Substituted 3-(5-imidazo[2,1- b ]thiazolylmethylene)-2-indolinones and analogues: Synthesis, cytotoxic activity, and study of the mechanism of action(1)

Article abstract of DOI:10.1021/jm2012694

The synthesis of substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2- indolinones and analogues is reported. Their cytotoxic activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. The action of selected compounds was examined for potential inhibition of tubulin assembly in comparison with the potent colchicine site agent combretastatin A-4. The most potent compounds also strongly and selectively inhibited the phosphorylation of the oncoprotein kinase Akt in cancer cells. The effect of the most interesting compounds was examined on the growth of HT-29 colon cancer cells. These compounds caused the cells to arrest in the G2/M phase of the cell cycle, as would be expected for inhibitors of tubulin assembly.

Full text of DOI:10.1021/jm2012694

Article
pubs.acs.org/jmc
Substituted 3-(5-Imidazo[2,1-b]thiazolylmethylene)-2-indolinones
and Analogues: Synthesis, Cytotoxic Activity, and Study of the
Mechanism of Action1
Aldo Andreani,*,† Massimiliano Granaiola,† Alessandra Locatelli,† Rita Morigi,† Mirella Rambaldi,†
Lucilla Varoli,† Natalia Calonghi,‡ Concettina Cappadone,‡ Giovanna Farruggia,‡ Claudio Stefanelli,‡
Lanfranco Masotti,‡ Tam L. Nguyen,§ Ernest Hamel,⊥ and Robert H. Shoemaker⊥
†Dipartimento di Scienze Farmaceutiche, Universita
‡Dipartimento di Biochimica “G. Moruzzi”, Universita
̀
di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
di Bologna, Via Irnerio 48, 40126 Bologna, Italy
̀
§Target Structure-Based Drug Discovery Group, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick,
Maryland 21702, United States
⊥Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, Frederick, Maryland 21702, United States
S
* Supporting Information
ABSTRACT: The synthesis of substituted 3-(5-imidazo[2,1-b]-
thiazolylmethylene)-2-indolinones and analogues is reported. Their
cytotoxic activity was evaluated according to protocols available at
the National Cancer Institute (NCI), Bethesda, MD. The action
of selected compounds was examined for potential inhibition of
tubulin assembly in comparison with the potent colchicine site
agent combretastatin A-4. The most potent compounds also
strongly and selectively inhibited the phosphorylation of the
oncoprotein kinase Akt in cancer cells. The effect of the most
interesting compounds was examined on the growth of HT-29
colon cancer cells. These compounds caused the cells to arrest in the G2/M phase of the cell cycle, as would be expected for
inhibitors of tubulin assembly.
well-known antitumor agents such as combretastatin A-4, col-
chicine, and podophyllotoxin.
INTRODUCTION
■
In the previous paper of this series,2 we described the synthesis
of substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indoli-
nones and 3-(5-imidazo[2,1-b]thiadiazolylmethylene)-2-indoli-
nones. The best results in terms of inhibition of cell prolifer-
ation were obtained with the derivatives bearing methyl groups
at positions 2 and 6 of the imidazothiazole or imidazothiadia-
zole system. Therefore, we planned the synthesis of new deriva-
tives (Scheme 1) bearing this scaffold condensed with indolinones,
benzoindolinone, or coumaranone (3−10).
CHEMISTRY
■
Compounds 3−42 (see Table 1) were prepared by means of a
single-step Knoevenagel reaction between 1 and 2 in methanol/
piperidine (triethylamine was used in place of piperidine for
compounds 6, 10, 15, and 20 to improve the yield). The struc-
tures of the final compounds were confirmed by means of IR and
1H NMR spectra. Most compounds were obtained as almost
pure geometrical isomers, which, according to the usual NOE
experiments described previously,3,4 were assigned the E con-
figuration. Compounds 10, 15, 20, 29, 30, and 37−39 were
obtained as E/Z mixtures and used as such in the biological
assays. The E/Z ratio in solution is time dependent and tends to
equilibrate at 50/50. In a previous paper, we described the
separation of the two isomers by fractional crystallization,2 but
no significant difference in pharmacological behavior was noted.
The indolinones 2b, 2k, 2l, and 2-coumaranone (2d) are
commercially available, whereas the other starting compounds
We also demonstrated2 that the methyl group at the 6 posi-
tion of the imidazothiazole or imidazothiadiazole system could
be replaced without loss of activity by p-methylphenyl and
p-chlorophenyl groups. To confirm the effect of these substi-
tuents on biological activity, a new series of derivatives modified
only in the indolinone portion was prepared (11−20).
From a further analysis of the previous results, we decided
that it would be interesting to study the effects of other sub-
stituents at the 6 position of the imidazothiazole system, such
as methoxy, pyridinyl, thienyl, and 4-nitrophenyl groups (26−
42). Moreover, the activity of 2,5-dimethoxyphenyl derivatives3
prompted us to prepare analogues bearing a trimethoxyphenyl
group (21−25), because it is present, free or hindered, in
Received: September 22, 2011
Published: January 30, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Article
a
Scheme 1.
a
CBR = condensed benzene ring.
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Journal of Medicinal Chemistry
Article
Table 1. Compounds 3−42
compd
formula
MW
mp, °C
compd
formula
MW
mp, °C
3
C18H18N4OS
338.42
329.80
345.41
296.34
314.34
339.41
346.40
297.33
401.48
385.48
415.50
441.93
392.85
422.88
410.86
435.93
492.93
393.84
463.50
542.40
223−226
>310
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
C25H23N3O5S
C24H20BrN3O4S
C25H22BrN3O5S
C21H14N4O3S
C19H13N3OS2
C19H12N4OS
477.53
526.40
556.43
402.42
363.45
344.39
378.83
378.83
374.41
388.44
297.33
331.77
331.77
331.77
315.33
327.35
341.38
311.35
447.50
298.31
220−223
270−273 dec.
196−200
>310
4
C16H12ClN3OS
C20H15N3OS
5
267−271
175−177
>310
6
C16H12N2O2S
C15H11FN4OS
C17H17N5OS
7
>310
8
264−265
>300
>310
9
C19H14N4OS
C19H11ClN4OS
C19H11ClN4OS
C20H14N4O2S
C21H16N4O2S
C15H11N3O2S
C15H10ClN3O2S
C15H10ClN3O2S
C15H10ClN3O2S
C15H10FN3O2S
C16H13N3O3S
C17H15N3O3S
C16H13N3O2S
C24H21N3O4S
C15H10N2O3S
>310
10
11
12
13
14
15
16
17
18
19
20
21
22
C15H11N3O2S
C23H19N3O2S
C23H19N3OS
176−178
>310
>310
286−288
301−303
214−216
267−269
263−265
267−269
260−262
216−218
188−190
187−189
172−173
180−182
227−230
200−204
>310
C24H21N3O2S
C25H16ClN3OS
C21H13ClN2O2S
C21H15ClN4O2S
C20H12ClFN4OS
C22H18ClN5OS
C24H17ClN4O4S
C20H12ClN3O2S
C24H21N3O5S
C24H20BrN3O5S
248−250
301−303
>310
>310
283−285
220−223
250−255
193−195
were prepared according to the literature,5−23 except indoli-
none 2o and aldehydes 1g, 1h, 1i, whose synthesis is reported
in the Supporting Information. The aforementioned aldehydes
were obtained by means of the Vilsmeier reaction on the
corresponding imidazo[2,1-b]thiazole, prepared in turn from
2-aminothiazole or 2-amino-5-methylthiazole and 2-bromo-1-
(3,4,5-trimethoxyphenyl)ethanone or 2-bromo-1-(2-bromo-
3,4,5-trimethoxyphenyl)ethanone.
thiazoles and imidazothiadiazoles confirmed that these moieties
can generate active derivatives. However, substitution of indo-
linone with coumaranone (6, 10) and the introduction of a
dimethylamino group in the indolinone system (3, 8) led to
compounds that had weak activity. The most active compounds
in this group bear a benzoindole system (5 and 9, with mean
GI50 values of 0.21 and 0.50 μM, respectively).
Considering the 4-chlorophenyl and 4-methylphenyl substituents
at the 6 position of the imidazole ring (11−20), it is clear that the
latter leads to increased activity (11−13). From the data obtained
with compounds 11−20, it is also clear that replacement of
indolinone with coumaranone (15, 20) led to inactive compounds.
Considering other substituents on the imidazole ring (21−42),
the 4-nitrophenyl and the 2-thienyl groups were detrimental for
antiproliferative activity, while the most active compounds had
either a 2-pyridyl (28−32) or a methoxy (33−42) group, with
mean GI50 values of 0.55 μM for the former and 2.56 μM for
the latter. In particular, compound 31 was the most active agent
in the entire series (GI50, 0.04 μM), although it had a relatively
high LC50 (32 μM).
BIOLOGY
■
1. Cell-Based Assays. Compounds 3−42 were initially
tested at a single high concentration (10−5 M) in the full NCI
60 cell panel (NCI 60 Cell One-Dose Screen). This panel is
organized into subpanels representing leukemia, melanoma,
and cancers of lung, colon, kidney, ovary, breast, prostate, and
central nervous system. Only compounds which satisfy pre-
determined threshold inhibition criteria in a minimum number
of cell lines will progress to the full 5-log concentration assay.
The threshold inhibition criteria for progression to the five-
concentration screen were selected to efficiently capture com-
pounds with antiproliferative activity based on the analysis of
historical DTP screening data. The result is expressed as the
percent growth of treated cells relative to the control following
a 48 h incubation. Twenty-two of forty evaluated compounds
were active in the preliminary test and progressed to the five-
concentration assay, ranging from 10−8 to 10−4 M. Table 2 reports
the results obtained (vincristine is reported for comparison
purposes), at three assay end points: 50% growth inhibition (GI50),
total cytostatic effect (TGI = total growth inhibition) and cytotoxic
effect (LC50, loss of 50% of the initial cell protein). For some com-
pounds, the 5-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.
2. Tubulin. A COMPARE analysis24 of a number of the
compounds indicated that tubulin was the likely target of the
most active agents. We therefore examined most of the com-
pounds with mean GI50 values less than 1 μM for potential
inhibition of tubulin assembly (Table 3) in comparison with the
potent colchicine site agent combretastatin A-4.25 In the assay
we used,26 the IC50 is defined as the concentration of compound
that inhibits by 50% the extent of assembly of 10 μM tubulin after a
20 min incubation at 30 °C. All compounds inhibited the reaction,
although an IC50 could not be obtained with compound 29. None
of the compounds was as active as combretastatin A-4, but most of
them had substoichiometric (i.e., <10 μM) IC50 values.
The compounds were also evaluated for their effects on
the binding of [3H]colchicine to tubulin26 (Table 3). As in the
assembly assay, none of the compounds was as active as com-
bretastatin A-4, but, except for the relatively inactive 29, the
other compounds had similar inhibitory effects on colchicine
binding, with inhibition ranging from 36% to 62%. With these
agents, there was relatively little correlation between the two
The tested compounds showed a mean GI50 range between
0.04 and 93 μM, and compounds 5, 29-31, 33−36, 38, and 39
were submitted to the Biological Evaluation Committee of the
NCI for possible future development.
The first group of compounds prepared for further study
of the biological activity of 2,6-dimethyl-substituted imidazo-
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Table 2. Nine Subpanels at Five Concentrations: Growth Inhibition and Cytostatic and Cytotoxic Activity (μM) of Selected
Compounds
a
b
compd
modes
leukemia
NSCLC
colon
CNS
melanoma
ovarian
renal
prostate
breast
MG-MID
4
5
GI50
TGI
GI50
TGI
LC50
GI50
TGI
GI50
TGI
LC50
GI50
TGI
GI50
TGI
GI50
TGI
GI50
GI50
TGI
GI50
TGI
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
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
GI50
TGI
1.07
37.15
0.21
53.70
−
2.29
−
0.98
66.07
0.11
58.88
−
2.40
25.70
0.17
1.58
57.54
0.14
50.12
−
7.41
−
4.79
70.79
0.23
2.29
−
0.21
16.22
−
1.20
15.85
0.18
2.00
51.29
0.21
c
0.31
28.18
93.33
19.50
−
0.87
15.49
74.13
18.62
72.44
23.99
89.13
14.79
85.11
−
0.85
14.13
74.13
33.88
−
0.91
11.75
56.23
10.47
63.10
32.36
89.13
56.23
−
5.37
12.02
70.79
26.92
91.20
0.83
5.62
19.95
81.28
15.14
91.20
0.50
74.13
20.89
87.10
0.39
50.12
6.31
7
9
2.34
4.17
26.92
−
−
−
−
−
57.54
0.30
c
0.17
3.31
66.07
52.48
83.18
7.08
63.10
4.37
56.23
−
0.35
6.61
48.98
60.26
−
0.68
16.98
70.79
33.88
−
17.38
87.10
52.48
−
0.38
5.13
15.14
21.38
−
4.90
10.47
56.23
18.20
−
15.14
85.11
4.79
6.31
8.51
46.77
11.75
91.20
19.05
75.86
39.81
−
69.18
20.42
79.43
23.44
54.95
0.81
75.86
12.59
63.10
13.49
75.86
12.88
72.44
95.50
36.31
95.50
19.05
63.10
0.59
58.88
22.91
83.18
16.60
83.18
13.80
74.13
93.33
37.15
93.33
28.84
77.62
1.15
11
12
13
20.42
6.46
−
−
5.37
−
−
−
6.03
33.11
−
70.79
81.28
26.30
72.44
30.90
75.86
2.34
16
17
−
66.07
−
21.88
25.12
60.26
38.02
−
36.31
−
−
−
−
−
−
24
28
32.36
89.13
1.58
70.79
−
26.30
75.86
0.49
52.48
−
29.51
85.11
2.45
91.20
−
39.81
85.11
2.88
37.15
91.20
1.62
17.38
50.12
0.27
11.48
36.31
0.04
2.19
16.98
0.44
20.42
−
4.68
43.65
97.72
3.47
46.77
0.65
21.38
89.13
5.62
44.67
0.10
12.02
−
30.20
85.11
1.02
−
0.35
14.79
−
0.28
7.08
69.18
0.06
4.57
−
0.02
0.58
−
0.50
21.38
60.26
2.19
64.57
−
1.32
26.30
0.14
5.37
89.13
3.39
52.48
0.02
4.57
91.20
0.16
4.90
79.43
3.89
43.65
0.10
15.85
20.89
89.13
0.71
95.50
−
1.15
36.31
−
0.45
50.12
97.72
0.83
−
c
c
c
c
c
29
30
31
33
34
1.78
18.20
58.88
0.33
28.84
48.98
0.11
6.46
39.81
0.43
51.29
93.33
7.08
70.79
−
4.27
89.13
1.29
79.43
−
6.03
66.07
0.16
44.67
−
0.52
10.47
33.88
0.11
21.88
46.77
0.02
1.35
15.14
0.29
13.80
89.13
3.72
91.20
−
1.41
64.57
0.33
22.39
79.43
3.16
61.66
0.04
7.59
66.07
0.35
23.99
87.10
2.24
60.26
0.10
3.98
0.68
11.75
51.29
0.12
15.14
38.90
0.02
6.76
38.90
0.47
40.74
95.50
4.90
60.26
−
3.72
40.74
0.21
34.67
93.33
6.03
47.86
0.04
20.89
81.28
0.79
72.44
91.20
8.71
77.62
0.16
7.94
1.26
19.05
47.86
0.15
31.62
−
0.03
3.98
−
22.39
66.07
0.17
10.47
43.65
0.25
7.76
12.88
50.12
0.l8
42.66
0.21
6.03
15.85
42.66
0.07
12.59
46.77
0.04
14.13
43.65
0.04
38.02
0.03
2.57
4.68
8.32
3.39
34.67
0.25
28.84
0.54
46.77
0.28
31.62
0.39
0.55
29.51
−
7.94
38.90
−
7.08
13.18
91.20
3.24
22.91
85.11
4.90
50.12
6.31
13.18
32.36
87.10
3.31
48.98
89.13
3.31
−
−
50.12
93.33
2.45
57.54
95.50
2.82
c
c
35
3.98
81.28
0.39
69.18
−
12.02
93.33
0.05
40.74
−
40.74
0.35
58.88
0.62
74.13
0.54
54.95
0.44
36
16.22
75.86
6.46
48.98
85.11
6.76
58.88
−
2.88
31.62
89.13
5.01
c
c
37
42.66
0.04
47.86
0.14
51.29
0.17
52.48
0.07
38
15.49
−
0.38
31.62
−
0.71
34.67
−
0.49
19.05
93.33
0.54
c
c
39
1.02
58.88
−
7.94
95.50
0.25
15.85
0.63
31.62
−
6.46
72.44
0.32
19.95
0.55
18.62
53.70
−
−
21.38
48.98
34.67
95.50
6.31
−
−
−
2.19
40
10.96
77.62
0.13
15.85
85.11
0.32
−
0.13
6.31
60.26
0.32
72.44
0.20
d
vincristine sulfate
6.31
19.95
7.94
10.00
a
b
Highest concn tested = 10−4 M. The GI50 and TGI values are reported only when <100 μM. The compound exposure time was 48 h. Mean Graph
c
d
MIDpoint: average value for all cell lines tested; i.e., mean GI50. Mean of two separate experiments. Highest concn tested = 10−3 M.
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Table 3. Inhibitory Effects of Selected Compounds on
Tubulin Assembly and Colchicine Binding to Tubulin
inhibition of
tubulin assembly,
IC50 (μM) SD
inhibition of colchicine
binding, % inhibition SD,
5 μM inhibitor
compd
combretastatin A-4
5 (NSC 743420)
9 (NSC 742500)
29 (NSC 748117)
30 (NSC 748119)
31 (NSC 748118)
33 (NSC 744489)
36 (NSC 748115)
38 (NSC 748113)
39 (NSC 748114)
1.1 0.1
5.3 0.3
99 0.06
49
45
13
48
36
48
53
62
45
2
4
3
4
5
3
4
2
5
16
1
>20
9.3 0.1
9.1
3.7 0.02
13
1
Figure 2. Binding model of the most active compound 38, with its
carbon atoms rendered in cyan. The carbon atoms of the binding site
amino acids are rendered in purple. Other colors as in Figure 1.
2
2.9 0.3
3.8 0.01
pocket, the imidazothiazole system is wedged against Tyr200,
with the sulfur atom potentially forming a weak hydrogen bond
to the hydroxyl group of Tyr200.
Compounds 5 and 9 are distinguishable from the others in
that they are characterized by a condensed benzene ring at
the R3,R4 positions and a methyl substituent at the R position.
The binding models showed that the condensed benzene ring
of 5 and 9 can be accommodated at the binding site and that
the condensed benzene ring forms stabilizing interactions with
the alipathic side chain of Lys350.
assays, except that compound 38 was the most active as both an
inhibitor of assembly and an inhibitor of colchicine binding.
Structural Basis for Tubulin Activity. The binding modes of
the compounds in Table 3 were modeled using the 3G2N
crystal structure of αβ-tubulin in complex with NSC 613863
as a template. These models revealed structural insights into
the in vitro activities of these compounds. The models show
that compounds where R is methyl or methoxy assume a
common binding mode relative to compounds where R is a
2-pyridyl group (Figure 1). Compounds that possess methyl
In contrast to compounds 5 and 9, the two most active com-
pounds, these being compounds 36 and 38, are characterized
by methoxy substituents at the R position. In the binding models,
these methoxy groups are favorably wedged against the side
chains of Leu240 and Leu253, and this provides more favorable
binding contacts than for the methyl groups of compounds
5 and 9. The binding models also indicate favorable binding
interactions of the R2-methoxy of 38 and the R3-Cl of 36
against the Lys350 side chain, and this is reflected in the similar
% inhibition of colchicine binding for 36 and 38 of 53% and
62%, respectively. In contrast, compound 33, which is unsubsti-
tuted at the R1−R4 positions and, accordingly, does not gain
favorable interactions with Lys350, is characterized by a weaker
inhibition of 48%. Additionally, the weaker inhibitory activity of
compound 39, which differs by only a N-methyl group from the
most active compound 38, may be explained by the loss of a
hydrogen bond to Ala315 due its offset binding mode.
3. Effect on Growth Inhibition of Cancer Cells. We
examined the effect of the very active compounds 38, 31, and 5
on the growth of HT-29 colon cancer cells. First, in order to
assess whether these compounds interfered with cell cycle pro-
gression, DNA profiles of cultured cells were examined by flow
cytometry. Figure 3A shows that compounds 38 and 31 induced
accumulation of HT-29 cells in the G2/M phase at 100 nM;
compound 5 produced a similar effect only at a higher concen-
tration, 500 nM.
Next, the expression levels of the p21 protein were evaluated
by immunofluorescence analysis, because this protein can induce
cell cycle arrest.27 Upon p21 staining with FITC-conjugated anti-
body, fluorescence was detected in entire cells by confocal micro-
scopy. Figure 3B shows a significant increase in p21 expression
after treatment with all examined compounds.
The cell cycle block was associated with a significant reduc-
tion in cell viability. Figure 4A shows that 38 and 31 triggered
cell death at 100 nM, while compound 5 elicited a cytotoxic
effect at 500 nM. This was similar to the effects observed with
G2/M accumulation. Moreover, propidium iodide (PI) staining
Figure 1. Two distinct orientations for the binding modes of the
compounds in β-tubulin. Carbon atoms of 5, 9, 33, 36, 38, and 39 are
colored cyan, and those of 30, 31, and 29 are purple. Sulfur, oxygen,
nitrogen, chlorine, and hydrogen atoms are colored yellow, red, blue,
green, and white, respectively.
and methoxy substituents include 5, 9, 33, 36, 38, and 39, and,
for these compounds, their binding poses exhibit a significant
overlap in their indolinone systems. The one exception is com-
pound 39, which is characterized by a N-methyl substitution
of its indolinone and accordingly, has a binding mode that is
slightly offset from the other compounds. The larger 2-pyridine
substituent at the R position of compounds 30, 31, and 29
results in alternative binding conformations for these com-
pounds. This may explain their weak activity against tubulin
relative to the other compounds, because they do not assume the
optimal binding poses represented by methyl- and methoxy-
substituted compounds.
As shown for compound 38 in Figure 2, the other active
compounds form a number of common stabilizing interactions
with β-tubulin. The first is a hydrogen bond between the indo-
lone oxygen atom and the backbone NH of Ala315. Addi-
tionally, the aryl portion of the indolone ring is packed against
the alipathic side chain of Lys350. At the interior of the binding
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and aggregates in mitochondria, producing a green fluores-
cence. Following ΔΨm collapse in apoptotic cells, DiOC6
no longer enters mitochondria, resulting in a decrease in green
fluorescence. Figure 5A shows that a 24 h treatment with 5
caused a marked disruption of ΔΨm. Because depolarization of
the mitochondrial membrane is associated with mitochondrial
production of reactive oxygen species (ROS), intracellular ROS
content was evaluated by using dihydroethidium (DHE).
DHE is a specific probe for superoxide species, because it
binds with superoxide to form the highly fluorescent derivative
2-hydroxyethidium. The treatment of colon cancer cells with
compound 5 caused a marked increase in superoxide formation,
as shown in Figure 5B. Finally, we studied the involvement of
the proapoptotic Bax protein by measuring its expression levels
and its translocation from cytosol to mitochondria in intact
cells. Bax was detected with a fluorescent FITC-conjugated
antibody, and the resulting fluorescence was observed in the
cells by confocal microscopy. Figure 5C shows that compound
5 caused a significant increase in the cellular content of Bax,
which was largely localized to mitochondria.
The activity of compounds 38, 31, and 5 on signal trans-
duction pathways was studied by examining effects on the acti-
vation of a panel of kinases involved in cell growth and survival.
In these studies, cervical carcinoma HeLa cells were treated
with 1 μM of the different agents for 3 h, and afterward kinase
activation was determined by detecting their specific phospho-
rylation by Western blotting (Figure 6). At this time point,
all cells were viable but committed to death. The most potent
compounds 38 and 31 strongly reduced Akt(Ser473) phos-
phorylation, whereas no significant effect was detectable on the
phosphorylation of the other oncoprotein mTOR(Ser2448),
master regulator of protein synthesis.30
Compounds 38 and 31 also slightly decreased phosphorylation/
activation of all three members of the mitogen-activated protein
kinase family ERK1/2(Thr202/Tyr204), JNK(Thr183/Tyr185),
and p38MAPK(Thr180/Tyr182) that, depending of the cir-
cumstances, can be associated either with cell growth or with
activation of apoptosis in cancer cells.31
Figure 3. Effects of 38, 31, and 5 on cell cycle distribution in HT-29
cells. (A) Cell cycle distribution was determined in cells treated with
the indicated concentration of compounds for 24 h. The graph depicts
the results obtained in one experiment representative of three. (B)
Effects of compounds on p21 cellular levels detected by immuno-
fluorescence confocal microscopy. Nuclei were stained with propidium
iodide (red fluorescence). p21 was stained with FITC antibody and
results in green fluorescence. The image is representative of three
experiments.
(Figure 3B) revealed chromatin condensation and nuclear frag-
mentation, suggesting the activation of apoptosis. However,
identification of the mode of cell death requires multiple assays.28
Therefore, to determine whether treatment with 38, 31, or 5
caused cell death by activation of the apoptotic program, the
activity of caspase proteases, a marker of apoptosis, was assayed.
In cells treated for 48 h with these compounds, the activity of
effector caspases was significantly increased (Figure 4B). The
exposure of phosphatidylserine from the inner to the outer side
of the plasma membrane is an early marker of apoptosis.28 To
further confirm the onset of apoptosis, we utilized the Annexin
V-FLUOS staining method. This technique measures the extent
of phosphatidylserine externalization. Annexin is a protein with
high affinity for phospholipids, and cells undergoing apoptosis are
marked from early stages with Annexin V-FLUOS, which binds to
externalized phosphatidylserine. On the other hand, PI enters cells
and stains DNA only if there is a permeabilized plasma membrane,
a later event in cell death. As shown in Figure 4C, after 24 h
of incubation, only 3.9% of control HT-29 cells were Annexin V
positive and PI negative (early apoptotic cells). In contrast, this
percentage rose to 27%, 23%, and 12.5% in cells incubated in the
presence of compounds 38, 31, or 5, respectively.
Similarly, the phosphorylation of AMPK(Thr172), a meta-
bolic stress-sensitive kinase often associated with cell survival,32
was only slightly inhibited by 38 and 31. In conclusion, at the
concentration used, compound 5 did not significantly influence
the signaling pathways examined, whereas 38 and 31 selectively
and strongly inhibited Akt activation.
CONCLUSION
■
We have described the synthesis of a new class of colchicine site
agents that inhibit tubulin polymerization. In the colon cancer
cell line HT-29, we studied compounds 5, 31, and 38 in detail.
These compounds caused the cells to arrest in the G2/M phase
of the cell cycle, as would be expected for inhibitors of tubulin
assembly. This cell cycle arrest was associated with activation of
apoptosis, as occurs with most inhibitors of tubulin assembly.
The apoptosis in the HT-29 cells was accompanied by caspase
activation and phosphatidylserine externalization. In these cells
there was disruption of the mitochondrial membrane potential,
increased ROS production, and Bax translocation into mito-
chondria. Interestingly, the most potent compounds 31 and 38
strongly inhibited the activation of the kinase Akt, an onco-
protein associated with cell survival and proliferation. Akt is
upregulated in several cancers and responsible for resistance to
cell death.30 Considering that the tubulin effects were relatively
modest, compared with combretastatin A-4, the cytotoxic effects
Next, the mechanisms involved in activation of apoptosis
were examined. In order to clarify whether the apoptotic path-
way triggered by the new derivatives was mediated by mito-
chondrial events,29 we investigated the effects of compound 5.
The mitochondrial membrane potential (ΔΨm) can be mea-
sured in intact cells by using the fluorescent probe 3,3′-dihexyl-
oxacarbocyanine iodide (DiOC6). This compound accumulates
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Figure 4. Induction of apoptosis in HT-29 cells after treatment with compounds 38, 31, or 5. (A) The cells were treated for 24 h with 100 or
500 nM compound, and then the percentage of viable cells was determined by the trypan blue exclusion assay. Data are means SEM of three
determinations. (B) The activity of caspase proteases acting on the peptide sequence Asp-Glu-Val-Asp (DEVD), indicated as DEVDase activity, was
measured in extracts obtained from cells treated for 48 h with the indicated compound (500 nM). Results are means SEM of four determinations.
(C) Cells treated for 24 h, as indicated (derivative concentration was 500 nM), were labeled with Annexin V-FLUOS and PI and analyzed by flow
cytometry. The exposure of phosphatidylserine on the outer side of the plasma membrane in PI-negative cells, marking early apoptotic cells, is
demonstrated by the cell population with high AnnexinV and low PI fluorescence (lower right section of each panel).
1 and piperidine (1 mL). The reaction mixture was refluxed for 3−5 h
(progress of the reaction followed by TLC), and the precipitate that
formed on cooling was collected by filtration with a yield of 20%
for compound 40 and 42, 40−50% for compounds 3, 7−9, 11−14,
16−19, 21−23, 27, 33−39, and 41 and 70−80% for compounds 4, 5,
24-26, 28, and 29−32.
For compounds 6, 10, 15, and 20 the yield was much lower (5%),
and an improvement (15−25%) was obtained by replacing piperidine
with triethylamine.
Compounds 6, 10, 15, and 37 were purified by column chromato-
graphy with petroleum ether/acetone as the eluent. All the crude prod-
ucts were crystallized from methanol except 6, 8, and 12 (acetone/
petroleum ether), 15, 20, and 38 (CHCl3/petroleum ether), and 42
(ethanol).
of compounds 38 and 31 may be substantially mediated by
effects on the AKT pathway.
EXPERIMENTAL SECTION
■
1. Chemistry. All the compounds prepared had a purity of at least
95%, as determined by combustion analysis. The melting points are
uncorrected. TLC was performed on Fluka plates (art. 99577) and
column chromatography on Silica gel 70−200 μ 60A (Fluorochem):
the eluent was a mixture of petroleum ether/acetone in various pro-
portions. The IR spectra were recorded in nujol on a Nicolet Avatar
320 E.S.P.; νmax is expressed in cm−1. The H NMR spectra were re-
1
corded on a Varian Gemini (300 MHz); the chemical shift (referenced
to solvent signal) is expressed in δ (ppm) and J in hertz (abbrevia-
tions: im = imidazole, th = thiazole, ind = indole, bzind = benzoindole,
coum = coumaranone, ph = phenyl, thio = thiophene, py = pyridine).
For spectra and elemental analyses that are not reported here, see
Supporting Information. The oxindoles 2b, 2k, 2l, 2-coumaranone
(2d), and 1-(3,4,5-trimethoxyphenyl)ethanone are commercially avail-
able. The following compounds were prepared according to the litera-
ture: 1a−f,5,8,10,14,15,18 1j−m,19,20,10,22 2a,6 2c,7 2e,9 2f−h,11−13 2i,16
2j,17 2m,21 and 2n.23
Data for 4. IR: 2707, 1706, 1607, 1156, 725. 1H NMR: 2.20 (3H, s,
CH3im), 2.39 (3H, d, CH3th, J = 1.2), 6.73 (1H, d, ind-4, J = 8.4), 6.89
(1H, d, ind-7, J = 2.1), 6.96 (1H, dd, ind-5, J = 8.4, J = 2.1), 7.52 (1H,
q, th, J = 1.2), 7.61 (1H, s, CH), 10.75 (1H, s, NH). Anal. Calcd for
C16H12ClN3OS (MW 329.80): C, 58.27; H, 3.67; N, 12.74. Found: C,
57.98; H, 3.72; N, 13.03.
1
Data for 5. IR: 2727, 1695, 1618, 818, 725. H NMR: 2.25 (3H, s,
CH3), 2.37 (3H, s, CH3), 6.99 (1H, d, ind-4/5, J = 8.7), 7.45 (1H, d,
ind-4/5, J = 8.7), 7.51 (2H, m, bzind), 7.55 (1H, s, th), 7.61 (1H, s,
CH), 7.86 (1H, m, bzind), 8.14 (1H, m, bzind), 11.38 (1H, s, NH).
General Procedure for the Synthesis of Compounds 3−42. The
appropriate compound 2 (10 mmol) was dissolved in methanol
(100 mL) and treated with the equivalent of the appropriate aldehyde
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bzind), 8.13 (1H, m, bzind), 11.39 (1H, s, NH). Anal. Calcd for
C19H14N4OS (MW 346.40): C, 65.88; H, 4.07; N, 16.17. Found: C,
66.02; H, 4.67; N, 15.96.
Data for 11. IR: 3151, 1696, 1614, 1189, 824. 1H NMR: 2.09 (3H, s,
CH3), 2.32 (3H, s, CH3), 2.39 (3H, d, CH3th, J = 1.4), 6.08 (1H, s,
ind), 6.60 (1H, s, ind), 7.14 (1H, q, th, J = 1.4), 7.25 (2H, d, ph, J =
8.0), 7.39 (1H, s, CH), 7.60 (2H, d, ph, J = 8.0), 8.77 (1H, s, OH),
10.30 (1H, s, NH). Anal. Calcd for C23H19N3O2S (MW 401.48): C,
68.81; H, 4.77; N, 10.47. Found: C, 69.01; H, 5.01; N, 10.73.
Data for 12. IR: 1695, 1242, 1149, 824, 765. 1H NMR: 2.30 (3H, s,
CH3ph), 2.38 (3H, d, CH3th, J = 1.2), 3.24 (3H, s, NCH3), 6.52 (1H,
d, ind-4, J = 7.5), 6.86 (1H, t, ind-5/6, J = 7.5), 7.06 (2H, d, ph, J =
7.6), 7.25 (1H, q, th, J = 1.2), 7.27 (3H, m, 2Hph+1Hind), 7.59 (2H,
m, CH+1Hind). Anal. Calcd for C23H19N3OS (MW 385.48): C, 71.66;
H, 4.97; N, 10.90. Found: C, 71.35; H, 5.06; N, 11.13.
Data for 13. IR: 1690, 1593, 1117. 1H NMR: 2.30 (3H, s, CH3ph),
2.40 (3H, d, CH3th, J = 1.4), 3.21 (3H, s, NCH3), 3.46 (3H, s, OCH3),
6.10 (1H, d, ind-4, J = 2.2), 6.86 (1H, dd, ind-6, J = 8.4, J = 2.2), 6.96
(1H, d, ind-7, J = 8.4), 7.23 (2H, d, ph, J = 7.6), 7.35 (1H, q, th, J =
1.4), 7.59 (2H, d, ph, J = 7.6), 7.66 (1H, s, CH). Anal. Calcd for
C24H21N3O2S (MW 415.50): C, 69.38; H, 5.09; N, 10.11. Found: C,
69.76; H, 4.89; N, 10.34.
Figure 5. Induction of apoptosis by compound 5 in HT-29 colon
cancer cells is accompanied by mitochondrial events. The cells were
treated for 24 h with 500 nM 5 before each evaluation. All panels depict
the result obtained in one experiment that was representative of three
to five. (A) Change in mitochondrial membrane potential as estimated
from DiOC6 fluorescence histograms. ΔΨm collapse is evidenced by a
decrease in DiOC6 fluorescence in whole cells. (B) Analysis of intra-
cellular ROS content measured by flow cytometry. Reaction of DHE
with superoxide radicals causes the formation of a derivative with in-
creased fluorescence. (C) Confocal laser scanning micrographs showing
staining for Bax (green fluorescence) and mitochondria (red fluores-
cence) in control (left) and treated cells (right). Localization of Bax with
mitochondria is demonstrated by orange pixels.
Data for 16. IR: 3140−2663, 1696, 1199, 717. 1H NMR: 2.73 (3H, s,
CH3), 3.38 (3H, s, OCH3), 6.20 (1H, s, ind-4), 6.74 (2H, s, ind-6 + 7),
7.44 (2H, d, ph, J = 8.4), 7.62 (1H, s, CH), 7.71 (2H, d, ph, J = 8.4),
10.47 (1H, s, NH). Anal. Calcd for C21H15ClN4O2S (MW 422.88): C,
59.64; H, 3.58; N, 13.25. Found: C, 59.83; H, 3.87; N, 13.67.
Data for 17. IR: 3124−2725, 1704, 1183, 721. 1H NMR: 2.74 (3H,
s, CH3), 6.42 (1H, dd, ind-4, J = 9.0, J = 2.4), 6.80 (1H, dd, ind-7 J =
9.0, J = 4.6), 7.00 (1H, dt, ind-6, J = 9.0, J = 2.4), 7.46 (2H, d, ph, J =
8.4), 7.65 (1H, s, CH), 7.69 (2H, d, ph, J = 8.4), 10.67 (1H, s, NH).
Anal. Calcd for C20H12ClFN4OS (MW 410.86): C, 58.61; H, 2.71; N,
13.67. Found: C, 58.38; H, 2.98; N, 13.98.
Data for 24. IR: 3300−2800, 1706, 1110, 999. 1H NMR: 2.44 (3H,
d, CH3, J = 1.3), 3.34 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.72 (3H, s,
OCH3), 6.54 (1H, d, ind-4, J = 7.5), 6.70 (1H, t, ind-5/6, J = 7.5), 6.79
(1H, d, ind-7, J = 7.5), 6.92 (1H, s, ph), 7.13 (1H, t, ind-5/6, J = 7.5),
7.43 (1H, s, CH), 7.57 (1H, q, th, J = 1.3), 10.56 (1H, s, NH). Anal.
Calcd for C24H20BrN3O4S (MW 526.40): C, 54.76; H, 3.83; N. 7.98.
Found: C, 54.91; H, 4.00; N, 8.04.
Data for 28. IR: 3250−2800, 1696, 1583, 717. 1H NMR: 6.28 (1H,
d, ind-4, J = 7.6), 6.80 (1H, t, ind-5/6, J = 7.6), 6.89 (1H, d, ind-7, J =
7.6), 7.20 (1H, t, ind-5/6, J = 7.6), 7.32 (1H, d, th, J = 4.5), 7.31 (1H,
m, py), 7.45 (1H, d, th, J = 4.5), 7.90 (1H, td, py, J = 7.7, J = 1.8), 8.16
(1H, d, py, J = 7.7), 8.18 (1H, s, CH), 8.63 (1H, m, py), 10.68 (1H, s,
NH). Anal. Calcd for C19H12N4OS (MW 344.39): C, 66.26; H, 3.51;
N, 16.27. Found: C, 66.72; H, 3.22; N, 15.99.
Data for 29. IR: 3200−2720, 1688, 1601, 721. 1H NMR: 6.16 (1H,
d, ind-4, J = 2.2), 6.88 (2H, m, ind-6 + 7), 7.25 (1H, m, py), 7.43 (1H,
d, th, J = 4.4), 7.52 (1H, d, th, J = 4.4), 7.90 (1H, t, py, J = 7.7), 8.16
(1H, d, py, J = 7.7), 8.26 (1H, s, CH), 8.61 (1H, m, py), 10.80 (1H, s,
NH). Anal. Calcd for C19H11ClN4OS (MW 378.83): C, 60.24; H,
2.93; N, 14.79. Found: C, 60.56; H, 3.03; N, 14.98.
Figure 6. Effect of 38, 31, and 5 on the activation of protein kinases
associated with cell growth and/or cell survival. HeLa cells were incu-
bated for 3 h in the presence of a 1 μM concentration of the indicated
compounds. The content and phosphorylation of the indicated protein
kinases in cell extracts were determined by Western blotting.
Data for 30. IR: 3200−2720, 1692, 1603, 722. 1H NMR: 6.27 (1H,
d, ind, J = 8), 6.87 (2H, m, ind), 7.33 (1H, m, py), 7.34 (1H, d, th, J =
4.4), 7.47 (1H, d, th, J = 4.4), 7.91 (1H, t, py, J = 7.7), 8.16 (1H, d, py,
J = 7.7), 8.23 (1H, s, CH), 8.63 (1H, m, py), 10.72 (1H, s, NH). Anal.
Calcd for C19H11ClN4OS (MW 378.83): C, 60.24; H, 2.93; N, 14.79.
Found: C, 60.57; H, 3.01; N, 14.42.
Anal. Calcd for C20H15N3OS (MW 345.41): C, 69.54; H, 4.38; N,
12.16. Found: C, 69.85; H, 4.47; N, 11.92.
Data for 31. IR: 3180−2660, 1689, 1034, 722. 1H NMR: 3.76 (3H,
s, OCH3), 6.81 (2H, m, ind), 7.33 (2H, m, py+ind), 7.34 (1H, d, th,
J = 4.4), 7.56 (1H, d, th, J = 4.4), 7.90 (1H, t, py, J = 7.7), 8.16 (1H, d,
py, J = 7.7), 8.33 (1H, s, CH), 8.65 (1H, m, py), 10.37 (1H, s, NH).
Anal. Calcd for C20H14N4O2S (MW 374.41): C, 64.16; H, 3.77; N,
14.96. Found: C, 64.59; H, 3.47; N, 15.02.
Data for 7. IR: 2714, 1706, 1633, 1233, 712. 1H NMR: 2.26 (3H, s,
CH3), 2.72 (3H, s, CH3), 6.74 (1H, dd, ind-4, J = 9.0, J = 2.4), 6.85
(1H, dd, ind-7, J = 9.0, J = 4.4), 7.07 (1H, td, ind-6, J = 9.0, J = 2.4),
7.57 (1H, s, CH), 10.66 (1H, s, NH). Anal. Calcd for C15H11FN4OS
(MW 314.34): C, 57.31; H, 3.53; N 17.82. Found: C, 56.98; H, 3.87;
N, 18.00.
Data for 33. IR: 3500−2800, 1692, 1598, 1081. 1H NMR: 4.05 (3H,
s, OCH3), 6.83 (1H, d, ind-4/7, J = 7.7), 6.93 (1H, t, ind-5/6, J = 7.7),
7.13 (1H, t, ind-5/6, J = 7.7), 7.33 (1H, d, th, J = 4.4), 7.52 (1H, s,
CH), 7.58 (1H, d, ind-4/7, J = 7.7), 7.77 (1H, d, th, J = 4.4), 10.47
1
Data for 9. IR: 3375−2715, 1699, 815, 723. H NMR: 2.27 (3H, s,
CH3), 2.70 (3H, s, CH3), 7.16 (1H, d, ind-4/5, J = 8.6), 7.44 (1H, d,
ind-4/5, J = 8.6), 7.51 (2H, m, bzind), 7.56 (1H, s, CH), 7.88 (1H, m,
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(1H, s, NH). Anal. Calcd for C15H11N3O2S (MW 297.33): C, 64.16;
H, 3.77; N, 14.96. Found: C, 63.99; H, 3.23; N, 15.03.
β-tubulin structure contains the full binding site for NSC 613863. The
missing segments in the β-tubulin crystal structure were modeled, and
the offset amino acid sequence of the crystal structure relative to the
uniprot code D0VWY9 was corrected to give a model of β-tubulin in
complex with NSC 613863. The D0VWY9−NSC 613863 complex
was prepared for modeling by addition of bond orders and hydrogen
atom and then energy minimized with limited atom movements of less
than 0.3 Å.
Data for 34. IR: 3400−2700, 1688, 1523, 722. 1H NMR: 4.06 (3H,
s, OCH3), 6.84 (1H, d, ind-5/7, J = 7.8), 6.98 (1H, d, ind-5/7, J = 7.8),
7.13 (1H, t, ind-6, J = 7.8), 7.34 (1H, d, th, J = 4.4), 7.65 (1H, d, th,
J = 4.4), 8.20 (1H, s, CH), 10.74 (1H, s, NH). Anal. Calcd for C15H10-
ClN3O2S (MW 331.77): C, 54.30; H, 3.04; N, 12.67. Found: C, 54.02;
H, 2.89; N, 12.86.
Data for 35. IR: 3300−2800, 1688, 1076, 723. 1H NMR: 4.07 (3H,
s, OCH3), 6.83 (1H, d, ind-7, J = 8.2), 6.96 (1H, d, ind-4, J = 2.1), 7.18
(1H, dd, ind-6, J = 8.2, J = 2.1), 7.43 (1H, d, th, J = 4.3), 7.67 (1H, s,
CH), 8.18 (1H, d, th, J = 4.3), 10.57 (1H, s, NH). Anal. Calcd for
C15H10ClN3O2S (MW 331.77): C, 54.30; H, 3.04; N 12.67. Found: C,
54.67; H, 2.97; N, 12.39.
Compound 38, which was the most active in the series, was selected
for the initial docking. The Glide program was used to flexible dock
compound 38. The top five binding poses were individually inspected,
and unfavorable intermolecular contacts were identified. Manual changes
to the position or the torsional bond angles of the ligands were used to
relieve unfavorable protein−ligand contacts, if possible. The binding
poses were subsequently refined using the Glide program using the
highest precision setting. A high scoring 38 pose was selected and
subjected to further stepwise refinement. First, with tubulin fixed
in Cartesian space, the conformation of 38 was energy minimized.
Second, with 38 fixed, β-tubulin was minimized. This was followed
by energy minimization of the complex in which all atoms were un-
constrained. The resulting binding model was evaluated for unfavor-
able intra- and intermolecular contacts. If unfavorable contacts were
evident, the refinement cycle was iterated. Upon determination of
satisfactory intra- and intermolecular contacts in the model, compound
38 was extracted from the pocket and a docking grid was created based
on the refined β-tubulin model. Using the Glide program, the com-
pound was redocked into the colchicine site. The final binding pose
scored a favorable binding Gscore of −6.0.
The docking grid based on the refined β-tubulin structure was used
as a common template for generating docking poses for the other com-
pounds. The binding scores (Gscores) for these compounds were as
follows: compound 5 = −4.3; 9 = −4.1; 29 = −3.9; 30 = −4.3; 31 =
−2.9; 33 = −5.4; 36 = −6.0; 38 = −6.0; 39 = −4.3.
The binding models indicate that the two most active compounds
in terms of % inhibition of colchicine binding (Table 3), 36 and 38,
have the most favorable binding scores of −6.0, while the least active
compounds, 29 and 31, have the least favorable binding scores of −3.9
and −2.9, respectively.
c. Cell Culture and Treatment. Human colon adenocarcinoma HT-
29 and cervical carcinoma HeLa cells were cultured in RPMI 1640
medium (Labtek Eurobio, Milan, Italy), supplemented with 10% fetal
bovine serum (PAA Laboratories GmbH, Pashing, Austria) and 2 mM
L-glutamine (Sigma-Aldrich, St Louis MO), in humidified air at 37 °C
with 5% CO2.
Compounds were dissolved in DMSO at 10 mmol/L and diluted
with medium to obtain the desired concentration. DMSO concentra-
tion in the medium was kept constant at 0.01%. The cells were plated
at 2 × 104 cells/cm2 in a plastic well (60 cm2) and treated for 24 h
in triplicate. In control cells, only DMSO was added to the culture
medium. After incubation for the indicated times, viable cells were
detected and counted by trypan blue exclusion.
Data for 36. IR: 3300−2700, 1712, 1004, 722. 1H NMR: 4.04 (3H,
s, OCH3), 6.85 (1H, s, ind), 6.96 (2H, m, ind), 7.41 (1H, d, th, J = 4.4),
7.62 (1H, s, CH), 8.10 (1H, d, th, J = 4.4), 10.58 (1H, broad, NH).
Anal. Calcd for C15H10ClN3O2S (MW 331.77): C, 54.30; H, 3.04; N
12.67. Found: C, 54.62; H, 2.96; N, 12.82.
Data for 37. IR: 3300−2700, 1693, 1599, 722. 1H NMR: 4.06 (3H,
s, OCH3), 6.79 (2H, m, ind), 6.95 (1H, m, ind), 7.43 (1H, d, th,
J = 4.5), 7.66 (1H, s, CH), 8.15 (1H, d, th, J = 4.5), 10.45 (1H, s, NH).
Anal. Calcd for C15H10FN3O2S (MW 315.33): C, 57.14; H, 2.71; N,
13.67. Found: C, 56.98; H, 2.97; N, 13.45.
Data for 38. IR: 3300−2700, 1707, 1687, 722. 1H NMR: 3.65 (3H,
s, OCH3ind), 4.04 (3H, s, OCH3th), 6.71 (1H, s, ind), 6.73 (2H, s,
ind), 7.55 (1H, s, CH), 7.72 (1H, d, th, J = 4.2), 8.04 (1H, d, th, J =
4.2), 10.25 (1H, s, NH). Anal. Calcd for C16H13N3O3S (MW 327.35):
C, 58.71; H, 4.00; N, 12.84. Found: C, 58.98; H, 4.37; N, 12.99.
Data for 39. IR: 1681, 1523, 1157, 1070, 722. 1H NMR: 3.19 (3H, s,
NCH3), 3.68 (3H, s, OCH3ind), 4.04 (3H, s, OCH3th), 6.64 (1H, d,
ind-4, J = 2.4), 6.82 (1H, dd, ind-6, J = 8.4, J = 2.4), 6.90 (1H, d, ind-7,
J = 8.4), 7.42 (1H, d, th, J = 4.4), 7.64 (1H, s, CH), 8.06 (1H, d, th, J =
4.4). Anal. Calcd for C17H15N3O3S (MW 341.38): C, 59.81; H, 4.43;
N, 12.31. Found: C, 60.01; H, 4.68; N, 12.57.
1
Data for 40. IR: 1701, 1603, 1153, 999, 717. H NMR: 3.21 (3H, s,
NCH3), 4.05 (3H, s, OCH3), 7.00 (2H, m, ind), 7.22 (1H, t, ind, J = 7.4),
7.33 (1H, d, th, J = 4.4), 7.56 (1H, s, CH), 7.65 (1H, d, ind, J = 7.4), 7.80
(1H, d, th, J = 4.4). Anal. Calcd for C16H13N3O2S (MW 311.35): C,
61.72; H, 4.21; N, 13.50. Found: C, 62.01; H, 4.43; N, 13.68.
2. Biology. a. Cell-Based Screening Assay. The NCI screening
process occurs in two stages,33 beginning with the evaluation of
all compounds against the 60 cell lines at 10−5 M. Compounds
exhibiting significant growth inhibition are subsequently eval-
uated against the 60 cell lines at five concentration levels ac-
cording to standard procedures (http://dtp.nci.nih.gov/branches/
btb/ivclsp.html). In both cases, the exposure time is 48 h.
b. Tubulin. Combretastatin A-4 was a generous gift of Dr. G. R.
Pettit, Arizona State University.
Bovine brain tubulin was purified as described previously.34 The
tubulin assembly assay was performed with 10 μM tubulin and varying
compound concentrations as described previously.26 The IC50 is the
compound concentration that inhibits extent of assembly after 20 min
at 30 °C.
The colchicine binding assay was performed with 1.0 μM tubulin,
5.0 μM [3H]colchicine, and 5.0 μM inhibitor. Incubation was for
10 min at 37 °C. At this time point, about 40−60% maximum
colchicine binding occurs in the control reactions. Details of the
method were described previously.35
Confocal Microscopy. Cells were seeded at 1 × 104 cells/cm2 on
glass coverslips and treated with test compounds for 24 h. They were
washed three times with phosphate-buffered saline (PBS), fixed with
3% paraformaldehyde, washed with 0.1 M glycine in PBS, and per-
meabilized with 70% ice-cold ethanol. Cells were then washed three
times with 1% bovine serum albumin in PBS and incubated with
the respective primary antibody (anti-Bax, anti-p21, antitubulin, and
antiactin) for 1 h at room temperature. The cells were washed again
and incubated with FITC-conjugated antibody (secondary antibody)
for 1 h at room temperature in the dark. In experiments in which Bax
intracellular localization was investigated, cells were first incubated
with 150 nM Mito-Tracker Red CM-H2 XRos in fresh medium at
37 °C for 10 min and then fixed. Finally, we used PI to stain the nuclei.
All preparations were embedded in Mowiol containing antibleaching
DABCO and analyzed by using a laser scanning confocal microscope
NIKON C1s, equipped with a NIKON Eclipse TE300.
Molecular Modeling. The Maestro 9 (Schrodinger, LLC, New
̈
York, NY) modeling software running on a Dell (Round Rock, TX)
Precision 690 with Red Hat (Raleigh, NC) Enterprise Linux 4 was
used to perform the modeling studies. Simulations were performed in
vacuo using a distance-dependent dielectric with a nonbonded inter-
action limited to within 13 Å in an OPLS 2005 force field. Minimi-
̀
zations involved up to 500 steps of Polak−Ribiere conjugate gradient.
The 3G2N crystal structure of αβ-tubulin in complex with NSC
613863 and a stathmin fragment was selected as the template for
docking studies. More precisely, the B-subunit (β-tubulin) of the
3G2N structure was extracted and utilized in the docking studies. The
Cell Cycle and Apoptosis. Cells were fixed with 70% ethanol at
−20 °C for at least 12 h. After two washes with PBS, the cells were in-
cubated in RNaseA/PBS (10 μg/mL) at 37 °C for 30 min. Intracellular
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Journal of Medicinal Chemistry
Article
DNA was labeled with PI (50 μg/mL). Cell cycle analysis was per-
formed using Modfit 5.0 software. Surface exposure of phosphatidyl-
serine in apoptotic cells was measured with an Annexin V-FLUOS
staining kit (Roche Diagnostics Deutschland GmbH, Mannheim,
Germany), following the manufacturer’s instructions. Briefly, cells were
washed twice with cold PBS and resuspended in binding buffer at a
concentration of 1 × 105 cells in a total volume of 100 μL. Next, 5 μL
of Annexin V-FLUOS and 5 μL of PI (50 μg/mL) solutions were
added. The cells and reagents were gently mixed and incubated for
20 min at room temperature. PI and Annexin V-FLUOS were quan-
titated by flow cytometry, with the data acquired with logarithmic
amplification. The enzymatic activity of caspases hydrolyzing the pep-
tide sequence Asp-Glu-Val-Asp (DEVD) is indicated as DEVDase
activity, which was measured in cell extracts by a fluorometric assay.36
Mitochondrial Membrane Potential (ΔΨm). To measure ΔΨm,
mitochondria were selectively probed with the potential-sensitive dye
DiOC6. After treatment, cells were incubated with medium containing
4 nM DiOC6 for 40 min at a cell concentration of 1 × 106 cell/mL
at 37 °C in the dark. Cells were counterstained by PI at 5 μg/mL to
permit elimination of dead cells. Fluorescence was analyzed by flow
cytometry with logarithmic amplification. Flow cytometric analysis was
performed with a Beckmann Coulter Epics XL MCL cytometer (USA)
equipped with a 15 mW argon ion laser.
ROS Measurement. In order to detect intracellular superoxide
levels,37 the cells were incubated with 5 μM DHE (Molecular Probes,
Leiden, The Netherlands), made as a 10 mM stock in DMSO, for
30 min at 37 °C. Cells were analyzed by measuring red fluorescence
with logarithmic amplification.
Western Blotting. The cells were lysed, and the homogenates were
centrifuged at 15 000g for 15 min. The supernatant was diluted in
loading buffer and then denatured by boiling for 4 min. Aliquots
corresponding to 80 μg of protein were analyzed by SDS-PAGE.
Proteins were transferred onto a nitrocellulose membrane and probed
with the specific primary antibodies (Cell Signaling Technology, Inc.,
Danvers, MA). After further washing, the membrane was incubated for
1 h with peroxidase-conjugated goat antirabbit IgG (Santa Cruz Bio-
technology, Santa Cruz, CA). Immunoreactive bands were visualized
by chemiluminescence with the ECL reagent (Amersham Biosciences
Corp., Piscataway, NJ).
in the Division of Cancer Treatment and Diagnosis of the
National Cancer Institute.
ABBREVIATIONS USED
■
NCI, National Cancer Institute; DTP, Developmental Ther-
apeutics Program; DMSO, dimethyl sulfoxide; GI, growth
inhibition; TGI, total growth inhibition; LC, lethal concen-
tration; PI, propidium iodide; DiOC6, 3,3′-dihexyloxacarbocya-
nine iodide; ROS, reactive oxygen species; DHE, dihydroethi-
dium; FITC, fluorescein isothiocyanate; PBS, phosphate-
buffered saline
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: +39-051-2099714, fax +39-051-2099734, e-mail aldo.
andreani@unibo.it.
ACKNOWLEDGMENTS
■
This work was supported in part by a grant from the University
of Bologna, Italy (RFO), and from MIUR (PRIN 2009). We
are grateful to the National Cancer Institute (Bethesda, MD)
for the anticancer tests. It has been funded in part with federal
funds from the National Cancer Institute, National Institutes of
Health, under contract N01-CO-12400. The content of this
publication does not necessarily reflect the views or policies of
the Department of Health and Human Services, nor does
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