Synthesis and biological evaluation of novel heterocyclic derivatives of combretastatin A-4

Article abstract of DOI:10.1016/j.bmcl.2012.09.047

A novel series of combretastatin A-4 heterocyclic analogues was prepared by replacement of the B ring with indole, benzofurane or benzothiophene, attached at the C2 position. These compounds were evaluated for their abilities to inhibit tubulin assembly: derivative cis 3b, having a benzothiophene, showed an activity similar to those of colchicine or deoxypodophyllotoxine. The antiproliferative and antimitotic properties of cis 3b against keratinocyte cancer cell lines were also evaluated and the intracellular organization of microtubules in the cells after treatment with both stereoisomers of 3b was also determined, using confocal microscopy.

Full text of DOI:10.1016/j.bmcl.2012.09.047

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7227–7231
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel heterocyclic derivatives
of combretastatin A-4
Thi Thanh Binh Nguyen a, Thierry Lomberget a, , Ngoc Chau Tran a, Evelyne Colomb b, Lore Nachtergaele b,
⇑
Sylviane Thoret c, Joëlle Dubois c, Joren Guillaume a, Rawad Abdayem b, Marek Haftek b, Roland Barret a
a Université de Lyon, Université Lyon 1, Faculté de Pharmacie, ISPB, EA 4446 Biomolécules, Cancer et Chimiorésistances, SFR Santé Lyon-Est CNRS UMS3453 - INSERM US7, 8
avenue Rockefeller, F-69373 Lyon cedex 08, France
b Université Lyon 1, Faculté de Médecine et de Pharmacie, EA 4169, Fundamental, clinical and therapeutic aspects of the skin barrier, 8 avenue Rockefeller, F-69373 Lyon cedex
08, France
c Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2012
Revised 14 September 2012
Accepted 16 September 2012
Available online 24 September 2012
A novel series of combretastatin A-4 heterocyclic analogues was prepared by replacement of the B ring
with indole, benzofurane or benzothiophene, attached at the C2 position. These compounds were evalu-
ated for their abilities to inhibit tubulin assembly: derivative cis 3b, having a benzothiophene, showed an
activity similar to those of colchicine or deoxypodophyllotoxine. The antiproliferative and antimitotic
properties of cis 3b against keratinocyte cancer cell lines were also evaluated and the intracellular orga-
nization of microtubules in the cells after treatment with both stereoisomers of 3b was also determined,
using confocal microscopy.
Keywords:
Combretastatin A-4
Antimitotic
Ó 2012 Elsevier Ltd. All rights reserved.
Benzoheterocycles
Inhibitors of tubulin assembly
Keratinocyte
Amongst all the chemotherapeutic classes that are used to cure
cancer, microtubule-binding agents (MBAs) became over the last
decades an important tool and an alternative to DNA-damaging
drugs.1
Microtubules, which are cylindrical polymeric structures, are
essential to maintain the cell shape and play a crucial role during
targeting neo-vascularized tumors.6 Using this strategy, several
derivatives such as CA-4P7 (fosbretabulin, Fig. 1) and AVE80628
(ombrabulin, Fig. 1) undergo clinical trials.
For these reasons, a large number of CA-4 analogues have been
synthesized and evaluated for their tubulin-binding abilities,9
therefore acting as mitotic spindle poisons and, sometimes, as vas-
cular targeting and disrupting agents (VTA and VDA).10
the cell cycle. They are composed of a- and b-tubulin heterodimers
and the inhibition of their dynamic assembly/disassembly into the
The structure–activity relationships (SAR) have demonstrated
that the cis configuration of the double bond and the presence of
a trimethoxy aryl unit were essential for the activity.9 In parallel,
many studies aimed to appreciate the effect of the replacement
of the cis double bond by various heterocycles (1, Fig. 1) such as
furan/thiophene,11 dioxolane,12 thiazole,13 isoxazole,14 imidazole,15
triazole16 or tetrazole.17 This approach represents a way to block a
putative in vivo isomerization of the double bond to the less active
trans alkene.18,19
In continuation with investigations performed by Simoni et al
on heterocyclic derivatives 2 (Fig. 1),20 we have chosen while
designing our new anti-cancer drugs to keep the cis configuration
of the double bond of the trimethoxystyrene unit and to make a
variation on the nature of the substitution of the alkene. The
replacement of the B ring by various heterocycles attached on their
2-position (3, Fig. 1), could allow to evaluate the influence of this
modulation (i.e., attachment on a different position of the hetero-
cycle) on the biological activity.
mitotic spindle leads to apoptotic cell death.2
Many academic and industrial research groups have focused
their efforts on the development of anti-cancer drugs based on this
strategy, thus leading to numerous MBAs. Natural products were
frequently the source of different series, such as combretastatin
A-4 (CA-4, Fig. 1), which was isolated from the bark of the South
African willow tree Combretum caffrum by Pettit et al.3,4
This stilbenoid derivative was proved to bind to the colchicine
site of tubulin5 and, regarding to very promising antimitotic activ-
ities and its very simple structure, there was an ever-growing
interest for the design of CA-4 analogues. Another reason lies in
the vascular targeting and disrupting properties of water-soluble
CA-4 derived compounds, which would present advantage of
⇑
Corresponding author. Tel./fax: +33 478 777 082.
E-mail address: thierry.lomberget@univ-lyon1.fr (T. Lomberget).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.047

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T. T. B. Nguyen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7227–7231
b
c
I
MeO
MeO
OH
OMe
MeO
MeO
Y
A
B
O
O
5
SiEt3
OMe
OMe
OMe
OMe
CA-4
OMe
OMe
d
CA4-P:
Y = OPO3Na2
AVE8062: Y = NH-serine, HCl
OMe
OMe
O
O
6a
MeO
Het
7a
6
e
f
MeO
MeO
R
MeO
MeO MeO Y
X
OMe
OMe
O
OMe
OMe
MeO
3a
1
2
R = OH or NH2
X = O, S, NMe
Y = OH, OPO3Na2
Scheme 2. Reagents and conditions: (b) (1) 2,20,6,60-tetramethylpiperidine/BuLi/
ZnCl2ꢁTMEDA 1.5/1.5/0.5 equiv, THF, 0 °C then rt, 2 h. (2) I2 1.5 equiv, 16 h, 99%. (c)
3,4,5-trimethoxy ethynylbenzene 1.14 equiv, PdCl2(PPh3)2 2 mol %, CuI 4 mol %,
Et3N 1.3 equiv, THF, rt, 5 h, 94%. (d) Et3SiH 3 equiv, PtO2 5 mol %, 60 °C, 4 h, 95%. (e)
TBAF 1 M solution in THF 1.1 equiv, 60 °C, 2.5 h, 84%, 62/38 Z/E ratio. (f) Pd(OAc)2
2 mol %, KOH 1.5 equiv, DMF, 145 °C, 6 h, 94%, 93/7 Z/E ratio.
2
X
X = O
X = S
3a
3b
MeO
MeO
X = NR 3c
OMe
The solution was finally brought by a more recent reduction
method: the use of potassium hydroxide and dimethylformamide,
in the presence of a catalytic amount of palladium acetate.30 In-
deed, these conditions provided the desired Z isomer of 3a as the
major product, isolated with a 54% overall yield31 (Scheme 2).
According to the same synthetic route, compound Z-3b was pre-
pared from benzothiophene with a better 58% overall yield.
In order to compare oxygen and sulfur heteroatoms with the
isosteric NH group on the bioactivity, the preparation of the indole
derivative 3c was then envisaged.
Figure 1. Representative synthetic combretastatins and CA-4 analogues synthe-
sized in this study.
Our preliminary approach to prepare the target compounds 3
was to use a Wittig reaction between commercially available ben-
zofuran or benzothiophene 2-carboxaldehydes and the phosponi-
um salt 4,21 derived from 3,4,5-trimethoxy benzaldehyde
(Scheme 1). Unfortunately, our conditions led to a mixture of cis
and trans isomers that were difficult to separate, yielding after a
flash chromatography the desired stereoisomers cis-3a,b in moder-
ate to low isolated amounts.22
To improve the poor selectivity of the reaction, another strategy
was tried: the reduction of an acetylenic derivative (Scheme 2).
After deprotonation of the benzofurane, thanks to the zincate re-
agent TMPLi/ZnCl2 developed by Mongin’s group,23 followed by
iodination, 2-iodo benzofurane 5 was obtained in a quantitative
yield. A Sonogashira coupling reaction24 of this substrate with
3,4,5-trimethoxy ethynylbenzene25 afforded compound 6a in an
excellent 94% yield (Scheme 2).
We first envisaged reducing 6a by known procedures such as
hydrogenation in the presence of Lindlar’s catalyst26 or by acidic
hydrolysis of a pre-formed vinylborane derivative,27 but these
attempts led to unsatisfactory results.
OMe
g,h
OMe
N
6c
H
OMe
j
BF3K
N
Boc
N
N
H
Boc
i
Br
MeO
MeO
MeO
OMe
OMe
MeO
We then decided to use an hydrosilylation/desilylation se-
quence28 on 6a to prepare the cis double bond of the target com-
pound but, after the fluoride mediated vinylsilyl group
deprotection of 7a, a Z/E mixture was obtained.29
3c
MeO
MeO
8
OMe
Scheme 3. Reagents and conditions: (g) Et3SiH 3 equiv, PtO2 5 mol %, 60 °C, 4 h,
78%. (h) TBAF 1 M solution in THF 1.1 equiv, 60 °C, 2.5 h, 80%, Z isomer only. (i)
Pd(PPh3)4 5 mol %, Na2CO3, DME/water 2/1, 90 °C, 18 h, 47%. (j) K2CO3 3 equiv,
MeOH/water, rt (22 h) then 60 °C (24 h), 14%.
Br
PPh3
OMe
OMe
CHO
Table 1
a
Inhibition of tubulin assembly by cis- and trans-3
+
X
X
Entry
Compound
Inhibitiona
OMe
MeO
MeO
1
2
3
4
5
6
7
Colchicine
Deoxypodophyllotoxine
trans 3a
cis 3a
trans 3b
IC50 = 1.7
IC50 = 1.7
l
l
M
M
4
X = O or S
23% at 32 lM
OMe
IC50 = 9.6
IC50 = 8.4
IC50 = 2.6
IC50 = 21
l
l
l
M
M
M
3 (Z)-isomer
+ (E)-isomer
cis 3b
cis 3c
lM
a
IC50: Compound concentration required to decrease the rate of microtubule
assembly by 50%.
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, ꢀ78 °C, 1 h then rt, 18 h;
global yields 65% for 3a (X@O) and 77% for 3b (X@S).

T. T. B. Nguyen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7227–7231
7229
The synthesis of 2-iodoindole was first carried out from indole,
using literature protocols: protection of the NH group as a phenyl-
sulfonamide,32 LDA-mediated iodination33 at position 2 (thanks to
the ortho-directing properties of SO2Ph) and removal of the pro-
tecting group using a fluoride source.34 This iodo derivative was
then engaged in a Sonogashira coupling reaction with 3,4,5-
trimethoxy ethynylbenzene to get alkyne 6c in a moderate 39%
yield.
After addition of trimethylsilyl hydride under the previously de-
scribed conditions, the protodesilylation surprisingly afforded the
pure Z-isomer in an 80% yield. The Z configuration of compound
3c was confirmed after 1H NMR spectra comparison with a com-
pound obtained after Suzuki coupling35,36 of Z-monobromotri-
methoxystyrene37 with N-Boc protected indole 2-potassium
trifluoroborate38 followed by tert-butyl carbamate basic deprotec-
tion39 of 8 (Scheme 3).
With these E and Z compounds 3 in hand, we then carried out
the evaluation of biological properties of these new heterocyclic
derivatives of CA-4.
The heteroaromatic derivatives 3 prepared above were first
evaluated for their ability to inhibit tubulin assembly (Table 1).
The tubulin assembly assay was realized according to a previously
described protocol, using colchicine and deoxypodophyllotoxine as
reference compounds, evaluated the same day under the same
conditions.40
Firstly, it is to note that the best activity was once more ob-
served for cis derivatives as described in previous literature stud-
ies. The benzothiophene analogues 3b are much more active than
the benzofurane 3a. This first indication shows that the more volu-
minous sulfur heteroatom in 3b may bring a beneficial effect, com-
pared to the oxygen in 3a (Table 1, entries 4 and 6). On the
contrary, the introduction of a nitrogen atom seems detrimental
to the activity as shown by the weaker inhibition of compound
3c on tubulin polymerization (Table 1, entry 7). Finally, the most
active compound is cis derivative 3b (3 times more active than
its trans stereoisomer),41 which tubulin binding properties are sim-
ilar to the reference compounds colchicine and deoxypodophyllo-
toxine (Table 1, entry 6).
We have then decided to further investigate the biological ef-
fects of the most promising 3b compounds on two different epithe-
lial cell lines.
Indeed, epidermal cancers are most common in Man due to the
mutagenic action of long-term exposure to solar radiation and the
large surface of exposed skin. Most of the skin cancers are treated
with surgery. However, multiple lesions, especially when situated
at locations rendering surgery difficult because of the functionality
or esthetics issues, may require efficient local treatments sparing
the surrounding healthy tissue. Also, the related pre-cancerous le-
sions, for example, actinic keratosis, are a good indication for top-
ical therapies.
Two human keratinocyte lines were selected for this study: a
spontaneously immortalized HaCaT line,42 showing no tumoro-
geneicity in animal models, and SKv-a line derived from a human
papillomavirus (HPV-16)-induced intraepithelial neoplasia.43 The
latter line was selected for its capacity of forming tumors after
Figure 2. Effect of compounds cis and trans 3b and colchicine (a positive control) on
the intracellular organization of microtubules in SKv-a cells (left) and HaCaT cells
(right). (a,e) vehicle-treated controls; (b,f) colchicine; (c,g) cis 3b, (d,d0,h) trans 3b.
Beta-tubulin was labelled with a specific monoclonal antibody (Amersham Life
Sciences; CA, USA) revealed with a goat anti-mouse FITC probe (Caltech; Carlsbad,
CA, USA). Confocal optical sections were recorded at the objective magnification of
ꢂ63. Scale bar corresponds to 10
lm.
Table 2
Anti-proliferative activities of cis CA-4, colchicine and cis 3b as measured with MTT
test in HaCaT keratinocyte cell line
experimental subcutaneous injection in immunocompromised
mice.
a
Entry
Compound
IC50 (nM)
MTT test evaluating metabolic activity of cells, directly related
to the number of viable cells remaining after 72 h exposure,
showed that the cis isomer of 3b derivative displayed an anti-
proliferative activity on the cultured cells comparable to that of
colchicine (Table 2, entries 2 and 3). Nevertheless, compound cis
1
2
3
cis CA-4
Colchicine
cis 3b
1
12
16
a
A sample’s concentration which produces a 50% reduction in cell activity. For
details, see Supplementary data.

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T. T. B. Nguyen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7227–7231
3b was much less active than cis CA-4 itself (Table 2, entries 1 and
3).
4 °C. FACS analysis was performed on 20,000 cells using human
keratinocyte cell lines HaCaT and SKv-a and showed cell cycle
arrest in mitosis. This effect was most pronounced in HaCaT cells
after 24 h treatment with compound cis 3b (Fig. 3).
We then decided to use a 10ꢀ6 M drug concentration for the cell
microtubule organization studies.
Compounds cis 3b and, to a much lesser degree, trans 3b had a
major impact on the microtubule network in SKv-a and HaCaT
cells, as visualized in Figure 2.
To conclude with these preliminary results, we have described
herein new CA-4 derivatives, designed after replacement of the B
ring with various heterocycles attached to the vicinal heteroatom
position. Stereoisomers of these compounds 3a–c were evaluated
for their ability to inhibit tubulin and the benzothiophene series
proved to be the most active.
Evaluations of the anti-proliferative and anti-mitotic properties
on cutaneous cancer cells and of the effect on the cytoskeleton
indicate that compound cis 3b seems to be a good candidate for
further developments and structural modifications. Several ana-
logues are currently being synthesized and assessed for their bio-
logical properties: these studies will be reported in due course.
Indeed, pictures in Figure 2 show representative changes in-
duced by the drugs applied at 10ꢀ6 M. Colchicine and cis 3b treat-
ment resulted in radical changes in cell morphology including cell
rounding, detachment, and loss of the radial distribution of struc-
tured microtubules (c–f). Trans 3b isomer was less active, leaving
several keratinocytes virtually intact (d) whereas the remaining
cells presented characteristic microtubule depolymerisation (d0).
These results indicate that the benzothiophene analogue of CA-
4
cis 3b shows anti-microtubule activity similar to that of
colchicine.
As cell cycle control is the major regulatory mechanism of cell
Acknowledgments
growth, we analyzed the influence of the 3b compounds on the cell
lines by flow cytometry. Cells at the exponential phase of growth
were treated with 5 ꢂ 10ꢀ6 M compounds for 24 h. Then, they
were detached from the plates with gentle Accutase treatment
(PAA, France) and exposed in suspension to propidium iodide at
0.05 mg/mL in a citrate buffer containing Nonidet P40 for 1 h at
T.T.B.N thanks the Ministère de l’Enseignement Supérieur et de
la Recherche for a PhD fellowship. L.N. and J.G. thank the European
Community for ERASMUS fellowships. N.C.T. thanks the Région
Rhône-Alpes for a MIRA fellowship.
Confocal laser scanning microscopy studies were performed at
the Centre Technologique des Microscopies at University Lyon 1
and cell cycle data were obtained at the Laboratoire de Cytologie
Analytique, Fédération de Recherche Santé Lyon Est).
Supplementary data
Supplementary data associated (these data include 1H and 13C
NMR spectra of the most important compounds described in this
article and anti-proliferative effects of cis CA-4, colchicine and cis
3b on HaCaT cells using MTT assay) with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.
047.
References and notes
1. Dumontet, C.; Jordan, M. A. Nat. Rev. Drug Disc. 2010, 9, 790.
2. Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Med. Res. Rev. 1998, 18,
259.
3. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D.
Experientia 1989, 45, 209.
4. Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Schmidt, J. M.;
Hogan, F. J. Med. Chem. 1995, 38, 1666.
5. Lin, C. M.; Singh, S. B.; Chu, P. S.; Dempcy, R. O.; Schmidt, J. M.; Pettit, G. R.;
Hamel, E. Mol. Pharmacol. 1988, 34, 200.
6. (a) Lippert, J. W., III Bioorg. Med. Chem. 2007, 15, 605; (b) Patterson, D. M.;
Rustin, G. J. S. Clin. Oncol. 2007, 19, 443; (c) McKeage, M. J.; Baguley, B. C. Cancer
2010, 116, 1859.
7. (a) Pettit, G. R.; Temple, C.; Narayanan, V. L.; Varma, R.; Simpson, M. J.; Boyd, M.
R.; Rener, G. A.; Bansal, N. Anti-Cancer Drug Des. 1995, 10, 299; (b) Siemann, D.
W.; Chaplin, D. J.; Walicke, P. A. Expert Opin. Investig. Drugs 2009, 18, 189.
8. (a) Ohsumi, K.; Nakagawa, R.; Fukuda, Y.; Hatanaka, T.; Morinaga, Y.; Nihei, Y.;
Ohishi, K.; Suga, Y.; Akiyama, Y.; Tsuji, T. J. Med. Chem. 1998, 41, 3022; (b)
Delmonte, A.; Sessa, C. Expert Opin. Investig. Drugs 2009, 18, 1541.
9. (a) Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. J. Med.
Chem. 2006, 49, 3033; (b) Chaudhary, A.; Pandeya, S. N.; Kumar, P.; Sharma, P.;
Gupta, S.; Soni, N.; Verma, K. K.; Bhardwaj, G. Mini-Rev. Med. Chem. 2007, 7,
1186; (c) Shan, Y.; Zhang, J.; Liu, Z.; Wang, M.; Dong, Y. Curr. Med. Chem. 2011,
18, 523.
10. Marrelli, M.; Conforti, F.; Statti, G. A.; Cachet, X.; Michel, S.; Tillequin, F.;
Menichini, F. Curr. Med. Chem. 2011, 18, 3035.
11. Theeramunkong, S.; Caldarelli, A.; Massarotti, A.; Aprile, S.; Caprioglio, D.;
Zaninetti, R.; Teruggi, A.; Pirali, T.; Grosa, G.; Tron, G. C.; Genazzani, A. A. J. Med.
Chem. 2011, 54, 4977.
12. Shirai, R.; Takayama, H.; Nishikawa, A.; Koiso, Y.; Hashimoto, Y. Bioorg. Med.
Chem. Lett. 1998, 8, 1997.
13. Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda, Y.; Nihei, Y.; Suga,
Y.; Morinaga, Y.; Akiyama, Y.; Tsuji, T. Bioorg. Med. Chem. Lett. 1998, 8, 3153.
14. Lee, S.; Kim, J. N.; Lee, H. K.; Yoon, K. S.; Shin, K. D.; Kwon, B.-M.; Han, D. C.
Bioorg. Med. Chem. Lett. 2011, 21, 977.
Figure 3. Induction of G2/M phase arrest and polyploid cells in HaCaT cell line
treated with compound cis 3b. Human keratinocyte line was treated with the
vehicle solvent (0.5% DMSO) (a) or with cis 3b (5 ꢂ 10ꢀ6 M) (b) for 24 h. Cells were
harvested, stained with propidium iodide and 20,000 stained cells were subjected
to FACS analysis to determine the distribution of cells throughout the G1, S, and G2/
M phases. Experiments were performed in triplicate and yield repeatable results.

T. T. B. Nguyen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7227–7231
7231
15. (a) Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey, R. W.; Hannick, S. M.;
Gherke, L.; Credo, R. B.; Hui, Y.-H.; Marsh, K.; Warner, R.; Lee, J. Y.; Zielinski-
Mozng, N.; Frost, D.; Rosenberg, S. H.; Sham, H. L. J. Med. Chem. 2002, 45, 1697;
(b) Bellina, F.; Cauteruccio, S.; Monti, S.; Rossi, R. Bioorg. Med. Chem. Lett. 2006,
16, 5757.
27. Lawrence, N. J.; Ghani, F. A.; Hepworth, L. A.; Hadfield, J. A.; McGown, A. T.;
Pritchard, R. G. Synthesis 1999, 9, 1656.
28. Giraud, A.; Provot, O.; Hamze, A.; Brion, J.-D.; Alami, M. Tetrahedron Lett. 2008,
49, 1107.
29. 1H NMR spectrum of the crude product showed
a 62/38 Z/E ratio.
16. Odlo, K.; Fournier-Dit-Chabert, J.; Ducki, S.; Gani, O. A. B. S. M.; Sylte, I.; Hansen,
T. V. Bioorg. Med. Chem. 2010, 18, 6874.
17. Romagnoli, R.; Baraldi, P. G.; Salvador, M. K.; Preti, D.; Tabrizi, M. A.; Brancale,
A.; Fu, X.-H.; Li, J.; Zhang, S.-Z.; Hamel, E.; Bortolozzi, R.; Basso, G.; Viola, G. J.
Med. Chem. 2012, 55, 475.
Replacement of the triethyl silyl group by a ethoxy dimethyl silyl one did
not improve the ratio in favor of the Z isomer, as a 57/43 Z/E ratio was obtained
in this case.
30. Li, J.; Hua, R.; Liu, T. J. Org. Chem. 2010, 75, 2966.
31. The Z/E ratio was estimated to be 93/7, by 1H NMR on the crude product: traces
of the E isomer were removed by flash chromatography on silica gel (pre-
treated with Et3N). Despite a careful purification, the stereopure compound
(Z)-3a was isolated with a 58% yield, together with another fraction (34%)
which contained a mixture of Z/E compounds.
18. Aprile, S.; Del Grosso, E.; Tron, G. C.; Grosa, G. Drug Metab. Dispos. 2007, 35, 2252.
19. Beside the design of these cis-restricted derivatives, other research groups has
replaced the alkene linker by an exo methylene group (thus leading to isoCA-4
derivatives) or by unsaturated carbocycles. For these two approaches, see
respectively: (a) Messaoudi, S.; Tréguier, B.; Hamze, A.; Provot, O.; Peyrat, J.-F.;
Rodrigo De Losada, J.; Liu, J.-M.; Bignon, J.; Wdzieczak-Bakala, J.; Thoret, S.;
Dubois, J.; Brion, J.-D.; Alami, M. J. Med. Chem. 2009, 52, 4538; (b)
Rasolofonjatovo, E.; Provot, O.; Hamze, A.; Rodrigo, J.; Bignon, J.; Wdzieczak-
Bakala, J.; Desravines, D.; Dubois, J.; Brion, J.-D.; Alami, M. Eur. J. Med. Chem.
2012, 52, 22.
20. Simoni, D.; Romagnoli, R.; Baruchello, R.; Rondanin, R.; Rizzi, M.; Pavani, M. G.;
Alloatti, D.; Giannini, G.; Marcellini, M.; Riccioni, T.; Castorina, M.; Guglielmi,
M. B.; Bucci, F.; Carminati, P.; Pisano, C. J. Med. Chem. 2006, 49, 3143.
21. 3,4,5-Trimethoxybenzyltriphenylphosphonium bromide was prepared in two
steps from 3,4,5-trimethoxybenzaldehyde, according to this reference:
Asakawa, Y.; Tanikawa, K.; Aratani, T. Phytochemistry 1976, 15, 1057.
22. (Z) and (E) benzofurane derivatives 3a were isolated with 26% and 35% yields,
respectively, whereas (Z) and (E) benzothiophenes 3b were isolated with 8%
and 46% yields, respectively.
23. L’Helgoual’ch, J.-M.; Seggio, A.; Chevallier, F.; Yonehara, M.; Jeanneau, E.;
Uchiyama, M.; Mongin, F. J. Org. Chem. 2008, 73, 177.
24. (a) Chinchilla, R.; Nájera, C. Chem. Soc. Rev. 2011, 40, 5084; (b) Chinchilla, R.;
Nájera, C. Chem. Rev. 2007, 107, 874; (c) Doucet, H.; Hierso, J.-C. Angew. Chem.,
Int. Ed. 2007, 46, 834.
25. Kaafarani, B. R.; Wex, B.; Strehmel, B.; Neckers, D. C. Photochem. Photobiol. Sci.
2002, 1, 942.
26. Fürstner, A.; Nikolakis, K. Liebigs Ann. 1996, 2107.
32. Mahboobi, S.; Uecker, A.; Sellmer, A.; Cénac, C.; Höcher, H.; Pongratz, H.;
Eichhorn, E.; Hufsky, H.; Trümpler, A.; Sicker, M.; Heidel, F.; Fischer, T.;
Stocking, C.; Elz, S.; Böhmer, F.-D.; Dove, S. J. Med. Chem. 2006, 49, 3101.
33. Ketcha, D. M.; Lieurance, B. A.; Homan, D. F. J.; Gribble, G. W. J. Org. Chem. 1989,
54, 4350.
34. Yasuhara, A.; Sakamoto, T. Tetrahedron Lett. 1998, 39, 595.
35. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
36. Gaukroger, K.; Hadfield, J. A.; Hepworth, L. A.; Lawrence, N. J.; McGown, A. T. J.
Org. Chem. 2001, 66, 8135.
37. Z-Monobromotrimethoxystyrene (91/9 Z/E mixture) was prepared in two steps
from 3,4,5-trimethoxybenzaldehyde, according to this reference: Herz, H.-G.;
Queiroz, M. J. R. P.; Maas, G. Synthesis 1999, 6, 1013.
38. Kassis, P.; Bénéteau, V.; Mérour, J.-Y.; Routier, S. Synthesis 2009, 14, 2447.
39. Chakrabarty, M.; Kundu, T.; Harigaya, Y. Synth. Commun. 2006, 36, 2069.
40. Lewin, G.; Aubert, G.; Thoret, S.; Dubois, J.; Cresteil, T. Bioorg. Med. Chem. 2012,
20, 1231.
41. Previous work reported that cis CA-4 was 30 times more potent than its trans
isomer for the inhibition of tubulin polymerization: Pettit, G. R.; Rhodes, M. R.;
Herald, D. L.; Hamel, E.; Schmidt, J. M.; Pettit, R. K. J. Med. Chem. 2005, 48, 4087.
42. Boukamp, P.; Petrussevska, R. T.; Breitkreutz, D.; Hornung, J.; Markham, A.;
Fusenig, N. E. J. Cell Biol. 1988, 106, 761.
43. Schneider-Manoury, S.; Pehau-Arnaudet, G.; Breitburd, F.; Orth, G. J. Gen. Virol.
1990, 71, 809.