Spirooxindole-derived morpholine-fused-1,2,3-triazoles: Design, synthesis, cytotoxicity and apoptosis inducing studies

Article abstract of DOI:10.1016/j.ejmech.2015.08.017

A series of new spirooxindole-derived morpholine-fused-1,2,3-triazole derivatives has been synthesized from isatin spiro-epoxides. The protocol involves regiospecific isatin-epoxide ring opening with azide nucleophile followed by sequential O-propargylation, and intramolecular 1,3-dipolar cycloaddition reaction. These compounds have been evaluated for their antiproliferative activity against selected human tumor cell lines of lung (A549), breast (MCF-7), cervical (HeLa), and prostate (DU-145). Among the tested compounds, 6i, 6n and 6p showed potent growth inhibition against A549 cell line with IC₅₀ values in the range of 1.87-4.36 μM, which are comparable to reference standards doxorubicin and 5-flourouracil. The compounds 6i and 6p treated A549 cells displayed typical apoptotic morphological features such as cell shrinkage, nuclear condensation, fragmentation, and decreased migration potential. Flow-cytometry analysis revealed that the compounds arrested the cells in G2/M phase of cell cycle. Hoechst and acridine orange/ethidium bromide staining studies also showed that the cell proliferation was inhibited through induction of apoptosis. Moreover, the compounds treatment led to collapse of the mitochondrial membrane potential (DΨm) and increased levels of reactive oxygen species (ROS) were noted in A549 cells.

Full text of DOI:10.1016/j.ejmech.2015.08.017

Accepted Manuscript
Spirooxindole-derived morpholine-fused-1,2,3-triazoles: Design, synthesis,
cytotoxicity and apoptosis inducing studies
Kishna Ram Senwar, Pankaj Sharma, T.Srinivasa Reddy, Manish Kumar Jeengar,
V.Lakshma Nayak, V.G.M. Naidu, Ahmed Kamal, Dr. Nagula Shankaraiah
PII:
S0223-5234(15)30201-4
10.1016/j.ejmech.2015.08.017
EJMECH 8053
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 June 2015
Revised Date: 4 August 2015
Accepted Date: 7 August 2015
Please cite this article as: K.R. Senwar, P. Sharma, T.S. Reddy, M.K. Jeengar, V.L. Nayak, V.G.M.
Naidu, A. Kamal, N. Shankaraiah, Spirooxindole-derived morpholine-fused-1,2,3-triazoles: Design,
synthesis, cytotoxicity and apoptosis inducing studies, European Journal of Medicinal Chemistry (2015),
doi: 10.1016/j.ejmech.2015.08.017.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
Spirooxindole-derived morpholine-fused-1,2,3-triazoles: Design, synthesis,
cytotoxicity and apoptosis inducing studies
a
a
b
c
Kishna Ram Senwar, Pankaj Sharma, T. Srinivasa Reddy, Manish Kumar Jeengar, V.
b
c
b,
a
Lakshma Nayak, V. G. M. Naidu, Ahmed Kamal, Nagula Shankaraiah *
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research
(NIPER) Hyderabad 500 037, India
b
Medicinal Chemistry & Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad
5
00 007, India
c
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and
Research (NIPER) Hyderabad 500 037, India

ACCEPTED MANUSCRIPT
Spirooxindole-derived morpholine-fused-1,2,3-triazoles: Design, synthesis,
cytotoxicity and apoptosis inducing studies
a
a
b
c
Kishna Ram Senwar, Pankaj Sharma, T. Srinivasa Reddy, Manish Kumar Jeengar, V.
b
c
b
a
Lakshma Nayak, V. G. M. Naidu, Ahmed Kamal, Nagula Shankaraiah *
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research
(
NIPER) Hyderabad 500 037, India
b
Medicinal Chemistry & Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500
0
07, India
c
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and
Research (NIPER) Hyderabad 500 037, India
*Corresponding author
Dr. Nagula Shankaraiah
Department of Medicinal Chemistry
National Institute of Pharmaceutical Education and Research
Hyderabad, 500037, India
Tel.: +91 40 23074750
Fax: (+91) 040 23073751
E-mail: shankar@niperhyd.ac.in
1

ACCEPTED MANUSCRIPT
Abstract
A series of new spirooxindole-derived morpholine-fused-1,2,3-triazole derivatives has been
synthesized from isatin spiro-epoxides. The protocol involves regiospecific isatin-epoxide ring
opening with azide nucleophile followed by sequential O-propargylation, and intramolecular 1,3-
dipolar cycloaddition reaction. These compounds have been evaluated for their antiproliferative
activity against selected human tumor cell lines of lung (A549), breast (MCF–7), cervical
(HeLa), and prostate (DU–145). Among the tested compounds, 6i, 6n and 6p showed potent
growth inhibition against A549 cell line with IC values in the range of 1.87–4.36 µM, which
5
0
are comparable to reference standards doxorubicin and 5-flourouracil. The compounds 6i and 6p
treated A549 cells displayed typical apoptotic morphological features such as cell shrinkage,
nuclear condensation, fragmentation, and decreased migration potential. Flow-cytometry
analysis revealed that the compounds arrested the cells in G2/M phase of cell cycle. Hoechst and
acridine orange/ethidium bromide staining studies also showed that the cell proliferation was
inhibited through induction of apoptosis. Moreover, the compounds treatment led to collapse of
the mitochondrial membrane potential (DΨm) and increased levels of reactive oxygen species
(ROS) were noted in A549 cells.
Key words: Spirooxindole, morpholine-fused-1,2,3-triazole, epoxide, 1,3-dipolar cycloaddition,
anticancer, apoptosis.
2

ACCEPTED MANUSCRIPT
1
.0. Introduction
Cancer remains a challenging problem and treatment usually involves a combination of
chemotherapy, surgery and radiotherapy. Chemotherapy is considered to be one of the effective
approaches in suppressing tumor growth and eradication of tumors [1,2]. The development of
resistance to chemotherapeutic agents and associated side effects are major obstacle to the
effective treatment of cancer [3]. In spite of recent advances in understanding the etiology of
cancer and a greater emphasis placed on early detection of disease, the overall mortality rate has
not been changed and is not expected to diminish. Therefore, it is necessary to identify and
develop new anti-cancer agents with improved efficacy and reduced side effects to complement
the present chemotherapeutic strategies. Hence, further research regarding the development of
new therapeutic drugs that more effectively combat cancer is needed.
The spirooxindole core is a kinase-privileged scaffold which has emerged as an attractive
synthetic target because of occurrence in numerous natural products and biologically active
molecules [4,5]. In particular, the spirooxindole alkaloids are inspiration for the development of
potential therapeutic agents [6,7]. Among these motifs, the synthesized spirooxindoles such as
MI-888 have been in preclinical research for the treatment of human cancers [8]. Specifically,
the spiro ring systems have been successfully incorporated into some of kinase (e.g. c-Met/ALK,
PLK4) inhibitors [9–11]. The key structural characteristic of these compounds is the spiro ring
fused at the C3-position of the oxindole core with varied heterocyclic motifs. The prevalence of
these structures has resulted in the production of diverse libraries of small molecules for in vitro
biological evaluation. As such, these spirooxindoles seem to be promising candidates for drug
discovery due to their inherent three-dimensionality and structural novelty [12]. Furthermore, the
spiro ring fusion tends to impose the conformational restriction on the oxindole scaffold [13].
3

ACCEPTED MANUSCRIPT
Additionally, these spirocyclic compounds have reduced conformational entropies upon binding
to a protein targets. As an outcome, there is a huge interest in both industry and academia to
develop novel spirooxindoles with interesting biological activities, particularly anticancer
potential.
On the other hand, the morpholine moiety has been found to be an excellent pharmacophore
in medicinal chemistry and a number of molecules possessing morpholine skeleton are the
clinically approved drugs [14]. It does form critical hydrogen bond with the target proteins. For
instance, the chemical inhibitors of most of the kinases usually carry analogous structural
heterocyclic motifs. The most common feature of these inhibitors is a critical hydrogen bond of
heterocyclic motifs to target receptors [15]. These phenomena served as a valuable chemical tool
for the development of new chemotherapeutic agents for cancer treatment. Likewise, the 1,2,3-
triazole moiety is also an attractive pharmacophoric unit because it is stable to metabolic
degradation and readily associate with several biological targets, through hydrogen-bonding and
dipole interactions [16–20]. Moreover, it does not protonate at physiological pH because of its
poor ba sicity, while thus, improves the solubility. The 1,2,3-triazole moiety is the part of well-
known drugs including β-lactum antibiotic (i.e., tazobactum) and the cephalosporine (i.e.,
cefatrizine).
In the drug discovery process, incorporation of medicinally active moieties into a core
bioactive natural product provides the means for accessing wider range of pharmacological
profiles. Despite the tremendous biological significance of spirooxindole (Fig. 1), morpholine
and 1,2,3-triazole derivatives, their fused-derivatives are not explored. Inspired by the synthetic
feasibility and biological profiles of spirooxindoles, it would thus be considering worthwhile to
fuse these moieties as a single molecular entity to evaluate their anticancer properties. The
4

ACCEPTED MANUSCRIPT
incorporation of a morpholine and 1,2,3-triazole moiety in oxindole scaffold might unleash the
potential to access a new dimension of structural diversity to the molecules via intramolecular
1
,3-dipolar cycloaddition reaction. We herein report the study of morpholine-spirooxindoles
containing an additional five-membered morpholine-fused 1,2,3-traizole ring. To the best of our
knowledge, this is the first report for the synthesis of spirooxindole-derived morpholine-fused-
1
,2,3-triazole derivatives. Hence, we have synthesized a library of novel spirooxindole-derived
morpholine-fused-1,2,3-triazoles containing different substituent’s around the oxindole moiety
and evaluated for their in vitro anticancer activity on selected human cancer cell lines.
<
insert Figure 1 here>
2
.0. Results and discussion
.1. Chemistry
2
The
tetracyclic
spirooxindole-derived
morpholine-fused-1,2,3-triazoles
derivatives)
(4,7-
were
dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
synthesized from isatin-epoxides via epoxide-ring opening with azide nucleophile followed by
sequential O-propargylation, and intramolecular 1,3-dipolar cycloaddition reaction. The isatin-
epoxides 4a–t were synthesized from the corresponding N-alkylated-isatins by treatment with
trimethylsulphoxonium ylide as illustrated in Scheme 1. Isatins were directly synthesized from
anilines by the Sandmeyer reaction with chloral hydrate under acidic conditions in a polar
medium [21–23]. Further, they were N-alkylated with alkyl iodide in the presence of NaH.
In particular, our strategy involved the regiospecific ring-opening of isatin-epoxides 4a–t with
sodium azide in water as reaction media, to furnish the corresponding oxindole-azido alcohols
5
a–t. The epoxide ring was opened from the less hindered side, affording the oxindole-azido
5

ACCEPTED MANUSCRIPT
alcohols regiospecifically. The azido alcohols 5a–t were then further utilized for the synthesis of
diverse range of spirooxindole-derived morpholine-fused-1,2,3-triazoles. Specifically, when
these oxindole-azido alcohols were subjected to O-propargylation with propargyl bromide
followed by intramolecular 1,3-dipolar cycloaddition achieved a variety of substituted
spirooxindole-derived morpholine-fused-1,2,3-triazole products 6a–t in good to excellent yields.
In this protocol, the tailor-made oxindole-derived azido-alkyne intermediates possessing alkyne
and azide moieties disposed in the same molecule when heated undergo intramolecular Huisgen
1
,3-dipolar cycloaddition. Therefore, without isolating these intermediates by column
chromatography, the crude reaction mixture were subjected to thermal cycloaddition (toluene,
o
1
10 C, 0.5˗2 h) to afford the tetracyclic spirooxindole-derived morpholine-fused-1,2,3-triazole
as title compounds 6a–t. Gratifyingly, isatin-epoxides with electron-rich and electron-poor
substituents underwent the desired reaction in good to excellent yields. As shown in Scheme 1,
there was a wide tolerance for several isatins varying in C5 and N-substitution around oxindole
core.
<
insert Scheme 1 here>
Herein, we were able to synthesize the oxindole-azido alcohols regiospecifically from isatin-
epoxides, therefore we have explored the conversion of these oxindole-azido alcohols into
tetracyclic[1,2,3]triazolo[1,5-d][1,4]oxazines. The intramolecular azide-alkyne cycloaddition
was carried out under mild reaction conditions in the absence of copper due to favourable
entropy effects on the corresponding transition state. The 1,5-disubstituted triazole moieties are
easy to access by simple azide-alkyne thermal cycloaddition, since the formation of 1,4-
regioisomer is less favorable in this case due to strain induced in the ring.
6

ACCEPTED MANUSCRIPT
Due to steric strain, the presence of a spiro carbon atom in the isatin-epoxides induces easy
rearrangements that can lead to different spirocyclic compounds. Under such a complex scenario,
isatins constitute perhaps the single class of heterocyclic compounds which has been employed
so extensively, either directly or via 3-substituted 2-oxindoles, in design and synthesis of spiro-
cyclic frameworks, both carbocyclic and heterocyclic, of diverse types. Introduction of
conformational restriction by spiro ring formation, can not only modulate binding potency and
specificity, but also potentially improve bioavailability and metabolic stability. However, a
centrally situated spiro ring significantly rigidifies the spirooxindole. The presence of a spiro ring
fusion tends to impose conformational restriction on a molecule resulting in reducing off-target
activities of the spirooxindoles.
The tetracyclic compounds 6a–t represent a combination of oxindole, morpholine, and 1,2,3-
1
13
triazole whose structures were deduced from H, C NMR, FT–IR and HRMS (ESI) analysis.
1
13
1
The NMR spectra ( H and C) are consistent with the planned structures. The H NMR spectrum
of 1'-methyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one, 6a showed
a sharp singlet at δ 7.64 near the aromatic region, typical for the 1,2,3-triazole hydrogen. In the
1
3
C NMR spectrum of 6a, the carbonyl carbon appeared at δ 171.6. The aromatic carbons were
observed in the range of δ 143.9–109.4. The signal attributed to the spiro C appeared at δ 73.6,
whereas other aliphatic carbon signals occurred in the range of δ 59.4–26.4. Almost similar
1
13
patterns were observed in H NMR and C NMR spectra of all synthesized compounds of this
series. Moreover, the absence of the azide peak in the FT–IR spectrum confirmed the structure
-1
assigned to it. In addition, the sharp band at 2229 cm confirmed (compounds 6q and 6r) the
-1
presence of cyano functionality. The stretching bands at ~1707 cm corresponds to the amide
7

ACCEPTED MANUSCRIPT
+
carbonyl group of oxindole moiety. The HRMS (ESI) of all the compounds showed an [M + H]
peak equivalent to their molecular formula.
2
2
.2. Biological Evaluation
.2.1. In vitro cell growth inhibitory activity
All the synthesized compounds 6a–t were evaluated for their in vitro antiproliferative activity
against selected human cancer cell lines of breast adenocarcinoma (MCF-7), lung
adenocarcinoma (A549), cervical carcinoma (HeLa) and prostate cancer (DU-145) cells using 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Doxorubicin and 5-
fluorouracil were used as reference standards and the screening results were summarized in
Table 1. The results indicated that the compounds 6i, 6n, 6p, 6r and 6s exhibited significant cell
growth inhibition with IC50 <10 µM which are comparable to reference standard 5-fluorouracil
against A549 and MCF-7 cells. Specifically, compounds 6i, 6n and 6p displayed potent
cytotoxicity against A549 cell line with IC50 values of 1.87–4.26 µM. Interestingly, the
compound 6i was the most active compound in the series which exhibited potent growth
inhibition on A549 cell line with IC of 1.87 µM, whereas the reference standards doxorubicin
5
0
and 5-fluorouracil displayed IC of 1.99 and 7.24 µM, respectively.
5
0
Structural activity relationship (SAR) studies for these compounds demonstrated that with N-
methyl substituent’s containing electron-withdrawing groups such as fluoro (6b), chloro (6c) and
th
bromo (6d) on the 5 -position of oxindole exhibited minimal activity compared to electron
donating groups. For instance, compound 6s having N-methyl and 5-methyl substituent showed
remarkable cytotoxicity against MCF-7, A549 and HeLa cancer cell lines with IC <10 µM.
5
0
Among N-alkyl substituted derivatives, increasing trend of activity was observed when increase
8

ACCEPTED MANUSCRIPT
the carbon chain length from N-methyl 6a–d to N-ethyl derivative 6i–l, may be due to the
increase in the lipophilicity. Compounds with the N-benzyl substituent’s, the order of inhibitory
activity against A549 and MCF-7 cells depends on the substituent on the aromatic ring of the
oxindole and varies in the order H > Cl > Br > F (6e–h). The presence of the N-(p-bromo)-benzyl
substituent’s 6m–p, on the oxindole core leads to increase the anticancer activity in comparison
with N-benzyl substituent’s 6e–h. Among N-(p-bromo)-benzyl substituted derivatives; the
compound 6p with 5-bromo substitution is most potent against all tested cancer cell lines. The
compound 6r with N-(p-cyano)-benzyl substituent is also equally potent as the N-(p-Br)-benzyl
substituent counterparts. Based on the promising cytotoxic activity, two most active compounds
from this series were 6i, and 6p taken-up for further mechanism of cell growth inhibition studies.
<
insert Table 1 here>
2
.2.2. Morphology
To examine whether the treatment with these compounds could lead to loss of cell viability and
induction of apoptosis, A549 cells were treated with the compounds 6i and 6p. Cells were
observed and photographs were taken with phase contrast microscope. It can be inferred from the
Figure 2 that the treatment with compounds results in reduced number of viable cells in
comparison to the vehicle treated control cells as observed by the distinctive morphological
features such as cells detachment and shrinkage of cell nucleus. This indicates that the
compounds induce marked morphological changes in A549 cells.
<
insert Figure 2 here>
2
.2.3. In vitro cell migration assay
Migration of cancer cell is a key feature of tumour progression and metastatic cascade [24].
Herein, we investigated the effect of these two compounds 6i and 6p on migration potential of
9

ACCEPTED MANUSCRIPT
human non-small cell lung cancer A549 cells by using in vitro wound healing assay. Wounds
were created in the confluent monolayer A549 cells using 200 µL sterile pipette tip and images
were captured at 0, 24 and 48 h after the treatment with the compounds. The results from Figure
3
demonstrated that the compounds 6i and 6p effectively suppressed the migration of lung cancer
cells in to the wound area in contrast to the vehicle (DMSO) treated cells. The migration ability
of the A549 cells was significantly reduced in both the compounds treated plates 6i and 6p (38.1
to 55.3 %). Interestingly, it was more prominent with the former compound which inhibits 54.9
and 56.1 % migration of A549 cells after 24 and 48 h, respectively.
<
insert Figure 3 here>
2
.2.4. Flow-cytometry analysis
The blockade of cell cycle progress by anti-cancer agents prevents the proliferation of cancer
cells, which is exploited for cancer chemotherapy. In vitro screening results revealed that the
compound 6i showed significant activity against A549 cells. Therefore, it was our interest to
understand whether this inhibition of A549 cells growth on account of cell cycle arrest. A549
cells were treated with compound 6i at concentration of 1 and 2 µM for 24 h, and stained with
propidium iodide, which was further analyzed by using Muse analyzer. The results from the
Figure 4 indicated that the control untreated cells exposed to DMSO showed 21.8% cells in
G2/M phase, whereas compound 6i treatment resulted in increased G2/M population to 35.6 % in
2
4 h. Moreover, the percentage of cells in S phases was 15.6 and 16.2% with 1 and 2 µM of the
compound respectively in comparison to the control cells. These results were clearly indicated
that treatment of A549 cells with the compound 6i resulted in G2/M cell cycle arrest.
<insert Figure 4 here>
1
0

ACCEPTED MANUSCRIPT
2
.2.5. Apoptosis detection studies
Apoptosis is one of the major pathways that lead to the process of cell death. Therefore, it was
considered of interest to investigate the apoptosis inducing effect of these compounds on A549
cells by the Hoechst nuclear staining and acridine orange/ethidium bromide (AO/EB) method.
2
.2.5.1. Hoechst staining
Chromatin condensation and fragmented nuclei are known as the classic characteristics of
apoptosis [25]. Therefore, we examined the apoptosis inducing ability of these 6i and 6p on
A549 cells using Hoechst nuclear staining. Hoechst 33242 is a cell permeable nuclear staining
dye, which emits blue fluorescence and stains the nucleus of the live cells as light blue, whereas
the apoptotic cell nuclei appear as bright blue due to chromatin condensation. A549 cells were
stained with Hoechst 33242 following the treatment with the compounds 6i and 6p and cells
were observed for nuclear morphological changes under confocal fluorescence microscope. The
results from Figure 5 demonstrated that the nuclear structure of untreated cells was intact
whereas compound 6i and 6p treated cells exhibited fragmented nuclei (white arrow), chromatin
condensation, pyknotic and horseshoe shaped nuclei which are the typical characteristics of
apoptosis.
<
insert Figure 5 here>
2
.2.5.2. Acridine orange/ethidium bromide (AO/EB) staining
The morphological abnormalities induced by the most active compound 6i in lung
adenocarcinoma A549 cells were further studied using acridine orange/ethidium bromide
(AO/EB) staining technique to understand whether the inhibition is due to apoptotic induction or
1
1

ACCEPTED MANUSCRIPT
nonspecific necrosis. Acridine orange permeates the intact cell membrane and stains the nuclei
green, whereas ethidium bromide is taken up only when the membrane integrity is deteriorated,
and stains the nucleus red. The compound 6i treated or untreated A549 cells were stained with
acridine orange and ethidium bromide and analyzed under fluorescence microscope. The early
signs of apoptosis characterized by extremely condensed chromatin and cell membrane blebbing
were observed at 0.5 µM concentration of compound 6i treated lung adenocarcinoma A549 cells,
whereas destructive fragmentation of the nuclei and irregular distribution of chromatin, which
marginated into a horseshoe-shaped structure were observed at 1 µM concentration. The late
apoptotic cells exhibited condensed chromatin and their nuclei, stained red with ethidium
bromide at 2 µM concentration, whereas these features were absent in the control lung
adenocarcinoma A549 cells.
<
insert Figure 6 here>
2
.2.5.3. Effect of compounds 6i and 6p on mitochondrial membrane potential (DΨm)
The maintenance of mitochondrial membrane potential (DΨm) is significant for mitochondrial
integrity and bioenergetic functions. Previous reports have suggested that collapse of
mitochondrial membrane potential has been correlated to the early events that occur during the
apoptotic process [26]. In order to further investigate the apoptotic inducing ability of the
compounds 6i and 6p, the effect on mitochondrial membrane potential was determined by using
spectroflourometer with rhodamine 123, a cell permeable cationic dye that emits green
fluorescence. The uptake rhodamine 123 into the mitochondria depends on mitochondrial
membrane potential (DΨm). Cells with intact DΨm retain rhodamine and disruption of this
mitochondrial membrane potential result in loss of rhodamine from the mitochondria and a
decrease in intracellular green fluorescence. Spectroflourimetric analysis of A549 cells after
1
2

ACCEPTED MANUSCRIPT
treatment with compounds 6i and 6p revealed that both the compounds significantly disrupted
the mitochondrial membrane potential in comparison to the untreated control cells (Fig. 7).
Among these, the compound 6i is more potent in inducing mitochondrial depolarization (48.2%).
These results suggest that the compounds induce apoptosis through collapse of mitochondrial
membrane potential in A549 cells.
<
insert Figure 7 here>
2
.2.5.4. Effect of compounds 6i and 6p on intracellular Reactive Oxygen Species (ROS)
Elevation of intracellular reactive oxygen species (ROS) levels is known to induce apoptosis by
affecting the mitochondrial membrane potential and membrane permeability [27]. To examine,
whether the collapse of mitochondrial membrane potential was due to the elevation of ROS
levels, we investigated the effect of these compounds on intracellular levels of ROS using a
peroxide-sensitive fluorescent dye, 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) in
A549 cells. DCFH-DA is a non-fluorescent cell permeable dye which upon oxidation by
intracellular esterase’s in the cytosol converts in to non-diffusible green fluorescent DCF. Thus
intracellular ROS levels can be expressed as mean fluorescence intensity of DCF. As shown in
Figure 8, the levels of intracellular ROS were higher in both the compound treated cells in
comparison to the control cells as observed by the increased green fluorescence. The results from
the quantitative analysis revealed that the compounds 6i and 6p induce elevation of ROS levels
by 2.6 and 2.3 times higher in comparison to the control. These results clearly demonstrate the
role of ROS generation in the apoptosis induction by these compounds.
<
insert Figure 8 here>
1
3

ACCEPTED MANUSCRIPT
3
.0. Conclusion:
In conclusion, we have developed a novel and concise method for the synthesis of spirooxindole-
derived morpholine-fused-1,2,3-triazole derivatives. The protocol involves regiospecific ring-
opening of isatin spiro-epoxides by azide nucleophile followed by sequential O-propargylation,
and intramolecular 1,3-dipolar cycloaddition reaction with good yields. These novel compounds
were evaluated for their antiproliferative activities on different human cancer cell lines. Among
them, the compounds 6i and 6p displayed potent growth inhibition on A549 cells. The exposure
of lung A549 cancer cells to the compounds 6i and 6p resulted in remarkable inhibition of in
vitro cell migration. Flow cytometry analysis indicated that the compound 6i arrested the cells at
G2/M phase of the cell cycle. The apoptosis inducing effect of these compounds was analyzed by
Hoechst nuclear staining and AO/EB staining. Moreover, the compounds 6i and 6p induces
apoptosis in A549 cells through collapse of mitochondrial membrane potential and elevation of
intracellular reactive oxygen species levels. Overall, the results demonstrate that these
compounds have the potential to be developed as leads and their further structural modifications
may produce promising anticancer agents for lung adenocarcinoma A549 cancer cells.
4
4
.0. Materials & Methods
.1. Chemistry
All the starting materials and other reagents were commercially available of the best grade and
were used without further purification. The progress of the reactions were monitored by thin
layer chromatography (TLC), performed on silica gel 60-F254 plates. Spots were visualized by
UV light. All melting points were taken and are uncorrected. FT–IR spectra for all the
1
13
compounds were recorded by using ATR method. H and C NMR spectra were recorded on
1
4

ACCEPTED MANUSCRIPT
3
00, and 500 MHz spectrometers using tetramethylsilane as the internal standard. Chemical
shifts are reported in parts per million (ppm) downfield from tetramethylsilane. Spin
multiplicities are described as s (singlet), brs (broad singlet), d (doublet), dd (double doublet), t
(triplet), q (quartet), or m (multiplet). Coupling constants are reported in hertz (Hz). HRMS
analyses were acquired on single quadruple and carried out in the ESI techniques at 70 eV.
Wherever required column chromatography was performed using silica gel of 60-120 µm with
hexane and ethyl acetate as eluent.
4
.1.1. General procedure for azidolysis of isatin spiro-epoxides in water (5)
A mixture of isatin-epoxide (1.0 mmol), sodium azide (1.1 mmol) and distilled water (5 mL) was
stirred at room temperature until complete conversion of the epoxide was observed by TLC. The
reaction mixture was extracted with EtOAc (3 X 10 mL) and the combined organic extracts were
2 4
washed with brine (10 mL). The organic layer was dried over anhydrous Na SO and filtered and
the solvent removed in vacuo to obtain the crude product which was used in the next step
without any further purification.
4
.1.2. General procedure for the synthesis of spirooxindole-derived morpholine-fused-1,2,3-
triazole derivatives (6a–t)
To a solution of azido alcohol (1.0 mmol) in anhydrous DMF (5 mL) was cooled to 0 ºC and
sodium hydride (1.5 mmol) was added. The reaction mixture was stirred at 0 ºC under nitrogen
atmosphere for 20 minutes. Then at same temperature, propargyl bromide (1.2 mmol) was added
and reaction mixture was further stirred at room temperature until the complete consumption of
azido alcohol (monitored by TLC). After disappearance of starting materials, reaction was
quenched by cold water and the solvent was removed under reduced pressure, extracted with
1
5

ACCEPTED MANUSCRIPT
ethyl acetate (3 × 10 mL) and water (10 mL). The combined organic layer was washed with brine
solution (10 mL), dried over anhydrous Na SO , filtered, concentrated under vacuum to afford
2
4
o
the crude residue. To the resulting residue, toluene was added and refluxed at 110 C (0.5–2 h) to
obtain the desired product. Evaporation of the toluene gave a crude product which was further
purified by column chromatography on silica gel (hexane/ethyl acetate as eluents) to obtain the
morpholine-fused-1,2,3-triazolo-spiooxindole derivatives 6a–t as title compounds in good to
excellent yield.
1
'-Methyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one (6a)
o
–1
1
White solid, yield 89%; Mp: 170–171 C; IR (ATR): 2985, 1708, 1613, 1024, 752 cm ; H
NMR (300 MHz, CDCl ): δ 3.24 (s, 3H), 4.58 (d, J = 13.6 Hz, 1H), 4.71 (d, J = 13.6 Hz, 1H),
3
5
1
.16 (d, J = 15.1 Hz, 1H), 5.49 (d, J = 15.1 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 7.00 (d, J = 7.5 Hz,
1
3
3
H), 7.09 (t, J = 7.5 Hz, 1H), 7.44 (t, J = 8.3 Hz, 1H), 7.64 (s, 1H); C NMR (75 MHz, CDCl ):
δ 26.4, 49.4, 59.4, 73.6, 109.4, 123.6, 124.4, 125.0, 128.1, 130.0, 131.5, 143.9, 171.6; HRMS
+
(
ESI): m/z calcd. for C H O N , 257.1033, found 257.1029 [M+H] .
1
3
13
2
4
5
'-Fluoro-1'-methyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6b).
o
–1 1
White solid, yield 87%; Mp: 174–175 C; IR (ATR): 2985, 1722, 1624, 1474, 865, 818 cm ; H
NMR (300 MHz, CDCl ): δ 3.23 (s, 3H), 4.56 (d, J = 13.6 Hz, 1H), 4.71 (d, J = 13.6 Hz, 1H),
3
5
4
4
.17 (d, J = 15.5 Hz, 1H), 5.51 (d, J = 15.5 Hz, 1H), 6.79 (dd, J = 2.6, 7.3 Hz, 1H), 6.88 (dd, J =
13
.1, 8.7 Hz, 1H), 7.16 (dt, J = 2.4, 8.7 Hz, 1H), 7.65 (s, 1H); C NMR (75 MHz, CDCl ): δ 26.4,
3
9.1, 59.4, 73.5, 110.1 (d, JCF = 7.7 Hz), 112.8 (d, JCF = 25.2 Hz), 117.8 (d, JCF = 23.1 Hz),
1
6

ACCEPTED MANUSCRIPT
1
26.3 (d, JCF = 7.7 Hz), 128.1, 129.7, 139.7, 159.3 (d, JCF = 244.2 Hz), 171.3; HRMS (ESI): m/z
+
calcd. for C H O N F, 275.0938, found 275.0936 [M+H] .
1
3
12
2
4
5
'-Chloro-1'-methyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6c)
o
–1 1
White solid, yield 87%; Mp: 171–172 C; IR (ATR): 1709, 1614, 1493, 822, 542 cm ; H NMR
300 MHz, CDCl ): δ 3.22 (s, 3H), 4.57 (d, J = 13.6 Hz, 1H), 4.71 (d, J = 13.6 Hz, 1H), 5.17 (d,
J = 15.5 Hz, 1H), 5.50 (d, J = 15.3 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 1.9 Hz, 1H),
(
3
1
3
7
1
.43 (dd, J = 2.1, 8.5 Hz, 1H), 7.65 (s, 1H); C NMR (75 MHz, CDCl ): δ 26.6, 49.2, 59.5, 73.5,
3
10.4, 125.0, 126.8, 128.3, 129.8, 131.5, 134.4, 142.4, 171.2; HRMS (ESI): m/z calcd. for
+
C H O N Cl, 291.0643, found 291.0640 [M+H] .
13
12
2
4
5
'-Bromo-1'-methyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6d)
o
–1
Light brown solid, yield 86%; Mp: 178–179 C; IR (ATR): 1723, 1488, 1338, 1236, 819 cm ;
1
H NMR (300 MHz, CDCl ): δ 3.22 (s, 3H), 4.57 (d, J = 13.6 Hz, 1H), 4.70 (d, J = 13.4 Hz, 1H),
3
5
1
7
.17 (d, J = 15.3 Hz, 1H), 5.49 (d, J = 15.1 Hz, 1H), 6.83 (d, J = 8.3 Hz, 1H), 7.17 (d, J = 1.7 Hz,
1
3
3
H), 7.58 (dd, J = 2.1, 8.5 Hz, 1H), 7.65 (s, 1H); C NMR (75 MHz, CDCl ): δ 26.6, 49.2, 59.5,
3.5, 110.9, 116.1, 127.2, 127.8, 128.3, 129.9, 134.5, 142.2, 171.0; HRMS (ESI): m/z calcd. for
+
C H O N Br, 335.0138, found 335.0136 [M+H] .
13
12
2
4
1
'-Benzyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one (6e)
o
–1 1
Light brown solid, yield 90%; Mp: 151–152 C; IR (ATR): 1721, 1488, 1363, 818, 758 cm ; H
3
NMR (500 MHz, CDCl ): δ 4.63 (d, J = 13.4 Hz, 1H), 4.76 (d, J = 13.6 Hz, 1H), 4.88 (d, J =
1
7

ACCEPTED MANUSCRIPT
1
6
5.6 Hz, 1H), 4.93 (d, J = 15.7 Hz, 1H), 5.20 (d, J = 15.3 Hz, 1H), 5.54 (d, J = 15.4 Hz, 1H),
1
3
.83 (d, J = 7.9 Hz, 1H), 7.02–7.07 (m, 2H), 7.28–7.36 (m, 6H), 7.65 (s, 1H); C NMR (75
MHz, CDCl ): δ 43.9, 49.4, 59.4, 73.5, 110.3, 123.5, 124.4, 125.0, 127.3, 128.0, 128.1, 129.0,
3
1
3
30.0, 131.4, 134.8, 143.0, 171.7; HRMS (ESI): m/z calcd. for C H O N , 333.1346, found
19 17 2 4
+
33.1345 [M+H] .
1
'-Benzyl-5'-fluoro-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6f)
o
–1 1
White solid, yield 89%; Mp: 145–146 C; IR (ATR): 1716, 1489, 1173, 995, 819, 705 cm ; H
NMR (500 MHz, CDCl ): δ 4.61 (d, J = 13.4 Hz, 1H), 4.76 (d, J = 13.6 Hz, 1H), 4.87 (d, J =
3
1
6
7
8
1
3
5.7 Hz, 1H), 4.91 (d, J = 15.7 Hz, 1H), 5.20 (d, J = 15.6 Hz, 1H), 5.55 (d, J = 15.6 Hz, 1H),
.74 (dd, J = 4.0, 8.7 Hz, 1H), 6.80 (dd, J = 2.3, 7.3 Hz, 1H), 7.02 (dt, J = 2.6, 8.8 Hz, 1H), 7.28–
1
3
3
.37 (m, 5H), 7.66 (s, 1H); C NMR (125 MHz, CDCl ): δ 44.1, 49.3, 59.6, 73.6, 111.2(d, JCF =
.2 Hz), 112.8(d, JCF = 25.4 Hz), 117.8(d, JCF = 23.6 Hz), 126.4, 126.5, 127.3, 128.2, 129.2,
29.9, 134.6, 138.9, 159.3 (d, JCF = 244.3) 171.6; HRMS (ESI): m/z calcd. for C H O N F,
19
16
2
4
+
51.1252, found 351.1250 [M+H] .
1
'-Benzyl-5'-chloro-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6g)
o
–1 1
White solid, yield 88%; Mp: 159–160 C; IR (ATR): 1707, 1488, 1176, 989, 814, 742 cm ; H
NMR (500 MHz, CDCl ): δ 4.61 (d, J = 13.6 Hz, 1H), 4.76 (d, J = 13.6 Hz, 1H), 4.86 (d, J =
3
1
6
5.6 Hz, 1H), 4.91 (d, J = 15.7 Hz, 1H), 5.20 (d, J = 15.4 Hz, 1H), 5.54 (d, J = 15.4 Hz, 1H),
1
3
.74 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 7.25–7.37 (m, 6H), 7.66 (s, 1H); C NMR (75
MHz, CDCl ): δ 44.1, 49.3, 59.7, 73.5, 111.4, 125.1, 126.8, 127.3, 128.2, 128.3, 129.1, 129.2,
3
1
8

ACCEPTED MANUSCRIPT
1
3
16 2 4
29.8, 131.4, 134.5, 141.5, 171.5; HRMS (ESI): m/z calcd. for C19H O N Cl, 367.0956, found
+
67.0958 [M+H] .
1
'-Benzyl-5'-bromo-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6h)
o
–1
1
Light brown solid, yield 85%; Mp: 169–170 C; IR (ATR): 1704, 1614, 1176, 711 cm ; H
NMR (300 MHz, CDCl ): δ 4.61 (d, J = 13.6 Hz, 1H), 4.76 (d, J = 13.6 Hz, 1H), 4.85 (d, J =
5.9 Hz, 1H), 4.91 (d, J = 15.9 Hz, 1H), 5.20 (d, J = 15.3 Hz, 1H), 5.54 (d, J = 15.3 Hz, 1H),
.69 (d, J = 8.5 Hz, 1H), 7.19 (d, J = 1.9 Hz, 1H), 7.25–7.38 (m, 5H), 7.44 (dd, J = 1.9, 8.3 Hz,
3
1
6
1
1
1
3
H), 7.66 (s, 1H); C NMR (75 MHz, CDCl ): δ 44.1, 49.3, 59.7, 73.5, 111.9, 116.3, 127.1,
3
27.3, 127.8, 128.3, 128.4, 129.2, 129.8, 134.3, 134.4, 142.0, 171.4; HRMS (ESI): m/z calcd. for
+
C
19
H
16
O
2
N
4
Br, 411.0451, found 411.0448 [M+H] .
1
'-Ethyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one (6i)
o
–1 1
White solid, yield 93%; Mp: 162–163 C; IR (ATR): 1705, 1614, 1372, 752 cm ; H NMR (500
MHz, CDCl ): δ 1.31 (t, J = 7.3 Hz, 3H), 3.71–3.83 (m, 2H), 4.58 (d, J = 13.4 Hz, 1H), 4.71 (d, J
3
=
13.4 Hz, 1H), 5.16 (d, J = 15.4 Hz, 1H), 5.49 (d, J = 15.4 Hz, 1H), 6.96 (d, J = 7.8 Hz, 1H),
7
.00 (dd, J = 0.9, 7.5 Hz, 1H), 7.07 (dt, J = 0.9, 7.6 Hz, 1H), 7.43 (dt, J = 1.2, 7.8 Hz, 1H), 7.64
1
3
(
s, 1H); C NMR (125 MHz, CDCl
3
): δ 12.6, 35.1, 49.4, 59.4, 73.5, 109.5, 123.4, 124.6, 125.3,
1
2
28.1, 130.1, 131.5, 143.0, 171.2; HRMS (ESI): m/z calcd. for C H O N , 271.1189, found
1
4
15
2
4
+
71.1186 [M+H] .
1
'-Ethyl-5'-fluoro-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6j)
1
9

ACCEPTED MANUSCRIPT
o
–1
1
White solid, yield 89%; Mp: 159–160 C; IR (ATR): 1706, 1615, 1487, 1176, 825 cm ; H
NMR (300 MHz, CDCl ): δ 1.29 (t, J = 7.2 Hz, 3H), 3.67–3.84 (m, 2H), 4.55 (d, J = 13.6 Hz,
3
1
2
H), 4.71 (d, J = 13.4 Hz, 1H), 5.16 (d, J = 15.5 Hz, 1H), 5.51 (d, J = 15.3 Hz, 1H), 6.78 (dd, J =
1
3
.6, 7.4 Hz, 1H), 6.89 (dd, J = 4.0, 8.5 Hz, 1H), 7.14 (dt, J = 2.6, 8.9 Hz, 1H), 7.64 (s, 1H); C
NMR (75 MHz, CDCl ): δ 12.5, 35.2, 49.3, 59.5, 73.5, 110.2 (d, J = 8.8 Hz), 113.0 (d, JCF
=
3
CF
2
6.3 Hz), 117.8 (d, JCF = 24.1 Hz), 126.7, 128.2, 129.8, 138.9, 159.2 (d, JCF = 243.7 Hz), 171.0;
+
HRMS (ESI): m/z calcd. for C14
H
14
O
2
N
4
F, 289.1095, found 289.1091 [M+H] .
5
'-Chloro-1'-ethyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6k)
o
–1
White solid, yield 90%; Mp: 166–167 C; IR (ATR): 1707, 1611, 1434, 1360, 1121, 824 cm ;
1
H NMR (500 MHz, CDCl ): δ 1.29 (t, J = 7.3 Hz, 3H), 3.69–3.81 (m, 2H), 4.56 (d, J = 13.6 Hz,
3
1
8
H), 4.70 (d, J = 13.4 Hz, 1H), 5.17 (d, J = 15.4 Hz, 1H), 5.50 (d, J = 15.4 Hz, 1H), 6.89 (d, J =
1
3
.4 Hz, 1H), 7.02 (d, J = 2.1 Hz, 1H), 7.41 (dd, J = 2.1, 8.4 Hz, 1H), 7.65 (s, 1H); C NMR (75
MHz, CDCl ): δ 12.5, 35.2, 49.2, 59.6, 73.4, 110.5, 125.2, 126.9, 128.2, 128.9, 129.8, 131.4,
3
+
1
41.6, 170.9; HRMS (ESI): m/z calcd. for C H O N Cl, 305.0799, found 305.0798 [M+H] .
1
4
14
2
4
5
'-Bromo-1'-ethyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6l)
o
–1
1
White solid, yield 87%; Mp: 164–165 C; IR (ATR): 1709, 1612, 1434, 1360, 826 cm ; H
NMR (500 MHz, CDCl ): δ 1.29 (t, J = 7.2 Hz, 3H), 3.68–3.80 (m, 2H), 4.56 (d, J = 13.6 Hz,
3
1
8
H), 4.69 (d, J = 13.6 Hz, 1H), 5.17 (d, J = 15.4 Hz, 1H), 5.50 (d, J = 15.3 Hz, 1H), 6.84 (d, J =
1
3
.4 Hz, 1H), 7.17 (d, J = 1.8 Hz, 1H), 7.56 (dd, J = 1.8, 8.4 Hz, 1H), 7.65 (s, 1H); C NMR (75
2
0

ACCEPTED MANUSCRIPT
MHz, CDCl
3
): δ 12.5, 35.2, 49.2, 59.6, 73.4, 110.9, 116.0, 127.3, 127.9, 128.2, 129.8, 134.4,
+
1
42.1, 170.8; HRMS (ESI): m/z calcd. for C H O N Br, 349.0295, found 349.0294 [M+H] .
1
4
14
2
4
1
'-(4-Bromobenzyl)-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6m)
o
–1 1
White solid, yield 88%; Mp: 174–175 C; IR (ATR): 1709, 1614, 1434, 1373, 826, 755 cm ; H
NMR (500 MHz, CDCl ): δ 4.63 (d, J = 13.6 Hz, 1H), 4.75 (d, J = 13.4 Hz, 1H), 4.80 (d, J =
5.9 Hz, 1H), 4.90 (d, J = 15.7 Hz, 1H), 5.19 (d, J = 15.4 Hz, 1H), 5.52 (d, J = 15.4 Hz, 1H),
.79 (d, J = 7.9 Hz, 1H), 7.02–7.08 (m, 2H), 7.18 (d, J = 8.5 Hz, 2H), 7.33 (dt, J = 1.4, 7.6 Hz,
3
1
6
1
1
1
3
H), 7.48 (d, J = 8.4 Hz, 2H), 7.65 (s, 1H); C NMR (75 MHz, CDCl ): δ 43.4, 49.4, 59.5, 73.6,
3
10.2, 122.2, 123.8, 124.6, 125.1, 128.2, 129.1, 130.0, 131.5, 132.3, 134.0, 142.7, 171.8; HRMS
+
(
ESI): m/z calcd. for C19
H
16
O
2
N
4
Br, 411.0451, found 411.0449 [M+H] .
1
'-(4-Bromobenzyl)-5'-fluoro-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-
indolin]-2'-one (6n)
White solid, yield 86%; Mp: 202–204 C; IR (ATR): 1709, 1613, 1486, 1361, 1173, 827, 805
o
–
1 1
cm ; H NMR (500 MHz, CDCl ): δ 4.60 (d, J = 13.6 Hz, 1H), 4.75 (d, J = 13.4 Hz, 1H), 4.79
3
(d, J = 15.7 Hz, 1H), 4.88 (d, J = 15.7 Hz, 1H), 5.20 (d, J = 15.4 Hz, 1H), 5.54 (d, J = 15.4 Hz,
1
7
4
1
H), 6.71 (dd, J = 4.0, 8.7 Hz, 1H), 6.81 (dd, J = 2.4, 7.3 Hz, 1H), 7.04 (dt, J = 2.6, 8.7 Hz, 1H),
1
3
.16 (d, J = 8.4 Hz, 2H), 7.47–7.49 (m, 2H), 7.66 (s, 1H); C NMR (75 MHz, CDCl ): δ 43.5,
3
9.3, 59.7, 73.6, 110.0 (d, JCF = 7.7 Hz), 113.0 (d, JCF = 25.3 Hz), 117.9 (d, JCF = 23.1 Hz),
22.3, 126.6 (d, JCF = 8.2 Hz), 128.3, 129.1, 129.8, 132.4, 133.7, 138.6, 159.5 (d, JCF = 244.3
+
Hz), 171.6; HRMS (ESI): m/z calcd. for C H O N BrF, 429.0357, found 429.0352 [M+H] .
1
9
15
2
4
2
1

ACCEPTED MANUSCRIPT
1
'-(4-Bromobenzyl)-5'-chloro-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-
indolin]-2'-one (6o)
Light brown solid, yield 85%; Mp: 224–226 C; IR (ATR): 2989, 1709, 1613, 1486, 1173, 826,
o
–
1 1
8
4
05 cm ; H NMR (500 MHz, CDCl ): δ 4.61 (d, J = 13.6 Hz, 1H), 4.75 (d, J = 13.6 Hz, 1H),
3
.79 (d, J = 15.7 Hz, 1H), 4.88 (d, J = 15.9 Hz, 1H), 5.20 (d, J = 15.4 Hz, 1H), 5.53 (d, J = 15.4
Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 7.31 (dd, J =
1
3
2
4
1
3
.0, 8.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.66 (s, 1H); C NMR (125 MHz, CDCl ): δ 43.5,
9.2, 59.7, 73.5, 111.2, 122.3, 125.2, 126.7, 128.3, 129.0, 129.4, 129.8, 131.4, 132.4, 133.5,
+
41.2, 171.4; HRMS (ESI): m/z calcd. for C H O N BrCl, 445.0061, found 445.0064 [M+H] .
1
9
15
2
4
5
'-Bromo-1'-(4-bromobenzyl)-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-
indolin]-2'-one (6p)
o
–1
Light brown solid, yield 85%; Mp: 225–225 C; IR (ATR): 1709, 1613, 1486, 1174, 826 cm ;
1
H NMR (500 MHz, CDCl ): δ 4.61 (d, J = 13.6 Hz, 1H), 4.74 (d, J = 13.6 Hz, 1H), 4.78 (d, J =
3
1
6
7
1
5.9 Hz, 1H), 4.87 (d, J = 15.7 Hz, 1H), 5.20 (d, J = 15.4 Hz, 1H), 5.53 (d, J = 15.4 Hz, 1H),
.66 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 1.8 Hz, 1H), 7.45–7.49 (m, 3H),
1
3
.66 (s, 1H); C NMR (75 MHz, CDCl ): δ 43.5, 49.2, 59.7, 73.4, 111.7, 116.6, 122.4, 127.2,
3
28.0, 128.3, 129.1, 129.8, 132.4, 133.5, 134.4, 141.7, 171.4; HRMS (ESI): m/z calcd. for
+
C
19
H
15
O
2
N
4
Br
2
, 490.9541, found 490.9537 [M+H] .
4
-((2'-Oxo-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-1'-
yl)methyl)benzonitrile (6q)
o
1
Light brown solid, yield 90%; Mp: 193–195 C; H NMR (300 MHz, CDCl ): δ 4.64 (d, J = 13.6
3
Hz, 1H), 4.76 (d, J = 13.4 Hz, 1H), 4.88 (d, J = 16.0 Hz, 1H), 5.03 (d, J = 16.2 Hz, 1H), 5.20 (d,
2
2

ACCEPTED MANUSCRIPT
J = 15.5 Hz, 1H), 5.52 (d, J = 15.5 Hz, 1H), 6.75 (d, J = 7.9 Hz, 1H), 7.05–7.13 (m, 2H), 7.32–
1
3
7
1
.42 (m, 3H), 7.64–7.67 (m, 3H); C NMR (75 MHz, CDCl ): δ 43.5, 49.3, 59.5, 73.5, 109.9,
3
12.2, 118.4, 124.1, 124.8, 125.0, 127.9, 128.2, 129.9, 131.6, 132.9, 140.2, 142.4, 171.8; HRMS
+
(
ESI): m/z calcd. for C H O N , 358.1304, found 358.1300 [M+H] .
2
0
16
2
5
4
-((5'-Chloro-2'-oxo-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-1'-
yl)methyl)benzonitrile (6r)
White solid, yield 86%; Mp: 205–207 C; H NMR (300 MHz, CDCl ): δ 4.63 (d, J = 13.6 Hz,
o
1
3
1
1
7
1
H), 4.76 (d, J = 13.6 Hz, 1H), 4.86 (d, J = 16.2 Hz, 1H), 5.01 (d, J = 16.2 Hz, 1H), 5.21 (d, J =
5.5 Hz, 1H), 5.53 (d, J = 15.5 Hz, 1H), 6.68 (d, J = 8.3 Hz, 1H), 7.08 (d, J = 1.9 Hz, 1H), 7.31–
1
3
.39 (m, 3H), 7.64–7.67 (m, 3H); C NMR (75 MHz, CDCl ): δ 43.6, 49.1, 59.7, 73.4, 111.0,
3
12.4, 118.3, 125.4, 127.0, 127.9, 128.3, 129.6, 129.7, 131.5, 133.0, 139.8, 140.9, 171.5; HRMS
+
(
ESI): m/z calcd. for C20
H
16
O
2
N
5
Cl, 392.0914, found 392.0902 [M+H] .
1
',5'-Dimethyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one (6s)
o
1
Light brown solid, yield 93%; Mp: 207–209 C; H NMR (300 MHz, CDCl ): δ 2.30 (s, 3H),
3
3
.21 (s, 3H), 4.56 (d, J = 13.4 Hz, 1H), 4.70 (d, J = 13.4 Hz, 1H), 5.16 (d, J = 15.3 Hz, 1H), 5.49
1
3
(
d, J = 15.5 Hz, 1H), 6.82 (d, J = 7.7 Hz, 2H), 7.23 (d, J = 8.5 Hz, 1H), 7.64 (s, 1H); C NMR
(75 MHz, CDCl
3
): δ 21.1, 26.4, 49.3, 59.4, 73.7, 109.1, 125.0, 125.1, 128.1, 130.0, 131.7, 133.4,
+
1
41.4, 171.5; HRMS (ESI): m/z calcd. for C H O N , 271.1195, found 271.1189 [M+H] .
1
4
15
2
4
1
'-Benzyl-5'-methyl-4,7-dihydrospiro[[1,2,3]triazolo[5,1-c][1,4]oxazine-6,3'-indolin]-2'-one
(6t)
2
3

ACCEPTED MANUSCRIPT
o
1
White solid, yield 91%; Mp: 147–148 C; H NMR (500 MHz, CDCl
13.6 Hz, 1H), 4.74 (d, J = 13.6 Hz, 1H), 4.85 (d, J = 15.7 Hz, 1H), 4.90 (d, J = 15.6 Hz, 1H),
.19 (d, J = 15.7 Hz, 1H), 5.54 (d, J = 15.4 Hz, 1H), 6.70 (d, J = 7.9 Hz, 1H), 6.86 (s, 1H), 7.11
3
): δ 2.26 (s, 3H), 4.61 (d, J
=
5
1
3
(
dd, J = 0.8, 7.9 Hz, 1H), 7.27–7.36 (m, 5H), 7.65 (s, 1H); C NMR (75 MHz, CDCl ): δ 21.1,
3
4
1
3.9, 49.4, 59.5, 73.7, 110.1, 125.1, 125.2, 127.3, 128.0, 128.2, 129.0, 130.1, 131.7, 133.4, 134.9,
+
40.5, 171.8; HRMS (ESI): m/z calcd. for C H O N , 347.1508, found 347.1505 [M+H] .
2
0
19
2
4
4
.2. Cell Culture
The A549 (human lung cancer), MCF-7 (Human breast cancer), HeLa (human cervical cancer)
and DU-145 cell line (human prostate cancer) were obtained from National center for Cell
science (NCCS), Pune, India. DMEM (Dulbecco’s Modified Eagles Medium), MTT [3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide], Trypsin, EDTA were purchased from
Sigma Chemicals Co (st. Louis, MO). RPMI 1640 medium were purchased from GIBCO-
Invitrogen. Fetal bovine serum was purchased from Arrow labs. The 6 and 96 well flat bottom
tissue culture plates were purchased from Tarson.
4
.2.1. MTT assay
4
The cytotoxic activity of the compounds (6a–t) was determined using MTT assay. 1 × 10 cells
per well were seeded in 100 µL DMEM, supplemented with 10% FBS in each well of 96-well
microculture plates and incubated for 24 h at 37 °C in a CO incubator. Compounds, diluted to
2
the desired concentrations in culture medium, were added to the wells with respective vehicle
control. After 48 h of incubation, 10 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
−
1
tetrazolium bromide) (5 mg mL ) was added to each well and the plates were further incubated
2
4

ACCEPTED MANUSCRIPT
for 4 h. Then, the supernatant from each well was carefully removed, formazon crystals were
dissolved in 100 µL of DMSO and absorbance was recorded at 570 nm wavelength.
4
.2.2. Morphology
5
A549 cells were plated in 6 well culture plates with a cell density of 1 × 10 cells/ml and allowed
to adhere for overnight. Cells were incubated with the IC50 concentrations of concentration of the
compounds 6i and 6p. After 48 h treatment, cells were observed for the morphological changes
and photographs were taken under a phase contrast microscope (Nikon).
4
.2.3. In Vitro Cell migration Assay/Wound healing assay
5
The wound-healing assay is a proven method to study in vitro cell migration. A549 cells (5 × 10
cells/well) were cultured in 6 well plates for 24 h. The confluent monolayers were then scratched
with 200 µL pipette tip. The wounded monolayers were washed twice with PBS to remove non-
adherent cells. Then, media containing the IC concentrations of the compounds 6i and 6p were
5
0
added to each well. Cells which migrated across the scratched wound were photographed under
the phase contrast microscope (Nikon) at 0, 24 and 48 h time interval after treatment. The cells
that migrated to the wounded areas were counted and the percentage of migrated cells in each
experimental condition was calculated in comparison to control cells (DMSO exposed cells).
4
.2.4. Flow cytometry analysis
To determine the effect of compound 6i on the cell cycle, cells were seeded in 6-well plates at a
5
density of 1 × 10 cells/mL for 24 h. After incubation, cells were treated with different
concentration of compound 6i for 24 h. Then the cells were collected, washed and fixed in 70%
o
ethanol in PBS at -20 C. After leaving overnight, the fixed cells were pelleted and stained with
Propidium Iodide (50 µg/mL) in the presence of RNase A (20 µg/mL) containing 0.1% Triton X-
2
5

ACCEPTED MANUSCRIPT
o
1
00 and EDTA (0.02 mg/mL) for 30 min at 37 C in dark, and about 5000 events were analyzed
on a Muse™ Cell Analyzer (Merck-Millipore, Germany).
4
.2.5. Hoechst staining
4
Human lung cancer A549 cells were grown on coverslips in a 6 well plates (5 × 10 cells/well)
and allowed to adhere for 24 h. The culture medium containing the compounds 6i and 6p at their
IC50 concentration were added to the cells. After 24 h incubation, culture medium was removed;
cells were washed with PBS and fixed with 4 % para formaldehyde solution at 4 ºC for 10 min.
Then, cells were washed twice with PBS and stained with Hoechst 33242 (5 µg/mL) for 30 min
at room temp. The excess dye was removed by washing twice with PBS and cell suspensions
were mounted on slides and were examined for morphological changes under confocal
fluorescence microscope using 350 nm excitation and 460 nm emission (Nikon, magnification
4
0X).
4
.2.6. Acridine Orange/Ethidium Bromide (AO/EB) staining
A549 cells were grown in 24 well plates (50,000 cells/well) for 24 h and were treated with and
with 0.5, 1 and 2 µM concentration of compound 6i for 24 h. After treatment, cells were washed
twice with phosphate buffer saline (PBS) and fixed with 4 % para formaldehyde at 4 ºC for 10
min. Then, the cells were incubated with 0.5 mL of PBS solution containing mixture of Acridine
Orange and Ethidium Bromide (1:1, 100 µg/mL) in dark for 10 min at 37 ºC. The stained cells
were washed with PBS to remove the excess dye and the cells were observed immediately under
fluorescence microscope with excitation (488 nm) and emission (550 nm) at 200X
magnification.
4
.2.7. Measurement of Mitochondrial Membrane Potential (MMP)
2
6

ACCEPTED MANUSCRIPT
5
A549 cells were cultured in 6 well plates at a density of 5 × 10 cells/mL and allowed to grow
over night. The cells were treated with IC concentrations of the compounds 6i and 6p. After 24
5
0
h incubation, the adherent cells were collected by trypsinsation, washed with PBS and
resuspended in a solution of PBS containing Rhodamine-123 (10 µg/mL). After incubation for
3
0 min at room temp, cells were washed twice with PBS and re-suspended in PBS. The samples
were analyzed for Rhodamine123 fluorescence using spectroflourimeter.
4
.2.8. Measurement of Reactive Oxygen Species (ROS) levels
Intracellular ROS levels were measured by using 2'-7'-dichlorodihydrofluorescein diacetate
5
(
DCFH-DA). A549 cells were plated in 6 well plates at a density of 5 × 10 cells/mL and
allowed to grow overnight. The cells were incubated with the IC50 concentrations of the
compounds 6i and 6p for 24 h. After treatment, the medium was replaced with the culture
medium containing DCFH-DA (10 µM) and further incubated for 30 min at room temperature in
4
dark. Cells were washed with PBS, collected from the plate and resuspended in PBS at 5 × 10
cells/mL. The fluorescence intensity of the 2’, 7’-dichlorofluorscein formed by the hydrolysis of
DCFH-DA from each sample was analyzed by spectroflourimeter at an excitation of 488 nm and
emission wavelength 525 nm. Qualitative cellular fluorescence images were captured by using
Nikon ECLIPSETE2000-S fluorescence microscope.
Acknowledgements
K.R.S. and P.S. are thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, New
Delhi, for the award of NIPER fellowship.
5
.0. References
2
7

ACCEPTED MANUSCRIPT
[1] M. Center, R. Siegel, A. Jemal, Global cancer facts & figures, Atlanta, Georgia: American
Cancer Society, (2011).
[2] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, 2014, CA: Cancer J. Clin., 64 (2014) 9–
2
9.
[3] S. Raguz, E. Yague, Resistance to chemotherapy: new treatments and novel insights into an
old problem, Br. J. Cancer, 99 (2008) 387–391.
[
4] D. Cheng, Y. Ishihara, B. Tan, C.F. Barbas III, Organocatalytic asymmetric assembly
reactions: Synthesis of spirooxindoles via organocascade strategies, ACS Catal., 4 (2014) 743–
62.
7
[5] L. Hong, R. Wang, Recent advances in asymmetric organocatalytic construction of 3,
3
′‐spirocyclic oxindoles, Adv. Synth. Catal., 355 (2013) 1023–1052.
[
6] C.V. Galliford, K.A. Scheidt, Pyrrolidinyl‐spirooxindole natural products as inspirations for
the development of potential therapeutic agents, Angew. Chem. Int. Ed. Engl., 46 (2007) 8748–
758.
8
[7] A.P. Antonchick, C. Gerding-Reimers, M. Catarinella, M. Schürmann, H. Preut, S. Ziegler,
D. Rauh, H. Waldmann, Highly enantioselective synthesis and cellular evaluation of
spirooxindoles inspired by natural products, Nat. Chem., 2 (2010) 735–740.
[8] Y. Zhao, S. Yu, W. Sun, L. Liu, J. Lu, D. McEachern, S. Shargary, D. Bernard, X. Li, T.
Zhao, A potent small-molecule inhibitor of the MDM2–p53 interaction (MI-888) achieved
complete and durable tumor regression in mice, J. Med. Chem., 56 (2013) 5553–5561.
2
8