Design, synthesis, and evaluation of novel cinnamic acid-tryptamine hybrid for inhibition of acetylcholinesterase and butyrylcholinesterase

Article abstract of DOI:10.1007/s40199-020-00346-9

Background: Acetylcholine deficiencies in hippocampus and cortex, aggregation of β-amyloid, and β-secretase over activity have been introduced as main reasons in pathogenesis of Alzheimer’s disease. Methods: Colorimetric Ellman’s method was used for determination of IC₅₀ value in AChE and BChE inhibitory activity. The kinetic studies, neuroprotective and β-secretase inhibitory activities, evaluation of inhibitory potency on β-amyloid (Aβ) aggregations induced by AChE, and docking study were performed for prediction of the mechanism of action. Result and discussion: A new series of cinnamic acids-tryptamine hybrid was designed, synthesized, and evaluated as dual cholinesterase inhibitors. These compounds demonstrated in-vitro inhibitory activities against acetyl cholinesterase (AChE) and butyryl cholinesterase (BChE). Among of these synthesized compounds, (E)-N-(2-(1H-indol-3-yl)ethyl)-3-(3,4-dimethoxyphenyl)acrylamide (5q) demonstrated the most potent AChE inhibitory activity (IC₅₀ = 11.51?μM) and (E)-N-(2-(1H-indol-3-yl)ethyl)-3-(2-chlorophenyl)acrylamide (5b) were the best anti-BChE (IC₅₀ = 1.95?μM) compounds. In addition, the molecular modeling and kinetic studies depicted 5q and 5b were mixed type inhibitor and bound with both the peripheral anionic site (PAS) and catalytic sites (CAS) of AChE and BChE. Moreover, compound 5q showed mild neuroprotective in PC12 cell line and weak β-secretase inhibitory activities. This compound also inhibited aggregation of β-amyloid (Aβ) in self-induced peptide aggregation test at concentration of 10?μM. Conclusion: It is worth noting that both the kinetic study and the molecular modeling of 5q and 5b depicted that these compounds simultaneously interacted with both the catalytic active site and the peripheral anionic site of AChE and BChE. These findings match with those resulted data from the enzyme inhibition assay. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s40199-020-00346-9

DARU Journal of Pharmaceutical Sciences
https://doi.org/10.1007/s40199-020-00346-9
RESEARCH ARTICLE
Design, synthesis, and evaluation of novel cinnamic acid-tryptamine
hybrid for inhibition of acetylcholinesterase
and butyrylcholinesterase
Shahrzad Ghafary1 & Roshanak Ghobadian1 & Mohammad Mahdavi2 & Hamid Nadri3 & Alireza Moradi3
&
Tahmineh Akbarzadeh1 & Zahra Najafi4 & Mohammad Sharifzadeh5 & Najmeh Edraki6
Farshad Homayouni Moghadam7 & Mohsen Amini1
&
Received: 18 April 2019 /Accepted: 3 April 2020
#
Springer Nature Switzerland AG 2020
Abstract
Background Acetylcholine deficiencies in hippocampus and cortex, aggregation of β-amyloid, and β-secretase over activity
have been introduced as main reasons in pathogenesis of Alzheimer’s disease.
Methods Colorimetric Ellman’s method was used for determination of IC50 value in AChE and BChE inhibitory activity. The
kinetic studies, neuroprotective and β-secretase inhibitory activities, evaluation of inhibitory potency on β-amyloid (Aβ)
aggregations induced by AChE, and docking study were performed for prediction of the mechanism of action.
Result and discussion A new series of cinnamic acids-tryptamine hybrid was designed, synthesized, and evaluated as dual
cholinesterase inhibitors. These compounds demonstrated in-vitro inhibitory activities against acetyl cholinesterase (AChE)
and butyryl cholinesterase (BChE). Among of these synthesized compounds, (E)-N-(2-(1H-indol-3-yl)ethyl)-3-(3,4-
dimethoxyphenyl)acrylamide (5q) demonstrated the most potent AChE inhibitory activity (IC50 = 11.51 μM) and (E)-
N-(2-(1H-indol-3-yl)ethyl)-3-(2-chlorophenyl)acrylamide (5b) were the best anti-BChE (IC50 = 1.95 μM) compounds. In addi-
tion, the molecular modeling and kinetic studies depicted 5q and 5b were mixed type inhibitor and bound with both the peripheral
anionic site (PAS) and catalytic sites (CAS) of AChE and BChE. Moreover, compound 5q showed mild neuroprotective in PC12
cell line and weak β-secretase inhibitory activities. This compound also inhibited aggregation of β-amyloid (Aβ) in self-induced
peptide aggregation test at concentration of 10 μM.
Conclusion It is worth noting that both the kinetic study and the molecular modeling of 5q and 5b depicted that these compounds
simultaneously interacted with both the catalytic active site and the peripheral anionic site of AChE and BChE. These findings
match with those resulted data from the enzyme inhibition assay.
.
.
.
.
Keywords Alzheimer’s disease Butyrylcholinesterase Docking study Cinnamic acid derivatives In-vitro assay
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s40199-020-00346-9) contains supplementary
material, which is available to authorized users.
3
* Mohsen Amini
Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid
Sadoughi University of Medical Sciences, Yazd, Iran
moamini@tums.ac.ir
4
5
6
7
Department of Medicinal Chemistry, School of Pharmacy, Hamedan
University of Medical Sciences, Hamedan, Iran
1
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
Department of Pharmacology and Toxicology, Faculty of Pharmacy,
Tehran University of Medical Sciences, Tehran, Iran
Medicinal and Natural Products Chemistry Research Center, Shiraz
University of Medical Science, Shiraz, Iran
2
Endocrinology and Metabolism Research Center, Endocrinology and
Metabolism Clinical Sciences Institute, Tehran University of Medical
Sciences, Tehran, Iran
Department of Cellular Biotechnology at Cell Science Research
Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

DARU J Pharm Sci
Introduction
Cinnamic acid derivatives has been recognized as a core
pharmacophor to develop for AD [10, 15]. There are several
reports that introduced different hybrid between cinnamic acid
derivative and other chemical groups including carbazole,
tacrin, rivasetigmine and tryptamine. A new series of coupled
cinnamic acid and donepezil has been introduced as ChE in-
hibitors (Fig. 1, B). Some of them showed low activity in
AChE inhibition and the more activity for inhibition of
BChE. On the other hands, tryptamine has been introduced
as a suitable scaffold for preparation of ChE inhibitors [3]. A
coumarin-amide conjugated tryptamine (Fig. 1, A) showed
inhibition of AChE and BChE with IC50 of 5.9 and 50.5 μM
respectively.
In the current work we prepared a new hybrid of cinnamic
acid-tryptamine in order to study their biological activity.
However cinnamic acid-tryptamine did not demonstrated
good activity for inhibition of AChE, our results showed that
this hybrid increased activity for inhibition of BChE in com-
parison to the coumarin-trypatime hybrid and could be con-
sidered for design more new active AChE and BChE
inhibitors.
Alzheimer’s disease (AD) is the most common form of pro-
gressive dementia in the elderly people, that is cause loss of
cognitive abilities such as memory, language skills, attention,
disorientation, and depression [1, 2]. The etiology of AD is
still not known and many factors such as reduced level of
cholinergic transmitters, accumulation of beta-amyloid pep-
tide (Aβ) plaque, oxidative stress, and hyper-
phosphorylation of microtubule-associated tau protein in-
volved in the progression of the disease [3]. The most com-
mon and important hypothesis about AD has been attributed
to the levels of acetylcholine [4, 5]. There is two important
types of catalytic enzyme, namely acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) which are able to
hydrolyzed cholinergic neurotransmitters. According to a re-
cent report on Alzheimer’s patient, AChE activity in a specific
region of the brain dramatically reduced, while BChE activity
increase to compensate for the lack of AChE. Based on the
important role of BChE in the hydrolysis of acetylcholine,
simultaneous inhibition of both enzymes can be effective to
the improvement of the symptoms in Alzheimer’s patients [6].
The X-ray crystallography studies have shown that AChE has
two binding sites containing the catalytic active site (CAS)
and the peripheral anionic site (PAS) [7, 8]. AChE inhibitors
act through the interaction with amino acid residues in the
CAS and PAS and consequently inhibition of this two site
increases the inhibitory activity.
Experimental
Chemistry
Melting points was taken on a Kofler hot stage apparatus and
are uncorrected. The 1H and 13C NMR spectra were recorded
on a Bruker FT-500, using TMS as an internal standard at 500
and 125 MHz respectively. The mass spectra were recorded on
an Agilent 5975C spectrometer. Elemental analysis was car-
ried out by a Perkin Elmer 2400 CHN Elemental Analyzer
using a tin capsule including 2–3 mg of sample. The analysis
was performed under excess oxygen exposure in the reactor
chamber, 990 °C and tungsten trioxide catalyst for complete
oxidation. Elemental analyses were within 0.4% of theoret-
ical values for C, H and N. IR spectra were obtained on a
Nicolet Magna FTIR 550 spectrophotometer (KBr disks).
All the commercially available reagents were purchased from
Merck or Sigma-Aldrich and used without further purifica-
tion. Electriceel (Torpedo California) 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 Donepezil hydrochloride
was obtained from Sigma-Aldrich (Steinheim, Germany).
Another most important cause of AD is the accumulation
of β-amyloid (Aβ) plaques in the brain. One of the ways to
prevent the accumulation of Aβ plaques is prevention the
hydrolysis of amyloid precursor protein (APP) by inhibition
of β-secretase (BACE1). There are also reports that show
accumulation of amyloid plaques and neurofibrillary tangles
in the brain increased by oxidative damage. Therefore,
preventing the formation of free radicals by antioxidant agents
can be effective for treating AD [9–11].
Even though researches were conducted to develop inhibi-
tors of AChE, there aren’t many drugs on the market due to the
adverse effects of therapeutic drugs [12]. In the recent years,
four AChE-inhibitors contains donepezil, tacrine, rivastigmine,
and galantamine have been approved by FDA, However, these
drugs only improve the symptoms in the short term, but not
treating. Tacrine was withdrawn from clinical usage due to
sever its hepatotoxicity by FDA at 2012. The efficacy and safe-
ty of donepezil encourages researches for the development of
donepezil-like compounds for the treatment of AD. In the liter-
ature, various compounds are presented in the article as inhib-
itors of acetyl cholinesterase such as carbamates, organophos-
phates, coumarin and cinnamic acids [11–14].
General procedure for the synthesis of compound 3
A solution of benzaldehyde derivatives 1 (5 mmol), malonic
acid 2 (5.5 mmol), in 1.25 mL of pyridine, 0.5 mL of piperi-
dine, and 5 mL of ethanol 95% was heated at 100 °C for 24 h.
After completion of the reaction, monitored by TLC, diluted
The aim of this study was to synthesize and investigate
cinnamic acids-tryptamine scaffold hybrids as anticholinester-
ase agents.

DARU J Pharm Sci
Fig. 1 Structure of ferulic acid,
cinnamic acid and coumarine-
triptamine based anti-AChE
agents as a lead compounds for a
newly designed AChE inhibitors
HCl solution (0.1 M) was added to the reaction mixture in an
ice bath. The resulting solid was filtered and dried. To obtain
pure product 3, the dried solid crystalized in ethanol.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(2-chlorophenyl)acrylamide
(5b)
Yield: 75%, mp: 140–142 °C. IR (KBr) cm−1 υ: 3444, 3295,
3053, 2921, 1650, 1603, 1547. 1HNMR (DMSO-d6,
500 MHz): δ = 2.91 (t, J = 7.5 Hz, 2H, CH2), 3.50 (q, J =
6.5 Hz, 2H, NH-CH2), 6.69 (d, J = 16.0 Hz, 1H, CHα), 6.98
(t, J = 6.5 Hz, 1H, H5), 7.07 (t, J = 7 Hz, 1H, H6), 7.17 (s, 1H,
H2), 7.34 (d, J = 7.5 Hz, 1H, H7), 7.38–7.40 (m, 2H, H3’, 5’),
7.52 (d, J = 5 Hz, 1H, H6’), 7.56 (d, J = 7.5 Hz, 1H, H4), 7.70
(t, J = 5.0 Hz, 1H, H4’), 7.74 (d, J = 16.0 Hz, 1H, CHβ), 8.28
(t, J = 5.5 Hz, 1H, NH-CH2), 10.78 (s, 1H, NH) ppm.
13CNMR (DMSO-d6, 125 MHz): δ = 25.1, 39.6, 111.2,
111.6, 118.1, 118.8, 120.8, 121.1, 122.5, 125.4, 127.1,
127.5, 129.8, 130.02, 130.6, 132.7, 133.1, 138.1,
164.3 ppm. Mass, m/z (%): 324(3), 165(5), 143(50),
130(36), 101(35), 86(100), 58(34). Anal. Calcd for
C19H17ClN2O: C, 70.26%; H, 5.28%; N, 8.62%. Found: C,
70.17%; H, 5.35%; N, 8.44%.
General procedure for the synthesis of compounds 5
A mixture of cinnamic-derived acid 3 (2 mmol), N-hydroxy
benzotriazole (HOBT) (2.4 mmol) and 1-ethyl-3-(3-dimethyl
aminopropyl) carbodiimide (EDC) (2.4 mmol) in 10 mL of
dry acetonitrile was stirred for 1 h at ambient temperature and
then tryptamine (2 mmol) was added. The reaction mixture
was stirred at ambient temperature for 48 h and completion of
the reaction monitored by TLC. The crude product was ex-
tracted using CH2Cl2 (50 mL) and NaCl 10% (10 mL),
NaHCO3 10% (10 mL) and citric acid 10% (10 mL). The
organic phase was dried (Na2SO4) and the solvent was evap-
orated under vacuum. All the compounds were purified by
flash chromatography on silica gel using petroleum ether/ eth-
yl acetate (6:1) to obtain products 5, and then were recrystal-
lized from ethyl acetate and hexane (1:1).
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(3-chlorophenyl)acrylamide
N-(2-(1H-indol-3-yl)ethyl)cinnamamide (5a)
(5c)
Yield: 73%, mp: 141–143 °C. IR (KBr) cm−1 υ: 3440, 3312,
Yield: 68%, mp: 153–156 °C. IR (KBr) cm−1 υ: 3444, 3244,
3052, 2923, 1647, 1601, 1512. 1H NMR (DMSO-d6,
500 MHz): δ = 2.92 (t, J = 7.5 Hz, 2H, CH2), 3.51 (q, J =
6.5 Hz, 2H, NH-CH2), 6.72 (d, J = 16.0 Hz, 1H, CHα), 6.98
(t, J = 7.5 Hz, 1H, H5), 7.07 (t, J = 7.5 Hz, 1H, H6), 7.18 (s,
1H, H2), 7.35 (d, J = 8.5 Hz, 1H, H7), 7.40–7.45 (m, 3H, CHβ,
H5’, 6’), 7.51 (d, J = 7.0 Hz, 1H, H4’), 7.57 (d, J = 7.5 Hz, 1H,
H4), 7.62 (s, 1H, H2’), 8.21 (t, J = 5.5 Hz, 1H, NH-CH2), 10.80
(s, 1H, NH) ppm. 13CNMR (DMSO-d6, 125 MHz): δ = 25.1,
39.7, 111.1, 111.7, 118.2, 119.0, 120.9, 122.6, 124.1, 126.0,
127.1, 129.0, 130.6, 132.4, 133.6, 136.12, 136.95, 137.29,
164.6 ppm. Mass, m/z (%): 324(2), 237(3), 182(5), 165(10),
143(40), 130(35), 101(29), 86(100), 58(33). Anal. Calcd for
1
1
2928, 2831, 1653, 1600, 1513 . HNMR (DMSO-d6,
500 MHz): δ 2.91 (t, J = 7.0 Hz, 2H, CH2), 3.50 (q, J =
7.0 Hz, 2H, NH-CH2), 6.66 (d, J = 15.5 Hz, 1H, CHα),
), 7.07 (t, J = 7.0 Hz, 1H, H6), 7.18 (s, 1H, H2), 7.35–7.42
(m, 4H, H3’, 4’, 5’, 7), 7.42 (d, J = 15.5 Hz, 1H, CHβ), 7.55–
7.58 (m, 3H, H2’,6’, 4), 8.21 (t, J = 5.5 Hz, 1H, NH-CH2),
10.82 (s, 1H, NH) ppm. 13CNMR (DMSO-d6, 125 MHz):
δ = 25.2, 39.6, 111.3, 111.76, 118.18, 11.63, 120.87, 122.4,
122.59, 127.2, 127.4, 128.8, 129.27, 134.9, 136.2, 138.4,
164.87 ppm. Mass, m/z (%): 290(33), 143(100), 130(95),
103(58), 77(45). Anal. Calcd for C19H18N2O: C, 78.59%; H,
6.25%; N, 9.65%. Found: C, 78.77%; H, 6.35%; N, 9.93%.

DARU J Pharm Sci
C19H17ClN2O: C, 70.26%; H, 5.28%; N, 8.62%. Found: C,
70.33%; H, 5.39%; N, 8.59%.
143(100), 130(99), 101(25), 86(55), 58(22). Anal. Calcd for
C19H17FN2O: C, 74.01% H, 5.56%; N, 9.08%. Found: C,
74.24%; H, 5.69%; N, 9.22%.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(4-chlorophenyl)acrylamide
(5d)
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(4-fluorophenyl)acrylamide
(5g)
Yield: 71%, mp: 165–169 °C. IR (KBr) cm−1 υ: 3408, 3287,
3072, 2918, 651, 1619, 1552. 1HNMR (DMSO-d6,
500 MHz): δ = 2.90 (t, J = 7.0 Hz, 2H, CH2), 3.49 (q, J =
7.0 Hz, 2H, NH-CH2), 6.65 (d, J = 16.0 Hz, 1H, CHα), 6.98
(t, J = 7.0 Hz, 1H, H5), 7.07 (t, J = 7.5 Hz, 1H, H6), 7.16 (s,
1H, H2), 7.34 (d, J = 8.0 Hz, 1H, H7), 7.42 (d, J = 16.0 Hz, 1H,
CHβ), 7.46 (d, J = 8.0 Hz, 2H, H3’, 5’), 7.55–7.58 (m, 3H, H2’,
6’, 4), 8.17 (t, J = 5.5 Hz, 1H, NH-CH2), 10.77 (s, 1H, NH)
ppm. 13CNMR (DMSO-d6, 125 MHz): δ = 25.5, 39.6, 111.2,
111.7, 118.1, 118.2, 120.8, 122.5, 123.2, 127.1, 128.9, 129.1,
133.7, 133.9, 136.2, 137.0, 164.6 ppm. Mass, m/z (%):
324(12), 165(9), 143(100), 130(82), 102(16), 77(8), 58(5).
Anal. Calcd for C19H17ClN2O: C, 70.26%; H, 5.28%; N,
8.62%. Found: C, 70.11%; H, 5.05%; N, 8.50%.
Yield: 69%, mp: 152–154 °C. IR (KBr) cm−1 υ: 3403, 3300,
3074, 2932, 1652, 1601, 1551. 1HNMR (DMSO, 500 MHz):
δ = 2.90 (t, J = 7.0 Hz, 2H, CH2), 3.49 (q, J = 7.0 Hz, 2H, NH-
CH2), 6.58 (d, J = 15.5 Hz, 1H, CHα), 6.99 (t, J = 7.5 Hz, 1H,
H5), 7.07 (t, J = 7.5 Hz, 1H, H6), 7.17 (s, 1H, H2), 7.24 (t, J =
7.5 Hz, 2H, H3’, 5’), 7.35 (d, J = 8.0 Hz, 1H, H7), 7.44 (d, J =
16.0 Hz, 1H, CHβ), 7.56 (d, J = 8.0 Hz, H4), 7.60–7.63 (m,
2H, H2’, 6’), 8.17 (t, J = 5.5 Hz, 1H, NH-CH2), 10.80 (s, 1H,
NH) ppm. 13CNMR (DMSO, 125 MHz): δ = 25.1, 39.5,
111.3, 111.7, 115.7 (JC-F = 21.5 Hz), 118.1, 118.2, 120.8,
122.3, 122.5, 127.2, 129.4 (JC-F = 8.7 Hz), 131.5, 136.2,
137.1, 161.5 (JC-F = 246 Hz), 164.7 ppm. Mass, m/z (%):
308(25), 143(100), 130(88), 121(20), 101(24), 77(15). Anal.
Calcd for C19H17FN2O: C, 74.01%; H, 5.56%; N, 9.08%.
Found: C, 74.19%; H, 5.39%; N, 8.89%.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(2-fluorophenyl)acrylamide
(5e)
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(o-tolyl)acrylamide (5h)
Yield: 72%, mp: 137–140 °C. IR (KBr) cm−1 υ: 3451, 3228,
3052, 2928, 1667, 1628, 1560. 1HNMR (DMSO-d6,
500 MHz): δ = 2.92 (m, 2H, CH2), 3.51 (m, 2H, NH-CH2),
6.33 (d, J = 16.0 Hz, 1H, CHα), 6.52 (t, J = 7.0 Hz, 1H, H5),
6.61(t, J = 7.0 Hz, 1H, H6), 6.71–6.75 (m, 3H, H4, 4’, 5’), 6.90–
6.92 (m, 2H, H7,3’), 7.11–7.15 (m, 3H, CHβ, H2, 6’), 7.88 (m,
1H, NH-CH2), 10.35 (s, 1H, NH) ppm. 13CNMR (DMSO-d6,
Yield: 68%, mp: 159–161 °C. IR (KBr) cm−1 υ: 3449, 3317,
3047, 2920, 1694, 1603, 1546. 1HNMR (DMSO-d6,
500 MHz): δ = δ = 2.37 (s, 3H, CH3), 2.89 (t, J = 7.5 Hz,
2H, CH2), 3.48 (q, J = 6.5 Hz, 2H, NH-CH2), 6.58 (d, J =
16.0 Hz, 1H, CHα), 6.99 (t, J = 7.5 Hz, 1H, H4’), 7.07 (t,
J = 8 .0 Hz, 1H, H5), 7.17 (s, 1H, H2), 7.213–7.26 (m, 3H,
H3’, H6, H5’), 7.34 (d, J = 8.0 Hz, 1H, H6’), 7.51 (d, J = 7.5 Hz,
1H, H7), 7.56 (d, J = 8.0 Hz, 1H, H4), 7.66 (d, J = 15.5 Hz, 1H,
Hβ), 8.213 (m, 1H, NH-CH2), 10.79 (s, 1H, NH) ppm.
13CNMR (DMSO-d6, 125 MHz): δ = 19.27, 25.15, 39.76,
111.21, 112.12, 118.12, 120.09, 122.58, 123.59, 125.72,
126.29, 127.16, 128.94, 129.80, 130.53, 133.76, 135.88,
136.51, 137.01, 164.2 ppm. Mass, m/z (%): 304(2), 230(11),
217(14), 162(13), 143(50), 115(57), 101(25), 86(100), 58(29).
Anal. Calcd for C20H20N2O: C, 78.92%; H, 6.62%; N, 9.20%.
Found: C, 78.88%; H, 6.41%; N, 9.01%.
125 MHz): δ = 25.5, 39.8, 111.3, 111.5, 111.8, 115.9 (JC-F
21.25 Hz), 118.3, 118.37, 120.9, 121.89, 122.6 (JC-F
=
=
11.25 Hz), 124.8, 125.2, 127.3, 128.9 (JC-F = 21.25), 130.9,
136.4, 137.16, 161.5 (JC-F = 248.75 Hz), 164.8 ppm. Mass, m/
z (%): 308(10), 160(11), 143(96), 130(100), 101(28), 86(99),
58(26). Anal. Calcd for C19H17FN2O: C, 74.01%; H, 5.56%;
N, 9.08%. Found: C, 74.19%; H, 5.39%; N, 8.84%.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(3-fluorophenyl)acrylamide
(5f)
Yield: 67%, mp: 149–151 °C. IR (KBr) cm−1 υ: 3276, 3066,
2933, 1663, 1619, 1586. 1HNMR (DMSO-d6, 500 MHz): δ =
2.90 (m, 2H, CH2), 3.49 (m, 2H, NH-CH2), 6.69 (d, J =
16.0 Hz, 1H, CHα), 6.99 (t, J = 7.0 Hz, 1H, H5), 7.07 (t, J =
7.0 Hz, 1H, H6), 7.18–7.22 (m, 2H, H2, 5’), 7.35 (d, J = 7.5 Hz,
1H, H7), 7.39–7.46 (m, 5H, CHβ, H2’, 4’, 5’, 6’), 7.57 (d, J =
7.5 Hz, 1H, H4), 8.24 (m, 1H, NH-CH2), 10.83 (s, 1H, NH)
ppm. 13CNMR (DMSO-d6, 125 MHz): δ = 25.2, 39.8, 111.4,
111.7, 113.76, 115.9, 118.3, 119.09, 120.9, 121.8, 122.6,
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(m-tolyl)acrylamide (5i)
Yield: 64%, mp: 122–124 °C. IR (KBr) cm−1 υ: 3312, 3284,
3051, 2922, 1653, 1607, 1547. 1HNMR (DMSO-d6,
500 MHz): δ = 2.32 (s, 3H, CH3), 2.93 (t, J = 7.0 Hz, 2H,
CH2), 3.53 (q, J = 7.0 Hz, 2H, NH-CH2), 6.66 (d, J =
16.0 Hz, 1H, CHα), 6.99 (t, J = 7.0 Hz, 1H, H5), 7.08 (t, J =
7.5 Hz, 1H, H6), 7.17 (s, 1H, H2’), 7.18 (s, 1H, H2), 7.29 (t,
J = 7.5 Hz, 1H, H5’), 7.34–7.37 (m, 3H, H7, 4, 4’), 7.43 (d, J =
15.5 Hz, 1H, CHβ), 7.51 (d, J = 8.0 Hz, 1H, H6’), 8.16 (t, J =
5.5 Hz, 1H, NH-CH2), 10.80 (s, 1H, NH) ppm. 13CNMR
123.4, 127.2, 130.7, 130.91, 136.2, 137.2, 161.4 (JC-F
=
242.5 Hz), 164.6 ppm. Mass, m/z (%): 308(8), 160(5),

DARU J Pharm Sci
(DMSO, 125 MHz): δ = 20.7, 25.1, 39.4, 111.2, 111.7, 118.1,
118.2, 120.8, 122.2, 122.5, 124.5, 127.1, 127.8, 128.6, 129.8,
134.8, 136.2, 137.9, 138.4, 164.8 ppm. Mass, m/z (%):
304(8), 143(100), 130(45), 115(14), 58(10). Anal. Calcd for
C20H20N2O: C, 78.92%; H, 6.62%; N, 9.20%. Found: C,
78.71%; H, 6.53%; N, 9.41%.
H4), 7.70 (t, J = 7.5 Hz, 1H, H5’), 7.99 (d, J = 7.5 Hz, 1H, H6’),
8.19 (d, J = 8.0 Hz, 1H, H4’), 8.23 (m, 1H, NH-CH2), 8.38 (s,
1H, H2’), 10.78 (s, 1H, NH) ppm. 13CNMR (DMSO-d6,
125 MHz): δ = 25.0, 39.6, 111.2, 111.6, 118.1, 120.8, 121.5,
122.5, 123.4, 123.6, 125.3, 127.1, 130.4, 133.8, 136.1, 136.2,
1336.8, 148.2, 164.2 ppm. Mass, m/z (%): 335(8), 176(3),
143(100), 132(64), 102(7), 77(5). Anal. Calcd for
C19H17N3O3: C, 68.05%; H, 5.11%; N, 12.53%. Found: C,
68.18%; H, 5.27%; N, 12.87%.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(p-tolyl)acrylamide (5j)
Yield: 72%, mp: 171–173 °C. IR (KBr) cm−1 υ: 3447, 3229,
2919, 2723, 1651, 1605, 1543. 1HNMR (DMSO-d6,
500 MHz): δ = 2.32 (s, 3H, CH3), 2.90 (t, J = 7.0 Hz, 2H,
CH2), 3.49 (q, J = 7.0 Hz, 2H, NH-CH2), 6.58 (d, J =
15.5 Hz, 1H, CHα), 6.98 (t, J = 7.0 Hz, 1H, H5), 7.07 (t, J =
7.0 Hz, 1H, H6), 7.17 (s, 1H, H2), 7.21 (d, J = 7.5 Hz, 2H, H3’,
5’), 7.34 (d, J = 7.5 Hz, 1H, H7), 7.42 (d, J = 15.5 Hz, 1H,
CHβ), 7.44 (d, J = 7.5 Hz, 2H, H2’, 6’), 7.56 (d, J = 7.5 Hz,
1H, H4), 8.14 (t, J = 5.5 Hz, 1H, NH-CH2), 10.78 (s, 1H, NH)
ppm. 13C NMR (DMSO, 125 MHz): δ = 20.8, 25.2, 39.6,
111.2, 111.7, 118.1, 118.2, 120.8, 121.4, 122.5, 127.2,
127.4, 129.4, 132.2, 136.2, 138.3, 139.0, 138.9, 164.9 ppm.
Mass, m/z (%): 304(24), 143(100), 130(83), 115(30), 103(14),
91(15), 77(13). Anal. Calcd for C20H20N2O: C, 78.92%; H,
6.62%; N, 9.20%. Found: C, 78.59%; H, 6.75%; N, 9.33%.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(4-nitrophenyl)acrylamide
(5 m)
Yield: 81%, mp: 173–175 °C. IR (KBr) cm−1 υ: 3382, 3268,
3052, 2931, 1666, 1625, 15441. 1HNMR (DMSO-d6,
500 MHz): δ = 2.91 (t, J = 7.0 Hz, 2H, CH2), 3.52 (q, J =
7.0 Hz, 2H, NH-CH2), 6.83 (d, J = 15.5 Hz, 1H, CHα), 6.98
(t, J = 7.0 Hz, 1H, H5), 7.07 (t, J = 7.5 Hz, 1H, H6), 7.17 (s,
1H, H2), 7.34 (d, J = 8.0 Hz, 1H, H7), 7.53–7.57 (m, 2H, CHβ,
H4), 7.82 (d, J = 8.5 Hz, 2H, H2’,6’), 8.25 (d, J = 8.5 Hz, 2H,
H3’, 5’), 8.32 (t, J = 5.5 Hz, 1H, NH-CH2), 10.78 (s, 1H, NH)
ppm. 13CNMR (DMSO-d6, 125 MHz): δ = 25.0, 39.5, 111.2,
111.6, 118.1, 120.8, 122.4, 123.8, 124.1, 126.7, 127.1, 128.4,
136.1, 136.2, 141.6, 147.4, 164.1 ppm. Mass, m/z (%):
335(14), 176(4), 143(100), 132(72), 102(11), 77(7). Anal.
Calcd for C19H17N3O3: C, 68.05%; H, 5.11%; N, 12.53%.
Found: C, 68.12%; H, 5.28%; N, 12.49%.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(2-nitrophenyl)acrylamide
(5k)
Yield: 79%, mp: 153–155 °C. IR (KBr) cm−1 υ: 3452, 3308,
3054, 2938, 1663, 1614, 15291. 1HNMR (DMSO-d6,
500 MHz): δ = 2.91 (t, J = 7.5 Hz, 2H, CH2), 3.50 (q, J =
7.0 Hz, 2H, NH-CH2), 6.64 (d, J = 15.5 Hz, 1H, CHα), 6.99
(t, J = 7.0 Hz, 1H, H5), 7.07 (t, J = 7.0 Hz, 1H, H6), 7.18 (s,
1H, H2), 7.34 (d, J = 8.0 Hz, 1H, H7), 7.56 (d, J = 7.5 Hz, 1H,
H4), 7.63 (t, J = 7.5 Hz, 1H, H4’), 7.70 (d, J = 15.5 Hz, 1H,
CHβ), 7.74–7.77 (m, 2H, H5’, 6’), 8.03 (d, J = 8.0 Hz, 1H, H3’),
8.3 (t, J = 5.5 Hz, 1H, NH-CH2), 10.78 (s, 1H, NH) ppm.
13CNMR (DMSO-d6, 125 MHz): δ = 25.0, 39.6, 111.3,
111.6, 118.1, 118.22, 120.8, 122.48, 122.6, 124.5, 127.1,
128.5, 129.9, 133.1, 133.2, 133.6136.2, 148.3, 164.0 ppm.
Mass, m/z (%): 335(7), 193(10), 176(15), 143(100),
132(69), 102(11), 86(56). Anal. Calcd for C19H17N3O3: C,
68.05%; H, 5.11%; N, 12.53%. Found: C, 68.28%; H,
5.31%; N, 12.21%.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(2-methoxyphenyl)acrylamide
(5n)
Yield: 71%, mp: 152–155 °C. IR (KBr) cm−1 υ: 3397, 3260,
3063, 2934, 1657, 1616, 1540. 1HNMR (DMSO-d6,
500 MHz): δ = 284 (t, J = 7.0 Hz, 2H, CH2), 3.10 (q, J =
7.0 Hz, 2H, NH-CH2), 3.78 (s, 3H, OCH3), 6.29 (d, J =
16.0 Hz, 1H, CHα), 6.49 (t, J = 7.0 Hz, 1H, H5), 6.53–6.56
(m, 2H, H3’,5’), 6.63 (t, J = 7.0 Hz, 1H, H6), 6.74 (s, 1H, H2),
6.86 (t, J = 7.5 Hz, 1H, H4’), 6.93 (d, J = 7.5 Hz, 1H, H7), 7.07
(d, J = 7.5 Hz, 1H, H6’), 7.14 (d, J = 7.5 Hz, 1H, H4), 7.33 (d,
J = 16.0 Hz, 1H, CHβ), 7.81 (t, J = 5.5 Hz, 1H, NH-CH2),
10.40 (s, 1H, NH) ppm. 13CNMR (DMSO-d6, 125 MHz):
δ = 25.3, 39.6, 55.5, 111.6, 111.9, 112.2, 118.2, 118.3,
120.7, 120.9, 122.6, 122.9, 123.4, 127.3, 127.6, 130.5,
133.8, 136.3, 157.5, 165.5 ppm. Mass, m/z (%): 320(8),
161(31), 143(100), 130(38), 118(6), 103(6), 77(7). Anal.
Calcd for C20H20N2O2: C, 74.98%; H, 6.29%; N, 8.74%.
Found: C, 74.88%; H, 6.31%; N, 8.98%.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(3-nitrophenyl)acrylamide (5l)
Yield: 73%, mp: 168–171 °C. IR (KBr) cm−1 υ: 3395, 3249,
3080, 2926, 1660, 1621, 1546. 1HNMR (DMSO-d6,
500 MHz): δ = 2.92 (t, J = 7.0 Hz, 2H, CH2), 3.51 (q, J =
6.5 Hz, 2H, NH-CH2),6.84 (d, J = 16.0 Hz, 1H, CHα), 6.99
(t, J = 7.0 Hz, 1H, H5), 7.07 (t, J = 7.5 Hz, 1H, H6), 7.18 (s,
1H, H2), 7.35 (d, J = 7.5 Hz, 1H, H7), 7.54–7.57 (m, 2H, CHβ,
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(3-methoxyphenyl)
acrylamide (5o)
Yield: 69%, mp: 169–170 °C. IR (KBr) cm−1 υ: 3442, 3232,
3053, 2921, 1655, 1613, 1551. 1HNMR (DMSOd-6,

DARU J Pharm Sci
500 MHz): δ = 2.81 (t, J = 7.5 Hz, 2H, CH2), 3.39 (q, J =
7.5 Hz, 2H, NH-CH2), 3.70 (s, 3H, CH3), 6.54 (d, J =
15.5 Hz, 1H, CHα), 7.00 (t, J = 7.0 Hz, 1H, H5), 7.12 (t, J =
7.5 Hz, 1H, H6), 7.19 (s, 1H, H2’), 7.25 (s, 1H, H2), 7.23 (t,
J = 7.5 Hz, 1H, H5’), 7.40–7.45 (m, 3H, H4,4’,7), 7.41 (d, J =
15.5 Hz, 1H, CHβ), 7.62 (d, J = 7.0 Hz, 1H, H6´), 8.20 (t, J =
5.5 Hz, 1H, NH-CH2), 10.78 (s, 1H, NH) ppm. 13CNMR
(DMSO-d6, 125 MHz): δ = 25.01, 39.6, 55.2, 111.3, 111.6,
115.1, 118.1, 120.8, 122.5, 124.0, 125.5, 127.1, 127.6, 128.2,
130.9, 132.9, 133.1, 134.5, 158.2, 164.2 ppm. Mass, m/z (%):
320(6), 279(7), 174(6), 161(97), 143(100), 130(39), 118(28),
105(20), 77(22). Anal. Calcd for C20H20N2O2: C, 74.98%; H,
6.29%; N, 8.74%. Found: C, 74.69%; H, 6.48%; N, 8.73%.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(4-hydroxy-3-methoxyphenyl)
acrylamide (5r)
Yield: 58%, mp: 168–170 °C. IR (KBr) cm−1 υ: 3359, 3294,
3084, 2931, 1649, 1575, 1420. 1HNMR (DMSO-d6,
500 MHz): δ = 2.89 (t, J = 7.5 Hz, 2H, CH2), 3.47 (q, J =
7.0 Hz, 2H, NH-CH2), 3.80 (s, 3H, OCH3), 6.46 (d, J =
15.5 Hz, 1H, CHα), 6.79 (d, J = 8.0 Hz, 1H, H5’), 6.96–6.99
(m, 2H, H5, 6’), 7.07 (t, J = 7.5 Hz, 1H, H6), 7.12 (s, 1H, H2’),
7.17 (s, 1H, H2), 7.32–7.35 (m, 2H, CHβ-H7), 7.56 (d, J =
7.5 Hz, 1H, H4), 8.02 (t, J = 5.5 Hz, 1H, NH-CH2), 9.36 (s,
1H, OH), 10.80 (s, 1H, NH) ppm. 13CNMR (DMSO,
125 MHz): δ = 25.2, 39.5, 55.5, 110.8, 111.3, 111.8, 115.6,
118.1, 118.2, 119.2, 120.8, 121.4, 122.6, 126.4, 127.2, 136.2,
138.8, 147.8, 148.1, 165.3 ppm. Mass, m/z (%): 336(18),
221(4), 194(6), 177(52), 143(100), 130(98), 117(15),
103(16), 77(20). Anal. Calcd for C20H20N2O3: C, 71.41%;
H, 5.99%; N, 8.33%. Found: C, 71.64%; H, 6.19%; N, 8.60%.
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(4-methoxyphenyl)acrylamide
(5p)
Yield: 65%, mp: 177–179 °C. IR (KBr) cm−1 υ: 3450, 3231,
3049, 2929, 1648, 1600, 1539. 1HNMR (DMSO-d6,
500 MHz): δ = 2.89 (t, J = 7.0 Hz, 2H, CH2), 3.47 (q, J =
7.0 Hz, 2H, NH-CH2), 3.78 (s, 3H, OCH3), 6.48 (d, J =
15.5 Hz, 1H, CHα), 6.98 (d, J = 8.5 Hz, 2H, H3’,5’), 6.99 (t,
J = 7.5 Hz, 1H, H5), 7.07 (t, J = 7.5 Hz, 1H, H6), 7.17 (s, 1H,
H2), 7.34 (d, J = 8.0 Hz, 1H, H7), 7.38 (d, J = 16.0 Hz, 1H,
CHβ), 7.50 (d, J = 8.5 Hz, 2H, H2’, 6’), 7.56 (d, J = 8.0 Hz, 1H,
H4), 8.10 (t, J = 5.5 Hz, 1H, NH-CH2), 10.80 (s, 1H, NH)
ppm. 13CNMR (DMSO-d6, 125 MHz): δ = 25.2, 39.5, 55.1,
111.2, 111.8, 114.3, 118.1, 118.2, 119.9, 120.8, 122.5, 127.2,
127.5, 128.9, 136.2, 138.0, 160.2, 165.1 ppm. Mass, m/z (%):
320(18), 161(41), 143(100), 130(51), 118(12), 103(13),
77(15). Anal. Calcd for C20H20N2O2: C, 74.98%; H, 6.29%;
N, 8.74%. Found: C, 74.74%; H, 6.52%; N, 9.04%.
AChE and BChE inhibition assay
To evaluate the potential inhibition of this series of com-
pounds, Ellman s method was used on AChE and BChE in-
hibitory test. Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type
V-S, lyophilized powder, from electric eel 1000 units), butyr-
ylcholinesterase (BChE, E.C. 3.1.1.8, from equine serum) and
butyrylthiocholine iodide were obtained from Sigma-Aldrich.
5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), potassium
dihydrogen phosphate, dipotassium hydrogen phosphate, po-
tassium hydroxide, sodium hydrogen carbonate,
acetylthiocholine iodine and butyrylthiocholine iodine were
purchased from Fluka. Donepezil hydrochloride was obtained
from Merck, Darmstadt, Germany. To prepare the stock solu-
tion of testing compounds, 10 mg of testing compounds were
dissolved in DMSO (10 μL) and ethanol 99% (400 μL). To
determine IC50 values, four different concentrations of each
compound were examined in the range of 20–80% for AChE
and BChE. All experiments were performed at 25 °C. The
assay was performed as the literature reported [9]. The IC50
values were determined [3, 16] graphically from inhibition
curve (log inhibitor concentration vs. percent of inhibition).
(E)-N-(2-(1H-indol-3-yl)ethyl)-3-(3,4-dimethoxyphenyl)
acrylamide (5q)
Yield: 76%, mp: 151–153 °C. IR 1 (KBr) cm−1 υ: 3376, 3299,
3054, 2939, 1648, 1609, 1545. 1HNMR (DMSO-d6,
500 MHz): δ = 2.89 (t, J = 7.5 Hz, 2H, CH2), 3.47 (q, J =
7.0 Hz, 2H, NH-CH2), 3.78 (s, 6H, 2-OCH3), 6.51 (d, J =
16.0 Hz, 1H, CHα), 6.96–6.99 (m, 2H, H5, 5’), 7.07 (t, J =
7.0 Hz, 1H, H6), 7.10 (dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H, H6’),
7.15 (dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H, H2’), 7.33–7.38 (m, 3H,
H2, CHβ-H7), 7.55 (d, J = 8.0 Hz, 1H, H4), 8.06 (t, J = 5.5 Hz,
1H, NH-CH2), 10.79 (s, 1H, NH) ppm. 13CNMR (DMSO-d6,
125 MHz): δ = 25.2, 39.5, 55.4, 55.5, 110.1, 111.3, 111.8,
112.0, 118.1, 118.2, 120.2, 120.8, 121.2, 122.5, 127.2,
127.8, 136.2, 138.4, 149.8, 150.0, 165.1 ppm. Mass, m/z
(%): 350(3), 272(3), 255(7), 225(5), 191(6), 143(100),
130(60), 103(7), 77(8). Anal. Calcd for C21H22N2O3: C,
71.98%; H, 6.33%; N, 7.99%. Found: C, 71.74%; H, 6.22%;
N, 7.69%.
Kinetic studies of BChE and AChE inhibition
For estimation of inhibition model and inhibition constant
(Ki), cal plots of 1/V versus 1/[S] was used. Three concentra-
tions of the inhibitor (compound 5b and 5q) were selected for
the analysis of BChE and AChE inhibition. The rate of enzy-
matic reaction was obtained with three different concentra-
tions of the inhibitor and in the absence of inhibitor. For each
of them, the initial velocity (V) of substrate hydrolysis was
measured at the different concentration of substrate (S) in the
range of 0.14–0.69 μM [6] and with the obtained data; double

DARU J Pharm Sci
reciprocal plots (1/V vs. 1/[S]) were made. By increasing the
inhibitor concentration, the resulting Lineweaver-Burk plot
showed increasing slopes and intercepts which are character-
ized by mixed-type inhibition. Slops of these reciprocal plots
were then plotted against the concentration of compound 5b
and 5q in a weighted analysis, and Ki was determined as the
intercept on the negative x-axis. All rate measurements were
performed in triplicate and data analysis was performed with
Microsoft Excel 2013 [17].
Molecular docking study
Based on the X-ray crystal structures of AChE and BChE
docking studies for compounds 5q and 5b were performed
using Autodock 4.2 program. The PDB structures of 1EVE
(for compound 5q) and 4BDS (for compound 5b) [6, 14, 17]
were retrieved from the Brookhaven protein database (http://
www.rcsb.org). Then the water molecules and the original
inhibitors were removed and the amino acid chain was kept.
Subsequently, the 3D structure of the compounds 5q and 5b
were prepared by using Marvin Sketch 5.8.3, 2012,
ChemAxon (http://chemaxon.com) and using Autodock 4.2
program converted to pdbqt format. Also, the pdbqt of
protein was provided by using the same software. It should
be noted that polar hydrogen atoms were added to the receptor
and resulting protein by using Autodock tools (ADT;
version1.5.6), Kullman charges were appointed to all atoms
of an enzyme. The resulting structure of enzyme was used as
an input format of the AUTOGRID software which is
performed a pre-calculated atomic affinity grid map. For the
docking calculation, the dimensions of grid box size 60 ×
60 × 60 Ao was set at the geometrical center of co-
crystalized ligand. The other parameters were placed by de-
fault. At the end, the conformation which has the lowest free
energy of binding was selected for analyzing the interactions
between AChE and the inhibitor. The results were visualized
using Discovery studio 4.0 Client [25]. The same method was
utilized in the case of BChE.
Neuroprotective assay
The neuroprotective activity of the synthesized com-
pounds was evaluated according to the literature [1].
Before treatment with H2O2 (300 mM), differentiated
PC12 cells were incubated with different concentrations
of the compound 5q for 3 h. The assay was performed as
the literature reported [9, 11, 18]. Results were adjusted
considering OD measured in the blank.
BACE1 enzymatic assay
By using fluorescence resonance energy transfer (FRET),
BACE1 enzyme assay kit was used (Invitrogen, former Pan
Vera Corporation, Madison, WI) and the assay was accom-
plished according to the manufacturer’s instructions
(Invitrogen. http://tools.com/content/sfs/manuals/L0724.pdf).
The sample preparation was performed as the literature
reported [9]. Fluorescence measurements were performed
with a multimode microplate reader (BMG Labtech) at
excitation and emission wavelength of 545 nm and 585 nm,
respectively. This test was repeated for 3 to 5 times eventually
average percent of enzyme inhibitory activities at 10 and
50 μM concentration of test compounds were calculated [9,
17, 19–21].
Computational methods
The log P values, tPSA, HBD, and HBA were calculated by
the means of MarvineSketch 5.8.3. RBC was calculated using
Autodock tools (ver.1.5.6).
Result and discussion
Determination of the inhibitory potency on Aβ1–42
Chemistry
self-aggregation induced
The cinnamic-derived acids 5a-r were synthesized through
the routes depicted in Scheme 1. The related benzaldehyde
(1) were reacted with malonic acid (2) in a mixture of pyri-
dine, piperidine and ethanol under refluxing condition. The
product was crystallized from ethanol to product cinnamic-
derived acids (3) in a good to excellent yield. The reaction
of compounds 3 and tryptamine (4) in the presence of N-
hydroxy benzotriazole (HOBT), 1-ethyl-3-(3-dimethyl
aminopropyl) carbodiimide (EDC) gave 5a-54 in dry acetoni-
trile at room temperature. The purification of the product was
carried out using flash chromatography using hexane and eth-
yl acetate (8:1). The final compounds were crystallized in
Inhibition of self-induced Ab (1–42) aggregation was mea-
sured using a Thioflavin T(ThT)-binding assay [22–24]. The
sample preparation and the assay were performed as the liter-
ature reported [5]. Each measurement was run in triplicate.
Fluorescence was measured on a Synergy HTX Multi-Mode
reader from BioTek instruments with excitation and emission
wavelengths at 440 nm and 485 nm, respectively. The percent
inhibition of aggregation was calculated by the expression (1-
IFi/IFO) × 100% in which IFi and IFO are the fluorescence
intensities obtained for Aβ in the presence and absence of
inhibitors, respectively.

DARU J Pharm Sci
O
Scheme 1 Synthesis pathway for
preparation of compounds 5.
Reagents and conditions: I:
pyridine, piperidine, ethanol,
100 °C, reflux, 24 h; II: HOBT,
EDCI, dray acetonitrile, r.t, 48 h
O
O
O
OH
I
H
HO
OH
+
+
R
R
3
1
2
NH2
O
HN
N
H
II
N
R3
R1
H
R2
5
4
5a: R1, R2, R3=H; 5b: R1=Cl, R2, R3=H; 5c: R1, R3= H, R2=Cl; 5d: R1, R2= H, R3=Cl; 5e: R1=F, R2, R3=H;
5f: R1, R3= H, R2=F; 5g: R1, R2= H, R3=Cl; 5h: R1=CH3, R2, R3=H; 5i: R1, R3= H, R2=CH3; 5j: R1, R2= H,
R3=CH3; 5k: R1=NO2, R2, R3=H; 5l: R1, R3= H, R2=NO2; 5m: R1, R2= H, R3=NO2; 5n: R1=OCH3, R2, R3=H;
5o: R1, R3= H, R2=OCH3; 5p: R1, R2= H, R3=OCH3; 5q: R1=H, R2, R3=OCH3; 5r: R1=H, R2=OCH3, R3=OH
ethyl acetate and hexane (1:1). These structures were charac-
terized by IR, 1H-NMR, 13C-NMR and elemental analysis.
In the 1HNMR spectra of compounds 5, there are several
specified signals that could be referred to the structure. For
example hydrogen of vinyl group, indol ring, methylen groups
in aliphatic bridge and also aromatic hydrogen of indol be-
tween amid group and indol ring are identified. In the more
derivatives, H2 indol appeared at about 7.2 ppm as singlet
with integration value of one proton. However, in some deriv-
atives this peak was overlapped with other aromatic hydro-
gens. Two separated peaks at about 7 and 7.1 ppm appeared as
triplet that could be attributed to 5-H and 6-H of indol ring
respectively. The two triplet signals at 2.9 and 3.5 ppm could
be referred to CH2 group of aliphatic side chain. In α, β-
unsaturated carbonyl group, two 15 Hz-spilited signals ap-
peared about 6.6 and 7.4 ppm. Other aromatic protons are
observable at a broad range (6.7–8 ppm) related to the
substituted groups on the cinnamic acid moiety.
atom on every third position of ortho, meta and para improved
AChE inhibitory activity (IC50 = 21.11, 32.63 and 27.2 μM),
while the insertion of fluorine atom instead of the chlorine
atom decreased anticholinesterase inhibitory activity (IC50
=
33.57, 31.89 and 48.14 μM). The methyl group at every third
position showed a mild increase in the AChE inhibitory activ-
ity (IC50 = 23.77, 31.34 and 39.22 μM). The presence of
electron-donating methoxy groups in every third position of
the aryl group of cinnamic-derived acids resulted from in-
creasing the AChE inhibitory activities (IC50 = 19.34, 27.85
and 14.36 μM). It should be noted that replacement of
Hydrogen in position R2 and R3 by methoxy in the aryl group
of cinnamic-derived acids (5q) showed the highest activity in
this group (IC50 = 11.51 μM). It was 5 times more potent than
parent compound 5a, while in compound 5r, that has a
methoxy group in position R2 and a hydroxyl group in posi-
tion R3, resulted in the reduction of anti-AChE activity with
IC50 = 21.92 μM compared with compound 5q. Insertion of a
nitro substituent on the ring compared with the parent com-
pound 5a led to the significant reduction of anti-AChE activity
(IC50 = 95.85, >100 and 65.36 μM). Another instructive point
that is a replacement of hydrogen by fluorine, methyl, chlorine
and methoxy group in the aryl group of cinnamic-derived
acids moieties respectively led to increasing of anti-AChE
activity. It seems that the AChEI activities of these synthesized
compounds were under the influenced of electronic effect of
substituent groups on the aromatic ring of cinnamic acids
moieties.
Pharmacology
In vitro inhibition of AChE, BChE
The in vitro AChE and BChE inhibitory activity of new
cinnamic acid-tryptamine hybrids (5a-5r) were studied using
the Ellman s method and donepezil as a reference drug.
Inhibition potency of the synthesized compounds under the
title of IC50 is presented in Table 1. Among these compounds,
5q (IC50 = 11.51 μM) showed the best AChE inhibitory activ-
ity. The simplest compound 5a, with no substitution on the
Based on our data, compound 5b depicted the best BChEI
activity (IC50 = 1.95 μM) which has chlorine in position R1, It
was 25 folds more potent than parent compound 5a. The ob-
served IC50 values of these designed compounds (5a-5r)
against BChE demonstrated that these compounds had mild
aryl groups, showed moderated activity against AChE (IC50
=
53.21 μM) and BChE (IC50 = 25.27 μM). Replacement of
hydrogen atom on the cinnamic acid moiety with chlorine

DARU J Pharm Sci
Table 1 AChE and BChE inhibition IC50 (μM) of compounds, Selectivity index IC50 (BChE)/IC50 (AChE)
5 a-r
Entry Compunds
R1
R2
R3
AChE inhibiꢀon BChE inhibiꢀon
Selecꢀvity
index
0.47
0.09
0.76
0.62
0.19
0.49
0.51
0.49
0.52
0.56
-
IC50 ( μM )
53.21
21.11
32.63
27.2
IC50 ( μM )
25.27
1.95
1
2
5a
5b
H
Cl
H
H
H
H
H
3
5c
H
Cl
24.81
17.1
4
5d
H
H
Cl
5
5e
F
H
H
33.57
31.89
48.14
23.77
31.34
39.22
95.85
>100
6.67
6
5f
H
F
H
15.77
24.94
11.8
7
5g
H
H
F
8
5h
CH3
H
H
H
9
5i
CH3
H
H
16.44
22.33
18.55
50.46
26.15
18.25
41.25
25.6
10
11
12
13
14
15
16
17
18
19
5j
H
CH3
H
5k
NO2
H
H
NO2
H
H
OCH3
H
5l
H
NO2
H
H
OCH3
-
5m
5n
H
65.36
19.34
27.58
14.36
11.51
21.92
0.039
0.4
OCH3
H
0.94
1.49
1.78
-
5o
5p
H
5q
H
OCH3 OCH3
OCH3 OH
>100
17.82
7.7
5r
H
0.81
193.43
Donepezil
-
-
-
to good activity against BChE and Compound 5q with two
methoxy group in position R2 and R3, showed no inhibitory
effect against BChE. It should be noted that compound 5q
acted as a selective AChE inhibitor. Replacement of hydrogen
in position R2 with the nitro group (IC50 = 50.46 μM) or
methoxy group (IC50 = 41.25 μM) led to decrease of inhibito-
ry activity against BChE significantly, and showed less anti-
BChE activity compared with parent compound 5a and other
with negligible difference in IC50 values. Also, compounds
5f with fluorine atoms in position R2 and 5i with the methyl
group in position R2 showed good inhibitory activity toward
BChE as IC50 = 15.77 μM and 16.44 μM respectively. In
compounds 5 h which had the methyl group in position R1
and 5e which had fluorine atom in position R1, we have seen
an increase in inhibitory activity of BChE as IC50 = 11.8 μM
and 6.67 μM respectively. However, the best inhibitory activ-
ity was obtained by compound 5b with IC5 0 = 1.95 μM which
had chlorine in position R1.
derivatives. Compounds 5n (IC50 = 18.25 μM), 5 k (IC50
=
18.55 μM) and 5r (IC50 = 17.81 μM) which respectively had
methoxy group or nitro group in position R1 or methoxy group
in position R2 and hydroxyl group in position R3 instead of
hydrogen depicted better inhibitory activity against BChE
With information obtained from IC50 values, the process of
anti-BChE activity did not follow anti-AChE activity as com-
pound 5q with the strongest inhibitory effect toward AChE,

DARU J Pharm Sci
Fig. 2 The Lineweaver-Burk secondary plots of compounds 5b (A) and 5q (B)
showed no inhibitory effect on BChE and it was exclusively
served for the inhibition of AChE and compounds 5 k and 5 l
were exclusive for BChE.
Parallel to the progression of AD, there is a significant
increase in BChE level in the most parts of the brain, therefore
inhibition of BChE could be important as well as AChE.
Recent studies shows selective BChE inhibitors enhance
learning in animal models [26].
and AChE and suggesting that these compounds have the
ability to bind both the peripheral anionic site (PAS) and the
catalytic anionic site (CAS) of BChE and AChE. As can be
seen in Fig. 3, for compound 5b the inhibition constant Ki
(Ki = 0.97 μM) and for compound 5q the inhibition constant
Ki (Ki = 6.53 μM) were calculated using the graph of the slope
versus concentration of synthesis inhibitors (Table 2).
Neuroprotective study
Kinetic studies
A useful model to assess the neuronal differentiation is the
PC12 cell line. For this aim, differentiated PC12 used as
in vitro model and hydrogen peroxide (H2O2) used as an
oxidative agent. Therefore subjecting PC12 to H2O2-in-
duced damage depicted the neuroprotective activity of the
most potent compound 5q toward AChE. The percent of cell
In order to obtain the kinetic model of BChE and AChE inhi-
bition by the most active compounds 5b and 5q, Ellman s
method has been used to achieve this aim. The graphical anal-
ysis of Lineweaver-Burk reciprocal plot showed in Fig. 2.
This graph depicted a mixed type of inhibition for BChE
Fig. 3 Steady state inhibition of BChE and AChE by compound 5b (A) and 5q (B)

DARU J Pharm Sci
Table 2 Ki value for compounds 5b and 5q
BACE1 inhibitory acivity
Compounds
Ki (μM, BChE)
Ki (μM, AChE)
To investigate the BACE-1 inhibitory activity of the most
potent compound toward AChE (5q) through the BACE-1
kit that includes of BACE-1 enzyme and specific APP
based peptide substrate and on the basis of fluorescence
resonance energy transfer (FRET) was done. All the ex-
periments were repeated three times and were compared
with the reference compound OM99–2, a hexapeptid de-
rivative (CAS Number 314266–76-7). The results in
Table 3 show that compound 5q demonstrated BACE1
5b
5q
0.97
–
–
6.53
viability in comparison to control group (Quercetin)
showed in Fig. 4 [1, 25]. On the basis of the result, com-
pound 5q showed mild neuroprotective activity at 10 μM
(P < 0.05 vs. H2O2 alone) but in concentration 1 and
100 μM showed no significant neuroprotective activity.
inhibitory activity weaker than OM99–2 with IC50
0.014 0.001 μM.
=
Fig. 4 Neuroprotective effect of
compound 5q on cell viability of
PC12 cells in H2O2-induced
damage. Data are expressed as
mean SD (n = 8) and one-way
analysis of variance (ANOVA)
followed by Tukeys multiple
comparision test was carried out
to determine the level of signifi-
cance. ####P < 0.0001 vs. control,
****P < 0.0001 vs. H2O2 and
*P < 0.05 vs. H2O2

DARU J Pharm Sci
Table 3 Inhibitory activity of compound 5q against BACE1 comparing with OM99–2
Entry
Compound
Inhibition at 50 μMa (%)
Inhibition at 10 μMa (%)
IC50 (μM)
1
2
5q
OM99–2b
43.73
27.81
–
–
–
0.014 0.001
a Values represent means standard error (S.E.) of three independent experiments. b OM99–2 was tested at 10, 1 and 0.1 nM
Inhibition of self-induced Aβ aggregation
(ThT) fluorescence method and compared with donepezil
and curcumin as reference compounds. The result of the inhi-
bition for compound 5q at 50 μM concentration was 10.12
1.23% that could be comparable with donepezil (14.70
2.35%).
The most potent compound of this series of synthesized com-
pound (5q) was selected to investigate its ability to inhibit
Aβ1–42 peptide self-aggregation utilizing the thioflavin T
Fig. 5 a Superimosition of the
most potent compound 5q
(purple) and donepezil (green) in
the active site of AChE. b The
binding mode of the most active
compound 5q in the active site of
AChE

DARU J Pharm Sci
Fig. 6 a Superimposition of the
most potent compound 5b
(purple), donepezil (green) and
tacrinne (blue) in the active site of
BChE. b The binding mode of the
most active compound 5b in the
active site of BChE
Docking studies
and the catalytic site (CAS) of AChE. As Fig. 5b, tryptamine
moiety occupied the CAS with two П-П stacking interaction
between Phe330 and Trp 84 residues respectively. There is an
extra polar interaction with Glu 199 residues in CAS and
cinnamic acid moiety that was located the PAS and there is
hydrophobic interaction between methoxy group and Tyr 121
and Tyr 70 residues which they are two key residues in PAS.
In addition, there is another hydrophobic interaction between
the methoxy group and Asn 85 and a hydrogen bonding be-
tween the oxygen of carboxyl group and Tyr130 led to stabi-
lization of resulting complex between compound 5q and the
active site of the enzyme.
In order to better understand the inhibition of AChE, BChE
and possible interaction between compounds 5q and 5b with
active site of the enzyme, the molecular modeling study was
carried out. It was done using the docking program, auto duck
4.2 and discovery studio 4.0 client. The results in Figs. 5a, b
and 6a, b depicted that compounds 5q and 5b with a proper
orientation located in the binding site of AChE and BChE.
These figure showed that compounds 5q and 5b are long
enough to accommodate in the peripheral anionic site (PAS)
On the other hand, docking result of compound 5b in
the active site of BChE was illustrated in Fig. 6b. As this
figure, cinnamic acid moiety of compound 5b formed П-
alkyl stacking interaction between Leu 286 and Val 288
residues. Furthermore, П-П stacking interaction between
Triptamine moiety and Trp 82 residue and a hydrogen
bonding with Asp 70 residue (in the PAS) could be ob-
served. Moreover, two П-sigma interactions with Trp 231
and Ala 328, one П-alkyl interaction with Phe 329 and
Table 4 Molecular descriptors of the compound 5q
Entry Compound HBD HBA Clog P tPSA (Ao2) MW
RBC
7
1
5q 3.55 59.59 350.42
2
3
ClogP: Calculated octaol-water partition coefficient, HBD: number of H-
bond donors, HBA: number of H-bond acceptor, RBC: rotatable bonds
count, tPSA: topological polar surface area

DARU J Pharm Sci
one conventional hydrogen bonding with Gly 78 residues
led to well fitted of compound 5b in the active site of
BChE.
These figures clear that compounds 5q and 5b were a dual
binding site inhibitors which could be simultaneously
interacted with both CAS and PAS of AChE and BChE.
References
1. Najafi Z, Mahdavi M, Saeedi M, Karimpour-Razkenari E, Asatouri
R, Vafadarnejad F, et al. Novel tacrine-1,2,3-triazole hybrids:
in vitro, in vivo biological evaluation and docking study of cholin-
esterase inhibitors. Eur J Med Chem. 2017;125:1200–12.
2. Ting Luo X, Wang C, Liu Y, Huang ZG. New multifunctional
melatonin-derived benzylpyridinium bromides with potent cholin-
ergic, antioxidant and neuroprotective properties as innovative
drugs for Alzheimer’s disease. Eur J Med Chem. 2015;103:302–11.
3. Ghanei-Nasab S, Khoobi M, Hadizadeh F, Marjani A, Moradi A,
Nadri H, et al. Synthesis and anticholinesterase activity of
coumarin-3-carboxamides bearing tryptamine moiety. Eur J Med
Chem. 2016;121:40–6.
4. Mostofi M, Mohammadi Ziarani G, Mahdavi M, Moradi A, Nadri
H, Emami S, et al. Synthesis and structure-activity relationship
study of benzofuran-based chalconoids bearing benzylpyridinium
moiety as potent acetylcholinesterase inhibitors. Eur J Med Chem.
2015;103:361–9.
5. Mollazadeh M, Mohammadi-Khanaposhtani M, Zonouzi A, Nadri
H, Najafi Z, Larijani B, et al. New benzyl pyridinium derivatives
bearing 2,4-dioxochroman moiety as potent agents for treatment of
Alzheimer’s disease: design, synthesis, biological evaluation, and
docking study. Bioorg Chem. 2019;87:506–15.
6. Khoobi M, Alipour M, Saakhteman A, Nadri H, Moradi A,
Ghanadi M, et al. Design, synthesis, biological evaluation and
docking study of 5-oxo-4,5-dihydropyrano[3,2-c]chromene deriv-
atives as acetylcholinesterase and butyrylcholinesterase inhibitors.
Eur J Med Chem. 2013;68:260–9.
Virtual calculation of pharmacokinetic properties
In order to evaluate the competence of compound 5q as a drug
candidate, it is necessary some pharmacokinetic properties
such as octanol-water partition coefficient (ClogP), number
of H-bond donors (HBD), number of H-bond acceptor
(HBA), rotatable bonds count (RBC) and topological polar
surface area (tPSA) calculated and the outcome of the assess-
ment for compound 5q shown in Table 4. Based on Lipinski’s
rule-of-five including MW ≤ 500, HBD ≤ 5, HBA ≤ 10,
ClogP≤5 and polar surface area ≤ 140Ao2, it seems that these
synthesized compound are the good candidate to orally active
drug in human.
7. Pb W, Zimmerman HJ, Knapp MJ, Gracon SI, Lewis KW.
Hepatotoxic effects of tacrine administration in patients with
Alzheimer s disease. JAMA. 1994;271:992–8.
8. Baharloo F, Moslemin MH, Nadri H, Asadipour A, Mahdavi M,
Firoozpour L, et al. Benzofuran-derived benzylpyridinium bro-
mides as potent acetylcholinesterase inhibitors. Eur J Med Chem.
2015;93:196–201.
9. Najafi Z, Saeedi M, Mahdavi M, Sabourian R, Khanavi M, Tehrani
MB, et al. Design and synthesis of novel anti-Alzheimer s agents:
Acridine-chroomenone and quinolone-chromenone hybrids.
Bioorg Chem. 2016;67:84–94.
10. Estrada M, Herrera-Arozamena C, Perez C, Vina D, Romero A,
Morales-Garcia JA, et al. New cinnamic-N-benzylpiperidine and
cinnamic-N,N-dibenzyl (N-methyl) amine hybrids as Alzheimer-
directed multitarget drugs with antioxidant, cholinergic, neuropro-
tective and neurogenic properties. Eur J Med Chem. 2016;121:376–
86.
11. Arab S, Sadat-Ebrahimi S, Mohammady-Khanaposhtani M,
Moradi A, Nadri H, Mahdavi M, et al. Synthesis and evaluation
of chroman-4-one linked to N-benzyl pyridinium derivatives as
new acethylcholinesterase inhibitors. Arch Pharm Chem.
2015;384:1–7.
12. Saeed A, Mahesar PA, Zaib S, Siraj Khan M, Matin A, Shahid M,
et al. Synthesis, cytotoxicity and molecular modeling studies of new
phenylcinnamide derivatives as potent inhibitors of cholinesterase.
Eur J Med Chem. 2014;78:43–53.
13. Hariri R, Afshar Z, Mahdavi M, Safavi M, Saeedi M, Najafi Z, et al.
Novel Tacrine-based Pyranol [3′,4′:5,6]pyrano[2,3-b]quinolinones:
synthesis and cholinesterase inhibitory activity. Arch Pharm Chem.
2016;349:1–10.
14. Xu W, Bing Wang X, Min Wang Z, Wu JJ, Li F, Wang J, et al.
Synthesis and evaluation of donepezil-ferulic acid hybrids as multi-
targeted ligands against Alzheimer s disease. Med Chem Commun.
2016;7:990–8.
Conclusion
In summary, we reported design and synthesis of a new series
of cinnamic acids-tryptamine hybrids as the new anti-AD
agent. The potency of these compounds for inhibition of
AChE was moderate to good. In this series, compound 5q
with IC50 = 11.51 μM was the most potent compound with
the inhibitory effect against AChE. The synthesized com-
pounds contained triptamine moiety and the cinnamic acid
group occupied the CAS and the PAS that was confirmed by
the kinetic and molecular modeling study. Furthermore, these
compounds was examined for anti-cholinesterase, neuropro-
tective, BACE1 inhibitory activity. It should be noted that the
cholinesterase inhibitory and neuro-protective effect of 5q
was acceptable; however it did not show potent BACE1 in-
hibitory activity. Docking study showed tryptamine group of
5b captures the peripheral anionic site of BChE. This interac-
tion is reinforced by cinnamic acid moiety that interacts with
catalytic site of enzyme. These findings shows that hybrid
between cinnamic acid and tryptamine increased the potency
of compounds 5 series via formation of a dual binding inter-
action with CAS and PAS. Based on Lipinski’s rule this com-
pound is the good candidate to orally active drug in human.
The results showed that this series with dual binding site for
interaction with AChE and BChE have a potential for the
treatment of AD.
Acknowledgments This work was supported by grants (9211302002)
from the Research Council of Tehran University of Medical Sciences.

DARU J Pharm Sci
15. Zhang X, He X, Chen Q, Lu J, Rapposelli S, Pi R. A review on the
hybrids of hydroxycinnamic acid as multi-targetdirected ligands
against Alzheimer’s disease. Bioorg Med Chem. 2016;26:543–50.
16. Khoobi M, Ghanoni F, Nadri H, Moradi A, Pirali Hamedani M,
Hmayouni Moghadam F, et al. New tetracyclic tacrine analogs con-
taining pyranol [2,3-c] pyrazole: efficient synthesis, biological as-
sessment and docking simulation study. Eur J Med Chem. 2015;89:
296–303.
17. Mohammadi-Khanaposhtani M, Mahdavi M, Saeedi M, Sabourian
R, Safavi S, Khanavi M, et al. Design, synthesis, biological evalu-
ation, and doking study of acetylcholinesterase inhibitors: new
acridone-1,2,4-oxadiazole-1,2,3-triazole hybrids. Chem Biol Drug
Des. 2015;86:1425–32.
18. Wang J, Cai P, Yang X, Li F, Wu JJ, Kong LY, et al. Novel
cinnamide-dibenzyl hybrids: potent neurogenic agents with antiox-
idant, cholinergic, and neuroprotective properties as innovative
drugs for Alzheimer s disease. Eur J Med Chem. 2017;139:68–83.
19. Bhakta HK, Park CH, Yokozowa T, Tanaka T, Jung AH, Choi JS.
Potent anti-cholinesterase and β-amyloid precursor protein cleav-
ing enzyme 1 inhibitory activities of cornuside and gallotannins
from cornus officinalis fruits. Arch Pharm Res. 2017;40:836–53.
20. Pietrzik C, Behl C. Concepts for the treatment of Alzheimer s dis-
ease: molecular mechanisms and clinical application. Int J Exp
Pathol. 2005;86:173–85.
linked to 1,2,3-triazole ring system: investigation of biological ac-
tivities against Alzheimer s disease. Bioorg Chem. 2017;70:86–93.
22. Bartolini M, Bertucci C, Bolognesi ML, Cavalli A, Melchiorre C,
Andrisano V. Insight into the kinetic of amyloid beta (1-42) peptide
self-aggregation: elucidation of inhibitors’ mechanism of action.
ChemBioChem. 2007;8:2152–61.
23. Zha GF, Zhang CP, Qin HL, Jantan L, Sher M, Amjad MW, et al.
Biological evaluation of synthetic α,β-unsaturated carbonyl based
cyclohexanone derivatives as neuroprotective novel inhibitors of
acetylcholinesterase, butyrylcholinesterase and amyloid-β aggrega-
tion. Bioorg Med Chem. 2016;24:2352–9.
24. Reinke AA, Gestwicki JE. Structure-activity relationships of amy-
loid beta-aggregation inhibitors based on curcumin: influence of
linker length and flexibility. Chem Biol Drug Des. 2007;70:206–15.
25. Gordone J, Amini S, White MK. General overview of neuronal cell
culture. Methods Mol Biol. 2013;1078:1–8.
26. Ghobadian R, Mahdavi M, Nadri H, Moradi A, Edraki N,
Akbarzadeh T, et al. Novel tetrahydrocarbazole benzyl pyridine
hybrids as potent and selective butryl cholinesterase inhibitors with
neuroprotective and b-secretase inhibition activities. Eur J Med
Chem. 2018;155:49–60.
Publisher’s note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
21. Saeedi M, Safavi M, Karimpour-Razkenari E, Mahdavi M, Edraki
N, Homayouni Moghadam F, et al. Synthesis of novel chromenons