Synthesis and biological evaluation of enantiomerically pure cyclopropyl analogues of combretastatin A4

Article abstract of DOI:10.1016/j.bmc.2012.11.056

To evaluate the influence of stereochemistry on biological activities of cis-cyclopropyl combretastatin A4 (CA4) analogues, we have prepared several cyclopropyl compounds in their pure enantiomeric forms. The key reactions in our synthesis are the cyclopropanation of a (Z)-alkenylboron compound bearing a chiral auxiliary, and the cross-coupling of both enantiomeric cyclopropyl trifluoroborate salts with aryl and olefinic halides. Three pairs of cis-cyclopropyl CA4 analogues were evaluated for their potential antivascular activities. The diarylcyclopropyl compounds with SR-configuration (-)-1b, (-)-2b and the cyclopropylvinyl enantiomer (+)-3a with RR-configuration were the most potent tubulin polymerization inhibitors. A correlation was noted between anti-tubulin activity and rounding up activity of endothelial cells. The cytotoxic activity on B16 melanoma cells was in the submicromolar range for most compounds, but unlike the anti-tubulin activity, there was no difference in cytotoxic activity between racemic and enantiomerically pure forms for the three series of compounds. Molecular docking studies within the colchicine binding site of tubulin were in good agreement with the tubulin polymerization inhibitory data and confirmed the importance of the configuration of the synthesized cis-cyclopropyl CA4 analogues for potential antivascular activities.

Full text of DOI:10.1016/j.bmc.2012.11.056

Bioorganic & Medicinal Chemistry 21 (2013) 1357–1366
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of enantiomerically pure
cyclopropyl analogues of combretastatin A4
c,d,
Nancy Ty a,b, Renée Pontikis a,b, , Guy G. Chabot
, Emmanuelle Devillers a,b, Lionel Quentin c,d
⇑
,
⇑
Stéphane Bourg e,f, Jean-Claude Florent a,b,
⇑
a Institut Curie, Centre de Recherche, 26 rue d’Ulm, Paris F-75248 Cedex 05, France
b CNRS, UMR 176, 26 rue d’Ulm, Paris F-75248 Cedex 05, France
c Université Paris Descartes, Faculté des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l’Observatoire, 75006 Paris, France
d Laboratoire de Pharmacologie Chimique, Génétique et Imagerie, INSERM U 1022-CNRS UMR 8151, 4 Avenue de l’Observatoire, 75006 Paris, France
e Institut de Chimie Organique et Analytique (ICOA), Université d’Orléans, CNRS, UMR 7311, rue de Chartres, 45067 Orléans Cedex 2, France
f Centre de Biophysique Moléculaire, CNRS, UPR 4301, Avenue Charles Sadron, 45071 Orléans Cedex 2, France
a r t i c l e i n f o
a b s t r a c t
Article history:
To evaluate the influence of stereochemistry on biological activities of cis-cyclopropyl combretastatin A4
(CA4) analogues, we have prepared several cyclopropyl compounds in their pure enantiomeric forms. The
key reactions in our synthesis are the cyclopropanation of a (Z)-alkenylboron compound bearing a chiral
auxiliary, and the cross-coupling of both enantiomeric cyclopropyl trifluoroborate salts with aryl and ole-
finic halides. Three pairs of cis-cyclopropyl CA4 analogues were evaluated for their potential antivascular
activities. The diarylcyclopropyl compounds with SR-configuration (ꢀ)-1b, (ꢀ)-2b and the cyclopropylvi-
nyl enantiomer (+)-3a with RR-configuration were the most potent tubulin polymerization inhibitors. A
correlation was noted between anti-tubulin activity and rounding up activity of endothelial cells. The
cytotoxic activity on B16 melanoma cells was in the submicromolar range for most compounds, but
unlike the anti-tubulin activity, there was no difference in cytotoxic activity between racemic and enan-
tiomerically pure forms for the three series of compounds. Molecular docking studies within the colchi-
cine binding site of tubulin were in good agreement with the tubulin polymerization inhibitory data and
confirmed the importance of the configuration of the synthesized cis-cyclopropyl CA4 analogues for
potential antivascular activities.
Received 25 September 2012
Revised 22 November 2012
Accepted 24 November 2012
Available online 21 December 2012
Keywords:
Vascular disrupting agents
Tubulin
Combretastatin
Cyclopropyl compounds
Palladium catalyzed reactions
Ó 2013 Published by Elsevier Ltd.
1. Introduction
platinum salts in advanced solid tumors, and a phase II/III clinical
trial in patients with advanced soft tissues sarcoma.7–9
Tumor vasculature represents an attractive target for cancer
therapy, because solid tumors require blood vessels for growth
and metastasis.1 The antivascular approach exploits differences be-
tween normal and immature tumor blood vessels and aims at
selectively destroying established tumor vasculature.2 Tumor-
vascular disrupting agents (VDAs) are essentially small molecules
that bind to tubulin at the colchicine site and act as tubulin poly-
By binding to the tubulin of tumor endothelial cells, VDAs cause
rapid microtubule depolymerization. This damage triggers disrup-
tion of the cell–cell junction involving the protein vascular endo-
thelial-cadherin10 and further cytoskeletal rearrangements
(assembly of actin stress fibers) through activation of the Rho sig-
naling pathway.2a,11 The net result of these effects is a rounding up
and a membrane blebbing of tumor endothelial cells, together with
an increased vessel permeability to macromolecules, leading to a
rapid blood flow shutdown in vivo. The consequent reduction in
nutrients and oxygen supply induces central necrosis of the tumor,
with a thin rim of viable cells that can be targeted with conven-
tional anti-cancer treatments.2a,12
The encouraging antivascular and antitumor profile of CA4 has
greatly contributed to the current interest in the design and
synthesis of several CA4 analogues.13 A key structural feature for
binding to tubulin is the cis-orientation of the two aromatic rings.
However, CA4 and other olefinic analogues are prone to
isomerization into their inactive trans-forms during storage and
merization inhibitors.2c,3 Combretastatin A4 (CA4),
a natural
cis-stilbene, is one of the most studied compounds (Fig. 1).4 Its
water-soluble phosphate prodrug CA4P (fosbretabulin) is, alone
or in combination with paclitaxel or carboplatin, currently under-
going clinical trials for the treatment of patients with anaplastic
thyroid carcinoma5 or other advanced cancers.6,7 Another CA4 ana-
logue, the serine amide AVE8062 (ombrabulin) is being investi-
gated in a phase II clinical trial in combination with taxanes and
⇑
Corresponding authors. Tel.: +33 (0)1 56 24 66 58 (J.-C.F.).
E-mail address: jean-claude.florent@curie.fr (J.-C. Florent).
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2012.11.056

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N. Ty et al. / Bioorg. Med. Chem. 21 (2013) 1357–1366
Figure 1. Structures of combretastatin A4 (CA4) and its water-soluble prodrugs.
Figure 3. Retrosynthetic approach.
have selected the diol 4, developed by the Pietruszka group20–22
which is proved to be an efficient chiral auxiliary.23
administration. Various strategies have been designed to lock the
compounds in the desired conformation, particularly by the
replacement of the double bond with heterocyclic moieties.14
Although the cyclopropyl group is generally considered as an al-
kene bioisostere, only few cyclopropyl CA4 analogues have been
described so far, and furthermore in their racemic forms (Fig. 2).
Phenol ( )-1 and aniline ( )-2, reported by Fürst et al., displayed
submicromolar cytotoxicity towards HeLa and MCF-7 cells.15 For
our part, compound ( )-3 with a (cis,E)-cyclopropylvinyl moiety,
was investigated as the analogue of a (Z,E)-diarylbutadiene that
we have previously reported as a potent tubulin polymerization
inhibitor.16,17 Compound ( )-3 exhibits only moderate anti-tubulin
and antiproliferative activity which could be due to the fact that
this product is a mixture of two cis-cyclopropyl enantiomers. In-
deed, as suggested by our docking study in the colchicine binding
site, only one enantiomer superimposed well to CA4.17 Conse-
quently, it was of interest to evaluate cyclopropyl CA4 analogues
in their optically pure forms. Herein, we report our results concern-
ing the synthesis of cis-cyclopropyl CA4 analogues in their both
enantiomeric forms. We also present biological evaluations linked
to their potential vascular disrupting activities, and propose their
tubulin binding mode by molecular docking investigations.
(Z)-Vinylboronic esters could be obtained from the correspond-
ing vinylbromides using the one pot sequence24 involving a lith-
ium–halogen exchange, a transmetallation with a trialkylborate
and the direct transesterification with a diol (Scheme 1). Borylation
of the required bromide 525 was first attempted with pinacol by
using the conventional procedure, that is, t-BuLi, tri-isopropylb-
orate and pinacol successively added to the bromide. Unfortu-
nately, the reaction failed, probably due to the unstability of the
vinyllithium species. However, application of the Lautens proce-
dure,26 which involved treatment of a cooled (ꢀ78 °C) mixture of
bromide 5 and tri-isopropylborate with t-BuLi, followed by the
addition of pinacol, cleanly afforded the pinacol boronate 6, but
in a modest 51% yield. This procedure was successfully applied to
the synthesis of the chiral (Z)-vinylboronic ester 7 in a quite good
yield, but with a longer transesterification step.
The chiral vinylboronate 7 was then submitted to a Simmons–
Smith cyclopropanation (diethylzinc and diiodomethane),27 but
the reaction failed whatever the operating conditions applied.
The cyclopropanation step was conveniently achieved by the palla-
dium-catalyzed decomposition of diazomethane20,28 (25 equiv
were required for a complete reaction). Unfortunately, the cis-
cyclopropylboronate formed was obtained as a mixture of two dia-
stereoisomers in a 55:45 ratio and the diastereoselectivity of the
reaction could not be improved by decreasing the reaction temper-
ature. These diastereoisomeric boronates were further separated
by chiral phase HPLC, but their respective absolute stereochemis-
try was undetermined. Nevertheless, by analogy to Pietruska’s pre-
vious work,21 we can presume that the first eluted compound
would be the RS-isomer 8a.29 This attribution being in agreement
with the biological data and the docking study (vide infra), it was
therefore chosen to assign the chemical structure 8a to the first
eluted compound. Since the chiral center CH-a (cf. Fig. 2) remained
untouched during the synthetic sequence, compounds synthesized
from the first eluted boronate were numbered ‘a’ and those coming
from the other boronate ‘b’.
2. Results and discussion
2.1. Chemistry
Enantiomerically pure cyclopropyl derivatives synthesis18 could
be efficiently achieved by a Simmons–Smith cyclopropanation in
the presence of a chiral ligand, such as the Charette’s dioxoboro-
lane.19 With this strategy, the olefinic substrate usually requires
a hydroxyl group to enhance both reactivity and stereocontrol.
One alternative method for the preparation of such cyclopropyl
derivatives involves olefins bearing a chiral auxiliary. Depending
on the substrate, the cyclopropanation is carried out according to
the Simmons–Smith conditions or the palladium-catalyzed decom-
position of diazomethane.
To access the desired cyclopropyl analogues of CA4, we opted
for a Suzuki–Miyaura cross coupling reaction between an aryl ha-
lide and a chiral cis-cyclopropylboronic ester which could result of
the cyclopropanation of a (Z)-alkenylboron compound bearing a
chiral auxiliary (Fig. 3). For our diastereoselective approach, we
Concerning the (Z)-alkenylboronate bearing a pinacol, it was
conveniently converted to the cis-cyclopropyl derivative 9 by using
7 equiv of diazomethane (79% yield).
According to Pietruszka’s previous works, cyclopropylboronic
esters 8a and 8b would not be suitable substrates for the Suzu-
ki–Miyaura cross-coupling due to steric hindrance of the chiral
auxiliary, contrary to the corresponding cyclopropyltrifluorobo-
rates.20,30 Thus, conversion of the pinacol boronate 9 to a trifluo-
roborate was attempted. The racemic salt 10 was obtained in
96% yield by treatment with 7 equiv of KHF2 in a methanol/water
mixture,31 at reflux during 3 h. Deprotection of the pure diastereo-
isomers 8a and 8b was achieved with 40 equiv KHF2 in 2 days. As
indicated by their opposite optical rotations, the resulting enantio-
mers (+)-10a and (ꢀ)-10b were obtained in pure form.
To date, only few examples of cross-coupling of cyclopropyltri-
fluoroborates with aryl halides have been reported.30,32 This proce-
dure allowed the synthesis of 1,2-disubstituted cyclopropanes in
Figure 2. Reported cyclopropyl CA4 analogues.15,17

N. Ty et al. / Bioorg. Med. Chem. 21 (2013) 1357–1366
1359
Scheme 1. Preparation of the trifluoroborate salts ( )-10, (+)-10a and (ꢀ)-10b. Reagents and conditions: (1) (a) B(OiPr)3, THF, ꢀ78 °C; (b) t-BuLi, ꢀ78 °C, 10 min; (c) pinacol,
ꢀ78 °C then rt, 1.5 h for boronate 6; diol 4, ꢀ78 °C then rt, 20 h for boronate 7; (2) CH2N2, Pd(OAc)2, Et2O, 0 °C, 1 h; chiral HPLC for boronates 8; (3) KHF2, MeOH/H2O, reflux,
3 h for ( )-10, 2 days for 10a and 10b.
good yields and with retention of the cyclopropane configuration.
For our part, the Suzuki–Miyaura reaction conditions were opti-
mized by using the racemic trifluoroborate ( )-10 and 4-bromoan-
isol 11a as model. We first used a protocol reported by Deng and
co-workers.32a and also applied by Pietruszka and co-workers:30
Pd(PPh3)4 as catalyst and K3PO4 as base, with a dual-solvent sys-
tem toluene/water (Table 1, entry 1). Thus, after heating at
100 °C for 15 h, the desired cross-coupling product 12 was formed,
but conversion was low (ratio coupling product 12/boronic acid 13
1:0.7).33 Combination of PdCl2(dppf)ꢁCH2Cl2 and K3PO4 was more
efficient, providing a 58% yield of 12 (entry 2). It should be noted
that both of these conditions led to the formation of the product
of protodeboronation 14. Since conversion was optimal by treat-
ment with PdCl2(dppf) and Cs2CO3 (entry 3), this catalyst system
was applied to the coupling with different aryl bromides.
To obtain the target CA4 analogue ( )-1 we first planned to pre-
pare the corresponding phenol derivative protected by a tert-butyl-
dimethylsilyl group (Table 2). By using bromide 11b,34 the cross
coupling adduct 15 was isolated in a 59% yield, then deprotected
with n-Bu4NF to give the desired phenol ( )-1 in 41% yield from
( )-10. Suzuki reaction was also attempted with the free phenol
11c,34 affording ( )-1 in 48% yield. 30-Amino CA4 analogues were
mainly obtained by reduction of the corresponding nitro deriva-
tives. Thus, a cross-coupling reaction was carried out with arylbr-
omide 11d,35 rapidly providing the nitro derivative ( )-16 in good
yield. It is noteworthy that the reaction was also effective with the
bromoaniline 11e36 affording ( )-2, the cyclopropyl analogue of
AVE8063, in 41% yield. Treatment of the trifluoroborate 10 with
the pure (E)-2-bromostyrene 1737 afforded the vinylcyclopropyl
derivative ( )-3 in a modest yield, but with retention of the double
bond stereochemistry.
Finally, coupling reactions with the enantiomeric trifluorobo-
rates (+)-10a and (ꢀ)-10b were carried out using aryl bromides
11c, 11d and (E)-bromostyrene 17, providing respectively products
(+)-1a, (+)-2a, (+)-3a and their corresponding (ꢀ)-enantiomers,
with yields ranging from 22% to 60% (see formulas Table 3).
Table 1
Optimization of the Suzuki couplinga
Br
CH3O
CH3O
CH3O
CH3O
CH3O
H3C O
2.2. Biological evaluation
+
OCH3
BF3K
R
[Pd]
100°C
The potential antivascular effects of the synthesized compounds
were assessed by their ability to inhibit tubulin polymerization
(fluorimetric assay)38 and to rapidly induce a rounding up of endo-
thelial cells.39 This morphological test is considered to be predic-
tive of potential in vivo antivascular activity, and it allows
determination of the lowest active concentration that causes
rounding of immortalized HUVEC (EAhy 926 cells) within 2 h.
Compounds were also evaluated for their growth inhibitory effects
against the murine B16 melanoma cell line. The biological activi-
ties of the cyclopropyl CA4 analogues with CA4 as reference are
presented in Table 3.
CH3O
CH3O
CH3O
toluene-H2O
OCH3
13: R = B(OH)
( )-14: R = H
( )-
2
( )-10
( )-12
b
Entry
Catalyst
Base
Product ratios
12 Yieldc
12
13
14
1
2
3
Pd(PPh3)4
PdCl2(dppf)
PdCl2(dppf)
K3PO4
K3PO4
Cs2CO3
1
1
1
0.7
0.2
0
0.1
0.1
0
—
58
75
a
All reactions were performed on a 0.1 mmol scale using 5 mol % of catalyst,
The CA4 analogue 1 in its racemic form showed an inhibition of
3 equiv of base, and 1.1 equiv of bromide 11a at reflux during 15 h.
b
Ratios were determined by 1H NMR analysis of the crude product.
tubulin polymerization (IC50 = 1.0
l
M) slightly higher than that of
M), and it could induce
c
Isolated yield based on trifluoroborate 10.
the reference compound CA4 (IC50 = 0.3
l

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N. Ty et al. / Bioorg. Med. Chem. 21 (2013) 1357–1366
Table 2
enantiomer (ꢀ)-1b, which displayed the same potency than CA4
with an ITP of 0.3 M. However, for the morphological activity
on endothelial cells, activity of (ꢀ)-1b was inferior to that of CA4
(0.12 M vs 0.006, respectively). Compound ( )-12 which did not
Synthesis of racemic cyclopropyl compoundsa
l
CH3O
l
BF3K
CH3O
bear the phenoxy group showed slightly lower biological values
CH3O
( )-10
compared to ( )-1.
Br
In the amino series, the enantiomer (ꢀ)-2b displayed the best
anti-tubulin activity compared to the (+)-2a (IC50 = 1.9 vs
Br
or
(dppf), Cs2CO ,
PdCl2
toluene-H2O, 100°C3
Ph
R
OCH3
11.2
l
M, respectively). Interestingly, compound (ꢀ)-2b clearly in-
duced rapid changes in the endothelial cell morphology (Fig. 4)
at very low concentration (rounding up at 60 nM). The presence
of a nitro group on the B-ring (compound ( )-16), led to a signifi-
cant loss of all the evaluated biological activities.
17
11a-e
CH3O
O
CH3
As previously reported, the racemic cyclopropylvinyl compound
( )-3 exhibited an anti-tubulin activity slightly weaker than that of
CH3O
CH3O
CH3O
CH3O
R
OCH3
CA4 (IC50 = 1.8 l
M).40 Contrary to what has been observed with the
foregoing compounds where the (ꢀ) enantiomers were the most
( ) -1,2,12,15,16
( )-3
active, the enantiomer (ꢀ)-3b displayed a reduced anti-tubulin
activity (IC50 = 4.5
(IC50 = 1.3 M).
For the cytotoxicity, most of the cyclopropyl compounds pre-
sented significant activities in the submicromolar range (IC50
0.27–0.62 M), although these values were superior to that of
the CA4 value (0.003 M). Nevertheless, compounds which display
lM) compared to the isomer (+)-3a
Bromide
R
Time (h)
Product
Yield (%)
l
11a
11b
11c
11d
11e
17
H
18
2
4
1
18
18
12
15
1
16
2
75
59
48
73
41
23
OTBDMS
OH
NO2
NH2
—
:
l
l
3
potent ability to inhibit tubulin assembly, while showing moder-
ated cytotoxicity, may be of interest in the search of new antivas-
cular agents.41 While compound (ꢀ)-1b is 20-fold more active than
its enantiomer (+)-1a in the ITP assay, it was only twofold more ac-
tive in the antiproliferative assay. Surprisingly, the enantiomer
(ꢀ)-1b displayed the same cytotoxic activity than its racemic form
a
All reactions were performed on a 0.1 mmol scale using 5 mol % of catalyst,
3 equiv of base, and 1.1 equiv of bromide.
change in the endothelial cell morphology (rounding up) at a great-
er concentration compared to CA4 (0.12 lM vs 0.006 for CA4).
( )-1 (IC50: 0.30 and 0.32 lM, respectively). We also observed sim-
ilar results for the amino compounds (ꢀ)-2b and ( )-2 (IC50: 0.27
While the anti-tubulin activity was significantly reduced with the
enantiomer (+)-1a, it was however increased threefold with the
and 0.29 M, respectively). In the olefinic series, there was no
l
Table 3
Inhibition of tubulin polymerization (ITP), morphological effects on endothelial cells (rounding up) and cytotoxicity of synthesized cyclopropyl CA4 analogues
Morphology HUVECb
(
lM)
Cytotoxicity B16 IC50 (lM)
a
c
Compd
R
ITP IC50
(l
M)
( )-1
(+)-1a
(ꢀ)-1b
12
( )-2
(+)-2a
(ꢀ)-2b
16
( )-3
(+)-3a
(ꢀ)-3b
CA4
OH
OH
OH
H
NH2
NH2
NH2
NO2
—
1.0
6.8
0.3
2.4
2.8
11.2
1.9
26.3
1.8
1.3
4.5
0.3
0.12
0.5
0.12
0.25
0.12
0.5
0.06
3.13
0.5
0.32 0.01
0.52 0.09
0.30 0.01
0.20 0.02
0.29 0.01
0.44 0.01
0.27 0.01
15.40 1.9
0.62 0.04
0.57 0.01
2.74 0.29
0.003
—
—
—
0.5
3.13
0.006
a
Compound concentration required to inhibit tubulin polymerization by 50%. In vitro microtubule assembly was monitored using the fluorescent probe DAPI.38
Morphological activity on immortalized HUVEC (EAhy 926 cells) was expressed at the lowest concentration at which cell rounding up was observed, following 2 h
b
incubation with the tested compound.
c
Compound concentration required to kill 50% of B16 murine melanoma cells after a 48 h incubation time (MTT assay). Mean SEM.

N. Ty et al. / Bioorg. Med. Chem. 21 (2013) 1357–1366
1361
difference in cytotoxic activity between the potent anti-tubulin
enantiomer (+)-3a and its racemic form ( )-3 (IC50: 0.57 and
0.62 M, respectively).
l
Contrary to what was observed in the anti-tubulin activity, we
noted that there was no difference in cytotoxic activity between
racemic and enantiomerically pure forms for the three series of
compounds. This apparent discrepancy could be due to the differ-
ence in the ITP assay and the cytotoxic assay, where in the former
we deal with pure tubulin in vitro, and in the latter case cells are
used. In the cellular assay, tubulin as well as other molecular tar-
gets could be involved in the cytotoxic effect. In addition, biotrans-
formation and/or bioisomerization of the cyclopropyl group42 may
occur in cells or in cell culture conditions.
These biological results show that for 1,2-diarylcyclopropyl
compounds, the enantiomers (ꢀ)-1b and (ꢀ)-2b are the most po-
tent tubulin polymerization inhibitors, whereas for the cyclopro-
pylvinyl compounds, the more active is the enantiomer (+)-3a.
Because this discrepancy could be due to a different binding mode
of these compounds with tubulin, we therefore performed docking
studies, as described hereafter.
2.3. Molecular docking
To determine the possible binding modes of these three pairs of
cyclopropyl enantiomers with tubulin, docking studies were car-
ried out by using the reported high-resolution crystal structure
of the tubulin/DAMA-colchicine complex (PDB ID: 1SA0).43 The
colchicine-binding site located at the
a–b interface of tubulin
heterodimers is mostly buried in the b-subunit. The ligand also
maintains few interactions with the T5 loop of the neighboring
a
-subunit. Several important residues for binding of colchicine
type ligands to tubulin have been identified, including Cysb241,
Metb259, Thr 179 and Val
181.44,45
a
a
Docking simulations were performed with GOLD software ver-
sion 5.146 with default parameters, as stated in our previous stud-
ies.47 To validate our docking process, DAMA-colchicine was first
extracted from the complex then re-docked. Superimposition of
our best docked pose on the crystal structure gave an RMSD devi-
ation of 0.92 Å (not shown). The carbonyl group of the tropolone
ring is involved in hydrogen bond with Vala181 (263 Å) and the
trimethoxyphenyl ring is positioned in proximity to Cysb241, but
the hydrogen atom of the cystein thiol group is not properly direc-
ted to form hydrogen bond with one of the methoxy groups. CA4
was found to adopt a very similar binding mode, the trimethoxy-
phenyl ring lies in a hydrophobic pocket close to Cys b241 and
the 40-methoxy group occupies the same position as the corre-
sponding (equivalent) group of the colchicine C ring. As previously
reported, CA4 interacts extensively with b-tubulin and with only
two residues in a
-tubulin (Thr179 and Val181).44,48
The best docked poses of investigated cyclopropyl enantiomers
were then selected to be superimposed to CA4. Enantiomeric phe-
nol 1b with SR-configuration overlaps perfectly with CA4 (Fig. 5a).
For both molecules, the 3-methoxy group of the TMP ring is 2.7 Å
(O–S) from Cysb241, and the phenol group forms a hydrogen bond
Figure 4. Morphological effects on endothelial cells. Typical rounding up of
endothelial cells (EAhy 926) exposed to compound (+)-2a (0.5
lM) and (ꢀ)-2b
with Vala181 (1.94 and 2.38 Å, respectively). This hydrogen bond
(0.06 M) for a 2 h exposure time. The solvent DMSO 1% was used as the control.
l
is missing with enantiomer 1a, due to a shift of the molecule to-
ward Cysb241. These results could be correlated to the significant
difference of anti-tubulin activity between the two enantiomeric
Original magnification, 200ꢃ.
compounds (0.3 vs 6.8 lM).
backbone oxygen atom of Thr
form strong hydrogen bond.
a
179 (ꢂ2.8 Å) but not enough to
Amino derivatives 2a and 2b docked in the colchicine site in a
similar fashion than the corresponding phenol compounds
(Fig. 5b). Nevertheless, compound 2b showed a slightly different
position relative to CA4. This shift could explain why amino com-
pound 2b is less effective than its phenolic analogue 1b (1.9 vs
The cyclopropylvinyl compound 3a occupies an almost identical
binding space as CA4, the lower part of the phenyl ring overlaying
with the methoxy methyl group of the CA4 B-ring (Fig. 5c). Its
phenyl ring is embedded in a hydrophobic pocket consisting of
the side chains of residues Alab316, Valb351 and Lysb352
0.3 l
M). The 30-amino groups of both enantiomers are near the

1362
N. Ty et al. / Bioorg. Med. Chem. 21 (2013) 1357–1366
Figure 5. Proposed binding mode of CA4 and synthesized cyclopropyl compounds in the colchicine binding site of tubulin. (A) Superimposition of CA4 (cyan), 1b (green), 1a
(gray); (B) CA4 (cyan), 2b (green), 2a (gray); (C) CA4 (cyan), 3b (green), 3a (gray); (D) CA4 (cyan), 3a (gray); hydrophobic region (brown). Only relevant residues are indicated.
(Fig. 5d). A different binding orientation is observed for enantiomer
3b shifting closer to the b-tubulin.
and the cyclopropylvinyl enantiomer (+)-3a were the most potent
tubulin polymerization inhibitors. There was a correlation between
anti-tubulin activity and rounding up activity of endothelial cells
for most of the compounds. The cytotoxic activity was in the sub-
micromolar range for most compounds, but unlike the anti-tubulin
activity, there was no difference in cytotoxic activity between race-
mic and enantiomerically pure forms for the three series of com-
pounds. Molecular docking studies confirmed the importance of
stereochemistry for interaction of the compounds with the colchi-
cine binding site of tubulin and were in good agreement with the
tubulin polymerization inhibitory data. These results highlight
the importance of stereochemistry in the tubulin interaction of
cyclopropyl CA4 analogues and could be valuable in the search of
more active tumor vascular disrupting agents.
This result differs from our previous assumption that enantio-
mer 3b would be better positioned in the colchicine site.17 We
can essentially explain this difference by the use of another protein
optimization protocol (Gold version 5.1 vs 3.1). Moreover in our
current model (Ref. 47 and herein) ligands were not constrained
on TMP and conformational space of each ligand within the recep-
tor site was more explored (100 poses instead of 10 in default anal-
ysis). In this manner, CA4 poses could be obtained without any
constraints. Contrary to our previous data, docking results pre-
sented herein are not biased by constraints and therefore are more
valuable.
For the diarylcyclopropyl compounds, enantiomers 1b and 2b
with SR-configuration show better superimposition with CA4 than
the RS-enantiomers (1a and 2a). With the cyclopropylvinyl com-
pounds, enantiomer 3a with RR-configuration is best positioned
in the colchicine binding site. Thereby, the docking results are in
good agreement with the observed anti-tubulin activities. Together
these data corroborate the previously assignment proposed on the
grounds of NMR data.
4. Experimental section
4.1. Chemistry
4.1.1. General methods
Unless otherwise noted, all materials were obtained from com-
mercial sources and were used without purification. THF was dried
over sodium/benzophenone. Diazomethane was prepared in ether
from N-methyl-N-nitrosourea and KOH.49 The resulting biphasic
solution was frozen at ꢀ78 °C, and the ether layer was quickly fil-
tered (large cotton-plugged funnel) and dropped directly into the
cooled reaction mixture. Aryl bromides 11b, 11c and 11d were pre-
pared as reported in the literature:33,34 phenol 11c was obtained by
a Baeyer–Villiger oxidation of commercially available 5-bromo-2-
methoxybenzaldehyde using m-chloroperoxybenzoic acid, nitro
compound 11d was obtained by selective monobromination of 2-
nitroanisole with NBS promoted by HBF4ꢁEt2O. CA4 was prepared
by decarboxylation of the appropriate cinnamic acid according to
Gaukroger et al.50 NMR spectra were recorded with a Bruker ACP
300 spectrometer at 300 MHz for 1H NMR and 75 MHz for 13C
NMR spectroscopy. Chemical shifts are given as d values in ppm
relative to the residual solvent peak (CHCl3) as the internal
3. Conclusions
In order to evaluate the influence of the stereochemistry on the
biological activities of cis-cyclopropyl CA4 analogues, we have syn-
thesized three pairs of cis-cyclopropyl CA4 analogues in their pure
enantiomeric forms. To obtain the desired compounds, we have at-
tempted an asymmetric cyclopropanation of a (Z)-alkenylboron
compound bearing a chiral auxiliary. Two diastereoisomeric com-
pounds were obtained, but could be separated by chiral HPLC.
Cross-coupling of the corresponding trifluoroborate salts with aryl
and olefinic halides yielded the wanted three pairs of cis-cyclopro-
pyl CA4 analogues.
The synthesized cyclopropyl compounds were evaluated for
their potential antivascular activities. Anti-tubulin activities
showed that the diarylcyclopropyl compounds (ꢀ)-1b, (ꢀ)-2b

N. Ty et al. / Bioorg. Med. Chem. 21 (2013) 1357–1366
1363
reference, coupling constants are given in Hertz. All 13C NMR spec-
tra were recorded with complete proton decoupling. In the 13C
NMR spectra of the potassium cyclopropyltrifluoroborates 10, the
signals of the carbon attached to the bore atom are not seen.31a
Peak assignment was unambiguously performed using HMQC,
HMBC and NOESY techniques. Melting points were determined
by the capillary method using an Electrothermal 9200 apparatus
and are uncorrected. Mass spectra were recorded on a Waters ZQ
2000 system using electrospray ionization (ESI). High-resolution
ESIMS data were acquired from the ‘imagif’ service (CNRS-ICSN,
91198 Gif-sur-Yvette, France) on a Waters LCT spectrometer. Reac-
tions were monitored by thin-layer chromatography (TLC) with
Merck silica gel 60 F254. Flash chromatography was carried out
on Merck silica gel (320–400 mesh). Representative structures
with indicative numbering for NMR assignments are shown:
(75 MHz, CDCl3): d 152.3 (C-3,C-5), 148.7 (CH-a), 140.9 and 140.7
(4 ꢃ C-Ph), 137.8 (C-4), 133.5 (C-1), 129.7 (CHPh), 29.6 (CHPh),
128.4 (CHPh), 127.8 (CHPh), 127.6 (CHPh), 127.2 (CHPh), ꢄ117
(C-b), 105.9 (C-2,C-6), 83.1 (C ꢃ 2), 77.9 (2 CHO), 60.9 (OCH3),
56.0 (OCH3 ꢃ 2), 51.7 (OCH3 ꢃ 2); MS (ESI+): m/z 679 [M+Na]+;
HRMS (ESI+): m/z 679.2823 [M+Na]+, requires 679.2843.
4.1.4. ( )-4,4,5,5-Tetramethyl-2-[2-(3,4,5-
trimethoxyphenyl)cyclopropyl]-[1,3,2]-dioxaborolane (9)
A freshly prepared ethereal solution of diazomethane (from
2.2 g of N-methyl-N-nitrosourea) was added to a suspension of
the vinylboronate 6 (965 mg, 3.0 mmol) and Pd(OAc)2 (34 mg,
0.15 mmol) in ether (15 mL) at ꢀ10 °C. The reaction mixture was
stirred at this temperature for 30 min and then allowed to warm
up to room temperature. The catalyst was removed by filtration
through Celite and the solvent evaporated under reduced pressure.
The brown residue was further purified by column chromatogra-
phy (cyclohexane/EtOAc/Et3N 9/1/0.01), affording 9 as a white
crystalline solid (795 mg, 79%); Mp 65–66 °C; 1H NMR (300 MHz,
CDCl3): d 6.52 (s, 2H, H-2, H-6), 3.85 (s, 6H, 2 ꢃ OCH3), 3.78 (s,
3H, OCH3), 2.32 (ddd, 1H, J = 10.2 Hz, J = 7.8 Hz, J = 6.0 Hz, H-a),
1.25 (ddd, 1H, J = 7.1 Hz, J = 6.0 Hz, J = 4.2 Hz, H-ctrans), 1.08
(ddd, 1H, J = 9.2 Hz, J = 7.8 Hz, J = 4.2 Hz, H-ccis), 1.03 (s, 6H,
CH3 ꢃ 2), 0.91 (s, 6H, CH3 ꢃ 2), 0.42 (ddd, 1H, J = 10.2 Hz,
J = 9.2 Hz, J = 7.1 Hz, H-b); 13C NMR (75 MHz, CDCl3): d 152.5 (C-
3, C-5), 136.4 (C-1), 136.2 (C-4), 105.8 (CH2, C-6), 82.9 (C-40, C-
50), 60.8 (OCH3), 56.0 (OCH3 ꢃ 2), 24.8 (CH3 ꢃ 2), 24.4 (CH3 ꢃ 2),
22.0 (CH-a), 8.9 (CH2-c), ꢄ3.0 (CH-b); MS (ESI+): m/z 357
[M+Na]+; HRMS (ESI+): m/z 357.1844 [M+Na]+, requires 357.1849.
4.1.2. 4,4,5,5-Tetramethyl-2-[(Z)-2-(3,4,5-trimethoxyphenyl)
ethenyl]-[1,3,2]-dioxaborolane (6)
To a solution of the Z-vinylbromide 5 (720 mg, 2.64 mmol) and
tri-iso-propylborate (670
lL, 2.90 mmol) in THF (6 mL) at ꢀ78 °C,
4.1.5. (4R,5R)-4,5-bis[Methoxydiphenylmethyl-2-[2-(30,40,50-
trimethoxyphenyl)cyclopropyl]-[1,3,2]-dioxaborolane (8a and
8b)
t-butyllithium (4.93 mL, 1.6 M in pentane, 7.88 mmol) was added
dropwise over 10 min. Then pinacol (345 mg, 2.90 mmol) in THF
(2 mL followed by 2 ꢃ 2 mL rinses) was added via cannula. The
reaction mixture was stirred at ꢀ78 °C for 15 min and then allowed
to warm to room temperature in 1.5 h. The reaction mixture was
then diluted with EtOAc, washed with saturated aqueous ammo-
nium chloride solution and brine. The organic layer was dried with
MgSO4, filtrated and concentrated in vacuo. Chromatography on
silica gel (cyclohexane/EtOAc/Et3N 90/10/0.1) gave compound 6
as a white amorphous solid (626 mg, 51%); 1H NMR (300 MHz,
CDCl3): d 7.10 (d, 1H, J = 15.1 Hz, H-a), 7.05 (s, 2H, H-2, H-6), 5.51
(d, 1H, J = 15.1 Hz, H-b), 3.88 (s, 6H, OCH3 ꢃ 2), 3.86 (s, 3H,
OCH3), 1.29 (s, 12H, CH3 ꢃ 4); 13C NMR (75 MHz, CDCl3): d 152.8
(C-3, C-5), 149.1 (CH-a), 138.2 (C-4), 133.8 (C-1), ꢄ117 (C-b),
106.5 (C-2,C-6), 83.5 (C ꢃ 2), 60.9 (OCH3), 56.1 (OCH3 ꢃ 2), 24.9
(CH3 ꢃ 4); MS (ESI+): m/z 343 [M+Na]+; HRMS (ESI+): m/z
343.1685 [M+Na]+, requires 343.1693.
A freshly prepared ethereal solution of diazomethane (from
2.8 g of N-methyl-N-nitrosourea) was added to a suspension of
the vinylboronate 7 (657 mg, 1.0 mmol) and Pd(OAc)2 (12 mg,
0.05 mmol) in ether (5 mL) at ꢀ15 °C. The reaction mixture was
stirred at this temperature for 30 min and then allowed to warm
up to room temperature. The catalyst was removed by filtration
through Celite and the solvent evaporated under reduced pressure
to give the cyclopropane derivative 8 (mixture of two diastereoiso-
mers) as a foam (650 mg, 97%). Both isomeric derivatives were sep-
arated by using
a
Phenomenex Lux-Amylose-2 column
(250 ꢃ 10 mm; eluent: hexane/iPrOH 95/5; flow rate; 5 mL/min;
detection at 254 nM). Their diastereoisomeric excess, determinat-
ed by analytical HPLC, are higher than 96% (retention times: isomer
8a: 9.9 min, isomer 8b: 13.8 min).
Isomer 8a: ½a 2D0
ꢅ
ꢀ120 (c 1.0 CH2Cl2); 1H NMR (300 MHz, CDCl3):
4.1.3. (4R,5R)-4,5-bis[Methoxydiphenylmethyl-2-[(Z)-2-(3,4,5-
trimethoxyphenyl)ethenyl]-[1,3,2]-dioxaborolane (7)
d 7.32–7.14 (m, 20H, 20 ꢃ Ph-CH), 6.17 (s, 2H, H-2, H-6), 5.22 (s,
2H, H-40,H-50), 3.88 (s, 3H, OCH3), 3.71 (s, 6H, OCH3 ꢃ 2), 2.91 (s,
6H, OCH3 ꢃ 2), 1.97 (ddd, 1H, J = 9.8 Hz, J = 8.2 Hz, J = 6.4 Hz, H-a),
1.06 (ddd, 1H, J = 9.8 Hz, J = 8.2 Hz, J = 4.0 Hz, H-ccis), 0.81 (ddd,
1H, J = 7.8 Hz, J = 6.4 Hz, J = 4.0 Hz, H-ctrans), 0.03 (dt, 1H,
J = 9.8 Hz, J = 7.8 Hz, H-b); 13C NMR (75 MHz, CDCl3): d 152.9 (C-
3, C-5), 141.8 (C-Ph ꢃ 2), 141.2 (C-Ph ꢃ 2), 137.5 (C-4), 136.4 (C-
1), 129.9 (CHPh),128.7 (CHPh), 128.1 (CHPh), 127.9 (CHPh), 127.6
(CHPh), 127.5 (CHPh), 104.9 (C-2, C-6), 83.6 (C ꢃ 2), 78.4
(CHO ꢃ 2), 61.3 (OCH3), 56.4 (OCH3 ꢃ 2), 52.2 (OCH3 ꢃ 2), 24.0
(CH-a), 13.5 (CH2-c), ꢄ4.0 (CH-b); MS (ESI+): m/z 693 [M+Na]+.
To a solution of the Z-vinylbromide 5 (784 mg, 2.87 mmol) and
tri-iso-propylborate (720
l
L, 3.12 mmol) in THF (7 mL) at ꢀ78 °C,
t-butyllithium (3.4 mL, 1.7 M in pentane, 5.78 mmol) was added
dropwise over 10 min. Then diol 4 (1.30 g, 2.87 mmol) in THF
(4 mL followed by 2 ꢃ 2 mL rinses) was added via cannula. The
reaction mixture was stirred at ꢀ78 °C for 15 min, allowed to
warm to room temperature in 1.5 h, then stirred for additional
20 h. The reaction mixture was diluted with EtOAc, washed with
saturated aqueous ammonium chloride solution and brine. The or-
ganic layer was dried with MgSO4, filtrated and concentrated in
vacuo. Chromatography on silica gel (cyclohexane/EtOAc/Et3N
Isomer 8b: ½a 2D0
ꢅ
ꢀ144 (c 1.0 CH2Cl2); 1H NMR (300 MHz, CDCl3):
d 7.30–7.12 (m, 20H, Ph-CH ꢃ 20), 6.14 (s, 2H, H-2, H-6), 5.16 (s,
2H, H-40,H-50), 3.63 (s, 6H, OCH3 ꢃ 2), 3.59 (s, 3H, OCH3), 2.89 (s,
6H, OCH3 ꢃ 2), 2.02 (m, 1H, H-a), 0.87 (ddd, 1H, J = 10.5 Hz,
J = 8.0 Hz, J = 4.1 Hz, H-ccis), 0.57 (m, 1H,, H-ctrans), 0.01 (m, 1H,
H-b); 13C NMR (75 MHz, CDCl3): d 152.6 (C-3, C-5), 141.5 (C-
Ph ꢃ 4), 136.5 (C-1), 136.0 (C-4), 129.8 (CHPh), 128.9 (CHPh),
90/10/0.1) gave compound 7 as a white foam (1.15 g, 61%); ½a D20
ꢅ
ꢀ211 (c 1.01, CHCl3); 1H NMR (300 MHz, CDCl3): d 7.32–7.18 (m,
20 H, Ph-H), 6.88 (d, 1H, J = 15.0 Hz, H-a), 6.42 (s, 2H, H-2, H-6),
5.36 (s, 2H, 2 ꢃ CH), 5.04 (d, 1H, J = 15.0 Hz, H-b), 3.83 (s, 3H,
OCH3), 3.68 (s, 6H, OCH3 ꢃ 2), 3.01 (s, 6H, OCH3 ꢃ 2); 13C NMR

1364
N. Ty et al. / Bioorg. Med. Chem. 21 (2013) 1357–1366
128.1 (CHPh), 127.9 (CHPh), 127.7 (CHPh), 127.6 (CHPh), 127.4
(CHPh), 105.5 (C-2, C-6), 83.6 (C ꢃ 2), 78.0 (CHO ꢃ 2), 61.3
(OCH3), 56.3 (OCH3 ꢃ 2), 52.1 (OCH3 ꢃ 2), 23.0 (CH-a), 10.2 (CH2-
c), ꢄ4.0 (CH-b); MS (ESI+): m/z 693 [M+Na]+.
CDCl3): d 6.56 (s, 2H, H-20, H-60), 6.42 (s, 1H, H-50), 6.11 (s, 2H,
H-2, H-6), 3.74 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.65 (s, 6H,
OCH3 ꢃ 2), 2.40 (m, 1H, H-a), 2.31 (m, 1H, H-b), 1.41 (m, 1H, H-
c), 1.21 (m, 1H, H-c), 0.90 (s, 9H, CH3 x3), -0.04 (s, 6H, Si(CH3) ꢃ 2).
4.1.6. ( )-[2-(30,40,50-Trimethoxyphenyl)cyclopropyl]
trifluoroborate de potassium (10)
4.1.7.3. 2-(4-Methoxy-3-hydroxyphenyl)-1-(30,40,50-trimethoxy-
phenyl)cyclopropane (1).
Compound ( )-1 was obtained
To a stirred solution of boronic ester 9 (749 mg, 2.24 mmol) in
methanol (12 mL) was added KHF2 (875 mg, 11.2 mmol) then
water (2.5 mL). The mixture was heated at 80 °C for 4 h, then con-
centrated to dryness. The resulting solid was collected by filtration,
rinsed with ether then hot CH3CN (3 ꢃ 10 mL). The combined fil-
trates were concentrated and then precipitated with CH3CN/Et2O
to give the desired pure product in 96% yield as a white solid
(678 mg): 1H NMR decoupled 19F (300 MHz, DMSO-d6): d 6.55 (s,
2H, H-2, H-6), 3.68 (s, 6H, OCH3 ꢃ 2), 3.58 (s, 3H, OCH3), 1.59
(ddd, 1H, J = 10.1 Hz, J = 8.0 Hz, J = 5.2 Hz, H-a), 0.67 (ddd,
J = 10.1 Hz, J = 8.0 Hz, J = 2.6 Hz, H-ccis), 0.39 (ddd, J = 7.9 Hz,
J = 5.2 Hz, J = 2.6 Hz, H-ctrans), ꢀ0.23 (td, J = 10.1 Hz, J = 7.9 Hz, H-
after column chromatography (cyclohexane/EtOAc 8:2) as a color-
less glass (16 mg, 48%): 1H NMR (300 MHz, CDCl3): d 6.66 (d, 1H,
J = 1.7 Hz, H-20), 6.62 (d, 1H, J = 8.2 Hz, H-60), 6.46 (dd, 1H,
J = 1.7 Hz, J = 8.2 Hz, H-50), 6.11 (s, 1H, H-2, H-6), 5.45 (s, 1H, OH),
3.80 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.65 (s, 6H, OCH3 ꢃ 2),
2.41 (m, 1H, H-a), 2.31 (m, 1H, H-b), 1.41 (m, 1H, H-c), 1.22 (m,
1H, H-c); 13C NMR (75 MHz, CDCl3): d 152.8 (C-3, C-5), 145.4 (C-
30), 145.1 (C-40), 136.2 (C-1), 135.0 (C-10), 131.9 (C-4), 121.0 (C-
60), 116.1 (C-20), 110.5 (C-50), 106.2 (C-2, C-6), 61.2 (OCH3-C4),
0
56.4 (OCH3-C4 ), 56.3 (OCH3-C3, OCH3-C5), 24.5 (CH-a), 24.4 (CH-
b), 12.4 (CH2-c); SM (ESI+): m/z 353 [M+Na]+; HRMS (ESI+) m/z
[M+H]+ calcd for C19H23O5: 331.1545, found 331.1552.
13
b); C NMR (75 MHz, DMSO-d6): d 152.0 (C-30, C-50), 141.5 (C-
Compound (+)-1a was obtained from trifluoroborate 10a
10), 134.6 (C-40), 105.9 (C-2, C-6), 60.3 (OCH3), 55.8 (OCH3 ꢃ 2),
20.8 (CH-a), 11.5 (CH2-c), CH-b not seen; 19F NMR (282 MHz,
DMSO-d6): d ꢀ131.88; MS (ESI+): m/z 291 [boronic acid + K]+;
(ESIꢀ): m/z 271 [boronic acid + Na]+.
(20 mg) in 60% yield; ½a D20
ꢅ
+5.2 (c 0.67, CH2Cl2).
Compound (ꢀ)-1b was obtained from trifluoroborate 10b
(20 mg) in 50% yield; ½a D20
ꢀ5.5 (c 0.80, CH2Cl2).
ꢅ
Trifluoroborate 10a: According to the procedure for racemic 10
(but heating for 2 days), enantiomer 10a was obtained from boro-
nate 8a (192 mg, 0.286 mmol) and KHF2 (893 mg, 11.45 mmol) as a
4.1.7.4. ( )-2-(4-Methoxy-3-nitrophenyl)-1-(30,40,50-trimethoxy-
phenyl)cyclopropane (16). Compound ( )-16 was obtained
after column chromatography (cyclohexane/EtOAc 8:2) as yellow
crystals (26 mg, 73%); mp: 78–80 °C; 1H NMR (300 MHz, CDCl3):
d 7.57 (d, 1H, J = 2.2 Hz, H-20), 7.04 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz,
H-60), 6.80 (d, 1H, J = 8.6 Hz H-50), 6.15 (s, 2H H-2, H-6), 3.86 (s,
3H, OCH3), 3.75 (s, 3H, OCH3), 3.69 (s, 6H, OCH3 ꢃ 2), 2.44 (m, 2H
H-a, H-b), 1.49 (m, 1H, H-c), 1.31 (m, 1H, H-c); 13C NMR
(75 MHz, CDCl3): d 153.1 (C-3, C-5), 151.5 (C-40), 139.3 (C-30),
136.8 (C-1), 134.9 (C-60), 133.5 (C-10), 131.6 (C-4), 126.3 (C-20),
white powder (80 mg, 88%): ½a D20
ꢅ
+12.1 (c 0.54, CH3CN); 1H NMR
(300 MHz, CD3CN): d 6.62 (s, 2H, H-2, H-6), 3.76 (s, 6H, OCH3 ꢃ 2),
3.66 (s, 3H, OCH3), 1.71 (m, 1H, H-a), 0.77 (m, 1H, H-c), 0.53 (m, 1H,
H-c), ꢀ0.11 (m, 1H, H-b).
Trifluoroborate 10b: Enantiomer 10b was obtained from boro-
nate 8b (205 mg, 0.305 mmol) and KHF2 (955 mg, 12.2 mmol) as
a white powder (80 mg, 83%): ½a D20
ꢀ11.9 (c 0.53, CH3CN).
ꢅ
0
113.3 (C-5’), 106.5 (C-2,C-6), 61.3 (OCH3-C4), 56.9 (OCH3-C4 ),
4.1.7. General procedure for the Suzuki cross coupling reaction
of trifluoroborates (10)
56.4 (OCH3-C3, OCH3-C5), 25.0 (CH-a), 23.3 (CH-b), 12.0 (CH2-c);
SM (ESI+): m/z 382 [M+Na]+; HRMS (ESI+) m/z [M+Na]+ calcd for
Preparation of ( )-12: A pressure glass tube charged with triflu-
oroborate ( )-10 (31 mg, 0.10 mmol), Cs2CO3 (98 mg, 0.30 mmol)
and PdCl2(dppf)ꢁCH2Cl2 (4 mg, 5 mol %) was alternatively evacu-
ated and sparged with argon three times. A solution of 4-bromoan-
isol (21 mg, 0.11 mol) in toluene (1.5 mL) previously flushed with
argon, then water (0.5 mL) were added by syringes. The reaction
mixture was stirred at 100 °C for 18 h and then cooled to room
temperature. Water (5 mL) was added and the mixture extracted
with EtOAc (5 mL ꢃ 2). The combined organic layers were washed
with water (5 mL ꢃ 2), dried (MgSO4) and concentrated in vacuo.
The residue was purified by column chromatography (cyclohex-
ane/EtOAc 9:1), affording 12 as a colorless oil (24 mg, 75%).
C
19H21NO6 + Na: 382.1267, found 382.1283.
4.1.7.5.
thoxyphenyl)cyclopropane (2).
( )-2-(3-Amino-4-methoxyphenyl)-1-(30,40,50-trime-
Amino compound ( )-2 was
obtained after column chromatography (cyclohexane/EtOAc 7:3)
as a pink oil (13 mg, 41%); 1H NMR (300 MHz, CDCl3): d 6.57 (dd,
1H, J = 8.1 Hz H-50), 6.43 (d, 1H, J = 1.9 Hz, H-20), 6.38 (dd, 1H,
J = 8.1 Hz, J = 1.9 Hz, H-60), 6.11 (s, 2H, H-2, H-6), 3.76 (s, 3H,
OCH3), 3.75 (s, 3H, OCH3), 3.65 (s, 6H, OCH3 ꢃ 2), 3.62 (s, 2H,
NH2), 2.38 (m, 1H, H-a), 2.27 (m, 1H, H-b), 1.41 (m, 1H, H-c),
1.16 (m, 1H, H-c); 13C NMR (75 MHz, CDCl3): d 152.7 (C-3, C-5),
146.1 (C-40), 136.1 (C-1), 136.0 (C-10), 135.5 (C-30), 131.2 (C-4),
119.6 (C-60), 116.8 (C-20), 110.4 (C-50), 106.0 (C-2, C-6), 61.2
4.1.7.1. ( )-2-(4-Methoxyphenyl)-1-(30,40,50-trimethoxyphenyl)
1H NMR (300 MHz, CDCl3): d 6.93 (m,
0
(OCH3-C4), 56.3 (OCH3-C3, OCH3-C5), 56.0 (OCH3-C4 ), 24.5 (CH-a),
cyclopropane (12).
24.2 (CH-b), 12.9 (CH2-c); SM (ESI+): m/z 352 [M+Na]+; HRMS
(ESI+) m/z [M+H]+ calcd for C19H24NO4: 330.1705, found 330.1709.
Compound (+)-2a was obtained from trifluoroborate 10a
2H, H-20, H-60), 6.69 (m, 2H, H-30, H-50), 6.07 (s, 2H, H-2, H-6),
3.75 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.63 (s, 6H, OCH3 ꢃ 2),
2.43 (m, 1H, H-b), 2.32 (m, 1H, H-a), 1.44 (m, 1H, H-c), 1.20 (m,
(20 mg) in 22% yield; ½a D20
ꢅ
+6.5 (c 0.57, CH2Cl2).
Compound (ꢀ)-2b was obtained from trifluoroborate 10b
13
1H, H-c); C NMR (75 MHz, CDCl3): d 157.8 (C-40), 152.4 (C-3, C-
5), 135.8 (C-4), 134.7 (C-1), 130.3 (C-10), 130.2 (C-20, C-60), 113.3
(20 mg) in 31% yield; ½a D20
ꢀ7.5 (c 0.56, CH2Cl2).
ꢅ
(C-3’, C-50), 105.8 (C-2, C-6), 60.8 (OCH3-C4), 55.8 (OCH3-C3,
0
OCH3-C5), 55.2 (OCH3-C4 ), 23.9 (CH-a or CH-b), 23.7 (CH-a or
CH-b), 12.4 (CH2-c); SM (ESI+): m/z 337 [M+Na]+; HRMS (ESI+) m/
z [M+H]+ calcd for C19H23O4: 315.1596, found 315.1591.
4.1.7.6. cis-2-(E-2-Styryl)-1-(3,4,5-trimethoxyphenyl)cyclopro-
pane (3). Olefinic compound ( )-3 was obtained after column
chromatography (toluene/EtOAc 98:2) as a colorless oil (10 mg,
32%). Its spectroscopic data are the same as those previously re-
ported by us.17
4.1.7.2. ( )-2-(3-tert-Butyldimethylsilyloxy-4-methoxyphenyl)-
1-(30,40,50-trimethoxyphenyl)cyclopropane (15).
Compound
Compound (+)-3a was obtained from trifluoroborate 10a
( )-15 was obtained after column chromatography (cyclohexane/
(30 mg) in 27% yield; ½a D20
ꢅ
+41 (c 0.62, CH2Cl2).
EtOAc 9:1) as a colorless oil (26 mg, 59%): 1H NMR (300 MHz,

N. Ty et al. / Bioorg. Med. Chem. 21 (2013) 1357–1366
1365
Compound (ꢀ)-3b was obtained from trifluoroborate 10b
2 h-incubation period, digital photographs were taken of represen-
tative centre areas of each well at a magnification of 100ꢃ and
200ꢃ. 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 a positive internal standard.
(35 mg) in 30% yield; ½a D20
ꢀ51 (c 1.04, CH2Cl2).
ꢅ
4.1.8. 5-Bromo-1-methoxyaniline (11e)
A mixture of 11d (515 mg, 2.2 mmol) and SnCl2ꢁ2H2O (2.1 g,
10 mmol) in absolute ethanol (30 mL) was heated at 70 °C under
argon for 2 h. The reaction mixture was allowed to cool to room
temperature and then poured into ice. The pH was adjusted to
7–8 by addition of 5% aqueous NaHCO3, and the mixture was ex-
tracted with EtOAc (2 ꢃ 30 mL). The combined organic phases
were washed with brine, dried over MgSO4 and concentrated.
The residue was purified by column chromatography (cyclohex-
ane/EtOAc/Et3N: 85/15/0.5) to give aniline 11e51 as a pink powder
(260 mg, 60%).
4.2.3. Cytotoxicity assay
Murine B16 melanoma cells were grown in DMEM medium
containing 2 mM
L-glutamine, 10% fetal bovine serum, 100 U/mL
penicillin and 100
lg/mL streptomycin (37 °C, 5% CO2). All com-
pounds were initially dissolved in DMSO at a stock concentration
of 2.5 mg/mL and were further diluted in cell culture medium.
Exponentially growing cells were plated onto 96-well plates at
4.2. Biological evaluations
5000 cells per well in 100
100 L of medium containing the compound at final concentra-
tions ranging from 0.01 to 100 M were added to the wells (in trip-
lL of culture medium. 24 h after plating,
4.2.1. Inhibition of tubulin polymerization
l
Tubulin assembly in microtubules was carried out using the
fluorescent dye DAPI (40,6-diamidino-2-phenylindole)52 in a 96-
well plate format as described.38b,53 The standard assay was per-
formed as follows: wells were charged with tubulin (Cytoskeleton,
97% pure, final concentration 1 mg/mL) in PME buffer (100 mM
PIPES (1,4-piperazinebis(ethanesulfonic acid); 1 mM MgSO4;
l
licate) 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 via-
bility was assayed using the MTT (methylthiazoletetrazolium)
test56 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 concen-
tration of compound that inhibited cell viability by 50% (inhibitory
concentration for 50% of cells, or IC50) was determined using the
GraphPad Prism software.
2 mM EGTA) with 10
test compounds using colchicine at 30
standard control. The final concentrations used for the test com-
pounds were started at 30 M and diluted in threefold decrements
lM DAPI and varying concentrations of the
l
M as a positive internal
l
until no inhibition was observed. Triplicate wells were run for each
concentration. After pre-incubation at room temperature for
4.3. Docking protocol
45 min, 1 mM GTP (5 lL) was added to each well to initiate tubulin
polymerization, and the plate was then transferred to a thermo-
stated Victor plate reader at 37 °C for an additional 2 h. Fluores-
cence was then read at the excitation wavelength of 360 nm and
emission of 450 nm. The percent inhibition was determined as fol-
Our previous docking work on analogues of combretastatin
A447 implied GOLD (Genetic Optimisation for Ligand Docking), so
we performed our study with this software in its new version.46
lows: 1 ꢀ (
D
F(sample/
D
F(control) ꢃ 100, where
D
F control = F(no
4.3.1. Ligands preparation
inhibition) ꢀ F(complete inhibition), and
D
F sample = F(sam-
All compounds, CA-4, 1a, 1b, 2a, 2b, 3a and 3b were initially
built within MarvinSketch 5.8.0 from Chemaxon57 next submitted
to Corina,58 a 3D structure generator. Obtained 3D conformations
were next submitted to Ligprep from the Schrodinger software
suite.59 In order to take into account flexibility of these structures,
conformational analyses, according to the OPLS2005 force field,60
were computed and the lowest energy conformations were kept
as input for the docking stage.
ple) ꢀ F(complete inhibition with colchicine). The IC50 for com-
pound-induced inhibition of tubulin polymerization is the
concentration at which the extent of inhibition of polymerization
is 50% of the maximum value, as determined from the semi-loga-
rithmic 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 inhibi-
tion of tubulin polymerization was 0.36
lM.
4.3.2. Protein preparation
3D structure of tubulin was retrieved from the Protein Data
Bank (http://www.rcsb.org, PDB ID: 1SA0).43 Subunits C, D and E
were removed. Only subunits A, B, corresponding to the colchicine
binding site, and small molecules DAMA-colchicine (CN2), GTP and
Mg2+ ion in this site were conserved.
4.2.2. Endothelial cell morphology
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 A549.54 The
EAhy 926 cell line is considered as one of the best immortalized
HUVEC cell lines because these cells express most of the biochem-
ical markers of parental HUVEC cells.55 EAhy 926 cells were grown
4.3.3. Molecular docking
Docking runs were performed with an exploration of the con-
formational 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. Gold-
score was chosen again as our fitness scoring function.
The binding-site was mapped following a procedure that oper-
ates in a manner analogous to Goodfoord’s GRID algorithm.61
Hydrophilic and hydrophobic regions are defined in a way that
considers both spatial proximity to the receptor and suitability
for occupancy by solvent water, not only receptor residues
properties.
in DMEM containing 2 mM
100 U/mL penicillin and 100
atmosphere (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
L-glutamine, 10% fetal bovine serum,
lg/mL streptomycin in a humidified
l
l
medium containing the test compound was added to the well con-
taining the cells (in triplicate) and incubated for 2 h (37 °C, 5%
CO2). The highest concentration used was 100 lM followed by se-
rial twofold dilutions down to low nanomolar concentrations, if
needed for the most morphologically active compounds. After the

1366
N. Ty et al. / Bioorg. Med. Chem. 21 (2013) 1357–1366
Org. Chem. 1990, 55, 4986; (b) Pietruszka, J.; Widenmeyer, M. Synlett 1997, 977;
Acknowledgements
(c) Pietruszka, J.; Witt, A. Synlett 2003, 91.
24. Dieck, H. A.; Heck, R. F. J. Org. Chem 1975, 1083; Brown, H. C.; Cole, T. E.
Organometallics 1983, 2, 1316.
This work was financially supported by the Centre National de
la Recherche Scientifique (CNRS), the Institut Curie, the Institut
National de la Santé et de la Recherche Médicale (Inserm), and
by a grant from the Institut National du Cancer (INCa, Boulogne
Billancourt, France). N.T. is grateful to the Ministère de l’Enseigne-
ment Supérieur et de la Recherche for a Ph.D. Fellowship Grant. We
thank N. Vanthuyne (UMR6263, Univ. Paul Cézanne, Marseille,
France) for preparative HPLC purification.
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