Synthesis of novel 3,5-disubstituted-2-oxindole derivatives as antitumor agents against human nonsmall cell lung cancer

Article abstract of DOI:10.1021/ml400162g

This study was aimed at investigating the antitumor activity of novel 2-oxindole derivatives against a well-characterized human nonsmall cell lung cancer (NSCLC) cell line. Test compounds produced an antiproliferative activity in the low micromolar/submicromolar range of concentrations and significantly induced typical apoptotic morphology with cell shrinkage, nuclear condensation and fragmentation, and rupture of cells into debris in a relatively low percentage of A549 cells. Cell cycle arrest occurred at the G1/S phase (1a and 2), and Akt phosphorylation was significantly inhibited at Thr308 and Ser473. The most active compound (1a) has an IC₅₀ 6-fold lower than the Akt inhibitor, perifosine. These data suggest that the new compounds may be cytostatic and may have maximum clinical effects in NSCLC patients who do not respond to EGFR inhibitors. These findings prompt us to further explore the oxindole structure as leading scaffold to design new molecules with potent antitumor activity against NSCLC.

Full text of DOI:10.1021/ml400162g

Letter
pubs.acs.org/acsmedchemlett
Synthesis of Novel 3,5-Disubstituted-2-oxindole Derivatives As
Antitumor Agents against Human Nonsmall Cell Lung Cancer
†
,∥
†,§,∥
‡
‡
§
‡
Giulia Nesi, Simona Sestito,
Valentina Mey, Simona Ricciardi, Marco Falasca, Romano Danesi,
†
†
†
,†
Annalina Lapucci, Maria C. Breschi, Stefano Fogli, and Simona Rapposelli*
†
Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
‡
Division of Pharmacology, Department of Internal Medicine, University of Pisa, 56126 Pisa, Italy
§
Queen Mary University of London, Barts, and The London School of Medicine and Dentistry, Blizard Institute, Centre for Diabetes,
Inositide Signalling Group, London, United Kingdom
*
S Supporting Information
ABSTRACT: This study was aimed at investigating the
antitumor activity of novel 2-oxindole derivatives against a
well-characterized human nonsmall cell lung cancer (NSCLC)
cell line. Test compounds produced an antiproliferative activity
in the low micromolar/submicromolar range of concentrations
and significantly induced typical apoptotic morphology with
cell shrinkage, nuclear condensation and fragmentation, and
rupture of cells into debris in a relatively low percentage of A549 cells. Cell cycle arrest occurred at the G1/S phase (1a and 2),
and Akt phosphorylation was significantly inhibited at Thr308 and Ser473. The most active compound (1a) has an IC50 6-fold
lower than the Akt inhibitor, perifosine. These data suggest that the new compounds may be cytostatic and may have maximum
clinical effects in NSCLC patients who do not respond to EGFR inhibitors. These findings prompt us to further explore the
oxindole structure as leading scaffold to design new molecules with potent antitumor activity against NSCLC.
KEYWORDS: Antiproliferative activity, nonsmall cell lung cancer, cell cycle, PDK1/Akt inhibitors, oxindole
ung cancer is one of the most common cancers worldwide
and about 85% of lung cancers are nonsmall cell lung
Compounds with an indole/oxindole moiety are promising
pharmacophore endowed of interesting biological and
L
12
cancer (NSCLC). Patients with NSCLC have a poor prognosis
and limited treatment options including conventional chemo-
therapy and targeted agents that inhibit epidermal growth
factor receptor (EGFR) activation. However, these drugs are
not curative because of both primary and acquired treatment
pharmacological properties as anticancer agents. Recently,
new compounds designed to target the activity of the serine/
13,14
threonine kinases PDK1 and/or Akt were described.
Moreover, the therapeutic potential of tetrahydroisoquinoline
1
5,16
(
THI) derivatives characterized by antineoplastic,
multi-
1
1
7,18
19
resistance highlighting the need to identify new molecules with
drug resistance reversal
and kinases inhibitor activities
potent antitumor activity against NSCLC cells.
have encouraged us to design and synthesize a new class of
novel chemotherapeutic agents active on EGFR-resistant
NSCLC cell line, by anchoring the THI-nucleus to the
oxindole scaffold. While providing further insights into the
structure−activity relationship, the introduction of different
nuclei could also be the starting point for the development of a
new class of cytotoxic agents. On this basis, we designed and
synthesized a series of 2-oxindole-derivatives (OXIDs, 1a−d)
characterized by the presence of different heterocycles (i.e.,
thiophene, imidazole, N-methylimidazole, and pyridine) in 3-
position and 3,4-dimethoxy-THI group anchored in 5-position
through an amidomethyl chain (Figure 1). The structure of
compounds 1a−d matches well with the common structure of
The role of the PI3K/Akt/mTOR pathway in the develop-
ment of the neoplastic phenotype has been extensively
2
reviewed. Noteworthy, activation of Akt was found in 51%
3
of NSCLC patient samples and in 74% of NSCLC cell lines.
Persistent activity of this pathway was associated with increased
4
resistance to multiple chemotherapeutic agents for gastric and
5
ovarian cancer and in the lack of sensitivity of NSCLC cell
6
lines to EGFR inhibitors.
After stimulation with growth factors and cytokines, Akt is
recruited from the cytosol to the plasma membrane and is
phosphorylated at two key regulatory sites, Thr308 (in the
activation domain) and Ser473 (in the carboxy-terminal
hydrophobic motif) by 3-phosphoinositide-dependent protein
20,21
7
,8
recently patented PDK1/Akt inhibitors.
kinase-1 (PDK1) and PDK2, respectively. Once activated,
Akt stimulates cell growth and survival by phosphorylating
9
numerous downstream substrates including caspase-9, glyco-
Received: April 30, 2013
Accepted: October 18, 2013
Published: October 18, 2013
gen synthase kinase-3 (GSK3), BCL antagonist of cell death
1
0
11
(
BAD), and forkhead box transcription factors (FOXO).
©
2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
1
a−d were obtained by reaction of 2 with 2-thiophencarbox-
aldehyde, 1-methyl-5-imidazolecarboxaldehyde, 1H-imidazole-
4
-carbaldehyde, and 2-pyridinecarboxaldehyde, respectively, in
the presence of a catalytic amount of pirrolidine. The
configuration of final products 1a−d was assigned by
1
comparison of their H NMR spectra with those reported in
22
literature for analogue structures.
The in vitro antitumor activity of compounds was evaluated
against A549 cells that have been reported to be gefitinib-
resistant due to the maintenance of an EGFR-independent
23
activity of the PI3K/Akt pathway. All tested compounds
produced a concentration and time-dependent cell growth
inhibition with IC50 mean values in the low micromolar/
submicromolar range (Figure 2).
Figure 1. General structures of new oxindole-derivatives (OXIDs).
Compound 1a−c showed 2- to 6-fold higher antitumor
activity than the Akt inhibitor perifosine, and 2.5- to 10-fold
higher than sunitinib (a 2-oxindole-based anticancer drug),
while compounds 1d and 2 showed IC50 mean values of 19.95
In the current study, we report findings clearly demonstrating
the potent antiproliferative activity of test compounds
associated to inhibition of Akt phosphorylation and cell cycle
arrest at the G1/S phase in human NSCLC cells.
±
1.28 and 16.22 ± 1.04 μM, respectively (Table 1).
The desired compounds 1a−d were synthesized according to
the procedure reported in Scheme 1. The 5-amino-2-oxindole 3
Table 1. IC50 Values for OXIDs Compounds 1a−d and 2,
Sunitinib, and the Akt Inhibitor Perifosine on A549 Cells
Scheme 1. Synthesis of OXIDs Derivatives 1a−d and 2
was obtained by catalytic hydrogenation of the commercial 5-
nitro-1,3-dihydro-2H-indol-2-one. The subsequent reaction of
3
with 2-chloroacetyl chloride afforded the 2-chloro-N-(2-oxo-
2,3-dihydro-1H-indol-5-yl)acetamide 4, which reacted with 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride in the
a
presence of K CO affording derivative 2. Final compounds
See ref 12.
2
3
Figure 2. Concentration- and time-dependent effect on A549 cell proliferation by test compounds and the Akt inhibitor, perifosine. Data were
expressed as mean values ± SD (n = 3).
1
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ACS Medicinal Chemistry Letters
Letter
The ability to escape apoptosis through the phosphorylation
of the above downstream substrates is a critical characteristic of
cancer cells that allow their uncontrolled growth and invasive
behavior. In the current study, the ability of tested compounds
to induce apoptosis on A549 cell line was assessed by
microscopy that remains the gold standard for measurement
2
4
of nuclear fragmentation. Upon exposure to OXIDs
derivatives at their corresponding IC50 values, A549 cells
presented typical apoptotic morphology with cell shrinkage,
nuclear fragmentation, and cellular rupture into debris. The
apoptotic index, determined by morphological metrics, was
significantly higher in cells treated with test compounds than
controls with values ranging from 12% (2) to 24% (1a). The
apoptotic index for the Akt inhibitor, perifosine, was 32%
Figure 4. (A) Effect of compound 1a and perifosine on the
distribution of events in the A549 cell cycle. (B) Percentage of cells
in the G1, S, and G2/M phases of the cell cycle in the presence or
absence of test compounds and the Akt inhibitor perifosine. Data were
expressed as mean values ± SD (n = 3). *P < 0.05, as compared with
control.
(
Figure 3). In addition to play a crucial role for cell survival, Akt
25
signaling pathway modulates cell cycle progression.
worth mentioning that pThr308 and pSer473 are two target
26
sites of the PDK1 and mTORC2 action, respectively. In our
experimental setting, compounds 1a,c and 2 and the Akt
inhibitor, perifosine, showed a similar ability to inhibit both
regulatory sites, whereas compounds 1b and 1d appeared to
have some selectivity toward pThr308 (see Supporting
Information).
In order to confirm selectivity of the compounds, we
evaluated the phosphorylation status of several important
proteins of the PDK1/Akt cascade by Western Blot analysis
(
Figure 5). While the OXIDs 1a and 1b did not affect EGFR
Figure 3. Percentage of A549 cells with damaged DNA after exposure
to test compounds and the Akt inhibitor perifosine. Upper panels,
morphological appearance of control and treated cells. Data were
expressed as mean values ± SD (n = 3). *P < 0.05, as compared with
control.
Therefore, cell-cycle distribution in A549 cells exposed to
OXIDs derivatives was assessed by flow cytometry, and results
were expressed as percentage variation on cells population in
ΔG1, ΔS, and ΔG2 phases (Figure 4).
Treatment with compounds 1a and 2 caused a significant
increase in the proportion of cells in the G1 phase of the cell
cycle (21.04 and 19.24%, respectively) associated to a
percentage decrease of S-phase and G2/M-phase cells, as a
result of the inhibition of DNA synthesis.
Similar cell cycle changes were observed when A549 was
treated with perifosine, while compounds 1b−d did not have
any effect (Figure 4). Therefore, the OXID derivatives 1a and 2
could affect cell survival and cell cycle progression, a
pharmacological profile similar to that observed for the Akt
inhibitor, perifosine.
To provide further insight into the molecular mechanism of
compounds’ action, we used a specific and sensitive
immunoenzymatic method to assess Akt activation in treated
and untreated cells. Specifically, we measured the phosphor-
ylation of the threonine and serine residues at position 308 and
Figure 5. Effect of 1a and 1b on protein phosphorylation of different
targets involved in the PDK1/Akt pathway. Data are representative of
3
independent experiments (see Supporting Information).
(Tyr992) and Her2 (Tyr1221/1222) phosphorylation, they
inhibited phosphorylation of Akt (Thr308) and FoxO1
(Thr24)/FoxO3a (Thr32), two downstream effectors of the
PDK1/Akt pathway. These findings suggest that tested
compounds are selective inhibitors of PDK1/Akt survival
pathway in NSCLC cells.
4
73 of the aminoacid sequence of the enzyme, respectively.
We also performed cytotoxicity experiments on MDA-MB-
231, a cell line reported to grow independently from PDK1/
OXIDs at their IC s for 72 h could significantly decrease the
phosphorylation of Akt by about 20−40%, with the maximal
effect observed for the compound 1a on the pThr308 site. It is
5
0
27,28
Akt pathway.
Our findings clearly demonstrated that
compounds 1a and 1b at 1 and 10 μM for 72 h do not affect
1
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ACS Medicinal Chemistry Letters
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MDA-MB-231 cell proliferation (see Supporting Information,
Figure S1), supporting the selectivity of the compounds on the
PDK1/Akt pathway.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
Phillips, G. B.; Yu, H.; Adler, M.; Whitlow, M.; Ho, E.; Lentz, D.;
Polokoff, M. A.; Subramanyam, B.; Wu, J. M.; Zhu, D.; Feldman, R. I.;
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*
Corresponding Author
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Author Contributions
These authors (G.N. and S.S.) contributed equally to this
work. All authors have given approval to the final version of the
manuscript.
∥
Phillips, G. B.; Yuan, S.; Adler, M.; Whitlow, M.; Lentz, D.; Polokoff,
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ABBREVIATIONS
■
845−5848.
PI3K, phosphoinositide-3-kinase; PDK1, 3-phosphoinositide-
dependent kinase-1; Akt, protein kinase B; OXIDs, 2-oxindole
derivatives; NSCLC, nonsmall cell lung cancer; EGFR,
epidermal growth factor receptor; CAC, chloroacetilchloride;
mTORC2, mammalian target of rapamycin complex 2
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