Synthesis and biological evaluation of some novel thiobenzimidazole derivatives as anti-renal cancer agents through inhibition of c-MET kinase

Article abstract of DOI:10.1016/j.bioorg.2019.01.006

Benzimidazole is an interesting scaffold constituting a main core in many anticancer agents against variable cell lines as Carbendazim (I) and Nocodazole (II). Accordingly, eighteen compounds of 2-((1H-benzoimidazol-2-yl)thio)-1-(aryl/heteroaryl)ethan-1-ones, in their sulfate salt and free forms, were designed and investigated as anticancer agents. In vitro preliminary screening of selected compounds by the National Cancer Institute (NCI) on a panel of 60 cell lines revealed renal cancer cell line (A498) as the most vulnerable cell line; accordingly, IC₅₀ values against A498 cell line were determined for compounds with the best results. The best inhibitory activity was for compound 4a with (IC₅₀ = 6.97 μM) compared to sunitinib as a reference drug (IC₅₀ = 6.99 μM). Compound 4a was further subjected to cell cycle analysis that indicated the decrease in cell population in the G2/M phase when compared to the untreated control cells. In addition, it showed significant increase in the late apoptosis in Annexin-V FTIC study compared to the control cells. An enzymatic inhibitory study on compound 4a against c-Met and MAP kinases revealed its better activity against c-Met kinase with (IC₅₀ = 0.27 μM) compared to sunitinib (IC₅₀ = 0.18 μM). Molecular docking study was conducted to reveal the interactions of compound 4a in the active site of c-Met kinase. Computational ADME study was performed to insure that compound 4a has proper pharmacokinetic and drug-likeness properties.

Full text of DOI:10.1016/j.bioorg.2019.01.006

Accepted Manuscript
Synthesis and biological evaluation of some novel thiobenzimidazole deriva-
tives as anti-renal cancer agents through inhibition of c-MET kinase
Hany S. Ibrahim, Mohamed E. Albakri, Walaa R. Mahmoud, Heba
Abdelrasheed Allam, Ahmed M. Reda, Hatem A. Abdel-Aziz
PII:
S0045-2068(18)31272-0
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.006
YBIOO 2710
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
5 November 2018
27 December 2018
6 January 2019
Please cite this article as: H.S. Ibrahim, M.E. Albakri, W.R. Mahmoud, H. Abdelrasheed Allam, A.M. Reda, H.A.
Abdel-Aziz, Synthesis and biological evaluation of some novel thiobenzimidazole derivatives as anti-renal cancer
agents through inhibition of c-MET kinase, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2
019.01.006
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Synthesis and biological evaluation of some novel thiobenzimidazole
derivatives as anti-renal cancer agents through inhibition of c-MET kinase
a,*
a
b
b
Hany S. Ibrahim, Mohamed E. Albakri, Walaa R. Mahmoud, Heba Abdelrasheed Allam,
c
d
Ahmed M. Reda, Hatem A. Abdel-Aziz.
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo
1
1829, Egyptb
b
c
d
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
Department of Biochemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo 11829, Egypt
Department of Applied Organic Chemistry, National Research Center, Dokki, Giza, P.O. Box 12622, Egypt
ABSTRACT.
Benzimidazole is an interesting scaffold constituting a main core in many anticancer
agents against variable cell lines as Carbendazim (I) and Nocodazole (II). Accordingly, eighteen
compounds of 2-((1H-benzoimidazol-2-yl)thio)-1-(aryl/heteroaryl)ethan-1-ones, in their sulphate
salt and free forms, were designed and investigated as anticancer agents. In vitro preliminary
screening of selected compounds by the National Cancer Institute (NCI) on a panel of 60 cell
lines revealed renal cancer cell line (A498) as the most vulnerable cell line; accordingly, IC50
values against A498 cell line were determined for compounds with the best results. The best
inhibitory activity was for compound 4a with (IC50= 6.97 µM) compared to sunitinib as a
reference drug (IC50= 6.99 µM). Compound 4a was further subjected to cell cycle analysis that
indicated the decrease in cell population in the G2/M phase when compared to the untreated
control cells. In addition, it showed significant increase in the late apoptosis in Annexin-V FTIC
study compared to the control cells. An enzymatic inhibitory study on compound 4a against c-
Met and MAP kinases revealed its better activity against c-Met kinase with (IC50 = 0.27 µM)
compared to sunitinib (IC50 = 0.18 µM). Molecular docking study was conducted to reveal the
interactions of compound 4a in the active site of c-Met kinase. Computational ADME study was
performed to insure that compound 4a has proper pharmacokinetic and drug-likeness properties.
_
______
Keywords: Benzimidazole; Renal cell carcinoma; c-Met kinase.
*
Corresponding author. Tel.: +20-1223871274; e-mail: Hany.s.ibrahim@gmail.com (HSI).
1

1
.
Introduction
Cancer is the second deadly disease worldwide according to WHO. Among different
types of cancer, kidney cancer is the world’s sixteenth reason of cancer death as it is responsible
for 1.7 % of all death rate worldwide and it shares 2.4 % of total cancer patients around the
world [1-3]. Kidney cancer is among the 10 most common cancers in both men and women and
it is has high incidence rate in many countries among them Czech Republic and North America
[
4, 5].
On the other hand, the anticancer activity of benzimidazole derivatives is well established
against different cancer types [6-8]. For example, methyl-2-benzimidazole carbamate
Carbendazim, FB642, I) (Figure 1), an anticancer agent in preclinical trials, exerted an apoptotic
(
effect by inhibition of microtubules against both colon cancer cell lines (HT-29) and murine B16
melanoma [6, 7]. Another methyl-2-benzimidazole carbamate derivative (Nocodazole, NSC-
2
38189, II) interferes with the microtubules and affects many cancer cells by inhibition of
several kinases as MET, AB1 and C-KIT [8]. Dovotinib, (III) can target multiple RTK enzymes
such as FLT3, VEFGR, C-Kit and PDGFR with IC50 values = 1, 65, 2 and 5 nM, respectively [9,
1
0]. It is used for patients with gastrointestinal stromal tumor resistant to imatinib [11]. A
bicyclic benzimidazole derivative (BMT-1, IV) (Figure 1) activates several mechanisms to
induce apoptosis in multiple myeloma cells including the activation of caspase-3, caspase-8 and
caspase-9 [12]. Similarly, compound (V) was found to be cytotoxic against UO31 renal cancer
cell line with inhibitory activity against c-MET kinase up to 82 % at conc.=10 µM [13]. 2-Tolyl
benzimidazole derivatives (VI) (Figure 1) displayed cytotoxicity and apoptotic activity against
MDA-MB-468 breast cancer cell line, with high expression for EGFR, by IC50 = 3.31 µM [12].
To be more specific in this aspect, incorporation of acetophenone moieties with 2-
thiobenzimidazole as shown in compounds VII and VIII (figure 1) resulted in significant
cytotoxic activity. Compound VII inhibited 50% of cell surface expression of CD-133 on HT-29
cancer cell line at conc.= 10 µM [14] while compound VIII caused cell cycle arrest and
apoptosis of MDA-MB-468 cancer cell line [15].
In this research, we report the synthesis of new 2-thiobenzimdazole derivatives combined
with different acetophenones (4a-b, 9a-c and 14a-d) and their corresponding sulfate salt
analogues (3a-b, 8a-c and 13a-d) to be investigated for their cytotoxic activity followed by
2

some mechanistic studies to explain their biological activity. Furthermore, molecular docking
study was conducted to draw the full picture about the mechanism of action of the anticancer
activity of the synthesized compounds.
Figure 1. Reported benzimidazole and 2-thiobenzimidazole anticancer agents, and the scaffold of the
target compounds
2
. Results and Discussion
2
.1. Chemistry
We report herein the synthesis of novel 2-substituted benzimidazol-3-ium sulfate compounds
and their free analogues following three different schemes starting from different ketones
Schemes 1-3). In scheme 1, cyclization of salicylaldehyde (1a) or 5-bromosalicylaldehyde (1b)
(
with chloroacetone in the presence of potassium hydroxide yielded the corresponding 2-
3

acetylbenzofurans 2a, b [16]. Reaction of compounds 2a, b with 2-mercaptobenzothiazole in the
presence of two equivalents of conc. sulfuric acid according to the modified method reported by
Abdel-Aziz et al. [14] gave 2-substituted thiobenzimidazole sulfate salts 3a, b in 88 and 90%
yields, respectively. The structure of the latter isolated sulfate salts was supported by the reported
X-ray crystal structure of similar salts [14, 17]. Liberation of the free compounds from the
sulfate salts was done by neutralization via stirring with sodium bicarbonate solution at room
temperature to furnish the corresponding free bases 4a, b.
Scheme 1. (i) chloroacetone, KOH, methanol, reflux, 15 min.; (ii) 2-mercaptobenzimidazole, H
AcOH, reflux 1 h; (iii) Na CO , stirring, rt., 24 h.
2
SO ,
4
2
3
Secondly, in scheme 2, 4-aminoacetophenone (5) is converted into the corresponding 4-
phenylurido-acetophenones 7a, b [18] by the reaction of the appropriate isocyanate 6a, b. The
reaction of 5 or 7a, b with 2-mercaptobenzimidazole gave the corresponding sulfate salts 8a-c and
then the corresponding free analogues 9a-c applying the same procedure adopted in scheme 1.
1
The H NMR spectra of 8a and 9a revealed the singlet signal at 5.34 and 4.87 ppm, respectively,
assigned for the two protons of the CH group. It’s worthy to mention that applying the same
2
steps using five equivalents of conc. sulfuric acid afford the previously reported
thiazolobenzimidazole sulfate salt [17] .
4

Scheme 2. (i) dry toluene, reflux, 24 h; (ii) 2-mercaptobenzimidazole, H
Na CO , stirring, rt., 24 h.
2
SO , AcOH, reflux 1 h; (iii)
4
2
3
In scheme 3, pyrrolidine (11a), morpholine (11b), 1H-imidazole (11c) or 1H-
benzo[d][1,2,3]triazole (11d) were reacted with 4-fluoroacetophenone (10) to yield 4-
substituted-acetophenones 12a-d [19] which were then subjected to the same procedure to afford
the sulfate salts 13a-d and then free bases 14a-d.
5

2
Scheme 3. (i) K CO
3
, DMSO, reflux, 3-5 h; (ii) 2-mercaptobenzimidazole, H
2
SO , AcOH, reflux 1 h; (iii)
4
Na CO , stirring, rt., 24 h.
2
3
2
2
.2. Biological evaluation
-
5
.2.1. In vitro anticancer activity as primary single high dose (10 M) screening
The structure of the synthesized compound 8c was selected by the NCI Developmental
Therapeutic Program (www.dtp.nci.nih.gov) for evaluation of its cytotoxic activity against a
-5
panel of sixty cancer cell lines at concentration 10 M. The anticancer assays were performed in
accordance with the protocol of the Drug Evaluation Branch, NCI, Bethesda [38-40].
The obtained results in figure 2 revealed that, compound 8c possessed the highest cytotoxic
activity against renal cancer cell line A498 with GI% = 59. This result was the starting point to
6

investigate the anticancer activity of the other synthesized compounds against A498 cancer cell
lines.
GI%
70
60
50
40
30
20
10
0
Figure 2. Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10 μM
concentration for compound 8c.
2
.2.1.1. In vitro cytotoxic activity against renal cancer cell line A498
The previous results motivated us to investigate the cytotoxic activity for all the
synthesized compounds against renal cancer cell line A498 as shown in table 1. Cell viability for
A498 was measured upon treatment with all the compounds except 8c (previously screened by
NCI) at conc. 10 µM. The results revealed that compounds 4a, 9a and 9c in addition to their
corresponding salts 3a, 8a and 8c displayed the most cytotoxic effects (table 1). Consequently,
IC50 values for these compounds were calculated to evaluate anticancer effect on A498 cancer
cell line. Compound 4a possessed the best IC50 value among all the tested compounds (IC50
.97 µM) with respect to the reference drug sunitinib (IC50 = 3.99 µM). According to the given
results, it was discovered that combination of 2-thiobenzimidazole with 2-acetylbenzofuran and
-aminoacetophenone will lead to the maximum cytotoxic activity against A498.
=
6
4
Table 1. Cytotoxic activity of the synthesized compounds against renal cancer cell line A498.
Cytotoxicity against A498
Compound % Cell viability at
*
IC50 (µM)
1
0 µM
3a
3b
4a
25.93
88.06
20.89
7.12 ± 0.08
-----
6.97 ± 0.87
7

4b
8a
8b
70.76
35.87
100
-----
11.64 ± 1.13
-----
8
c
----
36.87
100
36.77 ± 0.92
12.5 ± 0.76
-----
9
9
a
b
9
c
36.72
65.69
55.90
100
91.72
78.09
85.77
100
39.79 ± 1.04
-----
1
3a
3b
3c
3d
4a
4b
4c
4d
Sunitinib
1
1
-----
-----
-----
----
1
1
1
1
1
-----
------
-----
3.99 ± 0.14
100
--
a
IC50 values are the mean ± S.E. of three separate experiments.
2
.2.1.2. Structure activity relationship (SAR)
Cytotoxic activity on renal cancer cell line A498 revealed that both salt and free
compound for the same derivative showed near activity. Based on the used acetyl derivative in
each compound, SAR studies showed benzofused heterocyclic acetyl derivative, as 2-
acetylbenzofuran in compounds 3a and 4a yield the maximum cytotoxicity (4a; IC50 = 6.97 µM)
while addition of 5-bromo substituent to this group would abolish the activity. On the other hand,
according to different p-susbtituted acetophenones were combined with 2-thiobenzimidazoles,
using p-amino group, as compound 8a) would result a moderate activity with IC50 = 11.64 µM.
Cyclization of this amino group as in secondary aliphatic amines as in compounds (13a-d and
1
4a-d) would decrease or abolish the activity. Moreover, phenyl urea extension of this amino
group would abolish the activity as in compounds (8b and 9b). Addition of nitro group to phenyl
urea extension as in compounds (8c and 9c) would retain weak cytotoxic activity (8c; IC50
=
3
6.77 µM).
2
.2.2. Flow cytometric analysis and apoptotic studies
Compound 4a was the most cytotoxic compound against A498 cancer cell line;
accordingly, flow cytometric analysis was established to study its effect on the cell cycle phases
8

in the same cancer cell line. In addition, apoptotic activity were measured by Annexin V-based
flow cytometric analysis.
2
.2.2.1. Cell cycle analysis
In this part, cancer cell line A498 was treated with compound 4a with concentration
equals to its IC50 (6.97 µM). Figure 3 revealed that compound 4a could disrupt the normal cell
cycle by decreasing G0-G1 phase by approximately 0.4 folds and S phase by approximately 0.5
folds with respect to the control. On the other hand, Pre-G and G2/M phase of the treated cell
was increased by 9.5 folds and 4.6 folds relative to the control. As a result, this disruption in cell
cycle which leads to arrest in G2/M phases was considered as a significant marker for induction
of apoptosis [20-22].
(2)
(1)
(4)
(3)
Figure 3. (1) Flow cytometric analysis of A498 cancer cell lines treated with compound 4a at
concentration equals to its IC50 on the same cell line. (2) Pie chart to illustrate the % of each stage of the
9

cell cycle of the same case in (1). (3) Flow cytometric analysis of A498 cancer cell lines without any
treatment. (4) Pie chart to illustrate the % of each stage of the cell cycle of the same case in (3).
2
.2.2.2. Annexin-V FTIC apoptotic study
To assure the apoptotic effect of compound 4a, Annexin-V FTIC/PI dual staining assay
was established with IC50 concentration on A498 cancer cell line. Analysis of the results in figure
declared the apoptotic effect of compound 4a by showing a significant increase in the percent
of positive apoptotic cells in (UR + LR) corners from 1.4 % to 21.11 % which comprises about
4
1
5 fold with respect to control (Figure 4).
Control
Compound 4a
Figure 4. Apoptotic effect of compound 4a on renal cancer cell lines A498 that was illustrated through
Annexin V-FTIC positive staining technique. The four quadrants is known as (LL: viable, LR: early
apoptosis, UR: late apoptosis, UL: necrosis).
10

2
.2.3. Inhibitory activity of compound 4a against c-MET and MAP kinases
In order to acquire more details about the mechanism regarding the cytotoxic activity of
compound 4a against A498 cancer cell line, literature survey informed us with very interesting
information that c-Met (hepatocyte growth factor receptor which is encoded by the met gene)
and MAPK (mitogen activated protein kinase) are highly expressed in A498 cancer cells [23,
2
4]. For this reason, compound 4a was evaluated for its inhibitory activity against both enzymes
using sunitinib as a reference drug as shown in table 2. The given results revealed that the
inhibitory activity of compound 4a (IC50 = 0.27 µM) against c-MET kinase is very close to the
reference drug sunitinib (IC50 = 0.18 µM). On the other hand, compound 4a showed weak
activity against MAPK (IC50 = 1.01 µM) compared to the reference drug sunitinib (IC50 = 0.64
µM) (table 2).
Table 2. Inhibitory effect of both compound 4a and the reference drug sunitinib against both c-
MET and MAP kinases
IC50 (M)
Compound
c-MET
MAPK
1.01 ±8.7
0.645.05
4
a
0.27 ±4.28
0.18 ±1.99
Sunitinib
3
.
In silico studies
.1.Molecular docking study
Molecular docking simulations of compound 4a inside the active site of c-MET kinase
3
was established in order to get more information about the mode of interaction that leads to the
given inhibitory activity. Docking simulation was performed using Accelrys Discovery Studio 4
using the crystal structure of c-Met kinase with PDB code (3ZZE) (Res. 1.87 Å) in which the
main group of co-crystalized ligand was oxindole. Therefore, analogy of the structure of the co-
crystalized ligand [25] and compound 4a can be revealed by the interact of both of them with
two amino acids R1208 (1.9 Å) and D1222 with 2 hydrogen bonds (2.5 Å) (Figure 5a).
Additionally, the benzofuran moiety was engulfed by the hydrophobic pocket consisted of
V1092, A1108, M1160 and M1211 (Figure 5b).
11

(b)
(a)
Figure 5. (a) Alignment of co-crystallized ligand (carbons were colored as cyan) and compound 4a
carbons were colored as violet) in the active site of c-Met kinase (PDB code 3ZZE) showing the main
(
interaction with the key amino acids in the active site. (b) Compound 4a in the active site of c-Met kinase
surrounded by a pocket showing the hydrophobic interaction. Color gradient from brown (hydrophobic)
to blue (hydrophilic). Color scheme; hydrogen bonds, Pi-Pi interactions, hydrophobic interactions were
assigned as green, orange and purple dots, respectively.
3
.2. ADME study and pharmacokinetic properties prediction
In order to have an overview on the pharmacokinetic aspects of the biologically
promising candidate 4a, the physical properties, the ADME parameters as well as the
pharmacokinetic profile of compound 4a were computed using the freely accessible web server
SwissADME (http://swissadme.ch/index.php#undefined) where it revealed promising
physicochemical and pharmacokinetic properties (Figure 6). It exhibited a predicted consensus
log Po/w value of 3.48 with moderate water solubility, high GIT absorption and no BBB
permeability, which correlates with minimal predicted CNS adverse effects. In addition, figure 6
showed that compound 4a achieve all the conditions of drug-likeness characters however, it
cannot be classified as lead-like as XLOGP3 model is more than 3.5.
12

Figure 6. The pharmacokinetic profile of compound 4a from SwissADME web server
Figure 7. The BOILED-Egg chart for compound 4a developed by swissADME.
The BOILED-Egg chart of compound 4a relating WLOGP and TPSA [26] (figure 7) indicated
its high probability to be passively absorbed by the gastrointestinal tract with no BBB
permeability. The chart also clarified that compound 4a is not a substrate for the P-glycoprotein
(
PGP-) and thus eliminating the possibility of its resistance by tumor cell lines through efflux.
Figure 8 shows the oral bioavailability radar chart of compound 4a taking into consideration the
six key criteria controlling the oral bioavailability [27, 28] where the optimal values are
13

represented as a pink area with the tested compound is presented as red line therein. As shown in
figure 8, compounds which is nearly fully included in the pink area (except saturation (INSATU)
property) indicating its good predicted oral bioavailability, with promising pharmacokinetic
properties.
Figure 8. Bioavailability radar chart for compound 4a.The pink area represents the range of the optimal
property values for oral bioavailability and the red line is compound 4a predicted properties. Saturation
(
(
INSATU), size (SIZE), polarity (POLAR), solubility (INSOLU), lipophilicity (LIPO) and flexibility
FLEX).
5
. Conclusion
Eighteen derivatives of 2-substituted thiobenzimidazoles with different acetophenone
moieties were synthesized and screened for their anticancer activity against renal cancer cell line
A498. Among the tested compounds, the best IC50 value was for compound 4a with (IC50= 6.97
µM) compared to sunitinib as reference drug (IC50= 6.99 µM). Moreover, cell cycle analysis of
compound 4a revealed its relevant decrease in cell population in the G2/M phases as well as late
apoptotic induction effect as demonstrated from Annexin-V FTIC study. Applying an enzymatic
inhibitory study of compound 4a against c-Met and MAP kinases showed its superior activity
against c-Met kinase with (IC50 = 0.27 µM) compared to sunitinib (IC50 = 0.18 µM). Finally, the
obtained results were supported by a molecular docking study of compound 4a pointing out its
possible interactions at the active site of c-Met kinase enzyme. Compound 4a illustrated suitable
pharmacokinetic properties that was proved by swissADME server.
5
. Experimental
14

5
5
.1. Chemistry
.1.1. General
Melting points were recorded on a Stuart SMP10 digital melting point apparatus and were
uncorrected. Infrared and NMR spectra were performed at the Microanalytical Unit-Faculty of
Pharmacy, Cairo University. IR Spectra were recorded as KBr disks using a Shimadzu FT-IR
8
400S infrared spectrophotometer. Mass spectral data are given as m/z (Intensity %). NMR
1
13
1
spectra were recorded on a Bruker Ascend 400/ R ( H: 400, C: 100 MHz) spectrometer. H
1
3
NMR spectra were run at 400 MHz and C spectra were run at 100 MHz in deuterated
dimethylsulfoxide (DMSO-d ). Chemical shifts are expressed in  values (ppm) using the solvent
6
peak as internal standard. All coupling constant (J) values are given in hertz. The abbreviations
used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were carried out at
the Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt. Thin Layer
Chromatography (TLC) routinely monitored reaction progress on silica gel pre-coated F254
Merck plates. Unless otherwise noted, all solvents and reagents were commercially available and
used without further purification. The acetophenone intermediates 2a, 2b [16], 6a[18], 6b [18]
,
12a-d [19, 29] were synthesized according to the reported procedures.
5
.1.2. General procedure for preparation of sulfate salts:
To a solution of the appropriate acetophenone (5 mmol) in 10 mL glacial acetic acid, 2-
mercaptobenzimidazole (0.75 g, 5 mmol) and conc. sulphuric acid (10 mmol) were added. The
reaction mixture was heated under reflux for half to two hours. The solid product obtained upon
cooling was filtered off, washed with cold water then with petroleum ether and recrystallized
from ethanol to afford the corresponding sulfate salts.
5
.1.2.1. 2-[(2-(Benzofuran-2-yl)-2-oxoethyl)thio]-1H-benzo[d]imidazol-3-ium sulfate 3a.
-1
1
Buff powder, (yield 88%), m.p. 214-215°C; IR (KBr, ν cm ): 3400 (NH) and 1666 (C=O); H
NMR (DMSO-d )  ppm: 5.14 (s, 4H, 2CH ), 7.13-7.14 (m, 2H, Ar-H), 7.36-7.39 (m, 4H, Ar-H),
.41-7.45 (m, 2H, Ar-H), 7.59-7.63 (m, 4H, Ar-H), 7.77 (dd, 2H, Ar-H, J = 7.6, 7.8 Hz), 7.91 (d,
6
2
7
2
1
3
H, Ar-H, J = 7.8), 8.11 (s, 2H, Ar-H), 12.51 (s, 2H, 2NH, D
)  ppm: 40.61 (CH ), 109.94, 112.79, 113.93, 115.91, 122.77, 124.43, 124.57,
24.85, 127.12, 129.54, 132.71, 150.20, 151.02, 155.61, 168.59, 183.36 (C=O); Anal. Calcd. for
2
O exchangeable); C NMR
(
DMSO-d
6
2
1
15

C
34
H
26
N
4
O
8
S (714.79): C, 57.13; H, 3.67; N, 7.84; S, 13.46; Found C, 57.41; H, 3.81; N, 8.09;
3
S, 13.38.
5
.1.2.2. 2-[(2-(5-Bromobenzofuran-2-yl)-2-oxoethyl)thio]-1H-benzo[d]imidazol-3-ium sulfate
3
b.
-1
Light yellow powder, (yield 90%), m.p. 220-222°C; IR (KBr, ν cm ): 3348 (NH), 1678 (C=O);
1
H NMR (DMSO-d
6
)  ppm: 5.14 (s, 4H, 2CH ), 7.36-7.38 (m, 4H, Ar-H), 7.61-7.62 (m, 4H, Ar-
2
H), 7.73-7.79 (m, 4H, Ar-H), 8.05 (s, 2H, Ar-H), 8.16 (s, 2H, Ar-H), 12.51 (s, 2H, 2NH, D O
2
1
3
exchangeable); C NMR (DMSO-d
24.48, 126.71, 128.50, 129.28, 132.07, 135.46, 143.20, 150.05, 151.96, 153.20, 154.33, 183.44
C=O); Anal. Calcd. for C34 (872.58): C, 46.80; H, 2.77; N, 6.42; S, 11.02; Found
C, 47.07; H, 2.94; N, 6.58; S, 11.13.
6
)  ppm: 40.46 (CH ), 113.94, 114.94, 116.97, 121.13,
2
1
(
H24Br
2
N
4
O S
8 3
5
.1.2.3. 2-[(2-(4-Ammoniophenyl)-2-oxoethyl)thio]-1H-benzo[d]imidazol-3-ium sulfate 8a.
White powder, (yield 91%), m.p. 198-200°C; IR (KBr, ν cm-1): 3470, 3410 (NH) and 1678
1
(
C=O); H NMR (DMSO-d
6
)  ppm: 5.34 (s, 2H, CH ), 6.68 (d, 2H, Ar-H, J = 7.6 Hz), 7.48-
2
7
.51 (m, 2H, Ar-H), 7.70-7.73 (m, 2H, Ar-H), 7.82 (d, 2H, Ar-H, J = 8.4 Hz), 11.10 (s, 1H, NH,
1
3
D
2
O exchangeable); C NMR (DMSO-d
6
)  ppm: 41.99 (CH ), 113.49, 113.80, 123.09, 125.80,
2
1
27.20, 129.99, 130.80, 131.61, 132.57, 151.51, 154.17, 189.30 (C=O); Anal. Calcd. for
(381.43): C, 47.23; H, 3.96; N, 11.02; S, 16.81; Found C, 47.59; H, 4.12; N,
1.38; S, 16.95.
C
15
H
15
N
3
O S
5 2
1
5
.1.2.4. 2-[(2-Oxo-2-(4-(3 phenylureido)phenyl)ethyl)thio]-1H-benzo[d]imidazol-3-ium sulfate
8
b.
-1
Buff powder, (yield 87%), m.p. 263-265°C; IR (KBr, ν cm ): 3352, 3275 (NHs) and 1654
1
(
C=O); H NMR (DMSO-d
6
)  ppm: 5.36 (s, 4H, 2 CH ), 7.01 (t, 2H, Ar-H, J = 7.3 Hz), 7.31 (t,
2
4
8
2
4
1
H, Ar-H, J = 7.9 Hz), 7.42-7.44 (m, 4H, Ar-H), 7.48 (d, 4H, Ar-H, J = 7.6 Hz), 7.65-7.68 (m,
H, Ar-H), 8.04 (d, 4H, Ar-H, J = 8.8 Hz), 9.33 (s, 2H, 2NH, D O exchangeable), 9.75 (s, 2H,
2
1
3
NH, D
1.54 (CH
37.50, 139.78, 145.96, 151.02, 152.72, 191.01 (2 C=O);; ESI MS m/z: 903.31 [M] ; Anal.
(903.02): C, 58.52; H, 4.24; N, 12.41; S, 10.65; Found C, 58.76; H,
2
O exchangeable), 12.50 (s, 2H, 2NH, D
2
O exchangeable); C NMR (DMSO-d
6
)  ppm:
2
), 113.67, 117.51, 118.76, 122.69, 125.03, 127.79, 128.44, 129.31, 130.65, 134.13,
+
Calcd. for C44
H
38
N
8
O S
8 3
4
.33; N, 12.69; S, 10.80.
16

5
.1.2.5. 2-[(2-(4-(3-(4-Nitrophenyl)ureido)phenyl)-2-oxoethyl)thio]-1H-benzo[d]imidazol-3-ium
sulfate 8c.
-1
Dark yellow powder, (yield 88%), m.p. 277-280°C; IR (KBr, ν cm ): 3217, 3356 (NHs) and
1
1
7
2
689 (C=O); H NMR (DMSO-d
6
)  ppm: 5.29 (s, 4H, 2 CH ), 7.42-7.45 (m, 4H, Ar-H), 7.66-
2
.76 (m, 12 H, Ar-H), 8.04 (d, 4H, Ar-H, J = 8.8 Hz), 8.22 (d, 4H, Ar-H, J = 9.2 Hz), 9.60 (s,
1
3
H, 2 NH), 9.75 (s, 2H, 2 NH, D
)  ppm: 40.60 (CH
25.59, 129.87, 130.09, 130.42, 141.66, 146.76, 150.26, 152.42, 191.55 (2 C=O); Anal. Calcd.
(993.01): C, 53.22; H, 3.65; N, 14.11; S, 9.69; Found C, 53.49; H, 3.71; N,
2
O exchangeable), 12.51 (s, 2H, 2 NH, D
2
O exchangeable); C
NMR (DMSO-d
6
2
), 118.06, 118.15, 118.22, 118.27, 120.25, 121.71, 124.07,
1
for C44
H
36
N
10
O
S
12 3
1
4.45; S, 9.82..
5
.1.2.6. 2-[(2-Oxo-2-(4-(pyrrolidin-1-yl)phenyl)ethyl)thio]-1H-benzo[d]imidazol-3-ium sulfate
1
3a.
-1
1
Grey crystals, (yield 90%), m.p. 219-221°C; IR (KBr, ν cm ): 3421 (NHs) and 1650 (C=O); H
NMR (DMSO-d )  ppm: 1.97-2.00 (m, 8H, pyrrolidine Hs), 3.34-3.37 (m, 8H, pyrrolidine Hs),
.06 (s, 4H, 2 CH ), 6.62 (d, 2H, Ar-H, J = 8.9 Hz), 7.12-7.16 (m, 8H, Ar-H), 7.27-7.29 (m, 2H,
Ar-H), 7.54-7.56 (m, 2H, Ar-H), 7.89 (d, 2H, Ar-H, J = 8.9 Hz), 12.51 (s, 2H, 2NH, D
6
5
2
2
O
1
3
exchangeable); C NMR (DMSO-d
pyrrolidine CH ), 109.98, 111.34, 120.20, 122.61, 130.13, 132.67, 139.69, 145.05, 150.37,
51.57, 168.48, 190.88 (C=O); Anal. Calcd. for C38 (772.95): C, 59.05; H, 5.22; N,
0.87; S, 12.45; Found C, 58.89; H, 5.38; N, 10.95; S, 12.61.
6
)  ppm: 25.38 (pyrrolidine CH
2
), 43.18 (CH ), 47.78
2
(
2
1
1
H
40
N
6
O S
6 3
5
.1.2.7. 2-[(2-(4-Morpholinophenyl)-2-oxoethyl)thio]-1H-benzo[d]imidazol-3-ium sulfate 13b.
-1
1
Buff crystals, (yield 95%), m.p. 260-262°C; IR (KBr, ν cm ): 3421 (NH) and 1643 (C=O); H
NMR (DMSO-d )  ppm: 3.35-3.37 (m, 8H, morpholine Hs), 3.73-3.76 (m, 8H, morpholine Hs),
.25 (s, 4H, 2 CH ), 7.05 (d, 2H, Ar-H, J ꢀ ꢁꢂ1 ꢃꢄꢅ, ꢆꢂ11 ꢆ.18 (m, 8H, Ar-H), 7.45-7.47 (m, 2H,
Ar-H), 7.68-7.70 (m, 2H, Ar-H), 7.92 (d, 2H, Ar-H, J = 9.0 Hz), 12.56 (s, 2H, 2NH, D
6
5
2
ꢇ
2
O
1
3
exchangeable); C NMR (DMSO-d
6
)  ppm: 41.37 (CH
2
), 47.04, 66.25 (morpholine Cs),
1
09.98, 113.65, 114.93, 123.10, 125.41, 130.84, 131.05, 133.47, 151.30, 155.11, 168.48, 190.10
+
(
C=O); ESI MS m/z: 804 [M] ; Anal. Calcd. for C38
H
40
N
6
O
8
S (804.95): C, 56.70; H, 5.01; N,
3
1
0.44; S, 11.95; Found C, 56.87; H, 4.14; N, 10.67; S, 12.08.
17

5
.1.2.8. 2-[(2-(4-(1H-Imidazol-1-yl)phenyl)-2-oxoethyl)thio]-1H-benzo[d]imidazol-3-ium sulfate
1
3c.
-1
1
Buff powder, (yield 93%), m.p. 245-247°C; IR (KBr, ν cm ): 3410 (NH) and 1678 (C=O); H
NMR (DMSO-d )  ppm: 5.36 (s, 4H, 2 CH ꢅ, ꢆꢂ1ꢈ ꢆ.14 (m, 2H, Ar-H), 7.36-7.43 (m, 4H, Ar-
H), 7.63-7.68 (m, 4H, Ar-H), 7.99 (s, 2H, Ar-H), 8.08 (d, 4H, Ar-H, J = 8.6 Hz), 8.32 (d, 4H, Ar-
6
2
ꢇ
1
3
H, J = 8.7 Hz), 8.46 (s, 2H, Ar-H), 12.51 (s, 2H, 2NH, D
2
O exchangeable); C NMR (DMSO-
), 109.95, 113.88, 121.09, 121.88, 122.54, 122.77, 124.77, 130.88, 132.71,
34.82, 135.64, 135.77, 139.13, 150.56, 192.13 (C=O); Anal. Calcd. for C36 (766.87):
d
6
)  ppm: 41.28 (CH
2
1
H
30
N
8
O S
6 3
C, 56.38; H, 3.94; N, 14.61; S, 12.54; Found C, 56.62; H, 3.87; N, 14.89; S, 12.60.
5
.1.2.9.
2-[(2-(4-(1H-Benzo[d][1,2,3]triazol-1-yl)phenyl)-2-oxoethyl)thio]-1H-
benzo[d]imidazol-3- ium sulfate 13d.
-1
1
White powder, (yield 89%), m.p. 260-262°C; IR (KBr, ν cm ): 3414 (NH) and 1678 (C=O); H
NMR (DMSO-d )  ppm: 5.41 (s, 4H, 2 CH ), 7.42-7.44 (m, 4H, Ar-H), 7.57-7.61 (m, 2H, Ar-
H), 7.67-7.69 (m, 4H, Ar-H), 7.74-7.78 (t, 2H, Ar-H, J = 7.6 Hz), 8.07-8.11 (m, 2H, Ar-H), 8.20
6
2
(
(
d, 2H, Ar-H, J = 8.7 Hz), 8.26 (d, 2H, Ar-H, J = 8.4 Hz), 8.37 (t, 4H, Ar-H, J = 7.5 Hz), 8.56
1
3
d, 2H, Ar-H, J = 8.8 Hz), 12.52 (s, 2H, 2NH, D
2
O exchangeable); C NMR (DMSO-d
6
)  ppm:
4
1
1.15 (CH
2
), 111.73, 113.89, 118.89, 120.92, 122.82, 124.49, 125.67, 128.96, 129.76, 130.96,
31.96, 134.96, 140.87, 146.51, 150.59, 192.16 (C=O); Anal. Calcd. for C42
H
32
N
10
O S
6 3
(
868.96): C, 58.05; H, 3.71; N, 16.12; S, 11.07; Found C, 58.27; H, 3.89; N, 16.34; S, 11.21.
5
.1.3. General procedure for preparation of compounds4a, b, 9a-c and 14a-d:
A suspension of different 2-substituted-benzimidazol-3-ium sulfate salts 3a, b, 8a-c and
3a-d (2 mmol) in water (10 mL) was stirred at room temperature with an aqueous solution of
1
sodium bicarbonate overnight. The solid obtained was filtered, washed with water, dried and
then crystallized from ethanol to afford the corresponding free bases 4a, b, 9a-c and 14a-d,
respectively.
5
.1.3.1. 2-[(1H-benzo[d]imidazol-2-yl)thio]-1-(benzofuran-2-yl)ethanone 4a.
-1
1
Buff powder, (yield 91%), m.p. 170-172°C; IR (KBr, ν cm ): 3394 (NH) and 1678 (C=O); H
NMR (DMSO-d )  ppm: 4.96 (s, 2H, CH ), 7.09-7.14 (m, 2H, Ar-H), 7.41 7.42 (m, 3H, Ar-H),
.59 (t, 1H, Ar-H, J = 7.6 Hz), 7.75 (d, 1H, Ar-H, J = 8.3 Hz), 7.88 (d, 1H, Ar-H, J = 7.7 Hz),
6
2
ꢇ
7
18

1
3
8
.12 (s, 1H, Ar-H), 12.65 (s, 1H, NH, D
2
O exchangeable); C NMR (DMSO-d )  ppm: 40.62
6
(
CH ), 110.86, 114.96, 117.81, 122.18, 126.61, 129.38, 131.82, 136.03, 143.92, 149.49, 152.36,
2
+
1
54.29, 184.38 (C=O); ESI MS m/z: 308 [M] ; Anal. Calcd. for C17
H
12
N
2
2
O S (308.35): C, 66.22;
H, 3.92; N, 9.08; S, 10.40; Found C, 66.43; H, 4.15; N, 9.23; S, 10.59.
.1.3.2. 2-[(1H-benzo[d]imidazol-2-yl)thio)-1-(5-bromobenzofuran-2-yl)ethanone 4b.
5
-1
1
White powder, (yield 88%), m.p. 183-185°C; IR (KBr, ν cm ): 3380 (NH) and 1685 (C=O); H
NMR (DMSO-d
6
)  ppm: 4.98 (s, 2H, CH
2
), 7.16-7.18 (m, 2H, Ar-H), 7.44-7.47 (m, 2H, Ar-H),
7
.71-7.78 (m, 2H, Ar-H), 8.06 (s, 1H, Ar-H), 8.13 (s, 1H, Ar-H), 12.55 (s, 1H, NH, D O
2
1
3
exchangeable); C NMR (DMSO-d
6
)  ppm: 38.91 (CH ), 109.94, 114.93, 115.54, 121.92,
2
1
3
22.77, 124.69, 127.21, 132.72, 149.59, 151.40, 155.54, 168.60, 184.33 (C=O); ESI MS m/z:
+
87 [M] ; Anal. Calcd. for C17
H
11BrN
2
2
O S (387.25): C, 52.73; H, 2.86; N, 7.23; S, 8.28; Found
C, 52.91; H, 3.07; N, 7.45; S, 8.31.
.1.3.3. 2-[(1H-Benzo[d]imidazol-2-yl)thio]-1-(4-aminophenyl)ethanone 9a.
5
-1
White powder, (yield 92%), m.p. 117-120°C; IR (KBr, ν cm ): 3471, 3367 (NHs) and 1650
1
(
C=O); H NMR (DMSO-d
6
)  ppm: 4.87 (s, 2H, CH
2
), 6.20 (s, 2H, NH2,
2
D O exchangeable),
6
.60 (d, 2H, Ar-H, J = 8.7 Hz), 7.09-7.11 (m, 2H, Ar-H), 7.41-7.43 (m, 2H, Ar-H), 7.77 (d, 2H,
1
3
Ar-H, J = 8.7 Hz), 11.00 (s, 1H, NH, D
CH ), 113.04, 121.72, 123.29, 125.82, 127.31, 131.41, 150.49, 154.71, 190.71 (C=O); ESI MS
m/z: 283 [M] ; Anal. Calcd. for C15
Found C, 63.74; H, 4.80; N, 15.12; S, 11.55.
2
O exchangeable); C NMR (DMSO-d )  ppm: 40.62
6
(
2
+
H
13
N OS (283.35): C, 63.58; H, 4.62; N, 14.83; S, 11.32;
3
5
.1.3.4. 1-[4-(2-((1H-Benzo[d]imidazol-2-yl)thio)acetyl)phenyl]-3-phenylurea 9b.
-1
White powder, (yield 91%), m.p. 248-250°C; IR (KBr, ν cm ): 3352, 3278 (NHs) and 1654
1
(
C=O); H NMR (DMSO-d
6
)  ppm: 5.29 (s, 2H, CH ), 7.00 (t, 1H, Ar-H, J = 7.3 Hz), 7.31 (t,
2
2
H, Ar-H, J = 7.9 Hz), 7.37-7.39 (m, 2H, Ar-H), 7.48 (d, 2H, Ar-H, J = 7.6 Hz), 7.61-7.63 (m,
2
H, Ar-H), 7.66 (d, 2H, Ar-H, J = 8.8 Hz), 8.04 (d, 2H, Ar-H, J = 8.8 Hz), 9.29 (s, 1H, NH, D O
2
1
3
exchangeable), 9.69 (s, 1H, NH, D
2
O exchangeable), 12.52 (s, 1H, NH, D
2
O exchangeable); C
NMR (DMSO-d
6
)  ppm: 41.49 (CH
2
), 113.69, 117.49, 118.74, 122.67, 124.85, 128.47, 129.30,
+
1
30.64, 134.40, 139.80, 145.95, 150.97, 152.73, 191.07 (2C=O); ESI MS m/z: 402 [M] , 403
19

+
[
M+1] ; Anal. Calcd. for C22
H
18
N
4
2
O S (402.47): C, 65.65; H, 4.51; N, 13.92; S, 7.97; Found C,
6
5.89; H, 4.63; N, 14.08; S, 8.19.
5
.1.3.5. 1-[4-(2-((1H-Benzo[d]imidazol-2-yl)thio)acetyl)phenyl]-3-(4-nitrophenyl)urea 9c.
-1
Yellow powder, (yield 95%), m.p. 170-172°C; IR (KBr, ν cm ): 3479, 3356 (NHs) and 1662
1
(
C=O); H NMR (DMSO-d
6
)  ppm: 5.00 (s, 2H, CH ), 7.09-7.11 (m, 2H, Ar-H), 7.40-7.42 (m,
2
2
H, Ar-H), 7.67 (d, 2H, Ar-H, J = 8.6 Hz), 7.73 (d, 2H, Ar-H, J = 9.1 Hz), 8.04 (d, 2H, Ar-H, J =
.4 Hz), 8.20 (d, 2H, Ar-H, J = 9.1 Hz), 10.24 (br s, 2H, 2NH, D O exchangeable), 12.50 (s, 1H,
)  ppm: 41.28 (CH ), 113.69, 118.20, 118.31,
8
2
1
3
NH, D
2
O exchangeable); C NMR (DMSO-d
6
2
1
24.09, 125.30, 125.58, 129.08, 129.88, 130.57, 141.85, 145.23, 146.42, 152.19, 191.03 (2C=O);
+
ESI MS m/z: 447 [M] ; Anal. Calcd. for C22
H
17
N
5
4
O S (447.47): C, 59.05; H, 3.83; N, 15.65; S,
7
.17; Found C, 59.31; H, 4.09; N, 15.83; S, 7.24.
5
.1.3.6. 2-[(1H-benzo[d]imidazol-2-yl)thio]-1-[4-(pyrrolidin-1-yl)phenyl]ethanone 14a.
-1
1
Brown powder, (yield 88%), m.p. 130-132°C; IR (KBr, ν cm ): 3275 (NH) and 1635 (C=O); H
NMR (DMSO-d )  ppm: 1.97-2.00 (m, 4H, pyrrolidine Hs), 3.34-3.37 (m, 4H, pyrrolidine Hs),
.91 (s, 2H, CH ), 6.60 (d, 2H, Ar-H, J = 8.8 Hz), 7.11 7.14 (m, 2H, Ar-H), 7.37 (d, 1H, Ar-H,
J = 6.4 Hz), 7.47 (d, 1H, Ar-H, J = 7.0 Hz), 7.89 (d, 2H, Ar-H, J = 8.8 Hz), 12.56 (s, 1H, NH,
6
4
2
ꢇ
1
3
13
D
2
O exchangeable); C NMR (DMSO-d
6
)  ppm: C NMR (DMSO-d
6
)  ppm: 25.39
(
pyrrolidine CH ), 43.14 (CH
2
2
), 47.80 (pyrrolidine CH
2
), 109.94, 111.37, 120.28, 122.77,
+
1
30.17, 132.70, 144.98, 151.62, 168.19, 171.74, 190.52 (C=O); ESI MS m/z: 337 [M] ; Anal.
Calcd. for C19
H
19
3
N OS (337.44): C, 67.63; H, 5.68; N, 12.45; S, 9.50; Found C, 67.80; H, 5.89;
N, 12.61; S, 9.63.
.1.3.7. 2-[(1H-Benzo[d]imidazol-2-yl)thio]-1-(4-morpholinophenyl)ethanone 14b.
5
-1
1
White powder, (yield 86%), m.p. 241-243°C; IR (KBr, ν cm ): 3414 (NH) and 1658 (C=O); H
NMR (DMSO-d )  ppm: 3.32 (t, 4H, morpholine Hs, J = 4.8 Hz), 3.74 (t, 4H, morpholine Hs, J
4.7 Hz), 4.95 (s, 2H, CH ), 7.02 (d, 1H, Ar-H, J = 9.0 Hz), 7.11- 7.16 (m, 5H, Ar-H), 7.42-7.44
m, 1H, Ar-H), 7.93 (d, 1H, Ar-H, J = 8.9 Hz), 12.52 (s, 1H, NH, v); C NMR (DMSO-d
ppm: 40.61 (CH ), 47.14, 66.28 (morpholine carbons), 109.93, 113.41, 121.77, 125.54, 130.85,
32.71, 154.81, 168.59, 191.49 (C=O); ESI MS m/z: 353 [M] ; Anal. Calcd. for C19
353.44): C, 64.57; H, 5.42; N, 11.89; S, 9.07; Found C, 64.80; H, 5.63; N, 12.15; S, 9.23.
6
=
2
1
3
(
6
) 
2
+
1
H
19
N
3
2
O S
(
20

5
.1.3.8. 2-[(1H-Benzo[d]imidazol-2-yl)thio]-1-(4-(1H-imidazol-1-yl)phenyl)ethanone 14c.
-1
1
Buff powder, (yield 90%), m.p. 228-230°C; IR (KBr, ν cm ): 3379 (NH) and 1683 (C=O); H
NMR (DMSO-d )  ppm: 5.08 (s, 2H, CH ), 7.10- 7.17 (m, 3H, Ar-H), 7.41-7.43 (m, 2H, Ar-H),
.91 (t, 3H, Ar-H, J = 8.3 Hz), 8.21 (d, 2H, Ar-H, J = 8.3 Hz), 8.47 (s, 1H, Ar-H), 12.59 (s, 1H,
6
2
7
1
3
NH, D
2
O exchangeable); C NMR (DMSO-d
6
)  ppm: 40.61 (CH ), 109.94, 118.26, 121.86,
2
1
3
6
22.77, 130.89, 132.71, 133.89, 136.25, 141.04, 149.87, 168.59, 192.89 (C=O); ESI MS m/z:
+
34 [M] ; Anal. Calcd. for C18
H
14
4
N OS (334.39): C, 64.65; H, 4.22; N, 16.75; S, 9.59; Found C,
4.81; H, 4.39; N, 17.03; S, 9.71.
5
.1.3.9.
1-[4-(1H-Benzo[d][1,2,3]triazol-1-yl)phenyl]-2-((1H-benzo[d]imidazol-2-yl)thio)
ethanone 14d.
-
1
Light yellow powder, (yield 87%), m.p. 220-222°C; IR (KBr, ν cm ): 3325 (NH) and 1681
1
(
C=O); H NMR (DMSO-d
6
)  ppm: 5.09 (s, 2H, CH ), 7.07-7.12 (m, 2H, Ar-H), 7.39 (br s, 2H,
2
Ar-H), 7.57 (t, 1H, Ar-H, J = 7.1 Hz), 7.73 (t, 1H, Ar-H, J = 7.6 Hz), 8.07 (d, 1H, Ar-H, J = 8.2
Hz), 8.13 (d, 1H, Ar-H, J = 8.4 Hz), 8.24 (d, 1H, Ar-H, J = 8.4 Hz), 8.34 (br s, 2H, Ar-H), 8.49
1
3
(
d, 1H, Ar-H, J = 8.4 Hz), 12.52 (s, 1H, NH, D
2
O exchangeable); C NMR (DMSO-d
6
)  ppm:
4
1
0.60 (CH
31.98, 133.10, 140.30, 145.27, 146.46, 168.66 (C=O); ESI MS m/z: 385 [M] ; Anal. Calcd. for
OS (385.44): C, 65.44; H, 3.92; N, 18.17; S, 8.32; Found C, 65.80; H, 4.06; N, 18.45;
2
), 109.95, 111.71, 114.31, 118.86, 120.40, 121.68, 122.60, 125.58, 128.80, 129.65,
+
C
21
H
15
N
5
S, 8.49.
5
5
.2. Biological Evaluation
-
5
.2.1. In vitro anticancer activity as primary single high dose (10 M) screening
The cytotoxicity assays for compound 4a were performed according to the protocol of
National Cancer Institute (NCI), Bethesda, USA (against 60 cell lines) [30, 31]. Drug exposure
protocol was applied for 48 h using sulforhodamine B (SRB) protein assay [30] and cell viability
and growth was estimated, as reported earlier [22].
5
.2.2. In vitro cytotoxic activity against renal cancer cell line A498
21

A498 cell viability tesing of all the synthesized compounds at conc. 10 M and
consequently IC50 values on serial concentrations of the compounds with the best results namely
3
a, 4a, 8a, 8c, 9a and 9c were determined using MTT spectrophotometric assay. Initial
incubation of A498 renal cancer cells was done at 37°C for 24 hours followed by incubation of
the desired concentration of each tested compound with the cells for 48 h at 37ºC ,then the plates
were examined under the inverted microscope and proceeded for the MTT assay according to the
reported procedure [32, 33].
5
5
.2.3. Flowcytometric analysis
.2.3.1.Cell cycle analysis
Further exploration of the cytotoxic activity of compound 4a was performed using
propidium iodide (PI) flow cytomertric analysis to measure the extent of PI that that binds to
DNA of dead cells with permeable plasma membranes to determine cell cycle status in tissue
culture quantitate cell death at all cell phases [34]. After incubation of A498 cells with
compound 4a, cells were fixed with ethanol then dehydrated before staining with PI according
to the reported methodology [35].
5
.2.3.2. Annexin-V FTIC apoptotic study
An apoptotic study using Annexin V-FITC dual staining assay was carried out on
compound 4a at its IC50 concentration (6.97 µM) in three successive steps; incubation of cells
with Annexin V-FITC followed by quantification by flow cytometry and finally detection by
fluorescence microscope according to the published protocol [36].
5
.2.4.Inhibitory activity of compound 4a against c-MET kinase
An ADP-Gl kinase assay measures ADP formed from a kinase reaction. ADP is
converted into ATP, which is used to generate light in a luciferase reaction. The luminescence
generated correlates with the c-MET kinase activity of compound 4a. The enzyme and its
substrate with ATP and compound 4a all are buffered and added to the wells and incubated at
room temperature for 60 minutes followed by addition of the ADP-Glo reagent and finally the
kinase detection reagent following the steps the documented experimental steps [37].
22

5
.2.5.Inhibitory activity of compound 4a against MAP kinase
A time resolved fluorescence resonance energy transfer assay was carried out to
determine the MAP kinase inhibitory activity of compound 4a (LANCE) in two steps. Firstly,
kinase reaction step by mixing and incubation of MAP kinase with the ULight-substrate, ATP,
and compound 4a in a suitable buffer and secondly, a detection step according to reported [38].
5
5
.3. In silico studies
.3.1. Molecular docking simulations
Molecular docking studies were performed using Accelerys software (Discovery Studio 4)
for compound 4a against c-MET kinase (PDB code 3ZZE). Crystal structure (3ZZE) (resolution
.87 Å) was downloaded and prepared as reported [39]. Ligand was drawn using marvin sketch
1
and imported to the visualizer and prepared as reported [40]. Docking simulations were run using
CDOCKER for the co-crystallized ligand and then it was validated by the alignment of the co-
crystallized ligand and docked ligand to calculate RMSD value, which was 0.89Å. The docked
poses were ranked according to their cDOCKER interaction energy, and the top pose was chosen
for analysis of interactions for each compound.
5
.3.2. ADME study and pharmacokinetic properties prediction
The pharmacokinetic data relevant to compound 4a was achieved via the free online server
swissADME (http://swissadme.ch/index.php#undefined) where the SMILE of the compound was
inserted directly on the webpage followed by running the prediction process. A whole set of the
different physical properties, pharmacokinetic parameters ADME parameters along with the
BOILED-Egg chart were obtained online and explained [27, 28].
Funding: This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
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27

Synthesis and biological evaluation of some novel thiobenzimidazole
derivatives as anti-renal cancer agents through inhibition of c-MET kinase
a,*
a
b
b
Hany S. Ibrahim, Mohamed E. Albakri, Walaa R. Mahmoud, Heba Abdelrasheed Allam,
c
d
Ahmed M. Reda, Hatem A. Abdel-Aziz.
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo
1
1829, Egypt
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
cDepartment of Biochemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo 11829, Egypt
d
Department of Applied Organic Chemistry, National Research Center, Dokki, Giza, P.O. Box 12622, Egypt






2-Substituted thiobenzimidazoles were synthesized and screened against A498 cell line
The best IC50 value was for compound 4a with (IC50= 6.97 µM) compared to sunitinib
Compound 4a affected G2/M phases basically and was proved as late apoptotic inducer
Compound 4a revealed good inhibitory activity against c-Met kinase (IC50 = 0.27 µM)
Docking of 4a at c-Met kinase active site demonstrated good binding interactions
Compound 4a showed a suitable predicted ADME profile
28

Graphical Abstract
Synthesis and biological evaluation of some novel thiobenzimidazole
derivatives as anti-renal cancer agents through inhibition of c-MET kinase
1
,*
1
2
2
Hany S. Ibrahim, Mohamed E. Albakri, Walaa R. Mahmoud, Heba Abdelrasheed Allam,
3
4
Ahmed M. Reda, Hatem A. Abdel-Aziz.
(A498; IC50 = 6.79 µM)
G2/M phases arrest
total % apoptosis = 21.11%
(IC50 = 0.27 µM)
29