Design, Synthesis, and Cholinesterase Inhibition Assay of Coumarin-3-carboxamide-N-morpholine Hybrids as New Anti-Alzheimer Agents

Article abstract of DOI:10.1002/cbdv.201900144

A new series of coumarin-3-carboxamide-N-morpholine hybrids 5a–5l was designed and synthesized as cholinesterases inhibitors. The synthetic approach for title compounds was started from the reaction between 2-hydroxybenzaldehyde derivatives and Meldrum's acid to afford corresponding coumarin-3-carboxylic acids. Then, amidation of the latter compounds with 2-morpholinoethylamine or N-(3-aminopropyl)morpholine led to the formation of the compounds 5a–5l. The in vitro inhibition screen against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) revealed that most of the synthesized compounds had potent AChE inhibitory while their BuChE inhibitions are moderate to weak. Among them, propylmorpholine derivative 5g (N-[3-(morpholin-4-yl)propyl]-2-oxo-2H-chromene-3-carboxamide) bearing an unsubstituted coumarin moiety and ethylmorpholine derivative 5d (6-bromo-N-[2-(morpholin-4-yl)ethyl]-2-oxo-2H-chromene-3-carboxamide) bearing a 6-bromocoumarin moiety showed the most activity against AChE and BuChE, respectively. The inhibitory activity of compound 5g against AChE was 1.78 times more than that of rivastigmine and anti-BuChE activity of compound 5d is approximately same as rivastigmine. Kinetic and docking studies confirmed the dual binding site ability of compound 5g to inhibit AChE.

Full text of DOI:10.1002/cbdv.201900144

Title: Design, synthesis, and cholinesterase inhibition assay of
coumarin-3-carboxamide-N-morpholine hybrids as new anti-
Alzheimer agents
Authors: Maryam Mohammadi-khanaposhtani, Maliheh Barazandeh
Tehrani, Zahra Rezaei, Mehdi Asadi Asadi, Hossein
Behnammanesh, Hamid Nadri, Fatemeh Afsharirad, Alireza
Moradi, Bagher Larijani, and Mohammad Mahdavi
This manuscript has been accepted after peer review and appears as an
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201900144
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900144

10.1002/cbdv.201900144
Chemistry & Biodiversity
Chem. Biodiversity
Design, synthesis, and cholinesterase inhibition assay of coumarin-3-
carboxamide-N-morpholine hybrids as new anti-Alzheimer agents
Maliheh Barazandeh Tehrania, Zahra Rezaeia, Mehdi Asadia, Hossein Behnammanesha, Hamid Nadrib, Fatemeh
Afsharirada, Alireza Moradib, Bagher Larijanic, Maryam Mohammadi-Khanaposhtanid*, Mohammad Mahdavic*
a Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417653761, Iran
b Department of Medicinal Chemistry, Faculty of pharmacy and Pharmaceutical sciences research center, Shahid Sadoughi University of Medical Sciences,
Yazd 8915173160, Iran
c Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran
1417653761, Iran, e-mail: momahdavi@tums.ac.ir
dCellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol 4717647745, Iran, e-mail:
maryammoha@gmail.com
Abstract A new series of coumarin-3-carboxamide-N-morpholine hybrids 5a-l was designed and synthesized as cholinesterases inhibitors. The synthetic
approach for title compounds was started from the reaction between 2-hydroxybenzaldehyde derivatives and Meldrum’s acid for produce corresponding
coumarin-3-carboxylic acids. Then, amidation of the latter compounds with 2-morpholinoethylamine or N-(3-aminopropyl)morpholine led to the
formation of the compounds 5a-l. The in vitro inhibition screen against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) revealed that most
of the synthesized compounds had potent AChE inhibitory while their BuChE inhibitions are moderate to weak. Among them, propylmorpholin derivative
5g bearing an unsubstituented coumarin moiety and ethylmorpholine derivative 5d bearing a 6-bromocoumarin moiety showed the most activity against
AChE and BuChE, respectively. The inhibitory activity of the compound 5g against AChE was 1.78 times more than that of rivasetigmin and anti-BuChE
activity of compound 5d is approximately same with rivasetigmin. Kinetic and docking studies confirmed the dual binding site ability of compound 5g to
inhibit AChE.
Keywords: Coumarin • Acetylcholinesterase • Butyrylcholinesterase • N-Morpholine • Coumarin-3-carboxamide-N-morpholine
Introduction
Alzheimer's disease (AD) is a progressing neurodegenerative disorder of the central nervous system (CNS) that affects a large number of aging peoples.
This disease is the most common cause of dementia and death in elderly populations.[1, 2] AD is associated with fallowing disorders in brain: deficiency in
neurotransmission cholinergic system, aggregation of β-amyloid (Aβ) plaques, and formation neurofibrillary tangles.[3-5]
In order to enhance cholinergic neurotransmission in AD patients used of AChE inhibitors such as donepezil, galantamine, and rivastigmine. Recent
findings have demonstrated that in AD patients the BuChE activity in specific regions of the brain increases while in the same regions the AChE activity is
greatly reduced.[6-8] Based on the mentioned finding, presumably the inhibition of both types of cholinesterases (ChEs) is beneficial for treatment of AD
signs and symptoms.
Structural analysis of the X-ray crystallographic structure of AChE and BuChE revealed that these enzymes have two main substrate-binding sites: 1) the
peripheral anionic site (PAS) and 2) the catalytic site (CS) with the following subunits: the catalytic triad (CT) and the anionic site (AS).[9, 10] Several studies
have revealed that PAS area of AChE interacts with Aβ peptides and accelerates the polymerization of them.[11] Thus, AChE inhibitors that interact with
both CS and PAS areas could be useful for the management of AD.
Coumarin is a naturally occurring heterocycle that found in many plant species. Natural and synthetic coumarin derivatives exhibit a wide range of
biological activities such as anti-inflammatory, anti-cancer, hepatoprotective, anti-HIV-1, antimicrobial, anti-oxidant, anti-depressant, anti-diabetic.[12-19]
Furthermore, various derivatives of coumarin with potent AChE inhibitory activities have been reported.[20-23] For example, coumarin-3-carboxamides A
and B were found to be potent anti-AChE inhibitors that interact with both CS and PAS of AchE (Fig 1).[24, 25] On the other hands, N-morpholine moiety
was recently used in design and synthesis of new AChE inhibitors (Fig 1, C).[26]
Hence, in continuation to our interest in the development of potent anti-Alzheimer agents by molecular hybridization, in this work, a novel series of
coumarin-3-carboxamide-N-morpholine hybrids 5a-l has been presented (Fig 1).[27] Herein, the synthesis, AChE/BuChE inhibitory effects, kinetic, and
docking studies of these hybrids as new dual binding site ChE inhibitors are reported.
1
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10.1002/cbdv.201900144
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Chem. Biodiversity
Fig. 1. Structures of compounds A, B, and C as lead compounds for design of coumarin-3-carboxamide-N-morpholine hybrids 5a-l.
Results and Discussion
Chemistry
The designed coumarin-3-carboxamide-N-morpholine hybrids 5a-l were synthesized in two steps (Scheme 1). Firstly, a reaction between appropriate 2-
hydroxybenzaldehyde derivatives 1 and Meldrum’s acid 2 in the presence of piperidinium acetate as catalyst afforded corresponding 2-oxo-2H-
chromene-3-carboxylic acids 3. The target compounds 5a-l were prepared through the reaction of compounds 3 with morpholine derivatives 4 in the
presence of HOBt and EDCI in dry acetonitrile at room temperature.
Synthesized structures 5a-l were shown in Table 1 and assigned by IR, 1H, and 13C NMR. For example, the IR spectrum of 6-bromo-N-(2-morpholinoethyl)-
2-oxo-2H-chromene-3-carboxamide 5d showed an absorption band at 3458 cm-1 for the NH group and 2 sharp absorption bands at 1728 and 1691 cm-1 for
1
esteric and amidic carbonyl group respectively. The H NMR spectrum of this compound exhibited two broad multiple peak for the two methylenes
groups connected to N and O on the morpholine ring moiety (δ = 2.42, m, 4H,) and (δ = 3.58, m, 4H), respectively. As well as a broad signal (δ = 2.50) for
the linear methylene group connected to nitrogen atom of morpholine ring and a broad multiple signal (δ = 3.44) for the linear methylene group
connected to NH of amid group exist in aliphatic area. Characteristic multiples with the appropriate chemical shifts and coupling constants were appeared
for the four aromatic H-atoms of the coumarin ring and NH of amid group in the range of 7.45-8.84 of the spectrum. The 13C NMR spectrum of 5d showed
four signals at δ = 36.3, 53.0, 56.5, and 66.2 ppm due to the methylene groups of morpholine moiety and linear chain, together with other 10 distinct
resonances (4 × CH, 4 × C, and 2 × C=O) in agreement with the proposed structure. Partial assignments of these resonances are given in the support
information.
2
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Scheme 1. Reagents and conditions: (a) piperidinium acetate, ethanol, reflux, 1 h, (b) HOBT, EDCI, dry acetonitrile, rt, 24h.
Pharmacology
Inhibitory activity against AChE and BChE. The AChE/BuChE inhibitory activities of coumarin-3-carboxamide-N-morpholine hybrids 5a-l were determined
by the Ellman’s method and compared with rivastigmine as the reference drug (Table 1).[28]
Based on the IC50 values for AChE, all the synthesized compounds with the exception of 5b exhibited good inhibitory activity in the range of sub-
micromolar concentrations (IC50 values of 6.21-26.4 μM). It is notable that compounds 5g, 5i, 5l, and 5d were found to be more potent than rivastigmine
against AChE.
Structurally, the target compounds are divided into two series: ethylmorpholine derivatives 5a-f and propylmorpholine derivatives 5g-l. In each series, the
substituent on the coumarin moiety was altered to optimize the inhibitory activity against AChE.
The inhibitory activity of ethylmorpholine derivatives 5a-f against AChE demonstrated that 6-bromocoumarin derivative 5d (IC50 = 8.26 ± 0.03 μM)
showed the most potent activity, while 6-methoxycoumarin derivative 5b did not show activity (IC50 > 100 μM). In this series, introduction of 8-methoxy or
6-nitro substituent on coumarin moiety of unsubstituented coumarin derivative 5a cannot improve anti-AChE potency (compounds 5c and 5e vs. 5a).
Moreover, adding a phenyl group on coumarin moiety of compound 5a, as in compound 5f, has no significant effect on inhibitory activity against AChE.
In the propylmorpholine series (compounds 5g-l), compound 5g with unsubstituented coumarin ring was found as the most potent compound. This
compound was also the most potent compound among the synthesized compounds 5a-l. Adding 8-methoxy substituent or a phenyl group on coumarin
moiety of compound 5g led to a slight decrease in the inhibitory activity against AChE as observed in compounds 5i and 5l. In this series, the 6-methoxy
and 6-nitro compounds 5h and 5k showed approximately the same anti-AChE activities. In the propylmorpholine series, unlike ethylmorpholine, the less
potent compound was 6-bromocoumarin derivative (compound 5j vs. 5d).
The comparison of IC50 values of ethylmorpholine derivatives with their corresponding propylmorpholine analogs against AChE revealed that with the
exception of compound 5j, propylmorpholine analogs were more active than their analogs. Based on these findings, it seems that increasing the length of
the linker led to an increase in the inhibitory activity against AchE.
In the term of BuChE inhibitory activity, compound 5d (IC50 = 7.65 ± 0.01 µM) shows inhibitory activity similar to standard drug rivastigmine (IC50 = 7.72 ±
0.01 µM). Moreover, compounds 5i-l and 5f-g with IC50 values ranging from 10.34 ± 0.02 to 17.8 ± 0.01 µM are moderate inhibitors for BuChE and
compound 5b with IC50 = 40.75 ± 0.01 µM is a weak anti-BuChE. The remaining derivatives 5a, 5c, 5e, and 5h showed IC50 > 100, and thus considered as
inactive compounds.
In the of ethylmorpholine series, 6-bromocoumarin derivative 5d showed most inhibitory activity against BuChE. Replacement 6-bromo substituent with
6-methoxy group, as in compound 5b, led to a significant decrease in inhibitory activity. In this series, unsubstituented coumarin derivative 5a, the 8-
methoxycoumarin derivative 5c and 6-nitrocoumarin derivative 5e did not show inhibitory activity against BuChE (IC50 > 100 μM). The presence of a
phenyl group on coumarin moiety of compound 5a, as in compound 5f, led to a dramatically increase in the inhibitory activity.
In the propylmorpholine series, the unsubstituented, 8-methoxy, 6-bromo, and 6-nitro derivatives 5g, 5i, 5j, and 5k showed approximately the same
inhibitory activities against BuChE. Adding a phenyl group on coumarin moiety of compound 5g led to a slight decrease in the inhibitory activity as
observed in compound 5l. In this series, compound 5h with 6-methoxy substituent did not show anti-BuChE activity.
Table 1. Cholinesterase inhibitory activity of compounds 5a-l.a
Compound
Structure
IC50 AChE (µM)
15.92 ± 0.01
IC50 BuChE (µM)
5a
≥100
5b
5c
≥100
40.75 ± 0.01
≥100
25.7 ± 0.01
8.26 ± 0.03
5d
7.65 ± 0.01
3
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5e
26.4 ± 0.01
≥100
5f
13.67 ± 0.04
6.21 ± 0.03
16.4 ± 0.04
14.9 ± 0.05
12.9 ± 0.02
≥100
5g
5h
5i
7.64 ± 0.01
10.34 ± 0.02
5j
21.2 ± 0.04
15.97 ± 0.02
11.96 ± 0.02
12.4 ± 0.04
5k
5l
7.8 ± 0.03
17.8 ± 0.01
7.72 ± 0.01
Rivastigmine
-
11.07 ± 0.03
a Data are expressed as Mean ± SE (three independent experiments).
Kinetic study. To determination of inhibition mode of the synthesized coumarin-3-carboxamide-N-morpholines against AChE, a kinetic study was
performed on the most potent compound 5g. As shown in Fig 2a, analysis of reciprocal Lineweaver–Burk plot demonstrated that this compound acts as a
mixed type inhibitor against AChE. This finding revealed that compound 5g can bind to both CS and PAS of AChE. Plotting of the slopes versus
concentration of 5g gave an estimation of inhibition constant, Ki of 0.56 µM for AChE (Fig 2b).
(a)
(b)
Fig. 2. (a) Lineweaver–Burk plot for the inhibition of AChE by compound 5g; (b) secondary plot for steady-state inhibition constant (Ki) of compound 5g.
Docking study. Docking study was performed by Autodock Tools (1.5.6) to predict the interaction mode of the most potent compounds 5g and 5d in the
active site of AChE and BuChE, respectively. [29, 30] Several ligand-bounded crystallographic structures with various resolutions of AChE and BuChE are
available in the RCSB protein data bank (http://www.rcsb.org/pdb/home/home.do). Herein, PDB structure of 1EVE for AChE and 1P0I for BuChE were
retrieved for docking purpose. As can be seen in Fig 3a, compound 5g was well accommodated in the active site. The detailed binding mode of the
4
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compound 5g showed that this compound formed interactions with residues Asp72 (PAS), Tyr121 (PAS), Phe288 (acyl pocket), Phe330 (AS), and Phe331
(AS) (Fig 3b).[10] These finding revealed that compound 5g binds to both CS and PAS of AchE.
(a)
(b)
Fig. 3. (a) The best pose of the most potent compound 5g in the active site of AChE; (b) amino acids of the active site involved in ligand binding.
Compound 5d as the most potent compound against BuChE can interact with important amino acids of the active site (Fig 4). As depicted in Fig 4b, this
compound established interactions with residues Trp82 (AS), Gly116 (oxyanion hole), Ser198 (CT), Ala199 (oxyanion hole), Trp321, Leu286 (acyl pocket),
Val288 (acyl pocket), Ala328, Trp430, His438 (CT).[10]
(a)
(b)
Fig. 4. (a) The best pose of the most potent compound 5d in the active site of BuChE; (b) amino acids of the active site involved in ligand binding.
Drug likeliness parameters. An effective anti-Alzheimer agent should be able to cross the brain in addition to having drug-like properties. Therefore, drug
likeliness parameters and blood–brain barrier (BBB) permeability of the two most potent compounds 5g and 5d were evaluated by proper software. Some
of physicochemical and topological properties of these compounds such as molecular weight (MW), a number of H-bond donors (HBD), a number of H-
bond acceptors (HBA), octanol/water partition coefficients (Clog P), a number of rotatable bonds (RBC), and the polar surface area (tPSA) were calculated
and shown in Table 2. For prediction of BBB penetration, online BBB predictor (www.cbligand.org) was applied (Table 2, SVM_MACCSFP BBB Score >
0.02 were able to cross the BBB). According to Lipinski rule of 5, compounds 5g and 5d possessed good pharmacokinetic properties (MW < 500, HBD ˂5,
HBA ˂10, Clog P ˂ 5, and RBC ˂10).[31] Also, the latter compounds possessed tPSA = 67.87 Ǻ2 (Veber rule: tPSA ≤ 140 Ǻ2).[32] Furthermore, based on the
SVM_MACCSFP BBB Score, the most potent compounds 5g and 5d were suitable to cross the BBB.
5
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Table 2. Drug likeliness parameters of the compounds 5g and 5d.
Compound
MW
<500
316.35
381.22
HBD
<5
1
HBA
<10
4
Clog P
<5
1.53
RBC
<10
4
Lipiniski's violations
tPSA (Ǻ2)
BBB score a
-
+0.029
+0.024
Rule
5g
5d
<1
0
0
-
67.87
67.87
1
4
2.203
4
Conclusions
In conclusion, a series of new coumarin-3-carboxamide derivatives bearing morpholine moiety were designed, synthesized, and evaluated for AChE and
BuChE inhibitory activity. The obtained results revealed that most of the synthesized compounds exhibited good anti-AChE activity and moderate anti-
BuAChE activity. Among the synthesized derivatives, compounds 5g, 5i, 5l, and 5d were found to be more potent than standard drug rivastigmine against
AChE and compound 5d shows inhibitory activity similar to rivastigmine against BuChE. Kinetic analysis and docking study of most potent AChE inhibitor
5g demonstrated that this compound can fit into the CS and PAS areas of AchE active site.
Experimental Section
Chemistry
Melting points were taken on a Kofler hot stage apparatus. The IR spectra of the compound 5a-l were obtained using Nicolet FT-IR Magna 550
spectrograph (KBr disks). Also, NMR spectra were recorded on an NMR instrument Bruker 500 MHz. Elemental analyses of the title compounds for C, H
and N were performed using a Heraeus CHN-O-Rapid analyzer.
General procedure for the synthesis of compounds 3. Various 2-hydroxybenzaldehyde derivatives 1 (1mmol) and Meldrum’s acid 2 (1mmol) were incubated
in the presence of a catalytic amount of piperidinium acetate at room temperature and then followed by reflux in ethanol for 1h. After completion of the
reaction (monitored by TLC), the mixture was cooled down to room temperature. Then, the precipitate was filtered and washed with ethanol to afford
pure products 3.
General procedure for the synthesis of compounds 5a-l. A solution of compounds 3 (1 mmol), hydroxybenzotriazole (HOBt) (1 mmol) and 1-ethyl-3-(3
dimethylaminopropyl) carbodiimide (EDCI) (1.1 mmol) were dissolved in dry acetonitrile (5 mL) and were stirred at room temperature for 30 min. Then,
appropriate morpholin derivative 4 (1 mmol) was added to the mixture. The reaction was completed at 25 ºC for 24 hours. After, the solvent was
evaporated under vacuum and the resulting residue was dissolved in dichloromethane and washed with sodium carbonate (10%). Subsequently, the
organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the obtained compounds 5a-l were pure.
Biological Evaluation
In vitro AChE and BChE inhibition assay. Electric eel (Torpedo californica) AChE (type VI-S), BChE (E.C.3.1.1.8, from equine serum), and butyrylthiocholine
iodide were purchased from Sigma-Aldrich. Also, 5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB), dipotassium hydrogen phosphate, potassium hydroxide,
sodium hydrogen carbonate, potassium dihydrogen phosphate and acetylthiocholine iodide were purchased from Fluka. The AChE inhibitory activity of
derivatives was determined by the Ellman’s spectroscopic method. The temperature condition was adjusted at 25°C during all experiments. The stock
solutions of each compound were prepared by dissolving them in dimethyl sulfoxide (DMSO). Five different concentrations were prepared for each
compound (in ethanol) in triplicate in order to obtain the range of 20%-80% inhibition of AChE. The assay solution constituted of 2 mL phosphate buffer
(0.1 M, pH = 8), 30 μL test compound, 60 μL DTNB and 20 μL AChE solution (5 IU/mL). The rate of absorbance change was measured at 412 nm after the
initiation of the reaction by adding 20 μL of acetylthiocholine iodide as substrate to each well. To obtained IC50 values, inhibition curves (log inhibitor
concentration versus percent of inhibition) were drawn by Prism 6.01 (2012, http://www.graphpad.com). The same procedure was performed for BChE
inhibition assay.
Kinetic studies of AChE inhibition. To explore the inhibition mechanism of the synthesized compounds, Lineweaver–Burk plots were drawn using three
different concentrations of 5g (12.4, 6.2, 3.1 µM) and acetylthiocholine iodide (0.07, 0.17, and 0.34 mM) as the most potent AChE inhibitor and substrate
respectively. Correspondingly, the slopes of the Lineweaver–Burk plots were plotted against the inhibitor concentrations, and Ki was calculated.
Docking study. Docking study of the most potent compounds 5g and 5d in the active site of respectively AChE and BuChE were performed by Auto dock
Tools, using previously described method.[29]
6
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In silico molecular properties prediction. The Clog P values and tPSA of the compounds 5g and 5d were calculated using the ChemDraw Ultra 12.0. HBA and
HBD of the title compounds were calculated by the means of MarvineSketch 5.8.3 and RBCs were calculated using Autodock Tools (ver.1.5.6). BBB
penetration of the selected compounds was predicted by online BBB predictor (www.cbligand.org).
Author Contribution Statement
Maliheh Barazandeh Tehrani, Bagher Larijani, and Hossein Behnammanesh contributed reagents and materials, and analyzed the data. Zahra Rezaei
performed docking study. Mehdi Asadi and Fatemeh Afsharirad performed the synthesis of compounds. Hamid Nadri and Alireza Moradi performed the
biological assay. Maryam Mohammadi-Khanaposhtani and Mohammad Mahdavi conceived and designed the experiments.
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Graphical Illustration
Design, synthesis, and cholinesterase inhibition assay of coumarin-3-carboxamide-N-morpholine hybrids as new anti-Alzheimer
agents
Maliheh Barazandeh Tehrani, Zahra Rezaei, Mehdi Asadi, Hossein Behnammanesh, Hamid Nadri, Fatemeh Afsharirad, Alireza Moradi,
Bagher Larijani, Maryam Mohammadi-Khanaposhtani*, Mohammad Mahdavi*
9
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