Synthesis and antitumor activity of 1,5-disubstituted 1,2,4-triazoles as cis-restricted combretastatin analogues

Article abstract of DOI:10.1021/jm100245q

A series of 1-aryl-5-(3′,4′,5′-trimethoxyphenyl) derivatives and their related 1-(3′,4′,5′-trimethoxyphenyl)-5- aryl-1,2,4-triazoles, designed as cis-restricted combretastatin analogues, were synthesized and evaluated for antiproliferative activity, inhibitory effects on tubulin polymerization, cell cycle effects, and apoptosis induction. Their activity was greater than, or comparable with, that of the reference compound CA-4. Flow cytometry studies showed that HeLa and Jurkat cells treated with the most active compounds 4l and 4o were arrested in the G2/M phase of the cell cycle in a concentration dependent manner. This effect was accompanied by apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species, activation of caspase-3, and PARP cleavage. Compound 4l was also shown to have potential antivascular activity, since it induced endothelial cell shape change in vitro and disrupted the sprouting of endothelial cells in the chick aortic ring assay.

Full text of DOI:10.1021/jm100245q

4248 J. Med. Chem. 2010, 53, 4248–4258
DOI: 10.1021/jm100245q
Synthesis and Antitumor Activity of 1,5-Disubstituted 1,2,4-Triazoles as Cis-Restricted
Combretastatin Analogues
Romeo Romagnoli,*,† Pier Giovanni Baraldi,† Olga Cruz-Lopez,† Carlota Lopez Cara,† Maria Dora Carrion,†
Andrea Brancale,‡ Ernest Hamel,§ Longchuan Chen, Roberta Bortolozzi,^ Giuseppe Basso,^ and Giampietro Viola*,^
†Dipartimento di Scienze Farmaceutiche, Universitaꢀ 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, Department of
Pathology, VA Medical Center, Long Beach, California 90822, and ^Laboratorio di Oncoematologia, Dipartimento di Pediatria, Universitaꢀ di
Padova, 35131 Padova, Italy
Received February 24, 2010
A series of 1-aryl-5-(30,40,50-trimethoxyphenyl) derivatives and their related 1-(30,40,50-trimethoxyphenyl)-
5-aryl-1,2,4-triazoles, designed as cis-restricted combretastatin analogues, were synthesized and evaluated
for antiproliferative activity, inhibitory effects on tubulin polymerization, cell cycle effects, and apoptosis
induction. Their activity was greater than, or comparable with, that of the reference compound CA-4. Flow
cytometry studies showed that HeLa and Jurkat cells treated with the most active compounds 4l and 4o
were arrested in the G2/M phase of the cell cycle in a concentration dependent manner. This effect was
accompanied by apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species,
activation of caspase-3, and PARP cleavage. Compound 4l was also shown to have potential antivascular
activity, since it induced endothelial cell shape change in vitro and disrupted the sprouting of endothelial
cells in the chick aortic ring assay.
Introduction
It has been established by previous SAR studies that both
the 30,40,50-trimethoxy substitution pattern on the A-ring and
the cis-olefin configuration at the bridge were essential for
optimal activity, while B-ring structural modifications were
tolerated by the target.7 However, the cis configuration of
CA-4 is prone to isomerize to the thermodynamically more
stable trans form during storage and administration, produ-
cing a dramatic reduction in both antitubulin and antiproli-
ferative activities. Thus, to retain the cis-olefin configuration
of CA-4 required for bioactivity, several groups have reported
that the isomerization from cis- to trans-olefin can be avoided
by incorporating the double bond in five-member aromatic
heterocyclic rings, such as pyrazole,8 imidazole,9 thiazole,8
isoxazole,10 1,2,3-thiadiazole,11 isomeric triazoles,8,12,13 and
1,2,3,4-tetrazole.8
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 intracel-
lular transport.1 Inhibition of microtubule function using
tubulin targeting agents, many of which are natural products,
is a validated approach for anticancer therapy.2 One of the
most important antimitotic agents is combretastatin A-4
(CA-4,a 1; Chart 1). CA-4, isolated from the bark of the
South African tree Combretum caffrum,3 is one of the well-
known natural tubulin-binding molecules affecting microtu-
bule dynamics by binding to the colchicine site.4 CA-4 shows
potent cytotoxicity againsta wide varietyof human cancer cell
lines, including those that are multidrug resistant.5 A water-
soluble disodium phosphate derivative of CA-4 (named
CA-4P) has shown promising results in human cancer clinical
trials,6 thus stimulating significant interest in a variety of
CA-4 analogues.7
Welsh and co-workers reported a series of 3,4-diaryl-1,2,4-
triazoles (compounds 2a-c) with antiproliferative activity,
but this was 10-fold reduced relative to the activity of CA-4.
Nevertheless, these compounds had activity similar to that of
CA-4 as inhibitors of tubulin polymerization.13
In this article we reconfigured the substitution pattern
around the triazole ring by the preparation of two different
regioisomeric series of 1,5-diaryl-substituted 1,2,4-triazole
derivatives with general structures 3 and 4. In these two series
of designed analogues, obtained by interchanging the sub-
stitution pattern of rings A and B, we fixed one of the aryl
groups as a 30,40,50-trimethoxyphenyl moiety, identical with
the A-ring of CA-4, and examined several substitutions with
electron-withdrawing (F, Cl, and NO2) or electron-releasing
(Me, MeO, and EtO) groups (EWG and ERG, respectively)
on the other aryl moiety, corresponding to the B-ring of CA-4.
*To whom correspondence should be addressed. 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.
aAbbreviations: CA-4, combretastatin A-4; EWG, electron-withdrawing
group; ERG, electron-releasing group; SAR, structure-activity relation-
ship; CuI, copper iodide; CsCO3, cesium carbonate; NBS, N-bromo-
succinimide; CCl4, carbon tetrachloride; PI, propidium iodide; PS,
phosphatidylserine; HE, hydroethidine; H2DCFDA, 2,7-dichlorodi-
hydrofluorescein diacetate; DCF, dichlorofluorescein; PARP, poly
ADP-ribose polymerase; HUVEC, human umbilical vein endothelial
cell; GFP, green fluorescent protein; HBSS, Hank’s balanced salt
solution; PBS, phosphate buffered saline; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis.
r
pubs.acs.org/jmc
Published on Web 04/26/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4249
Scheme 1a
Chart 1. Inhibitors and Potential Inhibitors of Tubulin Poly-
merization
a Reagents and conditions: (a) HCONH2, 120 ꢀC, 18 h; (b, e) NBS,
benzoylperoxide (cat.), CCl4, reflux; (c, f) Pd(PPh3)4, K2CO3, PhMe,
reflux, 18 h; (d) 1-bromo-3,4,5-trimethoxybenzene, CsCO3, CuI, DMF,
120 ꢀC, 18 h; (g) H2, 10% Pd/C, DMF.
To compare the effect of para-, meta-, and ortho-substitu-
tion on compounds with general formula 4, the methoxy
and ethoxy groups were introduced at different positions of
the B-phenyl ring to furnish derivatives 4h-j and 4l-n,
respectively.
1,2,4-triazole nucleus. A positive NOE correlation was ob-
served between the signals at 8.489 (H-5) and 6.876 ppm, in
which this latter signal corresponds to the aromatic ortho-
protons of the 30,40,50-trimethoxyphenyl moiety, and at the
same time signals at 8.489 and 6.876 ppm did not show any
NOE with the signal at 8.085 ppm.
Chemistry
The target 1,5-diaryl-1,2,4-triazoles 3a-k and 4a-r were
synthesized as outlined in Scheme 1. 1-Aryl-1,2,4-triazoles
6a-i were prepared by the condensation of the substituted
arylhydrazine hydrochlorides 5a-i in formamide at 120 ꢀC.
The 1-(30,40,50-trimethoxyphenyl)-1,2,4-triazole 8 was ob-
tained by an efficient N-arylation procedure of 1,2,4-triazole
with 1-bromo-3,4,5-trimethoxybenzene catalyzed by CuI and
CsCO3.14 The intermediates 6a-i and 8 were chemoselectively
brominated at the 5-position of the 1,2,4-triazole ring by NBS
in refluxing CCl4 to yield derivatives 7a-i and 9, respectively.
The Suzuki cross-coupling reaction with the appropriate
arylboronic acid under heterogeneous conditions [Pd(PPh3)4,
K2CO3] in refluxing toluene furnished the 1-aryl-5-(30,40,50-
trimethoxyphenyl)- and 1-(30,40,50-trimethoxyphenyl)-5-aryl-
1,2,4-triazoles 3a-i and 4a-r, respectively. Starting from 3h,i,
the nitro group was reduced with hydrogen in the presence
of 10% Pd/C in DMF, affording amino derivatives 3j,k,
respectively.
Results and Discussion
In Vitro Antiproliferative Activities. The series of 1-aryl-
5-(30,40,50-trimethoxyphenyl) and their related 1-(30,40,50-
trimethoxyphenyl)-5-aryl-1,2,4-triazoles, corresponding to
compounds 3a-k and 4a-r, respectively, were evaluated
for their antiproliferative activity against a panel of six
different human tumor cell lines and compared with the
reference compound CA-4 (1). Data for inactive compounds
(IC50 > 10 μM) are not shown in Table 1. Two of the
synthesized compounds, 4l and 4o, had the best antiproli-
ferative activities against these cell lines and, overall, were as
active as CA-4. In particular, the 30-chloro-40-ethoxyphenyl
derivative 4o exhibited IC50 values ranging from 3 to 20 nM
against the cell lines, compared with the range of 4-370 nM
obtained with CA-4. It was less active than CA-4 only
against the K-562 cells.
The chemoselective bromination at the 5-position of the
1,2,4-triazole ring was unambiguously attributed by 1H-1H
NOE experiments on the representative compound 8. These
experiments permit assignment of the signals at 8.489
and 8.085 to the H-5 and H-3 protons, respectively, of the
Apparently, the relative positions of the two aromatic
rings on the 1,2,4-triazole moiety did not seem to be critical
for antiproliferative activity. With the exception of the weak

4250 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Romagnoli et al.
Table 1. In Vitro Cell Growth Inhibitory Effects of Compounds 3g, 4g,h,k,l,o,p, and CA-4 (1)
IC50 a(nM)
compd
HeLa
A549
HL-60
Jurkat
K562
MCF-7
3g
4g
>10000
250 ( 50
280 ( 60
150 ( 20
15 ( 4
6 ( 2
600 ( 20
4 ( 1
>10000
1100 ( 100
520 ( 90
800 ( 50
100 ( 20
10 ( 5
6200 ( 1200
90 ( 6
120 ( 10
500 ( 20
20 ( 3
3 ( 0.2
700 ( 200
1 ( 0.2
93 ( 30
300 ( 80
50 ( 10
50 ( 10
5 ( 0.2
3 ( 0.6
650 ( 60
5 ( 0.6
3600 ( 900
>10000
950 ( 40
340 ( 50
20 ( 8
20 ( 10
800 ( 100
5 ( 0.1
7400 ( 1200
540 ( 20
360 ( 50
390 ( 90
50 ( 9
17 ( 1
610 ( 10
370 ( 100
4h
4k
4l
4o
4p
>10000
180 ( 50
CA-4
a IC50: compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ( SE from the dose-response
curves of at least three independent experiments.
activity observed with the p-methoxyphenyl derivative 3g, all
the 1-aryl-1,2,4-triazole derivatives 3a-k were inactive (IC50
> 10 μM). Switching the position of the two aryls on the
1,2,4-triazole ring (3a vs 4a, 3b vs 4b, 3c vs 4c, 3d vs 4d, 3f vs
4f, and 3i vs 4r) did not yield active compounds except that
there was a considerable difference in potency observed
between the regioisomeric 40-methoxyphenyl derivatives 3g
and 4h (the latter was considerably more active than the
former in four out of the six cell lines).
In the series of 1-(30,40,50-trimethoxyphenyl)-1,2,4-triazole
analogues 4h-j and 4l-n, the position of methoxy or ethoxy
substituent on the 5-phenyl ring had a profound influence on
antiproliferative activity. Starting from compound 4h, mov-
ing the methoxy group from the para- to the meta- and
ortho-positions (compounds 4i and 4j, respectively) led to a
dramatic drop of potency. The same effect was observed for
the ethoxy substituent (4l vs 4m and 4n). The p-ethoxy
derivative 4l was 4- to 40-fold more potent than its methoxy
counterpart 4h. The enhanced effect on activity resulting
from replacement of the methoxy group with an ethoxy
moiety in colchicine site compounds was previously ob-
served by us and by others.15
Replacement of the p-methoxy group with a weak elec-
tron-releasing thiomethyl group resulted in derivative 4k,
which overall had activity similar to that of 4h against Jurkat
and MCF-7 cells. Similarly, except with the K562 cells,
replacing the 40-methoxy of 4h with a 40-methyl group (4g)
overall had only minor effects on antiproliferative activities.
Since the 40-ethoxy group of 4l was favorable for potency,
it is important to point out that introduction of an additional
EWG chlorine group at the 30-position of 40-ethoxyphenyl
ring, resulting in compound 4o, produced a 2- to 15-fold
increase in antiproliferative activity against four of the six
cell lines, while 4l and 4o were equipotent against the Jurkat
and K562 cells.
Finally, among the antiproliferative compounds, repla-
cing the 40-ethoxy group of 4l with the bulky isopropoxy
moiety (4p) caused a sharp drop in antiproliferative activity
in all cell lines, suggesting that an increase in steric bulk at
this position causes a decrease in potency.
Inhibition of Tubulin Polymerization and Colchicine Bind-
ing. To investigate whether the antiproliferative activities of
compounds 3g, 4g,h, 4k,l, and 4o,p derived from an interac-
tion with tubulin, they were evaluated for their inhibition of
tubulin polymerization and for effects on the binding of
[3H]colchicine to tubulin (Table 2).16 For comparison, CA-4
was examined in contemporaneous experiments. In the
assembly assay, compound 4l was found to be the most
active (IC50 = 0.76 μM), and it was almost twice as potent as
CA-4 (IC50 = 1.2 μM). While 4l was generally less potent
Table 2. Inhibition of Tubulin Polymerization and Colchicine Binding
by Compounds 3g, 4g,h,k,l,o,p, and CA-4
tubulin assembly,a
colchicine binding,b
compd
3g
4g
IC50 ( SD (μM)
% ( SD
16 ( 1
nd
3.9 ( 0.4
2.3 ( 0.0
3.6 ( 0.1
0.76 ( 0.1
1.5 ( 0.2
5.1 ( 0.8
1.2 ( 0.1
33 ( 6
55 ( 1
38 ( 3
86 ( 2
75 ( 0.1
37 ( 3
87 ( 3
4h
4k
4l
4o
4p
CA-4 (1)
a Inhibition of tubulin polymerization. Tubulin was at 10 μM. b In-
hibition of [3H]colchicine binding. Tubulin, colchicine, and tested
compound were at 1, 5, and 1 μM, respectively. nd: not determined.
than 4o as an antiproliferative agent, 4l was about twice as
active as 4o as an inhibitor of tubulin assembly. Derivative 4o
was also slightly less active than CA-4 as an inhibitor of
tubulin assembly. Compound 4h was the next most active
agent as an inhibitor of tubulin assembly (about half as
potent as CA-4). The remaining compounds with lower
antiproliferative effects on cancer cells were still less active
as inhibitors of tubulin assembly. For these seven com-
pounds and CA-4, the order of activity was 4l > CA-4 >
4o > 4h > 4k > 4g > 4p . 3g.
In the colchicine binding studies, derivative 4l was as
potent as CA-4, which in these experiments inhibited colchi-
cine binding by 87%, while 4o was slightly less potent (75%
inhibition). The potent inhibition observed with the two
compounds indicates that 4l and 4o bind to tubulin at a site
overlapping the colchicine site. Inhibition of colchicine
binding by compounds 4g,h, 4k, and 4p fell into the
33-55% range. In this series of seven compounds, inhibition
of [3H]colchicine binding correlated more closely with in-
hibition of tubulin assembly than with antiproliferative
activity. In conclusion, we note that CA-4 is one of the most
potent colchicine compounds yet described. It is thus sig-
nificant that two agents in the present series have activities
comparable to that CA-4 as inhibitors of tubulin assembly
and, less frequently observed, as inhibitors of colchicine
binding to tubulin.
Analysis of Cell Cycle. The effects of different concentra-
tions of compounds 4l and 4o on cell cycle progression were
examined with HeLa and Jurkat cells (Figure 1). Untreated
HeLa cells showed a classical pattern of proliferating cells
distributed in the G1 (55.2%), S (32.4%), and G2/M (12.4%)
phases. Both 4l and 4o caused a clear G2/M arrest pattern in
a concentration-dependent manner, with a concomitant
decrease of cells in other phases of the cell cycle (Figure 1).
In particular, as shown in Figure 2 (upper panels), the G2/M

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4251
Figure 1. Effect of 4l and 4o induced G2/M phase arrest in HeLa (upper panels) and Jurkat cells (lower panels). Cells were treated with
different concentrations ranging from 7 to 250 nM for 24 h. Then the cells were fixed and stained with PI to analyze DNA content by flow
cytometry. Data are presented as the mean ( SEM of three independent experiments.
Figure 2. Effect of 4l and 4o induced G2/M phase arrest in HeLa (upper panels) and Jurkat cells (lower panels). Cells were treated with a
concentration of 30 nM for 24 and 48 h. Then the cells were fixed and stained with PI to analyze DNA content by flow cytometry. Data are
presented as the mean ( SEM of three independent experiments.
cell population increased from 12% in the control to over
70% with 30 nM compounds 4l and 4o at 48 h.
is a Ca2þ-dependent phospholipid binding protein with high
affinity for PS. Annexin-V staining precedes the loss of
membrane integrity that accompanies the final stages of cell
death resulting from either apoptotic or necrotic processes.
Because the externalization of PSoccurs in the earlier stages of
apoptosis, annexin-V staining identifies apoptosis at an earlier
stage than the appearance of sub-G1 cells. These cells appear
at a later stage of cell death and indicate the occurrence of
nuclear changes such as DNA fragmentation.
In the leukemia T-cell Jurkat line, both compounds also
induced a significant block in the G2/M phase. With both cell
lines, the accumulation of G2/M cells increased to varying
extents in a time-dependent manner (Figure 2). In addition,
treatment of cells with 4l or 4o caused the appearance of a
hypodiploid peak (sub-G1) indicative of apoptosis (data not
shown).
Loss of Plasma Membrane Asymmetry during Apoptosis.
To better characterize drug-induced apoptosis, we performed
a biparametric cytofluorimetric analysis using propidium
iodide (PI) and annexin-V-FITC, which stain DNA and
phosphatidylserine (PS) residues, respectively.17 Annexin-V
After treatment with 4l and 4o at 50 nM for different times,
Jurkat cells were labeled with the two dyes, and the resulting
red (PI) and green (FITC) fluorescence was monitored by
flow cytometry. It can be observed from Figure 3 that 4l and
4o provoked a significant induction of apoptotic cells after

4252 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Romagnoli et al.
Figure 3. Flow cytometric analysis of apoptotic cells after treat-
ment of Jurkat cells with 4l and 4o. After different times of
treatment, cells were harvested and labeled with annexin-V-FITC
and PI and then analyzed by flow cytometry. The data are expressed
as the mean of percentage of annexin-V positive cells ( SEM for
four independent experiments.
24 h of treatment. The percentage of annexin-V positive cells
then further increased at 48 and 72 h. These findings
prompted us to further investigate the apoptotic process
after treatment with the two compounds.
Induction of Mitochondrial Depolarization. Mitochondria
play an essential role in the propagation of apoptosis.18 It is
well established that at an early stage apoptotic stimuli alter
the mitochondrial transmembrane potential (Δψmt). Δψmt
was monitored by the fluorescence of the dye JC-1.19 With
normal cells (high Δψmt), JC-1 displays a red fluorescence
(590 nm). This is caused by spontaneous and local formation
of aggregates that is associated with a large shift in the
emission. In contrast, when the mitochondrial membrane is
depolarized (low Δψmt), JC-1 forms monomers that emit at
530 nm. Treated Jurkat cells in the presence of derivatives 4l
and 4o (50 nM) exhibited a remarkable shift in fluorescence
compared with control cells, indicating depolarization of the
mitochondrial membrane potential (Figure 4, upper panels).
The percentage of cells with low Δψmt following treatment
increased in a time-dependent fashion (Figure 4, lower
panel). The disruption of Δψmt is associated with the appear-
ance of annexin-V positivity in the treated cells when they are
in an early apoptotic stage. In fact, the dissipation of Δψmt is
characteristic of apoptosis and has been observed with both
microtubule stabilizing and destabilizing agents, including
CA-4, in different cell types.20
Figure 4. Assessment of mitochondrial dysfunction after treatment
with compound 4l or 4o. Upper and middle panels show represen-
tative histograms of Jurkat cells treated with 50 nM 4l for the
indicated times and stained with PI and annexin-V-FITC. Lower
panel shows induction of loss of mitochondrial membrane potential
after 24, 48, and 72 h of incubation of Jurkat cells with 50 nM 4l or
4o. Cells were stained with the fluorescent probe JC-1 and analyzed
by flow cytometry. Data are expressed as the mean ( SEM for three
independent experiments.
Mitochondrial Generation of Reactive Oxygen Species
(ROS). Mitochondrial membrane depolarization is asso-
ciated with mitochondrial production of ROS.21 Therefore,
we investigated whether ROS production increased after
treatment with the test compounds. We utilized the fluores-
cence indicator hydroethidine (HE), whose fluorescence
appears if ROS are generated.22 HE is oxidized by super-
oxide anion into the ethidium ion, which fluoresces red.
Superoxide is produced by mitochondria because of a shift
from the normal four-electron reduction of O2 to a one-
electron reduction when cytochrome c is released from
mitochondria. ROS generation was also measured with the
dye 2,7-dichlorodihydrofluorescein diacetate (H2-DCFDA),
which is oxidized to the fluorescent compound dichloro-
fluorescein (DCF) by a variety of peroxides, including
hydrogen peroxide.22
agrees with the previously described dissipation of Δψmt
The amount of ROS produced increased over the entire 72 h
treatment time.
.
Caspase-3 Activation, Poly ADP-Ribose Polymerase
(PARP) Cleavage, and Bcl-2 Down-Regulation. Caspases
are the central executioners of apoptosis mediated by various
inducers.23 Caspases are synthesized as proenzymes that are
activated by cleavage. Caspases-2, -8, -9, and -10 are termed
apical caspases and are usually the first to be stimulated in
the apoptotic process. Their activation in turn leads to their
activation of effector caspases, in particular caspase-3.24
Exposure of Jurkat cells to compound 4l or 4o resulted in
the activation of caspase-3 in a time-dependent manner, as
shown in Figure 6.
The results are presented in Figure 5, where it can be
observed that 4l and 4o induced the production of large
amounts of ROS in comparison with control cells, which
PARP is a 116 kDa nuclear protein that appears to be
involved in apoptosis.25 This protein is one of the main
cleavage targets of caspase-3 both in vitro and in vivo.25

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4253
Figure 7. Western blot analysis for the cleavage of PARP and the
expression of Bcl-2 in Jurkat cells. Control lanes (Ctr) refer to
untreated cells. In the other lanes the cells were treated with 50 nM 4l
or 4o for the indicated times. Whole cell lysates were subjected to
SDS-PAGE, followed by blotting with an anti-PARP, anti-Bcl-2,
or anti-actin antibody.
Figure 5. Mitochondrial production of ROS in Jurkat cells. After
24, 48, and 72 h of incubation with 4l or 4o, cells were stained with
H2DCFDA (upper panel) or HE (lower panel) and analyzed by flow
cytometry. Data are expressed as the mean ( SEM of three
independent experiments.
Figure 8. Effect of compound 4l on endothelial cell shape in
cultured HUVECs (top panels) and chick aortic vascular sprouts
(lower panels). Cells or aortic arches from 14-day chick embryos
were treated with 4l and analyzed by confocal microscopy. The left-
hand panels are cells prior to compound addition, and the right-
hand panels show cells before and after 30 min of treatment with
10 μM 4l. In the lower panels the arrows indicate the disruption of
the aortic vascular sprout induced by drug treatment.
especially with 4o, after treatment for another 24 h, most
noticeable through disappearance of uncleaved PARP. Si-
milar results were obtained with CA-4 in HeLa cells.20b
Bcl-2 is a protein that has been extensively investigated as a
modulating agent of apoptosis and plays a major role as an
inhibitor of apoptosis. It does this by regulating the mito-
chondrial membrane potential, thus avoiding release of
cytochrome c and caspase activation.26 Therefore, we exami-
ned whether the induction of apoptosis by 4o and 4l is
associated with changes in the expression of this protein.
As depicted in Figure 7 (middle panel), immunoblot analysis
showed that treatment with either compound resulted in
decreased expression of Bcl-2. Altogether these data indicate
that the induction of apoptosis by these new derivatives is
associated with Bcl-2 down-regulation and caspase-3 activa-
tion that in turn stimulated PARP cleavage.
Figure 6. Caspase-3 induced activity by compound 4l and 4o.
Jurkat cells were incubated in the presence of 4l and 4o at 50 nM.
After 24, 48, and 72 h of treatment, cells were harvested and stained
with an antihuman active caspase-3 fragment monoclonal antibody
conjugated with FITC: (upper panels) representative histograms of
Jurkat cells incubated in the presence of 50 nM 4l and 4o for 48 h;
(lower panel) percentage of caspase-3 active fragment positive cells
after 24, 48, and 72 h of treatment. Data are expressed as the mean (
SEM of three independent experiments.
Antivascular Activity. CA-4 and its analogues in clinical
development have been shown to quickly and selectively shut
down the blood flow of tumors.6a,b This family of drugs is
therefore called antivascular or vascular disrupting agents.
The effect is thought to be mediated by inducing endothelial
As shown in Figure 7 (top panel), immunoblot analysis
demonstrated extensive formation of the typical 89 kDa
fragment of PARP following a 24 h treatment with either
50 nM 4l or 50 nM 4o. There was a further increase,

4254 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Romagnoli et al.
Conclusions
In conclusion, we proposed that the 1,5-diarylsubstituted
1,2,4-triazole ring could serve as a suitable mimic to retain the
bioactive configuration afforded by the cis-double bond pre-
sent in CA-4. In the present study, we fixed one of the aryl
groups as a 30,40,50-trimethoxyphenyl moiety, and the mod-
ifications were mainly focused on variation of the substituents
on the second phenyl ring. It is clear that the substitution
pattern on the phenyl at the 5-position of the 1,2,4-triazole
ring plays an important role for antitubulin and antiprolifera-
tive activities, and this was supported by the molecular dock-
ing studies. The results demonstrated that either the 40-ethoxy
(4l) substituent or the 40-ethoxy and 30-chloro (4o) substitu-
ents on the second phenyl ring could replace the B-ring of CA-
4, at least with a 1,2,4-triazole ring as the bridge. Both these
derivatives exhibited potent tubulinpolymerization inhibitory
activity as well as antiproliferative activity, comparable with
that of CA-4. Compound 4l was the most potent inhibitor of
tubulin polymerization and one of the most potent inhibitors
of colchicine binding (IC50 = 0.76 μM for assembly, 86%
inhibition of the binding of [3H]colchicine). We also showed
by flow cytometry that 4l and 4o had cellular effects typical for
microtubule-interacting agents, causing accumulation of cells
in the G2/M phase of the cell cycle. Further studies showed
that 4l and 4o are potent inducers of apoptosis in the Jurkat
cell line. Apoptosis induced by antimitotic agents has been
associated with alteration in a variety of cellular signaling
pathway. As with many antimitotic drugs, compounds 4l and
4o are able to induce Bcl-2 down-regulation just after 24 h of
treatment. Bcl-2 prevents the initiation of the cellular apopto-
tic program by stabilizing mitochondrial permeability. The
loss of Δψmt results in an uncoupling of the respiratory chain
and the efflux of small molecules such as caspase-9 and the
apoptosis-inducing factor (AIF), which in turn can stimulate
proteolytic activation of caspase-3. Our results confirm that
the induction of apoptosis by 4l and 4o is associated with
down-regulation of Bcl-2, dissipation of the mitochondrial
transmembrane potential, and activation of caspase-3, which
is coupled with terminal events of apoptosis such as PARP
cleavage. Finally, preliminary experiments have assessed the
potential antivascular activity of compound 4l. The ability of
this compound to inhibit vascular sprouting is consistent with
antivascular agent utility and warrants further testing in
preclinical in vivo cancer models.
Figure 9. Presumptive binding mode of compound 4l (green) and
compound 4o (orange). DAMA-colchicine is shown in gray, and
Cys 241 is shown on the right. All compounds and Cys 241 are in
stick representation.
cell shape change, possibly through disrupting microtubule
dynamics.27 We tested the derivative 4l for its ability to
induce rapid endothelial cell shape changes using a human
umbilical vein endothelial cell (HUVEC) culture assay and a
chick aortic ring assay.28 The HUVEC line expressed green
fluorescent protein (GFP). In these model systems, we found
that changes occurred rapidly and were extensive 30 min
after drug addition, corresponding to the quick in vivo effect
of shutting down tumor circulation. Like CA-4, 4l caused
spreading HUVECs to retract and form blebs on their
membranes at a concentration as low as 0.5 μM, and the
effect was prominent at 10 μM. The area of GFP positive
HUVECs was reduced to 30% of the area measured before
treatment (Figure 8). Compound 4l also disrupted formation
of vascular sprouts from chick aortic ring after only a 30 min
incubation. These observations suggest that 4l, like CA-4,
would most likely cause severe vascular disruption in vitro
and it could be considered as a new vascular disrupting
agent.
Molecular Modeling Studies. To rationalize the experi-
mental data obtained, molecular docking studies were per-
formed on this series of compounds. The docking pose
observed for 4l showed a very similar binding mode to the
cocrystallized DAMA-colchicine29 with the trimethoxyphe-
nyl ring in close contact to Cys 241 (residue numbering
derived from the crystal structure used) and the other aro-
matic moiety placed deep in the binding pocket (Figure 9).
These results are in accordance with the observed experi-
mental results; in particular, the ethoxy group is placed in a
tight hydrophobic region, which does not seem able to
accommodate a bulkier group, explaining the loss in activity
of compound 4p. It should also be noted that the methoxy
analogue 4h does not occupy this hydrophobic area as
efficiently as 4l, possibly explaining the reduced biological
activity observed for 4h. Furthermore, in the case of 4o, the
chlorine atom in position 3 of the aromatic ring is placed in
the same position as the carbonyl group of DAMA-colchi-
cine, and also in this case, only relatively small groups should
be tolerated in this position. In further support of this model
is the inactivity of compounds 4i and 4m, which have a
methoxy group and an ethoxy group, respectively, at posi-
tion 3 of the aromatic ring. These compounds could not be
docked successfully in the colchicine binding site.
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 deuter-
ated 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 are uncor-
rected. 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 Depart-
ment of Chemistry 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% 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

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4255
solvents. Reaction courses and product mixtures were routinely
monitored by TLC on silica gel (precoated F254 Merck plates),
and compounds were visualized with aqueous KMnO4. Flash
chromatography was performed using 230-400 mesh silica gel
and the indicated solvent system. Organic solutions were dried
over anhydrous Na2SO4. Arylhydrazine hydrochlorides 5a-i
and arylboronic acids are commercially available and used as
received.
General Procedure A for the Synthesis of 1-Aryl-1H-[1,2,4]-
triazoles 6a-i. A suspension of the appropriate arylhydrazine
hydrochloride (10 mmol) in 10 mL of formamide was heated at
120 ꢀC for 12 h. The mixture was cooled and dissolved in a
mixture of ethyl acetate (20 mL) and water (10 mL). The organic
layer was washed with water (3 ꢀ 10 mL), brine (10 mL), dried
(Na2SO4), filtered, and concentrated to give a residue purified
by column chromatography on silica gel.
and brine (10 mL). The organic layer was dried, filtered, and
evaporated, and the residue was purified by flash chromatography
on silica gel.
1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1-(4-methoxy-
phenyl)-1H-1,2,4-triazole (3g). Following general procedure C,
compound 3g was purified by chromatography, eluting with
petroleum ether-EtOAc (6:4). Yellow solid, yield 58%, mp
1
117-119 ꢀC. H NMR (CDCl3) δ: 3.67 (s, 6H), 3.85 (s, 3H),
3.86 (s, 3H), 6.74 (s, 2H), 6.96 (d, J = 9.0 Hz, 2H), 7.31 (d, J = 9.0
Hz, 2H), 8.05 (s, 1H). MS (ESI): [M]þ = 341.8. Anal.
(C18H19N3O4) C, H, N.
1-(3,4,5-Trimethoxyphenyl)-5-p-tolyl-1H-1,2,4-triazole (4g).
Following general procedure C, compound 4g was purified by
chromatography, eluting with petroleum ether-EtOAc (1:1).
White solid, yield 55%, mp 106-108 ꢀC. 1H NMR (CDCl3) δ:
2.36 (s, 3H), 3.73 (s, 6H), 3.88 (s, 3H), 6.56 (s, 2H), 7.22 (d, J =
8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 8.06 (s, 1H). **MS (ESI):
[M þ 1]þ=326.5. Anal. (C18H19N3O3) C, H, N.
1-(4-Methoxyphenyl)-1H-1,2,4-triazole (6g). Following gene-
ral procedure A, compound 6g was purified by chromatogra-
phy, eluting with petroleum ether-EtOAc (1:1). Brown solid,
yield 71%, mp 88-89 ꢀC. 1H NMR (CDCl3) δ: 3.88 (s, 3H), 7.03
(d, J = 9.0 Hz, 2H), 7.58 (d, J = 9.0 Hz, 2H), 8.10 (s, 1H), 8.47
(s, 1H).
1-(3,4,5-Trimethoxyphenyl)-5-(4-methoxyphenyl)-1H-1,2,4-tria-
zole (4h). Following general procedure C, compound 4h was
purified by chromatography, eluting with petroleum ether-
1
EtOAc (1:1). White solid, yield 56%, mp 72-74 ꢀC. H NMR
(CDCl3) δ: 3.75 (s, 6H), 3.86 (s, 3H), 3.88 (s, 3H), 6.58 (s, 2H), 6.90
(d, J = 9.0 Hz, 2H), 7.48 (d, J = 9.0 Hz, 2H), 8.04 (s, 1H). MS
(ESI): [M]þ=341.7. Anal. (C18H19N3O4) C, H, N.
Synthesis of 1-(3,4,5-Trimethoxyphenyl)-1H-1,2,4-triazole (8).
To a round-bottom flask charged with CuI (382 mg, 2 mmol)
were added CsCO3 (6.52 g., 20 mmol), 1,2,4-triazole (1 g, 14.5
mmol), 3,4,5-trimethoxyphenylboronic acid (2.47 g, 10 mmol),
and DMF (20 mL) under nitrogen. The system was then eva-
cuated twice, backfilled with nitrogen, and heated at 120 ꢀC for
24 h. The reaction mixture was then cooled to room temperature
and diluted with EtOAc (20 mL) and water (10 mL). The aqueous
phase was filtered on a pad of Celite, and the filtrate was washed
with EtOAc (2 ꢀ 10 mL). The combined organic extracts were
washed with brine (10 mL), dried over Na2SO4, and concen-
trated. The resulting residue was purified by column chromatog-
raphy on silica gel (EtOAc) to provide 8 as a white solid. Yield
78%, mp 119-121 ꢀC. 1H NMR (CDCl3) δ: 3.88 (s, 3H), 3.93 (s,
6H), 6.88 (s, 2H), 8.09 (s, 1H), 8.49 (s, 1H). Anal. (C17H17N3O3)
C, H, N.
General Procedure B for the Synthesis of 1-Aryl-5-bromo-1H-
[1,2,4]triazoles (7a-i) and 1-(3,4,5-Trimethoxyphenyl)-5-bromo-
1H-[1,2,4]triazoles (9). To a suspension of the appropriate
1-aryl-1H-[1,2,4]triazole 6a-i or 1-(3,4,5-trimethoxyphenyl)-
1H-1,2,4-triazole 9 (3 mmol) in 15 mL of CCl4 were added
NBS (1.07 g, 6 mmol) and a catalytic amount of benzoyl
peroxide (72 mg, 0.3 mmol). The solution was heated to reflux
for 12 h, then cooled and filtered through Celite, washing the
solids with warm CCl4 (5 mL). The solvent was removed in
vacuo and the residue purified by column chromatography on
silica gel.
5-Bromo-1-(4-methoxyphenyl)-1H-1,2,4-triazole (7g). Follow-
ing general procedure B, compound 7g was purified by chroma-
tography, eluting with petroleum ether-EtOAc (8:2). Gray solid,
yield 52%, mp 103-105 ꢀC. 1H NMR (CDCl3) δ: 3.88 (s, 3H),
7.02 (d, J = 6.8 Hz, 2H), 7.44 (d, J = 6.8 Hz, 2H), 8.01 (s, 1H).
5-Bromo-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole (9).
Following general procedure B, compound 9 was purified by
chromatography, eluting with petroleum ether-EtOAc (3:7).
Yellow solid, yield 68%, mp 182-184 ꢀC. 1H NMR (CDCl3) δ:
3.89 (s, 3H), 3.90 (s, 6H), 6.75 (s, 2H), 8.02 (s, 1H).
General Procedure C (Suzuki Coupling) for the Synthesis of
Compounds 3a-i and 4a-r. A mixture of 5-bromo-1-aryl-
1H-[1,2,4]triazoles 7a-i or 1-(3,4,5-trimethoxyphenyl)-5-bro-
mo-1H-1,2,4-triazole 9 (0.5 mmol), potassium carbonate (104 mg,
0.75 mmol, 1.5 equiv), the appropriate arylboronic acid (1 mmol,
2 equiv), and tetrakis(triphenylphosphine)palladium (13.5 mg,
0.012 mmol) in dry toluene (10 mL) was stirred at 100 ꢀC
under nitrogen for 18 h, cooled to ambient temperature, filtered
through Celite, and evaporated in vacuo. The residue was dissol-
ved with EtOAc (30 mL), and the resultant solution was
washed sequentially with 5% NaHCO3 (10 mL), water (10 mL),
1-(3,4,5-Trimethoxyphenyl)-5-(4-(methylthio)phenyl)-1H-1,2,4-
triazole (4k). Following general procedure C, compound 4k was
purified by chromatography, eluting with petroleum ether-
EtOAc (1:1). White solid, yield 86%, mp 123-125 ꢀC. 1H
NMR (CDCl3) δ: 2.48 (s, 3H), 3.74 (s, 6H), 3.88 (s, 3H), 6.56 (s,
2H), 7.18 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 8.02 (s,
1H). MS (ESI): [M]þ=357.3. Anal. (C18H19N3O3S) C, H, N.
1-(3,4,5-Trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-1,2,4-triazole
(4l). Following general procedure C, compound 4l was purified by
chromatography, eluting with petroleum ether-EtOAc(1:1).White
solid, yield 55%, mp 107-109 ꢀC. 1H NMR (CDCl3) δ: 1.42 (t, J =
7.0 Hz, 3H), 3.75 (s, 6H), 3.88 (s, 3H), 4.02 (q, J = 7.0 Hz, 2H), 6.58
(s, 2H), 6.84 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 8.04 (s,
1H). MS (ESI): [M þ H]þ = 356.0. Anal. (C19H21N3O4) C, H, N.
5-(3-Chloro-4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-
1,2,4-triazole (4o). Following general procedure C, compound
4o was purified by chromatography, eluting with petroleum
ether-EtOAc (1:1). Yellow solid, yield 56%, mp 107-109 ꢀC.
1H NMR (CDCl3) δ: 1.47 (t, J = 7.2 Hz, 3H), 3.76 (s, 6H), 3.88
(s, 3H), 4.12 (q, J = 7.2 Hz, 2H), 6.56 (s, 2H), 6.84 (d, J = 8.6
Hz, 1H), 7.30 (d, J = 8.6 Hz, 1H), 7.69 (s, 1H), 8.04 (s, 1H). MS
(ESI): [M þ H]þ = 390.6. Anal. (C19H20ClN3O4) C, H, N.
5-(4-Isopropoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-
triazole (4p). Following general procedure C, compound 4p was
purified by chromatography, eluting with petroleum ether-
EtOAc (1:1). White solid, yield 83%, mp 120-122 ꢀC. 1H
NMR (CDCl3) δ: 1.31 (d, J = 6.2 Hz, 6H), 3.74 (s, 6H), 3.88
(s, 3H), 4.57 (m, 1H), 6.58 (s, 2H), 6.85 (d, J = 9.2 Hz, 2H), 7.45
(d, J = 9.2 Hz, 2H), 8.03 (s, 1H). MS (ESI): [M þ 1]þ = 370.2.
Anal. (C20H23N3O4) C, H, N.
General Procedure D for the Synthesis of 3j-k. A solution of
the appropriate nitrophenyl derivative 7h,i (0.25 mmol) in DMF
(5 mL) was hydrogenated over 35 mg of 10% Pd/C at 60 psi for
2 h. The catalyst was removed by filtration on Celite, and the
filtrate was concentrated to give a residue which was purified by
column chromatography on silica gel.
Biology. Materials and Methods. Antiproliferative Assays.
Human T-leukemia (Jurkat), human promyelocytic leukemia
(HL-60), and human chronic myelogenous leukemia (K562) cells
were grown in RPMI-1640 medium (Gibco Milano, Italy). Breast
adenocarcinoma (MCF7), human nonsmall lung carcinoma
(A549) and human cervix carcinoma (HeLa) cells were grown
in DMEM medium (Gibco, Milano, Italy), all supplemented with
115 units/mL of penicillin G (Gibco, Milano, Italy), 115 μg/mL
streptomycin (Invitrogen, Milano, Italy) and 10% fetal bovine

4256 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Romagnoli et al.
serum (Invitrogen, Milano, Italy). Individual wells of a 96-well
tissue culture microtiter plate were inoculated with 100 μL of
complete medium containing 8 ꢀ 103 cells. The plates were
incubated at 37 ꢀC in a humidified 5% CO2 incubator for 18 h
prior to the experiments. After medium removal, 100 μL of the
drug solution, dissolved in complete medium at different con-
centrations, 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-diphenyltetrazolium bromide (MTT) test as previously
described.30 The IC50 was defined as the compound concentra-
tion required to inhibit cell proliferation by 50%.
Effects on Tubulin Polymerization and on Colchicine Binding
to Tubulin. To evaluate the effect of the compounds on tubulin
assembly in vitro,16a varying concentrations of compounds were
preincubated with 10 μM bovine brain tubulin in glutamate
buffer at 30 ꢀC and then cooled to 0 ꢀC. After addition of 0.4 mM
GTP, the mixtures were transferred to 0 ꢀC cuvettes in a
recording spectrophotometer and warmed to 30 ꢀC. Tubulin
assembly was followed turbidimetrically at 350 nm. The IC50
was defined as the compound concentration that inhibited the
extent of assembly by 50% after a 20 min incubation. The
capacity of the test compounds to inhibit colchicine binding to
tubulin was measured as described16b except that the reaction
mixtures contained 1 μM tubulin, 5 μM [3H]colchicine, and
1 μM test compound.
washed and analyzed by flow cytometry. Results are expressed
as percentage of caspase-3 active fragment positive cells.
Western Blot Analysis. Jurkat cells were incubated in the
presence of test compounds and, after different times, were
collected, centrifuged, and washed two times with ice cold
phosphate buffered saline (PBS). The pellet was then resus-
pended in lysis buffer. After the cells were lysed on ice for 30 min,
lysates were centrifuged at 15000g at 4 ꢀC for 10 min. The
protein concentration in the supernatant was determined using
the BCA protein assay reagents (Pierce, Italy). Equal amounts
of protein (20 μg) were resolved using sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) (7.5-15%
acrylamide gels) and transferred to a PVDF Hybond-p mem-
brane (GE Healthcare). Membranes were blocked with I-block
(Tropix), and the membrane was gently rotated overnight at
4 ꢀC. Membranes were then incubated with primary antibodies
against Bcl-2, PARP (all rabbit, 1:1000, Cell Signaling), or
β-actin (mouse, 1:10,000, Sigma) for 2 h at room temperature.
Membranes were next incubated with peroxidase-labeled goat
antirabbit IgG (1:100000, Sigma) or peroxidase-labeled goat
antimouse IgG (1:100000, 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 reprobed
with an anti-β-actin antibody.
Endothelial Cell Shape Change Assays. HUVECs expressing
GFP (green fluorescence protein) were cultured in EGM-2
media with endothelial cell growth supplement and transferred
into 18-well μ-Slide plates coated with collagen 1 day before
assay. Cell morphology and GFP fluorescence were recorded in
a Nikon confocal microscope with an imaging system before and
after drug additions at different time points. The area of
HUVECs with GFP was further analyzed by the imaging soft-
ware MetaMorph (Molecular Devices). The chick aortic ring
assay was performed as described in ref 28. Briefly, aortic arches
were dissected from day 14 chick embryos, cut into cross-
sectional slices, and implanted in 10 μL of Matrigel (Becton
Dickinson) in eight-well Lac-Tek chamber slides with complete
media. Endothelial cell sprouts or channels were formed in
24-48 h. Various concentrations of 4 L were added, and images
of the vascular channels were captured with a digital camera
before and 30 min after drug addition.
Flow Cytometric Analysis of Cell Cycle Distribution and
Apoptosis. For flow cytometric analysis of DNA content, 5 ꢀ
105 HeLa or Jurkat cells in exponential growth were treated with
different concentrations of the test compounds for 24 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 stained with PI. Samples were analyzed on a Cytomic
FC500 flow cytometer (Beckman Coulter). DNA histograms
were analyzed using MultiCycle for Windows (Phoenix Flow
Systems).
Annexin-V Assay. 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 and 585 nm, respectively.
Assessment of Mitochondrial Changes. The mitochondrial
membrane potential was measured with the lipophilic cation
5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolcarbocya-
nine (JC-1, Molecular Probes), as described.19,30 Briefly, after
different times of treatment, the cells were collected by centri-
fugation and resuspended in Hank’s balanced salt solution
(HBSS) containing 1 μM JC-1. The cells were then incubated
for 10 min at 37 ꢀC, centrifuged, and resuspended in HBSS. The
production of ROS was measured by flow cytometry using
either HE (Molecular Probes) or H2DCFDA (Molecular
Probes).
Molecular Modeling. All molecular modeling studies were
performed on a MacPro dual 2.66 GHz Xeon computer running
Ubuntu 8. The tubulin structure was downloaded from the PDB
data bank (http://www.rcsb.org/; PDB code 1SA0).30 Hydrogen
atoms were added to the protein, using Molecular Operating
Environment (MOE),31 and minimized keeping all the heavy
-1
atoms fixed until a rmsd gradient of 0.05 kcal mol-1
A
was
˚
reached. Ligand structures were built with MOE and minimized
using the MMFF94x force field until a rmsd gradient of 0.05
-1
kcal mol-1
A
was reached. The docking simulations were
˚
performed using PLANTS.32
After different times of treatment, cells were collected by
centrifugation and resuspended in HBSS containing the fluor-
escence probe HE or H2DCFDA at 2.5 or 0.1 μM, respectively.
The cells were then incubated for 30 min at 37 ꢀC, centrifuged,
and resuspended in HBSS. The fluorescence was directly re-
corded with the flow cytometer, using an excitation wavelength
of 488 nm and emission wavelengths of 585 and 530 nm for HE
and H2DCFDA, respectively.
Supporting Information Available: Additional information
for synthesis procedures and spectroscopic data for com-
pounds 6a-f,h,i, 7a-f,h,i, 3a-f,h-k, and 4a-f,h-j,m,n,q,r.
This material is available free of charge via the Internet at
http://pubs.acs.org.
References
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 Cytofix (BD Pharmingen) buffer for 20 min,
washed with Perm/Wash (BD Pharmingen), and then incubated
for 30 min with the antibody. After this period, cells were
(1) (a) Amos, L. A. Microtubule structure and its stabilisation. Org.
Biomol. Chem. 2004, 2, 2153–2160. (b) Walczak, C. E. Microtubule
dynamics and tubulin interacting proteins. Curr. Opin. Cell. Biol.
2000, 12, 52–56.
(2) (a) Mahindroo, N; Liou, J. P.; Chang, J. Y.; Hsieh, H. P. Anti-
tubulin agents for the treatment of cancer. A medicinal chemistry
update. Expert Opin. Ther. Pat. 2006, 16, 647–691. (b) Jordan, M. A.;
Wilson, L. Microtubules as a target for anticancer drugs. Nat. Rev.
Cancer 2004, 4, 253–265. (c) Hadfield, J. A.; Ducki, S.; Hirst, N.;

Article
McGown, A. T. Tubulin and microtubules as targets for anticancer
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4257
combretastatin A-4: synthesis, molecular modeling and evaluation
as cytotoxic agents and inhibitors of tubulin. Bioorg. Med. Chem.
2008, 16, 4829–4838.
drugs. Prog. Cell Cycle Res. 2003, 5, 309–325. (d) Honore, S.;
Pasquier, E.; Braguer, D. Understanding microtubule dynamics for
improved cancer therapy. Cell. Mol. Life. Sci. 2005, 62, 3039–3065.
(e) Pellegrini, F.; Budman, D. R. Review: tubulin function, action of
antitubulin drugs, and new drug development. Cancer Invest. 2005, 23,
264–273. (f) Attard, G.; Greystoke, A.; Kaye, S.; De Bono, J. Update on
tubulin binding agents. Pathol. Biol. 2006, 54, 72–84. (g) Lawrence,
N. J.; McGown, A. T. The chemistry and biology of antimitotic
chalcones and related enone systems. Curr. Pharm. Des. 2005, 11,
1679–1693.
(13) Zhang, Q.; Peng, Y.; Wang, X. I.; Keeman, S. M.; Aurora., S.;
Welsh, W. J. Highly potent triazole-based tubulin polymerization
inhibitors. J. Med. Chem. 2007, 50, 749–754.
(14) Zhu, L.; Guo, P.; Li, G.; Lan, J.; Xie, R.; You, J. Simple copper
salt-catalyzed N-arylation of nitrogen-containing heterocycles
with aryl and heteroaryl halides. J. Org. Chem. 2007, 72, 8535–
8538.
(15) (a) Romagnoli, R.; Pier Baraldi, P. G.; Carrion, M. D.; Cruz-Lopez,
O.; Lopez Cara, C.; Tolomeo, M.; Grimaudo, S.; Di Cristina, A.;
Pipitone, M. R.; Balzarini, J.; Zonta, N.; Brancale, A.; Hamel, E..
Design, synthesis and structure-activity relationship of 2-(30,40,50-
trimethoxybenzoyl)-benzo[b]furan derivatives as a novel class of
antitubulin agents. Bioorg. Med. Chem. 2009, 17, 6862–6871. (b)
Cushman, M.; He, H.-M.; Katzenellenbogen, J. A.; Hamel, E. Synthesis,
antitubulin and antimitotic activity and cytotoxicity of analogs of 2-meth-
oxyestradiol, an endogenous mammalian metabolite of estradiol that
inhibits tubulin polymerization by binding to the colchicine. J. Med.
Chem. 1995, 38, 2041. (c) Kim, S.; Min, S. Y.; Lee, S. K.; Cho, W.-J.
Comparative molecular field analysis study of stilbene derivatives active
against A549 lung carcinoma. Chem. Pharm. Bull. 2003, 51, 516–521.
(16) (a) Hamel, E. Evaluation of antimitotic agents by quantitative
comparisons of their effects on the polymerization of purified
tubulin. Cell Biochem. Biophys. 2003, 38, 1–21. (b) Verdier-Pinard,
P.; Lai, J.-Y.; Yoo, H.-D.; Yu, J.; Marquez, B.; Nagle, D. G.; Nambu, M.;
White, J. D.; Falck, J. R.; Gerwick, W. H.; Day, B. W.; Hamel, E.
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. 1998, 53,
62–67.
(3) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.;
Garcia-Kendall, D. Isolation and structure of the strong cell
growth and tubulin inhibitor combretastatin A-4. Experentia
1989, 45, 209–211.
(4) Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Antimitotic natural
products combretastatin A-4 and combretastatin A-2: studies on
the mechanism of their inhibition of the binding of colchicine to
tubulin. Biochemistry 1989, 28, 6984–6991.
(5) McGown, A. T.; Fox, B. W. Differential cytotoxicity of combre-
tastatins A1 and A4 in two daunorubucin-resistant P388 cell lines.
Cancer Chemother. Pharmacol. 1990, 26, 79–81.
(6) (a) Grosios, K.; Holwell, S. E.; McGown, A. T.; Pettit, G. R.;
Bibby, M. C. In vivo and in vitro evaluation of combretastatin A-4
and its sodium phosphate prodrug. Br. J. Cancer 1999, 81, 1318–
1327. (b) Vincent, L.; Kermani, P.; Young, L. M.; Cheng, J.; Zhang, F.;
Shido, K.; Lam, G.; Bompais-Vincent, H.; Zhu, Z.; Hicklin, D. J.;
Bohlen, P.; Chaplin, D. J.; May, C.; Rafii, S. Combretastatin A4
phosphate induces rapid regression of tumor neovessels and growth
through interference with vascular endothelial-cadherin signaling.
J. Clin. Invest. 2005, 115, 2992–3006. (c) Chaplin, D. J.; Hill, S. A.
The development of combretastatin A4 phosphate as a vascular target-
ing agent. Int. J. Radiat. Oncol., Biol., Phys. 2002, 54, 1491–1496. (d)
Young, S. L.; Chaplin, D. J. Combretastatin A-4 phosphate: background
and current clinical status. Expert Opin. Invest. Drugs 2004, 13, 1171–
1182. (e) Bilenker, J. H.; Flaherty, K. T.; Rosen, M.; Davis, L.;
Gallagher, M.; Stevenson, J. P.; Sun, W.; Vaughn, D.; Giantonio, B.;
Zimmer, R.; Scnall, M.; O'Dwyer, P. J. Phase I trial of combretastatin
A-4 phospate with carboplatin. Clin. Cancer Res. 2005, 11, 1527–
1533.
(17) Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger, C.
A novel assay for apoptosis. Flow cytometric detection of phos-
phatidylserine expression on early apoptotic cells using fluorescein
labelled annexin V. J. Immunol. Methods 1995, 184, 39–51.
(18) (a) Ly, J. D.; Grubb, D. R.; Lawen, A. The mitochondrial
membrane potential (Δψm) in apoptosis: an update. Apoptosis
2003, 3, 115–128. (b) Green, D. R.; Kroemer, G. The pathophysiology
of mitochondrial cell death. Science 2005, 305, 626–629.
(19) Salvioli, S.; Ardizzoni, A.; Franceschi, C.; Cossarizza, A. JC-1 but
not DiOC6(3) or rhodamine 123 is a reliable fluorescent probe to
assess ΔΨ changes in intact cells: implications for studies on
mitochondrial functionality during apoptosis. FEBS Lett. 1997,
411, 77–82.
(20) (a) Mollinedo, F.; Gajate, C. Microtubules, microtubule-interfer-
ing agents and apoptosis. Apoptosis 2003, 8, 413–450. (b) Vitale, I..;
Antoccia, A.; Cenciarelli, C.; Crateri, P.; Meschini, S.; Arancia, G.;
Pisano, C.; Tanzarella, C. Combretastatin CA-4 and combretastatin
derivative induce mitotic catastrophe dependent on spindle checkpoint
and caspase-3 activation in non-small cell lung cancer cells. Apoptosis
2007, 12, 155–166.
(21) Zamzami, N.; Marchetti, P.; Castedo, M.; Decaudin, D.; Macho,
A.; Hirsch, T.; Susin, S. A.; Petit, P. X.; Mignotte, B.; Kroemer, G.
Sequential reduction of mitochondrial transmembrane potential
and generation of reactive oxygen species in early programmed cell
death. J. Exp. Med. 1995, 182, 367–377.
(22) (a) Rothe, G.; Valet, G. Flow cytometric analysis of respiratory
burst activity in phagocytes with hydroethidine and 20,70-dichloro-
fluorescein. J. Leukocyte Biol. 1990, 47, 440–448. (b) Cai, J.; Jones,
D. P. Superoxide in apoptosis. Mitochondrial generation triggered
by cytochrome c loss. J. Biol. Chem. 1998, 273, 11401–11404. (c)
Nohl, H.; Gille, L.; Staniek, K. Intracellular generation of reactive
oxygen species by mitochondria. Biochem. Pharmacol. 2005, 69,
719–723.
(7) (a) Nam, N. H. Combretastatin A-4 analogues as antimitotic
antitumor agents. Curr. Med. Chem. 2003, 10, 1697–1722. (b)
Chaudari, A.; Pandeya, S. N.; Kumar, P.; Sharma, P. P.; Gupta, S.; Soni,
N.; Verma, K. K.; Bhardwaj, G. Combretastain A-4 analogues as
anticancer agents. Mini-Rev. Med. Chem. 2007, 12, 1186–1205. (c)
Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A.
A. Medicinal chemistry of combretastatin A4: present and future
directions. J. Med. Chem. 2006, 49, 3033–3044. (d) Gaukroger, K.;
Hadfield, J. A.; Lawrence, N. J.; Nlan, S.; McGown, A. T. Structural
requirements for the interaction of combretastatins with tubulin: how
important is the trimethoxy unit? Org. Biomol. Chem. 2003, 1, 3033–
3037. (e) Hatanaka, T.; Fujita, K.; Ohsumi, K.; Nakagawa, R.; Fukuda,
Y.; Nihei, Y.; Suga, Y.; Akiyama, Y.; Tsuji, T. Novel B-ring modified
combretastatin analogues: synthesis and antineoplastic activity. Bioorg.
Med. Chem. Lett. 1998, 8, 3371–3374.
(8) Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda,
Y.; Nihai, Y.; Suga, Y.; Morinaga, Y.; Akiyama, Y.; Tsuji, T.
Synthesis and antitumor activity of cis-restricted combretastatins
5-membered heterocyclic analogues. Bioorg. Med. Chem. Lett.
1988, 8, 3153–3158.
(9) (a) Bellina, F.; Cauteruccio, S.; Monti, S.; Rossi, R. Novel imida-
zole-based combretastatin A-4 analogues. Evaluation of their in
vitro antitumor activity and molecular modeling study of their
binding to the colchicine site of tubulin. Bioorg. Med. Chem. Lett.
2000, 16, 5757–5762. (b) Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.;
McCroskey, R. W.; Hannick, S. M.; Gherke, L.; Credo, R. B.; Hui,
Y.-H.; Marsh, K.; Warner, R.; Lee, J. Y.; Zielinski-Mozng, N.; Frost, D.;
Rosenberg, S. H.; Sham, H. L. Potent, orally active heterocycle-based
combretastatin A-4 analogues: synthesis, structure-activity relation-
ship, pharmacokinetics, and in vivo antitumor activity evaluation.
J. Med. Chem. 2002, 45, 1697–1711.
(23) (a) Thomberry, A. N.; Lazebnik, Y. Caspases: enemies within.
Science 1998, 281, 1312–1316. (b) Earshaw, W. C.; Martins, L. M.;
Kaufmann, S. H. Mammalian caspases: structure, activation, substrates
and functions during apoptosis. Annu. Rev. Biochem. 1999, 68, 383–
424. (c) Reed, J. C. Apoptosis-based therapies. Nat. Rev. Drug
Discovery 2002, 1, 111–121. (d) Denault, J.-B.; Salvesen, G. S.
Caspases: keys in the ignition of cell death. Chem. Rev. 2002, 102,
4489–4499.
(10) Kaffy, J.; Pontikis, R.; Carrez, D.; Croisy, A.; Monneret, C.;
Florent, J.-C. Isoxazole-type derivatives related to combretastatin
A-4, synthesis and biological evaluation. Bioorg. Med. Chem. 2006,
14, 4067–4077.
(24) Porter, A. G.; Janicke, R. U. Emerging role of caspase-3 in
apoptosis. Cell Death Differ. 1999, 6, 99–104.
(11) Wu, M.; Li, W.; Yang, C.; Chen, D.; Ding, J.; Chen, Y.; Lin, L.;
Xie, Y. Synthesis and activity of combretastatin A-4 analogues:
1,2,3-thiadiazoles as potent antitumor agents. Bioorg. Med. Chem.
Lett. 2007, 17, 869–873.
(12) Odlo, K.; Hentzen, J.; Fournier dit Chabert, J.; Ducki, S.; Gani,
O. A. B. S. M.; Sylte, I.; Skrede, M.; Florenes, V. A.; Hansen,
T. V. 1,5-Disubstituted 1,2,3-triazoles as cis-restricted analogues of
(25) Soldani, C.; Scovassi, A. I. Poly(ADP-ribose) polymerase-1 clea-
vage during apoptosis: an update. Apoptosis 2002, 7, 321–328.
(26) (a) Kluck, R. M.; Bossy-Wetzel, E.; Green, D. R. The release of
cytochrome c from mitochondria: a primary site for Bcl-2 regula-
tion of apoptosis. Science 1997, 275, 1132–1136. (b) Knudson, C. M.;
Korsmeyer, S. J. Bcl-2 and Bax function independently to regulate cell
death. Nat. Genet. 1997, 16, 358–363.

4258 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Romagnoli et al.
(27) Satchi-Fainaro, R.; Puder, M.; Davies, J.; Tran, H.; Greene,
A. K.; Corfas, G.; Folkman, J. Targeting angiogenesis with a
conjugate of HPMA copolymer and TNP-470. Nat. Med. 2004,
10, 255–261.
(28) Galbraith, S. M.; Chaplin, D. J.; Lee, F.; Stratford, M. R.; Locke,
R. J.; Vojnovic, B.; Tozer, G. M Effects of combretastatin A4
phosphate on endothelial cell morphology in vitro and relationship
to tumour vascular targeting activity in vivo. Anticancer Res. 2001,
21, 93–102.
(29) Ravelli, R. B. G.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar,
S.; Sobel, A.; Knossow, M. Insight into tubulin regulation from a
complex with colchicine and a stathmin-like domain. Nature 2004,
428, 198–202.
(30) Viola, G.; Fortunato, E.; Cecconet, L.; Del Giudice, L.; Dall’Acqua,
F.; Basso, G. Central role of p53 and mitochondrial damage in
PUVA-induced apoptosis in human keratinocytes. Toxicol. Appl.
Pharmacol. 2008, 227, 84–96.
(31) Molecular Operating Environment (MOE 2008.10); Chemical Com-
puting Group, Inc.: Montreal, Quebec, Canada; http://www.chemcomp.
com.
€
(32) Korb, O.; Stutzle, T.; Exner, T. E. PLANTS: Application of Ant
Colony Optimization to Structure-Based Drug Design. In Ant
Colony Optimization and Swarm Intelligence, 5th International Work-
shop, ANTS 2006, Brussels, Belgium, Sep 4-7, 2006; Dorigo, M.,
Gambardella, L. M., Birattari, M., Martinoli, A., Poli, R., St€utzle, T.,
Eds.; Springer: Berlin, 2006; LNCS 4150, pp 247-258.