In vitro and in vivo biological evaluation of new 4,5-disubstituted 1,2,3-triazoles as cis-constrained analogs of combretastatin A4

Article abstract of DOI:10.1016/j.ejmech.2012.04.017

To find new and better antivascular agents for cancer therapy, a series of combretastatin A4 (CA4) analogs were prepared from 1,3-diaryl-2-nitroprop-1-enes (6-12) obtained in a two-step synthesis from appropriate arylaldehydes and 2-aryl-1-nitroethanes (4 or 5). Treatment of these 1,3-diaryl-2-nitroprop-1-enes 6-12 by sodium azide in DMSO yielded the targeted compounds. The synthesized 1,2,3-triazoles disubstituted in 4- and 5-positions by one benzyl group and one aryl nucleus have also been tested for biological activities involved in antivascular action. It was found that several new compounds exhibited interesting biological activities in the nanomolar or low micromolar range, in terms of rounding up of endothelial cells, inhibition of tubulin polymerization, and cytotoxicity on B16 melanoma cancer cells. In silico docking studies of 11 and 19 within the active site of tubulin were also carried out in order to rationalize the inhibitory properties of these compounds and further understand their inhibition mechanism. In vivo evaluation of compounds 11 and 19 in mice bearing colon 26 carcinoma indicated modest anticancer activity.

Full text of DOI:10.1016/j.ejmech.2012.04.017

European Journal of Medicinal Chemistry 54 (2012) 22e32
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
In vitro and in vivo biological evaluation of new 4,5-disubstituted 1,2,3-triazoles
as cis-constrained analogs of combretastatin A4
,
,
d
,
c
,
,
Núria Mur Blanch a b, Guy G. Chabot c
, Lionel Quentin d, Daniel Scherman c d, Stéphane Bourg e,
**
,b,
Daniel Dauzonne a
*
a Institut Curie, Centre de Recherche, 26 rue d’Ulm, F-75005 Paris, France
b CNRS, UMR176, 26 rue d’Ulm, F-75005 Paris, France
c Paris Descartes University, Sorbonne Paris Cité, Faculty of Pharmacy, F-75006 Paris, France
d INSERM U1022, CNRS UMR8151, Chimie ParisTech, Chemical, Genetic and Imaging Pharmacology Laboratory, F-75006 Paris, France
e Fédération de Recherche Physique et Chimie du Vivant, Université d’Orléans-CNRS, FR 2708, avenue Charles Sadron, 45071 Orléans Cedex 2, France
g r a p h i c a l a b s t r a c t
Amongst the synthesized compounds, the best rounding up activity was obtained with compounds 11 and 19 (R1 ¼ R2 ¼ R3 ¼ R6 ¼ OCH3, R4 ¼ R5 ¼ H,
R7 ¼ OH) with concentrations of 0.13 and 0.2
m
M, respectively.
NO2
R7
N
R1
R3
HN
R4
14 - 20
N
R6
NaN3
DMSO, 85°C, 15 h
R2
R7
R6
R1
R4
6 - 12
R5
R3
R2
R5
a r t i c l e i n f o
a b s t r a c t
Article history:
To find new and better antivascular agents for cancer therapy, a series of combretastatin A4 (CA4) analogs
were prepared from 1,3-diaryl-2-nitroprop-1-enes (6e12) obtained in a two-step synthesis from appro-
priate arylaldehydes and 2-aryl-1-nitroethanes (4 or 5). Treatment of these 1,3-diaryl-2-nitroprop-1-enes 6
e12 by sodium azide in DMSO yielded the targeted compounds. The synthesized 1,2,3-triazoles disubsti-
tuted in 4- and 5-positions by one benzyl group and one aryl nucleus have also been tested for biological
activities involved in antivascular action. It was found that several new compounds exhibited interesting
biological activities in the nanomolar or low micromolar range, in terms of rounding up of endothelial cells,
inhibition of tubulinpolymerization, and cytotoxicity on B16 melanoma cancer cells. In silico docking studies
of 11 and 19 within the active site of tubulin were also carried out in order to rationalize the inhibitory
properties of these compounds and further understand their inhibition mechanism. In vivo evaluation of
compounds 11 and 19 in mice bearing colon 26 carcinoma indicated modest anticancer activity.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 19 December 2011
Received in revised form
11 April 2012
Accepted 12 April 2012
Available online 9 May 2012
Keywords:
Antivascular agents
Combretastatin analogs
Triazoles
Endothelial cells
Tubulin polymerization
Cytotoxicity
1. Introduction
Tumor vasculature is an attractive target for cancer therapy
because solid tumors require blood vessels to grow and metastasize
[1e4]. This requirement for neovascularization to allow tumors to
grow beyond a certain threshold size drew attention to the potential
therapeutic agents that may act on angiogenesis which occurs in
several steps of the cancer process and offers several targets for
intervention [1e10].
* Corresponding author. Institut Curie, Centre de Recherche (CNRS UMR176), 26
rue d’Ulm, F-75005 Paris, France. Tel.: þ33 0 1 56 24 66 61.
** Corresponding author. Faculty of Pharmacy, INSERM U1022 and CNRS
UMR8151, Paris Descartes University, 4 avenue de l’Observatoire, F75006 Paris,
France. Tel.: þ33 0 1 53 73 95 71.
E-mail addresses: guy.chabot@parisdescartes.fr (G.G. Chabot), daniel.dauzonne@
curie.fr (D. Dauzonne).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.017

N. Mur Blanch et al. / European Journal of Medicinal Chemistry 54 (2012) 22e32
23
Tumor vascularization is the result of pro-angiogenic factors and
inhibitors and involves the interaction between endothelial cells
and extracellular matrix. Various approaches have been developed
to improve tumor control through the vascular targeting agents
which can be divided into antiangiogenic and vascular-disrupting
agents (VDAs) [11e14].
Considering the VDAs [15,16], the best active compounds that
have been found to date are acting on tubulin polymerization. In
this context, the molecule that emerged as one of the most potent is
combretastatin A4 (CA4 1) which is a cis-stilbene derivative that
was selected among seventeen combretastatin compounds origi-
nally isolated from the South African bushwillow tree Combretum
caffrum Kuntze (Combretaceae) by Pettit and co-workers [17e19].
report here that several compounds of this series present inter-
esting biological activities in the nanomolar or low micromolar
range, in terms of rounding up of endothelial cells, inhibition
of tubulin polymerization, and cytotoxicity on cancer cells. In
silico docking studies of 11 and 19 within the active site of
tubulin were also carried out and we also performed a prelimi-
nary in vivo evaluation of compounds 11 and 19 which revealed
a modest anticancer activity in mice bearing colon 26 carcinoma
tumors.
2. Results and discussion
2.1. Chemistry
The title compounds were prepared starting from 1,3-diaryl-2-
H3CO
H3CO
OH
CA4
1
nitroprop-1-enes (6e12) obtained according to
a procedure
OCH3
OCH3
already described by one of us [43] from appropriate arylaldehydes
and some 2-aryl-1-nitroethanes (4 or 5) by refluxing in toluene in
the presence of dimethylammonium chloride and a small amount
of potassium fluoride. The required nitro derivatives 4 and 5 were
synthesized by a two-step procedure starting from suitable benz-
aldehydes via the corresponding 2-aryl-1-nitroethenes (2 or 3,
respectively), as depicted in Scheme 1.
CA4 (1) is a strong inhibitor of tubulin polymerization and can
induce an important cytotoxicity in the nanomolar range against
various cancer cell lines, including multi-drug resistant ones [20].
To be active, 1 requires the cis-configuration, but it is known that it
can easily isomerize into its trans inactive form [21,22].
In order to stabilize the active configuration, numerous teams
have synthesized a wide range of various cis-restricted analogs of 1.
The literature has been repetitively reviewed during the past
decade [23e32] and, among the wide range of published hetero-
cyclic compounds, the triazole derivatives have received little
attention. In this context, some 3,4-disubstituted 1,2,4-triazole
[33,34], 1,4-disubstituted 1,2,3-triazole [35e38], 1,5-disubstituted
1,2,3-triazole [36e40] and 4,5-disubstituted 1,2,3-triazole [40e42]
showed interesting activities in terms of tubulin polymerization
inhibition and/or cytotoxicity. To our knowledge, only one report
[37] deals with triazoles having vicinal benzyl and phenyl substit-
uents (in the relative 1,5-positions) designed in order to confer to
the molecule a higher degree of conformational freedom.
Subsequent treatment of these 1,3-diaryl-2-nitroprop-1-enes
6e12 by sodium azide in dimethylsulfoxyde at 85 ꢀC, following
a methodology reported by Quiclet-Sire and Zard [44], gave the
desired compounds as shown in Scheme 2, with good yield in most
cases (cf. Experimental part).
In a similar way, the indole derivative 21 has been prepared in
93% yield from the already known nitroethene 13 previously
synthesized according to reference [43] (Scheme 3).
The amino derivative 22 was easily obtained in 88% yield by
catalytic hydrogenation in THF from its nitro precursor 17 as
presented in Scheme 4.
The knowncompounds 23, 24 and 25 have beenprepared from the
appropriate 1,3-diaryl-2-nitropropenes according to reference [43].
23 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = Cl, R5 = Cl, R6 = H
R6
NO2
24 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R5
R4 = OCH3, R5 = OCH3, R6 = OCH3
25 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = OCH3, R5 = OH, R6 = OCH3
R1
R4
R2
R3
In the context of hitherto unknown 1,2,3-triazoles disubsti-
tuted in the 4- and 5-positions by one aryl group and one benzyl
substituent, the present study was undertaken to provide
information on the biological activities of these compounds. We
2.2. Biological activities
To evaluate the potential antivascular activity of the new cis-
constrained analogs of 1, we first assessed their rounding up
R1
R1
R1
NO2
CHCl3, iPrOH, r. t.
NO2
CH3NO2, KF
(CH3)2NH2+, Cl-
Na BH4
R2
CHO
R2
R2
R3
R3
R3
Toluene, reflux
2 : R1 = OH, R2 = OCH3, R3 = H
4 : R1 = OH, R2 = OCH3, R3 = H
5 : R1= R2 = R3 = OCH3
3 : R1= R2 = R3 = OCH3
Scheme 1.

24
N. Mur Blanch et al. / European Journal of Medicinal Chemistry 54 (2012) 22e32
R7
R6
R4
N
R1
R3
HN
N
R5
R1
R3
O2N
R2
R7
R6
NaN3
R4
DMSO, 85°C, 15 h
R2
R5
14 : R1 = OH, R2 = OCH3, R3 = H
6 : R1 = OH, R2 = OCH3, R3 = H
R4 = H, R5 = OCH3, R6 = OCH3, R7= OCH3
R4 = H, R5 = OCH3, R6 = OCH3, R7 = OCH3
15 : R1 = OCH3, R2 = OCH3, R3 = OCH3
7 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = H, R5 = H, R6- R7 = OCH2O
R4 = H, R5 = H, R6- R7 = OCH2O
16 : R1 = OCH3, R2 = OCH3, R3 = OCH3
8 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = Cl, R5 = H, R6 = Cl, R7 = H
R4 = Cl, R5 = H, R6 = Cl, R7 = H
17 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = H, R5 = H, R6 = OCH3, R7 = NO2
18 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = H, R5 = H, R6 = OCH3, R7 = F
19 : R1 = OCH3, R2 = OCH3, R3 = OCH3
9 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = H, R5 = H, R6 = OCH3, R7 = NO2
10 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = H, R5 = H, R6 = OCH3, R7 = F
11 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = H, R5 = H, R6 = OCH3, R7 = OH
12 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = H, R5 = H, R6 = OH, R7 = OCH3
R4 = H, R5 = H, R6 = OCH3, R7 = OH
20 : R1 = OCH3, R2 = OCH3, R3 = OCH3
R4 = H, R5 = H, R6 = OH, R7 = OCH3
Scheme 2.
N
N
HN
NO2
OCH3
OCH3
OCH3
NaN3
DMSO, 85°C, 15 h
OCH3
OCH3
HN
N
H
H3CO
21
13
Scheme 3.
activity on endothelial cells, which is considered to be predictive of
potential in vivo antivascular activity [45]. The compounds were
also tested for their inhibition of tubulin polymerization and their
cytotoxic activity on the murine B16 melanoma cell line.
2.2.2. Tubulin polymerization inhibition
The compound concentrations needed to inhibit tubulin
polymerization by 50% are also shown in Table 1. We observed that
the best compounds for tubulin activity were also the ones that
could elicit a rounding up of endothelial cells at low concentrations.
It is noteworthy that a very good linear correlation exists between
the inhibition of tubulin polymerization and the morphological
activity (rounding up) on endothelial cells for the most active
compounds presenting morphological activity at concentrations of
2.2.1. Morphological activity
Table 1 presents the rounding up activity data for the synthe-
sized combretastatin analogs from the most active to the less active
compounds. Compared to the reference compound (1) which could
elicit a rounding up of endothelial cells at very low concentrations
(6 nM), seven compounds of our series showed interesting
less than 10 mM (Fig. 2).
rounding up activity below 1
7, 6, 14 and 10). A representative photograph depicting the
rounding up activity of compound 19 at a concentration of 1 M is
shown in Fig. 1 and compared to CA4. It is noteworthy that
rounding up activity was observed at concentration as low as
m
M for a 2 h exposure time (19, 11, 22,
2.2.3. Cytotoxicity
Finally our new combretastatin analogs were also evaluated for
their cytotoxicity on the B16 melanoma cell line. For this biological
activity, the correlation with rounding up activity was less evident.
With the exception of compounds 22, 14 and 18, which appear to
be outliers, a relative good correlation was nonetheless observed
between cytotoxicity and rounding up activity for the best
m
0.13
mM for compound 19. Alteration of endothelial cell
morphology is a distinctive feature of the activity of VDAs which is
correlated with cytoskeleton disorganization [45].
morphologically active compounds (<10 mM).
N
N
N
N
HN
HN
H3CO
H3CO
H3CO
H2, Pd/C 10%
THF, RT, 1h
OCH3
OCH3
H3CO
NH2
NO2
OCH3
OCH3
17
22
Scheme 4.

N. Mur Blanch et al. / European Journal of Medicinal Chemistry 54 (2012) 22e32
25
Table 1
Biological activities of new analogues of combretastatin A4.a
Compound
numbers
Morphological
activity on
endothelial cells (
Inhibition of tubulin
polymerization
Cytotoxicity
IC50
M)d
(
m
m
M)b
IC50 (m
M)c
CA4 (1)
19
11
22
7
0.006
0.13
0.2
0.2
0.4
0.6
0.6
0.8
1.6
2.5
3.0
3.1
3.1
6.3
6.3
12.5
25
25
0.3
1.2
0.9
1.8
10.5
0.9
1.7
1.6
5.2
12.0
8.9
21.9
15.5
22.4
>30
23.4
> 30
>30
>30
>30
>30
0.003
1.6 ꢂ 0.1
4.0 ꢂ 1.1
14.8 ꢂ 1.2
3.0 ꢂ 0.2
2.9 ꢂ 0.02
22.9 ꢂ 0.9
2.9 ꢂ 0.1
5.5 ꢂ 0.4
31 ꢂ 2.6
3.5 ꢂ 0.1
2.6 ꢂ 0.2
4.6 ꢂ 0.4
3.3 ꢂ 0.2
2.6 ꢂ 0.3
3.2 ꢂ 0.2
38.0 ꢂ 3.4
50.1 ꢂ 10.7
10.7 ꢂ 0.4
41.7 ꢂ 1.3
>100
6
14
10
13
18
9
8
23
24
12
25
15
17
21
16
20
50
>100
>100
a
Compounds have been ranked in order of decreasing potency on endothelial cell
morphology, i.e., the minimum concentration needed to elicit a cell rounding up
after a 2 h exposure time.
b
Morphological activity was tested on immortalized HUVEC (EAhy 926 cells) and
is expressed at the lowest concentration at which cell rounding up was observed,
following 2 h incubation with the tested compound.
c
Concentration required to inhibit tubulin polymerization (ITP) by 50%.
Concentration required to kill 50% of B16 melanoma cells for a 48 h incubation
d
time (MTT assay). Mean ꢂ SEM.
to their analogs having a 3,4,5-trimethoxybenzyl substituent
(compare 19 vs 14, 11 vs 6).
Concerning the inversion of the relative position of methoxy and
hydroxy groups (compare 12 with 11 and 6, or 20 with 19 or 14)
we noted that biological activities were noticeably diminished, in
particular concerning tubulin polymerization inhibition. We also
noted that the presence of a second methoxy group on the
other meta position led to diminished biological activity (compare
25 vs 12).
Although they remain slightly less efficient than 11 or 19, those
derivatives in which the meta-hydroxy is replaced by a fluorine atom
(10 or 18), a nitro group (9 or 17) or an amino substituent (22) led,
particularly in this latter case, to fairly good results (NH2 > F > NO2).
Fig. 1. Morphological effects on endothelial cells. Typical rounding up of endothelial
cells (EAhy 926) exposed to CA4 1 (0.006 M) and compound 19 (1 M) for a 2 h
m
m
exposure time. The solvent DMSO 1% was used as the control. Original magnification,
200ꢁ.
2.2.4. Structureeactivity relationships
Beside the well-known requirement of the 3,4,5-
trimethoxyphenyl group for optimal biological activity of com-
bretastatin analogs [21e30], we also noted the following inter-
esting features for the new 4,5-disubstituted 1,2,3-triazoles and
related 1,3-diaryl-2-nitroprop-1-enes. With regards to the nitro-
alkene and triazole series, we noted that the compounds the most
similar to 1, i.e., those bearing a methoxyl group in the para position
together with a hydroxy substituent in the meta position, were
among the most potent derivatives (e.g.,19,11, 6,14). In this context,
it must be pointed out that compounds belonging to series B, where
the 3,4,5-trimethoxyphenyl nucleus is directly connected to the
double bond of the triazole ring, are relatively less active compared
Fig. 2. Correlation between rounding up activity on endothelial cells and inhibition of
tubulin polymerization (ITP). Compounds with good morphological activity (rounding up
concentration <10 mM) are plotted against the concentration needed to inhibit tubulin
polymerization by 50% (IC50). Equation of the straight line: Y ¼ 4.396X, r2 ¼ 0.834.

26
N. Mur Blanch et al. / European Journal of Medicinal Chemistry 54 (2012) 22e32
Fig. 3. Putative binding of our best cis-constrained analogues of CA4. For clarity non polar hydrogen atoms are not represented. Top: CA4 docking pose (green stick model) is
superimposed to crystallographic CN-2 (dama-colchicine, gray stick model), reproducing the binding mode highlighted in the colchicine domain of the crystallographic complex
(PDB ID: 1SA0).
a
and
b-tubulin are ribbons displayed according to their secondary structure. Residues of the three zones of interaction [49] are represented too (zone 1: cyan, zone
2: orange, zone 3: green). Val181
a
from zone 1 and Cys241 from zone 2 are highlighted (tube, element colored) as they are involved in H bonds represented in yellow dashed
b
lines. Down: superimposition of CA4 (green tube), 11 (pink tube) and the best docking scored tautomer of 19 (gray tube). Concerning the compound 19, similar conformations, not
shown, were obtained regardless the tautomer forms. The right part represents a 90ꢀ rotation about the vertical axis of the left view and highlights the conservation of the cisoid
arrangement of the two aromatic rings.
However, the replacement of the 4-methoxy-3-hydroxybenzene
nucleus by a 3,4-methylenedioxybenzene (compare 11 to 7 and 19
to 15), by a 3,4,5-trimethoxybenzene (compare 11e24), by dichlo-
robenzenes (compare 11 to 8 or 23 and 19 to 16) or by a 3-indolyl
(compare 6 to 13 and 14e21) were found unfavorable to biolog-
ical activities.
between the nitroalkene series (6e13) and the triazole compounds
(14e22) demonstrates that the nitroalkenes 6e13, having a higher
degree of conformational freedom, are more active than their
corresponding triazole derivatives.
Considering the relatively good in vitro activities of compounds
11 and 19 in terms of cytotoxicity, rounding up of endothelial cells
and inhibition of tubulin polymerization, the putative mechanism
of action of these compounds is probably close to that of CA4.
Finally, with the exception of the couple 11/19 where the
measured biological activities are very close, the comparison

N. Mur Blanch et al. / European Journal of Medicinal Chemistry 54 (2012) 22e32
27
2.3. Molecular modeling
In order to compare the putative binding mode of our cis-
constrained analogs of CA4 and combretastatin itself, docking
studies were carried out by using the reported high-resolution
crystal structure of the tubulin-DAMA-colchicine(CN-2) complex
(PDB ID: 1SA0) [46] as it was done on other heterocyclic analogs
of CA4 [47]. The colchicine site, well described in two recent
reviews [48,49], is a deep pocket located at the aeb interface of
tubulin heterodimers. Massaroti et al. [49] identified three zones
of interaction reproduced in Fig. 3. The trimethoxyphenyl ring
(TMP or A ring) of CN-2 lies in a hydrophobic pocket of zone 2
while the other side of the molecule (C ring) interacts in zone 1
by van der Waals contacts. Furthermore two residues are high-
lighted as they are often involved in hydrogen bond interactions
with ligands: Cys241
b and Val181 a. It should be noted that the
residue numbering system we used is derived from the crystal
structure [46].
Fig. 4. Antitumor effects of compounds 11 and 19. Mice bearing subcutaneously
implanted colon 26 carcinoma tumors were treated intraperitoneally with solvent
(control) or with compound 11 or 19 for 2 weeks on days 3e7, and days 10e14, as
describe in the Experimental protocols section.
Docking simulations were performed with GOLD software
version 5.1 [50] (see docking protocol for more details). Our
protocol was validated by its ability to reproduce the crystal
complex structure CN-2/tubulin by docking the ligand extracted
from the complex. Superimposition, not shown, of our best
docked pose on the crystal structure gave an RMSD deviation,
based on heavy atoms, of 0.92 Å. The C ring is involved in
hydrogen bonds and the absence of new ones could explain the
lower biological activity than the one observed with CA4.
hydrogen bond with Val181
a. The TMP ring is at a good distance
2.4. Evaluation of antitumor activity in colon 26-bearing mice
of Cys241 to be involved in hydrogen bond, but the polar
b
hydrogen atom of cystein is not properly directed to the methoxy
oxygen atoms of CN-2.
Based on their good in vitro biological activities, compounds 11
and 19 were selected for further in vivo testing in colon 26 tumor
bearing mice. The colon 26 tumor was chosen because this model
is responsive to antivascular agents [52]. Fig. 4 presents the tumor
volume of controls receiving the solvent and treated animals
receiving compound 11 or 19 at their highest non toxic dose, i.e.,
150 mg/kg for 11 and 100 mg/kg for 19, administered intraperito-
neally for two weeks on days 3e7 and 10e14 after tumor implan-
tation (day 0). Compounds 11 and 19 presented similar percent T/C
values of 52% and 60%, respectively, measured when control tumors
reached 1500 mm3. The tumor growth delay of treated mice
compared to controls (TeC) was 2 days for compound 11 and 2.7
days for compound 19.
Although 11 and 19 caused a significant delay in tumor growth,
this antitumor efficacy can be regarded as modest activity consid-
ering that a T/C value of 42% is considered the minimum level for
activity according to the National Cancer Institute criteria. It is
however possible that other schedules of administration and/or
doses could contribute to improve the antitumor activity of these
compounds.
The next step was the docking of CA4. The conformer with the
lowest energy resulting from a conformational analysis was chosen
as starting point. The pose, corresponding to the best docking score
of the 100 conformations generated during the docking process, is
superimposed to CN-2 crystal structure (Fig. 3). It exhibits a similar
binding mode, where the TMP rings are buried in the same
hydrophobic pocket and, where the oxygen atom of the hydroxyl
group is involved in hydrogen bond with Val181
with the C ring of CN-2.
a, in accordance
Initial structures of 11 and tautomers of 19 were prepared
similarly to CA4. The best docked poses were then selected to be
superimposed, on Fig. 3, on the CA4 conformation previously
obtained. 11 docked in a very similar position relative to CA4,
establishing again a hydrogen bond with Val181
a. Although the
oxygen acceptor atoms of nitro group are close to the hydrogen
atoms of Ala 250
hydrogen bonds.
b or Asp251 b, our model does not exhibit such
Among the three tautomers of 19, we can distinguish the one
were hydrogen is on the central nitrogen. It adopts a CN2-like
pose but not in the top 10 of scored conformations. In this case,
not shown, the triazole group points its hydrogen atom to the
3. Conclusion
amino group of Asn101
a without favorable interaction. The two
other tautomers poses differ only by the hydrogen atom position.
In the first case, not shown, this hydrogen interacts via
We have, for the first time to our knowledge, synthesized new
1,2,3-triazoles disubstituted in 4- and 5-positions by one benzyl
and one aryl groups. We also evaluated these new compounds for
potential antivascular and anticancer activities and found that
several compounds presented interesting biological activities, e.g.,
rounding up of endothelial cells (at concentrations in the sub-
micromolar range), inhibition of tubulin polymerization and cyto-
toxicity on B16 cancer cells (at concentrations in the low
micromolar range). In silico docking studies of 11 and 19 within the
active site of tubulin were also carried out in order to rationalize the
inhibitory properties of these compounds and further understand
their inhibition mechanism. In addition, the evaluation of
compounds 11 and 19 in tumor bearing mice indicated modest
in vivo anticancer activity.
a hydrogen bond with the carbonyl group of Asn258
b. In the
second case, represented on Fig. 3, the hydrogen is at the opposite
side of the ring and is not involved in a hydrogen bond. It should
be noted that the poses of those two last tautomers have a very
similar score. The three tautomers have in common the pockets,
where their aromatic rings are embedded but shifted regarding
those of CA4. This shift result is the loss of hydrogen bond with
Val181 a.
To conclude, we observed the non-coplanar cisoid arrangement
of the two aromatic rings, highlighted on Fig. 3, of CA4 and its
analogs. This arrangement is described as essential for the biolog-
ical activity [51], but it is not sufficient because the loss of some

28
N. Mur Blanch et al. / European Journal of Medicinal Chemistry 54 (2012) 22e32
4. Experimental protocols
1244 (AreO), 1130 (CeO) cmꢃ1; MS (ESI(þ)): m/z 262 [M þ Na]þ.
Anal calcd for C11H13NO5: C: 55.23; H: 5.48; N: 5.86. Found C: 55.42;
H: 5.35; N: 5.97.
4.1. Synthesis
Melting points were measured on a Köfler hot stage apparatus
and are uncorrected. Mass spectra were recorded on a Waters ZQ
2000 system using electrospray ionization (ESI) or on a Waters LCT
apparatus for High-resolution mass spectra (HRMSeESI). Infrared
spectra were obtained using an FT-IR PerkineElmer spectrometer as
chloroform solutions or KBr discs. NMR spectra were recorded with
a Bruker ACP 300 spectrometer at 300 MHz for 1H NMR and 75 MHz
for 13C NMR. Chemical shifts are expressed as parts per million
downfield from tetramethylsilane. Splitting patterns have been
designated as follows: s (singlet), d (doublet), dd (doublet of doublet),
m (multiplet), br. (broad signal). Coupling constants (J values) are
listed in hertz (Hz). Reactions were monitored by analytical thin layer
chromatography and products were visualized by UV light. Merck
Silica gel (230-400 Mesh ASTM) was used for column chromatog-
raphy. Methanol and CH2Cl2 employed as eluents were distilled on
a rotary evaporator prior to use. Dry THF was prepared by distillation
from benzophenone/sodium.
4.1.2. General procedure 2 for the synthesis of 2-aryl-1-
nitroethanes 4 and 5
To a flask containing the corresponding 2-aryl-1-nitroethene
were added silica gel (2 g per mmol of starting material), and
1.5 mL of 2-propanol and 8 mL of chloroform per gram of silica gel
[53]. To this mixture were added 4 mmol of NaBH4 per mmol of
starting material in small amounts and the resulting mixture was
allowed to stir at room temperature until the complete disap-
pearance of the 2-aryl-1-nitroethene (usually noted after a reaction
time of about 30 min). The reaction mixture was quenched with an
aqueous solution of 0.1 N HCl, filtered to eliminate the silica gel and
evaporated under vacuum to eliminate 2-propanol and chloroform.
The resulting aqueous mixture was then extracted by CH2Cl2 (3ꢁ).
The organic phase was dried over MgSO4, filtered and concentrated
under vacuum. Crude product was purified by chromatography on
silica gel in order to afford the desired compound.
4.1.2.1. 2-(3-Hydroxy-4-methoxyphenyl)-1-nitroethane (4). General
procedure 4.1.2. was applied to 2 (10 g, 50.0 mmol) with a reaction
time of 30 min. The crude product was purified by chromatography
on silica gel (CH2Cl2) to provide the corresponding 2-aryl-1-
nitroethane as pale yellow needles (6.92 g, 35.1 mmol, 68%).Mp:
4.1.1. General procedure 1 for the synthesis of 2-aryl-1-nitroethenes
2 and 3
To a solution of the corresponding arylaldehyde in toluene were
added nitromethane (11 equivalents), dimethylamine hydrochlo-
ride (4 equivalents) and potassium fluoride (0.15 equivalents). The
flask was equipped with a DeaneStark and the mixture was heated
at reflux temperature until the disappearance of the arylaldehyde
(usually noted after a reaction time of 4e5 h). After being cooled at
room temperature, the reaction mixture was evaporated under
vacuum to eliminate the toluene, then diluted with water and
extracted by CH2Cl2 (3ꢁ). In some cases, a filtration over Celite
surface was required in order to eliminate excess reagent or reac-
tion residues prior to extraction. The organic phase was then
washed with water, dried over MgSO4, filtered and concentrated
under vacuum. Crude product was purified by chromatography on
silica gel in order to afford the desired compound.
52e53 ꢀC (pentane); 1H NMR (300 MHz, CDCl3):
d
¼ : 3.21
(t, J ¼ 7.7 Hz, 2H, CH2-2), 3.96 (s, 3H, OCH3), 4.56 (t, J ¼ 7.7 Hz, 2H,
CH2-1), 5.62 (s, 1H, OH), 6.67 (dd, J1 ¼ 8.4 Hz, J2 ¼ 2.1 Hz, 1H, H-60),
6.77 (d, J ¼ 2.1 Hz, 1H, H-20), 6.78 (d, J ¼ 8.4 Hz, 1H, H-50); 13C NMR
(75 MHz, CDCl3):
d
¼ 33.9 (CH2, C-2), 55.8 (CH3, OCH3), 76.7 (CH2,
C-1), 110.9 (CH, C-50), 114.7 (CH, C-20), 120.3 (CH, C-60), 128.6 (C,
C-10), 145.8 (C, C-30), 145.9 (C, C-40); IR (NaCl): y0 3541 (OH), 1555
(NO2 as),1380 (NO2 ss),1273 (AreO),1029 (CeO) cmꢃ1; MS (ESI(þ)):
m/z 220 [M þ Na]þ. C9H11NO4: C: 54.82; H: 5.62; N: 7.10. Found C:
55.01; H: 5.59; N: 7.21.
4.1.2.2. 2-(3,4,5-Trimethoxyphenyl)-1-nitroethane (5). General proce-
dure 4.1.2. was applied to 3 (20 g, 83.6 mmol) with a reaction time of
about 30 min. The crude product was purified by chromatography on
silica gel (CH2Cl2 100%) to provide the corresponding 2-aryl-1-
nitroethane as white fluffy crystals (19.05 g, 79.0 mmol, 94%). Mp:
4.1.1.1. 2-(3-Hydroxy-4-methoxyophenyl)-1-nitroethene
(2). General procedure 4.1.1. was applied to iso-vanillin (3-hydroxy-
4-methoxybenzaldehyde) (46.0 g, 0.30 mol) with a reaction time of
3 h. The crude product was purified by chromatography on silica gel
(CH2Cl2 100%) to provide the corresponding 2-aryl-1-nitroethene as
an ocher-colored solid, which was afterward recrystallized to afford
bright orange crystals (30.0 g, 0.154 mol, 51%). Mp: 161e162 ꢀC
84e85 ꢀC (cyclohexane/benzene/); 1H NMR (300 MHz, CDCl3):
0
d
¼ 3.24 (t, J ¼ 8.0 Hz, 2H, CH2-2), 3.80 (s, 3H, OCH3eC4 ), 3.85 (s, 6H,
OCH3eC3 , OCH3eC5’), 4.59 (t, J ¼ 8.0 Hz, 2H, CH2-1), 6.37 (s, 2H, H-20,
0
H-60); 13C NMR (75 MHz, CDCl3):
d
¼ 33.8 (CH2, C-2), 56.0 (CH3 ꢁ 2,
(heptane/benzene); 1H NMR (300 MHz, CDCl3):
d
¼ 3.97 (s, 3H,
OCH3eC3 , OCH3eC5 ), 60.7 (CH3, OCH3eC4 ), 76.5 (CH2, C-1), 105.6
(CH ꢁ 2, C-20, C-60), 131.4 (C, C-10), 153.5 (C ꢁ 3, C-30, C-40, C-50); IR
(NaCl): y0 1555 (NO2 as), 1349 (NO2 ss), 1240 (AreO), 1131 (CeO)
cmꢃ1; MS (ESI(þ)): m/z 264 [M þ Na]þ. C11H15NO5: C: 54.77; H:
6.27; N: 5.81. Found C: 54.89; H: 6.08; N: 5.90.
0
0
0
OCH3), 5.73 (s, 1H, OH), 6.90 (d, J ¼ 9.4 Hz, 1H, H-50), 7.07
(d, J ¼ 1.9 Hz, 1H, H-20), 7.12 (dd, J1 ¼ 9.4 Hz, J2 ¼ 1.9 Hz, 1H, H-60),
7.48 (d, J ¼ 12.6 Hz, 1H, ¼CH-1), 7.92 (d, J ¼ 12.6 Hz, 1H, ¼CH-2); 13C
NMR (75 MHz, DMSO):
d
¼ 56.5 (CH3, OCH3), 112.6 (CH, C-20), 116.18
(CH, C-50), 124.4 (CH, C-60), 128.7 (C, C-10), 136.1 (CH, C-1), 140.4 (CH,
C-2), 147.5 (C, C-30), 152.3 (C, C-40); IR (NaCl): y0 3542 (OH), 3023
(¼CeH), 1616 (C]C), 1508 (NO2 as), 1340 (NO2 ss), 1275 (AreO),
1133 (CeO) cmꢃ1; MS (ESI(ꢃ)): m/z 194 [M-1H]. Anal calcd for
C9H9NO4: C: 55.39; H: 4.65; N: 7.18. Found C: 55.51; H: 4.55; N: 7.29.
4.1.3. General procedure for the synthesis of 1,3-diaryl-2-
nitropropenes 6e12, 23e25
To a solution of the corresponding 2-aryl-1-nitroethane in
xylene were added dimethylamine hydrochloride (2 equivalents),
potassium fluoride (0.2 equivalents) and the appropriate arylalde-
hyde (1 equivalent). The flask was equipped with a DeaneStark and
the mixture was heated at reflux temperature until the disap-
pearance of the arylaldehyde (usually noted after a reaction time of
24 h). After being cooled at room temperature, the reaction mixture
was evaporated under vacuum to eliminate the xylene, then diluted
with water and extracted by CH2Cl2 (3ꢁ). The organic phase was
then washed with water, dried over MgSO4, filtered and concen-
trated under vacuum. Crude product was purified by
4.1.1.2. 2-(3,4,5-Trimethoxyphenyl)-1-nitroethene (3) [43]. Mp:
124e125 ꢀC (isopropanol); 1H NMR (300 MHz, CDCl3):
d
¼ 3.67
0
0
0
(s, 9H, OCH3eC3 , OCH3eC4 , OCH3eC5 ), 6.75 (s, 2H, H-20, H-60), 7.53
(d, J ¼ 14.4 Hz, 1H, ¼CH-2), 7.93 (d, J ¼ 14.4 Hz, 1H,0 ¼CH-1); 130C
NMR (75 MHz, CDCl3):
d
¼ : 56.3 (CH3 ꢁ 2, OCH3eC3 , OCH3eC5 ),
61.1 (CH3, OCH3eC4 ), 106.5 (CH ꢁ 2, C-20, C-60), 125.3 (CH, C-2),
136.4 (C, C-10), 139.3 (CH, C-1), 153.7 (C ꢁ 3, C-30, C-40, C-50); IR
(NaCl): y0 3021 (¼CeH), 1633 (C]C), 1503 (NO2 as), 1328 (NO2 ss),
0

N. Mur Blanch et al. / European Journal of Medicinal Chemistry 54 (2012) 22e32
29
chromatography on silica gel in order to afford the desired
compound [43]. Compounds 7, 8, 11, 12, 23, 24, and 25 are already
described in ref [43].
J ¼ 20.1 Hz, C-200), 124.5 (C, J ¼ 7.2 Hz, C-100), 127.3 (CH, C-2), 131.5
(CH, C-600), 134.6 (C, C-10), 137.0 (C, C-1), 149.8 (C, J ¼ 20.1 Hz, C-400),
152.2 (C, J ¼ 246.8 Hz, C-300), 153.6 (C ꢁ 3, C-30, C-40, C-50); IR (NaCl):
y0 3022 (¼CeH), 1613 (C]C), 1513 (NO2 as), 1329 (NO2 ss), 1240
(AreO),1131 (CeO) cmꢃ1; MS (ESI(þ)): m/z 400 [M þ Na]þ.
C19H20FNO6: C: 60.47; H: 5.34; N: 3.71. Found C: 60.66; H: 5.29; N:
3.67.
4.1.3.1. 3-(3-Hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-
2-nitropropene (6). General procedure 4.1.3. was performed with
3,4,5-trimethoxybenzaldehyde (6.47 g, 33.0 mmol) and 4 (5.91 g,
30.0 mmol) with a reaction time of 24 h. The crude product was
purified by chromatography on silica gel (CH2Cl2/acetone 99/1) to
provide the corresponding 1,2-diarylnitroethene together with
excess benzaldehyde, which was afterward eliminated by treatment
with Girard’s T reagent [54]. The expected compound was obtained
as a yellow solid and was then recrystallized from a mixture hexane/
benzene to provide bright yellow crystals (5.91 g, 15.7 mmol, 52%).
Mp: 95e96 ꢀC (hexane/benzene); 1H NMR (300 MHz, CDCl3)
4.1.4. General procedure for the synthesis of 4-benzyl-5-
phenylriazoles 14e22
To a solution of the corresponding benzylphenylnitroethene in
DMSO (minimum volume: approximately 10 mL for 3 mmol) was
added NaN3 (2 equivalents) [42]. The resulting mixture, which
acquired a dark red color right after the addition, was allowed to
stir at 85 ꢀC under argon atmosphere until complete conversion of
the starting material (usually noted after a reaction time of 15 h).
After being cooled at room temperature, the reaction mixture was
diluted with water and then extracted by CH2Cl2 (4ꢁ). The organic
phase was dried over MgSO4, filtered and concentrated under
vacuum. Crude product was purified by chromatography on silica
gel. In order to eliminate remaining DMSO, the evaporated fractions
containing the desired compound were dissolved in ether and
washed with water (3ꢁ), dried over MgSO4, filtered and then
concentrated under reduced pressure.
d
¼ 3.70 (s, 3H, OCH3eC4"), 3.82 (s, 6H, OCH3eC3", OCH3eC5"), 4.20
(s, 2H, CH2eC1), 5.55 (s, 1H, OH), 6.66 (s, 2H, H-2", H-6"), 6.70 (s, 1H,
H-20), 6.80e6.81 (m, 2H, H-50, H-60), 8.24 (s, 1H, H-2). 13C NMR
0
(75 MHz, CDCl3):
d
¼0 32.6 (CH2, CH2eAr), 56.4 (CH3 ꢁ 3, OCH3eC3 ,
OCH3eC4’, OCH3eC5 ), 80.9 (CH3, OCH3eC4”), 107.6 (CH ꢁ 2, C-20,
C-60),110.9 (CH, C-600),114.1 (CH, C-500),118.7 (CH, C-200),127.2 (CH, C-
2), 129.4 (C, C-10), 136.0 (C, C-100), 140.0 (C, C-1), 145.5 (C, C-300), 145.9
(C, C-400), 148.7 (C, C-40), 153.7 (C ꢁ 2, C-30, C-50); IR (NaCl): y0 3541
(OH), 3023 (¼CeH), 1592 (C]C), 1554 (NO2 as), 1379 (NO2 ss), 1272
(AreO), 1130 (CeO) cmꢃ1; MS (ESI(þ)): m/z 398 [M þ Na]þ.
C19H21NO7: C: 60.79; H: 5.64; N: 3.73. Found C: 60.84; H: 5.65; N:
3.78.
4.1.4.1. 4-(3-Hydroxy-4-methoxybenzyl)-5-(3,4,5-trimethoxyphenyl)-
1,2,3-triazole (14). General procedure 4.1.4. was applied to 6 (1.13 g,
3.0 mmol) with a reaction time of 12 h. The crude product was
purified by chromatography on silica gel (CH2Cl2/MeOH 95/5) and
then treated to eliminate remaining DMSO according to general
procedure 4 to provide the expected compound as a white foamy
solid (0.94 g, 2.53 mmol, 84%). Mp: 82e83 ꢀC (heptane/benzene);
4.1.3.2. 1-(4-Methoxy-3-nitrophenyl)-3-(3,4,5-trimethoxyphenyl)-2-
nitropropene (9). General procedure 4.1.3. was performed with 4-
methoxy-3-nitrobenzaldehyde (5.80 g, 32.0 mmol) and 5 (7.60 g,
31.5 mmol) with a reaction time of 24 h. The crude product was
purified by chromatography on silica gel (CH2Cl2/acetone 99/1) to
provide the corresponding 1,2-diarylnitroethene as a yellow solid,
which was afterward recrystallized to provide bright yellow crys-
tals (6.78 g, 16.8 mmol, 53%). Mp: 158e159 ꢀC (heptane/benzene);
1H NMR (300 MHz, CDCl3):
d
¼ 3.77 (s, 6H, OCH3eC3 , OCH3eC5 ),
0
0
3.64 (s, 3H, OCH3eC4 ), 3.66 (s, 3H, OCH3eC400), 4.18 (s, 2H, CH2),
5.80 (bs, 1H, OH), 6.65 (dd, J1 ¼ 8.5 Hz, J2 ¼ 2.0 Hz, 1H, H-6000), 6.76
(d, J ¼ 8.5 Hz, 1H, H-5000), 6.78 (d, J ¼ 2.0 Hz, 1H, H-200), 6.81 (s, 2H,
0
1H NMR (300 MHz, CDCl3):
d
¼ 3.80 (s, 6H, OCH3eC3 , OCH3eC5 ),
H-20, H-60); 13C NMR (75 MHz, 0CDCl3):
d
¼030.6 (CH2, CH2eAr), 56.1
0
0
3.82 (s, 3H, OCH3eC4 ), 4.01 (s, 3H, OCH3eC400), 4.21 (s, 2H,
CH2eAr), 6.39 (s, 2H, H-20, H-60), 7.15 (d, J ¼ 9.1 Hz, 1H, H-500), 7.62
(dd, J1 ¼ 9.1 Hz, J2 ¼ 2.0 Hz, 1H, H-600), 8.01 (d, J ¼ 2.0 Hz, 1H, H-200),
(CH3 ꢁ 3, OCH3eC3 , OCH3eC4 , OCH3eC5 ), 60.9 (CH3, OCH3eC400),
104.8 (CH ꢁ 2, C-20, C-60), 111.0 (CH, C-500), 114.8 (CH, C-200), 119.7
(CH, C-600), 125.9 (C, C-5), 131.3 (C, C-10), 138.0 (C, C-4), 145.5
(C, C-300), 146.0 (C, C-400), 153.4 (C ꢁ 3, C-30, C-40, C-50); IR (NaCl): y0
0
0
8.26 (s, 1H, ¼CH-2); 13C NMR (75 MHz, CDCl3):
d
0
¼ 33.1 (CH2,
CH2eAr),0 56.1 (CH3
ꢁ
2, OCH3eC3 , OCH3eC5 ), 56.8 (CH3,
3541 (OH), 3009 (NH), 1590 (C]C), 1239 (AreO), 1129 (CeO) cmꢃ1
;
0
OCH3eC4 ), 60.8 (CH3, OCH3eC400), 104.6 (CH ꢁ 2, C-20, C-60), 114.3
(CH, C-500), 121.2 (C, C-100), 124.2 (CH, C-200), 131.1 (CH, C-600), 132.6
(C, C-10), 135.6 (CH, C-2), 137.2 (C, C-300), 149.8 (C, C-1), 153.7 (C ꢁ 3,
HRMS (ESI): m/z 372.1577 [M
372.1559.
þ
H]þ, C19H22N3O5 requires
C-30, C-40, C-50),154.3 (C, C-400); IR (NaCl):
y
0 3021 (¼CeH),1592 (C]
4.1.4.2. 5-(Benzo-[1,3]-dioxol-5-yl)-4-(3,4,5-trimethoxybenzyl)-1,2,3-
triazole (15). General procedure 4.1.4. was applied to 2-(benzo-[1,3]-
dioxol-5-yl)-1-(3,4,5-trimethoxybenzyl)-1-nitroethene (7) (1.00 g,
2.7 mmol) with a reaction time of 6 h. The crude product was
purified by chromatography on silica gel (CH2Cl2/MeOH 95/5) and
then treated to eliminate remaining DMSO according to general
procedure 4.1.4 to provide the expected compound as a light brown
C), 1535 (NO2 as), 1329 (NO2 ss), 1240 (AreO), 1130 (CeO) cmꢃ1; MS
(ESI(þ)): m/z 427 [M þ Na]þ. C19H20N2O8: C: 56.43; H: 4.99; N: 6.93.
Found C: 56.54; H: 5.01; N: 7.02.
4.1.3.3. 1-(3-Fluoro-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-
2-nitropropene (10). General procedure 4.1.3. was performed with
3-fluoro-4-methoxybenzaldehyde (2.50 g, 16.2 mmol) and
5
foamy solid (0.96 g, 2.60 mmol, 96%). Mp: 153e154 ꢀC (heptane/
benzene); 1H NMR (300 MHz, CDCl3):
d
¼ 3.76 (s, 6H, OCH3eC3 ,
0
(3.50 g, 14.6 mmol) with a reaction time of 24 h. The crude product
was purified by chromatography on silica gel (CH2Cl2/acetone 99/1)
to provide the corresponding 1,2-diarylnitroethene as an orange
solid, which was afterward recrystallized to afford bright ocher-
colored fine crystals (1.77 g, 4.7 mmol, 32%). Mp: 126e128 ꢀC
(heptane/benzene); 1H NMR (300 MHz, CDCl3): 0d ¼ 3.80 (s, 6H,
0
0
OCH3eC5 ), 3.81 (s, 3H, OCH3eC4 ), 4.14 (s, 2H, CH2eAr), 5.99
(s, 2H, CH2eO), 6.39 (s, 2H, H-20, H-60), 6.85 (d, J ¼ 7.7 Hz, 1H, H-700),
7.06 (d, J ¼ 2.1 Hz, 1H, H-400), 7.09 (dd, J1 ¼ 7.7 Hz, J2 ¼ 2.1 Hz, 1H, H-
600); 13C NMR (75 0MHz, CDCl3):
d
¼ 31.3 (CH2, C0H2eAr), 55.9
0
(CH3 ꢁ 2, OCH3eC3 , OCH3eC5 ), 60.9 (CH3, OCH3eC4 ), 101.3 (CH2,
CH2eO), 105.5 (CH ꢁ 2, C-20, C-60), 108.3 (CH, C-400), 108.6 (CH, C-700),
121.6 (CH, C-600), 124.0 (C, C-500), 134.1 (C, C-10), 136.4 (C, C-5), 142.0
(C, C-4), 147.7 (C, C-300a), 147.9 (C, C-700a), 153.2 (C ꢁ 3, C-30, C-40,
C-50); IR (NaCl): y0 3009 (NH), 1592 (C]C), 1240 (AreO), 1130 (CeO)
cmꢃ1; HRMS (ESI): m/z 370.1414 [M þ H]þ, C19H20N3O5 requires
370.1403.
0
0
OCH3eC3 , OCH3eC5 ), 3.83 (s, 3H, OCH3eC4 ), 3.93 (s, 3H,
OCH3eC400), 4.22 (s, 2H, CH2eAr), 6.39 (s, 2H, H-20, H-60), 6.98e7.03
(m, 1H, H-500), 7.23e7.30 (m, 2H, H-200, H-600), 8.23 (s, 1H, ¼CH-2);
13C NMR 0(75 MHz, CDCl3):
104.5 (CH ꢁ 2, C-20, C-60), 113.5 (CH, J ¼ 7.2 Hz, C-500), 117.4 (CH,
d
¼ 33.1 (CH2, 0CH2eAr), 56.1 (CH3 ꢁ 2,
0
OCH3eC3 , OCH3eC5 ), 56.3 (CH3, OCH3eC4 ), 60.8 (CH3, OCH3eC400),

30
N. Mur Blanch et al. / European Journal of Medicinal Chemistry 54 (2012) 22e32
4.1.4.3. 5-(2,4-Dichlorophenyl)-4-(3,4,5-trimethoxybenzyl)-1,2,3-tria-
zole (16). General procedure 4.1.4. was applied to 2-(2,4-
was purified by chromatography on silica gel (CH2Cl2/MeOH 95/5)
and then treated to eliminate remaining DMSO according to
general procedure 4 to provide the expected compound as
a whitish foamy solid (0.94 g, 2.53 mmol, 84%). Mp: 82e83 ꢀC
(heptane/benzene); 1H NMR (300 MHz, CDCl3): 0d ¼ 3.75 (s, 6H,
dichlorophenyl)-1-(3,4,5-trimethoxybenzyl)-1-nitroethene
(8)
(1.00 g, 2.5 mmol) with a reaction time of 12 h. The crude product
was purified by chromatography on silica gel (CH2Cl2/MeOH 95/5)
and then treated to eliminate remaining DMSO according to general
procedure 4 to provide the expected compound as an orange solid
0
0
OCH3eC3 , OCH3eC5 ), 3.79 (s, 3H, OCH3eC4 ), 3.90 (s, 3H,
OCH3eC400), 4.17 (s, 2H, CH2), 5.35 (br. s,1H, OH), 6.43 (s, 2H, H-20, H-
60); 6.88 (d, J ¼ 8.3 Hz, 1H, H-500), 7.11 (dd, J1 ¼8.3 Hz, J2 ¼ 1.2 Hz, 1H,
(0.96 g, 2.43 mmol, 97%). Mp: 127e128 ꢀC (hept0ane/benzene); 1H
NMR (300 MHz,0CDCl3):
d
¼ 3.74 (s, 6H, OCH3eC3 , OCH3eC5 ), 3.76
H-600), 7.22 (br. s, 1H, H-200); 13C NMR (75 MHz, CDCl3):
d
¼ 320.0
0
(s, 3H, OCH3eC4 ), 3.98 (s, 2H, CH2), 6.25 (s, 2H, H-20, H-60), 7.18 (d,
(CH2, CH2eAr), 56.5 (CH3 ꢁ 3, OCH3eC3 , OCH3eC4 , OCH3eC5 ),
60.8 (CH3, OCH3eC400), 106.4 (CH ꢁ 2, C-20, C-60), 113.2 (CH, C-500),
115.4 (CH, C-200),119.1 (CH, C-600),124.8 (C, C-5),135.5 (C, C-10),136.6
(C, C-100), 142.7 (C, C-300), 144.6 (C, C-4), 147.5 (C, C-400), 153.6 (C ꢁ 2,
C-30, C-50); IR (NaCl): y0 3541 (OH), 3009 (NH), 1590 (C]C), 1239
(AreO), 1129 (CeO) cmꢃ1; HRMS (ESI): m/z 372.1578 [M þ H]þ,
C19H22N3O5 requires 372.1559.
0
0
J ¼ 9.1 Hz, 1H, H-600), 7.25 (dd, J1 ¼ 9.1 Hz, J2 ¼ 1.8 Hz, 1H, H-500), 7.48
(d, J ¼ 1.8 Hz, 1H, H-300); 13C NMR (75 MHz, CDCl3):0 d ¼ 31.2 (CH2,
0
CH2eAr),0 56.8 (CH3
ꢁ
2, OCH3eC3 , OCH3eC5 ), 60.9 (CH3,
OCH3eC4 ), 105.9 (CH ꢁ 2, C-20, C-60), 127.1 (CH, C-300), 128.3 (C, C-5),
129.6 (CH, C-600), 132.7 (CH, C-500), 133.6 (C, C-4), 134.7 (C, C-100),
135.5 (C, C-200), 136.3 (C, C-400), 152.0 (C ꢁ 3, C-30, C-40, C-50); IR
(NaCl): y0 3009 (NH), 1593 (C]C), 1239 (AreO), 1121 (CeO), 754
35
(CeCl) cmꢃ1; HRMS (ESI): m/z 370.1414 [M þ H]þ, C18
H
Cl2N3O3
4.1.4.7. 4-(3,4,5-trimethoxybenzyl)-5-(4-Hydroxy-3-methoxyphenyl)-
1,2,3-triazole (20). General procedure 4.1.4. was applied to 12 (1.13 g,
3.0 mmol) with a reaction time of 14 h. The crude product was
purified by chromatography on silica gel (CH2Cl2/MeOH 95/5) and
then treated to eliminate remaining DMSO according to general
procedure 4 to provide the expected compound as a whitish foamy
solid (0.91 g, 2.45 mmol, 81%). Mp: 91e93 ꢀC (heptane/benzene); 1H
18
requires 394.0730.
4.1.4.4. 5-(4-Methoxy-3-nitrophenyl)-4-(3,4,5-trimethoxybenzyl)-
1,2,3-triazole (17). General procedure 4.1.4. was applied to 9 (1.68 g,
4.15 mmol) with a reaction time of 6 h. The crude product was
purified by chromatography on silica gel, using a gradient of MeOH
in CH2Cl2 from 99/1 to 95/5 as eluent, and then treated to eliminate
remaining DMSO according to general procedure 4 to provide the
expected compound as a yellow foamy solid, which was afterward
recrystallized to afford bright orange fine crystals (0.60 g, 1.5 mmol,
36%). Mp: 165e166 ꢀC (benzene/acetonitrile); 1H NMR (300 MHz,
0
0
NMR (300 MHz, CDCl3):
d
¼ 3.68 (s, 6H, OCH3eC3 , OCH3eC5 ), 3.69
(s, 3H, OCH3eC4 ), 3.77 (s, 3H, OCH3eC400), 4.14 (s, 2H, CH2), 5.77
(br. s, 1H, OH), 6.36 (s, 2H, H-20, H-60); 6.90 (d, J ¼ 8.0 Hz, 1H, H-500),
7.02 (d, J ¼ 1.7 Hz, 1H, H-200), 7.22 (dd, J1 ¼ 8.0 Hz, J2 ¼ 1.7 Hz, 1H,
0
H-600); 13C NMR (75 MHz, CDCl3):
(CH3, OCH3eC4)’ 56.4 (CH3
d
¼ 31.7 (CH2, CH2eA0r), 56.2
0
0
0
CDCl3):
d
¼ 3.80 (s, 6H, OCH3eC3 , OCH3eC5 ), 3.82 (s, 3H,
ꢁ
2, OCH3eC3 , OCH3eC5 ), 61.3
OCH3eC4 ), 4.00 (s, 3H, OCH3eC400), 4.19 (s, 2H, CH2eAr), 6.44 (s, 2H,
(CH3, OCH3eC300), 105.8 (CH ꢁ 2, C-20, C-60), 110.9 (CH, C-200), 115.2
(CH, C-500), 121.2 (CH, C-600), 122.4 (C, C-5), 134.7 (C, C-100), 136.7
(C ꢁ 2, C-10, C-40), 146.5 (C, C-400), 147.4 (C, C-4), 153.6 (C ꢁ 3, C-30,
C-50, C-300); IR (NaCl): y0 3540 (OH), 3440 (NH), 1593 (C]C), 1240
(AreO), 1130 (CeO) cmꢃ1; MS (ESI(þ)): m/z 394 [M þ Na]þ. HRMS
(ESI): m/z 372.1574 [M þ H]þ, C19H22N3O5 requires 372.1559.
0
H-20, H-60), 7.14 (d, J ¼ 9.4 Hz, 1H, H-500), 7.88 (dd, J1 ¼ 9.4 Hz,
J2 ¼ 1.5 Hz, 1H, H-600), 8.13 (d, J ¼ 1.5 Hz, 1H, H-200); 13C NMR
0
(75 MHz,0CDCl3):
d
¼ 31.6 (CH2, CH2eAr), 56.1 (CH3 ꢁ 2, OCH3eC3 ,
OCH3eC5 ), 56.7 (CH3, OCH3eC4 ), 60.9 (CH3, OCH3eC400), 105.5
(CH ꢁ 2, C-20, C-60), 113.9 (CH, C-500), 123.3 (C, C-100), 124.6 (CH,
C-200), 132.9 (C, C-5), 133.2 (CH, C-600 þ C, C-10), 136.8 (C, C-300), 139.5
(C, C-4), 152.9 (C, C-400, 153.5 (C ꢁ 3, C-30, C-40, C-50); IR (NaCl): y0
3017 (NH), 1592 (C]C), 1508 (NO2 as), 1353 (NO2 ss), 1239 (AreO),
1129 (CeO) cmꢃ1; HRMS (ESI): m/z 401.1470 [M þ H]þ, C19H21N4O6
requires 401.1461.
0
4.1.4.8. 4-(3-Indolemethyl)-5-(3,4,5-trimethoxyphenyl)-1,2,3-triazole
(21). General procedure 4.1.4. was applied to 1-(3-indolemethyl)-2-
(3,4,5-trimethoxybenzyl)-1-nitroethene (13) (1.00 g, 2.70 mmol)
with a reaction time of 12 h. The crude product was purified by
chromatographyon silicagel(CH2Cl2/MeOH95/5) and thentreated to
eliminate remaining DMSO according to general procedure 4 to
provide the expected compound as a brown solid (0.92 g, 2.52 mmol,
93%). Mp: 122e123 ꢀC (benzene/acetone); 1HNMR (300 MHz, CDCl3):
4.1.4.5. 5-(3-Fluoro-4-methoxyphenyl)-4-(3,4,5-trimethoxybenzyl)-
1,2,3-triazole (18). General procedure 4.1.4. was applied to 10
(1.00 g, 2.63 mmol) with a reaction time of 12 h. The crude product
was then purified by chromatography on silica gel (CH2Cl2/MeOH
99.5/0.5) and then treated to eliminate remaining DMSO according
to general procedure 4 to provide the expected compound as a light
0
0
0
d
¼ 3.52 (s, 6H, OCH3eC3 , OCH3eC5 ), 3.83 (s, 3H, OCH3eC4 ), 4.28 (s,
2H, CH2), 6.67 (s, 1H, H-200), 6.82 (s, 2H, H-20, H-60), 7.12 (t, J ¼ 7.4 Hz,
1H, H-600), 7.18(t, J¼ 7.4 Hz, 1H, H-500), 7.29(d, J¼ 8.0Hz,1H, H-700), 7.52
(d, J ¼ 8.2 Hz, 1H, H-400), 8.17 (s, 1H, NH-indole); 13C NMR (75 MHz,
brown solid (0.53 g, 1.42 mmol, 54%). Mp: 115e117 ꢀC (heptane/
benzene); 1H NMR (300 MHz, CD0Cl3):
d
¼ 3.74 (s, 6H, OCH3eC3 ,
DMSO): d
¼ 22.6 (CH2, CH2eAr), 55.9 (CH3 ꢁ 2, OCH3eC3 , OCH3eC5 ),
0
0
0
OCH3eC5 ), 3.79 (s, 3H, OCH3eC4 ), 3.89 (s, 3H, OCH3eC400), 4.14
60.5 (CH3, OCH3eC4 ), 104.8 (CH ꢁ 2, C-20, C-60), 111.9 (C, C-300), 112.4
(C, C-300a),118.8 (CH ꢁ 2, C-500, C-600),121.8 (CH, C-200),123.6 (CH, C-400),
127.2 (CH, C-700),133.0(C, C-10),136.7(C,C-5),142.8(C, C-700a),143.8(C,
0
0
(s, 2H, CH2eAr), 6.38 (s, 2H, H-20, H-60), 6.94e6.99 (m, 1H, H-500),
7.30e7.39 (m, 2H, H-200, H-600); 13C NMR (75 MHz, CDCl3):
d
¼ 31.4
0
0
(CH2, CH2eAr), 56.0 (CH3 ꢁ 2, OCH3eC3 , OCH3eC5 ), 56.3 (CH3,
C-4),153.7(Cꢁ3,C-30, C-40, C-50);IR(NaCl):
y
0 3478(NH-indole), 3009
OCH3eC4 ), 60.8 (CH3, OCH3eC400), 105.5 (CH ꢁ 2, C-20, C-60), 113.5
(NH), 1589 (C]C), 1239 (AreO), 1128 (CeO) cmꢃ1; HRMS (ESI): m/z
365.1625 [M þ H]þ, C20H21N4O3 requires 365.1614.
0
(CH, J ¼ 6.9 Hz, C-500), 115.5 (CH, J ¼ 20.3 Hz, C-200), 123.4
(C, J ¼ 6.9 Hz, C-100), 123.6 (CH, J ¼ 3.7 Hz, C-600), 133.7 (C, C-10),136.6
(C ꢁ 2, C-4, C-5), 147.7 (C, J ¼ 20.3 Hz, C-400), 152.3 (C, J ¼ 244.1 Hz,
C-300), 153.3 (C ꢁ 3, C-30, C-40, C-50); IR (NaCl): y0 3011 (NH), 1591
(C]C), 1239 (AreO), 1130 (CeO) cmꢃ1; HRMS (ESI): m/z 374.1527
[M þ H]þ, C19H21FN3O4 requires 374.1516.
4.1.5. 5-(3-Amino-4-methoxyphenyl)-4-(3,4,5-trimethoxybenzyl)-
1,2,3-triazole (22)
Compound 22 was obtained by reduction of the nitro group in 17
(0.43 g, 1.07 mmol) via catalytic hydrogenation with a reaction time
of 1 h, using 10% w/w of Pd/C as a catalyst and THF as the solvent.
The reaction mixture was filtered over a Celite surface to eliminate
the Pd/C and thoroughly rinsed with acetone. The solvents were
then evaporated under vacuum and the crude product was purified
4.1.4.6. 4-(3,4,5-Trimethoxybenzyl)-5-(3-hydroxy-4-methoxyphenyl)-
1,2,3-triazole (19). General procedure 4.1.4. was applied to 11
(1.13 g, 3.0 mmol) with a reaction time of 12 h. The crude product

N. Mur Blanch et al. / European Journal of Medicinal Chemistry 54 (2012) 22e32
31
by chromatography on silica gel (CH2Cl2/MeOH 96/4) to provide the
expected compound as a bright white foamy solid (0.35 g,
0.94 mmol, 88%). Mp: 112e113 ꢀC (benzene/acetonitrile); 1H NMR
polymerization is the concentration at which the extent of inhibition
of polymerization is 50% of the maximum value as determined from
the semi-logarithmic plot of percent inhibition as a function of the
logarithm of drug concentration fitted to a sigmoidal model with
variable slope using the nonlinear regression program SigmaPlot
(Jandel Scientific). In these conditions the IC50 value for colchicine
0
0
(300 MHz, CDCl3):
d
¼ 3.74 (s, 6H,0OCH3eC3 , OCH3eC5 ), 3.80 (s, 3H,
OCH3eC400), 3.86 (s, 3H, OCH3eC4 ), 4.15 (s, 2H, CH2eAr), 6.40 (s, 2H,
H-20, H-60), 6.79 (d, J ¼ 8.0 Hz, 1H, H-500), 6.92 (dd, J1 ¼ 8.0 Hz,
J2 ¼ 2.0 Hz, 1H, H-600), 6.98 (d, J ¼ 2.0 Hz, 1H, H-200); 13C NMR
inhibition of tubulin polymerization was 0.36 mM.
(75 MHz, CDCl3):
d
¼ 31.5 (CH2, CH2eAr), 55.6 (CH3, OCH3eC400),
0
0
0
56.2 (CH3 ꢁ 2, OCH3eC3 , OCH3eC5 ), 61.0 (CH3, OCH3eC4 ), 105.5
(CH ꢁ 2, C-20, C-60), 110.5 (CH, C-500), 114.4 (CH, C-200), 118.1 (CH,
C-600), 122.8 (C, C-100), 134.4 (C, C-10), 136.5 (C ꢁ 2, C-4, C-5), 143.1
(C, C-300), 147.7 (C, C-400), 153.2 (C ꢁ 3, C-30, C-40, C-50); IR (NaCl): y0
3007 (NH), 1592 (C]C), 1240 (AreO), 1129 (CeO) cmꢃ1; HRMS
(ESI): m/z 371.1729 [M þ H]þ, C19H22N3O5 requires 371.1719.
4.2.3. Cytotoxicity assay
Murine B16 melanoma cells were grown in DMEM medium
containing 2 mM -glutamine, 10% fetal bovine serum, 100 U/mL
penicillin and 100
g/mL streptomycin (37 ꢀC, 5% CO2). All
L
m
compounds were initially dissolved in DMSO at a stock concen-
tration of 2.5 mg/mL and were further diluted in cell culture
medium. Exponentially growing cells were plated onto 96-well
4.2. Biological evaluation procedures
plates at 5000 cells per well in 100
ml of culture medium. Twenty-
four hours after plating, 100
ml of medium containing the
4.2.1. Endothelial cell morphology
compound at final concentrations ranging from 0.01 to 100 mM
To assess the effects of the compounds on the morphology of
endothelial cells, we used the EAhy 926 endothelial cell line which
is derived from the fusion of human umbilical vein endothelial cells
(HUVEC) with the permanent human cell line A549 [55]. The EAhy
926 cell line is considered as one of the best immortalized HUVEC
cell lines because these cells express most of the biochemical
markers of parental HUVEC EAhy 926 cells [56]. EAhy 926 cells
were added to the wells (in triplicate) containing the cells, and
incubated for 48 h at 37 ꢀC and 5% CO2. After the 48 h exposure
period to the test compounds, cell viability was assayed using the
MTT test [60] and absorbance was read at 562 nm in a microplate
reader (BioKinetics Reader, EL340). Appropriate controls with
DMEM only and MTT were run to subtract background absorbance.
The concentration of compound that inhibited cell viability by 50%
(inhibitory concentration for 50% of cells, or IC50) was determined
using the GraphPad Prism software.
were grown in DMEM containing 2 mM
bovine serum, 100 U/mL penicillin and 100
(37 ꢀC, 5% CO2). Exponentially growing EAhy 926 cells were plated
onto 96 well plates at 5000 cells/100 L/well. Twenty-four hours
after plating, the medium was aspirated, and 100 L of medium
L-glutamine, 10% fetal
mg/mL streptomycin
m
4.2.4. Docking protocol
m
Our previous docking work on analogs of Combretastatin A4
[47] implied GOLD (Genetic Optimisation for Ligand Docking), so
we performed our study with this software in its new version [50].
containing the test compound was added to the well containing the
cells (in triplicate) and incubated for 2 h (37 ꢀC, 5% CO2). The highest
concentration used was 100
mM followed by serial two-fold
dilutions down to low nanomolar concentrations, if needed for
the most morphologically active compounds. After the 2 h-incu-
bation period, digital photographs were taken of representative
centre areas of each well at a magnification of 100ꢁ and 200ꢁ. The
results are presented as the minimum concentration that could
elicit the rounding up of more than 15% of cells in a field in order to
exclude normal mitotic cells which is less that 15% in control 1%
DMSO treated cells. Triplicate concentrations on the same plate
were routinely carried out. CA4 was included in the experiments as
internal standard.
4.2.4.1. Ligands preparation. Three compounds, CA4, 11 and 19
were initially built within MarvinSketch 5.8.0 from Chemaxon [61]
next submitted to Corina [62], a 3D structure generator. Obtained
3D conformations were next submitted to Ligprep from the
Schrödinger software suite [63] in order to generate tautomers
forms. Regarding a pH ¼ 7.0 ꢂ 2.0 three forms were found for 19
where H atom can be bound to the three N atoms of the triazole
ring. Due to the high level of flexibility of the previously structures
conformational analyses, according to the OPLS2005 force field
[64], were computed and the lowest energy conformations were
kept as input for the docking stage.
4.2.2. Inhibition of tubulin polymerization
Tubulin microtubule assembly in microtubules was carried out
using the fluorescent dye DAPI (40,6-diamidino-2-phenylindole) [57]
in a 96-well plate format as described [58,59]. The standard assay
was performed as follows: wells were charged with tubulin (Cyto-
skeleton, 97% pure, final concentration 1 mg/ml) in PME buffer
(100 mM PIPES (1,4-piperazinebis(ethanesulfonic acid); 1 mM
4.2.4.2. Protein preparation. 3D structure of tubulin was retrieved
from the Protein Data Bank (http://www.rcsb.org, PDB ID: 1SA0)
[46]. Subunits C, D and E were removed. Only subunits A, B, corre-
sponding to the colchicine binding site, and small molecules DAMA-
colchicine (CN2), GTP and Mg2þ ion in this site were conserved.
MgSO4; 2 mM EGTA) with 10
the test compounds using colchicine at 30
control. The final concentrations used for the test compounds were
started at 30 M and diluted in 3-fold decrements thereafter until no
inhibition was observed. Triplicate wells were run for each concen-
tration. After a preincubation of 45 min at room temperature, 5 l of
m
M DAPI and varying concentrations of
4.2.4.3. Molecular docking. Docking runs were performed with an
exploration of the conformational space of ligands more important
than with default parameters in that sense that 100 conformations
were kept for each ligand and early termination process (run will
stop after 3 conformations found within an RMSD of 1.5 Å) was
disabled. Goldscore was chosen again as our fitness scoring function.
mM as an internal positive
m
m
1 mM GTP was added to each well to initiate tubulin polymerization,
and the plate was then transferred to a thermostated Victor plate
reader at 37 ꢀC for an additional 2 h. Fluorescence was then read at
the excitation wavelength of 360 nm and emission of 450 nm.
4.2.5. Antitumor evaluation in mice
Female BALB/cJRj mice (average weight, 18 g) obtained from
Janvier (Le Genest St Isle, France) were supplied food and water ad
libitum. Colon 26 tumor fragments (30e60 mg) were implanted
bilaterally in the axillary region by subcutaneous injection on day
0 with a 12 gauge trocar [65]. Bilateral implants were used to insure
a more uniform tumor burden per mouse. The methods of protocol
The percent inhibition was determined as follows: 1 ꢃ (
F(control) ꢁ 100, where F control ¼ F(no inhibition) ꢃ F(complete
inhibition), and
F sample ¼ F(sample) ꢃ F(complete inhibition with
colchicine). The IC50 for compound-induced inhibition of tubulin
DF(sample/
D
D
D

32
N. Mur Blanch et al. / European Journal of Medicinal Chemistry 54 (2012) 22e32
[24] A. Cirla, J. Mann, J. Nat. Prod. Rep. 20 (2003) 558e564.
[25] K.G. Pinney, C. Jelinek, K. Edvardsen, D.J. Chaplin, G.R. Pettit, in: G.M. Cragg,
D.G.I. Kingston, D.J. Newman (Eds.), Anticancer Agents from Natural Products,
Taylor & Francis Group, Boca Raton, 2005, pp. 23e46.
[26] H.P. Hsieh, J.P. Liou, N. Mahindroo, Curr. Pharm. Design 11 (2005) 1655e1677.
[27] T. Brown, H. Holt Jr., M. Lee, in: Top Heterocycl. Chem, Vol. 2, Springer-Verlag,
Berlin Heidelberg, 2006, pp. 1e51.
design, chemotherapy techniques, and data analysis have been
presented in detail elsewhere [66,67]. Briefly, the animals were
pooled before distribution to the various treatment and control
groups (5 mice per group) and preliminary experiments in non
tumored animals allowed to determine the maximum tolerated
dose which was 150 mg/kg for 11 and 100 mg/kg for 19, when
administered intraperitoneally for two weeks on days 3e7 and
10e14 after tumor implantation (day 0). Compounds were freshly
prepared daily in saline containing 5% DMSO and 5% polysorbate 80
and injected intraperitoneally in a volume of 0.2 ml. Mice were
weighed daily and checked for clinical signs of toxicity. Tumors
were measured daily with a caliper and tumor volumes (mm3)
were estimated from two dimensional tumor measurements (mm):
Tumor volume (mm3) ¼ (length ꢁ width2 ꢁ 0.5). The end points
used to determine the antitumor activity were the percent T/C
value and the tumor growth delay (T-C value). The %T/C was
calculated when the tumor volume reached 1500 mm3 for the
controls (C). The T/C value in percent is calculated as follows: T/C
(%) ¼ (median tumor weight of the Treated/median tumor weight
of the Control) ꢁ 100. According to the National Cancer Institute
(NCI) standards, a T/C of 42% is the minimum level for activity. The
tumor growth delay (TeC value) was calculated based on the
median time (in days) required for the treatment group (T) and the
control group (C) tumors, to reach the size of 1500 mm3.
[28] G.C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Busacca, A. Genazzani, J. Med. Chem.
49 (2006) 3033e3044.
[29] A. Brancale, R. Silvestri, Med. Res. Rev. 27 (2007) 209e238.
[30] Anurag Chaudhary, S.N. Pandeya, P. Kumar, P.P. Sharma, S. Gupta, N. Soni,
K.K. Verma, G. Bhardwaj, Mini Rev. Med. Chem. 7 (2007) 1186e1205.
[31] Y. Shan, J. Zhang, Z. Liu, M. Wang, Y. Dong, Curr. Med. Chem. 18 (2011)
523e538.
[32] M. Marrelli, F. Conforti, G.A. Statti, X. Cachet, S. Michel, F. Tillequin,
F. Menichini, Curr. Med. Chem. 18 (2011) 3035e3081.
[33] K. Ohsumi, T. Hatanaka, K. Fujita, R. Nakagawa, Y. Fukuda, Y. Nihei, Y. Suga,
Y. Morinaga, Y. Akiyama, T. Tsuji, Bioorg. Med. Chem. Lett.
8 (1998)
3153e3158.
[34] Q. Zhang, Y. Peng, X.I. Wang, S.M. Keenan, S. Arora, W.J. Welsh, J. Med. Chem.
50 (2007) 749e754.
[35] F. Pagliai, T. Pirali, E. Del Grosso, R. Di Brisco, G.C. Tron, G. Sorba, A. Genazzani,
J. Med. Chem. 49 (2006) 467e470.
[36] K. Odlo, J. Hentzen, J. Fournier dit Chabert, S. Ducki, O.A.B.S.M. Gani, I. Sylte,
M. Skrede, V.A. Flørenes, T.V. Hansen, Bioorg. Med. Chem. 16 (2008)
4829e4838.
[37] K. Odlo, J. Fournier dit Chabert, S. Ducki, O.A.B.S.M. Gani, I. Sylte, T.V. Hansen,
Bioorg. Med. Chem. 18 (2010) 6874e6885.
[38] Ø.W. Akselsen, K. Odlo, J.-J. Cheng, G. Maccari, M. Botta, T.V. Hansen, Bioorg.
Med. Chem. 20 (2012) 234e242.
[39] L. Cafici, T. Pirali, F. Condorelli, E. Del Grosso, A. Massarotti, G. Sorba,
P.L. Canonico, G.C. Tron, A.J. Genazzani, J. Comb. Chem. 10 (2008) 732e740.
[40] Z. Demko, C. Borella, S. Chen, L. Sun, U.S. Patent 2007238699 A1, oct. 11, 2007.
[41] H.N. Pati, M. Wicks, H.L. Holt Jr., R. LeBlanc, P. Weisbruch, L. Forrest, M. Lee,
Heterocycl. Commun. 11 (2005) 117e120.
Acknowledgments
[42] O. Mezenzani, A. Massarotti, M. Giustiniano, T. Pirali, V. Bevilacqua,
A. Caldarelli, P. Canonico, G. Sorba, E. Novellino, A.A. Genazzani, G.C. Tron,
Bioorg. Med. Chem. Lett. 21 (2011) 764e768.
[43] D. Dauzonne, R. Royer, Chem. Pharm. Bull. 34 (1986) 1628e1633.
[44] B. Quiclet-Sire, S.Z. Zard, Synthesis (2005) 3316e3326.
[45] S.M. Galbraith, D.J. Chaplin, F. Lee, M.R. Stratford, R.J. Locke, B. Vojnovic,
G.M. Tozer, Anticancer Res. 21 (2001) 93e102.
This work was financially supported by the Centre National de la
Recherche Scientifique (CNRS), the Institut National de la Santé et
de la Recherche Médicale (Inserm), the Institut Curie and by a grant
from the Institut National du Cancer (INCa, Boulogne Billancourt,
France). We are also grateful to the University of Barcelone for
a studentship (to Núria Mur Blanch).
[46] R.B.G. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel,
M. Knossow, Nature 428 (2004) 198e202.
[47] M. Arthuis, R. Pontikis, G.G. Chabot, J. Seguin, L. Quentin, S. Bourg, L. Morin-
Allory, J.-C. Florent, Chem. Med. Chem. 6 (2011) 1693e1705.
[48] M. Botta, S. Forli, M. Magnani, F. Manetti, Top. Curr. Chem. 286 (2009)
279e328.
[49] A. Massaroti, A. Coluccia, R. Silvestri, G. Sorba, A. Brancale, Chem. Med. Chem.
7 (2012) 33e42.
[50] G. Jones, P. Willet, R.C. Glen, J. Mol. Biol. 245 (1995) 43e53.
[51] R. Álvarez, C. Álvarez, F. Mollinedo, B.G. Sierra, M. Medarde, R. Peláez, Bioorg.
Med. Chem. 17 (2009) 6422e6431.
[52] T.H. Corbett, M.C. Bissery, A. Wozniak, J. Plowman, L. Polin, E. Tapazoglou,
J. Dieckman, F. Valeriote, Invest. New Drugs 4 (1986) 207e220.
[53] A.K. Sinhababu, R.T. Bordchardt, Tetrahedron Lett. 24 (1983) 227e230.
[54] D. Dauzonne, P. Demerseman, Synthesis (1990) 66e70.
[55] C.J. Edgell, C.C. McDonald, J.B. Graham, Proc. Natl. Acad. Sci. U. S. A. 80 (1983)
3734e3737.
[56] D. Bouis, G.A. Hospers, C. Meijer, G. Molema, N.H. Mulder, Angiogenesis 4
(2001) 91e102.
[57] D. Bonne, C. Heusele, C. Simon, D. Pantaloni, J. Biol. Chem. 260 (1985)
2819e2825.
[58] D.M. Barron, S.K. Chatterjee, R. Ravindra, R. Roof, E. Baloglu, D.G. Kingston,
S. Bane, Anal. Biochem. 315 (2003) 49e56.
[59] S.L. Bane, R. Ravindra, A.A. Zaydman, in: J. Zhou (Ed.), Microtubule Protocols,
Humana Press, Totowa, New Jersey, 2007, pp. 281e288.
[60] D.A. Scudiero, R.H. Shoemaker, K.D. Paull, A. Monks, S. Tierney, T.H. Nofziger,
M.J. Currens, D. Seniff, M.R. Boyd, Cancer Res. 48 (1988) 4827e4833.
[61] Marvin 5.8.0 (2012).http://www.chemaxon.com.
[62] (a) J. Sadowski, J. Gasteiger, Chem. Rev. 93 (1993) 2567e2581;
(b) CORINA e Fast Generation of High-Quality 3D Molecular Models: http://
www.molecular-networks.com/products/corina.
References
[1] D.W. Siemann, D.J. Chaplin, M.R. Horsman, Cancer 100 (2004) 2491e2499.
[2] P.E. Thorpe, Clin. Cancer Res. 10 (2004) 415e427.
[3] D. Neri, R. Bicknell, Nat. Rev. Cancer 5 (2005) 436e446.
[4] D.J. Chaplin, M.R. Horsman, D.W. Siemann, Curr. Opin. Investig. Drugs 7 (2006)
522e528.
[5] C. Kanthou, G.M. Tozer, Expert Opin. Ther. Targets 11 (2007) 1443e1457.
[6] C.A. Honstvet, P.F. Jones, Comput. Math. Methods Med. 8 (2007) 1e9.
[7] C. Kanthou, G.M. Tozer, Int. J. Exp. Pathol. 90 (2009) 284e294.
[8] V.L. Heath, R. Bicknell, Nat. Rev. Clin. Oncol. 6 (2009) 395e404.
[9] M. Ham Fens, G. Storm, R.M. Schiffelers, Expert Opin. Ther. Targets 19 (2010)
1321e1338.
[10] L.G.M. Daenen, J.M.L. Roodhart, Y. Shaked, E.E. Voest, Curr. Clin. Pharmacol. 5
(2010) 178.
[11] G.M. Tozer, C. Kanthou, C.S. Parkins, S.A. Hill, Int. J. Exp. Pathol. 83 (2002)
21e38.
[12] G.M. Tozer, C. Kanthou, B.C. Baguley, Nat. Rev. Cancer 5 (2005) 423e435.
[13] D.W. Siemann, M.C. Bibby, G.G. Dark, A.P. Dicker, F.A. Eskens, M.R. Horsman,
D. Marmé, P.M. Lorusso, Clin. Cancer Res. 11 (2005) 416e420.
[14] R.S. Kerbel, N. Engl. J. Med. 358 (2008) 2039e2049.
[15] D.W. Siemann, Cancer Treat. Rev. 37 (2011) 63e74.
[16] R.P. Mason, D. Zhao, L. Liu, M.L. Trawick, K.G. Pinney, Integr. Biol. 3 (2011)
375e387.
[17] G.R. Pettit, G.M. Cragg, D.L. Herald, J.M. Schmidt, P. Lohavani-Jaya, Can. J.
Chem. 60 (1982) 1374e1376.
[18] G.R. Pettit, G.M. Cragg, S.B. Singh, J. Nat. Prod. 50 (1987) 386.
[19] G.R. Pettit, S.B. Singh, E. Hamel, C.M. Lin, D.S. Alberts, D. Garcia-Kendall,
Experientia 45 (1989) 209e211.
[63] Schrödinger Suite, Version 2011 Update 1, Schrödinger, Inc., New York, 2012.
[64] G.A. Kaminski, R.A. Friesner, J. Tirado-Rives, W. Jorgensen, J. Phys. Chem. B 105
(2001) 6474e6487.
[20] C.M. Lin, S.B. Singh, P.S. Chu, R.O. Dempey, J.M. Schmidt, G.R. Pettit, E. Hamel,
Mol. Pharmacol. 34 (1988) 200e208.
[65] T.H. Corbett, D.P. Griswold Jr., B.J. Roberts, J.C. Peckham, F.M. Schabel Jr.,
Cancer 40 (1977) 2660e2680.
[66] T.H. Corbett, W.R. Leopold, D.J. Dykes, B.J. Roberts, D.P. Griswold Jr.,
F.M. Schabel Jr., Cancer Res. 42 (1982) 1707e1715.
[67] M.C. Bissery, P. Vrignaud, F. Lavelle, G.G. Chabot, Ann. N. Y. Acad. Sci. 803
(1996) 173e180.
[21] I.G. Kirwan, P.M. Loadman, D.J. Swaine, D.A. Anthoney, G.R. Pettit,
J.W. Lippert III, S.D. Shnyder, P.A. Cooper, M.C. Bibby, Clin. Cancer Res. 10
(2004) 1446e1453.
[22] S. Aprile, E. Del Grosso, G.C. Tron, G. Grosa, Drug Metab. Dispos. 35 (2007)
2252e2261.
[23] N.H. Nam, Curr. Med. Chem. 10 (2003) 1697e1722.