Synthesis, computational studies and biological evaluation of new 1-acetyl-3-aryl thiourea derivatives as potent cholinesterase inhibitors

Article abstract of DOI:10.1007/s00044-017-1829-6

A new series of 1-acetyl-3-aryl thioureas (3f1–15) was synthesized by the reaction of acetyl isothiocyanate with a variety of suitably substituted aromatic anilines. The acetyl isothiocyanate was freshly prepared by reaction of corresonding acid chloride with potassium thiocyanate. The structural confirmation of all compounds was carried out by spectroscopic techniques and in case of 3a by X-ray diffraction study. The newly prepared compounds were subjected to computational studies and evaluated for their cholinesterase (acetylcholinesterase and butyrylcholinesterase) inhibition studies. Except 3f9 and 3f15, all the derivatives were found as selective inhibitor of acetylcholinesterase. Compound 3f2 (IC₅₀ ± SEM = 1.99 ± 0.11 μM) was found to be the most potent inhibitor of acetylcholinesterase exhibited ≈11 times greater inhibitory potential than reference inhibitor i.e. neostigmine (IC₅₀ ± SEM = 22.2 ± 3.2 μM). Compound 3f9 was found to be most potent butyrylcholinesterase inhibitor (IC₅₀ ± SEM = 1.33 ± 0.11 μM), exhibiting ≈four times greater selectivity for butyrylcholinesterase over acetylcholinesterase. Molecular docking studies were carried out to determine the binding site interactions of these potent inhibitors with cholinesterases and also supported the experimental observations.

Full text of DOI:10.1007/s00044-017-1829-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1829-6
ORIGINAL RESEARCH
Synthesis, computational studies and biological evaluation of new
1-acetyl-3-aryl thiourea derivatives as potent cholinesterase
inhibitors
Aamer Saeed1 Muhammad Shakil Shah2,3 Fayaz Ali Larik1 Shafi Ullah Khan2
●
●
●
●
Pervaiz Ali Channar1 Ulrich Flörke4 Jamshed Iqbal2
●
●
Received: 18 September 2016 / Accepted: 1 March 2017
© Springer Science+Business Media New York 2017
Abstract A new series of 1-acetyl-3-aryl thioureas (3f1–
15) was synthesized by the reaction of acetyl isothiocyanate
with a variety of suitably substituted aromatic anilines. The
acetyl isothiocyanate was freshly prepared by reaction of
corresonding acid chloride with potassium thiocyanate. The
structural confirmation of all compounds was carried out by
spectroscopic techniques and in case of 3a by X-ray dif-
fraction study. The newly prepared compounds were sub-
jected to computational studies and evaluated for their
cholinesterase (acetylcholinesterase and butyrylcholinester-
ase) inhibition studies. Except 3f9 and 3f15, all the
derivatives were found as selective inhibitor of acet-
ylcholinesterase. Compound 3f2 (IC50 SEM = 1.99
0.11 µM) was found to be the most potent inhibitor of
acetylcholinesterase exhibited ≈11 times greater inhibitory
potential than reference inhibitor i.e. neostigmine (IC50
SEM = 22.2 3.2 µM). Compound 3f9 was found to be
most potent butyrylcholinesterase inhibitor (IC50 SEM =
1.33 0.11 µM), exhibiting ≈four times greater selectivity
for butyrylcholinesterase over acetylcholinesterase. Mole-
cular docking studies were carried out to determine the
binding site interactions of these potent inhibitors with
cholinesterases and also supported the experimental
observations.
●
●
Keywords Cholinesterases Molecular docking
●
Potassium thiocynate Substituted aniline
Introduction
Thioureas are well known organic compounds having
thiocarbonyl group in their basic ring (Schroeder 1955;
Mertschenk et al. 2002). Moreover, thioureas serve as
valuable synthon in organic chemistry (Larik et al. 2016).
The presence of oxygen and sulfur atoms in thiourea pro-
vides a site for metals to coordinate and form complexes
(Koch 2001; Aly et al. 2007; Saeed et al. 2014). Different
derivatives of thiourea have been used as basic precursor for
the synthesis of various heterocyclic compounds and
showed a diverse range of biological importance (Saeed
et al. 2009). Among different classes of thiourea deriva-
tives, acyl thioureas have gained considerable interest in the
field of medicinal chemistry due to their numerous biolo-
gical applications (Nencki 1873). Acyl thioureas exhibit a
broad spectrum of bioactivities including antibacterial
(Saeed et al. 2010), antifungal (Eweis et al. 2006), anti-
tubercular (Sriram et al. 2007), herbicidal (Ke and Xue
2006), insecticidal (Brouwer and Grosscurt 1982,) as well
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1829-6) contains supplementary material,
which is available to authorized users.
* Aamer Saeed
aamersaeed@yahoo.com
* Jamshed Iqbal
drjamshed@ciit.net.pk
1
Department of Chemistry, Quaid-I-Azam University, 45320
Islamabad, Pakistan
2
Centre for Advanced Drug Research, COMSATS Institute of
Information Technology, Abbottabad 22060, Pakistan
3
Division of Analytical Chemistry, Institute of Chemical Science,
Bahauddin Zakariya University, 60800 Multan, Pakistan
4
Department Chemie, Fakultätfür Naturwissenschaften, Universität
Paderborn, Warburgerstrasse 100, 33098 Paderborn, Germany

Med Chem Res
as corrosion resistant, antioxidants, and basic component in
polymeric derivatives (Gopi et al. 2000). In addition to
aroylthioureas, acylthiourea derivatives are also well
known for their biological activities like bactericidal,
fungicidal, herbicidal, insecticidal and as regulator of
plant growth (Ren et al. 2000). However, thiourea may
cause liver toxicity when coupled to the pharmaceutical
drugs. It was shown in liver microsomes, in mammalian
cells in culture (Ziegler 1978; Ziegler-Skylakakis 1998),
and in intact rat liver (Krieter et al. 1984) that thiourea
can form S-oxygenated products such as the reactive
electrophiles formamidine sulfenic acid and formamidine
sulfinic acid. The latter has been shown to be genotoxic in
cultured mammalian cells (Ziegler-Skylakakis 1998).
Acetylcholinesterase (AChE, EC 3.1.1.7) and butyr-
ylcholinesterase (BChE, EC 3.1.1.8) are serine-hydrolase
enzymes that belong to a super family of estrases. Both
isozymes have substantial similarities in their
structures but show different specificities toward substrate
and inhibitors due to the differences in amino acid
sequences of their active sites (Marrs and Maynard 2013).
These enzymes are present in different conducting
tissues, however they are more active in motor neurons
than in sensory neurons (Dave et al. 2000).
BChE has higher activity in kidney, heart, intestine, liver,
and lungs while AChE is more abundant in muscles, brain
and erythrocyte membrane (Tougu 2001). The main
function of these isozymes (AChE and BChE) is to cata-
lyze the breakdown of neurotransmitter acetylcholine into
choline and acetic acid (Koelle 1994) and thus
terminate nerve impulse at cholinergic synapses (Shen
et al. 2009). Normally, AChE degrades acetylcholine
(ACh) to maximum extant and is responsible for the ter-
mination of signaling action (Terry and Buccafusco 2003).
Moreover, it has been observed that AChE and BChE are
found extensively higher in Alzheimer disease (AD). In
the view of cholinergic hypothesis, Alzheimer’s disease
results from a lack of cholinergic function in the brain
(Darvesh et al. 2003). A promising therapeutic strategy
should be adopted for stimulating the central cholinergic
functions. Recently, it has been reported that BChE act as
therapeutic target site for ameliorating cholinergic dys-
function associated with AD, thus resulting in the reg-
ulation of synaptic ACh levels (Hardy and Selkoe 2002;
Smith 2009).
Results and discussion
Chemistry
General procedure for the synthesis of 1-acetyl-3-aryl
thioureas (3f1–15)
A new series of thiourea derivatives (3f1–15) was synthe-
sized by forming isothiocyanate intermediate. This inter-
mediate was formed by the addition of acid chloride to the
solution of potassium thiocyanide in the presence of dry
acetone. The reaction was allowed to stand for 30 min. After
30 min, acetyl chloride was added to the potassium thio-
cyanide solution. Then the reaction mixture was allowed to
cool and different aromatic substituted amines were incor-
porated as given in Scheme 1.
The characterization of all the synthesized aromatic
substituted acyl thioureas was done on the basis of their
spectroscopic data. 1H and 13C NMR spectra were recorded
1
in deuterated DMSO-d6 solvent. In H NMR; two N–H
protons at 11.87 and 11.34 ppm were appeared as singlets.
These are the most deshielded signals because of intra
molecular hydrogen bonding that result in shifting the sig-
nals towards higher ppm value, thus justify the presence of
thio core in thiourea. The signal at 7–8 ppm region was
clearly indicating the aromatic ring. The methyl of acyl
group appeared at 2.35 ppm. The N–H proton lying in
between thiocarbonyl and carbonyl is most deshielded
due to intramolecular hydrogen bonding. The protons
attached with benzene ring appeared in the usual range. In
13C NMR, thiocarbonyl carbon appeared most deshielded at
about 180 ppm and carbonyl carbon appeared at around
172–175 ppm. The vibrational analysis of synthesized
compounds was investigated through IR spectroscopy. The
characteristic broad peak of N–H bond appeared at
3200 cm−1. The C–H sp2 stretching frequency appeared at
around 3000 cm−1
.
The intense peak at around
1700–1600 cm−1 confirmed the presence of carbonyl group.
The thiocarbonyl bond vibration appeared between 1050
and 1250 cm−1
.
In vitro cholinesterase inhibition studies and
structure–activity relationship (SAR)
The inhibition studies of these synthesized compounds
against AChE and BChE revealed that all compounds,
except compound 3f9 and 3f15, exhibited selective inhibi-
tion of AChE (Table 1). The compound 1-acetyl-3-(2,4-
dimethylphenyl) thiourea (3f2) with IC50 SEM value
1.99 0.11 μM was found to be the most potent inhibitor of
AChE. This compound exhibited up to 11-fold higher
inhibitory potential as compared to the reference
inhibitor i.e. Neostigmine (IC50 SEM = 22.2 3.2 μM).
Herein, we reported the synthesis of new series of 1-
acetyl-3-aryl thiourea with complete characterization (FTIR,
multinuclear NMR, single crystal XRD) along with their
inhibitory potential against both AChE and BChE. Fur-
thermore, docking studies were also carried out to predict
the most plausible binding site interactions of potent
inhibitors.

Med Chem Res
Scheme 1 Synthetic route to
new 1-acetyl-3-arylthiourea
(3f1–3f13 and 3f14 and 3f15)
3
3
2
(84%)
(86%)
(80%)
(77%)
(87%)
(83%)
(88%)
(82%)
(82%)
(62%)
(79%)
(74%)
(79%)
3
3
3
2
3
3
(yield:60%)
(yield:65%)
Table 1 Acetylcholinesterase and butyrylcholinesterase inhibition
studies in presence of the synthesized compounds
more as compared to 1-acetyl-3-mesitylthiourea (3f1) hav-
ing three methyl groups at ortho, para, and meta position. It
can be inferred that the activity of compound (3f2) is due to
the presence of the two methyl groups on benzene moiety
i.e. one at ortho and other at para position that activate the
ring by sigma pi conjugation. Other compounds exhibited
inhibitory values in the range of IC50 SEM = 2.01 0.11
to 78.5 3.56 μM. Only two compounds i.e. 1-acetyl-3-(4-
chlorophenyl) thiourea (3f9) and 1-acetyl 3-benzylthiourea
(3f15) were found as potential inhibitors of BChE having
the IC50 SEM value 1.33 0.21 and 3.02 0.12 μM,
respectively. The compound 3f9 exhibited up to four times
and compound 3f15 exhibited up to eight times greater
selectivity for BChE over AChE inhibition. The reason for
the maximum inhibition by these two compounds 3f9 and
3f15 might be due to presence of un-substituted benzene
ring in compounds or substituted ring by less electro-
negative group i.e methyl group
Code
AChE activity
BChE activity
IC50 (µM) SEM
3f1
3.27 0.44
1.99 0.11
2.17 0.56
78.5 3.56
4.09 0.26
25.8 2.52
35.1 3.11
2.01 0.11
5.38 0.62
4.79 0.63
19.8 1.04
16.9 0.98
18.2 1.03
11.2 1.32
24.6 2.09
22.2 3.2
<100
<100
3f2
3f3
<100
3f4
<100
3f5
<100
3f6
<100
3f7
<100
3f8
<100
3f9
1.33 0.21
<100
3f10
3f11
3f12
3f13
3f14
3f15
Neostigmine
<100
<100
<100
<100
3.02 0.12
49.6 6.11
Kinetics study
The IC50 is the concentration at which 50% of the enzyme activity is
inhibited. AChE and BChE activities were performed at the final
concentration of 800 µM
The potential inhibitors (3f2 and 3f9) with potent
IC50 values were then selected for the kinetics study in order
to determine their type of inhibition. Both compounds 3f2
and 3f9 exhibited non-competitive mode of inhibition
against their respective AChE (Fig. 1) and BChE (Fig. 2)
enzymes.
From SAR based comparative analysis of compound 3f2
with the other derivatives, it was found that the inhibitory
activity of 1-acetyl-3-(2,4-dimethylphenyl) thiourea (3f2) is

Med Chem Res
aromatic ring of the 3f2 is facing Trp86 and Tyr337 for
making pi–pi stacked interaction. Ala204 and Gly121 make
hydrogen bonding with the carbonyl group of 3f2. In
addition to this N6 also makes hydrogen bonding with the
His447.
The plausible binding mode of 3f9 within the active site
of BChE (Fig. 4) differs from the docked pose of compound
3f2 in the active site of AChE. The backbone of active
site of BChE consists of Trp82, Gly116, Gly117, Ala199,
Ser198, Glu197, and His438. The key interactions are in
agreement with already reported bonding interactions
such as aromatic stacking interaction to Trp82 and hydro-
gen bonding with the main chain carbonyl of His438.
Critical analysis also revealed some other key hydrogen
bonding interactions with Ser198 with Gly117 amino
acid.
Fig. 1 Double-reciprocal plot of the inhibition kinetics of AChE by
compound 3f2 indicating non-competitive inhibition. Changes in the
initial velocities of the reaction were measured at different con-
centrations of the inhibitors using substrate acetylthiocholine chloride
Conclusion
A new series of 1-acetyl-3-aryl thiourea derivatives was
synthesized and characterized. When evaluated for their
inhibitory potential against cholinesterases i.e. AChE and
BChE, except compound 3f9 and 3f15 all the remaining
derivatives were found to have selective inhibition of AChE
over BChE activity. Compound 3f2 (IC50 SEM = 1.99
0.11 µM) was found to be the most potent inhibitor of
AChE. While, compound 3f9 was found to be most potent
BChE inhibitor (IC50 SEM = 1.33 0.11 µM), exhibiting
≈four times greater selectivity for BChE over AChE. It was
also further validated from molecular docking studies that
were carried out to determine the binding site interactions of
potent inhibitors with cholinesterases.
Fig. 2 Double-reciprocal plot of the inhibition kinetics of BChE by
compound 3f9 indicating non-competitive inhibition. Changes in the
initial velocities of the reaction were measured at different con-
centrations of the inhibitors using substrate butyrylthiocholine chloride
Materials and methods
General
Molecular docking
Melting points were recorded using a digital Gallenkamp
(SANYO) model MPD.BM 3.5 apparatus. 1H NMR
spectra were determined in CDCl3 and DMSO solutions at
300 MHz using a Bruker AM-300 spectrophotometer
while TMS was used as an internal reference. Similarly,13C
NMR spectra were determined at 75 MHz using a
Bruker 75 MHz NMR spectrometer in CDCl3 solution.
FTIR spectra were recorded on an FTS 3000 MX spectro-
photometer. Elemental analyses were conducted
using a LECO-183 CHNS analyzer. The reactions were
monitored by TLC using Pre-coated Silicagel-60 HF254
TLC plates.
Molecular docking studies against AChE and BChE
The putative binding mode of compound 3f2 (most potent
inhibitor of AChE) was predicted inside the active site of
AChE (Fig. 3). Analysing the key interactions of 3f2 within
the active site of AChE revealed that compound 3f2 was
entrenched by aromatic ring containing amino acids residue
that is Trp86, Ala204, Phe297, Typ337, and Phe338
(Fig. 4). In its preferred docking pose, compound 3f2 is
docked at the bottom of the active site gorge and almost
coplanar with residues Trp86 and Tyr337. The central

Med Chem Res
Fig. 3 Putative binding mode of
3f2 (most potent inhibitor
AChE, green colored) in active
site of AChE (light cyan
colored) (color figure online)
Experimental data
1-Acetyl-3-(2, 4-dimethylphenyl) thiourea (3f2)
Synthesis of 1-acetyl-3-aryl thiourea (3f1–3f15)
The required chemicals were purchased from sigma Aldrich
and were dried and distilled according to standard proce-
dures. The progress of reaction was monitored by thin layer
chromatography (TLC). The synthesized derivatives were
filtered and washed with n-hexane. The crude product
obtained was dried and then recrystallized in ethanol solvent
to get the pure product.
Light yellow solid, m.p = 190 °C, yield = 86%, Rf =
0.62 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3182.1
(N–H, stretching), 2997.5 (Aromatic C–H, stretching),
1683.2 (C=O, stretching), 1573 (N–O, bending), 1509.5
(C–C, stretching), 1413.2 (C–H, bending), 1335.95 (N–O,
1
bending), 1255.3 (C=S, stretching). H NMR (DMSO-d6,
300 MHz,); δ (ppm) 11.66 (s, 1H, NH), 11.45 (s, 1H, NH),
6.9–7.39 (m, 3H Ar–H), 2.49 (s, 3H), 2.21 (s, 6H Ar 2 ×
CH3); 13C NMR (75 MHz DMSO-d6) δ (ppm) 178 (C=S),
171.9 (C=O), 138.9, 134.1, 133.5, 132.5, 128.7, 126.5
(Ar–C), 21.0, 18.20 (CH3), anal. calcd. for C11H14N2OS: C,
59.43; H, 6.35; N, 12.60; S, 14.42 found: C, 59.41; H, 6.37;
N, 12.63; S, 14.45.
1-Acetyl-3-mesitylthiourea (3f1)
White crystalline solid, m.p = 199 °C, yield = 84%, Rf =
0.58 (n-Hexane:ethyl acetate 1:1) FTIR υ (cm−1) 3188.3
(N–H, stretching), 2999.5 (Aromatic C–H, stretching),
1689.2 (C=O, stretching), 1578 (N–O, bending), 1507.5
(C–C, stretching), 1411.6 (C–H, bending), 1332.99 (N–O,
1-Acetyl-3-(2-methoxyphenyl) thiourea (3f3)
1
bending), 1250.8 (C=S, stretching). H NMR (DMSO-d6,
300 MHz,); δ (ppm) 11.66 (s, 1H, NH), 11.45 (s, 1H, NH),
6.90 (2H Ar–H), 2.51 (s, 3H), 2.24 (s, 6H Ar 2 × CH3),
2.10 (s, 3H Ar–CH3); 13C NMR (75 MHz DMSO-d6) δ
(ppm) 180 (C=S), 172.9(C=O), 136.9, 135.0, 133.8, 128.9
(Ar–C), 24.17, 21.0, 18.20 (CH3), anal. calcd. for
C12H16N2OS: C, 60.99; H, 6.82; N, 11.85; S, 13.57 found:
C, 60.97; H, 6.84; N, 11.84; S, 13.58.
Brown crystalline solid, m.p = 190 °C, yield = 80%, Rf
= 0.62 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3170.5
(N–H, stretching), 2915.5 (Aromatic C–H, stretching),
1670.5 (C=O, stretching), 1560 (N–O, bending), 1504.2
(C–C, stretching), 1419.2 (C–H, bending), 1310.61 (N–O,
1
bending), 1292.8 (C=S, stretching). H NMR (DMSO-d6,

Med Chem Res
Fig. 4 Putative binding mode of
3f9 (most potent inhibitor
BChE, maroon colored) in
active site of BChE (light cyan
colored) (color figure online)
300 MHz,); δ (ppm) 11.90 (s, 1H, NH), 11.70 (s, 1H, NH),
7.12 (d, 2H, J = 7.8 Hz ArH), 6.83(Ar–H, d, 2H, J = 7.7
Hz), 3.88 (s, 3H), 2.40 (s, 3H); 13C NMR (75 MHz DMSO-
d6) δ (ppm) 177 (C=S), 165.2 (C=O), 131.1, 130.1, 127.2,
124.8 (Ar–C), 23 (CH3), 55 (CH3), anal. calcd. for
C10H12N2O2S: C, 53.58; H, 5.41; N, 12.51; S, 14.31 found:
C, 53.43; H, 5.30; N, 12.32; S, 14.15.
White crystalline solid, m.p = 186 °C, yield = 87%, Rf
= 0.42 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3189.5
(N–H, stretching), 2995.5 (Aromatic C–H, stretching),
1685.5 (C=O, stretching), 1579 (N–O, bending), 1509.8
(C–C, stretching), 1419.5 (C–H, bending), 1338.93 (N–O,
1
bending), 1296.5 (C=S, stretching). H NMR (DMSO-d6,
300 MHz,); δ (ppm) 11.79 (s, 1H, NH), 11.65 (s, 1H, NH),
7.66 (d, 2H, J = 7.7 Hz ArH), 7.43(Ar–H, d, 2H, J = 7.7
Hz) 2.44 (s, 3H); 13C NMR (75 MHz DMSO-d6) δ (ppm)
182 (C=S), 169.2 (C=O), 132.1, 131.1, 128.2, 125.8
(Ar–C), 20 (CH3), anal. calcd. for C9H9BrN2OS:C, 39.55;
H, 3.38; N, 10.26; S, 11.71 found: C, 39.41; H, 3.28; N,
10.17; S, 11.65.
1-Acetyl-3-(2-chlorophenyl) thiourea (3f4)
1-Acetyl-3-(2, 3-dichlorophenyl) thiourea (3f6)
Light yellow solid, m.p = 188 °C, yield = 77%, Rf =
0.52 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3186.2
(N–H, stretching), 2994.3 (Aromatic C–H, stretching),
1687.2 (C=O, stretching), 1571 (N–O, bending), 1507.1
(C–C, stretching), 1415.3 (C–H, bending), 1337.91 (N–O,
1
bending), 1295.3 (C=S, stretching). H NMR (DMSO-d6,
300 MHz,); δ (ppm) 11.77 (s, 1H, NH), 11.55 (s, 1H, NH),
7.73 (d, 2H, J = 7.8 Hz ArH), 7.47 (Ar–H, d, 2H, J = 7.8
Hz) 2.43 (s, 3H); 13C NMR (75 MHz DMSO-d6) δ (ppm)
187 (C=S), 170.1 (C=O), 134.1, 132.5, 128.7, 126.5
(Ar–C), 23 (CH3), anal. calcd. for C9H9ClN2OS: C, 47.25;
H, 3.98; N, 12.25; S, 14.02 found: C, 47.28; H, 3.95; N,
12.23; S, 14.06.
Light yellow solid, m.p = 178 °C, yield = 83%, Rf =
0.48 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3180.2
(N–H, stretching), 2984.3 (Aromatic C–H, stretching),
1687.2 (C=O, stretching), 1565 (N–O, bending), 1502.1
(C–C, stretching), 1411.3 (C–H, bending), 1331.91 (N–O,
1
bending), 1290.3 (C=S, stretching). H NMR (DMSO-d6,
300 MHz,); δ (ppm) 11.79 (s, 1H, NH), 11.65 (s, 1H, NH),
7.74 (d, 2H, J = 7.9 Hz ArH), 7.47(Ar–H, d, 2H, J = 7.6
Hz) 2.40 (s, 3H); 13C NMR (75 MHz DMSO-d6) δ (ppm)
185 (C=S), 174.1 (C=O), 136.1, 133.5, 1297, 127.5
(Ar–C), 21 (CH3), anal. calcd. for C9H9Cl2N2OS:C, 47.25;
H, 3.98; N, 12.25; S, 14.02 found: C, 47.28; H, 3.95; N,
12.23; S, 14.06.
1-Acetyl-3-(4-bromophenyl) thiourea (3f5)

Med Chem Res
1-Acetyl-3-(4-methoxyphenyl) thiourea (3f7)
(C–C, stretching), 1421.5 (C–H, bending), 1341.57 (N–O,
bending), 1291.3 (C=S, stretching). H NMR (DMSO-d6,
1
300 MHz); δ (ppm) 12.53 (s, 1H, NH), 11.92 (s, 1H, NH),
7.26–6.8 (ArH, m, 4H), 2.29 (s, 3H); 2.11 (s, 3H); 13C
NMR (75 MHz DMSO-d6) δ (ppm) 183.7 (C=S), 168.9
(C=O), 137.4, 134.2, 131.7, 129.2, 128.2, 126.4 (Ar–C),
25, 20 (CH3), –. for C10H12N2OS: C, 47.29; H, 3.95; N,
12.24; S, 14.01 found: C, 47.13; H, 3.77; N, 12.16; S,
14.95.
Brown crystalline solid, m.p = 180 °C, yield = 88%, Rf
= 0.82 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3179.5
(N–H, stretching), 2985.5 (Aromatic C–H, stretching),
1675.5 (C=O, stretching), 1569 (N–O, bending), 1504.2
(C–C, stretching), 1416.2 (C–H, bending), 1337.61 (N–O,
1-Acetyl-3-(3-methoxyphenyl) thiourea (3f10)
1
bending), 1292.8 (C=S, stretching). H NMR (DMSO-d6,
300 MHz,); δ (ppm) 11.99 (s, 1H, NH), 11.77 (s, 1H, NH),
7.12 (d, 2H, J = 7.6 Hz ArH), 6.83(Ar–H, d, 2H, J = 7.6
Hz), 3.88 (s, 3H), 2.40 (s, 3H);13C NMR (75 MHz DMSO-
d6) δ (ppm) 181 (C=S), 169.2 (C=O), 131.1, 130.1, 127.2,
124.8 (Ar–C), 19 (CH3), 59(CH3), anal. calcd. for
C10H12N2O2S: C, 53.58; H, 5.41; N, 12.51; S, 14.31 found:
C, 53.43; H, 5.30; N, 12.45; S, 14.22.
Red solid, m.p = 212 °C, yield = 62%, Rf = 0.57 (n-
Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3188.2 (N–H,
stretching), 2985.7 (Aromatic C–H, stretching), 1687.4
(C=O, stretching), 1590 (N–O, bending), 1510.5 (C–C,
stretching), 1420.5 (C–H, bending), 1339.57 (N–O, bend-
1-Acetyl-3-(3-nitrophenyl) thiourea (3f8)
1
ing), 1289.3 (C=S, stretching). H NMR (DMSO-d6, 300
MHz,); δ (ppm) 12.11 (s, 1H, NH), 11.82 (s, 1H, NH),
7.35–6.9 (ArH, m, 4H), 3.89 (CH3), 2.33 (s, 3H); 13C NMR
(75 MHz DMSO-d6) δ (ppm) 185.1 (C=S), 175.5 (C=O),
136.4, 133.2, 130.7, 128.2, 127.2, 125.6 (Ar–C), 55 (CH3),
20 (CH3), anal. calcd. for C10H12N2O2S: C, 53.57; H, 5.37;
N, 12.49; S, 14.31 found: C, 53.49; H, 5.24; N, 12.38; S,
14.15.
Yellow crystalline solid, m.p = 215 °C, yield = 82%, Rf
= 0.61 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3199.1
(N–H, stretching), 2984.1 (Aromatic C–H, stretching),
1685.8 (C=O, stretching), 1589 (N–O, bending), 1509.8
(C–C, stretching), 1419.7 (C–H, bending), 1337.87
(N–O, bending), 1299.2 (C=S, stretching). 1H NMR
(DMSO-d6, 300 MHz,); δ (ppm) 12.80 (s, 1H, NH), 11.88
(s, 1H, NH), 7.89–7.32 (m, 4H, ArH), 2.33 (s, 3H); 13C
NMR (75 MHz DMSO-d6) δ (ppm) 189.1 (C=S), 170.5
(C=O), 146.4, 139.2, 136.7, 132.2, 129.2, 127.6 (Ar–C), 20
(CH3), anal. calcd. for C9H9N3O3S: C, 45.17; H, 3.76; N,
17.59; S, 13.41 found: C, 46.19; H, 4.77; N, 18.58; S,
14.43.
1-Acetyl-3-(3-chlorophenyl) thiourea (3f11)
White crystalline solid, m.p = 199.5 °C, yield = 79%, Rf
= 0.71 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3185.2
(N–H, stretching), 2998.3 (Aromatic C–H, stretching),
1691.2 (C=O, stretching), 1581 (N–O, bending), 1507.7
(C–C, stretching), 1415.8 (C–H, bending), 1337.51 (N–-O,
1-Acetyl-3-(4-methylphenyl) thiourea (3f9)
1
bending), 1297.2 (C=S, stretching). H NMR (DMSO–d6,
300 MHz,); δ (ppm) 11.78 (s, 1H, NH), 11.59 (s, 1H, NH),
7.75–6.92 (Ar–-H, m, 4H), 2.36 (s, 3H); 13C NMR (75 MHz
DMSO-d6) δ (ppm) 183 (C=S), 173.5 (C=O), 137.1,
135.5, 129.7, 128.2, 126.2 (Ar–C), 23 (CH3), anal. calcd.
for C10H10ClNOS: C, 52.71; H, 4.45; N, 6.17; S, 14.04
found: C, 52.63; H, 4.29; N, 5.97; S, 13.98.
Light yellow solid, m.p = 188.2 °C, yield = 82%, Rf =
0.64 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3178.8
(N–H, stretching), 2989.5 (Aromatic C–H, stretching),
1688.2 (C=O, stretching), 1595 (N–O, bending), 1515.5

Med Chem Res
1-Acetyl-3-(2, 6-dibromo-4-fluorophenyl) thiourea (3f12)
7.69–7.26 (d, 2H, ArH), 2.27 (s, 3H); 13C NMR (75 MHz
DMSO-d6) δ (ppm) 185.2 (C=S), 174.5 (C=O), 153.4,
146.2, 136.7, 127.6 (Ar–C), 22 (CH3), anal. calcd. for
C6H7N3OS2: C, 35.83; H, 3.53; N, 20.87; S, 31.83 found:
C, 35.75; H, 3.45; N, 20.70; S, 31.73.
1-Acetyl-3-benzylthiourea (3f15)
Brown solid, m.p = 209 °C, yield = 74%, Rf = 0.55 (n-
Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3189.5 (N–H,
stretching), 2991.8 (Aromatic C–H, stretching), 1689.8
(C=O, stretching), 1580 (N–O, bending), 1509.3 (C–C,
stretching), 1414.3 (C–H, bending), 1382.19 (N–O, bend-
1
ing), 1257.8 (C=S, stretching). H NMR (DMSO-d6, 300
Red solid, m.p = 179 °C, yield = 60%, Rf = 0.51 (n-
Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3169.8 (N–H,
stretching), 2949.7 (Aromatic C–H, stretching), 1676.5
(C=O, stretching), 1581 (N–O, bending), 1515.8 (C–C,
stretching), 1431.5 (C–H, bending), 1355.37 (N–O, bend-
MHz,); δ (ppm) 11.76 (s, 1H, NH), 11.55 (s, 1H, NH), 7.50
(Ar–H,2H), 2.51 (s, 3H); 13C NMR (75 MHz DMSO-d6) δ
(ppm) 185 (C=S), 178.9 (C=O), 139.9, 136.0, 134.8, 130.9
(Ar–C), 20 (CH3), anal. calcd. for C9H7Br2FN2OS:C,
29.24; H, 1.92; N, 7.57; S, 8.67 found: C, 29.17; H, 1.83; N,
7.44; S, 8.56.
1
ing), 1289.7 (C=S, stretching). H NMR (DMSO-d6, 300
MHz,); δ (ppm) 11.87 (s, 1H, NH), 11.53 (s, 1H, NH),
7.26–6.9 (ArH, dd, 3H), 3.95 (s, 2H), 2.27 (s, 3H); 13C
NMR (75 MHz DMSO-d6) δ (ppm) 188.7 (C=S), 173.5
(C=O), 143.4, 129.2, 127.6 (Ar–C), 47 (CH2), 22 (CH3),
anal. calcd. for C10H12N2OS: C, 57.65; H, 5.82; N, 13.47;
S, 15.41 found: C, 58.69; H, 6.83; N, 14.49; S, 15.45
1-Acetyl-3-(4-bromo-2-fluorophenyl) thiourea (3f13)
Structure determinationof 3f1
Yellow crystalline solid, m.p = 205 °C, yield = 79%, Rf
= 0.68 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3185.3
(N–H, stretching), 2996.4 (Aromatic C–H, stretching),
1685.3 (C=O, stretching), 1575 (N–O, bending), 1509.7
(C–C, stretching), 1417.3 (C–H, bending), 1345.97 (N–O,
C12H16N2OS, Mr = 236.3, colourless crystal, size 0.47 ×
0.21 × 0.18 mm3, triclinic space group P -1 with Z = 4, a =
8.2324, b = 11.2356, c = 13.7870 Å, α = 89.981(2)°, β =
84.542(2)°, γ = 85.393(2)°, V = 1265.3 (2) Å3; Dc = 1.241
Mg/m3, µ = 0.238 mm−1, F(000) = 504. The intensity data
was recorded using a Bruker SMART CCD area-detector
diffractometer with graphite monochromated MoKα radia-
tion (λ = 0.71073 Å) at T = 130(2) K. 7562 reflections
collected 1.5 > Θ > 27.9°; 5595 independent reflections I >
2σ (I), Rint = 0.010. Structure solution was carried out by
direct method (Sheldrick 2008) and full-matrix least squares
refinement was done based on F2 and 297 parameters.
Hydrogen atoms were refined anisotropically and were
clearly located from difference Fourier maps Moreover they
were also refined at idealized positions riding on the carbon
atoms with isotropic displacement parameters Uiso(H) =
1.2Ueq(C/N) or 1.5Ueq(CH3) and C–H 0.98–1.00 and N–H
0.88 Å. There are two crystallographically independent but
chemically equal molecules A and B per asymmetric
unit. Refinement converged at R1 = 0.039 [I > 2σ(I)],
wR2 = 0.107 [all data] and S = 1.03; min./max. ΔF -0.24/
0.37 e/Å3.
1
bending), 1259.3 (C=S, stretching). H NMR (DMSO-d6,
300 MHz,); δ (ppm) 11.61 (s, 1H, NH), 11.41 (s, 1H, NH),
7.35–6.9 (Ar–H, m, 3H), 2.45 (s, 3H); 13C NMR (75 MHz
DMSO-d6) δ (ppm) 182 (C=S), 175.6 (C=O), 140.9,
137.1, 135.1, 131.5, 127.7, 125.5 (Ar–C), 23 (CH3), anal.
calcd. for C9H8BrFN2OS:C, 37.15; H, 2.77; 6.55; N, 9.62S,
11.04 found: C, 37.99; H, 2.68; N, 9.51S, 10.95.
1-Acetyl-3-(thiazol-5-yl) thiourea (3f14)
Reddish brown solid, m.p = 218 °C, yield = 65%, Rf =
0.71 (n-Hexane:Ethyl acetate 1:1) FTIR υ (cm−1) 3189.8
(N–H, stretching), 2989.7 (Aromatic C–H, stretching),
1686.5 (C=O, stretching), 1591 (N–O, bending), 1511.8
(C–C, stretching), 1421.5 (C–H, bending), 1345.57 (N–O,
Crystallographic data (excluding structure factors) for the
structure reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
1
bending), 1289.3 (C=S, stretching). H NMR (DMSO-d6,
300 MHz,); δ (ppm) 11.80 (s, 1H, NH), 11.43 (s, 1H, NH),

Med Chem Res
publication no. CCDC-1437229. Copies of available
material can be obtained free of charge via www.ccdc.cam.
ac.uk/data_request/cif, by e-mailing data_request@ccdc.-
cam.ac.uk, or contacting the Cambridge Crystallographic
Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK;
fax: + 44 1223 336033.
and LUMO values (Table 2), which can be attributed to the
presence of methoxy group at the meta position of phenyl
ring. It can also be seen in Fig. 5 that the orbitals are
polarized to a much greater extent in 3f15, 3f14, 3f4 in
comparison to other thioureas, owing to the presence of a
more polar methoxy group on the phenyl ring. The calcu-
lated values were found to be well in agreement with the
experimental values calculated via cyclic voltametry.
Computational study
The geometry of the synthesized compounds was optimized
with DFT/B3LYP as implemented in Guassian-09 (Gaus-
sian09 2009) quantum chemistry package. Using the DFT/
B3LYP calculation method with 6–311++G(d,p) basis set
in the ground state, the optimized molecular geometric
parameters were performed. The energy gap between
highest occupied molecular orbital (HOMO) and lowest
unoccupied molecular orbital (LUMO) is a critical para-
meter to determine molecular electrical transport properties
(Fukui 1982; Gökce and Bahçeli 2013). By analyzing
HOMO and LUMO energy values, molecular properties,
such as the chemical reactivity, kinetic stability, optical
polarizability, chemical hardness and softness, and electro-
negativity can be accessed. HOMO–LUMO energies were
calculated and are summarized in Table 2 while the shape of
orbitals is shown in Fig. 5. The oxidation and reduction
potential of 3f10 is observed to be least among the other
thioureas and is in accordance with the computed HOMO
Table 2 Observed oxidation and reduction potentials in cyclic
voltametry and computed HOMO, LUMO values
Sample
code
Oxidation
potential (V)
HOMO (eV) Reduction
potential (V)
LUMO (eV)
3f1
−0.20621
−0.20695
−0.22519
−0.2236
−0.20938
−0.20893
−0.22429
−0.22225
−0.21329
−0.21126
−0.22928
−0.31348
−0.21561
−0.23830
−0.21572
−0.03583
−0.04322
−0.07404
−0.07361
−0.04746
−0.06241
−0.12118
−0.24852
−0.05457
−0.15385
−0.04717
−0.04381
−0.04561
−0.07301
−0.07250
−0.04835
−0.05342
−0.11107
−0.24943
−0.05647
−0.14294
−0.05818
3f2
3f3
3f4
3f5
−0.21438
−0.22135
−0.02391
−033457
−0.2246
3f7
3f8
3f10
3f12
3f14
3f15
−0.24740
−0.31563
Fig. 5 Orbital diagrams of
compound 3f1–14, where 1 = 3f
1, 2 = 3f2, WB 3rd = 3f3, 4 =
4f4, 5 = 3f5, 6 = 3f7, 7 = 3f8, 8
= 3f14, Gaussian09 = 3f15, 10
= 3f10, 11 = 3f12
(WB 3rd)
(1)
(2)
(4)
(6)
(5)
(8)
(7)
(Gaussian09)
(10)
(11)

Med Chem Res
Structure discussion
In the crystal packing (Fig. 7) N–H…S interactions
connect molecules with (N11)H..S1i (-x + 1, -y + 2, -z)
2.54Å, N11-H..S1i 160.8° and (N21)H…S2ii (-x + 1, -y, -z
+ 1) 2.53Å, N2-H…S2ii 165.3° into centrosymmetricAAi
and BBii dimers that are stacked along the b-axis
The asymmetric unit contains two crystallographically
independent but chemically equal molecules A and B with
numbering schemes X1nn for A and X2nn for B (Fig. 6).
Geometric parameters for both A and B exhibit no unex-
pected features, bond lengths and angles for both are equal,
but the thiourea moiety of B is somewhat more twisted than
that of A as may be seen from the torsion angles N22-C201-
N21-C202 −8.9(2)°, C201-N21-C202-O2 8.8(2)° for B and
N12-C101-N11-C102 0.6(2)°, C101-N11-C102-O1 −2.9
(2)° for A. The thiourea conformations are connected with
intramolecular N12-H…O1 and N22-H…O2 bonds that
form S(6) rings. The thiourea planes are almost perpendi-
cular to the aromatic ring planes with torsion angles C101-
N12-C104-C105 103.4(2)° and C201-N22-C204-C205
104.7(2)°, respectively.
Biochemical assays
Acetylcholinesterase and butyrylcholinesterase inhibition
assay
AChE and BChE inhibitory studies of synthesized deriva-
tives were measured by using the reported Ellman’s method
with slight modifications (Ellman et al. 1961). Briefly, total
reaction volume was kept as 100 μL, containing 60 μL of
phosphate buffer (50 mM KH2PO4 pH 7.7), 10 μL of test
compound (800 µM per well), followed by the addition of
Fig. 6 Molecular structure of
3f1 showing both independent
molecules A (1nn) and B (2nn).
Anisotropic displacement
ellipsoids are drawn at the 50%
probability level
Fig. 7 Crystal packing of 3f1
viewed along [010] with
intermolecular hydrogen bonds
as dotted lines. H-atoms not
involved are omitted

Med Chem Res
Acknowledgments J. Iqbal is thankful to the Organization for the
Prohibition of Chemical Weapons (OPCW), The Hague, The Neth-
erlands and Higher Education Commission of Pakistan for the finan-
cial support through Project No. 20-3733/NRPU/R&D/14/520 for the
financial support.
10 μL of AChE (0.015 unit per well) or 10 μL of BChE (0.1
unit per well). The contents of the well were mixed
homogenously and incubated at 37 °C for 10 min and
optical density was measured at 405 nm using 96-well plate
reader (Bio-TekELx 800TM, Instruments, Inc.). The reac-
tion was initiated by addition of 10 μL of acetylthiocholine
chloride or butyrylthiocholine chloride as substrate (0.5 mM
per well) followed by addition of 10 μL of coloring reagent
DTNB (5, 5/- dithiobis-2- nitro benzoic acid). The reaction
mixture was incubated at 37 °C for 20 min and optical
density was measured at 405 nm. Neostigmine was used as
a positive control. For the compounds which exhibited over
50% inhibition of either AChE or BChE activity, full con-
centration inhibition curves were produced to evaluate IC50
values. All experiments were carried out as triplicate. A
non-linear regression analysis of the program PRISM 5.0
(GraphPad, San Diego, California, USA) was used to fit the
dose–response curves and to calculate IC50 values.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
References
Aly AA, Ahmed EK, Mokadem KMEl, Hegazy MEAF (2007) Update
survey on aroyl substituted thioureas and their applications.
J Sulfur Chem 28:73–93
Brouwer MS, Grosscurt AC, Duphar International Research BV
(1982) Urea and thiourea compounds and insecticidal composi-
tions. U.S. Patent 4,350,706
Darvesh S, Hopkins DA, Geula C (2003) Neurobiology of butyr-
ylcholinesterase. Nat Rev Neurosci 4:131–138
Dave KR, Syal AR, Katyare SS (2000) Tissue cholinesterases. A
comparative study of their kinetic properties. Z Naturforsch C
55:100–108
Kinetics study
Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A new
and rapid colorimetric determination of acetylcholinesterase
activity. Biochem Pharmacol 7:88–95
Eweis M, Elkholy S, Elsabee M (2006) Antifungal efficacy of chitosan
and its thiourea derivatives upon the growth of some sugar-beet
pathogens. Int J Biol Macromol 38:1–8
Fukui K (1982) Chemical reactivity theory-its pragmatism and
beyond. Pure Appl Chem 54:1825–1836
Gaussian 09, Revision D.01, Frisch MJ, Trucks GW, Schlegel HB,
Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V,
Mennucci B, Petersson GA et al. (2009) Gaussian. Inc., Wall-
ingford CT
Gökce H, Bahçeli S (2013) FT-IR, Micro-Raman and UV–Vis spec-
troscopic and quantum chemical investigations of free 2, 2′-
dithiodipyridine and its metal (Co, Cu, and Zn) halide complexes.
Spectrochim Acta Mol Biomol Spectrosc 114:61–73
Gopi D, Bhuvaneswaran N, Rajeswarai S, Ramadas K (2000) Syner-
gistic effect of thiourea derivatives and non-ionic surfactants on
the inhibition of corrosion of carbon steel in acid environments.
Anti-Corrosion Method Mater 47:332–339
Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s
disease: progress and problems on the road to therapeutics. Sci-
ence 297:353–356
Ke SY, Xue SJ (2006) Synthesis and herbicidal activity of N-(o-
fluorophenoxyacetyl) thioureas derivatives and related fused
heterocyclic compounds. Arkivoc 10:63–68
Koch KR (2001) New chemistry with old ligands: N-alkyl-and N, N-
dialkyl-N′-acyl (aroyl) thioureas in co-ordination, analytical and
process chemistry of the platinum group metals. Coord Chem
Rev 216:473–488
Koelle GB (1994) Pharmacology of organophosphates. J Appl Toxicol
14:105–109
Krieter PA, Ziegler DM, Hill KE, Burk RF (1984) Increased biliary
GSSG efflux from rat livers perfused wth thiocarbamide sub-
strates for the flavin-containing monooxygenase. Mol Pharmacol
26:122–127
Larik FA, Saeed A, Channar PA, Ismail H, Dilshad E, Mirza B (2016)
New 1-octanoyl-3-aryl thiourea derivatives: solvent-free synth-
esis, characterization and multi-target biological activities. Ban-
gladesh J Pharmacol 11:894–902
For determination of mode of inhibition of most potent
inhibitor 3f2 against AChE and 3f9 against BChE, changes
in the initial velocities of the reaction were measured at
different concentrations of inhibitor (0, 0.5, 1.0, and 2.0
µM) using respective substrate concentrations i.e. 0, 0.25,
0.5, 1 and 1.5 mM. A double-reciprocal plot of the inhibi-
tion kinetics of AChE by 3f2 and BChE by 3f9 was mea-
sured by using PRISM 5.0 (GraphPad, San Diego,
California, USA).
Molecular docking
Molecular docking calculations were carried out using
FlexX utility of LeadIT from BioSolveIT GmbH, Germany
(LeadIT 2011). X-rays structures of equine BChE are cur-
rently not available and for electric eel AChE only low
crystallographic resolution (>4 Å) structures are available
(Saeed et al. 2014). Therefore, X-ray structures of human
AChE (PDB ID: 4BDT) and human BChE (PDB ID:
4BDS) were used as template structures and retrieved from
RSC Protein Data Bank and used for molecular docking
studies (Nachon et al. 2013). Prior to the performance of
docking studies the receptor active site was defined as the
amino-acid residues in 6.5 Å radius around the co-
crystallized ligands. Default parameters of amino acid
flips and solvent handling were used. Molecular docking of
compounds were then carried out using default docking
parameters and 50 top ranking poses were retained for
visual inspection in Discovery Studio Visualizer (Studio
and Insight 2009) to determine the putative binding mode.

Med Chem Res
LeadIT B (2011) http://www. biosolveit. de/LeadIT. Accessed 12th Mar
Marrs TC, Maynard R (2013) Neurotranmission systems as targets for
toxicants: a review. Cell Biol Toxicol 29:381–396
Mertschenk B, Beck F, Bauer W (2002) Thiourea and thiourea deri-
vatives. Ullmann’s encyclopedia of industrial chemistry. Wiley-
VCH Verlag GmbH & Co. KGaA, Berlin
Nachon F, Carletti E, Ronco C, Trovaslet M, Nicolet Y, Jean L,
Renard PY (2013) Crystal structures of human cholinesterases in
complex with huprine W and tacrine: elements of specificity for
anti-Alzheimer’s drugs targeting acetyl-and butyryl-cholinester-
ase. Biochem J 453:393–399
(fluorobenzoyl)-3-(fluorophenyl) thioureas. J Fluor Chem 130
(11):1028–1034
Schroeder DC (1955) Thioureas. Chem Rev 55:181–228
Sheldrick GM (2008) A short history of SHELX. Acta Cryst A
64:112–122
Shen Y, Zhang J, Sheng R, Dong X, He Q, Yang B, Hu Y (2009)
Synthesis and biological evaluation of novel flavonoid derivatives
as dual binding acetylcholinesterase inhibitors. J Enzyme Inhib
Med Chem 24:372–380
Smith DA (2009) Treatment of Alzheimer’s disease in the long-term-
care setting. AJHP 66(10):899–907
Nencki M (1873) Zur kenntniss des sulfoharnstoffs. Eur J Inorg Chem
6:598–600
Sriram D, Yogeeswari P, Dinakaran M, Thirumurugan R (2007)
Antimycobacterial activity of novel 1-(5-cyclobutyl-1, 3-oxazol-
2-yl)-3-(sub) phenyl/pyridylthiourea compounds endowed with
high activity toward multidrug-resistant Mycobacterium tuber-
culosis. J Antimicrob Chemother 59:1194–1196
Ren J, Diprose J, Warren J, Esnouf RM, Bird LE, Ikemizu S, Slater M,
Milton J, Balzarini J, Stuart DI (2000) Phenylethylthiazo-
lylthiourea (PETT) non-nucleoside inhibitors of HIV-1 and HIV-
2 reverse transcriptases structural and biochemical analyses. J
Biol Chem 275:5633–5639
Studio D, Insight I (2009) Accelrys Software Inc. San Diego, CA,
92121.
Saeed A, Flörke U, Erben MF (2014) A review on the chemistry,
coordination, structure and biological properties of 1-(acyl/aroyl)-
3-(substituted) thioureas. J Sulfur Chem 35:318–355
Saeed A, Khera RA, Abbas N, Latif M, Sajid I, Floerke U (2010)
Synthesis, characterization, crystal structures, and antibacterial
activity of some new 1-(3, 4, 5-trimethoxybenzoyl)-3-aryl
thioureasreas. Turk J Chem 34:335–346
Saeed A, Mahesar PA, Zaib S, Khan MS, Matin A, Shahid M, Iqbal J
(2014) Synthesis, cytotoxicity and molecular modelling studies of
new phenylcinnamide derivatives as potent inhibitors of choli-
nesterases. Eur J Med Chem 78:43–53
Saeed A, Shaheen U, Hameed A, Naqvi SH (2009) Synthesis, char-
acterization and antimicrobial activity of some new 1-
Terry AV, Buccafusco JJ (2003) The cholinergic hypothesis of age
and Alzheimer’s disease-related cognitive deficits: recent chal-
lenges and their implications for novel drug development. J
Pharm Exp Ther 306:821–827
Tougu V (2001) Acetylcholinesterase: mechanism of catalysis and
inhibition. Curr Med Chem Cent Nerv Syst Agents 1:155–170
Ziegler DM (1978) Intermediate metabolites of thiocarbamides,
thioureylenes and thioamides; mechanism of formation and
reactivity. Biochem Soc Trans 6:94–96
Ziegler-Skylakakis K, Nill S, Pan JF, Andrae U (1998) S-Oxygenation
of thiourea results in the formation of genotoxic products.
Environ Mol Mutagen 31:362–373