Antitumor activity of new substituted 3-(5-imidazo[2,1-b] thiazolylmethylene)-2-indolinones and 3-(5-imidazo[2,1-b]thiadiazolylmethylene)- 2-indolinones: Selectivity against colon tumor cells and effect on cell cycle-related events

Article abstract of DOI:10.1021/jm800827q

The synthesis of new 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and 3-(5-imidazo[2,1-b]thiadiazolylmethylene)-2-indolinones is reported. The antitumor activity was evaluated according to the protocols available at the National Cancer Institute (

Full text of DOI:10.1021/jm800827q

7508
J. Med. Chem. 2008, 51, 7508–7513
Antitumor Activity of New Substituted 3-(5-Imidazo[2,1-b]thiazolylmethylene)-2-indolinones and
3-(5-Imidazo[2,1-b]thiadiazolylmethylene)-2-indolinones: Selectivity against Colon Tumor Cells
and Effect on Cell Cycle-Related Events1
Aldo Andreani,*,† Silvia Burnelli,† Massimiliano Granaiola,† Alberto Leoni,† Alessandra Locatelli,† Rita Morigi,†
Mirella Rambaldi,† Lucilla Varoli,† Natalia Calonghi,‡ Concettina Cappadone,‡ Manuela Voltattorni,§ Maddalena Zini,‡
Claudio Stefanelli,‡ Lanfranco Masotti,‡ and Robert H. Shoemaker|
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, Centro Interdipartimentale di Ricerche Biotecnologiche (CIRB),
UniVersita` di Bologna, Via S. Donato, 15, 40127 Bologna, Italy, and Screening Technologies Branch, DeVelopmental Therapeutics Program,
DiVision of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, Frederick, Maryland 21702
ReceiVed July 8, 2008
The synthesis of new 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and 3-(5-imidazo[2,1-b]thia-
diazolylmethylene)-2-indolinones is reported. The antitumor activity was evaluated according to the protocols
available at the National Cancer Institute (NCI), Bethesda, MD. To investigate the mechanism of action of
the most potent antitumor agent of this series, its effect on growth of HT-29 colon carcinoma cells was
studied. Its ability to inhibit cellular proliferation was mediated by cell cycle arrest at the G2/M phase,
accompanied by inhibition of ornithine decarboxylase (ODC), the limiting enzyme of polyamine synthesis,
and followed by induction of apoptosis.
Chart 1
Introduction
This is our fourth paper devoted to the synthesis of antitumor
3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones.2-4 Chart
1 reports the most active derivatives so far obtained: A (mean
pGI50 ) 5.82)2 and B (mean pGI50 ) 6.42).4
We describe here new analogues with different substituents
in the indole as well in the imidazothiazole portion. The
synthesis of analogues bearing the imidazothiadiazole in place
of the imidazothiazole system was also performed. The antitu-
mor activity of all the new compounds was evaluated in a
preliminary test according to the protocols available at the
National Cancer Institute (NCIa), Bethesda, MD, and the active
compounds were further tested in the full five-dose assay. To
investigate the mechanism of action of the most potent antitumor
agent, its effect on growth of HT-29 colon carcinoma cells was
studied.
Scheme 1a
Chemistry
In the design of new derivatives (Scheme 1 and Table 1), we
took into account five approaches:
(1) With the same imidazothiazole portion (2,6-dimethyl
substituted) that gave the best results in the last two papers of
this series,3,4 different substituents have been considered at the
5-position of the indolinone portion (compounds 3-7). This
set of compounds includes N-methyl derivatives too (see point
4).
a For x-y, R, R1, R2, see Table 1.
(2) In additional tests performed by NCI on the compounds
published in the first paper of this series,2 a significant antitumor
activity was found in two compounds bearing a 2,5-dimethox-
yphenyl group at the 6-position of the imidazothiazole portion,
and one of them (compound A in Chart 1) was submitted to
the Biological Evaluation Committee (BEC) of the NCI
(unpublished results). On this basis, we planned new derivatives
with the same imidazothiazole portion (and its 2-methyl
derivative) but different indolinones, in particular those con-
sidered in the previous paper4 (compounds 8-11 and 19-22).
(3) With the same indolinone portion as above and with the
same 2-methyl group in the ring condensed with imidazole, the
effect of the introduction of an additional nitrogen was
considered (imidazothiadiazole derivatives 23-27).
* 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.
Centro Interdipartimentale di Ricerche Biotecnologiche (CIRB), Uni-
‡
§
versita` di Bologna.
|
National Cancer Institute at Frederick.
a Abbreviations: DTP, Developmental Therapeutics Program; NCI,
National Cancer Institute; GI, growth inhibition; TGI, total growth inhibition;
LC, lethal concentration; BEC, Biological Evaluation Committee; ODC,
ornithine decarboxylase; DMSO, dimethyl sulfoxide; DAPI, 4′,6-diamidino-
2-phenylindole; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate-
buffered saline; PI, propidium iodide.
10.1021/jm800827q CCC: $40.75
2008 American Chemical Society
Published on Web 11/12/2008

Antitumor ActiVity of Indolinones
Table 1. Compounds 3-28a
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7509
compd
x-y
R
R1
R2
formula
MW
mp, °C
3
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CH3CdCH
CHdCH
CHdCH
CHdCH
CHdCH
CH3CdN
CH3CdN
CH3CdN
CH3CdN
CH3CdN
CH3CdN
CH3
CH3
CH3
CH3
CH3
2,5-DMPh
2,5-DMPh
2,5-DMPh
2,5-DMPh
4-Tol
4-Tol
4-Tol
4-CPh
4-CPh
4-CPh
4-CPh
2,5-DMPh
2,5-DMPh
2,5-DMPh
2,5-DMPh
CH3
CH3
CH3
CH3
CH3
H
H
CH3
CH3
CH3
H
H
H
CH3
H
H
H
H
H
H
H
H
H
CH3
CH3
H
H
H
H
CH3
H
F
C16H12FN3OS
C17H13N3O3S
C17H15N3OS
313.35
339.37
309.39
343.83
325.39
447.51
451.93
433.48
461.54
401.48
405.90
387.46
391.88
421.90
426.32
407.88
437.90
419.46
417.48
447.51
296.35
326.38
330.79
312.35
340.40
392.86
314-315
4
COOH
H
Cl
OH
OCH3
Cl
OH
OCH3
OCH3
Cl
OH
H
OCH3
Cl
OH
Cl
OH
H
314-316 dec
180-182
5
6
C17H14ClN3OS
C17H15N3O2S
C24H21N3O4S
C23H18ClN3O3S
C23H19N3O4S
C25H23N3O4S
C23H19N3O2S
C22H16ClN3OS
C22H17N3O2S
C21H14ClN3OS
C22H16ClN3O2S
C21H13Cl2N3OS
C21H14ClN3O2S
C22H16ClN3O3S
C22H17N3O4S
C23H19N3O3S
C24H21N3O4S
C15H12N4OS
210-211
7
275-277 dec
224-226
8
9
230-232
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
317-319 dec
257-260
270-272
285-287
340-342 dec
314-316
264-266
342-344 dec
338-340
262-264
298-300 dec
100-105 dec
160-162
OCH3
H
OCH3
Cl
OH
OCH3
H
288-290
270-272
C16H14N4O2S
C15H11ClN4OS
C15H12N4O2S
C17H16N4O2S
C20H13ClN4OS
315-320 dec
285-287 dec
100-103 dec
316-318
4-CPh
a DMPh ) dimethoxyphenyl. Tol ) methylphenyl. CPh ) chlorophenyl.
Figure 2. Effects of compound 24 on microtubule networks in HT-
29 cells. Cells were treated for 24 h with compound 24 (1 µM) or
vincristine (30 nM). Afterward, the cells were fixed and processed for
double immunofluorescence labeling with antibody against R-tubulin
(upper panels) to assess the level of tubulin polymerization, and DAPI
(lower panels) to detect nuclear morphology. Staining was analyzed
by laser scanning confocal microscopy. All the images have been taken
at the same magnification and depict microscopic fields representative
of the whole cell population.
Figure 1. Effects of compound 24 on cell cycle-related events in HT-
29 cells. (A) Cell cycle distribution after 24 h of treatments with the
indicated concentration of compound 24 or vincristine. Results are mean
values ( SEM of three determinations. (B) Influence of compound 24
on ODC activity. Extracts were prepared from cells incubated for 24 h
in the presence of the indicated concentration of compound 24 or
vincristine. Results are mean values ( SEM of three determinations.
tion). Most of them were obtained as almost pure geometrical
isomers that, according to the usual NOE experiments described
in the previous papers,2,3 were assigned to the E configuration.
Compounds 3, 6, 13, 17, 21 were obtained as evident E/Z
mixtures and submitted as such to the biological tests. The E/Z
ratio in solution is time dependent and tends to 50/50. In our
experience with similar derivatives, we described the separations
of the two isomers by fractional crystallization2 but no
significant difference in the pharmacological behavior was
noticed.
(4) As was successfully done for one compound in the
previous paper,4 the N-methyl homologues of some derivatives
were also considered. These compounds (5-7, 11, 21, 22, 27)
show different features and therefore are reported in the three
groups described above.
(5) A number of derivatives were prepared to evaluate the
effect of different substituents on the phenyl ring at the
6-position of the 2-methylimidazothiazole/2-methylimidazothia-
diazole (compounds 12-18, 28).
Compounds 3-28 were prepared by means of the single step
Knoevenagel reaction between the aldehydes 1 and the indoli-
nones 2 in methanol/piperidine or acetic acid/hydrochloric acid.
The structures of the final compounds were confirmed by means
Biology
a. Antitumor Activity. Compounds 3-28 were preliminary
tested at a single high dose (10-5 M) in the full NCI 60 cell
panel. This panel is organized into subpanels representing
1
of IR and H NMR spectra (Table S1 in Supporting Informa-

7510 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Andreani et al.
Table 2. Sixty Cell Panel: Growth Inhibition and Cytostatic and Cytotoxic Activity of the Compounds That Passed the Preliminary Test
compda
modes
leukemia
NSCLC
colon
CNS
melanoma
ovarian
renal
prostate
breast
MG-MIDb
3
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pLC50
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
pGI50
pGI50
pTGI
pGI50
pTGI
pGI50
pTGI
pLC50
pGI50
pTGI
pLC50
5.34
4.63
5.30
4.68
5.84
4.93
5.09
4.37
4.75
4.12
5.10
4.38
5.62
4.39
5.34
4.16
5.57
4.42
4.08
5.41
4.25
4.48
4.09
4.89
4.18
4.73
4.36
4.10
4.99
4.67
4.41
4.61
4.12
5.22
4.48
5.06
4.29
5.50
4.54
4.10
5.34
4.43
4.62
4.13
4.84
5.20
4.45
4.88
4.14
5.26
4.20
5.14
4.35
4.81
4.13
5.42
4.35
4.94
4.39
4.65
4.01
5.22
4.21
5.02
4.39
4.67
5.35
4.63
5.26
4.26
5.17
4.48
5.22
4.46
4.98
4.20
5.37
4.43
4.02
5.16
4.27
4.58
4.09
4.90
4.12
4.86
4.43
4.14
5.11
4.6
4.44
4.81
4.29
4.04
4.60
4.16
4.02
4.69
4.21
4.01
4.49
5.51
4.23
4.47
4.03
4.81
4.43
4.32
6.70
5.00
3.60
5
6
6.00
5.36
7
5.47
4.56
4.80
4.20
5.39
4.29
4.64
4.14
4.89
4.17
4.54
4.05
4.84
4.11
4.87
4.43
4.16
5.09
4.75
4.42
4.78
4.26
4.04
4.56
4.13
5.13
4.20
4.60
4.25
4.79
4.26
4.83
4.50
4.18
5.02
4.72
4.49
4.82
4.24
4.05
4.71
4.37
4.10
4.71
4.32
4.06
4.34
5.20
4.16
4.38
4.01
4.70
4.45
4.32
6.80
5.10
3.60
5.04
4.28
4.59
4.04
4.74
4.01
4.84
4.46
4.13
5.00
4.69
4.40
4.86
4.35
4.05
4.59
4.13
4.88
4.07
4.51
4.01
4.88
4.17
4.90
4.49
4.19
5.48
4.73
4.48
4.82
4.30
4.06
4.53
4.06
4.02
4.56
4.14
4.90
5.34
4.42
4.54
4.02
4.89
11
12
13
4.51
4.01
5.33
5.18
4.69
4.26
5.17
4.82
4.52
4.88
4.45
4.09
4.60
4.16
4.65
4.26
4.90
4.41
4.12
5.19
4.75
4.45
4.80
4.25
4.02
4.66
4.18
15
18
19
21
4.74
4.35
5.01
4.77
4.54
4.82
4.30
4.10
4.47
4.90
4.41
4.02
4.58
4.09
4.60
4.18
4.04
4.63
4.17
4.02
5.03
6.16
4.38
4.85
5.13
4.35
4.01
4.79
5.94
4.42
4.85
4.09
4.75
4.57
4.13
4.67
4.24
4.01
4.31
5.60
4.42
4.53
4.01
5.49
4.78
4.41
6.90
5.20
3.70
4.53
4.09
4.60
4.04
4.80
4.27
23
4.41
5.27
4.25
4.27
4.05
4.72
4.34
4.30
5.30
4.14
4.28
4.21
5.21
4.04
4.25
4.32
5.41
4.64
5.58
4.12
4.55
4.08
4.77
4.37
24c
25
28
4.36
4.73
4.35
4.59
4.75
4.48
4.33
6.50
4.70
3.60
4.91
4.49
vincristine sulfated
7.00
4.80
3.20
6.60
4.80
3.60
7.00
5.40
4.10
6.50
4.70
3.50
6.90
5.20
3.50
6.50
5.10
3.50
a Highest concentration ) 10-4 M. Only modes showing a value >4.00 are reported. b Mean graph midpoint, i.e., the mean concentration for all cell
lines. c Mean of two different experiments. d Highest concentration ) 10-3 M.
leukemia, melanoma, and cancers of lung, colon, kidney, ovary,
breast, prostate, and central nervous system. Only compounds
that satisfy predetermined threshold inhibition criteria in a
minimum number of cell lines entered the full five-dose assay.
The one-dose data are reported as a mean graph of the percent
growth of treated cells (data not shown). Fifteen of twenty-six
compounds reported in Table 1 were active in this preliminary
test and passed on for evaluation in the full five-dose assay.
The test compounds were dissolved in dimethyl sulfoxide
(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 concentra-
tion at three assay end points: the 50% growth inhibitory power
(pGI50), the cytostatic effect (pTGI ) total growth inhibition),
and the cytotoxic effect (pLC50). Vincristine sulfate is reported
as the reference drug. In the Supporting Information a table is
available where the growth inhibition is expressed as micromolar
concentration (see Table S2).
b. Effect of Compound 24 on Growth of HT-29 Colon
Carcinoma Cells. Compound 24is the most active of this series
and is particularly active against colon carcinoma cell lines
(Table 2).
To investigate the mechanisms of its antiproliferative action,
we first examined its effect on the cell cycle profile of HT-29
colon carcinoma cells. The cells were incubated for 24 h in the
presence of compound 24 at concentrations ranging from 0.5
to 2 µM. Afterward the analysis of DNA profiles was performed
by flow cytometry. Figure 1A shows that compound 24 caused
a dose-dependent accumulation of HT-29 cells in the G2/M
phase, whereas most of the control cells were in the G0/G1
phase of the cell cycle. Actually, the G2/M cell population
increased from 18% in the control to 58% in HT-29 cells treated
with 2 µM compound 24 for 24 h. A similar effect was obtained
with vincristine, used as a reference compound, but at lower
concentration (30 nM). A cellular event strictly correlated with
cell cycle progression is represented by the induction of ornithine
decarboxylase (ODC), the limiting enzyme for the biosynthesis
of polyamines, which are absolutely required for cell prolifera-
tion.5 Figure 1B shows that, together with HT-29 cell arrest in
the G2/M phase, compound 24 also caused the dose-dependent
inhibition of ODC activity. Again, vincristine had a similar, but
more pronounced effect.
Next we examined the effect of compound 24 on cellular
microtubule networks by using immunofluorescence techniques.
As shown in Figure 2, the microtubules exhibit normal arrange-
ment and organization in control HT-29. However, after
treatment for 24 h with compound 24 (1 µM), we observed an
increase of cellular tubulin condensation, which represents a
marker of cytoskeleton reorganization, together with a great
number of mitotic spindles. These observations suggested that
compound 24 arrested the cells in mitosis, similar to vincristine.
Morphological evaluation using 4′,6-diamidino-2-phenylindole
(DAPI) staining revealed condensed chromatin along with
fragmented nuclei, suggestive of activation of apoptosis.

Antitumor ActiVity of Indolinones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7511
a relatively low toxicity (mean pLC50 < 4.00, not shown), and
a pGI50 of 6.16 against colon tumors. Even compound 23
showed a certain degree of selectivity against these tumor cells,
whereas compound 28 was most active against CNS tumors.
The antiproliferative action of the tested compounds can be
mediated through either cytotoxic or cytostatic effects. We found
that the effect of the very active compound 24 against HT-29
colon carcinoma cells was associated with a block in cell cycle
progression, with cell arrest in the G2/M phase. The ability of
24 to interfere with cell cycle in tumor cells was also evidenced
by inhibition of ODC, the first and rate limiting enzyme for
polyamine synthesis. It is known that ODC inhibitors can
interfere with the cell cycle at the G1-S and G2-M transitions.6
For this reason, ODC represents a potential target for anticancer
drug development.7 As expected, the arrest of the cell cycle is
then followed by a late induction of apoptosis, with activation
of caspase proteases. These observations show that compound
24 can interfere with proliferation of colon cancer cells by
multiple mechanisms.
From a quantitative standpoint, compounds 3, 5, 7, 23, 24,
and 25 showed the most similar profiles of activity in the NCI60
testing with Pearson correlation coefficients of 0.8 or greater
with each other (Table S3 of Supporting Information). Com-
pound 24 showed the strongest pattern of colon panel selectivity
and was tested twice in the NCI60 to confirm this pattern.
Compounds 5, 7, and 25 demonstrated very similar patterns of
activity against colon tumor panel. While not unprecedented in
the NCI60 screening experience, observation of this pattern
within an analogue series is quite striking and suggests a
common mechanism of action, targeting events particularly
important in colon tumors. As stated above for compound 24,
we believe that all the compounds of this series act by multiple
mechanisms. This theory was widely discussed and documented
for another series of compounds we recently published.8
Figure 3. Effects of compound 24 on growth and death of HT-29
cells. (A) Rate of cell growth determined as total cell number. The
cells were incubated in the presence of the indicated concentration of
compound 24 and counted daily. (B) Activation of apoptotic caspase
proteases. Caspase activity acting on the peptide sequence Asp-Glu-
Val-Asp (DEVD), indicated as DEVDase activity, was measured in
extracts obtained from cells treated for 24 or 48 h with the indicated
concentration of compound 24. Each panel represents the results
obtained in one typical experiment repeated at least twice with
comparable results.
As a consequence of its effects on the cell-division machinery,
depicted in Figures 1 and 2, compound 24 inhibited cell growth
and, at 2 µM, completely and irreversibly blocked HT-29 tumor
cell proliferation (Figure 3A). Following 48 h of cycle arrest,
the activity of caspase proteases acting on the amino acid
sequence Asp-Glu-Val-Asp (DEVD) began to increase (Figure
3B), confirming the onset of apoptosis.
Conclusion
From the biological results, it is possible to schematize the
following conclusions. The introduction of a carboxyl group at
the 5 position of the indolinone system led to loss of activity
(compound 4 did not pass the preliminary test). Good results
were instead obtained with the introduction, at the same position,
of fluorine, since compound 3 showed a mean growth inhibition
of 5.22 and was particularly effective against colon tumors
(pGI50 ) 5.62).
Compounds bearing a methyl group at the indole nitrogen
(5-7) were a little less active than the corresponding unsub-
stituted analogues (mean pGI50 of 5.80, 6.42, and 5.48,
respectively3,4), but compound 6 was especially active against
prostate tumors (pGI50 ) 6.00) and leukemias (pGI50 ) 5.84).
Compounds bearing a 2,5-dimethoxyphenyl group at the
6-position of the imidazothiazole (19-22) and 2-methylimida-
zothiazole system (8-11) did not lead to improvement of the
result obtained with previously published analogues such as
compound A (Chart 1).
Experimental Section
1. Chemistry. The melting points are uncorrected. Elemental
analyses were performed with a Fisons Carlo Erba EA1108
instrument, and results were within 0.4% of the theoretical values
(Table S4 of Supporting Information). TLC was performed on
Bakerflex plates (silica gel IB2-F) and column chromatography on
Kieselgel 60 (Merck). The eluent was a mixture of petroleum ether/
acetone in various proportions. The IR spectra were recorded in
Nujol on a Nicolet Avatar 320 E.S.P.; νmax is expressed in cm-1
.
The 1H NMR spectra were recorded 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 of Supporting Information. 5-Chloro-2-indolinone and
2-oxindole are commercially available, whereas the other substituted
2-indolinones (5-hydroxy,9 1-methyl-5-hydroxy,10 1-methyl-5-
methoxy,10 5-methoxy,11 5-fluoro,12 5-carboxy,13 1-methyl14) and
the starting aldehydes [(2,6-dimethyl-imidazo[2,1-b]thiazole-5-car-
boxaldehyde,15 6-(2,5-dimethoxyphenyl)imidazo[2,1-b]thiazole-5-
carboxaldehyde,16 2-methyl-6-(2,5-dimethoxyphenyl)imidazo[2,1-
b]thiazole-5-carboxaldehyde,16 2-methyl-6-p-methylphenylimida-
zo[2,1-b]thiazole-5-carboxaldehyde,17 2-methyl-6-p-chlorophenylimi-
dazo[2,1-b]thiazole-5-carboxaldehyde,18 2,6-dimethylimidazo[2,1-b]-
[1,3,4]thiadiazole-5-carboxaldehyde,19 2-methyl-6-p-chlorophenylimi-
dazo[2,1-b][1,3,4]thiadiazole-5-carboxaldehyde20] were prepared
according to the literature.
It is interesting that compound 21 (a N-methylindole deriva-
tive) was mainly active against leukemias (pGI50 ) 5.13) with
relatively low cytotoxicity (pLC50 ) 4.01).
Better results were obtained with the 2-methylimidazothiazole
system bearing, at position 6, a 4-methyphenyl (12-14) or a
4-chlorophenyl group (15-18). Some of these compounds (12,
13, 15, 18) were more active than two analogues where the
phenyl ring was unsubstituted: the analogue of 12 did not pass
the preliminary test and the analogue of 15 showed a mean pGI50
of 4.2.3 In particular compound 15 showed a mean pGI50 of
5.11 and was most active toward kidney tumors. Compound
12 was most active toward prostate cancers and leukemias, and
compound 13 was most active toward CNS tumors.
General Procedure for the Synthesis of Compounds 3 and
5-28. The appropriate aldehyde 1 (10 mmol) was dissolved in
methanol (100 mL) and treated with an equivalent of the indolinone
2 and piperidine (1 mL). The reaction mixture was refluxed for
3-5 h, according to a TLC test. The precipitate that formed on
cooling was collected by filtration with a yield of 45-55%.
Compound 21 was purified by column chromatography.
The results from the imidazothiadiazole derivatives (23-28)
are very interesting. Compound 24 was the most active of the
whole series presented in this paper, with a mean pGI50 of 5.51,

7512 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Andreani et al.
1
Data for 3. IR: 3068, 1713, 1183, 1001, 765. H NMR: 2.21
At the end of the incubation, the cells were washed twice with
PBS, fixed with 3% paraformaldehyde, subsequently washed with
0.1 M glycine in PBS, and permeabilized in 70% ice-cold ethanol.
Microtubules staining was performed, incubating overnight at 4 °C
with a mouse monoclonal anti-R-tubulin primary antibody (Upstate).
Cells were then washed, incubated with fluorescein isothiocyanate
conjugated antimouse IgG antibody (Sigma) for 1 h at room
temperature, and finally stained with DAPI. All preparations were
embedded in Mowiol and analyzed using a laser scanning confocal
microscope (Nikon C1s) equipped with Nikon eclipse TE300.
(3H, s, CH3), 2.40 (3H, d, CH3, J)1.4), 6.48 (1H, dd, ind, J ) 9.1,
J ) 2.5), 6.84 (1H, m, ind), 7.06 (1H, m, ind), 7.56 (1H, q, th, J
) 1.4), 7.87 (1H, s, CH), 10.64 (1H, s, NH). Anal. (C16H12FN3OS)
C, H, N.
Data for 6. IR: 1685, 1618, 1104, 881, 666. 1H NMR: 2.20 (3H,
s, CH3), 2.41 (3H, d, CH3, J ) 1.5), 3.23 (3H, s, NCH3), 6.69 (1H,
d, ind-4, J ) 1.9), 7.08 (1H, d, ind-7, J ) 9.6), 7.37 (1H, dd, ind-
6, J ) 9.6, J ) 1.9), 7.57 (1H, q, th, J ) 1.5), 7.75 (1H, s, CH).
Anal. (C17H14ClN3OS) C, H, N.
Data for 7. IR: 3104, 1698, 1117, 810, 661. 1H NMR: 2.19 (3H,
s, CH3), 2.39 (3H, d, CH3, J ) 1.4), 3.17 (3H, s, NCH3), 6.39 (1H,
d, ind-4, J ) 2.6), 6.69 (1H, dd, ind-7, J ) 8.4, J ) 2.6), 6.84 (1H,
d, ind-6, J ) 8.4), 7.56 (1H, q, th, J ) 1.4), 7.59 (1H, s, CH), 9.08
(1H, s, OH). Anal. (C17H15N3O2S) C, H, N.
Acknowledgment. This work was supported by a grant from
MIUR-COFIN 2006. We are grateful to NCI for the antitumor
tests and to CIRB for the use of confocal microscope and flow
cytometer.
1
Data for 15. IR: 3204, 1702, 1197, 833, 727. H NMR: 2.44
Supporting Information Available: IR and 1H NMR of the
new compounds (Table S1), antitumor activity expressed as
micromolar concentrations (Table S2), matrix compare analysis
results for compounds with correlation coefficient g0.8 (Table S3),
elemental analysis results (Table S4), and NSC numbers (Table
S5). This material is available free of charge via the Internet at
http://pubs.acs.org.
(3H, s, CH3), 6.88 (1H, d, ind, J ) 7.5), 6.98 (1H, t, ind, J ) 7.5),
7.24 (1H, t, ind, J ) 7.5), 7.41 (1H, s, th), 7.54 (2H, d, ph, J )
8.5), 7.73 (4H, m, 1H-ind + 2H-ph + CH), 10.58 (1H, s, NH).
Anal. (C21H14ClN3OS) C, H, N.
1
Data for 24. IR: 3200, 1703, 1033, 876, 768. H NMR: 2.25
(3H, s, CH3), 2.72 (3H, s, CH3), 3.60 (3H, s, OCH3), 6.54 (1H, d,
ind-4, J ) 2.2), 6.77 (1H, d, ind-7, J ) 8.4), 6.83 (1H, dd, ind-6,
J ) 8.4, J ) 2.2), 7.50 (1H, s, CH), 10.45 (1H, s, NH). Anal.
(C16H14N4O2S) C, H, N.
References
Procedure for the Synthesis of Compound 4. 2,6-Dimethylimi-
dazo[2,1-b]thiazole-5-carbaldehyde (10 mmol) was dissolved in
acetic acid (50 mL) and treated with an equivalent of 2-oxoindoline-
5-carboxylic acid and 37% hydrochloric acid (1 mL). The reaction
mixture was refluxed for 15-20 h, according to a TLC test, and
the precipitate that formed on cooling (yield 65%) was collected
by filtration. IR: 3310, 1709, 1163, 892, 765. 1H NMR: 2.19 (3H,
s, CH3), 2.39 (3H, d, CH3, J ) 1.2), 6.98 (1H, d, ind-7, J ) 8.2),
7.41 (1H, q, th, J ) 1.2), 7.62 (1H, d, ind-4, J ) 1.5), 7.65 (1H, s,
CH), 7.85 (1H, dd, ind-6, J ) 8.2, J ) 1.5), 11.07 (1H, s, NH).
Anal. (C17H13N3O3S) C, H, N.
2. Biology. 2.a. Antitumor Activity. The screening is a two-
stage process,21 beginning with the evaluation of all compounds
against the 60 cell lines at a single dose of 10-5 M. Compounds
that exhibit significant growth inhibition are evaluated against the
60 cell panel at five concentration levels by the NCI according to
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2.b. Effects on Growth of HT-29 Colon Carcinoma Cells.
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10% fetal bovine serum (Euroclone, Milan, Italy) and 2 mM
L-glutamine (Sigma-Aldrich, St. Louis, MO) at 37 °C and 5% CO2.
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mmol/L immediately before use. Cells were plated at 2 × 104 cells/
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different concentrations, was added. In control cells only DMSO
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0.01% nonidet P-40 (Sigma), 10 µg/mL RNase, 0.1% sodium citrate
(Sigma), and 50 µg/mL propidium iodide (PI) (Sigma) for 30 min
at room temperature in the dark. PI fluorescence was analyzed by
using a Beckman Coulter Epics XL MCL cytometer. Cell cycle
analysis was performed using Modfit 5.0 software.
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Antitumor ActiVity of Indolinones
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