Synthesis and anticholinesterase activity of coumarin-3-carboxamides bearing tryptamine moiety

Article abstract of DOI:10.1016/j.ejmech.2016.05.014

A number of N-(2-(1H-indol-3-yl)ethyl)-2-oxo-2H-chromene-3-carboxamides were synthesized and tested against AChE and BuChE. The in?vitro assessment of the synthesized compounds 4a-o revealed that most of them had significant activity toward AChE. The SAR study demonstrated that the introduction of benzyloxy moiety on the 7-position of coumarin scaffold can improve the anti-AChE activity. The best result was obtained with 7-(4-fluorobenzyl)oxy moiety in the case of compound 4o, displaying IC₅₀value of 0.16?μM. Based on the docking study of AChE, the prototype compound 4o was laid across the active site and occupied both peripheral anionic site (PAS) and catalytic anionic site (CAS).

Full text of DOI:10.1016/j.ejmech.2016.05.014

Accepted Manuscript
Synthesis and anticholinesterase activity of coumarin-3-carboxamides bearing
tryptamine moiety
Samaneh Ghanei-Nasab, Mehdi Khoobi, Farzin Hadizadeh, Azam Marjani, Alireza
Moradi, Hamid Nadri, Saeed Emami, Alireza Foroumadi, Abbas Shafiee
PII:
S0223-5234(16)30391-9
10.1016/j.ejmech.2016.05.014
EJMECH 8603
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 March 2016
Revised Date: 1 May 2016
Accepted Date: 3 May 2016
Please cite this article as: S. Ghanei-Nasab, M. Khoobi, F. Hadizadeh, A. Marjani, A. Moradi, H.
Nadri, S. Emami, A. Foroumadi, A. Shafiee, Synthesis and anticholinesterase activity of coumarin-3-
carboxamides bearing tryptamine moiety, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.05.014.
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ACCEPTED MANUSCRIPT
Synthesis and anticholinesterase activity of coumarin-3-carboxamides bearing
tryptamine moiety
Samaneh Ghanei-Nasab, Mehdi Khoobi, Farzin Hadizadeh, Azam Marjani, Alireza Moradi,
Hamid Nadri, Saeed Emami, Alireza Foroumadi, Abbas Shafiee*
O
N
H
O
O
O
N
H
F
4o
A
number of N-(2-(1H-indol-3-yl)ethyl)-2-oxo-2H-chromene-3-carboxamides were
synthesized and tested against AChE and BuChE. Compound 4o with IC50 value of 0.16 µM
was the most potent compound against AChE.

ACCEPTED MANUSCRIPT
Synthesis and anticholinesterase activity of coumarin-3-carboxamides
bearing tryptamine moiety
a
d
a
Samaneh Ghanei-Nasab , Mehdi Khoobi b,c, Farzin Hadizadeh , Azam Marjani , Alireza
Moradi e, Hamid Nadri e, Saeed Emami f, Alireza Foroumadi c, Abbas Shafiee c,*
a Department of Chemistry, Arak Branch, Islamic Azad University, Arak, Iran
bDepartment of Pharmaceutical Biomaterials and Medical Biomaterials Research Center,
Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
c
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medicinal Sciences, Tehran, Iran
dBiotechnology Research Center, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad, Iran
e
Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of
Medical Sciences, Yazd, Iran
f Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty
of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
Abstract:
A
number of N-(2-(1H-indol-3-yl)ethyl)-2-oxo-2H-chromene-3-carboxamides were
synthesized and tested against AChE and BuChE. The in vitro assessment of the synthesized
compounds 4a-o revealed that most of them had significant activity toward AChE. The SAR
study demonstrated that the introduction of benzyloxy moiety on the 7-position of coumarin
scaffold can improve the anti-AChE activity. The best result was obtained with 7-(4-
fluorobenzyl)oxy moiety in the case of compound 4o, displaying IC50 value of 0.16 µM.
Based on the docking study of AChE, the prototype compound 4o was laid across the active
site and occupied both peripheral anionic site (PAS) and catalytic anionic site (CAS).
Keywords: Acetylcholinesterase; butyrylcholinesterase; Alzheimer’s disease; coumarin;
indole.
Corresponding author. Tel.: +98 21 66406757; fax: +98-21-66461178.
E-mail address: shafieea@tums.ac.ir (A. Shafiee).
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1. Introduction
Alzheimer's disease (AD) is an age-related, progressive and multi-factorial neurodegenerative
disorder in the elderly population. The neuropathological changes in the brain of patients with
AD lead to decline in language skills, progressive cognitive impairment, and dementia [1,2].
This disease which depends on a combination of genetic and environmental factors, reaches
epidemic proportions, with a great social, and economic burden worldwide [3,4].
Although the etiopathogenesis of AD is complicated and unknown, several hallmarks
including acetylcholine (ACh) deficiency and aggregation of beta-amyloid peptide (Aβ) are
found to be involved in the onset and progression of this disease. In particular, the
presynaptic decrease of ACh which may be due to damage of cholinergic neurons in some
special parts of the brain results in a generalized cognitive decline [5-7]. Based on the
cholinergic hypothesis, the increase of ACh via inhibiting acetylcholinesterase (AChE) in the
brain is an effective approach to retard the AD’s symptoms [8,9]. The AChE is the main
enzyme responsible for hydrolysis of ACh in the central nervous system (CNS). Accordingly,
most studies have focused on AChE inhibition in the treatment of AD [10,11]. Therefore,
AChE inhibitors such as donepezil, rivastigmine, galantamine and tacrine have been
demonstrated to have great potential in the treatment of AD patients. All of these drugs with
the exception of tacrine are still in clinical use. Tacrine was withdrawn from the market due
to the hepatotoxicity [12,13]. Primarily, AChE inhibitors have been considered only as
symptomatic therapy for AD, however recent studies have suggested that AChE inhibitors
can act as disease-modifying agents by inhibition of the amyloid cascade, modulation of
various proteins' activity, regulation of cerebral blood flow and neuroprotection [14-16].
Coumarin moiety is an important aromatic ring with a broad spectrum of biological activity
[17]. Among the various classes of compounds studied for design of new AChE inhibitors,
coumarin scaffold has received great attention, due to its ability for binding to peripheral
anionic site (PAS) of AChE [18]. Recently, we have reported different series of coumarin-
based AChE inhibitors [19-23]. On the other hand, some authors have described the
usefulness of indole amine framework in the design of new AChE inhibitors or multi-target
agents for AD therapy. For example, Luo et al. synthesized a series of indole amine-based
benzylpyridinium bromides (I, Fig. 1) as multi-functional anti-AD agents with cholinesterase
inhibitory, antioxidant, and neuroprotective activities [24]. In a study by Cheng and co-
workers, some (−)-meptazinol–indole amine hybrids (II) were synthesized as potential multi-
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target-directed ligands against AD with dual inhibitory potency against cholinesterases and
effective inhibition of Aβ self-aggregation [25]. In order to find new lead candidate for AD
therapy, Peng et al. designed p-hydroxybenzamide derivatives III bearing indole amine
scaffold and used it for step-by-step optimization of the framework to obtaining potential
multi-site AChE inhibitors [26]. In this context and in continuation of our recent works [27-
29], it seems very interesting to develop some coumarin carboxamide derivatives bearing
indole amine (tryptamine) scaffold in order to find new AChE inhibitors. Thus, we report
here the synthesis and cholinesterases inhibitory activity of N-(2-(1H-indol-3-yl)ethyl)-2-oxo-
2H-chromene-3-carboxamides 4a-o (Fig. 1).
2. Chemistry
The synthetic routes to intermediates 3a-o and final compounds 4a-o were depicted in
Schemes 1 and 2, respectively. The cyclization reaction of salicylaldehyde derivatives 1a-d
with diethyl malonate in the presence of piperidine in ethanol afforded ethyl coumarin-3-
carboxylate 2a-d. The 7-hydroxy derivative 2b was reacted with alkyl halides or benzyl
halides by using potassium carbonate as a base in DMF to give O-alkyl or O-benzyl
derivatives 2e-o. The ethyl esters 2a-o was hydrolyzed in the presence of NaOH to give
corresponding coumarin-3-carboxylic acids 3a-o (Scheme 1). In the final step, the acids 3a-o
were converted to related acid chlorides by using thionyl chloride. Subsequently, the obtained
acid chlorides were reacted with tryptamine in the presence of potassium carbonate in dry
toluene to afford final compounds 4a-o (Scheme 2). The final step was also evaluated under
microwave irradiations in different solvents, as well as solvent-free condition, to decrease the
reaction time. The best result was obtained in acetonitrile as solvent. Interestingly, the time of
the reaction was decreased from about 15 hours under conventional condition to around five
minutes under microwave irradiation (Supplementary material, Table S1).
3. Results and discussion
3.1. In vitro anticholinesterase activity
The in vitro anticholinesterase activity of designed compounds 4a-o was determined against
AChE and BuChE. The obtained IC50 values (in µM) were presented in Table 1. The IC50s
against AChE revealed that most of compounds possess significant activity in the range of
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0.16 to 43.8 µM. Among them, the (4-fluorobenzyl)oxy derivative 4o with IC50 of 0.16 µM
showed the highest activity against AChE.
In the structure-activity point of view, the simplest compound 4a with no substitution on the
core structure exhibited good activity against AChE. Introduction of 6-bromo substituent on
the coumarin ring resulted in inactive compound 4b. In contrast, the 8-methoxy substitution
could improve the activity as observed in compound 4c. While the insertion of 7-hydroxy
group dramatically eliminated the anti-cholinesterase activity, but O-alkylation or O-
benzylation of 7-hydroxy could retained the activity. 7-hydroxy analog 4d was inactive (IC50
> 100 µM) however O-alkyl and O-benzyl derivatives (compounds 4e-h) showed mild to
good activity against AChE. In particular, the O-benzyl derivative 4h with IC50 value of 1.5
µM was about four times more potent than parent compound 4a. In the 7-alkoxy derivatives
4e-g, the best activity was observed with 7-methoxycoumarin derivative 4e. The activities of
7-ethoxy and 7-propoxy analogs (4f and 4g) were less than those of unsubstituted counterpart
4a and 7-methoxy derivate 4e. Thus, the elongation of O-alkyl group diminished the activity.
The comparison of 7-benzyloxy 4h and unsubstituted compound 4a demonstrated that the
insertion of 7-benzyloxy group could improve the anti-cholinesterase activity. A survey on
the IC50 values of O-benzyl derivatives 4h-o revealed that the introduction of one or two
chlorine atoms significantly decreased the anti-AChE activity. However, the fluoro
substituent was tolerated on the benzyl moiety as observed in compounds 4m-o. The 2-
fluoro- and 3-fluorobenzyl derivatives (4m and 4n) with IC50 values of 2.5 µM showed same
activity against AChE. The displacement of fluorine atom from ortho or meta positions to
para position of benzyl moiety greatly improved the activity against AChE. The most potent
compound 4o bearing a (4-fluorobenzyl)oxy residue was 15-fold more potent than
compounds 4m and 4n. Moreover, the activity of 4-fluorobenzyl derivative 4o was 9 times
superior to that of benzyl analog 4h.
The obtained data of test compounds 4a-o against BuChE demonstrated that the most of
designed compounds had mild or no activity against this enzyme. The O-benzyl derivative 4h
with IC50 of 16.2 µM was the most potent compound against BuChE. It was 3-fold more
potent than parent compound 4a. Furthermore, the 8-methoxy analog 4c showed suitable anti-
BuChE activity (IC50= 20.1 µM). The chloro-substituted benzyloxycoumarins 4i-l were
inactive against BuChE, while the fluorobenzyl congeners 4m-o could properly inhibit this
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enzyme (IC50s < 30 µM). In general, the inhibitory activity of all compounds against AChE
was higher over BuChE.
3.2. Kinetic study of AChE inhibition
To assess the kinetic mode of AChE inhibition displayed by target compounds, the most
active compound 4o was subjected to kinetic studies. For this purpose, the rate of enzyme
activity was measured at four different concentrations of inhibitor 4o (0, 0.069, 0.206 and
0.618 µM) in the presence of different concentrations of substrate (ATCh). For each inhibitor
concentration, the initial velocity was measured at different substrate concentrations (S) and
the reciprocal of the initial velocity (1/v) was plotted versus the reciprocal of substrate
concentration (1/[s]). As depicted in Fig. 2, the obtained double reciprocal (Lineweavere-
Burk) plot showed a mixed-type inhibition pattern for compound 4o. The Ki value was also
calculated using the secondary plot (Ki = 0.49 µM, Fig. 2). All experiments were performed
as same as cholinesterase inhibition test described in experimental section.
3.3. Docking simulation
As the target compounds showed moderate to high selective inhibitory activity against AChE,
the molecular modeling studies were performed to obtain the proposed binding mode of the
most active compound 4o in the active site of AChE. AutoDock Vina (ver. 1.1.2) was used
for all docking experiments. Docking validation was based on the co-crystallized ligand
(E2020) from crystal structure (1EVE) and it was shown that docking runs were able to
reproduce original arrangement of the ligand with low RMSD value below 1 Å. As depicted
in Fig. 3, the target compound was laid across the active site and occupied both PAS and
CAS (catalytic anionic site) of enzyme active site where the 4-fluorobenzyl moiety oriented
toward the bottom of active site. A closer examination of this binding mode was done to find
out the interactions of this compound with enzyme residues. As shown in Fig. 3, the 4-
fluorophenyl ring formed a face-to-face π-π stacking with Trp84. Another π-π stacking was
observed between coumarin ring and Tyr334. The indole ring of this compound also
interacted with PAS through a π-π interaction with Trp279.
4. Conclusion
We have synthesized some coumarin carboxamide derivatives bearing indole amine scaffold
(tryptamine) as new cholinesterase inhibitors. The in vitro assessment of synthesized
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compounds 4a-o against AChE/BuChE revealed that most of compounds had significant
activity toward AChE. The modification of substituent on the coumarin ring could modulate
the activity of designed compounds against cholinesterases. The best result was obtained with
7-(4-fluorobenzyl)oxy moiety against AChE in compound 4o, displaying IC50 value of 0.16
µM. The SAR study demonstrated that the introduction of O-benzyl moiety on the 7-position
of coumarin scaffold can improve the anti-AChE activity. The docking simulation study
showed the dual binding mode of the promising compound 4o. As a potent inhibitor of AChE
with dual binding to both PAS and CAS, this prototype compound could be a good candidate
for further studies and modifications.
5. Experimental
All commercially available reagents were purchased from Merck AG or Aldrich and used
without further purification. TLC was conducted on silica gel 250 micron. Melting points
were measured on the Buchi Melting point B-540. FT-IR spectra were run on a Nicolet FT-IR
Magna 550 spectrograph (KBr discs). 1H NMR spectra were recorded on a Bruker 300 MHz
NMR instrument. The chemical shifts (δ) and coupling constants (J) are expressed in parts
per million and Hertz, respectively. The final step of synthesis was alternatively carried out
under microwave irradiation using a microwave oven (MicroSYNTH, Microwave
Labstation).
5.1. General procedure for the synthesis of compounds 2a-d
A mixture of salicylaldehyde derivatives 1a-d (5 mmol), diethyl malonate (6 mmol) and
piperidine (0.2 ml) was refluxed in ethanol (10 ml) for 14-16 h. After completion of the
reaction (monitored by TLC), the reaction mixture was cooled, filtrated and washed with cold
ethanol to obtain white solid. The solid was recrystallised from ethanol to give pure
compounds 2a-d.
5.2. General procedure for the synthesis of compounds 2e-o
Ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (2b, 5 mmol) and potassium carbonate
(6 mmol) were mixed with DMF (10 ml). The mixture was stirred at room temperature for
several minutes and then appropriate alkyl halide or benzyl halide derivative (7 mmol) was
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added dropwise to the mixture. The reaction mixture was stirred at room temperature for 14-
16 h. After completion of the reaction (monitored by TLC), the mixture was diluted with
water. The precipitate was filtered, washed with water, and used without further purification.
5.3. General procedure for the synthesis of compounds 3a-o
NaOH solution (2N, 25 ml) was added to the ester derivatives 2a-o, and the mixture was
stirred at room temperature for 5-7 h. After cooling, hydrochloric acid (2N) was added to the
yellow solution until a white precipitate formed. The precipitated solid was isolated by
filtration, washed with water and dried to give compounds 3a-o.
5.4. General procedure for the synthesis of compounds 4a-o
Compounds 3a-o (1 mmol) were added to thionyl chloride (5 ml) and the mixture was
refluxed for 5-6 h. After completion of the reaction, thionyl chloride was removed with
simple distillation to give appropriate coumarin-3-carboxylic acid chloride. The crude
product was used directly without further purification. In the next step, the corresponding
acid chloride, tryptamine (1 mmol) and potassium carbonate (2 mmol) were suspended in dry
toluene (15 ml). The mixture was refluxed for 14-16 h under argon atmosphere. After
completion of the reaction (monitored by TLC), the solvent was removed under reduced
pressure and the residue was washed with water and dried to give compounds 4a-o.
Alternatively the same reaction could carry out under microwave irradiations at 85 ºC for 5
min.
5.4.1. N-(2-(1H-Indol-3-yl)ethyl)-2-oxo-2H-chromene-3-carboxamide (4a). Pale brown solid;
1
yield 98%; mp 184-188 ºC; IR (KBr, cm-1) νmax: 3500, 1710, 1680; H NMR (300 MHz,
Acetone-d6) δ: 10.09 (br, 1H, NH), 8.93 (s, 1H, H4 coumarin), 8.88 (br, 1H, NH), 7.98 (d, J =
7.8 Hz, 1H, H5 coumarin), 7.79 (t, J = 7.8 Hz, 1H, H6 coumarin), 7.71 (d, J = 7.8 Hz, 1H, H8
coumarin), 7.48 (t, J = 7.8 Hz, 1H, H7 coumarin), 7.47 (d, J = 8.0 Hz, 1H, H7 indole), 7.41 (d,
J = 8.0 Hz, 1H, H4 indole), 7.28 (s, 1H, H2 indole), 7.12 (t, J = 8.0 Hz, 1H, H5 indole), 7.04
(t, J = 8.0 Hz, 1H, H6 indole), 3.78 (t, J = 7.2 Hz, 2H, NH-CH2), 3.11 (t, J = 7.2 Hz, 2H,
13
indole-CH2). C NMR (75 MHz, Acetone-d6) δ: 160.9, 160.8, 154.4, 147.6, 136.9, 133.8,
130.1, 127.6, 125.1, 122.6, 121.2, 119.0, 118.8, 118.5, 118.4, 116.1, 112.3, 111.3, 40.2, 25.2.
Anal. Calcd for C20H16N2O3 (332.35): C, 72.28; H, 4.85; N, 8.43. Found: C, 72.01; H, 4.57;
N, 8.16.
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5.4.2. 6-Bromo-N-(2-(1H-indol-3-yl)ethyl)-2-oxo-2H-chromene-3-carboxamide (4b). Pale
1
brown solid; yield 82%; mp 221-224 ºC; IR (KBr, cm-1) νmax: 3500, 1710, 1680; H NMR
(300 MHz, CDCl3) δ: 8.85 (brs, 2H, NH and H4 coumarin), 8.06 (s, 1H, NH), 7.84 (t, J = 7.8
Hz, 1H, H7 coumarin), 7.76 (d, J = 7.8 Hz, 1H, H5 coumarin), 7.69 (d, J = 7.8 Hz, 1H, H8
coumarin), 7.41 (d, J = 8.1 Hz, 1H, H7 indole), 7.13-7.36 (m, 4H, H2, 4, 5, 6 indole), 3.83 (m,
2H, NH-CH2), 3.14 (t, J = 6.9 Hz, 2H, indole-CH2). 13C NMR (75 MHz, DMSO-d6) δ: 161.1,
160.36, 153.3, 146.6, 136.7, 136.6, 132.5, 127.5, 123.3, 121.4, 120.8, 120.56, 118.8, 118.8,
118.7, 117.1, 111.8, 40.8, 25.4. Anal. Calcd for C20H15BrN2O3 (411.25): C, 58.41; H, 19.43;
N, 6.81. Found: C, 58.63; H, 19.21; N, 6.48.
5.4.3. N-(2-(1H-Indol-3-yl)ethyl)-8-methoxy-2-oxo-2H-chromene-3-carboxamide (4c). Pale
1
brown solid; yield 89%; mp 176-179 ºC; IR (KBr, cm-1) νmax: 3500, 1710, 1680; H NMR
(300 MHz, Acetone-d6) δ: 10.07 (brs, 1H, NH), 8.89 (brs, 2H, H4 coumarin and NH), 7.72 (d,
J = 7.8 Hz, 1H, H5 coumarin), 7.50 (t, J = 7.8 Hz, 1H, H6 coumarin), 7.36-7.46 (m, 3H, H7
coumarin and H4,7 indole), 7.28 (s, 1H, H2 indole), 7.12 (t, J = 7.5 Hz, 1H, H5 indole), 7.04 (t,
J = 7.5 Hz, 1H, H6 indole), 4.01 (s, 3H, CH3), 3.78 (m, J = 7.2 Hz, 2H, NH-CH2), 3.11 (t, J =
7.2 Hz, 2H, indole-CH2).13C NMR (75 MHz, Acetone-d6) δ: 161.15, 160.66, 147.96, 146.96,
143.91, 136.78, 127.55, 125.13, 122.56, 121.23, 121.06, 119.39, 119.01, 118.53, 118.45,
115.81, 112.10, 111.29, 55.84, 40.28, 25.17. Anal.Calcd for C21H18N2O4 (362.38): C, 69.60;
H, 5.01; N, 7.73. Found: C, 69.46; H, 4.89; N, 7.50.
5.4.4. N-(2-(1H-Indol-3-yl)ethyl)-7-hydroxy-2-oxo-2H-chromene-3-carboxamide (4d). Pale
1
brown solid; yield 91%; mp 264-266 ºC; IR (KBr, cm-1) νmax: 3500, 1710, 1680; H NMR
(300 MHz, DMSO-d6) δ: 10.85 (brs, 1H, NH), 8.81 (s, 1H, H4 coumarin), 8.77 (t, J = 5.7 Hz,
1H, NH), 7.82 (d, J = 7.8 Hz, 1H, H5 coumarin), 7.62 (d, J = 7.8 Hz, 1H, H6 coumarin), 7.35
(d, J = 8.1 Hz, 1H, H4 indole), 7.21 (s, 1H, H8 coumarin), 7.08 (t, J = 8.1 Hz, 1H, H5 indole),
6.99 (t, J = 8.1 Hz, 1H, H6 indole), 6.88 (d, J = 8.7 Hz, 1H, H7 indole), 6.80 (s, 1H, H2
indole), 3.45 (m, 2H, NH-CH2), 2.96 (t, J = 7.2 Hz, 2H, CH2 indole). 13C NMR (75 MHz,
DMSO-d6) δ: 164.25, 161.95, 161.49, 156.76, 148.45, 136.77, 132.43, 127.61, 123.27,
121.44, 118.84, 118.71, 114.87, 114.03, 111.98, 111.84, 111.54, 102.26, 25.53, 19.03. Anal.
Calcd for C20H16N2O4 (348.35): C, 68.96; H, 4.63; N, 8.04. Found: C, 66.81; H, 4.90; N,
8.25.
5.4.5. N-(2-(1H-Indol-3-yl)ethyl)-7-methoxy-2-oxo-2H-chromene-3-carboxamide (4e). Pale
1
brown solid; yield 85%; mp 191-193 ºC; IR (KBr, cm-1) νmax: 3500, 1710, 1680; H NMR
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(300 MHz, CDCl3) δ: 8.86 (brs, 1H, NH), 8.84 (s, 1H, H4 coumarin), 8.07 (br, 1H, NH), 7.67
(d, J = 7.5 Hz, 1H, H5 coumarin), 7.56 (d, J = 8.7 Hz, 1H, H7 indole), 7.38 (d, J = 8.1 Hz, 1H,
H4 indole), 7.08-7.24 (m, 3H, H6, 8 coumarin and H 5 indole), 6.93 (m, 1H, H6 indole ), 6.84 (s,
1H, H2 indole), 3.91 (s, 3H, OCH3), 3.79 (q, J = 7.5 Hz, 2H, NH-CH2), 3.10 (t, J = 7.5 Hz,
2H, indole-CH2). 13C NMR (75 MHz, CDCl3) δ: 164.7, 161.9, 161.7, 156.63, 148.1, 136.4,
130.9, 127.3, 122.1, 119.4, 118.8, 115.0, 113.9, 113.1, 112.4, 118.1, 100.3, 56.0, 40.1, 25.3.
Anal. Calcd for C21H18N2O4 (362.38): C, 69.60; H, 5.01; N, 7.73. Found: C, 69.43; H, 4.84;
N, 7.55.
5.4.6. N-(2-(1H-Indol-3-yl)ethyl)-7-ethoxy-2-oxo-2H-chromene-3-carboxamide (4f). Pale
1
brown solid; yield 88%; mp 201-203 ºC; IR (KBr, cm-1) νmax: 3500, 1710, 1680; H NMR
(300 MHz, Acetone-d6) δ: 10.86 (brs, 1H, NH), 8.86 (s, 1H, H4 coumarin), 8.78 (brs, 1H,
NH), 7.91 (d, J = 8.7 Hz, 1H, H5 coumarin), 7.62 (d, J = 7.5 Hz, 1H, H4 indole), 7.35 (d, J =
7.5 Hz, 1H, H7 indole), 7.21 (s, 1H, H2 indole ), 6.94-7.15 (m, 4H, H6, 8 coumarin and H5, 6
indole), 4.19 (q, J = 7.2 Hz, 2H, OCH2), 3.61 (q, J = 6.9 Hz, 2H, NH-CH2), 2.97 (t, J = 6.9
Hz, 2H, indole-CH2), 1.38 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) δ: 164.5,
161.8, 161.3, 156.9, 148.5, 136.5, 136.9, 132.1, 127.4, 123.8, 123.1, 121.9, 118.2, 118.6,
115.9, 114.8, 112.4, 111.1, 101.4, 64.0, 25.3, 14.7. Anal. Calcd for C22H20N2O4 (376.41): C,
70.20; H, 5.36; N, 7.44. Found: C, 68.98; H, 5.06; N, 7.15.
5.4.7. N-(2-(1H-Indol-3-yl)ethyl)-2-oxo-7-propoxy-2H-chromene-3-carboxamide (4g). Pale
1
brown solid; yield 90%; mp 174-176 ºC; IR (KBr, cm-1) νmax: 3500, 1710, 1680; H NMR
(300 MHz, DMSO-d6) δ: 10.51 (brs, 1H, NH), 9.31 (s, 1H, Ar-H4), 9.28 (brs, 1H, NH), 8.31
(d, J = 8.7 Hz, 1H, H5 coumarin), 8.16 (d, J = 7.5 Hz, 1H, H4 indole), 7.86 (d, J = 7.8 Hz, 1H,
H7 indole), 7.72 (s, 1H, H2 indole ), 7.37-7.63 (m, 4H, H6, 8 coumarin and H5, 6 indole), 4.61 (t,
J = 6.6 Hz, 2H, OCH2), 4.21 (q, J = 7.2 Hz, 2H, NH-CH2), 3.55 (t, J = 7.2 Hz, 2H, indole-
CH2), 2.31 (sex, J = 7.2 Hz, 2H, CH3-CH2-CH2), 1.53 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (75
MHz, DMSO-d6) δ: 164.2, 161.8, 161.3, 156.6, 148.2, 136.7, 132.0, 127.6, 123.2, 121.4,
118.8, 118.7, 115.1, 114.3, 112.5, 111.9, 111.8, 101.1, 70.5, 25.5, 22.2, 10.7. Anal. Calcd for
C23H22N2O4 (390.43): C, 70.75; H, 5.68; N, 7.17. Found: C, 70.53; H, 5.39; N, 6.97.
5.4.8. N-(2-(1H-Indol-3-yl)ethyl)-7-(benzyloxy)-2-oxo-2H-chromene-3-carboxamide (4h).
1
Pale brown solid; yield 91%; mp 195-197 ºC; IR (KBr, cm-1) νmax: 3500, 1710, 1680; H
NMR (300 MHz, CDCl3) δ: 8.80-8.90 (brs, 1H, NH), 8.86 (s, 1H, H4 indole), 8.12 (brs, 1H,
NH), 7.70 (d, J = 7.5 Hz, 1H, H5 indole), 7.60 (d, J = 8.7 Hz, 1H, H6 coumarin), 7.34-7.53
9

ACCEPTED MANUSCRIPT
(m, 6H, H2 indole and 5H benzyl), 7.09-7.27 (m, 3H, H4,7,5 indole), 7.03-6.89 (m, 2H, H6
indole and H8 coumarin), 5.19 (s, 2H, CH2-O), 3.82 (q, J = 6.9 Hz, 2H, NH-CH2), 3.13 (t, J =
6.9 Hz, 2H, indole-CH2). 13C NMR (75 MHz, DMSO-d6) δ: 163.8, 161.7, 161.2, 156.4, 148.1,
136.7, 136.4, 132.0, 129.9, 127.6, 123.2, 121.4, 118.8, 118.7, 115.4, 114.6, 112.7, 111.9,
111.8, 101.6, 70.6, 25.5. Anal. Calcd for C27H22N2O4 (438.47): C, 73.96; H, 5.06; N, 6.39.
Found: C, 73.74; H, 4.88; N, 6.11.
5.4.9.
N-(2-(1H-Indol-3-yl)ethyl)-7-((2-chlorobenzyl)oxy)-2-oxo-2H-chromene-3-
carboxamide (4i). Pale brown solid; yield 85%; mp 189-192 ºC; IR (KBr, cm-1) νmax: 3500,
1
1710, 1680; H NMR (300 MHz, DMSO-d6) δ: 10.87 (brs, 1H, NH), 8.86 (s, 1H, H4
coumarin), 8.79 (brs, 1H, NH), 7.94 (d, J = 7.8 Hz, 1H, H5 coumarin), 7.72 (d, J = 7.8 Hz,
1H, H6 coumarin), 7.63 (m, 2H, H3,5 benzyl), 7.46 (t, J = 7.5 Hz, 1H, H4 benzyl), 7.33-7.38
(m, 2H, H4,7 indole), 7.23 (m, 2H, H2 indole and H8 coumarin), 7.04-7.17 (m, 2H, H5, 6
indole), 6.99 (t, J = 7.5 Hz, 1H, H6 benzyl), 5.28 (s, 2H, CH2-O), 3.63 (d, J = 5.7 Hz, 2H,
NH-CH2), 2.97 (t, J = 6.3 Hz, 2H, indole-CH2). 13C NMR (75 MHz, DMSO-d6) δ: 163.6,
161.8, 161.2, 156.4, 148.1, 136.7, 136.6, 135.2, 133.2, 132.1, 131.3, 131.1, 128.5, 127.5,
123.8, 123.2, 123.1, 127.4, 118.8, 118.7, 115.5, 114.4, 112.9, 111.9, 101.5, 70.5, 25.4. Anal.
Calcd for C27H21ClN2O4 (472.92): C, 68.57; H, 4.48; N, 5.92. Found: C, 68.38; H, 4.69; N,
5.70.
5.4.10.
N-(2-(1H-Indol-3-yl)ethyl)-7-(2,3-dichlorobenzyloxy)-2-oxo-2H-chromene-3-
carboxamide (4j). Pale brown solid; yield 82%; mp 219-222 ºC; IR (KBr, cm-1) νmax: 3500,
1
1710, 1680; H NMR (300 MHz, DMSO-d6) δ: 10.87 (brs, 1H, NH), 8.86 (s, 1H, H4
coumarin), 8.78 (brs, 1H, NH), 7.94 (d, J = 8.7 Hz, 1H, H5 coumarin), 7.79 (d, J = 7.8 Hz,
1H, H4 benzyl), 7.70 (d, J = 7.8 Hz, 1H, H5 benzyl), 7.62 (d, J = 7.8 Hz, 1H, H6 benzyl), 7.50
(d, J = 7.8 Hz, 1H, H7 indole), 7.35 (d, J = 7.8 Hz, 1H, H4 indole), 6.87-7.28 (m, 5H, H6, 8
coumarin and H5, 6, indole), 5.29 (s, J = 7.8 Hz, 2H, OCH2), 3.63 (q, J = 6.5 Hz, 2H, NH-
2
13
CH2), 2.97 (t, J = 6.5 Hz, 2H, indole-CH2). C NMR (75 MHz, DMSO-d6) δ: 163.4, 161.7,
161.2, 156.4, 148.1, 136.6, 136.4, 132.5, 132.1, 131.4, 131.1, 129.4, 128.9, 127.5, 123.1,
121.4, 118.8, 118.7, 115.6, 114.4, 113.0, 111.9, 111.8, 101.6, 68.6, 25.4. Anal. Calcd for
C27H20Cl2N2O4 (507.36): C, 63.92; H, 3.97; N, 5.52. Found: C, 63.75; H, 3.79; N, 5.23.
5.4.11.
N-(2-(1H-Indol-3-yl)ethyl)-7-(4-chlorobenzyloxy)-2-oxo-2H-chromene-3-
carboxamide (4k). Pale brown solid; yield 82%; mp 203-207 ºC; IR (KBr, cm-1) νmax: 3500,
1
1710, 1680; H NMR (300 MHz, CDCl3) δ: 8.83-8.94 (brs, 1H, NH), 8.87 (s, 1H, H4
10

ACCEPTED MANUSCRIPT
coumarin), 8.07 (brs, 1H, NH), 7.70 (d, J = 8.0 Hz, 1H, H5 coumarin), 7.62 (d, J = 8.0 Hz,
1H, H6 coumarin), 7.35-7.46 (m, 5H, H2,3,6,5 benzyl and H7 indole), 7.12-7.28 (m, 3H, H4,5
indole and H8 coumarin), 7.01 (dd, J = 8.7 Hz, J = 2.1 Hz, 1H, H6 indole), 6.91 (d, J = 2.4 Hz,
1H, H2 indole), 5.15 (s, 2H, CH2-O), 3.82 (q, J = 6.8 Hz, 2H, NH-CH2), 3.13 (t, J = 6.8 Hz,
2H, indole-CH2). 13C NMR (75 MHz, DMSO-d6) δ: 163.5, 161.7, 161.2, 156.4, 148.1, 136.7,
135.5, 133.3, 132.0, 130.3, 129.0, 127.6, 123.2, 121.7, 118.8, 118.7, 115.5, 114.5, 112.8,
111.9, 111.8, 101.6, 69.7, 25.5. Anal. Calcd for C27H21ClN2O4 (472.92): C, 68.57; H, 4.48;
N, 5.92. Found: C, 68.69; H, 4.67; N, 5.73.
5.4.12.
N-(2-(1H-Indol-3-yl)ethyl)-7-(3,4-dichlorobenzyloxy)-2-oxo-2H-chromene-3-
carboxamide (4l). Pale brown solid; yield 92%; mp 236-239 ºC; IR (KBr, cm-1) νmax: 3500,
1
1710, 1680; H NMR (300 MHz, DMSO-d6) δ: 10.87 (brs, 1H, NH), 8.88 (s, 1H, H4
coumarin), 8.79 (t, 1H, J = 8.7 Hz, NH), 7.96 (d, J = 8.7 Hz, 1H, H5 coumarin), 6.90-7.75 (m,
10H, H6,8 coumarin, H2,4,5,6,7 indole and H2,5,6 benzyl), 5.37 (s, 2H, OCH2), 3.64 (t, J = 6.9 Hz,
2H, NH-CH2), 2.97 (t, J = 6.9 Hz, 2H, indole-CH2). 13C NMR (75 MHz, DMSO-d6) δ: 163.4,
161.7, 161.2, 156.4, 148.1, 136.6, 136.4, 132.5, 132.1, 131.4, 131.1, 129.4, 128.9, 127.5,
123.1, 121.4, 118.8, 118.7, 115.6, 114.4, 113.0, 111.9, 111.8, 101.6, 68.6, 25.4. Anal. Calcd
for C27H20Cl2N2O4 (507.36): C, 63.92; H, 3.97; N, 5.52. Found: C, 63.78; H, 4.25; N, 5.73.
5.4.13. N-(2-(1H-Indol-3-yl)ethyl)-7-(2-fluorobenzyloxy)-2-oxo-2H-chromene-3-carboxamide
(4m). Pale brown solid; yield 89%; mp 173-176 ºC; IR (KBr, cm-1) νmax: 3500, 1710, 1680;
1H NMR (300 MHz, DMSO-d6) δ: 10.86 (brs, 1H, NH), 8.86 (s, 1H, H4 coumarin), 8.80 (t, J
= 6.0 Hz, 1H, NH), 7.94 (d, J = 8.7 Hz, 1H, H5 coumarin), 7.56-7.67 (m, 2H, H4,6 benzyl),
7.42-7.53 (m, 1H, H5 benzyl), 7.20-7.40 (m, 4H, H8 coumarin and H4,7,5 indole), 7.03-7.17 (m,
3H, H6 coumarin and H2, 6 indole), 6.92-7.03 (m, 1H, H3 benzyl), 5.31 (s, 2H, OCH2), 3.28 (q,
J = 7.2 Hz, 2H, NH-CH2), 2.68 (t, J = 7.2 Hz, 2H, indole-CH2). 13C NMR (75 MHz, CDCl3)
δ: 163.5, 161.8, 161.7, 156.4, 148.0, 136.4, 131.0, 130.5, 130.4, 1129.8, 129.8, 127.3, 124.51,
124.4, 122.6, 122.1, 122.1, 119.4, 118.8, 115.8, 115.5, 115.2, 114.3, 113.1, 112.8, 111.1,
101.3, 40.1, 25.3. Anal. Calcd for C27H21FN2O4 (456.47): C, 71.04; H, 4.64; N, 6.14. Found:
C, 70.82; H, 4.31; N, 5.89.
5.4.14.
N-(2-(1H-Indol-3-yl)ethyl)-7-((3-fluorobenzyl)oxy)-2-oxo-2H-chromene-3-
carboxamide (4n). Pale brown solid; yield 87%; mp 173-176 ºC; IR (KBr, cm-1) νmax: 3500,
1
1710, 1680; H NMR (300 MHz, CDCl3) δ: 8.83 (s, 1H, H4 coumarin), 8.75-8.93 (brs, 1H,
NH), 8.08 (brs, 1H, NH), 7.66 (d, J = 7.8 Hz, 1H, H5 coumarin), 7.59 (d, J = 8.7 Hz, 1H, H7
11

ACCEPTED MANUSCRIPT
indole), 7.32-7.45 (m, 2H, H4,5 benzyl), 6.94-7.24 (m, 7H, H8 coumarin, H2,4,5,6 indole and
H2,6 benzyl), 6.8 (d, J = 7.8 Hz, 1H, H6 coumarin), 5.15 (s, 2H, OCH2), 3.79 (q, J = 6.9 Hz,
2H, NH-CH2), 3.10 (t, J = 7.2 Hz, 2H, indole-CH2). 13C NMR (75 MHz, CDCl3) δ: 163.5,
161.8, 161.7, 156.4, 148.0, 136.4, 131.0, 130.5, 130.4, 129.8, 129.8, 127.3, 124.5, 124.4,
122.6, 122.1, 122.1, 119.4, 118.8, 115.8, 115.5, 115.2, 114.3, 113.1, 112.8, 111.1, 101.3,
40.1, 25.3. Anal. Calcd for C27H21FN2O4 (456.47): C, 71.04; H, 4.64; N, 6.14. Found: C,
71.14; H, 4.42; N, 5.85.
5.4.15.
N-(2-(1H-Indol-3-yl)ethyl)-7-((4-fluorobenzyl)oxy)-2-oxo-2H-chromene-3-
carboxamide (4o). Pale brown solid; yield 90%; mp 193-196ºC; IR (KBr, cm-1) νmax: 3500,
1
1710, 1680; H NMR (300 MHz, CDCl3) δ: 8.83 (s, 1H, H4 coumarin), 8.78-8.92 (brs, 1H,
NH), 8.10 (brs, 1H, NH), 7.66 (d, J = 8.2 Hz, 1H, H5 coumarin), 7.56 (d, J = 8.2 Hz, 1H, H6
coumarin), 7.31-7.47 (m, 3H, H2,6 benzyl and H7 indole), 7.04-7.24 (m, 5H, H3,5 benzyl, H4,5
indole and H8 coumarin), 6.98 (d, J = 8.6 Hz, 1H, H6 indole ), 6.88 (s, 1H, H2 indole), 5.10 (s,
2H, OCH2), 3.79 (q, J = 7.0 Hz, 2H, NH-CH2), 3.10 (t, J = 7.0 Hz, 2H, indole-CH2). 13C
NMR (75 MHz, CDCl3) δ: 163.5, 161.8, 161.7, 156.4, 148.0, 136.4, 131.0, 130.5, 130.4,
129.8, 129.8, 127.3, 124.5, 124.4, 122.6, 122.1, 122.1, 119.4, 118.8, 115.8, 115.5, 115.2,
114.3, 113.1, 112.8, 111.1, 101.3, 40.1, 25.3. Anal. Calcd for C27H21FN2O4 (456.47): C,
71.04; H, 4.64; N, 6.14. Found: C, 71.31; H, 4.37; N, 6.05.
5.5. Cholinesterases inhibition assay
The inhibitory potency of target compounds on AChE/BuChE was determined using
Ellman’s method [30]. The compounds 4a-o were dissolved in 1 ml DMSO and 9 ml ethanol
and then four different concentrations of each compound were tested to obtain the range of
20% to 80% enzyme inhibition for AChE and BuChE. The reaction mixture included 2 ml
phosphate buffer (0.1 M, pH= 8.0), 65 µl of DTNB 0.1 M, 35 µl of enzyme [2 U/mL of
AChE (E.C. 3.1.1.7, Type V-S, lyophilized powder, from electric eel) or BuChE (E.C.
3.1.1.8, from equine serum)] and 35 µl of inhibitor solution. The changing of the absorbance
was measured at 412 nm for 2 min (30s intervals) after addition of 10 µl of substrate
(acetylthiocholine iodide or butyrylthiocholine iodide, 0.15 M) to the reaction mixture. The
IC50 values were determined graphically from inhibition curves (log inhibitor concentration
vs. percent of inhibition). All experiments were performed in 24 well plates on asynergy
HTX microplate reader in quadruplicates.
5.6. Molecular modeling study
12

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All docking simulations were performed using Autodock Vina (ver. 1.1.2) [31]. The high
resolution crystal structure of acetylcholinesterase complexed with E2020 (code ID: 1EVE,
resolution: 2.5 Å) was retrieved from protein data bank (www.rcsb.org). Then, the co-
crystallized ligand and water molecules were removed and the protein was converted to pdbqt
format using Autodock Tools (1.5.6) [32]. The 2D structures of ligands were prepared using
MarvinSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com) and then converted to
3D format by Openbabel (ver. 2.3.1) [33]. Finally, pdbqt format of ligands was prepared
using Autodock Tools python script, prepare_ligand4.py. After preparation of ligands and
protein, the docking studies were performed using the following docking parameters:
center_x = 2.023; center_y = 63.295; center_z = 67.062; size_x = 20; size_y = 20; size_z =
20; exhaustiveness = 80; num_modes = 15. The other parameters were left as default. At the
end, the best docking solutions were selected for further analysis of enzyme-inhibitor
interactions. The graphics were depicted using Chimera 1.6 software [34] and PoseView
(http://poseview.zbh.uni-hamburg.de/poseview/wizard, 2016.).
Acknowledgments
This research has been supported by grants from the Research Council of Tehran University
of Medical Sciences and Iran National Science Foundation (INSF).
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Captions:
Figure 1. structures of previously reported anti-AChE compounds (I-III) bearing indole
amine moiety and newly designed compounds 4a-o.
Figure 2. Left: Lineweavere-Burk plot for the inhibition of AChE by compound 4o at
different concentrations of substrate (ATCh); Right: Secondary plot for calculation of steady-
state inhibition constant (Ki) of compound 4o.
Figure 3. 2D (left) and 3D (right) representation of interactions of compound 4o in the active
site of AChE.
Scheme 1. Synthesis of intermediates 3a-o. Reagents and conditions: (a) diethyl malonate,
piperidine, EtOH, reflux ; (b) NaOH (2N) and then HCl (2N); (c) R-X, K2CO3, DMF.
Scheme 2. Synthesis of final compounds 4a-o. Reagents and conditions: (a) SOCl2; (b)
Tryptamine, K2CO3, dry toluene, reflux.
16

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Table 1. The IC50 values of the target compounds 4a-o against AChE and BuChE.
O
R1
R2
N
H
O
O
N
H
R3
Compound
R1
H
Br
H
H
H
H
H
H
R2
R3
AChE
IC50 (µM)
5.9
BuChE
IC50 (µM)
50.5
H
H
4a
4b
4c
4d
4e
4f
H
H
>100
2.8
>100
20.1
H
CH3O
H
HO
>100
3.5
>100
34.6
CH3O
H
CH3CH2O
H
43.8
18.7
1.5
>100
>100
16.2
CH3CH2CH2O
H
4g
4h
H
O
H
H
H
H
22.5
>100
>100
4i
4j
O
Cl
>100
O
Cl
Cl
H
H
H
H
12.4
31.5
>100
>100
4k
4l
O
Cl
O
Cl
Cl
H
H
H
H
2.5
2.5
27.5
27.8
4m
4n
O
F
O
F
H
H
0. 16
29.7
7.4
4o
O
F
Donepezil
0.016
17

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Figure 1. Structures of previously reported anti-AChE compounds (I-III) bearing indole
amine moiety and newly designed compounds 4a-o.
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Figure 2. Left: Lineweavere-Burk plot for the inhibition of AChE by compound 4o at
different concentrations of substrate (ATCh); Right: Secondary plot for calculation of steady-
state inhibition constant (Ki) of compound 4o.
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Figure 3. 2D (left) and 3D (right) representation of interactions of compound 4o in the active
site of AChE.
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O
O
O
O
R1= R3= H
R2= OH
R1
R2
R1
R2
a
OEt
H
OEt
OH
O
O
c
RO
O
R3
R3
(2e-o)
(2a-d)
(1a-d)
R1= H, Br
R2= H, OH
b
b
R3= H, CH3O
O
O
R1
R2
OH
OH
O
O
RO
O
O
R3
(3e-o)
(3a-d)
R= H, Me, Et, n-Pr, benzyl,
substituted benzyl
Scheme 1. Synthesis of intermediates 3a-p. Reagents and conditions: (a) diethyl malonate,
piperidine, EtOH, reflux ; (b) NaOH (2N) and then HCl (2N); (c) R-X, K2CO3, DMF.
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Scheme 2. Synthesis of final compounds 4a-o. Reagents and conditions: (a) SOCl2; (b)
Tryptamine, K2CO3, dry toluene, reflux.
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Research Highlights
•
•
•
•
•
Some coumarin-3-carboxamides of tryptamine were synthesized as anticholinesterases
All compounds were tested against AChE and BuChE.
Most of the compounds had significant activity toward AChE.
The introduction of 7-benzyloxy moiety on coumarin moiety improved the anti-AChE activity.
The 7-(4-fluorobenzyl)oxy analog 4o was the most potent compound (IC50= 0.16 µM).