Synthesis and cytotoxic evaluation of cis-locked and constrained analogues of combretastatin and combretastatin A4

Article abstract of DOI:10.1016/j.tetlet.2010.07.120

A series of cis-locked stilbenic compounds related to combretastatin and combretastatin A4 has been designed and synthesized. The cytotoxic effects of these rigid analogues were evaluated as well as their abilities to inhibit tubulin polymerization.

Full text of DOI:10.1016/j.tetlet.2010.07.120

Tetrahedron Letters 51 (2010) 5146–5149
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and cytotoxic evaluation of cis-locked and constrained analogues
of combretastatin and combretastatin A4
Magali Lorion a,b, Vangelis Agouridas a,b, Axel Couture a,c, , Eric Deniau a,b, Pierre Grandclaudon a,b
*
a Univ Lille Nord de France, F-59000 Lille, France
b USTL, Laboratoire de Chimie Organique Physique, EA CMF 4478, Bâtiment C3(2), F-59655 Villeneuve d’Ascq, France
c CNRS, UMR 8181 ‘UCCS’, F-59655 Villeneuve d’Ascq, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of cis-locked stilbenic compounds related to combretastatin and combretastatin A4 has been
designed and synthesized. The cytotoxic effects of these rigid analogues were evaluated as well as their
abilities to inhibit tubulin polymerization.
Received 2 June 2010
Revised 15 July 2010
Accepted 21 July 2010
Available online 25 July 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Combretastatins
Isoindolinones
Oxygen heterocycle
Oxidation
Anticancer agents
1. Introduction
and B and double bond C (Fig. 1).6,8a Owing to the fact that the
3,4,5-trimethoxy substitution on the A ring, the cis orientation be-
The combretastatins represent a group of structurally simple
antimitotic agents known as potent inhibitors of tubulin assem-
bly,1 a research field that received a major impetus when Pettit
and co-workers isolated (À)-combretastatin 1 (Fig. 1) from the
bark of the South African bush willow tree Combretum caffrum.2
Owing to its high affinity to the colchicine site3 the most active
of these, combretastatin A4 (CA4) (2) acts as a potent inhibitor of
cancer cell growth inducing irreversible vascular shutdown
within solid tumors while sparing normal vasculature.4 Its more
available prodrug disodium phosphate analogue is now in Phase
III clinical trials.5 Consequently significant efforts have been
expended to develop combretastatin-type analogues and the
Z-stilbenoid molecular scaffold has provided a simple structural
template for the design of related compounds that retain the bio-
logical action of the parent molecules but provide improved phar-
macokinetic properties.6,7 Structure–activity relationship studies
(SAR) then led to the discovery of an array of biologically active
analogues that were assessed as potential anticancer agents, some
of which have recently been reviewed.6,8 The literature demon-
strates the impressive possibilities for structural variability in
analogues of this class of compounds and structure 2 has been
modified in each of the three elements, that is, aromatic rings A
tween the two aryl rings and the free hydroxyl functionality at
ring B are essential requirements for efficient binding to tubulin
and high levels of cytotoxicity16,9 structural variations have been
notably devoted to the synthesis and biological evaluation of
derivatives with carbo and heterocyclic moieties fused with one
of the aromatic units (Fig. 1D)6,8 or in place of the olefinic bridge
(Fig. 1E).10
In this regard a group of five-membered benzolactamic com-
pounds has been designed and reported with potent growth inhib-
itory activities on hormone-independent prostate and breast
cancer lines with IC50 values in low to subnanomolar range.11
However, these SAR studies have been confined to 3-benzylidene
OMe
A
OMe
MeO
MeO
A
C
B
D
A/B
B/A
OH
MeO
HO
MeO
HO
E
C
MeO
MeO
Combretastatin (1)
Combretastatin A4
(CA4, 2)
* Corresponding author. Tel.: +33 (0) 3 20 43 44 32; fax: +33 (0) 3 20 33 61 09.
E-mail address: axel.couture@univ-lille1.fr (A. Couture).
Figure 1. Combretastatins.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.07.120

M. Lorion et al. / Tetrahedron Letters 51 (2010) 5146–5149
5147
(Fig. 1, AD = isoxindole) have been ignored by the scientific com-
munity. This is probably attributable to the difficulties associated
with the assembly of Z-configured stilbene compounds which are
prone to double bond isomerization during storage, leading to
the E-isomers which display a dramatically reduced biological
activity.12
OMe
OMe
O
O
MeO
MeO
MeO
MeO
N
N
O
O
BnO
MeO
HO
MeO
In this Letter we report our exploration of the synthesis and bio-
logical evaluation of a number of combretastatin-like benzylidene-
isoindolinones and our attention was focused on a structural mod-
ification that maintained the cis-diaryl relationship of 2 (CA4). In
this view we targeted the synthesis of configurationally cis-locked
CA4 analogues 3a and 3b (Scheme 1) which are characterized by
the presence of a bridging olefinic moiety embedded in a morpho-
line unit. It has been well established that the presence of hetero-
atoms in a cyclic moiety around the bridge helps in retaining the
bioactive configuration of the molecule and leads to a broader vari-
ety of SAR studies.8a Furthermore the elaboration of these targeted
compounds offered a double advantage since we surmised that
such compounds could be accessed from the corresponding satu-
rated compounds 4a, 4b, respectively, which possess the main
structural feature of the inhibitor of cancer cell proliferation com-
bretastatin 1.13
4a
3a
O
O
MeO
BnO
MeO
MeO
MeO
HO
N
N
O
O
MeO
MeO
OMe
3b
4b
OMe
Scheme 1. Constrained analogues of combretastatin and combretastatin A4.
indolin-2-one derivatives (Fig. 1, AD = oxindole) and to the best of
our knowledge the corresponding 3-benzylidene-isoindolinones
1. NaH , DMF
O
2.
nBuLi (3 equiv)
TMEDA (3 equiv)
THF , -78 °C
R1
R1
R1
HN
O
O
O
N
MeO
R2
Li
MeO
R2
Br
MeO
R2
Br
O
O
Br
N
9c,d
7c,d
8
1. KHMDS (1 equiv)
THF , -78 °C
CHO
2.
R3
R1
R1
O
O
Cl
N
10, 11
MeO
R2
MeO
R2
OH
SOCl2
H3O
MeO
R4
N
5c,d
6c,d
R1
R1
1. -78 °C
to rt
O
O
MeO
R2
MeO
R2
Cl
4c R3 = OBn ; R1 = R2 = OMe ; R4 = H
4d R2 = OBn ; R1 = H ; R3 = R4 = OMe
N
N
2. H3O
H
O
H
H2
O
Pd/C
R3
MeO
R3
MeO
4a R3 = OH ; R1 = R2 = OMe ; R4 = H
4b R2 = OH ; R1 = H ; R3 = R4 = OMe
R4
R4
12
S8
,
tetraline , reflux
R1
O
N
O
MeO
R2
3c R3 = OBn ; R1 = R2 = OMe ; R4 = H
3d R2 = OBn ; R1 = H ; R3 = R4 = OMe
H2
Pd/C
R3
MeO
3a R3 = OH ; R1 = R2 = OMe ; R4 = H
3b R2 = OH ; R1 = H ; R3 = R4 = OMe
R4
Scheme 2. Synthesis of combretastatin analogues 3a,b and 4a,b.

5148
M. Lorion et al. / Tetrahedron Letters 51 (2010) 5146–5149
Table 1
Compounds 6c,d, 5c,d, 4a–d, 3a–d, produced via Scheme 2
R1
R2
R3
R4
Mp (yield)
6
5
4
3
a
b
c
OMe
H
OMe
H
OMe
OH
OMe
OBn
OH
H
OMe
H
—
—
—
—
194–196 °C (87%)
227–229 °C (65%)
165–167 °C (91%)
210–212 °C (56%)
170–172 °C (69%)
221–223 °C (55%)
152–154 °C (54%)
198–200 °C (52%)
OMe
OBn
OMe
146–148 °C (54%)
175–177 °C (52%)
113–115 °C (96%)
159–161 °C (97%)
d
OMe
bling of the highly fused models 4c,d could be fortunately per-
formed as a single one-pot reaction. For this purpose, compounds
5c,d were smoothly deprotonated with KHMDS in THF at À78 °C
and subsequently allowed to react with the appropriate aldehydes
10, 11. To trigger off the intramolecular O-alkylation process the
reaction mixture containing the transient oxanion 12 was warmed
to rt followed by standard aqueous workup. Gratifyingly conduct-
ing this reaction according to this procedure brought about the
intramolecular cyclization and afforded straightforwardly and so-
lely the annulated compounds 4c,d. The isolation of a single crystal
of 4c gave access to its X-ray analysis (Fig. 2). The ORTEP view
clearly established the trans configuration of the hydrogen atoms
at C-2 and C-19 with a dihedral angle of 176.08°.15
With these highly fused compounds in hand we were only one
deprotection away from the targeted compounds 4a,b which can
be regarded as constrained analogues of combrestatin 1. Thus the
treatment of 4c,d with Pd/C under H2 atmosphere under mild con-
ditions triggered off the cleavage of the benzyl-protecting group
and delivered excellent yields of the desired compounds 4a,b.
The synthesis of the strict structural analogues 3a,b of combretast-
atin A4 (2) from the corresponding mono-protected and saturated
compounds 4c,d, respectively, revealed itself to be much more
problematic than we had anticipated. Several attempts to ensure
the creation of the central double bond including treatment with
oxone, DDQ, Pd/C in refluxing nitrotoluene, NBS then AIBN, a mul-
tistep sequence involving metallation/capture with PhSeCl/oxida-
tion/elimination, and oxidation with neat Se failed. One can
reasonably assume that this elimination reaction is fraught with
difficulties associated with the trans configuration of benzylic
2. Results/discussion
The key step for the assembly of the targeted compounds 3a,b
was the preliminary elaboration of the suitably substituted N-chlo-
roethylisoindolinones 5c,d (Scheme 2). We surmised that these
compounds would be readily accessible from the corresponding
alcohols 6c,d which could be in turn conceivably assembled by
an anionic cyclization process applied to the N-bromobenzyl
substituted oxazolidinones 7c,d.14 Indeed we conjectured that
the interception of the aryllithiated species 8 derived from 7c,d
by the oxazolidinone ring system, that is, a cyclic carbamate acting
as the internal electrophile would provide the potential for a direct
access to a properly substituted isoindolinone with the concomi-
tant installation of the mandatory hydroxyethyl appendage, there-
fore providing the N-functionalized models 6c,d.
Initially the cyclic carbamate precursors 7c,d were readily
assembled by N-alkylation of 2-oxazolidinone with the suitably
substituted ortho-bromobenzylbromide derivatives 9c,d obtained
by the standard chemical manipulation of the corresponding ben-
zyl alcohols. Optimal conditions for the key ring-opening/ring -
closing reaction had to be determined and we found that adding
the parent compound (7c,d, 1 equiv) in THF to nBuLi (3 equiv)
and TMEDA (3 equiv) in degassed THF at À78 °C for 30 min led to
complete consumption of the starting material and isolation solely
of the targeted hydroxyethyl chain tethered isoindolinones 6c,d in
fairly good yields (Scheme 1, Table 1). Conversion of alcohols 6c,d
into the key intermediates 5c,d by treatment with thionyl chloride
proceeded uneventfully to deliver very satisfactory yields of the
requisite N-chloroethylisoindolinones 5c,d (Table 1). The assem-
Figure 2. ORTEP view of the crystal structure of 4c.

M. Lorion et al. / Tetrahedron Letters 51 (2010) 5146–5149
5149
Table 2
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c
Compound
Cytotoxicity IC50
[lM]
% ITPd
Combretastatin A4
0.003
0.16
>100
>100
>100
100
23
—
30
—
3a
3b
4a
4b
a
b
c
KB = cervical carcinoma cells.
Concentration: 10À5 M in DMSO.
IC50 is the concentration of compound inducing 50% cell growth after 72 h
incubation.
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d
Inhibition of tubulin polymerization of the tested compound compared to that
of CA4 (i.e., 100%).
7. For very recent publications on the synthesis and biological evaluation of
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hydrogen atoms of these rather congested models. At last we found
that this rather simple chemical transformation could be readily
secured by treatment of the saturated compounds 4c,d with sulfur
in refluxing tetraline for a short period (5 min). This operation
delivered quite satisfactory yields of the mono-protected oxidized
compounds 3c,d (Scheme 1, Table 1). Regeneration of the hydroxyl
phenolic function from 3c,d afforded excellent yields of the re-
quired Z-configured stilbenic derivatives 3a,b.
Table 2 displays the cytotoxicity on human KB cells and the
antitubulin activity values of the various analogues synthe-
sized.16,17 Despite their structural similarity with combretastatin
A4, we were surprised to notice that three of them (3b, 4a,b) were
devoid of any appreciable cytotoxic activity. Rather disappoint-
ingly, only compound 3a featuring an isoxindole fused with a tri-
methoxyaryl ring and incorporating a cis-locked alkene seemed
to gather the structural requirements for an improved cytotoxicity
(IC50 = 0.16 lM). This is probably a consequence of the ability of
this compound to inhibit tubulin polymerization.
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3. Conclusion
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P.; Grimaudo, S.; Jung, M. K.; Hamel, E.; Gebbia, N.; Crosta, L.; Abbadessa, V.; Di
Cristina, A.; Dusonchet, L.; Meli, M.; Tolomeo, M. J. Med. Chem. 2005, 48, 723–
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Day, B. W. Bioorg. Med. Chem. 1997, 5, 715–722; (e) Pirali, T.; Busacca, S.;
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G. C.; Sorba, G.; Genazzani, A. A. J. Med. Chem. 2006, 49, 5372–5376.
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Lewis, A.; Johnsamuel, J. Bioorg. Med. Chem. Lett. 2005, 15, 5382–5385; (b)
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We developed a new high-yielding synthetic route to highly
fused combretastatin derivatives incorporating a five-membered
lactamic unit and a morpholine moiety and we were also able to
prepare the environmentally diverse analogues of cis CA4 from
appropriately substituted isoindolinones.
Acknowledgments
The authors wish to thank Dr. O Mentré and Dr. F. Capet (UCCS,
CNRS, USTLille1) for their single crystal X-ray structure analysis.
We thank Drs. T. Cresteil and J. Dubois (ICSN, CNRS, Gif-sur-Yvette)
for the cytotoxicity and tubulin assays.
12. Pettit, G. R.; Toki, B. E.; Herald, D. L.; Boyd, M. R.; Hamel, E.; Pettit, R. K.;
Chapuis, J. C. J. Med. Chem. 1999, 42, 1459–1465.
13. Pinney, K. G.; Jelinek, C.; Edvardsen, K.; Chaplin, D. J.; Pettit, G. R. In Anticancer
Agents from Natural Products; Cragg, G. M., Kingston, D. G. I., Newmann, D. J.,
Eds.; CRC/Taylor and Francis: Boca Raton, 2005; pp 23–46.
Supplementary data
Supplementary data (copies of 1H and 13C NMR spectra for
tested compounds) associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.07.120.
14. Lorion, M.; Couture, A.; Deniau, E.; Grandclaudon, P. Synthesis 2008, 2141–2147.
15. X-ray data for 4c:
Dx = 1.263 g.cmÀ3
a = 11.0823 (5), b = 27.2073 (15), c = 8.5764 (5) Å,
= 90.00°, V = 2586.0(2) Å3. Further details of the X-ray structure data are
C
l
28H29NO7, M = 491.52, F(000) = 1040, colorless crystal,
(MoK ) = 0.091 cmÀ1
orthorhombic, Pna2, Z = 4,
= 90.00°, b = 90.00°,
,
a
a
c
available on request from the Cambridge Crystallographic Data Centre
(deposition number CCDC 776601).
References and notes
16. Experimental protocol: KB (human epidermoid carcinoma) cells were grown in
Dulbecco’s modified Eagle’s medium supplemented with 25 mM glucose, 10%
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(v/v) fetal calf serum, 100 UI penicillin, 100
fungizone and were kept under 5% CO2 at 37 °C. 96-well plates were seeded
with 500 KB cells per well in 200 medium. Twenty four hours later,
lg/ml streptomycin and 1.5 lg/ml
ll
chemicals dissolved in DMSO were added for 72 h at a final concentration
(10À5 M) in a fixed volume of DMSO (1% final concentration). Controls received
an equal volume of DMSO. The number of viable cells measured at 490 nm
with the MTS reagent (Promega, Madison, WI) and IC50 was calculated as the
concentration of compound eliciting a 50% inhibition of cell proliferation.
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