Design, synthesis, and in vitro evaluation of novel 1,3,4-oxadiazolecarbamothioate derivatives of Rivastigmine as selective inhibitors of BuChE

Article abstract of DOI:10.1007/s00044-019-02475-6

Rivastigmine has been prescribed for the therapy of Alzheimer’s disease (AD) symptoms. This drug is classified in the carbamate derivative group that has dual activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). According to the structure of Rivastigmine and its performance, a new series of 5-aryl-1,3,4-oxadiazole-2-carbamothioate compounds I–XI was synthesized using structure-based drug discovery approaches. For this purpose a set of these compounds were designed with computational docking method and their interactions with amino acid residues in the active sites of AChE and BuChE checked out. The structures of synthesized compounds were established by physicochemical and spectroscopic methods. The carbamoyl moiety of Rivastigmine structure was modified to carbamothioate and the effects of 1,3,4-oxadiazole heterocycle as a pharmacophoric nucleus were investigated. The potential of the synthesized compounds I–XI was evaluated against two most known agents of AD (AChE and BuChE) to determine their IC₅₀ values. The results of the docking showed the range of binding affinity for the best poses of ten individual conformers for any compounds (I–XI) was between ?7.81 (VI) and ?6.75 (II) kcal/mol. The results of biological experiments displayed that most synthetic compounds (I–VIII) showed moderate to excellent selective activity range against BuChE (0.51–69.44 μM). In vitro cytotoxicity evaluation of these compounds (I–XI) by MTT assay on human dermal fibroblast (HDF) cell line exhibited no activity against HDF. The compound VI [S-(5-(p-tolyl)-1,3,4-oxadiazol-2-yl) ethyl(methyl)carbamothioate] showed the most stable binding affinity (?7.81 kcal/mol) and the lowest IC₅₀ value (0.51 μM) in comparison with Rivastigmine with 7.72 μM and Donepezil with 5.20 μM against BuChE.

Full text of DOI:10.1007/s00044-019-02475-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02475-6
ORIGINAL RESEARCH
Design, synthesis, and in vitro evaluation of novel 1,3,4-
oxadiazolecarbamothioate derivatives of Rivastigmine as selective
inhibitors of BuChE
Akram Fallah1 Farajollah Mohanazadeh Maliheh Safavi2
1 ●
●
Received: 14 August 2019 / Accepted: 8 November 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Rivastigmine has been prescribed for the therapy of Alzheimer’s disease (AD) symptoms. This drug is classified in the
carbamate derivative group that has dual activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).
According to the structure of Rivastigmine and its performance, a new series of 5-aryl-1,3,4-oxadiazole-2-carbamothioate
compounds I–XI was synthesized using structure-based drug discovery approaches. For this purpose a set of these
compounds were designed with computational docking method and their interactions with amino acid residues in the active
sites of AChE and BuChE checked out. The structures of synthesized compounds were established by physicochemical and
spectroscopic methods. The carbamoyl moiety of Rivastigmine structure was modified to carbamothioate and the effects of
1
,3,4-oxadiazole heterocycle as a pharmacophoric nucleus were investigated. The potential of the synthesized compounds
I–XI was evaluated against two most known agents of AD (AChE and BuChE) to determine their IC50 values. The results of
the docking showed the range of binding affinity for the best poses of ten individual conformers for any compounds (I–XI)
was between −7.81 (VI) and −6.75 (II) kcal/mol. The results of biological experiments displayed that most synthetic
compounds (I–VIII) showed moderate to excellent selective activity range against BuChE (0.51–69.44 µM). In vitro
cytotoxicity evaluation of these compounds (I–XI) by MTT assay on human dermal fibroblast (HDF) cell line exhibited no
activity against HDF. The compound VI [S-(5-(p-tolyl)-1,3,4-oxadiazol-2-yl) ethyl(methyl)carbamothioate] showed the
most stable binding affinity (−7.81 kcal/mol) and the lowest IC50 value (0.51 µM) in comparison with Rivastigmine with
7
.72 µM and Donepezil with 5.20 µM against BuChE.
●
●
●
●
●
Keywords 1,3,4-oxadiazole Carbamothioate Rivastigmine Acetylcholinesterase Butyrylcholinesterase Selective
inhibitor
Introduction
cholinergic neurons and continued with irreversible deficit
of acetylcholine (Faraji et al. 2019; Zengin et al. 2019;
Najafi et al. 2016).
Alzheimer disease (AD) is a neural disturbance that relates
to aging and pace of life (Yazdani et al. 2019). AD is
characterized by symptoms like as continuous memory loss,
deterioration in language skills and other cognitive incom-
petency. AD is caused by progressive deficiency of
Scientists have provided several solutions to develop
therapeutic approaches and made many efforts to obtain
remedy for alleviating the symptoms of AD in the patients.
Due to the fact that the growth of AD is continual and
ascendant, until now the most preferential remedy for
therapeutic treatment of AD is taking medicine. However
diversity of prescribed cholinergic drugs are limited to some
acetylcholinesterase (AChE) and butyrylcholinesterase
*
Maliheh Safavi
m.safavi@irost.ir
(BuChE) inhibitors like as Rivastigmine, Donepezil and
1
2
Galantamine. These enzymes are responsible for hydrolysis
of acetylcholine in the brain with the prominent role in the
AD (Turkan et al. 2019; Zhang et al. 2019; Hariri et al.
2016).
Department of Chemical Technologies, Iranian Research
Organization for Science and Technology (IROST), Tehran, Iran
Department of Biotechnology, Iranian Research Organization for
Science and Technology (IROST), Tehran, Iran

Medicinal Chemistry Research
Donepezil and Galantamine are reversible and selective
drugs to inhibit AChE functionality. However, Rivas-
tigmine binds covalently to the active site of the receptors,
hence is known as a pseudo-irreversible drug, because of its
slow release of the substrates. Rivastigmine also has dual
inhibitory activity against both AChE and BuChE (Koola
et al. 2019; San Juan et al. 2016).
Rivastigmine is the only prescribed carbamate drug for
treatment the symptoms of AD in the patients. The other
significant role of carbamate group in the other pharma-
ceutical functions are documented as its chemical stability,
capability to increase permeability across cellular mem-
brane and taking part in serine hydrolase inhibitors for
asthma treatment as well as in pesticides in order to control
pest (Danish et al. 2019; Roy et al. 2019).
The resemblance of amino acid sequence between human
AChE and BuChE is about 65%. The main differences
between AChE and BuChE are resulted from their surface
specificity and gorge sensitivity as well as their volume of
the gorge to entrance inhibitors. According to X-ray spec-
troscopic analysis, the mouth of the BuChE gorge is clearly
wider than AChE, in addition the amino acid residues in the
peripheral site of AChE and BuChE are partially different.
Three amino acids containing aromatic moiety are absent in
peripheral site of BuChE which results in changes in
interactions between ligand and substrate (Shamsimey-
mandi et al. 2019; Bajda et al. 2018).
The importance of BuChE in the normal conditions of
the healthy brain activity is not considered, while the con-
centration of BuChE increases along with AD progression
in the advance stages of the disease. Surprisingly in these
conditions the amount of AChE starts to go down
several preferential properties like as electron-rich and
̈
thermally stable entity (Rohand et al. 2019; Bostrom et al.
2012). They are neutral hetero aromatic molecules, while
indicate maximum partial positive charge in the position 2.
1,3,4-oxadiazoles have more gentle lipophilicity than the
other regio-isomers, therefore they have been used as an
essential part of the pharmacophores. Indeed 1,3,4-oxadia-
zoles have drew attention in medicinal chemistry as isosters
of carboxylic acids, esters, and carboxamides. They display
a wide range of pharmaceutical and biological potency such
as antiviral, antimicrobial, anticancer, and antihypertensive
(Yan et al. 2019; Karabanovich et al. 2016). Currently there
are several 1,3,4-oxadiazole compounds with pharmaceu-
tical potential including Zibotentan with anticancer sus-
ceptibility and Raltegravir as an antiretroviral agent which
are in the late stage of clinical trials as well as launched onto
the market place; respectively (Sekhar et al. 2019; Gudi
et al. 2018).
Our literature survey ascertained that structural mod-
ifications of the 1,3,4-oxadiazoles can lead to quantitative
and qualitative changes in their biological activities. It was
documented that 1,3,4-oxadiazole derivatives containing
different substitution groups in the position 2 and 5 of the
ring were required for different biological and pharmaceu-
tical activities.
As demonstrated in Fig. 1, in the present study a set of
carbamothioate derivatives of Rivastigmine as isosteric
analogs of carbamate moiety was designed. A 1,3,4-oxa-
diazole heterocycle with three electronegative atoms was
introduced to improve the physicochemical properties and
hydrogen binding interactions of these compounds.
(
Makhaeva et al. 2018; Shi et al. 2018).
There is less structural information about interactions
Materials and methods
between BuChE as receptor and drugs as ligands. During
the progress of time, many causes like as ineffectiveness of
drugs, complication of disease progression, severity of
therapy, and detecting more valid causes of disease per-
suade scientists to synthesize and evaluate new compounds
in order to alleviate the symptoms of the disease (Nunes
et al. 2019; Mohammadi-Khanaposhtani et al. 2015).
Oxadiazoles among various heterocyclic compounds
have gained so much attention in medicinal chemistry.
These compounds increase bioactivity by taking part in
hydrogen bond interactions with proteins due to their three
electronegative atoms in the ring and their flat and aromatic
structures make them precious nucleus in pharmaceutical
approaches. Oxadiazoles can work as proper linker to place
various substitutions in appropriate positions (Bingul et al.
General
All chemical materials were purchased from Merck and
Sigma-Aldrich Companies and are used without further
purification. The cell-culture medium (RPMI 1640), fetal
bovine serum (FBS), streptomycin, and penicillin were
2
019; Simurova and Maiboroda 2019; Shingare et al. 2018).
There are structurally four oxadiazole regio-isomers;
,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, and
,2,5-oxadiazole. Among them 1,3,4-oxadiazoles have
1
1
Fig. 1 Structural schematic presentation of designed Rivastigmine
derivatives as selected cholinesterase inhibitors

Medicinal Chemistry Research
obtained from Gibco BRL (life technologies, Paisley,
Schotland). Melting points were determined in an open end
capillary tubes using Thermo Electrothermal lA9200 and
were uncorrected. Infrared (IR) spectra were recorded as
KBr disks on PU 9624 FTIR spectrometer. Spectro-
photometric measurements were performed in the range of
detected by exposure to UV-lamp at λ 254 nm for few
seconds. Solvents were evaporated with a BUCHI rotary
evaporator R-114 evaporator. Yields in the context refer to
isolated yields of compounds after recrystallization and
drying.
2
00–400 nm on a JASCO V-570 UV/VIS/NIR Spectro-
Synthesis steps
1
13
photometer. H and C NMR spectra were recorded on
00 MHZ and 75 MHZ BRUKER DRX-300 spectrometers,
3
Synthesis of desired compounds I–XI was carried out as
depicted in Scheme 1. This study was undertaken to synthe-
size the 5-aryl-1,3,4-oxadiazole-2-carbamothioate derivatives
I–XI, according to Scheme 1 in order to convert aromatic
carboxylic acids (1) to their ester forms (2) by esterification
procedure, we used sulfuric acid as a dehydrating agent in
methanol and followed by reacting with hydrazine hydrate.
Resulting appropriate acid hydrazide (3) in turn reacted with
respectively. Chemical shifts are accounted in parts per
million (δ) relative to the internal standard of tetra-
methylsilane. Data are given in the following order: δ value,
multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; br,
broad), number and kind of protons. The mass spectra
(
HRMS) of the compounds were recorded on an Agilent
Technology (HP) spectrophotometer model 5973 Network
Mass Selective Detector with Quadropole Analyzer by
Electrospray Ionization technique with Electron Impact of
CS in the presence of KOH as a basic reagent in refluxing
2
EtOH. The reaction was essentially completed within 4–5 h.
The aqueous solution of the crude 1,3,4-oxadiazoles as the
solution of its potassium salt (4) was acidified by using HCl
(0.1 M) to give the 1,3,4-oxadiazole-2-thiol derivative (5)
which could be isolated and used in the next step (Nayak and
Poojary 2019; Li et al. 2015). The 1,3,4-oxadiazole-5-aryl-2-
thiol derivative (5) was appropriately condensed with N-ethyl-
N-methylcarbamoylchloride (6) during reflux with DMF, in
7
0 eV. The elemental analysis (C, H, N) was performed on a
LECO FP 528 CHN analyzer using sulfanilic acid as a
standard, and all the values were within 0.4% of the theo-
retical compositions. Follow up of the reactions and
checking the homogeneity of the compounds were made by
Thin-layer chromatography (TLC) on silica gel-protected
aluminum sheets (Type 60 F254, Merck) and the spots were
Scheme 1 Synthetic route of the
synthesized compounds I–XI
2
2
2
2
2
4
2
2
2

Medicinal Chemistry Research
the presence of CH ONa as a basic reagent. The reaction was
TLC, the mixture triturated with water, resulting precipita-
tion filtrated off and washed with water.
Eventually the residue was recrystallized from EtOH and
3
generally completed within 4–5 h to give the corresponding 5-
aryl-1,3,4-oxadiazole-2-carbamothioate derivative I–XI. This
reaction not only preserved simple, but also consistently gave
the corresponding products in moderate to good yields.
identified by the results of spectroscopic data, FTIR, λ
max
1
13
(UV–vis), and log ε, H and C NMR, mass spectra, and
elemental analysis as well as its melting point was
measured.
Synthesis of esters (2) from aromatic carboxylic acids (1)
The mixture of corresponding carboxylic acid (2 mmol),
methanol (15 ml) in the presence of concentration sulfuric
acid (2 mmol) were refluxed until monitoring by TLC
indicated the end of the reaction. The excess of alcohol was
distilled off under reduced pressure. The reaction mixture is
solidified by cooling to room temperature. After filtering off
the crystal and washing with distilled water to remove the
sulfuric acid, the residue was recrystallized from ethanol
and distilled water.
Experimental data
S-(5-phenyl-1,3,4-oxadiazol-2-yl)
ethyl(methyl)carba-
mothioate (I) White powder (recrystallized from EtOH
and H O), Yield: 80%; MW 263.32 g/mol; m.p. 98–101 °C;
2
−
1
FT-IR (KBr) υ cm : 1279 (C–N), 1505 (C=N), 1623
(C=C), 1687 (C=O), 2970 (CH ), 3030 (C–H aromatic);
3
1
UV (Octanole) λmax (log ε) 205 (5.32) 275 (6.50) nm; H
NMR (300 MHz, CDCl ) δ [ppm] 1.24 (3H, dt, J = 0.023,
3
Synthesis of acid hydrazide derivatives (3) from esters (2)
One millimole of the corresponding ester was added in
small portion to a round bottom flask containing solution of
hydrazine hydrate (10 ml) and followed by stirring the
mixture under reflux conditions. When completion of the
reaction was monitored by TLC, the media was poured onto
ice bath and the resulting precipitation was isolated by fil-
tration. The corresponding acid hydrazide was afforded and
recrystallized from ethanol and water.
0.144; N–CH –CH ), 3.05 (3H, d, J = 0.063, CH –N), 3.43
2
3
3
13
(2H, m, N–CH –CH ), 7.53 (3H, ArH), 8.08 (2H, ArH);
C
2
3
NMR (75 MHz, DMSO) δ [ppm] 11.92, 12.47 (N–CH2–
CH ); 34.46, 34.63 (CH –N); 44.11, 44.51 (N–CH –CH );
3
3
2
3
117.24, 121.52, 125.34, 130.17 (phenyl); 159.29, 159.63
(C=O); 167.52 (HET-C-phenyl); 168.01 (HET-C-S);
C H N O S MS (ESI+) calculated: 263.32, found: m/e
1
2
13
3
2
+
+
234.00 [M-29] (100.0%), 263.00 [M] ; Anal. found for
C H N O S: C, 54.7034%, N, 15.7981%, H, 4.8784%.
1
2
13
3
2
Synthesis of 5-aryl-1,3,4-oxadiazole-2-thiol derivatives (5)
Acid hydrazid (1 mmol) in EtOH (10 ml) was added to a
flask containing KOH (1 mmol, dissolved in at least water),
after dissolving occurred, CS2 (1.5 mmol) was added
dropwise. The reaction mixture was stirred and heated to
reflux for 4–5 h, until most of the hydrogen sulfide evolved.
Completion of the reaction was monitored by TLC until the
starting materials were not detected.
S-(5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl) ethyl(methyl)
carbamothioate (II) White powder (recrystallized from
EtOH and H O), Yield: 81%; MW 279.31 g/mol; m.p.
2
−
1
109–111 °C; FT-IR (KBr) υ cm : 1275 (C–N), 1507
(C=N), 1614 (C=C), 2981 (CH ), 1685 (C=O), 3047 (C–H
3
aromatic), 3600 (O–H aromatic); UV (Octanole) λ (log ε)
max
1
206 (6.00) 263.5 (6.40) 313 (5.59) nm; H NMR (300 MHz,
CDCl ) δ [ppm] 1.26 (3H, dt, J = 0.023, 0.145; N–CH –
3
2
The mixture was cooled to room temperature and excess
solvent was removed under reduced pressure by rotary
evaporator to give the crude product as potassium salt.
Fifteen milliliters of water was added to flask and then
acidified by using HCl (0.1 M) dropwise.
CH ), 3.06 (3H, d, J = 0.061, CH –N), 3.45 (2H, m, N–
3
3
CH –CH ), 6.97–7.81 (4H, ArH, separately), 9.99 (1H, s,
2
3
1
3
OH); C NMR (75 MHz, DMSO) δ [ppm] 12.00, 12.79
(N–CH –CH ); 34.29, 34.46 (CH –N); 44.39, 44.50 (N–
2
3
3
CH –CH ); 109.29, 117.21, 119.68, 129.02, 133.88, 156.42
2
3
Resultant solid was isolated by filtration, washed with
water, and then recrystallized from EtOH and water to
afford respective 5-aryl-1,3,4-oxadiazole-2-thiol.
(phenyl); 156.80 (HET-C-phenyl); 159.16, 159.60 (C=O);
167.00 (HET-C-S); C H N O S MS (ESI+) calculated:
279.31, found: m/e 250.00 [M-29] (100.0%), 279.00 [M] ;
1
2
13
3
3
+
+
Anal found for C H N O S: C, 54.4902 %, N, 15.4982%,
1
2
13
3
3
Synthesis of 5-aryl-1,3,4-oxadiazole-2-carbamothioate I-
H, 4.8845%.
XI To
a
solution of 5-aryl-1,3,4-oxadiazole-2-thiol
(
(
1 mmol) in DMF (10 ml) was slowly added CH ONa
1 mmol, 0.054 g) at room temperature. When the addition
S-(5-(3-hydroxyphenyl)-1,3,4-oxadiazol-2-yl) ethyl(methyl)
carbamothioate (III) White powder (recrystallized from
3
was completed, the reaction mixture was cooled to
0–12 °C and N-ethyl-N-methylcarbamoylchloride
1.5 mmol) was added dropwise to the stirring solution
EtOH and H O), Yield: 65%; MW 279.31 g/mol; m.p.
105–107 °C; FT-IR (KBr) υ cm : 1288 (C–N), 1513
2
−
1
1
(
(C=N), 1628 (C=C), 1688 (C=O), 2965 (CH ), 3043 (C–H
3
and then was stirred and heated to reflux for the appropriate
time (4–5 h). After completion of the reaction monitored by
aromatic), 3600 (O–H aromatic); UV (Octanole) λ (log ε)
max
1
204.5 (5.17) 267.5 (6.18); H NMR (500 MHz, CDCl ) δ
3

Medicinal Chemistry Research
−
1
[
ppm] 1.16 (3H, dt, J = 0.023, 0.145; N–CH –CH ), 3.07
FT-IR (KBr) υ cm : 1269 (C–N), 1487 (C=N), 1618
2
3
(
3H, d, J = 0.063, CH –N), 3.35 (2H, m, N–CH –CH ),
(C=C), 1685 (C=O), 2967 (CH ), 3023 (C–H aromatic);
3
2
3
3
6
.63 (1H, ArH), 6.95 (1H, ArH), 7.12 (2H, ArH), 10.06
UV (Octanole) λ
H NMR (300 MHz, CDCl ) δ [ppm] 1.26 (3H, dt, J =
0.021, 0.145; N–CH –CH ), 2.43 (3H, s, ArCH ), 3.05
(log ε) 206.5 (5.70) 269.5 (6.00) nm;
max
1
3
1
(
1H, s, OH); C NMR (75 MHz, DMSO) δ [ppm] 12.02,
2.80 (N–CH –CH ); 34.30, 34.49 (CH –N); 44.23, 44.98
3
1
2
3
3
2
3
3
(
1
N–CH –CH ); 112.95, 117.38, 119.74, 123.81, 130.79,
57.32 (phenyl); 158.03 (HET-C-phenyl); 159.16, 159.61
(3H, d, J = 0.061, CH –N), 3.45 (2H, m, N–CH –CH ),
7.30 (2H, ArH), 7.98 (2H, ArH); C NMR (75 MHz,
2
3
3 2 3
1
3
(
C = O); 167.53 (HET-C-S); C H N O S MS (ESI+)
DMSO) δ [ppm] 11.97, 12.76 (N–CH –CH ); 21.05
(CH –aryl); 34.52, 34.43 (CH –N); 44.46, 44.91 (N–
3 3
CH –CH ); 120.03, 126.56, 129.98, 142.76 (phenyl);
2 3
12
13
3
3
2
3
+
calculated: 279.31, found: m/e 250.00 [M-29] (100.0%),
2
+
79.00 [M] ; Anal found for C H N O S: C, 51.4871%,
12
13
3
3
N, 15.1634%, H, 4.8213%.
159.13, 159.58 (C=O); 167.03 (HET-C-phenyl); 167.54
HET-C-S); C H N O S MS (ESI+) calculated: 277.34,
found: m/e 248.00 [M-29] (100.0%), 277.00 [M] ; Anal
(
1
3
15
3
2
+
+
S-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-yl) ethyl(methyl)
carbamothioate (IV) White powder (recrystallized from
found for C H N O S: C, 56.2034%, N, 15.0106%, H,
1
3
15
3
2
EtOH and H O), Yield: 85%; MW 279.31 g/mol; m.p.
5.6042%.
2
−
1
1
13–115 °C; FT-IR (KBr) υ cm : 1275 (C–N), 1521
(
C=N), 1613 (C=C), 1680 (C=O), 2967 (CH ), 3035 (C–H
S-(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl) ethyl(methyl)
3
aromatic), 3600 (O–H aromatic); UV (Octanole) λ (log ε)
carbamothioate (VII) Pale yellow powder (recrystallized
max
1
2
05 (6.00) 287 (6.18) nm; H NMR (500 MHz, DMSO d ) δ
from EtOH and H O), Yield: 80%; MW 293.34 g/mol; m.p.
6
2
−
1
[
ppm] 1.17 (3H, dt, J = 0.022, 0.146; N–CH –CH ), 2.87
98–101 °C; FT-IR (KBr) υ cm : 1295 (C–N), 1512
2
3
(
6
3H, d, J = 0.062, CH –N), 3.35 (2H, m, N–CH –CH ),
(C=N), 1628 (C=C), 1678 (C=O), 2870 (CH -O), 2970
3
2
3
3
3
1
.81 (2H, ArH), 7.57 (2H, ArH), 10.40 (1H, s, OH);
C
(CH ), 3030 (C-H aromatic); UV (Octanole) λ
206 (5.60) 274 (6.10) nm; H NMR (500 MHz, DMSO d ) δ
(log ε)
3
max
1
NMR (75 MHz, DMSO) δ [ppm] 12.03, 12.80 (N-CH2-
CH ); 34.32, 34.50 (CH –N); 44.35, 44.89 (N–CH –CH );
6
[ppm] 1.14 (3H, dt, J = 0.024, 0.146; N–CH –CH ), 3.09
3
3
2
3
2
3
1
(
33.21, 135.15, 145.62, 162.71 (phenyl); 159.15, 159.48
C=O); 161.02 (HET-C-phenyl); 167.35 (HET-C-S);
C H N O S MS (ESI+) calculated: 279.31, found: m/e
(3H, s, CH O), 3.30 (2H, m, N–CH –CH ), 3.73 (3H, d, J
3 2 3
13
= 0.061, CH –N), 7.02 (2H, ArH), 7.10 (2H, ArH);
C
3
NMR (75 MHz, DMSO) δ [ppm] 12.01, 12.81 (N–CH2–
CH ); 21.63 (CH O); 34.39, 34.57 (CH –N); 44.61, 44.76
1
2
13
3
3
+
+
2
50.00 [M-29] (100.0%), 279.00 [M] ; Anal found for
3
3
3
C H N O S: C, 51.5012%, N, 14.9241%, H, 4.7767%.
(N-CH -CH ); 121.10, 126.73, 129.72, 142.13 (phenyl);
1
2
13
3
3
2
3
159.21, 159.53 (C=O); 160.71 (HET-C-phenyl); 167.41
S-(5-(2,4-dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)
methyl)carbamothioate (V) White powder (recrystallized
from EtOH and H O), Yield: 85%; MW 295.31 g/mol; m.p.
ethyl
(HET-C-S); C H N O S MS (ESI+) calculated: 293.34,
found: m/e 264.00 [M-29] (100.0%), 293.00 [M] ; Anal
1
3
15
3
2
+
+
(
found for C H N O S: C, 53.3105%, N, 14.3101%, H,
2
13 15
3
2
−
1
1
12–114 °C; FT-IR (KBr) υ cm : 1283 (C–N), 1513
5.1721%.
(
C=N), 1617 (C=C), 1685 (C=O), 2975 (CH ), 3030 (C–H
3
aromatic), 3600 (O–H aromatic); UV (Octanole) λ (log ε)
S-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)
carbamothioate (VIII) White powder (recrystallized from
ethyl(methyl)
max
1
2
08 (5.68) 281.5 (6.18) 312 (5.33) nm; H NMR (500 MHz,
CDCl ) δ [ppm] 1.30 (3H, dt, J = 0.023, 0.146; N-CH -
EtOH and H O), Yield: 67%; MW 297.76 g/mol; m.p.
97–101 °C; FT-IR (KBr) υ cm : 725 (C–Cl), 1269 (C–N),
3
2
2
−
1
CH ), 3.51 (2H, m, N–CH –CH ), 3.10 (3H, d, J = 0.063,
3
2
3
CH -N), 6.30 (1H, m, ArH), 6.39 (1H, d, ArH), 7.07 (1H,
1509 (C=N), 1617 (C=C), 1679 (C=O), 2985 (CH ), 3035
3
3
1
3
expand, OH), 7.36 (1H, d, ArH), 9.86 (1H, s, OH);
C
(C–H aromatic); UV (Octanole) λ
274 (6.18) nm; H NMR (500 MHz, DMSO d ) δ [ppm]
(log ε) 203.5 (6.00)
max
1
NMR (75 MHz, DMSO) δ [ppm] 12.05, 12.83 (N–CH2–
CH ); 34.31, 34.47 (CH –N); 44.49, 44.91 (N–CH –CH );
6
1.13 (3H, dt, J = 0.023, 0.146; N–CH –CH ), 3.05 (3H, d,
3
3
2
3
2
3
1
1
00.68, 103.17, 108.47, 130.17, 155.61, 158.26 (phenyl);
59.35, 159.79 (C=O); 162.56 (HET-C-phenyl); 167.3
J = 0.065, CH –N), 3.33 (2H, m, N–CH –CH ), 7.02 (2H,
3 2 3
1
3
ArH), 7.15 (2H, ArH); C NMR (75 MHz, DMSO) δ
[ppm] 12.03, 12.79 (N–CH –CH ); 34.41, 34.59 (CH –N);
(
HET-C-S); C H N O S MS (ESI+) calculated: 295.31,
12 13 3 3
2
3
3
+
+
found: m/e 266.00 [M-29] (100.0%), 295.00 [M] ; Anal
44.73, 44.81 (N–CH –CH ); 128.11, 129.41, 131.94,
2 3
found for C H N O S: C, 48.7614%, N, 14.0368%, H,
137.25 (phenyl); 159.16, 159.47 (C=O); 160.72 (HET-C-
phenyl); 167.39 (HET-C-S); C H ClN O S MS (ESI+)
1
2
13
3
3
4
.5436%.
1
2
12
3
2
+
calculated: 297.76, found: m/e 234.00 [M-63] , 269.00 [M-
+
+
+
S-(5-(p-tolyl)-1,3,4-oxadiazol-2-yl)
ethyl(methyl)carba-
29] (100.0%), 297.00 [M] , 299.00 [M + 2] ; Anal found
mothioate (VI) White powder (recrystallized from EtOH
for C H ClN O S: C, 48.3503%, N, 14.0003%, H,
1
2
12
3
2
and H O), Yield: 80%; MW 277.34 g/mol; m.p. 98–101 °C;
4.1765%.
2

Medicinal Chemistry Research
(100.0%), 277.00 [M]+ Anal found for C H N O S: C,
;
S-(5-(3-chlorophenyl)-1,3,4-oxadiazol-2-yl)
ethyl(methyl)
13 15 3 2
carbamothioate (IX) White powder (recrystallized from
59.8308%, N, 15.9801%, H, 5.6706%.
EtOH and H O), Yield: 52%; MW 297.76 g/mol; m.p.
2
−
1
8
8–90 °C; FT-IR (KBr) υ cm : 748 (C–Cl), 1281 (C–N),
Biological and pharmacological methods
1
517 (C=N), 1632 (C=C), 1659 (C=O), 2983 (CH ), 3017
3
(
(
C–H aromatic); UV (Octanole) λmax (log ε) 203 (5.30) 263
General approaches
1
6.17) nm; H NMR (500 MHz, DMSO d ) δ [ppm] 1.15
6
(
0
3H, dt, J = 0.024, 0.145; N–CH –CH ), 3.01 (3H, d, J =
Literature survey revealed that minor modification in the
substitution groups of 1,3,4-oxadiazole ring can lead to
significant changes in their biological activities. Therefore
we were interested in the synthesizing a set of novel car-
bamothioate Rivastigmine derivatives containing 1,3,4-
oxadiazole ring. The biological activities as their half
2
3
.063, CH -N), 3.38 (2H, m, N–CH –CH ), 7.15–7.19 (4H,
3 2 3
1
3
ArH); C NMR (75 MHz, DMSO) δ [ppm] 12.05, 12.81
N–CH –CH ); 34.32, 34.48 (CH -N); 44.27, 44.65 (N–
(
2
3
3
CH –CH ); 115.12, 118.91, 121.32, 127.11, 138.72, 145.91
2
3
(
phenyl); 158.10 (HET-C-phenyl); 159.12, 159.35 (C=O);
1
2
67.32 (HET-C-S); C H ClN O S MS (ESI+) calculated:
maximum inhibitory concentration (IC ) against AChE and
12
12
3
2
50
+
+
97.76, found: m/e 234.00 [M-63] , 269.00 [M-29]
BuChE was evaluated and compared with Donepezil and
Rivastigmine as the reference drugs. General synthetic
routes leading to the novel 5-aryl-1,3,4-oxadiazole-2-car-
bamothioate compounds I–XI are outlined in Scheme 1.
Molecular Docking was prepared on ligand bound
BuChE (PDB code: 2Wjo) and AChE (PDB code: 2Ckm)
structures by using Auto Dock Tools program [Auto-
DockTools (Version 1.5.6 Sep-17-14)]. Molecular docking
was performed with a Lamarckian Genetic Algorithm
(LGA) concomitant with a rectangular box with dimension
of (44.00 Å × 60.30 Å × 67.10 Å) to cover whole of the
active site and gorge of AChE and BuChE. The grid spacing
as determined by the defaulted program remained in
0.375 Å. 150 runs was applied to LGA. The geometry of
enzymes presentation was selected as ribbon and ligands as
sticks and balls. Molecular docking program predicted and
estimated the binding modes between compounds as ligand
and essential amino acid residues into the active site of the
enzyme that is responsible for its activity. Some binding
modes are hydrogen bond interactions, π–S interactions, π–
π interactions and Van der Waals interactions. The design
compounds at first were separately drew, built, minimized
and optimized and finally saved as ligands with PDB format
+
+
(
100.0%), 297.00 [M] , 299.00 [M + 2] ; Anal found for
C H ClN O S: C, 42.1250%, N, 12.0168%, H, 3.4691%.
1
2
12
3
2
S-(5-(4-bromophenyl)-1,3,4-oxadiazol-2-yl)
carbamothioate(X) White powder (recrystallized from
EtOH and H O), Yield: 75%; MW 342.21 g/mol; m.p.
ethyl(methyl)
2
−
1
1
05–107 °C; FT-IR (KBr) υ cm : 718 (C–Br), 1270
(
(
C–N), 1518 (C=N), 1628 (C=C), 1692 (C=O), 2983
CH ), 3030 (C–H aromatic); UV (Octanole) λ
(log ε)
3
max
1
2
03.5 (6.00) 281.5 (5.70) nm; H NMR (500 MHz, DMSO
d ) δ [ppm] 1.19 (3H, dt, J = 0.023, 0.146; N–CH –CH ),
3
CH –CH ), 7.06 (2H, ArH), 7.39 (2H, ArH); C NMR
6
2
3
.03 (3H, d, J = 0.064, CH –N), 3.39 (2H, m, N–
3
13
2
3
(
75 MHz, DMSO) δ [ppm] 12.03, 12.81 (N–CH –CH );
2 3
3
1
4.42, 34.59 (CH –N); 44.62, 44.79 (N–CH –CH );
3 2 3
24.23, 125.11, 129.91, 132.23 (phenyl); 159.16, 159.51
(
C=O); 159.87 (HET-C-phenyl); 167.43 (HET-C-S);
C H BrN O S MS (ESI+) calculated: 342.21, found: m/e
1
2
12
3
2
+
+
+
3
13.00 [M-29] (100.0%), 342.00 [M] , 344.00 [M + 2] ;
Anal found for C H BrN O S: C, 42.1004%, N,
1
12
12
3
2
2.0317%, H, 3.7421%.
TM
S-(5-benzyl-1,3,4-oxadiazol-2-yl) ethyl(methyl)carbamothio-
by HyperChem program (Release 8.0.10 for Windows,
ate (XI) White powder (recrystallized from EtOH and
Molecular Modeling System). In the following, these
ligands were docked to BuChE and AChE to bind with their
prepared crystal structure by using Auto Dock Tools
respectively. This program generates several conformers of
any compounds and export data as binding affinity (kcal/
mol) as well as some interactions like as van der waals and
hydrogen bonds with amino acid residues into the active site
of the AChE and BuChE gorges. The obtained conformers
were loaded to Molegro Virtual Docker (MVD 2010.4.2.0-
2010-10-04-[WIN32] to prepare a complex of the best
respective conformer and enzymes with PDB format.
Finally the complex with PDB format was loaded in Lig-
Plus (LigPlot+v.1.4.5) to visualize the interaction manners
with BuChE and AChE.
H O), Yield: 52%; MW 277.34 g/mol; m.p. 76–77 °C; FT-
2
−
1
IR (KBr) υ cm : 1269 (C–N), 1685 (C=O), 1515 (C=N),
1
613 (C=C), 2950 (CH ), 3027 (C–H aromatic); UV
3
1
(
Octanole) λmax (log ε) 205 (5.32) 275 (6.38) nm; H NMR
(
500 MHz, DMSO d ) δ [ppm] 1.90 (3H, dt, J = 0.023,
6
0
.147; N–CH –CH ), 3.33 (2H, m, N–CH –CH ), 3.57 (3H,
2 3 2 3
d, J = 0.063, CH –N), 4.18 (2H, s, CH (benzylic)),
3
2
13
7
1
4
1
1
.33–7.38 (5H, ArH); C NMR (75 MHz, DMSO) δ [ppm]
1.71, 12.54 (N–CH –CH ); 34.46, 34.65 (CH –N); 44.21,
2
3
3
4.67 (N–CH –CH ); 62.71 (CH benzylic); 123.37,
2
3
2
29.17, 131.42, 133.05 (phenyl); 159.37, 159.71 (C=O);
66.91 (HET-C-phenyl); 167.54 (HET-C-S); C H N O S
13
15
3
2
+
MS (ESI+) calculated: 277.34, found: m/e 248.00 [M-29]

Medicinal Chemistry Research
Physicochemical study
In vitro cytotoxicity evaluation by MTT assay
Lipinski’s rule of five can predict that a chemical com-
pound with certain pharmacological or biological activ-
ities and molecular properties could be a candidate for
orally active drug in human. The rule was formulated
based on the observations that most orally administered
drugs are relatively small (MW < 500 daltons) and
moderately lipophilic molecules (Log P ≤ 5) as well as
have maximum five hydrogen bond donors and ten
hydrogen bond acceptors (Ghobadian et al., 2019),
MTT assay is one of the cell- based assay. Living cells with
metabolism ability reduce the tetrazolium dye in MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
mide) into purple colored formazan.
bro-
The in vitro cytotoxicity effects of our synthesized
compounds (I–XI) were assessed by MTT colorimetric
assay and HDF. After trypsinization the culture in the
6
exponential growth phase, the 95 µL cells (1 × 10 cells/ml)
were seeded into the 96-well plates (Nunc, Denmark). The
(
Mahajan et al., 2019). Lipophilicity of synthesized
plates were incubated overnight in a humidified air atmo-
compounds is calculated by using computational meth-
ods. Among available computational methods for deter-
mining lipophilicity, ChemDraw computer program is
one of the best to calculate logP of synthesized com-
pounds because according to some articles, there has
been an appropriate correlation between their calculated
and experimental Log P results (Morán-Díaz et al. 2019;
Bajda et al. 2018). These data are collected and presented
in Table 1.
sphere at 37 °C with 5% CO . After plating 5 µl of each
2
diluted compound was added per well in triplicate. Three
control wells (the same number of cells without any test
compounds) and three blank wells (the same number of
cells containing 0.5% DMSO as solvent) were applied for
checking out the cell viability. Synthetic compounds were
prepared in DMSO and it is necessary in all analysis to
reach a concentration not higher than 0.5%. After 48 h
treatment, the medium was removed and phenol red-free
medium (200 µl) containing MTT (1 mg/ml) was added to
each well which followed by 4 h incubation. In the fol-
lowing, culture medium was replaced with 100 µl of DMSO
and the absorbance of each well was measured by using a
microplate reader at 570 nm (Gen5, Epoch, BioTek,
America) (Abolhasani et al. 2018; Pordeli et al. 2017).
Molecular docking
One of the essential tools for drug design and discovery is
Molecular Docking program. It considers amino acid
residues in binding site of target protein. Phe286,
Phe388, Tyr72, Asp74, Trp86, Tyr341, Tyr124, His447,
Tyr337, Glu334, and Ser203 are some amino acids which
are present in the active site of AChE and Ala328, Asn67,
Gln119, Ser198, Glu325, Trp82, Asp70, Tyr332, His438
in the active site of BuChE (Özdemir et al. 2017; Saeed
et al. 2017). In the following molecular Docking program
checks out common interactions like as van der waals,
conventional hydrogen bond, π–π and π–σ interactions
between ligand and these amino acid residues (Yusufzai
et al. 2018; Saeedi et al. 2017). In order to understand and
estimate inhibitory activity of the design compounds I–XI
against AChE and BuChE as receptors, we decided to run
docking program using Auto Dock Tools and sure about
our hypothesis. Hence all designed compounds were
separately docked with the amino acid residues in the
active site of BuChE (PDB code: 2Wjo) and AChE (PDB
code: 2Ckm).
In vitro evaluation of AChE and BuChE- inhibiting activity
Encouraged by the results of molecular docking of the
designed 5-aryl-1,3,4-oxadiazole-2-carbamothioate scaf-
folds I–XI, the evaluation of the inhibitory potency of the
synthesized compounds as Rivastigmine analogs against
acetyl and BuChE was performed. The synthesized com-
pounds I–XI were dissolved in a solution of DMSO (5 ml)
and MeOH (5 ml), and then were added to a buffer mixture
consisting of KH PO /K HPO (pH 8.00). 50 µL potassium
2
4
2
4
phosphate buffer (KH PO /K HPO , 0.1 M, pH 8.00),
2
4
2
4
25 µL prepared compound, 25 µL solution of intended
enzyme (AChE or BuChE) in buffer with concentration of
0.22 U/ml were poured to each well. The plates were pre-
incubated for 15 min at room temperature, and followed by
adding 125 µL of 3 mM solution of DTNB. 25 µL aqueous
solution of acetylcholine (3 mM) was added to per well. The
change in the absorbance was surveyed at 405 nm by
interval of 15 min. After drawing the inhibition curve (log
inhibitor concentration versus percent of inhibition), the
IC50 values were obtained. The blank containing buffer,
water, DTNB and substrate without inhibitor were prepared
as a control experiment under the same condition. This
method was also used for the other enzyme (AChE or
BuChE).
Cell line and cell cultures
The human dermal fibroblast cell line (HDF) was purchased
from National Cell Bank of Iran (NCBI) and cultivated in
RPMI 1640 medium supplemented with 10% heat-
inactivated fetal calf serum. 100 mg/ml streptomycin and
1
00 U/ml penicillin incubated at 73 °C in a humidified
atmosphere with 5% CO in air.
2

Medicinal Chemistry Research
Table 1 Physicochemical properties of the synthesized compounds according to Lipinski’s rule
No.
structure
MW
Hydrogen bond
donor
Hydrogen bond
acceptor
LogPa
I
263.32
279.31
--
1
5
6
2.83
2.44
II
III
279.31
1
6
2.44
IV
V
279.31
295.31
1
2
6
7
2.44
2.05
VI
277.34
293.34
--
--
5
6
3.32
2.70
VII
VIII
297.76
--
5
3.39

Medicinal Chemistry Research
IX
297.76
--
5
3.39
X
342.21
277.34
--
--
5
5
3.66
3.21
XI
Rivastigmine
250.34
377.53
--
--
4
3
2.36
4.01
Donepezil
aLipophilicity calculated by ChemDraw Computer Program
Results and discussion
Chemistry
stretching peak at 725 cm− due to stretching of aromatic C–Cl
1
group while compound IX demonstrated the peak at
748 cm− . Compound X demonstrated the peak at 718 cm
1
−1
because of aromatic C–Br stretching.
On the basis of Rivastigmine structure and its functionality,
some novel 5-aryl-1,3,4-oxadiazole-2-carbamothioate com-
pounds I–XI were designed, synthesized and evaluated against
AChE and BuChE. The structures of compounds I–XI estab-
lished by physicochemical and spectroscopic methods were in
agreement with the assigned molecular structure confirmed by
Our results of H and 13C NMR spectra demonstrated
that there are two kinds of each group of protons as well as
carbon for the carbamothioate moiety in the synthesized
compounds, respectively. This fact states that the molecules
are rigid between the carbamothioate moiety and the rest of
the molecule (Fig. 2).
1
1
13
FTIR, H NMR, C NMR spectra, mass spectral data, and
elemental analysis. Melting point were measured and recorded
as well as λmax and log ε all synthesized compounds were
determined and calculated on a UV spectrophotometer. FTIR
spectra of synthesized compounds I–XI at υ cm−1 showed that
there are, in general for all compounds I–XI, some char-
We try to synthesize some compounds with electron
withdrawing substitution like as nitro on the aryl group
separately. We found the presence of nitro as a strong
electron withdrawing substitution on the aryl group caused
the reaction to stop in the last step, because the negative
charge on the thiolate intermediate was stable by nitro
group and had no tendency to attack positive center at
carbonyl moiety of the N-ethyl-N-methylcarbamoylchloride
reagent (Scheme 2).
−1
acteristic peaks at υ cm 1620, 1505, 2970, 1280, 1685, 3030
due to presence of C=C, C=N, CH , C–N, C=O and aro-
3
matic protons respectively. Compound II, III, IV, and V
−1
showed broad band around 3600 cm due to stretching of
As well as the presence of amino substitution on the aryl
group probably caused forming stable urea derivatives,
therefore a medley products obtained. According to Scheme
aromatic O–H. Compound VII displayed peak at 2870 cm−
1
due to stretching of CH –O. Compound VIII showed
3

Medicinal Chemistry Research
3
the reaction may be encountered with side reaction routes
and conducted to form stable but undesired products such as
2) and (3) besides (1). According to Scheme 3 the optimal
site of the enzymes to afford suitable hydrogen bonds between
oxygen atom of carbamothioate moiety and functional group
of Tyr 121 and 198, Arg 195, His 362 (Fig. 3a, b). The
aromatic and hetero aromatic ring bound to the residues in the
active site to create π–π stacking with Pro 232, Tyr 334, Phe
(
separation of the favorite product (1) from the other side
products (2 and 3) was impossible.
330, Trp 84, Tyr 121 at the peripheral anionic site (PAS).
Physicochemical studies
There is also π–S interaction between Sulphur atom in car-
bamothioate moiety and Phe 330 at peripheral anionic site of
the enzyme.
The optimistic conditions for drug delivery like as the
acceptable speed of drug metabolism in human body that is
directly related to its lipophilicity and hydrophobicity,
encouraged us to introduced an 1,3,4-oxadiazol heterocycle
and aryl group at position 5 of 1,3,4-oxadiazol with dif-
ferent substitutions to moderate final molecules lipophili-
city. The effect of the presence of Sulfur atom in
carbamothioate moiety can also improve lipophilicity.
According to ChemDraw Computer Program, Log P
values for synthesized compounds were in the range of 2.05
It seems that expanding the structure of Rivastigmine with
introducing an 1,3,4-oxadiazole ring and exchanging oxygen
atom in carbamoyl moiety with Sulphur were suitable to locate
conveniently into the gorge of BuChE rather than AChE
because of tightness mouth of the AChE gorge. After placing
and docking the compounds into the active site of the gorge,
presence of hydroxyl group on the aryl moiety also improved
the interactions with amino acid residues.
The docking results showed that localization of the com-
pounds I–XI into the active site of enzymes resulted in several
kinds of interactions between ligands and functional groups of
amino acid residues. These arrangements illustrated good
interactions and high activity of the compounds. The role of
heteroatoms in oxadiazole moiety as well as hydroxyl sub-
stitution on the phenyl ring were vital for hydrogen bond
interactions with functional groups of amino acids like as Tyr
130, Ser 122, Arg 235, Arg 289, Arg 194, Trp 76.
The main scaffold of our novel synthesized compounds
I–XI is almost the same with little differences among sub-
stitutions of their aryl group. Therefore binding affinity
obtained by molecular docking program were relatively close
together for the synthesized compounds I–XI. Their domain is
between –7.81 and –6.75 kcal/mol for VI and II, respectively.
The difference domain among the binding affinities of the
most stable conformers of any of the synthesized compounds
I–XI is equal to –1.6 kcal/mol that is not significant.
(
V)–3.66 (X). It is proved that all synthesized compounds
were agreed with Lipinski’s rule, their molecular weight
≤342.21 g/mol), Hydrogen bond donors and acceptors (0–2
and 3–7 respectively) as well as the strength of lipophilicity
logP) were in acceptable ranges. It could be observed that
(
(
introduction of a heterocyclic ring containing electro-
negative atoms like as nitrogen and oxygen as well as
hydroxyl substitutions on the phenyl ring decreased the
lipophilicity, while existence of sulfur atom in exocyclic
position of the ring at the carbamothioate moiety caused
increasing and balance the total lipophilicity of the syn-
thesized compounds I–XI (Table 1).
Docking study
In the following step we predicted the modification effects of
designed compounds by Molecular Docking program. In Fig.
3
interactions between ligand IV with active site of BuChE
(3A) and AChE (3B) are shown. The results were obtained by
Pharmacology
Auto Dock Tools program. The interactions are shown as
hydrogen bonds, π–π and van der waals interactions (Fig. 3)
and their potential of inhibition was reported as binding affi-
nity (kcal/mol) in Table 2. According to the results, our
compounds located and matched appropriately in the active
In vitro cytotoxicity assay
Cytotoxic effects of all the synthesized compounds I–XI
were assessed on a HDF. The in vitro cytotoxic activity
Fig. 2 Rigidity demonstration of
3
D-structure of compound (VI)

Medicinal Chemistry Research
evaluation of all synthesized compounds with different aryl
group in position 5 of 1,3,4-oxadiazole ring I–XI against
HDF determined by MTT colorimetric assay demonstrated
that none of them showed cytotoxic activity against HDF
till 100 mM applied concentration and hence reported as
safe compounds.
drugs e.g., Rivastigmine and Donepezil as reference drugs
were performed. The IC50 values of all our synthesized
compounds showed that these compounds despite of Riv-
astigmine had selective inhibitory activities and even in high
concentration (>100 µM) were not able to inhibit AChE.
In the experimental condition the size of the designed
compounds can be one cause of appropriate interactions
into the active site of BuChE, because the entrance and
volume of BuChE gorge are accountably larger than AChE,
hence the compounds with more volume can easily adopt to
the active site of BuChE, while the same ligands couldn’t
match and interact with residues in the AChE active site
(Kang et al. 2018; Yusufzai et al. 2018).
In vitro AChE and BuChE inhibitory assay
The potential of inhibitory activities of the synthesized
compounds against AChE from electric eel (AChE, E.C.
3
.1.1.7, Type V-S, lyophilized powder, 1000 unit) and
BuChE from equine serum (BuChE, E.C. 3.1.1.8) was
evaluated by widely used modified Ellman’s method. The
values of the activities were determined based on inhibitory
concentration of synthesized compounds at 50% inhibition
Compound VI with methyl substitution at the para
position of the aryl has the best inhibitory activity (IC =
50
0.51 µM). It seems that the interactions between compound
VI and amino acid residues reached to balance with its size.
This compound located conveniently into the active site of
BuChE. The compounds IX, X had no activity against
BuChE and none of our synthesized compounds I–XI could
inhibit activity of AChE. IC50 values of BuChE inhibition
assay revealed that the change in the substitution of the
synthesized compounds caused changing in their inhibitory
activity.
Differences between IC50 values of the compounds with
hydroxyl substitutions II–V and compound VII with mothoxy
substitution at the para position of the aryl group are due to
some factors like as size and possibility of forming hydrogen
bonds. Hydrogen bond interactions between hydroxyl group
of ligand and amino acid residues are necessary as found in
the compounds II–V. Besides the size of the substitutions are
smaller in the case of the compounds with hydroxyl sub-
stitution II–V. In fact both two factors are in favor of the
compounds with hydroxyl substitution II–V rather than
methoxy substitution VII. It appears that methoxy substitution
is larger than hydroxyl substitution and was not able to par-
ticipate to form hydrogen bond with other amino acid residues
in the active site of BuChE.
(
IC ) for all synthesized compounds I–XI (Table 2). The
50
results of biological activities demonstrated that the com-
pounds I–VIII had selective inhibitory activity against
BuChE in the range of 0.51 µM (VI) to 69.44 µM (VII)
while compounds IX and X had no activity against both
AChE and BuChE. The best inhibition of BuChE activity
(
IC = 0.51 µM) was obtained by compound VI possessing
50
methyl group at para position of the phenyl moiety. Sub-
stitution of different halogens such as chlorine and bromine
showed different inhibition of enzyme activity. Introduction
of chlorine into para position of the aryl moiety (compound
VIII) caused inhibition of BuChE activity (IC = 7.47 µM)
50
while existence of chlorine into meta position (compound
IX) or bromine into para position (compound X) showed no
inhibitory effect on the AChE and BuChE activity.
In this literature comparisons between our synthesized
compounds I–XI and two prescribed Alzheimer disease
2
2
The IC50 values of the other compounds with halogen
substitutions at the different positions of the aryl (VIII–X)
Scheme 2 Stabilization of negative charge on the thiolate by electron
withdrawing group of nitro
Scheme 3 Three possible
products from the reaction
between 5-(4-aminophenyl)-
1
,3,4-oxadiazole-2-thiol and N-
ethyl-N-methylcarbamoyl
chloride

Medicinal Chemistry Research
Fig. 3 Structural- based drug design with molecular docking: sche-
matic representation of the binding mode of compound IV with
BuChE (PDB code: 2Wjo) (a) and compound IV with AChE (PDB
code: 2Ckm) (b); The Tyr 121, Ser 122, Tyr 130, Tyr 198, Trp76, and
Glu 199 can form the crucial hydrogen bond interactions with
hydroxyl group of homo aromatic ring and electronegative atoms in
whole molecules, as well as possible interactions (π–π stacking, van
der waals, π–S and π–Sigma) with catalytic residues are shown
showed that only compound VIII with chlorine substitu-
tion at the para position was able to inhibit BuChE
activity. Bromine with bigger size than chlorine at the
para position (compound X) and chlorine at the meta
position were not able to inhibit BuChE activity.
electronegative rich atoms in 1,3,4-oxadiazole moiety as
well as carbonyl group of carbamothioate moiety with
functional group of amino acid residues; while its IC50
value is higher in comparison with compound VI because
of its unsuitable size.
The importance of balance between the compound’s
size and the tendency to interact with amino acid residues
into the active site of the enzyme is revealed during
There are some problems in prediction absolute bind-
ing affinity and IC50 values between small molecules as
ligands and target proteins as demonstrated in Table 2.
When preparing protein for Docking Tools Program, it is
necessary to remove some ions, co-factors and water
molecules that are concomitant with protein crystal
studying the IC50 value of compound I (IC50
1.40 µM). compound I like as compound VI can parti-
cipate to form hydrogen bond between their
=
1

Medicinal Chemistry Research
Table 2 Biological IC50 results as inhibition effect against BuChE and AChE and theoretical results as binding affinity to BuChE (kcal/mol) of 5-
aryl-1,3,4-oxadiazole-2-cabamothioate
a
b
Entry Product
R
AChE (IC , µM) BuChE (IC , µM) Domain of binding affinity to BuChE (kcal/mol) for ten conformers
50 50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
I
H
>100
>100
>100
>100
11.40
10.70
10.90
13.00
12.00
0.51
−7.22 to −5.53
−6.75 to −4.82
−6.99 to −5.29
−7.08 to −4.77
−7.27 to −5.18
−7.81 to −5.78
−7.74 to −5.71
−7.79 to −5.63
−7.78 to −6.52
−7.32 to −5.77
−7.75 to −6.01
−5.12 to −4.32
−4.44 to −3.29
−6.29 to −4.81
-8.77 to −7.93
II
2-OH
3-OH
4-OH
III
IV
V
2,4-diOH >100
VI
4-Me
>100
>100
>100
>100
>100
>100
>100
>100
11.07
0.079
VII
4-MeO
69.44
7.47
VIII
4-Cl
3-Cl
4-Br
–
IX
>100
>100
>100
>100
>100
7.72
0
1
2
3
4
5
X
XI
XII
–
XIII
–
Rivastigmine
donepezil
–
–
5.20
aInhibition concentration (mean ± SD of three experiments) required for 50% inhibition of AChE and BuChE
bEstimated binding free energy (kcal/mol) calculated by Auto Dock Tools software
structure. Hence there is less conformity between theo-
retical data as binding affinity and experimental data as
IC . It is noteworthy to mention that absence of water
environment in calculating binding affinity applies
essential effect on the docking results (Purgatorio et al.
Conclusions
In this report, we synthesized some novel 5-aryl-1,3,4-
oxadiazole-2-carbamothioate compounds (I–XI) as Rivas-
tigmine analogs. The molecular docking was used to ana-
lyze protein-ligand interactions. The correlations between
activity of the synthesized compounds I–XI and their lipo-
philicity by Chem Draw Program were obtained to show
susceptibility of the design compounds for synthesizing as
inhibitor of AChE and BuChE. The data obtained from
molecular docking were consistent with the dominant trends
of carbamate derivatives to inhibition of AChE and BuChE.
These information employed to synthesize a set of 5-aryl-
1,3,4-oxadiazole-2-carbamothioate compounds (I–XI) and
their affinity against AChE and BuChE. The docking results
and inhibitory activity of synthesized compounds in com-
parison with Rivastigmine and Donepezil revealed that most
of the synthesized compounds (I–VIII) showed moderate to
excellent potent inhibition activity against BuChE with high
selectivity for this enzyme. Compound VI [S-(5-(p-tolyl)-
1,3,4-oxadiazol-2-yl) ethyl(methyl)carbamothioate] with
methyl group on the para position of the phenyl ring
5
0
2
019; Yadav et al. 2019).
Regarding the docking and biological results and in
order to support our data, in the following we synthe-
sized and evaluated two compounds XII and XIII (Table
2
) to investigate the importance of heterocycle moiety
and size in the respective compounds. Compound XII
with aromatic ring but without hetero aromatic moiety
demonstrated no activity against inhibition of AChE and
BuChE (IC50 > 100 µM) also its binding affinity was
negligible (~4 kcal/mol). Compound XIII without any
aromatic and heteroaromatic ring also evaluated against
AChE and BuChE. According to the results no activity
(
IC > 100 µM) was observed, also its docking result
50
demonstrated low binding affinity (~4 kcal/mol). These
results were caused by the small size and insufficient inter-
communications with amino acid residues into the active site
of the enzyme.

Medicinal Chemistry Research
demonstrated the most stable binding affinity (−7.81 kcal/
mol) and excellent inhibitory activity against BuChE equal
to 0.51 µM in comparison with Rivastigmine with 7.72 µM
and Donepezil with 5.20 µM inhibitory activity. Recent
essay data survey indicates that BuChE plays a significant
interest role in AD, especially at advance stage of the dis-
ease, therefore these selective BuChE inhibitors can be
favorable drug candidates in the future.
cholinesterase inhibitory activity. Arch Pharm Chem Life Sci
3
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Technology (IROST) for financial support of this study.
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Li P, Shi L, Gao MN, Yang X, Xue W, Jin LH, Hu DY, Song BA
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Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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