Design, synthesis and broad spectrum antibreast cancer activity of diarylindoles via induction of apoptosis in aggressive breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2021.116252

Breast cancer is the second leading cause of cancer deaths in women with significant morbidity and mortality. Present study describes design, synthesis and detailed pharmacology of indole derivatives exhibiting remarkable broad spectrum antiproliferative

Full text of DOI:10.1016/j.bmc.2021.116252

Bioorg. Med. Chem. 42 (2021) 116252
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and broad spectrum antibreast cancer activity of
diarylindoles via induction of apoptosis in aggressive breast cancer cells
,
1
,
,
,
,
Yashveer Gautama 3, Sharmistha Dasb 1, Hamidullah Khanb 4, Nandini Pathaka c, Hina Iqbala,
Pankaj Yadava, Vijay Kumar Sirohib, Sana Khana c, Dushyant Singh Raghuvanshia,
,
Anila Dwivedib c, Debabrata Chandaa c, Karuna Shankera c, Feroz Khana
,
,
,
,
,c
,
c,
*
,
,c,*
Rituraj Konwarb
2, Arvind S. Negia
a CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Kukrail Picnic Spot Road, Lucknow 226 015, U.P., India
b Endocrinology Division, CSIR-Central Drug Research Institute, Jankipuram Extension, Lucknow 226031, U.P., India
c Academy of Scientific and Innovative Research (AcSIR), New Delhi 110001, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Breast cancer is the second leading cause of cancer deaths in women with significant morbidity and mortality.
Present study describes design, synthesis and detailed pharmacology of indole derivatives exhibiting remarkable
broad spectrum antiproliferative activity against breast cancer cells. Detailed mechanistic evaluations confirmed
induction of G0/G1 arrest, apoptosis induction, loss of mitochondrial integrity, enhanced ROS generation,
autophagy, estrogen receptor β-transactivation and increased tubulin polymerization. In in-vivo efficacy studies
in rodent model, these indole derivatives induced significant regression in mice mammary tumour on 21 days
daily oral dose. Moreover, compounds 19 and 23 were safe in Swiss albino mice in safety studies. These dia-
rylindoles may further be optimized for better efficacy.
Diarylindoles
Breast cancer
Apoptosis
In-vivo efficacy
Acute oral toxicity
1. Introduction
nucleus exhibiting diverse pharmacological activities. It is considered as
one of the most privileged structures by medicinal chemists due to its
Breast cancer is a complex and heterogeneous group of disease.
Broadly, there are hormone dependent, and hormone independent type
breast cancers. Hormone dependent breast cancer (estrogen receptor,
progesterone receptor and HER2 receptor positive, triple positive)
which comprises about two third of breast cancer cases has several op-
tions for treatment, but triple negative breast cancer (TNBC) constitutes
about 10–15% of all cases is more aggressive with limited options for
specific and effective therapy. Moreover, hormone dependent breast
cancer targeting drugs are mostly ineffective against triple negative
breast cancer cells.1 TNBC has been considered a disease with poor
response to molecular target therapy due to less validated biomarkers.2
Nitrogen heterocycles play a crucial role in drug discovery. Pres-
ently, 59% of all unique small-molecule drugs approved by the FDA have
some sort of nitrogen heterocycles.3 Indole is one of the most attractive
ability to bind multiple receptors with high affinity and eliciting wide
range of pharmacological activities.4 Indoles represent the most
important bioactive core of all structural classes in drug discovery.5 This
core has been reported to exhibit diverse pharmacological activities i.e.
anticancer, anti-inflammatory, analgesic, antitubercular, antihyperten-
sive, and antidiabetic etc.4–6 Many naturally occurring compounds
possess indole framework like reserpine from Rauwolfia serpentina as
antihypertensive drug,7 vinca alkaloids from Catharanthus roseus as
antileukemic and anti-Hodgkin’s agents,8 yahimbine from Pausinystalia
yahimbe as sedation reversal agent,9 and ajmalicine from Rauwolfia
serpentina as antiarrythmic agent 10 etc. Further, several clinical drugs
have been developed synthetically on indole core particularly, baze-
doxifene as anti-osteoporotic agent,11 indalpine as selective serotonin
re-uptake inhibitor,12 pravadoline as anti-inflammatory drug,13
* Corresponding authors at: Academy of Scientific and Innovative Research (AcSIR), New Delhi 110001, India.
E-mail addresses: rituraj.konwar@neist.res.in (R. Konwar), arvindcimap@rediffmail.com, arvindcimap@gmail.com (A.S. Negi).
1
2
Equal authorship.
Present address: CSIR-North East Institute of Science and Technology, Jorhat, Assam-785006, India.
3
Present address: Department of Chemistry, Pandit Prithi Nath PG College, 96/12, Mahatma Gandhi Marg, Kanpur 208001, Uttar Pradesh, India.
4
Present address: Department of Dermatology, University of Wisconsin-Madison, Madison, WI 53706, USA.
https://doi.org/10.1016/j.bmc.2021.116252
Received 19 March 2021; Received in revised form 23 May 2021; Accepted 28 May 2021
Available online 5 June 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
panobinostat, a histone deacetylase inhibitor as anticancer agent,14,15
and many more as potential bioactive lead compounds.4 Herein, we
report design and synthesis of 1-benzyl-2-arylindoles as possible anti-
breast cancer agents. We designed dual action hybrid molecules hav-
ing anti-estrogenic and anti-tubulin effect as well. For anti-estrogenic
property the molecule should have affinity to estrogen receptor and an
amino alkyl group should be placed either at C7 or C-11 position parallel
to estradiol which induces anti-estrogenic effect after binding to anti-
estrogenic binding site. Thus, we planned to have aminoalkyl chain at
C11 and also at both C7 and C11 positions which has not been tried so
far. Simultaneously, for anti-tubulin effect, we placed a 3,4,5-trimethox-
yphenyl fragment at C-2 position of indole pharmacophore to induce
anti-tubulin effect (Fig. 1).16 This fragment is supposed to inhibit tubulin
polymerization and induce cell cycle arrest. Thus, these dual acting
compounds are designed to have cytotoxicity not only against estrogen
receptor positive breast cancers via their anti-estrogenic component, but
also against ER-negative breast cancer cases through their co-existing
anti-tubulin effect. Thus a broad spectrum of breast cancer can be
addressed simultaneously.
system to afford 11 and 18 in excellent yields. Further, various amino-
alkyl chains were hooked-up at phenolic OH of 8 & 9 using dry acetone/
K2CO3, at reflux for 2–3 h, to achieve the desired product in excellent
yield (82–89%). (12–16 & 19–23). The indole core was further benzy-
lated with plain benzyl chloride to afford 1-benzyl-5-methoxy-2-(3,4,5-
trimethoxyphenyl)-1H-indole (4) in 40% yield and 1,3-dibenzyl-5-
methoxy-2-(3,4,5-trimethoxyphenyl)-1H-indole (6) in 58% yield. All
the intermediates and final products were confirmed by spectroscopy
[Supplementary information].
2.1.1. Purity profile of potent cytotoxic compounds
The purity of all active compounds 12, 14, 16, 19, and 20–23 was
checked by UPLC. All these compounds possessed purity 95.21% to
98.52%.
2.2. Biological evaluation
2.2.1. Indole derivatives induce loss of cell viability in cancer cell lines
Out of twenty compounds screened for anti-cancer activity against
human breast cancer cell lines (MCF-7 and MDA-MB-231), and prostate
cancer cell line (PC-3) nine compounds showed significant anticancer
activity against all the cell lines and rest of the compounds are moder-
ately active (Table 1). Further, compounds, 19, 20, 22 and 23 were also
investigated against non-malignant human epithelial kidney cell line
(HEK-293) to assess the safety aspect. Among the potent compounds, 19,
20, 22 and 23 possessed relatively better cytotoxicity in the series
against all the cancer cell lines whereas poor cytotoxicities against
normal cell line (HEK-293) (Table 1).
2. Results and discussion
2.1. Chemistry
As outlined in Scheme 1. 2-arylindole core was synthesized by using
modified Fischer indole synthesis. 4-methoxyphenylhydrazine hydro-
chloride (1) and 3,4,5-trimethoxyacetophenone (2) underwent
condensation reaction in presence of N,N’-dimethylurea (DMU) and L
(+) tartaric acid to afford 5-methoxy-2-(3,4,5-trimethoxyphenyl)-1H-
indole (3) in 35% yield.17 The indole core was benzylated at nitrogen
with benzyloxybenzyl chloride, using NaH in dry DMF in an ice-bath to
Structure activity relationship. In the two series of indole derivatives,
Series I possessed mono-N-benzylated indoles (10–16) and series II, 1,3-
dibenzylated indoles (7, 9, 17–23). In general in both series (I & II)
indole derivatives possessing aminoalkyl chains exhibited significant
anticancer activity against all four human cancer cell lines. Only com-
pound 8 with N-benzylated phenol derivatives showed moderate cyto-
toxicity against MDA-MB-231 and PC-3 cell lines.
get
1-(4-(benzyloxy)benzyl)-5-methoxy-2-(3,4,5-trimethoxyphenyl)-
1H-indole (4) in 41% yield. Interestingly, we got a dibenzylated product
also where C-benzylation also took place at C3 position along with N-
benzylation (1,3-bis-(4-(benzyloxy)benzyl)-5-methoxy-2-(3,4,5-trime-
thoxyphenyl)-1H-indole) (5) in 45% yield. The O-benzyl group (5 & 7)
was deprotected by hydrogenolysis using 10% Pd-C in dry THF and
hydrogen gas in a balloon for 4–5 h at RT to afford 4-(5-methoxy-2-
(3,4,5-trimethoxyphenyl)-1H-indol-1-yl)methyl)phenol (8) in 40%
yield and 1,3-bis(4-(hydroxy)benzyl)-5-methoxy-2-(3,4,5-trimethox-
yphenyl)-1H-indole (9) in 56% yield. Phenolic compounds 8 and 9 were
treated with ethyl bromoacetate in potassium carbonate dry acetone
In series I, only N-benzylated indoles with amino alkyl chains were
active against all three cancer cell lines. These compounds (12–16)
mainly differed at aminoalkyl chains. Compounds 12 and 14 with N,N’-
dimethyl chain and pyrrolidine chain were most active against MCF-7
cell line. Compounds 13 and 15 possessing aminoalkyl chains with
higher carbons N,N’-diethyl or piperidine possessed low cytotoxicities.
The corresponding dibenzylated derivatives of compounds 12 (i.e. 19)
N
O
N
O
OH
N
OH
HO
HO
Estradiol
Tamoxifen
Bazedoxifene
A
n
t
i
e
s
t
r
o
g
e
R
n
i
c
N
b
i
n
R
d
i
n
O
g
A
H3CO
OCH3
OCH3
OCH3
n
H3CO
HO
OCH3
OCH3
r
t
o
i
t
t
u
N
p
b
u
e
H3CO
c
l
i
e
r
g
n
n
i
H3CO
OCH3
e
n
OCH3
d
f
e
f
n
e
c
i
g
b
o
t
r
t
s
E
Trimethoxyphenyl motif
Combretastatin A4
Pharmacophore
Fig. 1. Structure of estradiol, tamoxifen, bazedoxifene, combretastatin A4, trimethoxyphenyl motif and designed pharmacophore.
2

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
Scheme 1. Synthesis of compounds 3–23. Reagents and conditions: i) DMU, L(+) tartaric acid, neat, 120 ◦C, 3–4 h; 35%; ii) For 4 & 6 benzylbromide/for 5 & 6
benzyloxybenzylchloride, NaH, dry DMF, 1 h, 40–58%; iii) Dry THF, 10% Pd-C, H2 balloon, 3 h, 8: 40%, 9: 56%; iv) For 10 & 17: Me2SO4, K2CO3, dry acetone, reflux,
86–91%, for 11 & 18: ethylbromoacetate, dry acetone, K2CO3, reflux, 3–4 h, 92–94%; v) various aminoalkyl chains, dry acetone, K2CO3, reflux, 4 h, 82–89%.
and 14 (i.e. 23) were more cytotoxic in series II against MCF-7 cells. In
series-II, compounds 20, 22 and 23 exhibited potential cytotoxicity
against MDA-MB-231 cells, which might be due to antitubulin effect of
these compounds.
and 23 retarded the progression of MDA-MB-231 cells at G0/G1 phase.
Compounds 19, 20, and 23 caused G0/G1 phase of cell cycle arrest and
subsequent reduction of G2/M phase. Whereas compound 22 also
induced G0/G1 phase of cell cycle arrest but without subsequent
decrease in cell population in G2/M phase of cell cycle. However,
compound 22 induced stronger reduction in S phase of cell cycle.
Overall, in case of MCF-7 cell line, in both the series (I & II) an
aminoalkyl chain is essentially required for potential cytotoxicity.
Among the various type of aminoalkyl chains hooked up, N,N-dimethyl
(12 and 19) and pyrrolidine (14 and 21) were relatively more effective
than the rest of the chains.
2.2.3. Compounds 19, 20, 22 and 23 induce mitochondria-mediated
apoptosis
The major therapeutic approaches of clinical oncology have been the
development of therapies promoting effective elimination of cancer cells
by apoptosis because dead tumour cells can contribute to clinical
response but not to tumour relapse. In order to determine whether
cytotoxic response is due to apoptosis or necrosis, we undertook
Annexin V-FITC experiment following treatment with indicated con-
centration of compounds in MDA-MB-231 cells using flow cytometry. As
shown in Fig. 3, compounds 19, 20, 22 and 23 induced early as well late
apoptosis in MDA-MB-231 cells. The apoptotic effects were more pro-
nounced with increasing concentration of test compounds. Whereas,
there was no significant difference in PI positive cell population, thus
indicating necrosis. There was an increased green fluorescence (JC-1
monomers, indication of depolarised mitochondria) and subsequent
2.2.2. Compounds 19, 20, 22 and 23 inhibit cell cycle progression
Cell cycle check-points play an important role in the regulation of
cell cycle by sensing defects that occur during essential processes and
inducing cell cycle arrest in response until the defects are repaired.18
Induction of cell cycle arrest in cancer cells account for one of the most
widespread strategies to stop or halt cancer spreading.19 Majority of
anticancer drugs exert their inhibitory effect on tumor cell growth by
interfering with cell cycle progression by arresting cells at different
phases to stop or limit cancer spreading.20 Therefore, to investigate ef-
fect of compounds 19, 20, 22 and 23 on the cell cycle progression, cell
cycle phase distribution was analysed following treatment with com-
pounds for 24 h in MDA-MB-231 cell line (Fig. 2). Compounds 19, 20, 22
3

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
augmented, endogenous antioxidant capacity.23 However, role of ROS
in the regulation of apoptosis is well established and accepted.23
Table 1
Cytotoxicity of the compounds against cancerous and non-cancerous cells in
terms of IC50 ± SE in µM.
2.2.5. Compounds 19, 20, 22 and 23 induce autophagy
S.
Compound
MCF-7
MDA-MB-
231
PC3
HEK-293
No.
Macro-autophagy also termed as autophagy is a catabolic and
evolutionarily conserved process required for maintenance of cellular
biosynthesis during nutrient deprivation or metabolic stress and cancer
chemotherapy. Therefore, targeting autophagy is considered as prom-
ising therapeutic strategies during development of new cancer ther-
apy.24 To investigate autophagy induction, cells were stained with
acidotropic dye, MDC following treatment with different concentrations
of compounds. Autophagy is characterized by formation of acidic
autolysosome under fluorescence microscopy when cells were stained
with MDC. Results showed that all the compounds increased consider-
able MDC staining in comparison to untreated vehicle control (Fig. 7). In
order to further delineate the cross regulation of autophagy on cell
viability, autophagy was inhibited with widely used autophagy inhibi-
tor, chloroquine and cell viability was evaluated by MTT assay. Results
showed that inhibition of autophagy led to inhibition of compounds 19
and 20 mediated loss of cell viability (Fig. 8). The effect was more
pronounced in case of 19 as compared to 20. Whereas inhibition of
autophagy led no significant difference in case of compounds 22 and 23.
Collectively, these results suggest that compounds 22 and 23 mediated
autophagy induction drive cancer cells toward cell death rather than cell
survival.
1
3
>30
>30
>30
—
—
—
—
—
—
—
—
—
—
2
4
>30
>30
>30
3
5
>30
>30
>30
4
6
>30
>30
>30
5
7
>30
>30
>30
6
8
>30
17.3 ± 0.01
>30
>30
7
9
>30
>30
8
10
11
12
>30
>30
>30
9
>30
>30
>30
10
6.67 ± 0.01
7.84 ± 0.01
6.87 ±
0.02
11
12
13
14
13
14
15
16
29.8 ± 0.05
5.08 ± 0.03
21.6 ± 0.01
14.7 ± 0.02
13.2 ± 0.01
12.4 ± 0.02
>30
15.0 ±
0.01
—
—
—
—
6.03 ±
0.01
21.8 ±
0.01
10.6 ± 0.02
13.3 ±
0.03
15
16
17
18
>30
>30
>30
>30
>30
—
—
17.6 ±
0.06
17
18
19
20
21
22
23
19
4.48
3.78 0.01
2.34 0.01
9.45 ± 0.05
2.42 0.01
3.88 0.03
10.11 ± 0.01
1.6 ± 0.03
5.82 ±
0.03
24.5 ±
0.01
0.06
20
6.36 ± 0.02
8.31 ±
0.01
25.4 ±
0.01
2.2.6. Compounds 19, 20, 22 and 23 induce cell death is caspase-
dependent cell death
21
4.25 ± 0.01
6.48 ± 0.03
7.06 ± 0.01
8.42 ± 0.02
0.48 ± 0.01
7.48 ±
0.03
N.D
Caspases are considered as primary drivers of apoptotic cell death.25
Therefore, to investigate the contribution of caspase in anticancer action
of these compounds, we blocked caspase using pan caspase inhibitor, Z-
VAD-FMK and evaluated the cell viability by MTT assay. Results showed
that inhibition of caspase activation leads to abrogation of compounds
20 and 22 induced loss of cell viability (Fig. 9). However, loss of cell
viability induced by compounds 19 and 23 in MDA-MB-231 cells was
not significantly perturbed by inactivation of caspase. Thus, compounds
20 and 22 induced caspase-dependent apoptosis in breast cancer,
whereas compounds 19 and 23 induced cell death was independent of
caspase action.
22
7.07 ±
0.01
30.5 ±
0.01
23
6.72 ±
0.02
21.8 ±
0.01
Tamoxifen
Paclitaxel
15.8 ±
0.03
22.4 ±
0.02
2.6 ± 0.01
5.8 ± 0.02
IC50 > 30 µM considered as inactive; incubation period = 24 h; — not
determined.
decreased red fluorescence (JC-1 aggregates, indication of polarized
mitochondria). Collectively, increased mitochondrial membrane depo-
larization clearly indicates that apoptosis in MDA-MB-231 cells was
possibly due to activation of intrinsic apoptotic pathway (Fig. 4).
However, the interaction of apoptosis pathways with other signalling
mechanisms can also affect cell death. Targeting mechanisms of
apoptosis remain a promising strategy in oncology that will continue to
evolve in future clinical practice.21
2.2.7. Compounds 19, 20 and 23 enhance ERβ trans-activation without
any effect on ERα trans-activation
None of the four compounds significantly altered trans-activation of
unlike estradiol or tamoxifen. However, compounds 19, 20, and 23
significantly enhanced ERβ trans-activation in MDA-MB-231 cells
ERα
(Fig. 10). Over-expression of ERα is considered responsible for devel-
opment of breast cancer26 while ERβ counteracts hyperproliferative ef-
2.2.4. Compounds 19, 20, 22 and 23 induce ROS-dependent cell death
Reactive oxygen species is a known mediator of apoptosis and play
important role in chemotherapeutics mediated cancer cell death.22
Therefore, to decipher the ROS generation MDA-MB-231 cells were
treated with different concentrations of test compounds for 24 h and
ROS generation was detected using ROS sensitive fluorescent probe
DCFH-DA using flow cytometer (Fig. 5). We found that all the com-
pounds increased the level of ROS in MDA-MB-231 cells and level of ROS
was positively correlated with concentrations of test compounds. To
further confirm the contribution of ROS generation on cell viability, ROS
level was scavenged with N-acetylcysteine (NAC), a widely used ROS
inhibitor, along with different compounds and cell viability was evalu-
ated with MTT assay (Fig. 6). Results showed that all the compounds
induced loss of cell viability in MDA-MB-231 cells whereas when ROS
level was scavenged using NAC compounds-mediated loss of cell
viability was abrogated. Collectively, these data support that ROS gen-
eration induced by compounds may be the plausible reason of cell death
in cancer cells. Nevertheless, many cancer cells become adapted to such
stress under persistent intrinsic oxidative stress and develop an
fect of ER
α
.
27 Hence, activation of ERβ is considered antiproliferative in
breast tumour cells.28 Compound 19 showed highest trans-activation of
ERβ and exhibited strong antiproliferative effect (IC50 = 4.48 µM)
against MCF-7 cells which is highest among the four evaluated com-
pounds. Compounds 22 neither influenced trans-activation of ERα nor
that of ERβ, possibly suggesting its action to be independent of ER.
2.2.8. Compounds 19, 20, 22 and 23 enhance tubulin polymerization
Targeting microtubule is an established anticancer target. Microtu-
bule targeting drugs disrupt microtubule dynamics either as a stabilizer
or as a destabilizer. Stabilizers promote microtubule assembly by ulti-
mately microtubule aggregation while destabilizers inhibit tubulin
polymerization and microtubule assembly.16 In our tubulin polymeri-
zation kinetics study, compounds 19, 20, 22 and 23 showed significant
enhancement of tubulin polymerisation as compared to vehicle
(Fig. 11). Compounds showed higher microtubule formation than pos-
itive control paclitaxel. Our results suggest that compounds 19, 20, 22
and 23 target MDA-MB-231 cells as stabilizers in tubulin assembly.
Modulation of tubulin-microtubules dynamics is one of the most
4

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
Fig. 2. Effects of compounds 19, 20, 22 and 23 on cell cycle in MDA-MB-231 cells. [As indicated above, treatments of compounds 19, 20, 22, and 23 were given in
MDA-MB-231 cells and after 24 h cells were harvested and stained with PI and data acquired by flowcytometry]
effective targets for cancer chemotherapeutics.29
sub-types except compound 22 which showed moderate affinity with
ER-β sub-type (Table 3). There were only one or two residual amino
acids common to these which indicated that indole derivatives did not
occupy the same binding pocket (Supplementary information).
2.2.9. Molecular docking studies
a) Interaction of indanone derivatives 19, 20, 22 and 23 with tubulin at
paclitaxel binding site
2.2.10. Compounds 19, 20, 22 and 23 induce tumor regression in 4T1-
induced syngeneic mouse mammary tumor model
Docking studies were performed to confirm the interaction of indole
derivatives with target protein microtubule. Crystal structure of tubulin
dimer was used from a 13-protofilament, paclitaxel stabilized microtu-
bule [PDB: 6WVR]. Among the four indole derivatives, compounds 19
and 22 exhibited very good affinity with target protein which were
comparable to paclitaxel (Table 2). There were twelve amino acid res-
idues common to all four indole derivatives and paclitaxel (VAL23,
LEU217, HIS229, ALA233, PHE272, LEU275, THR276, ARG278,
GLN281, PRO360, ARG369, & LEU371) which clearly indicates that all
these occupied similar binding pocket (Supplementary information).
In order to confirm the anti-tumor activity and efficacy of com-
pounds 19, 20, 22 and 23, we utilized orthotopically transplanted 4T1
cell line induced mouse mammary tumor model. Daily oral adminis-
tration of 10 mg/kg and 20 mg/kg bodyweight of compounds 19, 20, 22
and 23 led to significant suppression of tumor volume with all the
compounds at all tested concentrations as compared to untreated vehicle
control (Fig. 12). However, tumor volume regression was highly sig-
nificant in case of 10 mg/kg bodyweight dose of compound 19 and 20
mg/kg bodyweight dose of compound 20. Antitumor efficacies of all the
four compounds are either comparable or significantly better than
paclitaxel. Interestingly, both the compounds lack of any apparent
toxicity as no significant difference in body weights and other
biochemical parameters was observed. Furthermore, all the compounds
showed better survivability than untreated and paclitaxel treated
groups. Collectively, these data support that all the compounds show
significant anti-cancer effect in both in vitro as well as in in vivo tumor
model.
b) Interaction of indoles with α/β-estrogen receptors
All the four indole derivatives 19, 20, 22 and 23 were docked with
both the estrogen receptor subtypes alpha and beta. Estradiol was used
as standard ligand. Human estrogen receptor alpha was taken from
ligand binding domain in complex with estradiol [PDB: 2OCF] and es-
trogen receptor-beta bound to estradiol [PDB: 5TOA]. Estradiol exhibi-
ted good affinity with both the estrogen receptor sub-types. All the four
2.2.11. Safety studies of compounds 19 and 23 through acute oral toxicity
Indole derivatives did not show good affinity with both ER-
α
and ER-β
Acute oral toxicity describes the adverse effects of potentially
5

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
Fig. 3. Apoptosis induction by compounds 19, 20, 22
and 23 MDA-MB-231 cells. [After treatment for 24 h
by compounds 19, 20, 22, and 23 as indicated above,
cells were harvested and stained by Annexin V-FITC/
PI and data acquired by flow-cytometric analysis. The
percentage of annexin V-FITC-negative/PI-negative
(viable cells), annexin V-FITC-positive/PI-negative
(early apoptotic cells), annexin V-FITC-negative/PI
positive cells (late apoptotic) and annexin V-FITC-
positive/PI-positive (necrosis)]
bioactive compounds on exposure for a short period of time to get an
idea about the safety aspects. For safety evaluation compounds 19 and
23 were given once orally to Swiss albino mice at 5, 50, and 300 mg/kg
dose.30 No mortality was observed throughout the experimental period,
there were non-significant changes in gait, posture and response of an-
imals. Animals on gross pathological study did not show any significant
changes in any of the organs studied, including their absolute and
relative body-weights (Fig. 13 and Fig. 15). Blood and serum samples
upon analysis showed non-significant changes in all the parameters
studied like haemoglobin (Hb), Red Blood Cells (RBC), White Blood
Cells (WBC), differential leukocyte count (DLC) (Fig. 14 and Fig. 16),
Serum SGPT, Alkaline Phosphatase (ALP), creatinine, triglycerides,
cholesterol, albumin, serum protein (Tables 4A and 4B). Therefore, the
experiment showed that compounds 19 and 23 are well tolerated by the
Swiss albino mice up to the dose level of 300 mg/kg bodyweight as a
single acute oral dose for 7 days. However, sub-acute, chronic or sub-
chronic experiments with both the compounds (19 and 23) are needed
to be carried out for any adverse effect on repeated exposure.31
Safety is an essential aspect in the development of a drug candidate,
nowadays referred as ‘Pharmacovigilance’. It aims to enhance patient
care and safety in relation to use of medicine and to support public
health programmes by providing reliable, balanced information for
effective assessment of risk–benefit profile of medicines.32 Now it has
been legalised and streamlined existing responsibilities for regulators
and the pharmaceutical industry.
3. Conclusions
We have designed and synthesized two series of indole derivatives.
These compounds were evaluated as potential antitubulin anticancer
agents. Four of the derivatives (19, 20, 22 and 23) were extensively
evaluated for their pharmacological effect on breast cancer. All the four
compounds induced cell cycle arrest and apoptosis through multiple
mechanisms by induction of ROS generation, mitochondrial membrane
6

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
Fig. 4. Effect of compounds 19, 20, 22 and 23 on mitochondrial membrane depolarization in MDA-MB-231 cells. [MMP was evaluated using JC-1 dye following
treatment with 19, 20, 22 and 23 for 24 h, by flow cytometry]
Fig. 5. Induction of ROS generation by compounds 19, 20, 22 and 23 in MDA-MB-231 cells. [Cells were treated with 19, 20, 22 and 23 for 24 h and generation of
ROS is checked by DCFH-DA dye using flow cytometric analysis]
7

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
Fig. 6. Effect of compounds 19, 20, 22 and 23 on ROS dependent cell viability in MDA-MB-231 cells. [Cells were pre-treated with 10 mM NAC for 2 h and further
treated by 4 µM of 19, 20, 22 and 23 for 24 h and analysed by MTT Assay. Graphical plots derived from three independent experiments and their numerical values
represent mean ± S.E.M.(*P < 0.05,**P < 0.01,***P < 0.001)]
4. Experimental section
4.1. General
Melting points were determined in open glass capillaries and were
uncorrected. Reagents and other chemicals were procured from Sigma-
Aldrich, USA, Avra Synthesis India and Sigma-Merck India and were
used without purification. Reactions were monitored on pre-coated sil-
ica gel TLC-GF254 aluminium sheets and visualization of compounds was
done under UV light (254 nm and 365 nm) and further charring with 2%
ceric sulphate-10% sulphuric acid (aqueous). Purification of compounds
was done using glass column and silica gel (100–200 mesh) and char-
acterised by 1H and 13C NMR, ESI-MS, and ESI-HRMS. NMR spectra were
obtained with Bruker Avance-300/500 MHz spectrometer. Chemical
shifts are given in δ ppm values with respect to tetramethylsilane (TMS)
as an internal standard. 1H–1H coupling constant (J) values are given in
Hz. ESI mass spectra were recorded using an APC3000 LC-MS-MS
(Applied Biosystem) and High Resolution Mass (HRMS) on Agilent
6520Q-TOF after dissolving the compounds in methanol. Purity profile
of potent analogues was checked on Waters UPLC system.
Human breast cancer cell lines (MDA-MB-231 and MCF-7), prostate
cancer cell line (PC3), and non-malignant human embryonic kidney cell
line (HEK-293) were cultured in RPMI media at 37 0C with 5% CO2 in a
humidified chamber. RPMI media was supplemented with 10% fetal
bovine serum (GIBCO BRL Laboratories, New York, USA) and 1% pen-
icillin–streptomycin solution (Sigma Chemical Co., St. Louis, MO, USA).
Compounds were dissolved in DMSO (dimethyl sulfoxide) and further
diluted in media so that DMSO concentration would be less than
0.001%. Unless otherwise mentioned, all the chemicals were procured
from Sigma-Aldrich.
Fig. 7. Induction of autophagy by compounds 19, 20, 22 and 23 in MDA-MB-
231 cells. [Cells were treated with 19, 20, 22 and 23 for 24 h and autopha-
gosome formation is analysed by MDC staining using fluorescence microscopy.]
depolymerisation and caspase activation. All of the four compounds
induced autophagy in MDA-MB-231 cells, but only compounds 19 and
20 mediated autophagy is linked with cell death. Compounds 19, 20 and
23 also caused significant ERβ transactivation, indicating their efficacy
against MCF-7 cells through estrogen receptor pathways. All the four
compounds stabilized tubulin polymerization similar to paclitaxel. In in-
vivo efficacy, all these compounds exhibited anti-tumour activity com-
parable or better than tested regime of paclitaxel. Both the compounds
19 and 23 were well tolerable and safe up to 300 mg/kg oral dose.
Collectively, these findings suggest that these indole derivatives have
potential as cancer chemotherapeutics against both types of breast
cancer.
4.2. Chemical synthesis
4.2.1. Synthesis of 5-methoxy-2-(3,4,5-trimethoxyphenyl)-1H-indole (3)
A mixture of L(+)-tartaric acid (1.5 g) and DMU (3.5 g) in 30:70 were
heated at 90 ◦C to obtain a clear melt. To this melt, (174 mg, 1 mmol) of
4-methoxyphenyl hydrazine hydrochloride and 3,4,5-trimethoxyaceto-
phenone (210 mg, 1 mmol) were added and reaction mixture was
heated at 90 ◦C. After completion, the reaction mixture was quenched by
adding water and the aqueous layer was extracted with ethyl acetate (3
8

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
Fig. 8. Induction of autophagy mediated cell death by compounds 19, 20, 22 and 23 in MDA-MB-23 cells. [Cells were pre-treated with 10 mM CQ for 2 h and further
treated by 4 µM of 19, 20, 22 and 23 for 24 h and analysed by MTT Assay. Graphical plots derived from three independent experiments and their numerical values
represent mean ± S.E.M.(*P < 0.05,**P < 0.01)]
Fig. 9. Caspase mediated cell death in MDA-MB-23 cells by compounds 19, 20, 22 and 23. [Cells were pre-treated with Z-VAD-FMK(CI) for 2 h followed by 19, 20,
22 and 23 treatment for 24 h and cell viability was assessed with MTT assay. Graphical plots derived from three independent experiments and their numerical values
represent mean ± S.E.M. (**P < 0.01,***P < 0.001).]
Fig. 10. Effect of compounds 19, 20, 22 and 23 on
ERα and ERβ trans-activation. [Compounds 19, 20, 22
and 23 do not cause ER
pounds 19, 20 and 23 enhances ERβ trans-activation.
Estradiol (E2) was used as ER and β) trans-
activating positive control, whereas Tamoxifen
(TAM) was used as negative control of ER ( and β)
α trans-activation, but com-
(α
α
trans-activation. Results were described as percent of
normalized relative luciferase unit (RLU). Results
were expressed as mean ± SEM, n = 3. p values are
*** = p < 0.001, ** = p < 0.01 and * = p < 0.05
versus control]
× 10 mL). The organic layer was washed with water (2 × 10 mL), dried
over anhydrous Na2SO4 and concentrated under reduced pressure. The
crude compound was purified by column chromatography over silica gel
(100–200 mesh). On elution with acetone-hexane the pure product 3
was obtained as a crystalline solid (Brownish). Purity of synthesized
compounds was checked by UPLC and was found to be > 95%.
128.9, 130.1, 132.4, 138.3, 138.3, 154.1, 154.9 .
4.2.2. Synthesis of 1-(4-(benzyloxy)benzyl)-5-methoxy-2-(3,4,5-
trimethoxyphenyl)-1H-indole (5)
Compound 3 (313 mg, 1 mmol) was taken in dry DMF (10 mL) in
round bottom flask and kept on ice bath to stir about 10 min. Now pre-
washed sodium hydride (60 mg) was added to it and stirred. After 15
min, benzyloxybenzyl chloride (290 mg, 1.25 mmol) was added to it and
reaction mixture was stirred for an hour. After completion, reaction
mixture was quenched by adding cold water (5 mL) dropwise. Aqueous
layer was extracted with ethyl acetate (2 × 10 mL), the combined
organic layer was washed with water (3x5 mL), dried over anhydrous
◦
Yield: 35%, brownish solid; m.p.: 168–170 C; ESI-MS (MeOH) for
C
18H19NO4, 314 [M+H]+, 336.3 [M+Na]+, 352.2 [M+K]+; 1H NMR
(CDCl3, 300 MHz): δ3.90 (s,3H,OCH3), 3.93 (s, 3H, OCH3), 3.94 (s, 6H,
2 × OCH3), 6.7 (s,1H,CH, aromatic), 6.80 (s, 3H, 3xCH, aromatic), 7.10
(s, 1H, CH, aromatic), 7.30 (d, 1H, CH, aromatic, J = 8.7 Hz); 13C NMR
(CDCl3,75 MHz): δ 56.2, 56.6, 61.4, 100.1, 102.6, 103.1, 112.0, 112.8,
9

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
2
1.6
1.2
0.8
0.4
0
Control
Paclitaxel
19
20
22
23
0
5
10
15
20
25
30
35
40
45
50
55
60
Time (Minutes)
Fig. 11. Effect of compounds 19, 20, 22 and 23 on tubulin kinetics (tubulin polymerisation). [Compound 19, 20, 22 and 23 enhances tubulin polymerization in cell-
free system. Paclitaxel was used as positive control. Mean absorbance obtained from duplicate reactions of each group is plotted versus time in minutes.]
aromatic, J = 8.4 Hz), 7.14 (bd, 2H, 2xCH, aromatic); 13C NMR
Table 2
(CDCl3,75 MHz): δ 47.8, 48.0, 56.2, 56.2, 56.2, 61.3, 70.4, 101.9, 102.7,
Interaction studies of compounds 19, 20, 22, 23, and paclitaxel with β-tubulin
106.6, 106.6, 111.4, 112.5, 115.6, 115.6, 15.6, 127.3, 127.3, 127.3,
(PDB ID: 6WVR).
128.3, 128.3, 128.3, 128.3, 128.9, 131.3, 133.9, 137.3, 142.7, 153.5,
Name
Docking
Binding site residues within
4 Å region
Key amino
acid
155.0, 158.2.
binding energy
kcal/mol
residues
4.2.3. 5-Methoxy-2-(3,4,5-trimethoxyphenyl)-1,3-(4-dibenzyoloxybenzyl-
indole (7)
Paclitaxel
ꢀ 8.1
GLU22, VAL23, ASP26,
PHE83, LEU217, HIS229,
LEU230, ALA233, PHE272,
LEU275, THR276, SER277,
ARG278, GLN281, PRO360,
ARG369, GLY370, LEU371
LYS19, GLU22, VAL23,
THR276
Yield: 53%, white solid; m.p.: 149–151 ◦C; ESI-MS (MeOH) for
C46H43NO6: 706 [M+H]+; 1H NMR (CDCl3, 300 MHz): δ3.44 (s, 6H, 2 ×
OCH3), 3.85 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 4.04 (s, 2H, 3-CH2), 5.03
(s, 4H, 2 × OCH2, benzyloxy gps), 5.19 (s, 2H, N-CH2), 6.38 (s, H, CH,
aromatic), 6.83 (m, 8H, 8xCH, aromatic), 6.86 (d, H, CH, aromatic, J =
9 Hz), 6.97 (bd, 3H, CH, aromatic), 7.15 (s, 1H, CH, aromatic), 7.19 (bd,
10H, CH, aromatic).
Compound
ꢀ 7.9
19
ASP26, LEU217, LEU219,
ASP226, HIS229, LEU230,
ALA233, SER236, PHE272,
PRO274, LEU275, THR276,
ARG278, GLN281, ARG320,
PRO360, ARG369, LEU371
VAL23, GLU27, LEU217,
LEU219, HIS229, ALA233,
PHE272, PRO274, LEU275,
THR276, SER277, ARG278,
GLN281, LEU286, PRO360,
ARG369, GLY370, LEU371
VAL23, ASP26, GLU27,
LEU217, LEU219, HIS229,
ALA233, PHE272, PRO274,
LEU275, THR276, SER277,
ARG278, GLN281, LEU286,
PRO360, ARG369, GLY370,
LEU371
4.2.4. Synthesis of 5-methoxy-2-(3,4,5-trimethoxyphenyl)-1-(4-hydroxy
benzyl)-indole (8)
Compound
ꢀ 6.8
ꢀ 7.8
Compound 5 (509 mg, 1 mmol) was taken in dry THF (10 mL). To
this stirred solution, 10% Pd-C was added. The reaction was kept under
H2 supply through balloon for 2 h. After completion, it was filtered
through celite bed (filter aid) in sintered crucible (G2), washed with
acetone, organic layer was evaporated and residue was taken in ethyl
acetate, washed with water, dried over anhydrous sodium sulphate. On
evaporation a residue was obtained which was purified through silica
gel (100–200 mesh) column using hexane–ethyl acetate as eluants. The
pure product 8 was obtained as a white crystalline solid. Similarly,
compound 9 was also synthesized by this procedure.
20
Compound
22
Yield: 40%, white solid; m.p.: 140–142 ◦C; ESI-MS (MeOH) for
Compound
ꢀ 7.1
VAL23, ASP26, GLU27,
LEU217, LEU219, ASP226,
HIS229, LEU230, ALA233,
PHE272, PRO274, LEU275,
THR276, ARG278, GLN281,
LEU286, PRO360, ARG369,
GLY370, LEU371
25H25NO5: 420 [M+H]+; 1H NMR (Acetone‑d6,300 MHz): δ3.76 (s, 6H,
23
C
2 × OCH3), 3.78 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 5.39 (s, 2H, N-CH2),
6.62 (s,1H, 3-CH, aromatic), 6.80 (bd, 5H, 5xCH, aromatic), 6.93 (d, 2H,
2xCH, aromatic), 6.98 (d 1H, CH, aromatic), 7.17 (d, 1H, CH, aromatic),
7.24 (d, 1H,CH,aromatic, J = 9 Hz), 8.01 (s, 1H,exchangeable,OH); 13
C
NMR (Acetone‑d6, 75 MHz): δ47.3, 55.4, 55.8, 55.8, 60.1, 101.9, 102.5,
106.8, 106.8, 111.5, 112.0, 115.7, 115.7, 127.5, 127.5, 128.6, 129.1,
129.9, 132.2, 133.9, 142.5, 153.6, 153.6, 155.0, 156.9.
Na2SO4 and evaporated in vacuo. The crude mass was purified through
column chromatography over silica gel (100–200 mesh). On elution
with benzene-hexane the pure product 5 was obtained as a white crys-
talline solid in 45% yield. Similarly, compounds 4, 6, and 7 were also
synthesized by same reaction procedure.
4.2.5. 5-Methoxy-2-(3,4,5-trimethoxyphenyl)-1,3-(4,4-
dihydroxydibenzyl)-indole (9)
Yield: 56%, white solid; m.p.: 235–238 ◦C; ESI-MS (MeOH) for
C32H31NO6: 526 [M+H]+; 1H NMR(CDCl3,300 MHz): δ3.66 (s, 6H, 2 ×
OCH3), 3.81(s, 6H, 2 × OCH3), 4.05 (s,2H, 3-CH2), 5.29 (s, 2H, N-CH2),
6.63 (s, 2H, 2xCH, aromatic), 6.76 (bd, 7H, 7xCH, aromatic), 7.05 (bd,
3H, 3xCH, aromatic), 7.31(d, 1H, CH, aromatic, J = 8.1 Hz), 8.21 (s, 1H,
exchangeable, OH), 8.42 (s, 1H, exchangeable, OH); 13C NMR
Yield: 45%, white solid; m.p.:148–150 ◦C; ESI-MS (MeOH) for
C
32H31NO5: 510 [M+H]+,532 [M+Na]+, 548 [M+K]+; 1H NMR
(CDCl3,300 MHz): δ3.66 (s, 6H, 2 × OCH3), 3.90 (s, 6H, 2 × OCH3), 5.06
(s, 2H, OCH2), 5.31(s, 2H, N-CH2), 6.58 (s, 1H, CH, aromatic), 6.63 (s,
2H, 2xCH, aromatic), 6.85 (bd, 2H, 2xCH, aromatic), 6.94 (d, 2H, 2xCH,
10

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
4.2.6. General procedure for the synthesis of alkyloxy alkane and alkyloxy
ester derivatives
Table 3
Details about docking studies of indole derivatives with estrogen receptor alpha
and beta sub-types.
4.2.6.1. Synthesis of 1-(4-methoxybenzyl)-5-methoxy-2-(3,4,5-trimethox-
yphenyl)-1H-indole (10). 5-Methoxy-2-(3,4,5-trimethoxyphenyl)-1(4-
hydroxy benzyl)-indole (8) was taken in 10 mL of dry acetone and
anhydrous potassium carbonate (1 g, 7.24 mmol) was added to it. To this
refluxing mixture dimethylsulphate was added (0.15 mL, 1.35 mmol)
and further refluxed for an hour. After completion, reaction mixture was
filtered, washed with acetone and evaporated to dryness under reduced
pressure. The residue was taken in ethyl acetate, washed with water and
organic layer was dried over anhydrous sodium sulphate. It was evap-
orated to dryness to get crude compound which was purified through
column chromatography over silica gel (100–200 mesh) eluting with
ethyl acetate- hexane to get 10 in 84% yield (amorphous solid). Simi-
larly, compounds 11, 17 and 18 were also synthesized by this procedure.
Compd
code
Estrogen receptor-alpha (PDB
entry 2OCF)
Estrogen receptor-beta (PDB
entry 5TOA)
Binding
affinity
(Kcal/
mol)
Binding pocket
amino acids
Binding
affinity
(kcal/
mol)
Binding pocket
amino acids
Compound
ꢀ 6.5
LEU320,
ꢀ 6.0
GLY352, ASP363,
ARG364,
19
GLU323, ILE326,
GLU353,
GLU375,
GLY390,
ASP378,
TRP393,
MET379,
ARG394,
LEU381,
HIS398, LEU403,
PRO406,
ALA382,
THR383,
GLY442,
SER385,
GLU443
ARG388, HIS464
PRO351,
Compound
ꢀ 6.1
LEU320,
ꢀ 5.5
4.2.6.2. Synthesis of 1-(4-(2-(N,N’-dimethylamine-1-yl)-ethoxy)-benzyl)-
20
GLU323,
GLY352,
5-methoxy-2-(3,4,5-trimethoxyphenyl)-1H-indole (12). Compound
8
PRO325, ILE326,
LEU327,
ARG364,
(419 mg, 1 mmol) was taken in dry acetone (10 mL) and potassium
carbonate (1 g, 7.24 mmol) was added to it. To this refluxing mixture, N,
N’-dimethylethyl chloride hydrochloride was added (181 mg, 1.2 mmol)
and further refluxed for 2–3 h. After completion, the reaction mixture
was filtered, washed with acetone and solvent was evaporated to dry-
ness under reduced pressure. The residue was taken in ethyl acetate,
washed with water, organic layer was dried over anhydrous sodium
sulphate. It was evaporated to dryness to get crude mass which was
purified through column chromatography by using 100–200 mesh silica
gel, eluting with methanol-chloroform solvent to get pure compound as
amorphous solid in 85% yield. Similarly compounds 13–16 and 19–23
were also synthesized by this procedure.
ASP378,
GLU353,
MET379,
GLY390,
LEU381,
TRP393,
ALA382,
ARG394,
THR383,
MET396,
GLU397,
SER385,
ARG386, HIS464
GLY442,
PHE445, LYS449
LEU320,
Compound
ꢀ 6.9
ꢀ 6.6
ꢀ 10.9
ꢀ 8.0
ꢀ 6.9
ꢀ 11.0
LEU273,
22
GLU323,
GLU276,
PRO324, ILE326,
GLU353,
PRO277,
PRO278, HIS279,
GLU305,
TRP393,
ARG394,
MET309,
MET396,
GLU397,
VAL338,
4.2.6.3. 1,3-Bis[4-(2-(N,N’-dimethylamine-1-yl)ethoxy)benzyl]-5-
methoxy-2-(3,4,5-trimethoxyphenyl)-1H-indole (19). Yield: 86%, yellow
gummy; ESI-MS (MeOH) for C40H49N3O6: 668 [M+H]+; ESI-HRMS
LEU339,
GLN441,
TRP345,
GLY442,
ARG346, ILE348,
ASP349, ILE355,
PRO358, HIS394,
TYR397,
46H57N3O6 for [M+H]+, cald, 668.3699, found
GLU443
(MeOH) for
C
668.3645. 1H NMR (CDCl3,300 MHz): δ2.39 (s, 12H, 4 × N-CH3 of
chain), 2.79 (t, 4H, 2 × N-CH2 of chain), 3.50 (s, 6H, 2 × OCH3), 3.72 (s,
3H, OCH3), 3.73 (s, 3H, OCH3), 4.05 (bt, 4H, 2 × OCH2 & 3-CH2), 5.18
(s, 2H, N-CH2), 6.40 (s, 2H, 2xCH, aromatic), 6.77 (bd, 4H, 4xCH, aro-
matic), 6.83 (d, 1H, CH, aromatic), 6.90 (bs, 1H, CH, aromatic), 6.93 (d,
2H, 2xCH, aromatic, J = 8.4 Hz), 7.08 (bd, 3H, 3xCH, aromatic); 13C
NMR (CDCl3,75 MHz): δ30.0, 30.2, 45.6, 45.6, 45.6, 45.7., 47.7, 54.9,
56.1, 56.1, 56.1 ,56.2, 58.1, 61.2, 65.9, 101.8, 107.7, 107.7, 111.2,
112.0, 112.3, 114.8, 114.8, 114.8, 115.14, 115.1, 115.1, 127.3, 127.4,
129.1, 129.4, 129.4, 131.5, 132.8, 135.0, 139.7, 153.2, 153.27,
154.6,157.0,158.0.
LEU398, LYS401
LEU273,
Compound
GLU330,
TYR331,
ASP332,
23
GLU276,
PRO277,
ARG335,
PHE337,
SER338,
PRO278, HIS279,
GLU305,
VAL338,
ALA340,
SER341,
GLY342,
TRP345,
GLY344,
LEU345,
ASN348,
VAL534,
PRO535,
SER537
ARG346, ILE355,
PRO358,
TYR397, HIS394,
LEU398, LYS401
4.2.6.4. 1,3-Bis[4-(2-(N,N’-diethylamine-1-yl)ethoxy)benzyl]-5-methoxy-
2-(3,4,5-trimethoxyphenyl)-1H-indole (20). Yield: 83%, yellow viscous;
ESI-MS (MeOH) for C44H57N3O6: 724 [M+H]+; HRMS for C44H57N3O6,
[M+H]+, calcd, 724.4306, obsvd 724.4306; 1H NMR (CDCl3, 300 MHz):
δ 1.15 (m, 12H, 4 × CH3 of chain), 2.80 (bs, 8H, (CH2)4), 3.02 (bs, 4H, N-
(CH2)2), 3.51 (s, 6H, 2 × OCH3), 3.80 (s, 6H, 2 × OCH3), 4.04 (bd, 6H, 3
× CH2), 5.19 (s, 2H, 1-N-CH2), 6.41 (s, 2H, 2xCH, aromatic), 6.79 (bs,
5H, 5xCH, aromatic), 6.92 (bd, 3H, 3xCH, aromatic), 7.10 (s, 3H, 3xCH,
aromatic); 13C NMR (CDCl3, 75 MHz): δ10.6, 10.6, 10.8, 10.8, 32.0,
32.0, 32.0, 32.0, 34.2, 47.8, 47.8, 51.7, 56.1, 56.2 ,56.2, 61.2, 65.6,
65.9, 101.8, 107.7, 107.7, 111.9, 112.3, 114.4, 114.7, 115.0, 115.0,
116.2, 116.2, 127.5, 127.5, 129.1, 129.1,129.1, 129.4, 131.6, 132.8,
138.1, 139.7, 153.2, 153.2, 154.6, 156.8, 157.9.
β-estradiol
GLU353,
LEU384,
LEU387,
MET388,
LEU391,
ARG394,
PHE404,
MET421,
ILE424, LEU428,
GLY521,
HIS524, LEU525
LEU298,
LEU301,
GLU305,
MET336,
LEU339,
MET340,
LEU343,
ARG346,
PHE356, ILE373,
ILE376, LEU380,
GLY472, HIS475,
LEU476
(CDCl3,75 MHz): δ47.3, 55.4, 55.6, 55.6, 60.1, 101.6, 108.2, 108.2,
111.3, 111.8, 112.3, 115.3, 115.3, 115.3, 127.5, 127.7, 127.7, 127.7,
129.4, 129.4, 129.4, 129.4, 129.4, 130.1, 132.8, 133.3, 139.6, 153.6,
154.6, 155.7, 156.8.
4.2.6.5. 1,3-Bis[4-(2-(piperidin-1-yl)ethoxy)benzyl]-5-methoxy-2-(3,4,5-
trimethoxyphenyl)-1H-indole (22). Yield: 82%, yellow solid; m.p.:
170–173 ◦C; ESI-MS (MeOH) for C46H57N3O6: 748 [M+H]+, HRMS for
11

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
Fig. 12. Compounds 19, 20, 22 and 23 exhibit anti-tumour efficacy in 4 T1 orthotropic syngeneic mice mammary tumour model. [Compounds 19, 20, 22 and 23 at
the rate of 10 mg/kg and 20 mg/kg daily as oral gavage and paclitaxel at the rate of 10 mg/kg through intravenous route twice a week were administered for 21 days.
Effect of compounds 19, 20, 22 and 23 (top to bottom) on tumour volume, tumour weight, whole body weight and animal survival (left to right) show significant
anti-tumour efficacy]
Fig. 13. Effect of compound 19 as a single acute oral dose at 5, 50, 300 mg/kg on absolute and relative organ weight in Swiss albino mice. [Animals showed non-
significant changes up to 300 mg/kg single oral dose as compared to control group]
C46H57N3O6 for [M+H]+, calcd 748.4325, found 748.4314; 1H NMR
(CDCl3, 300 MHz): δ1.63 (bd,12H, 6 × CH2 of piperidine ring), 2.59 (bs,
8H, N-(CH2)4 of piperidine ring), 2.81 (t,4H, N-(CH2)2), 3.49 (s, 6H, 2 ×
OCH3), 3.73 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 4.02 (s, 2H, 3-CH2), 4.08
(bt, 4H, 2 × OCH2), 5.17 (s, 2H, N-CH2), 6.39 (s, 2H, 2xCH, aromatic),
6.76 (bd, 5H, 5xCH, aromatic), 6.89 (bd, 2H, 2xCH, aromatic), 6.93 (d,
H, CH, aromatic, J = 10 Hz), 7.07 (bd, 3H, 3xCH, aromatic); 13C NMR
(CDCl3,75 MHz): δ25.6, 25.7, 29.5, 29.5, 29.7, 29.9, 30.0, 47.7, 55.1,
55.1, 56.1, 56.1, 56.1, 56.2, 57.9, 57.9, 61.2, 65.7, 65.8, 101.7, 107.7,
107.7, 111.2, 112.0, 112.3, 114.4, 114.8, 114.8, 115.1, 115.1, 127.4,
12

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
Fig. 14. Effect of compound 19 as a single acute oral dose at 5, 50, 300 mg/kg body weight on differential leucocyte count in Swiss albino mice. [Animals showed
non-significant changes up to 300 mg/kg single oral dose as compared to control group]
Fig. 15. Effect of compound 23 as a single acute oral dose at 5, 50, 300 mg/kg on absolute and relative organ weight in Swiss albino mice. [Animals showed non-
significant changes up to 300 mg/kg single oral dose as compared to control group]
Fig. 16. Effect of compound 23 as a single acute oral dose at 5, 50, 300 mg/kg body weight on differential leucocyte count in Swiss albino mice. [Animals showed
non-significant changes up to 300 mg/kg single oral dose as compared to control group]
127.4, 127.4, 127.4, 129.1, 129.4, 131.5, 134.9, 138.1, 139.7, 153.2,
153.2, 154.6, 157.0, 158.0.
107.7, 111.3, 112.99, 112.9, 114.7, 114.7, 115.0, 115.0, 116.3, 116.5,
127.6, 127.6, 127.6, 129.5, 129.59, 129.59, 131.0, 131.0, 132.0, 135.4,
139.7 , 153.3, 154.6, 156.4.
4.2.6.6. 1,3-Bis-[4-(3-dimethylaminopropyl-2-oxy)benzyl]-5-methoxy-2-
(3,4,5-trimethoxy phenyl)-1H-Indole (23). Yield: 89%, yellow gummy;
ESI-MS (MeOH) for C42H53N3O6: 696 [M+H]+; HRMS for C42H53N3O6
4.3. Biological evaluation
1
for [M+H]+; cacld 696.4012, found, 696.4002; H NMR (CDCl3, 300
4.3.1. Cell viability assay
MHz): δ1.97 (d, 6H, 2 × CH3 of chain), 2.47 (dd,12H, N-(CH3)4 of chain),
3.49 (bd, 4H, 2xN-CH2), 3.50 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.84 (s,
3H, OCH3), 4.04 (bd, 2H,3-CH2), 4.04 (m, 2H, 2xCH of chain), 5.17 (s,
2H, N-CH2), 6.39 (s, 2H, 2xCH, aromatic), 6.75 (m, 8H, 8xCH, aro-
matic),), 6.90 (bd, 2H,2xCH, aromatic); 7.10 (d, H, CH aromatic. J = 9.6
Hz); 13C NMR (CDCl3,75 MHz): δ11.9, 14.5, 18.4, 22.6, 23.0, 44.8,
45.03, 47.7, 56.1, 56.1, 56.1, 56.1, 59.1, 59.1, 59.0, 61.2, 101.9, 107.7,
MTT assay was used to determine the effect of compounds on cell
viability as described previously.33 Briefly, MTT (3-[4,5-dimethylth-
iazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to deter-
mine the effect of compounds on cell viability. Cells were seeded in 96-
well plate at a density of 1 × 104 cells/well. After 24 h of growth, cells
were treated with different concentration of compounds. For inhibitor
studies, MDA-MB-231 were pre-treated with 10 mM NAC, 10 mM of CQ
13

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
and 50 µM of Z-VAD-FMK for 2 h followed by treatment with 2 µM of
compounds 19, 20, 22 and 23 for 24 h. At the end of treatment, 20 µL of
MTT (5 mg/mL) was added in each well. After incubation for 3 h, media
along with MTT was carefully removed. 200 µL DMSO was added to
dissolve the formazan crystal and absorbance was recorded at 540 nm
using ELISA plate reader. The assay was performed in triplicates having
three replica of each test compound.
Table 4A
Effect of compound 19 as a single acute oral dose at 5, 50, 300 mg/kg on body
weight, hematological and serum biochemical parameters in Swiss albino mice.
Parameters
Oral dose of compound 19 at mg/kg body weight as a single
oral dose
Control
5 mg/kg
50 mg/kg
300 mg/kg
Body weight (gm)
22.75 ±
0.93
21.71 ±
1.00
21.31 ±
0.81
21.88 ±
0.73
4.3.2. Cell cycle analysis
Haemoglobin
(gm/dL)
17.35 ±
0.30
16.59 ±
0.60
17.01 ±
0.47
15.96 ±
0.71
Cell cycle analysis was performed to evaluate effect of compounds in
cell cycle progression using propidium iodide (PI) staining by flow cy-
tometer against MDA-MB-231.34 Briefly, MDA-MB-231 cells (1 × 106
cells) were seeded in T-25 culture flask. After 24 h of growth, cells were
treated with different concentration of compounds for 24 h. At the end of
treatment, all the cells including floating were harvested, washed with
PBS and fixed in ice cold 70% ethanol for 1 h at 40 ◦C. Cells were then
centrifuged, re-suspended in 300 µL PBS containing 30 µg of RNAse A
and 15 µg of PI and incubated for 30 min at room temperature. Samples
were acquired and analyzed by flow cytometry using FACS Calibur in-
strument (BD Biosciences).
RBC (million/
9.21 ± 0.32
9.67 ± 0.74
8.72 ± 0.37
9.45 ± 0.63
mm3)
WBC(thousands/
mm3)
8.55 ± 2.71
6.73 ± 0.80
4.86 ± 0.35
5.53 ± 0.98
ALP (U/L)
123.22 ±
16.04
97.53 ±
19.48
106.88 ±
9.77
98.70 ±
13.96
SGOT (U/L)
SGPT (U/L)
22.94 ±
4.04
22.11 ±
2.82
21.92 ±
3.24
27.73 ±
3.16
12.65 ±
2.98
14.47 ±
0.84
15.07 ±
1.54
16.00 ±
1.64
Creatinine (mg/
dL)
0.11 ± 0.03
0.12 ± 0.02
0.14 ± 0.02
0.15 ± 0.03
Triglycerides (mg/
dL)
81.12 ±
7.75
63.83 ±
7.82
91.70 ±
91.20 ±
13.60
16.23
4.3.3. Apoptosis analysis
Bilirubin(mg/dL)
Cholesterol (mg/
dL)
0.44 ± 0.06
122.05 ±
8.12
0.30 ± 0.02
112.13 ±
6.90
0.35 ± 0.02
121.96 ±
11.26
0.36 ± 0.04
132.33 ±
10.57
Apoptosis analysis was carried out using annexinV-FITC/PI dual
staining kit using flow cytometry.33 In brief, MDA-MB-231 cells (1 ×
106) were seeded in six-well plates and allowed to grow for 24 h. The
medium was then replaced with medium containing different concen-
trations of compounds 19, 20, 22 and 23 and incubated for 24 h. At the
end of incubation, cells were harvested by trypsinization, washed with
PBS, re-suspended in binding buffer along with Annexin V-FITC and PI
for 10 min in dark at room temperature. Samples were analyzed using
FACS Calibur instrument (BD Biosciences).
Albumin (g/dL)
Protein (mg/ml)
1.62 ± 0.09
2.01 ± 0.11
1.52 ± 0.12
1.61 ± 0.16
1.43 ± 0.23
1.48 ± 0.21
1.94 ± 0.17
1.93 ± 0.31
Data are expressed as Mean ± SE (n = 6). SGOT: serum glutamic-oxaloacetic
transaminase , SGPT: Serum glutamic pyruvic transaminase ALP: Alkaline
phosphatise; gm/dL, gram per deciliter; U/L, units per litre; mg/dL, milligram
per deciliter. Tukey’s multiple comparison test was used to determine the sig-
nificance between compound 19 and control, P < 0.05.
4.3.4. Mitochondrial membrane potential (MMP) assay
Table 4B
MMP was measured by flow-cytometry using JC-1 (5,5,6,6-tetra-
chloro-1,1,3,3 tetraethylbenzimidazolecarbocyanine iodide) dye
against MDA-MB-231 cells.33 Briefly, MDA-MB-231 cells (1 × 106) were
seeded in six well plates for 24 h. Cells were treated with different
concentrations of compounds for 24 h. After 24 h, cells were harvested
by trypsinization, washed with PBS and incubated along with 5 µg/ml
JC-1 dye for 30 min in dark at room temperature. At the end of incu-
bation, cells were re-suspended in 300 µL of PBS and analyzed using
FACS Calibur instrument and FACSuite Software (BD Biosciences).
Effect of compound 23 as a single acute oral dose at 5, 50, 300 mg/kg on body
weight, haematological and serum biochemical parameters in Swiss albino mice.
Parameters
Oral dose of compound 23 at mg/kg body weight as a single
oral dose
Control
5 mg/kg
50 mg/kg
300 mg/kg
Body weight (gm)
22.75 ±
0.93
21.60 ±
0.86
23.48 ±
0.93
22.75 ±
0.63
Haemoglobin
(gm/dL)
17.35 ±
0.30
15.83 ±
1.26
15.21 ±
0.37
15.54 ±
0.65
RBC (million/
9.21 ± 0.32
9.12 ± 0.48
8.67 ± 0.55
7.96 ± 0.44
mm3)
4.3.5. ROS generation assay
WBC(thousands/
mm3)
8.55 ± 2.71
6.23 ± 1.39
7.50 ± 0.82
5.60 ± 0.98
ROS was measured using DCFH-DA (2,7-dichlorodihydrofluorescein
diacetate) dye by flow cytometer in MDA-MB-231 cell.33 Briefly, MDA-
MB-231 cells (1 × 106) were seeded in 6-well plates for 24 h. Cells were
treated with different concentrations of compounds for 24 h. At the end
of treatment, cells were harvested by trypsinization, fixed in cold
methanol and incubated along with 30 µg/mL DCFH-DA for 30 min in
dark at room temperature. At the end of incubation, cells were re-
suspended in 300 µL of PBS and analyzed using FACS Calibur instru-
ment (BD Biosciences).
ALP (U/L)
123.22 ±
16.04
87.76 ±
8.97
84.85 ±
14.25
74.12 ±
8.11
SGOT (U/L)
SGPT (U/L)
22.94 ±
4.04
28.86 ±
3.88
33.28 ±
3.50
33.25 ±
2.39
12.65 ±
2.98
11.82 ±
1.02
18.69 ±
2.55
19.12 ±
3.65
Creatinine (mg/
dL)
0.14 ± 0.02
0.12 ± 0.04
0.12 ± 0.02
0.14 ± 0.02
Triglycerides (mg/
dL)
81.12 ±
7.75
105.82 ±
14.59
83.99 ±
86.58 ±
8.99
8.66
Bilirubin(mg/dL)
Cholesterol (mg/
dL)
0.44 ± 0.06
122.05 ±
8.12
0.34 ± 0.04
117.25 ±
12.55
0.39 ± 0.02
108.65 ±
13.90
0.36 ± 0.02
108.72 ±
11.79
4.3.6. Monodansylcadaverine (MDC) staining
MDC staining was used to detect formation of autophagic vacuoles
by fluorescence microscopy in MDA-MB-231cells.33 MDA-MB-231 (1 ×
106) cells were seeded on cover slips for 24 h followed by treatment with
different concentrations of compounds for 24 h. After 24 h, cells were
gently washed with PBS and stained with 0.05 mM MDC for 30 min at
room temperature in dark. At the end of incubation, cells were washed
with PBS and samples were analyzed for autophagosome formation
using Nikon ECLIPSE 80i fluorescence microscope.
Albumin (g/dL)
Protein (mg/ml)
1.62 ± 0.09
2.01 ± 0.11
1.48 ± 0.06
1.57 ± 0.32
1.29 ± 0.11
1.28 ± 0.12
1.53 ± 0.08
1.66 ± 0.24
Data are expressed as Mean ± SE (n = 6). SGOT: serum glutamic-oxaloacetic
transaminase , SGPT: Serum glutamic pyruvic transaminase ALP: Alkaline
phosphatise; gm/dL, gram per deciliter; U/L, units per litre; mg/dL, milligram
per deciliter. Tukey’s multiple comparison test was used to determine the sig-
nificance between compound 19 and control, P < 0.05.
4.3.7. Tubulin polymerisation assay
Tubulin polymerisation assay was performed as per reported method
14

Y. Gautam et al.
Bioorganic & Medicinal Chemistry 42 (2021) 116252
using ‘assay kit’ from Cytoskeleton, USA.35 Paclitaxel (Sigma, USA) was
used as standard stabilizer of tubulin polymerisation. In brief, 96 well
microtiter plate was kept pre-warmed and tubulin protein 3 mg/mL in
tubulin polymerization buffer (80 mM PIPES, pH 6.9, 2 mM MgCl2, 0.5
mM EGTA, 1 mM GTP and 15% glycerol) was placed in it at 37 ◦C in the
presence of our test compounds 19, 20, 22 and 23 with desired con-
weighing between 20 and 25 g. The animals were maintained at 22 ± 5
0C with humidity control and also on an automatic dark and light cycle
of 12 h. The animals were fed with the standard mice feed and provided
ad libitum drinking water. Mice of group 1 (Group I) were kept as control
and animals of groups 2, 3, and 4 (Groups II, III, & IV) were kept as
experimental. The animals were acclimatized for 7 days in the experi-
mental environment prior to the actual experimentation. Test compound
was dissolved in minimum volume of DMSO, diluted with 0.7% CMC
(carboxymethylcellulose) and was given at 5, 50, and 300 mg/kg body
weight to animals of groups 2, 3, and 4 (Groups II, III & IV) respectively
once orally. Control animals received only vehicle. The animals were
checked for mortality and any signs of ill health at hourly interval on the
day of administration of drug and there after a daily general case side
clinical examination was carried out for changes in skin, mucous
membrane, eyes, occurrence of secretion and excretion and also re-
sponses like lachrymation, pilo-erection respiratory patterns etc. Also
changes in gait, posture and response to handling were also recorded. In
addition to observational study, body weights were recorded and blood
and serum samples were collected from all the animals on 7th day of the
experiment in acute oral toxicity. The samples were analyzed for total
RBC, WBC, differential leucocytes count, haemoglobin percentage and
biochemical parameters like ALP, SGPT, SGOT, total cholesterol, tri-
glycerides, creatinine, bilirubin, serum protein and tissue protein ac-
tivity. The animals were then sacrificed and were necropsied for any
gross pathological changes. Weights of vital organs like liver, heart,
kidney etc. were recorded.
centrations (10 μM). All samples were properly mixed and polymerisa-
tion was monitored in kinetic mode at 340 nm every min for 1 h using
Spectramax plate reader. Paclitaxel (taxol) was used as standard stabi-
lizer of tubulin polymerisation.
4.3.8. In vivo antitumor efficacy
In vivo antitumor studies were conducted in orthotopic syngenic
mice model in adult female Balb/c mice.36 Animal studies were con-
ducted with prior approval from the Institutional Animal Ethics Com-
mittee (IAEC), CSIR-CDRI, Lucknow, India. Adult female Balb/c mice
were used to develop 4 T1 syngenic mice mammary tumor model.
Briefly, 4 T1 cells (7 × 105) were injected in mammary fat pad of six
week old female Balb/c mice. After one week, when the tumors become
measurable, animals were randomly divided into ten groups. Com-
pounds were dissolved in ethanol and further diluted in PBS. Different
compounds were orally administered at the dose of 10 mg/kg body and
20 mg/kg of body weight daily for 21 days. 10 mg/kg of body weight of
paclitaxel was given intravenous twice a week. Control group animals
received vehicles only. The body weight of animals as well as tumor size
were measured twice a week. The tumor volume was calculated using
the formula (LW2) × 0.5, where L is the tumor length and W is the tumor
width. Test compounds 19, 20, 22 and 23 were administered as oral
gavage at the dose of 10 mg/kg and 20 mg/kg of bodyweight daily for
21 days. Paclitaxel at the dose of 10 mg/kg of bodyweight was admin-
istered intravenously twice a week for 21 days. Control group animals
received vehicles only. Oral administration of compounds 19, 20, 22
and 23 was preferred in order to enhance safety, versatility as well as
minimize pain. However, further pharmacokinetics optimization study
required for maximum bioavailability. Intravenous administration of
paclitaxel is widely used due its better efficacy and bioavailability.
CRediT authorship contribution statement
Yashveer Gautam, Nandini Pathak and Dushyant Singh raghuvanshi-
Chemical Synthesis Sharmistha Das and Hamidullah Khan-Cytotoxicity,
cellcycle analysis, Annexin-V, ROS exp., In-vivo efficacy etc. Hina Iqbal,
Pankaj Yadav and D. Chanda-Acute oral toxicity exp. Vijay Kumar Sirohi
and Anila Dwivedi- Estrogen Receptor trans activation exp. Sana Khan
and Feroz Phan- Docking exp. Karuna Shanker-Purity Profile Rituraj
Konwar-Detailed biology exp. planning, execution Arvind S. negi-
detailed chemistry exp., planning, execution.
4.3.9. ERα and ERβ transcriptional activation assay
Transcription activation studies were done in MDA-MB-231 cells
Declaration of Competing Interest
with ER
α
(pSG5 mER
α
) and ERβ (pSG5-hER β) plasmids.37 MDA-MB-231
cells were seeded in 24 well plate and allowed to attain a confluency of
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
80–90%. The cells were then transfected with 500 ng of expression
vector for ERα (pSG5 mERα) and ERβ (pSG5-hER β) plasmids (generous
gifts from Prof. M.G. Parker, Imperial Cancer Research Fund, London,
UK) using Lipofectamine 2 LTX transfection reagent (ThermoFisher) as
per manufacturer’s protocol. To normalize for transfection efficiencies,
pRL-SV40-luc (Promega) was used. After transfection, cells were tryp-
sinized and seeded (2 × 04 cells/well) into the 96-well plate. Cells were
Acknowledgement
Senior Research Fellowship to the authors (YG) from Council of
Scientific and Industrial Research (CSIR) is duly acknowledged. The
manuscript has CIMAP Communication No.: CIMAP/PUB/2017/25.
incubated with E2 (100 nM), 1
μ
M of compounds 19, 20, 22 and 23 and
tamoxifen 1
μ
M. Luciferase activity was quantified using Dual Luciferase
Assay System (Promega), according to the manufacturer’s protocol to
detect the transcriptional activity. The firefly luciferase intensity for
each sample was normalized based on transfection efficiency measured
by Renilla luciferase activity. The experiments were performed intri-
plicate with three replicates in each.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116252.
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approved by the Institutional Animal Ethics Committee (IAEC) of CSIR-
Central Institute of Medicinal and Aromatic Plants, Lucknow, India via
CIMAP/IAEC/2016–19/01 dated 09–02-2017.
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