Convergent synthesis and biological evaluation of 2-amino-4-(3′, 4′,5′-trimethoxyphenyl)-5-aryl thiazoles as microtubule targeting agents

Article abstract of DOI:10.1021/jm200392p

Combretastatin A-4, a potent tubulin polymerization inhibitor, caused us to synthesize a novel series of 2-amino-4-(3′,4′,5′- trimethoxyphenyl)-5-aryl thiazoles with the goal of evaluating the effects of substituents on the phenyl at the 5-position of the

Full text of DOI:10.1021/jm200392p

ARTICLE
pubs.acs.org/jmc
Convergent Synthesis and Biological Evaluation of
2-Amino-4-(30,40,50-trimethoxyphenyl)-5-aryl Thiazoles
as Microtubule Targeting Agents
Romeo Romagnoli,*,† Pier Giovanni Baraldi,† Andrea Brancale,‡ Antonio Ricci,‡ Ernest Hamel,§
Roberta Bortolozzi,|| Giuseppe Basso,|| and Giampietro Viola*,||
†Dipartimento di Scienze Farmaceutiche, Universitꢀa di Ferrara, 44100 Ferrara, Italy
‡The Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3NB, U.K.
§Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, United States
Dipartimento di Pediatria, Laboratorio di Oncoematologia, Universitꢀa di Padova, 35131 Padova, Italy
S Supporting Information
b
ABSTRACT: Combretastatin A-4, a potent tubulin polymeri-
zation inhibitor, caused us to synthesize a novel series of
2-amino-4-(30,40,50-trimethoxyphenyl)-5-aryl thiazoles with
the goal of evaluating the effects of substituents on the phenyl
at the 5-position of the thiazole skeleton on biological activities.
An ethoxy group at the para-position produced the most active
compound in the series, with IC50 values of 0.03ꢀ0.9 nM
against five of seven cancer cell lines. The most active com-
pounds retained full activity in multidrug resistant cancer cells
and acted through the colchicine site of tubulin. Treated cells
were arrested in the G2/M phase of the cell cycle, with cell death
proceeding through an apoptotic pathway that was only partially caspase-dependent. Preliminary results suggest that, in addition to
cell death by apoptosis, cells were also killed via mitotic catastrophe as an alternative cell death mechanism.
’ INTRODUCTION
tolerated by the target.8,9 However, the cis-configuration of CA-4
is prone to isomerize to the thermodynamically more stable
trans-form during storage and metabolism, resulting in a dramatic
decrease in antitumor activity. Thus, to retain the appropriate
geometry of the two adjacent aryl groups required for potent
bioactivity, chemically stable cis-restricted derivatives of CA-4
were obtained by incorporating the olefinic double bond into
vicinally diaryl-substituted five-member aromatic heterocyclic
rings such as pyrazole,10 imidazole,11 thiazole,12 furazan (1,2,5-
oxadiazole),13 isoxazole,14 oxazole,10 1,2,3-thiadiazole,15 triazole,16
and 1,2,3,4-tetrazole.12
In a previous study, Ohsumi and co-workers reported a linear
synthetic approach for the preparation of a small series of
molecules based on the thiazole ring (compounds 2aꢀd) sub-
stituted at the 2-position with a hydrogen (2a), methyl (2b),
amino (2c), or hydrazino (2d) group.12 The 2-amino derivative
2c retains activity similar to that of CA-4 as an inhibitor of tubulin
polymerization, but it is inferior to CA-4 with respect to anti-
proliferative activity against colon cancer cells.
The microtubule system of eukaryotic cells is a critical element
in a variety of fundamental cellular processes such as cell division,
formation, and maintenance of cell shape, regulation of motility,
cell signaling, secretion, and intracellular transport.1 Among the
various strategies developed to block mitosis, microtubules
represent an attractive target for numerous small natural and
synthetic molecules that inhibit the formation of the mitotic
spindle.2 One of the most important antimitotic agents is
combretastatin A-4 (CA-4, 1; Chart 1). CA-4, isolated from
the bark of the South African tree Combretum caffrum,3 is one of
the well-known natural molecules that strongly inhibits tubulin
polymerization by binding to the colchicine site.4 CA-4 shows
potent cytotoxicity against a wide variety of human cancer cell
lines, including those that are multidrug resistant,5 and CA-4 is
also a vascular disrupting agent.6 A water-soluble disodium
phosphate derivative of CA-4 (named CA-4P) has shown
promising results in human cancer clinical trials,7 thus stimulat-
ing significant interest in a variety of CA-4 analogues.8
Previous SAR studies have demonstrated that both the 30,40,50-
trimethoxy substitution pattern on the A-ring and the cis-olefin
configuration at the bridge were fundamental requirements for
optimal activity, while some B-ring structural modifications were
Received: April 2, 2011
Published: June 13, 2011
r
2011 American Chemical Society
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ARTICLE
Chart 1. Lead Structures of Tubulin Polymerization
Inhibitors
Scheme 1a
In our efforts to discover new potent antimitotic agents, we
developed an efficient and versatile convergent synthetic proce-
dure for the preparation of a new series of 2-amino-4-(30,40,50-
trimethoxyphenyl)-5-arylthiazoles with general structure 3, starting
from a common 2-amino protected-4-(30,40,50-trimethoxyphenyl)-
5-bromothiazole intermediate. In this series of designed analo-
trimethoxyphenyl moiety, identical with the A-ring of CA-4,
which was considered essential for maximal tubulin binding
activity.8 Modifications were focused on varying the other aryl
moiety at the 5-position of the thiazole skeleton, corresponding
to the B-ring of CA-4, by adding electron-withdrawing (CF3) or
electron-releasing (Me, Et, MeO, and EtO) substituents (EWG
and ERG, respectively). In addition, the B-ring was replaced with
naphth-1-yl, naphth-2-yl, or benzo[b]thienyl moieties. Because
the methoxy and ethoxy groups proved to be favorable for
bioactivity, we maintained one of these substituents at the para-
position and introduced additional substituents (F, Cl, Me, and
MeO) at the meta-position of the phenyl ring.
a Reagents. (a) thiourea, EtOH, reflux; (b) phthalic anhydride, AcOH,
reflux; (c) NBS, CHCl3, room temperature; (d) PdCl2(DPPF), ArB-
(OH)2, CsF, 1,4-dioxane, 65 °C; (e) NH2NH2, EtOH, reflux.
heterogeneous conditions [PdCl2(DPPF), CsF] in 1,4-dioxane at
65 °C to furnish derivatives 8aꢀo.17 The removal of the N-protected
phthaloyl group was accomplished by the use of hydrazine in
refluxing ethanol to afford final compounds 3aꢀo.
’ BIOLOGICAL RESULTS AND DISCUSSION
In Vitro Antiproliferative Activities. The 2-amino-4-(30,40,50-
trimethoxyphenyl)-5-aryl thiazoles 3aꢀo were evaluated for their
antiproliferative activity against a panel of seven human tumor cell
lines and compared with the reference compound CA-4 (1). The
data shown in Table 1 indicated the importance of substituents on
the phenyl ring at the 5-position of the thiazole system for activity
and selectivity against different cancer cell lines. In general, the
introduction of EWGs or ERGs increased activity compared with
the unsubstituted phenyl derivative 3a, with no clear difference in
effect on potency between EWGs and ERGs. The antiproliferative
activity of the most potent compounds was generally greater
against the cervical carcinoma HeLa cells as compared with the
other cell lines.
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
(P-gp) drug efflux pump. Moreover, a possible mechanism of
action for these compounds is described.
’ CHEMISTRY
The synthetic protocol employed for the preparation of
2-amino-4-(30,40,50-trimethoxyphenyl)-5-aryl thiazoles 3aꢀo is
shown in Scheme 1. 2-Amino-4-(30,40,50-trimethoxyphenyl)-
thiazole 5 was easily obtained in good yield by the Hantzsch
cyclocondensation of 2-bromo-1-(30,40,50-trimethoxyphenyl)-
ethanone 4 and thiourea in refluxing ethanol. The amino group
was temporarily protected as the phthalimide by treatment with
phthalic anhydride in refluxing acetic acid to afford 6 and then
regioselectively brominated with NBS in chloroform to yield the
5-bromothiazole 7. With our optimized conditions in hand, this
latter compound was subjected to Suzuki cross-coupling condi-
tions in the presence of the appropriate arylboronic acid under
The 40-ethoxyphenyl derivative 3n displayed the strongest
growth-inhibitory activity against HeLa, A549, and K562 cells,
with IC50 values of 0.03, 0.09, and 0.07 nM, respectively.
Compounds 3g (40-CF3 substituent) and 3j (40-OMe and 30-F
substituents) were the most active molecules against MCF-7 and
HT-29 cells, respectively (IC50s of 0.4 and 5.7 nM, respectively).
With the exception of compounds 3a, 3b, 3g, 3i, and 3m, the
tested compounds were distinctly more potent than CA-4, with
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Table 1. In Vitro Cell Growth Inhibitory Effects of Compounds 3aꢀo and CA-4 (1)
IC50a (nM)
compd
HeLa
A549
HL-60
Jurkat
K562
MCF-7
HT-29
3a
3b
3c
3094 ( 126
3865 ( 335
1.6 ( 0.6
2.8 ( 0.5
0.73 ( 0.2
1.0 ( 0.4
3.2 ( 0.4
2.3 ( 0.2
209 ( 0.2
2.0 ( 0.2
1.6 ( 0.3
2.6 ( 0.4
249 ( 13
0.03 ( 0.005
0.91 ( 0.32
4 ( 1
9600 ( 1490
6321 ( 2882
3.8 ( 0.6
3642 ( 132.5
8657 ( 2403
2.7 ( 0.3
0.9 ( 0. 1
3.9 ( 1.1
1.1 ( 0.3
5.1 ( 1.1
3.2 ( 1.2
118 ( 30
6.1 ( 2.1
2.8 ( 0.4
4.0 ( 0.3
301 ( 17
0.9 ( 0.3
0.27 ( 0.02
1 ( 0.2
3834 ( 443
4691 ( 325
3.3 ( 0.9
0.6 ( 0.01
3.9 ( 0.6
4.1 ( 1.1
8.5 ( 1.2
2.1 ( 0.4
48.4 ( 7.1
7.3 ( 1.1
2.9 ( 1.5
21.5 ( 7.5
319 ( 80
0.14 ( 0.05
0.06 ( 0.01
5 ( 0.6
8502 ( 840
9031 ( 891
22.1 ( 7.5
19.6 ( 9.1
58.7 ( 37.1
49.7 ( 9.5
7.6 ( 2.1
>10000
>10000
51.2 ( 9.2
7922 ( 1820
>10000
23.9 ( 3.9
19.1 ( 9.0
12.4 ( 5.1
54.1 ( 15.0
221 ( 50
3d
3e
3f
16.0 ( 2.1
42.8 ( 5.7
238 ( 88
47.0 ( 2.6
279.5 ( 69.8
117 ( 38
3g
3h
3i
357 ( 65
0.4 ( 0.1
20.2 ( 1.7
2374 ( 594
3.1 ( 0.6
5.1 ( 1.1
60.9 ( 8.4
476 ( 30
11.7 ( 3.9
239 ( 44
728 ( 122
6.0 ( 2.0
3j
18.6 ( 6.2
27.3 ( 9.6
58.7 ( 4.7
1170 ( 222
44.0 ( 6.3
14.1 ( 6.3
370 ( 100
5.7 ( 1.5
3k
3l
37.7 ( 5.0
305 ( 87
36.1 ( 18.6
67.6 ( 28.3
829 ( 181
0.07 ( 0.004
8.8 ( 1.0
12.8 ( 5.0
24.0 ( 10.4
432 ( 79
3m
3n
3o
CA-4
1797 ( 231
0.09 ( 0.01
11.2 ( 1.5
180 ( 50
38.2 ( 11.4
15.6 ( 7.9
3100 ( 100
5 ( 0.1
a IC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ( SE from the doseꢀresponse
curves of at least three independent experiments.
IC50 concentrations in the double-digit nanomolar range in the
CA-4 resistant HT-29 cells. However, compound 3n was the only
compound more active than CA-4 against all cell lines.
trifluoromethyl moiety (compounds 3h and 3g, respectively)
resulted in an 18- and 20-fold reduction in activity against A-549
and HT-29 cells, while 3h and 3g showed comparable potencies
against HeLa, HL-60, Jurkat, and K562 cells, but 3g was 150-fold
more active than 3h against MCF-7 cells and was the most active
compound of the series against this cancer cell line.
The phenyl (3a) and naphth-1-yl (3b) derivatives were the
least active compounds in the series, with IC50s over 3 μM
against all cell lines. Replacement of the naphth-1-yl moiety (3b)
with the naphth-2-yl group (3c) resulted in a marked increase in
activity, and 3c was more active than CA-4 in five of the seven cell
lines. Because the electronic properties of the 3b and 3c sub-
stituents are similar, the inactivity of 3b is presumably due to
steric factors caused by the naphthyl ring (see modeling section
below). Replacement of the naphth-2-yl ring (3c) by the bio-
isosteric benzo[b]thien-2-yl moiety (3d) had relatively minor
effects on antiproliferative activity in all cell lines.
Relative to the activity of 3h, the insertion of an additional
EWG or ERG group on the 30-position of the 40-methoxyphenyl
ring affected antiproliferative activity. In general, compounds 3j
and 3k, with the EWG fluorine and chlorine atoms, showed
stronger antiproliferative activities as compared with compounds
with electron-releasing methyl (3l) and, especially, methoxy
(3m) groups. Specific effects, however, seemed to vary with
the cell line tested. Thus, with compounds 3hꢀl, 3h had the
greatest activity with Jurkat and K562 cells, 3j with A549, MCF-7
and HT-29 cells, and 3k with HeLa and HL-60 cells.
In comparing the 40-methylphenyl (3e) and 4-ethylphenyl
(3f) with each other and with 3c and 3d, there were only minor
differences in IC50 values, except that 3f was the least active
against the A549 cells and 3e against the MCF-7 cells. Re-
placement of the methyl group (3e) with the methoxy group
(3h) increased antiproliferative activity in five of the cell lines,
with a 10-fold increase in the A549 and K562 cells.
Comparing the highly active 40-ethoxy derivative 3n with 3o,
which has an additional 30-chloro substituent on the phenyl ring,
activity was increased with 3o by 2ꢀ20-fold against HL-60,
Jurkat, MCF-7, and HT-29 cells but sharply reduced (30- to 125-
fold) against HeLa, A549, and K562 cells.
The number and position of methoxy substituents on the C5-
phenyl ring had a major influence on antiproliferative activity.
Moving the methoxy group from the para- (3h) to the meta-
position (3i) or the insertion of an additional methoxy group
(30,40-dimethoxy derivative 3m) led to a drop in potency. As
noted above, the 40-ethoxy homologue 3n generally had the
greatest antiproliferative activity of all compounds in the series.
Specifically comparing it with the 40-methoxy compound 3h, 3n
was less active only in the HT-29 cells. The greatest differences in
activity were 77-fold in the HeLa cells, 224-fold in the A549 cells,
and 73-fold in K562 cells.
Inhibition of Tubulin Polymerization and Colchicine Bind-
ing. To investigate whether the antiproliferative activities of com-
pounds 3cꢀh, 3jꢀl, and 3nꢀo 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 2).18 For comparison, CA-4 was examined in
contemporaneous experiments. In the assembly assay, compound
3e was found to be the most active (IC50, 0.44 μM), and it was
almost 3-fold more potent than CA-4 (IC50, 1.2 μM). With the
exception of compounds 3d and 3g, all tested molecules strongly
inhibited tubulin assembly, with activity superior to (3c, 3k, 3n,
and 3o) or comparable with (3e, 3f, 3h, 3j, and 3l) that of CA-4.
Compound 3d was half as active and 3g, about one-fourth as active
as CA-4, although 3d and 3g were more potent than CA-4 as
antiproliferative agents against MCF-7 and HT-29 cells.
In comparing the effects of strong ERGs and EWGs on the
phenyl at the C5 thiazole position, no consistent difference in
effects on antiproliferative activity occurred. For example, re-
placement of the ERG methoxy group with the EWG and bulkier
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Table 2. Inhibition of Tubulin Polymerization and Colchicine
Binding by Compounds 3cꢀh, 3jꢀl, 3nꢀo, and CA-4
tubulin assemblya
colchicine binding b
compd
3c
IC50 ( SD (μM)
% ( SD
0.74 ( 0.0
2.0 ( 0.2
0.44 ( 0.0
1.1 ( 0.0
4.0 ( 0.5
1.1 ( 0.0
1.2 ( 0.1
0.88 ( 0.1
1.1 ( 0.1
0.61 ( 0.07
0.89 ( 0.05
1.2 ( 0.1
94 ( 0.7
86 ( 2
3d
3e
88 ( 1
3f
73 ( 3
3g
25 ( 0.7
79 ( 0.3
81 ( 0.2
77 ( 0.2
72 ( 3
3h
3j
3k
3l
3n
95 ( 1
3o
87 ( 1
CA-4 (1)
98 ( 0.6
a Inhibition of tubulin polymerization. Tubulin was at 10 μM. b Inhibi-
tion of [3H]colchicine binding. Tubulin, colchicine and tested com-
pound were at 1, 5, and 5 μM, respectively.
Figure 1. Immunofluorescences images of HeLa cells stained with anti-
β-tubulin antibody FITC-conjugated and then observed by confocal
microscopy (A). Cells were exposed to 10 or 100 nM 3n for 24 or 48 h
and then fixed and analyzed by fluorescence microscopy. Magnification
60ꢁ. (B) Representative images of HeLa cells stained with phalloidin-
tetramethylrhodamine B isothiocyanate conjugate after a 24 h incuba-
tion in the presence of 3n. Cells were also counterstained with DAPI to
visualize the nuclei.
When comparing inhibition of tubulin polymerization versus
the growth inhibitory effect, we found a good correlation for most,
but not all, of the active compounds. While 3e was generally less
potent than 3d as an antiproliferative agent, 3d was about five
times less active than 3e as inhibitor of tubulin assembly.
In the colchicine binding studies, derivatives 3cꢀe and 3nꢀo
were almost (86ꢀ95% inhibition) as potent as CA-4, which in
these experiments inhibited colchicine binding by 98%. The
potent inhibition observed indicates that 3cꢀe and 3nꢀo bind
to tubulin at a site overlapping the colchicine site. Inhibition of
colchicine binding by compounds 3f, 3h, and 3jꢀl was slightly
lower (72ꢀ81%), while 3g inhibited colchicine binding by only
25%. It is thus significant that many agents in the present series
have activities superior to that of CA-4 as inhibitors of tubulin
assembly and are also highly active as inhibitors of colchicine
binding to tubulin.
While this group of compounds were all potent in the
biological assays (inhibition of cell growth, tubulin assembly,
and colchicine binding), correlations between the three assay
types were imperfect. Thus, while 3e was 5-fold more potent than
3d in the tubulin assembly assay, these two molecules were
equipotent as inhibitors of colchicine binding.
We further examined the effect of 3n, one of the most active
compounds, on the cellular microtubule network by immuno-
fluorescence analysis. As shown in Figure 1, the microtubule
network exhibited normal arrangement and organization in HeLa
cells in the absence of drug treatment. In contrast, 24 or 48 h of
exposure to 10 nM 3n caused microtubule disassembly, with
induction of a spherical morphology. Exposure to the compound
at 100 nM resulted in an almost complete loss of microtubules.
Nuclear changes were also observed. Exposure of the cells to 3n
resulted in the appearance of giant interphase cells with multiple
nuclei of different sizes. In contrast to the dramatic changes in
cellular microtubules, cells treated with 3n showed minimal effects
in the arrangement and amount of F-actin (Figure 1B), consistent
with a preferential effect on microtubules.
Figure 2. Docked pose of 3c (orange), 3e (magenta), and 3n (cyan)
overlapped with DAMA-colchicine (green) in the tubulin binding site.
mode observed for this series of compounds is very similar to the
one observed for the cocrystallized DAMA-colchicine (Figure 2).19
The trimethoxyphenyl group occupies the same position as the
corresponding moiety of the colchicine analogue, while the
aromatic ring in the 5 position of the thiazole ring establishes a
πꢀπ interaction with Asn258 and a series of nonpolar interactions
with Met259 and Lys352. Also consistent with our experimental
Molecular Modeling. To rationalize our experimental find-
ings, a series of molecular docking simulations on β-tubulin were
performed, using a procedure reported previously.16c The binding
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Table 3. In Vitro Cell Growth Inhibitory Effects of Compounds 3h, 3n, and 3o on Drug Resistant Cell Lines
IC50a(nM)
compd
LoVo
LoVoDoxo
CEM
CEM Vbl100
A549
A549-T12
3h
3n
3o
3.1 ( 1.0
0.7 ( 0.08
2.0 ( 1.0
120 ( 30
nd
0.8 ( 0.06 (0.25)b
0.6 ( 0.05 (0.9)
1.7 ( 0.7 (0.9)
13150 ( 210 (109.6)
nd
7.7 ( 3.7
0.9 ( 0.2
1.4 ( 0.6
nd
1.2 ( 0.6 (0.1)
0.8 ( 0.09 (0.9)
0.7 ( 0.08 (0.5)
nd
20.2 ( 1.7
0.09 ( 0.01
11.2 ( 2.5
nd
11.4 ( 2.3 (0.6)
0.2 ( 0.05 (2.2)
8.5 ( 1.2 (0.8)
nd
doxorubicine
vinblastine
taxol
4.1 ( 0.2
nd
230 ( 32 (56.1)
nd
nd
nd
nd
nd
7.2 ( 0.1
75.2 ( 12.5 (10.4)
a IC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ( SE from the doseꢀresponse
curves of at least three independent experiments. nd, not determined b Values in parentheses are fold resistance, indicating reduced potency of the
compounds in the resistant cell lines
data, while the 2-naphthyl analogue 3c binds in the way described
above (Figure 2), the different orientation of the naphthyl ring of
3b does not allow a suitable docking for this compound. Also, in
the cases of compounds 3i and 3m, steric hindrance by the
methoxy substituent in the meta position of the aromatic ring
does not allow a successful docking, while smaller groups like
F, Cl, and CH3 in the same position (3j, 3k and 3l) can be
accommodated in the binding pocket. This observation is in
accordance with the experimental data obtained for these
compounds as well as with the results obtained previously with
another series of compounds.16c
Effects of Compounds 3h, 3n, and 3o on Multidrug Resis-
tant Cell Lines. Although many anticancer drugs in clinical use are
effective in the treatment of different kinds of tumors, their
potential is limited by the development of drug resistance.20
Resistance can be intrinsic or acquired but, in either case, tumors
become refractory to a variety of structurally different drugs. Thus,
the antiproliferative effects of 3h, 3n, and 3o were evaluated in
human cancer cell lines derived from a lymphoblastic leukemia
(CEMVbl-100) and a colon carcinoma (LovoDoxo), both expressing
high levels of the 170-kDa P-glycoprotein (P-gp) drug efflux
pump.21,22 As shown in Table 3, the examined compounds were
equally potent toward parental cells and cells resistant to vinblas-
tine or doxorubicin.
Resistance to microtubule inhibitors is also mediated by
changes in the levels of expression of different β-tubulin isotypes
and by tubulin gene mutations that result in modified tubulin
with impaired polymerization properties. A-549-T12 is a cell line
with an R-tubulin mutation with increased resistance to taxol.23
This cell line, too, was sensitive to 3h, 3n, and 3o. Thus, the data
shown in Table 3 suggested that 3h, 3n, and 3o might be useful in
the treatment of drug refractory tumors, in particular those with
resistance to other antitubulin drugs.
Analysis of Cell Cycle. The effects of different concentrations
of compound 3n on cell cycle progression were examined in
stained with PI to analyze DNA content by flow cytometry. Data are
Figure 3. Effect of compound 3n on cell cycle distribution of HeLa
(A) and Jurkat cells (B). Cells were treated with different 3n concentra-
tions ranging from 1 to 100 nM for 24 h. Then the cells were fixed and
HeLa and Jurkat cells (Figure 3A,B). Compound 3n caused a
clear G2/M arrest pattern in a concentration-dependent manner,
starting at a concentration of 5 nM for both cell lines, a
concentration higher than that which induces cytotoxicity. In
parallel, we observed a concomitant decrease of cells in the G1
phase of the cell cycle (Figure 3A,B), while the percentage of S
phase cells declined slightly at 100 nM for HeLa cells and
extensively for Jurkat cells. Analogous behavior was also observed
after 48 h of treatment (data not shown).
presented as mean ( SEM of three independent experiments. ** p < 0.01
vs control. (C) Effect of 3n on 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.
could be explained by the crucial role that microtubules play
in maintaining normal cellular functions. Most antimitotic
drugs have an all or nothing effect on cell division in the sense
that they have no observable effect at low concentrations but
induce a significant mitotic arrest above critical concentrations
The apparent discrepancy between the different concentra-
tions that induce cell cycle arrest and the cytotoxic efficacy of 3n
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Figure 4. Flow cytometric analysis of apoptotic cells after treatment of
HeLa cells with 3n. (A) Percentage of cells found in the different regions
of the biparametric histograms obtained from cytofluorimetric analysis,
after incubation with 3n for 24 or 48 h (A, annexin-V; PI, propidium
iodide). (B) Percentage of cell viability after 48 h of incubation of HeLa
cells with 3n (100 nM) in the presence or in the absence of z-VAD.fmk
(100 μM). Mean ( SEM of three independent experiments. **P < 0.01
vs. 3n treated cells.
Figure 5. (A) Western blot analysis for the cleavage of PARP and the
expression of Bcl-2, Bax, pro-caspase-9, and pro-caspase-2 in HeLa cells.
The cells were treated with the indicated concentration of 3n for the
indicated times. Whole cell lysates were subjected to SDS-PAGE,
followed by blotting with the appropriate antibody. (B) Induction of
caspase-3 activity by compound 3n. HeLa cells were incubated in the
presence of 3n at the indicated concentrations. After a 24 or 48 h
treatment, cells were harvested and stained with an antihuman active
caspase-3 fragment monoclonal antibody conjugated with FITC. Data
are expressed as percentage of caspase-3 active fragment positive cells. **
p < 0.01 vs control.
and may induce cell death without an apparent block of the cell
cycle.24,25
We next studied the association between 3n-induced G2/M
arrest and alterations in expression of proteins that regulate cell
division. Cell cycle arrest at the prometaphase/metaphase to
anaphase transition is normally regulated by the mitotic
checkpoint.26 In eukaryotic cells, the activation of Cdc2 kinase
is necessary for occurrence of the G2/M transition of the cell
cycle. Activation of the kinase requires accumulation of the cyclin
B1 protein and its dephosphorylation at Tyr15 and Thr14.26 As
shown in Figure 3C in HeLa cells, 3n caused a concentration- and
time-dependent increase in cyclin B1 expression and a decreased
expression of p-Cdc2Y15, in particular, after 48 h of treatment.
In addition, slower migrating forms of phosphatase Cdc25c
were present, especially at the concentration of 100 nM, indicat-
ing changes in the phosphorylation state of this protein. The
phosphorylation of Cdc25c directly stimulates its phosphatase
activity, and this is necessary to activate Cdc2/Cyclin B on entry
into mitosis.26 These results indicate that arrest at G2/M induced
by 3n is accompanied by an increased expression of cyclin B1
and, at later times (48 h) for the highest concentration (100 nM),
(see Supporting Information). As depicted in Figure 4A, com-
pound 3n at 24 h had already induced an accumulation of
annexin-V positive cells in comparison with the control, and this
accumulation was concentration dependent. After a 48 h incuba-
tion, we observed a further decrease of cell viability along with a
marked increase in PI positive cells.
To evaluate if the cell death induced by 3n is caspase-
dependent, HeLa cells were treated with 3n in the absence or
presence of the pan-caspase inhibitor z-VAD.fmk. Inhibition of
caspases significantly increased cell viability (Figure 4,B), but this
caspase inhibition had only a partial impact on 3n-induced cell
death. The caspase inhibitor increased cell survival from about
20% to 40%, suggesting that other mechanism(s) may lead to cell
death following treatment with 3n.
by a marked decrease of Cdc25c and p-Cdc2Y15
.
Effect of 3n on the Activation of Caspases. To have better
insight into the mechanism of cell death induced by compound
3n, we analyzed the mitochondrial potential (ΔΨmt) after cell
treatment with the compound. No changes in ΔΨmt were
observed after treatment with 3n, as depicted in Figure 4s (see
Supporting Information). We also evaluated the mitochondrial
production of ROS by two fluorescent probes, HE and
H2DCFDA, by flow cytometry.29 In agreement with the ΔΨmt
results, only a slight increase of ROS production was observed for
cells treated with 3n (Supporting Information, Figure S2).
Compound 3n Induces Apoptosis That Is Partially Caspase-
Dependent. To characterize the mode of cell death induced by 3n,
a biparametric cytofluorimetric analysis was performed using PI,
which stains DNA and is permeable only to dead cells, and
fluorescent immunolabeling of the protein annexin-V, which binds
to PS in a highly selective manner.27
Dual staining for annexin-V and with PI permits discrimina-
tion between live cells (annexin-Vꢀ/PIꢀ), early apoptotic
cells (annexin-V+/PIꢀ), late apoptotic cells (annexin-V+/PI+),
and necrotic cells (annexin-Vꢀ/PI+),28 as shown in Figure 1s
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ARTICLE
usually occurs during mitosis in response to DNA damage or
antimitotic agents.33 Unlike apoptosis, which is basically depen-
dent on caspase activation, mitotic catastrophe may be mediated
in a caspase-independent or caspase-dependent fashion. In some
cases, mitotic catastrophe shares the same signaling pathways
with apoptosis. Cells undergoing mitotic catastrophe are char-
acterized by multinucleation, incomplete DNA synthesis and
premature chromosome condensation.32c,d
Morphological alterations were observed in both HeLa and Jurkat
cells after 24 and 48 h of exposure to 3n (Figure 6A,B). These
changes consisted mainly of DNA condensation, abnormal mitotic
figures, multinucleation, and formation of large cells. Furthermore,
large cytoplasmic vacuoles were also observed in Jurkat cells.
To determine whether cell death was induced because of DNA
damage, we examined the expression of phospho-γH2AX, a well-
known marker for cellular DNA double-strand breaks. As shown
in Figure 6C, a dramatic increase in expression of phospho-
γH2AX was observed after both 24 and 48 h treatments,
suggesting that DNA damage did occur after its replication was
stalled by compound 3n. In this context, p53 and p21waf/cip1 play
an essential role in inducing cell cycle arrest.33 The p53 protein
levels did not show any variation relative to the untreated cells,
except at 48 h, when expression was reduced in the presence of
3n (Figure 6C), bypassing the G2/M checkpoint stage to enter
mitotic catastrophe. Cells may proceed to mitosis again by the
degradation of p21. Our results showed that p21 expression was
strongly reduced after both 24 and 48 h treatments with 100 nM
3n. Downregulation of p21waf/cip1 protein by 3n might have
contributed to the formation of multinucleated cells, as pre-
viously reported.34
Figure 6. (A) Mitotic catastrophe. Representative images of Giemsa-
stained HeLa (A) and Jurkat cells (B), observed under a light micro-
scope after a 48 h treatment with compound 3n. Magnification 60ꢁ.
(C) Western blot analysis for the expression of p-γH2AXSer139, p53, and
p21waf/cip1 in HeLa cells. The cells were treated with the indicated
concentration of 3n for the indicated times. Whole cell lysates were
subjected to SDS-PAGE, followed by blotting with the appropriate
antibodies.
Next, we analyzed the effect of 3n on the expression of two
members of the Bcl-2 family, the antiapoptotic Bcl-2 and the
proapototic Bax. 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 mol-
ecules important for the cell death pathway.26a As shown in
Figure 5A, 3n did not significantly affect the expression of these
two proteins at 24 h, but there was a marked decrease at 48 h
following the death of a large number of cells. Altogether, these
data indicate that mitochondria were not involved in the
mechanism of cell death induced by 3n at 24 h.
As shown before, apoptosis is only partially caspase-depen-
dent. To determine which caspases were involved in 3n-induced
cell death, the expression of caspases was evaluated by immuno-
blot analysis and flow cytometry. We observed a clear activation
in a time-dependent manner, especially at 100 nM, of the effector
caspase-3 and cleavage of its substrate PARP (Figure 5A,B).
Compound 3n did not induce activation of caspase-9, the major
initiator caspase of the mitochondrial apoptotic pathway
(Figure 5A). Following treatment with 3n, caspase-2 seems to
be activated upstream of caspase-3. Recent evidence about the
functions and activation mechanisms of the caspases indicate that
caspase-2 is unique among these enzymes, displaying features of
both initiator and executioner.30 Many recent studies indicate
that activation of caspase-2 is fundamental for the induction of
apoptosis induced by antimitotic drugs.31
’ CONCLUSIONS
An efficient convergent synthesis and biological evaluation of a
new series of rigid analogues of CA-4 were described. The new
compounds contain the 2-amino thiazole ring system in place of
the ethylene bridge present in CA-4. We fixed one of the aryl
groups as a 30,40,50-trimethoxyphenyl moiety throughout the
present investigation, and the modifications were mainly focused
on variation of the substituents on the second aryl ring. Changes
in this second aryl moiety had unpredictable effects on biological
activity. The screening antiproliferative studies revealed that
most of the synthesized compounds strongly inhibited the
growth of all cancer cell lines examined, but specific effects were
highly dependent on the aryl substituent at the 5-position of the
thiazole ring, with no clear influence on activity observed by
comparing the effects of ERGs and EWGs. It is clear that the
substitution pattern on the phenyl moiety at the 5-position of the
2-amino thiazole ring plays an important role for antitubulin and
antiproliferative activities, and this was supported by the molecular
docking studies. As shown in Table 1, human cervical carcinoma
HeLa cells were, in general, more sensitive toward tested
compounds than the other cancer cell lines, with IC50 values
in the low nanomolar range. Among the evaluated compounds,
one of the most active analogues was found to be the 2-amino-
4-(30,40,50-trimethoxyphenyl)-5-(40-ethoxyphenyl)thiazole ana-
logue 3n. This compound displayed antiproliferative activity at
subnanomolar concentrations (IC50 = 0.03ꢀ0.9 nM) against all
tested cancer cell lines, with the exception of HT-29 and MCF-7
cells (IC50 = 39 and 44 nM, respectively). Compounds 3cꢀh,
3jꢀk, and 3nꢀo exhibited the strongest growth inhibition
activity, with some of their IC50 values much lower than those
Compound 3n Induces Mitotic Catastrophe. It was recently
demonstrated that antimitotic drugs, including CA-4, induce an
alternative form of cell death to that of apoptosis. This has been
called mitotic catastrophe or cell death occurring during
metaphase.32 Mitotic catastrophe is a type of cell death that
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Journal of Medicinal Chemistry
ARTICLE
of the reference compound CA-4. More importantly, these
compounds were also found to be active in cells overexpressing
P-gp, suggesting that these derivatives might be useful in treating
drug-refractory patients. We identified tubulin as the molecular
target of all compounds examined. With most of them, tubulin
polymerization was more potently inhibited than occurred with
the reference compound CA-4. Like CA-4, however, all com-
pounds inhibited colchicine binding to tubulin.
Compound 3e was the most potent inhibitor of tubulin
polymerization and one of the most potent inhibitors of colchi-
cine binding (IC50 = 0.44 μM for assembly, 88% inhibition of the
binding of [3H]colchicine). We also showed by flow cytometry
that 3n induced apoptosis and that apoptosis induced by 3n was
only partially dependent on caspase activation. Apoptosis in-
duced by 3n did not involve mitochondrial depolarization.
Preliminary experiments suggested that, in addition to apoptosis,
cells were killed by mitotic catastrophe, as indicated by morpho-
logical changes including formation of giant cells and multi-
nucleated cells. These observations were corroborated at the
molecular level by analysis of the expression of some proteins
associated with mitotic catastrophe. The ability of 3n to induce
different modes of cell death may contribute to its efficacy in
different types of cancer cells, and its properties warrant its
further testing in preclinical in vivo cancer models.
Synthesis of 2-(4-(3,4,5-Trimethoxyphenyl)thiazol-2-yl)-
isoindoline-1,3-dione (6). To a suspension of compound 5 (1.33 g,
5 mmol) in acetic acid (30 mL) was added phthalic anhydride (890 mg,
6 mmol). After stirring for 18 h at reflux, the solvent was evaporated and
the residue dissolved in CH2Cl2 (50 mL). The organic solution was
washed with a saturated solution of NaHCO3 (15 mL), water (10 mL),
and brine (10 mL), dried, and concentrated. The crude product was
purified by crystallization from ethyl ether, to afford 6 (1.5 g, 76%) as a
yellow solid, mp 202ꢀ203 °C. 1H NMR (CDCl3) δ: 3.88 (s, 3H), 3.93
(s, 6H), 7.18 (s, 2H), 7.42 (s, 1H), 7.84 (m, 2H), 8.03 (m, 2H). MS
(ESI): [M]+ = 396.4.
Synthesis of 2-(5-Bromo-4-(3,4,5-trimethoxyphenyl)thia-
zol-2-yl)isoindoline-1,3-dione (7). A solution of compound 6
(1.19 g, 3 mmol) in anhydrous chloroform (20 mL) was cooled to
0 °C and treated with NBS (746 mg, 3.3 mmol) under nitrogen. The
reaction was allowed to warm to room temperature after 15 min and
then stirred for 4 h. After quenching with saturated Na2S2O3 (20 mL),
the resulting mixture was diluted with water (20 mL) and extracted with
CH2Cl2 (2 ꢁ 30 mL). The combined organics were dried (MgSO4) and
evaporated to give a yellow solid, which was purified by crystallization
from ethyl ether, to afford 7 (1.1 g, 78%) as a yellow solid, mp 220ꢀ
1
221 °C. H NMR (DMSO-d6:) δ: 3.72 (s, 3H),3.83 (s, 6H), 7.21
(s, 2H), 7.95 (m, 4H). MS (ESI): [M]+ = 474.7, [M]+ = 476.7
General Procedure A for the Synthesis of Compounds
8aꢀo. A stirred suspension of 7 (237 mg, 0.5 mmol) and the
appropriate phenylboronic acid (0.75 mmol) in dioxane (6 mL contain-
ing 1 drop of water) was degassed under a stream of nitrogen over 10
min and then treated with PdCl2(DPPF) (41 mg, 0.05 mmol) and CsF
(190 mg, 1.25 mmol). The reaction mixture was heated under nitrogen
at 45 °C for 30 min and then at 75 °C for 5 h. The reaction mixture was
cooled to ambient temperature, diluted with CH2Cl2 (10 mL), filtered
on a pad of Celite, and evaporated in vacuo. The residue was dissolved
with CH2Cl2 (15 mL), and the resultant solution was washed sequen-
tially with water (5 mL) and brine (5 mL). The organic layer was dried
and evaporated, and the residue was purified by flash chromatography
on silica gel.
’ EXPERIMENTAL SECTION
1
Chemistry. Materials and Methods. H NMR spectra were
recorded on a Bruker AC 200 spectrometer. Chemical shifts (δ) are
given in ppm upfield from tetramethylsilane as internal standard, and the
spectra were recorded in appropriate deuterated solvents, as indicated.
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
1
are uncorrected. All products reported showed H NMR spectra in
General Procedure B for the Synthesis of Compounds
3aꢀo. A stirred suspension of a thiazole derivative (0.5 mmol) and
hydrazine monohydrate (58 μL, 0.6 mmol, 1.2 equiv) in abs EtOH
(10 mL) was refluxed for 1 h. The solvent was evaporated, the residue
was suspended with CH2Cl2 (10 mL), and the suspension filtered
through Celite. The filtrate was concentrated in vacuo to obtain a residue
that was purified by column chromatography.
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 Uni-
versity 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. Standard syringe techniques were used for transferring dry
solvents. Reaction courses and product mixtures were routinely mon-
itored by TLC on silica gel (precoated F254 Merck plates), and
compounds were visualized with aqueous KMnO4. Flash chromatogra-
phy was performed using 230ꢀ400 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).
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed characterization of
b
synthesized compounds 8aꢀo and 3aꢀo. Experimental proce-
dures for biological assays and computational methodology. This
material is available free of charge via the Internet at http://pubs.
acs.org.
Synthesis of 4-(3,4,5-Trimethoxyphenyl)thiazol-2-amine
(5). The mixture of 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone
(2.91 g, 10 mmol) and thiourea (836 mg, 11 mmol) in anhydrous
EtOH (30 mL) was heated at reflux for 1 h. After that, the solvent was
removed in vacuo, and saturated aqueous NaHCO3 was added to make
the mixture basic (pH = 8ꢀ9). Then the mixture was extracted with
CH2Cl2 (3 ꢁ 25 mL). The combined organic phases were washed with
brine (25 mL) and dried with anhydrous Na2SO4. After removal of the
solvent, the residue was stirred for 20 min with petroleum ether (40 mL)
and filtered to afford 5 (2.1 g, 79%) as a yellow solid, mp 170ꢀ171 °C.
1H NMR (CDCl3) δ: 3.86 (s, 3H), 3.92 (s, 6H), 5.42 (bs, 2H), 6.63 (s,
1H), 7.00 (s, 2H). MS (ESI): [M]+ = 266.4.
’ AUTHOR INFORMATION
Corresponding Author
*For R.R.: phone, 39-(0)532-455303; fax, 39-(0)532-455953;
E-mail, rmr@unife.it. For G.V.: phone, 39-(0)49-8211451; fax,
39-(0)49-8211462; E-mail, giampietro.viola1@unipd.it.
’ ACKNOWLEDGMENT
We thank Dr. Alberto Casolari for excellent technical
assistance.
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ARTICLE
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CA-4, combretastatin A-4; EWG, electron-withdrawing group; ERG,
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