New cholinesterase inhibitors for Alzheimer's disease: Structure activity relationship, kinetics and molecular docking studies of 1–butanoyl–3–arylthiourea derivatives

Article abstract of DOI:10.1016/j.ijbiomac.2018.05.001

Highly progressive neurodegenerative disorder generally known as Alzheimer's disease (AD), is a type of dementia, which is very common in elderly. The most common symptoms may include loss of memory along with disturbed behavioral and cognitive functions. Until now, only 4 cholinesterase (ChE) inhibitors are approved by FDA for symptomatic treatment of AD. Aroyl thiourea derivatives are well known bioactive organic molecules containing carbonyl and thiocarbonyl functional groups. Here, total 14 different thiourea derivatives (3a–3n) were synthesized and characterized by NMR, FTIR and X–ray crystallographic techniques. The synthesized compounds displayed varying inhibition activities on both acetylcholineterase (AChE) and butyrylcholinesterase (BuChE) enzymes. Among all compounds, 3b and 3e were potent inhibitors of AChE (IC₅₀ ± SEM = 8.92 ± 1.03 μM) and BuChE (IC₅₀ ± SEM = 6.96 ± 0.961 μM) respectively. Enzyme kinetic studies showed that 3b exhibited uncompetitive binding with AChE while 3e demonstrated a mixed inhibition of BuChE. Molecular docking studies on AChE showed that 3b got binding interaction with Trp86 and Tyr337 while 3e showed binding affinity with Trp82 and His438 when docked with BuChE. The obtained results indicated that these thiourea derivatives could be considered as potential candidates to treat AD.

Full text of DOI:10.1016/j.ijbiomac.2018.05.001

Accepted Manuscript
New cholinesterase inhibitors for Alzheimer's disease: Structure
activity relationship, kinetics and molecular docking studies of
1–butanoyl–3–arylthiourea derivatives
Fayaz Ali Larik, Muhammad Shakil Shah, Aamer Saeed, Hamid
Saeed Shah, Pervaiz Ali Channar, Michael Bolte, Jamshed Iqbal
PII:
DOI:
Reference:
S0141-8130(17)32892-1
doi:10.1016/j.ijbiomac.2018.05.001
BIOMAC 9602
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Revised date:
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3 August 2017
30 January 2018
1 May 2018
Please cite this article as: Fayaz Ali Larik, Muhammad Shakil Shah, Aamer Saeed, Hamid
Saeed Shah, Pervaiz Ali Channar, Michael Bolte, Jamshed Iqbal , New cholinesterase
inhibitors for Alzheimer's disease: Structure activity relationship, kinetics and molecular
docking studies of 1–butanoyl–3–arylthiourea derivatives. The address for the
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ACCEPTED MANUSCRIPT
New cholinesterase inhibitors for Alzheimer’s disease: structure activity relationship,
kinetics and molecular docking studies of 1–Butanoyl–3–Arylthiourea derivatives
Fayaz Ali Larik1, Muhammad Shakil Shah2,3, Aamer Saeed1*, Hamid Saeed Shah2, 4, Pervaiz
Ali Channar1, Michael Bolte5, Jamshed Iqbal2*
1Department of Chemistry, Quaid–i–Azam University, 45320, Islamabad, Pakistan.
2Centre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad 22060, Pakistan.
3Division of Analytical Chemistry, Institute of Chemical Science, Bahauddin Zakariya
University, 60800, Multan, Pakistan.
4College of Pharmacy, University of Sargodha Sargodha 40100, Pakistan.
5Institut für Anorganische Chemie, J.W.–Goethe–Universität, Max–von–Laue–Str. 7, D–
60438 Frankfurt, Germany.
Running Title: Arylthiourea derivatives as cholinesterase inhibitors
Correspondence:
Prof. Dr. Jamshed Iqbal
Centre for Advanced Drug Research, COMSATS Institute of Information
Technology, Abbottabad–22060, Pakistan
Tel: +92–992–383591–96; Fax: +92–992–383441; e–mail: drjamshed@ciit.net.pk
jamshediqb@googlemail.com
/
Dr. Aamer Saeed
Department of Chemistry, Quaid–i–Azam University, 45320, Islamabad, Pakistan
Tel: +92–51–9064–2128; e–mail: aamersaeed@yahoo.com/asaeed@qau.edu.pk
1

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Abstract
Highly progressive neurodegenerative disorder generally known as Alzheimer’s disease (AD), is
a type of dementia, which is very common in elderly. The most common symptoms may include
loss of memory along with disturbed behavioral and cognitive functions. Until now, only 4
cholinesterase (ChE) inhibitors are approved by FDA for symptomatic treatment of AD. Aroyl
thiourea derivatives are well known bioactive organic molecules containing carbonyl and
thiocarbonyl functional groups. Here, total 14 different thiourea derivatives (3a–3n) were
synthesized and characterized by NMR, FTIR and X–ray crystallographic techniques. The
synthesized compounds displayed varying inhibition activities on both acetylcholineterase
(AChE) and butyrylcholinesterase (BuChE) enzymes. Among all compounds, 3b and 3e were
potent inhibitors of AChE (IC50 ± SEM = 8.92 ± 1.03 μM) and BuChE (IC50 ± SEM = 6.96 ±
0.961 μM) respectively. Enzyme kinetic studies showed that 3b exhibited uncompetitive binding
with AChE while 3e demonstrated a mixed inhibition of BuChE. Molecular docking studies on
AChE showed that 3b got binding interaction with Trp86 and Tyr337 while 3e showed binding
affinity with Trp82 and His438 when docked with BuChE. The obtained results indicated that
these thiourea derivatives could be considered as potential candidates to treat AD.
Keywords: Thiourea/ Acetylcholinesterase/ Butyrylcholinesterase/ Alzheimer’s disease
1
Introduction
Among highly progressive neurodegenerative diseases, Alzheimer’s disease (AD), that is a
type of dementia frequently seen in elderly [1]. Every year, about 5 million people worldwide
have been diagnosed with AD where it is expected that by 2050 this number might be raised
remarkably to one among every eighty five individuals [2, 3]. As AD is closely associated with
aging, it might have potential to alter mood and behavior that badly affect functional
competences which may lead to the loss of cognitive and memory functions [4]. Likewise, AD
exerts financial burden that further adds more suffering for patient’s family [5]. In spite of
modern scientific development, till now there is no effective therapy to alleviate AD symptoms
[6]. AD patients are characterized by neurotransmitter imbalance at synaptic terminal due to the
loss of cholinergic neurons associated with memory functions [7]. AD displays extracellular
accumulation of insoluble amyloid β (Aβ) proteins, creating plaques and intracellular deposition
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of neurofibrillary tangles (NFTs) resulting from hyperphosphorylated tau proteins [8]. These
pathological signs trigger different neurotoxic pathways that result into synaptic malfunctioning
and neuronal loss [9]. Moreover, hereditary factors, oxidative stress, mitochondrial dysfunction,
as well as calcium and hormonal imbalance may also take part in AD progression. Numerous
enzymes are involved in the pathological process of AD e.g. lipooxygenases, secretases,
glycogen synthase kinase–3 (GSK3) and more specifically cholinesterases (ChEs). The
inhibition of these enzymes could play a role in stagnation of AD [10, 11].
Cholinesterases including acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are
found in mammalian brain [12]. Both enzymes normalize the concentration of acetylcholine
(Ach) in a variable capacity by deactivating neurotransmitter at synaptic cleft. These enzymes
have equal potential to disintegrate Ach expeditiously with 10,000 molecules in each second
[13]. The integrated course of signal transmission at neuromuscular juncture occurs particularly
in nanosecond including Ach liberation, its diffusion across synaptic gap, interaction with
nicotinic acetylcholine receptors (nAChRs) and lastly, hydrolysis by AChE that terminates Ach–
mediated neurotransmission [14].
The crystal structure of these enzymes describes an active hydrophobic gorge that is deep-rooted
(20Å) and receives Ach. This active site is further categorized into anionic and esteratic sub-sites
[15]. The positively charged quaternary amine of Ach binds to anionic sub-site while its
hydrolysis into choline and acetate occurs after binding with esteratic spot present on the surface
of both enzymes [16]. The amino acid sequence in AChE and BuChE is similar up to 65%
regardless of being encoded by distinct genetic material present on human chromosomes 7q22
and 3q26 correspondingly [17]. Furthermore, the deep–rooted gorge at the bottom of both
enzymes accommodates catalytic triad which consists of three amino acids to interact with varied
substrates. In acyl pocket of AChE, two amino acids Phe295 and Phe297 generally obstruct
substrate interaction caused by aromatic ring that bulge out into gorge. In contrast, the amino
acids in acyl pocket of BuChE are replaced by low molecular weight Valine (Val) and Leucine
(Leu) thus providing more space and permit the binding of large molecules. Functional
dissimilarity among AChE and BuChE is related to specific location and their ability to interact
with neuroactive peptides [18]. AChE is primarily located in neurons and hydrolyzes 80% ACh,
whereas, BuChE present in non-neuronal cells which is less substrate–specific and responsible
for the residual activity [19].
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Human AChE (hAChE) is comprised of a permanent core with 534 amino acids while its flexible
C–terminal and N–terminal peptide chains are composed of variable 14, 26 or 40 and 60–66
amino acids respectively [20]. AChE mainly works at neuromuscular junction where it is
involved in termination of impulse transmission at synaptic region through hydrolysis of Ach
into choline and acetate moieties [21]. The role of BuChE in regulating Ach levels has generally
been disregarded. Though, it is a viable sign that AChE and BuChE mutually contribute in
controlling Ach levels that is critical in evolution and advancement of AD [22]. Furthermore, it
is evident that BuChE protects AChE from natural toxins by distracting and reacting
simultaneously with these molecules [23].
Latest efforts for an efficient treatment of AD is particularly related to improve cholinergic
function, either by utilizing cholinergic receptor agonists or ChE inhibitors obtained from natural
and synthetic source. Aroyl thiourea derivatives are well known bioactive organic molecules
containing carbonyl and thiocarbonyl functional groups. The presence of sulfur and oxygen
atoms inducts hard and soft donor sites for metal complexation and finds numerous applications
like extraction of precious metals such as platinum and gold [24, 25]. Thiourea moiety contains
different sites for hydrogen bonding, which has made them suitable as organo–catalyst.
Moreover, these thiourea derivatives can serve as basic precursor for the synthesis of variety of
heterocycles [26, 27]. Synthesis of aryl thiourea derivatives, their coordination chemistry and
biological aspects have been described previously [24, 28-30]. Moreover, the role of thiourea
derivatives in therapeutic, their utilization in polymer synthesis and corrosion resistance are also
evident in previous reports [31, 32]. Moreover, metal containing bis–thiourea found applications
in optoelectronics [33].
Here we report a new series of thiourea derivatives as cholinesterase inhibitors, which can be of
great importance to treat AD with potent and selective inhibitors of AChE and BuChE.
2
Experimental methods
2.1 Materials
All chemicals, reagents and organic solvents were commercially purchased and utilized
without further purification. Furthermore, the enzymes (AChE and BuChE) and their substrates
acetylthiocholine chloride and butyrylthiocholine chloride were purchased from sigma–Aldrich
Germany.
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2.2 Chemistry
For melting point determination, a digital Gallenkamp (MPD.BM 3.5) instrument was
1
employed. The H NMR and 13C NMR spectra were recorded with the help of Bruker (AM–300)
set at 300 MHz frequency. FTIR studies of thiourea derivatives were conducted on FTS (3000
MX) spectrometer.
2.3 General synthesis of lead compound (1–butanoyl–3–mesitylthiourea (3a–n))
A novel series of thiourea derivatives (3a–n) was synthesized from 1–butanoyl
isothiocyanate. The latter was produced by adding butanoyl chloride to a solution of potassium
thiocyanate in presence of dry acetone for 30 min. After cooling, different aromatic substituted
amines were incorporated to get various derivatives of 1–butanoyl–3–arylthiourea (see
supplementary material).
2.4 Biochemical assay
2.4.1 AChE and BuChE inhibitory assay
A previously reported assay was followed to determine the inhibition potential of
thiourea derivatives against AChE and BuChE enzymes [34]. Briefly, the test compound (10 µL)
was mixed with assay buffer (60 µL) with pH 7.7 in a 96 well plate. A 10 µL from specified
units of enzymes (AChE or BuChE) were added into the mixture. After 10 min of incubation at
37oC the absorbance was measured at 405 nm and considered as pre–incubation reading. Lastly,
a 10 μL of specific substrate for each particular enzyme and 10 μL of coloring agent (2–Nitro
benzoic acid) were added into a 96 well plate. The plate was kept in incubator for at least 20 min.
Post–incubation absorbance was recorded at 405 nm employing Bio–Tek microplate reader
(ELx–800 USA). The absorbance data was further used to estimate inhibitory concentration
(IC50) for each potential inhibitor (≥50%) after suitable dilutions.
2.4.2 Kinetics Study
The mode of inhibition of test compounds 3b and 3e versus AChE and BuChE was determined
through enzyme kinetic studies carried out at different concentrations of substrate (0, 0.25, 0.5, 1
and 1.5 mM) and inhibitor (0, 0.5, 1, 2 µM). The change in initial velocity of reaction was
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calculated at each concentration with the help of double–reciprocal graph to obtain information
about the type of inhibition.
2.5 In silico studies
2.5.1 Molecular Docking
Initially 3D structures of selected compounds were drawn and protonated with the help of
molecular operating environment (MOE) [35]. Energy minimization of selected compounds was
performed with the help of MMFF94x force–field through adjustment of hydrogen atoms [36].
The crystal structure of h–AChE (PDB ID 4BDT) and BuChE (PDB ID 4BDS) were selected as
prototype for docking studies. Active site on each receptor was carefully chosen around 6.5Å
radius of co-crystallized ligand. The solvent handling and amino acid flip parameters were set as
default. By making use of LeadIT program, the best 50 scoring docked posed were nominated
for further analysis [37]. For visual analysis, the Discovery studio visualizer v4 was employed
[38]. The mode of binding of docked poses was evaluated with the help of HYDE assessment
tool of LeadIT [37]. Binding free energy (∆G) for each pose was determined to explore the
degree of interaction with receptor. The minimum energy value reflects high stability and affinity
of molecules to bind with receptor.
3
Results and discussion
3.1 Synthesis scheme of thiourea derivatives
The aromatic ring in synthesized intermediate 1–butanoyl–3–arylthiourea was further
utilized to prepare different derivatives by substituting various functional groups on aromatic
ring have been shown in scheme 1 below.
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Scheme 1 Synthetic pathway of novel thiourea derivatives
3.2 Characterization of thiourea derivatives
3.2.1 NMR analysis
1
The H NMR spectrum showed two types of methyl (–CH3) groups, i.e. –CH3 on acyl
group appeared at 2.35 ppm while another –CH3 attached with aromatic ring was slightly
1
shielded. In H NMR, it was found that –NH proton when positioned among phenyl ring and
C=S bond got maximum de–shielded effect and produced a single peak at 12 ppm (Figure 1(a)).
This effect was due to intra-molecular hydrogen bonding. When –NH proton located in between
C=O and C=S, it was appeared to be less de–shielded and gave signal in the range of 10–11 ppm
[25, 39]. Furthermore, the presence of signals between 7 and 8 ppm was due to aromatic ring
proton.
In 13C NMR, a signal of carbonyl carbon was found in the range of 172–175 ppm while C=S
showed a sharp peak at 180 ppm (Figure 1(b)). Aromatic carbon gave signal between 120–140
ppm while the signal of ipso carbon was weak in intensity because of NOE effect.
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(a)
(b)
1
Figure 1 (a) H NMR spectrum of 1–butanoyl–3–mesitylthiourea (b) 13C NMR spectrum of
compound 1–butanoyl–3–mesitylthiourea
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3.2.2 FTIR analysis
In FTIR spectrum, a broad peak appeared at 3200 cm−1 was due to hydrogen
bonding among –NH and oxygen atom of carbonyl group present in thiourea derivatives.
The strong peak in functional group region (1600–1700 cm–1) was due to the presence of
carbonyl group while broad peak at 3000 cm–1 was due to Ar–H stretching vibration.
The presence of C=S bond was confirmed with sharp and highly intense peaks in the region of
1050 cm–1 and 1250 cm−1 (Figure 2).
Figure 2 FTIR spectra of synthesized thiourea derivative 1–butanoyl–3–mesitylthiourea
3.2.3 X-ray crystallography
The crystal structure of intermediate 1–butanoyl–3–arylthiourea displayed monoclinic
system with vector length a = 10.0387(4) Å, b = 15.3739(5) Å and c = 19.3798(8) Å and bond
angles α = 90o, β= 97.442(3)o and γ = 90o with Z value equals to 8. There are two molecules in
the asymmetric unit (Figure. 3 (a)), which possesses very similar geometries. There were only
minor differences in the torsion angles of the mesityl ring and the propyl group. In the crystal,
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the molecules were connected by N–H...O and N–H...S hydrogen bonds with the chain running
along a-axis (Figure. 3 (b)).
(a)
(b)
Figure 3 (a) Crystal structure of the 1–butanoyl–3–mesitylthiourea. Anisotropic displacement
ellipsoids are drawn at the 50% probability level. (b) Crystal packing of 1–butanoyl–3–
mesitylthiourea viewed along a–axis with intermolecular hydrogen bonds as dotted lines. H
atoms not involved are omitted.
3.3 Biochemical assays
3.3.1 Enzyme inhibition studies
The enzymes (AChE and BuChE) inhibition studies of thiourea derivatives were
performed by a previously reported method with minor variations [34]. After initial screening,
the inhibitors (≥50 % inhibition) were further investigated to determine their IC50 values. All
synthesized molecules exhibited inhibitory values in the range of 8–158 µM and 6–150 µM
against AChE and BuChE respectively. The data was obtained in triplicate and IC50 of inhibitors
was calculated by non–linear regression analysis.
The phenyl ring in thiourea derivatives with the exception of 3n (1–butanoyl–3–benzylthiourea),
was undergone controlled electrophilic and nucleophilic substitutions. In comparison to
para– (p–) and meta– (m–) positions, an ortho– (o–) position was found to be more favorable to
inhibit AChE. In that context compound 3b (1–butanoyl–3–(2, 6–dimethylphenyl) thiourea),
having a methyl substitution at o– position, was produced excellent inhibition against AChE with
IC50 ± SEM = 8.92 ± 1.03 μM. The compound 3b showed 2.5 fold greater inhibition in
comparison to neostigmine i.e. IC50 ± SEM = 22.2 ± 3.21 μM used as standard inhibitor. In case
of electron withdrawing substituents, a p–position was found to be more favorable as confirmed
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by IC50 value in case of 3i (1–butanoyl–3–(4–chlorophenyl)thiourea) i.e. IC50 ± SEM = 11.7 ±
3.78 μM. As for as BuChE is concerned, compound 3e ((1–butanoyl–3–(3–methoxyphenyl)
thiourea) having an electron donating group at m–position, was found to be an excellent inhibitor
with IC50 ± SEM = 6.96 ± 0.961 μM. The SI between AChE and BuChE was calculated which
showed high selectivity of 3b against AChE with SI = 0.073 while compound 3e displayed
selectivity against BuChE with the highest SI (5.568) as compared with the series (see Table 1).
Table 1 The activities of potential inhibitors against AChE and BuChE enzymes
Code
AChE
BuChE
Selectivity index
(SI)
IC50 (µM) ± SEM
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
58.9 ± 3.56
8.92 ± 1.03
42.5 ± 3.56
49.1 ± 5.34
34.3 ± 8.82
96.6 ± 7.92
29.7 ± 4.25
26.4 ± 2.32
11.7 ± 3.78
42.7 ± 5.34
158.3 ± 15.7
40.3 ± 2.33
10.4 ± 1.858
132.5± 9.42
22.2 ± 3.21
43.9 ± 6.25
121 ± 27
1.341
0.073
1.16
36.6 ± 5.83
74.1 ± 8.09
6.96 ± 0.961
75.8 ± 13.6
68.1 ± 5.94
64.5 ± 4.12
16.2 ± 2.19
15.5 ± 2.77
150.1 ± 11.2
23.1 ± 2.09
36.5 ± 3.37
113.1 ± 14.5
49.6 ± 6.31
0.662
5.568
1.274
0.436
0.409
0.722
2.754
1.054
1.744
0.284
1.171
0.447
3n
Neostigmine
The initial screeningof compounds was done at 800µM against AChEand BuChE enzymes
Selectivity index= IC50(hAChE)/ IC50 (hBuChE)
3.3.2 Kinetic models
The kinetic studies of the most potent inhibitors 3b and 3e against AChE and BuChE were
performed at various concentration of substrate (0, 0.25, 0.5, 1 and 1.5 mM) and inhibitors
(0, 0.5, 1.0, 2.0 µM). The initial velocity of reaction at each concentration was determined to
observe the type of inhibition and presented as double–reciprocal plot of inhibition kinetics
(Figure 4). The data showed that 3b produced uncompetitive enzyme inhibition while compound
3e expressed as mixed inhibitor of BuChE enzyme
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Figure 4 A Double–reciprocal graph shows inhibition kinetics of AChE and BuChE. (a) an uncompetitive inhibition
of AChE after incubation with 3b (b) a mixed inhibition of BuChE by 3e
3.4 Molecular docking
Enzyme–inhibitor interactions were evaluated with the help of molecular docking where
binding free energy (∆G) was recorded at each possible position. The lowest ∆G value predicts
the stability of molecule with the highest affinity for receptor interaction [35, 37-38].
The predicted interaction of 3b with the active site of AChE revealed that it was entrenched in a
remarkable group of amino acids with aromatic ring including Trp86 and Tyr337 (Figure 5). The
compound 3b was docked with AChE with its lowermost active site available whereas 3b was
laid parallel to Trp86 and Tyr337. A central aromatic ring of F–4 was fronting Trp86 and Tyr337
for creating π–π stacking. Hydrogen attached to N3 atom making hydrogen bonding with –OH
group of His447 and another hydrogen bond also formed between hydrogen atom attached to N6
and the –OH group of Tyr337.
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Figure 5 Possible binding mode of 3b (pink colored) and AChE (gold colored)
Alternatively, the type of binding interaction between inhibitor 3e and BuChE was different from
interaction among 3b and AChE due to a difference of amino acid chain in active site of BuChE
(Figure 6). The chain was composed of Ala199, Glu197, Gly115–117, His438, Leu286, Phe329,
Phe398, Trp82 and Trp231 amino acid residues. The leading interactions were in accordance
with previous reports [40]. For instance, aromatic π–π stacking occurred between Trp82 and
hydrogen bonding with carbonyl group of His438. Molecular docking has also revealed some
other important interactions such as aromatic π–π stacking between Trp82 as well as hydrogen
bonding with Gly116 and Ala199 and carbonyl group of His438.
Figure 6 Expected binding mode of 3e (Blue colored) in active site of BuChE (gold coloured)
4
Conclusions
In summary, we have successfully synthesized new derivatives of thiourea and tested
biochemically at cholinesterases (AChE and BuChE) to evaluate their inhibition potential. All
these compounds proved to be cholinesterase inhibitors to a variable extent. Compound 3b was
established as the most potent and selective inhibitor of AChE (IC50 ± SEM = 8.92 ± 1.03 μM)
while 3e showed maximum inhibition (IC50 = 6.16 ± 0.98 μM) with highest SI value (5.568)
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against BuChE. Furthermore, in silico studies confirmed the results obtained from in vitro
experiments by explaining molecular interaction that may provide an insight into the enzyme–
inhibitor binding interaction.
Acknowledgements
J. Iqbal is thankful to the Organization for the Prohibition of Chemical Weapons (OPCW), The
Hague, The Netherlands and Higher Education Commission of Pakistan for the financial support
through Project No. 20–3733/NRPU/R&D/ 14/520 ."
Conflict of interest
The authors declare no competing financial interests.
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