Discovery and optimization of a series of 2-aryl-4-amino-5-(3′, 4′,5′-trimethoxybenzoyl)thiazoles as novel anticancer agents

Article abstract of DOI:10.1021/jm300388h

A new series of tubulin polymerization inhibitors based on the 2-aryl/heteroaryl-4-amino-5-(3′,4′,5′-trimethoxybenzoyl) thiazole scaffold was synthesized and evaluated for growth inhibition activity on a panel of cancer cell lines, cell cycle effects, and in vivo potency. Structure-activity relationships were elucidated with various substitutions at the 2-position of the thiazole skeleton. Hydrophobic moieties, such as phenyl and 3-thienyl, were well tolerated at this position, and variation of the phenyl substituents had remarkable effects on potency. The most active compound (3b) induced apoptosis through the mitochondrial pathway with activation of caspase-3. We also showed that it has potential antivascular activity since it reduced in vitro endothelial cell migration and disrupted capillary-like tube formation at noncytotoxic concentrations. Furthermore, compound 3b significantly reduced the growth of the HT-29 xenograft in a nude mouse model, suggesting that 3b is a promising new antimitotic agent with clinical potential.

Full text of DOI:10.1021/jm300388h

Article
pubs.acs.org/jmc
Discovery and Optimization of a Series of 2-Aryl-4-Amino-5-(3′,4′,5′-
trimethoxybenzoyl)Thiazoles as Novel Anticancer Agents
Romeo Romagnoli,*,† Pier Giovanni Baraldi,*,† Maria Kimatrai Salvador,† Delia Preti,†
Mojgan Aghazadeh Tabrizi,† Andrea Brancale,‡ Xian-Hua Fu,§ Jun Li,§ Su-Zhan Zhang,§ Ernest Hamel,∥
Roberta Bortolozzi,⊥ Elena Porcu,⊥ Giuseppe Basso,⊥ and Giampietro Viola*,⊥
̀
†Dipartimento di Scienze Chimiche e Farmaceutiche, Universita
̀
di Ferrara, 44121 Ferrara, Italy
‡School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, U.K.
§Cancer Institute, Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education,
The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province 310009,
People’s Republic of China
∥Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick,
Maryland 21702, United States
⊥Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Universita
̀
di Padova, 35131 Padova, Italy
ABSTRACT: A new series of tubulin polymerization inhibitors based on the 2-aryl/heteroaryl-4-amino-5-(3′,4′,5′-
trimethoxybenzoyl)thiazole scaffold was synthesized and evaluated for growth inhibition activity on a panel of cancer cell
lines, cell cycle effects, and in vivo potency. Structure−activity relationships were elucidated with various substitutions at the
2-position of the thiazole skeleton. Hydrophobic moieties, such as phenyl and 3-thienyl, were well tolerated at this position, and
variation of the phenyl substituents had remarkable effects on potency. The most active compound (3b) induced apoptosis
through the mitochondrial pathway with activation of caspase-3. We also showed that it has potential antivascular activity since it
reduced in vitro endothelial cell migration and disrupted capillary-like tube formation at noncytotoxic concentrations.
Furthermore, compound 3b significantly reduced the growth of the HT-29 xenograft in a nude mouse model, suggesting that 3b
is a promising new antimitotic agent with clinical potential.
inhibit tubulin polymerization.3 Numerous chemically diverse
INTRODUCTION
■
antimitotic agents, many of which are natural products, interact
specifically with tubulin.4 Among the naturally occurring
derivatives, combretastatin A-4 (CA-4, 1, Chart 1), isolated
from the bark of the South African tree Combretum caffrum,5 is
one of the well-known tubulin-binding molecules affecting
microtubule dynamics, and compound 1 strongly inhibits the
polymerization of tubulin by binding to the colchicine site.6
Compound 1 shows potent cytotoxicity against a wide variety of
human cancer cells, including those that are multidrug resistant.7
Besides being critical for cell architecture, the microtubule
system of eukaryotic cells is essential for cell division since micro-
tubules are key components of the mitotic spindle.1 Microtubules
are a dynamic cellular compartment in both neoplastic and normal
cells. This dynamicity is characterized by the continuous turnover
of αβ-tubulin heterodimers in the polymeric microtubules.
The microtubule system is also important in other fundamental
cellular processes, such as regulation of motility, cell signaling,
formation and maintenance of cell shape, secretion, and intra-
cellular transport.2
In the last decades, there has been a continuing interest in
the discovery and development of novel small molecules able to
Received: March 21, 2012
Published: May 14, 2012
© 2012 American Chemical Society
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small and that tubulin only tolerated the presence of
hydrophobic substituents, indicating the possibility for further
improvement in activity. Thus, once the 4-amino-5-(3′,4′,5′-
trimethoxybenzoyl) thiazole scaffold was identified as the
minimum structural requirement for activity in this new series
of compounds, our strategy for further development of active
antimitotic agents was to perform modifications at the
2-position of the thiazole ring. The first round of optimization
included replacement of the pyrrolidine ring of compound 2a
with a phenyl or bioisosteric 3-thienyl ring, to furnish
derivatives 3a and 3b, respectively. As shown in Table 1,
phenyl or 3′-thienyl substitutions at the C-2 position of the
thiazole ring significantly enhanced antiproliferative activity.
Encouraged by the increased potency obtained with 3a, we
then synthesized compounds 3c−o to determine whether
various electron-releasing (Me, Et, C(CH3)3, OMe, OEt, and
OCF3) or electron-withdrawing (F, Cl, CF3, CN, and NO2)
substituents on the para- or meta-positions of the phenyl ring
would further enhance activity. To compare the effects of
para- and meta-substitution, the chloro atom and the methoxy-
group were also introduced at the meta-position of the phenyl
ring. We are including 3b in this article because it had the
best antiproliferative activity (see below) of all the compounds
prepared in this study.
Chart 1. Chemical Structures of CA-4 and 2-Substituted-4-
amino-5-aroylthiazoles 2a and 3a−o
In the course of our search for new synthetic tubulin
inhibitors, we recently reported the synthesis and biological
characterization of a series of 2-alkylamino-4-amino-5-aroylth-
iazoles with general structure 2, prepared by an easy one-pot
four step procedure.8 Among the synthesized compounds, the
2-(pyrrolidin-1-yl)-4-amino-5-(3′,4′,5′-trimethoxybenzoyl)-
thiazole derivative 2a was the only compound of this series
active at submicromolar concentrations against a panel of five
cancer cell lines, with IC50 values ranging from 0.2 to 0.4 μM.
Compound 2a was 10- to 100-fold less active than the reference
compound 1 as an antiproliferative agent, while it was
comparable to 1 as an inhibitor of tubulin polymerization.
Structure−activity relationship studies indicated that the
pyrrolidine is the only substituent tolerated at the 2-position
of the thiazole scaffold. This finding led us to assume that the
tubulin binding pocket for this portion of the molecule is quite
We examined the efficacy of the newly synthesized com-
pounds on a panel of human cancer cell lines, including multi-
drug resistant lines overexpressing the 170-kDa P-glycoprotein
drug efflux pump, and we investigated in detail the modalities of
cell death induced by these derivatives. Since many antimitotic
drugs, such as compound 1, have been shown to possess anti-
angiogenic and antivascular activities,9,10 we also investigated
the effects of these compounds on in vitro assays with
HUVECs.
CHEMISTRY
■
The general synthesis of 2-aryl/heteroaryl-4-amino-5-(3′,4′,5′-
trimethoxybenzoyl)thiazoles 3a−o is outlined in Scheme 1.
These molecules were prepared by a one-step efficient synthetic
procedure, starting from an easily accessible common
Table 1. In Vitro Cell Growth Inhibitory Effects of Compounds 1 and 3a−o
a
IC50 (nM)
compd
HeLa
A549
HL-60
Jurkat
MCF-7
HT-29
3a
3b
3c
3d
3e
3f
7.6 1.7
367 11.0
210 38.1
1082 36.7
6730 202
326 14.8
1223 97.1
5175 135
>10,000
80.8 7.1
77.9 2.6
384 25.0
4445 68.2
137 27.2
295 27.0
1813 244
>10,000
58.0 5.6
26.4 2.3
582 15.4
1749 58.8
35.9 4.4
201 57.1
2328 577
>10,000
2.2 0.8
51.2 9.3
29.4 1.5
845 18.5
41.7 3.5
3.2 0.7
1583 267
508 123
>10,000
13.2 5.4
11.4 2.8
115 3.5
2083 50.5
24.0 5.8
53.1 14.8
1018 135
9067 158
>10,000
2.4 0.9
24.1 4.3
188 46.0
2.8 1.0
22.7 7.8
191 44.8
4148 75.4
1799 27.6
244 41.3
13.7 4.5
191 35.9
1981 36.0
>10,000
3g
3h
3i
>10,000
>10,000
>10,000
3j
>10,000
>10,000
2023 75.9
66.6 8.9
606 45.7
4829 64.1
>10,000
1255 25.7
11.3 5.1
257 65.4
>10,000
2806 72.2
22.3 4.4
220 18.6
>10,000
3k
3l
720 28.3
>10,000
41.2 5.5
1136 74.4
>10,000
3m
3n
3o
1
6253 318
>10,000
>10,000
>10,000
>10,000
570 10.2
6106 169.9
180 50
>10,000
>10,000
>10,000
4785 83.2
3100 100
4
1
1
0.2
5
0.6
370 100
a
IC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean SEM from the dose−
response curves of at least three independent experiments.
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a
minimal in HL-60 and HT-29 cells. Only in MCF-7 cells was
3b less active than 3a (IC50 values of 51 and 2.2 nM,
respectively). Excluding the A549 cells, compounds 3a and 3b
had IC50 values ranging from 24 to 80 nM against the cell lines,
compared with a range of 1−3100 nM obtained with 1.
The data shown in Table 1 demonstrated the importance of
substituents on the phenyl ring at the 2-position of the thiazole
system for activity and selectivity against the different cancer
cell lines. In general, all substituents caused a reduction in
antiproliferative activity relative to 3a, except in four cases. The
meta-chloro substituent of 3e resulted in enhanced antiprolifer-
ative activity against three cell lines (HeLa, A549, and Jurkat
cells), while the meta-methoxy substituent of 3k resulted
in enhanced activity in one cell line (HL-60 cells). Otherwise,
all substituents led to reduced activity, sometimes mild to
moderate (e.g., 3c, 3e, and 3f) and sometimes profound (e.g.,
3h−j and 3l−o).
Scheme 1
Turning specifically to the para-substituted phenyl deriva-
tives, these showed highly variable potencies. Generally, it was
found that most substituents in the para-position resulted in
lower activity as compared to the unsubstituted parent
compound 3a, with the least deleterious being fluorine and
methyl (compounds 3c and 3f, respectively). Comparing the
fluorine derivative (3c) with the methyl derivative (3f), the
latter was more active in five of the six cell lines we examined,
with the greatest difference observed in the MCF-7 cells.
Comparing the fluorine (3c) and the chlorine (3d) derivatives,
increasing the size of the halide led to a large loss of activity
with all six cell lines. In contrast, as noted above, the meta-
chloro derivative (3e) was much more active than its para-
congener 3d, including enhanced activity relative to 3a in three
cell lines. Indeed, the activity of 3e did not differ greatly from
the 3-thienyl analogue 3b in all cell lines.
Turning to the electronic characteristics of the para-sub-
stituents, the introduction of a weak electron-releasing methyl
group (3f) caused a 1.5−4-fold loss in antiproliferative activity
in the six cell lines relative to the unsubstituted 3a. A para-ethyl
group (compound 3g) caused a further, major decrease in
antiproliferative activity relative to 3a, and 3f was 14−700-fold
less active in the six cell lines. Little antiproliferative activity
was observed with still larger para-substituents (3h−j, 3m−o),
regardless of their electronic characteristics.
The antiproliferative activities of compounds 3j, 3k, and 3l
were influenced by the number and position of methoxy sub-
stituents on the phenyl ring. The meta-methoxy derivative 3k,
as noted above, was relatively active. Relative to the
unsubstituted 3a, 3k was 1.1−5-fold less active in five cell
lines and twice as active in HL-60 cells. In contrast, the para-
methoxy derivative 3j was 32−570-fold less active than 3a when
IC50 values could be determined. The 3,4-dimethoxy
substituted analogue 3l was generally intermediate in activity
between 3j and 3k.
In summary, comparing the effects of ERGs and EWGs on
the phenyl at the C2-thiazole position, no consistent difference
in effects on antiproliferative activity occurred. Overall, all
substituents at the para-position led to a loss of activity, and
profound loss occurred with larger substituents. The effects of
the two small meta-substitents examined were not dramatic,
leading, perhaps, to some enhancement of antiproliferative
activity. In different cell lines, moreover, different effects were
observed. For example, replacement of the electron-donating
methoxy group with the electron-withdrawing chlorine atom
(compounds 3k and 3e, respectively), resulted in a 3- and
a
Reagents: a, Na2S·9H2O, DMF, 70 °C, 2 h; b, 1-(3′,4′,5′-
trimethoxyphenyl)-2-bromoethanone, 50 °C, 2 h; c, K2CO3, 1 h; d,
Pd2dba3, Cu(I)Tc, TFP, ArB(OH)2, THF, 60 °C, 18 h.
intermediate 5.8 This latter compound was obtained in good
yield by a one-pot three-step sequential procedure, starting
from dimethyl cyanodithiocarbonate 4, which was reacted
successively with sodium sulfide, 2-bromo-1-(3,4,5-
trimethoxyphenyl)ethanone and potassium carbonate.11 The
Pd2dba3-catalyzed, Cu(I)Tc-mediated coupling of thiazole-2-
thiomethyl ether 5 with the appropriate aryl/heteroaryl boronic
acid, in the presence of TFP, furnished the 2-aryl/heteroaryl
substituted thiazoles 3a−o in high yields, avoiding the
protection/deprotection sequence of the amino group at the
4-position of the thiazole ring.12
BIOLOGICAL RESULTS AND DISCUSSION
■
In Vitro Antiproliferative Activities. The 2-aryl/hetero-
aryl-4-amino-5-(3′,4′,5′-trimethoxybenzoyl)thiazoles 3a−o were
evaluated for their antiproliferative activity against a panel of
six human tumor cell lines and compared with reference
compound 1. As shown in Table 1, the antiproliferative
activities of the tested compounds were generally more
pronounced against HeLa and MCF-7 cells as compared with
the other cell lines. With the exception of MCF-7 cells, the
3-thienyl derivative 3b was the most active compound in this
series, exhibiting IC50 values ranging from 2.4 to 78 nM against
five of the six cancer cell lines and an IC50 of 210 nM against
the A549 cells. Moreover, with the MCF-7 and HT-29 cells,
compounds 3a−c, 3e−f, and 3k were more potent than 1, with
IC50 values in the single- or double-digit nanomolar range.
Compounds 3b and 3e showed comparable potency to 1
against the HeLa cells. Of the 15 tested compounds, 3a−b, 3e,
and 3k possessed the highest overall potency, with IC50 values
of 2.4−140 nM against five of the six cancer cell lines and IC50
values of 200−700 nM against the A549 cells. With the
exception of MCF-7 and HT-29 cells, reference compound 1
possessed the highest potency in four of the six cell lines tested.
The bioisosteric replacement of the phenyl ring of
compound 3a with the 3-thienyl group (3b) produced a
1.5- to 3-fold increase of potency against A549, Jurkat, and
HeLa cells, while the differences between 3a and 3b were
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4-fold reduction in activity against HL-60 and MCF-7 cells,
while 3k and 3e showed comparable potencies against HT-29,
and 3e was 5- and 2-fold more active than 3k against HeLa and
Jurkat cells.
Effect of Compound 3b on Multidrug Resistant Cells.
To investigate whether these derivatives are substrates of drug
efflux pumps, one of the most active compounds (3b) was
When comparing the inhibition of tubulin polymerization
versus the growth inhibitory effects, we found a positive correla-
tion for most, but not all, of the active compounds. While 3f
was generally less potent than 3b as an antiproliferative agent,
3f and 3b showed equal potency as inhibitors of tubulin
assembly. Similarly, 3k is 1.5-fold less active than 3f in the
tubulin assay, but 3k is more active than 3f in five of the cell
lines. Such discrepancies in antitubulin versus antiproliferative
activity are not infrequently observed, but the reasons are
usually uncertain, as in our case. Among possible explanations is
that we used bovine brain tubulin in the former studies, and its
composition in terms of tubulin isotypes differs significantly
from that of different human cancer cell lines.16c
tested against a panel of drug resistant cell lines which either
13,14
overexpress P-glycoprotein (LovoDoxo and CemVbl100
)
or
are associated with tubulin gene mutations (A549-T12)15 that
result in modified tubulin with impaired polymerization
properties. As shown in Table 2, 3b exhibited cytotoxic activity
In the colchicine binding studies, derivatives 3a and 3b
potently inhibited the binding of [3H]colchicine to tubulin,
with 79% and 77% inhibition occurring with these agents.
Inhibition of colchicine binding by compounds 3c, 3e−f, and 3j
was lower, ranging from 45 to 63%. None, however, was quite
as potent as 1, which in these experiments inhibited colchicine
binding by 98%.
For the most active compounds 3a and 3b, a good correla-
tion was observed between antiproliferative activities, inhibition
of tubulin polymerization, and inhibition of colchicine binding.
In general, inhibition of [3H]colchicine binding to tubulin cor-
related more closely with antiproliferative activity than did
inhibition of tubulin assembly.
Table 2. In Vitro Cell Growth Inhibitory Effects of
Compound 3b on Drug Resistant Cell Lines
a
IC50 (nM)
b
compd
LoVo
LoVoDoxo
resistance ratio
3b
18.0 4.5
120 30
CEM
24.8 5.3
13150 210
CEMVbl100
1.4
c
doxorubicin
109.6
b
resistance ratio
3b
273.3 20.3
0.8 0.1
A549
666.7 12.0
205 46
2.4
vinblastine
256.2
b
A549-T12
resistance ratio
3b
209.6 38.1
7.2 0.1
201.5 46.7
75.2 12.5
0.96
10.4
c
taxol
Molecular Modeling. A series of molecular docking
simulations were performed to rationalize the observed SARs
for this series of compounds. The proposed binding mode that
emerged from these studies is virtually identical to the
pyrrolidine analogue 2a reported previously.8 Figure 1 shows
how the thienyl analogue 3b sits in a tight hydrophobic pocket
defined by β-tubulin residues Val181, Asn258, Met259, and
Lys352. It is interesting to note that this pocket can only
accommodate an aromatic ring with relatively small substituents,
like the ones present in 3a, 3c−f, and 3k (Figure 2), while
compounds with bulkier substituents (e.g., the t-butyl of 3h) do
not dock well in the binding site because of steric hindrance. In
addition to these nonpolar interactions, the trimethoxyphenyl
ring of 3b is also in contact with βCys241, while the amino
group establishes a hydrogen bond with βThr179. These latter
two interactions are often observed in models of the binding of
tubulin inhibitors in the colchicine site.17
Analysis of Cell Cycle Effects. The effect of 3a and 3b on
cell cycle progression was examined by flow cytometry in HeLa
and Jurkat cells. Treatment with the two compounds in HeLa
cells, resulted in the accumulation of cells in the G2/M phase,
with a concomitant reduction in cells in both the S and G1
phases. These changes occurred in a concentration-dependent
manner (Figure 3, panel A), but changes were observed even at
the lowest concentration (62 nM) used. A similar behavior was
observed also for Jurkat cells (Figure 3, panel B), except that for
compound 3a we detected a less pronounced G2/M arrest in
comparison with 3b in the same cell lines accompanied with a
reduction of cells in the G1 phase while cells in the S phase
remained constant.
a
IC50= compound concentration required to inhibit tumor cell
proliferation by 50%. Data are expressed as the mean SEM from
the dose−response curves of at least three independent experiments.
b
The values express the ratio between IC50 determined in resistant and
c
nonresistant cell lines. Data are from ref 8.
in all three of the drug resistant cell lines. The activity in the
two P-glycopprotein overexpressing cell lines demonstrated
that 3b is not a substrate for this important drug pump.
Inhibition of Tubulin Polymerization and Colchicine
Binding. To investigate whether the antiproliferative activities
of compounds 3a−c, 3e−f, and 3k derived from an interaction
with tubulin, these agents were evaluated for their inhibition of
tubulin polymerization and for effects on the binding of
[3H]colchicine to tubulin (Table 3).16a,b For comparison,
Table 3. Inhibition of Tubulin Polymerization and
Colchicine Binding by Compounds 1, 3a−c, 3e−f and 3k
a
b
compd tubulin assembly IC50 SD (μM) colchicine binding
%
SD
3a
3b
3c
3e
3f
1.6 0.2
1.5 0.2
2.7 0.2
1.7 0.2
1.5 0.1
2.2 0.3
1.2 0.1
78
77
45
63
59
51
6
4
6
3
4
6
3k
1
98 0.6
a
Inhibition of tubulin polymerization. Tubulin was at 10 μM.
b
Inhibition of [3H]colchicine binding. Tubulin, colchicine, and the
tested compound were at 1, 5, and 5 μM, respectively.
Next, we investigated the association between 3b-induced
G2/M arrest and alterations in G2/M regulatory protein
expression in HeLa cells. As shown in Figure 3 (panel C), 3b
caused an increase in cyclin B1 expression after 24 and 48 h,
indicating an activation of the mitotic checkpoint following
drug exposure. This effect was confirmed by the appearance of
slower migrating forms of phosphatase cdc25c at 24 h, followed
compound 1 was examined in contemporaneous experiments.
In the assembly assay, compounds 3a−b and 3e−f were found
to be the most active compounds (IC50 values, 1.5−1.7 μM),
with activity comparable with that of 1 (IC50, 1.2 μM).
Compounds 3c and 3k were half as active as 1, although they
were more potent than 1 as antiproliferative agents against
MCF-7 and HT-29 cells.
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Figure 1. Proposed binding mode of 3b in the colchicine site of β-tubulin.
Figure 2. Putative binding mode of 3c (in cyan), 3e (in magenta), and 3f (in orange) in the colchicine site of β-tubulin.
by a strong reduction at 48 h. The phosphorylation of cdc25c
directly stimulates its phosphatase activity, and this is necessary
to activate cdc2/cyclin B on entry into mitosis.18 In good
agreement, we also observed at 24 h of incubation, at 100 nM,
a remarkable dephosphorylation at Tyr15 of cdc2 kinase, and
this effect, although less pronounced, was also present after 48
h of incubation.
Compound 3b Induced Apoptosis. To evaluate the
mode of cell death induced by compound 3b, we performed a
biparametric cytofluorimetric analysis using propidium iodide
(PI) and annexin-V-FITC, which stain DNA and phosphati-
dylserine (PS) residues, respectively.
cytometry. As shown in Figure 4 (panel A), 3b caused a signifi-
cant induction of apoptotic cells after 24 h. The percentage
of annexin-V positive cells then further increased at 48 h
(Figure 4, panel B). These findings prompted us to further
investigate the apoptotic process after treatment of cells with 3b.
Compound 3b Induced Mitochondrial Depolariza-
tion and ROS Production. Mitochondria play an essential
role in the propagation of apoptosis.19 It is well established that,
at an early stage, apoptotic stimuli alter the mitochondrial
transmembrane potential (Δψmt). Δψmt was monitored by the
fluorescence of the dye JC-1.20 Treated HeLa cells in the
presence of 3b (50 and 100 nM) exhibited a marked shift in
fluorescence compared with control cells, indicating depolariza-
tion of the mitochondrial membrane potential (data not shown).
Following treatment, the percentage of cells with low Δψmt
After treatment with 3b at 50 or 100 nM for 24 or 48 h,
HeLa cells were labeled with the two dyes, and the resulting red
(PI) and green (FITC) fluorescence was monitored by flow
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Figure 3. Effect of compounds 3a and 3b on cell cycle phase arrest in HeLa (panel A) and Jurkat cells (panel B). Cells were treated with different
concentrations of 3a or 3b, as indicated, ranging from 62.5 to 500 nM for 24 h. Then, the cells were fixed and stained with PI to analyze DNA
content by flow cytometry. Data are presented as the mean SEM of three independent experiments. Panel C: effect of 3b on some G2/M
regulatory proteins. HeLa cells were treated for 24 or 48 h with the indicated concentration of the compound. The cells were harvested and lysed for
the detection of cyclin B, p-Cdc2Y15, and Cdc25c expression by Western blot analysis. To ensure equal protein loading, each membrane was stripped
and reprobed with anti-β-actin antibody.
increased in a time-dependent fashion (Figure 5, panel A). The
disruption of Δψmt is associated with the appearance of annexin-
V positivity in the treated cells when they are in an early
apoptotic stage. In fact, the dissipation of Δψmt is characteristic of
apoptosis and has been observed with both microtubule
stabilizing and destabilizing agents, including 1, in different cell
types.18
Mitochondrial membrane depolarization is associated with
mitochondrial production of ROS.21 Therefore, we investigated
whether ROS production increased after treatment with the test
compounds. We utilized the two fluorescence indicators
hydroethidine (HE) and 2,7-dichlorodihydrofluorescein diac-
etate (H2-DCFDA).22 The results are presented in Figure 5,
panels B and C, which show that 3b induced the production of
significant amounts of ROS in comparison with control cells, in
agreement with the dissipation of Δψmt described above.
Effect of 3b on Bcl-2 and Bax Expression and
Caspase-3 Activation. We evaluated the activity of caspase-
3 after treatment of HeLa cells with 3b since this enzyme is
essential for the propagation of the apoptotic signal after
exposure to many antimitotic compounds.23 We observed a clear
activation of caspase-3, as well as cleavage of the caspase-3
substrate PARP after 24 and 48 h of 3b exposure (Figure 6). In
addition, after a 48 h exposure to 100 nM, we also found an
increase in the expression of phospho-γH2AX, a well-known
marker for cellular DNA double strand breaks.24 This suggests
that DNA damage probably occurred in the unsegregated
chromosomes resulting from the stalled replication caused by
compound 3b. There is increasing evidence that regulation of the
Bcl-2 family of protein shares the signaling pathways induced by
antimicrotubule compounds.18,25 The proteins of the Bcl family
play a major role in controlling apoptosis through the regulation
of mitochondrial processes and the release of mitochondrial
proapoptotic molecules that are important for the cell death
pathway.18 Our results (Figure 6) showed that the expression of
the antiapoptotic protein Bcl-2 was strongly reduced after 24 h
of treatment with 100 nM 3b and after 48 h with either 50 or
100 nM 3b. In contrast, expression of Bax, a proapoptotic
protein of the Bcl-2 family, was unchanged.
In Vitro Evaluation of Antivascular Activity of 3b.
Many studies have shown that most microtubule binding drugs
possess vascular disrupting activity, and such data suggest that
this class of drugs could be useful as antiangiogenic com-
pounds.9,10 In this context, CA-4 and its analogues in clinical
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Figure 4. Panel A: representative flow cytometric histograms of apoptotic HeLa cells after 48 h treatment with 3b. The cells were harvested and
labeled with annexin-V-FITC and PI and analyzed by flow cytometry. Panel B: percentage of cells found in the different regions of the biparametric
histograms after incubation with 3b for 24 or 48 h. Data are expressed as the mean SEM for five independent experiments.
development have been shown to quickly and selectively shut
down the blood flow of tumors.26,27 The effect is thought to be
mediated by inducing endothelial cell shape change, possibly
through disruption of microtubule dynamics.9 We tested 3b for
its ability to induce rapid endothelial cell shape changes using a
human umbilical vein endothelial cell (HUVEC) culture assay.
We first investigated the effects of 3b on endothelial cell
migration using an in vitro scratch assay.28 We scraped confluent
monolayers of HUVECs to clear space for motile cells to move
into. As shown in Figure 7 (panel A), we observed that after
24 h nontreated cells migrated to completely fill the area that
was initially scraped. In contrast, 3b significantly inhibited
HUVEC migration in a concentration and time dependent
manner (Figure 7, panels A and B).
We also tested the effects of 3b in a tube formation assay.
After being seeded on Matrigel, HUVECs form a rich mesh-
work of branching capillary-like tubules with multicentric
junctions. After just 1 h in different concentrations (100−
500 nM) of 3b, the capillary-like tubes were interrupted. At the
two higher concentrations of 3b, most cells were spherical and
either isolated or aggregated in small clumps (Figure 7, panel C).
Quantitative image analysis (Figure 7, panel D) showed that 3b
markedly decreased in a concentration dependent manner
both dimensional (percent of area covered by HUVECs, total
length per field) and topological parameters (number of mesh
per field, number of branching points) of the capillary-like
network.
higher than that required for the inhibition of cell migration and
tube formation. Altogether, these preliminary observations
suggest that 3b, like CA-4, would most likely cause severe
vascular disruption in vitro, although at a concentration higher
than that of CA-4,26,27 and warrant further studies to evaluate in
depth its vascular disrupting properties.
In Vivo Antitumor Activity of Compound 3b. To
evaluate the in vivo antitumor activity of 3b, human colon
adenocarcinoma xenografts were established by subcutaneous
injection of HT29 cells into the backs of nude mice. Once the
HT-29 xenografts reached a size of ∼300 mm3, 12 mice were
randomly assigned to one of two groups. In one of the groups,
compound 3b dissolved in DMSO was injected intra-
peritoneally at doses of 100 mg/kg. The drug, as well as the
vehicle control, were administered three times a week for one
week. As shown in Figure 8 (panel A), compound 3b caused
a significant reduction in tumor growth (58%, p < 0.01) as
compared with the administration of vehicle only.
During the whole treatment period, no significant weight
changes or macroscopic signs of toxicity occurred in the treated
animals (Figure 8, panel B) suggesting that the administration
of 3b was well tolerated.
CONCLUSIONS
■
The structural refinement of compound 2a led to the discovery
of a novel series of synthetic inhibitors of tubulin polymer-
ization with general structure 3, based on the 2-aryl/heteroaryl-
4-amino-5-(3′,4′,5′-trimethoxybenzoyl)thiazole molecular skel-
eton, prepared in a one-step procedure by Liebeskind−Srogl
conditions starting from an easily accessible building block 5
bearing an unprotected amino group function. The anti-
proliferative activity of these compounds was highly dependent
To evaluate if the inhibition of cell migration and tube
formation was due to a cytotoxic action of 3b, we analyzed cell
proliferation of the HUVECs by the MTT assay. The calculated
GI50 after 72 h was 4.4
0.9 μM, indicating that this com-
pound inhibited proliferation of these cells only at concentrations
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Figure 6. Western blot analysis for caspase-3 activation, PARP
cleavage, and the expression of Bcl-2, Bax, and histone γH2AX in HeLa
cells. The cells were treated with the indicated concentration of 3b for
24 or 48 h. Whole cell lysates were subjected to SDS−PAGE, followed
by blotting with the appropriate antibody or an antiactin antibody.
their position on the phenyl ring had a profound influence on
potency. Moving the chloro group from the para- to the meta-
position (compounds 3d and 3e, respectively) led to a dramatic
increase in antiproliferative activity. The same effect was
observed for the methoxy substituent (3j vs 3k). In contrast,
the insertion of an additional methoxy group, to yield the m,p-
dimethoxy derivative 3l, substantially reduced antiproliferative
activity. These substituent effects are probably caused by
increased steric hindrance in the colchicine site, preventing
efficient binding, as observed in the molecular modeling studies.
Thus, a selected, single, and small EWG or ERG could be placed
on the phenyl ring with relatively minor effects on anti-
proliferative activity, but no modification improved activity
relative to the unsubstuted phenyl or 3-thienyl derivatives 3a and
3b, respectively. Moreover, several compounds, such as 3c and
3f, characterized by the presence of substituents with opposite
electronic effects showed approximately the same potency.
We identified tubulin as the molecular target of the com-
pounds since derivatives 3a and 3b, with the greatest inhibitory
effects on cell growth, strongly inhibited tubulin assembly
and the binding of colchicine to tubulin. Their potency for
inhibition of tubulin polymerization was comparable with that
of the reference compound 1.
We also showed that 3b had cellular effects typical for
microtubule interacting agents, causing accumulation of cells in
the G2/M phase of the cell cycle. Further studies showed that
3b was a potent inducer of apoptosis in HeLa cells. Apoptosis
induced by antimitotic agents has been associated with
alterations in a variety of cellular signaling pathways. As with
many antimitotic drugs, compound 3b induced Bcl-2 down-
regulation after a 24 h treatment. Bcl-2 prevents the initiation of
the cellular apoptotic program by stabilizing mitochondrial
permeability. Our results confirmed that the induction of
apoptosis by 3b was associated with down-regulation of Bcl-2,
dissipation of the mitochondrial transmembrane potential,
and activation of caspase-3, which is coupled with terminal
events of apoptosis, such as PARP cleavage. These effects were
in good agreement with the cytotoxic potency of this com-
pound and occur at lower concentrations than was observed
with compound 2a.8 Compound 3b was also active in
Figure 5. Assessment of HeLa mitochondrial dysfunction after
treatment with compound 3b. Panel A: induction of the loss of
mitochondrial membrane potential after 24 or 48 h incubations with
compound 3b. Cells were stained with the fluorescent probe JC-1 and
analyzed by flow cytometry. Data are expressed as the mean SEM
for three independent experiments. Panels B and C: mitochondrial
production of ROS in HeLa cells. After 24 or 48 h incubations with 3b,
cells were stained with H2-DCFDA (panel B) or HE (panel C) and
analyzed by flow cytometry. Data are expressed as the mean SEM of
three independent experiments.
on the structure modifications on the 2-position of the thiazole
ring. The substituents examined included phenyl, 3-thienyl, and
substituted phenyls bearing EWGs or ERGs.
The study revealed that phenyl (3a) and 3-thienyl (3b)
thiazole derivatives exhibited improved growth inhibition activity
as compared with the activity of the corresponding pyrrolidine
analogue 2a. Moreover, compounds 3a and 3b exhibited the best
antiproliferative activity among the compounds synthesized in
this study.
Overall, these compounds had greater potency than 1 against
MCF-7 and HT-29 cells, comparable activity against HeLa and
A549 cells, and less activity against HL-60 and Jurkat cells.
Comparing 3a with the bioisosteric 3-thienyl derivative 3b, the
latter was less active than 3a only in MCF-7 cells. In general, the
introduction of EWGs and ERGs reduced activity compared
with the unsubstituted phenyl derivative 3a, with no clear
difference in effect on potency between EWGs and ERGs.
At the para-position, only fluorine and methyl were tolerated,
while the introduction of all other EWGs (Cl, NO2, and CF3)
and ERGs (Et, C(CH3)3; OCF3 and OEt) significantly reduced
antiproliferative activity. For methoxy and chloro substituents,
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Figure 7. Antivascular activity of compound 3b. Panel A: 3b inhibits HUVEC migration. Confluent HUVEC monolayers were scratch wounded. The
cells were treated with various concentrations of 3b and at different times cells were photographed (40× magnification). The dotted lines define the
areas lacking cells. Panel B: the graph shows the quantitative effect of 3b. Data are presented as the mean SEM of three independent experiments.
*p < 0.05, **p < 0.01 vs control. Panel C: inhibition of endothelial cell capillary-like tubule formation by 3b. Tubule formation on Matrigel was
performed as described in Materials and Methods. Representative pictures (40× magnification) of HUVECs treated with the indicated
concentrations of 3b for 3 h. Panel D: the graph shows the quantitative effects of 3b on the dimensional and topological parameters of the HUVEC
network.
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. Standard
syringe techniques were used for transferring dry solvents. Reaction
courses and product mixtures were routinely monitored by TLC on
silica gel (precoated F254 Merck plates), and compounds were visualized
with aqueous KMnO4. Flash chromatography was performed using
230−400 mesh silica gel and the indicated solvent system. Organic
solutions were dried over anhydrous Na2SO4. Arylboronic acids are
commercially available and used as received. All chemicals and reagents
were purchased from Aldrich (Sigma-Aldrich) or Lancaster (Alfa Aesar,
Johnson Matthey Company).
suppressing the growth of drug resistant cells, and even more
importantly, it had significant in vivo activity in a colon cancer
xenograft model. Finally, preliminary experiments were
performed to assess the potential antivascular activity of
compound 3b. The ability of this compound to inhibit
HUVEC migration and to destroy pre-established vessels
formed by HUVECs is consistent with antivascular activity and
warrants further testing in in vivo cancer models.
EXPERIMENTAL SECTION
■
1
Chemistry. Materials and Methods. H and 13C NMR spectra
Synthesis of (4-Amino-2-(methylthio)thiazol-5-yl)(3,4,5-
trimethoxyphenyl)methanone (5). To a stirred suspension of
Na2S·9H2O (1.2 g., 5 mmol) in DMF (7 mL), dimethyl cyanodi-
thioimidocarbonate 4 (730 mg, 5 mmol) dissolved in DMF (5 mL)
was added, and the mixture was heated at 70 °C for 2 h. Then,
2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone (2.89 g., 10 mmol)
dissolved in DMF (5 mL) was slowly added dropwise at 50 °C. The
mixture was heated at 50 °C for 2 h, and potassium carbonate was
added (690 mg, 10 mmol). The reaction was stirred at 50 °C for an
additional hour. The mixture was poured onto water (100 mL) with
stirring. When a yellow precipitate appeared, it was filtered, washed
with water, and dried at room temperature until a constant weight
was reached. The isolated solid was purified by stirring in petroleum
ether for 1 h and filtered to afford (5) as a yellow solid. Yield 52%,
were recorded on a Bruker AC 200 and Varian 400 Mercury Plus
spectrometer, respectively. Chemical shifts (δ) are given in ppm, and
the spectra were recorded in appropriate deuterated solvents, as
indicated. Infrared spectra were recorded on a Perkin-Elmer Spectrum
100 FTIR spectrometer using the ATR technique. The main bands are
given in cm−1. 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. All products reported showed
1H NMR spectra in agreement with the assigned structures. 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
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(2C), 164.0, 166.4, 186.3. IR (ATR) ν: 3377, 1614, 1577, 1445, 1408,
1338, 1234, 1125, 989, 819, 763. MS (ESI): [M]+ = 376.8. Anal.
(C17H16N2O4S2) C, H, N.
( 4 - A m i n o - 2 - ( 4 - fl u o r o p h e n y l ) t h i a z o l - 5 - y l ) ( 3 , 4 , 5 -
trimethoxyphenyl)methanone (3c). Following the general procedure,
the crude residue, purified by flash chromatography using ethyl
acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3c as a yellow
1
solid (49% yield); mp 196−197 °C. H NMR (CDCl3) δ: 3.92 (s,
6H), 3.93 (s, 3H), 7.02 (bs, 2H), 7.12 (s, 2H), 7.19 (m, 2H), 7.94 (m,
2H). 13C NMR (100 MHz, CDCl3) δ: 56.4 (2C), 61.0, 102.4, 105.2
(2C), 116.3, 116.6, 128.5, 129.1, 129.2, 135.9, 141.0, 153.2 (2C),
163.8, 165.3 (d, J = 253.3 Hz), 170.8, 186.5. IR (ATR) ν: 3262, 1620,
1578, 1450, 1408, 1337, 1236, 1129, 994, 813, 759. MS (ESI): [M]+ =
388.9. Anal. (C19H17FN2O4S) C, H, N.
( 4 - A m i n o - 2 - ( 4 - c h l o r o p h e n y l ) t h i a z o l - 5 - y l ) ( 3 , 4 , 5 -
trimethoxyphenyl)methanone (3d). Following the general procedure,
the crude residue, purified by flash chromatography using ethyl
acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3d as a yellow
1
solid (48% yield); mp 146−148 °C. H NMR (CDCl3) δ: 3.92 (s,
6H), 3.93 (s, 3H), 6.82 (bs, 2H), 7.10 (s, 2H), 7.42 (d, J = 8.8 Hz,
2H), 7.88 (d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ: 56.4
(2C), 61.1, 102.6, 105.3 (2C), 110.2, 128.1 (2C), 129.5 (2C), 130.7,
135.9, 138.2, 153.2 (2C), 164.8, 170.7, 186.6. IR (ATR) ν: 3313, 2930,
1614, 1572, 1414, 1335, 1129, 818, 758. MS (ESI): [M]+ = 404.9.
Anal. (C19H17ClN2O4S) C, H, N.
Figure 8. Inhibition of human xenograft growth in vivo by compound
3b. Panel A: HT29 tumor-bearing nude mice were administered either
vehicle control or 100 mg/kg of 3b in vehicle intraperitoneally on days
0, 2, and 4 (indicated by arrows). The figure shows the tumor volume
(panel A) and body weight (panel B) recorded at the indicated days
( 4 - A m i n o - 2 - ( 3 - c h l o r o p h e n y l ) t h i a z o l - 5 - y l ) ( 3 , 4 , 5 -
trimethoxyphenyl)methanone (3e). Following the general procedure,
the crude residue, purified by flash chromatography using ethyl
acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3e as a yellow
after treatments. Data are expressed as the mean
SEM of tumor
volume and body weight at each time point for six animals per group.
*p < 0.01 vs the control.
1
solid (58% yield); mp 185−186 °C. H NMR (CDCl3) δ: 3.93 (s,
6H), 3.94 (s, 3H), 6.84 (bs, 2H), 7.11 (s, 2H), 7.35 (d, J = 7.8 Hz,
2H), 7.46 (dt, J = 7.2 and 1.6 Hz, 1H), 7.83 (dt, J = 7.2 and 1.6 Hz,
1H), 7.96 (t, J = 1.4 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 56.3
(2C), 60.9, 102.7, 105.2 (2C), 124.9, 126.7, 130.4, 131.7, 133.9, 135.3,
135.8, 141.0, 153.1 (2C), 164.7, 170.2, 186.6. IR (ATR) ν: 3307, 1614,
1577, 1339, 1236, 1133, 992, 820, 761. MS (ESI): [M]+ = 404.9. Anal.
(C19H17ClN2O4S) C, H, N.
1
mp 130−132 °C. H NMR (CDCl3) δ: 2.67 (s, 3H), 3.90 (s, 3H),
3.91 (s, 6H), 6.82 (bs, 2H), 7.03 (s, 2H). 13C NMR (100 MHz,
CDCl3) δ: 13.6, 56.3 (2C), 61.0, 104.7 (2C), 105.2, 137.1, 139.9,
153.0 (2C), 165.2, 172.2, 183.3. IR (ATR) ν: 3293, 1594, 1532, 1455,
1407, 1337, 1116, 998, 736.
(4-Amino-2-p-tolylthiazol-5-yl)(3,4,5-trimethoxyphenyl)-
methanone (3f). Following the general procedure, the crude residue,
purified by flash chromatography using ethyl acetate/petroleum ether
1:1 (v/v) as eluent, furnished 3f as a yellow solid (45% yield); mp
130−132 °C. 1H NMR (CDCl3) δ: 2.41 (s, 3H), 3.92 (s, 6H), 3.93 (s,
3H), 6.84 (bs, 2H), 7.11 (s, 2H), 7.26 (d, J = 8.2 Hz, 2H), 7.84 (d, J =
8.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ: 21.7, 56.3 (2C), 60.9,
101.9, 105.2 (2C), 126.9 (2C), 129.2, 129.9 (2C), 135.9, 140.9, 143.0,
153.1 (2C), 164.4, 172.2, 186.3. IR (ATR) ν: 3314, 2928, 1612, 1574,
1412, 1335, 1235, 1128, 991, 816, 759. MS (ESI): [M]+ = 384.9. Anal.
(C20H20N2O4S) C, H, N.
General Procedure for the Synthesis of (4-Amino-2-arylthiazol-5-
yl)(3,4,5-trimethoxyphenyl)methanone (3a−o). To a stirred solution
of (4-amino-2-(methylthio)thiazol-5-yl)(3,4,5-trimethoxyphenyl)-
methanone 5 (170 mg, 0.5 mmol) in THF (10 mL) were subsequently
added the appropriate arylboronic acid (1 mmol), TFP (19 mg,
0.08 mmol), and CuTC (0.125 g, 0.65 mmol). The reaction vessel was
flushed with argon, then Pd2-dba3·CHCl3 (20 mg, 0.02 mmol) was
added, and the reaction mixture was stirred at 70 °C for 18 h. After
cooling, the mixture was diluted with dichloromethane (5 mL) and
filtered through Celite, and the filtrate was evaporated. The residue was
dissolved in dichloromethane (15 mL), and the mixture was washed
with 25% aqueous ammonia (2 × 5 mL). The organic layer was dried
and the residue after evaporation purified by silica gel column
chromatography (gradient of petroleum ether and ethyl acetate), giving
the desired product.
(4-Amino-2-(4-ethylphenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)-
methanone (3g). Following the general procedure, the crude residue,
purified by flash chromatography using ethyl acetate/petroleum ether
1:1 (v/v) as eluent, furnished 3g as a yellow solid (56% yield); mp 86−
87 °C. 1H NMR (CDCl3) δ: 1.26 (t, J = 7.2 Hz, 3H), 2.69 (q, J = 7.2
Hz, 2H), 3.92 (s, 6H), 3.93 (s, 3H), 6.94 (bs, 2H), 7.12 (s, 2H), 7.29 (d,
J = 8.2 Hz, 2H), 7.88 (d, J = 8.2 Hz, 2H). 13C NMR (100 MHz, CDCl3)
δ: 15.3, 29.0, 56.4 (2C), 61.0, 102.2, 105.2 (2C), 127.6 (2C), 133.9,
135.9 (2C), 136.1, 140.9, 149.0, 153.1 (2C), 165.1, 172.6, 186.5. IR
(ATR) ν: 3273, 2931, 1621, 1574, 1453, 1412, 1334, 1237, 1128, 1004,
820, 759. MS (ESI): [M]+ = 398.8. Anal. (C21H22N2O4S) C, H, N.
(4-Amino-2-(4-tert-butylphenyl)thiazol-5-yl)(3,4,5-
trimethoxyphenyl)methanone (3h). Following the general procedure,
the crude residue, purified by flash chromatography using ethyl
acetate/petroleum ether 4:6 (v/v) as eluent, furnished 3h as a yellow
(4-Amino-2-phenylthiazol-5-yl)(3,4,5-trimethoxyphenyl)-
methanone (3a). Following the general procedure, the crude residue,
purified by flash chromatography using ethyl acetate/petroleum ether
4:6 (v/v) as eluent, furnished 3a as a yellow solid (52% yield); mp
156−158 °C. 1H NMR (CDCl3) δ: 3.93 (s, 6H), 3.94 (s, 3H), 6.94 (bs,
2H), 7.11 (s, 2H), 7.48 (m, 3H), 7.95 (dd, J = 8.0 and 2.0 Hz, 2H). 13C
NMR (100 MHz, CDCl3) δ: 56.3 (2C), 60.9, 105.2 (2C), 108.9, 127.0
(2C), 129.3 (2C), 131.2, 132.4, 133.4, 135.7, 141.1, 153.2 (2C), 163.7,
186.4. IR (ATR) ν: 3384, 1620, 1578, 1450, 1411, 1337, 1127, 999,
817, 760. MS (ESI): [M]+ = 370.7. Anal. (C19H18N2O4S) C, H, N.
(4-Amino-2-(thiophen-3-yl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)-
methanone (3b). Following the general procedure, the crude residue,
purified by flash chromatography using ethyl acetate/petroleum ether
1:1 (v/v) as eluent, furnished 3b as a yellow solid (58% yield); mp
1
solid (54% yield); mp 66−68 °C. H NMR (CDCl3) δ: 1.35 (s, 9H),
3.93 (s, 6H), 3.94 (s, 3H), 6.91 (bs, 2H), 7.12 (s, 2H), 7.47 (d, J = 8.4
Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ:
31.3 (3C), 35.2, 56.3 (2C), 61.1, 105.3 (2C), 110.9, 125.0, 126.2 (2C),
126.8 (2C), 128.5, 136.1, 142.1, 153.2 (2C), 155.9, 164.9, 186.5. IR
(ATR) ν: 2958, 1574, 1402, 1334, 1233, 1124, 1000, 818, 763. MS
(ESI): [M]+ = 426.7. Anal. (C23H26N2O4S) C, H, N.
1
198−200 °C. H NMR (CDCl3) δ: 3.92 (s, 6H), 3.93 (s, 3H), 6.98
(bs, 2H), 7.09 (s, 2H), 7.42 (d, J = 8.4 Hz, 1H), H), 7.51 (d, J = 8.4
Hz, 1H), 8.03 (s, 1H). 13C NMR (100 MHz, CDCl3) δ: 56.3 (2C),
60.9, 101.4, 105.2 (2C), 125.9, 127.6, 133.9, 134.2, 135.8, 141.0, 153.1
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(4-Amino-2-(4-trifluoromethylphenyl)thiazol-5-yl)(3,4,5-
trimethoxyphenyl)methanone (3i). Following the general procedure,
the crude residue, purified by flash chromatography using ethyl
acetate/petroleum ether 4:6 (v/v) as eluent, furnished 3i as a yellow
purified by flash chromatography using ethyl acetate/petroleum ether
6:4 (v/v) as eluent, furnished 3o as a red solid (42% yield); mp 234−
236 °C. 1H NMR (CDCl3) δ: 3.93 (s, 6H), 3.94 (s, 3H), 6.92 (bs, 2H),
7.11 (s, 2H), 8.15 (d, J = 9.0 Hz, 2H), 8.31 (d, J = 9.0 Hz, 2H). 13C
NMR (100 MHz, CDCl3) δ: 56.4 (2C), 61.1, 105.3 (2C), 106.5, 124.5
(2C), 127.6 (2C), 124.8, 135.7, 137.9, 149.4, 153.3 (2C), 160.2, 168.4,
186.9. IR (ATR) ν: 3337, 1593, 1577, 1519, 1415, 1331, 1234, 1130,
850, 816. MS (ESI): [M]+ = 415.8. Anal. (C19H17N3O6S) C, H, N.
Antiproliferative Assays. Human T-leukemia (Jurkat) and
human promyelocytic leukemia (HL-60) cells were grown in RPMI-
1640 medium, (Gibco, Milano, Italy). Breast adenocarcinoma (MCF-7),
human nonsmall cell lung carcinoma (A549), human cervix carcinoma
(HeLa), and human colon adenocarcinoma (HT-29) cells were grown
in DMEM medium (Gibco, Milano, Italy). Both media were
supplemented with 115 units/mL of penicillin G (Gibco, Milano,
Italy), 115 μg/mL of streptomycin (Invitrogen, Milano, Italy), and 10%
fetal bovine serum (Invitrogen, Milano, Italy). LoVoDoxo cells are a
doxorubicin resistant subclone of LoVo cells13 and were grown in
complete Ham’s F12 medium supplemented with doxorubicin (0.1 μg/mL).
CEMVbl‑100 cells are a multidrug-resistant line selected against
vinblastine.14 A549-T12 cells are a nonsmall cell lung carcinoma line
exhibiting resistance to taxol.15 They were grown in complete DMEM
medium supplemented with taxol (12 nM). 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 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)-2,5-diphenyl tetrazolium
bromide test as previously described.20 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.
Effects on Tubulin Polymerization and on Colchicine
Binding to Tubulin. To evaluate the effect of the compounds on
tubulin assembly in vitro,16a varying concentrations of compounds
were preincubated with 10 μM bovine brain tubulin in glutamate
buffer at 30 °C and then cooled to 0 °C. After the 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,16b
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 10.
The tubulin structure was downloaded from the Protein Data Bank
(http://www.rcsb.org/; PDB code 3HKC).29 Hydrogen atoms were
added to the protein, using the Protonate3D function of Molecular
Operating Environment (MOE).30 Ligand structures were built with
MOE and minimized using the MMFF94x forcefield until a rmsd
gradient of 0.05 kcal mol−1 Å−1 was reached. The docking simulations
were performed using PLANTS.31
1
solid (56% yield); mp 166−167 °C. H NMR (CDCl3) δ: 3.93 (s,
6H), 3.94 (s, 3H), 6.86 (bs, 2H), 7.11 (s, 2H), 7.71 (d, J = 8.2 Hz,
2H), 8.05 (d, J = 8.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ: 56.4
(2C), 61.0, 103.2, 105.3 (2C), 123.7 (q, J = 271.5 Hz), 126.2 (2C),
126.4, 127.1 (2C), 133.2 (q, J = 32.7 Hz), 135.5, 135.8, 141.2, 153.2
(2C), 164.9, 169.9, 186.8. IR (ATR) ν: 3314, 2930, 1613, 1575, 1322,
1237, 1127, 1104, 1067, 845, 817, 760. MS (ESI): [M]+ = 438.7. Anal.
(C20H17F3N2O4S) C, H, N.
(4-Amino-2-(4-methoxyphenyl)thiazol-5-yl)(3,4,5-
trimethoxyphenyl)methanone (3j). Following the general procedure,
the crude residue, purified by flash chromatography using ethyl
acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3j as a yellow
1
solid (46% yield); mp 196−198 °C. H NMR (CDCl3) δ: 3.87 (s,
3H), 3.92 (s, 6H), 3.93 (s, 3H), 6.82 (bs, 2H), 6.93 (d, J = 8.0 Hz,
2H), 7.11 (s, 2H), 7.90 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ: 55.8, 56.5 (2C), 61.2, 101.8, 105.3 (2C), 114.8 (2C), 124.9,
129.0 (2C), 136.3, 141.1, 153.2 (2C), 163.1, 164.8, 172.3, 186.4. IR
(ATR) ν: 3298, 1573, 1397, 1259, 1124, 1004, 825, 756. MS (ESI):
[M]+ = 400.8. Anal. (C20H20N2O5S) C, H, N.
(4-Amino-2-(3-methoxyphenyl)thiazol-5-yl)(3,4,5-
trimethoxyphenyl)methanone (3k). Following the general procedure,
the crude residue, purified by flash chromatography using ethyl acetate/
petroleum ether 1:1 (v/v) as eluent, furnished 3k as an orange solid
(47% yield); mp 167−168 °C. 1H NMR (CDCl3) δ: 3.87 (s, 3H), 3.92
(s, 6H), 3.93 (s, 3H), 6.84 (bs, 2H), 7.07 (m, 1H), 7.11 (s, 2H), 7.36
(t, J = 7.2 Hz, 1H), 7.54 (m, 2H). 13C NMR (100 MHz, CDCl3) δ:
55.7, 56.4 (2C), 61.1, 105.3 (2C), 111.7, 118.7, 119.5, 130.5, 132.9.
135.8, 141.1, 153.2 (2C), 160.2, 162.8, 170.5, 171.8, 186.5. IR (ATR) ν:
3275, 1615, 1579, 1503, 1409, 1337, 1235, 1129, 994, 826, 769, 674.
MS (ESI): [M]+ = 400.8. Anal. (C20H20N2O5S) C, H, N.
(4-Amino-2-(3,4-dimethoxyphenyl)thiazol-5-yl)(3,4,5-
trimethoxyphenyl)methanone (3l). Following the general procedure,
the crude residue, purified by flash chromatography using ethyl acetate/
petroleum ether 2:8 (v/v) as eluent, furnished 3l as a red solid (45%
1
yield); mp 176−177 °C. H NMR (CDCl3) δ: 3.92 (s, 6H), 3.93 (s,
3H), 3.95 (s, 3H), 3.98 (s, 3H), 6.80 (bs, 2H), 6.88 (d, J = 8.8 Hz, 1H),
7.10 (s, 2H), 7.51 (d, J = 8.8 Hz, 1H), 7.54 (s, 1H). 13C NMR (100
MHz, CDCl3) δ: 56.3, 56.5 (2C), 61.1, 62.3, 100.4, 105.2, 107.2 (2C),
107.6, 109.8, 111.4, 121.5, 135.3, 141.3, 149.7, 153.2 (2C), 169.2, 170.9,
186.0. IR (ATR) ν: 2933, 1579, 1454, 1411, 1333, 1261, 1121, 1101,
1006, 799. MS (ESI): [M]+ = 431.0. Anal. (C21H22N2O6S) C, H, N.
( 4 - A m in o - 2 - ( 4 - e t h o x y p h e n y l )t h i azo l- 5 - y l) (3 , 4, 5 -
trimethoxyphenyl)methanone (3m). Following the general proce-
dure, the crude residue, purified by flash chromatography using ethyl
acetate/petroleum ether 6:4 (v/v) as eluent, furnished 3m as an
orange solid (44% yield); mp 185−186 °C. 1H NMR (CDCl3) δ: 1.44
(t, J = 7.2 Hz, 3H), 3.92 (s, 6H), 3.93 (s, 3H), 4.11 (q, J=7.2 Hz, 2H),
6.80 (bs, 2H), 6.94 (d, J = 8.6 Hz, 2H), 7.11 (s, 2H), 7.90 (d, J = 8.6
Hz, 2H). 13C NMR (100 MHz, CDCl3) δ: 14.8, 56.4 (2C), 61.1, 63.9,
102.0, 105.6 (2C), 115.2 (2C), 124.6, 128.9 (2C), 136.1, 141.3, 153.2
(2C), 163.1, 164.6, 172.2, 186.0. IR (ATR) ν: 2996, 1597, 1573, 1406,
1337, 1235, 1119, 104, 1006, 820, 763. MS (ESI): [M]+ = 414.9. Anal.
(C21H22N2O5S) C, H, N.
(4-Amino-2-(4-trifluoromethoxyphenyl)thiazol-5-yl)(3,4,5-
trimethoxyphenyl)methanone (3n). Following the general procedure,
the crude residue, purified by flash chromatography using ethyl
acetate/petroleum ether 1:1 (v/v) as eluent, furnished 3n as a yellow
Flow Cytometric Analysis of Cell Cycle Distribution. For flow
cytometric analysis of DNA content, 5 × 105 HeLa or Jurkat cells in
exponential growth were treated with different concentrations of the
test compounds for 24 or 48 h. After the incubation, the cells were
collected, centrifuged, and fixed with ice-cold ethanol (70%). The cells
were treated with lysis buffer containing RNase A and 0.1% Triton
X-100 and stained with PI. Samples were analyzed on a Cytomic
FC500 flow cytometer (Beckman Coulter). DNA histograms were
analyzed using MultiCycl for Windows (Phoenix Flow Systems).
Annexin-V Assay. Surface exposure of PS on apoptotic cells
was measured by flow cytometry with a Coulter Cytomics FC500
1
solid (49% yield); mp 174−176 °C. H NMR (CDCl3) δ: 3.92 (s,
6H), 3.93 (s, 3H), 6.90 (bs, 2H), 7.11 (s, 2H), 7.28 (d, J = 9.0 Hz,
2H), 7.98 (d, J = 9.0 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ: 56.4
(2C), 61.1, 102.6, 105.3 (2C), 114.4, 120.4 (q, J = 257.8 Hz), 121.3
(2C), 128.7 (2C), 135.8, 151.9, 153.2 (2C), 162.6, 163.6, 170.1, 186.6.
IR (ATR) ν: 3315, 1614, 1576, 1337, 1236, 1127, 997, 816, 759. MS
(ESI): [M]+ = 454.7. Anal. (C20H17F3N2O5S) C, H, N.
(4-Amino-2-(4-nitrophenyl)thiazol-5-yl)(3,4,5-trimethoxyphenyl)-
methanone (3o). Following the general procedure, the crude residue,
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(Beckman Coulter) by adding annexin-V-FITC to cells according to
the manufacturer’s instructions (Annexin-V Fluos, Roche Diagnostic).
Simultaneously, the cells were stained with PI.
network per field) and topological parameters (number of meshes and
branching points per fields).32 Values were expressed as percent
change from control cultures grown with complete medium.
Assessment of Mitochondrial Changes. The mitochondrial
membrane potential was measured with the fluorescent dye JC-1
(Molecular Probes, USA) by flow cytometry, as described previously.20
The production of ROS was measured by flow cytometry using either
HE (Molecular Probes) or H2DCFDA (Molecular Probes). After
different times of treatment, cells were collected by centrifugation and
resuspended in HBSS containing the fluorescence probes HE or
H2DCFDA at 2.5 and 0.1 μM, respectively. The cells were incubated
for 30 min at 37 °C, centrifuged and resuspended in HBSS. The
fluorescence was directly recorded with the flow cytometer, using as
excitation wavelength 488 nm and emission at 585 and 530 nm for HE
and H2DCFDA, respectively.
Western Blot Analysis. HeLa cells were incubated in the presence
of test compounds and, after different times, were collected,
centrifuged, and washed two times with ice cold phosphate-buffered
saline (PBS). The pellet was 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 BCA protein assay reagents (Pierce, Italy). Equal
amounts of protein (20 μg) were resolved using sodium dodecyl
sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) (7.5−15%
acrylamide gels) and transferred to a PVDF Hybond-p membrane
(GE Healthcare). Membranes were blocked with I-block (Tropix), the
membrane being gently rotated overnight at 4 °C. Membranes were
incubated with primary antibodies against Bcl-2, cleaved PARP,
cdc25C, Bax, phospho-histone γH2AX and cdc2 (all rabbit, 1:1000,
Cell Signaling, Milano, Italy), cyclin B1 (mouse, 1:1000, BD, Milano,
Italy), or β-actin (mouse, 1:10,000, Sigma-Aldrich, Milano, Italy) for 2
h at room temperature. Membranes were next incubated with
peroxidase-labeled goat antirabbit IgG (1:100,000, Sigma-Aldrich) or
peroxidase-labeled goat antimouse IgG (1:100,000, Sigma-Aldrich)
for 60 min. All membranes were visualized using ECL Advance
(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.
Antivascular Activity. HUVECs were prepared from a primary
culture of human umbilical cord veins. The adherent cells were
maintained in M200 medium supplemented with LSGS (Low Serum
Growth Supplement), which contains FBS, hydrocortisone, hEGF,
bFGF, heparin, and gentamycin/amphotericin B (Invitrogen, Milano,
Italy), and seeded on collagen I (BD Biosciences). Once confluent, the
cells were detached by treatment with a trypsin−EDTA solution and
used in experiments from the third to seventh passages.
Motility Assay. Motility assay for HUVECs was based on “scratch”
wounding of a confluent monolayer.26 Briefly, HUVECs (1 × 105)
were seeded onto 0.3% collagen I-coated six well plates in complete
medium until a confluent monolayer was formed. The cells were
scratch wounded using a pipet tip. Then the cells were treated with the
test compounds, and at different times after the scratch, the cells were
photographed under a light microscope (40× magnification). Wound
closure was evaluated by Image-J software.
Antitumor Activity in Vivo. Four week old female BALB/c-nu
nude mice (15−18 g) were obtained from Shanghai SLAC Laboratory
Animal Co., Ltd. (Shanghai, China). The animals were maintained
under specific pathogen-free conditions with food and water supplied
ad libitum in Zhejiang University of Traditional Chinese Medicine
Laboratory Animal Center. Human colon adenocarcinoma HT-29 cells
in logarithmic growth phase were resuspended in RPMI 1640 without
fetal bovine serum at 1 × 107cells/mL and inoculated (0.2 mL) in the
hypodermis of the pars dorsalis of each mouse. Once the HT-29
xenografts reached a size of ∼300 mm3, 12 mice were randomly
assigned to two groups. For the first group, compound 3b was prepared
in DMSO and injected intraperitoneally at volumes of 0.01 mL/g body
weight to give a dose of 100 mg/kg to each mouse. The compound was
administered three times a week for one week. After completing the
treatment schedule and the evaluation period, tumor-bearing mice were
euthanized. Tumor volume was calculated by the following formula:
V = (L × W2)/2 where L is the length, and W is the width of the tumor
nodules measured by Vernier calipers. The study was approved by the
Institutional Animal Ethical Committee of the Second Affiliated
Hospital, School of Medicine, Zhejiang University (PRC).
Statistical Analysis. Unless indicated otherwise, results are
presented as the mean
SEM. The differences between different
treatments were analyzed using the two-sided Student’s t test. P values
lower than 0.05 were considered significant.
AUTHOR INFORMATION
Corresponding Author
■
*(R.R.) Phone: 39-(0)532-455303. Fax: 39-(0)532-455953.
E-mail:rmr@unife.it. (P.G.B.) Phone: 39-(0)532-455293. Fax:
39-(0)532-455953. E-mail: pgb@unife.it. (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 Dr. Alberto Casolari and Dr. Elisa Durini for
excellent technical assistance.
■
ABBREVIATIONS USED
■
CA-4, combretastatin A-4; HUVEC, human umbilical vein
endothelial cells; EWG, electron-withdrawing group; ERG,
electron-releasing group; SAR, structure−activity relationships;
CuTc, copper(I)thiophene-2-carboxylate; Pd2dba3·CHCl3, tris-
(dibenzylideneacetone)dipalladium complex with chloroform;
TFP, tris(2-furyl)phosphine; FITC, fluorescein isothiocyanate;
PI, propidium iodide; PS, phospholipid phosphatidylserine;
ROS, reactive oxygen species; HE, hydroxyethidine;
H2DCFDA, 2,7-dichlorodihydrofluorescein; PARP, poly-ADP-
ribose polymerase; HBSS, Hank’s Balanced Salt Solution; PBS,
phosphate-buffered saline; SDS−PAGE, sodium dodecyl
sulfate−polyacrylamide gel electrophoresis
Endothelial Cell Vessel Formation on a Matrigel Matrix. A
Matrigel matrix was kept at 4 °C for 16 h. Two hundred microliters of
Matrigel (BD Biosciences) 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. Different concentrations of test
compound were added to the cultures, which were incubated for
different times. To explore the effect of test compounds on pre-
established vasculature, HUVECs were seeded and incubated over
Matrigel for 6 h to allow the capillary tubes to form. Then the
compound was added and continuously incubated for additional times,
and the disappearance of existing vasculature was monitored by
recording phase contrast images (five fields for each well: the four
quadrants and the center) using a digital camera. The images were
saved as TIFF files. Image analysis was carried out using the ImageJ
image analysis software and evaluating both dimensional parameters
(percent area covered by HUVECs and total length of HUVECs
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