Synthesis, antimitotic and antivascular activity of 1-(3′,4′, 5′-trimethoxybenzoyl)-3-arylamino-5-amino-1,2,4-triazoles

Article abstract of DOI:10.1021/jm5008193

A new class of compounds that incorporated the structural motif of the 1-(3′,4′,5′-trimethoxtbenzoyl)-3-arylamino-5-amino-1,2, 4-triazole molecular skeleton was synthesized and evaluated for their antiproliferative activity in vitro, interactions with tubulin, and cell cycle effects. The most active agent, 3c, was evaluated for antitumor activity in vivo. Structure-activity relationships were elucidated with various substituents on the phenyl ring of the anilino moiety at the C-3 position of the 1,2,4-triazole ring. The best results for inhibition of cancer cell growth were obtained with the p-Me, m,p-diMe, and p-Et phenyl derivatives 3c, 3e, and 3f, respectively, and overall, these compounds were more or less as active as CA-4. Their vascular disrupting activity was evaluated in HUVEC cells, with compound 3c showing activity comparable with that of CA-4. Compound 3c almost eliminated the growth of syngeneic hepatocellular carcinoma in Balb/c mice, suggesting that 3c could be a new antimitotic agent with clinical potential.

Full text of DOI:10.1021/jm5008193

Article
pubs.acs.org/jmc
Synthesis, Antimitotic and Antivascular Activity of 1‑(3′,4′,5′-
Trimethoxybenzoyl)-3-arylamino-5-amino-1,2,4-triazoles
Romeo Romagnoli,*,† Pier Giovanni Baraldi,*,† Maria Kimatrai Salvador,† Filippo Prencipe,†
Valerio Bertolasi,† Michela Cancellieri,§ Andrea Brancale,§ Ernest Hamel,⊥ Ignazio Castagliuolo,∥
Francesca Consolaro,‡ Elena Porcu,‡ Giuseppe Basso,‡ and Giampietro Viola*,‡
̀
†Dipartimento di Scienze Chimiche e Farmaceutiche, Universita
̀
di Ferrara, Ferrara, Italy
‡Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Universita
̀
di Padova, Padova, Italy
§The Welsh School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, U.K.
∥Dipartimento di Medicina Molecolare, Universita
̀
di Padova, Padova, Italy
⊥Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick
Laboratory for Cancer Research, National Institutes of Health, Frederick, Maryland
S
* Supporting Information
ABSTRACT: A new class of compounds that incorporated the structural motif of the 1-(3′,4′,5′-trimethoxtbenzoyl)-3-
arylamino-5-amino-1,2,4-triazole molecular skeleton was synthesized and evaluated for their antiproliferative activity in vitro,
interactions with tubulin, and cell cycle effects. The most active agent, 3c, was evaluated for antitumor activity in vivo. Structure−
activity relationships were elucidated with various substituents on the phenyl ring of the anilino moiety at the C-3 position of the
1,2,4-triazole ring. The best results for inhibition of cancer cell growth were obtained with the p-Me, m,p-diMe, and p-Et phenyl
derivatives 3c, 3e, and 3f, respectively, and overall, these compounds were more or less as active as CA-4. Their vascular
disrupting activity was evaluated in HUVEC cells, with compound 3c showing activity comparable with that of CA-4. Compound
3c almost eliminated the growth of syngeneic hepatocellular carcinoma in Balb/c mice, suggesting that 3c could be a new
antimitotic agent with clinical potential.
Among the synthetic inhibitors of tubulin polymerization, we
previously described a series of 2-arylamino-4-amino-5-
(3′,4′,5′-trimethoxybenzoyl)thiazoles with general structure 2
that showed strong antiproliferative activity against a panel of
five cancer cell lines.10 These compounds also caused
accumulation of HeLa cells in the G2/M phase of the cell
cycle, as is typical for antimicrotubule agents. Derivatives 2a (R1
= H) and 2b (R1 = 4′-Me) were the most active as inhibitors of
tumor cell growth, with IC50 values of 6−23 and 15−86 nM,
respectively, in the five cell lines.
Ring bioisosterism is widely used as a rational approach for
the discovery of new anticancer agents, especially for finding
agents with optimal pharmacological properties.11 Continuing
our search strategy for novel and potent antimicrotubule
agents, we have underway a pharmacophore exploration and
optimization effort based on compounds with general formula
INTRODUCTION
■
The cellular microtubule system, established by an equilibrium
between the polymerization and depolymerization of αβ-
tubulin heterodimers, is essential in a variety of cellular
processes, including maintenance of cell shape, regulation of
motility, intracellular transport of vesicles and organelles, and
cell division.1 Because of the latter function in eukaryotic cells,
microtubules are a successful target for the development of
numerous small natural and synthetic molecules that inhibit the
formation of the mitotic spindle.2−4 Among the naturally
occurring antimicrotubule agents, one of the most active is the
cis-stilbene combretastatin A-4 (CA-4, 1a, Chart 1), isolated
from the African cape bushwillow Combretum caffrum.5 CA-4
inhibits tubulin assembly by strongly binding to the colchicine
site on β-tubulin.6 Its water-soluble prodrug, CA-4 disodium
phosphate (CA-4P, 1b),7 is in advanced clinical trials,8 and it
was found to have potent activity in reducing tumor blood flow,
thus acting as a vascular disrupting agent (VDA).9
Received: May 29, 2014
© XXXX American Chemical Society
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a
Chart 1. Inhibitors of Tubulin Polymerization
Scheme 1
a
Reagents: (a) ArNH2, i-PrOH, reflux; (b) NH2NH2·H2O, THF,
reflux; (c) 3',4',5'-(OMe)3C6H2COCl, pyridine, 0 °C.
2. Here we describe replacing the thiazole nucleus with the
more electron-rich 1,2,4-triazole bioisosteric ring12,13 to afford a
new series of 1-(3′,4′,5′-trimethoxybenzoyl)-3-arylamino-5-
amino 1,2,4-triazole analogues with general structure 3. Our
goal was to evaluate the steric and electronic effects of different
substituents on the benzene portion of the arylamino moiety.
Besides hydrogen (compound 3a), the examined substituents
included fluorine (3b) and electron donating alkyl and alkoxy
groups (compounds 3c−h and 3i−n, respectively). Because it is
well-known that the trimethoxyphenyl skeleton is the character-
istic structural requirement to maximize activity in a large series
of inhibitors of tubulin polymerization, such as colchicine, CA-
4, and podophyllotoxin,14 all newly prepared compounds 3a−n
retain the 3′,4′,5′-trimethoxybenzoyl group at the C-1 position
of the 1,2,4-triazole ring. The newly synthesized derivatives
were evaluated for their antiproliferative activity in a panel of
human cancer cell lines for their antitubulin activity (including
cell cycle and apoptotic effects) and their antivascular activity in
HUVEC cells. Finally, the antitumor activity of 3c, the most
potent member of the group in the in vitro studies, was
evaluated in vivo in a syngeneic hepatocellular carcinoma in
Balb/c mice in comparison with CA-4P.
7k−l, respectively, were also isolated in pure form and low
yields (5−12%).
X-ray crystallographic analysis of a representative compound
(3c) was determined to confirm the regioselective aroylation at
the less sterically hindered N-1 nitrogen of the 1,2,4-triazole
ring. Single crystals of 3c were grown by slow evaporation from
i-PrOH−MeOH solution, and the ORTEP view of compound
3c is shown in Figure 1. The molecule displays an
intramolecular N4−H···O1 hydrogen bond having N4···O1
and H···O1 distances of 2.773(3) and 2.12(3) Å, respectively,
and an N4−H···O1 angle of 128(2)°.
CHEMISTRY
■
Synthesis of compounds 3a−n was accomplished using a three-
step procedure described in Scheme 1. The condensation of
dimethyl cyanodithioimidocarbonate 415 with the appropriate
substituted aniline resulted in the formation of imidates 5a−n,
which were cyclized into the corresponding 5-amino-1H-
[1,2,4]-triazole derivatives 6a−n in the presence of hydrazine
hydrate in refluxing THF.16 Treatment of 6a−n with an
equimolar quantity of 3′,4′,5′-trimethoxybenzoyl chloride
resulted in the formation of compounds 3a−n as the major
regioisomers. In the synthesis of compounds 3a−c, 3g−h, and
3k−l, the corresponding minor regioisomers 7a−c, 7g−h, and
Figure 1. ORTEP view of compound 3c displaying the thermal
ellipsoids at 30% probability.
B
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Table 1. In Vitro Cell Growth Inhibitory Effects of Compounds 3a−n and CA-4 (1)
a
IC50 (nM)
compd
Jurkat
CCRF-CEM
SEM
HeLa
HT-29
A549
MCF-7
3a
3b
3c
1200 270
1900 400
0.81 0.03
42 16
1000 90
2200 400
0.21 0.04
330 60
0.81 0.10
3.0 0.09
1800 90
>10 000
1200 190
2400 210
0.51 0.10
30 10
>10 000
410 70
3.2 1.3
440 50
2.0 0.82
6.0 1.0
130 28
>10 000
6200 1200
2000 700
0.82 0.10
460 80
3.0 0.91
0.21 0.08
800 100
>10 000
>10 000
3800 160
0.51 0.22
870 30
1.0 0.82
0.92 0.51
550 70
>10 000
500 240
370 30
1.0 0.61
15 3.2
4.0 0.21
5.0 1.0
510 70
5200 110
260 80
11 4.2
>10 000
3d
3e
3f
2.0 0.11
1.0 0.09
520 40
>10 000
0.40 0.11
0.81 0.21
500 10
>10 000
3g
3h
3i
51 10
1000 200
120 30
9000 330
>10 000
44 16
120 30
30 1.2
>10 000
480 15
700 32
>10 000
2400 100
54 4.3
3j
7.2 2.0
>10 000
1.0 0.4
>10 000
3k
3l
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
3m
3n
CA-4
2500 220
24 7.2
2000 100
40 3.3
1900 170
2100 800
930 54
3700 400
60 10
6200 540
150 50
180 50
34 18
30
5
6
19 5.2
370 100
5
0.6
12 2.5
0.1
4
0.1
3100 100
a
IC50= compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean SE from the dose−response
curves of at least three independent experiments carried out in triplicate.
orders of magnitude, with double-digit nanomolar activity
against Jurkat, SEM, and MCF-7 cells. Because the para-
toluidino moiety of 3c was favorable for potency, it is important
to point out that the introduction of an additional methyl group
at the meta-position, resulting in the meta,para-dimethyl
derivative 3e, produced a 2−4-fold reduction in antiproliferative
activity against five of the six cancer cell lines, while 3c and 3e
were equipotent against SEM and HeLa cells.
The para-ethyl homologue 3f was 2−15-fold less active than
methyl counterpart 3c against four of the seven cancer cell
lines, with a minimal difference between the two compounds in
the Jurkat and SEM cells, while 3f was 4-fold more potent than
3c against HT-29 cells. Replacing the para-ethyl group with
branched (i-Pr) or larger (n-butyl) moieties (compounds 3g
and 3h, respectively) was detrimental for activity in all cell lines,
suggesting that an increase in steric bulk at this position caused
a decrease in potency.
The number and position of methoxy substituents on the
phenyl ring (compounds 3i−l) had a major influence on
antiproliferative activity. Replacement of the methyl moiety
with a more electron-releasing methoxy group at the para-
position of the phenyl ring (compound 3i) decreased
antiproliferative activity by 100-fold compared with 3c,
indicating that the methyl and methoxy groups are not
bioequivalent at the para-position of the phenyl ring. The
contribution of methyl or methoxy groups on the phenyl ring
to activity (3c vs 3d and 3i vs 3j, respectively) was position
dependent, with opposite effects. While for the methyl group,
as previously observed, the para-derivative 3c was considerably
more potent than meta-isomer 3d, an opposite effect was
observed for the two methoxy isomers 3i and 3j, with the meta-
isomer 3j from 4- to 44-fold more potent than the para-isomer
3i in six of the seven cancer cell lines, the exception being the
HT-29 cells. Either two or three methoxy substituents
(derivatives 3k and 3l, respectively) caused substantial loss in
antiproliferative activity relative to 3i and 3j, suggesting that
steric factors account for the loss of activity observed with these
two compounds. With the exception of the MCF-7 cells, the
para-ethoxy derivative 3m was 2−50-fold less potent than its
methoxy counterpart 3m. The 3′,4′-methylenedioxy derivative
3n, with IC50 values in the double-digit nanomolar range in five
BIOLOGICAL RESULTS AND DISCUSSION
■
In Vitro Antiproliferative Activities. Table 1 summarizes
the antiproliferative effects of 1-(3′,4′,5′-trimethoxybenzoyl)-3-
anilino-5-amino 1,2,4-triazoles 3a−n against a panel of seven
human cancer cell line, using CA-4 (1) as the reference
compound. The 1-(3′,4′,5′-Trimethoxybenzoyl)-3-amino-5-ani-
lino 1,2,4-triazole isomers 7a−c, 7g−h, and 7k−l were also
evaluated for their activities, but because they were all inactive
(IC50 > 10 μM), the data are not shown in Table 1. Three of
the synthesized compounds, corresponding to the p-Me, m,p-
di-Me, and p-Et phenyl analogues 3c, 3e, and 3f, respectively,
were significantly more active than the rest of derivatives, with
IC50 values of 0.21−3.2, 0.4−4.0, and 0.21−6.0 nM,
respectively, in the seven cell lines, as compared with 4−3100
nM for CA-4. With average IC50 values of 1.0, 1.9, and 2.4 nM
for 3c, 3e, and 3f, respectively, 3c appears to be the most active
compound in the series (for CA-4, the average value was 525
nM or, excluding the HT-29 cell line from the average, 96 nM).
Thus, these three compounds are substantially more active than
CA-4, and they are also more potent than their previously
described isosteres 2a and 2b.11 In addition to these highly
potent three derivatives, the m-OMe and m,p-methylenedioxy
phenyl derivatives 3j and 3n, respectively, were more active
than CA-4 against HT-29, A549 and MCF-7 cells. In short, the
data shown in Table 1 indicate the importance of substituents
and their relative position on the phenyl ring of the arylamino
moiety at the C-3 position of the 1,2,4-triazole skeleton for
activity and selectivity against different cancer cell lines.
The unsubstituted anilino derivative 3a was weakly active
(IC50 > 0.5 μM), and the introduction of a weak electron-
releasing fluorine atom at the para-position of the phenyl
(compound 3b) had the opposite effect, with slightly improved
antiproliferative activity with respect to 3a against HeLa, HT-
29, A549, and MCF-7 cells, while the activity was reduced
against Jurkat, CCRF-CEM, and SEM cells.
We found that the small methyl group at the para-position of
the phenyl ring, to furnish derivative 3c, improved significantly
antiproliferative activity relative to 3a. Moving the methyl
group from the para- to the meta-position, to furnish isomer
derivative 3d, reduced antiproliferative activity from 1 to 3
C
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of the seven cancer cell lines, showed an antiproliferative
activity intermediate between those of para- and meta-methoxy
analogues 3i and 3j, respectively.
Evaluation of Cytotoxicity in Human Noncancer Cells.
To obtain a preliminary indication of the cytotoxic potential of
these derivatives for normal human cells, two of the most active
compounds (3c and 3f) were assayed in vitro against peripheral
blood lymphocytes (PBL) from healthy donors (Table 2). Both
index (RI), which is the ratio between GI50 values of resistant
cells and sensitive cells, of 1.2 and 5.7 respectively, while
doxorubicin in LoVoDoxo and vinblastine in CEMVbl100 showed a
high RI of 118 and 193, respectively. Altogether, these results
suggest that this compound might be useful in the treatment of
drug refractory tumors.
Inhibition of Tubulin Polymerization and Colchicine
Binding. A subset of the compounds (3c−g, 3i−j and 3n)
were evaluated for their in vitro inhibition of tubulin
polymerization in comparison with CA-4. The same com-
pounds were also examined for inhibitory effects on the binding
of [3H]colchicine to tubulin (Table 4). In the assembly assay,
Table 2. Cytotoxicity of 3c and 3f in Human Noncancer
a
Cells
b
IC50 (μM)
cell line
3c
3f
Table 4. Inhibition of Tubulin Polymerization and
Colchicine Binding by Compounds 1a, 3c−g, 3i−j, and 3n
c
PBLresting
31.2 8.7
8.5 2.6
11.9 3.8
34.0 11.7
8.8 2.7
nd
d
PBLPHA
HUVEC
a
b
compd tubulin assembly IC50 SD (μM) colchicine binding
%
SD
3c
3d
3e
3f
0.75 0.1
1.8 0.0
1.2 0.0
1.4 0.0
13 0.7
3.7 0.4
2.5 0.0
2.0 0.0
1.2 0.1
92
67
83
80
2
1
1
2
a
Values are the mean SEM for three separate experiments. nd: not
b
determined. Compound concentration required to reduce cell growth
inhibition by 50%. PBL not stimulated with PHA. PBL stimulated
with PHA.
c
d
c
3g
3i
nd
48
2
compounds were practically ineffective in quiescent lympho-
cytes, with an IC50 of about 30 μM. In the presence of the
mitogenic stimulus phytohematoagglutinin (PHA), the IC50
decreased to about 8 μM for both compounds, a value that is
thousands of times higher than those observed against the
lymphoblastic cell lines Jurkat and CCRF-CEM. Furthermore,
compound 3c was also evaluated in human umbilical vein
endothelial cells (HUVECs), and again the IC50 value was
negligible compared with those found in the panel of cancer cell
lines. Altogether, these data suggest that these compounds may
have cancer cell selective antiproliferative properties.
Effect of Compound 3c on Drug-Resistant Cell Lines.
Drug resistance has become a serious problem in cancer
chemotherapy.17,18 One of the common mechanisms of
resistance so far identified both in preclinical and clinical
studies involves the overexpression of a cellular membrane
protein called P-glycoprotein (P-gp) that mediates the efflux of
various structurally unrelated drugs.17,18 We evaluated
3j
70 0.9
51 0.3
98 0.5
3n
1a
a
Inhibition of tubulin polymerization. Tubulin was at 10 μM.
b
Inhibition of [3H]colchicine binding. Tubulin, colchicine and tested
c
compound were at 1, 5, and 5 μM, respectively. nd: not determined
with 10 μM tubulin, compound 3c was highly potent, yielding
an IC50 of 0.75 μM, almost twice as active as CA-4. Derivatives
3e and 3f had IC50 values of 1.2 and 1.4 μM, comparable to the
value obtained with CA-4. Compounds 3d, 3i−j, and 3n were
less active as inhibitors of tubulin polymerization, with IC50
values of 1.8, 3.7, 2.5, and 2.0 μM, respectively, while 3g was,
relatively speaking, almost inactive. The order of inhibitory
effects on tubulin assembly was 3c > 3e = CA-4 > 3f > 3d > 3n
> 3j > 3i ≫ 3g. This order of activity as inhibitors of tubulin
assembly correlates well with their order of activity as
antiproliferative agents against Jurkat, CCRF-CEM, SEM, and
HeLa cells.
In the competition assay, compound 3c was again the most
active derivative, inhibiting colchicine binding by 92% versus
98% for CA-4. In the experiments summarized in Table 4, the
concentration of tubulin was 1.0 μM, while that of both the
inhibitors and [3H]colchicine was 5.0 μM. Inhibition of
colchicine binding by compounds 3e and 3f was lower, with
83% and 80% inhibition occurring with these agents. With the
exception of compounds 3j and 3n, a good correlation was
observed between antiproliferative activities, inhibition tubulin
polymerization, and inhibition of colchicine binding.
To further investigate if the new derivatives interfered with
the microtubule network, we examined the effects of 3c on
HeLa cells by immunofluorescence microscopy. Following a 24
h treatment with 3c at 50, 100, and 250 nM, the microtubule
network was substantially modified in comparison with the
untreated cells (Figure S1, Supporting Information). Alto-
gether, these results are consistent with the conclusion that the
antiproliferative activity of these compounds derived from an
interaction with the colchicine site of tubulin, and this
ultimately results in interference with microtubule assembly.
sensitivity of compound 3c in two multidrug-resistant cell
19
lines, one derived from a colon carcinoma (LovoDoxo
)
the
other derived from a lymphoblastic leukemia (CEMVbl‑100).20
Both these lines express high levels of the P-gp.19,20 As shown
in Table 3, compound 3c was almost equally potent toward
cells resistant to doxorubicin or vinblastine showing a resistance
Table 3. In Vitro Cell Growth Inhibitory Effects of
Compound 3c on Drug Resistant Cell Lines
a
IC50 (nM)
b
compd
LoVo
LoVoDoxo
resistance ratio
3c
0.9 0.1
95.6 43.2
CEM
1.1 0.5
11296 356
CEMVbl100
1.2
doxorubicin
118
b
resistance ratio
3c
0.21 0.4
1.0 0.3
1.2 0.5
193 39
5.7
vinblastine
193
a
IC50 = compound concentration required to inhibit tumor cell
proliferation by 50%. Data are presented as the mean SE from the
dose−response curves of two independent experiments performed in
triplicate. The values express the ratio between IC50 determined in
b
resistant and nonresistant cell lines.
D
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Molecular Modeling. To rationalize the experimental data
observed for 3a−n, we performed a series of molecular docking
simulations of these compounds in the colchicine site of
tubulin. The proposed binding mode for 3c is very similar to
the one presented by the co-crystallized DAMA−colchicine
(Figure 2A). In particular, it is possible to observe how the
the structure obtained experimentally by X-ray crystallography
(Figure 2C).
Analysis of 3-Arylamino 1,2,4-Triazole Derivatives for
Effects on the Cell Cycle. The effects of a 24 h treatment with
different concentrations of 3c on cell cycle progression in HeLa
and Jurkat cells were determined by flow cytometry (Figure
3A,B). The compound caused a significant G2/M arrest in a
Figure 3. Percentage of cells in each phase of the cell cycle in HeLa
(A) and Jurkat cells (B) treated with 3c at the indicated concentrations
for 24 h. Cells were fixed and labeled with PI and analyzed by flow
cytometry as described in the Experimental Section. Data are
represented as mean SEM of three independent experiments.
concentration-dependent manner in the cell lines tested, with a
rise in G2/M cells occurring at a concentration as low as 60
nM, while, at higher concentrations, more than 70% of the cells
were arrested in G2/M. The cell cycle arrest in the G2/M
phase was accompanied by a corresponding reduction in cells in
the other phases of the cell cycle. In particular, the G1 phase
decreased in both cell lines, whereas S phase reduction was
mainly evident in Jurkat cells.
We next studied the association between 3c-induced G2/M
arrest and alterations in expression of proteins that regulate cell
division. As shown in Figure 4 in HeLa cells, a 24 h treatment
with 3c at concentrations lower than 100 nM caused no
significant variation in cyclin B expression, which, in association
with cdc2, controls both entry into and exit from mitosis,21,22
while at 100 and 250 nM, we observed a clear increase in the
cyclin B1 band. After a 48 h treatment, cyclin B1 expression
decreased. More importantly, p-cdc2Tyr15 expression decreased
after either 24 or 48 h of treatment. However, no major
changes in the expression of phosphatase cdc25c were
observed. These results indicate that arrest at G2/M induced
by 3c is caused by an immediate block of cyclin B1 activity,
followed by its accumulation, leading to a persistent and
marked decrease of p-cdc2Tyr15 more detectable at the highest
concentrations (100−250 nM) examined.
Figure 2. (A) Proposed binding for 3c (in gray) in the colchicine site.
Co-crystallized DAMA−colchicine is shown in green. (B) Representa-
tion of the binding mode of 3c in the colchicine site, with a summary
of the SARs observed for the reported series of compounds. (C)
Superposition of the conformation obtained from the docking
simulations (in gray) and the crystal structure (in yellow) of 3c.
trimethoxyphenyl ring is in proximity of Cys241, while the
phenyl ring occupies a hydrophobic region deep in the binding
site, establishing a series of interactions with Met259, Thr314,
and Lys352. Indeed, this subpocket is relatively small and, while
the methyl substituent on the phenyl ring of 3c is able to fit in
properly, larger groups, such as the isopropyl (3g) or the n-
butyl (3h), cannot be accommodated into it. In these cases, the
docking simulations are not able to generate a reasonable
binding pose. The docking results are in accordance with the
experimental data, and they provide a possible structural
justification of the SARs observed (Figure 2B). Finally, it is
interesting to note that the 3c binding conformation generated
in the docking simulation is very similar to the conformation of
In addition to the analysis of proteins that control cell cycle
checkpoints, we also examined the expression of the tumor
suppressor p53 after treatment of Hela cells with 3c. It is well-
known that prolonged mitotic arrest induces DNA damage and,
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Figure 4. Effects of 3c on G2/M regulatory proteins (A) and on p53,
p21, and γH2AX expression (B). HeLa cells were treated for 24 or 48
h with the indicated concentration of 3c. The cells were harvested and
lysed for the detection of cyclin B1, p-cdc2Tyr15, and cdc25C (A) or
p53, p21, and γH2AX expression (B) by Western blot analysis. To
confirm equal protein loading, each membrane was stripped and
reprobed with anti-β-actin antibody.
consequently, p53 up-regulation.23,24 As shown in Figure 4B,
we detected in a concentration-dependent manner an increase
in p53 expression that is particularly evident after 48 h of
treatment. At the same time, we also observed a marked
increase in the expression of phosphorylated histone γH2A.X,
which is an early sensitive indicator of DNA damage.25
Interestingly, the expression of the cyclin-dependent kinase
inhibitor p21, which was previously demonstrated to have an
antiapoptotic role,26 decreased both after 24 and 48 h of
treatment.
Figure 5. Flow cytometric analysis of apoptotic cells after treatment of
HeLa cells (A) or Jurkat cells (B) with 3c at the indicated
concentrations after incubation for 24 or 48 h. The cells were
harvested and labeled with annexin-V-FITC and PI and analyzed by
flow cytometry. Data are represented as mean
SEM of three
independent experiments.
Compound 3c Induces Apoptosis. To characterize the
mode of cell death induced by 3c, a biparametric cytofluori-
metric analysis was performed using propidium iodide (PI),
which stains DNA and enters only dead cells, and fluorescent
immunolabeling of the protein annexin-V, which binds to
phosphatidylserine (PS) in a highly selective manner.27 Dual
staining for annexin-V and with PI permits discrimination
between live cells (annexin-V−/PI−), early apoptotic cells
(annexin-V+/PI−), late apoptotic cells (annexin-V+/PI+), and
necrotic cells (annexin-V−/PI+). As shown in Figure 5, both
HeLa (panel A) and Jurkat cells (panel B) treated with the two
compounds for 24 h showed an accumulation of annexin-V
positive cells that further increased after 48 h in comparison
with the untreated cells. Analogous results were also obtained
for compound 3e (see Figure S2, Supporting Information).
Compound 3c Induces Caspase-Dependent Apoptosis.
We then analyzed by Western blot which proteins are involved
on the triggered apoptotic pathway upon treatment with 3c.
HeLa cells were treated with different concentrations of 3c for
24 or 48 h (Figure 6). Interestingly, 3c induced activation of
the initiator caspase-9 in a time and concentration-dependent
manner. We observed also an activation of the effector caspase-
3 and cleavage of its substrate PARP. Furthermore, the
antiapoptotic protein Bcl-2 was decreased by treatment with
3c in a time dependent manner, while the expression of a pro-
apoptotic protein, Bax, was slightly increased only at 250 nM
and at 48 h.
Figure 6. Western blot analysis of caspase-3, cleaved caspase-9, PARP
Bcl-2, and Bax after treatment of HeLa cells with 3c at the indicated
concentrations and for the indicated times. To confirm equal protein
loading, each membrane was stripped and reprobed with anti-β-actin
antibody.
tubulin binding agents show antivascular effects against tumor
endothelium,30 including CA-4, and for that reason we
evaluated 3c for effects on endothelial cells in vitro. We used
human umbilical vein endothelial cells (HUVECs) as a model
for angiogenesis/vasculogenesis processes in vitro. Endothelial
cell migration to the tumor site is one of the described
mechanisms of angiogenesis.31 Inhibiting this mechanism could
be a strategy to arrest the development of tumor vasculature.
We evaluated cell motility by scratching a HUVEC monolayer
and monitoring the ability of cells to reclose the wound. As
shown in Figure 7A, 3c is very efficient in arresting cell motility.
The effect is statistically significant after a 24 h incubation, at all
the tested concentrations (5, 10, 25 nM), while after 6 h, 3c
significantly inhibited cell motility at 10 and 25 nM, with a
dose−response relationship observed (Figure 7B).
Derivative 3c has Antivascular Effects in Vitro. Tumor
growth requires an oxygen supply, so the tumor microenviron-
ment stimulates the development of additional blood vessels.28
Recent antitumor strategies are based on the use of
chemotherapeutics with antiangiogenic or antivascular drugs
in order to increase the efficacy of the treatment.29 Many
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Figure 7. Compound 3c has antivascular activity in vitro. (A) Confluent HUVECs in a monolayer were wounded, and cells treated with different
concentrations of 3c and at different times were photographed, 7× magnification; bar = 100 μm. The dotted lines define the areas lacking cells. (B)
The graph shows the quantitative effect of 3c. Migration was quantified by measuring the gap closure at the indicated times as shown in A. Data are
represented as mean SEM of three independent experiments. *p < 0.05, **p < 0.01 vs control. (C) Inhibition of endothelial cell capillary-like
tubule formation by 3c. Tubule formation on Matrigel was carried out as described in the Experimental Section. Representative pictures (10×
magnification; bar = 100 μm) of preformed capillary-like tubules treated with increasing concentrations of 3c for 1 or 3 h. (D) Quantitative analysis
of the effects of 3c on the dimensional and topological parameters of the preformed capillary-like tubule networks after a 3 h treatment. Data were
represented as mean SEM of three independent experiments. *p < 0.05, **p < 0.01 vs. control.
To support the antivascular activity of 3c, we evaluated the
ability of the compound to disrupt the “tubule-like” structures,
formed by HUVECs seeded on Matrigel. Matrigel is an
extracellular matrix, rich in pro-angiogenic factors that stimulate
single endothelial cells to assume an extended shape. The
overall effect results in a reticulum similar to a capillary
network.
As shown in Figure 7C, after a 1 h incubation, 25 nM 3c
visibly disrupted the network of HUVECs, as compared with
the control. After 3 h, all the tested concentrations were
effective in altering the tubule-like structures. An image
analysis32 was performed to obtain a quantitative measurement
of the total length of the tubules, the area and the number of
meshes, the percent of area covered by HUVECs, and the
number of branching points (Figure 7D) after a 3 h treatment.
The results indicate that the effects on endothelial cells
induced by 3c are similar to those observed after CA-4
treatment, in the same experimental conditions, carried out by
our group.33
Evaluation of Antitumor Activity of Compound 3c in Vivo.
To evaluate the in vivo antitumor activity of 3c, a syngeneic
hepatocellular carcinoma model in mice was used.34 Tumors
were established by subcutaneous injection of BNL 1ME
A.7R.1 cells into the backs of Balb/c mice. In preliminary
experiments in vitro, we determined that both compound 3c
and CA-4, used as a reference compound, showed similar,
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potent cytotoxic activity (3c IC50 = 3.4 1.1 nM; CA-4 IC50
=
compound CA-4 against all cancer cell lines. The para-position
tolerates small substituents, such as methyl (3c) or ethyl (3f)
groups, while the inactivity of i-Pr or n-Bu derivatives 3g and
3h, respectively, indicates that bulky substituents were
detrimental for activity. Placing the methyl group in the meta-
position (3d) led to a dramatic drop in potency as compared
with the para-isomer 3c, even though we observed excellent
activity with the meta,para-dimethyl derivative 3e. Compound
3c was the most potent inhibitor of tubulin polymerization and
of colchicine binding (IC50 = 0.75 μM for assembly, 92%
inhibition of the binding of 5 μM colchicine), and the
antiproliferative activity of 3c, in terms of IC50s, ranged from
0.21 to 3.2 nM in the seven cell tumor lines examined, values
lower than that of previously published isosteric analogues.11 In
addition, in preliminary experiment 3c had low toxicity in
nontumoral cells and is active also in drug-resistant cell lines.
Although 3c was almost twice as active as CA-4 as an inhibitor
of tubulin polymerization, these two compounds showed
similar activity as inhibitors of colchicine binding. Moreover,
in a detailed series of biological assays, we clearly demonstrated
that 3c induced caspase-dependent apoptosis and late DNA
damage and p53 induction. Compound 3c, in addition to its
ability to inhibit tubulin polymerization, efficiently targeted
endothelial cells, acting as a VDA. More importantly, in vivo
experiments showed that this compound was able to
significantly reduce the growth of a syngeneic tumor model
in mice, indicating that it is a very promising anticancer
compound that warrants further evaluation for its potential
clinical use.
1.1
0.5 nM) against BNL 1ME A.7R.1 cells. Once the
allografts reached a measurable size (about 100 mm3), 20 mice
were randomly assigned to one of four groups. In two of the
groups, compound 3c was injected intraperitoneally at doses of
5 and 10 mg/kg, respectively. In a third group, CA-4P was
injected at 5 mg/kg, while the fourth group was used as a
control. As depicted in Figure 8A, compound 3c caused a
Figure 8. Inhibition of mouse allograft tumor growth in vivo by
compound 3c. (A) Male mice were injected subcutaneously in their
dorsal region with 107 BNL 1MEA.7R.1 cells, a syngeneic
hepatocellular carcinoma cell line. Tumor-bearing mice were
administered the vehicle, as control, or the indicated doses of 3c or
CA-4P as reference compound at the concentration of 5 mg/kg. Daily
injections were given intraperitoneally starting on day 1. The figure
shows the average measured tumor volumes (A) and body weights of
the mice (B) recorded at the beginning and at the end of the
treatments. Data are presented as mean SEM of tumor volume and
body weight at each time point for five animals per group. *p < 0.05,
**p < 0.01 vs. control.
EXPERIMENTAL SECTION
■
Chemistry: Materials and Methods. 1H and 13C NMR data were
obtained with a Varian VXR 200 spectrometer and a Varian Mercury
Plus 400 spectrometer, respectively. Peak positions are given in parts
per million (δ) downfield, and J values are given in hertz. Positive-ion
electrospray ionization (ESI) mass spectra were recorded on a double-
focusing Finnigan MAT 95 instrument with BE geometry. Melting
points (mp) were determined on a Buchi-Tottoli apparatus and are
uncorrected. The purity of tested compounds was determined by
combustion elemental analyses conducted by the Microanalytical
Laboratory of the Chemistry Department of the University of Ferrara
with a Yanagimoto MT-5 CHN recorder elemental analyzer. All tested
compounds yielded data consistent with a purity of at least 95% as
compared with the theoretical values. All reactions were carried out
under an inert atmosphere of dry nitrogen unless otherwise indicated.
TLC was carried out using glass plates coated with silica gel 60 F254 by
Merck, and compounds were visualized by UV detection or with
aqueous KMnO4. Flash column chromatography was performed using
230−400 mesh silica gel and the indicated solvent system. Organic
solutions were dried over anhydrous Na2SO4. Solvents and reagents
that are commercially available were purchased from Aldrich (Sigma-
Aldrich) or Alfa Aesar (Johnson Matthey Company) and were used
without further purification unless otherwise noted.
significant reduction in tumor growth, as compared with
administration of vehicle, at 10 but not 5 mg/kg. The effect of 5
mg/kg of CA-4P was not as great as that of 10 mg/kg of 3c, but
the CA-4P effect was still significant relative to the control.
During the treatment period, only a small decrease in body
weight occurred in the 3c-treated animals (Figure 8B).
CONCLUSIONS
■
The bioisosteric equivalence between thiazole and 1,2,4-triazole
prompted us to synthesize a series of 1-(3′,4′,5′-trimethox-
ybenzoyl)-3-arylamino-5-amino 1,2,4-triazole derivatives with
general formula 3, in which the 1,2,4-triazole ring replaced the
thiazole system of previously published analogues with general
structure 2. The substitution pattern on the phenyl of the
arylamino moiety had variable effects.
General Procedure A for the Synthesis of Compounds 5a−n.
To a solution of the appropriate aniline derivative (3 mmol, 1 equiv)
in 2-propanol (10 mL) was added dimethyl cyanodithioimidocar-
bonate 4 (439 mg, 3 mmol), and the mixture was refluxed for 16 h.
After this time, the solvent was removed under reduced pressure, and
the resulting residue was suspended in ethyl ether (10 mL) and filtered
to furnish the final compound 5a−n used for the next reaction without
any purification.
Compound 3c, bearing a p-toluidino moiety at the C-3
position of 1,2,4-triazole ring, its p-ethyl homologue 3f, and the
m,p-dimethyl analogue 3e exhibited the greatest antiprolifer-
ative activity among the tested compounds, with IC50 values of
0.21−3.2, 0.21−6.0, and 0.4−4.0 nM, respectively. These
results were superior or comparable with those of the reference
(Z)-Methyl N′-Cyano-N-phenylcarbamimidothioate (5a). Synthe-
sized according to method A, derivative 5a was obtained as a white
1
solid (yield 71%); mp 192−193 °C. H NMR (CDCl3) δ: 2.46 (s,
3H), 7.38 (m, 5H), 7.92 (bs, 1H). MS (ESI): [M + 1]+ = 192.3.
H
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(Z)-Methyl N′-Cyano-N-(4-fluorophenyl)carbamimidothioate
(5b). Synthesized according to method A, compound 5b was obtained
General Procedure B for the Synthesis of Compounds 6a−n.
To a stirred suspension of derivative 5a−n (2 mmol) in dry THF (10
mL) was added hydrazine monohydrate (0.1 mL, 2 mmol, 1 equiv),
and the reaction mixture was heated under reflux for 6 h. After this
time, the suspension was evaporated in vacuo to dryness, and the
residue was suspended with ethyl ether (10 mL) and stirred for 10
min. The resultant solid was collected by filtration and then used for
the next reaction without any purification.
1
as a gray solid (yield 78%); mp 216−218 °C. H NMR (CDCl3) δ:
2.45 (s, 3H), 7.12 (t, J = 8.0 Hz, 2H), 7.29 (m, 2H), 7.94 (bs, 1H). MS
(ESI): [M + 1]+ = 210.3.
(Z)-Methyl N′-Cyano-N-(p-tolyl)carbamimidothioate (5c). Syn-
thesized according to method A, derivative 5c was isolated as a white
1
solid (yield 67%); mp 152−154 °C. H NMR (CDCl3) δ: 2.38 (s,
N3-Phenyl-1H-1,2,4-triazole-3,5-diamine (6a). Synthesized accord-
ing to general procedure B, derivative 6a was obtained as a white solid
(yield 74%); mp 160−161 °C. 1H NMR (DMSO-d6) δ: 5.83 (bs, 2H),
6.71 (t, J = 7.8 Hz, 1H), 7.14 (t, J = 7.8 Hz, 2H), 7.46 (d, J = 8.4 Hz,
2H), 8.57 (bs, 1H), 11.1 (bs, 1H). MS (ESI): [M + 1]+ = 176.1.
N3-(4-Fluorophenyl)-1H-1,2,4-triazole-3,5-diamine (6b). Synthe-
sized according to general procedure B, derivative 6b was obtained as a
3H), 2.43 (s, 3H), 7.14 (dd, J = 9.0 and 2.6 Hz, 2H), 7.19 (dd, J = 9.0
and 2.6 Hz, 2H), 7.97 (bs, 1H). MS (ESI): [M + 1]+ = 206.1.
(Z)-Methyl N′-Cyano-N-(3-methylphenyl)carbamimidothioate
(5d). Synthesized according to method A, derivative 5d was obtained
1
as a white solid (yield 52%); mp 148−150 °C. H NMR (CDCl3) δ:
2.39 (s, 3H), 2.44 (s, 3H), 6.81 (m, 1H), 6.93 (s, 1H), 7.03 (m, 2H),
7.82 (bs, 1H). MS (ESI): [M + 1]+ = 206.2.
( Z ) - M e t h y l N ′ - c y a n o - N - ( 3 , 4 - d i m e t h y l p h e n y l ) -
carbamimidothioate (5e). Synthesized according to method A,
derivative 5e was obtained as a white solid (yield 87%); mp 138−
1
white solid (yield 83%); mp 216−218 °C. H NMR (DMSO-d6) δ:
5.84 (bs, 2H), 6.98 (t, J = 8.8 Hz, 2H), 7.48 (m, 2H), 8.61 (bs, 1H),
11.1 (bs, 1H). MS (ESI): [M + 1]+ = 194.2.
1
140 °C. H NMR (CDCl3) δ: 2.27 (s, 6H), 2.43 (s, 3H), 7.05 (m,
N3-(p-Tolyl)-1H-1,2,4-triazole-3,5-diamine (6c). Synthesized ac-
cording to general procedure B, compound 6c was obtained as a white
2H), 7.14 (d, J = 7.8 Hz, 1H), 7.84 (bs, 1H). MS (ESI): [M + 1]+ =
220.2.
1
solid (yield 92%); mp 185−187 °C. H NMR (DMSO-d6) δ: 2.19 (s,
(Z)-Methyl N′-Cyano-N-(4-ethylphenyl)carbamimidothioate (5f).
3H), 5.81 (bs, 2H), 6.93 (d, J = 8.6 Hz, 2H), 7.36 (dd, J = 8.6 Hz, 2H),
8.42 (bs, 1H), 11.0 (bs, 1H). MS (ESI): [M + 1]+ = 190.2.
N3-(m-Tolyl)-1H-1,2,4-triazole-3,5-diamine (6d). Synthesized ac-
cording to general procedure B, compound 6d was obtained as a white
solid (yield >95%); mp 112−114 °C. 1H NMR (DMSO-d6) δ: 2.21 (s,
3H), 5.81 (bs, 2H), 6.51 (d, J = 7.2 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H),
7.24 (d, J = 7.2 Hz, 1H), 7.33 (s, 1H), 8.49 (bs, 1H), 11.1 (bs, 1H).
MS (ESI): [M + 1]+ = 220.2.
Synthesized according to method A, compound 5f was obtained as a
1
white solid (yield 68%); mp 159−161 °C. H NMR (CDCl3) δ: 1.25
(t, J = 7.6 Hz, 3H), 2.43 (s, 3H), 2.62 (q, J = 7.6 Hz, 2H), 7.17 (dd, J =
9.0 and 2.8 Hz, 2H), 7.24 (dd, J = 9.0 and 2.8 Hz, 2H), 7.90 (bs, 1H).
MS (ESI): [M + 1]+ = 220.4.
(Z)-Methyl N′-Cyano-N-(4-isopropylphenyl)carbamimidothioate
(5g). Synthesized according to method A, derivative 5g was obtained
1
as a white solid (yield 64%); mp 129−131 °C. H NMR (CDCl3) δ:
N3-(3,4-Dimethylphenyl)-1H-1,2,4-triazole-3,5-diamine (6e). Syn-
thesized according to general procedure B, derivative 6e was obtained
as a white solid (yield >95%); mp 180−182 °C. 1H NMR (DMSO-d6)
δ: 2.10 (s, 3H), 2.13 (s, 3H), 5.77 (bs, 2H), 6.86 (d, J = 8.2 Hz, 1H),
7.18 (d, J = 8.2 Hz, 1H), 7.26 (s, 1H), 8.35 (bs, 1H), 11.0 (bs, 1H).
MS (ESI): [M + 1]+ = 204.3.
1.26 (d, J = 7.0 Hz, 6H), 2.44 (s, 3H), 2.93 (m, 1H), 6.89 (dd, J = 8.4
and 2.4 Hz, 2H), 7.22 (dd, J = 8.4 and 2.4 Hz, 2H), 7.93 (bs, 1H). MS
(ESI): [M + 1]+ = 234.4.
(Z)-Methyl N-(4-n-Butylphenyl)-N′-cyanocarbamimidothioate
(5h). Synthesized according to method A, derivative 5h was obtained
1
as a white solid (yield 63%); mp 149−151 °C. H NMR (CDCl3) δ:
N3-(4-Ethylphenyl)-1H-1,2,4-triazole-3,5-diamine (6f). Synthe-
sized according to general procedure B, compound 6f was obtained
0.93 (t, J = 7.4 Hz, 3H), 1.33 (m, 2H), 1.59 (m, 2H), 2.43 (s, 3H),
2.63 (t, J = 7.8 Hz, 2H), 7.17 (dd, J = 8.8 and 2.4 Hz, 2H), 7.24 (dd, J
= 8.8 and 2.4 Hz, 2H), 7.93 (bs, 1H). MS (ESI): [M + 1]+ = 248.4.
(Z)-Methyl N′-Cyano-N-(4-methoxyphenyl)carbamimidothioate
(5i). Synthesized according to method A, compound 5i was obtained
as a purple solid (yield 91%); mp 193−195 °C. 1H NMR (CDCl3) δ:
2.41 (s, 3H), 3.83 (s, 3H), 6.89 (d, J = 8.8 Hz, 2H), 7.18 (d, J = 8.8
Hz, 2H), 7.97 (bs, 1H). MS (ESI): [M + 1]+ = 222.1.
1
as a white solid (yield 92%); mp 187−189 °C. H NMR (DMSO-d6)
δ: 1.13 (t, J = 7.6 Hz, 3H), 2.47 (q, J = 7.6 Hz, 2H), 5.80 (bs, 2H),
6.96 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 8.42 (bs, 1H), 11.0
(bs, 1H). MS (ESI): [M + 1]+ = 204.3.
N3-(4-Isopropylphenyl)-1H-1,2,4-triazole-3,5-diamine (6g). Syn-
thesized according to general procedure B, compound 6g was obtained
1
(Z)-Methyl N′-Cyano-N-(3-methoxyphenyl)carbamimidothioate
(5j). Synthesized according to method A, compound 5j was obtained
as a gray solid (yield 67%); mp 161−163 °C. 1H NMR (DMSO-d6) δ:
2.69 (s, 3H), 3.75 (s, 3H), 6.82 (ddd, J = 7.4, 2.4, and 1.2 Hz, 1H),
7.00 (m, 2H), 7.03 (m, 1H), 10.1 (bs, 1H). MS (ESI): [M + 1]+ =
222.1.
( Z ) - M e t hy l N′ - C y a n o - N - ( 3 , 4 - d i m e t h o xy p h e n y l ) -
carbamimidothioate (5k). Synthesized according to method A,
derivative 5k was obtained as a purple solid (yield 69%); mp 178−
180 °C. 1H NMR (CDCl3) δ: 2.42 (s, 3H), 3.89 (s, 3H), 3.90 (s, 3H),
6.86 (m, 3H), 7.95 (bs, 1H). MS (ESI): [M + 1]+ = 252.3.
(Z)-Methyl N′-Cyano-N-(3,4,5-trimethoxyphenyl)-
carbamimidothioate (5l). Synthesized according to method A,
compound 5l was obtained as a gray solid (yield 68%); mp 150−
151 °C. 1H NMR (CDCl3) δ: 2.45 (s, 3H), 3.81 (s, 3H), 3.86 (s, 6H),
6.54 (s, 2H), 7.96 (bs, 1H). MS (ESI): [M + 1]+ = 282.3.
as a white solid (yield 60%); mp 156−158 °C. H NMR (DMSO-d6)
δ: 1.17 (d, J = 6.8 Hz, 6H), 2.76 (m, 1H), 5.77 (bs, 2H), 6.99 (d, J =
8.6 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 8.44 (bs, 1H), 11.0 (bs, 1H).
MS (ESI): [M + 1]+ = 218.2.
N3-(4-n-Butylphenyl)-1H-1,2,4-triazole-3,5-diamine (6h). Synthe-
sized according to general procedure B, derivative 6h was obtained as a
1
white solid (yield 66%); mp 177−179 °C. H NMR (DMSO-d6) δ:
0.88 (t, J = 7.0 Hz, 3H), 1.26 (m, 2H), 1.48 (m, 2H), 2.41 (t, J = 7.6
Hz, 2H), 5.80 (bs, 2H), 6.94 (d, J = 8.2 Hz, 2H), 7.36 (dd, J = 8.2 Hz,
2H), 8.43 (bs, 1H), 11.1 (bs, 1H). MS (ESI): [M + 1]+ = 232.3.
N3-(4-Methoxyphenyl)-1H-1,2,4-triazole-3,5-diamine (6i). Synthe-
sized according to general procedure B, derivative 6i was obtained as a
gray solid (yield 88%); mp 200−201 °C. 1H NMR (DMSO-d6) δ: 3.67
(s, 3H), 5.76 (bs, 2H), 6.74 (dd, J = 7.0 and 2.2 Hz, 2H), 7.39 (dd, J =
7.0 and 2.2 Hz, 2H), 8.32 (bs, 1H), 11.0 (bs, 1H). MS (ESI): [M + 1]+
= 206.3.
(Z)-Methyl N′-Cyano-N-(4-ethoxyphenyl)carbamimidothioate
N3-(3-Methoxyphenyl)-1H-1,2,4-triazole-3,5-diamine (6j). Synthe-
sized according to general procedure B, derivative 6j was obtained as a
(5m). Synthesized according to method A, derivative 5m was obtained
1
as a gray solid (yield 82%); mp 165−167 °C. H NMR (CDCl3) δ:
1
red solid (yield >95%); mp 117−119 °C. H NMR (DMSO-d6) δ:
1.43 (t, J = 7.2 Hz, 3H), 2.41 (s, 3H), 4.02 (q, J = 7.2 Hz, 2H), 6.88
(dd, J = 7.0 and 2.2 Hz, 2H), 7.17 (dd, J = 7.0 and 2.2 Hz, 2H), 7.89
(bs, 1H). MS (ESI): [M + 1]+ = 236.2.
3.76 (s, 3H), 5.78 (bs, 2H), 6.88 (dd, J = 7.4 and 2.4 Hz, 1H), 7.02 (m,
2H), 7.05 (m, 1H), 8.47 (bs, 1H), 11.1 (bs, 1H). MS (ESI): [M + 1]+
= 206.2.
(Z)-Methyl N-Benzo[d][1,3]dioxol-5-yl-N′-cyanocarbamimido-
N3-(3,4-Dimethoxyphenyl)-1H-1,2,4-triazole-3,5-diamine (6k).
Synthesized according to general procedure B, derivative 6k was
thioate (5n). Synthesized according to method A, derivative 5n was
1
obtained as a white solid (yield 73%); mp 191−193 °C. H NMR
1
obtained as a purple solid (yield 72%); mp 158−160 °C. H NMR
(CDCl3) δ: 2.42 (s, 3H), 6.04 (s, 2H), 6.79 (m, 3H), 7.81 (bs, 1H).
MS (ESI): [M + 1]+ = 236.2.
(DMSO-d6) δ: 3.66 (s, 3H), 3.75 (s, 3H), 5.75 (bs, 2H), 6.74 (d, J =
I
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8.8 Hz, 1H), 6.98 (m, 1H), 7.23 (d, J = 2.4 Hz, 1H), 8.32 (bs, 1H),
11.2 (bs, 1H). MS (ESI): [M + 1]+ = 236.2.
(3-(3,4-Dimethylphenylamino)-5-amino-1H-1,2,4-triazol-1-yl)-
(3,4,5-trimethoxyphenyl) Methanone (3e). Synthesized according to
method C, compound 3e was obtained as a yellow solid (yield 48%);
mp 169−171 °C. 1H NMR (DMSO-d6) δ: 2.12 (s, 6H), 3.80 (s, 3H),
3.85 (s, 6H), 6.93 (d, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.35 (d, J = 8.0
Hz, 1H), 7.61 (s, 2H), 7.78 (bs, 2H), 9.14 (s, 1H). 13C NMR (DMSO-
d6) δ: 18.61, 19.75, 55.98 (2C), 60.22, 108.51 (2C), 114.01, 117.93,
127.45, 127.69, 129.42, 136.11, 138.74, 141.17, 152.03 (2C), 157.38,
158.21, 165.57. MS (ESI): [M]+ = 397.7. Anal. (C20H23N5O4) C, H,
N.
(3-(4-Ethylphenylamino)-5-amino-1H-1,2,4-triazol-1-yl)(3,4,5-tri-
methoxyphenyl) Methanone (3f). Synthesized according to method
C, compound 3f was obtained as a yellow solid (yield 54%); mp 194−
196 °C. 1H NMR (DMSO-d6) δ: 1.14 (t, J = 8.4 Hz, 3H), 3.51 (d, J =
8.4 Hz, 2H), 3.78 (s, 3H), 3.97 (s, 6H). 7.02 (d, J = 8.6 Hz, 2H), 7.46
(d, J = 8.6 Hz, 2H), 7.68 (s, 2H), 7.81 (bs, 2H), 8.22 (s, 1H). 13C
NMR (DMSO-d6) δ: 16.35, 31.15, 56.41 (2C), 61.68, 109.07 (2C),
127.14 (2C), 127.66, 128.22 (2C), 135.97, 139.08, 141.76, 152.50
(2C), 156.03, 158.74, 165.78. MS (ESI): [M]+ = 397.5. Anal.
(C21H26N5O4) C, H, N.
N3-(3,4,5-Trimethoxyphenyl)-1H-1,2,4-triazole-3,5-diamine (6l).
Synthesized according to general procedure B, derivative 6l was
1
obtained as a white solid (yield 68%); mp 241−243 °C. H NMR
(DMSO-d6) δ: 3.56 (s, 3H), 3.70 (s, 6H), 5.84 (bs, 2H), 6.90 (s, 2H),
8.43 (bs, 1H), 11.1 (bs, 1H). MS (ESI): [M + 1]+ = 266.3.
N3-(4-Ethoxyphenyl)-1H-1,2,4-triazole-3,5-diamine (6m). Synthe-
sized according to general procedure B, derivative 6m was obtained as
a purple solid (yield 94%); mp 200−201 °C. 1H NMR (DMSO-d6) δ:
1.28 (t, J = 7.0 Hz, 3H), 3.93 (q, J = 7.0 Hz, 2H), 5.76 (bs, 2H), 6.72
(dd, J = 7.0 and 2.0 Hz, 2H), 7.38 (dd, J = 7.0 and 2.0 Hz, 2H), 8.31
(bs, 1H), 11.0 (bs, 1H). MS (ESI): [M + 1]+ = 220.2.
N3-(Benzo[d][1,3]dioxol-5-yl)-1H-1,2,4-triazole-3,5-diamine (6n).
Synthesized according to general procedure B, compound 6n was
1
obtained as a brown solid (yield 73%); mp 206−208 °C. H NMR
(DMSO-d6) δ: 5.80 (bs, 2H), 5.88 (s, 2H), 6.69 (d, J = 8.2 Hz, 1H),
6.87 (dd, J = 8.2 and 2.2 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 8.45 (bs,
1H), 11.1 (bs, 1H). MS (ESI): [M + 1]+ = 220.2.
General Procedure C for the Synthesis of Compounds 3a−n
and 7a−n. To a stirred solution of the appropriate 1,2,4-triazole 6a−
n (1 mmol) in dry pyridine (10 mL) cooled at −5 °C was added
3′,4′,5′-trimethoxybenzoyl chloride (230 mg, 1.1 mol, 1.1 equiv) in
small portions. The reaction mixture was kept for 30 min at −5 °C and
then overnight at room temperature. Pyridine was then removed by
evaporation under reduced pressure. To the residue was added
CH2Cl2, and the organic phase was washed with saturated aq
NaHCO3, water, and brine and dried over Na2SO4. The reaction
mixture was filtered, and the solvent was removed in vacuo. The crude
residue was purified by column chromatography on silica gel using a
mixture of CH2Cl2−methanol (9.5:0.5) as eluent to separate the two
regioisomeric 3′,4′,5′-trimethoxybenzoyl triazoles 3a−n and 7a−n,
with the former compounds 3a−n characterized by the lower Rf.
(3-(Phenylamino)-5-amino-1H-1,2,4-triazol-1-yl)(3,4,5-trimethox-
yphenyl) Methanone (3a). Synthesized according to method C,
derivative 3a was obtained as a yellow solid (yield 56%); mp 181−183
°C. 1H NMR (DMSO-d6) δ: 3.79 (s, 3H), 3.86 (s, 6H), 6.86 (t, J = 7.2
Hz, 1H), 7.22 (t, J = 8.4 Hz, 2H), 7.56 (d, J = 7.6 Hz, 2H), 7.68 (s,
2H), 7.93 (bs, 2H), 9.35 (s, 1H). 13C NMR (DMSO-d6) δ: 55.96
(2C), 60.23, 108.66 (2C), 116.57 (2C), 120.37, 127.17, 128.57 (2C),
140.89, 141.37, 152.07 (2C), 157.58, 158.19, 165.37. MS (ESI): [M]+
= 369.8. Anal. (C18H19N5O4) C, H, N.
(3-(4-Isopropylphenylamino)-5-amino-1H-1,2,4-triazol-1-yl)-
(3,4,5-trimethoxyphenyl) Methanone (3g). Synthesized according to
method C, derivative 3g was obtained as a yellow solid (yield 52%);
mp 197−199 °C. H NMR (DMSO-d6) δ: 1.14 (d, J = 6.8 Hz, 6H),
2.80 (m, 1H), 3.77 (s, 3H), 3.87 (s, 6H), 7.06 (d, J = 8.2 Hz, 2H), 7.46
(d, J = 8.2 Hz, 2H), 7.68 (s, 2H), 7.81 (bs, 2H), 9.21 (s, 1H). 13C
NMR (DMSO-d6) δ: 24.06 (2C), 32.65, 55.95 (2C), 60.23, 108.65
(2C), 116.75 (2C), 120.39 (2C), 124.28, 126.24, 138.70, 140.25,
152.05 (2C), 157.58, 158.32, 165.32. MS (ESI): [M]+ = 411.7. Anal.
(C21H25N5O4) C, H, N.
1
(3-(4-Butylphenylamino)-5-amino-1H-1,2,4-triazol-1-yl)(3,4,5-tri-
methoxyphenyl) Methanone (3h). Synthesized according to method
C, compound 3h was obtained as a yellow solid (yield 54%); mp 134−
1
136 °C. H NMR (DMSO-d6) δ: 0.88 (t, J = 7.2 Hz, 3H), 1.29 (m,
2H), 1.50 (m, 2H), 2.44 (t, J = 7.6 Hz, 2H), 3.80 (s, 3H), 3.87 (s, 6H),
7.00 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.6 Hz, 2H), 7.68 (s, 2H), 7.81
(bs, 2H), 9.22 (s, 1H). 13C NMR (DMSO-d6) δ: 13.78, 21.66, 33.36,
34.09, 55.94 (2C), 60.23, 108.64 (2C), 116.67 (2C), 120.38, 127.21,
128.30 (2C), 134.05, 138.61, 152.05 (2C), 157.57, 158.29, 165.33. MS
(ESI): [M]+ = 425.8. Anal. (C22H27N5O4) C, H, N.
(3-(4-Methoxyphenylamino)-5-amino-1H-1,2,4-triazol-1-yl)-
(3,4,5-trimethoxyphenyl) Methanone (3i). Synthesized according to
method C, compound 3i was obtained as a yellow solid (yield 64%);
mp 196−197 °C. 1H NMR (DMSO-d6) δ: 3.67 (s, 3H), 3.77 (s, 3H),
3.84 (s, 6H), 6.77 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.66
(s, 2H), 7.79 (bs, 2H), 9.11 (s, 1H). 13C NMR (DMSO-d6) δ: 55.07,
55.85 (2C), 60.13, 108.53 (2C), 113.76 (2C), 127.81 (2C), 127.11,
134.24, 141.21, 151.95 (2C), 153.19, 157.48, 158.29, 165.16. MS
(ESI): [M]+ = 399.8. Anal. (C19H21N5O5) C, H, N.
(3-(3-Methoxyphenylamino)-5-amino-1H-1,2,4-triazol-1-yl)-
(3,4,5-trimethoxyphenyl) Methanone (3j). Synthesized according to
method C, compound 3j was obtained as a yellow solid (yield 42%);
mp 178−180 °C. 1H NMR (DMSO-d6) δ: 3.65 (s, 3H), 3.78 (s, 3H),
3.86 (s, 6H), 6.44 (m, 1H), 7.12 (m, 2H), 7.26 (s, 1H), 7.61 (s, 2H),
7.81 (bs, 2H), 9.32 (s, 1H). 13C NMR (DMSO-d6) δ: 54.76, 55.94
(2C), 60.19, 102.99, 105.19, 108.45 (2C), 109.37, 127.36, 129.29,
141.22, 142.01, 152.07 (2C), 157.45, 158.12, 159.78, 165.63. MS
(ESI): [M]+ = 399.6. Anal. (C19H21N5O5) C, H, N.
(3-(3,4-Dimethoxyphenylamino)-5-amino-1H-1,2,4-triazol-1-yl)-
(3,4,5-trimethoxyphenyl) Methanone (3k). Synthesized according to
method C, derivative 3k was obtained as a yellow solid (yield 51%);
mp 170−171 °C. 1H NMR (DMSO-d6) δ: 3.67 (s, 3H), 3.73 (s, 3H),
3.77 (s, 3H), 3.83 (s, 6H), 6.77 (d, J = 8.6 Hz, 1H), 7.08 (dd, J = 8.6
and 2.2 Hz, 1H), 7.22 (d, J = 2.2 Hz, 1H), 7.56 (s, 2H), 7.78 (bs, 2H),
9.09 (s, 1H). 13C NMR (DMSO-d6) δ: 55.16 (2C), 56.00 (2C), 60.20,
102.58, 108.25, 108.33 (2C), 112.61, 127.61, 134.95, 141.06, 142.85,
148.90, 152.02 (2C), 157.35, 158.28, 165.71. MS (ESI): [M]+ = 429.8.
Anal. (C20H23N5O6) C, H, N.
(3-(4-Fluorophenylamino)-5-amino-1H-1,2,4-triazol-1-yl)(3,4,5-
trimethoxyphenyl) Methanone (3b). Synthesized according to
method C, compound 3b was obtained as a yellow solid (yield
60%); mp 218−220 °C. 1H NMR (DMSO-d6) δ: 3.79 (s, 3H), 3.86 (s,
6H), 7.07 (t, J = 8.8 Hz, 2H), 7.56 (dd, J = 8.8 and 3.4 Hz, 2H), 7.61
(s, 2H), 7.83 (bs, 2H), 9.38 (s, 1H). 13C NMR (DMSO-d6) δ: 55.98
(2C), 60.22, 108.58 (2C), 114.99, 115.21, 117.81, 117.88, 127.15,
137.40, 141.35, 152.08 (2C), 155.17, 157.60, 158.19, 165.41. MS
(ESI): [M]+ = 387.7. Anal. (C18H18FN5O4) C, H, N.
(3-(4-Tolylamino)-5-amino-1H-1,2,4-triazol-1-yl)(3,4,5-trimethox-
yphenyl) Methanone (3c). Synthesized according to method C,
compound 3c was obtained as a yellow solid (yield 55%); mp 235−
1
237 °C. H NMR (DMSO-d6) δ: 2.22 (s, 3H), 3.79 (s, 3H), 3.88 (s,
6H), 6.97 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 7.68 (s, 2H),
7.81 (bs, 2H), 9.22 (s, 1H). 13C NMR (DMSO-d6) δ: 20.26, 95.96
(2C), 60.22, 108.63 (2C), 116.68 (2C), 120.36, 127.20, 128.87 (2C),
138.41, 141.32, 152.15 (2C), 157.56, 158.26, 165.33. MS (ESI): [M]+
= 383.5. Anal. (C19H21N5O4) C, H, N.
(3-(3-Tolylamino)-5-amino-1H-1,2,4-triazol-1-yl)(3,4,5-trimethox-
yphenyl) Methanone (3d). Synthesized according to method C,
derivative 3d was obtained as a yellow solid (yield 61%); mp 165−167
°C. 1H NMR (DMSO-d6) δ: 2.30 (s, 3H), 3.78 (s, 3H), 3.85 (s, 6H),
6.65 (d, J = 7.4 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.39 (d,
J = 7.8 Hz, 1H), 7.61 (s, 2H), 7.80 (bs, 2H), 9.26 (s, 1H). 13C NMR
(DMSO-d6) δ: 21.26, 55.99 (2C), 60.22, 108.52 (2C), 113.77, 117.16,
120.98, 127.40, 128.38, 137.70, 140.87, 141.20, 152.05 (2C), 157.40,
158.14, 165.63. MS (ESI): [M]+ = 383.5. Anal. (C19H21N5O4) C, H,
N.
(3-(3,4,5-Trimethoxyphenylamino)-5-amino-1H-1,2,4-triazol-1-
yl)(3,4,5-trimethoxyphenyl) Methanone (3l). Synthesized according
to method C, derivative 3l was obtained as a yellow solid (yield 48%);
J
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mp 172−174 °C. 1H NMR (DMSO-d6) δ: 3.56 (s, 3H), 3.58 (s, 3H),
3.75 (s, 6H), 3.79 (s, 6H), 6.91 (s, 2H), 7.41 (s, 2H), 7.75 (bs, 2H),
9.16 (s, 1H). 13C NMR (DMSO-d6) δ: 55.44 (2C), 56.08 (2C), 60.17
(2C), 94.92 (2C), 108.04 (2C), 128.01, 131.38, 137.16, 140.87, 151.98
(2C), 152.84 (2C), 157.09, 158.09, 166.29. MS (ESI): [M]+ = 459.8.
Anal. (C21H25N5O7) C, H, N.
2,5-diphenyltetrazolium bromide test as previously described.37 The
IC50 was defined as the compound concentration required to inhibit
cell proliferation by 50% in comparison with cells treated with the
maximum amount of DMSO (0.25%) and considered as 100%
viability.
Peripheral blood lymphocytes (PBL) from healthy donors were
obtained by separation on Lymphoprep (Fresenius KABI Norge AS)
gradient. After extensive washing, cells were resuspended (1.0 × 106
cells/mL) in RPMI-1640 with 10% fetal bovine serum and incubated
overnight. For cytotoxicity evaluations in proliferating PBL cultures,
nonadherent cells were resuspended at 5 × 105 cells/mL in growth
medium, containing 2.5 μg/mL PHA (Irvine Scientific). Different
concentrations of the test compounds were added, and viability was
determined 72 h later by the MTT test. For cytotoxicity evaluations in
resting PBL cultures, nonadherent cells were resuspended (5 × 105
cells/mL) and treated for 72 h with the test compounds, as described
above.
Effects on Tubulin Polymerization and on Colchicine
Binding to Tubulin. To evaluate the effect of the compounds on
tubulin assembly in vitro,38a varying concentrations of compounds
were preincubated with 10 μM bovine brain tubulin in 0.8 M
monosodium glutamate (pH adjusted to 6.6 with HCl in a 2.0 M stock
solution) at 30 °C and then cooled to 0 °C. After addition of 0.4 mM
GTP, the mixtures were transferred to 0 °C cuvettes in a recording
spectrophotometer and warmed to 30 °C. Tubulin assembly was
followed turbidimetrically at 350 nm. The IC50 was defined as the
compound concentration that inhibited the extent of assembly by 50%
after a 20 min incubation. The capacity of the test compounds to
inhibit colchicine binding to tubulin was measured as described,38b
except that the reaction mixtures contained 1 μM tubulin, 5 μM
[3H]colchicine, and 5 μM test compound.
Molecular Modeling. All molecular modeling studies were
performed on a MacPro dual 2.66 GHz Xeon running Ubuntu
12.04. The tubulin structure was downloaded from the PDB data bank
(http://www.rcsb.org/; PDB code 1SA0).39 Hydrogen atoms were
added to the protein, using the Protonate 3D routine of the Molecular
Operating Environment (MOE).40 Ligand structures were built with
MOE and minimized using the MMFF94x force field until a RMSD
gradient of 0.05 kcal mol−1 Å−1 was reached. The docking simulations
were performed using PLANTS.41
Flow Cytometric Analysis of Cell Cycle Distribution. HeLa or
Jurkat cells (5 ×105) were treated with different concentrations of the
test compounds for 24 h. After the incubation period, the cells were
collected, centrifuged, and fixed with ice-cold ethanol (70%). The cells
were then treated with lysis buffer containing RNase A and 0.1%
Triton X-100 and then stained with PI. Samples were analyzed on a
Cytomic FC500 flow cytometer (Beckman Coulter). DNA histograms
were analyzed using MultiCycle for Windows (Phoenix Flow
Systems).
Apoptosis Assay. Cell death was determined by flow cytometry of
cells double stained with annexin V/FITC and PI. The Coulter
Cytomics FC500 (Beckman Coulter) was used to measure the surface
exposure of PS on apoptotic cells according to the manufacturer’s
instructions (Annexin-V Fluos, Roche Diagnostics).
Western Blot Analysis. HeLa cells were incubated in the presence
of 3c and after different times were collected, centrifuged, and washed
two times with ice-cold phosphate buffered saline (PBS). The pellet
was then resuspended in lysis buffer. After the cells were lysed on ice
for 30 min, lysates were centrifuged at 15000g at 4 °C for 10 min. The
protein concentration in the supernatant was determined using the
BCA protein assay reagents (Pierce, Italy). Equal amounts of protein
(10 μg) were resolved using sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) (7.5−15% acrylamide gels) and trans-
ferred to PVDF Hybond-P membrane (GE Healthcare). Membranes
were blocked with a bovine serum albumin (BSA) solution (BSA 5%
in Tween PBS 1×), the membranes being gently rotated overnight at 4
°C. Membranes were then incubated with primary antibodies against
Bcl-2, Bax, PARP, cleaved caspase-9, cdc25c (Cell Signaling), caspase-
3 (Alexis), H2AX (Cell Signaling), p53 (Cell Signaling), cyclin B (Cell
Signaling), p-cdc2Tyr15 (Cell Signaling), p21 (Cell Signaling), or β-
(3-(4-Ethoxyphenylamino)-5-amino-1H-1,2,4-triazol-1-yl)(3,4,5-
trimethoxyphenyl) Methanone (3m). Synthesized according to
method C, compound 3c was obtained as a yellow solid (yield
1
54%); mp 176−178 °C. H NMR (DMSO-d6) δ: 1.29 (t, J = 6.8 Hz,
3H), 3.79 (s, 3H), 3.86 (s, 6H), 3.93 (q, J = 6.8 Hz, 2H), 6.77 (d, J =
9.0 H, 2H), 7.46 (d, J = 9.0 H, 2H), 7.68 (s, 2H), 7.80 (bs, 2H), 9.11
(s, 1H). 13C NMR (DMSO-d6) δ: 14.73, 55.95 (2C), 60.22, 63.09,
108.62 (2C), 114.44 (2C), 127.86 (2C), 127.22, 134.27, 141.29,
152.05 (2C), 152.51, 157.57, 158.38, 165.25. MS (ESI): [M]+ = 413.8.
Anal. (C20H23N5O5) C, H, N.
(5-Amino-3-(benzo[d][1,3]dioxol-5-ylamino)-1H-1,2,4-triazol-1-
yl)(3,4,5-trimethoxyphenyl) Methanone (3n). Synthesized according
to method C, compound 3n was obtained as a yellow solid (yield
46%); mp 200−201 °C. 1H NMR (DMSO-d6) δ: 3.78 (s, 3H), 3.86 (s,
6H), 5.93 (s, 2H), 6.76 (d, J = 8.4 Hz, 1H), 6.92 (dd, J = 8.4 and 2.2
Hz, 1H), 7.33 (d, J = 2.2 Hz, 1H), 7.65 (s, 2H), 7.81 (bs, 2H), 9.23 (s,
1H). 13C NMR (DMSO-d6) δ: 55.97 (2C), 60.22, 99.01, 100.67,
108.12, 108.51 (2C), 108.93, 127.25, 135.73, 140.71, 141.28, 147.23,
152.06 (2C), 157.53, 158.26, 165.34. MS (ESI): [M]+ = 413.8. Anal.
(C19H19N5O6) C, H, N.
X-ray Structure Determination. X-ray diffraction data for
compound 3c were collected at room temperature, 295 K, on a
Nonius Kappa CCD diffractometer with graphite monochromated Mo
Kα radiation (λ = 0.7107 Å). The structure was solved by direct
methods (SIR97)35 and refined (SHELXL-97)36 by full matrix least-
squares with anisotropic non-hydrogen atoms. The hydrogen atoms
were included on calculated positions, riding on their carrier atoms,
except for those bound to nitrogen. The latter were refined
isotropically.
Crystal Data. C19H21N5O4; orthorhombic, space group Pbca, a =
7.6945(1), b = 18.7364(4), c = 26.0711(5) Å, V = 3758.6(1) Å3, Z = 8,
Dc = 1.355 g cm−3. Intensity data collected with θ ≤ 26°; 3677
independent reflections measured; 2412 observed [I > 2σ(I)]. Final R
index = 0.0471 (observed reflections), Rw = 0.1395 (all reflections), S
= 1.022. Complete crystallographic data have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publication
number CCDC no. 984800. Copies of that data can be obtained, free
of charge, via www.ccdc.cam.ac.uk./conts/retrieving.html or on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K.
[fax: +44(0)-1223-336033, e-mail: deposit@ccdc.cam.ac.uk].
Cell Growth Conditions and Antiproliferative Assay. Human
T-leukemia (CCRF-CEM and Jurkat) and human B-leukemia (SEM)
cells were grown in RPMI-1640 medium (Gibco, Milano, Italy). Breast
adenocarcinoma (MCF-7), human cervix carcinoma (HeLa), and
human colon adenocarcinoma (HT-29) cells were grown in DMEM
medium (Gibco, Milano, Italy), all supplemented with 115 units/mL
penicillin G (Gibco, Milano, Italy), 115 μg/mL streptomycin
(Invitrogen, Milano, Italy), and 10% fetal bovine serum (Invitrogen,
Milano, Italy). CEMVbl‑100 cells are a multidrug-resistant line selected
against vinblastine.20 LoVoDoxo cells are a doxorubicin resistant
subclone of LoVo cells21 and were grown in complete Ham’s F12
medium supplemented with doxorubicin (0.1 μg/mL). LoVoDoxo and
CEMVbl‑100 were a kind gift of Dr. G. Arancia (Istituto Superiore di
̀
Sanita, Rome, Italy). Stock solutions (10 mM) of the different
compounds were obtained by dissolving them in DMSO. Individual
wells of a 96-well tissue culture microtiter plate were inoculated with
100 μL of complete medium containing 8 × 103 cells. The plates were
incubated at 37 °C in a humidified 5% CO2 incubator for 18 h prior to
the experiments. After medium removal, 100 μL of fresh medium
containing the test compound at different concentrations was added to
each well in triplicate and incubated at 37 °C for 72 h. The percentage
of DMSO in the medium never exceeded 0.25%. This was also the
maximum DMSO concentration in all cell-based assays described
below. Cell viability was assayed by the (3-(4,5-dimethylthiazol-2-yl)-
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Journal of Medicinal Chemistry
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actin (Sigma-Aldrich) for 2 h at room temperature. Membranes were
next incubated with peroxidase-labeled secondary antibodies for 60
min. All membranes were visualized using ECL Select (GE
Healthcare) and exposed to Hyperfilm MP (GE Healthcare). To
ensure equal protein loading, each membrane was stripped and
reprobed with anti-β-actin antibody.
and apoptosis assay on HeLa cells. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*For R.R.: phone, 39-(0)532-455303; fax, 39-(0)532-455953;
E-mail, rmr@unife.it.
■
Antivascular Activity. HUVECs were prepared from human
umbilical cord veins, as previously described.33 The adherent cells were
maintained in M200 medium supplemented with low serum growth
supplement (LSGS), containing FBS, hydrocortisone, hEGF, bFGF,
heparin, and gentamycin/amphotericin (Life Technologies, Monza,
Italy). Once confluent, the cells were detached by trypsin−EDTA
solution and used in experiments from the first to sixth passages.
The motility assay for HUVECs was based on “scratch” wounding
of a confluent monolayer.42 Briefly, HUVECs (1 × 105) were seeded
onto 0.1% collagen type I (BD Biosciences, Italy)-coated six-well
plates in complete medium until a confluent monolayer was formed.
The cells were wounded using a pipet tip, and wells were washed with
PBS to remove the detached cells. Then the cells were treated with the
test compounds, and at different times from the scratch, the cells were
photographed under a light microscope. At all indicated time points,
the wound width was measured in four areas and compared with the
initial width.
Matrigel matrix (Basement Membrane Matrix, BD Biosciences,
Italy) was kept at 4 °C for 3 h, when 230 μL of Matrigel solution was
added to each well of a 24-well plate. After gelling at 37 °C for 30 min,
gels were overlaid with 500 μL of medium containing 6 × 104
HUVECs. The cells were incubated over Matrigel for 6 h to allow
capillary tubes to form. Different concentrations of test compound
were added in the cultures and incubated for different times, and the
disappearance of existing vasculature was monitored and photo-
graphed (five fields for each well: the four quadrants and the center) at
a 10× magnification. Phase contrast images were recorded using a
digital camera and saved as TIFF files. Image analysis was carried out
using the ImageJ image analysis software, and the following
dimensional parameters (percent area covered by HUVECs and
total length of HUVECs network per field) and topological parameters
(number of meshes and branching points per fields) were estimated.32
Values were expressed as percent change from control cultures grown
with complete medium.
Antitumor Activity in Vivo. The in vivo cytotoxic activity of
compound 3c was investigated using a syngeneic murine hepatocel-
lular carcinoma cell line (BNL 1ME A.7R.1) in Balb/c mice.34 Male
mice, 8 weeks old, were purchased from Harlan (S. Pietro al Natisone
Udine, Italy), and tumors were induced by a subcutaneous injection in
their dorsal region of 107 cells in 200 μL of sterile PBS. Animals were
randomly divided into four groups, and, starting on the second day,
the first group was daily dosed intraperitoneally (ip) with 7 μL/kg of
free vehicle (0.9% NaCl containing 5% polyethylene glycol 400 and
0.5% Tween 80). Groups two and three were treated with compound
3c at the doses of 5 and 10 mg/kg body weight, respectively. The
fourth group received the reference compound CA-4P at the dose of 5
mg/kg body weight. Both compound 3c and CA-4P were dissolved in
free vehicle. Tumor sizes were measured daily for 7 days using a pair of
calipers. In particular, the tumor volume (V) was calculated by the
rotational ellipsoid formula: V = A × B2/2, where A is the longer
diameter (axial) and B is the shorter diameter (rotational). All
experimental procedures followed guidelines recommended by the
Institutional Animal Care and Use Committee of Padova University.
Statistical Analysis. Unless indicated otherwise, the results are
*For P.G.B.: phone, 39-(0)532-455293; fax, 39-(0)532-455953;
E-mail, pgb@unife.it.
*For G.V.: phone, 39-(0)49-8211451; fax, 39-(0)49-8211462;
E-mail, giampietro.viola1@unipd.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Alberto Casolari for technical assistance.
■
ABBREVIATIONS USED
■
CA-4, combretastatin A-4; VDA, vascular disrupting agent; PI,
propidium iodide; PS, phosphatidylserine; PARP, polyADP-
ribose polymerase; PBL, peripheral blood lymphocytes; PHA,
phytohemaglutinin; FITC, fluorescein isothiocyanate; BSA,
bovine serum albumin; HE, hydroxyethidine; PBS, phosphate-
buffered saline; SDS-PAGE, sodium dodecyl sulfate poly-
acrylamide gel electrophoresis; HUVEC, human umbilical vein
endothelial cell; LSGS, low serum growth supplement
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ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed characterization of synthesized compounds 7a−c, 7g−
h, and 7k−l. Immunofluorescence analysis of tubulin network
L
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