Antiproliferative activity of 2,3-disubstituted indoles toward apoptosis-resistant cancers cells

Article abstract of DOI:10.1016/j.bmcl.2013.03.110

Many types of cancer, including glioma, melanoma, NSCLC, among others, are resistant to apoptosis induction and poorly responsive to current therapies with propaptotic agents. We describe a series of 2,3-disubstituted indoles, which display cytostatic rat

Full text of DOI:10.1016/j.bmcl.2013.03.110

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiproliferative activity of 2,3-disubstituted indoles toward
apoptosis-resistant cancers cells
b
a
c
d
Igor V. Magedov a, , Florence Lefranc , Liliya V. Frolova , Laetitia Moreno Y. Banuls , Amanda S. Peretti ,
⇑
Snezna Rogelj d, Véronique Mathieu c, Robert Kiss c, Alexander Kornienko e,
a Department of Chemistry, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA
⇑
b Service de Neurochirurgie, Hôpital Erasme, Brussels, Belgium
c Laboratoire de Toxicologie, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), 1050 Brussels, Belgium
d Department of Biology, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA
e Department of Chemistry and Biochemistry, Texas State University—San Marcos, San Marcos, TX 78666, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Many types of cancer, including glioma, melanoma, NSCLC, among others, are resistant to apoptosis
induction and poorly responsive to current therapies with propaptotic agents. We describe a series of
2,3-disubstituted indoles, which display cytostatic rather than cytotoxic effects in cancer cells, and serve
as a new chemical scaffold to develop anticancer agents capable of combating apoptosis-resistant cancers
associated with dismal prognoses.
Received 31 December 2012
Revised 26 March 2013
Accepted 27 March 2013
Available online xxxx
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Cancer
Apoptosis resistance
Indole
Antiproliferative
Apoptosis-resistant cancers represent a major challenge in the
clinic as most of the currently available chemotherapeutic agents
work through the induction of apoptosis and, therefore, provide
very limited therapeutic benefits for the patients affected by these
malignancies. Such apoptosis-resistant cancers include the tumors
of the lung, liver, stomach, esophagus, pancreas as well as melano-
mas and gliomas.1 For example, patients afflicted by a type of gli-
with cytostatic agents and thus a search for novel cytostatic antican-
cer drugs that can overcome cancer cell resistance to apoptosis is an
important pursuit.10–13
Recently we described a series of 2-aryl-3-azoindoles A as anti-
bacterial agents active against MRSA (Scheme 1).14 We showed
that the metabolically labile azo group could be bioisosterically re-
placed by an ether or thioether functionalities leading to structures
B, which were potent antibacterial agents as well. Unfortunately,
omas, known as glioblastoma multiforme,2,3 have
a median
survival expectancy of less than 14 months when treated with a
standard protocol of surgical resection, radiotherapy and chemo-
therapy with temozolomide.4 Because these glioma cells display
resistance to apoptosis, they respond poorly to conventional che-
motherapy with proapoptotic agents.3,5
R
R
BF4
N2
N
N
(4 equiv)
In addition, it must be recalled that 90% of cancer patients die
from tumor metastases6 and that metastatic cancer cells have ac-
quired resistance to a process termed anoikis, that is, cell death
resulting from losing contact with extracellular matrix or neigh-
boring cells.6 This phenomenon results in apoptosis resistance by
metastatic cells making them unresponsive to a large majority of
proapoptoticagentsaswell.3,7–9 Onesolutiontoapoptosisresistance
entails the complementation of cytotoxic therapeutic regimens
N
H
AcONa, dioxane/H2O
R
N
H
A
R2
bioisosteric
substitution
X
R3
R1
N
H
⇑
Corresponding authors. Tel.: +1 575 835 6886; fax: +1 575 835 5364 (I.V.M.);
tel.: +1 512 245 3632; fax: +1 512 245 2374 (A.K.).
E-mail addresses: imagedov@nmt.edu (I.V. Magedov), a_k76@txstate.edu
(A. Kornienko).
B (X = O or S)
Scheme 1.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.110
Please cite this article in press as: Magedov, I. V.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.03.110

2
I. V. Magedov et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
these compounds were also found to inhibit the growth of human
cervical cancer cells HeLa at comparable concentrations and this
diminished our interest in pursuing them as novel antibacterials.
However, during the antiproliferative assays, we noticed that these
compounds do not kill cancer cells at GI50-related concentrations,
but rather display cytostatic effects. This observation prompted
our efforts to investigate the compounds of this type as a new class
of cytostatic agents potentially useful in the treatment of apopto-
sis-resistant cancers.
Because the antiproliferative effects of indoles A and B were
similar, we surmised that the position C-3 of the indole ring can
tolerate structurally diverse substituents. Previously, we devel-
oped synthetic pathways based on the Fisher indole reaction,
which in addition to C-3 ether and thioether indoles B (Scheme
2a) can be used to prepare C-3 amide indoles C (Scheme 2b), C-3
pyrazole indoles D (Scheme 2c) and C-3 dithiocarbamate indoles
E (Scheme 2d).15–18 Using this chemistry, a number of C-3 analogs
of each structural type were synthesized and their structures are
shown in Table 1.
The synthesized compounds were evaluated for in vitro growth
inhibition using the MTT colorimetric assay19 against a panel of
five cancer cell lines including those resistant to proapoptotic stim-
uli [human U373 glioblastoma,20 human A549 non-small-cell-lung
cancer (NSCLC),21 and human SKMEL-28 melanoma22] as well as
apoptosis-sensitive tumor models (human Hs683 anaplastic oligo-
dendroglioma20 and mouse B16F10 melanoma22). Analysis of the
data shown in Table 1 reveals that most of the synthesized com-
pounds exhibit antiproliferative properties in the double-digit
micromolar region and do not drastically differ in their potencies.
Indeed, it appears that the position C-3 of the indole ring tolerates
diverse substitution in this type of structure. Yet, C-3 ether and thi-
oether indoles B appear to the most potent, with ether indole B6
(a)
R3
(b)
R2
R1
R2
NH
R1
NH2
NH
O
R2
O
R1
NH2
NH
X
H
N
R1
X
R3
R2
O
HCl, EtOH
O
HCl, EtOH
N
X = O or S
N
R2
H
C
B (X = O or S)
Me
N
NR4R5
S
(c)
(d)
R3
S
R1
Me
R1
NH2
NH
R3
NR4R5
S
NH2
N
N
R1
N
N
S
R1
Me
R2
R2
HCl, EtOH
R2
O
Me
HCl, EtOH
O
N
R2
N
H
E
D
Scheme 2.
Table 1
Structures of C-3 derivatized 2-arylindoles and their antiproliferative activities toward cancer cells
#
Structure
GI50 in vitro valuesa
U373 Hs683 A549 SKMEL B16
(lM)
#
Structure
GI50 in vitro valuesa
U373 Hs683 A549 SKMEL B16
(lM)
Cl
Cl
O
O
N
N
A1
33
31
26
33
33
C5
66
38
37
48
32
NH
Br
N
H
N
H
Br
N
N
F
A2
30
24
23
31
26
D1
47
32
29
31
39
N
N
N
H
Br
N
H
Cl
N
N
O
B1
13
13
10
19
10
D2
10
29
26
33
40
Cl
N
Cl
H
N
H
Please cite this article in press as: Magedov, I. V.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.03.110

I. V. Magedov et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
Table 1 (continued)
#
Structure
GI50 in vitro valuesa
U373 Hs683 A549 SKMEL B16
(lM)
#
Structure
GI50 in vitro valuesa
U373 Hs683 A549 SKMEL B16
(lM)
Cl
N
N
Cl
S
O
B2
20
35
25
50
22
26
28
54
22
30
25
59
24
28
28
57
14
29
29
31
D3
32
43
84
90
32
50
67
58
40
42
61
38
35
46
84
80
36
F
N
H
Cl
Cl
N
H
Cl
N
N
O
B3
B4
B5
D4
D5
D6
65
Cl
Br
Cl
N
H
N
H
N
N
O
>100
N
Cl
H
N
H
Cl
N
N
S
O
73
MeO
Cl
N
H
Cl
N
H
O
N
S
S
O
B6
8
7
7
8
8
E1
43
32
31
35
20
Br
Cl
N
N
H
S
H
F
Cl
CO2Et
N
O
B7
35
24
28
28
20
E2
33
36
34
39
21
S
EtO2C
Cl
Cl
Br
N
H
N
H
CO2Et
Cl
N
S
S
B8
28
31
27
34
28
E3
68
34
37
52
37
S
Cl
Cl
Br
N
N
H
H
Cl
OH
N
S
S
S
B9
24
25
26
26
35
E4
39
31
29
34
37
Cl
Br
Br
N
N
H
H
(continued on next page)
Please cite this article in press as: Magedov, I. V.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.03.110

4
I. V. Magedov et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 1 (continued)
#
Structure
GI50 in vitro valuesa
U373 Hs683 A549 SKMEL B16
(lM)
#
Structure
GI50 in vitro valuesa
U373 Hs683 A549 SKMEL B16
(lM)
CO2Et
Cl
N
S
S
B10
28
31
27
34
28
E5
>100
85
72
78
50
S
Cl
Br
N
N
H
Me
N
S
S
O
O
Br
O
C1
66
45
37
55
27
E6
9
22
22
29
33
NH
Br
Cl
N
H
N
H
Cl
N
N
S
O
S
F
Br
O
C2
24
15
9
13
22
E7
36
26
31
28
40
NH
N
H
N
H
OH
O
N
S
NH
S
C3
38
69
38
67
44
E8
32
24
24
28
29
N
Br
H
N
H
BnS
CO2Et
O
NH
N
S
S
C4
47
33
30
30
35
E9
35
36
34
36
27
N
H
Br
N
H
a
U373 (ECACC code 89081403) cell line was cultured in MEM medium supplemented with 5% heat inactivated fetal bovine serum; Hs683 (ATCC code HTB-138), SKMEL-28
(ATCC code HTB-72), A549 (DSMZ code ACC107) and B16F10 (ATCC code CRL-6475) cell lines were cultured in RPMI medium supplemented with 10% heat inactivated fetal
bovine serum; MEM and RPMI cell culture media were supplemented with 4 mM glutamine, 100 g/mL gentamicin and penicillin–streptomycin (200 U/mL and 200 g/mL).
l
l
exhibiting single-digit micromolar GI50 values. Importantly, all
synthesized 2,3-disubstituted indoles do not discriminate between
the cancer cell lines based on the apoptosis sensitivity criterion
and display comparable potencies in both cell types, further indi-
cating that apoptosis induction may not the primary mechanism
responsible for antiproliferative activity in this series of com-
pounds, at least in solid cancers.
after 24, 48 and 72 h) to the number of cells present in the first im-
age (at 0 h). We divided this ratio obtained in the B10-treated
experiment by the ratio obtained in the control. The GGR values
of 0.1 and 0.3 correspondingly in these two cell lines indicate that
10% and 30% of cells grew in the B10-treated experiment as com-
pared to the control over a 72 h observation period. Thus, the
GGR calculations confirm the MTT colorimetric data in Table 1, that
We also employed computer-assisted phase-contrast micros-
copy10,22 (quantitative videomicroscopy) to analyze the principal
mechanism of action associated with indoles’ B in vitro growth
inhibitory effects, as first revealed by the MTT colorimetric assay.
Figure 1 shows that indole B10 inhibits cancer cell proliferation
without inducing cell death when assayed at its GI50 concentra-
tions (Table 1) in SKMEL-28 melanoma and A549 NSCLC cells.
Based on the phase contrast pictures obtained by means of quanti-
tative videomicroscopy, we calculated the global growth ratio
(GGR), which corresponds to the ratio of the mean number of cells
present in a given image captured in the experiment (in this case
is, 30 lM B10 exhibits marked growth inhibitory activity in SKMEL-
28 and A549 cells, which display resistance to apoptosis induction
(Fig. 2).
To confirm that indoles B do not induce cell death as suggested
by the videomicroscopy experiments, we employed flow cytomet-
ric propidium iodide staining, which detects necrotic and late
apoptotic cells that have lost the plasma membrane integrity.
The experiments performed with apoptosis-resistant A549 NSCLC
and SKMEL-28 cells indicate that B10 at its GI50 concentration of
30
lM does not induce any cell permeabilization even after 72 h
of treatment in both cell types. In contrast, 90% of ice-cold ethanol
Please cite this article in press as: Magedov, I. V.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.03.110

I. V. Magedov et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
5
Figure 1. Cellular imaging of B10 against melanoma SKMEL-28 and NSCLC A549 cells illustrating noncytotoxic, but cytostatic, antiproliferative mechanism at MTT
colorimetric assay-related GI50 concentrations after 72 h of cell culture with the drug.
fixed and permeabilized cells were positively stained and cisplatin,
a pro-apoptotic agent, induced an increase in the percentage of PI
positive cells even in these apoptosis-resistant models (increase
from 1% to 10% for A549 NSCLC and from 8% to 30% for SKMEL-
28 cells).
the C-3 ether and thioether indoles, which exhibit their antiprolif-
erative effects mainly through cytostatic mechanisms.
Acknowledgments
In conclusion, the anticancer evaluation of C-3 derivatized 2-
aryl indoles, accessible by a straightforward synthetic preparation
utilizing the Fisher indole reaction, revealed their promising activ-
ity against apoptosis-resistant cancers associated with dismal clin-
ical outcomes. The most promising structural type appears to be
This project was supported by grants from the National
Institute of General Medical Sciences(P20GM103451) and National
Cancer Institute (CA-135579) as well as Texas State University
start-up funding to A.K. The authors thank Thierry Gras for his
excellent technical assistance in cell culture. R.K. is a director of
research and L.M.Y.B. is a research assistant with the Fonds
National de la Recherche Scientifique (FRS-FNRS, Belgium).
References and notes
1. Brenner, H. Lancet 2002, 360, 1131.
2. Kleihues, P.; Cavenee, W. K. Pathology and Genetics of Tumors of the Nervous
System, International Agency for the Research on Cancer (IARC) and WHO Health
Organization; Oxford Press: Oxford, UK, 2000.
3. Lefranc, F.; Sadeghi, N.; Camby, I.; Metens, T.; De Witte, O.; Kiss, R. Expert Rev.
Anticancer Ther. 2006, 6, 719.
4. Stupp, R.; Hegi, M. E.; Mason, W. P.; van den Bent, M. J.; Taphoorn, M. J.; Janzer,
R. C.; Ludwin, S. K.; Allgeier, A.; Fisher, B.; Belanger, K., et al Lancet Oncol. 2009,
10, 459.
5. Giese, A.; Bjerkvig, R.; Berens, M. E.; Westphal, M. J. Clin. Oncol. 2003, 21, 1624.
6. Simpson, C. D.; Anyiwe, K.; Schimmer, A. D. Cancer Lett. 2008, 272, 177.
7. Savage, P.; Stebbing, J.; Bower, M.; Crook, T. Nat. Clin. Pract. Oncol. 2009, 6, 43.
8. Wilson, T. R.; Johnston, P. G.; Longley, D. B. Curr. Cancer Drug Targets 2009, 9,
307.
9. Lamoral-Theys, D.; Pottier, L.; Dufrasne, F.; Nève, J.; Dubois, J.; Kornienko, A.;
Kiss, R.; Ingrassia, L. Curr. Med. Chem. 2010, 17, 812.
10. Van Goietsenoven, G.; Andolfi, A.; Lallemand, B.; Cimmino, A.; Lamoral-Theys,
D.; Gras, T.; Abou-Donia, A.; Dubois, J.; Lefranc, F.; Mathieu, V.; Kornienko, A.;
Kiss, R.; Evidente, A. J. Nat. Prod. 2010, 73, 1223.
11. Lamoral-Theys, D.; Andolfi, A.; Van Goietsenoven, G.; Cimmino, A.; Le
Calvé, B.; Wauthoz, N.; Mégalizzi, V.; Gras, T.; Bruyère, C.; Dubois, J.;
Mathieu, V.; Kornienko, A.; Kiss, R.; Evidente, A. J. Med. Chem. 2009, 52,
6244.
Figure 2. Percentage of cells that lost plasma membrane integrity after treatment
with B10 as assessed by propidium iodide staining. Positive controls correspond to
fixed and permeabilized corresponding cells.
Please cite this article in press as: Magedov, I. V.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.03.110

6
I. V. Magedov et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
12. Evdokimov, N.; Lamoral-Theys, D.; Mathieu, V.; Andolfi, A.; Pelly, S.; van
Otterlo, W.; Magedov, I.; Kiss, R.; Evidente, A.; Kornienko, A. Bioorg. Med. Chem.
2011, 19, 7252.
13. Luchetti, G.; Johnston, R.; Mathieu, V.; Lefranc, F.; Hayden, K.; Andolfi, A.;
Lamoral-Theys, D.; Reisenauer, M. R.; Champion, C.; Pelly, S. C.; van Otterlo, W.
A. L.; Magedov, I. V.; Kiss, R.; Evidente, A.; Rogelj, S.; Kornienko, A.
ChemMedChem 2012, 7, 815.
14. Seth, D.; Hayden, K.; Malik, I.; Porch, N.; Tang, H.; Rogelj, S.; Frolova, L. V.;
Lepthien, K.; Kornienko, A.; Magedov, I. V. Bioorg. Med. Chem. Lett. 2011, 21, 4720.
15. Przheval’skii, N. M.; Skvortsova, N. S.; Magedov, I. V. Chem. Heterocycl. Compd.
2002, 38, 1055.
16. Przheval’skii, N. M.; Magedov, I. V.; Drozd, V. N. Chem. Heterocycl. Compd. 1997,
33, 1475.
17. Przheval’skii, N. M.; Skvortsova, N. S.; Magedov, I. V. Chem. Heterocycl. Compd.
2003, 39, 161.
18. Przheval’skii, N. M.; Skvortsova, N. S.; Magedov, I. V. Chem. Heterocycl. Compd.
2004, 40, 1435.
19. Janssen, T.; Darro, F.; Petein, M.; Raviv, G.; Pasteels, J. L.; Kiss, R.; Schulman, C.
C. Cancer 1996, 77, 144.
20. Branle, F.; Lefranc, F.; Camby, I.; Jeuken, J.; Geurts-Moespot, A.; Sprenger, S.;
Sweep, F.; Kiss, R. Cancer 2002, 95, 641.
21. Mathieu, A.; Remmelink, M.; D’Haene, N.; Penant, S.; Gaussin, J. F.; Van Ginckel,
R.; Darro, F.; Kiss, R.; Salmon, I. Cancer 2004, 101, 1908.
22. Mathieu, V.; Pirker, C.; Martin de Lasalle, E.; Vernier, M.; Mijatovic, T.; De Neve,
N.; Gaussin, J. F.; Dehoux, M.; Lefranc, F.; Berger, W.; Kiss, R. J. Cell. Mol. Med.
2009, 13, 3960.
Please cite this article in press as: Magedov, I. V.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.03.110