.6 (C5), 127.0 (C6), 134.2(C7), 135.3
(C4 ), 136.0 (tetrahydrocarbazole-C), 147.9 (C5 ), 148.0 (tetrahydrocarbazole -CN), 148.3(C3 )
՛
՛
՛
ppm.
1-(2-chlorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (5a)
Yellow solid; yield: 75%, m.p. = 225-226 °C. IR (KBr): 2986, 2852, 1678, 1464, 750 cm-1 .1H NMR
(DMSO, 500 MHz): δ = 1.81-1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.60-2.61 (m, 2H, H4),2.66-
2.68 (m, 2H, H1), 5.73 (s, 2H, N- CH2), 5.93 (s, 2H, N+- CH2), 7.03-7.04 (m, 2H, H6,7), 7.31 (d, J= 7
Hz, 1H, H8), 7.45-7.50 (m, 4H, H5, 4 ˝, 5˝, 6 ˝), 7.56-7.59 (m, 3H, H3 ,5 , 3 ˝
՛
՛
), 8.97 (d, J=5Hz, 2H, H2 ,6 )
՛
՛
ppm. 13C- NMR (DMSO, 500 MHz): δ = 20.6 (C4), 21.2 (C2), 22.5 (C3), 44.8 (CH2-N), 60.6 (CH2-N+),
109.1 (tetrahydrocarbazole-C), 109.9 (C8), 117.7 (C5), 119.2 (C6), 120.9 (C7), 125.3 (C5˝), 127.2
(C4˝), 128.1 (C3˝), 130.0 (C4׳, C5׳ ), 131.2 (C2˝), 131.5 (C-N), 131.7 (C-N), 144.9 (C1˝), 145.1 (C2׳, C6׳),
159.3 (C4׳) ppm.
1-(3-chlorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium bromide (5b)
Cream solid; yield: 76%, m.p. = 225-227 °C. IR (KBr): 2925, 2852, 1678, 1642, 1464 cm-1.1H NMR
(DMSO), 500 MHz): δ = 180-1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.59 (t, J= 6.0 Hz, 2H, H4),
2.67 (t, J= 6.0 Hz, 2H, H1), 5.73 (s, 2H, N- CH2), 5.90 (s, 2H, N+- CH2), 7.02-7.03 (m, 2H, H6,7), 7.30
(d, J= 7.5 Hz, 1H, H8), 7.43-7.47 (m, 3H, H5, 5՛՛,6՛՛), 7.54 (d, J=7 Hz, 1H, H4՛՛), 7.59 (d, J=6.5 Hz, 2H,
H3 ,5
՛
՛
), 7.71 (s, 1H, H2՛՛), 9.18 (d, J=7 Hz, 2H, H2 ,6
) ppm.13C- NMR (DMSO, 500 MHz): 20.5 (C4),
՛
՛
21.2 (C2), 22.5 (C3), 44.8 (CH2-N), 61.5 (CH2-N+), 109.1 (tetrahydrocarbazole-C), 109.8 (C8), 117.6
(C5), 119.1 (C6), 120.8 (C7), 125.3 (C4˝), 127.2 (C6˝), 127.7 (tetrahydrocarbazole-C), 128.9 (C2˝),
129.3 (C3׳, C5׳), 131.0 (C3˝), 133.5 (C-N), 135.3 C1˝), 136.2(C-N), 144.7(C2˝, C6˝), 159.0 (C4׳) ppm.
1-(4-chlorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (5c)
Cream solid; yield: 77%, m.p. = 225-227 °C. IR (KBr): 2928, 2852, 1678, 1643, 1463, 747cm-1.1H
NMR (DMSO, 500 MHz): δ = 1.76-1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.50-2.59 (m, 2H, H4),
2.66-2.68 (m, 2H, H1), 5.71 (s, 2H, N- CH2), 5.81 (s, 2H, N+- CH2), 7.01-7.03 (m, 2H, H6,7), 7.28-
7.30 (m, 1H, H8), 7.43-7.45 (m, 1H, H5), 7.51-7.55 (m, 4H, H 2 ,3 ,5 ,6
՛՛ ՛՛ ՛՛ ՛՛), 7.56 (d, J=6.5 Hz, 2H, H3 ,5 ),
՛
՛
18

ACCEPTED MANUSCRIPT
9.05 (d, J=7 Hz, 2H, H2 ,6
՛
՛
) ppm. 13C- NMR (DMSO, 500 MHz): δ = 20.6 (C4), 21.2 (C1), 22.5 (C2),
44.7 (C3), 61.7 (CH2-N), 109.1 (CH2-N+), 109.8 (tetrahydrocarbazole-C), 117.7 (C8), 119.2(C5),
120.9 (C6), 125.3 (C7), 127.2 (tetrahydrocarbazole-C), 129.1 (C3׳, C5׳), 130.9 (C3˝, C5˝), 132.9 (C2˝,
C6˝), 134.2(C4˝), 135.2 (C1˝), 135.9 (C-N), 144.6 (C-N), 144.8(C2׳, C6׳), 159.0 (C 4׳ ) ppm.
1-(2-methylbenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium bromide (5d)
Yellow solid; yield: 75%, m.p. = 205-206 °C. IR (KBr): 3035, 2925, 2852, 1638, 1463, 1424, 1373,
744cm-1.1H NMR (DMSO, 500 MHz): δ = 1.81 (t, J= 6.0 Hz, 4H, tetrahydrocarbazole-CH2), 2.27 (s,
3H, CH3), 2.62 (t, J= 6.0 Hz, 2H, H4), 2.68 (t, J= 6.0 Hz, 2H, H1), 5.75 (2H, s, N- CH2), 5.91 (s, 2H,
N+- CH2), 7.01-7.06 (m, 2H, H6,7), 7.19 (d, J= 7.5 Hz, 1H, H3՛՛), 7.25 (t, J=7 Hz, H5՛՛), 7.27 (d, J=7 Hz,
H6՛՛), 7.31-7.32 (m, 2H, H8,4՛՛),7.44 (d, J= 7 Hz, 1H, H5), 7.56-7.59 (d, J= 6Hz, 2H, H3 ,5
՛
՛
), 8.95 (d, J=5
Hz, 2H, H2 ,6
՛
՛
) ppm.13C- NMR (DMSO, 500 MHz): δ = 18.7 (CH3), 20.6 (C4), 21.2 (C1), 22.5 (C2), 44.8
(C3), 60.8 (CH2-N), 109.1 (CH2-N+), 109.8 (tetrahydrocarbazole-C), 117.6 (C8), 119.1 (C5), 120.9
(C6), 125.3 (C7), 126.6 (C4˝, C5˝), 127.2 (tetrahydrocarbazole-C), 129.4 (C3׳, C5׳), 130.8 (C6˝), 132.0
(C3˝), 135.3 (C-N), 135.9 (C2˝), 137.0 (C-N), 144.6 (C1˝), 144.8(C2׳, C6׳), 158.9 (C4׳) ppm.
1-(3-methylbenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (5e)
Yellow solid; yield: 77%, m.p. = 199-200 °C. IR (KBr): 2925, 2855, 1643, 1463, 1371 cm-1.1H NMR
(DMSO, 500 MHz): δ =1.80-1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.29 (s, 3H, CH3), 2.58-2.60
(m, 2H, H4), 2.65-2.68 (m, 2H, H1), 5.70 (2H, s, N- CH2), 5.75 (2H, s, N+- CH2), 7.01-7.03 (m, 2H,
H6,7), 7.22 (d, J=7 Hz, 1H, H4՛՛), 7.28-7.29 (m, 2H, H8,2՛՛), 7.32-7.33 (m, 2H, H5՛՛,6՛՛), 7.44 (d, J= 7 Hz,
1H, H5), 7.55 (d, J=5.5 Hz, 2H, H3 ,5
՛
՛
), 9.05 (d, J=5.5 Hz, 2H, H2 ,6
) ppm.13C- NMR (DMSO, 500
՛
՛
MHz): δ = 20.6 (C4), 20.8 (CH3), 21.2 (C1), 22.6 (C2), 44.8 (C3), 61.5 (N- CH2), 109.1 (N+- CH2) ,
109.9 (tetrahydrocarbazole-C), 117.6 (C8), 119.1(C5), 120.8 (C6), 125.4 (C7), 127.2 (C4˝, C6˝),
127.7, 128.9 (C
158.9 (C4 ), ppm.
1-(4-methylbenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (5f)
՛
3 , C5
՛
), 129.4(C2˝), 131.0 (C1˝), 134.0 (C-N), 135.4 (C-N), 136.2(C3˝), 144.8 (C2 ,
՛
C6 ),
՛
՛
Yellow solid; yield: 78%, m.p. = 195-196 °C. IR (KBr): 3027, 2928, 2850, 1638, 1615, 1464,
1
749cm-1. H NMR (DMSO, 500 MHz): δ = 1.81-1.84 (m, 4H, tetracarbazole-CH2), 2.29 (s, 3H,
CH3), 258-2.59 (m, 2H, H4), 2.66-2.68 (m, 2H, H1), 5.70 (s, 2H, N- CH2), 5.75 (s, 2H, N+- CH2), 7.03
19

ACCEPTED MANUSCRIPT
(t, J= 7.5 Hz, 2H, H6,7), 7.23 (d, J= 8 Hz, 2H, H3՛՛,5՛՛), 7.29 (d, J= 8 Hz, 1H, H8), 7.40 (d, J= 8 Hz, 2H,
H2՛՛,6՛՛), 7.45 (d, J= 7 Hz, 1H, H5), 7.55 (d, J= 6 Hz, 2H, H3 ,5
՛
՛
), 9.06 (d, J=6 Hz, 2H, H2 ,6
) ppm.13C-
՛
՛
NMR (DMSO, 500 MHz): δ = 20.6 (C4), 20.7(C1), 21.2 (C2), 22.5 (C3), 44.7(N- CH2), 62.5 (N+- CH2),
109.1 (tetrahydrocarbazole-C), 109.8 (C8), 117.7 (C5), 119.2 (C6), 120.9 (C7), 125.3
(tetrahydrocarbazole-C), 128.9 (C2˝, C3˝, C5˝, C6˝), 129.7 (C3׳, C5׳), 131.1 (C1˝), 135.2 (C4˝), 138.9
(C-CH3), 144.4 (C-N), 144.6 (C2׳, C6׳), 158.8 (C4׳) ppm.
1-(2-fluorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (5g)
Yellow solid; yield: 73%, m.p. = 231-232 °C. IR (KBr): 3039, 2925, 2854, 1641, 1463, 759 cm-1.1H
NMR (DMSO, 500 MHz): δ = 1.81-1.84 (m, 4H, tetracarbazole-CH2), 2.59-2.61 (m, 2H, H4), 2.66-
2.68 (m, 2H, CH1), 5.72 (s, 2H, N- CH2), 5.90 (s, 2H, N+- CH2), 7.01-7.03 (m, 2H, H6,7), 7.29-7.31
(m, 3H, H8, 4՛՛,6՛՛), 7.44-7.45 (m, 1H, H5), 7.50-7.51 (m, 1H, H5՛՛), 7.58-7.59 (m, 3H, H3 ,5 ,3
՛
՛
՛՛), 8.97 (d,
J=5Hz, 2H, H2 ,6
՛
՛
) ppm.13C- NMR (DMSO, 500 MHz): δ = 20.6 (tetrahydrocarbazole CH2) , 21.2
(tetrahydrocarbazole CH2) , 22.5 (tetrahydrocarbazole CH2, tetrahydrocarbazole CH2), 44.8 (N-
CH2), 57.3 (N+- CH2)), 109.1 (tetrahydrocarbazole-C), 109.9 (C8), 116.0 (JC-F= 7.5 Hz, C3˝), 117.7
(C5), 119.2 (C6), 120.9 (C7), 125.23 (JC-F= 15.5 Hz, C5˝), 125.3 (C1˝), 127.3 (C4˝), 127.6
(tetrahydrocarbazole-C), 128.9 (C3
՛
, C5
՛
), 131.54 (C6˝), 132.13 (C-N), 137.2 (C-N), 145.0 (C2
՛
, C6 ),
՛
159.2 (C4
՛
), 162.1 (JC-F= 223.75 Hz, C2˝) ppm.
1-(3-fluorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium bromide (5h)
Yellow solid; yield: 74%, m.p. = 231-232 °C. IR (KBr): 2924, 2854, 1740, 1643, 1463, 1151, 838,
750 cm1-.1H NMR (DMSO, 500 MHz): δ = 1.81-1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.60-2.62
(m, 2H, H4), 2.67-2.68 (m, 2H, H1), 5.71 (s, 2H, N- CH2), 5.82 (s, 2H, N+- CH2), 7.01-7.03 (m, 2H,
H6,7), 7.28-7.32 (m, 3H, H8, 5՛՛,6՛՛), 7.37 (d, J= 7.5 Hz, 1H, H5) 7.45-7.49 (m, 3H, H2՛՛,5), 7.50-7.51 (m,
1H, H4՛՛), 7.57 (d, J= 6.0 Hz, 2H, H3 ,5
՛
՛
), 9.09 (d, J= 6.0 Hz, 2H, H2 ,6
) ppm.13C- NMR (DMSO, 500
՛
՛
MHz): 20.7 (tetrahydrocarbazole-CH2), 20.8 (tetrahydrocarbazole-CH2), 21.5
δ
=
(tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2), 44.8 (N- CH2), 57.5 (N+- CH2), 109.2
(tetrahydrocarbazole-C), 110.0 (C8), 116.1 (C4՛՛), 117.7 (C2՛՛), 119.2 (C5), 120.9 (C6), 125.2 (C7),
125.3 (C6՛՛), 127.6 (tetrahydrocarbazole-C), 128.9 (C3׳, C5׳), 130.2 (C5՛՛),135. 4 (C-N), 136.0 (C1՛՛),
137.2 (C-N), 146.5 (C2׳, C6׳), 159.6 (C4׳), 162.8 (JC-F= 257.5 Hz) ppm.
20

ACCEPTED MANUSCRIPT
1-(4-fluorobenzyl)-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (5i)
Pink solid; yield: 75%, m.p. = 231-232 °C. IR (KBr): 2925, 2855, 1642, 1464, 839 cm-1.1H NMR
(DMSO, 500 MHz): δ = 1.81-1.83 (m, 4H, tetrahydrocarbazole-CH2), 2.59-2.60 (m, 2H, CH4), 2.66-
2.67 (m, 2H, CH1), 5.70 (s, 2H, N- CH2), 5.79 (s, 2H, N+- CH2), 7.03 (t, J= 7.5 Hz, 2H, H6,7), 7.26-
7.30 (m, 3H, H8, 2՛՛,6՛՛), 7.44 (d, J= 8.5 Hz,1H, H5), 7.56 (d, J= 6.5 Hz,2H, H3 ,5
՛
՛
), 7.61 (dd, J= 5.5 Hz,
J= 3.0 Hz, 2H, H3՛՛,5՛՛), 9.07 (d, J=5Hz, 2H, H2 ,6
՛ ՛
) ppm.13C- NMR (DMSO, 500 MHz): δ = 20.6
(tetrahydrocarbazole-CH2), 21.2 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2,
tetrahydrocarbazole-CH2), 44.7 (N- CH2), 61.8 (N+- CH2), 109.1 (tetrahydrocarbazole-C), 116.0
(C8), 116.1 (C3˝, C5˝), 117.7 (C5), 119.3 (C6), 121.0 (C7), 125.3 (tetrahydrocarbazole-C), 130.2 (C3
՛
,
C5 ), 131.6 (C1˝), 135.3 (C2˝, C6˝), 135.9 (C-N), 144.9 (C-N), 148.0 (C2 , C6 ), 159.2 (C3 ), 161.0 (JC-F=
՛
՛
՛
՛
223.75 Hz) ppm.
1-benzyl-4-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium bromide (5j)
1
Orange solid; yield: 77%, m.p. = 231-232 °C. IR (KBr): 3027, 2925, 2850, 1637, 1462 cm-1. H
NMR (DMSO, 500 MHz): δ = 1.80-1.83 (m, 4H, tetrahydrocarbazole-CH2), 2.60-2.61 (m, 2H, H4),
2.66-2.67 (m, 2H, H1), 5.77 (s, 2H, N- CH2), 5.92 (s, 2H, N+- CH2), 7.03 (t, J= 7.0 Hz, 2H, H6,7), 7.34
(d, J= 8.0 Hz, 1H, H8), 7.42-7.45 (m, 4H, H5,2 ,4 ,6
՛՛ ՛՛ ՛՛), 7.57-7.60 (m, 4H, H3՛՛,5՛՛, H3 ,5 ), 9.25(d, J=5Hz,
՛
՛
2H, H2 ,6
՛
՛
) ppm. 13C- NMR (DMSO, 500 MHz): δ = 20.5 (tetrahydrocarbazole-CH2), 21.2
(tetrahydrocarbazole-CH2), 22.3 (tetrahydrocarbazole-CH2, tetrahydrocarbazole-CH2), 44.8 (N-
CH2), 62.2 (N+-CH2), 109.1 (tetrahydrocarbazole-C), 109.8 (C8), 117.6 (C5), 119.1(C6), 120.8 (C7),
125.2 (C4˝), 127.1 (tetrahydrocarbazole-C), 128.9(C2˝, C6˝) , 129.1 (C3˝, C5˝), 134.1(C1˝), 135.3 (C-
N), 135.9 (C-N), 144.5(C3׳, C5׳), 144.6 (C2׳, C6׳), 158.9 (C4׳) ppm.
1-(2-chlorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (6a)
Yellow solid; yield: 76%, m.p. = 209-210 °C. IR (KBr): 2924, 2853, 1632, 1462, 751 cm-1.1H NMR
(DMSO, 500 MHz): δ = 1.78-1.82 (m, 4H, tetrahydrocarbazole-CH2), 2.50-2.63 (m, 4H,
tetrahydrocarbazole-CH2), 5.60 (s, 2H, N- CH2), 5.97 (s, 2H, N+- CH2), 7.01-7.02 (m, 2H, H6,7),
7.35-7.52 (m, 6H, H5,8,2 ,4 ,5 ,6
՛՛ ՛՛ ՛՛ ՛՛), 8.13 (m, 1H, H5
՛
), 8.20 (m, 1H, H4
՛
), 8.62 (s, 1H, H2
՛), 9.07(d,
J=5Hz, 1H, H6
՛
) ppm. 13C- NMR (DMSO, 500 MHz): δ = 20.6 (tetrahydrocarbazole-CH2), 21.4
21

ACCEPTED MANUSCRIPT
(tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2),
42.5 (N- CH2), 61.3 (N+- CH2), 109.0 (tetrahydrocarbazole-C), 109.8 (C8), 117.5 (C5), 117.8 (C6),
127.2 (C7), 128.2 (C5׳), 129.9 (C5˝), 130.9 (C4˝), 131.7 (tetrahydrocarbazole-C), 131.9 (C3˝), 135.1
(C6˝), 135.7 (C3׳), 139.9 (C6׳), 140 (C2˝), 142.5 (C-N), 144.1 (C-N), 145 (C2˝), 150 (C2׳), 153 (C4׳)
ppm.
1-(3-chlorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (6b)
Yellow solid; yield: 77%, m.p. = 209-210 °C. IR (KBr): 2925, 2854, 1630, 1465, 751, 713, 676 cm-
1.1H NMR (DMSO, 500 MHz): δ = 1.78-1.86 (m, 4H,tetrahydrocarbazole-CH2), 2.08 (t, J= 5.5 Hz,
2H, tetrahydrocarbazole-CH2), 2.64 (t, J= 5.5 Hz, 2H, carbazole-CH2), 5.58 (s, 2H, N- CH2), 5.87 (s,
2H, N+- CH2), 7.00-7.05 (m, 2H, H6,7), 7.37 (d, J= 7.5 Hz, 1H, H8), 7.41 (d, J= 7.5 Hz, 1H, H6՛՛), 7.44-
7.48 (m, 2H, H4՛՛,5՛՛), 7.50 (d, J= 7.5 Hz, 1H, H5), 7.66 (s, 1H, H2՛՛), 8.03 (d, J= 7.5 Hz, 1H, H5
՛
), 8.08
(t, J= 8.0 Hz, 1H, H4 ), 9.02 (s, 1H, H2 ), 9.17(d, J=5 Hz, 1H, H6
՛
՛
՛
) ppm. 13C- NMR (DMSO, 500 MHz):
δ = 20.8 (tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-
CH2), 22.6 (tetrahydrocarbazole-CH2), 42.6 (N- CH2), 63.3 (N+- CH2), 109.0 (tetrahydrocarbazole-
C), 109.3 (C8), 109.8 (C5), 117.5 (C6), 117.75(C7), 119.0 (tetrahydrocarbazole-C),127.2 (C6˝), 128.5
(tetrahydrocarbazole-C), 129.1 (C5׳), 129.32 (C5˝), 129.8 (C2˝), 133.9 (C4˝), 135.2 (C-N), 135.8 (C-
N), 138.5 (C3׳), 139.8 (C3˝), 140.1 (C4׳), 142.3(C6׳), 143.57 (C2׳) ppm.
1-(4-chlorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (6c)
Yellow solid; yield: 78%, m.p. = 200-201 °C. IR (KBr): 3021, 2921, 2851, 1498, 1400, 803 cm-1. 1H
NMR (DMSO, 500 MHz): δ = 1.78-1.84 (m, 4H, tettahydrocarbazole-CH2), 2.61-2.65 (m, 4H,
tetrahydrocarbazole-CH2), 5.60 (s, 2H, N- CH2), 5.58 (s, 2H, N- CH2), 5.87 (s, 2H, N+- CH2), 7.00-
7.02 (m, 2H, H6,7), 7.34 (d, J= 7.5 Hz, 1H, H8), 7.41 (d, J= 7 Hz, 1H, H5), 7.49 (d, J= 7.5 Hz, 2H,
H2՛՛,6՛՛), 7.52 (d, J= 7.5 Hz, 2H, H3՛՛,5՛՛), 8.03 (d, J= 8 Hz, 1H, H5
՛
), 8.08 (t, J= 6.5 Hz, 1H, H4 ), 8.92 (s,
՛
1H, H2
՛
), 9.07(d, J=6 Hz, 1H, H6
՛
) ppm.13C- NMR (DMSO, 500 MHz): δ = 20.6
(tettahydrocarbazole-CH2), 21.4 (tettahydrocarbazole-CH2), 22.5 (tettahydrocarbazole-CH2),
+
22.6 (tettahydrocarbazole-CH2), 42.6 (N-CH2), 62.4 (N-CH2 ), 109.0 (tetrahydrocarbazole-C),
109.3 (tettahydrocarbazole-CH), 109.8 (tettahydrocarbazole-CH), 117.5 (tettahydrocarbazole-
CH), 117.8 (tettahydrocarbazole-CH), 119.2 (tetrahydrocarbazole-C), 127.2 (C5 ), 128.3 (C3՛՛,5՛՛),
՛
22

ACCEPTED MANUSCRIPT
128.9 (H2՛՛,6՛՛), 129.2 (C4՛՛), 130.8 (C1՛՛), 131.0 (C-N), 132.8 (C-N), 134.2 (C3
՛
), 135.2 (C4
՛
), 135.7 (C2 ),
՛
139.9 (C
՛
6 ) ppm.
1-(2-fluorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (6d)
Yellow solid; yield: 75%, m.p. = 200-201 °C. IR (KBr): 2926, 2858, 1645, 1460, 1376 cm-1.1H NMR
(DMSO, 500 MHz): δ = 1.78-1.85 (m, 4H, tetrahydrocarbazole-CH2), 2.60-2.64 (m, 4H,
tetrahydrocarbazole-CH2), 5.59 (s, 2H, N- CH2), 5.94 (s, 2H, N+- CH2), 7.00-7.03 (m, 2H, H6,7),
7.27-7.30 (m, 2H, H4՛՛,6՛՛), 7.34 (d, J= 8.0 Hz, 1H, H8), 7.41 (d, J= 7.5 Hz, 2H, H5), 7.53-7.56 (m, 2H,
H3՛՛,5՛՛), 8.02-8.09 (m, 2H, H5 ,4
՛
՛
), 8.80 (s, 1H, H2
՛
), 9.10 (d, J=6 Hz, 1H, H6
) ppm. 13C- NMR (DMSO,
՛
500 MHz): δ = 20.6 (tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazole-CH2), 22.5
(tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2), 42.5 (N- CH2), 61.3 (N+- CH2), 109.0
(tetrahydrocarbazole-C)
,
109.8 (tetrahydrocarbazole-CH)
,
117.5 (C3˝), 117.8
(tetrahydrocarbazole-CH), 121.0 (C1˝), 127.2 (C5˝), 128.2 (tetrahydrocarbazole-C) , 130.9 (C4˝),
131.7 (C5׳), 131.9 (C6˝), 135.1 (C-N) , 135.7 (C-N), 140.0 (C3׳) , 142.5 (C4׳), 144.1 (C2׳), 145.12 (C6׳),
162.5 (JC-F= 235 Hz, C2˝) ppm.
1-(3-fluorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (6e)
Yellow solid; yield: 75%, m.p. = 200-201 °C. IR (KBr): 2925, 2867, 2837, 2724, 1639, 1457 cm1. 1H
NMR (DMSO, 500 MHz): δ = 1.79-1.83 (m, 4H, tetrahydrocarbazole-CH2), 2.64-2.65 (m, 4H,
tetrahydrocarbazole-CH2), 5.58 (s, 2H, N- CH2), 5.89 (s, 2H, N+- CH2), 7.03 (m, 2H, H6,7), 7.28 (t, J=
8.0 Hz, 2H, H5՛՛), 7.35 (m, 2H, H8, 6՛՛), 7.41 (m, 2H, H5, 2՛՛), 7.48 (d, J= 7.0 Hz, 1H, H4՛՛), 8.02 (d, J= 8
Hz, 1H, H5
՛
), 8.08 (t, J= 6.5 Hz, 1H, H4
՛
), 9.04 (s, 1H, H2
՛
), 9.18 (d, J=6 Hz, 1H, H6
) ppm. 13C- NMR
՛
(DMSO, 500 MHz): δ = 20.6 (tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazole-CH2), 22.5
(tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2), 30.6 (N- CH2), 42.6 (N+- CH2), 62.4
(tetrahydrocarbazole-C), 109.3 (tetrahydrocarbazole-CH), 110.0 (C4˝), 116.0 (C2˝), 117.5
(tetrahydrocarbazole-CH), 117.9 (tetrahydrocarbazole-CH), 119.2 (tetrahydrocarbazole-CH),
127.2 (C6˝), 128.3 (tetrahydrocarbazole-C), 130.1 (C5׳), 131.4 (C5˝), 131.6 (C-N), 135.2 (C1˝), 135.8
(C-N), 140.0 (C3׳), 142.4 (C4׳), 143.6 (C2׳), 144.0 (C6׳), 162.1 (JC-F= 240 Hz, C3˝) ppm.
1-(4-fluorobenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (6f)
1
Yellow solid; yield: 77%, m.p. = 200-201 °C. IR (KBr): 2930, 2860, 1638, 1456 cm-1. H NMR
(DMSO, 500 MHz): δ = 1.78-1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.62-2.66 (m, 4H,
23

ACCEPTED MANUSCRIPT
tetrahydrocarbazole-CH2), 5.58 (s, 2H, N- CH2), 5.87 (s, 2H, N+- CH2), 7.02-7.03 (m, 2H, H6,7), 7.27
(t, J= 8.0 Hz, 2H, H2՛՛,6՛՛), 7.35 (d, J= 7.0 Hz, 1H, H8), 7.41 (d, J= 7.5 Hz, 1H, H5), 7.59 (t, J= 8.0 Hz,
1H, H3՛՛,5՛՛), 8.02 (d, J= 8 Hz, 1H, H5
՛
), 8.08 (t, J= 6.5 Hz, 1H, H4
՛
), 8.95 (s, 1H, H2 ), 9.17 (d, J=6 Hz,
՛
1H, H
՛
6 ) ppm.13C- NMR (DMSO, 500 MHz): δ = 20.6 (tetrahydrocarbazole-CH2), 21.5
(tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2),
42.6 (N- CH2), 62.4 (N+- CH2), 109.2 (tetrahydrocarbazole-C), 109.8 (tetrahydrocarbazole-CH) ,
116.0 (tetrahydrocarbazole-CH), 117.5 (tetrahydrocarbazole-CH), 117.8 (JC-F= 25.0 Hz , C3˝,5˝),
119.2 (tetrahydrocarbazole-CH), 127.2 (C5׳), 128.4 (tetrahydrocarbazole-C), 130.1 (C1˝), 131.5
(JC-F= 8.75 Hz, C2˝,6˝ ), 135.2 (C-N), 135.7 (C-N), 139.9 (C3׳), 142.4 (C4׳), 143.5 (C2׳), 143.9 (C6׳),
162.5 (JC-F= 245 Hz, C4˝) ppm.
1-(2-methylbenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (6g)
Yellow solid; yield: 75%, m.p. = 200-201 °C. IR (KBr): 3067, 3011, 2928, 2848, 1629, 1467,
1
741cm-1. H NMR (DMSO, 500 MHz): δ = 1.76-1.81 (m, 4H, tetrahydrocarbazole-CH2), 2.14 (s,
3H, CH3), 2.59-2.63 (m, 4H, tetrahydrocarbazole-CH2), 5.61 (s, 2H, N- CH2), 5.93 (s, 2H, N+- CH2),
7.00-7.04 (m, 2H, H6,7), 7.13 (d, J= 7.0 Hz, 1H, H3՛՛), 7.21-7.24 (m, 2H, H5՛՛,6՛՛), 7.32-7.35 (m, 2H,
H4՛՛,8), 7.40 (d, J= 7.0 Hz, 2H, H5), 7.13 (t, J= 6.5 Hz, 1H, H5
՛
), 8.18 (d, J= 7.0 Hz, 1H, H4
՛), 8.50 (s,
1H, H2 ), 9.05 (d, J=6 Hz, 1H, H6
՛
՛
) ppm. 13C- NMR (DMSO, 500 MHz): δ = 18.6 (CH3), 20.5
(tetrahydrocarbazole-CH2) , 21.4 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2),
22.6 (tetrahydrocarbazole-CH2), 42.5 (N- CH2), 61.4 (N+- CH2), 109.0 (tetrahydrocarbazole-C),
109.3 (tetrahydrocarbazole-CH), 109.8 (tetrahydrocarbazole-CH), 117.5 (tetrahydrocarbazole-
CH), 117.8 (tetrahydrocarbazole-CH), 118.9 (C5׳), 119.2 (C4˝), 120.7 (tetrahydrocarbazole-C),
126.5 (C5˝), 127.2 (C6˝), 128.2 (C3˝), 129.4 (C3׳), 130.8 (C4׳), 131.5 (C-N), 135.1 (C2˝) , 135.6 (C-N),
137.0 (C1˝), 144.1 (C6׳), 143.2 (C2׳) ppm.
1-(3-methylbenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (6h)
Yellow solid; yield: 74%, m.p. = 200-201 °C. IR (KBr): 3049, 3006, 2933, 2831, 1629, 1499, 1466,
1375 cm-1. 1H NMR (DMSO, 500 MHz): δ = 1.79-1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.29 (s,
3H, CH3), 2.63-2.65 (m, 4H, tetrahydrocarbazole-CH2), 5.58 (s, 2H, N- CH2), 5.82 (s, 2H, N+- CH2),
24

ACCEPTED MANUSCRIPT
7.01-7.04 (m, 2H, H6,7), 7.24 (t, J= 7.0 Hz, 1H, H4՛՛), 7.27-7.29 (m, 2H, H2՛՛,5՛՛), 7.31 (d, J= 7.5 Hz,
1H, H8), 7.37 (d, J= 7.0 Hz, 1H, H6՛՛), 7.41 (d, J= 7.0 Hz, 2H, H5), 8.04-8.09 (m, 1H, H5 ,4
՛
՛), 8.99 (s,
1H, H2 ), 9.16 (d, J=6 Hz, 1H, H6
՛
՛
) ppm. 13C- NMR (DMSO, 500 MHz): δ = 20.6 (CH3), 20.8
(tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2),
22.6 (tetrahydrocarbazole-CH2), 42.6 (N- CH2), 63.3 (N+- CH2), 109.0 (tetrahydrocarbazole-C),
109.3 (C8), 109.8 (C5), 117.5 (C6), 117.75(C7), 119.0 (tetrahydrocarbazole-C),127.2 (C6˝), 128.5
(tetrahydrocarbazole-C), 129.1 (C5׳), 129.32 (C5˝), 129.8 (C2˝), 133.9 (C4˝), 135.2 (C-N), 135.8 (C-
N), 138.5 (C3׳), 139.8 (C3˝), 140.1 (C4׳), 142.3(C6׳), 143.6 (C2׳) ppm.
1-(4-methylbenzyl)-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium chloride (6i)
Yellow solid; yield: 76%, m.p. = 200-202 °C. IR (KBr): 3033, 2932, 2851, 1618, 1499, 1462, 1377
1
cm-1. H NMR (DMSO, 500 MHz): δ = 1.77-1.83 (m, 4H, tetrahydrocarbazole-CH2), 2.31 (s, 3H,
CH3), 2.60-2.64 (m, 4H, tetrahydrocarbazole-CH2), 5.58 (s, 2H, N- CH2), 5.82 (s, 2H, N+- CH2),
7.01-7.03 (m, 2H, H6,7), 7.22 (d, J= 7.0 Hz, 2H, H3՛՛,5՛՛), 7.35 (d, J= 7.0 Hz, 1H, H8), 7.37 (t, J= 7.0 Hz,
1H, H2՛՛,6՛՛), 7.41 (d, J= 7.0 Hz, 1H, H5), 8.02 (d, J= 8 Hz, 1H, H5
՛
), 8.07 (t, J= 6.5 Hz, 1H, H4
՛
), 8.95 (s,
1H, H2 ), 9.15 (d, J=6 Hz, 1H, H6
՛
՛
) ppm. 13C- NMR (DMSO, 500 MHz): δ = 20.6 (CH3), 20.7
(tetrahydrocarbazole-CH2), 21.4 (tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2),
22.6 (tetrahydrocarbazole-CH2), 42.5 (N- CH2), 63.1 (N+- CH2), 109.0 (tetrahydrocarbazole-C),
109.3 (C8), 109.8 (C5), 117.5 (C6), 117.7 (C7), 119.0 (tetrahydrocarbazole-C), 127.2 (C5׳), 128.3
(C2˝,C3˝, C5˝, C6˝), 128.7(C1˝), 129.0 (C-N), 129.6 (C4˝), 130.9 (C-N), 135.2 (C3׳), 135.7 (C4׳), 139.0
(C2׳), 139.8 (C6׳) ppm.
1-benzyl-3-((1,2,3,4-tetrahydro-9H-carbazol-9-yl)methyl)pyridin-1-ium bromide (6j)
Yellow solid; yield: 75%, m.p. = 198-200 °C. IR (KBr): 2924, 2850, 1560, 1460, 1371 cm-1. 1H NMR
(DMSO, 500 MHz): δ = 1.78-1.84 (m, 4H, tetrahydrocarbazole-CH2), 2.62-2.66 (m, 4H,
tetrahydrocarbazole-CH2), 5.60 (s, 2H, N- CH2), 5.90 (s, 2H, N+- CH2), 6.99-7.05 (m, 2H, H6,7), 7.38
(d, J= 7.5 Hz, 1H, H8), 7.40 (d, J= 7.5 Hz, 1H, H5), 7.40-7.43 (m, 3H, H2՛՛,4՛՛,6՛՛), 7.49 (m, 2H, H3՛՛,5՛՛),
8.02 (d, J= 8.0 Hz, 1H, H5
՛
), 8.08 (t, J= 6.5 Hz, 1H, H4
՛
), 9.08(s, 1H, H2
՛
), 9.19 (d, J= 6.0 Hz, 1H, H6 )
՛
ppm. 13C- NMR (DMSO, 500 MHz):
δ
= 20.6 (tetrahydrocarbazole-CH2), 21.4
25

ACCEPTED MANUSCRIPT
(tetrahydrocarbazole-CH2), 22.5 (tetrahydrocarbazole-CH2), 22.6 (tetrahydrocarbazole-CH2),
42.6 (N- CH2), 63.2 (N+- CH2), 109.1 (tetrahydrocarbazole-C), 109.3 (C8), 109.8 (C5), 117.5 (C6),
117.7 (C7), 119.2 (C4՛՛), 120.9 (tetrahydrocarbazole-C), 127.2 (C5׳), 128.4 (C3՛՛, C5՛՛), 128.6 (C2՛՛
,
C6՛՛), 128.9 (C1՛՛), 129.2 (C-N), 129.5 (C-N), 134.0 (C3׳), 135.2 (C4׳), 135.7 (C2׳), 139.8 (C6׳) ppm.
4.2. Cholinesterase inhibition assay
Anti-AChE and anti-BuChE activity of derivatives were obtained using the Ellman’s method [24,
27]. Sodium hydrogen carbonate, potassium hydroxide, dipotassium hydrogen phosphate and
potassium dihydrogen phosphate were purchased from Fluka. Electric eel (Torpedo californica)
AChE (type VI-S), BuChE (E.C.3.1.1.8, from equine serum), acetylthiocholine iodide,
butyrylthiocholine iodide, 5,5׳-dithiobis[2-nitrobenzoic acid] (DTNB) and Donepezile
hydrochloride was obtained from Sigma–Aldrich (Steinheim, Germany). It is necessary to note
that the last-mentioned drug was used as the reference drug. The stock solutions of the
derivatives were prepared by dissolving them in the dimethyl sulfoxide (DMSO). Afterwards,
they were diluted in the absolute ethanol to obtain three different assay concentrations and,
for each concentration, done in triplicate. The assay solution contained 2 mL of phosphate
buffer (0.1 M, pH = 8), 60 μL DTNB, 30 μL of inhibitor and 20 μL of 5 IU/mL butryl cholinesterase
solution. Then, the previous mixture was pre-incubated for 10 minutes at 25°C. The reaction
was then initiated by adding 20 μL of butyrylthiocholine iodide as the substrate to each of the
24 wells. The absorbance changes were scored at 412 nm for 5 min using a Synergy HTX
multimode plate reader. The IC50 values were calculated graphically from a concentration
inhibition curve for each derivative. All plots were obtained with Microsoft Excel 2010. Also, the
same assay was performed with AChE to obtain the anti-AChE activity of the derivatives.
4.3. Kinetic studies of BuChE inhibition
Derivative (6i) with the best BuChE IC50 value was selected to investigate the mechanism of
BuChE’s inhibition. Double reciprocal plots (1/v versus 1/s) were outlined [21] for three
different concentrations of 6i as the inhibitor (0.043, 0.087 and 0.174 µM) and
butyrylthiocholine iodide as the substrate (0.13, 0.32 and 0.69 µM). Correspondingly, the
26

ACCEPTED MANUSCRIPT
inhibition constant (Ki) was calculated with the interception of the slope versus the
concentration plot. All analyses were carried out using Microsoft Excel 2010.
4.4. Molecular dynamic simulation studies
Molecular dynamic simulation study was performed using Gromacs 5.1 (University of
Groningen, Netherlands) on an 8 core PC running Linux Ubuntu 14.0.1. According to tutorial of
Gromacs
(http://www.bevanlab.biochem.vt.edu/Pages/Personal/justin/gmx-
tutorials/complex/index.html) GROMOS96-43a1 force field was used for preparation of
topology file. The required format of ligand was prepared using PRODRG 2 online server [28].
To obtain 3D coordinates of ligand for simulation purpose, ligand docking was performed and
the coordinates of the best pose (considering the best docking energy) was retrieved. The
proper Gromacs input format (gro) was constructed by appending ligand coordinates to the end
of protein gro file (Human butyrylcholinesterase, PDBID: 4BDS). The above complex was
surrounded by a cubic box filled with simple point charge (SPC) water molecules and the
minimum distance of 1.0 nm. After solvation the system was neutralized by adding 6 chloride
ions (Cl-). Afterwards, the minimization step was performed using the following parameters:
maximum steps=50000, maximum force threshold < 10kJ/mole. The, system was equilibrated
for 100 ps at the temperature of 300 °K and then at 1 bar pressure for 100 ps. The MD
production phase was performed for 10 ns at 300 °K and 1 bar pressure using default
parameters.
4.5. Computational methods
The Clog P values, tPSA, HBD, and HBA were calculated by the means of MarvineSketch 6.2.0
Also, RBC was calculated using Autodock Tools 1.5.6.
4.6. MTT assay
To study the neuroprotective activity of compound 6i as the best inhibitor, MTT assay was
performed [31]. PC12 cell line was obtained from the Pasteur institute; in addition, all culture
media and supplements were purchased from Gibco. Quercetin (10 μM) was used as a positive
27

ACCEPTED MANUSCRIPT
control. Differentiated PC12 cells were incubated with 10 μl of 6i (10, 50 and 100 μM) in 96 well
culture plates (4000cells/well) for 3 hours before their treatment with H2O2 (375 μM).
Subsequently, MTT assay was performed after 12 hours. Briefly, 10 μl of MTT solution (5
mg/ml, Sigma) was added to the cell culture media and left for 3 hours. Then, the formazan
precipitates were dissolved by adding 100 μl of the 10% SDS (in 0.01 M HCl (w/v)) solution into
each well and the plates were incubated overnight. Finally, the optical density (OD) was
obtained at 570 nm on the ELISA reader and each experiment was performed in triplicate.
4.7. BACE1 enzymatic assay
The assay was carried out according to the manufacturers’ instructions and the previously
reported method [30]. A FRET-based BACE1 enzyme assay kit was obtained from Invitrogen
(former Pan Vera Corporation, Madison, WI). Also Stock solution of compound 6i was prepared
in DMSO and further diluted in assay buffer to prepare different concentrations of the test
compound. Subsequently 10 μL of different concentrations of the test compound was added to
10 μL of BACE1 substrate in separate wells of a black 96-well micro plate. Then 10 μL of BACE1
was added to each well to start the reaction. Plates were incubated for 90 minutes at 25 ºC.
Then, 10 μL of stop buffer (sodium acetate) was added to each well to stop the reaction and
fluorescence measurements were performed with a multimode micro plate reader (BMG
Labtech) at excitation and emission wavelength of 545 nm and 585 nm, respectively.
Experiments were repeated 3 times and mean percent of enzyme inhibitory activities at 10 and
50 μM concentrations of the compound 6i were calculated.
4.8. Determination of the inhibitory potency on Aβ1-42 self-aggregation and Aβ1-40 aggregatin
induced by AchE
ThT-based fluorometric assay method [32,33] 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
(6i) 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
28