Structure-based drug design, synthesis, In vitro, and In vivo biological evaluation of indole-based biomimetic analogs targeting estrogen receptor-α inhibition

Article abstract of DOI:10.1016/j.ejmech.2019.01.068

Offering novel scaffolds targeting estrogen receptor creates huge necessity to overcome the evolving resistance developed by tumors. Structure-based drug design coupled with ring opening strategy of the steroids skeleton revealed the potential of indole-based analogs to be synthesized targeting the ligand binding domain of estrogen receptor-α. In vitro studies revealed the potential of the total sub-classes of the synthesized analogs to show anti-proliferative activity against estrogen receptor-dependent cancer cell lines at IC₅₀ ranging from 28.23 to 57.13 μM. This was further validated by evaluating the potential of the synthesized analogs to compete along with estradiol via ER-α ELISA assay to show inhibitory profile at IC₅₀ ranging from 1.76 to 204.75 nM. Two analogs (YMA-005 and YMA-006) showed significant reduction in tumor size at two dose levels with extensive degeneration and necrosis. Both YMA-005 and YMA-006 showed in-situ reduction of ER-α Immunohistochemical expression at both dose levels. Ultimately, novel analogs of indole-based biomimetic of estrone scaffolds were offered as estrogen receptor-α inhibitors.

Full text of DOI:10.1016/j.ejmech.2019.01.068

Accepted Manuscript
Structure-based drug design, synthesis, In vitro, and In vivo biological evaluation of
indole-based biomimetic analogs targeting estrogen receptor-α inhibition
Moataz S. Hendy, Aya A. Ali, Lubna Ahmed, Reham Hossam, Alaa Mostafa,
Mohamed M. Elmazar, Bassem H. Naguib, Yasmeen M. Attia, Mahmoud Salama
Ahmed
PII:
S0223-5234(19)30088-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.068
EJMECH 11075
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 November 2018
Revised Date: 14 January 2019
Accepted Date: 28 January 2019
Please cite this article as: M.S. Hendy, A.A. Ali, L. Ahmed, R. Hossam, A. Mostafa, M.M. Elmazar, B.H.
Naguib, Y.M. Attia, M.S. Ahmed, Structure-based drug design, synthesis, In vitro, and In vivo biological
evaluation of indole-based biomimetic analogs targeting estrogen receptor-α inhibition, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.01.068.
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ACCEPTED MANUSCRIPT
Structure-Based Drug Design, Synthesis, In vitro, and In vivo Biological Evaluation of
Indole-Based Biomimetic Analogs Targeting Estrogen Receptor-α Inhibition
1
, 2
2, 3
4
4
4
Moataz S. Hendy , Aya A. Ali
, Lubna Ahmed , Reham Hossam , Alaa Mostafa ,
2
, 3
1,2, 5
2, 3*
Mohamed M. Elmazar , Bassem H. Naguib
, Yasmeen M. Attia
, Mahmoud Salama
1
, 2, 6*
Ahmed
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The British University in Egypt,
El-Sherouk, Cairo, Egypt
2
Center of Drug Research and Development (CDRD), The British University in Egypt, El-Sherouk, Cairo,
Egypt
3
Department of Pharmacology, Faculty of Pharmacy, The British University in Egypt,
El-Sherouk, Cairo, Egypt
4
Faculty of Pharmacy, The British University in Egypt, El-Sherouk, Cairo, Egypt
5
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
6
Department of Internal Medicine, Division of Cardiology, University of Texas Southwestern Medical
Center, Dallas, Texas, USA
*Corresponding Authors: Mahmoud Salama Ahmed; Email Address: Mahmoud.salama@bue.edu.eg,
Yasmeen M. Attia; Email Address: Yasmeen.attia@bue.edu.eg
Highlights:
•
•
Indole-based analogs mimicking steroids scaffold were designed and synthesized.
All newly synthesized analogs enrolled into in vitro screening against estrogen receptor
dependent cancer cell lines and estrogen receptor binding assays.
•
•
YMA-005 and YMA-006 showed significant reduction in the tumor size in the animal
model of Ehrlich ascites carcinoma solid tumor at two dose levels.
YMA-005 and YMA-006 showed reductions in the immune-histochemical expression of
ER-α expression at two different dose levels.
Graphical Abstract:

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Abstract:
Offering novel scaffolds targeting estrogen receptor creates huge necessity to overcome the
evolving resistance developed by tumors. Structure-based drug design coupled with ring opening
strategy of the steroids skeleton revealed the potential of indole-based analogs to be synthesized
targeting the ligand binding domain of estrogen receptor-α. In vitro studies revealed the potential
of the total sub-classes of the synthesized analogs to show anti-proliferative activity against
estrogen receptor-dependent cancer cell lines at IC50 ranging from 28.23 to 57.13 µM. This was
further validated by evaluating the potential of the synthesized analogs to compete along with
estradiol via ER-α ELISA assay to show inhibitory profile at IC ranging from 1.76 to 204.75
5
0
nM. Two analogs (YMA-005 and YMA-006) showed significant reduction in tumor size at two
dose levels with extensive degeneration and necrosis. Both YMA-005 and YMA-006 showed in-
situ reduction of ER-α Immunohistochemical expression at both dose levels. Ultimately, novel
analogs of indole-based biomimetic of estrone scaffolds were offered as estrogen receptor-α
inhibitors.

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Keywords:
Indole, Biologically Inspired Organic Synthesis, Estrogen Receptor-α, Breast Cancer
1
. Introduction:
Human Breast carcinoma is the second leading cause for mortality among women. According to
the latest statistics issued by American Cancer Society in 2017, reporting an estimate of 252,710
newly diagnosed cases of invasive breast cancer, 63,410 newly diagnosed cases of breast
carcinoma in situ, and 40,610 breast cancer deaths [1]. Breast cancer has been classified into four
main categories; where estrogen receptor-α (ER-α positive) sub-type is the principle cause for
6
0-80% of breast cancer cases [2,3]. ER is a nuclear receptor with known physiological functions
regulating normal growth, development of reproductive organs, protecting the cardiovascular
system by decreasing cholesterol level, and bone homeostasis [4]. The structural organization of
ER is composed of two main domains, DNA binding domain (DBD) and ligand-binding domain
(
LBD) linked by hinge region, in addition to N and C terminal domains [5].
ER is expressed in two isoforms, ER-α and ER-β, where they share up to 95% homology at the
DBD and 55% homology at the LBD [5]. Typically, 17 β-estradiol is acting as agonist for
induction favorable conformation for coactivator binding surface of the LBD called activation
function-2 (AF2), where helix-12 (H-12) harbors across helix-11 and helix-3 (H-11 and H-3)
leading to formation of one side of the AF2 surface, leading to promotion of ER-α gene
expression [6]. Estrogen and estradiol have been known for their promoter role for cancer
proliferation accompanied with increased tumor invasion. Therefore, the conceptual basics for
tackling ER dependent breast cancer are built on development of new chemical entities
inhibiting the binding of estrone family to ER.

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Several FDA approved drugs, such as Tamoxifen led to revolutionary step towards treatment
of ER dependent breast cancer, acting as agonist at low doses and antagonist at high doses to
be categorized as selective estrogen receptor modulator (SERM) [7]. In addition, Fulvestrant
showed potent anti-estrogenic activity in all tissues with no agonistic activity to be known as
selective estrogen receptor down regulator (SERD) [8]. Both of SERM and SERD showed
their antagonism via competing with the natural endogenous substrates at LBD to inhibit ER-
α gene expression [8]. However, the endocrine therapy/chemotherapeutic agents are typically
associated with the evolution of drug resistance; in other words, cancer cells develop
resistance against the administered drugs; leading to serious complications [9]. Different
scaffolds were synthesized to target estrogen receptor whether inspired from Estrone or
Tamoxifen skeleton lacking the typical bulky side chain [10-12], as shown in Figure 1.
Herein, two fundamental strategies in the drug discovery, biologically inspired organic
synthesis (BIOS) and ring opening based on the steroidal skeleton at B/C ring juncture were
explored to assemble novel indole-based scaffolds that can be categorized as conformers to
biomimic estradiol configuration targeting ER-α, as shown in Figure 2. The whole design
strategy focused on overcoming the rigidity present at the steroidal scaffold to offer structural
flexibility via assembly of indole ring along with substituted either sulfonamide or
carboxamide derivatives. This was followed by in vitro biological screening against ER-
dependent breast cancer cell lines, inhibitory profiles for the synthesized analogs against ER-
α. This was followed by selecting two top hits in vivo biological evaluation.
2
2
. Results and Discussion:
.1. In silico virtual screening:

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The initial hypothesis started with employment of structure-based drug design (SBDD) strategy
via design of a virtual library of energy-minimized analogs including amide and sulfonamide
analogs coupled with 6-amino indole. The library underwent multi-conformer generation for
molecular docking study against ER-α receptor (PDB ID: 1ERE), to be sorted out based on the
binding energy profiles related to the endogenous substrates of the receptors of interest [13].
The indole-based analogs exhibited promising binding affinity towards ligand binding domain
(
LBD) of ER-α via hydrophobic–hydrophobic interactions and interesting hydrogen bond
between –NH of indole ring along with Threonine (Thr) 347: A, as shown in Figure 3. Thr 347:
A is one of the featured amino acid residues for structural antagonism towards ER-α [14,15],
suggesting that the analogs will disrupt H-12 interaction, dislocating the co-activators, leading to
inhibition of gene expression.
2
.2. Synthetic strategy and chemistry:
The structural insights of inhibitors design showed the significance of employment of BIOS
strategy coupled with ring opening to end up with conformational mimetics to steroid
scaffolds while overcoming the known steroidal adverse effects. Synthetic schemes towards
synthesis of the best scoring indole-based ligands, therefore, were outlined.
2
.2.1. General synthetic pathway for indole-based analogs:
Ten final indole-based analogs were selected from the library and synthesized for initial
biological screening. Initially, reduction of 6-nitro indole to 6-amino indole was accomplished
using stannous chloride conditions [16]. Acylation and sulfonylation of 6-amino indole with
substituted benzoyl chlorides and substituted benzene sulfonyl chlorides in basic conditions and
aprotic solvents under nitrogen ended up with two main classes of substituted N-(1H-indol-6-yl)

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benzamide and substituted N-(1H-indol-6-yl) benzene sulfonamide, respectively, as shown in
schemes 1 and 2.
Coupling of substituted benzoyl chlorides with substituted 4- or 5-amino indole derivatives or
coupling of substituted benzene sulfonyl chlorides with substituted 4-amino indole derivatives
were previously reported [17-20]. The present methodology for the synthesis of substituted N-
(1H-indol-6-yl) benzamide and substituted N-(1H-indol-6-yl) benzene sulfonamide in basic
conditions targeting estrogen receptor, however, is newly reported. Wherein, the whole objective
of the synthesis is to offer novel non-steroidal scaffold based on the initial SBDD screening to
create focused library.
2
2
.3. Biological Screening:
.3.1. In vitro anti-proliferative MTT assay against estrogen receptor dependent cancer
cell lines: The synthesized indole-based analogs underwent cell viability assays against estrogen
receptor dependent cancer cell lines (MCF-7 and T47D cell lines) to exhibit inhibitory behavior
ranging from 28.23 to 57.13 µM, compared to Tamoxifen (34 µM), as shown in table 1. This
suggested the potential of the synthesized analogs to have inhibitory activities against
proliferation of breast cancer cell lines.
2
.3.2. ER-α competitive binding assay:
The synthesized analogs underwent ER-α ELISA competitive binding assays to evaluate the
potential of the analogs to compete along with estrogen on LBD, where they showed the
inhibitory competitive manner against ER-α ranging from 1.76 to 204.75 nM, as shown in table
1
. Two representative compounds from each class, YMA-005 and YMA-006, were further
selected for investigation of their in vivo biological behavior.
.3.3. In vivo anticancer activity and safety of YMA-005 and YMA-006:
2

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Two compounds, YMA-005 and YMA-006, were selected for further in vivo animal studies
based on their promising in vitro activity. The safety of the selected compounds was observed in
normal mice at a dose of 10 mg/mouse where they were observed for short-term acute signs of
toxicity. No toxic signs were observed and the animals showed no signs of behavioral changes.
Moreover, no mortality was seen in all groups treated with either YMA-005 or YMA-006. The
two compounds were tested at two dose levels, 5 and 10 mg/mouse, in an animal model of
Ehrlich ascites carcinoma (EAC) solid tumor and their efficacy was compared to that of
Fulvestrant (FULV). Both compounds, especially YMA-005, showed better reductions in tumor
size, compared to both control and FULV-treated groups. After nine and twelve days of
treatment, YMA-005 caused significant reductions in tumor size at both low and high doses,
compared to FULV. On day fifteen after treatment and just before scarification, low dose of
YMA-005 and high dose of YMA-006 showed reductions in tumor volume, compared to FULV,
whereas the remaining compounds showed only significant reductions compared to untreated
control group. In addition, there was no significant change in body weights among different
treated groups compared to control group, as shown in Figure 4.
2
.3.4. Histopathological examination and necrotic indices:
The effect of YMA-005 and YMA-006 treatments on the histopathological changes in the
present model was examined using hematoxylin and eosin (H&E) staining for necrotic changes /
areas of malignant viable cells. The group treated with a low dose of YMA-006 showed tumor
sections with the least areas of necrosis. However, YMA-006 (10 mg)-treated group showed
mixed viable and focal necrotic areas in the deeper parts of the tumor mass. YMA-005 (10 mg)-
treated group, on the other hand, demonstrated extensive necrotic areas as well as degenerated
tumor cells and less extensive areas of viable tumor cells in the outer zone of the tumor mass, as

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shown in Figures 5 and 6. The necrotic indices in H&E-stained tumor sections belonging to the
different groups were also estimated where low dose of YMA-005 showed the highest index
compared to both control and FULV-treated groups.
2
.3.5. Estrogen-α expression:
Primary monoclonal antibody against ER-α receptor was used to evaluate the effect of
compounds on ER-α expression in tumor sections of different groups. Both YMA-005 and
YMA-006 at the two dose levels showed reductions in ER-α expression, compared to control and
FULV-treated groups. YMA-005 showed 64.71% and 35.30% reduction in ER-α expression,
compared to control, and 57.75% and 22.54% reduction, compared to FULV at low and high
doses, respectively, as shown in Figure 7. Likewise, YMA-006 showed 58.24% and 37.65%
reduction, compared to control, as well as 50% and 25.35% reduction, compared to FULV at low
and high doses, respectively. Expression of ER-α in EAC animal model was reported to decrease
with estrogen receptor modulators.
2
.3.6. Structural activity relationship analysis:
Applying BIOS and ring opening strategies enabled us to offer novel pharmacophore as indole-
based biomimetic analogs to steroidal scaffolds with inhibitory activity against ER-α dependent
breast cancer. We managed to synthesize top ten compounds based on structure-based drug
design for indole-based analogs representing two main classes; (1) substituted N-(1H-indole-6-
yl) benzamide derivatives and (2) substituted N-(1H -indol-6-yl) benzene sulfonamide. In vitro
screening against ER--α dependent breast cancer cell lines and ER-α competitive binding assay
were executed to elucidate preliminary structural activity correlation. Generally, benzamide
analogs showed better inhibitory profiles against MCF-7 and T-47D cell lines as well as better
inhibitory competitive binding affinity towards ER-α.

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The substituted N-(1H-indole-6-yl) benzamide derivatives showed inhibitory profiles in the
following manner 3-trifluromethyl > 4-cyano > 4-bromo > 4-trifluromethyl > 4-fluoro. The
electron-withdrawing group showed promising binding affinity. Trifluromethyl at meta position
showed the best binding affinity with respect to the energy profile prior to the synthesis to show
the best biological activity against ER-α dependent breast cancer cell lines and ER-α competitive
binding assay.
The substituted N-(1H -indol-6-yl) benzene sulfonamide showed inhibitory profiles in the
following manner 4-fluoro > 4-methoxy > 4-bromo > 4-chloro > 4-methyl. This suggests that the
presence of electron withdrawing group could be important for the activity against ER-α
dependent breast cancer cell lines and ER-α competitive binding assay.
This study represents an introduction of novel synthetically feasible scaffolds targeting ER-α
dependent breast cancer.
3
. Conclusions:
Two sets of indole-based class were designed targeting estrogen receptor based on two main
approaches; BIOS starting with steroidal scaffolds with its known biological activity,
followed by ring opening strategy. Indole-based analogs were initially screened via SBDD
principles showing well-tailored binding affinity towards ER via hydrophobic-hydrophobic
interactions at the LBD and hydrogen bond along with THR 347:A. Ten indole-based
derivatives were synthesized via two step reactions; starting with reduction of 6-nitroindole,
followed by nucleophilic substitution reactions with benzoyl or benzene sulfonyl chlorides in
basic conditions. YMA-005 and YMA-006 s inhibit cell proliferation of ER-dependent cancer
cell lines, which was further confirmed by ER-α inhibitory assay. In vivo studies where they
exhibited significant reduction in tumor size of EAC-bearing mice at two dose levels with

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remarkable necrosis and reduction in ER-α expression in tumor sections. In the present study,
promising novel indole-based scaffolds as biomimetic analogs targeting estrogen receptor
were developed.
4
4
. Experimental Section:
.1. In silico virtual screening
A virtual library of indole-based analogs along with estradiol and estrone structures were
designed in the two-dimensional (2D) structures. The structures were exported to three-
dimensional (3D) configuration to be ready for energy minimized using MMFF94 force field.
The energy minimized compounds underwent semi-flexible docking using Omega and FRED
along with the box assigned for the receptor (PDB ID: 1ERE), as previously described [21-25].
4
.2. Chemistry
1
13
4
.2.1. General: Spectroscopic data ( H and C NMR spectra) were done using Bruker
AVANCE-400ꢀMHz and 100 MHz NMR spectrometer, in deuterated solvents of dimethyl
sulfoxide (DMSO-d ) and chloroform (CDCl ). Chemical shifts (δ) are reported in ppm and
6
3
coupling J-constants in Hz. The signals are reported as singlet (s), doublet (d), triplet (t), quartet
q), or multiplet (m). TLC analyses were performed using pre-coated silica gel sheets visualized
(
at short and long wavelengths. Mass spectra were processed at Al-Azhar University, Nasr City,
Cairo, Egypt, using Thermo-scientific GCMS ISQ. Chemicals and reagents were purchased
from Sigma Aldrich and used as received.
4
.2.2. Synthesis of 6-amino indole:

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To a solution of 6-nitro indole (1 eq.) in a mixture of acetic acid (0.15 M) and hydrochloric
1
acid (0.6 M), SnCl (10 eq.) was added. The reaction mixture proceeded and H NMR was
2
recorded, as previously reported [16].
4
.2.3. General Synthesis of substituted N-(1H-indole-6-yl) benzamide derivatives:
A flask was charged with 6-amino Indole (1eq.) in the presence of dichloromethane (DCM, 0.5
M) and E N (1.2 eq.) under nitrogen at 0 °C. This was followed by addition of substituted
3
benzoyl chlorides (1 eq.) to stir at room temperature overnight. The reaction mixture was
2 4
quenched by water to be extracted with ethyl acetate and dried over anhydrous Na SO . The
ethyl acetate layer was evaporated in vacuo. The crude product was purified using silica gel
column chromatography using ethyl acetate/hexane solvent mixture (50:50) to obtain substituted
N-(1H-indole-6-yl) benzamide derivatives.
1
4
.2.3.1. YMA-001: 4-fluoro-N-(1H-indol-6-yl) benzamide; white powder; yield, 90%; H NMR
(
400 MHz, DMSO-d ): δ 11.07 (s, 1H), 10.18 (s, 1H), 8.06 (m, 3H), 7.49 (d, J= 8.31 Hz ,1H),
6
7
.27-7.39 (m, 4H), 6.39 (d, J= 8.31 Hz, 1H). Addition of D O quenched -NH peaks at δ 11.07
2
1
3
and 10.18. C NMR (100 MHz, DMSO-d
6
): 165.5, 164.9, 163.6, 139.6, 131.9, 130.9, 130.8,
+
1
1
4
29.1, 120.9, 120.4, 115.9, 115.7; MS calcd for C15
00%]
H11FN
2
O, 254.09, found m/z: 253.26 [M -1,
.2.3.2. YMA-002: N-(1H-indol-6-yl)-4-(trifluoromethyl) benzamide, buff white powder;
1
yield, 75%. H NMR (400 MHz, DMSO-d ): δ 10.50 (s, 1H), 10.08 (s, 1H), 8.35 - 8.22 (m, 1H),
6
7
6
1
3
.65 (d, J = 8.07 Hz. 1H), 7.49 (d, J = 8.07 Hz, 1H), 6.96 - 6.86 (m, 3H), 6.70 -6.60 (m, 2H),
1
3
.50 (s, 1H). C (100 MHz, DMSO-d ): 166.5, 137.6, 135.9, 134.6, 130.8, 129.1, 126.7, 126.6,
6
24.9, 124.5, 120.4, 113.5, 104.6, 102.1; M.S. calcd for C16
11 3 2
H F N O, 304.08; found m/z:
+
03.26 [M -1, 100 %]

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1
4
.2.3.3. YMA-003: 4-cyano-N-(1H-indol-6-yl) benzamide, white powder; yield, 80%. H
NMR (400 MHz, DMSO-d ): δ 11.10 (s, 1H), 10.01 (s, 1H), 8.14 - 8.01 (m, 5H), 7.50 (d, J=
6
8
.31 Hz, 1H), 7.30 (d, J = 7.82 Hz, 2H), 6.39 (s, 1H). M.S. calcd for C16 H N O, 261.09;
11 3
+
found m/z: 260.28 [M -1, 100%]
4
.2.3.4. YMA-004: 4-bromo-N-(1H-indol-6-yl) benzamide, yellowish white powder; yield, 75
1
%
. H NMR (400 MHz, DMSO-d ): δ 11.50 (s, 1H), 10.20 (s, 1H), 8.06 (m, 3H), 7.49 (d, J =
6
1
3
8
1
6
.31 Hz, 1H), 7.27-7.39 (m, 4H), 6.39 (d, , J = 8.31 Hz, 1H). C NMR (100 MHz, DMSO-d ): δ
65.1, 135.6, 132.5, 131.7, 129.8, 125.2, 124.8, 120.2, 115.3, 115.01, 113.2, 102.5, 100.6. M.S.
+
calcd for C15
H
11BrN
2
O, 314.01; found m/z: 313.17 [M -1, 70%]
4
.2.3.5. YMA-005: N-(1H-indol-6-yl)-3-(trifluoromethyl) benzamide, buff white powder;
1
yield, 85%. H NMR (400 MHz, DMSO-d ): δ 11.06 (s, 1H), 10.35 (s, 1H), 8,59 – 7.60 (m, 4H),
6
1
3
7
.35 (d, J= 2.69 Hz, 2H), 7.09 (d, J= 2.69 Hz , 2H), 6.65 (s, 1H). C NMR (100 MHz, DMSO-
d6): δ 169.5, 135.3, 132.5, 131.2, 130.0, 129.9, 125.0, 124.8, 119.01, 115.09, 114.8, 112.9, 103.7,
+
1
01.5. M.S. calcd for C H F N O, 304.08; found m/z: 303.26 [M -1, 100%]
1
6
11
3
2
4
.2.4. General Synthesis of substituted N-(1H -indol-6-yl) benzene sulfonamide
derivatives:
A flask was charged with 6-amino indole (1 eq.) in the presence of mixture of anhydrous THF /
Ether (1:1, 0.5 M) and anhydrous pyridine (1.2 eq.) under nitrogen at 0 °C. This was followed by
addition of substituted benzene sulfonyl chloride (1eq.) to stir at room temperature overnight.
The reaction mixture was quenched by addition of water to be extracted with ethyl acetate and
dried over anhydrous Na SO . The ethyl acetate layer was allowed to evaporate in vacuo. The
2
4
crude product was purified using silica gel column chromatography using ethyl acetate / hexane
mixture (60:40) to obtain substituted N-(1H -indol-6-yl) benzene sulfonamide.

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4
9
2
1
.2.4.1. YMA-006: 4-fluoro-N-(1H-indol-6-yl) benzene sulfonamide; purple powder; yield,
1
5%; HNMR (400 MHz, CDCl ): δ 8.39 (s, 1H), 7.59 (d, J= 2.69 Hz, 2H), 7.30 (d, J= 2.69 Hz,
3
1
3
H), 7.22 (s, 1H), 6.83 – 6.78 (m, 2H), 6.20 (s, 1H). C NMR (100 MHz, CDCl ): δ 166.3,
3
63.8, 135.8, 134.8, 130.2, 130.1, 130.0, 126.3, 125.5, 121.1, 116.2, 116.2, 116.0, 106.8, 102.2;
+
M.S. calcd for C H FN O S, 290.05; found m/z: 289.31 [M -1, 100%]
1
4
11
2
2
4
8
,
.2.4.2. YMA-007: 4-chloro-N-(1H-indol-6-yl) benzene sulfonamide; dark tan powder; yield,
1
5%; HNMR (400 MHz, CDCl
3
): δ 8.25 (s, 1H), 7.55 (d, J = 8.31 Hz, 2H), 7.36 (d, J = 8.31 Hz
1
3
2H), 7.22 (s, 1H), 7.09 (m, 1H), 6.58 (d, J = 7.82 Hz, 1H), 6.39 (s, 1H ). C NMR (100 MHz,
CDCl
3
): δ 139.3, 137.4, 135.8, 130.1, 129.2, 128.7, 126.4, 125.3, 121.2, 116.3, 106.9, 102.4;
+
M.S. calcd for C H ClN O S, 306.02; found m/z: 306.00 [M , 100%]
1
4
11
2
2
4
9
2
.2.4.3. YMA-008: 4-Methyl-N-(1H-indol-6-yl) benzene sulfonamide; purple powder; yield,
1
0%; H NMR (400 MHz, CDCl ): δ 8.21 (s, 1H), 7.53 (d, J =7.82 Hz, 2H), 7.35 (d, J =8.07 Hz ,
3
1
3
H), 7.26 (s, 1H), 7.08 – 7.06 (m, 1H), 6.58 (d, J =8.07, 1H), 6.39 (s, 1H), 2.26 (s, 3H). C
NMR (100 MHz, CDCl ); δ 143.6, 136.1, 135.8, 130.7, 129.5, 127.3, 126.1, 125.0, 121.0, 116.1,
3
+
1
4
8
2
06.4, 102.4, 21.5; M.S. calcd for C15
H
14
N
2
O
2
S, 286.08; found m/z: 285.35 [M -1, 100%]
.2.4.4. YMA-009: 4-Bromo-N-(1H-indol-6-yl) benzene sulfonamide; purple powder; yield,
1
0%; H NMR (400 MHz, CDCl ): δ 8.29 (s, 1H), 7.47 (d, J =8.07 Hz, 2H), 7.36 (d, J =8.07 Hz ,
3
1
3
H), 7.22 (s, 1H), 7.07 (s, 1H), 6.59 (d, J =8.31, 1H), 6.38 (s, 1H). C NMR (100 MHz, CDCl ):
3
δ 138.0, 135.8, 132.2, 130.0, 129.1, 130.8, 128.8, 127.8, 126.4, 125.3, 121.2, 116.3, 106.9, 102.5;
+
M.S. calcd for C H BrN O S, 351.97; found m/z: 350.21 [M -2, 70%]
1
4
11
2
2
4
9
2
.2.4.5. YMA-010: 4-Methoxy-N-(1H-indol-6-yl) benzene sulfonamide; purple powder; yield,
1
0%; H NMR (400 MHz, CDCl
3
) δ 8.20 (s, 1H), 7.56 (d, J =7.82 Hz, 2H), 7.38 (d, J =8.07 Hz ,
1
3
H), 7.28 (s, 1H), 7.10 – 7.08 (m, 1H), 6.60 (d, J =8.07, 1H), 6.40 (s, 1H), 3.81 (s, 3H). C

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NMR (100 MHz, CDCl
3
); δ 143.6, 136.1, 135.8, 130.7, 129.5, 127.3, 126.1, 125.0, 121.0, 116.1,
+
1
4
4
06.4, 102.4, 57.5. M.S. calcd for C H N O S, 302.07; found m/z: 301.5 [M -1, 100%]
1
5
14
2
3
.3. Biological Screening:
.3.1. In vitro anti-proliferative MTT assay against MCF-7 and T47D cell lines:
Cell Lines: Cells were obtained from American Type Culture Collection (ATCC). The cell
lines (MCF-7 and T47D) were sub-cultured in a similar fashion mode, as previously reported
[
26], before being treated without or with 5- fold serial dilutions of YMA compounds in
triplicates for 24ꢀh. The cells were harvested, washed with PBS, followed by addition of 20ꢀµL
of MTT to each well, and incubated for 2ꢀh before 200ꢀµL DMSO was added. The absorbance
was measured on an ELISA reader (Multiskan EX, Lab systems) at a wavelength of 570ꢀnm.
4
.3.2. ER-α competitive binding assay:
The ER-α activity assay was carried out using Estrogen Receptor Alpha ELISA Kit (ABCAM,
Cambridge, MA), according to the manufacturer's instructions. The absorbance for the tested
compounds and controls (Tamoxifen) was measured on an ELISA plate reader at 450ꢀnm.
4
.3.3. In vivo anticancer activity and safety of YMA-005 and YMA-006:
Swiss albino female mice were used in the present experiment of Ehrlich ascites carcinoma
EAC) solid tumor animal model [27]. On the day of induction, EAC cells were collected from
(
the ascitic fluid of a female mouse bearing ascitic tumor, diluted with sterile saline to produce
6
~
12.5×10 per 1 mL. 0.2 mL was then injected intramuscularly in the left thigh of all female
3
mice. When mice developed palpable mass (~100 mm ), treatment was started (day 0). EAC-

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bearing mice received a once weekly i.p. injection of either, fulvestrant (FULV) at a dose of 5
mg/mouse as well as YMA-005 and YMA-006 at a dose of 5 and 10 mg/mouse for each.
Treatments were delivered in 10% Tween 80 starting 5 days after inoculation. Mice in the
control groups received drug vehicles. Tumor volume was measured using a digital caliper at
different times after treatment, according to the following equation:
3
2
Tumor volume (mm ) = Length (mm) x [Height (mm)] x 0.52
Mice belonging to different groups were overnight food-deprived before scarification under
anesthesia after 15 days of starting treatment. Tumor tissues were then collected for further
analysis.
The safety of YMA-005 and YMA-006 was observed in normal mice at a single dose of 10
mg/mouse, i.p., where they were monitored for short-term acute signs of toxicity or mortality for
7
days. All experimental procedures were carried out in accordance with the National Institutes
of Health guide for the care and use of Laboratory animals.
Histopathological analysis:
Tumors were collected, fixed in 10% formalin solution, and embedded in paraffin. They were
stained with hematoxylin and eosin (H&E) to examine the histopathological changes. Moreover,
the necrotic index for each group was determined in 10 random fields per section per animal in
the treated and control groups using Leica application computer analyzer system.
4
.3.4. Immunohistochemical analysis:
Immunohistochemistry was performed using an avidin-biotin complex immunoperoxidase
technique with anti-mouse primary monoclonal antibody against ER-α receptor diluted at 1:100,
in PBS. For detection, a streptavidin-biotin-peroxidase preformed complex and peroxidase- 3,3′-

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diaminobenzidine (DAB) were used, according to the manufacturer’s instructions. Sections were
counterstained with Mayer’s hematoxylin and mounted with DPX medium. Positive and negative
control slides were included. Tumor section with no primary antibody was used as negative
control. Percentage of positively stained cells correlated with the expression of ER-α was
estimated in 10 random fields per section per animal using Leica application computer analyzer
system [28].
4
.3.5. Statistical analysis: Data are presented as mean ± S.D., and analyzed by one-way
analysis of variance (One-way ANOVA) followed by Tukey Kramer post hoc test for multiple
comparisons using GraphPad Prism version 5 (GraphPad Software, CA, USA). Values of P<0.05
were considered to be statistically significant.
5
. Acknowledgments:
The British University in Egypt (BUE) funded this research; grant number [YIRG-2016–01].
The authors appreciate the support offered by OpenEye molecular modeling software for sharing
an academic license.
6
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List of Figures and Tables:

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Figure 1. Examples for selective estrogen receptor modulator (SERM), classical selective
estrogen receptor down regulator (SERD), and atypical SERD lacking side chain
Figure 2. Design strategy to assemble indole-based biomimetic analogs

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Figur
3.
Visua
e
A
B
l
Repre
sentat
ion of
the
bindi
ng
mode
of
indol
e-
based
analo
gs
(A)
amide
bond
and (B) Sulfonamide bond along with estrogen receptor alpha (PDB ID: 1ERE), showing the
hydrophobic interactions with the ligand binding domain and hydrogen bond interactions of –NH
of indole along with THR 347:A represented by green dotted lines.
Scheme 1. Assembly of YMA-001 to YMA-005 analogs

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Scheme 2. Assembly of YMA-006 to YMA-010 analogs
Table 1. Inhibitory activity of indole-based analogs against estrogen receptor dependent cancer
cell lines and ER-
α
ELISA competitive binding assays
Molecular
IC MCF-7
IC T-47D
IC50 (ER – α)
(nM)
5
0
50
Compound R
weight
g/mol)
(µM)
(µM)
(

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YMA-001
YMA-002
YMA-003
YMA-004
YMA-005
4-F
254.09
304.27
261.28
315.17
304.27
371.51
45.93 ± 0.98
63.43 ± 2.14
33.56 ± 0.46
38.14 ± 0.85
30.63 ± 0.72
42.40 ±0.78
35.06 ± 0.75
51.34 ± 0.98
35.75 ± 0.95
63.74 ± 2.30
28.23 ± 0.95
34.42 ± 0.83
69.46
8.15
2.40
4.71
1.76
1.50
4- CF3
4-CN
4-Br
3-CF
Tamoxifen
3
Molecular
weight
IC50 MCF-7
µM)
IC50 T-47D
(µM)
IC50 (ER – α)
(nM)
Compound R
(
(
g/mol)
290.31
306.76
286.35
351.22
YMA-006
YMA-007
YMA-008
YMA-009
F
30.89 ± 0.96
54.02 ± 0.84
37.2 ± 1.20
37.75 ± 1.08
32.96 ± 0.69
46.16 ± 1.76
57.13 ± 1.43
40.06 ± 1.53
3.31
44.43
204.75
22.67
Cl
CH
Br
3

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YMA-010
OCH3
302.35
57.22 ± 2.02
45.04 ± 0.89
7.53
Figure 4. (A) EAC solid tumor-bearing mice in control and YMA-005- and YMA-006-treated
3
groups at 15 days of treatment. (B) Tumor volume (mm ) in EAC-bearing mice of control and
3
treated groups at different time points. (C-G) Bar charts showing tumor volume (mm ) at 3, 6, 9,
1
2, and 15 days of treatment. (H) Body weight of animals in treated and control groups, at
different time points. Values are presented as means SD (n=10). One-way analysis of variance
One-way ANOVA) followed by Tukey Kramer post hoc test was applied for statistics.
Significant difference compared to control at *P<0.05, **P<0.01, ***P<0.001. Significant
ꢀ±ꢀ
(
#
##
difference compared to FULV at P<0.05, P<0.01

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Figure 5. Photomicrographs of H&E-stained tumor sections (X400) in (A) control group
showing widespread viable tumor cells sheets with many mitotic figures (arrows) and prominent
nucleoli in the outer zone of tumor mass. Limited small area of necrotic tissue was observed in

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the deeper zone of the examined sections (star), (B) Fulvestrant- and (C) YMA-005 (5 mg)-
treated groups showing moderate size areas of necrosis and degenerated cells (star), infiltrated by
viable sheets of tumor cells as well as inflammatory cells (arrow), (D) YMA-005 (10 mg)-
treated group demonstrating the most extensive necrotic areas (star) as well as degenerated tumor
cells (dashed arrow), (E) YMA-006 (5 mg)-treated group showing tumor sections with the least
areas of necrosis, and (F) YMA-006 (10 mg)-treated group showing mixed viable and focal
necrotic areas (star) in deeper parts of tumor mass. Scattered apoptotic cells were observed
(arrows) between viable active tumor cells.

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Figure 6. Photomicrographs of H&E-stained tumor sections (X100) showing areas of necrosis in
(A) control group and groups treated with (B) fulvestrant, (C) YMA-005 (5 mg), (D) YMA-005
(10 mg), (E) YMA-006 (5 mg), and (F) YMA-006 (10 mg). (G) Necrotic indices as assessed in
H&E-stained tumor sections in control and treated groups. Values are presented as means
n=10). One-way analysis of variance (One-way ANOVA) followed by Tukey Kramer post hoc
test was applied for statistics. Significant difference compared to control at *P<0.05,
ꢀ±ꢀSD
(
#
#
*
**P<0.001. Significant difference compared to FULV at P<0.01.

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Figure 7. Photomicrographs of immunohistochemically staining (immunostain, DAB, X400)
using primary monoclonal antibody against estrogen-alpha (ER-α) receptor in tumor sections
from (A) control group and groups treated with (B) fulvestrant, (C) YMA-005 (5 mg), (D)
YMA-005 (10 mg), (E) YMA-006 (5 mg), and (F) YMA-006 (10 mg). (G)
Immunohistochemical expression of ER-
α in tumor sections of control and treated groups.
Values are presented as means SD (n=10). One-way analysis of variance (One-way ANOVA)
ꢀ±ꢀ
followed by Tukey Kramer post hoc test was applied for statistics. Significant difference
compared to control at *P<0.05, ***P<0.001. Significant difference compared to FULV at
#
##
P<0.001.