Synthesis and Biological Evaluation of 3-thiazolocoumarinyl Schiff-base Derivatives as Cholinesterase Inhibitors

Article abstract of DOI:10.1111/j.1747-0285.2012.01435.x

On the basis of the observed biological activity of the coumarins, a new set of 3-thiazolocoumarinyl Schiff-base derivatives with chlorine, hydroxy and methoxy functional group substitutions were designed and synthesized. These compounds were tested against acetylcholinesterase from Electrophorus electricus and butyrylcholinesterase from horse serum and their structure-activity relationship was established. Studies revealed them as the potential inhibitors of cholinesterase (acetylcholinesterase and butyrylcholinesterase). The 3f was found to be most potent against acetylcholinesterase with K_i value of 1.05±0.3μm and 3l showed excellent inhibitory action against butyrylcholinesterase with K_i value of 0.041±0.002μm. The synthesized compounds were also docked into the active sites of the homology models of acetylcholinesterase and butyrylcholinesterase to predict the binding modes of these compounds. It was predicted that most of the compounds have similar binding modes with reasonable binding affinities. Our docking studies have also shown that these synthesized compounds have better interaction patterns with butyrylcholinesterase over acetylcholinesterase. The main objective of the study was to develop new potent and selective compounds, which might be further optimized to prevent the progression of the Alzheimer's disease and could provide symptomatic treatment.

Full text of DOI:10.1111/j.1747-0285.2012.01435.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01435.x
Chem Biol Drug Des 2012
Research Article
Synthesis and Biological Evaluation of
3-thiazolocoumarinyl Schiff-base Derivatives as
Cholinesterase Inhibitors
Rabia Raza1, Aamer Saeed2, Mubeen Arif2,
Received 14 March 2012, revised 9 May 2012 and accepted for publica-
tion 5 June 2012
Shamsul Mahmood2, Muhammad
Muddassar3,†, Ahsan Raza1 and Jamshed
Iqbal1,*
Alzheimer's disease (AD) is a chronic, neurodegenerative disorder
that causes an irreversible dementia among the elderly people. It is
characterized by the formation of extracellular amyloid-b peptide
plaques and intracellular neurofibrillary tangles within the brain of
afflicted individuals (1). With the progression of the disease, the
evident symptoms includes confusion, petulance, anger, long-term
memory loss, inability to perform body functions efficiently which
ultimately leads to death (2,3). Researchers have emphasized their
efforts to find new medicines for the treatment of AD for many
1Department of Pharmaceutical Sciences, COMSATS Institute of
Information Technology, Abbottabad 22060, Pakistan
2Department of Chemistry, Quaid-i-Azam University, Islamabad-
45320, Pakistan
3School of Computational Sciences, KIAS, Dongdaemun-gu, Seoul
130-722, South Korea
*Corresponding author: Jamshed Iqbal, drjamshed@ciit.net.pk
Present address: Zhang Initiative Research Unit, Advanced Science
Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
years. However, as the molecular theory of the disease is yet to be
discovered, its treatment is based on various biochemical theories
that describe the pathogenesis of AD (4–7). As a consequence, up
to now medicines can only delay the progression of the disease,
rather than to eradicate it completely. 'Cholinergic hypothesis' is
considered to be the most accepted one among the biochemical
theories for the progression of AD. It explains that diminished
motor and intellectual activity of the brain results owing to the loss
of cortical cholinergic neurotransmission (8–11). Acetylcholine is a
potent neurotransmitter released by presynaptic cholinergic termi-
nals, which activates nicotinic and muscarinic receptors postsynapti-
cally. Acetylcholine (ACh) is metabolically degraded by the enzymes
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).
Therefore, for the treatment of AD, acetylcholinesterase inhibitors
(AChEIs) are usually administered, which increases the level of ace-
tylcholine in the synaptic cleft (12,13). However, the AChEIs do not
uproot the disease but only minify the symptoms temporarily.
On the basis of the observed biological activity of
the coumarins, a new set of 3-thiazolocoumarinyl
Schiff-base derivatives with chlorine, hydroxy and
methoxy functional group substitutions were
designed and synthesized. These compounds were
tested against acetylcholinesterase from Elec-
trophorus electricus and butyrylcholinesterase
from horse serum and their structure–activity rela-
tionship was established. Studies revealed them
as the potential inhibitors of cholinesterase (ace-
tylcholinesterase and butyrylcholinesterase). The
3f was found to be most potent against acetylcho-
linesterase with Ki value of 1.05 0.3 lM and 3l
showed excellent inhibitory action against butyr-
ylcholinesterase with Ki value of 0.041 0.002 lM.
The synthesized compounds were also docked into
the active sites of the homology models of acetyl-
cholinesterase and butyrylcholinesterase to pre-
dict the binding modes of these compounds. It
was predicted that most of the compounds have
similar binding modes with reasonable binding
affinities. Our docking studies have also shown
that these synthesized compounds have better
interaction patterns with butyrylcholinesterase
over acetylcholinesterase. The main objective of
the study was to develop new potent and selective
compounds, which might be further optimized to
prevent the progression of the Alzheimer’s disease
and could provide symptomatic treatment.
Recent researches have shown that apart from improving cholinergic
neurotransmission, AChEIs also hamper the formation and deposition
of amyloid-b-peptides (14,15). Owing to this reason, AChEIs have
become first-line treatment choice for AD (16,17). AChEIs for
instance, donepezil, ensaculine, propidium, rivastigmine and tacrine
(Figure 1) have been used for many years for the treatment of AD
and have improved cognitive and psychological functions of the brain
(18). Recent studies have shown that among the examples mentioned
above, ensaculine, a coumarin derivative (18) slows down or to some
extend prevents the progression of the neurodegradation. Coumarins,
an important class of natural compounds, have been investigated
widely because of their significant role as antibacterial, antifungal,
(19) anticancer, (20) antitubercular, (21) antiacetylcholinesterase, (22)
antimutagenic, (23) anthelmintic, (24) anticoagulant, (25) anti-inflam-
matory, antihepatitis C (26) and analgesic activity (27). Moreover,
Key words: 3-thiazolocoumarinyl Schiff Base, acetylcholinesterase,
Alzheimer's disease, butyrylcholinesterase, coumarin derivative
1

Raza et al.
Figure 1: Some inhibitors of
Acetylcholiesterase used as Alzhei-
mer's disease therapeutics.
many of the coumarin derivatives are used as inhibitors of heat-shock
protein, (28) non-peptidic protease inhibitor, (29) inhibitor of 17b-hy-
droxysteroid dehydrogenase (17b-HSD) type 1, (30) Inhibitors of
tumour necrosis factor-alpha (TNF-a) (31) and monoamine oxidase
inhibitor (32). 4-methylcoumarins bearing different functionalities are
well-known antioxidant and radical scavengers (33).
Methods and Materials
For the synthesis of compounds, all chemicals were commercially
obtained and used without additional purification. These compounds
were confirmed by elemental analysis, UV spectroscopy, Fourier
transform-IR (FT-IR) spectroscopy and NMR spectra. Butyrylcholinest-
erase (EC 3.1.1.8, from horse serum), AChE (EC 3.1.1.7, type VI-S
from electric eel), S-butyrylthiocholine chloride, acetylthiocholine
iodide (ATCI), 5,5¢-dithio-bis(2-nitrobenzoic acid) (DTNB), neostigmine
methylsulphate and dimethylsulphoxide (DMSO) and donepezil
hydrochloride were purchased from Sigma–Aldrich (St. Louis, MO,
USA).
Substitution pattern in coumarins play a major role in their biochemi-
cal and pharmacological properties. For instance, the quinoline 4-
hydroxy-3-iodo-quinol-2-one has been found to have antimicrobial
activity against the bacterial MRSA clinical isolates, while 4-hydroxy-
3-iodo-quinol-2-one was more active against an Irish hospital MRSA-
1 strain (19). Similarly, stilbene–coumarin hybrid compounds are
found to be anticancer (20), while coumarin-4-acetic acid benzylidene
hydrazides show antitubercular activity (21). Coumarin analogues with
phenylpiperazine functions are found to be as AChE inhibitors (22),
while conjugation of benzimidazole with coumarin imparts it antihep-
atitis activity (26). Hence, the subsituent attached to coumarine
seems to decide its selevtivity towards a specicial target.
General
The Rf-values were determined using aluminium precoated silica gel
plates Kiesel 60 F254 from Merck (Darmstadt, Germany). Melting
points were determined using Gallenkamp melting point apparatus
(MP-D) and are uncorrected. Infrared spectra were recorded using on
FTS 3000 MS, Bio-Rad Merlin (Excalibur Model) spectrophotometer
1
(Bio-Rad, Cambridge, MA, USA). The H NMR spectra were obtained
Thiazole derivatives have been isolated and synthesized in view of
their versatile pharmacological activities. Some of thiazole analogues
are used as fungicides, (34) cardiotonic, (35) anti-arrhythmic (36) and
antitumour (37). Thiazoles are used as drugs for the treatment of HIV
infections and pain (38). Many of thiazoles are fibrinogen receptor
antagonists with antithrombotic activity, (39) inhibitors of bacterial
DNA gyrase B (40) and lipo-oxygenase inhibitor (41). Aminothiazoles
are known to be ligands of oestrogen receptors (42) as well as a
novel class of adenosine receptor antagonists. Thiazolin ring present
in vitamin B1 serves as an electron sink and its coenzyme form is
important for the decarboxylation of alpha-ketoacids (43).
using a Bruker 300 NMR MHz spectrometer in, DMSO-d6 and solu-
tions using TMS as an internal reference. Chemical shift is given in
d-scale (ppm). Abbreviations s, d, dd, t, at have been used for
singlet, doublet, double doublet, triplet, apparent triplet, respectively;
m stand for a multiplet. The 13C NMR spectra by Bruker NMR
spectrometer (75.4 MHz) in CDCl3, DMSO-d6 and C3D6O solutions.
General procedure for the synthesis of ( E)-3-(2-
(2-(substituted benzylidene)hydrazinyl)thiazol-4-
yl)-2H-chromen-2-ones (3a–l)
(E)-1-(substituted benzylidene) thiosemicarbazide (1a–l) (0.3 mmol)
was dissolved in 10–15 mL of absolute ethanol in a round bottom
flask, then added with 3-bromoacetylcoumarin (2) (0.3 mmol) and
the mixture was stirred at 30–35 ꢀC for 25–30 min. Progress of the
reaction was monitored with TLC using [Petroleum ether ⁄ Ethyl
The main purpose of the study was to synthesize a new set of cou-
marin analogues with different functional groups and to determine
their inhibitory potential. Newly synthesized compounds were com-
pared with the reference and conclusions were made.
2
Chem Biol Drug Des 2012

Thiazolocoumarinyl Derivatives as Cholinesterase Inhibitors
acetate (1:1)]. The solid separated was purified by recrystallization
using ethanol.
137.0, 134.1, 132.1, 131.1, 129.3, 129.2, 125.9, 125.4, 125.1, 120.9,
119.6, 116.3, 111.36; elemental analysis calculation for
C19H12ClN3O2S (381.84) (%): C 59.76, H 3.17, Cl 9.28, N 11.00, S
8.40; Found: C 59.61, H 3.06, Cl 9.20, N 10.85, S 8.24.
(E)-3-(2-(2-(3-methoxybenzylidene)hydrazinyl)
thiazol-4-yl)-2H-chromen-2-one (3a)
Yield: 86.3%; light yellow solid; m.p.: 243–244 ꢀC (decomp.). Rf:
0.73 (b); IR (pure per cm): 3289 (N-H), 3109 (Csp2-H), 1704 (C=O),
1609 (C=N), 1559 (C=C aromatic), 1168 (C-O), 2941, 2832 (Csp3-H,
(E)-3-(2-(2-(4-chlorobenzylidene)hydrazinyl)
thiazol-4-yl)-2H-chromen-2-one (3e)
Yield: 90%; dark yellow solid; m.p.: 286–287 ꢀC (decomp.). Rf: 0.62
(b); IR (pure per cm): 3289 (N-H), 3135 (Csp2-H), 1713 (C=O), 1596
(C=N), 1580 (C=C aromatic), 1177 (C-O), 780 (C-Cl); 1H NMR
(300 MHz, DMSO-d6): d 12.31 (s, 1H, NH), 8.52 (s, 1H, Ar-H), 8.04
(s, 1H, CH=N), 7.84 (d, J = 6.9 Hz, 2H, Ar-H)7.79 (s, 1H, thiazole H-
5), 7.71 (d, J = 7.4 Hz, 1H, Ar-H), 7.59–7.54 (m, 3H, Ar-H), 7.48–
7.41 (m, 2H, Ar-H); 13C NMR: (75 MHz, DMSO-d6): d 167.3, 159.4,
151.9, 144.3, 141.5, 139.1, 138.0, 134.5, 131.9, 131.2, 129.6, 128.5,
125.0, 124.4,122.4, 119.6, 111.36; elemental analysis calculation for
C19H12ClN3O2S (381.84) (%): C 59.76, H 3.17, Cl 9.28, N 11.00, S
8.40; Found: C 59.61, H 3.06, Cl 9.20, N 10.85, S 8.24.
1
methoxy) H NMR (300 MHz, DMSO-d6): d 12.25 (s, 1H, NH), 8.54
(s, 1H, Ar-H), 8.033 (s, 1H, CH=N), 7.86 (d, 1H, J = 7.5 Hz, Ar-H),
7.84 (s, 1H, thiazole H-5) 7.63 (at, 1H, Ar-H), 7.473–7.330 (m, 3H,
Ar-H), 7.262–7.271 (m, 2H, Ar-H), 6.96 (dd, J = 8.1, 2.1 Hz, 1H, Ar-
H); 13CNMR: (75 MHz, DMSO-d6): d 168.84, 160.0, 159.2, 152.78,
140.9, 138.6, 136.1, 132.1, 130.4, 129.3, 125.1, 120.9, 119.6, 119.3,
116.3, 1116.2, 115.7, 111.5, 111.1, 55.57; elemental analysis calcu-
lation for C20H15N3O3S (377.42) (%): C 63.65, H 4.01, N 11.13, S
8.50.; Found: C 63.32, H 4.15, N 11.02, S 8.27.
(E)-3-(2-(2-(4-methoxybenzylidene)hydrazinyl)
thiazol-4-yl)-2H-chromen-2-one (3b)
(E)-3-(2-(2-(3-bromobenzylidene)hydrazinyl)
thiazol-4-yl)-2H-chromen-2-one (3f)
Yield: 92%; light yellow solid; m.p.: 270–272 ꢀC (decomp.). Rf: 0.62
(b); IR (pure per cm): 3225 (N-H), 3138, (Csp2-H), 1698 (C=O), 1602
(C=N), 1574 (C=C aromatic), 11167 (C-O), 2936, 2838 (Csp3-H, meth-
Yield: 87%; light yellow solid; m.p.: 290–291 ꢀC (decomp.). Rf: 0.70
(b); IR (pure per cm): 3307 (N-H), 3143 (Csp2-H), 1712 (C=O), 1575
(C=N), 1557 (C=C aromatic), 1175 (C-O), 643 (C-Br) 1H NMR
(300 MHz, DMSO-d6): d 12.4 (s, 1H, NH), 8.58 (s, 1H, Ar-H) 8.21 (s,
1H, CH=N), 7.85–7.81 (m, 2H, Ar-H), 7.79 (s, 1H, thiazole H-5), 7.65
(s, 1H, Ar-H), 7.57–7.51 (m, 2H, Ar-H), 7.48–7.36 (m, 2H, Ar-H); 13C
NMR: (75 MHz, DMSO-d6): d 167.5, 161.4, 151.9, 145.8, 142.3,
139.2, 137.5, 135.3, 134.0, 132.2, 129.5, 129.0, 128.6, 126.7, 125.7,
123.8, 122.1, 120.7, 112.1; elemental analysis calculation for
C19H12BrN3O2S (426.29) (%): C 53.53, H 2.84, Br 18.74, N 9.86, S
7.52; Found: C 53.60, H 3.02, Br 18.68, N 9.71, S 7.41.
1
oxy) H NMR (300 MHz, DMSO-d6): d 12.03 (s, 1H, NH), 8.529 (s, 1H,
Ar-H), 8.013 (s, 1H, CH=N), 7.84 (d, J = 7.8 Hz, 1H, Ar-H), 7.747 (s, 1H,
thiazole H-5), 7.65–7.59 (m, 3H, Ar-H), 7.45–7.36 (m, 2H, Ar-H), 7.00
(d, 2H, Ar-H) 3.79 (s, 3H, methoxy);13C NMR: (75 MHz, DMSO-d6): d
168.2, 16.7, 159.2, 152.7, 144.4, 142.2, 138.5, 132.1, 129.2, 128.3,
127.3, 125.1, 121.0, 119.6, 116.3, 114.8, 110.83, 55.73; elemental
analysis calculation for C20H15N3O3S (377.42) (%): C 63.65, H 4.01, N
11.13, S 8.50; Found: C 63.32, H 4.15, N 11.02, S 8.27.
(E)-3-(2-(2-(2-chlorobenzylidene)hydrazinyl)
thiazol-4-yl)-2H-chromen-2-one (3c)
(E)-3-(2-(4-bromo-1-benzylidenehydrazine)
thiazol-4-yl)-2H-chromen-2-one (3g)
Yield: 95%; light yellow solid; m.p.: 276–277 ꢀC (decomp.). Rf: 0.73
(b); IR (pure per cm): 3281 (N-H), 3134 (Csp2-H), 1702 (C=O), 1591
(C=N), 1573 (C=C aromatic), 1174 (C-O), 785 (C-Cl); 1H NMR (300 MHz,
DMSO-d6): d 12.4 (s, 1H, NH), 8.55 (s, 1H, Ar-H), 8.42 (s, 1H, CH=N),
7.59–7.81 (m, 3H. Ar-H), 7.80 (s, 1H, thiazole H-5), 7.52–7.37 (m, 4H,
Ar-H); 13C NMR: (75 MHz, DMSO-d6): d 167.8, 159.2, 152.7, 138.7,
137.8, 132.7, 132.2, 131.9, 130.4, 129.3, 128.1, 126.6, 125.1, 124.5,
124.4, 120.9, 119.6, 116.3, 111.4; elemental analysis calculation for
C19H12ClN3O2S (381.84) (%): C 59.76, H 3.17, Cl 9.28, N 11.00, S 8.40;
Found: C 59.61, H 3.06, Cl 9.20, N 10.85, S 8.24.
Yield: 88%; dark yellow solid; m.p.: 220–222 ꢀC (decomp.). Rf: 0.65
(b); IR (pure per cm): 3292 (N-H), 3117 (Csp2-H), 1710 (C=O), 1589
(C=N), 1572 (C=C aromatic), 1174 (C-O), 639 (C-Br); 1H NMR
(300 MHz, DMSO-d6): d 12.3 (s, 1H, NH), 8.59 (s, 1H, Ar-H) 8.25 (s,
1H, CH=N), 7.81 (s, 1H, thiazole H-5), 7.79 (d, J = 7.4 Hz, 1H, Ar-H),
7.64–7.56 (m, 5H, Ar-H), 7.43–7.36 (m, 2H, Ar-H); 13C NMR:
(75 MHz, DMSO-d6): d 167.3, 161.5, 152.9, 146.3, 142.8, 138.7,
134.5, 132.6, 131.5, 129.6, 129.0, 126.2, 126.0, 123.4, 121.4, 119.8,
111.7; elemental analysis calculation for C19H12BrN3O2S (426.29)
(%): C 53.53, H 2.84, Br 18.74, N 9.86, S 7.52; Found: C 53.60, H
3.02, Br 18.68, N 9.71, S 7.41.
(E)-3-(2-(3-chloro-1-benzylidenehydrazine)
thiazol-4-yl)-2H-chromen-2-one (3d)
Yield: 91.4%; light yellow solid; m.p.: 282–284 ꢀC (decomp.). Rf:
0.67 (b); IR (pure per cm): 3309 (N-H), 3147 (Csp2-H), 1713 (C=O),
1597 (C=N), 1558 (C=C aromatic), 1175 (C-O), 779 (C-Cl); 1H NMR
(300 MHz, DMSO-d6): d 12.3 (s, 1H, NH), 8.15 (s, 1H, Ar-H), 8.03 (s,
1H, CH=N), 7.84 (dd, J = 7.5 0.9, Hz, 1H, Ar-H), 7.68 (s, 1H, Ar-H),
7.64–7.59 (m, 2H, Ar-H), 7.48–7.35 (m, 4H, Ar-H), 13C NMR:
(75 MHz, DMSO-d6): d 167.9, 159.1, 152.7, 144.4, 140.3, 138.6,
(E)-3-(2-(2-(4-nitrobenzylidene)hydrazinyl)
thiazol-4-yl)-2H-chromen-2-one (3h)
Yield: 89.6%; orange solid; m.p.: 327–328 ꢀC (decomp.). Rf: 0.82 (b);
IR (pure per cm): 3291 (N-H), 3129 (Csp2-H), 1706 (C=O), 1589 (C=N),
1564 (C=C aromatic), 1171 (C-O), 1508 (NO2) 1H NMR (300 MHz,
DMSO-d6): d 12.36 (s, 1H, NH), 8.52 (s, 1H, Ar-H), 8.021 (s, 1H, CH=N),
7.84 (d, J = 6.9 Hz 2H, Ar-H), 7.78 (s, 1H, thiazole H-5), 7.67–7.54 (m,
Chem Biol Drug Des 2012
3

Raza et al.
3H, Ar-H), 7.45–7.36 (m, 3H, Ar-H); 13C NMR: (75 MHz, DMSO-d6): d
167.9, 159.9, 152.7, 144.47, 140.27, 138.6, 137.24, 132.2, 131.45,
129.3, 128.8, 125.8, 125.5, 122.6, 119.6, 116.35, 111.36; elemental
analysis calculation for C19H12N4O4S (392.39) (%): C 58.16, H 3.08, N
14.28, S 8.17; Found: C 58.03, H 3.21, N 14.14, S 8.01.
(s, 1H, CH=N) 7.87–7.83 (m, 2H, Ar-H), 7.76, 129.3, 128.9, 128.3, 128.0,
125.4, 125.1, 123.2, 121.5, 120.9, 119.6, 116.3, 113.7, 111.0, 70.90. (s,
1H, thiazole H-5), 7.65–7.60 (m, 1H, Ar-H), 7.52 (d, J = 6.9 Hz, 2H, Ar-
H), 7.46–7.34 (m, 5H, Ar-H), 7.19 (d, J = 8.1 Hz, 1H, Ar-H), 7.03 (t,
J = 6.9 Hz, 1H, Ar-H) 5.21 (s, 2H, CH2); 13C NMR: (75 MHz, DMSO-d6):
d 168.1, 159.2, 156.6, 152.7, 144.4, 138.6, 137.8, 137.3, 132.1, 131.2;
elemental analysis calculation for C26H19N3O3S (453.51) (%): C 68.86,
H 4.22, N 9.27, S 7.07; Found: C 68.71, H 4.36, N 9.14, S 7.18.
(E)-3-(2-(2-(4-N,N-dimethylbenzylidene)
hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (3i)
Yield: 88%; yellowish brown solid; m.p.: 278–280 ꢀC (decomp.). Rf
0.73 (b); IR (pure per cm): 3219 (N-H), 3134 (Csp2-H), 1689 (C=O), 1601
(C=N), 1557 (C=C aromatic), 1174 (C-O), 2983,2907 (Csp3-H, methyl)
1H NMR (300 MHz, DMSO-d6): d 12.3 (s, 1H, NH), 8.57 (s, 1H, Ar-H),
8.15 (s, 1H, CH=N), 7.79 (s, 1H, thiazole H-5), 7.64–7.59 (m, 3H, Ar-H),
7.53 (dd, J = 7.8, 1.2 Hz, 1H, Ar-H), 7.42–7.37 (m, 2H, Ar-H), 7.15 (d,
J = 6.9 Hz, 2H, Ar-H), 3.34 (s, 6H, CH3); 13C NMR: (75 MHz, DMSO-
d6): d 167.4, 159.6, 152.7, 150.9, 144.0, 143.2, 138.9, 135.3, 131.3,
130.0, 128.6, 125.7, 123.6, 122.0, 117.8, 116.5, 111.4, 40.5; elemental
analysis calculation for C21H18N4O2S (390.46) (%): C 64.60, H 4.65, N
14.35, S 8.21; Found: C 64.73, H 4.39, N 14.22, S 8.05.
Determination of AChE and BuChE inhibitory
activity
Inhibitory activities of AChE and BuChE were determined using Ell-
man method (44). Newly synthesized coumarins derivatives were
tested as inhibitors of AChE and BuChE. For initial screening, each
compound was dissolved in DMSO (end concentration of DMSO was
<1% in assay) and tested at a final concentration of 1 mM. Com-
pounds with considerable inhibition (more than 50%) were subjected
to further analysis by making their 8–10 serial dilutions in assay
buffer (50 mM Tris–HCl, 0.1 M NaCl and 0.02 M MgCl2, pH 8.0).
Reaction mixture comprised of 20 lL assay buffer, 10 lL of test
compound and 10 ll of 0.031 U ⁄ mL of enzyme (0.5 and 3.4 U ⁄ mg
of AChE or BuChE, respectively). This mixture was incubated at
25 ꢀC for 10 min. After this preincubation, enzymatic reaction was
started by addition of 10 lL of 1 mM acetylethiocholine iodide or
butyrylthiocholine chloride (according to the respective enzyme) and
the mixture was incubated again for 15 min. The amount of enzy-
matic product was estimated by measuring change in absorbance at
405 nm using a microplate reader (Bio-Tek ELx 800ꢁ, Instruments
Inc., Winooski, VT, USA). Neostigmine and donepezil were used as
standard inhibitor. Enzyme dilution buffer consisted of 50 mM Tris–
HCl buffer containing 0.1% (w ⁄ v) bovine serum albumin (BSA) and
pH 8. The effect of DMSO on activity of enzyme was subtracted by
a negative control containing DMSO, instead of inhibitor. The Ki val-
ues were calculated from IC50 values using non-linear curve fitting
program PRISM 5.0 (GraphPad, San Diego, CA, USA).
(E)-3-(2-(2-(3,4,5-trimethoxybenzylidene)
hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (3j)
Yield: 70.8%; yellowish brown solid; m.p.: 279–280 ꢀC (decomp.).
Rf: 0.48 (b); IR (pure per cm): 3205 (N-H), 3110 (Csp2-H), 1704 (C=O),
1606 (C=N), 1579 (C=C aromatic), 1175 (C-O), 2943, 2827 (Csp3-H,
1
methoxy); H NMR (300 MHz, DMSO-d6): d 12.31 (s, 1H, NH), 8.58
(s, 1H, Ar-H), 8.07 (s, 1H, CH=N), 7.81 (d, J = 7.7 Hz, 1H, Ar-H),
7.75 (s, 1H, thiazole H-5), 7.63–7.56 (m, 2H, Ar-H), 7.24–7.28 (m,
3H, Ar-H), 3.87 (s, 9H, methoxy); 13C NMR: (75 MHz, DMSO-d6): d
167.9, 160.0, 152.9, 151.3, 146.1, 143.2, 142.3, 139.4, 131.4, 129.8,
128.3, 126.5, 124.0, 122.4, 1119.2, 117.4, 111.7, 56.8; elemental
analysis calculation for C22H19N3O5S (437.37) (%): C 60.40, H 4.38,
N 9.61, S 7.33; Found: C 60.55, H 4.25, N 9.74, S 7.45.
(E)-3-(2-(2-(4-hydroxy-3,5-dimethoxybenzylidene)
hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (3k)
Yield: 76.9%; yellowish brown solid; m.p.: 214–215 ꢀC (decomp.).
Rf: 0.32 (b); IR (pure per cm): 3302 (N-H), 3125 (Csp2-H), 1713 (C=O),
1590 (C=N), 1568 (C=C aromatic), 1165 (C-O), 2932, 2827 (Csp3-H,
Determination of type of inhibition
The type of inhibition was determined using the most potent inhibi-
tor against AChE (3f). The effect of different concentrations of
inhibitor (from 0–300 and 0–30 nM, were used for acetyl and Bu-
ChE, respectively) on initial velocities was investigated over a range
of substrate concentrations (from 200 to 2500 lM). Initial enzymatic
velocities were measured without inhibitor and with different con-
centrations of 3f inhibitor. Standard solutions of the inhibitor were
prepared in assay buffer. Enzyme kinetic studies were performed
under the same incubation conditions using ATCI and butyrylthiocho-
line chloride as substrates and DTNB was used as the chromo-
phoric reagent. A Lineweaver–Burk (1 ⁄ V versus 1 ⁄ [S]) plot was
constructed using GraphPad Prism software.
1
methoxy) H NMR (300 MHz, DMSO-d6): d 12.9 (s, 1H, OH), 12.1 (s,
1H, NH), 8.55 (s, 1H, Ar-H) 8.21 (s, 1H, CH=N), 7.82 (d, J = 7.8 Hz,
1H, Ar-H), 7.75 (s, 1H, thiazole H-5), 7.58–7.49 (m, 2H, Ar-H), 7.41–
7.34 (m, 3H, Ar-H), 3.84 (s, 6H, methoxy); 13C NMR: (75 MHz,
DMSO-d6): d 167.3, 159.8, 152.6, 151.8, 145.1, 142.7, 138.6, 137.2,
132.7, 129.3, 127.8, 125.5, 124.2, 121.9, 118.4, 113.2, 111.6; ele-
mental analysis calculation for C21H17N3O5S (423.44) (%): C 59.57,
H 4.05, N 9.92, S 7.57; Found: C 59.61, H 4.21, N 9.79, S 7.43.
(E)-3-(2-(2-(2-benzyloxybenzylidene)
hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (3l)
Yield: 78%; light yellow solid; m.p.: 297–299 ꢀC (decomp.). Rf: 0.70
(b); IR (pure per cm): 3302 (N-H), 3125 (Csp2-H), 1713 (C=O), 1590
Molecular docking studies
Homology models generation
The BuChE (EC 3.1.1.8, from horse serum) and AChE (EC 3.1.1.7, type
VI-S from electric eel) sequences were threaded using LOMETS (45)
1
(C=N), 1568 (C=C aromatic), 1166 (C-O), 2924, 2851 (Csp3-H OCH2) H
NMR (300 MHz, DMSO-d6): d 12.6 (s, 1H, NH), 8.53 (s, 1H, H-4) 8.50
4
Chem Biol Drug Des 2012

Thiazolocoumarinyl Derivatives as Cholinesterase Inhibitors
threading programs. These programs retrieved and threaded the PDB
spectra of synthesized hydrazide derivatives revealed the presence
of C=S stretching around 1602 (C=N), 1590 (Ar-C=C), 1073 per cm,
in addition to stretching at 3385 and 3229, indicated the presence
of NH2 and NH, respectively. The appearance of a singlet at d 8.11
for characteristic azomethine (CH=N) proton and singlets at d 10.4
IDs (3i6m, 1q83, 2pm8) as templates for AChE and (3i6m, 1ea5, 2xb6)
as possible templates for BuChE. Both AChE and BuChE have very
high sequence similarities and identities with their selected tem-
plates. For instance, the electric eel_AChE sequence has 66%, 60%
and 53.5% identities with template crystal structures 3i6m, 1q83 and
2pm8, respectively. While in case of BuChE from horse serum, it has
54.4%, 54.0% and 34.0% sequence identities with 3i6m, 1ea5 and
2xb6 crystal structures, respectively. After that continuous fragments
were generated from these templates and finally used to assemble
full-length models using modified replica-exchange Monte Carlo sim-
ulations (46). The loop regions were constructed by ab initio model-
ling implemented in I-TASSER. The simulation decoys were clustered
using SPICKER (47) and the cluster centroid was used as the next round
of I-TASSER reassembly. The structures with the lowest energy in the
second round of assembly simulation were selected and full-atomic
models were refined using fragment guided molecular dynamics. The
best model based on C-score was subjected for the molecular docking
simulations of the synthesized compounds. Before docking simula-
tions, hydrogen atoms were added in each structure and Gasteiger
charges were assigned using the chimera modelling tool.
1
for NH and 7.50 for in NH2, respectively, in H NMR and signals at
d 179.0 (C=S) and 153 (C=N) in 13C NMR confirmed formation of
the semicarabazones (Scheme 1).
3-(2-bromoacetyl)-2H-chromene-2-one was synthesized as key inter-
mediate by a-bromination of 3-acetyl-2H-chromen-2-one itself syn-
thesized by the base catalysed reaction of salicylaldehyde and ethyl
acetoacetate (Scheme 2).
1
The H NMR spectrum of 3-acetyl-2H-chromen-2-one shows H-4 in
the most deshielded region having d 8.51, characteristic of couma-
rins in addition to three protons singlet at d 2.72. The 13C NMR
shows characteristic signals at 195.5 for C=O indicative of acetyl
group and 159.2 ppm for lactone carbonyl and C-4 at 147.52. Bro-
mination to afford (2) was indicated by IR stretchings at 3024
(Csp2-H), 1728 (C=O ketone), 1684 (C=O lactone) besides 1684 (C=C
aromatic) and 633 per cm (C-Br). In 1H NMR spectrum, coumarin
proton (H-4) at 8.78 and a singlet for two protons at 4.87 ppm are
indicative of a-bromination. The 13C NMR shows characteristic sig-
nals d 36.2 (CH2Br) (Scheme 2) (36).
Compounds structures preparation
Before performing the molecular docking simulations of small mole-
cules inside the homology models of both protein structures, all
a
molecules were sketched and protonated using the MOE molecules
In view of the pharmacological, biochemical properties and therapeu-
tic applications of substituted coumarins and thiazoles, on one hand
and that of Schiff bases on the other, we wish to report the synthesis
of thiazolocoumarinyl Schiff bases containing a coumarin ring, thia-
zole nucleus and imine linkage combined in a single structural unit.
sketcher tool. The three-dimensional conformations of these mole-
cules were generated using the protonate three-dimensional tool
implemented in MOE. Finally, the molecules were minimized and
their charges were optimized using the MMFF94x force-field with
the adjustment of hydrogens.
Thus, (E)-3-(2-(2-(Substituted benzylidene)hydrazinyl)thiazol-4-yl)-2H-
chromen-2-ones (3a–l) were prepared by stirring semicarbazones
(1a–l) with 3-bromoacetylcoumarin (2) under mild conditions in
absolute ethanol (Scheme 3).
Docking protocol
The DOCK 6.3 (48) was utilized to dock the molecules into the homology
models of AChE and BuChE structures. The DMS program implemented
in UCSF Chimera was used to generate a molecular surface for each
protein structure. The SPHGEN algorithm was utilized to create a neg-
ative image of the binding pocket, which is complementary to the
molecular surface of the proteins. All the spheres within 10 ꢀ of the
ligand binding site were selected for docking. The receptor box was
generated using the SHOWBOX algorithm which calculates the binding
pocket with an additional box size so that it was >20 ꢀ in length,
which is large enough to contain these compounds. Potential grids
were calculated for each receptor using a 0.3 ꢀ spacing that fully
enclosed the spheres and were generated by the GRID program, which
increases the docking speed. The protein structures were treated as
rigid in the docking simulation, and the compounds were taken as flex-
ible, the number of ligand poses sampled was set to 100. All docking
experiments were carried out using the Supercomputer cluster.
The FT-IR spectral data of hybrid compounds (3a–l) showed the
characteristic peaks for N-H, Csp2-H, C=O and C=N stretching vibra-
tions. Thus, and NH, lactone (C=O) and imine (C=N) functional
groups with absorptions bands at 1690–1718 and 1609–1638 per
1
cm, respectively. The H NMR spectral data showed signals for NH
and HC=N in the range 12.6–12.0 ppm and 8.50–8.01 ppm, respec-
tively, and the thiazolyl proton (H-5¢¢) appeared in the range 7.90–
7.40 ppm. In 13C NMR, the signals are for C=N of thiazole moiety
and C=O of coumarin appeared in the range 167.0–168.9 and 158–
160 ppm, respectively.
Cholinesterase inhibition
Therapeutic efficacy of newly synthesized coumarin analogues for
the treatment of AD depend upon their anticholinesterase activities.
Inhibition of AChE and BuChE by coumarin derivatives is compared
by using neostigmine and donepezil reference drugs and Ki values
of individual compounds are listed in Table 1.
Results and Discussion
Chemistry
Schiff bases (1a–l) of semicarbazide with a variety of aromatic
aldehydes were prepared by acid-catalysed condensation. In IR
The basic structures of our compounds consist of a thiazole ring
attached to 3-position of coumarin nucleus and a substituted phenyl
Chem Biol Drug Des 2012
5

Raza et al.
Scheme 1: Synthesis of (E)-2-
(Subsituted bezylidene) hydra-
zinecarbothioamide (1a–l).
Scheme 2: Synthesis of 3-(2-Bromoacetyl)-2H-chromene-2-one (2) Reagents and conditions: (i) Piperidine, 0–5C (ii) Br2 ⁄ CHCl3.
Scheme 3: Synthesis of (E)-3-(2-(2-(Substituted benzylidene)hydrzinyl)thiazole-4-yl)-2H-chromen-2-ones (3a–l).
ring attached to thiazole at position 4 by hydrazinyl linkage. Varying
Ki values were observed by the attachment of different substituents
to the phenyl ring. Among the newly synthesized analogues, the
most potent compounds against BuChE were 3a and 3l having
strong electron donating methoxy (-OCH3) and benzyloxy (-OCH2Ph)
groups at meta and ortho positions, respectively. However, when (-
OCH3) group is attached at meta position, there was slight decrease
in the activity of compound as in case of 3b. Furthermore, if three
methoxy groups were substituted at 3¢, 4¢ and 5¢, Ki value was
increased to a greater extent, for example, 3j. Inhibition potency of
compounds against BuChE was also affected by substitution posi-
tion of –Cl. Ortho position of phenyl ring was much better choice
of -Cl attachment (e.g. 3c) as compared to meta and para positions
(3d and 3e). Bromine substitution to the parent structure also
imparted a good inhibition activity against BuChE as depicted in 3f
and 3g; however, substitution position merely affected activity of
compound.
siderable decrease in the activity of compound as can be noticed in
3a. Attachment of three -OCH3 groups also resulted in increased Ki
value (3j); however, replacement of one -OCH3 group with -OH
group resulted in a much improved activity as in case of 3k where
-OCH3 is substituted at position 3¢ and 5¢ and an -OH at position 4.
Substitution position of -Cl group also exhibited considerable role in
inhibitory tendency of compound. Highest activity was observed
when -Cl group was attached at para position (3e). However, when
nitrogen-containing electron donating group -NMe2 was at para
position activity against AChE was reduced as in the case of 3i.
Attachment of -OCH2Ph (in case of 3l) and NMe2 groups (in case
of 3i) showed relatively higher Ki values. On the other hand, strong
electron withdrawing -NO2 group at para position in 3h noticeably
enhanced inhibitory potency of compound against AChE. All of the
tested compounds were more potent than neostigmine, however,
relatively lesser potent on AChE as compared to donepezil but quite
interestingly more 10- to 90-fold more potent on BuChE.
All the tested compounds were also very good inhibitors of AChE.
The 3f was found to be the most potent inhibitor of AChE having
bromide attached at meta position of parent compound. Role of
electron donating -OCH3 group was very interesting. Its attachment
at para position attributed good inhibition potency to the compound
as in 3b. However, its shifting at meta position resulted in a con-
Molecular docking studies
Molecular docking studies of newly synthesized compounds were
performed in the predicted homology models of AChE and BuChE
to identify the plausible binding modes. The predicted binding
modes of these compounds in AChE and BuChE active sites can
6
Chem Biol Drug Des 2012

Thiazolocoumarinyl Derivatives as Cholinesterase Inhibitors
Table 1: AChE and BuChE inhibitory activities of the synthesized coumarin analogues
R
S
N
NH
N
O
O
AChE
BuChE
Selectivity for AChE ⁄ BuChE
No
Compound
R
Ki € SEM (lM)
1
2
3
4
5
6
7
8
3a
3b
3-OMe
4-OMe
2-Cl
3-Cl
4-Cl
7.17 € 0.8
1.25 € 0.06
5.41 € 1.0
3.09 € 0.6
1.62 € 0.3
1.05 € 0.3
2.47 € 0.2
1.81 € 1.5
9.96 € 1.2
5.61 € 0.2
1.55 € 0.1
7.47 € 0.12
23 € 3
0.041 € 0.008
0.064 € 0.002
0.061 € 0.005
0.131 € 0.02
0.181 € 0.07
0.071 € 0.003
0.074 € 0.01
0.081 € 0.01
0.072 € 0.002
0.212 € 0.04
0.072 € 0.006
0.041 € 0.002
48 € 6
179
20
90
24
9
15
33
23
138
27
3c
3d
3e
3f
3g
3-Br
4-Br
3h
3i
4-NO2
4-NMe2
3,4,5-tri-OMe
4-OH-3,5-di-OMe
2-OCH2Ph
9
10
11
12
13
14
3j
3k
22
3l
182
0.5
0.004
Neostigmine
Donepezil
0.026 € 0.003
6.32 € 0.42
AChE, acetylcholinesterase; BuChE, butyrylcholinesterase.
be seen in Figures 2 and 3, respectively. Our docking results
showed that all compounds possessed the similar binding modes
with the slightly different docking scores as shown in Table 2. It
has been observed that all the compounds have been gorged into
the catalytic amino acid triad (49) (Glu225, Ser226 and His466) of
BuChE and (Glu224, Ser225 and His 494) AChE, respectively. On
the basis of docking simulations, we can explain that the strong
binding affinity of 3l with the BuChE may be because of the
hydrogen bonding with the backbone carbonyl of the one of
catalytic residues His 466, (Figure 4). Similarly, t-type p–p interac-
tions between histidine and thiazole ring of the 3l can provide
the extra binding strength as compare to the least active 3j com-
pound, which has no such binding interactions with the side
chains of the enzyme. In AChE inhibitors docking, we did not
found any eminent interactions between the most active com-
pound and the enzyme (Figure 5), which might be due to the
wrong selection of binding confirmation of this compound.
Because, in this study, we have considered the top rank orienta-
tion as a plausible binding mode, which is not always correct in
docking simulations. We also conducted the comparison studies of
active sites of these model structures, which reveal that there are
only two residues differences in the binding sites of these
enzymes (Figure 6). AChE contains Tyr-146 and Phe-315, whereas
BuChE comprised of Gln-147 and Val-316 in the same three-
dimensional co-ordinate space. We assume that these structurally
Figure 2: Binding modes of acetylcholinesterase (AChE) inhibi-
tors, compounds are displayed with sticks and protein in cartoon.
The figure has been drawn with PYMOL program.
Chem Biol Drug Des 2012
7

Raza et al.
Figure 3: Binding modes of butyrylcholinesterase (BuChE) inhibi-
tors. Figure has same representation as in Figure 2.
Figure 4: Binding orientation of best ranked most active (3l in
yellow colour) and least active (3j in green) compound in the bind-
ing site of butyrylcholinesterase (BuChE) enzyme. Hydrogen bond is
indicated with green color line while hydrophobic interactions are
shown with bold white dotted lines.
Table 2: Docking scores of all synthesized coumarin analogues
in AChE and BuChE
AChE
BuChE
Sr. No.
Compound
Docking scores
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
)53.748
)54.048
)52.292
)50.649
)51.204
)50.977
)52.511
)54.413
)56.443
)59.416
)58.420
)67.878
)50.898
)50.315
)46.823
)48.717
)47.882
)49.128
)48.775
)51.448
)51.573
)55.115
)54.504
)58.816
3g
3h
3i
9
10
11
12
3j
3k
3l
AChE, acetylcholinesterase; BuChE, butyrylcholinesterase.
and chemically different amino acids might be responsible for the
difference of the activities and selectivity of this compound series
towards these enzymes.
Enzyme kinetics
Double reciprocal plots of initial velocity and substrate concentra-
tion (per micromolar) was plotted for the most potent inhibitor 3f,
against AChE and a very good inhibitor of BuChE (Figure 7). Lines
were drawn from a weighted least square analysis of data points.
Enzyme kinetics was determined in the presence of different con-
centrations of inhibitor, that is, 0, 100 and 300 nM for AChE and 0,
Figure 5: Binding orientation of best ranked most active (3f in
yellow colour) and least active (3i in green) compound in the bind-
ing site of acetylcholinesterase (AChE) enzyme. Bold white dotted
lines are showing the hydrophobic interactions.
8
Chem Biol Drug Des 2012

Thiazolocoumarinyl Derivatives as Cholinesterase Inhibitors
A
B
Figure 6: Binding site comparison of acetylcholinesterase (AChE)
(white) and butyrylcholinesterase (BuChE) (yellow) homology models.
Non conserved residues are shown with sticks.
10 and 30 nM for BuChE. It was observed that Km value depicted
direct relationship with the inhibitor concentration, that is, Km value
increased with the increase in concentration of inhibitor. Thus, it
was inferred that compound 3f exhibited competitive mechanism of
inhibition. However, value of Vmax remained almost same in the
presence or absence of the inhibitor. Plots drawn previously repre-
sent competitive mechanism of inhibition against AChE and BuChE.
Conclusions
Figure 7: Double-reciprocal plots of the inhibition kinetics of (A)
acetylcholinesterase (B) butyrylcholinesterase by compound 3f.
Changes in the initial velocities of the reaction were measured at
different concentrations of the inhibitor 3f using substrate [acetyl-
thiocholine iodide (ATCI)] concentrations of 0.2–2.5 mM.
Results from biological assays, SAR's study and enzyme kinetic
studies revealed similar outcomes, regarding inhibitory activity on
AChE and BuChE and selectivity for AChE ⁄ BuChE. All of the newly
synthesized coumarin derivatives were potent competitive inhibi-
tors of AChE and BuChE. The 3f was found to be the most
potent against AChE with Ki value of 1.05 € 0.3 lM and 3l
showed excellent inhibitory action against BuChE with Ki value of
0.041 € 0.002 lM. The discovery of these new coumarin analogues
can serve as the landmark in probing new therapies of AD. We
are looking forward that the current work could help in further
investigations on the potent ACh and BuCh inhibitors with excel-
lent inhibitory activity.
Conflicts of interest
All authors declared no financial or commercial conflicts of interest.
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