Synthesis and in vitro α-chymotrypsin inhibitory activity of 6-chlorobenzimidazole derivatives

Article abstract of DOI:10.1016/j.bmc.2016.05.018

A library of benzimidazole derivatives 1–20 were synthesized, and studied for their α-chymotrypsin (α-CT) inhibitory activity in vitro. Kinetics and molecular docking studies were performed to identify the type of inhibition. Compound 1 was found to be a good inhibitor of α-chymotrypsin enzyme (IC₅₀?=?14.8?±?0.1?μM, K_i?=?16.4?μM), when compared with standard chymostatin (IC₅₀?=?5.7?±?0.13?μM). Compounds 2–8, 15, 17, and 18 showed significant inhibitory activities. All the inhibitors were found to be competitive inhibitors, except compound 17, which was a mixed type inhibitor. The substituents (R) in para and ortho positions of phenyl ring B, apparently played a key role in the inhibitory potential of the series. Compounds 1–20 were also studied for their cytotoxicity profile by using 3T3 mouse fibroblast cells and compounds 3, 5, 6, 8, 12–14, 16, 17, 19, and 20 were found to be cytotoxic. Molecular docking was performed on the most active members of the series in comparison to the standard compound, chymostatin, to identify the most likely binding modes. The compounds reported here can serve as templates for further studies for new inhibitors of α-chymotrypsin and other chymotrypsin-like serine proteases enzymes.[Figure presented]

Full text of DOI:10.1016/j.bmc.2016.05.018

Accepted Manuscript
Synthesis and In vitro α-Chymotrypsin Inhibitory Activity of 6-Chlorobenzi-
midazole Derivatives
Hina Siddiqui, Rabia Farooq, Bishnu P. Marasini, Rizwana Malik, Naima Syed,
Syed Tarique Moin, Atta-ur-Rahman, M. Iqbal Choudhary
PII:
S0968-0896(16)30342-X
http://dx.doi.org/10.1016/j.bmc.2016.05.018
BMC 13002
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 February 2016
4 May 2016
12 May 2016
Please cite this article as: Siddiqui, H., Farooq, R., Marasini, B.P., Malik, R., Syed, N., Moin, S.T., Atta-ur-Rahman,
Iqbal Choudhary, M., Synthesis and In vitro α-Chymotrypsin Inhibitory Activity of 6-Chlorobenzimidazole
Derivatives, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.05.018
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Synthesis and In vitro α-Chymotrypsin Inhibitory Activity of 6-Chlorobenzimidazole Derivatives
Hina Siddiquia*, Rabia Farooqa, Bishnu P. Marasinia, Rizwana Malikb, Naima Syeda,
Syed Tarique Moina, Atta-ur-Rahmana, and M. Iqbal Choudhary a,b,c*
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi,
Karachi-75270, Pakistan
bDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan
cDepartment of Biochemistry, Faculty of Science, King Abdul Aziz University, Jeddah-21452, Saudi Arabia
ABSTRACT
A library of benzimidazole derivatives 1-20 were synthesized, and studied for their α-
chymotrypsin (α-CT) inhibitory activity in vitro. Kinetics studies and molecular docking studies
were performed to identify the type of inhibition. Compound 1 was found to be a good inhibitor
of α-chymotrypsin enzyme (IC50 = 14.8 µM, Ki = 16.4 µM), when compared with standard
chymostatin (IC50 = 5.7 0.13 µM). Compounds 2-8, 15, 17, and 18 showed significant inhibitory
activities. All the inhibitors were found to be competitive inhibitors, except compound 17, which
was a mixed type inhibitor. The substituents (R) in para and ortho positions of phenyl ring B,
apparently played a key role in the inhibitory potential of the series. Compounds 1-20 were also
studied for their cytotoxicity profile by using 3T3 mouse fibroblast cells and compounds 3, 5, 6,
8, 12-14, 16, 17, 19, and 20 were found to be cytotoxic. Molecular docking was performed on
the most active member of the series in comparison to the standard compound, chymostatin, to
show the most likely binding modes. The compounds reported here can serve as templates for
further studies of new inhibitors of α- chymotrypsin and other chymotrypsin-like serine proteases
enzymes.
Keywords: Benzimidazole; α-Chymotrypsin inhibition; Serine protease, docking
1

1.0
Introduction:
Serine proteases are important targets for the development of drugs against several diseases1. The
enzyme α-chymotrypsin (EC 3.4.21.1) belongs to the serine protease family, which plays a vital
role in the digestion of dietary proteins after food intake2. Premature activation of chymotrypsin
leads to chronic pancreatitis3. Chymotrypsin and cathepsin also catalyzes the cleavage of
interleukin 1-β (IL-1β) precursor into active IL-1β, which causes arthritis4. Chymotrypsin also
activates epithelial sodium channel (Enac) by proteolysis, and may cause cystic fibrosis5.
Benzimidazole is an important scaffold, known to possess various biological activities, such as
antimicrobial6, angiotensin II receptor antagonist7, phosphodiesterase inhibition, anti-HIV8, anti-
influenza9, antiparasitic10, anti-inflammatory11, topoisomerase I inhibition, inhibition of the
hepatitis C virus RNA polymerase, anti-fertility, anti-helmintic12, antihistaminic13 properties.
Therefore, the synthesis of new benzimidazole derivatives has been vigorously pursued in
medicinal chemistry. Several benzimidazole-based drugs, such as omeprazole, rabeprazole, and
timoprazole, are widely used as proton pump inhibitors in the treatment of ulcer, and gastritis.
Moreover, albendazole, and mebendazole are used against a variety of worm infestations
(Figure-1).
Figure-1: Structures of Some Common Drugs Based on Benzimidazole Pharmacophore.
2

Current study describes the synthesis of 6-chlorobenzimidazole derivatives 1-20, and evaluation
of their α-chymotrypsin inhibitory, and cytotoxic activities using biochemical and cellular in
vitro assays, respectively. All the compounds were found to be known except compound 10.
These compounds have been reported for antibacterial14, antiproliferative15, antimicrobial16,
modulators of ion channels17, analgesic18, antiinflammatory19, cyclooxygenase inhibitory19, and
antioxidant19 activities. However to the best of our knowledge compounds 1-20 have not been
reported for the α-chymotrypsin, and related enzyme inhibition activity. Modern spectroscopic
techniques were used to characterize the synthesized compounds.
Molecular level characterization of binding pattern of these compounds into α-chymotrypsin
active site has provided insights into mechanism of action of these benzimidazole derivatives.
Molecular docking was performed on the most active compound 1, in comparison to the standard
compound, chymostatin, to identify the most likely binding modes of benzimidazole derivatives
which could contribute for the inhibitory activity of these compounds.
2.0
2.1
Results and Discussion:
Chemistry:
6-Chlorobenzimidazoles derivatives 1–20 were synthesized by condensation reaction between 4-
chlorophenylenediamine and different aldehydes, by using a methodology described by Ridley et
al20 (Scheme-1). Resulting compounds were purified by solvent-solvent extraction, and obtained
in good to excellent yields. In few cases compounds were purified by using normal phase column
chromatography.
3

Scheme-1: Synthesis of Benzimidazole Derivatives 1-20.
Structure Elucidation of Representative Compound 1:
The structures of compounds 1-20 were deduced by using MS and NMR spectroscopic
techniques. Structure elucidation of a representative member (compound 1) is presented below;
6-Chloro-2-(3,4-dimethoxyphenyl)-1H-benzimidazole (1)
1
In the H-NMR, H-6' was found coupled with H-5' and H-2', and appeared as a doublet of a
doublet at δH 7.67 (J 6',5' = 6.3 Hz, J 6', 2' = 1.5 Hz). H-5' was coupled with H-6' at δH 7.12 (d, 1H,
J5', 6' = 6.3 Hz). H-5 resonated as a doublet at δH 7.66 (d, 1H, J5,7 = 1.5 Hz). In addition, H-8/ H-2'
appeared as a broad singlet at δH 7.53. H-7 showed coupling with H-8 and H-5 at δH 7.23 (dd,
1H, J7, 8 = 6.3 Hz, J7, 5 = 1.2 Hz).
13C-NMR spectrum showed characteristic C-2 carbon was resonated at δ 152.8. Other prominent
13
peaks in the C-NMR were δ 151.0 (C-3'and C-4'), 124.0 (C-4 and C-9), 123.2 (C-6 and C-1'),
121.1 (C-7 and C-6'), 112.9 (C-8 and C-5), 111.2 (C-2' and C-5'), and 56.5 (2OCH3 at C-3' and
C-4'). The HREI-MS was found to be m/z 288.0664 supporting the molecular formula
C15H13ClN2O2 (Exact Mass: 288.0666).
4

2.2
In Vitro α-Chymotrypsin Activity:
Compounds 1-20 were evaluated for their α-chymotrypsin inhibitory activity. Compounds which
showed more than 50% inhibition of the enzyme at 500 µM concentration were selected for IC50
value measurement, and kinetics studies. The IC50 values were found to be in the range of 15 to
97 µM.
Table-1 presents the α-chymotrypsin inhibitory activity of the compounds 1-20. Compound 1
was found to be the most active member of the series with an IC50 = 14.8
0.1 µM, in
comparison to standard drug chymostatin (IC50 = 5.7 0.13 µM). The activity of compound 1 may
be attributed to the presence of two electron donating methoxy groups at C-3' and C-4' of ring B.
Compounds 2 and 3 were also found to be active members of the series with IC50 values 15.9
0.5, and 20.9 0.1 µM, respectively. The activity was reduced with the removal of one of
methoxy group from C-4', as observed in compound 17 (IC50= 38.1 0.6 µM). Activity also
decreased two folds when dimethylamino group was present at the C-4' position, as in compound
4. Compound 5 showed good activity with IC50 value 34.6 0.5 µM, which may be because of
two electron donating hydroxyl groups at C-2', and C-4'. The removal of hydroxyl from C-4'
resulted in a complete loss of activity, as observed in compound 14. Substitution of chloro group
at C-2', and C-4' positions also resulted in a significant activity (compound 6), but changing the
sequence of substitution resulted in a loss of activity many folds, as in compound 8 (IC50= 96.4
2.6 µM). Introduction of chloro and nitro group at C-2' and C-5' respectively, resulted in
complete loss of activity as observed in the compound 10. Use of pyridine as substituent resulted
in reduced activity as observed in compound 7 IC50= 48.9 0.5 µM). In comparison replacement
of pyridine with furan substituent resulted in reduced activity in compound 15 (IC50= 93.1 3.2
µM). Similarly use of styrene group resulted in loss of activity by many folds in compound 13
5

(IC50= 486.1 9.8 µM).It is proposed that benzimidazole moiety apparently interfere with the
catalytic action of histidine-57 of α-chymotrypsin enzyme via accepting or donating hydrogen by
N-1’ or N-3’, and thus contributing towards the affinity of the compounds with the enzyme.
2.3
Kinetic studies
Kinetics studies were performed on the most active members of the series, i.e. compounds 1-8,
15, 17 and 18, to study the mode of inhibition. Table-1 shows the Ki values, and type of
inhibitions of these compounds. The pattern of inhibition at variable concentrations of compound
1, and substrate is graphically presented in Figures-2- 4.
No Inhibitor
250
3.9
7.8
µM
µM
100
80
60
40
20
0
200
150
100
50
15.6
µM
B
A
0
1 / [Substrate, µM]
[Compound 1, µM]
Figure-2: (A) Line-weaver Burk plot of various concentrations of compound 1. In this steady-state kinetics, all the
lines intersect each other at y-axis indicating a competitive-type of inhibition. (B) Secondary plot generated from
Line-weaver Burk plot. Ki value is equivalent to the meeting point of the straight line at x-axis.
6

250
200
150
100
50
Sub3200 uM
Sub1600 uM
Sub 800 uM
Sub 400 uM
A
8
6
4
2
0
B
0
-16
-8
0
8
16
1 / [Substrate, µM]
[Compound 1, µM]
Figure-3: (A) shows the Dixon plot of compound 1 with various concentrations of substrate. In this graph, these
lines meet at 2nd quadrant indicating a competitive type of inhibition. Ki value is equivalent to the meeting point of
the straight line at x-axis (B) Secondary plot produced from Dixon plot for compound 1. The line passes through
origin clarifies confusion with mixed-type of inhibition.
140
120
100
80
No inhibitor
Data set #1
Data set #2
3.9 µM
60
40
Data set #3
7.8 µM
Data set #4
15.6 µM
0
1000
2000
3000
4000
[Substrate, µM]
Figure-4: Hanes-Woolf plot of various concentrations of compound 1. These parallel lines indicate that the
compound is a competitive-type of inhibitor.
7

2.4
Molecular Docking Studies
Active site of chymostrypsin is burried in solvent-inaccessible pocket. The active site of
chymotrypsin contains Asp-102, His-57, and Ser-195, referred as catalytic triad which via a
series of reaction sequence produces nucleophilic Ser-19521. Inhibition of chymotrypsin activity
is linked with the blockage of nucleophilic catalytic reaction, carried out by Ser-195.
Chymostatin22, a standard compound was found to bind with Ser-195 and other residues,
including hydrophobic residues in its close proximity. These residues involve Asp-194, Cys-191,
Gly193, Met-192, Ser-217, and Tyr-228. Among these residues, backbone amide hydrogens of
Ser-195 and Gly-193 pointing into the active site cavity form a region in space which is named
as “oxyanion hole”. This space plays an important role to enhance the rate of the catalytic
reaction by several factors.
Chymostatin (CHY) is a large complex organic molecule, which is stabilized in the active site of
α-chymotrypsin by polar interactions between aromatic rings of CHY, and Ser-195 and Tyr-228,
as illustrated in Figure-5. In addition to these, His-57 and Ser-217 dynamically participate in
hydrogen bonding with amide hydrogens of CHY. Met-192 and Asp-194 also provide extra
stability to CHY and contribute in the inhibitory activity of the molecule. Nitrogen containing
aliphatic ring was found to partially occupy the space of the oxyanion hole region that may
causes influence on the rate of enzymatic activity. Figure-6 depicts the binding of compound 1
in the enzyme’s active site. Compared to CHY, compound 1 occupies less space of the active site
but it has a major interaction between methoxy substituted aromatic ring and Ser-195, whereas
His-57 was found to contribute in the stabilization effect via possible interaction with imidazole
ring and methoxy aromatic ring. Apart from these catalytic residues, amide hydrogen of Gly-193
forms H-bonding with imidazole nitrogen, whereas chlorine atom of the benimidazole ring was
8

partially exposed to solvent accessible region and thus could not find suitable residues to form
favourable polar interactions. This may lead to lesser inhibitory activity of the compound 1 as
compared to chymostatin. However, description of the binding pattern of compound 1 to the
enzyme’s active site may serve to rationalize its inhibitory potency in comparison to the standard
compound, chymostatin which has higher inhibition potential as deduced from a lower IC50
value, yet the newly synthesized benzimidazole derivatives may therefore, be considered as new
leads for further optimization.
Figure-5: Binding of compound 1 with active site residues of
chymotrypsin enzyme (hydrogens were removed for clarity)
9

Figure-6: Binding of chymostatin (standard) with active
site residues of chymotrypsin enzyme (hydrogens were
removed for clarity)
3.0
Conclusion:
We have identified a series of chloro-benzimidazole derivatives as new inhibitors οf α-
chymotrypsin inhibitors. This may serve as templates for the designing, and development of new
inhibitors of α- chymotrypsin and chymotrypsin-like serine proteases enzymes and associated
diseases.
4.0
4.1
Experimental
General Experimental Conditions:
All the chemicals were purchased from commercial suppliers and used for reaction without
10

purification. Reaction was carried out in flame dried flasks, and with chemically dried solvents.
EI-MS (Electron Impact Mass Spectra) were recorded on a JEOL JMS-600H mass spectrometer.
The data was analyzed by using MASPEC data system. The NMR spectra were recorded in
deuterated solvents on Avance Bruker 300, 400, 500, and 600 MHz NMR spectrometers. The
chemical shifts (δ) and coupling constants (J) were measured in ppm and Hz, respectively, SiMe4
was used as an internal standard. TLC analyses were performed on pre-coated ALUGRAM, SIL
G/UV254 aluminum plates (Kieselgel 60, 20×20, 0.5 mm thick, E. Merk, Germany).
Chromatograms developed on TLC plates were visualized under ultraviolet light at 254 nm for
fluorescence quenching spots, and 365 nm for florescent spots.
4.2
General Procedure for Synthesis of Compounds 1-20:
The synthesis of 6-chlorobenzimidazole derivatives 1-20 was carried out by reaction of 4-
chlorophenylenediamine with various aromatic aldehydes in DMF in very good yields. In a
general reaction, 4-chlorophenylenediamine (1.00 eq), and aromatic aldehydes (1.00 eq) were
dissolved in 10 mL DMF, followed by addition of sodium metabisulfite (Na2S2O5) (0.29 eq), and
refluxed till the reaction was completed. After the completion, the reaction mixture was cooled
to room temperature, followed by the addition of ice cold water. Resulting precipitates were
filtered, and purified through column chromatography. The structures of the products were
deduced by 1H-NMR spectroscopy. and EI-MS analysis.
4.2.1 6-Chloro-2-(3,4-dimethoxyphenyl)-1H-benzimidazole (1)
Yield: 0.29 gm (58 %): 1H-NMR: (300 MHz, CD3OD): δH 7.71 (d, 1H, J 5,7 = 1.5 Hz, H-5 ) 7.67
(dd, 1H, J 6',5' = 6.3 Hz, J 6', 2' = 1.5 Hz, H-6'), 7.53 (bs, 2H, H-8/ H-2'), 7.23 (dd, 1H, J5,4 = 6.3 Hz,
J7 ,5 = 1.2 Hz, H-7), 7.12 (d, 1H, J5',6'= 6.3 Hz, H-5'), 3.95 (s, 3H, OCH3-3'), 3.90 (s, 3H, OCH3-4')
11

13C-NMR: (125 MHz, CD3OD): δ 152.8 (C-2), 150.96 (C-3' and C-4'), 124.0 (C-4 and C-9),
123.2 (C-6 and C-1'), 121.1 (C-7 and C-6'), 112.9 (C-8 and C-5), 111.2 (C-2' and C-5'), 56.5
(OCH3-3', and C-4'); EI-MS: m/z (rel. abund %): 290 ([M+2]+, 95), 288 (M+, 100), 230 (15), 192
(19); HREI-MS: Calculated for C15H13ClN2O2 Exact Mass: 288.0666 found 288.0664.
4.2.2 4-(6-Chloro-1H-benzimidazol-2-yl)-2-ethoxyphenol (2)
Yield: 0.4 gm (80%): 1H-NMR: (300 MHz, CD3OD): δH 7.67 (d,1H, J5,7 = 2.1 Hz, H-5), 7.54 (m,
2H, H-8/H-6'), 7.50 (d, 1H, J2',6' = 1.8 Hz, H-2'), 7.21 (dd, 1H, J7,8 = 8.7 Hz, J7,5 = 1.8 Hz, H-7),
6.95 (d, 1H, J 5',6' = 8.4 Hz, H-5'), 4.23 (q, 2H, J2˝,3˝ = 6.9 Hz, H-2˝), 1.50 (t, 3H, J3˝,2˝ = 6.9 Hz, H-
3˝); EI-MS: m/z (rel. abund %): 290 ([M+2]+, 36), 288 (M+, 100), 260 (75); 13C-NMR: (125
MHz, CD3OD): δ 150.7 (C-2), 148.6 (C-3' and C-4'), 123.8 (C-7 and C-6'), 122.0 (C-4 and C-9),
121.3 (C-5 and C-8), 116.7 (C-5'), 112.5 (C-2'), 112.4 (C-6 and C-1'), 65.7 (C-2˝), 15.0 (C-3˝);
HREI-MS: Calculated for C15H13ClN2O2 Exact Mass: 288.0666 found 288.0671.
4.2.3 6-Chloro-2-(1-naphthyl)-1H-benzimidazole (3)
Yield: 0.28 gm (58%): 1H-NMR: (300 MHz, CD3OD): δH 8.47 (m, 1H, H-8'), 8.08 (d, 1H, J3'/2'/4'
= 8.4 Hz, H-3'), 8.00 (m, 2H, H-7'/ H-4'), 7.88 (dd, 1H, J2',3' = 7.2 Hz, J2',4'= 0.9 Hz, H-2'), 7.65
(m, 4H, H-7'/ H-6'/ H-5'/ H-8), 7.32 (dd, 1H, J7,8 = 8.7 Hz, J7,5 = 1.8 Hz, H-7); EI-MS: m/z (rel.
abund %): 279 ([M+2]+, 44), 277 (M+, 100), 278 (71), 242 (29), 141 (25); HREI-MS: Calculated
for C17H11ClN2 Exact Mass: 278.0611 found 278.0601.
4.2.4 4-(6-Chloro-1H-benzimidazol-2-yl)-N, N-dimethylaniline (4)
Yield: 0.25 gm (53%): 1H-NMR: (300 MHz, CD3OD): δH 7.92 (d, 2H, J2',3'/6',5' = 9.0 Hz, H-2'/H-
6'), 7.52 (d, 1H, J5,7 = 1.8 Hz, H-5), 7.50 (d, 1H, J8,7 = 8.7 Hz, H-8), 7.22 (dd, 1H, J7,8 = 8.7 Hz,
12

J7,5 = 1.8 Hz, H-7), 6.86 (d, 2H, J3',2'/5',6'= 9.0 Hz, H-3'/ H-5'), 3.05 (s, 6H, N(CH3)2); EI-MS: m/z
(rel. abund %): 273 ([M+2]+, 36), 271 (M+, 100), 255 (44), 221 (18); HREI-MS: Calculated for
C15H14ClN3 Exact Mass: 271.0876 found 271.0891.
4.2.5 4-(6-Chloro-1H-benzimidazol-2-yl)-1, 3-benzenediol (5)
1
Yield: 0.20 gm (44%): H-NMR: (300 MHz, CD3OD): δH 7.72 (d, 1H, J8,7 = 6.0 Hz, H-8), 7.53
(s, 1H, H-5), 7.49 (d, 1H, J6',5' = 6.3 Hz, H-6'), 7.19 (dd, 1H, J7,8 = 8.7 Hz, J7,5 = 1.8 Hz, H-7),
6.44 (d, 1H, J3',5' = 1.5 Hz, H-3'), 6.42 (overlapped, 1H, H-5'); EI-MS: m/z (rel. abund %): 262
([M+2]+, 47), 260 (M+, 100), 203 (23); HREI-MS: Calculated for C13H9ClN2O2 Exact Mass:
260.0353 found 260.0339.
4.2.6 6-Chloro-2-(2, 4-dichlorophenyl)-1H-benzimidazole (6)
Yield: 0.35gm ( 68%): 1H-NMR: (300 MHz, CD3OD): δH 7.84 (d, 1H, J6',5' = 8.4 Hz, H-6'), 7.70
(d, 1H, J3',5' = 2.1 Hz, H-3'), 7.63 (bs, 2H, H-5/ H-8), 7.53 (dd, 1H, J5',6' =8.4 Hz, J5',3' = 2.1 Hz, H-
5'), 7.29 (dd, 1H, J7,8 = 8.7 Hz, J7,5 = 1.8 Hz, H-7); EI-MS: m/z (rel. abund %): 298 ([M+2]+,
100), 296 (M+, 100); HREI-MS: Calculated for C13H7Cl3N2 Exact Mass: 295.9675 found
295.9674.
4.2.7 6-Chloro-2-(3-pyridinyl)-1H-benzimidazole (7)
1
Yield: 0.34 gm (65%): H-NMR: (300 MHz, CD3OD): δH 9.24 (d, 1H, J2',6' = 1.5 Hz), 8.68 (dd,
1H, J4',5' = 4.8 Hz, J4',6' =1.5 Hz, H-4'), 8.50 (dt, 1H, J6 ',5'= 8.1 Hz, J6'/2',4'=1.8 Hz, H-6' ), 7.63 (m,
3H, H-5'/ H-8/ H-5), 7.30 (dd, 1H, J7,8 = 8.7 Hz, J7,5 = 2.1 Hz, H-7); EI-MS: m/z (rel. abund %):
231 ([M+2]+, 94), 229 (M+, 100), 203 (60), 226 (45); HREI-MS: Calculated for C12H8ClN3 Exact
Mass: 229.0407 found 229. 0404.
13

4.2.8 6-Chloro-2-(2, 6-dichlorophenyl)-1H-benzimidazole (8)
1
Yield: 0.38 gm (74%): H-NMR: (300 MHz, CD3OD): δH 7.60 (bs, 1H, H-5), 7.60 (overlapped,
1H, H-8), 7.56 (m, 2H, H-3'/ H-5'), 7.52 (d, 1H, J4'/3',5' = 4.8 Hz, H-4'), 7.32 (dd, 1H, J7,8 = 8.7
Hz, J7,5 =1.8 Hz, H-7); EI-MS: m/z (rel. abund %): 298 ([M+2]+, 76), 296 (M+, 100), 271 (20),
226 (45); HREI-MS: Calculated for C13H7Cl3N2 Exact Mass: 295.9675 found 298.9680.
4.2.9 6-Chloro-2-phenyl-1H-benzimidazole (9)
1
Yield: 0.25 gm (64%): H-NMR: (300 MHz, CD3OD): δH 8.08 (m, 2H, H-5/8), 7.58 (m, 3H, H-
3'/4'/5'), 7.53 (m, 2H, H-2'/ H-6'), 7.25 (dd, 1H, J7,8 = 8.4 Hz, J7,5 =1.8 Hz, H-7); EI-MS: m/z
(rel. abund %): 230 ([M+2]+, 61), 228 (M+, 100), 229 (31); HREI-MS: Calculated for C13H9ClN2
Exact Mass: 228.0454 found 228.0452.
4.2.10 6-Chloro-2-(2-chloro-5-nitrophenyl)-1H-benzimidazole (10)
1
Yield: 0.35 gm (66%): H-NMR: (300 MHz, CDCl3): δH 10.9 (s, 1H, H-3), 8.75 (s. 1H, H-6'),
7.99 (d, 1H, J4',3' = 6.3 Hz, H-4'), 7.72 (s. 1H, H-5), 7.68 (d, 1H, J3',4' = 6.3 Hz, H-3'), 7.29 (d, 1H,
J8,7 = 6.3 Hz, H-8), 7.02 (d, 1H, J7,8 = 6.6 Hz, H-7); EI-MS: m/z (rel. abund %): 309 ([M+2]+,
100), 307 (M+, 100), 226 (85).
4.2.11 4-(6-Chloro-1H-benzimidazol-2-yl)benzonitrile (11)
1
Yield: 0.31 gm (70%): H-NMR: (300 MHz, CD3OD): δH 8.23 (d, 2H, J3',2'/5',6'= 8.4 Hz, H-3'/H-
5'), 7.91 (d, 2H, J2',3'/6',5' = 8.4 Hz, H-2'/H-6'), 7.62 (overlapped, 1H, H-8), 7.58 (s, 1H, H-5), 7.29
(dd, 1H, J7,8 = 8.7 Hz, J7,5 =1.8 Hz, H-7); EI-MS: m/z (rel. abund %): 255 ([M+2]+, 97), 253 (M+,
100), 217 (15); HREI-MS: Calculated for C14H8ClN3 Exact Mass: 253.0407 found 253.0406.
14

4.2.12 6-Chloro-2-(4-methylphenyl)-1H-benzimidazole (12)
1
Yield: 0.28 gm (66%): H-NMR: (300 MHz, CD3OD): δH 7.96 (d, 2H, J2',3'/6',5' = 8.4 Hz, H - 2'/
H-6'), 7.56 (Overlaped, 2H, H-5/ H-8), 7.36 (d, 2H, J 3',2'/5',6' = 8.1 Hz, H-3'/ H-5'), 7.23 (dd, 1H,
J7,8 = 8.4 Hz, J7,5 = 1.8 Hz, H-7), 2.413(s, 3H, CH3); EI-MS: m/z (rel. abund %): 244 ([M+2]+,
87), 242 (M+, 100), 207 (20); HREI-MS: Calculated for C14H11ClN2 Exact Mass: 242.0611
found 242.0602.
4.2.13
6-Chloro-2-[2-phenylethenyl]-1H-benzimidazole (13)
Yield: 0.24 gm (54%): 1H-NMR: (300 MHz, CD3OD): δH 7.66 (d, 1H, J1',2' = 16.5 Hz, H-1'), 7.63
(d, 2H, J2˝,3˝/6˝,5˝ = 7.2 Hz, H-2˝/ H-6˝), 7.53 (bs, 2H, H-5/ H-8), 7.39 (m, 3H, H-3˝/ H-4˝/ H-5˝),
7.22 (dd, 1H, J7,8 = 8.4 Hz, J7,5 = 1.8 Hz, H-7), 7.15 (d, 1H, J2',1' = 16.5 Hz, H-2'); EI-MS: m/z
(rel. abund %): 255 ([M+2]+, 39), 253 (M+, 100), 218 (22); HREI-MS: Calculated for
C15H11ClN2 Exact Mass: 254.0611 found 254.0587.
4.2.14
2-(6-Chloro-1H-benzimidazol-2-yl)phenol (14)
Yield: 0.3 gm (71%): 1H-NMR: (300 MHz, CD3OD): δH 7.92 (bs, 1H, H-5), 7.60 (m, 2H, H-8/H-
6'), 7.35 (t, 1H, J4',5' = 7.2 Hz, H-4'), 7.25 (dd, 1H, J7,8 = 8.7 Hz, J7,5 = 1.8 Hz, H-7), 7.02 (m, 2H,
H-3'/H-5'); EI-MS: m/z (rel. abund %): 264 ([M+2]+, 34), 244 (M+, 100), 216(35); HREI-MS:
Calculated for C13H9ClN2O Exact Mass: 244.0403 found 244.0408.
4.2.15
6-chloro-2-(furan-2-yl)-1-methyl-1H-benzimidazole (15)
Yield: 0.31 gm (77%): 1H-NMR: (300 MHz, CD3OD): δH 7.52 (bs, 2H, H-5/H-3'), 7.23 (dd, 1H,
J7,8 = 8.7 Hz, J7,5 = 1.8 Hz, H-7), 7.09 (d, 1H, J8,7 = 3.3 Hz, H-8), 6.28 (d, 1H, J5',4' = 3.3 Hz, H-
5'), 6.0 (m, 1H, H-4'), 2.43 (s, 3H, N-Me); EI-MS: m/z (rel. abund %): 234 ([M+2]+, 29),232
15

(M+, 100), 217(22), 202(25); HREI-MS: Calculated for C12H9ClN2O Exact Mass: 232.0403
found 232.0405.
4.2.16
6-Chloro-2-(4-isopropylphenyl)-1H-benzimidazole (16)
Yield: 0.32 gm (68%): 1H-NMR: (300 MHz, CD3OD): δH 8.00 (d, 2H, J2',3'/6',5' = 8.4 Hz, H-2'/H-
6'), 7.56 (bs, 2H, H-5/H-8), 7.43 (d, 2H, J3'2',/5',6' = 8.4 Hz, H-3'/H-5'), 7.23 (dd, 1H, J7,8 = 8.4 Hz,
J7,5 = 1.5 Hz, H-7), 7.29 (m, 1H, CH(CH3)2), 1.31 (d, 6H, J = 6.9 Hz); EI-MS: m/z (rel. abund
%): 272 ([M+2]+, 17), 270 (M+, 50), 255(100); HREI-MS: Calculated for C16H15ClN2 Exact
Mass: 270.0924 found 270.0913.
4.2.17
3-(6-Chloro-1H-benzimidazol-2-yl)phenyl methyl ether (17)
Yield: 0.34 gm (76%): 1H-NMR: (300 MHz, CD3OD): δH 7.66 (d, 1H, J8,7 = 2.4 Hz, H-8), 7.62
(s, 1H, H-5), 7.58 (bs, 2H, H-2′/ H-6′ ), 7.44 (t, 1H, J 5′/4′,6′ = 8.1, H-5′), 7.26 (dd, 1H, J7,8 = 8.4,
J7,5 = 3 Hz , H-7), 7.10 (dd, 1H, J4′,5′ = 8.4 Hz, J4′,2′ = 2.4 Hz, H-4′), 3.89 (s, 3H, OCH3-3'); EI-
MS: m/z (rel. abund %): 260 ([M+2]+, 36), 258 (M+, 100), 228 (40); HREI-MS: Calculated for
C14H11ClN2O Exact Mass: 258.0560 found 258. 0553.
4.2.18
6-Chloro-2-(2-fluorophenyl)-1H-benzimidazole (18)
1
Yield: 0.36 gm (83%): H-NMR: (300 MHz, CD3OD): δH 8.19 (td, 1H, J4′,6′ = 1.8 Hz, J4′/3′,5′
=
9.3 Hz, H-4′), 7.632 (m, 1H, H-3′), 7.53 (m, 2H, H-5′/ H-6′), 7.40 (overlapped, 2H, H-5/H-8),
7.27 (dd, 1H, J7,8 = 8.7, J7,5 = 1.8 Hz, H-7); EI-MS: m/z (rel. abund %): 248 ([M+2]+, 39), 246
(M+, 100); HREI-MS: Calculated for C13H8ClFN2 Exact Mass: 246.0360 found 246.0357.
16

4.2.19
2-(3-bromophenyl)-6-chloro-1H-benzimidazole (19)
Yield: 0.38 gm ( 71%): 1H-NMR: (300 MHz, CD3OD): δH 8.27 (s, 1H, H-5), 8.04 (d, 1H, J6',5'
=
7.8 Hz, H-6'), 7.68 (d, 1H, J8,7 = 7.8 Hz, H-8), 7.60 (m, 1H, H-4'), 7.55 (s, 1H, H-2'), 7.47 (t, 1H,
J5'/4',6' = 7.8 Hz, H-5'), 7.28 (dd, 1H, J7,8 = 8.7 Hz, J7,5 = 2.1 Hz, H-7); EI-MS: m/z (rel. abund %):
309 ([M+2]+, 26), 307 (M+, 100), 227 (72), 192 (82); HREI-MS: Calculated for C13H8BrClN2
Exact Mass: 307.9716 found 307.9712.
4.2.20
6-Chloro-2-(4-nitrophenyl)-1H-benzimidazole (20)
Yield: 0.42 gm (79%): 1H-NMR: (300 MHz, CD3OD): δH 8.42 (d, 2H, J3',2'/5',6' = 9 Hz, H - 3'/ H-
5'), 8.31 (d, 2H, J2',3'/6',5' = 9 Hz, H - 2'/ H-6'), 7.64 (d, 1H, J8,7 = 3.0 Hz, H-8), 7.60 (s, 1H, H-5),
7.30 (dd, 1H, J7,8 = 8.7 Hz, J7,5 = 1.8 Hz, H-7); EI-MS: m/z (rel. abund %): 275 ([M+2]+, 83),
273 (M+, 100), 227(90), 192(75); HREI-MS: Calculated for C13H8ClN3O2 Exact Mass: 273.0305
found 273.0303.
4.3
α-Chymotrypsin Inhibitory Assay
α-Chymotrypsin inhibitory activity was determined in 50 mM tris-HCl buffer pH 7.6 with 10
mM CaCl2. α-Chymotrypsin (12 units/mL, in buffer) with the various concentrations of test
compounds in DMSO was incubated at 30 °C for 25 min. The reaction began with the addition of
the substrate, N-succinyl-L-phenylalanine-p-nitroanilide (SPpNA; 0.4 mM final, in the buffer).
The variations in the absorbance with the released p-nitroaniline was monitored at 410 nm23. All
the experiments were performed in triplicate in a final volume of 200 µL, using a micro plate
reader (SpectraMax M2, Molecular Devices, CA, USA).The % of inhibition was calculated as:
⁄
OD min of test compound
% Inhibition Ɣ 100 Ǝ ʦ
ꢀ ꢁ 100
⁄
OD min of positive control
17

IC50 (Inhibition of enzymatic hydrolysis of the substrate SPpNA by 50%) values were calculated
using EZ-Fit enzyme kinetics program (Perellela Scientific, Inc., Amherst, USA). α-Chymostatin
(Standard) was used as the positive control, whereas DMSO was added in the negative control
instead of compounds
4.4
α-Chymotrypsin Inhibition Kinetic Study
Per minute change in optical density (OD/min) was measured by incorporating various
concentrations of test compound with various concentrations (between 0.4 and 3.2 mM) of
substrate (SPpNA). Reciprocal of the rate of the reactions against the reciprocal of the substrate
concentration as Lineweaver–Burk plot (and its secondary plot; slope vs compound
concentration); the Dixon plot (and its secondary plot; slope vs reciprocal of compound
concentration), and then Hanes-Woolf plot were plotted24. Graphs were plotted using GraFit 4
(Erithacus Software Limited, Surrey, UK), and Graph Pad Prism5 (Graph Pad Software,
California, USA). Graphical views of Dixon plots, Lineweaver-Burk plots, and their secondary
plots, as well as Hanes-Woolf plot were used to determine the types of inhibition. The Ki values,
obtained directly from the software, were cross-checked also in these graphs. Final
concentration of DMSO was maintained at 5.5 %.
4.5
Cytotoxicity Assay:
The cytotoxicity of all compounds (1-20) was evaluated by using 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) assay against 3T3 (Mouse fibroblast) cells25. Briefly 3T3
18

cells were grown in DMEM (containing 100 µg/mL streptomycin, 5% of FBS and 100 IU/mL
o
penicillin), and incubated at 37 C in 5% CO2. Cells were maintained at 5x104 cells/ mL
concentration, and introduced into 96-well plates and allowed to stand for overnight period.
Medium was removed, and 200 µL of fresh medium was added, various concentrations of the
test compounds were also added and allowed to react for 48 hrs. 0.5 mg/mL of MTT was added
to each well and incubated for 4 more hours. DMSO was added and reduction of MTT to
formazan dye was calculated by recording the absorbance at 540 nm using a micro plate reader
(Spectra Max plus, Molecular Devices, CA, USA). Cycloheximide was used as positive control
whereas DMSO was added in the negative control instead of compounds. The percent inhibition
was calculated for each cell by using the following formula:
% Inhibition = 100-{(mean of O.D. of test compound – mean of O.D. of negative control)/
(mean of O.D. of positive control – mean of O.D. of negative control) x 100}
4.6
Molecular Docking Studies:
Crystal structure of chymotrypsin complexed with benzohydroxamic acid/vanadate (BVA) (PDB
ID: 2P8O)26 was used for the docking studies using AutoDock 4.2, executed with the help of
2
7
graphical user interface, AutoDockTools 1.4.5 (ADT) . Docking studies were performed using
the Lamarckian Genetic Algorithm (LGA) as a search engine. The GUI (Graphical User
Interface) helped to prepare the protein and ligands structure files for the grid and docking
calculations. The free active site was obtained after removing coordinates of co-crystallized
ligand as well as water molecules were also removed. Coordinates of ligands i.e. compound 1,
19

chymostatin (standard compound), and BVA were built and quantum mechanically optimized
using Gaussian 0928 at Density Functional Theory level with B3LYP functional and 6-31G (d, p)
basis sets were utilized for all atoms. Point atomic charges were applied to ligands which were
adopted from the Mulliken Population Analysis. The docking protocol was validated by
reproducing binding pose of the BVA inhibitor into the active site on the basis of root mean
square deviation, and that came out to be around 1.00 Å, as shown in Figure-7. The resolution
and dimensions of three-dimensional energy scoring grids were set to 0.375 Å and 40 × 40 × 40
Å dimensions to carry out docking of BVA ligand, whereas the dimensions of other ligands were
increased to 44× 44 × 44 Å in order to keep the ligand inside the grid. Other parameters for LGA
consist of default setting for each docking calculation with a total of 30 runs with a maximum of
2,500,000 energy evaluations, and cluster analysis using a root–mean–square (rms) tolerance of
1.5 Å.
Figure-7 Superposition of co-crystallized (yellow) and re-docked (green) conformation
into the protein active site
20

Table-1: Inhibition of α-chymotrypsin by 6-chlorobenzimidazole derivatives 1-20, and their
cytotoxicity.
Cytotoxicity
IC50 SD
(µM)
Compound
IC50 SEM
(µM)
Ki SEM
(µM)
Type of
inhibition
-Ar
-R
1
H
14.8 0.1
16.4 0.9
Competitive
Competitive
>30
>30
2
3
4
5
6
H
H
H
H
H
15.9 0.5
20.9 0.1
24.2 2.1
34.6 0.5
35.6 2.2
17.3 0.8
25.31 3.1
17.5 0.8
33.3 1.1
39.6 2.7
Competitive 11.577 0.467
Competitive
>30
Competitive 28.146 1.731
Competitive 13.794 0.970
21

7
8
H
H
H
H
H
H
48.9 0.5
96.4 2.6
>500
50.1 2.9
99.8 7.4
NC
Competitive
>30
Competitive 25.180 0.384
9
NC
NC
NC
NC
>30
>30
1'
6'
10
11
12
>500
NC
3'
2
4'
>500
NC
>30
>500
NC
25.99 0.573
13
H
486.1 9.8
NC
NC
12.460 0.240
14
15
H
>500
NC
NC
3.792 0.200
>30
CH3
93.1 3.2
71.4 5.9
Competitive
22

16
17
18
19
20
21
22
H
H
H
H
H
-
427.6 10.2
38.1 0.6
118.2 6.1
457.5 8.8
470.6 8.2
5.7 0.13
-
NC
NC
7.913 0.242
36.5 5.8
Mixed
18.371 0.668
>30
79.3 8.7
Competitive
NC
NC
-
NC
NC
-
6.903 0.084
7.218 0.199
Chymostatin
(Standard)
-
Cycloheximide
(Standard)
-
-
-
0.26 0.1
S.E.M. = Standard Error of Mean at n=3
SD = Standard Deviation
** = Anomalous Type of Inhibition
NC = Not calculated (because of % of inhibition shown was less than 50% at 500 µM.
5.0.
Conflict of Interest:
The authors declare no conflict of interest.
23

6.0
References:
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26

Graphical Abstract
Synthesis and In vitro α-Chymotrypsin Inhibitory Activity of 6-Chlorobenzimidazole
Derivatives
Hina Siddiquia*, Rabia Farooqa, Bishnu P. Marasinia, Rizwana Malikb, Naima Syeda,
Syed Tarique Moina, Atta-ur-Rahmana, and M. Iqbal Choudhary a,b,c*
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi,
Karachi-75270, Pakistan
bDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan
cDepartment of Biochemistry, Faculty of Science, King Abdul Aziz University, Jeddah-21452, Saudi Arabia
R
O
N
NH2
NH2
Na2S2O3
A
H
B
R
DMSO, reflux
N
H
Cl
Cl
1-20
Binding of chymostatin (standard),
competitive inhibitor, at the active site residues
of chymotrypsin enzyme
a
Binding of compound 1, a competitive inhibitor at the
active site residues of chymotrypsin enzyme
IC50 =14.8 0.1 µM
IC50 =5.7 0.13 µM
Ki 16.4 0.9 µM
27