Synthesis and Biological Evaluation of 4-Arylcoumarin Analogues of Combretastatins

Article abstract of DOI:10.1021/jm030903d

A series of A-ring polymethoxylated neoflavonoids was prepared by ligand coupling reactions involving either Suzuki or Stille reactions. Cytotoxicity studies indicated a potent activity against a CEM leukemia cell line for the compounds presenting a subst

Full text of DOI:10.1021/jm030903d

J . Med. Chem. 2003, 46, 5437-5444
5437
Syn th esis a n d Biologica l Eva lu a tion of 4-Ar ylcou m a r in An a logu es of
Com br eta sta tin s
Christian Bailly,*,† Christine Bal,† Pascale Barbier,‡ Se´bastien Combes,§ J ean-Pierre Finet,*,§
Marie-Paule Hildebrand,† Vincent Peyrot,‡ and Nicole Wattez†
INSERM U-524 et Laboratoire de Pharmacologie antitumorale du Centre Oscar Lambret, Institut de Recherches sur le Cancer,
Place de Verdun, 59045 Lille, France, UMR 6032 CNRS - Universite´ d’Aix-Marseille 2, “Interaction entre Syste`mes Prote´iques
et Diffe´renciation dans la Cellule Tumorale”, Faculte´ de Pharmacie de Marseille, 27 boulevard J ean Moulin, 13385 Marseille
Cedex 5, France, and UMR 6517 CNRS - Universite´s d’Aix-Marseille 1 et 3, “Chimie, Biologie et Radicaux Libres” Faculte´ des
Sciences St J e´roˆme, case 541, 13397 Marseille Cedex 20, France
Received May 22, 2003
A series of A-ring polymethoxylated neoflavonoids was prepared by ligand coupling reactions
involving either Suzuki or Stille reactions. Cytotoxicity studies indicated a potent activity
against a CEM leukemia cell line for the compounds presenting a substitution pattern related
to that of combretastatin A-4. The two compounds having a 3′-OH and a 4′-OCH3 substituents
on the 4-phenyl B-ring have no effect on human topoisomerases I and II but potently inhibit,
in vitro, microtubule assembly. At the cell level, the active compounds were characterized as
proapoptotic agents, but they can also trigger cell death via a nonapoptotic pathway.
Among the various antimitotic agents inhibiting tu-
bulin polymerization by interaction with the colchicine
site,1 combretastatin derivatives constitute one of the
most extensively investigated group since the discovery
of combretastatin A-4 (Scheme 1).2 A number of deriva-
tives or analogues have been prepared to improve the
solubility, stability, or therapeutic index. A key struc-
tural factor for the cytotoxic activity is the presence
of the cis double bond, forcing the two aromatic
rings to be noncoplanar and within an appropriate
distance.2b,3,4
In the past recent years, a number of neoflavonoid
(4-phenylcoumarin) derivatives isolated from various
plant sources have revealed cytotoxic properties.5 Chemo-
preventive activity against cancer without cytotoxicity
was observed for some derivatives.6 As the two aromatic
rings (A and B) of 4-phenylcoumarin derivatives adopt
also a conformation in which they are not coplanar,7 the
structural similarity between combretastatin and neofla-
vonoid can be expected to lead to an activity on the
colchicine site of tubulin polymerization in the case of
conveniently substituted neoflavonoids. Moreover, such
a structure will avoid inactivation resulting from cis-
to-trans isomerization of the double bond of combret-
astatin derivatives. We therefore decided to investigate
the synthesis of neoflavonoid analogues of combretasta-
tin A-4 and to study their biological efficiency toward
tubulin polymerization as well as potential topo-
isomerase inhibitors,8 in view of the antitopoisomerase-
II activity that has been reported for the structurally
related isoflavonoid, genistein, and for podophyllotoxin
derivatives.9
Resu lts a n d Discu ssion
Ch em istr y. Synthesis of 4-arylcoumarin derivatives
by ligand coupling reaction of two aromatic units, the
preformed coumarin ring and a conveniently activated
phenyl derivative, constitutes an efficient access to the
polymethoxylated derivatives.10 Among the various
synthetic methods (Stille coupling or Suzuki coupling),
the method of Boland et al.11 can be used for the
synthesis of neoflavonoids containing a number of
alkoxy or hydroxy groups,12 by C-4 arylation of 4-acti-
vated coumarins with arylboronic acids in the presence
of a palladium catalyst and copper(I) iodide as a
cocatalyst. We used this method for the synthesis of the
5,6,7-trimethoxycoumarin derivatives 1-4 as well as for
the synthesis of the 5,7-dimethoxy analogues 6-9.
The 4-trifluoromethylsulfonyloxycoumarins 10 and 11
were easily prepared in 87-90% yield by treatment of
the appropriate 4-hydroxycoumarins13,14 with triflic
anhydride.11 Use of the benzyl-protected arylboronic
acids 12-15 in the palladium-copper-catalyzed Suzuki
coupling reaction led to the protected hydroxycoumarin
derivatives 16-23 in good to high yields (69-96%, Table
1).
In the presence of the easily reduced benzopyrone,
deprotection of the benzyloxy group by the reported
selective palladium-catalyzed hydrogenolysis proved
unreliable. However, selective removal of the benzyl
group was performed successfully by treatment with
hydrobromic acid, leading to the expected hydroxylated
4-arylcoumarins 1-4 and 6-9 in good yields (62-96%,
Table 2). On the other hand, the trimethoxylated
benzodioxane derivative 5 was prepared in 63% yield
by the Stille palladium-catalyzed coupling of the cou-
marin derivative 10 with tributyl-3,4-methylenedi-
oxyphenyltin.15
* Corresponding authors. C.B.: Tel, (+) 33 3 20 16 92 18; Fax, (+)
33 3 20 16 92 29; e-mail, bailly@lille.inserm.fr. J .-P.F.: Tel, (+) 33 4
91 28 89 27; Fax, (+) 33 4 91 28 87 58; e-mail, finet@srepir1.
univ-mrs.fr.
Biology. Cytotoxicity. A tetrazolium-based assay
was applied to determine the drug concentration re-
quired to inhibit cell growth by 50% after incubation in
† Institut de Recherches sur le Cancer.
‡ Universite´ d’Aix-Marseille 2.
§ Universite´s d’Aix-Marseille 1 et 3.
10.1021/jm030903d CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/31/2003

5438 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Bailly et al.
Sch em e 1
Sch em e 2
Ta ble 1. Structures and Yields of the Products of the Suzuki
Ta ble 3. Cytotoxicity
Coupling Reaction
compd
1
2
3
4
5
6
7
8
9
yield
(%)
IC50 (µM)a 47 54 0.52 62 >50 16 10.6 0.083 >75
substrate ArB(OH)2 product
Ar
4-BnOC6H4
4-BnO-3-MeOC6H3
3-BnO-4-MeOC6H3
4-BnO-3,5-(MeO)2C6H2
a
Drug concentration that inhibits the growth of CEM by 50%
5,6,7-OMe Substituted
after incubation in liquid medium for 72 h. Each drug concentra-
tion was tested in triplicate, and the SE of each point is <10%.
10
10
10
10
12
13
14
15
16
17
18
19
69
89
96
90
amounts to 83 nM with 8, which reflects a very
pronounced antiproliferative activity. Parenthetically,
it is worth mentioning that a similar low IC50 value of
81 nM was measured with compound 3 when using
CEM/C2 cells selected for resistance to the topo-
isomerase I inhibitor camptothecin.16 The mutation of
the top1 gene in these cells does not affect the action of
the drug, and this is consistent with a nontopoisomerase
dependent cytotoxicity (see below).
5,7-OMe Substituted
11
11
11
11
12
13
14
15
20
21
22
23
94
93
96
86
4-BnOC6H4
4-BnO-3-MeOC6H3
3-BnO-4-MeOC6H3
4-BnO-3,5-(MeO)2C6H2
Ta ble 2. Structures and Yields of the Debenzylation Reaction
Products
substituent
The position of the hydroxy and methoxy substi-
tutents is essential for the cytotoxic effect. A priori, the
A-ring dimethoxy compounds are more cytotoxic than
the A-ring trimethoxy (compare 6 vs 1 and 8 vs 3), but
the most important position effect concerns the 4-phenyl
B-ring. A 3′-hydroxy group is critical to maintain the
cytotoxic activity. The two potent compounds 3 and 8
both bear 3′-OH and 4′-OCH3 groups. The reverse
situation 4′-OH and 3′-OCH3, as it is the case for 2 and
7, results in a very significant loss of activity. This is
further evidenced with compounds 4 and 9, which have
also the unfavorable 4′-OH and 3′-OCH3 and are totally
inactive. The bridge conformation with the methylene
dioxy compound 5 is also detrimental to the cytotoxic
action. It is absolutely clear that the correct substitution
product
R1
R2
R3
R4
yield (%)
6
7
8
9
1
2
3
4
H
H
H
H
OMe
OMe
OMe
OMe
H
H
H
OMe
H
H
H
OMe
OH
OH
OMe
OH
OH
OH
OMe
OH
H
OMe
OH
OMe
H
OMe
OH
69
62
74
96
80
83
79
88
OMe
the culture medium for 72 h. The calculated IC50 values
with the CEM human leukemia cell line are collated in
Table 3. Compounds 1, 2, 4, 5, and 9 were found to be
inactive and a modest cytotoxic effect was observed with
6 and 7. In sharp contrast, the two molecules 3 and 8
exhibited potent cytotoxic activities. The IC50 value

4-Arylcoumarin Analogues of Combretastatins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5439
pattern for the 4-phenyl B-ring is 3′-OH and 4′-OCH3,
providing thus a precise structure-activity relationship.
The next step of our medicinal chemistry program
consisted of identifying the potential target(s) for the
two highly cytotoxic compounds 3 and 8. On the basis
of the structural similarity between the 4-arylcoumarin
and combretastatin A-4, it was logical to investigate the
effects of the compounds on tubulin polymerization. But
in parallel, we also studied the effect of the different
molecules against topoisomerases I and II, as different
coumarin derivatives, such as genistein, have been
shown to act as poisons for these DNA-manipulating
enzymes.17,18 Colchicine derivatives can also function as
topoisomerase II inhibitors.19
Effects on Top oisom er a ses. A conventional DNA
relaxation assay was used to investigate the effect of
all compounds on human DNA topoisomerases. In these
experiments, supercoiled DNA was treated with either
topoisomerase I or topoisomerase II in the presence of
the test drug at 10 or 50 µM and the DNA relaxation
products were then resolved by agarose gel electro-
phoresis. None of the coumarin derivatives was able to
promote DNA cleavage by topoisomerases (data not
shown). The amount of nicked (for topoI) or linear (for
topoII) DNA species remains very weak in the presence
of the arylcoumarin, in contrast to what was observed
with the reference drugs camptothecin and etoposide,
which produce a high level of single or double strand
breaks, respectively. Therefore, the 4-arylcoumarin
derivatives cannot be considered as a topoisomerase
poisons.
Effects on tu bu lin p olym er iza tion . Figure 1A-C
shows the effects of 3, 8, and 2 on the turbidimetry time
course of microtubule assembly from pure tubulin. A
clear inhibition was noted, and the rate of assembly as
well as the final amount of microtubules was lower in
the presence of 3 and 8 than in the control experiment.
Compound 2 had no effect on tubulin polymerization
until 26 µM (Figure 1C). In the presence of all three
drugs, when the samples were cooled to 10 °C, the
polymers depolymerized as well as the control as-
sembled without drugs. The insets of parts A and B of
Figure 1 show that the extent of inhibition by 3 and 8,
respectively, increased monotonically with the mole
ratio of the total ligand to total tubulin in the solution
(R). In these figures, 50% inhibition occurred at a mole
ratio of 1.45 mol of 3 per mol of tubulin and at a mole
ratio of 0.28 mol of 8 per mol of tubulin. A similar
experiment performed with combretastatin A-4 showed
that a mole ratio of 0.07 mol of combretastatin A-4 per
mol of tubulin was necessary to halve the microtubule
formation (Figure 1D). Although less potent in tubulin
polymerization inhibition, 8 shows, like combretastatin
A-4, a substoichiometric mode of inhibition, while 3 is
stoichiometric.
F igu r e 1. Effect of 3, 8, and 2 on the turbidity time course of
in vitro microtubule assembly. The reaction was started by
warming the solution from 4 to 37 °C. Panel A shows (a)
tubulin at 16 µM and (b-e) aliquots of the same solution with
7, 9, 21, and 28 µM of 3. Panel B shows (a) tubulin at 16 µM
and (b-e) aliquots of the same solution with 1.6, 3.2, 4.8, and
6.4 µM of 8. Panel C shows (a) tubulin at 16 µM and (b-d)
aliquots of the same solution with 6.5, 13, 19.5, and 26 µM of
2. Panel D shows (a) tubulin at 30 µM and (b-e) aliquots of
the same solution with 1.2, 1.8, 2.3, 2.9, and 4.0 µM of
combretastatin A-4. At the time indicated by the arrow, the
samples were cooled to 10 °C. The insets show the percentage
of turbidity inhibition as a function of the mole ratio of the
total ligand to total tubulin in the solution (R).
seems however plausible that tubulin represents a
privileged target for these compounds. Having identified
one of the targets, we extended our pharmacology study
at the cell level to determine the type of cell death
induced by these compounds, principally the two cyto-
toxic hits 3 and 8. Different cellular assays were
deployed to analyze the effects of the drugs on the cell
machinery.
Cell Cycle Effects. Treatment of CEM cells with 3
and 8 for 24 h led to profound changes of the cell cycle
profiles, whereas the other compounds had no effect
(Figure 2). The flow cytometry analysis of propidium
iodide-labeled cells indicates that the two cytotoxic
drugs induce a massive accumulation of cells in the
G2/M phase. The G2 cell population increases from 23%
in the control to 54%, while the G1- and S-phase cell
populations gradually decrease from 42% and 33% to
5% and 9%, respectively, in the presence of 1 µM 8. The
G2 block is dose-dependent, as illustrated with 3 in
Figure 2b.
Electron microscopy of the polymers remaining with
3 and 8 showed identical normal microtubule structure
(data not shown).
The two cytotoxic compounds 3 and 8 inhibit tubulin
assembly, whereas the inactive compound 2 shows very
little effect in this assay. Interestingly, the most potent
compound against tubulin is also, by far, the most
potent cytotoxic agent in the series. Although a direct
correlation cannot yet be established at this stage, it
In parallel to the G2 block, a characteristic hypodip-
loid DNA content peak (sub-G1, 32%) can be detected
with 3 and 8. Here again the effect is clearly dose-
dependent (Figure 2b). The progressive generation of
cells having a hypodiploid DNA content (sub-G1 mate-
rial) is characteristic of apoptosis and reflects frag-

5440 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Bailly et al.
F igu r e 3. (a) Variations of the mitochondrial membrane
potential (∆ψmt) measured with the dye DiOC6 (3,3-dihexyl-
oxacarbocyanine iodide, 25 nM) in CEM cells treated for 24 h
with increasing concentrations of 3. Parallel experiments were
performed with the uncoupling agent CCCP (carbonyl cyanide
p-chlorophenylhydrazone, 5 and 50 µM for 10 min at 37 °C).
(b) The histograms indicate the percentage of depolarized cells
measured in the presence of 3 or 8 at the indicated concentra-
tion.
F igu r e 2. Cell cycle distribution determined by flow cytom-
etry in CEM cells treated for 24 h with (a) 1 or 5 µM
compounds 6-9 or (b) graded concentration of 3. Cells were
analyzed with the FACScan flow cytometer. Results show a
typical experiment which has been repeated three times.
mented DNA. It is not common to see simultaneously
the accumulation of cells in the G2- and sub-G1-phases,
as these effects are generally sequential.20 This may
reflect an action on two different targets and/or the
activation of two independent cell death pathways (as
discussed below). Nevertheless, the cell cycle analysis
suggests that CEM leukemia cells accomplish apoptosis
under treatment with 3 and 8. Therefore, we went on
to examine a variety of apoptotic events using several
complementary cytometric and biochemicals methods.
In d u ction of Ap op tosis. (1) Va r ia tion s of th e
Mitoch on d r ia l Mem br a n e P oten tia l. Mitochondria
plays an essential role in the propagation of apoptosis.
We used the ampholitic cationic fluorochrome DiOC6 to
monitor the changes of the mitochondrial transmem-
brane potential (∆Ψmt) induced by 3 and 8. CEM cells
were treated with graded concentrations of the drugs
(50 nM to 10 µM) for 24 h and then analyzed by flow
cytometry after DiOC6 labeling. The results are pre-
sented in Figure 3.
F igu r e 4. Drug-induced activation of caspase-3 in CEM cells.
Cells were exposed to graded concentrations of 3 or 8 (0.05-
10 µM) or to etoposide (10 µM) for 24 h prior to the addition of
the PhiPhiLux-G1D2 probe containing a rhodamine-tagged
GDEVDGI peptide motif substrate for caspase-3. Cells were
analyzed with the FACScan flow cytometer.
With both drugs, a population of depolarized cells,
characterized by a significant reduction of the cellular
uptake of the fluorochrome, appeared gradually as the
drug concentration was raised. The decrease of the
fluorescence intensity measured with the DiOC6 probe
reflects the collapse of ∆Ψmt, which is a signature for
the opening of the mitochondrial megachannels (the so-
called permeability transition pores). The effect is
reminiscent to that observed with the mitochondrial
uncoupling agent CCCP (carbonyl cyanide p-chlorophe-
nylhydrazone), a protonophore that abolishes ∆Ψmt
(Figure 3). The dissipation of ∆Ψmt observed with 3 and
8 reflects the opening of the mitochondrial permeability
transition pores. This effect, which has been commonly
observed with other anticancer drugs irrespective of the
cell type, generally defines an early but already ir-
reversible stage of apoptosis.
(2) Ca sp a ses Activa tion . Many studies have impli-
cated cysteine proteases (caspases) as key participants
in the sequence of events that results in the dismantling
of the cell during apoptosis.21 Therefore, it was interest-
ing to evaluate the effect of 3 and 8 on the activity of
caspase-3, one of the major effector caspases. We
explored the proteolytic cleavage of the rhodamine-
tagged substrate which contains a heptapeptide GDEVD-
GI recognition sequence for caspase-3 and possibly for
other caspases such as caspase-7. As shown in Figure
4, CEM cells treated with 3 showed a massive activation
of caspases. The effect is comparable to that of the well-
established anticancer proapoptotic drug etoposide.
Strong cleavage of the rhodamine-based substrate car-

4-Arylcoumarin Analogues of Combretastatins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5441
F igu r e 5. Western blot analysis for the cleavage of PARP.
Control lanes (cont) refer to untreated cells. In the other lanes,
the cells were treated with the indicated compound at 1 µM
for 24 h. In each case, whole cell lysates were subjected to
SDS-PAGE followed by blotting with an anti-PARP mono-
clonal antibody. Etoposide (Etop.) was used at 10 µM. The two
arrows point to the full-length PARP (116-kDa band) and the
89-kDa cleavage fragment.
rying the DEVD peptide backbone was also detected
with 8 (Figure 4).
Caspase activation was also evidenced by Western
blot analysis to monitor the cleavage of poly(ADP-ribose)
polymerase (PARP), an enzyme involved in DNA repair
that serves as a substrate for caspase-3. As shown in
Figure 5, the 116-kDa PARP protein was cleaved into
its characteristic 89-kDa fragment upon treatment of
the cells with 1 µM 3 and 8 (and with the control drug
etoposide) but not with the other compounds. The
cleavage of PARP was detected upon treatment of the
cells with concentrations of 3 and 8 g 0.5 µM (data not
shown), i.e. the concentrations for which the hypodiploid
peak (sub-G1) starts to appear in the cell cycle experi-
ments. It must be noted, however, that the extent of
PARP cleavage always remained limited to about 30-
50%, as we could never detect quantitative cleavage of
the protein. We are inclined to believe that two cell
death mechanisms coexist: one related to apoptosis and
the other possibly arising from necrosis. This hypothesis
is supported by the following cytometry data.
(3) Loss of P la sm a Mem br a n e Asym m etr y d u r -
in g Ap op tosis. To further characterize the cell death
mechanism induced by 3 and 8, we performed a bipara-
metric cytofluorimetric analysis using propidium iodide
and annexin V-FITC, which stain DNA and phospha-
tidylserine (PS) residues, respectively. Annexin V is a
Ca2+-dependent phospholipid binding protein with a
high affinity for PS. After the drug treatment, CEM cells
were labeled with these two dyes and washed, and the
resulting red (PI) and green (FITC) fluorescence was
monitored by flow cytometry. As indicated in Figure 6,
the percentage of annexin V positive cells slightly
increased upon treatment with 3 and 8. At 2 µM, about
11% of the cells stained positively with annexin V and
this cell population, characteristic of an early stage of
apoptosis, always remained <17%, even with a higher
drug concentration (data not shown). Rapidly, we de-
tected the accumulation of cells doubly stained in green
with annexin V and in red by PI, corresponding to late
stages of apoptosis and/or dead or necrotic cells. The loss
of membrane asymmetry and the ensuing increased
accessibility of PS residues to annexin V-FITC is
another criteria indicating that the two cytotoxic drugs
induce apoptosis, but it is clear from these experiments
that compounds 3 and 8 can also cause cell death via a
nonapoptotic pathway.
F igu r e 6. Drug-induced externalization of phosphatidylserine
residues. CEM cells were treated with 0.5 or 2 µM 3 or 8 for
24 h and then stained with propidium iodide (PI) and an
annexin V-FITC conjugate specifically detecting the exposure
of phosphatidylserine (PS) residues at the cell surface. Fluo-
rescence intensities are shown as log scales. In each case, the
percentage of annexin-V+/IP- cells (lower right square) and
annexin-V+/IP+ cells (upper right square) is given ((3%).
Results show a typical experiment that has been repeated
three times.
Altogether, the results give a very consistent picture
of the drug mechanism of action. On one hand, there is
absolutely no doubt that the newly discovered inhibitors
of tubulin assembly 3 and 8 induce apoptosis of the
CEM human leukemia cells. The induction of apoptosis
is associated with (i) characteristic cell cycle changes
(appearance of the sub-G1 population), (ii) loss of the
mitochondrial potential membrane (∆Ψmt), (iii) activa-
tion of caspases-3, and (iv) the loss of membrane
asymmetry (annexin V positivity). But in parallel, there
is apparently another cell death pathway activated by
the two cytotoxic arylcoumarin compounds. This second
pathway may explain why 8 is significantly more
cytotoxic than 3, whereas the two compounds perform
more or less similarly in terms of drug-induced apop-
tosis. The high cytotoxic potential of 8 is not directly
related to its proapoptotic properties, and another mode
of cell killing, as yet undefined, must be implicated.
Con clu sion
The two 4-phenylcoumarin derivatives 3 and 8 have
been identified as potent cytotoxic agents. The 3′-OH
and a 4′-OCH3 substituents on the 4-phenyl C-ring play
an essential role in the cytotoxic action. Compounds 3
and 8 have no effects on human topoisomerases I and
II but potently inhibit microtubule assembly. They can
therefore be considered as functional analogues of
combretastatin A-4, validating thus the design strategy.
At the cell level, the two compounds were characterized
as proapoptotic agents, but they can also trigger cell
death via a nonapoptotic pathway. Compounds 3 and 8

5442 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25
Bailly et al.
128.7, 129.0, 131.5, 136.8, 139.6, 151.2, 151.8, 155.2, 156.8,
158.9, 160.8.
represent interesting candidates for a preclinical devel-
opment of novel anticancer agents.
4-(4′-Ben zyloxy-3′-m et h oxyp h en yl)-5,6,7-t r im et h oxy-
ch r om en -2-on e 17: fine white needles, 89%; mp 109 °C
(MeOH); δH 3.27 (3H, s), 3.80 (3H, s), 3.89 (3H, s), 3.94 (3H,
s), 5.22 (2H, s), 6.07 (1H, s), 6.72 (1H, s), 6.83 (1H, dd, J 8.3
and 2), 6.88 (1H, d, J 2), 6.92 (1H, d, J 8.3), 7.31-7.48 (5H,
m); δC 56.1, 56.3, 61.1, 61.3, 71.0, 96.3, 107.3, 111.8, 113.1,
114.0, 119.8, 127.3, 127.9, 128.6, 132.1, 136.9, 139.5, 148.1,
148.7, 151.1, 151.8, 155.1, 156.8, 160.7.
4-(3′-Ben zyloxy-4′-m et h oxyp h en yl)-5,6,7-t r im et h oxy-
ch r om en -2-on e 18: fine light yellow needles, 96%; mp 70 °C
(MeOH); δH 3.17 (3H, s), 3.78 (3H, s), 3.93 (3H, s), 3.95 (3H,
s), 5.17 (2H, s), 6.01 (1H, s), 6.70 (1H, s), 6.90-6.92 (3H, m)
and 7.27-7.44 (5H, m); δC 56.1, 56.3, 61.1, 61.2, 71.2, 96.4,
107.4, 110.8, 114.1, 114.3, 120.8, 127.4, 128.0, 128.6, 131.5,
137.0, 139.6, 147.1, 149.9, 151.2, 151.8, 155.1, 156.8, 160.8.
4-(4′-Ben zyloxy-3′,5′-d im eth oxyp h en yl)-5,6,7-tr im eth -
oxych r om en -2-on e 19: fine yellow needles, 90%; mp 105 °C;
δH 3.29 (3H, s), 3.80 (3H, s), 3.83 (6H, s), 3.94 (3H, s), 5.10
(2H, s), 6.10 (1H, s), 6.52 (2H, s), 6.71 (1H, s), 7.27-7.53 (5H,
m); δC 56.1, 56.2, 61.0, 61.1, 74.9, 96.1, 104.8, 107.2, 113.8,
127.9, 128.1, 128.5, 134.6, 136.4, 137.6, 139.4, 151.0, 151.6,
152.7, 155.2, 156.8, 160.6.
4-(3′-Be n zyloxy-4′-m e t h oxyp h e n yl)-5,7-d im e t h oxy-
ch r om en -2-on e 22: fine white needles, 96%; mp 184-185 °C
(lit.12 mp 100-102 °C); δH 3.33 (3H, s), 3.87 (3H, s), 3.95 (3H,
s), 5.13 (2H, s), 5.96 (1H, s,), 6.20 (1H, d, J 2.3), 6.51 (1H, d,
J 2.3), 6.84-6.92 (3H, m), 7.32-7.45 (5H, m).
4-(4′-Ben zyloxy-3′,5′-d im eth oxyp h en yl)-5,7-d im eth oxy-
ch r om en -2-on e 23: colorless plates, 86%; mp 155 °C; δH 3.43
(3H, s), 3.81 (6H, s), 3.87 (3H, s), 5.09 (2H, s), 6.04 (1H, s),
6.23 (1H, d, J 2.3), 6.47 (2H, s), 6.52 (1H, d, J 2.3), 7.30-7.53
(5H, m); δC 55.3, 55.7, 56.2, 74.9, 93.6, 95.7, 103.4, 104.7, 112.3,
127.8, 128.1, 128.5, 135.3, 136.4, 137.7, 152.7, 155.4, 157.1,
158.1, 160.7, 163.3.
Rem ova l of th e Ben zyl Gr ou p sGen er a l P r oced u r e. A
suspension of the substrate (0.5 mmol) in 47% aqueous HBr
was stirred at 50 °C for the indicated time. The mixture was
then poured into water (50 mL) and extracted with dichlo-
romethane (4 × 20 mL). The organic layers were combined,
washed with brine, and dried over Na2SO4, and the solvent
was distilled off under reduced pressure. The residue was
purified by column chromatography (eluant Et2O) or directly
by crystallization from ether to give the corresponding aryl-
coumarin.
4-(4′-Hyd r oxyp h en yl)-5,6,7 tr im eth oxych r om en -2-on e
1: reaction time 12 h; crystallized from ether as light pink
plates, 80%; mp 232 °C (AcOEt-Et2O); δH 3.29 (3H, s), 3.81
(3H, s), 3.94 (3H, s), 5.11 (1H, s), 6.06 (1H, s), 6.73 (1H, s),
6.88 (2H, d, J 8.5) and 7.24 (2H, d, J 8.5); δC 56.4, 6.13, 96.5,
107.4, 114.1, 114.5, 129.2, 131.3, 139.6, 151.3, 151.8, 155.4,
156.0, 156.9, 161.0. Anal. (C18H16O6) C, H.
4-(4′-H yd r oxy-3′-m e t h oxyp h e n yl)-5,6,7-t r im e t h oxy-
ch r om en -2-on e 2: reaction time 3 h; purified by column
chromatography (Et2O) as fine white needles, 83%; mp 154
°C (from AcOEt-Et2O); δH 3.32 (3H, s), 3.81 (3H, s), 3.90 (3H,
s), 3.94 (3H, s), 5.72 (1H, s), 6.08 (1H, s), 6.72 (1H, s), 6.85-
6.89 (2H, m), 6.96 (1H, d, J 8.5); δC 56.1, 56.3, 61.2, 61.3, 96.4,
107.4, 110.7, 113.6, 114.1, 120.7, 130.9, 139.6, 145.7, 145.9,
151.2, 151.8, 155.3, 156.9, 160.8. Anal. (C19H18O7) C, H.
4-(3′-H yd r oxy-4′-m e t h oxyp h e n yl)-5,6,7-t r im e t h oxy-
ch r om en -2-on e 3: reaction time 3 h; purified by column
chromatography (Et2O) as fine white needles, 79%; mp 157
°C (AcOEt-Et2O-pentane 1:2:1); δH 3.36 (3H, s), 3.80 (3H,
s), 3.94 (3H, s), 3.95 (3H, s), 6.06 (1H, s), 6.71 (1H, s), 6.83
(1H, dd, J 8.2 and 2.1), 6.99 (1H, d, J 8.2) and 6.92 (1H, d, J
2.1); δC 56.0, 56.3, 61.1, 61.2, 96.3, 107.4, 109.8, 114.1, 114.2,
119.3, 132.3, 139.6, 144.7, 146.7, 151.3, 151.8, 155.2, 156.8,
160.8. Anal. (C19H18O7) C, H.
Exp er im en ta l Section
Ch em istr y. Melting points were taken on a Bu¨chi capillary
apparatus and are uncorrected. NMR spectra were obtained
on a Bruker AC 300 spectrometer. Chemical shifts (δ) are
reported in ppm for a solution of the compound in CDCl3 with
internal reference Me4Si, and J values are in hertz. Combus-
tion analyses were performed at the Laboratoire de Microanal-
yse of the Centre National de la Recherche Scientifique,
Vernaison, France. Separation by column chromatography was
performed using Merck Kieselgel 60 (70-230 mesh). Ether
refers to diethyl ether, and petroleum spirit refers to the
fraction with distillation range 40-65 °C. All solvents were
purified by standard techniques. Arylboronic acids 12-15 were
prepared by reported procedures involving reaction of the
appropriate aryllithium with triisopropylborate.12,22 5,7-
Dimethoxy-4-trifluoromethylsulfonyloxychromen-2-one 11,11
4-(4′-benzyloxyphenyl)-5,7-dimethoxychromen-2-one 20,12 and
4-(4′-benzyloxy-3′-methoxyphenyl)-5,7-dimethoxychromen-2-
one 2112 were prepared as previously reported.
5,6,7-T r i m e t h o x y -4-t r i flu o r o m e t h y ls u lfo n y lo x y -
ch r om en -2-on e 10. Trifluoromethanesulfonic anhydride (3.29
mL, 1.3 equiv) was added dropwise over 10 min to a mixture
of the 4-hydroxy-5,6,7-trimethoxycoumarin13 (3.78 g, 15 mmol)
and triethylamine (2.72 mL, 1.3 equiv) in dry dichloromethane
(100 mL) at 0 °C. Then the mixture was stirred for 1 h at 0
°C. After dilution with 50% ether-petroleum spirit (200 mL)
and filtration through a short pad of silica, the solvent was
distilled off under reduced pressure to a small volume (10 mL)
and the residue kept at -15 °C overnight. The precipitate was
collected and washed with petroleum spirit to afford 10 as fine
white needles (5.01 g, 87%): mp 149 °C (EtOH); δH 3.88 (3H,
s), 3.96 (3H, s), 4.03 (3H, s), 6.14 (1H, s), 6.70 (1H, s); δC 56.6
(6-OMe), 61.3, 61.8, 96.3, 102.3, 104.7, 118.6, 139.6, 149.6,
151.5, 157.6, 158.7, 160.1.
4-(3′,4′-Me t h y le n e d io x y p h e n y l)-5,6,7-t r im e t h o x y -
ch r om en -2-on e 5. A mixture of 10 (384 mg, 1 mmol), tetrakis-
(triphenylphosphine)palladium(0) (25 mg, 2 mol %), lithium
chloride (126 mg, 3 equiv), and tributyl-3,4-methylenediox-
yphenyltin15 (452 mg, 1.1 equiv) in dioxane (10 mL) was
refluxed overnight. After cooling at room temperature, pyridine
(1 mL) was added and the mixture stirred for a further 7 h.
The mixture was diluted with ether (50 mL), filtered through
a short path of silica, washed with 10% aqueous HCl (3 × 20
mL) and brine (20 mL), and dried over Na2SO4. Distillation of
the solvent under reduced pressure afforded a white solid
which was recrystallized from ethanol to yield 5 as fine white
needles (63%): mp 192-193 °C; δH 3.37 (3H, s), 3.81 (3H, s),
3.94 (3H, s), 6.03 (2H, s), 6.06 (1H, s), 6.72 (1H, s), 6.81-6.85
(3H, m); δC 56.3, 61.1, 61.2, 96.3, 101.3, 107.3, 107.6, 108.5,
114.1, 120.9, 132.7, 139.5, 146.9, 147.6, 151.1, 151.7, 154.9,
156.9, 160.6. Anal. (C19H16O7) C, H.
Cou p lin g of 4-Tr iflu or om eth ylsu lfon yloxycou m a r in s
w ith Ar ylbor on ic Acid ssGen er a l P r oced u r e. A mixture
of 4-trifluoromethylsulfonyloxycoumarin (1 mmol), tetrakis-
(triphenylphosphine)palladium(0) (46 mg, 4 mol %), copper(I)
iodide (210 mg, 1.1 equiv), sodium carbonate (742 mg, 7 equiv),
and arylboronic acid (1.5 equiv) in dry benzene (10 mL) and
absolute ethanol (3 mL) was refluxed overnight. Then the
reaction mixture was diluted with chloroform (40 mL) and
filtered through Celite. The filtrate was washed with
a
saturated aqueous solution of sodium bicarbonate (3 × 20 mL),
and the combined aqueous layers were extracted with chloro-
form (3 × 20 mL). The organic phases were combined, washed
with brine, and dried over Na2SO4, and the solvent was
distilled under reduced pressure. The residue was crystallized
from ether to afford the 4-arylchromen-2-one derivative.
4-(4′-Ben zyloxyp h en yl)-5,6,7-t r im et h oxych r om en -2-
on e 16: colorless plates, 69%; mp 130 °C; δH 3.27 (3H, s), 3.81
(3H, s), 3.94 (3H, s), 5.13 (2H, s), 6.06 (1H, s), 6.72 (1H, s),
7.01 (2H, d, J 8.7), 7.29 (2H, d, J 8.7), 7.38-7.48 (5H, m); δC
56.3, 61.1, 61.2, 70.1, 96.4, 107.4, 114.0, 114.1, 127.6, 128.1,
4-(4′-H yd r oxy-3′,5′-d im et h oxyp h en yl)-5,6,7-t r im et h -
oxych r om en -2-on e 4: reaction time 1 h; crystallized from
ether as fine white needles, 88%; mp 205 °C (CH2Cl2-Et2O);
δH 3.36 (3H, s), 3.81 (3H, s), 3.90 (6H, s), 3.94 (3H, s), 5.62

4-Arylcoumarin Analogues of Combretastatins
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 25 5443
(1H, s), 6.10 (1H, s), 6.58 (2H, s), 6.72 (1H, s); δC 56.2, 56.4,
61.0, 61.2, 96.2, 104.7, 107.2, 113.9, 129.9, 134.8, 139.5, 146.3,
151.0, 151.7, 155.3, 156.8, 160.6. Anal. (C20H20O8) C, H.
4-(4′-Hyd r oxyp h en yl)-5,7-d im eth oxych r om en -2-on e 6.
Reaction time 12 h; crystallized from ether as fine white
needles, 69%, mp 204 °C (lit.12 mp 210-213 °C).
4-(4′-H y d r o x y -3′-m e t h o x y p h e n y l)-5,7-d im e t h o x y -
ch r om en -2-on e 7. Reaction time 3 h; purified by column
chromatography (Et2O) as white needles, 62%, mp 172 °C
(CHCl3-Et2O) (lit.12 mp 172-173 °C).
4-(3′-H y d r o x y -4′-m e t h o x y p h e n y l)-5,7-d im e t h o x y -
ch r om en -2-on e 8: reaction time 48 h; purified by column
chromatography (Et2O) as white needles, 74%; mp 152 °C
(CHCl3-Et2O) [lit.12 mp 153 °C (from EtOH)].
Mitoch on d r ia l En er giza tion . Mitochondrial energization
was determined as the retention of the fluorescent dye DiOC6
(3,3-dihexyloxacarbocyanine iodide, Molecular Probes, Inc.).
After the drug treatment, 106 cells in 1 mL of complete RPMI
1640 medium were loaded with the probe DiOC6 (usually 25
nM unless otherwise stated) during 30 min at 37 °C prior to
the flow cytometric analysis. The same incubation time was
applied to the controls and the drug-treated samples. Control
experiments were performed by incubating cells with CCCP
(carbonyl cyanide p-chlorophenylhydrazone) at 5 and 50 µM
for 10 min at 37 °C. DiOC6 was excited at 488 nm and
fluorescence analyzed at 525 nm (Fl-1) after logarithmic
amplification. Forward scattering (FSC) and side scattering
(SSC) were analyzed after linear amplification.
Ca sp a se Activa tion (DEVD cleavage activity). After the
drug treatment, the cells were centrifuged, washed with PBS,
and resuspended in 50 µL of PhiPhiLux-G1D2 substrate
(OncoImmunin Inc, MD) for 1 h at 37 °C following the
manufacturer’s protocol. Cleavage of the peptide linker of
PhiPhiLux (sequence DEVD) separates the rhodamine moi-
eties and results in fluorescence detectable by flow cytometry.
P ARP Clea va ge. Briefly, 7 × 105 exponentially growing
CEM cells in complete medium were treated with the test drug
at the indicated concentration for 24 h at 37 °C. Cells were
pelleted by centrifugation, washed with ice-cold PBS, and
resuspended in 100 µL of lysis buffer containing 10 mM Tris
pH 7.4, 1 mM sodium vanadate, 1% sodium dodecyl sulfate,
0.1 mM PMSF (added extemporaneously), and the protease
inhibitor cocktail (1/1000 dilution, Sigma). Cell lysis was per-
formed for 10 min at 4 °C. The viscosity of the resulting
solution was reduced by passage through a 26 gauge needle,
prior to measuring the protein content (Bradford method). The
protein sample (15 µg), mixed with the loading dye (0.01%
bromophenol blue and glycerol), was then boiled to 100 °C for
3 min, prior to electrophoresis on a 7.5% polyacrylamide gel
containing 0.1% SDS. Next, for Western blotting, the proteins
are transferred onto a Hybond-C nitrocellulose membrane
(Amersham) for 90 min at 100 V with a wet transfer system
(Biorad). Membranes were blocked with 10% nonfat milk in
PBST (0.1% Tween-20, 25 mM phosphate buffer, pH 6.5) for
60 min followed by incubation with anti-PARP polyclonal
antibody (Santa Cruz Biotechnology) (dilution 1:1000 in PBST
supplemented with 1% nonfat milk) for 60 min. The blots were
washed three times (5 min each with PBST) and incubated
with a donkey anti-rabbit IgG conjugated to horseradish
peroxidase (Santa Cruz Biotechnology, 1:10 000 dilution in
PBST containing 1% nonfat milk) for 45 min. After three
successive washes with PBST, the Western blot chemilumi-
nescence reagent from NEN (Boston, MA) was used for the
detection. Bands were visualized by autoradiography.
4-(4′-H yd r oxy-3′,5′-d im et h oxyp h en yl)-5,7-d im et h oxy-
ch r om en -2-on e 9: reaction time 12 h; crystallized from ether
as fine white needles, 96%; mp 215 °C (CHCl3-Et2O); δH 3.51
(3H, s), 3.88 (3H, s), 3.89 (6H, s), 5.62 (1H, s), 6.04 (1H, s),
6.26 (1H, d, J 2.5), 6.52 (2H, s), 6.54 (1H, d, J 2.5); δC 55.5,
55.8, 56.4, 93.6, 95.8, 103.5, 104.5, 112.5, 130.8, 134.8, 146.3,
155.6, 157.2, 158.2, 160.9, 163.3. Anal. (C19H18O7) C, H.
Biology: Top oisom er a ses In h ibition . The experimental
procedure has been previously detailed.23
Cell Cu ltu r es a n d Su r viva l Assa y. Human CEM leuke-
mia cells were obtained from the American Tissue Culture
Collection. Cells were grown at 37 °C in a humidified atmo-
sphere containing 5% CO2 in RPMI 1640 medium, supple-
mented with 10% fetal bovine serum, l-glutamine (2 mM), 1.5
g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1 mM
sodium pyruvate, penicillin (100 IU/mL), and streptomycin
(100 µg/mL). The cytotoxicity of the test compounds was
assessed using a cell proliferation assay developed by Promega
(CellTiter 96 AQueous one solution cell proliferation assay).
Briefly, 3 × 104 exponentially growing cells were seeded in
96-well microculture plates with various drug concentrations
in a volume of 100 µL. After 72 h incubation at 37 °C, 20 µL
of the tetrazolium dye was added to each well, and the samples
were incubated for a further 2 h at 37 °C. Plates were analyzed
on a Labsystems Multiskan MS (type 352) reader at 492 nm.
P r ep a r a tion of La m b Br a in Tu bu lin . Tubulin was
purified from lamb brain by ammonium sulfate fractionation
and ion-exchange chromatography. The protein was stored in
liquid nitrogen and prepared as described.24-26 Protein con-
centrations were determined spectrophotometrically with a
Perkin-Elmer spectrophotometer Lambda 800 and an extinc-
tion coefficient at 275 nm of 1.07 L g-1 cm-1 in 0.5% SDS in
neutral aqueous buffer or 1.09 L g-1 cm-1 in 6 M guanidine
hydrochloride.
Tu bu lin P olym er iza tion . Microtubule assembly was per-
formed in 20 mM sodium phosphate buffer, 1 mM EGTA, 10
mM MgCl2, and 3.4 M glycerol, pH 6.5. The reaction was
started by warming the samples at 37 °C in thermostated
cuvettes (1 × 0.2 cm), and the mass of polymer formed was
monitored by turbidimetry at 350 nm with a Beckman DU7400
spectrophotometer. Samples containing the compound and
their controls had less than 2% residual Me2SO.
Ack n ow led gm en t. This work was done under the
support of research grants (to C.B.) from the “Ligue
Nationale Franc¸aise Contre le Cancer (Equipe labellise´e
LA LIGUE)”. It was also partially supported by grants
from “la Ligue Contre le Cancer” and grants no. 7264
from ARC (to P.B. and V.P.).
Electr on Micr oscop y. Small aliquots of the solution were
adsorbed to carbon-coated Formvar films on copper grids,
stained for 1 min in 2% uranyl acetate, and observed with a
J EOL 1200 electron microscope.
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