Synthesis, characterization, and pharmacological evaluation of thiourea derivatives

Article abstract of DOI:10.1515/chem-2020-0139

Thioureas and their derivatives are organosulfur compounds having applications in numerous fields such as organic synthesis and pharmaceutical industries. Symmetric thiourea derivatives were synthesized by the reaction of various anilines with CS2. The synthesized compounds were characterized using the UV-visible and nuclear magnetic resonance (NMR) spectroscopic techniques. The compounds were screened for in vitro inhibition of α-amylase, α-glucosidase, acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) enzymes and for their antibacterial and antioxidant potentials. These compounds were fed to Swiss male albino mice to evaluate their toxicological effects and potential to inhibit glucose-6-phosphatase (G6Pase) inhibition. The antibacterial studies revealed that compound 4 was more active against the selected bacterial strains. Compound 1 was more active against 2,2-diphenyl-1-picrylhydrazyl and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, AChE, BuChE, and α-glucosidase. Compound 2 was more potent against α-amylase and G6Pase. Toxicity studies showed that compound 4 is safe as it exerted no toxic effect on any of the hematological and biochemical parameters or on liver histology of the experimental animals at any studied dose rate. The synthesized compounds showed promising antibacterial and antioxidant potential and were very active (both in vitro and in vivo) against G6Pase and moderately active against the other selected enzymes used in this study.

Full text of DOI:10.1515/chem-2020-0139

Open Chemistry 2020; 18: 764–777
Research Article
Sumaira Naz, Muhammad Zahoor*, Muhammad Naveed Umar, Saad Alghamdi,
Muhammad Umar Khayam Sahibzada*, Wasim UlBari
Synthesis, characterization, and
pharmacological evaluation of thiourea
derivatives
https://doi.org/10.1515/chem-2020-0139
experimental animals at any studied dose rate. The
received November 20, 2019; accepted May 27, 2020
synthesized compounds showed promising antibacterial
and antioxidant potential and were very active (both in vitro
and in vivo) against G6Pase and moderately active against
the other selected enzymes used in this study.
Abstract: Thioureas and their derivatives are organosulfur
compounds having applications in numerous fields such as
organic synthesis and pharmaceutical industries. Symmetric
thiourea derivatives were synthesized by the reaction of
various anilines with CS2. The synthesized compounds
were characterized using the UV-visible and nuclear
magnetic resonance (NMR) spectroscopic techniques. The
compounds were screened for in vitro inhibition of
α-amylase, α-glucosidase, acetylcholinesterase (AChE), and
butyrylcholinesterase (BuChE) enzymes and for their anti-
bacterial and antioxidant potentials. These compounds were
fed to Swiss male albino mice to evaluate their toxicological
effects and potential to inhibit glucose-6-phosphatase
(G6Pase) inhibition. The antibacterial studies revealed that
compound 4 was more active against the selected bacterial
strains. Compound 1 was more active against 2,2-diphenyl-1-
picrylhydrazyl and 2,2’-Azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid) (ABTS) free radicals, AChE, BuChE, and α-
glucosidase. Compound 2 was more potent against α-amylase
and G6Pase. Toxicity studies showed that compound 4 is safe
as it exerted no toxic effect on any of the hematological and
biochemical parameters or on liver histology of the
Keywords: thiourea derivatives, pharmacological evalua-
tion, acetylcholinesterase, glucose-6-phosphatase, bio-
chemical and histopathological evaluation, in silico
modeling
1 Introduction
Thiourea or thiocarbamide is an organosulfur compound with
the formula SC(NH2)2. The term “thioureas” is used for the
class of compounds with the general formula (R1R2N)(R3R4N)
C═S [1]. None of any other class of organic compounds has
such a wide diversity and multiple applications as thioureas
have. Indeed, in almost every branch of chemistry, thioureas
have played their exceptional role [2]. Commercially,
industrially, and academically, they are of huge importance.
On commercial scale, thioureas are frequently used in
photographic film, plastics, dyes, elastomers, and textiles
[3,4]. Several derivatives of thioureas are used as pharma-
ceuticals, preservatives, rodenticides, and insecticides [5–8].
Thioureas have valuable uses in organic synthesis and are
used as intermediates in several organic synthetic reac-
tions [9].
Another very important, diverse, and effective area of
thiourea applications is their biological activities. Again,
none other class of organic compounds has as much
biological activities as thioureas. These have been reported
to have antifungal, antiviral, antibacterial, catalytic, anti-
tubercular, analgesic, insecticidal, anti-inflammatory, herbi-
cidal, anticonvulsant, anti-cancer, anti-thyroid, anthelmintic,
and anti-phenoloxidase activities [9–16]. They also act as
rodenticide and have anti-HIV, antiviral, high density
lipoprotein (HDL)-elevating, antibacterial, analgesic,

* Corresponding author: Muhammad Zahoor, Department of
Chemistry, University of Malakand, Chakdara, Dir Lower, 18800
KPK, Pakistan, e-mail: mohammadzahoorus@yahoo.com,
tel: +92 3416062388
* Corresponding author: Muhammad Umar Khayam Sahibzada,
Department of Pharmacy, Sarhad University of Science and
Information Technology, Peshawar, KPK 25000, Pakistan,
e-mail: umar.sahibzada@gmail.com, tel: +92 3338331556
Sumaira Naz, Muhammad Naveed Umar, Wasim UlBari: Department
of Chemistry, University of Malakand, Chakdara, Dir Lower, 18800
KPK, Pakistan
Saad Alghamdi: Department of Laboratory Medicine, Faculty of
Applied Medical Sciences, Umm Al-Qura University, P.O. Box 715,
Makkah 21955, Saudi Arabia
Open Access. © 2020 Sumaira Naz et al., published by De Gruyter.
This work is licensed under the Creative Commons Attribution 4.0 Public
License.

Synthesis, characterization, and pharmacological evaluation of thiourea derivatives

765
antidiabetic, anti-hypertensive, anti-epileptic, anticancer, of diabetes mellitus, the main aim is to reduce blood glucose
DNA-binding, hypnotic, and anesthetic properties [9–16]. concentrations to normal range. One of the several strategies
Free radicals with unpaired electrons are very unstable is to use such drugs that can control blood glucose level. One
and reactive molecules. The most reactive molecules are class of such drugs having the ability to inhibit two
different atomic and molecular forms of oxygen, which are important enzymes involved in carbohydrate metabolism,
called reactive oxygen species (ROS). Being unstable, they namely, α-amylase and α-glucosidase, has been proved to be
attack nearby molecules, snatching their electrons and thus the most effective treatment [17–20].
making them reactive and unstable free radicals, initiating a
In polysaccharides, the α-1,4-glycosidic bond is
free radical chain reaction. Free radicals are normally and hydrolyzed by the α-amylase enzyme to yield oligosac-
continually produced in the body because of biochemical charides and disaccharides, which are further hydrolyzed
reactions, and they are normally scavenged by the body by α-glucosidase (EC 3.2.1.20), an intestinal enzyme that
scavenging mechanism. If the body’s self-defense system splits their α-1,4-glycosidic linkages, yielding α-D-glucose
becomes incapable of eliminating them, then ROS can molecules, which are then absorbed into the blood.
damage DNA, unsaturated lipids, and proteins and can lead Inhibition of these enzymes delays the digestion of
to death of the cell. ROS are the cause of a variety of carbohydrates and thus their consequent absorption,
pathologic processes such as cancer, aging, inflammatory ultimately lowers the postprandial blood glucose concen-
disease, reperfusion injury, diabetes, stroke, and dementia tration [17–20].
and several other lethal diseases [17–20].
Glucose-6-phosphatase (G6Pase) is an important enzyme
Alzheimer’s disease (AD) is a type of dementia that of carbohydrate metabolism, which catalyzes the last step of
causes memory loss, problems with cognitive abilities, gluconeogenesis and glycogenolysis, where it converts
thinking, and behavior. Usually, its symptoms develop slowly glucose-6-phosphate to glucose and inorganic phosphate
and get worse with the passage of time, becoming serious [20,21]. Both glycogenolysis and gluconeogenesis result in the
enough to interfere with routine life and usually accounts for formation of glucose-6-phosphate from gluconeogenic pre-
about 60–80% of dementia cases [19]. The two main types of cursors in liver and kidney and from glycogen in liver. It not
cholinesterases present in the mammalian brain are acet- only catalyzes the hydrolysis of glucose-6-phosphate but also
ylcholinesterase (AChE) and butyrylcholinesterase (BuChE). catalyzes its resynthesis from glucose and a phosphoryl
In patients with AD, a remarkable decrease in the concentra- donor. This enzyme, therefore, plays an important role in the
tion of acetylcholine (ACh) was observed. One possible way to regulation of glucose homeostasis. Consequently, its insuffi-
stop and maintain proper ACh hydrolysis is inhibiting AChE, ciency prompts glycogen storage disorder, which is an
which is the enzyme responsible for its hydrolysis [17–20].
acquired metabolic condition, described by poor resistance
BuChE has a minor role in the normal, healthy brain, to fasting, development impediment, and hepatomegaly,
but its activity gradually increases in patients with AD. In the which results in amassing of glycogen and fat in the liver.
advanced stages of AD, when AChE is inhibited, BChE G6Pase also has an important role in diabetes mellitus, where
usually can compensate for AChE and hydrolyze the its activity is increased in liver, leading to high blood glucose
remaining ACh present, thus making the situation worse. level. Therefore, its inhibition can decrease hepatic glucose
Recently, experimental evidence from the dual inhibition of output. Recently, this enzyme has been identified as an
both AChE and BChE show enhanced therapeutic benefits in important antidiabetic target [20,21].
AD and other related dementias [17–20].
Many compounds such as 4,4′-di-diisothiocyanatostil-
Diabetes mellitus is a disorder of carbohydrate meta- bene-2,2′-disulfonic acid, which is a well-known inhibitor of
bolism, where the body is unable to either produce insulin or anion transporter, 2-hydroxy-5-nitrobenzaldehyde, and
respond to it, which results in impairment of maintaining chlorogenic acid derivatives inhibit the translocases of
normal blood glucose levels. Insulin is a hormone secreted G6Pase system, but only a few compounds have inhibitory
by β cells of islets of Langerhans in the pancreas. It triggers activities on the G6Pase enzyme complex [22–24]. Recently, a
the uptake of glucose by the cells, thereby ensuring a proper series of 4-, 5-, 6-, 7-tetrahydrothienopyridines have been
blood glucose level and making it available to the cells to be reported as potent competitive inhibitors of G6Pase [20].
used as an energy source. People with diabetes either have There is a need to find out a compound that competitively
inactive β cells that cannot secrete insulin (type 1 diabetes inhibit the G6Pase; hence, it will be an alternative to the
mellitus) or have unresponsive muscles and adipose cells drugs that inhibit T1 translocase [20,21].
that are resistant to the actions of insulin (type 2 diabetes
An organism’s hematological and biochemical para-
mellitus). In both cases, the concentration of glucose in meters of blood are indicative of its overall physiological
blood increases, resulting in hyperglycemia. In the treatment status. A normal healthy organism without any toxic

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Sumaira Naz et al.
metabolites in blood should maintain its blood indices in recrystallized with ethanol, purified by column chromato-
relatively normal or constant state. A disorder, disease, or graphy, and characterized using UV-visible and NMR
toxicity may be expected if the blood indices are found to be techniques (Figure 1S).
significantly altered or a tissue damage is detected [25,26].
In vivo studies of certain synthetic compounds (metribuzin,
etc.) in albino mice have shown marked alterations in
hematological and biochemical parameters [27]. It is
necessary that a chemical compound that is a drug or
intended to be used for therapeutic purpose should be
assessed for its possible toxicity or adverse effects through
examining the changes it may or may not bring in blood
parameters and in function or structure of tissues [25,26].
The aim of this study was to synthesize novel thiourea
derivatives that have antidiabetic, antibacterial, antioxidant,
and anticholinesterase properties. The synthesized com-
pounds were also tested in in vivo using albino mice whether
they can inhibit G6Pase or not. Furthermore, various
hematological and biochemical parameters were studied and
histopathological examinations were also carried out to find
out the effect of the synthesized compounds on the overall
metabolic status and on liver tissue to tell that whether they are
safe enough to be used therapeutically or not.
Cl
Cl
S
Cl
N
H
N
H
Cl
1,3-bis(3,4-dichlorophenyl)thiourea
Compound 1
2.1.2 Compound 2
Carbon disulfide (5 mmol) and 2,6-dimethylaniline (10 mmol)
were added to 5 mL of water in a 50-mL round-bottom flask
without using magnetic bar or stirring. The flask was corked
and placed in the sunlight. After 5 h, the white solid was
obtained in water. Thin-layer chromatography (10:90% ethyl
acetate:n-hexane) was used to check the progress of the
reaction. The product obtained was recrystallized with ethanol
and further purified by column chromatography and char-
acterized by UV-visible and NMR techniques (Figure 2S).
S
N
H
N
H
1,3-bis(2,6-dimethylphenyl)thiourea
2 Materials and methods
Compound 2
Chemicals such as 3,4-dichloroaniline, 2,6-dimethylani-
line, ethyl 4-aminobenzoate, 4-nitroaniline, 4-methyl-
aniline, and CS2 were acquired from Sigma-Aldrich,
Merck, and Alpha Acer and were used without further
purification. Solvents including n-hexane, methanol,
dichloromethane, ethanol, ethyl acetate, and chloroform
were purchased from Sigma-Aldrich. The solvents
were distilled before use. Thin layer chromatography
(TLC) plates were used to monitor the reaction at regular
intervals under ultraviolet irradiation.
2.1.3 Compound 3
Carbon disulfide (5 mmol) and ethyl 4-aminobenzoate
(10 mmol) were added to 5 mL water in a 50-mL round-bottom
flask without using magnetic bar or stirring. The flask was
corked and placed in the sunlight. After 5 h, the white solid
was obtained in water. Thin-layer chromatography (10:90%
ethyl acetate:n-hexane) was used to check the progress of the
reaction. The product obtained was recrystallized with
ethanol, purified by column chromatography, and character-
ized by UV-visible and NMR techniques (Figure 3S).
S
2.1 Synthesis
C2H5OOC
N
H
N
H
COOC2H5
2.1.1 Compound 1
diethyl 4,4'-(thiocarbonylbis(azanediyl))dibenzoate
Compound 3
Carbon disulfide (5 mmol) and 3,4-dichloroaniline (10 mmol)
were added to 5 mL of water in a 50-mL round-bottom flask
without using magnetic bar stirring, and the flask was
corked and placed in the sunlight. After 7 h, the white solid
was obtained in water. Thin-layer chromatography (10:90%
ethyl acetate:n-hexane used as a mobile phase) was used to
check the progress of the reaction. The product obtained was
2.1.4 Compound 4
Carbon disulfide (5 mmol) and 4-nitroaniline (10 mmol) were
added to 5 mL of water in a 50 mL round-bottom flask
without using magnetic bar or stirring. The flask was corked

Synthesis, characterization, and pharmacological evaluation of thiourea derivatives

767
and placed in the sunlight. After 7 h, a white solid was Klebsiella pneumoniae ATCC 13883, Enterococcus faecalis
obtained. Thin-layer chromatography (10:90% methanol:- ATCC 29212, and Salmonella typhi ATCC 6539 were used in
chloroform) was used to check the progress of the reaction. this study. Nutrient agar and the Petri dishes were sterilized
The product obtained was recrystallized with ethanol, at 121°C for 15 min using autoclave (Jisico: J-NAS 45, Korea).
purified by column chromatography, and characterized by Nutrient agar was poured into the sterilized Petri dishes
UV-visible and NMR techniques (Figure 4S).
under aseptic environment just before solidification. In this
study, DMSO and ceftriaxone were used as negative and
positive controls, respectively. Sample solutions of the
synthesized thiourea compounds were prepared in DMSO
(30 µg/mL). At the end of the incubation period, the average
zones of inhibition were acquired and were compared with
the positive control. Triplicate analysis was performed for the
antibacterial assay.
S
O2N
N
H
N
H
NO2
1,3-bis(4-nitrophenyl)thiourea
Compound 4
To determine the MIC of the synthesized compounds,
they (10–100 µg/mL) were added to the test tubes containing
nutrient broth and inoculated with 5 × 105 cfu/mL of the
selected bacterial strains and incubated at 37°C for 20 h. After
20 h incubation time, the bacterial growth in each tube was
noted.
2.1.5 Compound 5
Carbon disulfide (5 mmol) and ethyl-4-methylaniline
(10 mmol) were added to 5 mL of water in a 50 mL round-
bottom flask without using magnetic bar or stirring. The
flask was corked and placed in the sunlight. After 5 h, the
white solid was obtained in water. Thin-layer chromato-
graphy (10:90% ethyl acetate:n-hexane) was used to check
the progress of the reaction. The product obtained was
washed with a mixture of ethyl acetate:n-hexane, purified
by column chromatography, and characterized by UV-
visible and NMR techniques (Figure 5S).
2.3.2 Antioxidant activity by DPPH method
Antioxidant activity of the synthesized compounds was
determined using the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) and ABTS assays, described by Naz et al. [28].
The stock solution of DPPH having concentration of
20 mg/100 mL of methanol was prepared, while stock
solution of ABTS was prepared by dissolving 7 mM of
ABTS and 2.45 mM of potassium persulfate separately in
100 mL of methanol. About 3 mL from each stock
solutions was taken and diluted with methanol to adjust
its absorbance of 0.7 at 515 nm. The stock solutions were
covered with aluminum foil and placed in a cold and
dark place for 24 h to ensure the formation of free
radical. Sample solutions were prepared by dissolving
5 mg of each compound in 5 mL methanol (sample stock
solution). A dilution series having concentrations of 62.5,
125, 250, 500, and 1,000 µg/mL were also prepared from
stock compound solutions. Specified amount from
working standards was mixed with specified amount of
DPPH/ABTS solutions and incubated for specified
interval of time, as described by Naz et al. [28].
Percentage of free radical inhibition was calculated
using the following equation:
S
H3C
N
H
N
H
CH3
1,3-di-p-tolylthiourea
Compound 5
2.2 Instrumentation
Electrothermal melting point instrument (Model BioCote
Stuart-SMP-10, Japan) was used to determine the
melting point of the compounds in a sealed capillary
1
tube. The H-NMR spectra were recorded using an NMR
Bruker apparatus at 300 MHz in CDCl3.
2.3 In vitro biological activities of the
synthesized compounds
2.3.1 Antibacterial assay
A − B
Minimum inhibitory concentration (MIC) and agar well
diffusion method were used to evaluate the antibacterial
potential of the synthesized compounds. Four different
bacterial strains Pseudomonas aeruginosa ATCC 10145,
% inhibition =
× 100,
(1)
A
where A is the absorbance of pure DPPH in oxidized
form and B is the absorbance of the sample.

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Sumaira Naz et al.
2.3.2 Anticholinesterase activities of the synthesized
compounds
percentage (w/v) of maltose generated using the
following formula:
(maltose) test
% inhibition =
× 100,
(4)
Anticholinesterase activities of the synthesized compounds
were evaluated using AChE and BuChE enzymes using
Ellman’s assay [14,29], which is based on acetylthiocholine
iodide and butyrylthiocholine iodide hydrolysis by respec-
tive enzymes. The resultant product of hydrolysis is then
combined with 5-thio-2-nitrobenzoate anion to from a
yellow colored compound, 5,5′-dithiobis-(2-nitrobenzoic
acid) (DTNB). The DTNB is then detected by the spectro-
(maltose) control
where (maltose) test is the percentage (w/v) of maltose
generated by the sample solution and (maltose) control
is the percentage (w/v) of maltose generated by the
control solution. Acarbose was used as the standard.
photometer (T60 PG Instruments, UK) after 15 min of the 2.3.3.2 Alpha-glucosidase inhibition
reaction.
Phosphate buffer, DTNB, AChE and BuChE, and acetyl- The potential alpha-glucosidase inhibition was deter-
choline iodide and butyrylcholine iodide solutions were mined according to the procedure of Naz et al. [32]. One
prepared as described in Naz et al. [32]. From working hundred microliters of the enzyme solution in phosphate
dilution, 1 mL was added to a series of test tubes, and 100 μL buffer (0.50 unit/mL) was mixed with compound
of enzyme and DTNB solutions were added following 15 min working dilutions and incubated for 15 min at room
incubation at 25°C. At the end of incubation, 100 μL of the temperature. About 100 µL of p-nitrophenyl-α-D-gluco-
substrate AChEI/BChEI solution was added to the test tube pyranoside was added to start the reaction, while 80 µL
and was left for 15 min to get the reaction completed. The Na2CO3 (0.2 M) was added to stop the reaction. The
absorbance of the mixture was monitored at 412 nm. absorbance of the resultant mixture of the reaction was
Galantamine was used as a positive control.
Percent activities of AChE and BChE and their lated using the following equation:
percent inhibition were calculated using the following
recorded at 405 nm. The percent inhibition was calcu-
Percent inhibition
equations:
(5)
Control absorbance − sample absorbance
=
,
ΔAbs
Control absorbance
(2)
(3)
V =
,
ΔT
where control absorbance is the absorbance of the
control solution and sample absorbance is the absor-
bance of the sample solution.
V
% enzyme activity =
× 100,
Vmax
where V is the rate of reaction in the presence of
inhibitor, ΔAbs is the change in absorbance, ΔT is the
change in time, and Vmax is the rate of reaction without
inhibitor.
2.3.3.3 G-6Pase inhibition
The G-6Pase activity was calculated on the basis of the rate
of phosphate release under experimental conditions
described by Fiske and Subbarow [21]. For calculating
the enzyme activity, the same protocol was adopted as
followed by Naz et al. [28]. The concentration of inorganic
phosphate in each sample was determined from compar-
ison with the standard phosphate calibration curve.
2.3.3 Antidiabetic property of the synthesized
compounds
2.3.3.1 Alpha-amylase inhibition
Alpha-amylase inhibition assay was performed ac-
cording to the procedure of Naz et al. [28]. Enzyme
solution in phosphate buffer (25 μL) after 10 min of
incubation at 25°C was mixed with starch dissolved in
phosphate buffer (25 μL) and again incubated at the
same interval of time and temperature. Then, 50 μL DNS
(3,5-dinitrosalicylic acid) was added to the mixture to
stop the reaction, and absorbance was measured at
540 nm. The percent inhibition was determined from the
2.4 In vivo experiments
2.4.1 Experimental animals and their treatment
Swiss male albino mice, 24 weeks old, weighing 30–35 g,
were obtained from the National Institute of Health,

Synthesis, characterization, and pharmacological evaluation of thiourea derivatives

769
Islamabad, and were housed in plastic cages having good group 2 died after 22 h. In the light of the acute toxicity,
space and sawdust containing bedding. The animals were dose range for the subchronic toxicity was set as
given free access to water and food. The room temperature 0.5–1.5 mg/kg body weight.
was maintained at 25°C. All experiments and overall
handling of the animals were performed in accordance
with the guidelines, “Ethics Committee for Animal Care & 2.4.2 G6Pase inhibition
Use, Pakistan” and ARRIVE guidelines. Approval was
obtained from the University Ethical Committee vide letter Mice were sacrificed on the 22nd day of the experiment, their
NO: UOM-EC/PHARM & BIOCHEM/2018/1.
livers were collected, and fast frozen, each in a separate flask
(Labconco Corporation, Kansas City, MO, USA). Liver
homogenates were prepared in iced water, and enzyme
extract was prepared by placing the homogenate in a flask at
a shaker (Stuart SF1, Cole Parmer, UK) (maintained at 0°C by
continuous addition of ice) for an hour, and the supernatant
obtained is filtered and used as a source of G6Pase. Then,
1 mL of the obtained liver homogenate (enzyme source),
2.5 mL of 0.01 M glucose-6-phosphate (substrate,) and 1.5 mL
Tris(hydroxymethyl)aminomethane buffer (pH 6.7) were
taken in a flask and incubated for an hour at 33°C. The
reaction was stopped by the addition of 1 mL of 10%
trichloroacetic acid. Inorganic phosphate released (one of the
products) during the reaction was measured by the method
of Fiske and Subbarow [21].
2.4.1.1 Groups
The animals were divided into six groups. The group
numbers were actually the arbitrary names of compounds 1,
2, 3, 4, and 5 fed to each group. Based on the quantity of
dose the mice received, each of the five groups were
subdivided in three groups (each having five mice): A
(received 0.5 mg of dose/kg body weight of mice), B (1 mg
of dose/kg body weight of mice), and C (received 1.5 mg of
dose/kg body weight of mice). Following are the groups
and subgroups:
Group CL (having five mice): The group receiving
only the normal food and water.
Group 1 (fed with compound 1): Subgroups: 1A, 1B, 1C
Group 2 (fed with compound 2): Subgroups: 2A, 2B, 2C
Group 3 (fed with compound 3): Subgroups: 3A, 3B, 3C
Group 4 (fed with compound 4): Subgroups: 4A, 4B, 4C
Group 5 (fed with compound 5): Subgroups: 5A, 5B, 5C
2.4.3 Hematological studies
About 0.2 mL of blood sample was drawn in 10% EDTA
(anticoagulant) tube. Hematological parameters such as
hematocrit, RBCs, hemoglobin, and leukocyte and platelet
concentration were determined by using an automated
hematology analyzer (Abbot CELL-DYN 3200, USA).
2.4.1.2 Collection of blood and tissue samples
Blood samples were taken at the start of the experiment and
on days 7, 14, and 21 for evaluation of the hematological
and biochemical parameters. On the 22nd day, the mice
were sacrificed by first euthanizing them with isoflurane
and then killed through cervical dislocation, and the livers
were collected for histopathology studies.
2.4.4 Biochemical studies
Another 0.2 mL of blood sample was collected without any
EDTA to obtain serum after centrifugation. Serum was
studied for biochemical parameters such as blood sugar,
total cholesterol, triacylglycerol, alanine transaminase (ALT),
and HDL using the HUMAN kit (HUMAN Diagnostic,
Germany).
2.4.1.3 Acute toxicity
Mice (n = 90) were taken, fasted overnight, and divided
into five major groups (for the five compounds); each
had further three subgroups and each sub-group had six
mice. The subgroups were based on the three different
doses they received (0.8, 1.6, and 2.4 mg/kg body
weight). It was recorded that two of the six mice in the
subgroup 3 (fed with the highest dose) of the major
2.4.5 Histopathology studies
Specimens from the collected liver dissected, fixed in
10% formalin, and dehydrated using ethanol. Thin
sections (about 8–10 µm) were cut from the specimens
and studied under a light microscope (Olympus BH-2,
Shinjuku, Tokyo, Japan) after staining [30].

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Sumaira Naz et al.
2.5 Statistical analysis
scale (ppm). Abbreviations s, d, t, and m represent singlet,
doublet, triplet, and multiplet, respectively.
The IC50 for % DPPH, ABTS, α-amylase, α-glucosidase, AChE,
and BuChE was measured by linear regression analysis
against the different concentrations of the tested compounds
using Excel 2013 version. All results were presented as mean
SEM (standard error of mean). Student’s t-test and one-way
ANOVA followed by Dunnett’s post hoc multiple comparison
test were used to determine P values. P values less than 0.05
were considered as significant.
3.2.1 Compound 1
1,3-Bis(3,4-dichlorophenyl)thiourea: IUPAC name, 1,3-bis
(3,4-dichlorophenyl)thiourea, yield = 84%, m.p. = 174–
1
180°C, H-NMR (300 MHz, CDCl3): δ ppm 7.20 (d, 2H), 6.70
(d, 2H), 6.40(d, 2H). Since this is a known compound, its
structure elucidation data were already reported [31].
3.2.2 Compound 2
3 Results and discussion
1,3-Bis(2,6-dimethylphenyl)thiourea: IUPAC name, 1,3-bis
(2,6-dimethylphenyl)thiourea, yield = 82%, m.p. = 180–
1
184°C, H-NMR (300 MHz, CDCl3): δ ppm 6.90 (d, 4H), 6.70
3.1 General mechanism for the synthesis of
thiourea
(d, 4H), 2.10 (d, 4H). Since this is a known compound, its
structure elucidation data were already reported [31].
General Mechanism:
S
C
S
C S
H2
N
step 1:
step 2:
step 3:
R
R
NH2
R
S
S
C
H2
N
S
H transfer
H
N
S
R
C SH
S
C
H
N
R
H2N
S
R
SH
R
N
N
H
R
H
+
H2S
3.2.3 Compound 3
In the first step, amine or amine derivative attacks as a
nucleophile on the electrophilic carbon of CS2 to
form thiocarbamate intermediate. In the second step,
H+ transfer to form thiocarbamate. In the third
step, another amine molecule attacks on the electro-
philic center of carbamate to form thiourea. The H2S is
evolved during this mechanism.
Diethyl 4,4-thiocarbonylbis(azanediyl)dibenzoate: yield =
78%, m.p. = 177–183°C, H-NMR (300 MHz, CDCl3): δ ppm
7.60 (d, 4H), 6.50 (d, 4H), 4.30 (d, 2H) 1.30 (d, 2H). Since this
is a known compound, its structure elucidation data were
already reported [31].
1
3.2 Spectral analysis of the synthesized
compounds
3.2.4 Compound 4
1,3-Bis(4-nitrophenyl)thiourea: IUPAC name, 1,3-bis(4-
The spectral details of the synthesized compounds are nitrophenyl)thiourea, yield = 80%, m.p. = 188–193°C,
1
described below. H-NMR spectra of compounds 1–5 are 1H-NMR (300 MHz, CDCl3): δ ppm 8.0 (d, 4H), 6.60 (d,
shown in Figures 1S–5 S. As an internal reference, tetra- 4H). Since this is a known compound, its structure
methyl silane was used. Chemical shifts are given in a delta elucidation data were already reported [31].

Synthesis, characterization, and pharmacological evaluation of thiourea derivatives

771
3.2.5 Compound 5
Table 1: Antibacterial spectrum of the synthesized compounds
Sample Bacterial strain Zone of Zone of MIC
1,3-Di-p-tolylthiourea: IUPAC name, 1,3-bis(4-methylphenyl)
thiourea, yield = 86%, m.p. = 181–185°C, 1H-NMR (300 MHz,
CDCl3): δ ppm 6.90 (d, 4H), 6.30 (d, 4H), 2.30 (d, 2H) 1.30 (d,
2H). Since this is a known compound, its structure
elucidation data were already reported [31].
inhibition of inhibition of (µg/
standard*
(mm)
sample*
(mm)
mL)
1
Enterococcus
faecalis
Pseudomonas
aeruginosa
Salmonella
typhi
30
28
25
23
27
24
22
20
40
50
60
60
3.3 Antibacterial spectrum of the
synthesized compounds
Klebsiella
pneumoniae
E. faecalis
P. aeruginosa
S. typhi
K. pneumoniae
E. faecalis
P. aeruginosa
S. typhi
K. pneumoniae
E. faecalis
P. aeruginosa
S. typhi
K. pneumoniae
E. faecalis
P. aeruginosa
S. typhi
2
3
4
5
31
29
24
29
22
26
24
28
21
29
24
30
19
28
26
32
24
40
40
40
50
50
40
40
50
40
40
40
50
50
50
40
60
The synthesized compounds were evaluated for their anti-
bacterial potential against K. pneumoniae, Pseudomonas
aeruginosa, S. typhi, and Enterococcus faecalis. Compounds 1
and 5 showed moderate antibacterial activities. The anti-
bacterial studies are shown as zone of inhibition in
millimeters in Table 1. Compound 2 showed substantial
activities against the selected bacterial strains, whereas
compounds 3 and 4 showed significant antibacterial
activities; compound 4 was the highly potent one. Against
E. faecalis, P. aeruginosa, S. typhi, and K. pneumoniae,
compound 4 showed 29 mm (standard 30 mm), 24 mm
(standard 25 mm), 30 mm (standard 31 mm), and 19 mm
(standard 20 mm) zone of inhibitions, respectively.
27
33
24
28
25
30
22
30
25
31
20
32
28
35
26
K. pneumoniae
The MIC values of each compound (in µg/mL) are
shown in Table 1. The MIC values were in the range of *Antibacterial activity expressed as zone of inhibitions expressed in
millimeters.
40–60 µg/mL, indicating the high antibacterial potency
of the synthesized compounds. Here, again compounds 3
and 4 were more potent, and less amount was required
Table 2: Antioxidant activities of the synthesized compounds
to inhibit the bacterial growth.
Compound
1
2
3
4
5
Ascorbic acid
DPPH IC50 (μg/mL) 45 100 50 105 112 20
ABTS IC50 (μg/mL) 52 103 56 109 120 30
Ascorbic acid was used as a positive control.
3.4 Antioxidant activity of the synthesized
compounds
The synthesized compounds were investigated for their
antioxidant potential using the DPPH and ABTS assays. In
the DPPH assay, compound 1 was found to have higher
antioxidant activity, with the IC50 value of 45 μg/mL,
followed by compound 3 (IC50 = 50 μg/mL). The IC50 values
of compounds 2, 4, and 5 were 100, 105, and 112 μg/mL
(Table 2), respectively. As a positive control, ascorbic acid
was used with the IC50 value of 20 μg/mL.
radical scavenging (Table 2). Compound 3 was also a potent
antioxidant (IC50 = 56 μg/mL), followed by compounds 2, 4,
and 5 (IC50 of 103, 109, and 120 μg/mL, respectively). Ascorbic
acid was used as a positive control (IC50 = 30 μg/mL).
3.5 AChE inhibition by the thiourea
derivatives
Comparable antioxidant activities of the synthesized
compounds against ABTS free radical were observed. For
compound 1, the IC50 value was 52 μg/mL, almost similar to
its IC50 value in the antioxidant activity using DPPH free
In AD, the inhibition of AChE is required to inhibit the ACh
hydrolysis in the brain and hence to preserve the neuro-
transmitter acetyl choline. Many other thiourea derivatives

772
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Sumaira Naz et al.
Table 3: Thiourea derivatives inhibitory potential against AChE and
BuChE
Table 4: Percent alpha-amylase and alpha-glucosidase inhibition
by the synthesized compounds
Compound
1
2
3
4
5
Galantamine
Compound
1
2
3
4
5
Acarbose
AChE IC50 (µg/mL) 130 200 160 475 300 40
BChE IC50 (µg/mL) 125 300 123 490 495 55
Alpha-amylase IC50
(µg/mL)
Alpha-glucosidase IC50 126 200 250 450 275 60
(µg/mL)
122 115 250 200 130 50
Galantamine was used as a positive control.
Acarbose was used as a positive control.
that could inhibit the activity of AChE and BChE have also
been investigated previously by several researchers [8–11].
The AChE inhibitory activity of the synthesized compounds
as their IC50 values is shown in Table 3. Among the different
synthesized compounds, compound 1 showed maximum
inhibition of AChE with IC50 value of 130 µg/mL, followed by
compound 3 with IC50 value of 160 µg/mL. Compound 2
showed substantial inhibition with IC50 of 200 µg/mL,
followed by compounds 5 and 4 with the IC50 of 300 and
475 µg/mL, respectively. The AChE percent inhibitions were
insulin antidiabetic drug, works on the principle of
alpha-amylase inhibition. The synthesized thiourea
derivatives were investigated for their α-amylase inhibi-
tory potentials, and the results are shown in Table 4.
Compounds 1 and 2 were found to be highly potent
against the selected enzyme, showing promising inhibi-
tory potential with IC50 of 122 and 115 µg/mL, respec-
tively. Compound 5 was ranked the third with IC50 of
130 µg/mL, followed by compounds 4 and 3 with IC50 of
200 and 250 µg/mL, respectively (Table 5). The observed
inhibitory potentials of the compounds were compared
with that of the standard acarbose (IC50 = 36 µg/mL).
compared with that of the standard galantamine (IC50
45 μg/mL).
=
3.6 BuChE inhibition by thiourea derivatives
Currently, the treatment available for the treatment of
AD is only symptomatic and 90% of the medications are
cholinesterase inhibitors. The patients receiving only
AChE inhibitors as medicines could not completely give
response to the treatment as the activity of BChE
becomes high in such patients to compensate for the
loss of AChE activity; the ideal and required approach,
therefore, is to develop such drug that could inhibit both
the enzymes. The compounds were thus also tested for
their potential BChE inhibition. BChE inhibitory activity
of the synthesized compounds as their IC50 values is
shown in Table 3. Compound 3 showed maximum
BChE inhibitory activity among the group with the IC50
value of 123 µg/mL compared with the other four
compounds. Compounds 2, 4, and 5 showed compara-
tively moderate inhibitory potential with the IC50 values
of 300, 490, and 495 µg/mL, respectively. In this study,
galantamine (IC50 = 60 µg/mL) was used as a positive
control.
Table 5: Effect of the synthesized compounds on the activity of
glucose-6-phosphatase
Compound
1
Concentration (µg/mL)
Total activity*
Control
1,000
500
250
25.60
29.80
32.70
38.10
04.20
09.20
08.20
10.40
18.50
22.50
24.70
28.40
15.10
15.70
17.30
22.70
12.20
13.10
14.50
17.50
91.0
125
2
3
4
5
1,000
500
250
91.0
91.0
91.0
91.0
125
1,000
500
250
125
1,000
500
250
125
1,000
500
250
3.7 Alpha-amylase inhibition by thiourea
derivatives
125
Alpha-amylase is an important enzyme of glucose
metabolism. It hydrolyzes polysaccharides, starch, and
glycogen to glucose and maltose. Acarbose, a non-
*Activity expressed as mg of inorganic phosphate (P) released from
the potassium salt of glucose-6-phosphate per hour at 33°C and
pH 6.7.

Synthesis, characterization, and pharmacological evaluation of thiourea derivatives

773
3.8 Alpha-glucosidase inhibition by
Table 6: Effect of thiourea derivatives on the activity of glucose-6-
phosphatase in Swiss albino mice
thiourea derivatives
Description of groups
Total activity*
Alpha-glucosidase is another important enzyme of glucose
metabolism, which is found in the small intestine and
hydrolyzes starch and maltose to glucose. The results are
presented in Table 4, which shows that compound 1 was
most potent against the enzyme with IC50 of 126 µg/mL,
followed by compounds 2 and 3 with IC50 of 200
and 250 µg/mL, respectively. The least activity was recorded
Group 1
1A
1B
1C
67.99 1.735
58.3 1.904
46.11 0.6416
46.37 1.412
38.38 1.649
32.07 1.113
56.04 0.8536
47.42 0.5892
37.59 2.428
53.38 1.896
44.55 1.38
39.25 1.616
63.95 2.39
51.58 1.633
41.22 2.027
84.55 3.213
Group 2
Group 3
Group 4
Group 5
Control
2A
2B
2C
3A
3B
3C
4A
4B
4C
5A
5B
5C
for compound
4
with IC50 of 450 µg/mL. The
inhibitory potentials of the synthesized compounds were
compared with that of acarbose used as standard (IC50
34 µg/mL).
=
3.9 G6Pase inhibition by synthesized
thioureas
The values are expressed as mean
subgroup.
*Activity here is expressed as mg of inorganic phosphate (P)
released from the potassium salt of glucose-6-phosphate/hour at
pH 6.7 and temperature 33°C.
The enzyme’s activity is significantly decreased by oral adminis-
tration of each of the five compounds when measured at the 22nd
day of the experiment.
SEM of n = 5 mice per
Inhibition of G6Pase by the synthesized compounds is
shown in Table 5. Compound 2 was highly potent,
followed by compound 4, and compound 5 also showed
very good inhibition. Compounds 1 and 3 exhibited
moderate inhibition of the enzyme. The inhibitory
pattern seemed to be competitive as the activity of the
enzyme significantly decreased with increasing concen-
tration of the compounds.
Therefore, all these can securely be presumed as potential
inhibitors of G6Pase.
3.10 In vivo effect of the synthesized
3.11 Effect on hematological parameters
compounds on the activity of G6Pase
Table 7 shows the effect of the compounds on various
The synthesized compounds were tested for their G6Pase hematological parameters. Packed cell volume (PCV) was
inhibitory potential in vivo using male Swiss Albino mice. significantly decreased (P < 0.001) at all dose rates by
Table 6 illustrates the effect of the compounds on the activity compounds 1, 2, 3, and 5, and compound 4 had no
of G6Pase. It is clear from the table that the enzyme’s activity significant effect on any dose. Erythrocyte concentration
is significantly decreased after oral administration of each of remains unaffected at all dose rates by all the test
the five compounds when measured on the 22nd day of the compounds. Hemoglobin was significantly decreased at
experiment. Highest inhibitory activity was shown by dose rates of 0.5, 1.0 (P < 0.01), and 1.5 (P < 0.01) mg/kg
compound 2 at all doses but the effect was maximum at body weight by compound 1; a significant decrease (P <
1.5 mg/kg body weight of mice. Likewise, compounds 3 and 0.01) by compound 2 at dose rates 0.5, 1.0, and 1.5 (p <
4 also showed very good inhibition. Compounds 1 and 5 also 0.001)/kg body weight and a significant (P < 0.05 and 0.01)
inhibited the enzyme and decreased its activity significantly. decrease by compound 3 at 0.5, 1.0, and 1.5 mg/kg body
The compounds were generally found to suppress the weight, respectively, were observed. Compound 4 had no
enzyme activity at one level or other. The mechanism of significant effect on PCV. WBC count was significantly
inhibition is not known, but the increase in inhibition decreased (P < 0.001) at a dose rate of 1.5 mg/kg body weight
with an increase in the amount of dose and time suggests by compound 1 and at all dose rates for compound 2,
that it may be competitive inhibition. Compounds 2 and 4 whereas compound 5 significantly (P < 0.05) increased
possessed the maximum potential, followed by compound 3. leukocyte count at higher doses. No significant effect was
A moderate inhibition was observed for compounds 1 and 5. observed at any dose of compounds 3 and 4. Monocyte

774
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Sumaira Naz et al.

Synthesis, characterization, and pharmacological evaluation of thiourea derivatives

775
Table 8: Effect of thiourea derivatives on biochemical parameters in mice after 30 days of administration
Compounds
1
Subgroups
Glucose (mg/dL)
Triglyceride (mg/dL)
Cholesterol (mg/dL)
95.26 1.86###
99 1.58###
HDL (mg/dL)
ALT (µ/L)
62 1.81###
1A
1B
1C
86.43 1.69
85.6 2.04
87.4 2.15
85.65 1.12
87.43 3.41
89 2.91
84.43 2.23
86.75 2.26
86.91 1.53
73.52 2.96***
70.2 1.94***
69.8 3.78***
83.34 1.95
84.6 2.48
85.84 3.07
87.2 2.49
115.7 2.34
40.23 2.21***
38.16 3.61***
38.79 1.68***
37.53 3.17***
35.27 1.77***
35.05 1.86***
39.1 1.44***
36.31 1.81***
35.6 1.17***
51.10 1.43###
54.70 1.51###
57.20 0.87###
37.78 2.45***
38.07 2.17***
38.86 1.73***
45.4 1.63
117 1.61###
119 2.56###
118.1 1.39###
118.4 4.36###
121.4 2.48###
115.9 3.94
71.14 2.79###
77.4 4.87###
65.82 2.22###
71 3.162###
81.6 2.702###
61.42 1.26###
59.73 1.97###
68.2 2.58###
48.65 1.49
106.8 1.43###
103.12 1.69###
108.04 3.36###
110.08 2.76###
97 1.81###
2
2A
2B
2C
3A
3B
3C
4A
4B
4C
5A
5B
5C
3
117.8 1.37###
119.7 1.19###
106.1 2.41***
97.14 2.29***
92.2 3.24***
115.3 1.74
117.7 4.19###
119 3.06###
110.4 2.68
101 1.47###
105.2 1.94###
77.08 2.41***
74.49 2.19***
73.6 2.48***
96.25 1.86###
98.15 2.13###
102.34 1.68###
82.6 3.78
4
50.14 2.79
48 2.91
62.26 1.83###
63.4 1.17###
69.45 1.88###
49.2 2.87
5
Control
Data are expressed as mean SD of n = 6. Numbers 1–5 represent compounds 1–5; A, 0.5 mg/kg body weight; B, 1 mg/kg body weight; and
C, 1.5 mg/kg body weight. Significant difference was measured using one-way analysis of variance, followed by Bonferroni’s post hoc test.
* P < 0.05, ** P < 0.01, *** P < 0.001 vs control group, and ###P > 0.001 means significant increase compared to the control.
count was significantly (P < 0.01) increased at higher doses compounds 1 and 3 and at all dose rates by compounds 2
by compound 5. Platelet count was significantly increased and 5; a significant decrease (P < 0.001) in TG levels was
(P < 0.01) at all dose rates by all compounds except caused by compound 4. Cholesterol level was increased
compound 4. It is essential that the safety of a compound to (P < 0.001) at all doses by compounds 1, 2, 3, and 5, and
be used as drug must properly be assessed, as metabolites of compound 4 at all dose rates significantly (P < 0.001)
synthetic drugs may cause cellular destruction and tissue decreased its level. HDL level was decreased (P < 0.001) by
damage, which may lead to several immunological, bio- compounds 1, 2, 3, and 5, while it was increased by
chemical, and hematological responses. Hence, along with compound 4 at all doses. ALT level was significantly
enzyme inhibitory studies, the effect of the compounds on increased (P < 0.001) by all compounds except com-
various blood indices and histology of the liver was also pound 4.
examined, and as the alterations in blood indices or tissue
The increased levels of TG, cholesterol, and ALT in
damage can occur either rapidly or slowly, the assays were the presence of compounds 1, 2, 3, and 5 compared with
carried out for different duration of time (10, 20, and 30 days) that of the control propose an underlying infection/
and at different dose levels (0.5, 1.0, and 1.5 mg/kg body inflammation caused by the administration of the
weight of mice). In this study, only the results of 30-day compounds.
exposure are represented. The effects observed are sugges-
tive of the inflammation/infection occurred during exposure
of the animals to the aforementioned compounds.
3.13 Effect on liver histology
Figure 1 shows the effect of the compounds on histology of
3.12 Effect on biochemical parameters
the mice liver after 30 days of oral administration.
Compound 1 has caused a moderate degree of necrosis
Table 8 represents the effect of the five compounds on and inflammation (slide 1C) at 1.5 mg/kg body weight of
hematological parameters of mice after 30 days of mice. Compound 2 at 1.5 mg/kg body weight (slide 2C)
administration. We can see that blood glucose level after shows inflammation, necrosis, swelling, and vacuolization
30 days of dose administration was significantly (P < of hepatocytes. Compound 3 has caused a higher degree of
0.001) reduced by compound 4 at all doses; other inflammation and necrosis (slide 3C). Almost normal
compound did not have any significant effect. An increase hepatocyte and parenchyma can be seen for compound 4
in triglycerides (TG) levels was observed at higher doses by at 1.5 mg/kg body weight of mice. Compound
5

776
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Sumaira Naz et al.
Control
1C
2C
4C
5C
3C
Figure 1: Effect of thiourea derivatives on liver (1, 2, 3, 4, and 5 indicate the respective thiourea derivative. C indicates the amount of dose
given which is 1.5 mg/kg body weight. Livers were collected at day 30 of the experiment).
shows necrosis, inflammation, and hepatocyte swelling at specific. The in vitro inhibition of G6Pase was further
1.5 mg/kg body weight of mice.
confirmed and supported in the in vivo inhibition study,
The liver histology was studied for all the three which also showed that the activity of the enzyme was
studied doses, but in this study, the results of the highest appreciably inhibited by the compounds, whereas the
dose administration (1.5 mg/kg body weight) are activity of both α-amylase and α-glucosidase were also
presented. The histology findings indicate liver tissue effectively inhibited by compounds 1 and 2, respectively.
damage in case of compounds 2 and 3 at all doses and Compounds 1 and 3 exhibited considerable antioxidant
compounds 1 and 5 at higher doses, and compound 4 potentials and also inhibited the activities of AChE and
showed no or less damage to the tissue. The findings of BuChE in an effective way. This provides a clue that these
these three studies suggest that compound 2 is most compounds may be effective in symptomatic treatment of
toxic of all, the toxicity is highest at 1.5 mg/kg body AD. Furthermore, a notable antibacterial activity was shown
weight of mice and 30 days, followed by compound 3, by compounds 2 and 4 pointing toward their possibility to
which is also highly toxic. Compound
5 showed be used in treating various disease caused by bacteria. The
moderate toxicity, and compound 1 had mildly toxic compounds were also found to have antilipidemic and anti-
effects. Compound 4 can be generally assumed as safe, hyperglycemic properties in vivo. To find out whether these
but it would be safer if used in lesser or moderate compounds are safe for the systemic use or not, they were
quantities.
evaluated for their toxic effect on different blood indices and
histology of liver. The in vivo studies showed that only
compound 4 can be assumed to be safe (safer in moderate
doses); compound 1 is mildly toxic and could be used only
in lower doses; and compounds 2, 3, and 5 are toxic and
should not be used at all. Further research and clinical trials
are needed to fully determine their biological spectrum.
4 Conclusions
The study was mainly devised to address three different yet
tangentially interlinked biological conditions, viz., type 2
diabetes, oxidative stress, and AD. The synthesized com-
pounds exhibited promising inhibitory activities against the
studied free radicals and enzymes. Of all the five enzymes,
highest in vitro inhibition against G6Pase was shown by all
the compounds in general and by compounds 2 and 4 in
Acknowledgment: The authors extend their appreciation
to the Umm Al-Qura University for providing funds.
Conflict of interest: The authors declare no conflicts of
interest.

Synthesis, characterization, and pharmacological evaluation of thiourea derivatives

777
References
[17] Zahoor M, Shafiq S, Ullah H, Sadiq A, Ullah F. Isolation of
quercetin and mandelic acid from Aesculus indica fruit and
their biological activities. BMC Biochem. 2018;19:5.
[18] Zahoor M, Zafar R, Rahman NU. Isolation and identification of
phenolic antioxidants from Pistacia integerrima gall and their
anticholine esterase activities. Heliyon. 2018;4:e01007.
[19] Nazir N, Zahoor M, Nisar M, Khan I, Karim N, Abdel-Halim H,
et al. Phytochemical analysis and antidiabetic potential of
Elaeagnus umbellata (Thunb.) in streptozotocin-induced
diabetic rats: pharmacological and computational approach.
BMC Compl Alt Med. 2018;18:332.
[1] Favre HA, Powell WH. Nomenclature of organic chemistry:
IUPAC recommendations and preferred names 2013.
Cambridge, UK: Royal Society Chemistry. 2013.
[2] Schroeder DC. Thioureas. Chem Rev. 1955;55:181–228.
[3] Mertschenk B, Knott A, Bauer W. Thiourea and thiourea
derivatives. Ullmann’s Encyclo Ind Chem. 2000; Jun 15:1–15.
[4] Schreiner PR. Metal-free organocatalysis through explicit
hydrogen bonding interactions. Chem Soc Rev.
2003;32:289–96.
[20] Zahoor M, Jan MR, Naz S. Investigation of repressive and
enhancive effects of fruit extracts on the activity of glucose-6-
phosphatase. Pak J Pharm Sci. 2016;29:1985–91.
[5] KILCIGİL GA, Altanlar N. Synthesis and antifungal properties of
some benzimidazole derivatives. Turk J Chem.
2006;30:223–8.
[21] Fiske CH, Subbarow Y. The colorimetric determination of
phosphorus. J Biol Chem. 1925;66:375–400.
[6] Fengling C, Yanrui C, Hongxia L, Xiaojun Y, Jing F, Yan L.
Interaction of APT with BSA or HSA. Chin Sci Bull.
2006;51:2201–7.
[7] Kaneko C, Sugimoto A, Tanaka S. A facile one-step synthesis
of cis-2-cyclopentene- and cis-2-cyclohexene-1,4-diols from
the corresponding cyclodienes. Synthesis. 1974;1974:876–7.
[8] Gupta D, Soman R, Dev S. Thiourea, a convenient reagent for
the reductive cleavage of olefin ozonolysis products.
Tetrahedron. 1982;38:3013–8.
[9] Alcolea V, Plano D, Karelia DN, Palop JA, Amin S, Sanmartín C,
et al. Novel seleno- and thio-urea derivatives with potent in
vitro activities against several cancer cell lines. Eur J Med
Chem. 2016;113:134–44.
[10] Rivera A, Maldonado M, Ríos-Motta J. A facile and efficient
procedure for the synthesis of new benzimidazole-2-thione
derivatives. Molecules. 2012;17:8578–86.
[11] Kulakov I, Nurkenov O, Akhmetova S, Seidakhmetova R,
Zhambekov Z. Synthesis and antibacterial and antifungal
activities of thiourea derivatives of the alkaloid anabasine.
Pharm Chem J. 2011;45:15–8.
[12] Mishra A, Batra S. Thiourea and guanidine derivatives as
antimalarial and antimicrobial agents. Curr Top Med Chem.
2013;13:2011–25.
[13] Hasanen J, El-Deen I, El-Desoky R, Abdalla A. Synthesis of
some nitrogen heterocycles and in vitro evaluation of their
antimicrobial and antitumor activity. Res Chem Interm.
2014;40:537–53.
[14] Pattan S, Kedar M, Pattan J, Dengale S, Sanap M, Gharate U,
et al. Synthesis and evaluation of some novel 2,4-thiazolidi-
nedione derivatives for antibacterial, antitubercular and
antidiabetic activities. India: Indian Journal of Chemistry;
2012. ISSN: 0975-0983.
[22] Zoccoli MA, Karnovsky ML. Effect of two inhibitors of anion
transport on the hydrolysis of glucose 6-phosphate by rat liver
microsomes. Covalent modification of the glucose 6-P
transport component. J Biol Chem. 1980;255:1113–9.
[23] Arion WJ, Canfield WK, Ramos FC, Su ML, Burger H-J, Hemmerle H,
et al. Chlorogenic acid analogue S 3483: a potent competitive
inhibitor of the hepatic and renal glucose-6-phosphatase systems.
Arch Biochem Biophys. 1998;351:279–85.
[24] Hemmerle H, Burger H-J, Below P, Schubert G, Rippel R,
Schindler PW, et al. Chlorogenic acid and synthetic chloro-
genic acid derivatives: novel inhibitors of hepatic glucose-6-
phosphate translocase. J Med Chem. 1997;40:137–45.
[25] Arika W, Nyamai D, Musila M, Ngugi M, Njagi E. Hematological
markers of in vivo toxicity. J Hematol Thrombo Dis. 2016;4:236.
[26] Sahibzada MUK, Sadiq A, Zahoor M, Naz S, Shahid M,
Qureshi NA. Enhancement of bioavailability and hepatopro-
tection by Silibinin through conversion to nanoparticles
prepared by liquid antisolvent method. Arab J Chem.
2020;13(2):3682–89.
[27] Khogali FA, Sheikh JB, Rahman SA, Rahim AA, Daghestani MH.
Histopathological and hematological effects of dimethoate 40EC
on some organs of albino mice. J King Saud Univ. 2005;18:73–87.
[28] Naz S, Zahoor M, Umar MN, Ali B, Ullah R, Shahat AA, et al.
Enzyme inhibitory, antioxidant and antibacterial potentials of
synthetic symmetrical and unsymmetrical thioureas. Drug Des
Dev Ther. 2019;13:3485.
[29] Peng L, Rong Z, Wang H, Shao B, Kang L, Qi H, et al. A novel
assay to determine acetylcholinesterase activity: the appli-
cation potential for screening of drugs against Alzheimer’s
disease. Biomed Chromatogr. 2017;31:e3971.
[30] Elalfy M, Aboumosalam M, Ali F. Biochemical, hematological
and pathological effects of bispyribac sodium in female albino
rats. J Vet Sci Technol. 2017;8:467.
[31] Zhang Z, Wu H-H, Tan Y-J. A simple and straightforward
synthesis of phenyl isothiocyanates, symmetrical and unsym-
metrical thioureas under ball milling. RSC Adv. 2013;3:16940–4.
[15] Ganesh A. Biological activities of some Pyrazoline derivatives.
Int J Pharm Bio Sci. 2013;4:727–33.
[16] Arden C, Petrie JL, Tudhope SJ, Al-Oanzi Z, Claydon AJ,
Beynon RJ, et al. Elevated glucose represses liver glucokinase
and induces its regulatory protein to safeguard hepatic
phosphate homeostasis. Diabetes. 2011;60:3110–20.