Thieno[2,3-b]pyridine amines: Synthesis and evaluation of tacrine analogs against biological activities related to Alzheimer's disease

Article abstract of DOI:10.1002/ardp.202000101

In search of safer tacrine analogs, various thieno[2,3-b]pyridine amine derivatives were synthesized and evaluated for their inhibitory activity against cholinesterases (ChEs). Among the synthesized compounds, compounds 5e and 5d showed the highest activity towards acetylcholinesterase and butyrylcholinesterase, with IC₅₀ values of 1.55 and 0.23 μM, respectively. The most active ChE inhibitors (5e and 5d) were also candidates for further complementary assays, such as kinetic and molecular docking studies as well as studies on inhibitory activity towards amyloid-beta?(βA) aggregation and β-secretase 1, neuroprotectivity, and cytotoxicity against HepG2 cells. Our results indicated efficient anti-Alzheimer's activity of the synthesized compounds.

Full text of DOI:10.1002/ardp.202000101

Received: 1 April 2020
Revised: 10 June 2020
Accepted: 15 June 2020
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|
DOI: 10.1002/ardp.202000101
F U L L P A P E R
Thieno[2,3‐b]pyridine amines: Synthesis and evaluation of
tacrine analogs against biological activities related to
Alzheimer's disease
1
,2
3
4
2
Mina Saeedi
| Maliheh Safavi | Emad Allahabadi | Arezoo Rastegari
|
4
3
5
6
Roshanak Hariri | Sanaz Jafari | Syed N. A. Bukhari | Seyedeh S. Mirfazli
|
7
7
8
Omidreza Firuzi | Najmeh Edraki | Mohammad Mahdavi
|
4
,2
Tahmineh Akbarzadeh
1
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran
Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Department of Pharmaceutical Chemistry, College of Pharmacy, Aljouf University, Aljouf, Saudi Arabia
Department of Medicinal Chemistry, Iran University of Medical Sciences, Tehran, Iran
2
3
4
5
6
7
8
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
Correspondence
Tahmineh Akbarzadeh, Department of
Medicinal Chemistry, Faculty of Pharmacy,
Tehran University of Medical Sciences, Tehran
Abstract
In search of safer tacrine analogs, various thieno[2,3‐b]pyridine amine derivatives
were synthesized and evaluated for their inhibitory activity against cholinesterases
14117, Iran.
Email: abarzad@tums.ac.ir
(
ChEs). Among the synthesized compounds, compounds 5e and 5d showed the
Funding information
highest activity towards acetylcholinesterase and butyrylcholinesterase, with IC50
values of 1.55 and 0.23 µM, respectively. The most active ChE inhibitors (5e and 5d)
were also candidates for further complementary assays, such as kinetic and mole-
cular docking studies as well as studies on inhibitory activity towards amyloid‐
beta (βA) aggregation and β‐secretase 1, neuroprotectivity, and cytotoxicity against
HepG2 cells. Our results indicated efficient anti‐Alzheimer's activity of the syn-
thesized compounds.
Research Council of Tehran University of
Medical Sciences, Grant/Award Number: 95‐
01‐33‐31760
K E Y W O R D S
Alzheimer's disease, amyloid beta (βA), BACE1, cytotoxicity, thieno[2,3‐b]pyridine
1
| INTRODUCTION
dysfunction, psychiatric and behavioral disturbances, and serious diffi-
[1]
culties in performing daily activities. The exact underlying cause of AD
is obscure, and various factors are believed to play important roles in
the onset and progression of the disease. In this respect, reduced
Alzheimer's disease (AD) is a progressive neurodegenerative disorder
that accounts for about 70% of all dementia cases. It occurs as people
get older, and the main symptoms of the disease include cognitive
[2]
synaptic levels of acetylcholine (ACh), abnormal deposits of amyloid
This paper is dedicated to the memory of our unique teacher in Chemistry and Medicinal Chemistry, Professor Abbas Shafiee (1937–2016).
Arch Pharm. 2020;e2000101
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000101

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SAEEDI ET AL.
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[
3]
[4]
β plaques, the presence of neurofibrillary tangles, and dyshomeos-
lipophilicity and polarity affects multiple interactions with com-
plementary sites of ChEs and suppresses toxicity profile, compared with
tacrine.
2+
2+
2+
tasis and miscompartmentalization of metal ions (Fe , Cu , and Zn
)
[5]
in the brain have been comprehensively discussed in the literature.
Currently, there is no definite cure for the treatment of AD due to the
complicacy of AD pathology, and available treatments are merely pal-
liative. Hence, the design and synthesis of multifactorial compounds,
considering various mechanisms involved in AD, have occupied a special
2.2 | Chemistry
[
6]
position in drug discovery endeavors.
Synthesis of the target compound 5 is demonstrated in Scheme 1.
Initially, an appropriate substrate 4 possessing β‐amino nitrile moiety
was prepared by the condensation of various cycloalkanones (1),
malononitrile (2), and sulfur (S8, 3) in the presence of L‐proline in
Currently available FDA‐approved anti‐AD drugs, including done-
pezil, rivastigmine, and tacrine, profit from the ability of the inhibition of
acetylcholinesterase (AChE) and increasing the level of ACh in the brain.
Therefore, the cholinergic hypothesis of AD is still important
dimethylformamide (DMF) at 60°C for 24 hr through the Gewald
[20]
[7]
in drug discovery developments. 1,2,3,4‐Tetrahydroacridin‐9‐amine,
known as tacrine (Figure 1), was the first biologically important com-
pound for the treatment of AD, which was removed from the market due
to serious clinical shortcomings instigating hepatotoxicity probably by
mediating the stimulation of reactive oxygen species production and
reaction.
It should be mentioned that the reaction was examined
in the presence of other reagents, such as morpholine and triethy-
lamine, using different solvents, including dimethyl sulfoxide and
acetonitrile, under various conditions. However, using L‐proline in
DMF at 60°C afforded a higher yield of the reaction. Lewis acid‐
catalyzed Friedländer condensation reaction of compound 4 and
reactive methylene derivative 1 led to the formation of pyridine ring
fused with the thiophene moiety. For this purpose, according to our
[8]
glutathione depletion.
Focusing on the striking effect of tacrine on cognitive defects, a
wide range of research has been devoted to modify the structure of
tacrine to decrease toxicity and involvement of other approaches in
[
16]
previous experience reported in the literature,
reaction of com-
in 1,2‐
[
9]
the treatment of AD. In this respect, various tacrine analogs such
pound 4 and cycloalkanone 1 in the presence of AlCl
3
[
10]
as tacrine–tryptophan (Figure 2a),
tacrine–8‐hydroxyquinoline
dichloroethane under reflux conditions for 24 hr gave the desired
[
11]
[12]
(Figure 2b),
tacrine–chromene (Figure 2c,d),
and tacrine–1,2,
product 5.
[
13–15]
3
‐triazole–coumarin (Figure 2e–f)
hybrids were found to be
versatile compounds against AD through various mechanisms, along
with inhibitory activity against ChEs. Herein, in continuation of our
2.3 | Biology
[13–17]
research program on the development of tacrine,
we planned
to synthesize some tacrine analogs possessing different activities
against AD.
2.3.1 | Anticholinesterase (AChE) activity
Anti‐AChE and anti‐butyrylcholinesterase (BChE) activities of syn-
thesized compound 5 were evaluated according to the modified Ell-
[
21]
2
| RESULTS AND DISCUSSION
.1 | Compound design
man's method,
comparing with tacrine as the reference drug. As
discussed, different modifications were achieved to afford multi-
target anti‐AD compounds with lower hepatotoxicity. In this respect,
four different classes of tacrine hybrids were prepared (5a–c, 5d–f,
5g–i, and 5j/5k) according to the ring size of cycloalkyl and cy-
cloalkylthiophene moieties inserted into the target compounds.
As reported in Table 1, compound 5e was the most active
AChE inhibitor (IC50 = 1.55 µM). It possessed cyclohexyl moiety
connected to both thiophene and pyridine moieties m = 1, n = 1).
Keeping cyclohexylthiophene moiety intact (n = 1) and changing
the ring size of cycloalkyl moiety connected to the pyridine moiety
(m = 0 and 2) led to a lower inhibitory activity against AChE as
compounds 5d and 5f showed IC50 values = 3.47 and 22.52 µM,
respectively (entries 4–6). Changing the ring size of cycloalk-
ylthiophene moiety (n = 0, 2) resulted in a different activity (entries
2
Design of target compounds (Figure 3) is related to the potent ChE
inhibitory activity of the tacrine scaffold. Herein, we tried to keep the
main moiety intact, whereas the aromatic moiety was replaced
by the cycloalkylthiophene moiety, as thiophene core had shown a
[
18]
significant BACE1 inhibitory activity (compound h, Figure 3)
and
[19]
amyloid‐beta aggregation inhibition (compound i, Figure 3)
to afford
multitarget anti‐AD agents. The cyclohexane moiety of tacrine was also
replaced by cyclopentane and cycloheptane moieties; however, all
alterations were considered to investigate how a change in the
1
–3 and 7–9). In both series, compounds with m = 1 showed higher
activity. Compound 5b compared with 5a and 5c as well as com-
pound 5h compared with 5g and 5i showed the following order:
5
b > 5c > 5a and 5 h > 5 g > 5i. It should be noted that in the series
–3, the lowest activity was related to compound 5a (m = 0, n = 0);
1
FIGURE 1 The structure of tacrine
however, the lowest activity in the series 7–9 was observed for

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FIGURE 2 The structures of tacrine hybrids and the designed compounds
compound 5i (m = 2, n = 2). It seems that the replacement of both
aromatic ring and cyclohexyl moieties of tacrine by smaller or
larger size rings together led to a weak inhibitory activity. Ap-
parently, both steric hindrance and reduction of lipophilicity could
deteriorate the AChEI activity.
can be confirmed by the selectivity index calculated in Table 1. There
was an exception related to the most active AChE inhibitor 5e, which
possessed a lower anti‐BChE activity.
The best BChEI activity was obtained by compound 5d having cy-
clopentyl moiety connected to the pyridine ring (m = 0) and cyclohex-
ylthiophene (n = 1) with IC50 = 0.23 µM. The increase in the size of
cycloalkyl moiety (m = 1 and 2) led to the reduction of activity, and
compounds 5e and 5f showed an anti‐BChE activity with IC50 va-
lues = 2.33 and 3.08 µM, respectively. In the series of 5a–c (n = 0 and
m = 0, 1, and 2), a weaker activity was obtained as compared with other
compounds, IC50 values = 8.94, 3.09, 7.81 µM, respectively; however,
the order of BChEI activity, 5b > 5c > 5a, indicated that the cyclohexyl
moiety connected to the pyridine ring is effective. In the third category,
5g–i, the same results were observed and the order of BChEI activity
was 5h > 5i > 5g (IC50s = 1.63, 1.81, and 2.07 µM, respectively), which
highlighted the efficacy of cyclohexyl moiety (m = 1). Our results in the
Another bioisosteric replacement was performed using benzyl-
piperidine moiety (n = 1, X = N) connected to the thiophene moiety
(entries 10 and 11). Compound 5j possessing cyclohexyl moiety
connected to the pyridine ring (m = 1) was found to be five‐fold more
potent than compound 5k having cycloheptyl moiety (m = 2). Our
result revealed that a good AChEI activity of tacrine analogs 5 was
usually achieved by the compounds with m = 1 (compounds 5b, 5e,
5h, and 5j), which mostly mimic the tacrine scaffold from both
binding interactions in the active site and lipophilicity points of view.
In the case of BChEI activity of compound 5 derivatives, most
compounds showed much better activity than AChEI activity. This

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FIGURE 3 The design of the target compounds
case of compounds 5j and 5k, which were provided by the connection of
BChE, respectively. Graphical analysis of the reciprocal
Lineweaver–Burk plot of compound 5d described a mixed‐type
inhibition pattern (Figure 4a), indicating that 5e may bind to
AChE, even if it is already bound to the substrate. In addition, the
benzylpiperidine moiety to the thiophene ring, showed that compound
5k possessing cycloheptyl moiety showed a more potent activity
(IC50 = 2.25 µM) than compound 5j having cyclopentyl moiety (IC50
=
3.74 µM). However, there was no definite rule for the effect of cy-
K
i
value was calculated using the secondary plot as 2.13 µM
cloalkyl moieties on the BChEI activity.
(Figure 4b). The kinetic study of compound 5d toward BChE de-
Comparing AChEI and BChEI activity of synthesized compound 5
derivatives showed that cycloalkyl substituents apparently played a more
important role in AChE inhibition as mostly compounds possessing
cyclohexyl moiety (m = 1) depicted a better inhibitory activity. However,
benzothienopyridine moiety was an important scaffold for the desired
interactions with BChE amino acid residues, and definite
structure–activity relationships (SARs) could not be established for the
anti‐BChE activity.
i
picted the same results (Figure 5a). Also, the K value was cal-
culated as 0.55 µM (Figure 5b).
2.3.3 | BACE1 enzymatic assay
β‐Secretase (BACE1) inhibitors have emerged as important agents in
the treatment of AD due to the role of BACE1 in the proteolytic
[
22]
cleavage of the amyloid protein precursor (APP).
Accordingly, the
BACE1 inhibitory activities of the most potent ChE inhibitors, com-
pounds 5d and 5e, were examined via the fluorescence resonance
emission transfer (FRET) method. The used kit consisted of BACE1
enzyme and APP peptide‐based substrate (Rh‐EVNLDAEFK‐
quencher), compared with OM99‐2 (IC50 = 0.014 μM) as the
2
.3.2 | Kinetic study
The kinetic studies were performed to determine the mechanism
of inhibition by compounds 5e and 5d against AChE and
SCHEME 1 The synthesis of the thieno[2,3‐b]pyridine amine derivative 5

SAEEDI ET AL.
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a
TABLE 1 The anticholinesterase activity (IC50, μM) of compound 5
b
Entry
Compound 5
5a
m
n
X
R
AChEI (IC50 [μM])
BChEI (IC50 [μM])
SI
1
0
0
C
H
28.57 ± 0.04
8.94 ± 0.17
3.2
1.6
2
3
4
5
6
7
8
9
5b
5c
1
2
0
1
2
0
1
2
1
2
0
0
1
1
1
2
2
2
1
1
C
C
C
C
C
C
C
C
N
N
H
4.85 ± 0.20
18.92 ± 0.77
3.47 ± 0.20
1.55 ± 0.04
22.52 ± 0.22
17.73 ± 0.31
2.01 ± 0.07
30.71 ± 0.73
5.50 ± 0.03
30.44 ± 0.49
0.151 ± 0.0035
3.09 ± 0.03
7.81 ± 0.19
0.23 ± 0.01
2.33 ± 0.25
3.08 ± 0.33
2.07 ± 0.62
1.63 ± 0.05
1.81 ± 0.01
3.74 ± 0.23
2.25 ± 0.55
0.005 ± 0.0003
H
2.4
5d
5e
H
15.1
0.7
H
5f
H
7.3
5g
H
8.6
5h
5i
H
1.2
H
17.0
1.5
10
11
12
5j
Benzyl
Benzyl
5k
13.5
Tacrine
Abbreviation: AChE, acetylcholinesterase; BChE, butyrylcholinesterase.
aData are represented in terms of mean ± standard deviation.
bThe selectivity index (SI) is calculated as the ratio of IC50 AChE/IC50 BChE.
reference drug. It was found that these compounds induced a weak
cells up to 50 μM. It can be understood that bioisosterical
replacement of cycloalkyl and cycloalkylthiophene induced no
desired neuroprotectivity.
inhibitory activity (8–10% inhibition) at the concentrations of 50 µM.
2
.3.4 | Neuroprotective effect against Aβ‐induced
damage measured in PC12 cells
2.3.5 | Inhibition of AChE‐induced and self‐induced
Aβ aggregation
Compounds 5d and 5e were selected to study the neuroprotec-
tive ability using PC12 cell injury induced by Aβ25–35 by
MTT assay, compared with rutin as the reference drug. They
demonstrated negligible neuroprotectivity on Aβ‐induced PC12
Aβ peptide is the major constituent of senile plaques in the brains
[
3]
of patients with AD.
In this respect, the effect of the most
potent anti‐BChE compound 5d was assessed for the inhibition
(
a)
(b)
i
FIGURE 4 (a) Kinetic study of anticholinesterase inhibition by compound 5e. (b) Inhibition constant (K ) of compound 5e

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(
a)
(b)
i
FIGURE 5 (a) Kinetic study of butyrylcholinesterase inhibition by compound 5d. (b) Inhibition constant (K ) of compound 5d
2
.3.6 | Cytotoxic activity
TABLE 2 Inhibitory activities of compound 5d against Aβ1–42
aggregation
The clinical use of tacrine was restricted due to its poor oral bioavail-
a
[23]
ability and significant side effects mainly related to its hepatotoxicity.
In this regard, the design of new nonhepatotoxic analogs is receiving
great attention. Compounds 5d and 5e were selected to be evaluated in
vitro toward HepG2 (Figure 6). It was found that the cytotoxicity of those
compounds was comparable with that of tacrine. Compound 5d de-
monstrated lower toxicity, as cell viability values were calculated as 100,
%
Inhibition
Self‐induced Aβ1–42
aggregation
AChE‐induced Aβ
aggregation
Compound
b
c
5
d
29.5 ± 1.2
36.6 ± 1.4
Tacrine
7.9 ± 0.5
7.0 ± 1.4
100, 100, and 90.2% at concentrations of 2.5, 5, 10, and 20 µg/ml, re-
Donepezil
15.9 ± 1.2 (10 µM)
28.4 ± 3.6 (100 µM)
spectively. Compound 5e showed cell viability (%) of 100, 100, 97.6, and
65.5% at the same concentrations. It is worth mentioning that tacrine
showed higher cytotoxicity against HepG2 than compounds 5d and 5e, as
cell viability values (%) were obtained as 100, 100, 89.4, and 65.8% at
concentrations of 2.5, 5, 10, and 20 µg/ml, respectively.
aValues are expressed as mean ± standard error of the means of three
experiments.
bThe inhibition of self‐induced Aβ1–42 aggregation (25 mM) produced by
the tested compound at 10 µM concentration.
cThe co‐aggregation inhibition of Aβ1–40 and AChE (2 µM; ratio, 100:1) by
the tested compound at 100 µM concentration.
2
.4 | Molecular docking
against Aβ1–42 aggregations and AChE‐induced Aβ1–40 peptide
aggregation using the thioflavin T assay, compared with done-
pezil and tacrine as the reference compounds. Compound 5d was
more potent than both controls against Aβ1–42 self‐aggregation,
as it depicted 29.5% inhibition at 10 µM (Table 2). Furthermore,
compound 5d inhibited AChE‐induced Aβ aggregation by 36.6%
at 100 μM.
The best binding modes for both AChE and BChE were ranked
according to the minimized affinity values (Table 3).
The proposed binding mode of compound 5e is illustrated in
Figure 7. The interactions were dominated in the region of Phe331,
Phe330, and Trp84 amino acid residues due to pronounced existence of
the π–alkyl interaction at the anionic site and the π–alkyl interaction with
Tyr334 at the edge of the peripheral site region.
FIGURE 6 Cytotoxicity of compounds 5d and
5e against HepG2 cells

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TABLE 3 Docking results based on hydrogen bonding, hydrophobic interaction, binding free energy (ΔG
b
) of 5d and 5e
Bonds between atoms of compounds and amino acids
a
̊
Compound
5d
ΔG
b
(kcal/mol)
Atom of the ligand
NH
Binding mode
Amino acid
Distance (A)
−9.37 to −7.92
2
H‐bond
His438
Trp82
Trp82
Trp82
Trp82
Trp430
Ala328
2.05
Thienopyridine ring
Four π–π stacking
3.81 and 4.92
3.86 and 4.57
4.44 and 4.94
5.15
Two π–alkyl interactions
π–alkyl
Cyclopentane moiety
Two π–alkyl interactions
Alkyl (hydrophobic interaction)
5.17 and 4.90
3.71
5
e
−9.78 to −9.11
Cyclohexane moiety
Cyclopentane moiety
Two π–alkyl interactions
π–alkyl interactions
Trp84
4.88 and 4.68
4.74
Phe331
Tyr334
Phe330
4.41
4.66
aBinding free energy.
FIGURE 7 Two‐ and three‐dimensional representation of the docked pose of compound 5e at the 1EVE active site
FIGURE 8 Two‐ and three‐dimensional representation of the docked pose of compound 5d at the 1P0P active site
As observed in Figure 8, compound 5d was able to form a
hydrogen bond with His438 in the catalytic site through the NH
group. Benzothienopyridine moiety was trapped in the anionic site
These results were in good agreement with those obtained ex-
perimentally, indicating the efficacy of compounds 5e and 5d. It
seems that benzothienopyridine moiety is more significant for de-
sired interactions with BChE amino acid residues, whereas it is not
2
[
24]
through π–π stacking and π–alkyl interactions with Trp82.

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significant for those of AChE. In this respect, the AChEI activity was
more dependent on cycloalkyl substituents than the BChEI activity.
was added to the mixture and it was basicified with NaOH (10%). The
mixture was stirred at room temperature for 30 min, and the crude
product was extracted using CH
2
Cl
2
(2 × 50 ml) and washed with
SO and the
brine (2 × 50 ml). The organic phase was dried over Na
2
4
3
| CONCLUSION
solvent was evaporated under vacuum. All compounds were re-
crystallized from EtOH to obtain pure product 5.
In conclusion, various thieno[2,3‐b]pyridine amines were synthesized as
tacrine analogs and evaluated for their anti‐ChE activity. They showed a
good inhibitory activity; however, the inhibitory activity toward BChE
was mostly more significant. It should be mentioned that only compound
1,2,3,6,7,8‐Hexahydrocyclopenta[e]cyclopenta[4,5]thieno[2,3‐
b]pyridin‐9‐amine (5a)
Cream solid, yield: 85%, m.p.: >280°C. IR (KBr): 3,474, 3,293, 2,953,
−
1 1
5
e showed a better AChEI activity (IC50 = 1.55 µM) than the BChEI ac-
2,897, 2,843, 1,641, and 1,575 cm
δ = 4.29 (s, 2H, NH ), 3.07 (t, J = 6.5 Hz, 2H, CH
2 × CH ), 2.76 (t, J = 7.0 Hz, 2H, CH ), 2.49 (t, J = 7.0 Hz, 2H, CH
2.19–2.16 (m, 2H, CH ) ppm. C NMR (CDCl , 125 MHz): δ = 163.1,
159.2, 143.6, 137.7, 133.4, 115.5, 110.9, 34.3, 29.7, 29.6, 27.7, 26.7,
.
H NMR (CDCl
), 3.03–2.95 (m, 4H,
), and
3
, 500 MHz):
tivity (IC50 = 2.33 µM). Also, comprehensive investigations indicated that
the most potent AChE and BChE inhibitors (compounds 5e and 5d)
showed a good activity against AChE‐induced and self‐induced Aβ ag-
gregation, whereas they exhibited a negligible BACE1 inhibitory activity
and neuroprotectivity against Aβ‐induced damage in PC12 cells. It is
worth mentioning that these compounds showed a lower toxicity than
tacrine against the HepG2 cell line.
2
2
2
2
2
1
3
2
3
+
+
and 22.9 ppm. MS: m/z (%) = 231 [M+1] (100), 230 [M] (82), 216
(35), 198 (11), 115 (12), 89 (14), 71 (17), and 45 (31). Anal. calcd. for
13 14 2
C H N S: C, 67.79; H, 6.13; N, 12.16. Found: C, 67.58; H, 6.31;
N, 12.38.
4
4
4
| EXPERIMENTAL
2,3,6,7,8,9‐Hexahydro‐1H‐cyclopenta[4,5]thieno[2,3‐b]quinolin‐10‐
amine (5b)
.1 | Chemistry
Cream solid, yield 80%, m.p.: >280°C. IR (KBr): 3,459, 3,326, 2,930,
−
1 1
2
,849, 1,619, 1,570, and 1,524 cm
.
H NMR (CDCl
), 3.07 (t, J = 7.0 Hz, 2H, CH ), 2.96 (t, J = 7.0 Hz,
), and 2.5–2.49 (m, 4H, 2 × CH ),
, 125 MHz):
3
, 500 MHz):
.1.1 | General
δ = 4.35 (s, 2H, NH
2
2
2H, CH
2
), 2.91 (t, J = 6.0 Hz, 2H, CH
2
2
1
3
Melting points were taken on a Kofler hot‐stage apparatus and were
and 1.90–1.86 (m, 4H, 2 × CH
2
) ppm. C NMR (CDCl
3
1
13
uncorrected. H‐ and C‐nuclear magnetic resonance (NMR) spectra
were recorded on Bruker FT‐500, using tetramethylsilane as an in-
ternal standard. The infrared (IR) spectra were obtained on a Nicolet
Magna FTIR 550 spectrophotometer (in KBr). Mass spectra were
determined on an Agilent Technology (HP) mass spectrometer op-
erating at an ionization potential of 70 eV. The elemental analysis
was performed with an Elementar Analysensystem GmbH Vario EL
CHNS mode. All reagents and solvents were obtained from Merck
and Aldrich and used without purification.
δ = 162.5, 154.1, 146.2, 137.3, 134.9, 118.1, 111.0, 33.1, 29.7, 29.6,
+
27.7, 23.0, 22.9, and 22.8 ppm. MS: m/z (%) = 245 [M+1] (100), 244
+
[M] (79), 230 (29), 202 (10), 89 (11), and 45 (27). Anal. calcd. for
14 16 2
C H N S: C, 68.82; H, 6.60; N, 11.46. Found: C, 68.62; H, 6.42;
N, 11.27.
1,2,3,6,7,8,9,10‐Octahydrocyclohepta[e]cyclopenta[4,5]thieno[2,3‐
b]pyridin‐11‐amine (5c)
Cream solid, yield 85%, m.p.: >280°C. IR (KBr): 3,481, 3,287,
−
1
1
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
2,959, 2,841, 1,646, 1,568, and 1,522 cm
500 MHz): δ = 4.39 (s, 2H, NH ), 3.09 (t, J = 6.5 Hz, 2H, CH
t, J = 5.0 Hz, 2H, CH ), 2.97 (t, J = 7.0 Hz, 2H, CH ), 2.67–2.65 (m,
H, CH ), 2.51–2.48 (m, 2H, CH ), 1.86–1.85 (m, 2H, CH ),
1.72–1.71 (m, 2H, CH ) ppm. C NMR
.
H NMR (CDCl
3
,
2
2
), 3.02
(
2
2
2
2
2
2
1
3
4
.1.2 | General procedure for the synthesis of
2
), and 1.64 (m, 2H, CH
2
compounds 5a–k
(dimethyl sulfoxide [DMSO]‐d
6
, 125 MHz): δ = 159.3, 157.9,
1
47.8, 136.4, 136.1, 116.4, 116.0, 37.8, 32.0, 29.6, 29.5, 27.8,
+
First, a mixture of cycloalkanone (1) (1 mmol), malononitrile (2)
27.4, 26.7, and 24.8 ppm. MS: m/z (%) = 259 [M+1] (100), 258
+
(1.1 mmol), sulfur (S8, 3) (1.5 mmol), and L‐proline (0.1 mmol) in DMF
(10 ml) was stirred at 60°C for 24 hr. After completion of the reaction
(checked by thin layer chromatography [TLC]), the mixture was
[M] (96), 230 (84), 205 (47), 190 (17), 115 (14), 89 (17), and 45
18 2
(38). Anal. calcd. for C15H N S: C, 69.73; H, 7.02; N, 10.84.
Found: C, 69.57; H, 6.85; N, 10.61.
poured into water and the precipitated product was filtered off and
recrystallized from ethyl acetate to afford pure compound 4. Then, a
mixture of compound 4 (1 mmol) and cycloalkanone (1) (1.5 mmol)
1,2,3,6,7,8,9‐Heptahydrocyclopenta[e]cyclohexa[4,5]thieno[2,3‐
b]pyridin‐10‐amine (5d)
was added to the suspension of AlCl
2 mmol in 30 ml) and heated at reflux for 24 hr. After completion of
the reaction (checked by TLC), a mixture of H O/THF (1:1, 100 ml)
3
in dry 1,2‐dichloroethane
Cream solid, yield 70%, m.p.: 246–248°C. IR (KBr): 3,491, 3,393,
−1 1
(
2,925, 2,834, 1,644, 1,615, 1,567, and 1,532 cm
3
. H NMR (CDCl ,
2
500 MHz): δ = 4.43 (s, 2H, NH ), 3.03–3.00 (m, 4H, 2 × CH ),
2
2

SAEEDI ET AL.
9 of 11
|
2
.80–2.73 (m, 4H, 2 × CH
2
), 2.21–2.15 (quint, J = 7.5 Hz, 2H, CH
2
),
30.6, 30.0, 29.0, 27.2, 26.5, 23.1, 22.9, and 22.8 ppm. MS: m/z
1
3
+
+
and 1.90–1.88 (m, 4H, 2 × CH
2
) ppm. C NMR (CDCl
3
, 125 MHz):
(%) = 273 [M+1] (100), 272 [M] (49), 244 (94), 218 (47), 202 (17),
115 (13), 77 (14), and 41 (32). Anal. calcd. for C16 S: C, 70.55; H,
7.40; N, 10.28. Found: C, 70.68; H, 7.55; N, 10.41.
δ = 162.7, 160.8, 144.8, 131.7, 125.8, 118.1, 116.1, 34.2, 26.8, 26.6,
20 2
H N
+
2
6
1
5.6, 25.7, 22.8, and 22.6 ppm. MS: m/z (%) = 244 [M] (25), 216 (14),
16 2
6 (15), and 41 (16). Anal. calcd. for C14H N S: C, 68.82; H, 6.60; N,
1.46. Found: C, 68.66; H, 6.45; N, 11.28.
1,2,3,4,5,8,9,10,11,12‐Decahydrocyclohepta[e]cyclohepta[4,5]‐
thieno[2,3‐b]pyridin‐13‐amine (5i)
1
,2,3,4,7,8,9,10‐Octahydrobenzo[4,5]thieno[2,3‐b]quinolin‐11‐
Cream solid, yield 87%, m.p.: 247–249°C. IR (KBr): 3,481, 3,319, 2,916,
−
1 1
amine (5e)
2,846, 1,643, and 1,558 cm . H NMR (CDCl
NH ), 3.13 (t, J = 5.0 Hz, 2H, CH ), 3.01 (t, J = 5.0 Hz, 2H, CH
J = 5.0 Hz, 2H, CH ), 2.67 (t, J = 5.0 Hz, 2H, CH ), 1.90–1.78 (m, 8H,
4 × CH ), 1.71 (quint, J = 5.5 Hz, 2H, CH ), and 1.68 (quint, J = 5.5 Hz, 2H,
CH ) ppm. C NMR (CDCl , 125 MHz): δ = 159.8, 153.7, 147.8, 132.1,
130.0, 119.2, 107.9, 38.9, 32.1, 30.8, 29.9, 29.1, 27.2, 27.1, 26.7, 26.5, and
3
, 500 MHz): δ = 4.45 (s, 2H,
Cream solid, yield 76%, m.p.: 259–261°C. IR (KBr): 3,475, 3,368, 2,930,
2
2
2
), 2.86 (t,
−1 1
2
,847, 1,610, 1,566, and 1,531 cm . H NMR (CDCl
s, 2H, NH ), 3.00 (m, 2H, CH ), 2.94–2.92 (m, 2H, CH
CH ), 2.84–2.46 (m, 2H, CH ), and 1.90–1.87 (m, 8H, 4 × CH
NMR (CDCl , 125 MHz): δ = 159.5, 153.9, 146.7, 131.9, 125.7, 118.0,
10.9, 33.0, 26.7, 25.6, 23.0, 22.9, 22.8, 22.7, and 22.6 ppm. MS: m/z
3
, 500 MHz): δ = 4.68
2
2
(
2
2
2
), 2.78 (m, 2H,
2
2
13
13
2
2
2
) ppm.
C
2
3
3
+
+
1
25.4 ppm. MS: m/z (%) = 287 [M+1] (100), 286 [M] (52), 258 (88), 232
+
+
(
%) = 259 [M+1] (100), 258 [M] (65), 231 (82), 216 (23), 202 (15), 179
22 2
(33), 216 (21), 190 (11), and 41 (29). Anal. calcd. for C17H N S: C, 71.29;
(
11), 71 (14), and 45 (32). Anal. calcd. for C15 S: C, 69.73; H, 7.02; N,
18 2
H N
H, 7.74; N, 9.78. Found: C, 71.45; H, 7.91; N, 9.56.
10.84. Found: C, 69.56; H, 6.90; N, 10.71.
2
‐Benzyl‐1,2,3,4,6,7,8,9‐octahydropyrido[4′,3′:4,5]thieno[2,3‐
b]quinolin‐5‐amine (5j)
Cream solid, yield 78%, m.p.: >280°C. IR (KBr): 3,480, 3,225, 2,920, 2,845,
1
,2,3,4,7,8,9,10,11‐Nonahydrocyclohepta[e]cyclohexa[4,5]thieno‐
[
2,3‐b]pyridin‐12‐amine (5f)
−
1 1
Cream solid, yield 83%, m.p.: 273–275°C. IR (KBr): 3,492, 3,492,
1,660, 1,643, and 1,558 cm . H NMR (CDCl
(m, 5H, Ph), 4.56 (s, 2H, NH ), 3.74–3.69 (m, 4H, 2 × CH
CH ), 2.93–2.88 (m, 4H, 2 × CH ), 2.48–2.45 (m, 2H, CH ), and 1.89–1.85
(m, 4H, 2 × CH ) ppm. C NMR (CDCl , 125 MHz): δ = 158.1, 154.0,
3
, 500 MHz): δ = 7.39–7.27
−
1 1
3
5
2
2
,301, 2,914, 2,843, 1,644, 1,568, and1,527 cm
00 MHz): δ = 4.52 (s, 2H, NH ), 3.02–3.00 (m, 4H, 2 × CH
.80–2.78 (m, 2H, CH ), 2.66–2.64 (m, 2H, CH ), 1.91–1.84 (m, 4H,
× CH ), 1.72–1.70 (m, 2H, CH ), and 1.64–1.59 (m, 4H, 2 × CH
, 125 MHz): δ = 160.3, 157.4, 147.6, 130.2,
27.6, 118.5, 116.5, 38.7, 32.1, 27.7, 26.9, 26.5, 25.7, 24.8, 22.8, and
.
H NMR (CDCl
3
,
2
2
), 3.10 (s, 2H,
2
2
),
2
2
2
13
2
2
2
3
2
2
2
)
146.9, 137.8, 129.5, 129.1, 128.4, 127.4, 124.2, 117.5, 111.1, 61.9, 52.3,
13
+
ppm. C NMR (DMSO‐d
6
49.8, 32.8, 29.7, 26.8, 22.9, and 22.7 ppm. MS: m/z (%) = 350 [M+1] (25),
+
1
2
2
1
349 [M] (10), 259 (40), 231 (75), 216 (15), 91 (100), 65 (18), and 45 (14).
+
+
2.6 ppm. MS: m/z (%) = 273 [M+1] (100), 272 [M] (71), 244 (59),
16 (31), and 41 (23). Anal. calcd. for C16 S: C, 70.55; H, 7.40; N,
0.28. Found: C, 70.39; H, 7.28; N, 10.50.
23 3
Anal. calcd. for C21H N S: C, 72.17; H, 6.63; N, 12.02. Found: C, 72.38;
20 2
H N
H, 6.45; N, 11.85.
2‐Benzyl‐1,2,3,4,6,7,8,9,10‐nonahydrocyclohepta[e]pyrido‐
1
,2,3,6,7,8,9,10‐Octahydrocyclopenta[e]cyclohepta[4,5]thieno[2,3‐
[4′,3′:4,5]thieno[2,3‐b]pyridin‐5‐amine (5k)
b]pyridin‐11‐amine (5g)
Cream solid, yield 85%, m.p.: >280°C. IR (KBr): 3,482, 3,215, 2,920,
−
1
1
Cream solid, yield 81%, m.p.: 211–214°C. IR (KBr): 3,493, 3,300,
2,845, 1,660,1,645, and 1,555 cm
δ = 7.39–7.27 (m, 5H, Ph), 4.59 (s, 2H, NH
2 × CH ), 3.11 (s, 2H, CH ), 3.04–3.02 (m, 2H, CH
CH ), 2.64–2.63 (m, 2H, CH
and 1.63 (m, 2H, CH
.
H NMR (CDCl
), 3.74–3.70 (m, 4H,
), 2.90–2.88 (m, 2H,
), 1.70 (m, 2H, CH ),
2 3
) ppm. C NMR (CDCl , 125 MHz): δ = 159.1,
3
, 500 MHz):
−
1
1
2
,915, 2,843, 1,644, 1,567, and 1,525 cm
00 MHz): δ = 4.34 (s, 2H, NH ), 3.11–3.09 (m, 2H, CH
), 2.87–2.85 (m, 2H, CH
.19–2.16 (m, 2H, CH
.
H
NMR (CDCl
3
,
2
5
2
2
), 3.01 (t,
2
2
2
J = 7.5 Hz, 2H, CH
2
2
), 2.77–2.75 (m, 2H, CH
2
),
2
2
), 1.85 (m, 2H, CH
2
2
1
3
13
2
2
), and 1.89–1.69 (m, 6H, 3 × CH
2
) ppm.
C
NMR (DMSO‐d
6
, 125 MHz): δ = 161.8, 159.4, 146.4, 133.5, 132.6,
154.3, 146.2, 137.8, 130.1, 129.1, 128.4, 127.4, 124.5, 118.4, 116.7,
119.0, 116.7, 34.1, 31.0, 29.4, 29.1, 27.7, 27.4, 26.9, and 22.8 ppm.
61.8, 52.3, 49.8, 38.5, 32.1, 27.1, 26.9, 26.5, and 25.2 ppm. MS: m/z
+
+
+
+
MS: m/z (%) = 259 [M+1] (100), 258 [M] (37), 230 (90), 204 (49),
(%) = 364 [M+1] (42), 363 [M] (14), 273 (53), 245 (88), 216 (23), 91
(100), 65 (23), and 45 (17). Anal. calcd. for C22 S: C, 72.69; H,
6.93; N, 11.56. Found: C, 72.44; H, 6.70; N, 11.71.
1
68 (11), 115 (16), 57 (23), and 41 (40). Anal. calcd. for C15
18
H N
2
S: C,
25 3
H N
6
9.73; H, 7.02; N, 10.84. Found: C, 69.57; H, 6.84; N, 10.63.
2
,3,4,7,8,9,10,11‐Octahydro‐1H‐cyclohepta[4,5]thieno[2,3‐
b]quinolin‐12‐amine (5h)
Cream solid, yield 73%, m.p.: 230–233°C. IR (KBr): 3,498, 3,301,
4.2 | Pharmacological/biological assays
4.2.1 | Anticholinesterase assay
−
1
1
2
5
,916, 2,849, 1,624, 1,560, and 1,523 cm
.
H
NMR (CDCl
), 3.11–3.09 (m, 2H, CH ), 2.89 (t,
), 2.48 (t, J = 6.0 Hz,
3
,
00 MHz): δ = 4.45 (s, 2H, NH
2
2
J = 6.0 Hz, 2H, CH
2
), 2.85 (t, J = 5.0 Hz, 2H, CH
2
The in vitro anticholinesterase activity of the synthesized compound
1
3
2
H, CH
2
), and 1.90–1.78 (m, 10H, 5 × CH
2
) ppm. C NMR (CDCl
3
,
5 was assayed using modified Ellman's method, exactly according to
[
21]
125 MHz): δ = 158.7, 153.8, 146.4, 136.1, 130.6, 119.3, 111.3, 33.1,
our previous study.

10 of 11
SAEEDI ET AL.
|
4
.2.2 | Kinetic study
complex with the anti‐Alzheimer drug (1EVE) and butyrylcholinesterase
in complex with the substrate analog butyrylthiocholine (1P0P) were
fetched and prepared for docking. The accuracy of the docking software
was measured using the root mean squared deviations between re-
The kinetic study for the inhibition of AChE and BChE by compounds 5e
and 5d was carried out according to Ellman's method reported in our
[14]
[27]
previous work,
using four different concentrations of inhibitors. For
docked proteins and cocrystal ligands.
the kinetic study of AChE, compound 5e was used at the concentrations
of 0, 1.94, 3.87, and 7.75 µM. The Lineweaver–Burk reciprocal plot was
constructed by plotting 1/V against 1/[S] at variable concentrations of the
substrate acetylthiocholine (187.5, 750, 1,500, and 3,000 µM). The in-
ACKNOWLEDGMENTS
This study was supported by grants from the Research Council of
Tehran University of Medical Sciences with Project No. 95‐01‐33‐
31760.
i
hibition constant K was achieved by the plot of slopes versus the cor-
responding concentrations of the compound 5e. The same method was
performed for the kinetic study of BChE using four different con-
centrations of compound 5d (0, 0.12, 0.49, and 0.98 µM) and butyr-
ylthiocholine at concentrations of 187.5, 750, 1,500, 3,000 µM.
CONFLICT OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
Mohammad Mahdavi
Tahmineh Akbarzadeh
http://orcid.org/0000-0002-4171-7310
http://orcid.org/0000-0002-0212-7297
4
.2.3 | BACE1 inhibition
A FRET‐based BACE1 enzyme assay kit was used to evaluate the
inhibitory activity of the selected compounds toward BACE1. The kit
was purchased from Invitrogen (formerly Pan Vera Corporation,
Madison, WI) and the evaluation procedure was conducted according
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