Synthesis and antitumor properties of 2,5-bis(3′-indolyl)thiophenes: Analogues of marine alkaloid nortopsentin

Article abstract of DOI:10.1016/j.bmcl.2007.01.065

A series of 11 bis-indolylthiophenes of type 8-10 were obtained by cyclization of diketones 4 and 7 using Lawesson's reagent. Derivatives 8c, 9c, 9d, and 10c were selected to be evaluated in the full panel of about 60 human tumor cell lines derived from nine human cancer cell types and showed antiproliferative activity generally in the micromolar range. The most sensitive cell lines were: CCRF-CEM, MOLT-4, HL60 (TB), and RPMI-8226 of the leukemia subpanel, HT29 and HCC-2998 cell lines of the colon sub-panel, NCI-H522 of the non-small cell lung cancer sub-panel, LOX IMVI of the melanoma sub-panel, and UO-31 of the renal cancer sub-panel.

Full text of DOI:10.1016/j.bmcl.2007.01.065

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2342–2346
Synthesis and antitumor properties of 2,5-bis(30-indolyl)thiophenes:
Analogues of marine alkaloid nortopsentin
Patrizia Diana,a,* Anna Carbone,a Paola Barraja,a Alessandra Montalbano,a
Annamaria Martorana,a Gaetano Dattolo,a Ornella Gia,b
Lisa Dalla Viab and Girolamo Cirrincionea
a
`
Dipartimento Farmacochimico Tossicologico e Biologico, Universita degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
bDepartment of Pharmaceutical Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy
Received 7 November 2006; revised 15 January 2007; accepted 17 January 2007
Available online 26 January 2007
Abstract—A series of 11 bis-indolylthiophenes of type 8–10 were obtained by cyclization of diketones 4 and 7 using Lawesson’s
reagent. Derivatives 8c, 9c, 9d, and 10c were selected to be evaluated in the full panel of about 60 human tumor cell lines derived
from nine human cancer cell types and showed antiproliferative activity generally in the micromolar range. The most sensitive cell
lines were: CCRF-CEM, MOLT-4, HL60 (TB), and RPMI-8226 of the leukemia subpanel, HT29 and HCC-2998 cell lines of the
colon sub-panel, NCI-H522 of the non-small cell lung cancer sub-panel, LOX IMVI of the melanoma sub-panel, and UO-31 of the
renal cancer sub-panel.
Ó 2007 Elsevier Ltd. All rights reserved.
Bis(indolyl)alkaloids are recognized as one of the rapidly
growing groups of sponge metabolites because of their
broad spectrum of biological properties including antimi-
crobial, antiviral, and antitumor activities. Nortopsentins
A–C, having a characteristic 2,4-bis(30-indolyl)imidazole
skeleton, exhibited in vitro cytotoxicity against P388 cells
(IC50 1.7, 7.6, and 7.8 lg/mL, respectively). Their N-in-
dolyl methylated derivatives showed significant improve-
ment in P388 activity compared with that of the parent
compounds (IC50 0.34–0.90 lg/mL).1–3
A great limitation in the use of the reservoir of marine
organisms for therapy is that only very small amounts
of the biologically active substances are isolated from
the natural material. Due to the interesting biological
activities, different analogues of the marine nortopsen-
tins have been synthesized.
Thus, many bis(indolyl)alkaloids in which the imidazole
moiety of nortopsentins was replaced by thiazole,
pyrimidine, pyrazine, and pyrazinone rings were report-
ed.4–8
N
2,4-Bis(30-indolyl)thiazole analogues exhibited cytotoxic
activities against a wide range of human tumor cell lines
at micromolar concentration.4,5 Also 2,5-bis(30-indo-
N
H
lyl)pyrazines
and
3,6-(30-indolyl)2-(1H)pyrazinone
R
N
H
N
H
R
1
showed inhibitory activity against a variety of human
tumor cell lines with GI50 values that reached submicro-
molar level. In these series the pyrazinone derivative was
less active than the pyrazine one, whereas the N-indolyl
methylated compound was the most active showing
GI50 values between 0.058 and 7.19 lM.5,6
Nortopsentin A R = R1 = Br
Nortopsentin B R = Br, R1 = H
Nortopsentin C R = H, R1 = Br
In our effort to search for novel antitumor compounds,
we designed new analogues with further modification of
indole alkaloids to get more potent and selective agents.
Thus we thought to synthesize several different series of
bis-indolyl-5-membered heterocycles in order to verify
Keywords: 2,5-bis(30-indolyl)thiophenes; Antitumor activity; Topoiso-
merase II; Nortopsentin analogues.
*
Corresponding
+390916169999; e-mail: diana@unipa.it
author.
Tel.:
+390916161606;
fax:
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.065

P. Diana et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2342–2346
2343
R1
S
N
N
N
N
H
N
R
N
R
N
H
R
R
2,4-bis(3'-indolyl)pyrimidines
2,4-bis(3'-indolyl)thiazoles
R
N
H
N
H
N
O
N
N
N
N
N
H
R
2,5-bis(3'-indolyl)pyrazines
3,6-bis(3'-indolyl)-2(1H)pyrazinone
the influence of the central heterocyclic ring on the anti-
neoplastic activity.
tion with phosphorus oxychloride and dimethylformam-
ide (85–99%). These latter compounds were protected
with the benzensulfonyl group at the NH to give N-ben-
zensulfonyl-indole-3-carboxaldehydes 3 (95–99%). The
next step involves a Stetter reaction between the elec-
tron-deficient indole aldehyde and divinyl sulfone using
sodium acetate and thiazolium chloride as catalyst in
refluxing ethanol to afford 1,4-diketones 4 (55–66%)9
(Scheme 1).
In this paper, we report the synthesis of bisindolylthi-
ophene derivatives of type 8–10, in which the imidazole
moiety of the nortopsentin was replaced by a thiophene
ring, and the NCI’s in vitro disease-oriented antitumor
screen of the most potent in this series.
1,4-Bis-indolyl-diketones appeared valuable and versa-
tile intermediates for the synthesis of bis(indolyl)thioph-
enes. Indole derivatives of type 1 were converted into
indole-3-carboxaldehydes 2 by a Vilsmeier–Haack reac-
Alternatively, the treatment of the indole derivatives 1
with potassium tert-butoxide, TDA-1 as catalyst, and
methyl iodide in benzene yielded the corresponding
CHO
CHO
R
R
R
ii
i
N
N
N
H
SO2Ph
H
2a-c
1a-c
3a-c
iii
O
O
R
R
a R = H,
b R = Me,
c R = OMe
N
N
SO2Ph
SO2Ph
4a-c
Scheme 1. Synthesis of intermediates 4a–c. Reagents and condition: (i) POCl3/DMF, NaOH; (ii) NaH/THF, ClSO2Ph; (iii) thiazolium salt/EtOH,
NaOAc/Divinyl sulfone, reflux.

2344
P. Diana et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2342–2346
N-methyl derivatives 5 (96–98%). These latter com-
pounds were converted into 1,4-diketones of type 7 by
Vilsmeier–Haack reaction using phosphorus oxychlo-
ride and tetramethylsuccinamide 6 (56–70%) (Scheme 2).
All the bis(indolyl)thiophene compounds 8–1010 were
submitted to the National Cancer Institute (Bethesda,
MD); four of them were selected (8c, 9c, 9d, and 10c),
for evaluation the full panel of about 60 human cancer
cell lines derived from nine human cancer cell types, that
have grouped in disease sub-panels including leukemia,
non-small-cell lung, colon, central nervous system, mel-
anoma, ovarian, renal, prostate, and breast tumor cell
lines. The compounds were tested at five concentrations
at 10-fold dilution the highest being 10ꢀ4 M and the oth-
ers 10ꢀ5, 10ꢀ6, 10ꢀ7, and 10ꢀ8 M.11
The resulting diketones 4a–c and 7a–e were converted
into the corresponding bis(indolyl)thiophenes 8a–c and
9a–e in excellent yields with Lawesson’s reagent in reflux-
ing toluene (88–99%). Hydrolysis of compounds 8a–c,
with KOH in refluxing ethanol, gave the corresponding
thiophenes of type 10a–c (65–86%) (Scheme 3).
O
R
Me
Me
Me
N
i
+
Me
N
1a-e
N
O
Me
5a-e
6
ii
O
O
R
a R = H,
R
b R = Me,
c R = OMe,
d R = Cl,
e R = Br
N
N
Me
Me
7a-e
Scheme 2. Synthesis of intermediates 7a–e. Reagents: (i) t-BuOK/TDA-1, benzene, CH3I; (ii) POCl3/DMF.
R
R
S
i
4a-c
N
N
SO2Ph
SO2Ph
8a-c
a R = H,
b R = Me,
c R = OMe,
d R = Cl,
e R = Br
ii
R
R
S
i
7a-e
N
N
R1
R1
9a-e R1 = Me;
10a-c R1 = H
Scheme 3. Synthesis of 2,5-bis-indolylthiophenes. Reagents and conditions: (i) Lawesson’s reagent/toluene, reflux; (ii) KOH/EtOH, reflux.

P. Diana et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2342–2346
2345
Table 2. Inhibition of in vitro cancer cell lines by compound 10ca
The most active compound is derivative 10c. The antitu-
mor activity is given by three parameters for each cell
line; pGI50 value, pTGI value, and pLC50 value. More-
over, a mean graph midpoint (MG_MID) is calculated
for each of the mentioned parameters, giving an average
activity parameter over all cell lines.
Cell lines
GI50 (lM)b
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
0.34
2.27
3.54
2.83
1.91
RPMI-8226
MOLT-4
An evaluation of the data reported in Tables 1 and 2
revealed that compound 10c was cytotoxic showing
GI50 values against the total number of cell lines investi-
gated at micromolar concentration. Moreover positive
TGI and LC50 values were observed with respect to a con-
gruous number of cell lines (88% and 46%, respectively).
Non-small cell lung cancer
A549/ATCC
EKVX
3.66
13.8
9.88
4.64
16.2
11.1
21.8
3.24
1.31
HOP-62
HOP-92
NCI-H226
NCI-H23
Derivative 10c was particularly efficacious against the
leukemia sub-panel having GI50 in the range 0.34–
3.54 lM. The most sensitive leukemia cell lines are
CCRF-CEM (GI50 0.34 lM), MOLT-4 (GI50
1.91 lM), HL60 (TB) (GI50 2.27 lM), and RPMI-
8226 (GI50 2.83 lM).
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO-205
HCC-2998
HCT-15
HT29
10.6
2.83
3.17
2.79
3.39
3.66
Compound 10c showed good selectivity with respect to
the HT29 (GI50 2.79 lM) and HCC-2998 (GI50
2.83 lM) cell lines of the colon sub-panel. It also showed
selectivity with respect to NCI-H522 (GI50 1.31 lM) of
the non-small cell lung cancer sub-panel, LOX IMVI
(GI50 2.55 lM) of the melanoma sub-panel, and UO-
31 (GI50 2.66 lM) of the renal cancer sub-panel.
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
13.0
3.03
5.81
19.0
Ndc
3.91
At TGI and LC50 level, the best responses were
observed in the case of the HCC-2998 (TGI 6.50 lM
and LC50 27.2 lM,) colon cancer cell line and LOX
IMVI (TGI 7.08 lM and LC50 38.3 lM), melanoma cell
lines.
Melanoma
LOX IMVI
MALME-3M
M14
2.55
17.3
13.5
16.4
14.5
5.69
18.0
15.8
In order to discern the mechanism of action of deriva-
tive 10c we performed COMPARE computations
against the NCI ‘Standard Agents’ database.12 Com-
pound 10c had a Pearson correlation coefficient (PCC)
<0.6 suggesting that the antiproliferative activity would
be mechanistically unrelated to that of any known drug.
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
10.8
10.8
9.04
12.9
6.35
19.2
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Table 1. Overview of the results of the in vitro antitumor screening for
compound 10ca
No.e
No.f
Range
MG_MIDg
pGI50b
pTGIc
pLC50d
56
56
57
56
49
26
6.46–4.66
5.40–4.18
4.57–4.01
5.2
4.55
4.1
Renal cancer
786-0
A498
3.29
3.29
4.21
12.0
3.98
14.0
11.4
2.66
a Data obtained from the NCI’s in vitro disease-oriented human tumor
cell screen.
ACHN
CAKI-1
RXF 393
SN12C
TK-10
b pGI50 is the ꢀlog of the molar concentration that inhibits 50% net
cell growth.
c pTGI is the ꢀlog of the molar concentration giving total growth
inhibition.
UO-31
d pLC50 is the ꢀlog of the molar concentration leading to 50% net cell
death.
Prostate cancer
PC-3
DU-145
e No. is the number of cell lines investigated.
f No. is the number of cell lines giving positive pGI50, pTGI, and
pLC50.
4.51
6.05
Breast cancer
MCF7
NCI/ADR-RES
g MG_MID = mean graph midpoint = arithmetical mean value for all
tested cancer cell lines. If the indicated effect was not attainable
within the used concentration interval, the highest tested concen-
tration was used for the calculation.
6.71
3.76
(continued on next page)

2346
P. Diana et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2342–2346
Table 2 (continued)
cellular molecular target contributes to the antiprolif-
erative ability of 10c.
Cell lines
GI50 (lM)b
MDA-MB-231/ATCC
HS 578T
4.20
11.7
13.7
11.0
11.1
Acknowledgments
This work was financially supported by Ministero
MDA-MB-435
BT-549
T-47D
`
dell’Istruzione dell’Universita e della Ricerca. We thank
a Data obtained from NCI’s in vitro disease-oriented tumor cell screen.
b The cytotoxicity GI50 values are the concentrations corresponding to
50% growth inhibition of tumor cells.
the National Cancer Institute (Bethesda, MD) and espe-
cially Dr V. L. Narayanan and his team for the antitu-
mor tests reported in this paper.
c Not determined.
References and notes
1. Alvarez, M.; Salas, M. Heterocycles 1991, 32, 1391.
2. Sakem, S.; Sun, H. H. J. Org. Chem. 1991, 56, 4304.
3. Kawasaki, I.; Yamashita, M.; Ohta, S. Chem. Pharm. Bull.
1996, 44, 1831.
Relaxed DNA•
4. Gu, X.; Wan, X.; Jiang, B. Bioorg. Med. Chem. Lett. 1999,
9, 569.
Supercoiled DNA•
5. Jiang, B.; Gu, X. Bioorg. Med. Chem. Lett. 2000, 8, 363.
6. Jiang, B.; Xiong, X.; Yang, C. Bioorg. Med. Chem. 2001,
9, 1149.
7. Jiang, B.; Xiong, X.; Yang, C. Bioorg. Med. Chem. Lett.
2001, 11, 475.
a
b
c
d
e
f
g
Figure 1. Effect of derivative 10c on the relaxation of supercoiled
plasmid DNA by human recombinant Topoisomerase II.14
8. Xiong, W.; Yang, C.; Jiang, B. Bioorg. Med. Chem. 2001,
9, 1773.
Moreover, we performed experiments on the ability of
derivative 10c to interfere with the catalytic activity of
DNA Topoisomerase II.
9. Jones, R. A.; Karatza, M.; Voro, T. N.; Civeir, P. U.;
Franck, A.; Ozturk, O.; Seaman, J. P.; Whitmore, A. P.;
Williamson, D. J. Tetrahedron 1996, 52, 8707.
10. H and 13C NMR, IR, elemental analysis (C, H, and N),
and melting point determination. For example, for com-
Indeed, it was widely demonstrated that a number of
anticancer drugs exert their antiproliferative effect by
inhibiting this nuclear enzyme.13
pound 10c: mp 201–202 °C; IR 3402 (NH) cmꢀ1
;
1H
NMR (CDCl3): d 3.84 (s, 3H, OCH3), 6.85 (dd, J = 2.0,
8.8 Hz, 1H, H-6), 7.31 (s, 1H, H-30), 7.37 (d, J = 8.8 Hz,
1H, H-7) 7.39 (s, 1H, H-2), 7.68 (d, J = 2.0 Hz, 1H, H-4),
11.3 (s, 1H, NH); 13C NMR (CDCl3): d 55.3 (q), 100.9 (d),
109.8 (s), 111.9 (d), 112.7 (d), 121.9 (d), 123.5 (d), 125.0 (s),
131.7 (s), 134.2 (s), 154.0 (s).
Figure 1 shows the relaxation of supercoiled plasmid
DNA mediated by Topoisomerase II in the absence
(lane b) and in the presence of increasing concentra-
tions of 10c (lanes c–f). The results obtained indicate
a somewhat weaker inhibitory capacity at the lower
concentrations taken into consideration (1 and
10 lM, lanes c and d, respectively). The inhibitory ef-
fect increases in a dose-dependent manner and at
200 lM concentration is comparable with that induced
by the well-known Topoisomerase II inhibitor m-am-
sacrine, used at 8 lM concentration (compare lanes f
and g). Considering that the antiproliferative effect
exerted by 10c for a number of cell lines is in the
0.34–21.8 lM range (Tables 1 and 2), the inhibition
of Topoisomerase II does not seem the main cause
of cell death, even though an undoubtable effect on
the enzyme occurs. Topoisomerase II modifies the
topological states of DNA and a number of antiprolif-
erative drugs, which affect its catalytic activity, are
known to be DNA binders.13 Therefore, it appeared
of interest to investigate on the ability of 10c to form
a molecular intercalative complex by means of flow
linear dichroism experiments, performed with salmon
testes DNA. The spectra of DNA alone and in pres-
ence of 10c do not evidence any detectable difference
suggesting the incapacity of the test derivative to
intercalate inside the macromolecule (data not shown).
In conclusion, it is conceivable that more than one
11. (a) Grever, M. R.; Sherpartz, S. A.; Chabner, B. A. Semin.
Oncol. 1992, 19, 622; (b) Monks, A. P.; Scudiero, D. A.;
Skehan, P.; Shoemaker, R.; Paull, K. D.; Vistica, D.; Hose,
C.; Langley, J.; Croniste, P.; Vaigro-Woiff, A.; Gray-
Goodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. R.
J. Natl. Cancer Inst. 1991, 83, 757; (c) Weinstein, J. N.;
Meyers, T. G.; O’Connor, P. M.; Friend, S. H.; Fornace, A.
J., Jr.; Kohn, K. W.; Fojo, T.; Bates, S. E.; Rubinstein, L. V.;
Anderson, N. L.; Boulamwini, J. K.; van Osdol, W. W.;
Monks, A. P.; Scudiero, D. A.; Sausville, E. A.; Zaharevitz,
D. W.; Bunow, B.; Viswanadhan, V. N.; Johnson, G. S.;
Wittes, R. E.; Paull, K. D. Science 1997, 275, 343.
12. Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.;
Scudiero, D. A.; Rubistein, L.; Plowman, J.; Boyd, M. R.
J. Natl. Cancer Inst. 1989, 81, 1088.
13. D’Arpa, P.; Liu, L. F. Biochim. Biophys. Acta 1989, 989,
163.
14. Supercoiled pBR322 DNA (0.25 lg, lane a) was incubated
for 60 min at 37 °C with Topoisomerase II (1 U) in the
absence (lane b) or presence of 10c at 1, 10, 100, and
200 lM (lanes c–f, respectively). Addition of m-amsacrine
8 lM (lane g) was used as reference. DNA samples were
separated by electrophoresis on a 1% agarose gel. The gel
was stained with ethidium bromide 1 lg/mL in TAE
buffer, transilluminated by UV light, and fluorescence
emission visualized using a CCD camera coupled to a Bio-
Rad Gel Doc XR apparatus.