Design, synthesis and docking studies of benzimidazole derivatives as potential EGFR inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.04.012

In this study, a series of benzimidazoles bearing thiosemicarbazide chain or triazole and thiadiazole rings were designed and synthesized. Crystal and molecular structure of the compound 5c has been characterized by single crystal X-ray crystallographic a

Full text of DOI:10.1016/j.ejmech.2019.04.012

Accepted Manuscript
Design, synthesis and docking studies of benzimidazole derivatives as potential
EGFR inhibitors
İsmail Çelik, Gülgün Ayhan-Kılcıgil, Berna Güven, Zümra Kara, A. Selen Gurkan-Alp,
Arzu Karayel, Arzu Onay-Beşikci
PII:
S0223-5234(19)30312-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.012
EJMECH 11247
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 December 2018
Revised Date: 1 March 2019
Accepted Date: 4 April 2019
Please cite this article as: İ. Çelik, Güü. Ayhan-Kılcıgil, B. Güven, Zü. Kara, A.S. Gurkan-Alp, A.
Karayel, A. Onay-Beşikci, Design, synthesis and docking studies of benzimidazole derivatives as
potential EGFR inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.04.012.
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ACCEPTED MANUSCRIPT
R
NH
S
S
R
NH
O
N
N
N
NH
NH
NHR
S
N
A
A
N
N
N
A
N
N
N
R= methyl; ethyl; 2-piperidinoethyl; 2-morpholinoethyl
A= H; 4-Cl; 4-methoxy; 3,4-dibenzyloxy

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Design, Synthesis and Docking Studies of Benzimidazole Derivatives as Potential EGFR
Inhibitors
İsmail Çelik1,2, Gülgün Ayhan-Kılcıgil1*, Berna Güven3, Zümra Kara3, A. Selen Gurkan-Alp1, Arzu Karayel4,
Arzu Onay-Beşikci3
1 Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Tandoğan-Ankara/Turkey
2 Erciyes University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 38039 Kayseri, Turkey
3 Ankara University, Faculty of Pharmacy, Department of Pharmacology, 06100 Tandoğan-Ankara/Turkey
4 Hitit University, Faculty of Arts and Science Department of Physics 19030 Çorum, Turkey
Abstract
In this study, a series of benzimidazoles bearing thiosemicarbazide chain or triazole and thiadiazole rings were
designed and synthesized. Crystal and molecular structure of the compound 5c has been characterized by
single crystal X-ray crystallographic analysis. EGFR kinase inhibitory potencies of synthesized compounds
were compared with erlotinib in vitro and most of the compounds exhibited significant activities. Cell culture
studies were also carried out for selected compounds and 12b was found to be the most active compound. To
understand the binding mode of synthesized benzimidazoles, three compounds (12b, 16, 16c) were selected
and placed on the binding site of EGFR tyrosine kinase based on their kinase inhibitor potencies and cell
culture studies. By docking study that compound 12b indicated two-hydrogen bonding interactions with
residues of LYS721 and THR830 at the binding pocket.
Key words: Benzimidazole, Thiosemicarbazide, Triazole, Thiadiazole, EGFR Inhibitory Activity, X-Ray,
Docking
1. Introduction
Cancer is still one of the most serious diseases in the world. According to the World Health Organization
(WHO), cancer is the second most important cause of deaths worldwide that had reached 8.8 million in 2015.
The number of new cases per year is projected to increase from 14.1 billion in 2012 to 21.6 billion in 2030.
The epidermal growth factor receptor (EGFR) belongs to ErbB family of receptor tyrosine kinases. It is
upregulated in many cancers such as breast cancer and head and neck squamous cell carcinoma. Moreover, in
more than half of the patients with non-small cell lung cancer (NSCLC), EGFR is overexpressed [1, 2].
Therefore, EGFR is an attractive target for anticancer therapy and a larger number of EGFR tyrosine kinase
inhibitors (TKIs) have been developed [3-7]. They exert their action through competitive inhibition for
adenosine triphosphate (ATP) binding in the tyrosine kinase domain. Benzimidazoles have various activities
and have versatile use in medicinal chemistry. Various studies reported that a third generation EGFR inhibitor
nazartinib [8], which has benzimidazole structure and the core structure of numerous biologically active
compounds, always possess a wide range of bioactivities including antimicrobial [9], antiparasitic [10],
antihistaminic [11], antiallergic [12], anticancer [13-16] and antioxidant [17-19] . Other third generation
EGFR TKIs, avitinib [20] and osimertinib [21], that bear indolopirimidine and indol rings, respectively, are
bioisosteres to benzimidazole (Figure 1) and are indicated for EGFR-mutated NSCLC.

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O
NH
O
N
F
N
O
N
N
N
N
NH
N
N
NH
NH
O
H
N
O
NH
N
N
N
N
Osimertinib
N
N
Avitinib
O
Nazartinib
Figure 1. Third generation EGFR Inhibitors
In this study, a series of benzimidazoles with thiosemicarbazide side chain or triazole and thiadiazole rings
were designed and synthesized. Next, their inhibitory potencies of EGFR kinase activity were compared to
erlotinib in vitro.
R
NH
NH
S
S
R
NH
O
N
N
N
NH
NHR
A
N
S
A
N
N
N
A
N
N
N
R= methyl; ethyl; 2-piperidinoethyl; 2-morpholinoethyl
A= H; 4-Cl; 4-methoxy; 3,4-dibenzyloxy
Figure 2. Designed benzimidazole derivatives

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NH
2
CHCOOEt
2
H
N
NH
2
A
A
N
b
a
A
N
N
Ia-IVa
I-IV
c
CHO
CHCONHNH
2
2
NH
S
R
O
A
N
N
NH
NH
d1
A
N
N
Ib-IVb
d2
e
1, 4-5, 8-9, 12-13, 16
f
S
R
N
NH
N
N
N
A
N
NHR
S
N
A
N
N
1-16c
1b, 4-5b, 8-9b, 12b
Scheme. Synthesis of benzimidazole derivatives
Reagents and conditions (a) Na2S2O5 (b) ClCH2COOEt/KOH–DMSO (c) NH2NH2.H2O/EtOH (d1) methyl/ethyl isothiocyanate-abs
EtOH 3h (d2) 2-piperidino/morpholinoethylisothiocyanate-abs EtOH 3h (e) H2SO4 (f) NaOH
2. Results and Discussion
2.1. Synthesis of compounds
The reaction sequences of the synthesized compounds are outlined in the Scheme above. Benzimidazoles (I-
IV) were synthesized via oxidative condensation of o-phenylenediamine and benzaldehyde derivatives. 2-(2-
substitutedphenyl)-1H-benzo[d]imidazol-l-yl) acetates (Ia-IVa) were obtained from the reaction of compounds
I-IV with ethyl chloroacetate in KOH/DMSO. Ester compounds (Ia-IVa) were treated with hydrazine hydrate
to give the hydrazides (Ib-IVb). The thiosemicarbazides were obtained from reaction of acid hydrazide with
alkyl isothiocyanates in ethanol. Cyclization reaction of these compounds with sulfuric acid or sodium
hydroxide resulted in the formation of thiadiazole and triazole derivatives, respectively. Surprisingly, reaction
of the hydrazide derivatives with 2-piperidino and 2-morpholinoethylisothiocyanates leads directly to the
triazole derivatives, in which no thiosemicarbazide derivatives can be isolated. Therefore, thiadiazole
compounds containing this side chain have not been obtained. All newly synthesized compounds were
characterized by 1H NMR,13C NMR, MS and elemental analysis data.

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2.2. Biological evaluations
2.2.1. EGFR Kinase Inhibitory Activity
EGFR kinase activity was measured in vitro by using ADP-Glo™ assay kit. [22]. This assay determines
luminescence obtained in a phosphorylation reaction where EGFR kinase, a substrate (PolyE4Y1), and ATP
were present in the reaction medium. Inhibitory efficiency of each new compound was determined by
comparing the enzyme activity in the presence of the inhibitors with the maximum enzyme activity. Inhibitory
efficiencies of the newly synthesized compounds were then evaluated by comparing them with the known
inhibitor, erlotinib (Figure 3). New compounds inhibited EGFR kinase to varying degrees (6-42%). Next, cell
culture studies were carried out for selected compounds that showed an inhibitory effect of ≥30%.
Figure 3. Percent inhibition by new compounds and erlotinib of EGFR kinase activity.
Control experiments only included DMSO and erlotinib – an inhibitor of EGFR activity- was used to validate the assay
conditions
.
Structure activity relationships of the synthesized compounds as EGFR kinase inhibitors have shown
that the benzimidazoles having 3,4-dibenzyloxyphenyl substituents in the second position are more
active than the unsubstituted/4-Cl/4-OCH3 phenyl derivatives. This can be interpreted as bulky
groups increasing activity.
It has also been found that those carrying alkyl groups on the “N” such as methyl or ethyl are more
active than those carrying piperidinyl or morpholinyl ethyl groups. This has shown that increasing the
polarity reduces effectiveness.
When compared with the thiosemicarbazide, thiadiazole and triazole structures, it was found that
most of the molecules carrying triazole structure were more active than the ones bearing the
thiosemicarbazide and thiadiazole counterparts.
2.2.2. Cell Proliferation Inhibitory Activity

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The MCF7 line represents differentiated mammary epithelium by its ability to process estradiol via
cytoplasmic estrogen receptors in addition to expressing epidermal growth factor receptors [23, 24]. Therefore,
this cell line was selected to investigate whether the new compounds affect cell proliferation and how they
compare with erlotinib. In line with the superior structural relationship with EGFR receptor, only compound
12b which contains phenyl ring in the second position of benzimidazole and N-metilamino group of 1,3,4-
thiadiazole ring inhibited cell proliferation by 25%.. Compound 12c inhibited cell proliferation by about 7%
however other compounds had no effect. At a concentration where EGFR kinase activity was inhibited by
nearly 92%, erlotinib’s inhibitory effect on cell proliferation was around 60%. This indicates that in vitro
inhibitory activity of EGFR kinase may not be fully translated to the inhibition of proliferation when tested on
cells.
Some selected compounds were also tested against cervical cancer represented by HeLA cell line. This cell
line also includes EGFR receptor and erlotinib (1 µM) inhibited kinase activity by 93% . This confirmed the
sensitivity of EGFR kinase of HeLa cells to the inhibition by erlotinib. However, none of the compounds
inhibited EGFR kinase activity significantly, although some inhibitory effect was observed within the
range of 4-28%. (results not shown).
Figure 4. Percent inhibition of cell proliferation as calculated based on the amount of viable cells after
inhibitor treatment.
*vs DMSO-treated cells, p<0.05
2.3. Molecular docking studies
In order to understand the binding mode of benzimidazole linked thiosemicarbazide, substituted triazole and
thiadiazole derivatives, docking studies were carried out by using the X-ray structure of EGFR tyrosine kinase
(PDB ID: 1M17), which was previously used for the development of some novel substituted benzimidazole-
linked oxadiazoles [14] and some pyrazolo[3,4-d]pyrimidines [25] as cytotoxic agents. Three compounds were
selected and docked into the binding site of EGFR tyrosine kinase (PDB ID: 1M17) based on their kinase
inhibitor potencies (16 and 16c) and cell culture studies (12b). Amino acids in the active site of EGFR
tyrosine kinase and the interaction of the best docking pose of compounds 12b, 16 and 16c are shown in
Figure 5. The docking analysis of the compounds revealed that hydrogen bonding represented by green line

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with LYS721 (for all compounds studied), THR830 (only for compound 12b) and van der Waals interactions
(cyan-maroon) observed in the active site stabilize the molecules in the protein. One hydrogen bond (2.26 Å)
was observed between C=O of the compound 16 and LYS721 side chain. It was observed that the compound
16 was compatible with the active binding site thanks to van der Waals interactions between the residues
LEU694, LYS721, PHE699, THR766, ASP831, MET769 at the binding pocket and the molecule (Figure 5a).
One hydrogen bond (2.36 Å) was also observed between NH of the triazole ring of the compound 16c and
LYS721 side chain. It was shown that compound 16c was also settled down with the formation of van der
Waals interactions between the molecule and the residues LEU694, VAL702, LYS721, PHE699, ARG817,
THR766 at the active site of the enzyme (Figure 5b). Compound 12b showed two-hydrogen bonding
interactions; one hydrogen bond between S of the thiadiazole ring of the compound and LYS721, one
hydrogen bond between N4 of the thiadiazole and THR830 side shain with the distances 3.74 Å and 3.15 Å,
respectively. In addition, the compound 12b was inside the protein due to van der Waals interactions between
the residues LEU694, LEU768, MET769 HIS811, LEU820, GLU738, ARG817, PHE699 at the binding site
and the molecule (Figure 5c).

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a
b
c
Figure 5. Binding mode of the most active compounds 16 (a), 16c (b) and 12b (c) into EGFR (PDB ID: 1M17) active site showing hydrogen bonds (green
line) with LYS721 and THR830 side chain (only for compound 12b) and van der Waals interactions (cyan-maroon). One hydrogen bond interaction between
C=O (16) and LYS721. One hydrogen bond between triazole nitrogen (16c) and LYS721. Two-hydrogen bonding interactions between S of the thiadiazole
(12b) and LYS721, and between N4 of the thiadiazole (12b) and THR830 side chain.

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2.4. X-Ray Structure Determination
We analyzed both molecular and crystal structure of the compound 5c (5-[(2-(4-methoxyphenyl)-1H-
benzimidazole-1-yl)methyl]- 2,4-dihidro-4-ethyl-3H-1,2,4-triazole-3-thione). We determined the parameters
regarding to molecular structure including the unit cell dimensions, atomic positions, bond length, angular
positions of bonds and the relative displacements. In Table 1, we tabulated all relevant parameters as well as
settings of X-ray analysis data. In Figure 6, we show the Ortep diagram [26] and crystal packing diagram of
5c in unit cell.
Figure 6. The ORTEP diagram of 5c (left) and the crystal packing of 5c (right) along [010]
containing N5―H1•••N2 hydrogen bond.
The main molecule in building blocks of the compound 5c consists of 1, 2, 4-triazole ring with ethyl
and methoxyphenylbenzimidazole groups at fourth and at fifth location of this ring, respectively.
Relevant molecule was crystallized in thione form. The hydrogen atoms are bound to the N5 atom
instead of the S1 atom in thione tautomeric form. Moreover, the bond distances of N5−C16 (1.340(3)
Å), N4−C16 (1.370(3) Å) and C16−S1(1.676(3) Å) are in value what we expected single and double
bond distances for this tautomeric form.
To describe three dimensional structure of compound 5c, we found the torsion angles of ethyl and
methoxyphenylbenzimidazole groups by the triazole ring. We determined the rotation of the ethyl
with respect to triazole ring with the torsion angle of C16−N4−C17−C18 which is found to be [-
87.7(5)º]. The rotation of benzimidazolemethyl group with respect to triazole ring can be described
by two torsion angles; N1−C14−C15−N4 (-63.8(4)º) and C1−N1−C14−C15 (-59.0(3)º). Additionally,
the angular positioning of the methoxyphenyl ring with respect to benzimidazole ring is given by the

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torsion angles of N2−C7−C8−C9 (-148.2(3)º). We listed some of the specific geometric parameters in
Table 2.
We can say that the benzimidazole counterpart of the structure is almost planar as the dihedral angle
connecting the imidazole and the adjacent phenyl rings is 0.90(9)º. While the dihedral angle joining
benzimidazole ring and the methoxyphenyl ring is 34.94(8)º, the dihedral angle between triazole ring
plane and benzimidazole group is 87.59(8)º. This result clearly demonstrates that the triazole part of
the molecule is almost perpendicular to the benzimidazole moieties.
In addition, triazole ring is also planar. The deviation of C16 atom from this plane is 0.0034 Å, while
the deviation of the thione group bonded to C16 from the triazole plane is 0.0223 Å. The deviation
of C17 bonded to triazole ring is 0.1020 Å. The maximum deviation from the mean plane of the
benzimidazole ring for C2 is -0.0106 Å (for C4 0.0158 Å). The deviation of C9 from the mean plane
of phenyl ring connected to benzimidazole ring is -0.0098 Å, while the deviation of O1 bonded to
phenyl ring is 0.0415 Å.
Table 1. The list of parameters of compound 5c related to crystal data and structure refinement.
Crystal data
5c
Chemical Formula
System, sp. gr., Z
a, b, c, Å
C19H19N5O1S1
Triclinic, P -1, 2
8.5407(6),9.0142(6),13.2882(11)
102.055(4),95.180(5),112.763(4)
905.8
α, β, γ, deg
V, Å3
Dx, g cm−3
1.34
Radiation, λ, Å
MoKα, 0.71073
0.197
µ, mm-1
T, K
296(2)
Sample size, mm
Diffractometer
Scan mode
0.69 x 0.52 x 0.32
Bruker APEX2
ω - 2θ
Absorption correction,Tmin, Tmax
θmax, deg
Psi-scan, 0.876, 0.9396
28.57
-11≤ h ≤11,-12 ≤ k ≤12,
-16≤ l ≤17
h, k, l ranges
Number of
reflections:measured/unique(N1)
Rint/with I > 2σ(I) (N2)
Refinement method
Number of refined parameters
wR2 relative to N1
20512/4551
0.0455/3280
full-matrix least square in F2
257
0.218
R1 relative to N2
0.069
1.058
0.531/-0.581
Goodness-of-fit on F2
∆ρmax/∆ρmin, e/Å3

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Table 2. Bond lengths (Å) , bond angles and torsion angles (
º
) of current molecule.
Bond distances
1.424(4)
1.676(3)
1.381(3)
1.392(4)
1.457(3)
1.324(3)
1.388(3)
1.340(3)
1.370(3)
1.298(3)
1.367(3)
1.370(3)
1.364(4)
C19 − O1
S1 − C16
N1− C7
N1 − C1
N1 − C14
N2 − C7
N2 − C6
N5 − C16
N5 − N3
N3 − C15
N4 − C15
N4 − C16
O1 − C11
Bond angles
106.6(2)
123.5(2)
106.4(2)
113.1(2)
107.7(2)
106.0(2)
117.5(2)
131.4(2)
130.5(2)
111.6(2)
122.4(2)
103.7(2)
128.9(2)
111.4(2)
116.5(3)
C7 − N1 − C1
C1 − N1 − C14
C7 − N2 − C6
C16 − N5 − N3
C15 − N4 − C16
N1− C1 − C6
C11 − O1 − C19
N1 − C1 − C2
Torsion angles
C16 − N5 − N3 − C15
N3 − N5 − C16 − N4
N3 − N5 − C16 − S1
C9 − C8 − C7 − N2
C13 − C8 − C7 − N1
N2 − C6 − C5
N2 − C7 − N1
N2 − C7 − C8
N5 − C16 − N4
N5 − C16 − S1
N3 − C15 − N4
O1 − C11 − C10
-0.5(3)
0.6(3)
-179.0(2)
-148.2(3)
-141.8(3)
173.8(3)
121.2(3)
179.0(3)
-0.2(3)
C19 − O1 − C11 − C10
N1 − C14 − C15 − N3
O1 − C11 − C10 − C9
C7 − N1 − C1 − C6
Hydrogen-bonding analysis revealed that current molecule has an intermolecular N−H···N hydrogen
bond between N2 atom of imidazole ring and N5 atom of triazole ring of neighbor molecule shown in
Table 3. We conclude N−H···N type hydrogen bond is the most effective one for crystal stabilization
(Figure 6).
Table 3. Hydrogen-bonding analysis of the relevant molecule (Å, º).
H ···A
D ···A
D − H ···A
D − H
D − H ···A
0.95(4)
1.87(4)
2.808(4)
172(4)
N(5) − H(1)….N(2)(i)
Symmetry code: (i) x, 1+y, z

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Conclusion
A series of benzimidazole derivatives bearing thiosemicarbazide side chain or triazole and thiadiazole rings
were synthesized. Their EGFR kinase inhibitory efficiency was determined by comparing them with a known
kinase inhibitor erlotinib in vitro and most of the compounds exhibited significant activities. Cell culture
studies were also carried out for selected compounds (12b, 12c, 13c, 14c, 15c, 16 and 16c) which exhibited
EGFR kinase inhibitory activities over ≥30%) against MCF7 cell and 12b was found to be the most active
compound. This situation also supported by docking study that compound 12b indicated two-hydrogen
bonding interactions with residues of LYS721 and THR830 at the binding pocket while other compounds
show only one interaction with LYS721 side chain.
3. Experimental
3.1. Chemistry
Uncorrected melting points were measured on a Büchi B-540 and an Electrothermal capillary melting point
1
13
apparatus. All chemical reagents were purchased from E. Merck and Aldrich. H and C NMR spectra were
recorded on Varian Varian Mercury 400 MHz instrument (Varian Inc., Palo Alto, CA, USA) at a proton
resonance frequency of 400.1779 and 100.6243 MHz for carbon . Chemical shifts are given in parts per
million (ppm) relative to tetramethylsilane as an internal standard. The following abbreviations are used to
identify peak splitting patterns; br = broad, s = singlet, d = doublet, t = triplet, q = quartet, dd =doublet of
doublet, m =multiplet. Coupling constant (J) are reported in Hz. ES-MS were taken with a Waters ZQ
Micromass LC–MS spectrometer with positive electrospray ionization method. Elemental analyses (C, H,
N,and S) were performed by Leco CHNS 932 instrument. All instrumental analyses were carried out at The
Central Instrumentation Laboratory of the Pharmacy Faculty of Ankara University, Ankara, Turkey.
Compounds I-IV, Ia-IVa, Ib-IIIb have been synthesized [27] and compound IVb in our laboratory previously.
3.1.1. Preparation of thiosemicarbazides N-methyl/ethyl-2-(2-(2-(phenyl/substitutedphenyl-1H-
benzo[d]imidazol-1-yl)acetyl)hydrazine-1-carbothioamides (1, 4-5, 8-9, 12-13, 16).
The mixture of acid hydrazide (2.03 mmol) and alkyl isothiocyanate (3.05 mmol) in absolute ethanol (20mL)
were refluxed for 3 h. The precipitate formed by cooling was filtered and crystallized from ethanol or
isopropanol.
3.1.1.1. 2-{[2-(4-Chlorophenyl)-1H-benzimidazol-1-yl]acetyl}-N-ethylhydrazinecarbothioamide (1)
1
Yield 88%, Mp: 234°C. H NMR (400 MHz, DMSO-d6, ppm)
δ
: 1.09(t, 3H, CH2CH3), 3.47-3.51 (m, 2H,
CH2CH3), 4.97 (s, 2H, CH2), 7.26-7.32 (m, 2H, Ar-H), 7.53-7.79 (m, 6H, Ar-H), 8.08, 8.71, 9.32, 9.54, 9.73,
10.30 (brs, NH). MS (ESI+) m/z: 388.01 (M+H), 390.15 (M+2+H). Anal. for C18H18ClN5OS (%) Calcd/Found:
C 55.74/55.40; H 4.68/4.84; N 18.06/17.76; S 8.27/8.104

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3.1.1.2. 2-{[2-(4-Chlorophenyl)-1H-benzimidazol-1-yl]acetyl}-N-methylhydrazinecarbothioamide (4)
1
Yield 83%, Mp: 244°C. H NMR (400 MHz, DMSO-d6, ppm)
δ
: 2.91(d, 3H, CH3), 4.96 (s, 2H, CH2), 7.26-
7.33 (m, 2H, Ar-H), 7.52-7.79 (m, 6H, Ar-H), 8.10, 8.65, 9.40, 9.61, 9.77, 10.32 (brs, NH). MS (ESI+) m/z:
373.94 (M+H), 375.95 (M+2+H). Anal. for C17H16ClN5OS (%) Calcd/Found: C 54.61/54.38; H 4.31/4.46; N
18.73/18.42; S 8.58/8.481
3.1.1.3. N-Ethyl-2-{[2-(4-methoxyphenyl)-1H-benzimidazol-1-yl]acetyl}- hydrazinecarbothioamide (5)
1
Yield 78%, Mp: 215°C. H NMR (400 MHz, DMSO-d6, ppm)
δ
: 1.09 (t, 3H, CH2CH3), 3.47-3.52 (m, 2H,
CH2CH3), 3.85 (s, 3H, OCH3), 4.94 (s, 2H, CH2), 7.10 (d, 2H, Jo= 8.8 Hz, Ar-H), 7.21-7.27 (m, 2H, Ar-H),
7.42-7.52 (m, 1H, Ar-H), 7.62-7.73 (m, 3H, Ar-H), 7.97, 8.60, 9.26, 9.63, 10.22 (brs, NH). MS (ESI+) m/z:
384.01(M+H). Anal. for C19H21N5O2S (%) Calcd/Found: C 59.51/59.13; H 5.52/5.81; N 18.26/18.02; S
8.36/8.21
3.1.1.4. 2-{[2-(4-Methoxyphenyl)-1H-benzimidazol-1-yl]acetyl}-N-methylhydrazinecarbothioamide (8)
Yield 87%, Mp: 221°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 2.92 (d, 3H, CH3), 3.85 (s, 3H, OCH3), 4.94
(s, 2H, CH2), 7.11 (dd, 2H, Jo=8.8Hz, Jm=2.4 Hz, Ar-H), 7.22-7.28 (m, 2H, Ar-H), 7.49-7.51 (m, 1H, Ar-H),
7.61-7.71 (m, 3H, Ar-H), 8.08, 8.63, 9.40, 9.61, 9.74, 10.29 (brs, NH). MS (ESI+) m/z: 370.03 (M+H). Anal.
for C18H19N5O2S.0.9 H2O (%) Calcd/Found: C 56.07/55.88; H 5.39/5.70; N 18.17/17.97; S 8.31/8.339.
3.1.1.5. N-Ethyl-2-[(2-phenyl-1H-benzimidazol-1-yl)acetyl]hydrazinecarbothioamide (9)
1
Yield 81%, Mp: 220°C. H NMR (400 MHz, DMSO-d6, ppm)
δ
: 1.09 (t, 3H, CH2CH3), 3.47-3.51 (m, 2H,
CH2CH3), 4.97 (s, 2H, CH2), 7.26-7.29 (m, 2H, Ar-H), 7.52-7.58 (m, 4H, Ar-H), 7.69-7.77 (m, 3H, Ar-H),
8.08, 8.69, 9.33, 9.55, 9.71, 10.22 (brs, NH). MS (ESI+) m/z: 354.02 (M+H). Anal. for C18H19N5OS (%)
Calcd/Found: C 61.17/61.06; H 5.38/5.55; N 19.83/19.42; S 9.07/8.92.
3.1.1.6. N-Methyl-2-[(2-phenyl-1H-benzimidazol-1-yl)acetyl]hydrazinecarbothioamide (12)
1
Yield 80%, Mp: 210°C. H NMR (400 MHz, DMSO-d6, ppm)
δ
: 2.91(d, 3H, CH3), 4.96 (s, 2H, CH2), 7.24-
7.32 (m, 2H, Ar-H), 7.52-7.59 (m, 4H, Ar-H),7.67-7.76 (m, 3H, Ar-H), 8.08, 8.64, 9.41, 9.61, 9.74, 10.29 (brs,
NH). MS (ESI+) m/z: 340.05 (M+H). Anal. for C17H17N5OS (%) Calcd/Found: C 60.16/59.76; H 5.05/5.28; N
20.63/20.23; S 9.45/9.39.
3.1.1.7. 2-{[2-(3,4-Dibenzyloxyphenyl)-1H-benzimidazol-1-yl]acetyl}-N-ethylhydrazinecarbothioamide
(13)
1
Yield 69%, Mp: 170°C. H NMR (400 MHz, DMSO-d6, ppm)
δ
: 1.05 (t, 3H, CH2CH3), 3.41-3.48 (m, 2H,
CH2CH3), 4.70 (s, 2H, CH2), 5.18 (s, 2H, OCH2Ph), 5.22 (s, 2H, OCH2Ph), 7.19-7.23 (m, 4H, Ar-H), 7.28-7.84

ACCEPTED MANUSCRIPT
(m, 16H, Ar-H, NH), MS (ESI+) m/z: 565.98 (M+H). Anal. for C32H31N5O3S-0.5H2O (%) Calcd/Found: C
66.87/66.78; H 5.61/5.72; N 12.19/12.32; S 5.58/5.41.
3.1.1.8. 2-{[2-(3,4-Dibenzyloxyphenyl)-1H-benzimidazol-1-yl]acetyl}-N-methylhydrazinecarbothioamide
(16)
Yield 72%, Mp: 160°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 3.89 (d, 3H, CH3), 4.98 (s, 2H, CH2), 5.18 (s,
2H, OCH2Ph), 5.24 (s, 2H, OCH2Ph), 7.20-7.49 (m, 15H, Ar-H), 7.54 (d, Jm=2 Hz, Ar-H),7.67 (dd, Jo=6.8
Hz, Jm=2.0 Hz, Ar-H), 8.03, 9.38, 9.59, 9.69, 10.29 (brs, NH). MS (ESI+) m/z: 551.97 (M+H). Anal. for
C31H29N5O3S. H2O (%) Calcd/Found: C 65.37/65.29; H 5.44/5.02; N 12.30/12.18; S 5.69/5.93
3.1.2. Preparation of the N-alkyl-5-((2-phenyl/substitutedphenyl-1H-benzo[d]imidazol-1-yl)methyl)-
1,3,4-thiadiazol-2-amines (1b, 4b-5b, 8b-9b, 12b).
Thiosemicarbazides (3.4 mmol) were stirred in concentrated sulfuric acid (10 mL) for 10 min in ice bath and
then continued stirring at room temperature for another 10 min. At the end of this period the solution was
poured slowly into ice water and pH was made alkaline (pH=8) with the aqueous ammonia. The precipitate
was separated by filtration and washed with water. The crude product was purified through crystallization with
ethanol or ethanol / isopropanol (10:1)
3.1.2.1. 5-{[2-(4-Chlorophenyl)-1H-benzimidazol-1-yl]methyl}-N-ethyl-1,3,4-thiadiazol-2-amine (1b)
Yield 42%, Mp: 176-177°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 1.10 (t, 3H, CH2CH3), 3.16-3.23 (m, 2H,
CH2CH3), 5.75 (s, 2H, CH2), 7.27-7.34 (m, 2H, Ar-H), 7.64-7.73 (m, 5H, Ar-H, N-H), 7.88 (d, 2H, Jo=8.8 Hz,
Ar-H). MS (ESI+) m/z: 370.03 (M+H), 372.4 (M+2+H). Anal. for C18H16ClN5S (%) Calcd/Found: C
58.45/58.17; H 4.36/4.05; N 18.93/18.82; S 8.67/8.48
3.1.2.2. 5-{[2-(4-Chlorophenyl)-1H-benzimidazol-1-yl]methyl}-N-methyl-1,3,4-thiadiazol-2-amine (4b)
Yield 51%, Mp: 234-237°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 2.79 (d, 3H, CH3), 5.75 (s, 2H, CH2),
7.14-7.34 (m, 2H, Ar-H), 7.65-7.73 (m, 5H, Ar-H, N-H), 7.87 (d, 2H, Jo=8.8 Hz, Ar-H). MS (ESI+) m/z: 356.2
(M+H), 358.3 (M+2+H). Anal. for C17H14ClN5S-1.6H2O (%) Calcd/Found: C 53.07/52.78; H 4.50/4.68; N
18.20/18.70; S 8.33/7.93.
3.1.2.3. N-Ethyl-5-{[2-(4-methoxyphenyl)-1H-benzimidazol-1-yl]methyl}-1,3,4-thiadiazol-2-amine (5b)
Yield 38%, Mp: 189-191°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 1.09 (t, 3H, CH2CH3), 3.19-3.23 (m, 2H,
CH2CH3), 3.84 (s, 3H, OCH3), 5.71 (s, 2H, CH2), 7.13 (d, 2H, Jo=8.8 Hz, Ar-H), 7.23-7.30 (m, 2H, Ar-H),
7.60-7.71 (m, 3H, Ar-H, N-H), 7.80 (d, 2H, Jo=8.4 Hz, Ar-H). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 14.06,
39.41, 43.15, 55.27, 110.76, 114.24, 118.96, 121.84, 122.27, 122.45, 130.65, 135.42, 142.53, 152.59, 152.80,

ACCEPTED MANUSCRIPT
160.47, 168.97. MS (ESI+) m/z: 352.0 (M+H). Anal. for C19H19N5OS (%) Calcd/Found: C 62.44/62.10; H
5.24/5.51; N 19.16/18.81; S 8.77/8.74.
3.1.2.4. 5-{[2-(4-Methoxyphenyl)-1H-benzimidazol-1-yl]methyl}-N-methyl-1,3,4-thiadiazol-2-amine (8b)
Yield 35%, Mp: 194°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 2.78 (d, 3H, CH3), 3.84 (s, 3H, OCH3), 5.71
(s, 2H, CH2), 7.13 (d, 2H, Jo=8.4 Hz, Ar-H), 7.23-7.30 (m, 2H, Ar-H), 7.60-7.69 (m, 3H, Ar-H, N-H), 7.79 (d,
2H, Jo=8.8, Ar-H). MS (ESI+) m/z: 352.00 (M+H). Anal. for C18H17N5OS-1.0 H2O (%) Calcd/Found: C
58.51/58.42; H 5.18/5.11; N 18.95/19.35; S 8.68/9.43.
3.1.2.5. N-Ethyl-5-[(2-phenyl-1H-benzimidazol-1-yl)methyl]-1,3,4-thiadiazol-2-amine (9b)
Yield 44%, Mp: 105-107.5°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 1.09 (t, 3H, CH2CH3), 3.18-3.21 (m,
2H, CH2CH3), 5.73 (s, 2H, CH2), 7.30-7.31 (m, 2H, Ar-H), 7.58-7.71 (m, 6H, Ar-H, N-H), 8.84 (s, 2H, Ar-H).
MS (ESI+) m/z: 336.5 (M+H). Anal. for C18H17N5S (%) Calcd/Found: C 64.45/64.47; H 5.10/5.32; N
20.87/20.48; S 9.55/9.22.
3.1.2.6. N-Methyl-5-[(2-phenyl-1H-benzimidazol-1-yl)methyl]-1,3,4-thiadiazol-2-amine (12b)
Yield 53%, Mp: 109-112.5°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 2.79 (d, 3H, CH3), 5.73 (s, 2H, CH2),
7.26-7.33 (m, 2H, Ar-H), 7.57-7.59 (m, 3H, Ar-H, N-H), 7.62 (d, 2H, Jo=7.2 Hz, Ar-H), 7.72 (d, 1H, Jo=7.2
Hz, Ar-H), 7.82-7.85 (m, 2H, Ar-H). MS (ESI+) m/z: 322.4 (M+H). Anal. for C17H15N5S-0,7H2O (%)
Calcd/Found: C 61.13/60.97; H 4.94/4.82; N 20.96/21.36; S 9.60/9.98.
3.1.3. Preparation of 4-alkyl-5-((2-phenyl/substitutedphenyl -1H-benzo[d]imidazol-1-yl)methyl)-2,4-
dihydro-3H-1,2,4-triazole-3-thione (1c-16c).
3.1.3.a. Compounds 1c, 4c-5c, 8c-9c, 12c-13c, 16c
Thiosemicarbazides (3.4 mmol) (1,4-5,8-9,12-13,16) in 1 N sodium hydroxide (10 mL) were refluxed for 1 h,
then the mixture was cooled and acidified to pH 6 with 1 N hydrochloric acid. Precipitate formed was filtered,
washed with water. The solid was crystallized from ethanol.
3.1.3.a.1. 5-{[2-(4-Chlorophenyl)-1H-benzimidazol-1-yl]methyl}-4-ethyl-2,4-dihydro-3H-1,2,4-triazole-
3-thione (1c)
Yield 48%, Mp: 279-280°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 1.00 (t, 3H, CH2CH3), 3.85-3.90 (m, 2H,
CH2CH3), 5.75 (s, 2H, CH2), 7.26-7.32 (m, 2H, Ar-H), 7.54-7.64 (m, 6H, Ar-H), 13.64 (s, 1H, N-H). MS
(ESI+) m/z: 369.95 (M+H), 371.95 (M+2+H). Anal. for C18H16ClN5S-1.25 H2O (%) Calcd/Found: C
55.22/54.89; H 4.73/4.13; N 17.88/17.79; S 8.19/7.95.

ACCEPTED MANUSCRIPT
3.1.3.a.2. 4-Methyl-5-{[2-(4-chlorophenyl)-1H-benzimidazol-1-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-
3-thione (4c)
Yield 61%, Mp: 270°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 3.42 (s, 3H, CH3), 5.75 (s, 2H, CH2), 7.32-
7.34 (m, 2H, Ar-H), 7.64-7.68 (m, 3H, Ar-H), 7.74-7.79 (m, 3H, Ar-H), 13.67 (s, 1H, N-H). MS (ESI+) m/z:
356.2 (M+H), 358.3 (M+2+H). Anal. for C17H14ClN5S (%) Calcd/Found: C 57.38/57.19; H 3.97/4.14; N
19.68/19.26; S 9.01/8.76.
3.1.3.a.3. 4-Ethyl-5-{[2-(4-methoxyphenyl)-1H-benzimidazol-1-yl]methyl}-2,4-dihydro-3H-1,2,4-
triazole-3-thione (5c)
Yield 53%, Mp: 275-276°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 0.93 (t, 3H, CH2CH3), 3.79-3.85 (m, 2H,
CH2CH3), 3.87 (s, 3H, OCH3), 5.65 (s, 2H, CH2), 7.09-7.14 (m, 2H, Ar-H), 7.28-7.46 (m, 2H, Ar-H), 7.47-
7.51 (m, 1H, Ar-H), 7.63-7.69 (m, 3H, Ar-H). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 11.95, 29.26, 39.93,
54.56, 110.38, 114.18, 118.38, 120.97, 122.86, 123.15, 130.55, 135.31, 141.85, 148.17, 154.06, 161.67,
168.26. MS (ESI+) m/z: 366.03 (M+H). Anal. for C19H19N5OS-0,15H2O (%) Calcd/Found: C 61.98/61.70; H
5.28/5.29; N 19.02/19.49; S 8.70/8.85.
3.1.3.a.4. 5-{[2-(4-Methoxyphenyl)-1H-benzimidazol-1-yl]methyl}-4-methyl-2,4-dihydro-3H-1,2,4-
triazole-3-thione (8c)
Yield 56%, Mp: 284°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 3.42 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 5.74
(s, 2H, CH2), 7.14-7.16 (m, 2H, Ar-H), 7.33-7.35 (m, 2H, Ar-H), 7.65-7.75 (m, 4H, Ar-H), 13.67 (s, 1H, N-H).
13C NMR (100 MHz, DMSO-d6, ppm) δ: 29.86, 55.30, 110.84, 114.31, 118.96, 121.82, 122.32, 122.43,
130.44, 135.87, 142.49, 148.50, 153.19, 160.49, 167.53. MS (ESI+) m/z: 352.5 (M+H). Anal. for C18H17N5OS-
0.2H2O (%) Calcd/Found: C 60.89/60.88; H 4.93/5.11; N 19.72/19.77; S 9.03/9.01.
3.1.3.a.5. 4-Ethyl-5-[(2-phenyl-1H-benzimidazol-1-yl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione
(9c)
1
Yield 88%, Mp: 290°C. H NMR (400 MHz, DMSO-d6, ppm)
δ
: 0,98 (t, 3H, CH2CH3), 3.83-3.88 (m, 2H,
CH2CH3), 5.74 (s, 2H, CH2), 7.27-7.31 (m, 2H, Ar-H), 7.54-7.58 (m, 4H, Ar-H), 7.71-7.73 (m, 3H, Ar-H). 13C
NMR (100 MHz, DMSO-d6, ppm) δ: 12.86, 38.29, 39.90, 110.81, 119.21, 122.32, 122.80 128.76, 128.86,
129.50, 129.95, 135.77, 142.35, 147.67, 153.00, 168.85. MS (ESI+) m/z: 336.3 (M+H). Anal. for C18H17N5S-
0,2H2O (%) Calcd/Found: C 63.76/63.98; H 5.17/5.64; N 20.66/20.58; S 9.45/9.55.
3.1.3.a.6. 4-Methyl-5-[(2-phenyl-1H-benzimidazol-1-yl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione
(12c)
Yield 38%, Mp: 262-263°C. 1H NMR (400 MHz, DMSO-d6, ppm)
7.26-7.28 (m, 2H, Ar-H), 7.55-7.58 (m, 4H, Ar-H), 7.71-7.75 (m, 3H, Ar-H), 13.62 (s, 1H, N-H). 13C NMR
δ: 2.49 (s, 3H, CH3), 5.70 (s, 2H, CH2),

ACCEPTED MANUSCRIPT
(100 MHz, DMSO-d6, ppm) δ: 29.78, 39.99, 110.93, 119.13, 122.30, 122.66, 128.75, 129.55, 129.86, 135.78,
142.38, 148.35, 153.10, 167.44. MS (ESI+) m/z: 322.3 (M+H). Anal. for C17H15N5S (%) Calcd/Found: C
63.52/63.48; H 4.70/4.63; N 21.79/21.61; S 9.97/9.75.
3.1.3.a.7. 5-{[2-(3,4-Dibenzyloxyphenyl)-1H-benzimidazol-1-yl]methyl}-4-ethyl-2,4-dihydro-3H-1,2,4-
triazole-3-thione (13c)
Yield 36%, Mp: 268-270°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 0,93 (t, 3H, CH2CH3), 3.79-3.84 (m,
2H, CH2CH3), 5.15 (s, 2H, OCH2Ph), 5.22 (s, 2H, OCH2Ph), 5.71 (s, 2H, CH2), 7.24-7.50 (m, 14H, Ar-H),
7.55-7.57 (m, 2H, Ar-H), 7.70-7.73 (m, 1H, Ar-H), 13.72 (s, 1H, N-H). MS (ESI+) m/z: 548.7 (M+H). Anal.
for C32H29N5O2S-1.5H2O (%) Calcd/Found: C 66.87/66.84; H 5.61/5.236; N 12.18/12.18; S 5.57/5.33.
3.1.3.a.8. 5-{[2-(3,4-Dibenzyloxyphenyl)-1H-benzimidazol-1-yl]methyl}-4-methyl-2,4-dihydro-3H-1,2,4-
triazole-3-thione (16c)
Yield 34%, Mp: 238°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 3.43 (s, 3H, CH3), 5.15 (s, 2H, OCH2Ph),
5.26 (s, 2H, OCH2Ph), 5.86 (s, 2H, CH2), 7.30-7.58 (m, 15H, Ar-H), 7.83-7.91 (m, 2H, Ar-H), 13.77 (s, 1H,
N-H). MS (ESI+) m/z: 534.4 (M+H). Anal. for C31H27N5O2S-2,55H2O (%) Calcd/Found: C 64.24/64.30; H
5.58/5.37; N 12.08/11.85; S 5.53/5.43.
3.1.3.b. Compounds 2c-3c, 6c-7c, 10c-11c, 14c-15c
Acid hydrazide (2.03 mmol) in absolute ethanol:N,N-dimethylformamide (1:1) (20 mL) and 2-piperidino/2-
morpholinoethyl isothiocyanate (3.05 mmol) were refluxed for 3 h. Precipitate formed was cooled, filtered and
recrystallized from ethanol or isopropanol.
3.1.3.b.1. 5-{[2-(4-Chlorophenyl)-1H-benzimidazol-1-yl]methyl}-4-[2-(piperidin-1-yl)ethyl]-2,4-dihydro-
3H-1,2,4-triazole-3-thione (2c)
Yield 80%, Mp: 173°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 1.28 (brs, 6H, aliphatic H), 2.32 (brs, 4H,
aliphatic H), 2.48 (t, 2H, aliphatic H), 4.09 (t, 2H, aliphatic H), 5.76 (s, 2H, CH2), 7.28-7.30 (m, 2H, Ar-H),
7.50-7.53 (m, 1H, Ar-H), 7.64 (d, 2H, Jo= 8.4 Hz, Ar-H), 7.74 (d, 3H, Jo= 8.8 Hz, Ar-H), 13.64 (s, 1H, N-H).
13C NMR (100 MHz, DMSO-d6, ppm) δ: 23.67, 25.28, 40.22, 41.43, 54.38, 56.57, 110.94, 119.37, 122.55,
122.93, 128.50, 129.04, 130.69, 135.06, 135.78, 142.46, 148.75, 152.28, 167.36. MS (ESI+) m/z: 453.7(M+H),
455.5 (M+2+H). Anal. for C23H25ClN6S-0.1H2O (%) Calcd/Found: C 60.73/60.39; H 5.58/5.77; N
18.47/18.08; S 7.05/7.05.
3.1.3.b.2. 5-{[2-(4-Chlorophenyl)-1H-benzimidazol-1-yl]methyl}-4-[2-(morpholin-4-yl)ethyl]-2,4-
dihydro-3H-1,2,4-triazole-3-thione (3c)

ACCEPTED MANUSCRIPT
Yield 57%, Mp: 270-272°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 2.37 (brs, 4H, aliphatic H), 3.30 (brs,
6H, aliphatic H), 4.09 (s, 2H, aliphatic H), 5.76 (s, 2H, CH2), 7.26-7.29 (m, 2H, Ar-H), 7.51-7.52 (m, 1H, Ar-
H), 7.62-7.65 (m, 2H, Ar-H), 7.72-7.74 (m, 3H, Ar-H), 13.64 (s, 1H, N-H). 13C NMR (100 MHz, DMSO-d6,
ppm) δ: 40.25, 40.91, 53.40, 56.26, 65.89, 110.86, 119.31, 122.48, 122.91, 128.47, 128.96, 130.63, 134.97,
135.81, 142.42, 148.54, 152.15, 167.47. MS (ESI+) m/z: 455.5(M+H), 457.9(M+2+H). Anal. for
C22H23ClN6OS (%) Calcd/Found: C 58.08/58.08; H 5.10/5.32; N 18.47/18.08; S 7.08/6.93.
3.1.3.b.3. 5-{[2-(4-Methoxyphenyl)-1H-benzimidazol-1-yl]methyl}-4-[2-(piperidin-1-yl)ethyl]-2,4-
dihydro-3H-1,2,4-triazole-3-thione (6c)
Yield 75%, Mp: 158-159°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 1.27 (brs, 6H, aliphatic H), 2.31 (brs,
4H, aliphatic H), 2.46 (t, 2H, aliphatic H), 3.81 (s, 3H, OCH3), 4.09 (t, 2H, aliphatic H), 5.75 (s, 2H, CH2),
7.12 (d, 2H, Jo=9.2 Hz, Ar-H), 7.21-7.27 (m, 2H, Ar-H), 7.46-7.48 (m, 1H, Ar-H), 7.63-7.70 (m, 3H, Ar-H),
13.66 (s, 1H, N-H). 13C NMR (100 MHz, DMSO-d6, ppm) δ: δ 24.16, 25.74, 40.69, 41.84, 54.84, 55.78,
57.02, 111.20, 114.81, 119.50, 122.28, 122.74, 122.87, 130.82, 136.22, 143.01, 149.38, 153.84, 161.06,
167.74. MS (ESI+) m/z: 449.5 (M+H). Anal. for C24H28N6OS-0,95H2O (%) Calcd/Found: C 61.89/61.57; H
6.47/6.23; N 18.04/17.82; S 6.88/6.81.
3.1.3.b.4. 5-{[2-(4-Methoxyphenyl)-1H-benzimidazol-1-yl]methyl}-4-[2-(morpholin-4-yl)ethyl]-2,4-
dihydro-3H-1,2,4-triazole-3-thione (7c)
Yield 49%, Mp: 220°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 2.38 (brs, 4H, aliphatic H), 3.32 (brs, 6H,
aliphatic H), 3.82 (s, 3H, OCH3), 4.11 (brs, 2H, aliphatic H), 5.76 (s, 2H, CH2), 7.12 (d, 2H, Jo= 8.8 Hz, Ar-
H), 7.24-7.26 (m, 2H, Ar-H), 7.49-7.51 (m, 1H, Ar-H), 7.66 (d, 2H, Jo= 8.8 Hz, Ar-H), 7.68-7.71 (m, 1H, Ar-
H), 13.69 (s, 1H, N-H). MS (ESI+) m/z: 451.7 (M+H). Anal. for C23H26N6O2S-1,7H2O (%) Calcd/Found: C
57.40/57.59; H 6.15/5.98; N 17.46/16.77; S 6.66/6.28.
3.1.3.b.5. 5-[(2-Phenyl-1H-benzimidazol-1-yl)methyl]-4-[2-(piperidin-1-yl)ethyl]-2,4-dihydro-3H-1,2,4-
triazole-3-thione (10c)
Yield 72%, Mp: 252-254°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 1.27 (brs, 6H, aliphatic H), 2.31 (brs,
4H, aliphatic H), 2.46 (t, 2H, aliphatic H), 4.09 (t, 2H, aliphatic H), 5.79 (s, 2H, CH2), 7.26-7.30 (m, 2H, Ar-
H), 7.49-7.57 (m, 4H, Ar-H), 7.72-7.74 (m, 3H, Ar-H), 13.66 (s, 1H, N-H). 13C NMR (100 MHz, DMSO-d6,
ppm) δ: 23.67, 25.29, 40.21, 41.38, 54.37, 56.51, 109.53, 110.89, 119.28, 122.41, 122.71, 128.88, 129.66,
130.01, 135.74, 142.52, 148.83, 153.35, 167.26. MS (ESI+) m/z: 419.3 (M+H). Anal. for C23H26N6S (%)
Calcd/Found: C 64.88/64.73; H 6.34/6.44; N 19.73/19.49; S 7.53/7.49.
3.1.3.b.6. 5-[(2-Phenyl-1H-benzimidazol-1-yl)methyl]-4-[2-(morpholin-4-yl)ethyl]-2,4-dihydro-3H-1,2,4-
triazole-3-thione (11c)

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Yield 49%, Mp: 135-137.5°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 2.36 (brs, 4H, aliphatic H), 3.33 (brs,
6H, aliphatic H), 4.10 (t, 2H, aliphatic H), 5.77 (s, 2H, CH2), 7.26-7.29 (m, 2H, Ar-H), 7.51-7.58 (m, 4H, Ar-
H), 7.72-7.74 (m, 3H, Ar-H), 13.68 (s, 1H, N-H). 13C NMR (100 MHz, DMSO-d6, ppm) δ: 40.79, 41.41,
53.94, 56.83, 66.42, 110.00, 111.38, 119.75, 122.87, 123.22, 129.36, 130.14, 130.51, 136.27, 142.98, 149.24,
153.76, 167.85. MS (ESI+) m/z: 421.4 (M+H). Anal. for C22H24N6OS-1.7H2O (%) Calcd/Found: C
58.56/58.21; H 6.12/6.23; N 18.62/18.34; S 7.10/7.02.
3.1.3.b.7. 5-{[2-(3,4-Dibenzyloxyphenyl)-1H-benzimidazol-1-yl]methyl}-4-[2-(piperidin-1-yl)ethyl]-2,4-
dihydro-3H-1,2,4-triazole-3-thione (14c)
Yield 63%, Mp: 127°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 1.23 (brs, 6H, aliphatic H), 2.26 (brs, 4H,
aliphatic H), 2.45 (t, 2H, aliphatic H), 4.06 (t, 2H, aliphatic H), 5.04 (s, 2H, OCH2Ph), 5.21 (s, 2H, OCH2Ph),
5.71 (s, 2H, CH2), 7.24-7.25 (m, 4H, Ar-H), 7.31-7.49 (m, 12H, Ar-H), 7.69 (s, 1H, Ar-H). MS (ESI+) m/z:
631.6 (M+H). Anal. for C37H38N6O2S-0,1H2O (%) Calcd/Found: C 70.24/69.84; H 6.08/6.42; N 13.28/13.06; S
5.06/5.38.
3.1.3.b.8. 5-{[2-(3,4-Dibenzyloxyphenyl)-1H-benzimidazol-1-yl]methyl}-4-[2-(morpholin-4-yl)ethyl]-2,4-
dihydro-3H-1,2,4-triazole-3-thione (15c)
Yield 38%, Mp: 187°C. 1H NMR (400 MHz, DMSO-d6, ppm)
δ: 3.23 (brs, 6H, aliphatic H), 3.59 (brs, 4H,
aliphatic H), 4.59 (t, 2H, aliphatic H), 5.21 (s, 2H, OCH2Ph), 5.27 (s, 2H, OCH2Ph), 6.18 (s, 2H, CH2), 7.29-
7.49 (m, 12H, Ar-H), 7.52-7.56 (m, 2H, Ar-H), 7.62 (s, 1H, Ar-H), 7.86 (d, 1H, Jo= 6.8 Hz, Ar-H), 8.03 (d,
1H, Jo= 7.6 Hz, Ar-H), 13.99 (s, 1H, N-H). MS (ESI+) m/z: 633.8 (M+H). Anal. for C36H36N6O3S-0.3 H2O
(%) Calcd/Found: C 67.75/67.85; H 5.78/6.15; N 13.16/12.52; S 5.02/4.76.
3.2. Pharmacology
3.2.1. EGFR Kinase Assays
ADP-Glo™ assay kit [22] evaluates kinase activity by quantifying the amount of ADP produced during a
kinase reaction. The assays contained several consecutive steps. Preliminary assays were performed to
determine the amount of kinase and ATP (not shown). Next a kinase reaction was performed with 5 ng EGFR
kinase and 50 µM ATP. As recommended by the manufacturer, kinase reaction was the total volume of the
kinase reaction was 25 µLand the amount of EGFR substrate, PolyE4Y1, was 1 µg based on the previously
published manuscripts in the field. To terminate the kinase reaction, 25 µL ADP-Glo™ reagent was added to
terminate the reaction and deplete the unused ATP. Finally, a kinase detection reagent was added to convert
the ADP produced during the kinase reaction to ATP. The amount of newly synthesized ATP was determined
by a luciferase/luciferin reaction.
Maximum kinase activity (100% kinase) was determined as the luminescence obtained in the kinase reaction
where EGFR kinase, PolyE4Y1, ATP and diluting agent, 5% DMSO were present in the reaction medium.
Inhibitory efficiency of each compound (with expected inhibitory activity) was determined by comparing the

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enzyme activities in the presence of the inhibitors with the maximum enzyme activity. Percent inhibition was
calculated based on the luminescence of the maximum activity.
3.2.2. Screening of Inhibitor Activity
Kinase reactions were performed in 25 µl reaction medium where 50 µM ATP, 5 ng EGFR kinase ± 1 µM
inhibitor were present in each reaction. To confirm dose-dependency of its inhibitory activity, erlotinib was
tested in two different concentrations: 100nM and 1µM to confirm of the inhibitory activity. DMSO (solvent,
is used as a control in all experiments. These reactions constitute the maximum kinase activity (100%
activity).
3.2.3. Cell Culture Studies
The cells were kept in high glucose (4.5 mM) DMEM that was supplemented with 10% fetal bovine serum
(FBS), 2 mM L-glutamine, and 1% penicillin–streptomycin (10.000 U/mL penicillin–10.000 µg/mL
streptomycin). Cells were seeded onto the 24-well tissue culture plates and kept in a 37°C incubator. MTT (3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were employed to assess the effects of
compounds 12b, 12c, 13c, 14c, 15c, 16 and 16c and erlotinib on cell viability. When the cells reached 80%
confluency, the inhibitors (10µM) and erlotinib (1µM) were dissolved in DMSO and added to cells. To test for
possible effects of DMSO on viability, 1 µl of DMSO was added to control cells. In addition, some wells were
left untreated and incubated with regular cell culture media. MTT in phosphate buffer saline (PBS) solution
was added to all cells. After incubation at 37°C for 2 h, the media was carefully removed, purple formazan
crystals were dissolved in DMSO and the absorbance of the solution was determined at 550 nm. The viability
of the control cells was defined as 100% and the relative cell viability (%) was calculated based on the
absorbance.
3.3. Molecular Docking
AutoDock vina v.1.1.2 [28] was used to dock the ligands into receptor binding site. The X-ray
structure of EGFR tyrosine kinase [29] in complex with erlotinib, PDB ID: 1M17, was downloaded
from protein data bank (http://www.rcsb.org/pdb/home/home.do). The ligand erlotinib and other non-
protein molecules were initially removed from the binding pocket. MGL Tools v.1.5.6. [30] was used
to arrange the receptor and the ligands that were saved in .pdbqt format for docking. Docking of the
compounds 16 and 16c were carried out in a grid box determined by MGL Tools v.1.5.6 [30]. The
most suitable poses of the docked ligands were selected based on the benzimidazole pharmacophore.
Docked ligands were analyzed with UCSF Chimera-1.7 package [31].

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3.4. X-Ray Diffraction
We determined the crystal structure of compound 5c by X-ray diffraction. We collected the intensities of
Bragg reflections which are used in determining the structure on Bruker APEX2 [32]. We used WinGX [33]
program for the structural solution (SHELXS-97 [34]) and the full-matrix least squares refinement (SHELXL-
97 [35]). We did the refinement of all atoms anisotropically, except for hydrogens. We found the position of
the hydrogen atom of N5 in triazole ring using a difference map and refined it isotropically, on the other hand,
we used riding model to calculate other hydrogen atoms idealized positions.
Crystallographic structural data of 5c molecule have been deposited at the Cambridge Crystallographic Data
Centre (CCDC) with CCDC 1854601 deposition number.
Acknowledgement: This study is partially funded by Ankara University-BAP 17H0237002 and TUBITAK
214S574. The authors acknowledge Hitit University-Scientific Research Unit (BAP) with project
number FEF19004.17.001 and the Aksaray University Science and Technology Application and
Research Center, Aksaray, Turkey, for the use of the Bruker SMART BREEZE CCD diffractometer
(purchased under grant No. 2010K120480 of the State of Planning Organization).
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30 New compounds with benzimidazole scaffolds were designed and synthesized.
Crystal and molecular structure of the compound 5c has been characterized by single crystal X-ray
crystallographic analysis.
EGFR kinase inhibitory potencies of synthesized compounds were compared with erlotinib in vitro.
Docking was carried for three compounds by using the X-ray structure of EGFR tyrosine kinase.