Novel combretastatin analogues endowed with antitumor activity

Article abstract of DOI:10.1021/jm0510732

We studied the anticancer activity of a series of new combretastatin derivatives with B-ring modifications. The structure-activity relationship (SAR) information confirmed the importance of cis-stereochemistry and of a phenolic moiety in B-ring. We select

Full text of DOI:10.1021/jm0510732

J. Med. Chem. 2006, 49, 3143-3152
3143
Novel Combretastatin Analogues Endowed with Antitumor Activity
Daniele Simoni,*,† Romeo Romagnoli,† Riccardo Baruchello,† Riccardo Rondanin,† Michele Rizzi,† Maria Giovanna Pavani,†
Domenico Alloatti,‡ Giuseppe Giannini,‡ Marcella Marcellini,‡ Teresa Riccioni,‡ Massimo Castorina,‡ Mario B. Guglielmi,‡
Federica Bucci,‡ Paolo Carminati,‡ and Claudio Pisano*,‡
Dipartimento di Scienze Farmaceutiche, UniVersita` di Ferrara, Via Fossato di Mortara 17/19, 44100 Ferrara, Italy, and
R&D, Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Pomezia, Italy
ReceiVed October 24, 2005
We studied the anticancer activity of a series of new combretastatin derivatives with B-ring modifications.
The structure-activity relationship (SAR) information confirmed the importance of cis-stereochemistry and
of a phenolic moiety in B-ring. We selected the benzo[b]thiophene and benzofuran combretastatin analogues
11 (ST2151) and 13 (ST2179) and their phosphate prodrugs (29 and 30) for their high antitumor activity in
in vitro and in vivo models. Cell exposure to IC50 of 11, 13, and CA-4 led to the arrest of various cell types
in the G2/M phase of the cell cycle and induction of apoptosis. Mainly, 11 and 13 induced the formation of
multinucleated cells with abnormal chromatin distribution, with only a minimal effect on the microtubule
organization, with respect to CA-4. Interestingly, both the pharmacokinetic profile of 29 and its in vivo
antitumor effect and those of 30, active even after oral administration, suggest additional pharmacological
differences between these compounds and CA-4P.
Introduction
report that the B-ring of CA-4 can be replaced with naphtha-
lene,22 quinoline, or a variety of six-membered heterocycles20
(pyridine, pyrimidine, and pyridazine) without significant loss
of antimitotic and cytotoxic activity.
Vascular targeting agents (VTAs) induce occlusion of pre-
existing tumor blood vessels, producing tumor cell death from
ischemia and extensive hemorrhagic necrosis.1 The potential of
vascular targeting as a therapeutic approach to cancer has been
firmly established in several experimental studies.2 In fact,
preclinical studies show that VTAs exert an impressive antitu-
mor activity when used as single chemotherapeutic agents.3
When combined, they enhance the effects of conventional
chemotherapeutic agents4 such as radiation, hyperthermia,
radioimmunotherapy, and antiangiogenic agents.5-8
Combretastatin A-4 (CA-4, compound 1), a natural stilbenoid
isolated from Combretum caffrum, is a new VTA molecule
known for its antitumor activity due to its anti-tubulin
properties.9-15 The corresponding sodium phosphate prodrug
(CA-4P, 2) is water-soluble and currently showing promising
results in phase I human cancer clinical trials.16,17 Moreover,
other derivatives that carry the phosphate ester moiety are of
particular interest in the quest for new anticancer drugs.18,19 In
the case of CA-4, its structural simplicity, along with its ability
to selectively damage tumor neovascularization, makes it very
interesting from a medicinal chemistry point of view, stimulating
the search for new and more potent compounds with improved
pharmacological properties.20 Previous structure-activity rela-
tionship (SAR) studies on CA-4 established that the cis-
configuration of two benzene rings and methoxy groups at
positions 3, 4, and 5 in the A-ring and at position 4 in the B-ring
are essential for biological activity.20
Therefore, in an ongoing project aimed at developing novel
apoptosis inducer agents, we set out to study a novel class of
stilbenes, structurally related to CA-4, focusing our attention
on different ring modifications. Since the 3,4,5-trimethoxy
substituent in the A-ring is essential for CA-4 activity, we
maintained this substituted pattern throughout the present
investigation, examining the effects due to replacement of the
B-ring with different benzoheterocycles, obtaining a small
library of novel combretastatins (Figure 1). We concentrated
on benzofuran and benzothiophene rings as the most interesting
substitutions but also synthesized indole, indazole, and naph-
thalene derivatives. An additional oxygenated function (methoxy
or hydroxy) was always present and both cis- and trans-
stereoisomers were obtained. Benzofuran and benzothiophene
moieties have already been reported in the literature as bridge
modifications of combretastatin-like derivatives maintaining the
antitubulin activity.23,24
After evaluation of a primary cytotoxicity screening, the most
interesting derivatives were considered for further investigation.
In particular, we selected the benzo[b]thiophene and the
benzofuran molecules 11 (ST2151) and 13 (ST2179) and their
phosphate prodrugs 29 (ST2495) and 30 (ST2496),25 respec-
tively (Table 1), for their high antitumor activity against different
cancer models. A very important feature emerging from the
detailed biological investigation into these compounds, reported
here, is their potent antitumor activity, which could represent
the basis for development of a novel chemotherapeutic drug.
Recently, we started a study aimed at evaluating the apoptotic
activity of natural and synthetic stilbenoids structurally related
to CA-4.21 The alkenyl motif of CA-4 was replaced by five-
and six-membered rings, leading in some cases to compounds
with potent apoptotic activity. However, an investigation of cell
cycle distribution and tubulin interaction suggested mechanisms
not involving the mitotic spindle. Moreover, several publications
Chemistry
Scheme 1 outlines the synthetic route followed for the
synthesis of the novel combretastatins (see Table 1). The
heteroaromatic aldehydes 3a-j (Figure 2) were transformed into
the corresponding benzoheterocycles 4a-j (Figure 3) by a
Stobbe condensation using diethyl succinate, followed by
cyclization as reported in the literature.26 The phenol functions
* To whom correspondence should be addressed: Phone: +39-(0)532-
291291. Fax: +39-(0)532-291296. E-mail: smd@unife.it.
† Universita` di Ferrara.
‡ SIGMA-TAU Industrie Farmaceutiche Riunite S.p.A.
10.1021/jm0510732 CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/06/2006

3144 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Simoni et al.
Figure 1. Natural and synthetic combretastatins.
Scheme 1a
Table 1. IC50 on BMEC Cell Line Determined for All New
Combretastatin Derivativesa
B
compd ring
X
E or Z
R
R2
IC50, µM
CA-4
CA-4P
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
C
C
C
C
D
D
D
D
E
E
E
E
-
-
Z
Z
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
Z
Z
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
H
-
-
0.0037 ( 0.0003
0.056 ( 0.0024
0.087 ( 0.001
38.7 ( 9.6
PO3Na2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
PO3Na2
PO3Na2
-
-
-
-
H
H
CH3
S
S
O
O
S
S
O
O
S
S
O
O
S
S
S
S
O
O
S
O
S
S
O
O
-
H
H
H
H
2-CH3
2-CH3
2-CH3
2-CH3
0.049 ( 0.001
27.7 ( 9.4
0.023 ( 0.0074
0.84 ( 0.09
0.093 ( 0.0069
12.3 ( 0.83
3-phenyl >2
3-phenyl >20
2-phenyl >2
a Reagents and conditions: (a) (CH2COOEt)2, t-BuOH, KOBu, reflux,
2 h; (b) Ac2O, AcONa, reflux, 5 h; (c) K2CO3, EtOH, reflux; (d) Me2SO4,
K2CO3, THF; (e) imidazole, TBDMSCl, CH2Cl2; (f) LiAlH4, THF; (g)
MnO2, CCl4; (h) 3,4,5-trimethoxybenzyltriphenylphosphonium bromide,
NaH, THF, 0 °C; (i) TBAF, CH2Cl2.
2-phenyl >20
2-phenyl 23 ( 3.2
2-phenyl 38 ( 4.5
H
H
H
H
H
H
-
-
-
-
-
-
-
-
-
8.1 ( 0.3
53 ( 6.5
7.5 ( 0.9
75 ( 6.7
0.64 ( 0.04
0.34 ( 0.01
0.35 ( 0,02
24.8 ( 2.6
0.054 ( 0.004
8.4 ( 1.6
0.67 ( 0.045
56.4 ( 9.8
10 ( 0.7
-
-
-
-
-
-
-
CH3
70 ( 10
-
-
-
-
20 ( 6
-
-
-
32 ( 4
0.028 ( 0.0055
1.9 ( 0.229
a Cytotoxic activity was assayed by exposure for 24 h to substances and
expressed as concentration required to inhibit tumor cell proliferation by
50% (IC50). Data are expressed as mean ( SE from the dose-response
curves of at least three independent experiments.
Figure 2. Heterocyclic aldehydes as starting material in Scheme 1.
reported procedure. Finally, aldehydes 7a-c, 8a-i, 10b,c and
commercial indole-6-carbaldehyde were coupled with 3,4,5-
trimethoxybenzyltriphenylphosphorane following a standard
Wittig procedure. The phenolic moieties of the E/Z crude
mixtures of silyl ethers were revealed by treatment with TBAF.
The 1:1 mixture of the Z and E stilbene isomers was separated
by silica gel column chromatography and the isomers identified
were protected with TBDMSCl, to give compounds 6a-i, or
methylated with dimethyl sulfate, to obtain compounds 5a-c.
Then, the carboxylic esters were converted into aldehydes 7a-c
and 8a-i by reduction with LiAlH4 and subsequent mild
oxidation using MnO2. Naphthalene aldehyde analogues 10b,c
were obtained similarly from the known esters 9a,c,27-29 whose
synthesis was also carried out by benzaldehyde using the above-

NoVel Antitumor Combretastatin Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3145
Scheme 2a
a Reagents and conditions: (a) AcCl, C5H5N, CH2Cl2; (b) MeOH,
mercury lamp; (c) K2CO3, EtOH, reflux; (d) (BnO)2P(O)H, DIEA, CH3CN,
CCl4, -25 °C; (e) TMSCl, NaI, CH3CN; (f) NaOCH3, CH3OH.
Table 2. In Vitro Tubulin Polymerization Inhibition (TPI) and
Colchicine Tubulin Binding Competition Assay (CTBC) of CA-4, 11,
and 13
IC50 ( SE (µM)
compd
TPI
CTBC
CA-4
11
13
4.92 ( 0.2
24.6 ( 1.5
10.3 ( 0.05
0.0129 ( 0.009
1.16 ( 0.0485
2.5 ( 0.0796
it is well-documented in the literature.33,34 The phenolic moiety
seems to have an important role in this class of compounds;
indeed, the synthesized methoxy derivatives, 25 and 27,
demonstrated a dramatic loss of potency compared to their
corresponding phenols, 11 and 13. Furthermore, even the indolic
Z-derivative, 41, shows a very low IC50, suggesting that another
hydrogen-bond donor could replace the hydroxy group. In the
hydroxylated benzofuran and benzothiophene series of deriva-
tives, activity does not seem greatly influenced by the position
of the endocyclic heteroatom (see 11-14 vs 31-34).
Subsequently, in an effort to better understand the structure-
activity relationships of this class of derivatives, we studied the
influence of a substituent at the 2- and 3-positions of the
heterocyclic system of compounds 15-24, through alteration
of its steric bulk, seeking a potentially positive lipophilic
interaction capable of enhancing the observed activities. The
synthesized series, with a methyl or phenyl group at the said
positions, offered the interesting information (Table 1) that
methyl derivatives 15 and 17 show almost unchanged activity
compared to 11 and 13. On the contrary, a phenyl ring, either
at the 2- or 3-position (compounds 19, 21, and 23), causes an
important loss of potency. Thus, only small substituents seem
to be allowed for the 2- and 3-positions, in some cases showing
slight improvement in activity.
Among synthesized compounds we selected two new mol-
ecules, 11 and 13, and their phosphate prodrugs, 29 and 30,
respectively, for their antitumor activity in different cancer
models. Preliminary screening was carried out, analyzing the
activity of the new compounds in both in vitro tubulin
polymerization inhibition (TPI) and colchicine tubulin binding
competition assay (CTBC). The results show that 11 and 13
determine a relatively lower inhibition of tubulin polymerization
compared to that of CA-4 (Table 2). On the other hand, 11 and
13 show a 100-200-fold reduction in affinity to the colchicine
binding site compared to CA-4.
Figure 3. Synthetic intermediates as reported in Scheme 1.
by NMR for their different coupling constants between alkenyl
proton signals (about 12 Hz for Z-isomers and 16 Hz for
E-isomers).30 Following initial results from biological assays,
and in order to optimize the yield of the more active cis
compounds, we exploited a photochemical isomerization31 that
converted the O-acetylated E-isomers from 12 (ST2152) and
14 (ST2180)25 into the corresponding Z-isomers; then, by
detachment of the acetyl group with ethanolic K2CO3, we
obtained the desired phenolic derivatives 11 and 13 (Scheme
2). In an attempt to prepare derivatives having enhanced aqueous
solubility that can be more readily formulated, compounds 11
and 13 were transformed into the corresponding sodium
phosphate prodrugs 29 and 30, respectively, using a previously
published three-step phosphorylation protocol.32
Biological Results and Discussion
All the synthesized compounds were tested in a preliminary
cytotoxicity assay on a BMEC cell line, the results being
reported in Table 1. At first glance, it is evident that there is a
difference in activity between E- and Z-stereoisomers, the latter
being unambiguously more potent, which is unsurprising, since

3146 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Simoni et al.
Table 3. Antiproliferative Effect of CA-4, CA-4P, 11, 13, and Their Phosphate Prodrugs against the Proliferation of Several Cell Linesa
IC50 (nM)
cell line
cell type
CA-4
CA-4P
11
29
13
30
HUVEC
H460
HCT116
MeWo
MCF-7
MCF-7 dx
A2780
human endothelium
1.5 ( 0.4
1.24 ( 0.02
1.9 ( 0.01
4 ( 0.0001
2 ( 0.064
2.3 ( 0.018
2.19 ( 0.08
1.66 ( 0.1
34 ( 0.34
37 ( 1
16 ( 0.13
10 ( 1.2
23 ( 1.1
9.4 ( 21
nd
49 ( 0.64
74 ( 2.9
84 ( 3.8
68 ( 5
83 ( 2.06
620 ( 65
3000 ( 102
830 ( 0.13
639 ( 21
487 ( 14.8
500 ( 20
750 ( 50
31 ( 1.7
53 ( 1.3
54 ( 9
85 ( 1.2
780 ( 3.4
1680 ( 70
820 ( 77
646 ( 29
357 ( 9.4
260 ( 6
human lung carcinoma
human colon carcinoma
human melanoma
human breast carcinoma
human breast carcinoma
human ovarian carcinoma
doxorubicin-resistant
human ovarian carcinoma
human ovarian carcinoma
71 ( 17
38 ( 3
66 ( 2.7
54 ( 2.1
70 ( 2
28 ( 0.5
50 ( 2
46 ( 4.4
A2780 dx
22 ( 0.6
95 ( 7
290 ( 16
IGROV-1
10.3 ( 0.55
nd
280 ( 4
1610 ( 67
110 ( 6
680 ( 60
a Cytotoxic activity was assayed by exposure for 24 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50%
(IC50). Data are expressed as mean ( SE from the dose-response curves of at least three independent experiments; nd ) not determined.
Table 4. Effects of CA-4, 11, and 13 on Cell Cycle
evaluated their activity on the alteration of the microtubule
network by tubulin immunostaining, comparing it to that of CA-
4. To this end, we added the drugs to human endothelial cells
(HUVEC) and human lung carcinoma cells (H460) at different
concentrations for 24 h. As expected, tubulin immunostaining
reveals that cells exposed to CA-4 at IC50, as well as at IC80
(data not shown), produce complete disruption of the microtu-
bule structure in endothelial and tumor cells, compared to
untreated cells (Figure 4, panels A, B). Instead of the clear
antimicrotubule effect of CA-4, 11 and 13 determine, at their
IC50 and IC80 levels, the formation of giant aberrant multinucle-
ated cells with abnormal chromatin distribution and a mild effect
on microtubule organization.
Further analysis, by counting the number of nuclei per cell,
reveals that 24-h exposition to IC80 of 11 and 13 in H460 cells
induces a significant 40-50% of multinucleated cells (p <
0.0001 vs control, Figure 5). This phenomenon is less evident
in cells treated with equitoxic concentrations of CA-4 (IC80)
and becomes more relevant only when starting from higher
concentrations (2 × IC80) of the molecule (p < 0.0001 vs
control). The peculiar cell phenotype, induced by 11 and 13
treatment, is retained by their prodrugs 29 and 30, when used
at the equitoxic concentration of parental compounds (data not
shown).
To investigate the antitumor activity of these compounds in
vivo, we first analyzed the pharmacokinetic behavior of
compound 29 (11 phosphate prodrug). After single intravenous
administration of compound 29 to healthy mice (100 mg/kg iv),
plasma samples were collected over 24 h and the plasma
concentrations of compounds 29 and 11 were simultaneously
determined by HPLC. Table 5 presents the pharmacokinetic
parameters, while Figure 6 shows the mean profile of plasma
levels. Plasma concentrations of both compounds are not
measurable 24 h after treatment. The apparent terminal elimina-
tion half-life (t1/2) of compound 29 is 1.32 h, similar to that
described for CA-4P,35 and there is a similar t1/2 (1.72 h) for
compound 11. However, the plasma concentrations of the more
G
1/0 (%)
S (%)
G2/M (%)
Apo (%)
24-h treatment
untreated
CA-4
11
46
8
16
8
29
12
27
25
26
79
57
67
0
19
40
26
13
24-h treatment + 24-h recovery
untreated
CA-4
11
58
62
36
13
29
22
38
43
13
16
27
44
0
30
30
24
13
24-h treatment + 48-h recovery
untreated
CA-4
11
76
55
57
52
13
18
13
22
11
27
30
26
0
47
40
35
13
The lower antitubulin activity of 11 and 13 correlates well
with their reduced cytotoxic potency with respect to CA-4. In
fact, although 11 and 13 determine cell growth inhibition in
various cell lines, their IC50 is about 20 to 50 times higher than
that of reference compound CA-4 (Tables 1 and 3). The
phosphate prodrugs 29 and 30 are 10-20 times less active than
their respective drugs in all cell lines, except for HUVEC, in
which the activity is only two times lower and similar to that
of CA-4P. This unexpected result might be related to a rapid
formation in HUVEC conditioned medium of more active
compounds 11 and 13 from phosphate hydrolysis of their
prodrugs 29 and 30. Alternatively, 29 and 30 might be
intrinsically more active against human endothelial cell with
respect to tumor cell lines.
The 4-methoxy Z-isomers 25 and 27, along with all E-
isomers, show dramatic reduction in cytotoxicity (IC50 > 5 µM,
data not shown) when tested on endothelial cells. These results
indicate that substitution of the 4-hydroxy with a 4-methoxy
group is detrimental to cytotoxic activity.
To compare the effects of both 11 and 13 on the cell cycle
with those of CA-4, NCI-H460 cells were exposed to the various
molecules for 24 h and allowed to recover in drug-free medium
for a further 24 and 48 h. There is a quite similar perturbation
of cell cycle progression in the H460 cell line after treatment
with equitoxic concentrations of 11 (160 nM), 13 (100 nM),
and CA-4 (3 nM), corresponding to their respective IC80 values
(Table 4). In these cells, there is an increase of sub-G1 cells,
consistent with the occurrence of apoptotic DNA fragmentation.
In addition, after 24-h treatment and during the next 48 h
following drug removal, there is a persistent accumulation of
cells in G2/M phases.
active compound 11 (AUC0-t ) 5.00 µg·h/mL) are ap-
z
proximately 39 times lower than those for the prodrug 29
(AUC0-t ) 282.43 µg·h/mL), thus suggesting that the release
z
kinetics of 11 from 29 differ from those of CA-4P to CA-4.34
In the in vivo experiments, 29 and 30, administered intrave-
nously at a dose of 60 mg/kg (q2dx6), produce the same
antitumor effects as the same CA-4P dose and schedule in a
human non-small lung cancer model (H460) xenografted in nude
mice (Figure 7). Interestingly, 29 and 30 significantly reduce
tumor growth even when administered orally (60 mg/kg; bid,
qdx5x3w).
In an attempt to identify the cellular effects that may be
relevant to the antiproliferative activity of 11 and 13, we
In conclusion, in an effort to obtain novel derivatives of
natural combretastatin A-4, we synthesized and tested a series

NoVel Antitumor Combretastatin Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3147
Figure 4. HUVEC (panel A) and H460 cells (panel B) were exposed at different concentrations of drugs (brackets) for 24 h and immunostained
with mAb anti-R-tubulin (sigma) as shown in a-k and with Hoechst to stain nuclei b-l.
Table 5. Pharmacokinetic Parameters of Compounds 29 and 11 in
Female Mice after Intravenous Bolus Injection of Prodrug 29 (100
mg/kg)
parametersa
C0 (µg/mL)
max (µg/mL)
max (h)
tz (h)
1/2 (h)
29
11
778.88
319.60
0.25
8
1.32
0
C
t
8.16
0.25
8
1.72
5.00
5.02
t
AUC0-t (µg h/mL)
282.43
282.63
z
AUC0-∞ (µg h/mL)
a C0, initial plasma concentration obtained by extrapolation to time 0;
max, highest measured concentration corresponding to tmax; tmax, time of
C
the maximum observed concentration; tz, time of last measurable concentra-
tion; t1/2, apparent terminal elimination half-life; AUC0-t , area under the
z
plasma concentration-time curve up to time tz; AUC0-∞, area under the
plasma concentration-time curve extrapolated to infinity.
of giant multinucleated cells. On the contrary, this pattern was
specular to that obtained by equal pharmacological active
concentrations of CA-4, which is characterized mostly by
microtubules damage. These observations suggest that the
activity of 11 and 13 might not be related solely to a
microtubule-damaging mechanism and that an additional mech-
anism(s) involved in mitosis control should be considered.
Interestingly, pharmacokinetic studies reveal that only a small
fraction of the more active drug 11 forms from compound 29.
Since 29 is as active on human endothelial cells as 11, it is
argued that both compounds may act as an antivascular agent.
Alternatively, the activity of 29 is realized through a complex
profile of metabolites (including 11) that can be produced after
intravenous injection of the prodrug, as described in the literature
for CA-4P.
Figure 5. Evaluation of multinucleated cells after 24-h exposure to
CA-4, 11, and 13. Values are mean ( SD from two independent
experiments. ∧p ) 0.07, *p < 0.05, **p < 0.0001 vs control; °p <
0.0001 compared to CA-4 IC80 treatment (two-tailed Student’s t-test).
of compounds modified at the B-ring. Several aromatic and
heteroaromatic rings were introduced, bearing an oxygenated
appendage. Our results suggest that benzofuran and ben-
zothiophene may be considered interesting compounds for the
development of potent antitumoral CA-4 derivatives. Further-
more, a hydroxylated function always produced higher activity
than did the corresponding methoxyl group. A hydrophobic
substitution at the 2- or 3-position of the heterocycle moiety
was tolerated only by the addition of a little steric bulk. All
trans isomers are definitely less active than the corresponding
cis isomers.
Materials and Methods
Concerning the in vitro results, we suggest that the tumor
cell lines treated with 11 and 13 undergo cell death mainly
through mitotic catastrophe, as demonstrated by the presence
Chemistry. Melting points were obtained with a Kofler apparatus
and are uncorrected. Reaction courses and product mixtures were
routinely monitored by thin-layer chromatography (TLC) on Merck

3148 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Simoni et al.
added. After 1 h at room temperature, the solution was diluted with
methylene chloride (5 mL), washed with water (3 × 5 mL) and
brine (5 mL), and dried (Na2SO4). After concentration, the residue
was purified by flash chromatography on silica gel.
Starting from 8a the following compounds were produced.
6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]benzo[b]thiophen-
4-ol (11). Yield: 41% (280 mg), colorless solid. Mp: 145-147
1
°C. H NMR (CDCl3) δ: 3.64 (s, 6H), 3.83 (s, 3H), 5.34 (s, 1H),
6.50 (d, J ) 12.6 Hz, 1H), 6.54 (s, 1H), 6.60 (d, J ) 12.6 Hz, 1H),
6.69 (s, 1H), 7.27 (s, 1H), 7.32 (d, J ) 5.6 Hz, 1H), 7.41 (d, J )
5.6 Hz, 1H), 7.42 (s, 1H). 13C NMR (CDCl3) δ: 56.0, 106.3, 109.5,
115.8, 119.8, 125.5, 129.6, 130.3, 153.0. Anal. (C19H18O4S) C, H,
S.
6-[(E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]benzo[b]thiophen-
4-ol (12). Yield: 43% (295 mg), yellow solid. Mp: 67-69 °C. 1H
NMR (CDCl3) δ: 3.88 (s, 6H), 3.92 (s, 3H), 5.50 (s, 1H), 6.74 (s,
2H), 6.93 (s, 1H), 7.03 (s, 2H), 7.35 (d, J ) 5.2 Hz, 1H), 7.43 (d,
J ) 5.2 Hz, 1H), 7.56 (s, 1H). 13C NMR (CDCl3) δ: 56.2, 61.1,
103.6, 106.6, 114.1, 119.9, 125.5, 128.1, 128.8, 129.1, 133.2, 135.2,
137.9, 142.4, 150.8, 153.5. Anal. (C18H19O4S) C, H, S.
Starting from 8b the following compounds were produced.
6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]benzofuran-4-ol (13).
Yield: 34% (220 mg), colorless solid that solidifies at 4 °C. Mp:
134-136 °C. 1H NMR (CDCl3) δ: 3.64 (s, 6H), 3.83 (s, 3H), 5.94
(s, 1H), 6.73 (s, 2H), 6.82 (d, J ) 6.0 Hz, 1H), 6.85 (d, J ) 2.2
Hz, 1H), 6.99 (s, 2H), 7.24 (d, J ) 6.0 Hz, 1H), 7.55 (d, J ) 2.2
Hz, 1H). 13C NMR (CDCl3) δ: 55.9, 61.0, 103.5, 105.2, 106.1,
108.6, 115.9, 129.8, 130.1, 132.5, 134.6, 144.3, 148.8, 152.9, 156.8.
Anal. (C19H18O5) C, H.
Figure 6. Mean profile ((SEM; n ) 3) of 29 and 11 plasma levels
(µg/mL) in female mice after intravenous bolus injection of prodrug
29 (100 mg/kg).
6-[(E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]benzofuran-4-ol (14).
1
Yield: 37% (240 mg), light yellow solid. Mp: 142-143 °C. H
NMR (CDCl3) δ: 3.89 (s, 6H), 3.92 (s, 3H), 5.50 (s, 1H), 6.74 (s,
2H), 6.93 (s, 1H), 7.03 (s, 2H), 7.35 (d, J ) 5.2 Hz, 1H), 7.43 (d,
J ) 5.2 Hz, 1H), 7.56 (s, 1H). Anal. (C19H18O5) C, H.
Starting from 8e the following compounds were produced.
6-[(Z)-2-(3,4,5-Trimethoxypheyl)ethenyl]-2-methylbenzo[b]-
thiophen-4-ol (15). Yield: 31% (220 mg), colorless solid. Mp:
118-120 °C. 1H NMR (CDCl3) δ: 2.56 (s, 3H), 3.65 (s, 6H), 3.84
(s, 3H), 5.20 (br, 1H), 6.47 (d, J ) 12 Hz, 1H), 6.54 (s, 2H), 6.57
(d, J ) 12 Hz, 1H), 6.64 (d, J ) 0.8 Hz, 1H), 7.03 (d, J ) 2 Hz,
1H), 7.30 (s, 1H). 13C NMR (CDCl3) δ: 16.2, 55.9, 60.9, 103.4,
106.0, 109.5, 115.4, 117.4, 128.2, 129.6, 129.8, 132.5, 133.7, 137.2,
140.1, 141.1, 152.9. MALDI-TOF: 357.6 [M+]. Anal. (C20H20O4S)
C, H, S.
Figure 7. Antitumor activity of CA-4P, 29, and 30 on human NCI-
H460 lung carcinoma. Tumor volume was determined on day 20 after
tumor cell injection. Values represent mean ( SD from two independent
experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. Neither
mortality nor reduction of body weight was observed.
precoated silica gel F254 plates. Nuclear magnetic resonance (1H
and 13C NMR) spectra were determined, with a Bruker AC-200
spectrometer, and peak positions are given in parts per million
downfield from tetramethylsilane as internal standard. Mass spectra
were obtained with a Bruker Daltonics MALDI-TOF (Omniflex
mod.). Photoisomerization was performed with a medium-pressure
mercury Hanau TQ 120 K lamp.
All drying operations were performed over anhydrous sodium
sulfate. Column chromatography (medium pressure) was carried
out with 60-200 mesh silica gel, using the flash technique. Yields
were given after chromatographic purification. Pure samples were
obtained after recrystallization from 2-propanol. Microanalysis of
all new synthesized compounds agreed within (0.4% of calculated
values.
General Procedure for the Synthesis of Stilbenes 11-28 and
31-42. 3,4,5-Trimethoxybenzyltriphenylphosphonium bromide (2
mmol, 1.05 g, 2 equiv) was added to a solution of aldehyde (7a-
c, 8a-i, or 10b,c, 2 mmol) in 10 mL of anhydrous THF. The
suspension thus obtained was cooled in an ice bath and then NaH
(50% in mineral suspension, 2.2 mmol, 1.1 equiv., 110 mg) was
added. The reaction was stirred at room temperature for 24 h,
filtered on a Celite bed, and washed with THF. After solvent
evaporation, the residue was extracted with methylene chloride (15
mL), washed with water (5 mL) and brine (5 mL), dried, and
evaporated again.
6-[(E)-2-(3,4,5-Trimethoxypheyl)ethenyl]-2-methylbenzo[b]-
thiophen-4-ol (16). Yield: 33% (235 mg), colorless solid. Mp:
200-202 °C. 1H NMR (CDCl3) δ: 2.59 (s, 3H), 3.87 (s, 3H), 3.92
(s, 6H), 5.18 (br, 1H), 6.73 (s, 2H), 6.89 (s, 1H), 7.0 (s, 2H), 7.04
(d, J ) 12 Hz, 1H), 7.45 (s, 1H). 13C NMR (CDCl3) δ: 16.3, 56.2,
61.1, 103.5, 106.8, 113.7, 117.4, 128.2, 128.4, 140.3, 149.9, 153.5,
207.2. MALDI-TOF: 356.6 [M+]. Anal. (C20H20O4S) C, H, S.
Starting from 8f the following compounds were produced.
6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-2-methylbenzofu-
ran-4-ol (17). Yield: 29% (195 mg), colorless solid. Mp: 114-
1
116 °C. H NMR (CDCl3) δ: 2.42 (s, 3H), 3.65 (s, 6H), 3.84 (s,
3H), 5.18 (s, 1H), 6.38 (s, 1H), 6.47 (d, J ) 12 Hz, 1H), 6.53 (s,
2H), 6.57-6.60 (m, 2H), 6.97 (d, J ) 1.2 Hz, 1H). 13C NMR (75
MHz, CDCl3) δ: 14.0, 55.9, 60.9, 99.4, 104.4, 106.1, 108.5, 117.4,
129.4, 130.1, 132.7, 133.2, 137.0, 148.0, 152.8, 154.7, 156.3.
MALDI-TOF: 340.6 [M+]. Anal. (C20H20O5) C, H.
6-[(E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-2-methylbenzofu-
ran-4-ol (18). Yield: 33% (225 mg), colorless solid. Mp: 65-70
1
°C. H NMR (CDCl3) δ: 2.46 (s, 3H), 3.87 (s, 3H), 3.92 (s, 6H),
4.98 (br, 1H), 6.41 (s, 1H), 6.97 (d, J ) 16.6 Hz, 1H), 7.01 (d, J
) 16.6 Hz, 1H), 7.16 (s, 1H), 6.57-6.60 (m, 2H), 6.97 (d, J ) 1.2
Hz, 1H). 13C NMR (75 MHz, CDCl3) δ: 14.2, 56.2, 61.1, 99.5,
102.6, 103.5, 106.1, 118.1, 128.0, 128.5, 133.3, 133.9, 148.3, 153.5,
155.1, 156.8. MALDI-TOF: 340.6 [M+]. Anal. (C20H20O5) C, H.
Starting from 8i the following compounds were produced.
6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-3-phenylbenzo[b]-
For derivatives in which the phenolic moiety is protected as the
TBDMS ether, the residue was dissolved in methylene chloride
(10 mL) and tetrabutylammonium fluoride (6 mmol, 3 equiv) was
1
thiophen-4-ol (19). Yield: 36% (300 mg), oil. H NMR (CDCl3)

NoVel Antitumor Combretastatin Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3149
δ: 3.66 (s, 6H), 3.84 (s, 3H), 5.21 (br, 1H), 6.62 (d, J ) 12.4 Hz,
1H), 6.57 (s, 2H), 6.62 (d, J ) 12.4 Hz, 1H), 6.79 (d, J ) 1.2 Hz,
1H), 7.14 (s, 1H), 7.45 (m, 1H), 7.50-7.52 (m, 4H). 13C NMR
(CDCl3) δ: 16.2, 55.9, 60.9, 103.4, 106.0, 109.5, 115.4, 117.4,
128.2, 129.6, 129.8, 132.5, 133.7, 137.2, 140.1, 141.1, 152.9.
MALDI-TOF: 418.1 [M+]. Anal. (C25H22O4S) C, H, S.
6-[(E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-3-phenylbenzo[b]-
thiophen-4-ol (20). Yield: 40% (335 mg), colorless solid. Mp:
138-140 °C. 1H NMR (CDCl3) δ: 3.88 (s, 3H), 3.93 (s, 6H), 5.34
(s, 1H), 6.76 (s, 2 H), 7.02 (d, J ) 1.2 Hz, 1H), 7.05 (d, J ) 16
Hz, 1H), 7.10 (d, J ) 16 Hz, 1H), 7.16 (s, 1H), 7.48-7.56 (m, 5
H), 7.58 (d, J ) 1.2 Hz, 1H). 13C NMR (CDCl3) δ: 20.6, 56.2,
61.1, 103.6, 108.2, 114.0, 123.8, 127.8, 128.8, 129.1, 129.3, 129.5,
133.1, 135.9, 153.5. MALDI-TOF: 418.2 [M+]. Anal. (C25H22O4S)
C, H, S.
7.54 (d, J ) 2.2 Hz, 1H). MALDI-TOF: 340.6 [M + 1]. Anal.
(C20H20O5) C, H.
Starting from 8c the following compounds were produced.
5-[(Z)-2-(3,4,5-Trimethoxypheyl)ethenyl]benzo[b]thiophen-7-
ol (31). Yield: 31% (210 mg), brown solid. Mp: 152-154 °C; 1H
NMR (CDCl3) δ: 3.63 (s, 6H), 3.83 (s, 3H), 5.51 (s, 1H), 6.48 (d,
J ) 12.2 Hz, 1H), 6.52 (s, 1H), 6.64 (d, J ) 12.2 Hz, 1H), 6.73 (s,
1H), 7.26 (s, 1H), 7.29 (d, J ) 3.2 Hz, 1H), 7.41 (d, J ) 3.2 Hz,
1H), 7.43 (s, 1H). Anal. (C19H18O4S) C, H, S.
5-[(E)-2-(3,4,5-Trimethoxypheyl)ethenyl]benzo[b]thiophen-7-
ol (32). Yield: 38% (260 mg), yellow solid. Mp: 172-174 °C;
1H NMR (CDCl3) δ: 3.89 (s, 6H), 3.92 (s, 3H), 5.63 (s, 1H), 6.74
(s, 2H), 6.94 (s, 1H), 7.02 (d, J ) 2.8 Hz, 1H), 7.32 (d, J ) 5.2
Hz, 1H), 7.45 (d, J ) 5.2 Hz, 1H), 7.53 (s, 1H). Anal. (C19H18O4S)
C, H, S.
Starting from 8h the following compounds were produced.
6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-2-phenylbenzofu-
ran-4-ol (21). Yield: 27% (215 mg), colorless solid. Mp: 60-62
°C. 1H NMR (CDCl3) δ: 3.67 (s, 6H), 3.85 (s, 3H), 5.13 (br, 1H),
6.51 (d, J ) 12 Hz, 1H), 6.55 (s, 2 H), 6.60 (d, J ) 12 Hz, 1H),
6.62 (s, 1H), 7.05 (d, J ) 1.2 Hz, 1H), 7.32-7.36 (m, 1 H), 7.41-
7.46 (m, 3H), 7.81-7.84 (m, 2 H). 13C NMR (CDCl3) δ: 56.0,
61.0, 98.3, 105.1, 106.1, 108.9, 123.8, 117.9, 124.8, 128.6, 128.9,
129.9, 130.0, 148.6, 152.9, 156.5. MALDI-TOF: 401.9 [M+]. Anal.
(C25H22O5) C, H.
Starting from 8d the following compounds were produced.
5-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]benzofuran-7-ol (33).
Yield: 36% (235 mg), colorless solid. Mp: 140-141 °C; 1H NMR
(CDCl3) δ: 3.63 (s, 6H), 3.83 (s, 3H), 5.33 (s, 1H), 6.46 (d, J )
12.4 Hz, 1H), 6.52 (s, 1H), 6.57 (d, J ) 12.4 Hz, 1H), 6.69 (d, J
) 2.2 Hz, 1H), 6.82 (s, 1H), 7.12 (s, 1H), 7.26 (s, 1H), 7.58 (d, J
) 2.2 Hz, 1H). 13C NMR (CDCl3) δ: 55.9, 61.0, 106.2, 107.3,
111.4, 114.4, 129.1, 129.5, 130.0, 132.6, 133.3, 137.2, 140.7, 145.4,
152.9. Anal. (C19H18O5) C, H.
5-[(E)-2-(3,4,5-Trimethoxypheyl)ethenyl]benzofuran-7-ol (34).
Yield: 42% (275 mg), colorless solid. Mp: 173-175 °C; 1H NMR
(CDCl3) δ: 3.89 (s, 6H), 3.92 (s, 3H), 6.01 (s, 1H), 6.74 (s, 2H),
6.97 (s, 1H), 7.06 (d, J ) 3.2 Hz, 1H), 7.26 (d, J ) 5.2 Hz, 1H),
7.45 (d, J ) 5.2 Hz, 1H), 7.60 (s, 1H). 13C NMR (CDCl3) δ: 56.2,
61.1, 103.5, 107.5, 108.5, 112.1, 128.0, 128.5, 129.4, 133.3, 133.8,
137.8, 141.1, 143.3, 145.5, 153.5. Anal. (C19H18O5) C, H.
Starting from 10b the following compounds were produced.
3-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]naphthalen-1-ol (35).
6-[(E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-2-phenylbenzofu-
ran-4-ol (22). Yield: 35% (280 mg), colorless solid. Mp: 180-
182 °C. 1H NMR (CDCl3) δ: 3.89 (s, 3H), 3.93 (s, 6H), 5.18 (br,
1H), 6.75 (s, 2 H), 6.84 (d, J ) 0.8 Hz, 1H), 7.03 (s, 2H), 7.08 (d,
J ) 0.8 Hz, 1H), 7.28 (s, 1H), 7.34-7.37 (m, 1 H), 7.44-7.47 (m,
2 H), 7.84-7.86 (m, 2H). 13C NMR (CDCl3) δ: 56.2, 62.1, 98.4,
102.9, 103.6, 106.5, 124.8, 128.4, 128.9, 130.3, 149.9, 153.5.
MALDI-TOF: 401.9 [M+]. Anal. (C25H22O5) C, H.
1
Starting from 8g the following compounds were produced.
6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-2-phenylbenzo[b]-
Yield: 33% (220 mg), yellow solid. Mp: 163-165 °C; H NMR
(CDCl3) δ: 3.62 (s, 6H), 3.84 (s, 3H), 5.56 (s, 1H), 6.53 (d, J )
12.4 Hz, 1H), 6.56 (s, 2H), 6.68 (d, J ) 12.4 Hz, 1H), 6.79 (s,
1H), 7.38 (s, 1H), 7.45 (m, 2H), 7.72 (dd, J ) 9.8 and 3.6 Hz,
1H), 8.11 (dd, J ) 9.8 and 3.6 Hz, 1H).
1
thiophen-4-ol (23). Yield: 31% (260 mg), oil. H NMR (CDCl3)
δ: 3.60 (s, 6H), 3.78 (s, 3H), 5.22 (br, 1H), 6.44 (d, J ) 12.4 Hz,
1H), 6.50 (s, 2H), 6.55 (d, J ) 12.4 Hz, 1H), 6.61 (s, 1H), 7.27-
7.40 (m, 4 H), 7.57-7.66 (m, 3 H). MALDI-TOF: 418 [M+]. Anal.
(C25H22O4S) C, H, S.
3-[(E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]naphthalen-1-ol (36).
1
Yield: 40% (270 mg), yellow solid. Mp: 176-178 °C; H NMR
6-[(E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-2-phenylbenzo[b]-
thiophen-4-ol (24). Yield: 30% (250 mg), colorless solid. Mp:
218-220 °C. 1H NMR (CDCl3) δ: 3.88 (s, 3H), 3.93 (s, 6H), 5.32
(br, 1H), 6.75 (s, 2H), 6.95 (s, 1 H), 7.04 (s, 1H), 7.33-7.37 (m,
2 H), 7.42-7.46 (m, 2 H), 7.65 (s, 1 H) 7.66 (s, 1H), 7.70-7.73
(m, 2 H). MALDI-TOF: 418 [M+]. Anal. (C25H22O4S) C, H, S.
Starting from 7a the following compounds were produced.
4-Methoxy-6-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]benzo[b]-
thiophene (25). Yield: 42% (300 mg), yellow oil. 1H NMR
(CDCl3) δ: 3.65 (s, 6H), 3.76 (s, 3H) 3.84 (s, 3H), 6.55 (s, 1H),
6.58 (d, J ) 11.2 Hz, 1H), 6.64 (d, J ) 11.2 Hz, 1H), 6.70 (s, 1H),
7.27 (s, 1H), 7.31 (d, J ) 5.0 Hz, 1H), 7.42 (d, J ) 5.0 Hz, 1H),
7.44 (s, 1H). MALDI-TOF: 356.4 [M + 1]. Anal. (C20H20O4S) C,
H, S.
(CDCl3) δ: 3.90 (s, 3H), 3.93 (s, 6H), 5.73 (s, 1H), 6.77 (s, 2H),
7.07 (m, 3H), 7.46 (m, 3H), 7.80 (dd, J ) 9.6 and 2.8 Hz, 1H),
8.14 (dd, J ) 9.6 and 2.8 Hz, 1H).
Starting from 10c the following compounds were produced.
1-Methoxy-3-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]naph-
thalene (37). Yield: 36% (250 mg), oil. 1H NMR (CDCl3) δ: 3.63
(s, 6H), 3.75 (s, 3H), 3.83 (s, 3H), 6.57 (s, 1H), 6.66 (d, J ) 13.2
Hz, 1H), 6.71 (d, J ) 13.2 Hz, 1H), 6.75 (s, 1H), 7.44 (m, 4H),
7.69 (m, 1H), 8.12 (m, 1H). MALDI-TOF: 350.3 [M + 1].
1-Methoxy-3-[(E)-2-(3,4,5-trimethoxyphenyl)ethenyl]naph-
thalene (38). Yield: 40% (280 mg), yellow solid. Mp: 166-168
1
°C. H NMR (CDCl3) δ: 3.89 (s, 3H), 3.95 (s, 6H), 4.09 (s, 3H),
6.80 (s, 2H), 7.06 (s, 1H), 7.16 (s, 2H), 7.46 (m, 3H), 7.76 (dd, J
) 9.2 e 1.8 Hz, 1H), 8.20 (dd, J ) 9.2 and 1.8 Hz, 1H). MALDI-
TOF: 350.3 [M + 1].
4-Methoxy-6-[(E)-2-(3,4,5-trimethoxyphenyl)ethenyl]benzo-
[b]thiophene (26). Yield: 44% (315 mg), yellow solid; mp 171-
Starting from 7c the following compounds were produced.
7-Methoxy-1-methyl-5-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]-
1H-indazole (39). Yield: 29% (205 mg), colorless solid. Mp: 182-
1
173 °C. H NMR (CDCl3) δ: 3.89 (s, 6H), 3.94 (s, 3H), 4.03 (s,
3H), 6.78 (s, 2H), 6.95 (s, 1H), 7.10 (s, 2H), 7.33 (d, J ) 5.6 Hz,
1H), 7.47 (d, J ) 5.6 Hz, 1H), 7.58 (s, 1H). MALDI-TOF: 356.3
[M + 1]. Anal. (C20H20O4S) C, H, S.
1
183 °C; H NMR (CDCl3) δ: 3.64 (s, 3H), 3.67 (s, 3H), 3.82 (s,
3H), 4.23 (s, 3H), 6.51 (d, J ) 12.5 Hz, 1H), 6.53 (s, 2H), 6.59 (d,
J ) 12.5 Hz, 1H), 7.19 (s, 1H), 7.80 (s, 2H).
Starting from 7b the following compounds were produced.
4-Methoxy-6-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]benzo-
7-Methoxy-1-methyl-5-[(E)-2-(3,4,5-trimethoxyphenyl)ethenyl]-
1H-indazole (40). Yield: 35% (250 mg), oil. 1H NMR (CDCl3) δ:
3.86 (s, 3H), 3.91 (s, 6H), 4.01 (s, 3H), 4.28 (s, 3H), 6.73 (s, 2H),
6.94 (d, J ) 15.8 Hz, 1H), 7.06 (d, J ) 15.8 Hz, 1H), 7.31 (s, 1H),
7.86 (s, 2H).
1
furan (27). Yield: 38% (260 mg), yellow oil. H NMR (CDCl3)
δ: 3.65 (s, 6H), 3.74 (s, 3H), 3.83 (s, 3H), 6.52 (s, 1H), 6.55 (d,
J ) 11.2 Hz, 1H), 6.62 (d, J ) 11.2 Hz, 1H), 6.63 (s, 1H), 6.80 (s,
1H), 7.27 (s, 1H), 7.10 (s, 1H), 7.51 (s, 1H). MALDI-TOF: 340.6
[M + 1]. Anal. (C20H20O5) C, H.
Starting from commercial indole-6-carboxaldehyde the following
compounds were produced.
6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-1H-indole (41).
Yield: 18% (110 mg), oil. 1H NMR (CDCl3) δ: 3.60 (s, 6H), 3.83
(s, 3H), 6.43-6.51 (m, 2H),, 6.53 (s, 2 H), 6.70 (d, J ) 12.2 Hz,
4-Methoxy-6-[(E)-2-(3,4,5-trimethoxyphenyl)ethenyl]benzo-
furan (28). Yield: 41% (280 mg), yellow solid. Mp: 152-153
1
°C. H NMR (CDCl3) δ: 3.88 (s, 6H), 3.94 (s, 3H), 4.00 (s, 3H),
6.76 (s, 2H), 6.84 (s, 2H), 7.08 (s, 2H), 7.28 (d, J ) 2.2 Hz, 1H),

3150 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Simoni et al.
1
1H), 7.10 (dd, J ) 8.2 Hz, 1.2 Hz, 1H), 7.16-7-19 (m, 1H), 7.33
(s, 1H), 7.51 (d, J ) 8.2 Hz, 1H), 8.09 (br, 1H). 13C NMR (CDCl3)
δ: 55.9, 61.0, 102.7, 106.0, 111.4, 120.3, 121.5, 124.9, 128.7, 131.2,
133.2, 152.9. MALDI-TOF: 309.3 [M+]. Anal. (C19H19NO3) C,
H.
6-[(E)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-1H-indole (42).
Yield: 28% (175 mg), oil. 1H NMR (CDCl3) δ: 3.87 (s, 3H), 3.92
(s, 6H), 6.74 (s, 2 H), 6.84 (d, J ) 0.8 Hz, 1H), 7.03 (s, 2H), 7.08
(d, J ) 0.8 Hz, 1H), 7.28 (s, 1H), 7.34-7.37 (m, 1 H), 7.44-7.47
(m, 2 H), 7.84-7.86 (m, 2H), 8.21 (br, 1H). 13C NMR (CDCl3) δ:
56.2, 62.1, 98.4, 102.9, 103.6, 106.5, 124.8, 128.4, 128.9, 130.3,
149.9, 153.5. MALDI-TOF: 309.2 [M+]. Anal. (C19H19NO3) C,
H.
General Procedure for the Synthesis of Phosphate Prodrugs
29 and 30. CCl4 (580 µL, 6 mmol, 5 equiv) was added to a solution
of 1.2 mmol of compound 11 or 13 in 5 mL of anhydrous CH3CN
cooled to -25 °C. After approximately 10 min, 440 µL (2.52 mmol,
2.1 equiv) of diisopropylethylamine, 15 mg (0.12 mmol, 0.1 equiv)
of (dimethylamino)pyridine, and 380 µL (1.74 mmol, 1.45 equiv)
of dibenzyl phosphate were added to the solution. After 2 h at -10
°C, when the reaction was completed, 20 mL of KH2PO4 (0.5 M)
was added and the aqueous phase was extracted with ethyl acetate
(3 × 10 mL). The organic phases were dried over anhydrous
Na2SO4, and the crude intermediate product was purified by
chromatography.
NaI (36 mg, 2.4 mmol, 2 equiv) and 300 µL (2.4 mmol, 2 equiv)
of Me3SiCl dissolved in 1 mL of anhydrous CH3CN were added at
room temperature to a solution of 1.2 mmol of dibenzyl ester
dissolved in 7 mL of anhydrous CH3CN. After 2 h, when the
reaction was completed, a small amount of water was added to
dissolve the salts. A solution of 10% Na2S2O3 was added until
decolorization of the reaction mixture. The solution thus obtained
was extracted with ethyl acetate, the organic phases were dried over
Na2SO4, and solvent was removed in vacuo. The crude oil obtained
was dissolved in 4 mL of anhydrous MeOH, and 130 mg (2.4 mmol,
2 equiv) of NaOMe was added to the solution. The mixture was
left to stand at room temperature for 20 h. Then, solvent was
removed in vacuo and the residue washed with Et2O to give the
desired product.
solid. Mp: 212 °C. H NMR (CD3OD) δ: 3.60 (s, 6H), 3.76 (s,
3H), 6.48 (d, J ) 12.1 Hz, 1H), 6.61 (s, 2H), 6.73 (d, J ) 12.1 Hz,
1H), 7.00 (s, 1H), 7.04 (d, J ) 2.2 Hz, 1H), 7.44 (s, 1H), 7.53 (d,
J ) 2.2 Hz, 1H). 13C NMR (D2O) δ: 54.4, 59.2, 103.2, 104.3,
105.6, 113.3, 118.9, 119.1, 128.2, 130.2, 132.7, 133.5, 136.3, 142.8,
148.4, 148.5, 152.1, 155.5. MS-IS [M - 1]- ) 403. Anal. (C19H17-
Na2O8P) C, H, S.
General Procedure for Photochemical Isomerization. Step
1: Acetylation of E-Isomers. Acetyl chloride (1.8 mL, 20 mmol)
was added to an ice-cooled solution of E-isomer 12 or 14 (10 mmol)
dissolved in methylene chloride (20 mL) containing pyridine (1.6
mL, 20 mmol) and the mixture stirred until completion of reaction
(10 min). Water (10 mL) was added, the organic layer was
separated, washed with saturated aqueous NaHCO3, and dried. The
solvent was evaporated to give a residue, which was purified by
silica gel column chromatography.
4-Acetyloxy-6-[(E)-2-(3,4,5-trimethoxyphenyl)ethenyl]benzo-
[b]thiophene. Yield: 71% (2.73 g), colorless solid. Mp: 136-
138 °C. H NMR (CDCl3) δ: 2.43 (s, 3H), 3.89 (s, 6H), 3.92 (s,
3H), 6.69 (d, J ) 1.4 Hz, 1H), 6.78 (s, 2H), 7.03 (s, 2H), 7.19 (s,
1H), 7.50 (s, 1H), 7.60 (d, J ) 1.4 Hz, 1H).
4-Acetyloxy-6-[(E)-2-(3,4,5-trimethoxyphenyl)ethenyl]benzo-
furan. Yield: 74% (2.73 g), yellow oil. 1H NMR (CDCl3) δ: 2.41
(s, 3H), 3.88 (s, 6H), 3.92 (s, 3H), 6.67 (d, J ) 1.2 Hz, 1H), 6.75
(s, 2H), 7.06 (s, 2H), 7.23 (s, 1H), 7.52 (s, 1H), 7.61 (d, J ) 1.2
Hz, 1H).
Step 2: Irradiation. A stirred solution of the O-acetylderivatives
(1 mmol) dissolved in 300 mL of MeOH was irradiated with a
mercury lamp for 2 h. After removal of the methanol in vacuo, the
product was purified by silica gel column chromatography.
4-Acetyloxy-6-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]benzo-
[b]thiophene. The unreacted starting material E-isomer (32%) and
the corresponding Z-isomer (64%, 245 mg) were separated by silica
gel column chromatography to give a brown oil. 1H NMR (CDCl3)
δ: 2.20 (s, 3H), 3.63 (s, 6H), 3.82 (s, 3H), 6.51 (s, 2H), 6.58 (d,
J ) 12.2 Hz, 1H), 6.92 (s, 1H), 6.98 (s, 2H), 7.30 (s, 1H), 7.58 (d,
J ) 2.0 Hz, 1H).
4-Acetyloxy-6-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]benzo-
furan. The unreacted E-isomer (24%) and the corresponding
Z-isomer (67%, 250 mg) were separated by silica gel column
chromatography to give a yellow oil. 1H NMR (CDCl3) δ: 1H NMR
(CDCl3) δ: 2.18 (s, 3H), 3.65 (s, 6H), 3.84 (s, 3H), 6.50 (s, 2H),
6.58 (d, J ) 6.0 Hz, 1H), 6.96 (s, 1H), 6.99 (s, 2H), 7.34 (s, 1H),
7.58 (d, J ) 2.0 Hz, 1H).
1
Starting from 11 the following compounds were produced.
6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-1-benzo[b]thiophen-
4-ol 4-O-Dibenzyl Phosphate. The product was purified by flash
chromatography on silica gel using hexane:ethyl acetate 8:2.
1
Yield: 86% (625 mg), yellow oil. H NMR (CDCl3) δ: 3.6 (s,
6H), 3.8 (s, 3H), 5.05 (s, 2H), 5.1 (s, 2H), 6.5 (s, 2H), 6.6 (bs,
2H), 7.2-7.4 (m, 11H), 7.6 (s, 1H). 13C NMR (CDCl3) δ: 56.1,
61.1, 70.3, 106.4, 115.9, 119.7, 120.4, 127.1, 128.2, 128.8, 128.9,
129.0, 131.1, 131.6, 132.2, 134.9, 135.6, 153.2. MS-IS: [M + H]+
) 603.2. Anal. (C33H31O7PS) C, H.
Disodium 6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-1-ben-
zothiophen-4-ol 4-O-Phosphate (29). Yield: 92% (515 mg),
colorless solid. Mp: 226 °C. 1H NMR (D2O) δ: 3.57 (s, 6H), 3.75
(s, 3H), 6.50 (d, J ) 12.1 Hz, 1H), 6.63 (s, 2H), 6.74 (d, J ) 12.1
Hz, 1H), 7.30 (d, J ) 5.5 Hz, 1H), 7.39 (s, 1H), 7.57 (s, 1H), 7.66
(d, J ) 5.5 Hz, 1H). 13C NMR (D2O) δ: 54.4, 59.2, 105.7, 113.8,
121.2, 122.8, 128.5, 129.9, 131.6, 131.8, 132.5, 134.1, 136.3, 139.7,
150.1, 150.2, 152.1. MS-IS: [M - 1]- ) 419. Anal. (C19H17Na2O7-
PS) C, H, S.
Step 3: Hydrolysis of Z-Acetyl Derivatives. Compounds 11
(63% yield) and 13 (72% yield) were obtained following the
O-deacetylation method reported in the general procedure for the
synthesis of 4a-j.
Biology. Cell Culture and Cytotoxicity Assay. Primary cultures
of bovine microvascular endothelial cells (BMEC) were obtained
from bovine adrenal glands as described by Folkman.36 BMEC were
maintained in DMEM supplemented with 20% fetal calf serum
(FCS), 50 units/mL heparin (Sigma, St. Louis, MO), 50 µg/mL
bovine brain extract, and 100 units/mL gentamycin.
HUVEC (human umbilical vein endothelial cells) were obtained
from BioWhittaker (Walkersville, MD) and grown in EGM-2
(BioWhittaker). The following cell lines were purchased from
ATCC and cultured according to manufacturer’s instructions: NCI-
H460 human non-small cell lung carcinoma, MCF-7 human
hormone-dependent breast carcinoma, and MeWo human mela-
noma. Wild-type and doxorubicin-resistant A2780 ovarian carci-
noma and doxorubicin-resistant MCF-7 (both gifts of Dr. F. Zunino,
Istituto Nazionale dei Tumori, Milan, Italy) were grown in RPMI
1640 containing 10% FBS and 50 µg/mL gentamycin sulfate.
IGROV-1 ovarian carcinoma cells, also obtained from Dr. Zunino,
were cultured in D-MEM containing 10% FBS. HCT-116 human
colon carcinoma cells, purchased from “Istituto Zooprofilattico”
(Brescia, Italy), were grown in McCoy’s medium containing 10%
FBS and 50 µg/mL gentamycin sulfate.
Starting from 13 the following compounds were produced.
6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-1-benzofuran-4-
ol 4-O-Dibenzyl Phosphate. The product was purified by flash
chromatography on silica gel using hexane:ethyl acetate 7:3.
1
Yield: 88% (620 mg), yellow oil. H NMR (CDCl3) δ: 3.6 (s,
6H), 3.8 (s, 3H), 5.05 (s, 2H), 5.1 (s, 2H), 6.45 (s, 2H), 6.55 (bs,
2H), 6.75 (bs, 1H), 7.05 (s, 1H), 7.2-7.4 (m, 11H), 7.5 (bs, 1H).
13C NMR (CDCl3) δ: 56.1, 61.1, 70.3, 98.8, 104.3, 106.4, 109.1,
115.1, 119.9, 128.2, 128.8, 128.9, 129.2, 130.9, 132.3, 134.7, 135.5,
145.5, 153.2, 156.5. MS-IS: [M + H]+ ) 587.2. Anal. (C33H31O8P)
C, H.
Disodium 6-[(Z)-2-(3,4,5-Trimethoxyphenyl)ethenyl]-1-ben-
zofuran-4-ol 4-O-Phosphate (30). Yield: 91% (490 mg), colorless
To test the effect of the drugs on cell growth, the above cell
lines were resuspended at different concentrations in 200 µL of

NoVel Antitumor Combretastatin Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3151
appropriate growth medium and seeded in 96-well plates. Twenty-
four hours after plating, scalar concentrations of each drug were
added to the cells. The drugs were removed after 24 h and, after a
further 48 h of recovery, the number of surviving cells was
determined by staining with sulforhodamine B test, as previously
described.37 Optical density was measured at 564 nM. Results,
expressed as concentrations that inhibit 50% of cell growth (IC50),
were calculated by the ALLFIT program.
Tubulin Polymerization Inhibition. The tubulin polymerization
test was performed by CytoDINAMIX Screen (Cytoskeleton Inc.,
Denver, CO). One hundred microliters of 3 mg/mL HTS tubulin
in G-PEM buffer plus 5% glycerol at 4 °C was pipetted into wells
of the 96-well plate and incubated at 37 °C in the presence or
absence of single compounds. Tubulin polymerization was detected
by measuring the absorbance of the solution (340 nm) for 60 min.
Because the amount of tubulin polymerized is directly proportional
to the AUC (area under the curve), we used AUC to determine the
concentration that inhibited tubulin polymerization by 50% (IC50).38
The AUC of the untreated control was set to 100% polymerization
(maximal attainable polymerization), and the IC50 was calculated
by nonlinear regression.
Colchicine Tubulin Binding Competition. Competition-binding
SPAs (Scintillation Proximity Assay) were conducted in 96-well
plates. Radiolabeled [3H]colchicine (final concentration 0.08 µM),
unlabeled compound, and 0.5 mg of special long-chain biotin-
labeled tubulin (Cytoskeleton, Denver, CO) were incubated together
in 100 µL of binding buffer [80 mM PIPES, 1 mM EGTA, 1 mM
MgCl2, 1 mM GTP (pH 6.8)] for 2 h at 37 °C. Streptavidin-labeled
yttrium SPA beads (80 µg suspended in 20 µL of binding buffer;
Amersham, Arlington Heights, IL) were added to each well, and
the bound radioactivity was determined using a scintillation counter.
Nonspecific binding was determined in the presence of excess
unlabeled colchicines. The IC50 was calculated as the concentration
of compounds required for 50% inhibition of [3H]colchicine-
binding; each data point is a result of the average of triplicate wells
and analyzed by nonlinear regression analysis with the Prism
GraphPad software program.
Cell Cycle. NCI-H460 cells were exposed to the various
molecules (at the IC80 dose) for 24 h and allowed to recover in
drug-free medium for 24 and 48 h. Cells were then harvested with
trypsin/EDTA, pooled with the respective supernatant, and fixed
in cold 70% ethanol at 4 °C. On the day of analysis, ethanol was
removed, cells were treated with RNAse (75 kU/mL) for 30 min
at 37 °C, and propidium iodide (PI) was added (50 µg/mL) to stain
cellular DNA. Samples (2 × 104 cells) were processed by a
FACScan flow cytometer to assess cell cycle distribution and
apoptosis (Apo). Apo was evaluated by measuring the percentage
of cells with hypodiploid DNA content (sub-G1/0 population) with
CELLQuest software, while cell cycle analysis was performed with
ModFit software.
Immunofluorescence. For tubulin immunostaining, the cells,
cultured on gelatin-coated cover slips, were treated with the
compounds at their respective IC50, 2 × IC50, and 4 × IC50 values.
After 24 h the cells were washed with PBS and then fixed in cold
methanol for 5 min and acetone for 1 min. Then, the fixed cells
were incubated in a PBS solution containing 0.1% Triton X-100
and 0.5% acetic acid for 10 min and, after three washings in PBS,
in a solution containing 2 µg/mL of the monoclonal anti-R-tubulin
antibody (Sigma Chemicals Co., St. Louis, MO) for 1 h at room
temperature in a humidified chamber. Following primary antibody
incubation, the cells were washed three times in PBS and incubated
with an AlexaFluor-conjugated goat anti-mouse secondary antibody
(Molecular Probes, Leiden, The Netherlands) at a concentration of
20 µg/mL for 1 h at room temperature, in the dark. For nuclear
staining, cells were incubated with Hoechst 33342 (Sigma Chemi-
cals) at a concentration of 10 µg/mL for 10 min at room
temperature, in the dark. Cover slips were then mounted with 50%
glycerol in PBS. Cells were analyzed with an epifluorescent
microscope (Polyvar, Zeiss AG, Germany) and imaged with a
cooled CCD camera (Photometrics, Roper Scientific Inc., CA).
Images were then processed with Metamorph software (Universal
Imaging, Downingtown, PA). Quantification of multinucleated cells
was performed by counting cells containing more than two nuclei
(including micronuclei) in 20 randomly chosen microscope fields
at 100×. These counts were expressed as percentage of the total
R-tubulin stained cells in identical fields.
In Vivo Study. For the in vivo xenograft model, 3.0 × 106 NCI-
H460 cells, resuspended in 100 µL of Ca2+/Mg2+-free Hank’s
balanced solution (HBSS), were inoculated subcutaneously into the
right flank of CD-1 nude female mice. Five days after cell injection,
tumors attained a mass of about 30 mg and the mice were assigned
to treatment groups, eight mice per group. Animals were treated
with 29 and 30 either orally (po), at a dose of 60 mg/kg twice a
day, 5 days a week over a three-week period (qdx5x3w), or
intravenously (iv) at the same dose of 60 mg/kg every other day
for a total of six administrations (q2dx6). Control groups received
CA-4P at a dose of 60 mg/kg iv (q2dx6) or were treated with vehicle
only. Animals were size-matched for palpable tumors (20-30 mm3)
and tumor size was measured twice a week with a Vernier caliper.
Tumor volume (TV, mm3) was estimated by the formula: length
× (width)2/2, where width and length are the shortest and longest
diameters of each tumor, respectively. When tumors reached a mass
of about 1.4 g, mice were sacrificed. Toxicity of the different drug
treatments was determined as body weight loss percentage (% max
BWL) ) 100 - (mean BWx/mean BW1 × 100), where BWx is the
mean BW at the day of maximal loss during treatment and BW1 is
the mean BW on the first day of treatment.
The care and husbandry of animals were in accordance with
European Directives 86/609 and with the Italian Regulatory system
(D.L. vol. 116, Art. 6, 27 Jan 1992). Statistical comparison of data
was performed by the nonparametric Mann-Whitney U test.
Probability values of p e 0.05 were considered significant. Dixon’s
test was eventually used for exclusion of abnormal values at the
end of treatment.
In Vivo Pharmacokinetics. Healthy CD-1 female mice (three
mice/group) were treated with 100 mg/kg of 29 by intravenous bolus
as a 5% glucose solution. Blood samples were taken at various
time points up to 24 h postdose from the retro-orbital plexus under
terminal anesthesia and collected in Eppendorf tubes containing
2% EDTA. Plasma was separated immediately by centrifugation
at 1000g at 4 °C for 10 min and deproteinized by adding two
volumes of methanol kept at -20 °C. Samples were placed on ice
for 10 min, followed by centrifugation at 10 000g at 4 °C for 10
min, and transferred into vials for high-performance liquid chro-
matography (HPLC). This method permitted the simultaneous
extraction of the phosphate prodrug 29 and of the corresponding
water-insoluble combretastatin analogue 11. Indeed, the estimated
recovery factor (R) was 96 ( 3% for 29 and 80 ( 7% for 11,
evaluated by adding primary stock solutions of the compounds,
respectively, in water or methanol containing up to 2% DMSO to
untreated plasma sample processed as described. Concentrations
of 29 and 11 were calculated from the respective calibration curves,
which were linear over the range tested (from 0.0624 to 2 µg/mL
for 11; from 0.0624 to 8 µg/mL for 29). The mean of plasma
concentrations ((SE) was calculated and used to estimate the
pharmacokinetic parameters by using a noncompartmental model
(WinNonlin Enterprise software, Version 4.0.1).
HPLC-fluorimetric analysis and sample quantification were
carried out on a System Gold HPLC Systems (Beckman Coulter)
coupled with a Shimadzu fluorescence detector (RF-10AXL), fixed
at wavelengths of 295 nm (λex) and 390 nm (λem), using 32 Karat
Software (Beckman Coulter). A Symmetry C18 column (5 µm, 4.6
× 150 mm, Waters) was used in gradient mode with a flow rate of
1 mL/min. The mobile phase consisted of 20 mM ammonium
acetate in water and acetonitrile (75/25) for the elution of 29. After
20 min, the proportion of acetonitrile was programmed to linearly
increase to 90% over 15 min and held for an additional 5 min for
the elution of 11. Then, the percentage of acetonitrile was decreased
within 5 min to 25%. For reequilibration of the column, the
proportion was subsequently held at this concentration for 20 min.

3152 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Simoni et al.
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Piccagli, L.; Baruchello, R.; Rossi, R.; Romagnoli, R.; Invidiata, F.
P.; Grimaudo, S.; Jung, M. K.; Hamel, E.; Gebbia, N.; Crosta, L.;
Abbadessa, V.; Di Cristina, A.; Dusonschet, L.; Meli, M.; Tolomeo,
M. Heterocyclic and phenyl double-bond-locked combretastatin
analogues possessing potent apoptosis-inducing activity in HL60 and
in MDR cell lines. J. Med. Chem. 2005, 48, 723-736.
(22) Maya, A. B. S.; Perez-Melero, C.; Mateo, C.; Alonso, D.; Fernandez,
J. L.; Gajate, C.; Mollinedo, F.; Pelaez, R.; Caballero, E.; Medarde,
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Acknowledgment. Funding for this study was provided by
the Ministero dell’Universita` e della Ricerca Scientifica e
Tecnologica, Rome, Italy.
Supporting Information Available: Experimental procedures
for intermediate compounds and elemental analysis results of target
derivatives. This material is available free of charge via the Internet
at http://pubs.acs.org.
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