One-pot synthesis and biological evaluation of 2-pyrrolidinyl-4-amino-5- (3′,4′,5′-trimethoxybenzoyl)thiazole: A unique, highly active antimicrotubule agent

Article abstract of DOI:10.1016/j.ejmech.2011.10.013

A wide variety of small molecules with diverse molecular scaffolds inhibit microtubule formation. In this article we report a one-pot procedure for the preparation of a novel 2-(N-pyrrolidinyl)-4-amino-5-(3′,4′,5′- trimethoxybenzoyl)thiazole in which the size of the substituent at the C-2 position of the thiazole ring plays an essential role in compound activity. The most active agent (3f) inhibited at submicromolar concentrations the growth of tumor cell lines. It also inhibited tubulin polymerization with an activity quantitatively similar to that of CA-4, and treatment of HeLa cells resulted in their arrest at the G2-M phase of the cell cycle. Furthermore, 3f was effective against multidrug resistant cancer cells and inhibited the growth of the HT-29 xenograft in a nude mouse model. This indicated that 3f is a promising new antimitotic agent with encouraging preclinical potential.

Full text of DOI:10.1016/j.ejmech.2011.10.013

European Journal of Medicinal Chemistry 46 (2011) 6015e6024
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
One-pot synthesis and biological evaluation of 2-pyrrolidinyl-4-amino-5-(30,40,50-
trimethoxybenzoyl)thiazole: A unique, highly active antimicrotubule agent
,
a
a
a
Romeo Romagnoli a , Pier Giovanni Baraldi , Carlota Lopez Cara , Maria Kimatrai Salvador ,
**
*
Roberta Bortolozzi b, Giuseppe Basso b, Giampietro Viola b , Jan Balzarini , Andrea Brancale ,
***
,
c
d
Xian-Hua Fu e, Jun Li e, Su-Zhan Zhang e , Ernest Hamel
,
f
a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy
b Dipartimento di Pediatria, Laboratorio di Oncoematologia, Università di Padova, 35131 Padova, Italy
c Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium
d The Welsh School of Pharmacy, Cardiff University, CF10 3NB Cardiff, UK
e 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
f Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A wide variety of small molecules with diverse molecular scaffolds inhibit microtubule formation. In this
article we report a one-pot procedure for the preparation of a novel 2-(N-pyrrolidinyl)-4-amino-5-
(30,40,50-trimethoxybenzoyl)thiazole in which the size of the substituent at the C-2 position of the
thiazole ring plays an essential role in compound activity. The most active agent (3f) inhibited at sub-
micromolar concentrations the growth of tumor cell lines. It also inhibited tubulin polymerization with
an activity quantitatively similar to that of CA-4, and treatment of HeLa cells resulted in their arrest at the
G2-M phase of the cell cycle. Furthermore, 3f was effective against multidrug resistant cancer cells and
inhibited the growth of the HT-29 xenograft in a nude mouse model. This indicated that 3f is a promising
new antimitotic agent with encouraging preclinical potential.
Received 8 August 2011
Received in revised form
4 October 2011
Accepted 6 October 2011
Available online 15 October 2011
Keywords:
Microtubules
Structure-activity relationship
Thiazole
Ó 2011 Elsevier Masson SAS. All rights reserved.
In vivo and in vitro activity
Tubulin
1. Introduction
is one of the well-known natural tubulin-binding molecules
affecting microtubule dynamics by binding to the colchicine site
Antimitotic agents are one of the major classes of cytotoxic
drugs for cancer treatment, and tubulin is the target for
numerous small natural and synthetic molecules that inhibit the
formation of the mitotic spindle [1]. Besides mitosis, the micro-
tubule system of eukaryotic cells is a critical element in a variety
of essential cellular processes, including formation and mainte-
nance of cell shape, regulation of motility, cell signaling, secretion
and intracellular transport [2]. Among the microtubule depoly-
merizing agents, combretastatin A-4 (CA-4, 1; Chart 1) [3], iso-
lated from the bark of the South African tree Combretum caffrum,
on tubulin [4]. Because of its simple structure, a wide number of
CA-4 analogs have been developed and evaluated in structure-
activity relationships (SAR) studies [5].
Among the synthetic inhibitors of tubulin polymerization, we
have recently described
a series of 2-arylamino-4-amino-5-
aroylthiazoles with general structure 2 that showed strong anti-
proliferative activity against tumor cell lines and inhibited tubulin
polymerization by interfering with the colchicine site [6]. These
compounds also caused cancer cells to arrest in the G2-M phase of
the cell cycle, and all possessed a 4-aminothiazole nucleus in which
substitution either at the C-2 or C-5 position played an essential
role in potency.
As a part of our continuing search for novel antimitotic agents,
these findings prompted us to synthesize by a one-pot four-step
procedure a new series of 2-alkylamino-4-amino-5-aroylthiazole
derivatives with general structure 3 in which the 2-anilino
* Corresponding author. Tel.: þ39 (0) 532 455303; fax: þ39 (0) 532 455953.
** Corresponding author. Tel.: þ39 (0) 49 8211451; fax: þ39 (0) 49 8211462.
*** Corresponding author. Tel.: þ86 571 8731 5002; fax: þ86 571 8702 2776.
E-mail addresses: rmr@unife.it (R. Romagnoli), giampietro.viola1@unipd.it
(G. Viola), zhangsirhao@gmail.com (S.-Z. Zhang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.013

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R. Romagnoli et al. / European Journal of Medicinal Chemistry 46 (2011) 6015e6024
Scheme 1. Reagents. a: Na2S.9H2O, DMF, 70 ꢀC, 2 h; b: 1-(30,40,50-trimethoxyphenyl)-2-
bromoethanone, 50 ꢀC, 2 h; c: K2CO3, 1 h; d: NH3 for 3a, (CH3)2NH in EtOH for 3b,
(C2H5)NH for 3c; e: appropriate secondary amine, DMF, 70 ꢀC, 1 h; f: appropriate
a-
bromoacetophenone, 2 h, 50 ꢀC.
3. Biological results and discussion
3.1. Antiproliferative effects of derivatives 3aeo
Table 1 summarizes the antiproliferative effects of derivatives
3aeo against a panel of three human and two rodent cancer cell
lines. These are murine leukemia (L1210) and mammary carcinoma
(FM3A) lines and the human T-leukemia lines Molt/4 and CEM and
the human cervix carcinoma HeLa line. Only 3f showed sub-
micromolar antiproliferative activity against all tested lines, while
other derivatives generally had little activity, with IC50 values
Chart 1. Inhibitors of Tubulin Polymerization.
moiety of compounds 2 was replaced by an amino moiety or
various cyclic or acyclic alkyl amino substituents. SAR between the
substitutions at the C-2 and C-5 positions of the thiazole core were
evaluated.
usually greater than 10 mM. Derivative 3f was 10- to 100-fold less
active than the reference compound CA-4.
Extensive exploration of SAR of this novel series of compounds
showed that the presence of pyrrolidin-1-yl and 30,40,50-trime-
thoxybenzoyl at the C-2 and C-5 positions of the 4-aminothiazole
scaffold were essential for the biological activity of 3f. Consid-
ering substitutions at the C-2 position, even minor modifications,
such as expanding the pyrrolidine (3f) to a piperazine (3g) ring,
caused a large reduction in antiproliferative activity.
Comparing 3f with 3ieo, all with a pyrrolidin-1-yl moiety at the
C-2 position of the thiazole skeleton, demonstrated that the 30,40,50-
trimethoxbenzoyl moiety at the C-5 position (3f) was essential for
activity. Its replacement with a 30,40-dimethoxybenzoyl (3i), several
isomeric methoxybenzoyl (3jel), 40-chloro- and 40-bromobenzoyl
(derivatives 3m and 3n, respectively) or the unsubstituted benzoyl
(3o) moiety all led to inactive compounds.
Since it is well known that the trimethoxyphenyl group is the
characteristic structural requirement to maximize the potent
activity in a large series of inhibitors of tubulin polymerization,
such as colchicine, CA-4 and podophyllotoxin [7], we prepared
compounds 3aeh, all of which retain the 30,40,50-trimethox-
ybenzoyl group at the C-5 position of the thiazole ring, allowing us
to evaluate the effect of substitution at the C-2 position by cyclic
and acyclic alkyl amines. The pyrrolidin-1-yl substituent (3f)
resulted in a compound that was superior to the others. The potent
activity of 3f led us to synthesize a second series of derivatives 3ieo
in which we maintained the pyrrolidin-1-yl group at the C-2
position, but replaced the 5-(30,40,50-trimethoxybenzoyl) moiety of
3f with other benzoyl groups.
Conversely, when the 30,40,50-trimethoxbenzoyl group was at
the C-5 position of the thiazole skeleton (derivatives 3aef and 3gh),
significant loss of activity occurred upon changing the pyrrolidin-1-
yl ring at the C-2 position to any other substituent that we evalu-
ated. Alternate groups examined were the amine (3a), dimethyl-
amine (3b), diethylamine (3c), N-methyl-N’-n-butyl amine (3d), N-
methyl-N’-benzylamine (3e), piperidin-1-yl (3g) and morpholin-4-
yl (3h). Thus, we speculate that the tubulin binding pocket for this
portion of the molecule is quite small and allowed the presence
only of the pyrrolidin-1-yl moiety.
2. Chemistry
The effectiveness of the methylsulfanyl group as a leaving group
was employed for the preparation of compounds 3aec, obtained by
the condensation of the appropriate volatile amine with 2-
methylsulfanyl-4-amino-5-(30,40,50-trimethoxybenzoyl)thiazole
5
(Scheme 1). This latter derivative was obtained by a one-pot three-
step sequential procedure, starting from the condensation of
dimethyl cyanodithioimidocarbonate 4 and sodium sulfide, fol-
lowed by the addition of 1-(30,40,50-trimethoxyphenyl)-2-
bromoethanone and cyclization with potassium carbonate
(Scheme 1) [8]. Compounds 3deo were prepared by one-pot four
step procedure from 4, which was condensed successively with the
3.2. Molecular modeling studies
This hypothesis was supported by molecular docking experi-
ments that we performed on this series of compounds. Figure 1s
(see Supplementary data) shows how compound 3f is placed
with the trimethoxyphenyl ring in proximity to Cys241, while the
appropriate secondary amine, sodium sulfide, the corresponding a-
bromoacetophenone and finally cyclized with potassium
carbonate.
amino group establishes
a
hydrogen bond with Thr179.

R. Romagnoli et al. / European Journal of Medicinal Chemistry 46 (2011) 6015e6024
6017
Table 1
In vitro inhibitory effects of compounds 3a-o and CA-4.
a
Compd.
IC50
(m
M)
L1210
FM3A
Molt4
CEM
HeLa
3a
3b
3c
3d
3e
3f
3g
3h
3i
48 ꢁ 7.2
11 ꢁ 2.4
62 ꢁ 5.4
53 ꢁ 0
63 ꢁ 11
12 ꢁ 3.2
115 ꢁ 43
47 ꢁ 0
n.d.
n.d.
n.d
42 ꢁ 0.0
11 ꢁ 0.2
78 ꢁ 1.7
42 ꢁ 18
>200
0.32 ꢁ 0.04
22 ꢁ 12
25 ꢁ 1.3
19 ꢁ 1.3
>200
31 ꢁ 6.3
8.2 ꢁ 1.1
38 ꢁ 11
31 ꢁ 1.8
>200
0.34 ꢁ 0.02
11 ꢁ 5.1
8.8 ꢁ 1.1
36 ꢁ 6.2
>200
32 ꢁ 15
>200
>200
>200
0.37 ꢁ 0.07
43 ꢁ 10
38 ꢁ 23
60 ꢁ 20
>200
0.18 ꢁ 0.02
39 ꢁ 16
24 ꢁ 2.2
112 ꢁ 38
>200
0.26 ꢁ 0.09
16 ꢁ 3.2
22 ꢁ 17
31 ꢁ 6.2
>200
3j
3k
3l
3m
3n
3o
CA-4
>200
>200
>200
>200
>200
0.003 ꢁ 0.001
>200
>200
>200
>200
>200
0.042 ꢁ 0.006
>200
>200
>200
>200
>200
0.016 ꢁ 0.001
>200
>200
171 ꢁ 74
185 ꢁ 4
>200
>200
>200
>200
0.002 ꢁ 0.001
>200
>200
0.002 ꢁ 0.001
a
IC50 ¼ compound concentration required to inhibit tumor cell proliferation by 50%.
Furthermore, the pyrrolidine ring can be placed in close contact
with Val181 and Met259 deep in the binding pocket.
that the antiproliferative activity of 3f derives from an interaction
with the colchicine site of tubulin, and this results in interference
with microtubule assembly.
Because only compound 3f had submicromolar activity as an
antiproliferative agent against cancer cell lines, further evaluation
was limited to this agent.
This hydrophobic region does not seem able to accommodate
more voluminous substituents, without affecting the overall
binding pose of the compounds. In support of this observation, the
docking results for more sterically hindered compounds, such as 3c,
3g and 3h, did not produce a satisfactory binding pose (Figure 2s,
Supplementary data).
3.4. Effects of 3f on multidrug resistant cell lines
3.3. In vitro inhibition of tubulin polymerization and colchicine
binding
Drug resistance is an important therapeutic problem caused by
the emergence of tumor cells possessing different mechanisms
which confer resistance against a variety of anticancer drugs [9,10].
Among the more common mechanisms are those related to the
overexpression of glycoproteins capable of mediating the efflux of
various drugs [9,10]. Therefore, we investigated whether 3f
inhibited the growth of two drug-resistant cell lines, one derived
from a lymphoblastic leukemia (CEMVblꢂ100), the other derived
from a colon carcinoma (LovoDoxo). Both these lines express high
levels of the 170-kDa P-glycoprotein (P-gp) drug efflux pump
[11,12]. As shown in Table 2, compound 3f was equally potent
toward parental cells and cells resistant to vinblastine or
doxorubicin.
To further characterize the interaction with the microtubule
system of this novel series of 4-aminothiazole derivatives,
a selected series of compounds (3aec and 3feh) were evaluated for
their in vitro inhibition of tubulin polymerization. For comparison,
CA-4 was examined in contemporaneous experiments. In this
assembly assay, compound 3f was slightly less active than the
reference compound CA-4, with IC50 values of 1.3 ꢁ 0.1 and
1.0 ꢁ 0.1
mM, respectively. In agreement with the antiproliferative
data, compounds 3aec and 3geh were inactive as inhibitors of
tubulin polymerization and did not inhibit tubulin assembly at
concentrations as high as 20
mM.
Resistance to microtubule inhibitors is also mediated by
Compound 3f was also evaluated for inhibitory effects on the
binding of [3H]colchicine to tubulin. In this assay, 67% inhibition
occurred with equimolar concentrations of 3f and radiolabeled
changes in the levels of expression of different b-tubulin iso-
types and by tubulin gene mutations that result in modified
tubulin with impaired polymerization properties. A-549-T12 is
colchicine (5
mM each) in the reaction mixture. Compound 3f was
a cell line with an a-tubulin mutation with increased resistance
somewhat less potent than CA-4, which inhibited colchicine
binding by 99%. These results are consistent with the conclusion
to taxol [13]. Compound 3f had greater relative activity than
taxol in this cell line, suggesting that 3f might be useful in the
Table 2
In vitro cell growth inhibitory effects of 3f on drug resistant cell lines.
a
Compd.
IC50
(m
M)
LoVo
LoVoDoxo
CEM
CEMVbl100
A549
A549-T12
3f
0.10 ꢁ 0.04
0.12 ꢁ 0.03
n.d.
0.43 ꢁ 0.19 (4.3)b
0.02 ꢁ 0.006
n.d.
0.01 ꢁ 0.002 (0.2)
0.037 ꢁ 0.013
n.d.
n.d.
0.0072 ꢁ 0.0001
0.16 ꢁ 0.09 (4.3)
Doxorubicin
Vinblastine
Taxol
13.1 ꢁ 0.21 (109.6)
n.d.
n.d.
n.d.
n.d.
0.004 ꢁ 0.0002
0.23 ꢁ 0.032 (56.1)
n.d.
n.d.
n.d.
n.d.
0.075 ꢁ 0.012 (10.4)
Data are expressed as the mean ꢁ SE from the doseeresponse curves of at least three independent experiments. n.d. not determined.
a
IC50 ¼ compound concentration required to inhibit tumor cell proliferation by 50%.
b
Values in parentheses are fold resistance, indicating reduced potency of the compounds in the resistant cell lines.

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R. Romagnoli et al. / European Journal of Medicinal Chemistry 46 (2011) 6015e6024
treatment of drug refractory tumors resistant to other anti-
tubulin drugs.
More importantly, we observed a concentration-dependent
increase of the cell population with a hypodiploid DNA content
peak (subG1), representing those cells with a DNA content less than
G1, which are usually considered to be apoptotic cells (Fig. 1, panel
B). This suggests that 3f may induce apoptosis.
3.5. Analysis of cell cycle effects
We next studied the association between 3f-induced G2/M
arrest and alterations in expression of proteins that regulate cell
division. Cell arrest at the prometaphase/metaphase to anaphase
transition is normally regulated by the mitotic checkpoint [14]. 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 [14]. As shown in Fig. 1
(panel C) in HeLa cells, 3f caused an increase in cyclin B1 expres-
sion after 24 h of treatment that then decreased by 48 h. At the
same time, there was a marked reduction in Tyr15 phosphorylation
at 24 h, with partial recovery by 48 h.
The effects of different concentrations of compound 3f after 24 h
of treatment on cell cycle progression were examined in HeLa cells
(Fig. 1, panel A). Compound 3f caused a remarkable G2/M arrest
pattern in a concentration-dependent manner, starting at 0.125 mM.
In parallel we observed a concomitant decrease of cells in the G1
phase of the cell cycle (Fig. 1, panel A), while the percentage of S
phase cells declined only at the highest concentration used
(0.5 mM).
In addition, slower migrating forms of phosphatase Cdc25c
appeared at 24 h, followed by partial disappearance at 48 h, indi-
cating 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 [14].
3.6. Compound 3f induces apoptosis
To characterize the mode of cell death induced by 3f, a bipara-
metric cytofluorimetric analysis was performed using propidium
iodide (PI), which stains DNA and enters only dead cells, and fluo-
rescent immunolabeling of the protein annexin-V, which binds to
phosphatidylserine (PS) in a highly selective manner [15]. Dual
staining for annexin-V and with PI permits discrimination between
live cells (annexin-Vꢂ/PIꢂ), earlyapoptotic cells (annexin-Vþ/PIꢂ), late
apoptotic cells (annexin-Vþ/PIþ) and necrotic cells (annexin-Vꢂ/PIþ)
[16]. As depicted inFig. 2, compound 3fat 24 h had already induced an
accumulation of annexin-V positive cells in comparison with the
control, and this accumulationwas concentration-dependent, in good
agreement with the appearance of the sub-G1 cells described above.
After 48 h incubation, we observed a further decrease of cell viability
along with a marked increase in PI positive cells.
3.7. Effect of 3f on mitochondrial depolarization
Mitochondria play an essential role in the propagation of
apoptosis [17]. It is well established that, at an early stage, apoptotic
stimuli alter the mitochondrial transmembrane potential (Djmt).
Djmt was monitored by the fluorescence of the dye 5,50,6,60
Fig. 1. Effect of compound 3f on G2/M phase arrest in Hela cells (panel A). Cells were
treated with different concentrations ranging from 0.06 to 0.5 M for 24 h. Then the
m
cells were fixed and stained with PI to analyze DNA content by flow cytometry. Data
are presented as mean ꢁ SEM of three independent experiments. Panel B. Percentage
of cells presenting a SubG1 peak (apoptotic cells) after 24 h of treatment of HeLa cells
with the indicated concentration of 3f. Panel C. Effect of 3f 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
Fig. 2. Flow cytometric analysis of apoptotic cells after treatment of HeLa cells with 3f.
The cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow
cytometry. Percentage of cells found in the different regions of the biparametric
histograms after incubation with 3f for 24
h or 48 h. Data are expressed as
membrane was stripped and reprobed with anti-b-actin antibody.
mean ꢁ S.E.M. for four independent experiments.

R. Romagnoli et al. / European Journal of Medicinal Chemistry 46 (2011) 6015e6024
6019
tetrachlo-1,10,3,30-tetraethylbenzimidazol-carbocyanine (JC-1) [18].
With normal cells (high Djmt), JC-1 displays a red fluorescence
(590 nm). This is caused by spontaneous and local formation of
aggregates that are associated with a large shift in the emission. In
contrast, when the mitochondrial membrane is depolarized (low
Djmt), JC-1 forms monomers that emit at 530 nm. As shown in
Fig. 3 (panel A), 3f induced a time and concentration-dependent
increase in cells with depolarized mitochondria.
Mitochondrial membrane depolarization is associated with
mitochondrial production of reactive oxygen species (ROS) [19].
Therefore, we investigated whether ROS production increased after
treatment with 3f. We utilized two fluorescence indicators:
hydroethidine (HE), whose fluorescence appears if ROS are gener-
ated [19a] and the dye 2,7-dichlorodihydrofluorescein diacetate
(H2-DCFDA), which is oxidized to the fluorescent compound DCF by
a variety of peroxides, including hydrogen peroxide [19a].
of Djmt. Altogether, these results indicate that compound 3f induced
apoptosis through the mitochondrial pathway.
3.8. Effect of 3f on Bcl-2, Bax expression and caspase-3 activation
Next, we analyzed the effect of 3f on the expression of two
members of the Bcl-2 family, the anti-apoptotic Bcl-2 and the pro-
apoptotic 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 proapoptotico mole-
cules important for the cell death pathway [20]. As shown in Fig. 4
(panel B), 3f did not significantly affect the expression of these two
proteins, except for a marked decrease at 48 h of the anti-apoptotic
protein Bcl-2.
To determine if caspases were involved in 3f-induced cell death,
the expression of caspase-3 was evaluated by flow cytometry. We
observed a clear activation in a time-dependent manner, of
caspase-3 and cleavage of its substrate polyADP-ribose polymerase
(PARP) (Fig. 4, panels A and B).
The results presented in Fig. 3 (panels B and C) show that 3f
induced the production of large amounts of ROS in comparison with
control cells, which agrees with the previously described dissipation
3.9. In vivo antitumor activity of compound 3f
To evaluate the in vivo antitumor activity of 3f, human colon
adenocarcinoma xenografts were established by subcutaneous
injection of HT-29 cells in the backs of nude mice. In preliminary
experiments in vitro, we determined that compound 3f showed
remarkable cytotoxic activity (IC50 ¼ 0.02 ꢁ 0.002
mM) against HT-
Fig. 3. Assessment of mitochondrial dysfunction after treatment with compound 3f.
Panel A. Induction of loss of mitochondrial membrane potential after 24 or 48 h of
incubation of HeLa cells with compound 3f. Cells were stained with the fluorescent
probe JC-1 and analyzed by flow cytometry. Data are expressed as mean ꢁ S.E.M. for
three independent experiments. Panels B and C. Mitochondrial production of ROS in
HeLa cells. After 24 or 48 h of incubation with 3f, cells were stained with HE (panel B)
or H2-DCFDA (panel C) and analyzed by flow cytometry. Data are expressed as
mean ꢁ S.E.M. of three independent experiments.
Fig. 4. Panel A. Caspase-3 induced activity by compound 3f. HeLa cells were incubated
in the presence of 3f at the indicated concentrations. After 24 or 48 h of treatment, cells
were harvested and stained with an anti-human active caspase-3 fragment monoclonal
antibody conjugated with FITC. Data are expressed as percentage of caspase-3 active
fragment positive cells. Panel B. Western blot analysis for the cleavage of PARP and the
expression of Bcl-2 and Bax, in HeLa cells. The cell were treated with the indicated
concentration of 3f for 24 or 48 h. Whole cell lysates were subjected to SDS-PAGE, fol-
lowed by blotting with the appropriate antibody or an anti-actin antibody.

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R. Romagnoli et al. / European Journal of Medicinal Chemistry 46 (2011) 6015e6024
29 cells, while CA-4 showed only moderate cytotoxicity
M).
C-5 substitutions at the thiazole ring was essential for the activity.
Of all substituents examined, the pyrrolidin-1-yl moiety at the C-2
position was required. These results suggested a strict spatial
requirement for the substituent at the C-2 position on the thiazole
scaffold at the tubulin binding site, which was confirmed in
molecular docking studies. A similar phenomenon was observed
with the substituent at the C-5 position, where the 30,40,50-trime-
thoxybenzoyl moiety was the only tolerated group of all those
examined and was crucial for potent biological activity.
Compound 3f was also active in suppressing the growth of drug
resistant cells, and. even more importantly, it had significant in vivo
activity in a colon cancer xenograft. These findings suggest that 3f is
a promising new antimitotic compound for the potential treatment
of cancer.
(IC50 ¼ 3.1 ꢁ 0.1
m
Once the HT-29 xenografts reached a size of w300 mm3, eighteen
mice were randomlyassigned to one of the three groups. In two of the
groups, compound 3f or CA-4, prepared in DMSO, were injected
intraperitoneally at doses of 100 mg/kg. All drugs, as well as a vehicle
control, were administered three times a week for one week. As
shown in Fig. 5, compound 3f caused a remarkable reduction in
tumor growth (58%) as compared with administration of vehicle only.
The effect on tumor volume reduction by 3f was better than that of
CA-4, which caused a 43.7% of reduction at day 24, despite the greater
antiproliferative and antitubulin activities of CA-4.
Although the effects of the two compounds are similar as
inhibitor of tubulin polymerization, it is interesting to note that only
compound 3f exerted a statistically significant reduction of the
tumor mass in comparison with the group treated with vehicle only.
During the whole treatment period, no significant weight
changes occurred in the treated animals (Fig. 5, panel B). However,
in the CA-4 group two animal died on day 7 and one animal died in
the 3f on day 19. Thus, 3f is potentially less toxic in vivo than CA-4
despite equivalent or superior in vivo antitumor activity.
5. Experimental protocols
5.1. Chemistry
5.1.1. Materials and methods
1H NMR spectra were recorded on a Bruker AC 200 spectrom-
eter. Chemical shifts (d) are given in ppm upfield from tetrame-
thylsilane as internal standard, and the spectra were recorded in
appropriate deuterated solvents, as indicated. Positive-ion elec-
trospray 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 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. Reac-
tion 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 230e400 mesh silica gel and the indicated solvent
system. Organic solutions were dried over anhydrous Na2SO4.
4. Conclusions
In conclusion,
a
series of 2-alkylamino-4-amino-5-
aroylthiazoles was synthesized by a one-pot procedure. This effi-
cient method afforded a readily accessible compound, 2-(pyrroli-
dinin-1-yl)-4-amino-5-(30,40,50-trimethoxybenzoyl)thiazole, dis-
playing submicromolar IC50 values in all cancer cell lines examined.
Compound 3f was comparable to CA-4 as an inhibitor of tubulin
polymerization through an interaction at the colchicine site. SAR
studies demonstrated that an appropriate combination of C-2 and
A
2000
1500
1000
500
0
Ctr
CA-4
3f
*
*
*
*
*
5.1.2. Synthesis of [4-amino-2-(methylsulfanyl)-1,3-thiazol-5-
yl](30,40,50-trimethoxyphenyl)methanone (5)
*
*
*
*
0
5
10
Days
15
20
To a stirred suspension of Na2S$9H2O (1.2 g, 5 mmol) in DMF
(7 mL), dimethyl cyanodithioimidocarbonate 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
(2890 mg, 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 reactionwas
stirred at 50 ꢀC for 1 h more. The mixture was poured onto water
(100 mL) with vigorous stirring. When a yellow precipitate appeared,
the reaction mixture was filtered, and the filtrate was 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 the mixture was filtered to afford 5 as a yellow solid.
25
20
15
10
5
B
Ctr
CA-4
3f
0
0
7
16
19
Yield. 52%, mp 130e132 ꢀC 1H NMR (CDCl3)
d: 2.67 (s, 3H), 3.90 (s,
Days after treatment initiation
3H), 3.91 (s, 6H), 6.82 (bs, 2H), 7.03 (s, 2H). MS (ESI): [M]þ ¼ 340.6.
Fig. 5. Inhibition of human xenograft growth in vivo by compound 3f. Panel A. HT-29
tumor-bearing nude mice were administered vehicle alone or 100 mg/kg of 3f or CA-4
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 after
treatments. Data are expressed as mean ꢁ SEM of tumor volume and body weight at
each time point for six animals per group. *p < 0.01 vs. control.
5.1.3. (2,4-Diamino-1,3-thiazol-5-yl)(3,4,5-trimethoxyphenyl)
methanone (3a)
A stirred suspension of 5 (102 mg, 0.3 mmol) in a saturated
solution of ammonia in absolute ethanol (10 mL) was heated in

R. Romagnoli et al. / European Journal of Medicinal Chemistry 46 (2011) 6015e6024
6021
a steel bomb for 18 h at 100 ꢀC. After this time, the solvent was
evaporated and the residue purified by column chromatography on
silica gel (4% MeOH in EtOAc) to yield 3a as a yellow solid. Yield 62%,
using N,N-dimethyl(phenyl)methanamine (255
m
L) and 2-bromo-1-
(3,4,5-trimethoxyphenyl)ethanone (1156 mg), compound 3e was iso-
lated as a yellow solid. Yield 69%, mp 159e161 ꢀC 1H NMR (CDCl3)
d:
mp 211e213 ꢀC 1H NMR (DMSO-d6)
d
: 3.42 (bs, 2H), 3.81 (s, 3H),
2.02 (bs, 2H), 3.09 (s, 3H), 3.88 (s, 3H), 3.91 (s, 6H), 4.73. (s, 2H), 7.00 (s,
2H), 7.34 (m, 5H). MS (ESI): [M]þ ¼ 413.5. Anal. calcd for C21H23N3O4S:
C, 61.00; H, 5.61; N, 10.16; found: C, 60.88; H, 5.47; N, 9.88.
3.87 (s, 6H), 6.94 (s, 2H), 8.05 (bs, 2H). MS (ESI): [M]þ ¼ 309.7. Anal.
calcd for C13H15N3O4S: C, 50.47; H, 4.89; N, 13.58; found: C, 50.23;
H, 4.68; N: 13.33.
5.1.6.3. (4-Amino-2-pyrrolidin-1-yl-1,3-thiazol-5-yl)(3,4,5-trimethox-
5.1.4. [4-Amino-2-(dimethylamino)-1,3-thiazol-5-yl](3,4,5-
trimethoxyphenyl)methanone (3b)
yphenyl)methanone (3f). Following the general procedure, using pyr-
rolidine (170
mL) and 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone
A mixture of 5 (102 mg, 0.3 mmol) in a 33% solution of dime-
thylamine in absolute ethanol (10 mL) was heated in a steel bomb
for 18 h at 100 ꢀC. After this time, the solvent was evaporated and
the residue stirred with petroleum ether. The precipitate was
removed by filtration to furnish 3b as a yellow solid. Yield 55%, mp
(1156 mg), compound 3f was isolated as a yellow solid. Yield 72%, mp
228e230 ꢀC 1H NMR (CDCl3)
d: 2.07 (m, 4H), 3.66 (m, 6H), 3.86 (s, 3H),
3.98 (s, 6H), 7.00 (s, 2H). MS (ESI): [M]þ ¼ 363.6. Anal. calcd for
C17H21N3O4S: C, 56.18; H, 5.82; N, 11.56; found: C, 55.97; H, 5.61; N,
11.41.
180e182 ꢀC 1H NMR (CDCl3)
d: 2.02 (bs, 2H), 3.13 (s, 6H), 3.88 (s,
3H), 3.89 (s, 6H), 7.02 (s, 2H). MS (ESI): [M]þ ¼ 337.8. Anal. calcd for
C15H19N3O4S: C, 53.40; H, 5.68; N, 12.45; found: C, 53.18; H, 5.46; N,
12.31.
5.1.6.4. (4-Amino-2-piperidin-1-yl-1,3-thiazol-5-yl)(3,4,5-trimethox-
yphenyl)methanone (3g). Following the general procedure, using
piperidine (200
none (1156 mg), compound 3g was isolated as a yellow solid. Yield
74%, mp 176e178 ꢀC 1H NMR (CDCl3)
: 1.68 (m, 6H), 3.54 (m, 4H),
mL) and 2-bromo-1-(3,4,5-trimethoxyphenyl)etha-
5.1.5. [4-Amino-2-(diethylamino)-1,3-thiazol-5-yl](3,4,5-
d
trimethoxyphenyl)methanone (3c)
3.88 (s, 3H), 3.98 (s, 6H), 4.02 (bs, 2H), 7.00 (s, 2H). MS (ESI):
[M]þ ¼ 377.6. Anal. calcd for C18H23N3O4S: C, 57.28; H, 6.14; N, 11.13;
found: C, 57.02; H, 5.98; N, 11.01.
The thiazole 5 (102 mg, 0.3 mmol) was added to a mixture of
diethylamine (5 mL) in absolute ethanol (10 mL) and the resulting
solution was heated in a steel bomb for 18 h at 100 ꢀC. After this
time, the solvent was evaporated, the residue dissolved in
dichloromethane (10 mL) and the solution washed with water
(5 mL) and brine (5 mL) and dried over Na2SO4. The solvent was
removed by evaporation under reduced pressure. The residue
purified by column chromatography on silica gel (40% EtOAc in
petroleum ether) furnished 3c as a brown solid. Yield 58%, mp
5.1.6.5. (4-Amino-2-morpholin-4-yl-1,3-thiazol-5-yl)(3,4,5-trimethox-
yphenyl)methanone (3h). Following the general procedure, using
morpholine (180
none (1156 mg), compound 3h was isolated as a yellow solid. Yield
68%, mp 186e188 ꢀC 1H NMR (CDCl3)
mL) and 2-bromo-1-(3,4,5-trimethoxyphenyl)etha-
d: 3.56 (t, J ¼ 5.0 Hz, 4H), 3.76 (t,
J ¼ 5.0 Hz, 4H), 3.86 (s, 3H), 3.89 (s, 6H), 4.14 (bs, 2H), 7.00 (s, 2H). MS
(ESI): [M]þ ¼ 379.8. Anal. calcd for C17H21N3O5S: C, 53.81; H, 5.58; N,
11.07; found: C, 53.64; H, 5.38; N, 10.91.
99e101 ꢀC 1H NMR (CDCl3)
3.53 (t, J ¼ 7.0 Hz, 4H), 3.89 (s, 3H), 3.91 (s, 6H), 6.99 (s, 2H). MS
(ESI): [M]þ ¼ 365.7. Anal. calcd for C17H23N3O4S: C, 55.87; H, 6.34;
N, 11.50; found: C, 55.64; H, 6.11; N, 11.31.
d
: 1.26 (t, J ¼ 7.0 Hz, 6H), 1.88 (bs, 2H),
5.1.6.6. (4-Amino-2-pyrrolidin-1-yl-1,3-thiazol-5-yl)(3,4-dimethoxy-
phenyl)methanone (3i). Following the general procedure, using pyr-
5.1.6. General procedure for the synthesis of compounds (3deo)
To a stirred solution of dimethyl cyanodithioimidocarbonate 4
(292 mg, 2 mmol) in DMF (5 mL) was added the appropriate
secondary amine (2 mmol), and the mixture was heated at 70 ꢀC for
1 h. After this time, Na2S$9H2O (2 mmol) was added, and the
rolidine (170
(1040 mg), compound 3i was isolated as a yellow solid. Yield 75%, mp
218e220 ꢀC 1H NMR (CDCl3)
: 2.06 (m, 4H), 3.44 (m, 6H), 3.92 (s,
3H), 3.94 (s, 3H), 6.85 (d, J ¼ 8.0 Hz, 2H), 7.36 (s, 1H), 7.38 (d,
J ¼ 8.0 Hz, 2H),. MS (ESI): [M]þ ¼ 333.5. Anal. calcd for C16H19N3O3S:
C, 57.64; H, 5.74; N, 12.60; found: C, 57.51; H, 5.52; N, 12.48.
mL) and 2-bromo-1-(4,5-dimethoxyphenyl)ethanone
d
mixture was heated for 90 min at 70 ꢀC. Then, the appropriate
a-
bromoacetophenone (4 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 (276 mg, 2 mmol). The reac-
tion was stirred at 50 ꢀC for another 1 h. The mixture was poured
onto water (20 mL) and the resulting suspension was extracted
with dichloromethane (3 ꢃ 15 mL). The combined organic phases
were washed with water (2 ꢃ 15 mL) and brine (20 mL), dried over
Na2SO4 and concentrated under reduced pressure. The resulting
residue was purified by stirring in ethyl ether for 1 h and filtered to
furnish the final compound.
5.1.6.7. (4-Amino-2-pyrrolidin-1-yl-1,3-thiazol-5-yl)(4-methoxyphenyl)
methanone (3j). Following the general procedure, using pyrrolidine
(170
mL) and 2-bromo-1-(4-methoxyphenyl)ethanone (920 mg),
compound 3j was isolated as a yellow solid. Yield 74%, mp 203e205 ꢀC
1H NMR (CDCl3)
d: 2.05 (t, J ¼ 6.6 Hz, 4H), 3.48 (m, 6H), 3.85 (s, 3H),
6.90 (d, J ¼ 8.4 Hz, 2H), 7.74 (d, J ¼ 8.4 Hz, 2H). MS (ESI): [M]þ ¼ 303.5.
Anal. calcd for C15H17N3O2S: C, 59.38; H, 5.65; N,13.85; found: C, 59.13;
H, 5.44; N, 13.71.
5.1.6.8. (4-Amino-2-pyrrolidin-1-yl-1,3-thiazol-5-yl)(3-methoxyphenyl)
5.1.6.1. {4-Amino-2-[butyl(methyl)amino]-1,3-thiazol-5-yl}(3,4,5-trime-
methanone (3k). Following the general procedure, using pyrrolidine
thoxyphenyl) methanone (3d). Following the general procedure,
(170
mL) and 2-bromo-1-(3-methoxyphenyl)ethanone (920 mg),
using N,N-dimethylbutan-1-amine (240
trimethoxyphenyl)ethanone (1156 mg), compound 3d was isolated
as a yellow solid. Yield 72%, mp 156e158 ꢀC 1H NMR (CDCl3)
: 0.95 (t,
J ¼ 7.2 Hz, 3H), 1.42 (m, 2H), 1.64 (m, 2H), 2.02 (bs, 2H), 3.10 (s, 3H),
3.48 (m, 2H), 3.88 (s, 3H), 3.92 (s, 6H), 7.01 (s, 2H). MS (ESI):
[M]þ ¼ 379.5. Anal. calcd for C18H25N3O4S: C, 56.97; H, 6.64; N, 11.07;
found: C, 56.78; H, 6.43; N, 10.89.
mL) and 2-bromo-1-(3,4,5-
compound 3k was isolated as a yellow solid. Yield 69%, mp
180e182 ꢀC 1H NMR (CDCl3)
d
: 2.06 (t, J ¼ 6.6 Hz, 4H), 3.46 (m, 6H),
d
3.85 (s, 3H), 6.99 (m, 1H), 7.32 (m, 3H). MS (ESI): [M]þ ¼ 303.4. Anal.
calcd for C15H17N3O2S: C, 59.38; H, 5.65; N, 13.85; found: C, 59.21; H,
5.48; N, 13.74.
5.1.6.9. (4-Amino-2-(pyrrolidin-1-yl)thiazol-5-yl)(2-methoxyphenyl)
methanone (3l). Following the general procedure, using pyrrolidine
5.1.6.2. {4-Amino-2-[benzyl(methyl)amino]-1,3-thiazol-5-yl}(3,4,5-tri-
methoxyphenyl) methanone (3e). Following the general procedure,
(170
mL) and 2-bromo-1-(2-methoxyphenyl)ethanone (920 mg),
compound 3l was isolated as a yellow solid. Yield 74%, mp 204e206 ꢀC

6022
R. Romagnoli et al. / European Journal of Medicinal Chemistry 46 (2011) 6015e6024
1H NMR (CDCl3)
d
: 2.02 (t, J ¼ 6.6 Hz, 4H), 3.43 (m, 6H), 3.84 (s, 3H),
Molecular Operating Environment (MOE) [25], and minimized
keeping all the heavy atoms fixed until a RMSD gradient of
0.05 kcal molꢂ1 Åꢂ1 was reached. Ligand structures were built with
MOE and minimized using the MMFF94x forcefield until a RMSD
gradient of 0.05 kcal molꢂ1 Åꢂ1 was reached. The docking simula-
tions were performed using PLANTS [26].
6.92 (t, J ¼ 8.6 Hz, 2H), 7.35 (m, 2H). MS (ESI): [M]þ ¼ 303.3. Anal. calcd
for C15H17N3O2S: C, 59.38; H, 5.65; N, 13.85; found: C, 59.18; H, 5.38; N,
13.70.
5.1.6.10. (4-Amino-2-pyrrolidin-1-yl-1,3-thiazol-5-yl)(4-chlorophenyl)
methanone (3m). Following the general procedure, using pyrrolidine
(170
m
L) and 2-bromo-1-(4-chlorophenyl)ethanone (934 mg),
5.2.4. Antiproliferative assays on MDR cells
compound 3m was isolated as a yellow solid. Yield 68%, mp
LoVoDoxo is a doxorubicin resistant subclone of LoVo cells and
were grown in complete HAM’s F12 medium supplemented with
206e208 ꢀC 1H NMR (CDCl3)
d: 2.06 (t, J ¼ 6.6 Hz, 4H), 3.49 (m, 6H),
7.36 (d, J ¼ 8.4 Hz, 2H), 7.69 (d, J ¼ 8.4 Hz, 2H). MS (ESI): [M]þ ¼ 307.8.
Anal. calcd for C14H14ClN3OS: C, 54.63; H, 4.58; N, 13.65; found: C,
54.38; H, 4.41; N, 13.52.
doxorubicin (0.1 m
g/ml). CEMVblꢂ100 is a multidrug-resistant line
selected against vinblastine. A549-T12 is a non-small cell lung
carcinoma cells exhibiting resistance to taxol. They were grown in
complete DMEM medium supplemented with taxol (12 nM). Indi-
vidual wells of a 96-well tissue culture microtiter plate were
5.1.6.11. (4-Amino-2-pyrrolidin-1-yl-1,3-thiazol-5-yl)(4-bromophenyl)
methanone (3n). Following the general procedure, using pyrrolidine
inoculated with 100 mL of complete medium containing
(170
mL) and 2-bromo-1-(4-bomophenyl)ethanone (1112 mg),
8 ꢃ 103 cells. The plates were incubated at 37 ꢀC in a humidified 5%
compound 3n was isolated as a yellow solid. Yield 73%, mp
CO2 incubator for 18 h prior to the experiments. After medium
removal,100 mL of the drug solution, dissolved in complete medium
230e232 ꢀC 1H NMR (CDCl3)
d
: 2.08 (t, J ¼ 6.6 Hz, 4H), 3.56 (m, 6H),
7.53 (d, J ¼ 8.4 Hz, 2H), 7.60 (d, J ¼ 8.4 Hz, 2H). MS (ESI): [M]þ ¼ 352.4.
Anal. calcd for C14H14BrN3OS: C, 47.74; H, 4.01; N, 11.93; found: C,
47.58; H, 3.89; N, 11.74.
at different concentrations, was added to each well and incubated
at 37 ꢀC for 72 h. Cell viability was assayed by the (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT)
test as described previously [18]. The IC50 was defined as the
compound concentration required to inhibit cell proliferation by
50%.
5.1.6.12. (4-Amino-2-pyrrolidin-1-yl-1,3-thiazol-5-yl)(phenyl)meth-
anone (3o). Following the general procedure, using pyrrolidine
(170
mL) and 2-bromo-1-phenylethanone (800 mg), compound 3o was
isolated as a yellow solid. Yield 72%, mp 176e178 ꢀC 1H NMR (CDCl3)
d:
5.2.5. Flow cytometric analysis of cell cycle distribution
2.05 (m, 4H), 3.46 (m, 6H), 7.43 (m, 3H), 7.74 (m, 2H). MS (ESI):
[M]þ ¼ 273.5. Anal. calcd for C14H15N3OS: C, 61.51; H, 5.53; N, 15.37;
found: C, 61.36; H, 5.37; N, 15.16.
For flow cytometric analysis of DNA content, 5 ꢃ 105 HeLa cells
in exponential growth were treated with different concentrations
of the test compounds for 24 and 48 h. After an incubation period,
the cells were collected, centrifuged and fixed with ice-cold ethanol
(70%). The cells were then treated with lysis buffer containing
RNAse A and 0.1% Triton X-100, and then stained with PI. Samples
were analyzed on a Cytomic FC500 flow cytometer (Beckman
Coulter). DNA histograms were analyzed using MultiCycleÒ for
Windows (Phoenix Flow Systems).
5.2. Biological assays and computational studies
5.2.1. Cell growth inhibitory activity
Murine leukemia L1210, murine mammary carcinoma FM3A,
human T-lymphocyte Molt 4 and CEM and human cervix carcinoma
(HeLa) cells were suspended at 300,000e500,000 cells/mL of
culture medium, and 100
mL of the cell suspension was added to
5.2.6. Annexin-V assay
100 L of an appropriate dilution of the test compounds in wells of
m
Surface exposure of PS on apoptotic cells was measured by flow
cytometry with a Coulter Cytomics FC500 (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. Excitation was set at 488 nm, and the
emission filters were at 525 nm and 585 nm, for measurement of
FTIC and PI fluorescence, respectively.
96-well microtiter plates. After incubation at 37 ꢀC for two days, the
cell number was determined using a Coulter counter. The IC50 was
defined as the compound concentration required to inhibit cell
proliferation by 50%. Data are expressed as the mean ꢁ SE from the
doseeresponse curves of at least three independent experiments.
5.2.2. Effects on tubulin polymerization and on colchicine binding
to tubulin
5.2.7. Assessment of mitochondrial changes
Bovine brain tubulin was purified as described previously [21].
To evaluate the effect of the compounds on tubulin assembly
in vitro [22], varying concentrations were preincubated with 10 mM
The mitochondrial membrane potential was measured with the
lipophilic cation JC-1, while the production of ROS was followed by
flow cytometry using the fluorescent dyes HE and H2DCFDA (the
three compounds were from Molecular Probes, USA) as previously
described [18]. Briefly, after different times of treatment, the cells
were trypsinized, collected by centrifugation and resuspended in
tubulin in glutamate buffer at 30 ꢀC and then cooled to 0 ꢀC. After
addition of GTP, the mixtures were transferred to 0 ꢀC cuvettes in
a recording spectrophotometer and warmed to 30 ꢀC, and the
assembly of tubulin was observed turbidimetrically. 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
Hank’s Balanced Salt Solution (HBSS) containing the JC-1 (1 mM), HE
(2.5 mM) or H2DCFDA (0.1 mM). The cells were then incubated for
10 min at 37 ꢀC, centrifuged and resuspended in HBSS and analyzed
by flow cytometry.
as described [23], except that the reaction mixtures contained 1
mM
tubulin, 5 M test compound.
m
M [3H]colchicine and 5
m
5.2.8. Caspase-3 assay
Caspase-3 activation in Jurkat cells was evaluated by flow
cytometry using a human active caspase-3 fragment antibody
conjugated with FITC (BD Pharmingen). Briefly, after different
incubation times in the presence of test compounds, the cells were
collected by centrifugation and resuspended in CytofixÔ (BD
Pharmingen) buffer for 20 min, washed with Perm/WashÔ (BD
5.2.3. 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 PDB data bank (http://www.rcsb.org/ - PDB
code: 1SA0) [24]. Hydrogen atoms were added to the protein, using

R. Romagnoli et al. / European Journal of Medicinal Chemistry 46 (2011) 6015e6024
6023
Pharmingen) and then incubated for 30 min with the antibody.
Appendix. Supplementary data
After this period, cells were washed and analyzed by flow cytom-
etry. Results are expressed as percentage of caspase-3 active frag-
ment positive cells.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.10.013.
5.2.9. Western blot analysis
References
HeLa cells were incubated in the presence of test compounds
and, after different times, were collected, centrifuged and washed
two times with ice cold PBS. The pellet was then resuspended in
lysis buffer. After the cells were lysed on ice for 30 min, lysates
were centrifuged at 15,000ꢃ g 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
[1] A.L. Risinger, F.J. Giles, S.L. Mooberry, Microtubule dynamics as a target in
oncology, Cancer Treat. Rev. 35 (2008) 255e261.
[2] S. Honore, E. Pasquier, D. Braguer, Understanding microtubule dynamics for
improved cancer therapy, Cell. Mol. Life Sci. 62 (2005) 3039e3056.
[3] G.R. Pettit, S.B. Singh, E. Hamel, C.M. Lin, D.S. Alberts, D. Garcia-Kendall,
Isolation and structure of the strong cell growth and tubulin inhibitor com-
bretastatin A-4, Experientia 45 (1989) 209e211.
[4] C.M. Lin, H.H. Ho, G.R. Pettit, E. Hamel, Antimitotic natural products com-
bretastatin A-4 and combretastatin A-2: studies on the mechanism of their
inhibition of the binding of colchicine to tubulin, Biochemistry 28 (1989)
6984e6991.
[5] A. Chaudari, S.N. Pandeya, P. Kumar, P.P. Sharma, S. Gupta, N. Soni, K.K. Verma,
G. Bhardwaj, Combretastain A-4 analogues as anticancer agents, Mini Rev.
Med. Chem. 12 (2007) 1186e1205.
[6] R. Romagnoli, P.G. Baraldi, M.D. Carrion, O. Cruz-Lopez, C. Lopez-Cara,
G. Basso, G. Viola, M. Khedr, J. Balzarini, S. Mahboobi, A. Sellmer, A. Brancale,
E. Hamel, 2-Arylamino-4-Amino-5-Aroylthiazoles. “One-Pot” synthesis and
biological evaluation of a new class of inhibitors of tubulin polymerization,
J. Med. Chem. 52 (2009) 5551e5555.
(20
mg) were resolved using sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE) (7.5e15% acrylamide
gels) and transferred to PVDF Hybond-p membrane (GE Health-
care). Membranes were blocked with I-block (Tropix) the
membrane being gently rotated overnight at 4 ꢀC. Membranes
were then incubated with primary antibodies against Bcl-2, Bax,
cleaved PARP, (all rabbit, 1:1000, Cell Signaling), cyclin B, p-
cdc2Tyr15, cdc25c, or
b-actin (mouse, 1:10,000, Sigma) for 2 h at
room temperature. Membranes were next incubated with
peroxidase-labeled goat anti-rabbit IgG (1:100,000, Sigma) or
peroxidase-labeled goat anti-mouse IgG (1:100,000, Sigma) 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
[7] K. Gaukroger, J.A. Hadfield, N.J. Lawrence, S. Nlan, A.T. McGown, Structural
requirements for the interaction of combretastatins with tubulin:
how important is the trimethoxy unit? Org. Biomol. Chem. 1 (2003) 3033e3037.
[8] (a) D. Thomae, E. Perspicace, S. Hesse, G. Kirsch, P. Seck, Synthesis of
substituted [1,3]thiazolo[4,5-d][1,2.3]triazines, Tetrahedron 64 (2008)
9306e9314;
(b) D. Thomae, E. Perspicace, Z. Xu, D. Henryon, S. Schneider, S. Hesse,
G. Kirsch, P. Seck, One-pot synthesis of new 2,4,5-trisubstituted 1,3-thiazoles
and 1,3-selenazoles, Tetrahedron 65 (2009) 2982e2988.
reprobed with anti-b-actin antibody.
[9] G. Szakács, J.K. Paterson, J.A. Ludwig, C. Booth-Genthe, M.M. Gottesman, Tar-
5.2.10. Antitumor activity in vivo
geting multidrug resistance in cancer, Nat. Rev. Drug Discov.
5 (2006)
Four weeks old, Female BALB/c-nu nude mice (15e18 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 the Zhejiang University of Traditional Chinese Medicine Labo-
ratory Animal Center. Human colon adenocarcinoma HT-29 cells in
logarithmic growth phase were suspended in fetal bovine serum-
free RPMI 1640 at 1 ꢃ 107cells/mL and inoculated in 0.2 mL into
the hypodermis of the pars dorsalis of each mouse. Once the HT-29
xenografts reached a size of w300 mm3, eighteen mice were
randomly assigned either to the control group or one of two
treatment groups: Compound 3f and CA-4 were prepared in DMSO
and injected intraperitoneally at 0.01 mL/g body weight to give
a dose of 100 mg/kg. The drugs or vehicle were administered three
times a week for one week. After completing the treatment
schedule (23 days), except when death occurred earlier, tumor-
bearing mice were euthanized. Tumor volume was calculated by
the formula: V ¼ ꢃ (L ꢃ W2)/2, where L is the length and W is the
width of the tumor nodules measured by vernier caliper. The study
was approved by the Institutional Animal Ethical Committee of the
Second Affiliated Hospital, School of Medicine, Zhejiang University
(PRC).
219e234.
[10] B.C. Baguley, Multidrug resistance mechanism in cancer, Mol. Biotechnol. 46
(2010) 308e316.
[11] M. Dupuis, M. Flego, A. Molinari, M. Cianfriglia, Saquinavir induces stable
and functional expression of the multidrug transporter P-glycoprotein
in human CD4 T-lymphoblastoid CEM rev cells, HIV Med.
338e345.
4 (2003)
[12] G. Toffoli, A. Viel, I. Tuimoto, G. Bisconti, G. Rossi, M. Baoiocchi, Pleiotropic-
resistant phenotype is a multifactorial phenomenon in human colon carci-
noma cell lines, Br. J. Cancer 63 (1991) 51e56.
[13] L.A. Martello, P. Verdier-Pinard, H.J. Shen, L. He, K. Torres, G.A. Orr,
S.B. Horwitz, Elevated level of microtubule destabilizing factors in a taxol-
resistant/ dependent A549 cell line with an alpha-tubulin mutation, Cancer
Res. 63 (2003) 448e454.
[14] F. Mollinedo, C. Gajate, Microtubules, microtubule-interfering agents and
apoptosis, Apoptosis 8 (2003) 413e450.
[15] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutelingsperger, A novel assay
for apoptosis. Flow cytometric detection of phosphatidylserine expression on
early apoptotic cells using fluorescein labelled annexin V, J. Immunol.
Methods 184 (1995) 39e51.
[16] S.J. Martin, C.P. Reutelingsperger, A.J. Mc Gahon, J.A. Rader, R.C. van Schie,
D.M. Laface, D.R. Green, Early redistribution of plasma membrane phospha-
tidylserine is a general feature of apoptosis regardless of the initiating stim-
ulus: inhibition by overexpression of Bcl-2 and Abl, J. Exp. Med. 182 (1995)
1545e1556.
[17] (a) J.D. Ly, D.R. Grubb, A. Lawen, The mitochondrial membrane potential
(
Djm) in apoptosis: an update, Apoptosis 3 (2003) 115e128;
(b) D.R. Green, G. Kroemer, The pathophysiology of mitochondrial cell death,
Science 305 (2005) 626e629.
[18] G. Viola, E. Fortunato, L. Cecconet, L. Del Giudice, F. Dall’Acqua, G. Basso,
Central role of mitochondria and p53 in PUVA-induced apoptosis in
human keratinocytes cell line NCTC-2544, Toxicol. Appl. Pharm. 227 (2008)
84e96.
[19] (a) G. Rothe, G. Valet, Flow cytometric analysis of respiratory burst activity in
phagocytes with hydroethidine and 20,70-dichlorofluorescein, J. Leukoc. Biol.
47 (1990) 440e448;
5.2.11. Statistical analysis
Unless indicated differently, the results are presented as
mean ꢁ S.E.M. The differences between different treatments were
analyzed using the two-sided Student’s t test. P values lower than
0.05 were considered significant.
(b) J. Cai, D.P. Jones, Superoxide in apoptosis. Mitochondrial generation trig-
gered by cytochrome c loss, J. Biol. Chem. 273 (1998) 11401e11404;
(c) H. Nohl, L. Gille, K. Staniek, Intracellular generation of reactive oxygen
species by mitochondria, Biochem. Pharmacol. 69 (2005) 719e723.
[20] (a) R.M. Kluck, E. Bossy-Wetzel, D.R. Green, The release of cytochrome c from
mitochondria: a primary site for Bcl-2 regulation of apoptosis, Science 275
(1997) 1132e1136;
Acknowledgments
Financial support was provided by GOA (Krediet no. 10/014) of
the K.U.Leuven. The authors would like to thank Mrs. Lizette van
Berckelaer and Dr. Alberto Casolari for technical assistance.
(b) C.M. Knudson, S.J. Korsmeyer, Bcl-2 and Bax function independently to
regulate cell death, Nat. Genet. 16 (1997) 358e363.

6024
R. Romagnoli et al. / European Journal of Medicinal Chemistry 46 (2011) 6015e6024
[21] E. Hamel, C.M. Lin, Separation of active tubulin and microtubule-associated
proteins by ultracentrifugation, and isolation of a component causing the
formation of microtubule bundles, Biochemistry 23 (1984) 4173e4184.
[22] E. Hamel, Evaluation of antimitotic agents by quantitative comparisons of
their effects on the polymerization of purified tubulin, Cell Biochem. Biophys.
38 (2003) 1e21.
[23] P. Verdier-Pinard, J.-Y. Lai, H.-D. Yoo, J. Yu, B. Marquez, D.G. Nagle, M. Nambu,
J.D. White, J.R. Falck, W.H. Gerwick, B.W. Day, E. Hamel, Structure-activity
analysis of the interaction of curacin A, the potent colchicine site antimitotic
agent, with tubulin and effects of analogs on the growth of MCF-7 breast
cancer cells, Mol. Pharmacol. 53 (1998) 62e67.
[24] R.B.G. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel,
M. Knossow, Insight into tubulin regulation from a complex with colchicine
and a stathmin-like domain, Nature 428 (2004) 198e202.
[25] Molecular Operating Environment (MOE 2010.10). Chemical Computing
Group, Inc. Montreal, Quebec, Canada. http://www.chemcomp.com.
[26] O. Korb, T. Stützle, T.E. Exner, PLANTS: application of ant colony opti-
mization to structure-based drug design. in: M. Dorigo, L.M. Gambardella,
M. Birattari, A. Martinoli, R. Poli, T. Sttzle (Eds.), Ant Colony Optimization
and Swarm Intelligence, 5th International Workshop, ANTS 2006, Brus-
sels, Belgium. Springer, Berlin, Sep 4ꢂ7, 2006, pp. 247e258 2006; LNCS
4150.