Synthesis, biological evaluation, and molecular docking analysis of phenstatin based indole linked chalcones as anticancer agents and tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104447

A library of new phenstatin based indole linked chalcone compounds (9a-z and 9aa-ad) were designed and synthesized. Of these, compound 9a with 1-methyl, 2- and 3-methoxy substituents in the aromatic ring was efficacious against the human oral cancer cell

Full text of DOI:10.1016/j.bioorg.2020.104447

Journal Pre-proofs
Synthesis, biological evaluation, and molecular docking analysis of phenstatin
based indole linked chalcones as anticancer agents and tubulin polymerization
inhibitors
Jyoti Kode, Jeshma Kovvuri, Burri Nagaraju, Shailesh Jadhav, Madan
Barkume, Subrata Sen, Nirmal Kumar Kasinathan, Pradip Chaudhari,
Bhabani Shankar Mohanty, Jitendra Gour, Dilep Kumar Sigalapalli, C.
Ganesh Kumar, Trupti Pradhan, Manisha Banerjee, Ahmed Kamal
PII:
S0045-2068(20)31745-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.104447
YBIOO 104447
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
1 April 2020
27 October 2020
28 October 2020
Please cite this article as: J. Kode, J. Kovvuri, B. Nagaraju, S. Jadhav, M. Barkume, S. Sen, N. Kumar
Kasinathan, P. Chaudhari, B. Shankar Mohanty, J. Gour, D. Kumar Sigalapalli, C. Ganesh Kumar, T. Pradhan,
M. Banerjee, A. Kamal, Synthesis, biological evaluation, and molecular docking analysis of phenstatin based
indole linked chalcones as anticancer agents and tubulin polymerization inhibitors, Bioorganic Chemistry (2020),
doi: https://doi.org/10.1016/j.bioorg.2020.104447
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Synthesis, biological evaluation, and molecular docking analysis of phenstatin based indole
linked chalcones as anticancer agents and tubulin polymerization inhibitors
Jyoti Kode*,a,b,c@, Jeshma Kovvurid,e,j,@, Burri Nagarajud,e,@, Shailesh Jadhav , Madan Barkume ,
a
a
a
a
c,f
f
Subrata Sen , Nirmal Kumar Kasinathan , Pradip Chaudhari , Bhabani Shankar Mohanty ,
g
g
d,e
b
Jitendra Gour , Dilep Kumar Sigalapalli , C. Ganesh Kumar , Trupti Pradhan , Manisha
c,h
,,d,e,g,i,@
Banerjee and Ahmed Kamal*
a
b
Anti-Cancer Drug Screening Facility (ACDSF), Tumor Immunology & Immunotherapy Group,
Advanced Centre for Treatment, Research & Education in Cancer (ACTREC), Tata Memorial
Centre, Kharghar, Navi Mumbai 410210, India
cHomi Bhabha National Institute (HBNI), Training School Complex, Anushakti Nagar, Mumbai
4
00085, India
dDepartment of Organic Synthesis and Process Chemistry, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007,India
eAcademy of Scientific and Innovative Research (AcSIR), New Delhi 110025, India
fSmall Animal Imaging Facility, Advanced Centre for Treatment, Research & Education in
Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India
gDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
hMolecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai- 400
0
85, India.
iSchool of Pharmaceutical Education and Research (SPER), Jamia Hamdard, New Delhi 110062,
India
jDepartment of Humanities and Sciences, Vardhaman College of Engineering (Autonomous),
Shamshabad, Hyderabad, Telangana, 501218, India
@The authors contributed equally.
*
Corresponding authors
1

ABSTRACT:
A library of new phenstatin based indole linked chalcone compounds (9a-z and 9aa-ad) were
designed and synthesized. Of these, compound 9a with 1-methyl, 2- and 3-methoxy substituents
in the aromatic ring was efficacious against the human oral cancer cell line SCC-29B, spheroids,
and in a mouse xenograft model of oral cancer AW13516. Compound 9a exhibited anti-cancer
activity through disrupting cellular integrity and affecting glucose metabolism-which is a
hallmark of cancer. The cellular architecture was affected by inhibition of tubulin polymerization
as observed by an immunofluorescence assay on 9a-treated SCC-29B cells. An in vitro tubulin
polymerization kinetics assay provided evidence of direct interaction of 9a with tubulin. This
physical interaction between tubulin and compound 9a was further confirmed by Surface
Plasmon Resonance (SPR) analysis. Molecular docking experiments and validations revealed
that compound 9a interacts and binds at the colchicine binding site of tubulin and at active sites
of key enzymes in the glucose metabolism pathway. Based on in silico modeling, biophysical
interactions, and pre-clinical observations, 9a consisting of phenstatin based indole-chalcone
scaffolds, can be considered as an attractive tubulin polymerization inhibitor candidate for
developing anti-cancer therapeutics.
KEYWORDS:
Phenstatin based indole-linked chalcones; in vitro cell cytotoxicity; Cancer stem- like cells.
1
8
Spheroid assay, oral cancer xenograft model; PET-CT imaging, F-fluorodeoxyglucose uptake,
glucose metabolism; in silico analysis; molecular docking; tubulin polymerization inhibition;
Surface Plasmon Resonance, multi-target metabolic inhibitor; anti-cancer efficacy,
immunofluorescence, laser confocal microscopy, transmission electron microscopy.
2

INTRODUCTION:
Oral cancer is a debilitating cancer that ranks sixth in global occurrence. Oral squamous cell
carcinoma (OSCC) is the most common malignant epithelial neoplasm affecting the oral cavity
1
and it accounts for more than 90% of all oral cancers. Microtubules are dynamic polymers of α-
and β-tubulin dimers that play a pivotal role in the cell architecture, cellular processes, and cell
division. Microtubule disruption can induce cell cycle arrest in G /M phase and abnormal mitotic
2
2
3-5
spindle formation. Moreover, tubulin inhibitors can also act as vascular disrupting agents.
Hence tubulin polymerization inhibitors are one of the most important tools for drug discovery.
6
,7
Anti-tubulin agents such as vinca alkaloids, taxane and colchicine (Figure 1, (1)) bind to one
of the three binding sites on tubulin subunits. It is believed that colchicine binding site tubulin
inhibitors may be better than the other two site binders in overcoming drug resistance.8-10 They
1
1
exhibit antiangiogenic property either by microtubule stabilization (e.g. docetaxel, taxanes) or
1
2
by destabilization (e.g. colchicine, combretastatins). The present study is focused on the design
of new tubulin polymerization inhibitors that act through blocking active colchicine binding site.
O
H CO
3
Z
NH
H CO
OH
3
H CO
3
A
B
O
OCH3
H CO
3
OCH3
OH
OCH3
H CO
3
OCH3
OCH3
OCH3
H CO
3
Z = O, Phenstatin (3)16, 17
Colchicine (1)13
Combretastatin A-4 (2)14,15
16
Z =CH , Isocombretastatin A-4 (4)
2
R4
R3
O
O
O
R2
H CO
3
H CO
3
R
O
X
N
H CO
3
H CO
3
H CO
3
OCH3
OCH3
N
R1
H CO
3
OCH
3
X=O, (5)18
Indole-Chalcone (8)27
Target compounds (9a-z & 9aa-ad)
X= NH, R = 6-OMe SCB01A, BPR0L075(6)19-25
X =S, (7)26
3

Figure 1. Chemical structures of some pharmaceutically important tubulin polymerization inhibitors
including target compounds 9a-z and 9aa-ad.
2
8
Combretastatin A-4 (CA-4) (Figure 1, (2)) is one of the most important naturally occurring
tubulin polymerization inhibitor that binds to the colchicine site (Figure 1, (2)). CA-4 analogs
were synthesized and evaluated for their activity because of its simple structure. Based on the
2
previous reports, the replacement of double bond in CA-4 with sp hybridized carbonyl group
provides molecules like phenstatin (Figure 1, (3)), that will maintain the cis orientation of the
two rings, a characteristic feature of CA-4, which is responsible for exhibiting better biological
activities.17 Several phenstatin related compounds were synthesized, while retaining 3,4,5-
timethoxyphenyl ring and by changing either B-ring with different heteroaromatic rings such as
benzofuranyl (Figure 1, (5)), indolyl (Figure 1, (6)), benzothiophenyl (Figure 1, (7)) or by
bridging with a methylene functionality (4). Among them, a compound with carbonyl bridge and
indolyl B-ring (Figure 1, (6)) is known to efficiently inhibit cancer cell growth as well as tubulin
polymerization.29-31 Moreover, indole is a well-known privileged scaffold and its derivatives
have been reported to exhibit a number of biological properties including anticancer activity.32
On the other hand, chalcone is the privileged scaffold and one of the important core moieties of
many naturally occurring biologically active compounds and has attracted much research
attention in the recent past. In recent trend analysis, chalcone was reported by Zdrazil and Guha
3
3
(
2018) , as on the third position as a popular choice in the medicinal chemistry literature, after
biphenyl and diphenyl ether. Several synthetic (Figure 1, (8)) and naturally occurring chalcones
have been reported to possess significant anti-tubulin activities.27,34-38 These compounds are
known to exhibit various biological activities including anti-malarial, anti-tubercular, anti-HIV
3
9
apart from anticancer. As a crucial example, penta-OMe-chalcone was found to overcome the
drug resistance against the Vismodegib-resistant mutant G protein-coupled receptor
Smoothened.40 In the last few years, we have previously designed and synthesized a large
number of conjugates and hybrids involving heterocyclics particularly based on combretastatin
A-4, chalcones, and indoles in pursuit of newer, more effective and safer anticancer agents.41-44
Considering these modifications and the biological importance of phenstatin, indole, and
4

chalcone scaffolds, we designed and synthesized phenstatin based indole linked chalcones to
conserve the privileged structures of indole and trimethoxyphenyl. The resulted compounds were
evaluated for their antiproliferative activity.
Increased glucose metabolism is considered a hallmark of cancer growth and progression.45,46
Cancer cells utilize aerobic glycolysis rather than oxidative phosphorylation termed as “Warburg
effect”, which leads to faster glucose metabolism for ATP generation. This property of rapid
glucose uptake is utilized for evaluating the effect of potential anti-cancer compounds as a
1
8
18
response to therapy by tumor visualization using [ F]-fluorodeoxyglucose ( F-FDG) positron
emission tomography/computed tomography (PET-CT) imaging. Hexokinase-2 (HK2) enzyme
which marks the first catalytic step of glucose metabolism, is considered an important target of
4
5
metabolic inhibitors that are designed as potential anti-cancer compounds. Fenbendazole, a
new microtubule interfering agent was found to display anti-neoplastic efficacy in vitro as well
as in vivo through blocking the activity of HK2 enzyme- a key glycolytic enzyme. Fenbendazole
was further found to act through multiple cellular pathways leading to the effective elimination
4
7
of cancer cells, hence was considered as a potential therapeutic agent for cancer . Besides the
glycolytic pathway, the tricarboxylic acid cycle (TCA) cycle also contributes to altered
bioenergetic metabolism. Hence current efforts are focused on designing and identifying
chemical compounds that have inhibitory action against multiple enzymes in glycolytic pathway
and alternative energy pathway viz. TCA cycle. Based on this, in current study, biologically
efficacious compounds were also evaluated for their efficacy to target glucose uptake by PET-
CT study. Further advanced laser confocal microscopy imaging, in silico molecular docking
tools, in vitro tubulin polymerization assay, and a biophysical tool like Surface Plasmon
Resonance (SPR) were used to characterize molecular interactions of potential anti-cancer
compounds with the active site of tubulin.
1
2
. RESULTS AND DISCUSSION:
.1. Chemistry
Phenstatin based indole linked chalcones (9a-z and 9aa-ad) were synthesized as shown in
Scheme 1. Initially, the N-substituted-5-bromoindoles 11a-c were synthesized by N-alkylation
5

reaction of 5-bromoindole (10) with MeI and EtBr or by protecting with TBDMSCl in the
presence of sodium hydride and DMF. This upon treatment with n-BuLi and 3,4,5-
trimethoxybenzaldehyde in dry THF at -78˚C yielded the corresponding secondary alcohols 12a-
c as shown in Scheme 1. Oxidation of 12a-c with IBX in DMSO provided the keto compounds
(13a-c). Thereafter the 13a compound TBDMS group was deprotected using TBAF. Later 13a-c
compounds were subjected to Vilsmeier formylation to afford 1-methyl-5-(3,4,5-
trimethoxybenzoyl)-1H-indole-3-carbaldehydes (14a-c) in good yields. Finally, the synthesized
aldehydes were subjected to Claisen-Schmidt condensation with respective substituted
acetophenones to afford the desired products, 9a-z and 9aa-ad. All the synthesized compounds
1
13
were characterized by H, C NMR, and HRMS spectral analysis.
OH
O
Br
H CO
d
H3CO
H CO
Br
3
a or b
c (i, ii)
N
N
R1
N
N
H CO
3
3
H
R1
1a-c
1a: R =CH
R1
OCH3
OCH3
13a-c
1
12a-c
10
R =TBDMSCl
1
e
1
1
1
1
3
1
1
1
3a: R =H
1
f
1b: R =C H
1
2 5
3b: R =CH
1
3
1c: R =TBDMSCl
1
3c: R =C H
1
2 5
O
CHO
H CO
3
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a, R = CH , R , R = OCH , R = H 9p, R = C H , R , R , R = OCH
1 3 2 3 3 4 1 2 5 2 3 4 3
b, R = CH , R , R = H, R , = NO
9q, R = C H , R -R = C H , R = H
1 2 5 2 3 4 6 4
1
3
2
4
3
2
N
H CO
3
c, R = CH , R , R , R = OCH ,
9r, R = C H , R , R = H, R = OCH
1
3
2
3
4
3
1 2 5 2 4 3 3
R1
OCH3
d, R = CH , R -R = C H , R = H
9s, R = C H , R , R = H, R = CH
1 2 5 2 4 3 3
1
3
2
3
4
6
4
e, R = CH , R , R = H, R = OCH 9t, R = C H , R , R , R = H,
14a-c
1
3
2
4
3
3
1
2
5
2
3
4
f, R = CH , R , R = H, R = CH
9u, R = C H , R , R = H, R = Cl
1
3
2
4
3
3
1 2 5 2 4 3
g, R = CH , R , R , R = H,
9v, R = C H , R = H, R -R = CH O
2
g
1
3
2
3
4
1
2
5
2
3
4
2
h, R = CH , R , R = H, R = Cl
9w, R = C H , R = NO , R , R = H
1
3
2
4
3
1 2 5 2 2 3 4
i, R = CH , R = Cl, R , R = H
9x, R = C H , R ,R = Cl, R = H
1 2 5 2 3 4
1
3
2
3
4
R2
j, R = CH , R = H, R -R = CH O 9y, R = C H , R , R = H, R = F
1
3
2
3
4
2
2
1
2
5
2
4
3
k, R = CH , R = NO , R , R = H
9z, R =H, R , R = OCH , R = H
1
3
2
2
3
4
1 2 3 3 4
O
R3
l, R = CH , R ,R = Cl, R = H
9aa, R =H, R , R = H, R , = NO
1 2 4 3 2
1
3
2
3
4
m, R = CH , R , R = H, R = F
9ab, R =H, R , R = H, R = OCH
1
3
2
4
3
1 2 4 3 3
O
R4
n, R = C H , R , R = OCH , R = H 9ac, R =H, R , R , R = H,
1
2
5
2
3
3
4
1
2
3
4
H CO
o, R = C H , R , R = H, R , = NO 9ad, R =H, R , R = H, R = Cl
3
1
2
5
2
4
3
2
1
2
4
3
N
H CO
3
R1
OCH3
9a-z and 9aa-ad
Scheme 1: Reagents and conditions: (a) TBDMSCl , NaH, THF, 0 °C-rt, 3 h, 82%; (b) MeI/EtBr, NaOH,
DMSO, 0 °C-rt, 3 h, 91-93%; (c) i) THF, -78 °C, n-BuLi, 1 h; ii) 3,4,5-trimethoxybenzaldehyde (in THF),
4
h, 72˗76%; (d) IBX, DMSO, 0 °C-rt, 3 h, 92-95%; (e) TBAF, THF, 0 °C-rt, 4 h, 84% (f) POCl , DMF,
3
CHCl , reflux, 12 h, 76-84%; (g) substituted acetophenone (1 eq), EtOH, 10% NaOH (aq.), rt, 6 h, 78-
3
8
9%.
6

2
2
.2.
Biology
.2.1. Cytotoxicity assay
To evaluate the biological usefulness of phenstatin based indole linked chalcones, these
compounds were tested against some cancer cell lines of different tissue origins. The in-vitro
testing of all these compounds was conducted at concentrations 0.1, 1, 10, and 100 µM by
standard Sulforhodamine B (SRB) assay. Percent control growth was calculated and
chemosensitivity response parameters GI50 (50% growth inhibition), TGI (total growth
inhibition), and LC (50% lethal concentration) were extrapolated for all the compounds. Cell
5
0
control was considered as 100% cell growth, Adriamycin (ADR) was considered as positive
control and exhibited a lytic effect on cells seeded i.e. -50% cell growth.
-6
As per NCI guidelines GI value of ≤ 10 M (i.e. 1 µM) was considered to demonstrate highly
5
0
significant inhibitory activity in case of pure compounds. Compound 9b (R is methyl and R is
1
3
nitro) demonstrated significant anti-proliferative activity against Hep-G2 liver hepatoma cells,
compound 9a (R is methyl, R and R are methoxy substituents Supplementary Figure S1)
1
2
3
,
against SCC-29B oral cancer cells and compounds 9e (R is methoxy) and 9l (R and R are
3
2
3
chloro substituents) against HT-29 colon cancer cells (Table 1). The structure activity
relationship of the synthesized compounds indicated that the pesence of trimethoxy ring and
carbonyl group essential for tubulin activity and methoxy substituents at R and R enhanced
2
3
cytotoxic activity (Supplementary Figure S1). We observed a few compounds exhibited
moderate activity with GI50 ≤ 10µM and few demonstrated low activity with GI50 at ≤100µM.
It was observed that SCC-29 B oral cancer cell line was found to be sensitive target for most of
the synthesized compounds (21 of 30) exhibiting low, moderate, or significant anti-proliferative
activity. Hence this oral cancer cell line model was further utilized for spheroid assay too.
7

_
__________________________________________________________________________
Table 1: In vitro cytotoxicity testing of the synthetic compounds against a panel of cancer cell
lines by SRB assay
Sample
Code
GI (µM)
5
0
GI (µM)
50
Sample Code
HEP-G2 SCC-29B HT-29
HEP-G2 SCC-29B HT-29
9
a
>100
0.018
6.8
< 0.1
88
51
9q
9r
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
NT@
< 0.1
>100
>100
>100
59
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
NT
9
b
43
9
c
1.4
1.6
1.2
1
15
9s
9
d
6.5
5.2
9t
9
e
11
< 0.1
>100
>100
>100
>100
>100
>100
<0.1
>100
>100
>100
>100
9u
>100
>100
>100
32
9
f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9v
9
g
6.4
17
9w
9x
9
h
9
i
>100
19
9y
64
9
j
9z
>100
56
9
k
>100
59
9aa
9ab
9ac
9ad
CA-4
ADR
9
l
8.9
9
m
11
44
9
n
72
41
9
o
27
< 0.1
< 0.1
9
p
13
< 0.1
*
GI50 value is considered to demonstrate highly significant activity at ≤ 1 µM (yellow highlighted),
moderate activity at ≤ 10µM (blue highlighted), low activity at ≤100µM (green highlighted) and no
activity (>100 µM) for synthetic compounds.
@
ND: Not tested.
The data reflects GI50 calculated from mean percent growth value at four different concentrations of
three different experiments. Individual experiment was performed with each drug dilution in three
replicates. Mean Growth percent data with standard error, along with individual three experiment percent
growth values is provided in Supplementary Tables S1-S3.
CA-4: Combretastatin A-4 and ADR: Adriamycin employed as positive controls for SRB assay.
8

We had performed in vitro cytotoxicity studies for 10 cancer cell lines. However, it was observed from
this data that these class of compounds are active mostly against oral cancer cell line, presumably in view
of its selectivity towards this cell line.
_
____________________________________________________________________________
Supplementary Figure S2 demonstrates growth curve profiles of SCC-29B oral cancer cells post
treatment with phenstatin bearing indole linked chalcones (9a-z and 9aa-9ad) for 48 hr. The
comparative growth curve profiles of cancer cell lines HT-29 (colon), Hep-G2 (liver), and SCC-
2
9B (oral) after treatment with compounds 9a, 9f, 9e, 9l, and 9b are shown in Supplementary
Figure S3 . Cell growth response lines to compounds Adriamycin (Supplementary Figure S2),
e, and 9l (Supplementary Figure S3) are flat and not descending, hence slope and intercept are
9
not feasible for standard calculations. However, as we see, at 0.1µM cell growth was below 50%,
hence we report GI50 as below 0.1µM. Only compound 9a exhibited significant anti-proliferative
activity against SCC-29B oral squamous cancer cells. Hence this compound was evaluated
further for its anti-cancer efficacy in cancer stem-like cells (CSC) or spheroid assay and in-vivo
NOD-SCID immunodeficient animal models.
2
.2.2. Evaluation of anti-cancer potential using oral cancer spheroids
To evaluate the efficacy of compound 9a to inhibit the growth of cancer stem-like cells, it was
tested in a cytotoxicity assay using oral squamous cancer spheroids (SP) cells. Oral CSC were
+
lo
characterized by flow cytometry and were found to have increased populations of CD44 CD24
as compared to that found in an adherent SCC-29B cell line (Supplementary Figure S4, panels B
and D). ADR drug uptake was also found to be significantly reduced in oral CSC as compared to
adherent cell line (Supplementary Figure S4, Panel C). The drug uptake increased with time in
the case of adherent as well as spheroid SP cells. Once characterized, both adherent oral cancer
cell line and spheroid cells were tested against compound 9a in the cytotoxicity assay. As evident
in Figure 2A, compound 9a affected the percent growth of oral cancer bulk (adherent) cells as
well as cancer stem-like SP cells.
9

Figure 2: Chemosensitivity of compound 9a against oral cancer spheroids and adherent cells. SCC-
9B cells were tested against 9a and Adriamycin (ADR) in 48-hr SRB assay. Panel A exhibits line
2
graphs for percent growth of SCC-29B adherent cells (intact line) and spheroids (SP, dashed line) for
compound 9a and ADR. This data is mean of three experiments. Standard error in each set was less than
<
3%. Spheroids cells exhibited reduced sensitivity to compounds compared to adherent counterparts.
Panel B shows mean values of lethal concentration 50 (LC ), total growth inhibition (TGI) and growth
5
0
inhibition 50 (GI ) of three experiments. It is evident that GI value is higher for spheroids in
5
0
50
comparison with that shown by adherent cells.
Oral CSC exhibited chemoresistance to compound 9a as higher LC , TGI, and GI
50
5
0
concentrations (Figure 2) of 9a were required for growth inhibition in comparison to that shown
by their adherent counterparts (SCC-29B GI , CSC: 29 µM; adherent: 0.96 µM). Thus, the
5
0
compound 9a was found to be effective in growth inhibition against CSC as well as bulk oral
4
8
cancer cells. The previous reports showed that compounds which act on CSC besides non-CSC
or bulk cells have better potential to reduce the tumor in xenograft study.
2
.2.3.Evaluation of anti-tumor efficacy using an in vivo animal model
The in vivo antitumor property of this lead compound 9a was further evaluated in the human
xenograft carcinoma model employing adriamycin as a reference antitumor agent. The maximum
tolerated dose (MTD) determination study for compound 9a was carried out by studying acute
toxicity. Compound 9a when injected intravenous (i.v.) as one-time dose in NOD/SCID strain
mice at 50 mg/kg and 25 mg/kg dose, exhibited very high toxicity with immediate mortality.
Hence, the next dose of 12.5 mg/kg was tested i.v. as one time dose. At this dose, 50% of mice
1
0

died at the end of the 5 day period. It was observed that the compound was found to be well
tolerated at 7.5 mg/kg dose by i.v. route (Supplementary Table S4). This study indicated that
compound 9a at 7.5 mg/kg (i.v. route) dose did not affect mice body weight significantly
(
Supplementary Figure S6). Therefore, 7.5 mg/Kg dose was considered as MTD for i.v. route
Supplementary Table S4). Based on these observations, for efficacy testing, the dosing
(
protocols were designed as 2.5 mg/kg intravenously once a week for 4 weeks Mice per group
n=6. The vehicle used for adriamycin was water. Vehicle used for 9a dilution was DMSO for
the first dilution and then water was used for further dilutions. Tumour volume, body weight, and
signs of toxicity were monitored and recorded on respective days: 1, 5, 9, 12, 15, 18, 21, 24, 27,
and 30.
It was interesting to note that the compound 9a demonstrated substantial tumor reduction with
respect to tumor-bearing control in a time-dependent manner (Table 2 and Figure 3). It was
observed that compound 9a via intravenous route exhibited better efficacy against AW13516
oral cancer xenograft (Figure 3). As evident from relative tumor volume and T/C ratio (Mean
relative tumor volume for compound 9a or standard drug Adriamycin treated groups over that of
tumor-bearing control group on individual day data), compound 9a exhibited a significant
reduction in tumor volume by day 21 in i.v. dosing protocol and continued till the end of the
study (percent regression 60%; T/C=0.4; Table 2 and Figure 3).
_
__________________________________________________________________________
Table 2. Effect of compound 9a on tumor reduction of AW13516 oral cancer xenograft
Days
T/C Ratio*
ADR
1.00
0.85
0.54
0.46
0.57
0.55
9a
1
5
9
1.00
0.93
0.67
0.52
0.51
0.45
1
1
1
2
5
8
1
1

2
2
2
3
1
4
7
0
0.54
0.51
0.36#
0.32
0.41
0.35
0.44
0.40
*
T/C ratio: Ratio of mean relative tumor volume for compound 9a or standard drug Adriamycin treated
groups over that of tumor bearing control group on an individual day. Mice per group n=6.
The vehicle used for adriamycin was water. The vehicle used for 9a dilution was DMSO for the first
dilution and then water was used for further dilutions.
Dosing protocols: 9a, 2.5mg/kg once a week i.v. for 4 weeks; Adriamycin, 2.5mg/kg at day 1,5,9 i.v.
i.v. intravenous; ADR: Adriamycin.
#
As per NCI guidelines, biological activity was considered as significant when T/C values were ≤ 0.42
(yellow highlighted and bold).
_
__________________________________________________________________________
There was no significant body weight loss (Figure 3) and all experimental mice in treated groups
survived till the end of the study. This indicated that compound 9a did not exhibit toxicity for the
provided dosing protocols and can be safely followed for further validation.
The tumor size seen in the animal groups was found to be concordant with the tumor volume
recorded (Supplementary Figure S7 panel A). Median relative tumor volume for intravenous
dosing protocol was found to be 4 cc for ADR-treated group and 2.32 cc for 9a treated group as
compared to 12 cc for tumor-bearing control (Supplementary Figure S7 panel B). Shrinking of
oral cancer xenograft with compound 9a treatment was found to have been initiated earlier than
that shown by Adriamycin (Supplementary Figure S7 panel B).
1
2

Figure 3: Effect of compound 9a on relative tumor volume, body weight and survival of
experimental mice. The line graphs show (A) relative tumor volume (RTV), (B) mean body weight
values in grams and (C) percent survival for treatment of AW13516 oral cancer xenograft bearing mice
by an intravenous route at different time points in days for all groups studied. Figure legend in the
rightmost panel depicts three groups in the study i.e. Control: tumor bearing control, ADR: Adriamycin
treated and 9a: 9a treated mice group. Each group consisted of 6 mice. RTV was measured as tumour
volume (TV) on the day of measurement/ tumour volume on day 1. RTV mean and Standard error (S.E.)
values for each day are provided in Supplementary Table S5.
2
.2.3.1. Histopathology studies
Xenograft tumors harvested at the end of the study at day 30 were processed for Hematoxylin &
Eosin (H & E) staining. Microscopic images revealed that tumor-bearing control had increased
angiogenesis and compact tissue architecture (Figure 4A). While the treated tumor sections
demonstrated that angiogenesis was significantly reduced with increased necrosis. Masson-
Trichrome stained (MTS) tumor sections showed increased collagen matrix (blue) in tumor-
bearing control which got significantly reduced upon treatment with adriamycin and compound
9
a (Figure 4A).
Apgar et al (1998)49 demonstrated that Masson’s Trichrome stained sections can be better
analyzed under fluorescence microscopy with higher sensitivity and recognition of special
protein structures. They reported that red blood cells were stained bright red-orange, while
cytoplasmic proteinaceous material was stained pale red.
1
3

Therefore in our study, MTS staining was followed to visualize cytoplasmic proteinaceous
microtubule network distinctly in tumor tissue architecture before and after compound treatment.
Tumor section stained by MTS were observed under laser confocal fluorescence microscope and
it demonstrated that Adriamycin and compound 9a were efficient in disrupting cytoskeleton
components (stained pale red) as observed by cell body shrinkages, shortening of cell migratory
processes and diffused microtubule protein staining; versus distinct microfilament staining and
compact tissue architecture observed in tumor bearing control sections (Figure 4B and 4C). It
was observed that bright red-stained red blood cells and fibrin components were also reduced in
number after treatment with adriamycin and compound 9a (Figure 4C). It was evident that in
tumor sections of compound 9a-treated animals, intercell adhesion was almost lost. Cell shape
and integrity were also found to be greatly disrupted as compared to those observed in
adriamycin-treated group tumor sections. This data clearly signifies the involvement of
compound 9a as microtubulin inhibitors.
1
4

Figure 4: Histopathological changes in oral cancer xenograft tumors treated with 9a and
Adriamycin. Panel A shows bright-field microscopic images of H & E stained (upper section) and
Masson-Trichrome stained (MTS) (lower section) sections of oral cancer xenograft tumors from
representative mice. Scale bar of 100 m is shown at the right bottom of each image. Panel B and C
shows MTS-stained laser confocal microscopic images of xenograft tumors along with a 3D plot of the
signal intensity. Panel C shows magnified regions of representative sections from each animal experiment
group. OMA-organized microtubule assembly; DM-disorganized microtubular structure and DRM-
disorganized and reduced microtubular cytoskeletal proteins.
2
.2.4. Effect of 9a on microtubule cytoskeleton of oral cancer cell line
1
5

Effect of 9a on microtubule assembly of SCC-29B oral cancer cells was observed in an
immunofluorescence assay. SCC-29B cells demonstrated normal cytoskeletal architecture with
the presence of distinct Microtubule Organizing Centre (MTOC) near the nucleus (Figure 5A).
Leading and trailing edges were clearly seen. Treatment with known tubulin destabilizer CA-4
caused tubulin destabilization in these cells and tubulin was evident in amorphous form. Further,
MTOC was lost in CA-4 treated SCC-29B cells (Figure 5). It was interesting to note that the
effect of 9a on tubulin polymerization was confirmed as it caused destabilization of tubulin
leading to the disorientation of microtubules, loss of MTOC, and cell integrity. Cells lost
polygonal shape and got rounded due to loss of microtubule dynamics. 9a at 2.5µM
concentration also caused nuclear degradation and apoptotic blebbing (Figure 5A-a and Figure
5
A-c). These observations were further confirmed at the ultrastructural level by transmission
electron microscopy. Compound 9a and positive control CA-4 caused retractions and breakage
of cell protrusions which are formed by microtubules and intermediate filaments50,51 (Figure 5B)
Compound 9a at concentration 5µM caused the complete destruction of tubulin architecture of
SCC-29B cells (Data not shown).
Figure 5: Effect of compound 9a on tubulin assembly in oral cancer cell line SCC-29B. SCC-29B
cells were treated with compound 9a (1 and 2.5 µM) and known tubulin destabilizer CA-4 (1 µM) at
1
6

given concentrations for 24 hrs. Panel A depicts cell cultures treated with anti-tubulin antibody and
observed by immunofluorescence assay. The control panel shows untreated cells with organized
microtubules and intact polygonal cell shape. Treatment with 9a caused the loss of tubulin polymerization
leading to loss of cell integrity, reduction in cell size and induced apoptosis. Subpanels b, c, and d are
magnified images. Scale bar is shown at the right bottom of each panel (a 20µm, b-c 10µm, and d 5µm).
Panel B shows electron microscopy images of cultures treated with compound 9a at 2.5µM and CA-4 at
1
µM. Arrows indicate intercellular tubular interactions that were found damaged after treatment with
compounds. Subpanels b, and c are magnified images. Scale bar is shown at the right bottom of each
micrograph (a 10µm, b 1µm, and c 500nm,1µm).
2
.3. Biophysical interaction of tubulin with compound 9a
To evaluate the direct interaction of 9a with tubulin and its effect on tubulin polymerization, in
vitro polymerization assay and SPR assay were performed. 9a demonstrated concentration-
dependent increase in the inhibition of tubulin polymerization (Figure 6). Paclitaxel stabilized
5
2
tubulin polymerization while CA-4, known tubulin assembly disruptor , inhibited tubulin
polymerization. This data was validated using transmission electron microscopy. At the
ultrastructural level, tubulin filaments were found to be destabilized in presence of 9a and CA-4
(Figure 6C). CA-4 at 5μM and 9a at 10μM concentration exhibited typical microtubule
intermediates and tubulin rings50,51. Paclitaxel stabilized tubulin filaments (Figure 6C).
To further ascertain physical interaction between Tubulin and 9a, SPR spectroscopic analysis
was performed. Tubulin protein (0.13µM ) was immobilized while the compound 9a was present
in the mobile phase. With an increasing concentration of 9a ranging from 6.25 µM to 100 µM, a
gradual increase in the association was observed in SPR response suggesting physical interaction
5
3
between the two (Figure 6B). Compound CA-4 (well-characterized tubulin interactor) was
used as a positive control to validate the interaction (Figure 6B). This result gave us evidence
for the direct interaction of compound 9a with tubulin.
1
7

Figure 6: Effect of compound 9a on tubulin polymerization by direct physical interaction with
tubulin in cell-free assays. A] In vitro tubulin polymerization kinetics assay. Tubulin in reaction
buffer was incubated at 37°C in the buffer, vehicle control (DMSO at 0.0001%), compound 9a (5 and
1
0μM), Paclitaxel (10μM) and CA-4 (5μM). The tubulin assembly was quantitated by
spectrophotometry. The experiments were performed twice, and representative data is shown. B] Physical
binding of 9a to tubulin by Surface Plasmon Resonance. Panel B provides validation of the physical
interaction of immobilized tubulin at different concentrations of the compound 9a (present in the mobile
phase). Compound Combretastatin (CA-4, well-characterized tubulin interactor) was used as a positive
1
8

control to validate the interaction with tubulin. An increase in response unit was observed with an
incresing concentration of 9a and CA-4. C] Effect of 9a on cell-free tubulin polymerization at
ultrastructure level by transmission electron microscopy. Panel C shows electron micrographs after
negative staining of tubulin treated with compounds for 90 min. Except for Buffer control (neat), other
samples were diluted as neat samples had concentrated tubulin structures (Paclitaxel (1:100 dilution),
CA-4 and 9a (1:50 dilution)). Scale bar is shown at the right bottom of each micrograph (500nm, 100nm).
2
.4. Micro-PET-CT imaging
Tubulins and microtubules function as key modulators of mitochondrial metabolism. In
particular, tubulins associate with enzymes of the glycolysis, preferential interactions between
glycolytic enzymes and either the soluble or polymerized tubulin pool may also influence
metabolic activity and microtubule dynamics.50
Metabolic reprogramming especially glycolysis is one of the hallmarks of cancer. Cancer cells
1
8
exhibit faster glucose consumption which can be visualized and monitored with [ F]-
1
8
fluorodeoxyglucose ( F-FDG) positron emission tomography/computed tomography (PET/CT)
4
5
scanning. This method has broad applications in clinical oncology including diagnostics and
1
8
monitoring therapy response. F-FDG uptake study was performed in tumor-bearing control and
compound 9a treated tumor mice at the end of the experiment at day 40 for evaluating the effect
1
8
on tumor metabolism. It was observed that F-FDG uptake (Figure 7) was reduced by 75.8% in
compound 9a treated tumor (mean SUV 487 KBq/cc, Supplementary Videos S5 and S6) which
was comparable to reduction of 68% as observed in adriamycin treated tumor (Mean SUV 644
KBq/cc, Supplementary Videos S3 and S4) over that shown by control tumor (mean SUV 2015
KBq/cc, Supplementary Videos S1, and S2).
1
9

Figure 7: 18F-FDG PET-CT imaging for metabolic changes in compound 9a-treated AW13516
1
8
xenograft bearing mice. F-FDG PET-CT exhibits the distribution of glucose uptake in tumor tissue. A]
Immunodeficient oral cancer xenograft AW13516 bearing mice were imaged 4-hr after 18F-FDG
injection. Images shown are representative 2 mice from each group. Values on the top left of each panel
exhibit the maximum standardized uptake values (SUVsKBq/cc) of tumors imaged at the end of the study
(40 days). Yellow dotted circles mark the tumor boundary in individual mice. Glucose uptake was
distinctly reduced in tumors from compound 9a-treated mice as compared to control tumors. The color
panel next to each group indicates uptake intensity from high (red) to low (blue). B] Bar graph depicts a
quantitative analysis of maximum standardized uptake values (SUVsKBq/cc) of tumors imaged at the end
of the study (tumor area marked with yellow dotted lines). Values represent mean  standard error of
glucose update in two mice per group.
The vehicle used for adriamycin was water. The vehicle used for 9a dilution was DMSO for the first
dilution and then water was used for further dilutions.
Dosing protocols: 9a, 2.5mg/kg once a week i.v. for 4 weeks; Adriamycin, 2.5mg/kg at day 1,5,9 i.v.
i.v. intravenous; ADR: Adriamycin.
2
0

Glucose uptake relates to active proliferation in xenograft tumor tissues. Further, Vergez et al54
1
8
reported that F-FDG PET-CT imaging provided an early indication of head and neck squamous
cancer xenograft treatment which was well correlated with clinical outcome in a human clinical
trial. Thus, a significant decrease in glucose uptake indicated that compound 9a successfully
abrogated proliferation and glycolysis metabolism of tumor cells in oral cancer xenograft in an
animal model which may have immense potential as well in human clinical trials.
2
.5. Molecular docking studies
The studies suggest that these synthesized compounds exert their effect by acting as microtubule
inhibitors. For better understanding and to know how these phenstatin based indole linked
chalcone compounds interact with tubulin, the potential binding modes for two of the most
potent compounds, 9a and 9e, were investigated at the colchicine binding site in the tubulin
dimer, the colchicines bind at the interface of α β-tubulin subunits, both the chains were
considered for molecular docking studies. The tubulin crystal structures were obtained from
Protein Data Bank (PDB ID code: 3UT5).55 The protein preparation was done using the
Schrodinger’s tool (PPrep). Both 9a and 9e were sketched by using 2D Sketcher and
Schrödinger’s LigPrep program was used to generate different conformations of ligands.
Molecular docking studies were performed using a GLIDE docking module of Schrödinger suite
and the results were analyzed on the basis of the GLIDE docking score and molecular
recognition interactions. The 3D figures were obtained using Schrödinger Suite 2017-1.56
Figure 8 illustrated the overview of the potential binding site of compounds 9a and 9e, which
binds at the interface of α- and β-tubulin. Molecular docking results along with hydrogen
bonding as well as hydrophobic results for compound 9a, 9e with tubulin protein was depicted in
Supplementary Table S6. According to previous studies, the Asnα101 could also play a role as
strong hydrogen bond donors. The binding modes with the active site were observed to be very
similar to previous results as reported for colchicine and various other colchicine binding site
5
7
inhibitors. Figures 8A and 8B showed that the trimethoxy rings of both compounds 9a and 9e
were well-positioned in proximity to the Lysβ254, Glnα183 and Ala180 residues, and they adopt
an orientation very similar to that of the trimethoxy ring of colchicine in the crystallized
2
1

structure. The interaction was strongly stabilized by hydrogen bonds. Hydrogen bonding
interactions of compounds 9a and 9e include C=O of trimethoxybenzoyl moiety with ASNα101
at distance of 1.85 Å and C=O of chalcone with Serα178 at distance of 2.19 Å, similar to that
compound 9e formed with ASNα101 at a distance of 2.18 Å and with Serα178 at distance of
2
.10 Å. In addition, compound 9e has formed one more hydrogen bond between methoxy of
trimethoxybenzoyl moiety and Tyrα224 at distance of 2.04 Å. Whereas, compound 9a
highlighting the pi-cation interaction with Lysβ254 at the colchicine binding site. Moreover,
compounds 9a (turquoise) and 9e (grey) overlapped well with the native ligand colchicine
(green) in Supplementary Figure S8 and bind at the colchicine binding site. The indole ring
occupied the site where the 7-membered ring in colchicine binds, while the 3,4,5-
trimethoxybenzoyl group of compounds 9a and 9e overlapped very well with the 3,4,5-
trimethoxyphenyl ring in colchicine. Most interactions of compounds 9a and 9e were driven
mainly by hydrophobic interactions with Gln11, Thr179, Ala180, Gln183, Arg221 and Tyr224,
amino acids of the α-tubulin and Cys241, Leu248, Lys254, Leu255, Asn258, Met259, Val315,
Ala316, Ile318, Met325 and Lys 352 amino acids of the β-tubulin.
2
2

Figure 8. Proposed binding mode of (A) compound 9a (turquoise) and (B) compound 9e (turquoise) in
the colchicine binding site of tubulin (PDB ID code: 3UT5) shown as a ball and stick. Interacting residues
of α- and β-tubulin are presented as tube and wire. Hydrogen bonds are represented with pink dotted lines
(in Å). Images were generated using Maestro, Schrodinger, LLC.
The validation of docking was conducted by redocking of the bound ligand colchicine in the
colchicine binding site of tubulin protein. The bound and docked conformations of colchicine
showed similar interactions and binding pose at the binding site with a root mean square
deviation (RMSD) of 1.7230 Å (Supplementary Figure S9).
The bioenergetic and biosynthetic pathways are primarily altered in cancer. Recently efforts are
being focused to identify inhibitors that are capable of affecting multiple enzymes
4
6
simultaneously in tumor metabolism pathways. Since we observed a significant effect of 9a on
glucose uptake in an oral cancer xenograft model, we analyzed 9a for its effect on a series of
enzymes in the glycolytic pathway and TCA cycle.
2
3

The crystal structures of some of the key metabolic regulatory enzymes described by Yadav et
4
6
58
59
60
al. i.e. GAPDH (PDB ID: 1U8F) , HK 2 (PDB ID: 2NZT) , LDH (PDB ID: 1I0Z) , PGK
6
1
62
63
(
PDB ID: 4AXX) , PDH (PDB ID: 3EXE) and IDH1 (PDB ID: 4UMX) were obtained from
RCSB-Protein Data Bank. The grid box was generated around the active site residues present in
5
7
the receptors. Finally, the 3D structure of compound 9a was docked into the active sites of
55
GAPDH, HK 2, LDH, PGK, PDH and IDH1 enzymes using Schrödinger Suite 2017-1. We
observed that the resolution of crystal structures of enzymes of the glycolytic pathway and TCA
cycle was found to be in the range of 1.5-2.5 Å, which is considered optimal for docking. Hence
crystal structures of these PDBs were considered for our modelling work.
Supplementary Table S7 demonstrates the result of the molecular docking along with hydrogen
bonding as well as arene-arene interactions of compound 9a with the different metabolic
regulatory enzymes. Thus, these molecular docking data provided a detailed account of
interactions of compound 9a with that of targeted receptors. Compound 9a exhibited good
binding properties with the key metabolic regulatory enzymes and these studies intended for
some important directions for future structural modifications.
6
4
Rai et al. have observed that inhibitors of LDH, a key glycolytic enzyme that catalyzes the final
step in the glycolytic pathway, significantly affected the growth of pancreatic cancer cell line
Mia-Pa-Ca-2 and sarcoma cell line A673 in vitro cytotoxicity assay. They also predicted robust
cell-based inhibition of target. Hence reduced glucose uptake observed in our PET-CT imaging
study, after treatment of xenograft with 9a can be justified that it must be through inhibition of
target metabolic enzymes of the glycolytic pathway and TCA cycle.
Overall, these molecular docking results gave us a detailed explanation of interactions of
compound 9a with colchicine binding site of tubulin and also key metabolic regulatory enzymes
of glycolysis pathway.
3
.
CONCLUSION:
In conclusion, we have synthesized a series (9a-z and 9aa-ad) of phenstatin bearing indole
linked chalcones and evaluated them for their antiproliferative activity on few cancer cell lines.
2
4

Compound 9a demonstrated significant anti-proliferative activity against oral cancer squamous
cancer cells as well as oral cancer stem-like spheroids. It was further tested in oral cancer
xenograft AW13516 in an immunodeficient mouse model. It was efficacious in significantly
reducing tumor volume without causing any toxicity in experimental mice. Interestingly, it also
reduced angiogenesis in mice xenografts, reduced collagen levels and caused significant
reduction in cell processes, cellular integrity as well as cytoskeletal organization. Moreover, it
successfully abrogated glucose uptake in tumor xenografts. In vitro tubulin polymerization
kinetics assay revealed direct interaction of 9a with tubulin and inhibition of tubulin
polymerization leading to destabilization, profilament bending and generation of tubulin ring
intermediates. SPR assay further confirmed the physical binding of 9a with tubulin. In silico
molecular docking of 9a to the colchicine binding site of tubulin and metabolic enzymes of
glycolysis/ TCA cycle, demonstrated a better binding affinity to target binding site and
consequently inhibition of microtubule polymerization and glucose metabolism. Based on these
in silico and pre-clinical observations, compound 9a consisting of phenstatin based indole-
chalcone scaffolds can be considered as an attractive tubulin polymerization inhibitor candidate
for developing anti-cancer therapeutics. Further investigations on safety and acute/ chronic
toxicity of compound 9a are warranted to proceed under the drug-development pipeline.
4
4
.
EXPERIMENTAL SECTION:
Chemistry
.1.
All solvents and reagents were purchased from commercial sources and were used without
further purification. Reactions progress was routinely monitored by TLC on silica gel glass plate
1
containing 60 GF-254, and compounds visualized with UV light, iodine vapor and H SO . H
2
4
and 13C NMR spectra were recorded on Bruker UXNMR/XWIN-NMR (300 MHz) or
InnovaVarian-VXR-Unity (400, 500 MHz) instruments. Chemical shifts (δ) were reported in
parts per million (ppm) downfield from TMS expressed as internal standard and coupling
1
constants are expressed in Hz. H NMR spectral data were reported in the following order:
multiplicity (s, singlet; brs, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; m,
2
5

multiplet), coupling constants in Hz, and a number of protons. ESI mass spectra were recorded
on Micromass Quattro LC (ESI+ software with capillary voltage 3.98 kV and positive ion trap
detector). QSTAR XL Hybrid MS-MS mass spectrometer was used to record High-resolution
mass spectra. Melting points were determined with an electrothermal digital melting point
apparatus IA9100 and are uncorrected. The purity of the compounds was determined by HPLC
performed on a Shimadzu LC-20AD apparatus equipped with a SPD-M20A diode array detector
and a Shimadzu SIL- 20AC auto-injector using C18 column (waters 5 μM C18, 4.6 mm 250 mm
column). Elution conditions: mobile phase A (85%)-acetonitrile; mobile phase B(15%)-water,
pH 4.3 adjusted with 0.1% formic acid + 10 mM NH OAc. The flow rate was 1.0 mL/min and
4
the injection volume was 5 μL at 25 °C and detection at 254 nm.
4
.1.1. General procedure for the synthesis of compounds (11a–c)
To a stirred solution of 5-bromo-1H-indole (1 mmol) in dry THF, was added NaH (1.5 equiv,
0% dispersion in mineral oil) portion wise at 0 °C and stirring was continued at room
6
temperature for 30 min. Then, methyl iodide/ethyl bromide/tert-butyldimethylsilyl chloride (1.2
equiv) were added at 0 °C and the reaction mixture was allowed to warm to room temperature
with continuous stirring for 3 h. Then ice cold water was added carefully and extracted using
CH Cl . The combined organic extracts were washed with water and saturated brine solution,
2
2
dried over Na SO and concentrated under vacuum. Then the obtained crude mixture was
2
4
purified by column chromatography using ethyl acetate and petroleum ether as an eluent to give
pure compounds 11a-c.
4
.1.2. General reaction procedure for the synthesis of compounds (12a-c)
To a solution of the above compounds (11a-c, 1 equiv) in dry THF, n-BuLi (1.2 mmol, 1.6 M in
o
hexane) was added at -78 C under argon atmosphere and stirred for 1 h. Then 3,4,5-
trimethoxybenzaldehyde (1 equiv) was added slowly to the reaction mixture and the reaction
mixture was allowed slowly to reach the room temperature and stirred for 4 h. Then reaction
mixture was poured into ice-cold water and extracted with CH Cl , washed with water and
2
2
saturated brine solution. Then the combined organic layer was dried over Na SO and
2
4
concentrated under vacuum. The resulted residue was purified by column chromatography using
ethyl acetate and petroleum ether as an eluent over silica gel to give pure compounds 12a-c.
2
6

4
.1.3. General reaction procedure for the synthesis of compounds (13a-c)
o
To a stirred solution of compounds (12a-c, 1 equiv) in DMSO, IBX (2 eqiv) was added at 0 C
and stirred at room temperature for 3 h. After completion of the reaction, monitored by using
TLC the reaction mixture was poured into ice water and extracted with CH Cl , the combined
2
2
organic extracts were washed with water and saturated brine solution. The combined organic
extracts were dried over Na SO and concentrated under vacuum. The obtained crude was
2
4
purified by column chromatography using ethyl acetate and petroleum ether as an eluent over
silica gel to afford pure compounds 13a-c.
4
.1.4. General reaction procedure for the synthesis of compounds (14a-c)
To a stirred solution of DMF (2.5 equiv) in CHCl (5 mL) was added POCl (2 equiv) dropwise
3
3
at 0 °C for preparing Vilsmeier reagent. To this reagent, compound 13a-c (1 equiv) dissolved in
CHCl (60 mL) was added dropwise, the reaction mixture was warmed to 27 °C and stirred for 2
3
h, then refluxed for 12 h. The solvent was removed under vacuum. After completion of the
reaction, the reaction mixture was poured onto ice and neutralized by saturated sodium
bicarbonate solution. The resulting crude solid was filtered and recrystallized using ethanol to
obtain aldehydes 14a-c in high purity.
4
.1.5. General reaction procedure for the synthesis of compounds (9a-9z and 9aa-9ad)
To a solution of aldehydes 14a-c (1 equiv) in ethanol, substituted acetophenone (1 equiv) and
0% aqueous sodium hydroxide (3 mL/100mg) were added and the reaction mixture was stirred
1
for 6 h at room temperature. After completion of the reaction monitored by TLC, the solvent was
evaporated under vacuum. The resulted crude product was dissolved in ethyl acetate and washed
with water and brine solution. The organic extract was dried over Na SO and concentrated in
2
4
vacuo. The residue was finally purified by column chromatography using ethyl acetate and
petroleum as a solvent system over silica gel to obtain the pure product.
4
.1.6.
(E)-1-(3,4-dimethoxyphenyl)-3-(1-methyl-5-(3,4,5-trimethoxybenzoyl)-1H-indol-3-
yl)prop-2-en-1-one (9a)
The compound was synthesized according to the general procedure using 14a (100 mg, 0.28
mmol), acetophenone (51 mg, 0.28 mmol) and 10% aqueous sodium hydroxide solution (3 ml) to
1
obtain pure product 9a as a pale yellow solid. Yield: 133 mg (78%); mp: 239-241 ˚C. H NMR
2
7

(
500 MHz, CDCl ) δ 8.56 (s, 1H), 8.04 (d, J = 15.5 Hz, 1H), 7.79 (s, 1H), 7.65 (d, J = 1.9 Hz,
3
1
7
H), 7.62 (d, J = 15.5 Hz, 1H), 7.55 (s, 1H), 7.49 (d, J = 5.6 Hz, 1H), 7.47 (d, J = 5.6 Hz, 1H),
1
3
.14 (s, 2H), 7.01 (d, J = 8.4 Hz, 1H), 3.96 (d, J = 5.5 Hz, 9H), 3.88 (s, 9H); C NMR (101
MHz, CDCl ) δ 195.92, 195.69, 153.07, 152.91, 152.88, 149.24, 141.56, 140.21, 139.86, 135.39,
3
1
5
5
32.64, 130.92, 126.03, 125.50, 124.90, 122.61, 117.74, 114.52, 110.06, 107.96, 107.61, 61.02,
+
6.34, 56.31, 33.97; ESI-MS: m/z 516 (M + H) ; HRMS (ESI) m/z for C H O N calculated m/z:
3
0
30
7
16.2017, found m/z: 516.2005; HPLC: t 5.63 min, purity 97.4%.
R
The characterization data for intermediates and other 9-series compounds including HPLC data
is provided in the supporting file.
4
4
4
.2.
Biology
.2.1. In vitro cytotoxicity of compounds by SRB assay
.2.1.1. Cell culture
Cancer cell line HT-29 (colon) was procured from NCI, USA; SCC-29B (oral) was provided by
Dr Susanne M. Gollin, University of Pittsburgh, Pennsylvania, USA; and Hep-G2 (liver) was
procured from NCCS, Pune. All the cell lines were maintained in RPMI or DMEM medium
(
Life Technologies, USA) in presence of 10% fetal calf serum, 2 mM L-glutamine and 1%
antibiotics.
.2.1.2. SRB assay
4
In vitro cytotoxicity, SRB assay was performed as described previously.65,66 SRB assay is a
rapid, sensitive, cost-effective and high-throughput method used for the quantification of cellular
proteins and forms an integral part of in vitro drug screening in the Developmental Therapeutic
Program of the National Cancer Institute (NCI), USA. For this assay, the cells were dispensed at
a density of 5000 cells/ well/ 100 µL in 96-well flat-bottom microtiter plates (Eppendorf Inc,
USA). Cells were allowed to adhere by incubating plates at 37 °C in 95% humidified air with 5
%
CO for 24 h. Compounds 9a-9z and 9aa-9ad were dissolved in dimethyl sulfoxide (DMSO)
2
and further diluted with a plain medium. Compounds were added to cells in triplicates at final
concentrations of 0.1, 1, 10 and 100 µM and plates were further incubated for 48 hours in 37 °C
incubator. Only cells served as cell control, while wells with the addition of Adriamycin (ADR)
2
8

served as a positive control. Each experiment was performed three times. At the end of 48 hours
of incubation, morphological changes in cell cultures were observed and cells treated at the
highest drug concentration were captured using Phase Contrast Inverted Microscope (Model
Eclipse Ti-S, NIKON Co., Japan) fitted with the digital camera to the computer. Further, the cell
proteins were fixed using 10% trichloroacetic acid. Proteins were stained with SRB dye and
bound stain in the cell proteins was subsequently eluted with 10mM Trizma base. The
absorbance was read on a plate reader (Model Sunrise, Tecan Inc., USA), at a wavelength of 540
nm with a 690 nm reference wavelength. Percent growth was calculated set wise and was
expressed as the ratio of average absorbance (O.D.) of the test well to the average absorbance of
the control wells.
Percentage of control cell growth = Mean OD sample at 48hr- Mean OD 0 hr × 100
Mean OD cell control at 48hr- Mean OD 0 hr
The following values which implicates the cytotoxicity of each drug on each cell line, namely
LC (Concentration of drug causing 50% cell kill), GI (Concentration of drug causing 50%
5
0
50
inhibition of cell growth) and TGI (Concentration of drug causing total inhibition of cell growth)
were calculated. GI value of ≤1µM was considered to demonstrate activity in case of synthetic
5
0
compounds.
4
.2.2. Oral cancer spheroid cell culture (CSC) assay
It was observed that spheroid cultures consisting of cancer-stem-like cells exhibit increased drug
resistance or less chemosensitivity as compared to their adherent cell counterparts.67 To
determine if compounds that showed activity against bulk cancer cells have any growth
inhibition efficacy against stem-like cells of oral cancer, compounds were tested against oral
cancer spheroids in CCK-8 dye assay. Oral cancer cell line SCC-29B cells were dispensed at a
seeding density of 5000 per well in matrigel coated 96-well Flat bottom plates (Eppendorf India
Ltd). MEM serum-free medium reconstituted with 1% B27 supplement, 20 ng/ml of Epidermal
Growth factor and 20 ng/ml of Basic Fibroblast Growth Factor (Life Technologies, USA) was
added every 48 hr and repeated for two cycles. Sphere-like structures were visible after 6 days.
+
lo
Spheroids were characterized for a stem-like population by flow cytometry for CD44 CD24
2
9