2-((Benzimidazol-2-yl)thio)-1-arylethan-1-ones: Synthesis, crystal study and cancer stem cells CD133 targeting potential

Article abstract of DOI:10.1016/j.ejmech.2015.09.023

In order to develop a potent anti-tumor agent that can target both cancer stem cells and the bulk of tumor cells, a series of 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a-o was synthesized. All compounds were evaluated for their anti-proliferative activity towards colon HT-29 cancer cell line. In addition, their inhibitory effect against cell surface expression of CD133, a potent cancer stem cells (CSCs) marker, in the same cells was evaluated by flow cytometry at 10 μM. Compound 5l emerged as the most active anti-proliferative analog against HT-29 (IC₅₀ Combining double low line 18.83 ± 1.37 μM), that almost equipotent as 5-fluorouracil (IC₅₀ Combining double low line 15.83 ± 1.63 μM) with 50.11 ± 4.05% inhibition effect on CD133 expression, suggested dual targeted effect. Also, compounds 5h, 5j, 5k and 5m-o inhibited the expression of CD133 with more than 50%. The SAR study pointed out the significance of substitution of the pendent phenyl group with lipophilic electron-donating groups or replacing it by 2-thienyl or 2-furyl groups.

Full text of DOI:10.1016/j.ejmech.2015.09.023

Accepted Manuscript
2-((Benzimidazol-2-yl)thio)-1-arylethan-1-ones: Synthesis, crystal study and cancer
stem cells CD133 targeting potential
Hatem A. Abdel-Aziz, Hazem A. Ghabbour, Wagdy M. Eldehna, Sara T.A. Al-
Rashood, Khalid A. Al-Rashood, Hoong-Kun Fun, Mays Al-Tahhan, Abdullah Al-
Dhfyan
PII:
S0223-5234(15)30267-1
10.1016/j.ejmech.2015.09.023
EJMECH 8120
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 April 2015
Revised Date: 26 June 2015
Accepted Date: 15 September 2015
Please cite this article as: H.A. Abdel-Aziz, H.A. Ghabbour, W.M. Eldehna, S.T.A. Al-Rashood, K.A.
Al-Rashood, H.-K. Fun, M. Al-Tahhan, A. Al-Dhfyan, 2-((Benzimidazol-2-yl)thio)-1-arylethan-1-ones:
Synthesis, crystal study and cancer stem cells CD133 targeting potential, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.09.023.
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ACCEPTED MANUSCRIPT
Graphical abstract
2-((Benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a-o were synthesized to evaluate their activities
towards colon HT-29 cancer cell line and the cell surface expression of CD133, a potent cancer
stem cells (CSCs) marker.

ACCEPTED MANUSCRIPT
2-((Benzimidazol-2-yl)thio)-1-arylethan-1-ones: Synthesis, crystal study and
cancer stem cells CD133 targeting potential
Hatem A. Abdel-Aziza,b,*, Hazem A. Ghabboura, Wagdy M. Eldehnac, Sara T. A. Al-
Rashooda, Khalid A. Al-Rashooda, Hoong-Kun Funa,d, Mays Al-Tahhane, Abdullah
Al-Dhfyane
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box
2457, Riyadh 11451, Saudi Arabia.
bDepartment of Applied Organic Chemistry, National Research Centre, Dokki, Giza, P.O. Box
12622, Egypt.
cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,
Badr City, Cairo, P.O. Box 11829, Egypt.
dX-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, 11800, USM,Penang,
Malaysia
eStem Cell & Tissue Re-Engineering Program, Research Center, King Faisal Specialized Hospital
& Research Center, MBC-03, PO Box 3354, Riyadh 11211, Saudi Arabia
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Abstract
In order to develop a potent anti-tumor agent that can target both cancer stem cells and the bulk
of tumor cells, a series of 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a-o was synthesized.
All compounds were evaluated for their anti-proliferative activity towards colon HT-29 cancer cell
line. In addition, their inhibitory effect against cell surface expression of CD133, a potent cancer
stem cells (CSCs) marker, in the same cells was evaluated by flow cytometry at 10µM. Compound
5l emerged as the most active anti-proliferative analog against HT-29 (IC50 = 18.83±1.37 µM), that
almost equipotent as 5-fluorouracil (IC50 = 15.83±1.63 µM) with 50.11±4.05 % inhibition effect on
CD133 expression, suggested dual targeted effect. Also, compounds 5h, 5j, 5k and 5m-o inhibited
the expression of CD133 with more than 50 %. The SAR study pointed out the significance of
substitution of the pendent phenyl group with lipophilic electron-donating groups or replacing it by
2-thienyl or 2-furyl groups.
Keywords: Synthesis; Anti-proliferative; Cancer stem cells; CD133 (prominin-1); X-Ray.
*Corresponding author: Department of Pharmaceutical Chemistry, College of Pharmacy, King
Saud University, P.O. Box. 2457, Riyadh 11451, Saudi Arabia. Tel: +966-146-73764; Fax: +966-
146-76220. E-mail address: hatem_741@yahoo.com
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1. Introduction
Cancer stem cells (CSCs) are a specific subpopulation of tumor cells that selectively possess
tumor initiation and self-renewal capacity, as well as, the ability to give rise to bulk populations of
non-tumorigenic cancer cell progeny through differentiation [1, 2]. These cancer stem cells show
bidirectional interactions with the cellular constituents of their individual niches, which enfold
distinct developmental signaling networks, soluble mediators, and/or cell matrix processes. These
interactions are essential for the establishment of a stem cell–permissive microenvironment and
provide a crucial regulatory balance between self-renewal and differentiation and between
quiescence and proliferation [3]. To be resilient to electromagnetic and chemical insults, to
colonize other parts of the body and to be able to slumber for prolonged periods of time, CSCs are
tacitly thought to have acquired the molecular armaments of normal stem cells: CSCs can renew
themselves, and they are built to last a lifetime. [4] So, an intrinsic resistance to a variety of
traditional chemo- or radiotherapies has been displayed by the CSCs [5, 6], which elucidates the
failure of many cancer therapies that kill the bulk of tumor cells but fail to eliminate CSCs which
survive to regenerate new tumors. There are numerous studies which have identified cancer stem
cells in leukemia [7], breast [8], brain [9], lung [10], colon [11], and others. CSCs in solid tumors
are identified using an extensive list of markers, to name just a few, CD44, CD24, CXCR4,
CDCP1 and CD133 [12]. CD133, also known as prominin-1, represents one of the most popular
CSC markers. CD133 is a member of the prominin family of proteins and is a surface protein with
five transmembrane domains initially discovered as a marker of hematopoietic stem cells [13] and
of bone marrow-derived circulating endothelial progenitors involved in postnatal angiogenesis,
inflammation and tissue regeneration [14, 15] . Although being a reliable marker of
stem/progenitor cells in normal adult tissues, such as the brain [16], liver [17], kidney [18] and
prostate [19], CD133 antigen has been used as a CSC marker in various cancers, including those of
the brain [20], pancreas [21], lung [22], ovarian [23], prostate [24] and colon [12].
According to the World Health Organization statistics, colon cancer is one of the leading causes
of cancer related deaths worldwide and is more common in developed countries. Both drug
resistance to chemotherapy and metastases are the major concerns in colon cancer therapy [25-27].
Regarding the colon cancer, the disease aggressiveness and evolution [28-30], treatment resistance
[31-33], and survival [28] in colorectal cancer were found to be associated with high expression of
CD133. Also, the prognostic value of circulating CD133 mRNA levels in colorectal cancer has
been also reported [33-35]. All these findings suggest a crucial role of CD133 expressing cells in
the initiation and evolution of human colon tumorigenesis and support its role as important CSC
marker. Noteworthy, Many treatment protocols have been applied to colorectal cancer, but they
have not resulted in a complete cure, most probably, due to the resistant colon CSCs. Therefore, it
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is important to develop therapeutics that target bulk tumor cells and CSCs simultaneously, in order
to eliminate all cancer cells.
Among the heterocycles, benzimidazole has emerged as a promising class that possesses
interesting biological activities including anticancer activity [36-40].
In view of the facts mentioned above and as part of our ongoing effort towards developing
effective anticancer agents [41–43], herein we report the synthesis of certain 2-((benzimidazol-2-
yl)thio)-1-arylethan-1-ones and biological evaluation of their anti-proliferative activity towards
colon HT-29 cancer cell line, in addition to, their inhibitory activity against cell surface expression
of CD133 in HT-29 cancer cells by flow cytometry.
2. Results and Discussion
2.1. Chemistry
Although A.A.O. Sarhan et al. [44] reported the reaction of 2-mercaptobenzimidazole 2 with
aromatic ketones in acidified acetic acid, they did not separate the salts product, and directly
neutralized the reaction mixture to obtain 2-benzimidazolylthioacetophenone derivatives. With the
aim to separate the salt product in a good yield and to determine its definite structure, we modified
the reported procedure [44]. While A.A.O. Sarhan et al. used few drops of concentrated H2SO4, we
increased the used amount of concentrated H2SO4 up to two equivalents to obtain a quantitative
yield of the salt after a short duration, about 30 minutes. X-ray crystallographic analysis for a salt
product unequivocally revealed that its structure is 2-((2-oxo-2-phenylethyl)thio)-1H-
benzo[d]imidazol-3-ium sulfate salt (figure 1). Subsequent neutralization of the prepared sulfate
salts 4a-o afforded the target 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a-o in an excellent
yield 85-94 % (Scheme 1).
Another synthetic strategy was utilized to prepare the target compounds 5a-o. Bromination of
aromatic ketones 3a-o with copper (II) bromide in a refluxing mixture of chloroform and ethyl
acetate afforded the corresponding phenacyl bromides 7a-o. Then the reaction of 7a-o with the
potassium salt of 2-mercaptobenzimidazole 6, furnished the target derivatives 5a-o in a moderate
yield 69-81 % (Scheme 1).
Scheme 1
All the prepared compounds were confirmed by spectral analyses which were in full agreement
with the proposed structures.
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2.2. X-ray crystallographic analysis
Crystals of compounds 4b and 5n were obtained by slow evaporation from solutions of ethanol
(Figures 1 and 2). The measurements of the crystals were performed on a Bruker SMART APEX II
D8 Venture diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) at 150
K. The structures were solved by direct method and refined with SHELXTL [45]. E-maps provided
the positions of all the non-H-atoms. The full-matrix least-squares refinement was carried out on
F2’s using anisotropic temperature factors for all non-H-atoms. Crystallographic data for the
structure reported in this paper have been deposited at the Cambridge Crystallographic Data Center
and allocated with the deposition numbers: CCDC 1048412-1048413 and 1047318-1047319 for
compounds 4b and 5n, respectively. The crystallographic data and refinement for the two crystals
are presented in table 1. Also, selected geometric parameters of compounds 4b and 5h are
presented in the supplementary data.
Figure 1
Figure 2
Table 1
2.3. Biological Evaluation
2.3.1. In vitro anti-proliferative activity against HT-29
Anti-proliferative activity of the prepared 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a-o
was evaluated against human colon cancer cell line HT-29 using the WST-1 assay as described by
Ngamwongsatit et al. [46]. The broad spectrum anticancer drug 5-fluorouracil was used as a
positive control. The anti-proliferative activities were expressed as growth inhibitory concentration
(IC50) values which represent the compounds concentrations required to produce a 50% inhibition
of cell growth after 48 hours of incubation compared to untreated controls (Table 2). From the
obtained results, it is evident that most the tested compounds displayed excellent to moderate
growth inhibitory activity against the tested HT-29 cancer cell line. Compounds 5f, 5j and 5l were
found to be the most active analogs against HT-29 (IC50 = 19.22±1.12, 19.11±1.11 and 18.83±1.37
µM, respectively) and almost equipotent as 5-fluorouracil (IC50 = 15.83±1.63 µM). Besides,
compounds 5h, 5n and 5o with IC50 = 25.19±2.02, 24.57±2.06 and 23.93±2.18 µM, respectively
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displayed good anti-proliferative activity. Moreover, compounds 5a, 5b, 5g, 5i and 5k were
moderately active against HT-29 with IC50 values ranging from 33.05±2.32 to 38.46±2.59 µM.
Table 2
Examining the IC50 values in table 2, we could conclude worthy data about the structure
activity relationships. Firstly, we explored the impact of grafting different substituents on the
pendent phenyl ring on the prepared 2-benzimidazolylthioacetophenones 5a-o activities.
Incorporation of unsubstituted phenyl group led to compound 5a with moderate activity against
HT-29 (IC50 = 38.46±2.59 µM). The substitution of a hydrogen atom by a fluorine atom is one of
the most commonly employed classical bioisosteric replacements [47]. Compound 5c bears
fluorine substituent at the 4-position displayed sharp decrease in activity (IC50 = 78.70±7.08 µM).
Also, introduction of bromine atom in the same position slightly decreased the activity (IC50 =
42.95±3.13 µM), suggesting that incorporation of halogens, electron-withdrawing groups, is not
appropriate for the anti-proliferative activity against HT-29. Likewise, grafting an electron-
withdrawing nitro group as in compound 5e resulted in a partial loss of activity the activity (IC50 =
57.00±5.14 µM).
Contrariwise, substitution with an electron-donating group in the 4-position as methyl group,
compound 5b, improved the activity (IC50 = 34.46±3.02 µM). Also, grafting 4-OH and 4-OCH3
substituents afforded 5f and 5h analogs, respectively, with an enhanced activity (IC50 = 19.22±1.12
and 25.19±2.02 µM, respectively) compared to the unsubstituted derivative 5a, hinting that
introduction of electron-donating groups (methyl, hydroxyl and methoxy) at the 4-position is
essential to enhance the activity. Moving the methoxy group from the 4- to the 2-position, 5g,
decreased the activity (IC50 = 36.12±3.08µM), suggesting that substitution at the 4-position is more
beneficial to activity than the 2-position. Moreover, di- and tri-substitution with 3,4-(OCH3)2 and
2,3,4-(OCH3)3 groups afforded the potent members 5j and 5l, respectively, with superior activity
towards HT-29 (IC50 = 19.11±1.11 and 18.83±1.37 µM, respectively), while di-substitution with 3-
OH-4-OCH3 resulted in a slight activity improvement as in 5i analog (IC50 = 33.05±2.32 µM). In
contrast, incorporation of the large electron-donating ethoxy group, compound 5m, diminished the
activity.
Interestingly, attaching 2-thienyl or 2-furyl groups (compounds 5n and 5o) as bioisosteres to
the phenyl one improved the activity (IC50 = 24.57±2.06 and 23.93±2.18 µM, respectively).
Finally, we can deduce that the type of the terminal aryl group and the substitution pattern for
analogs entailed with phenyl group are crucial elements for the antitumor activity. Bioisosteric
replacement of the phenyl group with 2-thienyl or 2-furyl groups and substitution with electron-
donating groups as methyl, hydroxyl and methoxy groups greatly enhanced the activity. While,
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incorporation of electron-withdrawing groups as halogens and nitro groups is not favorable for the
activity.
2.3.2. CD133 expression measure by flow cytometry
The inhibitory effect of the prepared 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a-o on
cell surface expression of CD133 was evaluated at 10 µM via flow cytometry (Figure 3). The
analysis was performed on a BD LSR II™ (BD Biosciences). The results expressed as a side
population inhibition (%) (Table 3). The obtained data revealed that tested compounds have
distinctive pattern of potency against CD133. Compound 5n emerged as the most active one in this
study with 56.82 inhibition %. Also, compounds 5h, 5j-m and 5o elicited more than 50 %
inhibition against CD133 expression. Moreover, compounds 5b, 5g and 5i inhibited the expression
of CD133 with more than 30 %. All the remaining analogs did not displayed significant activity
against the cell surface expression of CD133.
Figure 3
Observing the results, it is obvious that incorporation of lipophilic electron-donating groups as
methyl, methoxy and ethoxy groups to the terminal phenyl group, also, replacing the phenyl group
with heteroaryl group as 2-thienyl or 2-furyl groups are indispensable for the inhibitory activity
against CD133.
Table 3
3. Conclusion
In summary, we have synthesized fifteen 2-((benzimidazol-2-yl)thio)-1-arylethan-1-one
derivatives 5a-o and evaluated their anti-proliferative activities against colon HT-29 cancer cell
line. In particular, compounds 5f, 5j and 5l emerged as the most active analogs against HT-29 (IC50
= 19.22±1.12, 19.11±1.11 and 18.83±1.37 µM, respectively) and nearly equipotent as 5-
fluorouracil (IC50 = 15.83±1.63 µM). Moreover, the inhibitory activity of the prepared derivatives
5a-o against cell surface expression of CD133 in HT-29 cancer cells was analyzed by flow
cytometry. Compound 5n was found to be the most potent inhibitor of CD133 expression with
56.82 inhibition %. Also, compounds 5h, 5j-m and 5o displayed more than 50 % inhibition against
CD133 expression. From the structure activity relationships (SARs) we can deduce that
substitution of the pendent phenyl group with lipophilic electron-donating groups or replacing it by
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2-thienyl or 2-furyl groups were needful for the inhibition of the both bulk tumor cells and CSCs.
In addition, single crystal X-ray diffraction has also been recorded for compounds 4b and 5h.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were measured with a Gallenkamp melting point apparatus and were
uncorrected. Infrared (IR) Spectra were recorded as KBr disks using the Perkin Elmer FT-IR
Spectrum BX apparatus. Mass spectral data are given as m/z (Intensity %). NMR Spectra were
1
recorded on a Bruker NMR spectrometer. H spectrum was run at 500MHz and 13C spectrum was
run at 125MHz in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts are expressed in
δ
values (ppm) using the solvent 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. Reaction
courses and product mixtures were routinely monitored by thin layer chromatography (TLC) on
silica gel precoated F254 Merck plates. Unless otherwise noted, all solvents and reagents were
commercially available and used without further purification. Compounds 5a [50], 5b [50], 5d
[51], 5e [51], 5f [52], 7h [53], 7j [52], 7l [54], 7o [51] are previously reported.
4.1.2. 1H-benzo[d]imidazole-2-thiol 2
To a solution of 1,2-phenylene diamine 1 (1.08 g, 10 mmol) in 10 mL of ethanol, potassium
hydroxide (0.56 g,10 mmol) and carbon disulfide (0.76 g, 10 mmol) were added and the contents
were refluxed for 3 h. The reaction mixture was acidified with 0.5 M HCl and the precipitate
obtained upon cooling was filtered off, washed with water and recrystallized from ethanol to give
compound 2 [48].
4.1.3. General procedure for preparation of 2-((2-oxo-2-arylethyl)thio)-1H-benzo[d]imidazol-3-
ium sulfate salts 4a-o.
To a solution of the appropriate acetophenone 3a-o (5 mmol) in glacial acetic acid (10 mL), 2-
mercaptobenzimidazole 2 (0.75 g, 5 mmol) and conc. Sulphuric acid (10 mmol) were added. The
reaction mixture was heated under reflux for 0.5 h. 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 4a-o, respectively.
4.1.3.1. 2-((2-Oxo-2-phenylethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4a). White crystals,
m.p. 201-204°C; IR (KBr, ν cm-1): 3467 (NH) and 1680 (C=O); 1H NMR (DMSO-d6)
(s, 4H, 2CH2), 7.46 7.48 (m, 4H, H-5 and H-6 of 2-mercaptobenzimidazoles), 7.61 (t, 4H, H-3 and
δ ppm: 5.38
̶
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̶ 7.71 (m, 4H, H-4 and H-7 of 2-mercaptobenzimidazoles), 7.73
H-5 of 2[C6H5], J = 7.5 Hz), 7.69
(s, 2H, H-4 of 2[C6H5], J = 7.5 Hz), 8.07 (d, 4H, H-2 and H-6 of 2[C6H5], J = 7.5 Hz), 10.72 (s,
2H, 2NH), 12.63 (s, 2H, 2NH); 13C NMR (DMSO-d6)
ppm: 41.64 (CH2), 113.71, 125.44, 129.02,
129.45, 133.47, 134.72, 135.22, 151.00, 192.66 (C=O).
δ
4.1.3.2. 2-((2-Oxo-2-(p-tolyl)ethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4b). White crystals,
m.p. 228-231°C; IR (KBr, ν cm-1): 3435 (NH) and 1668 (C=O); 1H NMR (DMSO-d6)
ppm: 2.37
(s, 6H, 2CH3), 5.33 (s, 4H, 2CH2), 7.42 (d, 4H, H-3 and H-5 of 2[4-CH3C6H4], J = 8.0 Hz), 7.44
7.47 (m, 4H, H-5 and H-6 of 2-mercaptobenzimidazoles), 7.66 7.70 (m, 4H, H-4 and H-7 of 2-
mercaptobenzimidazoles), 7.97 (d, 4H, H-2 and H-6 of 2[4-CH3C6H4], J = 8.0 Hz), 10.68 (s, 2H,
2NH), 12.57 (s, 2H, 2NH); 13C NMR (DMSO-d6)
ppm: 21.75 (CH3), 41.54 (CH2), 113.72,
δ
̶
̶
δ
125.29, 129.13, 129.97, 132.74, 133.75, 145.36, 151.01, 192.24 (C=O).
4.1.3.3. 2-((2-(4-Fluorophenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4c). White
1
crystals, m.p. 179-181°C; IR (KBr, ν cm-1): 3435 (NH) and 1670 (C=O); H NMR (DMSO-d6)
δ
ppm: 5.24 (s, 4H, 2CH2), 7.32
4H, Ar-H, J = 8.5 Hz), 7.58
̶
7.35 (m, 4H, H-5 and H-6 of 2-mercaptobenzimidazoles), 7.43 (t,
̶
7.61 (m, 4H, H-4 and H-7 of 2-mercaptobenzimidazoles), 8.15 ̶ 8.18
(m, 4H, Ar-H), 10.43 (s, 2H, 2NH), 12.59 (s, 2H, 2NH); 13C NMR (DMSO-d6)
δ ppm: 56.49
(CH2), 113.91, 116.40, 116.57, 124.17, 132.03, 132.11, 132.30, 135.79, 150.55, 162.78, 164.92,
166.93, 191.78 (C=O).
4.1.3.4. 2-((2-(4-Bromophenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4d). White
1
crystals, m.p. 225-228°C; IR (KBr, ν cm-1): 3430 (NH) and 1685 (C=O); H NMR (DMSO-d6)
δ
ppm: 5.28 (s, 4H, 2CH2), 7.38 ̶ 7.41 (m, 4H, H-5 and H-6 of 2-mercaptobenzimidazoles), 7.62 ̶ 7.65
(m, 4H, H-4 and H-7 of 2-mercaptobenzimidazoles), 7.83 (d, 4H, H-3 and H-5 of 2[4-BrC6H4], J =
8.5 Hz), 7.99 (d, 4H, H-2 and H-6 of 2[4-BrC6H4], J = 8.5 Hz), 10.53 (s, 2H, 2NH), 12.62 (s, 2H,
2NH); 13C NMR (DMSO-d6)
δ ppm: 41.16 (CH2), 109.95, 113.83, 122.78, 124.75, 128.75, 130.95,
132.50, 134.43, 134.75, 150.64, 192.26 (C=O).
4.1.3.5. 2-((2-(4-Nitrophenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfates (4e). White
1
crystals, m.p. 245-248°C; IR (KBr, ν cm-1): 3440 (NH) and 1675 (C=O); H NMR (DMSO-d6)
δ
ppm: 5.33 (s, 4H, 2CH2), 7.37 ̶ 7.39 (m, 4H, H-5 and H-6 of 2-mercaptobenzimidazoles), 7.61 ̶ 7.63
(m, 4H, H-4 and H-7 of 2-mercaptobenzimidazoles), 8.29 (d, 4H, H-2 and H-6 of 2[4-NO2C6H4], J
= 9.0 Hz), 8.41 (d, 4H, H-3 and H-5 of 2[4-NO2C6H4], J = 8.5 Hz), 10.63 (s, 2H, 2NH), 12.75 (s,
2H, 2NH); 13C NMR (DMSO-d6)
δ ppm: 41.32 (CH2), 113.89, 124.44, 124.56, 127.96, 130.41,
135.16, 140.15, 150.38, 150.77, 162.80, 192.37 (C=O).
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4.1.3.6. 2-((2-(4-Hydroxyphenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4f). White
1
crystals, m.p. 241-244°C; IR (KBr, ν cm-1): 3428 (NH) and 1664 (C=O); H NMR (DMSO-d6)
δ
ppm: 5.31 (s, 4H, 2CH2), 6.93 (d, 4H, H-3 and H-5 of 2[4-OHC6H4], J = 9.0 Hz), 7.48 ̶ 7.49 (m,
4H, H-5 and H-6 of 2-mercaptobenzimidazoles), 7.70-7.72 (m, 4H, H-4 and H-7 of 2-
mercaptobenzimidazoles), 7.95 (d, 2H, H-2 and H-6 of 2[4-OHC6H4], J = 8.5 Hz), 10.22 (s, 2H,
2OH), 10.58 (s, 2H, 2NH), 12.61 (s, 2H, 2NH); 13C NMR (DMSO-d6)
δ ppm: 41.08 (CH2), 112.54,
113.65, 116.02, 116.31, 122.54, 125.66, 126.38, 131.42, 131.74, 133.51, 160.54, 163.56, 190.57
(C=O).
4.1.3.7. 2-((2-(2-Methoxyphenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4g). White
1
crystals, m.p. 195-198°C; IR (KBr, ν cm-1): 3413 (NH) and 1685 (C=O); H NMR (DMSO-d6)
δ
ppm: 3.99 (s, 6H, 2OCH3), 5.32 (s, 4H, 2CH2), 6.95 (d, 2H, Ar-H, J = 8.5 Hz), 7.19 (t, 2H, Ar-H, J
= 8.0 Hz), 7.49 7.58 (m, 6H, H-5, H-6 of 2-mercaptobenzimidazoles and 2H, Ar-H), 7.68 7.69 (m,
̶
̶
4H, H-4 and H-7 of 2-mercaptobenzimidazoles), 7.86 (d, 2H, Ar-H, J = 8.0 Hz), 10.61 (s, 2H,
13
2NH), 12.59 (s, 2H, 2NH); C NMR (DMSO-d6)
δ ppm: 44.33 (CH2), 56.08 (OCH3), 112.23,
113.36, 113.68,121.19, 121.56, 125.77, 130.94, 132.57, 133.45, 136.09, 151.25, 158.04, 159.81,
191.57 (C=O).
4.1.3.8. 2-((2-(4-Methoxyphenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4h). White
1
crystals, m.p. 263-265°C; IR (KBr, ν cm-1): 3485 (NH) and 1675 (C=O); H NMR (DMSO-d6)
δ
ppm: 3.88 (s, 6H, 2OCH3), 5.33 (s, 4H, 2CH2), 7.12 (d, 4H, H-3 and H-5 of 2[4-OCH3C6H4], J =
8.5 Hz), 7.47 7.49 (m, 4H, H-5 and H-6 of 2-mercaptobenzimidazoles), 7.69 7.73 (m, 4H, H-4
and H-7 of 2-mercaptobenzimidazoles), 8.05 (d, 4H, H-2 and H-6 of 2[4-OCH3C6H4], J = 8.5 Hz),
10.55 (s, 2H, 2NH), 12.73 (s, 2H, 2NH); 13C NMR (DMSO-d6)
ppm: 41.63 (CH2), 56.22 (OCH3),
̶
̶
δ
113.69, 114.68, 125.58, 128.03, 131.49, 132.37, 151.19, 164.45, 190.98 (C=O).
4.1.3.9. 2-((2-(3-Hydroxy-4-methoxyphenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate
1
(4i). White crystals, m.p. 218-220°C; IR (KBr, ν cm-1): 3346 (NH) and 1675 (C=O); H NMR
(DMSO-d6)
OCH3C6H3], J = 8.5 Hz), 7.43
2[3-OH-4-OCH3C6H3], 7.61 (d, 2H, H-6 of 2[3-OH-4-OCH3C6H3], J = 8.5 Hz), 7.65
δ
ppm: 3.89 (s, 6H, 2OCH3), 5.24 (s, 4H, 2CH2), 7.11 (d, 2H, H-5 of 2[3-OH-4-
7.44 (m, 6H, H-5, H-6 of 2-mercaptobenzimidazoles and H-2 of
7.66 (m, 4H,
̶
̶
H-4 and H-7 of 2-mercaptobenzimidazoles), 9.75 (s, 2H, 2OH), 10.27 (s, 2H, 2NH), 12.33 (s, 2H,
13
2NH); C NMR (DMSO-d6)
δ ppm: 40.58 (CH2), 56.34 (OCH3), 111.87, 113.74, 115.09, 122.35,
125.29, 128.31, 147.04, 151.40, 153.45, 191.88 (C=O).
4.1.3.10. 2-((2-(3,4-Dimethoxyphenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4j).
White crystals, m.p. 271-273°C; IR (KBr, ν cm-1): 3434 (NH) and 1670 (C=O); 1H NMR (DMSO-
10

ACCEPTED MANUSCRIPT
d6)
2[3,4-(OCH3)2C6H3], J = 8.5 Hz), 7.45
7.52 (s, 2H, H-2 of 2[3,4-(OCH3)2C6H3]), 7.69
mercaptobenzimidazoles), 7.77 (d, 2H, H-6 of 2[3,4-(OCH3)2C6H3], J = 8.5 Hz), 10.55 (s, 2H,
2NH), 12.50 (s, 2H, 2NH); 13C NMR (DMSO-d6)
ppm: 41.32 (CH2), 56.14 (OCH3), 56.41
δ
ppm: 3.84 (s, 6H, 2OCH3), 3.89 (s, 6H, 2OCH3), 5.31 (s, 4H, 2CH2), 7.16 (d, 2H, H-5 of
7.47 (m, 4H, H-5 and H-6 of 2-mercaptobenzimidazoles),
7.71 (m, 4H, H-4 and H-7 of 2-
̶
̶
δ
(OCH3), 110.99, 111.56, 113.74, 124.17, 125.37, 127.98, 133.27, 149.20, 151.07, 154.43, 191.15
(C=O).
4.1.3.11. 2-((2-(Benzo[d][1,3]dioxol-4-yl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate
1
(4k). White crystals, m.p. 227-230°C; IR (KBr, ν cm-1): 3444 (NH) and 1668 (C=O); H NMR
(DMSO-d6)
7.44 (m, 4H, H-5 and H-6 of 2-mercaptobenzimidazoles), 7.66
mercaptobenzimidazoles), 7.73 (d, 2H, Ar-H, J = 8.0 Hz), 10.37 (s, 2H, 2NH), 12.80 (s, 2H, 2NH);
13C NMR (DMSO-d6)
ppm: 41.31 (CH2), 102.83, 104.78, 108.18, 108.81, 110.02, 113.76,
122.79, 124.34, 125.86, 129.80, 133.48, 148.47, 150.55, 152.76, 191.75 (C=O).
δ
ppm: 5.25 (s, 4H, 2CH2), 6.19 (s, 4H, 2-O-CH2-O-), 7.11
̶ 7.16 (m, 4H, Ar-H), 7.43 ̶
̶
7.67 (m, 4H, H-4 and H-7 of 2-
δ
4.1.3.12. 2-((2-Oxo-2-(2,3,4-trimethoxyphenyl)ethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4l).
White crystals, m.p. 232-234°C; IR (KBr, ν cm-1): 3440 (NH) and 1664 (C=O); 1H NMR (DMSO-
d6)
δ
ppm: 3.78 (s, 6H, 2OCH3), 3.88 (s, 12H, 4OCH3), 5.29 (s, 4H, 2CH2), 7.37
̶
7.42 (m, 8H, H-5,
7.66 (m, 4H, H-4
H-6 of 2-mercaptobenzimidazoles and H-5, H-6 of 2[2,3,4-(OCH3)3C6H2]), 7.65
̶
and H-7 of 2-mercaptobenzimidazoles), 10.56 (s, 2H, 2NH), 12.66 (s, 2H, 2NH); 13C NMR
(DMSO-d6) ppm: 40.98 (CH2), 56.73 2(OCH3), 60.76 (OCH3), 106.74,113.86, 124.82, 130.56,
δ
134.69, 143.15, 150.65, 153.37, 191.91(C=O).
4.1.3.13. 2-((2-(4-Ethoxyphenyl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4m). White
1
crystals, m.p. 240-243°C; IR (KBr, ν cm-1): 3350 (NH) and 1670 (C=O); H NMR (DMSO-d6)
δ
ppm: 1.36 (t, 6H, 2CH2CH3, J = 7.0 Hz), 4.15 (q, 4H, 2CH2CH3, J = 7.0 Hz), 5.29 (s, 4H, 2CH2),
7.11 (d, 4H, H-3 and H-5 of 2[4-OC2H5C6H4], J = 8.5 Hz), 7.43 7.45 (m, 4H, H-5 and H-6 of 2-
̶
mercaptobenzimidazoles), 7.66
̶
7.69 (m, 4H, H-4 and H-7 of 2-mercaptobenzimidazoles), 8.03 (d,
13
4H, H-2 and H-6 of 2[4-OC2H5C6H4], J = 9.0 Hz), 10.51 (s, 2H, 2NH), 12.64 (s, 2H, 2NH); C
NMR (DMSO-d6)
δ ppm: 14.92 (OCH2CH3), 41.32 (SCH2), 64.25 (OCH2CH3), 113.73, 115.03,
125.17, 127.93, 131.47, 133.94, 151.10, 163.71, 191.02 (C=O).
4.1.3.14. 2-((2-(Furan-2-yl)-2-oxoethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4n). White
1
crystals, m.p. 158-160°C; IR (KBr, ν cm-1): 3413 (NH) and 1670 (C=O); H NMR (DMSO-d6)
δ
ppm: 5.03 (s, 4H, 2CH2), 6.82 ̶ 6.83 (m, 2H, H-4 furans), 7.40 ̶ 7.43 (m, 4H, H-5 and H-6 of 2-
mercaptobenzimidazoles), 7.63
̶
7.66 (m, 6H, H-4, H-7 of 2-mercaptobenzimidazoles and H-3
11

13
ACCEPTED MANUSCRIPT
furans), 8.12 (d, 2H, H-5 furans, J = 3.5 Hz), 10.54 (s, 2H, 2NH), 12.67 (s, 2H, 2NH); C NMR
(DMSO-d6) ppm: 56.49 (CH2), 113.48, 113.84, 120.61, 124.99, 134.40, 149.24, 150.48, 150.72,
δ
172.54, 180.97, 189.57 (C=O).
4.1.3.15. 2-((2-Oxo-2-(thiophen-2-yl)ethyl)thio)-1H-benzo[d]imidazol-3-ium sulfate (4o). White
1
crystals, m.p. 187-190°C; IR (KBr, ν cm-1): 3412 (NH) and 1654 (C=O); H NMR (DMSO-d6)
δ
ppm: 5.18 (s, 4H, 2CH2), 7.34 (d, 2H, H-4 thiophenes, J = 4.0 Hz), 7.39
of 2-mercaptobenzimidazoles), 7.63 7.66 (m, 4H, H-4 and H-7 of 2-mercaptobenzimidazoles),
8.14 (d, 2H, H-3 thiophenes, J = 4.0 Hz), 8.16 (d, 2H, H-5 thiophenes, J = 4.0 Hz), 10.48 (s, 2H,
2NH), 12.62 (s, 2H, 2NH); 13C NMR (DMSO-d6)
ppm: 40.56 (CH2), 113.87, 122.90, 124.84,
̶ 7.42 (m, 4H, H-5 and H-6
̶
δ
129.55, 134.66, 135.37, 136.75, 141.72, 150.50, 185.87 (C=O).
4.1.4. substituted 2-bromo-1-arylethanone 7a-o.
A solution of the appropriate ketone 3a-o (25 mmol) in hot chloroform (25 mL) was added to a
preheated, stirred suspension of copper (II) bromide (50 mmol) in ethyl acetate (25 mL). The
resulting reaction mixture was refluxed with vigorous stirring till completion of the reaction which
detected by TLC monitoring. The reaction mixture was filtered while hot to remove copper (I)
bromide then concentrated under reduced pressure. After cooling, the separated solid was filtered,
washed with petroleum ether and dried to give compounds 7a-o [49].
4.1.5. General procedure for preparation of 2-((1H-benzo[d]imidazol-2-yl)thio)-1-arylethan-1-one
derivatives 5a-o.
Route A:
To a suspension of the appropriate 2-((2-oxo-2-arylethyl)thio)-1H-benzo[d]imidazol-3-ium
sulfate salts 4a-o (2 mmol) in water (10 mL), an aqueous solution of sodium bicarbonate was
added. The reaction mixture was stirred at room temperature for 2 h. The solid formed was
collected by filtration, washed with water, dried and crystallized from ethanol to afford compounds
5a-o.
Route B:
Solution of potasium hydroxide (10 mmol) and 2-mercaptobenzimidazole 2 (5 mmol) in
ethanol (20 mL) was refluxed for 1 h. After cooling, 2-bromo-1-phenylethanone 7a-o (5 mmol)
was added and the refluxing was continued for another 3 h. Then, the reaction mixture was cooled
and poured into water. The solid product was filtrated, washed with water crystallized from
ethanol.
12

ACCEPTED MANUSCRIPT
4.1.5.1. 2-((1H-benzo[d]imidazol-2-yl)thio)-1-phenylethan-1-one (5a). White crystals (yield 91%),
1
m.p. 162-164°C (reported: 168-170°C [50]); IR (KBr, ν cm-1): 3410 (NH) and 1690 (C=O); H
NMR (DMSO-d6)
δ
ppm: 5.23 (s, 2H, CH2), 7.12
̶
7.14 (m, 2H, H-5 and H-6 of 2-
7.63
mercaptobenzimidazole), 7.30
̶
7.32 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 7.56
̶
(m, 3H, H-3, H-4 and H-5 of C6H5), 8.07 (d, 2H, H-2 and H-6 of C6H5, J = 7.5 Hz), 12.52 (s, 1H,
13
NH); C NMR (DMSO-d6)
δ ppm: 40.51 (CH2), 111.57, 113.96, 114.38, 123.84, 128.96, 129.39,
134.47, 135.54, 150.53, 193.21 (C=O); ESI MS m/z: 269 [M+1]+.
4.1.5.2. 2-((1H-benzo[d]imidazol-2-yl)thio)-1-(p-tolyl)ethan-1-one (5b). White crystals (yield
88%), m.p. 197-200°C (reported: 184-186°C [50]); IR (KBr, ν cm-1): 3413 (NH) and 1684 (C=O);
1H NMR (DMSO-d6)
of 2-mercaptobenzimidazole), 7.37
H-5 of 4-CH3C6H4), 7.97 (d, 2H, H-2 and H-6 of 4-CH3C6H4, J = 8.0 Hz), 12.60 (s, 1H, NH); 13C
NMR (DMSO-d6) ppm: 21.69 (CH3), 40.50 (CH2), 112.77, 114.09, 121.85, 129.01, 129.84,
δ
ppm: 2.41 (s, 3H, CH3), 5.03 (s, 2H, CH2), 7.09
̶ 7.13 (m, 2H, H-5 and H-6
̶
7.41 (m, 4H, H-4, H-7 of 2-mercaptobenzimidazole and H-3,
δ
133.42, 144.68, 150.00, 193.41(C=O); ESI MS m/z: 283 [M+1]+.
4.1.5.3. 2-((1H-benzo[d]imidazol-2-yl)thio)-1-(4-fluorophenyl)ethan-1-one (5c). White crystals
(yield 93%), m.p. 148-150°C; IR (KBr, ν cm-1): 3413 (NH) and 1670 (C=O); 1H NMR (DMSO-d6)
δ
ppm: 5.05 (s, 2H, CH2), 7.09
̶
7.13 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.39 ̶ 7.42
13
(m, 4H, Ar-H), 8.15
̶
8.18 (m, 2H, Ar-H), 12.65 (s, 1H, NH); C NMR (DMSO-d6)
δ
ppm: 40.57
(CH2), 116.26, 116.44, 121.90, 131.93, 132.01, 132.71, 132.73, 149.89, 164.75, 166.75, 192.60
(C=O); ESI MS m/z: 287 [M+1]+, 288 [M+2]+.
4.1.5.4. 2-((1H-benzo[d]imidazol-2-yl)thio)-1-(4-bromophenyl)ethan-1-one (5d). White crystals
(yield 94%), m.p. 187-190°C (reported: 187-189°C [51]); IR (KBr, ν cm-1): 3400 (NH) and 1680
1
(C=O); H NMR (DMSO-d6)
δ
ppm: 5.04 (s, 2H, CH2), 7.10
̶ 7.14 (m, 2H, H-5 and H-6 of 2-
mercaptobenzimidazole), 7.40
̶ 7.44 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 7.79 (d,
2H, H-3 and H-5 of 4-BrC6H4, J = 8.0 Hz), 8.00 (d, 2H, H-2 and H-6 of 4-BrC6H4, J = 8.5 Hz),
12.60 (s, 1H, NH); 13C NMR (DMSO-d6)
ppm: 40.57 (CH2), 121.93, 122.50, 124.10, 128.29,
δ
130.91, 132.38, 134.98, 149.83, 158.50, 193.30 (C=O); ESI MS m/z: 347 [M]+, 349 [M+2]+.
4.1.5.5. 2-((1H-benzo[d]imidazol-2-yl)thio)-1-(4-nitrophenyl)ethan-1-one (5e). White crystals
(yield 91%), m.p. 189-192°C (reported: 168-170°C [51]); IR (KBr, ν cm-1): 3413 (NH) and 1670
1
(C=O); H NMR (DMSO-d6)
δ
ppm: 5.12 (s, 2H, CH2), 7.11
̶ 7.15 (m, 2H, H-5 and H-6 of 2-
mercaptobenzimidazole), 7.41
̶ 7.44 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 8.29 (d,
2H, H-2 and H-6 of 4-NO2C6H4, J = 9.0 Hz), 8.39 (d, 2H, H-3 and H-5 of 4-NO2C6H4, J = 9.0 Hz),
13

13
ACCEPTED MANUSCRIPT
δ ppm: 50.19 (CH2), 110.19, 118.61, 122.16, 122.53,
12.73 (s, 1H, NH); C NMR (DMSO-d6)
124.37, 127.85, 130.32, 140.74, 147.66, 149.70, 150.59, 193.40 (C=O); ESI MS m/z: 314 [M+1]+.
4.1.5.6. 2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(4-hydroxyphenyl)ethan-1-one (5f). White crystals
(yield 90%), m.p. 241-244°C (reported: 248-250°C [52]); IR (KBr, ν cm-1): 3324 (NH) and 1648
(C=O); 1H NMR (DMSO-d6)
= 8.5 Hz), 7.07 7.11 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.36
and H-7 of 2-mercaptobenzimidazole), 7.54 (d, 2H, H-2 and H-6 of 4-OHC6H4, J = 8.5 Hz), 10.07
(s, 1H, OH), 12.59 (s, 1H, NH); 13C NMR (DMSO-d6)
ppm: 40.47 (CH2), 107.56, 111.89,
δ
ppm: 4.97 (s, 2H, CH2), 6.89 (d, 2H, H-3 and H-5 of 4-OHC6H4, J
7.38 (m, 2H, H-4
̶
̶
δ
115.85, 116.17, 119.13, 119.81, 120.74, 123.49, 130.18, 130.95, 131.55, 134.13, 148.66, 150.17,
156.97, 159.50, 193.77 (C=O); ESI MS m/z: 285 [M+1]+.
4.1.5.7. 2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(2-methoxyphenyl)ethan-1-one (5g). White crystals
(yield 85%), m.p. 195-198°C; IR (KBr, ν cm-1): 3412 (NH) and 1665 (C=O); 1H NMR (DMSO-d6)
δ
ppm: 3.89 (s, 3H, OCH3), 5.00 (s, 2H, CH2), 7.07 (t, 1H, H-5 of 2-OCH3C6H4, J = 7.5 Hz), 7.25
(d, 1H, H-3 of 2-OCH3C6H4, J = 7.5 Hz), 7.29 7.30 (m, 2H, H-5 and H-6 of 2-
mercaptobenzimidazole), 7.55 7.60 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 7.62 (t,
1H, H-4 of 2-OCH3C6H4, J = 7.5 Hz), 7.71 (d, 1H, H-6 of 2-OCH3C6H4, J = 7.5 Hz), 12.65 (s, 1H,
NH); 13C NMR (DMSO-d6)
ppm: 44.52 (CH2), 56.57 (OCH3), 113.20, 113.96, 121.16, 123.77,
̶
̶
δ
125.57, 130.80, 135.56, 150.72, 159.43, 193.71 (C=O); ESI MS m/z: 299 [M+1]+.
4.1.5.8. 2 -((1H-Benzo[d]imidazol-2-yl)thio)-1-(4-methoxyphenyl)ethan-1-one (5h). White crystals
(yield 86%), m.p. 228-230°C (reported: 217-218°C [53]); IR (KBr, ν cm-1): 3460 (NH) and 1669
(C=O); 1H NMR (DMSO-d6)
5, H-6 of 2-mercaptobenzimidazole and H-3, H-5 of 4-OCH3C6H4), 7.38
7 of 2-mercaptobenzimidazole), 8.05 (d, 2H, H-2 and H-6 of 4-OCH3C6H4, J = 7.0 Hz), 12.60 (s,
1H, NH); 13C NMR (DMSO-d6)
ppm: 40.47 (CH2), 56.11 (OCH3), 110.78, 114.52, 121.60,
δ
ppm: 3.87 (s, 3H, OCH3), 5.01 (s, 2H, CH2), 7.09
̶ 7.10 (m, 4H, H-
̶
7.45 (m, 2H, H-4 and H-
δ
122.07, 128.75, 131.30, 150.07, 164.01, 192.25 (C=O); ESI MS m/z: 299 [M+1]+.
4.1.5.9. 2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(3-hydroxy-4-methoxyphenyl)ethan-1-one (5i).
1
White crystals (yield 91%), m.p. 195-198°C; IR (KBr, ν cm-1): 3420 (NH) and 1654 (C=O); H
NMR (DMSO-d6)
δ
ppm: 3.87 (s, 3H, OCH3), 4.97 (s, 2H, CH2), 7.06 (d, 1H, H-5 of 3-OH-4-
7.12 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.43 7.44
OCH3C6H3, J = 8.0 Hz), 7.11
̶
̶
(m, 3H, H-4, H-7 of 2-mercaptobenzimidazole and H-2 of 3-OH-4-OCH3C6H3), 7.61 (d, 1H, H-6
13
of 3-OH-4-OCH3C6H3, J = 8.5 Hz), 9.51 (s, 1H, OH), 12.67 (s, 1H, NH); C NMR (DMSO-d6)
δ
ppm: 40.46 (CH2), 56.24 (OCH3), 111.79, 115.06, 121.86, 122.12, 128.84, 146.95, 150.15, 153.06,
192.29 (C=O); ESI MS m/z: 315 [M+1]+.
14

ACCEPTED MANUSCRIPT
4.1.5.10. 2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(3,4-dimethoxyphenyl)ethan-1-one (5j). White
crystals (yield 87%), m.p. 251-253°C (reported: >250°C [52]); IR (KBr, ν cm-1): 3413 (NH) and
1
1664 (C=O); H NMR (DMSO-d6)
δ ppm3.84 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.93 (s, 2H,
CH2), 7.15 (d, 1H, H-5 of 3,4-(OCH3)2C6H3, J = 8.5 Hz), 7.50 (s, 1H, H-2 of 3,4-(OCH3)2C6H3),
7.71 (d, 1H, H-6 of 3,4-(OCH3)2C6H3, J = 8.5 Hz), 7.09 7.11 (m, 2H, H-5 and H-6 of 2-
mercaptobenzimidazole), 7.38
̶ 7.40 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 12.73 (s,
1H, NH); ESI MS m/z: 312 [M]+.
4.1.5.11. 2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(benzo[d][1,3]dioxol-4-yl)ethan-1-one (5k). White
1
crystals (yield 89%), m.p. 185-188°C; IR (KBr, ν cm-1): 3415 (NH) and 1675 (C=O); H NMR
(DMSO-d6)
mercaptobenzimidazole and 2H, Ar-H), 7.39
mercaptobenzimidazole), 7.72 (t, 1H, Ar-H, J = 7.5 Hz), 12.71 (s, 1H, NH); 13C NMR (DMSO-d6)
ppm: 43.97 (CH2), 102.66, 105.69, 108.21, 108.68, 110.54, 116.54, 121.81, 122.57, 124.67,
δ
ppm: 4.80 (s, 2H, CH2), 6.16 (s, 2H, -O-CH2-O-), 7.10
̶ 7.11 (m, 4H, H-5, H-6 of 2-
̶
7.41 (m, 2H, H-4 and H-7 of 2-
δ
125.57, 132.88, 148.36, 150.27, 152.36, 191.94 (C=O); ESI MS m/z: 313 [M+1]+.
4.1.5.12. 2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(2,3,4-trimethoxyphenyl)ethan-1-one (5l). White
crystals (yield 93%), m.p. 166-169°C (reported: 118-119°C [54]); IR (KBr, ν cm-1): 3330 (NH) and
1
1654 (C=O); H NMR (DMSO-d6)
δ ppm: 3.76 (s, 3H, OCH3), 3.85 (s, 6H, OCH3), 5.04 (s, 2H,
CH2), 7.11
̶
7.13 (m, 2H, H-5 and H-6 of 2-mercaptobenzimidazole), 7.39
̶
7.42 (m, 4H, H-4, H-7
13
of 2-mercaptobenzimidazole and H-5, H-6 of 2,3,4-(OCH3)3C6H2), 12.75 (s, 1H, NH); C NMR
(DMSO-d6)
δ ppm: 40.56 (CH2), 56.59 2(OCH3), 60.67 (OCH3), 106.58, 121.92, 131.12, 142.73,
149.87, 153.29, 192.94 (C=O); ESI MS m/z: 359 [M+1]+.
4.1.5.13. 2-((1H-Benzo[d]imidazol-2-yl)thio)-1-(4-ethoxyphenyl)ethan-1-one (5m). White crystals
(yield 90%), m.p. 182-185°C; IR (KBr, ν cm-1): 3390 (NH) and 1661 (C=O); 1H NMR (DMSO-d6)
δ
ppm: 1.35 (t, 3H, CH2CH3, J = 7.0 Hz), 4.13 (q, 2H, CH2CH3, J = 7.0 Hz), 5.00 (s, 2H, CH2),
7.07 7.12 (m, 4H, H-5, H-6 of 2-mercaptobenzimidazole and H-3, H-5 of 4-OC2H5C6H4), 7.40
7.42 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 8.03 (d, 2H, H-2 and H-6 of 4-
OC2H5C6H4, J = 8.5 Hz), 12.73 (s, 1H, NH); 13C NMR (DMSO-d6)
ppm: 14.94 (OCH2CH3),
̶
̶
δ
40.57 (SCH2), 60.67 (OCH2CH3), 114.88, 121.81, 127.35, 128.59, 131.31, 150.09, 161.24, 163.32,
192.20 (C=O); ESI MS m/z: 313 [M+1]+.
4.1.5.14. 2-((1H-benzo[d]imidazol-2-yl)thio)-1-(furan-2-yl)ethan-1-one (5n). White crystals (yield
1
85%), m.p. 148-150°C; IR (KBr, ν cm-1): 3409 (NH) and 1684 (C=O); H NMR (DMSO-d6)
δ
ppm: 4.80 (s, 2H, CH2), 6.78 ̶ 6.79 (m, 1H, H-4 furan), 7.10 ̶ 7.12 (m, 2H, H-5 and H-6 of 2-
mercaptobenzimidazole), 7.40
̶
7.42 (m, 2H, H-4 and H-7 of 2-mercaptobenzimidazole), 7.65 (d,
15

1H, H-3 furan, J = 3.5 Hz), 8.08 (d, 1H, H-5 furan, J = 3.5 Hz), 12.70 (s, 1H, NH); 13C NMR
(DMSO-d6) ppm: 38.46 (CH2), 113.24, 120.06, 122.40, 127.60, 128.50, 139.70, 148.81, 149.69,
ACCEPTED MANUSCRIPT
δ
151.19, 182.16 (C=O); ESI MS m/z: 259 [M+1]+.
4.1.5.15. 2-((1H-benzo[d]imidazol-2-yl)thio)-1-(thiophen-2-yl)ethan-1-one (5o). White crystals
(yield 87%), m.p. 169-172°C (reported: 168-169°C [51]); IR (KBr, ν cm-1): 3419 (NH) and 1664
1
(C=O); H NMR (DMSO-d6)
δ
ppm: 4.96 (s, 2H, CH2), 7.10
̶
7.12 (m, 2H, H-5 and H-6 of 2-
7.42 (m, 2H, H-4 and H-7
of 2-mercaptobenzimidazole), 8.08 (d, 1H, H-3 thiophene, J = 5.0 Hz), 8.17 (d, 1H, H-5 thiophene,
J = 4.5 Hz), 12.63 (s, 1H, NH); 13C NMR (DMSO-d6)
ppm 40.58 (CH2), 111.26, 121.96, 129.40,
mercaptobenzimidazole), 7.30 (t, 1H, H-4 thiophene, J = 5.0 Hz), 7.40
̶
δ
134.86, 136.09, 142.48, 149.77, 186.93 (C=O); ESI MS m/z: 275 [M+1]+.
4.2 Biological evaluation
4.2.1. In vitro anti-proliferative activity
Anti-proliferative activity of the synthesized compounds 5a-o was evaluated at Stem Cell
Therapy and Tissue Reengineering Program, King Faisal Specialized Hospital and Research
Center, P.O. 3354, Riyadh 11211, Saudi Arabia. In vitro anti-proliferative activity was measured
by the cell growth inhibition assay. This assay was conducted by use WST-1 reagent for
determination of IC50 for each compound and the results are given in Table 2. HT-29 colon cancer
cell line was purchased from the American Type Culture Collection. Cells were maintained in
RPMI 1640 (Sigma), supplemented with 10% FBS (Lonza), 100 IU/mL penicillin, 100 mg/mL
streptomycin and 2 mmol/L L-glutamine (Sigma). Cells were seeded into 96-well plates at
0.4*104/well and incubated overnight. The medium was replaced with fresh one containing the
desired concentrations of the test compounds. After 48 h, 10 ꢀl of the WST-1 reagent were added
to each well and the plates were re-incubated for 4 h at 37°C. The amount of formazan was
quantified using ELISA reader at 450 nm.
4.2.2. CD133 expression measure by flow cytometry
HT-29 Cells harvested, washed and then the cells were stained with conjugated monoclonal
antibodies CD133-APC (Miltenyi Biotec). The analyses were performed on a BD LSR II™ (BD
Biosciences). Debris and cell clusters were excluded during side-scatter and forward-scatter
analyses.
Acknowledgements
16

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The authors would like to extend their sincere appreciation to the Deanship of Scientific
Research at King Saud University for its funding of this research through the Research Group
Project no. RGP-VPP-321.
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Captions page
Figure 1. An ORTEP diagram of final X-ray structure of compound 4b.
Figure 2. An ORTEP diagram of final X-ray structure of compound 5h.
Figure 3. Flow cytometric histograms of CD133 expression of untreated HT-29 cells and treated
cells with 5a-o compounds at 10 ꢀM. The analysis showed inhibitory effect of compounds 5b
and 5g-o on CD133 expression compared with untreated cells.
Scheme 1. Synthesis of the target 2-((benzimidazol-2-yl)thio)-1-arylethan-1-ones 5a-o and their
corresponding sulfate salts 4a-o.
Table 1. Crystallographic data and refinements for compounds 4b and 5h.
Table 2. In vitro anti-proliferative activity of compounds 5a-o against colon HT-29 cancer cell
line.
Table 3. Inhibition (%) of cell surface expression of CD133 on HT-29 cancer cells at 10 µM.
22

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Table 1. Crystallographic data and refinements for compounds 4b and 5h.
Compound
4b
5h
Crystal data
Chemical formula
Mr
C34H36N4O7S3
708.85
C16H16N2O3S
316.37
Crystal system, space group
Temperature (K)
a, b, c (Å)
Tetragonal, P43
150
16.0351 (8), 16.0351 (8),
Monoclinic, C2/c
150
22.2407 (15), 10.5049 (5),
13.3731 (6)
90.00
3438.6 (4)
4
12.8922 (7)
90.947 (3)
3011.7 (3)
8
â (°)
V (Å3)
Z
Radiation type
µ (mm−1)
Crystal size (mm)
Mo K
0.27
0.28 × 0.27 × 0.25
α
Mo Ká
0.23
0.57 × 0.41 × 0.35
Data collection
Diffractometer
Bruker APEX-II D8
venture diffractometer
Multi-scan
Bruker APEX-II D8
venture diffractometer
Multi-scan
Absorption correction
SADABS V2014/3
0.86, 0.92
SADABS V2014/3
0.880, 0.925
Tmin, Tmax
No. of measured, independent 16797, 16797, 11711
and observed [I > 2ó(I)]
75023, 4626, 3612
reflections
Rint
0.108
0.060
Refinement
R[F2 > 2ó(F2)], wR(F2), S
No. of reflections
No. of parameters
No. of restraints
0.065, 0.125, 1.05
16797
439
1
0.041, 0.101, 1.04
4626
213
0
H-atom treatment
H-atom parameters
constrained
H atoms treated by a
mixture of independent and
constrained refinement
0.37, −0.32
Äñmax, Äñmin (e Å−3)
0.56, −0.45

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Table 2. In vitro anti-proliferative activity of compounds 5a-o against colon HT-29 cancer
cell line.
Comp.
Ar
IC50 (µM)a
38.46±2.59
C6H5
5a
4-Me-C6H4
4-F-C6H4
4-Br-C6H4
4-NO2-C6H4
4-OH-C6H4
2-OCH3-C6H4
4-OCH3-C6H4
3-OH-4-OCH3-C6H3
3,4-(OCH3)2-C6H3
3,4-O-CH2-O-C6H3
2,3,4-(OCH3)3-C6H2
4-OC2H5-C6H4
furan-2-yl
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
34.46±3.02
78.70±7.08
42.95±3.13
57.00±5.14
19.22±1.12
36.12±3.08
25.19±2.02
33.05±2.32
19.11±1.11
36.83±3.05
18.83±1.37
NAb
24.57±2.06
23.93±2.18
15.83±1.63
thiophen-2-yl
-
5-fluorouracil
a IC50 values are the mean ± S.D. of three separate experiments.
b NA: Compounds having IC50 value > 100 µM.

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Table 3. Inhibition (%) of cell surface expression of CD133 on HT-29 cancer cells at 10 µM.
Comp.
Inhibition (%)±SD a
-
Untreated
NAb
5a
5b
5c
5d
5e
5f
40.00±2.35
NAb
NAb
NAb
NAb
5g
5h
5i
5j
5k
5l
5m
5n
5o
34.90±1.97
52.55±3.82
49.66±2.91
52.57±3.80
51.16±3.92
50.11±4.05
55.15±3.26
56.82±4.44
55.59±3.88
aValues are the mean ± S.D. of three separate experiments.
b

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Figure 1. An ORTEP diagram of final X-ray structure of compound 4b.

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Figure 2. An ORTEP diagram of final X-ray structure of compound 5h.

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Figure 3. Flow cytometric histograms of CD133 expression of untreated HT-29 cells and treated cells with
5a-o compounds at 10 µM. The analysis showed inhibitory effect of compounds 5b and 5g-o on CD133
expression compared with untreated cells.