Benzimidazole tethered thioureas as a new entry to elastase inhibition and free radical scavenging: Synthesis, molecular docking, and enzyme inhibitory kinetics

Article abstract of DOI:10.1002/jhet.4318

The porcine pancreatic elastase inhibition and free-radical scavenging play a crucial role in age progression. All the series of 10 newly synthesized benzimidazole thioureas (4a-j) were assessed for elastase inhibition and radical scavenging activity to identify the suitable anti-aging ingredient for cosmetics products. The compounds 4e, 4f, 4g, and 4h showed inhibition better than the standard, while compound 4f showed the most significant elastase inhibition with the IC₅₀ value of 1.318 ± 0.025 μM compared with oleanic acid IC₅₀ 13.451 ± 0.014 used ±1.989 and 41.563 ± 0.824, respectively, as standard. Molecular docking studies were performed and the compound 4f showed binding energy of 7.2 kcal/mol. Kinetics studies revealed inhibition of the pancreatic elastase in a competitive manner. The relative binding energy and structure activity relationship (SAR) identified compound 4f as an effective inhibitor of porcine pancreatic elastase. Compounds 4e and 4i showed remarkable free-radical scavenging activity with SC₅₀ values of 26.421.

Full text of DOI:10.1002/jhet.4318

Benzimidazole Tethered Thioureas as a New Entry to Elastase inhibition and Free
Radical Scavenging: Synthesis, Molecular docking, and Enzyme Inhibitory Kinetics
Rabail Ujana, Pervaiz Ali Channarb, Aamer Saeedb*, Qamar Abbasc, Hummera Rafiqued,
Saba Ashrafb, Mahboob Ali Rinda, Abbas Hassanb, Anwar Ul-Hamide, Mubashar Hassang
Hussain Razaf, Sung-Yum Seof
a Dr. M. A. Kazi Institute of Chemistry, University of Sindh, Jamshoro, Pakistan
b Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
cDepartment of Biology, College of Science, University of Bahrain, Sakhir, Kingdom of Bahrain
d Department of Chemistry, University of Gujrat, Gujrat, Pakistan
dDepartment of Chemistry, University of Agriculture, Faisalabad, 38040, Pakistan
e Core Research Facilities, King Fahd University of Petroleum & Minerals, Dhahran 31261, Saudi Arabia.
fDepartment of Biological Sciences, College of Natural Sciences, Kongju National University, 56 Gongjudehak-
Ro, Gongju, Chungnam 314-701, Republic of Korea
g Institute of Molecular Biology and Biotechnology/(IMBB), The University of Lahore, 1-KM, Defense Road,
Bhubtian Chowk, Lahore, Pakistan
* Address for Correspondence
Aamer Saeed
Email: asaeed@qau.edu.pk, aamersaeed@yahoo.com
Contact number: +92-51-9064-2128, Fax: +92-51-9064-2241
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1002/jhet.4318
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Graphical Abstract
Benzimidazole Tethered Thioureas as a New Entry to Elastase inhibition and Free
Radical Scavenging: Synthesis, Molecular docking, and Enzyme Inhibitory Kinetics
Rabail Ujana, Pervaiz Ali Channarb, Aamer Saeedb*, Qamar Abbasc, Hummera Rafiqued, Saba Ashrafb,
Mahboob Ali Rinda, Abbas Hassanb, Anwar Ul-Hamide, Mubashar Hassang Hussain Razaf, Sung-Yum Seof
This article is protected by copyright. All rights reserved.

Benzimidazole Tethered Thioureas as a New Entry to Elastase inhibition and Free
Radical Scavenging: Synthesis, Molecular docking, and Enzyme Inhibitory Kinetics
Rabail Ujana, Pervaiz Ali Channarb, Aamer Saeedb*, Qamar Abbasc, Hummera Rafiqued,
Saba Ashrafb, Mahboob Ali Rinda, Abbas Hassanb, Anwar Ul-Hamide, Mubashar Hassang
Hussain Razaf, Sung-Yum Seof
a Dr. M. A. Kazi Institute of Chemistry, University of Sindh, Jamshoro, Pakistan
b Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
cDepartment of Biology, College of Science, University of Bahrain, Sakhir, Kingdom of Bahrain
d Department of Chemistry, University of Gujrat, Gujrat, Pakistan
dDepartment of Chemistry, University of Agriculture, Faisalabad, 38040, Pakistan
e Core Research Facilities, King Fahd University of Petroleum & Minerals, Dhahran 31261, Saudi Arabia.
fDepartment of Biological Sciences, College of Natural Sciences, Kongju National University, 56 Gongjudehak-
Ro, Gongju, Chungnam 314-701, Republic of Korea
g Institute of Molecular Biology and Biotechnology/(IMBB), The University of Lahore, 1-KM, Defense Road,
Bhubtian Chowk, Lahore, Pakistan
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Abstract
The porcine pancreatic elastase inhibition and free-radical scavenging play a crucial role in
age progression. All the series of ten newly synthesized benzimidazole thioureas (4a-j) were
assessed for elastase inhibition and radical scavenging activity to identify the suitable anti-
aging ingredient for cosmetics products. The compounds 4e, 4f, 4g, and 4h showed inhibition
better than the standard whilst compound 4f showed the most significant elastase inhibition
with the IC50 value of 1.318 ± 0.025 µM compared with oleanic acid IC50 13.451 ± 0.014
used ± 1.989 and 41.563 ± 0.824, respectively.as standard. Molecular docking studies were
performed and the compound 4f showed binding energy of 7.2 Kcal/mol. Kinetics studies
revealed inhibition of the pancreatic elastase in a competitive manner. The relative binding
energy and SAR identified compound 4f as an effective inhibitor of porcine pancreatic
elastase. Compounds 4e and 4i showed remarkable free-radical scavenging activity with SC50
values of 26.421
Keywords: Elastase inhibition; benzimidazole thioureas; radical scavenging activity;
structure-property relationship.
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1. Introduction
Skin aging is a common occurrence witnessed with the increase in human age. It is reflected
by wrinkling, loss of elasticity, laxity, rough-textured look together with phenotypic changes.
Various cosmetic products are used to break this process. Elastin is a major part of the elastic
fibers in animal tissues primarily responsible for the skin elasticity. The skin elasticity is
reduced by the inhibition of elastin fibers by the enzyme Elastase. Therefore Inhibition of this
enzyme can assist to avoid the skin aging process [1]. On the contrary, Exposure of skin to
sunlight and UV radiations can cause free radicals to react with the skin epidermal cells
resulting in oxidative damage [2,3]. Therefore, elastase inhibition and free-radical scavenging
processes are vital to the skin health [4].
Amongst the most widely employed for both inhibition of porcine pancreatic elastase as well
as free radical scavenging are natural polyphenolic compounds such as phenolic acids,
flavonoids, stilbenes and lignans [5-7]. Peel and seed extract of Passiflora edulis were tested
against porcine pancreatic elastase and found to be potent anti-aging ingredients for
cosmetics [8]. Similarly, flavonoids Scutellarin and apigenin and other phytochemicals have
also been employed [9].
Over and above natural products, several synthetic molecules have also been used for both
purposes. We have already identified densely substituted piperidines and 1,4-
dihydropyridines as innovative skin shielding and anti-aging agents [10-11]. Alternatively,
quinolyl based acyl thioureas, and valeroyl-3-arylthioureas have been confirmed as efficient
free radical scavengers. [12-14]. Benzimidazole is a bicyclic heterocyclic motif having a
range of beneficial uses cover antitumor, antifungal, antiparasitic, analgesics, antiviral,
antihistamine, along with cardiovascular, neurological, and ophthalmological diseases [15].
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Keeping in view the importance of elastase inhibitors and free-radical scavengers in the anti-
aging treatment of skin on one hand and the biological significance of benzimidazoles and
thioureas on the other, we designed and synthesized benzimidazole based thioureas as a novel
entry to elastase inhibitors and free-radical scavengers. These compounds contain
benzimidazole as main core along with thiourea linker and the aryl or alkyl group for
structural variation in a single structural unit to obtain compounds as dual inhibitors of
elastase as well as free radicals (Figure1).
Figure 1
Molecular architecture of target compounds
Newly prepared benzimidazole fused thioureas were obtained in good yields, purified by
recrystallization, and characterized by spectroscopic techniques and evaluated for the
aforesaid biological potential.
2. Results and discussion
2.1
Synthesis
Scheme1, indicates the synthetic pathway adopted for the preparation of benzimidazole
linked thioureas. Accordingly, freshly prepared acid chlorides were added to a solution of
potassium thiocyanate in anhydrous acetone to yield isothiocyanates intermediates (2a-j).
After cooling, a solution of benzimidazolyl amine (3) in dry acetone was added dropwise and
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the mixture under reflux to afford benzimidazole-thiourea derivatives (4a-j) in good yields.
FTIR analysis revealed that distinctive absorption bands for N-H at 3215-3350 cm-1, C-H
aromatic at 3033-3080 cm-1, C=O of carbonyl at 1680-1748 cm-1, C=N of imine at 1542-1694
1
cm-1 and C=C (Ar) 1532-1575. The H NMR spectrum of a specific compound 4j signifies
three distinctive broad singlets for N-H protons at δ 12.79, 11.66 ppm, 10.98 ppm, and
13
multiplets for aromatic protons at δ 8.02-7.93 and δ 7.68-7.21 ppm. C NMR spectrum of
compound 4j revealed signals for thiocarbonyl at δ 179.34, and carbonyl carbon at δ 168.75
ppm, while the imino carbon resonated at δ 151.15 ppm.
Scheme 1: Synthetic route of benzimidazole fused thioureas (4a-j)
The molecular structures of benzimidazole-thioureas (4a-j) are shown in Figure 2.
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Figure 2: Molecular structures of benzimidazole-thioureas (4a-j)
2.2 Porcine pancreatic elastase inhibition
Table.1 lists the Elastase and Free radical scavenging inhibitory activity of the synthesized
derivatives (4a-j). Compounds 4e 4f, 4g and 4h indicated improved inhibition than the
standard whilst compound 4f having a C-8 alkyl side chain showed considerable elastase
inhibition with IC50 value of 1.318 ± 0.025 µM compared to the Oleanolic Acid, with IC50
value 13.451 ± 0.014 µM. The presence of a C-8 alkyl side chain adopts a conformation
suitable for binding to active site of enzyme. Thus, varying the alkyl chain length on the
hydrophobic tail can selectivity toward inhibition of elastase. Increasing or decreasing the
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length of the hydrophobic tail away from eight carbons leads to a decrease enzyme inhibition.
It can be seen that C-8 chain interacts with histidine and valine residues in the docking
complex (Fig S6). The relative binding energy and SAR identified compound 4f as a potent
inhibitor of elastase.
2.3 Free radical scavenging
All the synthesized (4a-j) compounds were evaluated for DPPH free radical scavenging
ability. The compounds 4e and 4h showed some activity, however rest of the compounds did
not show significant radical scavenging activity even at the higher concentration (100µg/mL)
(Table 1). These molecules 4e having a phenyl ring substituted at 3 and 5 positions with
electron withdrawing NO2 and 4h possessing a C-5 alkyl side chain showed noteworthy free-
radical scavenging activity with SC50 value of 26.421 ± 1.989 and 41.563 ± 0.824 µM
respectively compared to Ascorbic acid. It indicates that presence of strong electron
withdrawing NO2 group helps in free radical scavenging potential.a C-5 alkyl side chain
adopts a conformation suitable for DPPH free radical scavenging. Similarly, presence of
electron withdrawing NO2 at 3 and 5 position of the phenyl ring can enhance the potency of
synthesized derivatives.
Table.1 Porcine pancreas elastase and Free radical scavenging activity of derivatives (4a-j)
Elastase IC50± SEM
(µM)
Compound
Free radical
scavenging
%
(100 µg/mL)
9.428 ± 0.745
16.136 ± 0.991
12.737 ± 1.558
6.032 ± 0.887
30.159 ± 0.112
39.005 ±0.841
4.502 ± 0.095
14.988 ± 0.967
4a
4b
4c
4d
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54.307 ± 1.719
1.318 ± 0.025
3.769 ± 0.068
8.355 ± 0.081
67.806 ± 2.81
10.532 ± 1.083
13.451 ± 0.014
26.421 ± 1.989
4.303 ± 0.259
8.386 ± 0.554
41.563 ± 0.824
2.656 ± 0.985
16.915 ± 0.519
----------------
4e
4f
4g
4h
4i
4j
Oleanolic
Acid
----------------
94.823 ± 0.046
Vitamin C
SEM = Standard error of the mean; values are expressed in mean ± SEM.
2.4
Kinetic Mechanism
To understand the inhibitory style of the synthetic compounds versus elastase inhibition,
kinetic study was carried out. On the basis of IC50 the most potent compound 4 f was chosen
to determine the type of inhibition and inhibition constant. The kinetic results of the enzyme
by the Lineweaver-Burk plot of 1/V versus substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide
1/[S] in the presence of different inhibitor concentrations gave a series of straight lines.
Lineweaver-Burk plot of compound 4f indicated that Vmax remains the equal without much
effecting the slopes. Km increases with increasing concentration while Vmax remains the same
with insignificant difference. This behavior indicates that 4f compound inhibits the enzymes
in a competitive manner (Figure. 3a), second plot (Figure. 3b) of slope against concentration
of 4f showed EI dissociation constant. Ki was calculated from inhibitor concentration of 4f
versus the slope and Ki was 1.9 µM.
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Figure 3. Lineweaver–Burk plots for inhibition of elastase from porcine pancreas in the
presence of Compound 4f (A) Concentrations of 4f were 0.00, 0.659, 1.318 and 2.636 µM,
Substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide concentrations were 2, 1, 0.5, 0.25, 0.125
and 0.0625 mM. (B) The insets represent the plot of the slope.
4.0
4.1
Molecular docking analysis
Docking energy analyses of synthesized compounds
To envisage the best tailored conformation of compounds (4a-j) within the active zone of
protein molecular docking analysis was executed. The docked complexes were evaluated
based on minimum energy values (Kcal/mol) as H- and hydrophobic interactions. Docking
results indicated that all compounds have good energy values (Fig 3). Nonetheless, all
compounds revealed good binding energy values (Kcal/mol) against receptor with slight
variations. The standard error mean of docking energy for Autodock is 2.5 Kcal/mol.
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Fig 3. The graphical depiction of docking energy values.
5.
Binding pocket and ligand conformational analysis
The docked complexes were analyzed on the basis of hydrogen and hydrophobic bonding
interactions. The best in vitro result compound was designated to check best conformational
position inside active site of the protein. The binding pocket analysis showed that 4f binds
within the active site of protein (Fig 4).
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Figure 4. Binding pocket depiction of target protein
The comprehensive docking analysis showed that in 4f docking complex three H-bonds in the
active region of protein. The amino group made good interactive bond against Thr41 with
bond distances 2.73 Å. Even though, oxygen atom of 4f formed couple of hydrogen bonds
Asp60 and Arg61 with bond distances 2.31 and 2.95 Å, respectively (Figure 5). Literature
data also confirmed the value of these residues in bonding with other elastase inhibitors
which strengthens the docking results [25]. The relative binding energy and SAR showed 4f
to be considered as potent inhibitor. The other docking complexes are mentioned in the
supplementary data (Fig S1-S10).
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Figure 5. Molecular docking interaction of 4f. A) The general overview of docking
depiction. B) The closer view of docking complex. The residues involve in binding
interactions are shown in brown color. Red dotted lines with distance stated in angstrom (Å)
are justified for H-bond distances.
In-silico Methodology
3.1
Retrieval of porcine pancreatic elastase structure
The retrieval of porcine pancreatic elastase structure was in line with procedure reported
earlier [21-23].
3.2
Molecular docking of synthesized compounds
The synthesized chemical structures (4a-j) were sketched in ACD/ChemSketch and
minimized by UCSF Chimera 1.6rc tool. PyRx docking tool [24] was employed to perform
molecular docking experiment. All the synthesized ligands (4a-j) were docked separately
against elastase. Docking analysis was done based on lowest binding energy (Kcal/mol)
values and structure activity relationship (SAR) analyses. The three dimensional (3D)
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graphical depictions of all the docked complexes were accomplished by Discovery Studio
(2.1.0).
In-vitro Methodology
3.3
Elastase inhibition assay
The inhibition of Elastase from porcine pancreas was achieved following reported method
[16-17]. The elastase inhibition activities were calculated according to the following formula:
Elastase inhibition activity (%) = (ODcontrol – ODsample × 100) / ODcontrol
Where ODcontrol and ODsample represents the optical densities in the absence and presence of
sample, respectively using oleanolic acid as the standard.
3.6
Protocol for Kinetics
Kinetic analysis was done to determine the mode of inhibition using reported method [18].
The compound 4f was selected because of most potent IC50 values. Kinetics were carried out
by varying the concentration of N-succinyl-Ala-Ala-Ala-p-nitroanilide the presence of
different concentrations of compound 4f (0.00, 0.659, 1.318 and 2.636 µM). The inhibition
mode of enzyme was evaluated by Lineweaver-Burk plot of inverse of velocities (1/V) versus
inverse of substrate concentration 1/ [S] mM-1. The EI dissociation constant Ki was
determined by secondary plot of 1/V versus inhibitor concentration and processed using
SoftMaxPro.
3.7
Free radical scavenging assay
Radical scavenging activity was determined by reported method [19-20] by 2, 2-diphenyl-1
picryl hydrazyl (DPPH) assay.
4. Conclusions
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A new series of the benzimidazole-thioureas (4a-j) were prepared, characterized, and
appraised for elastase inhibition and antioxidant activities. Compound 4f having a C-8
hydrophobic alkyl chain showed maximum elastase inhibition (IC50 = 1.318 ± 0.025 µM)
compared to other compounds of the series. Kinetic analysis revealed the competitive mode
of inhibition of the elastase enzyme with the Ki value of 1.9 µM. The molecular docking of
the compound 4f showed good binding energy value of 7.3 Kcal/mol against receptor. The
presence of benzimidazole and alkyl chain with the acyl thiourea play crucial role in elastase
inhibition. Compound 4f was recognized as potent elastase inhibitor that could be used a
potential anti-aging ingredient in cosmetics.
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