Synthesis and apoptosis inducing studies of triazole linked 3-benzylidene isatin derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.09.009

In our venture towards the development of effective cytotoxic agents, a panel of triazole linked 3-benzylidene isatin hybrids were synthesized and characterized by IR,¹H NMR,¹³C NMR and Mass spectral analysis. All the newly synthesiz

Full text of DOI:10.1016/j.ejmech.2016.09.009

Accepted Manuscript
Synthesis and apoptosis inducing studies of triazole linked 3-benzylidene isatin
derivatives
Atulya Nagarsenkar, Lalita Guntuku, Sravanthi Devi Guggilapu, K. Danthi Bai,
Gannoju Srinivasulu, V.G.M. Naidu, Bathini Nagendra Babu
PII:
S0223-5234(16)30737-1
10.1016/j.ejmech.2016.09.009
EJMECH 8875
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 August 2016
Revised Date: 1 September 2016
Accepted Date: 3 September 2016
Please cite this article as: A. Nagarsenkar, L. Guntuku, S.D. Guggilapu, K. Danthi Bai, G. Srinivasulu,
V.G.M. Naidu, B.N. Babu, Synthesis and apoptosis inducing studies of triazole linked 3-benzylidene
isatin derivatives, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.009.
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Graphical Abstract
Synthesis and apoptosis inducing studies of triazole linked 3-benzylidene isatin
derivatives
Atulya Nagarsenkar,a Lalita Guntuku,b Sravanthi Devi Guggilapu,a K. Danthi Bai,a Gannoju
Srinivasulu,a V. G. M. Naidu,b,* Bathini Nagendra Babua,*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bPharmacology and Toxicology Division, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India

ACCEPTED MANUSCRIPT
Synthesis and apoptosis inducing studies of triazole linked 3-benzylidene isatin
derivatives
Atulya Nagarsenkar,a Lalita Guntuku,b Sravanthi Devi Guggilapu,a K. Danthi Bai,a Gannoju
Srinivasulu,a V. G. M. Naidu,b,* Bathini Nagendra Babua,*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bPharmacology and Toxicology Division, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
*Corresponding author:
Dr. Bathini Nagendra Babu
Department of Medicinal Chemistry
National Institute of Pharmaceutical Education and Research
Hyderabad, 500037, India
E-mail: bathini@niperhyd.ac.in; bnniper@gmail.com
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Abstract
In our venture towards the development of effective cytotoxic agents, a panel of triazole
1
13
linked 3-benzylidene isatin hybrids were synthesized and characterized by IR, H NMR, C
NMR and Mass spectral analysis. All the newly synthesized target compounds were assessed
against DU145 (prostate), PC-3 (prostate), MDA-MB-231 (breast), BT549 (breast), A549
(lung) and HeLa (cervical) human cancer cell lines by employing MTT assay for their
cytotoxic potential. Significantly, compound Z-8l was found to be most potent amongst all
the tested compounds with an IC50 value of (3.7±0.05 µM) on DU145 cells. The most active
compound (Z-8l) was also tested on RWPE-1 (normal prostate) cells and was found to be
safe compared to the DU145 cells. The influence of the cytotoxic compound Z-8l on the cell
cycle distribution was assessed on the DU145 cell line, exhibiting a cell cycle arrest at the
G2/M phase. Additionally, treatment with compound Z-8l caused collapse of mitochondrial
membrane potential (DΨm) in DU145 cells. Moreover, acridine orange/ethidium bromide
staining, DAPI nuclear staining, DCFDA staining and annexin V binding assay confirmed
that compound Z-8l can induce cell apoptosis in DU145 cells. Western blotting was
performed to examine the appearance of active forms of cytochrome c, Bax, Bcl2 and PARP
(Poly ADP ribose polymerase), indicator proteins of apoptosis in DU145 cells; the study
confirmed the triggering of mitochondrial mediated apoptotic pathway upon exposure of
compound Z-8l.
Keywords: Triazole, oxindole, 3-benzylidene isatin, anticancer, Knoevenagel condensation,
apoptosis.
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1.0 Introduction
Cancer is one of the foremost global health burden and most severe clinical problems in the
world with increasing frequency every year [1]. Regardless of avoiding behavioral risk
factors such as tobacco, obesity, and preventive managements like dietary, medication and
vaccination, the disease still affects millions of patients worldwide [2]. Many of the current
anticancer drugs usually act on rapidly proliferating cells, and may have poor selectivity
between normal and cancerous cells. The high toxicity and poor tolerance of the existing
anticancer drugs, stress on the need to identify novel molecules with potent antitumor
activity, low toxicity and minimum side effects. Thus, the design and synthesis of new
chemical entities for the effective and safe cure of cancer is a dynamic area of research in
medicinal chemistry.
The oxindole is a privileged scaffold, which represents a vital class of heterocyclic
compounds bestowed with interesting pharmacological activities [3] such as inhibitors of the
MDM2-p53 interaction [4], cholinesterases [5], antimicrobial [6], histone deacetylases [7],
and anticancer properties [8, 9]. In addition, sunitinib and nintedanib (Figure 1) are the
representative drugs emerged from this class and are in clinical use for targeted anticancer
therapies [10]. The FDA approval of sunitinib and nintedanib paved the way to develop
various indolin-2-one based molecules against cancer [11, 12].
Principally, the structural modifications at the C3- and C5-positions of the oxindole ring have
led to many derivatives possessing increased antitumor activity [1].
<Insert Figure 1 here>
It is well-known that substituted 3-benzylidene isatin scaffold is strongly related with
anticancer activity [13]. It has several targets, most of which are directly connected to
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oncogenesis [14, 15]. Moreover, Zhang et. al, have proposed that functionalized 3-
benzylidene isatins may be included as compounds with chemopreventive potential [15]. The
presence of the Michael acceptor motif makes the 3-benzylidene isatin scaffold a probable
candidate for such activity. If antitumour 3-benzylidene isatins hold chemopreventive
properties, this may mean that when deployed for their effects on cancer cells, the same agent
would bestow cytoprotection to normal/non-metastasized cells, a motivating proposition with
potential clinical benefits.
On the other hand, the chemistry of 1,2,3-triazoles has received considerable attention due to
their synthetic and effective biological importance [16]. Triazole ring is a potential
pharmacophore that has been significantly acknowledged over the past few decades [17].
Even though 1,2,3-triazole moiety itself does not occur in nature, there are several examples
in literature that show assorted biological activities associated with this system, such as anti-
HIV activity [18], antimicrobial activity [19], β3-selective adrenergic agonism [20], kinase
inhibitory [21] and other enzyme inhibitory activities [22]. 1,2,3-Triazole moieties have been
incorporated into a wide diversity of therapeutically interesting drug candidates; for example
(Figure 1), antibacterial agents β-lactam (tazobactam), cefatrizine (Trizicef) and anticancer
agents (carboxyamidotriazole CAI).
Molecular hybridization is an efficient tool to design more active and new chemical entities
by covalently combining two or more drug pharmacophores into a single molecule [23]. Of
late, it has been observed that chromone-pyrazole, chromone-pyrimidine, chromone-
indolinone, indole-indolinone, indole-pyrimidine and indole-pyrazole conjugates
demonstrated profound growth inhibitory activity against different cancer cells [24, 25].
In this context, we desire to exploit 1-benzyl-4-(phenoxymethyl)-1H-1,2,3-triazoles as the
diversity surrogate to condense with various substituted oxindoles to explore the dual
pharmacophoric potential (Figure 2). These triazole linked 3-benzylidene isatin hybrids may
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have the advantages of tunable anticancer activity and structural diversity. Additionally, the
chemical processes and reactions drawn in are facile and concise, which make it feasible for
the bulk production of important isatin derivatives.
<Insert Figure 2 here>
2.0 Results and discussion
2.1 Chemistry
The synthesized 3-(3-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)indolin-2-one
[E-8(a-b, d, f-m) and Z-8(a-c, e, i, k-m)] compounds of this study comprise two core
structural elements: (i) 3-benzylidene isatin, and (ii) a substituted 1,2,3-triazole moiety.
Initially, for the preparation of the substituted triazole aldehyde 6 required for the synthesis of
proposed target compounds, we have utilized commercially available benzyl bromide 1 as the
starting material (Scheme 1). The benzyl azide 2(a,b) was synthesized via bimolecular
displacement of the halide substituent of benzyl halide 1(a,b) precursor with an azide
nucleophile [26]. Meanwhile, 3-hydroxy benzaldehyde 3 was subjected to N-propargylation
for the synthesis of respective alkyne 5 [27]. This terminal alkyne bearing substrate 5 was
further subjected to [3+2] cycloaddition with benzyl azide 2(a,b) to afford substituted triazole
aldehyde 6(a,b) [28]. Knoevenagel condensation was employed on oxindole 7a by using
triazole
aldehyde
6a
to
afford
3-(3-((1-phenyl-1H-1,2,3-triazol-4-
yl)methoxy)benzylidene)indolin-2-ones in an equal mixture of E/Z isomeric forms (E-8a+Z-
8a) [29]. The reaction was also tried by using dimethoxy triazole aldehyde 6b to afford
consequent final compound. This too was obtained in an equal mixture of E/Z isomeric forms
(E-8b+Z-8b).
Next, various substituted oxindoles (7b-g) were subjected to Knoevenagel condensation by
using triazole aldehyde 6a to get subsequent condensed products. Surprisingly, the target
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compounds from 5-Cl (7b) and 5-Br (7d) oxindoles were obtained as mixture of E/Z isomeric
forms in 20:80 ratios, whereas in case of other oxindoles (7c, e-g), the target compounds were
obtained as mixture of E/Z isomeric forms in 70:30 ratios. Encouraged by such interesting
observations, 5-acylated oxindoles (7h-i) were used and upon Knoevenagel condensation
gave corresponding target compounds. 5-Acetyl oxindole bearing product was obtained in an
equal mixture of E/Z isomeric forms (E-8i+Z-8i), whereas 5-benzoyl oxindole bearing
compound was obtained as E/Z mixture in 90:10 ratios. Furthermore, 5-alkylated (7j-l)
oxindoles were subjected to Knoevenagel condensation using triazole aldehyde 6a. However,
the target molecules were obtained in an equal mixture of E/Z isomeric forms [E-(8k-m) and
Z-(8k-m)]. Finally, these E/Z isomers were separated by using the conventional column
chromatography. It has to be noted that in cases where the target compounds were obtained in
unequal ratios, we were only able to isolate the major isomer in pure form.
<Insert Scheme 1 here>
The earlier literature reports suggest that the ortho protons (C2'- or C6'-protons) of the
benzylidene ring in Z isomer are deshielded relative to those in E isomer [13, 30]. This
deshielding of C2'- or C6'-protons could be due to the presence of nearby C2-position
carbonyl of indolin-2-one ring in the Z isomer form. So, the two isomer forms can be easily
distinguished by 1H NMR analysis.
The stereochemistry was assigned for one of the representative compounds Z-8a by detailed
1
13
NOE studies. The H and C NMR assignments for compound Z-8a were carried out using
gDQFCOSY and ROESY experiments in DMSO-d6 at 27 °C Bruker 500 MHz (for 1H NMR)
and 125 MHz (for 13C NMR), respectively [see ESI†]. The presence of NOE between H5-H6
and H6-H7 in ROESY experiment confirms the geometry at double bond and the compound
was characterized as Z-isomer as depicted in Figure 3 and 4.
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<Insert Figure 3 here>
<Insert Figure 4 here>
2.2 Pharmacology
2.2.1 In vitro anticancer activity
All synthesized compounds were evaluated for their in vitro cytotoxic activity against
six human cancer cell lines DU145 (prostate), PC-3 (prostate), MDA-MB-231
(breast), BT549 (breast), A549 (lung) and HeLa (cervical) cancer cell lines by
employing MTT assay [31]. Sunitinib was taken as the reference in this study.
Concentration response course analysis was executed to determine drug concentrations
required to inhibit the growth of cancer cells by 50% (IC50) after incubation for 48 h.
The results of in vitro anticancer activity revealed that, some of the synthesized
compounds exhibited different levels of anticancer properties (Table 1).
From the close analysis of the IC50 values, it was observed that, that the Knoevenagel
product
and A549 cell lines, while its
PC-3 and HeLa. Target compound
aldehyde (6b) showed moderate activity against PC-3, A549 and HeLa; whereas its
isomer -8b) displayed remarkable activity against MDA-MB-231 cells along with
moderate activity towards other tested cell lines.
(
Z
-8a) of oxindole and aldehyde (6a) was moderately active only against PC-3
isomer -8a) was found to be active against DU145,
-8b) synthesized using dimethoxy substituted
E
(E
(
Z
E
(
E
Target compounds with 5-chloro (Z-8c), 6-chloro (E-8d) and 5-bromo (Z-8e) substitutions on
oxindole were specifically active against DU145, whereas compound with 6-bromo (E-8f)
substitution displayed significant activity towards DU145 and A549 cell lines. The
compounds with 5-fluro (E-8g) and 5-nitro (E-8h) substitutions on oxindole were found to be
inactive towards all the tested cell lines. Compound bearing 5-acetyl (Z-8i) substitution on
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oxindole exhibited moderate activity selectively against A549, whereas its E isomer (E-8i)
was inactive. Compound containing 5-benzoyl (E-8j) substitution was inactive towards all
tested cell lines.
The target molecule with 5-p-methoxy benzyl substitution on oxindole (Z-8k) displayed
striking activity towards DU145 and HeLa cell lines; on the other side its E isomer (E-8k)
was moderately active against PC-3. Target compound holding bulky benzhydryl substitution
at 5th position of oxindole (Z-8l) demonstrated remarkable activity against all the tested cell
lines except MDA-MB-231 cells. However, its E isomer (E-8l) was found to be inactive
towards all tested cell lines. The compound having p-methyl substitution on diphenyl
propargyl moiety (Z-8m) was found to be inactive against all the tested cell lines, though its
E isomer (E-8m) showed moderate activity against DU145, A549 and HeLa.
The most active compound (Z-8l) was also tested on normal prostate (RWPE-1) cells and was
found to be safe compared to the DU145 prostate cancer cells. Additionally, aldehyde (6a)
was screened for its anticancer potential and was found to be inactive against all tested cell
lines.
Analysis of the MTT assay results suggests DU145 cells are more sensitive towards the
synthesized target compounds. The impact of modification of the “R1” group on 5th position
of oxindole derivatives is interesting in the light of the results of the SAR study, which
suggests that bulky aryl groups at 5rd position of oxindole is optimal for anticancer activity.
However, it has to be noted that Z isomers of 5-p-methoxy benzyl and 5-benzhydryl
substituted oxindole compounds showed better results than its E isomers. On the other hand
simple halogen substitutions at this position demonstrated increase in selectivity towards
DU145 cell lines. Further derivatization has mostly resulted in a significant loss of activity.
Among all compounds synthesized, compound (Z-8l) showed the most significant
cytotoxicity towards most of the tested cancer cell lines. These primary results persuade
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further assessment on the synthesized compounds aiming to the development of novel
potential anticancer agents.
<Insert Table 1 here>
2.2.2 Acridine orange–ethidium bromide (AO–EB) staining
Acridine orange/ethidium bromide (AO/EB) fluorescent staining assay was performed to
distinguish the live, apoptotic and necrotic cells [32]. AO permeates the intact cell membrane
and stains the nuclei green, while EB can stain the cells that have lost their membrane
integrity and tinge the nucleus red. It can be interpreted from Figure 5 that the control cells
showed the normal healthy morphology with intact nuclear architecture and appeared green
in colour. Fluorescence microscopic images of Z-8l treated DU145 cells have clearly
demonstrated morphological changes which are the characteristic features of apoptotic cells
such as cell shrinkage, membrane blebbing and apoptotic body formation. This confirms that
the compound Z-8l induced cell death in DU145 cancer cells.
<Insert Figure 5 here>
2.2.3 DAPI Nucleic Acid Staining
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye that binds sturdily to nucleus and
detect the nuclear damage or chromatin condensation. The DAPI stains the apoptotic cells as
bright colored owing to the condensed nucleus which is a distinctive apoptotic characteristic.
Hence, it was of our interest to detect nuclear damage or chromatin condensation persuaded
by the compound Z-8l in DU145 cells. DAPI staining technique was performed according to
the earlier reported method [33]. The results from the Figure 6 illustrated that the nuclear
structure of untreated cells was intact whereas compound Z-8l treated cells exhibited
condensed, pyknotic, or fragmented nuclei.
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<Insert Figure 6 here>
2.2.4 Cell cycle analysis
Many of the cytotoxic compounds exert their growth inhibitory effect by arresting the cell
cycle at a specific phase of a cell cycle. In vitro screening results revealed that the compound
Z-8l showed significant activity against DU145 cells. Thus, we herein examined the effect of
the compound Z-8l on cell cycle using propidium iodide staining method [34].
DU145 cells were treated with designated concentrations of compound Z-8l for 24 h. They
were stained with propidium iodide and samples were further analysed by flow cytometry.
Treatment with the compound Z-8l at 2.5 and 5 µM in DU145 cells displayed rise in G2/M
population from 29.66% (control) to 43.74% and 48.02% respectively in a dose dependent
manner (Figure 7). The results suggest clear induction of G2/M cell cycle arrest by the test
compound. Furthermore, increase in sub G1 population compared to control also indicates the
induction of apoptotic death in the treated cells.
<Insert Figure 7 here>
2.2.5 Annexin V binding assay
The apoptosis inducing effect of compound Z-8l on DU145 cancer cells was further
investigated using annexin V-FITC/propidium iodide staining assay [35]. DU145 cells were
treated with 1.25, 2.5 and 5 µM of compound Z-8l for 24 h and stained with Annexin V-FITC
and propidium iodide, and samples were analysed by flow cytometry.
As depicted in Figure 8, Z-8l treated DU145 cells demonstrated rise in the total percentage of
apoptotic (early and late apoptotic cells- Annexin V +ve cells) and dead cells from 9.68%
(untreated) to 23.53%, 35.46% and 54.1% respectively in a dose dependent manner.
<Insert Figure 8 here>
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2.2.6 Measurement of reactive oxygen species (ROS) levels
The reactive oxygen species generation is one of well characterised mechanisms of many
anticancer drugs. Hence, in next array of experiments, we assessed the generation of
intracellular reactive oxygen species by compound Z-8l in DU145 cells using DCFDA
staining method [36].
Treatment with compound Z-8l for 6 h resulted in significant increase in DCFDA
fluorescence in a dose dependent manner, signifying ROS accumulating property of
compounds (Figure 9). Treatment with N-acetyl cysteine (NAC) prior to compound
treatment has decreased DCFDA fluorescence intensity; this indicated that compounds
induced cytotoxicity by ROS generation.
<Insert Figure 9 here>
2.2.7 Analysis of Mitochondrial Membrane Potential (DΨm)
Increase in intracellular ROS can cause oxidative stress and lead to the loss of mitochondrial
membrane potential. Thus, the effect of compound Z-8l on mitochondrial membrane potential
(DΨm) was determined by staining with lipophilic cationic JC-1 dye [37]. Healthy polarised
mitochondria stains red due to potential dependent formation of J-aggregates, while
depolarised mitochondria in apoptotic cells stains green because of J-monomers DU145 cells
were treated with 1.25, 2.5 and 5 μM of Z-8l for 24 h and stained by JC-1 dye .
Flowcytometric analysis of the treated cells clearly displayed increase in depolarised cell
population (P2) from control (5.39%) to 64.28%, 85.24% and 99.7% respectively in conc.
dependent manner (Figure 10). Thus, the results clearly indicate loss of mitochondrial
membrane potential by the compound Z-8l and suggest the involvement of mitochondria
dependent apoptotic pathway in their mechanism of action.
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<Insert Figure 10 here>
2.2.8 Western blotting analysis
Cytochrome c, Bax, Bcl2 and PARP are some of the proteins whose expression plays a
critical role in the apoptotic process. Particularly, PARP (Poly ADP ribose polymerase) is
116 kda protein that gets cleaved into 89 kda protein fragment during caspases activation and
this cleavage is prominent hallmark of the apoptosis induction. Intrinsic or mitochondria
mediated apoptotic pathway can be evidenced with loss of mitochondrial membrane potential
usually preceded by the release of cytochrome c into cytosol. Herein, to investigate the
molecular mechanisms of compound Z-8l on apoptosis, we have checked the expression of
Bcl2, Bax, PARP and cytochrome c by using western blot method [38]. Results from the
Figure 11 indicated that compound Z-8l treatment led to the dose dependent increased
expression of cleaved PARP and cytochrome c in DU145 cells, which is a hallmark feature of
apoptosis. Moreover, the compound Z-8l treatment resulted in decreased expression of anti-
apoptotic Bcl2 and increased expression of proapoptotic Bax proteins in a dose dependent
manner. Collectively, these results illustrate that compound Z-8l induced apoptosis through
apoptosis-related protein expression.
<Insert Figure 11 here>
3.0 Conclusion
In the current study, novel triazole linked benzylidene-isatin derivatives were synthesized,
and were further evaluated for their in vitro anticancer potentials. An initial screening was
performed against DU145, PC-3, MDA-MB-231, BT549, A549 and HeLa human cancer cell
lines. In MTT assay, the compound Z-8l was found to be the most active against the DU145
(prostate cancer) cell line. Most importantly, the compound Z-8l was found to be
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comparatively safe towards normal cell line RWPE-1. The detailed studies like AO/EB
staining and Annexin V binding assay and DAPI nuclear staining suggested that compound
Z-8l induced apoptosis in DU145 cells. The cell cycle analysis confirmed that the compound
Z-8l target the G2/M phase of DU145 cell cycle in a dose-dependent manner. Moreover, the
compound Z-8l treatment resulted in collapse of mitochondrial membrane potential and
elevated intracellular ROS levels in DU145 cells. Investigation of expression levels of
apoptotic proteins cytochrome c, Bax, Bcl2 and cleaved PARP in DU145 cells revealed that
compound Z-8l trigger mitochondrial mediated intrinsic pathway thereby inducing apoptosis
in cancer cells. Overall, the current studies demonstrated that dual pharmacophores strategy
can be useful in the synthesis of promising new chemical entities for the development of
cancer therapeutics.
4.0 Experimental protocols
4.1 Chemistry
All reagents and solvents were obtained from commercial suppliers and were used without
further purification. Analytical thin layer chromatography (TLC) was performed on MERCK
precoated silica gel 60-F-254 (0.5 mm) aluminium plates. Visualization of the spots on TLC
1
13
plates was achieved UV light. H and C NMR spectra were recorded on bruker 500 MHz
1
spectrometer using tetramethyl silane (TMS) as the internal standard. Chemical shifts for H
and 13C are reported in parts per million (ppm) downfield from tetramethyl silane. Spin
multiplicities are described as s (singlet), bs (broad singlet), d (doublet), dd (double douplet),
t (triplet), q (quartet), and m (multiplet). Coupling constant (J) values are reported in hertz
(Hz). IR spectra were recorded on a Perkin Elmer, FT-IR spectrometer using KBr discs.
HRMS were determined with Agilent QTOF mass spectrometer 6540 series instrument.
Column chromatography was performed using silica gel 60-120.
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4.2 General procedure for the synthesis of 3-(prop-2-yn-1-yloxy)benzaldehyde (5)
To a stirred solution of 3-hydroxy benzaldehyde 3 (1 equiv.) in acetonitrile was added
propargyl bromide 4 and potassium carbonate (5 equiv.) and refluxed for 6 h. Completion of
the reaction was checked by TLC (n-hexane/EtOAc; 7:3). Solvent was evaporated under
reduced pressure. Compound was extracted using ethyl acetate and water. Organic layer was
separated and concentrated. The compound was purified using column chromatography (n-
hexane/ethyl acetate) to give pure compound 5 in good yield.
4.3 General procedure for the synthesis of substituted triazole aldehyde 6(a,b)
To a stirred solution of 3-(prop-2-yn-1-yloxy)benzaldehyde 5 (1 equiv.) in t-butanol/water
(1:1) was added CuI (0.01 equiv.) and azide 2 (1 equiv.) and heated at 40 °C for 48 h.
Completion of the reaction was checked by TLC (n-hexane/EtOAc; 5:5). Solvent was
evaporated under reduced pressure. Compound was extracted using ethyl acetate and water.
Organic layer was separated and concentrated. The compound was purified using column
chromatography (n-hexane/ethyl acetate) to give pure compound 6(a,b) in good yields.
4.4 General procedure for the synthesis of triazole linked benzylidene-isatin derivatives
[E-8(a-b, d, f-m) and Z-8(a-c, e, i, k-m)]
To a stirred solution of oxindole 7 (1 equiv) in ethanol was added substituted triazole
aldehyde 6 (1 equiv) and piperidine (0.01 equiv) and refluxed for 3 h. Completion of the
reaction was checked by TLC (n-hexane/EtOAc; 3:7). The product was filtered and was
purified using column chromatography (n-hexane/ethyl acetate) to give pure compound [E-
8(a-b, d, f-m) and Z-8(a-c, e, i, k-m)] in good yields.
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4.4.1 (Z)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)indolin-2-one (Z-8a)
Yellow solid; yield: 45%; mp 198-200 °C. IR (KBr): 3320, 2781, 1705, 1610, 1453, 1075,
1
788 cm–1. H NMR (500 MHz, DMSO-d6): δ = 10.60 (s, 1H), 8.33 (t, J = 2.0 Hz, 1H), 8.29
(s, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.77 (s, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.36-7.28 (m, 6H),
7.21 (t, J = 7.6 Hz, 1H), 7.11 (dd, J = 8.0 2.0, Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H), 6.82 (d, J =
13
7.6 Hz, 1H), 5.60 (s, 2H), 5.18 (s, 2H) ppm. C NMR (125 MHz, DMSO-d6): δ = 166.9,
157.5, 142.7, 140.6, 136.5, 135.8, 135.1, 129.1, 128.9, 128.6, 128.0, 127.8, 126.9, 125.1,
124.7, 124.6, 121.0, 119.7, 117.2, 117.0, 109.2, 61.0, 52.7 ppm. HRMS (ESI): m/z calcd. for
C25H20N4O2 [M+H]+ 409.1665; found 409.1663.
4.4.2 (E)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)indolin-2-one (E-8a)
Yellow solid; yield: 39%; mp 185-189 °C. IR (KBr): 3318, 2793, 1703, 1604, 1445, 1058,
811 cm–1. 1H NMR (500 MHz, DMSO-d6): δ = 10.59 (s, 1H), 8.29 (s, 1H), 7.59 (s, 1H), 7.52
(d, J = 7.7 Hz, 1H), 7.44 (t, J = 7.7 Hz, 1H), 7.36 (d, J = 7.2 Hz, 2H), 7.34-7.29 (m, 5H),
7.25 (t, J = 7.6 Hz, 2H), 7.14 (d, J = 8.2 Hz, 1H), 6.89-6.82(m, 1H), 5.62 (s, 2H), 5.20 (s,
2H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 168.4, 158.0, 142.8, 142.7, 135.8, 135.7,
135.4, 130.0, 129.8, 128.6, 128.0, 127.8, 127.7, 124.6, 122.4, 121.6, 121.0, 120.7, 116.2,
115.1, 110.0, 61.1, 52.7 ppm. HRMS (ESI): m/z calcd. for C25H20N4O2 [M+H]+ 409.1665;
found 409.1691.
4.4.3
(Z)-3-(3-((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)
indolin-2-one (Z-8b)
Yellow solid; yield: 31%; mp 223-225 °C. IR (KBr): 3298, 2883, 1706, 1610, 1448, 1212,
1
1068, 813 cm–1. H NMR (500 MHz, DMSO-d6): δ = 10.62 (s, 1H), 8.34 (s, 1H), 8.31 (s,
1H), 7.84 (d, J = 7.7 Hz, 1H), 7.77 (s, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H),
7.25 (t, J = 7.7 Hz, 1H), 7.14 (dd, J = 2.1, 8.0 Hz, 1H), 7.02 (t, J = 7.7 Hz, 1H), 6.84 (d, J =
7.7 Hz, 1H), 6.48-6.43 (m, 3H), 5.52 (s, 2H), 5.19 (s, 2H), 3.70 (s, 6H) ppm. 13C NMR (125
15

ACCEPTED MANUSCRIPT
MHz, DMSO-d6): δ = 166.9, 160.5, 157.5, 142.7, 140.6, 137.9, 136.5, 135.1, 129.1, 128.9,
126.9, 125.1, 124.7, 124.7, 121.0, 119.7, 117.2, 117.0, 109.2, 105.9, 99.5, 60.9, 55.1, 52.7
ppm. HRMS (ESI): m/z calcd. for C27H25N4O4 [M+H]+ 469.1876; found 469.1892.
4.4.4
(E)-3-(3-((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)
indolin-2-one (E-8b)
Yellow solid; yield: 36%; mp 211-213 °C. IR (KBr): 3356, 3065, 2923, 1703, 1690, 1587,
1456, 1219, 1076, 696 cm–1. 1H NMR (500 MHz, DMSO-d6): δ = 10.60 (s, 1H), 8.29 (s, 1H),
7.58 (s, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.34-7.28 (m, 2H), 7.24 (t, J =
7.6 Hz, 1H), 7.15-7.12 (m, 1H), 6.88-6.81 (m, 2H), 6.49-6.44 (m, 3H), 5.52 (s, 2H), 5.19 (s,
2H), 3.70 (s, 6H) ppm.13C NMR (125 MHz, DMSO-d6): δ = 160.5, 158.0, 142.8, 142.7,
137.9, 136.5, 135.7, 135.4, 130.1, 129.8, 127.7, 124.7, 122.4, 121.6, 121.0, 120.7, 116.2,
115.1, 110.0, 105.9, 99.5, 61.0, 55.1, 52.7 ppm. HRMS (ESI): m/z calcd. for C27H25N4O4
[M+H]+ 469.1876; found 469.1879.
4.4.5 (Z)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)-5-chloroindolin-2-
one (Z-8c)
Yellow solid; yield: 60%; mp 198-202 °C. IR (KBr): 3338, 2884, 1704, 1610, 1462, 1462,
1218, 1028, 741 cm–1. 1H NMR (500 MHz, DMSO-d6): δ = 10.74 (s, 1H), 8.36 (s, 1H), 8.30
(d, J = 3.5 Hz, 1H), 7.91 (s, 1H), 7.85 (m, 1H), 7.66 (s, 1H), 7.50-7.23 (m, 6H), 7.18-7.13 (m,
13
1H), 6.89 (d, J = 8.3Hz, 1H), 6.84 (d, J = 8.3Hz, 1H), 5.61 (s, 2H), 5.20 (s, 2H) ppm. C
NMR (125 MHz, DMSO-d6): δ = 170.2, 158.1, 157.5, 142.6, 141.6, 137.3, 135.8, 129.9,
129.5, 128.6, 128.0, 127.8, 125.4, 125.3, 124.6, 121.9, 121.5, 116.6, 115.1, 111.4, 110.6,
59.6, 52.7 ppm. HRMS (ESI): m/z calcd. for C25H19ClN4O2 [M+H]+ 443.1275; found
443.1295.
16

ACCEPTED MANUSCRIPT
4.4.6 (E)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)-6-chloroindolin-2-
one (E-8d)
Yellow solid; yield: 57%; mp 198-202 °C. IR (KBr): 3210, 3064, 2923, 1704, 1690, 1455,
1219, 1054, 696 cm–1. 1H NMR (500 MHz, DMSO-d6): δ = 10.77 (s, 1H), 8.30 (m, 1H), 7.63
(s, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 7.40-7.26 (m, 6H), 7.16 (dd, J =
13
2.1, 8.0 Hz, 1H), 6.93-6.87 (m, 2H), 5.61 (s, 2H), 5.19 (s, 2H) ppm. C NMR (125 MHz,
DMSO-d6): δ = 168.4, 158.0, 144.2, 142.7, 136.3, 135.9, 13.40, 129.9, 129.2, 128.6, 128.0,
127.8, 126.6, 124.6, 123.7, 121.7, 120.9, 119.6, 116.6, 115.1, 110.0, 61.1, 52.7 ppm. HRMS
(ESI): m/z calcd. for C25H19ClN4O2 [M+H]+ 443.1275; found 443.1277.
4.4.7 (Z)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)-5-bromoindolin-2-
one (Z-8e)
Yellow solid; yield: 63%; mp 201-203 °C. IR (KBr): 3351, 2924, 1703, 1580, 1462, 1253,
1218, 1028, 696 cm–1. 1H NMR (500 MHz, DMSO-d6): δ = 10.76 (s, 1H), 8.36 (s, 1H), 8.31
(s, 1H), 7.98-7.95 (m, 1H), 7.92 (s, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.42-7.29 (m, 7H), 7.17
(dd, J = 1.8, 7.7 Hz, 1H), 6.80 (d, J = 8.2 Hz, 1H), 5.62 (s, 2H), 5.19 (s, 2H) ppm. 13C NMR
(125 MHz, DMSO-d6): δ = 168.7, 158.2, 148.5, 142.6, 141.5, 139.1, 135.9, 134.9, 130.1,
128.7, 128.0, 127.8, 126.5, 124.7, 122.0, 121.1, 117.6, 117.4, 115.0, 110.1, 109.3, 61.2, 52.7
ppm. HRMS (ESI): m/z calcd. for C25H19BrN4O2 [M+H]+ 487.0771; found 487.0773.
4.4.8 (E)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)-6-bromoindolin-2-
one (E-8f)
Yellow solid; yield: 55%; mp 196-199 °C. IR (KBr): 3310, 2870, 1704, 1604, 1445, 1240,
1
1057, 693 cm–1. H NMR (500 MHz, DMSO-d6): δ = 10.76 (s, 1H), 8.29 (s, 1H), 7.65 (s,
1H), 7.44 (t, J = 8.0 Hz, 2H), 7.38-7.27 (m, 7H), 7.15 (dd, J = 2.2, 8.0 Hz, 1H), 7.06 (dd, J =
13
1.8, 8.2 Hz, 1H), 7.02 (d, J = 1.8 Hz, 1H), 5.62 (s, 2H), 5.19 (s, 2H) ppm. C NMR (125
MHz, DMSO-d6): δ = 168.2, 158.0, 142.2, 142.7, 136.5, 135.8, 135.4, 129.9, 128.6, 128.0,
17

ACCEPTED MANUSCRIPT
127.8, 126.7, 124.6, 123.9, 123.7, 122.5, 121.6, 120.0, 116.6, 115.0, 112.7, 61.1, 52.7 ppm.
HRMS (ESI): m/z calcd. for C25H19BrN4O2 [M+H]+ 487.0771; found 487.0763.
4.4.9 (E)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)-5-fluoroindolin-2-
one (E-8g)
Yellow solid; yield: 62%; mp 188-190 °C. IR (KBr): 3296, 2919, 1694, 1594, 1469, 1209,
1
1155, 784 cm–1. H NMR (500 MHz, DMSO-d6): δ = 10.65 (s, 1H), 8.30 (s, 1H), 7.67 (s,
1H), 7.47 (t, J = 7.8 Hz, 1H), 7.40-7.26 (m, 7H), 7.21 (d, J = 9.2 Hz, 1H), 7.16 (d, J = 8.2
Hz, 1H), 7.12-7.06 (m, 1H), 6.90-6.85 (m, 1H), 5.61 (s, 2H), 5.19 (s, 2H) ppm. 13C NMR
(125 MHz, DMSO-d6): δ = 168.5, 158.1, 142.7, 139.2, 137.2, 135.9, 135.3, 134.9, 130.0,
129.3, 128.7, 128.1, 127.9, 125.4, 124.7, 121.5, 117.5, 117.4, 116.5, 116.4, 115.3, 110.9,
110.8, 61.1, 52.8 ppm. HRMS (ESI): m/z calcd. for C25H19FN4O2 [M+H]+ 427.1574; found
427.1569.
4.4.10 (E)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)-5-nitroindolin-2-
one (E-8h)
Yellow solid; yield: 59%; mp 189-191 °C. IR (KBr): 3300, 2870, 1704, 1604, 1444, 1209,
1
693 cm–1. H NMR (500 MHz, DMSO-d6): δ = 11.37 (s, 1H), 8.44-8.27 (m, 2H), 8.24-8.14
(m, 2H), 7.52-7.27 (m, 7H), 7.24 (t, J = 6.5 Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 8.2
13
Hz, 1H), 5.62 (s, 2H), 5.21 (s, 2H) ppm. C NMR (125 MHz, DMSO-d6): δ = 168.7, 158.2,
148.5, 142.6, 141.5, 139.1, 135.9, 130.1, 129.3, 128.7, 128.0, 127.8, 126.5, 124.7, 122.0,
121.1, 117.6, 117.4, 115.0, 110.1, 109.3 ppm. HRMS (ESI): m/z calcd. for C25H19N5O4
[M+H]+ 454.1515; found 454.1513.
4.4.11 (Z)-5-Acetyl-3-(3-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)indolin-2-
one (Z-8i)
Yellow solid; yield: 30%; mp 210-212 °C. IR (KBr): 3320, 2902, 1693, 1583, 1512, 1241,
1207, 1034, 809 cm–1. 1H NMR (500 MHz, DMSO-d6): δ = 10.62 (s, 1H), 8.35 (s, 1H), 8.31
18

ACCEPTED MANUSCRIPT
(s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.77 (s, 1H), 7.70 (d, J =7.4 Hz, 1H), 7.39-7.35 (m, 2H),
7.33-7.30 (m, 3H), 7.24 (dt, J = 0.9, 7.6 Hz, 1H), 7.14 (dd, J = 2.1, 7.9 Hz, 1H), 7.01 (dt, J =
13
0.7, 7.6 Hz, 1H), 6.84 (d, J =7.6 Hz, 1H), 5.61 (s, 2H), 5.19 (s, 2H), 2.52 (s, 3H) ppm. C
NMR (125 MHz, DMSO-d6): δ = 198.7, 166.7, 157.3, 142.5, 140.4, 136.2, 135.6, 134.8,
128.8, 128.7, 128.4, 127.7, 127.5, 126.6, 124.8, 124.5, 124.4, 120.7, 119.4, 116.9, 116.8,
109.0, 60.7, 52.4, 28.4 ppm. HRMS (ESI): m/z calcd. for C27H22N4O3 [M+H]+ 451.1770;
found 451.1772.
4.4.12 (E)-5-Acetyl-3-(3-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)indolin-2-
one (E-8i)
Yellow solid; yield: 42%; mp 178-180 °C. IR (KBr): 3323, 2989, 1704, 1613, 1588, 1221,
1054, 713 cm–1. 1H NMR (500 MHz, DMSO-d6): δ = 10.64 (d, J = 10.5 Hz, 1H), 8.32 (d, J =
7.4 Hz, 1H), 7.59 (s, 1H), 7.46-7.28 (m, 7H), 7.24 (td, J = 1.0, 7.5 Hz, 1H), 7.15-7.11 (m,
1H), 7.01-6.97 (m, 1H), 6.88-6.81 (m, 2H), 5.63 (d, J = 5.0 Hz, 2H), 5.19 (s, 2H), 2.53 (s,
3H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 198.2, 168.4, 158.0, 142.8, 142.7, 140.6,
136.5, 135.8, 135.4, 130.1, 129.8, 128.6, 128.0, 127.8, 124.6, 122.4, 121.0, 120.9, 117.2,
116.2, 115.1, 110.0, 61.1, 52.7, 28.6 ppm. HRMS (ESI): m/z calcd. for C27H22N4O3 [M+H]+
451.1770; found 451.1778.
4.4.13 (E)-5-Benzoyl-3-(3-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)indolin-
2-one (E-8j)
Yellow solid; yield: 63%; mp 196-198 °C. IR (KBr): 3401, 3136, 1704, 1613, 1220, 713 cm–
1
1. H NMR (500 MHz, DMSO-d6): δ = 11.10 (s, 1H), 8.37-8.25 (m, 1H), 8.07-7.98 (m, 1H),
7.79-7.47 (m, 6H), 7.42-7.25 (m, 8H), 7.23-6.79 (m, 3H), 5.62 (s, 2H), 5.19 (s, 2H) ppm. 13C
NMR (125 MHz, DMSO-d6): δ = 194.2, 168.8, 158.0, 146.8, 142.8, 142.7, 137.5, 137.2,
135.8, 135.4, 135.2, 132.9, 131.8, 128.9, 128.6, 128.3, 128.0, 127.8, 124.6, 122.4, 121.5,
19

ACCEPTED MANUSCRIPT
121.0, 120.6, 116.5, 115.1, 109.9, 61.1, 52.7 ppm. HRMS (ESI): m/z calcd. for C32H24N4O3
[M+H]+ 513.1927; found 513.1927
4.4.14
(Z)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)-5-(4-
methoxybenzyl) indolin-2-one (Z-8k)
Yellow solid; yield: 32%; mp 198-200 °C. IR (KBr): 3415, 3029, 1704, 1510, 1478, 1244,
1
1032, 691 cm–1. H NMR (500 MHz, DMSO-d6): δ = 10.54 (s, 1H), 8.33 (s, 1H), 8.30 (s,
1H), 7.85 (d, J = 7.7 Hz, 1H), 7.73 (s, 1H), 7.58 (s, 1H), 7.39-7.29 (m, 6H), 7.17 (d, J = 8.6
Hz, 2H), 7.12 (dd, J = 2.2, 8.2 Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 6.86 (d, J = 8.6 Hz, 2H),
6.75 (d, J = 7.7 Hz, 1H), 5.61 (s, 2H), 5.18 (s, 2H), 3.84 (s, 2H), 3.70 (s, 3H) ppm. 13C NMR
(125 MHz, DMSO-d6): δ = 167.0, 157.5, 157.4, 142.7, 138.8, 136.3, 135.8, 135.1, 134.4,
133.5, 129.3, 129.1, 128.6, 128.0, 127.8, 127.0, 125.1, 124.9, 124.6, 120.0, 117.2, 117.0,
113.7, 109.1, 61.0, 54.9, 52.7, 39.7 ppm. HRMS (ESI): m/z calcd. for C33H28N4O3 [M+H]+
529.2240; found 529.2238
4.4.15
(E)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)-5-(4-
methoxybenzyl) indolin-2-one (E-8k)
Yellow solid; yield: 39%; mp 138-140 °C. IR (KBr): 3385, 2901, 1703, 1588, 1461, 1221,
1
1054, 713 cm–1. H NMR (500 MHz, DMSO-d6): δ = 10.52 (s, 1H), 8.29 (s, 1H), 7.54 (s,
1H), 7.40-7.29 (m, 8H), 7.22 (d, J = 7.4 Hz, 1H), 7.12-7.07 (m, 2H), 7.04 (d, J = 8.3 Hz, 2H),
6.80-6.75 (m, 3H), 5.61 (s, 2H), 5.16 (s, 2H), 3.71 (s, 2H), 3.67 (s, 3H) ppm. 13C NMR (125
MHz, DMSO-d6): δ = 168.6, 158.0, 157.4, 142.7, 140.9, 135.8, 135.6, 135.2, 134.4, 132.9,
130.2, 129.6, 129.4, 128.6, 128.0, 127.8, 124.6, 122.7, 121.5, 120.8, 116.2, 115.1, 113.7,
109.8, 61.3, 54.8, 52.7, 39.7 ppm. HRMS (ESI): m/z calcd. for C33H28N4O3 [M+H]+
529.2240; found 529.2234
20

ACCEPTED MANUSCRIPT
4.4.16
(Z)-5-Benzhydryl-3-(3-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)
indolin-2-one (Z-8l)
Yellow solid; yield: 38%; mp 245-247 °C. IR (KBr): 3392, 2901, 1685, 1578, 1223, 1052,
1
785 cm–1. H NMR (500 MHz, DMSO-d6): δ = 10.60 (s, 1H), 8.29 (s, 2H), 7.83 (d, J = 7.7
Hz, 1H), 7.68 (s, 1H), 7.57 (s, 1H), 7.39-7.28 (m, 11H), 7.25-7.19 (m, 2H), 7.18-7.13 (m,
3H), 7.11 (dd, J = 2.2, 8.2 Hz, 1H), 6.93 (d, J = 7.9 Hz, 1H), 6.78 (d, J = 7.9 Hz, 1H), 5.61 (s,
2H), 5.59 (s, 1H), 5.17 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 168.6, 157.9,
143.6, 142.6, 141.1, 136.5, 135.8, 135.4, 135.3, 130.9, 129.5, 128.9, 128.7, 128.6, 128.2,
128.0, 127.8, 127.7, 126.0, 124.5, 123.2, 121.0, 120.7, 116.2, 115.0, 109.8, 61.0, 55.2, 52.7
ppm. HRMS (ESI): m/z calcd. for C38H30N4O2 [M+H]+ 575.2447; found 575.2440
4.4.17
(E)-5-Benzhydryl-3-(3-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)
indolin-2-one (E-8l)
Yellow solid; yield: 35%; mp 220-222 °C. IR (KBr): 3380, 3136, 2921, 1685, 1578, 1446,
1223, 1012, 785 cm–1. 1H NMR (500 MHz, DMSO-d6): δ = 10.57 (s, 1H), 8.22 (s, 1H), 7.52
(s, 1H), 7.39-7.28 (m, 8H), 7.26-7.19 (m, 4H), 7.18-7.11 (m, 3H), 7.09-6.99 (m, 6H), 6.83 (d,
13
J = 8.0 Hz, 1H), 5.60 (s, 2H), 5.47 (s, 1H), 5.07 (s, 2H) ppm. C NMR (125 MHz, DMSO-
d6): δ = 168.6, 157.9, 143.7, 142.6, 141.1, 136.5, 135.9, 135.4, 135.4, 130.9, 129.6, 128.9,
128.7, 128.6, 128.2, 128.0, 127.8, 127.7, 126.0, 124.5, 123.2, 121.0, 116.2, 115.0, 109.8,
61.0, 55.2, 52.7 ppm. HRMS (ESI): m/z calcd. for C38H30N4O2 [M+H]+ 575.2447; found
575.2440
4.4.18 (Z)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)-5-(3-phenyl-1-(p-
tolyl)prop-2-yn-1-yl)indolin-2-one (Z-8m)
Yellow solid; yield: 33%; mp 202-204 °C. IR (KBr): 3324, 3137, 1704, 1690, 1588, 1462,
1220, 1028, 740 cm–1. 1H NMR (500 MHz, DMSO-d6): δ = 10.63 (s, 1H), 8.32 (s, 1H), 8.30
(s, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.78-7.75 (m, 1H), 7.52-7.49 (m, 1H), 7.40-7.27 (m, 12H),
21

ACCEPTED MANUSCRIPT
7.26 (dd, J = 2.7, 8.0 Hz, 1H), 7.17 (d, J = 7.9 Hz, 2H), 7.13 (dd, J = 2.2, 8.0 Hz, 1H), 7.08
(d, J = 7.7 Hz, 1H), 6.82 (d, J = 7.9 Hz, 1H), 5.61 (s, 2H), 5.32 (s, 1H), 5.18 (s, 2H), 2.26 (s,
3H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 167.0, 157.5, 142.8, 139.5, 139.0, 135.9,
135.8, 135.0, 135.0, 131.3, 131.2, 129.1, 129.1, 128.6, 128.5, 128.4, 128.3, 128.0, 127.8,
127.2, 127.1, 126.8, 125.2, 125.0, 124.6, 122.6, 119.0, 109.4, 91.0, 84.0, 61.0, 52.7, 41.9,
20.5 ppm. HRMS (ESI): m/z calcd. for C41H32 N4O2 [M+H]+ 613.2604; found 613.2604
4.4.19 (E)-3-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)benzylidene)-5-(3-phenyl-1-(p-
tolyl)prop-2-yn-1-yl)indolin-2-one (E-8m)
Yellow solid; yield: 45%; mp 183-185 °C. IR (KBr): 3358, 2901, 1702, 1605, 1445, 1253,
1
1048, 691 cm–1. H NMR (300 MHz, DMSO-d6): δ = 10.59 (s, 1H), 8.23 (s, 1H), 7.77 (m,
1H), 7.57 (s, 1H), 7.40-7.26 (m, 13H), 7.26-7.21 (m, 3H), 7.09 (d, J = 7.9 Hz, 1H), 7.05-7.02
(m, 2H), 6.85 (d, J = 8.0 Hz, 1H), 5.59 (s, 2H), 5.25 (s, 1H), 5.13 (s, 2H), 2.21 (s, 3H) ppm.
13C NMR (75 MHz, DMSO-d6): δ = 168.5, 158.0, 142.6, 141.6, 138.9, 135.8, 135.6, 135.5,
134.8, 131.1, 129.5, 129.3, 129.1, 128.6, 128.4, 128.2, 128.0, 127.8, 127.6, 127.1, 124.5,
122.4, 121.5, 121.4, 121.0, 116.2, 115.1, 109.9, 90.7, 84.0, 61.1, 52.7, 41.4, 20.4 ppm.
HRMS (ESI): m/z calcd. for C41H32 N4O2 [M+H]+ 613.2604; found 613.2602
4.5 Pharmacology
4.5.1 Cell culture
Cells were obtained from National Centre for Cell Science (NCCS) Pune, India and stocks
were maintained in the laboratory. Breast (BT549 and MDA-MB-231), prostate (PC-3 and
DU145), lung (A549) and cervical (HeLa) cancer cells were grown in tissue culture flasks in
DMEM (Dulbecco modified Eagle medium, Sigma) or RPMI (Roswell Park Memorial
Institute medium) supplemented with 10% fetal bovine serum with 1X stabilized antibiotic-
22

ACCEPTED MANUSCRIPT
o
antimycotic solution (Sigma) in a CO2 incubator at 37 C with 5% CO2 and 90% relative
humidity.
4.5.2 MTT assay
The anticancer activity of all newly synthesized compounds was determined using MTT
assay. 3-5x103 cells per well were seeded in 100 µL DMEM or RPMI, supplemented with
10% FBS in each well of 96-well microculture plates and incubated for 48 h at 37 °C in a
CO2 incubator. Compounds, diluted to the desired concentrations in culture medium, were
added to the wells with respective vehicle control. After 48 h of incubation, drugs containing
media was removed and 100 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) (0.5mg/mL) was added to each well and the plates were further incubated for 4 h.
Then, the supernatant from each well was carefully removed, formazon crystals were
dissolved in 200 µL of DMSO and absorbance was recorded at 570 nm wavelength.
4.5.3 Acridine orange–ethidium bromide (AO–EB) staining
DU145 cells were plated at a concentration of 1×106 cell/ml and treated with different
concentration of compound Z-8l. Plates were incubated in an atmosphere of 5% CO2 at 37 °C
for 48 h. 10 µL of fluorescent dyes containing Acridine Orange (AO) and Ethidium Bromide
(EB) added into each well in equal volumes (10 ꢀg/mL) respectively and within 10 min the
cells were visualized under fluorescence microscope (Nikon, Inc. Japan) with excitation (488
nm) and emission (550 nm) at 200x magnification.
4.5.4 DAPI Nucleic Acid Staining
Nuclear morphological changes were observed through DAPI staining. After treatment with
Z-8l for 48 h in DU145 cells, cells were washed with PBS and permeabilized with 0.1 %
Tween 20 for 10 min followed by staining with 1 µM DAPI. Control and Treated cells were
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observed with fluorescence microscope (Model: Nikon, Japan) with excitation at 359 nm and
emission at 461 nm using DAPI filter at 200X magnification.
4.5.5 Cell cycle analysis
Flow cytometric analysis (FACS) was performed to calculate the distribution of the cell
population through the cell cycle phases. DU145 cancer cells were incubated with compound
Z-8l at varied concentration of 2.5 µM, and 5.0 µM for 24 h. Untreated and treated cells
were harvested, washed with PBS, fixed in ice-cold 70% ethanol and stained with propidium
iodide (50 µg/mL, sigma aldrich) in the presence of RNase A (20 µg/mL) containing 0.1%
o
Triton X-100 for 30 min at 37 C in dark, and about 10000 events were analyzed by flow
cytometer (BD FACSVerseTM, USA).
4.5.6 Annexin V binding assay
DU145 cells (1x106) were seeded in six-well plates and allowed to grow overnight. The
medium was then replaced with complete medium containing compound Z-8l at 1.25, 2.5 and
5.0 µM concentrations. After 24 h of treatment, cells from the supernatant and adherent
monolayer cells were harvested by trypsinization, washed with PBS at 3000 rpm. Then the
cells were processed with Annexin V-assay kit (FITC Annexin V Apoptosis Detection Kit,
BD PharmingenTM) according to the manufacturer’s instruction. Further, flow cytometric
analysis was performed using a flow cytometer (BD FACSVerseTM, USA).
4.5.7 Measurement of reactive oxygen species (ROS) levels
DU145 cells were plated in 6 well plates at a density of 1×106 cells/mL and allowed to adhere
for overnight. The cells were treated with 1.25, 2.5 and 5 µM concentrations of the compound
Z-8l for 24 h. The medium was replaced with culture medium containing DCFDA dye (10
µM) and further incubated for 30 min at room temperature in dark. The fluorescence intensity
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from each sample was analysed by spectrofluorometer at an excitation and emission
wavelength of 488 and 525 nm, respectively.
4.5.8 Measurement of Mitochondrial Membrane Potential
DU145 cells (1×106 cells/mL) were seeded in 6 well plates and allowed to adhere for
overnight. The cells were incubated with 1.25, 2.5 and 5 µM concentrations of the compound
Z-8l for 24 h. Cells were collected, washed with PBS and resuspended in solution of JC-1
°
(2.5 µg/mL) and incubated for 45 min in incubator at 37 C . The cells were washed twice
with PBS and cells were trypsinized, centrifuged and analysed by flow cytometer (BD
FACSVerseTM, USA).
4.5.9 Western blotting analysis
Primary antibodies against Bax, Bcl-2, PARP, Cyt c, β-actin and HRP conjugated antirabbit
secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).
Equal amounts of protein sample (25 µg/lane) from each experimental group and were
separated by SDS–polyacrylamide gel electrophoresis. Then the protein bands were
transferred to polyvinylidene fluoride membrane (Pierce Biotechnology, Rockford, IL, USA)
for western blotting. Membranes were blocked with 3% bovine serum albumin in TBS buffer
°
(pH-7.4) and incubated with the indicated antibodies overnight at 4 C. β-actin (1:1000) was
used for equal loading. After washing the membranes in TBST buffer, membranes were
exposed to the HRP-conjugated secondary antibodies (1:5000) for 2 h at room temperature.
The reactive bands were visualized with chemiluminescent detection reagents (Supersignal
West Pico, Pierce Biotechnology, Rockford, IL, USA). The densitometry analysis of the blots
was performed by using Image J software, NIH, USA.
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Acknowledgements
Authors are thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, New Delhi,
for the award of NIPER fellowship.
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