Identification of novel indole based heterocycles as selective estrogen receptor modulator

Article abstract of DOI:10.1016/j.bioorg.2018.04.002

In the present study, we have designed and synthesized indole derivatives by coalescing the indole nucleus with chromene carbonitrile and dihydropyridine nucleus. Two compounds 5c and 6d were selected from series I and II after sequential combinatorial li

Full text of DOI:10.1016/j.bioorg.2018.04.002

Bioorganic Chemistry 79 (2018) 72–88
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification of novel indole based heterocycles as selective estrogen
receptor modulator
a
a
b
c
b
a,
⇑
Ramit Singla , Kunal Prakash , Kunj Bihari Gupta , Shishir Upadhyay , Monisha Dhiman , Vikas Jaitak
a
Department of Pharmaceutical Sciences and Natural Products, Central University of Punjab, Bathinda, India
Department of Biochemistry and Microbial Sciences, Central University of Punjab, Bathinda, India
Department of Animal Sciences, Central University of Punjab, Bathinda, India
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, we have designed and synthesized indole derivatives by coalescing the indole
nucleus with chromene carbonitrile and dihydropyridine nucleus. Two compounds 5c and 6d were
selected from series I and II after sequential combinatorial library generation, docking, absorption, distri-
Received 9 March 2018
Revised 4 April 2018
Accepted 6 April 2018
Available online 24 April 2018
bution, metabolism and excretion (ADME) filtering, anti-proliferative activity, cytotoxicity, and ER-
competitor assay kit by utilizing estrogen receptor- (ER- ) dominant T47D BC cells line and PBMCs
Peripheral Blood Mononuclear Cells). Cell imaging experiment suggested that both the compounds suc-
a
a
a
(
Keywords:
Breast cancer
Chromene
Estrogen receptor
Indole derivatives
Dihydropyridine
cessfully cross cellular biomembrane and accumulate in nuclear, cytoplasmic and plasma membrane
region. Semiquantitative RT-PCR and Western blotting experiments further supported that both com-
pounds reduced the expression of mRNA and receptor protein of ER-
a, thereby preventing downstream
transactivation and signaling pathway in T47D cells line. Current findings imply that 5c and 6d represent
novel ER-
a
antagonists and may be used in the development of chemotherapy for the management of BC.
Ó 2018 Elsevier Inc. All rights reserved.
1
. Introduction
Breast cancer (BC) is the second most widespread cancer and
osteoporosis and BC [5,6]. Bazedoxifene is a third generation
indole-based SERM, in phase II clinical trial for the treatment of
BC [7].
The search for a new and safer SERMs with a simple structure
and high efficiency remains desideratum for a better treatment
of BC. It is widely accepted that compounds bearing heteroatoms
increase the stability of complex by forming hydrogen bonds with
the cancer-related protein targets [8]. It has been observed that
nitrogen-containing heterocyclic ring imparts a polarized character
one of the leading causes of mortality among women globally. In
clinical practice, a variety of molecular factors are considered for
predicting the prognosis and response to therapy of BC patients.
Abnormal expression of estrogen receptor (ER) is a salient patho-
logical phenotype in the majority of BC cases. Preeminent levels
of estrogens are thought to be associated with the incidence and
proliferation of BC. ER-
a
is a critical growth regulator in BC, and
and helps in establishing an efficient interaction with ER-a [9], and
its expression in BC cells is essential for tumor progression [1].
Selective estrogen receptor modulators (SERMs) are the com-
pounds used for thwarting the effect of estrogens in breast tissues.
These compounds bind to the ER similar to natural estrogens. How-
ever, their effects range from anti-estrogenic in some cases to
estrogenic in others depending upon the target site tissues [2–4].
Since the introduction of tamoxifen as first generation SERM, sub-
sequently second and third generation SERMs have been devel-
oped with higher efficacy and safety. Currently available SERMs
include raloxifene, lasofoxifene, and bazedoxifene which are useful
pharmacological agents for the prevention and treatment of
thus have a potential for providing a new paradigm for maintaining
the health of women [8,10]. Later, a fusion of two or more hetero-
cyclic rings in a single molecular frame has become an appealing
method for drug design. The outcome being a combined pharma-
cophore having structural features of two or more active sub-
stances leading to synergism, enhancement or modulation of the
desired characteristics of individual components [11,12]. Another
exciting feature of this approach is a generation of a diverse array
of pharmacophore which has an extensive application in the field
of medicinal chemistry. These molecules can also be modulated
for combating the incidence of drug resistance as a single molecule
can have different modes of action at the same time thereby reduc-
ing the chances of drug resistance [13–15]. In recent years, there
has been an arising interest in an indole-fused heterocyclic ring
structure in cancer drug discovery [16–19].
⇑
Corresponding author at: Department of Pharmaceutical Sciences and Natural
Products, Central University of Punjab, Bathinda, Pb 151001, India.
E-mail address: vikasjaitak@gmail.com (V. Jaitak).
https://doi.org/10.1016/j.bioorg.2018.04.002
0
045-2068/Ó 2018 Elsevier Inc. All rights reserved.

R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
73
It has been reported that 2-arylindole derivative is selective
towards the ER- and antagonizes the action of estradiol in BC cells
amino-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene
was recognized as a potent apoptosis inducer and has shown the
a
and uterus tissues.
EC50 value of 73 nM in T47D cells [25,34]. a-Mangostin isolated
Owing to the importance of N-heterocycles in the present study
we have designed and synthesized Series I (indole-chromene car-
bonitrile derivative) and II (indole-dihydropyridine derivative)),
from Garcinia mangostana has been reported to be effective in BC
by the mechanism of arresting the cell cycle, elevating the caspases
and aromatase inhibition [35].
a-Mangostin has been found to
as putative SERM for targeting ER-
a
for the management of
reduce the expression of ER- in MCF-7 cell lines [36]. Hence these
a
hormone-dependent BC (HDBC). The synthesized molecules were
characterized by NMR and HRMS. Compounds were analyzed for
derivatives could be used as a template for the development of
pharmacophore targeting BC. Hence, by rationally combining the
structural features of Indole core with chromene and DHP we have
designed indole-chromene carbonitrile and indole-dihydropyridine
derivatives for targeting BC (Fig. 1).
the anticancer activity using ER-
binding assay, semiquantitative RT-PCR, Western blotting and
confocal microscopy. Molecular docking was used to identify the
possible binding modes of indole derivatives with ER-
a expressing T47D cell lines, ER-
a
a
.
2.2. Selection of indole derivatives
2
. Results and discussion
.1. Design of indole derivatives
Indole based compounds have a tremendous potential for the
Molecular simulation techniques supported the selection of
compounds for synthesis. Initially, a combinatorial library for the
2
lead structure of Series I and II was generated. The combination
1
2
of the database of reagents substituting R and R group (Fig. 1)
generated 4086 and 4,618,200 compounds for Series I and II
respectively.
development of chemotherapeutic agents [20,21]. Structural anal-
ysis of the available literature suggests that 1-benzyl-indole-3-
carbinol, an analog of indole-3-carbinol was found to be thousand
times more potent in comparison to indole-3-carbinol in the sup-
pression of both estrogen-dependent and independent BC cell lines
The generated set of compounds were then evaluated in the
two-step procedure. In the first step, ADME was applied for filter-
ing of compounds with the most promising drug-like properties
(Table 1) and next step docking to the active site of ER-a for a final
[
22]. Hence, N-benzylation significantly improves the anti-
selection of compounds with the highest potential activity
(Table 2). With the help of qikprop module used during ADME
analysis only top-ranked 500 molecules were retained. Calculation
of molecular descriptors and ADME analysis minimizes the
chances of failure of compounds in early pre-clinical/phase I clini-
cal trials during the drug development process.
proliferative activity as compared to its counterpart. It has been
also reported that 2-aryl indole derivative exerts its anti-
estrogenic effect by targeting ER-
another indole derivative having an anti-estrogenic activity which
targets ER- and is equally effective as tamoxifen and can over-
a [19,23]. Similarly, ERA923 is
a
come the resistance associated with tamoxifen [24] (Fig. 1).
Bazedoxifene is another indole-based SERM which is known to
The acceptable limits of QP logPo/w (octanol/water partition
coefficient) value were taken in the range of ꢀ2.0 to 6.5 and with
maximum possible oral bioavailability, thus possessing desirable
characteristic absorption parameters for a lead drug molecule.
Higher than 6.5 value indicates that compounds will tend to have
higher lipophilicity and will not be able to solubilize in the aqueous
phase for systemic distribution. Log Po/w value lower than ꢀ2.5
indicates higher hydrophilicity which in result will hinder the
compound from crossing the cellular biomembrane and acting on
the target receptor [37]. During absorption, the molecules must
cross the epithelial and endothelial cell barriers to reach the target
cells. Caco and MDCK prediction assays have been successfully uti-
lized in the study of drug transport. Caco cells have comparable
permeability with transport in vivo human jejunum, hence estab-
lishes a correlation between bioavailability and absorption. But
Caco has suffered a disadvantage of variable expression of trans-
porters like P-gp (a P-glycoprotein, a well-recognized efflux trans-
porter in many tissues including brain, kidney, and intestine).
Therefore, MDCK (Madin-Darby Canine Kidney) cells are used to
study drug efflux and active transport. Numerous studies have
demonstrated that human oral drug absorption and permeability
coefficient have good correlations. QPP Caco and QPP MDCK have
a recommended acceptable value greater than 500 nM/s. Larger
the calculated value better the compound permeability through
the gut membrane and higher the oral absorption [38]. Thereby,
could be absorbed effectively for next phase of blood plasma distri-
bution. Upon absorption, drugs exist in equilibrium binding with
plasma albumin for the distribution. The unbound drug is active
and reaches the site of action and gets metabolized and/or
excreted. QP logKhsa predicts the binding of the compound to
human serum albumin and the acceptable limit was taken as
ꢀ1.5 to 1.5. Predicted value more than 1.5 signifies higher albumin
binding thus lower the fraction of available drug reaching the site
of action. On the contrary value less than -1.5 indicates lower
binding and hence reduced plasma half-life of the drug. Hence,
bind ER-a with a higher affinity as compared to ER-b. In recent
years, chromene and acridine derivatives have attracted consider-
able interest in the development of drug candidates for cancer
chemotherapeutics. SAR studies have demonstrated that the aryl
substitution at the 4th position of chromenes and acridine com-
pound further potentiates the activity [25]. Among the wide range
of biological properties of 1,4-dihydropyridines (DHP) as an anti-
cancer moiety, they have also been recognized as multidrug
resistance reversing agents in cancer therapy [26,27]. Several
reports are published for different derivatives of DHPs including
dexniguldipine, niguldipine, nicardipine, and nitrendipine regard-
ing their potent activity in the management of BC by breast cancer
receptor protein (BCRP) inhibition [28]. It is well established that
slight structural modifications of the DHP ring may result in
remarkable changes in pharmacological effects, thereby DHP core
has revitalized the interest of the synthetic community. Among
different derivatives of 1,4-DHPs, 1,8-acridinedione is a known
scaffold with a broad spectrum of biological effects such as DNA
intercalating, anti-tumor, and cytotoxic activity [28–31]. Recently,
a 1,8-acridinedione derivative (3,3,6,6-Tetramethyl-9-[1-(4-fluoro-
benzyl)-2-(methylthio)-5-imidazolyl]-2,3,4,5,6,7,9,10-octahydro-
1
1
,8-acridinedione) have been reported to be effective at as low as
nm in MDR reversal and tamoxifen resistant T47D cell lines
[
30]. A chromene based derivative, 12-(4-hydroxyphenyl)-9,9-
dimethyl-7a,8,9,10,11a,12-hexahydro-11H-benzo[a]xanthen-11-one
6.7 mg/mL) was found to possess more effective anti-proliferative
(
activity than tamoxifen in MCF-7 cells line [32]. In another exam-
ple, phenyl substituted dioxooctahydroxanthenes derivatives have
been found to be active in MCF-7 cell line with an IC50 value of
0
.02 lmol/l [33]. Recently, EM-652 which is an active chromene
metabolite of EM-800 has been shown to exhibit antiestrogen
profile and as in clinical trials for the treatment and prevention
of BC. Another chromene derivative, 2-amino-3-cyano-7-dimethyl-

7
4
R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
Fig. 1. Designing strategy of indole derivatives.
Table 1
ADME characteristics of indole derivatives selected for synthesis.
Series
S. No
Code
QPlog Po/w
QPlog HERG
QPP Caco (nM/s)
QPP MDCK (nM/s)
QPlogKhsa
Percent human oral absorption
Series I
1
2
3
4
5
6
5a
5b
5c
5d
5e
5f
4.90
4.89
4.64
5.95
4.25
5.50
ꢀ5.68
ꢀ5.54
ꢀ5.79
ꢀ7.17
ꢀ5.48
ꢀ6.71
812.92
793.24
567.79
570.05
748.69
570.04
595.47
685.14
868.32
1069.47
1261.81
969.47
1.01
1.01
0.94
1.40
0.74
1.22
100
100
100
100
100
82.56
Series II
7
8
9
6a
6b
6c
6d
6e
6f
6g
6h
6i
6.19
6.31
5.94
4.88
4.21
4.16
5.20
4.12
3.98
6.12
6.20
6.44
ꢀ5.18
ꢀ5.24
ꢀ6.97
ꢀ5.80
ꢀ8.31
ꢀ8.10
ꢀ6.93
ꢀ7.67
ꢀ7.90
ꢀ7.57
ꢀ7.28
ꢀ7.21
2502.12
2730.50
2387.68
3612.42
2409.08
2407.88
2370.33
2627.63
2410.40
2474.20
2436.51
2362.54
1333.13
1465.13
1267.35
1982.73
1279.63
7796.06
1257.40
1405.56
1280.39
3251.73
3198.20
3093.37
1.48
1.63
1.41
1.30
1.49
1.34
1.16
1.06
1.20
1.58
1.33
1.35
100
100
100
100
100
100
100
100
100
100
100
100
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
6j
6k
6l
Standard
Bazedoxifene
–
6.06
ꢀ7.45
227.66
110.54
ꢀ3.68
91.71
acceptable limit is considered to be optimum in between ꢀ1.5 to
.5. Upon distribution, the drug candidate must be devoid of car-
diotoxicity [39]. Several non-cardiovascular drugs have been
shown to precipitate cardiotoxicity by blocking the hERG (human
ether-a-go-go-related gene) channels which prolongs the QT inter-
val. Thus, in drug discovery and in clinical practice hERG channel is
viewed as primary anti-target [40]. QP log hERG model has been
developed for the prediction of IC50 value for blockage of this
channel. The recommended value is above ꢀ5.0, which signifies
compounds having a value lower than ꢀ5 will tend to activate
hERG receptor and may lead to torsade de pointes [41]. The ADME
analysis identified top 500 compounds which are devoid or have
1

R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
75
Table 2
Docking score, H-bond score, Lipophilic EvdW and Electrostatic interaction score of indole derivatives selected for synthesis.
Series
S. No
Code
Docking score (kcal/mol)
XP HBond (kcal/mol)
XP Lipophilic EvdW (kcal/mol)
XP Electro (kcal/mol)
Series I
1
2
3
4
5
6
5c
5e
5b
5a
5f
ꢀ4.38
ꢀ4.31
ꢀ3.33
ꢀ2.53
ꢀ0.95
ꢀ0.12
ꢀ0.70
ꢀ6.37
ꢀ3.71
ꢀ6.17
ꢀ6.24
ꢀ4.26
ꢀ2.47
ꢀ0.19
ꢀ0.15
ꢀ0.17
ꢀ0.10
0.08
0
0
ꢀ0.60
ꢀ0.59
0
5d
ꢀ0.32
Series II
7
8
9
6a
6b
6c
6d
6g
6h
6l
6e
6i
6k
6f
ꢀ8.05
ꢀ7.62
ꢀ5.95
ꢀ4.41
ꢀ3.94
ꢀ3.38
ꢀ2.95
ꢀ2.72
ꢀ2.68
ꢀ2.49
ꢀ1.22
ꢀ0.07
ꢀ0.70
ꢀ0.70
ꢀ0.41
0
ꢀ0.54
ꢀ0.35
ꢀ0.35
ꢀ0.35
ꢀ0.51
ꢀ0.44
0
ꢀ7.00
ꢀ6.88
ꢀ5.73
ꢀ4.02
ꢀ3.93
ꢀ3.53
ꢀ3.49
ꢀ3.22
ꢀ3.97
ꢀ3.27
ꢀ3.51
ꢀ2.18
ꢀ0.25
ꢀ0.40
ꢀ0.25
ꢀ0.09
ꢀ0.42
ꢀ0.38
ꢀ0.41
ꢀ0.34
0.07
10
11
12
13
14
15
16
17
18
ꢀ0.07
ꢀ0.40
ꢀ0.39
6j
ꢀ0.64
Standard
Bazedoxifene
–
ꢀ9.33
ꢀ0.98
ꢀ9.53
ꢀ0.75
a minimal predicted cardiotoxicity. These 500 molecules were then
screened by docking with ER- for identifying the compounds
showing H-bond or Van der Waals interaction with Leu 346. Leu
46 has been reported as an essential amino acid in Helix 12 of
ER- for the antagonistic activity [42]. In addition to the above
criteria, cut off docking score was set up as ꢀ0.08 kcal/mol. This
cutoff score eliminated the compounds which were not showing
impressive interaction profile with the target receptor. From the
docking filtration criteria, top 50 molecules were selected for fur-
ther evaluation. Further, 50 molecules were evaluated using the
complexity based synthetic accessibility [43]. In this approach,
the molecules were evaluated for the availability of synthetic pro-
cedures, reagents and building blocks for the synthesis. Collec-
tively on the basis of glide score, favorable ADME profile, and
synthesis accessibility six compounds 5a–f for the lead structure
I and twelve compounds 6a-l for lead structure II were selected
for the synthesis and further evaluation for biological studies.
cyano group affords formation of cyclized indole-chromene car-
bonitrile derivative (Fig. 2).
In the compound 5e, the functional presence of the functional
2
group including amino (NH ) at C-2, cyanonitrile (CN) at C-3 and
a
3
a
carbonyl (C@O) at C-5 was indicated by the appearance of a peak
at d 163.08, 62.20 and 196.12 respectively. The carbon of the
cynonitrile group was confirmed by the peak at d 118.45. The
unsaturation between C-4a, 8a was designated by the shift at d
114.13 and 157.93. Proton for the amino group appears at d 5.74
and the linkage of the indole with the chromene carbonitrile was
confirmed by the d 27.38 and singlet proton at d 4.65. HRMS of
+
+
21 3 2
5e showed the peak matching to [M] for C25H N O at m/z
395.1633. Similarly, other products of the series were confirmed
by NMR and HRMS.
The indole-dihydropyridine derivatives (6a–l) were prepared by
ascorbic acid catalyzed multicomponent Hantzsch dihydropyridine
reaction of 1-benzyl-1H-indole-2-carbaldehyde (4), dimedone and
4
NH OAc in the presence of ethanol at 10 °C. The practical yield was
good (65–80%). The reaction proceeds via the enolization of dime-
done and 1-benzyl-1H-indole-2-carbaldehyde (4) facilitated by
ascorbic acid. In the next step intermediated formed by Knoeve-
nagel condensation between aldehyde and dimedone undergoes
Michael addition by second molecules of enol form of dimedone
2
.3. Synthesis of indole derivatives
The indole-chromene derivatives (5a–f) were synthesized by
the general route with high to moderate yields as described in
Scheme 1. The N-position of the starting material ethyl indole-2-
carboxylate (1) was benzylated using benzyl bromide in the
presence of potassium hydroxide using DMSO as a polar aprotic
solvent to give ethyl-1-benzyl-1H-indole-2-carboxylate (2). Com-
pound 2 was reduced to (1-benzyl-1H-indol-2-yl) methanol (3)
in the presence of LiAlH
was oxidized to 1-benzyl-1H-indole-2-carbaldehyde (4) by using
activated manganese dioxide in dry CH Cl . A series of indole-
chromene derivatives (5a–f) were synthesized by one-pot
three-component cyclo-condensation reaction of 1-benzyl-1H-
indole-2-carbaldehyde (4), malononitrile and dimedone. The above
mixture upon refluxing in ethanol gives moderate to a good yield,
i.e., 69–86%. The plausible mechanism in the literature [44] suggest
that the formation of chromene carbonitrile derivative proceeds
by in-situ initial formation heterylidenenitrile in the presence
of ethanol as a basic catalyst [45]. Heterylidenenitrile formed by
the Knoevenagel condensation between 1-benzyl-1H-indole-2-
carbaldehyde (4) and malononitrile with subsequent loss of water
molecules contains electron deficient C@C double bond. In the next
step, the Michael addition of dimedone to the unsaturated nitrile
which involves the nucleophilic attack of hydroxyl moiety to the
(
See Fig. 3). The resulting intermediate upon reaction with ammo-
nium acetate yield amine which undergoes intramolecular cyclisa-
tion and subsequently dehydration to form indole-dihydropyridine
derivative [46].
In compound 6b the peak at d 190.10 helped in confirming the
carbonyl functional group at C-1 and C-8. The introduction of
unsaturation between C-8a,4b and C-9a,4a upon cyclisation was
confirmed by the peak at d 144.88 and 115.41. The characteristic
NH proton were observed to give a broad signal at d 12.16. The
bond between indole moiety and dihydropyridine was indicated
4 .
under dry THF conditions Compound 3
2
2
1
3
1
by the C signal for C-9 at d 22.76 and H signal at d 4.13. HRMS
+
+
of 6b showed the peak conforming to [M + 2H] for C32
36 2 2
H N O
at m/z 480.2717. Likewise, other congener of the series was spec-
1
13
troscopically identified by H, C NMR and HRMS. It should be
noted that compounds 5a–f and 6a–l are novel and were purified
using flash chromatography.
2.4. Biological evaluation of indole derivatives
All the synthesized compounds (5a–f, 6a–l) were screened for
antiproliferative activity using T47D cell lines which are known

7
6
R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
Scheme 1. Reagent and conditions (a) Benzyl bromide, KOH, DMSO, r.t. 1.5 h (b) LiAlH
4 2 2 2
, dry THF, 0 °C, 30 min (c) activated MnO , dry CH Cl , r.t., 5 h, (d) malononitrile,
dimedone, ethanol, reflux 12–15 h. (e) ammonium acetate, ascorbic acid, dimedone, 10 °C, 9–10 h.
Fig. 2. Plausible mechanistic pathway for the synthesis of indole-chromene carbonitrile derivatives.
for their dominant expression of ER-
a
(9/1 ratio of ER-
a
/ER-b
the structural characteristics of the compound, it was observed
that alkyl substituent as side chain as in compound 5a, 5b, 5c,
6a, 6b, 6c and 6d was more active then compounds having classical
aryl group as in 5d, 6e, 6f, 6j, 6k and 6l. Such observation indicates
that presence of bulky side chains is detrimental and renders the
compound inactive at T47D cells line. Majority of chemotherapeu-
tic drugs lacks specificity towards cancerous cells and hence, gets
expression) [47,48]. All the synthesized compounds (5a–f, 6a–l)
were screened for anti-proliferative activity by MTT assay and tak-
ing bazedoxifene as positive control using these cells line. Upon
analyzing the results of the results from antiproliferative assay, it
was found that ten compounds (5a, 5b, 5c, 6a, 6b, 6c, 6d, 6g, 6h
and 6i) had IC50 in the range of 3.4–24.75 mM (Table 3). On analyzing

R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
77
Fig. 3. Plausible mechanistic pathway for the synthesis of indole-dihydropyridine derivatives.
failed in clinical trials. To evaluate the effect of synthesized com-
pounds we further screened the compound of series I and II for
cytotoxicity utilizing human PBMCs (Peripheral Blood Mononu-
clear Cells) MTT assay. Bazedoxifene (Baz) was also included in
the study as a reference standard. PBMC assay indicated that com-
pounds were showing non-significant cytotoxicity even at higher
Docking score analysis of these compounds showed that presence
of the heterocyclic ring structure adds the factor of hydrogen bond-
ing at the receptor site, thus counteracting the steric hindrance
imposed by the bulkiness of the unsaturated ring system. Com-
pounds 5c and 6d in series I and II were escalated for further bio-
logical evaluation as this exhibit most potent anti-proliferative
activity and binding affinity. Compounds 5c and 6d were examined
by confocal laser scanning microscopy (CLSM), quantitative RT-PCR
and Western blotting for gene expression studies.
concentration of 100 lM (Fig. 4). Therefore, all the compounds
were considerably selective towards the cancerous cells line.
In the expeditious search for the hits targeting BC, the com-
pounds should have binding capacity towards the ER-
binding affinity of all the synthesized compounds was determined
by utilizing ER- competitor assay kit (Polar Screen ER- Competi-
tor Assay Kit, Green, Life Technology). The principle of the assay is
based on the detection of a change in the fluorescence upon com-
a
. The ER-
a
With the advancement in technology, there has been a consid-
erable evolution in the development of non-invasive methodolo-
gies for determining the distribution of drug molecules in
intracellular compartments within intact, living single cells and
inside viable cell populations [50]. CLSM is one such technique
which eliminates the out of focus light and focuses only narrow
regions of the specimen for imaging. An image is built from the flu-
orescence emitted from the treatment cells and appropriate filtra-
tion for background noise. CLSM was used for determining the
distribution of the compound 5c and 6d inside the T47D cells.
The compounds 5c and 6d have blue emission, so it was possible
to detect the compound using the DAPI excitation-emission chan-
nel. The confocal images obtained after the treatment of the com-
pounds 5c and 6d after 24 h incubation at 5 and 10 mM respectively
indicated that compounds were profusely distributed throughout
the nucleoplasm and cytoplasm. Both the compounds were able
to cross the cellular bio-membrane, enter the nucleus region of
T47D cells. At low biological relevant concentration, compounds
were accumulated predominantly in the intracellular region and
partially in the intranuclear region. Thus, the confocal images
undoubtedly indicate that the compounds were able to interact
with proteins present in the nucleoplasm, cytoplasm and plasma
membrane (Fig. 5). Thereby, such protein interactions support
our concept of anti-cancer activity of these compounds.
a
a
petitive displacement of the fluorochrome tagged estrogen by the
TM
molecule under investigation from the ER-a-Fluormore ES2 com-
plex [49]. This change in the fluorescence is calculated for obtain-
ing the binding affinity of the compound for ER- . Bazedoxifene
was included as the reference standard for comparative study
Table 3). Anti-proliferative bioassay using T47D cells line and
a
(
binding affinity assay helped in revealing biologically significant
structural characteristics present in the synthesized compounds.
It was found that compounds having at least one methyl or
dimethyl substitution on ring D, F or both were showing best affin-
ity and anti-proliferative activity (Table 3). A similar observation
was also observed during the in silico screening, the presence of
methyl (5c, 6c) or dimethyl (5a, 6a, 5b, 6b, 6d) substituents
enables easy ingress of compounds to the receptor site as reflected
by higher docking score, higher lipophilic score as compared to
other compounds (Table 2, XP lipophilic EvdW). On the other hand,
the presence of a classical lipophilic group like phenyl (5d, 6e) and
chloro-phenyl (6f, 6j) group were expected to increase the
hydrophobic interactions with the receptor. But, on the contrary,
these groups renders the compound inactive at both the cellular
as well as at the receptor level as indicated by the MTT assay
and binding affinity (Table 3). In-depth analysis of the binding con-
firmation of these compounds revealed that the compounds were
It is reported that there is elevated levels of mRNA for ER-a in
BC progression is one of the important factor [51]. A decrease in
the mRNA level can be expected if synthesized molecules are active
and have a high affinity for ER-a. Since, the compounds 5c and 6d
not able to bind efficiently at the receptor site of ER-
a
due to steric
have shown effective anti-proliferative activity, strong binding
affinity and were able to enter and profusely distribute in the cyto-
plasm, nucleoplasm and cell membrane of the T47D cells line
hence were further evaluated for assessing their effect of on the
hindrance of the bulky side chains (Table 2). Such observations
were supported by the lower lipophilic score obtained upon dock-
ing of these compound (Table 2, XP lipophilic EvdW). Furthermore,
compounds having heterocyclic ring structure such as furan (6g, 6i,
abundance of mRNA of ER-
level of ER- was determined by semi-quantitative RT-PCR using
specific primers chosen from human DNA sequences and amplified
a in human T47D cells line. The mRNA
6
k) and benzo[d][1,3]dioxole (5f, 6h, 6l) as a side chain on D and F
a
ring improved the binding affinity and anti-proliferative activity.

7
8
R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
Table 3
Structure and biological evaluation of the synthesized indole derivatives.
S. No
1
Code
Anti-proliferative activity using T47D cell line, IC50
4.8 ± 0.71
=
lM
Binding affinity with ER-
153.04 ± 4.39
a, IC50 = nM
Structure
5a
2
3
4
5b
5c
5d
3.4 ± 0.67
3.8 ± 0.54
ND
65.96 ± 0.91
64.8 ± 0.11
ND
5
6
5e
5f
ND
ND
ND
60.78 ± 0.11
7
8
6a
6b
18.22 ± 0.88
15.76 ± 1.78
74.12 ± 0.29
73.15 ± 1.56
9
6c
24.75 ± 0.04
94.72 ± 2.63
1
1
0
1
6d
6e
14.63 ± 1.07
52.74 ± 1.33
ND
ND

R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
79
Table 3 (continued)
S. No Code
Anti-proliferative activity using T47D cell line, IC50
ND
=
lM
Binding affinity with ER-
ND
a
, IC50 = nM
Structure
1
2
6f
1
1
3
4
6g
6h
23 ± 0.02
65.91 ± 0.06
67.58 ± 7.73
23.98 ± 0.48
1
1
5
6
6i
6j
22.72 ± 0.56
90.46 ± 4.03
ND
ND
1
7
6k
ND
96.27 ± 9.12
1
1
8
9
6l
ND
87.92 ± 6.16
31.71 ± 1.41
Baz
16.43 ± 0.94
ND: Not determined as IC50 > 25 lM on T47D cell line and binding affinity IC50 > 1000 nM.
in simpliAmp thermal cycler (Applied biosystem). Upon analyzing
the agarose gel image and densitometric analysis (Fig. 6), it can be
confirmed that the expression of the mRNA of ER-a is completely
inhibited in the T47D cells treated with bazedoxifene, 5c, and 6d
as compared to the control (untreated) cells. Thus, it can be con-
cluded that compounds 5c and 6d decreases the expression of
In our earlier experimental results from semi-quantitative RT-
PCR confirm the potent activity of the compounds at mRNA level.
Therefore, for extending and confirming the results of RT-PCR,
Western blotting was performed. Subsequent analysis obtained
from the Western blot image and the densitometry analysis
revealed that expression of the ER-a protein was reduced to
ER-a
at the transcription level effectively similar to the standard
20.94% and 28.17% in the T47D cells treated with compound 5c
bazedoxifene, thereby downregulating the signaling and transacti-
and 6d respectively (Fig. 7). While comparing the mRNA level
vation pathways.
and the protein level of ER-
pounds completely inhibited the mRNA of ER-
reduced protein levels of ER- . The results suggest that receptor
degradation by the compounds was without the contribution of
the neosynthesized ER- pool (pre-formed ER- ). The compounds
may be effective only in the newly synthesized ER- , as the recep-
a
, it was observed that both the com-
Western blot has been widely for understanding the effect of
the investigational drugs on the target protein levels. Herein we
also utilized for quantifying the effect of lead molecules on the
expression of ER-
ated with the propagation and survival of BC cells. Non-genomic
and genomic pathways rely on the activation mediated by co-
a
but slightly
a
a
. Frequently, elevated levels of ER-
a
are associ-
a
a
a
tor undergoes post-translational modification which can result in
activators affiliated with ER-a [52]. Hence, it could be anticipated
reduced drug-receptor interaction. Similar results were obtained
that reduction in the inherent levels of ER-
a
leads to superior
with naringenin on the mRNA and protein levels of ER-
a [53]. It
management of BC.
signifies that compound 5c and 6d not only downregulate the

8
0
R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
Fig. 4. Human peripheral blood mononuclear cells assay for compound of Series I and II.
Fig. 5. CLSM analysis of T47D cells after treating the cells with compound 5c and 6d for 24 h and untreated cells as a control (Scale bar, 10 mM).
mRNA of the ER-
Therefore, synthesized compounds 5c and 6d exerts its anticancer
activity in T47D cells by targeting the expression of mRNA and pro-
a
but also reduces its protein levels in BC cells.
redocking the co-crystallized bazedoxifene present in the receptor
and RMSD was calculated which was found to be 0.02 Å. The sim-
ilar occupancy as reflected by RMSD and superposition diagram
(Fig. 8) signifies that bazedoxifene upon docking recapitulates
the confirmation resembling its natural antagonistic confirmation
tein of ER-
.5. Structural investigation of lead compounds by induced fit docking
The lead compounds and bazedoxifene were analyzed for the
a.
with the ER-a.
2
The superposition diagram revealed that the lead compounds
and bazedoxifene bind in similar confirmation and binding cavity
of the receptor (Fig. 9a, c). The compounds were able to form H-
mechanistic insight with receptor using induced fit docking
approach. The validation of the docking protocol was done by
bond interaction with Glu 353, Arg 394 and
p-p stacking with

R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
81
Fig. 6. mRNA expression profile of endogenous ER-a in the T47D cell line after treatment with bazedoxifene and synthesized compounds 5c and 6d. The cells were incubated
with 15, 5 and 10 mM bazedoxifene, 5c and 6d for 48 h respectively. Data were normalized with GAPDH as an internal control, the results shown here are the representatives
of three different experiments.
Fig. 7. Western blot analysis of endogenous ER-a protein in the T47D cells after treatment with bazedoxifene, 5c, and 6d. The cell line was incubated with 15, 5 and 10 mM of
bazedoxifene, 5c, and 6d respectively for 48 h. Data were normalized with b-actin as an internal control, the results shown here are the representatives of three different
experiments.
Fig. 8. (a) Superposition of the docked structure of bazedoxifene (blue) with the recrystallized experimental pose of bazedoxifene (orange) in 4XI3 with 0.02 Å rmsd (b)
Bazedoxifene ligand interaction diagram at the receptor site of ER-a.

8
2
R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
Phe 404 (Fig. 9b, d). In addition, 5c and 6d were able to form van
der Waals contacts with Met 343, Leu 346, Leu 349, Leu 354, Trp
alkyl substituent as in case of 5a, 5b, 5c, 6a, 6b, 6c, and 6d were
found to be active at both cellular and receptor level as they can
effectively enter the narrow cavity of the receptor and can form
H-bonding/Van der Waals contact at the receptor site. On the
contrary compound, 5e which was devoid of any alkyl sub-
3
5
83, Met 388, Met 421, Ile 424, Leu 428, Met 522, Leu 525, Cys
30, Val 533 and Pro 535 which were like the amino acid residues
present in the ligand interaction diagram of bazedoxifene. This fur-
ther signifies that compound was able to occupy the binding cavity
in an antagonistic confirmation analogous to bazedoxifene.
Thereby, indicating that compounds 5c and 6d upon binding to
the active site of ER-a by extensive H-bond and van der Waals con-
tacts causes a reduction in its expression.
stituent was neither having anti-proliferative activity nor ER-
a
binding affinity. Another noteworthy finding was that the pres-
ence of bulky aryl groups such as phenyl and chloro-phenyl
and as in case of compounds 5d, 6e, 6f and 6j reduces the activ-
ity of the compound. This can be due to restricted accesses of
The development of effective drugs for the treatment of
hormone-dependent cancer is emerging as an important field
of research in medicinal chemistry. The major strategies for hor-
monal therapy are to block the estrogens signaling pathways.
The molecular docking in addition to the anti-proliferative activ-
ity and binding affinity illuminated some key structural aspects
the compound to the binding cavity of ER-a, thereby, reducing
the bioactivity. In addition, presence of heteroaryl side chains
as in compounds 5f, 6g, 6h, 6i, 6k, and 6l adds an additional fac-
tor of H-bonding at the receptor site attributed to the heteroa-
tom. The enhanced bonding at the receptor site counters the
steric hindrance and hence improves the interaction at the
receptor site. Biological activity and in silico investigation imply
that anticancer potential of 5c and 6d was supported by strong
of the compound for targeting BC. The binding cavity of ER-
a
is narrow and lined by hydrophobic amino acids. The compound
must possess a lipophilic character for binding successfully to
the receptor. Hence, the structure-activity relationship revealed
some interesting findings. I was found that compounds having
binding affinity and confirmation to ER-
a of these compounds
similar to bazedoxifene which leads to reduced protein and
mRNA expression. Hence, 5c and 6d can be identified as a
Fig. 9. (a) Superposition of bazedoxifene (brown) with compound 5c (blue) at receptor site (b) ligand interaction diagram of compound 5c (c) Superposition of bazedoxifene
brown) with compound 6d (blue) at receptor site (d) ligand interaction diagram of compound 6d. (Green outline corresponds to Van der Waals contacts, cyan outline polar
interactions, pink arrow pointer H-bond interaction, green pointers stacking).
(
p-p

R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
83
potential lead molecule which might be of therapeutic impor-
tance for BC treatment.
the binding site (Site), metal binding terms (Metal), penalty for
buried polar groups (BuryP) and freezing rotatable bonds (RotB).
G ꢀ score ¼
D
H
bond
þ
D
Lipo
þ
D
D
Metal
þ Site þ 0:130Coul
D
3
. Conclusion
þ 0:065vdW ꢀ
BuryP
ꢀ
D
RotB
In summary, herein we have described the design, synthesis
and in vitro biological evaluation of novel indole derivatives for tar-
geting ER- . Indole based chromene and dihydropyridine deriva-
tives were developed, and the combinatorial library was
generated, screened by favorable drug-like ADME characteristics,
docking profile and synthetic accessibility. All the synthesized
derivatives were evaluated for the antiproliferative activity, cyto-
toxicity, and ER-a binding affinity. From these studies, it was found
that methyl and dimethyl substituted compounds were more
active then aryl substituted compounds. Compounds 5c and 6d
4
4
.3. Chemistry
a
.3.1. Chemistry
Starting materials and solvents were purchased from commer-
cial suppliers and used without further purification. Reaction pro-
gress was monitored by TLC using silica gel 60 F254 (0.040e0.063
mm) with detection by UV. Silica gel 60–120 and 200–400 was
used for column chromatography and flash chromatography
respectively. Yields refer to purified materials and are not opti-
were showing most promising antiproliferative and ER-
a
binding
1
13
mized. H NMR and C NMR spectra were recorded on a Jeol
00 MHz and Bruker 400 MHz spectrometer using the residual sig-
affinity. Therefore, 5c and 6d were forwarded for gene expression
studies. CLSM experiment indicated that resultant anti-
proliferative activity of the compounds on T47D cells line was
probably due to the accumulation of compound in the nucleo-
plasm, cytoplasm, and biomembrane. From the semi-quantitative
RT-PCR and Western blotting, it was found that compounds 5c
and 6d were able to decrease the mRNA and protein expression
4
nal of the deuterated solvent as internal standard. Splitting pat-
terns are described as singlet (s), doublet (d), triplet (t) and
broad (br); chemical shifts (d) are given in ppm. High-resolution
mass spectra (HRMS) were recorded on a Q-ToF Micro Waters.
Melting points were recorded on Stuart melting point apparatus
(
SMP-30) with open glass capillary tube and were uncorrected.
of the target ER-
obtained the study were encouraging considering that compound
c and 6d represents an example of indole derivatives which could
a, thereby reducing the ER-a activity. The result
4.3.2. General procedure for the synthesis of intermediates 2, 3 and 4
5
Potassium hydroxide (4 mmol) was stirred in dried dimethyl
be used for the lead optimization studies and can be expanded for
in vivo testing as a potent compound for the management of BC.
sulfoxide (25 mL) for 10 min in a round-bottom flask. Ethyl 1H-
indole-2-carboxylate 1 (1 mmol) was added to the solution, and
the mixture was stirred for 0.5 h. Benzyl bromide (1.3 mmol) was
added to the stirring mixture. After the completion of the reaction
4
. Experimental
(
1.5 h) as monitored by TLC, the reaction mixture was cooled to 0
4
.1. Combinatorial library enumeration and ADME screening
°C. The product was extracted with ethyl acetate; the organic layer
was washed using brine solution (40 mL). The combined organic
layer was dried over anhydrous sodium sulfate, filtered, concen-
trated and was purified by flash column chromatography (petro-
leum ether: ethyl acetate = 9: 1, v/v) on silica gel (200–400) to
get pure compound 2 with 95% yield.
For the synthesis of 3, lithium aluminum hydride (2 mmol) was
stirred in dry THF at 0 °C. Then 2 (1 mmol) was dissolved in THF
and added dropwise to the solution of lithium aluminum hydride
at 0 °C. Then, the mixture was slowly warmed to room tempera-
ture till the completion (0.5 h) of reaction as monitored by TLC. It
was then cooled to 0 °C, 10% aqueous sodium hydroxide (20 mL)
and 40 mL of water were added successively. The reaction mixture
was allowed to stir for 0.5 h, filtered through Celite and washed
with dry ethyl acetate, dried over anhydrous sodium sulfate, con-
centrated and purified by flash column chromatography (petro-
leum ether:ethyl acetate = 8:2, v/v) on silica gel (200–400) to get
pure compound 3 with 98% yield.
A combinatorial library was generated with the use of the Com-
biGlide software [33] by an iterative combination of an in-house
database of reagents substituting R and R group (Fig. 1) with core
structures. The compounds were subjected to ADME filtering and
the following criteria were applied for filtering the compounds
with favorable profiles log octanol/water ratio (QPlog Po/w, limit:
1
2
ꢀ
2.0 to 6.5), QPlog hERG (limit: above-5.0), QPP Caco (acceptable
value: poor ꢁ25, great ꢁ500(nm/s) QPPMDCK (acceptable limit:
poor ꢁ25, great ꢁ500(nm/s)), QPlogKhsa (limit: ꢀ1.5 to 1.5) and
percent oral absorption.
4
.2. Docking with ER-a
The 2D structure of ligands was constructed using the Maestro
.6 software and saved in sdf format (standard data format). The
D structures were converted to the 3D structure using the Ligprep
9
2
For the synthesis of 4, compound 3 (1 mmol) was dissolved in
module of Maestro 9.6. This module adds hydrogens and elimi-
nates any discrepancies between bond length and angle. The 3-
2 2
dry CH Cl (25 mL) was stirred briefly. Activated manganese diox-
ide (7.3 mmol) was added, stirred at room temperature for 4 h.
Another portion of activated manganese dioxide (5.9 mmol) was
added and stirred at room temperature for 0.5 h. After completion
of the reaction, as monitored by TLC, the reaction mixture was fil-
tered through Celite and washed with ethyl acetate, concentrated
and purified by flash chromatography (petroleum ether:ethyl acet-
ate 9.5:0.5, v/v) on silica gel (200–400) to afford pure compound 4
with 70% yield. The structure of the compounds 2, 3 and 4 were
confirmed by comparing the spectroscopic data with the reported
data.
dimensional and X-ray structure coordinates of ER-a was obtained
from protein data bank with PDB id code 4XI3 (www.rcsb.org).
Protein preparation wizard of maestro 9.6 was used for protein
preparation. Protein preparation begins with the application of
Impact molecular mechanics program for correcting the bond
orders, adding missing hydrogens and removing the crystallized
waters that are not present in the active site. Docking procedure
was performed using GLIDE (Grid-based Ligand Docking with Ener-
getics) software 9.6. Docking score was taken into consideration
for comparing the results, more the negative value of docking score
more potent is the compound and indicates the more binding
potential. Docking score has several components such as hydrogen
bonds (H-bond), hydrophobic contacts (Lipo), van der-waals inter-
actions (vdW), coulombic interactions (Coul), polar interactions in
4.3.3. General procedure for the synthesis of compounds 5a–f
For the synthesis of 5a–f, a mixture of compound 4 (1 mmol),
corresponding dimedone (1 mmol) and malononitrile (1 mmol)

8
4
R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
was refluxed with stirring for 12 h and was monitored for comple-
tion using TLC. The reaction was generally completed in 12–15 h.
After the completion of the reaction, excess of ethanol was
removed under reduced pressure. The product was extracted using
ethyl acetate and organic layer was washed with distilled water.
The combined organic layer was dried over anhydrous sodium sul-
fate, filtered, concentrated and purified by flash column chro-
matography (petroleum ether:ethyl acetate = 9:1, v/v) on silica
gel (200–400) to afford compounds 5a–f in 50–70% yields. The
structures were confirmed by NMR and HRMS.
7.30–7.25 (2H, m), 7.23–7.19 (1H, m), 7.16–7.14 (1H, d, J = 8 Hz),
7.10–7.02 (4H, m), 6.32–6.31 (1H, m), 5.74–5.52 (2H, m), 4.65
(1H, s), 4.59 (2H, s), 2.54–2.44 (1H, m), 2.37–2.32 (1H, m), 2.24–
2.18 (1H, m), 2.04–1.80 (2H, m), 1.41–1.24 (1H. m); 13C NMR
(100 MHz, DMSO-d6) d 196.12, 163.08, 157.93, 142.33, 137.93,
137.34, 128.70 (2C), 127.80, 127.19, 126.21 (2C), 121.64, 120.30,
119.83, 118.45, 114.15, 110.13, 100.23, 62.20, 46.98, 35.08, 28.25,
+
[M]+
27.38, 20.60. HRMS (ESI): m/z calcd for
C
25
H
21
N
3
O
2
395.1634; found: 395.1633.
4.3.3.6. 2-amino-7-(benzo[d][1,3]dioxol-5-yl)-4-(1-benzyl-1H-indol-
4
.3.3.1.
2-amino-4-(1-benzyl-1H-indol-2-yl)-7,7-dimethyl-5-oxo-
2-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile
white solid; yield 69%; mp 234–235 °C; H NMR (400 MHz, CDCl )
3
(5f).
1
5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (5a). yellow solid;
1
yield 50%; mp 191–193 °C; H NMR (400 MHz, CDCl
.50 (1H, m), 7.30–7.26 (2H, m), 7.22–7.18 (1H, m), 7.16–7.14
1H, d, J = 8 Hz), 7.11–7.09 (2H, m), 7.07–7.02 (2H, m), 6.30 (1H,
3
) d 7.52–
d 7.56–7.51 (1H, m), 7.32–7.23 (4H, m), 7.17–7.14 (1H, m), 7.10–
7.03 (4H, m), 6.77–6.71 (1H, m), 6.64–6.54 (2H, m), 5.94 (2H, s),
5.74–5.51 (2H, m), 4.70 (1H, s), 4.56 (2H, s), 3.30–2.86 (1H, m),
7
(
s), 5.73–5.53 (2H, m), 4.69 (2H, s), 4.63 (1H, s), 2.31–2.30 (2H,
m), 2.17–2.13 (1H, m), 2.05–2.01 (1H, m), 1.04 (3H, s), 1.01 (3H,
s). C NMR (100 MHz, CDCl
1
1
4
C
2.66–2.59 (2H, m), 2.53–2.39 (1H, m), 2.24–2.04 (1H, m). 13
C
3
NMR (100 MHz, CDCl ) d 195.34, 161.83, 157.64, 148.09, 146.85,
141.15, 138.06, 137.60, 135.63, 128.91 (2C), 127.78, 127.28,
126.16 (2C), 121.78, 120.50, 119.92, 119.77, 119.84, 118.30,
1
3
3
) d 196.42, 161.77, 158.05, 142.52,
37.95, 137.30, 128.77 (2C), 127.87, 127.23, 126.32 (2C), 121.62,
20.36, 119.82, 118.72, 113.09, 110.16, 101.44, 61.60, 50.56,
7.03, 40.68, 32.18, 28.42, 28.20, 27.56. HRMS (ESI): m/z calcd for
109.97, 108.57, 107.02, 102.30, 101.27, 62.09, 46.94, 44.01, 38.54,
+
[M + H]+
34.93, 28.21. HRMS (ESI): m/z calcd for C32
H
26
N
3
O
4
+
+
27
H
26
N
3
O
2
[M + H] 424.2025; found: 424.2023.
516.1923; found: 516.1924.
4.3.3.2.
2-amino-4-(1-benzyl-1H-indol-2-yl)-8,8-dimethyl-5-oxo-
4.3.4. General procedure for the synthesis of compounds 6a–l
5
,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (5b). yellow solid;
For the synthesis of 6a–l, a mixture of compound 4 (1 mmol),
corresponding dimedone (2 mmol), ammonium acetate (1 mmol)
and ascorbic acid (10 mol%) was stirred for at 10 °C and was mon-
itored for completion using TLC. The reaction was generally com-
pleted in 9–10 h. After the completion of the reaction, excess of
ethanol was removed under reduced pressure. The product was
extracted using ethyl acetate and organic layer was washed with
distilled water. The combined organic layer was dried over anhy-
drous sodium sulfate, filtered, concentrated and purified by iso-
cratic flash column chromatography (petroleum ether:ethyl
acetate = 9:1, v/v) on silica gel (200–400) to afford compounds
1
yield 69%; mp 116–118 °C; H NMR (400 MHz, DMSO-d6) d 7.53–
.51 (1H, m), 7.29–7.25 (2H, m), 7.22–7.18 (1H, m), 7.14–7.12
1H, m), 7.10–7.03 (4H, m), 6.31–6.25 (1H, m), 5.73–5.52 (2H,
7
(
m), 4.69 (2H, s), 4.63 (1H, s), 2.49–2.46 (1H, m), 2.38–2.30 (1H,
m), 1.78–1.73 (1H, m), 1.72–1.57 (1H, m), 1.04 (3H. s), 1.03 (3H,
s). C NMR (100 MHz, CDCl3) d 196.20, 161.53, 158.01, 142.71,
1
1
4
C
1
3
38.00, 137.31, 128.75 (2C), 127.92, 127.19, 126.25 (2C), 121.51,
20.32, 119.75, 118.65, 112.45, 110.22, 101.34, 61.77, 47.04,
0.55, 34.79, 33.28, 28.04, 25.95, 24.37. HRMS (ESI): m/z calcd for
+
+
27
H
26
N
3
O
2
[M + H] 424.2025; found: 424.2027.
6
a–l in 60–70% yields. The structures were confirmed by NMR
4.3.3.3. 2-amino-4-(1-benzyl-1H-indol-2-yl)-7-methyl-5-oxo-5,6,7,8-
and HRMS.
tetrahydro-4H-chromene-3-carbonitrile (5c). pale yellow solid;
1
yield 70%; mp 203–204 °C; H NMR (400 MHz, DMSO-d6) d 7.52–
4.3.4.1. 9-(1-benzyl-1H-indol-2-yl)-3,3,6-trimethyl-3,4,6,7,9,10-hex-
7
7
5
2
.51(1H, d, J = 8 Hz), 7.30–7.25 (2H, m), 7.24–7.18 (1H, m),
.16–7.14(1H, d, J = 8 Hz), 7.09–7.02 (4H, m), 6.31–6.30 (1H, m),
.73–5.51 (2H, m), 4.64 (1H, s), 4.58 (2H, s), 2.53–2.44 (1H, m),
ahydroacridine-1,8(2H,5H)-dione (6a). off white solid; yield 77%;
1
3
mp 175–177 °C; H NMR (400 MHz, CDCl ) d 12.17 (1H, s), 7.58–
7.57 (1H, m), 7.30–7.27 (4H, m), 7.18–7.16 (2H, m), 6.93–6.89
(2H, m), 6.48 (1H, s), 5.55 (1H, s), 5.13 (2H, s), 2.49–2.40 (2H, m),
2.32–2.27 (2H, m), 2.20–2.15 (2H, m), 2.05–2.01 (2H, m), 1.92–
1
3
.36–2.18 (2H, m), 2.03–1.79 (2H, m), 1.04–1.03 (3H, m).
C
NMR (100 MHz, DMSO-d6) d 196.10, 163.06, 157.91, 142.31,
1
3
1
1
4
C
37.91, 137.38, 128.68 (2C), 127.78, 127.17, 126.19 (2C), 121.62,
20.28, 119.81, 118.43, 114.13, 110.11, 101.44, 62.18, 46.96,
4.83, 35.06, 34.80, 27.36, 20.58. HRMS (ESI): m/z calcd for
1.78 (1H, m), 1.05 (6H, s), 1.04–1.03 (3H, m), C NMR (100 MHz,
CDCl ) d 191.25, 190.43, 146.55 (2C), 138.23, 137.85, 136.94,
3
128.23 (2C), 127.76, 125.77 (2C), 125.70, 121.63, 120.36, 119.94,
115.43 (2C), 109.59, 102.81, 47.98, 47.03, 46.16, 40.05, 39.35,
+
+
26
24 3 2
H N O [M + H] 410.1869; found: 410.1868.
3
C
8.56, 37.11, 27.75, 22.77, 22.69, 20.83. HRMS (ESI): m/z calcd for
H N O [M + 2H] 466.2620; found:466.2629.
31 34 2 2
+
+
4.3.3.4. 2-amino-4-(1-benzyl-1H-indol-2-yl)-5-oxo-7-phenyl-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile (5d). bright yellow solid;
1
yield 64%; mp 141–142 °C; H NMR (400 MHz, CDCl
3
) d 7.57–
4.3.4.2. 9-(1-benzyl-1H-indol-2-yl)-3,3,6,6-tetramethyl-3,4,6,7,9,10-
7
.51 (1H, m), 7.35–7.24 (6H, m), 7.16–7.14 (2H, m), 7.10–7.04
hexahydroacridine-1,8(2H,5H)-dione (6b). yellow solid; yield 45%;
mp 220–222 °C; H NMR (400 MHz, CDCl3) d 12.16 (1H, s), 7.57
(1H, m), 7.27 (1H, m), 7.25 (1H, m), 7.19 (2H, m), 7.08 (1H, m),
1
(
(
5H, m), 6.42–6.31 (1H, m), 5.74–5.54(2H, m), 4.72 (1H, s), 4.65
2H, s), 3.37–2.93 (1H, m), 2.71–2.63 (2H, m), 2.57–2.45 (2H, m).
1
3
3
C NMR (100 MHz, CDCl ) d 195.27, 162.87, 158.03, 141.97,
7.06 (2H, m), 6.95 (1H, m), 6.48 (1H, s), 5.13 (2H, s), 4.13 (1H, s),
1
3
1
1
1
41.68, 138.11, 137.54, 129.06 (2C), 128.91(2C), 128.86, 127.88,
27.50, 126.70 (2C), 126.20 (2C), 121.85, 120.48, 120.01, 118.44,
14.40, 110.19, 101.96, 62.22, 47.00, 43.79, 38.68, 37.64, 27.96.
3
2.20 (4H, s), 1.25 (4H, s), 0.95 (12H, s); C NMR (100 MHz, CDCl )
d 190.10 (2C), 144.88 (2C), 138.21, 137.84, 136.93, 128.91 (2C),
128.21, 127.08 (2C), 125.80, 121.66, 120.37, 119.93, 115.41 (2C),
+
+
HRMS (ESI): m/z calcd for C31
H
26
3
N O
2
[M + H] 472.2025; found:
109.58, 102.79, 47.02, 46.85 (2C), 46.23 (2C), 31.97, 29.81, 28.45,
+
4
72.2028.
27.92, 27.73, 27.40, 22.76. HRMS (ESI): m/z calcd for C32
H
36
N
2
O
2
+
[
M + 2H] 480.2777; found: 480.2717.
4.3.3.5. 2-amino-4-(1-benzyl-1H-indol-2-yl)-5-oxo-5,6,7,8-tetrahy-
dro-4H-chromene-3-carbonitrile (5e). red solid; yield 53%; mp
4.3.4.3. 9-(1-benzyl-1H-indol-2-yl)-3,6-dimethyl-3,4,6,7,9,10-hexahy-
droacridine-1,8(2H,5H)-dione (6c). off white solid; yield 55%; mp
1
1
89–190 °C; H NMR (400 MHz, DMSO-d6) d 7.53–7.52 (1H, m),

R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
85
1
90–193 °C; 1H NMR (400 MHz, CDCl
m), 7.49 (1H, m), 7.24 (1H, m), 7.08 (2H, m), 7.01 (1H, m), 6.96
3
) d 12.45 (1H, s), 7.57 (1H,
(4H, s), 5.94 (2H, s), 5.62–5.53 (2H, m), 5.20 (2H, s), 5.06 (1H, s),
2.72–2.66 (4H, m), 1.26–1.20 (4H, m), 0.86–0.76 (2H, m). 13
NMR (100 MHz, CDCl ) d 190.07 (2C), 155.33 (2C), 141.79 (2C),
138.15, 137.98, 137.74 (2C), 137.70 (2C), 136.66, 128.90 (2C),
127.47, 125.61 (2C), 125.51, 121.79, 121.69, 120.38 (2C), 120.08,
116.51 (2C), 110.14 (2C), 109.63 (2C), 109.79, 104.80 (2C),
C
(
(
(
2H, m), 6.90 (1H, m), 6.49 (1H, s), 5.54 (2H, s), 5.15 (1H, s), 2.41
4H, m), 1.95 (4H, m), 1.30–1.22 (2H, m), 0.90 (6H, m); C NMR
100 MHz, CDCl3) d 191.26 (2C), 147.61 (2C), 141.86, 137.78,
3
1
3
1
1
2
C
37.15, 128.82 (2C), 128.70, 127.08 (2C), 125.66, 121.57, 120.31,
19.93, 116.79 (2C), 109.65, 102.73, 54.03, 47.06 (2C), 40.97 (2C),
102.92, 47.03, 38.48 (2C), 37.44 (2C), 30.88 (2C), 28.35. HRMS
+
6.79 (2C), 26.67 (2C), 20.54. HRMS (ESI): m/z calcd for
(ESI): m/z calcd for C42
36
H N
2
O
6
+ [M + 2H] 664.2573; found:
+
30
H
32
N
2
O
2
+ [M + 2H] 452.2464; found: 452.2461.
664.2525.
4
.3.4.4. 9-(1-benzyl-1H-indol-2-yl)-4,4,5,5-tetramethyl-3,4,6,7,9,10-
4
.3.4.9.
9-(1-benzyl-1H-indol-2-yl)-3-(furan-2-yl)-6-phenyl-
hexahydroacridine-1,8(2H,5H)-dione (6d). light yellow solid; yield
1
3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (6i). light brown
5
7
7
3
4%; mp 170–172 °C; H NMR (400 MHz, CDCl ) d 12.69 (1H, s),
1
solid; yield 80%; mp 135–137 °C; H NMR (400 MHz, CDCl
2.85 (1H, s), 7.32–7.24 (10H, m), 7.10–7.07 (3H, m), 6.99–6.95
2H, m), 6.28 (1H, s), 6.03–5.95 (1H, m), 5.68–5.53 (1H, m), 5.22
(2H, s), 5.10 (1H, s), 2.55–2.49 (4H, m), 1.26–1.21 (4H, m), 0.86–
3
)
.58–7.57 (1H, m), 7.56–7.55 (1H, m) 7.52–7.49 (1H, m), 7.30–
.27 (2H, m), 7.21–7.19 (1H, m), 7.10–7.08 (2H, m), 7.06–7.05
1
(
(
1H, m), 7.03–7.02 (1H, m), 5.49 (2H, s), 4.94 (1H, s), 1.38 (4H,
13
m), 1.23 (4H, m), 1.07 (12H, m). C NMR (100 MHz, CDCl3) d
96.62 (2C), 166.77 (2C), 142.22, 137.76, 137.52, 129.14, 128.79
2C), 127.20, 125.59 (2C), 121.49, 120.30, 119.88, 115.14 (2C),
1
3
0
1
1
.81 (2H, m).
3
C NMR (100 MHz, CDCl ) d 190.42, 190.15,
1
(
1
2
55.46, 151.61 (2C), 141.90, 140.18, 137.73, 137.65, 136.69,
28.88 (2C), 128.78 (2C), 128.72, 127.05 (2C), 126.58 (2C), 125.55
09.84, 102.51, 51.07, 38.17 (2C), 35.63 (2C), 33.25 (2C), 28.03,
36 2 2
6.24 (2C), 24.43 (2C). HRMS (ESI): m/z calcd for C32H N O +
(
2C), 121.60, 120.22, 119.93, 116.41 (2C), 109.77 (2C), 109.51,
102.75, 51.07, 46.99, 46.85, 40.88, 39.80, 36.86, 29.69, 28.26. HRMS
ESI): m/z calcd for
[M + 2H]+ 566.2569;
found:566.2534.
+
[
M + 2H] 480.2777; found:480.2719.
(
38 34 2 3
C H N O +
4.3.4.5. 9-(1-benzyl-1H-indol-2-yl)-3,6-diphenyl-3,4,6,7,9,10-hexahy-
droacridine-1,8(2H,5H)-dione (6e). light brown solid; yield 40%; mp
1
1
(
10–112 °C; H NMR (400 MHz, CDCl
1H, m), 7.60–7.58 (1H, m), 7.33–7.32 (4H, m), 7.29–7.26 (8H,
m), 7.20–7.18 (2H, m), 7.14–7.10 (2H, m), 7.04–6.99 (1H, m),
.62 (1H, s), 5.26 (2H, s), 3.30 (1H, s), 2.54 (4H, m), 1.63–1.55
3
) d 12.90 (1H, s), 7.64–7.62
4.3.4.10.
9-(1-benzyl-1H-indol-2-yl)-3-(4-chlorophenyl)-6-phenyl-
3
,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (6j). yellow solid;
1
yield 92%; mp 117–119 °C; H NMR (400 MHz, CDCl3) d 12.84
1H, s), 7.61–7.56 (1H, m), 7.31–7.22 (10H, m), 7.13–7.08 (4H,
m), 7.02–6.97 (4H, m), 5.67 (1H, s) 5.23 (2H, s), 2.65–2.58 (4H,
6
(
13
(
(
(
2H, m), 1.22 (4H, m);
2C),150.14 (2C), 142.07 (2C), 140.07, 137.90, 136.86, 128.88
6C), 127.22, 126.75 (6C), 125.72 (3C), 121.77, 120.39, 120.10,
3
C NMR (100 MHz, CDCl ) d 190.59
1
3
m), 2.55–2.47 (4H, m), 1.57–1.42 (2H, m). C NMR (100 MHz,
CDCl ) d 189.07, 188.83, 149.13 (2C), 140.98, 139.84, 139.28,
36.70, 135.50, 131.78, 127.85 (4C), 127.77 (2C), 127.07, 126.92
3
1
16.57 (2C), 109.94, 102.92, 47.16, 41.05 (2C), 39.97 (2C), 37.03
1
+
(
36 2 2
2C), 28.43. HRMS (ESI): m/z calcd for C40H N O + [M + 2H]
(
(
3
6
4C), 126.61 (2C), 124.57 (2C), 120.68, 119.22, 119.00, 115.40
2C), 108.76, 101.77, 52.96, 45.96, 45.83, 39.72, 38.69, 35.64,
5
76.2777; found: 576.2721.
2 2
5.28, 27.22. HRMS (ESI): m/z calcd for C40H35ClN O
+ [M + 2H]+
4.3.4.6. 9-(1-benzyl-1H-indol-2-yl)-3,6-bis(4-chlorophenyl)-3,4,6,7,9,10-
10.2387; found:610.2374.
hexahydroacridine-1,8(2H,5H)-dione (6f). yellow solid; yield 50%;
mp 122–124 °C; H NMR (400 MHz, CDCl
1
3
) d 12.86 (1H, s), 7.61–
7
.60 (1H, m), 7.31–7.24 (8H, m), 7.19–7.09 (2H, m), 7.02–6.97
4
.3.4.11. 9-(1-benzyl-1H-indol-2-yl)-3-(4-chlorophenyl)-6-(furan-2-
(
(
6H, m), 6.58–6.45 (1H, m), 5.67 (1H, s), 5.23 (2H, s), 2.64–2.55
yl)-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (6k). off white
4H, m), 1.63–1.41 (2H, m), 1.29–1.20 (4H, m). 13C NMR (100
1
solid; yield 55%; mp 190–193 °C; H NMR (400 MHz, CDCl
2.84 (1H, s), 7.61–7.55 (1H, m), 7.30–7.25 (9H, m), 7.02–6.95
4H, m), 6.56 (1H, s), 6.34–6.28 (2H, m), 5.22 (2H, s), 5.09 (1H, s),
3
) d
MHz, CDCl
3
) d 190.23 (2C), 150.29 (2C), 142.14 (2C), 140.44,
1
(
2
1
1
4
37.85, 136.66, 132.94 (2C), 129.01 (6C), 128.23, 128.08 (6C),
25.73, 121.84, 120.38, 120.15, 116.56 (2C), 109.92, 102.93,
7.12, 40.87 (2C), 39.85 (2C), 36.43 (2C), 28.38. HRMS (ESI): m/z
13
.75–2.65 (4H, m), 1.59–1.52 (2H, m), 0.87–0.82 (4H, m).
C
NMR (100 MHz, CDCl ) d 190.20, 189.94, 155.35 (2C), 146.56,
3
+
calcd for C40
H34Cl
2
N
O
2 2
+ [M + 2H] 644.1997; found: 644.1864.
1
1
1
4
41.82, 141.79, 140.47, 136.64, 136.41, 132.91, 129.01 (2C),
28.91 (2C), 128.21 (2C), 128.05 (2C), 127.76, 125.75, 121.81,
20.38, 120.12, 116.54 (2C), 110.15 (2C), 109.85, 102.93, 46.98,
4.3.4.7.
9-(1-benzyl-1H-indol-2-yl)-3,6-di(furan-2-yl)-3,4,6,7,9,10-
hexahydroacridine-1,8(2H,5H)-dione (6g). brown solid; yield 95%;
mp 142–144 °C; H NMR (400 MHz, CDCl3) d 12.84 (1H, s), 7.60–
0.87, 39.86, 38.55, 37.40, 30.94, 29.83, 28.38. HRMS (ESI): m/z
1
+
calcd for C38
2
H33ClN O
3
+ [M + 2H] 600.2180; found: 600.2131.
7
.56 (1H, m), 7.31–7.24 (4H, m), 7.18–7.07 (3H, m), 6.97–6.96
(
(
2H, m), 6.78–6.72 (2H, m), 6.56–6.51 (4H, m), 5.93 (2H, s), 5.28
1H, s), 2.59–2.43 (4H, m), 1.23–1.20 (4H, m), 0.86–0.82 (2H, m).
4.3.4.12. 3-(benzo[d][1,3]dioxol-5-yl)-9-(1-benzyl-1H-indol-2-yl)-6-
(4-chlorophenyl)-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione
(6l). yellow solid; yield 45%; mp 140–142 °C; H NMR (400 MHz,
1
3
C NMR (100 MHz, CDCl
2C), 142.77 (2C), 138.60, 138.52, 137.56, 129.59 (2C), 127.92,
27.45 (2C), 126.42, 122.47, 121.09, 120.80, 117.28 (2C), 110.64
2C), 110.38 (2C), 109.06, 103.62, 53.89, 47.86 (2C), 41.75 (2C),
3
) d 191.29 (2C), 156.33 (2C), 152.48
1
(
1
(
4
5
CDCl3) d 12.82 (1H, s), 7.61–7.59 (1H, m), 7.33–7.25 (7H, m),
7.11–7.09 (2H, m), 7.08–6.97 (4H, m), 6.75–6.73 (1H, m), 6.57
(2H, s), 5.94 (2H, s), 5.65 (1H, s), 5.23 (2H, s), 2.64–2.58 (4H, m),
+
30 2 4
0.67, 37.73, 29.13. HRMS (ESI): m/z calcd for C36H N O + [M]
1
3
54.2206; found: 554.2231.
2.50–2.44 (4H, m), 1.33–1.21 (2H, m); C NMR (100 MHz, CDCl3)
d 195.23, 195.00, 157.52 (2C), 147.97, 146.73, 141.75, 141.04,
137.95, 137.48, 135.51, 135.45, 128.80 (2C), 128.72 (2C), 127.67,
127.25, 127.16 (2C), 126.05 (2C), 121.67, 120.39, 119.81, 119.72,
118.19 (2C), 110.04, 109.85, 108.45, 102.19, 101.16, 46.83, 43.95,
4
3
.3.4.8. 3,6-bis(benzo[d][1,3]dioxol-5-yl)-9-(1-benzyl-1H-indol-2-yl)-
,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (6h). off white
1
solid; yield 55%; mp 190–193 °C; H NMR (400 MHz, CDCl
2.86 (1H, s), 7.31–7.25 (6H, m), 7.09–7.08 (2H, m), 7.01–6.98
1H, m), 6.95–6.93 (1H, m), 6.91–6.89 (1H, m), 6.55 (1H, s), 6.27
3
) d
1
(
43.89, 38.42, 37.33, 34.82, 32.88, 28.10. HRMS (ESI): m/z calcd for
+
C
41
H35ClN
2
O
4
+ [M + 2H] 654.2285; found: 654.2275.

8
6
R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
4.4. Biological assay methods
followed by adding 40 mL human recombinant ER-a/fluoromone
TM complex solution. After mixing and allowing at least 2 h incu-
bation at room temperature with light protection of the reagents.
The fluorescence of each well was measured using the Synergy 2
multi-mode microplate reader (BioTek) with 535 nm excitation fil-
ters and 590 nm emission filters. Three controls were included in
the assay: (1) assay maximum polarization control (40 mL of ER/flu-
oromone complex solution and 40 mL of ER green assay buffer with
4% DMSO, provides maximum polarization value for assay); (2)
assay minimum polarization control (40 mL of ER/fluromone com-
plex solution and 40 mL of oestradiol (20 mM), provides bottom
baseline); (3) free fluoromone tracer control (40 mL Fluoromone
tracer and 40 mL of ER green assay buffer with 4% DMSO, provides
absolute minimum polarization value). The polarization values
were normalized to percent inhibition using the following
equation.
The biological methodologies and techniques utilized herein
corresponds to our recent published reports with slight modifica-
tions [20,21].
4
.4.1. Culturing and maintaining of cancer cell line
Human BC cell line T47D were procured from National Centre
for Cell Sciences (NCCS) Pune. T47D cells were grown in the Ros-
well Park Memorial Institute-1640 (RPMI-1640) medium supple-
mented with 10% heat inactivated (30 min at 56 °C) FBS (Fetal
Bovine Serum), 1X penicillin and streptomycin in a tissue culture
2
flask at 37 °C in a humidified atmosphere of 5% CO incubator. At
7
0–80% confluence the cells were trypsinized using 1X trypsin-
EDTA and sub-cultured in a new sterile tissue culture flask for fur-
ther experiments.
I% ¼ ðA
I% = the percent inhibition, A
0
ꢀ AÞ=ðA
0
ꢀ A100Þ ꢂ 100
= Absorbance at 0% inhibition, A100 =
4
.4.2. Anti-proliferative activity
The anti-proliferative activity of the compounds was evaluated
0
using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo
lium bromide) assay. It is a colorimetric assay for assessing cell
metabolic activity. Viable cells with active metabolism convert
the MTT into a purple colored formazan product with an absor-
bance maximum near 570 nm. When cells die, they lose the ability
to convert MTT into formazan, thus color formation serves as a use-
ful and convenient marker of only the viable cells. Human BC cell
line T47D were seeded in 96 wells plate at cell density 1x10 per
well and after 24 h, cells were treated with different concentration
of synthesized compounds (1, 5, 25 lM) and incubated for 48 h in
Absorbance at 100% inhibition, and A = observed Absorbance value.
The percent inhibition values are plotted against the concentra-
tion of test compounds and analyzed by a non-linear regression
curve fitting. The concentration of the test compounds needed to
displace half of the bond ligand equals IC50 of the test compound.
4.4.5. Confocal microscopy
4
Cell imaging is an important technique for locatisation of the
compound inside the cell. As the ER is located in the plasma mem-
brane, cytosol, and nucleoplasm, live cell imaging enables to track
the compound and site of action. Cells were seeded in sterile glass
FBS-free media. MTT solution (final concentration of MTT was 0.5
mg/ml) was added followed by incubation for 4 h at 37 °C. After 4
h; the purple color formazone crystals were solubilized using 100
4
coverslip at 5 ꢂ 10 cells/ml in a 6 well plate. After 24 h of seeding
the cells were treated with 5 and 15 mM of 5c and 6d for 48 h after
that cells were washed with 1X PSB. After that cells were fixed
with 2% formaldehyde solution for 10 min in the dark, again cells
were washed thrice with 1X PBST. Then cells were mounted on
glass cover by using mounting media. Cells were visualized an
image was captured under an Olympus FV1200 Laser Scanning
Microscopes fluorescence confocal microscope; the captured
image was analyzed with Olympus Fluoview software of Olympus
version 4.2a. The CLSM experiment was performed in duplicate.
lL of acidified DMSO with 0.6% acetic acid and absorbance was
measured at both 570 nm and 620 nm. The absorbance reading is
directly proportional to a number of living cells [54]. The cell via-
bility of each group was calculated with their respective control.
The Anti-proliferative experiment was performed in triplicate.
4
.4.3. Human peripheral blood mononuclear cells (PBMCs) culture and
MTT assay
Majority of the chemotherapeutic agents available having a side
effect of inducing apoptosis in the normal cell along with the can-
cer cells. Therefore, in order to analyze the side effect of the com-
pounds on the normal cells at higher concentrations, human PBMC
was cultured and MTT assay was performed. Fresh blood was
drawn from healthy individual as per the protocol no. CUPB/
cc/14/IEC/4483 approved by Institutional Ethics Committee of Cen-
tral University of Punjab, Bathinda. The protocol used was standard
operating procedure, provided by Institutional Ethics Committee of
Central University of Punjab according to guidelines issued by
Indian Council of Medical Research (ICMR), Govt. of India. PBMC
were isolated from whole blood discarding the RBCs (Red Blood
Cells) after treatment with RBC lysis buffer. The PBMCs were
counted on the automated cell counter (Invitrogen). The cells were
then suspended in RPMI-1640 media supplemented with 10% fetal
4.4.6. Preparation of total cell lysates
To determine target specificity of the compounds, there is a
need to establish the expression of target proteins. Cell lysates
were prepared from both treated and untreated cells of T47D
Briefly, cells were first rinsed twice gently with ice-cold 1X PBS,
then 300–500 lL of modified RIPA buffer [50 mM Tris-HCl (pH
7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and protease inhibitor
cocktail (1 mL per 100 mL of lysis buffer)] was added per plate, kept
on ice for 5 min, and cells were mechanically scraped using plastic
cell scraper, and cell suspension was transferred into pre-cooled
1.5 mL microcentrifuge tube [55]. Following centrifugation, the
pellet was re-suspended in the cell lysis buffer with intermediate
vortexing every 5 min for 20 min at 4 °C. Then, the mixture was
centrifuged at 12,000g for 20 min at 4 °C. The supernatant was col-
lected in prechilled sterile microcentrifuge tubes and stored at
ꢀ20 °C for further experiments. The concentration of total protein
in this supernatant was estimated by using standard Bradford
method with BSA as standard [56].
bovine serum (FBS), 1ꢂ antibiotic solution and incubated at 37 °C
4
2
for 24 h in a humidified chamber of 5% CO . Approximately 10
cells were seeded in each well of the 96 well plate. MTT assay
was performed as described in Section 4.4.2. The cytotoxicity of
the compounds was evaluated at 25, 50 and 100 mM concentration
and the experiment was performed in triplicate.
4.4.7. Western blot analysis
50 lg of total protein samples were resolved on 10% denatura-
4
.4.4. Estrogen receptor competitive binding assay
A stock solution of each compound was serially diluted using
tion SDS-PAGE and transferred to a nitrocellulose membrane.
Membranes were blocked for 1 h with 5% nonfat dry milk (NFDM)
in 1X PBST. All antibody dilutions were made in 2.5% NFDM-PBST.
Membranes were incubated (4 °C overnight) with primary
DMSO, and the 40 mL of each concentration was transferred to
the assay plate (Grenier, 384-well high volume flat bottom)

R. Singla et al. / Bioorganic Chemistry 79 (2018) 72–88
87
antibodies of rabbit
PBST, incubated with horseradish peroxidase-conjugated sec-
ondary antibody (Invitrogen) for 1 h. The image was captured by
using Bio-Rad ChemiDoc MP imaging system after applying
enhanced chemiluminescence (Clarity Western ECL Substrate of
Bio-Rad), and densitometric analysis was done using Image Lab
software of Bio-Rad version 5.2. The Western blotting experiment
was performed in triplicate.
a
-ER-
a
(HC-20. Membranes were washed with
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_
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4.4.8. Total RNA isolation and cDNA synthesis
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Total RNA was isolated from T47D cells from both treated and
untreated cells by using TRIzolÒ (Invitrogen) and followed the
3320–3328.
manufacturer instructions, and finally, RNA pellet was resus-
pended in nuclease-free water. Quality of isolated RNA was
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turing agarose gel. Traces of DNA contamination was removed by
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the quality of DNA free RNA was checked, followed by synthesis
TM
of the first strand of cDNA by using the SuperScript IV First-
Strand Synthesis System (Invitrogen) as per the instructions by
the manufacturer.
4.4.9. Semi-quantitative RT-PCR
cDNA was used as a template for the PCR reaction with gene-
0
0
0
specific primer pairs of ER-
a
(5 GTGCCTGGCTAGAGATCCTG 3 , 3
0
GATGTGGGAGAGGATGAGGA 5 ) along with a housekeeping gene
(
GAPDH) as a loading control. The PCR product was separated on
1
1
.2% agarose gel containing ethidium bromide (EtBr) along with
TM
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agents for breast cancer therapy: a review, Anti-Cancer Agents Med. Chem. –
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Acknowledgment
estrogen receptor alpha (ER-a), Eur. J. Med. Chem. 146 (2018) 206–219.
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for indole-3-carbinol, J. Nutr. Biochem. 17 (10) (2006) 659–664.
a as a target
Authors are grateful to Department of Science and Technology,
New Delhi, India for providing financial assistance during the work.
Ramit Singla acknowledges CSIR-India for the award of SRF.
Authors acknowledge Mr. Ashish Pandey of CIL in the Central
University of Punjab, for outstanding work on CLSM. Authors are
also thankful to the Honourable Vice-Chancellor for providing the
necessary facilities at Central University of Punjab, Bathinda, India.
[23] J. Singh Sidhu, R. Singla, V. Jaitak, Indole derivatives as anticancer agents for
breast cancer therapy: a review, Anti-Cancer Agents in Medicinal Chemistry
Formerly Current Medicinal Chemistry-Anti-Cancer Agents) 16(2) (2016)
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(
1
A
phenyl)-3-methyl-1-[4-(2-piperidin-1-yl-ethoxy)-benzyl]-1H-indol-5-ol
hydrochloride (ERA-923), inhibits the growth of tamoxifen-sensitive and-
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Appendix A. Supplementary material
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a cell- and caspase-based Anti-cancer
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9
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