Antitumor activity of bis-indole derivatives

Article abstract of DOI:10.1021/jm800194k

This paper reports the synthesis of compounds formed by two indole systems separated by a heterocycle (pyridine or piperazine). As a primary screening, the new compounds were submitted to the National Cancer Institute for evaluation of antitumor activity in the human cell line screen. The pyridine derivatives were far more active than the piperazine derivatives. For the study of the mechanism of action, the most active compounds were subjected to COMPARE analysis and to further biological tests including proteasome inhibition and inhibition of plasma membrane electron transport. The compound bearing the 5-methoxy-2-indolinone moiety was subjected to the first in vivo experiment (hollow fiber assay) and was active. It was therefore selected for the second in vivo experiment (human tumor xenograft in mice). In conclusion we demonstrated that this approach was successful, since some of the compounds described are much more active than the numerous, so far prepared and tested 3-indolylmethylene-2-indolinones.

Full text of DOI:10.1021/jm800194k

J. Med. Chem. 2008, 51, 4563–4570
4563
Antitumor Activity of Bis-indole Derivatives1
Aldo Andreani,*,† Silvia Burnelli,† Massimiliano Granaiola,† Alberto Leoni,† Alessandra Locatelli,† Rita Morigi,†
Mirella Rambaldi,† Lucilla Varoli,† Laura Landi,‡ Cecilia Prata,‡ Michael V. Berridge,§ Carole Grasso,§ Heinz-Herbert Fiebig,|
Gerhard Kelter,| Angelika M. Burger, and Mark W. Kunkel#
Dipartimento di Scienze Farmaceutiche, UniVersita` di Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Dipartimento di Biochimica “G.
Moruzzi”, UniVersita` di Bologna, Via Irnerio 48, 40126 Bologna, Italy, Malaghan Institute of Medical Research, P.O. Box 7060, Wellington,
New Zealand, Oncotest GmbH, Am Flughafen 12-14, 79108 Freiburg, Germany, Department of Pharmacology and Experimental Therapeutics,
Marlene and Stewart Greenebaum Cancer Center, Bressler Research Building, Room 9-039, UniVersity of Maryland School of Medicine,
655 West Baltimore Street, Baltimore, Maryland 21201, and DeVelopmental Therapeutics Program, Information Technology Branch, DiVision
of Cancer Treatment and Diagnosis, National Cancer Institute, 6130 ExecutiVe BouleVard, Room 8008, RockVille, Maryland 20892-7444
ReceiVed February 24, 2008
This paper reports the synthesis of compounds formed by two indole systems separated by a heterocycle
(pyridine or piperazine). As a primary screening, the new compounds were submitted to the National Cancer
Institute for evaluation of antitumor activity in the human cell line screen. The pyridine derivatives were far
more active than the piperazine derivatives. For the study of the mechanism of action, the most active
compounds were subjected to COMPARE analysis and to further biological tests including proteasome
inhibition and inhibition of plasma membrane electron transport. The compound bearing the 5-methoxy-
2-indolinone moiety was subjected to the first in vivo experiment (hollow fiber assay) and was active. It
was therefore selected for the second in vivo experiment (human tumor xenograft in mice). In conclusion
we demonstrated that this approach was successful, since some of the compounds described are much more
active than the numerous, so far prepared and tested 3-indolylmethylene-2-indolinones.
Chart 1
Introduction
In 19842 we published the first of a series of papers devoted
to the synthesis of compounds with positive inotropic activity.
In 19903 we introduced in this project new derivatives arising
from the Knoevenagel reaction between an indole aldehyde and
a compound containing an active methylene group. Later we
found that analogous compounds showed antitumor activity too.
In our first paper on 3-(2-chloro-3-indolylmethylene)-1,3-
dihydroindol-2-ones as antitumor agents,4 we acknowledged that
the first synthesis of the unsubstituted 3-indolylmethylene-1,3-
dihydroindol-2-one was performed by a German team in 19695
and the first attempt to search an antitumor potential in this
class of compounds was made in 1977.6 We also reported that
a team at Farmitalia-Carlo Erba was working on the antitumor
activity of analogue compounds.7 In the subsequent years we
developed our project based on 3-(2-chloro-3-indolylmethylene)-
1,3-dihydroindol-2-ones8–11 whereas the project at Farmitalia-
Carlo Erba, passing through Pharmacia, Upjohn, Sugen, and
finally Pfizer, led to Sutent (see Chart 1 and http://www.sutent.
com/) which was approved by FDA in 2006.
structures show similarity to TAS-20214 and RITA15 (Chart 1)
for their molecular symmetry.
Chemistry
The Knoevenagel reaction we employed in the previous
papers involved the condensation of a carbonyl group with a
methylene activated by a neighboring electron withdrawing
group. The two classes of compounds reported in Scheme 1
present our continued synthetic efforts using the Knoevenagel
reaction. In the first case (3a-q) the carbonyl groups were in
the core (a pyridine ring) whereas the active methylene groups
were in the indole wings. In the second case (7-9) this situation
was inverted; the active methylene groups were in the core (a
piperazine ring), whereas the carbonyl groups were in the indole
wings. Moreover, the core may be connected to the wings by a
double bond (7a,b) or through a methine bridge (8a,b, 9a,b),
as in compounds 3a-q.
We also prepared compounds similar to 3-(2-chloro-3-
indolylmethylene)-1,3-dihydroindol-2-ones but with the two
indole systems separated by an heterocycle instead of a methine
bridge. These compounds were first tested as positive inotropic
agents12 and later as antitumor agents but only on HeLa cells.13
In this paper we describe several aspects of the antitumor activity
of this class of compounds, reported in Scheme 1, whose
* To whom correspondence should be addressed. Phone: +39-051-
2099714. Fax: +39-051-2099734. E-mail: aldo.andreani@unibo.it.
†
Dipartimento di Scienze Farmaceutiche, Universita` di Bologna.
Dipartimento di Biochimica “G. Moruzzi”, Universita` di Bologna.
Malaghan Institute of Medical Research.
Oncotest GmbH.
Most of the new derivatives 3 have been prepared with -
the same procedure described for the previously published
compounds:12,13 the appropriate indolinone 1 in methanol has
been treated with pyridine-2,6-dicarbaldehyde 2 in the presence
of piperidine (see Scheme 1 and Table 1, method 1). Only
‡
§
|
University of Maryland School of Medicine.
National Cancer Institute.
#
10.1021/jm800194k CCC: $40.75
2008 American Chemical Society
Published on Web 07/04/2008

4564 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Andreani et al.
Table 1. Compounds 3 and 7-9
Scheme 1
mp (°C)
or ref
starting material
ref
compd
formula
MW method
3a
3b
3c
3d
3e
3f
3g
3h
3i
C23H15N3O2
C23H13Cl2N3O2 434.28
C23H13Cl2N3O2 434.28
C25H19N3O4
C23H15N3O4
C31H23N3O8
C27H25N5O2
C23H13F2N3O2 401.37
C25H15N3O6 453.41
365.39
1
1
1
1
1
2
1
1
2
2
2
1
1
1
1
1
1
a
b
b
a
425.44
397.39
565.53
451.52
335-340
210-215 dec
340-350
347-350 dec
>360
355 dec
325-330
332 dec
c
d
e
f
g
h
3j
3k
3l
C23H13Br2N3O2 523.18
C23H15N3O8S2 525.52
C23H13Cl2N3O2 434.28
3m
3n
3o
3p
3q
7a
7b
8a
8b
9a
9b
C25H19N3O4
C27H23N3O4
C25H19N3O2
C27H23N3O4
C25H19N3O4
C20H12N4O4
C22H16N4O6
C26H20N4O4
C28H24N4O6
C22H16N4O2
C24H20N4O4
425.44
453.49
393.44
453.49
425.44
372.34
432.39
452.46
512.52
368.39
428.44
>360
i
j
314-315 dec
a
218-220
260-262 dec
>360
k
k
>360
302-305 dec
344-346 dec
355-358 dec
>360
a Reference 12. b Reference 13. c References 26–31. d References 26–31.
e References 26–31. f References 26–31. g References 26–31. h References
26–31. i Reference 9. j References 32, 33. k References 32, 33.
employed in place of the isatine 4 for the synthesis of
compounds 8a,b, this procedure gave poor yields whereas a
better result was obtained with acetic anhydride/sodium acetate.
Compounds 9a,b were obtained by alkaline hydrolysis of the
corresponding acetyl derivatives 8a,b.
All compounds 3 were obtained as pure geometrical isomers
and the configuration was assigned by means of 1H NMR spectra
(Table S1). We performed nuclear Overhauser enhancement
(NOEa) analysis on compound 3n. The spectrum shows that
the geometrical configuration of the two possible centers is the
same, since the protons at the positions 3 and 5 of the pyridine
ring give a single signal. The same happens for the couple of
NH groups and for the other couples of indole protons/
substituents: H-4, H-7, OCH3-5, CH3-6. The first NOE analysis
was devoted to the identification of H-4 and H-7 in the
indolinone system. The irradiation of the NH groups (10.39
ppm) and of the 6-methyl groups (2.04 ppm) produced NOE at
6.62 ppm (peak that was assigned to H-7), whereas the
irradiation of the 5-methoxy groups (3.33 ppm) produced NOE
at 8.01 ppm (H-4). Therefore, the remaining singlet of the
aromatic region (7.67 ppm) was assigned to the methine groups.
When this peak was irradiated, NOE was observed only at the
protons in positions 3 and 5 of the pyridine ring (7.90 ppm);
therefore, compound 3n belongs to the E configuration.
Also compounds 7-9 were obtained as pure isomers, and
for the study of the geometrical configuration of these deriva-
tives, NOE analyses were performed on compound 8a. As
compounds 3f, 3i-k have been prepared in a mixture of acetic
acid/hydrochloric acid (method 2).
1
observed in the previous case, the H NMR spectrum shows a
For the synthesis of compound 3k, ethyl 2-oxoindoline-5-
sulfonate was the starting indolinone. It was prepared by treating
2-oxoindoline-5-sulfonyl chloride16 with ethanol. The condensa-
tion with pyridine-2,6-dicarbaldehyde under acidic conditions
led to the simultaneous hydrolysis of the ester and to the
formation of the expected sulfonic acid 3k.
The synthesis of compounds 7a,b from the appropriate isatine
4 and 1,4-diacetylpiperazine-2,5-dione 6 has been performed
in dimethylformamide in the presence of triethylamine.17 In this
reaction the condensation took place with contemporaneous
hydrolysis of the acetyl group. When the indolaldehyde 5 was
symmetrical molecule; i.e., the two centers have the same
configuration. The irradiation of the NH group (10.44 ppm) gave
NOE at the indole protons in position 2 (8.39 ppm), thus
a Abbreviations: NOE, nuclear Overhauser enhancement; DTP, Devel-
opmental Therapeutics Program; NCI, National Cancer Institute; GI, growth
inhibition; TGI, total growth inhibition; LC, lethal concentration; DAVID,
Database for Annotation, Visualization and Integrated Discovery; PMET,
plasma membrane electron transport; mPMS, 1-methoxyphenazinemetho-
sulfate; WST-1, water soluble tetrazolium salt-1; MTT 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide; CHX, cycloheximide; RSI,
relative signal intensity; MTD, maximum tolerated dose.

Bis-indole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4565
Table 2. Sixty Cell Panel (Growth Inhibition, Cytostatic and Cytotoxic Activity of the Selected Compounds)
compounda
modes
leukemia
NSCLC
colon
CNS
melanoma
ovarian
renal
prostate
breast
MG-MIDb
3a
3b
3c
3d
3e
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pGI50
pTGI
pLC50
pGI50
pTGI
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pGI50
pTGI
pGI50
pGI50
pGI50
pTGI
pLC50
6.63
5.71
6.09
5.50
4.65
6.17
5.57
4.46
5.76
5.36
4.70
5.77
4.97
4.36
4.47
6.55
6.11
5.19
6.38
5.30
4.72
6.03
5.59
4.82
6.10
5.33
4.51
4.41
6.24
5.74
5.15
6.22
5.55
4.63
6.05
5.56
4.83
5.79
4.89
4.20
4.39
6.52
6.03
5.33
6.23
5.50
4.80
6.16
5.72
5.10
6.05
5.40
4.87
4.08
6.23
5.75
5.18
6.30
5.55
4.51
5.90
5.38
4.75
5.95
5.03
4.33
4.08
6.24
5.79
5.27
6.37
5.82
5.12
5.90
5.53
5.12
5.84
5.37
4.62
4.46
4.16
4.02
6.20
5.57
4.61
6.36
5.68
4.80
5.82
5.18
4.61
5.78
5.03
4.16
4.70
6.31
5.70
4.24
6.41
5.48
4.40
5.95
5.38
4.64
5.83
4.83
4.09
4.39
4.07
6.34
5.79
4.85
6.34
5.55
4.63
5.97
5.47
4.79
5.92
5.15
4.40
4.40
4.06
4.01
4.12
4.02
5.27
4.39
4.04
5.49
4.93
4.22
4.14
4.08
4.05
5.08
4.59
4.25
4.01
5.69
5.06
4.40
4.16
4.01
4.29
4.02
7.04
5.72
4.35
6.18
5.31
4.32
6.21
5.42
4.24
4.76
4.26
4.03
4.81
3f
4.52
4.21
5.36
4.35
4.05
5.42
4.94
4.27
4.14
4.06
3g
5.63
5.02
5.28
4.36
5.17
4.41
5.36
4.52
4.22
5.69
5.31
4.44
5.12
5.06
4.30
5.45
4.53
5.22
4.16
3h
3i
5.93
5.11
5.14
4.58
4.10
5.67
5.27
4.40
5.38
4.60
4.09
5.38
4.89
4.29
5.46
4.91
4.13
5.53
4.84
4.30
5.21
4.67
4.46
5.01
3j
4.96
4.55
4.20
4.98
4.61
4.28
5.43
4.85
4.39
5.00
4.63
4.28
4.02
5.75
5.21
4.67
4.06
5.01
4.59
4.23
5.10
4.66
4.29
4.07
5.97
5.48
4.70
4.17
5.19
4.68
4.26
5.21
4.74
4.27
3k
3l
4.03
5.84
5.09
4.02
4.56
5.43
4.83
4.24
4.07
5.76
5.31
4.60
4.14
5.65
5.11
4.44
5.52
4.71
4.20
4.12
5.47
4.39
4.15
4.18
5.64
4.87
4.28
4.15
4.06
4.13
4.03
3m
3n
5.36
4.22
4.04
5.51
4.80
4.30
5.93
4.42
4.08
4.31
4.05
4.10
4.05
4.28
4.47
3o
3p
4.92
4.52
4.30
5.61
4.72
4.13
4.07
4.07
5.00
4.55
4.30
5.84
5.47
4.93
5.12
4.53
4.30
5.49
4.83
5.31
4.65
4.36
5.98
5.42
5.05
5.00
4.53
4.30
5.59
4.50
5.00
4.63
4.33
5.82
5.28
4.68
4.08
4.53
4.30
4.15
4.87
4.40
4.30
5.68
5.05
4.48
4.30
5.79
4.70
4.20
4.07
4.18
5.09
4.57
4.31
5.76
4.91
4.36
4.05
4.13
4.04
4.09
4.03
6.70
5.00
3.60
7a
8a
4.13
4.12
4.01
4.10
4.03
4.06
9a
9b
4.01
4.19
4.26
4.14
6.80
5.10
3.60
4.19
4.10
6.50
5.10
3.50
4.05
6.50
4.70
3.50
vincristine sulfatec
7.00
4.80
3.20
6.60
4.80
3.60
7.00
5.40
4.10
6.90
5.20
3.70
6.50
4.70
3.60
6.90
5.20
3.50
a Highest concentration ) 10-4 M unless otherwise reported; only modes showing a value greater than 4.00 are reported. b Mean graph midpoint, i.e., the
mean concentration for all cell lines. c Highest concentration ) 10-3 M.
allowing us to confirm that the other singlet in the aromatic
region (6.99 ppm) was due to the methine bridges. When these
protons were irradiated, NOE was observed only at 7.74 ppm
(which is a doublet of the indole protons at the 4 position; as a
consequence, the doublet at 8.38 ppm was attributed to the
indole protons at the 7-position). This is in agreement with the
Z configuration. The irradiation of the CH3CO groups (2.77
ppm) produced NOE only at the indole protons in position 2
(8.39 ppm) and not at the methine bridges, thus confirming the
Z configuration.
MCF-7 breast cancer, and SF-268 glioma) a compound is
considered active when it reduces the growth of any of the cell
lines to 32% or less. Most of the compounds reported in Scheme
1 were active and passed on for evaluation in the full panel of
60 human tumor cell lines. This panel is organized into
subpanels representing leukemia, melanoma, and cancers of
lung, colon, kidney, ovary, breast, prostate, and central nervous
system.
The test compounds were dissolved in DMSO and evaluated
using five concentrations at 10-fold dilutions, the highest being
10-4 M. Table 2 reports the results obtained, expressed as the
negative log of the molar concentration at three assay end points:
the 50% growth inhibitory power (pGI50), the cytostatic effect
(pTGI ) total growth inhibition), and the cytotoxic effect
(pLC50). Some of the compounds were reassayed on the basis
of the results of the first screen: compounds 3d, 3h, and 3n
Antitumor Activity. As a primary screening, compounds 3
and 7-9 were submitted to the Developmental Therapeutics
Program (DTP) at the National Cancer Institute (NCI) (http://
dtp.nci.nih.gov) for evaluation of antitumor activity in the human
cell line screen. In a preliminary screen at a single concentration
(100 µM) against three human cell lines (NCI-H460 lung cancer,

4566 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Andreani et al.
were assayed twice, compounds 3b and 3i were assayed three
times, and all other compounds were assayed once. The pyridine
derivatives (3) were far more active than the piperazine
derivatives (7-9). Compounds 3a-d,g,h,j,l,o,p were potent
growth inhibitors with mean pGI50 ranging from 5.08 to 6.34
and cytotoxicity from 4.04 to 4.85. The difference between the
average concentration that caused 50% growth inhibition and
the concentration that killed 50% of the cells was around 1.2
log with only 0.78 for 3o and 1.71 for 3b. Within each of the
nine cell panels there was a difference of 0.9 between the least-
sensitive and the most-sensitive (leukemia) cell lines in response
to 3b.
A comparison of the activities of the compounds reported in
this paper shows that the weak activity of 7-9 is not related to
the methoxy group in the indole system, to the acetyl group in
the piperazine ring, or to the methine bridge. Even with this
limited number of compounds, it seems clear enough that the
core is important, since in the pyridyl derivatives 3a-q the
antitumor activity is evident and related to the substituents in
the indole system. The presence of acidic groups led to loss of
activity probably because the resulting compounds (3f,i,k) are
too hydrophilic (in particular, compounds 3f,i showed their
highest activity on leukemia cells). This hypothesis is confirmed
by the weak activity of the hydroxy derivatives 3e,m,q.
Interesting results were obtained with halogens, methoxy, and
dimethylamino groups. The compound containing chlorine at
the 4 position of the indole (3b) was very active; the activity
decreased by shifting the halogen from the 4 to the 5 (3c) and
6 positions (3l).
Figure 1. Antiproliferative activity of 3b (black lines) in a panel of
breast cancer cell lines in comparison to the proteasome inhibitor MG-
132 (red lines). Growth curves are shown for effects of drugs after 5
days of continuous exposure. Proliferation was measured by MTT assay,
and data are displayed as percent growth inhibition compared to the
growth of vehicle treated control cells.
correlations to some related compounds previously submitted,
suggest commonalities among them. It may be that the bis-indole
derivatives are affecting several different cellular processes at
the same time, producing cell response patterns that do not
correlate with any public compounds previously submitted to
COMPARE. It is also possible that these compounds do not
satisfy the important assumptions when using COMPARE to
search for a mechanism of action; compounds with similar
mechanism(s) or the relevant molecular target(s) must already
have been assayed and included in the database, and the standard
48 h exposure time in the screening assay must have been
sufficient for a compound’s effects to manifest themselves, and
in addition, the cell lines in the screen must have exhibited a
range of sensitivities to the compounds or a range of expression
of the molecular targets so that there are useful patterns for
COMPARE to work with.
Further Studies for Compounds 3b and 3d. Compounds
3b and 3d, with pGI50 values of 6.34 and 5.92, respectively,
were among the most active of the whole series. In a collabora-
tion with Oncotest (Freiburg, Germany) these compounds were
subjected to a further in vitro evaluation on a panel of 36 human
tumor cell lines (bladder, colon, CNS, gastric, head-neck, lung,
breast, melanoma, ovarian, pancreas, prostate, pleuramesothe-
lioma, renal, uterus body), and the result was subjected to
COMPARE analysis against the company’s own database of
compound screening data. This is a similar methodology to the
NCI COMPARE above but used a separate set of screening
data obtained from a distinct set of cell lines.
Compound 3b was the closest match with 3d. Overall, 3b
was more potent than 3d (mean IC70 0.35 versus 0.56 µg/mL)
and has a more pronounced selectivity. Compounds 3b and 3d
were similar in the COMPARE analyses (r ) 0.60), and 3b
was correlated with tyropeptin A, a proteasome inhibitor, with
a correlation of r ) 0.54. This result prompted us to test whether
compound 3b was a proteasome inhibitor. The study was
performed at the University of Maryland after taking into
account the high growth inhibitory effect of compound 3b
against breast cancer cells and its comparable in vitro growth
inhibitory activity to the known proteasome inhibitor MG-132
(Z-Leu-Leu-Leu-al)asshowninFigure1.Theubiquitin-proteasome
system regulates cellular protein degradation and homeostasis.
COMPARE Analyses. We used the COMPARE tool from
the DTP at the NCI to analyze the screening results of the most
active compounds for indications of mechanism of action.
COMPARE uses the pattern of relative sensitivity/insensitivity
of the cell lines in the screen to identify other compounds with
similar patterns of activity or to identify molecular targets whose
patterns of expression correlate with the pattern of compound
activity.
Matrix COMPARE was used to determine the pairwise
correlation of the cell response pattern for every combination
of two compounds in the set of nine compounds for which we
had data at the GI50 end point. In addition, the individual
compound screening results at the GI50 end point were also
submitted for COMPARE analyses against the NCI standard
agent compound set, against all of the NCI public compound
screening data and against all of the NCI public microarray
expression data. COMPARE correlations of 0.6 or greater
between two compounds suggest that the compounds affect the
same mechanism(s) within cells.18
In the matrix COMPARE analysis of the nine compounds,
the highest correlation was 0.74 between 3c and 3h and the
second highest correlation of 0.65 was between 3c and 3o. The
direct correlation between 3h and 3o was 0.48. Qualitative
inspection of the relative sensitivities in the cell line panels
indicates that these correlations are driven by greater-than-
average activity against leukemia and melanoma cell lines
coupled with less-than-average activity against non-small-cell
lung and renal cell lines. Within the matrix COMPARE results,
correlations other than those above had an average of about
0.19 with a standard deviation of 0.19 and were not considered
further.
COMPARE has not identified any single strong candidates
for mechanism of action or molecular targets that may be
targeted by the bis-indole derivatives, though the correlations
among a few compounds in this series, along with high

Bis-indole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4567
Table 3. Summary of in Vitro Growth Data
different when we examined the compounds’ ability to influence
the stability of endogenous BCA2, via inhibition of the
proteasome. While MG-132 can stabilize the protein, in the
presence of the protein synthesis inhibitor cycloheximide (CHX),
3b had only very limited effects. However, some potential to
stabilize BCA2 was seen at 16 h (lanes 3 and 5, Figure 2) in
that 3b treatment led to an approximately 2-fold increase of
BCA2 protein compared to CHX treatment. However, MG-132
was able to stabilize BCA2 more than 10-fold compared to the
CHX treatment (Figure 2). In particular at 8 h a second, a smaller
band appeared in the Western blot. This band is likely a form
of BCA2 that is phosphorylated by AKT as shown before,
suggesting that 3b might modulate the stability of kinases such
as AKT.20
IC50, µM
breast cancer cell line
3b
MG-132
MCF-7
MDA-MB-231
Hs578T
0.23
0.3
0.4
0.3
0.25
not done
No effects of 3b were found on ꢀ-actin (used as loading
control) or the oncogenic receptor C-erbB2, a protein that is
degraded via the lysosomal pathway (Figure 2).
PMET Inhibition. Another potential mechanism of action
of anticancer agents is the inhibition of plasma membrane
electron transport (PMET), a stress adaptation pathway that has
been associated with tumor activity.21,22 Since these compounds
could exhibit a potential redox activity due to the presence of
ketone groups, we thought it was useful to determine whether
the in vitro activity was associated with the ability to inhibit
PMET. Therefore, compounds active against HL60 cells and
some inactive compounds were selected (3b-e,g-i,l-o,8a,9a)
and tested at the Malaghan Institute of Medical Research (New
Zealand) in human leukemic HL60 cells and mitochondrial
gene-knockout HL60F0 cells which have defective mitochondrial
electron transport and therefore exclude possible respiratory
effects. PMET was assayed by measuring the extracellular
reduction of the tetrazolium dye, WST-1, in the presence of
the intermediate electron acceptor, 1-methoxyphenazinemetho-
sulfate (mPMS). Compounds 3g, 3i, 3o, 8a, and 9a were inactive
at 200 µM while IC50 values for the other compounds were
117 µM or greater in both cell lines. In contrast, the IC50 values
for cell proliferation as determined in a 2-day MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell pro-
liferation assay for compounds 3b and 3c were 3-8 µM for
both HL60 and HL60F0 cells. Other experiments, performed at
the Biochemistry Department, University of Bologna, showed
that inhibition of PMET occurred in different human leukemic
cell lines, such as B1647, HEL, and M07e, only in the presence
of 3b (IC50 ) 15-30 µM) (Figure 3), 3d (IC50 ) 30-45 µM),
and to a lesser extent 3e (IC50 ) 40-50 µM). These data
correlate with effects on mitochondrial complex I and on total
respiratory chain activity. In these cell lines the MTT assay
confirmed the antiproliferative activity of all tested compounds.
Results here reported emphasize that PMET inhibitory activity
of compounds 3b, 3d, and 3e may contribute to their antipro-
liferative effect in some leukemic cell lines, but in general,
PMET inhibitory activity does not correlate closely with
antiproliferative effect in the leukemic cells tested.
Figure 2. Effects of 3b on the stability of the ubiqutin E3 ligase BCA2
in MCF-7 breast cancer cells (VC, vehicle control). The protein
synthesis inhibitor CHX alone and in combination with the proteasome
inhibitor MG-132 and 3b were evaluated for effects on the protein
stability of BCA2 (which is degraded in the proteasome), the stability
of C-erbB2 (a receptor tyrosine kinase that is degraded in the lysosome),
and the stable housekeeping gene ꢀ-actin. Expression levels of BCA2
relative to ꢀ-actin are shown below each lane of the BCA2 blot.
Compound 3b treated MCF-7 cells lysates exhibit approximately double
the amount of BCA2 compared to CHX alone treated cells. The
expression levels of C-erbB2 relative to ꢀ-actin ranged from 1.1 to 0.8
RSI (relative signal intensity) as determined by ImageJ software.
Figure 3. Effect of compound 3b on PMET activity in acute leukemic
cell lines. Cells (105 cells per well in 0.1 mL) were pretreated for 30
min with 3b compound dissolved in DMSO prior to adding WST-1/
mPMS (final concentration of 500 and 20 µM, respectively). WST-1
reduction was measured in real time at 450 nm in a BMG Fluostar
microplate reader. Results are averages ( SEM of four independent
experiments.
In Vivo Experiments. Further studies continued at the NCI
where the maximum tolerated dose (MTD) was determined for
compounds 3b and 3d and it was found high enough (400 mg/
kg) to warrant further testing which started with 3d only. The
first in vivo experiment was the “hollow fiber assay”.23
Compound 3d was tested against a panel of six tumor cell lines
consisting of the leukemia cell lines HL-60, K562, MOLT-4,
CCRF-CEM, RPMI-8226, SR. A compound is considered for
xenograft testing if it has a combined ip + sc score of 10 or
greater, has a sc score of 4 or greater, or produces cell kill of
any cell line at either dose level evaluated. Compound 3d
In fact, many of the known E3 ligases that tag a given protein
with ubiquitin targeting it for degradation in the proteasome
play a role in various diseases, including cancer development
and progression. In particular, breast cancer seems to be
associated with the aberrant expression of several RING E3
ligases including BCA2.19 E3 ligases are very unstable because
of autoubiquitination, a hallmark of E3 activity, and their rapid
degradation in the proteasome.19 Although, compound 3b and
MG-132 had very similar activity in the considered breast cancer
cell line panel (Figure 1 and Table 3), their behavior was very

4568 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Andreani et al.
6.86 (2H, d, ind-7, J ) 8.5 Hz), 7.49 (2H, dd, ind-6, J ) 8.5 Hz, J
) 1.2 Hz), 8.01 (2H, s, -CHd), 8.07 (2H, d, ind-4, J ) 1.2 Hz),
8.27 (1H, t, py-4, J ) 7.5 Hz), 8.65 (2H, d, py-3 + 5, J ) 7.5 Hz),
11.11 (2H, s, NH). Anal. (C23H13Br2N3O2) C, H, N.
Synthesis of 3,6-Bis(2-oxo-1,2-dihydroindol-3-ylidene)pipera-
zine-2,5-diones (7a,b). The appropriate isatine (4a,b, 5 mmol) was
added to a solution of 1,4-diacetylpiperazine-2,5-dione 6 (2.5 mmol)
in DMF (10 mL). After the addition of triethylamine (5.5 mmol),
the reaction mixture was stirred at room temperature for 3-5 h.
The brown precipitate thus formed was collected by filtration with
a yield of 90% and used without further purification.
resulted in a ip + sc score of 22, and it was therefore examined
for distal site antitumor activity in an appropriate human tumor
xenograft model in nude mice24,25 (Table S2). As shown in
Table S3 it did not show significant activity in the tumor
employed (HL-60-TB leukemia).
In conclusion we demonstrated that this new approach was
successful, since some of the compounds here described are
much more active than the numerous, so far prepared and tested
3-indolylmethylene-2-indolinones which have been mentioned
in the introduction as the rationale for this search.
Synthesis of 1,4-Diacetyl-3,6-bis(indol-3-ylmethylene)piperazine-
2,5-diones (8a,b). The appropriate indolealdehyde 5 (10 mmol) was
treated with 1,4-diacetylpiperazine-2,5-dione 6 (5 mmol), anhydrous
sodium acetate (20 mmol), and acetic anhydride (10 mL). The
reaction mixture was refluxed for 3-5 h (according to a TLC test),
and acetic anhydride was removed under reduced pressure. The
residue was treated with ice-water, and the resulting yellow to
orange precipitate was collected by filtration with a yield of 70%
and used without further purification.
Synthesis of 3,6-Bis(indol-3-ylmethylene)piperazine-2,5-diones
(9a,b). The appropriate compound 8 (1 mmol) was dissolved in
ethanol (250 mL) and refluxed for 5 h with 2 N KOH (3 mL). The
yellow to orange precipitate formed after cooling was collected by
filtration and used without further purification. The yield was 60%
for 9a and 30% for 9b.
Experimental Section
(A) Chemistry. The melting points are uncorrected. Elemental
analyses were performed with a Fisons Carlo Erba Instrument
EA1108 and resulted within 0.4% of the theoretical values (Table
S4). Bakerflex plates (silica gel IB2-F) were used for TLC; the
eluent was petroleum ether/acetone in various proportions. The IR
spectra were recorded in Nujol on a Nicolet Avatar 320 E.S.P.;
ν
max is expressed in cm-1. The 1H NMR spectra were recorded in
(CD3)2SO or CF3COOD on a Varian Gemini (300 MHz); the
chemical shift (referenced to solvent signal) is expressed in δ (ppm)
and J in Hz. For the spectra that are not reported here, see Table
S1.
Pyridine-2,6-dicarbaldehyde and all the starting indolinones 1,
except ethyl 2-oxoindoline-5-sulfonate, are commercially available
or have been prepared as described in the literature (see Table
1).9,12,13,26–33 Compounds 7-9 were prepared from commercially
available starting material.
(B) Biology. (a) In Vitro Growth Inhibition and Cytotoxicity. It
was determined on the 3 and on the 60 cell line panel by the NCI
according to standard procedures.34
(b) 36 Cell Line Assay. The 36 cell line assay was performed at
Oncotest; 24 of the 36 cell lines were established from patient-
derived tumor xenografts passaged subcutaneously in nude mice.35
The origin of the donor xenografts has been already described.36,37
The other 12 cell lines were commercially available. All cells were
grown at 37 °C in a humidified atmosphere (95% air, 5% CO2) in
RPMI 1640 medium (PAA, Co¨lbe, Germany) supplemented with
10% fetal calf serum (PAA) and 0.1 mg/mL gentamicin (PAA). A
modified propidium iodide assay38 was used to assess the effects
of compounds. Tumor derived cell lines were incubated in
96-multiwell plates. After 1 day, the compounds under test were
added to the plates at five concentrations in the range from 0.001–10
µg/mL and left for further 4 days. The inhibition of proliferation
was determined by measuring the DNA content using an aqueous
propidium iodide solution (7 mg/mL). Fluorescence was measured
using the Cytofluor 4000. All compounds were tested in three
independent experiments. In each experiment, all data points were
determined in triplicate.
The COMPARE algorithm uses in vitro activity data to obtain
clues to the mechanism of action of a test compound. The individual
IC70 values of the test compounds in 36 test cell lines obtained in
the monolayer assay were correlated to the corresponding IC70
values for more than 100 standard agents determined in exactly
these 36 cell lines. Data for these standard agents are available in
the Oncotest database.
(c) Analysis of BCA2 Protein Stability. To determine the stability
of endogenous BCA2, 1 × 106 MCF-7 cells were seeded into six-
well plates and grown overnight. The protein synthesis inhibitor
cyclohexamide (Sigma) was added alone (100 µmol/L) or in
combination with the proteasome inhibitor MG-132 (10 µmol/L,
Calbiochem) or compound 3b (1 µmol/L) for a total of 8 and 16 h.
Cell lysates were generated and Western blots performed as
described before.39 We used the polyclonal anti-BCA2 antibody
(diluted 1:800) that has been generated.19 The ꢀ-actin monoclonal
mouse antibody was from Sigma (St. Louis, MO, diluted 1:5000),
and the monoclonal anti-C-erbB2 antibody (diluted 1:2000) was
from EMD Bioscienes, La Jolla, CA.
Synthesis of Ethyl 2-Oxoindoline-5-sulfonate. 2-Oxoindoline-5-
sulfonyl chloride (6 mmol) was refluxed for 30 min with 30 mL of
ethanol. The white crystalline precipitate formed after cooling was
separated by filtration with a yield of 90% and used without further
purification. C10H11NO4S, MW 241.27, mp 150 °C. IR: 1716, 1614,
1004, 922, 784. NMR: 1.19 (3H, t, CH3-CH2, J ) 7.2) 3.61 (2H,
s, ind-3), 4.04 (2H, q, CH3-CH2), 7.01 (1H, d, ind-7, J ) 8), 7.69
(1H, s, ind-4), 7.73 (1H, d, ind-6, J ) 8), 10.90 (1H, s, NH).
Synthesis of 3,3′-[Pyridine-2,6-diylbis(methylene)]bis(1,3-dihy-
droindol-2-ones) (3a-q). Two different methods were employed
according to the substituents in the indole system (Table 1).
Method 1 (Compounds 3e,g,h,l-n,p,q). This method was em-
plyed even for the published compounds 3a-d,o.12,13 The ap-
propriate indolinone 1 (10 mmol) was dissolved in methanol and
treated with pyridine-2,6-dicarbaldehyde 2 (5 mmol) and piperidine
(2 mL). The reaction mixture was refluxed for 3-5 h (according
to a TLC test), cooled, and if necessary, concentrated at reduced
pressure. The yellow to orange precipitate thus formed was collected
by filtration with a yield of 80% for compounds 3n and 50-60%
for the others. They were used as such without further purification.
Data for 3g. IR: 3283, 1710, 1677, 1214, 1091. 1H NMR: 2.52
(12H, s, CH3), 6.60 (2H, dd, ind-6, J ) 8.4 Hz, J ) 2.2 Hz), 6.66
(2H, d, ind-7, J ) 8.4 Hz), 7.67 (2H, s, -CHd), 7.87(2H, d, ind-4,
J ) 2.2 Hz), 7.89 (2H, d, py-3,5, J ) 7.8 Hz), 8.09 (1H, t, py-4, J )
7.8 Hz), 10.28 (2H, s, NH). Anal. (C27H25N5O2) C, H, N. Data for
1
3h. IR: 3100, 1695, 1617, 1194, 912. H NMR: 6.76 (2H, m, ind),
6.91 (2H, m, ind), 7.81 (2H, s, -CHd), 7.98 (4H, m, ind + py-3,5),
8.15 (1H, t, py-4, J ) 7 Hz), 10.65 (2H, s, NH). Anal. (C23H13F2N3O2)
1
C, H, N. Data for 3l. IR: 3201, 1721, 1611, 1070, 917. H NMR:
6.48 (2H, dd, ind-5, J ) 8.1 Hz, J ) 1.5 Hz), 6.81 (2H, d, ind-7, J
) 1.5 Hz), 7.44 (2H, s, -CHd), 7.94 (2H, d, py-3,5, J ) 7.8 Hz),
8.11 (1H, t, py-4, J ) 7.8 Hz), 8.12 (2H, d, ind-4, J ) 8.1 Hz),
10.78 (2H, s, NH). Anal. (C23H13Cl2N3O2) C, H, N.
Method 2 (Compounds 3f,i-k). Pyridine-2,6-dicarbaldehyde 2
(10 mmol) was dissolved in acetic acid (50 mL) and treated with
the appropriate indolinone 1 (20 mmol) and 37% hydrochloric acid
(1 mL). The reaction mixture was refluxed for 3-5 h (according
to a TLC test) and cooled. The yellow to orange precipitate thus
formed was collected by filtration with a yield of 80% for 3k and
50-60% for the others. They were used as such without further
purification. Data for 3j. IR: 3170, 1706, 1609,1301, 717. 1H NMR:
Signals were quantified using the NIH ImageJ software by
measuring the mean signal intensity (gray scale) value and its
integrated density. Resulting absolute intensities were determined,
and a relative intensity value was obtained by dividing the absolute
intensity by the absolute intensity of the control sample. MTT

Bis-indole DeriVatiVes
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cytotoxicity assays were performed with the breast cancer cell lines
in Table 3 as described before.39
(d) PMET and MTT Cell Proliferation Assays. Inhibition of
PMET was determined essentially as described22 using a microplate
format with 100 000 cells per well in 0.1 mL. Cells were pretreated
for 30 min with compound dissolved in DMSO (<0.1% final
concentration) prior to adding WST-1/mPMS, and WST-1 reduction
was measured in real time at 450 nm in a BMG Fluostar microplate
reader. MTT reduction was measured following incubation of cells
with compound for 48 h.
(e) MTD Assay (http://dtp.nci.nih.gov). Basically what is done
is to dose a mouse with a single injection of 400 mg/kg, ip. If the
animal survives 14 days, the MTD is 400. If the animal dies, the
dose is lowered to 200 mg/kg. The dose keeps being lowered by
50% until a tolerated dosage is determined, and that will be the
MTD.
(f) Hollow Fiber Assay and Xenograft. These tests were
performed by the NCI according to standard procedures (http://
dtp.nci.nih.gov/branches/btb/hfa.html).24,25
Acknowledgment. This work has been supported by a grant
from MIUR-COFIN 2006. We are grateful to the National
Cancer Institute (Bethesda, MD) for the anticancer tests and to
Professor Romana Fato for the studies on mitochondrial complex
I and on total respiratory chain.
Note Added after ASAP Publication. This manuscript was
released on July 4, 2008 with an error in the Experimental Section.
The correct version posted on July 10, 2008.
Supporting Information Available: IR and 1H NMR of the
new compounds (Table S1), hollow fiber data for compound 3d
(Table S2), response of advanced stage SC HL-60 (TB) leukemia
xenografts to 3d (Table S3), elemental analyses (Table S4), and
NSC numbers (Table S5). This material is available free of charge
via the Internet at http://pubs.acs.org.
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