Synthesis, cholinesterase inhibition and molecular modelling studies of coumarin linked thiourea derivatives

Article abstract of DOI:10.1016/j.bioorg.2015.09.009

Alzheimer's disease is among the most widespread neurodegenerative disorder. Cholinesterases (ChEs) play an indispensable role in the control of cholinergic transmission and thus the acetylcholine level in the brain is enhanced by inhibition of ChEs. Coum

Full text of DOI:10.1016/j.bioorg.2015.09.009

Bioorganic Chemistry 63 (2015) 58–63
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, cholinesterase inhibition and molecular modelling studies
of coumarin linked thiourea derivatives
Aamer Saeed a, Sumera Zaib b, Saba Ashraf a, Javeria Iftikhar b, Muhammad Muddassar c,d, Kam Y.J. Zhang c,
Jamshed Iqbal b,
⇑
a Department of Chemistry, Quaid-I-Azam University, Islamabad, Pakistan
b Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
c Structural Bioinformatics Team, Division of Structural and Synthetic Biology, Center for Life Science Technologies, RIKEN, 1-7-22 Suehiro, Yokohama, Kanagawa 230-0045, Japan
d Department of Biosciences, COMSATS Institute of Information Technology, Park Road, Islamabad, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2015
Revised 14 September 2015
Accepted 28 September 2015
Available online 30 September 2015
Alzheimer’s disease is among the most widespread neurodegenerative disorder. Cholinesterases (ChEs)
play an indispensable role in the control of cholinergic transmission and thus the acetylcholine level in
the brain is enhanced by inhibition of ChEs. Coumarin linked thiourea derivatives were designed, synthe-
sized and evaluated biologically in order to determine their inhibitory activity against acetyl-
cholinesterases (AChE) and butyrylcholinesterases (BChE). The synthesized derivatives of coumarin
linked thiourea compounds showed potential inhibitory activity against AChE and BChE. Among all the
synthesized compounds, 1-(2-Oxo-2H-chromene-3-carbonyl)-3-(3-chlorophenyl)thiourea (2e) was the
Keywords:
Acetylcholinesterase
Alzheimer’s disease
Butyrylcholinesterase
Coumarin
most potent inhibitor against AChE with an IC50 value of 0.04 0.01
mene-3-carbonyl)-3-(2-methoxyphenyl)thiourea (2b) showed the most potent inhibitory activity with
an IC50 value of 0.06 0.02 M against BChE. Molecular docking simulations were performed using the
lM, while 1-(2-Oxo-2H-chro
l
homology models of both cholinesterases in order to explore the probable binding modes of inhibitors.
Results showed that the novel synthesized coumarin linked thiourea derivatives are potential candidates
to develop for potent and efficacious acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
inhibitors.
Thioureas
Homology models
Molecular docking
Ó 2015 Published by Elsevier Inc.
1. Introduction
hence BChE enhances progression of AD. Therefore, synchronous
inhibition achieved by AChE and BChE may provide additional ben-
Alzheimer’s disease (AD), progressively developing neurode-
generative disorder characterized by which includes cognitive
insufficiency and memory loss, as its major side effects [1] are
specified by cholinergic neurons and with their cortical extension
deficiency mainly from the nucleus basalis and related areas of
basal forebrain [2]. Cholinesterases being an extensively dispersed
enzymes, classified as acetyl and butyryl belongs to class of hydro-
lases enzyme [3,4]. Acetylcholinesterase (AChE) terminates signal-
ing action following degradation of acetylcholine (ACh) [5,6],
whereas butyrylcholinesterase (BChE) has got a major role in the
metabolism of different molecules, most commonly the distinct
neuroactive peptides [7]. Hence AChE inhibitors (AChEI) are con-
sidered to be one of the most useful approach for the treatment
of AD by enhancing cholinergic objectives in AD patients. In normal
healthy brain, ACh metabolic degradation is also caused by BChE,
efits for AD treatment [6,8–10].
Cholinesterase inhibitors can be prescribed as a co-drug for the
therapy of addicts as they have potential to overcome the side
effects caused by addictive drugs [11]. Hence subsequently, devel-
opment of ChEIs plays an important role in determining the cholin-
ergic transmission disorders mechanism [12,13]. ChEIs can also be
used for the treatment of glaucoma, myasthenia gravis and in case
of phosphate compounds poisoning as they act as competitors for
cholinesterase sites in case of poisoning [14]. Since last few years,
the discovery of several anti-AChE factors like tacrine, donepezil,
rivastigmine and galantamine has been observed [15–19]. Despite
the fact that AChEIs are used extensively for the treatment of AD
for the beneficiary effects on central nervous system, they still have
certain detrimental consequences on peripheral organs [19]. Hence
the design of selective AChEIs, with minimum hazardous effect
remains a challenging role still to be achieved.
Coumarin derivatives have sustained efficacy as they inhibit
both acetyl and butyryl cholinesterases and help to decelerate
⇑
Corresponding author.
E-mail address: drjamshed@ciit.net.pk (J. Iqbal).
http://dx.doi.org/10.1016/j.bioorg.2015.09.009
0045-2068/Ó 2015 Published by Elsevier Inc.

A. Saeed et al. / Bioorganic Chemistry 63 (2015) 58–63
59
formation of amyloidogenic compounds [20,21]. Substitution on
their benzopyran ring results a change in the biological activity.
Activities like antibacterial [22], anti-HIV [23], anti-cancer [24],
monoamaine oxidase inhibition [25] and anticoagulant [26] have
been reported. Coumarins exhibit different biological and
pharmacological properties like 3-benzylideneamino coumarins
have inflammatory property [27], N-substituted 4-aminometyl
coumarins have nervous system stimulating activity [22], 4-3-
coumarinyl-3-cyclohexyl-4-thiozoline-2-one benzylidene hydrazone
derivatives have anti-tubercular activity [28], N-substituted-3-
carboxamido-coumarin derivatives have antibacterial and
anti-inflammatory activities [29], 3-carboxamido-7-substituted cou-
marins displayed inhibition against human monoamine oxidase A
and B [30]. These all suggest that selectivity towards specific target
is affected by substitution of the pharmacophore. Thioureas are
organosulphur compounds [31,32]. 1-2-Aminoethoxy-3-aroyl-
thioureas possess acetylcholinesterases inhibitory activity [33].
Several other properties like anticancer [34], anti-microbial,
anti-malarial [35], antibacterial [36] and anti-tuberculosis [37]
have been reported for thioureas and their derivatives. Similarly,
halogenated thioureas have anticholinesterase potential [19].
2,4-Disubstituted thiazoles having coumarin rings displayed
different pharmacological properties like, antimicrobic activity,
cytotoxicity, and hMAO inhibition [38]. The available treatments
for the AD have got short half-life with hazardous side effects,
thus demands the need to introduce new therapies. In the current
study, different analogues of coumarin linked thiourea were
screened to explore the effect of different functional groups that
are attached on the activity towards AChEIs in terms of their bio-
logical activity and pharmacological properties. On the basis of
structural attractiveness and synthetic accessibility, we began to
investigate SAR of novel thiourea derivatives having a bulkier R
substituents. Herein, we describe the efficient and versatile synthe-
sis and cholinesterase inhibitory effects of coumarin linked
thioureas.
added slowly with constant stirring to the reaction mixture, fol-
lowed by 1–2 h reflux. The progress of the reaction was monitored
by thin layer chromatography. After the completion of the reaction,
the reaction mixture was poured into crushed ice. The thiourea
formed was precipitated as solid which was then filtered off,
washed well with cold distilled water, dried and recrystallized
from ethanol.
2.1.1.1.
1-(2-Oxo-2H-chromene-3-carbonyl)-3-(3-methylphenyl)
thiourea (2a). Pale yellow solid; yield: 69%; R⁄f : 0.53; M.P.:
148 °C; IR (pure, cmꢀ1): 3260 (NH), 1720, 1685 (2C@O), 1568
(C@C), 1253 (C@S); 1H NMR (300 MHz, DMSO-d6): d 12.61 (s, 1H,
NH), 11.85 (s, 1H, NH), 9.28 (s, 1H, CAH, H-4) 8.57–8.52 (m, 1H,
ArAH), 8.10–8.06 (m, 1H, ArAH), 7.89–7.83 (m, 1H, ArAH), 7.65–
7.62 (m, 1H, ArAH), 7.55–7.50 (m, 1H, ArAH), 7.30–7.24 (m, 1H,
ArAH), 7.17–7.15 (m, 1H, ArAH), 7.05–7.00 (m, 1H, ArAH), 2.35
(s, 3H, CH3); 13C NMR (75.5 MHz, DMSO-d6): d 181.3, 168.3,
159.5, 157.7, 150.2, 138.4, 128.4, 127.5, 126.8, 125.6, 122.6,
121.4, 114.6, 24.3; Anal. Calc. For C18H14N2O3 S (338.38): C 63.9,
H 4.2, N 8.3, S 9.5; found: C 62.9, H 4.1, N 7.9, S 9.1.
2.1.1.2.
1-(2-Oxo-2H-chromene-3-carbonyl)-3-(2-methoxyphenyl)
thiourea (2b). Light yellow solid; yield: 66%; R⁄f : 0.48; M.P.:
243 °C; IR (pure, cmꢀ1): 3280 (NH), 1725, 1683 (2C@O), 1558
(C@C), 1253 (C@S); 1H NMR (300 MHz, DMSO-d6): d 12.81 (s, 1H,
NH), 11.80 (s, 1H, NH), 9.18 (s, 1H, CAH, H-4) 8.67–8.65 (m, 1H,
ArAH), 8.10–8.08 (m, 1H, ArAH), 7.89–7.83 (m, 1H, ArAH), 7.62–
7.59 (d, 1H, J = 8.7 Hz, ArAH), 7.55–7.50 (m, 1H, ArAH), 7.30–7.24
(m, 1H, ArAH), 7.19–7.17 (d, 1H, J = 7.2 Hz, ArAH), 7.05–7.00 (m,
1H, ArAH), 3.91 (s, 3H, OCH3); 13C NMR (75.5 MHz, DMSO-d6): d
181.3, 168.3,159.5, 157.7, 150.2, 138.4, 128.4, 127.5, 126.8, 125.6,
122.6, 121.4, 114.6, 55.9. Anal. Calc. for C18H14N2O4S (354.38): C
61.0, H 3.9, N 7.9, S 9.0; found: C 60.9, H 3.8, N 7.7, S 8.9.
2.1.1.3.
1-(2-Oxo-2H-chromene-3-carbonyl)-3-(3-fluorophenyl)
thiourea (2c). Light yellow solid; yield: 70%; R⁄f : 0.54; M.P:
202 °C; IR (pure, cmꢀ1): 3275 (NAH), 1735, 1685 (2C@O), 1558
(C@C), 1255 (C@S); 1H NMR (300 MHz, DMSO-d6): d 10.80 (s, 1H,
NH), 8.92 (s, 1H, CAH, H-4), 8.03–8.00 (m, 1H, ArAH), 7.82–7.75
(m, 2H, ArAH), 7.58–7.55 (m, 1H, ArAH), 7.51–7.38 (m, 3H, ArAH),
7.03–6.96 (m, 1H, ArAH); 13C NMR (75.5 MHz, DMSO-d6): d 163.4,
161.6, 140.7, 138.7, 131.4, 130.8, 129.1, 128.3,127.9; Anal. Calc. for
2. Materials and methods
2.1. Chemistry
The Rf-values were determined using aluminum pre-coated sil-
ica gel plates Kiesel 60 F254 from Merck (Darmstadt, Germany).
Melting points of the compounds were measured in open capillar-
ies using Stuart melting point apparatus (SMP3) and are uncor-
rected. The IR spectra were recorded on FTS 3000 MX, Bio-Rad
Merlin (Excalibur Model) spectrophotometer as pure compounds.
The 1H NMR and 13C NMR spectra were run on a Bruker 300 MHz
NMR spectrometer in DMSO-d6 solutions using TMS as an internal
standard. Mass spectra were recorded on Agilent technologies
6890N gas chromatograph and an inert mass selective detector
5973 mass spectrometer and the elemental analyses were con-
ducted using a LECO-183 CHNS analyser.
C17H11FN2O3S (342.34): C 59.6, H 3.2, N 8.2, S 9.4; found: C 58.8, H
3.0, N 7.9, S 9.1.
2.1.1.4. 1-(2-Oxo-2H-chromene-3-carbonyl)-3-(2,3-dichlorophenyl)
thiourea (2d). Off white solid; yield: 72%; R⁄f : 0.51; M.P: 217 °C;
IR (pure, cmꢀ1): 3251 (NAH), 1710, 1692 (2C@O), 1568 (C@C),
1223 (C@S); 1H NMR (300 MHz, DMSO-d6): d 12.38 (s, 1H, NH),
11.94 (s, 1H, NH), 9.16 (s, 1H, CAH, H-4) 8.11–8.08 (dd, 1H, ArAH),
7.98–7.95 (dd, 1H, ArAH), 7.89–7.83 (m, 1H, ArAH), 7.66–7.59 (m,
2H, ArAH), 7.55–7.40 (m, 3H, ArAH); 13C NMR (75.5 MHz, DMSO-
d6): d 179.2, 162.0, 161.0, 154.7, 150.9, 137.8, 136.0, 132.4, 131.4,
129.3, 128.5, 127.3, 126.1, 118.9, 117.5, 116.9; Anal. Calc. for
2.1.1. General procedure for the synthesis of Coumarinyl thioureas
(2a–2l)
C
17H10Cl2N2O3 S (393.24): C 51.9, H 2.6, N 7.1, S 8.1; found: C
50.6, H 2.2, N 6.9, S 7.8.
Coumarin-3-carboxylic acid (2.6 mM) was placed in a 100 mL
two necked round bottom flask, fitted with a reflux condenser
and a gas trap. Then (0.23 mL, 0.003 mol) thionyl chloride was
added and the reaction mixture was heated under reflux for
2–3 h to give acid chloride. To a stirred solution of potassium thio-
cyanate in 20 mL dry acetone, placed in a 250 mL two necked
round bottom flask fitted with a reflux condenser, freshly prepared
acid chloride (2.0 mM) was added drop wise and the mixture was
refluxed for 30 min. After the initial reaction has subsided, a solu-
tion of substituted aniline (0.0026 mol) in 20 mL dry acetone was
2.1.1.5.
1-(2-Oxo-2H-chromene-3-carbonyl)-3-(3-chlorophenyl)
thiourea (2e). Yellow solid; yield: 68%; R⁄f : 0.51; M.P.: 217 °C; IR
(pure, cmꢀ1): 3265 (NH), 1735, 1695 (2C@O), 1590 (C@C), 1245
(C@S); 1H NMR (300 MHz, DMSO-d6): d 12.31 (s, 1H, NH), 11.70
(s, 1H, NH), 9.08 (s, 1H, CAH, H-4) 8.68–8.65 (m, 1H, ArAH),
8.10–8.02 (m, 1H, ArAH), 7.89–7.81 (m, 1H, ArAH), 7.65–7.62 (d,
1H, J = 8.7 Hz, ArAH), 7.55–7.50 (m, 1H, ArAH), 7.30–7.24 (m, 1H,
ArAH), 7.17–7.15 (d, 1H, J = 7.2 Hz, ArAH), 7.06–7.02 (m, 1H,
ArAH); 13C NMR (75.5 MHz, DMSO-d6): d 179.3, 166.3, 158.5,

60
A. Saeed et al. / Bioorganic Chemistry 63 (2015) 58–63
157.7, 153.2, 136.4, 128.4, 127.5, 126.8, 125.6, 123.6, 121.4, 116.6;
Anal. Calc. for C17H11ClN2O3S (358.80): C 56.9, H 3.1, N 7.8, S 8.9;
found: C 55.8, H 2.9, N 7.2, S 8.3.
(C@C), 1253 (C@S); 1H NMR (300 MHz, DMSO-d6): d 12.81 (s, 1H,
NH), 11.80 (s, 1H, NH), 9.18 (s, 1H, CAH, H-4) 8.67–8.65 (m, 1H,
ArAH), 8.10–8.08 (m, 1H, ArAH), 7.89–7.83 (m, 1H, ArAH), 7.62–
7.59 (m, 1H, ArAH), 7.55–7.50 (m, 1H, ArAH), 7.30–7.24 (m, 1H,
ArAH), 7.19–7.17 (m, 1H, ArAH), 7.05–7.00 (m, 1H, ArAH), 3.91
(s, 3H, OCH3); 13C NMR (75.5 MHz, DMSO-d6): d 181.3, 168.3,
159.5, 157.7, 150.2, 138.4, 128.4, 127.5, 126.8, 125.6, 122.6,
121.4, 114.6, 55.9; Anal. Calc. for C18H14N2O4S (354.38): C 61.0, H
3.9, N 7.9, S 9.0; found: C 60.7, H 3.6, N 7.2, S 8.5.
2.1.1.6.
1-(2-Oxo-2H-chromene-3-carbonyl)-3-(3-chloro-4-fluo-
rophenyl)thiourea (2f). Bright yellow solid; yield: 72%; R⁄f : 0.41;
M.P.: 184 °C; IR (pure, cmꢀ1): 3246 (NH), 1712, 1695 (2C@O),
1558 (C@C), 1253 (C@S); 1H NMR (300 MHz, DMSO-d6): d 12.20
(s, 1H, NH), 11.84 (s, 1H, NH), 9.10 (s, 1H, CAH, H-4), 8.11–8.06
(m, 1H, ArAH), 8.03–7.96 (m, 1H, ArAH), 7.89–7.76 (m, 1H, ArAH),
7.69–7.57 (m, 2H, ArAH), 7.55–7.42 (m, 3H, ArAH); 13C NMR
(75.5 MHz, DMSO-d6): d 178.9, 170.5, 161.8, 154.7, 150.6, 136.0,
131.4, 127.6, 126.0, 118.8, 117.7, 117.1, 116.9; Anal. Calc. for
2.1.1.12. 1-(2-Oxo-2H-chromene-3-carbonyl)-3-(cyclohexyl)thiourea
(2l). Bright yellow solid; yield: 68%; R⁄f : 0.46; M.P.: 146 °C; IR
(pure, cmꢀ1): 3250 (NH), 1725, 1685 (2C@O), 1558 (C@C), 1253
(C@S); 1H NMR (300 MHz, DMSO-6): d 12.20 (s, 1H, NH), 11.84 (s,
1H, NH), 10.75 (s, 1H, CAH, H-4), 7.70 (d, 1H, J = 7.5 Hz, H-8),
7.43–7.37 (m, 2H, H-7, H-6), 6.89 (m, 1H, H-5), 2.43–2.62 (m, 1H,
CH), 1.78–1.53 (m, 4H, 2CH2), 1.49–1.39 (m, 6H, 3CH2);
13C NMR (75.5 MHz, DMSO-d6): d 187.0, 168.3, 159.5, 150.2,
138.6, 128.4, 126.6, 125.0, 121.8, 54.3, 34.5, 28.4, 23.5; Anal. Calc.
for C17H18N2O3S (330.40): C 61.8, H 5.5, N 8.5, S 9.7; found:
C 60.9, H 5.1, N 8.0, S 9.2.
C17H10ClFN2O3S (376.79): C 54.2, H 2.7, N 7.4, S 8.5; found: C
53.8, H 2.1, N 6.8, S 8.1.
2.1.1.7.
1-(2-Oxo-2H-chromene-3-carbonyl)-3-(2-chlorophenyl)
thiourea (2g). Light yellow solid; yield: 66%; R⁄f : 0.37; M.P.: 231 °C;
IR (pure, cmꢀ1): 3260 (NH), 1714, 1677 (2C@O), 1562 (C@C), 1277
(C@S); 1H NMR (300 MHz, DMSO-d6): d 12.44 (s, 1H, NH), 11.92 (s,
1H, NH), 9.17 (s, 1H, CAH, H-4), 8.13–8.07 (m, 1H, ArAH), 7.89–
7.83 (m, 1H, ArAH), 7.64–7.57 (m, 2H, ArAH), 7.53–7.50 (m, 1H,
ArAH), 7.47–7.33 (m, 3H, ArAH); 13C NMR (75.5 MHz, DMSO-d6):
d 178.8, 150.9, 136.0, 135.6, 131.4, 130.0, 128.8, 128.5, 127.9,
126.1, 118.9, 117.5, 116.9; Anal. Calc. for C17H11ClN2O3S (358.80):
C 56.9, H 3.1, N 7.8, S 8.9; found: C 55.6, H 2.8, N 7.1, S 8.4.
2.2. Determination of AChE and BChE inhibitory activities
Newly synthesized coumarin thioureas were tested against
electric eel AChE and horse serum BChE. The cholinesterase
inhibitory activity was measured using standard protocol [39,40].
Donepezil and neostigmine were used as standard references in
the assay. The compounds were initially tested against these
enzymes at 1 mM concentration. The derivatives showing P50%
inhibition were selected for further determination of dose–
response curves against all compounds. For this purpose 8–10
serial dilutions of each compound were prepared in enzyme assay
buffer and their dose response curves were obtained by following
the same method used for initial screening. All experiments were
repeated in triplicate. Results reported are mean of three indepen-
dent experiments ( SEM) and expressed as percent inhibitions
calculated by the formula:
2.1.1.8. 1-(2-Oxo-2H-chromene-3-carbonyl)-3-(2,4-dichlorophenyl)
thiourea (2h). Light yellow solid; yield: 73%; R⁄f : 0.54; M.P.:
244 °C; IR (pure, cmꢀ1): 3236 (NAH), 1727, 1672 (2C@O), 1595
(C@C), 1245 (C@S); 1H NMR (300 MHz, DMSO-d6): d 12.01 (s, 1H,
NH), 11.61 (s, 1H, NH), 10.13 (s, 1H, CAH, H-4) 8.54–8.50 (m, 1H,
ArAH), 8.11–8.08 (m, 1H, ArAH), 7.85–7.80 (m, 1H, ArAH), 7.61–
7.58 (m, 1H, ArAH), 7.53–7.45 (m, 4H, ArAH); 13C NMR
(75.5 MHz, DMSO-d6): d 179.2, 162.0, 161.0, 154.7, 150.9, 137.8,
136.0, 132.4, 131.4, 129.3, 128.5, 127.3, 126.1, 125.4, 123.3,
121.6; Anal. Calc. for C17H10Cl2N2O3S (393.24): C 51.9, H 2.6, N
7.1, S 8.1; found: C 50.5, H 2.3, N 6.9, S 7.9.
Inhibition ð%Þ ¼ ½100 ꢀ ðAbs of test compound=Abs of controlÞ
ꢁ 100ꢂ
2.1.1.9.
1-(2-Oxo-2H-chromene-3-carbonyl)-3-(4-chlorophenyl)
thiourea (2i). Orange yellow solid; yield: 70%; R⁄f : 0.51; M.P.:
210 °C; IR (pure, cmꢀ1): 3265 (NH), 1704, 1695 (2C@O), 1541
(C@C), 1241 (C@S); 1H NMR (300 MHz, DMSO-d6) d 12.31 (s, 1H,
NH), 11.70 (s, 1H, NH), 9.08 (s, 1H, CAH, H-4) 8.68–8.65 (m, 1H,
ArAH), 8.10–8.02 (m, 1H, ArAH), 7.89–7.81 (m, 1H, ArAH), 7.65–
7.62 (m, 1H, ArAH), 7.55–7.50 (m, 1H, ArAH), 7.30–7.24 (m, 1H,
ArAH), 7.17–7.15 (m, 1H, ArAH), 7.06–7.02 (m, 1H, ArAH); 13C
NMR (75.5 MHz, DMSO-d6): d 180.3, 169.3, 158.5, 157.7, 153.2,
136.4, 128.4, 127.5, 126.8, 125.6, 123.6, 121.4, 117.6; Anal. Calc.
for C17H11ClN2O3S (358.80): C 56.9, H 3.1, N 7.8, S 8.9; found: C
55.7, H 3.0, N 7.5, S 8.1.
IC50 values of selected compounds exhibiting >50% activity
were calculated after dilutions using computer program GraphPad,
San Diego, California, USA.
2.3. Molecular modeling
2.3.1. Data set preparation
The molecular construction and modeling of the synthesized
compounds were performed using the ‘‘Build” tool available in
Maestro 2011 (Schrödinger Inc.). Then these molecules were con-
verted from 2D to 3D conformations using the LigPrep [41] module
implemented in molecular modeling Schrodinger package. This
algorithm generates accurate energy minimized molecular struc-
tures with their tautomers, ionization states, ring conformations,
and stereoisomers to produce broad chemical and structural diver-
sity from a single input structure. Hence we have also used all its
utilities to prepare our compounds for molecular docking
simulations.
2.1.1.10.
1-(2-Oxo-2H-chromene-3-carbonyl)-3-(2-nitrophenyl)
thiourea (2j). Light yellow solid; yield: 66%; R⁄f : 0.37; M.P.:
231 °C; IR (pure, cmꢀ1): 3260 (NH), 1714, 1677 (2C@O), 1562
(C@C), 1277 (C@S); 1H NMR (300 MHz, CDCl3): d 12.58 (s, 1H,
NH), 9.02 (s, 1H, CAH, H-4), 8.79–8.76 (m, 1H, ArAH), 8.25–8.22
(m, 1H, ArAH), 7.77–7.68 (m, 3H, ArAH), 7.49–7.41 (m, 1H, ArAH),
7.32–7.27 (m, 2H, ArAH); 13C NMR (75.5 MHz, DMSO-d6): d 154.7,
149.8, 134.9, 134.7, 133.3, 130.0, 125.7, 125.4, 124.3, 124.1, 118.5,
116.8; Anal. Calc. for C17H11N3O5S (369.35): C 55.3, H 3.0, N 11.4, S
8.7; found: C 54.6, H 2.7, N 10.6, S 8.1.
2.3.2. Protein preparation and docking simulations
The homology models of acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) [42] were used for molecular docking
simulations of synthesized compounds. First hydrogen atoms were
added and OPLS_2005 force field-based charges were assigned to
2.1.1.11. 1-(2-Oxo-2H-chromene-3-carbonyl)-3-(3-methoxyphenyl)
thiourea (2k). Light yellow crystals; yield: 66%; R⁄f : 0.48; M.P.:
243 °C; IR (pure, cmꢀ1): 3280 (NH), 1725, 1683 (2C@O), 1558

A. Saeed et al. / Bioorganic Chemistry 63 (2015) 58–63
61
Table 1
the model structures. A brief minimization was also performed to
In vitro AChE and BChE inhibitory activity of the compounds (2a–2l).
relieve steric clashes among the atoms using the protein prepara-
tion wizard option in Maestro. This restrained partial minimization
was terminated when the root-mean-square deviation (rmsd)
reached a maximum value of 0.3 Å from the native structures.
For molecular docking simulations, Glide [43] module imple-
mented in Schrodinger suite 2011 (Schrodinger, Inc.) was used.
The amino acid residues located within 16 Å from the catalytic
triad were defined as the possible binding site for the docking sim-
ulations. In the grid, enclosing box and a scaling factor of 1.00 was
set to van der Waals (VDW) radii of receptor atoms with the partial
atomic charge <0.25. All other parameters were used with their
default settings. In the docking studies, standard precision (SP)
mode was used and three docking poses for each of the compound
was retained in final solution. To select the final best pose, Glide
scoring function (G-score) and visual inspection of binding mode
were carried out.
Entry
IC50 SEM (lM)
AChEa
BChEb
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
0.86 0.05
13.3 1.34
17.2 1.21
1.13 0.24
0.04 0.01
0.75 0.06
7.28 1.58
6.21 1.47
5.10 0.12
0.23 0.08
8.43 1.21
6.97 0.59
0.03 0.01
22.2 3.2
2.15 0.07
0.06 0.02
16.2 1.07
31.6 0.54
1.25 0.06
0.84 0.02
11.1 0.01
13.2 0.54
42.2 1.84
26.1 1.58
53.2 2.52
12.20 3.01
6.41 0.34
49.6 6.11
Donepezil
Neostigmine
a
AChE from electric eel.
BChE from horse serum.
b
3. Results and discussion
3.1. Chemistry
for AChE and BChE inhibition were calculated as shown in Table 1.
The results presented in the table suggest that most of the tested
compounds exhibit moderate to good inhibitory activities in low
micromolar range. The basic structure was 1-(2-Oxo-2H-chro
mene-3-carbonyl)-3-(substituted phenyl)thioureas having diverse
range of substituents at phenyl ring, resulting in different IC50 val-
ues. Compound 2e was the most potent exhibiting IC50 value of
Synthetic pathway to 1-(2-oxo-2H-chromene-3-carbonyl)-3-
aryl thiourea is shown in Scheme 1. Coumarin-3-carbonyl chloride
was freshly prepared from coumarin-3-carboxylic acid using
thionyl chloride. Then it was added to a solution of potassium thio-
cyanate in dry acetone to obtain coumarin-3-carbonyl isothio-
cyanate as an intermediate via stirring at room temperature
followed by reflux. Substituted aniline derivatives was then sepa-
rately reacted with isothiocyanate intermediate to afford the cou-
marinyl thioureas (2a–l).
0.04 0.01 lM and it has 3-chloro substituent at phenyl ring.
Interestingly, the replacement of the flouro group at the same posi-
tion at phenyl ring in compound 2c resulted in the loss of activity
by a factor of approximately 400, as it exhibit IC50 value of
17.2 1.21 lM. The inhibitory potency of the compound 2j
reflected that nitro group substituent in the core of molecule is
3.2. In vitro evaluation of cholinesterase inhibition
of great value in terms of its potency. It has IC50 value of
0.23 0.08
showed IC50 value of 0.86 0.05
envisaged in order to increase the binding of inhibitors in the pres-
l
M. Methyl substituent in case of compound 2a
The newly synthesized thiourea derivatives (2a–l) were
screened for their anti-cholinesterase activity using donepezil
and neostigmine as standard references [39,40]. The IC50 values
lM. The screened series was
Scheme 1. Synthesis of 1-(2-Oxo-2H-chromene-3-carbonyl)-3-(substituted phenyl)thioureas.

62
A. Saeed et al. / Bioorganic Chemistry 63 (2015) 58–63
Fig. 1. Probable binding modes of the compounds (colored sticks) in the active sites of both cholinesterase enzymes. (a) Binding models of the synthesized compounds in
AChE enzyme active site. (b) Binding models of the synthesized compounds in BChE enzyme active site. Yellow dashed lines in (a) showing hydrogen bonding between Tyr-
146 and one of thioureas hydrogen atom from different compounds. Protein is represented in cartoon with green color.
(Glu225, Ser226, His466) and BChE (Glu224, Ser225, His 494).
Docking simulations showed that most of the compounds of
Table 2
Glide docking scores of the compounds (2a–2l) in AChE and BChE enzymes active
2a–2l series having thiourea group, form hydrogen bonding with
Tyr-146 through O--HAN interaction shown in Fig. 1a. Similarly
it has also been observed that most of the synthesized compounds
have very similar probable binding modes in AChE enzyme active
site shown in Fig. 1a. However docking simulations in BChE homol-
ogy model showed that these compounds have slightly different
binding modes (Fig. 1b) with no eminent hydrogen bonding inter-
action patterns, which might be due to their slightly different
active site architecture. From docking scores energy terms, the
hydrophobic interactions were dominant over the electrostatic
interactions energy terms, which is very likely to SAR of these
compounds.
sites.
Compounds
Glide docking score
AChE
BChE
2a
2b
2c
2d
2e
2f
2g
2h
2i
ꢀ8.12
ꢀ6.83
ꢀ7.87
ꢀ7.69
ꢀ8.71
ꢀ7.85
ꢀ7.66
ꢀ8.44
ꢀ8.56
ꢀ8.87
ꢀ8.08
ꢀ7.22
ꢀ6.32
ꢀ6.86
ꢀ6.81
ꢀ7.19
ꢀ7.57
ꢀ6.94
ꢀ6.72
ꢀ6.68
ꢀ6.36
ꢀ7.09
ꢀ8.26
ꢀ7.21
2j
2k
2l
4. Conclusion
ence of phenyl group and thereby increasing the inhibitory
potency.
The BChE inhibition result also showed that most of the com-
pounds exhibit inhibitory activity in the low micromolar range of
concentration. The IC50 value of 0.06 0.02 lM, by compound 2b
suggested that presence of methoxy substituent at ortho position
on phenyl ring has increased inhibitory activity. Whereas the same
group showed IC50 value of 53.2 2.52 lM, at meta position on the
phenyl ring in case of 2k. The most active AChE inhibitor 2e having
We report the synthesis and in vitro inhibitory activity of cou-
marin linked thiourea (2a–2l) derivatives towards acetyl-
cholinesterase and butyrylcholinesterase. The substituents on
coumarins had a profound influence on the cholinesterases inhibi-
tory activity. The most potent compound against acetyl-
cholinesterase was 2e, with an IC50 value of 0.04 0.01
for butyrylcholinesterase was 2b, with an IC50 value of
0.06 0.02 M. Computational modeling showed that most of the
lM and
l
compounds in the series possessed similar binding modes against
each enzyme with different docking scores. Main theme of the
study was to investigate new coumarin derivatives, which may
serve as a base to find new therapies for Alzheimer disease. The
findings of the present study suggest to focus the attention of
researchers on designing new coumarin derivatives as potent inhi-
bitors of acetyl and butyrylcholinesterases. The most active com-
pounds can be further evaluated for in vivo study and might be
useful for the design of pharmacologically important new drug
candidates.
chloro substituent attached to phenyl ring showed IC50 value of
1.25 0.06
flouro group showed further increase in BChE inhibitory activity
i.e., IC50 value of 0.84 0.02 M. Selected concentration–response
lM against BChE, whereas 2f having an additional
l
curves of some inhibitors against acetylcholinesterase and butyryl-
cholinesterase are shown in Fig. S11 (supporting information).
3.3. Docking studies
3.3.1. Predicted binding modes of most active compounds against AChE
and BChE enzymes
Conflict of interest
In order to envisage the probable binding modes of the synthe-
sized compounds against AChE and BChE, these were first docked
into the binding sites of both enzymes. The plausible binding
modes of most active compounds against both enzymes can be
seen in Fig. 1a and b, respectively. Our docking results showed that
most of the compounds possessed similar binding modes with dif-
ferent docking scores shown in Table 2. It has been observed that
these compounds docked well near the catalytic triad of AChE3
The authors have declared no conflict of interest.
Acknowledgments
This work was financially supported by COMSTECH–TWAS and
German-Pakistani Research Collaboration Programme.

A. Saeed et al. / Bioorganic Chemistry 63 (2015) 58–63
63
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