Novel A-ring and B-ring modified combretastatin A-4 (CA-4) analogues endowed with interesting cytotoxic activity

Article abstract of DOI:10.1021/jm8005004

A novel class of combretastatins, modified at A-ring or both A- and B-rings, mainly by replacement with benzofuran or benzo[b]thiophene, were synthesized. The new heterocombretastatins showed good cytotoxic activity on BMEC and H-460 cell lines. The aminocombretastatin 9f potently inhibits cell growth of BMEC and combretastatin-resistant HT-29 cell lines, with potential interest to treat colon carcinoma. Heterocombretastatins 9a,b inhibit tubulin polymerization similarly to CA-4 by having a binding to colchicine site five times stronger.

Full text of DOI:10.1021/jm8005004

J. Med. Chem. 2008, 51, 6211–6215
6211
Brief Articles
Novel A-Ring and B-Ring Modified Combretastatin A-4 (CA-4) Analogues Endowed with
Interesting Cytotoxic Activity
Daniele Simoni,*,† Romeo Romagnoli,† Riccardo Baruchello,† Riccardo Rondanin,† Giuseppina Grisolia,† Marco Eleopra,†
Michele Rizzi,† Manlio Tolomeo,‡ Giuseppe Giannini,§ Domenico Alloatti,§ Massimo Castorina,§ Marcella Marcellini,§ and
Claudio Pisano§
Dipartimento di Scienze Farmaceutiche, UniVersita` di Ferrara, Via Fossato di Mortara 17/19, 44100 Ferrara, Italy,
Centro Interdipartimentale di Ricerca in Oncologia Clinica (C.I.R.O.C.) e SerVizio AIDS, Palermo, Italy, R&D,
Sigma-Tau SpA Industrie Farmaceutiche Riunite, Pomezia, Italy
ReceiVed April 30, 2008
A novel class of combretastatins, modified at A-ring or both A- and B-rings, mainly by replacement with
benzofuran or benzo[b]thiophene, were synthesized. The new heterocombretastatins showed good cytotoxic
activity on BMEC and H-460 cell lines. The aminocombretastatin 9f potently inhibits cell growth of BMEC
and combretastatin-resistant HT-29 cell lines, with potential interest to treat colon carcinoma. Heterocom-
bretastatins 9a,b inhibit tubulin polymerization similarly to CA-4 by having a binding to colchicine site five
times stronger.
Introduction
Combretastatins are natural antimitotic agents isolated from
the bark of the South African tree Combretum caffrum. Among
these compounds, combretastatin A-4 (CA-4,a 1, Figure 1)
possesses the most potent and interesting antitumor activity.1,2
CA-4 is an exceptionally strong inhibitor of tubulin polymer-
ization and potently cytotoxic against murine lymphocytic
leukemia and human ovarian and colon cancer cell lines.3 Its
mechanism of action is believed to be related to tubulin-binding
properties that result in rapid tumor endothelial cell damage,
neovascular shutdown, and subsequent hemorrhagic necrosis.4
CA-4P, a combretastatin-A4 phosphate prodrug, is a vascular
disrupting agent (VDA) that was tested in clinical studies.5
Interestingly, CA-4P does not induce the common side effects
of existing chemotherapy such as alopecia and bone marrow
Figure 1. Natural and synthetic combretastatins.
study for the purpose of evaluating the apoptotic activity of
natural and synthetic stilbenoids. Several active stilbenes were
identified and, among them, the cis-3,4′,5-trimethoxy-3′-ami-
nostilbene (3) proved highly potent against various tumor cells,
selectively suppressing tumor vascular perfusion without damaging
normal vascular perfusion in a DCE-MRI study.7 Most importantly,
mice treated with five daily injections of stilbene 3 did not show
any compromise in heart function.8,9 Recently, we also examined
the effects regarding replacement of the B-ring of CA-4 with
different benzoheterocycles.10,11The phosphate prodrugs ST2495
(5) and ST2496 (6) demonstrated high antitumor activity in in
vitro and in vivo models, notwithstanding a minimal effect on
microtubule organization, with respect to CA-4. Interestingly,
despite a lower in vitro cytotoxic potency of the precursor drugs,
compared to CA-4, the prodrugs were active in vivo in a manner
comparable to CA-4P. The prodrugs 5 and 6 were also effective
after oral administration, thus suggesting an additional mitotic
molecular target or, alternatively, a different binding site or
mode of interaction with tubulin for ST molecules. In a more
recent work, aminoindole cycles have been also introduced into
phenstatin derivatives active as tubulin assembly inhibitors.12
In this study, we further investigated stilbenes by extending our
toxicity. However, cardiovascular toxicity and neurotoxicity
were dose limiting for CA-4P.6 These significant side effects
currently represent the main obstacles to broad clinical applica-
tion of CA-4P. For this reason, it is necessary to develop other
CA-4 structurally related compounds with more specificity for
tumor endothelial cells than normal endothelial cells to avoid
cardiac toxicity from endothelial damage. In this context, from
a medicinal chemistry point of view, the structural simplicity
of CA-4 offers a stimulating premise for the design of new and
more potent compounds with improved pharmacological proper-
ties.3
In an ongoing project aimed at developing novel apoptosis
inducers incorporating the stilbene motif, we recently started a
* To whom correspondence should be addressed. Phone: +39-0532-
455923. Fax: +39-0532-455953. E-mail: smd@unife.it.
†
Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
Centro Interdipartimentale di Ricerca in Oncologia Clinica (C.I.R.O.C.)
‡
e Servizio AIDS.
§
R&D, Sigma-Tau SpA Industrie Farmaceutiche Riunite.
a Abbreviations: CA-4, combretastatin A-4; CA-4P, combretastatin-A4
phosphate prodrug; VDA, vascular disrupting agent; CTBC, colchicine
tubulin binding competition assay; TPI, tubulin polymerization inhibition
assay.
10.1021/jm8005004 CCC: $40.75
2008 American Chemical Society
Published on Web 09/11/2008

6212 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Brief Articles
Scheme 1a
a Reagents and conditions: (i) NaH, THF; (ii) TBAF, CH2Cl2; (iii) AcOH, Zn, MeOH, 1N HCl.
earlier studies on benzoheterocyle-based combretastatins. We
describe the effects due to replacement of the A-ring of CA-4
with different benzoheterocycles, concentrating mainly on
benzofuran and benzo[b]thiophene rings (compounds 9a-c,f).
The novel structural modifications are, in part, in agreement
with a pharmacophoric model recently described in the literature.
Additionally, compounds structurally related to 5 and 6
(derivatives 13a-d and 14a–d) were obtained by replacement
of the A-ring with the 3,4-methylendioxy ring system, the
characteristic moiety of natural combretastatin A-2 (4), as well
as with the dimethoxy group, characteristic of 3. To corroborate
our SAR investigation, we further explored combretastatins
where both the A- and B-rings were replaced with the ben-
zo[b]thiophene ring (compounds 13e, 14e) and derivatives 9e
and 10e bearing the 1,2,3,4-tetrahydro-1,1,4,4-tetramethylnaph-
thalene hydrophobic system.
We describe the evaluation of a primary screening of cy-
totoxicity on BMEC cell lines for all synthesized compounds.
Moreover, preliminary screening was carried out by analyzing
the activity of the new active compounds in both in vitro tubulin
polymerization inhibition (TPI) and colchicine tubulin binding
competition assay (CTBC).
The interesting biological results obtained with compounds
9a-c and 9f, the high cytotoxic activity as well as tubulin
polymerization inhibition properties, further demonstrated the
importance of the benzofuran and benzo[b]thiophene heteroaro-
matic rings in conferring activity on combretastatin molecules,
proving them ideal bioisosteres not only for replacement of the
B-ring of CA-4 but also, and possibly more important, as
bioisosters of the A-ring.
Chemistry. The synthetic route followed for the synthesis
of the desired novel combretastatins is outlined in Scheme 1a,b.
The phosphonium salts 11j,k13,14 and the (1,2,3,4-tetrahydro-
1,1,4,4-tetramethylnaphthalen-6-yl)methyl-triphenylphosphoni-
um bromide (7z)15 were prepared as reported in the literature.
The phosphonium salts 7w,x,y were obtained by starting from
the corresponding benzyl alcohols by reaction with carbon
tetrabromide and triphenylphosphine to obtain the corresponding
benzyl bromides, which were, in turn, reacted with triph-
enylphosphine in acetonitrile at 100 °C.11 The aldehydes 12
were achieved through a previously communicated procedure.11
Stilbenes 9a-g, 13a-e and the relative trans isomers 10a-g
and 14a-e were prepared in good yields through a Wittig
reaction between the appropriate aldehyde and phosphonium
salt in the presence of NaH. The phenolic moieties of the crude
stilbenes were, in turn, revealed by treatment with TBAF. The
1:1 mixture of the Z and E stilbene isomers was finally separated
by careful silica gel column chromatography and the single
isomers identified by NMR for their different coupling constants
between alkenyl proton signals (about 12 Hz for Z and 16 Hz
for E isomers). The nitro groups of derivatives 9d and 10d were
reduced with zinc in acetic acid to give amines 9f and 10f in
good yields.
Biological Results. A pharmacophore model for colchicine
site inhibitors was recently described, indicating for combret-
astatins a positive electrostatic potential region favoring activity

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6213
Table 1. Cytototoxicity and Tubulin Interactions of Synthesized Compounds
IC50 [nM] ( SD
compd
E/Z
IC50 [nM] ( SD
BMECa
HT-29b
H-460c
TP
CTBC
colchicine
CA-4
CA-2
9a
9b
9c
9e
9f
9g
10b
13a
13b
13c
13d
13e
1.7 ( 0.6
Z
Z
Z
Z
Z
Z
Z
Z
E
Z
Z
Z
Z
Z
3.7 ( 0.3
48 ( 1.3
17 ( 0.2
35 ( 1.8
35 ( 0.3
>10000
20 ( 2.3
123 ( 33
>10000
110 ( 11
800 ( 40
880 ( 52
>3000
118 ( 33
>1000
260 ( 32
420 ( 57
590 ( 39
1.2 ( 0.2
40 ( 0.4
13 ( 2.5
19 ( 5
4.9 ( 0.2
0.1 ( 0.02
2.9 ( 0.09
5.1 ( 0.07
3.9 ( 0.1
21.5 ( 1.9
3.6 ( 0.28
0.02 ( 0.002
0.02 ( 0.003
0.05 ( 0.002
NT
12 ( 0.8
21 ( 1.3
19 ( 1.3
110 ( 8.5
6.2 ( 0.12
>100
6.2 ( 0.3
3.8 ( 0.6
4.0 ( 0.09
4.9 ( 0.07
7.7 ( 0.1
NT
12.3 ( 0.5
7.1 ( 0.08
14.2 ( 0.9
16.6 ( 0.7
24.3 ( 0.8
250 ( 0.5
312 ( 53
a Bovine microvascular endothelial cells. b Colon carcinoma cells. c Non-small-cell lung carcinoma cells. TP: tubulin polymerization; CTBC: colchicine
tubulin binding competition; IC50: concentration of drug required to produce an inhibitory effect of 50%; SD: standard deviation; NT: not tested.
in A- and B-rings. This region is located in the outer zone of
the trimethoxy groups in the A-ring,16,17 suggesting that other
hydrogen bond acceptors could replace the trimethoxy groups
giving active compounds. Therefore, heteroaromatic rings, such
as 9a-c, 9f,g, and 13c-e, containing electron-rich atoms, may
contribute to increase the activity of novel combretastatins. At
the same time, the benzoheterocycle skeleton could also be
favorable for steric interaction with a large hydrophobic pocket
at the active site, as described in the pharmacophoric model. In
this context, we synthesized a novel class of heterocombretastins
bearing different substitutions at the A-ring as well as at both
A- and B-rings. In particular, we synthesized compounds where:
(a) The A-ring is replaced by a benzofuran ring, 9b
(ST2897),18 10b, 9c, 10c; the benzo[b]thiophene ring, 9a
(ST2899), 10a, 9f,g, 10f,g, as well as the 1,2,3,4-tetrahydro-
1,1,4,4-tetramethylnaphthalene portion, 9e and 10e.
(b) The B-ring is replaced by a benzofuran or a benzo[b-
]thiophene heterocycle, whereas the A-ring is replaced by a
benzo[b]thiophene, a methylendioxobenzene ring as well as the
dimethoxy phenyl group: 13a (ST2900), 14a, 13b (ST2902),
14b, 13c (ST2892), 14c (ST2891), 13d (ST2933), 14d (ST2934),
13e, 14e.
All the synthesized compounds were tested in a preliminary
cytotoxicity assay on a BMEC cell line (see Table 1).19 Some
of the most active compounds were also tested on lung H-460
and colon HT-29 carcinoma cells, the latter naturally resistant
to CA-4. 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 because it
is well documented in the literature and also by us.20,21
Compounds 9a-c, in which the A-ring is replaced by a
benzofuran or benzo[b]thiophene ring system, proved of great
importance. The new heterocombretastatins showed cytotoxic
activity at a low nanomolar level on the BMEC cell line as
well as on H-460 cell lines. Interestingly, 9a activity on the
combretastatin-resistant HT-29 cell line is comparable to the
natural CA-4. The activity of 9a-c also does not seem greatly
influenced by the position of the endocyclic heteroatom because
the two regioisomers 9b and 9c possess the same IC50 inhibition
on BMEC cell lines. The benzo[b]thiophene derivative 9a
therefore, has the lowest IC50 on both BMEC and HT-29 cell
lines, making it a lead for further investigations.
interactions at the active site. A further dramatic loss of activity
was observed by replacement of the characteristic isovanillic
moiety of compounds 9a-c and of CA-2 with a benzohetero-
cycle, as Table 1 shows. The only exception is compound 13e,
bearing the double benzo[b]thiophene substitution, which,
although less potent than the nonactive compounds, shows
cytotoxic activity against BMEC and H460 cell lines (IC50 value
about 250 and 312 nM, respectively). Accordingly, the com-
pound still affects tubulin polymerization (IC50 ) 24.3 nM).
The most active compounds described above, 9a-c, were
further examined for their effects on tubulin assembly. Prelimi-
nary 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).
Interestingly, the heterocombretastatins 9a-c potently inhibit
tubulin polymerization with an IC50 similar to or better than
that of CA-4 and, quite surprisingly, the binding of compounds
9a-b to the colchicine binding site resulted five times higher
than that of natural CA-4. Regarding the dimethoxylated
derivatives 13a and 13b, these are less active than the parent
trimethoxy derivatives. This was also quite surprising, since we
previously demonstrated the importance of the dimethoxy group
for compound 3. Because both compounds showed inhibition
to tubulin polymerization and to binding at the colchicine site,
the lack of cytotoxic activity may be due to the difficulty in
reaching the cellular target.
Finally, further SAR information was obtained by structural
alterations of the isovanillic portion of 9a by removal of the
OH moiety as well as by its replacement with an amino group
as in compounds 9f,g. The amino compound 9f seems of great
importance in inhibiting cell growth of BMEC and H-460 at
low nanomolar level, with a profile of activity partly comparable
with the amino derivatives of CA-2, recently described by
Pettit,22 and, quite surprisingly, the compound is also active on
the HT-29 cell line. Colon carcinoma cells are not particularly
responsive toward antimitotic agents, including combretastatins,
and generally this class of chemotherapic agents is not used
for the treatment of colon carcinoma. As displayed in Table 1,
combretastatin A-4 showed in HT-29 cells an IC50 of 118 nM
and the most active compounds of the series 9a-c were still
less active than combretastatin A-4. In contrast, 9f displayed
an IC50 of only 21 µM, which was similar to that obtained on
non-small-cell lung carcinoma H-460 cell line. Compound 9f
showed a tubulin polymerization IC50 similar to that of CA-4
(4.9 µM vs 3.6 µM), although the colchicine tubulin binding
Substitution of the A-ring with the 1,2,3,4-tetrahydro-1,1,4,4-
tetramethylnaphthalene gave derivative 9e, lacking any activity
notwithstanding the potential improvement due to hydrophobic

6214 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Brief Articles
silica gel (eluent: 5-20% ethyl acetate/light petroleum). Stereoi-
someric purity of separated compounds was confirmed through
NMR.
2-Methoxy-5-[(Z)-2-(4-methoxy-benzo[b]thiophen-6-yl)-ethenyl]-
phenol (9a). Yield 43%, oil. 1H NMR δ 3.74 (s, 3H), 3.87 (s, 3H),
5.50 (s, 1H), 6.52 (d, J ) 12 Hz, 1H), 6.60 (d, J ) 12. Hz, 1H),
6.68- 6.72 (m, 2H), 6.78 (d, J ) 2 Hz, 1H), 6.91 (d, J ) 2 Hz,
1H), 7.29 (d, J ) 5.4 Hz, 1H), 7.37-7.44 (m, 2H). Anal.
(C18H16O3S) C, H, S.
2-Methoxy-5-[(Z)-2-(4-methoxy-benzofuran-6-yl)-ethenyl]-phe-
1
nol (9b). Yield 40%, oil. H NMR δ 3.76 (s, 3H), 3.86 (s, 3H),
Figure 2. Effects of compound 9f on DNA content/cell. Cells were
cultured without (a), or with 0.5 µM 9f (b). Cell cycle distribution was
analyzed after 24 h treatment by the standard propidium iodide
procedure as described in the Experimental Section of Supporting
Information. Sub-G0-G1 (A), G0-G1, S, and G2-M cells are indicated in
the control panel (a).
5.52 (br, 1H), 6.50 (d, J ) 12 Hz, 1H), 6.56-6.64 (m, 2H), 6.73
(s, 1H), 6.78-6.80 (m, 2H), 6.91 (d, J ) 2 Hz, 1H), 7.01 (s, 1H),
7.49 (d, J ) 2 Hz, 1H). MALDI-TOF: 297.2 [M+]. Anal.
(C18H16O4) C, H.
2-Methoxy-5-[(Z)-2-(7-methoxy-benzofuran-5-yl)-ethenyl]-phe-
1
nol (9c). Yield 38%, oil. H NMR δ 3.81 (s, 3H), 3.86 (s, 3H),
5.47 (s, 1H), 6.48 (d, J ) 12 Hz, 1H), 6.60 (d, J ) 12.2 Hz, 1H),
6.68 (d, J ) 2 Hz, 1H), 6.72 (s, 1H), 6.75-6.76 (m, 2H), 6.88 (d,
J ) 2 Hz, 1H), 7.1 (s, 1H), 7.57 (d, J ) 2 Hz, 1H). Anal. (C18H16O4)
C, H.
6-[(Z)-2-(4-Methoxyphenyl)-ethenyl]-4-methoxybenzo[b]thio-
phene (9g). Yield 40%. 1H NMR δ 3.72 (s, 3H), 3.78 (s, 3H),
6.57 (d, J ) 12.0 Hz, 1H), 6.60 (d, J ) 12.0 Hz, 1H), 6.67-6.69
(m, 1H), 6.75-6.79 (m, 2H), 7.22-7.23 (m, 1H), 7.24-7.26 (m,
1H), 7.29 (d, J ) 5,6 Hz, 1H), 7.38 (s, 1H), 7.42 (dd, J ) 5.6 Hz,
J ) 0.8 Hz 1H). 13C NMR δ 55.3, 105.0, 113.7, 115.4, 120.5,
124,9, 129.0, 129.6, 129.8, 130.4, 135.1, 141.1, 158.8. Anal.
(C18H16O2S) C, H, S.
General Procedure for the Synthesis of Amino Derivatives 9f
and 10f. Zinc powder (100 mmol, 6.5 g) was added portionwise to
a solution of nitrostilbenes 9d, 10d (1 mmol) in acetic acid (15
mL). The suspension was stirred for 2 h at room temperature. The
reaction mixture was filtered over celite and concentrated. The crude
material was dissolved in ethyl acetate (15 mL) and washed with
sodium bicarbonate 5% (5 mL) and brine (5 mL), dried, and
concentrated to afford the desired crude amino compound used for
the next reaction without any purification. To a suspension of amino
stilbene derivative solubilized in methanol, 1 N HCl was added,
the mixture stirred at room temperature for 10 min and concentrated
to afford the solid stilbene salt.
6-[(Z)-2-(3-Amino-4-methoxyphenyl)-ethenyl]-4-methoxyben-
zo[b]thiophene Hydrochloride (9f). Yield 70%, mp 159-161 °C.
1H NMR (CD3OD) δ 3.75 (s, 3H), 3.95 (s, 3H), 6.62 (d, J ) 12.4
Hz, 1H), 6.67 (s, 1H), 6.77 (d, J ) 12.4 Hz, 1H), 7.13 (d, J ) 8.4
Hz, 1H), 7.25 (d, J ) 2.0 Hz, 1H), 7.34 (d, J ) 1.2 Hz, 1H),
7.36-7.37 (m, 1H), 7.11-7.15 (m, 1H), 7.43 (d, J ) 5.6 Hz, 1H).
13C NMR (CD3OD) δ 55.7, 56.9, 105.8, 113.2, 116.1, 121.3, 125.6,
126.4, 129.1, 131.1, 132.1, 132.3, 135.7, 153.1, 156.0. Anal.
(C18H18ClNO2S) C, H, N, S.
competition (CTBC) IC50s of 9f and CA-4 were markedly
different (0.1 µM vs 6.2 µM). This might suggest that 9f acts
on tubulin polymerization like combretastatins but on a site
different from colchicine. To confirm that the main mechanism
of action of 9f was antimitotic, we evaluated its effect on cell
cycle. As shown in Figure 2, compound 9f induced a prevalent
block of cells in G2/M phase of cell cycle. Therefore, 9f is an
unique analogue displaying a profile of activity comparable to
combretastatin but endowed with therapeutical potential in colon
carcinoma.
In conclusion, we describe in this paper a novel class of
combretastatins bearing the replacement of the A-ring or both
A- and B-rings by a benzoheterocycle (benzofuran or benzo[b]-
thiophene) and methylenedioxobenzene as well as dimethoxy-
benzene or 1,2,3,4-tetrahydro-1,1,4,4-tetramethylnaphthalene
fragments. Our data evidence that derivatives of CA-4, bearing
a benzofuran or benzo[b]thiophene heterocycle, which replaces
the A-ring, possess potent cytotoxic activity while also showing
potent binding to tubulin and inhibition of tubulin polymerization.
Moreover, compounds bearing the double benzo[b]thiophene
substitution appears of interest as it produces good cytotoxic
activity. It appears to be particularly interesting that the amino
derivative 9f is able to inhibit growth of colon carcinoma HT-
29 cells at low nM concentration.
The interesting biological results obtained with structural
changes at A-ring of CA-4, a region of the natural compound
hitherto scarcely considered, demonstrate that structural alter-
ation at this portion of the natural compound offers interesting
premises for the design of novel and potent CA-4 analogues.
Experimental Section
Acknowledgment. This work was financially supported in
part by Ministero dell’Universita` e della Ricerca Scientifica e
Tecnologica (PRIN 2006), Rome, Italy.
General Procedure for the Synthesis of Stilbenes 9a-e,g,
10a-e,g, 13a-e, 14a-e. To a solution of aldehyde 8, 12 (2 mmol),
solubilized in 10 mL of anhydrous THF, the opportune triph-
enylphosphonium salt 7w,x,y,z, 11j,k (2.2 mmol) was added. The
suspension thus obtained was cooled in an ice bath, then NaH (50%
in mineral suspension, 2.2 mmol, 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), the extracts washed
with water (5 mL) and brine (5 mL), then dried and concentrated
in vacuo.
For derivatives in which the phenolic moiety is protected as
TBDMS ether, the residue was dissolved in methylene chloride
(10 mL) and tetrabutyl ammonium fluoride (6 mmol) was 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), then dried. After concentration, E and Z stereoisomers
were separated and purified by careful flash chromatography on
Supporting Information Available: Synthetic procedures and
characterizations of intermediates and less active compounds,
experimental procedures for biological assays, and elemental
analysis of target derivatives. This material is available free of
charge via the Internet at http://pubs.acs.org.
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