Design, synthesis and anticancer/antiestrogenic activities of novel indole-benzimidazoles

Article abstract of DOI:10.1016/j.bioorg.2020.103929

Indole-benzimidazoles have recently gained attention due to their antiproliferative and antiestrogenic effects. However, their structural similarities and molecular mechanisms shared with selective estrogen receptor modulators (SERMs) have not yet been investigated. In this study, we synthesized novel ethylsulfonyl indole-benzimidazole derivatives by substituting the first (R₁) and fifth (R₂) positions of benzimidazole and indole groups, respectively. Subsequently, we performed ¹H NMR, ¹³C NMR, and Mass spectral and in silico docking analyses, and anticancer activity screening studies of these novel indole-benzimidazoles. The antiproliferative effects of indole-benzimidazoles were found to be more similar between the estrogen (E2) responsive cell lines MCF-7 and HEPG2 in comparison to the Estrogen Receptor negative (ER-) cell line MDA-MB-231. R₁:p-fluorobenzyl group members were selected as lead compounds for their potent anticancer effects and moderate structural affinity to ER. Microarray expression profiling and gene enrichment analyses (GSEA) of the selected compounds (R₁:p-fluorobenzyl: 48, 49, 50, 51; R₁:3,4-difluorobenzyl: 53) helped determine the similarly modulated cellular signaling pathways among derivatives. Moreover, we identified known compounds that have significantly similar gene signatures to that of 51 via queries performed in LINCS database; and further transcriptomics comparisons were made using public GEO datasets (GSE35428, GSE7765, GSE62673). Our results strongly demonstrate that these novel indole-benzimidazoles can modulate ER target gene expression as well as dioxin-mediated aryl hydrocarbon receptor and amino acid deprivation-mediated integrated stress response signaling in a dose-dependent manner.

Full text of DOI:10.1016/j.bioorg.2020.103929

Journal Pre-proofs
Design, Synthesis and Anticancer/Antiestrogenic Activities of Novel Indole-
benzimidazoles
Fikriye Zengin Karadayi, Murat Yaman, Mehmet Murat Kisla, Ayse G.
Keskus, Ozlen Konu, Zeynep Ates-Alagoz
PII:
S0045-2068(20)30673-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.103929
YBIOO 103929
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
27 March 2020
6 May 2020
8 May 2020
Please cite this article as: F. Zengin Karadayi, M. Yaman, M. Murat Kisla, A.G. Keskus, O. Konu, Z. Ates-
Alagoz, Design, Synthesis and Anticancer/Antiestrogenic Activities of Novel Indole-benzimidazoles, Bioorganic
Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103929
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Design, Synthesis and Anticancer/Antiestrogenic Activities of Novel Indole-benzimidazoles
Fikriye Zengin Karadayia,#, Murat Yamanc,#, Mehmet Murat Kislaa,$, Ayse G. Keskusc,$, Ozlen
Konub,c,d,*, Zeynep Ates-Alagoza,*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey, bDepartment of
Molecular Biology and Genetics, Bilkent University, 06800, Ankara, Turkey, cInterdisciplinary Program in
Neuroscience, Bilkent University, 06800, Ankara, Turkey, dUNAM-Institute of Materials Science and Nanotechnology,
Bilkent University, 06800, Ankara, Turkey
#,$Equal Contributors
*Corresponding Authors:
Zeynep Ates-Alagoz, zeynep.ates@pharmacy.ankara.edu.tr
Ozlen Konu, konu@fen.bilkent.edu.tr
Abstract
Indole-benzimidazoles have recently gained attention due to their antiproliferative and antiestrogenic
effects. However, their structural similarities and molecular mechanisms shared with selective estrogen
receptor modulators (SERMs) have not yet been investigated. In this study, we synthesized novel
ethylsulfonyl indole-benzimidazole derivatives by substituting the first (R1) and fifth (R2) positions of
benzimidazole and indole groups, respectively. Subsequently, we performed 1H NMR, 13C NMR, and Mass
spectral and in silico docking analyses, and anticancer activity screening studies of these novel indole-
benzimidazoles. The antiproliferative effects of indole-benzimidazoles were found to be more similar
between the estrogen (E2) responsive cell lines MCF-7 and HEPG2 in comparison to the Estrogen Receptor
negative (ER-) cell line MDA-MB-231. R1:p-fluorobenzyl group members were selected as lead compounds
for their potent anticancer effects and moderate structural affinity to ER. Microarray expression profiling
and gene enrichment analyses (GSEA) of the selected compounds (R1:p-fluorobenzyl: 48, 49, 50, 51; R1:3,4-
difluorobenzyl: 53) helped determine the similarly modulated cellular signaling pathways among
derivatives. Moreover, we identified known compounds that have significantly similar gene signatures to
that of 51 via queries performed in LINCS database; and further transcriptomics comparisons were made
using public GEO datasets (GSE35428, GSE7765, GSE62673). Our results strongly demonstrate that these
novel indole-benzimidazoles can modulate ER target gene expression as well as dioxin-mediated aryl
hydrocarbon receptor and amino acid deprivation-mediated integrated stress response signaling in a dose-
dependent manner.
Keywords: Indole-benzimidazole, molecular docking, comparative transcriptomics, estrogen signaling
1.Introduction
Breast cancer, which is among the most prevalent cancer types affecting women all over the world, can be
conventionally subtyped according to the presence of estrogen receptor (ER), progesterone receptor (PR),
and/or human epidermal growth factor receptor 2 (HER2/ERBB2) activity. These subtypes possess
differential characteristics regarding prognosis, incidence, therapeutic response and tumor aggressiveness.

The heterogeneous nature and adverse effects associated with therapeutic targeting of such diverse and
crucial pathways bring challenges into the therapy and hence makes the discovery of novel, more effective,
and subtype specific anticancer molecules invaluable [1].
Estrogens (E2) play crucial roles in breast cancer development, consequently a considerable amount of
research has been done either to block their synthesis or to modulate their activity [2]. Therefore, drugs that
function as antiestrogens in mammary tissue have been frequently used for the treatment of hormone-
dependent breast cancers. Nuclear receptors ERɑ and ERβ, through E2 binding, take part in multiple cellular
activities such as proliferation and differentiation. In addition, they can be found at an equilibrium [2-4] and
differentially regulate their downstream elements upon exposure to selective estrogen receptor modulators
(SERMs) [5]. Moreover, their expression levels differ among various tissues while the expression of ERɑ
is tightly associated with breast cancer physiology [6] as well as prognosis of breast tumors [7]. ERβ on the
other hand has been implicated in tumor suppression and breast carcinogenesis [8].
Multiple SERMs have been designed and assessed over the years for breast cancer treatment [9]. Moreover,
ERα and ERβ binding affinities and downstream effects of these SERMs might differ leading to variable
outcomes [5, 10, 11]. Accordingly, tamoxifen (Fig. 1) belonging to the first generation of SERMs has been
shown to significantly reduce the incidence of breast cancer. Raloxifene (Fig. 1) is a second-generation
SERM exhibiting a role similar to tamoxifen yet it functions as a pure antagonist in the uterus and a partial
agonist against tamoxifen-resistant breast cancers [12]. ICI 182,780 acts antagonistically in ER positive
(ER+) MCF-7 cells and can outperform raloxifene [13]. A third-generation SERM called bazedoxifene (Fig.
1) that has been introduced for the treatment of breast cancer and osteoporosis [14] is based on the
pharmacophore of raloxifene. Indole based derivatives (bazedoxifene, melatonin and KB9520), as well as
methyl and naphthyl-substituted benzimidazole derivatives also exhibit different modes of actions on breast
cancer cell lines some of which could be through actions similar to SERMs [2, 15-17]. Accordingly, a
combination of affinity studies with toxicological approaches as well as molecular profiling could be highly
beneficial to help identify more selective/effective breast cancer therapeutic agents [18-21].
OH
HO
S
OH
O
N
N
N
O
N
O
O
OH
Raloxifene
Bazedoxifene
Tamoxifen
Fig. 1. Chemical structure of tamoxifen, raloxifene and bazedoxifene.
Indole and benzimidazole rings, which are bioavailable molecules, constitute structures found in current
drugs. These two ring structures are also isosteres of DNA bases that carry purine and pyrimidine cores, and
they can as well be purine antimetabolites. For this reason, indole and benzimidazole rings are thought to
interact easily with biopolymers in biosystems [22]. Benzimidazole and its derivatives are effective agents
against cancer [23, 24], inflammation [25] and oxidative stress [26, 27] while also having antiviral [28] and
antibacterial [28-30] effects. Indole core has already been used to obtain novel derivatives with
antiproliferative activity [31, 32]. Aside from several crucial bioactive compounds (tryptophan, serotonin

and melatonin), some of the antineoplastic compounds, such as vinblastine sulfate, vincristine sulfate,
vinorelbine ditartrate and lanreotide carry indole ring systems [33]. In addition, phenyl-indole derivatives
have been shown to inhibit breast cancer development through different mechanisms [34-36]. Similarly,
recent studies on benzimidazoles reveal that different heterocycles at 2-position yield to potent anticancer
agents for various carcinoma cell lines [37, 38]. Furthermore, indole-benzimidazole hybrids have been
designed and synthesized by fusing the indole nucleus with benzimidazole to develop novel selective ER
modulators. These indole-benzimidazoles can represent novel potent ERα antagonist properties and provide
promising insight into the discovery of novel SERMs for the management of breast cancer [39]. For
instance, in our previous studies, we have discovered a small molecule with benzene sulfonyl structure
exhibiting selectivity toward breast cancer cells while sparing normal surrounding cells [40]. Also, benzene
sulfonyl structures have been shown to exhibit higher anticancer activity than doxorubicin in breast and
prostate cancers [41, 42]. However, the molecular mechanism of action of novel indole-benzimidazoles
carrying benzene sulfonyl structures has not yet been assessed. Because of the above and the need for new
compounds with better anticancer and antiestrogenic properties, we designed, synthesized and tested a series
of indole-benzimidazoles possessing ethylsulfonyl moiety (Scheme 1).
X
NH2
NO2
NH2
CAMPTOTHECIN
BAZEDOXIFENE
O
S O
C2H5
NH
O
S O
C2H5
e
t
r
h
u
y
l
b
e
n
e
z
e
n
e
s
u
l
f
o
n
y
l
s
t
c
t
u
r
C2H5
O
S
O
R2
N
N
Ar
N
N
H
R1
HN
Scheme 1. Scheme showing previous studies and starting point of the new syntheses.
2. Material and Methods
2.1. Chemistry
Melting points were determined with Buchi SMP-20 (BuchiLabortechnik, Flawil, Switzerland) and
Electrotermal 9100 capillary melting point apparatus (Electrothermal, Essex, U.K.) and are
uncorrected. The 1H NMR spectra in DMSO-d6 using Varian Mercury-400 FT-NMR spectrometer
(Varian Inc., Palo Alto, CA, USA), and the Mass spectra based on ESI(+) method using Waters ZQ
micromass LC-MS spectrometer (Waters Corporation, Milford, MA, USA) were recorded. For
elemental analysis we used LECO 932 CHNS (Leco-932, St. Joseph, MI, USA) instrument. Silica
gel 60 (40–63 mm particle size) was used for column chromatography.

2.1.1. General procedure for synthesis of 3-12
To a solution of 4-(Ethylsulfonyl)-1-chloro-2-nitrobenzene (2) (5 mmol) in ethanol (5 mL), amine
derivative (15 mmol) was added and heated under reflux, until the starting material was consumed
(determined by TLC, 8–48 h). Upon cooling the mixture, water was added. The resultant yellow
residue was crystallized from ethanol or purified by column chromatography (cc) by using a mixture
of hexane and ethyl acetate in varying concentrations as eluent (Table 1) [43].
2.1.2. General procedure for synthesis of 13-22
Compounds 3–12 (3.5 mmol) in EtOH (75 mL) reduced by hydrogenation using 40 psi of H2 and
10% Pd/C (40 mg) until cessation of H2 uptake to obtain the catalyst before filtering off on a bed of
celite and washing with EtOH; and concentrating the filtrate in vacuo [44]. The crude amine was
used without purification (Table 1).
R1
R1
HN
HN
NO2
NH2
Pd/C
H2
SO2C2H5
SO2C2H5
3-12
13-22
M.p. Yield
Comp. R1
1H NMR
Comp. Mass
(oC)
%
1.29 (t, 3H), 3.11 (q, 2H), 6.96 (d, J=9.2 Hz, 1H), 7.77 (dd, J=9.2 Hz, J=2 Hz, 1H),
8.68 (d, J=2 Hz, 1H).
1.12 (t, 3H), 3.03 (d, 3H), 3.28 (q, 2H), 7.18 (d, J=8.8 Hz, 1H), 7.89 (dd, J=9 Hz, J=2.4
Hz, 1H), 8.45 (d, J=2 Hz, 1H), 8.68 (d, 1H, NH).
1.09 (t, 3H), 1.2 (t, Hz, 3H), 3.25 (q, 2H), 3.46 (m, 2H), 7.22 (d, J=8.8 Hz, 1H), 7.83
(dd, J=9.2 Hz, J=2.4 Hz, 1H), 8.42 (d, J=1.6 Hz, 1H), 8.59 (t, 1H, NH).
0.94 ( t,3H), 1.12 (t, 3H), 1.64 (m, 2H), 3.28 (q, 2H), 3.40 (q, 2H), 7.25 (d, J=9.2Hz,
1H), 7.84 (dd, J=9.2 Hz, J=2.4 Hz, 1H), 8.44 (d, J=2.4 Hz, 1H), 8.61 (t,1H, NH).
0.92 (t,3H), 1.11 (t, 3H), 1.38 (m, 2H), 1.61 (m, 2H), 3.26 (q, 2H), 3.44 (q, 2H), 7.25
3
4
5
6
-H
143
88
13
14
15
16
201
215
229
243
-CH3
-C2H5
-C3H7
138
125
90
83
84
81
7
8
-C4H9
(d, J=8.8 Hz, 1H), 7.85 (dd, J=8.8 Hz, J=2 Hz, 1H), 8.44 (d, J=2 Hz, 1H), 8.60 (t,1H, 76
NH).
1.11 (t, 3H), 1.26 (m, 1H), 1.43 (m, 4H), 1.61 (d, 1H), 1.71 (m, 2H), 1.95 (m, 2H), 3.27
(q, 2H), 3.75 (m, 1H), 7.34 (d, J=9.6 Hz, 1H), 7.85 (dd, J=9.2 Hz, J=2.4 Hz, 1H), 8.32 154
(d, J=8 Hz, 1H), 8.55 (d, J=2.4 Hz, 1H).
82
80
17
18
257
283
-cyclohexyl
-benzyl
1.09 (t, 3H), 3.25 (q, 2H), 4.72 (d, 2H), 7.10 (d, J=9.2 Hz, 1H), 7.30 (m, 5H), 7.79 (dd,
J=9.4 Hz, J=2.4 Hz, 1H), 8.47 (d, J=2 Hz, 1H), 9.18 (t,1H, NH).
9
120
75
73
71
19
20
21
291
309
357
1.08 (t, 3H), 3.26 (q, 2H), 4.70 (d, 2H), 7.09 (d, J=9.2 Hz, 1H), 7.17 (m, 2H), 7.43 (m,
2H), 7.79 (dd, J=9.2 Hz, J=1.6 Hz, 1H), 8.46 (d, J=2.4 Hz, 1H), 9.19 (t,1H, NH).
10
11
114
-p-fluorobenzyl
1.08 (t, 3H), 3.26 (q,2H), 4.70 (d, 2H), 7.07 (d, J=9.2 Hz, 1H), 7.25 (m, 1H), 7.37-7.52
(m, 2H), 7.79 (dd, J=9.2 Hz, J=2 Hz, 1H), 8.46 (d, J=2.4 Hz, 1H), 9.20 (t,1H, NH).
-3,4-difluorobenzyl
121
1.28 (t, 3H), 3.09 (q, 2H), 4.58 (d, 2H), 6.85 (d, J=8.8 Hz, 1H), 7.18 (dd, J=8 Hz, J=1.2
12
-3,4-dichlorobenzyl Hz, 1H), 7.43 (m, 2H), 7.80 (dd, J=9 Hz, J=2 Hz, 1H), 8.73 (d, J=2 Hz, 1H), 8.79 145
76
22
390
(t,1H, NH)
Table 1. Physicochemical data for compounds 3-22
2.1.3. General procedure for synthesis of 23-59
A mixture of the appropriate o-phenylenediamine (1 mmol), related indole derivative (1 mmol) and
Na2S2O5 (40%) (2 mL) in EtOH (4 mL), was refluxed until starting materials were consumed
(determined by TLC, 4-12 h). The precipitate was obtained upon pouring the reaction mixture and

then filtering and washing. The residue was purified by column chromatography to give final
product [45].
2.1.3.1. 5-(ethylsulfonyl)-2-(1H-indole-3-yl)-1H-benzo[d]imidazole (23)
Compound 23 was prepared according to general methods starting from 4-ethylsulfonyl-benzene-
1,2-diamine (1.35 mmol, 0.27 g) and indole-3-carboxaldehyde (1.35 mmol, 0.195 g). The residue
was purified by cc using the mixture of ethyl acetate-hexane (1:1) as eluent to give a light yellow
solid, m.p. 157 oC (0.058 g, 13% yield).1H NMR (400 MHz, DMSO-d6): δ ppm 1.10 (t, 3H), 3.27
(q, 2H), 7.23 (dd, J=8.8 Hz, J=2 Hz, 1H), 7.53 (d, J=8.8 Hz, 1H), 7.64 (m, 2H), 7.83-8.26 (m, 3H),
8.51 (d, J=1.6 Hz, 1H), 11.91 (brd s, 1H, NH), 12.99 (brd d, 1H, NH).13C NMR (CD3OD): 8.02,
52.02, 106.93, 113.20, 114.42, 121.25, 123.03, 124.36, 125.67, 127.47, 128.20, 129.74, 132.78,
132.90, 136.59, 136.96, 154.36. MS (ESI+) m/z: 326.C17H15N3O2S.0.9H2O: C, 59.77; H, 4.95; N,
12.30; S, 9.38 and found C, 59.42; H, 5.23; N, 11.91; S, 9.10.
2.1.3.2. 2-(5-bromo-1H-indol-3-yl)-5-(ethylsulfonyl)-1H-benzo[d]imidazole (24)
Compound 24 was prepared according to general methods starting from 4-ethylsulfonyl-benzene-
1,2-diamine (0.87 mmol, 0.175 g) and 5-bromo-indole-3-carboxaldehyde (0.87 mmol, 0.195 g). The
residue was purified by cc using the chloroform/ethyl acetate (1:1) as eluent to give a white solid,
o
1
m.p. 192 C (0.128 g, 36% yield). H NMR (400 MHz, DMSO-d6): δ ppm 1.11 (t, 3H), 3.29 (q,
2H), 7.37 (d, J=8.4Hz, 1H), 7.51 (d, J=8.8 Hz 1H), 7.63-7.71 (m, 2H), 7.88 (m, 1H), 8.27 (s, 1H),
8.68(s, 1H), 11.97 (brd d, 1H, NH), 13.04 (brd d, 1H, NH). MS (ESI+) m/z: 404.Anal. calcd. For
C17H14BrN3O2S.H2O: C, 48.35; H, 3.82; N, 9.95; S, 7.59 and found C, 48.16; H, 3.86; N, 9.68; S,
7.45.
2.1.3.3. 5-(ethylsulfonyl)-2-(1H-indol-3-yl)-1-methyl-1H-benzo[d]imidazole (25)
Compound 25 was prepared according to general methods starting from N1-methyl-4-ethylsulfonyl-
benzene-1,2-diamine (0.99 mmol, 0.211 g) and indole-3-carboxaldehyde (0.99 mmol, 0.143 g). The
residue was purified by cc using the ethyl acetate /hexane (1:1) as eluent to give a white solid, m.p.
o
1
273 C (0.095 g, 28% yield). H NMR (400 MHz, DMSO-d6): δ ppm 1.11 (t, 3H), 3.31 (q, 2H),
4.06 (s, 3H), 7.19-7.28 (m, 2H), 7.54 (d, J=7.6 Hz, 1H), 7.72 (dd, J=8.4 Hz, J=2 Hz, 1H), 7.84 (d,
J=8.4 Hz, 1H), 8.13 (d, J=1.6 Hz, 1H), 8.20 (d, J=2.8 Hz, 1H), 8.44 (d, J=7.6Hz, 1H), 11.93 (brd
13
s, 1H, NH). C NMR (DMSO-d6): 7.43, 32.01, 49.77, 104.33, 110.39, 111.81, 118.21, 120.52,
120.61, 121.53, 122.54, 126.29, 127.76, 131.26, 136.08, 139.35, 142.54, 152.90. MS (ESI+) m/z:
340. Anal. calcd. For C18H17N3O2S-0.3 H2O: C, 62.69; H, 5.14; N, 12.18; S, 9.29 and found C,
62.57; H, 5.06; N, 12.21; S, 9.08.
2.1.3.4. 5-(ethylsulfonyl)-2-(5-methoxy-1H-indol-3-yl)-1-methyl-1H-benzo[d]imidazole (26)
Compound 26 was prepared according to general methods starting from N1-methyl-4-ethylsulfonyl-
benzene-1,2-diamine (0.92 mmol, 0.2197 g) and 5-methoxy-indole-3-carboxaldehyde (0.92 mmol,
0.161 g). The residue was purified by cc using the ethyl acetate /hexane (1:1) as eluent to give a
light yellow solid, m.p. 198 oC (0.125 g, 37% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 1.11
(t, 3H), 3.30 (q, 2H), 3.82 (s, 3H), 4.05 (s, 3H), 6.90 (dd, J=8.8 Hz, J=2 Hz, 1H), 7.43 (d, J=8.8
Hz, 1H), 7.71 (dd, J=8.4 Hz, J=2 Hz, 1H), 7.82 (d, J=8.4 Hz 1H), 7.97 (d, J=2.4 Hz 1H), 8.14 (d,
J=2 Hz, 1H), 8.15 (s, 1H), 11.80 (brd s, 1H, NH). 13C NMR (DMSO-d6): 7.44, 32.03, 49.77, 55.38,

103.24, 104.10, 110.30, 112.55, 112.69, 118.21, 120.55, 126.93, 128.08, 131.13, 131.19, 139.33,
142.53, 153.07, 154.53. MS (ESI+) m/z: 370. Anal. calcd. For C19H19N3O3S: C, 61.77; H, 5.18;
N, 11.37; S, 8.67 and found C, 61.21; H, 5.43; N, 11.52; S, 8.63.
2.1.3.5. 2-(5-chloro-1H-indol-3-yl)-5-(ethylsulfonyl)-1-methyl-1H-benzo[d]imidazole (27)
Compound 27 was prepared according to general methods starting from N1-methyl-4-ethylsulfonyl-
benzene-1,2-diamine (1.15 mmol, 0.247 g) and 5-chloro-indole-3-carboxaldehyde (1.15 mmol,
0.206 g). The residue was purified by cc using the ethyl acetate /hexane (1:2) as eluent to give a
light yellow solid, m.p. 264 oC (0.098 g, 23% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 1.11
(t, 3H), 3.32 (q, 2H), 4.07 (s, 3H), 7.26 (dd, J=8.8 Hz, J=2.4 Hz, 1H), 7.56 (d, J=8.4 Hz, 1H), 7.72
(dd, J=8.4 Hz, J=2 Hz, 1H), 7.85 (d, J=8.8 Hz, 1H), 8.17 (d, J=1.6 Hz, 1H), 8.30 (s, 1H), 8.50 (d,
J=2 Hz, 1H). 13C NMR (DMSO-d6): 7.44, 32.02, 49.70, 104.08, 110.42, 113.58, 118.33, 120.69,
120.71, 122.53, 125.20, 127.47, 129.35, 131.37, 134.74, 139.27, 142.41, 152.34. MS (ESI+) m/z:
374. Anal. calcd. For C18H16ClN3O2S.0,4 H2O: C, 56.73; H, 4.44; N, 11.02; S, 8.41; Found: C,
56.48; H, 4.38; N, 11.02; S, 8.26.
2.1.3.6. 2-(5-bromo-1H-indol-3-yl)-5-(ethylsulfonyl)-1-methyl-1H-benzo[d]imidazole (28)
Compound 28 was prepared according to general methods starting from N1-methyl-4-ethylsulfonyl-
benzene-1,2-diamine (1.65 mmol, 0.228 g) and 5-bromo-indole-3-carboxaldehyde (1.65 mmol,
0.238 g). The residue was purified by cc using the ethyl acetate /hexane (1:1) as eluent to give a
light yellow solid, m.p. 259 oC (0.052 g, 8% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 1.11
(t, 3H), 3.31 (q, 2H), 4.06 (s, 3H), 7.35 (d, J=8.4 Hz, 1H), 7.54 (d, J=8 Hz, 1H), 7.71 (d, J=8.8 Hz,
1H), 7.83 (d, J=8.4 Hz, 1H), 8.17 (s, 1H), 8.26 (s, 1H), 8.64 (s, 1H). 13C NMR (DMSO-d6): 7.54,
31.96, 49.72, 103.76, 110.30, 113.05, 114.14, 118.22, 120.59, 123.60, 124.81, 128.16, 129.38,
131.35, 135.22, 139.24, 142.43, 152.42. MS (ESI+) m/z: 418. Anal. calcd. For
C18H16BrN3O2S.0,35 H2O: C, 50.91; H, 3.96; N, 9.89; S, 7.55; Found: C, 50.85; H, 3.94; N, 10.27;
S, 7.45.
2.1.3.7. 1-ethyl-5-(ethylsulfonyl)-2-(1H-indol-3-yl)-1H-benzo[d]imidazole (29)
Compound 29 was prepared according to general methods starting from N1-ethyl-4-ethylsulfonyl-
benzene-1,2-diamine (1 mmol, 0.240 g) and indole-3-carboxaldehyde (1 mmol, 0.152 g). The
residue was purified by cc using the ethyl acetate /metanol (4:0.5) as eluent to give a white solid,
m.p. 254 oC(0.130 g, 37% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 1.13 (t, 3H), 1.42 (t, 3H),
3.32 (q, 2H), 4.56 (q, 2H), 7.19-7.28 (m, 2H), 7.54 (d, J=8 Hz, 1H), 7.73 (dd, J=8.4 Hz, J=1.6 Hz,
1H), 7.86 (d, J=8.8 Hz, 1H), 8.09 (d, J=1.6Hz, 1H), 8.15 (d, J=1.6 Hz, 1H), 8.40 (d, J=8 Hz, 1H),
11.90 (brd s, 1H, NH). 13C NMR (DMSO-d6): 7.36, 14.61, 49.69, 104.04, 110.39, 111.79, 118.30,
120.43, 120.70, 121.44, 122.46, 126.35, 126.73, 131.40, 136.04, 138.33, 142.65, 151.89. MS
(ESI+) m/z: 354. Anal. calcd. For C19H19N3O2S: C, 64.57; H, 5.41; N, 11.88; S, 9.07; Found: C,
64.67; H, 5.14; N, 11.57; S, 8.84.
2.1.3.8. 1-ethyl-5-(ethylsulfonyl)-2-(5-methoxy-1H-indol-3-yl)-1H-benzo[d]imidazole (30)
Compound 30 was prepared according to general methods starting from N1-ethyl-4-ethylsulfonyl-
benzene-1,2-diamine (1.22 mmol, 0.280 g) and 5-methoxy-indole-3-carboxaldehyde (1.22 mmol,
0.214 g). The residue was purified by cc using the ethyl acetate as eluent to give a light yellow solid,

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m.p. 249 C (0.165 g, 36% yield). H NMR (400 MHz, DMSO-d6): δ ppm 1.12 (t, 3H), 1.41 (t,
3H), 3.30 (q, 2H), 3.82 (s, 3H), 4.55 (q, 2H), 6.90 (dd, J=8.8 Hz, J=2.4 Hz, 1H), 7.43 (d, J=8.8 Hz,
1H), 7.71 (dd, J=8.8 Hz, J=1.6 Hz, 1H), 7.85 (d, J=8.8 Hz, 1H), 7.95 (d, J=2.4 Hz, 1H), 8.04 (d,
J=3.2 Hz, 1H), 8.15 (d, J=1.6 Hz,1H), 11.76 (brd d, 1H, NH). 13C NMR (DMSO-d6): 7.43, 14.66,
49.76, 55.35, 103.19, 103.87, 110.37, 112.57, 112.72, 118.36, 120.71, 127.05, 127.13, 131.14,
131.41, 138.39, 142.69, 152.13, 154.52. MS (ESI+) m/z: 384. Anal. calcd. For
C20H21N3O3S.0,9H2O: C, 60.10; H, 5.74; N, 10.51; S, 8.02; Found: C, 60.05; H, 5.75; N, 10.12; S,
7.85;
2.1.3.9. 2-(5-chloro-1H-indol-3-yl)-1-ethyl-5-(ethylsulfonyl)-1H-benzo[d]imidazole (31)
Compound 31 was prepared according to general methods starting from N1-ethyl-4-ethylsulfonyl-
benzene-1,2-diamine (1.5 mmol, 0.342 g) and 5-chloro-indole-3-carboxaldehyde (1.5 mmol, 0.269
g). The residue was purified by cc using the ethyl acetate as eluent to give a light yellow solid, m.p.
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280 C (0.273 g, 47% yield). H NMR (400 MHz, DMSO-d6): δ ppm 1.10 (t, 3H), 1.40 (t, 3H),
3.29 (q, 2H), 4.55 (q, 2H), 7.25 (dd, J=8.8 Hz, J=2 Hz, 1H), 7.54 (d, J=7.6 Hz, 1H), 7.71 (dd, J=8.4
Hz, J=1.6 Hz, 1H), 7.84 (d, J=8 Hz, 1H), 8.17 (t, 2H), 8.47 (d, J=2 Hz, 1H), 12.07 (brd s, 1H, NH).
13C NMR (DMSO-d6): 7.41, 14.63, 49.69, 103.92, 110.46, 113.50, 118.51, 120.79, 120.88, 122.62,
125.28, 127.57, 128.29, 131.62, 134.63, 138.34, 142.57, 151.31. MS (ESI+) m/z: 388.Anal. calcd.
For C19H18ClN3O2S: C, 58.83; H, 4.67; N, 10.83; S, 8.26; Found: C, 58.56; H, 4.67; N, 10.64; S,
8.13.
2.1.3.10. 2-(5-bromo-1H-indol-3-yl)-1-ethyl-5-(ethylsulfonyl)-1H-benzo[d]imidazole (32)
Compound 32 was prepared according to general methods starting from N1-ethyl-4-
ethylsulfonyl)benzene-1,2-diamine (1.8 mmol, 0.406 g) and 5-bromo-indole-3-carboxaldehyde (1.8
mmol, 0.401 g). The residue was purified by cc using the ethyl acetate as eluent to give a light
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yellow solid, m.p. 288 C (0.370 g, 48% yield). H NMR (400 MHz, DMSO-d6): δ ppm 1.10 (t,
3H), 1.40 (t, 3H), 3.29 (q, 2H), 4.55 (q, 2H), 7.36 (dd, J=8.8 Hz, J=2 Hz, 1H), 7.50 (d, J=9.2 Hz,
1H), 7.71 (dd, J=8.4 Hz, J=1.6 Hz, 1H), 7.84 (d, J=8.4 Hz, 1H), 8.17 (t, 2H), 8.62 (d, J=2 Hz, 1H),
12.08 (brd s, 1H, NH). 13C NMR (DMSO-d6): 7.36, 14.57, 49.63, 103.74, 110.40, 113.23, 113.89,
118.47, 120.84, 123.74, 125.10, 128.06, 128.14, 131.57, 134.81, 138.28, 142.50, 151.22. MS
(ESI+) m/z: 434. Anal. calcd. For C19H18BrN3O2S: C, 52.78; H, 4.19 ; N, 9.71; S, 7.41; Found:
C, 52.48; H, 3.98; N, 9.58; S, 7.39.
2.1.3.11. 5-(ethylsulfonyl)-2-(1H-indol-3-yl)-1-propyl-1H-benzo[d]imidazole (33)
Compound 33 was prepared according to general methods starting from N1-(propyl)-4-
ethylsulfonyl-benzene-1,2-diamine (1.04 mmol, 0.253 g) and indole-3-carboxaldehyde (1.04 mmol,
0.152 g). The residue was purified by cc using the chloroform/ethyl acetate/hexane (2:1:1) as eluent
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to give a white solid, m.p. 182 C (0.199 g,52% yield). H NMR (400 MHz, DMSO-d6): δ ppm
0.89 (t, 3H), 1.13 (t, 3H), 1.78-1.84 (m, 2H), 3,32 (q, 2H), 4.49 (t, 2H), 7.18-7.27 (m, 2H), 7.53-
7.55 (m, 1H), 7.72 (dd, J=8.4 Hz, J=1.6 Hz, 1H), 7.78 (d, J=8.8 Hz, 1H), 8.07 (s, 1H), 8.14 (d, J=1.6
Hz, 1H), 8.39 (m, 1H), 11.85 (brd s, 1H). 13C NMR (DMSO-d6): 7.40, 10.92, 22.36, 45.57, 49.74,
104.24, 110.73, 111.85, 118.37, 120.48, 120.71, 121.46, 122.49, 126.45, 126.75, 131.43, 136.05,
138.91, 142.55, 152.20. MS (ESI+) m/z: 368. Anal. calcd. For C20H21N3O2S: C, 65.22; H, 6.03;
N, 11.54; S, 8.68; Found: C, 65.37; H, 5.76; N, 11.44; S, 8.72.

2.1.3.12. 5-(ethylsulfonyl)-2-(5-methoxy-1H-indol-3-yl)-1-propyl-1H-benzo[d]imidazole (34)
Compound 34 was prepared according to general methods starting from N1-(propyl)-4-
ethylsulfonyl-benzene-1,2-diamine (1.06 mmol, 0.258 g) and 5-methoxy-indole-3-carboxaldehyde
(1.06 mmol, 0.187 g). The residue was purified by cc using the chloroform/ethyl acetate/hexane
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(2:1:1) as eluent to give a white solid, m.p. 159 C (0.175 g, 41% yield). H NMR (400 MHz,
DMSO-d6): δ ppm 0.90 (t, 3H), 1.13 (t, 3H), 1.79-1.84 (m, 2H), 3.32 (q, 2H), 3.82 (s, 3H), 4.48 (t,
2H), 6.90 (dd, J=8.4 Hz, J=2.4 Hz, 1H), 7.44 (d, J=8.4 Hz, 1H), 7.71 (dd, J=8,8 Hz, J=1.6 Hz, 1H),
7.87 (d, J=8.8 Hz, 1 H), 7.95 (d, J=2.4 Hz, 1H), 8.02 (d, J=2.8 Hz, 1H), 8.15 (d, J=2 Hz, 1H) 11.8
(brd s, 1H) 13C NMR (DMSO-d6): 7.40, 10.93, 22.34, 45.57, 49.73, 55.35,103.20, 103.99, 110.62,
112.55, 112.67, 118.35, 120.66, 127.08, 131.08, 131.36, 138.91, 142.51, 152.36, 154.51. MS(ESI+)
m/z: 398. Anal. calcd. For C21H23N3O3S: C, 63.46; H, 5.83; N, 10.57; S, 8.07; Found: C, 63.18; H,
5.99; N, 10.50; S, 7.93.
2.1.3.13. 5-(ethylsulfonyl)-2-(5-chloro-1H-indol-3-yl)-1-propyl-1H-benzo[d]imidazole (35)
Compound 35 was prepared according to general methods starting from N1-(propyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.82 mmol, 0.199 g) and 5-chloro-indole-3-carboxaldehyde
(0.82 mmol, 0.147 g). The residue was purified by cc using the chloroform/ethyl acetate/hexane
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(2:1:1) as eluent to give a white solid, m.p. 232 C (0.050 g, 15% yield). H NMR (400 MHz,
DMSO-d6): δ ppm 0.88 (t, 3H), 1.10 (t, 3H), 1.76-1.82 (m, 2H), 3.30 (q, 2H), 4.48 (t, 2 H), 7.24
(dd, J=8.8 Hz, J=2 Hz, 1H), 7.54 ( d, J=8.8 Hz, 1H), 7.70 (dd, J=8.8 Hz, J=1.6 Hz, 1H), 7.86 (d,
J=8.4 Hz, 1H), 8.15-8.17 (m, 2H), 8.46 (d, J=2 Hz, 1H), 12.03 (brd s, 1H). 13C NMR (DMSO-d6):
7.34, 10.87, 22.30, 45.48, 49.60, 103.97, 110.69, 113.45, 118.46, 120.71, 120.78, 122.54, 125.20,
127.56, 128.19, 131.50, 134.51, 138.82, 142.34, 151.49. MS (ESI+) m/z: 402. Anal. calcd. For
C20H20ClN3O2S: C, 59.77; H, 5.02; N, 10.46; S, 7.98; Found: C, 59.85; H, 5.20; N, 10.54; S, 7.77.
2.1.3.14. 5-(ethylsulfonyl)-2-(5-bromo-1H-indol-3-yl)-1-propyl-1H-benzo[d]imidazole (36)
Compound 36 was prepared according to general methods starting from N1-(propyl)-4-
ethylsulfonyl-benzene-1,2-diamine (1.11 mmol, 0.269 g) and 5-bromo-indole-3-carboxaldehyde
(1.11 mmol, 0.249 g). The residue was purified by cc using the chloroform/ethyl acetate/hexane
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(2:1:1) as eluent to give a white solid, m.p. 234 C (0.079 g, 16% yield). H NMR (400 MHz,
DMSO-d6): δ ppm 0.87 (t, 3H), 1.11 (t, 3H), 1.76-1.82 (m, 2H), 3.29 (q, 3H), 4.47 (t, 2H) 7.35 (d,
J=8 Hz, 1H), 7.49 (d, J=8.8 Hz, 1H), 7.7 (d, J=8.4 Hz, 1H), 7.84 (d, J=8.4 Hz, 1H), 8.11 (s, 1H),
8.16 (s, 1H), 8.6 (s, 1H), 11.98 (brd s, 1H). 13C NMR (DMSO-d6): 7.39, 10.92, 22.35, 45.54, 49.66,
103.93, 110.76, 113.28, 113.95, 118.52, 120.85, 123.77, 125.14, 128.09, 128.24, 131.59, 134.81,
138.87, 142.40, 151.52. MS (ESI+) m/z: 446. Anal. calcd. For C20H20BrN3O2S: C, 53.81; H, 4.51;
N, 9.41; S, 7.18; Found: C, 53.26; H, 4.51; N, 9.56; S, 6.98.
2.1.3.15. 5-(ethylsulfonyl)-2-(1H-indol-3-yl)-1-butyl-1H-benzo[d]imidazole (37)
Compound 37 was prepared according to general methods starting from N1-(butyl)-4-ethylsulfonyl-
benzene-1,2-diamine (0.89 mmol, 0.228 g) and indole-3-carboxaldehyde (0.89 mmol, 0.129 g). The
residue was purified by cc using the chloroform/ethyl acetate/hexane (2:1:1) as eluent to give a
white solid, m.p. 177 oC (0.044 g, 13% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 0.82 (t, 3H),
1.10 (t, 3H), 1.29 (m, 2H), 1.71-1.75 (m, 2H), 3.30 (q, 2H), 4.50 (t, 2H), 7.15-7.24 (m, 2H), 7.51 (d,

J=7.6Hz, 1H), 7.70 (dd, J=8.8 Hz, J=1.6 Hz, 1H), 7.85 (d, J=8.4 Hz, 1H), 8.06 (d, J=2.4 Hz, 1H),
8.12 (d, J=1.6 Hz, 1H), 8.34 (d, J=8 Hz, 1H), 11.84 (brd s, 1H). 13C NMR (DMSO-d6): 7.33, 13.42,
19.31, 31.02, 43.90, 49.65, 104.16, 110.62, 111.79, 118.31, 120.41, 120.67, 121.36, 122.43, 126.36,
126.71, 131.35, 135.96, 138.77, 142.50, 152.10. MS (ESI+) m/z: 382. Anal. calcd. For
C21H23N3O2S-0,2 H2O: C, 65.49; H, 6.12; N, 10.91; S, 8.30; Found: C, 65.20; H, 6.11; N, 11.10; S,
8.30.
2.1.3.16. 5-(ethylsulfonyl)-2-(5-chloro-1H-indol-3-yl)-1-butyl-1H-benzo[d]imidazole (38)
Compound 38 was prepared according to general methods starting from N1-(butyl)-4-ethylsulfonyl-
benzene-1,2-diamine (1.08 mmol, 0.277 g) and 5-chloro-indole-3-carboxaldehyde (1.08 mmol,
0.194g). The residue was purified by cc using the chloroform/ethyl acetate/hexane (2:1:1) as eluent
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to give a white solid, m.p. 221 C (0.065 g, 14% yield). H NMR (400 MHz, DMSO-d6): δ ppm
0.87 (t, 3H), 1.13 (t, 3H), 1.32-1.37 (m, 2H), 1.75-1.79 (m, 2H), 3.30 (q, 2H), 4.51 (t, 2H), 7.28 (dd,
J=8.4 Hz, J=2.4 Hz, 1H), 7.58 (d, J=8.4 Hz, 1H), 7.74 (dd, J=8.4 Hz, J=2 Hz, 1H), 7.88 (d, J=8.8
Hz, 1H), 8.2 (s, 2H), 8.48 (d, J=2.4 Hz, 1H), 12.08 (brd s, 1H). 13C NMR (DMSO-d6): 7.45, 13.58,
19.44, 31.14, 44.01, 49.69, 104.05, 110.76, 113.57, 118.57, 120.78, 120.90, 122.65, 125.31, 127.63,
128.33, 131.59, 134.60, 138.84, 142.45, 151.55. MS (ESI+) m/z: 416. Anal. calcd. For
C21H22ClN3O2S: C, 60.64; H, 5.33; N, 10.10; S, 7.71; Found: C, 60.23; H, 5.37; N, 10.38; S, 7.62.
2.1.3.17. 5-(ethylsulfonyl)-2-(5-bromo-1H-indol-3-yl)-1-butyl-1H-benzo[d]imidazole (39)
Compound 39 was prepared according to general methods starting from N1-(butyl)-4-ethylsulfonyl-
benzene-1,2-diamine (0.86 mmol, 0.220 g) and 5-bromo-indole-3-carboxaldehyde (0.86 mmol,
0.194g). The residue was purified by cc using the chloroform/ethyl acetate/hexane (2:2:1) as eluent
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to give a white solid, m.p. 235 C (0.040 g, 10% yield). H NMR (400 MHz, DMSO-d6): δ ppm
0.86 (t, 3H), 1.13 (t, 3H), 1.30-1.39 (m, 2H), 1.73-1.80 (m, 2H), 3.33 (q, 2H), 4.54 (t, 2H), 7.38 (dd,
J=8.8 Hz, J=1.6 Hz, 1H), 7.53 (d, J=8.4 Hz, 1H), 7.78 (dd, J=8.4 Hz, J=1.6 Hz, 1H), 7.88 (d, J=8.8
13
Hz, 1H), 8.17 (s, 1H), 8.2 (d, J=1.6 Hz, 1 H), 8.62 (d, J=2 Hz, 1H), 12.06 (brd s, 1H). C NMR
(DMSO-d6): 7.40, 13.52, 19.39, 31.09, 43.98, 49.66, 103.92, 110.72, 113.28, 113.96, 118.54,
120.86, 123.73, 125.14, 128.11, 128.23, 131.59, 134.81, 138.79, 142.42, 151.48. MS (ESI+) m/z:
460. Anal. calcd. For C21H22BrN3O2S: C, 54.78; H, 4.81; N, 9.12; S, 6.96; Found: C, 54.28; H,
4.67; N, 9.51; S, 6.96.
2.1.3.18. 1-cyclohexyl-5-(ethylsulfonyl)-2-(1H-indol-3-yl)-1H-benzo[d]imidazole (40)
Compound 40 was prepared according to general methods starting from N1-cyclohexyl-4-
ethylsulfonyl-benzene-1,2-diamine (0.94 mmol, 0.265 g) and indole-3-carboxaldehyde (0.94 mmol,
0.136 g). The residue was purified by cc using the chloroform/ethyl acetate (1:1) as eluent to give a
light yellow solid, m.p. 250 oC (0.263 g, 69% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 1.14
(t, 3H), 1.32-1.43 (m, 3H), 1.66 (d, 1H), 1.85-1.98 (m, 4H), 2.29-2.37 (m, 2H), 3.30 (q, 2H), 4.62-
4.68 (m, 1H), 7.15-7.26 (m, 2H), 7.55 (d, J=8.4 Hz, 1H), 7.68 (dd, J=8.8 Hz, J=1.6 Hz, 1H), 7.84
(d, J=2.8 Hz, 1H), 7.97 (d, J=8 Hz, 1H), 8.10 (d, J=8.8 Hz, 1H), 8.14 (d, J=1.6Hz, 1H), 11.79 (brd
s, 1H, NH). 13C NMR (DMSO-d6): 7.28, 24.32, 25.47, 30.49, 49.70, 56.58, 104.18, 111.96, 113.11,
118.84, 120.12, 120.29, 122.27, 126.46, 127.13, 131.29, 136.05, 136.85, 143.42, 152.21. MS
(ESI+) m/z: 408. Anal. calcd. For C23H25N3O2S.0,3H2O: C, 66.89; H, 6.25; N, 10.18; S, 7.74;
Found: C, 66.57; H, 5.95; N, 9.94; S, 7.97.

2.1.3.19. 1-cyclohexyl-5-(ethylsulfonyl)-2-(5-methoxy-1H-indol-3-yl)-1H-benzo[d]imidazole (41)
Compound 41 was prepared according to general methods starting from N1-cyclohexyl-4-
ethylsulfonyl-benzene-1,2-diamine (1.05 mmol, 0.297 g) and 5-methoxy-indole-3-carboxaldehyde
(1.05 mmol, 0.184 g). The residue was purified by cc using the chloroform/ethyl acetate (1:1) as
eluent to give a light yellow solid, m.p. 163 oC (0.061 g, 13% yield). 1H NMR (400 MHz, DMSO-
d6): δ ppm 1.13 (t, 3H), 1.32-1.44 (m, 3H), 1.66 (d, 1H), 1.85-1.98 (m, 4H), 2.30-2.36 (m, 2H), 3.31
(q, 2H), 3.77 (s, 3H), 4.60-4.66 (m, 1H), 6.89 (dd, J=8.8 Hz, J=2.4 Hz, 1H), 7.45 (m, 2H), 7.67 (dd,
J=8.8 Hz, J=1.6 Hz, 1H), 7.79 (d, J=2.8 Hz, 1H), 8.09 (d, J=8.8 Hz, 1H), 8.14 (d, J=1.6 Hz, 1H),
11.68 (brd s, 1H, NH). 13C NMR (DMSO-d6): 7.37, 14.04, 24.38, 25.56, 30.55, 49.72, 55.24, 56.63,
59.71, 101.51, 104.01, 112.74, 112.82, 113.17, 118.88, 120.31, 126.95, 127.66, 131.10, 131.25,
136.92, 143.49, 152.45, 154.43. MS (ESI+) m/z: 438. Anal. calcd. For C24H27N3O3S. 0,9H2O: C,
63.52; H, 6.40; N, 9.26; S, 7.05; Found: C, 63.60; H, 6.40; N, 8.86; S, 6.81.
2.1.3.20. 2-(5-chloro-1H-indol-3-yl)-1-cyclohexyl-5-(ethylsulfonyl)-1H-benzo[d]imidazole (42)
Compound 42 was prepared according to general methods starting from N1-cyclohexyl-4-
ethylsulfonyl-benzene-1,2-diamine (1.12 mmol, 0.315 g) and 5-chloro-indole-3-carboxaldehyde
(1.12 mmol, 0.200 g). The residue was purified by cc using the chloroform/ethyl acetate (2:1) as
eluent to give a white solid, m.p. 182 oC (0.201 g, 41% yield). 1H NMR (400 MHz, DMSO-d6): δ
ppm 1.14 (t, 3H), 1.38-1.43 (m, 3H), 1.67-2.00 (m, 5H), 2.30-2.35 (m, 2H), 3.33 (q, 2H), 4.63-4.69
(m, 1H), 7.26 (dd, J=8.8 Hz, J=2 Hz, 1H), 7.59 (d, J=9.2 Hz, 1H), 7.71-7.73 (m, 1H), 7.98 (d, J=2
Hz, 1H), 8.05 (d, J=1.6 Hz, 1H), 8.14-8.19 (m, 2H), 12.06 (brd s, 1H, NH). 13C NMR (DMSO-d6):
7.35, 24.33, 25.43, 30.42, 49.65, 56.94, 103.06, 113.64, 113.78, 118.48, 119.64, 120.88, 122.60,
125.31, 127.56, 129.22, 131.93, 134.68, 136.47, 141.93, 151.22. MS (ESI+) m/z: 442. Anal. calcd.
For C23H24ClN3O2S: C, 62.50; H, 5.47; N, 9.51; S, 7.25; Found: C, 62.23; H, 5.71; N, 8.94; S, 7.12.
2.1.3.21. 2-(5-bromo-1H-indol-3-yl)-1-cyclohexyl-5-(ethylsulfonyl)-1H-benzo[d]imidazole (43)
Compound 43 was prepared according to general methods starting from N1-cyclohexyl-4-
ethylsulfonyl-benzene-1,2-diamine (1.10 mmol, 0.311 g) and 5-bromo-indole-3-carboxaldehyde
(1.10 mmol, 0.246 g). The residue was purified by cc using the chloroform/ethyl acetate (1:1) as
eluent to give a white solid, m.p. 184 oC (0.335 g, 79% yield). 1H NMR (400 MHz, DMSO-d6): δ
ppm 1.13 (t, 3H), 1.40 (m, 3H), 1.67 (s, 1H), 1.86-1.98 (m, 4H), 2.29-2.35 (m, 2H), 3.31 (q, 2H),
4.63-4.69 (m, 1H), 7.36 (dd, J=8.4 Hz, J=1.6 Hz, 1H), 7.53 (d, J=8.4 Hz, 1H), 7.68 (dd, J=8.4 Hz,
J=2 Hz, 1H), 7.93 (d, J=2.8 Hz, 1H), 8.10 (d, J=8.8 Hz, 1H), 8.20 (dd, J=11.2 Hz, J=1.6 Hz, 2H),
13
11.99 (brd s, 1H, NH). C NMR (DMSO-d6): 7.27, 24.32, 25.41, 30.45, 49.65, 56.65, 103.78,
113.05, 113.21, 114.00, 118.91, 120.40, 122.70, 124.92, 128.30, 128.40, 131.47, 134.83, 136.76,
143.23, 151.43. MS (ESI+) m/z: 488. Anal. calcd. For C23H24BrN3O2S.0.45 H2O: C, 55.86; H,
5.07; N, 8.49; S, 6.48; Found: C, 55.85; H, 4.85; N, 8.15; S, 6.43.
2.1.3.22. 1-benzyl-5-(ethylsulfonyl)-2-(1H-indol-3-yl)-1H-benzo[d]imidazole (44)
Compound 44 was prepared according to general methods starting from N1-benzyl-4-ethylsulfonyl-
benzene-1,2-diamine (0.80 mmol, 0.230 g) and indole-3-carboxaldehyde (0.80 mmol, 0.115 g). The
residue was purified by cc using the chloroform/ethyl acetate (1:1) as eluent to give a white solid,

o
m.p. 252 C (0.066 g, 20% yield).1H NMR (400 MHz, DMSO-d6): δ ppm 1.14 (t, 3H), 3.31 (q,
2H), 5.85 (s, 2H), 7.09 (d, J=7.2 Hz, 2H), 7.19-7.34 (m, 5H), 7.49 (d, J=7.2 Hz, 1H), 7.68-7.78 (m,
13
3H), 8.19 (s, 1H), 8.44 (d, J=7.2 Hz, 1H), 11.77 (brd s, 1H, NH). C NMR (DMSO-d6): 7.38,
47.38, 49.68, 103.92, 110.74, 111.87, 118.48, 120.63, 121.09, 121.50, 122.63, 125.96, 126.33,
126.84, 127.48, 128.93, 131.90, 135.98, 136.57, 139.12, 142.69, 152.61. MS (ESI+) m/z: 416.Anal.
calcd. For C24H21N3O2S.0,5C4H8O2-0,5H2O: C, 66.65; H, 5.59; N, 8.97; S, 6.84; Found: C, 66.68;
H, 5.40; N, 8.98; S, 6.90.
2.1.3.23. 1-benzyl-5-(ethylsulfonyl)-2-(5-methoxy-1H-indol-3-yl)-1H-benzo[d]imidazole (45)
Compound 45 was prepared according to general methods starting from N1-benzyl-4-ethylsulfonyl-
benzene-1,2-diamine (0.70 mmol, 0.203 g) and 5-methoxy-indole-3-carboxaldehyde (0.70 mmol,
0.123 g). The residue was purified by cc using the chloroform/ethyl acetate (1:1) as eluent to give a
white solid, m.p. 296 oC (0.036 g, 12% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 1.13 (t, 3H),
3.32 (q, 2H), 3.80 (s, 3H), 5.83 (s, 2H), 6.88 (dd, J=8.8 Hz, J=2.4 Hz, 1H), 7.09 (d, J=7.2 Hz, 2H),
7.23-7.39 (m, 4H), 7.67-7.74 (m, 3H), 7.96 (d, J=2.4 Hz, 1H), 8.19 (d, J=1.2 Hz, 1H), 11.64 (brd
13
s, 1H, NH). C NMR (DMSO-d6): 7.89, 47.89, 60.18, 55.81, 103.62, 104.17, 111.15, 113.09,
113.29, 118.98, 121.52, 126.47, 127.44, 127.72, 127.96, 129.43, 131.47, 132.32, 137.08, 139.62,
143.19, 153.31, 155.07. MS (ESI+) m/z: 446. Anal. calcd. For C25H23N3O3S: C, 67.39; H, 5.20;
N, 9.43; S, 7.19; Found: C, 67.29; H, 5.45; N, 9.30; S, 7.16.
2.1.3.24. 1-benzyl-2-(5-chloro-1H-indol-3-yl)-5-(ethylsulfonyl)-1H-benzo[d]imidazole (46)
Compound 46 was prepared according to general methods starting from N1-benzyl-4-
ethylsulfonyl)benzene-1,2-diamine (0.85 mmol, 0.246 g) and 5-chloro-indole-3-carboxaldehyde
(0.85 mmol, 0.152 g). The residue was purified by cc using the chloroform/ethyl acetate (1:1) as
eluent to give a white solid, m.p. 265 oC (0.139 g, 36% yield). 1H NMR (400 MHz, DMSO-d6): δ
ppm 1.15 (t, 3H), 3.32 (q, 2H), 5.86 (s, 2H), 7.09 (d, J=7.6 Hz, 2H), 7.25-7.34 (m, 4H), 7.52 (d,
J=8.8 Hz, 1H), 7.70-7.78 (m, 2H), 7.8 (s, 1H), 8.25 (s, 1H), 8.51 (d, J=2 Hz, 1H), 11.95 (brd s, 1H,
13
NH). C NMR (DMSO-d6): 7.31, 47.33, 49.62, 103.71, 110.71, 113.48, 118.61, 120.70, 121.16,
122.66, 125.34, 125.90, 127.45, 128.24, 128.89, 132.05, 134.46, 136.38, 139.05, 142.53, 151.95.
MS (ESI+) m/z: 450. Anal. calcd. For C24H20ClN3O2S: C, 64.06; H, 4.48; N, 9.33; S, 7.12; Found:
C, 63.47; H, 4.46; N, 9.19; S, 7.05.
2.1.3.25. 1-benzyl-2-(5-bromo-1H-indol-3-yl)-5-(ethylsulfonyl)-1H-benzo[d]imidazole (47)
Compound 47 was prepared according to general methods starting from N1-benzyl-4-ethylsulfonyl-
benzene-1,2-diamine (0.83 mmol, 0.240 g) and 5-bromo-indole-3-carboxaldehyde (0.83 mmol,
0.185 g). The residue was purified by cc using the chloroform/ethyl acetate (1:1) as eluent to give a
white solid, m.p. 267 oC (0.226 g, 55% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 1.13 (t, 3H),
3.33 (q, 2H), 5.87 (s, 2H), 7.08 (d, J=7.2 Hz, 2H), 7.25-7.38 (m, 4H), 7.48 (d, J=8.8 Hz, 1H),7.69-
7.78 (m, 2H), 7.87 (s, 1H), 8.25 (s, 1H), 8.66 (d, J=1.6 Hz, 1H), 11.97 (brd s, 1H, NH). 13C NMR
(DMSO-d6): 7.38, 47.34, 49.63, 103.61, 110.75, 113.41, 113.97, 118.68, 121.23, 123.77, 125.25,
125.94, 127.50, 128.10, 128.13, 128.95, 132.05, 134.73, 136.43, 139.10, 142.55, 151.95. MS
(ESI+) m/z: 496. Anal. calcd. For C24H20BrN3O2S.0,3H2O: C, 57.67; H, 4.15; N, 8.40; S, 6.41;
Found: C, 57.66; H, 4.12; N, 8.17; S, 6.13.

2.1.3.26. 5-(ethylsulfonyl)-1-(4-fluorobenzyl)-2-(1H-indol-3-yl)-1H-benzo[d]imidazole (48)
Compound 48 was prepared according to general methods starting from N1-(4-fluorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.68 mmol, 0.210 g) and indole-3-carboxaldehyde (0.68 mmol,
0.099 g). The residue was purified by cc using the chloroform/ethyl acetate (2:1) as eluent to give a
white solid, m.p. 234 oC (0.080 g, 27% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 1.13 (t, 3H),
3.31 (q, 2H), 5.82 (s, 2H), 7.08-7.24 (m, 6H), 7.48 (d, J=7.2 Hz, 1H), 7.69 (dd, J=8.4 Hz, J=1.6
Hz, 1H), 7.71 (d, J=8.8 Hz, 1H), 7.80 (d, J=2.4 Hz, 1H), 8.17 (d, J=1.2 Hz, 1H), 8.41 (d, J=8.4 Hz,
13
1H), 11.73 (brd s, 1H, NH). C NMR (DMSO-d6): 7.31, 46.68, 49.62, 103.80, 110.66, 111.82,
115.69 (d, J=21.3 Hz), 118.46, 120.57, 121.07, 121.45, 122.57,126.26, 126.81, 128.04 (d, J=8.4
Hz), 131.90, 132.65 (d, J=3.1 Hz), 135.94, 138.95, 142.66, 152.47, 161.32 (d, J=242.3 Hz), 170.23.
MS (ESI+) m/z: 434. Anal. calcd. For C24H20FN3O2S.0,5C4H8O2: C, 65.39; H, 5.06; N, 8.79; S,
6.71; Found: C, 65.18; H, 5.02; N, 8.71; S, 6.68.
2.1.3.27. 5-(ethylsulfonyl)-1-(4-fluorobenzyl)-2-(5-methoxy-1H-indol-3yl)-1H-benzo[d]imidazole (49)
Compound 49 was prepared according to general methods starting from N1-(4-fluorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.54 mmol, 0.168 g) and 5-methoxy-indole-3-carboxaldehyde
(0.54 mmol, 0.095 g). The residue was purified by cc using the chloroform/ethyl acetate (2:1) as
eluent to give a light yellow solid, m.p. 260 oC (0.044 g, 18% yield). 1H NMR (400 MHz, DMSO-
d6): δ ppm 1.13 (t, 3H), 3.32 (q, 2H), 3.80 (s, 3H), 5.82 (s, 2H), 6.89 (dd, J=8.8 Hz, J=2.4 Hz, 1H),
7.10-7.18 (m, 4H), 7.39 (d, J=8.8 Hz, 1H), 7.67-7.77 (m, 3H), 7.96 (d, J=2.4 Hz, 1H), 8.19 (d,
13
J=1.6 Hz, 1H), 11.66 (brd s, 1H, NH). C NMR (DMSO-d6): 7.31, 46.68, 49.60, 55.24, 103.08,
103.55, 110.55, 112.53, 112.72, 115.68 (d, J=21.7 Hz), 118.43, 120.98, 126.87, 127.16, 128.03 (d,
J=8.4 Hz), 130.92, 131.84, 132.64 (d, J=3.5 Hz), 138.93, 142.64, 152.64, 154.51, 161.30 (d,
J=242.3 Hz). MS (ESI+) m/z: 464. Anal. calcd. For C25H22FN3O3S.0,2H2O: C, 64.27; H, 4.83;
N, 9.00; S, 6.85; Found: C, 64.02; H, 4.98; N, 8.69; S, 6.62.
2.1.3.28. 2-(5-chloro-1H-indol-3-yl)-5-(ethylsulfonyl)-1-(4-fluorobenzyl)-1H-benzo[d]imidazole (50)
Compound 50 was prepared according to general methods starting from N1-(4-fluorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.52 mmol, 0.162 g) and 5-chloro-indole-3-carboxaldehyde
(0.52 mmol, 0.094 g). The residue was purified by cc using the chloroform/ethyl acetate (2:1) as
eluent to give a white solid, m.p. 230 oC (0.097 g, 40% yield). 1H NMR (400 MHz, DMSO-d6): δ
ppm 1.12 (t, 3H), 3.32 (q, 2H), 5.83 (s, 2H), 7.08-7.15 (m, 4H), 7.24 (dd, J=8.8 Hz, J=2 Hz, 1H),
7.50 (d, J=8.4 Hz, 1H), 7.69 (dd, J=8.4 Hz, J=2 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.9 (s, 1H), 8.22
13
(d, J=1.6 Hz, 1H), 8.48 (d, J=2.4 Hz, 1H), 11.96 (brd s, 1H, NH). C NMR (DMSO-d6): 7.31,
46.44, 49.55, 103.61, 110.68, 113.49, 115.72 (d, J=21.1 Hz), 118.62, 120.69, 121.20, 122.66,
125.33, 127.41, 128.03 (d, J=7.7 Hz), 128.28, 132.04, 132.52 (d, J=2.6Hz), 134.43, 138.92, 142.51,
151.82, 161.32 (d, J=240 Hz). MS (ESI+) m/z: 468.Anal. calcd. For C24H19ClFN3O2S: C, 61.60;
H, 4.09; N, 8.98; S, 6.85; Found: C, 61.51; H, 4.10; N, 9.00; S, 6.86.
2.1.3.29. 2-(5-bromo-1H-indol-3-yl)-5-(ethylsulfonyl)-1-(4-fluorobenzyl)-1H-benzo[d]imidazole (51)
Compound 51 was prepared according to general methods starting from N1-(4-fluorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.66 mmol, 0.202 g) and 5-bromo-indole-3-carboxaldehyde
(0.66 mmol, 0.146 g). The residue was purified by cc using the chloroform/ethyl acetate/hexane
o
1
(2:1:1) as eluent to give a white solid, m.p. 240 C (0.099 g, 29% yield). H NMR (400 MHz,

DMSO-d6): δ ppm 1.13 (t, 3H), 3.32 (q, 2H), 5.86 (s, 2H), 7.10-7.18 (m, 4H), 7.38 (dd, J=8.8 Hz,
J=2 Hz, 1H), 7.48 (d, J=8.4 Hz, 1H), 7.71 (dd, J=8.4 Hz, J=1.6 Hz, 1H), 7.78 (d, J=8.4 Hz, 1H),
7.91 (d, J=2.8 Hz, 1H), 8.25 (d, J=1.6 Hz, 1H), 8.66 (d, J=2 Hz, 1H), 11.99 (brd s, 1H, NH). 13C
NMR (DMSO-d6): 7.31, 46.64, 49.64, 103.49, 110.68, 113.34, 113.92, 115.72 (d, J=21.2 Hz),
118.63, 121.20, 123.70, 125.20, 127.97, 128.02 (d, J=8.3 Hz), 128.113, 132.05, 132.51 (d, J=3.2
Hz), 134.67, 138.91, 142.50, 151.79, 161.32 (d, J=241.5 Hz). MS (ESI+) m/z: 514. Anal. calcd.
For C24H19BrFN3O2S: C, 56.26; H, 3.74; N, 8.20; S, 6.25; Found: C, 56.51; H, 4.02; N, 7.72; S,
5.84.
2.1.3.30. 5-(ethylsulfonyl)-1-(3,4-difluorobenzyl)-2-(1H-indol-3-yl)-1H-benzo[d]imidazole (52)
Compound 52 was prepared according to general methods starting from N1-(3,4-difluorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.64 mmol, 0.209 g) and indole-3-carboxaldehyde (0.64 mmol,
0.093g). The residue was purified by cc using the chloroform/ethyl acetate/hexane (2:1.5:1) as
eluent to give a white solid, m.p. 262 oC (0.175 g, 61% yield). 1H NMR (400 MHz, DMSO-d6): δ
ppm 1.11 (t, 3H), 3.31 (q, 2H), 5.82 (s, 2H), 6.77 (d, J=8.4 Hz, 1H), 7.16-7.36 (m, 4H), 7.48 (d,
J=7.6 Hz, 1H), 7.69 (dd, J=8.8 Hz, J=1.6 Hz, 1H), 7.75 (d, J=8.4 Hz, 1H), 7.80 (d, J=2.8 Hz, 1H),
8.18 (d, J=1.6 Hz, 1H), 8.41 (d, J=8.4 Hz, 1H), 11.77 (brd s, 1H). MS (ESI+) m/z: 452. Anal. calcd.
For C24H19F2N3O2S: C, 63.85; H, 4.24; N, 8.42; S, 7.10; Found: C, 63.61; H, 4.41; N, 8.97; S, 6.91
2.1.3.31. 5-(ethylsulfonyl)-1-(3,4-difluorobenzyl)-2-(5-methoxy-1H-indol-3-yl)-1H-benzo[d]imidazole
(53)
Compound 53 was prepared according to general methods starting from N1-(3,4-difluorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.71 mmol, 0.233 g) and 5-methoxy-indole-3-carboxaldehyde
(0.71 mmol, 0.125g). The residue was purified by cc using the chloroform/ethyl acetate/hexane
o
1
(2:1:1) as eluent to give a white solid, m.p. 271 C (0.151 g, 44% yield). H NMR (400 MHz,
DMSO-d6): δ ppm 1.11 (t, 3H), 3.30 (q, 2H), 3.79 (s, 3H), 5.80 (s, 2H), 6.78 (d, 1H), 6.87 (dd, J=9
Hz, J=2.4 Hz, 1H), 7.24-7.39 (m, 3H), 7.67-7.76 (m, 3H), 7.94 (d, 1H), 8.19 (d, J=1.2 Hz, 1H),
11.65 (brd s, 1H).MS (ESI+) m/z: 482. Anal. calcd. For C25H21F2N3O3S: C, 62.36; H, 4.40; N,
8.73; S, 6.66; Found: C, 61.94; H, 4.60; N, 8.61; S, 6.68.
2.1.3.32. 5-(ethylsulfonyl)-1-(3,4-difluorobenzyl)-2-(5-chloro-1H-indol-3-yl)-1H-benzo[d]imidazole
(54)
Compound 54 was prepared according to general methods starting from N1-(3,4-difluorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.89 mmol, 0.293 g) and 5-chloro-indole-3-carboxaldehyde
(0.89 mmol, 0.160 g). The residue was purified by cc using the chloroform/ethyl acetate/hexane
o
1
(2:1:1) as eluent to give a white solid, m.p. 258 C (0.209 g, 48% yield). H NMR (400 MHz,
DMSO-d6): δ ppm 1.11 (t, 3H), 3.30 (q, 2H), 5.83 (s, 2H), 6.76 (d, 1H), 7.22- 7.36 (m, 3H), 7.50
(d, J=8.4 Hz, 1H), 7.70 (dd, J=8.6 Hz, J=1.6 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.9 (d, J=1.6 Hz, 1H),
8.23 (d, J=1.6 Hz, 1H), 8.47 (d, J=2.4 Hz, 1H), 11.96 (brd s, 1H). MS (ESI+) m/z: 486. Anal. calcd.
For C24H18ClF2N3O2S: C, 59.32; H, 3.73; N, 8.65; S, 6.60; Found: C, 59.01; H, 3.74; N, 8.45; S,
6.45
2.1.3.33. 5-(ethylsulfonyl)-1-(3,4-diflorobenzyl)-2-(5-bromo-1H-indol-3-yl)-1H-benzo[d]imidazole
(55)

Compound 55 was prepared according to general methods starting from N1-(3,4-difluorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.72 mmol, 0.234 g) and 5-bromo-indole-3-carboxaldehyde
(0.72 mmol, 0.160 g). The residue was purified by cc using the chloroform/ethyl acetate/hexane
o
1
(2:2:1) as eluent to give a white solid, m.p. 248 C (0.141 g, 37% yield). H NMR (400 MHz,
DMSO-d6): δ ppm 1.14 (t, 3H), 3.33 (q, 2H), 5.86 (s, 2H), 6.80 (d, 1H), 7.29-7.39 (m, 3H), 7.49 (d,
J=8.8 Hz, 1H), 7.75 (dd, J=8.4 Hz, J=1.6 Hz, 1H), 7.79 (d, J=8.8 Hz, 1H), 7.91 (d, J=2.8 Hz, 1H),
8.26 (d, J=1.2 Hz, 1H), 8.65 (d, J=2 Hz, 1H), 12.00 (brd s, 1H). MS (ESI+) m/z: 532. Anal. calcd.
For C24H18BrF2N3O2S: C, 54.35; H, 3.42; N, 7.92; S, 6.04; Found: C, 54.43; H, 3.20; N, 7.84; S,
6.01.
2.1.3.34. 5-(ethylsulfonyl)-1-(3,4-diclorobenzyl)-2-(1H-indol-3-yl)-1H-benzo[d]imidazole (56)
Compound 56 was prepared according to general methods starting from N1-(3,4-dichlorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.44 mmol, 0.158 g) and indole-3-carboxaldehyde (0.44 mmol,
0.064 g). The residue was purified by cc using the chloroform/ethyl acetate (2:1) as eluent to give a
white solid, m.p. 247 oC (0.070 g, 33% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 1.15 (t, 3H),
3.34 (q, 2H), 5.88 (s, 2H), 6.90 (dd, J=8.2 Hz, J=2.4 Hz, 1H), 7.20-7.27 (m, 2H), 7.49-7.60 (m, 3H),
7.71 (dd, J=8.2 Hz, J=2 Hz, 1H), 7.78 (d, J=8.8 Hz, 1H), 7.82 (d, J=2.8 Hz, 1H), 8.21 (d, J=1.6 Hz,
1H), 8.45 (d, J=7.2 Hz, 1H), 11.81 (brd s, 1H).13C NMR (DMSO-d6): 7.30, 46.30, 49.61, 103.62,
110.58, 111.84, 118.52, 120.62, 121.45, 122.63, 126.04, 126.85, 128.37, 130.07, 130.80, 131.09,
131.41, 132.07, 135.95, 137.73, 138.88, 142.65, 152.43, 161.22. MS (ESI+) m/z: 484. Anal. calcd.
For C24H19Cl2N3O2S-0.5 H2O: C, 58.42; H, 4.08; N, 8.51; S, 6.49; Found: C, 58.30; H, 4.31; N,
8.78; S, 6.04.
2.1.3.35. 5-(ethylsulfonyl)-1-(3,4-diclorobenzyl)-2-(5-methoxy-1H-indol-3-yl)-1H-benzo[d]imidazole
(57)
Compound 57 was prepared according to general methods starting from N1-(3,4-dichlorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (1.01 mmol, 0.363 g) and 5-methoxy-indole-3-carboxaldehyde
(1.01 mmol, 0.177 g). The residue was purified by cc using the chloroform/ethyl acetate (2:1) as
1
eluent to give a white solid, m.p. 242 oC (0.065 g, 12% yield). H NMR (400 MHz, DMSO-d6): δ
ppm 1.11 (t, 3H), 3.31 (q, 2H), 3.79 (s, 3H), 5.83 (s, 2H), 6.89 (m, 2H), 7.37 (d, J=8.8 Hz, 1H), 7.46
(d, J=2 Hz, 1H), 7.53 (d, J=8.4 Hz, 1H), 7.67-7.76 (m, 3H), 7.95 (d, J=2.4 Hz, 1H), 8.19 (d, J=1.6
Hz, 1H), 11.65 (brd s, 1H).13C NMR (DMSO-d6): 7.31, 46.30, 49.61, 55.25, 103.08, 103.39,
110.50, 112.57, 112.79, 118.52, 121.15, 126.06, 126.87, 127.20, 128.37, 130.05, 130.94, 131.09,
131.40, 132.08, 137.74, 138.87, 142.64, 152.61, 154.57. MS (ESI+) m/z: 514. Anal. calcd. For
C25H21Cl2N3O3S C, 58.37; H, 4.11; N, 8.17; S, 6.23; Found: C, 58.04; H, 4.06; N, 7.83; S, 5.98.
2.1.3.36. 5-(ethylsulfonyl)-1-(3,4-dichlorobenzyl)-2-(5-chloro-1H-indol-3-yl)-1H-benzo[d]imidazole
(58)
Compound 58 was prepared according to general methods starting from N1-(3,4-dichlorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.56 mmol, 0.202 g) and 5-chloro-indole-3-carboxaldehyde
(0.56 mmol, 0.101 g). The residue was purified by cc using the chloroform/ethyl acetate (2:1) as
eluent to give a white solid, m.p. 278 oC (0.045 g, 15% yield). 1H NMR (400 MHz, DMSO-d6): δ
ppm 1.12 (t, 3H), 3.32 (q, 2H), 5.86 (s, 2H), 6.86 (dd, J=8.4 Hz, J=2 Hz, 1H), 7.24 (dd, J=8.8 Hz,
J=2 Hz, 1H),7.47-7.53 (m, 3H), 7.71 (dd, J=8.2 Hz, J=1.6 Hz, 1H),7.77 (d, J=8.4 Hz, 1H), 7.90 (d,

J=3.2 Hz, 1H),8.24 (d, J=1.6 Hz, 1H), 8.49 (d, J=2 Hz, 1H), 11.96 (brd s, 1H).13C NMR (DMSO-
d6): 7.31, 46.26, 49.55, 103.44, 110.61, 113.52, 118.69, 120.69, 121.35, 122.72, 125.39, 125.99,
127.39, 128.32, 128.39, 130.09, 131.11, 131.42, 132.21, 134.45, 137.60, 138.85, 142.51, 151.80.
MS (ESI+) m/z: 518. Anal. calcd. For C24H18Cl3N3O2S:C, 55.56; H, 3.50; N, 8.10; S, 6.18; Found:
C, 55.19; H, 3.35; N, 7.92; S, 5.98.
2.1.3.37. 5-(ethylsulfonyl)-1-(3,4-dichlorobenzyl)-2-(5-bromo-1H-indol-3-yl)-1H-benzo[d]imidazole
(59)
Compound 59 was prepared according to general methods starting from N1-(3,4-dichlorobenzyl)-4-
ethylsulfonyl-benzene-1,2-diamine (0.78 mmol, 0.280 g) and 5-bromo-indole-3-carboxaldehyde (0.78
mmol, 0.174 g). The residue was purified by cc using the chloroform/ethyl acetate (2:1) as eluent to give a
white solid, m.p. 156 oC (0.055 g, 12% yield). 1H NMR (400 MHz, DMSO-d6): δ ppm 1.13 (t, 3H), 3.33
(q, 2H), 5.89 (s, 2H), 6.88 (dd, J=8.4 Hz, J=1.6 Hz, 1H), 7.38 (dd, J=8.8 Hz, J=2 Hz, 1H),7.48-7.56 (m,
3H), 7.73 (dd, J=8.2 Hz, J=1.6 Hz, 1H),7.80 (d, J=8.8 Hz, 1H), 7.91 (d, J=3.2 Hz, 1H),8.27 (d, J=0.8 Hz,
1H), 8.66 (d, J=1.6 Hz, 1H), 12.00 (brd s, 1H). 13C NMR (DMSO- d6): 12.45, 47.26, 56.68, 103.65, 110.68,
113.47, 118.45, 120.69, 121.03, 122.65, 125.31, 125.87, 127.40, 127.42, 128.23, 128.88, 132.60, 134.41,
136.37, 139.00, 142.47, 151.90. MS (ESI+) m/z: 564. Anal. calcd. For C24H18BrCl2N3O2S-0,5H2O:C,
50.52; H, 3.35; N, 7.37; S, 5.60; Found: C, 50.14; H, 3.05; N, 7.12; S, 5.35.
2.2. Biological Activity Assays
2.2.1 Cytotoxic assays on human cancer lines
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) (Molecular Probes) was
used to measure cell viability. Cell lines (MCF-7, MDA-MB-231 and HEPG2) were seeded onto
96-well plates with 10000 cells/well in phenol-free media (DMEM-low-glucose, GIBCO). After 24
hours, the cells were exposed to compounds listed in Table 2 with different concentrations for
another day. All compounds were tested first at 0.25 µM, 2 µM, 16 µM and 40 µM doses using
MCF-7 cells. At each dose, percent cell viability was calculated in relationship to the DMSO control
for each concentration. Selected compounds were further studied using three different cell lines
(MCF-7, MDA-MB-231, and HEPG2) at eight different concentrations to calculate IC50 values.
Camptothecin was used as a positive control (0.25 and 2 µM) as there was a DMSO group for
calibration for each drug concentration. Cells were then fixed according to the manufacturer’s
instructions and intensities were measured spectrophotometrically (BIO-TEK/µQuant Universal
Microplate Spectrophotometer and BIO-TEK/KC junior software (v.1.418)). Percent viability was
calculated at each dose, separately, by dividing the blank subtracted average OD values of each
treated sample with the blank subtracted average ODs of corresponding DMSO treated counterparts;
and the resulting values were multiplied by 100 to obtain percentile viabilities. One-way ANOVA
followed by multiple comparisons (MATLAB R2016a) were used to test differences in group means
between the drug and DMSO control groups at each concentration. For clustering the MTT data,
percentiles were divided by 100 and logarithmically transformed at base two before performing
hierarchical clustering. For testing the significance of mean differences between groups from the
MCF-7 four-concentration screening, raw data from each plate of compounds were statistically
compared with respect to their corresponding DMSO control values at each concentration,
separately. For wider dose screens, IC50 values for each cell line were calculated using GraphPad

Prism (v. 6.05). Further statistical analyses were performed by using the viability values obtained
from MCF-7 and other cell lines, to determine any relationship between the viability and R1 or R2
status of the derivatives. n-way ANOVA analyses with log2 transformed viability values (in R
environment), and one-sided Wilcoxon-rank sum test and logIC50 (GRcalculator [46]) were
performed by taking into account the triplicate values of viability scores and corresponding
treatment concentrations. In GRcalculator analyses, sigmoidal fit and capping GR values below 1
were used. Additionally, two-way ANOVA with Tukey’s multiple comparisons was performed to
test the significance of difference between specific groups of compounds in GraphPad Prism (v.
6.05), by using cell viability values in triplicates. Principal component analysis (PCA) was used for
further investigating the effect of cell line and concentration; and log2 transformed cell viability was
used for the analysis.
2.3. Molecular docking analyses with multiple targets
ERα ligand-binding domain (PDB ID:1a52, resolution: 2.8 Å) file was obtained from the RCSB
Protein database website [47]. Additional proteins were tested to analyze the selectivity of
compounds against ERα. These compounds were Protein kinase C beta II (PDB ID:1pfq, resolution:
1.9 Å), glycogen synthase kinase 3 (PDB ID:1io9, resolution: 2.7 Å), Platelet-derived growth factor
receptor beta (PDB ID:3mjg, resolution: 2.3 Å), tubulin (PDB ID:1sa0, resolution: 3.58 Å) and
vEGFR2 kinase domain (PDB ID:2xir, resolution:1.5 Å), respectively. Proteins were prepared with
Maestro’s Protein Preparation Wizard [48] and the gridbox was prepared via the Receptor Grid
Generation module of Maestro [49]. Binding sites of co-ligands were used for gridbox generation.
2D builder was used to draw the ligands and same ligands were minimized and prepared with the
LigPrep module [50]. Tautomers and conformers were generated to maximize the number of
conformers. For all the complexes, bound ligands were used. Structures of these compounds were
procured from DrugBank [51], and were subjected to the identical LigPrep procedure. After this,
Ligand Docking process of the Glide program was initiated [52]. Precision was set to SP (Standard
precision) and Ligand Sampling was set to Flexible. 10 poses were generated for each ligand and
poses having the least binding energies amongst them were evaluated. 2D-interaction diagrams were
visualized via Ligand Interactions. Additionally, molecular descriptors of these compounds were
calculated via the QikProp module and assessed accordingly [53].
2.4. Microarray analyses of novel-indole benzimidazole derivatives and comparative
transcriptomics
MCF-7 cells were exposed to compounds 48, 49, 50, 51, and 53 for 24 hours at a dose of 20 µM.
Total RNA was extracted from each sample where DMSO control and 51, each, had two biological
replicates (RNeasy Mini Kit (QIAGEN)) before performing microarray experiments using HuGene
2.0 ST platform (Affymetrix). Data were normalized via Transcriptome Analysis Console Software
(V3.0.0.466) using default Affymetrix analysis parameters and rma using affy package [54]. For
differential expression analysis of 51 (n=2) in comparison with DMSO (n=2), the limma toolbox of
R was used [55]). Volcano plot of statistical significance against fold change between control and
51 treated MCF-7 cells was generated in MATLAB. For multiple probes hitting the same gene, the
probe with the lowest adjusted p-value was used.

GSEA was performed for each compound separately with default parameters to calculate the KEGG
pathway enrichment using MSigDB [56]. Significantly enriched pathways were chosen (false
discovery rate (FDR) q value <0.25); and commonly enriched KEGG pathways were reported.
LINCS database was used to identify compounds with the most and least similar expression profiles
to significantly up- and down-regulated gene lists obtained from 51 (top 150 and bottom 150 ranked
genes according to their logFC values) [57].
Limma analyses were performed between expression profiles of 48-49 and those of 50-51-53
compound series to identify the significantly differentially expressed genes at the adjusted p-value
< 0.05. Pathway enrichment was done on the significantly up- and down-regulated genes between
groups via STRING database with Reactome Pathways option while Venn Diagrams of unique and
variably affected pathways were also shown [58, 59].
For comparative transcriptomics, GSE35428, GSE7765 and GSE62673 were retrieved and
normalized with rma [60]. Differential expression analyses of normalized dataset were done using
limma between groups as follows: for GSE35428: E2, tamoxifen (4OHT), ICI 182,780,
Bazedoxifene or Raloxifene and EtOH (control) treatments; for GSE7765: Dioxin and DMSO
(control) treatments; and for GSE62673: AA depletion (AA (-)) and control samples. For GSE7765,
the results from hgu133A and hgu133B were merged. For multiple probes hitting the same gene,
the probe with the lowest adjusted p-value was used. For GSE35428 and for GSE62673 best jetset
probesets were selected for further analysis [61].
Venn diagrams were generated to represent the expression pattern (i.e., log2 fold changes) of the
significantly altered genes (N=2177, p-value < 0.05 between 51 & E2; N=111, p-value < 0.05
between 51 & Dioxin; N=1480, p-value < 0.05 between 51 & AA (-)). KEGG pathway enrichment
analysis was performed using the STRING database; and Venn diagrams were generated based on
the lists of significantly enriched pathways. Obtained diagrams were further utilized to form
contingency tables where counts of shared and unique upregulated or downregulated genes were
used in performing Fisher’s exact test in R.
Genes altered more than one-fold (FDR adj p-value < 0.05), in response to treatment with 51, were
selected for the correlation analysis. The Pearson’s correlation coefficient between each pair of
treatments was used for the hierarchical clustering and heatmap was performed using
ComplexHeatmap toolbox in R [62].
2.5 RT-QPCR assays for validation of treatment effects in MCF-7
Differential effects of candidate compounds on selected genes, known to be modulated by E2,
dioxin, AA depletion, and/or to have roles in cell cycle, DNA damage/repair, drug metabolism were
evaluated via RT-QPCR (LightCycler 480 II–Roche) in MCF-7 breast cancer cells exposed to 40
µM of each compound for 24 h. Following exposure, total mRNA was isolated and collected using
the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. Total RNA was then
converted into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific).
Logarithmically transformed relative expression (-ΔΔCt) levels were calculated based on TPT1 as
the reference gene and DMSO treatment as the control group. The results were analyzed via either

One-way ANOVA followed by Tukey’s multiple comparisons to evaluate the compound-based
effects or a Two-way ANOVA to assess dose-dependent effects (GraphPad Prism (v. 6.05)).
ComplexHeatmap toolbox in R was utilized; and GSE35428 (E2), GSE7765 (dioxin), and
GSE62673 (AA (-)) logFC data for the tested genes were annotated on top of the RT-QPCR data,
for comparative representation. A list of primers was given in Table A.1.
3. Results
3.1. Design and synthesis of indole-benzimidazole derivatives
The synthesis of compounds (Scheme 2) was initiated from 4-chloro-benzenesulfonyl chloride. 4-
(ethylsulfonyl)-1-chlorobenzene (1) and 4-(ethylsulfonyl)-1-chloro-2-nitrobenzene (2) were
synthesized according our previous publication [42]. To a solution of 4-(ethylsulfonyl)-1-chloro-2-
nitrobenzene (2) (5 mmol) in ethanol (5 mL), amine derivative (15 mmol) was added and heated
under reflux until the starting material was consumed (determined by TLC, 8–48 h). Upon cooling
the mixture, water was added. The resultant yellow residue was crystallized from ethanol or purified
by column chromatography (cc) by using a mixture of hexane and ethyl acetate in varying
concentrations as eluent (Table 1) [43]. 5-methoxy-indole-3-carboxaldehyde was synthesized from
5-methoxy-indole, N,N-dimethylformamide, and phosphorus oxychloride [63].
Compounds 3–12 (3.5 mmol) in EtOH (75 mL) were reduced by hydrogenation using 40 psi of H2
and 10% Pd/C (40 mg) until cessation of H2 uptake to obtain the catalyst before filtering off on a
bed of celite and washing with EtOH, and concentrating the filtrate in vacuo [44]. The crude amine
was used without purification (13-22) (see for details Experimental Section). A mixture of the
appropriate o-phenylenediamine (1 mmol), related indole derivative (1 mmol) and Na2S2O5 (40%)
(2 mL) in EtOH (4 mL), was refluxed until starting materials were consumed (determined by TLC,
4-12 h). The precipitate was obtained upon pouring the reaction mixture and then filtering and
washing. The residue was purified by cc to obtain the final product (23-59) [45]. The synthesis
details of the compounds were provided in the Experimental Section.

Scheme 2. Synthesis of new indole-benzimidazoles (23-59).
3.2. Biological evaluation of indole-benzimidazole derivatives
3.2.1 Anti-cancer activity of novel indole-benzimidazole compounds in MCF-7 cell line
All ethylsulfonyl derivatives were analyzed for their cytotoxicity using MTT assays. A four-dose
(0.25 µM, 2 µM, 16 µM and 40 µM) screening panel in MCF-7, an ER+ and TP53 (p53) wild-type
breast cancer cell line, was used to identify highly effective compounds. This allowed us to screen
large numbers of derivatives before pursuing selected compounds in more detail. As a result, the
primary anticancer activity screening in MCF-7 showed that most of the compounds exhibited
significance at one or more of the concentrations (Table 2). Hierarchical clustering of the compound
relative cell viabilities (at log2 scale) helped summarize similarities between activities across doses
(Fig. 2). Accordingly, molecules numbered 23, 35, 53, 36, 27, 29, 45, 37, 50 and 51 clustered
together, since they were highly effective at the highest dose, and one or more of the other three
concentrations. The remaining compounds were less effective than the above-mentioned
compounds with respect to their level of activity. In addition, compound 49 was highly effective at
the highest dose, i.e., 40 µM, yet was not effective at lower doses (Fig. 2). None of the molecules
exhibited activity at the lowest dose (0.25 µM).

Fig. 2. Hierarchical clustering of anti-cancer activity of the novel indole-benzimidazoles. Darker tones of
blue indicate stronger inhibition of cell growth. Euclidean distance and complete linkage were used for
clustering (MATLAB®).
R2
O
S
C2H5
N
N
O
HN
R1
% Relative viabilities
p-values
2 µM
No
23
R1
R2
40 µM
24.36
67.53
64.68
63.70
16 µM
67.57
85.44
72.12
65.96
2 µM
100
0.25 µM 40 µM
16 µM
0.25 µM
0.2498
-H
-H
-Br
-H
110.16
0.0000
0.0005
0.0391
0.0293
0.0002
1.0000
0.2914
0.9361
0.1572
24
25
26
-H
90.04
94.01
87.64
105.73
0.0055
0.9979
0.9238
0.9931
-CH3
-CH3
95.39
0.0000
-OCH3
111.74
0.0028

27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
-CH3
-Cl
40.38
69.58
44.16
72.80
66.03
58.80
69.52
69.76
26.90
32.85
43.76
89.88
62.15
42.19
64.12
63.80
68.96
89.26
39.58
80.60
79.31
59.55
52.00
40.52
45.69
73.53
33.53
65.13
66.23
84.14
86.39
73.06
81.31
48.75
79.32
45.09
74.36
79.80
58.13
98.64
99.09
68.60
48.06
55.21
81.38
88.91
83.28
85.42
82.62
80.98
95.42
54.85
109.71
85.37
92.02
92.06
46.74
46.37
92.77
43.94
70.69
75.12
91.57
90.78
72.70
71.81
60.56
94.45
77.02
93.02
85.35
85.41
101.48
91.76
92
86.70
0.0001
0.0000
0.0000
0.0112
0.0000
0.0012
0.0000
0.0000
0.0000
0.0000
0.0000
0.0760
0.0009
0.0005
0.0000
0.0025
0.0000
0.0957
0.0000
0.0700
0.0456
0.0012
0.0000
0.0000
0.0000
0.0190
0.0000
0.0000
0.0000
0.1014
0.0110
0.0001
0.0016
0.0000
0.0089
0.0000
0.0000
0.0102
0.0000
0.9871
0.9961
0.0003
0.0000
0.0000
0.0021
0.4355
0.8736
0.0008
0.0498
0.0001
0.5882
0.0000
0.3435
0.0000
0.4955
0.0124
0.0000
0.0000
0.8907
0.0000
0.0003
0.0009
0.6394
0.2980
0.0022
0.0018
0.0000
0.0426
0.0150
0.3659
0.0001
0.0862
0.9979
0.7629
0.7776
0.0089
0.3874
0.6895
0.9990
0.1878
0.3503
0.9865
0.1768
0.3247
0.0585
0.7116
0.2589
0.6626
0.1202
0.3942
0.5654
0.3750
0.0748
0.3661
0.5591
0.9074
0.3872
1.0000
0.7792
0.1723
0.5252
0.2749
0.9644
0.1001
0.9742
0.1720
0.4445
0.9773
0.3313
0.9919
0.2854
0.9993
0.9742
0.9805
0.9742
0.7885
0.9979
0.9010
0.9931
0.4536
0.9979
0.7249
0.3171
0.6023
0.9918
0.8098
0.4076
0.8262
0.9998
0.8039
0.2061
0.9997
-CH3
-Br
92.97
-C2H5
-C2H5
-C2H5
-C2H5
-C3H7
-C3H7
-C3H7
-C3H7
-C4H9
-C4H9
-C4H9
-H
87.82
-OCH3
-Cl
97.33
86.52
-Br
111.86
112.94
91.25
-H
-OCH3
-Cl
97.79
-Br
77.12
91.36
94.29
100.98
103.84
91.05
97.92
88.39
104.85
84.41
93.24
91.90
92.69
105.67
96.32
97.05
85.60
86.09
91.71
93.55
104.71
90.94
100.18
94.90
89.86
-H
101.55
110.80
101.15
108.25
102.61
105.45
93.54
-Cl
-Br
-H
-cyclohexyl
-cyclohexyl
-cyclohexyl
-OCH3
-Cl
-Br
-cyclohexyl
-benzyl
-H
99.40
-benzyl
-OCH3
-Cl
95.28
-benzyl
92.42
-benzyl
-Br
91.11
-p-fluoro benzyl
-p-fluoro benzyl
-p-fluoro benzyl
-p-fluoro benzyl
-3,4-difluorobenzyl
-3,4-difluorobenzyl
-3,4-difluorobenzyl
-3,4-difluorobenzyl
-3,4-dichlorobenzyl
-3,4-dichlorobenzyl
-3,4-dichlorobenzyl
-3,4-dichlorobenzyl
-H
93.24
-OCH3
-Cl
105.33
109.39
106.44
101.92
95.01
-Br
-H
-OCH3
-Cl
90.78
-Br
95.20
-H
99.27
-OCH3
-Cl
106.51
115.78
100.67
-Br
Table 2. Relative cell viability from four-dose screening with the ethylsulfonyl derivatives in MCF-7 cells.
p-values were calculated using One-Way ANOVA followed by multiple comparisons.
Synthesized compounds had either –H, -OCH3, -Cl, or –Br at their R2 position for each of the R1
(Table 2). Therefore, the most active molecule could be determined for each of the R1. According
to the n-way ANOVA, molecular substitutions by R1 and R2 resulted in alterations on cytotoxic
activities of the sulfonylethyl structures (p-value < 2e-16) where the R1 group was the major
predictor (p-value < 2e-16) of anticancer activity rather than the R2 group (p-value: 0.0885).
However, there was a significant interaction between R1 and R2 groups based on the cell viability
scores (R1x2 interaction p-value < 2e-16) suggesting that substitution on indoles could modify the
activity of benzimidazoles differentially. Analysis by GRcalculator tool indicated that p-
fluorobenzyl R1 group was one of the most effective R1 moiety outstanding from the rest of the
substitutions (p-value: 0.023) and other cyclic aromatic side chain groups (p-value: 0.012)
(

Fig. 3; Fig A.1). In addition to the p-fluorobenzyl, the substitutions of methyl (as in compound 27)
and propyl on R1 exhibited anti-proliferative trends.

Fig. 3. Log10(IC50) based representation and comparison of R1 carrying derivatives (GRcalculator tool was
used for this purpose and comparisons between all derivatives versus p-fluorobenzyl substituted compounds
were made with a built-in one-sided Wilcoxon rank-sum test)

3.2.2 Anti-cancer activity of selected compounds on different cell lines
Upon analysis of Table 2, we selected, for further screening, several compounds that were highly
effective in reducing viability at the highest dose 40 µM (compounds: 23 (24.36%); 27 (40.38%);
29 (44.16%); 35 (26.90%); 36 (32.85%); and 37 (43.76%)); 40 (42.19%); 45 (39.58%); 48
(59.55%); 49 (52.00%); 50 (40.52%); 51(45.69%); 53 (33.53%) and a control molecule with
relatively less cytotoxic activity (compound 46 (80.60%)). Among these, 48-51 spanning the full -
p-fluorobenzyl series exhibited similar activity at 40 µM whereas 50 and 51 were also significantly
antiproliferative at a relatively lower concentration of 16 µM along with another related compound
53 containing 3,4-difluorobenzyl group. In the wider dose panel, IC50 values of these 13 molecules
across multiple cell lines (Table 3) were studied along with n-way ANOVA. Overall, R1 chain (p-
value < 2e-16) had significant effects on viability while the effect of the R2 side chain was also
significant (p-value < 2e-16) and varied depending on the type of R1 (R1x2 interaction p-value < 2e-
16). Moreover, there was also a significant cell line effect (p-value: 2.62e-08) as well as a treatment
effect (p-value < 2e-16). Additional analyses with two-way ANOVA and multiple comparison tests
have
implied
possible
trends
by
cell
line
and
R2
(Table
3;
Fig. 4; Fig. A. 3; Fig. A. 4). Cell line specific effects in response to treatments were observable via
Principal Component Analysis (PCA) where both MCF-7 and HEPG2 lines interestingly yielded
parallel
(
profiles
in
comparison
to
MDA-MB-231

Fig. 4). PCA showed that E2 responsive cell lines MCF-7 and HEPG2 were more similar to each
other than they were to the ER- MDA-MB-231 cells at lower concentrations (up to 16 μM) while
at the highest dose tested (40 μM) each cell line assumed a relatively distinct response profile. In
particular, the compound 53 exhibited low IC50 values for the TP53 wild-type MCF-7 and HEPG2
cells (19.23 µM and 24.10 µM, respectively) while it was not as effective in MDA-MB-231, a cell
line with a mutant TP53 allele. In accord with two-way ANOVA comparisons, most of the candidate
compounds exhibited a cell-line dependency, but not compound 37 with butyl (R1) and -H (R2)
substitutions (Table 3; Fig. A. 4). Nonetheless, GRcalculator assessments showed that MCF-7 was
the cell line that seemed to be affected the most by the compounds, whereas -Br carrying R2 moieties
on the Table 2 compounds were also observed to have more effect on viability (Fig. A. 2; Fig. A.
3). After obtaining the toxicity data, we continued with docking studies and transcriptomic analyses
in order to get an understanding on the mechanisms of action.
Cell line
effect
Cell line
effect
IC50
IC50
Comp.
Comp.
MCF-7
42.9536
5.71
MDA-MB-231 HEPG2
p-value
< 0.0001
MCF-7
32.2849
43.4510
MDA-MB-231 HEPG2
p-value
< 0.0001
23
27
51.4043
NA
47.9733
NA
45
46
22.3872
10.9396
9.9540
89.54
< 0.0001
< 0.0001
< 0.0001
< 0.0001
29
35
36
37
40
89.3305
54.4503
15.7398
30.4089
40.2717
NA
73.7904
32.7341
7.8163
31.5500
NA
48
49
50
51
53
27.2270
39.5367
18.0717
35.1560
19.2309
20.8450
44.2588
36.1410
38.2825
NA
78.70
< 0.0001
< 0.0001
0.3538 (ns)
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
126.7652
49.8884
66.6807
76.9130
41.11
58.6138
17.2584
24.0991
Table 3. IC50 values and two-way ANOVA cell line specific p-value for each selected candidate tested on
MCF-7, MDA-MB-231 and HEPG2 cells (NA: Unmeasurable IC50 values, ns: not significant).

Fig. 4. PCA representation on cell viabilities of the cell lines upon exposure to varying concentrations of
novel derivatives.
3.3. Molecular docking studies
Structurally related R1 groups with relatively high potencies were taken into docking analyses. On the basis
of the literature on indoles and benzimidazoles as well as PCA clusters in this study, we primarily focused
on ERα, and assessed dockings of R1:p-fluorobenzyl derivatives and 53. Compound based statistical
comparisons between the cell lines were also in accord with these observations (Fig. A. 2). Our indole-
benzimidazole derivatives tended to exhibit increased affinity to ERα, vEGFR2, and tubulin rather than the
other ones which were discussed in section 2.3, such as Protein kinase C beta II, glycogen synthase kinase
3, Platelet-derived growth factor receptor beta.

Fig. 5. 2D diagram of aminoacid interactions of bazedoxifene with ERα ligand-binding domain.
Hydrophobic interactions are shown as green, whereas the red line represents Pi-cation interactions. H-bond
interactions are depicted as purple. Red-blue represents salt bridge interaction.
Based on the structural analysis (Fig. 5) ERα ligand binding domain mainly consists of hydrophobic
residues. Therefore, utilization of hydrophobic moieties such as indole and benzimidazole may play a key
role in inhibiting or activating this receptor. The binding mode of 4-hydroxytamoxifen with ERα suggested
that a hydrogen donator group could be important for H-bond interaction with polar residue Gly521 in this
cavity. This interaction’s distance was 2.28 Å. In the literature, these residues including Glu353, Arg394,
Phe404 and Lys529 take part in the modulation of this receptor. Hydrophobic interactions with Phe404 and
Trp383, H-bond interactions with Glu353 and Arg394, also a salt bridge interaction with Asp351 are
important according to both bazedoxifene and 4-hydroxytamoxifen’s patterns [64]. List of molecular
properties and ERα docking energies for all compounds were given in Table A.10.

51
53
Fig. 6. 2D interaction diagrams of the two most potent compounds against MCF-7 and microarray analyses.
Brown arrow indicates halogen bond interaction and purple one indicate hydrogen bond interaction, whilst
green line represents Pi-Pi steric interaction.
One of the prominent compounds that stood out in transcriptomic analyses, compound 51, created halogen
bond interactions with both Glu353 and Arg394. In the case of the another potent ligand 53, Phe404 joins a
Pi-Pi interaction with an indole ring while the sulfonyl group acts as the hydrogen bond donor (Fig. 6). Both
ligands have provided necessary interactions in the reference study. Their energy values were relatively
close to that of standard compound bazedoxifene.
According to the glide docking score results in Table 4, compounds 48, 49 and 51 have exhibited favorable
affinity value against ER when compared with those against tubulin and vEGFR2.
vEGFR2 kinase
Compounds
ERα
Tubulin
domain
-6.348
-6.786
-6.435
-6.131
-6.813
-
48
-7.776
-5.851
49
50
-7.726
-5.575
Unsuccessful binding
Unsuccessful binding
51
-7.802
-5.458
53
-6.610
-5.662
Vincristine
Tivozanib
Bazedoxifene
-
-
-8.1
-
-
-10.265
-
-9.852
Table 4. Data showing the glide scores of microarray compounds against different proteins.

3.4. Gene level alterations upon exposure to indole-benzimidazoles
3.4.1. Transcriptomics analysis of Compounds 48-51 and 53
Based
on
Fig. 3, derivatives with p-fluorobenzyl and the structurally related compound 53 represented strong
candidates for understanding the molecular mechanisms of action of the effective novel indole-
benzimidazoles. For that purpose, we initiated gene level analyses in a parallel line with molecular docking
studies. Limma analysis of expression data obtained upon exposure to compound 51 demonstrated that
MCF-7 transcriptome was significantly modulated leading to upregulation and downregulation of a
considerable number of genes (
Fig. 7; Table 5).