Substituted E-3-(3-Indolylmethylene)-1,3-dihydroindol-2-ones with antitumor activity. In depth study of the effect on growth of breast cancer Cells 1

Article abstract of DOI:10.1021/jm1007165

The synthesis of new substituted E-3-(3-indolylmethylene)-1,3-dihydroindol- 2-ones is reported. The antitumor activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. Structure-activity relationships ar

Full text of DOI:10.1021/jm1007165

J. Med. Chem. 2010, 53, 5567–5575 5567
DOI: 10.1021/jm1007165
Substituted E-3-(3-Indolylmethylene)-1,3-dihydroindol-2-ones with Antitumor Activity. In Depth Study
of the Effect on Growth of Breast Cancer Cells1
Aldo Andreani,*,† Stefania Bellini,† Silvia Burnelli,† Massimiliano Granaiola,† Alberto Leoni,† Alessandra Locatelli,†
Rita Morigi,† Mirella Rambaldi,† Lucilla Varoli,† Natalia Calonghi,‡ Concettina Cappadone,‡ 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, and §Screening Technologies Branch, Developmental
Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, Frederick, Maryland 21702
ꢀ
Received March 1, 2010
The synthesis of new substituted E-3-(3-indolylmethylene)-1,3-dihydroindol-2-ones is reported. The
antitumor activity was evaluated according to protocols available at the National Cancer Institute
(NCI), Bethesda, MD. Structure-activity relationships are discussed. The action of selected com-
pounds was investigated in MCF-7 breast cancer cells. The ability of these derivatives to inhibit cellular
proliferation was accompanied by increased level of p53 and its transcriptional targets p21 and Bax,
interference in the cell cycle progression with cell accumulation in the G2/M phase, and activation of
apoptosis.
Introduction
was weak, but when it was tested according to the
NCIa protocols it showed mean GI50 of 1.26 μM.
This prompted us to reconsider the possibility of bis-
methylation, and compounds 13-15 were prepared
accordingly.
In the previous papers of this series2-6 we described several
substituted E-3-(2-chloro-3-indolylmethylene)-1,3-dihydroindol-
2-ones prepared from an indolealdehyde and an oxindole.
Compounds with potent cytotoxic activity were obtained
when the aldehyde was 2-chloro-5-methoxy-6-methylindole-
3-carbaldehyde.4 Moreover, we noticed6 that in some cases
N-methylation increased the activity. Even preliminary at-
tempts to introduce substituents at positions 4, 5, and 6 of the
oxindole portion had been performed.4
The rationale for the synthesis of new analogues described
in this paper (Scheme 1, Table 1) is the following:
1. Evaluation of the effects induced by different substitu-
ents in the oxindole portion (bromine, dimethylamino,
and succinyl groups 4, 5, and 6) and by the position
of chlorine (7) was done: the compounds bearing chlo-
rine at the position 4 or 5 were described in previous
papers.5,6
2. Compound 8 was prepared to evaluate whether the
methylation of the nitrogen in the chloroindole portion
could lead to increase of activityas previously noticed.5,6
This kind of methylation was also performed in four
compounds mentioned in the above group (4-7) and
gave rise to the derivatives 9-12.
3. The synthesis of one compound bearing a methyl group
at both nitrogens and its activity on HeLa cells was
described in the first paper of this series.2 Its cytotoxicity
4. Compound 16 was prepared to evaluate the effect of
shifting methyl and methoxy group from the chloro-
indole to the oxindole portion.
5. In the compounds so far described, the best results were
obtained when the aldehyde was bearing a methoxy
group at the 5 position and a methyl group at the
6 position. Compounds 17-22 were prepared to inves-
tigate if they could be replaced by different substi-
tuents and in particular with a group that can be easily
protonated.
6. In the previous paper6 good results were obtained with
an additional condensed benzene ring in the oxindole
portion. Compounds 23-28 bear an additional con-
densed benzene ring but in the chloroaldehyde portion.
7. With the synthesis of compounds 29-34 we were
planning to compare the activity of the 2-chloro with
that of the unsubstituted derivatives.
8. The introduction of a hydrophilic and electron attract-
ing group was considered in compounds 35 and 36.
9. Compound 37 does not belong to the same series
considered so far but could be the first term of a new
series (devoid of the methine bridge) if it should display
significant antitumor activity.
*To whom correspondence should be addressed. Phone: þ39-51-
2099714. Fax: þ39-51-2099734. E-mail: aldo.andreani@unibo.it.
aAbbreviations: NCI, National Cancer Institute; DTP, Develop-
mental Therapeutics Program; GI, growth inhibition; TGI, total growth
inhibition; LC, lethal concentration; BEC, Biological Evaluation Com-
mittee; MTD, maximum tolerated dose; DMSO, dimethyl sulfoxide;
FITC, fluorescein isothiocyanate; EDTA, ethylenediaminetetraacetic;
FBS, fetal bovine serum; PBS, phosphate-buffered saline; PI, propidium
iodide.
Chemistry
Most of the compounds were prepared by means of
the single step Knoevenagel reaction between the indolinones
1 and the aldehydes 2 in methanol/piperidine. For com-
pounds 4, 6, 11, 24-26, 35, and 37 a mixture of acetic acid/
hydrochloric acid has been employed.
r
2010 American Chemical Society
Published on Web 07/14/2010
pubs.acs.org/jmc

5568 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Andreani et al.
Scheme 1
Table 1. Compounds 4-37
compd
formula
MW
mp, °C
compd
formula
MW
mp, °C
4
C
C
19H14BrClN2O2
21H20ClN3O2
417.69
381.86
438.86
373.23
396.87
431.71
395.88
452.89
387.26
366.84
382.84
410.90
352.82
372.25
372.25
353.80
367.83
325-328 dec
295-298 dec
338-340
21
22
23
24
25
26
27
28
29
30
31
32
33
34
36
37
C20H18ClN3O2
C21H21ClN4O
C21H12Cl2N2O
C21H12Cl2N2O
C21H12ClFN2O
C22H15ClN2O2
C23H17ClN2O2
C22H15ClN2O2
C18H14N2O2
C19H16N2O2
C21H20N2O3
C19H16N2O2
C20H18N2O2
C20H18N2O3
C20H16N2O4
C18H14N2O4
367.83
380.87
379.24
379.24
362.79
374.82
388.85
374.82
290.32
304.34
348.40
304.34
318.37
334.37
348.35
322.32
200-205 dec
340-343 dec
278-281 dec
310-312 dec
330-332 dec
185-187 dec
317-320 dec
335-338 dec
255-258
288-290
273-275
272-275
277-280
5
6
C23H19ClN2O5
C19H14Cl2N2O2
C22H21ClN2O3
7
285-290 dec
284-286 dec
290-292 dec
230-233 dec
340-345 dec
250-253 dec
277-280 dec
348-350 dec
225-228
220-222 dec
335-340 dec
200-204 dec
345-350 dec
180-185 dec
8
9
C
20H16BrClN2O2
C22H22ClN3O2
24H21ClN2O5
C20H16Cl2N2O2
21H19ClN2O2
C21H19ClN2O3
10
11
12
13
14
15
16
17
18
19
20
C
C
C
C
23H23ClN2O3
20H17ClN2O2
C19H15Cl2N3O
C19H15Cl2N3O
C19H16ClN3O2
280-282
175-180
340-345
C
20H18ClN3O2
Most compounds were obtained as almost pure geometrical
isomers that, according to the usual NOE experiments described
in the previous papers,7,8 were assigned to the E configuration.
Compounds 18 and 34 were obtained as evident E/Z mixtures
(around 70/30) and submitted as such to the biological tests.
The E/Z ratio in solution is time dependent and tends to be

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5569
50/50. In our experience with similar derivatives, we described
the separations of the two isomers by fractional crystallization,7
but no significant difference in the pharmacological behavior
was noticed.
The oxindoles 1d, 1j, 1o, the aldehyde 2h, and the isatin 3 are
commercially available, whereas the other starting com-
pounds (oxindoles andaldehydes)havebeen preparedaccord-
ing to the literature4,6,9-22 except 1h, 2d, 2e, and 2j, reported in
the Experimental Section.
24 h after treatment, and the viable cell mass in each fiber is
determined.
A point system was developed to assess the activity.
A value of 2 is assigned for each treatment dose that results
in a 50% or greater reduction in viable cell mass in any fiber.
Compounds with a combined ip and sc score of 20 or higher,
an sc score of 8 or higher, or cell kill of one or more cell lines
are considered for further in vivo testing. Compound 30
resulted in a combined ip þ sc score of 16, a sc score of 6, and
gave cell kill. Nevertheless, at this time BEC decided to
suspend further testing.
Biology
a. Cell-Based Assays. In a preliminary test, compounds
were assayed at a single high concentration (10-5 M) in the
full NCI 60 cell panel. This panel is organized into sub-
panels representing leukemia, melanoma, and cancers of lung,
colon, kidney, ovary, breast, prostate, and central nervous
system. Onlycompoundsthatsatisfy predeterminedthreshold
inhibition criteria in a minimum number of cell lines progress
to the full five-concentration assay. The threshold inhibition
criteria for progression to the five-concentration screen was
selected to efficiently capture compounds with antiprolifera-
tive activity based on the analysis of historical DTP screening
data. The result is expressed as the percent growth of treated
cells at the test concentration of 10-5 M following 48 h of
incubation (unpublished results).
Compounds 5, 6, 11, 17, 22, 31, 35, 36, and 37 were not
considered active enough to enter the five-concentration
test, whereas all the others were subjected to this screen.
The compounds were dissolved in dimethyl sulfoxide
(DMSO) and evaluated using five concentrations at 10-
fold dilutions (the highest being 10-4 M) following 48 h of
incubation.
Table 2 reports the results obtained (vincristine is reported
for comparison purposes), expressed as μM at three assay
end points: the 50% growth inhibitory power (GI50), the
cytostatic effect (TGI = total growth inhibition), and the
cytotoxic effect (LC50).
For some compounds the five-concentration test was
repeated and no significant differences were found. For these
compounds the data reported in Table 2 are the mean values
of the two experiments.
The tested compounds showed a mean GI50 between
23.99 and 0.03 μM, and compounds 7, 12, 13, 14, 16, 24,
27, 28, and 30 were submitted to BEC (Biological Evaluation
Committee) for possible future development. Further studies
continued at the NCI where the maximum tolerated dose
(MTD) was determined for compounds 16, 28, and 30. It was
found to be 100 mg/kg for compound 16, whereas it was
found to be 400 mg/kg for compounds 28 and 30, adminis-
tered ip in DMSO for 16 and 28 and in 10% DMSO in saline/
Tween 80 for 30. Compound 30 was then chosen for the first
in vivo experiment, i.e., the “hollow fiber assay”,23 which is a
rapid and efficient initial in vivo screening model including
multiple cell lines in a single assay. Therefore, it was sub-
jected to four experiments against a panel of three tumor cell
lines each consisting of the breast, non-small-cell lung, colon,
ovarian, CNS, and melanoma cell lines (administered ip in
10% DMSO and saline/Tween 80). The cancer cell lines were
loaded into biocompatible polyvinylidene fluoride hollow
fibers. Duplicate sets of fibers were implanted into mice
intraperitoneally (ip) and subcutaneously (sc) such that each
mouse carried three fibers ip and three fibers sc, representing
three distinct cell lines. The mice were treated ip with
compound 30 once daily for 4 days, the fibers are collected
In light of the NCI 60 results, the following may be
considered: shifting of chlorine from position 4/5 to 6 of
the oxindole portion is accompanied by an activity increment
which was more evident when the indole portion was
N-methylated. In fact compound 12 showed mean GI50 of
0.03 μM vs 5.37 μM of the 4-chloro isomer and 0.45 μM of
the 5-chloro isomer.6
The comparison between the activity of compounds 7 and
12 (mean GI50 of 0.21 and 0.03 μM, respectively) confirms
the activity increment observed for some analogues after
N-methylation of the chloroaldeyde. To a lesser extent an
increment was observed even in the comparison of 4 (mean
GI50 of 2.14 μM) vs 9 (1.66 μM) and 5 (inactive in the
preliminary test) vs 10 (11.22 μM), whereas the same does
not happen in the comparison of 8 (23.99 μM) vs the
previously reported NH free analogue (2.00 μM)4 and in
the comparison of 13-15 vs the monomethyl derivatives.4,5
Shifting of the methyl and methoxy groups from the
chloroindole to the oxindole portion (16) did not lead to a
significant difference in the biological behavior: mean GI50,
TGI, LC50 of 0.45, 10.96, 56.23 μM, respectively, vs 0.40,
12.59, 79.43 μM of the previously reported analogue.4
The introduction of the dimethylamino group was accom-
panied by loss of activity with respect to the 5-methoxy-
6-methyl analogues;5 compound 22, bearing two dimethyl-
amino groups, was inactive in the preliminary test. Other
attempts were unsuccessful such as the introduction of
hydrophilic/electron attracting groups or the lack of the
methine bridge.
The evaluation of the antitumor activity of the compounds
lacking chlorine at the 2-position of the indole portion
(29-34) did not indicate that this class of compounds is
superior to the 2-chloro derivatives: only compound 29 was
more active than the corresponding 2-chloro analogue (mean
GI50 of 4.57 vs 77.62 μM 3), whereas 30-34 were about as
active as the chloro analogues. Compound 30 was selected by
NCI for in vivo studies.
The replacement of the indole by a benzoindole portion
led to the most interesting compounds. In particular, 28 was
the most active of the whole series described in this paper,
thus confirming that the bulky benzoindole system is a
suitable pharmacophoric group in this class of compounds.
b. Effect on Growth of MCF-7 Breast Cancer Cells.
A subset of compounds was used for further assays in a
biological model. First, we examined the influence on the
growth and death of MCF-7 breast cancer cells of com-
pounds 12, 28 (which in the 60-cell panel were very active),
and 16, a less-active compound.
Figure 1A shows that even at a low concentration
(200 nM), 12 and 28 blocked MCF-7 cell growth within
48 h. At the same concentration, the less effective compound
16 initially decreased cell proliferation; however, after 24 h,
the cells recovered and started to grow. In some preliminary

5570 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Andreani et al.
Table 2. Nine Subpanels at Five Concentrations: Growth Inhibition and Cytostatic and Cytotoxic Activity (μM) of the Selected Compounds (See
Supporting Information for the Complete List of Cell Lines Employed)
compda
modes
leukemia
NSCLC
colon
CNS
melanoma
ovarian
renal
prostate
breast
MG-MIDb
4
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
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
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
1.62
6.17
3.02
14.13
53.70
0.43
1.95
6.76
1.58
8.91
2.14
9.55
2.00
8.13
2.45
10.96
37.15
0.23
2.24
7.94
2.14
10.47
50.12
0.15
2.14
9.33
37.15
0.14
4.79
23.44
0.17
3.55
35.48
0.12
4.37
41.69
0.23
38.90
0.28
30.90
0.22
5.25
39.81
0.21
6.61
7c
11.48
52.48
34.67
97.72
11.48
35.48
15.14
45.71
57.54
1.55
14.79
48.98
30.20
91.20
7.08
46.77
41.69
2.63
58.88
18.20
60.26
97.72
2.51
25.70
22.91
87.10
41.69
26.92
87.10
41.69
22.39
97.72
39.81
18.62
77.62
95.50
0.95
41.69
23.99
77.62
91.20
1.66
8c
9c
1.91
9.33
1.55
6.03
0.93
7.08
3.47
13.49
43.65
10.47
77.62
91.20
0.03
1.70
6.76
1.55
8.91
20.89
7.41
4.90
8.32
37.15
18.20
91.20
29.51
12.30
70.79
38.90
10.72
60.26
31.62
10.23
67.61
93.33
0.02
26.92
11.22
69.18
26.30
20.42
97.72
31.62
6.92
54.95
36.31
11.22
67.61
97.72
0.03
10
10.00
44.67
87.10
0.02
12c
13c
14c
15
0.07
11.48
93.33
0.58
0.02
0.95
0.02
1.20
0.04
15.85
70.79
0.33
0.02
3.02
0.05
5.62
1.74
7.08
50.12
0.34
2.34
52.48
0.38
3.80
48.98
0.32
39.81
0.25
4.07
25.12
0.20
8.51
23.44
0.20
4.47
21.38
0.39
72.44
0.28
33.11
14.13
69.18
0.11
18.20
95.50
0.26
10.23
79.43
0.11
10.00
66.07
0.10
13.18
95.50
0.06
11.48
77.62
0.10
46.77
0.05
0.47
72.44
0.07
3.55
74.13
0.04
1.74
0.22
6.03
7.41
11.75
47.86
18.62
60.26
89.13
0.60
4.27
3.16
1.70
40.74
6.76
3.39
19.95
6.92
56.23
42.66
19.50
69.18
93.33
0.58
26.92
20.42
83.18
97.72
0.37
29.51
10.47
26.92
61.66
0.32
33.11
16.22
67.61
36.31
15.85
41.69
77.62
0.62
23.99
35.48
67.61
33.88
14.13
52.48
87.10
0.45
36.31
83.18
0.30
16c
18c
19
0.26
7.41
0.71
18.20
51.29
2.69
0.32
21.38
69.18
2.45
12.30
57.54
2.57
9.33
7.59
11.75
61.66
3.31
12.88
56.23
1.78
8.32
10.96
56.23
2.09
69.18
1.62
46.77
2.09
7.24
47.86
1.58
5.50
57.54
1.32
9.12
17.78
91.20
2.24
10.00
46.77
5.13
11.22
37.15
5.25
11.75
41.69
7.24
8.13
29.51
5.13
13.49
46.77
8.91
9.77
38.90
4.37
22.91
3.89
18.20
3.80
36.31
54.95
2.88
33.88
13.49
31.62
95.50
3.02
20.42
69.18
1.55
25.70
79.43
3.55
52.48
33.88
91.20
3.02
53.70
29.51
91.20
2.29
20c
21
1.62
10.23
72.44
16.60
2.09
7.94
3.39
15.14
50.12
8.32
2.88
19.05
50.12
12.88
1.15
8.51
15.49
58.88
6.61
9.55
30.20
4.90
15.49
44.67
4.57
13.80
52.48
4.37
12.02
48.98
6.03
32.36
6.03
54.95
3.80
30.90
46.77
85.11
9.55
20.89
61.66
4.90
33.88
85.11
3.63
26.30
66.07
4.57
54.95
95.50
2.51
31.62
87.10
8.91
38.02
83.18
5.50
23c
24c
25c
26
5.50
25.70
75.86
0.19
10.23
31.62
63.10
0.38
5.01
19.95
67.61
0.38
28.18
72.44
0.87
17.38
47.86
0.49
17.78
53.70
0.36
19.05
46.77
0.66
13.18
57.54
0.59
23.44
56.23
0.55
20.89
58.88
0.49
7.24
12.59
58.88
0.65
6.17
5.37
11.22
40.74
0.56
11.22
61.66
0.33
8.51
52.48
0.63
8.91
5.62
8.32
72.44
0.49
8.71
35.48
0.35
6.92
47.86
0.40
6.46
38.02
0.39
9.77
54.95
0.40
6.76
51.29
0.47
8.91
12.59
70.79
7.94
10.96
44.67
6.46
7.41
41.69
9.12
10.96
43.65
8.71
87.10
3.31
38.02
57.54
6.61
51.29
8.71
51.29
5.37
70.79
3.72
56.23
6.31
30.20
81.28
0.63
20.89
61.66
0.31
24.55
67.61
0.31
20.89
57.54
0.56
46.77
87.10
0.40
33.11
75.86
0.49
26.30
74.13
0.25
19.05
69.18
0.23
26.92
72.44
0.37
27c
28c
29
0.19
1.00
19.05
0.01
0.08
5.75
2.34
7.76
43.65
0.09
2.88
64.57
9.33
2.88
3.39
7.94
7.24
8.13
3.02
4.37
4.79
47.86
0.06
4.07
18.62
0.02
0.71
30.90
0.02
1.95
32.36
0.03
4.68
33.88
0.04
4.68
35.48
0.04
5.62
25.70
0.01
0.68
47.86
0.02
1.32
30.90
0.03
1.78
32.36
5.13
9.12
2.24
22.39
6.17
27.54
6.61
27.54
6.31
51.29
5.75
16.22
5.62
66.07
3.47
23.99
4.57
25.12
77.62
0.11
15.49
70.79
0.09
28.84
75.86
0.15
27.54
75.86
0.12
30.20
81.28
0.18
26.30
83.18
0.23
23.99
83.18
0.07
18.20
91.20
0.12
21.38
74.13
0.13
30c
16.98
77.62
3.98
22.91
3.63
53.70
7.41
50.12
7.76
58.88
6.92
53.70
12.88
63.10
6.76
75.86
6.46
54.95

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5571
Table 2. Continued
compda
modes
leukemia
NSCLC
colon
CNS
melanoma
ovarian
renal
prostate
breast
MG-MIDb
32
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
8.71
54.95
13.49
43.65
89.13
6.31
8.13
52.48
95.50
0.76
10.00
33.88
95.50
1.07
13.18
37.15
81.28
3.09
12.59
45.71
97.72
7.76
11.48
38.02
85.11
4.17
13.80
36.31
91.20
4.17
11.75
40.74
91.20
1.78
11.22
42.66
91.20
2.75
33c
2.00
35.48
93.33
3.47
66.07
93.33
6.03
60.26
97.72
2.95
42.66
95.50
3.72
74.13
97.72
3.24
60.26
60.26
81.28
54.95
57.54
97.72
4.07
34
5.62
31.62
95.50
0.32
5.89
28.18
93.33
0.32
3.55
15.85
2.75
16.22
63.10
0.32
36.31
21.88
91.20
0.25
24.55
23.99
67.61
0.13
15.14
66.07
0.16
22.91
83.18
0.20
vincristine sulfate d
0.10
15.85
630.96
0.10
3.98
0.13
6.31
15.85
251.19
6.31
199.53
7.94
251.19
19.95
316.23
19.95
251.19
7.94
316.23
10.00
251.19
79.43
316.23
a Highest concentration = 10-4 M unless otherwise reported. Only values <100 μM are reported. The compound exposure time was 48 h. b Mean
graph midpoint, i.e., the calculated panel mean. c Mean of two separate experiments. d Highest concentration = 10-3 M.
Figure 1. Effects on growth and death of MCF-7 cells. (A) Rate of
cell growth determined as total cell number. The cells were incu-
bated in the presence of the indicated compounds (200 nM), and
viable cells were counted daily. The differences of all treated cells vs
control cells were significant (P < 0.05) at either 24 and 48 h.
(B) Caspase activity acting on the peptide sequence DEVD (mainly
caspase 7) was measured in extracts obtained from cells treated for
24 or 48 h with the indicated compounds (5 μM). The reported
data are mean values ( SEM obtained in five determinations.
The differences of all treated cells vs control cells were significant
(P < 0.05) only at 48 h.
experiments, apoptotic cell death was observed after a longer
treatment time (72-96 h). At shorter times, a significant
toxic effect became evident only at concentrations in the
micromolar range, and cell death was detectable after a 48 h
incubation (data not shown). To understand whether the
toxicity of these new derivatives was associated with acti-
vation of apoptosis, the cells were treated with a 5 μM con-
centration of the compounds, a concentration that caused
about 30-40% of cell death, and the activation of caspases
acting on the peptide substrate sequence Asp-Glu-Val-Asp
(DEVD) was determined. This sequence is the optimal
substrate for effector caspases 3 and 7, activated as the result
of all caspase cascades.24 However, since MCF-7 cells lack
caspase 3, the assay measures mainly the activation of
caspase 7, which is highly expressed in these cells.25 The
activation of the caspase proteases, considered the molecular
effectors of apoptosis,26 was significant only after 48 h of
treatment (Figure 1B).
We next examined the effect of the derivatives on some
biochemical pathways correlated with cell proliferation
and cell death (Figure 2). In these studies, the cells were
treated with the different agents at 5 μM for 20 h. First, we
determined whether the antiproliferative effect was accom-
panied by changes in the expression of the tumor suppressor
p53. Figure 2A shows that compounds 12, 16, and 28 caused
a p53 increase. Next, we investigated the effect on ERK1/2
Figure 2. Effect on biochemical pathways correlated with cell
growth in MCF-7 cells. (A) The cells were incubated for 20 h in
the presence of 5 μM of the indicated compound. Then the content
and phosphorylation status of the indicated proteins were deter-
mined in cell extracts by Western blotting (80 μg of protein/lane).
Immunoblots reported are from one experiment representative of at
least three that gave similar results. (B) Quantitative determination
of p53 and p21 proteins by densitometry of immunoblotting in cells
incubated 24 h in the presence of 5 μM compound 12. Results are
mean values ( SEM obtained in four separate determinations.
Differences between treated cells and control cells were significant
(P < 0.05) for both p53 and p21. (C) Effect of compound 12 (5 μM
for 24 h) on cellular Bax detected by immunofluorescence confocal
microscopy. Nuclei were evidenced by incubation with propidium
iodide (red fluorescence). Bax was stained with FITC antibody and
is evidenced as green fluorescence. The image is representative of
three experiments.

5572 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Andreani et al.
progression, with cell arrest in the G2/M phase. The arrest of
the cell cycle probably causes the late induction of apoptosis,
as indicated by activation of caspase proteases and appear-
ance of a sub-G1 cell population. From the biochemical
point of view, these events can be explained by the induction
of the tumor suppressor p53, which is known to exert cell
cycle blocking effect and eventual activation of apoptosis
directly and by means of several effectors such as p21 or
Bax,31 which were also increased.
Experimental Section
Chemistry. All the compounds prepared have a purity of at
least 95% as determined by combustion analysis.
Figure 3. Effects on cell cycle in MCF-7 cells. Cells were treated
with 200 nM of the indicated compound for 24 h. Afterward cell
cycle distribution was determined by flow cytometry. Results are
mean values ( SEM of three determinations.
The melting points are uncorrected. 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
.
mitogen-activated protein kinases, which are generally asso-
ciated with tumor cell growth.27 In this case, the three
compounds had different effects. In fact, 12 and especially
16 decreased ERK phosphorylation which, on the contrary,
was increased by compound 28. The three derivatives
had minimal effects on the kinase Akt, an oncoprotein up-
regulated in several cancers and responsible for resistance
to cell death,28 Akt-phosphorylation was only slightly
decreased by compound 12.
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 (abbreviations: ar = aromatic,
bzind=benzoindole, ind=indole, ox=oxindole). For the spectra
that are not reported here, see Supporting Information. The
oxindoles 1d, 1j, 1o, the aldehyde 2h and the isatin 3 are commer-
cially available. The following compounds were prepared accord-
ing to the literature: 1a,9 1b,11 1c,12 1e,10 1f,13 1g14 1i,16 1k,17 1l,18
1m,19 1n,4 2a,10 2b,6 2c,15 2f,20 2g,21 and 35.22
Synthesis of 5-Methoxy-1,6-dimethyl-1,3-dihydro-2H-indol-
2-one (1h). 5-Methoxy-6-methyl-1,3-dihydro-2H-indol-2-one10
(4 mmol) in acetone (40 mL) was treated with dimethyl sulfate
(9.5 mmol) in the presence of anhydrous potassium carbonate
(6.5 mmol). The reaction mixture was refluxed for 15 h, cooled,
and filtered. The solution was evaporated and the expected
compound was purified by column chromatography (petroleum
ether/acetone, 70/30), with a yield of 52%. It was crystallized
from acetone/petroleum ether: mp 93-95 °C. IR: 1697, 1240,
These data indicate that interference in the function of
growth-related kinases ERKs and Akt does not represent
a common mechanism of the antiproliferative effect of the
tested compounds, whereas p53 appears correlated to the
action of these derivatives. To confirm the involvement of
p53, the effects of the potent compound 12 on the levels of the
p53 transcriptional target p2129 were determined. In cells
incubated 24 h with 12, together with a 3-fold induction of
p53, p21 was also increased by about 90% (Figure 2B).
Another important protein associated with the tumor sup-
pressive role of p53 is Bax, which is an effector of p53-
induced apoptosis and is a transcriptional target of p53 and
a partner of p53 in its nontranscriptional actions.30 The cel-
lular content of the Bax protein was assayed by immuno-
fluorescence microscopy. Figure 2C shows that treatment
with compound 12 caused a considerable Bax accumula-
tion, evidenced by a large increase in green fluorescence.
Densitometric analysis of bands obtained by Western blot-
ting (n=3) indicated an increase of 2.8 times (datanot shown).
The p53 tumor suppressor can cause apoptosis and in-
fluence the cell cycle.31 The increase of p21 caused by 12
suggested that the anticancer action of the novel compounds
could also be associated with interference in cell cycle
progression. Therefore, we examined the effect of some
derivatives on the cell cycle profile of MCF-7 cells. The cells
were incubated for 24 and 48 h in the presence of 200 nM 12
and 28, and the analysis of DNA profiles was performed by
flow cytometry. As previously detailed, at this concentration
both compounds strongly inhibit cell growth without caus-
ing massive cell death. Figure 3 shows that they caused a
marked accumulation of MCF-7 cells in the G2/M phase,
whereas most of the control cells were in the G0/G1. More-
over, after treatment with the analogues, a notable cell
population was observed in a sub-G1 peak caused by frag-
mented DNA in apoptotic cells. This population increased
with incubation time, confirming activation of apoptosis.
In conclusion, the most active compounds tested on
MCF-7 breast cancer cells gave rise to a block in cell cycle
1
1151, 1069. H NMR: 2.17 (3H, s, CH3), 3.06 (3H, s, N-CH3),
3.47 (2H, s, CH2), 3.73 (3H, s, OCH3), 6.79 (1H, s, ar), 6.94 (1H, s,
ar). Anal. Calcd for C11H13NO2 (MW 191.23): C, 69.09; H, 6.85;
N, 7.32. Found: C, 69.12; H, 6.78; N, 7.36.
Synthesis of 5-(Dimethylamino)-3-[(dimethylamino)methylene]-
2-oxoindoline-1-carbaldehyde (1p). The Vilsmeier reagent was
prepared at 0-5 °C by dropping POCl3 (32 mmol) into a stirred
solution of DMF (43 mmol) in CHCl3 (5 mL). 5-(Dimethyl-
amino)-1,3-dihydroindol-2-one11 (1b) (6 mmol) was dissolved in
CHCl3 and treated with the Vislmeier reagent. After 3 h at room
temperature, CHCl3 was evaporated under reduced pressure and
the residue was poured into ice-water. After neutralization with
NaHCO3 the expected compound was collected by filtration and
crystallized from EtOH with a yield of 82%: mp 154-157 °C. IR:
1671, 1703, 1177, 1108. 1H NMR: 2.89 (6H, s, 2CH3), 3.37 (3H, s,
CH3), 3.56 (3H, s, CH3), 6.40 (1H, dd, ind-6, J=8.6, J= 2.6), 6.85
(1H, d, ind-4, J = 2.6), 7.69 (1H, d, ind-7, J = 8.6), 7.85 (1H, s,
CH), 9.14 (1H, s, CH). Anal. Calcd for C14H17N3O2 (MW 259.31):
C, 64.85; H, 6.61; N, 16.20. Found: C, 64.87; H, 6.58; N, 16.23.
Synthesis of 2-Chloro-5-(dimethylamino)indole-3-carbaldehyde
(2d). Compound 1p (2 mmol) was refluxed for 15 min with POCl3
(22 mmol). The mixture was cooled, poured into ice-water, and
basified with 30% NH4OH.32 The aldehyde 2d was collected by
filtration with a yield of 30%: mp 180-190 °C dec. IR: 3125,
1634, 830, 787. 1H NMR: 2.92 (6H, s, 2CH3), 6.87 (1H, dd, ind-6,
J = 8.7, J = 2.4), 7.28 (1H, d, ind-7, J = 8.7), 7.39 (1H, d, ind-4,
J = 2.4), 9.95 (1H, s, CHO), 12.72 (1H, broad, NH). Anal. Calcd
for C11H11ClN2O (MW 222.67): C, 59.33; H, 4.98; N, 12.58.
Found: C, 59.29; H, 5.01; N, 12.60.
Synthesis of 2-Chloro-1H-benzo[g]indole-3-carbaldehyde (2e)
and 3-Formyl-5-methoxy-6-methyl-1H-indole-2-carboxylic Acid
(2j). The Vilsmeier reagent was prepared at 0-5 °C by dropping
POCl3 (54 mmol) into a stirred solution of DMF (65 mmol) in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5573
CHCl3 (5 mL). The 1,3-dihydro-2H-benzo[g]indol-2-one, pre-
pared as reported in the literature,33 or 5-methoxy-6-methyl-
indole-2-carboxylic acid34 (5 mmol) was suspended in CHCl3
(20 mL) and treated with the Vislmeier reagent. The reaction
mixture was kept under reflux for 10-15 h, according to a TLC
test. Chloroform was removed under reduced pressure, and the
resulting oil was poured into ice. The crude aldehyde thus
obtained was collected by filtration with a yield of 70%.
(1H, s, NH-ox), 12.60 (1H, broad, NH-ind). Anal. (C19H15-
Cl2N3O) C, H, N.
Data for 20. IR: 3135, 1683, 1608, 1199, 810. 1H NMR: 2.72
(6H, s, N(CH3)2), 3.43 (3H, s, OCH3), 6.25 (1H, d, ind/ox-4, J =
2.2), 6.39 (1H, s, ind/ox), 6.77 (2H, s, ind/ox), 6.89 (1H, dd,
ind/ox-6, J = 9, J = 2.2), 7.31 (1H, d, ind/ox-7, J = 9), 7.65 (1H,
s, CH), 10.36 (1H, s, NH-ox), 12.70 (1H, broad, NH-ind). Anal.
(C20H18ClN3O2) C, H, N.
Data for 27. IR: 3408, 3172, 1721, 1674, 1086. 1H NMR: 2.11
(3H, s, CH3), 3.17 (3H, s, OCH3), 6.49 (1H, s, ox), 6.68 (1H, s,
ox), 7.31 (1H, d, bzind, J = 8.1), 7.48 (2H, t, bzind, J = 8.1), 7.61
(2H, m, bzind þ CH), 7.96 (1H, d, bzind, J = 8.1), 8.42 (1H,
d, bzind, J = 8.1), 10.35 (1H, s, NH-ox), 13.46 (1H, broad,
NH-bzind). Anal. (C23H17ClN2O2) C, H, N.
Data for 2e. Crystallized from ethanol: mp 220-225 °C. IR:
3160, 1649, 856, 805. H NMR: 7.52 (1H, t, ar, J = 7.9), 7.64
1
(1H, t, ar, J = 7.9), 7.73 (1H, d, ar, J = 7.9), 8.00 (1H, d, ar, J =
7.9), 8.16 (1H, d, ar, J = 7.9), 8.38 (1H, d, ar, J = 7.9), 10.08
(1H, s, CHO), 13.77 (1H, s, NH). Anal. Calcd for C13H8ClNO
(MW 229.66): C, 67.99; H, 3.51; N, 6.10. Found: C, 68.02; H,
3.49; N, 6.13.
1
Data for 28. IR: 3200, 1666, 1583, 805, 671. H NMR: 3.19
(3H, s, CH3), 6.39 (1H, d, ox-4, J = 2.3), 6.68 (1H, dd, ox-6, J =
8.4, J = 2.3), 6.84 (1H, d, ox-7, J = 8.4), 7.28 (1H, d, bzind, J =
8.1), 7.50 (1H, t, bzind, J = 8.1), 7.60 (1H, d, bzind, J = 8.1),
7.62 (1H, t, bzind, J = 8.1), 7.70 (1H, s, CH), 7.99 (1H, d, bzind,
J = 8.1), 8.39 (1H, d, bzind, J = 8.1), 8.82 (1H, s, OH), 13.45
(1H, s, NH-bzind). Anal. (C22H15ClN2O2) C, H, N.
Data for 2j. Crystallized from methanol: mp 260 °C dec. IR:
3247, 1672, 1643, 1102, 1025. 1H NMR: 2.26 (3H, s, CH3), 3.83
(3H, s, OCH3), 7.30 (1H, s, ind-4/7), 7.64 (1H, s, ind-4/7), 10.60
(1H, s, CHO), 12.52 (1H, s, NH), 13.20 (1H, broad, COOH).
Anal. Calcd for C12H11NO4 (MW 233.22): C, 61.80; H, 4.75; N,
6.01. Found: C, 61.82; H, 4.73; N, 5.98.
Data for 30. IR: 3364, 3131, 1684, 1603, 1100. 1H NMR: 2.27
(3H, s, CH3), 3.93 (3H, s, OCH3), 6.83 (1H, d, ox-4/7, J = 7.5),
6.98 (1H, t, ox-5/6, J = 7.5), 7.12 (1H, t, ox-5/6, J = 7.5), 7.27
(1H, s, ind), 7.67 (1H, s, ind), 7.92 (1H, d, ox-4/7), 8.14 (1H, s,
CH), 9.33 (1H, s, ind-2), 10.47 (1H, s, NH-ox), 11.78 (1H, broad,
NH-ind). Anal. (C19H16N2O2) C, H, N.
General Procedure for the Synthesis of Compounds 4, 6, 11,
24-26, and 37. The appropriate oxindole 1 (5 mmol) was
dissolved in acetic acid (25 mL) and treated with an equivalent
of indole-3-carbaldehyde 2 or 5-methoxyindole-2,3-dione 3 (for
compound 37) and 37% hydrochloric acid (1 mL). The reaction
mixture was refluxed for 24 h, and after it was cooled, the
precipitate was collected by filtration with a yield of 35-40% for
compounds 4 and 24-26 and 70-80% for compounds 6, 11,
and 37.
General Procedure for the Synthesis of Compounds 5,
7-10, 12-23, 27-34, and 36. The appropriate 2-indolinone 1
(10 mmol) was dissolved in methanol (100 mL) and treated with
the equivalent of the appropriate indole-3-carbaldehyde 2 and
piperidine (1 mL). The reaction mixture was refluxed for 3-5 h
(according to a TLC test), and the precipitate formed on cooling
was collected by filtration with a yield of 20% for compounds 5,
7, 28, and 33, 45-50% for compounds 13, 15, 16, 23, 27, and 31,
and 75-85% for compounds 8-10, 12, 14, 29, 30, 32, 34, and 36.
Compounds 17-22 were purified by column chromato-
graphy with a yield of 20%.
1
Data for 7. IR: 3355, 1667, 1593, 1207, 917. H NMR: 2.25
(3H, s, CH3), 3.56 (3H, s, OCH3), 6.47 (1H, s, ind-4), 6.76 (1H, d,
ox-4, J = 8.2), 6.89 (1H, d, ox-7, J = 1.8), 6.95 (1H, dd, ox-5,
J = 8.2, J = 1.8), 7.23 (1H, s, ind-7), 7.69 (1H, s, CH), 10.71 (1H,
s, NH-ox), 12.60 (1H, broad, NH-ind). Anal. (C19H14Cl2N2O2)
C, H, N.
Data for 4. IR: 3124, 1672, 1604, 1108, 1021. 1H NMR: 2.28
(3H, s, CH3), 3.62 (3H, s, OCH3), 6.48 (1H, s, ind-4), 6.86 (1H, d,
ox-7, J = 8.2), 6.90 (1H, d, ox-4, J = 1.5), 7.27 (1H, s, ind-7),
7.36 (1H, dd, ox-6, J = 8.2, J = 1.5), 7.73 (1H, s, CH), 10.74
(1H, s, NH-ox), 12.77 (1H, broad, NH-ind). Anal. (C19H14-
BrClN2O2) C, H, N.
1
Data for 9. IR: 3150, 1692, 1594, 1298, 922. H NMR: 2.32
(3H, s, CH3), 3.62 (3H, s, N-CH3), 3.86 (3H, s, OCH3), 6.48 (1H,
s, ind-4), 6.86 (2H, m, ind þ ox), 7.35 (1H, d, ox, J = 8.4), 7.52
(1H, s, ox), 7.74 (1H, s, CH), 10.73 (1H, s, NH-ox). Anal.
(C20H16BrClN2O2) C, H, N.
Data for 24. IR: 3426, 1698, 1623, 1198, 809. 1H NMR: 6.79
(1H, d, ox-4, J = 2.3), 6.89 (1H, d, ox-7, J = 8.3), 7.22 (1H, dd,
ox-6, J = 8.3, J = 2.3), 7.28 (1H, d, bzind, J = 8.2), 7.51 (2H, t,
bzind, J = 8.2), 7.63 (1H, d, bzind, J = 8.2), 7.77 (1H, s, CH),
8.00 (1H, d, bzind, J = 8.2), 8.43 (1H, d, bzind, J = 8.2), 10.76
(1H, s, NH-ox), 13.55 (1H, broad, NH-bzind). Anal. (C21H12-
Cl2N2O) C, H, N.
Data for 12. IR: 3140, 1714, 1611, 1040, 799. 1H NMR: 2.29
(3H, s, CH3), 3.56 (3H, s, N-CH3), 3.82 (3H, s, OCH3), 6.50 (1H,
s, ind-4), 6.74 (1H, d, ox-4, J = 7.5), 6.89 (1H, d, ox-7, J = 1.2),
6.92 (1H, dd, ox-5, J = 7.5, J = 1.2), 7.48 (1H, s, ind-7), 7.68 (1H,
s, CH), 10.74 (1H, s, NH-ox). Anal. (C20H16Cl2N2O2) C, H, N.
1
Data for 13. IR: 1709, 1605, 1040, 912, 851. H NMR: 2.30
1
Data for 25. IR: 3200, 1607, 1170, 815, 682. H NMR: 6.57
(3H, s, CH3), 3.34 (3H, s, N-CH3), 3.53 (3H, s, N-CH3), 3.84
(3H, s, OCH3), 6.52 (1H, s, ind-4), 6.83 (1H, d, ox-4/7, J = 7.2),
6.94 (1H, t, ox-5/6, J = 7.2), 7.05 (1H, d, ox-4/7, J = 7.2), 7.29
(1H, t, ox-5/6, J = 7.2), 7.49 (1H, s, ind-7), 7.73 (1H, s, CH).
Anal. (C21H19ClN2O2) C, H, N.
(1H, dd, ox, J = 9, J = 2.7), 6.88 (1H, dd, ox, J = 8, J = 4.5),
7.04 (1H, td, ox, J = 9, J = 2.7), 7.30 (1H, d, bzind, J = 8.3),
7.53 (2H, m, bzind), 7.63 (1H, d, bzind, J = 8.3), 7.77 (1H, s,
CH), 8.00 (1H, d, bzind, J = 8.3), 8.44 (1H, d, bzind, J = 8.3),
10.65 (1H, s, NH-ox), 13.56 (1H, broad, NH-bzind). Anal.
(C21H12ClFN2O) C, H, N.
Biology. a. Cell-Based Screening Assay. The NCI screening is
a two-stage process,35 beginning with the evaluation of all
compounds against the 60 cell lines at a single concentration
of 10-5 M. Compounds exhibiting significant growth inhibition
were evaluated against the 60-cell panel at five concentration
levels by the NCI according to standard procedures (http://
dtp.nci.nih.gov/branches/btb/ivclsp.html). In both cases the
exposure time was 48 h.
b. Acute Toxicity. To a single female athymic nude mouse
(APA) was given a single injection of 400 mg/kg compounds 16,
28, and 30. A second mouse received a dose of 200 mg/kg
compounds 16, 28, and 30, and a third mouse received a dose
of 100 mg/kg compounds 16, 28, and 30. The mice were allowed
ad libitum feed and water, and they were observed for 2 weeks.
Data for 14. IR: 3155, 1667, 1592, 1119, 912. 1H NMR: 2.31
(3H, s, CH3), 3.19 (3H, s, N-CH3), 3.60 (3H, s, N-CH3), 3.84
(3H, s, OCH3), 6.40 (1H, d, ox-4, J = 2.3), 6.57 (1H, s, ind-4),
6.68 (1H, dd, ox-6, J = 8.2, J = 2.3), 6.83 (1H, d, ox-7, J = 8.2),
7.48 (1H, s, ind-7), 7.68 (1H, s, CH), 8.94 (1H, s, OH). Anal.
(C21H19ClN2O3) C, H, N.
1
Data for 16. IR: 1686, 1602, 1052, 897, 739. H NMR: 2.17
(3H, s, CH3), 3.20 (3H, s, N-CH3), 3.25 (3H, s, OCH3), 6.43 (1H,
s, ox), 6.88 (1H, s, ox), 7.22 (3H, m, ind), 7.46 (1H, d, ind, J =
7.8), 7.64 (1H, s, CH), 12.78 (1H, broad, NH-ind). Anal.
(C20H17ClN2O2) C, H, N.
Data for 18. IR: 3165, 1692, 1600, 1215, 804. 1H NMR: 2.74
(6H, s, N(CH3)2), 6.20 (1H, d, ox/ind-4, J = 2.2), 6.70 (1H, d,
ox/ind-4, J = 2.2), 6.89 (1H, d, ox/ind-7, J = 8.4), 6.92 (1H,
dd, ox/ind-6, J = 8.4, J = 2.2), 7.21 (1H, dd, ox/ind-6, J = 8.4,
J = 2.2), 7.34 (1H, d, ox/ind-7, J = 8.4), 7.74 (1H, s, CH), 10.69

5574 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Andreani et al.
The doses of compound 16 were administered ip in DMSO. After
14 days the mice treated with 400 and 200 mg/kg were sacrificed,
since they had lost more than 20% of their body weight. There-
fore, the MTD was 100 mg/kg. The doses of compound 28 and 30
were administered ip in DMSO and DMSO in saline/Tween 80,
respectively. After 14 days no mice showed weight loss or other
signs of significant toxicity; therefore, MTD was 400 mg/kg.
c. Hollow Fiber Assay. Twelve tumor cell lines (NCI-H23,
NCI-H522, MDA-MB-231, MDA-MB-435, SW-620, COLO
205, LOX, UACC-62, OVCAR-3, OVCAR-5, U251, and SF-
295) were cultivated in RPMI-1640 containing 10% FBS and
2 mM glutamine. On the day preceding hollow fiber prepara-
tion, the cells were given a supplementation of fresh medium
to maintain log phase growth. For fiber preparation, the cells
were harvested by standard trypsinization technique and resus-
pended at the desired cell density. The cell suspension was
flushed into 1 mm (internal diameter) polyvinylidene fluoride
hollow fibers with a molecular weight exclusion of 500 000 Da.
The hollow fibers were heat-sealed at 2 cm intervals, and the
samples generated from these seals were placed into tissue
culture medium and incubated at 37 °C in 5% CO2 for 24-
48 h prior to implantation. A total of three different tumor
lines were prepared for each experiment so that each mouse
received three intraperitoneal implants (one of each tumor line)
and three subcutaneous implants (one of each tumor line). On
the day of implantation, samples of each tumor cell line pre-
paration were quantitated for viable cell mass by a stable end
point MTT assay so that the time zero cell mass is known. Mice
were treated with compound 30 starting on day 4 after fiber
implantation and continuing daily for 4 days. The compound
was administered by ip injection at two dose levels: 150 and
100.5 mg/kg. The vehicle was 10% DMSO in saline/0.05%
Tween 80. The fibers were collected from the mice on the day
following the fourth treatment and subjected to the stable end
point MTT assay. The optical density of each sample was
determined spectrophotometrically at 540 nm, and the mean
of each treatment group was calculated. The percent net growth
for each cell line in each treatment group was calculated and
compared to the percent net growth in the vehicle treated
controls. A 50% or greater reduction in percent growth of the
treated samples compared to the vehicle control samples was
considered a positive result. Each positive result was given a
score of 2, and all of the scores were totaled. The maximum
possible score for the described experiment is 96 (12 cell lines ꢀ
2 sites ꢀ 2 dose levels ꢀ 2 [score]).
two cycles of freeze-thawing. The cell proteins in the homo-
genate were extracted, separated by SDS-PAGE, and detected
as described.36 Primary antibodies against total and phosphory-
lated ERK 1/2 and Akt were from Cell Signaling Technology,
Danvers, MA; anti-p53, anti-p21, and anti-β-actin antibodies
were from Santa Cruz Biotechnology, Santa Cruz, CA. Quanti-
tative assay of immunoblotting was obtained by densitometry
with a Fluor-S Max Multilmager instrument (Bio-Rad).
g. Immunofluorescence Confocal Microscopy. MCF-7 cells
were seeded at 1ꢀ104 cells/cm2 on glass coverslips. At the end
of the incubation, cells were washed twice and fixed with 3%
p-formaldehyde, washed with 0.1 M glycine in PBS, and per-
meabilized in 70% ice-cold ethanol. Bax staining was obtained
by incubation for 1 h at room temperature with a mouse mono-
clonal anti-Bax antibody (BioSource). Cells were then washed,
incubated with FITC-coniugated antimouse IgG antibody
(Sigma) for 1 h, and finally stained with propidium iodide. All
preparations were embedded in Mowiol and analyzed using
a Nikon C1s confocal laser-scanning microscope equipped with
Nikon eclipse TE300. Multiple images were acquired by using
sequential laser excitations at 488 and 568 nm to reduce spectral
bleed-through artifacts.
h. Caspase Activity. The activity of caspase enzymes hydro-
lyzing the peptide sequence DEVD, indicated as DEVDase
activity, was measured in cell extracts by a fluorometric assay.36
i. Data Analysis. All the experiments on the effects of studied
compounds on MCF-7 cells were performed independently
three or four times with comparable results. The results are
expressed as mean values ( SEM of the data obtained in the
indicated numbers of independent experiments. When statistical
analysis was applicable, data were compared by the Student
t test. Differences were considered significant for P < 0.05.
Acknowledgment. This work was supported by a grant
from the University of Bologna, Italy (RFO). We are grateful
to the National Cancer Institute (Bethesda, MD) for the
anticancer tests.
Supporting Information Available: Additional IR and 1H
NMR spectra, elemental analysis results (Table S1), NSC num-
bers (Table S2), antitumor activity expressed as the negative log
of the molar concentration at three assay end points (Table S3),
and mean graph of compound 30 where all the cell lines employed
are reported (Figure S1). This material is available free of charge
via the Internet at http://pubs.acs.org.
d. Cell Culture and Treatment. The human breast adenocar-
cinoma cell line MCF-7 was maintained in RPMI 1640 medium
(Euroclone, Italy), supplemented with 10% FBS (Euroclone,
Italy) and 2 mM L-glutamine (Sigma) at 37 °C and 5% CO2.
Working solutions of compounds under test were prepared in
DMSO at 10 mmol/L immediately before use. Cells were plated
at 2 ꢀ 104 cells/cm2 in plastic culture wells, and after 24 h the
derivatives were added to the medium to obtain the indicated
concentrations. In control cells, DMSO was added to the culture
medium. At the end of the treatment, cells were harvested using
0.11% trypsin (Sigma) and 0.02% EDTA (Sigma) and counted.
Cell viability was determined by trypan blue exclusion.
e. Cell Cycle Analysis. MCF-7 control and treated cells were
detached with 0.11% trypsin (Sigma-Aldrich, St. Louis, MO)
and 0.02% EDTA, washed twice in PBS, and centrifuged. The
pellet was suspended in 0.01% nonidet P-40 (Sigma), 10 μg/mL
RNase, and 0.1% sodium citrate (Sigma), and 50 μg/mL
propidium iodide (PI) (Sigma) for 30 min at room temperature
in the dark. Propidium iodide fluorescence was analyzed using a
Bryte flow cytometer (Biorad), and cell cycle analysis was
performed by means of a Coulter Epics XL MCL cytometer.
Cell cycle analysis was performed using Modfit 5.0 software.
f. Western Blotting. The cells were collected in 5 mM dithio-
threitol, 2 mM EDTA, 0.1% CHAPS, 0.1% Triton X-100, and
protease inhibitors in 20 mM HEPES, pH 7.5, and subjected to
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