Synthesis and anticancer activity studies of indolylisoxazoline analogues

Article abstract of DOI:10.1016/j.bmcl.2018.07.035

A new library of thirteen indolylisoxazolines 6a–m has been synthesized by the treatment of indolylchalcones with hydroxylamine hydrochloride. Evaluation of anticancer activity of indolylisoxazolines 6a–m led to the identification of potent compounds 6c–d

Full text of DOI:10.1016/j.bmcl.2018.07.035

Accepted Manuscript
Synthesis and anticancer activity studies of indolylisoxazoline analogues
M.V.S.K. Chaitanya, P.O. Venkataramana Reddy, Nikhil Kumar, Anil Kumar,
Kavita Shah, Dalip Kumar
PII:
S0960-894X(18)30616-4
https://doi.org/10.1016/j.bmcl.2018.07.035
BMCL 25966
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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26 June 2018
24 July 2018
25 July 2018
Please cite this article as: Chaitanya, M.V.S.K., Reddy, P.O.V., Kumar, N., Kumar, A., Shah, K., Kumar, D.,
Synthesis and anticancer activity studies of indolylisoxazoline analogues, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.07.035
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Bioorganic & Medicinal Chemistry Letters
Synthesis and anticancer activity studies of indolylisoxazoline analogues
Chaitanya MVSKa, Venkataramana Reddy P Oa, Kumar Nikhilb, Anil Kumar,a Kavita Shahb,
and Dalip Kumara,
a Department of Chemistry, Birla Institute of Technology and Science, Pilani 333031, Rajasthan, India
b Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A new library of thirteen indolylisoxazolines 6a-m has been synthesized by the treatment of
indolylchalcones with hydroxylamine hydrochloride. Evaluation of anticancer activity of
indolylisoxazolines 6a-m led to the identification of potent compounds 6c-d, 6i and 6l, with IC50
ranging 2.5-5.0 µM against the tested cancer cell lines. Using a number of complementary
techniques such as acridine orange/ethidium bromide staining, PARP1 cleavage and DNA strand
breaks assay, we show that the compounds 6c and 6i induce apoptosis in highly aggressive C4-2
cells. Our data further revealed that 6c and 6i inhibited C4-2 cells proliferation without inducing
ROS. Finally, we show that compounds 6c and 6i also potently inhibit cell migration, indicating
these compounds have the potential to serve as effective anti-cancer agents.
Keywords:
Indolylisoxazolines
Isoxazolines
Indole heterocycles
Anticancer activity
Apoptosis
2009 Elsevier Ltd. All rights reserved.
Indole scaffold is frequently present in many important synthetic
and natural drug molecules including anticancer, anti-oxidants,
anti-inflammatory, analgesics and anti-pyretic drugs.1,2 Greater
versatility and biodiversity of indole nucleus makes it highly
privileged motif for the target-based drug design and
development of anticancer agents. In the last decade, importance
of indole motif in the anticancer drug development is reflected by
the identification of many indole-based natural and synthetic
anticancer agents with distinct mechanism of actions.3-5 Among
the indole-based compounds, indolylazoles containing five- and
six-membered heterocycles linked indole have received greater
attention due to their unique properties such as stability and
hydrophilic nature, which improves aqueous solubility and thus
simplify the formulation and in vivo uses (Fig 1).6 5-(3'- Indolyl)
oxazoles isolated from different micro-organisms are reported to
display interesting biological activities. For example, Labradorins
1 and 2 were found to be potential inhibitors (GI50 = 9.6-9.8
μg/mL) of lung cancer cell line.7,8 Inspired by naturally occurring
indolylazoles, we identified 5-(3-indolyl)-1,3,4-oxadiazoles 2 and
indolyl-1,2,4-triazoles 3 as potent anticancer agents.9 Some of the
indolylazoles 3 are reported to show selective cytotoxicity
against prostate cancer cell line (IC50 = 0.8 µM) and found to
inhibits tubulin polymerization.5 Compound A–289099 (4) with
oxazoline ring was recognized as an orally active antimitotic
Fig. 1. Representative examples of cytotoxic indolyl(aryl)azoles
In recent years, accumulating evidences have revealed that
isoxazolines possess important biological properties such as
anticancer, antimicrobial, fungicidal, anticonvulsant, anti-
inflammatory.11 Some of the isoxazolines containing molecules
have been found to elicit interesting anticancer activities with
improved pharmacokinetics profile. For example, diaryl
analogues 5 demonstrated potent cytotoxic activity by blocking
most of the cancer cells in G2 phase.12 Isoxazoline linked
dihydro-quinazolinones significantly reduced the growth of
cancer cell lines, and disrupted tubulin polymerization.13
As a part of our search for novel anticancer agents, in the present
study, we designed indole-based heterocycles by linking indole
and (hetero) aryl moieties through stable isoxazoline scaffold
found in many biologically active molecules and drugs.14-17 Many
anticancer agents with substantial cytotoxicity have been
agent with most promising anticancer property (IC50 = 6.2 nM)
10
a—ga—ins—t NCI-H460 cells.
 Corresponding author. Tel.: +91-1596-515238; fax: +91-1596-244183; e-mail: dalipk@pilani.bits-pilani.ac.in (Dalip Kumar)
 Corresponding author. Tel.: +001-7654969470; fax: +001-7654940239; e-mail: shah23@purdue.edu (Kavita Shah)

prepared by replacing alkenyl bond of Combrastatin with
isoxazoline moiety.10 With the broad spectrum activities observed
for indole derivatives and isoxazolines, we envisioned linking
two heterocyclic units in single molecule that could lead to
compound with improved efficacy and selectivity.
purity of the synthesized compounds 6a-m was found to be
greater than 97%.
Synthesized indolylisoxazolines 6a-m were screened for in vitro
cytotoxicity against six human cancer cell lines: castration-
resistant prostate cancer cell lines (22Rv1 & C4-2), PTEN-/-
prostate cancer cell line (PC3), human embryonic kidney cell line
(HEK293), pancreatic cancer cell line (BxPC3) and triple
negative breast cancer cell line (MDA-MB-231) using the MTT
assay. The IC50 values (in μM) were used to determine the
growth inhibition in the presence of indolylisoxazolines 6a–m
against the tested cancer cell lines and Doxorubicin was used as a
positive control. In our previous report, 3,4,5-trimethoxyphenyl
and 4-pyridyl bearing chalcones were found to be potent against
a panel of cancer cells.18,19 In light of favourable effects observed
with these derivatives on antitumor activity, isoxazolines 6a-i
with similar structural motifs were prepared.18 Compound 6a
without any substituent on indole ring was found to be
moderately active against the tested cancer cell lines. Next,
introduction of a methoxy group at position-5 of indole ring
resulted in analogue 6b with reduced activity. Interestingly, by
shifting the methoxy group from position-5 to position-6 in
indole ring led to 6c (IC50 = 3.5-16.5 μM) with substantial
improvement in cytotoxicity. Replacement of the methoxy group
with a fluorine atom was tolerated (MeO: 6c IC50 = 3.5–16 μM vs
F: 6d IC50 = 3.66–5 μM). Insertion of a methyl group at position-
2 of indole ring (compound 6e) displayed moderate to good
cytotoxicity. Recently, chlorobenzylindole and pyridyl motifs
have been reported to be beneficial for the cytotoxicity.4 By
incorporating these important structural units, we prepared
isoxazolines 6f-i with cytotoxicity in micromolar range. The
analogue 6i with (4-chlorobenzyl)-2-methylindole and pyridyl
moieties was found to be the most potent compound with IC50 of
2.5 μM against C4-2 and HEK293 cell lines. In subsequent
modifications, we prepared derivatives 6l and 6m possessing
3,4,5-trimethoxyphenyl fragment which is vital in many
anticancer agents, especially in various tubulin binding agents.18
Compound 6l with indole and 3,4,5-trimethoxyphenyl moieties
displayed significant cytotoxicity (IC50 ~ 4.5 μM). Incorporation
of C5-methoxy in indole ring (compound 6m) or alteration of any
methoxy group in trimethoxyphenyl fragment (compounds 6j &
6k) are detrimental for the activity.
Indolylisoxazolines 6a-m were synthesized from the reaction
of appropriate indolylchalcones 9a-m with hydroxylamine hydro-
chloride in ethanol under refluxing conditions as outlined in
Scheme 1. Firstly, substituted indole-3-carboxaldehydes 8 were
achieved from Vilsmeier-Haack formylation of commercially
available indoles, followed by the N-alkylation of 7 with an
appropriate alkyl halide in the presence of sodium hydride. This
afforded substituted indole-3-carboxaldehydes 8 in excellent
yields by incorporating necessary modifications in reported
procedures.18 Next, condensation reaction of aldehyde 8 with
diverse acetophenones under basic conditions led to indolyl
chalcones 9a-m in good to excellent yields. Finally, treatment of
9a-m with hydroxylamine hydrochloride in the presence of a
catalytic amount of anhydrous sodium sulfate in ethanol afforded
indolylisoxazolines 6a-m in 60–80% yields.
Scheme 1. Synthesis of indolylisoxazolines 6a-m
The prepared indolylisoxazolines 6a-m was fully
characterized using IR, NMR (1H & 13C) and mass spectral data.
1
In H NMR spectra of 6a-m revealed the appearance of a triplet
at δ ~ 6.10 ppm (methyne proton of isoxazoline) in addition to
two doublet of doublet at δ ~ 3.82 ppm and 3.65 ppm (methylene
protons of isoxazoline). The 13C NMR spectra of 6a-m exhibited
two characteristic signals at δ ~ 40 ppm and ~ 80 ppm assigned to
methyne and methylene carbons of isoxazoline ring. The HRMS
spectra of isoxazolines 6a-m showed molecular ion peak in
agreement with the calculated mass. Based on HPLC analysis,
Table 1. In vitro anticancer activity studies of indolylisoxazolines 6a-m (IC50 µM)
O
N
3
R
R
Ar
4
2
N
R
R
1
6a-m
Compd
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
R1
H
H
H
H
H
4-ClBz
4-ClBz
4-ClBz
4-ClBz
H
H
H
H
R2
H
H
H
H
CH3
H
H
H
CH3
H
R3
H
OMe
H
H
H
H
OMe
H
H
H
R4
H
H
OMe
F
H
H
H
OMe
H
H
Ar
22Rv1
> 100
35.5
16.32
3.66
17.33
9.6
10.6
17.3
8.33
C4-2
12.75
> 100
3.5
HEK293 BxPC-3 MDAMB-231 PC3
4-pyridyl
4-pyridyl
4-pyridyl
4-pyridyl
4-pyridyl
4-pyridyl
4-pyridyl
4-pyridyl
4-pyridyl
3,4-(OCH2O)C6H3
3,4-(OCH3)2C6H3
3,4,5-(OCH3)3C6H2 4.51
3,4,5-(OCH3)3C6H2 11.33
6.32
> 100
> 100
4.16
9.5
30.5
68.5
9.5
19.66
> 100
7.38
5
> 100
> 100
10.45
12.9
8
9.5
45.1
11.42
> 100
> 100
2.5
15.81
65.5
5
> 100
10.16
14.16
37.16
2.5
> 100
> 100
5
95.5
> 100
> 100
> 100
13.5
27
> 100
13.5
> 100
9.12
26.6
13.66
26
> 100
6.66
26.6
> 100
11.66
26
21.2
25.69
55.21
95.1
11.2
> 100
16.66
> 100
70.14
15.5
42.58
9.58
H
H
H
H
H
OMe
H
H
H
6m
Doxorubicin
71.2
3.25
45.4
3.9
6.25
*The activity data represent mean values of experiments conducted in triplicates at three independent times

Structure–activity relationship studies of the prepared
indolylisoxazolines 6a-m suggested that isoxazolines substituted
Previous studies have shown that compounds bearing indole
scaffolds can induce cell death by increasing reactive oxygen
species (ROS). ROS can include peroxides, superoxides,
hydroxyl radicals and singlet oxygen. ROS are formed as a
natural byproduct of the oxygen metabolism. Therefore, we
measured whether compounds 6c and 6i induce ROS
accumulation using DCFDA staining in C4-2 cells. H2O2-treated
C4-2 cells were used as a positive control. ROS levels were
quantified in untreated and C4-2 cells treated with H2O2 (10 µM),
6c (10 µM) or 6i (10 µM) for 48 h. While H2O2-treated cells
displayed high DCFDA staining, 6c and 6i-treated cells shows
similar DCFDA staining, indicating that these compounds do not
induce apoptosis via ROS upregulation (Fig. 4)
as
6-methoxyindole,
6-fluoroindole,
N-chlorobenzyl-2-
methylindole and indole at position-5, and at position-3 with
pyridyl and trimethoxyphenyl are favourable for the cytotoxicity
(Fig. 2)
Fig. 2. Identified potent indolylisoxazolines
To investigate the mechanism of cell death induced by these
compounds, we conducted various assays. Acridine orange (AO)
stains both live and dead cells, whereas ethidium bromide (EB)
stains only dead cells. Therefore, AO/EB staining was used to
examine whether cell death occur via apoptosis or necrosis.
Effect of indolylisoxazolines on the morphological changes of
C4-2 cells is illustrated in Fig 3A. Incubation of compounds 6i
(IC50 = 2.5μM) and 6c with C4-2 cells (IC50 = 3.5 μM) for 48 h
resulted in typical nuclear fragmentation (red), whereas no visible
changes in cell nucleus and cell integrity was observed for the
control cells. The results of AO/EB staining revealed the
compounds 6c and 6i triggered apoptosis in C4-2 cells.20
Fig. 4. Quantification of ROS levels induced by 6c and 6i
PARP is a eukaryotic DNA-binding protein that specifically
recognizes the single stranded breaks formed during DNA
damage. Once PARP detects the single stranded breaks it binds
to the DNA, undergoes the structural change and begins the
synthesis of a polymeric adenosine diphosphate ribose (poly
(ADP – ribose) or PAR) chain which acts as a signal for the other
DNA-repairing enzymes.14 PARP1 cleavage assay (Fig. 3B)
shows that indolylisoxazolines 6c and 6i induced apoptosis in
C4-2 cells. C4-2 cells were treated with compounds 6c and 6i and
level of cleaved PARP was observed. The results revealing the
possible occurrence of DNA damage and apoptosis.10
In solid cancers, metastasis is responsible for 90% of
mortality. Therefore, we investigated whether these compounds
inhibit cell migration. Wound healing assay is a classical and
commonly used method for studying cell migration, which
mimics wound healing in vivo.23 In this assay, a scratch is
introduced in a monolayer of cells and then images are taken at
the beginning and at regular intervals to access cell migration
rates. After cells were wounded, they were further incubated for
12h, 24h and 48h. The cells were fixed and images were
captured, which showed that both compounds 6c and 6i potently
inhibit C4-2 cell migration (Fig. 5). These results indicate that
compounds 6c and 6i have the potential to serve as effective anti-
cancer agents.
DNA fragmentation is a key feature of apoptosis, a type of
programmed cell death. Apoptosis is characterized by the
activation of caspase-3 activated DNase (CAD).20 CAD is
normally inhibited by the protein, inhibitor of Caspase Activated
DNase (ICAD). During apoptosis, the apoptotic effector caspase,
caspase-3, cleaves ICAD and causes CAD to become activated.21
Thus, we conducted DNA ladder assay22 to verify that the cell
death triggered by compounds 6c and 6i was indeed apoptosis.
As shown in Fig. 3C, treatment of C4-2 cells with compounds 6c
and 6i at 10 µM concentration for 48 h, triggered the formation
of DNA ladder, which confirms that these compounds induce
apoptosis.
Fig. 5. Inhibition of cell migration on C4-2 cells effected by 6c and 6i
In summary, we have prepared indolylisoxazolines and some
of them showed high potency in inhibiting the growth of 22Rv1,
C4-2, PC3, HEK293, BxPC-3, and MDA-MB-231 cancer cell
lines at micromolar concentrations. Compounds 6c-d, 6i and 6l
were significantly cytotoxic against all six cancer cell lines with
selectivity towards C4-2 and HEK293 cells. Importantly, two of
the tested compounds 6c and 6i also exhibited strong anti-cell
motility property in C4-2 cells. The preliminary anticancer
activity studies of indolylisoxazolines revealed that CH3 group at
second position of the indole ring, protection of indole nitrogen
with chlorobenzyl group and insertion of OMe group at sixth
position of indole ring are beneficial for anticancer activity and
selectivity. Identified indolylisoxazolines are potent anticancer
agents and could be explored for specific biological effects at
molecular level.
Fig. 3. Apoptosis inducing effects of 6c and 6i in C4-2 cells.

Acknowledgments
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Authors are highly thankful to Council of Scientific &
Industrial Research (CSIR, No. 02(0239)/17/EMR-II), New
Delhi, India for financial support. KS would like to thank
National Institute of Health (R03 CA 166912) for financial
support. Also, we would like to acknowledge BITS Pilani,
Rajasthan and DST-FIST, New Delhi for NMR and HRMS
facility.
12. Kamal A, Bharathi E V, Reddy J S, Ramaiah M J, Dastagiri D, Reddy
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Graphical Abstract
Synthesis and anticancer activity studies of
indolylisoxazoline analogues
Leave this area blank for abstract info.
Chaitanya MVSKa, Venkataramana Reddy P O a, Kumar Nikhilb, Anil Kumar,a Kavita Shah*b
and Dalip Kumar*a

Highlights
 A series of indolylisoxazolines were prepared
and assessed for their cytotoxicity.
 Isoxazoline derivatives 6c-d, 6i and 6l
displayed potent cytotoxicity (IC50 = 2.5-5.0
µM).
 Isoxazolines 6c and 6i induced apoptosis and
inhibited C4-2 cell migration.