Enzyme inhibitory, antioxidant and antibacterial potentials of synthetic symmetrical and unsymmetrical thioureas

Article abstract of DOI:10.2147/DDDT.S225311

Background: In this study, 2 symmetrical and 3 unsymmetrical thioureas were synthesized to evaluate their antioxidant, antibacterial, antidiabetic, and anticholinesterase potentials. Methods: The symmetrical thioureas were synthesized in aqueous media in

Full text of DOI:10.2147/DDDT.S225311

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O R I G I N A L R E S E A R C H
Enzyme Inhibitory, Antioxidant And Antibacterial
Potentials Of Synthetic Symmetrical And
Unsymmetrical Thioureas
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
1
Background: In this study, 2 symmetrical and 3 unsymmetrical thioureas were synthesized
Sumaira Naz
1
to evaluate their antioxidant, antibacterial, antidiabetic, and anticholinesterase potentials.
Muhammad Zahoor
Muhammad Naveed Umar1
Methods: The symmetrical thioureas were synthesized in aqueous media in the presence of
1,2
3
sunlight, using amines and CS2 as starting material. The unsymmetrical thioureas were
synthesized using amines as a nucleophile to attack the phenyl isothiocyanate (electrophile).
The structures of synthesized compounds were confirmed through H1 NMR. The antioxidant
potential was determined using DPPH and ABTS assays. The inhibition of glucose-6-
phosphatase, alpha amylase, and alpha glucosidase by synthesized compounds was used as
an indication of antidiabetic potential. Anticholinesterase potential was determined from the
inhibition of acetylcholinesterase and butyrylcholinesterase by the synthesized compounds.
Results: The highest inhibition of glucose-6-phosphatase was shown by compound V (03.12
mg of phosphate released). Alpha amylase was most potently inhibited by compound IV with
IC50 value of 62 µg/mL while alpha glucosidase by compound III with IC50 value of
75 µg/mL. The enzymes, acetylcholinesterase, and butyrylcholinesterase were potently inhib-
ited by compound III with IC50 of 63 µg/mL and 80 µg/mL respectively. Against DPPH free
radical, compound IV was more potent (IC50 = 64 µg/mL) while ABTS was more potently
scavenged by compound I with IC50 of 66 µg/mL. The antibacterial spectrum of synthesized
compounds was determined against Gram-positive bacteria (Staphylococcus aureus) and
Gram-negative bacteria (Agrobacterium tumefaction and Proteus vulgaris). Compound I
and compound II showed maximum activity against A. tumefaction with MIC values of 4.02
and 4.04 µg/mL respectively. Against P. vulgaris, compound V was more active (MIC =
8.94 µg/mL) while against S. aureus, compound IV was more potent with MIC of 4.03 µg/mL.
Conclusion: From the results, it was concluded that these compounds could be used as
antibacterial, antioxidant, and antidiabetic agents. However, further in vivo studies are
needed to determine the toxicological effect of these compounds in living bodies. The
compounds also have potential to treat neurodegenerative diseases.
Barkat Ali
Riaz Ullah
3,4
Abdelaaty A Shahat
Hafiz Majid Mahmood
Muhammad Umar
Khayam Sahibzada
1Department of Chemistry, University of
Malakand Chakdara, Dir Lower, Kpk
18800, Pakistan; 2Department of
Chemistry, GC University Faisalabad,
Faisalabad, Punjab, Pakistan; 3Medicinal,
Aromatic and Poisonous Plants Research
Center (MAPRC), College of Pharmacy,
King Saud University, Riyadh 11451, Saudi
Arabia; 4Phytochemistry Department,
National Research Centre, Giza, Egypt;
5Department of Pharmacology, College
of Pharmacy, King Saud University, Riyadh
11451, Saudi Arabia; 6Department of
Pharmacy, Sarhad University of Science
and Information Technology, Peshawar,
Kpk 25000, Pakistan
5
6
Keywords: picolylamine, symmetrical thioureas, enzyme inhibition, anti-diabetic,
antioxidant, Alzheimer’s disease, antibacterial
Correspondence: Muhammad Umar
Khayam Sahibzada
Department of Pharmacy, Sarhad
University of Science and Information
Technology, Peshawar, KPK 25000,
Pakistan
Introduction
Thioureas are an important class of compounds that are used as intermediates/
precursors in synthesis of many synthetic drugs.1,2 A number of substituted deri-
vatives of thioureas can be synthesized by simple condensation of different primary
and secondary amines (aliphatic and/or aromatic) with iso-thiocyanate or its
derivatives.3 They can also be prepared by reaction of carbon disulphide and
Email umar.sahibzada@gmail.com
Muhammad Zahoor
Department of Chemistry, University of
Malakand Chakdara, Dir Lower, KPK
18800, Pakistan
Email mohammadzahoorus@yahoo.com
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amines (with or without catalyst).4 N,N′-disubstituted inhibited by medicines, its therapeutic importance increases
thioureas, whether aliphatic, aromatic or heterocyclic can
be used as building blocks in the synthesis of heterocyclic
compounds (having a variety of applications in different
fields). In organic synthesis, they played a very important
role as catalysts in important organic reactions (Mannich
and Biginelli reactions) and as chelating agents.5 The
diverse applications of these compounds in industry, agri-
culture, and medicine made them unique. Due to the pre-
sence of substituted sites in their structures, they have been
used in drug design and synthesis.6 Literature on thiourea
derivatives shows that they possess biological activities
like anti-bacterial,7 anti-fungal, anti-cancer, analgesic,
and antimalarial.8–13 They have also been used as inhibi-
tors of certain enzymes like acetylcholinesterase, butyryl-
cholinesterase, carbonic anhydrase etc.14–17
as it could hydrolyze the available acetylcholine, minimizing
the effect of the medicines, thus exaggerates the disease and
its inhibition is then desired.19,23
Diabetes mellitus is a metabolic disorder characterized
by hyperglycemia, impairment of carbohydrate, lipid and
protein metabolism, which is either due to insufficient
secretion of insulin or insulin resistance. It is considered
to be one of the chronic diseases after cancer and cardio-
vascular diseases. Among the different strategies used to
control hyperglycemia, inhibition of the key enzymes of
glucose metabolism like alpha amylase, alpha glucosidase
and glucose-6-phosphatase is considered to be the most
effective strategy.20 α-Amylase cleaves polysaccharides at
α-1,4 glycosidic linkages and the products (oligo and dis-
accharides) are further hydrolyzed to free glucose by
intestinal α-glucosidase. Controlling/lowering activity of
these two enzymes slows down the digestion of carbohy-
drates, absorption of glucose hence lowering the overall
blood glucose level.24,25 Glucose-6-phosphatase catalyses
the last step of both glycogenolysis (breakdown of glyco-
gen) and gluconeogenesis (synthesis of glucose from non-
carbohydrate sources) pathways, as a result glucose level
is maintained during fasting condition. Inhibition of this
enzyme is also considered to be a good therapeutic target
in disorders of carbohydrate metabolism and would be
helpful in minimizing the severity of fasting hyperglyce-
mia occurring in diabetic patients.26
Oxidative stress, diabetes mellitus, neurodegenerative
diseases, and antibacterial resistance are the burning issues
of this era. Free radicals are continuously produced during
normal metabolism and nearly 1/4th of the inhaled oxygen
is converted into free radicals, which have deteriorating
effects on biologically important molecules like protein
and DNA. Although human bodies are equipped with
efficient systems to cope with these free radicals, for the
last two decades, humans' dependence on synthetically
processed food has complicated the issue and the amount
of free radicals now produced is high as compared to the
body's free radical scavenging capacities. Therefore, there
is a need for certain potent compounds to be taken addi-
tionally, to help the body in scavenging the free radicals.
As mentioned earlier, thioureas have two substation sites
that could be useful in such type of research.18–21
Keeping in mind the therapeutic uses of thioureas, an
attempt was made to synthesize novel symmetric and
unsymmetric derivatives of thiourea and use them as
remedy for oxidative stress, diabetes mellitus, and neu-
rodegenerative disorders. The synthesized derivatives
were also evaluated for their antibacterial potential
against Gram positive and Gram negative bacterial
strains.
In neurodegenerative diseases, there is maximum hydro-
lysis of acetylcholine, the substance responsible for the trans-
mission of nerve impulses at synoptic gaps between the two
nerves. A practical approach applied to relieve the symptoms
of neurodegenerative diseases is the use of inhibitors
of acetylcholinesterase and butyrylcholinesterase (the
enzymes responsible for hydrolyses of neurotransmitter
acetylcholine).22 An example of neurodegenerative disease
is Alzheimer’s disease (AD) where an uncontrolled hydro-
lysis of the mentioned neurotransmitter occurs, rendering the
neurotransmission difficult or almost impossible. If acetyl-
cholinesterase is inhibited, the hydrolysis of acetylcholine
can be controlled which can be helpful in the symptomatic
relief of dementia or AD.18 Butyrylcholinesterase normally
has a less significant role in hydrolysis of acetylcholine, but
Experimental
Materials
All the chemicals, reagents, and solvents used were of
analytical grade and were used without any further purifi-
cation. They were purchased from Sigma Aldrich Co, St
Louis, MO, USA. The reaction progress was monitored
using thin layer chromatography (TLC). The structures of
1
synthesized compounds were confirmed by H-NMR in
in the diseased condition, when acetylcholinesterase is CDCl3 using NMR-Bruker apparatus.
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was obtained which was then reduced into amine. The
amine was purified further by column chromatography
using ethyl acetate and hexane as solvent system.27 The
product was obtained as yellowish viscous oil with 68%
yield.
Methods
Cinchona alkaloids are a group of naturally occurring
compounds with promising biological activities due to
the presence of primary and tertiary nitrogens in their
structures. Keeping in mind the importance of these alka-
loids, the simplest commercially available analog 2-picolyl
amine was taken and a simpler symmetrical thiourea pre-
sented as compound II was synthesized using CS2 (1
equiv) in water and in the presence of sunlight. After
confirmation that 2-picolyl amine has the capability of
forming thiourea, the starting material 2-picolyl amine
was derivatized to dibenzyl picolyl amine (Precursor P).
Compound I and III were then synthesized using dibenzyl
picolyl amine as starting material by two different methods
described in the following section. Compound IV was
prepared from 2,4-dimethyl aniline and phenyl isothiocya-
nate. Phenyl isothiocyanate is a commercially available
compound, while 2,4-dimethyl aniline was prepared from
2, 4-dimethyl nitrobenzene by reduction of nitro group
into amines by heterogenous catalyst under high pressure
in the presence of hydrogen. Compound V was prepared
from phenyl isothiocyanate and 2-fluoro aniline. The 2-
fluoro aniline was prepared by reduction of the nitro group
as described previously.
Synthesis Of Symmetrical Thioureas
(Compounds I And II)
Picolylamine/Picolylamine derivatives (1.0 mmol, 2.0
equiv) were mixed with carbon disulphide (0.5 mmol,
1.0 equiv) in 10 mL EtOH/H2O (1:1) and placed in sun-
light for 6–8 hrs. After 8 hrs, white precipitates were
formed which were confirmed by NMR as symmetrical
thioureas. The reaction mixture was kept at room tempera-
ture for 12 hrs until crystal formation.
Characterization Of Compound I
Brown crystals (yield 79%).
1HNMR (300 MHz, CDCl3-d) δ ppm: 4.5–4.7 (m, 8H),
5.2 (m, 2H), 5.4 (m, 2H), 6.8 (m, 4H), 6.9–7.1 (m, 8H), 7.2
(m, 8H), 7.5 (m, 2H), 7.7 (m, 2H), 8.1 (m, 2H), 8.5 (m,
2H), 8.9 (s, 2H). The chemical structure of compound I is
given in Figure 2.
Characterization Of Compound II
Needle-like crystals (81% yield).
1HNMR (300 MHz, CDCl3-d) δ ppm 3.2 (d, 4H), 6.4 (d,
4H), 6.5 (d, 2H), 7.3 (m, 4H), 8.2 (s, 2H). The structural
formula of compound II has been presented in Figure 3.
Preparation Of Dibenzyl Picolyl Amine (P)
The precursor dibenzyl picolyl amine was synthesized
starting from acetylpyridine as more readily available
starting material (Figure 1). The sodium hydride (NaH)
was used to abstract proton from acetyl pyridine to make
enolate which then attack on benzyl bromide (added drop-
wise). As a result, first substitution takes place. With
reaction progress under same condition, a second substitu-
tion takes place. The reaction product was then treated
with hydroxyl amine hydrochloride. As a result, oxime
NH2
N
Figure 2 1,3-bis(2-benzyl-3-phenyl-1-(pyridine-2-yl)propyl)thiourea (Compound I).
S
N
N
N
H
C
N
H
Figure 1 Chemical structure of 2-Benzyl-3-phenyl-1-(pyridin-2-yl)propan-1-amine
(Precursor P).
Figure 3 1,3-bis(1-(pyridin-2-ylmethyl)thiourea (Compound II).
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Synthesis Of Unsymmetrical Thioureas
(Compound III-V)
The required amines were mixed with phenyl iso-thiocya-
nate (both at the ratio of 1.0 equiv at a concentration of 1.0
mmol, 1.0) in 6 mL anhydrous acetone. The reaction
mixture was then refluxed for 8–10 hrs at 52–56°C.
Progress of the reaction was checked through TLC with
EtOAc/n-hexane solution (3:7). The reaction mixture was
then cooled using crushed ice. Precipitates formed were
then filtered. Filtered precipitates were washed with water,
dried and recrystallized using ethanol.
Figure 5 1-(2,4-dimethylphenyl)-3-phenylthiourea (Compound IV).
Characterization Of Compound III
Yellow crystals (yield 87%).
Acetylcholinesterase and butyrl choliesterase hydrolyses
their respective substrates acetylthiocholine iodide and butyr-
ylthiocholine iodide. The resulting product reacts with
5-thio-2-nitrobenzoate anion formed from DTNB resulting
in the formation of yellow color product. The color changes
were measured spectrophotometrically which were then con-
verted into enzyme activity and percent inhibition.
1HNMR (300 MHz, CDCl3-d) δ ppm: 2.32 (m, 1H), 2.59
(m, 2H), 2.77 (m, 2H), 3.08 (s, 1H), 7.04–7.28 (m, 15H), 7.37
(m, 2H), 7.65 (m, 2H), 7.82 (s, H, –NH), 8.47 (s, H, –NH).
Compound III is presented in Figure 4.
Characterization Of Compound IV
Yield: 93%.
1HNMR (300 MHz, CDCl3-d) δ ppm 2.30 (s, 3H), 2.34
(s, 3H), 7.05 (d, 1H), 7.11 (d, 1H), 7.20 (s, 1H), 7.25
(m, 1H), 7.35–7.42 (m, 4H), 7.97 (s, 2H). Compound IV
is presented in Figure 5.
Briefly, 205 µL compound dilutions (1000–125 µL/mL)
were mixed with 5 µL of AChE (0.03 U/mL)/BChE (0.01 U/
mL) and 5 µL DTNB and incubated in water bath at 30°C for
15 min. Then, 5 µL acetyl choline iodide/butyryl choline
iodide (substrate). As a result, formation of yellow color
anion (5-Thio-2-nitro benzoate) took place. The color change
was measured after 4 mins at 412 NM using a double beam
spectrophotometer (Thermo electron-corporation; USA). A
blank solution was considered as a control. Galanthamine
was used as positive control. Activity and inhibition of the
selective enzymes were calculated by following relations:
Characterization Of Compound V
Yield: 96%.
1HNMR (300 MHz, CDCl3-d) δ ppm 7.0–7.49 (m, 9H),
7.8 (s, 1H, –NH), 7.9 (s, 1H, –NH). The chemical structure
of compound V is given in Figure 6.
V ¼ Δ Abs=Δ t
(1)
Anticholinesterase Assay
The acetylcholinesterase inhibitory potential of the synthe-
sized compounds was examined using Ellman’s assay.28
V
% enzyme activity ¼
ꢀ 100
(2)
Vmax
% enzyme inhibition ¼ 100 ꢁ % enzyme activity (3)
Figure 4 1-(2-benzyl-3-phenyl-1-(pyridine-2-yl) propyl)-3-phenylthiourea (Compound
III).
Figure 6 1-(2-fluorophenyl)-3-phenylthiourea (Compound V).
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Where, V is the inhibitor dependent rate of reaction while, the reaction, and inorganic phosphate released during the reac-
Vmax is the inhibitor independent rate of reaction.
tion was measured by method of Fiske and Subbarow.29
Free Radical Scavenging Activities Of
Synthesized Compounds
Antidiabetic Potential Of The Synthesized
Compounds
The free radical scavenging activities of synthesized
compounds were determined following standard DPPH
and ABTS protocols.10 DPPH stock solution was pre-
pared by dissolving 20 mg of DPPH in 100 mL metha-
nol. Blank (control) solution was prepared by taking 3
mL from the DPPH solution and its absorbance was
adjusted to 0.75 at 515 nm. For the development of
free radicals in stock solution, it was covered and kept
in the dark for about 24 hrs. Stock solution (in 5 mL of
methanol) of each compound was prepared and a series
of dilutions (1000, 500, 250, 125, 62.5 µg/mL) were
then prepared from it, using dilution formula. About 2
mL from each dilution was mixed with 2 mL DPPH and
allowed to react for 15 mins in the dark. The percent
inhibition by DPPH was calculated with the following
equation:
The antidiabetic potential of synthesized compounds was
checked by monitoring their inhibitory effects on three
important enzymes of carbohydrate metabolism viz alpha
amylase, alpha glucosidase, and glucose-6-phosphatase.
The stock solutions of the compounds were prepared in
10% DMSO and phosphate buffer (pH 6.9) having a concen-
tration of 20 mm. Different dilutions (1000–125 mg/mL) were
prepared for each compound. About 200 µL of these dilutions
was mixed with 200 µL of porcine α-amylase (2 U/mL) and
pre-incubated for 10 mins at 30°C. Added to the mixture, was
200 µL of starch solution (1% w/v, made in 20 mM phosphate
buffer, pH 6.9) and it was further incubated for 3 mins at 30°C.
The reaction was stopped by adding 1 mL of 96 mM 3,5dini-
trosalicylic acid (DNS). Again, the reaction mixture was incu-
bated for 10 mins in boiling water bath (85–90°C) and then
cooled at room temperature. Acarbose was used as positive
control. Absorbance was monitored at 540 nm and percent
inhibition was calculated using the following equation:
A ꢁ B
% inhibition ¼
X 100
(5)
A
Where; A = absorbance of pure DPPH in oxidized form.
B = absorbance of sample taken after 15 mins of
reaction with DPPH.
Control sample absorbance ꢁ Test
sample absorbance
% inhibition ¼
ꢂ 100
Control absorbance
(4)
Ascorbic acid (5 mg/5 mL) was taken as a standard and its
different solutions (1000, 500, 250, 125 µg/mL) were
prepared.
The α-glucosidase inhibition was carried out by mixing 100 µL
of enzyme stock solution (0.5 unit/mL) with 600 µL stock
phosphate buffer (pH 6.9) and 50 µL of each dilution prepared
in the previous step and incubated at 37°C for 15 mins. To start
the enzymatic reaction, 100 μL p-nitro-phenyl-α-D-glucopyr-
anoside (5 mM) was added to the mixture which reached
completion after 15 mins incubation at 37°C. To stop the
reaction, 400 μL sodium carbonate (0.2 M) solution was
added to the mixture. The absorbance of resulting mixture
was measured at 405 nm. Percent inhibition of glucosidase
was calculated using Formula 4. Acarbose was used as positive
control and the same dilution was prepared by serial dilution.
The activity of the glucose-6-phosphatase was measured,
which is based on the release of inorganic phosphate when the
substrate glucose-6-phosphate is hydrolyzed by the enzyme.
About 1 mL of 1 mM of the enzyme was taken, to which 1.5
mL tris (hydroxy methyl) amino methane buffer of pH 6.7 and
The ABTS free radical scavenging was carried out
using standard protocol of Re et al.30 About 7 mM of
ABTS and 2.45 mM of potassium per sulphate were dis-
solved, each in 100 mL of methanol and were then mixed
together. To develop free radicals of ABTS, the reaction
mixture was kept in the dark for 12 hrs. Absorbance of
blank control (3 mL) was adjusted to 0.75 at 745 nm by
diluting it with 50% methanol. About 300 μL of each
sample was taken, mixed with 3 mL of ABTS solution,
and incubated for 15 mins at 25°C. Absorbance of the
incubated mixture was measured at 745nm. The same
procedure was done to prepare various dilutions of ascor-
bic acid (positive control). Percent free radical inhibition
was calculated using Equation (5).
2.5 mL of 0.01 mM glucose-6-phosphate were added. The Anti-Bacterial Activities
reaction mixture was incubated at 33°C for 1 hr. After the The anti-bacterial activities of the synthesized compounds
incubation, 1 mL 10% trichloroacetic acid was added to stop were
determined
against
Staphylococcus
aureus
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Table 2 Effect Of The Compounds On The Activity Of
Butrylcholinesterase Enzyme
(Gram-positive bacteria), Agrobacterium tumefaction, and
Proteus vulgaris (Gram-negative bacteria) bacterial strain.
Agar well diffusion method was used following the standard
protocol.31
Compound Concentration
(µg/mL)
%BChE Inhibition
(mean SEM)
IC50
µg/mL
I
1000
500
250
125
57.81 0.54
56.26 1.66
51.40 2.05
48.13 1.08
200
Statistical Analysis
All the experiments were performed in three replicates
and the results have been presented as mean SEM.
II
1000
500
250
125
70.13 1.51
62.31 2.11
59.15 1.60
65.71 1.28
82
Results And Discussion
Effect Of Synthesized Compounds On
Acetylcholinesterase And
Butyrylcholinesterase
The effectiveness of the synthesized compounds as inhi-
bitors of the selected cholinesterases was tested. The
activities of both the enzymes were suppressed to a
lesser or greater extent. The results are presented in
Tables 1 and 2 respectively, for acetylcholinesterase
III
1000
500
250
125
68.55 1.07
63.83 1.53
61.81 1.54
58.26 1.06
80
IV
1000
500
250
125
76.14 1.90
60.50 1.61
53.55 1.07
49.83 1.53
150
100
V
1000
500
250
125
62.67 1.22
59.07 1.76
55.09 1.04
53.07 0.48
Table 1 Effect Of The Compounds On The Activity Of
Acetylcholinesterase Enzyme
Compound Concentration
(µg/mL)
%AChE Inhibition
(mean SEM)
IC50
µg/mL
Galantamine
1000
500
250
125
92.30 0.74
87.16 1.23
78.42 0.75
64.81 0.73
55
I
1000
500
250
125
68.11 1.40
55.59 1.23
46.10 1.85
38.63 1.33
1002
II
1000
500
250
125
78.67 1.20
72.16 2.01
59.21 1.21
52.55 1.73
82
and butyrylcholinesterase. As is clear from Table 1,
compound III was more potent against acetylcholines-
terase with IC50 value of 63 µg/mL, followed by com-
pound II (IC50 = 82 µg/mL). Compound V was
moderately active with IC50 value of 182 µg/mL while
compound VI and I were merely active (IC50 = 503 and
1002 µg/mL respectively).
III
1000
500
250
125
84.31 1.12
75.66 1.15
66.47 2.08
62.22 1.12
63
The butyrylcholinesterase inhibitory activities of the
synthesized compounds are presented in Table 2. Almost the
same trend observed for acetylcholinesterase was also
observed here with little variations. Compound III was more
active with IC50 value of 80 µg/mL, followed by compound II
with IC50 = 82 µg/mL. Comparatively improved activities
were observed for compound I, IV, and V with IC50 values
of 200, 150, and 100 µg/mL respectively.
IV
1000
500
250
125
64.02 1.70
50.04 1.88
45.15 1.83
41.99 1.01
503
182
V
1000
500
250
125
70.25 1.09
68.15 1.25
56.34 2.14
51.78 1.72
Galantamine
1000
500
250
125
93.22 0.81
88.14 0.52
78.44 0.61
64.53 0.52
Based on the results obtained it can be concluded that
compound III and II have some therapeutic values in the
treatment of neurodegenerative diseases. However, in vivo
studies regarding their toxicology are needed.
40
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Table 4 Effect Of The Compounds On Activity Of Enzyme
Alpha Glucosidase
Effect Of Synthesized Compounds On
The Activity Of Alpha Amylase
Compound Concentration
(µg/mL)
%Inhibition
IC50
Alpha amylase is a key enzyme of carbohydrate metabo-
lism that makes glucose available for the absorption in the
small intestine from starch sources. Inhibition of this
enzyme would be helpful in lessening the burden of
blood glucose in diabetic patients. Table 3 shows the
inhibitory potential of the compounds on alpha amylase.
Compound IV with IC50 value of 62 µg/mL was the most
potent, followed by compound I (IC50 = 84 µg/mL).
Compound II, III, and V were also quite potent, but less
than IVand I with IC50 values of 135, 115, and 100 µg/mL
respectively. All these compounds have inhibitory effects
of alpha amylase which is clear from their IC50 values.
(mean SEM)
µg/mL
I
1000
500
250
125
51.13 0.15
47.81 2.25
46.04 1.58
40.04 0.10
1000
II
1000
500
250
125
72.02 1.70
71.04 1.88
70.15 1.83
69.99 1.01
80
III
1000
500
250
125
84.67 1.20
81.16 2.01
79.21 1.21
75.67 1.20
75
Effect Of Synthesized Compounds On
The Activity Of Alpha Glucosidase
Glucosidase is another important enzyme for carbohydrate
metabolism and it helps in glucose release from starch
IV
1000
500
250
125
65.92 0.23
60.46 0.083
55.88 2.04
48.00 1.34
180
500
60
V
1000
500
250
125
57.18 1.46
52.14 3.93
40.45 1.59
32.49 2.07
Table 3 Effect Of The Compounds On The Activity Of Alpha
Amylase Enzyme
Compound Concentration
(µg/mL)
%Inhibition
IC50
(mean SEM)
µg/mL
Acarbose
1000
500
250
125
94.02 1.23
79.11 2.12
68.14 2.18
59.83 2.04
I
1000
500
250
125
70.29 0.31
68.08 0.87
63.15 1.41
60.23 1.03
84
II
1000
500
250
125
59.13 0.15
57.81 2.25
56.04 1.58
50.04 0.10
135
115
62
sources. In diabetic patients, its inhibition relieves the
blood glucose burden. Compound III and II were more
effective inhibitors of alpha glucosidase with IC50 values
of 75 and 80 µg/mL respectively. Compound IV appeared
moderately active with IC50 value of 180 µg/mL.
Compound I and V were merely active with IC50 values
of 500 and 1000 µg/mL respectively (Table 4).
III
1000
250
250
125
65.82 1.13
62.49 1.24
57.91 0.43
50.07 2.11
IV
1000
500
250
125
80.12 2.02
79.18 1.09
75.63 0.63
69.04 1.19
Effect Of Synthesized Compounds On
The Activity Of Glucose-6-Phosphatase
Glucose-6-phosphatase is another important enzyme of
carbohydrate metabolism that plays a very important role
in glycogenolytic and gluconeogenic pathways. Its inhibi-
tion is needed in case of diabetes mellitus and enhance-
ment of its activity is desired in patients suffering from
glycogen storage disease. The inhibition of this enzyme is
interpreted from the mg of inorganic phosphate released
during the reaction. The smallest amount of phosphate
released in a given reaction indicates high inhibition.
V
1000
500
250
125
62.82 1.13
59.49 1.24
55.91 0.43
52.07 2.11
100
50
Acarbose
1000
500
250
125
88.09 1.13
72.98 1.34
68.04 1.88
58.13 1.34
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Table 5 Effect Of The Compounds On Activity Of Enzyme
Glucose-6-Phosphatase
from Table 6. Compound IV was a more potent inhibitor
of DPPH free radical (IC50 = 64 µg/mL) followed by
compound I (IC50 = 67 µg/mL). Compound V and III
were also quite effective in scavenging the DPPH radical
and exhibited IC50 values of 70 and 79 µg/mL respectively.
The least effective compound was compound II.
Compound Concentration
µg/mL
Total
Control
Activity*
I
1000
500
250
125
21.20
25.31
31.62
34.11
72.0
A totally different inhibitory pattern was observed in
case of ABTS free radical. Compound I was more potent
with IC50 value of 66 µg/mL, followed by compound III
and V (IC50 = 80 and 85 µg/mL respectively). Compound
IV was moderately effective while compound II was the
least effective one.
II
1000
500
250
125
08.78
10.07
13.10
15.60
72.0
72.0
72.0
72.0
III
IV
V
1000
500
250
125
24.23
27.10
28.35
32.04
Anti-Bacterial Activities Of The
Compounds
The antibacterial spectrum of the synthesized compounds
was determined against A. tumefaction, P. vulgaris, and
S. aureus. The results have been presented in Table 7.
Among the tested compounds, compound I was highly
active against A. tumefaction with MIC value of
1000
500
250
125
10.10
11.77
13.58
17.70
1000
500
250
125
03.12
04.16
05.21
05.66
4.02 µg/mL followed by compound II (MIC
=
4.02 µg/mL). Against the same bacteria, compound III,
IV and V were also quite effective with MIC values of
11.62, 13.49, and 11.59 µg/mL respectively.
Note: *The activity has been expressed as mg of P released from the potassium salt
of glucose-6-phosphate per hour (at 33°C, pH 6.7).
Against P. vulgaris, compound V was more effective
followed by compound I (MIC = 8.94 and 9.76 µg/mL
respectively). The rest of the compounds were also quite
effective against this bacterial strain.
Compound V and II were the most potent inhibitors of this
enzyme as the amount of phosphate released was compara-
tively less than that of other compounds (Table 5).
Compound I, III, and V were also quite potent when
compared to control experiments. As stated earlier, enhan-
cers of this enzyme are also required in case of glycogen
storage disease patients, however, even though all of these
compounds were inhibitors of this enzyme, none of them
exhibited the enhanced role.
The growth of S. aureus was more effectively inhibited
by compound IV (MIC= 4.03 µg/mL). The rest of the
compounds were moderately active. Comparatively, all
these compounds exhibited good antibacterial actions
against all the selected bacterial strains.
Conclusion
The wide applicability of thioureas in various fields, espe-
cially in the field of medicines, makes it quite practical to
study biological activities of an already synthesized com-
pound or to synthesize some more and determine their ther-
apeutic significance, if any. In this study, five new thioureas
were synthesized from chiral amines in the presence of sun-
light (hence eco-friendly and economic procedure) which
were then used as starting material in the synthesis of N-
heterocyclic thiourea compounds. The compounds were
tested for their antibacterial, antioxidant, anticholinesterase,
and antidiabetic potentials. Glucose-6-phosphatase was
Free Radical Scavenging Activities Of The
Compounds
Free radicals are the chemically reactive substances with
singlet electron. Free radicals are continuously produced in
the human body and are promptly scavenged by the body's
defense system. However, when their production increases,
supplementation of antioxidants are needed from outside,
otherwise they damage the biologically important molecules
(DNA and protein). All these synthesized compounds
showed scavenging activities against the commercially
available free radicals DPPH and ABTS, which is clear potently inhibited by compound V and II, α-amylase by
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Table 6 Estimation Of Free Radical Scavenging Potential Through DPPH And ABTS Assays
Compound
Concentration μg/mL DPPH % I
(mean S.E.M)
DPPH IC50 μg/mL ABTS % I
(mean S.E.M)
ABTS IC50 μg/mL
I
1000
500
250
125
86.67 1.20
67
120
79
64
70
35
81.67 1.20
66
82.16 1.01
80.21 1.21
78.55 1.73
80.16 2.01
78.21 1.21
77.55 1.93
II
1000
500
250
125
57.59 1.23
54.10 1.85
52.63 1.33
50.02 1.99
55.67 1.12
52.07 1.16
49.09 1.04
47.07 0.46
400
80
III
IV
V
1000
500
250
125
67.45 0.095
66.07 0.283
64.74 0.267
61.14 0.177
69.11 0.234
68.27 0.532
66.25 0.123
64.77 0.087
1000
500
250
125
79.15 1.23
74.13 1.51
69.31 2.11
66.15 1.70
78.15 1.21
74.99 1.51
70.13 0.05
64.81 1.05
100
85
1000
500
250
125
73.14 1.10
71.50 1.61
70.55 1.27
68.83 1.53
75.04 0.10
73.18 1.46
71.14 1.90
69.45 1.29
Ascorbic acid 1000
90.14 0.76
85.11 1.64
76.13 1.45
67.40 1.18
88.17 0.43
85.45 1.36
70.14 1.16
55.30 1.75
55
500
250
125
Table 7 Evaluation Of Antibacterial Potential Of The Synthesized Compounds
Samples
Bacterial Species
Conc. (μg/mL)
Agrobacterium tumefaction
Proteus vulgaris
Staphylococcus aureus
ZI (mm)
MIC µg/mL
ZI (mm)
MIC µg/mL
ZI (mm)
MIC µg/mL
Compound I
Compound II
Compound III
Compound IV
Compound V
10
20
30
21
24
26
4.02
15
22
24
9.76
13
16
18
19.58
10
20
30
21
23
26
4.04
12
22
26
12.22
14.96
11.92
8.94
12
19
23
14.83
14.93
4.03
10
20
30
15
20
26
11.62
13.49
11.59
12
16
22
15
16
19
10
20
30
14
18
23
14
18
23
21
23
26
10
20
30
15
20
25
16
21
24
12
14
16
24.11
Abbreviations: ZI, Zone of inhibition; MIC, Minimum inhibitory concentration.
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Author Contributions
S.N. and M.Z. conceived and designed the experiments; M.
N.U., B.A., R.U. and M.U.K.S. performed the experiments;
M.Z., A.A.S and H.M.M. analyzed the data; M.Z., and M.
U.K.S contributed reagents and materials. All authors con-
tributed to data analysis, drafting or revising the article,
gave final approval of the version to be published, and
agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work.
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