Substituted chloroacetamides as potential cancer stem cell inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1002/ddr.21628

Cancer kills, irrespective of geographical and cultural origin. Novel modalities for treating cancer are desperately needed. Cancer stem cells (CSCs), main culprits behind chemoresistance and tumor relapse, are one of the few logical choices. Herein, we r

Full text of DOI:10.1002/ddr.21628

Received: 9 August 2019
DOI: 10.1002/ddr.21628
Revised: 24 October 2019
Accepted: 9 November 2019
R E S E A R C H A R T I C L E
Substituted chloroacetamides as potential cancer stem cell
inhibitors: Synthesis and biological evaluation
1
2
2
Komal N. Padhariya | Maithili Athavale | Sangeeta Srivastava
|
1
Prashant S. Kharkar
1
Shobhaben Pratapbhai Patel School of
Abstract
Pharmacy & Technology Management,
SVKM's NMIMS, Mumbai, India
Cancer kills, irrespective of geographical and cultural origin. Novel modalities for
treating cancer are desperately needed. Cancer stem cells (CSCs), main culprits behind
chemoresistance and tumor relapse, are one of the few logical choices. Herein, we
report the synthesis and biological evaluation of small molecules with chloroacetamide
war-head. These molecules were screened for viability against various breast, prostate,
and oral cancer cell lines using MTT and soft-agar assays. Further, promising hits were
screened in sphere-forming assay with the aim of discovering potential anti-CSC
agents. Our optimism yielded four hits inhibiting self-renewal of cancer cells with
stem-like characters in vitro. Finally, the hits were evaluated for in vitro toxicity against
human peripheral blood mononuclear cells and mouse embryonic fibroblast cell line.
Overall, these preliminary investigations yielded three hits exhibiting promising anti-
CSC potential with little or no toxicity against normal cells.
2
Department of Cancer Biology, Godavari
Biorefineries Ltd., Mumbai, India
Correspondence
Prashant S. Kharkar, Institute of Chemical
Technology (ICT), Nathalal Parekh Marg,
Matunga, Mumbai 400 019, India.
Email: ps.kharkar@ictmumbai.edu.in
K E Y W O R D S
breast cancer, cancer stem cells, chloroacetamides, CSCs, oral cancer, prostate cancer, soft-
agar assay, sphere-forming assay
1
|
INTRODUCTION
conventional clonal hypothesis of cancer development and paved a
way toward preventing tumor relapse and metastasis (Yu, Pestell,
Cancer, one of the most dreadful diseases in the history of mankind, is
characterized by uncontrolled, abnormal cell growth, and metastasis.
Despite the availability of efficacious drugs in the anticancer arma-
mentarium and other treatment modalities such as surgery and radia-
tion therapy, cancer is mostly incurable; metastasis being the most
notable reason (Anderson et al., 2019). Recent statistics on cancer is
horrifying. In the United States alone, 1.7 million new cases were reg-
istered in 2018 and by year 2030, more than 20 million cases will be
prevailing worldwide, as speculated (Cancer Statistics, 2019). It is a
growing cause of concern in both the developing and the developed
nations affecting millions of people.
Lisanti, & Pestell, 2012). The identification of highly chemoresistant,
cancer-initiating CSCs responsible for the growth, metastasis, and
relapse of the disease has rekindled the spirit to battle the disease
(Hanahan & Weinberg, 2011). Compared to the phenotypical stem
cells, the CSCs exhibit unique characteristics (self-renewal, differentia-
tion, and tumorigenicity), which enable them to divide asymmetrically
producing an identical daughter cell and a more differentiated cell,
which later forms the bulk of the tumor (Ratajczak, Bujko, Mack,
Kucia, & Ratajczak, 2018; Yu et al., 2012). Being less susceptible to
chemo- and radiotherapies compared to the bulk tumor, CSCs are
responsible for the treatment failure and cancer recurrence. Thus,
based on the characteristic nature of CSCs, targeting and exterminat-
ing CSCs represents a budding approach for significantly improving
clinical outcome or for possibly achieving complete eradication of
cancer.
Apart from the adverse effects associated with the treatment
which drain the patient mentally, physically, and financially, the
chances of relapse in majority of the cases are quite high. The recently
proposed cancer stem cell (CSC) hypothesis challenged the
Drug Dev Res. 2019;1–10.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
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PADHARIYA ET AL.
The research on CSCs has matured to an extent that it gives us
the tiny nature of these molecules and differential activity despite the
presence of a “war-head,” we were pretty convinced that these mole-
cules were hitting some target systematically (as inferred from the
structure–activity relationship). These targets could be kinases, ion
channels, or enzymes, critical for the survival of the cancer, and/or
CSC. Intrigued by the biological results of 2 and the literature reports, a
full-fledged design, synthesis, and biological evaluation of a focused
library based on chloroacetamide core structure was undertaken.
Around the same time, Roberts et al. (2017) published an interest-
ing study featuring chemoproteomic screening of cysteine-reactive
fragment-based covalent ligands library belonging to acrylamide and
2-chloroacetamide series in search of antipancreatic cancer therapeu-
tics. The authors started off with the sole aim of impairing pancreatic
cancer cells along with identification of druggable hotspots. The study
resulted in successful identification of a covalent ligand with pancreatic
cancer cell killing in vitro and inhibiting tumor growth in vivo. The ligand
covalently modified the catalytic cysteine of the ubiquitin-like modifier
activating enzyme 5 (UBA5), which was involved in activating the
ubiquitin-like protein UFM1 to UFMylate proteins. In a similar study,
anticancer natural product withaferin A and its cysteine-reactive cova-
lent ligand selectively targeted breast cancer signaling, proliferation,
and in vivo tumor growth (Grossman et al., 2017). The authors screened
a library of cysteine-reactive covalent ligands against 231MFP breast
cancer cells in order to discover compounds with phenotypes similar to
withaferin A. The screening process identified few hits, the best one
being a synthetically easily tractable chloroacetamide derivative.
These investigations, along with our in-house efforts and literature,
motivated us to further evaluate the medicinal chemistry-intuitive,
designed compounds in the ongoing anti-CSC therapeutics programme
to supplement our efforts to develop a novel anti-CSC therapeutic for
breast and prostate cancers, two major types of cancers occurring glob-
ally. In this study, we report a focused library of 14 structurally-diverse
small molecules containing chloroacetamide war-head.
fairly complete picture of the association between CSCs and the
deadly disease including its relapse (Soundararajan, Paranjape, Maity,
Aparicio, & Mani, 2018). We now have far better idea about the path-
ways and molecular targets associated with the CSCs. This informa-
tion can help us in differentiating CSCs from the normal stem cells
and normal cells of the body (Ratajczak et al., 2018). Despite the iden-
tification and targeting of many pathways that control cancer growth,
we are still far from complete eradication of the disease. Even though
several regulatory signaling pathways are known, the major challenge
lies in identifying the underlying targets unique to CSCs making up
the druggable hotspot. Significantly large number of research groups
(
including the authors') worldwide has been working on the develop-
ment of anti-CSC therapeutics (Kaur, Sharma, Dogra, & Singh, 2018;
Kharkar, 2017; Luo & Wicha, 2019; Saeg & Anbalagan, 2018; Skoda
et al., 2018; Yadav et al., 2018). The most advanced anti-CSC thera-
peutics, napabucasin (BBI-608), an orally administered small-molecule
investigational agent proposed to be an inhibitor of cancer stemness
pathways, is currently in Phase III clinical trials in combination with
5
-fluorouracil (NCT02753127) for the treatment of previously treated
metastatic colorectal cancer (mCRC; Canstem 303c Trial, 2019). The
search for novel, effective anti-CSC therapeutic is on, for the radical
change it is expected to bring about in the cancer therapy arena.
In the author's lab, while working on a series of 4-aryl-3-chloro-
2
-azetidinones (1, 1) as potential anti-CSC agents, we tested an inter-
mediate of the title compounds represented by 2, in a battery of assays
comprising of MTT, soft-agar, and sphere formation, against a couple of
breast and prostate cancer cell lines. To our surprise, intermediate
2
exhibited quite potent, submicromolar cellular activity against the cell
lines tested. Further, we searched DrugBank (Wishart et al., 2018) for
molecules similar to 2 (substructure search with default settings) for
understanding their potential macromolecular targets. The hits are
listed in Table 1. We reasoned that the electrophilic “war-head,” that is,
the chloroacetamide moiety, could potentially modify certain biological
macromolecules covalently as long as they selectively bound to them.
Further, it was hypothesized that if these chloroacetamides were to
behave as general toxins (owing to the presence of war-head), they
should exhibit potent cell-killing activity irrespective of the nature of
the aryl/arylalkyl group attached to the amide N. On the other hand, if
these molecules were to bind selectively (even if covalently) to their
putative molecular target(s), then the nature of the N-aryl/arylalkyl
group would affect the biological activity due to underlying ligand–
receptor binding kinetics. Also, the aryl/arylalkyl group may potentially
influence the cell permeability with implications on the activity. Given
2
|
MATERIALS AND METHODS
| Chemistry
2.1
2.1.1
|
General
All common reagents or materials were obtained from commercial
sources and used without further purification. All solvents were dried
prior to use with appropriate drying agents. Column chromatography
was performed using silica gel 60 (#230-400). Analytical thin-layer
chromatography (TLC) was carried out on Merck silica gel plates with
QF-254 indicator and visualized under short UV (λ254 nm). Proton
1
nuclear magnetic resonance ( H-NMR) spectra were obtained on
Bruker NMR 400 instrument (Billerica, MA), and chemical shifts were
given in ppm from tetramethylsilane (TMS) as internal standard. The
purity of the compounds (≥95%) were determined using HPLC con-
ducted on Agilent 1220 Infinity system (Santa Clara, CA), using a
1
2
CHART 1 Serendipitous design strategy of the 2-chloroacetamide
®
series
C18Kromasil column (25 cm × 4.6 mm, 5 μl, 100 Å pore size). Mass

PADHARIYA ET AL.
3
TABLE 1 Hits from substructure search based on query 2 from DrugBank
Sr. No.
DrugBank ID
Structure
Status
Molecular target
1
DB07217
Experimental drug
MAP kinase 10
2
3
DB07398
DB06790
Experimental drug
Experimental drug
Serine/threonine-protein kinase PknG
UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase
Catalogue No.: F029125), epidermal growth factor (EGF; Sigma-
Aldrich, Catalogue No.: E9644), insulin (Sigma, Catalogue No.: 19278),
Dulbecco's Modified Eagle Medium/F12 (DMEM-F12; HiMedia, Cata-
logue No.:AL139-6), minimum essential media (HiMedia, Catalogue
No.: AL255G), Rosewell Park Memorial Institute medium 1640 (RPMI
SCHEME 1 Synthesis of substituted chloroacetamides (5a-m)
1640; HiMedia, Catalogue No.: AL223A), Dulbecco's phosphate buff-
spectral data were collected on Shimadzu 8040 LC-MS/MS system
ered saline (HiMedia, Catalogue No.: TL1006), Trypan Blue (TCI 93),
prostate epithelial media (LONZA, Catalogue No.: CC-3166), MEGM
(LONZA, Catalogue No.: CC-3051), Heparin (Sigma, Catalogue No.:
H3393), Penstrep (HiMedia, Catalogue No.: A002).
(
Kyoto, Japan). The FT-IR spectra were recorded on Perkin Elmer RX1
instrument (Waltham, MA).
2
.1.2
|
General procedure for the syntheses of
chloroacetamide derivatives (2, 5a-m)
2.2.2
|
MTT assay
A 100-ml round-bottomed flask was charged with anhydrous THF
Briefly, the cells were plated in 96-well plate as per predetermined plat-
(
10 ml) and substituted amine 3a-m (1 mmol; Scheme 1) was stirred and
ing efficiency (Table S2). The plates were then incubated for 24 hr in
ꢀ
cooled in ice-bath till complete dissolution of the solid material.
Triethylamine (TEA; 1.5–3 mmol, 1.5–3 equiv.) was added to the flask,
followed by dropwise addition of cold solution of chloroacetyl chloride
5% CO
2
atmosphere at 37 C. Appropriate concentrations of the test
compounds were then added to the plate and further incubation was
ꢀ
carried out for 48 hr (in 5% CO
2
atmosphere at 37 C). The assay plate
4
(1.5 mmol, 1.5 equiv.) in anhydrous THF (10 ml) while maintaining the
was then centrifuged twice at 3,000 rpm for 3 min and supernatant
ꢀ
temperature below 5 C. The resulting solution was allowed to stir at RT
overnight. On completion of reaction, as monitored by TLC, the solvent
was evaporated under reduced pressure and the residue was washed
with ice-cold water and the precipitated solid was filtered off at the suc-
tion pump. The crude product was then quickly purified by syringe col-
umn chromatography using 20–30% ethylacetate/hexane as eluent.
was then discarded. A total of 100 μl of MTT solution (0.5 mg/ml) was
then added to each well of the plate and it was further incubated for
ꢀ
4 hr (in 5% CO
2
atmosphere at 37 C). Following 4 hr incubation, the
plate was then centrifuged twice, and supernatant was aspirated off
very carefully. Then, 200 μl of DMSO was then added to each well to
solubilize MTT crystals and mixed well by shaking the plate. The XY
graph of log % viability was then plotted against log drug concentration.
The IC50 (test compound concentration inhibiting 50% of cell popula-
tion) was then calculated by regression analysis.
2
2
.2
|
Biology
| Cell lines and reagents
.2.1
2
.2.3
|
Soft-agar assay
The cells were obtained from National Centre for Cell Science (NCCS;
Pune, India). Cells used were in culture for no more than 3 months.
Briefly, the cells were maintained in complete media with 10% fetal
bovine serum (Gibco Life Technologies, Grand Island, NY). Unless oth-
erwise noted, cells were seeded at 40% confluency for all assays.
Briefly, a mixture of 50 μl of 2× medium (taken appropriately as per cell
line) and 50 μl of 1.2% Bacto™ Agar were plated onto each well of
96-well microtiter assay plate. A 10 μl of cells (of specific plating effi-
ciency prestandardized for respective cell line, Table S3) were mixed with
20 μl of 2× medium and 30 μl of 0.8% of Bacto™ Agar and 1.6 μl of test
compound (of appropriate concentration) in a vial and transferred to the
50X B27 Supplement (Life Technologies, Invitrogen, Catalogue
No.: 17502-044), fibroblast growth factor (FGF; Sigma-Aldrich,

4
PADHARIYA ET AL.
solidified pre-layers of the assay plates. The cells were then allowed to
ꢀ
grow and form colonies at 37 C and 5% CO
2
for 1 week. An intermittent
feeding with 50 μl of appropriate 2× medium was performed after
®
3
days of experimental set up. Then l6 μl of alamarBlue (1.5 mg/ml) was
then added to all the wells to quantify the developed colonies. The plates
ꢀ
were incubated for 24 hr at 37 C. Absorbance was then measured at
6
30 nm. The XY graph of log % viability was then plotted against log drug
concentration. The IC50 (test compound concentration inhibiting 50% of
cell population) was then calculated by regression analysis.
2
.2.4
|
Sphere assay
Serum-free media under low attachment condition induces sphere
formation. Only CSCs (prepared as single-cell suspension) can survive
this condition and grow in the form of spheres. Depending upon the
cell line used the serum-free media was prepared as under:
mammosphere media preparation (for 100 ml): methyl cellulose (1 g)
added to plain media (mammosphere epithelial basal medium; 100 ml)
and dissolved under magnetic stirring. After complete dissolution,
FGF-80Ε, EGF-40 μL, Penstrep-1 ml, Heparin-400 μl were added.
Prostosphere media preparation (for 100 ml): methyl cellulose
(
1 g) added to plain media (prostate epithelial basal medium; 100 ml)
and dissolved under magnetic stirring. After complete dissolution,
Insulin-40 μl, B27-2 ml, EGF-80 μl, Penstrep-1 ml was added.
Briefly, the cells were trypsinized and made into single-cell sus-
pension by passing through cell strainers (100 μl and 40 μl, respec-
tively). The cells were diluted at a concentration of 2,000 cells/100 μl
and suspended in either mammosphere (for breast cell lines) or pros-
tosphere (for prostate cell lines) medium. Then 100 μl of this suspen-
FIGURE 1 Images of (a) mammospheres (MDA-MB-231)
b) prostatospheres (PC-3) under 20× magnification
(
sion was added into each well of 96-well suspension plates and
ꢀ
incubated at 37 C, 5% CO
2
for 24 hr. Appropriate concentrations of
the test compounds (2 μl) were added into the respective wells with
2.2.5
|
hPBMC assay
ꢀ
1
00 μl of stem cell culture medium. Plates were incubated at 37 C,
5
% CO
2
for 72 hr. After incubation 2.5 μl of the respective test com-
This assay was performed using freshly isolated lymphocytes from blood
of a healthy donor. Briefly, diluted defibrinated fresh blood was overlaid
gradually on HiSep™ LSM1077 and centrifuged at low speed for 30 min.
The lymphocyte layer (the buffy coat) was carefully removed in a new
collection tube. The buffy coat was given another wash, by the diluent
buffer, to reduce the platelet contamination. The supernatant was dis-
carded, and the pellet was resuspended in diluent buffer. The viability
was checked by hemocytometer. Viable cells with at least 95% purity
were considered for the assay. The MTT assay was performed with these
cells as described above with plating efficiency of 0.7 million/ml.
pound concentration and 50 μl of stem cell culture medium were
ꢀ
added into each well and plates were further incubated at 37 C, 5%
CO for 72 hr. Then 3 μl of the respective test compound concentra-
2
tion was added with 50 μl of stem cell culture medium again after
ꢀ
incubation and plates were incubated again for 72 hr at 37 C, 5%
2
CO . The number of primary spheres formed for each concentration
was counted. Live spheres appear to be bright and transparent under
phase contrast microscopy (Figure 1). The spheres were counted/
scored manually at the end of experimental period and % viability for
each concentration was calculated by using following formula
Number of spheres formedperwell × 100
Number of spheres formed in Control well
3
3
|
RESULTS AND DISCUSSION
| Chemistry
%
viability =
.1
A comparative graph of the number of spheres formed was then
plotted against the concentration and the growth curve was com-
pared with the positive control.
The title compounds were synthesized by reacting various aliphatic
and (hetero)aromatic amines with chloroacetyl chloride in presence of

PADHARIYA ET AL.
5

6
PADHARIYA ET AL.

PADHARIYA ET AL.
7
a
TABLE 3 Median effect of chloroacetamide derivatives on viability in human breast, prostate, and oral cancer cell lines in soft-agar assay
b
c
IC50 values (μM) in cell lines
R
Compound
Cisplatin
Sunitinib
2
MDA-MB-231
35.56 ± 0.15
2.8 ± 0.07
1.83 ± 0.05
1.46 ± 0.08
2.22 ± 0.13
2.18 ± 0.04
2 ± 0.12
DU 145
PC-3
KB
KB-3-1
KB-Ch -8-5
21.57 ± 0.11
1.74 ± 0.05
33.11 ± 0.16
1.13 ± 0.04
0.27 ± 0.04
4.14 ± 0.03
2.39 ± 0.2
4.5 ± 0.13
2.71 ± 0.06
1 ± 0.08
25.82 ± 0.24
2.64 ± 0.25
4.2 ± 0.12
nt
33.73 ± 0.08
3.71 ± 0.09
nt
41.4 ± 0.02
4.13 ± 0.01
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
3.09 ± 0.05
nt
3.09 ± 0.08
1.97 ± 0.2
3.98 ± 0.03
2.63 ± 0.06
>100
14.93 ± 0.03
22.38 ± 0.11
>100
1.59 ± 0.12
2.07 ± 0.11
>100
1.91 ± 0.09
30.48 ± 0.18
3.01 ± 0.05
3.42 ± 0.07
2.53 ± 0.07
1.16 ± 0.06
h
i
28.84 ± 0.22
1.41 ± 0.16
2.75 ± 0.15
2.09 ± 0.14
2.11 ± 0.29
3.52 ± 0.17
nt
11.53 ± 0.05
11.43 ± 0.07
9.68 ± 0.07
4.46 ± 0.05
4.71 ± 0.01
>100
2.82 ± 0.21
8.93 ± 0.06
3.18 ± 0.12
3.24 ± 0.13
j
k
m
>100
3.28 ± 0.05
7.64 ± 0.02
Abbreviation: nt, not tested.
a
Determined by soft-agar assay.
b
Compounds tested in triplicate.
c
Cancer cell line origin: Breast cancer cell lines: MDA-MB-231; prostate cancer cell lines: DU145, PC-3, and oral cancer cell lines: KB, KB-3-1, and
R
KB-Ch -8-5.
a base in anhydrous THF to yield 2, 5a-m (70–90% yield) as per previ-
ously described procedure in Section 2.1.2. The purified and structur-
ally characterized molecules were taken up for biological screening in
a battery of assays.
extremely aggressive prostate cancer unresponsive to hormonal ther-
apy (Tai et al., 2011).The oral cancer cell lines, KB, KB-3-1, and KB-
R
Ch -8-5, were used to further assess the anticancer potential of the
title compounds, since oral cancer is among the top 10 most com-
monly affecting cancer types. These cells represent highly drug-(col-
chicine, vinblastine, actinomycin
D
or adriamycin)-resistant
3
3
.2
|
Biology
| MTT assay
phenotypes owing to abundance of multidrug-resistant efflux trans-
porters such as P-gp, an ideal scenario for anti-CSC activity evaluation
(Richert, Akiyama, Shen, Gottesman, & Pastan, 1985; Shen et al.,
.2.1
1986). Very recently, Fang et al. (Fang et al., 2019) have established
Initially, the title compounds were screened in cell viability (MTT)
KB cell line overexpressing P-gp for understanding structural require-
ments of flavonoids undergoing P-gp mediated efflux.
assay against breast (MDA-MB-231, MCF7, and T-47D), prostate
R
(
DU145, LNCaP, and PC-3), and oral (KB, KB-3-1, and KB-Ch -8-5)
Table 2 lists the IC50 (μM) values for the title compounds 5a-m
against the breast, prostate and oral cancer cell lines. Quite a few
compounds—5e, 5i, 5k, and 5m (MDA-MB-231) and 2 (T-47D)
exhibited submicromolar potency (better than sunitinib). Other rela-
tively potent compounds included 2 (MCF7), 5f (DU145), 5m, 5g, 5l,
and 2. None of the tested compounds were more potent than sun-
itinib against the oral cancer cell lines. Moving down Table 2 starting
from the original lead structure 2, replacement of 3-CN (2) with
3-COOEt (5a) on the thiazole core decreased potency. Further con-
traction of the cyclohexane ring (5a) with cyclopentyl ring (5b) led to
slight gain in potency in breast cancer cell lines while the potency at
prostate cancer cell lines decreased marginally. Further optimization
of potency yielded 5e (MDA-MB-231, IC50 = 0.314 ± 0.05 μM). Care-
ful observation of the results indicated the requirement of limited bulk
on the amine. Extended aromatic substituent (e.g., 5l), presence of a
polar group on the aromatic ring of amine (e.g., 5h) had detrimental
cancer cell lines. The optimal plating densities of each cell line are
given in Table S2. Cisplatin and sunitinib were used as positive con-
trols and the vehicle DMSO as a negative control. Cisplatin represen-
ted conventional chemotherapy modality to which CSCs are resistant,
while sunitinib was used as a representative anti-CSC drug (Yuan
et al., 2016). The choice of cell line for biological evaluation was
governed by several factors, particularly, their aggressiveness, tumori-
genic potential, invasive behavior, in addition to others. MDA-MB-
2
31 is a perfect model for chemotherapy while MCF7 is a suitable
model for hormone therapy. T-47D is quite similar to MCF7 except
that it is quite susceptible to progesterone (Yu, Kim, Yoo, & Kang,
2
017). In addition, T-47D cells form tightly cohesive mass structures
in 3D cultures owing to extensive cell–cell adhesion (Holliday &
Speirs, 2011). In case of prostate cancer cell lines, PC-3 is highly meta-
static and is characteristic of prostatic small cell carcinoma, an

8
PADHARIYA ET AL.
FIGURE 3 % Cell viability of the hits against NIH/3T3 cell line
(
data are mean ± SD, n = 3). (Asterisk above columns indicate
statistically significant difference compared to control.
*p < .01, ***p < .001)
*
TABLE 4 Median effect of the hits on lymphocyte viability
(
hPBMC assay)
Sr. No.
Compound code
IC50 (μM)
98 ± 1.3
2.8 ± 0.07
0.33 ± 0.02
>100
1
2
3
3
4
5
Cisplatin
Sunitinib (+ve control)
2
5e
5i
>100
5m
>100
Abbreviation: hPBMC, human peripheral blood mononuclear cell.
trends in the activity profiles of the title compounds. Some of these
activity patterns could be due to the impaired cellular permeability
owing to unfavorable molecular properties in less potent compounds.
Overall, presence of a halogen on the aromatic ring alone or with a
heteroaromatic ring bearing amine contributed positively to impaired
cell viability.
3
.2.2
|
Soft-agar assay
Majority of the title compounds (except 5g and 5l) exhibiting relatively
potent activity in MTT assay were further taken up for evaluation in
soft-agar assay (SAA). Also known as anchorage-independent assay,
SAA is widely used to understand the effect of test compounds on
the ability of cancer cell's proliferation and migration to form colonies
in semisolid matrix mimicking in vivo condition, that is, tumor suppres-
sion in vitro. It is indeed the test of a cell to undergo “unlimited” divi-
sion. If a compound inhibits colony formation in a dose-dependent
manner, it can be concluded that it is an inhibitor of tumorigenicity
in vitro, an important characteristic of CSCs. Previously, Horibata, Vo,
Subramanian, Thompson, and Coonrod (2015) have used SAA for
FIGURE 2 Results from sphere-forming assay of the hits. (a) %
Viability of mammospheres (MDA-MB-231); (b) % viability of
prostatospheres (PC-3); (c) % viability of prostatospheres (DU 145)
compared to the untreated. (The data are expressed as mean ± SEM
[n = 6]. Asterisk above columns indicate statistically significant
difference compared to control. ***p < .001)
effect on potency in some (MDA-MB-231) cell line(s), while beneficial
in others (LNCaP). Overall, our concern that these molecules would
otherwise be general toxins was invalid since there were definitive

PADHARIYA ET AL.
9
identifying inhibitors of tumorigenicity in breast cancer cells. In a very
recent report, Seyfrid et al. (2019) have described SAA as one of the
inexpensive and convenient assays for evaluating the stemness prop-
erties of brain tumor stem cells.
have used NIH/3T3 cell line for evaluating cytotoxicity of acrylamide.
All the hits (2, 5e, 5i, and 5m) along with doxorubicin, representing
cytotoxic drugs as positive control and vehicle as negative control,
were evaluated in MTT assay using hPBMCs and NIH/3T3 cell line.
The hits 5e, 5i, and 5m were found to be significantly less toxic
(~fourfold) compared to doxorubicin at 10 μM dose against NIH/3T3
cell line (Figure 3). In a contrasting scenario, 2, the original hit, which
was one of the best hits in cell viability assays toxic at the dose level
tested (10 μM). Similar trend was observed during screening of the
hits against hPBMCs. The molecules were screened (highest concen-
tration tested 100 μM) and IC50 values were calculated. While 5e, 5i,
and 5m showed no activity (IC50 > 100 μM) toward hPBMCs, hit
2 was found to be relatively toxic (IC50 = 0.33 μM; Table 4) compared
to the positive control sunitinib (IC50 = 2.8 μM). In summary, the hits
5e, 5i, and 5m were relatively nontoxic to the normal cells.
In the present investigation, representative cell lines from breast
(
MDA-MB-231), prostate (DU 145, PC-3), and oral (KB, KB-3-1, KB-
ChR-8-5) cancer types were used. The IC50 (μM) values were calcu-
lated based on the capacity of the molecules to inhibit 50% colony
formation compared to the control (Table 3). Since the number of
formed colonies varies with cell type, the cell plating density was opti-
mized for each cell type used (Table S3).
Compounds 5a (MDA-MB-231), 2 (DU 145), 5i (PC-3 and KB-
R
3
-1), 5f (KB), and 5e (KB-Ch -8-5) were the most potent against the
indicated cell line. Several of the tested compounds were more potent
than sunitinib (Table 3). The results were encouraging to warrant fur-
ther evaluation of the pan-active compounds 2, 5e, 5i and 5 m
(
Table 3) in sphere formation or sphere assay to understand the
potential effects of these hits on CSCs. These hits were selected on
the basis of combined activity data from MTT and SAA.
4
|
CONCLUSION
Design and development of anti-CSC therapeutics is likely to offer a
promise for successful clinical outcome of cancer treatment modalities.
These agents are expected to act in conjunction with the standard che-
motherapy regimen(s), depleting CSCs, the root cause of relapse. Given
the complex nature of cancer as a heterogeneous disease, anti-CSC
agents must to act on CSC-specific pathways to be efficacious. The
basic research establishing the utility of these approaches in clearing
CSCs is crucial for validation of the concept. This is only possible with
the availability of unique agents acting via distinct mechanism(s) of
action. In this study, our hypothesis to use a war-head to direct activity
against CSCs led to interesting results. The chloroacetamide derivatives
incorporating structurally varied amines exhibited definitive potential as
anti-CSC therapeutics against breast and prostate cancers. The rela-
tively lesser incidence of toxicity against normal cell at the doses tested
ensured us that these hits were not general toxins. Rather, there was a
distinct structure–activity pattern. These preliminary results are likely
to serve as a foundation for more elaborate, distinctive, definitive, and
confirmatory studies. The possible mode of action of these compounds
may involve covalent modification of their target(s). Moving forward,
extensive experimental investigations are likely to prove the utility of
this promising, potential anti-CSC therapeutics.
3
.2.3
|
Sphere-formation assay
Sphere-formation assay (SFA) is widely utilized for identification of CSCs
in vitro based on the very characteristic of CSCs to grow in serum-free
gel matrix. Under the nonadherent conditions, only CSCs will survive and
proliferate to form spheres (Pastrana, Silva-Vargas, & Doetsch, 2011).
Very recently, Bahmad et al. (2018), used SFA for assessing the presence
and self-renewal potential of prostate CSCs in different models as an
alternative to various in vivo methods. In this study, we evaluated
selected hits from MTT and SAA against breast (MDA-MB-231) and
prostate (DU 145 and PC-3) CSCs at five different concentrations
(
0.025, 0.25, 2.5, 25, 250 μM; Figure 2a–c). Surprisingly, all the hits (2,
e, 5i, and 5 m) exhibited moderate to high activity at 0.025–2.5 μM
5
inhibiting 50% or more number of spheres in all cell lines tested. In total-
ity, the tested hits were more active against PC-3 compared to the other
two cell lines. Hits 2 and 5m were even better at 25 nM concentration
than sunitinib, the positive control (Figure 2b). Overall, 2 and 5m turned
out to be the clear winners in SFA against breast (MDA-MB-231) and
prostate (PC-3 and DU 145) CSCs, respectively.
ACKNOWLEDGMENTS
3
.2.4
|
hPBMC assay
K.P. is grateful to SVKM's NMIMS (Deemed to be University), Mum-
bai, for the financial support in the form of fellowship and to Dr. Sona
Warrier, Dr. Chetan Shah along with Dr. Nitesh Sahu for their help
during the synthetic work and critical comments during preparation of
the manuscript. M.A. is thankful to Ms. Smera Satish, Ms. Gayatri
Gore, and Mr. Akshay Ganpule, for their help during the biological
evaluation of all the compounds.
Given the nature of the hits, we intended to evaluate the in vitro tox-
icity of the hits on normal cells. Two cell lines—human peripheral
blood mononuclear cells (hPBMCs) and mouse embryonic fibroblasts
(
NIH/3T3) were used. The hPBMC assay is used by the scientific com-
munity as a cost-effective tool for demonstrating the toxicity, in par-
ticular immunotoxicity, on humans (Pourahmad & Salimi, 2015).
Similarly, NIH/3T3 cell line was another choice representing the
mesenchyme-derived cells forming the most basic unit of the connec-
tive tissues. Previously, Sahinturk, Kacar, Vejselova, and Kutlu (2018)
CONFLICT OF INTEREST
Authors declare no conflict of interests from a financial or commercial
standpoint.

1
0
PADHARIYA ET AL.
AUTHOR CONTRIBUTIONS
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https://orcid.org/0000-0003-1955-7223
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