Design, synthesis and docking study of novel coumarin ligands as potential selective acetylcholinesterase inhibitors

Article abstract of DOI:10.1080/14756366.2016.1250753

New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC₅₀ value of 43 nM) was the best AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c showed negligible cell death. Molecular docking studies were also carried out to clarify the inhibition mode of the more active compounds. Best pose of compound 6c is positioned into the active site with the coumarin ring wedged between the residues of the CAS and catalytic triad of AChE. In addition, the coumarin ring is anchored into the gorge of the enzyme by H-bond with Tyr130.

Full text of DOI:10.1080/14756366.2016.1250753

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis and docking study of novel
coumarin ligands as potential selective
acetylcholinesterase inhibitors
Fatih Sonmez, Belma Zengin Kurt, Isil Gazioglu, Livia Basile, Aydan Dag,
Valentina Cappello, Tiziana Ginex, Mustafa Kucukislamoglu & Salvatore
Guccione
To cite this article: Fatih Sonmez, Belma Zengin Kurt, Isil Gazioglu, Livia Basile, Aydan
Dag, Valentina Cappello, Tiziana Ginex, Mustafa Kucukislamoglu & Salvatore Guccione
(
2017) Design, synthesis and docking study of novel coumarin ligands as potential selective
acetylcholinesterase inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1,
85-297, DOI: 10.1080/14756366.2016.1250753
2
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1250753
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Date: 24 January 2017, At: 01:41

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 285–297
http://dx.doi.org/10.1080/14756366.2016.1250753
RESEARCH ARTICLE
Design, synthesis and docking study of novel coumarin ligands as potential
selective acetylcholinesterase inhibitors
a
b
b
c
b
c
d
Fatih Sonmez , Belma Zengin Kurt , Isil Gazioglu , Livia Basile , Aydan Dag , Valentina Cappello , Tiziana Ginex ,
e
c
Mustafa Kucukislamoglu and Salvatore Guccione
a
b
Pamukova Vocational High School, Sakarya University, Sakarya, Turkey; Department of Analytical and Pharmaceutical Chemistry, Faculty of
c
Pharmacy, Bezmialem Vakif University, Istanbul, Turkey; Department of Drug Sciences, University of Catania, Citt ꢀa Universitaria, Catania, Italy;
d
e
Molecular Modelling Laboratory, Department of Food Science, University of Parma, Parma, Italy; Faculty of Arts and Science, Department of
Chemistry, Sakarya University, Sakarya, Turkey
ABSTRACT
ARTICLE HISTORY
New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or
the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-
Received 9 June 2016
Revised 13 October 2016
Accepted 16 October 2016
(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC50 value of 43 nM) was the best
AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed
that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c
showed negligible cell death. Molecular docking studies were also carried out to clarify the inhibition
mode of the more active compounds. Best pose of compound 6c is positioned into the active site with
the coumarin ring wedged between the residues of the CAS and catalytic triad of AChE. In addition, the
coumarin ring is anchored into the gorge of the enzyme by H-bond with Tyr130.
KEYWORDS
Acetamide; acetylcholin-
esterase; Alzheimer’s
disease; coumarin;
selectivity
Introduction
Tricyclic and heterocyclic compounds, such as tacrine, quinolizi-
dinyl and coumarin derivatives, are able to bind both PAS and cata-
lytic anionic site (CAS) of the enzyme by hydrophobic interactions
and T-stacking with the aromatic residues of the enzyme gorge in
Alzheimer’s disease (AD) is a progressive neurodegenerative dis-
ease and the most common form of dementia that affects aged
1
,2
people . Currently, there is no cure for AD and the cholinergic
strategy, in which the acetylcholine (ACh) level in brain has been
increased by inhibiting acetylcholinesterase (AChE), remains the
2
0–23
AChE
. Accordingly, the coumarin scaffold has been considered
8
,24,25
to design new AChE inhibitors
, and many efforts were
3
,4
addressed to synthesize dual binding site inhibitors of AChE by
hybridizing a catalytic site interacting moiety with the coumarin
most effective therapeutic approach for the treatment of AD
.
Acetylcholinesterase (AChE; EC 3.1.1.7) is a hydrolase involved in
the termination of impulse transmission at cholinergic synapses by
rapid hydrolysis of the neurotransmitter ACh in the central and per-
ipheral nervous system. AChE inhibitors (AChEI) inhibit the hydrolysis
of ACh improving both the level and of duration of neurotransmit-
2
6
scaffold through an appropriate spacer . Amidic or imidic substitu-
ents are key functionalities acting as hydrogen bond donors at the
catalytic triad (Ser203-Glu334-His447) of the active site residues of
the human acetylcholinesterase by the unpaired electron of N and
2
7–30
5,6
O atoms
. Thiazolo-triazine derivatives are considered efficient
ter . Another cholinesterase, butyrylcholinesterase (BuChE; EC
.1.1.8), primarily localized in plasma, liver and muscle tissues, able of
acetylcholinesterase inhibitors for the ability to form a hydrogen
3
3
1
bond with Tyr124 and T-stacking interaction with Trp286 .
Benzofuran and coumarin derivatives bearing thiazole ring and
arylurea/thiourea moieties were synthesized as ChE inhibitors;
however, they exhibited moderate inhibitory activities against
hydrolyzing ACh and other acylcholines, differs from AChE for tissue
distribution and sensitivity to substrates and inhibitors . AChE inhibi-
tors such as galantamine, rivastigmine and donepezil are the main
stay drugs for the clinical management of AD in the early-to-moder-
ate stage (Figure 1) . Also, in clinical treatment of AD, selective
AChE inhibitors have shown better therapeutic effects, compared
with no selective inhibitors
AChE inhibitors could represent a successful therapeutic strategy for
the symptomatic treatment of AD and its progression .
7
3
2,33
8–12
AChE in our previous studies
. Several studies have presented
that benzofuran or coumarin molecules interacted with CAS and
PAS of AChE via the steric and T-stacking interactions; the H-bond-
ing and T-stacking interactions were determined between thiazole
ring or amide moiety and PAS; the phenyl ring of urea moiety
6,13,14
. Therefore, the design of selective
1
5
3
4,35
The analysis of the 3D structure of AChE revealed the presence interacted with CAS by T-stacking
. It has also been reported
of a deep and narrow gorge at the active site mainly composed of that the cation–p interactions were observed between N-alkyl
2
2,36
dual binding sites: the Ser-His-Glu catalytic site located at the bot- chains or heterocyclic moieties and mid-gorge site of AChE
.
tom of gorge, and the peripheral anionic binding site (PAS)
2-Aminothiazoles can be progressed to generate useful com-
pounds is testified by a number of marketed drugs, including anti-
biotics such as dopamine agonists for the treatment of Parkinson’s
1
6–19
located at the gorge entrance
.
CONTACT Fatih Sonmez
gmail.com, liviabasile@unict.it
fsonmez@sakarya.edu.tr
Pamukova Vocational High School, Sakarya University, 54900, Sakarya, Turkey; Livia Basile
basilelivia@
Department of Drug Sciences, University of Catania, Viale A. Doria 6 Ed. 2, Citta Universitaria, I-95125, Catania, Italy
Supplemental data for this article can be accessed here
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

2
86
F. SONMEZ ET AL.
Figure 1. Structures of well-known cholinesterase inhibitors and designed compounds.
Figure 2. Development strategy and interactions of fragment of the synthesized compounds.
disease, and riluzole, which is the 2-aminobenzothiazole
Based on the above consideration, we hypothesized that the
presence of acetamide moiety, bearing N-alkyl chains and/or
3
7–39
derivative used to treat acute myeloid leukemia (AML)
.
Conversely, 2-aminothiazoles have displayed cytotoxicity and heterocycle instead of phenylurea, contributes to inhibitory
metabolic instability as antimycobacterial and antiplasmodial activity of designed molecules via a cation–p interactions with
3
9
agents . It has been presented that the 2-amido and heterocyclic active sites of AChE (Figure 2). In this study, a novel series of
thiazoles show little or no binding to any of the proteins (apical 20 coumarin derivatives (6a-t) was synthesized, and their inhibi-
membrane antigen AMA1, the E3 ubiquitin ligase adapter protein tory effects on AChE and BuChE were evaluated. Additionally,
SPSB2, two DsbA oxidoreductases from different bacterial species kinetic study of AChE inhibition and molecular docking studies
(
oxidoreductase 1 and 2), carbonic anhydrase II and a kinase) and were carried out to clarify the inhibition mode of the more
3
9
do not appear to be inherently promiscuous . However, some active compounds. Moreover, the potential toxicity effect on the
docking studies showed that the nitrogen atom of the thiazole hepatoma cell line H4IIE was also examined for the most potent
ring might form a hydrogen bond and interact with active sites of compound 6c.
3
1,32
AChE
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
287
Methods
2-Chloro-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (5)
To a solution of 3-(2-amino-1,3-thiazol-4-yl)coumarin (4) (6.5 mmol)
in THF (30 mL), chloroacetylchloride (8.125 mmol) was added fol-
Chemistry
All solvents, reagents and starting materials were obtained from lowed by a catalytic amount of Et N. The mixture was refluxed for
3
commercial sources unless otherwise indicated. Melting points 10 h, then cooled and evaporated in vacuo. The residue was washed
ꢀ
were taken on a Barnstead Electrothermal 9200 (Staffordshire, UK). with water and dried in a vacuum-oven at 40 C. Greenish powder,
IR spectra were registered on a Shimadzu Prestige-21 (200 VCE) 96% yield, IR: 3171, 3134, 2981, 1707, 1658, 1568, 1316, 1094, 737;
1
13
1
spectrometer (Columbia, MD). H and C NMR spectra were regis-
6
H NMR (DMSO-d , 300 MHz) d/ppm: 4.41 (2H, s), 7.36–7.47 (2H, m),
tered on a Varian Infinity Plus spectrometer (Palo Alto, CA) at 300 7.63 (1H, t, J ¼ 7.02 Hz), 7.83 (1H, d, J ¼ 7.9 Hz), 8.04 (1H, s), 8.57 (1H,
1
13
þ
and at 75 Hz, respectively. H and C chemical shifts are refer- s), 12.68 (1H, s, NH); LC-MS-MS(ESI-) (m/z) 320 [M ].
enced to the internal deuterated solvent. Mass spectra were
obtained using MICROMASS Quattro LC-MS-MS spectrometer
(
Milford, MA). The elemental analyses were carried out with a Leco N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)-2-(RN-1-yl)acetamide
CHNS-932 instrument (St. Joseph, MI). Spectrophotometric analyses (6a-t)
were performed by a BioTek Power Wave XS (Winooski, VT). The To a solution of 2-chloro-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-
electric eel acetylcholinesterase (AChE, Type-VI-S, EC 3.1.1.7, yl)acetamide (5) (1 mmol) in DMF, 1.25 mmol Et N and 1.85 mmol
3
4
25.84 U/mg, Sigma) and horse serum butyrylcholinesterase (BuChE,
EC 3.1.1.8, 11.4 U/mg, Sigma) were purchased from Sigma
Steinheim, Germany). The other chemicals and solvents were pur-
chased from Fluka Chemie (Taufkirchen, Germany), Merck
amine derivatives were added. The mixture was refluxed over-
night, poured on crushed ice then extracted with CH Cl . The
organic layer was washed with water, dried over Na SO and con-
centrated in vacuo. The products were recrystallized from ethanol
over 95% purity. 6a-t were obtained with 20–80% yields.
2
2
(
2
4
(
(
Darmstadt, Germany), Alfa Aesar (Ward Hill, MA), and Sigma-Aldrich
Taufkirchen, Germany). Reactions were monitored by thin-layer
chromatography (TLC) on 0.25mm E. Merck silica gel plates (60F-
54) and visualized under UV lamp (Camag, Switzerland). Thin-layer
chromatography was provided by Merck (Darmstadt, Germany).
2
-(Methylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)aceta-
2
ꢀ
mide (6a) Yellow powder, 60% yield, mp. 168–170 C; IR: 3291,
ꢁ
1
1
3138, 1708, 1689, 1557, 1250, 1178, 1032, 783 cm
; H NMR
(
7
CDCl , 300 MHz) d/ppm: 2.55 (3H, s), 3.50 (2H, s, CO-CH -N),
3
2
General procedures of synthesis and spectral data
1
3
.28–7.38 (2H, m), 7.50–7.61 (2H, m), 8.15 (1H, s), 8.58 (1H, s);
C
3
-Acetylcoumarin (2)
NMR (CDCl
3
, 75 MHz) d/ppm: 36.9, 54.1, 114.8, 116.3, 119.5, 121.0,
A mixture of salicylaldehyde (1) (3 mmol), ethyl acetoacetate 124.8, 128.4, 131.6, 138.8, 142.6, 152.9, 156.9, 159.7, 170.5; LC-MS-
þ
(
3 mmol) and piperidine (0.1 mmol) was stirred at room tempera- MS(ESIþ) (m/z): 315.08 [M ]. Anal. Calcd for C15
H N O S: C, 57.13;
13 3 3
ture for 30 min. After completion of the reaction, the mixture was H, 4.16; N, 13.33; found: C, 57.10; H, 4.19; N, 13.31.
recrystallized from ethanol to get pure crystalline 3-acetylcoumarin
(
2) in 95% yields. Spectral data of this compound matched with
N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)-2-(propylamino)aceta-
4
0
1
the literature . H NMR (CDCl , 300 MHz) d/ppm: 2.73 (3H, s),
3
ꢀ
1
3
mide (6b) Light yellow powder, 71% yield, mp. 138–140 C; IR:
7
7
1
3
.33–7.39 (2H, m), 7.64–7.69 (2H, m), 8.52 (1H, s); C NMR (CDCl ,
ꢁ
1
1
3304, 3178, 2958, 1710, 1653, 1542, 1253, 1091, 784 cm
; H
5 MHz) d/ppm: 30.9, 116.9, 118.4, 124.6, 125.2, 130.5, 137.0, 147.8,
55.5, 159.5, 195.7. TLC: Hexane:Ethylacetate (4:1).
NMR (CDCl , 300 MHz) d/ppm: 0.99 (3H, t, J ¼ 7.3 Hz), 1.59–1.66
3
(
(
(
2H, m), 2.69 (2H, t, J ¼ 7.3 Hz), 3.52 (2H, s, CO-CH
2H, m), 7.50–7.61 (2H, m), 8.14 (1H, s), 8.57 (1H, s); C NMR
CDCl , 75 MHz) d/ppm: 11.8, 23.4, 52.1, 52.5, 115.3, 116.6, 119.7,
21.1, 124.8, 128.5, 131.6, 138.9, 142.7, 153.1, 156.8, 159.9, 170.7;
2
-N), 7.27–7.36
1
3
3
-(Bromoacetyl)coumarin (3)
3
To a solution of 3-acetylcoumarin (2) (0.01 mol) in 20 mL chloro-
form, a solution of bromine (0.01 mol) in 5 mL chloroform was
added. The mixture was stirred at 50 C for 15 min and then
cooled. The obtained precipitate was filtered and washed with
ether. The product was recrystallized from acetic acid. 3-
1
þ
17 3 3
LC-MS-MS(ESI-) (m/z): 343.03 [M ]. Anal. Calcd for C17H N O S: C,
ꢀ
59.46; H, 4.99; N, 12.24; found: C, 59.41; H, 4.95; N, 12.28.
2
-(Diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)aceta-
(Bromoacetyl)coumarin (3) was obtained in 85% yields. Spectral
4
1
1
ꢀ
mide (6c) Light yellow powder, 59% yield, mp. 150–152 C; IR:
data of this compound matched with the literature . H NMR
ꢁ
1
(
DMSO-d , 300 MHz) d/ppm: 4.83 (2H, s), 7.28–7.49 (2H, m), 3269 3148, 2975, 1726, 1703, 1528, 1435, 1269, 1092, 781 cm
;
6
1
7
.60–7.81 (2H, m), 8.56 (1H, s).
H NMR (CDCl
q, J ¼ 7.02 Hz), 3.29 (2H, s, CO-CH
t, J ¼ 8.4 Hz), 7.60 (1H, d, J ¼ 7.6 Hz), 8.14 (1H, s), 8.59 (1H, s);
NMR (CDCl , 75 MHz) d/ppm: 12.4, 49.1, 57.0, 115.4, 116.6, 119.7,
3
, 300 MHz) d/ppm: 1.13 (6H, t, J ¼ 7.02 Hz), 2.70 (4H,
2
-N), 7.27–7.37 (2H, m), 7.52 (1H,
1
3
C
3
-(2-Amino-1,3-thiazol-4-yl)coumarin (4)
3
To a solution of 3-(bromoacetyl)coumarin (3) (5 mmol) in boiling
ethanol (20 mL), thiourea (5 mmol) was added. The mixture was
refluxed for 1 h, then cooled and neutralized with aqueous ammo-
nia. The precipitate was filtered off, washed with ethanol and used
directly without re-crystallization or other purification. 3-(2-Amino-
1
21.1, 124.8, 128.4, 131.6, 138.9, 142.8, 153.1, 156.8, 159.9, 170.7;
þ
LC-MS-MS(ESI-) (m/z): 357.05 [M ]. Anal. Calcd for C18
H
19
N
3
O
3
S: C,
60.49; H, 5.36; N, 11.76; found: C, 60.47; H, 5.39; N, 11.72.
1
,3-thiazol-4-yl)coumarin (4) was obtained in 80% yields. Spectral 2-(Dibutylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)aceta-
4
2
ꢀ
data of this compound matched with the literature . mp. mide (6d) Light yellow powder, 70% yield, mp. 166–168 C; IR:
ꢀ
1
ꢁ 1
1
2
26–228 C; IR: 3376, 3310, 1694, 1642, 1504, 1376, 1093, 758; H 3289, 3142, 2956, 1724, 1700, 1525, 1269, 1093, 782 cm
; H
NMR (DMSO-d , 300 MHz) d/ppm: 7.16 (2H, s, NH ), 7.33–7.43
6
2
NMR (CDCl
3
, 300 MHz) d/ppm: 0.95 (6H, t, J ¼ 7.3 Hz), 1.34–1.56
(
8
2H,m), 7.49 (1H,s), 7.59 (1H, t, J ¼ 8.2 Hz), 7.82 (1H, d, J ¼ 7.9 Hz),
(
7
8H, m), 2.59 (4H, t, J ¼ 7.3 Hz, N-CH ), 3.30 (2H, s, CO-CH -N),
2
2
1
3
.49 (1H, s); C NMR (DMSO-d
6
, 75 MHz) d/ppm: 109.4, 116.5,
.28–7.37 (2H, m), 7.52 (1H, t, J ¼ 8.2 Hz), 7.61 (1H, d, J ¼ 7.6 Hz),
13
1
19.9, 121.1, 125.3, 129.3, 132.1, 138.7, 143.9, 152.8, 159.4, 168.1.
3
8.14 (1H, s), 8.59 (1H, s); C NMR (CDCl , 75 MHz) d/ppm: 14.2,

2
88
F. SONMEZ ET AL.
ꢀ
2
1
4
1
0.8, 29.5, 55.7, 58.2, 115.3, 116.6, 119.7, 121.2, 124.8, 128.5, 131.6, (6j) Light yellow powder, 80% yield, mp. 198–200 C; IR: 3341,
ꢁ
1 1
39.0, 142.8, 153.2, 156.7, 159.9, 170.7; LC-MS-MS(ESI-) (m/z): 3157, 2930, 1715, 1528, 1270, 1092, 766 cm
;
H NMR (CDCl ,
3
þ
13.21 [M ]. Anal. Calcd for C22
H
27
N
3
O
3
S: C, 63.90; H, 6.58; N, 300 MHz) d/ppm: 2.64 (4H, s, N-CH
3.85 (4H, s, O-CH
, br), 7.26–7.39 (2H, m), 7.55 (1H, t, J ¼ 7.3 Hz),
.62 (1H, d, J ¼ 7.3 Hz), 8.17 (1H, s), 8.60 (1H, s), 10.27 (1H, s, NH);
2
, br), 3.29 (2H, s, CO-CH2-N),
0.16; found: C, 63.93; H, 6.56; N, 10.14.
2
7
1
3
C NMR (CDCl , 75 MHz) d/ppm: 54.1, 61.7, 67.1, 115.5, 116.6,
3
2
-(Diisopropylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)ace-
ꢀ
ꢁ
119.6, 121.0, 124.8, 128.5, 131.7, 138.9, 142.8, 153.1, 156.6 159.8,
168.5; LC-MS-MS(ESI-) (m/z): 371.13 [M ]. Anal. Calcd for
C H N O : C, 58.21; H, 4.61; N, 11.31; found: C, 58.24; H, 4.63; N,
18 17 3 4
tamide (6e) Yellow powder, 20% yield, mp. 160–162 C; IR: 3342,
þ
1
1
3
141, 2984, 1717, 1651, 1562, 1468, 1094, 747 cm
; H NMR
(
(
CDCl , 300 MHz) d/ppm: 1.31–1.42 (8H, m), 3.59–3.66 (6H, m), 4.05
2H, s, CO-CH
3
11.30.
2
-N), 7.22–7.33 (2H, m), 7.46 (1H, t, J ¼ 7.3 Hz), 7.53 (1H,
13
d, J ¼ 7.3 Hz), 8.06 (1H, s), 8.81 (1H, s); C NMR (CDCl þ DMSO-d6,
3
2
-((2,3-Dihydro-1H-inden-2-yl)amino)-N-(4-(2-oxo-2H-chromen-3-
yl)thiazol-2-yl)acetamide (6k) Yellow powder, 60% yield, mp.
56–158 C; IR: 3306, 3137, 2900, 1712, 1692, 1554, 1267, 1101,
7
1
5 MHz) d/ppm: 7.9, 53.7, 61.1, 114.2, 116.0, 120.1, 121.7, 124.5,
28.1, 130.7, 138.2, 141.9, 152.6, 160.4, 166.1, 168.1; LC-MS-
þ
ꢀ
1 1
1
7
1
3
MS(ESIþ) (m/z): 387.07 [MH ]. Anal. Calcd for C20
23 3 3
H N O S: C, 62.32;
ꢁ
42 cm ; H NMR (CDCl , 300 MHz) d/ppm: 2.85 (2H, dd, J ¼ 4.68,
H, 6.01; N, 10.90; found: C, 62.35; H, 6.04; N, 10.87.
3
5.81 Hz), 3.23 (2H, dd, J ¼ 6.73, 15.81), 3.58 (2H, s, CO-CH
2
-N),
.69–3.73 (1H, m), 7.20–7.35 (6H, m), 7.51 (1H, t, J ¼ 7.6 Hz), 7.58
2
-(Cyclohexylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)ace-
1
3
(
1H, d, J ¼ 7.6 Hz), 8.12 (1H, s), 8.53 (1H, s); C NMR (CDCl
3
,
ꢀ
tamide (6f) Dark yellow powder, 77% yield, mp. 156–159 C; IR:
7
1
1
5 MHz) d/ppm: 40.0, 50.5, 60.2, 115.3, 116.6, 119.7, 121.1, 124.8,
ꢁ
1 1
3
301, 3160, 2988, 1712, 1651, 1557, 1254, 1089, 753 cm ; H NMR
25.2, 127.0, 128.4, 131.6, 139.0, 141.1, 142.8, 153.1, 156.6, 159.9,
70.5; LC-MS-MS(ESI-) (m/z): 416.89 [M ]. Anal. Calcd for
C H N O S: C, 66.17; H, 4.59; N, 10.07; found: C, 66.14; H, 4.57; N,
23 19 3 3
(CDCl , 300 MHz) d/ppm: 1.14–1.31 (4H, m), 1.67–1.97 (6H, m),
þ
3
2
7
2
.45–2.52 (1H, m), 3.54 (2H, s, CO-CH -N), 7.28–7.39 (2H, m),
.51–7.63 (2H, m), 8.15 (1H, s), 8.61 (1H, s); C NMR (CDCl þ DMSO-
1
3
3
10.09.
d6, 75 MHz) d/ppm: 25.1, 26.4, 33.9, 49.8, 58.17, 115.4, 116.6, 119.7,
1
21.1, 124.8, 128.5, 131.6, 139.0, 142.8, 153.1, 156.8, 159.9, 171.3;
þ
N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)-2-((2-(pyrrolidin-1-yl)e-
LC-MS-MS(ESI-) (m/z): 383.01 [M ]. Anal. Calcd for C20
21
H N
3
O
3
S: C,
thyl)amino)acetamide (6 l) Yellow powder, 35% yield, mp.
6
2.64; H, 5.52; N, 10.96; found: C, 62.60; H, 5.56; N, 10.93.
ꢀ
1
7
2
7
30–135 C; IR: 3283, 3152, 2954, 1710, 1654, 1553, 1257, 1091,
ꢁ
1
1
57 cm
;
H NMR (CDCl , 300 MHz) d/ppm: 1.91 (4H, s, br),
3
N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)-2-(pyrrolidin-1-yl)aceta-
mide (6g) Yellow powder, 20% yield, mp. 160–161 C; IR321, 3140,
2
.63–2.67 (6H, m), 2.88 (2H, t, J ¼ 6.1 Hz), 3.56 (2H, s, CO-CH
2
-N),
ꢀ
13
.28–7.38 (2H, m), 7.51–7.57 (2H, m), 8.13 (1H, s), 8.58 (1H, s);
C
ꢁ
1 1
953, 1709, 1529, 1268, 1092, 760 cm ; H NMR (CDCl
3
, 300 MHz)
-N),
NMR (CDCl
3
, 75 MHz) d/ppm: 23.8, 48.7, 52.4, 54.0, 55.3, 115.2,
d/ppm: 1.91 (4H, s, br), 2.74 (4H, s, N-CH2, br), 3.43 (2H, s, CO-CH
2
1
1
16.6, 119.7, 121.3, 124.8, 128.3, 131.5, 138.7, 142.7, 153.1, 157.5,
59.9, 171.9; LC-MS-MS(ESI-) (m/z): 398.07 [M ]. Anal. Calcd for
1
3
7
.27–7.38 (2H, m), 7.51–7.61 (2H, m), 8.15 (1H, s), 8.58 (1H, s);
C
þ
3
NMR (CDCl , 75 MHz) d/ppm: 24.3, 55.0, 58.0, 115.5, 116.6, 119.7,
20 22 4 3
C H N O S: C, 60.28; H, 5.56; N, 14.06; found: C, 60.25; H, 5.54; N,
1
21.1, 124.8, 128.4, 131.7, 138.9, 142.7, 153.1, 156.9, 159.9, 169.7;
14.09.
þ
LC-MS-MS(ESI-) (m/z): 355.03 [M ]. Anal. Calcd for C18
17 3 3
H N O S: C,
6
0.83; H, 4.82; N, 11.82; found: C, 60.85; H, 4.84; N, 11.80.
2
2
3
-((2-Morpholinoethyl)amino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-
ꢀ
-yl)acetamide (6m) Yellow powder, 50% yield, mp. 150–153 C; IR:
N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)-2-(piperidin-1-yl)aceta-
mide (6h) Grey powder, 80% yield, mp. 167–170 C; IR: 3273, 3145,
ꢁ 1
1
359, 3181, 2934, 1702, 1681, 1536 1253, 1095, 739 cm ; H NMR
ꢀ
(
CDCl , 300 MHz) d/ppm: 2.53 (8H, t, J ¼ 5.8 Hz), 2.85 (2H, t,
3
ꢁ
1 1
2
3
CH
931, 1719, 1682, 1545, 1251, 1093, 756 cm
00 MHz) d/ppm: 1.52 (2H, s, br), 1.68–1.75 (4H, m), 2.58 (4H, s, N-
, br), 3.22 (2H, s, CO-CH -N), 7.27–7.39 (2H, m), 7.54 (1H, t,
J ¼ 8.4 Hz), 7.62 (1H, d, J ¼ 7.9 Hz), 8.15 (1H, s), 8.61 (1H, s); C NMR
CDCl , 75 MHz) d/ppm: 23.7, 26.3, 55.3, 62.0, 115.4, 116.6, 119.7,
21.1, 124.8, 128.5, 131.7, 138.9, 142.7, 153.1, 156.8, 159.9, 169.6;
3
; H NMR (CDCl ,
J ¼ 5.5 Hz), 3.55 (2H, s, CO-CH -N), 3.86 (2H, t, J ¼ 4.6 Hz), 7.27–7.49
2
13
(
(
2H, m), 7.52–7.60 (2H, m), 8.12 (1H, s), 8.57 (1H, s); C NMR
CDCl
2
2
, 75 MHz) d/ppm: 46.5, 52.1, 53.9, 57.9, 66.9, 115.2, 116.5,
3
1
3
1
1
19.7, 121.1, 124.8, 128.5, 131.6, 138.9, 142.7, 153.1, 157.1, 159.9,
71.3; LC-MS-MS(ESI-) (m/z): 414.22 [M ]. Anal. Calcd for
S: C, 57.96; H, 5.35; N, 13.52; found: C, 57.94; H, 5.37; N,
þ
(
1
3
20 22 4 4
C H N O
þ
19 3 3
LC-MS-MS(ESI-) (m/z): 369.16 [M ]. Anal. Calcd for C19H N O S: C,
6
13.54.
1.77; H, 5.18; N, 11.37; found: C, 61.74; H, 5.16; N, 11.39.
2
-((2-(Cyclohex-1-en-1-yl)ethyl)amino)-N-(4-(2-oxo-2H-chromen-3-
2
-(4-Methylpiperazin-1-yl)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-
yl)thiazol-2-yl)acetamide (6n) Yellow powder, 40% yield, mp.
ꢀ
ꢀ
yl)acetamide (6i) Yellow powder, 60% yield, mp. 189–191 C; IR: 164–166 C; IR: 3295, 3177, 3150, 2987, 1710, 1650, 1550, 1258,
ꢁ
1
1
ꢁ 1
1
3
291, 3144, 2934, 1721, 1705, 1528, 1170, 1088, 753 cm
;
H
3
1094, 761 cm ; H NMR (CDCl , 300 MHz) d/ppm: 1.64 (4H, s, br),
NMR (CDCl , 300 MHz) d/ppm: 2.36 (3H, s), 2.58 (4H, s, N-CH , br), 1.96 (2H, s, br), 2.07 (2H, s, br), 2.20 (2H, t, J ¼ 6.15 Hz), 2.79 (2H, t,
3
2
2
7
.70 (4H, s, N-CH
2
, br), 3.29 (2H, s, CO-CH
2
-N), 7.32 (1H, td, J ¼ 1.17, J ¼ 6.44 Hz), 3.51 (2H, s, CO-CH -N), 5.54 (1H, s, br), 7.29–7.39 (2H,
2
1
3
.3 Hz), 7.36 (1H, d, J ¼ 8.2 Hz), 7.54 (1H, td, J ¼ 1.75, 7.3 Hz), 7.62 m), 7.51–7.61 (2H, m), 8.15 (1H, s), 8.60 (1H, s); C NMR (CDCl ,
3
1
3
(
7
1
1H, dd, J ¼ 1.75, 7.9 Hz), 8.15 (1H, s), 8.59 (1H, s); C NMR (CDCl
3
,
75 MHz) d/ppm: 22.7, 23.0, 25.5, 28.3, 38.4, 48.0, 52.0, 115.5, 119.7,
5 MHz) d/ppm: 46.2, 53.8, 55.2, 61.1, 115.5, 116.6, 119.6, 121.0, 121.1, 123.9, 124.8, 128.4, 131.6, 134.9, 138.9, 142.8, 153.1, 156.8,
þ
24.8, 128.4, 131.7, 138.9, 142.7, 153.1, 156.7, 159.8, 168.9; LC-MS- 159.9, 170.7; LC-MS-MS(ESIþ) (m/z): 411.03 [MH ]. Anal. Calcd for
þ
MS(ESI-) (m/z): 384.12 [M ]. Anal. Calcd for C19
20
H N
4
O
3
S: C, 59.36; C H N O S: C, 64.53; H, 5.66; N, 10.26; found: C, 64.50; H, 5.64; N,
2
2 23 3 3
H, 5.24; N, 14.57; found: C, 59.38; H, 5.20; N, 14.53.
10.29.
2
-Morpholino-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide
2-((3,4-Dimethoxyphenethyl)amino)-N-(4-(2-oxo-2H-chromen-3-

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
289
1
ꢁ
1
yl)thiazol-2-yl)acetamide (6o) Yellow powder, 60% yield, mp. IR: 3359, 3181, 2934, 1702, 1681, 1536 1253, 1095, 739 cm ; H
ꢀ
9
9
0–92 C; IR: 3288, 3143, 2993, 1714, 1654, 1513, 1258, 1091, 123, NMR (CDCl
3
, 300 MHz) d/ppm: 2.32–2.87 (12H, m), 3.28 (2H, s),
ꢁ
1
1
24, 754 cm
;
H NMR (CDCl , 300 MHz) d/ppm: 2.82 (2H, t, 7.34–7.44 (2H, m), 7.61 (1H, t, J ¼ 7.0 Hz), 7.82 (1H, d, J ¼ 7.6 Hz)
3
1
3
J ¼ 6.15 Hz), 2.99 (2H, t, J ¼ 6.7 Hz), 3.51 (2H, s, CO-CH
2
3
-N), 3.83 (3H, 7.98 (1H, s), 8.58 (1H, s); C NMR (CDCl , 75 MHz) d/ppm: 35.2,
s), 3.87 (3H, s), 6.76–6.85 (3H, m), 7.27–7.35 (2H, m), 7.52 (2H, t, 53.2, 57.4, 60.9, 114.9, 116.6, 119.7, 121.0, 125.4, 129.5, 132.5,
1
3
J ¼ 8.4 Hz), 7.70 (1H, d, J ¼ 7.3 Hz), 8.11 (1H, s), 8.61 (1H, s);
NMR (CDCl
C
139.2, 142.7, 153.1, 159.4, 161.7, 169.6; LC-MS-MS(ESI-) (m/z):
þ
, 75 MHz) d/ppm: 36.0, 51.6, 52.0, 56.0, 56.1, 111.6, 415.07 [M ]. Anal. Calcd for C20
H
N
23 5
O
3
S: C, 58.09; H, 5.61; N,
3
1
1
(
11.8, 115.2, 116.5, 119.8, 120.9, 121.0, 124.8, 128.6, 131.6, 131.8, 16.94; found: C, 58.07; H, 5.63; N, 16.91.
39.2, 142.8, 147.9, 149.4, 153.1, 156.6, 159.9, 170.4; LC-MS-MS(ESI-)
þ
m/z): 465.20 [M ]. Anal. Calcd for C H N O S: C, 61.92; H, 4.98;
2
4 23 3 5
Anticholinesterase activity assays
Acetyl- (AChE) and butyryl-cholinesterase (BuChE) inhibitory activ-
-((Benzo[d][1,3]dioxol-5-ylmethyl)amino)-N-(4-(2-oxo-2H-chromen- ities of the synthesized compounds were determined according to
N, 9.03; found: C, 61.94; H, 4.97; N, 9.05.
2
3
1
9
4
3
-yl)thiazol-2-yl)acetamide (6p) Yellow powder, 73% yield, mp. Ellman’s method . The IC50 was determined by constructing an
ꢀ
68–170 C; IR: 3291, 3139, 2887, 1715, 1604, 1535, 1243, 1094, absorbance and/or inhibition (%) curve and examining the effect
ꢁ
1 1
24, 754 cm ; H NMR (CDCl
-N), 3.80 (2H, s, N-CH ), 5.95 (2H, s, O-CH
1H, s), 7.31–7.37 (2H, m), 7.51 (1H, t, J ¼ 7.0 Hz), 7.61 (1H, dd, half of the maximum biological response of the substrate. The
J ¼ 1.46, 7.6 Hz), 8.13 (1H, s), 8.59 (1H, s); C NMR (CDCl
, 75 MHz) substrates of the reaction were acetylthiocholine iodide and butyr-
d/ppm: 51.31, 54.0, 101.3, 108.5, 108.8, 115.3, 116.5, 119.7, 121.0, ylthiocholine iodide. 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) was
3
, 300 MHz) d/ppm: 3.53 (2H, s, CO- of five different concentrations. IC50 values were calculated for a
-O), 6.79 (2H, s), 6.88 given inhibitor by determining the concentration needed to inhibit
CH
2
2
2
(
1
3
3
1
1
21.8, 124.8, 128.5, 131.6, 132.6, 139.0, 142.7, 147.3, 148.2, 153.1, used to measure anticholinesterase activity. Stock solutions of the
56.6, 159.9, 170.2; LC-MS-MS(ESI-) (m/z): 435.18 [M ]. Anal. Calcd compounds and galanthamine in methanol were prepared at a
þ
for C22
N, 9.63.
H
17
N
3
O
5
S: C, 60.68; H, 3.93; N, 9.65; found: C, 60.65; H, 3.96; concentration of 4000 mg/mL. Aliquots of 150 mL of 100 mM phos-
phate buffer (pH 8.0), 10 mL of sample solution and 20 mL AChE
ꢁ
4
ꢁ 4
(
2.476 ꢂ 10
U/mL) (or 3.1813 ꢂ 10
U/mL BuChE) solution were
ꢀ
mixed and incubated for 15 min at 25 C. About 10 mL of DTNB
solution was prepared by adding 2.0 mL of pH 7.0 and 4.0 mL of
pH 8.0 phosphate buffers to a mixture of 1.0 mL of 16 mg/mL
2
-(Morpholinoamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)ace-
ꢀ
tamide (6q) Yellow powder, 66% yield, mp. 212–214 C; IR: 3332,
140, 2820, 1710, 1534, 1268, 1090, 757 cm
ꢁ
1 1
3
3
(
; H NMR (CDCl3,
3
DTNB and 7.5 mg/mL NaHCO in pH 7.0 phosphate buffers. The
00 MHz) d/ppm: 3.30–3.33 (8H, m), 3.76 (4H, t, J ¼ 4.6 Hz), 7.06
reaction was initiated by the addition of 10 mL (7.1 mM) acetylthio-
choline iodide (or 0.79 mM butyrylthiocholine iodide). In this
method, the activity was measured by following the yellow colour
produced as a result of the thio anion produced by reacting the
enzymatic hydrolysis of the substrate with DTNB. Also, methanol
was used as a control solvent. The hydrolysis of the substrates
1H, s), 7.35–7.46 (2H, m), 7.62 (1H, t, J ¼ 8.4 Hz), 7.80 (1H, d,
13
J ¼ 1.1 Hz), 7.98 (1H, s), 8.62 (1H, s), 11.78 (1H, s, NH); C NMR
CDCl , 75 MHz) d/ppm: 50.9, 65.9, 70.7, 114.9, 116.6, 119.7, 123.4,
25.4, 129.4, 132.5, 139.1, 142.8, 153.1, 158.2 159.4, 162.9; LC-MS-
(
3
1
þ
MS(ESI-) (m/z): 387.06 [M ]. Anal. Calcd for C18
18 4 4
H N O S: C, 55.95;
H, 4.70; N, 14.50; found: C, 55.93; H, 4.72; N, 14.53.
4
3
was monitored using a BioTek Power Wave XS at 412 nm .
2
-((4-Methylpiperazin-1-yl)amino)-N-(4-(2-oxo-2H-chromen-3-yl)th-
iazol-2-yl)acetamide (6r) Yellow powder, 62% yield, mp. Kinetic study of AChE inhibition
ꢀ
1
7
92–194 C; IR: 3288, 3136, 2946, 1715, 1708, 1520, 1160, 1085,
The kinetic study of AChE was performed according to Ellman’s
method with three different concentrations (20, 40 and 60 nM)
ꢁ
1 1
55 cm ; H NMR (CDCl
3
, 300 MHz) d/ppm: 1.79 (3H, s), 2.37 (2H,
43
s, br), 2.60 (4H, t, J ¼ 5.2 Hz), 3.39 (4H, t, J ¼ 4.9 Hz), 6.80 (1H, s),
of compound 6c. Lineweaver–Burk reciprocal plots were con-
structed by plotting 1/velocity against 1/[substrate] at varying con-
centrations of the substrate acetylthiocholine (0.05–0.5 mM). The
plots were assessed by a weighted least-squares analysis that
assumed the variance of velocity (v) to be a constant percentage
of v for the entire data set. The inhibition constant Ki was calcu-
lated by plots of the slopes of these reciprocal plots versus the
concentrations of compound 6c in a weighted analysis.
7
(
1
1
.26–7.38 (2H, m), 7.50–7.59 (2H, m), 7.11 (1H, s), 8.56 (1H, s), 9.84
1
3
1H, s); C NMR (CDCl , 75 MHz) d/ppm: 46.2, 50.3, 54.0, 115.0,
3
16.5, 119.7, 121.1, 124.8, 128.4, 131.6, 138.7, 142.7, 153.1, 157.3,
59.9, 162.5; LC-MS-MS(ESI-) (m/z): 401.01 [M ]. Anal. Calcd for
C H N O S: C, 57.13; H, 5.30; N, 17.53; found: C, 57.10; H, 5.34; N,
19 21 5 3
þ
1
7.51.
N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)-2-(piperidin-1-ylamino)a-
cetamide (6s) Yellow powder, 66% yield, mp. 158–159 C; IR: 3301,
160, 2988, 1712, 1651, 1557, 1254, 1089, 753 cm
CDCl , 300 MHz) d/ppm: 1.63–1.83 (4H, m), 2.17 (2H, s), 3.46–3.52
2H, m), 3.73–3.77 (2H, m), 4.23 (2H, s), 6.58 (2H,s), 7.29–7.35 (2H,
ꢀ
ꢁ
1
1
In vitro cytotoxicity assay
3
(
(
; H NMR
3
The cytotoxicity effect of test compound on hepatoma cell H4IIE
cells was evaluated by MTT assay according to described meth-
1
3
m), 7.48–7.57 (2H, m), 7.97 (1H, s), 8.81 (1H, s);
C NMR
44
ods . Briefly, H4IIE cells were seeded in a flat-bottomed 96-well
(CDCl
3
þ DMSO-d6, 75 MHz) d/ppm: 20.3, 21.6, 63.2, 69.3, 113.7,
4
plate at a density of 5 ꢂ 10 cells/well in DMEM containing 10%
1
1
16.4, 120.2, 121.8, 125.2, 129.3, 131.7, 138.1, 142.0, 152.8, 159.8,
ꢀ
FBS. The plate was incubated at 37 C with 5% CO
2
for 24 h, and
þ
67.3, 167.9; LC-MS-MS(ESI-) (m/z): 387.05 [M ]. Anal. Calcd for
C H N O S: C, 59.36; H, 5.24; N, 14.57; found: C, 59.38; H, 5.22; N,
9 20 4 3
then 6c was prepared and added to make a final concentration of
1
2
.5, 1.25, 0.625, 0.312, 0.156, 0.078 mM, respectively, in serum-free
1
4.54.
ꢀ
DMEM. Cells were further incubated for 24 h at 37 C with 5% CO
2
;
then, the medium was replaced with DMEM containing 10% FBS.
N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)-2-((2-(piperazin-1-yl)ethy- About 10 mL of filter-sterilized MTT (3–(4,5 dimethylthiazol-2-yl)-2,5-
ꢀ
l)amino)acetamide (6t) Yellow powder, 64% yield, mp. 151–153 C; diphenyltetrazolium bromide) solution (5 mg/mL in PBS) was

2
90
F. SONMEZ ET AL.
ꢀ
1
13
added to each well and further incubated at 37 C with 5% CO
2
All the new compounds were characterized by H NMR,
C
1
13
for 4 h. At the end of incubation, media was aspirated from the NMR, IR, MS and elemental analysis. H NMR, C NMR and MS
wells and 100 mL of DMSO was added to dissolve insoluble formo- spectra of the synthesized compounds are given in supplementary
san crystals formed. The absorbance was measured at 570 nm materials.
using a microtiter plate reader. The relative % cell viability was cal-
culated from the following equation: Relative percent cell
Cholinesterase inhibitory activity
viability ¼ (Atest/Acontrol) ꢂ 100%. (A
is the absorbance of the
test
The inhibitory activities of the target compounds (6a-t) on AChE
sample treated cells and a control is the absorbance of the
untreated cells. Each absorbance was taken to be the mean of
triplicate measurements.) The cell viability was represented as a
percentage relative to untreated cells as a control.
4
3
and BuChE were determined by the Ellman’s method using gal-
antamine as the reference compound. The IC50 values for AChE
and BuChE inhibitions are summarized in Table 1. IC50 values
against AChE ranged from nanomolar to micromolar units (43 nM-
1
3.53 mM). High AChE selectivity (7.32–4151.16) over BuChE was
Docking study
observed. Compound 6c exhibited the strongest inhibition against
AChE with an IC50 value of 43 nM, which was 56-fold more than
that of galantamine (IC50 ¼2.41 mM). Furthermore, 11 of the syn-
thesized compounds (6d, 6e, 6 g, 6 h, 6i, 6k, 6 l, 6m, 6p, 6r and
Ligands were sketched and energy-minimized using Sybyl v8.1
(Tripos, Inc., St. Louis, MO) on an Intel (Xeon 4 core, HP Z820)
using Linux 6 operating system. Protonation states at physiological
pH were calculated and considered during molecular editing pro-
cedure; in any case, the most abundant protomer was saved.
AutoDock Vina software (version 1.1.2) was used to perform dock-
ing (standard options) of coumarin derivatives into 1ACJ, 1EVE and
6
s) exhibited better AChE inhibition (IC50 ¼0.09–2.36 mM) than the
positive control, galantamine, by 1.4–26.7-fold. Most of the synthe-
sized compounds showed lesser inhibitory activity against BuChE
than galantamine, except of four compounds 6i, 6 l, 6r and 6s.
Compound 6 l exhibited the strongest inhibition against BuChE
with an IC50 value of 2.35 mM, which was 2- and 7.5-fold more
than that of donepezil (IC50 ¼4.66 mM) and galanthamine
1
P0M crystallographic structures. To validate the docking program,
the co-crystallized ligand (donepezil) was redocked on the target
enzyme. A RMS (Root Mean Square) value of 0.531 was found for
donepzil-bound acetylcholinesterase. A ligand-centred grid box,
defined with a size of 50 ꢂ 60 ꢂ 50 Å3 and a regular space of
(IC50 ¼17.38 mM), respectively.
Some acetylcholinesterase inhibitors such as donepezil, rivastig-
mine and galantamine are currently used to treat the cognitive
problems of AD
0
.375 Å, able to cover the whole binding site, was considered for
9,22,47
. Tacrine is also a well-known class of AChE
docking. Nine poses (docking solutions) were generated for each
ligand into each model and then energetically scored. A total of
1
inhibitors with an IC50 value of 167 nm . Among the synthesized
compounds in this work, 6c showed the strongest inhibition
against AChE with an IC50 value of 43 nM, which was almost 4-,
and 70-fold more than that of tacrine (IC50 ¼167 nM) and rivastig-
mine (IC50 ¼3.01 mM), respectively. Also, it approached that of
donepezil (IC50 ¼30 nM), which is one of the most used AChE
inhibitor. The AChE selectivity of 6c increased 26-, 28828- and
9
ꢂ 20 ꢂ 3 ligand–protein complexes were analyzed to identify the
best solution from both a geometrical and energetic point of
view .
4
5
Results and discussion
4
1511-fold when compared to donepezil (26 versus 155.3), tacrine
Chemistry
(28828 versus 0.144) and rivastigmine (41511 versus 0.10), respect-
The synthetic procedures to obtain the target compounds 6a-t are ively (Table 1).
depicted in Scheme 1. 3-acetylcoumarin (2) was synthesized from
A plethora of literature papers has presented the newly synthe-
4
6
36
salicylaldehyde (1) according to the literature , and then it was sized coumarin derivatives as AChE inhibitors. Xie et al. and Nam
2
3
brominated by molecular bromine in chloroform. 3–(2-Amino-1,3- et al. designed and synthesized novel tacrine-coumarin hybrids
thiazol-4-yl)coumarin (4) was obtained by reacting 3-(bromoace- and aminoalkyl-subsitituted coumarin derivatives as cholinesterase
tyl)coumarin (3) with thiourea. The reaction of 4 with chloroacetyl- inhibitors. In these studies, the strongest inhibitors had IC50 value
chloride in THF gave 2-chloro-N-(4–(2-oxo-2H-chromen-3-yl)thiazol- of 92 nm and 2.87 mM, respectively. These IC50 values are 2- and
2
-yl)acetamide (5). Coumarylthiazole-substituted acetamido deriva- 67-fold less than that of 6c, which is the best AChE inhibitor in
1
8
tives (6a-t) were obtained by the reaction between compound 5 here, respectively. On the other hand, Asadipour et al. and Catto
and various amine derivatives in DMF.
4
7
et al. synthesized various coumarin-3-carboxamide derivatives as
O
O
O
O
O
O
O
H
OH
iii
i
ii
Br
1
2
3
O
O
O
O
O
O
Cl
R
iv
O
v
O
N
N
N
NH2
NH
NH
S
S
S
4
5
6a-t
ꢀ
3
, CHCl , 50 C,
Scheme 1. Synthesis of new coumarylthiazole-substituted acetamide derivatives. Reaction conditions: (i) Ethylacetoacetate, piperidin, rt, 30 min; (ii) Br
1
2
ꢀ ꢀ ꢀ
3 2
5 min; (iii) Thiourea, EtOH, 80 C, 2 h; (iv) Chloroacetylchloride, Et N, THF, 70 C, 8 h; (v) RNH , DMF, 60 C, 12 h.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
291
Table 1. In vitro inhibition IC50 values (lM) and selectivity of compounds 6a-t
Table 1. Continued
for AChE and BuChE.
AChE
IC50 (lM)
BuChE a Selectivity
a
b
Compound
R
IC50 (lM)
index
O
O
R
O
HN
N
O
NH
S
O
AChE a
BuChE
IC50 (lM)
a
Selectivity
index
b
6p
0.74 ± 0.001
60.35 ± 0.03
81.55
Compound
R
IC50 (lM)
6
a
2.36 ± 0.003 121.72 ± 0.02
51.58
HN
O
O
HN
6
b
2.15 ± 0.002 189.30 ± 0.05
88.05
6
6
6
6
q
r
2.52 ± 0.005 152.25 ± 0.75
60.42
7.32
HN
HN
6
6
c
0.043 ± 0.002 178.50 ± 0.03 4151.16
N
O
1.12 ± 0.003
0.82 ± 0.002
8.2 ± 0.59
N
HN
N
N
d
1.71 ± 0.002 180.30 ± 0.10
105.44
19.66
s
t
15.2 ± 0.82
18.54
14.00
N
HN
N
8.25 ± 0.057 115.48 ± 0.87
HN
6
e
2.29 ± 0.001
45.02 ± 0.01
N
NH
N
Galantamine
Donepezil
–
–
–
2.41 ± 0.01
0.03 ± 0.0005
3.01 ± 0.21
17.38 ± 0.56
4.66 ± 0.503 155.30
0.30 ± 0.01
7.21
c
d
Rivastigmine
0.10
0.144
e
6
6
f
3.05 ± 0.002 168.18 ± 0.40
55.14
67.95
Tacrine
a
0.167 ± 0.0119 0.024 ± 0.004
IC50 values represent the means ± SEM of three parallel measurements
b(p < 0.05).
HN
g
1.41 ± 0.001
0.73 ± 0.001
95.81 ± 0.36
22.90 ± 0.12
Selectivity index ¼ IC (BuChE)/IC50 (AChE).
50
c
47
From Ref. .
d
e
22
N
From Ref. .
1
From Ref. .
6
6
h
i
31.37
12.16
potent AChE inhibitors. These new compounds showed potent
activity in the range of sub-micromolar concentrations
N
1
8
47
(0.3–633 nm and 7.6–6600 nm ).
0.61 ± 0.001
7.42 ± 0.23
When compared to the best AChE inhibitors in our previously
works, 6c exhibited better AChE inhibition (IC50 ¼43 nM) than the
N
3
2
benzofuran derivative (PBI-1) (IC50 ¼3.85 mM)
and coumarin
3
3
N
derivative (PBI-2) (IC50 ¼4.58 mM) by almost 90- and 107-fold,
respectively. This increase can relate to the various interactions (a
cation–p interaction, especially) between each of the fragments at
the designed molecules and the catalytic triad of AChE. This find-
ing supports that the interactions with the catalytic triad of AChE
6
j
3.78 ± 0.003 182.70 ± 0.03
0.92 ± 0.001 175.30 ± 0.15
48.33
N
O
play
molecules.
The following structure–activity relationship (SAR) observations
a crucial role for efficient inhibitory activities of the
6
6
k
l
190.54
13.82
HN
can be drawn from data of Table 1: (i) Replacing the methyl (6a)
on the acetamide moiety by a propyl (6b) or a cyclohexyl (6f) did
not cause significant changes on the AChE inhibitory activity; (ii)
the increase of steric hindrance on the N atom of the acetamide
0.17 ± 0.001
2.35 ± 0.65
HN
N
moiety in compound 6c (IC50 ¼43 nM) by
a dibutyl (6d:
6
6
6
m
0.09 ± 0.001 157.29 ± 2.27 1747.67
IC50 ¼1.71 mM) or a propyl (6e: IC50 ¼2.29 mM) group led to a
decline of the inhibitory activity against AChE; (iii) the expansion
of the pyrrolidine ring of compound 6g (IC50 ¼1.41 mM against
AChE; IC50 ¼95.81 mM against BuChE) to a piperidine ring (com-
HN
O
N
n
10.24 ± 0.001 186.70 ± 0.36
18.23
pound 6h: IC50 ¼0.73 mM against AChE; IC ¼22.90 mM against
50
HN
BuChE) and 4-methylpiperazine ring (6i: IC50 ¼0.61 mM against
AChE; IC50 ¼7.42 mM against BuChE) increased the inhibitory activ-
ity against both ChEs. On the contrary, the presence of the mor-
o
13.53 ± 0.001 189.40 ± 0. 06
14.00
(
continued)

2
92
F. SONMEZ ET AL.
pholine ring (6j: IC50 ¼3.78 mM against AChE; IC50 ¼182.70 mM
against BuChE) strongly decreased the AChE and BuChE inhibitory
activity. The reason for this decrease can be explained by a nega-
tive inductive effect of the oxygen, on the morpholine moiety
which lowers the electron density of the amine nitrogen, finally
leading to a decrease of its H-bonding capability;(iv) The presence
of an ethyleneamine group as a spacer between the acetamide
moiety and the morpholine or pyrrolidine ring positively affected
the inhibitory activity against both ChEs. On the contrary, the eth-
yleneamine group on the piperazine ring caused an opposite
effect on the inhibitory activity against both AChE and BuChE as
in the case of compound 6i (IC50 ¼0.61 mM against AChE;
IC50 ¼7.42 mM against BuChE) and 6t (IC ¼8.25 mM against AChE;
50
IC50 ¼115.48 mM against BuChE); (v) the presence of an amine
group between the acetamide moiety and the piperidine or mor-
pholine ring did not significantly affect AChE and BuChE inhibition Figure 3. Kinetic study on the mechanism of AChE inhibition by compound 6c.
Overlaid Lineweaver–Burk reciprocal plots of AChE initial velocity at increasing
as showed by compound 6h (IC50 ¼0.73 mM against AChE;
substrate concentration (0.05–0.50 mM) in the absence of inhibitor and in the pre-
IC50 ¼22.90 mM against BuChE) when compared to 6s
sences of different concentrations of 6c are shown.
(
IC50 ¼0.82 mM against AChE; IC ¼15.20 mM against BuChE). On
50
the contrary, the same amino group caused a decrease in the
analysis of the reciprocal Lineweaver–Burk plot (Figure 3) showed
both increased slopes (decreased Vmax) and intercepts (higher
Km) at higher inhibitor concentration. This pattern indicated a
mixed-type inhibition and therefore revealed that compound 6c
might be able to bind to the catalytic active site (CAS) and periph-
eral anionic site (PAS) as well as catalytic triad of AChE, which
were consistent with our design strategy. The Ki value of 31 nM
was determined by plots of the slopes of the Lineweavere–Burk
reciprocal plots versus concentrations of 6c.
AChE and BuChE inhibitory activity, as showed by compound 6i
(
IC50 ¼0.61 mM against AChE; IC50 ¼7.42 mM against BuChE) on
respect to compound 6r (IC ¼1.12 mM against AChE;
5
0
IC50 ¼8.20 mM against BuChE). An opposite trend was observed for
compounds 6j (IC50 ¼3.78 and 182.70 mM for AChE and BuChE,
respectively) and 6q (IC50 ¼2.52 mM against AChE; IC50 ¼152.25 mM
against BuChE) where the introduction of an amine group
between the acetamide moiety and the morpholine ring nega-
tively affects the activity against both AChE and BuChE; (vi) The
replacement of the ethyleneamine group (6o: IC50 ¼13.53 mM
against AChE; IC50 ¼189.40 mM against BuChE) between the aceta- Docking study
mide moiety and the aromatic ring by a methyleneamine (6p:
Docking studies have been performed in order to analyze the bind-
IC50 ¼0.74 mM against AchE; IC50 ¼60.35 mM against BuChE) and
the acetalization of the dimethoxy group to a phenyl ring led to
an effective increase on the activity against both ChEs.
The obtained IC50 values against BuChE demonstrated that all
compounds act as selective AChE inhibitors, more than galant-
amine. The amine functional group on alkyl side chain or lipophilic
moieties and a tertiary amino group can represent key require-
ment for an anti-AChE activity.
ing profile of the new synthetized coumarin derivatives into AChE
and BuChE enzymes. Both cholinesterases (AChE and BuChE) are
structurally similar and they share 65% of the amino acid
45
sequence . The main difference substantially is on the replacement
of aromatic with aliphatic amino acids in BuChE, which is crucial for
22
the selectivity against different inhibitors of the two enzymes .
The two PDB structures 1ACJ of TcAChE co-crystallized with
tacrine and 1EVE of TcAChE co-crystallized with Donepezil have
been taken into account to model the binding to AChE. The
choice of these two structures is not casual since they are repre-
sentative of two peculiar ligand-induced local conformational
Besides inhibitory activity of coumarin compounds against
AChE, they are a well-known class of the carbonic anhydrase
4
8–50
inhibitors
. Supuran et al. investigated that both the simple
lead coumarin for its interaction with the CA active site by means
of high resolution X-ray crystallography as well as a series of cou-
marin derivatives possessing various moieties substituting the cou-
marin ring in the 3-, 6-, 7-, 3,6-, 4,7-, 3,8-, 6,7- and 7,8-
57
changes (see Figure 4) .
Analysis of the crystallographic structures for TcAChE deposited
in PDB reveals two important conformational changes located at
CAS and PAS, which can drastically affect ligand accommodation
during docking. In 1EVE crystal structure (Figure 4(A)), the con-
formation adopted by Phe330 (CAS) and Trp279 (PAS) opens the
gorge allowing the placement of dual binding site inhibitors as
Donepezil (E20). Conversely, in 1ACJ, Phe330 is stacked against
tacrine thus closing the gorge.
Proceeding through the species, analysis of pairwise sequence
alignment reveals the replacement of Phe330 with Tyr337 in
hAChE (see Figure 4). However, superposition of folded structures
for Torpedo californica and human AChE (Figure 4(B); 1EVE in blue
and 4EY7 in brown) allows to see that Tyr337 in hAChE (4EY7)
assumes the same orientation of Phe330 in TcAChE (1EVE).
Considering the structure of our ligands and the low accuracy in
energy predictions of scoring functions, we can suppose that the
additional hydroxyl in Tyr337 should not affect significantly our
5
1,52
positions
. According to these studies, the 3-substituted couma-
rin derivatives with bulky moieties had a poor CA inhibitory activ-
ity and the best positions would be 4-, 5-, 6-, 7-, 8- and 7,8-. These
5
1,52
coumarin derivatives were highly active as CAIs
studies
. Our previous
3
3,53
showed that 3-substituted coumarin derivatives bear-
ing thiazole ring and urea moieties had higher inhibitory activities
against AChE than CA I and II and the moderate AChE selectivity
(almost 1–10-fold) with respect to CA I and II was observed. Many
studies have also supported that most of synthesized compounds
as AChE and CA inhibitors have high AChE selectivity with respect
3
0,54–56
to CAs
.
Kinetic study of AChE inhibition
In order to explore the inhibition mechanism of compound 6c docking simulations.
with AChE, an enzyme kinetic study was carried out. Graphical

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
293
Figure 4. (A) Superposition of crystallographic structures for TcAChE co-crystallized with tacrine (1ACJ, in green) and Donepezil (1EVE, in blue). (B) Superposition of
TcAChE (1EVE residue Phe330, Ile287, in blue) and hAChE (4EY7 residue Tyr337, Val294, in brown), both in complex with Donepezil. Red circles highlight the most
important ligand-induced conformational changes. Pairwise sequence alignment for T. californica (PDB ID: 1ACJ, 1EVE) and human (PDB ID: 4EY7) AChE is also provided.
The catalytic triad is reported in red.
Figure 5. Comparison of TcAChE (1EVE residue Ile287, Phe288, Phe290, Val400, Trp279, Tyr70, Phe330, in blue) and hBuChE (1P0M residue Pro285, Leu286, Val288,
Phe398, Ala277, Asn68, Ala328, in gold). The most important changes in aminoacids sequence are highlighted by blue, green and red areas. As shown, modifications
generally affect both CAS (1, in blue) and PAS (2, in green and 3, in red).
Figure 6. Qualitative representation of correlation between binding energies coming from docking and experimental data. Regarding the TcAChE model, correlation
2
2
obtained by using 1ACJ/1EVE is reported in blue (R ¼ 0.06)/red (R ¼ 0.6). Correlation for hBuChE (1P0M) is reported in green.

2
94
F. SONMEZ ET AL.
Figure 7. Docking poses for compound 6i, 6j, 6o and 6p into TcAChE (1EVE; A–D) and BuChE (1P0M; E–H) binding sites. Aminoacids relevant for ligand interactions are
indicated and reported in white sticks.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
295
In addition, given the limited “crystallographic” mobility of ami- benzodioxo- moiety with this last also involved in H-bonding with
noacids within the binding site emerged by structural analysis, we the backbone nitrogens of Phe288 and Arg289.
can conclude that a satisfactory covering the conformational
ensemble for AChE would be reached by using the two structures pounds into the hBuChE binding site have been also reported in
ACJ and 1EVE which are indicative of the most important Figure 7(E–H) for comparative purposes. In principle, the binding
Docking-predicted protein–ligand complexes for the same com-
1
changes in side-chain orientation of residues Phe330 and Trp279. profile of coumarin derivatives into BuChE seems to be generally
For this, both the two structures of TcAChE (1ACJ and 1EVE) have less stable than that observed for AChE. In this regard, the replace-
been selected for docking studies. Moreover, the impact of ment of Phe330 and Trp279 with respectively Ala328 and Ala277
Phe330 and Trp279 side-chains orientation on ligand binding and in hBuChE could be in part responsible for inversion of the bind-
ranking performances has been analyzed in detail.
ing profile observed in case of AChE (the coumarin moiety stacked
against Trp82 in CAS) and lowering in binding affinity.
The BuChE system
H4IIE hepatoma cell toxicity
Comparative analysis of the binding site for TcAChE (Figure 5,
1
EVE in blue) and hBuChE (Figure 5, 1P0M in gold) allows seeing
On basis of the screening results above, the most potent com-
pound 6c was selected to further examine the potential toxicity
effect on the hepatoma cell line H4IIE. After exposing the cells to
this compound for 24 h, the cell viability was evaluated by the 3-
important changes in aminoacid sequence generally identifiable as
aromatic-to-aliphatic switch. As shown in Figure 5, modifications
affect both CAS and PAS. Among them, Phe330-to-Ala328 (CAS)
and Trp279-to-Ala277 (PAS) mutations seem to remarkably contrib-
ute to differentiate proteins fingerprint, thus helping in explan-
ation of ligand selectivity profile.
For docking purposes, the PDB structure 1P0M of hBuChE
bound to a substrate analogue (CHT) has been selected.
A qualitative representation of correlation between binding
energies coming from docking (expressed as kcal/mol) and experi-
mental data (reported here as pIC50; mM) is given in Figure 6.
Keeping in mind the (i) limitations of docking in energetic evalua-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. The
result indicated that 6c caused negligible cell death and was non-
toxic to H4IIE cell at 0.07–2.5 mM (cell viability: 0.07 mM: 130 ± 5.1%;
0
5
.15 mM: 106 ± 2.4%; 0.3 mM: 101 ± 0.9%; 0.6 mM: 78 ± 7.3%; 1.25 mM:
6 ± 5.0%; 2.5 mM: 38 ± 4.1%).
Conclusions
tions and (ii) the limited range of activity for AChE (2.5 logarithmic A series of 20 novel acetamide substituted coumarylthiazoles
units) and BuChE (about 2 logarithmic units) inhibitors, this repre- derivatives (6a-t) was synthesized, and their inhibitory activities on
sentation aims to be purely indicative and wants to show the AChE and BuChE were evaluated. All the synthesized compounds
impact of the structural model on docking results.
are selective inhibitors of AChE. Among them, 6c exhibited the
As expected from the previously performed structural analysis strongest inhibition against AChE with an IC50 value of 43 nM,
of the two structures of TcAChE, best results in term of qualitative which was 4-, 56- and 70-fold more than that of tacrine
correlation between in silico-determined binding affinities and (IC50 ¼167 nM), galantamine (IC50 ¼2.41 mM) and rivastigmine
experimental data are obtained by using the 1EVE structure (IC50 ¼3.01 mM), respectively. The selectivity of 6c towards AChE is
2
(
1
Figure 6: R of 0.6; in red) as structural model with respect to 26-, 575-, 28826- and 41511-fold compared with donepezil, galant-
2
ACJ (Figure 6: R of 0.06; in blue), with binding affinities that amine, tacrine and rivastigmine, respectively. Kinetic study of AChE
range between ꢁ9 and ꢁ12 kcal/mol. This is not surprising given inhibition revealed that 6c was a mixed-type inhibitor. Moreover,
the Donepezil-like features of our coumarin derivatives.
the result of H4IIE hepatoma cell toxicity assay indicated that 6c
A good correlation between experimental and in silico-pre- caused negligible cell death and was non-toxic at 0.07–2.5 mM
dicted data is also observed for the BuChE model (Figure 6: 1P0M,
The presence of N-alkyl and/or heterocyclic moiety instead of
in green; R2 of 0.6) with predicted binding affinities that range an aryl group increased the AChE inhibition by almost 107-fold
through the cation–p interactions with the catalytic triad of AChE.
between ꢁ7 and ꢁ10 kcal/mol.
Binding modes for dual binding site inhibitors 6i, 6j, 6o and This finding can provide guidance for researches to design new
p into TcAChE (1EVE model, A–D) and hBuChE (1P0M model, efficient ChEs inhibitors in the future works.
6
E–H) are reported in Figure 7. Compound 6i (Figure 7; in
Overall these derivatives could be recommended as new che-
green), one of the most potent AChE inhibitors of the tested motypes to develop new AChE inhibitors for the treatment of AD
coumarin series, shows a good disposition into the binding site, disease by suitably modulating the substitution pattern also in the
being able to give H-bonding interactions with Phe288, Arg289 perspective of multifunctional anti AD agents.
main chains and Tyr121. Moreover, an interesting cation–p inter-
action can be observed between the protonated piperazine and
Disclosure statement
Trp84. Loss of cation–p due to replacement of piperazine with
morfoline moiety could explain the reduction in inhibitory activ-
ity for compound 6j (Figure 7(B); in pink). T-stacking interaction
between the coumarin moiety and Trp279 is also observed in
both cases.
The authors report no declarations of interest. The authors alone
are responsible for the content and writing of the paper.
Going to the less active compound 6o (Figure 7(C); in light
red), a double T-stacking interaction can be observed for the cou-
Funding
marin and the 3,4-di-MeO-phenyl- moiety with Trp279 (PAS) and This work was supported by the Sakarya Research Fund of the
Trp84 (CAS) residues, respectively. H-bonding interactions also Sakarya University, 10.13039/501100004473. Project Number: 2014-
involve Tyr121 and Phe330 (main chain). Another interesting case 28-00-001.
is represented by compound 6p (Figure 7(D); in blue marine). For
this compound, an inversion of the binding mode is observed. The
coumarin moiety is stacked against Trp84 (CAS) and the 2H-1,3-

2
96
F. SONMEZ ET AL.
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with improved acetylcholinesterase inhibitory activity and
lower hepatotoxicity as a potential lead compound for the
treatment of Alzheimer’s disease. J Enzyme Inhib Med Chem
J
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