Substituted E-3-(2-Chloro-3-indolylmethylene)1,3-dihydroindol-2-ones with antitumor activity

Article abstract of DOI:10.1016/j.bmc.2003.11.031

The synthesis and antitumor activity of a new series of E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones is described. Several compounds were active on the primary test (three human cell lines) and entered the second level (60 human cell lines). All of them were potent growth inhibitors with GI₅₀ ranging from -5.32 to -7.27. Four are now under review by BEC (Biological Evaluation Committee of the NCI). The most potent antitumor derivatives were also evaluated as cardiotonic agents (in view of a possible coanthracyclinic activity). In order to find a possible mechanism of action their effects on cell cycle progression in an adenocarcinoma cell line (HT29) were tested, evidencing that these molecules are able to block HT29 in mitosis. The introduction of new substituents in the indolinone moiety while maintaining the same chloroindole portion generated interesting derivatives. 3-(2-Chloro-5-methoxy-6-methyl-3-indolylmethylene)5-hydroxy-1, 3-dihydroindol-2-one was the most active of the whole series. It was more potent than vincristine against seven of the nine tumors considered. Moreover it was selective towards some cell lines such as MDA-MB-435 (breast), OVCAR-3 (ovarian) and SK-MEL-28 (melanoma). Even the introduction of a benzyl ring at the nitrogen of the chloroindole portion, gave rise to potent compounds.

Full text of DOI:10.1016/j.bmc.2003.11.031

Bioorganic & Medicinal Chemistry 12 (2004) 1121–1128
Substituted E-3-(2-chloro-3-indolylmethylene)1,
3-dihydroindol-2-ones with antitumor activity§
Aldo Andreani,a,* Massimiliano Granaiola,a Alberto Leoni,a
Alessandra Locatelli,a Rita Morigi,a Mirella Rambaldi,a Vida Garaliene,b
Giovanna Farruggiac and Lanfranco Masottic
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
bLithuanian Institute of Cardiology, Kaunas, LT-3007, Lithuania
cDipartimento di Biochimica ‘G. Moruzzi’, Universita’ di Bologna,
Via Irnerio 48, 40126 Bologna, Italy
Received 28 October 2003;accepted 28 November 2003
Abstract—The synthesis and antitumor activity of a new series of E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones is
described. Several compounds were active on the primary test (three human cell lines) and entered the second level (60 human cell
lines). All of them were potent growth inhibitors with GI50 ranging from ꢀ5.32 to ꢀ7.27. Four are now under review by BEC
(Biological Evaluation Committee of the NCI). The most potent antitumor derivatives were also evaluated as cardiotonic agents (in
view of a possible coanthracyclinic activity). In order to find a possible mechanism of action their effects on cell cycle progression in
an adenocarcinoma cell line (HT29) were tested, evidencing that these molecules are able to block HT29 in mitosis. The introduc-
tion of new substituents in the indolinone moiety while maintaining the same chloroindole portion generated interesting derivatives.
3-(2-Chloro-5-methoxy-6-methyl-3-indolylmethylene)5-hydroxy-1,3-dihydroindol-2-one was the most active of the whole series. It
was more potent than vincristine against seven of the nine tumors considered. Moreover it was selective towards some cell lines such
as MDA-MB-435 (breast), OVCAR-3 (ovarian) and SK-MEL-28 (melanoma). Even the introduction of a benzyl ring at the
nitrogen of the chloroindole portion, gave rise to potent compounds.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
the chloroindole portion, while leaving the
indolinone nitrogen as NH since in a previous
paper2 we demonstrated that at least one of the
two nitrogens should be unsubstituted.
We report the synthesis and antitumor activity of a new
series of E-3-(2-chloro-3-indolylmethylene)1,3-dihydro-
indol-2-ones (3, see Scheme 1). It is our fourth paper on
this topic.2ꢀ4 Our previous paper4 pointed out that all
the most active compounds were derivatives of 5-meth-
oxy-6-methyl-2-chloroindol-3-carbaldehyde 1 (R¼H).
The growth inhibition of all the new compounds (3a–q)
was tested on three human tumor cell lines according to
the protocols developed at the National Cancer Insti-
tute (NCI, Bethesda, MD). The active compounds were
tested on the sixty tumor cell lines.
At the light of this finding we planned the synthesis of
new compounds 3 according to the following rationale:
The most potent antitumor derivatives (3c,f,m,o) were
also evaluated in their effects on cell proliferation and
cell cycle progression in the colon adenocarcinoma cell
line HT29, because it has been demonstrated that sev-
eral indole-derivatives are able to induce a G2/M
arrest5,6 and apoptosis in different cell lines.7 Finally
they were tested as positive inotropic agents in search
for a potential coanthracyclinic activity. We suggested
this term to indicate the pharmacological behavior of
a molecule endowed with both antitumor activity
1. introduction of new substituents in the indoli-
none moiety (3a–g) while maintaining the same
chloroindole portion,
2. introduction of an unsubstituted (3h–n) or
substituted (3o–q) benzyl ring at the nitrogen of
* Corresponding author. Tel.: +39-051-2099714;fax: +39-051-
2099734;e-mail: aldo.andreani@unibo.it
§See Ref. 1.
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.11.031

1122
A. Andreani et al. / Bioorg. Med. Chem. 12 (2004) 1121–1128
Scheme 1.
Table 1. Compounds 3a–q (Bn=benzyl)
Comp
R
R1
R2
R3
R4
Formula
MW
Mp, ꢁC
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
H
H
H
H
H
H
H
Bn
Bn
Bn
Bn
Cl
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
Cl
H
H
H
H
CH3
H
H
H
H
CH3
H
H
H
CH3
H
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
H
H
H
H
C19H14Cl2N2O2
C19H14Cl2N2O2
C19H15ClN2O3
C21H19ClN2O3
C20H17ClN2O3
C20H17ClN2O3
C21H19ClN2O3
C26H21ClN2O2
C27H23ClN2O3
C28H25ClN2O3
C26H20Cl2N2O2
C26H20Cl2N2O2
C26H21ClN2O3
C27H23ClN2O3
C26H20Cl2N2O2
C27H23ClN2O3
C26H20ClN3O4
373.2
373.2
354.8
382.8
368.8
368.8
382.8
428.9
458.9
473.0
463.4
463.4
444.9
458.9
463.4
458.9
473.9
335–337 dec.
215–220 dec.
285–287 dec.
204–206
300–302 dec.
340–345 dec.
285–289 dec.
197–200
OH
OC2H5
OH
OH
OCH3
H
OCH3
OCH3
H
189–190
211–214 dec.
270–272 dec.
190–192 dec.
200–205 dec.
293–295 dec.
215–217 dec.
258–260 dec.
221–223 dec.
Bn
Bn
Bn
Cl
OH
OH
H
H
H
4Cl-Bn
4CH3O-Bn
2NO2-Bn
H
(in order to reduce the anthracycline toxicity by
reducing its dosage) and positive inotropic activity (in
order to counteract the heart depression induced by
anthracyclines).8
3. Pharmacological results
3.1. In vitro growth inhibition and cytotoxicity9
As a primary screening, compounds 3a–q were eval-
uated for their cytotoxic potency on three human cell
lines (NCI-H460 lung cancer, MCF7 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 (negative numbers indicate cell kill). Eleven of the
compounds reported in Scheme 1 were active and pas-
sed on for evaluation in the full panel of 60 human
tumor cell lines.
2. Chemistry
The reaction between a 2-chloroindolaldehyde 1 and the
equivalent of an oxindole 2 was performed in methanol
in the presence of piperidine (see Scheme 1 and Table 1).
Only for compound 3f triethylamine was employed
since piperidine gave nucleophilic displacement of
chlorine at the 2 position with the formation of the 2-
piperidinyl derivative. This behavior was previously
noticed for analogue compounds.2,4
This panel is organised into subpanels representing leu-
kemia, melanoma and cancers of lung, colon, kidney,
ovary, breast, prostate and central nervous system.
The NMR spectra are in agreement with those pre-
viously published for analogue E-derivatives and are
reported in Table 2. A common feature in this series is
the hydrogen at position 4 in the 2-chloroaldehydes
(7.5–7.6 ppm in compounds 1) which is shielded by the
indolinone portion (6.5–6.9 ppm for the same hydrogen
in compounds 3).
The test compounds were dissolved in DMSO and
evaluated using five concentrations at 10-fold dilutions,
the highest being 10ꢀ4 M and the others 10ꢀ5, 10ꢀ6
10ꢀ7, 10ꢀ8 M.
,
Table 3 reports the results obtained expressed as log10 of
the molar concentration, taking into consideration the
50% growth inhibitory power (GI50), the cytostatic
effect (TGI=Total Growth Inhibition) and the cyto-
toxic effect (LC50).
All compounds
3 were obtained as pure geo-
metrical isomers. In order to confirm that they belong
(as the previous ones) to the E configuration, the usual
NOE experiments were performed on compounds
3e,h,q.
The four most active compounds (3c,f,m,o) have been
analyzed by means of COMPARE.10 This program

A. Andreani et al. / Bioorg. Med. Chem. 12 (2004) 1121–1128
Table 2. IR and 1H NMR of compounds 3a–q
1123
Comp
IR: nmax cmꢀ1
1H NMR:ad, ppm in DMSO-d6
3a
1665, 1614, 1209, 717
2.25 (3H, s, CH3), 3.75 (3H, s, OCH3), 6.83 (1H, dd, ox, J=7.9, J=1),
6.85 (1H, s, ind-4), 7.01 (1H, dd, ox, J=7.9, J=1), 7.15 (1H, s, ind-7),
7.19 (1H, t, ox, J=7.9), 8.42 (1H, s, CH), 10.77 (1H, s, NH-ox),
12.54 (1H, s, NH-ind)
3b
3c
1665, 1591, 1301, 1204
1679, 1596, 1199, 1132
2.28 (3H, s, CH3), 3.61 (3H, s, OCH3), 6.49 (1H, s, ind-4), 6.77 (1H, d, ox-4, J=2.2),
6.90 (1H, d, ox-7, J=8.2), 7.24 (1H, dd, ox-6, J=8.2, J=2.2), 7.27 (1H, s, ind-7),
7.73 (1H, s, CH), 10.72 (1H, s, NH-ox), 12.74 (1H, s, NH-ind)
2.27 (3H, s, CH3), 3.61 (3H, s, OCH3), 6.38 (1H, d, ox-4, J=2.1), 6.56 (1H, s, ind-4),
6.60 (1H, dd, ox-6, J=8.4, J=2.1), 6.67 (1H, d, ox-7, J=8.4),
7.23 (1H, s, ind-7), 7.58 (1H, s, CH), 8.85 (1H, s, OH), 10.27 (1H, s, NH-ox),
12.56 (1H, s, NH-ind)
3d
3e
3f
1664, 1593, 1281, 1189
1670, 1598, 1322, 1183
1664, 1588, 1209, 1143
1685, 1590, 1289, 723
1716, 1609, 1245, 1045
1.13 (3H, t, CH3CH2, J=7), 2.26 (3H, s, CH3), 3.58 (3H, s, OCH3),
3.71 (2H, q, CH3CH2, J=7), 6.41 (1H, s, ox-4), 6.54 (1H, s, ind-4), 6.77 (2H, s, ox-6,7),
7.24 (1H, s, ind-7), 7.64 (1H, s, CH), 10.37 (1H, s, NH-ox),
12.50 (1H, broad, NH-ind)
2.10 (3H, s, CH3), 2.26 (3H, s, CH3), 3.62 (3H, s, OCH3), 6.47 (1H, s, ox-4),
6.57 (1H, s, ox-7), 6.62 (1H, s, ind-4), 7.21 (1H, s, ind-7),
7.50 (1H, s, CH), 8.67 (1H, s, OH),
10.19 (1H, s, NH-ox), 12.46 (1H, s, NH-ind)
2.27 (3H, s, CH3), 3.19 (3H, s, N-CH3),
3.60 (3H, s, OCH3), 6.43 (1H, d, ox-4, J=2.5),
6.55 (1H, s, ind-4), 6.68 (1H, dd, ox-6, J=8.4, J=2.5), 6.83 (1H, d, ox-7, J=8.4),
7.24 (1H, s, ind-7), 7.67 (1H, s, CH), 8.96 (1H, s, OH), 12.59 (1H, broad, NH)
2.26 (3H, s, CH3), 3.22 (3H, s, N-CH3), 3.49 (3H, s, OCH3), 3.55 (3H, s, OCH3),
6.46 (1H, d, ox-4, J=2.3), 6.51 (1H, s, ind-4), 6.88 (1H, dd, ox-6, J=8.5, J=2.3),
6.95 (1H, d, ox-7, J=8.5), 7.25 (1H, s, ind-7), 7.73 (1H, s, CH),
12.62 (1H, broad, NH)
2.25 (3H, s, CH3), 3.56 (3H, s, OCH3), 5.58 (2H, s, CH2), 6.60 (1H, s, ind-4),
6.81 (2H, m, ox-4,5), 6.89 (1H, d, ox-7, J=7.6), 7.15 (2H, d, ph, J=6.8),
7.19 (1H, m, ox-6), 7.31 (3H, m, ph), 7.52 (1H, s, ind-7), 7.67 (1H, s, CH),
10.63 (1H, s, NH)
3g
3h
3i
3j
1711, 1619, 1204, 1035
1685, 1593, 1260, 1091
2.25 (3H, s, CH3), 3.40 (3H, s, OCH3), 3.60 (3H, s, OCH3), 5.58 (2H, s, CH2),
6.41 (1H, s, ox-4), 6.65 (1H, s, ind-4), 6.79 (2H, s, ox-6,7), 7.15 (2H, d, ph, J=6.9),
7.30 (3H, m, ph), 7.52 (1H, s, ind-7), 7.67 (1H, s, CH), 10.44 (1H, s, NH)
2.12 (3H, s, CH3), 2.25 (3H, s, CH3), 3.18 (3H, s, OCH3), 3.62 (3H, s, OCH3),
5.57 (2H, s, CH2), 6.38 (1H, s, ox-4), 6.68 (1H, s, ind-4), 6.72 (1H, s, ox-7),
7.14 (2H, d, ph, J=6.7), 7.31 (3H, m, ph), 7.51 (1H, s, ind-7), 7.60 (1H, s, CH),
10.35 (1H, s, NH)
3k
3l
1696, 1655, 1593, 697
1706, 1609, 1301, 697
1675, 1606, 1199, 692
2.24 (3H, s, CH3), 3.76 (3H, s, OCH3), 5.55 (2H, s, CH2), 6.84 (1H, d, ox, J=7.8),
6.90 (1H, s, ind-4), 7.02 (1H, d, ox, J=7.8),
7.16 (2H, d, ph, J=6.3), 7.20 (1H, t, ox-6, J=7.8),
7.31 (3H, m, ph), 7.40 (1H, s, ind-7), 8.44 (1H, s, CH), 10.80 (1H, s, NH)
2.26 (3H, s, CH3), 3.64 (3H, s, OCH3), 5.59 (2H, s, CH2), 6.62 (1H, s, ind-4),
6.78 (1H, d, ox-4, J=2.1), 6.90 (1H, d, ox-7, J=8.5), 7.18 (2H, d, ph, J=7.3),
7.25 (1H, dd, ox-6, J=8.5, J=2.1), 7.34 (3H, m, ph),
7.54 (1H, s, ind-7), 7.77 (1H, s, CH), 10.77 (1H, s, NH)
2.26 (3H, s, CH3), 3.61 (3H, s, OCH3), 5.58 (2H, s, CH2),
6.39 (1H, d, ox-4, J=2.1),
3m
6.62 (1H, dd, ox-6, J=8, J=2.1), 6.62 (1H, s, ind-4), 6.69 (1H, d, ox-7, J=8),
7.17 (2H, d, ph, J=7.3), 7.32 (3H, m, ph), 7.50 (1H, s, ind-7),
7.62 (1H, s, CH), 8.87 (1H, s, OH), 10.29 (1H, s, NH)
2.11 (3H, s, CH3), 2.26 (3H, s, CH3), 3.61 (3H, s, OCH3),
5.58 (2H, s, CH2), 6.47 (1H, s, ox-4),
6.60 (1H, s, ox-7), 6.66 (1H, s, ind-4), 7.17 (2H, d, ph, J=7.2), 7.31 (3H, m, ph),
7.49 (1H, s, ind-7), 7.54 (1H, s, CH), 8.67 (1H, s, OH), 10.23 (1H, s, NH)
2.25 (3H, s, CH3), 3.57 (3H, s, OCH3), 5.58 (2H, s, CH2), 6.61 (1H, s, ind-4),
6.84 (3H, m, ox), 7.16 (2H, d, ph, J=8.5),
3n
3o
3p
1670, 1598, 1322, 1183
1711, 1609, 1245, 738
1706, 1609, 1511, 1240
7.17 (1H, m, ox), 7.41 (2H, d, ph, J=8.5),
7.52 (1H, s, ind-7), 7.66 (1H, s, CH), 10.62 (1H, s, NH)
2.26 (3H, s, CH3), 3.56 (3H, s, OCH3), 3.70 (3H, s, OCH3), 5.49 (2H, s, CH2),
6.58 (1H, s, ind-4),
6.84 (3H, m, ox), 6.90 (2H, d, ph, J=8.6),
7.14 (2H, d, ph, J=8.6), 7.21 (1H, m, ox),
7.53 (1H, s, ind-7), 7.67 (1H, s, CH), 10.61 (1H, s, NH)
2.20 (3H, s, CH3), 3.60 (3H, s, OCH3),
5.97 (2H, s, CH2), 6.35 (1H, m, ph-6),
3q
1698, 1606, 1524, 1245
6.68 (1H, s, ind-4),
6.90 (3H, m, ox), 7.21 (1H, m, ox), 7.51 (1H, s, ind-7),
7.60 (2H, m, ph-4,5) 7.70 (1H, s, CH),
8.25 (1H, m, ph-3), 10.63 (1H, s, NH)
The NH and OH groups give broad bands in the range 3270–3030 cmꢀ1
a Abbreviations: ind=chloroindole, ox=oxindole, ph=phenyl.
.

1124
A. Andreani et al. / Bioorg. Med. Chem. 12 (2004) 1121–1128
compared the patterns evinced by the test compounds
with the patterns produced by the standard anticancer
agents in the NCI database.11 Among these drugs we
chose vincristine since its structure contains two indoles
and we reported in Table 3 the available data for com-
parison purposes (the highest dose tested was 10ꢀ3
instead of 10ꢀ4).
cules caused a marked cytotoxic effect, with the death of
the whole culture (data not shown). It was observed that
all the compounds at the doses 10ꢀ5 and 10ꢀ6M induced
a decrease of cell growth after 24 (Fig. 1A) and 48 h
(Fig. 1B) of treatment, but compounds 3c and 3f were
more effective and were able to decrease cell prolifera-
tion at lower doses than compound 3m and 3o. Fur-
thermore compound 3m at the lower doses did not show
any effect, and after 48 h seemed to stimulate cell
growth. In order to clarify this proliferative arrest in
more detail the analysis of DNA profiles was performed
using propidium iodide staining and flow cytometry,
and Figure 2 shows the profile of DNA of the cells
treated with 3c for 24 and 48 h. In Figure 3 the percen-
tage of cell in G2/M phase obtained with all the treat-
ments are reported. All the four compounds were able
to induce a block in G2/M phase, with different effi-
ciency: after 24 h (Fig. 3A) of 10ꢀ6M 3c treatment,
more than 90% of the cells were in G2/M phase, while
the percentages of cells in this cell cycle phase for com-
pounds 3f, 3m and 3o were respectively around 70%,
7% and 55% confirming the lower potency of these
compounds and the lack of activity of 3m at this con-
centration. However the 10ꢀ5 M dose was able to
induce over than 80% of the cells to arrest in the G2/M
phase with all the tested compounds. 48 h of treatments
All the compounds were potent growth inhibitors with
GI50 ranging from ꢀ4.90 to ꢀ7.27. Four of them
(3c,f,m,o) are now under review by BEC (Biological
Evaluation Committee of the NCI).
3.2. Growth inhibition and cell cycle arrest in colon ade-
nocarcinoma HT29 cell line
In order to investigate the mechanism of action of these
drugs, the antiproliferative effects of the four most
potent antitumor agents (3c,f,m,o) were studied by
monitoring their effects on cell proliferation and cell
cycle in a colon adenocarcinoma cell line, HT29. This
line was chosen because all these compounds demon-
strated a high activity on colon cancer cell lines as
shown in Table 3. The effects of treatments in the range
of concentrations between 10ꢀ7 and 10ꢀ5 are shown in
Figure 1. The highest dose (10ꢀ4M) of all these mole-
Table 3. Sixty cell panel (growth inhibition, cytostatic and cytotoxic activity of the selected compounds)
NSC
Comp a
Modes Leukemia NSCLC Colon CNS Melanoma Ovarian Renal Prostate Breast MGꢀMIDb
723558
3a
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
GI50
TGI
LC50
GI50
TGI
LC50
ꢀ5.88
ꢀ5.50
ꢀ5.08
ꢀ6.40
ꢀ5.58
ꢀ4.50
ꢀ7.22
ꢀ5.17
ꢀ4.08
ꢀ6.18
ꢀ5.54
ꢀ4.67
ꢀ6.60
ꢀ5.13
ꢀ4.11
ꢀ5.18
ꢀ
ꢀ5.61
ꢀ5.08
ꢀ4.42
ꢀ5.88
ꢀ4.89
ꢀ4.12
ꢀ6.38
ꢀ5.15
ꢀ4.22
ꢀ5.49
ꢀ4.82
ꢀ4.27
ꢀ6.10
ꢀ4.99
ꢀ4.30
ꢀ5.22
ꢀ4.31
ꢀ
ꢀ5.73 ꢀ5.71
ꢀ5.39 ꢀ5.25
ꢀ5.03 ꢀ4.75
ꢀ6.29 ꢀ6.42
ꢀ5.12 ꢀ5.17
ꢀ4.33 ꢀ4.35
ꢀ7.23 ꢀ7.12
ꢀ5.79 ꢀ5.29
ꢀ4.34 ꢀ4.31
ꢀ5.61 ꢀ5.56
ꢀ5.07 ꢀ4.96
ꢀ4.53 ꢀ4.39
ꢀ6.45 ꢀ6.39
ꢀ5.37 ꢀ5.56
ꢀ4.46 ꢀ4.34
ꢀ5.61 ꢀ5.56
ꢀ4.47 ꢀ4.30
ꢀ5.66
ꢀ5.15
ꢀ4.66
ꢀ6.23
ꢀ4.91
ꢀ4.30
ꢀ7.27
ꢀ5.46
ꢀ4.43
ꢀ5.57
ꢀ4.83
ꢀ4.36
ꢀ6.49
ꢀ5.12
ꢀ4.32
ꢀ5.46
ꢀ4.57
ꢀ4.05
ꢀ5.44
ꢀ4.68
ꢀ4.18
ꢀ6.18
ꢀ4.62
ꢀ4.11
ꢀ6.38
ꢀ5.28
ꢀ4.39
ꢀ5.12
ꢀ4.29
ꢀ5.16
ꢀ4.58
ꢀ4.14
ꢀ6.80
ꢀ5.10
ꢀ3.60
ꢀ5.89
ꢀ5.13
ꢀ4.49
ꢀ6.13
ꢀ5.03
ꢀ4.22
ꢀ7.03
ꢀ5.86
ꢀ4.28
ꢀ5.53
ꢀ4.91
ꢀ4.37
ꢀ6.26
ꢀ5.28
ꢀ4.38
ꢀ5.02
ꢀ4.07
ꢀ4.02
ꢀ5.64
ꢀ5.21
ꢀ4.18
ꢀ6.18
ꢀ4.88
ꢀ4.37
ꢀ6.45
ꢀ5.40
ꢀ4.71
ꢀ5.30
ꢀ4.21
ꢀ5.04
ꢀ4.40
ꢀ4.09
ꢀ6.50
ꢀ4.70
ꢀ3.50
ꢀ5.63
ꢀ5.12
ꢀ4.62
ꢀ6.06
ꢀ4.92
ꢀ4.34
ꢀ6.78
ꢀ5.15
ꢀ4.34
ꢀ5.49
ꢀ4.95
ꢀ4.28
ꢀ6.23
ꢀ4.91
ꢀ4.32
ꢀ5.49
ꢀ4.31
ꢀ
ꢀ5.65
ꢀ5.09
ꢀ4.56
ꢀ6.08
ꢀ5.15
ꢀ4.60
ꢀ6.66
ꢀ5.10
ꢀ4.40
ꢀ5.53
ꢀ4.82
ꢀ4.24
ꢀ5.83
ꢀ4.71
ꢀ4.19
ꢀ4.96
ꢀ
ꢀ5.80
ꢀ5.35
ꢀ4.74
ꢀ6.27
ꢀ5.21
ꢀ4.22
ꢀ7.24
ꢀ5.66
ꢀ4.66
ꢀ5.66
ꢀ4.99
ꢀ4.29
ꢀ6.49
ꢀ5.48
ꢀ4.51
ꢀ5.44
ꢀ4.53
ꢀ
ꢀ5.73
ꢀ5.23
ꢀ4.71
ꢀ6.19
ꢀ5.09
ꢀ4.30
ꢀ6.99
ꢀ5.42
ꢀ4.34
ꢀ5.62
ꢀ4.99
ꢀ4.38
ꢀ6.34
ꢀ5.18
ꢀ4.34
ꢀ5.36
ꢀ4.33
ꢀ4.01
ꢀ5.50
ꢀ4.84
ꢀ4.19
ꢀ6.19
ꢀ4.80
ꢀ4.17
ꢀ6.44
ꢀ5.60
ꢀ4.61
ꢀ5.22
ꢀ4.20
ꢀ5.14
ꢀ4.44
ꢀ4.08
ꢀ6.70
ꢀ5.00
ꢀ3.60
723557
723559
723560
723554
723565
723566
723564
723568
3b
3c
3e
3f
3k
3l
ꢀ
ꢀ
ꢀ4.02
ꢀ
ꢀ5.54
ꢀ4.94
ꢀ4.12
ꢀ6.28
ꢀ4.45
ꢀ
ꢀ5.52
ꢀ4.77
ꢀ4.15
ꢀ5.98
ꢀ4.66
ꢀ4.15
ꢀ6.23
ꢀ5.53
ꢀ4.47
ꢀ5.05
ꢀ4.12
ꢀ5.05
ꢀ4.40
ꢀ4.05
ꢀ6.60
ꢀ4.80
ꢀ3.60
ꢀ5.39 ꢀ5.52
ꢀ4.53 ꢀ4.93
ꢀ4.01 ꢀ4.26
ꢀ6.41 ꢀ6.37
ꢀ5.43 ꢀ5.09
ꢀ4.34 ꢀ4.19
ꢀ6.51 ꢀ6.49
ꢀ5.84 ꢀ5.59
ꢀ4.65 ꢀ4.32
ꢀ5.45 ꢀ5.31
ꢀ4.38 ꢀ4.12
ꢀ5.39 ꢀ4.90
ꢀ4.64 ꢀ4.43
ꢀ4.04 ꢀ4.11
ꢀ7.00 ꢀ6.90
ꢀ5.40 ꢀ5.20
ꢀ4.10 ꢀ3.70
ꢀ5.61
ꢀ5.22
ꢀ4.42
ꢀ6.03
ꢀ4.49
ꢀ4.07
ꢀ6.43
ꢀ5.63
ꢀ4.53
ꢀ5.19
ꢀ4.07
ꢀ5.06
ꢀ4.48
ꢀ4.10
ꢀ6.50
ꢀ4.70
ꢀ3.60
ꢀ5.32
ꢀ4.72
ꢀ4.14
ꢀ5.97
ꢀ4.29
ꢀ
ꢀ5.41
ꢀ4.56
ꢀ4.18
ꢀ6.23
ꢀ5.22
ꢀ4.24
ꢀ6.48
ꢀ5.48
ꢀ4.67
ꢀ5.28
ꢀ4.36
ꢀ5.25
ꢀ4.46
ꢀ4.07
ꢀ6.50
ꢀ5.10
ꢀ3.50
3m
3o
ꢀ6.62
ꢀ6.20
ꢀ5.28
ꢀ5.33
ꢀ4.02
ꢀ5.32
ꢀ4.04
ꢀ
ꢀ6.46
ꢀ5.44
ꢀ4.54
ꢀ4.97
ꢀ
723570
723569
3p
3q
ꢀ5.23
ꢀ4.29
ꢀ
67574
Vincristine sulfate c
ꢀ7.00
ꢀ4.80
ꢀ3.20
ꢀ6.90
ꢀ5.20
ꢀ3.50
a All values are log10. Highest conc.=10ꢀ4M;only modes showing value >ꢀ4.00 are reported.
b Calculated mean panel.
c Highest conc.=10ꢀ3M.

A. Andreani et al. / Bioorg. Med. Chem. 12 (2004) 1121–1128
1125
(Fig. 3B) did not significantly modify this distribution,
indicating that for 3c,f,o it was sufficient to incubate
cells in presence of a concentration of 10ꢀ6 M to achieve
a block in the cell cycle progression, whereas compound
3m needed a 10-fold increase in concentration to show
its synchronizing effects. The analysis of DNA profiles
did not show an appreciable presence of nuclei with
fragmented DNA, suggesting that these compounds
induce G2/M arrest but not apoptosis.
mitotic block increased after 48 h. The same results were
obtained with the dose 10ꢀ6 M of compound 3c,f,o. An
example of these results is shown in Figure 4, where the
photomicrographs of the control and of the cells treated
for 24 and 48 h with 3c 10ꢀ5 M are reported. Further-
more microscopic evaluation confirmed that a sig-
nificant increase of apoptotic cells in treated samples did
not occur.
3.3. Positive inotropic activity
To determine whether cells were arrested in G2 or in
mitosis after treatments, chromatin was visualized by
Hoechst 33432 staining. Results showed that after 24 h
of treatments with all the four compounds, a high per-
centage of cells had condensed chromosomes and the
The positive inotropic activity of the most potent anti-
tumor agents (3c,f,m,o) was evaluated in view of a pos-
sible coanthracyclinic8 activity. With the procedure
described, compound 3m showed a weak but significant
Figure 1. Relative cell growth after 24 h (A) and 48 h (B) treatment, calculated respect to control, taken as 100%.
3c, 3f, 3m, 3o
10ꢀ8M, 10ꢀ7M, 10ꢀ6M and 10ꢀ5M.
control cells, compounds
Figure 2. DNA profiles of cells treated for 24 h with compound 3c: A, control cells;B, 3c 10ꢀ8 M;C, 3c 10ꢀ7 M;D, 3c 10ꢀ6 M;E, 3c 10ꢀ5 M. F, cell
treated with 3c 10ꢀ5 M for 48 h.

1126
A. Andreani et al. / Bioorg. Med. Chem. 12 (2004) 1121–1128
Figure 3. Percentage of cell in G2/M phase after 24 h (A) and 48 h (B) of treatments.
10ꢀ7M, 10ꢀ6M and 10ꢀ5 M. The distribution of HT29 cells within the cell cycle was analyzed by flow cytometry as described in
Section 5.
control cells, compounds 3c, 3f, 3m, 3o
10ꢀ8M,
Figure 4. Photographs of control cells (a), cells treated with 3c 10ꢀ5 M for 24 h (b) and 48 h (c). Cells were stained by Hoechst 33432 as reported in
Section 5.
cardiotonic activity at 10ꢀ7M. The well known positive
inotropic agent amrinone, at the same concentration,
did not produce any effect. In our opinion compound
3m could be a promising candidate for testing its effect
on the antitumor/cardiotoxic activity of anthracyclines.
Unfortunately, as far as we know, in this moment there
are no laboratories performing this test.
Even the second approach, concerning also the intro-
duction of a benzyl ring at the nitrogen of the chloro-
indole portion, gave rise to potent compounds selected
by BEC (3m and 3o).
This study points out that the substituent at the 5 posi-
tion of the indolinone portion is much more critical
than the substituents considered in all the other posi-
tions and from the results up to now obtained it seems
that an hydrogen4 or a small substituent represents the
best solution.
4. Conclusions
The first approach (introduction of new substituents in
the indolinone moiety while maintaining the same
chloroindole portion) has been successful since gener-
ated interesting derivatives such as 3a–c,f. Compounds
3a, bearing a chlorine at position 4, was less active than
the 5-chloro analogue (3b) but was selective towards the
ovarian cancer OVCAR-4 (GI50 ꢀ7.11). Compounds 3c
(bearing a OH group at the 5 position) and 3f (which,
compared to 3c, bears a methyl group in position 1)
were selected by BEC for a possible further develop-
ment. Compound 3c was the most active of the whole
series. It was more potent than vincristine against seven
of the nine tumors considered. Moreover it was selective
towards some cell lines such as MDA-MB-435 (breast),
OVCAR-3 (ovarian) and SK-MEL-28 (melanoma).
Flow cytometric analysis and microscopic evaluation of
the effects on cell cycle progression induced by com-
pounds 3c,f,m,o showed that these molecules are able to
arrest adenocarcinoma HT29 cells in mitosis, even if
with different potency. The most active compound was
3c, whereas the presence of a benzyl ring at the nitrogen
of the chloroindole portion decreased this activity. The
arrest in mitosis suggest that these compounds could act
by inhibiting tubulin polymerization as other bis-indole
derivatives and inducing the so called ‘mitotic cata-
strophe’.6 They demonstrated to be more active than
vincristine, which is not able to completely synchronize
several cell lines in G2/M phase.5 Furthermore, vincris-
tine induces apoptosis in several tumor lines, including

A. Andreani et al. / Bioorg. Med. Chem. 12 (2004) 1121–1128
1127
colon carcinoma in vivo and in vitro,12 but as far as we
know its behavior on HT29 cells has never been reported
in the literature.
(2H, d, ph, J=8.4), 7.11 (2H, d, ph, J=8.4), 7.47 (1H, s,
ind-7), 7.54 (1H, s, ind-4), 9.96 (1H, s, CHO).
5.6.
2-Chloro-5-methoxy-6-methyl-1-(2-nitro-benzyl)-
indole-3-carbaldehyde (1, R¼2NO2Bn)
5. Experimental
5.1. Chemistry
C18H15ClN2O4 (358.8). Mp 220–222 ꢁC nmax cmꢀ1
:
1659, 1524, 1258, 1047 d, ppm: 2.17 (3H, s, CH3), 3.85
(3H, s, OCH3), 5.93 (2H, s, CH2), 6.34 (1H, m, ph-6),
7.48 (1H, s, ind-7), 7.59 (2H, m, ph-4,5), 7.62 (1H, s,
ind-4), 8.26 (1H, m, ph-3), 10.04 (1H, s, CHO).
The melting points are uncorrected. Analyses (C,H,N)
were within ꢂ0.4% of the theoretical values. Bakerflex
plates (silica gel IB2-F) were used for TLC: the eluent
was petroleum ether/acetone in various proportions.
Kieselgel 60 (Merck) was used for column chromato-
graphy. The IR spectra were recorded in Nujol on a
5.7. General procedure for the synthesis of compounds
3a–q
Nicolet Avatar 320 E.S.P.; nmax is expressed in cmꢀ1
.
The appropriate 2-chloroaldehyde 1 (10 mmol) was
dissolved in methanol (100 mL) and treated with the
equivalent of the appropriate indolinone
1
The H NMR spectra were recorded in (CD3)2SO on a
Varian Gemini (300 MHz);the chemical shift (refer-
enced to solvent signal) is expressed in d (ppm) and J in
Hz. (Table 2). 2-Indolinone is commercially available
whereas the substituted 2-indolinones4,13ꢀ16 and 2-chloro-
5-methoxy-6-methylindole-3-carbaldehyde17 were pre-
pared according to the literature. The synthesis of four
new aldehydes is reported below.
2 and
piperidine (1 mL). Only for compound 3f the same
amount of triethylamine was employed instead of
piperidine. The reaction mixture was refluxed for 3–5 h
(according to a TLC test) and the precipitate formed
on cooling was collected by filtration. Compounds
3a–b were purified by column chromatography and
all the others by crystallization from ethanol or tolu-
ene. The yield was 20–30% for compounds 3a–f,m,
50–60% for compounds 3g,n,q and 70–80% for
compounds 3h–l,o–p.
5.2. General procedure for the synthesis of the four new
aldeydes
2-chloro-5-methoxy-6-methylindole-3-carbaldehyde (10
mmol) was dissolved in DMF (10 mL) and treated por-
tionwise, under stirring, with NaH (15 mmol). The
reaction mixture was stirred at room temperature for 10
min, treated with the appropriate benzyl chloride (60
mmol) and maintained at 100 ꢁC for 1–3 h (according to
a TLC test). The reaction mixture was poured onto ice
and the resulting precipitate was recovered by filtration
with a yield of 70–80%. It was then purified by crystal-
lization with ethanol.
5.8. In vitro growth inhibition and cytotoxicity
It was determined by the NCI according to standard
procedures.9
5.9. Cell cycle analysis
HT29 cells (Istituto Zooprofilattico di Brescia, Italy)
were routinely cultured in RPMI 1640 medium
(Gibco,UK) supplemented by 10% heat-inactivated
fetal calf serum (Euroclone, UK) and l-glutamine
(Sigma, USA) 2 mmol, at 37 ꢁC in 5% CO2 atmosphere.
Compounds under test (3c,f,m,o) were dissolved in
DMSO (Sigma, USA) at 10 mg/mL immediately
before use, and the different drugs concentrations
were prepared in complete medium. In control cells
only DMSO was added to the fresh culture medium.
For treatments, cells were seeded in tissue culture plates
(Orange, Belgium) at 20ꢃ103 cell/cm2 and after 24 h
the medium was removed and fresh medium
containing the drugs at concentrations ranging
between 10ꢀ4 and 10ꢀ8M was added. After 24 and
48 h cells were trypsinized and counted;dead cells were
stained by diluting cell samples 1:1 with 5% tripan blue
solution. Growth was plotted as percentage of the
control.
5.3. 1-Benzyl-2-chloro-5-methoxy-6-methylindole-3-car-
baldehyde (1, R¼Bn)
C18H16ClNO2 (313.8). Mp 139–140 ꢁC nmax cmꢀ1: 1660,
1511, 1255, 1045 d, ppm: 2.22 (3H, s, CH3), 3.83 (3H, s,
OCH3), 5.55 (2H, s, CH2), 7.15 (2H, d, ph, J=7.6), 7.32
(3H, m, ph), 7.48 (1H, s, ind-7), 7.58 (1H, s, ind-4),
10.00 (1H, s, CHO).
5.4. 2-Chloro-1-(4-chlorobenzyl)-5-methoxy-6-methyl-
indole-3-carbaldehyde (1, R¼4Cl-Bn)
C18H15Cl2NO2 (348.2). Mp 176–179 ꢁC dec. nmax cmꢀ1
:
1640, 1500, 1035, 715 d, ppm: 2.23 (3H, s, CH3), 3.84
(3H, s, OCH3), 5.57 (2H, s, CH2), 7.16 (2H, d, ph,
J=6.6), 7.42 (2H, d, ph, J=6.6), 7.49 (1H, s, ind-7),
7.59 (1H, s, ind-4), 10.00 (1H, s, CHO).
At the indicated time points, nuclei were isolated
according to Nusse et al.18 Isolated nuclei were kept at
4 ꢁC in the dark. At the moment of the analysis, cells
were stained with propidium iodide (Sigma, USA) 50
mg/mL. Cell cycle profiles were determined using an
Epics Elite (Beckman Coulter, USA) and analyzed by
Modfit software (Verity).
5.5. 2-Chloro-5-methoxy-1-(4-methoxybenzyl)-6-methyl-
indole-3-carbaldehyde (1, R¼4CH3O-Bn)
C19H18ClNO3 (343.8). Mp 133-135 ꢁC nmax cmꢀ1: 1650,
1510, 1250, 1030 d, ppm: 2.20 (3H, s, CH3), 3.67 (3H, s,
OCH3), 3.80 (3H, s, OCH3), 5.43 (2H, s, CH2), 6.86

1128
A. Andreani et al. / Bioorg. Med. Chem. 12 (2004) 1121–1128
For microscopical evaluation, cells were grown on glass
coverslip, washed 3 times in phosphate-buffered saline
(PBS), fixed with 4% paraformaldehyde (Sigma, USA)
in PBS and permeabilized with 0.5% Triton X-100
(Sigma, USA) in PBS for 5 min at 4 ꢁC. After 2 washes
in PBS, sample were incubated in presence of Hoechst
33432 (Sigma USA) 0,1 mg/mL, washed and analyzed by
fluorescence microscopy.
3. Andreani, A.;Locatelli, A.;Leoni, A.;Morigi, R.;Chier-
icozzi, M.;Fraccari, A.;Galatulas, I.;Salvatore, G. Eur.
J. Med. Chem. 1998, 33, 905.
4. Andreani, A.;Granaiola, M.;Leoni, A.;Locatelli, A.;
Morigi, R.;Rambaldi, M.;Garaliene, V. J. Med. Chem.
2002, 45, 2666.
5. Comin-Anduix, B.;Agell, N.;Bachs, O.;Ovadi, J.;Cas-
cante, M. Mol. Pharm. 2001, 60, 1235.
6. Mahboobi, S.;Pongratz, H.;Hufsky, H.;Hockmeyer, J.;
Frieser, M.;Lyssenko, A.;Paper, D. H.;Burgermeister,
J.;Bohmer, F.-D.;Fiebig, H.-H.;Burger, A. M.;Baasner,
S.;Beckers, T. J. Med. Chem. 2001, 44, 4535.
7. Fan, M.;Goodwin, M. E.;Birrer, M. J.;Chambers, T. C.
Cancer Res. 2001, 61, 4450.
5.10. Positive inotropic activity
The experiments were carried out on the guinea-pig
papillary muscles according to a procedure previously
described.8
8. Andreani, A.;Leoni, A.;Locatelli, A.;Morigi, R.;Ram-
Bioorg. Med.
baldi, M.;Recanatini, M.;Garaliene, V.
Chem. 2000, 8, 2359.
9. Monks, A.;Scudiero, D.;Skehan, P.;Shoemaker, R.;
Paull, K.;Vistica, D.;Hose, C.;Langley, J.;Cronise, P.;
Vaigro-Wolff, A. J. Natl. Cancer Inst. 1991, 83, 757.
10. http://dtp.nci.nih.gov/docs/compare/compare_methodology.
html
Acknowledgements
This work has been supported by a grant from MIUR-
COFIN 2002, contract number 2002033121_003. We
are grateful to the National Cancer Institute (Bethesda,
MD) for the antitumor tests.
11. http://dtp.nci.nih.gov/docs/cancer/searches/standard_agent_
table.html
12. Hannun, Y. A. Blood 1997, 89, 1845.
13. Koelsch, C. F. J.Amer. Chem. Soc. 1944, 66, 2019.
14. Romeo, A.;Corrodi, H.;Hardegger, E. Helv. Chim. Acta
1955, 38, 463.
References and notes
15. Andreani, A.;Rambaldi, M.;Bonazzi, D.;Greci, L.;
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16. Beer, R. J. S.;Davenport, H. F.;Robertson, A. J. Chem.
Soc. 1953, 1262.
1. Potential Antitumor Agents 35. For part 34 see Andreani,
A.;Granaiola, M.;Leoni, A.;Locatelli, A.;Morigi, R.;
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17. Porter, J. C.;Robinson, R.;Wyler, M.
1941, 620.
J. Chem. Soc.
2. Andreani, A.;Locatelli, A.;Rambaldi, M.;Leoni, A.;
Anticancer Res.
Bossa, R.;Fraccari, A.;Galatulas, I.
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18. Nusse, M.;Beisker, W.;Hoffman, C.;Tarnok, A.
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Cyto-