Design, synthesis, in vivo and in vitro studies of 1,2,3,4-tetrahydro-9H-carbazole derivatives, highly selective and potent butyrylcholinesterase inhibitors

Article abstract of DOI:10.1007/s11030-019-09943-6

Abstract: Inhibition of butyrylcholinesterase (BChE) might be a useful therapeutic target for Alzheimer’s disease (AD). A new series of 1,2,3,4-tetrahydro-9H-carbazole derivatives were designed synthesized and evaluated as BChE inhibitors. While all of the derivatives have shown for AChE IC₅₀ values below the detectable limit (> 100?μM), they were selective potent BChE inhibitors. 1-(2-(6-fluoro-1,2,3,4-tetrahydro-9H-carbazole-9-yl)ethyl)piperidin-1-ium chloride (15?g) had the most potent anti-BChE activity (IC₅₀ value = 0.11?μM), the highest BChE selectivity and mixed-type inhibition. Pharmacokinetic properties were accordant to Lipinski rule and compound 15g demonstrated neuroprotective and inhibition of β-secretase (BACE1) activities. Furthermore, in vivo study of compound 15g in Morris water maze task has confirmed memory improvement in scopolamine-induced impairment. All results suggest that new sets of potent selective inhibitors of BChE have a therapeutic potential for the treatment of AD. Graphical abstract: A new series of 1,2,3,4-tetrahydro-9H-carbazole derivatives were designed synthesized and evaluated as BChE inhibitors. While all of the derivatives have shown for AChE IC50 values below the detectable limit, they were selective potent BChE inhibitors. Compound 15g had the most potent anti-BChE activity. All results suggest that new sets of potent selective inhibitors of BChE have a therapeutic potential for the treatment of AD.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-019-09943-6

Molecular Diversity
https://doi.org/10.1007/s11030-019-09943-6
ORIGINAL ARTICLE
Design, synthesis, in vivo and in vitro studies
of 1,2,3,4‑tetrahydro‑9H‑carbazole derivatives, highly selective
and potent butyrylcholinesterase inhibitors
Roshanak Ghobadian1 · Roghaieh Esfandyari1 · Hamid Nadri2 · Alireza Moradi2 · Mohammad Mahdavi3 ·
Tahmineh Akbarzadeh1,4 · Hossein Khaleghzadeh‑Ahangar5 · Najmeh Edraki6 · Mohammad Sharifzadeh7 ·
Mohsen Amini1
Received: 7 January 2019 / Accepted: 20 March 2019
© Springer Nature Switzerland AG 2019
Abstract
Inhibition of butyrylcholinesterase (BChE) might be a useful therapeutic target for Alzheimer’s disease (AD). A new series
of 1,2,3,4-tetrahydro-9H-carbazole derivatives were designed synthesized and evaluated as BChE inhibitors. While all of
the derivatives have shown for AChE IC50 values below the detectable limit (>100 µM), they were selective potent BChE
inhibitors. 1-(2-(6-fuoro-1,2,3,4-tetrahydro-9H-carbazole-9-yl)ethyl)piperidin-1-ium chloride (15 g) had the most potent
anti-BChE activity (IC50 value=0.11 μM), the highest BChE selectivity and mixed-type inhibition. Pharmacokinetic proper-
ties were accordant to Lipinski rule and compound 15g demonstrated neuroprotective and inhibition of β-secretase (BACE1)
activities. Furthermore, in vivo study of compound 15g in Morris water maze task has confrmed memory improvement in
scopolamine-induced impairment. All results suggest that new sets of potent selective inhibitors of BChE have a therapeutic
potential for the treatment of AD.
Graphical abstract
A new series of 1,2,3,4-tetrahydro-9H-carbazole derivatives were designed synthesized and evaluated as BChE inhibitors.
While all of the derivatives have shown for AChE IC50 values below the detectable limit, they were selective potent BChE
inhibitors. Compound 15g had the most potent anti-BChE activity. All results suggest that new sets of potent selective inhibi-
tors of BChE have a therapeutic potential for the treatment of AD.
Keywords Synthesis · Carbazole · Alzheimer’s disease · Butyrylcholinesterase · Neuroprotective activity
Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-019-09943-6) contains
During the last decades, the number of patients suffer-
ing from AD has increased [1]. AD is characterized by
neurofbrillary tangle formation, neuronal loss, the pres-
ence of plaques of amyloid β-peptide (Aβ) and decreasing
supplementary material, which is available to authorized users.
* Mohsen Amini
moamini@tums.ac.ir
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Molecular Diversity
acetylcholine in some regions of the brain such as cortex and
hippocampus [2, 3]. According to the cholinergic hypothe-
sis, the dysfunction of cholinergic neurons in the brain leads
to the cognitive decline observed in those with AD [4–9].
Acetylcholinesterase (AChE) is an important enzyme reg-
ulating amount of acetylcholine (Ach) in the brain. There-
fore, recently there was an interest for development of AChE
inhibitors [10–14]. Though butyrylcholinesterase (BChE)
works as a co-regulator of cholinergic neurotransmission,
recent studies show that there is an obvious increase in
BChE activity in the most afected areas of the brain. Also,
in progressive form of AD, the loss of AChE-activity is com-
pensated by BChE [15]. It is reported that AChE knockout
mouse model shows that mice did not sufer from increas-
ing ACh in the absence of AChE, cause of controlling the
hydrolysis of ACh by BChE [1]. On the other hand, it is
claimed that selective BChE inhibitors may circumvent clas-
sical cholinergic toxicity [3]. Therefore, development of new
selective BChE inhibitors can provide additional benefts in
the treatment of AD. In recent years, there is an interest in
development of selective inhibitors of BChE [1, 2, 16–23].
However, selective targeting of BChE over AChE is a
challenging task, as both AChE and BChE share about 70%
of homology with the main diferences in the acyl pocket and
the peripheral site. Two aromatic residues (Phe295, Phe297)
in the acyl pocket of AChE are replaced with two aliphatic
residues (Leu286, Val288) in BChE. This led to access of
bulkier inhibitors in the catalytic site of BChE [17].
R2
N
N
N
N
AChE IC50> 100 µM
BChE IC50 = 7.9 µM
15(a-o)
Scheme 1 Design of compounds 15a–o based on 2-methyl-9-phene-
thyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole structure
Results and discussion
Cholinesterase inhibition assay
The synthesized compounds (15a–o) were tested for their
inhibitory activity toward Electric eel (Torpedo califor-
nica) AChE (type VI-S) and BChE (E.C.3.1.1.8, from
equine serum), using the Elman assay [25–27]. Also, gal-
antamine hydrobromide was used as the reference drug.
The IC50 values are reported in Table 1.
According to Table 1, BChE IC50 values were ranging
from 0.11 to 27.84 µM. However, all of the derivatives
have shown for AChE IC50 values below the detectable
limit (> 100 µM). Thus, all designed compounds were
selective BChE inhibitor (SIBChE, IC50 of AChE/IC50 of
BChE). This might be a therapeutic advantage for the
treatment of AD, because in progressive form of AD, the
loss of AChE-activity could be compensated by unaf-
fected BChE [10]. The three most efective BChE inhibi-
tors were compounds 15g, 15d and 15m (IC50: 0.11 0.01,
0.15 0.02 and 0.17 0.03 μM, respectively) with great
BChE selectivity (SIBChE, respectively: 892.86, 645.16 and
581.39). According to Table 1, anti-BChE activities of
all derivatives except 15b, 15k and 15l were more potent
and selective than galantine (BChE IC50 of galantamine
hydrobromide: 9.4 2.5 μM).
N1-phenetylnorcymserine, phenothiazine derivatives
and some quinazolinimine-based compounds are selective
BChE inhibitors with tri- or polycyclic structures that have
been reported previously [16, 24–26]. Also recently, selec-
tive indole-containing tricycle BChE inhibitors has been
reported as a potential AD treatment [27]. But multistep
synthesis of indolo-N-substituted carboline derivatives does
not seem to be interesting attempt. Therefore, 2-methyl-
9-phenethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole [27]
was taken as template, and for simpler synthesis instead of
2-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole, similar
tricyclic ring named 1,2,3,4-tetrahydro-9H-carbazole were
used. Furthermore, as shown in Scheme 1, our derivatives
have diferent nitrogen-containing heterocycles at the end
site of hydrocarbon chain to gain chemical modifcations of
the heterocyclic core. Also, substitutions on aromatic part
of tricyclic ring (R1) were investigated. We have synthesis
carbazole-based BChE inhibitors previously [28, 29]. In all,
considering simultaneously increase selectivity and anti-
BChE activity of indole-containing tricycle derivatives, we
have designed a new series of tricycle derivatives.
It is realized that compound 7a (2,3,4,9-tetrahydro-
1H-carbazole BChE IC50: 89.13 0.01) did not show
potent anti-BChE activity in comparison with 15a–o deriv-
atives. So insertion of 2-(heterocycle-1-yl) ethyl moiety
seems to be necessary for anti-BChE activity. It seems
that anti-BChE activities have increased by replacement
of hydrogen in six position of 2, 3, 4, 4a, 9, 9a-hexahydro-
1H-carbazole moiety with fuorine, chlorine and methyl.
However, introduction of 6-methoxy led to decreasing of
anti-BChE activity.
Furthermore, introduction of 2-(piperidin-1-yl)ethyl
moiety led to more potent anti-BChE activity than 2-(pyro-
lidine-1-yl)ethyl and 2-(morpholine-1-yl)ethyl (BChE
1 3

Molecular Diversity
Table 1 The IC50 values of the compounds 15a–o against AChE and BChE
Entry
Compounds
R1
R2
AChE inhibition [IC50 BChE inhibition [IC50 SI BChE
(μM)]
(μM)]
(IC50 AChE/
IC50BuChE)
1
2
3
4
5
6
7
8
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
H
H
H
Me
Me
Me
F
F
F
OMe
OMe
OMe
Cl
Cl
Cl
CH2–CH2
CH2–O
CH2
CH2–CH2
CH2–O
CH2
CH2–CH2
CH2
CH2–O
CH2–CH2
CH2–O
CH2
CH2–CH2
CH2–O
CH2
–
–
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.7 0.9
0.41 0.04
21.80 0.01
1.88 01
0.15 0.02
8.20 03
1.70 0.01
0.11 0.01
1.18 0.02
7.68 0.01
0.85 0.02
27.84 0.01
22.97 0.02
0.17 0.03
2.64 0.01
0.73 0.01
89.130 0.01
9.4 2.5
242.72
4.59
53.19
645.16
12.19
58.82
892.86
84.74
13.02
117.65
3.592
4.35
581.39
37.88
136.24
1.12
9
10
11
12
13
14
15
16
17
15m
15n
15o
7a
H
–
Galantamine
0.18
Data are expressed as mean SE (three independent experiments)
IC50 15b,e,I,k,n > 15c,f,h,l,o > 15a,d,g,j,m). This was in
correlation with higher Pka value for nitrogen in piperi-
dine cycle (PKa: 10.042, 9.945 and 8.006 for piperidine,
pyrolidine and morpholine, respectively). Thus, it seems
that nitrogen of 2-(heterocycle-1-yl) ethyl moiety should
be protonated before interacting with active site of BChE.
Overall, derivatives having piperidine in 2-(heterocycle-
1-yl)ethyl moiety were more potent BChE inhibitors in com-
parison with those having other heterocyclic rings and also
the F substituent at six position of 2,3,4,4a,9,9a-hexahydro-
1H-carbazole moiety due to its hydrogen binding was the
most favorable one for BChE inhibition (15 g has the best
BChE IC50 =0.11 0.01 µM).
Lineweaver–Burk curves (Fig. 1) [30]. Three diferent con-
centrations of 15g 0, 0.06, 0.14 and 0.25 µM) and butyryl-
choline iodide 0.13, 0.32 and 0.69 mM) were used as
inhibitor and substrate, respectively. It seems that a mixed-
type inhibition could be attributed to the compound 15g.
Binding of compound 15g to BChE changed both Vmax
and Km values, a trend that is generally ascribed to mixed-
type inhibition. Furthermore, the inhibition constant Ki for
compound 15g was calculated (Ki =0.05 µM) using plot of
slope versus inhibitor concentration (Fig. 2). As Fig. 1, the
Lineweaver–Burk plot reveals that the type of inhibition is
mixed (competitive and non-competitive) (variable Km and
Vmax). This fact implies that the inhibitor could interact with
substrate–enzyme complex and intact enzyme as well. When
it binds to intact enzyme, the inhibitor can occupy both cata-
lytic triad and peripheral site. In complex, the catalytic triad
is occupied by substrate and then the inhibitor just interacts
with another site within the active site.
Kinetic studies of AChE and BChE inhibition
The mechanism of the BChE inhibition for the most
potent derivative (15g) was determined by plotting
1 3

Molecular Diversity
calculated. According to Table 2, MW <500, HBD <5, HBA
<10 and Clog P <5 were reported. Thus, pharmacokinetic
properties of 15 g are accordant to Lipinski’s rule [31] and
would have satisfactory pharmacokinetics after the oral admis-
sion as drug candidate.
Neuroprotective efect against H2O2‑induced cell
death in PC12 neurons
Additional study was performed to evaluate the neuroprotec-
tive activity of compound 15g as the most potent BChE inhibi-
tor, using in vitro MTT assay. H2O2 and diferentiated PC12
cells were considered as oxidative agent and in vitro model,
respectively, and neuroprotective activity of the desired com-
pound was utilized by subjecting PC12 cells to H2O2-induced
damage. As shown in Fig. 3, 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 signifcance. Also, the percent of
cell viability was calculated in comparison with control group.
According to Fig. 3, compound 15g has demonstrated good
neuroprotective activity at 10 μM (cell viability=67%, respec-
tively, and P<0.01 versus H2O2 treatment alone). But there
was no neuroprotective activity at 1 and 100 μM.
Fig. 1 Kinetic study of compound 15g on the inhibition mechanism
of BChE by Lineweaver–Burk plot (1/v) sec.DA−1 versus (1/[s])
mM−1
β‑secretase inhibitory activity of compound 15g
β-secretase inhibitory activity of 15g as the most potent BChE
inhibitor was evaluated via a fuorescence resonance energy
transfer (FRET)-based BACE-1 kit including β-secretase
enzyme and specifc APP-based peptide substrate (Rh-EVNL-
DAEFK-quencher). Also, experiments were repeated for three
times and compared with OM99-2 as the reference compound.
Compound 15g has shown inhibition against β-secretase
at the concentration of 50 µM. The non-peptide structure of
compound 15g is desired and acts as dual inhibitor of BChE
and β-secretase (Table 3).
Fig. 2 The plot of the slope versus the concentration of compound
15 g (µM) for calculating Ki
The in vivo study
The 10 and 20 mg/kg doses of compound 15g signifcantly
increased the time spent in target quadrant in the probe day in
compare with control and vehicle groups (p<0.001 vs. sco-
polamine 4 mg/kg and scopolamine 4 mg/kg+DMSO 5%)
but not as the donepezil positive control group (Fig. 4). The
10 mg/kg dose of compound 15g decreased signifcantly the
Screening of pharmacokinetic properties
The polar surface area (tPSA), a number of H-bond acceptors
(HBA), a number of H-bond donors (HBD), octanole/water
partition coefcients (Clog P) and a number of rotatable bonds
(RBC) for compound 15g as the most potent inhibitor were
Table 2 Molecular descriptors
Entry
7
Compound
15 g
HBD
0
HBA
3
ClogP
5.53
tPSA[Å2]
6.48
MW
RBC
3
of the compounds 15 g
300.42
HBD H-bond donors, HBA H-bond acceptors, Clog P calculated octanol–water partition coefcient, tPSA
topological polar surface area, MW molecular weight (g mol−1), RBC rotatable bond count
1 3

Molecular Diversity
might be a therapeutic advantage for the treatment of AD,
because in progressive form of AD the loss of AChE-activity
could be compensated by unafected BChE [10]. Also, most
of derivatives were more potent and selective BChE inhibi-
tors than galantamine as the reference drug. Compound 15g
(BChE IC50 = 0.11 0.01 μM) was the most potent BChE
inhibitor. Compound 15g also has shown neuroprotective
efect at 10 μM against H2O2-induced cell death in PC12
neurons. Also, compound 15g has shown β-secretase activ-
ity. Furthermore, in vivo study of compound 15g in Morris
water maze task confrmed memory improvement in sco-
polamine-induced impairment. Pharmacokinetic properties
of 15g are accordant to Lipinski rule and would have satis-
factory pharmacokinetics after the oral admission as drug
candidate. All results suggest that new selective inhibitors of
BChE have a therapeutic potential for the treatment of AD.
Fig. 3 Neuroprotective efect of compound 15g on the cell viability
of the PC12 cells in H2O2-induced damage. Data are expressed as
mean SEM (n=8) and one-way analysis of variance (ANOVA) fol-
lowed by the Newman–Keuls test was carried out to determine the
level of signifcance. ****P<0.0001; ***P<0.001, **P<0.01 all
versus H2O2 and +P<0.05 versus H2O2 (negatively signifcant)
Experimental
Chemistry
Table 3 BACE1 inhibitory activitya of compound 15g
All 1H and 13C nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker FT-500, using TMS as an inter-
nal standard and also IR spectra were obtained on a Nicolet
Magna FTIR 550 spectrophotometer (in KBr). All reagents
and solvents were obtained from Sigma-Aldrich and Merck.
2,3,4,9-tetrahydro-1H-carbazole was synthetized according
to Fischer Indole Synthesis [29]. Also, all of melting points
were determined by a Kofer hot-stage apparatus (Reichert,
Vienna, Austria).
Compound
Inhibitionb at 50 μM (%)
Inhibitionb at 10 μM (%)
15 g
18.8 0.1
3.2 0.1
aThe IC50 of standard drug OM99-2 was 3 nM
bValues represent mean standard error (S.E.) of three independent
experiments
mean training period scape latency (p<0.0001 versus sco-
polamine 4 mg/kg); The 20 mg/kg dose of compound 15g
decreased signifcantly the mean training period scape latency
(p<0.05 versus scopolamine 4 mg/kg; Fig. 4). The 10 mg/
kg dose of compound 15g decreased signifcantly the mean
training period travelled distance (p<0.001 versus scopola-
mine 4 mg/kg) but not as the donepezil positive control group
(Fig. 4). The 20 mg/kg dose of compound 15g decreased sig-
nifcantly the mean training period travelled distance (p<0.05
versus scopolamine 4 mg/kg). No signifcant diference was
observed for diferent treatments on swimming speed (Fig. 4).
Thus, in vivo study of compound 15g in Morris water maze
task confrmed memory improvement in scopolamine-induced
impairment [32].
Synthesis of 2,3,4,9‑tetrahydro‑1H‑carbazole
derivatives (compounds 7a–e)
As shown in Scheme 2, to synthesize diferent 2,3,4,9-tet-
rahydro-1H-carbazole derivatives (compounds 7a–e), Fis-
cher Indole Synthesis was utilized. Separately to obtain each
of derivatives (7a–e), a mixture of cyclohexanone (10 mol
of compound 1) and acetic acid (60 mmol) was collected,
heated under refux and stirred while one of phenyl hydra-
zine hydrochlorides (10 mol of compounds 2a–e) was added
during 1 h. After heating each of mixtures under refux for
an additional hour, they were cooled to room temperature.
The resulting solids were separately fltered with suction,
washed with 50 ml of water and fnally with 50 ml of 75%
ethanol. Recrystallization for each solid was performed
using 100 ml of methanol (yield 71–75%).
Conclusion
In conclusion, a novel series of 2,3,4,9-tetrahydro-1H-car-
bazole derivatives were synthesized and evaluated for their
anti-BChE activity. In vitro assay revealed that all of the
designed compounds were selective BChE inhibitors. This
Synthesis of compounds 15a–o
To synthesize compounds 15a–o, TBAB (Tetrabutyl ammo-
nium bromide) (2.73 mmol) was dissolved in an aqueous
1 3

Molecular Diversity
Fig. 4 The in vivo study; Morris water maze. Efects of i.p. infusion
for diferent doses of compound 15g, vehicle (DMSO), and positive
control (donepezil) on the time spent in target quadrant in the probe
day (A), scape latency (B), travelled distance (C), and swimming
speed (D). *P<0.05; **P<0.01; ***P<0.001 and ****P<0.0001
diferent from the scopolamine 4 mg/kg control group
solution of NaOH 50% (15 ml) and toluene (15 ml), then
stirred for 15 min. Subsequently, a solution of compound
7a–e (5.45 mmol) in toluene (15 ml) was added to the mix-
ture. Compound 12a–c (16.3 mmol) was added to this mix-
ture and stirred at refux for 6 h [33, 34]. Then, the solvent
was evaporated, poured into water and extracted with chlo-
roform (50 ml). The organic layer was dried over anhydrous
Na2SO4, fltered and the solvent removed by evaporation.
The resulting residue was purifed on a silica gel plates
using petroleum ether/ethyl acetate (3/1) tank to give solid
(50–53% yield) (compound 15a–o).
tetrahydrocarbazole-CH2), 7.05–7.09 (m, 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. 13C NMR (CDCl3, 125 MHz): δ = 21.10,
22.20, 23.27, 23.33, 108.59, 109.42, 117.77, 118.60, 120.50,
127.47, 135.28, 136.11 ppm.
6‑methyl‑2,3,4,9‑tetrahydro‑1H‑carbazole (7b) Yellow solid,
yield: 69% mp=118 °C. IR (KBr): 3408, 3022, 2934, 2849,
1
1680, 1617, 1472, 1442, 1373 cm−1. H NMR (CDCl3,
500 MHz): δ=1.84–1.87 (m, 4H, tetrahydrocarbazole-CH2),
2.43 (s, 3H, CH3), 2.65–2.67 (m, 4H, tetrahydrocarbazole-
CH2), 6.92 (d, J=8 Hz, 1H, H7), 7.11 (d, J=8 Hz, 1H, H8),
7.23 (s, 1H, H5), 7.45(s, 1H, NH) ppm. 13C NMR (CDCl3,
125 MHz): δ = 20.9, 21.44, 23.23, 23.31, 109.65, 109.95,
117.52, 122.34, 128.06, 133.93, 134.21 ppm.
2,3,4,9‑tetrahydro‑1H‑carbazole (7a) White solid; yield:
70% mp=117 °C. IR (KBr): 3455, 3048, 2931, 1612, 1580,
1468 cm−1.1H NMR (CDCl3, 500 MHz): δ = 1.85–1.92
(m, 4H, tetrahydrocarbazole-CH2), 2.69–2.72 (m, 4H,
1 3

Molecular Diversity
Scheme 2 Synthesis of com-
pounds 15a–o
6‑fouro‑2,3,4,9‑tetrahydro‑1H‑carbazole (7c) Yellow solid,
yield: 70% mp=120 °C. IR (KBr): 3433, 2938, 1626, 1587,
1479,1429,1371,1143 cm−1. 1H NMR (CDCl3, 500 MHz):
δ=1.81–1.94 (m, 4H, tetrahydrocarbazole-CH2), 2.64–2.72
(m, 4H, tetrahydrocarbazole-CH2), 6.85 (d, J = 7 Hz, 1H,
H7), 7.09 (d, J=7 Hz, 1H, H8), 7.25 (s, 1H, H5), 8 (s, 1H,
NH) ppm. 13C NMR (CDCl3, 125 MHz): δ=20.96, 22.24,
23.10, 23.14, 102.86 (JC–F =22.5), 108.405 (JC–F =26.25),
108.95 (JC-F = 10), 109.62, 127.65, 132.58, 137.02, 157,
158.52 (JC–F =231.25) ppm.
9‑(2‑(piperidin‑1‑yl)ethyl)‑2,3,4,9‑tetrahydro‑1H‑carbazole
(15a) Cream solid, yield: 60% mp=54–55.5 °C. IR (KBr):
3459, 3048, 2931, 2850, 2775, 1612, 1580, 1468, 1446,
1
1375, 738 cm−1. H NMR (CDCl3, 500 MHz): δ = 1.44
(m, 2H, H4׳), 1.46-1.62 (m, 4H, H3׳, H5׳), 1.84–1.87 (m,
2H, tetrahydrocarbazole-CH2), 1.91–1.95 (m, 2H, tetrahy-
drocarbazole-CH2), 2.46 (t, J=4.5 Hz, 4H, H2׳, H6׳), 2.57
(t, J = 7.5 Hz, 2H, piperidin-N-CH2), 2.72–2.73 (m, 4H,
tetrahydrocarbazole-CH2), 4.14 (t, J=7.5 Hz, 2H, tetrahy-
drocarbazole-N-CH2), 7.02–7.13 (m, 2H, H6, 7), 7.25 (d,
J=8.5 Hz, 1H, H8), 7.43 (d, J=7.5 Hz, 1H, H5) ppm. 13
C
6‑methoxy‑2,3,4,9‑tetrahydro‑1H‑carbazole (7d) Red solid,
NMR (CDCl3, 125 MHz): δ = 21.10, 22.20, 23.27, 23.33,
24.31, 26.03, 40.87, 55.12, 58.52, 108.59, 109.42, 117.77,
118.60, 120.50, 127.47, 135.28, 136.11 ppm, MS(EI)
m/z = 282(69, M+), 198(5), 184(14), 168(16), 156(11),
128(16), 99(32), 98(100), 77(3), 55(15).
yield: 69% mp=135 °C. IR (KBr): 3416, 2851, 1617, 1584,
1
1479, 1222 cm−1. H NMR (CDCl3, 500 MHz): δ= 1.86–
1.90 (m, 4H, tetrahydrocarbazole-CH2), 2.66–2.71 (m, 4H,
tetrahydrocarbazole-CH2), 3.84 (s, 3H, OCH3), 6.76 (d,
J=8.5 Hz, 1H, H7), 6.93 (d, J=8.5 Hz, 1H, H8), 7.24 (s, 1H,
H5), 7.54 (s, 1H, NH) ppm. 13C NMR (CDCl3, 125 MHz):
δ = 20.96, 23.21, 23.31, 23.35, 56.01, 100.38, 110.07,
110.53, 110.90, 128.25, 130.77, 135.08, 153.91 ppm.
4‑(2‑(1,2,3,4‑tetrahydro‑9H‑carbazol‑9‑yl)ethyl)morpholine
(15b) Cream solid, yield: 62% mp=101–102 °C. IR (KBr):
3446, 3044, 2923, 2855, 2830, 1609, 1577, 1468, 1374,
1
1113, 750 cm−1. H NMR (CDCl3, 500 MHz): δ = 1.85–
6‑chloro‑2,3,4,9‑tetrahydro‑1H‑carbazole (7e) Yellow solid,
yield: 68% mp=128 °C. IR (KBr): 3427, 2935, 1611, 1574,
1467,785 cm−1. 1H NMR (CDCl3, 500 MHz): δ=1.85–1.92
(m, 4H, tetrahydrocarbazole-CH2), 2.64–2.66 (t, 2H, tetrahy-
drocarbazole-CH2), 2.70–2.73 (m, 2H, tetrahydrocarbazole-
CH2), 7.04 (d, J=8.5 Hz, 1H, H7), 7.16 (d, J=8.5 Hz, 1H,
H8), 7.4 (s, 1H, H5), 7.67(s, 1H, NH) ppm. 13C NMR (CDCl3,
125 MHz): δ=20.89, 22.18, 23.06, 23.11, 109.26, 109.50,
117.33, 120.54, 124.36, 128.46, 134.43, 136.86 ppm.
1.86 (m, 2H, tetrahydrocarbazole-CH2), 1.94–1.95 (m, 2H,
tetrahydrocarbazole-CH2), 2.51 (t, J=4 Hz, 4H, H3׳, H5׳),
2.70 (t, J = 6.5 Hz, 2H, morpholine–N–CH2), 2.72–2.73
(m, 4H, tetrahydrocarbazole-CH2), 3.72 (t, J = 4 Hz, 4H,
H2׳, H6׳), 4.16 (t, J = 6.5 Hz, 2H, tetrahydrocarbazole-N-
CH2), 7.06–7.28 (m, 2H, H6, 7), 7.45 (d, J = 8.5 Hz, 1H,
H8), 7.46 (d, J = 7.5 Hz, 1H, H5) ppm. 13C NMR (CDCl3,
125 MHz): δ = 21.02, 22.19, 23.24, 40.51, 54.02, 58.01,
66.84, 108.47, 109.68, 117.85, 118.74, 120.60, 127.45,
1 3

Molecular Diversity
135.16, 135.94 ppm, MS(EI) m/z=284(66, M+), 184(30),
mp = 71–72 °C. IR (KBr): 3433, 2923, 2849, 2801, 2752,
2673, 1615, 1574, 1476, 1446, 1371 cm−1. 1H NMR (CDCl3,
500 MHz): δ = 1.79 (t, J = 4.5 Hz, 4H, H3׳, H4׳), 1.91 (m,
4H, tetrahydrocarbazole-CH2), 2.43 (s, 3H, CH3), 2.57
(t, J = 4.5 Hz, 4H, H2׳, H5׳), 2.68 (t, J = 7 Hz, 2H, pyrro-
lidin-N-CH2), 2.69 (m, 4H, tetrahydrocarbazole-CH2),
4.12 (t, J = 7 Hz, 2H, tetrahydrocarbazole-N-CH2), 6.94
(d, J=7.5 Hz, 1H, H 7), 7.15 (d, J=7.5 Hz, 1H, H8), 7.23
(s, 1H, H5) ppm. 13C NMR (CDCl3, 125 MHz): δ=21.03,
21.37, 22.12, 23.23, 23.28, 23.48, 42.19, 54.45, 55.66,
108.19, 108.92, 117.59, 121.93, 127.59, 127.71, 134.38,
135.17 ppm, MS(EI) m/z=282(76, M+), 198(26), 182(14),
170(12), 128(10), 84(100), 77(4), 55(14).
168(15), 156(11), 128(9), 100(100), 77(3), 55(21).
9‑(2‑(pyrrolidin‑1‑yl)ethyl)‑2,3,4,9‑tetrahydro‑1H‑carba-
zole (15c) Liquid in room temperature, yield: 61%. IR
(KBr): 3607, 3388, 3051, 2924, 2847, 1612, 1587, 1464,
1372, 738 cm−1. 1H NMR (CDCl3, 500 MHz): δ=1.80 (t,
J=4.5 Hz, 4H, H3׳, H4׳), 1.92 (m, 4H, tetrahydrocarbazole-
CH2), 2.57 (t, J=4.5 Hz, 4H, H2׳, H5׳), 2.67 (t, J=7 Hz, 2H,
pyrrolidin-N-CH2), 2.70 (m, 4H, tetrahydrocarbazole-CH2),
4.12 (t, J=7 Hz, 2H, tetrahydrocarbazole-N-CH2), 6.79 (m,
2H, H6, 7), 7.25 (d, J=8 Hz, 1H, H8), 7.46 (d, J=7 Hz, 1H,
H5) ppm. 13C NMR (CDCl3, 125 MHz): δ = 21.13, 22.25,
23.29, 23.37, 24.39, 42.01, 55.40, 58.55, 109.03, 109.46,
118.60, 122.50, 127.03, 127.59, 136.01, 136.15 ppm,
MS(EI) m/z=268(11, M+), 184(3), 168(3), 156(2), 128(1),
84(100), 77(1), 55(13).
6‑fuoro‑9‑(2‑(piperidin‑1‑yl)ethyl)‑2,3,4,9‑tetrahydro‑1H‑car-
bazole (15g) Liquid in room temperature, yield: 60%.
IR (KBr): 3433, 2938, 2857, 1626, 1587, 1479, 1429,
1
1371 cm−1. H NMR (CDCl3, 500 MHz): δ = 1.42 (m,
6‑methyl‑9‑(2‑(piperidin‑1‑yl)ethyl)‑2,3,4,9‑tetrahydro‑1H‑car-
bazole (15d) Cream solid, yield: 60% mp= 54.5–55.5 °C.
IR (KBr): 3408, 3302, 2934, 2849, 2782, 2706, 1617, 1585,
2H, H4׳), 1.60 (m, 4H, H3׳, H5׳), 1.82–1.85 (m, 4H, tet-
rahydrocarbazole-CH2), 2.45 (t, J = 4.5 Hz, 4H, H2׳, H6׳),
2.56 (t, J=7 Hz, 2H, piperidin-N-CH2), 2.64–2.70 (m, 4H,
tetrahydrocarbazole-CH2), 4.12 (t, J = 7 Hz, 2H, tetrahy-
drocarbazole-N-CH2), 7.05 (d, J = 7.5 Hz, 1H, H7), 7.15
(d, J = 7.5 Hz, 1H, H8), 7.40 (s, 1H, H5) ppm. 13C NMR
(CDCl3, 125 MHz): δ=20.96, 22.24, 23.10, 23.14, 24.13,
25.84, 29.68, 40.89, 55.10, 58.29, 102.87 (JC–F = 22.5),
108.404 (JC–F =26.25), 108.93 (JC–F =10), 109.61, 127.62,
132.58, 137.03, 157, 158.55 (JC–F =231.25) ppm, MS(EI)
m/z=300(6, M+), 185(4), 137(6), 98(100), 69(45), 55(11).
1
1472, 1373 cm1−. H NMR (CDCl3, 500 MHz): δ = 1.44
(m, 2H, H4׳), 1.46–1.62 (m, 4H, H3׳, H5׳), 1.83–1.92 (m,
4H, tetrahydrocarbazole-CH2), 2.43 (t, J=4.5 Hz, 4H, H2׳,
H6׳), 2.44 (s, 3H, CH3), 2.57 (t, J=7.5 Hz, 2H, piperidin-
N-CH2), 2.68-2.69 (m, 4H, tetrahydrocarbazole-CH2),
4.11 (t, J=7.5 Hz, 2H, tetrahydrocarbazole-N-CH2), 6.94
(d, J = 7 Hz, 1H, H 7), 7.15 (d, J= 7 Hz, 1H, H8), 7.23 (s,
J=s, 1H, H5) ppm. 13C NMR (CDCl3, 125 MHz): δ=21.03,
21.37, 22.12, 23.23, 24.17, 25.84, 40.62, 54.98, 58.36,
108.19, 108.84, 117.57, 121.89, 127.58, 127.70, 34.33,
135.27 ppm, MS(EI) m/z=296(71, M+), 198(17), 182(11),
170(10), 156(8), 128(7), 98(100), 77(2), 55(9).
6‑fuoro‑9‑(2‑(pyrrolidin‑1‑yl)ethyl)‑2,3,4,9‑tetrahydro‑1H‑car-
bazole (15h) Cream solid, yield: 61% mp= 43.2–44.3 °C.
IR (KBr): 3404, 3023, 2959, 2927, 2878, 2809, 1621, 1579,
1
1477, 1428, 1372 cm−1. H NMR (CDCl3, 500 MHz):
4‑(2‑(6‑methyl‑1,2,3,4‑tetrahydro‑9H‑carbazol‑9‑yl)ethyl)mor-
pholine (15e) Yellow solid, yield: 62% mp=76–77 °C. IR
(KBr): 3026, 2928, 2849, 2806, 2684, 1583, 1468, 1446,
δ=1.82 (t, J=4.5 Hz, 4H, H3׳, H4׳), 1.93 (m, 4H, tetrahy-
drocarbazole-CH2), 2.60 (t, J=4.5 Hz, 4H, H2׳, H5׳), 2.66
(t, J = 7 Hz, 2H, pyrrolidin-N-CH2), 2.72–2.75 (m, 4H,
tetrahydrocarbazole-CH2), 4.16 (t, J = 7 Hz, 2H, tetrahy-
drocarbazole-N-CH2), 6.85 (d, J=7 Hz, 1H, H7), 7.08 (d,
J=7 Hz, 1H, H8), 7.25 (s, 1H, H5) ppm. 13C NMR (CDCl3,
125 MHz): δ = 20.96, 22.24, 23.10, 23.14, 23.50, 42.32,
54.49, 55.62, 102.88 (JC–F =22.5), 108.403 (JC–F =26.25),
108.95 (JC–F =10), 109.62, 127.65, 132.58, 137.02, 157.00,
158.54 (JC–F = 231.25) ppm, MS(EI) m/z = 286(60, M+),
216(6), 202(12), 186(15), 146(10), 84(100), 55(18).
1
1370, 1114 cm−1. H NMR (CDCl3, 500 MHz): δ= 1.84–
1.85 (m, 2H, tetrahydrocarbazole-CH2), 1.92–1.93 (m,
2H, tetrahydrocarbazole-CH2), 2.44 (s, 3H, CH3), 2.48 (t,
J=4.5 Hz, 4H, H3׳, H5׳), 2.61 (t, J=7 Hz, 2H, morpholine-
N-CH2), 2.67–2.72 (m, 4H, tetrahydrocarbazole-CH2), 3.71
(t, J=4.5 Hz, 4H, H2׳, H6׳), 4.11 (t, J=7 Hz, 2H, tetrahy-
drocarbazole-N-CH2), 6.95 (d, J=8 Hz, 2H, H 7), 7.14 (d,
J = 8 Hz, 1H, H8), 7.24 (d, J = 7.5 Hz, 1H, H5) ppm. 13C
NMR (CDCl3, 125 MHz): δ = 21.04, 21.40, 22.22, 23.24,
23.29, 40.56, 54.03, 58.02, 66.85, 108.16, 109.07, 117.69,
122.00, 127.67, 127.89, 134.32, 135.26 ppm, MS(EI)
m/z = 298(19, M+), 198(15), 182(4), 170(4), 128(2),
100(100), 77(1), 55(7).
4‑(2‑(6‑fuoro‑1,2,3,4‑tetrahydro‑9H‑carbazol‑9‑yl)ethyl)mor-
pholine (15i) Yellow solid, yield: 62% mp= 78–79 °C. IR
(KBr): 2962, 2921, 2852, 1622, 1576, 1479, 1366 cm−1.
1H NMR (CDCl3, 500 MHz): δ = 1.81–1.86 (m, 2H, tet-
rahydrocarbazole-CH2), 1.91–1.95 (m, 2H, tetrahydro-
carbazole-CH2), 2.46 (t, J =4 Hz, 4H, H3׳, H5׳), 2.57-2.60
(m, 2H, tetrahydrocarbazole-CH2), 2.65 (t, J=6.5 Hz, 2H,
6‑methyl‑9‑(2‑(pyrrolidin‑1‑yl)ethyl)‑2,3,4,9‑tetrahy-
dro‑1H‑carbazole (15f) Cream solid, yield: 61%
1 3

Molecular Diversity
morpholine-N-CH2), 2.70-2.72 (m, 2H, tetrahydrocarbazole-
CH2), 3.69 (t, J = 4 Hz, 4H, H2׳, H6׳), 4.09 (t, J = 6.5 Hz,
2H, tetrahydrocarbazole-N-CH2), 6.85 (d, J = 7 Hz, 1H,
J = 8 Hz, 1H, tetrahydrocarbazole-CH), 6.90 (s, 1H, H5),
7.15 (d, J = 8 Hz, 1H, tetrahydrocarbazole-CH) ppm. 13C
NMR (CDCl3, 125 MHz): δ = 22.19, 23.21, 23.47, 42.22,
54.45, 55.70, 55.99, 100.36, 109.15, 110.14, 127.60, 131.26,
135.88, 153.65 ppm, MS(EI) m/z= 298(17, M+), 214(6),
171(4), 143(2), 115(2), 84(100), 56(5).
H7), 7.09 (d, J=7 Hz, 1H, H8), 7.15 (s, 1H, H5) ppm. 13
C
NMR (CDCl3, 125 MHz): δ = 20.64, 20.93, 22.26, 23.09
(JC-F = 5), 29.64, 40.84, 53.83 (JC–F = 55), 58.02, 102.85
(JC–F = 22.5), 108.33 (JC–F = 26.25), 108.87 (JC–F = 10),
109.55 (JC–F =3.75), 127.65 (JC–F =8.75), 132.52, 137.06,
156.67, 158.51 (JC–F =231.25) ppm, MS(EI) m/z=302(10,
M+), 202(7), 184(3), 174(3), 133(2), 100(100), 55(6).
6‑chloro‑9‑(2‑(piperidin‑1‑yl)ethyl)‑2,3,4,9‑tetrahydro‑1H‑car-
bazole (15m) Yellow solid, yield: 60% mp = 73 °C. IR
(KBr): 3427, 2935, 2847, 2807, 1611, 1574, 1467, 1364,
1304, 1271, 1231, 1123, 854, 785 cm−1. 1H NMR (CDCl3,
500 MHz): δ=1.45 (m, 2H, H4׳), 1.59–160 (m, 4H, H3׳, H5׳),
1.82–1.85 (m, 2H, tetrahydrocarbazole-CH2), 1.90-1.93 (m,
2H, tetrahydrocarbazole-CH2), 2.46 (t, J=4.5 Hz, 4H, H2׳,
H6׳), 2.56 (t, J = 7 Hz, 2H, piperidin-N-CH2), 2.64-2.72
(m, 4H, tetrahydrocarbazole-CH2), 4.13 (t, J = 7 Hz, 2H,
tetrahydrocarbazole-N-CH2), 7.05 (d, J=8.5 Hz, 1H, H7),
7.15 (d, J=8.5 Hz, 1H, H8), 7.40 (s, 1H, H5) ppm. 13C NMR
(CDCl3, 125 MHz): δ=20.89, 22.18, 23.06, 23.11, 24.13,
25.84, 40.88, 55.05, 58.29, 109.26, 109.50, 117.33, 120.54,
124.36, 128.46, 134.43, 136.86 ppm, MS(EI) m/z=318(13,
M + 2)+, 316(38, M+), 218(10), 190(7), 154(10), 127(4),
98(100), 77(1), 55(14).
6‑methoxy‑9‑(2‑(piperidin‑1‑yl)ethyl)‑2,3,4,9‑tetrahy-
dro‑1H‑carbazole (15j) Liquid in room temperature, yield:
60%. IR (KBr): 3416, 2935, 2851, 1617, 1584, 1479, 1429,
1
1375, 1222 1151 cm−1. H NMR (CDCl3, 500 MHz):
δ=1.48 (m, 2H, H4׳), 1.60–163 (m, 4H, H3׳, H5׳), 1.85–1.88
(m, 2H, tetrahydrocarbazole-CH2), 1.90–1.93 (m, 2H, tet-
rahydrocarbazole-CH2), 2.50 (t, J = 4.5 Hz, 4H, H2׳, H6׳),
2.57 (t, J=7 Hz, 2H, piperidin-N-CH2), 2.65-2.73 (m, 4H,
tetrahydrocarbazole-CH2), 4.15 (t, J = 7 Hz, 2H, tetrahy-
drocarbazole-N-CH2), 7.10 (d, J = 8.5 Hz, 1H, H7), 7.16
(d, J = 8.5 Hz, 1H, H8), 7.41 (s, 1H, H5) ppm. 13C NMR
(CDCl3, 125 MHz): δ=20.88, 22.17, 23.07, 23.11, 24.14,
25.85, 40.88, 55.05, 58.28, 109.25, 109.51, 117.33, 120.54,
124.36, 128.47, 134.42, 136.85 ppm, MS(EI) m/z=312(61,
M+), 214(14), 171(10), 143(6), 98(100), 77(2), 55(13).
4‑(2‑(6‑chloro‑1,2,3,4‑tetrahydro‑9H‑carbazol‑9‑yl)ethyl)mor-
pholine (15n) Dark orange solid, yield: 62% mp=88–89 °C.
IR (KBr): 3445, 2938, 2907, 2852, 2804, 1607, 1576, 1464,
1422, 1366, 1302, 1112, cm−1. 1H NMR (CDCl3, 500 MHz):
δ = 1.84 (m, 2H, tetrahydrocarbazole-CH2), 1.92 (m, 2H,
tetrahydrocarbazole-CH2), 2.46 (t, J=4 Hz, 4H, H3׳, H5׳),
2.59 (t, J = 7 Hz, 2H, morpholine-N-CH2), 2.65–2.70 (m,
4H, tetrahydrocarbazole-CH2), 3.69 (t, J = 4 Hz, 4H, H2׳,
H6׳), 4.09 (t, J = 7 Hz, 2H, tetrahydrocarbazole-N-CH2),
7.05 (d, J = 7.5 Hz, 1H, H7), 7.15 (d, J = 7.5 Hz, 1H,
H8), 7.34 (s, 1H, H5) ppm. 13C NMR (CDCl3, 125 MHz):
δ=20.85, 22.21, 23.02, 23.08, 29.66, 40.82, 54.04, 57.96,
66.86, 109.43, 117.37, 120.56, 124.41, 128.48, 134.38,
136.80 ppm, MS(EI) m/z=320(10, M+2)+, 318(28, M+),
218(81), 190(5), 167(6), 127(2), 100(100), 77(1), 55(18).
4‑(2‑(6‑methoxy‑1,2,3,4‑tetrahydro‑9H‑carbazol‑9‑yl)ethyl)
morpholine (15k) Cream solid, yield: 62% mp=94 °C. IR
(KBr): 3431, 2992, 2954, 2826, 1618, 1582, 1482, 1443,
1
1380, 1222, 1148 cm−1. H NMR (CDCl3, 500 MHz):
δ=1.83–1.88 (m, 2H, tetrahydrocarbazole-CH2), 1.91-1.94
(m, 2H, tetrahydrocarbazole-CH2), 2.48 (t, J=5 Hz, 4H, H3׳,
H5׳), 2.60 (t, J=7.5 Hz, 2H, morpholine-N-CH2), 2.67-2.72
(m, 4H, tetrahydrocarbazole-CH2), 3.70 (t, J=5 Hz, 4H, H2׳,
H6׳),3.85 (s, 3H, OCH3), 4.10 (t, J = 7.5 Hz, 2H, tetrahy-
drocarbazole-N-CH2), 6.79 (d, J=9 Hz, 1H, tetrahydrocar-
bazole-CH), 6.93 (s, 1H, H5), 6.92 (d, J = 9 Hz, 1H, tet-
rahydrocarbazole-CH) ppm. 13C NMR (CDCl3, 125 MHz):
δ=21.08, 22.28, 23.22, 40.72, 54.07, 56.02, 58.14, 66.90,
100.41, 109.14, 110.19, 127.68, 131.25, 135.98, 53.71 ppm,
MS(EI) m/z = 314(81, M+), 214(62), 171(15), 143(8),
100(100), 77(2), 55(14).
6‑chloro‑9‑(2‑(pyrrolidin‑1‑yl)ethyl)‑2,3,4,9‑tetrahydro‑1H‑car-
bazole (15o) Liquid in room temperature, yield: 61µ%. IR
(KBr): 3420, 2942, 2851, 1577, 1469, 1375, 1331 cm−1.
1H NMR (CDCl3, 500 MHz): δ=1.85 (t, J=6 Hz, 4H, H3׳,
H4׳), 1.91–1.94 (m, 4H, tetrahydrocarbazole-CH2), 2.65 (t,
J = 6 Hz, 4H, H2׳, H5׳), 2.72 (t, J = 7 Hz, 2H, pyrrolidin-
N-CH2), 2.84 (m, 4H, tetrahydrocarbazole-CH2), 4.20 (t,
J=7 Hz, 2H, tetrahydrocarbazole-N-CH2), 7.07 (d, J=7 Hz,
1H, H7), 7.21 (d, J=7 Hz, 1H, H8), 7.26 (s, 1H, H5) ppm.
13C NMR (CDCl3, 125 MHz): δ = 20.86, 22.20, 23.00,
23.08, 23.47, 41.68, 54.38, 55.18, 109.51, 117.46, 120.78,
124.57, 128.56, 134.36, 136.68 ppm, MS(EI) m/z=304(8,
6‑methoxy‑9‑(2‑(pyrrolidin‑1‑yl)ethyl)‑2,3,4,9‑tetrahy-
dro‑1H‑carbazole (15l) Cream solid, yield: 61% mp=66.5–
66.9 °C. IR (KBr): 3429, 2991, 2948, 2924, 2835, 2775,
1618, 1579, 1484, 1455, 1221, 1146 cm−1. 1H NMR (CDCl3,
500 MHz): δ = 1.79 (t, J = 4.5 Hz, 4H, H3׳, H4׳), 1.91 (m,
4H, tetrahydrocarbazole-CH2), 2.58 (t, J=4.5 Hz, 4H, H2׳,
H5׳), 2.67 (t, J = 7 Hz, 2H, pyrrolidin-N-CH2), 2.70 (m,
4H, tetrahydrocarbazole-CH2), 3.83 (s, 3H, OCH3), 4.12
(t, J = 7 Hz, 2H, tetrahydrocarbazole-N-CH2), 6.77 (d,
1 3

Molecular Diversity
M + 2)+, 302(25, M+), 218(6), 190(5), 168(6), 154(7),
127(4), 84(100), 77(1), 55(13).
Computational methods
The Clog P values, tPSA, HBD, and HBA were calculated
by the means of Marvine Sketch 6.2.0. Also, RBC was cal-
culated using AutoDockTools-1.5.6.
Cholinesterase inhibition assay
AChE and BChE IC50 values of all derivatives were
obtained using Ellman’s method [35–37]. Galantamine
hydrobromide was applied as reference drug, purchased
from Sigma-Aldrich. 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
carbonate were obtained from Fluka. To obtain desired
concentrations, the stock solutions were prepared by dis-
solving each derivative in dimethyl sulfoxide (DMSO) and
then diluted to three diferent concentrations in absolute
ethanol. This assay was performed in triplicate for each
concentration to obtain 20–80% enzyme inhibitions. The
assay solution consisted of 2-mL phosphate bufer (0.1 M,
pH = 8), 60 μL DTNB, 30 μL test compound and 20 μL
of 5 IU/mL 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 butyrylthiocholine iodide
as substrate to each well (24-well plates). Blank assays
were also carried out comprising all ingredients excluding
substrate. The absorbance changes were scored at 412 nm
for 5 min using a Synergy HTX multimode plate reader.
In order to determine IC50 values, inhibition curves were
drawn (log inhibitor concentration versus percent of inhi-
bition) by Microsoft Excel 2010. Also, the same assay was
performed to obtain anti-AChE-activity of all derivatives.
MTT assay
Neuroprotection assay for compound 15g against
H2O2-induced cell death in PC12 cells was performed.
PC12 cell line was obtained from Pasteur Institute (Iran),
and all culture media and supplements were purchased from
Gibco (Europe). MTT was purchased from Sigma–Aldrich
(Europe). Diferentiated PC12 cells were provided as previ-
ously described method [26]. Quercetin (10 μM) was used as
a positive control. Diferentiated PC12 cells were incubated
with 10, 50 and 100 μM of 15 g in the volume of 10 μL for
3 h. Subsequently, 375 μM of H2O2 was added and incu-
bated for half day. To perform MTT assay 10 all of MTT
solution (5 mg/ml) was added and left for 3 h. Then, 100 μL
of the 10% SDS in 0.01 M HCl (w/v) as solubilization solu-
tion was added into each well and the plates were incubated
overnight. Finally, optical density (OD) was measured at
570 nm with a 96-well ELISA plate reader. All MTT assays
were repeated three times.
Study of β‑secretase inhibition
β-secretase inhibition study was performed using a FRET-
based BACE1 enzyme assay kit that was obtained from Inv-
itrogen (former Pan Vera Corporation, Madison, WI). The
stock solution of compound 15g was prepared in DMSO and
further diluted in assay bufer to prepare diferent concentra-
tions of it. To 10 µL of BACE1 substrate in separate wells
of a black 96-well microplate were added 10 μL of diferent
concentrations of 15 g solutions and mixed slowly. In order
to start the reaction, 10 of μL of BACE1 was added to each
well and the plates were incubated for 90 min at 25 °C. For
quenching the reaction, 10 μL of sodium acetate bufer was
added. A multimode microplate reader (BMG Labtech) at
excitation and emission wavelength of 545 nm and 585 nm,
respectively, was used for fluorescence measurements.
Experiments were repeated triplicate and percentage of
enzyme inhibitory activity at 10 and 50 μM concentrations
of the compound 15g were calculated.
Kinetic studies of BuChE inhibition
In order to the determination of the mechanisms of BuChE
inhibitory efect of the 15a–o series, 15g was studied as
the most potent compound (IC50 = 0.11 0.01 µM). Four
diferent concentrations (0, 0.06, 0.14 and 0.25 µM) were
used for preparation of graph. Reciprocal plots of 1/V
versus 1/[S] were drawn using for test compound and
butyrylthiocholine iodide in there concentrations (0.13,
0.32 and 0.69 mM) as substrate. A secondary plot was
obtained using slopes of Lineweaver–Burk plots versus
diferent concentrations of inhibitor (15 g). The intercept
of the negative X-axis was used for calculation of inhibi-
tion constant (Ki) value. All kinetic studies were studied
in triplicate.
The in vivo study for compound 15g
In vivo study for compound 15g as the most potent BChE
inhibitor was performed. In total, 56 adult male albino
Wistar rats weighing 200–250 g were used in this experi-
ment. Animals were housed in groups of three per cage in a
12/12 h light/dark cycle with free access to food and water.
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Molecular Diversity
They were kept in animal room in which the temperature
was maintained at about 24 °C. The animals were randomly
divided into diferent groups. Each animal was used only
once. Rats were habituated to their new environment and
handled for 3 days before the experimental procedure was
started. 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 Com-
mittee of Tehran University of Medical Sciences.
Procedure
The single training session consisted of four trials with dif-
ferent 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 fnd the
hidden platform located in the target quadrant of the maze.
Each of four starting positions was used once in four train-
ing sessions; their order was randomized. During each trial,
each rat was given 90 s to fnd the hidden platform. If the rats
could not fnd the platform during this procedure, they were
manually guided to the platform. After fnding 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 four
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 s. After the probe trial,
the platform was elevated above the water surface and placed
in the diferent position and the rats were allowed to swim
freely for 120 s in order to test their visual ability.
Drug
The i.p. 4 mg/kg dose of scopolamine hydrobromide (Sigma-
Aldrich, Steinheim, Germany) was used to induce the AD.
The i.p. 2.5 mg/kg dose of donepezil hydrochloride (Sigma-
Aldrich) was used to treat the AD as the positive control
group. The 5% dimethyl sulfoxide, DMSO was used as
vehicle.
Behavioral test: Morris water maze (MWM)
Apparatus
Efects of i.p. infusion of 5, 10, 20, and 35 mg/kg doses
of compound 15g on spatial memory (time spent on target
quadrant, scape latency, travelled distance, swimming speed)
were investigated through this method [28].
Briefy, the Morris water maze consisted of a dark circular
pool (136 cm in diameter and 55 cm high) flled 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 northeast (NE),
southeast (SE), southwest (SW) or northwest (NW) quad-
rants. The platform provided the only escape from the water
and was fxed in its position. Many extra-maze cues were
embedded where the water maze was performed such as
racks, windows, door, bookshelves, wall color and pictures
on the walls surrounded the room. By these fxed cues, the
rat could locate the platform to escape from the water. The
movement of the animal was monitored by a camera that was
mounted above the pool. Animal displacement and its time
was recorded using a 3CCD camera (Panasonic Inc., Japan)
placed above the MWM pool and locomotion was tracking
and measured by etho-vision software (Noldus Information
Technology, the Netherlands).
Acknowledgements The authors thank the equipment supports from
Department of Medicinal Chemistry, Shahid Sadoughi University of
Medical Sciences, Yazd, Iran, and Department of Cellular Biotech-
nology at Cell Science Research Center, Royan Institute for Biotech-
nology, ACECR, Isfahan, Iran. This work was supported by Grants
(9211302001) from the Research Council of Tehran University of
Medical Sciences.
Compliance with ethical standards
Conflict of interest The authors declare no confict of interest, fnan-
cial or otherwise.
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Afliations
Roshanak Ghobadian1 · Roghaieh Esfandyari1 · Hamid Nadri2 · Alireza Moradi2 · Mohammad Mahdavi3 ·
Tahmineh Akbarzadeh1,4 · Hossein Khaleghzadeh‑Ahangar5 · Najmeh Edraki6 · Mohammad Sharifzadeh7 ·
Mohsen Amini1
1
4
5
6
7
Department of Medicinal Chemistry, Faculty of Pharmacy,
and Drug Design & Development Research Center, The
Institute of Pharmaceutical Sciences (TIPS), Tehran
University of Medical Sciences, Tehran, Iran
Persian Medicine and Pharmacy Research Center, Tehran
University of Medical Sciences, Tehran, Iran
Department of Physiology, School of Medicine, Babol
University of Medical Sciences, Babol, Iran
2
3
Pharmaceutical Science Research Center and Faculty
of Pharmacy, Shahid Sadoughi University of Medical
Sciences, Yazd 8915173143, Iran
Medicinal and Natural Products Chemistry Research Center,
Shiraz University of Medical Sciences, Shiraz, Iran
Department of Pharmacology and Toxicology, Faculty
of Pharmacy, Tehran University of Medical Sciences, Tehran,
Iran
Endocrinology and Metabolism Research Center,
Endocrinology and Metabolism Clinical Sciences Institute,
Tehran University of Medical Sciences, Tehran, Iran
1 3