Design, synthesis and biological evaluation of 3-substituted-2-oxindole hybrid derivatives as novel anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2017.03.089

The 2-oxindole nucleus is the central core to develop new anticancer agents and its substitution at the 3-position can effect antitumor activity. Utilizing a pharmacophore hybridization approach, a novel series of antiproliferative agents was obtained by the modification of the structure of 3-substituted-2-oxindole pharmacophore by the attachment of the α-bromoacryloyl moiety, acting as a Michael acceptor, at the 5-position of 2-oxindole framework. The impact of the substituent at the 3-position of 2-oxindole core on the potency and selectivity against a panel of seven different cancer cell lines was examined. We found that these hybrid molecules displayed potent antiproliferative activity against a panel of four cancer cell lines, with one-to double digit nanomolar 50% inhibitory concentrations (IC₅₀). A distinctive selective antiproliferative activity was obtained towards CCRF-CEM and RS4; 11 leukemic cell lines. In order to study the possible mechanism of action, we observed that the two most active compounds namely 3(E) and 6(Z) strongly induce apoptosis that follow the mitochondrial pathway. Interestingly a decrease of intracellular reduced glutathione content (GSH) and reactive oxygen species (ROS) production was detected in treated cells compared with controls suggesting that these effects may be involved in their mechanism of action.

Full text of DOI:10.1016/j.ejmech.2017.03.089

Accepted Manuscript
Design, synthesis and biological evaluation of 3-substituted-2-oxindole hybrid
derivatives as novel anticancer agents
Romeo Romagnoli, Pier Giovanni Baraldi, Filippo Prencipe, Paola Oliva, Stefania
Baraldi, Maria Kimatrai Salvador, Luisa Carlota Lopez-Cara, Roberta Bortolozzi,
Elena Mattiuzzo, Giuseppe Basso, Giampietro Viola
PII:
S0223-5234(17)30229-5
10.1016/j.ejmech.2017.03.089
EJMECH 9382
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 December 2016
Revised Date: 24 March 2017
Accepted Date: 25 March 2017
Please cite this article as: R. Romagnoli, P.G. Baraldi, F. Prencipe, P. Oliva, S. Baraldi, M.K. Salvador,
L.C. Lopez-Cara, R. Bortolozzi, E. Mattiuzzo, G. Basso, G. Viola, Design, synthesis and biological
evaluation of 3-substituted-2-oxindole hybrid derivatives as novel anticancer agents, European Journal
of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.089.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of 3-Substituted-2-Oxindole
Hybrid Derivatives as Novel Anticancer Agents
Romeo Romagnoli*a, Pier Giovanni Baraldia, Filippo Prencipea, Paola Olivaa,
Stefania Baraldia, Maria Kimatrai Salvadorb, Luisa Carlota Lopez-Carab,
Roberta Bortolozzic, Giuseppe Bassoc and Giampietro Viola*c
aDipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara,
44121 Ferrara, Italy;
bDepartamento de Quimica Organica y Farmaceutica, Facultad de Farmacia,
Campus de Cartuja s/n, 18071, Granada, Spain;
cDipartimento di Salute della Donna e del Bambino, Laboratorio di
Oncoematologia, Università di Padova, 35131 Padova, Italy

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of 3-Substituted-2-Oxindole
Hybrid Derivatives as Novel Anticancer Agents
Romeo Romagnoli*a, Pier Giovanni Baraldia, Filippo Prencipea, Paola Olivaa, Stefania
Baraldia, Maria Kimatrai Salvadorb, Luisa Carlota Lopez-Carab, Roberta Bortolozzic, Elena
Mattiuzzoc, Giuseppe Bassoc and Giampietro Viola*c
aDipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, 44121 Ferrara,
Italy;
bDepartamento de Química Farmaceútica y Orgánica Facultad de Farmacia, Campus de
Cartuja s/n, 18071, Granada, Spain;
cDipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia,
Università di Padova, 35131 Padova, Italy
*
To whom correspondence should be addressed. E-mail:rmr@unife.it; Phone: 39-(0)532-
455303. Fax: 39-(0)532-455953. (R.R.); E-mail:giampietro.viola.1@unipd.it Phone: 39-
(0)49-8215485. Fax: 39-(0)49-8211462. (G.V.).
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Abstract: The 2-oxindole nucleus is the central core to develop new anticancer agents and its
substitution at the 3-position can effect antitumor activity. Utilizing a pharmacophore
hybridization approach, a novel series of antiproliferative agents was obtained by the
modification of the structure of 3-substituted-2-oxindole pharmacophore by the attachment of
the α-bromoacryloyl moiety, acting as a Michael acceptor, at the 5-position of 2-oxindole
framework. The impact of the substituent at the 3-position of 2-oxindole core on the potency
and selectivity against a panel of seven different cancer cell lines was examined. We found
that these hybrid molecules displayed potent antiproliferative activity against a panel of four
cancer cell lines, with one- to double digit nanomolar 50% inhibitory concentrations (IC50). A
distinctive selective antiproliferative activity was obtained towards CCRF-CEM and RS4;11
leukemic cell lines. In order to study the possible mechanism of action, we observed that the
two most active compounds namely 3(E) and 6(Z) strongly induce apoptosis that follow the
mitochondrial pathway. Interestingly a decrease of intracellular reduced glutathione content
(GSH) and reactive oxygen species (ROS) production was detected in treated cells compared
with controls suggesting that these effects may be involved in their mechanism of action.
Keywords. 2-Oxindole derivatives, α-bromoacryloyl, Michael acceptor, in vitro
antiproliferative activity, GSH depletion.
1. Introduction
The 2-oxindole nucleus is a promising pharmacophore present in a wide series of compounds
endowed of interesting biological properties as anticancer agents [1-7]. The fascinating
properties of molecules bearing the 3-substituted-2-oxindole scaffold has attracted the
attention of numerous researchers and 2-oxindole conjugates with heterocycles such as
pyrrole and thiophene have been recognized as antineoplastic agents with a broad spectrum of
activity against many cancer cell lines [8-10]. A wide number of 3-[(substituted-1H-pyrrol-2-
yl)methylidenyl]-2-oxindole derivatives, such as SU 5402 (1a), SU 5416 or Semaxinib (1b),
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SU 6668 (1c) and Sunitinib (1d) [11], have demonstrated promising antitumor activity and
identified as receptor tyrosine kinase (RTK) inhibitors (Chart 1) [12]. By the modification of
Sunitinib, Henise and Tauton identified an irreversible Never in mitosis gene A related
Kinase 2 (Nek2) inhibitor (IC50: 920 nM) through the introduction of the electrophilic
propiolamide moiety at the 5-position of 2-oxindole ring (compound 1e) [13a]. Analysis of
the crystal structure of Nek2 in complex with Sunitinib-like derivatives revealed that the 5-
position of the 2-oxindole nucleus was located in close proximity to Cys22 within the kinase
[13b], such that the Michael acceptor moiety might covalently interact with the residue.
Noteworthy, the oxindole in the central nucleus of different Akt pathway inhibitors [7]. Nesi
et al. have reported a series of 3,5-disubstituted-2-oxindole derivatives, in which the presence
of the thiophene ring characterized the most active compound of the series. The 3-[(thiophen-
2-yl)methylidenyl]-2-oxindole analogue 1f was identified as an inhibitor of Akt
phosphorylation and inhibited A549 cell proliferation with an IC50 of 0.66 µM [14].
The α-bromoacrylic acid is an alkylating moiety of low chemical reactivity and devoid of
cytotoxic effects (IC50>120 µM against murine leukemia L1210 cells) [15]. The α-
bromoacryloyl moiety has provided the inspiration for the design and development of several
covalently acting synthetic compounds. This chemical function was present in a series of
potent anticancer distamycin-like minor groove binders, including PNU-166196 (brostallicin,
compound 2), which was evaluated as first-line single agent chemotherapy in patients with
advanced or metastatic soft tissue sarcoma [16, 17]. Nowadays the research is primarily
focused on the usage of brostallicin in combination with other chemotherapeutic drugs to
target triple-negative breast cancer [18]. Regarding the chemical reactivity of the α-
bromoacrylic moiety, we speculated that an intracellular biological nucleophile could perform
a first-step Michael attack on the double bond, possibly followed by a further reaction of the
alpha halogen to the carbonyl, leading to a beta-elimination or a second nucleophilic
substitution [15]. Several findings suggest that GSH levels affect the antitumor activity of
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brostallicin, supporting the hypothesis concerning the ability of brostallicin to react with
GSH, giving a reactive compound able to bind DNA [19].
The most common class of reactive functional groups for selective alkylation of cysteine
residues within the ATP-binding domain of their kinase targets are Michael acceptors [20]. In
view of the importance of 3-substituted-2-oxindole as privileged structures for the preparation
of anticancer agents [21-24], we decided to explore the synthesis and biological evaluation of
novel molecular conjugates reported for the first time and obtained by the structural
modification of 3-substituted-2-oxindole framework through the insertion at its 5-position of
the α-bromoacryloylamido moiety, as a potential site of alkylation with a nucleophilic
aminoacid residue within the kinase or with cellular nucleophiles such as glutathione. All
hybrid molecules were characterized by a common 5-α-bromoacryloylamido-2-oxindole
skeleton, anchored at its 3-position with two different heterocycles (pyrrole and thiophene) by
an exocyclic double bond, to furnish pyrrol-2-yl [3(Z)], 2’-thienyl [4(Z) and 4(E)], 3’-thienyl
[5(Z) and 5(E)], the isomeric 3’-methylthien-2’-yl [6(Z) and 6(E)] and 5’-methylthien-2’-yl
[7(Z) and 7(E)] derivatives reported in the Chart 2. For compound 3, the Z-form was favored
due to the presence of an intramolecular hydrogen bond between the proton at N-1 position of
pyrrole ring and the oxygen atom of the carbonyl group at the C-2 position of 2-oxindole core
[12]. For all compounds (4-7) with thiophene ring at the 3-position of 2-oxindole nucleus, the
two E and Z isomers were obtained and separated by flash chromatography. For derivatives 4
and 5, the 3-exocyclic double bond was linked at the 2’- and 3’-position of thiophene ring,
respectively, while analogues 6 and 7 were characterized by the presence of a methyl group at
the 3’- and 5’-position, respectively, of the thiophen-2-yl moiety. This study deals with (a) the
Structure-Activity Relationship (SAR) of the hybrid compounds 3-7 against a panel of seven
different human cancer cells, (b) characterization of the mechanism of action through their
evaluation of Nek2 inhibitory activity, (c) exploration of the effects induced by selected lead-
compounds 3(Z) and 6(E) on apoptosis, mithocondrial depolarization and caspase-9 activation
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and finally (d) capability of derivatives 3(Z) and 6(E) to induce depletion of glutathione
(GSH) as well as Reactive Oxygen Species (ROS) production.
2. Chemistry
The designed 5-α-bromoacryloylamido-3-substituted indolin-2-one derivatives 3-7 were
synthesized according to the procedure reported in Scheme 1. 5-Nitro-3-substituted-2-
oxindole derivatives 8-12 were prepared by the Knoevenagel aldolic condensation between an
equimolar mixture of the appropriate aldehyde with 5-nitro-2-oxindole in DMF at 80 °C in
the presence of a catalytic amount of piperidine [9]. The pyrrole analogue 8 and the 2’-thienyl
derivatives 9, 11 and 12 existed exclusively as thermodynamically stable Z-isomer forms at
the C-3 exocyclic double bond, while the thien-3’-yl derivative 10 (as a product of
condensation with thiophen-3-carboxaldehyde) was isolated as a mixture of Z and E isomers.
The 5-amino-2-oxindole derivatives 13-17 were generated starting from the corresponding
analogues 8-12 by reduction of the nitro group with iron and ammonium chloride in a
refluxing mixture of water and ethanol (1:4, v/v). While for the pyrrole derivative 8, the
corresponding amino analogues 13 was isolated exclusively in the Z configuration, the pure Z
isomers of nitro derivatives 9, 11 and 12 with 2-thienyl ring undergo partial isomerization to
E isomers during the reduction of the nitro group and the related amino analogues 14, 16 and
17, respectively, were obtained as an inseparable mixture of both Z and E isomer forms. The
5-amino-2-oxindole derivatives 13-18 were converted to the hybrid compounds 3-7 and 19 by
condensation
with
α-bromoacrylic
acid
using
1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide hydrochloride (EDCI) in dimethylformamide.
For hybrid molecules 4-7, the E/Z isomers were separated by silica gel flash chromatography
and the configuration of the exocyclic double bond at the 3-position of the 2-oxindole core
was assigned on results of NMR COSY and NOESY experiments. In general, Z-configurated
compounds showed a strong interaction (NOE effect) between the vinylic proton and the
hydrogen at the C-4 position of the 2-oxindole ring, while this effect was not observed for the
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E-configurated compounds. For these latter isomers, a NOE effect was observed between the
proton at the C-4 position of the benzene portion of the 2-oxindole nucleus and the proton(s)
or methyl on the thiophene ring.
3. Biological results and discussion.
3.1. In vitro antiproliferative activities.
Table 1 summarizes the antiproliferative effects of the novel series of 5-α-
bromoacryloylamido-3-substituted-2-oxindole derivatives 3-7 against the proliferation of a
panel of seven different human cancer cells, using the propiolamide analogue 1e and the 5-(α-
bromoacryloylamido)-2-oxindole derivative 19 as reference compounds. We found that most
of the new hybrid compounds displayed high antiproliferative activity towards a panel of
seven cancer cell lines, with one-digit to double-digit nanomolar IC50 values. In general, the
two leukemia cell lines CCRF-CEM and RS4;11 were the most sensitive to the influence of
conjugates 3-7, with compounds 3(Z), 6(Z) and 6(E) which were active in the single-digit
nanomolar range. Both the amino derivatives 13(Z) and 15(E/Z) did not exhibit tumor cell
growth inhibitory activity (IC50>10 µM), indicating that the α-bromoacryloyl moiety was
essential for the antiproliferative effects of related compounds 3(Z) and 4(Z), respectively,
confirming the validity of molecular hybridization approach. The 3’-methylthien-2’-yl
derivative 6 with Z-configuration [6(Z)] resulted the most active compound of the series
against CCRF-CEM, RS4;11 and HeLa, with IC50 values of 0.5, 0.4 and 420 nM,
respectively, resulting equipotent with the unsubstituted thien-2’-yl and 5’-methylthien-2’-yl
analogues 4(E) and 7(E), respectively, against HL-60 cells.
Based on the biological data reported in the Table 1, many structure-activity relationships
(SARs) could be deduced. We have found that the potency and selectivity of hybrid
compounds 3-7 against the different cancer cell lines depends on the substituent and
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configuration of the double bond at the C-3 position of the 2-oxindole core, with the E- and Z-
isomers which displayed different antiproliferative activities.
This suggests that the pyrrole substituent at the 3-position of 2-oxindole system was
favourable for antiproliferative activity. The pyrrole derivative 3(Z) resulted considerably
more potent against CCRF-CEM and RS4;11 cells, with IC50 values in the single digit
nanomolar range. The comparison between the two pyrrole derivatives 1e and 3(Z) revealed
different potency trends depending on the electrophile at the 5-position of the 2-oxindole core.
Thus, the propinamide derivative 1e resulted from 3- to 70-fold less potent than the α-
bromoacryloylamido counterpart 3(Z). The reduction of activity was more evident against
HL-60 and RS4;11 cell lines, with a 41- and 70-fold reduction of potency, respectively. By
comparison of compounds 8(Z) and 3(Z), the electrophilic α-bromoacryloyl moiety was
essential for a potent antiproliferative activity, as revealed by the sharply reduced activity of
the corresponding amino derivative 8(Z).
The 5-(α-bromoacryloylamido)-2-oxindole derivative 19 was from 2- to 220-fold less potent
than the corresponding 3-((1H-pyrrol-2’-yl)methylene) hybrid conjugate 3(Z). This data
suggests that the hybridization of compound 19 with pyrrole-2-methylene unit, to generate
3(Z), lead to an enhanced antiproliferative activity and further validate the concept that
anticancer activity can be increased by the hybridization of two different pharmacophoric
functional groups.
With the exception of HT-29 cells, the bioisosteric replacement of pyrrole with 2’-thiophene
[compounds 3(Z) and 4(Z), respectively] decreased potency from 1.5- to 12-fold compared
with 3(Z) on six of the seven cancer cell lines, indicating that pyrrole and thiophene are not
bioequivalent at the 3-position of the 2-oxindole ring. For the 2’-thienyl derivative 4, E-
isomer showed improved antiproliferative activity with respect to the Z-form against HL-60,
Jurkat and CCRF-CEM cell lines, with IC50 values of 15, 16 and 10 nM, respectively, while
the potency was reduced on RS4;11, HT-29 and MCF-7 cancer cells. The E and Z isomers
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were equipotent on HeLa cells. Comparing the two regioisomeric thiophene derivatives 4 and
5, in general the 2’-thienyl derivatives were more potent the 3’-thienyl counterpart [4(Z) vs.
5(Z) and 4(E) vs. 5(E)]. In comparing the two isomeric forms of the thien-3’-yl derivative 5
with each other, there was only minor difference in IC50 values against CCRF-CEM, RS4;11,
HeLa and MCF-7 cells, except than Z-isomer was 2-fold more active against the HL-60 cells,
while the E-isomer was 3-, 1.5- and 2- fold more active against Jurkat, HeLa and HT-29 cells.
For the thien-3’-yl derivative 5, comparing the Z- with the E-isomeric form, the latter was
more active against Jurkat and HT-29 cells, in contrast Z-isomer was 2-fold more active than
E-isomer in HL-60 and HT-29 cells, while the two compounds were equipotent against
RS4;11, HeLa and MCF-7 cells.
The data reported in the Table 1 indicated that substitution changes in the thiophene at the 3-
position of 2-oxindole nucleus triggered significantly changes in the antiproliferative activity.
Comparing compounds which shared common geometry at the 3-exocyclic double bond [4(Z)
and 5(Z) vs. 6(Z) and 7(Z), 4(E) and 5(E) vs. 6(E) and 7(E), in general, the introduction of the
small methyl group at the 3’- or 5’-position of the thophene ring improved significantly the
antiproliferative activity, with single digit nanomolar activity against CCRF-CEM cells. The
contribution of the methyl group on thiophene ring to activity [6(Z) vs. 7(Z), 6(E) vs. 7(E)]
was position dependent.
For the Z-isomer of 3’-methylthiophen-2’-yl derivative 6, moving the methyl group from the
3’- to the 5’- position, to furnish derivative 7(Z), resulted in 1.5- to 125-fold of reduction in
antiproliferative activity against five of the seven cancer cell lines, while the two compounds
were equipotent against MCF-7 cells, but compound 7(Z) was 2-fold more active than 6(Z)
against HT-29 cells. By comparison of the two derivatives 6 and 7 which shared common E-
geometry at the exocyclic double bond, the 3’-methyl derivative 6 was from 4- to 5-fold less
potent than 5’-methyl counterpart 7 on four of the seven cancer cell lines, while the two
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compounds were equipotent against CCRF-CEM and HT-29 cells. Only in HL-60 cells,
compound 7 was 3-fold more potent than 6.
Compound 7(Z) was from 2- to 9-fold fold less active than the corresponding E-isomer
against five of the seven cancer cell lines, while the difference between E and Z-form was
minimal in HeLa cells. Only in MCF-7 cells, Z-isomer was more potent than E-isomer (IC50 3
and 4.4 µM). An opposite effect was observed for the regioisomeric 3’-methylthiophen-2’-yl
derivative 6, where the Z-isomeric form had greater potency (from 1.5- to 10-fold) than
compound with E configuration against HL-60, Jurkat, CCRF-CEM and RS4;11 cells,
comparable activity against HeLa and MCF-7 cells and less activity against HT-29 cells.
Derivative 6(Z) exhibited potent activity, with subnanomolar IC50 values against both the
CCRF-CEM and RS4;11 cell lines.
Comparing the E-isomers of 3’- and 5’-methythiophen-2’-yl derivatives 6 and 7, the 3’-
methyl derivative 6(E) was 3-5-fold less active than 5’-methyl analogue 7(E) against HL-60
cells, with a minimal difference in potency between the two derivatives in the CCRF-CEM,
MCF-7 and HT-29 cells, while 6(E) was more potent than 7(E) against Jurkat, RS4;11 and
HeLa cells. With the exception of HeLa, HT-29 and MCF-7 cancer cells, compounds 4(E),
6(Z) and 6(E) were the most active derivatives in the series of thiophene analogues 4-7,
exhibiting IC50 values in the single or double-digit nanomolar range against four of the seven
cancer cell lines. With the exception of both the isomeric forms of thien-3’-yl derivative all
the conjugates prepared were more active than the reference compound 1e.
This study suggested that the two key pharmacophoric elements, namely the α-bromoacryloyl
amido and the heterocycle as well as the configuration of the double bond at the 5- and 3-
positions of 2-oxindole skeleton are important to achieve potent antiproliferative agents.
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3.2 Cytotoxicity of compounds 3(Z) and 6(E) in peripheral blood lymphocytes (PBL)
To obtain a preliminary indication of the cytotoxic potential of these derivatives in normal
human cells, two of the most active compounds [3(Z) and 6(E)] were evaluated in vitro
against peripheral blood lymphocytes (PBL) from healthy donors. Compounds 3(E) and 6(Z)
showed an IC50 of 1.5 µM and 0.45 µM respectively, in quiescent lymphocytes (Table 2),
whereas in lymphocytes in an active phase of proliferation induced by phytohematoagglutinin
(PHA) a mitogenic stimulus showed similar results having an IC50 of 0.85 and 1.22 µM
respectively. Anyway these values are higher than that observed against Jurkat and CCRF-
CEM lymphoblastic cell lines and the results indicate that these compounds have low toxicity
in normal cells in comparison to tumor cells, suggesting a potential for a good therapeutic
index.
3.3 Evaluation of Nek2 inhibitory activity
Since the introduction of the electrophilic propargylamide moiety at the 5-position of 2-
oxindole ring (1e) produce compounds able to inhibit Nek2 [13a], we evaluated if also our
new derivatives were able to inhibit the enzyme. As reference compound 1e was also runned.
As depicted in Figure 1 (panel A, the enzymatic activity evaluated in the presence of test
compounds at the concentration of 1 µM, is reduced by about 50% only in the presence of
compound 3(Z) and in well agreement with previous work also by 1e [13a], while all the other
derivatives present a weak activity (5-20% inhibition). These results suggest that apart
compound 3(Z) in which a inhibition of the enzymatic activity is conserved, although lower
respect to compound 1e, the introduction of the α-bromoacryloyl mojety causes the
disappearance of the inhibitory activity on the kinase.
In addition we also evaluated if the test compounds induce variation of the cell cycle in
particular if they are able to delay mitotic exit. As showed in Figure 1 (Panel B) two of the
most active antiproliferative compounds [3(Z) and 6(E)] were analyzed after 24 h of treatment
in Hela cells. The result indicate that both compounds along with the reference compound 1e
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induce only slight variation of the cell cycle. In particular a decrease of G1 phase of about
20% was observed for compounds 3(Z) and 6(E) at the highest concentration used (1 µM),
while the reference compound 1e do not show any significant variations.
3.4 Compounds 3(E) and 6(Z) induce apoptosis in both Jurkat and Hela cells
With the purpose to investigate the mode of cell death induced by the new derivatives the
annexin-V assay was used in both HeLa and Jurkat cells treated with compounds 3(Z) and
6(E) at different concentrations. As depicted in Figure 2, HeLa cells (Panels A and B), treated
with the two compounds at the concentration of 0.5 and 1 µM for 24 h or 48 h showed an
accumulation of annexin-V positive cells in comparison with the control, in a concentration
and time-dependent manner, and this is indicative of the occurrence of apoptosis. In well
agreement with the antiproliferative potency found in leukemic cells, both compounds induce
apoptosis in Jurkat cells (Panels C and D) but at concentration ten fold lower (100 nM) than
that used in HeLa cells.
3.5 Compounds 3(E) and 6(Z) induce mitochondrial depolarization and caspase-9 activation
in Hela cells
It is well known that an event recognized to be important in apoptosis is the depolarization of
mitochondrial membrane due to mitochondrial permeability transition (MPT) [25, 26]. Thus,
we determined whether 3(E) and 6(Z) induced an alteration of the mitochondrial
transmembrane potential (∆ψmt). ∆ψmt was monitored by flow cytometry using the dye
5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine (JC-1) [27]. As showed in
Figure 3 (Panels A and B), the two compounds induce a significant depolarization starting
after 12-24 h of treatment indicative of mitochondrial pore opening. In particular the effect is
more evident in HeLa (panel A) cells respect to Jurkat (panel B), although in these last we
observed a marked depolarization already after 12 h of treatment.
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To further study the apoptotic pathway we analyzed the cleavage of caspase-9 an effector
caspase involved in inducing mitochondrial damage during apoptosis [28]. As showed in
Figure 3 (panel C) in HeLa cells treated with the two compounds, we observed the appearance
of cleaved fragment suggesting activation of caspase-9.
3.6 Compounds 3(E) and 6(Z) induce ROS production and GSH depletion
To better understand the mechanism of action of the test compounds, we analyzed if they
induce ROS production. Both HeLa and Jurkat cells were treated for 12 h and 24 h with 3(E)
and 6(Z), and the levels of intracellular ROS were monitored by flow cytometry with the
fluorescent probes, 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) [29]. As shown in
Figure 4 (panels A and B), flow cytometric analysis showed a modest increase in the DCF-
positive cells, in the first times of treatment, but increase later with time in a manner similar
to what was observed with the mitochondrial potential. In particular the two compounds do
not present significant differences between them. Given that the mitochondrial membrane
depolarization has been associated with mitochondrial production of ROS [30,31], these
findings suggest that ROS, detected by H2DCFDA, could be produced as a consequence of
mitochondrial damage.
Since it is well known the chemical reactivity of the α-bromoacrylic moiety toward biological
nucleophiles including reduced glutathione (GSH) [17,32,33], we determined whether these
compounds caused a decrease in intracellular GSH content. Therefore, we analyzed HeLa and
Jurkat cells for changes in their GSH levels, by flow cytometry using the fluorescent probe 5-
chloromethylfluorescein diacetate (CMFDA) [34]. As shown in Figure 4 (Panels C and D),
incubations with either 3(Z) or 6(E) for 12 h and 24 h, reduced CMFDA fluorescence, in both
cell lines indicating GSH depletion.
Although intracellular GSH loss is an early feature in the progression of cell death in response
to different apoptotic stimuli and because of its action as a primary intracellular antioxidant in
the cells, a reduction in intracellular GSH content is generally believed to reflect generation of
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ROS [35]. Thus, to prove that ROS are involved in the mechanism of cell death of
compounds 3(Z) and 6(E) we analyzed cell viability in the presence of tocopherol acetate
(TOC), and N-acetylcysteine (NAC), two well known antioxidant. As shown in Figure 5
NAC, but not TOC significantly protected HeLa cells from the antiproliferative effect of the
two compounds increasing cell viability. This suggests that ROS may be involved in the
antiproliferative effects observed with 3(Z) and 6(E).
We also investigated whether 3(Z) and 6(E) may induce DNA damage by examining the
expression of phosphorylated histone H2AX at Ser139 (γH2A.X) upon treatment in HeLa
cells. γH2A.X phosphorylation occurs after DNA double strand break (DSB) induction, thus
identifying γH2A.X as an early sensitive indicator of DSBs [36]. As shown in Figure 5, after
a 24 h treatment both compounds induced γH2A.X expression indicating that they induced
DNA damage probably by oxidative stress.
4. Conclusions
Molecular hybridization, which covalently combines two pharmacophores in a single
molecule, is an effective tool to design highly active novel entities. The α-bromoacryloyl
moiety is an efficient and versatile electrophilic warhead that can be combined with a
recognition moiety to address specific enzymatic target. Several laboratories reported that the
2-oxindole is one of the most effective and newly emerging scaffold that has been frequently
used as pharmacophore to generate molecules that possesses anticancer activity against
various human tumor cell lines. In this context and in continuation of our previous works, the
insertion of a α-bromoacryloyl moiety at the 5-position of the 3-substituted-2-oxindole
framework allowed a higher number of sequential interactions with cellular nucleophiles
which contribute significantly to the increased antiproliferative activity showed by these
hybrid molecules. The structure-activity relationship study highlights the importance of
generate hybrid structures from these two pharmacophores, where biological activity was
achieved due to the effect of each entity in the unique structure resulted of the molecular
13

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hybridization. Our findings indicate the importance of substituent at the 3-position of 2-
oxindole skeleton for activity and selectivity against different cancer cell lines. In general,
there was a trend that the antiproliferative activities of conjugates 3-7 were somewhat more
pronounced against HL-60, Jurkat, CCRF-CEM and RS4;11 as compared with HeLa, HT-29
and MCF-7 cancer cells. Specific effects seemed to vary with the cell line tested. Thus,
compounds 4(E), 6(Z) and 7(E) had the greatest activity on HL-60 cells, 4(E) with Jurkat
cells, 6(Z) with CCRF-CEM, RS4;11 and HeLa cells, 4(Z) with HT-29 cells and 3(Z) with
MCF-7.
Comparing the antiproliferative activities of compounds 1e and 3(Z), the propiolamide moiety
was less efficacious than the α-bromoacryloylamido group as electrophile with biological
nucleophiles. Importantly, 1e is an irreversible inhibitor of the Nek2 kinase, whereas 3(Z) is
endowed with lower activity in the enzymatic activity. Thus while the insertion of the α-
bromoacryloyl moiety dramatically reduced the inhibitory activity on this enzyme at the same
time significantly increase the cell growth inhibitory activity.
Preliminary studies devoted to the elucidation of mechanism of action demonstrated that the
most potent hybrid compounds strongly induce apoptosis that follows the mitochondrial
pathway, as demonstrated by mitochondrial depolarization and caspase-9 cleavage. More
precisely their activity seems to be related to depletion of intracellular GSH and consequent
imbalance in the antioxidant defense of the cells that lead to ROS production [37]. If
intracellular GSH depletion occur through a direct chemical reaction or through interfering
with the enzymatic synthesis of GSH remains to be determined. Considering that human
tumors often show increased GSH content compared to normal tissues, the interaction of
compounds 3(E) and 6(Z) with glutathione suggest that these molecules may have a potential
value for the treatment of cancers characterized by constitutive or therapy-induced
overexpression of glutathione/glutathione-S-tranferase (GST) levels. The potent anticancer
activity and synthetic accessibility strongly encourage further optimization of hybrid
14

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compound 3(Z) as lead to develop more potent irreversible Nek2 kinase inhibitors. Further,
we believe that with interesting in vivo activity this series of new molecules is important and
should be pursued.
5. Experimental protocols
5.1. Chemistry
5.1.1. Materials and Methods
1H experiments were recorded on either a Bruker AC 200 or a Varian 400 Mercury Plus
spectrometer, while 13C NMR spectra were recorded on Varian 400 Mercury Plus
spectrometer. Chemical shifts (δ) are given in ppm upfield from tetramethylsilane as internal
standard, and the spectra were recorded in appropriate deuterated solvents, as indicated.
Positive-ion electrospray ionization (ESI) mass spectra were recorded on a double-focusing
Finnigan MAT 95 instrument with BE geometry. Melting points (mp) were determined on a
1
Buchi-Tottoli apparatus and are uncorrected. All products reported showed H and 13C NMR
spectra in agreement with the assigned structures. The purity of tested compounds was
determined by combustion elemental analyses conducted by the Microanalytical Laboratory
of the Chemistry Department of the University of Ferrara with a Yanagimoto MT-5 CHN
recorder elemental analyzer. All tested compounds yielded data consistent with a purity of at
least 95% as compared with the theoretical values. Standard syringe techniques were used for
transferring dry solvents. Reaction courses and product mixtures were routinely monitored by
TLC on silica gel (precoated F
254
Merck plates), and compounds were visualized with
aqueous KMnO . Flash chromatography was performed using 230-400 mesh silica gel and
4
the indicated solvent system. Organic solutions were dried over anhydrous Na2SO4.
15

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5.1.2. General procedure A for the synthesis of compounds 8-12.
A reaction mixture of 5-nitroindolin-2-one (356 mg, 2 mmol), the appropriate aldehyde (2.4
mmol, 1.2 equiv.) and piperidine (21 µL, 0.2 mmol, 0.1 equiv,) in DMF (5 mL) was stirred at
80 °C for 4h. The solvent was removed under reduced pressure, the resulting residue was
suspended in ethyl ether (15 mL) and filtered, to furnish the target compound used without
any further purification for the next reaction.
5.1.2.1. (Z)-3-((1H-pyrrol-2-yl)methylene)-5-nitroindolin-2-one (8). Following general
procedure A, compound 8(Z) was isolated as a brown solid. Yield: 91%, mp 184-186 ºC. 1H-
NMR (200 MHz, d6-DMSO) δ: 6.43 (m, 1H), 6.97 (m, 1H), 7.03 (d, J=8.8 Hz, 1H), 7.47 (m,
1H), 8.06 (dd, J=8.8 and 2.6 Hz, 1H), 8.17 (s, 1H), 8.59 (d, J=2.2 Hz, 1H), 11.2 (bs, 1H),
13.15 (s, 1H). MS (ESI): [M+1]+=256.2.
5.1.2.2. (Z)-5-Nitro-3-(thiophen-2-ylmethylene)indolin-2-one (9). Following general
procedure A, compound 9(Z) was obtained as a brown solid. Yield: >95%, mp 241-243 ºC.
1H-NMR (200 MHz, d6-DMSO) δ: 7.01 (m, 1H), 7.03 (d, J=8.8 Hz, 1H), 7.47 (m, 1H), 8.03
(m, 2H), 8.17 (dd, J=8.8 and 2.4 Hz, 1H), 8.56 (s, 1H), 8.66 (d, J=2.2 Hz, 1H), 11.2 (bs, 1H).
MS (ESI): [M+1]+=273.3.
5.1.2.3. (Z)-and (E) isomer mixture of 5-nitro-3-(thiophen-3-ylmethylene)indolin-2-one (10).
Following general procedure A, compound 10 (E/Z mixture) were obtained as a brown solid.
Yield: >95%, mp 226-232 ºC. 1H-NMR (200 MHz, d6-DMSO) δ: 6.99 (d, J=8.8 Hz, 1H), 7.01
(m, 1H), 7.08 (d, J=8.8 Hz, 1H), 7.47 (m, 1H), 7.67 (m, 2H), 7.84 (m, 2H), 8.12 (m, 2H), 8.18
(s, 1H), 8.28 (s, 1H), 8.51 (d, J=2.2 Hz, 1H), 8.65 (d, J=2.2 Hz, 1H), 11.3 (bs, 2H). MS (ESI):
[M+1]+=273.4.
5.1.2.4. (Z)-3-((3-Methylthiophen-2-yl)methylene)-5-nitroindolin-2-one (11). Following
general procedure A, compound 11(Z) was obtained as a brown solid. Yield: 91%, mp 283 ºC.
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1H-NMR (200 MHz, d6-DMSO) δ: 2.58 (s, 3H), 7.01 (d, J=8.6 Hz, 1H), 7.09 (d, J=5.0 Hz,
1H), 7.87 (d, J=5.0 Hz, 1H), 8.09 (dd, J=8.6 and 2.2 Hz, 1H), 8.24 (s, 1H), 8.82 (d, J=2.2 Hz,
1H), 10.8 (bs, 1H). MS (ESI): [M+1]+=287.3.
5.1.2.5. (Z)-3-((5-methylthiophen-2-yl)methylene)-5-nitroindolin-2-one (12). Following
general procedure A, compound 12(Z) was obtained as a purple solid. Yield: 92%, mp 252-
254 ºC. 1H-NMR (200 MHz, d6-DMSO) δ: 2.56 (s, 3H), 7.03 (m, 2H), 7.81 (d, J=4.0 Hz, 1H),
8.11 (dd, J=8.6 and 2.2 Hz, 1H), 8.62 (d, J=2.2 Hz, 1H), 8.62 (d, J=2.2 Hz, 1H), 11.2 (bs,
1H). MS (ESI): [M+1]+=287.3.
5.1.3. General procedure B for the synthesis of compounds 13-18.
To a suspension of 5-nitro-3-substituted-2-oxindole derivatives 8-12 or commercially
available 5-nitroindolin-2-one (1 mmol) in a mixture of ethanol and water (10 mL, 4:1 v/v)
was added iron powder (560 mg. 10 mmol, 10 equiv.) and NH4Cl (134 mg, 2.5 mmol, 2.5
equiv.). The mixture was refluxed for 2 h, cooled and filtered through Celite. The filtrate was
diluted with water (5 mL) and extracted with dichloromethane (3 x 10 mL). The combined
organic layers were washed with water (10 mL) and brine (10 mL), dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The crude residue was purified by
crystallization with ethyl ether.
5.1.3.1. (Z)-3-((1H-pyrrol-2-yl)methylene)-5-aminoindolin-2-one (13). Following general
1
procedure B, compound 13(Z) was isolated as a red solid. Yield: 58%, mp 148-150 ºC. H-
NMR (200 MHz, d6-DMSO) δ: 4.66 (bs, 2H), 6.29 (m, 1H), 6.41 (dd, J=8.0 and 1.2 Hz, 1H),
6.54 (d, J=8.0 Hz, 1H), 6.79 (m, 2H), 7.28 (d, J=1.2 Hz, 1H), 7.44 (s, 1H), 10.4 (s, 1H), 13.4
13
(s, 1H). C NMR (100 MHz, d6-DMSO) δ: 168.97, 143.42, 129.97, 129.48, 125.70, 125.07,
124.96, 119.58, 118.09, 113.23, 111.09, 109.91, 104.74. MS (ESI): [M+1]+=226.2.
17

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5.1.3.2. (Z) and (E) isomer mixture of 5-amino-3-(thiophen-2-ylmethylene)indolin-2-one (14).
Following general procedure B, the mixture of the two isomers (Z) and (E) was obtained as a
1
brown solid. Yield: 65%, mp 180-182 ºC. H-NMR (200 MHz, d6-DMSO) δ: 4.70 (bs, 4H),
6.44 (d, J=8.0 Hz, 1H), 6.54 (m, 2H), 6.59 (d, J=8.0 Hz, 1H), 6.87 (s, 1H), 7.18 (t, J=2.0 Hz,
1H), 7.27 (t, J=2.0 Hz, 1H), 7.63 (d, J=1.4 Hz, 1H), 7.74 (d, J=1.4 Hz, 1H), 7.81 (bs, 2H),
7.87 (d, J=1.4 Hz, 1H), 7.95 (d, J=2.2 Hz, 1H), 8.01 (d, J=2.2 Hz, 1H), 10.1 (bs, 2H),. MS
(ESI): [M+1]+=243.3.
5.1.3.3. (Z) and (E) isomer mixtutre of 5-amino-3-(thiophen-3-ylmethylene)indolin-2-one
(15). Following general procedure B, the mixture of the two isomers (Z) and (E) was obtained
1
as a red solid. Yield: 58%, mp 64-66 ºC. H-NMR (200 MHz, d6-DMSO) δ: 4.75 (bs, 4H),
6.48 (m, 3H), 6.60 (d, J=8.0 Hz, 1H), 6.86 (d, J=1.4 Hz, 1H), 7.12 (d, J=2.0 Hz, 1H), 7.49 (d,
J=8.0 Hz, 1H), 7.51 (s, 1H), 7.58 (m, 2H), 7.71 (dd, J=8.0 and 2.4 Hz, 1H), 8.06 (d, J=1.4 Hz,
1H), 8.09 (m, 1H), 8.80 (d, J=2.4 Hz, 1H),10.1 (bs, 1H), 10.2 (bs, 1H). MS (ESI):
[M+1]+=243.3.
5.1.3.4. (Z) and (E) isomers of 5-amino-3-((3-methylthiophen-2-yl)methylene)indolin-2-one
(16). Following general procedure B, the mixture of the two isomers (Z) and (E) was obtained
1
as a yellow oil. Yield: 62%. H-NMR (200 MHz, d6-DMSO) δ: 2.38 (s, 3H). 2.40 (s, 3H).
4.77 (bs, 4H), 6.52 (m, 4H), 6.96 (d, J=2.0 Hz, 1H), 7.05 (d, J=5.2 Hz, 1H), 7.14 (d, J=5.2
Hz, 1H), 7.49 (d, J=2.0 Hz, 1H), 7.63 (d, J=6.0 Hz, 2H), 7.71 (d, J=4.6 Hz, 1H), 7.84 (d,
J=5.4 Hz, 1H), 10.1 (bs, 1H), 10.2 (s, 1H). MS (ESI): [M+1]+=287.3.
5.1.3.5. (Z) and (E) isomers of 5-amino-3-((5-methylthiophen-2-yl)methylene)indolin-2-one
(17). Following general procedure B, the mixture of the two isomers (Z) and (E) was obtained
1
as a brown solid. Yield: 65%, mp 180-182 ºC. H-NMR (200 MHz, d6-DMSO) δ: 2.58 (s,
3H). 2.59 (s, 3H). 4.66 (bs, 2H), 4.80 (bs, 2H), 6.42 (d, J=2.0 Hz, 1H), 6.46 (d, J=2.0 Hz, 1H),
6.51 (m, 1H), 6.52 (d, J=3.4 Hz, 1H), 6.55 (d, J=3.4 Hz, 1H), 6.61 (s, 1H), 6.86 (d, J=3.4 Hz,
18

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1H), 6.91 (dd, J=8.6 and 2.2 Hz, 1H), 7.02 (dd, J=8.6 and 2.2 Hz, 1H), 7.52 (d, J=3.4 Hz,
1H), 7.57 (dd, J=8.6 and 2.2 Hz, 1H), 7.70 (m, 1H), 10.1 (bs, 2H). MS (ESI): [M+1]+=287.3.
5.1.3.6. 5-Aminoindolin-2-one (18). Following general procedure B, starting from the
commercially available 5-nitroindolin-2-one, the corresponding 5-aminoindolin-2-one 18 was
1
isolated as a brown solid. Yield: 78%, mp 137-138 ºC. H-NMR (200 MHz, d6-DMSO) δ:
3.31 (s, 2H). 4.75 (bs, 2H), 6.32 (dd, J=8.6 and 2.2 Hz, 1H), 6.48 (m, 2H), 9.93 (bs, 1H). MS
(ESI): [M+1]+=149.2.
5.1.4. General procedure C for the synthesis of compounds 3-7 and 19.
To an ice-cooled solution of amino derivative 13-18 (1.00 mmol) in dry DMF (5.0 mL) were
added a mixture of EDCI (383 mg, 2.00 mmol) and α-bromoacrylic acid (2.00 mmol, 306
mg). The reaction mixture was stirred at room temperature for 18 h and then concentrated
under reduced pressure. The residue was dissolved with a mixture of CH2Cl2 (15 mL) and
water (5 mL), and the organic phase was washed with brine (5 mL), dried over Na2SO4 and
evaporated to dryness in vacuo. The resulting crude residue was purified by column
chromatography on silica gel.
5.1.4.1. (Z)-N-(3-((1H-pyrrol-2-yl)methylene)-2-oxoindolin-5-yl)-2-bromoacrylamide (3).
Following general procedure C, after workup as described previously, the residue was
purified by flash chromatography on silica gel using light petroleum ether: EtOAc 1:1 as
1
eluent, affording compound 3(Z) as an orange solid. Yield: 56%, mp 219-221 ºC. H-NMR
(400 MHz, d6-DMSO) δ: 6.29 (d, J=3.2 Hz, 1H), 6.35 (dd, J=1.8 and 1.2 Hz, 1H), 6.75 (d,
J=3.2 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 6.90 (d, J=1.8 Hz, 1H), 7.27 (dd, J=8.4 and 2.0 Hz,
1H), 7.37 (d, J=1.2 Hz, 1H), 7.66 (s, 1H), 7.92 (d, J=2.0 Hz, 1H), 10.1 (s, 1H), 10.9 (s, 1H),
13.3 (s, 1H). 13C NMR (100 MHz, d6-DMSO) δ: 109.30, 111.40, 111.97, 116.52, 120.25,
120.75, 125.07, 125.18, 125.48, 125.87, 126.39, 129.39, 132.02, 135.74, 160.59, 169.21. MS
19

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(ESI): [M]+=358.09, [M+2]+=360.20. Anal. (C16H12BrN3O2) C, H, N. Anal. calcd for
C16H12BrN3O2. C, 53.65; H, 3.38; N, 11.73; found: C, 53.48; H, 3.21; N, 11.58.
5.1.4.2. (Z)-2-Bromo-N-(2-oxo-3-(thiophen-2-ylmethylene)indolin-5-yl)acrylamide (4) and
(E)-2-bromo-N-(2-oxo-3-(thiophen-2-ylmethylene)indolin-5-yl)acrylamide (4). Following
general procedure C, after workup as described previously, the residue was purified by
gradient flash chromatography on silica gel using EtOAc-light petroleum ether from 4:6 to
1:1 as eluent, affording compound 4(Z) (EtOAc-light petroleum ether 4:6) and 4(E) (EtOAc-
light petroleum ether 1:1).
Compound 4(Z). Yellow solid. Yield: 43%, mp 186-188 ºC. 1H-NMR (400 MHz, d6-DMSO)
δ: 6.29 (d, J=2.8 Hz, 1H), 6.76 (d, J=2.8 Hz, 1H), 6.82 (d, J=8.4 Hz, 1H), 7.22 (dd, J=5.2 and
3.6 Hz, 1H), 7.30 (dd, J=8.4 and 2.0 Hz, 1H), 7.89 (d, J=5.2 Hz, 1H), 7.97 (m, 2H), 8.05 (s,
13
1H), 10.2 (s, 1H), 10.6 (s, 1H). C NMR (100 MHz, d6-DMSO) δ: 109.25, 113.18, 121.39,
121.99, 124.23, 125.16, 125.59, 127.41, 128.45, 131.75, 134.63, 137.14, 137.30, 138.08,
160.64, 167.35. MS (ESI): [M]+=375.19, [M+2]+=377.17. Anal. calcd for C16H11BrN2O2S. C,
51.21; H, 2.95; N, 7.47; found: C, 51.03; H, 2.76; N, 7.35.
Compound 4(E). Yellow solid. Yield: 21%, mp 167-169 ºC. 1H-NMR (400 MHz, d6-DMSO)
δ: 6.29 (d, J=3.2 Hz, 1H), 6.73 (d, J=3.2 Hz, 1H), 6.87 (d, J=8.0 Hz, 1H), 7.32 (dd, J=5.2 and
3.6 Hz, 1H), 7.46 (dd, J=8.0 and 2.0 Hz, 1H), 7.79 (s, 1H), 7.83 (d, J=3.6 Hz, 1H), 8.05 (d,
13
J=5.2 Hz, 1H), 8.64 (d, J=2.0 Hz, 1H), 10.3 (s, 1H), 10.6 (s, 1H). C NMR (100 MHz, d6-
DMSO) δ: 109.68, 116.22, 120.58, 122.45, 123.24, 125.05, 125.54, 127.50, 128.69, 132.09,
132.35, 136.08, 136.98, 139.14, 160.82, 169.23. MS (ESI): [M]+=375.31, [M+2]+=377.17.
Anal. calcd for C16H11BrN2O2S. C, 51.21; H, 2.95; N, 7.47; found: C, 50.98; H, 2.78; N, 7.28.
5.1.4.3. (Z)-2-Bromo-N-(2-oxo-3-(thiophen-3-ylmethylene)indolin-5-yl)acrylamide (5) and
(E)-2-bromo-N-(2-oxo-3-(thiophen-3-ylmethylene)indolin-5-yl)acrylamide (5). Following
general procedure C, after workup as described previously, the residue was purified by
20

ACCEPTED MANUSCRIPT
gradient flash chromatography on silica gel using EtOAc-light petroleum ether from 4:6 to
1:1 as eluent, affording compound 5(Z) (EtOAc-light petroleum ether 4:6) and 5(E) (EtOAc-
light petroleum ether 1:1).
Compound 5(Z). Yellow solid. Yield: 24%, mp 204-206 ºC. 1H-NMR (400 MHz, d6-DMSO)
δ: 6.29 (d, J=2.8 Hz, 1H), 6.75 (d, J=2.8 Hz, 1H), 6.80 (d, J=8.4 Hz, 1H), 7.31 (dd, J=8.0 and
2.0 Hz, 1H), 7.62 (dd, J=4.8 and 2.8 Hz, 1H), 7.78 (s, 1H), 7.95 (d, J=2.0 Hz, 1H), 8.15 (d,
13
J=5.2 Hz, 1H), 8.86 (d, J=2.8 Hz, 1H), 10.2 (s, 1H), 10.6 (s, 1H). C NMR (100 MHz, d6-
DMSO) δ: 109.72, 113.61, 122.57, 124.51, 125.23, 125.69, 126.14, 126.59, 129.79, 131.79,
132.37, 134.23, 136.78, 137.82, 161.18, 167.87. MS (ESI): [M]+=375.31, [M+2]+=377.29.
Anal. calcd for C16H11BrN2O2S. C, 51.21; H, 2.95; N, 7.47; found: C, 50.96; H, 2.81; N, 7.30.
Compound 5(E). Orange solid. Yield: 36%, mp 185-187 ºC. 1H-NMR (400 MHz, d6-DMSO)
δ: 6.27 (d, J=2.8 Hz, 1H), 6.69 (d, J=2.8 Hz, 1H), 6.85 (d, J=8.4 Hz, 1H), 7.49 (dd, J=8.4 and
1.6 Hz, 1H), 7.56 (dd, J=5.2 and 1.2 Hz, 1H), 7.58 (s, 1H), 7.76 (m, 1H), 8.16 (m, 2H), 10.2
(s, 1H), 10.6 (s, 1H). 13C NMR (100 MHz, d6-DMSO) δ: 110.18, 116.38, 121.48, 122.94,
125.57, 126.05, 126.15, 127.99, 129.13, 130.20, 131.02, 132.53, 136.08, 139.71, 161.29,
169.65. MS (ESI): [M]+=375.31, [M+2]+=377.29. Anal. calcd for C16H11BrN2O2S. C, 51.21;
H, 2.95; N, 7.47; found: C, 51.02; H, 2.77; N, 7.32.
5.1.4.4. (Z)-2-Bromo-N-(3-((3-methylthiophen-2-yl)methylene)-2-oxoindolin-5-yl)acrylamide
(6) and (E)-2-bromo-N-(3-((3-methylthiophen-2-yl)methylene)-2-oxoindolin-5-yl)acrylamide
(6). Following general procedure C, after workup as described previously, the residue was
purified by gradient flash chromatography on silica gel using EtOAc-light petroleum ether
from 4:6 to 1:1 as eluent, affording compound 6(Z) (EtOAc-light petroleum ether 4:6) and
6(E) (EtOAc-light petroleum ether 1:1).
Compound 6(Z). Yellow solid. Yield: 38%, mp 206-208 ºC. 1H-NMR (400 MHz, d6-DMSO)
δ: 2.49 (s, 3H), 6.29 (d, J=3.2 Hz, 1H), 6.76 (d, J=3.2 Hz, 1H), 6.81 (d, J=8.0 Hz, 1H), 7.09
(d, J=5.2 Hz, 1H), 7.39 (dd, J=8.4 and 2.0 Hz, 1H), 7.79 (d, J=5.2 Hz, 1H), 7.82 (s, 1H), 7.92
21

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(d, J=2.0 Hz, 1H), 10.1 (s, 1H), 10.6 (s, 1H). 13C NMR (100 MHz, d6-DMSO) δ: 15.14,
109.63, 113.57, 120.80, 122.34, 124.62, 125.07, 125.71, 126.17, 130.65, 131.33, 132.23,
133.13, 137.67, 146.46, 161.15, 167.93. MS (ESI): [M]+=398.17, [M+2]+=400.22. Anal. calcd
for C17H13BrN2O2S. C, 52.45; H, 3.37; N, 7.20; found: C, 52.28; H, 3.18; N, 7.02.
Compound 6(E). Orange solid. Yield: 33%, mp 189-191 ºC. 1H-NMR (400 MHz, d6-DMSO)
δ: 2.42 (s, 3H), 6.28 (d, J=2.8 Hz, 1H), 6.72 (d, J=2.8 Hz, 1H), 6.86 (d, J=8.4 Hz, 1H), 7.18
(d, J=4.8 Hz, 1H), 7.44 (d, J=8.4 and 2.0 Hz, 1H), 7.76 (s, 1H), 7.95 (d, J=4.8 Hz, 1H), 8.62
13
(d, J=2.0 Hz, 1H), 10.3 (s, 1H), 10.6 (s, 1H). C NMR (100 MHz, CDCl3) δ: 15.07, 110.13,
116.94, 121.16, 122.96, 123.32, 125.60, 126.05, 126.14, 130.53, 131.15, 131.81, 132.53,
139.63, 145.38, 161.33, 169.86. MS (ESI): [M]+=398.17, [M+2]+=400.22. Anal. calcd for
C17H13BrN2O2S. C, 52.45; H, 3.37; N, 7.20; found: C, 52.33; H, 3.22; N, 7.01.
5.1.4.5. (Z)-2-Bromo-N-(3-((5-methylthiophen-2-yl)methylene)-2-oxoindolin-5-yl)acrylamide
(7) and (E)-2-bromo-N-(3-((5-methylthiophen-2-yl)methylene)-2-oxoindolin-5-yl)acrylamide
(7). Following general procedure C, after workup as described previously, the residue was
purified by gradient flash chromatography on silica gel using EtOAc-light petroleum ether
from 4:6 to 1:1 as eluent, affording compound 7(Z) (EtOAc-light petroleum ether 4:6) and
7(E) (EtOAc-light petroleum ether 1:1).
Compound 7(Z). Orange solid. Yield: 52%, mp 216-218 ºC. 1H-NMR (400 MHz, d6-DMSO)
δ: 2.48 (s, 3H), 6.29 (d, J=3.2 Hz, 1H), 6.75 (d, J=3.2 Hz, 1H), 6.80 (d, J=8.0 Hz, 1H), 6.95
(dd, J=4.0 and 2.8 Hz, 1H), 7.27 (dd, J=8.0 and 2.0 Hz, 1H), 7.76 (d, J=4.0 Hz, 1H), 7.92 (m,
2H), 10.1 (s, 1H), 10.6 (s, 1H). 13C NMR (100 MHz, CDCl3) δ: 15.92, 109.66, 113.44,
120.46, 122.14, 124.95, 125.72, 126.10, 126.81, 129.36, 132.21, 135.83, 137.60, 139.39,
149.64, 161.14, 167.96. MS (ESI): [M]+=398.31, [M+2]+=400.20. Anal. calcd for
C17H13BrN2O2S. C, 52.45; H, 3.37; N, 7.20; found: C, 52.29; H, 3.16; N, 6.98.
Compound 7(E). Orange solid. Yield: 24%, mp 208-210 ºC. 1H-NMR (400 MHz, d6-DMSO)
δ: 2.50 (s, 3H), 6.29 (d, J=2.8 Hz, 1H), 6.73 (d, J=2.8 Hz, 1H), 6.86 (d, J=8.0 Hz, 1H), 7.05
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(dd, J=3.6 and 1.6 Hz, 1H), 7.48 (dd, J=8.0 and 2.0 Hz, 1H), 7.66 (d, J=3.6 Hz, 1H), 7.69 (s,
13
1H), 8.52 (d, J=2.0 Hz, 1H), 10.3 (s, 1H), 10.6 (s, 1H). C NMR (100 MHz, d6-DMSO) δ:
16.09, 110.09, 116.57, 121.29, 122.42, 122.79, 125.60, 126.05, 128.08, 128.34, 132.53,
135.62, 137.26, 139.46, 147.47, 161.39, 169.88. MS (ESI): [M]+=398.31, [M+2]+=400.20.
Anal. calcd for C17H13BrN2O2S. C, 52.45; H, 3.37; N, 7.20; found: C, 52.33; H, 3.15; N, 7.03.
5.1.4.6. 2-Bromo-N-(2-oxoindolin-5-yl)acrylamide (19). Following general procedure C, after
workup as described previously, the residue was purified by flash chromatography on silica
gel using light petroleum ether: EtOAc 6:4 as eluent, affording compound 19 as a colour
1
cream solid. Yield: 73%, mp 194-195 ºC. H-NMR (400 MHz, d6-DMSO) δ: 3.48 (s, 2H),
6.26 (d, J=2.8 Hz, 1H), 6.69 (d, J=2.8 Hz, 1H), 6.75 (d, J=8.4 Hz, 1H), 7.38 (d, J=8.4 Hz,
1H), 7.51 (s, 1H), 10.2 (s, 1H), 10.4 (s, 1H). 13C NMR (100 MHz, d6-DMSO) δ: 35.90,
108.72, 117.64, 119.97, 125.13, 125.35, 125.96, 132.08, 140.15, 160.61, 176.22. MS (ESI):
[M]+=281.3, [M+2]+=283.3. Anal. calcd for C11H9BrN2O2. C, 47.00; H, 3.23; N, 9.97; found:
C, 46.81; H, 3.15; N, 9.78.
5.2. Biological assays
5.2.1. Cell growth conditions and antiproliferative assay.
Human T-leukemia (CCRF-CEM and Jurkat) and human B-leukemia (RS4;11) and human
promielocytic leukemia (HL-60) cells were grown in RPMI-1640 medium (Gibco, Milano,
Italy). Breast adenocarcinoma (MCF-7), human cervix carcinoma (HeLa), and human colon
adenocarcinoma (HT-29) cells were grown in DMEM medium (Gibco, Milano, Italy), all
supplemented with 115 units/mL penicillin G (Gibco, Milano, Italy), 115 µg/mL streptomycin
(Invitrogen, Milano, Italy), and 10% fetal bovine serum (Invitrogen, Milano, Italy). Stock
solutions (10 mM) of the different compounds were obtained by dissolving them in DMSO.
Individual wells of a 96-well tissue culture microtiter plate were inoculated with 100 µL of
complete medium containing 8 × 103 cells. The plates were incubated at 37 °C in a humidified
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5% CO2 incubator overnigth prior to the experiments. After medium removal, 100 µL of fresh
medium containing the test compound at different concentrations was added to each well in
triplicate and incubated at 37 °C for 72 h. Cell viability was assayed by the (3-(4, 5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test as previously described [38].
Some experiments were also performed in the presence of DL-α-tocopherol acetate (TOC) or
N-acetyl-L-cysteine (NAC), all purchased from Sigma-Aldrich (Milano, Italy).
Peripheral blood lymphocytes (PBL) from healthy donors were obtained from human
peripheral blood (leucocyte rich plasma- buffy coats) from healthy volunteers using the
Lymphoprep (Fresenius KABI Norge AS) gradient density centrifugation.
Buffy coats were obtained from the Blood Transfusion Service, Azienda Ospedaliera of
Padova and provided at this institution for research purposes. Therefore, no informed consent
was further needed. In addition, buffy coats were provided without identifiers. The
experimental procedures were carried out in strict accordance with approved guidelines.
After extensive washing, cells were resuspended (1.0 x 106 cells/mL) in RPMI-1640 with
10% FBS and incubated overnight. For cytotoxicity evaluations in proliferating PBL cultures,
non-adherent cells were resuspended at 5 x 105 cells/mL in growth medium, containing 2.5
µg/mL PHA (Irvine Scientific). Different concentrations of the test compounds were added,
and viability was determined 72 h later by the MTT test. For cytotoxicity evaluations in
resting PBL cultures, non-adherent cells were resuspended (5 x 105 cells/mL) and treated for
72 h with the test compounds, as described above.
5.2.2. Nek2 kinase activity assay.
Kinase assay was performed using the bioluminescent ADP-GloTM kinase assay (Promega,
Milano Italy), following manufacturer’s instructions. Assay was performed with the test
compounds at the final concentration of 1 µM
.
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5.2.3. Flow Cytometric Analysis of Cell Cycle Distribution.
5 × 105 HeLa or Jurkat cells were treated with different concentrations of the test compounds
for 24 h. After the incubation period, the cells were collected, centrifuged, and fixed with ice-
cold ethanol (70%). The cells were then treated with lysis buffer containing RNase A and
0.1% Triton X-100 and then stained with PI. Samples were analyzed on a Cytomic FC500
flow cytometer (Beckman Coulter). DNA histograms were analyzed using MultiCycle for
Windows (Phoenix Flow Systems).
5.2.4. Apoptosis Assay.
Cell death was determined by flow cytometry of cells double stained with annexin V/FITC
and PI. The Coulter Cytomics FC500 (Beckman Coulter) was used to measure the surface
exposure of PS on apoptotic cells according to the manufacturer’s instructions (Annexin-V
Fluos, Roche Diagnostics).
5.2.5. Western Blot Analysis.
HeLa cells were incubated in the presence of test compounds and after different times, were
collected, centrifuged, and washed two times with ice cold phosphate buffered saline (PBS).
The pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 30 min,
lysates were centrifuged at 15000 x g at 4 °C for 10 min. The protein concentration in the
supernatant was determined using the BCA protein assay reagents (Pierce, Italy). Equal
amounts of protein (10 µg) were resolved using sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) (7.5−15% acrylamide gels) and transferred to Immobilon-P
membrane (Millipore, Milano, Italy). Membranes were blocked with a bovine serum albumin
(BSA) solution (BSA 3%, Tween 0.1% in PBS), the membranes being gently rotated for two
hours at room temperature. Membranes were then incubated overnight at 4 °C with primary
antibodies against caspase-9, and γH2AX (Cell Signaling), or β-actin (Sigma-Aldrich) for 2 h
at room temperature. Membranes were next incubated with peroxidase labeled secondary
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antibodies for 60 min. All membranes were visualized using ECL Select (GE Healthcare) and
images were acquired using an Uvitec-Alliance imaging system (Uvitec, Cambridge, UK). To
ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin
antibody. Quantitative analysis of western blot was performed by Image J software and the
results were normalized to β-actin.
5.2.6. Assessment of mitochondrial changes and ROS production.
The mitochondrial membrane potential was measured with the lipophilic cation JC-1
(Molecular Probes, Eugene, OR, USA), while the production of ROS was followed by flow
cytometry using the fluorescent dye H2DCFDA (Molecular Probes), as previously
described[39].
5.2.7. Detection of the intracellular GSH content.
Cellular GSH levels were analyzed using CMFDA (Molecular Probes) [40]. Cells were
treated with the test compound for different times. Cells were harvested, centrifuged and
incubated in the presence of a solution of 5 µM CMFDA at 37 °C for 30 min. Cytoplasmic
esterases convert non-fluorescent CMFDA to fluorescent 6-chloromethylfluorescein, which
can then react with GSH. Fluorescence intensity was determined by flow cytometry.
Acknowledgments.
The authors gratefully acknowledged Alberto Casolari for the excellent technical assistance.
Supplementary data. Spectra of compounds 3-7. Supplementary data associated with this
article can be found in the online version.
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