New microtubule polymerization inhibitors comprising a nitrooxymethylphenyl group

Article abstract of DOI:10.1016/j.bmc.2011.05.031

We have designed cancer antiproliferative compounds, starting from aniline or phenol derivative, which comprise one or two nitrooxymethylphenyl groups as do the hybrid drugs NCX4040 and NCX530. Compound 2a with p- nitrooxymethylbenzoyl-oxy and -amino grou

Full text of DOI:10.1016/j.bmc.2011.05.031

Bioorganic & Medicinal Chemistry 19 (2011) 3995–4003
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New microtubule polymerization inhibitors comprising
a nitrooxymethylphenyl group
Yasuyuki Kawaratani a, Tomohiko Harada a, Yoshiyuki Hirata a, Yasuo Nagaoka a, Susumu Tanimura b,
Makio Shibano c, Masahiko Taniguchi c, Masahide Yasuda c, Kimiye Baba c, Shinichi Uesato a,
⇑
a Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680, Japan
b Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
c Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed cancer antiproliferative compounds, starting from aniline or phenol derivative, which
comprise one or two nitrooxymethylphenyl groups as do the hybrid drugs NCX4040 and NCX530. Com-
pound 2a with p-nitrooxymethylbenzoyl-oxy and -amino groups as well as 8a with a p-nitrooxymethyl-
benzoylamino group showed more promising effects than NCX4040 against human colon and breast
cancer cells. Since 2a and 8a, but not NCX4040, arrested human colon carcinoma HCT116 cells in the M
phase, the former two compounds may inhibit cell growth differently from NCX4040. Merged images of
Received 5 April 2011
Revised 16 May 2011
Accepted 16 May 2011
Available online 23 May 2011
Keywords:
immunofluorescence-stained a-tubulin and Hoechst-stained nuclei in human fibrosarcoma HT1080 cells
Tubulin polymerization inhibitor
Nitrooxymethylphenyl group
M phase regulator
Phospho-histone H3
HCT116 xenograft
showed that 2a and 8a disrupted microtubule formation just as did vincristine, the tubulin polymerization
inhibitor. In experiments in vivo, the intraperitoneal administration of 8a at 80 mg/kg/day reduced the
growth of HCT116 xenografts in nude mice to T/C 55%.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
of the histone H3 as did vincristine.11 Thus, we concluded that 2a
and 8a are a new M phase regulator. Merged images of immunoflu-
NO-donating NSAIDs, developed as hybrid drugs to overcome
the gastrointestinal and renal toxicity of traditional NSAIDs, have
a protective effect on the gastric mucosa by releasing NO from
their nitrooxymethylphenyl group. Two examples are NCX4040
(NO-aspirin)1–6 and NCX530 (NO-indomethacin)7-9 (Fig. 1). The
former drug has attracted the attention of many medicinal chem-
ists because it showed promising antitumor activity against colon
cancers. Later, however, Hulsman et al.10 proposed that NCX4040 is
converted to a quinone methide, which in turn binds to glutathione
as an alkylating agent. This causes a reduction in oxidative stress
tolerance, leading to the death of cancer cells.
In the present work, we designed aniline or phenol derivatives
which comprise a nitrooxymethylphenyl group as do NCX4040
and NCX530, in expectation of diverse activities1–6,10 derived from
its group. Among the new compounds, 2a and 8a had a more po-
tent effect than NCX4040 against human colon cancer cells, as well
as breast cancer cells. Cell cycle tests revealed that 2a and 8a, but
not NCX4040, arrested human colon carcinoma HCT116 cells in the
G2/M phase. Therefore, 2a and 8a may inhibit cell growth differ-
ently from NCX4040. Western blot and flow cytometric analyses
showed that these compounds strongly induced phosphorylation
orescence-stained a-tubulin and Hoechst-stained nuclei in human
fibrosarcoma HT1080 cells showed that 2a and 8a disrupted micro-
tubule formation similar to vincristine, a tubulin polymerization
inhibitor.12 In experiments in vivo, the intraperitoneal administra-
tion of 8a at 80 mg/kg/day reduced the growth of HCT116 xeno-
grafts in nude mice to T/C 55%.
2. Chemistry
The bis(nitrooxymethylbenzoyl) derivatives 2a, 2b, 4, and 6
were synthesized as illustrated in Scheme 1. 2-Aminophenol was
reacted with p- or m-(chloromethyl)benzoyl chloride in the pres-
ence of pyridine, yielding the p-chloromethyl (1a) or m-chloro-
methyl derivative (1b). These compounds were then treated with
AgNO3, giving 2a and 2b, respectively. Similarly, compounds 4
and 6 were synthesized via the chloromethyl derivatives 3 and 5,
respectively.
The mononitrooxymethylbenzoyl derivatives 8a, 8b, and 13, as
well as 9, 10, and 11, were synthesized as shown in Scheme 2. Ani-
line was reacted with p- or m-(chloromethyl)benzoyl chloride to
give the p-chloromethyl (7a) or m-chloromethyl derivative (7b),
respectively. These compounds were treated in the same way as
1a and 1b, yielding 8a and 8b, respectively. Aniline was also con-
verted into the monobenzoyl derivatives 9 and 10, respectively.
⇑
Corresponding author. Tel.: +81 6 6368 0834; fax: +81 6 6388 8609.
E-mail address: uesato@ipcku.kansai-u.ac.jp (S. Uesato).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.031

3996
Y. Kawaratani et al. / Bioorg. Med. Chem. 19 (2011) 3995–4003
O
O
H3CO
ONO2
O
ONO2
H3C
O
O
N
O
O
Cl
NCX4040
NCX530
Figure 1. Structures of NO-donating NSAIDs, NCX4040 and NCX530.
O
O
O
OH
NH2
H
N
O
H
N
a or b
c
O2NO
ONO2
Cl
Cl
O
O
1a: p-chloromethyl (59.7%)
1b: m-chloromethyl (56.9%)
2a: p-nitrooxymethyl (25.7%)
2b: m-nitrooxymethyl (43.0%)
HO
NH2
a
ONO2
H
Cl
H
c
O2NO
Cl
O
N
O
N
O
O
O
O
3 ((84.6%)
4 (83.3%)
NH2
O
O
NH2
ONO2
NH
NH
H
Cl
H
N
c
a
N
O
Cl
O2NO
O
5 (37.6%)
6 (48.1%)
Scheme 1. Reagents and conditions: (a) p-(chloromethyl)benzoyl chloride, Py, CHCl3; (b) m-(chloromethyl)benzoyl chloride, Py, CHCl3; (c) AgNO3, CH3CN.
H
N
H
N
c
ONO2
O
Cl
O
a or b
d or e
8a: p-nitrooxymethyl (56.3%)
8b: m-nitrooxymethyl (38.4%)
7a: p-chloromethyl (90.6%)
7b: m-chloromethyl (63.9%)
NH2
f
R
OH
H
N
H
N
O
O
11 (49.6%)
9: H
(34.3%)
10: CH2CH3 (97.3%)
Cl
ONO2
OH
O
O
a
c
O
O
13 (70.8%)
12 (55.7%)
Scheme 2. Reagents and conditions: (a) p-(chloromethyl)benzoyl chloride, Py, CHCl3; (b) m-(chloromethyl)benzoyl chloride, Py, CHCl3; (c) AgNO3, CH3CN; (d) benzoyl
chloride, Py, CHCl3; (e) p-ethylbenzoyl chloride, Py, CHCl3; (f) LiOH, 1,4-dioxane.

Y. Kawaratani et al. / Bioorg. Med. Chem. 19 (2011) 3995–4003
3997
Table 1
Inhibitory activities of nitrooxymethylphenyl-possessing compounds and derivatives
against the growth of cancer cells
Compound
Colon cancer cells (IC50
)
Breast cancer cells (IC50)
HCT116 SW620 SW480 SKBR-3 MCF7 MDA-MB-231
p-histone H3
1a
0.6
0.8
6.9
0.6
58.1
0.8
2a
2b
4
6
7a
8a
8b
9
10
11
13
NCX4040
0.7
47.6
6.2
>100
1.2
0.6
19.3
>100
>100
24.8
17.9
68.0
0.8
25.8
7.6
>100
0.8
0.8
3.8
22.2
8.4
>100
0.9
0.8
13.3
>100
>100
>100
80.7
19.1
0.7
18.5
6.3
>100
0.7
0.7
>100
>100
>100
>100
54.3
>100
>100
>100
>100
>100
>100
>100
1.4
30.0
6.7
>100
0.8
0.8
histone H3
Figure 3. Mitotic checkpoint analysis by Western blotting. HCT116 cells were
treated with 2a, 8a, NCX4040, or vincristine for 24 h. Cells were lysed to extract
crude histone proteins. The Western blot analysis was performed with crude
histone samples using an antibody specific to phospho-histone H3 (Ser-10), or total
histone H3. Results are representative of five independent experiments.
8.4
8.2
8.4
>100
>100
>100
16.9
15.1
>100
>100
9.8
50.1
30.5
>100
>100
>100
47.7
14.4
cancer cells; 0.7–0.8 lM for the breast cancer cells) as 2a. In con-
trast, 13, with a p-nitrooxymethylbenzoyloxy group, exhibited
only weak effects. Furthermore, the synthetic intermediates com-
prising a chloromethylbenzoylamino group, 1a and 7a, showed
comparatively strong activities, whereas 9, 10 and 11, not having
a leaving group such as Cl and ONO2, had little or no effect. The
IC50 values of 2a and 8a for the growth of normal human fibroblast
Furthermore, 7a was hydrolyzed with LiOH to afford the alcohol
11. Phenol was converted, via the chloromethyl derivative 12, into
13 in the conventional manner.
CCD-1059SK cells could not be determined even at 200 lM.
3. Results and discussion
These findings suggest that the framework of 8a and the para
orientation of its nitrooxymethyl group on the benzene ring as well
as an electronegative substituent such as ONO2 and Cl are essential
to the antiproliferative activities. Additional introduction of a
nitrooxymethylbenzoyl-oxy (2a, 2b and 4) or -amino (6) group
seems to be unnecessary. The impotency of 13 could be explained
by a higher rotatability of its p-nitrooxymethylbenzoyloxy chain
on a benzene ring than that of the p-nitrooxymethylbenzoylamino
chain in 8a; this might diminish the interaction between 13 and an
active site.
3.1. Antiproliferative activities and structure–activity
relationships of the nitrooxymethylbenzoyl derivatives and
related compounds
Compound 2a, comprising p-nitrooxymethylbenzoyl-oxy and -
amino groups, inhibited significantly the growth of the human co-
lon (IC50: 0.7–3.8 lM) and breast (IC50: 0.7–1.4 lM) cancer cells
except for human breast MCF7 cells as shown in Table 1. However,
6, possessing two p-nitrooxymethylbenzoylamino groups, as well
as 2b, having m-nitrooxymethylbenzoyl-oxy and -amino groups,
had greater IC50 values than 2a. Interestingly, 8a, with a p-nitro-
oxymethylbenzoylamino group, inhibited the growth of these can-
3.2. Effect of 2a and 8a on the cell cycle
Both 2a and 8a regulated HCT116 cells in the G2/M phase of the
cer cells to nearly the same extent (IC50: 0.6–0.8
lM for the colon
cell cycle at 20
lM in 24 h as did vincristine, and the percentage
control
NCX4040
vincristine
G0/G1: 35.9 %
G2/M: 28.6 %
G0/G1: 53.7 %
G0/G1: 76.5 %
G2/M: 12.0 %
S: 18.8 %
Sub-G1: 23.0 %
S: 12.5 %
G2/M: 10.9 %
Sub-G1: 15.3 %
Sub-G1: 6.2 %
S: 8.5 %
DNA content
2a
DNA content
DNA content
8a
G2/M: 51.8 %
G0/G1: 30.4 %
G0/G1: 52.4 %
G2/M: 29.3 %
Sub-G1: 10.4 %
Sub-G1: 11.0 %
S: 7.4 %
S: 7.3 %
DNA content
DNA content
Figure 2. Effects of 2a and 8a on the cell cycle in HCT116 cells. Cells were treated with a BD CycletestTM Plus DNA reagent Kit. Data shown are those for treatment for 24 h
with 2a, 8a (each at 20 lM), NCX4040 (at 40 lM), or vincristine (at 150 nM) or for the control (no treatment) for 24 h. They are representative of five independent
experiments.

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Y. Kawaratani et al. / Bioorg. Med. Chem. 19 (2011) 3995–4003
control
vincristine
NCX4040
25.3%
1.4%
1.7%
DNA content
DNA content
DNA content
8a
2a
26.4%
46.2%
DNA content
DNA content
Figure 4. Mitotic checkpoint analysis by flow cytometry. HCT116 cells were treated with 2a, 8a (each at 20 lM), NCX4040 (at 40 lM), or vincristine (at 150 nM) for 24 h.
Cells in mitosis were determined by staining with propidium iodide and phospho-histone H3 (Ser-10) antibody followed by FITC-conjugated secondary antibody. The
percentage of cells with tetraploid DNA and a high level of phospho-histone H3 was determined by FACS. Numbers in the upper right corners indicate percentages of mitotic
cells in each sample. They are representative of three independent experiments.
ratio (170.4%) of the G2/M phase to G0/G1 phase cells on the incu-
bation with 8a was much higher than the ratio (55.9%) on the incu-
bation with 2a (Fig. 2). However, NCX4040 did not arrest the cells
NCX4040. Furthermore, in the flow cytometric analysis, the
proportion of phospho-histone H3 positive cells was increased from
1.4% to 26.4% by 2a and 46.2% by 8a (at 20
lM in 24 h), but not in-
in the G2/M phase even at 40
l
M. These compounds induced apop-
creased by NCX4040 (1.7% at 40 M in 24 h). The findings indicated
l
tosis as suggested by the appearance of a sub G1 population in the
DNA histograms. These results suggested that 2a and 8a exhibited
inhibitory activities different from NCX4040.
that HCT116 cells accumulated in the M phase on the incubation with
2a or 8a and the M phase population was much larger in the cells
given 8a than in those given 2a.
To determine whether these two compounds arrest HCT116 cells
in the G2 or M phase, the phosphorylation of histone H3 at Ser 10
wasanalyzedbyWesternblotting (Fig. 3) as wellas by flow cytometry
(Fig. 4) using an anti-phospho-histone H3 (Ser 10) antibody. Histone
H3 phosphorylated atSer10 is a well-established mitoticmarker.13–16
In the Western blot assay, the expression of the phospho-histone H3
was enhanced by 2a and 8a as in the case of vincristine, but not by
3.3. Determination of apoptosis induced by 2a and 8a
The appearance of a sub G1 population among the HCT116 cells
treated with 2a and 8a (Fig. 2) suggested apoptosis, which was
confirmed by a TUNEL assay. As shown in Fig. 5, the percentage of
TUNEL-positive cells was 36.4% for 2a and 24.2% for 8a at 20 lM
vincristine
NCX4040
control
31.6%
3.6%
7.3%
FITC-A
FITC-A
FITC-A
8a
2a
24.2%
36.4%
FITC-A
FITC-A
Figure 5. FITC–TUNEL staining cell analysis by flow cytometry. HCT116 cells were incubated with 2a, 8a (each at 20 lM), NCX4040 (at 40 lM), or vincristine (at 150 nM) for
48 h and then treated as indicated in Materials and methods. Percentages of FITC–TUNEL-positive cells are indicated. Values are representative of five independent
experiments.

Y. Kawaratani et al. / Bioorg. Med. Chem. 19 (2011) 3995–4003
3999
control
taxol
vincristine
2a
8a
Figure 6. Examination of the ability of 2a and 8a to affect the microtubule structures. HT1080 cells were cultured with 2a (10
l
M), 8a (10
lM), taxol (100 nM), or vincristine
(100 nM) for 4 h and then treated as reported previously.17 Cells were imaged using a fluorescence microscope.
in 48 h, whereas it was 3.6% for NCX4040 at 40
l
M. The values were
260
240
220
200
180
160
140
120
4–5% for all these compounds at 24 h (data not shown). These re-
sults, as well as the effects on the cell cycle, suggested that 8a might
have a more cytostatic effect on HCT116 cells than 2a though these
compounds suppressed cell growth to the same extent.
3.4. Effect of 2a and 8a on microtubule organization
control
The effects of 2a, 8a, vincristine and taxol on the microtubule
structures were compared in HT1080 human fibrosarcoma cells
to distinguish whether the former two compounds inhibited the
polymerization of tubulin (to destabilize microtubule structures)
or the de-polymerization of tubulin (to stabilize microtubule struc-
tures).17 Fig. 6 illustrates merged images of immunofluorescence-
2a 20 µM
8a 20 µM
vincristine 10 µM
stained
a-tubulin (green) and Hoechst-stained nuclei (blue) in
HT1080 cells. It was indicated that both 2a and 8a, like vincristine,
but unlike taxol,18 disrupted microtubule formation. In the next
experiment, we thus tried to examine how 2a and 8a inhibited
microtubule development. A tubulin polymerization assay was
conducted using porcine brain tubulin by measuring the intensity
of fluorescence emission at 460 nm of microtubule-bound DAPI.19
As shown in Figure 7, 2a and 8a, though not strongly, reduced a
polymer mass, respectively. This result suggested that the interac-
tion of 2a or 8a with tubulin might be, at least in part, responsible
for the disruption of tubulin-polymerization. We assumed that 2a
and 8a bound to tubulin as alkylating agents after or with the re-
moval of -ONO2. It seems however necessary to examine the
in vivo behavior of these compounds in understanding their mode
of action in more detail.
0
1000
2000
3000
4000
5000
6000
Time (s)
Figure 7. Effects of 2a and 8a on tubulin assembly. Tubulin (2.0 mg/mL) was
dissolved in General Tubulin Buffer. The reaction was performed with 2a, 8a,
vincristine or no reagent (control). The mixture was incubated at 37 °C. Polymer-
ization was measured over 100 min by reading the increase in fluorescent emission
at 460 nm in the kinetic mode with excitation at 340 nm. Values are representative
of four independent experiments.
therapeutic agent with few side effects. We thus conducted anti-
tumor tests of 8a using xenografts of HCT116 cells in nude mice.
The compound was administered intraperitoneally at a dose of
50 or 80 mg/kg/day over 16 days according to the schedules indi-
cated in Figure 8A. Compound 8a suppressed the growth of the
xenografts to T/C: 55% in 21 days. Weight loss was similar between
the 8a-treated and control groups and was not significant through-
out the treatment period (Fig. 8B).
3.5. In vivo evaluation of 8a
Compound 8a, having comparatively low IC50 values as well as a
cytostatic effect on the HCT116 cells, could be applied as a cancer

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Y. Kawaratani et al. / Bioorg. Med. Chem. 19 (2011) 3995–4003
A
B
9.0
8.0
1.3
1.2
1.1
1.0
0.9
0.8
0.7
control
8a 50 mg/kg
8a 80 mg/kg
7.0
6.0
5.0
4.0
3.0
2.0
control
*
*
0.6
8a 50 mg/kg
8a 80 mg/kg
*
*
*
0.5
*
*
1.0
0.0
0.4
0.3
*
0
5
10
15
20
25
0
5
10
15
20
25
day
day
Figure 8. Effects of 8a on HCT116 cell-inoculated xenografts in vivo. The effects of 8a (j 50 mg/kg; N 80 mg/kg) on tumor volume (8A) as well as relative body weight
changes (8B) were examined as described in Materials and methods. The relative tumor volume (RTV) was calculated by dividing the tumor volume at each day by the tumor
volume at day 0. The relative body weight (RBW) was calculated as the ratio of body weight at each day to that at day 0. (ꢀ) represents the control group. Compounds were
intraperitoneally administered to mice at days 0, 2, 4, 7, 9, 11, 14 and 16. Vertical bars indicate standard errors. The asterisks denote significant difference (p <0.01 by two-
tailed Student t-test) for tumor volume with 8a versus the control.
001), Tubulin (porcine brain) (Cat. #T240-A) (Cytoskeleton) and
DAPI solution (DOJINDO). Female BALB/c-nu.nu nude mice (5
weeks of age) were purchased from Japan SLC Inc. Animals were
housed and experiments were performed according to guidelines
stipulated by the Osaka University of Pharmaceutical Sciences Ani-
mal Care and Use Committee.
4. Conclusion
Compounds 2a and 8a, each possessing a p-nitrooxymethylphe-
nyl group, showed stronger antiproliferative activities than
NCX4040 against the human cancer cell lines. Both compounds ar-
rested HCT116 cells in the M phase of the cell cycle and inhibited
microtubule formation. The intraperitoneal administration of 8a
reduced the growth of human HCT116 cancer xenografts in nude
mice to T/C 55%. Thus, 8a warrants further tests with other human
cancer xenografts to identify its effectiveness as an antitumor
agent.
5.2. Chemistry
5.2.1. 2-(4-(Chloromethyl)benzamido)phenyl 4-(chloromethyl)
benzoate (1a)
To a solution of o-aminophenol (0.33 g, 3.00 mmol) in CHCl3
(5 mL) were added pyridine (0.97 mL, 12.00 mmol) and p-(chloro-
methyl)benzoyl chloride (1.13 g, 6.00 mmol), and the mixture was
stirred at 0 °C for 2 h. The mixture was successively diluted with
CHCl3, washed with 1% HCl and satd. NaHCO3 and brine, dried,
and concentrated. The resulting solid was recrystallized from
CHCl3 and n-hexane to give 1a (0.74 g, 1.79 mmol, 59.7%) as color-
less needles. Mp 151.0–152.5 °C. IR: 1722.3, 1672.2, 1454.2,
5. Experimental
5.1. General
Melting points were determined on a Yanaco MP-500D micro-
melting point apparatus and are uncorrected. IR spectra in KBr
were recorded on a Shimadzu FTIR-8400 infrared spectrophotom-
eter. Low-resolution (LR)-FAB-MS spectra were measured on a
JEOL JMS-HX 100, whereas high-resolution (HR)- and LR-FAB-MS
spectra, on a JEOL Tandem MStation JMS-700. 1H NMR spectra
were recorded on a JEOL EX-400 (400 MHz), unless otherwise sta-
ted, in CDCl3 with tetramethylsilane as an internal standard. Ana-
lytical TLC and PLC were performed using Silica gel 60 F254
(Merck, 0.25 and 1 mm, respectively) glass plates. Column chroma-
tography was performed using Silica Gel 60 (70–230 mesh ASTM).
All the solvents were dried over Na2SO4, and evaporated in vacuo.
The HCT116, SW480, SW620 (human colon carcinoma), SKBR-3,
MCF7, MDA-MB-231 (human breast carcinoma) and CCD-1059SK
(human normal fibroblast) cell lines were purchased from Ameri-
can Tissue Culture Collection. The Cell Counting Kit for the WST-
1 assay was obtained from DOJINDO.20 NCX4040 was prepared as
reported prior.10 BD CycletestTM Plus DNA reagent Kit was pur-
chased from Becton Dickinson and Company. The TUNEL assay
reagent, DeadEndTM Fluorometric TUNEL System, was obtained
from Promega. Results of flow cytometry were collected with a
FACSCant II flow cytometer (Becton Dickinson and Company).
The tubulin polymerization assay was conducted according to
the catalogue (Tubulin Polymerization Assay, Cat. #BK011P) of
Cytoskeleton, using General Tubulin Buffer (Cat. #BST01-001),
Tubulin Glycerol Buffer (Cat. #BST05-001), GTP (Cat. #BST06-
1253.6, 1178.4, 750.3 cmꢀ1 1H NMR: d 5.50 (2H, s), 5.53 (2H, s),
.
7.03–7.06 (1H, m), 7.43–7.44 (2H, m), 7.54 (2H, d, J = 8.0 Hz),
7.56 (2H, s), 7.75 (1H, t, J = 1.4 Hz), 7.86 (1H, s), 7.90 (2H, d,
J = 8.0 Hz), 8.24 (2H, d, J = 8.4 Hz). FAB-MS m/z: 414 (M+H) +. HR-
FAB-MS m/z: (M+H)+calcd for C22H18Cl2NO3, 414.0662; found,
414.0670.
5.2.2. 2-(4-(Nitrooxymethyl)benzamido)phenyl 4-(nitrooxymeth
yl)benzoate (2a)
A suspension of AgNO3 (0.41 g, 2.40 mmol) in CH3CN (2 mL)
was added to a solution of 1a (0.21 g, 0.50 mmol) in CH3CN
(5 mL), and the mixture was stirred at 60 °C for 4 h under light-
shielding. The mixture was filtered off through Celite to separate
insoluble materials, and the filtrate was concentrated. The result-
ing solid was purified on silica gel column with EtOAc: CHCl3:
n-hexane (2:1:1) as an eluent and recrystallized from EtOAc and
n-hexane, affording 2a (0.06 g, 0.13 mmol, 25.7%) as colorless nee-
dles. Mp 136.2 °C. IR: 3346.3, 1720.4, 1674.1, 1537.2, 1259.4,
887.2, 750.3 cmꢀ1 1H NMR: d 4.92 (2H, s), 5.10 (2H, s), 7.36 (4H,
.
m), 7.43 (2H, d, J = 8.8 Hz), 7.57 (2H, d, J = 8.4 Hz), 7.77 (2H, d,
J = 5.2 Hz), 7.97 (1H, br s), 8.25 (2H, d, J = 4.4 Hz). FAB-MS m/z:
468 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for C22H18N3O9,
468.1044; found, 468.1046.

Y. Kawaratani et al. / Bioorg. Med. Chem. 19 (2011) 3995–4003
4001
5.2.3. 2-(3-(Chloromethyl)benzamido)phenyl 3-(chloromethyl)
1280.6, 856.3, 754.1 cmꢀ1
.
1H NMR: d 5.50 (4H, s), 7.05–7.07 (2H,
benzoate (1b)
m), 7.45–7.47 (2H, m), 7.54 (4H, d, J = 7.6 Hz), 8.01 (4H, d,
J = 8.0 Hz), 9.19 (2H, s). FAB-MS m/z: 467 (M+H)+. HR-FAB-MS m/z:
(M+H)+ calcd for C22H19N4O8, 467.1204; found, 467.1205.
Compound 1b was synthesized using o-aminophenol (0.11 g,
1.00 mmol), pyridine (0.50 mL, 6.21 mmol), and m-(chloro-
methyl)benzoyl chloride (0.34 mL, 2.40 mmol) in the same way
as 1a (colorless needles, 0.24 g, 0.57 mmol, 56.9%). Mp 154.7–
5.2.9. 4-(Chloromethyl)-N-phenylbenzamide (7a)
157.3 °C. IR: 2399.3, 1215.1, 754.1 cmꢀ1
.
1H NMR: d 4.52 (2H, s),
Compound 7a was synthesized using aniline (0.27 mL,
3.00 mmol), pyridine (0.29 mL, 3.60 mmol), and p-chloro-
methylbenzoyl chloride (0.68 g, 3.60 mmol) in the conventional
manner (colorless needles, 0.67 g, 2.72 mmol, 90.6%). Mp 164.5–
4.66 (2H, s), 7.37–7.41 (3H, m), 7.49–7.56 (2H, m), 7.72 (2H, d,
J = 8.0 Hz), 7.77 (1H, s), 8.04 (1H, br s), 8.20 (2H, d, J = 7.6 Hz),
8.26 (1H, s), 8.32 (1H, d, J = 7.6 Hz). FAB-MS m/z: 414 (M+H)+.
HR-FAB-MS m/z: (M+H)+ calcd for C22H18Cl2NO3, 414.0662; found,
414.0669.
166.3 °C. IR: 3346.3, 1654.8, 1598.9, 1529.4, 1438.8, 1325.0 cmꢀ1
.
1H NMR: d 4.64 (2H, s), 7.17 (1H, t, J = 8.0 Hz), 7.39 (2H, t,
J = 8.0 Hz), 7.52 (2H, d, J = 8.0 Hz), 7.63 (2H, d, J = 8.0 Hz), 7.78
(1H, br s), 7.87 (2H, d, J = 8.0 Hz). FAB-MS m/z: 246 (M+H)+. HR-
FAB-MS m/z: (M+H)+ calcd for C14H13ClNO, 246.0686; found,
246.0684.
5.2.4. 2-(3-(Nitrooxymethyl)benzamido)phenyl 3-(nitrooxymeth
yl)benzoate (2b)
Compound 2b was synthesized using AgNO3 (3.92 g,
23.0 mmol) and 1b (0.87 g, 2.11 mmol) in the same way as 2a (col-
orless needles, 0.42 g, 0.90 mmol, 43.0%). Mp 119.6–123.6 °C. IR:
5.2.10. 4-(Phenylcarbamoyl)benzyl nitrate (8a)
Compound 8a was synthesized using AgNO3 (1.70 g,
5.00 mmol) and 7a (0.54 g, 1.97 mmol) in the conventional manner
(colorless needles, 0.30 g, 1.11 mmol, 56.3%). Mp 168.0–170.9 °C.
3018.4, 2399.3, 1215.1, 754.1, 669.3, cmꢀ1 1H NMR: d 5.38 (2H,
.
s), 5.50 (2H, s), 7.30–7.33 (2H, m), 7.37 (1H, t, J = 6.8 Hz), 7.46
(1H, t, J = 7.6 Hz), 7.54 (1H, d, J = 7.2 Hz), 7.60 (1H, t, J = 7.6 Hz),
7.72 (1H, d, J = 7.6 Hz), 7.78 (2H, d, J = 6.0 Hz), 8.00 (1H, br s),
8.27 (3H, d, J = 4.8 Hz). FAB-MS m/z: 468 (M+H)+. HR-FAB-MS m/z:
(M+H)+ calcd for C22H18N3O9, 468.1044; found, 468.1052.
IR: 3018.4, 2399.3, 1215.1, 786.9, 754.1, 669.3 cmꢀ1 1H NMR: d
.
5.50 (2H, s), 7.18 (1H, t, J = 6.8 Hz), 7.39 (2H, t, J = 8.0 Hz), 7.53
(2H, d, J =7.6 Hz), 7.63 (2H, d, J = 8.0 Hz), 7.80 (1H, br s), 7.91
(2H, d, J = 8.0 Hz). FAB-MS m/z: 273 (M+H)+. HR-FAB-MS m/z:
(M+H)+ calcd for C14H13N2O4, 273.0876; found, 273.0876.
5.2.5. 3-(4-(Chloromethyl)benzamido)phenyl 4-(chloromethyl)
benzoate (3)
5.2.11. 3-(Chloromethyl)-N-phenylbenzamide (7b)
Compound 3 was synthesized using m-aminophenol (0.50 g,
4.58 mmol), pyridine (0.88 mL, 11.00 mmol), and p-(chloro-
methyl)benzoyl chloride (2.08 g, 11.00 mmol) in the same way as
1a (colorless needles, 1.60 g, 3.87 mmol, 84.6%). Mp 130.0–
Compound 7b was synthesized using aniline (0.46 mL,
5.00 mmol), pyridine (0.48 mL, 6.00 mmol), and m-(chloro-
methyl)benzoyl chloride (0.85 mL, 6.00 mmol) in the conventional
manner (colorless needles, 0.78 g, 3.20 mmol, 63.9%). Mp 127.7–
131.9 °C. IR: 1737.7, 1267.1, 1253.6, 1215.1, 1151.4 cmꢀ1 1H
.
129.2 °C. IR: 3018.4, 1222.8, 1207.4, 790.8, 727.1, 667.3 cmꢀ1 1H
.
NMR: d 4.64 (2H, s), 4.66 (2H, s), 7.02–7.05 (1H, m), 7.45–7.47
(2H, m), 7.52–7.56 (4H, m), 7.73 (1H, t, J = 6.0 Hz), 7.89 (2H, d,
J = 2.0 Hz), 7.88 (1H, s), 8.20 (2H, d, J = 4.4 Hz). FAB-MS m/z: 414
(M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for C22H18Cl2NO3,
414.0662; found, 414.0677.
NMR: d 4,66 (2H, s), 7.18 (1H, t, J = 8.0 Hz), 7.38 (2H, t, J = 7.6 Hz),
7.50 (1H, t, J = 7.6 Hz), 7.59 (1H, d, J = 7.6 Hz), 7.65 (2H, d,
J = 7.6 Hz), 7.82 (1H, d, J = 7.6 Hz), 7.90 (1H, s). FAB-MS m/z: 246
(M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for C14H13ClNO, 246.0686;
found, 246.0690.
5.2.6. 3-(4-(Nitrooxymethyl)benzamido)phenyl 4-(nitrooxymeth
yl)benzoate (4)
5.2.12. 3-(Phenylcarbamoyl)benzyl nitrate (8b)
Compound 8b was synthesized using AgNO3 (1.36 g,
8.00 mmol) and 7b (0.50 g, 2.04 mmol) in the conventional manner
(colorless needles, 0.21 g, 0.78 mmol, 38.4%). Mp 94.1–95.3 °C. IR:
Compound 4 was synthesized using AgNO3 (0.77 g, 9.68 mmol)
and 3 (1.00 g, 2.42 mmol) in the same way as 2a (colorless needles,
0.94 g, 2.02 mmol, 83.3%). Mp 130.4–133.1 °C. IR: 1643.2, 1280.6,
3018.4, 1209.3, 785.0, 731.0, 669.3 cmꢀ1 1H NMR: d 5.51 (2H, s),
.
1213.1, 850.5, 779.2 cmꢀ1 1H NMR: d 5.50 (2H, s), 5.53 (2H, s),
.
7.18 (1H, t, J = 7.2 Hz), 7.39 (2H, t, J = 7.6 Hz), 7.54 (1H, t,
J = 7.6 Hz), 7.60 (1H, d, J = 7.2 Hz), 7.64 (2H, d, J = 8.0 Hz), 7.79
(1H, br s), 7.89 (1H, d, J = 7.6 Hz), 7.92 (1H, s). FAB-MS m/z: 273
(M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for C14H13N2O4, 273.0876;
found, 273.0875.
7.03–7.06 (1H, m), 7.41–7.46 (2H, m), 7.53–7.56 (4H, m), 7.75
(1H, s), 7.86 (1H, s), 7.90 (2H, d, J = 8.0 Hz), 8.24 (2H, d,
J = 8.0 Hz). FAB-MS m/z: 468 (M+H)+. HR-FAB-MS m/z: (M+H)+
calcd for C22H18N3O9, 468.1044; found, 468.1055.
5.2.7. N,N0-(1,2-Phenylene)bis(4-(chloromethyl)benzamide) (5)
Compound 5 was synthesized using o-phenylene diamine
(0.22 g, 2.00 mmol), pyridine (0.39 mL, 4.80 mmol), and p-(chloro-
methyl)benzoyl chloride (0.91 g, 4.80 mmol) in the usual way (col-
orless needles, 0.94 g, 0.75 mmol, 37.6%). Mp 189.9–191.4 °C. IR:
5.2.13. N-Phenylbenzamide (9)
Compound
9
was synthesized using aniline (0.09 mL,
1.00 mmol), pyridine (0.08 mL, 1.00 mmol), and benzoyl chloride
(0.14 mL, 1.20 mmol) in the conventional manner (colorless nee-
dles, 0.68 g, 0.34 mmol, 34.3%). Mp 158.4–162.2 °C. IR: 3301.9,
1643.2, 1504.4, 1305.7, 1272.9, 754.1, 707.8, 675.0 cmꢀ1 1H
.
1656.7, 1535.2, 1438.8, 750.3 cmꢀ1 1H NMR: d 7.16 (1H, t,
.
NMR: d 4,65 (4H, s), 6.95–7.00 (2H, m), 7.41–7.43 (2H, m), 7.53
(4H, d, J = 8.4 Hz), 7.98 (4H, d, J = 8.4 Hz), 9.25 (2H, s). FAB-MS m/
z: 413 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for C22H19Cl2N2O2,
413.0824; found, 413.0821.
J = 7.6 Hz), 7.38 (2H, t, J = 7.6 Hz), 7.48–7.58 (3H, m), 7.65 (2H, d,
J = 7.6 Hz), 7.82 (1H, br s), 7.88 (2H, d, J = 7.2 Hz). FAB-MS m/z:
198 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for C13H12NO,
198.0920; found, 198.0923.
5.2.8. (4,40-(1,2-Phenylenebis(azanediyl))bis(oxomethylene)bis
(4,1-phenylene))bis(methylene) dinitrate (6)
Compound 6 was synthesized using AgNO3 (0.68 g, 4.00 mmol)
and 5 (0.41 g, 1.00 mmol) in the conventional manner (colorless
needles, 0.22 g, 0.48 mmol, 48.1%). Mp 152.5–158.2 °C. IR: 1629.7,
5.2.14. 4-Ethyl-N-phenyl benzamide (10)
Compound 10 was synthesized using aniline (0.09 mL,
1.00 mmol), pyridine (0.08 mL, 1.00 mmol), and p-ethylbenzoyl
chloride (0.18 mL, 1.20 mmol) in the conventional manner (color-
less needles, 0.22 g, 0.97 mmol, 97.3%). Mp 119.9–123.4 °C. IR:

4002
Y. Kawaratani et al. / Bioorg. Med. Chem. 19 (2011) 3995–4003
3300.0, 1649.0, 754.1, 518.8 cmꢀ1
.
1H NMR:
d
1.28 (3H, t,
with serum-free medium (2 ꢁ 1 mL) and immersed in serum-free
medium (5 mL) for 24 h. After removal of the medium, the cells
were washed with serum-free medium (2 ꢁ 1 mL) and incubated
J = 8.0 Hz), 2.73 (2H, q, J = 8.0 Hz), 7.14 (1H, t, J = 7.2 Hz), 7.31
(2H, d, J = 8.0 Hz), 7.36 (2H, t, J = 7.6 Hz), 7.64 (2H, d, J = 8.0 Hz),
7.79 (2H, d, J = 8.0 Hz). FAB-MS m/z: 226 (M+H)+. HR-FAB-MS
m/z: (M+H)+ calcd for C15H16NO, 226.1233; found, 226.1233.
with 2a, 8a (each 20 lM), NCX4040 (40 lM), vincristine
(150 nM), or control (0.4% DMSO) in the serum-free medium
(5 mL) for another 24 h. The medium (about 5 mL) was transferred
to Centrifuge Tubes (IWAKI). The adhered cells, after being washed
twice with cold PBS (each 1 mL), were treated with trypsin 0.25%
5.2.15. 4-(Hydroxymethyl)-N-phenyl benzamide (11)
To a solution of 7a (0.10 g, 0.41 mmol) in 1,4-dioxane (5 mL)
was added aq LiOH (5 mL, 5.00 mmol), and the mixture was stirred
at room temperature for 72 h. The reaction mixture was succes-
sively diluted with CHCl3, washed with satd. NaHCO3 and brine,
dried, and concentrated. The resulting solid was purified on silica
gel column with EtOAc/toluene (1:1) as an eluent and recrystal-
lized from CHCl3 and n-hexane, affording 11 (0.05 g, 0.20 mmol,
49.6%) as colorless needles. Mp 143.2–146.3 °C. IR: 3290.3,
(1ꢁ) (Invitrogen) (200
lL) for 3 min and transferred to the above
tubes. The cells in the tubes were treated with a Cycle Test Plus
DNA reagent Kit (Cat. #340242) according to the procedure recom-
mended by Becton Dickinson and Company. DNA content was
measured with a FACSTMCant II.
5.6. Western blot analysis of phospho-histone H3 levels
2945.1, 2833.2, 2356.9, 2337.6, 1028.0 cmꢀ1 1H NMR (DMSO-
.
d6): d 4.80 (2H, s), 7.16 (1H, t, J = 6.3 Hz), 7.38 (2H, t, J = 7.8 Hz),
7.50 (2H, d, J = 7.8 Hz), 7.65 (2H, d, J = 8.3 Hz), 7.79 (1H, br s),
7.88 (2H, d, J = 7.8 Hz). ESI-MS m/z: 228.2 (M+H)+. HR-FAB-MS m/z:
(M+H)+ calcd for C14H14NO2, 228.1025; found, 228.1023.
HCT116 cells were plated onto 60 mm diameter dishes
(1.0 ꢁ 106/dish). After incubating for 24 h, the cells were treated
and incubated with 2a, 8a, NCX4040 (each 20 and 40 lM), vincris-
tine (150 nM), or control (0.4% DMSO) in the same way as for the cell
cycle test. The medium was discarded, and floating and adheredcells
were washed with PBS (2 ꢁ 1 mL), scraped,, and resuspended in
5.2.16. Phenyl 4-(chloromethyl)benzoate (12)
Compound 12 was synthesized using phenol (0.09 g, 1.00 mmol),
pyridine (0.10 mL, 1.20 mmol), and p-(chloromethyl)benzoyl chlo-
ride (0.23 g, 1.20 mmol) in the conventional manner (colorless nee-
dles, 0.14 g, 0.56 mmol, 55.7%). Mp 72.2–76.6 °C. IR: 1730.0, 1274.9,
400 lL of modified Covance Research lysis buffer (pH 8.0 and 1%
Nonidet P-40 were adopted in place of pH 7.4 and 1% Triton-X 100,
respectively) at 0 °C for 1 h. The lysates were centrifuged at 14000
ꢁg for 15 min at 4 °C. The protein concentration was determined
with a BCA protein assay Kit (Thermo Fisher Scientific). An equal
1195.8, 1072.3, 742.5 cmꢀ1 1H NMR: d 4.63 (2H, s), 7.21 (2H, d,
.
J = 8.4 Hz), 7.27 (1H, t, J = 7.6 Hz), 7.42 (2H, t, J = 8.4 Hz), 7.52 (2H,
d, J = 8.0 Hz), 8.19 (2H, d, J = 8.4 Hz). FAB-MS m/z: 247 (M+H)+. HR-
FAB-MS m/z: (M+H)+ calcd for C14H12ClO2, 247.0527; found,
247.0527.
amount (7.5 lg) of protein was then resolved by SDS–PAGE and
transferred to a PVDF membrane. The blots (1 ꢁ Tris–HCl buffer sal-
ine, 5% nonfat milk and 0.05% Tween 20 or 1 ꢁ Tris–HCl buffer saline,
12.5 mM NaF, 1% nonfat milk, 1% BSA, and 0.05% Tween 20) were
probed with an antibody specific to each protein and were detected
by using ECL chemiluminescence.
5.2.17. Phenyl 4-(nitrooxymethyl)benzoate (13)
Compound 13 was synthesized using AgNO3 (0.34 g,
2.03 mmol) and 12 (0.10 g, 0.41 mmol) in the conventional manner
(colorless needles, 0.08 g, 0.29 mmol, 70.8%). Mp 108.3–109.2 °C.
5.7. Flow cytometric analysis of phospho-histone H3 levels21
IR: 1735.8, 1618.2, 1280.6, 881.4, 752.2, 688.5 cmꢀ1 1H NMR: d
.
5.52 (2H, s), 7.22 (1H, t, J = 8.2 Hz), 7.30 (2H, d, J = 8.0 Hz), 7.44
(2H, t, J = 8.0 Hz), 7.55 (2H, d, J = 8.0 Hz), 8.25 (2H, d, J = 6.4 Hz).
FAB-MS m/z: 274 (M+H)+. HR-FAB-MS m/z: (M+H)+ calcd for
HCT116 cells were plated onto 60 mm diameter dishes
(1.0 ꢁ 106/dish). After incubation for 24 h, the cells were treated
and incubated with 2a, 8a (each 20 lM), NCX4040 (40 lM), vin-
C14H12NO5, 274.0716; found, 274.0714.
cristine (150 nM), or control (0.4% DMSO) in the same way as for
the examination of cell cycle regulation. The cells were centrifuged
at 190ꢁg for 7 min, washed with PBS (2 ꢁ 5 mL), and fixed with
PBS (1 mL) and 80% ice-cold ethanol (5 mL) for 2 h at ꢀ20 °C. The
cells were centrifuged at 190ꢁg for 7 min, washed twice with
PBS (containing 1% BSA) (5 mL) and permeabilized by PBS (contain-
ing 1% BSA, 0.25% TritonX-100) (1 mL) for 5min at 0 °C. The cells
were washed with PBS (5 mL), blocked with PBS (containing 1%
5.3. Cells, animals, and antibodies
HCT116 and SW620 cells were cultured in McCoy’s 5A medium.
SW480, SKBr-3, MCF7 and MDA-MB-231 cells were cultured in
RPMI1640 medium. CCD-1059SK cells were cultured in MEM.
HT1080 cells were cultured in DMEM. All the culture media were
supplemented with 10% fetal bovine serum (FBS), 50
cillin G, and 50 g/mL streptomycin sulfate (Invitrogen) in an
atmosphere of 5% CO2 and 95% air at 37 °C. The mice were used
at 6 weeks of age. Antibodies specific to anti- -tubulin and b-actin
lg/mL peni-
BSA) (200 ll) for 2 h at room temperature, and incubated overnight
l
at 4 °C with anti-body (phospho-histone H3 (1:100)). The cells
were washed with PBS (containing 1% BSA)) (5 mL) twice, and
incubated for 1 h at 4 °C with secondary anti-body (rabbit
(1:40)). The cells were further washed with PBS (containing BSA)
a
were purchased from Sigma to Aldrich, and anti-phospho-histone
H3 (Ser 10) was from Cell signaling.
(1 mL), and incubated with propidium iodide (5 lg/ml in PBS)
(500 lL) and RNase A (10 mg/ml) (25 lL) for 30 min at 37 °C. Dual
5.4. Cell growth inhibition assay
label cell sorting was performed by FACSTMCant II.
Compounds were screened for cytotoxicity against seven mam-
malian cell lines representing different tissue types: cells were
grown in the presence of the compounds for 72 h before growth
was quantified using a Cell Counting Kit. The compounds were
5.8. TUNEL assay by flow cytometry
HCT116 cells were plated onto 60 mm diameter dishes
(1.0 ꢁ 106/dish). After incubating for 24 h or 48 h, the cells were
tested at final concentrations of 1, 10, 25, 50, 100, and 200 lM.
treated and incubated with 2a, 8a (each 20 lM), NCX4040
5.5. Examination of cell cycle regulation by flow cytometry
(40 lM), vincristine (150 nM), or control (0.4% DMSO) in the usual
way. The floating and adhered cells were subjected to a TUNEL as-
say using the DeadEndTM Fluorometirc TUNEL System according to
the procedure (Cat. #G3250) by Promega.
HCT116 cells were plated onto 60 mm diameter dishes
(1.0 ꢁ 106/dish). After incubating for 24 h, the cells were washed

Y. Kawaratani et al. / Bioorg. Med. Chem. 19 (2011) 3995–4003
4003
5.9. Immunofluorescent staining of tubulin
Acknowledgments
The effects of the test compounds on microtubule structures
were examined in HT1080 human fibrosarcoma cells as reported
previously.17
Part of this work was supported by the Strategic Project to
Support the Formation of Research Bases at PrivateUniversities. This
is a product of research financially supported in part by Kansai
University Research Grants: Grant-in-aid for Joint Research,
2009–2010.
5.10. Tubulin in vitro polymerization assay
Compounds 2a, 8a or vincristine was dissolved in General Tubu-
References and notes
lin Buffer containing 22% DMSO (220
vincristine). For vehicle, General Tubulin Buffer containing 22%
DMSO was used. An aliquot (5 L) of each solution was transferred
to a 96-well plate and allowed to warm at 37 °C. The tubulin solu-
tion was prepared by dissolving a purified tubulin (120 L of
10 mg/mL General Tubulin Buffer) in the tubulin reaction mixture
lM for 2a and 8a; 110 lM for
1. Rosetti, M.; Tesei, A.; Ulivi, P.; Fabbri, F.; Vannini, I.; Brigliadori, G.; Amadori, D.;
Bolla, M.; Zoli, W. Apoptosis 2003, 8, 1321.
2. Nilima Shukla, N.; Angelini, G. D.; Ascione, R.; Talpahewa, S.; Capoun, R.;
Jeremy, J. Y. Ann. Thorac. Surg. 2003, 75, 1437.
3. Tesei, A.; Ulivi, P.; Fabbri, F.; Rosetti, M.; Leonetti, C.; Scarsella, M.; Zupi, G.;
Amadori, D.; Bolla, M.; Zoli, W. J. Trans. Med. 2005, 3, 7.
4. Fabbri, F.; Brigliadori, G.; Ulivi, P.; Tesei, A.; Vannini, I.; Rosetti, M.; Bravaccini,
S.; Amadori, D.; Bolla, M.; Zoli, W. Apoptosis 2005, 10, 1095.
5. Bratasz, A.; Selvendiran, K.; Wasowicz, T.; Bobko, A.; Khramtsov, V.; Ignarro, L.
J.; Kuppusamy, P. J. Trans. Med. 2008, 6, 9.
6. Tesei, A.; Zoli, W.; Fabbri, F.; Leonetti, C.; Rosetti, M.; Bolla, M.; Amadori, D.;
Silvestrini, R. Nitric Oxide 2008, 19, 225.
7. Takeuchi, K.; Mizoguchi, H.; Araki, H.; Komoike, Y.; Suzuki, K. Dig. Dis. Sci. 2001,
46, 1805.
8. Huguenin, S.; Vacherot, F.; Fleury-Feith, J.; Riffaud, J. P.; Chopin, D. K.; Bolla, M.;
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9. Rao, C. V.; Reddy, B. S.; Steele, V. E.; Wang, C.-X.; Liu, X.; Ouyang, N.; Patlolla, J.
M. R.; Simi, B.; Kopelovich, L.; Rigas, B. Mol. Cancer Ther. 2006, 5, 1530.
10. Hulsman, N.; Medema, J. P.; Bos, C.; Jongejan, A.; Leurs, R.; Smit, M. J.; de Esch, I.
J.; Richel, D.; Wijtmans, M. J. Med. Chem. 2007, 50, 2424.
11. Juan, G.; Traganos, F.; James, W. M.; Ray, J. M.; Roberge, M.; Sauve, D. M.;
Anderson, H.; Darzynkiewicz, Z. Cytometry 1998, 32, 71.
12. Himes, R. H.; Kersey, R. N.; Heller-Bettinger, I.; Samson, F. E. Cancer Res. 1976,
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13. Hans, F.; Dimitrov, S. Oncogene 2001, 20, 3021.
14. Giet, R.; Glover, D. J. Cell Biol. 2001, 152, 669.
15. Adams, R. R.; Maiato, H.; Earnshaw, W. C.; Carmena, M. J. Cell Biol. 2001, 153,
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16. Wang, Y.; Zhang, W.; Jin, Y.; Johansen, J.; Johansen, K. M. Cell 2001, 105,
433.
l
l
(368
fer, 6
l
l
L of General Tubulin Buffer, 99.6
L of 100 mM GTP stock solution, and 6
L) of this tubulin solution (2.0 mg/mL)
was added to the solutions of 2a, 8a (20 M), vincristine (10 M)
l
L of Tubulin Glycerol Buf-
lL of 1 mg/mL DAPI
solution). An aliquot (50
l
l
l
or control (2.0% DMSO) prewarmed in the 96-well plate at 37 °C.
The polymerization was measured over 100 min by reading the in-
crease in fluorescent emission at 460 nm in the kinetic mode with
excitation at 340 nm.
5.11. In vivo antitumor activity
HCT116 cells (1.0 ꢁ 106/100
lL medium) were injected subcu-
taneously into the back of nude mice (6 weeks of age). Compound
8a (1.0 mg or 1.6 mg/200
l
L) and vehicle (10% castor oil in 1 ꢁ PBS,
200 L) were intraperitoneally administered to the mice with
l
54.1–108.6 mm3 (n = 6, respectively) at days 0, 2, 4, 7, 9, 11, 14,
and 16, respectively. Tumor length and width as well as weights
of mice were monitored for 21 days. Tumor volume (mm3) was cal-
culated by measuring length and width (in mm) as described.22 T/C
was calculated by dividing tumor volume for treated mice by
tumor volume for control mice.
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5.12. Statistical analysis
The statistical significance of the effects of drugs versus the con-
trol was analyzed with Student’s t-test.