A novel series of substituted 1,2,3-triazoles as cancer stem cell inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1002/ddr.21723

An alarming increase in global death toll resulting from cancer incidents, particularly due to multidrug resistance and reduced efficacy as a consequence of target mutations, has compelled us to look for novel anticancer agents. Cancer stem cells (CSCs),

Full text of DOI:10.1002/ddr.21723

Received: 8 March 2020
DOI: 10.1002/ddr.21723
Revised: 2 July 2020
Accepted: 6 July 2020
R E S E A R C H A R T I C L E
A novel series of substituted 1,2,3-triazoles as cancer stem cell
inhibitors: Synthesis and biological evaluation
Komal N. Padhariya1 | Maithili Athavale2 | Sangeeta Srivastava2
Prashant S. Kharkar1
|
1Shobhaben Pratapbhai Patel School of
Abstract
Pharmacy and Technology Management,
SVKM's NMIMS, Mumbai, 400056, India
An alarming increase in global death toll resulting from cancer incidents, particu-
larly due to multidrug resistance and reduced efficacy as a consequence of target
mutations, has compelled us to look for novel anticancer agents. Cancer stem cells
(CSCs), contributing majorly to the chemoresistance and tumor relapse, seem to
the main culprits. In the present investigation, new chemical entities (NCEs)
belonging to four novel chemical series (A: 40-allyl-20-methoxyphenoxymethyl-1,-
2,3-triazoles; B: 40-acetamidophenoxymethyl-1,2,3-triazoles; C: naphthalene-10-
yloxymethyl-1,2,3-triazoles, and D: naphthalene-20-yloxymethyl-1,2,3-triazoles)
were synthesized via Copper (I)-catalyzed alkyne-azide cycloaddition reaction and
evaluated for in vitro anticancer activity. A total of 30 NCEs (39–68) were screened
at 10 μM concentration in cell viability assay against cancer cell lines such as breast
(MDA-MB-231), prostate (PC-3), glioma (U87 MG), along with cervical (SiHa) and
lung (A549). The NCEs from Series C (56–60) and D (61–68) were more potent
than those in Series A (39-45) and Series B (46–55) at the tested concentration.
Furthermore, NCEs with >80% inhibition at 10 μM were evaluated for dose
response. A total of five NCEs, 48, 56, 61, 65 and 66, were further assessed in
soft-agar assay and found to be relatively potent (IC50 < 10 μM). Finally, the hits
were screened in sphere assay to identify potential CSC inhibitors against
mammospheres (MDA-MB-231) and prostatospheres (PC-3). More so, the hits
were also evaluated to understand in vitro cytotoxicity against normal cells using
mouse embryonic fibroblast cell line (NIH/3T3) and human peripheral blood mono-
nuclear cells (hPBMCs). Overall, hits 56 and 61 exhibited potent anticancer as well
as CSC inhibitory activities with notably less toxicity toward NIH/3T3 and
hPBMCs. On the whole, our arduous study led to the identification of potential
hits with anticancer and CSC inhibitory activities, with minimal or no toxicity to
normal cells.
2Department of Cancer Biology, Godavari
Biorefineries Ltd, Mumbai, 400001, 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
1,2,3-triazoles, breast cancer, cancer stem cells, click chemistry, CSCs, prostate cancer, soft-
agar assay, sphere assay
Drug Dev Res. 2020;1–18.
wileyonlinelibrary.com/journal/ddr
© 2020 Wiley Periodicals LLC
1

2
PADHARIYA ET AL.
1
|
INTRODUCTION
relapse. Due to the unique nature of these CSCs, exterminating the
evil at its root will possibly help us in winning the battle against
cancer.
Cancer, a group of heterogeneous diseases, is characterized by uncon-
trolled growth of supposedly abnormal cells having the potential to
invade and migrate to nearby tissues or other body parts culminating
into metastasis. The battle to conquer cancer is one of the prime chal-
lenges faced by modern medicine. Some forms of cancer are better
treated due to the availability of enhanced diagnostics tools. Recent
chemotherapeutic regimens, including hormonal therapy along with
adjuvant therapies like radiation and surgical proficiency, have shown
remarkable success against primary tumors. Despite these, the battle
against the swiftly growing disease is yet to be won. In spite of using
the costliest drug or treatment available in the market, the patients'
suffering is nowhere near the end. The horrifying statistics of 2018
proves the urgency of the matter. In 2018 alone, 17 million new cases
were registered worldwide while the death toll due to various cancer
was up to 9.6 million (Cancer Research UK, 2020). More so, 62%
increase in incidence rate projected for the next decade is a significant
cause of concern for the developed, developing and underdeveloped
nations.
In light of the role of CSCs in metastasis and relapse, many groups
throughout the research fraternity worldwide (including the authors')
are working on developing anti-CSC therapeutics (Yadav et al, 2019;
Gavade et al., 2020). Table S1 lists few promising clinical candidates
(alone or in combination) at various stages of development. The sea-
rch for an effective anti-CSC agent continues. In the author's lab,
while working on a lead from 2-azetidinones as potential anti-CSC
agents, we tested a focussed library series of structurally diverse mol-
ecules having chloroacetamide warhead with potential anti-CSC activ-
ities (Padhariya, Athavale, Srivastava, & Kharkar, 2020). The positive
result in breast and prostate CSCs motivated us further to modify the
molecules to gain the upper hand over the activity given the nature of
the warheads.
Continuing with our efforts to design more potent anti-CSC
agents, we adopted few strategies to speed up the process of
designing and constructing a diverse library of new chemical enti-
ties (NCEs). Designing of molecules containing two or more
pharmacophoric features or privileged substructures is an impor-
tant strategy in drug design and development. More so, the inclu-
The two primary concerns confound our ability to combat this
dreadful disease. Despite the availability of advanced treatment,
resistance to recent line of chemotherapeutics agents has resulted in
an increased incidence of therapy failure. However, the primary
nemesis remains the phenomenon of metastasis and tumor recur-
rence leading to reduced survival rate. Drug resistance has majorly
been attributed to the genetic and biochemical mechanisms includ-
ing mutations in the target proteins. Thus, the target identification
technique has become one of the recent trends in drug discovery for
cancer chemotherapy (Hati et al., 2016; Kumar et al., 2017). How-
ever, recent awareness of intratumoral heterogeneity has piqued the
interest to understand resistance, recurrence and relapse better. It is
now known that a tumor consists of a complex ecosystem of hetero-
geneous cell mass that controls the function of the tumor as a whole.
Majority of this cell mass has limited self-renewal capacity; however,
a small subpopulation of these heterogeneous cell mass, known as
cancer stem cells (CSCs) or cancer-initiating cells (CICs), has been
the focus of cancer research in last few decades (Kharkar, 2017).
These CICs are capable of self-renewal and have multilineage differ-
entiation potential to initiate and sustain tumor growth. Having
exceptional properties such as slow cell cycle, high expression of
drug-resistant transporters, increased ability to efflux cytotoxic
agents, high DNA repair capacity, and so on; CSCs are inherently
resistant to recent line of chemo/radio therapies. Being less suscep-
tible compared to the bulk tumor, CSCs are the prime reason for
conventional treatment failure and increased incidence of relapse.
The extensive ongoing research provides a robust platform on hall-
marks of CSCs that differentiates it from normal stem cells and other
normal cells of the body. These unique features of CSCs can be used
as a leverage to selectively inhibiting CSCs while preserving the
functionality of normal stem cells, thus, making it an essential target
in anticancer therapy. Specifically targeting CSCs would attenuate
their ability to survive conventional chemotherapy resulting in
sion of heterocyclic pharmacophore linked with
a promising
framework could overall improve the efficacy of the series. The
best way forward was to use click chemistry. Sharpless et al.
defined click chemistry as “the regiospecific reactions that can be
performed easily to yield pure compounds with less efforts” (Kolb,
Finn, & Sharpless, 2001). The most widely used click chemistry
reaction is the Cooper (I)-catalyzed azide alkyne cycloaddition reac-
tion to obtain 1,4-disubstituted 1,2,3-triazole ring from a terminal
alkyne and an azide (He, Xie, Tang, Li, & R Chen, 2012). The
1,2,3-triazole ring is an attractive pharmacophore for medicinal
chemists, due to its stability to extreme oxidative or reducing con-
ditions and to metabolic degradations (Dheer, Singh,
&
Shankar, 2017). Naturally, a variety of molecules bearing this N-
containing heterocyclic ring, including anticancer agents, were syn-
thesized by click chemistry as key step (Sahu, Purushothaman,
Thiruvenkatam, & Kharkar, 2019). The triazole ring is reported to
enhance anticancer activity due to its noncovalent interactions,
namely, hydrophobic interactions, dipole–dipole interactions, van
der Waals interaction, H-bonding, and so on with the putative bio-
logical target (Xu, Zhao, & Liu, 2019).
Intrigued by the results of 1,2,3-triazole ring containing moie-
ties, a new series was conceptualized to target the CSCs. Based on
our knowledge and previous experience with the chloroacetamide
derivatives, we designed molecules belonging to four different
Series A, B, C, and D (Scheme 1). In Series A, eugenol was attempted
to explore potential anti-CSC activity. Eugenol is a primary constitu-
ent of the essential oil clove and is traditionally used to relieve mild
pain, to reduce sepsis as well as an antibacterial agent (Ghosh
et al., 2005). Derivatives of eugenol and combination of eugenol
with a chemotherapeutic agent have been reported to suppress
~
malignant cell lines (Bezerra, Militao, De Morais, & De Sousa, 2017;

PADHARIYA ET AL.
3
SCHEME 1 General scheme for synthesis
substituted 1,2,3-triazole derivatives (39–68).
Reagents and conditions: a, propargyl chloride,
K2CO3, DMF, RT; b, chloroacetyl chloride, TEA,
THF 0ꢀC to RT; c, NaN3, DMF, RT; d, 5–8,
Cu(OAc)2.H2O, t-BuOH:H2O (3:1). TEA,
triethylamine; THF, tetrahydrofuran
a
1,5
2,6:
3,7
4,8:
: Ar = 4-Allyl-2-methoxyphenyl
Ar = 4-Acetamidophenyl
: Ar = Napthalen-1-yl
Ar
O
Ar OH
1 - 4
5 - 8
Ar: Napthalen-2-yl
O
O
N
N
N
O
Ar'/R' NH2
9 -18
R/Ar
N
H
39 - 45
(Series A)
b
O
HN
H
N
H
N
c
Ar'/R'
N3
O
d
N
Ar'/R'
Cl
N
N
O
O
O
R'/Ar'
N
H
19 - 28
29 - 38
46 - 55
O
(Series B)
N
N
N
O
R'/Ar'
N
H
56 - 60
O
(Series C)
N
N
N
O
R'/Ar'
N
H
61 - 68
(Series D)
Carrasco et al., 2008; Hussain et al., 2011; Pisano et al., 2007). Islam
et al. reported the increase in anti-CSCs activity of cisplatin in pres-
ence of eugenol in breast CSCs (Islam et al., 2018). Series B involved
2
|
MATERIAL AND METHODS
| Chemistry
2.1
4-acetylaminophenoxymethyl
derivatives
linked
to
the
1,2,3-triazole moiety. Acetaminophen or paracetamol is reported to
enhance antiproliferative and apoptosis-inducing potential in combi-
nation with anticancer agents in primary cancer cells (Bundscherer,
A straight forward route to synthesize 1,2,3-triazole derivatives was
adapted using click chemistry, a copper-catalyzed addition reaction
between, prop-2-ynyl derivatives and the 2-azido-N-substituted acet-
amide derivatives (Scheme 1).
Malsy, Gruber, Graf,
& Sinner, 2018; Posadas, Santos, &
Ceña, 2012). More so, it is also reported to suppress CSCs in human
breast carcinoma cell line MDA-MB-231 (Takehara, Hoshino,
Namba, Yamakawa, & Mizushima, 2011). In both Series C and D, we
incorporated α- and β-naphthol core structures, respectively, based
on our earlier findings (Sahu et al., 2019) and the reported literature
of naphthol derivatives as anticancer agents (Abdelrahim, Mohan,
2.1.1
|
General
All reagents or materials procured were of the highest commer-
cially available purity from commercial sources such as Sigma-
Aldrich (Steinheim, Germany), Spectrochem (Mumbai, India), Alfa
Aesar (Karlsruhe, Germany), or Merck (Darmstadt, Germany) and
were used without any further purification unless explicitly men-
tioned. Solvents procured commercially were dried as per
reported procedures prior to use. The reactions were performed
under inert (N2) atmosphere and progress was monitored by thin-
layer chromatography (aluminium plate coated with silica gel
60F254; Merck Millipore, Billerica, MA) and visualized under
short ultraviolet light (λ 254). All intermediates were purified by
recrystalization using appropriate solvents and final compounds
were purified by performing column chromatography using silica
Albarrati, Makeen,
Mamidyala, Kumar, 2012; Osowole, Kempe, Schobert,
Effenberger, 2011).
&
Safhi, 2016; Das, Reddy, Kashanna,
&
&
Thus, based on reported cytotoxicity of acetaminophen, naph-
thols, eugenol, and our in-house results of chloroacetamide deriva-
tives as well as molecules with 1,2,3-triazoles moiety; it encouraged
and prompted us to explore and screen the designed 1,2,3-triazole
hybrid molecules with aforementioned substructures for their poten-
tial anti-cancer and anti-CSC activities. Herein, we report NCEs con-
taining 1,2,3-triazole derivatives exhibiting comparative anti-CSC
activity against breast and prostate cancer.

4
PADHARIYA ET AL.
gel 60 (#230–400) using
a
mixture of 5% methanol in dic-
2.1.4
|
General procedure for the syntheses of
hloromethane as eluent. Melting points of the final compounds
were determined on Veego VMP DS (General Trading Co., Nag-
pur, India) and are uncorrected. The ATR spectra were recorded
using Fourier transform infrared with UATR on Perkin Elmer
spectrum 2 instrument (Waltham, MA). The purity of the com-
pounds (≥95%) was determined using Agilent 1220 Infinity HPLC
2-azido-N-substituted acetamide derivatives (29–38)
To the stirred solution of choroacetamide derivatives 19–28
(10 mmol) in anhydrous DMF, sodium azide (25 mmol) dissolved in
minimum DMF, was carefully transferred and the reaction was contin-
ued to stir overnight at RT. Upon reaction completion as indicated by
TLC, cold brine solution was added to the reaction and the crude
obtained was filtered at suction pump and washed with cold water.
Yield 80–90%.
system (Santa Clara, CA), using
a C18 Kromasil® column
(25 cm × 4.6 mm, 5 μm, 100 Å pore size) using isocratic mobile
phase (A) acetonitrile and (B) water (80:20, vol/vol) with flow rate
of 1 ml/min. For NMR spectra, DMSO-d6 was used as solvent
and tetramethylsilane as an internal standard. 1H-NMR spectra
were recorded on Bruker Advance 400 (400 MHz) spectrometer;
13C-NMR spectra were obtained on Bruker Advance
400 (100 MHz) instrument with chemical shifts expressed in δ
(ppm). Mass spectra (MS) were recorded on a Shimadzu 8040
LC–MS/MS system (Japan), using electrospray ionization (ESI)
mode or other ionization mode, as stated explicitly and high-
resolution mass spectra (HRMS) were recorded on Bruker
microTOF QII high-resolution mass spectrophotometer using
ESI mode.
2.1.5
|
General procedure for the syntheses of
1,2,3-triazole derivatives (39–68)
To the mixture of t-butanol: water (3:1, 5–10 mL), 2-azido-N-
substituted acetamide derivative 29–38 (1 equiv.) was dissolved and
stirred for 30 mins (Sahu et al., 2019). To the above stirred solution,
prop-2-ynyl derivative 5–8 (1 equiv.) and Cu(OAc)2 (20 mol%) were
added and stirred further for about 8 hr at RT with continuous TLC
monitoring using 5% methanol in dichloromethane as mobile phase.
The reaction was then quenched using crushed ice and diluted with
cold brine solution. Using separating funnel, the diluted mixture was
extracted twice with ethylacetate (30 ml). The organic layers were col-
lected and then dried by passing through the bed of anhydrous
sodium sulphate and further concentrated in vacuo to yield the
corresponding crude compounds 39–68. The compounds were then
purified by passing them through silica column using mixture of 5%
MeOH in DCM as eluent.
2.1.2
|
General procedure for the syntheses of
prop-2-ynyl derivatives (5–8)
To the stirred solution of substituted phenols 1–4 (10 mmol) in dry
DMF (20 ml), anhydrous K2CO3 (30 mmol) was added along with cata-
lytic amount of KI, and the mixture was stirred further for 30 min at
RT. To the stirred solution, propargyl chloride (15 mmol) was added
and reaction was stirred for additional 12 hr. The reaction was moni-
tored by TLC using 7:3 petroleum ether/EtOAc as the mobile phase.
After completion, the reaction was quenched with crushed ice (10 g).
The solid was filtered followed by recrystallization from absolute eth-
anol. Yield: 70–80%.
Synthesis of 2-(4-((4-allyl-2-methoxyphenoxy)methyl)-1H-
1,2,3-triazol-1-yl)-N-(4-chloro-phenyl)acetamide (39)
The title compound was synthesized from 2-azido-N-(4-chlorophenyl)
acetamide
(0.3
g,
1.485 mmol),
4-allyl-2-methoxy-1-prop-
2-ynyloxybenzene (0.31 g, 1.485 mmol), and Cu(OAc)2 (20 mol%)
according to the general procedure 2.1.5 to yield 39 as white solid.
Yield: 76.89%; TLC Rf = 0.43 (DCM:MeOH, 95:5); mp: 251–253ꢀC;
purity (HPLC): >99%; ATR cm−1 3,267.35 (amide N H, str), 3,192.89
2.1.3
|
General procedure for the syntheses of
chloroacetamide derivatives (19–28)
(Ar
C
H, str), 1,694.25 (amide
C
O, str), 1,549.43 (Ar
C C, str),
1,400.58 (N N, str), 1,254.64 (Ar
O
CH2, str), 854.14 (C Cl, str);
To the stirred solution of substituted amines 9–18 (1 mmol) and
triethylamine (TEA) (1.5–3 mmol) in anhydrous tetrahydrofuran
(THF) (10 ml) on ice-bath, cold solution of chloroacetyl chloride
(1.5 mmol) in anhydrous THF (5 ml) was supplemented dropwise.
The temperature was maintained below 5ꢀC throughout addition.
The resulting solution was stirred further for 12–15 hr at room tem-
perature. After completion of reaction, as monitored by TLC,
triethylammonium chloride was filtered off and the filtrate was con-
centrated under reduced pressure and quenched with ice-cold
water. The solid precipitated was filtered at suction and washed to
neutral filtrate using cold water. The crude was then quickly purified
by column chromatography using 20–30% EtOAc/petroleum ether
as eluent. Yield: 80–90%.
1H-NMR (400 MHz, DMSO-d6) δ ppm 10.62 (s, 1H), 8.22 (s, 1H),
7.64–7.56 (m, 2H), 7.42–7.34 (m, 2H), 7.05 (d, J = 8.2 Hz, 1H), 6.79 (d,
J = 2.0 Hz, 1H), 6.68 (dd, J = 8.2 and 2.0 Hz, 1H), 5.94 (ddt, J = 16.8,
10.0, and 6.7 Hz, 1H), 5.35 (s, 2H), 5.11–4.97 (m, 4H), 3.71 (s, 3H),
3.29 (d, J = 6.9 Hz, 3H); MS (ESI +ve): m/z 413.0 [M + H]+, 435.0
[M + Na]+.
Synthesis of 2-(4-((4-allyl-2-methoxyphenoxy)methyl)-1H-
1,2,3-triazol-1-yl)-N-(4-fluoro-phenyl)acetamide (40)
The title compound was synthesized from 2-azido-N-(4-fluorophenyl)
acetamide
(0.3
g,
1.546 mmol),
4-allyl-2-methoxy-1-prop-
2-ynyloxybenzene (0.31 g, 1.546 mmol), and Cu(OAc)2 (20 mol%)
according to the general procedure 2.1.5 to yield 40 as off-white solid.

PADHARIYA ET AL.
5
Yield: 71.67%; TLC Rf = 0.34 (DCM:MeOH, 95:5); mp: 246–248ꢀC;
str), 2,944.66 (Ar
C
H, str), 1,672.08 (NH
C
O, str), 1,514.85
purity (HPLC): >99%; ATR cm−13,285.39 (amide N H, str), 3,136.05
(Ar C, str), 1,418.04 (N N, str), 1,233.88 (Ar O
C
CH2, str); 1H-
(Ar
C
H, str), 1,682.42 (amide
C
O, str), 1,508.31 (Ar
C
C, str),
NMR (400 MHz, DMSO-d6) δ ppm 10.35 (s, 1H), 8.21 (s, 1H), 7.48 (s,
1H), 7.28 (d, J = 8.2 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 7.05 (d, J = 8.2 Hz,
1H), 6.79 (s, 1H), 6.68 (d, J = 8.2 Hz, 1H), 5.94 (ddt, J = 16.9, 10.3, and
6.9 Hz, 1H), 5.31 (s, 2H), 5.05 (d, J = 32.0 Hz, 4H), 3.72 (s, 3H), 3.29
(d, J = 6.8 Hz, 2H), 2.80 (q, J = 8.1 Hz, 4H), 1.99 (p, J = 7.4 Hz, 2H);
MS (ESI +ve): m/z 419 [M + H]+, 441[M + Na]+.
1,400.58 (N N, str), 1,215.88 (Ar
O
CH2, str), 833.20 (C F, str);
1H-NMR (400 MHz, DMSO-d6) δ ppm 10.54 (s, 1H), 8.22 (s, 1H), 7.60
(dd, J = 8.8 and 5.0 Hz, 2H), 7.17 (t, J = 8.7 Hz, 2H), 7.05 (d, J = 8.2 Hz,
1H), 6.79 (d, J = 2.0 Hz, 1H), 6.69 (dd, J = 8.2 and 1.9 Hz, 1H), 5.94
(ddt, J = 17.0, 10.1, and 6.8 Hz, 1H), 5.34 (s, 2H), 5.11–4.98 (m, 4H),
3.72 (s, 3H), 3.29 (d, J = 6.7 Hz, 2H); MS (ESI +ve): m/z 397 [M + H]+,
419 [M + Na]+.
Synthesis of 2-(4-((4-allyl-2-methoxyphenoxy)methyl)-1H-
1,2,3-triazol-1-yl)-N-(2-(4-bromo-phenyl)propan-2-yl)acetamide (44)
The title compound was synthesized from 2-azido-N-
[1-(4-bromophenyl)-1-methylethyl]acetamide (0.4 g, 1.352 mmol),
4-allyl-2-methoxy-1-prop-2-ynyloxybenzene (0.27 g, 1.352 mmol),
and Cu(OAc)2 (20 mol%) according to the general procedure 2.1.5 to
yield 44 as off-white solid. Yield: 78.26%; TLC Rf = 0.35 (DCM:
MeOH, 95:5); mp: 197–199ꢀC; purity (HPLC): >98%; ATR cm−1
Synthesis of 2-(4-((4-allyl-2-methoxyphenoxy)methyl)-1H-
1,2,3-triazol-1-yl)-N-(3,4-dichloro-phenyl)acetamide (41)
The title compound was synthesized from 2-azido-N-(3,-
4-dichlorophenyl)acetamide (0.4 g, 1.63 mmol), 4-allyl-2-methoxy-
1-prop-2-ynyloxybenzene (0.33 g, 1.63 mmol), and Cu(OAc)2 (20 mol
%) according to the general procedure 2.1.5 to yield 41 as off-white
solid. Yield: 21.23%; TLC Rf = 0.39 (DCM:MeOH, 95:5); mp: 223–
225ꢀC; purity (HPLC): >98%; ATR cm−1 3,337.67 (amide N H, str),
3,310.17 (amide
N H, str), 2,974.73 (Ar C H, str), 1,693.51
(NH
(Ar
C
O, str), 1,508.68 (C C, str), 1,400.58 (N N, str), 1,260.30
CH2, str), 546.93 (C Br, str); 1H-NMR (400 MHz, DMSO-d6)
3,144.27 (Ar C C, str), 1,688.60 (NH
1,481.38 (N N, str), 1,260.12 (Ar
C
O, str), 1,511.89 (C C, str),
O
O
CH2, str), 542.86 (C Cl, str);
δ ppm 8.68 (s, 1H), 8.08 (s, 1H), 7.45 (d, J = 8.2 Hz, 2H), 7.28 (d,
J = 8.2 Hz, 2H), 7.02 (d, J = 8.2 Hz, 1H), 6.77 (s, 1H), 6.66 (d, J = 8.2 Hz,
1H), 6.00–5.85 (m, 1H), 5.13 (s, 2H), 5.10–4.97 (m, 4H), 3.69 (s, 3H),
3.28 (d, J = 6.8 Hz, 2H), 1.55 (s, 6H); MS (ESI +ve): m/z 499 [M + H]+,
521 [M + Na]+.
1H-NMR (400 MHz, DMSO-d6) δ ppm δ 10.78 (s, 1H), 8.22 (s, 1H),
7.95 (d, J = 2.4 Hz, 1H), 7.60 (dd, J = 8.8, 2.4 Hz, 1H), 7.47 (dt, J = 8.9,
2.4 Hz, 1H), 7.05 (dd, J = 8.2, 2.4 Hz, 1H), 6.79 (d, J = 2.1 Hz, 1H),
6.69 (dd, J = 8.2, 2.2 Hz, 1H), 5.93 (dtd, J = 17.2, 6.8, 3.8 Hz, 1H), 5.37
(d, J = 2.4 Hz, 2H), 5.12–4.98 (m, 4H), 3.72 (d, J = 2.4 Hz, 3H), 3.29 (d,
J = 6.8 Hz, 2H); MS (ESI +ve): m/z 447 [M + H]+, 469 [M + Na]+.
Synthesis of 2-(4-((4-allyl-2-methoxyphenoxy)methyl)-1H-
1,2,3-triazol-1-yl)-N-(2-bromo-6-nitrophenyl)acetamide (45)
Synthesis of 2-(4-((4-allyl-2-methoxyphenoxy)methyl)-1H-
The title compound was synthesized from 2-azido-N(2-bromo-
6-nitrophenyl)acetamide (0.3 g, 1.007 mmol), 4-allyl-2-methoxy-
1-prop-2-ynyloxybenzene (0.2 g, 1.007 mmol), and Cu(OAc)2 (20 mol
%) according to the general procedure 2.1.5 to yield 45 as white solid.
Yield: 79.13%; TLC Rf = 0.37 (DCM:MeOH, 95:5); mp: 184–186ꢀC;
purity (HPLC): >98%; ATR cm−1 3,214.64 (amide N H, str), 2,944.66
1,2,3-triazol-1-yl)-N-(2-hydroxy-5-methylphenyl)acetamide (42)
The title compound was synthesized from 2-azido-N-(2-hydroxy-
5-methylphenyl)acetamide (0.3 g, 1.456 mmol), 4-allyl-2-methoxy-
1-prop-2-ynyloxybenzene (0.29 g, 1.456 mmol), and Cu(OAc)2
(20 mol%) according to the general procedure 2.1.5 to yield 42 as off-
white solid. Yield: 86.13%; TLC Rf = 0.41 (DCM:MeOH, 95:5); mp:
178–180ꢀC; purity (HPLC): >98%; ATR cm−1 3,274.38 (amide N H,
(Ar C C, str), 1,682.83 (NH
(N N, str), 1,258.84 (Ar
C
O, str), 1,512.33 (C C, str), 1,459.23
O
CH2, str), 794.83 (C Br, str); 1H-NMR
str), 3,274.38 (O H, str), 2,974.73 (Ar C C, str), 1,677.01 (NH
C
O,
(400 MHz, DMSO-d6) δ ppm 10.78 (s, 1H), 8.17 (s, 1H), 8.09 (d,
J = 8.1 Hz, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.48 (t, J = 8.1 Hz, 1H), 7.04
(d, J = 8.1 Hz, 1H), 6.78 (d, J = 1.9 Hz, 1H), 6.67 (d, J = 8.3 Hz, 1H),
5.93 (ddt, J = 17.0, 10.3, 6.8 Hz, 1H), 5.42 (s, 2H), 5.04 (d, J = 29.9 Hz,
4H), 3.70 (d, J = 1.6 Hz, 3H), 3.28 (d, J = 6.9 Hz, 2H); MS (ESI +ve): m/
z 502 [M + H]+, 524 [M + Na]+.
str), 1,511.84 (C C, str), 1,419.40 (N N, str), 1,260.65 (Ar
O
CH2,
str); 1H-NMR (400 MHz, DMSO-d6) δ ppm 9.68 (s, 2H), 8.19 (d,
J = 1.7 Hz, 1H), 7.65 (s, 1H), 7.05 (dd, J = 8.2, 1.7 Hz, 1H), 6.77 (dd,
J = 13.6, 2.1 Hz, 3H), 6.68 (dd, J = 8.2, 2.0 Hz, 1H), 5.93 (dddd,
J = 13.2, 10.0, 8.1, 5.8 Hz, 1H), 5.41 (s, 2H), 5.11–4.98 (m, 4H), 3.72
(m, 3H), 3.36–3.26 (m, 2H), 2.15 (s, 3H); MS (ESI +ve): m/z 409 [M
+ H]+, 431[M + H]+.
Synthesis of 2-(4-((4-acetamidophenoxy)methyl)-1H-1,2,3-triazol-
1-yl)-N-(4-chlorophenyl)- acetamide (46)
Synthesis of 2-[4-(4-allyl-2-methoxy-phenoxymethyl)-[1,2,3]triazol-
1-yl]-N-indan-5-yl-acetamide (43)
The title compound was synthesized from 2-azido-N-
(4-chlorophenyl)- acetamide (0.1 g, 0.476 mmol), N-(4-prop-
2-ynyloxyphenyl)acetamide (0.09 g, 0.476 mmol), and Cu(OAc)2
(20 mol%) according to the general procedure 2.1.5 to yield 46 as
white solid. Yield: 71.28%; TLC Rf = 0.43 (DCM:MeOH, 95:5); mp:
240–242ꢀC; purity (HPLC): >99%; ATR cm−1 3,322.83 (amide N H,
The title compound was synthesized from 2-azido-N-(2,3-dihydro-
1H-indan-5-yl)acetamide (0.3 g, 1.387 mmol), 4-allyl-2-methoxy-
1-prop-2-ynyloxybenzene (0.25 g, 1.387 mmol), and Cu(OAc)2
(20 mol%) according to the general procedure 2.1.5 to yield 43 as off-
white solid. Yield: 76.56%; TLC Rf = 0.31 (DCM:MeOH, 95:5); mp:
202–204ꢀC; purity (HPLC): >99%; ATR cm−1 3,282.05 (amide N H,
str), 2,953.92 (C H str), 1,677.28, 1,655.28 (NH
(C C, str), 1,402.45 (N N, str), 1,224.21 (Ar
C
O, str), 1,510.58
CH2, str), 830.32
O

6
PADHARIYA ET AL.
(C Cl, str); 1H-NMR (400 MHz, DMSO-d6) δ ppm 10.75 (s, 1H), 9.75
(s, 1H), 8.25 (s, 1H), 7.6 (d, 2H), 7.4 (dtd, 4H), 6.9 (d, 2H), 5.3 (s, 2H),
5.1 (s, 2H), 2.0 (s, 3H); MS (ESI –ve): m/z 398.0 [M-H]−.
Synthesis of 2-(4-((4-acetamidophenoxy)methyl)-1H-1,2,3-triazol-
1-yl)-N-(2,3-dihydro-1H-inden-5-yl)acetamide (50)
The title compound was synthesized from 2-azido-N-(2-hydroxy-
5-methylphenyl)acetamide
(0.1
g,
0.463 mmol),
N-(4-prop-
Synthesis of 2-(4-((4-acetamidophenoxy)methyl)-1H-1,2,3-triazol-
1-yl)-N-(4-fluorophenyl)-acetamide (47)
2-ynyloxyphenyl)acetamide (87 mg, 0.463 mmol), and Cu(OAc)2
(20 mol%) according to the general procedure 2.1.5 to yield 50 as
white solid. Yield: 85.43%; TLC Rf = 0.32 (DCM:MeOH, 95:5); mp:
192–194ꢀC; purity (HPLC): >99%; ATR cm−1 3,300.23 (amide N H,
The title compound was synthesized from 2-azido-N-(4-fluorophenyl)-
acetamide (0.1 g, 0.515 mmol), N-(4-prop-2-ynyloxyphenyl)acetamide
(97 mg, 0.515 mmol), and Cu(OAc)2 (20 mol%) according to the gen-
eral procedure 2.1.5 to yield 47 as white solid. Yield: 82.91%; TLC
Rf = 0.26 (DCM:MeOH, 95:5); mp: 233–235ꢀC; purity (HPLC): >99%;
str), 2,944.20 (Ar
C
H str), 1,674.93, 1,660.07 (amide
C
O, str),
1,515.53 (Ar
C
C, str), 1,416.99 (N N, str), 1,249.74 (Ar
O CH2,
str); 1H-NMR (400 MHz, DMSO-d6) δ ppm 10.34 (s, 1H), 9.78 (s, 1H),
8.22 (s, 1H), 7.48 (s, 3H), 7.28 (d, J = 8.2 Hz, 1H), 7.15 (d, J = 8.1 Hz,
1H), 6.97 (d, J = 8.5 Hz, 2H), 5.31 (s, 2H), 5.11 (s, 2H), 2.80 (d, 4H),
1.99 (s, 5H); MS (ESI +ve): m/z 406.0 [M + H]+, 428.0 [M + Na]+.
ATR cm−1 3,279.04 (amide
N
C
H, str), 3,091.79 (Ar
C
H
str),
1,679.23, 1,656.99 (amide
O, str), 1,513.76 (Ar
C
C, str),
1,412.19 (N N, str), 1,230.18 (Ar
O H
CH2, str), 511.00 (C F, str); 1
NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.79 (s, 1H), 8.23 (s, 1H),
7.58 (d, J = 8.1 Hz, 2H), 7.51–7.44 (m, 2H), 7.17 (t, J = 7.9 Hz, 2H),
6.97 (d, J = 8.4 Hz, 2H), 5.33 (s, 2H), 5.11 (s, 2H), 1.99 (s, 3H); MS
(ESI + ve): m/z 384.0 [M + H]+.
Synthesis of 2-(4-((4-acetamidophenoxy)methyl)-1H-1,2,3-triazol-
1-yl)-N-(2-(4-bromo-phenyl)propan-2-yl)acetamide (51)
The title compound was synthesized from 2-azido-N-
(2-(4-bromophenyl)propan-2-yl)acetamide (0.1 g, 0.336 mmol), N-
(4-prop-2-ynyloxyphenyl)-acetamide (87 mg, 0.336 mmol), and
Cu(OAc)2 (20 mol%) according to the general procedure 2.1.5 to yield
51 as white solid. Yield: 79.42%; TLC Rf = 0.31 (DCM:MeOH, 95:5);
mp: 185–186ꢀC; purity (HPLC): >99%; ATR cm−1 3,292.58 (amide
Synthesis of 2-(4-((4-acetamidophenoxy)methyl)-1H-1,2,3-triazol-
1-yl)-N-(3,4-dichloro-phenyl)acetamide (48)
The title compound was synthesized from 2-azido-N-(3,-
4-dichlorophenyl)acetamide (0.3 g, 1.224 mmol), N-(4-prop-
2-ynyloxyphenyl)acetamide (0.23 g, 1.224 mmol), and Cu(OAc)2
(20 mol%) according to the general procedure 2.1.5 to yield 48 as
white solid. Yield: 84.83%; TLC Rf = 0.42 (DCM:MeOH, 95:5); mp:
210–212ꢀC; purity (HPLC): >99%; ATR cm−1 3,298.85 (amide N H,
N
H, str), 3,065.53 (Ar
str), 1,507.78 (Ar
(Ar
CH2, str), 522.71 (C Br, str); 1H-NMR (400 MHz, DMSO-d6)
C
H str), 1,677.67, 1,661.94 (amide
C O,
C
C, str), 1,315.78 (N N, str), 1,222.26
O
δ ppm 9.77 (s, 1H), 8.68 (s, 1H), 8.08 (s, 1H), 7.45 (ddd, J = 7.7, 5.1,
2.6 Hz, 4H), 7.32–7.24 (d, 2H), 6.98–6.86 (d, 2H), 5.13 (dd, 4H), 1.99
(s, 3H), 1.55 (s, 6H); MS (ESI +ve): m/z 486 [M + H]+, 510 [M + Na]+.
str), 2,924.75 (Ar
C
H str), 1,681.87, 1,657.37 (NH
C
O, str),
1,514.82 (Ar C, str), 1,474.18 (N N, str), 1,245.33 (Ar
C
O CH2,
str), 817.99 (C Cl, str); 1H-NMR (400 MHz, DMSO-d6) δ ppm 10.79
(s, 1H), 9.79 (s, 1H), 8.23 (s, 1H), 7.94 (s, 1H), 7.59 (d, J = 8.7 Hz, 1H),
7.48 (d, J = 8.3 Hz, 3H), 6.97 (d, J = 8.5 Hz, 2H), 5.37 (s, 2H), 5.11 (s,
2H), 2.00 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ ppm 167.719,
164.809, 153.749, 142.648, 138.438, 132.841, 131.121, 130.873,
126.177, 125.272, 120.439, 119.296, 114.640, 61.113, 52.155,
23.789; MS (ESI +ve): m/z 434.0 [M + H]+, 456.0 [M + Na]+. HRMS
Synthesis of 2-(4-((4-acetamidophenoxy)methyl)-1H-1,2,3-triazol-
1-yl)-N-(2-bromo-6-nitro-phenyl)acetamide (52)
The title compound was synthesized from 2-azido-N-(2-bromo-
6-nitrophenyl)acetamide
(0.1
g,
0.336 mmol),
N-(4-prop-
2-ynyloxyphenyl)acetamide (87 mg, 0.336 mmol), and Cu(OAc)2
(20 mol%) according to the general procedure 2.1.5 to yield 52 as
white solid. Yield: 87.26%; TLC Rf = 0.33 (DCM:MeOH, 95:5); mp:
176–178ꢀC; purity (HPLC): >99%; ATR cm−1 3,254.21 (amide N-H,
(ESI) calculated for
456.0604.
C
19H17Cl2N5O3 [M + Na]+ 456.0708, found:
str), 3,078.53 (Ar O, str),
C
H str), 1,682.90, 1,661.82 (NH
C
Synthesis of 2-(4-((4-acetamidophenoxy)methyl)-1H-1,2,3-triazol-
1-yl)-N-(2-hydroxy-5-methylphenyl)acetamide (49)
1,509.94 (Ar C, str), 1,414.63 (N N, str), 1,232.04 (Ar O CH2,
C
str), 512.33 (C Br, str); 1H-NMR (400 MHz, DMSO-d6) δ ppm 10.78
(bs, 1H), 9.78 (s, 1H), 8.19 (s, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.99 (d,
J = 8.2 Hz, 1H), 7.48 (t, J = 8.7 Hz, 3H), 6.96 (d, J = 8.5 Hz, 2H), 5.42
(s, 2H), 5.09 (s, 2H), 1.99 (s, 3H); MS (ESI −ve): m/z 487.0 [M–H]−.
The title compound was synthesized from 2-azido-N-(2-hydroxy-
5-methylphenyl)acetamide
(0.1
g,
0.485 mmol),
N-(4-prop-
2-ynyloxyphenyl)-acetamide (91.7 mg, 0.485 mmol), and Cu(OAc)2
(20 mol%) according to the general procedure 2.1.5 to yield 49 as
white solid. Yield: 81.21%; TLC Rf = 0.36 (DCM:MeOH, 95:5); mp:
168–170ꢀC; purity (HPLC): >99%; ATR cm−1 3,256.61 (amide N H,
Synthesis of 2-(4-((4-acetamidophenoxy)methyl)-1H-1,2,3-triazol-
1-yl)-N-(6-chloropyrazin-2-yl)acetamide (53)
str), (C H str), 1,673.8, 1,654.12 (NH
C
O, str), 1,563.63 (C C, str),
The title compound was synthesized from 2-azido-N-
(6-chloropyrazin-2-yl)acetamide (0.1 g, 0.472 mmol), N-(4-prop-
2-ynyloxyphenyl)acetamide (89 mg, 0.472 mmol), and Cu(OAc)2
(20 mol%) according to the general procedure 2.1.5 to yield 53 as
white solid. Yield: 73.21%; TLC Rf = 0.42 (DCM:MeOH, 95:5); mp:
228–230ꢀC; purity (HPLC): >99%; ATR cm−1 3,338.74 (amide N-H,
1,402.96 (N N, str), 1,238.36 (Ar
O
CH2, str), 820.02 (C Cl, str);
1H-NMR (400 MHz, DMSO-d6) δ ppm 9.79 (s, 1H), 9.64 (d, J = 10.0 Hz,
2H), 8.21 (s, 1H), 7.65 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H), 6.97 (d,
J = 8.4 Hz, 2H), 6.75 (s, 2H), 5.41 (s, 2H), 5.10 (s, 2H), 2.15 (s, 3H),
1.99 (s, 3H); MS (ESI +ve): m/z 394.0 [M–H]−.

PADHARIYA ET AL.
7
str), 2,945.75 (Ar
C
H str), 1,706.91, 1,652.33 (amide -C O, str),
CH2,
105.745, 61.607, 52.214; MS (ESI +ve): m/z 393.0 [M + H]+, 415.0
[M + Na]+. HRMS (ESI) calculated for C21H17ClN4O2 [M + Na]+
415.1040, found: 415.0946.
1,544.12 (Ar C C, str), 1,415.17 (N N, str), 1,225.16 (Ar
O
str), 834.95 (C Cl, str); 1H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H),
9.78 (s, 1H), 9.21 (s, 1H), 8.54 (s, 1H), 8.24 (s, 1H), 7.48 (d, J = 8.4 Hz,
2H), 6.97 (d, J = 8.4 Hz, 2H), 5.46 (s, 2H), 5.12 (s, 2H), 1.99 (s, 3H); MS
(ESI +ve): m/z 402.0 [M + H]+, 424 [M + Na]+.
Synthesis of N-(4-fluorophenyl)-2-(4-((naphthalen-1-yloxy)methyl)-
1H-1,2,3-triazol-1-yl)acetamide (57)
The title compound was synthesized from 2-azido-N-(4-fluorophenyl)
acetamide (0.3 g, 1.546 mmol), 1-(prop-2-yn-1-yloxy)naphthalene
(0.28 g, 1.546 mmol), and Cu(OAc)2 (20 mol%) according to the gen-
eral procedure 2.1.5 to yield 57 as white solid. Yield: 78.94%; TLC
Rf = 0.33 (DCM:MeOH, 95:5); mp: 189–191ꢀC; purity (HPLC): >99%;
Synthesis of 2-(4-((4-acetamidophenoxy)methyl)-1H-1,2,3-triazol-
1-yl)-N-(5-(trifluoro-methyl)-1,3,4-thiadiazol-2-yl)acetamide (54)
The title compound was synthesized from 2-azido-N-
(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acetamide (0.1 g, 0.386 mmol),
N-(4-prop-2-ynyloxyphenyl)acetamide (75 mg, 0.386 mmol), and
Cu(OAc)2 (20 mol%) according to the general procedure 2.1.5 to yield
54 as white solid. Yield: 75.36%; TLC Rf = 0.31 (DCM:MeOH, 95:5);
mp: 237–240ꢀC; purity (HPLC): >99%; ATR cm−1 3,274.77 (amide
ATR cm−1 3,271.03 (amide N H, str), 3,093.39 (Ar
1,662.08 (amide -C O, str), 1,507.36 (Ar C, str), 1,338.16 (N N,
str), 1,242.76 (Ar
CH2, str), 519.35 (C F, str); 1H-NMR
C H, str),
C
O
(400 MHz, DMSO-d6) δ ppm 10.55 (s, 1H), 8.37 (s, 1H), 8.12 (d,
J = 8.1 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.60 (dt, J = 7.9 and 3.8 Hz,
2H), 7.48 (tdd, J = 16.6, 13.5, 11.4, and 7.4 Hz, 4H), 7.24–7.13 (m,
3H), 5.37 (s, 4H); MS (ESI + ve): m/z 377.0 [M + H]+, 399.0 [M + Na]+.
N
H, str), 3,154.99 (Ar
str), 1,511.55 (Ar
(Ar
CH2, str), 1,045.93 (C F, str); 1H NMR (400 MHz, DMSO-d6)
C
H str), 1,679.01, 1,660.37 (amide
C O,
C
C, str), 1,413.79 (N N, str), 1,232.93
O
δ 13.8 (bs, 1H), δ 9.78 (s, 1H), 8.25 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H),
6.97 (d, J = 8.6 Hz, 2H), 5.60 (s, 2H), 5.21 (s, 2H), 1.98 (s, 3H); MS (ESI
+ve): m/z 442 [M + H]+, 464 [M + Na]+.
Synthesis of N-(3,4-dichlorophenyl)-2-(4-((naphthalen-1-yloxy)
methyl)-1H-1,2,3-triazol-1-yl)acetamide (58)
The title compound was synthesized from 2-azido-N-(3,-
4-dichlorophenyl)acetamide (0.3 g, 1.224 mmol), 1-(prop-2-yn-
1-yloxy)naphthalene (0.22 g, 1.224 mmol), and Cu(OAc)2 (20 mol%)
according to the general procedure 2.1.5 to yield 58 as white solid.
Yield: 87.13%; TLC Rf = 0.33 (DCM:MeOH, 95:5); mp: 167–169ꢀC;
purity (HPLC): >99%; ATR cm−1 3,345.60 (amide N-H, str), 2,922.08
Synthesis of methyl 2-(2-(4-((4-acetamidophenoxy)methyl)-1H-
1,2,3-triazol-1-yl)acetamido)-thiophene-3-carboxylate (55)
The
title
compound
was
synthesized
from
methyl
2-(2-azidoacetamido)thiophene-3-carboxylate (0.1 g, 0.416 mmol), N-
(4-prop-2-ynyloxyphenyl)-acetamide (78 mg, 0.416 mmol), and
Cu(OAc)2 (20 mol%) according to the general procedure 2.1.5 to yield
55 as white solid. Yield: 81.23%; TLC Rf = 0.48 (DCM:MeOH, 95:5);
mp: 190–192ꢀC; purity (HPLC): >99%; ATR cm−1 3,301.69 (amide
(Ar
C
H, str), 1,691.00 (amide
C
O, str), 1,594.46 (C C, str),
CH2, str), 769.58 (C Cl, str);
1,393.07 (N N, str), 1,239.48 (Ar
O
1H-NMR (400 MHz, DMSO-d6) δ ppm 10.81 (s, 1H), 8.37 (s, 1H), 8.12
(d, J = 8.1 Hz, 1H), 7.96 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.60 (d,
J = 8.7 Hz, 1H), 7.56–7.40 (m, 5H), 7.21 (d, J = 7.4 Hz, 1H), 5.39 (d,
J = 8.9 Hz, 4H); MS (ESI + ve): m/z 427.0 [M + H]+, 449.0 [M + Na]+.
N
H, str), 3,186.75 (C H str), 1,734.66 (ester
C
O, str), 1,676.18
O, str), 1,512.28 (C C, str), 1,305.28 (N N, str), 1,237.54
CH2, str); 1H-NMR (400 MHz, DMSO-d6) δ ppm 11.12 (s, 1H),
(NH
(Ar
C
O
9.78 (s, 1H), 8.28 (s, 1H), 7.52–7.44 (d, 2H), 7.20 (d, J = 5.7 Hz, 1H),
7.08 (d, J = 5.4 Hz, 1H), 7.01–6.94 (d, 2H), 5.68 (s, 2H), 5.13 (s, 2H),
3.83 (s, 3H) 2.00 (s, 3H); MS (ESI −ve): m/z 428.0 [M–H]−.
Synthesis of N-(2-hydroxy-5-methylphenyl)-2-(4-((naphthalen-
1-yloxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide (59)
The title compound was synthesized from 2-azido-N-(2-hydroxy-
5-methylphenyl)acetamide (0.3 g, 1.456 mmol), 1-(prop-2-yn-1-yloxy)
naphthalene (0.26 g, 1.456 mmol), and Cu(OAc)2 (20 mol%) according
to the general procedure 2.1.5 to yield 59 as white solid. Yield:
71.91%; TLC Rf = 0.35 (DCM:MeOH, 95:5); mp: 173–175ꢀC; purity
(HPLC): >98%; ATR cm−1 3,276.34 (amide N H, str), 3,051.64 ( OH,
Synthesis of N-(4-chlorophenyl)-2-(4-((naphthalen-1-yloxy)methyl)-
1H-1,2,3-triazol-1-yl)acetamide (56)
The title compound was synthesized from 2-azido-N-(4-chlorophenyl)
acetamide (0.3 g, 1.428 mmol), 1-(prop-2-yn-1-yloxy)naphthalene
(0.26 g, 1.428 mmol), and Cu(OAc)2 (20 mol%) according to the gen-
eral procedure 2.1.5 to yield 56 as white solid. Yield: 89.12%; TLC
Rf = 0.31 (DCM:MeOH, 95:5); mp: 191–192ꢀC; purity (HPLC): >98%;
str), 1,677.40 (amide
C
O, str), 1,554.54 (Ar
C C, str), 1,357.91
(N N, str), 1,241.65 (Ar
O
CH2, str); 1H-NMR (400 MHz, DMSO-
ATR cm−1 3,262.53 (amide N H, str), 3,131.44 (Ar
1,665.56 (NH O, str), 1,549.40 (Ar C, str), 1,396.97 (N N,
str), 1,242.64 (Ar
CH2, str), 827.44 (C Cl, str); 1H-NMR
C
H, str),
d6) δ ppm 9.64 (s, 2H), 8.35 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.87 (d,
J = 7.9 Hz, 1H), 7.66 (s, 1H), 7.56–7.41 (m, 4H), 7.21 (d, J = 7.1 Hz,
1H), 6.76 (s, 2H), 5.45 (s, 2H), 5.37 (s, 2H), 2.16 (s, 3H); MS (ESI +ve):
m/z 389.0 [M + H]+, 411.0 [M + Na]+.
C
C
O
(400 MHz, DMSO-d6) δ ppm 10.64 (s, 1H), 8.38 (s, 1H), 8.12 (d,
J = 8.1 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.56–
7.40 (m, 4H), 7.39 (d, J = 8.5 Hz, 2H), 7.20 (d, J = 7.5 Hz, 1H), 5.39 (s,
Synthesis of N-(2,3-dihydro-1H-inden-2-yl)-2-(4-((naphthalen-
1-yloxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide (60)
4H); 13C-NMR (100 MHz, DMSO-d6)
δ ppm 164.398, 153.483,
142.629, 137.353, 134.031, 128.829,127.453, 127.351, 126.449,
126.210, 126.135, 125.309, 124.882, 121.491, 120.775, 120.281,
The title compound was synthesized from 2-azido-N-(indan-2-yl)acet-
amide (0.25 g, 1.157 mmol), 1-(prop-2-yn-1-yloxy)naphthalene

8
PADHARIYA ET AL.
(0.21 g, 1.157 mmol), and Cu(OAc)2 (20 mol%) according to the gen-
eral procedure 2.1.5 to yield 60 as white solid. Yield: 75.46%; TLC
Rf = 0.32 (DCM:MeOH, 95:5); mp: 204–206ꢀC; purity (HPLC): >99%;
(HPLC): >99%; ATR cm−1 ATR cm−1 3,307.53 (amide N H, str),
3,273.55 (Ar
C
H, str), 1,693.82 (amide
C
O, str), 1,538.66
(Ar C, str), 1,473.53 (N N, str), 1,212.48 (Ar
C
O
CH2, str),
ATR cm−1 3,263.79 (amide N H, str), 3,054.08 (Ar
C
C, str),
829.91 (C Cl, str); 1H-NMR (400 MHz, DMSO-d6) δ ppm 10.79 (s,
1H), 8.31 (s, 1H), 7.95 (s, 1H), 7.83 (d, J = 7.9 Hz, 3H), 7.60 (dd, J = 8.8
and 3.0 Hz, 1H), 7.56–7.42 (m, 3H), 7.35 (t, J = 7.6 Hz, 1H), 7.20 (dd,
J = 8.9 and 3.1 Hz, 1H), 5.38 (d, J = 3.1 Hz, 2H), 5.29 (d, J = 3.2 Hz,
2H); MS (ESI +ve): m/z 427 [M + H]+.
2,954.68 (C H str), 1,666.24 (NH
1,397.77 (N N, str), 1,271.29 (Ar
C
O, str), 1,539.01 (C C, str),
CH2, str); 1H-NMR (400 MHz,
O
DMSO-d6) δ ppm 10.36 (s, 1H), 8.36 (s, 1H), 8.12 (d, J = 8.1 Hz, 1H),
7.87 (d, J = 8.0 Hz, 1H), 7.49 (s, 4H), 7.44 (s, 1H), 7.29 (d, J = 8.2 Hz,
1H), 7.18 (dd, J = 21.0 and 7.7 Hz, 2H), 5.36 (d, J = 11.5 Hz, 4H), 2.81
(q, J = 8.4 Hz, 4H), 2.03–1.95 (m, 2H); MS (ESI +ve): m/z 399.0 [M
+ H]+, 421.0 [M + Na]+.
Synthesis of N-(2-hydroxy-5-methylphenyl)-2-(4-((naphthalen-
2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide (64)
The title compound was synthesized from 2-azido-N-(2-hydroxy-
5-methylphenyl)acetamide (0.3 g, 1.46 mmol), 2-(prop-2-yn-1-yloxy)
naphthalene (0.26 g, 1.46 mmol), and Cu(OAc)2 (20 mol%) according
to the general procedure 2.1.5 to yield 64 as white solid. Yield:
78.21%; TLC Rf = 0.32 (DCM:MeOH, 95:5); mp: 171–173ꢀC; purity
(HPLC): >97%; ATR cm−1 3,052.49 (O H, str), 2,922.66 (amide N H,
Synthesis of N-(4-chlorophenyl)-2-(4-((naphthalen-2-yloxy)methyl)-
1H-1,2,3-triazol-1-yl)acetamide (61)
The title compound was synthesized from 2-azido-N-(4-chlorophenyl)
acetamide (0.3 g, 1.428 mmol), 2-(prop-2-yn-1-yloxy)naphthalene
(0.26 g, 1.428 mmol), and Cu(OAc)2 (20 mol%) according to the gen-
eral procedure 2.1.5 to yield 61 as white solid. Yield: 76.21%; TLC
Rf = 0.35 (DCM:MeOH, 95:5); mp: 224–226ꢀC; purity (HPLC): >99%;
str), 2,852.96 (Ar
C
H str), 1,654.10 (amide
C
O, str), 1,560.27
O
CH2, str); 1H-
(Ar C, str), 1,300.67 (N N, str), 1,255.62 (Ar
C
ATR cm−1 3,261.85 (amide N H, str), 3,081.49 (Ar
1,672.63 (amide O, str), 1,550.50 (Ar C, str), 1,306.28 (N N,
str), 1,258.44 (Ar
CH2, str), 740.21 (C Cl, str); 1H-NMR
C
H str),
NMR (400 MHz, DMSO-d6) δ ppm 9.64 (d, J = 3.8 Hz, 2H), 8.28 (s,
1H), 7.83 (dd, J = 9.0, 4.0 Hz, 3H),7.65 (s, 1H), 7.53 (s, 1H), 7.46 (d,
J = 8.1 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.20 (d, J = 8.8 Hz, 1H), 6.75
(s, 2H), 5.43 (s, 2H), 5.28 (s, 2H), 2.15 (s, 3H); MS (ESI +ve): m/z 389
[M + H]+, 411 [M + Na]+.
C
C
O
(400 MHz, DMSO-d6) δ ppm 10.63 (s, 1H), 8.32 (s, 1H), 7.83 (d,
J = 8.3 Hz, 3H), 7.65 (d, 2H), 7.56 (s, 1H), 7.45 (t, 1H), 7.35 (m, 3H),
7.24 (d, 1H), 5.37 (s, 2H), 5.30 (s, 2H); 13C-NMR (100 MHz, DMSO-
d6) δ ppm 164.377, 155.926, 142.398, 137.338, 134.182, 129.348,
128.826, 128.576, 127.500, 127.360, 126.737, 126.407, 123.679,
120.775, 118.663, 107.142, 61.057, 52.197; MS (ESI +ve): m/z 393.0
[M + H]+, 415.0 [M + Na]+; HRMS (ESI) calculated for C21H17ClN4O2
[M + H]+ 393.1040, found: 393.1104.
Synthesis of N-(2,3-dihydro-1H-inden-2-yl)-2-(4-((naphthalen-
2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide (65)
The title compound was synthesized from 2-azido-N-(2,3-dihydro-1H-
inden-2-yl)acetamide (0.25 g, 1.157 mmol), 2-(prop-2-yn-1-yloxy)
naphthalene (0.21 g, 1.157 mmol), and Cu(OAc)2 (20 mol%) according
to the general procedure 2.1.5 to yield 65 as white solid. Yield:
84.12%; TLC Rf = 0.29 (DCM:MeOH, 95:5); mp: 208–210ꢀC; purity
(HPLC): >99%; ATR cm−1 3,195.24 (amide N H, str), 3,037.44
Synthesis of N-(4-fluorophenyl)-2-(4-((naphthalen-2-yloxy)methyl)-
1H-1,2,3-triazol-1-yl)acetamide (62)
The title compound was synthesized from 2-azido-N-(4-fluorophenyl)
acetamide (0.3 g, 1.546 mmol), 2-(prop-2-yn-1-yloxy)naphthalene
(0.28 g, 1.546 mmol), and Cu(OAc)2 (20 mol%) according to the gen-
eral procedure 2.1.5 to yield 62 as white solid. Yield: 74.78%; TLC
Rf = 0.36 (DCM:MeOH, 95:5); mp: 220–222ꢀC; purity (HPLC): >98%;
(Ar
C
H, str), 2,920.09 (C H str), 1,662.07 (amide
C
O, str),
O CH2,
1,552.66 (Ar
C
C, str), 1,297.69 (N N, str), 1,261.60 (Ar
str); 1H-NMR (400 MHz, DMSO-d6) δ ppm 10.40 (s, 1H), 8.31 (s, 1H),
7.83 (d, J = 8.5 Hz, 3H), 7.56–7.42 (m, 3H), 7.33 (dd, J = 25.7, 8.0 Hz,
2H), 7.18 (dd, J = 19.8, 8.6 Hz, 2H), 5.29–5.34 (dd, 4H), 2.80 (q,
J = 8.3 Hz, 4H), 1.98 (p, J = 7.5 Hz, 2H); 13C-NMR (100 MHz, DMSO-
d6) δ ppm 163.824, 155.936, 144.294, 142.335, 139.012, 136.583,
134.187, 129.342, 128.572, 127.498, 126.734, 126.402, 126.344,
124.296, 123.671, 118.661, 117.385, 115.452, 107.127, 61.061,
52.224, 32.445, 31.758, 25.102; MS (ESI +ve): m/z 399 [M + H]+, 421
[M + Na]+. HRMS (ESI) calculated for C24H22N4O2 [M + H]+
399.1743, found: 399.1816.
ATR cm−1 3,272.84 (amide N H, str), 3,222.18 (Ar
1,668.30 (amide O, str), 1,507.43 (Ar C, str), 1,308.84 (N N,
str), 1,217.13 (Ar
CH2, str), 475.65 (C F, str); 1H-NMR
C H, str),
C
C
O
(400 MHz, DMSO-d6) δ ppm 10.54 (s, 1H), 8.31 (s, 1H), 7.83 (t,
J = 6.3 Hz, 3H), 7.60 (dd, J = 8.6, 4.6 Hz, 2H), 7.55–7.42 (m, 2H), 7.36
(d, J = 7.9 Hz, 1H), 7.23–7.12 (m, 3H), 5.35 (d, J = 2.9 Hz, 2H), 5.29 (d,
J = 3.0 Hz, 2H); MS (ESI +ve): m/z 377.0 [M + H]+, 399.0 [M + Na]+.
Synthesis of N-(3,4-dichlorophenyl)-2-(4-((naphthalen-2-yloxy)
methyl)-1H-1,2,3-triazol-1-yl)acetamide (63)
Synthesis of N-(2-(4-bromophenyl)propan-2-yl)-2-(4-((naphthalen-
2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide (66)
The title compound was synthesized from 2-azido-N-(3,-
4-dichlorophenyl)acetamide (0.3 g, 1.22 mmol), 2-(prop-2-yn-1-yloxy)
naphthalene (0.22 g, 1.22 mmol), and Cu(OAc)2 (20 mol%) according
to the general procedure 2.1.5 to yield 63 as white solid. Yield:
82.36%; TLC Rf = 0.31 (DCM:MeOH, 95:5); mp: 173–175ꢀC; purity
The title compound was synthesized from 2-azido-N-
(2-(4-bromophenyl)propan-2-yl)acetamide (0.3 g, 1.013 mmol),
2-(prop-2-yn-1-yloxy)naphthalene
(0.185 g,
1.013 mmol),
and
Cu(OAc)2 (20 mol%) according to the general procedure 2.1.5 to yield
66 as white solid. Yield: 76.45%; TLC Rf = 0.34 (DCM:MeOH, 95:5);

PADHARIYA ET AL.
9
mp: 168–170ꢀC; purity (HPLC): >99%; ATR cm−1 3,303.41 (amide
carcinoma), A549 (lung adenocarcinoma) and NIH/3T3 (mouse
embroyonic fibroblast) were purchased from National Centre for Cell
Science (Pune, India). On receipt, the cell lines were passaged in our
lab and the earliest passaged cells were cryopreserved in liquid nitro-
gen container for future use. The cell lines used in culture were pas-
saged for fewer than 8 weeks and were carefully maintained as
described. The cells were maintained in Dulbecco's modified Eagle
medium/F12 (HiMedia, Catalogue No.: AL223A) complete media with
10% fetal bovine serum (Gibco Life Technologies, Grand Island, NY)
and penicillin–streptomycin (50 U/ml, 50 mg/ml; HiMedia, Catalogue
No.: A002) at 37ꢀC, CO2 (5%) and air (95%). Around 40–50% con-
fluency of cultured cells were used for seeding during the assays.
Other reagents used during cell line studies included fibroblast
growth factor (Sigma-Aldrich, Catalogue No.: F029125), epidermal
growth factor (Sigma-Aldrich, Catalogue No.: E9644), insulin (Sigma,
Catalogue No.: 19278), Dulbecco's phosphate-buffered saline
(HiMedia, Catalogue No.: TL1006), trypan blue (TCI 93).
N
H, str), 3,085.60 (Ar C C, str), 2,920.22 (C H str), 1,676.57
(NH
(Ar
C
O, str), 1,557.69 (C C, str), 1,466.84 (N N, str), 1,255.59
CH2, str), 625.83 (C Br, str); 1H-NMR (400 MHz, DMSO-d6)
O
δ ppm 8.69 (s, 1H), 8.17 (s, 1H), 7.85–7.77 (m, 3H), 7.53–7.41 (m, 4H),
7.39–7.25 (m, 3H), 7.18 (dd, J = 8.6, 2.8 Hz, 1H), 5.24 (s, J = 2.9 Hz,
2H), 5.16 (s, J = 3.0 Hz, 2H), 1.55 (s, J = 3.0 Hz, 6H); 13C-NMR
(100 MHz, DMSO-d6) δ ppm 164.302, 155.918, 146.622, 142.163,
134.162, 130.764, 129.308, 128.543, 127.479, 127.146, 126.707,
126.378, 126.179, 123.644, 119.128, 118.640, 107.068, 61.002,
54.924, 51.820, 28.980; MS (ESI +ve): m/z 479 [M + H]+, 481 [M + H
+ 2]+. HRMS (ESI) calculated for C24H23BrN4O2 [M + Na]+ 501.1004,
found: 501.0920.
Synthesis of N-(2-bromo-6-nitrophenyl)-2-(4-((naphthalen-2-yloxy)
methyl)-1H-1,2,3-triazol-1-yl)acetamide (67)
The title compound was synthesized from 2-azido-N-[1-(4-
bromophenyl)-1-methylethyl]acetamide (0.3 g, 1.002 mmol), 2-(prop-
2-yn-1-yloxy)-naphthalene (0.183 g, 1.002 mmol), and Cu(OAc)2
(20 mol%) according to the general procedure 2.1.5 to yield 67 as
white solid. Yield: 89.56%; TLC Rf = 0.31 (DCM:MeOH, 95:5); mp:
223–225ꢀC; purity (HPLC): >99%; ATR cm−1 3,262.17 (amide N H,
2.2.2
|
Cell viability assay
Cytotoxic activity of the compounds was evaluated by colorimetric
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay on the above-mentioned cell lines using doxorubicin HCl, cis-
platin and sunitinib as controls. Briefly, logarithmically growing cells
were seeded in 96-well plate (seeding density: MDA-MB-231:10,000
cells/well, PC-3: 8,000 cells/well, U87 MG: 5,000 cells/well, SiHa:
10,000 cells/well, A549: 5,000 cells/well, NIH-3T3: 10,000 cells/well)
and incubated for 24 hr in humidified condition (5% CO2) at 37ꢀC and
then observed under microscope. Test compounds with appropriate
dilution were then added to the wells in triplicate along with DMSO as
vehicle control. After 48 hr of test compound exposure in 5% CO2
humidified condition at 37ꢀC, the assay plate was centrifuged twice at
3,000 rpm for 3 min and the supernatant was discarded. Later, each
well was treated with 100 μl of MTT reagent (5 mg/ml) and it was fur-
ther incubated for 4 hr (in 5% CO2 atmosphere at 37ꢀC). Following
incubation, the plate was centrifuged and the supernatant was aspi-
rated off and 200 μl of DMSO was added in each well to solubilize the
formazan crystals. The absorbance was then measured at 540 and
630 nm (background scan) using EPOCH 2 Biotek microplate reader. A
concentration of 10 μM of test compounds was used for initial screen-
ing in all cell lines. The ones with >80% inhibition were taken forward
for IC50 determination (test compound concentration inhibiting 50%
of the cell population). To determine the IC50 value of test compounds
in respective cell lines, a total of six concentrations ranging from 0.01
to 100 μM in triplicate were used. The IC50 was then calculated by
regression analysis and expressed in μM using a mean of triplicate.
str), 2,941.54 (Ar
(C C, str), 1,457.21 ( NO2, str), 1,352.95 (N N, str), 1,252.95
(Ar
CH2, str), 470.34 (C Br, str); 1H-NMR (400 MHz, DMSO-d6)
C H, str), 1,688.61 (NH C O, str), 1,513.21
O
δ ppm 10.80 (s, 1H), 8.27 (d, J = 1.7 Hz, 1H), 8.13–8.06 (m, 1H), 8.02–
7.95 (m, 1H), 7.82 (dd, J = 8.1, 4.8 Hz, 3H), 7.55–7.42 (m, 3H), 7.35 (t,
J = 7.5 Hz, 1H), 7.19 (dt, J = 9.1, 2.1 Hz, 1H), 5.44 (s, 2H), 5.27 (s, 2H);
MS (ESI +ve): m/z 482 [M + H]+.
Synthesis of N-(6-chloropyrazin-2-yl)-2-(4-((naphthalen-2-yloxy)
methyl)-1H-1,2,3-triazol-1-yl)acetamide (68)
The title compound was synthesized from 2-azido-N-(6-
chloropyrazin-2-yl)acetamide (0.3 g, 1.415 mmol), 2-(prop-2-yn-
1-yloxy)naphthalene (0.25 g, 1.415 mmol), and Cu(OAc)2 (20 mol%)
according to the general procedure 2.1.5 to yield 67 as white solid.
Yield: 84.39%; TLC Rf = 0.38 (DCM:MeOH, 95:5); mp: 203–205ꢀC;
purity (HPLC): >98%; ATR cm−1 3,050.66 (amide N H, str), 3,019.67
(Ar
1,459.82 (C
(Ar
CH2, str), 832.974 (C Cl, str); 1H-NMR (400 MHz, DMSO-d6)
C
H, str), 1,697.29 (NH
C O, str), 1,545.58 (C C, str),
N
in ring, str), 1,408.98 (N N, str), 1,216.95
O
δ ppm 11.67 (s, 1H), 9.21 (s, 1H), 8.54 (d, J = 2.0 Hz, 1H), 8.32 (d,
J = 2.1 Hz, 1H), 7.83 (t, J = 7.4 Hz, 3H), 7.55 (s, 1H), 7.42 (t, 1H), 7.35
(t, J = 7.8 Hz, 1H), 7.20 (dd, J = 8.9, 2.6 Hz, 1H), 5.48 (s, 2H), 5.30 (s,
2H); MS (ESI +ve): m/z 395.0 [M + H]+, 417.0 [M + Na]+.
2.2
|
Biology
Cell lines and reagents
2.2.1
|
2.2.3
|
Soft-agar assay
All the cell lines: MDA-MB-231 (breast adenocarcinoma), PC-3 (pros-
tate carcinoma), U87 MG (glioblastoma astrocytoma), SiHa (cervical
Briefly, a 100 μL mixture of 1:1 dilution of 2× complete media and
1.2% Bacto™ agar were transferred onto each well of the 96-well

10
PADHARIYA ET AL.
microtiter assay plate. The cells (10 μl with seeding efficiency: MDA-
MB-231: 7,500 cells/well, and PC-3: 5,000 cells/well) were mixed
with 2× medium (20 μl), 0.8% of Bacto™ agar (30 μl) and appropriate
concentration of test compound (1.6 μl) in a vial and transferred onto
the solidified prelayers in the plates to incubate it further for (5%
CO2, 37ꢀC) 1 week to grow and form colonies. Midway through the
week, a feeding layer consisting of 50 μl of suitable 2× medium was
added. Once the incubation period was over, 16 μl of alamarBlue®
(1.5 mg/ml) was added to all the wells to quantify the developed colo-
nies and the plates were placed in the incubator for 24 hr at 37ꢀC
before measuring absorbance at 630 nm. The IC50 (test compound
concentration inhibiting 50% of cell population) was determined by
regression analysis.
% viable spheres formed at the given concentration to the positive
control was then plotted.
Number of spheres formedperwell × 100
Number of spheres formed in control well
%Viability =
:
ð1Þ
2.2.5
|
Human peripheral blood mononuclear cells
assay
The assay was performed on lymphocytes obtained from human
blood, stabilized by ethylenediaminetetraacetic acid, of healthy donors
upon approval from the Institutional Ethics Committee (Reg. No:
ECR/138/Inst/MH/2013). Briefly, the fresh blood was defribinated
and laid gradually on HiSep™ LSM1077 and centrifuged for half an
hour at low speed. The buffy-colored lymphocyte layer was carefully
transferred to a new collection tube and washed with a diluent buffer
solution to reduce platelet contamination. After discarding the super-
natant, the diluent buffer was used to resuspend the pellet and viabil-
ity was determined using hemocytometer. % Viability compared to
the control group was determined by MTT assay as described above
(seeding density 0.7 million/ml).
2.2.4
|
Sphere assay
One of the unique properties of CSCs is that it can survive and can be
cultured in serum-free media. Declustered suspension of CSCs under
nonadherent conditions in the serum-free medium can grow and form
spheres. For both the cell lines used in the assay, two different media
mammosphere media (MDA-MB-231) and prostosphere media (PC-3)
were used. The media were prepared as per our previously reported
procedure. (Padhariya et al., 2020).
Briefly, the trypsinized cells were declustered using both 100-
and 40-μm cell strainer to make a uniform single-cell suspension.
Dilution of 2,000 cells/100 μl in respective mammosphere (for
MDA-MB-231) and prostatosphere (for PC-3) medium was prepared
and plated onto each well of 96-well suspension plates and incu-
bated under humidified condition (37ꢀC, 5% CO2) for 24 hr. Once
the media was set, 2 μl of test compounds per 100 μl of the respec-
tive medium was added to each well and again incubated under the
same condition for 72 hr. After incubation, the spheres formed were
again exposed to the test compounds in two rounds of 2.5 μl/50 μl
and 3 μl/50 μl of the respective culture media and incubated for
72 hr each time at 37ꢀC, 5% CO2. At the end of 7 days of the experi-
mental period, live primary spheres were counted manually under
phase-contrast microscope (Figure 1) to determine % viability for
each concentration by using Formula (1) and a comparative graph of
2.3
|
Statistical analysis
All results were expressed as mean SD from experiments carried
out in triplicates. Statistical analysis was performed using a two-way
analysis of variance followed by Bonferroni post-test with the help of
GraphPad Prism software (Version 6.0).
3
|
RESULTS AND DISCUSSION
| Chemistry
3.1
In extension to our previous work on the synthesis of small molecules
as anti-CSC agents, we adopted a simple and efficient synthetic route
FIGURE 1 Phase contrast
microscopy images (×20
magnification) of
(a) mammospheres (MDA-MB-
231) (b) prostatospheres (PC-3)

PADHARIYA ET AL.
11

12
PADHARIYA ET AL.

PADHARIYA ET AL.
13

14
PADHARIYA ET AL.
TABLE 2 Median effect of hits on viability in human breast and
CSC drug (Yuan et al., 2016). The results are shown in Table 1. Mole-
cules displaying >80% inhibition at 10 μM were further taken up for
IC50 determination. Overall, at 10 μM most of the molecules exhibited
better activity against breast (MDA-MB-231), prostate (PC-3), glioma
(U87 MG) compared to cervical (SiHa) and lung (A549) cell line
(Table 1). More so, molecules in Series C (56–60) and Series D
(61–68) were found to more active than Series B (46–55) while Series
A (39–45) were comparatively less active at tested concentration.
In Series A, molecules displayed comparative activity in breast
and prostate cell line. The presence of electron-withdrawing substitu-
ent on the phenyl ring such as 4-Cl (39) and 4-F (40) showed
improved activity in all cell lines compared to 3,4-Cl2 (41) with an
exception in U78 MG (76%). The activity improved when the phenyl
ring was substituted with an indanyl ring (43). In Series B as well, mol-
ecules showed preference to inhibit breast and prostate cancer cell
lines. Similar to Series A, molecules with phenyl ring substitution as
4-Cl (46), 4-F (47) and 3,4-Cl2 (48) were found to be more active over-
all. More so, compound 48 exhibited around 80% inhibition at 10 μM
in both breast (82%) and prostate (80%) cell line. While the IC50 value
was comparable to sunitinib in MDA-MB-231 (3.5 μM), it was fourfold
less in case of PC-3 (8.6 μM). Replacing substituted phenyl ring with
heterocylic ring (54) clearly diminished the activity. A similar trend
was observed in Series C, where 4-Cl phenyl substituted molecule
(56) displayed >80% inhibition at 10 μM in both MDA-MB-231 and
U87 MG cell line. In the case of MDA-MB-231, the IC50 value was
found to be less than sunitinib (2.1 μM). However, in the case of Ser-
ies D, three molecules 61, 65, and 66 exhibited >80% inhibition at
10 μM concentration in three cell lines: breast, prostate, and glioma.
In case of MDA-MB-231, two molecules exhibited improved activity
compared to sunitinib (61:1.4 μM, 66:1.6 μM).
prostate cancer cell lines in soft-agar assaya
IC50 (μM)b
Compounds
MDA-MB-231c
33.19 0.11
1.08 0.09
2.8 0.07
PC-3d
Cispaltin
26.18 0.08
1.48 0.21
2.64 0.25
1.57 0.18
2.9 0.22
Doxorubicin
Sunitinib
48
4.68 0.08
6.34 0.05
>100
56
61
12.13 0.18
14.49 0.28
24.21 0.05
65
7.26 0.07
11.67 0.05
66
aDetermined by soft-agar assay.
bCompounds tested in triplicate.
cBreast adenocarcinoma: MDA-MB-231.
dProstate carcinoma: PC-3.
to synthesize title compounds 39–68. The substituted phenols 1–4
were O-alkylated using propargyl chloride in the presence of anhy-
drous K2CO3 in dry DMF to yield corresponding prop-2-ynyl deriva-
tives 5–8 according to general procedure mentioned in Section 2.1.2.
In a separate set of reactions as per general procedures in Sec-
tions 2.1.3 and 2.1.4, various substituted amines 9–18 bearing
electron-donating and -accepting groups were converted to their
respective 2-azido-N-substituted acetamide derivatives 19–28 by
acetylation with chloroacetyl chloride followed by reaction with NaN3
in anhydrous DMF. Finally, the 2-azido-N-substituted acetamide and
prop-2-ynyl derivatives were used to carry out 1,3-dipolar cycloaddi-
tion reaction using a catalytic amount of Cu(OAC)2.H2O in t-BuOH:
H2O (3:1) mixture to yield title compounds 39–68 (80–90% yield) as
per previously described procedure in Section 2.1.5. Although an
effort was made to synthesize 1,2,3-triazoles from all possible combi-
nations of 2-azido-N-substituted acetamide 19–28 and prop-2-ynyl
derivatives 5–8, few reactions did not undergo as expected and purifi-
cation of the same did not yield the desired product. The final sets of
purified and structurally characterized molecules (Data S1) were
screened in a battery of assays.
3.2.2
|
Soft-agar assay
Title compounds exhibiting >80% inhibition at 10 μM in the respec-
tive cell line were carried forward for evaluation in soft-agar assay
(SAA), also known as anchorage-independent assay (Fukazawa,
Mizuno, & Uehara, 1995). A normal cell line in culture differs from a
transformed cell line in various aspects with respect to growth param-
eters. Normal cells require adhesion to extracellular matrix and base-
ment membrane for survival and growth and undergo apoptosis in
suspension media. On the other hand, tumor cells can flourish without
cell-substratum support and evade anoikis resulting in uncontrolled
cell growth. A normal cell can survive, grow, and differentiate only in
proper tissue surrounding; however, tumor cell is competent for sur-
vival irrespective of their original environment. To understand the
hallmark characteristic of cell transformation, that is, uncontrolled cell
growth and tumorigenecity, an important characteristic of CSCs, in a
semisolid media without attachment to substratum, in vitro
anchorage-independent growth is the best way to correlate with their
tumorigenic nature in vivo (Shin, Freedman, Risser, & Pollack, 1975).
The superiority of testing anticancer molecules in three-
dimensional (3D) format like SAA over monolayer culture to validate
3.2
|
Biology
| Cell viability in MTT assay
3.2.1
Initially, all the 30 molecules were screened at 10 μM concentration in
cell viability (MTT) assay against various cancer cell lines: breast
(MDA-MB-231), prostate (PC-3), glioma (U87 MG), cervical (SiHa),
and lung (A549). The optimal plating densities of each cell line are
mentioned in Section 2.2.2. Doxorubicin HCl, cisplatin and sunitinib
were used as positive controls and the vehicle DMSO as a negative
control. While doxorubicin HCl and cisplatin represented conventional
chemotherapy modality, sunitinib was used as a representative anti-

PADHARIYA ET AL.
15
Sphere analysis of MDA-MB-231
(a)
Cisplatin
Doxorubicin
Sunitinib
48
100
50
0
56
61
65
66
Concentration (µM)
(b)
Sphere analysis of PC-3
Cisplatin
Doxorubicin
Sunitinib
48
56
61
65
100
50
0
66
Concentration (µM)
FIGURE 2 Results from sphere assay of the hits. (a) % Viability of mammospheres (MDA-MB-231); (b) % viability of prostatospheres (PC-3)
compared to the untreated. (The data are expressed as mean SD [n = 4]. Asterisk above columns indicate statistically significant difference
compared to control. ***p < .001)
cell transformation is used since 1977 in clonogenic assay developed
by Hamburger and Salmon (1977) and Hamburger (1987). Since then,
SAA is widely being used to identify potential antitumor agents and to
evaluate the stemness properties of CSCs (Fukazawa, Noguchi,
Murakami, & Uehara, 2002; Horibata, Vo, Subramanian, Thompson, &
Coonrod, 2015; Seyfrid et al., 2019). More so, the traditional method

16
PADHARIYA ET AL.
Median effect of hits on cell viability of NIH/3T3
free gel matrix supports the growth of only CSCs influencing the for-
mation of spheres under such stringent conditions (Pastrana, Silva-
Vargas, & Doetsch, 2011). The Sphere assay is widely used 3D-assay
to evaluate the anti-CSC effect of NCEs (Bahmad et al., 2018; Morales
et al., 2013).
100
***
***
***
50
0
In the present study, the hits from MTT and SAA were screened
at five different concentrations (0.0025, 0.025, 0.25, 2.5 and 25 μM)
against breast (MDA-MB-231) and prostate (PC-3) CSCs. (Figure 2a,
b). Interestingly, most of the hits (48, 56, 61, 65, and 66) demon-
strated moderate activity at 2.5 μM by inhibiting >50% spheres in
both the cell lines. In particular, compounds 61, 65, and 66 inhibited
>50% spheres at 25 nM concentration against the MDA-MB-231 cell
line. Notably, 65 exhibited better inhibition profile than sunitinib (Fig-
ure 2a). In the case of PC-3 cell line, except 61, all four NCEs exhibited
moderate activity with >50% sphere inhibition at 2.5 μM, while only
66 displayed high activity at 0.25 μM. Overall, the hits were found to
be more active against mammosphere than prostatosphere.
***
***
***
***
Compounds
FIGURE 3 % Cell viability of the hits against NIH/3T3 cell line
(IC50 SD, n = 3) (Asterisk above columns indicate statistically
significant difference compared to control. ***p < .05)
TABLE 3 Median effect of the hits on lymphocyte viability
(hPBMC assay)
3.2.4
assay
|
Human peripheral blood mononuclear cells
Compound code
IC50 (μM)
11.07 0.12
59.43 0.07
2.8 0.07
4.84 0.07
>100
Doxorubicin
The primary side effect of anticancer therapy is the destruction of
normal cells. While a potential drug candidate should be toxic to the
target cell, it should be nontoxic to the normal body cells. To under-
stand the toxic effect on normal cells, potential hits (48, 56, 61, 65,
and 66) were also screened against human peripheral blood mononu-
clear cells (hPBMCs) in lymphocyte assay and NIH/3T3 (mouse
embryonic fibroblast) cell line by MTT assay in two different
experiments.
Cisplatin
Sunitinib
48
56
61
>100
65
10.21 0.06
7.31 0.07
66
In NIH/3T3 cell line, the hits (48, 56, 61, 65, and 66) were
screened at five different concentrations (0.01, 0.1, 1, 10, and
100 μM) using cisplatin and doxorubicin as positive control. Hits 48,
56, and 61 found to have IC50 > 75 μM, while 65 and 66 had
IC50 < 45 μM. Overall, 48, 56, and 61 were nearly threefold less toxic
than doxorubicin under the same condition against NIH/3T3 cell line
(Figure 3).
is also modified with time to overcome time-consuming, labour inten-
sive process with HTS to meet current oncology drug-target screening
demand (Fukazawa et al., 1995; Ke et al., 2004).
In the current study, representative cell lines from the breast
(MDA-MB-231) and prostate (PC-3) cancer types were used with
their optimized cell plating density. The IC50 (μM) values were calcu-
lated based on the capacity of the molecules to inhibit 50% colony-
formation compared to the control (Table 2).
In lymphocyte assay, the hits were screened against hPBMCs
using both doxorubicin and sunitinib as positive controls and IC50
values were calculated (highest concentration tested 100 μM)
(Table 3). While 56 and 61 showed no toxicity toward hPBMCs
(IC50 > 100 μM); hit 48 (IC50 = 4.84 μM), 65 (IC50 = 10.21 μM), and 66
(IC50 = 7.31 μM) were relatively toxic compared to the positive con-
trols sunitinib (IC50 = 2.8 μM) and doxorubicin (IC50 = 11.07 μM).
Compound 48 (MDA-MB-231:4.6 μM and PC-3:1.5 μM) was the
most potent against the indicated cell line. Compound 56 also showed
activity comparable to sunitinib in the case of PC-3 (2.9 μM). Overall,
most of the tested compounds showed IC50 < 10 μM. The positive
results encouraged us to evaluate the hits in sphere-formation or
sphere assay to understand their potential effect on CSCs.
4
|
CONCLUSIONS
3.2.3
|
Sphere assay
Our original hypothesis to use a hybrid structure with more than one
pharmacophoric group to direct activity against CSCs led to interest-
ing results. In the present investigation, a total of 30 (39–68)
1,2,3-triazole ring containing derivatives belonging to four related
chemical series, were designed and synthesized using click chemistry
3D bioassays mimic and simulate in vivo conditions better than the
known 2D monolayer cell culture assays (Chen et al., 2012). To study
the effect of test molecule specifically on CSCs, more harsh conditions
are used to eliminate any chance of growth of cancer cells. A serum-

PADHARIYA ET AL.
17
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approach. While most of the synthesized molecules were active
against breast, prostate and glioma cell lines, five hits displayed single-
digit micromolar activity. Further screening the hits in sphere assay,
the hits were also found to be active against mammospheres and
prostatospheres with IC50 ranging from as low as 0.025 to 2.5 μM.
Among all, 56 exhibited potent anticancer as well as anti-CSCs activity
without affecting the tested normal cells NIH/3T3 and human lym-
phocytes. Further studies to improve pharmacokinetic and drug-like
properties needs to be addressed. Overall, combining small
pharmacophoric groups with the triazole functionality helped us to
identify potential hits as anti-CSC agents.
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ACKNOWLEDGMENTS
K. P. is grateful to SVKM's NMIMS (Deemed to be University), Mum-
bai, for the financial support in the form of fellowship; Dr Sona War-
rier, Dr Chetan Shah, and Dr Nitesh Sahu for their help during the
synthetic work; and to Mr Eknath Gadekar for analyzing all the com-
pounds using LC–MS. M. A. is thankful to Ms Smera Satish, Ms
Gayatri Gore, and Mr Akshay Ganpule for their help during the biolog-
ical evaluation of all the compounds.
Ghosh, R., Nadiminty, N., Fitzpatrick, J. E., Alworth, W. L., Slaga, T. J., &
Kumar, A. P. (2005). Eugenol causes melanoma growth suppression
through inhibition of E2F1 transcriptional activity. Journal of Biological
Chemistry, 280(7), 5812–5819.
Hamburger, A. W., & Salmon, S. E. (1977). Primary bioassay of human
tumor stem cells. Science, 197, 461–463.
CONFLICT OF INTEREST
Authors declare no conflict of interest from a financial or commercial
standpoint.
AUTHOR CONTRIBUTIONS
Hati, S., Tripathy, S., Dutta, P. K., Agarwal, R., Srinivasan, R., Singh, A., …
Sen, S. (2016). Spiro [pyrrolidine-3,3-oxindole] as potent anti-breast
cancer compounds: Their design, synthesis, biological evaluation and
cellular target identification. Scientific Reports, 6(1), 1–10.
He, X.-P., Xie, J., Tang, Y., Li, J., & R Chen, G. (2012). CuAAC click chemis-
try accelerates the discovery of novel chemical scaffolds as promising
protein tyrosine phosphatases inhibitors. Current Medicinal Chemistry,
19(15), 2399–2405.
Prashant S. Kharkar envisaged the project. Prashant S. Kharkar and
Sangeeta Srivastava monitored the progress of the project. Komal
N. Padhariya synthesized, characterized, purified, and partly carried
out the biological evaluation. Maithili Athavale and Sangeeta
Srivastava carried out the majority of the biological evaluation.
Prashant S. Kharkar and Sangeeta Srivastava contributed to the manu-
script compilation and critical reading.
Heamburger, A. W. (1987). The human tumor clonogenic assay as a model
system in cell biology. The International Journal of Cell Cloning, 5(2),
89–107.
Horibata, S., Vo, T. V., Subramanian, V., Thompson, P. R., & Coonrod, S. A.
(2015). Utilization of the soft agar colony formation assay to identify
inhibitors of tumorigenicity in breast cancer cells. Journal of Visualized
Experiments, 99, e52727.
ORCID
Prashant S. Kharkar
https://orcid.org/0000-0003-1955-7223
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