3,5-Bis(3′-indolyl)pyrazoles, analogues of marine alkaloid nortopsentin: Synthesis and antitumor properties

Article abstract of DOI:10.1016/j.bmcl.2007.09.042

A series of 10 bis-indolylpyrazoles of type 9, 10 were obtained by cyclization of diketones 8 using hydrazine monohydrate or methylhydrazine in refluxing acetic acid/THF. Derivatives 9a,c,d were selected, by the National Cancer Institute (NCI, Bethesda, USA), to be evaluated against the full panel of about 60 human tumor cell lines derived from nine human cancer cell types and showed antiproliferative activity in the micromolar range. In particular, 9d, the most active compound was effective against all the tested cell lines with a GI₅₀ mean value of 3.23 μM; TGI and LC₅₀ values were 14.5 and 58.9 μM having positive response on 91% and 41% of the tested cell lines, respectively.

Full text of DOI:10.1016/j.bmcl.2007.09.042

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6134–6137
3,5-Bis(30-indolyl)pyrazoles, analogues of marine alkaloid
nortopsentin: Synthesis and antitumor properties
Patrizia Diana,a,* Anna Carbone,a Paola Barraja,a Annamaria Martorana,a
Ornella Gia,b Lisa DallaViab 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 26 July 2007; revised 7 September 2007; accepted 8 September 2007
Available online 14 September 2007
Abstract—A series of 10 bis-indolylpyrazoles of type 9, 10 were obtained by cyclization of diketones 8 using hydrazine monohydrate
or methylhydrazine in refluxing acetic acid/THF. Derivatives 9a,c,d were selected, by the National Cancer Institute (NCI, Bethesda,
USA), to be evaluated against the full panel of about 60 human tumor cell lines derived from nine human cancer cell types and
showed antiproliferative activity in the micromolar range. In particular, 9d, the most active compound was effective against all
the tested cell lines with a GI50 mean value of 3.23 lM; TGI and LC50 values were 14.5 and 58.9 lM having positive response
on 91% and 41% of the tested cell lines, respectively.
Ó 2007 Elsevier Ltd. All rights reserved.
N
Marine sponge of the genera Spongosorites has been
reported to yield bisindole alkaloids. The interest in
this class of compounds has been stimulated by both
N
H
R
N
H
N
H
R1
their unique chemical structure and the wide range
of biological properties including antiviral, antimicro-
bial, and antitumor activity.1 Nortopsentins A–C, with
its 2,4-bis(30-indolyl)imidazole skeleton, exhibited
in vitro cytotoxicity against P388 cells (IC50 4.5–
20.7 lM) and antifungal activity against Candida albi-
cans (Fig. 1). Their N-indolyl methylated derivatives
showed significant improvement in P388 activity if
compared to the activity of parent compounds (IC50
0.8–2.1 lM).2–4 Due to their interesting biological
activities, nortopsentins have been considered impor-
tant lead compounds for the discovery of new biolog-
ical active derivatives.
Nortopsentin A R = R1 = Br
Nortopsentin B R = Br, R1 = H
Nortopsentin C R = H, R1 = Br
Figure 1.
Het
R1
R
N
R2
N
R2
R3
S
N
;
;
2
4
Het =
Het =
Het =
Het =
1
3
N
N
Various analogues of the marine nortopsentins have
been reported. Many bis(indolyl)alkaloids where the
imidazole moiety of nortopsentin was replaced by thia-
zole (1), pyrimidine (2), pyrazine (3), and pyrazinone
(4) rings were designed and synthesized (Fig. 2). These
H
N
N
N
O
;
N
Figure 2.
Keywords: Nortopsentins; 3,5-Bis(30-indolyl)pyrazoles; Antitumor
activity; Topoisomerase II.
analogues presented strong inhibitory activity against
a wide range of human tumor cell lines (GI50 <0.01–
89.4 lM).5–9
*
Corresponding author. Tel.: +39 09 16161606; fax: +39 09
16169999; e-mail: diana@unipa.it
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.042

P. Diana et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6134–6137
6135
R1
R1
R1
N N
S
R
R
R
N
R
N
R
5
N
H
N
Me
N
Me
Figure 3.
9a-e R1 = H;
10a-e R1 = Me
6a-e
a R = H; b R = Me;
c R = OMe; d R = Cl;
e R = Br
We have recently reported the synthesis and the antitu-
mor activity of 2,5-bis(30-indolyl)thiophenes of type 5
where the imidazole moiety of the nortopsentin was re-
placed by a thiophene ring (Fig. 3). Such derivatives
showed antiproliferative activity generally in the micro-
molar range, and proved to be particularly effective
against the leukemia sub-panel (GI50 0.34–3.54 lM).10
i
iii
O
O
R
R
R
ii
N
N
N
Me
Me
Me
In the attempt of looking for novel antitumor com-
pounds, we designed new analogues with further modi-
fication of indole alkaloids in order to get more potent
and selective agents. We therefore planned to synthesize
a new series of bis-indolyl-5-membered heterocycles so
as to verify the influence of the central heterocyclic ring
on the antineoplastic activity.
7a-e
8a-e
Scheme 1. Reagents and conditions: (i) t-BuOK, MeI, TDA-1/benzene
rt, 24 h; (ii) malonyldichloride/DCM, rt, 2 h; (iii) NH2NH2ÆH2O or
NH2NHMe, THF/AcOH, reflux, 24 h.
parameters, giving an average activity parameter over
all cell lines.
In this paper, we report the synthesis of 3,5-bis(30-indo-
lyl)pyrazoles 9, 10, where a pyrazole central ring substi-
tuted the imidazole ring of nortopsentin, and the NCI’s
in vitro disease-oriented antitumor screen of the most
potent compounds of this series.
Data evaluation of the data reported in Table 1 pointed
out that compounds 9a,d exhibited antineoplastic activ-
ity against most of the human cell lines. Compound 9d,
bearing a chloro group, is more active than the unsubsti-
tuted derivative 9a both in terms of GI50 (mean value
3.23 and 18.2, respectively) and of percentage of sensi-
tive cell lines out of the total number of cell lines inves-
tigated (100% and 90%, respectively). Derivative 9d was
1,3-Bis-indolyl-diketones 8 appeared as valuable and
versatile intermediates for the synthesis of bis(indo-
lyl)pyrazoles. The synthetic approach to the pyrazole
compounds involved the indole derivatives of type 6,
which were converted into the corresponding N-methyl
derivatives 7 using potassium t-butoxide, tris(3,6-dioxa-
heptyl)amine (TDA-1) as a catalyst and methyl iodide in
dry benzene (96–98%). Friedel-Craft reaction of the
N-methylindoles 7 with malonyldichloride in dichloro-
methane yielded the expected 1,3-diketones of type 8
(45–70%). The resulting symmetrical 1,3-diketones 8
were converted into corresponding 3,5-bis(30-indolyl)-
pyrazoles 9, 10 using hydrazine monohydrate or meth-
ylhydrazine in refluxing acetic acid/THF (54–92%)
(Scheme 1).
Table 1. Overview of the results of the in vitro antitumor screening for
compounds 9a,da
Compound
9a
9d
Noe
Nf
58
52
58
58
b
GI50
Range
MG_MIDg
1.95 to 93.1
18.2
1.55 to 9.60
3.23
No
N
TGIc
59
16
58
53
All the bis(indolyl)pyrazoles 9, 1011 were submitted to
the National Cancer Institute (Bethesda, MD). Three
of them were selected (9a,c,d) for evaluation against
the full panel of about 60 human cancer cell lines de-
rived from nine human cancer cell types, grouped into
disease sub-panels including leukemia, non-small-cell
lung, colon, central nervous system, melanoma, ovarian,
renal, prostate, and breast tumors cell lines. The com-
pounds were tested at five concentrations at 10-fold dilu-
Range
MG_MID
2.06 to >100
79.4
1.63 to 94.8
14.5
No
N
59
4
59
24
d
LC50
Range
MG_MID
59.7 to 74.3
97.7
6.62 to 96.8
58.9
a Data obtained from the NCI’s in vitro disease-oriented human tumor
cells screen.
b GI50 is the lM concentration that inhibits 50% net cell growth.
c TGI is the lM molar concentration giving total growth inhibition.
d LC50 is the lM molar concentration leading to 50% net cell death.
e No is the number of the cell lines investigated.
tion, the highest being 10ꢀ4M and the others 10ꢀ5, 10ꢀ6
10ꢀ7, 10ꢀ8 M.12
,
In this series bis(indolyl)pyrazoles 9a,d resulted the most
active compounds. The antitumor activity is given by
three parameters for each cell line; GI50 value, TGI va-
lue, and LC50 value. Moreover, a mean graph midpoint
(MG_MID) was calculated for each of the mentioned
f N is the number of cell lines giving positive GI50, TGI and LC50
.
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.

6136
P. Diana et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6134–6137
Table 2. Inhibition of in vitro cancer cell lines by compound 9a,da
cytotoxic against the total number of cell lines investi-
gated at micromolar concentration, and in particular it
proved to be selective with respect to the melanoma
sub-panel, having all the sub-panel cell lines GI50 in
the range 1.63–9.64. The most sensitive cell lines were
UACC-62, LOX IMVI, and SK-MEL-5 GI50 1.63,
1.70, and 1.79, respectively). It also showed selectivity
with respect to MOLT-4, SR, and K-562 (GI50 1.55,
2.36, and 2.78, respectively) of leukemia sub-panel,
HCC-2998 and COLO 205 (GI50) 1.71 and 2.22, respec-
tively) of colon cancer, CAKI-1 (GI50 1.70) of renal can-
cer, BT-549 (GI50 2.03) of breast cancer, and SF-539
(pGI50 1.81) of CNS sub-panel. Derivative 9a was par-
ticularly effective against the colon sub-panel having
GI50 in the range 4.58–19.0. The most sensitive colon
cell lines were KM12 and HCC-2998 (GI50) 4.58 and
4.74, respectively). Compound 9a showed good selectiv-
ity with respect to the HOP-92 (GI50 2.06) and NCI-
H460 (GI50 4.48) of the non-small-cell lung cancer
sub-panel and MCF7 (GI50 3.95) of the breast cancer
sub-panel (Table 2).
b
Cell line
GI50
9a
9d
Leukaemia
CCRF-CEM
HL-60 (TB)
K-562
14.4
15.4
42.1
37.8
6.40
4.45
2.78
6.79
1.55
2.36
RPMI-8226
MOLT-4
SR
4.75
3.46
Non-small cell lung cancer
A549/ATCC
EKVX
16.3
3.24
3.15
7.98
1.86
5.29
2.49
5.73
2.35
1.75
27.2
17.3
HOP-62
HOP-92
2.06
NCI-H226
NCI-H23
19.8
22.0
>100
4.48
22.1
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO-205
HCC-2998
HCT-116
HCT-15
HT29
7.98
4.74
19.0
8.68
5.35
4.58
>100
2.22
1.71
3.82
3.01
3.57
3.45
3.52
Moreover, as to 9d derivative, positive TGI and LC50 re-
sponses were observed with respect to a noticeable num-
ber of cell lines (91% and 41%, respectively).
KM12
SW-620
The best responses were observed in the case of the SF-
539 (TGI 3.47 and LC50 6.62) CNS cancer, HCC-2998
(TGI 3.84 and LC50 8.63) colon cancer, and LOX IMVI
(TGI 3.80 and LC50 8.53) melanoma cell lines (data not
shown).
CNS Cancer
SF-268
SF-295
16.0
17.1
16.2
27.0
18.4
12.9
5.29
2.52
1.81
4.09
4.25
3.37
SF-539
SNB-19
SNB-75
U251
In order to discern the mechanism of action of bis(indo-
lyl)pyrazoles, we performed COMPARE computations
for derivatives 9a,d against the NCI ‘Standard Agents’
database.13 Compound 9a,d had a Pearson Correlation
Coefficient (PCC) <0.6 suggesting that the antiprolifera-
tive activity of 3,5-bis(30-indolyl)pyrazoles would be
mechanistically unrelated to that of any known drug.
Melanoma
LOX IMVI
MALME-3M
M14
5.38
93.1
>100
57.5
>100
13.1
80.6
16.4
1.70
9.64
2.15
2.34
4.80
1.79
4.27
1.63
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Experiments aiming at the evaluation of 9a,d deriva-
tives’ capability of interacting with DNA—a crucial tar-
get for most of the known antitumor drugs—revealed
that such derivatives were unable to form a molecular
complex with the macromolecule. In particular, linear
flow dichroism spectra obtained by salmon testes
DNA, where different concentrations of 9a,d were pres-
ent, resulted practically overlapping to those recorded
without those test compounds (spectra not shown). Fur-
thermore, the ability of interfering with the activity of
the nuclear enzyme topoisomerase II, which catalyzes
the interconversion of different topological forms of
DNA,14 was assayed. Figure 4A and B show the relaxa-
tion of supercoiled plasmid DNA mediated by the en-
zyme, when 9a,d were absent (lanes b) and when 9a,d
were present in increasing concentrations (lanes c–e).
As to 9a, the results obtained point out that the inhibi-
tory capability becomes detectable at about 50 lM
(Fig. 4A, lane d), while in the case of 9d it happens at
100 lM (Fig. 4B, lane e). Furthermore, both test com-
pounds present a scored inhibition which is significantly
weaker than the one of m-amsacrine used at 8 lM con-
centration (lane f).
Ovarian cancer
IGROV1
8.90
28.4
34.9
>100
28.8
31.6
2.55
4.41
5.82
3.11
4.03
5.81
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
A498
14.1
19.6
7.67
>100
14.7
1.95
37.4
37.6
3.41
2.44
3.23
1.70
3.03
Ndc
5.16
3.01
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
8.11
15.6
3.05
9.60

P. Diana et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6134–6137
6137
Table 2 (continued)
activity as testified by the GI50 at lM level observed
for the 58 tested cell lines.
b
Cell line
GI50
9a
9d
Breast cancer
MCF7
Acknowledgments
3.95
27.6
8.06
2.64
2.25
2.95
3.27
2.99
2.03
4.06
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
This work was financially supported by Ministero
`
dell’Istruzione dell’Universita e della Ricerca. We thank
18.5
Nd
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.
MDA-MB-435
BT-549
T-47D
15.9
79.7
a Data obtained from the NCI’s in vitro disease-oriented human tumor
cells screen.
b GI50 is the lM concentration that inhibits 50% net cell growth.
c Not determined.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.09.042.
A
a
b
c
d
e
f
References and notes
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1
11. Melting points, spectral data (IR, H and 13C NMR) and
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These results point out that DNA cannot be considered
the main cause of cell death, suggesting that other cellu-
lar molecular targets participate in the antiproliferative
effect of 9a,d.
In conclusion, the substitution of the imidazole core of
nortopsentin with a pyrazole ring displayed a similar po-
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