Caulerpenyne-colchicine hybrid: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2006.04.024

The synthesis of an analog of caulerpenyne having a trimethoxyaryl moiety was achieved in 11% overall yield over 11 steps. Its biological activity has been evaluated as inhibitor of in vitro tubulin polymerization or angiogenesis.

Full text of DOI:10.1016/j.bmc.2006.04.024

Bioorganic & Medicinal Chemistry 14 (2006) 5540–5548
Caulerpenyne–colchicine hybrid: Synthesis and biological
evaluation
a,b
a,
b
b
*
Julien Bourdron, b Laurent Commeiras, Pascale Barbier, V e´ ronique Bourgarel-Rey,
c
d
b,
*
Eddy Pasquier, Nicolas Vanthuyne, Jean-Claude Hubaud, Vincent Peyrot and
a,
*
Jean-Luc Parrain
a
UMR-CNRS 6178 SymBio, e´ quipe Synth e` se par voie Organom e´ tallique, Universit e´ Paul C e´ zanne (Aix-Marseille III),
Avenue Escadrille Normandie Niemen, bo ˆı te D12, 13397 Marseille cedex 20, France
b
FRE-CNRS 2737, CISMET, Facult e´ de Pharmacie, 27 bd Jean Moulin, 13385 Marseille Cedex 05, France
UMR 6180 ‘‘Chirotechnologies: Catalyse et Biocatalyse’’, Laboratoire de St e´ r e´ ochimie Dynamique et Chiralit e´ ,
c
Universit e´ Paul C e´ zanne (Aix-Marseille III), France
DIPTA (SA), zac pichaury 2, 415 rue Pierre Berthier, 13290 Aix-en-Provence, France
d
Received 17 February 2006; revised 13 April 2006; accepted 20 April 2006
Available online 22 May 2006
Abstract—The synthesis of an analog of caulerpenyne having a trimethoxyaryl moiety was achieved in 11% overall yield over 11
steps. Its biological activity has been evaluated as inhibitor of in vitro tubulin polymerization or angiogenesis.
ꢀ
2006 Elsevier Ltd. All rights reserved.
AcO
AcO
R
1
. Introduction
Marine algae of the order Caulerpales are known for
their chemical defense against predators by producing
secondary metabolites. The majority of these com-
pounds are sesquiterpenoids and diterpenoids, often
acyclic. The terminal 1,4-diacetoxybutadiene moiety is
a common functional group to most of these metabolites
and uniquely found in this group of marine algae. Now-
adays, more than 30 toxins with this moiety have been
isolated from the Udoteaceae and Caulerpaceae families
such as caulerpenyne, flexiline, dihydrorhipocephaline,
OAc
caulerpenyne
OAc
OAc
R = H; flexiline
R = OAc; dihydrorhipocephaline
MeO
OAc
OAc
NHCOCH3
MeO
MeO
AcO
O
OCH3
colchicine
crispatenine
O
MeO
MeO
MeO
1
and crispatenine (Fig. 1). The 1,4-Diacetoxybutadiene
moiety represents an acetylated bis-enol form of the
,4-dialdehyde constellation, to which a high degree of
biological activity is generally attributed what we have
O
MeO
O
MeO
OMe
MeO
1
O
OH
O
hybrid A
combretastatin
2
O
recently confirmed.
OO
ON
MM ee OO
OAc
MeO
MeO
OAc
MM ee OO
MM ee OO
O
H
O
AcO
Keywords: Caulerpenyne; Colchicine; Biological evaluation; Tubulin
polymerization; Cell cytotoxicity; In vitro angiogenesis.
OO
OCH3
OMe
O
*
Corresponding authors. Tel.: +33 4 91 28 91 26; fax: +33 4 91 28 89
4 (L.C.); tel.: +33 4 91 28 89 14; fax: +33 4 91 28 89 14 (J.-L.P.); tel.:
33 4 91 83 55 05; fax: +33 4 91 83 55 05 (V.P.); e-mail addresses:
hybrid B
Super-imposition of
colchicine
1
+
and hybrid A
laurent.commeiras@univ-cezanne.fr;
univ-mrs.fr; jl.parrain@univ-cezanne.fr
vincent.peyrot@pharmacie.
Figure 1.
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.024

J. Bourdron et al. / Bioorg. Med. Chem. 14 (2006) 5540–5548
5541
1,0
0,8
0,6
0,4
0,2
0,0
During the search of new colchicine analogs derivatives
or not from the natural product, a series of compounds,
which interact with the colchicine-binding site of tubu-
lin, were discovered. For example, Combretastatin A-4
phosphate, the most promising antitumor agent, recent-
ly passed phase II clinical trials.
2
0
15
1
0
5
0
0
20
40 60
80 100 120 140
In a similar way, to find a new potential molecule which
can inhibit the microtubule formation process, we
thought about a compound possessing the more biolog-
ically active fragment of caulerpenyne (diacetoxybutadi-
ene moiety) and the cycle A (trimethoxybenzene moiety
recognized by tubuline) of colchicine instead of the ter-
minal unsaturated alkyl chain of CYN.
[iso-1 (+)] µM
0
10
20
30
40
50
60
time (min)
+)-iso-18
Herein, we report our investigation on the synthesis and
the biological evaluation of two colchicine–caul-
erpenyne hybrids A and B (Fig. 1). Our interest for the
synthesis of hybrid A was justified by a perfect superim-
position with colchicine in which all heteroatoms or ba-
sic functions overlap. Moreover, there is an obvious
flexibility of the alkyl chain compared to the rigidity
of the seven-membered ring of the colchicine. A good
reason for the synthesis of hybrid B is that it includes
a bis-allylic acetate function which could be easily re-
placed by a thio- or amino-moiety during binding with
the amino-acids of the tubulin. Moreover, numerous
analogs furnishing a library of colchicine analogs could
be easily prepared by allylic substitution.
(
Figure 2. Effect of (+)-iso-18 on tubulin assembly in microtubules.
Indeed, some metabolites containing this moiety have
been implicated in chemical defense against grazing fish-
es and invertebrates in herbivore-rich tropical waters
and this has, for example, been proposed to explain
the proliferation from Italy to Spain of Caulerpa taxifo-
lia, a tropical green seaweed accidentally introduced in
the Mediterranean sea. From C. taxifolia were isolated
3
nine mono- and sesquiterpenes such as caulerpenyne
(
CYN) which represents the main toxin of this algae
4
and well known for its important biological activity.
We have also demonstrated that CYN has an antiprolif-
erative activity on tumor cell line SK-N-SH, modifies
their microtubule network, and inhibits the brain puri-
fied tubulin and microtubule proteins polymerization
2. Results and discussion
2.1. Synthesis
5
with IC50 in a range of 20–50 lM.
For a ready access, synthesis of hybrid B was investigat-
ed first. Our strategy for synthesizing hybrid B from the
commercially available cinnamic acid and but-2-yn-1,4-
diol is outlined in Scheme 1 and was derived to the one
we applied in our recent synthesis of caulerpenyne and
dihydrorhipocephaline. Hybrid B was obtained by a
coupling reaction between the vinylstannanes (east seg-
ment) (tin–lithium exchange) derived from butynediol
and trimethoxycinnamaldehyde (west segment).
Tubulin, the major structural component of microtu-
bules, participates actively in mitotic spindle formation
and chromosomal organization during cell division.
Thus, this protein is a target of anti-mitotic drugs called
anti-tubulin agents. Since only a few molecules like tax-
oids or Vinca-alkaloids are used for cancer chemothera-
py, a lot of energy is being spent in finding new anti-
mitotic drugs. One of the approaches consists in the syn-
thesis and analysis of numerous analogs of colchicine
2
,7
(
inhibits the polymerization of microtubule with an IC50
of 1.7 ± 0.09 lM. However, the toxicity of colchicine
has hampered its use in cancer chemotherapy so far.
Fig. 1), a well-known natural cytotoxic product which
Synthesis of the vinyl segment 3 began with palladium
8
complex catalyzed hydrostannation of but-2-yn-1,4-di-
ol 1 to give (E)-vinyltin reagent 2 in which the more
accessible alcohol function was selectively protected as
6
O
OP
OAc
MeO
MeO
MeO
MeO
OAc
+
Bu Sn
3
AcO
HO
OMe
Hybrid B
OMe
west fragment 6
east fragment 3
O
OH
MeO
MeO
OH
HO
OMe
Scheme 1.

5542
J. Bourdron et al. / Bioorg. Med. Chem. 14 (2006) 5540–5548
tert-butyldimethylsilyl ether in 64% yield over two
9
steps.
with MnO gave the desired west fragment 6 in 93%
2
yield. (75% yield over three steps).
11
Synthesis of west fragment 6 was obtained over three
steps and is resumed in Scheme 2. Trimethoxycinnamic
acid was converted into the corresponding ester 4 in 81%
yield. Reduction of 4 using 2 equiv of Dibal-H in
DCM at ꢀ78 ꢁC furnished the alcohol 5 in quantitative
yield. Finally, oxidation of the allylic alcohol function
The assembly of the fragments 3 and trimethoxycinnam-
aldehyde 6 and the construction of carbon skeleton of
the hybrid B are described in Scheme 2. The coupling
reaction between west segment 6 and the carbanion gen-
erated by tin–lithium exchange reaction on the east
segment 3 gave diol 7 in fair yield (54%). At this stage,
1
0
9
a
Synthesis of 3
OH
OH
a
b
OAc
OH
HO
65 %
TBDMSO
SnBu3
MeO
MeO
OH
9
9 %
OH
SnBu3
AcO
1
2
east fragment 3
OMe
Synthesis of 6 and hybrid B
OR
OR
MeO
MeO
MeO
MeO
f
MeO
MeO
OTBS
MeO
MeO
d
e
OH
O
O
1
00 %
54 %
9
3 %
RO
OMe
OMe
OMe
OMe
5
6
R =H
R = H (7)
c
g
R = CH ( 81%) (4)
R = OAc ( 96%) 8
3
OMe
OH
OH
MeO
MeO
OH
MeO
MeO
OAc
j
MeO
MeO
OH
h
i
8
8
8
5
0 %
32 %
28 %
AcO
AcO
AcO
OMe
OMe
10
OMe
9
1
1
Synthesis of new west fragment 13 and 18 (hybrid A)
OR
MeO
MeO
MeO
MeO
MeO
MeO
l
m
MeO
MeO
OTBS
OH
k
OH
O
8
6 %
93 %
60 %
RO
OMe
OMe
OMe
OMe
1
2
13
5
R = H (14)
n
R = OAc ( 89%, 15)
OAc
OAc
MeO
OAc
OAc
MeO
AcO
1
8
OMe
OMe
MeO
MeO
OH
p
MeO
MeO
O
q
76 %
o
5 %
+
6
100 %
AcO
AcO
OAc
OMe
OMe
17
MeO
MeO
OAc
1
6
AcO
iso-18
a) Bu3SnH, PdCl2(PPh3)2, THF; b) TBDMSCl, imidazole, DMF, 0˚C; c) MeOH, H2SO4, Δ; d) Dibal-H, CH2Cl2, - 78 ˚C; e) MnO2, EtOAc; f) (i) MeLi.LiBr, - 35 ˚C; (ii) 3, - 35 ˚C; g)
Ac2O, DMAP, Pyridine; h) MeOH, NH4F; i) TBAF, THF; j) AcOH, THF, H2O k) nickel acetate tetrahydrated, NaBH4, EtOH; l) Dess-Martin Periodinane, CH2Cl2; m) (i) MeLi.LiBr, - 35
˚
C; (ii) 3, - 35 ˚C; n) Ac2O, DMAP, Pyridine; o) HF.Pyridine, THF; p) Dess-Martin Periodinane, CH2Cl2; q) Ac2O, DMAP, NEt3, 80 ˚C
Scheme 2.

J. Bourdron et al. / Bioorg. Med. Chem. 14 (2006) 5540–5548
5543
the two hydroxyl groups of 7 were protected as acetates
using acetic anhydride and a catalytic amount of DMAP
in pyridine to give crude clean bis-acetate 8 (96%). The
cells). Results are consigned in Table 1 and Figure 3.
Figure 3 shows the percentage of living cells, deter-
mined by the MTT assay, after treatment with isomers
of 18 and iso-18 (1–10 lM) during 72 h. No significant
effects were observed for concentrations lower than
1 lM. After 72 h of treatment, the IC50 were
2.8 ± 0.2, 2.0 ± 0.5, 4.0 ± 0.1, and 1.4 ± 0.1 lM for
(+)-18, (ꢀ)-18, (+)-iso-18, and (ꢀ)-iso-18, respectively.
We conclude that these CYN–colchicine hybrids are
less active than colchicine but induced a concentra-
tion-dependent inhibition of HaCaTs cells which is
of the same order as the IC observed for CYN on
1
2
next step is the desilylation of 8 by the complex HF-
pyridine to provide the corresponding alcohol. Unfortu-
nately the desired alcohol was never observed, only
unidentified degradation products were obtained. Sever-
al conditions were tested but no one gave expected re-
sults. Using ammonium fluoride in THF only starting
material was recovered at the end of reaction. The same
reaction conducted in tert-butanol furnished several
unidentified products. Ammonium fluoride in MeOH
underwent to the deprotection of the silyl ether but the
secondary acetate was replaced by methoxy group fur-
nishing 9 in 50% yield. Migration of the acetate function
from the secondary to the primary alcohol was observed
using anhydrous TBAF in THF giving 10 in 32% yield.
Same results were already described for the synthesis of
5
0
1
5
the same cells.
2.2.2. Tubulin polymerization. Because colchicine and
CYN inhibit the microtubule formation in vitro, it was
interesting to determine the effect of the four isomers
on this biological activity. The in vitro purified tubulin
polymerization process was investigated by turbidimetry
(Fig. 2). Increase in absorption at 350 nm results in an
increase of microtubule mass. Tubulin was incubated
for 35 min at 37 ꢁC without (control) or with various
concentrations of (+)-18, (ꢀ)-18, (+)-iso-18, and (ꢀ)-
7
a
caulerpenyne. The last trial was to use a mixture of
acetic acid in aqueous THF. One more time these condi-
tions gave poor results and diol 11 was obtained in only
2
8% yield. All these results show that the bis-allylic ace-
tate between the double bond is very sensitive to the
reaction conditions used and can undergo to the corre-
sponding allylic carbocation which can react with vari-
ous nucleophiles.
2
+
iso-18 in a Mg -free polymerization buffer. After
35 min, the polymerization process was induced by add-
2
+
ing 10 mM Mg . In the presence of drugs, we observed
only slight decrease in turbidity and concentration up to
100 lM was necessary to reduce the polymerization by
Due to the very low yields obtained, we turned our
attention to the hybrid A in which the central double
bond was replaced by a single carbon–carbon bond.
From the allylic alcohol 5, the double bond was re-
duced using nickel acetate, sodium borohydride, and
hydrogen to give the desired compound 12 in 86%
yield. Oxidation of the primary alcohol using Dess–
0
20%. The IC50 of all isomers of 18 and 18 are higher
than 100 lM, whereas the IC50 of natural CYN is
around 20 lM and colchicine 2 lM. On the contrary
to what we excepted, this molecule, which possesses
both colchicine and CYN moiety known to be involved
in the biological activity on tubulin, was not a powerful
microtubule formation inhibitor in vitro.
1
3
Martin periodinane furnished the new west fragment
3 in 93% yield (65% yield over four steps from tri-
methoxycinnamic acid).
1
2.2.3. In vitro angiogenesis. Angiogenesis (the formation
of new blood vessels from pre-existing ones) is a key
process in both tumor growth and metastasis. This can
be inhibited by conventional anti cancer drugs such as
anti-tubulin agent. Thus, the effect of one CYN hybrid
was investigated on the in vitro angiogenesis by measur-
ing the inhibition of capillary network formation on
As above the assembly between west segment 13 and
the carbanion generated by tin–lithium exchange reac-
tion on the east segment 3 gave diol 14 in 60% yield.
Protection of diol 14 into diacetates using standard
conditions afforded 15 in 89% yield. This time, depro-
tection of the silyl ether function with HF-pyridine
furnished the desired alcohol 16 in 65% yield. The
alcohol function was then oxidized with Dess–Martin
periodinane to give the sensitive aldehyde 17 in quan-
titative yield. To achieve the synthesis of the colchi-
cine–caulerpenyne hybrid, the last step was the
construction of the diacetoxybutadiene moiety. We
TM
Matrigel with HMEC-1 cells (Human Microvascular
Endothelial Cell line). At low concentration (up to
1 lM), there was no significant inhibition of angiogene-
sis (Fig. 4). High concentrations (10 lM) were necessary
to observe more than 50% inhibition. In comparison,
paclitaxel inhibited in vitro angiogenesis with a concen-
tration near by 1 nM,
16
concentration.
a
10,000-fold lower
7
b
used the conditions developed by our group (NEt3,
DMAP, Ac O at 80 ꢁC) and our target was obtained
2
in 76% yield as a 1:1 mixture of two racemic diaste-
0
reomers 18 and 18 . Separation of each enantiomer
was realized by semi-preparative chiral HPLC to give
Table 1. Cytotoxicity on HaCaTs cells and inhibition of polymeriza-
tion of tubulin of (+)-18, (ꢀ)-18, (+)-iso-18, and (ꢀ)-iso-18
1
4
(
+)-18, (ꢀ)-18, (+)-iso-18, and (ꢀ)-iso-18.
Products
Cytotoxicity
Tubulin polymerization
2
2
.2. Biology
IC50 (lM)
IC50 (lM)
(
+)-18
(ꢀ)-18
+)-iso-18
(ꢀ)-iso-18
2.8 ± 0.2
2.0 ± 0.5
4.0 ± 0.1
1.4 ± 0.1
>100
>100
>100
>100
.2.1. Cell cytotoxicity. Since CYN and colchicine are
4
cytotoxic in several tumor cell lines, we determined
the cytotoxicity of the different isomers of 18 and
1
(
0
8 on HaCaTs cells (keratynocytes, immortalized skin

5544
J. Bourdron et al. / Bioorg. Med. Chem. 14 (2006) 5540–5548
Figure 3. Cytotoxicity on HaCaTs cells of (+)-18, (ꢀ)-18, (+)-iso-18, and (ꢀ)-iso-18.
Control
1 µM
10 µM
Figure 4. Percentage of total capillary tube length in function of (+)-18 concentrations.
3
. Conclusion
Hexane and ethanol, HPLC grade, were degassed
and filtered on a 0.45 lm membrane before use.
The column used is Chiralcel OD-H (250 · 4.6 mm)
from ChiralTechnologies Europe (Illkirch, France).
Retention times tR in minutes, retention factors
We synthesized a new hybrid compound of caul-
erpenyne and colchicine in 11 steps with 11% yield. Since
the different isomers of this hybrid were not powerful
inhibitor of in vitro tubulin polymerization process
and angiogenesis, they showed an acceptable cytotoxic-
ity on HaCaTs cells (around 2.5 lM).
k
i
¼ ðtRi ꢀ tR0 Þ=tR0
,
and enantioselectivity factor
a = k /k are given. t was determined by injection
2
1
R0
of tri-tertio-butyl benzene. The sign given by the
on-line circular dichroism is the sign of the product
in the solvent used for the chromatographic separa-
tion. The two analyses were performed at 25 ꢁC,
with hexane/ethanol (95/5) as mobile phase and
1 mL/min as flow-rate, with UV and CD at
4
. Experimental
4
.1. Generality
2
54 nm.
18
t ðꢀÞ ¼ 14:58,
t ðþÞ ¼ 17:87,
R1
R2
All reactions sensitive to oxygen and moisture were car-
ried out in oven-dried glassware under a slight positive
pressure of argon unless otherwise noted. H NMR
k (ꢀ) = 3.78, k (+) = 4.86, a = 1.29. iso-18: t
R
1
ðꢀÞ ¼
1
2
12:84,
t
R
ðþÞ ¼ 16:52, k (ꢀ) = 3.21, k (+) = 4.42,
2
1
2
1
a = 1.38. Semi-preparative separations were per-
formed on an unit composed of Merck D-7000 sys-
1
3
and C NMR spectra were determined on a Bruker,
AC300.
tem
manager,
Merck-Hitachi
L-6000
pump,
Rheodyne valve with a 500 lL loop, and a Merck-
Hitachi L-4000 UV-detector. For 18 and iso-18, Chi-
ralcel OD-H (250 · 10 mm) was used with hexane/
ethanol (95:5) as mobile phase, 1 mL/min as flow-
rate, and UV at 254 nm. HMEC-1 were obtained
from the Cell Culture Laboratory in the H oˆ pital
de la Conception (Assistance publique des H oˆ pitaux
de Marseille, France). They were routinely main-
1
Chemical shifts for H NMR were reported in parts per
million (ppm) downfield from tetramethylsilane as the
internal standard and coupling constants are in hertz
1
3
(
Hz). Chemical shifts for C NMR were reported in
ppm relative to the central line of a triplet at
7.16 ppm for deuteriochloroform. Reagents and sol-
7
vents were of commercial grades and were used as sup-
plied. Dichloromethane, benzene, and toluene were
distilled from calcium hydride and stored over molecu-
tained in culture at 37 ꢁC and 5% CO . HMEC-1
2
were grown in MDCB-131 medium (Life Technolo-
gies) containing 10% heat-inactivated fetal bovine
serum, 2 mmol/L glutamine, 1% penicillin and strep-
tomycin, 1 lg/ml hydrocortisone (Pharmacia & Up-
john, St-Quentin-Yvelines, France), and 10 ng/ml
epithelial growth factor (R&D Systems, Minneapolis,
MN). HaCaTs were obtained from ATCC. They
were routinely maintained in culture at 37 ꢁC and
˚
lar sieves 4 A. THF and diethyl ether were distilled from
sodium benzophenone prior to use. N,N-Dimethylform-
amide was purchased anhydrous and stored over molec-
˚
ular sieves 4 A under argon. Mass spectra (MS) were
performed with a Sciex API III Plus triple quadrupole
system with a pneumatically assisted electrospray inter-
face (Sciex, Thornhill, Canada). The analytical chiral
HPLC experiments were performed on a unit composed
of a Merck D-7000 system manager, Merck-Lachrom L-
5% CO . HaCaTs were grown in DMEM (Dul-
2
becco’s modified Eagle’s medium) containing 10% fe-
tal bovine serum, 1% penicillin and streptomycin,
and 2% glutamine. The drugs were prepared in
DMSO and maintained frozen at ꢀ20 ꢁC.
7
100 pump, Merck-Lachrom L-7360 oven, Merck-La-
chrom L-7400 UV-detector, and on-line Jasco CD-
595 circular dichroism.
1

J. Bourdron et al. / Bioorg. Med. Chem. 14 (2006) 5540–5548
5545
8
1
4
.2. (E)-2-Tributylstannylbut-2-ene-1,4-diol (2)
in quantitative yield. H NMR (CDCl , 300 MHz) d
3
3.82 (s, 3H), 3.84 (s, 6H), 4.29 (dd, 2H, J = 5.7,
To a THF solution (15 mL) of but-2-yn-1,4-diol (2.58 g,
0 mmol) and PdCl (PPh ) (420 mg, 2 mol%) was add-
1.3 Hz), 6.25 (dt, 1H, J = 5.7, 15.9 Hz), 6.50 (d, 1H,
J = 15.9 Hz), 6.58 (s, 2H).
3
2
3 2
ed dropwise a THF solution (20 mL) of tributyltin hy-
dride (9.68 mL, 36 mmol) over a period of 1 h. The
originally light yellow solution abruptly turned orange-
brown. After stirring over 15 min, THF was evaporated
under vacuum. The crude product was then purified by
1
8
4.6. (E)-3-(3,4,5-Trimethoxy-phenyl)-prop-2-enal (6)
To a solution of MnO (7.17 g, 82.5 mmol) in ethyl ace-
2
tate (120 ml) was added a solution of 5 (1.85 g,
8.25 mmol) in ethyl acetate (40 ml). The solution was
stirred overnight, filtered over Celiteꢂ, and concentrat-
flash chromatography (light petroleum/Et O, 6:4–2:8) to
2
1
give 2 in 99% yield. H NMR (300 MHz, CDCl ) d 0.84–
3
0
3
.93 (m, 15H; 3 · CH , 3 · CH ), 1.23–1.35 (m, 6H;
ed. The crude product 6 was obtained in 93% yield.
3
2
1
· CH ), 1.43–1.53 (m, 6H; 3 · CH ), 1.64 (br t,
H NMR (CDCl , 300 MHz) d 3.90 (s, 9H), 6.64 (dd,
1H, J = 15.9, 7.8 Hz), 6.79 (s, 2H), 7.4 (d, 1H,
J = 15.9 Hz), 9.69 (d, 1H, J = 7.8 Hz).
2
2
3
J = 5.6 Hz, 1H; OH), 1.73 (br t, J = 5.2 Hz, 1H; OH),
.18 (br t,3 J = 5.4 Hz, 2H; CH2), 4.36 (br d,
J = 3.7 Hz, JSn,H = 37 Hz, 2H; CH ), 5.77 (br t,
4
2
3
J = 5.4 Hz, J
= 67 Hz, 1H; CH).
4.7. 2-[2-(1,1,2,2-Tetramethyl-propoxy)-ethylidene]-5-
(3,4,5- trimethoxy-phenyl)-pent-4-ene-1,3-diol (7)
Sn,H
4
but-2-en-1-ol (3)
.3. (E)-4-tert-Butyldimethylsilyloxy-2-tributylstannyl
9
a
To a solution of 3 (2 g, 4.07 mmol) in THF (50 ml) was
added, at ꢀ35 ꢁC and under argon, dropwise MeLiÆLiBr
To a solution of 2 (3.8 g, 10 mmol) in DMF (100 mL) at
(1.5 M in Et O, 5.7 ml, 8.547 mmol). The solution was
2
0
ꢁC were added imidazole (0.68 g, 10 mmol) and tert-
butyldimethylsilylchloride (1.5 g, 10 mmol). The solution
kept at ꢀ35 ꢁC for 2 h and then 6 (995 mg, 4.477 mmol)
was added. The solution was kept at ꢀ35 ꢁC for 24 h
was stirred at 0 ꢁC for 6 h and then crushed ice (2.5 g) was
added. The solution was diluted with Et O (200 mL),
washed with saturated aqueous NH Cl solution, dried
over MgSO , and evaporated. The crude product was
4
then purified by flash chromatography (light petroleum/
Et O, 10:0–9:1) to give 3 in 65% yield. H NMR
and then quenched with a NH Cl solid (650 mg). The
4
mixture was filtered, concentrated, and then purified
by flash chromatography (light petroleum/Et O,
2
4
2
1
7:3 ! 3:7 ! 0/1) to give 7 in 54% yield. H NMR
(CDCl , 300 MHz) d 0.10 (s, 6H), 0.91 (s, 9H), 2.37
3
1
(br s, OH), 2.52 (br s, OH), 3.84 (s, 3H), 3.87 (s, 6H),
4.27 (m, 2H), 4.33 (d, 2H, J = 5.9 Hz), 4.90 (m, 1H),
5.83 (t, 1H, J = 5.9 Hz), 6.18 (dd, 1H, J = 15.9,
2
(
(
300 MHz, CDCl ) d 0.06 (s, 6H; 2 · CH ), 0.84–0.92
3
3
m, 15H; 3 · CH , 3 · CH ), 0.88 (s, 9H, 3 · CH ),
3
2
3
1
3
1
OH), 4.20 (br d, J = 5.4 Hz, J
.23–1.53 (m, 12H; 6 · CH ), 1.80 (br t, J = 5.5 Hz, 1H;
6.04 Hz), 6.59 (d, 1H, J = 15.9 Hz), 6.61 (s, 2H);
C
2
4
= 15 Hz, 2H; CH ),
2
= 37 Hz, 2H; CH ), 5.68
2
NMR (CDCl , 75 MHz) d ꢀ5.2 (2 · CH ), 18.3 (C),
Sn,H
3
3
3
.32 (br d, J = 5.5 Hz, J
4
(
25.9 (3 · CH ), 56.1 (2 · CH ), 58.5 (CH ), 59.6 (CH ),
Sn,H
3
3
2
2
3
br t, J = 5.4 Hz, J
= 69 Hz, 1H; CH).
60.9 (CH ), 76.3 (CH), 103.6 (2 · CH), 129.4 (CH),
Sn,H
3
1
29.6 (CH), 130.7 (CH), 132.4 (C), 137.8 (C), 140.9
+
4
.4. 3,4,5-Trimethoxymethylcinnamat (4)
(C), 153.3 (2 · C); MS: m/z (ESI+) 442 [M+NH ] .
4
Compound 4 was prepared according to a modified pro-
1
cedure of Peterson et al. A solution of cinnamic acid
4.8. Acetic acid 2-acetoxymethyl-4-(1,1,2,2-tetramethyl-
propoxy)-1-[2-(3,4,5-trimethoxy- phenyl)-vinyl]-but-2-
enyl ester (8)
0
(
(
5 g, 21 mmol) and H SO4 (five drops) in MeOH
2
42 ml) was warmed to reflux overnight. The solution
was then cooled to room temperature and the product
precipitated. The solid was filtered off, dried under
A solution of 7 (420 mg, 0.990 mmol), acetic anhydride
(0.374 ml, 3.959 mmol), and DMAP (6 mg) in pyridine
(5 ml) under argon was stirred overnight. The mixture
4
1
vacuum to give 4 in 81% yield. H NMR (CDCl3,
00 MHz) d 3.79 (s, 3H), 3.87 (br s, 9H), 6.34 (d, 1H,
J = 16.1 Hz), 6.74 (s, 1H), 7.59 (d, 1H, J = 16.1 Hz).
3
was quenched with a saturated aqueous NaHCO solu-
3
tion and the aqueous layer was extracted with ethyl
ether. The combined organic layers were washed with
saturated aqueous CuSO solution and water, dried over
1
7
4
.5. (E)-3-(3,4,5-Trimethoxy-phenyl)-prop-2-en-1-ol (5)
4
MgSO , and concentrated under vacuum to give 8 in
4
1
To a solution of 4 (3 g, 11.89 mmol) in CH Cl (80 ml)
2
96% yield. H NMR (CDCl , 300 MHz) d 0.08 (s, 6H),
2
3
at ꢀ78 ꢁC, under argon, was added dropwise Dibal-H
17.45 ml, 26.16 mmol, 1.5 M in toluene). The mixture
was stirred for 1 h and half at ꢀ78 ꢁC and MeOH
15 ml) was added. The solution was warmed to room
temperature. A saturated aqueous potassium sodium
tartrate solution was added and the mixture was stirred
for 2 h. The aqueous layer was extracted with ethyl ace-
tate and the organic layers were washed with a saturated
0.90 (s, 9H), 2.03 (s, 3H), 2.10 (s, 3H), 3.84 (s, 3H),
3.87 (s, 6H), 4.37 (d, 2H, J = 5.8 Hz), 4.63 (d, 1H,
J = 12.5 Hz), 4.70 (d, 1H, J = 12.5 Hz), 5.90 (d, 1H,
J = 7.2 Hz), 5.97 (t, 1H, J = 5.8 Hz), 6.05 (dd, 1H,
J = 15.7, 7.2 Hz), 6.57 (d, 1H, J = 15.7 Hz), 6.60 (s,
(
(
1
3
2H); C NMR (CDCl , 75 MHz) d ꢀ5.1 (2 · CH ),
3
3
18.4 (C), 21.0 (3 · CH ), 21.3 (CH ), 26.0 (CH ), 56.2
3
3
3
(2 · CH ), 59.6 (CH ), 59.8 (CH ), 61.0 (CH ), 75.5
3
2
2
3
aqueous NaCl solution, dried over MgSO , and concen-
4
(CH), 103.9 (2 · CH), 125.1 (CH), 131.9 (C), 132.4
trated. The crude product was then purified by flash
chromatography (light petroleum/Et O, 2:8) to give 5
(C), 133.5 (CH), 134.5 (CH), 138.4 (C), 153.4 (2 · C),
+
169.8 (C), 170.8 (C); MS: m/z (ESI+) 526 [M+NH ] .
2
4

5546
J. Bourdron et al. / Bioorg. Med. Chem. 14 (2006) 5540–5548
1
9
4
.9. 3-(3,4,5-Trimethoxy-phenyl)-propan-1-ol (12)
(1 ml), under argon, was stirred overnight and quenched
with a saturated aqueous NaHCO solution. The aque-
3
In an Erlenmeyer, were placed nickel acetate tetrahydrat-
ed (4.06 g, 16.3 mmol) and 95% ethanol (110 ml). The
solution was purged with H , then a solution of NaBH
ous layer was extracted with ethyl ether. The combined
organic layers were washed with saturated aqueous
CuSO solution and water, dried over MgSO , and con-
2
4
4
4
(
16 mmol, 1 M in absolute ethanol, 16 ml) was added.
centrated under vacuum to give crude 15 in 89% yield.
1
The black mixture was stirred for 30 min and then a solu-
tion of 5 (3.66 g, 16.3 mmol) in 95% ethanol (110 ml) was
added. The mixture was stirred overnight, filtered on Cel-
iteꢂ, and concentrated. Diethyl ether was added and the
organic layer was washed with a saturated aqueous NaCl
H NMR (CDCl , 300 MHz) d 0.04 (s, 6H), 0.87 (s,
3
9H), 1.90–2.03 (m, 2H), 2.00 (s, 3H), 2.03 (s, 3H),
2.49–2.58 (m, 2H), 3.79 (s, 3H), 3.82 (s, 6H), 4.30 (d,
2H, J = 5.9 Hz), 4.61 (s, 2H), 5.27 (br t, 1H,
1
3
J = 5.9 Hz), 5.84 (t, 1H, J = 5.9 Hz), 6.35 (s, 2H).
C
solution, dried over MgSO , and concentrated to give the
4
NMR (CDCl , 75 MHz) d ꢀ5.1 (2 · CH ), 18.3 (C),
3
3
1
crude product 12 in 86% yield. H NMR (CDCl3,
20.9 (CH ), 21.2 (CH ), 25.9 (3 · CH ), 32.3 (CH ),
3
3
3
2
300 MHz) d 1.9 (m, 3H), 2.65 (m, 2H), 3.69 (t, 2H,
J = 6.4 Hz), 3.82 (s, 3H), 3.84 (s, 6H), 6.42 (s, 2H).
35.1 (CH ), 56.1 (2 · CH ), 59.5 (CH ), 59.6 (CH ),
2
3
2
2
60.9 (CH ), 75.3 (CH), 105.35 (2 · CH), 132.7 (C),
3
1
(C), 170.6 (C); MS: m/z (ESI+) 528 [M+NH ] .
34.7 (CH), 136.3 (C), 136.9 (C), 153.2 (2 · C), 170.1
1
9
+
4
.10. 3-(3,4,5-Trimethoxy-phenyl)-propan-1-al (13)
4
To a solution of 12 (3.1 g, 13.7 mmol) in CH Cl
2
4.13. Acetic acid 2-acetoxymethyl-4-hydroxy-1-[2-(3,4,5-
trimethoxy-phenyl)-ethyl]-but-2- enyl ester (16)
2
(
380 ml), under argon, at 0 ꢁC, was added Dess–Martin
periodinane (6.97 g, 16.4 mmol). The reaction mixture
was stirred at room temperature and monitored by
TLC. After disappearance of the starting material, the
mixture was poured into a saturated aqueous solution
of Na S O /NaHCO (700 ml, 1:1) and shaken vigor-
To a solution of 15 (76 mg, 0.149 mmol) in THF (2.3 ml)
was added an excess of HFÆPyridine (0.136 ml,
1.161 mmol). The mixture was stirred at room tempera-
ture and monitored by TLC. After disappearance of the
starting material, the solution was concentrated and
2
2
3
3
ously for 5 min. The aqueous layer was extracted with
diethyl ether. The combined organic layers were washed
with a saturated aqueous NaHCO solution, dried over
then purified by flash chromatography (light petro-
leum/Et O, 2:8 ! 0:1) to give 16 in 65% yield.
1
H
3
2
MgSO , and concentrated under vacuum. The crude
4
NMR (CDCl , 300 MHz) d 1.87–2.06 (m, 2H), 2.02 (s,
3
product was purified by flash chromatography (light
petroleum/Et O, 6:4) to give 13 in 93% yield.
3H), 2.05 (s, 3H), 2.30 (br s, OH), 2.47–2.64 (m, 2H),
3.80 (s, 3H), 3.83 (s, 6H), 4.26 (br d, 2H, J = 6.4 Hz),
4.62 (d, 1H, J = 12.5 Hz), 4.71 (d, 1H, J = 12.5 Hz),
5.26 (dd, 1H, J = 8.0, 5.4 Hz), 5.93 (t, 1H, J = 6.4 Hz),
1
H
2
NMR (CDCl , 300 MHz) d 2.75–2.92 (m, 4 H), 3.81
3
(
s, 3H), 3.84 (s, 6H), 6.40 (s, 2H), 9.82 (s, 1H).
1
3
6
.36 (s, 2H). C NMR (CDCl , 75 MHz) d 21.0
3
4
(
.11. 2-[2-(1,1,2,2-Tetramethyl-propoxy)-ethylidene]-5-
3,4,5- trimethoxy-phenyl)-pentane-1,3-diol (14)
(CH ), 21.2 (CH ), 32.3 (CH ), 35.2 (CH ), 56.1
3 3 2 2
(2 · CH ), 58.5 (CH ), 59.4 (CH ), 60.9 (CH ), 75.2
3
2
2
3
(
CH), 105.4 (2 · CH), 133.1 (CH), 134.6 (C), 136.4
To a solution of 3 (150 mg, 0.305 mmol) in THF (5 ml),
under argon, at ꢀ35 ꢁC, was added dropwise MeLiÆLiBr
(C), 136.8 (C), 153.3 (2 · C), 170.3 (C), 171.0 (C); MS:
+
m/z (ESI+) 414 [M+NH ] .
4
(
1.5 M in Et O, 0.427 ml, 0.641 mmol). The solution was
2
kept at ꢀ35 ꢁC for 2 h and then 13 (75 mg, 0.336 mmol)
4.14. Acetic acid 2-acetoxymethyl-4-oxo-1-[2-(3,4,5-tri-
methoxy-phenyl)-ethyl]-but-2-enyl ester (17)
was added. The solution was kept at ꢀ35 ꢁC for 3 h and
quenched with a NH Cl solid (50 mg). The mixture was
4
filtered on Celiteꢂ, concentrated, and purified by
To a solution of 16 (38 mg, 0.096 mmol) in CH Cl
2
2
flash
1
chromatography
(light
petroleum/Et O,
(2.6 ml) under argon at 0 ꢁC was added Dess–Martin
periodinane (49 mg, 0.115 mmol). The mixture was add-
ed at room temperature and monitored by TLC. After
disappearance of the starting material, the mixture was
poured into a separating funnel containing a saturated
aqueous Na S O / NaHCO solution (5 ml, 1:1) and
2
1
:1 ! 3:7 ! 0:1) to give 14 in 60% yield. H NMR
(
CDCl , 300 MHz) d 0.08 (s, 6H), 0.89 (s, 9H), 1.89–
3
1
6
2
96 (m, 2H), 2.53–2.74 (m, 4H), 3.81 (s, 3H), 3.83 (s,
H), 4.18 (br t, 1H, J = 6.6 Hz), 4.23 (s, 2H), 4.28 (d,
H, J = 6.0 Hz), 5.70 (t, 1H, J = 6.0 Hz), 6.41 (s, 2H).
C NMR (CDCl , 75 MHz) d ꢀ5.1 (2 · CH ), 18.4
2
2
3
3
1
3
shaken vigorously for 5 min. The aqueous layer was
extracted with diethyl ether (3 · 5 ml). The combined
organic layers were washed with saturated aqueous
NaHCO solution, dried over MgSO , and concentrated
3
3
(
(
(
(
C), 26.0 (3 · CH ), 32.7 (CH ), 37.4 (CH ), 56.2
3
2
2
2 · CH ), 58.5 (CH ), 59.6 (CH ), 60.9 (CH ), 75.9
3
2
2
3
CH), 105.4 (2 · CH), 129.0 (CH), 136.2 (C), 137.7
C), 142.6 (C), 153.2 (2 · C); MS: m/z (ESI+) 444
3
4
under vacuum to give crude product 17 in a quantitative
+
1
[
M+NH ] .
yield. H NMR (CDCl , 300 MHz) d 1.97–2.10 (m, 2H),
4
3
2
.04 (s, 3H), 2.10 (s, 3H), 2.51–2.71 (m, 2H), 3.80 (s,
4
.12. Acetic acid 2-acetoxymethyl-4-(1,1,2,2-tetramethyl-
3H), 3.83 (s, 6H), 4.98 (d, 1H, J = 13.9 Hz), 5.08 (d,
1H, J = 13.9 Hz), 5.33 (br t, 1H, J = 6.3 Hz), 6.11 (d,
1H, J = 7.4 Hz), 6.35 (s, 2H), 10.08 (d, 1H,
propoxy)-1-[2-(3,4,5-trimethoxy- phenyl)-ethyl]-but-2-
enyl ester (15)
1
3
J = 7.4 Hz). C NMR (CDCl , 75 MHz) d 20.7 (CH ),
3
3
A solution of 14 (71 mg, 0.166 mmol), acetic anhydride
0.0633 ml, 0.666 mmol), and DMAP (1 mg) in pyridine
21.0 (CH ), 32.2 (CH ), 35.1 (CH ), 56.2 (2 · CH ),
3
2
2
3
(
59.3 (CH ), 60.9 (CH ), 73.3 (CH), 105.4 (2 · CH),
2
3

J. Bourdron et al. / Bioorg. Med. Chem. 14 (2006) 5540–5548
5547
1
(
4
28.7 (CH), 136.2 (C), 136.5 (C), 153.4 (2 · C), 154.9
C), 170.0 (C), 170.2 (C), 190.5 (CH); MS: m/z (ESI+)
4.18. In vitro Capillary network formation
A 24-well culture plate was coated with 250 lL of
+
12 [M+NH ] .
4
TM
5
.25 mg/mL Matrigel (BD Sciences) at 4 ꢁC. Matri-
TM
4
(
.15. Acetic acid 4-acetoxy-2-acetoxymethylene-1-[2-
3,4,5-trimethoxy-phenyl)-ethyl]-but-3- enyl ester (18)
gel was then allowed to solidify for 1 h at 37 ꢁC before
cell seeding. One hour later, the drug was added for 4 h
at 37 ꢁC and photographs were taken after incubation
using Metamorph software (Universal Imaging Corp.,
Downingtown, PA) and a DM-IRBE microscope (Lei-
ca, Bensheim, Germany) coupled with a digital camera
(CCD camera coolsnap FX, Princeton Instruments,
Trenton, NJ). The formation of capillary networks
was quantitatively evaluated by measuring the total cap-
illary tube length in 10 view fields per well using Meta-
view software. Sigma Stat software (Jandel Scientific,
San Rafael, CA) was used to perform statistical analysis.
Data were expressed as means ± SD.
In a dry Schlenk tube, a solution of 17 (38 mg,
.096 mmol), DMAP (12 mg, 0.096 mmol), NEt3
3 ml), and acetic anhydride (0.027 ml, 0.289 mmol)
0
(
was stirred under argon at 80 ꢁC and monitored by
TLC. After disappearance of the starting material, the
mixture was concentrated under vacuum. The crude
product was then purified by flash chromatography
(light petroleum/Et O, 1:1) to give two isomers (1/1
2
E,Z/Z,Z) 18 and iso-18 in 76% yield. H NMR of 18
1
(
CDCl , 300 MHz) d 1.88–2.30 (m, 4H), 2.08 (s, 3H),
3
2
3
1
.09 (s, 3H), 2.15 (s, 3H), 2.53–2.60 (m, 2H), 3.81 (s,
H), 3.85 (s, 6H), 5.83 (d, 1H, J = 12.6 Hz), 5.92 (t,
H, J = 7.3 Hz), 6.39 (s, 2H), 7.23 (s, 1H), 7.63 (d, 1H,
Acknowledgments
1
J = 12.6 Hz). H NMR of iso-18 (CDCl , 300 MHz) d
3
1
2
3
.63–1.89 (m, 2H), 1.99–2.28 (m, 2H), 2.06 (s, 3H),
.12 (s, 3H), 2.21 (s, 3H), 2.48–2.63 (m, 2H), 3.81 (s,
H), 3.85 (s, 6H), 5.22 (d, 1H, J = 7.2 Hz), 5.94 (t, 1H,
J.B. thanks DIPTA and La region PACA for financial
support.
J = 7.2 Hz), 6.39 (s, 2H), 7.25 (d, 1H, J = 7.2 Hz), 7.84
(
1
3
s, 1H); C NMR (CDCl , 75 MHz) d 20.6 (CH ),
3 3
References and notes
2
0.7 (CH ), 20.8 (CH ), 20.9 (CH ), 21.1 (CH ), 21.2
3 3 3 3
(
(
1
1
(
1
(
CH ), 31.8 (CH ), 32.0 (CH ), 34.1 (CH ), 34.4
3 2 2 2
1
2
. (a) Paul, V. J.; Fenical, W. Mar. Ecol. Prog. Ser. 1986, 34,
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1
. Commeiras, L.; Bourdron, J.; Douillard, S.; Barbier, P.;
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2
3
3
57–169; (b) Paul, V. J.; Fenical, W. Bioorg. Mar. Chem.
987, 29, 1–29.
03.7 (CH), 105.4 (4 · CH), 109.3 (CH), 117.1 (C),
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C), 136.8 (C), 137.2 (CH), 137.9 (CH), 153.3 (4 · C),
67.27 (C), 167.3 (C), 167.4 (C), 167.9 (C), 170.2
+
2 · C); MS: m/z (ESI+) 454 [M+NH ] .
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3
8, 3593–3596.
4
.16. Growth inhibition assays
4
. (a) Lem e´ e, R.; Pesando, D.; Durand-Clement, M.; Dub-
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bated for 24 h before adding different concentrations of
drug. After a 72-h drug treatment, cells were incubated
for 2 h with 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) which allows us to
measure growth inhibition. The stain was eluted with
1
(
00 lL DMSO and absorbance was measured at 550 nm
MR-7000, Dynatech, Denkendorf, Germany). Percent-
6
. Blokhin, A. V.; Yoo, H-D.; Geralds, R. S.; Nagle, D. G.;
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.17. Tubulin polymerization
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2
0
matography. Tubulin was stored in liquid nitrogen
2
1
and prepared for use as described. Tubulin concentra-
tion was determined spectrometrically in 6 M guanidine
ꢀ
1
ꢀ1
HCl (E275nm = 1.09 L g cm ). The buffer used to
polymerize tubulin is 20 mM sodium phosphate buffer,
1
1
1
1
6
mM EGTA, 3.4 M glycerol, and 0.1 mM GTP, pH
.95. After 35 min incubation at 37 ꢁC with drug, the
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2
by turbidimetry at 350 nm in a Beckman DU 7400
spectrophotometer.
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7277–7287.

5548
J. Bourdron et al. / Bioorg. Med. Chem. 14 (2006) 5540–5548
1
1
1
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